Continental J. Pharmacology and Toxicology Research 2: 1 - 5, 2008.

© Wilolud Online Journals, 2008.

A COMPARATIVE EVALUATION OF 0.3% EYE DROPS AND IN SITU FORMING GEL OF PEFLOXACIN
MESYLATE IN EXPERIMENTALLY INDUCED PSEUDOMONAS CONJUNCTIVITIS

Suman Yadav1 and Nayyar Parvez2

1
Department of Chemistry, Advanced Institute of Technology, MD University Rohtak, Distt. Faridabad, Haryana, India.
2
Department of Pharmaceutics, Advanced Institute of Pharmacy, MD University Rohtak, Distt. Faridabad, Haryana, India.

ABSTRACT
The authors evaluated and compared the efficacies of eye drops and in situ forming gel of pefloxacin
mesylate in pseudomonas induced conjunctivitis in 40 rabbits. The in-situ forming gel of pefloxacin
mesylate was formulated as a liquid and when administered in the cul-de-sac, it gets converted to gel.
The objective of the present research work was to demonstrate the in vivo activity of in situ gelling
formulation and compared with marketed eye drops. No difference was obtained in all parameters of
conjunctivitis on day 0, between in situ gelling formulation (Group I) and marketed eye drops (Group
II), indicating that severity of conjunctivitis was similar initially between the two groups. Comparison
of scores obtained with marketed eye drops and in situ gel formulation indicated that in situ gelling
formulation was faster in relieving symptoms of conjunctivitis. The findings of the present study
indicate that in situ forming gel of pefloxacin mesylate is a potential delivery system for the treatment
of pseudomonas induced conjunctivitis.

KEY WORDS: Pefloxacin mesylate, conjunctivitis, in situ forming gel.

INTRODUCTION
Pseudomonas aeruginosa (P.aeruginosa) causes severe and rapid ocular infection and is one of the most common cause of
bacterial conjunctivitis (Wilson and Ahearn, 1977).

The mainstay of medical therapy for P.aeruginosa induced conjunctivitis is aminoglycosides. But they suffer from
disadvantages of limited spectrum, poor ocular penetration, high cost and side effects (Fuginele et al, 1965, Bowe et al,
1991). Emergence of aminoglycoside resistant pseudomonas strain is an added drawback for their use in P. aeruginosa
conjunctivitis Flouroquinolones are one of the promising group of antibiotics, already demonstrated to possess good anti-
pseudomonas activity. This group includes enoxacin, norfloxacin, ciprofloxacin, ofloxacin, and more recently gatifloxacin.
These antibiotics are currently being used topically to treat conjunctivitis and corneal ulcers, but suffers from disadvantages
of having less ocular penetration on the topical use when corneal epithelium is intact and low intra-ocular penetration when
given orally or parenterally.

Pefloxacin is newer flouroquinolone bactericidal antibiotic (1-ethyl, 6-flouro, 7-[4-methyl] –1-piperanyzyl-4-oxo-1, 4-

dihydro-3-quinolen carboxylic acid), and has been proved to possess superior antibacterial activity in vivo and has better
pharmacokinetic propereties as compared to other quinolones, including norfloxacin, ciprofloxacin and ofloxacin. Although
pefloxacin has been advocated primarily for oral and parenteral uses, it has the potential for topical ophthalmic use to treat
ocular infections (Gonzalez et al, 1989; Hobden et al, 1990; Sultana et al, 2004).

The authors evaluated and compared efficacies of pefloxacin mesylate eye drops and in situ forming gels in rabbit model of
experimentally induced pseudomonas conjunctivitis.

MATERIALS AND METHODS

Permission for the use of animals for the study was obtained from Jamia Hamdard Animal Ethics Committee. Forty healthy
adult albino rabbits of either sex, weighing 1 to 2 kg, having sterile cultures of conjunctival swabs taken from both eyes were
selected for the study. The rabbits were observed for one week to rule out any systemic and ocular diseases, particularly
infections. They were divided randomly into two groups of 20 rabbits each. The research was carried out at Jamia hamdard,
New Delhi, India. ( P<0.05)

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 Suman Yadav and Nayyar Parvez: Continental J. Pharmacology and Toxicology Research 2: 1 - 5 , 2008

Table 1: Mean score ± S.D. for grading of parameters of conjunctivitis viz. Redness, Lacrimal secretion, Mucoid discharge,
Response to ocular therapy and Swelling of eyelid in Group I of rabbits treated with marketed eye drops, n = 20.

S.No Parameter Mean score + S.D.

Day 0 Day 3 Day 5
1. Redness 4±0 0.947 ± 0.458 0.421 ± 0.51
2 Lacrimal secretion 3±0 0.75 ± 0.436 0.2 ± 0.402
3 Mucoid discharge 3±0 0.4 ± 0.656 0.15 ± 0.366
4 Response to 2±0 0.3 ± 0.477 0.15 ± 0.366
ocular stimulus
5 Swelling of eyelid 2±0 0.15 ± 0.307 0.05 ± 0

Table 2: Mean score ± S.D. for grading of parameters of conjunctivitis viz. Redness, Lacrimal secretion, Mucoid discharge,
Response to ocular therapy and Swelling of eyelid in Group II of rabbits treated with in situ forming gel, n = 20.

S.No Parameter Mean score + S.D.

Day 0 Day 3 Day 5
1. Redness 4±0 0.157 ± 0.41 0.05 ± 0
2 Lacrimal secretion 3±0 0.2 ± 0.402 0.05 ± 0
3 Mucoid discharge 3±0 0.15 ± 0.366 0.05 ± 0
4 Response to 2±0 0.2 ± 0.41 0.05 ± 0
ocular stimulus
5 Swelling of eyelid 2±0 0.05 ± 0 0.05 ± 0

Table. 3 Significance “t” test results of Group I (marketed eye drops) vs. Group II (in-situ forming gel) for improving the
symptoms of conjunctivitis in rabbit eye, viz. Redness, Lacrimal secretion, Mucoid discharge, Response to ocular therapy
and Swelling of eyelid.
S.No Parameter Significance t test results of Group I vs. Group II.
Day 0 Day 3 Day 5
1. Redness P > 0.999 P < 0. 0001 P = 0.0024
t=0 t = 5.747 t = 3.253
NS ES ES
2 Lacrimal secretion P > 0.999 P = 0.0002 P = 0.1034
t=0 t = 4.148 t = 1.669
NS ES NS
3 Mucoid discharge P > 0.999 P = 0.1449 P = 0.2293
t=0 t = 1.488 t = 1.222
NS NS NS
4 Response to P > 0.999 P = 0.4814 P = 0.2293
ocular stimulus t=0 t = 0.7710 t = 1.222
NS NS NS
5 Swelling of eyelid P > 0.999 P = 0.1534 P > 0.999
t=0 t = 1.457 t=0
NS NS NS

ES = Extremely significant ; NS = Non significant.

Inoculation and Induction of conjunctivitis

A sub-culture from a recently isolated strain of P.aeruginosa sensitive to pefloxacin was obtained from B.V.Patel Chest
Institute, Delhi University, New Delhi, India. Bacterial conjunctivitis was induced in rabbits eye by exposing them to
bacterial strains.

