Indian Journal of Research in Pharmacy and Biotechnology (IJRPB) Vol-1 Issue-2

Indian Journal of Research in Pharmacy and Biotechnology ISSN: 2320-3471 (Online)
Editor
B.Pragati Kumar, M.Pharm, Assistant Professor, Nimra College of Pharmacy
Consulting editor
Dr. S Duraivel, M.Pharm, Ph.D., Principal, Nimra College of Pharmacy
Associate Editors
Dr. A. Ravi Kumar, M.Pharm., Ph D. Dr. Emdad Hossain, M. Pharm., Ph D. Dr. S. Sangeetha, M.Pharm., Ph.D. Dr. Ramana Reddy, M.Pharm., Ph.D., F.I.C. Dr. M. Janardhan, M.Pharm., Ph.D. Mr. Lokesh Deb, M.Pharm.,(Ph.D) Mr. Debjit Bowmick, M.Pharm., (Ph.D) Mr. Harish Gopinath, M.Pharm., (Ph.D) Dr. Sankhadip Bose, M.Pharm., Ph.D. Dr. K.P.Sampath Kumar, M. Pharm., Ph D.
Editorial Advisory Board

Dr.Y.Narasimaha Reddy, M. Pharm., Ph D. Dr.V.Gopal, M. Pharm., Ph D. Dr. J.Balasubramanium, M. Pharm., Ph D. Dr.P.Ram Reddy, M. Pharm., Ph D. Dr. V.Prabhakar Reddy, M. Pharm., Ph D. Dr. S.D.Rajendran, M. Pharm., Ph D. Dr. Chinnala Krishnamohan, M. Pharm., Ph D. Dr.T.Venkateswara Rao, M.Pharm., Ph.D. Dr. Vijay Kumar, M.Pharm., Ph.D. Mr. A.Madhusudhan Reddy, M.Pharm.,(Ph.D) Prof.M.Ravi, M.Pharm, (Ph.D) Mr. C. Narendhar, M.Pharm. Mr.Subhashis Debnath, M.Pharm.,(Ph.D) Mr. Diptanu Biswas, M.Pharm. Mr.Digpati Roy, M.Pharm.,(Ph.D) Mr.Praneta Desale, M.Pharm. Mr.Nikhil P Jogad, M.Pharm.,(Ph.D) Mr.Shambaditya Goswami, M.Pharm.,(Ph.D) Mr.Samaresh Pal Roy, M.Pharm.,(Ph.D) Mr. Pulak Majumder, M.Pharm.,(Ph.D) Dr. R.Margret Chandira, M.Pharm., Ph.D. Mr.Akhilesh Prasad Yadav, M.Pharm.,(Ph.D) Mr.Rajnish Kumar Singh, M.Pharm. Mr. Supriya Das, M.Pharm.,(Ph.D) Mr.Pradip Das, M.Pharm. Mr. Vinod Raghuwanshi, M.Pharm. Mr. Shravan Kumar Paswan, M.Pharm. Mr. Praveen Khirwadkar M.Pharm.,(Ph.D)
Volume 1 Issue 2
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Indian Journal of Research in Pharmacy and Biotechnology ISSN: 2320-3471 (Online)

Indian Journal of Research in Pharmacy and Biotechnology is a bimonthly journal, developed and published in collaboration with Nimra College of Pharmacy, Ibrahimpatnam, Vijayawada, Krishna District, Andhra Pradesh, India-521456 Printed at: F. No: 501, Parameswari Towers, Ibrahimpatnam, Vijayawada, India -521456
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Volume 1 Issue 2
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ISSN: 2320 3471(Online)
Indian Journal of Research in Pharmacy and Biotechnology

Contents Antidepressant activity of ethanolic extract of plant Kalanchoe pinnata (lam) pers in mice Shashank Matthew, Ajay Kumar Jain, Cathrin Matthew, M.Kumar, Debjit Bhowmik Antinociceptive and anti-inflammatory activity of Tecoma stans leaf extracts V Lakshmi Prasanna, K Lakshman, Medha M Hegde and Vinutha Bhat Recent trends in usage of polymers in the formulation of dermatological gels Shaik Arif Bhasha, Syed Abdul Khalid, S.Duraivel, Debjit Bhowmik, K.P.Samapth Kumar Recent trends of polymer usage in the formulation of orodispersible tablets J.Preethi, MD Farhana, B.Chelli Babu, MD.Faizulla, Debjit Bhowmik, S.Duraivel Design and development of amlodipine besylate fast dissolving tablets by using natural superdisintegrants N.Narasimha Rao, B.Radha Krishna Murthy, D.Rajasekhar, P. Suri Babu, K. Phaneendra Babu, Srinivasa Babu. P Formulation optimization and evaluation of liposomal gel of prednisolone by applying statistical design Varde Neha M, Thakor Namita M, C.Sini Srendran, Shah Viral H ADR monitoring in hypertension outpatient department of hospital Sreenu Thalla, K.Venkatta Ramana, Sk.Sheherbanu, Sk.Ashya, A.Manikanteswara Reddy, B.Lakshmi Evaluation of the antioxidant and hepatoprotective activity of Madhuca longifolia (koenig) leaves Arun Kumar, Kaushik Biswas, S Ramachandra Setty Formulation and evaluation of sustained release matrix tablets of Metformin hydrhocloride A Madhusudhan Reddy, Ayesha Siddika, P Surya Bhaskara Rao, Herbal medicine Atheeq-ur-Rahman, Ismail Shaik , K.P.Samapth Kumar Microsponge drug delivery system SK Shafi, S.Duraivel, Debjit Bhowmik, K.P.Sampath Kumar Nanotherapeutics an era of drug delivery system in nanoscience Bhargavi, Ch.Anil, Debjit Bhowmik, Praneta Desale, K.P.Sampath Kumar A validated RP-HPLC method for the estimation of Baclofen in bulk drug and pharmaceutical formulations Rajesh M, Manzoor Ahmed, Maanasa Rajan BN A validated RP-HPLC method for the estimation of Cisapride in bulk drug and pharmaceutical formulations Maanasa Rajan.B.N, Manzoor Ahmd, Rajesh M Review article on antimicrobial resistance Maryam Bincy Thomas, Suruchi Singh Urinary tract infection: causes, symptoms, diagnosis and its management M. Komala, K.P.Sampath Kumar Diabetes epidemic in India: risk factors, symptoms and treatment Abhinov T, Md Aasif Siddique Ahmed Khan, Ashrafa, Shabana Parveen, K.P.Samapth Kumar Simultaneous estimation of Olmesartan and Atorvastatin in bulk and fromulation by using UV spectroscopy and RP-HPLC Revanth Reddy B, Aravind G Development and validation of RP-HPLC method for the determination of Cefdinir in bulk and capsule dosage form P.Ravisankar, G.DevalaRao, M.KrishnaChaitanya Simultaneous separation of six Fluoroquinolones using an isocratic hplc system with uv detection: application to analysis of levofloxacin in pharmaceutical formulations Ravisankar Panchumarthy, Devala Rao Garikapati, Krishna Chaitanya Manukonda, Sandhya Rani Nagabhairava Page Nos. 153-155 156-160 161-168 169-174 175-179
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Volume 1 Issue2
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ISSN: 2320 3471(Online)
Indian Journal of Research in Pharmacy and Biotechnology
INDIAN JOURNAL OF RESEARCH IN PHARMACY AND BIOTECHNOLOGY Instructions to Authors

Manuscripts will be subjected to peer review process to determine their suitability for publication provided they fulfill the requirements of the journal as laid out in the instructions to authors. After the review, manuscripts will be returned for revision along with reviewers and/or editors comments. Kindly follow the below guidelines for preparing the manuscript: 1. Prepare the manuscript in Times New Roman font using a font size of 12. There shall not be any decorative borders anywhere in the text including the title page. 2. Dont leave any space between the paragraphs. 3. Divide the research article into a. Abstract b. Introduction c. Materials and Methods d. Results e. Discussion f. conclusion g. References 4. References should include the following in the same order given below a) Author name followed by initials b) Title of the book/ if the reference is an article then title of the article c) Edition of the book/ if the reference is an article then Journal name d) Volume followed by issue of the journal e) Year of publication followed by page numbers 5. Download the author declaration form from the web site www.ijrpb.com, fill it and submit it after signing by corresponding and co-authors to IJRPB. You can send the filled in form by post or scanned attachment to ijrpb@yahoo.com. 6. Keep in touch with the editor through mail or through phone for further clarifications as well as for timely publication of your article.
Volume 1 Issue2
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Shashank Mathew et.al
ISSN: 2320 3471(Online) Indian Journal of Research in Pharmacy and Biotechnology
ANTIDEPRESSANT ACTIVITY OF ETHANOLIC EXTRACT OF PLANT KALANCHOE PINNATA (LAM) PERS IN MICE
Shashank Matthew1*, Ajay Kumar Jain2, Cathrin Matthew2, M.Kumar3, Debjit Bhowmik4 1. Department of Pharmaceutical Sciences, Sardar Patel College of technology, Balaghat 2. Government district hospital, Balaghat 3. Vinayaka missions college of Pharmacy, Salem 4. Karpagam University, Coimbatore * Corresponding author: Email: sheak1980@gmail.com ABSTRACT The main objective of present work is to find out good pharmacological activities in herbal source with their preliminary phytochemical study, and also it is aimed to investigate anti-diabetic activity of ethenolic and aqueous extracts of dried stem of plant Kalanchoe pinnata (LAM)PERS against CNSDepressant activity in rats. Normally herbal products are free from side effects/adverse effects and they are low cost medicines, which will be beneficial for human being. The main objective of this work is to develop potent CNS-Depressant agent having no or minimum side effects from indigenous plants for the therapeutic management. KEYWORDS: Phytochemical study, Kalanchoe pinnata, 1. INTRODUCTION The World Health Organization (WHO) defined health as a complete state of physical, mental, and social well-being and not merely the absence of disease or infirmity. So during the past decade, traditional systems of medicine have become a topic of global importance. Current estimate suggest that, in many developing countries a large proportion of the population relies heavily on traditional practitioners and medicinal plants to meet primary health care needs. Although modern medicine may be available in these countries, herbal medicines (phytomedicines) have often maintained popularity for historical and cultural reasons. Concurrently, many people in developed countries have begun to turn to alternative or complementary therapies, including medicinal herbs. 2. MATERIAL AND METHODS 2.1. Collection and authentication of plant material: The specimen copy (Herbarium) of selected plant collected in month of july-2007 from ABS Botanical garden, Karripatty, Distt. - Salem, Tamil Nadu Mr.A. Balsubramnian, (Consultant-central siddha research) Executive Director ABS botanical garden, Salem, authenticated the plant as Kalanchoe Pinnata (LAM) PERS (Family- Crassulaceae). 2.2. Preparation of extract: The stem of Kalanchoe Pinnata (LAM) PERS were dried under shade and then powdered with a mechanical grinder. The powder was passed through sieve No. 30 and stored in an airtight container for further use. 2.3. Solvent for extraction: Petroleum Ether (60-80o C) Alcohol (95% v/v) Distilled water with chloroform (0.25%) 2.4. Extraction procedure: The dried powders of stem of Kalanchoe pinnata were defatted with petroleum ether (60-80c) in a Soxhlet Apparatus by continuous hot- percolation. The defatted powder material (marc) thus obtained was Further extracted with ethanol (95% v/v) with same method and fresh powder used for aqueous extraction by Cold maceration method. The solvent was removed by distillation under low pressure and evaporation. The resulting semisolid mass was vacuum dried by using rotary flash evaporator. The resultant dried extracts were used for further study. 2.5. Procurement of experimental animals: Swiss albino mice (20-25 g) and albino Wister rats (150-200 g) of either sex and of approximate same age are used in the present studies were procured from listed suppliers of Sri Venkateswara Enterprises, Bangalore, India. The animals were fed with standard pellet diet (Hindustan lever Ltd. Bangalore) and water ad libitum. All the animals were housed in polypropylene cages. The animals were kept under alternate cycle of 12 hours of darkness and light. The animals were acclimatized to the laboratory condition for 1 week before starting the experiment. The animals were fasted for at least 12 hours before the onset of each activity. The experimental protocols were approved by Institutional Animal Ethics Committee (IAEC No.-P.Col. / /2007) after scrutinization. The animals received the drug treatments by oral gavage tube. 2.6. CNS- depressant activity: Most of the central nervous system acting drugs influence the locomotor activities in man and animals. The CNS - Depressant drugs such as barbiturates and alcohols reduce the motor activity,
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while the stimulants such as caffeine and amphetamine increase the activity, in other words, the locomotor activity can be an index of wakefulness (alertness) of mental activity. In the present study the attempt has been focused to evaluate the CNS-Depressant activity of extracts of stem of plant Kalanchoe pinnata (LAM) PERS on the locomotor activity of mice using actophotometer. Table.1. Treatment design Group I Normal control (Normal saline 0.9%) Group II Positive control (Chlorpromazine 3 mg / kg i.p.) Group III Ethanolic extract (300mg / kg) Group IV Ethanolic extract (600mg / kg) Group V Aqueous extract (300mg / kg) Group VI Aqueous extract (600mg / kg) Procedure:1. Albino mice weighing between 150-200 gm was purchased from Venkateshwara Enterprises, Bangalore 2. Animals are divided into 6 groups. 3. The equipment was turned on and each animal are placed in activity cage and for 10 min. and the basal activity score is noted down. 4. Normal saline is administrated in the dose of 2 ml / kg to the first group (normal control). 5. Chlorpromazine (3mg / kg) was administered to II group of animal. 6. The animals of group III, IV, were treated with ethanolic extracts and V, VI with aqueous extracts. 7. After 45 min of the treatment, once again the animals were placed in the activity cage and the score was noted. The difference in the activity before and after treatment was noted. The percent decrease in motor activity was calculated and compared with control group of animals (Kulkarni, S. K- 2005). 8. CNS -- Depressant activity of alcoholic and aqueous extract of dried stem of plant kalanchoe pinnata (LAM.)PERS., on the locomotor activity of mice using actophotometer. Table 2. Evaluations of CNS-depressant activity Treatment design Dose Locomotor activity in 10mins. Before treatment after treatment Normal control 2ml/kg 224.5 1.88 220.5 1.75 Standard 3mg/kg 280.33 0.55* * 106.00 1.03* * (Chlorpromazine) Ethanolic extract 300mg/kg 216.83 0.74* * 110.16 0.65* * Ethanolic extract 600mg/kg 209.00 0.57* * 94.8 0.83* * Aqueous extract 300mg/kg 223.00 0.41 170.00 1.53* * Aqueous extract 600mg/kg 229.160 0.98* * 176.5 1.89* *
Group 1 2 3 4 5 6
% decrease in activity ----62.18 49.19 54.62 23.76 32.98
Values are expressed by Mean SEM P values: * * P< 0.01; * P <0.05. One way ANOVA followed by DUNNETTS, multiple comparison tests 3. CONCLUSION Alcoholic extract of plant Kalanchoe pinnata (LAM) PERS have more CNS-depressant activity as compared to aqueous extract but as compare to standard drug it shows near about same action.
REFERENCES Dhanurkar RA, Kulkarni NN, Pharmacology of medicinal plants and natural products, Ind J Pharmacology, 32, 2000, 81-118. Gupta SS, Prospects and perspective of natural plant products in medicine, Indian J of pharmacology 1994, 26, 1-12. John AO, Ojewole, Antinociceptive, anti-inflammatory and antidiabetic effects of Bryophyllum pinnatum (Crassulaceae) leaf aqueous extract, Journal of Ethnopharmacology 99, 1, 13-19.
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Joshi Shashank R, Shah Siddhart N, Rising global burden of Diabetes, The Asian J of Diabetology, 1(3), 1999, 13-15. Lenzen S, and Munday R, Thiol-group reactivity, hydrophilicity and stability of alloxan, its reduction products and its N-methyl derivatives and a comparison with ninhydrin, Biochemical Pharmacology, 42, 1991, 1385-1391. Lenzen S, Panten U, Alloxan: history and mechanism of action, Diabetologia, 31, 1988, 337-342. Lipnick RL, Cotruvo JA, Hill RN, Comparison of the up and down method and the fixed dose procedure acute toxicity procedures, Fd Chen, Toxic, 33, 1995, 223-231. Pincus I J, Hurwitz, Scott M E, effect of rate of injection of alloxan on development of diabetes in Rabbits, J Am Physio Soc, 86, 1954, 553-555. Ramachandran A, Snehlata C, Viswanthan V, burden of type 2 Diabetes and its complications, The Indian scenario, Current science, 83, 2002, 1471-1476. Salahdeen HM and Yemitan OK, Neuropharmacological effects of aqueous leaf extract of Bryophyllum Pinnatum in mice, African journal of biomedical research, 9, 2006, 101-107. Shukla R, Sharma S B, Puri D, Prabhu M K, and Murth P S, Medicinal plants useful in diabetes, Indian J Clinic Biochem, 15, 2002, 169. Siddharta P, Chaudhuri AKN, Further studies on the anti-inflammatory profile of the methanolic fraction of the fresh leaf extract of Bryouphyllum pinnatum, Fitoterapia, 63(5), 1992, 451-459. V Babu, Gangadevi T, Subramonian A, Antihyperglycemic activity of cassia Kleinl leaf extract in glucose fed normal rats and alloxan induced diabetic rats; Indian Journal of Pharmacology 2002, 34, 409-415.
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V Lakshmi Prasanna et.al
ANTINOCICEPTIVE AND ANTI-INFLAMMATORY ACTIVITY OF TECOMA STANS LEAF EXTRACTS

V Lakshmi Prasanna1*, K Lakshman2, Medha M Hegde2 and Vinutha Bhat2 1.Department of Pharmacognosy, Creative Educational Societys College of Pharmacy, Kurnool. A.P. 2. Department of Pharmacognosy, PES College of Pharmacy, Bangalore, Karnataka. *Corresponding author: E-mail:vlakshmi.prasanna.14@gmail.com
ABSTRACT Tecoma stans (Bignoniaceae) is used in the treatment of diabetes, stomach pains, rheumatism, diuretic, vermifuge and tonic in traditional medicine. The present study has been design to evaluate antinociceptive and anti-inflammatory activities of alcohol and aqueous extracts of Tecoma stans leaves and to determine total phenolic and flavonoid contents. Both extracts shows dose dependent activity. The antinociceptive activity was investigated using hot plate, acetic acid induced writhing and formalin induced paw licking methods. Anti-inflammatory activity was evaluated using carrageenan induced paw oedema method. Total phenolic and flavonoid content determined using standard chemical methods. Alcohol extract (500 mg/kg) showed highest 76.92% inhibition of inflammation after 24 hrs. Both the extracts produced increased in latency time compared to vehicle but alcohol extract showed highest activity after 150 min in hot plate method (4.63 0.08 sec) and inhibit nocipeptive response in both phase. Extracts also produced significant inhibition of writhing. Content of total phenolic and flavonoid also found more in alcoholic extract. These findings demonstrate that the alcohol leaf extract of Tecoma stans have excellent antinociceptive and anti-inflammatory activity, which may due to presence of higher phenolic and flavonoid content.
Key-words: Tecoma stans leaves; antinociceptive; anti-inflammatory; phenolic content; flavonoid content. 1. INTRODUCTION Tecoma stans (Bignoniaceae) is a fast growing small evergreen shrub tree, that grows to a height of 7.5meters. Leaves are compound and imparipinnate with 2 to 5 pairs of leaflets. Flowers occur in clusters at the ends of the branches and are trumpet shaped with 5 rounded lobes, 6 cm long, pale to bright yellow in color. Fruits are narrow, slightly flattened to pointed capsules, up to 20 cm long (Orwa C, 2009). The chemical constituents of Tecoma stans are triterpenes, hydrocarbons, resins, volatile oil. Leaves and stems contain flavonoids, chrysoeriol, luteolin, hyperoside, indole oxygenase. Alkaloids like tecomanine, tecostanine, 4noractinidine, 4-norskytanthine and boschniakine. Flowers contain -carotene and Zeaxanthin. Roots are used as vermifuge, diuretic, tonic (Yoganarasimhan, 1904). The leaves are claimed to be useful in the treatment of inflammation and pain. Even though, Tecoma stans was reported to be useful in many ailments, there are no reports regarding its antinociceptive and antiinflammatory activity. Hence in the current study, the anti-inflammatory and antinociceptive activity of alcohol and aqueous extracts of Tecoma stans leaves was studied using different animal models. This study is a scientific approach to validate the traditional use of the leaves of Tecoma Stans. 2. MATERIALS AND METHODS 2.1. Plant material: Dried leaves of Tecoma stans (Bignoniaceae) were collected from GKVK and authenticated by Dr.Rajanna from GKVK, Bangalore. A voucher specimen has been deposited at departmental herbarium for future reference (TS-10-03).The plant material was dried, powdered and stored in air tight containers for further studies. The powdered material weighing 500 g was extracted by Soxhlet using alcohol and water. The solvent was completely removed by using a rotary flash evaporator to get a semisolid mass. The Alcohol and aqueous extract yield is 10.80 and 21.75% W/W. 2.2. Animals: Albino mice and Wistar rats of either sex weighing 18-24 g and 150-200 g respectively, housed under standardized animal house conditions were used in all the experiments. They had access to standard pellet and water ad libitum. Animals were divided into six groups of six each. All the experiments are approved by Institutional Animal Ethics Committee, PES College of Pharmacy, India. 2.3. Preliminary phytochemical studies: Alcoholic & aqueous extracts of Tecoma stans were investigated for qualitative chemical examination which gives an idea regarding the chemical constituents present in the extracts. Phytochemical screening was done as explained in literature (Ikhiri, 1992). Volume 1(2) March-April 2013 Page 156
2.4. Determination of total phenolic content: The amount of phenol in the alcohol and aqueous leaf extract of Tecoma stans was determined with Folin-Ciocalteu reagent using the method (Olayinka A Aiyegoro, Anthony I Okoh, 2010). Gallic acid (0-0.5 mg/ml) was used as standard and results were expressed as mg/g gallic acid equivalent of dry extract. 2.5. Estimation of total flavonoids: Aluminum chloride colorimetric method was used for flavonoids determination (Hole K, Hunskaar S, 1987). The content was determined from extrapolation of calibration curve which was made by preparing quercetin solution (0-0.8 mg/ml) in distilled water. The concentration of flavonoid was expressed in terms of mg/g quercetine equivalent. 2.6. Antinociceptive activity 2.6.1. Hot plate method: The animals were placed on Eddy's hot plate maintained at a temperature of 55 0.5C. A cut-off period of 15 s was observed to avoid damage to the paw. Reaction time and the type of response were noted using a stopwatch. The response is in the form of jumping, withdrawal of the paws or the licking of the paws. Pentazocine was used as standard (10 mg/kg) which was administered i.p. The alcohol and aqueous extracts of Tecoma stans (250 and 500 mg/kg) were administered orally (Koster R, 1959). The response was observed at 0, 30, 60, 120 and 150 min. 2.6.2. Formalin induced paw licking model: One hour after oral administration of test compounds (250 and 500 mg/kg alcohol and aqueous extracts of Tecoma stans), 20 l of 1% formalin was injected into the paw of each animal. Duration of paw licking was monitored 0-5min (first phase) and 15-30min (second phase) after formalin injection. Pentazocine was used as a standard (10mg/kg) which was administered i.p (Winter CA, 1962). 2.6.3. Acetic acid induced writhing test: Albino mice were administered with different treatments orally one hour before acetic acid injection. Control group received only vehicle, and animals under standard group received Diclofenac sodium (10 mg/kg, p.o.). One hour after drug administration, 1% v/v acetic acid (0.1ml/10 g, i.p.) was injected. Five minutes after the intraperitoneal injection of acetic acid, number of writhing were counted for the period of 20 minutes (Ahamed KN, 2005). 2.7. Anti-inflammatory activity 2.7.1. Carrageenan-induced rat paw edema: Acute inflammation was produced by injecting 0.1ml of (1%) carrageenan (in a normal saline solution) into plantar surface of rat hind paw. The alcohol and aqueous extracts (250 and 500 mg/kg, orally), Diclofenac sodium (10 mg/kg, orally) as a reference agent were administered 60min before carrageenan injection. The paw edema volume was recorded using a plethysmometer at a different time intervals (Tjolsen A, 1992). 2.7.2. Statistical analysis: The results and data obtained in this study were evaluated using one-way analysis of variance (ANOVA) followed by Dunnetts multiple comparison test. The values are expressed as mean + SEM and p < 0.05 was considered significant. 3. RESULTS 3.1. Preliminary phytochemical analysis: Our qualitative preliminary phytochemical tests showed the presence of alkaloids, glycosides, saponins, phenols, tannins, proteins, carbohydrates, phytosterols in alcoholic extract of Tecoma stans, and aqueous extract of the plant showed similar constituents positive except phyosterols, while fixed oils & fats are absent in both the extracts. 3.2. Total phenolic and total flavonoid content: The total phenolic & flavonoid content is more in alcohol extract (72.3 mg/g GAE and 49.6 mg/g QE) than aqueous extract (64.2 mg/g GAE and 38.5 mg/g QE) (Table 1). Table 1: Presence of total phenolic and flavonoid content in the extracts Extract Total phenolic content Total flavonoid content (mg/g gallic acid equivalent) (mg/g quercetine equivalent) Alcohol extract 72.31.23 49.60.99 Aqueous extract 64.21.02 Results were expressed as meanSEM (n=3). 3.3. Antinociceptive activity: The extracts of Tecoma stans has shown significant dose dependent antinociceptive activity, however the alcohol extract (500 mg/kg) produced better activity than the aqueous extract. The alcohol extract (500 mg/kg) showed highest activity after 150 min in hot plate method as latency time increases to 4.63 0.08 sec after 150 min compare to 1.08 0.08 of control (Table 2). Volume 1(2) March-April 2013 Page 157 38.50.80
Table 2: Effect of alcohol and aqueous leaf extract of Tecoma stans on Hot plate model Treatment Dose(mg/kg) Initial 30min 60min 120min 150min Control 10 1.020.09 0.940.05 1.160.07 1.270.01 1.080.08 Pentazocine 10 1.140.04 2.420.19*** 4.060.03*** 5.360.08*** 5.800.06*** Alcohol extract 250 0.970.01 1.080.05 1.940.01*** 2.620.06*** 3.260.03*** Alcohol extract 500 0.930.04 1.170.01* 2.690.04*** 3.740.07*** 4.630.08*** Aqueous extract 250 1.010.02 1.090.05 1.180.01 2.100.01*** 2.350.06*** Aqueous extract 500 0.950.07 1.150.02* 2.060.03*** 3.170.07*** 3.980.03*** Values are mean SE, n=6, ***P < 0.001, **P < 0.01 and *P < 0.05 using one-way ANOVA followed by Dunnetts test. Oral administration of alcohol extract (250 and 500 mg/kg) produced inhibition of 28.48% and 37.43% pain response in first phase and inhibition of 53.41% and 74.56% paw licking response in second phase. Aqueous extract produce comparatively less effect than the alcohol extract and produced inhibition of 23.98% and 29.73% pain response in first phase and inhibition of 40.33% and 59.46% pain response in second phase at a dose of 250 mg and 500 mg/kg respectively (Table 3). Table 3. Effect of alcohol and aqueous leaf extract of Tecoma stans on Formalin induced paw licking model Treatment Dose %inhibition of Paw licking in %inhibition of Paw licking in mg/kg early phase 0-5min late phase 15-30min Control 10 Pentazocine 10 47** 80.22*** Alcohol extract 250 28.48* 53.41*** Alcohol extract 500 37.43** 74.56*** Aqueous extract 250 23.98 40.33*** Aqueous extract 500 29.73* 59.46*** Values are mean SE, n=6, ***P < 0.001, **P < 0.01 and *P < 0.05 using one-way ANOVA followed by Dunnetts test. Alcohol extract produced 36.5% and 53.5% inhibition of writhing response in low and high dose respectively. Aqueous extract has produced less inhibition than the alcohol extract (Table 4). Table 4: effect of alcohol and aqueous leaf extract of Tecoma stanus on acetic acid induced writhing model Treatment Dose (mg/kg) Number of writhing % inhibition Control 70.5 Diclofenac sodium 3 25.8 63.4*** Alcohol extract 250 44.7 36.5** Alcohol extract 500 32.9 53.3*** Aqueous extract 250 50.2 28.7* Aqueous extract 500 39.3 44.2*** 3.4. Anti-inflammatory activity: The test extracts at doses of 250 and 500 mg/kg as well as diclofenac sodium (10 mg/kg), showed significant inhibition of edema in dose dependent manner 3 h after carrageenan-induced inflammation, when compared with the control. Both alcohol extract and aqueous extract of Tecoma stans produced dose dependent inhibition of paw edema, The percentage inhibition of edema was 63.3%, 76.92%, 57.05% and 64.74% against alcohol extract (250 mg/kg and 500 mg/kg) and aqueous extract (250 mg/kg and 500 mg/kg) respectively after 24h (Table 5).
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Table 5: Effect of alcohol and aqueous leaf extract of Tecoma stans on Carrageenan induced rat paw edema model. Treatment Dose (mg/kg) % inhibition of paw edema Inflammation 3h 24h Control 0.00 0.00 Diclofenac sodium 10 35.60*** 79.48*** Alcohol extract 250 23.48 63.34*** Alcohol extract 500 30.06*** 76.92*** Aqueous extract 250 11.36 57.05*** Aqueous extract 500 21.20 64.74*** Values are mean SEM, n=6, ***P < 0.001, **P < 0.01 and *P < 0.05 using one-way ANOVA followed by Dunnetts test. 4. DISCUSSION Both the extracts showed activity in a dose dependent manner. The alcohol extracts showed potent antinociceptive and anti-inflammatory activity compare to aqueous extract. The hot plate test is considered to be selective for opioid like compounds, which are centrally acting analgesic in several animal species. The hot plate method has been found to be suitable for evaluation of centrally acting analgesic (Shibata, 1989).The alcoholic and aqueous extracts at low and high doses (250 and 500 mg/kg) increase the reaction time in dose dependent manner to the thermal stimulus. The highest antinociception of thermal stimulus was exhibited at higher dose of alcohol extract than aqueous extract. This could be the possible explanation for its central analgesic activity observed in hot plate test. The formalin induced paw licking test is a valid and reliable model for analgesic activity and it is sensitive for various classes of analgesic drugs. Formalin test produces a distinct biphasic response and different analgesics may act differently in the early and late phases of this test. Therefore, the test can be used to clarify the possible mechanism of the antinociceptive effect of a proposed analgesic (Rosland, 1990). Centrally acting drugs such as opioids inhibit both phases equally (Taesotikul, 2003). But peripherally acting drugs such aspirin; indomethacin and dexamethasone only inhibit the late phase. The late phase seems to be anti inflammatory response with inflammatory pain that can be inhibited by anti-inflammatory drugs (Marsha, 2002). The alcoholic and aqueous extracts exhibited a significant antinociception in both early and late phase of the formalin test. Acetic acid induced writhing test, a model of chemo-nociception and it induced pain by increasing fluids of PGE2 and PGE2. Acetic acid also induces sympathetic nervous system mediators, which are found in high level at first 30 min after acetic acid injection. This probably indicates that the analgesic activity of the extracts was mediated by inflammatory as well as neurogenic mechanisms. The alcohol and aqueous extract exhibited significant, dose-dependent decrease in the number of abdominal constrictions. However alcohol extract has shown good activity compare to aqueous extract. Carrageenan induced paw edema is characterized by a biphasic events, with involvement of different inflammatory mediators (Marsha KMG, 2002). In first phase (during the first 2h after carrageenan injection) chemical mediators such as histamines and serotonin play a role (Marsha, 2002) while in second phase (3-5h) after carrageenan infection kinin and prostaglandins are also released. Administration of alcohol extract at 250 and 500 mg/kg inhibited the edema from the 3h after carrageenan challenge, and aqueous extract at dose of 250 and 500 mg/kg inhibited the edema from 4h after carrageenan challenge, which probably inhibits the different aspects and chemical mediators of inflammation. The phytochemical analysis of various extracts showed the presence of carbohydrates, alkaloids, glycosides, tannins, saponins, phytosterols, phenolic compounds, proteins, amino acids, flavonoids, gums and mucilage. Our result also showed that extract contain significant amount of total phenolic and flavonoid content. The anti-inflammatory effect of Tecoma stans may be due to the presence of flavonoids. It has been reported that flavonoids possess anti-inflammatory and analgesic activity. Flavonoids are known to target prostaglandins which are involved in the late phase of acute inflammation and pain perception. Hence, the presence of flavonoids may be contributory to the anti-inflammatory and analgesic activities of Tecoma stans. This study confirms the antinociceptive and anti-inflammatory activity of leaves of Tecoma stans. Both activities were found to be comparable with reference drug. Further studies need to be done to identify and separate the Volume 1(2) March-April 2013 Page 159
group of active constituents responsible for antinociceptive and anti-inflammatory activity from alcohol and aqueous extracts. ACKNOWLEDGEMENT Authors are thankful to the Management, Director, Principal, Dr.S.Mohan, PES college of Pharmacy for providing necessary facilities to carry out this work & Dr.Rajanna, GKVK, Bangalore, for identifying the plant. REFERENCES Ahamed KN, Kumar V, Raja S, Mukherjee K, Mukherjee PK. Anti- nociceptive and anti- inflammatory activity of Araucaria bidwilli Hook, IJPT, 1, 2005, 105-109. Hole K, Hunskaar S, The formalin test in mice dissociation between inflammatory and non inflammatory pain. Pain, 30, 1987, 103-111. Ikhiri K, Boureima D, Dan-Kouloudo D. Chemical screening of medicinal plants used in traditional pharmacopoeia of Niger, Int J Pharmacog, 30, 1992, 251262. Koster R, Anderson M, De-Beer EJ, Acetic acid analgesic screen Fed Proc Fed Am Soc Exp Biol, 18, 1959, 418-420. Marsha KMG, Everton TA, Oswald SR. Preliminary investigation of the Anti-inflammatory properties of an aqueous extract from Morinda citrifolia (Noni), Proc West Pharmacol Soc, 45, 2002, 76-78. Olayinka A Aiyegoro, Anthony I Okoh, Preliminary phytochemical screening and In vitro antioxidant activities of the aqueous extract of Helichrysum longifolium, BMC Complementary and Alternative Medicine, 10, 2010, 1472-6882. Orwa C, Mutua A, Kindt R, Jamnadass R, Anthony S, Agroforestry Database:a tree reference and selection guide version. Available at: http://www.worldagroforestry.org/sites/treedbs/treedatabases.asp.2009, Accessed on 14 June 2010. Rosland JH, Tjoisen A, Maehle B, Hole K, The formalin test in mice: effect of formalin concentration. Pain, 1990, 42, 235. Shibata M, Ohkubo T, Takahashi H, Inoki R. Modified formalin test characteristic biphasic pain response. Pain, 38, 1989, 347. Taesotikul T, Panthong A, Kanjanapothi D, Verpoorte R, Scheffer JJC. Anti- inflammatory,antipyretic and antinoceceptive activities of Tabernaemontaa pandacaqui Poir, J of Epharmacol, 84, 2003, 31-35. Tjolsen A, Berge OG, Hunskaar S, Rosland JH, Hole K, The formalin test: an evaluation of the method. Pain, 51, 1992, 5. Winter CA, Risley EA, Nuss GW, Carragenan induced edema in hind paw of the rat as assay for anti inflammatory drugs, Proceedings of the society for experimental biology and medicine, 1962, 11, 544-547. Yoganarasimhan SN, Medicinal plants of India, Bangalore, India, 10th ed, Interline publisher pvt limited. 1904, 81.
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Shaik Arif basha et.al
RECENT TRENDS IN USAGE OF POLYMERS IN THE FORMULATION OF DERMATOLOGICAL GELS

Shaik Arif Bhasha1, Syed Abdul Khalid1, S.Duraivel1, Debjit Bhowmik1, K.P.Samapth Kumar2* 1. Nimra college of pharmacy, Vijayawada, India 2. Department of Pharmaceutical Sciences, Coimbatore medical college, Coimbatore, India Corresponding author: Email:debjit_cr@yahoo.com ABSTRACT Topical preparations can be applied directly to an external body surface by spreading, rubbing, and spraying. The topical route of administration has been utilized either to produce local effect for treating skin disorder or to produce systemic drug effects. Within the major group of semisolid preparations, the use of transparent gels has expanded both in cosmetics and in pharmaceutical preparations. Gels often provide a faster release of drug substance, independent of the water solubility of the drug, as compared to creams and ointments. They are highly biocompatible with a lower risk of inflammation or adverse reactions, easily applied and do not need to be removed. Gels for dermatological use have several favorable properties such as being thixotropic, greaseless, easily spreadable, easily removed, emollient, non-staining, and compatible with several excipients and water soluble or miscile. Dosage form selection should include those delivery systems that are noncomedogenic. Gels tend to be most effective as they have faster absorption than creams. Gels containing only water tend to be slow to dry; so the addition of ethyl or isopropyl alcohol to the gel hastens their drying to a film. But some patients may need the less drying lotions or creams for dry or sensitive skin or for use during dry winter weather. INTRODUCTION Gels are semisolid systems in which a liquid phase is constrained within a three-dimensional polymeric matrix (consisting of natural or synthetic gums) in which a high degree of physical (or sometimes chemical) crosslinking has been introduced. Some of these systems are as clear as water in appearance, visually aesthetically pleasing as in gelatin deserts and other are turbid. The clarity range is from clear to a whitish translucent. The polymers are used between 0.5-15% and in most of the cases they are usually at the concentration between 0.5-2%. Gels are usually clear, transparent, semisolids, containing the solubilised active substances (Lachman, 1987). The term Gel was introduced in the late 1800 to name some semisolid material according to pharmacological, rather then molecular criteria. The U.S.P. defines gels as a semisolid system consisting of dispersion made up of either small inorganic particle or large organic molecule enclosing and interpenetrated by liquid. The inorganic particles form a three-dimensional house of cards structure. Gels consist of two-phase system in which inorganic particles are not dissolved but merely dispersed throughout the continuous phase and large organic particles are dissolved in the continuous phase, randomly coiled in the flexible chains. Advantages: Avoidance of first pass metabolism. Convenient and easy to apply. Avoidance of the risks and inconveniences of intravenous therapy and of the varied conditions of absorption, like pH changes, presence of enzymes, gastric emptying time etc. Achievement of efficacy with lower total daily dosage of drug by continuous drug input. Avoids fluctuation in drug levels, inter- and intrapatient variations. Ability to easily terminate the medications, when needed. A relatively large area of application in comparison with buccal or nasal cavity Ability to deliver drug more selectively to a specific site. Avoidance of gastro-intestinal incompatibility. Providing utilization of drugs with short biological half-life, narrow therapeutic window. Improving physiological and pharmacological response. Improve patient compliance. Provide suitability for self-medication. Disadvantages: Skin irritation of contact dermatitis may occur due to the drug and/or excipients. Poor permeability of some drugs through the skin.
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Possibility of allergenic reactions. Can be used only for drugs which require very small plasma concentration for action Enzyme in epidermis may denature the drugs. Drugs of larger particle size not easy to absorb through the skin. Characteristics of gels: 1. Ideally gelling agents for pharmaceutical and cosmetic use should be inert, safe and nonreactive with other formulation components. A potential incompatibility is illustrated by the combination of cationic drug, preservative or surfactant with an anionic gel former. For example sodium alginate has been shown to reduce the concentration of cationic preservatives in solution as well as complex with chlorpheniramine, reduce the drug release rate from gelled formulation. Polyether has been shown to interact with phenols and carboxylic acids, leading to loss of potency. 2. The inclusion of a gelling agent in a formulation should provide a reasonable solid like nature during storage that can be broken when subjected to the shear force generated in shaking a bottle, squeezing a tube, or during topical application. A cost consideration requires a low concentration of gallant to produce the desired characteristics. 3. The gel should exhibit little viscosity change under the temperature variations of normal use and storage. For e.g. plastic base exhibits a lesser decrease in consistency than petrolatum over the some temperature range. This minimizes unacceptable changes in the products characteristics. 4. The gels particularly those of polysaccharide nature are susceptible to microbial degradation. Incorporation of a suitable preservative may prevent contamination and subsequent loss of gel characteristics due to microbial attack. 5. The gel characteristics should match the intended use. A topical gel should not be tacky. Too high a concentration of gel former or the use of an excessive molecular weight may produce a gel difficult to dispense or apply. An ophthalmic gel must be sterile. The aim in to produce a stable elegant, economic gel product adequately suited for its intended use. 6. Swelling: Gels can swell, absorbing liquid with an increase in volume (e.g. Xerogels). This is referred to as swelling and the pressure developed is known as swelling pressure. The swelling can be looked on as the initial phase of dissolution as osmosis occurs, where solvent penetrates the gel matrix. Gel-gel interactions are replaced by gelsolvent interactions. Limited swelling is usually the result of some degree of cross linking in the gel matrix that prevents total dissolution. Such gels swell considerably when the solvent mixture posses a solubility parameter comparable to that of the gallant. 7. Syneresis: Many gels systems undergo a contraction upon standing. The interstitial liquid is expressed, collecting at the surface of the gel. This process is referred to as syneresis or bleeding. Syneresis is not limited to organic hydrogels but has been seen in organogels and inorganic hydrogels. Typically syneresis becomes more pronounced as the concentration of polymer decreases. 8. Structure: Inorganic particles are capable of gelling a vehicle due to formation of a house of card structure. Clays e.g. bentonite or kaolin posses a lamellar structure that can be extensive hydrated. The flat surface of bentonite particles are negatively charged while the edges are positively hydrated. The flat surface of bentonite particles are negatively charged while the edges are positively charged. The attraction of face to edge of these colloidal lamellae creates a three-dimension network of particles throughout the liquid, immobilizing the solvent. The interactions between the particles are fairly weak, being broken by stirring or shaking. 9. Rheology: Solutions of gelling agents and dispersions of flocculated solids are typically pseudo plastic, exhibiting non-Newtonian flow behavior characterized by decreasing viscosity with increasing shear rate. Such behavior is due to progressive breakdown of the structure of the system. The tenuous structure of inorganic particles dispersed in water is disrupted by an applied shear stress. As shear stress is increased, more and more interparticulate associations are broken, resulting in a greater tendency to flow. Similarly, for macromolecules dispersed in a solvent, the applied shear tends to align the molecules in the direction of flow. The molecules straighten out, becoming less entangled as shear increases, thus lessening the resistance to flow. CLASSIFICATION: Based on colloidal phases: Gels are classified into inorganic (two phase system) and organic (single phase) gels on the basis of their nature of colloidal phase present. In a two phase system, if the particle size of the dispersed phase is relatively large and forms the three dimensional house of cards structures throughout the gel, then the gel mass sometime is referred to as magma (eg.bentonite magma). A gel with two phase system generally consists of floccules of small particles rather than large molecules and gel structure in such a system is not always stable. Both gels and magma may be thixotropic forming semisolids on standing and becomes liquid an agitation.
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A single phase gel consists of large organic molecules existing on twisted matted strands, dissolved in a continuous phase, such that no apparent boundaries exist between the dispersed macromolecules and the liquid. this large organic molecule either natural or synthetic polymers often referred as gel formers, tends to entangle with each other due to their random motion or bounded together by stronger types of vander-waals force so as to form crystalline or amorphous regions throughout the entire system. Single phase in macro sense considers the molecule to be dissolved in continuous solvent phase; however the unique behaviors of long macromolecules in solutions, leading to fairly high viscosities and gel formation makes it possible to consider such a system as two phase, at micro level- the dispersion of lyophillic colloidal polymer molecule and the continuous phase solvent. Single phase gels may be made from synthetic macromolecules (E.g. carbomer), semi-synthetic natural polymer (E.g. Cellulose derivatives) or natural gums (tragacanth). The later preparations are also called aqueous, alcohol and oils may be used as the continuous phase. For ex. Mineral oil can be combined with a polyethylene resin to form an oleaginous ointment base. Based on nature of solvent: Gels may be classified even as hydrogels (water based) or organo gels (with a nonaqueous solvent) based on the type of solvent used as continuous liquid phase. Bentonite magma, gelatin, cellulose derivative, carbomer, polaxomer gels are example of hydrogel. Examples of hydrogels are plastibase (low molecular weight polyethylene dissolved in mineral oil and stock cooled), olag (aerosil) gel and dispersion of metallic stearates in oils. Organogels: Organogels contain a nonaqueous solvent as the continuous phase. Example of organogel are plastibase(low molecular weight polyethylene dissolved in mineral oil and stock cooled) and dispersions of metallic stearates in oils. An organogel is a non-crystalline, non-glassy thermo reversible (thermoplastic) solid material composed of a liquid organic phase entrapped in a three-dimensionally cross-linked network. The liquid can be e.g. an organic solvent, a mineral oil or a vegetable oil. The solubility and particle dimensions of the structurant are important characteristics for the elastic properties and firmness of the organogel. Often, these systems are based on self-assembly of the structurant molecules. Organogels have potential for use in a number of applications, such as in pharmaceuticals, cosmetics, art conservation, and food. An example of formation of an undesired thermo reversible network is the occurrence of wax crystallization in crude oil. Sorbitan monostearate, a hydrophobic nonionic surfactant, gels a number of organic solvents such as hexadecane, isopropyl myristate, and a range of vegetable oils. Gelation is achieved by dissolving/dispersing the organogelator in hot solvent to produce an organic solution/dispersion, which, on cooling sets to the gel state. Cooling the solution/dispersion causes a decrease in the solvent-gelator affinities, such that at the gelation temperature, the surfactant molecules self-assemble into inverse toroidal vesicles. Further cooling results in the conversion of the toroids into rod-shaped tubules. Once formed, the tubules associate with others, and a threedimensional network is formed which immobilizes the solvent. An organogel is thus formed. Sorbitan monostearate gels are opaque, thermoreversible semisolids, and they are stable at room temperature for weeks. Such organogels are affected by the presence of additives such as the hydrophilic surfactant, polysorbate 20, which improves gel stability and alters the gel microstructure from a network of individual tubules to star-shaped "clusters" of tubules in the liquid continuous phase. Another solid monoester in the sorbitan ester family, sorbitan monopalmitate, also gels organic solvents to give opaque, thermoreversible semisolids. Like sorbitan monostearate gels, the microstructure of the palmitate gels comprises an interconnected network of rod like tubules. Unlike the stearate gels, however, the addition of small amounts of a polysorbate monoester causes a large increase in tubular length instead of the clustering effect seen in stearate gels. The sorbitan stearate and palmitate organogels may have potential applications as delivery vehicles for drugs and antigens. Xerogels: solid gels with low solvent concentration are known as xerogels. Xerogels are often produced by evaporation of the solvent, leaving the gel framework behind. They can be returned to the gel state by introduction of an agent that,on imbition, swells the gel matrix. Example of xerogels include dry gelatin,tragacanth ribbons and acacia tears and dry cellulose and polystyrene. A xerogel is a solid formed from a gel by drying with unhindered shrinkage. Xerogels usually retain high porosity (25%) and enormous surface area (150900 m2/g), along with very small pore size (1-10 nm). When solvent removal occurs under hypercritical (supercritical) conditions, the network does not shrink and a highly porous, lowdensity material known as an aerogel is produced. Heat treatment of a xerogel at elevated temperature produces viscous sintering (shrinkage of the xerogel due to a small amount of viscous flow) and effectively transforms the porous gel into a dense glass.
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Hydrogel: Hydrogel (also called Aquagel) is a network of polymer chains that are water-insoluble, sometimes found as a colloidal gel in which water is the dispersion medium. Hydrogels are superabsorbent (they can contain over 99% water) natural or synthetic polymers. Hydrogels also possess a degree of flexibility very similar to natural tissue, due to their significant water content. Common uses for hydrogels include Currently used as scaffolds in tissue engineering. When used as scaffolds, hydrogels may contain human cells in order to repair tissue. Environmentally sensitive hydrogels. These hydrogels have the ability to sense changes of pH, temperature, or the concentration of metabolite and release their load as result of such a change. As sustained-release delivery systems Provide absorption, desloughing and debriding capacities of necrotics and fibrotic tissue. Hydrogels that are responsive to specific molecules, such as glucose or antigens can be used as biosensors as well as in DDS. Used in disposable diapers where they "capture" urine, or in sanitary napkins Contact Lenses (silicone hydrogels, polyacrylamides) Medical Electrodes using hydrogels composed of cross linked polymers (polyethylene oxide, polyAMPS and polyvinylpyrrolidone) Water gel explosives Other, less common uses include breast implants granules for holding soil moisture in arid areas Dressings for healing of burn or other hard-to-heal wounds. Wound gels are excellent for helping to create or maintain a moist environment. Reservoirs in topical drug delivery; particularly ionic drugs, delivered by iontophoresis (see ion exchange resin) Common ingredients are e.g. polyvinyl alcohol, sodium polyacrylate, acrylate polymers and copolymers with an abundance of hydrophilic groups. Natural hydrogel materials are being investigated for tissue engineering, these materials include agarose, methylcellulose, hylaronan, and other naturally derived polymers. Based on Rheological properties: Gels are considers to exhibit non-Newtonian properties. Gels may be classified based a rheological properties as plastic, pseudo plastic and thixotropic gels. Plastic gels for example Bingham bodies, flocculated suspension of Al (OH) 2 exhibit a plastic floe and the plot of rheogram gives yield value of gels above which the elastic gels distorts and begin to flow. Pseudo plastic gels e.g. liquid dispersion of natural gums like tragacanth, sodium alginate, methyl cellulose, sodium CMC, exhibit pseudo plastic flow. The viscosity of pseudo plastic gels decreases with increasing rate of shear, with no yield value. The rheogram for pseudo plastic material results from a shearing action on the long chain molecules of the linear polymers. As the shearing stress is increased, the disarranged molecules begin to align their long axis in the direction of flow with release of solvent from gel matrix. Thixotropic gels: The bonds between particles in these gels are very weak and can be broken down by a shaking or stirring. The resultant sol will revert back to gel due to the particle colliding and linking together again the reversible isothermal sol gel transformation is termed thixotropy. It is most likely to occur in colloidal system with nonspherical particles, to build up a scaffold like structure eg. Bentonite, kaolin and agar 0.5%. Based on the physical nature: Based on the physical nature i.e. consistency of gel they are classified as elastic and rigid gels. Elastic gel: Gel of agar, pectin, gaur gum, gelatin, and alginate exhibit a elastic behavior. The fibrous molecules being linked at the point of junction by relatively posses weak bounds such as hydrogen bounds and dipole attraction. If the molecule posses free COOH group then additional bounding takes by salt bridge of type COO-X2 +-COO between two adjacent strands network (ex. Alginate and carbopols) where X is linking atom/molecule. The type of link imparts elastic behavior to the gel and builds coulombs force, hydrogen bounding, and vander-waals force of attraction between gelling polymers. Rigid gels: It can be formed from macromolecule in which the framework linked by primary valence bound e.g. in solid silica gel, silicic acid molecules are held by Si-O-Si-O bound to give a polymer structure possessing a network
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of pores. The rigid gels are even formed in case of polaxomers by thermal changes or reaction between poly vinyl alcohols with glycidyl ether or toluene disocyanate or methane diphenyl isocynate. GEL FORMING SUBSTANCES: A number of polymers are used to provide the structural network that is the essence of a gel system. These include natural gums, cellulose derivatives, and carbomers. Although most of these function in aqueous media, several polymers that can gel nonpolar liquids are also available. Certain colloidal solids behave as gallants as a result of asymmetric flocculation of the particles. High concentration of some nonionic surfactants can be used to produce clear gel in systems containing up to about 15% mineral oil. These are employed mostly as hair dressings. Gel forming polymers are classified as follows: A. Natural polymer 1. Agar 2. Alginates 3. carageenan 4. Tragacanth 5. Pectin 6. Xanthan 7. Gellan Gum 8. Guar Gum 9. Other gums 10. Chitosan etc. B. Semi synthetic polymers 1. Cellulose derivatives 2. Carboxymethyl cellulose 3. Methylcellulose 4. Hydroxypropyl cellulose 5. Hydroxy propyl (methyl cellulose) 6. Hydroxyethyl cellulose etc. C. Synthetic polymers 1. Carbomer 2. Carbopol 934 3. Carbopol 940 4. Carbopol 980 etc. 5. Poloxamer/surfactants 6. Polyacrylamide 7. Polyethylene and its co-polymers D. Inorganic substances 1. Microcrystalline silica 2. Clays E. Other gallants 1. Beeswax 2. Cetyl ester wax 3. Aluminum staerate etc. A. Natural polymers: Natural gums have been used in commerce since the beginning of recorded history. Typically, they are branched-chain polysaccharides. Most are anionic (negative charged in aqueous solution or dispersion), although a few, such as gaur, are neutral molecules. Differences in proportion of the sugar building blocks that make up these molecules and their arrangement and molecular weight result in significant variations in gum properties. Because of their chemical makeup, neutral gums are subjected to microbial degradation and support microbial growth. Aqueous systems containing gums should contain a suitable preservative. As mentioned earlier, cationic antimicrobials are not generally compatible with the anionic gums and should usually be avoided. Although many of the most familiar gums are plant exudates of extracts, other sources are also used. 1. Alginates: These polysaccharides containing varying proportion of D-mannuronic and L-guluronic acids are derived from brown seaweed in the form of monovalent and divalent salts. Although other alginate salts are available commercially, sodium alginate is by far the most widely used. Gelation occurs by reduction of pH or reaction with
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divalent cations. Reduction of pH converts the carboxylate ions to free carboxyl groups. This reduces hydration of polymer segments as well as the repulsion between them. Generally, some calcium must be present; the small amounts contributed by the alginate may be sufficient. The pH at which gelation occurs calcium begins to gel below a pH 4. Gel strength is a function of alginate concentration; 0.5% is a practical minimum. 2. Carrageenan: All the carrageenans are anionic. Carrageenan, the hydrocolloid extracted from red seaweed, is a variable mixture of sodium, potassium, ammonium, calcium and magnesium sulfate esters of polymerized galactose, and 3,6-anhydrogalactose. The main copolymer types are labeled kappa-, iota-, and lambda-carrageenan. Kappa and iota fraction form thermally reversible gels in water. This has been ascribed to a temperature-sensitive molecular rearrangement. At high temperature, the copolymers exist as random coils; cooling result in formation of double helices that act as cross-links. 3. Tragacanth: Trgacanth is defined in the NFas the dried gummy exudation from Astragalus gummifer Labillardiere, or other Asiatic species of Astragalus. Tragacanth is a complex material composed of chiefly of acidic polysaccharide (tragacanth acid) containing calcium, magnesium, and potassium, and a smaller amount of a neutral polysaccharide, tragacanthin. The gum swells in water; concentrations of 2 % or above a high -quality gum produce a gel. 4. Pectin: Pectin, the polysaccharide extracted from the inner skin of citrus fruit or apple pomance, may be used in pharmaceutical jellies as well as in foods. The gel is formed at an acid pH in aqueous solutions containing calcium and possibly another agent that acts to dehydrate the gum. 5. Xanthan gum: Although xanthan gum is used most frequently as a stabilizer in suspensions and emulsions at concentrations below 0.5%, higher concentrations in aqueous media yield viscid solutions that are jellylike in nature. Xanthan gum is produced by bacterial fermentation, and other its availability and quality are not subject to many of the uncertainties that affect other natural products, particularly those that are extracted from plants whose habitat falls within politically unsettled part of the world. Thermally reversible gels result from combinations of xanthan with gaur or locust bean gum. 6. Gellan gum: Gellan gum is another polysaccharide produced by fermentation that has FDA clearance for use in foods. The gum is highly efficient; as little as 0.05% is required for gel formation. Gels will not form in the absence of free cations. While both monovalent and divalent ions can include gelation, the divalent ions are required in much lower concentration, roughly 1/25 the concentration of monovalent ions. To produce a uniform gel, the gum is first dissolved in deionized water heated to 70-75C. 7. Guar gum: Guar gum is a nonionic polysaccharide derived from seeds. Aqueous guar solutions can be crosslinked by several polyvalent cations to form gels. The mechanism is believed to involve chelate formation between groups in different polymer chains. A disadvantage of these gels is the presence of insoluble plant residue. 8. Other gums: Gelatin is used widely as a bodying agent and gel former in the food industry, and occasionally in pharmaceutical products. Agar can be used to make firm gels, it is most frequently used in culture media. 9. Chitosan: Chitosan is a natural biopolymer derived from the outer shell of crustaceans. Chitin is extracted and partially deacetylated to produce chitosan. Unlike most gums, chitosan carries a positive charge and is thus attracted to a variety of biological tissues and surfaces that are negatively charged. Various derivatives are being explored for specific applications. Concentrated aqueous solutions have a gel-like consistency. Firmer gels result from interaction with polysaccharides, such as alginate. B. Semi synthetic polymers Cellulose derivatives: Many useful derivatives are fashioned from cellulose, a natural structure polymer found in plants. Treatment in the presence of various active substances results in breakdown of the cellulose backbone as well as substitution of a portion of its hydroxyl moieties. The major factors affecting rheological properties of the resultant material are the nature of the substitution(s), degree of substitution, and average molecular weight of the resultant polymer. Carboxymethylacellulose: Carboxymethylcellulose, also known as sodium carboxymethylcellulose, CMC, and cellulose gum, is an anionic polymer available in a variety of grades that differ in molecular weight and degree of substitution. Gelation requires addition of an electrolyte with a polyvalent cation to a solution of the polymer; aluminum salts are proffered. Methylcellulose: Methylcellulose is an example of a polymer whose solubility in water decreases as the temperature is raised. If an aqueous solution is heated, viscosity increases markedly at a certain point as the result of aqueous solution is heated, viscosity increases markedly at a certain point as the result of formation of gel structure. This property, known as thermal gelation, is a function of polymer chemistry and the presence of additives. The gelation
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temperature range for methocel type A is 50-55 C. Salts and sugars with a high affinity for water lower the gelation temperature whereas alcohol and propylene glycol have the opposite effect. Other cellulose derivatives: Hydroxypropyl cellulose is soluble in water as well as many polar organic solvents. Consequently, it is useful as a gelling agent for such liquids and for mixture of water and various organic liquids, such as alcohol, that adversely affect the rheological properties of gums and certain other hydrophilic agents. High molecular weight grades of hydroxypropyl cellulose and hydroxyethyl cellulose, though highly viscous, behave as fluids and do not exhibit a yield value. C. Synthetic polymers Carbomer: Carbopol polymers, along with Pemulen polymeric emulsifiers are all cross-linked. They swell in water up to 1000 times their original volume (and ten times their original diameter) to form a gel when exposed to a pH environment between 4.0 - 6.0. Since the pKa of these polymers is 6.0 0.5, the carboxylate groups on the polymer backbone ionize, resulting in repulsion between the negative charges, which adds to the swelling of the polymer. Cross-linked polymers do not dissolve in water. The glass transition temperature of Carbopol polymer is 105C in powder form. However, the glass transition temperature drops dramatically as the polymer comes into contact with water. The polymer chains starts gyrating and the radius of gyration becomes larger. Macroscopically, this phenomenon manifests itself as swelling. Carbopol polymers and co-polymers are used mainly in liquid or semisolid pharmaceutical formulations as suspending or viscosity increasing agents. Formulations include creams, gels and ointments. Carbopol polymers are also employed as emulsifying agents in the preparation of o/w emulsions for external use and are also employed in cosmetics (C. Rowe, 2003). Poloxamer/surfactants: Poloxamer is a synthetic block copolymer of ethylene oxide and propylene oxide. Their molecular weight ranges from 1000-15000. In a molecule the hydrophilic poly (oxyethylene) sand witches the hydrophilic poly (oxypropylene) thereby the polo oxypropylene occupies a central position in the molecule and it is flanked by two hydrophilic polyoxyethylene blocks. The differences in the chain length of the polyoxyethylene and polyoxypropylene chains in different products are responsible for the divergences in their physical, chemical and practical properties. Polyethylene and its co-polymers: Various forms of polyethylene and its copolymers are used to gel hydrophobic liquids. The result is a soft, easily spreadable semisolid that forms a water-resistant film on the skin surface. Polyethylene itself is a suitable gellant for simple aliphatic hydrocarbon liquids but may lack compatibility with many other oils found in personal care products. For, these, copolymers with vinyl acetate and acrylic acid may be used, perhaps with the aid of a co-solvent. To form the gels, it is necessary to disperse the polymer in the oil at elevated temperature (above 80 C) and then shock cool to precipitate fine crystals that make up the matrix. D. Inorganic Substances: Certain finely divided solids can function efficiently as thickening agents in various liquid media. Gel formation depends on establishment of a network in which colloidal particles of the solid are connected in an asymmetric fashion. This requires mutual attraction of the particles (flocculation) and partial wetting by the liquid. Microcrystalline silica: Microcrystalline silica can functions as a gallant in a wide range of liquids. Network formation results from attraction of the particles by polar forces, principally hydrogen bonding. An important commercial application of silica is its use in dentifrices. Microcrystalline silica acts as a bonding agent that provides thixotropy to the formation; at the same time, the required concentration of polishing agents is required. Clays: Montmorillonite clays are capable of swelling in water as the result of hydration of exchangeable cations and electrostatic repulsion between the negatively charged faces. At high concentration in water, thixotropic gels are fromed because the particles combine in a flocculated structure in which the face of one particle is attracted to the edge of another. METHOD OF PREPARATION OF GELS Gels are normally in the industrial scale prepared under room temperature. However few of the polymers need special treatment before processing. The gel preparation can be categorized under the following headings: Gel prepared by thermal change: The solubility of most lyophilic colloids e.g. Gelatin, agar is reduced on lowering the temperature, so that cooling a concentrated hot sol will often produce a gel. In contrast to this, some material such as the cellulose ethers owe their water solubility to hydrogen bonding with the water. Raising the temperature of these sols will disrupt the hydrogen bonding and the reduced solubility will cause gelation. Gel prepared by flocculation with neutralizers: Gelation is produced by adding just sufficient precipitation to produce the gel state but insufficient to bring about complete precipitation. It is necessary to ensure rapid mixing to avoid local high concentrations of precipitant. Solutions of ethyl cellulose, polystyrene in benzene can be gelled by rapid mixing with suitable amounts of a nonsolvent such as petroleum ether. The additions of salts to hydropholic
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sols bring coagulation, and gelation is rarely observed. The additions of suitable proportion of salts to moderately hydrophilic sols such as aluminum hydroxide, bentonite, etc. produce gels. The gels formed are frequently thixotropic in behavior. Hydrophilic colloids such as gelatin and acacia are only affected by high concentration of electrolytes, when the effect is to salt out the colloid and gelation does not occur. Gel prepared by chemical reaction: In the preparation of sols by precipitation from solution e.g. aluminum hydroxide sol prepared by interaction in aqueous solution of an aluminum salt and sodium carbonate, an increased concentration of reactants will produce a gel structure. USES The uses of gels and gelling are quite widespread, but discussion here is limited to the pharmaceutical and cosmetic fields only. Gels find use as delivery system for oral administration as gels proper or as capsule shells made from gelatin; for topical drug applied directly to the skin,mucous membranes, or eye ; and for long-acting forms of drug injected intramuscularly or implanted into the body. Geliing agents are useful as binders in tablet granulations,protective colloids in suspensions, thickeners in oral liquids, and suppository bases. Cosmetically, gels have been employed in a wide variety of products,including shampoos,fragrance products,dentifrices, and skin and hair-care preparation. CONLUSION Dermatological formulations are among the most frequently compounded products because of their wide range of potential uses. These include solutions (i.e., collodions, liniments, aqueous and oleaginous solutions), suspensions and gels, emulsions, lotions, and creams. Lotions can be either suspensions or emulsions but are fluid liquids that are typically used for their lubricating effect. Creams are emulsions and are typically opaque, thick liquids or soft solids used for their emollient properties. Creams also have the added feature that they tend to "vanish" or disappear with rubbing. REFERENCES Alfred Martin, James Swarbrick, Arthur Cammarala, Physical Pharmacy 3rd Edition, 1983, 56-569, 522, 542. C. Rowe, P. J. Sheskey, P. J. Weller, Handbook of Pharmaceutical Excipients 4th Edition, Pharmaceutical Press, London, UK, 2003, 89 - 92. Fresno,M. J. C. Ramrez A. D., Jimnez M. M..Systematic study of the flow behaviour and mechanical properties of Carbopol hydroalcoholic gels. European Journal of Pharmaceutics and Biopharmaceutics, 2002, 54, 329 - 335. Herbert A Libermen, Martin M Rieger, Gilbert S Banker, Gels: In Pharmaceutical Dosage Forms, Dispersed Systems, Informa Health Care,1996, 399-419 Herbert A. Libermen,Martin M.Rieger, Gilbert S. Banker, Gels. In Pharmaceutical Dosage Forms, Dispersed Systems, Informa Health Care, 1996, 399-419. Herbert A. Libermen,Martin M.Rieger, Gilbert S. Banker. Gels. In Pharmaceutical Dosage Forms, Informa Health Care, 1996, 399-419. Lachman HP, Lieberman JL, Kanig, Theory and Practice of Industrial Pharmacy, 3rd Edition, Varghese Publishing House, Bombay, 1987, 534-548. MN Nutimer, Chromatograph, Biomed App, 420, 1987, 228-230.
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RECENT TRENDS OF POLYMER USAGE IN THE FORMULATION OF ORODISPERSIBLE TABLETS

