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Journal of Ethnopharmacology 90 (2004) 99–103 Evaluation of antiplaque activity of Azadirachta indica leaf extract
Journal of Ethnopharmacology 90 (2004) 99–103 Evaluation of antiplaque activity of Azadirachta indica leaf extract

Journal of Ethnopharmacology 90 (2004) 99–103

Journal of Ethnopharmacology 90 (2004) 99–103 Evaluation of antiplaque activity of Azadirachta indica leaf extract

Evaluation of antiplaque activity of Azadirachta indica leaf extract gel—a 6-week clinical study

M. Raveendra Pai ,1 , Leelavathi D. Acharya, N. Udupa

Department of Pharmaceutics, College of Pharmaceutical Sciences, Manipal Academy of Higher Education, Manipal 576119, India

Received 30 January 2003; accepted 22 September 2003

Abstract

Various chemical agents have been evaluated over the years with respect to their antimicrobial effects in the oral cavity; however, all are associated with side effects that prohibit regular long-term use. Therefore, the effectiveness of neem (Azadirachta indica A. Juss) leaf extract against plaque formation was assessed in males between the age group of 20–30 years over a period of 6 weeks. Present study includes formulation of mucoadhesive dental gel containing Azadirachta indica leaf extract (25 mg/g). A 6-week clinical study was conducted to evaluate the efficacy of neem extract dental gel with commercially available chlorhexidine gluconate (0.2% w/v) mouthwash as positive control. Microbial evaluation of Streptococcus mutans and Lactobacilli species was carried out to determine the total decrease in the salivary bacterial count over a period of treatment using a semi-quantitative four quadrant streaking method. The results of the study suggested that the dental gel containing neem extract has significantly (P < 0.05) reduced the plaque index and bacterial count than that of the control group. © 2003 Published by Elsevier Ireland Ltd.

Keywords: Gels; Neem; Azadirachta indica; Chlorhexidine; Plaque index; Salivary microbial count

1. Introduction

Dental diseases are recognized as major public health problem throughout the world. Numerous epidemiological studies showed that the diseases such as tooth decay and dis- eases of the periodontum are among the most common afflic- tions of mankind (Mcdougall, 1963). The studies conducted throughout the world indicated a high correlation between poor oral hygiene, dental plaque, prevalence and the severity of periodontal diseases. Teeth and their supporting structure, the gums (gingiva) are subjected to infection by Streptococ- cus bacteria that causes cavities and pyorrhea which, if left untreated, can eventually lead to gingivitis. Recent studies suggest that such chronic low grade localized infections such as gingivitis or pyorrhea contribute to heart disease (Hujoel et al., 2002) and coronary heart disease rate was found to increase drastically with the patients suffering from chronic periodontitis (Beck et al., 1996). So, treatment of localized oral infections gained more priority in the modern world.

Corresponding author. Present address: NDDS Department, Zydus Re- search Centre, Sarkhej-Bavla NH 8A, Moraiya, Changodhar, Ahmedabad 382213, India. Tel.: +91-79-3750802; fax: +91-79-3750606. E-mail address: raveendrapai@yahoo.co.in (M.R. Pai). 1 Tel.: +91-820-2571201x2482.

0378-8741/$ – see front matter © 2003 Published by Elsevier Ireland Ltd.

