Soumya Project Final

Faculty of Biological Engineering Page 1
INTRODUCTION
CHAPTER 1


INTRODUCTION
1.1 Arthritis
Arthritis[1] is a form of joint disorder that involves inflammation of one or more
joints. There are over 100 different forms of arthritis. The most
commonform, osteoarthritis (degenerative joint disease), is a result of trauma to the joint,
infection of the joint, or age.
Other arthritis forms are rheumatoid arthritis, psoriatic arthritis, and related autoimmune
diseases. Septic arthritis is caused by joint infection[1].Various drugs are used for the
treatment of arthritis but a controlled drug delivery is closely associated with a polymer
system that is degraded while the digestion and increases the effectiveness of the drug as
such the drug Tinosporacordifolia, which is known by the common name Guduchi, is an
herbaceousvine of the family Menispermaceae indigenous to the tropical areas of India,
Myanmar and Sri Lanka that cures the arthritis and Studies on induced oedema and arthritis
and on human arthritis proved anti-inflammatorypotency of the water extract of plant.
It also has antipyretic action. This drug relaxes the intestinal and uterine smooth muscles. It
is proved effective in prevention of fibrosis and in stimulating regeneration in hepatic tissue.

1.2 Drug Delivery
Drug delivery[2] is the method or process of administering a pharmaceutical compound to
achieve a therapeutic effect in humans or animals.Drug delivery technologies modify drug
release profile, absorption, distribution and elimination for the benefit of improving product
efficacy and safety, as well as patient convenience and compliance. Drug release is from:
diffusion, degradation, swelling, and affinity-based mechanisms.Most common routes of
administration include the preferred non-invasive peroral (through the mouth), topical
(skin), transmucosal (nasal, buccal/sublingual, vaginal, ocular and rectal)[3] and inhalation
routes. Many medications such as peptide and protein, antibody, vaccine and gene based
drugs, in general may not be delivered using these routes because they might be susceptible
to enzymatic degradation or can not be absorbed into the systemic circulation efficiently due
to molecular size and charge issues to be therapeutically effective. For this reason many
protein and peptide drugs have to be delivered by injection or a nanoneedle array. For
example, many immunizations are based on the deliver it refers to approaches,
formulations, technologies, and systems for transporting a pharmaceutical compound in the
body as needed to safely achieve its desired therapeutic effect. It may involve scientific site-
targeting within the body, or it might involve facilitating systemic pharmacokinetics; in any
case, it is typically concerned with both quantity and duration of drug presence. Drug
delivery is often approached via a drug's chemical formulation, but it may also involve
medical devices or drug-device combination products. Drug delivery is a concept heavily

integrated with dosage form and route of administration, the latter sometimes even being
considered part of the definition.

1.3 Ayurveda
Ayurveda[4] is a complete or holistic system that integrates the mind, body and spirit.
For a few centuries, the tradition of Ayurveda was dimmed due to the natural and human
calamities and also by the invasion of foreign cultures into India. The sacred texts were
either destroyed or stolen. However there were many vaidyas or doctors in India who
managed to preserve some of the knowledge available in these Holy Scriptures. Divine
plants that sustain long life and good health are now being rediscovered. Many renowned
families of Vaidyas, who are specialized in certain branches of Ayurveda, have started
functioning again in India. Today there is a revival of the ancient culture and traditions
inherent to Ayurveda, which is a true gift of the ancient civilization to the modern world.
The true history of Ayurveda starts from the time of the Holy books, the Vedas. Ancient
mythology contends that the concept and essence of Ayurveda was revealed by the creator
of the world himself Lord Brahma.
There are four Vedas. They are
Rigveda
Yajurveda
Samaveda
Atharvaveda
The Vedas date back to about five thousand years. They preach the philosophy of life.
The Atharvaveda contains the principles of healing on which Ayurveda is based. 'Ayur'
means 'life' in Sanskrit. Ayurveda is the most ancient science of healing which enhances
longevity. It has influenced many of the older traditional methods of healing including
Tibetan, Chinese and Greek medicine. Hence, Ayurveda is considered by many as the
'mother of healing.'
The practical tenets of Ayurveda are divided into eight sections or branches. These
sections include:
Internal medicine,
Surgery,
Organic medicine,
Pediatrics,
Toxicology,
Rejuvenating remedy,
Aphrodisiac remedies and
Spiritual healing.
These eight sections are called "Astanga Ayurveda".


1.4 Giloy( Tinospora Cordifolia)

Tinosporacordifolia[5], which is known by the common name Guduchi, is an
herbaceousvine of the family Menispermaceae indigenous to the tropical areas of India,
Myanmar and Sri Lanka.
The plant is a glabrous climbing shrub found throughout India, typically growing in
deciduous and dry forests. The leaves are heart shaped. The succulent bark is creamy white
to grey in color, with deep clefts spotted with lenticels. It puts out long, slender aerial roots,
and is often grown on mango or neem trees Flowers are yellow, growing in lax racemes
from nodes on old wood. Fruits are drupes, turning red when ripe.
Various name of giloy in India
Hindi Name
Giloya, Guduchi (Hindi)
Bengali Name
Gulancha/palo(Bengali),
Telugu Name
Tippaatigo (Telugu)
Tamil Name
Shindilakodi (Tamil)
Scientific classification
Kingdom:
Plantae
Division:
Magnoliophyta
Class:
Magnoliopsida
Order:
Ranunculales
Family:
Menispermaceae
Genus:
Tinospora
Species: T. cordifolia
Binomial name
Tinosporacordifolia


Uses of Giloy:

Immunity Disorders:
Giloy or Amrita carries anti-inflammatory and antipyretic properties. This herb has been
used in Ayurvedicrasayanas since centuries which is very helpful in building up the immune
system and the body's confrontation against definite infecting organisms. In a scientific
study conducted using human WBC (white blood corpuscles), the Ayurvedic herb helps in
increasing the killing ability of macrophages, the resistant cell those are accountable for
fighting foreign materials as well as microorganisms

Stomach Ulcer:
Giloy use for soothing inflamed and injured mucous membranes in the digestive tract. This
herb protect the stomach and duodenum by increasing production of mucin, a substance that
protects the lining of these organs against stomach acid and other harmful substances.

Urinary disorder :
This herb is used in urinary affections. The juice of the roots is very much effective in
Urinary problems.

Mental Disorder :
The whole plant and the juice of the leaves is traditionally used in various mental disorders.
This is regarded as one of the best psychotropic drugs in India.
Fig-1.2

Other uses :
There are many uses of Tinosporacordifolia. The stem is used in conditions like generalized
weakness, dyspepsia, pyrexias of unknown origin (fevers), swine flu and many urinary tract
infections. The bitter properties present in the drug show antiperiodic and antispasmodic
properties which is again helpful in preventing swine flu.
It is also used as an immune-modulator in immune-suppression of certain ailments like as
obstructive jaundice, hepatic fibrosis, peritonitis and sepsis. The Tinosporaor Giloy or
amrita has been exposed very effective in preventing fibrous changes and promoting
regeneration of the liver against CCl4 induced hepatic toxicity.
The herb is also used in stomach ulcer, urinary affections, vitalizer and remedy for diabetes
and metabolic disorders.

Studies on induced oedema and arthritis and on human arthritis proved anti-
inflammatorypotency of the water extract of plant. It also has antipyretic action. This drug
relaxes the intestinal and uterine smooth muscles. It is proved effective in prevention of
fibrosis and in stimulating regeneration in hepatic tissue.

FIG-1.3 GILOY- HERB

Medicinal properties of giloy:

External uses: Guduchi is very effective in skin disorders. It helps to boost resistance of skin
to microbes and is found effective in leprosy and ulcers formed due to gout. This herb heals
the herpes lesions and also accelerates the healing process of wounds in venereal diseases.

Digestive system: Usage of Guduchi is indicated in conditions like indigestion, excess thirst,
acidity, irritable bowel syndrome (IBS), dysentery and vomiting. It is a good anti spasmodic
and reduces stomach pain which arises due to intestinal spasms. Giloy is a very good
hepatotonic .Preparations of this herb is very effective in jaundice, hepatic fibrosis and other
diseases related to liver. This herb expels toxins accumulated in liver.