J.Preethi, MD Farhana, B.Chelli Babu, MD.Faizulla, Debjit Bhowmik*, S.Duraivel Nimra College Pharmacy, Nimranagar, Vijayawada, Andhra Pradesh, India *Corresponding author: debjit_cr@yahoo.com ABSTRACT Disintegrants are an essential component to tablet formulations. While rapidly disintegrating tablets do not necessarily ensure fast bioavailability, slowly disintegrating tablets almost always assure slow bioavailability. The ability to interact strongly with water is essential to disintegrant function. Combinations of swelling and/or wicking and/or deformation are the mechanisms of disintegrant action. Super disintegrants offer significant improvements over starch. But hygroscopicity may be a problem in some formulations. Tablet disintegration has received considerable attention as an essential step in obtaining fast drug release. Disintegration remains a powerful influence and precursor for drug absorption. Disintegration of tablet or capsule is depending upon the type and quantity of disintegrants. The development of Orodispersible tablets provides an opportunity to take an account of tablet disintegrants. Therefore, there is a huge potential for the evaluation of new disintegrants or modification of an existing disintegrants into superdisintegrants, so as to formulate Orodispersible tablets. The present study comprises the various kinds of disintegrants and superdisintegrants, which are being used in the formulation to provide the safer, effective drug delivery with patient's compliance. Key words: Super disintegrants, Polysorbate, Modified starches, Modified cellulose, Crospovidone 1. INTRODUCTION Bioavailability of a drug depends in absorption of the drug, which is affected by solubility of the drug in gastrointestinal fluid and permeability of the drug across gastrointestinal membrane. The drugs solubility mainly depends on physical chemical characteristics of the drug. However, the rate of drug dissolution is greatly influenced by disintegration of the tablet. The drug will dissolve at a slower rate from a non-disintegrating tablet due to exposure of limited surface area to the fluid. The disintegration test is an official test and hence a batch of tablet must meet the stated requirements of disintegration. Disintegrants are substances or mixture of substances added the drug formulation that facilitates the breakup or disintegration of tablet or capsule content into smaller particles that dissolve more rapidly than in the absence of disintegrants. Superdisintegrants are generally used at a low level in the solid dosage form, typically 1 to 10 % by weight relative to the total weight of the dosage unit. Examples of Superdisintegrants are crosscarmelose, crosspovidone, sodium starch glycolate which represent example of a crosslinked cellulose, crosslinked polymer and a crosslinked starch respectively. Superdisintegrants -an economical alternative: Orally disintegrating tablets are an emerging trend in formulation, gaining popularity due to ease of administration and better patient compliance for geriatric and pediatric patients. Disintegrating agents are substances routinely included in tablet formulations and in some hard shell capsule formulations to promote moisture penetration and dispersion of the matrix of the dosage form in dissolution fluids. An oral solid dosage form should ideally disperse into the primary particles from which it was prepared. Although various compounds have been proposed and evaluated as disintegrants, relatively few are in common usage today. Traditionally, starch has been the disintegrant of choice in tablet formulations, and it is still widely used. However, starch is far from ideal. For instance, starch generally has to be present at levels greater than 5% to adversely affect compactibility, especially in direct compression. Moreover, intragranular starch in wet granulations is not as effective as dry starch. In more recent years, several newer disintegrants have been developed. Often called super disintegrants, these newer substances can be used at lower levels than starch. Because they can be a sma ller part of the overall formulation than starch, any possible adverse effect on fluidity or compactibility would be minimized. These newer disintegrants may be organized into three classes based on their chemical structure (Table 1). Method of addition of disintegrants: The requirement placed on the tablet disintegrant should be clearly defined. The ideal disintegrant has1. Poor solubility 2. Poor gel formation 3. Good hydration capacity 4. Good molding and flow properties 5. No tendency to form complexes with the drugs
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Disintegrants are essentially added to tablet granulation for causing the compressed tablet to break or disintegrate when placed in aqueous environment. There are two methods of incorporating disintegrating agents into the tablet: I. Internal Addition (Intragranular) II.External Addition (Extragranular) III.Partly Internal and External In external addition method, the disintegrant is added to the sized granulation with mixing prior to compression. In Internal addition method, the disintegrant is mixed with other powders before wetting the powder mixtures with the granulating fluid. Thus the disintegrant is incorporated within the granules. When these methods are used, part of disintegrant can be added internally and part externally. This provides immediate disruption of the tablet into previously compressed granules while the disintegrating agent within the granules produces further erosion of the granules to the original powder particles. The two step method usually produces better and more complete disintegration than the usual method of adding the disintegrant to the granulation surface only. Table 1 Classification of super disintegrants (partial listing) Structural type (NF name) Description Trade name (manufacturer) Modified starches (Sodium Sodium carboxymethyl starch; the Explotab(Edward Mendell Co.) starch glycolate, NF) carboxymethyl groups induces Primojel (Generichem Corp.) Tablo hydrophilicity and cross-linking (Blanver, Brazil) reduces solubility. Modified cellulose Sodium carboxymethyl cellulose AcDiSol (FMC Corp.) Nymcel ZSX (Croscarmellose, NF) which has been cross-linked to render (Nyma, Netherlands) Primellose the material insoluble. (Avebe, Netherlands) Solutab (Blanver, Brazil) Cross-linked polyCross-linked polyvinylpyrrolidone; Crospovidone M (BASF Corp.) vinylpyrrolidone the high molecular weight and crossKollidon CL (BASF Corp.) (Crospovidone, NF) linking render the material insoluble Polyplasdone XL (ISP Corp.) in water. Factors affecting action of disintegrants: 1. Percentage of disintegrants present in the tablets. 2. Types of substances present in the tablets. 3. Combination of disintegrants. 4. Presence of surfactants. 5. Hardness of the tablets. 6. Nature of Drug substances. 7. Mixing and Screening. Effect of fillers:The solubility and compression characteristics of fillers affect both rate and mechanism of disintegration of tablet. If soluble fillers are used then it may cause increase in viscosity of the penetrating fluid which tends to reduce effectiveness of strongly swelling disintegrating agents and as they are water soluble, they are likely to dissolve rather than disintegrate. Insoluble diluents produce rapid disintegration with adequate amount of disintegrants. Chebli and cartilier proved that tablets made with spray dried lactose (water soluble filler) disintegrate more slowly due to its amorphous character and has no solid planes on which the disintegrating forces can be exerted than the tablet made with crystalline lactose monohydrate. Effect of binder: As binding capacity of the binder increases, disintegrating time of tablet increases and this counteract the rapid disintegration. Even the concentration of the binder can also affect the disintegration time of tablet. Effect of lubricants: Mostly lubricants are hydrophobic and they are usually used in smaller size than any other ingredient in the tablet formulation. When the mixture is mixed, lubricant particles may adhere to the surface of the other particles. This hydrophobic coating inhibits the wetting and consequently tablet disintegration. Lubricant has a strong negative effect on the water uptake if tablet contains no disintegrants or even high concentration of slightly swelling disintegrants. On the contrary, the disintegration time is hardly affected if there is some strongly swelling disintegrants are present in the tablet. But there is one exception like sodium starch glycolate whose effect remains unaffected in the presence of hydrophobic lubricant unlike other disintegrants.
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Effect of surfactants: Sodium lauryl sulphate increases absorption of water by starch. Surfactants are only effective within certain concentration ranges. Surfactants are recommended to decrease the hydrophobicity of the drugs because the more hydrophobic the tablet the greater the disintegration time. Table.2. Effects of various surfactants on the disintegration of tablets containing various drugs SURFACTANT REMARKS Sodium lauryl sulfate Good-various drugs Poor - various drugs Polysorbate 20 Good Polysorbate 40 & 60 Poor Polysorbate 80 Good Tweens Poor Poly ethylene glycol Poor (Good decrease in disintegration time, Poor increase in disintegration time) Superdisintegrants used in formulation of orodispersible tablets: Disintegrating agents are substances routinely included in the tablet formulations to aid in the breakup of the compacted mass when it is put into a fluid environment. They promote moisture penetration and dispersion of the tablet matrix. In recent years, several newer agents have been developed known as Superdisintegrants. These newer substances are more effective at lower concentrations with greater disintegrating efficiency and mechanical strength. On contact with water the superdisintegrants swell, hydrate, change volume or form and produce a disruptive change in the tablet. Effective superdisintegrants provide improved compressibility, compatibility and have no negative impact on the mechanical strength of formulations containing high-dose drugs. The commonly available superdisintegrants along with their commercial trade names are briefly described herewith. Modified starches: Sodium starch glycolate is the sodium salt of a carboxymethyl ether of starch. It is effective at a concentration of 2-8%. It can take up more than 20 times its weight in water and the resulting high swelling capacity combined with rapid uptake of water accounts for its high disintegration rate and efficiency. It is available in various grades i.e. Type A, B and C, which differ in pH, viscosity and sodium content. Other special grades are available which are prepared with different solvents and thus the product has a low moisture (<2%) and solvent content (<1%), thereby being useful for improving the stability of certain drugs. Modified celluloses Carboxymethylcellulose and its derivative (Croscarmellose Sodium): Cross-linked sodium carboxymethylcellulose is a white, free flowing powder with high absorption capacity. It has a high swelling capacity and thus provides rapid disintegration and drug dissolution at lower levels. It also has an outstanding water wicking capability and its cross-linked chemical structure creates an insoluble hydrophilic, highly absorbent material resulting in excellent swelling properties. Its recommended concentration is 0.52.0%, which can be used up to 5.0% L-HPC (Low substituted Hydroxy propyl cellulose) It is insoluble in water, swells rapidly and is used in the range of 1-5%. The grades LH- 11 and LH-21 exhibit the greatest degree of swelling. Cross-linked polyvinylpyrrolidone: It is a completely water insoluble polymer. It rapidly disperses and swells in water but does not gel even after prolonged exposure. The rate of swelling is highest among all the superdisintegrants and is effective at 1-3%. It acts by wicking, swelling and possibly some deformation recovery. The polymer has a small particle size distribution that imparts a smooth mouth feel to dissolve quickly. Varieties of grades are available commercially as per their particle size in order to achieve a uniform dispersion for direct compression with the formulation. Soy polysaccharide: It is a natural super disintegrant that does not contain any starch or sugar so can be used in nutritional products. Cross-linked alginic acid: It is insoluble in water and disintegrates by swelling or wicking action. It is a hydrophilic colloidal substance, which has high sorption capacity. It is also available as salts of sodium and potassium. Gellan gum: It is an anionic polysaccharide of linear tetrasaccharides, derived from Pseudomonas elodea having good superdisintegrant property similar to the modified starch and celluloses. Xanthan gum: Xanthan Gum derived form Xanthomonas campestris is official in USP with high hydrophilicity and low gelling tendency. It has low water solubility and extensive swelling properties for faster disintegration. Calcium Silicate: It is a highly porous, lightweight superdisintegrant, which acts by wicking action. Its optimum concentration range is 20-40%
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Ion exchange resins: The INDION 414 has been used as a superdisintegrant for ODT. It is chemically cross-linked polyacrylic, with a functional group of COO and the standard ionic form is K+. It has a high water uptake capacity. Others: Although there are many superdisintegrants, which show superior disintegration, the search for newer disintegrants is ongoing and researchers are experimenting with modified natural products, like formalincasein, chitin, chitosan, polymerized agar acrylamide, xylan, smecta, key-jo-clay, crosslinked carboxymethylguar and modified tapioca starch. Mechanism of action of superdisintegrating agent: Disintegrants are agents added to tablet (and some encapsulated) formulations to promote the breakup of the tablet (and capsule slugs) into smaller fragments in an aqueous environment thereby increasing the available surface area and promoting a more rapid release of the drug substance. There are three major mechanisms and factors affecting tablet disintegration as follows: A: Swelling: Although not all effective disintegrants swell in contact with water, swelling is believed to be a mechanism in which certain disintegrating agents (such as starch) impart the disintegrating effect. By swelling in contact with water, the adhesiveness of other ingredients in a tablet is overcome causing the tablet to fall apart. B: Porosity and Capillary Action (Wicking): Effective disintegrants that do not swell are believed to impart their disintegrating action through porosity and capillary action. Tablet porosity provides pathways for the penetration of fluid into tablets. The disintegrant particles (with low cohesiveness & compressibility) themselves act to enhance porosity and provide these pathways into the tablet. Liquid is drawn up or wicked into these pathways through capillary action and rupture the interparticulate bonds causing the tablet to break apart. C: Deformation: Starch grains are generally thought to be elastic in nature meaning that grains that are deformed under pressure will return to their original shape when that pressure is removed. But, with the compression forces involved in tableting, these grains are believed to be deformed more permanently and are said to be energy rich with this energy being released upon exposure to water. In other words, the ability for starch to swell is higher in energy rich starch grains than it is for starch grains that have not been deformed under pressure. It is believed that no single mechanism is responsible for the action of most disintegrants. But rather, it is more likely the result of inter-relationships between these major mechanisms. The classical example of the earliest known disintegrant is Starch. Corn Starch or Potato Starch was recognized as being the ingredient in tablet formulations responsible for disintegration as early as 1906 (even though tablet disintegration was itself not given much importance in tablet formulations until much later). Until fairly recently, starch was the only excipient used as a disintegrant. To be effective, corn starch has to be used in concentrations of between 5-10%. Below 5%, there is insufficient channels available for wicking (and subsequent swelling) to take place. Above 10%, the incompressibility of starch makes it difficult to compress tablets of sufficient hardness. Although the connection between bioavailability of drug and tablet disintegration took some time to become appreciated, it is now accepted that the role of the disintegrant is extremely important. In a direct compression process, drug is blended with a variety of excipients, subsequently lubricated and directly compressed into a tablet. A disintegrant used in this type of formulation, simply has to break the tablet apart to expose the drug substance for dissolution. Pregelatinized Starch (Starch 1500): Pregelatinized starch is a directly compressible form of starch consisting of intact and partially hydrolyzed ruptured starch grains. Pregelatinized starch has multiple uses in formulations as a binder, filler and disintegrant. As a disintegrant, its effective use concentration is between 5-10%. Its major mechanism of action as a disintegrant is thought to be through swelling. Microcrystalline Cellulose (Avicel): Like pregelatinized starch, microcrystalline cellulose is widely used in formulations because of its excellent flow and binding properties. It is also an effective tablet disintegrant when used in a concentration of between 10-20%. Others: Sodium Bicarbonate in combination with citric or tartaric acids is used as an effervescent disintegrant.Alginic Acid at a concentration of between 5-10% is an effective, but very expensive disintegrant.Ion Exchange Resins (Amberlite 88) has disintegrant properties at a concentration of between 1-5%. But this type of disintegrant is rarely used. Super disintegrants: Because of the increased demands for faster dissolution requirements, there are now available, a new generation of Super Disintegrants in addition to the disintegrants discussed earlier. Three major groups of compounds have been developed which swell to many times their original size when placed in water while producing minimal viscosity effects:
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1. Modified Starches: Sodium Carboxymethyl Starch (Chemically treated Potato Starch) i.e. Sodium Starch Glycolate (Explotab, Primogel) Mechanism of Action: Rapid and extensive swelling with minimal gelling. Effective Concentration: 4-6%. Above 8%, disintegration times may actually increase due to gelling and its subsequent viscosity producing effects. 2. Cross-linked polyvinylpyrrolidone: water insoluble and strongly hydrophilic. i.e. crospovidone (Polyplasdone XL, Kollidon CL) Mechanism of Action: Water wicking, swelling and possibly some deformation recovery. Effective concentration: 2-4% dified Cellulose: Internally cross-linked form of Sodium carboxymethyl cellulose. i.e. Ac-Di-Sol (Accelerates Dissolution), Nymcel Mechanism of Action: Wicking due to fibrous structure, swelling with minimal gelling. Effective Concentrations: 1-3% (Direct Compression), 2-4% (Wet Granulation) ADVANTAGES: Effective in lower concentrations than starch Less effect on compressibility and flow ability More effective intragranularly DISADVANTAGES: More hygroscopic (may be a problem with moisture sensitive drugs) Some are anionic and may cause some slight in-vitro binding with cationic drugs (not a problem in-vivo). Table.3. List of disintegrants
Disintegrants Starch USP Starch 1500 Avicel(PH 101, PH 102) Solka floc Alginic acid Na alginate Explotab Polyplasdone(XL) Amberlite (IPR 88) Methyl cellulose, Na CMC, HPMC AC-Di-Sol 2-4 Concentration in granules (%w/w) 5-20 5-15 10-20 5-15 1-5 2.5-10 2-8 0.5-5 0.5-5 5-10 1-3 Wet granulation Special comments Higher amount is required, poorly compressible Lubricant properties and directly compressible Purified wood cellulose Acts by swelling Acts by swelling Sodium starch glycolate, superdisintegrant. Crosslinked PVP Ion exchange resin Direct compression
CONCLUSION: Disintegrants, an important excipient of the tablet formulation, are always added to tablet to induce breakup of tablet when it comes in contact with aqueous fluid and this process of desegregation of constituent particles before the drug dissolution occurs, is known as disintegration process and excipients which induce this process are known as disintegrants.The objectives behind addition of disintegrants are to increase surface area of the tablet fragments and to overcome cohesive forces that keep particles together in a tablet. One of the challenges every formulator of oral solid dosage forms must address is drug solubility. Drugs must dissolve efficiently to be absorbed by the body, but this is a special challenge fo rpoorly soluble drugs. The choice of formulation ingredients can have a significant effect on the rate and extent of drug dissolution. Superdisintegrants used as enhance solubililty of poorly water soluble drugs.
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SUPERDISINTEGRANTS Crosscarmellose, Ac-Di-Sol, Nymce ZSX, Primellose, Solutab, Vivasol Crosspovidone, Crosspovidon M, Kollidon, Polyplasdone
Sodium starch glycolate Explotab, Primogel Alginic acid NF Satialgine
Table.4. List of superdisintegrants EXAMPLE MECHANISM OF OF ACTION Crosslinked Swells 4-8 folds in < 10 cellulose seconds. Swelling and wicking both. Crosslinked Swells very little and PVP returns to original size after ompression but act by capillary action Crosslinked Swells 7-12 folds in <30 starch seconds Crosslinked alginic acid Natural super disintegrant Wicking action Rapid swelling in aqueous medium or wicking action
SPECIAL COMMENT Swells in two dimensions, Direct compression or granulation, Starch free Water insoluble and spongy in nature so get porous tablet
Swells in three dimensions and high level serve as sustain release matrix Promote disintegration in both dry or wet granulation Does not contain any starch or sugar. Used in nutritional products. Highly porous, light weight optimum concentration is between 20-40%
Soy polysaccharides Emcosoy Calcium silicate
REFERENCES Bi Y, Sunada H, Yonezawa Y, Preparation and evaluation of a compressed tablet rapidly disintegrating in the oral cavity, Chem Pharm Bull (Tokyo), 44, 1996, 2121-2127. Bi YX, Sunada H, Yonezawa Y, Danjo K.Evaluation of rapidly disintegrating tablets prepared by a direct compression method, Drug Dev Ind Pharm, 25, 1999, 571-581. Chaudhari K.P.R, and Rao Rama N, Indian Drugs, 35 (6), 1988, 368 to 371, Chudhari K. P.R, and Radhika, Int. J. Pharm. Excipts, 2000 (4), 181-184 Grasono Alesandro et al, US Patent 6, 1997, 336 2001 Grasono, Alessandro et al, U S Patent 6,197,336 2001 Ihang J. A., & Christensen J. M., Drug Dev Ind Pharn, 22 (8), 1996, 833-839 Korunubhum S. S., Batopak S. B., J. Pharm Sci, 62 (1), 1973, 43-49 Liberman H.A., Lachman L. and Schawstr J.B., Pharmaceutical Dosage forms, tablets, vol 2, 1989, 173-177 Sallam E, Ibrahim H, Abu Dahab R, Shubair M, Khalil E.Evaluation of fast disintegrants in terfenadine tablets containing a gas-evolving disintegrant, Drug Dev Ind Pharm, 24, 1998,501-507. Sallem E, Ibrahim H, Dahab R. A, Drug Dev. Ind. Pharm, 24 (6), 1998, 501-507 Schimidt P.C and Brogramann B, Acta. Pharm. Technol, 1988, 34, 22.
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N Narasimha Rao et.al
DESIGN AND DEVELOPMENT OF AMLODIPINE BESYLATE FAST DISSOLVING TABLETS BY USING NATURAL SUPERDISINTEGRANTS
N.Narasimha Rao*, B.Radha Krishna Murthy, D.Rajasekhar, P. Suri Babu, K. Phaneendra Babu, Srinivasa Babu. P
Department of Pharmaceutics, Vignan Pharmacy College, Vadlmudi, Guntur (dt), A.P, India
Corresponding author: Email:narasimhampharm@gmail.com ABSTRACT Amlodipine besylate is a long-acting calcium channel blocker used in the treatment of chronic stable angina, vasospastic angina and hypertension. Amlodipine besylate is a sparingly soluble orally administered drug and the rate of absorption is often controlled by the rate of dissolution. The rate of dissolution can be increased by incorporating the drug in a fast disintegrating dosage form. Mucilage of natural origin is preferred over semisynthetic and synthetic substances because they are comparatively cheaper, abundantly available, nonirritating and nontoxic in nature. In this study, we are planning to develop the fast dissolving tablets of the amlodipine besylate, using different concentrations of natural super disintegrating agents like Fenugreek seeds mucilage, Treated Agar, Modified Tragacanth. Key Words: Fast Dissolving Tablets, Amlodipine Besylate, Natural Superdisintegrants. INTRODUCTION The proper choice of superdisintegrant and its consistency of performance are of critical importance to the formulation of a rapidly disintegrating dosage form. The choice of superdisintegrant for a tablet formulation depends largely on the nature of the drug being used. The orally disintegrating property of tablet is attributed to a quick ingress of water into the tablet matrix, which creates porous structure and result in rapid disintegration. Some tablets are design to dissolve in saliva remarkably fast, within a few seconds, and are true fast-dissolving tablets. Others contain agents to enhance the rate of tablet disintegration in oral cavity, and are more appropriately termed fast-disintegrating tablets, as they may take up to a minute to completely disintegrate. When put on tongue, these tablets disintegrates instantaneously, releasing the drug, this dissolves or disperses in saliva. Some drugs are absorbed from mouth, pharynx and esophagus as the saliva passes down into the stomach. To prepare tablets with different natural superdisintegrants and to select the best superdisintegrant and optimize its concentration. To compare the dissolution efficiency of marketed tablets and prepared tablets. MATERIALS AND METHODS Amlodipine besylate procured from Orchid Pharmaceuticals, Hyderabad, Fenugreek gum Purchased from Local market, Ponnur (Seeds), Agar gum, Tragacanth gum Purchased from Loba fine chem, Mumbai. MCC, Mannitol gift sample from Natco Pharma Ltd, Hyderabad Magnesium stearate purchased from S.D. Fine chem Ltd, Mumbai FORMULATION STUDIES Preparation of amlodipine besylate tablets: Tablets containing 200 mg of Amlodipine besylate were prepared by direct compression method. Drug was passed through sieve no 100. Amlodipine besylate along with other excipientswere mixed in a mortar. The resulting blend was lubricated with magnesium stearate and compressed into tablets using the Cadmach single punch (round shaped, 9mm thick) machine. The composition of the different tablets formulated was shown in the following tables. Extraction of mucilage from fenugreek seeds: The seeds are powdered using pestle and mortar and 100 g of the powder is extracted with hexane to remove lipophilic compounds using a soxhelet apparatus. To remove pigments and to deactivate enzyme, the defatted powder is boiled in ethanol for 20 min. This treated powder is then soaked in 10 liters water and the pH is adjusted to 3.5 using 0.5 M Hydrochloric acid. The mixture is stirred by a mechanical stirrer for 12 h and then filtered through filtration paper. The filtrate is centrifuged (5000 RPM) and the supernatant is concentrated in vacuum to 50% of its initial volume. The resulting solution is mixed with the same volume of 96% ethanol and stored in a refrigerator for 4 hours. The precipitated mucilage is separated by centrifugation (5000 RPM). The collected mucilage is re-suspended in distilled water, agitated for 20 min and reprecipitated one more time to eliminate chloride ions and other impurities. Finally the residue is washed with diethyl ether and acetone and dried overnight at 45C, resulting in an off-white powder. Preparation of treated agar: Agar suitable quantity of agar powder (5-10 g) weighed and added in distilled water (100ml). Agitation is done continuously by a stirrer for one day to swell. The swollen contents are dried on Volume 1(2) March-April 2013 Page 175
a tray for 3 days at room temperature. The dried powders are grinded by mortar and pestle. Thengrinded powder is passed through sieve no.100. Preparation of modified tragacanth: Tragacanth(5g), tween80(0.05g) and hydrogen peroxide(30% w/v) solution(1ml) were taken in 100ml of purified water and boiled for 15min. The mixture was allowed to cool and settle. The clear supernatant fluid was decanted and the sediment was washed repeatedly with water. Finally the sediment was collected by centrifuging at 2500RPM and dried at 80oC for 4hrs. The dried product was ground to fine powder and passed through mesh no.200. Preformulation studies: Preformulation studies are the first step in the rational development of dosage form of a drug substance. The objective of Preformulation studies are to develop a portfolio of information about the drug substance, so that this information useful to develop formulation. Preformulation can be defined as investigation of physical and chemical properties of drug substance alone and when combined with excipients. Preformulation investigations are designed to identify those physicochemical properties and excipients that may influence the formulation design, method of manufacture, and pharmacokinetic-biopharmaceutical properties of the resulting product Organoleptic Characteristics: The color, odor, and taste of the drug were characterized and recorded using descriptive terminology; the results were shown in the Table No 1. Table.1.organoleptic characteristics Properties Results Description Crystalline powder Taste Taste less Odour Odourless Colour White Sieve Analysis: Standard sieves of different meshes were available as per the specifications of USP; sieves were arranged in a nest with courses at the top. A sample of the 40 mesh passed powder is placed on top sieve. This sieve set was fixed to the mechanical shaker apparatus and shaken for a certain period of times. The powder retain on each sieve was weighed and percentage of powder retained on each sieve was calculated using the initial weight taken. Bulk Density: An accurately weighed quantity of powder, which was previously passed through sieve # 40 [USP] and carefully poured into graduated cylinder. Then after pouring the powder into the graduated cylinder the powder bed was made uniform without disturbing. Then the volume was measured directly from the graduation marks on the cylinder as ml. The volume measure was called as the bulk volume and the bulk density is calculated by following formula; Bulk density = Weight of powder / Bulk volume Tapped Density: After measuring the bulk volume the same measuring cylinder was set into tap density apparatus. The tap density apparatus was set to 300 taps drop per minute and operated for 500 taps. Volume was noted as (Va) and again tapped for 750 times and volume was noted as (Vb). If the difference between Va and Vb not greater than 2% then Vb is consider as final tapped volume. The tapped density is calculated by the following formula Tapped density = Weight of powder / Tapped volume Carrs Index [Compressibility Index] and Hausers Ratio: Carrs index and Hausners ratio measure the propensity of powder to be compressed and the flowability of powder. Carrs index and Hausners ratio can be calculated from the bulk and tapped density. Carrs index = Tapped density Bulk density / Tapped density 100 Hausners ratio = Tapped density / Bulk density Angle of repose: The angle of repose of powder was determined by the funnel method. The accurately weighed powder was taken in a funnel. The height of the funnel was adjusted in such a way that the tip of the funnel just touches the apex of the heap of the powder. The powder was allowed to flow through the funnel freely onto the surface. The diameter of the powder cone was measured and angle of repose was calculated using the following equation = tan-1 h / r Where, h and r are the height and radius of the powder cone, respectively. Volume 1(2) March-April 2013 Page 176
Table No 2:- Physical parameters of Atenolol powder (pure) Parameter Atenolol 2 Bulk Density (gm/cm ) 0.418 Tapped Density (gm/cm2) Compressibility Index Hausers Ratio Angle of repose 0.633 32.43 1.5 32.76
Table.3. Formulation of amlodipine besylate fast dissolving tablets

Ingredients Amlodipine Besylate Fenugreek gum Treated agar gum Modified tragacanth gum Mannitol MCC Talc Mg stearate F1 10 4 82.5 100 2 1.5 F2 10 6 80.5 100 2 1.5 F3 10 8 78.5 100 2 1.5 F4 10 10 76.5 100 2 1.5 F5 10 4 82.5 100 2 1.5 F6 10 6 80.5 100 2 1.5 F7 10 8 78.5 100 2 1.5 F8 10 10 76.5 100 2 1.5 F9 10 4 82.5 100 2 1.5 F10 10 6 80.5 100 2 1.5 F11 10 8 78.5 100 2 1.5 F12 10 10 76.5 100 2 1.5
Table.4. Micrometric properties for formulation blend 7

Formulation code F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 Bulk density (gm/cm3) 0.520 0.521 0.523 0.524 0.525 0.532 0.542 0.534 0.526 0.526 0.526 0.522 Tapped Density (gm/cm3) 0.618 0.652 0.612 0.636 0.615 0.625 0.625 0.616 0.617 0.618 0.615 0.610 Carrs index (%) 15.56 15.25 15.65 15.96 15.58 15.47 15.15 15.26 15.89 15.84 15.25 15.47 Hausners Ratio 1.185 1.168 1.178 1.196 1.145 1.158 1.126 1.148 1.159 1.162 1.165 1.158 Angle of repose () 26.8 25.3 26.4 25.6 26.5 26.8 26.6 25.9 24.6 28.4 25.9 25.7
Table.5.Postcompression parameters of formulations F1 to F12

Parameters Average weight (mg) Drug content (%) Disintegration time (min) Friability (%) Hardness (kg/sqcm) Wetting time (sec) Water absorption Ratio In-vitro dispersion time (min) F1 198 + 0.41 98.66 +0.34 3.1 + 0.12 0.66 + 0.23 3.2 + 0.45 153 + 0.32 60 + 0.43 3.7 + 0.11 F2 198 + 0.25 99.1 + 0.13 2.7 + 0.14 0.76 + 0.21 3.1 + 0.32 122 + 0.15 66 + 0.21 3.1 + 0.21 F3 198 + 0.86 99.41 +0.28 1.5 + 0.21 0.62 + 0.12 2.9 + 0.45 68 + 0.22 76 + 0.29 2.5 + 0.32 F4 198 + 0.41 98.26 +0.32 0.91+ 0.17 0.90 + 0.11 2.6 + 0.34 34 + 0.13 88 + 0.12 1.9 + 0.26 F5 199 + 0.51 99.5 + 0.23 1.8+ 0.28 0.88 + 0.12 2.5 + 0.36 74 + 0.61 75 + 0.43 2.4 + 0.38 F6 199+ 0.14 98.39 +0.32 1.4 + 0.17 0.90 + 0.11 2.6+ 0.34 65 + 0.13 88+ 0.12 1.9 + 0.26 F7 200+ 0.28 101.8 +0.21 1.1+ 0.19 0.88 + 0.18 2.8 + 0.21 42 + 0.53 90 + 0.35 1.5+ 0.11 F8 200+ 0.22 99.98+ 0.12 0.80+ 0.23 0.89+ 0.24 2.9+ 0.27 25+ 0.56 92+ 0.43 0.95+ 0.24 F9 197 + 0.54 97.5+ 0.16 1.9 + 0.10 0.85 + 0.02 2.9 + 0.13 64 + 0.22 88 + 0.32 2.3 + 0.11 F10 199 + 0.29 98.6 +0.23 1.4 + 0.13 0.92 +0.08 3.1 + 0.17 51+ 0.19 72 + 0.31 1.8 + 0.16 F11 198+ 0.32 99.4 + 0.31 0.92 + 0.18 0.76 + 0.11 2.8 + 0.19 39+ 0.27 69 + 0.26 1.3 + 0.13 F12 196 + 0.38 99.4+ 0.27 0.71+ 0.24 0.69+ 0.19 2.7+ 0.23 24+ 0.42 74+ 0.32 0.96+ 0.33
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Sampling Time (min) 0 5 10 15 20 25 30
Table.6.Percentage of drug released from formulations F1 to F12