doi:10.1016/j.jep.2003.09.035

The most essential type of dental care begins at home. Daily oral hygiene plays a vital role in maintaining healthy teeth and gums. Since dental diseases are chronic, long-term treatment is often necessary. Neem has been used in India and south Asia for thousands of years as the preferred tool for maintaining healthy teeth and gums. Brushing with neem twigs and chewing neem leaves and seeds after a meal has been the traditional dental care practice in this area. With available modern preparations many people are now using commercial products that contain the same basic neem com- ponents. The antibacterial activity of neem has been eval- uated and known from ancient times (Chaurasia and Jain, 1978; Chawla et al., 1994). Neem has been considered to have various activities such as astringent, antiseptic, insecti- cidal, anti ulcer and for cleaning the teeth in pyorrhoea and other dental diseases. Other than this leaf extract of the neem showed superior antiviral and antihyperglycemic activity in vitro and in vivo on animals (Chattopadhyay, 1999; Parida et al., 2002). Leaves of the neem have been used in the treat- ment of gingivitis and periodontitis (Husain et al., 1992). Neem has also showed better efficacy in the treatment of oral infections and plaque growth inhibition in treating pe- riodontal disorders (Patel and Venkatakrishna, 1988). Neem had showed good in vitro broad range antibacterial activity (Rao et al., 1986).

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M.R. Pai et al. / Journal of Ethnopharmacology 90 (2004) 99–103

Conventionally chlorhexidine gluconate (0.2%, w/v) mouthwash is available in the market for the treatment of oral infections. Chlorhexidine gluconate has good an- tibacterial efficacy against the microbes responsible for the oral infections (Emilson, 1994; Bowden, 1996). Although antibacterial effect of chlorhexidine gluconate is generally undisputed and well acknowledged, the mode of treatment and delivery system for maximal effectiveness is not yet fully clear. Evidently, therapeutic doses of the agent must be delivered at tooth surfaces with an established microbial flora for a sufficient period of time (Loesche, 1984). Lo- cal application of chlorhexidine gluconate in the form of gel showed greater efficacy (Emilson, 1981; Sennel et al., 2000). Based on the assumption of obtaining better efficacy of neem extract in the oral cavity when delivered in the form of gel, the present study was planned to develop a mucoad- hesive gel containing leaf extract. The study was planned to evaluate antiplaque activity by clinical and microbial evaluation of neem extract gel with commercially available chlorhexidine gluconate mouthwash as reference drug.

2. Materials and methods

Carbopol (934 P) for the preparation of the neem extract gel was procured from, The BF Goodrich Co., Cleveland. Neem extract was prepared from the dried leaves of neem collected from the medicinal garden of College of Pharma- ceutical Sciences, Manipal, India and dried under controlled parameters. The botonical identification of the leaves was done by Prof. K.K. Srinivasan (Department of Phytochem- istry, College of Pharmaceutical Sciences Manipal). The voucher specimen is conserved at phyto-medicinal herbar- ium of College of Pharmaceutical Sciences, Manipal, India, under the accession number UD109/01. Neem extract was prepared by macerating 20.0 g of dry powder of neem leaves with 100 ml of 70% (w/v) ethyl alcohol for a week in a round bottom flask with occasional shaking. The flask was kept under dark to avoid effect of light on the active ingredients of the neem. The extract was then filtered through a muslin cloth for coarse residue and finally through Whatman No. 1 filter paper, measured and kept in an airtight amber col- ored container. Gel formulation included neem extract 25%, Carbopol 934P 0.6%, Sorbitol 20.0% (sweetener), and pep- permint oil (<0.1%) as flavor and amaranth red color. The concentration of the neem extract in the gel formulation was restricted to 25 mg/g of the gel to fulfill the organoleptic (for patient compliance) properties of the final formulation, as neem is bitter drug. Stability study to evaluate the consistency of the gel over a period of 2 months was conducted by keeping the formu- lation at different conditions (4 C, 37 C and room temper- ature) and measuring the viscosity of the gel formulation at regular intervals. The viscosity was measured by Brooke- field synchro lectric viscometer. The TD bar spindle of LV series was employed for the measurement. The study indi-

cated that the viscosity of the carbopol gel did not change significantly throughout the stability period in the specified conditions.