Circulatory system: Texts of Ayurveda eulogize this herb as a cardiac tonic as it strengthens
the heart. Guduchi helps to purify blood and expel the toxins which are circulating in it.
Because of this property it is useful in gout to control blood uric acid level.

Respiratory system: It strengthens the lungs and reduces chronic cough

Reproductive system: Few herbal aphrodisiac preparations contain guduchi as their
ingredient. Due to aphrodisiac properties it increases sperm count and sperm motility. Its
adaptogen properties revitalize the male reproductive system and help in conditions like
erectile dysfunction and premature ejaculation.

Excretory System: Guduchi strengthens the urinary system and increases the resistance of
inner layers of bladder and urethra to fight repeated urinary tract infections.

Giloy is very effective in diabetes and reduces fever. It boosts immunity and increases body
energy level. Due to its anti microbial property the growth of micro organisms is inhibited in
body. This boosts body resistance to diseases. Herbal preparations of this wonder herb help
immensely in diseases of joints like arthritis and gout.
1.5 Tamarind Seed Powder

The tamarind seed powder is a complex mixture containing a galactoxyloglucan
polysaccharides, proteins, lipids and certain minor constituents e.g. Fibers, Sugar etc. It
forms a uniform solution on heating with water while stirring. It is used majorly in Paper
Industry, Textile Industry, Dyeing and Printing Mills, Jute Mills, Card Board Industry and
Explosive Plants.

REVIEW OF LITERATURE

CHAPTER 2


REVIEW OF LITERATURE

2.1 Pharmacological review

PahadiyaS et al [7] has evaluated the radioprotective effect of an aqueous extract of
Tinosporacordifolia (TC) against Co(60) gamma radiation in the dose of 5 mg/kg body
wt to Swiss albino mice. It has shown significant protection in terms of survival
percentage.

Grover JK et al [8] has evaluated the extract of M. charantia (200 mg/kg), E. jambolana
(200 mg/kg), M. pruriens (200 mg/kg) and T. cordifolia (400 mg/kg) was administered
for 50 days in STZ induced diabetic mice, the plasma glucose concentration was reduced
by 24.4, 20.84, 7.45 and 9.07% respectively.

Mary NK et al [9] has evaluated T. cordifolia as antioxidant, anticoagulant, platelet
antiaggregatory, lipoprotein lipase releasing, anti-inflammatory and hypolipidaemic
activity in rats in the dose of 5 mg/kg. The extract has significantly (p<0.001) enhanced
release of lipoprotein lipase enzyme.

Gupta R. S. et al [10] Oral administration of 70% methanolic extract of T. cordifolia
stem to male rats at the dose level of 100 mg/rat/day for 60 days did not cause body
weight loss but decreased the weight of testes, epididymis, seminal vesicle and ventral
prostate in a significant manner. The stem extract brought about an interference with
spermatogenesis. These results suggested antifertility effects of the stem extract of T.
cordifolia in male rats.

Umamaheswari S. et al [11 ]has evaluated the antihyperglycemic effect of 'Ilogen-
Excel' in streptozotocin induced diabetic rats in the dose of 50 mg/kg and 100 mg/kg .It
has shown significantly lowered levels of blood glucose and significantly increased
levels of plasma insulin, hepatic glycogen and total hemoglobin.

Grover JK et al [12]has evaluated Tinosporacordifolia (TC) are used as a household
remedy for diabetes in the dose of 21-120 days. It has found that extract has shown
percent reduction in glucose decreased significantly in the moderate and severe diabetes;
55.62 and 17.72% for EJ and 48.81 and 0% for TC at the similar time intervals.

Mathew S et al [13]has evaluate reducing the chemotoxicity induced by free radical

forming chemicals in the dose of 25 mg/kg b.wt, 10 days in mice. It has found extract of
Tinosporacordifolia has been shown to inhibit the lipid peroxidation and superoxide and
hydroxyl radicals in vitro.

Singh SM et al [14]has evaluate influence of T. cordifolia on myeloid differentiation of
bone marrow progenitor cells and the recruitment of macrophages in response to tumor
growth in situ. It has found extract of Tinosporacordifolia can influence the myeloid
differentiation of bone marrow progenitor cells and the recruitment of macrophase in
response to tumor growth in situ.

Thatte UM et al [15] has evaluate the protective effects of Asparagus racemosus (AR)
and Tinosporacordifolia (TC) against myelosuppression induced by single doses of
cyclophosphamide (CP) 200 mg/kg. It has found that extract of Tinosporacordifolia is
potent immunostimulant, with effects comparable to lithium and glucan.

Chaudhary R et al [16] Tinosporacordifolia (Guduchi), an Indian medicinal plant, was
used to explore antitumor promoting activity in a two-stage skin carcinogenesis model at
100 mg/kg body weight/day for 16 weeks. It has been observed that cumulative number
of papillomas, tumor yield, tumor burden, and tumor weight showed significant
reduction along with significant elevation of phase II detoxifying enzymes, and
inhibition of lipid peroxidation in liver and skin in the animals administered with such
plant extract concomitant to carcinogen exposure.

Kar A et al [17] has evaluate hypoglycaemic activities of the experimental herbal
samples in the dose of 250 mg/kg . It has found that extract of Tinosporacordifolia
shows the blood glucose lowering effect within 2 weeks in alloxan diabetic albino rats.

Nemmani KV et al [18] has evaluate cell proliferation and natural killer cell activity by
polyherbal formulation, Immu-21containing Ocimum sanctum, Withaniasomnifera,
Emblicaofficinalis and Tinosporacordifolia in mice in the dose of 30 mg/kg once a day
for 14 and 21 days. The results indicate that pretreatment with Immu-21 selectively
increased the profileration of splenic leukocyte to B cell mitogen, Lipopolysaccharide
(LPS), and cytotoxic activity against K 562 cells in mice.

.Dhuley JN et al[19] The effect of Indian herbs namely, Asparagus racemosus,
Tinosporacordifolia, Withaniasomnifera and Picrorhizakurrooa on the functions of
macrophages obtained from mice treated with the carcinogen ochratoxin A (OTA) was
investigated. The chemotactic activity of murine macrophages was significantly
decreased by 17 weeks of treatment with OTA compared with controls. Production of

interleukin-1 (IL-1) and tumor necrosis factor (TNF) was also markedly reduced.
Treatment with Asparagus racemosus, Tinosporacordifolia, Withaniasomnifera and
Picrorhizakurrooa significantly inhibited OTA-induced suppression of chemotactic
activity and production of IL-1 and TNF-alpha by macropahges. Moreover, we found
that Withaniasomnifera treated macrophage chemotaxis and that Asparagus racemosus
induced excess production of TNF-alpha when compared with controls.

Wadood N et al [20] The aqueous, alcoholic, and chloroform extracts of the leaves of
Tinosporacordifolia were administered in doses of 50, 100, 150 and 200 mg/kg body
weight to normal and alloxan-diabetic rabbits. The blood glucose and total lipid levels
were estimated before and 2, 4, 6, and 8 hours after administration of the extract. The
extract exerted a significant (P less than 0.5) hypoglycaemic effect in normal as well as
in alloxan-treated rabbits
Singh N et al[21] This article presents evidence to show that an alcoholic extract of
Tinosporacordifolia (ALTC) enhances the differentiation of TAM to dendritic cells (DC)
in response to granulocyte/macrophage-colony-stimulating factor, interleukin-4, and
tumor necrosis factor. DC differentiated in vitro from TAM that were harvested from
tumor-bearing mice after i.p. administration of ALTC (200 mg/kg body weight) 2 days
post tumor transplantation shows an enhanced tumor cytotoxicity and production of
tumoricidal soluble molecules like TNF, IL-1, and NO. Adoptive transfer of these TAM-
derived DC to Dalton's lymphoma-bearing mice resulted in prolongation of survival of
tumor-bearing mice. This is the first report regarding the differentiation and antitumor
functions of TAM-derived DC obtained from tumor-bearing host administered with
ALTC. The possible mechanisms involved also are discussed.