F1 0.000 28.72 +0.02 37.025 +0.04 47.426 +0.02 53.821 +0.05 62.951 +0.08 72.162 +0.05 F2 0.000 38.915 +0.08 45.255 + 0.02 50.267 +0.08 64.832 +0.04 74.713 +0.08 88.049 +0.02 F3 0.000 47.061 +0.05 52.125 +0.08 80.279 +0.02 86.845 +0.05 90.724 +0.02 F4 0.000 60.679 +0.08 67.829 + 0.04 90.653 +0.08 98.301 +0.02 Percentage of drug released ( S.D.) F5 F6 F7 F8 F9 0.000 0.000 0.000 0.000 0.000 35.525 39.587 46.421 67.503 35.495 +0.04 +0.02 +0.02 +0.05 +0.06 41.815 +0.02 47.500 +0.04 53.809 +0.02 67.765 +0.04 71.581 +0.02 48.652 +0.04 53.023 +0.04 56.717 +0.05 71.996 +0.02 83.956 +0.05 52.772 +0.02 64.650 +0.05 90.861 +0.02 96.783 +0.05 73.299 +0.02 95.490 +0.05 99.426 +0.02 42.612 +0.03 50.819 +0.07 57.329 +0.08 67.749 +0.02 74.329 +0.04 F10 0.000 41.640 +0.09 50.721 +0.05 57.795 +0.06 66.290 +0.05 70.739 +0.08 84.031 +0.05 F11 0.000 53.895 +0.09 58.948 +0.08 66.759 +0.06 75.291 +0.08 98.164 +0.04 F12 0.000 68.163 +0.08 81.459 +0.05 94.869 +0.06 99.56 +0.07 -
CONCLUSION Amlodipine besylate showed PH dependent solubility. The solubility was found to be influenced by the particle size of pure drug. The solubility can be improved by reducing the particle size. The drug and excipients were found to be compatible with each other. The dissolution rate was found to be influenced by nature and concentration of natural super disintegrants. Thus by changing the excipients and their concentrations, suitable formulations of amlodipine besylate fast dissolving tablets was developed. Thus major objective of the investigation were fulfilled. By studying and comparing, we can conclude modified tragacanth shows excellent super disintegrating property and treated agar gum shows better super disintegrating property. Fenugreek seed mucilage can be used as disintegrant which shows less disintegrant property in the formulation of fast dissolving tablets when compared to other natural polymers, since the primary ingredients are in expensive, devoid of toxicity, biocompatible, biodegradable and easy to manufacture, they can be used in place of currently marketed superdisintegrants. These gums could be used for different applications in tablet dosage forms and may be explored as high functionality excipients for future applications. REFERENCES Arun Shirwaikar, Novel co-processed Excipients of Mannitol and Microcrystalline cellulose For preparing fast dissolving tablet of Glipizide, Indian Journal of Pharmaceutical Sciences, Sept-Oct 2007, 633-639. Ashutosh Mohapatra Rajesh K Parikh, Formulation, development and evaluation of patient friendly dosage forms of metformin, Part-III: Soluble effervescent tablets, Asian Journal of pharmaceutics, July-September 2008, 177-181. Balakrishnan Prabagar, Enhancement of bioavailability of poorly water soluble Clotrimazole by inclusion complex with - cyclodextrin; Indain Journal of Pharmaceutical Sciences, Jan-Feb 2006, 89-94 Kaushik D, Dureja H and Saini TR, Development of Melt in Mouth Tablets by Sublimation Technique, Indian Drugs, 41 (4), 2004, 503-508. Mukesh C Gohel, Formulation, development and evaluation of patient friendly dosage forms of metformin, Part-I: Orally disintegrating tablets, Asian Journal of pharmaceutics, July-september 2008, 167-171. Nazma Inamdar, Kiran Bhise, Shakeel Memon; enhancement of solubility of Meloxicam and Development of Dispersible Tablet, Asian Journal of pharmaceutics, April 2008 ,128-132. Omaima A Sammour, Mohammed A Hammad, Nagia A Megrab, and Ahmed S Zidan, Formulation and Optimization of Mouth Dissolve Tablets Containing Rofecoxib Solid Dispersion, AAPS Pharm Sci Tech, 7 (2) 2006; Article 55; E1-E9. P.V.Swami, Studies to Enhance Dissolution Properties of Carbamazepine, Indain Journal of Pharmaceutical Sciences, May-June 2007, 427-430. Volume 1(2) March-April 2013 Page 178
Shailesh Sharma, G.D.Gupta, Formulation and characterization of fast dissolving tablet of Promethzine theoclate. Asian Journal of pharmaceutics, Jan- 2008, 70-74. Shishu and ashima Bhatti, Optimization of Fast Dissolving Tablet of Etoricoxib Prepared by sublimation technique, Indian Journal of Pharmaceutical Sciences, Jan-Feb 2008,71-76. Shishu and ashima Bhatti, Preparation of Tablets Rapidly Disintegrating in Saliva containing bitter taste masked granules by compression method, Indain Journal of Pharmaceutical Sciences, Jan-Feb 2008, 80-84.
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FORMULATION OPTIMIZATION AND EVALUATION OF LIPOSOMAL GEL OF PREDNISOLONE BY APPLYING STATISTICAL DESIGN
Varde Neha M*, Thakor Namita M, C.Sini Srendran, Shah Viral H Sigma Institute of Pharmacy, Bakrol, Vadodara *Corresponding Address: Email id: varde_neha@yahoo.com, Phone no. : +919558326014 ABSTRACT Liposomal carriers, well known for their potential in topical drug delivery have been chosen to help Prednisolone molecules in the skin layers. In the present work statistical study for the formulation of liposomes for topical delivery of Prednisolone using the factorial design approach was undertaken. Amount of Soya Lecithin, DPPS and cholesterol (CH) were taken at three different levels and liposomes were prepared using film hydration technique. Gels containing liposomes (optimized batch) were prepared in Carbopol 940P and were characterized for rheology, spreadability and permeation through the rat skin. Results of regression analysis revealed that vesicle size and entrapment efficiency were dependant on the cholesterol and lipid concentration. Rheological studies of all liposomal gels prepared with 1%, 1.5%, and 2% w/w carbopol gave a clear idea of concentration of carbopol required. Liposomal dispersion and gels were found to increase the skin permeation and deposition compared to control and marketed gel. Liposome dispersion and gel formulation were found to be stable for 60 days. Key words: Prednisolone, Thin Film Hydration method, Liposomes, Box Benhken design, Carbopol Gel. 1. INTRODUCTION Topical drug delivery is an attractive route for local and systemic treatment. The delivery of drugs onto the skin is recognized as an effective means of therapy for local dermatologic diseases and other diseases. Rheumatoid arthritis is a chronic auto immune disease characterized by joint synovial inflammation and progressive cartilage, bone destruction leading to gradual immobility. Drugs useful in treatment of rheumatoid arthritis are classified as first line agents having Non-steroidal anti inflammatory drugs and steroidal anti inflammatory drugs. Prednisolone (11)-11, 17, 21-trihydroxypregna-1, 4-diene-3,20-dione is a steroidal drug with predominant glucocorticoid and low mineral corticoid activity. It is mainly used for the treatment of a wide range of inflammatory and autoimmune diseases such as asthma, multiple sclerosis, rheumatoid arthritis, autoimmune hepatitis etc. Prednisolone is also known as disease modifying antiarthritic drugs because of its anti inflammatory action by inhibiting gene transcription for COX-2, cytokines, cell adhesion molecules, and inducible NO synthetase. When steroidal anti inflammatory drugs such as prednisolone are given orally results in systemic side effects like bone loss, increased susceptibility to infection, osteoporosis, peptic ulcers and buffalo hump. Parental route of administration results in rapid clearance rate of drug which ultimately compels invasive and frequent administration of drug. Attempts will be made in developing and characterizing a specific drug delivery system targeting drugs to synovium or specific tissues which in turn increase drug efficacy with minimum extra synovial toxicity in arthritis. The main objective of the study is to formulate and evaluate prednisolone liposomal gel formulation for effective topical pharmacotherapy in treatment of rheumatoid and other diseases. 2. MATERIALS AND METHODS 2.1 Materials: Prednisolone (Mercury Laboratories Pvt. Ltd., Vadodara) , DPPS(Lipoid GmbH, Germany) and saturated soya lecithin were a generous gift from Novastell,France. Cholesterol (CHOL), Carbopol 940P was purchased from S.D. Fine Chemicals, Mumbai, India. All other chemicals used were of HPLC or analytical grade. 2.2 Liposome preparation: Aqueous liposomal formulations were prepared by conventional lipid film hydration method. Different weight ratio of phospholipids: choleseterol were weighed and dissolved in chloroform: methanol mixture (2: 1 v/v) in 250 ml round bottom flask. A thin film was formed on the inner side of round bottom flask by evaporating organic solvent under vacuum in rotary evaporator at 45-50C. Subsequently, the flask was kept overnight under vacuum to ensure the complete removal of residual solvent. The dry lipid film was hydrated with 20 ml phosphate buffer solution (pH 7.4) containing at phase transition temperature of different lipids. The dispersion was left undisturbed at room temperature for 2-3 h to allow complete swelling of the lipid film and hence to obtain vesicular dispersion.
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2.3 Effect of variables: To study the effect of variables on liposome performance and characteristics, different batches were prepared using 33 Box Behnken design. Amount of Soya Lecithin, DPPS and Cholesterol were selected as three independent variables. Mean Vesicle size, %EE and %Cumulative drug release in the rat skin were selected as dependent variables. Amount of Prednisolone was kept constant. 2.3.1 Preparation of Liposomal Dispersion Formulation Using Experimental Design: Prednisolone liposomal formulations were prepared by conventional thin-layer hydration or rotary evaporation technique using BoxBehnken design. A three-factor, three-level Box- Behnken design was used for constructing a second-order polynomial models using Design Expert (Version 8.0.7.1; Stat-Ease Inc, Minneapolis, Minnesota). A design matrix comprising 15 experimental runs was constructed, for which the nonlinear computer-generated quadratic model is defined as: Y = b0 + b1X1 + b2X2 + b3X3 + b12X1X2 + b13X1X3 + b23X2X3 + b11X12 + b22X22 + b33X32 where Y is the measured response associated with each factor level combination; b0 is constant; b1, b2, b3 are linear coefficients, b12, b13, b23 are interaction coefficients between the three factors, b11, b22, b33 are quadratic coefficients computed from the observed experimental values of Y from experimental runs; and X 1, X2 and X3 are the codes of independent variables. The terms X1X2 (i = 1, 2 or 3) represent the interaction effect. The independent variables selected were the amount of the Soya lecithin(X1), 1,2-Dipalmatoyl-sn-glycero-3-phospho-Lserine(DPPS) (X2) and Cholesterol (X3). The dependent variables were Mean Particle size (Y1), EE % (Y2) and percentage cumulative drug permeated (Y3) with constraints applied on the formulation of liposome. The concentration range of independent variables under study is shown in Table 1 along with their low, medium and high levels, which were selected based on the results from preliminary experimentation. The concentration range of soya lecithin (X1), DPPS (X2) and Cholesterol (X3) used to prepare the 15 formulations and the respective observed responses are given in Table 2. Table 1: Variables in Box- Behnken design for preparation of Prednisolone liposome Code Independent variables Low (-1) Medium (0) High (+1) X1 Soya lecithin (mg) 30 60 90 X2 DPPS (mg) 120 160 200 X3 Cholesterol (mg) 5 10 15 Dependent variables Goal Y1 Mean vesicle size (nm) In Range Y2 %Entrapment Efficiency Maximum Y3 %Cumulative drug Release Maximum Formulation table: Ingredients(mg) F1 Prednisolone 10 Soya lecithin 30 DPPS 120 Cholesterol 10 Organic solvent (ml): Chloroform 20 Methanol 10 Table 2: Formulation composition of batch F1 to F15 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 10 10 10 10 10 10 10 10 10 10 90 30 90 30 90 30 90 60 60 60 120 200 200 160 160 160 160 120 200 120 10 10 10 5 5 15 15 5 5 15 20 10 20 10 20 10 20 10 20 10 20 10 20 10 20 10 20 10 20 10 F12 10 60 200 15 20 10 F13 10 60 160 10 20 10 F14 10 60 160 10 20 10 F15 10 60 160 10 20 10
2.4 Characterization of liposomes: 2.4.1 Mean Vesicles shape, size, and size distribution and zeta potential: Liposome vesicles were visualized using optical microscope. Digital micrograph and soft imaging viewer software were used for image capture and analysis. The vesicles size, size distribution and zeta potential were determined using a computerized inspection system with zetasizer (dynamic light scattering method, HAS 3000; Malvern Instruments, Malvern, United Kingdom). 2.4.2 Lamellarity: The lamellarity of the liposomes was determined by CLSM (Confocal laser scanning microscopy) study. Volume 1(2) March-April 2013 Page 181
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2.4.3 Encapsulation efficiency: Liposome encapsulation efficiency was determined from the amount of entrapped drugs using the ultracentrifugation technique. Briefly, total amount of drug was determined after having dissolved and disrupted drug-loaded liposomes in ethanol or Triton X-100 using an ultrasound bath for 10 min. Then, sample was centrifuged at 20,000 rpm for 50 min. The free drug was determined in the supernatant at 242.5 nm with a UVvisible spectrophotometer. The drug encapsulation efficiency (EE %) was calculated as follows: EE %= Total amount of drug incorporated Free amount of drug (supernant) 100 Total amount of drug 2.4.4 In- vitro drug release study: In-vitro drug release of drug from the liposomal formulation was evaluated using the franz diffusion cell technique. 5 ml aliquot of liposomal suspension was placed in the donor compartment. Hairless Rat skin was used as the diffusion membrane. Perfect sink conditions prevailed during the drug release studies and the entire system was kept at 320.5 C under continuous magnetic stirring at 50 rpm. Samples (5 ml) of the receptor compartment was taken at various time intervals and assayed for drug concentration by spectrophotometric method. 2.5 Preparation of liposomal gel: The appropriate amount of carbopol 940P was weighted and added slowly in a citrate buffer solution (pH 5.0), under constant stirring by a paddle stirrer. After addition of the full amount of solid material, the gel was allowed to swell under moderate stirring for at least 24 h or until fully swollen and transparent. Other ingredients, such as 15% w/v polyethylene glycol-400 (PEG-400) and triethanolamine (0.5% w/v), were added to obtain homogeneous dispersion of gel and sodium benzoate (0.5% w/v) was added in the buffer used for gel preparation. Liposomal gel formulations were prepared by mixing the liposomal dispersions with the gels in the ratio of 1:5 (w/w) (liposome dispersion/gel). 2.6 Characterization of liposomal gel: Optimized formulation of the liposomal suspension is converted to the gel formulation and its physical and chemical characterization is carried out by following tests. The carbopol 940P gel formulations were prepared using 1%, 1.5% and 2% carbopol concentration. 2.6.1 Physical examination: The prepared gel formulations were inspected visually for their colour, homogeneity, consistency and spreadability. Clarity was determined by using clarity chamber with black and white background. 2.6.2 pH: The pH values of 1% aqueous solutions of the prepared gels were measured by a pH meter. The glass electrode was calibrated with two standard buffers (pH of 4.00 and 9.00).The preparation was left for about 15 min for attaining equilibrium while measuring. 2.6.3Viscosity: Viscosity of prepared gels was measured by Brookfield Viscometer. Apparent viscosity was measured at 25C and rotating the spindle at 1.5 rpm. 2.6.4Content uniformity: Gel formulation (100 mg) was dissolved in methanol and filtered. The volume was made to 100 ml with methanol. The resultant solution was suitably diluted with methanol and absorbance was measured at 242.5 nm of drug using Shimadzu 1700 UV Visible spectrophotometer. 2.6.5 In-vitro drug permeation study: An essential parameter in the evaluation of drug delivery is the rate at which the drug is released from the carrier. Skin permeation study with drug-containing liposomal formulation was carried out using modified Franz diffusion cell. Full thickness abdominal skin of male Wister albino rats weighing 140 to 200 g was used for the skin permeation. Briefly, to obtain skin, animal was sacrificed. Hair from the abdominal region was carefully removed and an excision in the skin was made. The dermal side of the skin was thoroughly cleaned of any adhering tissues. Dermis part of the skin was wiped 3 to 4 times with a wet cotton swab soaked in isopropanol to remove any adhering fat. The skin specimen was cut into appropriate size after carefully removing subcutaneous fat and washing with normal saline. Skin was mounted in a modified Franz diffusion cell, kept at 320.5C. The known quantity of gel equivalent to 10 mg of drug was spread uniformly on the skin on donor side. pH 7.4 phosphate buffer was used as the acceptor medium, from which samples were collected at regular intervals. An equal amount of acetonitrile was added to the collected samples to precipitate skin protein prior to estimate with UV spectroscopy. All permeation experiments were repeated three times and data were expressed as mean of three experiments standard deviation (SD). 2.7 Stability study: Stability studies of liposomal suspension and gel were done for 3 months under conditions required by guidelines of the ICH. Accelerated stability studies were performed by keeping the temperature 48C, 250.5 C and 60 5 %RH (relative humidity). The samples were taken at the interval of 0, 30, 60 days. The stability was evaluated by comparing the particle size and %Entrapment Efficiency for suspension. The parameters like viscosity and percentage cumulative permeation of drug were evaluated for gel. Volume 1(2) March-April 2013 Page 182
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3. RESULT AND DISCUSSION
Figure 1: Microscopic Image of Liposome (a) Yellow box: MLV, (b)Brwon box: SUV and (c)Blue box: LUV
A B C Figure 2: Microphotographs corresponding to multilamellar liposomes by CLSM using transmitted channel: (A) Five-six lamellae; (B) three lamellae (C) unilamellar 3.1.Statistical data: Responses of different batches obtained using box benhken design are shown in Table 3 obtained data were subjected to multiple regression analysis using Design Expert (Version 8.0.7.1; Stat-Ease Inc, Minneapolis, Minnesota). Y = b0 + b1X1 + b2X2 + b3X3 + b12X1X2 + b13X1X3 + b23X2X3 + b11X12 + b22X22 + b33X32 Table 3: Responses obtained for studied parameters from experimental batches (n=3)
Formulation code F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15 Mean Particle size (nm) 213.217.26 266.115.16 394.614.81 266.815.33 237.216.22 277.513.79 246.714.49 272.516.74 276.413.89 255.313.89 286.312.33 243.118.68 173.616.65 169.116.74 181.415.89 Polydispersity Index 0.2310.012 0.2420.023 0.2250.006 0.2040.021 0.2060.011 0.2230.018 0.2190.013 0.2010.013 0.2800.009 0.2330.012 0.1920.018 0.2000.015 0.2170.022 0.1940.015 0.2210.010 %Entrapment Efficiency 65.23.76 72.31.82 68.92.00 67.30.44 69.64.94 69.43.25 63.73.82 65.41.60 67.22.18 63.92.96 63.73.73 68.40.82 76.91.48 79.91.63 78.20.98 %CDR 85.82.5 83.33.1 85.62.8 83.41.9 89.71.4 79.82.2 79.32.6 79.31.3 81.52.5 77.41.7 81.42.1 82.33.5 95.71.7 97.32.1 96.51.1
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100 80 %CDR 60 40 20 0 0
Diffusion study of Liposomal Suspension at 7.4 pH
Diffusion study of Liposomal Suspension at 7.4 pH 100

80 %CDR F1 F2 F3 F4 F5 60 40 20 0 0 5 Time(hr) 10 F6 F7 F8 F9 F10 15
5 Time(hr) 10
15
150 100
Diffusion study of Liposomal Suspension at 7.4 pH

F11 F12 F13
%CDR
50 0 0 2 4 6 8 Time(hr) 10 12
F14 F15 14
Figure 3: %cumulative release of drug content of all formulation batches F1-F15 Table 4: Summary of regression analysis results for measured responses Parameters b0 b1 b2 b3 b12 b13 b23 b11 b22 3.47 0.28 -22.67 -3.62 -5.58 40.59 47.39 Mean Particle size 174.70 10.15 78.33 0.87 0.012 -0.11 -2.18 0.47 2.00 -4.34 -5.57 %Entrapment Efficiency 96.5 0.0 -0.44 -1.26 0.075 -1.48 1.25 -4.67 -7.30 %CDR
b33 43.19 -6.97 -8.55
3.1.1Effect of variables on particle size: The most important parameter, which needs to monitor during liposome preparation its best performance, is the vesicle size and size distribution of liposomes.
(a) (b) (c) Figure 4: Response surface plots of (a) Soya Lecithin (X1) and DPPS (X2), (b) Soya Lecithin (X1) and Cholesterol(X3) and (c) DPPS(X2) and Cholesterol (X3) on the Mean Particle/Vesicle Size. Volume 1(2) March-April 2013 Page 184
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From the number of reports, it was observed that the size and size distribution of the liposome determines their invivo or ex-vivo performance. There are some reports, which showed the effect of liposome size on the drug release as well as drug deposition in the skin. Thus for the effective delivery, the selected method should result in optimum size range and homogeneous population. In the present study, film hydration technique found to produce polydispersity index of less than 0.400 indicates obtained liposome population have narrow size distribution (Table 3). It was observed that the relative amount of DPPS, Soya Lecithin and Cholesterol was found to play important role in vesicle size (Fig.4). 3.1.2 Effect of variables on entrapment efficiency: Determination of EE is an important parameter in case of liposomes as it may affect the drug release and skin deposition. EE is expressed as the fraction of drug incorporated into liposomes relative to total amount of drug used. In the present study, the observed EE for all batches were in the range of 60 80%. A positive correlation was observed for both variables X1, X2 and X3. Thus with increase in the concentration of Soya Lecithin, DPPS and Cholesterol entrapment efficiency found to be increased (Fig. 5). Among all the batches, which had minimum vesicle size and intermediate EE%, selected for the further study of gel formulation. Small particle size liposomes can cover the skin surface more compared to larger particle size.
(a)
(b) (c) Figure 5: Response surface plots of (a) Soya Lecithin (X1) and DPPS (X2), (b) Soya Lecithin (X1) and Cholesterol(X3) and (c) DPPS(X2) and Cholesterol (X3) on the %Entrapment Efficiency. 3.1.3 Effect of variables on %Cumulative Drug Release: In-vitro permeation profile of the Prednisolone from the different liposomal suspension formulations in 7.4 pH phosphate buffer was studied. In vitro permeation of Prednisolone from the liposomal formulation was found to be in the range of 70-97% during a period of 12 h. Thus, the liposomal formulations release the drug for prolonged period.
(a)
(b) (c) Figure 6: Response surface plots of (a) Soya Lecithin (X1) and DPPS (X2), (b) Soya Lecithin (X1) and Cholesterol(X3) and (c) DPPS(X2) and Cholesterol (X3) on the %CDR. Volume 1(2) March-April 2013 Page 185
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Y3= 96.5 + 0.0X1 -0.44X2 -1.26X3 + 0.075X1X2 1.48X1X3 + 1.25X2X3-4.67X12 -7.30X22 8.55X32 3.2. Characterization of liposomal gel 3.2.1 Physical examination, pH, Viscosity, Content uniformity of gel: All liposomal gel formulations were found to be clear and transparent. The pH of the liposomal gel was found to be in the range of 6-7.The range of content uniformity was from 89.75% to 99.44%. All the formulations were found to be Clear and transparent. 3.2.2 Skin permeation and drug deposition studies: Results obtained from in-vitro drug permeation studies conducted with different formulations of prednisolone are shown in Significant augmentation in the skin permeation of prednisolone has been observed. Table 5: Result of all the carbopol 940P formulations (Batch F14) Formulation pH % Content uniformity Viscosity (cps) %Cumulative code drug Release CF14 (1%) 6.970.24 98.451.13 4873830 69.641.34 CF 14 (1.5%) 6.850.20 89.753.72 4751668 76.471.53 CF 14 (2%) 7.160.16 99.442.47 6672552 97.042.90 3.2.3 Stability Studies: Stability of liposome dispersion as well as 2% w/w carbopol liposomal gel containg equivalent 10mg prednisolone was carried out for 60 months at 4-8 C and room temperature. Responses obtained for different parameters for liposomal dispersion and liposomal gel during stability period are as shown in Table 5. Liposomes were found to be reasonably stable in terms of aggregation, fusion and/or vesicle disruption tendencies, over the studied storage period. From results it can be concluded that at room temperature and freeze temperature there was slightly but insignificantly decrease in % entrapment efficiency and increase in particle size for liposomal batch. Deposition of prednisolone in rat skin from liposomal gel was found insignificantly decreased during stability period. Result suggests that keeping the liposomal product in refrigeration conditions minimizes stability problems of liposomes. Table 6: Effect on Mean Particle/vesicle size, entrapment efficiency and drug deposition from liposomal gel during stability No. Of Mean Particle size %Entrapment Efficiency %CDR days 4-8C RT 4-8C RT 4-8C RT 0 169.116.74 169.116.74 79.91.63 79.91.63 97.32.1 97.32.1 30 184.713.83 205.317.46 68.2 2.86 67.30.44 93.11.5 84.82.4 60 212.711.62 234.512.29 61.30.44 57.81.24 86.31.9 79.32.5 4. DISCUSSION Preparation of liposomes using statastical design was found to be well suited and sound approach to obtain stable liposomal formulation. Variables such as amount of phospholipids have a profound effect on the vesicle size and entrapment efficiency. Rheological studies of all liposomal gels prepared with 1%, 1.5%, and 2% w/w carbopol gave a clear idea of concentration of carbopol required i.e. 2 % carbopol 940P. Liposomal dispersion and gels were found to increase the skin permeation and deposition compared to control. Stability studies performed for liposomal dispersion and Liposomal gel indicates the prepared liposomes have more stability at freezing temperature than that of room temperature. Prednisolone molecules could be successfully entrapped in liposomes with reasonable drug loading. Hence from results obtained it can be concluded that liposomal gel containg Prednisolone has potential application in topical delivery. 6. ACKNOWLEDGEMENT Athors are thankful to Mercury Laboratories Pvt. Ltd, Vadodara; Lipoid LMBH, Germany and Novastell, France for providing Prednisolone gift sample and Phospholipid respectively and Dr. Reddy, Hyderabad for kind help in characterisation.
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REFERENCES Brahmankar DM, Jaiswal SB. Biopharmaceutics and pharmacokinetics: A treatise; 1 st Edn; Vallabh prakashan, 2007, 335. Guo J, Ping Q, Sun G, Lecithin vesicular carriers for transdermal delivery of cyclosporine, Int J Pharm, 2000, 194,201-207. Jain NK. Advances in controlled and novel drug delivery; 1st Edn; CBS Publication and distributors, New Delhi, 2005, 309. Jain S, Jain P, Umamaheshwari RB, Transfersomesa novel vesicular carrier for enhanced transdermal delivery: development, characterization, and performance evaluation, Drug Dev Ind Pharm, 2003, 29, 10131026. Kim MK, Chung SJ, Lee MH, Delivery of hydrocortisone from liposomal suspensions to the hairless mouse skin following topical application under non-occlusive and occlusive conditions, J Microencapsul, 1998, 15, 2129. Kokare CR, Mitkari BV, Korde SA, Mahadik KR, Formulation and evaluation of topical liposomal gel for Fluconazole, Int J. Pharm. Educ. Res, 2010, 44, 324-333. Korting HC, Schmid MH, A review-Therapeutic progress with topical liposome drugs for skin Disease, Advanced Drug Delivery Reviews, 1996, 18, 335-342. Kurakula M, Srinivas C, Kasturi N, Diwan PV, Formulation and Evaluation of Prednisolone Proliposomal gel for effective Topical Pharmacotherapy, Inter J Pharm Sci & Drug Research, 2012, 4, 35-43. Mishra D, Garg M, Dubey V, Elastic liposomes mediated transdermal delivery of an anti-hypertensive agent: propranolol hydrochloride, J Pharm Sci, 2007, 96, 145-155. Pokharkar VB, Padamwar MN, Development of vitamin loaded topical liposomal formulation using factorial design approach: Drug deposition and stability, Int J Pharm, 2006, 320, 3744. Seth AK, Misra A, Umrigar D, Topical liposomal gel of idoxuridine for the treatment of herpes simplex: pharmaceutical and clinical implications, Pharm Dev Technol, 2004, 9, 277289. The United State Pharmacopoeia, 28 (USP NF), The Official Compendia of Standards, Asian Edition, 2006, 1641. Vyas SP, Khar RK. Targeted & Controlled Drug Delivery; CBS Publication, Delhi, 2002, 173. Yamada S, Hanato J, Kuriyama K, Liposomal formulations of glucagon-like peptide-1: Improved bioavailability and anti-diabetic effect. Int J Pharm, 2009, 382,111116.
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Sreenu Thalla et.al
ADR MONITORING IN HYPERTENSION OUT PATIENT DEPARTMENT OF HOSPITAL

Sreenu Thalla*, K.Venkatta Ramana, Sk.Sheherbanu, Sk.Ashya, A.Manikanteswara Reddy, B.Lakshmi A.S.N Pharmacy College, Burripalem road, Tenali, Guntur (Dt), Andhra Pradesh, India. * Corresponding author: sreenuthalla87@gmail.com ABSTRACT Adverse drug reactions (ADRs) to anti-hypertensive agents are common and can lead to noncompliance or even discontinuation of therapy. There is paucity of such data in the Indian context. We deemed it worthwhile to assess the suspected ADR profile of anti-hypertensive drugs in an ambulatory setting in a public teaching hospital. A longitudinal observational study was conducted in the outpatient department (OPD) of the concerned unit. Twenty consecutive patients per day, irrespective of their hypertensive diagnosis, were screened for suspected ADRs, 2 days in a week, over 6 months. Adverse event history, medication history and other relevant details were captured in a format as adopted. We screened 2000 patients (68.69% males, median age 38 years), of whose 429 were suspected of having at least one ADR; 84 cases had insufficient evidence about causality and were excluded from further analysis. Thus, 17.25% (95% confidence interval: 15.59-18.91%) of our study population reported ADRs with at least possible causality. Of 352 events recorded, 327 (92.90%) were probable and the rest possible. None was labeled certain as re -challenge was not performed. Patients received a median of 3.2 anti-hypertensive drugs each. Thirty-three different kinds of ADRs were noted, including tremor (19.60%), weight gain (15.34%) and constipation (14.49%). Among the incriminated drugs, anti-hypertensive represented the majority (57.10%) of the list. This study offers a representative profile of ADRs to be expected in anti-hypertensive out-patients in an Indian public hospital. Establishment of an anti-hypertensive drug ADR database can be a worthy longterm goal in the Indian context. Key Words: anti-hypertensive, out-patient department, adverse drug reactions INTRODUCTION Hypertension is an important worldwide public-health challenge because of its high frequency and concomitant risks of cardiovascular and kidney disease. It has been identified as the leading risk factor for mortality, and is ranked third as a cause of disability-adjusted life-years. The prevalence of hypertension in various regions of the world has been widely reported; 49 however, no information has been compiled for its prevalence and absolute burden around the world (Laura, 2008). Accurate estimates of the worldwide prevalence of this condition are essential as a source of primary information and for rational planning of health services. Measurement of the global burden of hypertension would allow international public-health policy-makers to assign sufficient priority and resources to its management and prevention. National representative studies of the prevalence of this condition have been done in some countries, whereas in others, published data are from regional or local population-based samples. Anti-hypertensive drugs are plentiful in number and their use is increasing day by day. These drugs are capable of causing a number of adverse drug reactions (ADR), some of which may be fatal. ADRs associated with anti-hypertensive drugs can lead to noncompliance, and at times discontinuation of therapy. Pharmacovigilance in anti-hypertensive units can play a vital role in detecting ADRs and alerting physicians to the possibility and circumstances of such events, thereby protecting the user population from avoidable harm (Patricia, 2005). In India, pharmacovigilance activities are still in nascent stage and there are few reports available on the ADR profile of medicines in general and anti-hypertensive agents in particular. This prompted us to evaluate the ADR profile of anti-hypertensive drugs used by ambulatory patients in a teaching hospital. MATERIALS AND METHODS A longitudinal observational study was undertaken in the anti-hypertensive out-patient department (OPD) of Health Hospitals, Tenali; between July 2012 and January 2013. This hospital caters mostly to non-affluent sections of the community, like other public hospitals in Tenali. It was part of ongoing pharmacovigilance activity at the Institute which had the necessary administrative and Institutional Ethics Committee clearance. Twenty consecutive patients per day, irrespective of their hypertensive diagnosis, were screened for suspected ADRs, for two fixed days in a week, barring public holidays. The screening was carried out by three hypertensive and three pharmacology residents trained in the department for interviewing patients(Judith, 2003). Subjects and their accompanying family members were interviewed, and past prescriptions and case notes, where available, were Volume 1(2) March-April 2013 Page 188
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reviewed. A senior Physician was available for consultation in the event of any difficulty. Patients who were known substance abusers and subjects not accompanied by a family caregiver were not included in the study. Patient details (age, sex, body weight), adverse event history, history of medication suspected of having caused the ADR and details of concomitant medication use were recorded in the format followed in the Pharmacovigilance Programme. It was decided a prior to that events of only mild severity along with considerable confusion in differentiating from disease symptomatology will not be counted as suspected ADRs. Causality of the event was assessed and analysis was conducted by two pharmacology residents in consultation with a senior pharmacologist. Suspected ADRs with causality status less than possible were not considered for further analysis. RESULTS AND DISCUSSION A total of 2000 patients were screened for the study, of whom 429 (21.45%) were suspected of having at least one ADR. On causality assessment, 84 of these 429 cases (19.58%) were considered to have insufficient evidence about causality and they were excluded from further analysis. From the remaining 345 subjects, 352 ADRs were tabulated. Thus, 17.25% (95% confidence interval: 15.5918.91%) of our study subjects reported ADRs with at least possible causality. Males represented 68.69% of the cases. Notably, on an average day, about 60% of the patients attending the concerned OPD were males. The median age of the subjects was 38 years. Bipolar affective disorder (27%) was the commonest clinical diagnosis among these cases. Patients received 3.2 (1.5) anti-hypertensive drugs per prescription. A few subjects were taking concomitant medicines for other disorders such as started before their hypertensive medication, or OTC medicines casually for minor ailments. The drug history was taken very carefully in such cases before attributing suspected ADRs to the psychotropic medicines concerned. Different kinds of treatment emergent ADRs were encountered in the patients as listed. Tremor (19.60%) was the commonest ADR noted, closely followed by weight gain (of 5 kg or more over baseline weight; 15.34%) and constipation (14.49%). Causality assessment revealed that 327 ADRs (92.90%) belonged to probable category, whereas 25 (7.10%) were of possible type according to scale. No case could be labeled certain, as re-challenge was not attempted by the attending Physician, once a drug was withdrawn. Among the drugs incriminated, anti-hypertensives were the commonest group of agents causing ADRs (57.10%). Some interesting ADRs were noted during the course of study. One case of diabetes mellitus was noted in a 43-year-old female hypertensive patient. She was on therapy for last 2 years and developed symptoms. She had no history of raised blood sugar prior to starting her fasting blood glucose level was 237 mg/dL at the time of the study but returned to normal. No ADR encountered turned out to be fatal, life-threatening or required hospitalization for management. Some of the events were temporarily disabling but were managed by the clinicians with corrective medication or dose modification. The present study has reported the incidence and attempted to profile suspected ADRs to antihypertensive drugs in the OPD setting in the Indian context. In contrast to reports of ADR profiles of individual drugs, there is a dearth of pharmacovigilance profiling of anti-hypertensive agents in general, not only in India but also worldwide. A Brazilian study, conducted in 2001, analyzed 219 notifications of suspected ADRs of anti-hypertensive medicaments and incriminated as the commonest group responsible for ADRs, followed by anti-hypertensive. A Bulgarian study reported that the ADR frequency of individual anti-hypertensive drugs studied is less than 1%. A knowledge, attitude and practice based study conducted in Norway found that ADRs can be prevented by collecting reliable information about their frequencies and possible risk factors. In our study, which is based on active surveillance rather than spontaneous reporting, we found anti-hypertensive to be most commonly responsible; this could be partly related to the frequency and duration of their use in the hospital setting. Antihypertensive was frequently prescribed in our setting, as it was dispensed from the hospital pharmacy. Although several new anti-hypertensive drugs have been introduced in the Indian pharmaceutical market over the last few years, they were not included in our report because they are relatively expensive and not dispensed from the hospital pharmacy. Hence, they were seldom prescribed in our setting a public hospital catering mostly to economically weaker sections of society. Regarding causality assessment, our study had no certain cases since the suspected ADRs were mostly of mild to moderate severity and hence did not require withdrawal of therapy. In cases where de-challenge was done, re-challenge was not attempted with the offending drug. This is in contrast to the Brazilian study where 24 cases were found to be definite after re-challenge was attempted (Muzaffar, 2011). Regarding the particularly interesting ADRs noted and should be used with caution in patients requiring long-term therapy. Our study had limitations. For logistical reasons, we screened patients on Volume 1(2) March-April 2013 Page 189
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two fixed days of each week, rather than rotating days, and this could introduce potential bias in the sample. Being an OPD-based study, it is likely that we have missed ADRs that were transient or too mild to have inconvenienced the patient to an extent sufficient to report to the doctor on the next hospital visit. Although routine hematological and clinical chemistry (e.g., blood sugar, lipids) reports were available, we could not generally order tests like ECG screening of patients for QT interval prolongation or blood sampling to determine serum prolactin concentration. There was no access to therapeutic drug monitoring (TDM). However, though TDM of anti-hypertensive agents has been employed, there is lack of consensus regarding its optimum use in clinical practice. Although this post-marketing surveillance study cannot provide true incidence or prevalence figures, it offers a representative idea of the ADR profile of anti-hypertensive drugs likely to be encountered in ambulatory patients in an Indian public hospital. Compliance with therapy is a major issue in hypertensive patients. Constant vigil in detecting ADRs and subsequent dose adjustments can make therapy with antihypertensive drugs safer and more effective. This, in turn, should improve compliance. ADRs can perhaps also be reduced by using less medication and with adequate knowledge of drug interactions. An anti-hypertensive drug ADR database built up on the basis of such studies conducted across multiple centers, through active collaboration of Physicians and pharmacologists can be a worthy long-term goal in the Indian context. Such a database can provide early warning signals of drug-reaction links if kept under active scrutiny CONCLUSION This study offers a representative profile of ADRs to be expected in anti-hypertensive out-patients in an Indian public hospital. Establishment of an anti-hypertensive drug ADR database can be a worthy long-term goal in the Indian context. REFERENCES Hypertension, Federal bureau of prisons of clinical practicle guidelines, June 2004, 1-22. Judith A. Whitworth, World health organisation (WHO) international society of hypertension (ISH) statement on management of hypertension, 21, 2003, 11. Laura A. Magee, Michael Helewa, Diagnosis, Evaluation and management of hypertensive disorders of pregnancy Journal of obstetrics and gynaecology, 30(3), 2008, S1-S47. Muzaffar iqbal, Clinical perspective on the management of hypertension, Indian journal of clinical medicine, libertas academica, 2011, 258-267. Patricia M Kearney, Megan Whelton, Globar burden of hypertension, analysis of worldwide data, 365, 2005, 217223.
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Arun Kumar et.al
EVALUATION OF THE ANTIOXIDANT AND HEPATOPROTECTIVE ACTIVITY OF MADHUCA LONGIFOLIA (KOENIG) LEAVES
Arun Kumar1*, Kaushik Biswas2, S Ramachandra Setty3 1. Anand College of Pharmacy, Keetham, Agra, U.P. 2. Jadavpur University, Kolkata 3. Government College of Pharmacy, Bangalore *Corresponding author: jvsarun@gmail.com ABSTRACT The study was designed to evaluate the antioxidant and hepatoprotective activity of 70% ethanolic extract of Madhuca longilolia (Koenig) in experimental liver injury produced by carbon tetrachloride. The effects observed were compared with the standard silymarin. The Plant has reduced the elevated levels of serum biochemicals such as SGPT, SGOT, ALP, total and direct bilirubin, cholesterol and triglycerides to near normal levels in a dose dependant manner. The extract was also screened for GSH estimation and lipid peroxidation. It is evident by the results obtained that the plant posses hepatoprotective activity. INTRODUCTION The human beings are exposed to environmental, occupational and xenobiotics challenges due to modern life style. Enormous free radicals are generated during the exposure to such stressful challenges. The inbuilt free radical scavenging system like glutathione, superoxide dismutase (SOD), catalase etc. are involved in scavenging of the excessive free radicals. Since liver is involved in the metabolism of xenobiotics, it is more prone to produce excessive hazardous free radicals and causing the toxicity of the liver. In one of our field survey we found a tree with beautiful tender leaves by name Madhuca longifolia (Koenig) belongs to family Sapotaceae. The phytochemical profile of this plant reveals the presence of bassian, -sitosterol, -D-glucoside, stigmasterol, carotene, oleanolic acid, quercetine, dihydro quericetine, triterpenoids, amyrin acetate, myricetine, palmitic acid, saponin A and B. Flavonols and tannins are also reported in leaves (Karnick, 1994). It was also found on survey that various parts of the plants were used for anthelmintic, epilepsy, scorpion sting, analgesic, hepatoprotective and gastroprotective purposes (Varier, 1995) (Nadkarni, 1954) (Yosioka, 1974) (Dinesh Chandra, 2001). Hence, the study was conducted to know the antioxidant and Hepatoprotective activity of the leaves of this plant. MATERIALS AND METHODS Experimental animals: Albino rats (Wistar) weighing 150-200g and albino mice weighing 20-25g of either sex were used in this study. They were procured from Sri Venkateshwara Enterprises, Bangalore. The animals were acclimatized for one week under laboratory conditions. They were housed in polypropylene cages and maintained at 27C 2C under 12 hrs dark / light cycle. They were fed with standard rat feed (Gold Mohur Lipton India Ltd.) and water ad libitum was provided. The litter in the cages was renewed thrice a week to ensure hygeinity and maximum comfort for animals. Ethical clearance for handling the animals was obtained from the Institutional animals ethical committee prior to the beginning of the project work. Plant Material: Madhuca longifolia (Koenig) leaves were collected from the fields of Harapanahalli. The plant was identified and authenticated by Prof. K. Prabhu, Department of Pharmacognosy, S.C.S. College of Pharmacy, Harapanahalli. A herbarium specimen is deposited in our college museum. The leaves were shade dried at room temperature and pulverized. The powder obtained from leaves was subjected to successive soxhlet extraction with the solvents with increasing order of polarity i.e. petroleum ether (60 80), chloroform (59.561.5), ethanol (64.5-65.5) and water. In addition, the shade-dried powder was extracted directly with 70% ethanol (hydroalcoholic extract), which was used for biological investigations. Determination of acute toxicity: The acute toxicity for 70% ethanolic extracts of Madhuca longifolia (Koenig) leaves were determined on albino mice, maintained under standard conditions. The animals were fasted overnight prior to the experiment. Fixed dose method of OCED Guideline No. 420 given by CPCSEA(Aykae, 1985) were adopted for toxicity studies. Antioxidant Activity: A. In-vitro antioxidant activity: The reducing power of aqueous and 70% ethanolic extracts of Madhuca longifolia (Koenig) leaves was determined by the method of Oyaizu and the absorbance was measured at 700nm. Increased absorbance of the reaction mixture indicates increase in reducing power. The results are compiled in
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Table No.1.Measurement of super oxide anion scavenging activity of Madhuca longifolia(Koenig) leaves was done by using the method explained by Nishimiki (Nishimiki, 1972) and modified by Ilhami et al (Kyung, 2004) and the absorbance at 560 nm was measured against blank. The results are complied in the Table No. 2 Hydroxyl radical scavenging activity was measured by the assay of Hathwell and Gutteridge (Hye Gwang, 1999) and absorbance was taken at 532 nm. The results are compiled in Table No.3 B. In-vivo antioxidant activity: An attempt was made to assess the influence of pre-treatment with 70% alcoholic extract of Madhuca longifolia (Koenig) leaves on the levels of glutathione and lipid per oxidation in CCl4 induced hepatotoxicity. Glutathione measurements were performed using dithiobisnitrobenzoate by the modification of the Ellamn procedure (Barry hatchwell, 1981) and the absorbance at 412 nm were measured. The results are complied in Table No. 4. In- vivo carbon tetra chloride induced lipid peroxidation was performed by the method of John Buege et al (Be-Jen Wang, 2004). The results are compiled in Table No. 5 Evaluation of Hepatoprotective Activity in CCl4-Induced Hepatotoxicity: The method of Suja SR. et al (Suja, 2004) was followed. In the dose response experiment, albino rats were randomly assigned into 5 groups of 6 individuals each. Group-I Animals (-ve control) were administered 1ml distill water p.o., for 5 days Group-II Group-III Group-IV Group-V Animals (+ve control) were administered 1ml distill water p.o., for 5 days Animals were administered with silymarin 100 mg/kg p.o., for 5 days.
Animals were administered with 70% ethanolic extract 20 mg/kg p.o., for 5 days. Animals were administered with 70% ethanolic extract 40 mg/kg p.o., for 5 days.
Animals were sacrificed 24 hrs after the administration of CCl4 under mild ether anesthesia. Blood samples were collected for evaluating the serum biochemical parameters like SGPT, SGOT, ALP, serum direct bilirubin, serum total bilirubin, serum cholesterol and serum Triglycerides. The results are shown in Table No. 6 Statistical analysis: Results were expressed as mean SEM, (n=6). Statistical analysis were performed with one way analysis of variance (ANOVA) followed by studentt test. P value less than <0.05 was considered to be statistically significant. *P<0.05, **<0.01 and ***<0.001, when compared with control and toxicant group as applicable. RESULTS Preliminary phytochemical screening of Madhuca longifolia(Koenig) leaves: It was observed from the preliminary phytochemical screening of the leaves that alkaloids, glycosides and proteins are absent in all the extracts. Steroids are present in petroleum ether and chloroform extracts. Moreover it was found that carbohydrates, flavonoids, tannins and saponins are present in ethanolic, aqueous and hydro-alcoholic (30:70) extracts. Determination of acute toxicity (LD50): The acute toxicity studies reveal that the 70% alcoholic extract of the leaves of Madhuca longifolia were found to be lethal (3/3 mice were died) at dose of 2000 mg/kg and 300 mg/kg b.w. But the extracts were found to be safe at dose of 50 mg/kg. Hence 200 mg/kg was LD50 cutoff value considered for the 70% alcoholic extract. Therefore 1/10th and 1/5th dose (i.e. 20mg/kg and 40mg/kg) were selected for all further in vivo studies. Antioxidant activity of Madhuca longifolia (Koenig) leaves: Reducing power activity of extracts of Madhuca longifolia (Koenig) leaves: It was observed that the 70% ethanolic extracts of the leaves have demonstrated dose dependent increase in the reducing property. Whereas 25g sodium metabisulphate (std.) has 73.09% reducing property. However, 100g of the 70% ethanol extract has shown maximum reducing power i.e. 93.56%. The results are summarized in table no.1. Superoxide anion scavenging activity of extracts of Madhuca longifolia (Koenig) leaves: It is observed that 70% ethanolic extracts leaves have demonstrated dose dependent increase in the superoxide anion scavenging activity. Whereas 25g sodium metabisulphate (std.) has 73.89% activity. However, the test extracts even at 100g have shown lesser inhibition than standard.The results are summarized in Table No. 2
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Hydroxyl radical scavenging activity of extracts of Madhuca longifolia (Koenig) leaves: It was observed that the 70% ethanolic extract has demonstrated dose dependent increase in the hydroxical radical scavenging activity. Whereas 25g sodium metabisulphate (std.) has 66.47% and 55.61% scavenging activity in 1 hr and 4 hrs incubation respectively. However, 70% leaves extract at 100g has shown more activity at both 1 hr and 4 hrs incubation as compared to the standard (sodium metabisulphate). The results are summarized in Table No. 3 Influence of 70% ethanolic extract of Madhuca longifolia (Koenig) leaves on GSH levels in CCl4 induced hepatotoxicity in rats: There was a marked reduction in the tissue GSH levels i.e. to the extent of 44.41% upon CCl4 treatment. Treatment with 70% ethanolic extract has brought back the decreased GSH levels in a dose dependant manner to a near normal level. The test extract was found to be statistically significant at both lower and higher doses in normalizing tissue GSH levels. However test extract at 40 mg/kg (highest dose studied) was found to be less effective than standard (silymarin). The results are summarized in Table No. 4 Effect of 70% ethanolic extract Madhuca longifolia (Koenig) leaves on in vivo lipid peroxidation in CCl4 induced hepatotoxicity : There is a dose dependent inhibition of invivo lipid peroxidation by both the doses (20 mg/kg and 40 mg/kg) of 70% ethanolic extract. 100 mg/kg silymarin has 67.45% inhibition whereas 40 mg/kg 70% ethanolic extract of the leaves has 50.84% inhibition which is statistically significant when compared to the CCl4 treatment but have show lesser inhibition of lipid peroxidation than the standard silymarin. The results are summarized in Table No. 5 Effects 70% ethanolic extract of Madhuca longifolia (Koenig) leaves on biochemical markers in CCl4 induced hepatotoxicity : There is a marked increase in SGPT (U/L) levels observed in CCl 4 treated group (312.420). However the SGPT levels were reversed to near normal levels (64.276) with the treatment of 40 mg/kg of 70% ethanolic extracts of MLK. In addition the standard silymarin 100 mg/kg has restored the SGPT levels significantly i.e. 65.395. Serum SGOT levels have been also elevated in the CCl4 treated (318.412). Treatment with standard silymarin 100 mg/kg has brought back the SGOT levels to the near normal levels. However treatment with the 70% ethanolic extract of MLK has restored the SGOT levels in a dose dependent manner at both the doses (20 mg/kg and 40 mg/kg) upto 139.120 and 92.70 respectively, which satistically significant. In case of total and direct bilirubin there is a noticeable rise (4.892and 1.950 respectively) in serum levels on CCl 4 treatment observed. Treatment with 40 mg/kg has reversed the toal and direct bilirubin serum levels to 1.331 and 0.282 respectively which is statistically significant, when compared with CCl 4 treated group. The reversal by standard silymarin 100 mg/kg was also significant i.e. 1.546 and 0.251 in case of total and direct bilirubin respectively. There was a little rise observed in the total chloesterol levels and the treatment with the 70% ethanolic extract of MLK reversed it in a dose dependant manner. Rise in ALP IU/L serum levels was remarkable (235.86) and brought back significantly by the 20 mg/kg and 40 mg/kg doses of 70% ethanolic extract of MLK to 176.60 and 112.78 respectively, whereas standard silymarin 100 mg/kg responded well and restored the ALP levels to 95.68. Moreover, a slight rise in triglycerides levels which was reversed by 70% ethanolic extract of MLK 20 mg/kg and 40 mg/kg in a dose dependent manner. The reversal with the treatment of silymarin 100 mg/kg was found to be satistically significant (145.48) when compared with CCl4 treated group. The results are summarized in Table No. 6 DISCUSSION Administration of CCl4 has caused hepatotoxicity as indicated by the enhanced levels of biochemical markers of hepatotoxicity, e.g. SGPT, SGOT, ALP, total and direct bilirubin, cholesterol and triglycerides levels. Upon pre-treatment with 70% MLK extract has decreased the elevated levels of biochemical markers like SGPT, SGOT, ALP, total and direct bilirubin, total cholesterol and triglycerides levels in a dose dependent manner. This observation suggests that the 70% ethanolic MLK extract possess hepatoprotective activity against CCl 4 induced liver toxicity. Hepatic injury induced by CCl4 is due to its conversion to highly reactive radical trichloromethoxy radical which further reacts with O2 to give trichloromethylperoxy radical by cytochrome P450 2E1 enzyme. This trichloromethylperoxy radical is the main culprit in causing hepatotoxicity. This particular radical forms a covalent bond with sulfhydryl group of various membrane molecules like glutathione, protein thiol, lipids or unsaturated lipids. This covalent binding of free radical with cellular macromolecule is considered as initial step to chain events which leads to lipid peroxidation (Be-Jen wang, 2004) (Vacheva, 2004) (Hwa Kyung, 2000) (Simopolous, 1992).
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The treatment with 70% ethanolic MLK has normalized the CCl4 induced biochemical and tissue aberrations, therefore it may be suggested that hepatoprotective activity against CCl 4 challenge is probably due to its free radical scavenging activity and prevention of lipid peroxidation. The restoration of tissue GSH levels by the treatment with test extract is indicating that the inbuilt protective mechanism is being restored. This hepatoprotective activity may be attributed to the anti-oxidant activity of the plant. In addition, there are some reports that the leaves of this plant contain polyphenolic compounds like tannins and flavonoids (Kirtikar, 1998) (Yosioka, 1974). These polyphenolic compounds have anti-oxidant property and anti-oxidants have known to possess hepatoprotective activity (Sannomiya, 2005) (Cholbi, 1991) (Di carlo, 1999) (Fauconneau, 1997). Table No. 1. Reducing power activity of extracts of Madhuca longifolia (Koenig) leaves.
Groups Control Control + standard 25 g Control + 70% Ethanolic Extract 20 g Control + 70% Ethanolic Extract 40 g Control + 70%.Ethanolic Extract 60 g Control + 70% Ethanolic Extract 80 g Control + 70% Ethanolic Extract 100 g Absorbance Mean SEM 0.171 0.001 0.296 0.012*** 0.193 0.001 0.237 0.003*** 0.269 0.005*** 0.302 0.001*** 0.331 0.002*** % Increase -73.09 12.86 38.59 57.30 76.60 93.56
Values are the mean S.E.M., n=3, Significance *** P<0.001 and *P<0.05, compared to control. Std : Sodium metabisulphate Table No. 2. Superoxide anion scavenging activity of extracts of Madhuca longifolia (Koenig) leaves
Groups Control Control + standard 25 g Control + 70% Ethanolic Extract 20 g Control + 70% Ethanolic Extract 40 g Control + 70%. Ethanolic Extract 60 g Control + 70% Ethanolic Extract 80 g Control + 70% Ethanolic Extract 100 g Absorbance Mean SEM 0.862 0.020 0.225 0.001*** 0.453 0.002*** 0.417 0.002*** 0.362 0.002*** 0.311 0.001*** 0.249 0.002*** % Increase -73.89 47.44 51.62 58.00 63.92 71.11
Values are the mean S.E.M., n=3, Significance *** P<0.001 compared to control, Std : Sodium metabisulphate Table No. 3. Hydroxyl ion radical scavenging activity of extracts of Madhuca longifolia (Koenig) leaves
Group Absorbance Mean SEM (after 1 hr.) 0.352 0.001 0.118 0.003*** 0.271 0.002*** 0.244 0.002*** 0.201 0.002*** 0.155 0.002*** 0.109 0.003*** Percentage inhibition -66.47 23.00 30.00 42.80 55.90 69.00 Absorbance Mean SEM (after 4 hr.) 0.405 0.001 0.182 0.003*** 0.254 0.002*** 0.208 0.001*** 0.179 0.001*** 0.155 0.002*** 0.116 0.002*** Percentage inhibition -55.61 37.28 48.64 55.80 61.72 71.35
Control Control + standard 25 g Control +70% Ethanolic Extract 20 g Control +70% Ethanolic Extract 40 g Control +70%. Ethanolic Extract 60 g Control +70% Ethanolic Extract 80 g Control + 70% Ethanolic Extract 100 g
Values are the mean S.E.M., n=3, Significance *** P<0.001 compared to control, Std : Sodium metabisulphate
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Table No. 4. Effect of 70% ethanolic extract of Madhuca longifolia (Koenig) leaves on tissue GSH levels in CCl4 induced hepatotoxicity
Treatment Negative Control (1ml vehicle) Positive Control CCl4 + Liq. Paraffin (1:1) (2 ml/kg s.c.) CCl4 + Standard (Silymarin) (2 ml/kg s.c.+ 100 mg/kg p.o.) CCl4 + 70% ethanolic extract (2 ml/kg s.c.+ 20 mg/kg p.o.) CCl4 + 70% ethanolic extract (2 ml/kg s.c.+ 40 mg/kg p.o.) Absorbance Mean SEM 0.905 0.002 0.503 0.005 0.902 0.006*** 0.701 0.022*** 0.808 0.016*** % Increase ---
79.32 39.36 60.63
Values are the mean S.E.M. of six rats/treatment, Significance ***P<0.001, compared to CCl4 treatment. Table No. 5 Effect of 70% ethanolic extract of Madhuca longifolia (Koenig) tender leaves on in vivo lipid peroxidation in CCl4 induced hepatotoxicity.
Treatment Negative Control (1ml vehicle) Positive Control CCl4 + Liq. Paraffin (1:1) (2 ml/kg s.c.) CCl4 + Standard (Silymarin) (2 ml/kg s.c.+ 100 mg/kg p.o.) CCl4 + 70% ethanolic extract (2 ml/kg s.c.+ 20 mg/kg p.o.) CCl4 + 70% ethanolic extract (2 ml/kg s.c.+ 40 mg/kg p.o.) Absorbance Mean SEM 0.228 0.005 0.590 0.011 0.192 0.013*** 0.375 0.013*** 0.293 0.007*** % inhibition ---
67.45 36.44 50.84
Values are the mean S.E.M. of six rats/treatment, Significance ***P<0.001 compared to CCl4 treatment Table No. 6. Effects of 70% ethanolic extract of Madhuca longifolia (Koenig) leaves on biochemical markers in CCl4 induced hepatotoxicity
Treatment SGPT U/L Negative control (1ml dist. water p.o.+1ml/kg liq.paraffin s.c.) CCl4 treated (positive control) (1ml dist.water p.o.+2ml/kg s.c.) 52.558 3.331 312.420 14.275 SGOT U/L 52.216 5.617 318.412 13.543 Biochemical parameters Mean SEM Total Direct Total Bilirubin Bilirubin Cholesterol mg/dl mg/dl mg/dl 0.926 0.193 110.88 0.029 0.019 10.771 4.892 0.451 1.950 0.219 173.28 10.107 ALP IU/L 122.29 6.486 235.86 8.207 Triglycerides mg/dl 171.22 7.198 190.36 7.516
CCl4+Silymarin 65.395 71.212 1.546 0.251 118.25 95.68 145.48 (2ml/kg, s.c.+ 100mg/kg, p.o.) 8.700*** 6.843*** 0.301*** 0.025*** 8.978*** 9.485*** 8.253*** CCl4 +70% ethanolic extract 118.06 139.120 2.363 0.611 150.180 176.60 174.47 (2ml/kg s.c.+ 20mg/kg p.o.) 5.888*** 5.224*** 0.159*** 0.026*** 2.698 2.832*** 0.937 CCl4 +70% ethanolic extract 64.276 92.70 1.331 0.282 127.68 112.78 153.36 (2ml/kg s.c.+ 40mg/kg p.o.) 3.012*** 2422*** 0.106*** 0.009*** 2.463** 2.620*** 1.820** Values are the mean S.E.M. of six rats/treatment, Significance *P <0.05, **P <0.01 and *** P<0.001, compared to CCl 4 treatment.
REFERENCES Karnick CR, Pharmacopoeial standards of herbal drugs, Sri Satguru Publications Indological and Oriental Publishers, 1, 1994, 57-58. Varier PSV, Indian Medicinal Plants, Arya Vaidyasala Kottakkal, Orient Longman, 3, 1995, 362. Nadkarni AK, Indian Materia Medica, Bombay Popular Book Dept, 3(1), 1954, 181.
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Kirtikar KR, Basu BD, Indian Medicinal Plants, Lalit Mohan Babu, 2, 1998, 490-91. Yosioka I, Indada A, Kitagawa I. Structures of a genuine sapogenol protobasis acid and a prosapogenol of seed kernels of Madhuca longifolia, Tetrahedron, 30(6), 1974, 707-714. Dinesh Chandra, Analgesic effect of aqueous and alcoholic extract of Madhuca longifolia (Koeing). Indian J Pharmacol, 33, 2001, 108-111. Aykae G, Vysal M, Yalein AS, Kocak-Toker N, Sivas A, Oz H, The effect of chronic ethanol ingestion on hepatic lipid peroxide, Glutathione, glutathione peroxidase and glutathione transferase in rats, Toxicology, 36, 1985, 7176. Kyung Jin Lee, Eun-Rhan Woo, Chul Yung Choi, Dong Weon Shin, Dong Gun Lee, Ho Jin You, et al. Protective effect of Acteoside on carbon tetrachloride-induced hepatotoxicity, Life Sciences, 74, 2004, 1051-1064. Hye Gwang Jeong, Inhibiton of cytochrome P450 2E1 expression by oleanolic acid: hepatoprotective effects against carbon tetrachloride-induced hepatic injury, Toxicol Letter, 105, 1999, 215-222. Barry hathwell, John Gutteridge MC, Formation of a thiobarbituric acid reactive substance from deoxyribose in the presence of Iron salts, FEBS Letters, 128 (2), 1981, 347-52. Be-Jen Wang, Chu-ting Liu, Chin-Yin Tseng, Chien-Ping Wu. Hepatoprotective and antioxidant effects of Bupleurum kaoi Liu (Chao et Chuang) extract and its fractions fractionated using supercritical CO2 on CCl4induced liver damage, Food and Chemical Toxicology, 42, 2004, 609-617. Suja SR, Latha PG, Pushpangadan P, Rajasekharan S, Evaluation of hepatoprotective effects of Helminthostachyes zeylanica (L.) Hook against carbon tetrachloride-induced liver damage in Wister rats, J. Ethnopharmacol, 92, 2004, 61-66. Be-Jen Wang, Chu-ting Liu, Chin-Yin Tseng, Chien-Ping Wu. Hepatoprotective and antioxidant effects of Bupleurum kaoi Liu (Chao et Chuang) extract and its fractions fractionated using supercritical CO2 on CCl4induced liver damage. Food and Chemical Toxicology, 42, 2004, 609-617. Vlacheva-Kuzmanova, Borisova P, Galunska B, Krasnaliev I,Belcheva A. Hepatoprotective effect of the natural fruit juice from Asonia melanocarpa on carbon tetracholoride-induced acute liver damage in rats, Experimental and Toxicological Pathology, 56, 2004, 195-201. Hwa-Kyung Lim, Hack-seang Kim, Hong-Serck Choi, Seikwan Oh, Jongwon Choi. Hepatoprotective effects of bergenin, a major constitute of Mallotus japonicus, on carbon tetrachloride-intoxicated rats. J Ethnopharmacol, 72, 2000, 469-474. Simopoulous AP, Norman HA, Gillaspy JE, Duke JA, Common purslane: a source of omega-3-fatty acids and anti-oxidants, J Ame Col Nut, 11(4), 1992, 374-82. Kirtikar KR, Basu BD, Indian Medicinal Plants, Lalit Mohan Babu, 2, 1998, 490-91. Yosioka I, Indada A, Kitagawa I. Structures of a genuine sapogenol protobasis acid and a prosapogenol of seed kernels of Madhuca longifolia, Tetrahedron, 30(6), 1974, 707-714. Sannomiya M, Vitor B Fonseca, da Silva MA, Rocha LRM, dos Santos LC, Hiruma-Lima CA, et al. Flavonoids and antiulcerogenic activity from Byrsonima crassa leaves extracts, J Ethnopharmacol, 97, 2005, 1-6. Cholbi MR, Paya M, Alcaraz MJ, Inhibitory effects of phenolic-compounds on CCl4-induced microsomal lipidperoxidation, Experientia, 47, 1991, 195-99. Di Carlo G, Mascolo N, Izzo AA, Capasso F, Flavonoids: old and new aspects of a class of natural therapeutic drugs, Life Sci 1999, 65, 337-353. Fauconneau B, Waffo-Teguo P, Huguet F, Barrier L, Decendit A, Merillon JM. Comparative study of radical scavenger and anti-oxidant properties of phenolic compounds from Vitis viniefera cell cultures using in vitro tests, Life Sci, 61, 1997, 2103-10.
A Madhusudhan Reddy et.al
FORMULATION AND EVALUATION OF SUSTAINED RELEASE MATRIX TABLETS OF METFORMIN HYDRHOCLORIDE