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 Suman Yadav and Nayyar Parvez: Continental J. Pharmacology and Toxicology Research 2: 1 - 5, 2008

1
0.9
Scores of conjunctivitis

0.8 m a rk e t e d e y e
0.7 d ro p s
0.6
in s it u g e llin g
0.5 fo rm u la t io n s
0.4
0.3
0.2
0.1
0
Re dn e s s L a c r im a l M u c o id Res p on s e S w e llin g o f
s e c r e tio n d is c h a r g e to o c u la r e y e lid
s tim u lu s

P a r a m e te r s o f c o n j u n c ti v i ti s

F i g u r e 1 S c o r e s o f c o n j u n c ti v i ti s o f m a r k e te d e y e
d r o p s a n d i n si tu g e l l i n g fo r m u l a ti o n s o n d a y 3 o f
th e r a p y .

Grading of conjunctivitis and confirmation of diagnosis

Conjunctival swabs were taken from animals and sent to microbiology laboratory of our institution for confirmation of
P.aeruginosa as causitive microorganism

Parameters of conjunctivitis were graded as follows: Redness of the mucous membrane of the eye was observed visually and
grades were given from 0 to 4 i.e., 0 = absent; 1 = mild; 2 = moderate; 3= severe; 4= extensive; Lacrimal secretion: It was
graded from 0 to 3 as 0 = normal; 1= slightly more than normal and 2 = more than normal; 3= severe; Mucoidal discharge:
Whitish to yellowish white semi-solid discharge if any was noted and recorded as a grade of 0 to 3 in which 0 = absent; 1 =
little; 2 = more and 3 = extensive ; Response to ocular stimulus : It was assessed by throwing torch light on the eye from a
particular distance and noticing the response to this stimulus. It was graded from 0 to 2 as 0 = normal; 1 = fast; 2 = very fast;
Swelling of eye lid: It was graded from 0 to 2 as 0 = absent; 1 = slight; 2 = prominent.

Grading of parameters of conjunctivitis in Group I and II of animals are shown in Tables 1 and 2.

Drug formulation and therapy

Group I rabbits were treated with marketed eye drops of pefloxacin mesylate containing 0.3% w/v of drug and group II rabbit
were treated with in situ forming gel of pefloxacin mesylate containing 0.18 % of drug, 0.6 % of polymer Gelrite, 0.010 %
propyl paraben, 0.05 % methyl paraben in boric acid buffer of pH 4.7.

Criteria of conjunctivitis response to drug therapy

Decrease in redness, mucoid discharge, lacrimal secretion, response to ocular stimulus and swelling of eyelid were taken as
positive response to therapy. Observations were made to note any ocular or systemic side effects in all the rabbits.

Treatment effects were compared with those of the marketed formulations, and significance was determined using unpaired t
test. Significance levels were determined for P< 0.05, for two tailed test. The theoretical ‘t’ value is 2.306 (i.e., the table ‘t’
value) at this level of degrees of freedom. Treatment was given significance (S) if “t” value exceeded the table “t” value and
if it did not exceed then treatment was considered as non significant (NS). Results of the unpaired t test are shown in Table 3.

RESULTS
No difference was obtained in all parameters of conjunctivitis on day 0, between in situ gelling formulation (Group I) and
marketed eye drops ( Group II), indicating that severity of conjunctivitis was similar initially between the two groups.
Comparison of scores obtained with marketed eye drops and in situ gel formulation indicated that in situ gelling formulation
was faster in relieving symptoms of conjunctivitis. Results are shown in Tables 1 and 2.

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 Suman Yadav and Nayyar Parvez: Continental J. Pharmacology and Toxicology Research 2: 1 - 5, 2008

Significantly better responses in redness were obtained on day 3 (P < 0. 0001) and 5 ( P = 0.0024 ) with in situ gelling
formulation compared to marketed eye drops. Lacrimal secretion also showed significantly better response on day 3 (P =
0.0002 ), however on day 5 no significant difference was obtained with in situ gelling formulation compared to marketed eye
drops with respect to mucoid discharge, response to ocular therapy and swelling of eyelid. Result is shown in table 3.

The severity of conjunctivitis was appreciably lowered at day 3 with in-situ gelling formulation compared to marketed eye
drops.( figure 1)

Results of the unpaired t test of significance between two groups indicated that in situ gelling formulation showed better
response in treatment of conjunctivitis compared to marketed eye drops.

The in situ forming gels can be used as delivery systems for sustained release of incorporated drugs into the cul-de-sac.

DISCUSSION
The objective of the present research work was to demonstrate the in vivo activity of in situ gelling formulation and
compared with marketed eye drops. An improvement in the symptoms was observed for the in situ gelling

formulation. The infection was successfully treated with in situ gelling formulation in a much shorter time compared with
marketed eye drops.

Flouroquinolones are one of the promising group of antibiotics, already demonstrated to possess good anti-pseudomonas
activity. This group includes enoxacin, norfloxacin, ciprofloxacin, ofloxacin, and more recently gatifloxacin. These
antibiotics are currently being used topically to treat conjunctivitis and corneal ulcers, but suffers from disadvantages of
having less ocular penetration on the topical use when corneal epithelium is intact and low intra-ocular penetration when
given orally or parenterally.

Pefloxacin is a newer flouroquinolone bactericidal antibiotic and has similar activity on deoxyribonucleic acid (DNA)
gyrases to inhibit bacterial DNA replication as other flouroquinolones. It has wider antibacterial spectrum, better
bioavailability, better ocular penetration, and having least chances of developing resistance as compared to antibiotic of other
groups. For P.aeruginosa its in vitro minimum inhibitory concentration recorded were 4 mg per L for various strains that was
only second to ciprofloxacin.

The in situ forming gel of pefloxacin mesylate was formulated as a liquid and when administered in the cul-de-sac, it get
converted to gel. Significant quantities of gel were observed in the rabbit conjunctival sacs through 12 h. It was also observed
that the quantity of gel formed in the rabbit slowly diminished with time. Therefore drug is probably made available for
absorption partly by diffusion through the bulk of the gel and partly by uptake from surface of gel as it continously erodes. In
either case, the fact that the gel is well retained allows the drug to be absorbed over an extended time period, resulting in a
large increase in bioavailability.

ACKNOWLEDGEMENT
The authors are grateful to Late Hakeem Abdul Hameed Sahib, founder, Hamdard University, New Delhi, India. for the
facilities provided by him at the institute.

REFERENCES
Bowe, B.E., Snyden, J.W., Eiferman, R.A.(1991) An in vitro study of the potency and stability of fortified ophthalmic
antibiotic preparations. Am J Ophthalmol.,111 : 686-689.

Fuginele, F.P., Kiesel, R., Martyn, L.(1965) Pseudomonas infections of the rabbit cornea treated with gentamicin: a
preliminary report. Am.J.Ophthalmol.,60 : 818-822.

Gonzalez, J.P., (1989) Henwood, J.M., Peloxacin : a review of its antibacterial activity, pharmacokinetic properties and
therapeutic uses.Drugs.,37, 628-668.

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 Suman Yadav and Nayyar Parvez: Continental J. Pharmacology and Toxicology Research 2: 1 - 5, 2008

Hobden, J.A., Reidy, J.J., O’Callaghan, R.J., Insler, M.S. and Hill, J.M.(1990) Quinolones in collagen shields to treat
aminoglycoside- resistant pseudomonas keratitis. Invest. Ophthalmol.Vis.Sci.,31, 2241-2243.

