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Journal of Ethnopharmacology 90 (2004) 99103

Evaluation of antiplaque activity of Azadirachta indica


leaf extract gela 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 2030 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 efcacy 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 signicantly (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 periodontumare among the most common afic-
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 lowgrade 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.
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 efcacy 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).
0378-8741/$ see front matter 2003 Published by Elsevier Ireland Ltd.
doi:10.1016/j.jep.2003.09.035
100 M.R. Pai et al. / Journal of Ethnopharmacology 90 (2004) 99103
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 efcacy 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
ora for a sufcient period of time (Loesche, 1984). Lo-
cal application of chlorhexidine gluconate in the form of
gel showed greater efcacy (Emilson, 1981; Sennel et al.,
2000). Based on the assumption of obtaining better efcacy
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 identication 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 ask with occasional shaking. The ask was kept
under dark to avoid effect of light on the active ingredients
of the neem. The extract was then ltered through a muslin
cloth for coarse residue and nally through Whatman No.
1 lter 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 avor and amaranth red color. The
concentration of the neem extract in the gel formulation was
restricted to 25 mg/g of the gel to fulll the organoleptic (for
patient compliance) properties of the nal 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-
eld 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
signicantly throughout the stability period in the specied
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
specied 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) 99103 101
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 signicant 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
signicance 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 signicant 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
Baseline mean plaque
index S.D.
I Placebo gel 12 1.550 0.46
II Chx mouth wash (positive control) 12 1.604 0.26
III Neem-gel 12 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
subjects
Mean plaque
index S.D.
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
subjects
Mean plaque
index S.D.
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.
0
0.5
1
1.5
2
2.5
3
3.5
Placebo gel Chx. Mouthwash Neem extract gel
A
v
e
r
a
g
e

q
u
a
d
r
a
n
t

g
r
o
w
t
h
Pre-interventional After 6weeks After 3weeks
Fig. 1. Streptococcus mutans count in the stimulated saliva before and
after treatment with the formulations.
dex value. There is also a signicant 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 signicant (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 signicant 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 efcacy 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 signicant (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 signicantly 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 lm of bacteria on the surface
102 M.R. Pai et al. / Journal of Ethnopharmacology 90 (2004) 99103
0
0.5
1
1.5
2
2.5
3
3.5
Placebo gel Chx. Mouthwash Neem extract gel
A
v
e
r
a
g
e

q
u
a
d
r
a
n
t

g
r
o
w
t
h
Pre-interventional After 6weeks After 3weeks
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 lms
(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 efcacy 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 avor
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 efcacy of the gel formula-
tion, carbopol gel containing neemextract was prepared. The
efcacy 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 efcacy 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) signicant 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 13). Microbial count in the saliva was
found to be reduced signicantly by the neem extract gel
(Figs. 1 and 2). The observed efcacy of the neem extract
gel could be attributed to decreased ow 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 reex 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 efcacy 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 signicantly 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-
M.R. Pai et al. / Journal of Ethnopharmacology 90 (2004) 99103 103
port in successfully conducting the clinical and microbio-
logical studies.
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