Leyon PV et al [22] The antiangiogenic activity of Tinosporacordifolia was studied
using in vivo as well as in vitro models. In vivo antiangiogenic activity was studied
using B16F10 melanoma cell-induced capillary formation in animals. Intraperitoneal
administration of the extract at a concentration of 20 mg/kg significantly inhibited the
tumour directed capillary formation induced by melanoma cells. Analysis of the serum
cytokine profile showed a drastic increase of proinflammatory cytokines such as IL-
1beta, IL-6, TNF-alpha, granulocyte monocyte-colony stimulating factor (GM-CSF) and
the direct endothelial cell proliferating agent vascular endothelial cell growth factor
(VEGF) in the angiogenesis-induced control animals. Administration of Tinospora
extract could differentially regulate these cytokine's elevation. The differential regulation
is further evidenced by the increased production of antiangiogenic agents IL-2 and tissue
inhibitor of metalloprotease-1 (TIMP-1) in the B16F10-injected, extract-treated animals.
Moreover, using an in vitro rat aortic ring assay, it was observed that the extract at
nontoxic concentrations inhibited the production of proangiogenic factors from B16F10

melanoma cells. Direct treatment of the extract also inhibits the microvessel outgrowth
from the aortic ring. Hence, the observed antiangiogenic activity of the plant T.
cordifolia is related, at least in part, to the regulation of the levels of these cytokines and
growth factors in the blood of the angiogenesis-induced animal.

Jagetia GC et al [23] Exposure of HeLa cells to 0, 5, 10, 25, 50 and 100 microg/ml of
guduchi extracts (methanol, aqueous and methylene chloride) resulted in a dose-
dependent but significant increase in cell killing, when compared to non-drug-treated
controls. The effects of methanol and aqueous extracts were almost identical However,
methylene chloride extract enhanced the cell killing effect by 2.8- and 6.8-fold when
compared either to methanol or aqueous extract at 50 and 100 microg/ml, respectively.
Conversely, the frequency of micronuclei increased in a concentration-dependent
manner in guduchi-treated groups and this increase in the frequency of micronuclei was
significantly higher than the non-drug-treated control cultures and also with respect to 5
microg/ml guduchi extract-treated cultures, at the rest of the concentrations evaluated.
Furthermore, the micronuclei formation was higher in the methylene chloride extract-
treated group than in the other two groups. The dose response relationship for all three
extracts evaluated was linear quadratic. The effect of guduchi extracts was comparable
or better than doxorubicin treatment. The micronuclei induction was correlated with the
surviving fraction of cells and the correlation between cell survival and micronuclei
induction was found to be linear quadratic. Our results demonstrate that guduchi killed
the cells very effectively in vitro and deserves attention as an antineoplastic agent.

Singh N et al [24] The present investigations were under taken to study whether the
tumor-associated macrophages (TAM) of Dalton's lymphoma (DL), a spontaneous
transplantable T cell lymphoma, can be activated by the alcoholic extract of medicinal
plant Tinosporacordifolia (ALTC). Intraperitoneal administration of ALTC in DL-
bearing mice not only augments the basic function of macrophages such as Phagocytosis
as well as their antigen presenting ability and secretion of IL-1, TNF and RNI. The
results of the present investigation also indicate that the intraperitoneal administration of
ALTC slow down the tumor growth and increases the life span of tumor bearing host,
thus showing its anti tumor effect through destabilizing the membrane integrity of DL
cells directly or indirectly. This is the first study of it's kind regarding the effect of
alcoholic extract of Tinosporacordifolia on the activation of tumor associated
macrophages and showing the antitumor effect on the spontaneous T-cell lymphoma
(DL), thus may have clinical implications.

Atal CK et al[25] The immunobiological activity was investigated of certain medicinal

plants widely used in the Ayurvedic and Unani systems of medicine for treatment of
chronic infections and immunological disorders. The effect of an ethanolic extract of
each drug was studied on delayed type hypersensitivity, humoral responses to sheep red
blood cells, skin allograft rejection, and phagocytic activity of the reticuloendothelial
system in mice. Picrorhizakurroa was found to be a potent immunostimulant, stimulating
both cell-mediated and humoral immunity. Tylophoraindica, Aconitum heterophyllum
and Holarrhenaantidysenterica appeared to stimulate phagocytic function while
inhibiting the humoral component of the immune system. Tinosporacordifolia and
Ocimumgratissimum appeared to improve the phagocytic function without affecting the
humoral or cell-mediated immune system. Hemidesmusindicus suppressed both the cell-
mediated and humoral components of the immune system.

StanelyMainzen, Prince P etal [26] We undertook the present study to evaluate the
hypolipidaemic effect of an aqueous extract of Tinosporacordifolia roots, an indigenous
plant used in Ayurvedic medicine in India. Administration of the extract of T. cordifolia
roots (2.5 and 5.0 g/kg body weight) for 6 weeks resulted in a significant reduction in
serum and tissue cholesterol, phospholipids and free fatty acids in alloxan diabetic rats.
The root extract at a dose of 5.0 g/kg body weight showed highest hypolipidaemic effect.
The effect of T. cordifolia roots at 2.5 and 5.0 g/kg body weight was better than
glibenclamide. Insulin restored all the parameters to near normal values.

2.2 Phytochemical Review

Gangan VD et al [27] Several glycosides were isolated, as polyacetates, from the n-
BuOH fraction of the Tinosporacordifolia stems. The structures of three new
norditerpene furan glycosides cordifoliside A, B and C have been established by 1D and
2D NMR spectroscopy.

JahfarM et al [28] Polysaccharide from Tinosporacordifolia was isolated, purified,
methylated, hydrolyzed, reduced and acetylated. The partially methylated alditol acetate
(PMAA) derivative thus obtained was subjected to GC-MS studies. The following types
of linkages were noticed: terminal-glucose, 4-xylose, 4-glucose, 4,6-glucose and 2,3,4,6-
glucose.


AIMS AND OBJECTIVES
CHAPTER 3


AIM AND OBJECTIVES

To design the tsp blended polymeric system for controlled drug delivery of
anti-arthritic drug giloy that can be used as a drug for curing arthritic disesases
and studying the antimicrobial activitites of the drug.
Several steps are to be carried out for estimating the release of the drug from
the matrices at gastro-intestinal P.H for successful degradation of the
polymeric substance and easy with quick adsorption of the drug at the mean
time.[29]
To determine the moisture contain analysis of giloy.
To determine the total ash content in giloy.
To determine the acid insoluble ash of the sample.
To determine the alcohol soluble extractive in giloy.
To determine the soluble ash of giloy.
To determine the percentage of sodium chloride content in giloy.
To determine the percentage of borax content present in giloy
To determine the mercury content in giloy.
To determine the percentage of Iron content in giloy
To perform the separation of two phases using TLC.
Preparation of giloy films blended with TSP (tamarind seed powder) for
control drug delivery and anti- microbial studies.
To perform Drug Loading and Dissolution experiments:
To check anti-microbial activity of 5% composition giloy film blended
with TSP.


MATERIALS AND METHODS
CHAPTER-4


MATERIALS AND METHODS
4.1 CHEMICAL PARAMETERS[30]
4.1.1 MOISTURE CONTAIN & ANALYSIS
AIM OF THE EXPERIMENT:-
To determine the moisture contain analysis of giloy.
APPARATUS REQUIRED:-
Hot air oven
Electronic balance
Petri dish
Spatula
Desiccator
Motor and pistle

PRINCIPLE:-
Excess of water in food , spices, medicine, will encourage the microbial growth of fungi or
insect detoriation following hydrolysis.
Limit of water contain should therefore be set for every given food or medicinal plant
material. Although moisture in an important component of food and drugs it should be
eliminate as per required. The objective of drying of fresh material are to add to their
preservative to fix their constituents to check enzymatic &hydrolytic reaction that might
effect that chemical composition of drugs, to reduce their bulk and weight. To facilate
subsequent grinding into powder form. The test for loss or drying determines both water and
volatile matter. Drying can be carried out by heating at a temp. 100-105C inside the hot air
oven and then cooling for 30 min. inside the dessicator and wt. it.

PROCEDURE:-
5-10gms. of freshly prepared sample is taken (W) in pre weight petridish (W1) .
The petridish containing the sample is placed inside the oven for 1-2 hour at a
temp. between 100C-105C.
The sample is taken out from oven and place inside the dessicator for 30 mins. for
cooling.

Immediately take the weight (W2) in the balance and calculate the loss of wt. of
the given sample.