A Madhusudhan Reddy*, Ayesha Siddika, P Surya Bhaskara Rao, P Manasa1, J Siddaiah1, P Srinivasa Babu1 1. Department of pharmaceutics, Vignan Pharmacy College, Vadlamudi, Andhra Pradesh
* Corresponding author: Email: msreddympharm@gmail.com ABSTRACT Diabetes is the major disease in the world. Metformin is used to treat the type II diabetes. Sustained tablets of metformin were prepared by using of different proportion of polymers, xanthan gum, and guar gum by wet granulation technique. The mixture of drug and excipients has shown good flow properties and tablets were proven that it has sufficient post compression properties. The sustained matrix tablets prepared with natural excipients are having better sustainability than marketed Metformin hydrochloride tablet that is prepared with synthetic polymers. The dissolution studies proved that our matrix tablets have shown 87.02% of drug release in 8hrs where as the marketed drug has shown 105.6% drug release in 8hrs. Keywords-Sustain release tablets, Metformin hydrochloride, Natural Polymers, Wet granulation Technique. INTRODUCTION Diabetes is one of the major causes of death and disability in the world. World Health Organization (WHO) estimate for the number of people with diabetes worldwide, in 2000, is 171 million, which is likely to be at least 366 million by 2030. Metformin is an oral diabetes medicine that helps to control blood sugar levels metformin is used for the people with type II diabetes. Sometimes it is given in combination that is metformin and insulin. The aim of the study is to formulate and evaluate sustained released matrix tablets of metformin hydrochloride using natural polymers (Xanthan gum, guar gum) and evaluate its sustain release capability. MATERIALS AND METHODS Metformin HCL, Microcrystalline cellulose, Xanthan gum, Guar gum and Magnesium stearate were procured from Loba Chemical, Cochin. METHOD OF PREPARATION OF METFORMIN HCL MATRIX TABLETS Different Metformin HCL formulations were prepared by Wet granulations technique (F-1 to F-7) by using PVP K-30 as binding agent. Wet granulation was done in Rapid Mixing Granulator (RMG). The polymers xanthan gum, guar gum and microcrystalline cellulose (MCC) were added in the blending stage along with the lubricating agent magnesium stearate. Tabl.1.Formulation of Metformin Hcl matrix tablets Ingredients(mg or ml) F1 F2 F3 F4 F5 F6 F7 500 500 500 500 500 500 500 Metformin 250 200 150 100 50 0 125 Xanthan Gum 0 50 100 150 200 250 125 Gar gum 22 22 22 22 22 22 22 MCC 10 10 10 10 10 10 10 Mg.Sterate 18 18 18 18 18 18 18 PVP 5 5 5 5 5 5 5 IPA 800 800 800 800 800 800 800 Total PREFORMULATION STUDY Color, odor, taste and appearance: The drug sample was evaluated for its color and odor (Table.2). Melting point determination: Melting point of the drug sample was determined by capillary method by using melting point apparatus (Table.3). Determination of solubility: The solubility of the Metformin HCl was determined by adding excess amount of drug in the solvent (Table.4) and equilibrium solubility was determined by taking supernatant and analyzing it on Shimadzu UV 2501PC, double beam spectrophotometer. The solubility of the Metformin HCl was found to be slightly soluble in water and freely soluble in acetone.
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Table.2. Results of identification of drug Parameter Drug Color White Odour Odorless Taste Tasteless Appearance Crystalline powder Table.3. Results of melting point determination of drug Reported melting point Observed Melting point 222-226C 222-224C
Table: 4 Solubility of Metformin HCL in different solvents Calibration curves: Medium found %Found Mg/100ml Water 99.58% 99.58 0.1N HCL 100.10% 100.10 Phosphate buffer Ph(6.8) 99.65% 99.65 EVALUATION OF BLEND Angle of Repose, Bulk density, Tap density, Compressibility index, Hausners ratio were found and reported in Table 5. Table.5. Precompression studies of formulations F1-F7 Formula Angle of repose () Bulk density (gm/ml) Tap density (gm/ml) Compressibility index (%) Hausners ratio F1 21.8 0.5 0.6 16.66 1.2 F2 27.9 0.5 0.6 16.66 1.2 F3 26.1 0.36 0.53 32 1.47 F4 23.7 0.46 0.53 13.2 1.15 F5 22.8 0.44 0.57 22.8 1.29 F6 24.7 0.39 0.52 25 1.33 F7 18.4 0.53 0.66 1.33 1.24
EVALUATION OF TABLETS Weight variation, hardness and friability were calculated and reported in Table.6. Table.6. Postcompression studies of formulations F1-F7 Hardness Kg/cm2 Friability% Formula Weight variation(mg) F1 8002.54 5 Pass F2 8001.1 6 Pass F3 8000.2 4 Pass F4 8002.0 5 Pass F5 8001.27 3 Pass F6 8001.6 4 Pass F7 8000.88 3 Pass DISSOLUTION STUDIES The in-vitro release of Metformin HCl from formulated tablets was carried out for 8 hours in 6.8pH phosphate buffer. The studies were performed in USP dissolution apparatus II (Electrolab, Mumbai, India) at37 0.5 C and 100 rpm speed. Samples were collected at the end of each hour up to 8 hours and diluted to suitable concentration and analyzed for Metformin HCl content at 233.0 nm by using UVvisible spectrophotometer. Dissolution in Multimedia: The optimized batch then evaluated for multimedia dissolution study. The 4.5pH phosphate buffer and 0.1NHCl are used as dissolution media. Then the drug release was compared with the marketed drug release respectively. Drug release kinetics: Dissolution data of above two methods was fitted in Zero order, First order and Higuchi equations. The mechanism of drug release was determined by using Higuchi equation.
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Zero-Order Kinetics: Zero order as cumulative amount of drug released Vs time, C = K0 t. Where K0 is the zeroorder rate constant expressed in units of concentration/time and t is the time in hours. A graph of concentration Vs time would yield a straight line with a slope equal to K0 and intercept the origin of the axis. First order kinetics: First order as log cumulative percentage of drug remaining vs time, LogC = LogCo kt / 2.303. Where C0 is the initial concentration of drug, k is the first order constant, and t is the time. Higuchi Model: Higuchis model as cumulative percentage of drug released vs square root of time, Q = Kt1/2. Where K is the constant reflecting the design variables of the system and t is the time in hours. Hence, drug release rate is proportional to the reciprocal of the square root of time. Korsmayer Peppas equations: To evaluate the mechanism of drug release from Disulfiram implant, data for the first 60% of drug release were plotted in Korsmeyer et als equation log cumulative percentage of drug released vs log time, and the exponent n was calculated through the slope of the straight line, Mt / M = Ktn. Where Mt/M is the fractional solute release, t is the release time, K is a kinetic constant characteristic of the drug/polymer system, and n is an exponent that characterizes the mechanism of release of tracers. For cylindrical matrix tablets, if the exponent n = 0.45, then the drug release mechanism is Fickian diffusion, and if 0.45 <n < 0.89, then it is nonFickian or anomalous diffusion. An exponent value of 0.89 is indicative of Case-II Transport or typical zero-order release.
120 100 80 60 40 20 0 0 2 4 6 8 10
LOG % UN DISSOLVED
% Drug dissolved
f1 f2 f3 f4 f5 f6 f7
3 2 1 0 0 5 10
F1 F2 F3 F4 F5 F6 F7
2.5 1.5 0.5
Time
% Drug dissolved % Drug dissolved
Figure.3.Comparative zero order graph from F1-F7 and marketed drug F8 120 F1 100 80 60 40 20 0 0.00 1.00 2.00 F2 F3 F4 F5 F6 3.00 -1 -0.5
TIME
Figure. 4. Comparitive first order kinetics from F1-F7 and marketed drug F8 2.5 2 1.5 1 0.5 0 F1 F2 F3 F4 F5 F6 0 0.5 1
Square root of time

Figure.5. Comparative zero order graph from (F1-F7)
Log time
Figure: 6 Comparative Higuchi Plots of Formulation F1F7 and Marketed Tablet
CONCLUSION Tablets are formulated with different proportions of guar gum and xanthan gum, these formulation have been shown in good flow properties along with good drug release but it does not shows better sustain action. When tablets are formulated only with guar gum, these tablets are shown better flow properties better drug release and also show best sustained action even than the Innovator product. F7 contain xanthan gum and guar gum of ratio 1:1 maintained constantly throughout formulation F1-F7. From the dissolution studies it is observed that increasing the concentration of guar gum there was decrease in drug release. Among all the prepared metformin sustained released tablets formulation F6 with complete guar gum exhibited good sustained release and follows good zero order kinetics. From the kinetics of drug released studies it is observed that F6 shows regression value of 0.999. This gives the evidence that the drug release follows higuchi model conforming the effect of matrix tablets.
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REFERENCES Costa P, Modelling and comparission of dissolution profiles, European Journal of Pharmaceutical Sciences, 13, 2001, 123-133. Herman GA, Stein PP, Thornberry NA, wagner JA, Dipeptidyl peptidase-4 Inhibitors for the treatment of type 2 Diabetes, Clin Pharmacol Ther, 81 (5), 2007, 761-767. Higuchi W I, Diffusional models useful biopharmaceutics drug release rate processes, J Pharm Sci, 56, 1967, 315324. Korsmuyer RW, Gurny R, Doelker E, Mechanism of solute release from porous hydrophilic polymers, Int J Pharm, 15, 1983, 25-35. MA Kalam, M Humayun, N Parvez, S Yadav, A Garg, S Amin, Y Sultana and A Ali, Continental J Pharmaceutical Sciences, 1, 2007, 30-35. Peppas NA, Analysis of Fickian and Non-fickian drug release from polymers, Pharm Acta Helv, 60, 1985, 110111. R Colombo, PS Bettini, NA Peppas, Swellable matrixes for controlled drug delivery: gel-layer behavior, mechanisms and optimal performance, Pharm Sci Technol Today, 3, 2000, 198204. The British Pharamacopoeia, British Pharamacopoeia Commission Office, London, 3, 2009, 8980.
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Sampath Kumar et.al
HERBAL MEDICINE
Atheeq-ur-Rahman1, Ismail Shaik1 , K.P.Samapth Kumar2* 1. Nimra college of pharmacy, Nimranagar, Vijayawada 2. Department of Pharmaceutical Sciences, Coimbatore medical college, Coimbatore *Corresponding author: kp_sampath@rediffmail.com ABSTRACT Herbal medicines have become popular for healthcare. The consumption of such formulations and botanicals has increased in recent years. Herbal products are defined as herbal medicines that are administered to patients and are mixtures of herbal substances. In the preparation of herbal formulation various parts of plants are used such as roots, bark, stem, seeds, fruit, leaves etc. The various parts of the plants contain different constituents which have different pharmacological effects. Recently, the traditional use of plants for wound healing has received attention by the scientific community. Approximately one-third of all traditional medicines in use are for the treatment of wounds and skin disorders, compared to only 1-3 % of modern drugs. Medicinal plants are coming into prominence because of the conventional medicine such as antibiotics which have developed resistance to many of the infection organisms which no longer responsive to conventional medicines. Herbal preparation can be more effective and safer than conventional medicines. Key words: Herbal medicine, alternative medicine, Ayurvedic medicine, Integrative medicine, Chiropractic INTRODUCTION During the past decade, traditional systems of medicine have become a topic of global importance. Current estimate suggest that, in many developing countries a large proportion of the population relies heavily on traditional practioners and medicinal plants to meet primary health care needs. Although modern medicine may be available in these countries, herbal medicines (phytomedicines) have often maintained popularity for historical and cultural reasons. Concurrently, many people in developed countries have begun to turn to alternative or complementary therapies, including medicinal herbs. In the context the contribution made by the traditional medicine to modern system of medicine is worth noting. The well-established drugs developed by the scientists after analyzing the chemical constituent of plants which are traditionally used by the tribal and villagers. It is important to note that there is no doubt about the efficacy of herbal medicine. Traditional medicine constitutes an extremely fascinating and attractive world under many points of view, and for a wide range of users in developing and developed countries. For example, traditional medicine may be highly secretive, mystical, and extremely localized, or codified or very well regulatated. As a consequence, traditional medical knowledge may be passed on orally from generation to generation or may be openly taught in officially recognized universities. However, the WHO has delineated a working definition of traditional medicine as including diverse health practices, approaches, knowledge, and beliefs incorporating plant, animal, and/or mineral based medicines, spiritual therapies manual techniques and exercise applied singularly or in combination to maintain well-being, as well as to treat, diagnose or prevent illness. Furthermore traditional medicine therapies can be categorized as medication therapies, when they use herbal medicines, animal parts and/or minerals, and as non-medication therapies, if carried out mainly without the use of medication, as in the case of acupuncture or manual therapies. The term traditional medicine is sometime replaced by other terms, as complementary, alternative or non-conventional medicine. Traditional herbal medicines in drug discovery: Traditional medicines, mainly herbal in nature are now a days used for treating the disease. Various herbal formulations are existing now a day. The WHO recognized this fact in the early 1970s and encouraged government to use herbal medicines for disease prevention and health promotion .Drugs used in medicine today are either obtained from nature or synthetic origin. Therapeutic use of plants for the treatment of human illnesses dates back over many millennia. Evidence of their effectiveness in the diagnosis, cure and prevention of disease states exists in every culture throughout the world. Today traditional medicine, characterized by the use of herbs and other natural products, still remains a regular component of health care in the world. Thereview covered all aspects of traditional medicines and revealed that a detailed experimental as well as standardization study is required to explore the plantsand their uses to treat serious complication. It is hoped that the more efficient and effective application of natural products will improve the drug discovery.
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Table.1. Some of the modern drugs given in table which are developed from traditionally known drugs. Modern drug Traditional medicinal use Plant source Aspirin Reduces pain and inflammation Filipendula ulmaria Pilocarpine Reduces pressure in the eye Pilocarpus jaborandi Quinine Combats malaria Cinchona pubescens Reserpine Lowers blood pressure Rauwolfia serpentina Diosgenin contraceptive Dioscorea floribunda Digitoxin Dropsy, relives heart congestion Digitalis purpurea
SYSTEMS OF MEDICINE EXPLAINED: CONVENTIONAL, ALTERNATIVE, INTEGRATIVE, COMPLEMENTARY AND MORE Complementary medicine: Is a term that defenders of conventional medicine like to use to claim intellectual ownership over alternative medicine. Complementary medicine means combining conventional therapies with alternative therapies, but the alternative therapies are almost always dismissed from being the primary treatments as they are routinely relegated to supportive roles. For example, complementary medicine's supporters might say that ginger is great for reducing nausea following chemotherapy, but they would never say ginger or garlic are anticancer herbs in their own right. Promoters of "complementary medicine" are usually closet drugs-and-surgery pushers who use this phrase to avoid appearing totally out of touch with health trends. The complementary medicine movement is largely an attempt by conventional medicine promoters to prevent their harmful system of medicine from appearing completely irrelevant as the public turns to safer, more effective and natural alternatives. Conventional doctors refer to it as CAM, or Complementary and Alternative Medicine. (D.R.Laurence, et al., clinical pharmacology 7th edition) Alternative medicine: Is a somewhat outdated term that refers to everything outside the realm of conventional medicine. It's outdated because alternative medicine is now mainstream medicine. Most people use it, and the only reason more people don't is because health-insurance refuses to cover most of the therapies in alternative medicine. This term will probably fade away as the use of natural healing therapies becomes even more popular with the general public. Conventional medicine: Refers to the classic medical training offered through mainstream medical schools. This is a drugs-and-surgery approach to medicine that largely excludes nutrition, wellness, mind-body medicine, patient education, and other natural therapies. Western medicine: Refers to the type of medicine practiced in the West; the United States, Western Europe, and so on. It's based on the philosophical foundations of Western thinking, which maintains that a body is only a collection of its parts, and that by isolating the parts and studying them separately, you can understand the whole. Eastern medicine: Looks at the whole patient, the whole body, the whole experience, and never believes that just treating one organ or using one chemical, drug, or herb is the answer to any health condition. Traditional Chinese medicine: Sometimes shortened to TCM, involves the treatment of patients using the fundamental approaches of healing developed over the last 4000 years in China. The treatments in Chinese medicine include acupuncture, Chinese herbs, and Tui-Na, which resembles massage therapy combined with therapeutic touch. Ayurvedic medicine: Is a system of holistic medicine practiced widely in India and throughout Southeast Asia. It is also gaining popularity and recognition in the United States, Europe, Australia and many other areas of the world as people come to recognize the inherent wisdom and innate safety of Ayurvedic medicine. Based on thousands of years of development and use, Ayurvedic medicine is organized around the energy patterns of individuals (the way they use their bodies, what they eat, how they digest, levels of body heat, etc.) and treatment using a wide array of medicinal herbs and substances (like essential oils, coconut oil, and so on). There is also a prominent recognition of the mind/body link in Ayurvedic medicine. (Ayurvadic pharmacopoeia of India 1 st edition) Integrative medicine: Is a relatively recent term that typically describes a more balanced, welcoming approach to using natural therapies alongside conventional ones. While this system of medicine still uses conventional medicine therapies such as drugs and surgeries, it usually recommends them only as a last resort, instead attempting to prevent or treat health conditions using natural therapies first. The descriptive phrase "integrative medicine," coined in the late twentieth century, characterizes a new model of health care rooted both in
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conventional and alternative medicine. In the broadest sense, integrative medicine employs modalities drawn from all medical therapeutic paradigms, providing patients with individualized treatment plans optimized for their specific clinical situations. The underlying philosophy recognizes and relies upon the innate healing capacity of the human body and emphasizes the importance of the relationship between practitioner and patient in fostering this capacity. Advanced medicine: Refers to the future of medicine, based on supporting the patient's health and wellness rather than attacking the patient with various chemicals or procedures like radiation and surgery. Advanced medicine is the first step to the new age of understanding about the true underlying causes of health and wellness. Advanced medicine makes conventional medicine and Western medicine obsolete, and it includes therapies like phototherapy, sunlight, nutrition, sound therapy, vibrational medicine, electromedicine, mind-body medicine, energy healing, and other similar modalities that were once considered experimental, but are now well-known to be both safe and effective at supporting the health of the patient. TRADITIONAL HERBAL MEDICINE IN THE WORLD The most widespread system of traditional medicine in the world: Chinese medicine: The earliest record of traditional medicine can be trace back to the 8th century B.C., Diagnosis and treatment are based on a holistic view of the patients symptoms, expressed in terms of the yin and yang. Yin represents the earth, cold, feminity; Yang represents the sky, heat and masculinity. The action of Yin and Yang influence the interaction if the five elements composing the universe: metal, wood, water, fire and earth. Practioners of Chinese medicines seek to control the levels of Yin and Yang through 12 meridians, which bring energy to the body. Acupuncture for example that is one of the most widely used Chinese medicine practices, is based on the meridian theory. Herbal medicines are also a consistent part of such a traditional system. The Chinese herbs were classified to their perceive ability to affect any of manifestation of the living body. Herbs were also characterized pharmacologically in terms of their taste: bitter, sweet, salty and sour this being seen to denote particular properties. In China, doctors were practicing relatively advanced medicine before the birth of Christ. Western medicine, in response, dismisses everything under Chinese medicine, ignoring the long history of safe and effective use of Chinese herbs, acupuncture and other philosophies espoused by traditionally Chinese medicine .Chinese medicine is not limited to China, by the way. It is practiced throughout Asia, including Korea, Japan, Taiwan, Thailand, and many other countries. Ayurveda: Ayurvedas origin can be dated back earlier, in the 10th century B.C. However, its current form looks shape between 5th century A.D. The term Ayurveda is a Sanskrit term meaning science of life, and in fact, Ayurveda is not only system of medicine, but also a way of living. Ayurvedic philosophy is linked to sacred texts, the Vedas, and based on the theory of Panchmahabhutas. All objects and living bodies are composed of the five elements: earth, water, fire, air, sky. Furthermore, the environment is perceived as a macrocosm, and the individual as a microcosm, reciprocally acting on each other. In Ayurveda, drugs (Vegetable, Mineral and Animal) are broadly describe and classified under five properties, viz.1. Rasa 2. Guna 3. Veerya 4. Vipaka 5. Prabhava Ayurveda had never been static. Its practitioners had been innovative and dynamic in the therapeutic practice and carried on clinical trials out of the local flora and discovered newer medicine with same therapeutic values as the classical drugs which might have been than either locally un-available or perhaps demanding heavy prices. These newer drugs have been accepted by the practicing profession as substitutes. In fact on study of Ayurvedic literature, one comes across several references of permitting the use of a substitute drug when the classical drug is not available. This is based on its therapeutic equivalence and clinical efficacy. Recent scientific studies have shown popular Ayurvedic herbs to exhibit powerful medicinal effects, even from a Western point of view. Turmeric, for example, halts the growth of cancer tumors. Cinnamon stabilizes blood sugar, and gymnema sylvestre helps block dietary sugars while supporting the regeneration of insulin-producing beta cells in the pancreas. Chiropractic: Chiropractic was more recently founded at the end of the 19th century. Than founder was a magnetic therapist practicing in Lowa, USA, named Daniel David Palmer. This therapy is based on an association between the spine and nervous system and on the self-healing properties of the human body. It diagnoses and treats mechanical disorder of the joints, muscles and ligaments of the body by manual adjustments. Homeopathy: Homeopathy was first mentioned by Hippocrates (462-377B.C.), but it was a German physician, Hahnemann (1755-1843), who established homeopathys basic principles among which the most important is simila similibus curentur. The founder wrote that in order to radically heal a certain kind of chronic infections,
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it is necessary to find remedies which normally cause in the human body a similar disease, as much similar as possible basically this means that a substance which absorbed in a certain quantity in a healthy person may cause a disease, may also cure it if taken in a different dose. Other principles are direction of cure, principle of single remedy, the theory of minimum diluted dose, and the theory of chronic diseases. Homeopathy today is a rapidly growing system and is being practiced almost all over the world. In India it has become a household name give the safety of its pills and gentleness of its cure. A rough study indicates that about 10% of the Indian population solely depends on homeopathy for their health care needs. It is more than a century and a half now that homeopathy is being practiced in India. It has blended so well into the roots and tradition of the country that it has been recognized as one of the national system of medicine and plays an important role in providing healthcare to a large number of people. Its strength lies in its evident effectiveness as it takes a holistic approach towards the sick individual through promotion of inner balance at mental, emotional, spiritual, and physical levels. Unani: Unani is based on Hippocrates theory of the four bodily humors: blood, phlegm, yellow bile, and black bile, Galen (850-925A.D.) and Avicenna (980-1037A.D.) heavily influenced Unanis foundation and formed its structure. Unani is therefore Greek medicine which developed throughout the Arabic civilization. It draws from the traditional systems of medicine of China, Egypt, India, Iraq, Persia, and the Syrian Arabi Republic. It is also called Arabic medicine. The use of traditional medicine in the prevention, diagnosis and treatment of an extensive range of diseases, has been increasing, overall in the last 20years, both in developing and developed countries. Unani medicine originated in ancient Greece and has been influenced by Persian, Egyptian and African medicine. With the spread of Islam, the practice of unani became widespread in the Indian subcontinent, maintaining its Muslim origins. This admitted that the Unani system in its purest form did not suit local Indian condition, partly because the climate was unsuitable and partly because the drugs in the Unani system were not always available in the Indian subcontinent. THE ROLE OF INDIAN SYSTEM OF MEDICINE IN GLOBAL HEALTHCARE Since the ultimate objective of any system of medicine is to provide healthcare to the needy, it is important to assess whether the Indian systems of medicine are fulfilling their legitimate role in the Global healthcare scene. Among the various components of the Ayurvedic system, classical preparations manufactured according to classical texts are unlikely to gain a foothold in the major markets of the developed Countries, which follow the standards set by leading Regulatory Agencies such as the US FDA, in the matter of approval of drugs for human use. The current methods of their manufacture and the studies associated with them, including those concerned with safety and efficacy are unlikely to be approved by the regulatory agencies in these countries. Protection of the traditional knowledge (TK) of the local and indigenous communities seems to be one of the most contentious and complicated issues. The new technological development, particularly in biotechnology, clearly demonstrates the significance and usefulness of traditional knowledge for the development of new products of commercial importance. KNOWLEDGE ON TRADITIONAL MEDICINES FOR DRUG DISCOVERY During the last half a century, ever since modern chemotherapy emerged, there have been two major approaches to source chemicals for testing, the synthetic route or natural products. Both have been successfully employed and these continue to be relevant for the future of new drug discovery and advances in medicine. The problem of cost in new drug development is related to the very high cost of failures in new drug discovery. Recent efforts by the major R&D-based companies have been directed to minimizing the high costs and risks of drug research. One of the meaningful approaches will be recourse to the knowledge base and heritage available in traditional system of medicine in India and other countries. That natural products could be a major source of new drug leads in obvious. A study reported in 1997 mentioned that 85% of the treatment regimes of 80% of the worlds population are based on natural products. Almost half of the 29 top selling drugs in 1999 were derived from or developed on the basis of leads available from knowledge of properties of natural products either from traditional use or from modern studies. About 80%of the worlds population relies on herbal medicines, and government of Third- world countries, unable to sustain a complete coverage with western-type drug, have encouraged the rational development of traditional treatments. At present the world Health Organization is taking an official interest in such developments in order to facilitate its aim of making health care available for all. Traditional medicine is based on the needs of individual. Different people may receive different treatmen ts even if, according to modern medicine, they suffer from the same disease. Traditional medicine is based on a belief that each individual has his or her on constitution and social circumstances which result in different reaction to causes
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of disease and treatment. For this reason the increasing use of traditional medicine arises from the dissatisfaction caused by the allopathic medicine approach. CONCLUSION Medicinal plants have been a major source of cure of human diseases since time immemorial. Today, one fourth of the world population depends on traditional medicines. Despite the introduction of antibiotics since the 1940's, even 80 per cent of the population today relies on indigenous medicinal plants and the drugs. It is estimated that the global traditional medicine market is growing at the rate of 7 - 15 per cent annually. The medicinal plant value is about Rs.5000 crores in India and it is estimated that the country exports about Rs.550 crore worth of herbal drugs. But with the rich and diverse botanical resources in our country, this is not an impressive export performance considering the worldwide herbal market worth US 60 billion dollars.The Indian manufacturers have to stress on various standardization processes to establish their products in the global markets. Standardization starting from production of quality materials, analysis of raw materials for authentication, foreign matter, organoleptic evaluation, microscopic examination, extractive values, chromatographic profiles, pesticides residue, heavy metal detection etc. is necessary for standardization of drugs. Similarly, the standardization methods of medicinal plants and its extracts have great importance in the fields of cosmetics and neutraceuticals, which are emerging as two most important segments in the global markets. REFERENCES Aiyer MN, Namboodiri AN and Kolammal M, Pharmacognosy of Ayurvedic Drugs, Trivandrum, 5, 1957, 49-55. Akhtar N, Ali M and Alam MS, Herbal drugs used in dental care, The Pharma Review, 10, 2005, 61-68. Ansari FZ, Alam S, Jain P, Akhter S and Ansari MZH, Vitiligo and its herbal treatment, The Pharma Review, 12, 2008,137-13. Basu NK and Lamsal P, Investigation on Indian medicinal Plants II: Hydrocotyle asiatica, Quart.J. Pharm, 20, 1947, 137. Kathrin K, Eike R and Anne B (2003), Validation of standardized high performance thin layer chromatographic methods for quality and stability testing of medicines, Journal of AOAC International, 86(5), 2003, 909-915. Kuhn MA, Herbal remedies: drug-herb interactions, Critical Care Nurse, 22, 2002, 22-32. Nandakishore D, Shubhangi G, Prakash I, Pallavi S, Parimal K and Shishupal B, Herbal plants with antifertility activity, The Pharma Review, 8, 2007, 131-135. Singh P, Yadav RJ & Pandey A, Utilization of indigenous systems of medicine & homoeopathy in India, Indian J.Med Res, 122, 2005, 137-142. Sukhdev S, Arun N and Kalia AN, Patentability of herbal products: A review, The Pharma Review, 4, 2008, 118-124. Thakur AK, Prasad NAV and Ladha KS, Stability testing of herbal products, The Pharma Review, 4, 2008, 109-112.
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MICROSPONGE DRUG DELIVERY SYSTEM