Sultana, Y., Jha, M.C., Ali A., and Aqil, M. (2004) A three-way comparative study on the efficacy of twin sol to gel systems
and marketed eye drops of pefloxacin mesylate. J.Ocul.Pharmacol. therap.20(4) : 363-371.

Wilson, L.A., Ahearn, D.G. (1977) Pseudomonas induced corneal ulcers associated with contaminated eye mascara. Am. J.
Ophthalmol. 84 : 112-119.

Received for Publication: 10/03/2008

Accepted for Publication: 07/04/2008

Corresponding Author:
Dr Suman Yadav,
Department of Chemistry, Advanced Institute of Technology and Management, Palwal, Faridabad, Haryana, India.
Email: newchemistry2007@yahoo.com

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Continental J. Pharmacology and Toxicology Research 2: 6 - 11, 2008.
© Wilolud Online Journals, 2008.

INFLUENCE OF HONEY ON ADVERSE REACTIONS DUE TO ANTI-TUBERCULOSIS DRUGS IN PULMONARY

TUBERCULOSIS PATIENTS

Manju Sharma1, Khalid U. Khayyam2*, Vinay Kumar1, Faisal Imam1, KK Pillai1, D. Behera2
1
Department of Pharmacology, Faculty of Pharmacy Jamia Hamdard, New Delhi-110062 India.
2
Lala Ram Sarup Institute of Tuberculosis and Respiratory Diseases, New Delhi-110030, India.

ABSTRACT
The aim was to assess the influence of honey on adverse drug reactions induced by Anti-tuberculosis
(Anti-TB) drugs in newly diagnosed sputum acid fast bacilli (AFB) positive pulmonary tuberculosis
patients of category I receiving directly observed treatment short course (DOTS) under revised national
tuberculosis control programme (RNTCP) for a period of two months (i.e. intensive phase). A high
percentage of ADRs was experienced in control (69.88%) as compared to case (47.06%). Most of the
ADRs were mild to moderate and transient in nature. The most common adverse reactions reported
among control patients were anemia (21.16%), joint pains (15.6%), itching (18%) and nausea (14.4%).
The least percentage of adverse reactions among control patients reported was diarrhoea (2.4 %), while
there was no diarrhea observed in patients receiving honey along with anti-tubercular treatment (ATT).
Honey with ATT minimizes the adverse drug reactions induced by Anti-TB drugs in AFB positive
pulmonary positive tuberculosis patients. Thus, honey can be used as an adjuvant along with ATT in
pulmonary TB patients.

KEY WORDS: Tuberculosis, Adverse Drug Reactions, Honey, Direct Treatment Outcome, Revised
National Tuberculosis Control Program and Acid Fast Bacilli.

INTRODUCTION
According to World Health Organization (WHO) one third of the world’s population is infected with Mycobacterium
tuberculosis resulting in 8.4 million new tuberculosis cases in 1999 (WHO, 2001). India accounts for a fifth of the world’s
new TB cases and 2/3rd of the cases in South- East Asia. In India, pulmonary tuberculosis is one of the major causes of adult
deaths (Garner et al., 2004). This makes India the highest TB burden country in the world. As per WHO estimates in 2004,
370,000 persons in India died of tuberculosis (mortality rate 30 per 100,000 persons), which was estimated at over 500,000
annually prior to 2000. The increase in incidence of infection leads to increase in number of morbidity and mortality and is
more or less because of serious adverse reactions induced by Anti-TB drugs (Kopanoff et al., 1978; Burman and Reves.,
2001).

The ADRs induced by Anti-TB drugs is the matter of concern in many communities. Hepatotoxicity is one of the serious
ADRs reported in various studies. Rate of hepatotoxicity reported differs from different studies (British Thoracic and
Tuberculosis Association., 1975; Tanaja and Kaur., 1990; Snider et al., 1984). The type of reaction depends upon the
genotype of patients receiving Anti-TB drugs e.g. rapid-acetylator patients are more susceptible for isoniazid induced
hepatotoxicity. Studies show that the risk of hepatotoxicity in patients from India is higher than those reported in West
(11.5% versus 4.3%) (Sharma et al., 2002). Taking in to account the significant difference reported between Asian and
Western people in developing Anti-TB drugs induced hepatotoxicity, there is a need to detect the rate of Anti-TB drugs
induced ADRs with emphasize on hepatotoxic reactions in Indian patients.
Nutritional supplements are needed to help the body regain strength and fight the illness. Apitherapy or therapy with the bee
products as honey is an old tradition. Honey has potent bactericidal activity against many pathogenic organisms. A mixture of
honey and aged butter is said to be especially curative of TB transferred by cold temperatures. TB of the neck is treated with
honey, milk and herbs (Al-Jabri et al., 2003).

Avicenna, the great Iranian scientist and physician, almost 1000 years ago, had recommended honey as one of best remedies
in the treatment of tuberculosis (Avicenna., 1991). Therapeutic effects of honey include its use in the treatment of infantile
gastroenteritis (Haffejee and Moosa., 1985), ulcers, wound healing (Oryan and Zaker., 1998), laxative action, cough and sore
throat, eye diseases, topical antisepsis (Bansal et al., 2005). The immunomodulatory and antioxidant effects of honey has also
been recently reported (Tonks et al., 2003; Gheldof et al., 2003).

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 Manju Sharma et al: Continental J. Pharmacology and Toxicology Research 2: 6 - 11, 2008.

Table 1: Study patients enrolled from different locality of South Delhi.

S.No. Controls (Group I) Cases (Group II)
1. Mahipalpur Tigri
2. Safdarjung Dakshinpuri
3. Malviya Nagar SangamVihar I-block
4. Ber Sarai, New Delhi SangamVihar J-block
5. - SangamVihar G-block

Considering protective effects of honey on gastric and hepatic function, we designed present study to investigate whether
honey with Anti TB drugs has any influence on ADRs profiles in pulmonary TB patients receiving Direct Observed
Treatment Short-course (DOTS) under Revised National Tuberculosis Control Programm (RNTCP).

MATERIALS AND METHODS

Study was performed on pulmonary new sputum acid-fast bacilli positive patients of tuberculosis registered under Revised
National Tuberculosis Control Program for Direct Observed Treatment Short-course during three months (January to March
2007). These patients were diagnosed at RNTCP- Lala Ram Sarup Institute of Tuberculosis and Respiratory, Sri Aurobindo
Marg, New Delhi-110030, INDIA.

A total of 185 patients were enrolled in this study, were divided into two groups (Group-I and Group-II) on the basis of
treatment. Group-I served as controls, consist of 83 patients, received short course chemotherapy as per RNTCP guidelines
i.e. four drugs (Isoniazid, Rifampicin, Pyrazinamide and Ethambutol) thrice weekly for two months in the intensive phase.
Group II served as cases, consist of 102 patients, received short course chemotherapy as per RNTCP guidelines i.e. four
drugs (Isoniazid, Rifampicin, Pyrazinamide and Ethambutol) and one teaspoonful (5ml) honey [Wings Pharmaceuticals Pvt.
Ltd.,] 5 minutes prior to ATT, thrice weekly for two months in the intensive phase. Patients included in this study were taken
from different areas of South Delhi (Table 1).