4.1.2 .TOTAL ASH
To determine the total ash content in giloy.
APPRATUS REQUIRED:-
Muffle furnance
Desiccators
Crucible,
Tongs,
Electronic balance.
PROCEDURE:-
A clean dry crucible of platinum, nickel or silica was taken.
The blank weight of crucible measured (w1).
Let sample weight be w ( 2-4 gms sample weigh accurately in the crucible) .
Then ignite (incinerate) the sample with crucible inside the muffle furnace at 600-
650C for 1 to 2 hour (until it is white).
The crucible was taken out and cooled inside the desiccator and weight
If the carbon free ash cannot be obtained in this manner then the crucible was cooled
and moisture with 2ml of water over a saturated solution of ammonium nitrate.
Then it was dried inside a water bath, then on hot plate.
Ignition was done inside the muffle furnace up to a constant weight.
Then the residue was allowed to cool inside the desiccators for30 minutes and then
the weight was taken.


4.1.3 ACID INSOLUBLE ASH
APPRATUS REQUIRED:-
Muffle furnance
Desicator
Crucible
Tong
Hot plate
Chemicals required:- 7% HCL.
PROCEDURE:-
Transfer the ash obtained above in 250 ml beaker without loss of a ash and 100 ml of
dil.hydrochloric acid.
Wash the crucible with 10 ml of acid and transfer the washings to the beaker.
Heat the beaker till the liquid boils.
Filter the solution and collect the insoluble matter on ashless-filter paper (Whatman
filter paper No.4).
Wash with hot water until the filtrate is neutral.
Transfer the filter paper containing the insoluble matter to the original crucible.
Dry on a hot plate and ignite at around 600C in a muffle furnace(until it become
white ash).
Allow the residue to cool in suitable desiccator for 30 min. and weight without delay.
Repeated the process until the constant weight is obtained.
Calculate the insoluble ash with reference to the air dried drug.

4.1.4 WATER SOLUBLE EXTRACTIVE
To determine the water soluble extractive in giloy.

conical flask
Hot air oven
Water bath
Desiccator
Electronic balance
PROCEDURE:-
Macerate (powder) 5 grams of the air dried drug coarsely powdered with 100 ml of
chloroform water in a closed flask (stpppered conical flask) for 24 hour.
Shaking frequently during 6 hours and allowing to stand for 18 hours.
Filter rapidly taking precautions against loss of solvent.
Evaporate 25 ml of the filtrate to dryness in a tared flat bottom shallow
dish(petridish) and dry at 105C to constant weight and weigh it.

4.1.5 ALCOHOL SOLUBLE EXTRACTIVE
To determine the alcohol soluble extractive in giloy.
conical flask
Hot air oven
Water bath
Desicator
Electronic balance
PROCEDURE:-
Macerate(powered form) 5 grams of the air dried drug coarsely powered with 100 ml
of alcohol(90% ethyl alcohol) of the specified strength, in a closed flask for 24 hour.
Shaking frequently during 6 hour and allowing to stand for 18 hour.
And filter rapidly ,taking the precursions against the loss of solvent.

Evaporate 25 ml of the filtrate to dryness in a tarred flat bottom shallow
disc(petridish) and then dry at 105C to constant weight and to weight.

4.1.6 WATER SOLUBLE ASH
To determine the soluble ash of giloy.
APPRATUS REQUIRED:-
Muffle furnace
Desicator
Crucible
PROCEDURE:-
Prepare ash as given under total ash.
Boil the total ash for 5 min. with 25 ml of water.
Collect insoluble matter in a Gooch Crucible or on an ash-less filter paper.
Wash with hot water ignite for 15 min. at a temperature not exceeding 600C.
Subtract the weight of the insoluble matter from the weight of ash.
The difference in weight represents the water soluble ash.
Calculate the percentage of the water soluble ash with reference to the Air-dried
drug.
4.2 ASSAY
4.2.1 SODIUM CHLORIDE ASSAY
AIM OF THE EXPERIMENT:-To determine the percentage of sodium chloride content in
giloy.
APPRATUS REQUIRED:-
Muffle furnace
Silica crucible
Volumetric flask

Filter paper
Electronic balance
Conical flask
Titration stand
CHEMICALS REQUIRED:-
0.1AgNo
KCrO Indicator
PROCEDURE:-
Dissolve about 2-3 grams of accurately weighed drug in 25 ml of purified water and
left for 30 minutes then filtered.
Wash the filter paper completely with perfect water and the filtration made 100 ml in
volumetric flask and make the solution homogeneous
Titrate 25 ml of the solution with 0.1N AgNo using KCrO as indicator
The end point shows the light brick red colour.
Each ml of 0.1N AgNO is equivalent to 0.005845 gram NaCl.
Dissolve about 2-3 grams of accqurately weigh drug sample and dissolve it in 25 ml
of purified water and leave for 30 min and filter.
Wash the filter paper completely with purified water and the filtrate is made 100 ml
in volumetric flask and make the solution homogenous.
Titrate 25 ml of this solution in 0.1N AgNo3 (silver Nitrite) as indicator.
The end point shows light brick red colour.
4.2.2 BORAX
AIM OF THE EXPERIMENT:-To determine the percentage of borax content present in
giloy


APPRATUS REQUIRED :-
Muffle furnace
Conical flask
Filter paper
Silica Crucible
Desiccators
0.5N Hcl
Methyl orange Indicator
PROCEDURE :-
Powder 5-6 grams of drugs i.e liv-52 and Ignited at 450c for 3 hours in muffle
furnace to get it ash and after that put it on desiccator for cooling.
Then 20 ml of distilled water put in ash and rest it for 15 minutes and filtration will
be done
Finally titration of sample will occur against 0.5N Hcl by using methyl orange
Indicator.
Each ml of 0.5N Hcl is equivalent to 0.09536 gram of NaBO10HO.
4.2.3 MERCURY
AIM OF THE EXPERIMENT:-To determine the mercury content in giloy.
APPRATUS REQUIRED:-
RB Bottle
Dessicator
Muffle furnace
Condensor
Conical flask

Electronic balance machine
Concentrated Hcl
2) Nitric acid (HNO)
3) 0.02N Potassium permanganate
4) HO
5) Thiocyanine
6) Ferricallum indicator

PROCEDURE:-
Take 0.5 grams of sample in reflux bottle.
Add 15 ml of Concentrated NO to the sample.
Then reflux it at temperature (35-40c) for 1 hour and after reflux cool it for some
time.
After cooling add 50 ml of HNO then again it is heated for colourless.
Then 100 ml of destiled water put on the solution using condenser.
Then add 0.02N potassium permanganate(KMNO) till the solution looks like pink
colour.
Then decolourised it by adding 6% HO
Again add 3ml of Concentrated Nitric acid and titrate the sample against
theocyanine by using ferricallum indicator.
4.2.4 IRON
AIM OF THE EXPERIMENT :-
To determine the percentage of Iron content in giloy.
APPRATUS REQUIRED:-
Reflux
Beaker

Silica crucible
Muffle furnish
Volumetric flask
Titration strand
Conical flask

Mercury Chloride
Phosphoric acid
Stannous Chloride
Barium diphenyl amine sulphonate Indicator
0.1N KCrO
HSO
PROCEDURE :-
Take 2 grams of the Sample in a pre-weighed silica crucible.
Heat in muffle furnish at 600c until the colour changing to white ash
Reflux the ash with 100ml of 1:1 Hcl for 30 minutes.
Cool and filtered the sample and make up the volume of filterd sample to 250 ml in a
volumetric flask with the help of distilled water.
Pipette out 25 ml aliquot of the solution and boil it.
Add hot stannous chloride in drops in sample till the yellow colour disappears.
Then cool it and add 10 ml of mercuric Chloride.
Then the silver colour pipette occurs.
Add 10 ml of Phosphoric acid-sulphuric acid mixture.
Add 1ml of barium diphenyl amine sulphonate indicator to the sample.
Finally Titrate the sample against 0.1N KCrO and the end point seems to be violet
blue colour. Repeat titration for concordant values.
Avoid excess of stannous chloride solution so, that solution does not become black.