SK Shafi1, S.Duraivel, Debjit Bhowmik2, K.P.Sampath Kumar3* 1.Nimra College of Pharmacy. Vijayawada 2.Karpagam University,Coimbatore 3.Department of Pharmaceutical Sciences, Coimbatore Medical college, Coimbatore Corresponding author: Email:kp_sampath@rediffmail.com ABSTRACT Microsponges are porous, polymeric microspheres that are mostly used for prolonged topical administration. Microsponges are designed to deliver a pharmaceutically active ingredient efficiently at minimum dose and also to enhance stability, reduce side effects, and modify drug release profiles. The Microsponge Delivery System (MDS) is a unique technology for the controlled release of topical agents and consist of macro porous beads, typically 10-25 microns in a diameter, loaded with active agent. When applied to the skin, the Microsponge releases its active ingredient on a time mode and also in response to other stimuli (rubbing, pH, etc.). MDS technology is being used currently in cosmetics, over -the counter (OTC) skin care, sunscreens and prescription products. Conventional preparations have some disadvantages like unpleasant odour, greasiness and skin irritation. These problems are overcome by microsponge delivery system. Microsponge based drug delivery system produces controlled released action. It also produces site specific and target organ action produced. Microsponge (MDS) mainly developed in topical drug delivery as well as oral controlled delivery system. It also used in cosmetic formulations. Key words- Microsponge Delivery System, Trans dermal penetration, Polymeric delivery systems INTRODUCTION To control the delivery rate of active agents to a predetermined site in human body has been one of the biggest challenges faced by drug industry. Several predictable and reliable systems were developed for systemic drugs under the heading of transdermal delivery system (TDS) using the skin as portal of entry. It has improved the efficacy and safety of many drugs that may be better administered through skin. But TDS is not practical for delivery of materials whose final target is skin itself.Controlled release of drugs onto the epidermis with assurance that the drug remains primarily localized and does not enter the systemic circulation in significant amounts is an area of research that has only recently been addressed with success. No efficient vehicles have been developed for controlled and localized delivery of drugs into the stratum corneum and underlying skin layers and not beyond the epidermis. Application of topical drugs suffers many problems such as ointments, which are often aesthetically unappealing, greasiness, stickiness etc. that often results into lack of patient compliance. These vehicles require high concentrations of active agents for effective therapy because of their low efficiency of delivery system, resulting into irritation and allergic reactions in significant users. Other drawbacks of topical formulations are uncontrolled evaporation of active ingredient, unpleasant odour and potential incompatibility of drugs with the vehicles. Thus the need exists for system to maximize amount of time that an active ingredient is present either on skin surface or within the epidermis, while minimizing its transdermal penetration into the body. The microsponge delivery system fulfills these requirements. A Microsponge Delivery System (MDS) is Patented, highly cross-linked, porous, polymeric microspheres polymeric system consisting of porous microspheres that can entrap wide range of actives and then release them onto the skin over a time and in response to trigger (Kilicarslan, 2003). It is a unique technology for the controlled release of topical agents and consists of microporous beads, typically 10-25 microns in diameter, loaded with active agent. When applied to the skin, the MDS releases its active ingredient on a time mode and also in response to other stimuli (rubbing, temperature, pH, etc). MDS technology is being used in cosmetics, over-the-counter (OTC) skin care, sunscreens and prescription products.Delivery system comprised of a polymeric bead having network of pores with an active ingredient held within was developed to provide controlled release of the active ingredients whose final target is skin itself. The system was employed for the improvement of performance of topically applied drugs. The common methods of formulation remains same; the incorporation of the active substance at its maximum thermodynamic activity in an optimized vehicle and the reduction of the resistance to the diffusion of the stratum corneum. Application Solubility enhancement Site specific action produced on the target organ Increase stability of drug Targeted drug delivery Controlled release drug delivery Topical drug delivery Oral drug delivery Bone tissue engineering Cardiovascular engineering Reconstruction of vascular wall.
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CHARACTERISTICS OF MICROSPONGE Microsponges formulations are stable over range of pH 1-11; Microsponge formulations are stable at temperature up to 130C; Microsponge formulations are self-sterilizing as their average pore size o.25 m where bacteria cannot penetrate; Microsponge formulation have higher payload (50-60%), still free flowing and can be cost effective. The Microsponge technology is a proprietary system of microparticles that can entrap a very wide range of pharmaceutical and cosmetic active ingredient to enhance their performance in topically applied dermatological products. This technology has been introduced in topical drug products to ensure the controlled release of active drug into the skin in order to reduce systemic availability and reduce local cutaneous reaction to active drug.MDS is highly cross linked, patented, porous, polymeric microsphere that acquire the flexibility to entrap a wide variety of active ingredient such as emollients, fragrances, sunscreens, essential oils, anti-ineffective, antifungal and antiinflammatory agents etc. and are used as topical carrier system. Conventional formulation of topical drugs is intended to work on the outer layers of the skin. Typically, such products release their active ingredients upon application, producing a highly concentrated layer of active ingredient that is rapidly absorbed. Thus the need exists for system to maximize the amount of time that an active ingredient is present either on the skin surface or within the epidermis, while minimizing its trans dermal penetration in to the body. Microsponges are microscopic spheres capable of absorbing skin secretions, therefore reducing oiliness and shine from the skin. Microsponge particles are extremely small, inert, indestructible spheres that do not pass through skin. Drug loading in microsponges can take place in 2 ways, one or two step process. 1. Liquid liquid suspension polymerization 2. Quasi emulsion solvent diffusion techniques (Kawashima, 1991). The oral antifungal agents have some disadvantages like, headache, gastrointestinal disturbance, urticaria, diarrhea and photosensitivity. And also nausea, anorexia and vomiting some oral antifungal drug should be avoided during pregnancy. These disadvantages of antifungal drug may be overcome by loading the drug into microsponge. So, present study is designed to minimize tolerability problems like erythema, dryness, irritation caused due to topical administration. To overcome this problem the drug is incorporated into microsponge. Microsponge formulation can avoid systemic uptake of the drug. The application of microsponge delivery system to the skin distributes the active ingredient gradually and has demonstrated the ability to reduce the irritation. THE MICROSPONGE DELIVERY SYSTEM-A NOVEL DRUG DELIVERY SYSTEM The MDS has advantages over other technologies like microencapsulation and liposomes. Microcapsules cannot usually control the release rate of actives. Once the wall is ruptured the actives contained within microcapsules will be released. Liposomes suffer from lower payload, difficult formulation, limited chemical stability and microbial instability. While microsponge system in contrast to the above systems are stable over range of pH 1 to 11, temperature up to 130oC; compatible with most vehicles and ingredients; self sterilizing as average pore size is 0.25m where bacteria cannot penetrate; higher payload (50 to 60%), still free flowing and can be cost effective. Most liquid or soluble ingredients can be entrapped in the particles. Actives that can be entrapped in microsponges must meet following requirements: 1. It should be either fully miscible in monomer or capable of being made miscible by addition of small amount of a water immiscible solvent. 2. It should be water immiscible or at most only slightly soluble. 3. It should be inert to monomers. 4. It should be stable in contact with polymerization catalyst and conditions of polymerization. ADVANTAGES: 1. Controlled drug release 2. Site specific action produce on target organ 3. Enhanced drug stability 4. High drug loading capacity Improve therapy 5. Compatible with vehicle and ingredients 6. Stable over the range 1 -11 ph. 7. Solution Free flowing and cost effective 8. Improve thermal,physical, and chemical stability 9. Flexibility to develop novel product forms
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APPLICATIONS OF MICROSPONGE SYSTEMS Microsponges are porous, polymeric microspheres that are used mostly for topical and recently for oral administration. It offers the formulator a range of alternatives to develop drug and cosmetic products. Microsponges are designed to deliver a pharmaceutical active ingredient efficiently at the minimum dose and also to enhance stability, reduce side effects and modify drug release. 1. Microcapsules cannot usually control the release rate of the active pharmaceutical ingredients (API). Once the wall is ruptured the API contained within the microcapsules will be released 2. Pay load is up to 50 60%. 3. Free flowing and cost effective. 4. Microsponges are microscopic spheres capable of absorbing skin secretions, therefore, reducing oiliness and shine from the skin. PROPERTIES OF THE ACTIVES FOR THE ENTRAPMENT INTO MICROSPONGES 1. It should be either fully miscible in a monomer or capable of being made miscible by the addition of a small amount of a water-immiscible solvent. 2. It should be water immiscible or at most only slightly soluble. 3. It should be inert to monomers and should not increase the viscosity of the mixture during formulation. 4. It should be stable when in contact with the polymerization catalyst and under conditions of polymerization. 5. The spherical structure of the microsponges should not collapse. METHOD OF PREPARATION: Liquid Liquid suspension method: Quasi Emulsion solvent diffusion: CONCLUSION Microsponges are polymeric delivery systems composed of porous microspheres. They are tiny spongelike spherical particles with a large porous surface. Moreover, they may enhance stability, reduce side effects and modify drug release favorably. Microsponge technology has many favorable characteristics, which make it a versatile drug delivery vehicle. Microsponge Systems are based on microscopic, polymer-based microspheres that can suspend or entrap a wide variety of substances, and can then be incorporated into a formulated product such as a gel, cream, liquid or powder. The outer surface is typically porous, allowing a sustained flow of substances out of the sphere. Microsponges are porous, polymeric microspheres that are used mostly for topical use and have recently been used for oral administration. Microsponges are designed to deliver a pharmaceutical active ingredient efficiently at the minimum dose and also to enhance stability, reduce side effects, and modify drug release. REFERENCES Martin A, Swarbrick J & Cammarrata A, Chapter 19, In: Physical Pharmacy- Physical Chemical Principles in Pharmaceutical Sciences, 3rd Ed, 1991, 527. Emanuele A D, Dinarvand R, Preparation, Characterization and Drug Release from Thermo responsive Microspheres, International Journal of Pharmaceutics, 1995, 237-242. Kilicarslan M, Baykara T, The effect of the drug/polymer ratio on the properties of Verapamil HCl loaded microspheres, Int. J. Pharm, 252, 2003, 99109. Kawashima Y, Niwa T, Takeuchi H, Hino T, Itoh Y, Furuyama S. Characterization of polymorphs of tranilast anhydrate and tranilast monohydrate when crystallized by two solvent change spherical crystallization techniques, J. Pharm. Sci, 81, 1991, 472-478. Bodmeier R, Chen H. Preparation and characterization of microspheres containing the anti-inflammatory agents, indomethacin, ibu-profen, and ketoprofen, J. Control. Release, 10, 1989, 167-175. Jones DS, Pearce KJ, Investigation of the effects of some process variables on, microencapsulation of propranolol HCl by solvent evaporation method, Int. J. Pharm. 1995; 118, 1995, 99-205. Wakiyama N, Juni K, Nakano M, Preparation and evaluation in vitro of polylactic acid microspheres containing local anesthetic, Chem. Pharm. Bull, 29, 1981, 3363-3368.
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Barkai A, Pathak V, Benita S, Polyacrylate (Eudragit retard) microspheres for oral controlled release of nifedipine. I. Formulation design and process optimization. Drug Dev, Ind. Pharm, 16, 1990, 2057-2075. Shah V.P, Determination of In-vitro Release from Hydrocortisone Creams, International Journal of Pharmaceutics, 53, 1989, 53-59. Sato T, Kanke M, Schroeder G, Deluca P. Porous biodegradable microspheres for controlled drug delivery. I: Assessment of processing conditions and solvent removal techniques. Pharm Res, 5, 1988, 21 -30. Draize J.H, Woodard J & Calvary H.O, Methods for the Study of Irritation & Toxicity of Substances Applied Topically to the Skin & Mucous Membranes. Journal of Pharmacology & Experimental Therapeutics, 82, 1944, 377-389. Franz T. J, Percutaneous absorption, On the relevance of in vitro date, J. Invest. Dermatol, 45, 1975, 498-503. Khopade A. J., Jain Sanjay, Jain N.K., March 1996, The Microsponge; Eastern Pharmacist, 49-53. Yazici, E, Kas H. S, Hincal A. A, Microsponges, Farmasotik Bilimler Dergisi (Turkey), 19, (3), 1994, p121-128. Wester R, Patel R, Natch S, Leyden J, Melendres J, Maibach H, Controlled release of benzoyl peroxide from a porous microsphere polymeric system can reduce topical irritancy, J. Am. Acad. Derm, 24, 1991, 720-726. Fincham J. E, Karnik K. A, Patient Counseling and Derm Therapy, US Pharmacist, 19, 1994, 56-57, 61-62, 66, 71-72, 74, 77-78, 81-82. Grimes P. E, A microsponge formulation of hydroquinone 4% and retinol 0.15% in the treatment of melasma and post-inflammatory hyperpigmentation, Cutis, 74(6), 2004, 362-368. James J. Leyden , Alan Shalita, Diane Thiboutot, Kenneth Washenik, and Guy Webster, Topical Retinoids in Inflammatory Acne; A Retrospective, Investigator-Blinded, Vehicle-Controlled, Photographic Assessment, Clin. Therapeutics, 27, 2005, 216-224.
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NANOTHERAPEUTICS AN ERA OF DRUG DELIVERY SYSTEM IN NANOSCIENCE Bhargavi1, Ch.Anil1, Debjit Bhowmik2, Praneta Desale3, K.P.Sampath Kumar4* 1. Nimra College of Pharmacy. Vijayawada 2. Karpagam University, Coimbatore 3. CMJ University, Shillong, Megalaya 4. Department of Pharmaceutical Sciences, Coimbatore Medical College, Coimbatore * Corresponding author: Email: kp_sampath@rediffmail.com ABSTRACT Nanotherapeutics is likely to make increasing claims on a particular area of health care regulatory strength: scientific cost-effectiveness assessment of innovation in medical products. An important and exciting aspect in the newly developing field of nanomedicine is the use of nanoparticle drug delivery systems allowing for innovative therapeutic approaches. Due to their small size, these drug delivery systems are promising tools in therapeutic approaches such as selective or targeted drug delivery towards a specific tissue or organ, enhanced drug transport across biological barriers, and intracellular drug delivery. KEYWORDS- Nanomedicine, cost-effectiveness, innovative therapeutic approaches, Drug delivery INTRODUCTION Nanotechnology is a revolutionary field of micro-manufacturing involving manipulation, by chemical or physical processes, of individual atoms and molecules. Nanotechnology-enabled drug delivery will rise to $26 billion by 2012 from its current size of $3.39 billion, representing a compound annual growth rate of 37%.But this is just the beginning; the market could steeply rise after 2012, reaching potentially $220 billion by 2015 for these nano-enabled compounds. Nanotechnologies have already begun to change the scale and methods of drug delivery and hold huge potential for future developments in this area. New formulations and routes for drug delivery have the potential to broaden the therapeutic potential of administered treatments by allowing the delivery of new types of medicine to previously inaccessible sites in the body. In contrast to developing completely new drug compounds, introducing upgraded formulations greatly reduces the risk, time and capital invested in new drug development. Nanoparticle drug delivery technology can enable reformulation of existing drugs to increase product lifecycle, increase profitability, expand intellectual property estate and discourage competition during a drugs most valuable years. Nanomedicine is new concept in combining nanotechnology and medicine. Nanotherapeutics is the use of nanomedicine in therapy.Nanotechnology represents a cluster of technologies with different characteristic and applications. Nanomedicine related applications are under development and this is a long process of converting nanomedicine in to commercially viable products. The ultimate goal of nanotherapeutic is comprehensive monitoring repair and improvement of all human biologic system. There is great concern over the environmental and health risk with the use of nanomedicine. The protection and maintenance of health information of the patients is the ethical issue and ensuring privacy & confidentiality is of utmost importance while using nanomedicine. The global nanomedicine market reached $63.8 billion in 2010 and $72.8 billion in 2011. The market is expected to grow to $130.9 billion by 2016 at a compound annual growth rate (CAGR) of 12.5% between years 2011 and 2016.The central nervous system (CNS) products market reached $11.7 billion in 2010 and $14.0 billion in 2011. It is expected to grow to $29.5 billion by 2016, a CAGR of 16.1% between years 2011 and 2016.The anticancer products market reached $25.2 billion in 2010 and $28.0 billion in 2011. It is expected to reach $46.7 billion by 2016, a CAGR of 10.8% between years 2011 and 2016. NANOTEHNOLOGY BASED DRUG DELIVERY SYSTEM Nanomedicines: Nanomedicines are pharmaceuticals prepared by manipulating matter at the nanoscale (< 1000 nm); i.e. manipulations at less than 1000th of a millimetre, and for drug delivery applications this typically takes the form of creating nanoparticles (5 ~ 800 nm) which are then used to package drug molecules and genes. Alternatively some nanomedicines are created from the drug or more typically the pro-drug itself. However the vast majority of nanomedicines are the result of the packaging of pharmacologically active compounds within nanoparticles. Nanomedicines are attractive in pharmacy and medicine simply because it is possible to control drug biodistribution and achieve therapeutic benefit with these nanomedicines. The chemical compounds used to construct these nanomedicines are as follows: low molecular weight self assembling amphiphiles, self assembling amphiphilic polymers, polymer drug conjugates, water insoluble
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polymers/ cross-linked polymers, dendrimers, inorganic chemistries and carbonnanotubes. Sophisticated engineering of these particles has produced nanomedicines which target drugs to tumours and the brain. Targeting is achieved by passive means in which the nanomedicine simply exploits normal endogenous transport mechanisms or alternatively is achieved by using targeting ligands to direct therapeutics to areas of pathology. To date the former strategy has proven to be more effective than the latter. Nanomedicines are also capable of promoting oral drug absorption and drug transport across other biological barriers such as the cornea and skin. Only a few of these technologies are commercially available at present, such as:liposomes, low molecular weight micelles and polymer drug conjugates, but the therapeutic benefits being observed in both preclinical studies and early clinical testing suggest that more of these technologies will emerge into the patient arena in the future. Nanomedicine, by definition, is the use of nanotechnology for medical purposes. Nanotechnology, or nanotech for short, is the study of the control of matter on an atomic scale (Nanotechnology). The field of nanotechnology evolved through molecular engineering. Nanotechnology focuses on the design and creation of devices within the atomic scale, usually no larger than 100 nanometers, which can perform a wide variety of tasks (Nanotechnology). In 2000, the United States National Nanotechnology Initiative was founded to coordinate Federal nanotechnology research and development. Since then, the field of molecular engineering has developed vastly. Scientists and researchers have been able to take the field in a direction that allows for the use of this technology in the human body. This was the birth ofNanomedicine. Nanomedicines are far more beneficial than modern medicine is today and will revolutionize the field of health care in the years to come. Modern medicine today does not help the human body heal faster; it merely allows the patient a more comfortable experience during their time of healing (Cell Repair Machines). With nanomedicines, we can accomplish a wide variety of tasks (including healing faster) that, with medicine today, would be impossible APPLICATIONS IN PHARMACY Nanomedicines used as targeted drug delivery system: The most advanced nanomedicines are multifunctional nanomedicines, capable of simultaneously diagnosing and targeting drug to specific molecular targets by incorporating active molecules, targeting ligands and imaging agents. The National Nanotechnology Initiative defines nanotechnology as the 'understanding and control of matter at dimensions of roughly 1-100 nm, where unique phenomena enable novel applications', enabling fabrication of devices on the nanoscale. By contrast, Albert Franks defined nanotechnology as 'that area of science and technology where dimensions and tolerances are in the range of 0.1 to 100 nm'. Although primary nanotechnology defines the size of the nanoparticles in nanomedicines between 1 and 100 nm in diameter, the size of the individual particles tested for drug delivery of therapeutic agents may range from 10 to 1000 nm. Recently, attempts have been made to introduce a comprehensive definition for nanoparticles in nanomedicines. For pharmaceutical purposes: 'Nanoparticles are solid colloidal particles ranging in size from 10 to 1000 nm (1 m), they consist of macromolecular materials in which the active principle (drug or biologically active material) is dissolved, entrapped, encapsulated and/or to which the active principle is adsorbed or attached'. Particles larger than 200 nm can activate the human complement system and can be cleared from the blood by Kupffer cells. Additionally, splenic filtration captures particles that exceed slit size (200-250 nm) and liver filtration captures particles greater than 150 nm. Also, tumor capillaries rarely exceed 300 nm in diameter. For these reasons, current research focuses on nanoparticle size of less than 200 nm. Even though, in some cases, nanomedicine containing particles are of less than 100 nm, some, such as magnetic nanoparticles, may accumulate in substantial amount in the reticuloendothelial system (RES) system. The outer surface of most recently developed nanomedicines for targeted drug delivery are modified by attachment of various ligands onto the surface (i.e., specific antigens, antibodies and receptor targeting ligands) to facilitate targeted delivery to specific targets. Although development of efficient nanomedicines extends into all therapeutic classes of pharmaceuticals, the development of effective treatment modalities for cancer, AIDS and brain disorders remains a therapeutically significant need. The information regarding the use of targeted nanomedicines for effective treatment of these diseases has not been well classified and documented. However, there are many reviews on specific nanomedicines. Role of nanomedicines in cancer: Currently in cancer therapy there is the lack of selectivity of anticancer drugs towards neoplastic cells. Generally rapidly proliferating cells such as those of bone marrow or the gastrointestinal tract are affected by the cytotoxic action of these drugs. This results in the narrow therapeutic index of most anticancer compounds. Also the emergence of resistant cell sublines during the chemotherapeutic treatment may require the use of higher doses of these drugs. Thismay enhance the toxicity of the treatment. In order to decrease
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the toxicity of the drugs and enhance the selectivity many drug delivery systems have been developed This includes nanoparticles that have received a greater interest for drug targeting, as they can be easily prepared with well defined biodegradable polymers. The reason of targeting tumors with nanoparticles is because certain neoplstic cells show enhanced endocytotic activity. In addition since some particular tumors are blood supplied by capillaries having an increased vascular permeability, it can be anticipated that nanoparticles will gain access to extravascular tumor cells. The ultimate goal of nanomedicines is to create medically useful nanodevices that can function inside the body. Among the newly developed nanomedicine and nanodevices quantum dots, nanowires, nanotubes, nanocantilevers, nanoshells are the most promising applications for various cancer treatments. They would serve the two important purposes of localized drug delivery and specific targeting. Nanoscale devices have the potential to radically change cancer therapy by increasing the number of highly effective therapeutic agents. Nanoparticles can serve as customizable, targeted drug delivery vehicles capable of ferrying chemotherapeutic agents or therapeutic genes into malignant cells while sparing healthy cells. This may allow for small doses of toxic substances as the drugs are delivered directly into the target tissue. Targeting: Currently, cancer - fighting drugs are toxic to both tumor and normal cells, thus the efficacy of chemotherapy is always limited by the side effects of the drug. Some nanoscale devices can be targets to the cancer cells. This increases the selectivity of the drugs toward the cancer cells and will reduce the toxicity for normal tissue. Attaching monoclonal antibodies that bind specifically to the cancer cells does this. Surface modification of nanoparticles can also enhance the permeability of the drugs to create high permeability nanoparticle based cancer therapeutics. Barriers to the cancer drugs can be in the form of the cells plasma membrane or endothelial layers of the cell. Research on the covalent attachment of peptide- membrane translocation sequences, peptides with the ability to pass through the membrane, to nanoparticles has shown increased permeability through membranes. With improved cell permeability, nanoparticles can become more therapeutically effective drug transport vehicles. The activity of nanomedicines atvarious target sites is discussed in the further points Targeting macrophage with nanoparticles: macrophage isa specialized host defense cell wherein any alterations in its clearance may contribute to disorders like atherosclerosis, autoimmunity, andmajor infections. Passive targeting of nanoparticulate vehicles with encapsulated antimicrobial agents to infected macrophages is hence a logical strategy for effective microbial killing. Endothelium as the target: endothelium plays an importantrole in various pathological processes including cancer, inflammation, oxidative stress and thrombosis. Recent studies have shown that cationicliposomes are eternalized into endosomes and lysosomes of endothelialcells in a characteristic organ and vessel specific manner Extravasation: targeting of solid tumors: there are regulatory approved formulations of long circulating liposomes withentrapped doxorubicin for management of AIDS related Kaposis sarcoma,refractory ovarian cancer and metastatic breast cancer. Nanoparticles: Nanoparticles can be in the form of nanospheres (matrix systemsin which drugs are dispersed throughout the particle) and nanocapsulesdrug is confined in an aqueous or oily cavity surrounded by asingle polymeric membrane. They have the potential to overcome biological biophysical and biomedical barriers currently facedby conventional administration of the cancer drugs. They can selectivelytarget tumors protecting the drug from inactivation during transport. Experiments have shown that nanospheres loaded withanticancer drugs successfully increase drug concentration in cancertissues. Some nanospheres are made of poly (isohexylcyanoacrylate),poly (methylcyanoacrylate) and biodegradable poly (ethylcyanoacrylate).Nanoparticles can also be used as tumor biomarkers. They help thedetection process by concentrating and protecting a marker from degradationso that the analysis is more sensitive. Streptavidin coated fluorescentpolystyrene nanospheres used in flow cytometry to detect biological molecules have shown greater sensitivity as compared to conventional dyes. Nanoshells: Nanoshells have a core of silica coated with an ultra thin metallic layer, normally gold37. By adjusting the core and shell thickness, nanoshells can be designed to absorb and scatter lightat a desired wavelength. The metal shell converts the absorbed light into with greater efficacy and stability. In addition, biomaterial nanoshells arecomposed of elements that is biocompatible. Due to their small size, nanoshells are preferentially concentrated in cancer cells by enhanced permeation retention. By supplying a NIR from a laser, the particle heats up and kill the tissue. Research has shown that the temperature within the nanoshell treated tumors rose by about40 degrees Celsius compared to a rise in 10C in tissues that was treated with NIR light alone. Thus by usinga NIR laser, cancer tissue can be destroyed by local thermal heating around the nanoshells.Nanoshell assisted photo thermal therapy is a simple non invasive procedure for selective photo thermal tumor removal.
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Fighting cancer: Nanotechnology can be used in many different ways to aid the field of oncology. First, we can use quantum dots, combined with an MRI, to compose detailed images of cancer sites. Unlike expensive organic dyes, quantum dots are capable of emitting bright light once excited in order to fully map the location of a cancer site. Plus it is cheaper than the dyes. Or, we can use Kanzius RF therapy. It uses nanoparticles that attach themselves to the cancerous cells. The particles then emit radio waves in order to burn away the cancerous cells. This method has been used on mice with great results. Also, it does not harm the surrounding tissue. Finally, we can use the nanoparticles to delivery cancer fighting drugs to the site of the tumor. Surgery: At Rice University, a flesh welder is used to fuse two pieces of chicken meat into a single piece (Nanomedicine). With the use of coated nanoshells, researchers have been able to fuse together tissue. This procedure can be useful during surgery in which immediate stitching is required in order to keep blood loss minimal. Since infrared lasers are used to heat the coated nanoshells that cause the fusion of the tissues, welding of the flesh can occur without the fear of burning the patient's organs. Cell repair machines: Foreseeable developments include the use of synthetic growth factors and morphogens for inducing complex tissue regeneration, and even the introduction of novel genetic programs for reversing cellular and tissue injuries for which natural healing mechanisms do not exist (Cell Repair Machines). This new "ultimate medicine" will allow us to: modify cell DNA in sophisticated ways to achieve virtually any desired growth objectives, repair cellular injuries caused by the hours of absent blood flow, increase our physical strength hundreds of times, and theoretically expanding our lifespan to up to 600 years.minimal. Since infrared lasers are used to heat the coated nanoshells that cause the fusion, welding of the flesh can occur without the fear of burning the patients organs. Liposomal Nanomedicines: Liposomal nanoparticles (LNs) encapsulating therapeutic agents, or liposomal nanomedicines (LNMs), represent one of the most advanced classes of drug delivery systems, with several currently on the market and many more in clinical trials. During the past 20 years, a variety of techniques have been developed for encapsulating both conventional drugs and the new genetic drugs (plasmid DNA containing therapeutic genes, anti-sense oligonucleotides, and small, interfering RNA [siRNA]) within LNs encompassing a very specific set of properties: a diameter centered on 100 nm, a high drug-to-lipid ratio, excellent retention of the encapsulated drug, and a long (> 6 hours) circulation lifetime. Particles with these properties tend to accumulate at sites of disease, such as tumors, where the endothelial layer is leaky and allows extravasation of particles with small diameters. Thus, LNs protect the drug during circulation, prevent it from reaching healthy tissues, and permit its accumulation at sites of disease. We will discuss recent advances in this field involving conventional anticancer drugs as well as gene-delivery, immunostimulatory, and gene-silencing applications involving the new genetic drugs. LNMs have the potential to offer new treatments in such areas as cancer therapy, vaccine development, and cholesterol management.Novel technology in the nanomedicine field is expected to develop innovative products as targeted drug-delivery approaches. Targeted drug delivery of various drugs for the treatment of cancer, AIDS and brain disorders is the primary research area in which nanomedicines has a major role and need. This review is concerned with emerging targeted nanomedicines (polymeric nanoparticles, solid lipid nanoparticles, polymeric micelles, dendrimers, liposomes, gold nanoparticles and magnetic nanoparticles) and multifunctional carriers capable of combining targeted drug delivery and imaging (polymeric micelles, dendrimers and magnetic nanoparticles) in the field of pharmaceutical applications. The significant toxicity issues associated with these nanomedicines are also explored here. CONCLUSION Nanotechnology in medicine involves applications of nanoparticles currently under development, as well as in longer range research that involves the use of manufactured nano-robots to make repairs at the cellular level. One application of nanotechnology in medicine currently being developed involves employing nanoparticles to deliver drugs, heat, light or other substances to specific types of cells (such as cancer cells). Particles are engineered so that they are attracted to diseased cells which allow direct treatment of those cells. This technique reduces damage to healthy cells in the body and allows for earlier detection of disease. REFERENCES Cherukuri P, Bachilo SM, Litovsky SH, Near-infrared fluorercence microscopy of single-walled carbon nanotubes in phagocytic cells, Am J Chem Soc, 126 (48), 2004, 15638-9.
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Cyrus T, Winter PM, Caruthers SD, Magnetic resonance nanoparticles for cardiovascular molecular imaging and therapy, Expert Rev Cardiovasc Ther, 3(4), 2005, 705-15. Dahan M, Levi S, Luccardini C et al. Diffusion dynamics of glycine receptors revealed by single-quantum dot tracking. Science, 302, 2003, 442-5. Dugan LL, Lovett EG, Quick KL, Fullerence-based antioxidants and neurodegenerative disorders. Parkinsonism & Related Disorders, 7, 2001, 243-6. Gelperina SE, Khalansky AS, Skidan IN, Toxicological studies of doxorubicin bound to polysorbate 80-coated poly (butyl cyanoacrylate) nanoparticles in healthy rats and rats with intracranial glioblastoma. Toxicol Lett, 126, 2002, 131-41. Gelperina SE, Khalansky AS, Skidan IN, Toxicological studies of doxorubicin bound to polysorbate 80-coated poly (butyl cyanoacrylate) nanoparticles in healthy rats and rats with intracranial glioblastoma. Toxicol Lett, 126, 2002, 131-41. Hseih PC, MacGillivray C, Gannon J et al. Local controlled intramyocardial delivery of platelet-derived growth factor improves postinfarction ventricular function without pulmonary toxicity. Circulation, 114, 2006, 637-644. Huang X, El-Sayed IH, Qian W, Cancer cell imaging and photothermal therapy in the near-infrared region by using gold nanorods, Am J Chem Soc, 128(6), 2006, 2115-20. Liu P, Konstam MA, Force T, Highlights of the 2004 scientific sessions of the Heart Failure Society of America, Toronto, Canada, Spetember 12 to 15, 2004, Am J Coll Cardiol, 45(4), 2005, 617-25. Moxon KA, Kalkhoran NM, Markert M, Nanostructured surface modification of ceramic-based microelectrodes to enhance biocompatibility for a direct brain-machine interface, IEEE Trans Biomed Eng, 51, 2004, 881-9. Panchapakesan B, Lu S, Sivakumar K, Single-wall carbon nanotube nanobomb agents for killing breast cancer cells, Nanobiotechnology, 2005, 133-9. Roby A, Erdogan S, Torchilin VP, Solubilization of poorly soluble PDT agent, mesotetraphenylporphin, in plain or immunotargeted PEG-PE micells results in dramatically improved cancer cell killing in vitro. Eur J Pharm Biopharm, 62(3), 2006, 235-40. Sahoo SK, Labhassetwar V, Nanotech approaches to drug delivery and imaging, Drug Discov Today, 8, 2003, 1112-20. Service RF, Nanotechnology takes aim at cancer, Science 2005, 310:1134. Silva GA, Czeisler C, Niece KL, Selective differentiation of neural progenitor cells by high-epitope density nanofibers, Science, 303, 2004, 1352-5. Vu TQ, Chowdhury S, Muni NJ et al. Activation of membrane receptors by a neurotransmitter conjugate designed for surface attachment. Biomatrials, 26, 2005, 1895-903.
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A VALIDATED RP-HPLC METHOD FOR THE ESTIMATION OF BACLOFEN IN BULK DRUG AND PHARMACEUTICAL FORMULATIONS
Rajesh M*, Manzoor Ahmed, Maanasa Rajan BN Department of Pharmaceutical Analysis, National College of Pharmacy, Shimoga-577201, (K.S.), India. *Corresponding author: Rajesh.sdnr@gmail.com ABSTRACT A new, simple, specific, sensitive, rapid, accurate and precise RP-HPLC method was developed and validated for the estimation of Baclofen in bulk drug and pharmaceutical formulations. Baclofen was chromatographed on a reverse phase C18 column (150x4.6mm I.D., particle size 5 m) in a mobile phase consisting of ammonium acetate buffer (pH 5.0 adjusted with orthophosphoric acid) and methanol in the ratio 40:60 v/v. The mobile phase was pumped at a flow rate of 1.0 mL/min with detection at 220 nm. The detector response was linear in the concentration of 10-50g/mL. The limit of detection and limit of quantitation was found to be 1.025 and 3.107g/mL, respectively. The intra day and inter day variation was found to be less than 1%. The mean recovery of the drug from the solution was 100.1%. The proposed method is simple, fast, accurate, precise and reproducible hence, it can be applied for routine quality control analysis of Baclofen in bulk drug and pharmaceutical formulations. Keywords: RP-HPLC, Baclofen, Estimation, Tablets. INTRODUCTION Baclofen is an orally administered synthetic antispastic agent or muscle relaxant. It reduces spasticity in many neurological disorders like multiple sclerosis, amyotrophic lateral sclerosis, spinal injuries and flexor spasms but is relatively ineffective in stroke, cerebral palsy, rheumatic and traumatic muscle spasms and parkinsonism (Tripathi, 1991). It may act as an agonist at GABA-B receptors (Ghosh, 1991). Baclofen is chemically -(amino methyl)-4-chlorobenzene propanoic acid (fig.1) and it is used as antispastic agent or muscle relaxant. The molecular formula of Baclofen is C 10H12ClNO2(Indian Pharmacopoeia, 2007), the molecular mass of Baclofen is 213.67g/mol. It is freely soluble in water, 0.1N HCl and 0.1N NaOH, slightly soluble in methanol, very slightly soluble in ethanol. It is official drug in I.P, B.P and U.S.P (Indian Pharmacopoeia, 2007) (British Pharmacopoea, 2005) (US Pharmacopoeia, 2004). Literature survey reveals that, only bioanalytical methods by HPLC and few Spectrophotometric methods were found using human plasma and urine for the quantitative estimation of Baclofen in bulk drug and pharmaceutical formulations (Laurette, 1996) (Wuis, 1985) (Mohammed, 2003) (Zhu, 2003) (Pesez, 1974).
NH2CH2 - CH-CH2-COOH
Cl
Figure.1. Chemical structure of Baclofen MATERIALS AND METHODS Quantitative HPLC was performed on a isocratic high pressure liquid chromatography (shimadzu HPLC class VP-Series) with one LC-10 AT VP pump, UV/VIS detector SPD-10A VP, CTO-10 AS VP column oven (shimadzu), SCL-10A VP system controller (shimadzu), a disposable guard column LC-18 (PELLIGUARD)TM, LC-18, 2 cm, supelco, inc., Bellefonte, and a Reverse Phase C-18 Column (150mm x 4.6 mm i.d.particle size 5 m) was used . The HPLC system was equipped with the software class, N-2000 CHROMTECK (Shimadzu). Reagents and chemicals: Ammonium acetate and orthophosphoric acid of AR grade were obtained from Qualigens Fine Chemicals Ltd., Mumbai. Methanol of HPLC grade was purchased from E.Merck (India) Ltd., Mumbai. Baclofen was a gift sample by Unicare Pvt. Ltd., Gujarat. The commercially available Baclofen tablets were procured from the local market. Preparation of buffer: (0.005 M) Ammonium acetate buffer was prepared by dissolving 0.3854 g of Ammonium acetate in 1000 mL of milli-Q water and the pH was adjusted to 5.0 with orthophosphoric acid. Chromatographic conditions: The mobile phase consisting of ammonium acetate buffer (pH 5.0 adjusted with orthophosphoric acid) and methanol was in the ratio of 40:60 v/v filtered through 0.45 membrane filter before use, degassed and pumped from the solvent reservoir into the column at a flow rate of 1.0 mL/min. The detection Volume 1(2) March-April 2013 Page 215
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was monitored at 220nm and the run time was 10 minutes. The volume of injection loop was 20 L prior to the injection of the drug solution, the column was equilibrated for at least 30 minutes with the mobile phase flowing through the system. The column and the HPLC system were kept in ambient temperature. Procedure: Stock solution of Baclofen was prepared by dissolving 10 mg of Baclofen in 10 mL standard volumetric flask containing 2.5 mL of mobile phase and the solution was sonicated for 20 min. and then made up to the mark with mobile phase to get a concentration of 1000 g/mL. Subsequent dilutions of this solution were made with mobile phase to get concentration of 10-50 g/mL. The standard solutions prepared as above were injected into the 20 L loop and the chromatogram was recorded and shown in Figure 2. The retention time of Baclofen was found to be 5.345 min. The calibration curve was constructed by plotting concentration versus peak area ratio. The amount of Baclofen present in sample was calculated through the standard calibration curve. The linearity experiment was carried out in triplicate to ascertain accuracy and precision of the method. The peak area ratios of the drug versus concentration were found to be linear and the results are furnished in Table 1. Assay: Twenty tablets each containing 10 mg were weighed accurately and powdered. A quantity equivalent to100 mg of Baclofen was weighed accurately and transferred to 100 mL volumetric flask containing 30 mL of mobile phase. The contents were sonicated for 20 min. and made up to the mark with the mobile phase. The resulting solution is filtered through a membrane filter. The solution obtained was diluted with the mobile phase so as to obtain a concentration in the range of linearity previously for the pure drug determined. Sample solution was injected under the chromatographic conditions and the chromatogram was recorded. The amount of Baclofen present in tablet formulation was determined by comparing the peak area from the standard. The results were furnished in Table 2. Validation of proposed method: Selectivity of the method was assessed on the basis of elution of Baclofen using the above mentioned chromatographic conditions. To study the linearity, limit of detection, limit of quantitation and correlation co-efficient, retention time, theoretical plates, tailing factor has been validated for the determination of Baclofen. The results are furnished in Table 3. Linearity: The standard curve was obtained in the concentration range of 10-50 g/mL. The linearity was evaluated by linear regression analysis using the least square method. It was found that correlation coefficient and regression analysis are within the limits. Precision: The precision of the assay was determined in terms of intra-day and inter-day precision. The intra-day and inter-day variation in the peak area of drug solution was calculated in terms of coefficient of variation (C.V.) obtained by multiplying the ratio of standard deviation to mean with 100. The results are furnished in Table 4. Limit of detection (LOD) and limit of quantitation (LOQ): The LOD and LOQ for Baclofen were predicted basing on the parameters of standard error of estimate and slope, calculated from linearity of the response data of Baclofen. Robustness: The robustness was checked by changing the flow rate to 0.8 and 1.2 mL/min and the wavelength 218 and 222nm the method suits best. Accuracy: The accuracy of the HPLC method was assessed by adding known amount of drug solution to a drug solution of known concentration and subjecting the samples to the proposed HPLC method. The recovery studies were replicated 3 times. The accuracy was expressed in terms of recovery and calculated by multiplying the ratio of measured drug concentration to the expected drug concentration with 30 g/mL so as to give the percentage recovery. The results are furnished in Table 5. RESULTS AND DISCUSSION By applying the proposed method, the run time of the method was set at 10 min and Baclofen appeared on the typical chromatogram at 5.345 min, which indicates a good base line. When the same drug solution was injected 3 times, the retention time of the drug was same. Linearity range was observed in the concentration range of 10-50 g/mL. The regression equation of Baclofen concentration over its peak area ratio was found to be Y=33706.00+94203.00x (r = 0.9993) where Y is the peak area ratio and X is the concentration of Baclofen (g/mL). The proposed HPLC method was also validated for intra-day and inter-day variation. The coefficient of variation in the peak area of the drug for 3 replicate injections was found to be less than 1%. The tailing factor was found to be 1.285, which indicates good shape of peak. The numbers of theoretical plates were found to be 6889.163, which indicate efficient performance of the column. The limit of detection and limit of quantitation was found to be 1.025 g/mL and 3.107 g/mL, indicates the sensitivity of the method. To optimize the chromatographic conditions, various combinations of ammonium acetate buffer and methanol were tested. The Volume 1(2) March-April 2013 Page 216
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use of ammonium acetate buffer and methanol in the ratio of 40:60 v/v resulted in peak with good shape and resolution. The high percentage of recovery of Baclofen ranging from 99.90 to 100.40 indicates that the proposed method is highly accurate. No interfering peaks were found in the chromatogram indicating that excipients used in tablet formulation did not interfere with the estimation of the drug by proposed HPLC method.
Figure.2. Typical chromatogram of Baclofen Table.1. Calibration data of the method Concentration,g/mL 10 20 30 40 50 Peak area (n=5) 244882.703 593024 894065.813 1252336.75 1600501.75
Formulation Brand-1 Brand-2
Table 2. Assay of Baclofen Label claim(mg) Amount found(mg) 10 10.03 10 10.01
% Amount found 100.3 100.1
Table.3. System suitability parameters Parameter Result Linearity, g/ml 10-50, g/ml Correlation coefficient 0.9993 Retention time, min 5.345 Theoretical plates (N) 6889.163 Tailing factor 1.285 LOD, g/ml 1.025 LOQ, g/ml 3.107 Table.4. Precision of the proposed HPLC method Concentration of Baclofen Intra-day Precision Inter-day Precision g/ml Mean area %C.V Mean area %C.V. (n=3) (n=3) 80 708423.8 0.98 706383.9 0.92 100 896317.6 0.32 897090.7 0.16 Volume 1(2) March-April 2013 Page 217
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120
1072514.0
0.75
1063113.0
0.71
Table.5. Recovery studies of the proposed HPLC method. Amount of Amount of Concentration Baclofen Baclofen % Recovery Added, mg Found, mg 80%,24g/ml 100%,30g/ml 120%,36g/ml 81.6 102 122.4 81.9 102.1 122.2 100.4% 100.1% 99.9%
Mean Recovery
100.1%
CONCLUSION The proposed HPLC method was found to be simple, rapid, sensitive, precise and accurate for the estimation of Baclofen in pharmaceutical formulations. Hence, this method can easily and conveniently adopt for routine quality control analysis of Baclofen in bulk drug and its pharmaceutical formulations. ACKNOWLEDGEMENT The authors are highly thankful to the National education society and Principal National college of Pharmacy, Shimoga for proving all the facilities to carry out the research work. REFERENCES British Pharmacopoeia, 2005, 1, 202. Ghosh.R, Text book of Modern Concepts on Pharmacology and Therapeutics, Hilton and co, Calcutta, 1991, 24, 206. Indian Pharmacopoeia, 2007, 1, 2, 144, 768-770. Laurette M, Murielle B, Virginie G, High-Performance Liquid Chromatographic determination of baclofen in human plasma, J of Chrom A, 1996, 729(1-2), 309-314. Mohamed MH and Hassan YA, Enantioselective high- Performance liquid chromatographic method for the determination of baclofen in Human plasma, Talanta, 2003, 61(5), 667-673. Pesez M, Bartos J, Colorimetric and Fluorimetric analysis of Organic Compounds and Drugs, Marcel Dekker, INC, Newyork, 1974, 129, 131, 137-139, 200-201. Tripathi K D, Essentials of Medical Pharmacology, Jaypee brothers medical publishers, 2003, 5, 316-318. US Pharmacopoeia NF, 2004, 23, 207. Wuis E.W, Dirks R.J, Vree T.B, et al; High-Performance Liquid Chromatographic Analysis of baclofen in plasma and urine of man after precolumn Extraction and derivatization with O- phthaldialdehyde, J. of Chrom B, 1985, 337, 341-350. Zhu Z and Neirinck, Chiral separation and determination of R-(-)-and S-(+) - Baclofen in human plasma by highperformance liquid chromatography, J of Chrom B, 2003 March; 785(2), 277-283.
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A VALIDATED RP-HPLC METHOD FOR THE ESTIMATION OF CISAPRIDE IN BULK DRUG AND PHARMACEUTICAL FORMULATIONS
Maanasa Rajan.B.N.*, Manzoor Ahmd, Rajesh M Department of Pharmaceutical Analysis, National College of Pharmacy, Shimoga-577201, (K.S.), India. *Corresponding author:Lakshmi4natarajan@gmail.com ABSTRACT A new, simple, specific, sensitive, rapid, accurate and precise RP-HPLC method was developed and validated for the estimation of Cisapride in bulk drug and pharmaceutical formulations. Cisapride was chromatographed on a reverse phase C18 column (150x4.6mm I.D., particle size 5 m) in a mobile phase consisting of methanol (pH 3.0 adjusted with orthophosphoric acid) and 1-octane sulphonic acid in the ratio 90:10 v/v. The mobile phase was pumped at a flow rate of 1.5 mL/min with detection at 234nm. The detector response was linear in the concentration of 2-10 g/mL. The limit of detection and limit of quantitation was found to be 0.06 and 0.20 g/mL, respectively. The intra day and inter day variation was found to be less than 1%. The mean recovery of the drug from the solution was 100.1%. The proposed method is simple, fast, accurate, precise and reproducible hence, it can be applied for routine quality control analysis of Cisapride in bulk drug and pharmaceutical formulations. Keywords: RP-HPLC, Cisapride, Estimation, Tablets. INTRODUCTION Cisapride is a gastro prokinetic (Bertram, 2004) drug which act as an agonist at the pre-synaptic 5HT4 (Bertram, 2004) (Seth, 1998) receptor & act as antagonist at 5HT3 (Bertram, 2004) (Seth, 1998) receptors. The stimulation mediates release of acetyl-choline & its prokinetic action is blocked by atropine (Bertram, 2004). Cisapride is chemically cis-4-amino-5-chloro-N-[1-(3-(p-florophenyl)propyl]-3-methoxy-4piperidinyl]-oanisamide (O Neil, 2001)(Fig.1). Its molecular formula is C23H29ClFN3O4 and its molecular weight is 465.95.it is freely soluble in acetone, methanol and insoluble in water. Its melting point is 109.8 0C (ONeil, 2001) (Sweetman, 2002). It is official in E.P & B.P. Cisapride is officially not available in U.S. as it is withdrawn on January, 2000, (ONeil, 2001) (British Pharmacopoeia, 2005) (Sweetman, 2002) due to its side effects in the patients suffering from the cardiac arrthymias and hepato disorders, but it is official drug in Europe and U.K. In 1998 U.K. committee of safety medicine (ONeil, 2001) (British Pharmacopoeia, 2005) (Sweetman, 2002) as contraindicated to the patients with arrthymias. European society has recommended using cisapride for gastro-oesophageal reflux disease. Regulatory authorities of India have not been announced to with draw because of its therapeutic use. Its very efficient the patients who are not responding to the metoclopramide. Cisapride does not have any dopaminergic ((Bertram, 2004) effect. Literature survey reveals that, only bioanalytical methods by HPLC and few Spectrophotometric methods were found using human plasma and urine for the quantitative estimation of Cisapride in bulk drug and pharmaceutical formulations (Schirmer, 1991) (Sastry, 1997) (Abdine, 2000) (Hassan, 2001) (Revana Siddappa, 2002).
OMe NH O F N OMe NH2 Cl
Figure.1. Chemical structure of Cisapride MATERIALS AND METHODS Quantitative HPLC was performed on a isocratic high pressure liquid chromatography (shimadzu HPLC class VP-Series) with one LC-10 AT VP pump, UV/VIS detector SPD-10A VP, CTO-10 AS VP column oven (shimadzu), SCL-10A VP system controller (shimadzu), a disposable guard column LC-18 (PELLIGUARD)TM, LC-18, 2 cm, supelco, inc., Bellefonte, and a Reverse Phase C-18 Column (150mm x 4.6 mm i.d.,particle size 5 m) was used. The HPLC system was equipped with the software class, N-2000 CHROMTECK (Shimadzu). Volume 1(2) March-April 2013 Page 219
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Reagents and chemicals:1-octane sulphonic acid and orthophosphoric acid of AR grade were obtained from Qualigens Fine Chemicals Ltd., Mumbai. Methanol of HPLC grade was purchased from E.Merck (India) Ltd., Mumbai. Cisapride was a gift sample by Intas Labs Pvt. Ltd., Dehradun. The commercially available cisapride tablets were procured from the local market. Chromatographic conditions: The mobile phase consisting of 1-octane sulphonic acid(pH 3.0 adjusted with orthophosphoric acid) and methanol was in the ratio of 10:90 v/v filtered through 0.45 membrane filter before use, degassed and pumped from the solvent reservoir into the column at a flow rate of 1.5mL/min. The detection was monitored at 234nm and the run time was 10 minutes. The volume of injection loop was 20 L prior to the injection of the drug solution, the column was equilibrated for at least 30 minutes with the mobile phase flowing through the system. The column and the HPLC system were kept in ambient temperature. Procedure: Stock solution of Cisapride was prepared by dissolving 10 mg of Cisapride in 10 mL standard volumetric flask containing 2.5 mL of mobile phase and the solution was sonicated for 20 min. and then made up to the mark with mobile phase to get a concentration of 1000 g/mL. Subsequent dilutions of this solution were made with mobile phase to get concentration of 2-10 g/mL. The standard solutions prepared as above were injected into the20 L loop and the chromatogram was recorded and shown in Figure 2. The retention time of Cisapride was found to be 5.630 min. The calibration curve was constructed by plotting concentration versus peak area ratio. The amount of Cisapride present in sample was calculated through the standard calibration curve. The linearity experiment was carried out in triplicate to ascertain accuracy and precision of the method. The peak area ratios of the drug versus concentration were found to be linear and the results are furnished in Table 1. Assay: Twenty tablets each containing 10 mg were weighed accurately and powdered. A quantity equivalent to100 mg of Cisapride was weighed accurately and transferred to 100 mL volumetric flask containing 30 mL of mobile phase. The contents were sonicated for 20 min. and made up to the mark with the mobile phase. The resulting solution is filtered through a membrane filter. The solution obtained was diluted with the mobile phase so as to obtain a concentration in the range of linearity previously for the pure drug determined. Sample solution was injected under the chromatographic conditions and the chromatogram was recorded. The amount of Baclofen present in tablet formulation was determined by comparing the peak area from the standard. The results were furnished in Table 2. Validation of proposed method: Selectivity of the method was assessed on the basis of elution of Cisapride using the above mentioned chromatographic conditions. To study the linearity, limit of detection and limit of quantitation has been validated for the determination of Cisapride. The results are furnished in Table 3. Linearity: The standard curve was obtained in the concentration range of 2-10 g/mL. The linearity was evaluated by linear regression analysis using the least square method. It was found that correlation coefficient and regression analysis are within the limits. Precision: The precision of the assay was determined in terms of intra-day and inter-day precision. The intra-day and inter-day variation in the peak area of drug solution was calculated in terms of coefficient of variation (C.V.) obtained by multiplying the ratio of standard deviation to mean with 100. The results are furnished in Table 4. Limit of detection (LOD) and limit of quantitation (LOQ): The LOD and LOQ for Cisapride were predicted basing on the parameters of standard error of estimate and slope, calculated from linearity of the response data of Cisapride. Robustness: The robustness was checked by changing the flow rate to 1.4 and 1.6 mL/min and the wavelength 232 and 236nm the method suits best. Accuracy: The accuracy of the HPLC method was assessed by adding known amount of drug solution to a drug solution of known concentration and subjecting the samples to the proposed HPLC method. The recovery studies were replicated 3 times. The accuracy was expressed in terms of recovery and calculated by multiplying the ratio of measured drug concentration to the expected drug concentration with 6 g/Ml so as to give the percentage recovery. The results are furnished in Table 5. RESULTS AND DISCUSSION By applying the proposed method, the run time of the method was set at 10 min and Cisapride appeared on the typical chromatogram at 5.630 min, which indicates a good base line. When the same drug solution was injected 3 times, the retention time of the drug was same. Linearity range was observed in the concentration range of 2-10 g/mL. The regression equation of Cisapride concentration over its peak area ratio was found to be Y=13750.00+668.33x (r = 0.9998) where Y is the peak area ratio and X is the concentration of Cisapride(g/mL). Volume 1(2) March-April 2013 Page 220
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The proposed HPLC method was also validated for intra-day and inter-day variation. The coefficient of variation in the peak area of the drug for 3 replicate injections was found to be less than 1%. The tailing factor was found to be 1.621, which indicates good shape of peak. The numbers of theoretical plates were found to be 9407.160, which indicate efficient performance of the column. The limit of detection and limit of quantitation was found to be 0.06 g/mL and 0.20 g/mL, indicates the sensitivity of the method. To optimize the chromatographic conditions, various combinations of 1-octane sulphonic acid and methanol were tested. The use of 1-octane sulphonic acid and methanol in the ratio of 10:90 v/v resulted in peak with good shape and resolution. The high percentage of recovery of Cisapride ranging from 100.10to 100.30 indicates that the proposed method is highly accurate. No interfering peaks were found in the chromatogram indicating that excipients used in tablet formulation did not interfere with the estimation of the drug by proposed HPLC method.
Figure.2.Typical chromatogram of Cisapride Table.1. Calibration data of the method Concentration g/ml Peak area (n=5) 2 28191.051 4 55339.25 6 83148.25 8 111605 10 137558.203 Table.2. Assay of Cisapride Label claim(mg) Amount found(mg) 10 10.04 10 10.02 Table.3. System suitability parameters Parameter Result Linearity, g/ml 2-10g/ml Correlation coefficient 0.9998 Retention time, min 5.630 Theoretical plates (N) 9407.160 Tailing factor 1.621 LOD, g/ml 0.06 LOQ, g/ml 0.20
Formulation Brand-1 Brand-2
% Amount found 100.4 100.2
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Concentration of Cisapride g/ml 80 100 120
Table.4. Precision of the proposed HPLC method Intra-day Precision Inter-day Precision Mean area %C.V. Mean area %C.V. (n=3) (n=3) 66472.02 0.71 66299.76 0.63 82894.16 0.39 82920.51 0.21 99529.82 0.69 98983.77 0.63 Table.5. Recovery studies of the proposed HPLC method
Concentration 80%,4.8g/ml 100%,6g/ml 120%,7.2g/ml
Amount of Cisapride Added, mg 113.6 142.0 170.4
Amount of Cisapride Found, mg 113.9 142.1 170.5
% Recovery 100.3% 100.1% 100.1%
Mean Recovery 100.1%
CONCLUSION The proposed HPLC method was found to be simple, rapid, sensitive, precise and accurate for the estimation of Cisapride in pharmaceutical formulations. Hence, this method can easily and conveniently adopt for routine quality control analysis of Cisapride in bulk drug and its pharmaceutical formulations. ACKNOWLEDGEMENT The authors are highly thankful to the National education society and principal National college of pharmacy, Shimoga for proving all the facilities to carry out the research work. REFERENCES Bertram.G.katzung, Basic and clinical pharmacology 9th Edition, 2004, 1045 Biologicals, 13thEdition, 2001, 2341 British pharmacopoeia, 2005(1), 488. Charles R Craig, Robert E Stitzel, Modern pharmacology 3rded, 1990, 974 E.M.Hassan, M.E.M.Haggo and H.I.Al Johar.Determination of cisapride in pharmaceutical Preparations using derivative spectrophotometry, Journal of pharmaceutical and biomedical Analysis, 2001, 24(4), 659-665. European pharmacopoeia, 2005, 1303. H.H.Abdine, Alexandrian Journal of pharmaceutical sciences, 2000, 14(1), 75-78. ONeil M J, The Merck Index, An encyclopedia of chemicals, Drugs and Biologicals, 13th Edition, 2001, 2341. Revana siddappa HD, B.Manju, Spectrophotometric determination of some chemotherapeutic agents using acetyl acetone, Drug development and industrial pharmacy, 2002, 28(5), 515-521 S D Seth, Text book of pharmacology, 2nded; 1998, 408 Sastry CSP, Srinivas.Y, Subbarao.P.V, Assay of Cisapride in pharmaceutical formulations by Extraction spectroscopy, Talanta , 1997, 44(4), 517-526. Schirmer RE, Modern Methods of Pharmaceutical analysis.2nd ed. Bastin : C.R.C. Press, 1991, 83. Sweetman SC, Martindale, The Complete Drug Reference, 33rded, Pharmaceutical press, London, 2002, 1220.
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Maryam Bincy et.al
REVIEW ARTICLE ON ANTIMICROBIAL RESISTANCE

Maryam Bincy Thomas*, Suruchi Singh Sunder Deep Pharmacy College, Ghaziabad, UP, India *Corresponding author Email: maryambincythomas@gmail.com ABSTRACT Antimicrobial resistance (AMR) is not a recent phenomenon, but it is a critical health issue today. Over several decades, to varying degrees, bacteria causing common infections have developed resistance to each new antibiotic, and AMR has evolved to become a worldwide health threat. With the dearth of new antibiotics coming into market, the need for action to avert a developing global crisis in health care is increasingly urgent. Much of the AMR problem stems from the misuse of antibiotic, particularly excessive use. The eradication of AMR is neither a realistic nor a desirable goal. The aim should therefore be to contain resistance, to optimize the balance between the effective use of antimicrobials against infections, thus reducing morbidity, mortality and further spread of infection. Effective antibiotic stewardship is required to ensure that antibiotics are prescribed and used responsibly. This also requires a multistakeholder approach including the governments, policy-makers and planners, pharmaceutical industry, World Health Organization, health care professionals, public and the patients. WHO is engaged in guiding the response to AMR through: policy guidance, support for surveillance, technical assistance, knowledge generation and partnerships, including through disease prevention and control programmes; essential medicine quality; supply and rational use; infection prevention and control; patient safety; and laboratory quality assurance. Keywords: Antimicrobial resistance, Antibiotics, WHO INTRODUCTION The discovery and development of antimicrobials, during the 20th century, revolutionized the treatment of infectious diseases. The ability to use antimicrobials against infections caused by microorganisms introduced the golden age of antimicrobials and led to the notion that premature death due to infectious diseases would become a thing of the past. However the introduction of antimicrobials into clinical practice has invariably led to the evolution of antimicrobial-resistant pathogens, thus rendering the drugs concerned ineffective for the treatment of the disease. Antimicrobial resistance (AMR) is the resistance of a microorganism to an antimicrobial medicine to which it was previously sensitive. Antimicrobial resistance is not a new phenomenon; however, the current magnitude and the speed with which the new resistance phenotypes have emerged elevate the public health significance of this issue. WHO is engaged in guiding the response to AMR through: policy guidance, support for surveillance, technical assistance, knowledge generation and partnerships, including through disease prevention and control programmes; essential medicine quality; supply and rational use; infection prevention and control; patient safety; and laboratory quality assurance. This also requires a multi-stakeholder approach including the governments, policy-makers and planners, pharmaceutical industry, World Health Organization, health care professionals, public and the patients. The obstacle for a few new antimicrobials on the horizon and the increasing frequency of AMR mean that we must redouble our efforts to preserve the agents at hand, while intensifying the search for new therapeutics. Its high time that we recognize Antibiotics true value: Precious Resource or A Giveaway Marketing Tool. CAUSES FOR ANTIBIOTIC RESISTANCE The main cause of antibiotic resistance is overuse, abuse or misuse, due to incorrect diagnosis. Another cause is the use of counterfeit drugs. Increased globalization also causes the spread of drug resistance. MECHANISMS OF ANTIBIOTIC RESISTANCE Resistance to antibiotic can be: Natural or intrinsic resistance caused by a spontaneous gene mutation in the lack of selective pressure due to presence of antibiotic. Acquired resistance is often caused by mutation in the chromosomal genes or by the acquisition of mobile genetic elements such as plasmids or transposons, which carry the antibiotic resistant gene. Genetic resistance is caused by the transfer of genetic material among bacteria by several means like conjugation, transduction and transformation. Phenotypic resistance is caused due to the changes in the bacterial physiological state such as the stationary phase, antibiotic persisters and the dormant phase. Volume 1(2) March-April 2013 Page 223
ISSN: 2320 3471(Online) Maryam Bincy et.al Indian Journal of Research in Pharmacy and Biotechnology Biological mechanism of resistance may be due to reduced permeability or uptake, enhanced efflux, enzymatic inactivation, alteration or over-expression of the drug target, loss of enzymes involved in the drug inactivation. METHODS FOR DETERMINING ANTIBOTIC RESISTANCE Methods routinely used for testing of antibiotic susceptibility of bacteria include Kirby-Bauer (disk diffusion) method, Stokes method, E-test, agar and broth dilution method for the determination of minimum inhibitory concentration (MIC). The E-test is a popular quantitative technique for the determination of antimicrobial susceptibility. MEASURES TO COMBAT ANTIBIOTIC RESISTANCE The eradication of AMR is neither a realistic nor a desirable goal. The aim should therefore be to contain resistance, to optimize the balance between the effective use of antimicrobials against infections, thus reducing morbidity, mortality and further spread of infection. Strategies for containing emergence of resistance: Education of professionals and patients Rapid diagnosis of bacterial infections Antimicrobial policies Restriction of drug availability Control of sensitivity data related to prescribers Regulation of use of antimicrobials in agriculture Use of drug combinations Choosing optimal agent, dose and dosage frequency for different infections. Using probiotics as alternatives to antimicrobials Increasing vaccinations Improving nutrition to increase immune competence Strategies for containing transmission of resistance: Rapid diagnostic techniques Screening of patients and staff Improving immunity by vaccinations Isolation Handwashing Improving nutrition On World Health Day 2011, WHO issued an international call for concerted action to halt the spread of antimicrobial resistance and recommended a six-point policy package for governments to: 1. Commit to a comprehensive, financed national plan with accountability and civil social engagement. 2. Strengthen surveillance and laboratory capacity. 3. Ensure uninterrupted access to essential medicines of assured quality. 4. Regulate and promote rational use of medicines in animal husbandry and to ensure proper patient care. 5. Enhance infection prevention and control. 6. Foster innovations and research and development of new tools. DRUG RESISTANCE WEBSITES: Alliance for the Prudent use of Antibiotics (APUA): http://www.apua.org Global Alliance of Antibiotic Resistance Data (GAARD): http://www.apua.org/Miscellaneous/GaardDesc.pdf Reservoirs of Antibiotic Resistance (ROAR): http://www.apua.org/ROAR/roarhome.htm World Health Organization, Antituberculosis drug resistance in the world: http://www.who.int/gtb/publications/drugresistance/2004/drs_report_1.pdf CONCLUSION It is profound that the antimicrobial resistance will continue to develop to the currently available antimicrobials by either new mutations or the exchange of genetic information. The obstacle for a few new antimicrobials on the horizon and the increasing frequency of AMR mean that we must redouble our efforts to preserve the agents at hand, while intensifying the search for new therapeutics. Its high time that we recognize Antibiotics true value: Precious Resource or A Giveaway Marketing Tool. Volume 1(2) March-April 2013 Page 224
Maryam Bincy et.al REFERENCES
Ying Zhang, Mechanisms of Antibiotic Resistance in the microbial world, Clin Pharmacol Ther, 2007, 82:595600. Alfonso J Alanis, Resistance to Antibiotics: Are we in the Post-Antibiotic Era? Archives of Medical Research 2005, 36, 697-705. Ashraf R, Shah NP, Antibiotic resistance of probiotic organisms and safety of probiotic dairy products, International Food Research Journal 2011, 18(3), 837-853. Richard D Smith, Joanna Coast, Antimicrobial Resistance- A Global Response, Bulletin of the World Health Organization, 2002, 80, 126-133. The Evolving Threat of Antimicrobial Resistance- Options for Action. World Health Organization 2012, (www.who.int). Stuart B Levy, Bonnie Marshall, Antibacterial resistance worldwide: causes, challenges and responses. Nature Medicine Supplement, 2004, 10(12), 122-129. Fred C Tenover, Mechanism of Antimicrobial Resistance in Bacteria, The American Journal of Medicine 2006, 119(6A), S3-S10.
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M.Komala and Sampath Kumar
URINARY TRACT INFECTION: CAUSES, SYMPTOMS, DIAGNOSIS AND ITS MANAGEMENT