2.1. Clinical evaluation of neem extract gel and

chlorhexidine mouthwash

Clinical evaluation of the products on the selected subjects was carried out with the help of a dentist in College of Den- tal Sciences, Manipal according to the guidelines of Decla- ration of Helsinki for biomedical research involving human subjects. Institutional ethical committee permission was ob- tained prior to commencing the study and all the individuals signed the informed patient consent form. The study includes assessing the baseline plaque index followed by use of the specified products. The study involved 36 subjects who un- der went inclusion/exclusion criteria were divided into three different groups containing twelve members in each group by restricted randomization, in such a way that the average baseline plaque index of each group remains fairly same. The study consisted of assessing the baseline plaque in- dex status according to criteria given by Silness and Loe (1964). Then the subjects were supplied with neem extract gel, chlorhexidine mouthwash and a placebo gel for appli- cation for a period of 6 weeks. After 3 and 6 weeks use of their assigned product, the examining dentist scored the subjects for plaque index. The same examiner at each ex- amination, to avoid inter-examiner variations scored all the subjects. The subjects were also asked about past systemic history to rule out any complications in the dental probing and treatment procedures. During the study period the sub- jects continued their normal daily hygiene practices. The groups were treated as follows:

Group I: placebo gel. Group II: chlorhexidine gluconate mouthwash (positive control). Group III: neem extract gel.

Subjects of the group II were asked to rinse 10 ml of chlorhexidine gluconate (0.2%, w/v) mouthwash in the oral cavity for 1 min and then to spit it out. Subjects of the groups I and III were asked to apply approximately one gram of the gel thoroughly in the oral cavity. All the subjects were asked to apply the dosage form after the breakfast in the morning and just before going to bed. The subjects were given demonstrations and trained regarding the application of the respective formulation to minimize the variation.

2.2. Microbiological evaluation

Streptococcus mutans and Lactobacilli species are the most common bacteria associated with the plaque forma- tion (Emilson, 1994; Emilson and Westergren, 1979). In the present study, reduction in the salivary bacterial count be- fore and after treatment was measured. The microbiological evaluation for Streptococcus mutans and Lactobacilli count

M.R. Pai et al. / Journal of Ethnopharmacology 90 (2004) 99–103

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was carried out in the Department of Microbiology, Kas- turba Medical College, Manipal. For recording the Streptococcus and Lactobacilli count, stimulated saliva was used. The subjects were asked to sim- ulate chewing action with sterile cotton rolls and asked to swallow the saliva thus collected over the next 1 min. This procedure was carried out in order to clear the mouth of any

residual saliva. The subjects were then made to chew the cotton roll for next 4 min and then made to expectorate into sterile penicillin bottles.

A semi-quantitative, four quadrant streaking method was

adopted. Using a standard loop, the samples were streaked

on to

1.

Mitis salivarius agar with bacitracin (for Streptococcus mutans);

2.

Lactobacillus MRS agar (for Lactobacilli).

The growth in all the four quadrants was recorded. Growth

in

each quadrant was recorded a score of >1 and with a

maximum score of >4 if all the quadrants showed growth.

3. Results

All the subjects entered the study have completed the

6-week clinical evaluation. Baseline plaque index of all the three groups is given in the Table 1. All the three groups were well balanced in relation to number of subjects, age and plaque index. No significant difference in plaque index was observed between three groups in the beginning of the study.

A two way ANOVA was applied to observe the statistical

significance of the study.

A comparison of mean plaque index for the three groups

after 3 weeks use of the products is given in Table 2. There

is a significant difference (P < 0.05) between the con-

trol group (1.396) and the treated groups in the plaque in-

Table 1 The mean baseline plaque index and standard deviations for the three study groups

Group no. Formulation

No. of

subjects index ± S.D.

Baseline mean plaque

I

II

III

12

Chx mouth wash (positive control) 12

Placebo gel

Neem-gel

12

1.550 ± 0.46 1.604 ± 0.26 1.588 ± 0.33

Chx: chlorhexidine gluconate.

Table 2 The mean 3-week plaque index and standard deviations for the three study groups

Group no.

Formulation

No. of

Mean plaque index ± S.D.

 

subjects

I

Placebo gel

12

1.396 ± 0.22

II

Chx mouth wash (positive control)

12

1.191 ± 0.25 a

III

Neem-gel

12

0.916 ± 0.28 a,b

a P < 0.05 vs. group I. b P < 0.05 vs. group II.