Each ml of 1N KCrO is equivalent to 0.05585 grams of Fe

4.3 TLC(THIN LAYER CHROMATOGRAPHY)
To perform the separation of two phases using TLC.
APPRATUS REQUIRED:-
Flat glass plates
Aligning tray or flat surface
Spreader
Storage rack
Developing chamber
Graduated micro pipette
Reagent sprayer
U.V light
REAGENTS/CHEMICALS REQUIRED:-
Ninhydrin
Ether
Lime water
Silica gel
PROCEDURE:-
a. Preparation of plates:-
Prepare a suspension of the coating substance in a accordance with the instructions of the
supplier and using the spreading device designed for the purpose.
Spread a uniform layer of the suspension 0.25 0.30 mm thick on a flat glass plate 20 c.m
long.
Allow the coated plates to dry in air , heat at 100 - 105C for at least 1 hour.

Note- Except in the case of plates prepared with cellulose when heating for 10 minutes is
normally sufficient and allowed to cool, protected from moisture , store the plates protected
from moisture and use within three days of preparation .At the time of use dry the plates
again if necessary as presented
.
SPREADER USED FOR TLC METHOD

b Method:-
Unless un-saturated condition are prescribed prepare the tank by lining the walls
with sheets of filter paper , pour into the tank, saturating the filter paper in the
process sufficient of the mobile phase to form a layer of solvent 5-10 mm deep [n-
butanol:acetic acid : water(80:20:20)].
Close the tank and allowed to stand for 1 hour at room temperature.

c. Extraction:-
Weigh 100 gram of solid sample or 10 ml of liquid sample.
For solid sample add Ca(OH) 1 gram diturate with motor pistle and transfer the
contents to a 100 ml beaker, dilute it to 200 ml with lime water (5-10%) and shake
well for 30 minutes.
Filter it to collect in a separating funnel 80 ml of filtrate by filter paper on a Gooch
crucible.
It is twice washed with solvent ether 25 ml each for removing resin, glycosides etc.
Fig2.1

The ether layer is washed with 5 ml of lime water and 5 ml of water.
After washing remove the ether layer and collect the rest as sample.
Add 2 gram of ammonium sulphate to the whole extract.
First extraction is done with alcohol and chloroform in 1:1 dilution ratio.
Second extraction is done with alcohol and chloroform in 1:2 ratio, during this add
Barium hydroxide for clear distinction in layers.
The whole mixture is evaporated at 100C using water bath for 30 minutes.
After evaporation the residue is dissolved with 2 ml of alcohol and 25 ml of 0.02 N
Sulphuric acid and it is warmed.
It is cooled and titrated with 0.02 N NaOH with methyl orange/red as indicator, it
gives red yellow colour end point.
Remove a narrow strip of the coating substance or the stationary phase about 5 mm
of the vertical sides of the lead.
Apply the solution being examined in the form of circular spots about 2-6 mm in
diameter or in the form of bands (10-20 mm 2-6 mm).On a line parallel with and
20 mm from one end of the plate and not nearer than 20 mm of the sizes, the spots
could be 50 mm apart.
If necessary the solution may be applied in portions drying between applications.
Mark the sides of the plates 15 cm or the distance specified in the monograph.
Allow the solvent to evaporate and place the plate in tank, ensuring that it is nearly
vertical as possible and that the spots or bands are above the level of the mobile
phase.
Close the tank and allow to stand at room temperature until the mobile phase has
ascended to the marked line.
Remove the plates and dry. Visualize as directed in monograph. Where as a spraying
technique is prescribed. It is essential that the reagent be evenly applied as a fine
spray.
For 2-D chromatography dry the plate after the first development and carry out the
second development in a direction perpendicular to the first.
When the method prescribed in the monograph specifies protected from light or in
subdued light it is intended that the entire procedure is carried out under this
conditions.

SEPARATING FUNNEL USED FOR EXTRACTION METHOD

d. Visualization:-
The phrases U.V light (254 nm) and U.V light(365nm) indicates that the plates
should be examined under an U.V light having a maximum out put at about 254 nm
or at about 365 nm , as the case may be.
The term secondary spot means any spot other than the principal spot, similarly a
secondary band is any band other than principal band.Both the terms are applicable
for 2-D chromatography.
Fig-2.2

IDENTIFICATION OF PROTEIN

4.4 SYNTHESIS OF TSP BLENDED WITH GILOY


Preparation of giloy films blended with TSP (tamarind seed powder) for control drug
delivery and anti- microbial studies.
MATERIAL REQUIRED:-
Magnetic stirrer
Beaker
Hot air oven
Petriplates
PROCEDURE:-
Fig-2.3

1 ) ONLY TSP FILM
0.5gm of TSP was taken and made it soluble in25ml of distilled water at 50
0
c with
continuous stirring in magnetic stirrer.
After about 3
1/2
hours add 5-6 drops of glycerol.
Then after 4 hours add the mixture onto a petriplate.
Keep the petriplate in hot air oven at 50-55
0
c for turning into thin film.
After about 24 hours a thin film of TSP is taken out from the petriplate.

TSP FILM

2) 1% COMPOSITION FILM
0
c with
Then add 1ml of the extract to the polymer.
After that add 5-6 drops of glycerol to the mixture.Then after 4 hours of continuous
stirring add the mixture onto a petriplate.Keep the petriplate in hot air oven at 50-
55
0
After about 24 hours a thin film of TSP blended with 1ml of giloy extract is taken
out from the petriplate.
Fig-2.4

Fig-2.5- 1% COMPOSITION FILM
3) 5% COMPOSITION
0
c with
After that add 5-6 drops of glycerol to the mixture.
Then after 4 hours of continuous stirring add the mixture onto a petriplate.
0

Fig-2.6- 5% COMPOSITION FILM
4)7.5% COMPOSITION
0
c with
Then add 7.5ml of the extract to the polymer.

0
After about 24 hours a thin film of TSP blended with 7.5ml of giloy extract is taken

Fig-2.7- 7.5% COMPOSITION FILM
5) 10% COMPOSITION
0
c with
0

Fig-2.8 -10% COMPOSITION FILM

4.5 CONTROL DRUG DELIVERY[31]

Drug Loading :

Giloy blended with TSP(tamarind seed powder nanocomposites were prepared by
emulsion/solvent evaporation method. In short, giloy of different loadings, i.e., 1 wt%, 5
wt%, 7.5% and 10wt% were dissolved in glycerol. The formed solution was poured into a
labeled Petri dish and allowed to evaporate the solvent overnight at room temperature. This
compound was used for drug delivery purposes.

Dissolution experiments:

Dissolution experiments were performed at 37
o
C using the dissolution tester (Disso test,
Lab India, Mumbai, India) equipped with six paddles at a paddle speed of 100 rpm. About
900 ml of phosphatebuffer solution (pH 5.8,6 & 7) was used as the dissolution media to
stimulate gastrointestinal tract (GIT) conditions. A 5 ml aliquot was used each time for
analyzing the giloy content at a fixed time interval. The dissolution media was replenished
with a fresh stock solution. The amount of giloy released was analyzed using a UV
spectrophotometer (Systronics, India) at the max value of 380 nm.

Drug release mechanism from matrices:

From time to time, various authors have proposed several types of drug release mechanisms
from matrices. It has been proposed that drug release from matrices usually implies water
penetration in the matrix, hydration, swelling, diffusion of the dissolved drug (polymer
hydro fusion), and/or the erosion of the gelatinous layer. Several kinetic models relating to
the drug release from matrices, selected from the most important mathematical models, are
described over here.
However, it is worth mention that the release mechanism of a drug would depend on the
dosagefrom selected, pH, nature of the drug and, of course, the polymer used.

(i) Zero - Order Kinetics [29].
W = k1 t . (1)
(ii) First - Order Kinetics [29,32].
ln (100- W) = ln 100 - k2 t (2)
(iii) Hixon-Crowels Cube- Root Equation (Erosin Model) [32].
(100- W) 1/3 = 100 1/3 k3 t .. (3)
(iv) Higuchis Square Root of Time Equation (Diffusion Model) [30].
W = k4 t . (4)
(v) Power Law Equation (Diffusion/ Relaxation model) [31].
Mt / M = k5 t n . (5)
Mt / M is the fractional drug release into dissolution medium and k5 is a constant
incorporating the structural and geometric characteristics of the drug.


Analysis:

The term n is the diffusional constant that characterizes the drug release transport
mechanism. When n = 0.5, the drug diffused and released from the polymeric matrix with a
quasi-Fickian diffusion mechanism. For n =0.5, an anomalous, non-Fickian drug diffusion
occurs. When n =1, a non-Fickian, case II or Zero- order release kinetics.