M. Komala1*, K.P.Sampath Kumar2 1.Karpagam University, Coimbatore 2.Department of Pharmaceutics, Coimbatore medical college, Coimbatore-114, Tamil Nadu, India *Corresponding author: E.Mail:komala.pharmacy@gmail.com
ABSTRACT A urinary tract infection is an infection in any part of your urinary system - kidneys, ureters, bladder and urethra. Most infections involve the lower urinary tract the bladder and the urethra. Women are at greater risk of developing a UTI than men are. Infection limited to your bladder can be painful and annoying. However, serious consequences can occur if a UTI spreads to your kidneys. Antibiotics are the typical treatment for a UTI. But you can take steps to reduce your chance of getting a UTI in the first place. Antibacterial agents are a major part of the treatment for UTI. Due to the side effects associated with antibiotic and drug resistance a need for an alternative therapy is emphasised. A major error in management of UTI has been that most clinicians give too many drugs for too longer period. KEY WORDS: Urinary tract infection, Herbs, Antibiotic, Infection 1. INTRODUCTION Urinary tract infections are infections of the urethra, bladder, ureters, or the kidneys, which comprise the urinary tract.E. coli bacteria cause the majority of UTIs, but many other bacteria, fungi, and parasites may also cause UTIs.Females have a higher risk for UTIs than most males, probably because of their anatomy; other risk factors for UTIs include any condition that may impede urine flow (e.g., enlarged prostate, congenital urinary tract abnormalities, and inflammation). Patients with catheters or those who undergo urinary surgery and men with enlarged prostates are at higher risk for UTIs.Symptoms and signs of UTI vary somewhat depending on sex, age, and the area of the urinary tract that is infected; some unique symptoms develop depending on the infecting agent.UTIs are diagnosed usually by isolating and identifying the urinary pathogen from the patient; there are some home tests available for presumptive diagnosis.There are home remedies for UTI, but most may, at best, help reduce the risk or discomfort of UTIs. They are not considered cures for the disease. There can be many complications of urinary tract infections, including dehydration, sepsis, kidney failure, and death.If treated early and adequately, the prognosis is good for most patients with a UTI.Although there is no vaccine available for UTIs, there are many ways a person may reduce the chance of getting a UTI. Urinary Tract Infection (UTI): UTI is a bacterial infection affecting urinary tract. When bacteria from the rectal area enter the urinary tract via the urethra to the bladder and multiply in the urine, an infection occurs. In many cases bacteria first travel to the urethra. When bacteria multiply an infection can occur. An infection limited to the urethra is called urethritis. If bacteria move to the bladder and multiply, a bladder infection called cystitis. If the infection is not treated promptly, bacteria may then travel further up the ureters to multiply and infect the kidneys, called pyelonephritis. The urinary tract is comprised of the kidneys, ureters, bladder, and urethra. A urinary tract infection (UTI) is an infection caused by pathogenic organisms (for example, bacteria, fungi, or parasites) in any of the structures that comprise the urinary tract. However, this is the broad definition of urinary tract infections; many authors prefer to use more specific terms that localize the urinary tract infection to the major structural segment involved such as urethritis (urethral infection), cystitis (bladder infection), ureter infection, and pyelonephritis (kidney infection). Other structures that eventually connect to or share close anatomic proximity to the urinary tract (for example, prostate, epididymis, and vagina) are sometimes included in the discussion of UTIs because they may either cause or be caused by UTIs. Technically, they are not UTIs and will be only be briefly mentioned in this article.UTIs are common, leading to between seven and 10 million doctor visits per year. Although some infections go unnoticed, UTIs can cause problems that range from dysuria (pain and/or burning when urinating) to organ damage and even death. The kidneys are the active organs that produce about 1.5 quarts of urine per day. They help keep electrolytes and fluids (for example, potassium, sodium and water) in Volume 1(2) March-April 2013 Page 226
ISSN: 2320 3471(Online) M.Komala and Sampath Kumar Indian Journal of Research in Pharmacy and Biotechnology balance, assist in the removal of waste products (urea), and produce a hormone that aids in the formation of red blood cells. If kidneys are injured or destroyed by infection, these vital functions can be damaged or lost. There is general agreement that sexual intercourse can cause a UTI. This is mostly thought to be a mechanical process whereby bacteria are introduced into the urinary tracts during the sexual act. There is no dispute about the transmission of UTIs caused by sexually transmitted disease (STD) organisms; these infections (for example, gonorrhoea and Chlamydia) are easily transmitted between sex partners and are very contagious. Some of the symptoms of UTIs and sexually transmitted diseases can be similar (pain and foul smell). A urinary tract infection, or UTI, is an infection that can happen anywhere along the urinary tract. Urinary tract infections have different names, depending on what part of the urinary tract is infected. Bladder -- an infection in the bladder is also called cystitis or a bladder infection Kidneys -- an infection of one or both kidneys is called pyelonephritis or a kidney infection Urethras -- the tubes that take urine from each kidney to the bladder are only rarely the site of infection Urethra -- an infection of the tube that empties urine from the bladder to the outside is called urethritis Causes of UTI: Normally urine is sterile. It is usually free of bacteria, viruses and fungi but does contain fluids, salts and waste products. An infection occurs when tiny organisms, usually bacteria from the digestive tract, cling to the opening of the urethra and begin to multiply. Most infections arise from one type of bacteria, E.coli which normally lies in the colon. The organisms most commonly responsible for catheter-associated UTIs are E.coli, Proteus mirabilis, P.aeruginosa, and Streptococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Mycobacterium tuberculosis, Actinomycetes, Nocardia, Candida etc can cause UTI. In addition Mycoplasma and Chlamydia may be associated with sexually transmitted UTI.
Figure.1. Diagram showing contribution of various microbes for causing the UTI: E. coli 79%, S. Saprophyticus 11%, Klebsiella 3%, Mixed 3%, Proteus 2%, Enterococcus 2%, others 2%. SYMPTOMS

Common urinary tract infection (UTI) symptoms in women include: Urge to urinate frequently, often in small amounts Burning with urination Cloudy urine Strong unpleasant smell of urine Dark or bloody urine Pelvic pain Flank or back pain (kidney infection) Fever, chills (usually with kidney infection) Other possible symptoms include bloating, vaginal discharge Common urinary tract infection (UTI) symptoms in men include: Urge to urinate frequently, often in small amounts Burning with urination March-April 2013 Page 227
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ISSN: 2320 3471(Online) M.Komala and Sampath Kumar Indian Journal of Research in Pharmacy and Biotechnology Cloudy urine Strong unpleasant smell of urine Dark or bloody urine Rectal pain (kidney infection) Flank or back pain (kidney infection) Other symptoms may include penile, testicular and abdominal pain, and penile discharge Common urinary tract infection (UTI) symptoms in children include: Urge to urinate frequently, often in small amounts Burning with urination Cloudy urine Strong unpleasant smell of urine (not as reliable in children) Dark or bloody urine Abdominal pain Fever Vomiting Other symptoms (especially in newborns and infants) may include hypothermia, diarrhea, jaundice, poor feeding and in some children, bedwetting The following also increase chances of developing a UTI: Diabetes Advanced age (especially people in nursing homes) Problems emptying your bladder completely (urinary retention) A tube called a urinary catheter inserted into your urinary tract Bowel incontinence Enlarged prostate, narrowed urethra, or anything that blocks the flow of urine Kidney stones Staying still (immobile) for a long period of time (for example, while you are recovering from a hip fracture) Pregnancy Surgery or other procedure involving the urinary tract Factors that may make increase the risk for UTI: Infrequent voiding The bacteria spends a greater amount of time in the bladder allowing it time to replicate and take hold. Incomplete voiding An excess amount of urine is left in the bladder and the bacteria is not completely flushed out with each void. Personal Hygiene Perineal contamination with faeces increases the risk of coliform bacteria in the vagina and near the urethra will increase the risk of urinary tract infections. Sexual Activity Trauma to the urethra and surrounding tissue may increase susceptibility to infection and also the bacteria can be mechanically pushed into the urethra. Use of spermicidal contraception The actual spermicide changes the normal flora in the vagina and more coliform bacteria colonize the area. The presence of these strains of bacteria leads to a greater risk of a UTI. Genetics Certain cells on the vaginal mucosa and the urethra can express receptors that actually allow certain bacteria to attach and pull themselves into the bladder causing an increase risk of a UTI. Hormonal Status - A lack of estrogen allows for thinning and deficiency of the tissue in the vagina and urethral that may allow for greater susceptibility to UTIs. This lack of estrogen also changes the pH of the vagina which allows for colonization with more coliform bacteria and increases the risk of UTIs. Diabetes Persistently high blood sugar levels cause immunosuppression which allows for greater susceptibility to UTIs. Immunosuppression There are a variety of causes of immunosupression which decreases a person's ability to fight off infections. Urinary tract problems: If you are not able to control when you urinate, your risk for a UTI increases. Your risk also increases if you have had a UTI or if you have had surgery on your urinary tract in the past. Volume 1(2) March-April 2013 Page 228
ISSN: 2320 3471(Online) M.Komala and Sampath Kumar Indian Journal of Research in Pharmacy and Biotechnology Blockages: A blockage in your urinary tract stops your urine from flowing freely. Causes of a blockage include kidney and bladder stones. Not being circumcised: The foreskin of the penis makes it easier for germs to get trapped and enter your urinary tract. Prostate problems: An enlarged prostate gland or an infected prostate (prostatitis) increases your UTI risk. Sexual intercourse: You are more likely to get an infection if your sex partner has an infection. Anal sex also increases your UTI risk. Weak immune system: Your immune system is your body's defense against infection and disease. A weak immune system may not be able to fight the germs that can cause a UTI. Your immune system may become weak when you have a long-term illness, such as HIV or diabetes Diagnosis: The caregiver should obtain a detailed history from the patient, and if a UTI is suspected, a urine sample is usually obtained. The best sample is a midstream sample of urine placed in a sterile cup because it usually contains only the pathogenic organisms instead of the transient organisms that may be washed from adjacent surfaces when the urine stream begins. Male patients with foreskin should retract the foreskin before providing a midstream urine sample. In some patients who cannot provide a midstream sample, a sample can be obtained by a catheter. The urine sample is then sent for urinalysis. Patients with a "discharge," or possibility of having an STD, usually will have the discharge tested for STD organisms (for example, Neisseria and Chlamydia). A positive urinalysis is usually detection of about two to five leukocytes (white blood cells), about 15 bacteria per high-power microscopic field, and a positive nitrite test and/or positive leukocyte esterase test. Some clinicians and labs consider a positive test at least two of the above findings; still others report a positive for bacteria as >1,000 bacteria cultured per milliliter of urine. At best, the initial urinalysis, depending on the various criteria used by clinicians and labs, provides a presumptive positive test for a UTI. Most clinicians believe this presumptive test is adequate enough to begin treatment. A definitive test is usually considered to be isolation and identification of the infecting pathogen at a level of about 100,000 bacteria per cc of urine with the genus of the pathogen (usually bacterial) identified and antibiotic sensitivity determined by lab studies. This test takes 24-48 hours to obtain the results and your health care professional will usually start treatment before this result is available. Sometimes blood in the urine is a sign of a UTI but it may also indicate other problems, such as a urinary calculus or stone.In young children, infants, and some elderly patients, the best urine specimen is obtained by catheterization, as they are unable to deliver a clean catch" urine sample as described above. Urine can also be collected from "bags" placed over the urethral outlet (genital area), but these bagged specimens are only used for presumptive urinalysis as they are unreliable for culture. Some investigators consider any bagged urine samples as unreliable. Urine samples not processed within an hour of collection should either be discarded or be refrigerated before an hour passes because bacterial growth in urine at room temperature can yield false-positive tests. Special culture media and other tests are done for the infrequent or rare pathogens (for example, fungi and parasites).Other tests may be ordered to further define the extent of a UTI. They may include blood cultures, a complete blood count (CBC), intravenous pyelogram, a CT scan, or other specialized tests. Treatment: Treatment for a UTI should be designed for each patient individually and is usually based on the patient's underlying medical conditions, what pathogen(s) are causing the infection, and the susceptibility of the pathogen(s) to treatments. Patients who are very ill usually require intravenous (IV) antibiotics and admission to a hospital; they usually have a kidney infection (pyelonephritis) that may be spreading to the bloodstream. Other people may have a milder infection (cystitis) and may get well quickly with oral antibiotics. Still others may have a UTI caused by pathogens that cause STDs and may require more than a single oral antibiotic. The caregivers often begin treatment before the pathogenic agent and its antibiotic susceptibilities are known, so in some individuals, the antibiotic treatment may need to be changed. In addition, pediatric patients and pregnant patients should not use certain antibiotics that are commonly used in adults. For example,ciprofloxacin (Cipro) and other related quinolones should not be used in children or pregnant patients due to side effects. However, penicillins and cephalosporins are usually considered safe for both groups if the individuals are not allergic to the antibiotics. Volume 1(2) March-April 2013 Page 229
ISSN: 2320 3471(Online) M.Komala and Sampath Kumar Indian Journal of Research in Pharmacy and Biotechnology Patients with STD-related UTIs usually require two antibiotics to eliminate STD pathogens. The less frequent or rare fungal and parasitic pathogens require specific antifungal or antiparasitic medications; these more complicated UTIs should often be treated in consultation with an infectious disease expert.All antibiotics prescribed should be taken even if the person's symptoms disappear early. Reoccurrence of the UTI and evenantibiotic resistance of the pathogen may happen in individuals who are not adequately treated.Over-the-counter (OTC) medicines offer relief from the pain and discomfort of UTIs but they don't cure UTIs. OTC products like AZO or Uristat contain the medicine,phenazopyridine (Pyridium and Urogesic), which works in the bladder to relieve pain. This medication turns urine an orange-red color, so patients should not be worried when this occurs. This medication can also turn other body fluids orange, including tears, and can stain contact lenses. Most commonly used Antibiotics for UTIs and its possible side effects: 1. Macrodantin (Macrobid or nitrofurantoin) Side effects of long-term use may include fibrosis or scarring of the lungs and peripheral neuropathy. Generally, the medication is considered safe during pregnancy, except with rare genetic metabolic deficiencies. 2. Bactrim (Septra or sulfa/TMP) This drug should not be taken early during pregnancy and may affect the effectiveness of oral contraceptives. 3. Trimethoprim It should not be taken during pregnancy. 4. Quinolones (Levaquin, Levofloxacin, or Cipro) - This drug should not be taken during pregnancy. 5. Cephalosporin (Keflex) This may affect the effectiveness of oral contraceptives. 6. Doxycycline It is not safe during pregnancy or breastfeeding. Table.1. Parenteral antibiotics used for the treatment of pyelonephritis and its side effects Antibiotic Dose (Adult) Side effects Cefuroxime 750mg i.v every 8hr Diarrhoea, nausea, allergic reactions Ceftazidime 1g i.v every 8hr Diarrhoea, nausea, allergic reactions Co-amoxiclav 1.2g i.v every 8hr Nausea, diarrhoea, rashes, hepatitis, erythemamultiforme Gentamicin 80-120mg i.v every 8hr Vestibular and hearing damage, nephrotoxicity Ciprofloxacin 200mg i.v every 12hr Nausea ,vomiting, dizziness, headache, convulsions, hallucination, hepatitis, photosensitivity, blood disorder Imipenem 500mg i.v.every 8hr Nausea, vomiting, diarrhoea, allergic reactions, convulsions, confusion Table.2. Traditional Indian herbs used to treat urinary tract infection Herbs Applications in treating UTI Agrimony, Couch grass, General healing support for the urinary system. Elder flowers, Plantain, Yarrow, Juniper (not with inflammation), Horsetail, Lady's mantle, Saw palmetto Juniper berry Juniper relieves pain and is antiseptic, diuretic, and stimulant Couch grass. Diuretic with a soothing, anti-inflammatory healing effect on the lining of the bladder. Useful when there is mucus discharge from the bladder with painful and frequent urination Yarrow Anti-inflammatory, Antipyretic, Spasmolytic, Diaphoretic, Astringent, tonic. It regulates many urination problems and soothes and heals mucous membranes. It clears heat and congestion by aiding elimination via the kidneys through its diuretic effect Nettles, Red clover, Super blue-green algae, Astragulus, The ginsengs, Acidophilus, Aurdock Overall nutritional and adaptogenic support.
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ISSN: 2320 3471(Online) M.Komala and Sampath Kumar Indian Journal of Research in Pharmacy and Biotechnology Marshmallow root Increases the acidity of the urine thus inhibiting bacterial growth. It helps to strengthen and cleanse the bladder. It is a Demulcent, Emollient, and Diuretic. Marshmallow is the best source of easily digested vegetable mucilage which lubricates the body, protecting it against irritation and dryness. It soothes the urinary system and is usually combined with other diuretic herbs to treat kidney and bladder inflammations, difficult or painful urination and kidney stones or gravel. It stops bleeding in the urine. Marshmallow, Comfrey, Help soothe and coat irritated, inflamed tissue. Plantain, Violet, Mullein, Cornsilk Marshmallow, Hops, Red Helping the body reduce muscular spasms along the urinary tract. raspberry, Scullcap, Chamomile sarsaparilla, peppermint, Alkalize the urine marshmallow, comfrey root., plantain, ginger Echinacea, Goldenseal, Destroy pathogenic bacteria and strengthen the immune responses. Myrrh, Burdock, Garlic, Bilberry, Uva ursi, Feverfew, Honeysuckle, Barberry. Uva ursi For irritable bladder or an atonic boggy bladder, bacterial vaginosis and ulcerative cystitis Uva ursi is a strong, non-irritating diuretic and urinary antiseptic for bladder and kidney infections. When combined with marshmallow it helps to eliminate stones from the kidney and bladder. It strengthens and tones the urinary passages and is effective to treat blood in the urine. Buchu Urinary antiseptic, diuretic, urinary disinfectant. Its volatile oil stimulates urination and is excreted virtually unchanged by the kidneys, rendering the urine slightly antiseptic. Goldenseal Good for bladder infections if there is bleeding. It is an effective antimicrobial and choleretic. Dandelion, Cornsilk, Stimulate the kidney and bladder and increase the flow of urine. Sassafras, Juniper berry, Fennel, Cleavers, Uva ursi, Horsetail, Goldenrod, Meadowsweet, Pipsissewa, Plantain, Shepherd's purse Plantain It has cooling diuretic properties that make it beneficial for kidney and urinary bladder infections. Dandelion root It increases the flow of urine and has a laxative effect. Herbal diuretics help to cleanse the system. By promoting the release of fluids from the tissues it helps to relieve the false sensations of urgency characteristic of cystitis. Cleavers Soothing diuretic which is useful for acute or chronic cystitis with swollen lymph nodes and uterine inflammation. Goldenrod A mildly antiseptic and stimulating diuretic which is good to use if there is pain in the kidneys and scanty, dark urine Ginger, Echinacea, Yellow Assist in lessening the pain and discomfort dock, Licorice, Gotu kola, Comfrey, Chamomile, Marshmallow
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ISSN: 2320 3471(Online) M.Komala and Sampath Kumar Indian Journal of Research in Pharmacy and Biotechnology Chamomile flowers Chamomile flowers reduce muscle spasms and pains, reduce inflammation and are antiseptic. These compounds have a sedative and relaxing effect Beth root, Horsetail, For cystitis with weakness and exhaustion Hydrangea, Corn silk, Barberry, Black haw Hydrangea Good for stimulating the kidneys and flushing them clean Barberry It kills microorganisms (E. coli, staphylococci) that cause urinary tract infections Gravel root, Marshmallow For kidney involvement including kidney stones leaf, Couch grass, Barberry, Stone root, Hydrangea, Corn silk, Corn silk Soothing, Anti-inflammatory Diuretic that directly reduces painful symptoms and swelling due to inflammation. It is a Diuretic and urinary demulcent. Shepherd's Purse Bleeding Cinnamon Suppresses completely' the cause of most urinary tract infections (E. coli) and the fungus (Candida albicans) responsible for vaginal yeast infections Burdock Contains chemicals (polyacetylenes) that kill disease causing bacteria and fungi. It has been used traditionally for urinary tract infections Prevention: Lifestyle changes may help prevent some UTIs. After menopause, a woman may use estrogen cream in the vagina area to reduce the chance of further infections. Bathing and hygiene: Choose sanitary pads instead of tampons, which some doctors believe make infections more likely. Change the pad each time you use the bathroom. Do not douche or use feminine hygiene sprays or powders. As a general rule, do not use any product containing perfumes in the genital area. Take showers instead of baths. Avoid bath oils. Keep your genital area clean. Clean your genital and anal areas before and after sexual activity. Urinate before and after sexual activity. Wipe from front to back after using the bathroom. Clothing: Avoid tight-fitting pants. Wear cotton-cloth underwear and pantyhose, and change both at least once a day. Diet: Drink plenty of fluids (2 to 4 quarts each day). Drink cranberry juice or use cranberry tablets, but NOT if you have a personal or family history of kidney stones. Do not drink fluids that irritate the bladder, such as alcohol and caffeine. 2. CONCLUSION
Research on the pharmacological effectiveness of many herbal treatments has been indicated. But unfortunately the side effects produced are often untenable by modern standards. Many of the plants used in herbal medicine contain principles whose can be demonstrated pharmacologically and the action of the whole plant extract can usually be related to that of the isolated constituents.It appears to be a genarl notation that, by and large, herbal remidies, being natural products are inherently safer than the potent synthetic drugs, which often produce undesirable side effects. In treating uncomplicated UTI,herbal formulations are effective ,safe ,free from side effects and economical.Some of the complicated UTI,which is not respond with herbal formulation need antimicrobial
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agents.Thus it can be concluded that combination of herbal formulation with antibiotics are favourable for treating complicated UTIs.
REFERENCES Craig JC, Simpson JM, Williams GJ, Antibiotic prophylaxis and recurrent urinary tract infection in children. N Engl J Med, 361, 2009, 1748-1759. Gupta K, Hooton TM, Naber KG, International clinical practice guidelines for the treatment of acute uncomplicated cystitis and pyelonephritis in women: A 2010 update by the Infectious Diseases Society of America and the European Society for Microbiology and Infectious Diseases, Clin Infect Dis, 2011, 52(5), 103-120. Hooton TM, Bradley SF, Cardenas DD, Diagnosis, prevention, and treatment of catheter-associated urinary tract infection in adults: 2009 International Clinical Practice Guidelines from the Infectious Diseases Society of America, Clin Infect Dis. 2010, 50(5), 625-663. Lin K, Fajardo K, U.S. Preventive Services Task Force, Screening for asymptomatic bacteriuria in adults: evidence for the U.S. Preventive Services Task Force reaffirmation recommendation statement, Ann Intern Med.2008, 149(1), 20-24. Little P, Moore MV, Turner S, Effectiveness of five different approaches in management of urinary tract infection: randomised controlled trial, BMJ. 2010, 340. Shaikh N, Morone NE, Lopez J, Chianese J, Sangvai S, D'Amico F, Hoberman A, Wald ER. Does this child have a urinary tract infection? JAMA, 2007, 298, 2895-2904. Skoog SJ, Peters CA, Arant BS Jr, Pediatric Vesicoureteral Reflux Guidelines Panel summary report: clinical practice guidelines for screening siblings of children with vesicoureteral reflux and neonates/infants with prenatal hydronephrosis. J Urol, 184, 2010, 1145-1151. White B, Diagnosis and treatment of urinary tract infection in children, Am Fam Physician, 2011, 83, 409-415. Williams G, Craig JC, Long-term antibiotics for preventing recurrent urinary tract infection in children. Cochrane Database Syst Rev, 2011, (3), CD001534.
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DIABETES EPIDEMIC IN INDIA: RISK FACTORS, SYMPTOMS AND TREATMENT

Abhinov T1, Md Aasif Siddique Ahmed Khan1, Ashrafa2, Shabana Parveen2, K.P.Samapth Kumar3* 1. Shadan Institute of Medical Sciences and Research Centre, Hyderabad. 2. Nimra College of Pharmacy,Nimranagar,Vijayawada 2. Department of Pharmaceutical Sciences, Coimbatore Medical College, Coimbatore *Corresponding author: kp_sampath@rediffmail.com ABSTRACT Diabetes is a metabolism disorder in which the pancreas in the human body fails to produce insulin, or is unable to use the insulin produced in an effective manner. Most of the food we eat is broken into glucose, which is a form of sugar in the blood. When the food we eat is digested, the glucose enters our blood stream and is the main source of energy for the human body. But this glucose cannot enter our cells without sufficient insulin being present in our body. Insulin is a hormone produced by the pancreas and is needed to convert sugar, starch and other food into energy. After eating, the pancreas automatically releases the required amount of insulin to move the glucose present in our blood to our cells to regulate our blood sugar level. Insufficient secretion of insulin by the pancreas results in excess glucose levels in the blood stream, resulting in diabetes, which eventually damages various organs in the body..India has 61 million diabetics between 20-79 years according to the International Diabetes Federation. By 2030, this figure is estimated to go up to 101.1 million. President of the International Diabetes Federation Professor Jean Claude Mbanya said, 'Major number of diabetics are in the middle and low income countries.In fact by 2030, according to the estimates, 8.4 per cent of India's adult population will have diabetes. Key words: Diabetes, Insulin, Sulfonyl ureas, Biguanides, Thiazolidenediones INTRODUCTION Diabetes is a disease in which your blood glucose, or sugar, levels are too high. Glucose comes from the foods you eat. Insulin is a hormone that helps the glucose get into your cells to give them energy. With type 1 diabetes, your body does not make insulin. With type 2 diabetes, the more common type, your body does not make or use insulin well. Without enough insulin, the glucose stays in your blood. Over time, having too much glucose in your blood can cause serious problems. It can damage your eyes, kidneys, and nerves. Diabetes can also cause heart disease, stroke and even the need to remove a limb. Pregnant women can also get diabetes, called gestational diabetes. A blood test can show if you have diabetes. Exercise, weight control and sticking to your meal plan can help control your diabetes. You should also monitor your glucose level and take medicine if prescribed. A person with diabetes has no control over his blood sugar as his body either does not produce enough insulin, or produces no insulin or has cells that do not respond effectively to the insulin produced by the pancreas. This leads to too much glucose level in the blood stream which eventually damages various organs in the body. Persons suffering from diabetes show symptoms like fatigue, hazy vision, excessive thirst, weight loss, frequent urination and increase in appetite. Diabetes has emerged as a major healthcare problem in India. According to Diabetes Atlas published by the International Diabetes Federation (IDF), there were an estimated 40 million persons with diabetes in India in 2007 and this number is predicted to rise to almost 70 million people by 2025. The countries with the largest number of diabetic people will be India, China and USA by 2030. It is estimated that every fifth person with diabetes will be an Indian. Due to these sheer numbers, the economic burden due to diabetes in India is amongst the highest in the world. The real burden of the disease is however due to its associated complications which lead to increased morbidity and mortality. WHO estimates that mortality from diabetes, heart disease and stroke costs about $210 billion in India in the year 2005. Much of the heart disease and stroke in these estimates was linked to diabetes. WHO estimates that diabetes, heart disease and stroke together will cost about $ 333.6 billion over the next 10 years in India alone. 2. Current Status of Diabetes: In 2006, according to the World Health Organization, at least 171 million people worldwide suffer from diabetes. Its incidence is increasing rapidly, and it is estimated that by the year 2030, this number will double. Diabetes mellitus occurs throughout the world, but is more common (especially type 2) in the more developed countries. The greatest increase in prevalence is, however, expected to occur in Asia and Africa, where most patients will likely be found by 2030. The increase in incidence of diabetes in developing countries follows the trend of urbanization and lifestyle changes, perhaps most importantly a "Western-style" diet. This has suggested an environmental (i.e.,
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dietary) effect, but there is little understanding of the mechanism(s) at present, though there is much speculation, some of it most compellingly presented. Diabetes is in the top 10, and perhaps the top 5, of the most significant diseases in the developed world, and is gaining in significance there and elsewhere (see big killers). For at least 20 years, diabetes rates in North America have been increasing substantially. In 2005 there are about 20.8 million people with diabetes in the United States alone. According to the American Diabetes Association, there are about 6.2 million people undiagnosed and about 41 million people that would be considered prediabetic. However, the criteria for diagnosing diabetes in the USA means that it is more readily diagnosed than in some other countries. The Centers for Disease Control has termed the change an epidemic. The National Diabetes Information Clearinghouse estimates that diabetes costs $132 billion in the United States alone every year. About 5%10% of diabetes cases in North America are type 1, with the rest being type 2. The fraction of type 1 in other parts of the world differs; this is likely due to both differences in the rate of type 1 and differences in the rate of other types, most prominently type 2. Most of this difference is not currently understood. The American Diabetes Association point out the 2003 assessment of the National Center for Chronic Disease Prevention and Health Promotion (Centers for Disease Control and Prevention) that 1 in 3 Americans born after 2000 will develop diabetes in their lifetime. According to the American Diabetes Association, approximately 18.3% (8.6 million) of Americans age 60 and older have diabetes. Diabetes mellitus prevalence increases with age, and the numbers of older persons with diabetes are expected to grow as the elderly population increases in number. The National Health and Nutrition Examination Survey (NHANES III) demonstrated that, in the population over 65 years old, 18% to 20% have diabetes, with 40% having either diabetes or its precursor form of impaired glucose tolerance. 3. General consideration: Diabetes mellitus-often simply diabetes, is a syndrome characterized by disordered metabolism and inappropriately high blood sugar (hyperglycemia) resulting from either low levels of the hormone insulin or from abnormal resistance to insulin's effects coupled with inadequate levels of insulin secretion to compensate. The characteristic symptoms are excessive urine production (polygraph), excessive thirst and increased fluid intake (polydipsia), and blurred vision; these symptoms are likely absent if the blood sugar is only mildly elevated. Most of the food we eat is broken down by the digestive juices into a simple sugar called glucose. Glucose is the main source of fuel for the body. After digestion, the glucose passes into our bloodstream where it is available for body cells to use for growth and energy. For the glucose to get into the cells, insulin must be present. Insulin is a hormone produced by the pancreas, a large gland behind the stomach. When we eat, the pancreas is supposed to automatically produce the right amount of insulin to move the glucose from our blood into our cells. If your body doesn't make enough insulin or the insulin doesn't work right, the sugar cannot get into the cells. It stays in the blood. This makes your blood sugar level high, causing you to have diabetes. As a result, glucose builds up in the blood, overflows into the urine, and passes out of the body. Thus, the body loses its main source of fuel even though the blood contains large amounts of glucose. (www.google.com) The World Health Organization recognizes three main forms of diabetes mellitus: type 1, type 2, and gestational diabetes (occurring during pregnancy), which have different causes and population distributions. While, ultimately, all forms are due to the beta cells of the pancreas being unable to produce sufficient insulin to prevent hyperglycemia, the causes are different. Type 1 diabetes is usually due to autoimmune destruction of the pancreatic beta cells. Type 2 diabetes is characterized by insulin resistance in target tissues, this causes a need for abnormally high amounts of insulin and diabetes develops when the beta cells cannot meet this demand. Gestational diabetes is similar to type 2 diabetes in that it involves insulin resistance; the hormones of pregnancy can cause insulin resistance in women genetically predisposed to developing this condition. Gestational diabetes typically resolves with delivery of the child, however types 1 and 2 diabetes are chronic conditions. All types have been treatable since insulin became medically available in 1921. Type 1 diabetes, in which insulin is not secreted by the pancreas, is directly treatable only with injected or inhaled insulin, although dietary and other lifestyle adjustments are part of management. Type 2 may be managed with a combination of dietary treatment, tablets and injections and, frequently, insulin supplementation. While insulin was originally produced from natural sources such as porcine pancreas, most insulin used today is produced through genetic engineering, either as a direct copy of human insulin, or human insulin with modified molecules that provide different onset and duration of action. Insulin can also be delivered continuously by a specialized pump which subcutaneously provides insulin through a changeable catheter.
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Diabetes can cause many complications. Acute complications (hypoglycemia, ketoacidosis or nonketotic hyperosmolar coma) may occur if the disease is not adequately controlled. Serious long-term complications include cardiovascular disease (doubled risk), chronic renal failure, retinal damage (which can lead to blindness), nerve damage (of several kinds), and micro vascular damage, which may cause impotence and poor healing. Poor healing of wounds, particularly of the feet, can lead to gangrene, which may require amputation. Adequate treatment of diabetes, as well as increased emphasis on blood pressure control and lifestyle factors (such as not smoking and keeping a healthy body weight), may improve the risk profile of most aforementioned complications. In the developed world, diabetes is the most significant cause of adult blindness in the non-elderly, the leading cause of non-traumatic amputation in adults, and diabetic nephropathy is the main illness requiring renal dialysis in the United States. 4. Diagnosis: The diagnosis of type 1 diabetes and many cases of type 2, is usually prompted by recent-onset symptoms of excessive urination (polyuria) and excessive thirst (polydipsia), and often accompanied by weight loss. These symptoms typically worsen over days to weeks; about a quarter of people with new type 1 diabetes have developed some degree of diabetic ketoacidosis by the time the diabetes is recognized. The diagnosis of other types of diabetes is usually made in other ways. These include ordinary health screening; detection of hyperglycemia during other medical investigations; and secondary symptoms such as vision changes or unexplainable fatigue. Diabetes is often detected when a person suffers a problem that is frequently caused by diabetes, such as a heart attack, stroke, neuropathy, poor wound healing or a foot ulcer, certain eye problems, certain fungal infections, or delivering a baby with macrosomia or hypoglycemia. Diabetes mellitus is characterized by recurrent or persistent hyperglycemia, and is diagnosed by demonstrating any one of the following: Fasting plasma glucose level at or above 126 mg/dL (7.0 mmol/l). Plasma glucose at or above 200 mg/dL (11.1 mmol/l) two hours after a 75 g oral glucose load as in a glucose tolerance test. Random plasma glucose at or above 200 mg/dL (11.1 mmol/l). A positive result, in the absence of clinical symptoms of diabetes, should be confirmed by another of the above-listed methods on a different day. Most physicians prefer to measure a fasting glucose level because of the ease of measurement and the considerable time commitment of formal glucose tolerance testing, which takes two hours to complete. According to the current definition, two fasting glucose measurements above 126 mg/dL (7.0 mmol/l) are considered diagnostic for diabetes mellitus. Patients with fasting glucose levels between 110 and 125 mg/dL (6.1 and 7.0 mmol/l) are considered to have impaired fasting glycemia. Patients with plasma glucose at or above 140 mg/dL or 7.8 mmol/l two hours after a 75 g oral glucose load are considered to have impaired glucose tolerance. Of these two pre-diabetic states, the latter in particular is a major risk factor for progression to full-blown diabetes mellitus as well as cardiovascular disease. While not used for diagnosis, an elevated level of glucose irreversibly bound to hemoglobin (termed glycosylated hemoglobin or HbA1c) of 6.0% or higher (the 2003 revised U.S. standard) is considered abnormal by most labs; HbA1c is primarily used as a treatment-tracking test reflecting average blood glucose levels over the preceding 90 days (approximately). However, some physicians may order this test at the time of diagnosis to track changes over time. The current recommended goal for HbA1c in patients with diabetes is <7.0%, which is considered good glycemic control, although some guidelines are stricter (<6.5%). People with diabetes who have HbA1c levels within this range have a significantly lower incidence of complications from diabetes, including retinopathy and diabetic nephropathy. 5. Classification of Diabetes: The term diabetes, without qualification, usually refers to diabetes mellitus, which is associated with excessive sweet urine, (known as 'glycosuria') but there are several rarer conditions also named diabetes. The most common of these is diabetes insipidus in which the urine is not sweet (insipidus meaning "without taste" in Latin); it can be caused by either kidney (nephrogenic DI) or pituitary gland (central DI) damage. The principal two idiopathic forms of diabetes mellitus are known as types 1 and 2. The term "type 1 diabetes" has universally replaced several former terms, including childhood-onset diabetes, juvenile diabetes, and insulin-dependent diabetes (IDDM). Likewise, the term "type 2 diabetes" has replaced several former terms, including adult-onset diabetes, obesity-related diabetes, and non-insulin-dependent diabetes (NIDDM). Beyond these two types, there is no agreed-upon standard nomenclature. Various sources have defined "type 3 diabetes"
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as, among others, gestational diabetes,. There is also maturity onset diabetes of the young (MODY) which is a single gene disorder with strong family history that presents as type 2 diabetes before 30 years of age. Type 1 diabetes mellitus: Type 1 diabetes mellitus is characterized by loss of the insulin-producing beta cells of the islets of Langerhans in the pancreas, leading to a deficiency of insulin. The main cause of this beta cell loss is a T-cell mediated autoimmune attack. There is no known preventative measure that can be taken against type 1 diabetes, which comprises up to 10% of diabetes mellitus cases in North America and Europe (though this varies by geographical location). Most affected people are otherwise healthy and of a healthy weight when onset occurs. Sensitivity and responsiveness to insulin are usually normal, especially in the early stages. Type 1 diabetes can affect children or adults but was traditionally termed "juvenile diabetes" because it represents a majority of cases of diabetes affecting children.(K.D.Tripathi, Clinical Pharmacology) Type 2 Diabetes Mellitus- Type 2 diabetes mellitus is due to insulin resistance or reduced insulin sensitivity, combined with reduced insulin secretion. The defective responsiveness of body tissues to insulin almost certainly involves the insulin receptor in cell membranes. In the early stage the predominant abnormality is reduced insulin sensitivity, characterized by elevated levels of insulin in the blood. At this stage hyperglycemia can be reversed by a variety of measures and medications that improve insulin sensitivity or reduce glucose production by the liver. As the disease progresses the impairment of insulin secretion worsens, and therapeutic replacement of insulin often becomes necessary. There are numerous theories as to the exact cause and mechanism in type 2 diabetes. Central obesity (fat concentrated around the waist in relation to abdominal organs, but not subcutaneous fat) is known to predispose individuals for insulin resistance. Abdominal fat is especially active hormonally, secreting a group of hormones called adipokines that may possibly impair glucose tolerance. Obesity is found in approximately 55% of patients diagnosed with type 2 diabetes. Other factors include aging (about 20% of elderly patients in North America have diabetes) and family history (type 2 is much more common in those with close relatives who have had it). In the last decade, type 2 diabetes has increasingly begun to affect children and adolescents, likely in connection with the increased prevalence of childhood obesity seen in recent decades in some places. Gestational diabetes: Gestational diabetes mellitus (GDM) resembles type 2 diabetes in several respects, involving a combination of inadequate insulin secretion and responsiveness. It occurs in about 2% 5% of all pregnancies and may improve or disappear after delivery. Gestational diabetes is fully treatable but requires careful medical supervision throughout the pregnancy. About 20% 50% of affected women develop type 2 diabetes later in life. Even though it may be transient, untreated gestational diabetes can damage the health of the fetus or mother. Risks to the baby include macrosomia (high birth weight), congenital cardiac and central nervous system anomalies, and skeletal muscle malformations. Increased fetal insulin may inhibit fetal surfactant production and cause respiratory distress syndrome. Hyperbilirubinemia may result from red blood cell destruction. In severe cases, perinatal death may occur, most commonly as a result of poor placental profusion due to vascular impairment. Induction may be indicated with decreased placental function. A cesarean section may be performed if there is marked fetal distress or an increased risk of injury associated with macrosomia, such as shoulder dystocia. Other types:There are several rare causes of diabetes mellitus that do not fit into type 1, type 2, or gestational diabetes; attempts to classify them remain controversial. Some cases of diabetes are caused by the body's tissue receptors not responding to insulin (even when insulin levels are normal, which is what separates it from type 2 diabetes); this form is very uncommon. Genetic mutations (autosomal or mitochondrial) can lead to defects in beta cell function. Abnormal insulin action may also been genetically determined in some cases. Any disease that causes extensive damage to the pancreas may lead to diabetes (for example, chronic pancreatitis and cystic fibrosis). Diseases associated with excessive secretion of insulin-antagonistic hormones can cause diabetes (which is typically resolved once the hormone excess is removed). Many drugs impair insulin secretion and some toxins damage pancreatic beta cells. The ICD-10 (1992) diagnostic entity, malnutrition-related diabetes mellitus (MRDM or MMDM, ICD-10 code E12), was deprecated by the World Health Organization. (KD.Tripathi, clinical pharmacology, R.S.Satoskar, et.al., pharmacology and pharmacotherapeutics,XIth edition) 6. Signs and Symptoms: The classical triad of diabetes symptoms is polyuria, polydipsia and polyphagia, which are, respectively, frequent urination; increased thirst and consequent increased fluid intake; and increased appetite. Symptoms may develop quite rapidly (weeks or months) in type 1 diabetes, particularly in children. However, in type 2 diabetes the symptoms develop much more slowly and may be subtle or completely absent. Type 1
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diabetes may also cause weight loss (despite normal or increased eating) and irreducible fatigue. These symptoms can also manifest in type 2 diabetes in patients whose diabetes is poorly controlled. When the glucose concentration in the blood is raised beyond the renal threshold, reabsorption of glucose in the proximal renal tubuli is incomplete, and part of the glucose remains in the urine (glycosuria). This increases the osmotic pressure of the urine and inhibits the reabsorption of water by the kidney, resulting in increased urine production (polyuria) and increased fluid loss. Lost blood volume will be replaced osmotically from water held in body cells, causing dehydration and increased thirst. Prolonged high blood glucose causes glucose absorption, which leads to changes in the shape of the lenses of the eyes, resulting in vision changes. Blurred vision is a common complaint leading to a diabetes diagnosis; type 1 should always be suspected in cases of rapid vision change whereas type 2 is generally more gradual, but should still be suspected. Patients (usually with type 1 diabetes) may also present with diabetic ketoacidosis DKA), an extreme state of metabolic dysregulation characterized by the smell of acetone on the patient's breath; a rapid, deep breathing known as Kussmaul breathing; polyuria; nausea; vomiting and abdominal pain; and any of many altered states of consciousness or arousal (such as hostility and mania or, equally, confusion and lethargy). In severe DKA, coma may follow, progressing to death. Diabetic ketoacidosis is a medical emergency and requires hospital admission. A rarer but equally severe possibility is hyperosmolar nonketotic state, which is more common in type 2 diabetes and is mainly the result of dehydration due to loss of body water. Often, the patient has been drinking extreme amounts of sugar-containing drinks, leading to a vicious circle in regard to the water loss. (Ramington19th edition) 7. Pathophysology: Mechanism of insulin release in normal pancreatic beta cells. Insulin production is more or less constant within the beta cells, irrespective of blood glucose levels. It is stored within vacuoles pending release, via exocytosis, which is triggered by increased blood glucose levels. Insulin is the principal hormone that regulates uptake of glucose from the blood into most cells (primarily muscle and fat cells, but not central nervous system cells). Therefore deficiency of insulin or the insensitivity of its receptors plays a central role in all forms of diabetes mellitus. Much of the carbohydrate in food is converted within a few hours to the monosaccharide glucose, the principal carbohydrate found in blood and used by the body as fuel. Some carbohydrates are not so converted. Notable examples include fruit sugar (fructose), usable as cellular fuel but it is not converted to glucose, and which therefore does not participate in the insulin/glucose metabolic regulatory mechanism. Additionally, the carbohydrate cellulose (though it is actually many glucose molecules in long chains) is not converted to glucose, as humans and many animals have no digestive pathway capable of breaking up cellulose. Insulin is released into the blood by beta cells (-cells), found in the Islets of Langerhans in the pancreas, in response to rising levels of blood glucose after eating. Insulin is used by about two-thirds of the body's cells to absorb glucose from the blood for use as fuel, for conversion to other needed molecules, or for storage. Insulin is also the principal control signal for conversion of glucose to glycogen for internal storage in liver and muscle cells. Lowered glucose levels result both in the reduced release of insulin from the beta cells and in the reverse conversion of glycogen to glucose when glucose levels fall. This is mainly controlled by the hormone glucagons which acts in an opposite manner to insulin. Glucose thus recovered by the liver re-enters the bloodstream; muscle cells lack the necessary export mechanism. Higher insulin levels increase many anabolic (building up) processes such as cell growth and duplication, protein synthesis, and fat storage. Insulin (or its lack) is the principal signal in converting many of the bidirectional processes of metabolism from a catabolic to an anabolic direction, and vice versa. In particular, a low insulin level is the trigger for entering or leaving ketosis (the fat burning metabolic phase). If the amount of insulin available is insufficient, if cells respond poorly to the effects of insulin (insulin insensitivity or resistance), or if the insulin itself is defective, then glucose will not be absorbed properly by those body cells that require it nor will it be stored appropriately in the liver and muscles. The net effect is persistent high levels of blood glucose, poor protein synthesis, and other metabolic derangements, such as acidosis. (Remington 19th Ed.) 8. Diabetic Complication: Diabetes is associated with long-term (developing over many years) complications that affect almost every major part of the body. Diabetes causing stiffening and narrowing of very small blood vessels carrying oxygen to body cells and organs. Diabetes contributes to: blindness heart disease strokes kidney failure
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amputations nerve damage Uncontrolled diabetes can complicate pregnancy, and birth defects are more common in babies born to women with diabetes. Class of diabetic complication can be categorized as follows; Acute complications: Diabetic ketoacidosis: Diabetic ketoacidosis (DKA) is an acute and dangerous complication that is always a medical emergency. Lack of insulin causes the liver to turn fat into ketone bodies, a fuel mainly used by the brain. Elevated levels of ketone bodies in the blood decrease the blood's pH, leading to most of the symptoms of DKA. On presentation at hospital, the patient in DKA is typically dehydrated and is breathing rapidly and deeply. Abdominal pain is common and may be severe. The level of consciousness is typically normal until late in the process, when lethargy may progress to coma. Ketoacidosis can become severe enough to cause hypotension, shock, and death. Analysis of the urine reveals significant levels of ketone bodies present (which spill over from the blood when the kidneys filter blood). Prompt proper treatment usually results in full recovery, though death can result from inadequate or delayed treatment, or from complications. Ketoacidosis is much more common in type 1 diabetes than type 2. Nonketotic hyperosmolar coma: The hyperosmolar nonketotic state (HNS) is an acute complication with many symptoms in common with DKA, but an entirely different cause and different treatment. In a person with very high blood glucose levels (usually considered to be above 300 mg/dl (16 mmol/l)), water is drawn out of cells into the blood by osmosis and the kidneys dump glucose into the urine. This results in loss of water and an increase in blood osmolality. If fluid is not replaced (by mouth or intravenously), the osmotic effect of high glucose levels combined with the loss of water will eventually lead to dehydration. The body's cells become progressively dehydrated as water is taken from them and excreted. Electrolyte imbalances are also common and dangerous. As with DKA, urgent medical treatment is necessary, especially volume replacement. Lethargy may ultimately progress to a coma, which is more common in type 2 diabetes than type 1. Hypoglycemia: Hypoglycemia, or abnormally low blood glucose, is a complication of several diabetes treatments. It may develop if the glucose intake does not cover the treatment. The patient may become agitated, sweaty, and have many symptoms of sympathetic activation of the autonomic nervous system resulting in feelings similar to dread and immobilized panic. Consciousness can be altered or even lost in extreme cases, leading to coma, seizures, or even brain damage and death. In patients with diabetes, this can be caused by several factors, such as too much or incorrectly timed insulin, too much or incorrectly timed exercise (exercise decreases insulin requirements) or not enough food (specifically glucose-producing carbohydrates). In most cases, hypoglycemia is treated with sugary drinks or food. In severe cases, an injection of glucagon (a hormone with the opposite effects of insulin) or an intravenous infusion of glucose is used for treatment, but usually only if the person is unconscious. In hospital, intravenous dextrose is often used. Chronic complications Vascular disease: Chronic elevation of blood glucose level leads to damage of blood vessels (angiopathy). The endothelial cells lining the blood vessels take in more glucose than normal, since they don't depend on insulin. They then form more surface glycoproteins than normal, and cause the basement membrane to grow thicker and weaker. In diabetes, the resulting problems are grouped under "micro vascular disease" (due to damage to small blood vessels) and "macro vascular disease" (due to damage to the arteries).
Image of fundus showing scatter laser surgery for diabetic retinopathy The damage to small blood vessels leads to a microangiopathy, which can cause one or more of the following:
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Diabetic retinopathy, growth of friable and poor-quality new blood vessels in the retina as well as macular edema (swelling of the macula), which can lead to severe vision loss or blindness. Retinal damage (from microangiopathy) makes it the most common cause of blindness among non-elderly adults in the US.(Ramington19th ed.) Diabetic neuropathy, abnormal and decreased sensation, usually in a 'glove and stocking' distribution starting with the feet but potentially in other nerves, later often fingers and hands. When combined with damaged blood vessels this can lead to diabetic foot. Other forms of diabetic neuropathy may present as mononeuritis or autonomic neuropathy. Diabetic amyotrophy is muscle weakness due to neuropathy.(Ramington-19th ed.) Diabetic nephropathy, damage to the kidney which can lead to chronic renal failure, eventually requiring dialysis. Diabetes mellitus is the most common cause of adult kidney failure worldwide in the developed world. Macrovascular disease leads to cardiovascular disease, to which accelerated atherosclerosis is a contributor: Coronary artery disease, leading to angina or myocardial infarction ("heart attack") Stroke (mainly the ischemic type) Peripheral vascular disease, which contributes to intermittent claudication (exertion-related leg and foot pain) as well as diabetic foot. Diabetic myonecrosis ('muscle wasting') Diabetic foot, often due to a combination of neuropathy and arterial disease, may cause skin ulcer and infection and, in serious cases, necrosis and gangrene. It is why diabetics are prone to leg and foot infections and why it takes longer for them to heal from leg and foot wounds. It is the most common cause of adult amputation, usually of toes and or feet, in the developed world. 9. Treatment and management: Diabetes mellitus is currently a chronic disease, without a cure, and medical emphasis must necessarily be on managing/avoiding possible short-term as well as long-term diabetes-related problems. There is an exceptionally important role for patient education, dietetic support, sensible exercise, self glucose monitoring, with the goal of keeping both short-term blood glucose levels, and long term levels as well, within acceptable bounds. Careful control is needed to reduce the risk of long term complications. This can be achieved with combinations of diet, exercise and weight loss (type 2), various oral diabetic drugs (type 2 only), and insulin use (type 1 and increasingly for type 2 not responding to oral medication). Insulin: Insulin is the main hormone controlling intermediary, having actions on liver, muscle and fats. Its overall effect is to conserve fuel by facilitating the uptake and storage of glucose, amino acids and fats after a meal. Actually, it reduces blood sugar. Consequently a fall in plasma insulin increases blood glucose. Insulin is essential for the treatment of type-1 diabetes. Diet is the corner stone, combined with increase exercise. Oral agents are used to control symptoms from hyperglycemia, as well as to limit microvascular complications. Insulin is often needed as increasing age. Dietary measures to prevent atheromatous disease (specially limitations of saturated fat consumption) are crucial. Insulin for clinical use was once either porcine or bovine but is now almost entirely human made by recombinant DNA technology. Preparation of Insulin: Diabetes mellitus may be managed from a choice of four types of insulin (animal or human) preparations, having :( db, ins, oada, obes) Short duration of action (and rapid onset): soluble insulin (natural insulin). The most recent addition to this class of insulin, insulin lispro (Humalog), is modified human insulin in which the reversing of two amino acids has resulted in a very rapid onset of action (within 15 mins. Of injection). Insulin aspart is similar. Intermediate duration of action (and slower onset): Isophane Insulin, a suspension with protamine; Insulin Zinc suspensions, amorphous or a mixture and crystalline. Longer duration of action: Insulin Zinc suspension, crystalline or Protamine Zinc Insulin (Insulin in suspension with both Zinc and Protamine). A mixture of soluble and isophane insulin, officially called biphasic insulin. Mechanism of Action: Insulin binds to specific receptor on the surface of its target cells. Receptor is a large transmembrane glycoprotein complex consisting of two - and two -subunits. The -subunits are entirely Extra cellular and each carries an insulin binding site, where as the -subunits are transmembrane proteins with tyrosine kinase activity. This activity is suppressed by the -subunits, but insulin binding causes a conformational change that depresses (activates) the tyrosine kinase activity of the -subunits, which act on each other (autophosphorylation) and on other target proteins. At concentration of insulin that produces maximum effects, less than 10 percent of receptors are occupied. Occupied receptors aggregate into clusters, which are sub sequentially vesicles, resulting in down regulation. Internalized receptor is degraded in lysozomes, but the receptors are recycled to the plasma membrane.
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Adverse effects: The main undesirable effect of insulin is hypoglycemia. This is common and, if very severe, can cause brain damage. Rebound hyperglycemia (Somogyi effect) can fallow insulin -induced hypoglycemia, because of the release of counter regulatory hormones. Allergy to human insulin is unusual but can occur. It may take the form of local or systemic reaction. Insulin resistance as a consequence of antibody formation is rare.(Rang&Dale,2003) Oral Hypoglycemic Agents Sulfonylureas: Mechanism of Action: Sulfonylureas cause hypoglycemia by stimulating insulin release from pancreatic cells. Their effects in the treatment of diabetes, however, are more complex. The acute administration of sulfonylurea to type 2 diabetes mellitus patients increases insulin release from the pancreas. Sulfonylureas also my further increase insulin levels by reducing hepatic clearance of the hormone. In the initial months of the sulfonylurea treatment, fasting plasma insulin level and insulin responses to oral glucose challenges are increased. With chronic administration, circulating insulin levels decline to those that existed before treatment, but, despite this reduction in insulin levels, reduced plasma glucose levels are maintained. The effects of the sulfonylurea are initiated by binding to and blocking an ATP-sensitive K+ conductance causes membrane depolarization and influx of Ca2+ through voltage sensitive Ca2+ channels. Adverse Reactions: Adverse effects of sulfonylurea are infrequent, occurring in about 4% of patients taking firstgeneration drugs and perhaps slightly less often in patients receiving second-generation drugs. Not unexpectedly, sulfonylurea my cause hypoglycemic reactions, including coma. Other side effects of sulfonylurea may include nausea, vomiting, cholestatic jaundice, hemolytic anemias and dermatological reactions.(Goodman&Gilman,10th ed.) Other Drugs that Stimulate Insulin Secretion: Several other drugs that lack the sulphonylureas urea moiety but stimulate insulin secretion have recently been developed. This includes repaglinide and nateglinide. This act, like the sulphonylureas, by blocking the sulphonylureas receptors on K ATP channels in pancreatic -cell membranes. II. Biguanides: Metformin is the only drug of this class presently available. Mechanism of Action: Biguanides lower blood glucose. Their mechanisms are complex and incompletely understood. They increase glucose uptake and utilization in skeletal muscle (there by reducing insulin resistance) and reduce hepatic glucose production (gluconeogenesis). Metformin, as well as lowering blood glucose, additionally reduces low density and very low-density lipoproteins (LDL and VLDL respectively). Adverse Effects: The commonest unwanted effects of metformin are dose related gastrointestinal disturbances (e.g. anorexia, diarrhea, nausea). Lactic acidosis is a rare but potentially fatal toxic effect. (Goodman&Gilman, 10th Ed.) III. Thiazolidenediones (Glitazones): Thiazolidenediones reduce hepatic output and increase glucose uptake in muscle, enhancing the effectiveness of endogenous insulin and reducing the amount of exogenous insulin needed to maintain given level of blood glucose by approximately 30%. The reduction in blood glucose level often accompanied by reductions in circulating insulin and free fatty acids (FFA). Triglycerides may decline, while LDL and HDL are either unchanged or slightly increased with little alteration in LDL to HDL ratio. The Proportion of small dense LDL particles is reduced. Mechanism of Action: Thiazolidenediones bind to nuclear receptor called the Peroxisome Proliferator-activated receptor-gamma (PPAR), which is complexed with retinoid X recepor. PPAR occurs mainly in adipose tissue, but also in muscle and liver. I mediates differrentiation of adipocytes, increase lipogenesis, and enhance uptake of fatty acids and glucose. Thiazolidenedions are exogenous agonist. They change the PPAR -RXR complex so that it binds DNA and promotes transcription of several genes with products that are important in insulin signaling, including lipoprotein lipase, fatty acids transporter protein, adipocytes fatty acid-binding protein, Glut-4, Phosphoenolpyruvate carboxykinase, malic enzyme and others. Adverse Effects: Serious hepatotoxicity is associated with ciglitazones. The commonest unwanted effects of roziglitazones and piogliatzones are weight gain and fulid retention. Fluid retention is a substantial concern since it can precipitate heart failure, which contraindicates their use. Symptoms of uncertain cause including headache, fatigue, which contraindicates their use. Symptoms of uncertain cause including headache, fatigue, and gastrointestinal disturbances have also been reported. Thizolidinediones are contraindicated in pregnant and breast-feeding women and in children. (Charles R. Craig, modern pharmacology 4th Ed.) IV. -Glucosidase Inhibitors: Acarbose, an inhibitor of intestinal -glucosidase, is used in type 2 patients inadequately controlled by diet with or without other agents. It delays carbohydrates absorption, reducing
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postprandial increase in blood glucose. The commonest adverse effects are related to its main action and consist of flatulence, loose stools or diarrhoea and abdominal pain and bloating. Potential new antidiabetic drugs: Several agents are currently being studied including antagonists and inhibitors of fatty acid oxidation. Lipolysisin fat cell is controlled by adrenoreceptors of 3 subtype. The possibility of using selective 3- agonists, currently in development, in the treatment of obese patients with type 2 diabetes is being investigated. There is great interest in inhibitors of protein kinase C (e.g. LY33353, a specific inhibitor specific for the Isofrom), because of evidence implicating activation of this pathway in the development of vascular complications.(Rang & Dale) 10. Current Diabetes Research: In recent years, advances in diabetes research have led to better ways to manage diabetes and treat its complications. Major advances include: New forms of purified insulin, such as human insulin produced through genetic engineering. Better ways for doctors to monitor blood glucose levels and for people with diabetes to test their own blood glucose levels at home. Development of external and implantable insulin pumps that deliver appropriate amounts of insulin, replacing daily injections. Laser treatment for diabetic eye disease, reducing the risk of blindness. Successful transplantation of kidneys in people whose own kidneys fail because of diabetes. Better ways of managing diabetic pregnancies, improving chances of successful outcomes. New drugs to treat type 2 diabetes and better ways to manage this form of diabetes through weight control. Evidence that intensive management of blood glucose reduces and may prevent development of microvascular complications of diabetes. Demonstration that antihypertensive drugs called ACE-inhibitors prevents or delay kidney failure in people with diabetes. Government agencies that sponsoring diabetes programs, as well as collecting and analyzing statistics about diabetes, include the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), the Centers for Disease Control and Prevention (CDC), the Indian Health Service, the Health Resources and Services Administration, the Bureau of Veterans Affairs, and the Department of Defense. University research centers and hospitals throughout the United States are also involved in diabetes research. Many organizations outside of the Government support diabetes research and education activities. These organizations include the American Diabetes Association, the Juvenile Diabetes Foundation International, and the American Association of Diabetes Educators. 11. Potential Future Treatments: In the future, it may be possible to administer insulin through nasal sprays or in the form of a pill or patch. Devices that can "read" blood glucose levels without having to prick a finger to get a blood sample are also being developed. Researchers continue to search for the cause or causes of diabetes and ways to prevent and cure the disorder. Scientists are looking for genes that may be involved in type 2 diabetes and type 1 diabetes. Some genetic markers for type 1 diabetes have been identified, and it is now possible to screen relatives of people with type 1 diabetes to see if they are at risk for diabetes. Transplantation of the pancreas or insulin-producing beta cells offers the best hope of cure for people with type 1 diabetes. Some pancreas transplants have been successful. However, people who have transplants must take powerful drugs to prevent rejection of the transplanted organ. These drugs are costly and may eventually cause serious health problems. Scientists are working to develop less harmful drugs and better methods of transplanting pancreatic tissue to prevent rejection by the body. Using techniques of bioengineering, researchers are also trying to create artificial islet cells that secrete insulin in response to increased sugar levels in the blood. For type 2 diabetes, the focus is on ways to prevent diabetes. Preventive approaches include identifying people at high risk for the disorder and encouraging them to lose weight, exercise more, and follow a healthy diet. The Diabetes Prevention Program, another new NIDDK project, will focus on preventing the disorder in high-risk populations.
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CONCLUSION The World Health Organisation (WHO) estimates that nearly 200 million people all over the world suffer from diabetes and this number is likely to be doubled by 2030. Even as nations prepare to mark World Diabetes Day on November 14, WHO says about 80% of the diabetes deaths occur in middle-income countries.In India, there are nearly 50 million diabetics, according to the statistics of the International Diabetes Federation. As the incidence of diabetes is on the rise, doctors say, there is a proportionate rise in the complications that are associated with diabetes. They point out that it is a very crucial stage and awareness on the part of people and administration about diabetes is very essential, adding that people should be made aware and educated about their health and fitness level to reduce the number of patients in India. REFERENCES Peter H. Bennett and William C. Knowler, Joslins Diabetes Mellitus, Defination, Diagnosis and classification of diabetes mellitus and glucose homeostasis 14th Ed, Boston, Joslin Diabetes Center, 2005. Melinda Downie Maryniuk, Handbook of diabetes medical nutrition therapy, eclectic issues in diabetes nutrition therapy 2nd Ed, An Aspen Publication,1996. Garfinkel D, Role of magnesium in carbohydrate oxidation, Magnesium, 1988, 7, 249-61. Grafton G, Baxter MA, Sheppard MC, Effects of magnesium on sodium dependant Inositol transport, Diabetes, 1992, 41, 35-9. Nadler JC, Rude RK. Disorders of magnesium metabolism, Endocrinol Metab, Clinic. North. Am, 1995, 24, 623 41. Ma J, Folsom AR, Melnick SL, Eckfeldt JH, Sharret AR, Nabulsi AA, Associations of serum and dietary magnesium with cardiovascular disease, hypertension, diabetes, insulin and carotid arterial wall thickness, The Atherosclerosis Risk In Communities (ARIC) Study, J. Clin. Epidemiol, 1995, 48, 927-40. Rude RK. Magnesium deficiency and diabetes mellitus causes and effects, Postgrad Med J, 1992, 92, 217-24. Nadler JL, Buchnan T, Natarajan R, Antonipillai I, Bergman R, Rude RK. Magnesium deficiency produces insulin resistance and increased thromboxane synthesis, Hypertension, 1993, 21, 1024-9. McNair P, Christiansen C, Madsbad S, Lauritzen E, Faber O, Binder C, et al. Hypomagnesemia a risk factor in diabetic retinopathy, Diabetes, 1978, 27, 1075-7. Mather HM, Levin GE, Nisbet JA. Hypomagnesemia and ischemic heart disease in diabetes, Diabetes care, 1982, 5, 452-3. Andrea C, Ricardo L, Michele B, Domenico C, Vittorio NM, Salvatore M et al. Serum ionized magnesium levels in type-2 diabetic patients with microalbuminuria or clinical proteinuria. Am J of Nephrology, 2000, 20, 187-92. Kao WH, Folsom AR, Nieto FJ, Mo JP, Watson RL, Brancati FL. Serum and dietary magnesium and the risk of type 2 diabetes mellitus, The ARIC study, Arch Intern Med, 1999, 159, 2151-9. Girish Dwivedi And Shridhar Dwivedi, History Of Medicine Sushruta The Clinician Teacher Par Excellence, Indian J Chest Dis Allied Sci, 2007, 49, 243-244.
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SIMULTANEOUS ESTIMATION OF OLMESARTAN AND ATORVASTATIN IN BULK AND FROMULATION BY USING UV SPECTROSCOPY AND RP-HPLC
Revanth Reddy B*, Aravind G Nimra college of pharmacy, Vijayawada *Corresponding author: E.mail: revanth.reddy7@gmail.com ABSTRACT Simple, rapid and accurate UV Spectroscopic (Simultaneous Equation method and First order derivative method) and an isocratic RP HPLC methods showed excellent sensitivity, reproducibility, accuracy, and repeatability. In simultaneous UV method, the overlaid spectra of mixture of Olmesartan medoxomil and Atorvastatin calcium were recorded. From the spectra, 256.5nm for Olmesartan medoxomil and 247nm for Atorvastatin calcium was selected as wavelength to construct simultaneous equation. The percentage label claim present in tablet formulation was found to be 100.187 1.074% and 101.511 1.841% for Olmesartan medoxomil and Atorvastatin calcium respectively. In second method, the same spectrums were derivatised and 247 nm selected for detection of Olmesartan medoxomil where Atorvastatin calcium shows zero crossing and also 310 nm selected for detection of Atorvastatin calcium where Olmesartan medoxomil shows zero crossing. The percentage label claim present in formulation was found to be 100.254 1.6281 and 99.968 0.000054 for Olmesartan medoxomil and Atorvastatin calcium respectively. In RP-HPLC method, mobile phase used is acetonitrile: phosphate buffer pH 3.0 (60:40 V/V) with flow rate of 0.9 mL per min, the retention time of Olmesartan medoxomil and Atorvastatin calcium were found to be 3.19 and 4.63, respectively at 252 nm. The percentage purity was found to be 101.12 0.8107 and 101.83 0.4684 for Olmesartan medoxomil and Atorvastatin calcium, respectively. The low % RSD values for recovery indicated that the method was found to be accurate. Key words: Olmesartan medoxomil, Atorvastatin calcium, RP-HPLC, Simultaneous estimation INTRODUCTION Olmesartan medoxomil is (5-methyl-2-oxo1, 3-dioxolen-4-yl) methoxy-4- (1-hydroxy-1-methylethyl)-2propyl-1-{4-[2-(tetrazol-5-yl)phenyl]phenyl}methylimidazo-5-carboxylate). it is Angiotension II receptor antagonist drug, solid white in color and it is Practically insoluble in water and sparingly soluble in methanol. Atorvastatin Calcium is [R-(R*, R*)]-2-(4-fluorophenyl)-, -dihydroxy-5 (1-methylethyl)-3-phenyl-4[(phenylamino) carbonyl]-1H-pyrrole-1-heptanoic acid, calcium salt (2:1) trihydrate, category of HMG-CoA Reductase Inhibitor. It is white to off-white crystalline powder, soluble in methanol, ethanol and acetonitrile. Nilesh jain et al., (2010), reported Development and Validation of a Rapid RP-HPLC for the Determination of Amlodipine Besylate and Olmesartan Medoxomil in their Combined Tablet Formulation. Ritesh N. Sharma et al., (2010), reported RP-HPLC-DAD Method for Determination of Olmesartan Medoxomil in Bulk and Tablets Exposed to Forced Conditions. This method involves the study of drugs in UV and HPLC. UV/VISIBLE SPECTROSCOPY SIMULTANEOUS EQUATION METHOD DERIVATIVE SPECTROSCOPY METHOD Simultaneous equation method: If a sample contains two absorbing drugs (X and Y) each of which absorbs at max of the others it may be possible to determine both drugs by the technique of simultaneous equation ( Vierodts method) provided that criteria apply. 2 1 1 2 2 1 1 2 2 1 1 2 = 2 1 1 2 Criteria for obtaining maximum precision, based upon the absorbance ratios, have been suggested (Glenn, 1960) that place limits on the relative concentrations of the components of the mixture. The criteria are the ratios =
a y / a y1 A2 / A1 and 2 a x2 / a x1 A2 / A1
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ISSN: 2320 3471(Online) Revanth and Aravind Indian Journal of Research in Pharmacy and Biotechnology Derivative spectrophotometric method: This method involves the conversion of the normal spectrum into first, second or higher derivative spectrum. The transformation that occurs in the derivative spectrum are understood by reference to a Gaussian band which represents an ideal absorption band. The first order derivative spectrum of absorption band is characterized by a maximum, a minimum and a cross-over at a max of the absorption band. These spectral transformations confer two main advantages on derivative spectrophotometry. Firstly an even order spectrum is of narrower spectral band width than its fundamental spectrum. Derivative spectrum shows better resolution of overlapping bands than the fundamental spectrum and may permit the accurate determination of max of the individual bands. Secondly, derivative spectroscopy discriminates in favours of the substances of narrow spectral bandwidth against broad band width substances. High performance liquid chromatography: The high performance liquid chromatography is thus a method of separation in which the stationary phase is contained in a column one end which is attached to a source of pressurized liquid eluent (mobile phase ). The choice of mobile phase is very important in HPLC and the eluting power of the mobile phase is determined by its overall polarity of the stationary phase and the nature of sample components for normal phase separation eluting power increases with increasing polarity of the solvent ,but for reversed phase separations, eluting power decreases with increasing solvent polarity. OLMESARTAN MEDOXOMIL
O O O
ATORVASTATIN CALCIUM
H3C O CH3 OH OH 3H2O
O CH3 HO H3C N
CH3
H N N
N N
Ca
2+
NH
HO F
CH3
O 2
MATERIALS AND METHODS