Patients with history of allergy to honey and / or related products were excluded from the study. Patients taking over the
counter (OTC) drugs or drugs for co-morbidity (such as asthma, diabetes mellitus), serious or hospitalized patients, pregnant
women or women taking oral contraceptives and patients of paediatric age group were also excluded from the study.

All mentally retarded, drug addicts, unconscious and patients unable to respond to verbal questions were also excluded from
the study. A signed written informed consent was taken from the patients prior to enrollment in the study. The study was
initiated after the approval of the study protocol by Institutional Research Committee of Lala Ram Sarup Institute of TB and
Respiratory Disease, New Delhi.

The adverse drug reactions experienced by the patients were documented on ADR monitoring form designed on the basis of
CDSCO guidelines. The form includes data like age, sex, demographic details, past medical history, present drug treatment,
description of adverse drug reaction, its assessment and treatment for the drug reaction. The ADR definition used in this
study is that of the WHO “Any noxious and unintended response to a drug, which occurs at doses normally used in human
for the prophylaxis, diagnosis or treatment of disease or for the modification of physiological function” (WHO, 1972; WHO,
1996).

Serious: A serious adverse event (AE) or reaction is any untoward medical occurrence that at any dose:
- results in death,
- requires inpatient hospitalization or prolongation of existing hospital stay,
- results in persistent or significant disability/incapacity,
- is life threatening.

The term “severe” is not synonymous with serious. Words “severe” is used to describe the intensity of specific event (as in
mild, moderate or severe); the event itself, however, may be relatively minor medical significance (such as severe headache).
Seriousness (not severe) which is based on patient/event outcome or action criteria (Venulet and Ham., 1996).

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 Manju Sharma et al: Continental J. Pharmacology and Toxicology Research 2: 6 - 11, 2008.

Detection and monitoring was done by interviewing patients and consulting the physicians about the patient’s clinical
problems and ADRs were recorded routinely. Patients developed ADRs were kept under supervision until recovery.

Table 2: Distributions of adverse reactions on the basis of severity

Group Severity of ADR No. of Patients with ADR (%)

Mild ADRs 36 (62.07)

Control (group
I) Moderate ADRs 22 (37.93)
Mild ADRs 29 (60.42)
Case (group II)
Moderate ADRs 19 (39.58)

RESULTS AND DISCUSSION

The present study confirmed that intolerance of anti-tuberculosis standard therapy due to adverse effects is still a serious
problem of patients with tuberculosis and there is a need for the monitoring of patients receiving anti-tubercular treatment.
During this study, a total of 185 patients were diagnosed with positive pulmonary TB, enrolled for the study. Out of 185
patients 131 (71%) were male as compared to 54 (29%) female. A high percentage of ADRs was experienced in control
(69.88%) as compared to case (47.06%). In gender distribution of patients; the prevalence of ADR were found to be more in
male as compared to female in both, control group (60.34%) and case group (56.25%). Among the case group, about 48
(47%) patients showed at least one adverse reaction while in control group 58 (70%) patients showed at least one adverse
reaction. This relatively high percentage of adverse reactions among patients of control group indicates that there is a need
for extensive evaluation of ADRs in these patients. In case group, honey was administered to the patients along with standard
DOTS therapy under RNTCP, which shows that honey improves the adverse reaction profile of pulmonary tuberculosis
patients. These effects of honey are probably due to its protective effects on human gastrointestinal system such as its
influence on human gastric and hepatic function (Baltukeviius and Eksteryte., 1998) and its use in oral rehydration products.
Honey is also useful in the treatment of various ophthalmic conditions (Al-Waili., 2004). Honey is reported to stimulate
inflammatory cytokines production from monocytes (Tonks et al., 2003) and also known to increase serum antioxidant
capacity in human (Gheldof et al., 2003). Most of the ADRs were mild to moderate and transient in nature. Out of 58 ADRs,
36 (62.07%) were mild and 22 (37.93%) were moderate in patients of control group, whereas 29 (60.42%) were mild and 19
(39.58%) were moderate in patients of case group (Table 2). The rate of adverse reactions was varies in different age groups.
The majority of ADRs were found be in patients of age group of 25 to 54 years and in elderly age group i.e. 65 years and
above (Table 3). The reasons of more ADRs in these age group patients are due to, high disease prevalence in these age
group patients. It is reported that, the patients with age group of 25-50 years and elderly patients are more vulnerable to
develop ADRs (Sharma et al., 2007). Among the 185 patients, about 109 (59%) were smokers while 76 (41%) were
nonsmokers. Smokers were found to have more risk of ADR in both groups, i.e. 38 (20.54 %) in case as well as 45 (24.32%)
in control group as compare to nonsmoker 10 (05.41%) in case and 13 (07.03%) in control group (Table 4). Smokers had a
73 percent increased risk of becoming infected with tuberculosis and were more than twice as likely as to develop active
tuberculosis than the nonsmokers (American Thoracic Society, 2007).

The most common adverse reactions reported among control patients were anemia (21.16%), joint pains (15.6%), itching
(18%) and nausea (14.4%), while in case the most common adverse reactions reported were anemia (10.78%), itching (7.8%)
and nausea (6.8%). The least percentage of adverse reactions reported was diarrhea (2.4 %) among control patients while
there was no diarrhea observed in patients receiving honey along with ATT (Table 5). However some studies reported
hepatotoxicity as most frequent ADRs followed by constipation (Gholami et al., 2006; Gulbay et al., 2006). This difference
in ADRs among the patients of control group and patients of case group was due to honey which is reported to improve
appetite, gastrointestinal disorders, anemia, diarrhea, headache and tuberculosis (Haffejee et al., 1985; Bansal et al., 2005;
Oryan et al., 1998; Tonks et al., 2003).

CONCLUSION
The results of the present study provide evidence for potential corrective effect of honey on adverse drug reactions. Further
investigations with more properly conducted clinical trials are warranted to explore the full potential of

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 Manju Sharma et al: Continental J. Pharmacology and Toxicology Research 2: 6 - 11, 2008.

honey. Honey minimizes the adverse drug reactions induced by Anti- TB drugs in AFB +ve pulmonary tuberculosis patients.
Therefore, honey can be given as an adjuvant to pulmonary TB patients as it may improve patient’s compliance and thus may
reduce the cases of resistance.

ACKNOWLEDGMENTS: The project was supported by financial assistance from the University Grant Commission (UGC),
New Delhi (INDIA).

REFERENCES
Al-Jabri AA, Nzeako B, Al- Mahrooqi Z, Al-Naqdy A, Nsanza H (2003). In vitro antibacterial activity of Omani and
African Honey. Br J Biomed Sci; 60(1): 1-4.
Table 3: Distribution of adverse reactions among TB patients according to age group
Age group Control Group Case Group
Patients Patients with Patients Patients with
ADR (%) ADR (%)
14-24 9 06 (10.34) 15 06 (12.50)
25-34 18 12 (20.68) 22 10 (20.83)
35-44 21 15 (25.86) 25 12 (25.00)
45-54 13 09 (15.51) 17 08 (16.67)
55-64 7 04 (6.89) 8 03 (6.25)
65 and 15 12 (20.68) 15 09 (18.75)
more
83 58 (100) 102 48 (100)

Al-Waili NS (2004). Investigating the antimicrobial activity of natural honey and its effects on the pathogenic
bacterial infections of surgical wounds and conjunctiva. J Med Food; 7: 210-22.