4.6 ANTI-MICROBIAL ACTIVITY

To check anti-microbial activity of 5% composition giloy film blended with TSP.

MATERIALS REQUIRED:-
Hot plate
Magnetic stirrer
Auto clave
Laminar air flow
Hot air oven
Filter paper
Forceps
Inoculating wire loop
Beaker
Conical flask
Chemicals required for preparation of LB (Luria Media)
100ml distilled water
Peptone water-1.5gm
Yeast dextrose agar-3.5gm
Agar-agar-0.5gm
PROCEDURE
Water, peptone water, yeast dextrose water and agar-agar is taken in a conical flask
and mixed well in a magnetic stirrer.
0.5gm of 5% composition of the film is properly mixed with 7.5ml distilled water on
a magnetic stirrer for about 1 hour.
Conical flask with the LB media is wrapped with paper and kept in auto clave along
with a petriplate wrapped in a paper inside a polythene.

After the sterilization is done in the petriplate the conical flask and the petriplate is
kept in the laminar air flow.
2 small 1cm filter paper is taken.
One filter paper is kept in 90% ethyl alcohol and the other is kept in the film.
Then the culture is taken in a wire loop from the sterile culture and a smear is made
on the LB media which is spread with a glass spreader all around the LB media.
With the help of a forcep the filter paper which was in 90% ethyl alcohol is kept on
one side of the petriplate and the other filter paper is kept on the other side.
The petriplate is inverted and kept in the hot air oven.

4.6.1 ANTI-FUNGAL ACTIVITY

To check anti-fungal activity of 5% composition giloy film blended with TSP.

MATERIALS REQUIRED:-
Hot plate
Magnetic stirrer
Auto clave
Laminar air flow
Hot air oven
Filter paper
Forceps
Inoculating wire loop
Beaker
Conical flask
PROCEDURE:
Water, peptone water, yeast dextrose water and agar-agar is taken in a conical flask
and mixed well in a magnetic stirrer.
0.5gm of 5% composition of the film is properly mixed with 7.5ml distilled water on
a magnetic stirrer for about 1 hour.
Conical flask with the LB media is wrapped with paper and kept in auto clave
alongwith a petriplate wrapped in a paper inside a polythene.
After the sterilization is done in the petriplate the conical flask and the petriplate is
kept in the laminar air flow.

2 small 1cm filter paper is taken.
One filter paper is kept in 90% ethyl alcohol and the other is kept in the film.
Then the culture is taken in a wire loop from the sterile
culture and a smear is made on the LB media which is spread with a glass spreader
all around the LB media.
With the help of a forcep the filter paper which was in 90% ethyl alcohol is kept on
one side of the petriplate and the other filter paper is kept on the other side.
The petriplate is inverted and kept in the hot air oven.

Antifungal activity

Fig-2.9

RESULTS
CHAPTER 5


RESULT

TOTAL MOI STURE
Percentage of moisture= (Initial weight-final weight/sample weight)*100
Weight of petriplate=44.02gm
Weight of sample=6.55gm
Initial weight(weight of petriplate + weight of sample)=50.57gm
Final weight=50.41gm
% of moisture=(50.57-50.41/6.55)*100
=2.44%
Therefore the total % of moisture =2.44%

TOTAL ASH
Volume of total ash= (final weight-blank weight/sample weight)*100
Blank weight of crucible=22.41gm
Sample weight=2.10gm
Volume of total ash=(22.58-22.41/2.10)*100
=8%
Therefore, the total ash %=8%

ACI D I NSOLUBLE ASH
Percentage of acid insoluble ash=(final weight blank weight/sample weight)*100

Blank weight of crucible=22.42gm
Final weight =22.53gm
% of acid insoluble ash=(22.53-22.42/3.04)*100
=3.6%
Therefore, the total acid insoluble ash percentage=3.6%

WATER SOLUBLE EXTRACTI VE
Percentage of water soluble extractive= (final weight-blank weight/sample weight)*100
Blank weight of petriplate=44.85gm
Sample weight=2.5gm
% water soluble extractive=(45.01-44.85/2.5)*100*1/4
( because Ive used 2.5gm sample and 25ml distilled water)
=1.6%
Therefore, the total % water soluble extractive=1.6%

ACI D SOLUBLE EXTRACTI VE
Percentage of alcohol soluble extractive= (final weight blank weight/sample weight)*100
Blank weight of petriplate=44.25gm
Sample weight=2.5gm
% alcohol soluble extractive=(44.37-44.25/2.5)*100*1/4
=1.2%

Therefore, the total alcohol soluble extractive=1.2%

WATER SOLUBLE ASH
Percentage of water soluble ash={final weight of the total ash with crucible- final
weight(water insoluble ash)/sample weight}*100
Final weight of total ash=22.58gm
Final weight of water insoluble ash=22.50gm
% water insoluble ash= (22.58-22.50/2.10)*100
=3.8%
Therefore, water soluble ash=3.8%
Assay:
SODI UM CHLORI DE
Sample taken = 2.14gm
Initial Burette reading=0
Final burette reading=3ml
Burette Reading =(3-0) = 3ml
Each ml of 0.1N AgNo is equivalent to 0.00584 gm of NaCl.
Calculation:-
1 ml of 0.1N AgNo is to 0.005845 gmNacl
3 ml of 0.1N AgNO
3
is = 0.0058453=0.0175gmNaCl
So,25 ml of sample solution contains 0.0175gm of Nacl
100 ml sample solution contain =(0.01754)gmNacl = 0.07gm Nacl
So,2.14gm of sample contain 0.07gm Nacl

Therefore % of Nacl=(Nacl content/sample wt)100
=(0.07/2.14)100 =3.27% of Nacl
From the above calculation It is found that giloy contains 3.27% of Nacl.
BORAX
Initial Burette Reading = 0
Final Burette Reading = 4
Burette Reading = (4-0) = 4 ml
Each ml of 0.5N Hcl = 0.09536gm og NaBO10 HO
Calculation:-
Each ml of 0.5N Hcl = 0.09536gm NaBO10 HO
Each ml of 0.094N Hcl = (0.09536gm of NaBO10 HO/0.50.094)gm of Borax
=0.017927gm of Borax
4ml of 0.094N Hcl contains Borax = (0.0179274)gm of Borax
= 0.07170gm Borax
% of Borax = (0.07170gm.Borax/3.01)100 = 2.38% of Borax
From the above calculation it is found that giloy contain 2.38% of Borax
MERCURY:-
Final Burette Reading = 4 ml
Burette Reading = 4-0 = 4ml
Each ml of 0.1N NHScN = 0.01003g of H

Calculation:-
0.1N NHScN = 0.01003gm of Hg
4ml of 0.1N NHScN=(0.010034)gm of Hg =0.04012gm of Hg
0.5gm of Sample Contain Hg=(0.04012/0.5)100 =8.024%
So, from the above calculation it is found that giloy contain 8.024% 0f Hg.
I RON ASSAY:-
Sample taken = 2gm
Final Burette Reading = 3.5 ml
Burette Reading = 3.5-0 = 3.5ml
Each ml of 1N KCrO is equivalent to 0.05585 gm of Iron
Calculation:-
Each ml of 1N KCrO = 0.05585gm of Fe
Each ml of 0.1N KCrO = (0.055850.1)gm of Fe
3.5ml of 0.1N KCrO = (0.05585gm Fe3.50.1)gm Fe.
=0.01954gm of Fe
So,250 ml of sample contain (0.01954 10)gm of Fe = 0.1954gm of Fe
So,2gm of sample contain 0.1954gm of Fe.
Therefore % of Fe =( Amount of Fe in 2gm of sample/sample wt)100
=( 0.1954/2)100 = 9.77%
So From the above calculation it is found that giloy contain 9.77% of Iron.