Materials used: Olmesartan Medoxomil and Atorvastatin Calcium and the official drug 10mg tablets available in natco pharmaceuticals. All the chemicals used were of analytical grade and HPLC grade procured from Qualigens, India Ltd. The chemicals used for the study were methanol (HPLC grade), acetonitrile (HPLC grade), water (HPLC grade), methanol (Analytical grade) and ortho phosphoric acid (Analytical grade) Instruments Specifications: Shimadzu UV Visible spectrophotometer: Shimadzu, UV-1700; Double beam UV - Visible spectrophotometer. ELICO SL 210; Double beam UV - Visible spectrophotometer. Shimadzu High Performance Liquid Chromatography: Deuterium Arc lamp. UV Spectrophotometric method Simultaneous equation method First order derivative method Selection of solvent: The common solvent was found to be methanol for the analysis of Olmesartan medoxomil and Atorvastatin Calcium for proposed method. Preparation of standard stock solution: 10 mg of standard Olmesartan medoxomil and Atorvastatin calcium were weighed and transferred into10 mL volumetric flasks separately and dissolved in methanol and made up to the volume with methanol. These solutions were observed to contain 1000 g mL-1. Selection of wavelengths for estimation and stability studies: 10g mL-1 of Olmesartan medoxomil and Atorvastatin calcium solutions were scanned between 200 400 nm by using methanol as blank. The wavelengths selected were 256.5 nm and 247 nm. The Stability was performed by measuring the absorbance of same solution at different time intervals. It was observed that Olmesartan medoxomil and Atorvastatin calcium in methanol were stable for more than 4 hours at all the selected wavelengths. For derivative Spectroscopic method, the zero
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ISSN: 2320 3471(Online) Revanth and Aravind Indian Journal of Research in Pharmacy and Biotechnology order spectrums was derivatised to first order and the wavelength 247 was selected for the estimation of Olmesartan medoxomil which is zero crossing for Atorvastatin calcium where as 310 nm was selected for the estimation of Atorvastatin calcium which is zero crossing for Olmesartan medoxomil. Preparation of calibration graph: The aliquots of stock solution of Olmesartan medoxomil (1 5 ml of 80 g mL-1) and Atorvastatin calcium (1 5 ml of 40 g mL-1) were transferred into 10 mL volumetric flasks and made up to the volume with methanol. The absorbance of different concentration solutions were measured at 256.5 nm and 247 nm for Olmesartan medoxomil and Atorvastatin calcium. The calibration curve was plotted at their corresponding wavelengths. The two drugs, Olmesartan medoxomil and Atorvastatin calcium were linear with the concentration range of 8 40 g mL-1 and 4 -20 g mL-1 respectively, at their respective wavelengths. Quantification of formulation: Ten tablets of formulation (Containing Olmesartan medoxomil 20 mg and Atorvastatin calcium 10 mg) were weighed accurately. The average weight of tablets was found and powdered. The tablet powder equivalent to 50 mg of Olmesartan medoxomil was weighed and transferred into 50 mL volumetric flask added a minimum quantity of methanol to dissolve the substance by using ultra sonication for 15 minutes and made up to the volume with the same (1000 g mL-1). The content filtered through Whatmann filter paper No. 41. From the clear solution, further dilutions were made by diluting 2mL to 25 mL with methanol in 25ml volumetric flask, further dilute 1mL to 10mL to obtain 16 g mL-1 of Olmesartan medoxomil and 8 g mL-1 Atorvastatin calcium respectively. The absorbance measurements were made 6 times for the formulation at 256.5 nm and 247 nm. From the absorptivity values of Olmesartan medoxomil and Atorvastatin calcium at 256.5 nm and 247 nm the amount of Olmesartan medoxomil and Atorvastatin calcium could be determined by using simultaneous equation method. In derivative method the same zero order spectrums derivatised and A/ values noted at 247 nm and 310 nm for Olmesartan medoxomil and Atorva statin calcium, respectively. The calibration curve was constructed absorbance Vs concentration for the simultaneous equation method, A/ Vs concentration for first order derivative method. Olmesartan medoxomil and Atorvastatin calcium were linear with the concentration range of 8 40 g mL-1 and 4 20 g mL-1 , respectively in both the methods at their respective wavelengths. Recovery studies: The recovery experiment was done by adding known concentrations of Olmesartan medoxomil and Atorvastatin calcium raw material to the 50% preanalyzed formulation. Standard Olmesartan medoxomil and Atorvastatin calcium in the range of 80%, 100% and 120% to the 50% preanalyzed formulation into a series of 10 mL volumetric flasks and dissolved with methanol and the contents were sonicated for 15 minutes then the solution was made up to mark with methanol. After sonication the solutions were filtered through Whatmann filter paper No. 41. The absorbances of the resulting solutions were measured at their selected wavelengths for determination of Olmesartan medoxomil and Atorvastatin calcium respectively. The amount of each drug recovered from the formulation was calculated for the drugs by simultaneous equation method. The procedure was repeated for three times. Validation of developed method Linearity: Olmesartan medoxomil was linear with the concentration range of 8 40 g mL-1 and Atorvastatin calcium showed the linearity in the range of 4 20 g mL-1 at selected wavelengths for both the methods. Accuracy (Recovery studies): To the preanalyzed formulation, a known quantity of raw materials of Olmesartan medoxomil and Atorvastatin calcium were added and the procedure was followed as per the analysis of formulation. Precision: The repeatability of the method was confirmed by the analysis of formulation was repeated for 6 times with the same concentration. The percentage RSD was calculated. The intermediate precision of the method was confirmed by intraday and interday analysis. The amount of drugs was determined and percentage RSD also calculated. Ruggedness: Ruggedness of the method was confirmed by the analysis of formulation was done by the different analysts. The amount and % RSD were calculated. LOD and LOQ: The linearity study was carried out for six times. The LOD and LOQ were calculated by using the average of slope and standard deviation of intercept. RP-HPLC method Selection of mobile phase and max: Solutions of Olmesartan medoxomil and Atorvastatin calcium(10 g mL-1) were prepared in the mobile phase [Acetonitrile:Phosphate Buffer pH 3.0 (60:40 v/v)] and scanned in the
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ISSN: 2320 3471(Online) Revanth and Aravind Indian Journal of Research in Pharmacy and Biotechnology UV region of 200 400 nm and recorded the spectrums. It was found that two drugs have marked absorbance at 252 nm and can be effectively used for estimation of two drugs without interference. Stability of sample solutions: Solutions of Olmesartan medoxomil and Atorvastatin calcium (10 g mL-1) absorbance were checked for their stability at 252 nm and it was found that two drugs were stable for approximately three hours. Selection of mobile phase: A mobile phase contains Acetonitrile:25 mM Potassium dihydrogen phosphate pH 3.0 (60:40 v/v) was selected. Preparation of standard Olmesartan medoxomil solution: 50 mg of standard Olmesartan medoxomil was weighed accurately and transferred into 25 mL volumetric flask and dissolved in methanol, after dissolution the volume was made up to the mark with methanol (2000 g mL-1). Further dilution was made by pipetting 4 mL of standard stock into 50 mL to acquire 40 g mL-1 solution. Preparation of standard Atorvastatin calcium solution: 50 mg of standard Atorvastatin calcium was weighed accurately and transferred into 25 mL volumetric flask and dissolved in methanol, after dissolution the volume was made up to the mark with methanol (2000 g mL-1). Further dilution was made by pipetting 2 mL of standard stock into 50 mL to acquire 20 g mL-1 solution. Preparation of Calibration graph: In this progression, the aliquots of stock solution of Olmesartan medoxomil (0.5 2.5 mL of 40 g mL-1) and Atorvastatin calcium (0.5 2.5 mL of 20 g mL-1) individually were transferred into 10 ml volumetric flasks and made up to the mark with mobile phase, solutions containing the concentrations of 8, 16, 24, 32, and 40 g mL-1 of Olmesartan medoxomil and 4, 8, 12, 16 and 20 g mL-1 Atorvastatin calcium. The solutions were injected and the chromatograms were recorded at 252 nm. The above concentration range was found to be linear and obeys Beers law. Estimation of Olmesartan medoxomil and Atorvastatin calcium in tablet formulation: Ten tablets of formulation (Containing Olmesartan medoxomil 20 mg and Atorvastatin calcium 10 mg) were weighed accurately. The average weight of tablets was found and powdered. The tablet powder equivalent to 50 mg of Olmesartan medoxomil was weighed and transferred into 50 mL volumetric flask and added a minimum quantity of methanol to dissolve the substance and the content was sonicated for 15 minutes, and made up to the volume with methanol then filtered through Whatmann filter paper No. 41. From the clear solution, the dilutions were made by diluting 2 mL into 25 mL with methanol. This solution is used for further analysis. Assay Procedure: From the above solution 2 mL was pipetted into six different 10 mL volumetric flasks and made up the volume with mobile phase to obtain 16 g mL-1 solution of Olmesartan medoxomil and 8 g mL-1 of Atorvastatin calcium. A steady base line was recorded with optimized chromatographic conditions. After the stabilization of base line for 30 minutes, six test solutions of formulation were injected and recorded the chromatograms. The concentration of each test solution was determined by using slope and intercept values from the calibration graph. Recovery Experiments Preparation of Olmesartan medoxomil and Atorvastatin calcium raw material stock solution: An accurately weighed quantity of 50 mg of Olmesartan medoxomil and 25 mg of Atorvastatin calcium were transferred in to 50 mL volumetric flask and added sufficient methanol to dissolve the substance and made up to the mark with same. The above solutions were further diluted to get final concentration of 64 g mL -1 of Olmesartan medoxomil and 32 g mL-1 of Atorvastatin calcium Procedure: To each 1 mL of 50 % preanalyzed formulation solution (8 g mL-1 of Olmesartan medoxomil and 4 g mL-1) added 2, 2.5 and 3 mL of Olmesartan medoxomil and Atorvastatin calcium raw material stock solutions into 10 mL volumetric flasks and made up to the mark with mobile phase. The procedure was repeated as per analysis of formulation. The quantity of drug recovered was calculated by using slope and intercept values from the calibration graph. System suitability studies: The system suitability studies conceded as per ICH guidelines and USP. The parameters like capacity factor, tailing factor, asymmetry factor and number of theoretical plate and resolution were calculated. Mobile Phase: The mobile phase used for RP HPLC was Acetonitrile:Phosphate Buffer pH 3.0 (60:40 v/v)
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ISSN: 2320 3471(Online) Revanth and Aravind Indian Journal of Research in Pharmacy and Biotechnology RESULTS AND DISCUSSION UV spectrophotometric methods Simultaneous equation method: The overlaid spectra of 10 g mL-1 Olmesartan medoxomil and Atorvastatin calcium were recorded as shown in Figure 1. From the spectrum, 256.5 nm was max of Olmesartan medoxomil and 247 nm was max of Atorvastatin calcium, these two wavelengths used for Simultaneous estimation of Olmesartan medoxomil and Atorvastatin calcium respectively. Different aliquots of Olmesartan medoxomil in methanol were prepared in the concentration range of 8 40 g mL-1. The absorbances of solutions were measured at 256.5 nm and 247nm. The calibration curve was plotted using concentration against absorbance. The optical parameters like, Sandells sensitivity, Molar absorptivity, correlation coefficient, slope, intercept, LOD, LOQ and Standard error were calculated. The correlation coefficient for the two drugs was found to be above 0.999. This indicates that all the drugs obey Beers law in the selected concentration range. Hence the concentrations were found to be linear. The formulated tablets containing 20mg of Olmesartan medoxomil and 10mg of Atorvastatin calcium was selected for analysis. The nominal concentration of Olmesartan medoxomil from linearity (16 g mL-1) was prepared and also contains 8 g mL-1 of Atorvastatin calcium; the absorbances of the solution were measured at their respective wavelengths. The percentage label claim present in tablet formulation was found to be 100.187 1.074 and 101.511 1.841 for Olmesartan medoxomil and Atorvastatin calcium, respectively. The amount present in tablet formulation was in good concord with the label claim and the % RSD values were found to be 1.079 and 1.841 for Olmesartan medoxomil and Atorvastatin calcium, respectively. The low % RSD values indicate that the method has good precision. The results of analysis are shown in Table 2. The % RSD value of intraday and inter day analysis were found to be and 0.9754 and 0.9747 for Olmesartan medoxomil, 1.8560 and 1.3925 for Atorvastatin calcium. The results showed that the precision of the method was confirmed. The developed method was validated for Ruggedness. The % RSD value by analyst 1 and analyst 2 were found to be 0.714431 and 0.682101 for Olmesartan medoxomil and 0.52293 and 1.618648 for Atorvastatin calcium, respectively. The low % RSD values indicate that the developed method was more rugged. The accuracy of the method was performed by recovery studies. The percentage recovery was found to be in the range of 99.744 100.33% for Olmesartan medoxomil and 99.6109 100.7005% for Atorvastatin calcium. The low % RSD value for the two drugs indicates that this method is very accurate. Derivative spectrophotometric method: The zero order spectrums were derivatised into first order derivative spectrum. The overlaid first order derivative spectrum of 10 g mL-1 Olmesartan medoxomil and Atorvastatin calcium was recorded. At 310 nm, Olmesartan medoxomil has zero absorbance. At 247 nm, Atorvastatin calcium has zero absorbance value. Hence these two wavelengths were selected for the analysis of Olmesartan medoxomil and Atorvastatin calcium, respectively. Different aliquots of these solutions were measured at 247 nm and 310 nm in the first order derivative spectrum for Olmesartan medoxomil and Atorvastatin calcium, respectively. The plotted graphs are shown in Figure 2. The optical parameters like, Sandells sensitivity, Molar absorptivity, correlation coefficient, slope, intercept, LOD, LOQ and Standard error were calculated for the two drugs. The correlation coefficient for the two drugs was found to be above 0.999. This indicates that the two drugs obey Beers law in the selected concentration range. Hence the concentrations were found to be linear. The results are shown in Table 3. Formulated tablets containing 20mg of Olmesartan medoxomil and 10mg of Atorvastatin calcium was selected for analysis. The solution contains 16 g mL-1 of Olmesartan medoxomil was prepared (nominal concentration in the calibration curve of Olmesartan medoxomil), which is also contains 8 g mL-1 of Atorvastatin calcium, the absorbance of these solutions were measured at 247 nm and 310 nm. The amount of six test solutions was determined. The percentage label claim present in tablet formulation was found to be 100.254 1.6281 and 99.968 0.000054 for Olmesartan medoxomil and Atorvastatin calcium, respectively. The amount present in tablet formulation was in good concord with the label claim and the % RSD values were found to be 1.6282 and 0.000054 for Olmesartan medoxomil and Atorvastatin calcium, respectively. The low % RSD values indicate that the method has good precision. Further the precision of the method was confirmed by Intraday and Inter day analysis. The % RSD value of Intraday and Inter day analysis are 0.8736 and 0.1491 for Olmesartan medoxomil, 0.0411 and 0.4130 for Atorvastatin calcium, respectively. The results showed that the precision of the method was further confirmed.
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ISSN: 2320 3471(Online) Revanth and Aravind Indian Journal of Research in Pharmacy and Biotechnology The developed method was validated for Ruggedness. The % RSD value by analyst 1 and analyst 2 were found to be 1.285006 and 1.061925 for Olmesartan medoxomil and 0.045865 and 0.04941 for Atorvastatin calcium, respectively. The low % RSD values indicate that the developed method was more rugged. The accuracy of the method was performed by recovery studies. To the preanalyzed formulation, a known quantity of Olmesartan medoxomil and Atorvastatin calcium raw material solutions were added at different levels. The absorbance of the solutions was measured and the percentage recovery was calculated. The percentage recovery was found to be in the range of 98.776 - 99.1912% for Olmesartan medoxomil, and 101.225 101.987% for Atorvastatin calcium. The low % RSD value for the two drugs indicates that this method is very accurate. The recovery data is shown in Table 4. RP HPLC Method: The solutions of 10 g mL-1 of Olmesartan medoxomil and Atorvastatin calcium in mobile phase [Acetonitrile:Phosphate buffer pH 3.0 (60:40% v/v)] were prepared and the solutions were scanned in the range of 200 400 nm. It was found that the two drugs have marked absorbance at 252 nm and can be effectively used for estimation of two drugs without interference Acetonitrile: Phosphate buffer pH 3.0 with the ratio of 60:40% v/v at a flow rate of 0.9 mL/min was selected and the optimized chromatogram is shown in Figure 3. The retention time of Olmesartan medoxomil and Atorvastatin calcium, were found to be 3.19 and 4.63, respectively. The retention time between two drugs indicate that the drugs were separated with better resolution of 2.6181 Olmesartan medoxomil and Atorvastatin calcium. The system suitability parameters for the optimized chromatogram are shown in Table 5 Olmesartan medoxomil and Atorvastatin calcium were prepared in the concentration range of 8 - 40 g mL-1 of Olmesartan medoxomil, 4 20 g mL-1 of Atorvastatin calcium. 20 L of each solution was injected and recorded the chromatograms at 252 nm. The correlation co-efficient was found to be above 0.999 for the two drugs. The calibration graph of Olmesartan medoxomil and Atorvastatin calcium are shown in Figure 23 and 24, respectively. The optical characteristics of Olmesartan medoxomil and Atorvastatin calcium are shown in Table 6 Formulated tablets containing 20mg of Olmesartan medoxomil and 10mg of Atorvastatin calcium was selected for analysis. The ostensible concentration 16 g mL-1 of Olmesartan medoxomil, which is also contains 8 g mL-1 of Atorvastatin calcium in the mobile phase was prepared. 20 L of each solution was injected and chromatograms were recorded. The percentage purity was found to be 101.12 0.8107 and 101.83 0.4684 for Olmesartan medoxomil and Atorvastatin calcium, respectively. The precision of the method was confirmed by repeatability of formulation for six times and the chromatograms are shown in Figure 25 - 30. The % RSD was found to be 0.8107 and 0.4684 for Olmesartan medoxomil and Atorvastatin calcium, respectively. It indicates that the method has good precision. The data is shown in Table 7. The accuracy of the method was performed by recovery studies. To the preanalyzed formulation, a known quantity of Olmesartan medoxomil and Atorvastatin calcium raw material solutions were added at different levels, injected the solutions. The chromatograms were recorded as shown in the Figure 4. The percentage recovery was found to be in the range between 99.95 100.70 % for Olmesartan medoxomil and 99.8578 100.54% for Atorvastatin calcium. The % RSD was found to be 1.5880 and 1.7550 for Olmesartan medoxomil and Atorvastatin calcium, respectively. The low % RSD values for recovery indicated that the method was found to be accurate. All the above parameters with the ease of operation ensure that the projected methods could be applied for the routine analysis of Olmesartan medoxomil and Atorvastatin calcium in pure form and in tablet dosage forms.
ATC OLM
Figure.1.Overlaid absorption spectra of Olmesartan Medoxomil and Atorvastatin calcium in methanol
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ATC
OLM
Figure.2.Overlaid first order derivative UV spectrum of Olmesartan calcium in methanol
medoxomil and Atorvastatin
Figure.3.Optimized chromatogram for olmesartan medoxomil and atorvastatin calcium
Figure.4.Chromatogram for 100% recovery of formulation
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ISSN: 2320 3471(Online) Revanth and Aravind Indian Journal of Research in Pharmacy and Biotechnology Table.1.Optical characteristics of Atorvastatin calcium by simultaneous equation method Parameters Beers law limit (g/ml) Molar absorptivity (L mol-1 cm-1) Sandells sensitivity (g/cm2/0.001 A.U) Correlation coefficient (r) Regression equation (y=mx+c) Slope (m) Intercept (c) LOD (g/ml) LOQ (g/ml) Standard Error At 247 nm* 4-20 21421.7446 0.026016 0.99984 y=0.03844x+0.026014 0.03844 0.026014 0.1691342 0.5125324 0.004836 *Mean of six observations Table.2.Analysis of tablet formulation by simultaneous equation method Sample Labeled Amount Percentage Average S.D % R.S.D. No. amount found Obtained* (%) * (mg/tab) (mg/tab) 1 20 20.1988 100.99 2 20 19.9486 99.74 3 20 20.2929 101.46 100.187 1.081 1.079 4 20 19.6817 98.415 5 20 19.9853 99.92 6 20 20.1206 100.60 1 10 10.35 103.5 2 10 10.0972 100.97 3 10 10.4490 102.49 4 10 9.8252 98.25 101.511 1.8693 1.841 5 10 10.1132 101.13 6 10 10.2730 102.73 *Mean of six observations At 256.5 nm* 4-20 19611.5634 0.028514 0.99980 y=0.03507x+0.00025 0.03507 0.00025 0.3070915 0.093058 0.005138
Drug
S.E.
0.36154
OLM
ATC
0.0519
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ISSN: 2320 3471(Online) Revanth and Aravind Indian Journal of Research in Pharmacy and Biotechnology Table.3.Optical characteristics of Olmesartan medoxomil and Atorvastatin calcium by first order derivative spectrophotometric method
Parameters Beers law limit (g/ml) Molar absorptivity (L mol-1 cm-1) Sandells sensitivity (g/cm2/0.001 A.U) Correlation coefficient (r) Regression equation (y=mx+c) Slope (m) Intercept (c) LOD (g/ml) LOQ (g/ml) Standard Error Olmesartan medoxomil at 247nm* 8-40 857.8262 1.404208 0.9997 y=0.00071x+0.00005396 0.00071 0.00005396 0.11574329 0.35078643 0.000206 Atorvastatin calcium at 310nm* 4-20 331.2159 2.489676 0.9997 y=0.00040x+0.00000285 0.00040 0.00000285 0.19495642 0.59061364 0.0000112
*Mean of six observations Table .4.Quantification of formulation by first order derivative spectrophotometric method
Drug Sample No. 1 2 3 4 5 6 1 2 3 4 5 6 Labeled amount (mg/tab) 20 20 20 20 20 20 10 10 10 10 10 10 Amount found* (mg/tab) 20.1628 20.5152 19.9867 20.3390 19.8160 20.6913 9.9967 9.9987 9.9987 9.9967 9.9987 9.9967 Percentage Obtained* 100.8140 102.5761 99.9338 101.6953 99.05 103.4569 99.9676 99.9686 99.9686 99.9676 99.9686 99.9676 Average (%) S.D % R.S.D S.E
0LM
101.254
1.6486
1.6281
0.04579
ATC
99.968
0.0005
0.000054
0.000015
*Mean of six observations Table .5.System suitability parameters for the optimized chromatogram by RP - HPLC
Parameters Tailing factor Asymmetrical factor Theoretical plates Capacity factor Theoretical plate per unit Length Resolution Olmesartan medoxomil 1.21 1.29 5841 1.26 5841.63 2.6181 Arovastatin calcium 1.05 1.02 5666 1.96 5666.27
*Mean of three observations
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ISSN: 2320 3471(Online) Revanth and Aravind Indian Journal of Research in Pharmacy and Biotechnology Table .6.Optical characteristics of Olmesartan medoxomil and Atorvastatin calcium by RP HPLC Parameters max (nm) Beers law limit (g/ml) Correlation coefficient Regression equation (y=mx+c) Slope (m) Intercept LOD (g/ml) LOQ (g/ml) Standard Error Olmesartan medoxomil* 252 8 -40 0.9997 y= 523755.4929x + (111875.52) 523755.4929 -111875.52 0.1084768 0.571141911 552416.5435 *Mean of three observations Table .7. analysis of tablet formulation by RP-HPLC Drug Sample Labeled Amount Percentage Average (%) S.D % R.S.D. No. amount found Obtained* (mg/tab) (mg/tab)* 1 20 20.1667 100.8335 2 20 20.3334 101.66717 OLM 3 20 19.9456 99.728 101.12 0.8199 0.8107 4 20 20.3552 101.7764 5 20 20.375 101.8626 6 20 20.1750 100.8753 1 10 10.1851 101.85139 2 10 10.2288 102.2885 3 10 10.1415 101.4517 101.83 1.2479 1.2254 ATC 4 10 10.3365 103.3631 5 10 9.9653 99.6532 6 10 10.2417 102.4171 Atorvastatin calcium* 252 4 20 0.9998 y=481171.0167 x + (40954.66667) 481171.0167 -40954.66667 0.26788817 0.84208537 247882.9652
S.E.
0.02277
0.03466
*Mean of six observations SUMMARY AND CONCLUSION Simple, rapid, precise and accurate UV Spectrophotometric method and RP-HPLC method were developed and validated for the estimation of Olmesartan medoxomil and Atorvastatin calcium in tablet dosage form. Hence it is suggested that the proposed UV Spectroscopic and an isocratic RP-HPLC methods can be effectively applied for the routine analysis of Olmesartan medoxomil and Atorvastatin calcium in bulk and in tablet formulations.
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Altuntas a T.G, Krk B N, Liquid Chromatographic Determination of Atorvastatin in Bulk Drugs, Tablets and human plasma. Journal of Liquid Chromatography, 2004, 27(1), 83 93 Anonymous, Elico Instruction manual Pharamaspec SL 210 series operation guide, Elico Limited, Hyderabad, India, 2009, C-2. Anonymous, Martendale, 34th edition, The complete Drug Reference, Pharmaceutical Press, publication Division Of The Royal Pharmaceutical Society Of Great Britain, 2009, 975. Anonymous. Shimadzu Instruction Manual AX-200 Digital balance. Shimadzu Corporation, Kyoto, Japan, 2001, 42. Anonymous. Shimadzu Instruction manual Pharamaspec UV-1700 series operation guide. Shimadzu Corporation, Kyoto, Japan, 2001, 6.2. Anonymous. Shimadzu LC-10 ATVP High performance liquid chromatography Instruction Manual. Shimadzu Corporation, Kyoto, Japan, 2001, 11-2. Anonymous. Shimadzu SPD-10 ATVP High performance liquid chromatography Instruction Manual. Shimadzu Corporation, Kyoto, Japan, 2001, 11-2. Anonymous. The Indian Pharmacopoeia. The Indian Pharmacopoeia commission, Ghaziabad, 2007, Volume I, 751. Anonymous. The Merck Index. 14th edition, Merck research Laboratories, Merck & co., White House station, NJ, USA, 2006, 864, 6839. Anonymous. The United States Pharmacopoeia. 22nd Revision United States of Inc., Rock villa, MC, USA, 1995, 1776 -1777, 1983. Pharmacopoeia Convention,
Beckett A. H., and Stenlake J. B. Practical Pharmaceutical Chemistry. 4 th edition, CBS Publishers and Distributors, New Delhi, 2007, 278 - 300, 307 - 312. Bharat G. Chaudari., Ashok B. Patel. Simultaneous Spectrophotometric Estimation of Atorvastatin calcium and Amodipine Besylate in Tablet Dosage Form. International Journal of ChemTech Research, 2010, 2(1), 633 639. Bhusari K. P., Khedekar P. B., Seema Dhole and Banode V. S. Derivative and Q-Analysis Spectrophotometric Methods for Estimation of Hydrochlorothiazide and Olmesartan Medoxomil in tablets. Indian Journal of Pharmaceutical Sciences, 2009, 71(5), 505 508. Celebier M., Altinoz S. Determination of Olmesartan Medoxomil in tablets by UV- Spectroscopy. Govi Verlag, 2007, 10(6), 419 422. Chaudhari B. G., Patel N.M., Shah P.B. and Modi L.P. Development and Validation of a HPTLC Method for the Simultaneous Estimation of Atorvastatin Calcium and Ezetamide. Indian Journal of Pharmaceutical Sciences , 2006, 68(6), 793 797.
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DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR THE DETERMINATION OF CEFDINIR IN BULK AND CAPSULE DOSAGE FORM P.Ravisankar*1, 2, G.DevalaRao3, M.KrishnaChaitanya1 1 Department of Pharmaceutical Analysis and Quality Assurance, Vignan Pharmacy College, Guntur 522213, A.P., India. 2 Faculty of Science, Sri Chandrasekharendra Saraswathi Viswa Maha Vidyalaya (SCSVMV University), Enathur, Kanchipuram 631561, T.N., India. 3 Department of Pharmaceutical Analysis, KVSR Siddhartha College of Pharmaceutical Sciences, Vijayawada, A.P., India. *Corresponding Author: E-mail: banuman35@gmail.com, Mobile: +91-9059994000. ABSTRACT A simple, specific, accurate, rapid, inexpensive isocratic Reversed Phase-High Performance Liquid Chromatography (RP-HPLC) method was developed and validated for the quantitative determination of Cefdinir in pharmaceutical capsule dosage forms. RP-HPLC method was developed by using Welchrom C18 Column (4.6 X 250mm, 5m), Shimadzu LC-20AT Prominence Liquid Chromatograph. The mobile phase composed of10mM Phosphate buffer (pH-3.0, adjusted with triethylamine): acetonitrile (50:50v/v).The flow rate was set to 1.0 mL.min-1 with the responses measured at 235nm using Shimadzu SPD-20A Prominence UV-Vis detector. The retention time of Cefdinir was found to be 2.523 min. Linearity was established for Cefdinir in the range of 2-10g.mL-1 with correlation coefficient 0.999. The validation of the developed method was carried out for specificity, linearity, precision, accuracy, robustness, limit of detection, limit of quantitation. The developed method can be used for routine quality control analysis of Cefdinir in pharmaceutical capsule dosage form. KEY WORDS- Cefdinir, Isocratic RP-HPLC, UV-Vis detector, Method Validation. 1. INTRODUCTION Cefdinir is chemically 8-[2-(2-amino-1,3-thiazol-4-yl)-1-hydroxy-2-nitroso-ethenyl]amino-4-ethenyl7-oxo-2-thia-6-azabicyclo[4.2.0]oct-4-ene-5-caroxylic acid (Fig. 1). Cefdinir belongs to therapeutic class of 3rd generation of cephalosporins. It is generally used for acquired pneumonia, otitis media, pharyngitis, tonsillitis, lower respiratory tract infections, acute maxillary sinusitis, switch therapy, acute exacerbation of chronic bronchitis, uncomplicated skin and skin structure infections (Drug Today, 2012). Cefdinir is bactericidal and have the same mode of action as other beta-lactam antibiotics (such as penicillins) but are less susceptible to penicillinases. Cefdinir disrupt the synthesis of the peptidoglycan layer of bacterial cell wall synthesis. The peptidoglycan layer is important for cell wall structural integrity. The final transpeptidation step in the synthesis of the peptidoglycan is facilitated by transpeptidases known as penicillin-binding proteins (PBPs). PBPs bind to the D-Ala-D-Ala at the end of muropeptides (peptidoglycan precursors) to crosslink the peptidoglycan. Cefdinir mimic the D-Ala-D-Ala site, thereby competitively inhibiting PBP crosslinking of peptidoglycan. Literature survey reveals that for the determination of Cefdinir and its related substances in biological fluids like plasma, blood, urine and pharmaceutical dosage forms by spectrophotometry (Gouda A, 2012) (Shah P.B, 2006), High Performance Liquid Chromatography (Li J, 2012) (Khan A, 2011) (Rao KV, 2007) (Okamoto Y, 1996) (Narala S, 2011) (Mashelkar U.C, 2010) (Hamrapurkar P, 2011) (Hashem H, 2012), Electrochemical(Jain R,2007), Complexation (Babita K.S., 2010) methods. However, very few analytical methods were reported in literature for the determination of cefdinir in bulk and pharmaceutical dosage forms. The reported HPLC methods so far in the literature are considered to be uneconomical, time consuming and have poor symmetry. In fact there is a need for the development of a novel, simple, rapid, efficient RP-HPLC analytical method with reproducibility for determination of cefdinir in bulk and pharmaceutical dosage forms. The present study illustrates development and validation of a novel, simple, rapid and efficient RP-HPLC analytical method with reproducibility for determination of cefdinir in bulk and pharmaceutical tablet dosage form. The established method was validated with respect to specificity, linearity, precision, accuracy, robustness, LOD and LOQ according to ICH guidelines (ICH, 1997). 2. MATERIALS AND METHODS Chemicals and Reagents: The reference sample of Cefdinir standard was kindly supplied as gift sample by Hetero Drugs Ltd., Hyderabad, Andhra Pradesh, India. All the chemicals were analytical grade. Potassium
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dihydrogen orthophosphate and phosphoric acid from Rankem Ltd., Mumbai, India, while acetonitrile (HPLC grade) and triethylamine (HPLC grade) from Merck Pharmaceuticals Private Ltd., Mumbai, India. Ortho phosphoric acid used was of HPLC grade and purchased from Merck Specialties Private Ltd., Mumbai, India. Commercial capsules of Cefdinir formulation was procured from local market. OMNICEF capsules 300mg are manufactured by Abbot Laboratories Pvt. Ltd., India. Instruments: Quantitative HPLC was performed on a isocratic high performance liquid chromatograph (Shimadzu LC-20AT Prominence Liquid Chromatograph) with a LC-20AT VP pump, manual injector with loop volume of 20 L (Rheodyne), programmable variable wavelength Shimadzu SPD-20A Prominence UVVis detector and Welchrom C18 Column (4.6 X 250mm, 5m particle size). The HPLC system was equipped with Spinchrom software. In addition an electronic balance (Shimadzu TX223L), digital pH meter (Systronics model 802), a sonicator (spectra lab, model UCB 40), UV-Visible Spectrophotometer (Systronicsmodel-2203) were used in this study. Chromatographic conditions: Cefdinir was analyzed by various reversed phase columns like C8and C18 columns. Among C8 and C18 columns, C18 (250mmX4.6mm, 5m) column was selected. Various combinations of acetonitrile, phosphate buffer and methanol with triethylamine as column modifier were tested. The mixture of 10mM Phosphate buffer (pH adjusted to 3.0 using triethylamine) and Acetonitrile in ratio of 50:50 v/v was selected as mobile phase and UV detection wavelength was 235nm with a flow rate of 1mL.min-1. Injection volume was 20L, with ambient temperature, run time was 6min. and retention time was 2.523min. The resulting HPLC chromatogram was shown in Fig. 3. Preparation of mobile phase: A 10mM Phosphate buffer was prepared by dissolving 6.056 g of potassium dihydrogen orthophosphate in 445 mL of HPLC grade water. To this 55mL of 0.1M phosphoric acid was added and pH was adjusted to 3.0 with triethylamine. The above prepared buffer and acetonitrile were mixed in the proportion of 50:50 v/v and was filtered through 0.45 m nylon membrane filter and degassed by sonication. Preparation of Standard solution: About 100 mg of pure Cefdinir was accurately weighed and dissolved in 100 mL of mobile phase at to get 1 mg.mL-1 stock solution. Working standard solution of Cefdinir was prepared with mobile phase. The final volume was made with the mobile phase. The standard solution was filtered through 0.45 m nylon membrane filter and degassed by sonication. Preparation of Sample solution: The content of 20 capsules of Cefdinir (OMNICEF) were accurately weighed and transferred into a mortar and triturated to a fine powder. From this, capsule powder which is equivalent to 100 mg of Cefdinir was taken and the drug was extracted in 100 mL of mobile phase. The resulting solution was filtered using Whatman Grade No.1 filter paper and degassed by sonication. This solution was further suitably diluted for chromatography. Selection of detection wavelength: The UV spectra of various diluted solutions of Cefdinir in mobile phase were recorded using UV spectrophotometer. The peak of maximum absorbance was observed at 235nm. This wavelength was used for detection of Cefdinir. Calibration curve for Cefdinir: Replicates of each calibration standard solutions (2, 4, 6, 8, 10 g.mL-1) were injected using a 20L fixed loop system and the chromatograms were recorded. Calibration curves were constructed by plotting concentration of Cefdinir on X-axis and peak areas of standard Cefdinir on Y-axis and regression equations were computed for Cefdinir. The calibration data is presented in Table 3. 3. VALIDATION OF THE PROPOSED METHOD The developed method of analysis was validated as per the ICH for the parameters like system suitability, specificity, linearity, precision, accuracy, robustness and system suitability, limit of detection (LOD) and limit of quantitation (LOQ). System suitability: System suitability tests are an integral part of chromatographic method which was used to verify reproducibility of the chromatographic system. To ascertain its effectiveness, certain system suitability test parameters were checked by repetitively injecting the drug solution at the concentration level 10g.mL-1 for Cefdinir to check the reproducibility of the system. At first the HPLC system was stabilized for 40 min. One blank followed by six replicates of a single calibration standard solution of Cefdinir was injected to check the system suitability. To ascertain the systems suitability for the proposed method, the parameters such as theoretical plates, peak asymmetry, retention time and parameters were taken and results were presented in Table 1.
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Specificity: The effect of wide range of excipients and other additives usually present in the formulations of Cefdinir in the determinations under optimum conditions was investigated. The specificity of the RP-HPLC method was established by injecting the mobile phase and placebo solution in triplicate and recording the chromatograms. The common excipients such as lactose anhydrous, microcrystalline cellulose, purified talc and magnesium stearate have been added to the placebo solution and injected and tested. The representative chromatogram of placebo was shown in Fig. 2. The specificity results were presented in Table 5. Linearity: The linearity graphs for the proposed assay methods were obtained over the concentration range of 2-10 g.mL-1of Cefdinir. Method of least square analysis was carried out for getting the slope, intercept and correlation coefficient, regression data values and the results were presented in Table 2 and Table 3. The representative chromatograms indicating the Cefdinir were shown in Fig. 5 to 9. A calibration curve was plotted between concentration and area response and statistical analysis of the calibration curve is shown in Fig. 10. Precision: Intra-day and inter-day precision study of Cefdinir was carried out by estimating corresponding responses 3 times on the same day and on 3 different days for the concentration of 10g. The percent relative standard deviation (% RSD) was calculated which is within the acceptable criteria of not more than 2.0. The results for intra-day and inter-day precision were presented in Table 6 and Table 7 respectively. Accuracy (Recovery studies): The accuracy of the method was determined by calculating recovery of Cefdinir by the method of addition. Known amount of Cefdinir at 50%, 100% and 150% was added to a pre quantified sample solution. The recovery studies were carried out in the tablet in triplicate each in the presence of placebo. The mean percentage recovery of Cefdinir at each level was not less than 99% and not more than 101%. The results were presented in Table 8. Robustness: The Robustness was evaluated by the analysis of Cefdinir under different experimental conditions such as making small changes in flow rate ( 0.2 ml/min), detection wavelength (5 nm) and Mobile phase composition (5%). The results were presented in Table 9. LOD and LOQ: Limit of Detection is the lowest concentration in a sample that can be detected, but not necessarily quantified under the stated experimental conditions. The limit of quantitation is the lowest concentration of analyte in a sample that can be determined with acceptable precision and accuracy. Limit of Detection and Limit of Quantitation were calculated using following formula LOD= 3.3(SD)/S and LOQ= 10 (SD)/S, where SD=standard deviation of response (peak area) and S= slope of the calibration curve. The LOD and LOQ values are presented in Table 10. 4. RESULTS AND DISCUSSION The mobile phase consisting of 10mM phosphate buffer (pH-3.0): acetonitrile (50:50% v/v at1mL.min-1 flow rate was optimized which gave sharp peak, minimum tailing factor with short runtime for Cefdinir. The retention time for Cefdinir was 2.523min. UV spectra of Cefdinir showed that the drug absorbed maximum at 235 nm, so this wavelength was selected as the detection wavelength. System suitability parameters &optimized chromatographic conditions are shown in Table1.The calibration curve for Cefdinir was found to be linear over the range of 2-10g.mL-1. The data of regression analysis of the calibration curve is shown in Table 2 and Table 3. The developed method was applied to the assay of Cefdinir capsules. The experimental results are given in Table 4. The results were very close to labeled value of commercial capsules. The representative standard and sample chromatograms of Cefdinir are shown in Fig. 3 and 4 respectively. The regression equation was found to be Y=121.5x + 4.2674 with correlation coefficient is r2=0.999which indicates this method has good linearity. The representative chromatograms indicating the Cefdinir are shown in Fig. 5 to 9. The linearity of the graph is shown in Fig.10.The specificity was studied for the examination of the presence of interfering components, while the comparison of chromatograms there was no interference from placebo (Fig 2) with sample peak. They do not disturb the elution or quantification of Cefdinir, furthermore the well-shaped peaks also indicate the specificity of the method. Therefore, it was concluded that the method is specific. The specificity results are summarized in Table 5. Precision was studied to find out intra-day and inter-day variations in the test methods of Cefdinir for the three times on the same day and different day. The intra-day and inter-day precision obtained was %RSD (<2.0) indicates that the proposed method is quite precise and reproducible and results are shown in Tables6 and 7.Recovery studies of the drug were carried out for the accuracy parameter at three different concentrations levels i.e., multiple level recovery studies. A known amount of Cefdinir standard was added into pre-analyzed sample and subjected them to the
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proposed HPLC method. The % recovery was found to be within the limits as listed in Table 8.Generally the mean percentage recovery of Cefdinir at each level was not less than 99% and not more than 101%. In this case percentage recovery of Cefdinir was found to be in the range of 99.73 % to 100.26%.The method precision was done and the low % RSD (0.706) values indicates that the proposed method which was in good agreement with precision. Robustness was done by small changes in the chromatographic conditions like mobile phase flow rate, temperature, mobile phase composition etc., It was observed that there were no marked changes in the chromatograms. Infact the parameters are within the limit which indicates that the method has robustness and suitable for routine use. The Robustness results are presented in Table 9. The limit of detection (LOD) and limit of quantitation (LOQ) was calculated based on the standard deviation (SD) of the response and the slope (S) of the calibration curve at levels approximating the LOD and LOQ. The limit of detection (LOD) was 0.067045 g.mL-1 and the limit of quantitation (LOQ) was 0.203160 g.mL-1 which shows that this method is very sensitive. The results are presented in Table 10. 5. CONCLUSION A New validated RP-HPLC method has been developed for the quantitative determination of Cefdinir in bulk and pharmaceutical tablet dosage forms. Statistical analysis of the results shows that the proposed procedure has good precision and accuracy. The method was completely validated shows satisfactory results for all the method validation parameters tested and method was free from interference of the other active ingredients and additives used in the formulation. In fact, results of the study indicate that the developed method was found to be simple, reliable, accurate, linear, sensitive, economical, and reproducible and have short run time which makes the method rapid. Hence it can be concluded that this method may be employed for the routine quality control analysis of Cefdinir in active pharmaceutical ingredient (API) and pharmaceutical capsule preparations. 6. ACKNWOLEDGEMENT The authors thank to Hetero Labs Limited, Jeedimetla, Hyderabad for providing Cefdinir as gift sample for this work. They also thank chairman Dr.L.Rathaiah, Vignan Pharmacy College for providing necessary facilities to carry out this research work. Table 1: Optimized chromatographic conditions and system suitability parameters for proposed HPLC method for Cefdinir Parameter Chromatographic conditions Instrument SHIMADZU LC-20AT prominence liquid chromatograph Column WELCHROM C18 Column (4.6 X 250mm, 5m) Detector Diluents Mobile phase Flow rate Detection wave length Run time Column back pressure Temperature Volume of injection loop Retention time (Rt) Theoretical plates[th.pl] (Efficiency) Theoretical plates per meter[t.p/m] Tailing factor (asymmetry factor) SHIMADZU SPD-20A prominence UV-Vis detector 10mM Phosphate Buffer(pH-3): Acetonitrile (50:50 v/v) 10mM Phosphate Buffer(pH-3): Acetonitrile (50 : 50 v/v) 1mL.min-1. By UV at 235nm. 6 minutes 128-130(kgf) Ambient temperature(25oC) 20L 2.523min 6,001 120026 1.054
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Table 2: Linear regression data of the proposed HPLC method of Cefdinir Parameter Method Detection wavelength( max) By UV at 235nm Linearity range (g/ml) 2-10g.mL-1 Regression equation (Y=a+bx) Y=4.2674+121.5X Slope(b) 121.5 Intercept(a) 4.2674 Standard deviation of slope (Sb) 0.390288987 Standard deviation of intercept (Sa) 2.588884259 Standard error of estimation (Se) 2.468404289 Correlation coefficient (r2) 0.999 % Relative standard deviation* i.e., 1.054274126 Coefficient of variation(CV) Percentage range of errors* (Confidence limits) 0.005significance level 1.473353832 0.001 significance level 2.311061734 * Average of 6 determinations; Acceptance criteria < 2.0. Table 3: Calibration data of the proposed HPLC method of Cefdinir Concentration, g.mL-1. Retention time,(Rt) min. Peak area, mV.s. 0 0 2 2.523 249.538 4 2.527 494.096 6 2.527 732.446 8 2.527 978.724 10 2.523 1215.834 Slope 121.5 Intercept 4.2674 Correlation Coefficient [CC] (r) 0.999984358 Squared CC (R2) 0.999968717 Residual sum of squares 18.2790592 Table 4: Assay results of Cefdinir formulation Formulations OMNICEF (Abbot Laboratories Pvt. Ltd.,) Labeled amount 300mg Amount found 298.20mg % Assay RSD* 99.481.4733
S.No 1. 2. 3. 4. 5. 6.
*Average of 6 determinations; SD is standard deviation. Table 5: Specificity study Name of the solution Mobile phase Placebo Cefdinir 10g.mL-1 Retention time (Rt) min. No peaks No peaks 2.523 min.
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Sample Cefdinir