 

Table 3 The mean 6-week plaque index and standard deviations for the three study groups

Group no.

Formulation

No. of

Mean plaque index ± S.D.

 

subjects

I

Placebo gel

12

1.302 ± 0.28

II

Chx mouth wash (positive control)

12

0.823 ± 0.45 a

III

Neem-gel

12

0.423 ± 0.48 a,b

a P < 0.05 vs. group I. b P < 0.05 vs. group II.

 
3.5 3 2.5 2 1.5 1 0.5 0 Placebo gel Chx. Mouthwash Neem extract gel
3.5
3
2.5
2
1.5
1
0.5
0
Placebo gel
Chx. Mouthwash Neem extract gel
Pre-interventional
After 3weeks
After 6weeks
Average quadrant growth

Fig. 1. Streptococcus mutans count in the stimulated saliva before and after treatment with the formulations.

dex value. There is also a significant difference between the groups treated with neem extract dental gel (0.916) and chlorhexidine gluconate mouthwash (1.191) in reducing the plaque index. A comparison of mean plaque index for the three groups after 6 weeks of use of the products is given in the Table 3. There is a statistically significant (P < 0.05) difference be- tween the control group and the treated group as it was after 3-week study. The table clearly indicates a significant de- crease in the plaque index in the group treated with the neem extract gel (0.423) than that of the group treated with the commercially available chlorhexidine mouthwash (0.823). This suggests better efficacy of the neem extract applied in the form of mucoadhesive gel in reducing the plaque index than that of the commercially available chlorhexidine glu- conate mouth wash. The total decrease in salivary Streptococcus mutans and Lactobacillus species bacterial count is shown in the Figs. 1 and 2, respectively. The results of bacterial growth showed a significant (P < 0.05) difference between the pre-interventional bacterial count, and after 3 and 6 weeks treatment in both the groups. It can also be observed that the bacterial count was found to be reduced significantly in the groups treated with the neem extract gel compared to the chlorhexidine gluconate mouthwash.

4. Discussion

The Beginning of the periodontal disease occurs through the accumulation of a thin film of bacteria on the surface

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M.R. Pai et al. / Journal of Ethnopharmacology 90 (2004) 99–103

3.5 3 2.5 2 1.5 1 0.5 0 Placebo gel Chx. Mouthwash Neem extract gel
3.5
3
2.5
2
1.5
1
0.5
0
Placebo gel
Chx. Mouthwash
Neem extract gel
Average quadrant growth
Pre-interventional After 3weeks After 6weeks

Pre-interventional

Pre-interventional After 3weeks After 6weeks

After 3weeks

Pre-interventional After 3weeks After 6weeks

After 6weeks

Fig. 2. Lactobacilli count in the stimulated saliva before and after treatment with formulations.

of the teeth called plaque. Novel approaches were tried to deliver the drugs in different ways in treating such human ailments. Many antimicrobial agents were tried as mouth rinses and mouthwashes to control oral infections with poor to moderate degrees of success, except chlorhexidine which was proved to be dependable in reducing gingivitis and plaque formation (Emilson, 1994; Bowden, 1996). Conven- tionally 0.2% (w/v) chlorhexidine gluconate mouthwash is used for the treatment of oral infections. Chlorhexidine glu- conate gels (Emilson, 1981; Sennel et al., 2000) and films (Natalie et al., 1999) were evaluated as better delivery sys- tems for treating the local infections in the oral cavity. Though chlorhexidine was discovered in 1950s, is still con- sidered one of the most effective antiplaque agents in den- tistry. However, long term use of chlorhexidine is limited by its disagreeable taste and propensity to stain the teeth brown (Fardal and Turnbull, 1986). Therefore, the effective- ness of Azadirachta indica, leaf extract against plaque for- mation was assessed. Azadirachta indica, commonly known as “neem” belongs to the family Meliaceae and is widely dis- tributed in Asia and Africa. Almost every part of the tree was used in indigenous systems of medicine for the treatment of a variety of human ailments, particularly against diseases of bacterial and fungal origin (Randhawa and Parmar, 1996). Patel and Venkatakrishna (1988) studied the therapeutic use neem in periodontal disorders in India. Neem showed better efficacy in reducing the human plaque cultures and gram-negative bacteria compared to the commercially avail- able dentifrice. Rao et al. (1986), described the in vitro antibacterial activity of the neem oil on different bacterial pathogens isolated from varied clinical sources. Due to bit- ter taste of the drug the over all usage of the neem in various commercial preparations was restricted. So, the neem ex- tract gel was formulated along with the sweetener and flavor to increase the patient compliance and acceptability. In the present study, carbopol was used as a gelling poly- mer due to its mucoadhesive property. Novel approaches were tried using carbopol as gelling agent in delivering the drug through oral mucosa (Ishida et al., 1983; Bremecker et al., 1984). To compare the efficacy of the gel formula- tion, carbopol gel containing neem extract was prepared. The