American Thoracic Society (2007). Smokers may be a risk factor for tuberculosis. JAMA; Archived Journal March
1.

Avicenna (1991). The Cannon of Medical. Translated from Arabic into Persian by Abdul rahman Sharaf- kandi.
Book III, IRIB Publication, Teheran, (4th ed.); 489-03.

Baltukeviius A, Eksteryte V (1998). Influence of monofloral honey on human gastric and hepatic functions. Acta
Zoologica Lituanica Entomologia; 8 (3): 89-91.

Bansal V, Medhi B, Pandhi P (2005). Honey- A remedy rediscovered and its therapeutic utility. Kathmandu
University Medical Journal; 3: 305-09.

British Thoracic and Tuberculosis Association (1975): Short course chemotherapy in pulmonary tuberculosis.
Lancet; 119-24.

Burman WJ and Reves RR (2001). Hepatotoxicity from Rifampin plus Pyrazinamide. Lessons for Policymakers and
Messages for Care Providers. Am J Respir Crit Care Med; 164: 1112-3.

Garner P, Holmes A, Ziganshina L (2004). Tuberculosis. Clin Evid; 11: 1081-93.

Gheldof N, Wang XH, Engeseth NJ (2003). Buckwheat honey increases serum antioxidant capacity in humans. J
Agric Food Chem; 51(5): 1500-05.

Gholami K, Kamali E, Hajiabdolbaghi M, Shalviri G (2006). Evaluation of anti-tuberculosis induced adverse

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 Manju Sharma et al: Continental J. Pharmacology and Toxicology Research 2: 6 - 11, 2008.

reactions in hospitalized patients. Pharmacy Practice; 4(3): 134-8.

Gulbay BE, Gurkan OU, Yildiz OA, Onen ZP, Erkekol FO, Baccioglu A, Acican T (2006). Side effects due to

primary antituberculosis drugs during initial phase of therapy in 1149 hospitalised patients for tuberculosis.
Respiratory Medicine; 100: 1834-42.

Table 4: Distribution of patients and adverse reactions among smoker and non-smoker
Addiction Number of Patients (%) ADRs (%)
Control Case
Smokers 109 (59%) 45 (24.32%) 38 (20.54%)
Nonsmokers 76 (41%) 13 (07.03%) 10 (05.41%)

Table 5: Distribution of type of adverse reactions in TB Patients

Adverse reactions Case Group Control Group
Frequency Percentage of Frequency Percentage of
ADRs ADRs
Nausea 7 6.8% 12 14.4%
Loss of appetite 4 3.9% 8 9.6%
Icterus 6 5.8% 9 10.8%
Itching 8 7.8% 15 18%
Rash 1 0.09% 3 3.6%
Eye symptoms 1 0.09% 4 4.8%
Joint pains 5 4.9% 13 15.6%
Vomiting 4 3.9% 8 9.6%
Anemia 11 10.78% 18 21.16%
Fever 3 2.9% 6 7.2%
Headache 5 4.9% 7 8.4%
Dizziness 7 6.8% 9 10.8%
Drowsiness 15 7.8% 5 6.02%
Diarrhoea 0 0% 2 2.4%

Haffejee IE, Moosa A (1985). Honey in the treatment of infantile gastroenteritis. Br Med J (Clin Res Ed); 290:
1966-67.

Kopanoff DE, Snider DE, Caras GJ (1978). Isoniazid-related hepatitis. Am Rev Respir Dis; 117: 991-1001.

Oryan A, Zaker SR (1998). Effects of Topical application of Honey on Cutaneous Wound Healing in Rabbits.
Zentralbl. Veterinar Med. A; 45(3): 181-88.

Sharma H, Aqil M, Imam F, Alam MS, Kapur P, Pillai KK (2007). A pharmacovigilance study in the department of
medicine of a university teaching hospital. Pharmacy Practice; 5 (1): 46-49.

Sharma SK, Balamurgan A, Saha PK, Pandey RM, Mehra NK (2002). Evaluation of clinical and immunogenetic
risk factors for the development of hepatotoxicity during Antituberculosis treatment. Am J Respir Crit Care Med.;
166: 916-19.

Snider DE, Long MW, Cross FS, Farer LS (1984). Six months Isoniazid and Rifampin therapy for pulmonary
tuberculosis: report of a United States Public Health Service cooperative trial. Am Rev Respir Dis; 77: 233-42.

10
 Manju Sharma et al: Continental J. Pharmacology and Toxicology Research 2: 6 - 11, 2008.

Tanaja DP, Kaur D (1990). Study on hepatotoxicity and other side effects of antituberculosis drugs. J Indian Med
Assoc; 88: 278-80.

Tonks AJ, Cooper RA, Jones KP, Blair S, Parton J, Tonks A (2003). Honey Stimulates Inflammatory Cytokine
Production from Monocytes. Cytokine; 21(5): 242-47.

Venulet J and Ham MT (1996). Method for monitoring and documenting adverse drug reactions. Int J Clin
Pharmacol and Therap; 34(3): 112-29.

World Health Organization (1972). International Drug Monitoring: The roles of National centers. Tech Rep Ser
Who; No. 498. Geneva: WHO.

World Health Organization (1996). Uppsala Monitoring Center. Safety monitoring of medicinal products, guidelines
for setting up and running pharmacovigilance center, Geneva.

World Health Organization (2001). Global Tuberculosis Control. WHO Report. Geneva, Switzerland:
WHO/CDS/TB; 287.

Received for Publication: 28/03/2008

Accepted for Publication: 24/06/2008

Corresponding Author
Dr. Khalid U Khayyam
HOD, Epidemiology and Public Health, LRS Institute of TB & Resp. Diseases, Sri Aurobindo Marg New Delhi-110030.
India
E-mail: dr.khalidukhayyam@yahoo.co.in

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Continental J. Pharmacology and Toxicology Research 2: 12 - 18, 2008.
© Wilolud Online Journals, 2008.

ACTION OF PORTULACA OLERACEA AGAINST STREPTOZOTOCIN-INDUCED OXIDATIVE STRESS IN

EXPERIMENTAL DIABETIC RATS

Alok Sharma1, G.D. Reddy3, M.Vijayakumar1, M.K. Unnikrishnan2 and Ch.V. Rao1*
1
Pharmacognosy and Ethno Eharmacology Division, National Botanical Research Institute, Lucknow,
Uttar Pradesh- 226 001, India. 2 College of Pharmaceutical Sciences, Manipal University, Manipal- 576 119, India
3
Pharmacology Department, Central Research Institute (Ayurveda),
Kolkata- 700 091, India

ABSTRACT
Antidiabetic treatment with extract of Portulaca oleracea leaves (100mg/kg and 250mg/kg body
weight) for three weeks showed significant reduction in thiobarbituriuc acid reactive substances
(TBRAS) and increase in glutathione reductase (GSH-R) in both liver and kidney of STZ diabetic rats.
The treatment with P. oleracea significantly altered the glutathione and GSH-R to be comparable with
the control group. P. oleracea and tolbutamide treated rats showed decreased lipid peroxidation that is
associated with increased activity of superoxide dismutase (SOD) and catalase (CAT).The ability of P.
oleracea on tissue lipid peroxidation and antioxidant status in diabetic animals has not been studied
before. The result of this study thus shows that though, P. oleracea extract possesses moderate
antidiabetic activity, but it exhibits potent antioxidant potential in diabetic conditions. Since Portulaca
oleracea has been used traditionally but the antioxidant properties was not reported before. However
there are no reports on the antidiabetic and antioxidant activity of the plant. Hence, the present study
was designed to verify the claims of the native practitioners.