Table 6: Results of Precision study (Intra-day) Concentration Injection no. Peak area (g.mL-1) 10 1 1215.834 2 1228.641 3 1207.974 4 1209.692 5 1218.048 6 1216.743 # Acceptance criteria < 2.0. Table 7: Results of Precision study (Inter-day) Concentration Injection no. Peak area (g.mL-1) 10 1 1202.880 2 1216.534 3 1212.348 4 1219.562 5 1214.824 6 1222.861 # Acceptance criteria < 2.0. %RSD(acceptance criteria< 2.0) 0.706146
Sample Cefdinir
%RSD (acceptance criteria< 2.0) 0.573550
Table 8: Recovery data of the proposed Cefdinir by RP-HPLC method Recovery level 80% Amount added (mg) 79.92 80.12 79.78 100.24 99.86 99.94 119.28 120.22 119.86 Amount Amount % Mean % %RSD# found recovered recovery Recovery (mg) (mg) SD 179.11 79.63 99.63 99.68 0.0438 0.043 179.35 79.87 99.68 179.04 79.56 99.72 199.52 100.04 99.80 99.82 0.0461 0.046 199.14 99.66 99.79 199.3 99.82 99.87 218.62 119.14 99.88 100.03 0.1332 0.133 219.85 120.37 100.12 219.46 119.98 100.10 *SD is standard deviation # % RSD is percentage of relative standard deviation. Table 9: Robustness results of Cefdinir Optimized Used Peak Retention Plate Peak area time(Rt), min count asymmetry 0.8 1284.634 2.946 6342 1.254 1.0 1.0 1219.462 2.523 6001 1.054 mL.min-1 1.2 1179.724 2.324 5924 1.083 230nm 1188.546 2.527 6016 1.132 235 nm 235nm 1219.462 2.523 6001 1.054 240nm 1156.826 2.524 6094 1.154 50:50v/v 55:45v/v 1202.203 2.628 6028 1.102 50:50v/v 1219.462 2.523 6001 1.054 45:55v/v 1210.432 2.420 5991 1.167 Total amount (mg) 179.4 179.6 179.26 199.72 199.34 199.42 218.76 219.7 219.34
100%
120%
Parameter
Flow rate (0.2mL.min-1) Detection wavelength (5nm) Mobile phase composition (5%)
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Table 10: Limit of Detection (LOD) and Limit of Quantitation (LOQ) Limit of Detection(LOD) 0.0670 g.mL-1 Limit of Quantitation(LOQ) 0.2031 g.mL-1 Fig. 1: Structure of Cefdinir Fig 2: Chromatogram of placebo
Fig. 3: A typical chromatogram of Cefdinir standard
Fig. 4: Chromatogram of market formulation (OMNICEF 300mg Capsules)of Cefdinir
Fig. 5: Standard chromatogram of Cefdinir (2 g.mL-1)
Fig. 6: Standard chromatogram of Cefdinir (4g.mL-1)
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Fig. 9: Standard chromatogram of Cefdinir (10g.mL-1)
Fig. 10: Calibration plot of Cefdinir
cefdinir
1400 1200 peak area, mV.s 1000 800 600 400 200 0 0 5 10 15 Series1 Linear (Series1) y = 121.5x + 4.2674 R = 0.999
concentration, ppm
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REFERENCES
Drug today medical journal, Lorina publication (India) Inc., Delhi-91, 2012, DT 78, 1,479. Gouda, A. A., Hashem, H., Hassan, W., Spectophotometric methods for determination of cefdinir in pharmaceutical formulations via derivatization with 1,2naphthoquinone 4sulfonate and 4chloro7nitrobenzo2oxa1, 3diazole. Drug Testing and Analysis, 4(12), 2012, 991-1000. Shah, P. B., Pundarikakshudu, K., Difference spectroscopic and reverse phase HPLC methods for the estimation of cefdinir in pharmaceutical dosage forms. Indian journal of pharmaceutical sciences, 68(1), 2006, 90-93. Li J, Wang L, Chen Z, Xie R, Li Y, Hang T, Fan G, Development and validation of a rapid HPLC method for the determination of cefdinir in beagle dog plasma integrated with an automatic on-line solid-phase extraction following protein precipitation in the 96-well plate format, J Chromatogr B Analyt Technol Biomed Life Sci., 83(8), 2012, 895-896. Khan A, Iqbal Z, Khan MI, Javed K, Khan A, Ahmad L, Shah Y, Nasir F, Simultaneous determination of cefdinir and cefixime in human plasma by RP-HPLC/UV detection method: Method development, optimization, validation, and its application to a pharmacokinetic study, J Chromatogr B Analyt Technol Biomed Life Sci., 879(24), 2011, 2423-2429. Rao KV, Rani A, Reddy AV, Bharathi CH, Dandala R, Naidu A., Isolation, structural elucidation and characterization of impurities in Cefdinir, J Pharm Biomed Anal., 43(4), 2007, 1476-1482. Okamoto Y, Itoh K, Namiki Y, Matsushita J, Fujioka M, Yasuda T., Method development for the determination of cefdinir and its related substances by high-Performance Liquid Chromatography J Pharm Biomed Anal., 14(6), 1996, 739-48. Narala, S. R., Saraswathi, K., RP-HPLC methods for the determination of cephalosporins (Cefditoren Pivoxil and Cefdinir) in pharmaceutical dosage forms. J Pharm Sci Res, 3, 2011, 1002-1004. Mashelkar, U. C., Renapurkar, S. D., A LC-MS Compatible Stability Indicating HPLC Assay Method for Cefdinir. Int. J. Chem. Tech. Res, 2, 2010, 114-121. Hamrapurkar, P., Patil, P., Phale, M., Gandhi, M., Pawar, S., A developed and validated stability-indicating reverse-phase high performance liquid chromatographic method for determination of cefdinir in the presence of its degradation products as per International Conference on Harmonization guidelines, Pharmaceutical Methods, 2(1), 2011, 15-20. Hashem, H., Gouda, A. A., Hassan, W., Development and Validation of a rapid Stability Indicating chromatographic Determination of Cefdinir in Bulk Powder and Dosage Form Using Monolithic Stationary Phase, Journal of Liquid Chromatography & Related Technologies, 35(12), 2012, 1638-1648. Jain, R., Radhapyari, K., Jadon, N., Electrochemical evaluation and determination of cefdinir in dosage form and biological fluid at mercury electrode. Journal of the Electrochemical Society, 154(11), 2007, F199-F204. Babita K.S., Srivastava D.V., Seema S., Sudhir K., Selective and non-extractive spectrophotometric determination of cefdinir in formulations based on donor-acceptor complex formation, Qum. Nova., vol.33(7), 2010, 1471-1475. ICH, Harmonized Tripartite Guideline. Validation of Analytical Procedure: Methodology (Q2B). International Conference on Harmonization, 1997.
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ISSN: 2320 3471(Online) Ravisankar et.al. Indian Journal of Research in Pharmacy and Biotechnology SIMULTANEOUS SEPARATION OF SIX FLUOROQUINOLONES USING AN ISOCRATIC HPLC SYSTEM WITH UV DETECTION: APPLICATION TO ANALYSIS OF LEVOFLOXACIN IN PHARMACEUTICAL FORMULATIONS Ravisankar Panchumarthy*1,2, Devala Rao Garikapati3, Krishna Chaitanya Manukonda2, Sandhya Rani Nagabhairava2 1 Faculty of Science, Sri Chandrasekharendra Saraswathi Viswa Mahavidyalaya ( SCSVMV University), Enathur, Kanchipuram 631561 ( T.N.) India. 2 Department of Pharmaceutical Analysis and quality Assurance, Faculty, Vignan Pharmacy College, Vadlamudi, Guntur 522213 (A.P) india. 3 Department of Pharmaceutical Analysis, KVSR Siddhartha College of Pharmaceutical Sciences,Vijayawada (A.P) india. *Address for Correspondence: E-mail: banuman35@gmail.com, Mobile: 09059994000. ABSTRACT A simple, precise, specific, highly sensitive, an accurate and reproducible isocratic reversed phase HPLC method was developed for the separation of six fluoroquinolones and validated for determination of Levofloxacin in pure and in pharmaceutical formulations. One of the prominent aims for the method development is to achieve constant, reproducible separation. The selection of good method is essential if this goal is to be attained. RP-HPLC method was developed by using WELCHROM C18 Column (4.6 X 250mm, 5m), SHIMADZU LC-20AT prominence liquid chromatograph. The mobile phase consisting of phosphate buffer (pH-3.1) and acetonitrile in the proportion of 70:30 v/v under isocratic elution at a flow rate of 1mL/min was employed. The responses were measured at 293 nm using SHIMADZU SPD-20A prominence UV-Vis detector. The retention time of Levofloxacin was found to be 3.607 min. The developed method was according to ICH guidelines. The average recoveries ranged from 99.518 - 100.20%, with %RSD less than 2%. The concentration of calibration solutions was in the range of 2-10 g/mL and LOD and LOQ were 0.1234 and 0.3740 g/mL respectively. The method was successfully applied to analysis of Levofloxacin in pharmaceutical formulation. It can also be extended for the determination of other five fluoroquinolones or their combinations. KEYWORDS: Levofloxacin, prulifloxacin, gatifloxacin, sparfloxacin, moxifloxacin, balofloxacin 1.INTRODUCTION The quinolones are a family of synthetic broad-spectrum antibacterial drugs (P. Ravisankar, 2010) (Noura H. Abou-Taleb, 2011). The majority of quinolones in clinical have a fluorine atom attached to the central ring system, typically at the C6 or C7 position. Quinolones inhibit the topoisomerase II ligase domain, leaving the two nuclease domains intact. This modification, coupled with the constant action of the topoisomerase II in the bacterial cell, leads to DNA fragmentation. For many Gram-negative bacteria, DNA gyrase is the target, whereas topoisomerase IV is the target for many Gram-positive bacteria (Madhuri D, 2011) (Mahesh Attimarad, 2012) (Manisha Puranik, 2010). These six fluoroquinolones are broad spectrum antimicrobials with potent activity against Gram +ve and Gram ve bacteria. Levofloxacin is (S)-9-fluoro-2,3-dihydro-3-methyl-10-(4-methylpiperazin-1-yl)-7-oxo-7H-pyrido[1,2,3de]-1,4-benzoxazine-6-carboxylic acid (Fig. 1), a third-generation synthetic antibacterial agent of the fluoroquinolone class and is the S(-) enantiomer of the racemic drug substance Ofloxacin. It is used in the treatment of acute sinusitis, acute exacerbation of chronic bronchitis, community-acquired pneumonia and complicated urinary tract infection including pyelonephritis, skin and soft tissue infections. Literature survey revealed that very few methods have been reported for the analysis of various fluoroquinolones which include spectrophotometry(P. Ravisankar, 2010) (Noura H. Abou-Taleb, 2011) (Madhuri D, 2011) (Mahesh Attimarad, 2012), HPTLC (Manisha Puranik, 2010), RP-HPLC-UV (Ravisankar, 2013) (Samanidou, 2003) (Sun, 2008) (Shervington, 2005) (Du, 2003) (Espinosa-Mansilla, 2005), RP-HPLC with fluorescence detection (ICH, 1996). Several HPLC methods had been developed for determination of these drugs individually or in combination with other drugs but no HPLC method for simultaneous estimation of these six drugs using C 18 column with isocratic conditions has been reported till date. It can also be applied for routine analysis of either alone or of any combinations of the above mentioned drugs in dosage forms. 2. MATERIALS AND METHODS 2.1. Instruments and Chromatographic conditions: Chromatographic separations were achieved by using Shimadzu LC-20AT Prominence Liquid Chromatograph comprising a LC-20AT VP pump, Shimadzu SPD-20A Volume 1(2) March-April 2013 Page 264
ISSN: 2320 3471(Online) Ravisankar et.al. Indian Journal of Research in Pharmacy and Biotechnology Prominence UV-Vis detector and Welchrom C18 column (4.6 mm i.d. X 250 mm, 5 micron particle size). 20 L of sample was injected into the HPLC system. The HPLC system was equipped with Spinchrom data acquisition software. Separations were performed on the reversed phase column using a mobile phase consisting of phosphate buffer (pH-3.1) and acetonitrile in the proportion of 70:30 v/v. Triethylamine was used as column modifier. The mobile phase was delivered at a flow rate of 1 mL/min. Eluate was monitored at 293 nm. In addition, an electronic balance (Shimadzu TX223L), digital pH meter (Systronics model 802), a sonicator (spectra lab, model UCB 40), UV-Visible Spectrophotometer (Systronics model 2203) were used in this study. 2.2. Standards and chemicals used: Fluoroquinolone samples of Levofloxacin (LEV) and Gatifloxacin (GAT) were provided by Aristo Pharma, Prulifloxacin (PRF) and Balofloxacin (BLF) by Hetero Labs, Sparfloxacin (SPA) by Ananth Pharmaceuticals, and Moxifloxacin (MOX) by Torrent Pharmaceuticals. All chemicals were analytical grade. Potassium dihydrogen orthophosphate and phosphoric acid purchased from S.D Fine-Chem. Ltd., Mumbai, India. Acetonitrile (HPLC grade) and triethylamine (HPLC grade) are obtained from Merck Pharmaceuticals Private Limited, Mumbai, India. Commercial tablets of LEV were purchased from local market. GLEVO tablets containing 500mg of LEV are manufactured by Majesta (Glenmark) Pharmaceuticals Ltd., Mumbai, India. 2.3. Preparation of mobile phase: A 10 mM phosphate buffer was prepared was prepared by dissolving 6.056 g of potassium dihydrogen orthophosphate in 445 mL of HPLC grade water and adding 55ml of 0.1M phosphoric acid to make up the volume to 500mL. then pH was adjusted to 3.1 with triethylamime. The above prepared buffer and acetonitrile were mixed in the proportion of 70: 30 v/v and was filtered through 0.45 m nylon membrane filter and degassed by sonication. 2.4. Preparation of calibration standards: About 100 mg of pure LEV was accurately weighed and dissolved in 100 mL of mobile phase to get 1 mg/mL stock solution. Working standard solution of LEV was prepared with mobile phase. To a series of 10mL volumetric flasks, standard solutions of LEV in the concentration range of 2, 4, 6, 8, 10g/mL were transferred. The final volume was made with the mobile phase and similarly 10g/mL of each other standard fluoroquinolones were prepared from 1 mg/mL stock solutions of BLF, PRF, GAT, SPA and MOX respectively into each 10mL volumetric flask. 2.5. Calibration curve of Levofloxacin: Replicates of each calibration standard solutions 2, 4, 6, 8 and 10 g/mL were injected into the chromatograph, the retention times and average peak areas were recorded. Calibration graph was plotted by taking concentration of LEV on X-axis and peak areas of standard LEV on Y-axis (Fig. 11) and the regression equation data is computed (Table 2). The mixture of six fluoroquinolones was injected and the retention times, peak areas, asymmetry and efficiency values of six fluoroquinolones in mixture were presented in Table 9. The individual standard fluoroquinolones were injected and the individual experimental results of the six fluoroquinolones were described in Table 10. 2.6. Assay of Marketed Formulations of Levofloxacin: The content of twenty tablets was transferred into a mortar and ground to a fine powder. From this tablet powder a quantity equivalent to 100 mg of LEV was taken and the drug was extracted in 100 ml of mobile phase. The resulting solution was filtered through 0.25 m nylon membrane filter and degassed by sonication. This solution was further suitably diluted for chromatography. The test solutions were injected into the system by filling a 20 L fixed volume loop manual injector. The chromatographic run time of 6 min was maintained for the elution of the drug from the column. The elutes were monitored with UV detector at 293 nm. The amount of drug present in sample was computed from the calibration graph. The results were presented in Table 4. 3. METHOD VALIDATION The developed method of analysis was validated as per the ICH for the parameters like system suitability, specificity, linearity, precision, accuracy, robustness and system suitability, limit of detection (LOD) and limit of quantitation (LOQ). 3.1. System suitability: Set up the chromatographic system, allow the HPLC system to stabilize for 40 min. Inject blank preparation (single injection) and standard preparation (six replicates) and record the chromatograms to evaluate the system suitability parameters like resolution (NLT 2.0), tailing factor (NMT 1.5), theoretical plate count (NLT 3000) and % RSD for peak area of six replicate injections of LEV standard (%RSD NMT 2.0). If system suitability parameters are met, then inject sample (GLEVO) preparation in duplicate and record the chromatograms. Volume 1(2) March-April 2013 Page 265
ISSN: 2320 3471(Online) Ravisankar et.al. Indian Journal of Research in Pharmacy and Biotechnology 3.2. Specificity: The specificity of the proposed method was established by reviewing the effect of various excipients and other additives usually present in the preparations of LEV in the determinations under ideal conditions. The blank, standard, placebo, placebo spiked with analyte and test preparations were analyzed as per the method to examine the interference of blank and placebo with LEV peaks. The common excipients such as lactose anhydrous, microcrystalline cellulose, purified talc and magnesium stearate have been added to the placebo solution and injected and tested. Furthermore the well-shaped peaks also indicate the specificity of the method. The chromatogram for placebo indicating the specificity of developed method is presented in Fig. 2. 3.3. Linearity: Linearity for LEV was determined by fixing standard solutions at different concentrations from 50% to 150% of the test concentration. The linearity graphs for the suggested assay methods were acquired over the concentration range of 2-10 g/mL of LEV. Method of least square analysis was carried out for getting the slope, intercept and correlation coefficient, regression data values. A calibration curve was designed between concentration and peak area response and statistical analysis of the calibration curve was accomplished. 3.4. Precision: Intra-day and inter-day precision of the procedure were determined by performing six determinations at the same concentration (10g/mL) of LEV during the same day, un der the same experimental conditions and on a different day respectively. The percent relative standard deviation (% RSD) was calculated which is within the acceptable criteria of not more than 2.0. 3.5. Accuracy/Recovery: The accuracy of the method was assessed in triplicate at 3 different concentrations equivalent to 80%, 100% and 120% of the active ingredient, by adding a known amount of LEV standard to a sample with pre-determined amount of LEV. The recovered amount of LEV, %RSD of recovery, % recovery of each concentration is calculated to determine the accuracy. 3.6. Robustness: The Robustness of developed analytical method was proven by the analysis of LEV under different experimental conditions such as making deliberate changes in chromatographic conditions like flow rate ( 0.2 ml/min), detection wavelength (5 nm) and Mobile phase composition (5%). 3.7. LOD and LOQ: Limit of Detection is the lowest concentration in a sample that can be detected, but not necessarily quantified under the stated experimental conditions. The limit of quantitation is the lowest concentration of analyte in a sample that can be determined with acceptable precision and accuracy. Limit of Detection and Limit of Quantitation were calculated using following formula LOD= 3.3/S and LOQ= 10/S, where SD=standard deviation of response (peak area) and S= slope of the calibration curve. 4. RESULTS AND DISCUSSION The present study was designed to develop a rapid, accurate and precise HPLC method for the separation of six fluoroquinolones and subsequent determination of LEV in pharmaceutical dosage forms. In order to set up analysis of the component peaks under isocratic conditions, mixtures of phosphate buffer, acetonitrile in different combinations were tested as mobile phase on a C18 stationary phase. A combination of phosphate buffer (pH - 3.1, adjusted using o-phosphoric acid) and acetonitrile in a ratio of 70:30, v/v, and triethylamine as column modifier, was used as a mobile phase at a flow rate of 1mL/min was proved to be the most suitable of all combinations of mobile phase tried since the chromatographic peak obtained was well shape symmetrical peak. The wave length of detection was set at 293 nm from UV overlain spectra of drugs under study. The system suitability was carried out on freshly prepared LEV standard solution for the evaluation of system suitability parameters such as retention time, peak area, peak tailing and number of theoretical plates, LOD and LOQ. Six replicate injections for system suitability test were injected into the chromatographic system and system suitability results are given in Table 1. The specificity results were found that there was no interference due to excipients in the tablet formulation and also that there is good correlation between the retention times of the standard and sample. The specificity results are shown in Table 2 and chromatogram for placebo is depicted in Figure 2. Under the conditions described above separation of the fluoroquinolone mixture was achieved with a run time of 8 min, with an elution window of 3 min for all six analytes. The retention times for LEV, PRF, GAT, SPA, MOX and BLF in mixture were found to be 3.613 min, 4.250 min, 4.703 min, 5.497min, 5.880 min and 6.253 min respectively. The separation chromatogram of mixture is shown in Figure 3.The retention times and peak areas of six fluoroquinolones in combination were shown in Table 3. In Figures 4 9, the individual chromatograms obtained from six fluoroquinolone standards injected are shown. The individual chromatogram results of each analyte are listed in Table 4. These results indicated excellent peak shape, phenomenal plate count and shorter run-time. The calibration curve obtained by concentration on X-axis and peak area on Y-axis shows the linearity in the concentration range of 2-10 g/mL of LEV and the linearity graph is shown in Figure 11. The calibration data was presented in Table 5. The regression equation was found to be Y = 2.8847 + 53.493X with regression coefficient of R2 = 0.9998 which indicates this method had good linearity. The linear Volume 1(2) March-April 2013 Page 266
ISSN: 2320 3471(Online) Ravisankar et.al. Indian Journal of Research in Pharmacy and Biotechnology regression data of the calibration curve for determination of LEV is shown in Table 6. The developed method was applied to the assay of LEV tablets. The results were very close to labeled value of commercial tablets. The representative standard and sample chromatograms of LEV are shown in Figure 4 and Figure 10 respectively and the assay results are presented in Table 7. Precision was studied to find out intra-day and inter-day variations in the test methods of LEV for three times on the same day and on different days. The %RSD for intra-day and inter-day precision variations studied at 10g/mL obtained was 0.5915 and 0.7083 respectively which is within the acceptable criteria of NMT 2.0. This reveals that the proposed method is quite precise and reproducible and the precision results for intra-day and inter-day are shown in Table 8. The % recoveries of the drug solutions were studied at 3 different concentration levels. The % individual recovery and the % RSD values at each level were within the acceptance limits. Generally the mean percentage recovery of LEV at each level was not less than 99% and not more than 101%. Satisfactory recoveries ranging from 99.51 - 100.20% were obtained by the proposed method. The % recovery and % RSD were calculated and results are presented in Table 9. Robustness was done by deliberate changes in the chromatographic conditions like mobile phase flow rate, temperature, mobile phase composition etc., Deliberate changes in developed method had not much affected the peak tailing, theoretical peaks and % assay which demonstrated that the developed method was Robust in nature. The results of robustness study are shown in Table 10. 5. CONCLUSION A validated RP-HPLC method has been developed for the determination of LEV and Rapid separation of selected six fluoroquinolones with a relatively short retention time, provides phenomenal resolution, excellent peak shape, gave consistent and highly reproducible results on C18 HPLC column. The method overall proved to be economical, simple, rapid, precise, very sensitive, cost-effective, time saving, robust and accurate. It can be reliably used for determination of the said six fluoroquinolones in short period and even in small concentrations. Its short run-time allows the analysis of large volume of samples in short period of time. By using this method one can elute all the six drugs within eight minutes. This method was completely validated shows excellent results and also free from interference of the other additives used in the formulations. Under the conditions described above, separation of the six fluoroquinolone agent mixture was achieved with a total run time of 8 minutes with an elution window of 3 minutes for all six analytes. The ease in preparation of mobile phase and economy of the components of mobile phase make this method the best choice in routine analysis of LEV, BLF, GAT, PRF, SPA and MOX in bulk and their pharmaceutical formulations. This method provides decorous resolution power with short runtimes, good retention, superior peak shape and excellent reproducibility of the results achieved. Therefore it is suitable for routine analysis of above said anti-bacterial agents. So it could be used for rapid and reliable determination of the above described anti-bacterial fluoroquinolone agents, irrespective of their concentration levels or in combinations. ACKNWOLEDGEMENT The authors would like to acknowledge Hetero Labs for providing the samples of Prulifloxacin and Balofloxacin. They also thank Aristo Pharma for proving the gift samples of Levofloxacin and Gatifloxacin. We whole heartedly thank Ananth Pharmaceuticals, Torrent pharmaceuticals for their samples drugs Sparfloxacin and Moxifloxacin. We are highly grateful to Dr.L.Rathaiah, Honorable Chairman, Vignan group of institutions, and principal of Vignan pharmacy college, Dr. P.Srinivasa Babu for providing the necessary facilities to carry out this research work.
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ISSN: 2320 3471(Online) Ravisankar et.al. Indian Journal of Research in Pharmacy and Biotechnology Table 1: Optimized chromatographic conditions and system suitability parameters of proposed RP-HPLC method for Levofloxacin Parameter Chromatographic conditions Instrument SHIMADZU LC-20AT prominence liquid chromatograph Column WELCHROM C18 Column (4.6 mm i.d. X 250 mm, 5 m particle size) Detector SHIMADZU SPD-20A prominence UV-Vis detector Diluents Phosphate buffer (pH-3.1): Acetonitrile = 70:30, v/v. Mobile phase Phosphate buffer (pH-3.1): Acetonitrile = 70:30, v/v. Column modifier Triethylamine Flow rate 1 mL/min. Detection wave length UV at 293 nm. Run time 6 minutes Column back pressure 118 - 119 kgf Temperature Ambient temperature(25oC) Volume of injection loop 20 L Retention time (tR) 3.607 min Theoretical plates [th.pl] 12,261 (Efficiency) Theoretical plates per meter [t.p/m] 245210 Tailing factor (asymmetry factor) 1.106 LOD 0.1234 LOQ 0.3740 Table 2: Specificity study for Levofloxacin Name of the solution Mobile phase Placebo Levofloxacin, 10 g/mL Retention time, (tR) min. No peaks No peaks 3.607 min.
Table 3: Chromatogram results of proposed combination of Fluoroquinolones: Sample Retention time, Assymmetry Efficiency(theoretical Resolution (tR)min. plates) Levofloxacin 3.613 1.178 12306 Prulifloxacin 4.250 1.167 12354 4.508 Gatifloxacin 4.703 1.120 13115 2.866 Sparfloxacin 5.497 1.069 14711 4.604 Moxifloxacin 5.880 1.032 14912 2.056 Balofloxacin 6.253 1.063 15916 2.017
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ISSN: 2320 3471(Online) Ravisankar et.al. Indian Journal of Research in Pharmacy and Biotechnology Table 4: Individual chromatogram results of six Fluoroquinolone standards. Name of the Efficiency Retention time (tR) Assymmetry Compound (theoretical plates) Levofloxacin 3.607 1.106 12261 Prulifloxacin 4.230 1.159 12256 Gatifloxacin 4.687 1.119 12957 Sparfloxacin 5.497 1.067 14698 Moxifloxacin 5.870 1.035 14845 Balofloxacin 6.253 1.063 15916 Table 5: Calibration data of the proposed HPLC method for estimation of Levofloxacin S.No Concentration, g/mL. Retention time, (tR) min. Peak area, mV.s. 1. 0 0 2. 2 3.613 111.736 3. 4 3.608 219.634 4. 6 3.610 324.742 5. 8 3.607 427.668 6. 10 3.607 538.324 Slope 53.4932 Intercept 2.8846 Correlation Coefficient [CC] (r) 0.9999 Squared CC (R2) 0.9998 Residual sum of squares 30.5723 Table 6: Linear Regression data of the proposed method of Levofloxacin: Parameter Method Detection wavelength( max) By UV at 293nm Linearity range (g/mL) 2-10 g/ml Regression equation (Y = a+bx) Y = 2.8847 + 53.493X Slope(b) 53.493 Intercept(a) 2.8847 Standard error of slope (Sb) 0.3304 Standard error of intercept (Sa) 2.0008 Correlation coefficient (r) 0.9999 2 Regression coefficient (R ) 0.9998 % Relative standard deviation* i.e., 0.9850 Coefficient of variation (CV) Limit of detection (g/mL) 0.1234 Limit of quantitation(g/mL) 0.3740 Percentage range of errors*(Confidence limits) 0.005significance level 1.2374 0.001 significance level 1.9410 * Average of 6 determinations; Acceptance criteria < 2.0. Table 7: Assay results of Levofloxacin formulation Formulations
GLEVO tablets (Majesta (Glenmark) Pharmaceuticals Ltd., Mumbai, India)
Labelled amount 500 mg/tablet
Amount found* 496.588 mg/tablet
% AssaySD* 99.317 0.990 %
*Average of 6 determinations; SD is standard deviation. Volume 1(2) March-April 2013 Page 269
ISSN: 2320 3471(Online) Ravisankar et.al. Indian Journal of Research in Pharmacy and Biotechnology Table 8: Results of precision study (intra-day and inter-day) for Levofloxacin Precision Sample Injection no.1 Injection no.2 Injection no.3 Injection no.4 Injection no.5 Injection no.6 % RSD Inter-day Levofloxacin (10 g/mL) Peak area, mV.s Peak area, mV.s 538.324 534.281 536.268 532.337 538.112 530.274 539.302 539.128 532.446 533.548 531.963 528.190 0.5915 0.7083 Intra-day
Table 9: Recovery data of the proposed RP-HPLC method for Levofloxacin Recovery level 80% Amount added (mg) 80.24 80.11 79.82 100.34 99.86 100.21 119.94 120.42 119.78 Total amount (mg) 179.55 179.42 179.13 199.65 199.17 199.52 219.25 219.73 219.09 Amount Amount % found recovered recovery (mg) (mg) 90.11 80.02 99.725 90.13 80.04 99.912 90.05 79.96 100.175 110.35 100.26 99.920 110.01 99.92 100.060 109.78 99.69 99.481 130.3 120.21 100.225 130.41 120.32 99.916 129.83 119.74 99.966 # acceptance criteria < 2.0. Mean % Recovery SD 99.937 0.225 99.820 0.302 100.036 0.165 %RSD#
0.2259
100%
0.3026
120%
0.1653
Table 10: Robustness results of Levofloxacin Parameter a Optimized 1.0 mL/min Used 0.8 mL/min 1.2 mL/min 288 nm 293 nm 50:50, v/v
#
Retention time (tR), min 3.848 3.325 3.612 3.604 3.794 3.425
$
Plate count$ 12562 12054 12242 12274 12628 12106
Peak asymmetry# 1.126 1.097 1.114 1.108 1.115 1.102
Remark *Robust *Robust Robust Robust *Robust *Robust
Flow rate (0.2 mL/min) Detection wavelength (5 nm) Mobile phase composition (5 %)
298 nm 55:45, v/v 45:55, v/v
Acceptance criteria (Limits): Peak Asymmetry < 1.5, Plate count > 3000, * Significant change in Retention time
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Fig. 1: Structures of fluoroquinolones investigated in the present study
Fig. 2: Chromatogram of placebo
Fig. 3: Chromatogram of mixture of six Fluoroquinolones
Fig. 4: Standard Chromatogram of Levofloxacin (10g/ml)
Fig. 5: Standard chromatogram of Prulifloxacin (10 g/ml)
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Fig. 6: Standard chromatogram of Gatifloxacin (10 g/ml)
Fig. 7: Standard chromatogram of Sparfloxacin (10 g/ml)
Fig 8: Standard Chromatogram of Moxifloxacin (10g/ml)
Fig. 9: Standard chromatogram of Balofloxacin (10 g/ml)
Levofloxacin y = 53.493x + 2.8847

peak area, mV.s 600 400 200 0 0 5 10 15 concentration, g/ml R = 0.9998 Series1 Linear (Series1)
Fig. 10: Chromatogram of market formulation (GLEVO 500mg Capsules) of Levofloxacin.
Fig 11: Calibration Plot of Levofloxacin
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Ravisankar et.al. REFERENCES
Du, L., Wei, H., Zhang, J., & Zhang, Q., Separation and Determination of Six Fluoroquinolones by ReversedPhase High Performance Liquid Chromatography. Chinese Journal of Chromatography, 21(5), 2003, 503-506. Espinosa-Mansilla, A., Pea, A., Gmez, D. G., & Salinas, F. HPLC determination of enoxacin, ciprofloxacin, norfloxacin and ofloxacin with photoinduced fluorimetric (PIF) detection and multiemission scanning: Application to urine and serum. Journal of Chromatography B, 822(1), 2005, 185-193. ICH, Q2B, Harmonized Tripartite Guideline, Validation of Analytical Procedure: Methodology, IFPMA, in: Proceedings of the International Conference on Harmonization, Geneva, March 1996. Madhuri D. Game, D. M. Sakarkar., Simultaneous Spectrophotometric Estimation of Nitazoxanide and Ofloxacin in Tablets. indian J Pharm Sci. 73(1), 2011, 7074. Mahesh Attimarad, Bander E Al-Dhubiab, Ibrahim A Alhaider, Anroop B Nair, Harsha N Sree, Ahmed K Mueen., Simultaneous determination of moxifloxacin and cefixime by first and ratio first derivative ultraviolet spectrophotometry. Chem Cent J. 6, 2012, 105. Manisha Puranik, D. V. Bhawsar, Prachi Rathi, P. G. Yeole., Simultaneous Determination of Ofloxacin and Ornidazole in Solid Dosage Form by RP-HPLC and HPTLC Techniques. Indian J Pharm Sci. ; 72(4), 2010, 513517. Noura H. Abou-Taleb, Dina T. El-Sherbiny, Dalia R. El-Wasseef, Mohamed A. Abu El-Enin, Saadia M. ElAshry., Simultaneous Determination of Norfloxacin and Tinidazole Binary Mixture by Difference Spectroscopy. Int J Biomed Sci. 7(2), 2011, 137-144. P. Ravisankar, Ch. Devadasu, G. Devala Rao, P. Srinivasa Babu, G. Sudhakar Saibabu, P. Venkateswar Reddy, A validated RP-HPLC method for the assay of balofloxacin in bulk and pharmaceutical dosage forms. Int. J. Chem. Sci.: 11(1), 2013, 553-568. P. Ravisankar, Ch. Devadasu, P. Srinivas Babu, G. Devala Rao, S. Gananathamu. Development and validation of new RP-HPLC Method for the estimation of prulifloxacin in Pharmaceutical dosage forms. Int. J. Chem. Sci.: 8(1), 2010, 433-444. P. Ravisankar, Ch. Devadasu, P. Srinivasa Babu, G. Devala Rao, S. Gananathamu, S. Sowjanya., new spectrophotometric methods for the Quantitative analysis of prulifloxacin in Pharmaceutical dosage forms. Int. J. Chem. Sci.: 8(4), 2010, 2309-2324. P. Ravisankar, G. Devala Rao, CH. Devadasu, G. Sudhakar Saibabu, P. Srinivasa Babu., A validated RPHPLC method for the assay of Prulifloxacin in marketed drug product using Levofloxacin as an internal standard. Int. J. Chem. Sci.: 11(1), 2013, 95-105. Ravisankar Panchumarthy, Devala Rao Garikapati, Devadasu Chapala, Afzal Basha SK, Sandhya Rani Nagabhairava, Puttagunta Srinivasa Babu., A validated RP-HPLC method for the determination of gemifloxacin in bulk and pharmaceutical dosage forms. Journal of Chemical and Pharmaceutical Sciences., 6(1), 2013, 46-54. Ravisankar Panchumarthy, Devala Rao Garikapati, Gopala Reddy Padala., rapid simultaneous separation of fluoroquinolone antibacterial - levofloxacin, sparfloxacin and balofloxacin by isocratic RP-HPLC: application to sparfloxacin determination in pharmaceutical dosage forms. Journal of Chemical and Pharmaceutical Sciences., 6(2), 2013, 120-133. Samanidou, V. F., C. E. Demetriou, I. N. Papadoyannis., Direct determination of four fluoroquinolones, enoxacin, norfloxacin, ofloxacin, and ciprofloxacin, in pharmaceuticals and blood serum by HPLC. Analytical and bioanalytical chemistry 375(5), (2003), 623-629 Volume 1(2) March-April 2013 Page 273
ISSN: 2320 3471(Online) Ravisankar et.al. Indian Journal of Research in Pharmacy and Biotechnology Shervington, L. A., Abba, M., Hussain, B., Donnelly, J. The simultaneous separation and determination of five quinolone antibotics using isocratic reversed-phase HPLC: Application to stability studies on an ofloxacin tablet formulation. Journal of pharmaceutical and biomedical analysis, 39(3), 2005, 769-775. Sun, H., Qiao, F., Liu, G., & Liang,S., Simultaneous isolation of six fluoroquinolones in serum samples by selective molecularly imprinted matrix solid-phase dispersion. Analytica chimica acta, 625(2), 2008, 154-159. Syed Naeem Razzaq, Islam Ullah Khan, Irfana Mariam, Syed Saleem Razzaq., Stability indicating HPLC method for the simultaneous determination of moxifloxacin and prednisolone in pharmaceutical formulations. Chem Cent J. 6, 2012, 94.
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% decrease in activity ----62.18 49.19 54.62 23.76 32.98

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RECENT TRENDS IN USAGE OF POLYMERS IN THE FORMULATION OF DERMATOLOGICAL GELS

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RECENT TRENDS OF POLYMER USAGE IN THE FORMULATION OF ORODISPERSIBLE TABLETS

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Sodium starch glycolate Explotab, Primogel Alginic acid NF Satialgine

Soy polysaccharides Emcosoy Calcium silicate

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Table.3. Formulation of amlodipine besylate fast dissolving tablets