efficacy of the neem extract gel was evaluated by clinical and microbiological study with the commercially available chlorhexidine mouthwash. The present study was found to support the earlier similar studies conducted to determine the efficacy of the gel formulations over conventional dosage forms delivering the drug locally in the oral cavity. Clin- ical evaluation of the neem extract gel over a 6-week pe- riod showed (P < 0.05) significant reduction in the plaque index and was found to show better activity than that of the placebo group and the group treated with chlorhexidine mouthwash (Tables 1–3). Microbial count in the saliva was found to be reduced significantly by the neem extract gel (Figs. 1 and 2). The observed efficacy of the neem extract gel could be attributed to decreased flow of the saliva during overnight (Edgar, 1992) and due to slow release of the drug from the viscous matrix of the gel formulation maintaining the drug concentration well above the therapeutic concen- tration. This could also be due to mucoadhesive property of the carbopol that stays in the oral cavity for a prolonged period prolonging the drug action. Chlorhexidine gluconate mouthwash was also successful in reducing the plaque in- dex and microbial count compared to the placebo group. It is clear from the study that by regularly applying the gel formulation the total duration of the therapy can be reduced to a greater extent with high patient compliance. This study also showed certain advantages relative to conventional ther- apy such as maintaining effective levels of an antimicrobial agent locally for a prolonged period and can be useful for application in children below the age group of 6 years, who cannot control their swallowing reflex effectively and may swallow anywhere ranging from 100% of the mouthwash. So, the gel formulation can be used with high success rates for such patients in treating oral infections. This study showed greater efficacy of the neem against the local oral infections when applied in the gel form. However, for commercialization of this product, further studies are required with large number of patient populations.

5. Conclusion

This study establishes the use of neem in treating the oral infections by inhibiting the plaque growth as claimed by the traditional medicine. Neem extract gel formulated with a mucoadhesive polymer can significantly reduce the duration of the therapy in treating the oral infections and controlling the microbes responsible for the dental disorders. Present study provided more insights on its activity for dental care.

Acknowledgements

We are thankful to Prof. Mahalinga Bhat, Head Depart- ment of Periodontics and Dr. P. Sugandhi Rao, Additional Professor, Department of Microbiology, Manipal Academy of Higher Education, Manipal, India for their immense sup-

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port in successfully conducting the clinical and microbio- logical studies.

References

Beck, J., Garcia, R., Heiss, G., Vokonas, P.S., Offenbacher, S., 1996. Peri- odontal disease and cardiovascular disease. Journal of Periodontology 67, 1123–1137. Bowden, G.H., 1996. Mutans streptococci caries and chlorhexidine. Jour- nal of Canadian Dental Association 62, 703–707. Bremecker, K.D., Strempel, H., Klein, G., 1984. Novel concept for a mucosal adhesive ointment. Journal of Pharmaceutical Sciences 73,

548–552.