KEYWORDS- Portulaca oleracea, Portulacaceae, Antioxidant enzymes, Diabetes

INTRODUCTION
Diabetes mellitus is considered one of the five leading causes of death in the world. The World Health Organization (WHO)
has predicted that the worldwide number of patients with diabetes will double by the year 2025, from the current number of
approximately 150 million to 300 million. (World Health Organization, 2004) It has been assumed that the etiology of the
complication of diabetes involves oxidative stress perhaps as a result of hyperglycemia (Hunt et al., 1990). The elevated
levels of blood glucose in diabetes produce oxygen free radicals which cause membrane damage due to peroxidation of
membrane lipids and protein glycation (Baynes, 1991). As the diabetogenic action can be prevented by the SOD, CAT, and
other hydroxyl radical scavengers such as ethanol, dimethyl urea, there is evidence to suggest that the incidence of diabetes
involves superoxide anion and hydroxyl radicals can be counteracted by antioxidant enzymes such as SOD, CAT, and
glutathione peroxides (GSH-px. There is clear cut evidence to show the role of free radicals in diabetes and studies indicates
that tissues injury in diabetes may be due to free radicals (Grankvist et al., 1981). Thus Antioxidant and free radical
scavengers may help in the regeneration of beta cells and protect pancreatic islets.

Portulaca oleracea (Family Portulacaceae) is a succulent, prostrate or erect annual, with green or purple stems swollen at the
nodes, up to 50 cm long. The herb is considered to possess cooling, alternative, antiscorbutic, aperient and diuretic properties,
the diuretic property probably due to its high content of potassium salts. The fresh leaf juice is considered an effective thirst-
quencher, leaves and tops are used in anti-haemorrhagic poultices. (Nadkarni and Nandkarni, 1954). It exhibits a wide range
of pharmacological effects, including antibacterial (Zhang et al., 2002), analgesic, anti-inflammatory (Chan et al., 2000),
skeletal muscle-relaxant (Parry et al., 1987 and Parry et al., 1993) wound-healing (Rashed et al., 2003) and antiulcer (Karimi
et al., 2004) activities. It is also consumed as a vegetable and has been reported to be rich in α-linolenic acid, β-carotene and
Omega -3- fatty acids (Liu et al., 2000, Simopoulose et al., 1986). In addition to flavonoids, coumarins (Awad, 1994),
monoterpene glycoside (Sakai et al., 1996) and alkaloids have also been reported to be important chemical constituents of
this plant (Xiang et al., 2005). Since Portulaca oleracea has been used traditionally but the antioxidant properties was not
reported before. Thus the present study was designed to study the level of the antioxidant enzymes SOD, GSH, GSH-R and
CAT along with lipid peroxidation in STZ induced diabetic rats.

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 Alok Sharma et al: Continental J. Pharmacology and Toxicology Research 2: 12 - 18, 2008.

MATERIALS AND METHODS

Plant materials- Plant material of Portulaca oleracea were collected in the month of September, 2006 from the Lucknow,
U.P. India. The plant material were identified taxonomically and authenticated by National Botanical Research Institute,
Lucknow. A voucher specimen of the collected sample was deposited in the departmental herbarium for future reference.

Preparation of extract - The shade dried whole plant materials were air-dried at room temperature and coarsely powdered.
Air-dried powdered material of P. oleracea (1000 g) was exhaustively extracted with 10 volumes of 50% ethanol. This
process of extraction was repeated for four times, filtered, concentrated on rotavapour (Buchi, USA) and then freeze-dried
(Freezone® 4.5, Labconco, USA) at high vacuum (133 x 10-3 m Bar) and at temperature – 40 ± 2 °C (Yield 9.45% w/w).

Animals- Sprague-Dawley rats (150-175g) and albino mice (20 - 25 g) were obtained from the animal colony of National
Laboratory Animal Centre, Lucknow. They randomly distributed into various groups and housed in cages (6 per cage) and
maintained under standard conditions i.e. 26 ± 2 °C and relative humidity 44 - 56% and 10 h light: 14 h dark cycles each day
for one week before and during the experiments. All animals were fed standard rodent pellet diet (Amrut, India) and drinking
water ad libitum. All studies were performed in accordance with the guide for the care and use of laboratory animals, as
adopted and promulgated by the Institutional Animal Care Committee, CPCSEA, India (Reg. No. 222/2000/CPCSEA).

Induction of experimental diabetes- A freshly prepared solution of streptozotocin (STZ, 50 mg/ kg, body weight) in 0.1 M
citrate buffer pH 4.5 was injected intra peritonealy in a volume of 1 ml/ kg. STZ injected animals exhibited massive
glycosuria and hyperglycaemia within two days (Vijayakumar et al, 2006). Diabetes was confirmed in STZ rats by measuring
the fasting blood glucose concentration after 96 h after the injection of STZ. The rats with blood glucose level above 200 mg/
dl were considered to be diabetic and were used in the experiment.

Experiment design and Treatment - After induction of diabetes, the rats was divided into 5 groups of 6 animals each as
follows: Group I animals served as control rats received 2% gum acacia as vehicle solution. Group II constituted STZ
diabetic rats and received 2% gum acacia. Group III and IV animals are STZ diabetic rats received P. oleracea extracts (100
and 250 mg/kg b.w) and Group V received tolbutamide (10mg/kg b.w). The vehicle and the drugs were administered orally
using an intragastric tube daily for three weeks. After three weeks of treatment the rats were fasted overnight and sacrificed
by cervical decapitation. Blood samples were analyzed for serum glucose content by commercial glucose kit (Qualigens
Diagnostics, Mumbai, India). The liver and kidney was exposed and perfused with cold phosphate buffer saline of pH
7.4.Blood free liver was taken out and homogenized in a glass Teflon homogenizer(10%w/v).Incubation were done at 370C
under controlled conditions. Liver and kidney homogenates were subjected for antioxidant enzymes estimation.

Antioxidant activities - Lipid peroxidation in liver and kidney were estimated colorimetrically by thiobarbituric acid reactive
substances (TBRAS) (Okhawa et al., 1979) and hydroperoxides by the method of (Jamall and smith 1985).Glutathione
(GSH) was estimated using Butler et al. (1967), Glutathione reductase (GSH-R) was estimated using standard method of
Horn (1963). Superoxide dismutase (SOD) was measured by using the methos of Kakkar et al. (1984). Catalse (CAT)
activity was measured by using the rate of decomposition of H2O2 by the method of Aebi (1974). All these estimations were
made in both liver and kidney.