CONTROL DRUG DELIVERY
As 4-5 ml of aliquot is used as a replenished medium so the below graphs estmiates the
plot of time interval with the release of drug.The absorbance is recorded by U.V
spectrophotometer denoted by n[32].
pH 5.8 of 1% giloy
n=1.4205

pH 6 of 1% giloy
n=1.8148
0
0.5
1
1.5
2
2.5
3
3.5
4
0 0.2 0.4 0.6 0.8 1 1.2
l
o
g
(
m
t
)

log(t)
pH 5.8 0f 1% giloy film

Fig-3.0
pH 7 of 1% giloy
n=1.7366

Fig-3.1
pH 5.8 of 5% giloy
n=1.4544
0
0.5
1
1.5
2
2.5
3
3.5
4
0 0.2 0.4 0.6 0.8 1 1.2
l
o
g
(
m
t
)

log(t)
pH 6 of 1% giloy film
0
0.5
1
1.5
2
2.5
3
3.5
0 0.2 0.4 0.6 0.8 1 1.2
l
o
g
(
m
t
)

log(t)

Fig-3.2
pH 6 of 5% giloy
n=1.7866(absorbance)

Fig-3.3
pH 7 of 5%
n=1.66999(
0
0.5
1
1.5
2
2.5
3
3.5
4
0 0.2 0.4 0.6 0.8 1 1.2
l
o
g
(
m
t
)

log(t)
pH 5.8 of 5% giloy film
0
0.5
1
1.5
2
2.5
3
3.5
4
0 0.2 0.4 0.6 0.8 1 1.2
l
o
g
(
m
t
)

log(t)

Fig-3.4
pH 5.8 of 7.5% giloy
n=1.4829

Fig-3.4
pH 6 of 7.5% giloy
n=1.7718
0
0.5
1
1.5
2
2.5
3
3.5
0 0.2 0.4 0.6 0.8 1 1.2
l
o
g
(
m
t
)

log(t)
0
0.5
1
1.5
2
2.5
3
3.5
4
0 0.2 0.4 0.6 0.8 1 1.2
l
o
g
(
m
t
)

log(t)
pH 5.8 OF 7.5% GILOY FILM

Fig-3.5

Fig-3.6
pH 7 of 7.5% giloy
n=2.5048
0
0.5
1
1.5
2
2.5
3
3.5
0 0.2 0.4 0.6 0.8 1 1.2
l
o
g
(
m
t
)

log(t)
pH 6 of 7.5% giloy film
0
0.5
1
1.5
2
2.5
3
3.5
0 0.2 0.4 0.6 0.8 1 1.2
l
o
g
(
m
t
)

log(t)
pH 7 of 7.5% giloy film

Fig-3.7
pH 5.8 of 10% giloy
n=1.4826

pH 6 of 10% giloy
n=1.7859
0
0.5
1
1.5
2
2.5
3
3.5
4
0 0.2 0.4 0.6 0.8 1 1.2
l
o
g
(
m
t
)

log(t)
pH 5.8 OF 10% GILOY FILM
-5000
0
5000
10000
15000
20000
25000
30000
35000
0 0.2 0.4 0.6 0.8 1 1.2
l
o
g
(
m
t
)

log(t)

Fig-3.5 pH 7 of 10% giloy
n=1.9828

Where n= absorbancy

0
0.5
1
1.5
2
2.5
3
3.5
0 0.2 0.4 0.6 0.8 1 1.2
l
o
g
(
m
t
)

log(t)

DISCUSSION AND CONCLUSION
CHAPTER 6


DISCUSSION AND CONCLUSION

Giloy( Tinospora cordifolia) was used for treatment of various skin dieases ,piles ,dibetes
swine flu,malaria,various other fever conjunctivitis ,Immunity Disorders, Stomach
UlcerUrinary disorder Mental Disorder enzymes.
So it was very important for human being. During the above experiments when the
dissolution experiments were carried out and are observed with the absorbancy methods.the
rates of dissolution that are termed at least possible P.H dissolved and disintegrated at a
faster rate. The total ash value is an indicative of total amount of inorganic material after
complete incineration and the acid insoluble ash value is an indicative of silicate impurities,
which might have arisen due to improper washing of drug.The loss on drying value obtained
is an indicative of amount of moisture content present in the drug. The extractive values
names water soluble and alcohol soluble indicates the amount of active constituent in given
amount of plant material when extracted with respective solvent, values obtained supports
the fact that drug is unexhausted which is contrary to lower extractive value[6].
In summary, a very simple, basic method was developed to fabricate drug-loaded
biocompatible polymeric films by directly spreading polymer/drug solution on the
nonsolvent surface. Tamarind polymer were used as the model polymer and drug,
respectively.
By controlling the weight ratio of giloy, different drug loading percentage films can be
prepared. The drug release behaviors of the as-prepared products show their potential
applications in a drug controlled delivery system. The release rate of drug(giloy) from these
films into phosphate buffered solutions appeared to depend on a number of factors including
drug loading content and the pH of the release mediums. This method can easily be scaled
up and potentially extended to the fabrication of other drug-loaded composites for the
application in drug delivery systems.Also giloy has shown anti-microbial activity that
extends the purpose of being only a preparative medicine in various parts of world it is used
extensively for curing illness by simply warming the tuber roots of the plant or chewing the
stem and leaf of the plant.Present study simply enlightens the fact that the herbal drug can be
formulated in different ways and can be pelleted by using different polymer systems and
based on which the coating and pelleting can be done[32].
As we are discussing the In-vitro release rate of sublingual tablets will becarried out using
United State Pharmacopoeia (USP) XXIV dissolution testing apparatus (Paddle method). A
aliquot sample of the solution is withdrawn from the dissolution apparatus. The samples are
replaced with fresh dissolution medium of same quantity. The samples are filtered through
Whatman filter paper No 40 and analyzed in UV spectrophotometer. The percentage drug
release is calculated using an equation obtained from the calibration curve[5]


CHAPTER 7
REFERNCES


REFERENCES
[1].Wikipedia.The free Encyclopedia http://en.wikipedia.org/wiki/Arthritis

[2] Wikipedia.The free Encyclopedia
http://en.wikipedia.org/wiki/Drug_delivery

[3] An Overview on: Sublingual Route for Systemic Drug Delivery K. Patel
Nibha1 and SS. Pancholi2*1Department of Pharmaceutics, BITS Institute of
Pharmacy, Gujarat Technological university, Varnama, Vadodara, Gujarat, Ind.

[4]Standardization and phytochemical evaluation of tinospora cordifolia
(willd.) miers. (menispermaceae) shivani tanwar1*, jainendra jain1, sristi
verma1, deepa solanki2

[5]Quality control methods for herbal materials.Updated edition of Quality
control methods for medicinal plant materials, 1998 1. Plants, Medicinal. 2.
Medicine, Herbal. 3. Medicine, Traditional. 4. Quality control. 5. Manuals. I.
World Health Organization.
ISBN 978 92 4 150073 9
[6]Evaluation and Standardization of Marketed Polyherbal Formulation
Arogya Vati Yogendr Bahuguna*, Jaseem Saqib, Praveen Kumar, Ashutosh
Badola

[7].Alteration of lethal effects of gamma rays in Swiss albino mice by
Tinospora cordifolia. Pahadiya S, Sharma J. Phytother Res. 2003
May;17(5):552-4
[8]Evaluation of the antineoplastic activity of guduchi (Tinospora cordifolia) in
Ehrlich ascites carcinoma bearing mice. Grover J.K, Rao SK

[9].Tinospora cordifolia induces enzymes of carcinogen/drug metabolism

and antioxidant system, and inhibits lipid peroxidation in mice.Singh RP,
Banerjee S, Kumar PV, Raveesha KA, Mary N.K. Phytomedicine. 2006
Jan;13(1-2):74-84. Epub 2005 Jun 29

[10] Sharma.A.Antifertility effect of Tinospora cordifolia (Willd.) stem extract
in male rats. GuptaRS,IndianJExpBiol.2003Aug;41(8):885-9.
[11] Radioprotective potential of an herbal extract of Tinospora cordifolia.
Goel HC, Prasad J, Singh S, Sagar RK, Agrawala PK, Bala M, Sinha AK,
Umamaheswari.S. J Radiat Res (Tokyo). 2004 Mar;45(1):61-8.

[12] Anti-hyperglycemic effect of Eugenia jambolana and Tinospora cordifolia
in experimental diabetes and their effects on key metabolic enzymes involved
in carbohydrate metabolism.J K Grover, V Vats, S S Rathi

[13] Restoration of antioxidant defence by ethanolic Tinospora cordifolia root
extract in alloxan-induced diabetic liver and kidney.Prince PS, Padmanabhan
M, Mathew S.