Chattopadhyay, R.R., 1999. Possible mechanism of antihyperglycemic ef- fect of Azadirachta indica leaf extract: part V. Journal of Ethnophar- macology 67, 373–376. Chaurasia, S.C., Jain, P.C., 1978. Antibacterial activity of essential oils of four medicinal plants. Indian Journal of Hospital Pharmacy, 166–

167.

Chawla, A.S., Kumar, M., Bansal, I., 1994. Chemical constituents and biological activity of neem—a review. Indian Drugs 32, 57–

62.

Edgar, W.M., 1992. Saliva: its secretion, composition and functions. British

Dental Journal 25, 305–312. Emilson, C.S., 1981. Effect of chlorhexidine gel treatment on Streptococ- cus mutans population in human saliva and dental plaque. Scandanav- ican Journal of Dental Research 86, 239–246. Emilson, C.G., 1994. Potential efficacy of chlorhexidine against mutans streptococci and human dental caries. Journal of Dental Research 73,

682–691.

Emilson, C.G., Westergren, G., 1979. Effect of chlorhexidine on the relative proportions of Streptococcus mutans and Streptococcus sanguis in hamster plaque. Scandanavican Journal of Dental Research 87, 288–

295.

Fardal, D., Turnbull, R.S., 1986. A review of the literature on use of chlorhexidine in dentistry. Journal of American Dental Association 112, 863–869. Hujoel, P.P., Drangsholt, M., Spiekerman, C., DeRouen, T.A., 2002. Pre-existing cardiovascular disease and periodontitis: a follow-up study. Journal of Dental Research 81, 186–191. Husain, A., Virmani, O.P., Popli, S.P., Misra, L.N., Gupta, M.M., Sri- vastava, G.N., Abraham, Z., Singh, A.K., 1992. Dictionary of Indian Medicinal Plants. Vap Enterprises, New Delhi, pp. 62–63. Ishida, M., Nambu, N., Nagai, T., 1983. Highly viscous gel ointment containing carbopol for application to the oral mucosa. Chemical and Pharmaceutical Bulletin 31, 4561–4564. Loesche, W.J., 1984. Antimicrobials. Can they be effective? In: Guggen- heim, B. (Ed.), Cardiology Today. Karger, Basel, pp. 293–300. Mcdougall, H.A., 1963. Studies on the dental plaque. The histology of dental plaque and its attachment. Australian Dental Journal 8, 261–265.

Natalie, J., Dough, W.H., Michael, J., Rathbone, , Jones, D.S., Tucker, I.G.,

1999. Local delivery of chlorhexidine using a tooth-bonded delivery

system. Journal of Controlled Release 61, 337–343. Parida, M.M., Upadhyay, C., Pandya, G., Jana, A.M., 2002. Inhibitory potential of neem (Azadirachta indica Juss) leaves on Dengue virus type-2 replication. Journal of Ethnopharmacology 79, 273–278. Patel, V.K., Venkatakrishna, B.H., 1988. Folklore therapeutic indigenous plants in periodontal disorders in India (review). International Journal of Clinical Pharmacology and Therapeutic Toxicology 26, 176–184. Randhawa, N.S., Parmar, B.S., 1996. Neem, second ed. New age Inter- national Pvt. Ltd. Publishers, India, pp. 77–111. Rao, D.V.K., Sing, I., Chopra, P.C., Chhabra, , Ramanujalu, G., 1986. In vitro antibacterial activity of neem oil. Indian Journal of Medical Research 84, 314–316.

Sennel, S., Ikinci, G., Kas, S., Yousefi, R.A., Sargon, M.F., Hincal, A.A.,

2000. Chitosan films and hydrogels of chlorhexidine gluconate for oral

mucosal delivery. International Journal of Pharmaceutics 193, 197–203. Silness, J., Loe, H., 1964. Periodontal in children. Periodontal disease in pregnancy. II. Correlation between oral hygiene and periodontal condition. Acta Odontologica Scandinavica 22, 121–135.