Statistical analysis- All the data are presented as mean ± S.E.M. and one-way analysis of variance (ANOVA) followed by
Newman-Keuls Multiple Comparision Test was applied for determining the statistical significance between different groups.

RESULTS
There was a moderate decrease in the blood glucose level of STZ diabetic rats upon administration P. oleracea (Table 1).
Table 2 shows the activities of the enzymatic antioxidants SOD and CAT in liver and kidney (P<0.001). Activities of these
enzymes decreased significantly in the diabetic control rats as compared to the normal control (P<0.001). Oral administration
of P. oleracea extract (100mg and 250mg/kg b. w) for three weeks significantly reversed these enzymes to near normal
values.

Table 3 shows the levels of TBARS, GSH and GSH-R in liver and kidney of control and experimental animals (P<0.001). A
significant elevation in tissues TBARS and significant reduction in GSH, and GSH-R was observed in the diabetic control
rats as compared to the normal control rats. Oral administration of P. oleracea extract (100 and 250mg/kg body weight) for

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 Alok Sharma et al: Continental J. Pharmacology and Toxicology Research 2: 12 - 18, 2008.

three weeks shows significant reduction in TBARS and increase in GSH-R in both liver and kidney (P<0.001). With respect
to GSH there was a significant increase in the glutathione in the liver and kidney.

DISCUSSION
Streptozotocin is often used to induce diabetes mellitus in experimental animals through its toxic effects on pancreatic beta
cells. STZ induced diabetes mellitus is associated with the generation of reactive oxygen species causing oxidative damage
(Kamalakkannan and Prince, 2006). Lipid peroxidation is a free radical induced Process leading to oxidative deterioration of
polyunsaturated fatty acids. Under Physiologic condition, low concentrations of lipid peroxides are found in tissues (Mertz,
1984) . Karpen et al., (1982) observed an elevated level of lipid peroxides in the plasma of diabetic rats and lipid
peroxidation as one of the characteristic features of chronic diabetes. Lipid Peroxidation has also been observed in the
development of both type I and II diabetes. Increased levels of lipid peroxides has also been reported in the kidney of
diabetic rats and increased levels of TBARS as an index of lipid peroxidation (Nakakimura and Mizuno, 1980). The
involvement of free radicals in diabetes and the role of these toxic species in LPO and the antioxidant defence system have
been studied. Depletion of tissue glutathione and increase in lipid peroxidation has been observed in diabetes (Mukherjee et.
al., 1994). It has been proposed that antioxidants that maintain the concentration of reduced glutathione may restore the
cellular defence mechanisms, block lipid peroxidation and thus protect the tissue damage against oxidative damage (Rauschar
et al., 2000). Our results showed that in diabetic control animals the level of TBARS was high due to increased lipid
peroxidation. P. oleracea reduced the TBARS levels in both liver and kidney, which may be due to the free radical
scavenging action of the active ingredients present the P. oleracea extract inhibited the lipid peroxidation process
effectively and that could be attributed to the ability to scavenge the free radicals involved in the initiation and propagation
steps.

One of the consequences of hyperglycaemia is increased metabolism of glucose by sorbitol pathway. Besides this, other
pathways such as fatty acid and cholesterol biosynthesis also compete for NADPH with GSH. The decrease in GSH level in
liver during diabetes is probably due to its increased utilization by the hepatic cells which could be the result of decreased
synthesis or increased degradation of GSH by oxidative stress in diabetes (Loven et al., 1986). We have also observed the
decrease in GSH in liver and kidney. The activities of GPx and GST were observed to decrease significantly in diabetic rats.
GPx an enzyme with selenium and GST catalyses the reduction of hydrogen peroxide to non toxic compounds (Illing et al.,
1991). The treatment with P. oleracea significantly altered the GSH and GSH-R to be comparable with the control group.
SOD scavenges the superoxide ions produced as cellular by-products. SOD is a major defence for aerobic cells in combating
the toxic effects of superoxide radicals (McCrod et al., 1976). CAT reduces Hydrogen per oxide produced by disputation
reaction and preventing generation of hydroxyl radicals thereby protecting the cellular constituents from oxidative damage in
peroxisomes. SOD and CAT are two major scavenging enzymes that remove the toxic free radical in vivo. Reduced activities
of SOD and CAT in liver and kidney have been observed during diabetes and this may result in a number of deleterious
effects due to the accumulation of superoxide radicals and hydrogen peroxide (Santhakumari et al ., 2003). P. oleracea and
tolbutamide treated rats showed decreased lipid peroxidation that is associated with increased activity of SOD and CAT.

The ability of P. oleracea on tissue lipid peroxidation and antioxidant status in diabetic animals has been studied. The result
of this study thus shows though, P.oleracea possesses moderate antidiabetic activity and exhibits potent antioxidant potential
in diabetic conditions which may be presence of flavanoids in the plant. Several studies indicate that food supplementation
with antioxidant properties may be a complement to traditional therapies for treating diabetic complications. Since the plant
is source of nutrients like α-linolenic acid, β-carotene and Omega -3- fatty acids, flavonoids and coumarins, it may be served
as a diabetic diet. Further experimental and clinical studies are needed to validate the use of the plant in the treatment of
diabetes and diabetic complication.

ACKNOWLEDGEMENT
This study was supported by Council of Scientific and Industrial Research, New Delhi. Authors are highly thankful to
Director NBRI, Lucknow, for providing the necessary facilities.

REFERENCES
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Awad, N.E., Lipid content and antimicrobial activity of phenolic constituents of cultivated Portulaca oleracea L., Bull. Fac.
Pharm. Cairo Univ. 32, 137–142 (1994).