[14] Effect of alcoholic extract of Ayurvedic herb Tinospora cordifolia on the
proliferation and myeloid differentiation of bone marrow precursor cells in a
tumor-bearing host.Singh SM, Singh N, Shrivastava P.

[15] Immune stimulating properties of a novel polysaccharide from the
medicinal plant Tinospora cordifolia. Nair PK, Rodriguez S, Ramachandran R,
Alamo A, Thatte.U.L Int Immunopharmacol. 2004 Dec 15;4(13):1645-59.

[16] Rubia cordifolia, Fagonia cretica linn and Tinospora cordifolia exert
neuroprotection by modulating the antioxidant system in rat hippocampal slices
subjected to oxygen glucose deprivation.Rawal AK, Muddeshwar MG,
Chaudhry R.

[17] Comparative evaluation of hypoglycaemic activity of some Indian
medicinal plants in alloxan diabetic rats.Ajit Kar,B K Choudhary,N G
Bandyopadhyay Satsang Herbal Research and Analytical Laboratories, PO
Satsang-814 116 Deoghar, India. Journal of Ethnopharmacology (Impact
Factor: 2.76). 02/2003; 84(1):105-8.
[18] Nemmani KV, Jena GB, Dey CS, Kaul CL, Ramarao P (2002) Cell
proliferation and natural killer cell activity by poly herbal formulation, Immu-
nity in mice. Indian J Exp. bioi. 40 (3): 282-287.

[19] Antioxidant and Lipophilic Constituents of Tinospora crispa. Cavin A,
Hostettmann K, Dyatmyko W, Dhuley J.N. Planta Med. 1998 Jun;64(5):393-6.

[20]. Hepatoprotective and immunomodulatory properties of Tinospora
cordifolia in CCl4 intoxicated mature albino rats. Bishayi B, Roychowdhury S,
Ghosh S, Wadood N. J Toxicol Sci. 2002 Aug;27(3):139-46

[21] Effect of Tinospora cordifolia on the antitumor activity of tumorassociated
macrophages-derived dendritic cells.Singh N, Singh SM, Shrivastava P Int
Immunopharmacol. 2004 Dec 15;4(13):1569-75.

Effect of Tinospora cordifolia on the cytokine profile of angiogenesisinduced
animals.Leyon PV, Kuttan G.

[22] Tinospora cordifolia induces enzymes of carcinogen/drug metabolism and
antioxidant system, and inhibits lipid peroxidation in mice. Singh RP, Banerjee
S, leyon PV, Raveesha KA, Rao AR.

[23] Evaluation of the antineoplastic activity of guduchi (Tinospora cordifolia)
in Ehrlich ascites carcinoma bearing mice.Jagetia GC, Rao SK.

[24] Effect of Tinospora cordifolia on the antitumor activity of tumorassociated
macrophages-derived dendritic cells.
Singh N, Singh SM, Shrivastava P


.[25]. A study of drug release from homogeneous PLGA microstructures. J
Contr Release. Acharya G, Shin CS, Atal C,K, et al 2010;146:201206.

[26] Restoration of antioxidants by ethanolic Tinospora cordifolia in
alloxaninduced diabetic Wistar rats.Prince P, Kamalakkannan N, Stanley
Manzen.

[27] The influence of ph on drug release from metformin hcl matrices
containing different grades of hydroxypropyl methyl cellulose Masheer Ahmed
Khan,Gangan V.D.

[28] ] Effect of alcoholic extract of Ayurvedic herb Tinospora cordifolia on the
proliferation and myeloid differentiation of bone marrow precursor cells in a
tumor-bearing host.Singh SM, Singh N, Jaffer N.

[29] Quality control methods for herbal materials.Updated edition of Quality
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[30] Evaluation and Standardization of Marketed Polyherbal Formulation
Arogya Vati Yogendr Bahuguna*, Jaseem Saqib, Praveen Kumar, Ashutosh
Badola

[31] Standardization and phytochemical evaluation of tinospora cordifolia
(willd.) miers. (menispermaceae) shivani tanwar1*, jainendra jain1, sristi
verma1, deepa solanki2

[32] Modeling and comparison of dissolution profilesPaulo Costa*,
JoseManuel Sousa Lobo


APPENDIX
REAGENTS AND SOLUTIONS

1) 0.1N AgNo:-Dissolve 17gm of AgNo in sufficient amount of destiled water and make
the volume 100 ml.
Standardisation:- Titrate the above prepared solution with 0.1N Nacl using KCrO
(potassium dichromate) as an indicator to slight and Red pipette is found.
Calculation:- Actual Normality of AgNo = Normality of Nacl 10/Burette Reading.
2).1N KCrO:- A 7% w/v solution of KCrO dissolve 4.9gm.of Potassium dichromate in
sufficient water to produce 100 ml.
Standardisation:- 20ml of 0.1N sodium thiosulphate add in 1gm.of potassium iodide and 7ml
of 2M Hcl add in 250ml of water and titrate with above prepared solution using 3ml of
starch indicator until the colour changes from blue to light green or green to blue.
Calculation:-Actual Normality of KCro =
Normality of NaSO20
3) 0.1M EDTA:-Dissolve 37.225gm of disodium EDTA in a small amount of destiled water
and make up to 1000ml of destiled water.
Standardisation:-Weigh 200mg or 2gms of CaCo in a 250ml of conical flask and add 50 ml
of HO and then 2ml of dilute Hcl added till solution dissolves then it is boiled on a hot plate
till effervescence(CaCo gas) evolved, then cooled and 10ml of dilute ammonia solution
along with 15ml of ammonia ammonium chloride buffer(NHClNH) was added to render
the solution highly Alkalines then a pinch of 10ml of Erechrome black Tea indicator was
added and immediately titrated above prepared EDTA solution.
The end point sharp change in the colour mostly pink to blue or blue to pink.
Calculation:-1ml of 0.01M EDTA = 1mg.CaCo
Actual Normality of EDTA = The artificial Normality(0.02N)Mass of neutralizing
sample(2gm CaCo)/Eqquivalence factor of sample(0.001gm)Actual burette reading

4) Ammonia Ammonium chloride buffer:-Dissolve 67.5gm.of Ammonium chloride in
200ml of water and add 570ml of strong ammonia solution and dilute with HO to 1000ml.
5) Ammonia Buffer(pH 10.0):-Dissolve 5.4 gm of Ammonium chloride in 20ml of HO, and
35 ml of 10M Ammonia solution and dilute with water to 100ml.
6 )0.1N NaSO:-Dissolve 24.82 or 25gm of NaSO in small amount of water along with
0.1gm of sodium carbonate as preservative and make upto 1000 ml destiled water.
Standardisation of NaSO:-weighy .2gm of Pottasium dichromate and add 100ml of
destiled water then 7ml concentrated Hcl are added and again added 25ml of 10% of
potassium iodide,finally titrate with the sodium theosulfate above prepared solution using
starch indicator.
Calculation:-Normality of NaSO = Weight of KCrO/.0490Burette reading.
7) 0.1N NaOH:-Dissolve 4gm of pure NaOH in sufficient destiled water and make it volume
1000ml.
Standardisation:-Take 20 ml of above prepared solution in 200ml of conical flask and titrate
with 0.1N Hcl using Phenopthaline as an indicator.
Calculation:-Normality of NaoH = Normality of HclBurette Reading/20
8)Methyl Orange Indicator:-Dissolve .1gm of methyl orange in some of HO and add
sufficient ethanol(95%) to produced 100ml.
9)Methyl Red Indicator:-Dissolve 50mg(0.05) of Methyle red in a mixture of 1.86ml of .1M
NaOH and 50ml of ethanol(95%).Add sufficient amount of HO to produce 100ml.
10)Ferricallum Indicator:-Prepare a saturated solution of Ferric ammonium sulfate in HO
about 40% and add a few drops of 6N HNO.
11)Phenopthaline Indicator:-Dissolve 1gm of purest grade phenopthaline in 50ml of Alcohol
and dilute with water to 100ml.Add .1N NaOH solution until a faint pink colour
appears.Remove this colour with ,1N Acid(HSo).
12)Starch Indicator:-Dissolve 1gm of arrow root starch tapioca starch in small amount of
water and add this content to 100ml of boiled distilled water contains stirring and then
cooled.
13) Triethanol amine 20%:-Dissolve 20 ml of Triethanol amine in a small amount of
distilled water make upto 100ml volume.

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