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Baynes, J.W., Perspectives in diabetes, role of oxidative stress on development of complications in diabetes. Diabetes 40,
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the rat heart: a possible mechanism of cadmium cardiotoxicity. Toxicol. Appli. Pharmacol. 80, 33-42 (1985).
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130-132, (1984).
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streptozotocin induced diabetic wistar rats. Basic Clin. Pharmacol. Toxicol. 98, 97-103 (2006).
Karimi1, G., Hosseinzadeh, H., and Ettehad, N., Evaluation of the gastric Antiulcerogenic effects of Portulaca oleracea L.
Extracts in Mice. Phytother. Res. 18, 484–487 (2004).
Karpen, C.W., Pritchard, K.A., Merola, A.J., and Ranganamala, R.V., Alterations of the prostaglandin-thromboxin ratio in
streptozotocin induced diabetes rats. Prostagland. Leukotrien. Med. 8, 93-103 (1982).
Liu, L.X., Howe, P.Y., Zhou, F., Xu, Zh.Q., Hocart, C., Zhang, R., Fatty acids and β-carotene in Australian purslane
(Portulaca oleracea) varieties, J. Chromatogr. A. 893, 207–213 (2000).
Loven, D., Schedl, H., Wilson, H., Daabees, T.T., Stegink, L.D., Diekus, M., Effect of insulin and oral glutathione on
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superoxide dismutase. Proc. Natl. Acad.Sci., 68, 1024-1027 (1976).
Mukherjee, B., Mukherjee, J.H., and Chatterjee, M., Lipid peroxidation, gluthione levels and change in gluthione related
enzymes activities in streptozotocin- induced diabetic rats. Immunol. Cell. Biol. 72, 109-111 (1994).
Nadkarni, K.M, Nadkarni, A.K, Indian Materia Medica, Popular Pakashan, New Delhi, 1995, pp.669.
Nakakimura, H., and Mizuno, K., Studies on lipid per oxidation in biological system II. Hyperlipoperoxidemia in mice
induced by alloxan. Chem.. pharm Bull. 28, 2207-2211 (1980).
Okhawa, H., Oshishi, N., and yagi, K., Assay for lipid peroxised in animals tissues by thiobarbituric acid reaction. Anal.
Biochem. 95, 351-355 (1979).
Parry, O., Okwuasaba, F., and Ejike, C., Skeletal muscle relaxant action of an aqueous extract of Portulaca oleracea in the
rat. J. Ethnopharmacol. 19, 247–253 (1987).
Parry, O., Marks, J.A., and Okwuasaba, F., The skeletal muscle relaxant action of Portulaca oleracea: role of potassium ions.
J. Ethnopharmacol. 40, 187–194. 1993.
Rashed, N., Afifi, F.U., and Disi, A.M., Simple evaluation of the wound healing activity of a crude extract of Portulaca
oleracea L. (growing in Jordan) in Mus musculus JVI-1. J. Ethnopharmacol. 88, 131–136 (2003).
Rauscher, F.M.,Sanders, R.A., and Watkins, J.B., Effects of new antioxidant compounds PNU-104067F and PNU-74389G
on antioxidant defense in normal and diabetic rats. J. Biochem. Mol.Toxicol.14, 189-194 (2000).
Sakai, N., Inada, K., Okamoto, M., Shizuri, Y., and Fukuyama, Y., Portuloside A, a monoterpene glucoside from Portulaca
oleracea, Phytochem. 42, 1625–1628 (1996).
Santhakumari, P., Prakasam, A., and Pugalendi, K.V., Modulation of oxidative stress parmeters by treatment with piper betel
leaf in streptozotocin induced diabetic rats. Indian J. Pharmacol. 35, 373-378 (2003).
Simopoulos AP, Salem H Jr. Purslane: a terrestrial source of omega-3 fatty acids. N. Engl. J. Med. 315, 833 (1986).
Vijayakumar, M., Govindarajan, R., Rao, G.M.M., Rao. Ch.V., Shirwaikar, A., Mehrotra, S., Pushpangadan, P., Action of
Hygrophila auriculata against streptozotocin induced oxidative stress. J. Ethnopharmacol. 104, 356-361 (2006).

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World Health Organization, Diabetes action Now: An Initiative of the World Health Organization and International Diabetes
Federation, WHO Publication, Geneva. (2004).
Xiang, L., Xing, D., Wang, W., Wang, F., Ding, Y., and Du, L., Alkaloids from Portulaca oleracea L.Phytochem. 66, 2595-
2601 (2005).
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Received for Publication: 21/08/2008

Accepted for Publication: 28/08/2008

Corresponding Author
Alok Sharma
Pharmacognosy and Ethno pharmacology Division, National Botanical Research Institute, Lucknow, Uttar Pradesh- 226
001, India.
E-mail address: alokaloksharma@rediffmail.com

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 Alok Sharma et al: Continental J. Pharmacology and Toxicology Research 2: 12 - 18, 2008.

Table 1. Effect of various treatments on blood glucose level

Blood glucose level (mg/dl)
Group
0day 7th day 21st day
Group 1 90.95 ± 5.12 91.71 ± 6.84 89.16 ± 4.52
Group II 236.28 ± 12.46† 231.48 ± 13.26 † 223.83 ± 12.35 †
Group III 237.86 ± 14.25 197.87 ± 11.42* 136.48 ± 9.46***
Group IV 236.19 ± 13.82 183.49 ± 10.86** 115.27 ± 6.34***
Group V 235.09 ± 12.24 155.79 ± 8.12*** 89.83 ± 7.52***

Results are expressed as mean ±S.E.M. for six rats.

†
P < 0.001 as compared to normal group 1, * P < 0.05, ** P < 0.01, *** P < 0.001 as compared to diabetic group.

Table 2. Effect of various treatments on super oxide dismutase and catalase

SOD Catalase
Group (unit/mg protein) (unit/mg protein)
Liver Kidney Liver Kidney
Group 1 9.32±0.75 7.2±0.48 57.70±3.71 28.59±2.48
Group II 4.36±0.61† 3.26±0.42† 32.13±2.42† 9.68±1.09†
* *
Group III 6.56±0.02 4.34±0.12 47.82±3.83** 18.14±1.74**
*** **
Group IV 8.36±0.22 5.82±0.10 60.71±4.18*** 25.58±2.21***
** **
Group V 7.29±0.04 6.3±0.14 59.72±4.07*** 25.31±3.14***

Results are expressed as mean ±S.E.M. for six rats.

†
P < 0.001 as compared to normal group 1, * P < 0.05, ** P < 0.01, *** P < 0.001 as compared to diabetic group.

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 Alok Sharma et al: Continental J. Pharmacology and Toxicology Research 2: 12 - 18, 2008.

Table 3. Effect of various treatments on lipid peroxidation and activities of glutathione

Groups TBARS Glutathione Gluthione transferase

(Nmoles of MDA/mg protein) (nM of DTNB (nM of NADPH oxidized/mg
conjugated/mg protein) protein)
Liver Kidney Liver Kidney Liver Kidney
Group 1 6.86 ± 1.26 7.47 ± 0.86 115.61 ± 4.11 104.70 ± 4.95 240.82 ± 6.6 256.85 ± 5.04
Group II 15.97 ± 1.56† 14.92 ± 1.31† 62.7 ± 5.28† 48.80 ± 2.15† 117.38 ± 5.13† 199.60 ± 9.30†

Group III 10.30±1.48** 12.62± 1.07* 98.4 ± 6.71** 168.15 ± 3.35** 181.47±6.96** 154.26± 1.16***
Group IV 8.64± 0.14*** 9.72 ± 0.63** 118.9± 5.41*** 94.70 ± 4.16*** 235.71±5.29*** 247.86 ± 4.07***
Group V 7.25± 0.48*** 8.99± 0.62*** 114.62± 4.5*** 92.80 ± 4.45*** 234.19±7.72*** 245.13 ± 7.73***

Results are expressed as mean ±S.E.M. for six rats.

†
P < 0.001 as compared to normal group 1, * P < 0.05, ** P < 0.01, *** P < 0.001 as compared to diabetic group

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Continental J. Pharmacology and Toxicology Research 2: 19 - 26, 2008.
© Wilolud Online Journals, 2008.

COMPARATIVE EVALUATION OF HERBAL MOUTH RINSE, HERBAL TOOTHPASTE GEL AND

CHLORHEXIDINE MOUTH RINSE FOR CARCINOGENIC BACTERIA

Aftab Alam1, Nayyar Parvez2 , Suman Yadav3, Raman Dang1 and JP Shethy4
1
Dept of Pharmacognosy, Al-Ameen College of Pharmacy Lalbagh road, Bangalore-560027, 2Department
of Pharmaceutics,Advanced Institute of Pharmacy, Palwal, Distt. Faridabad, Haryana, India, 3Department
of Chemistry, Swami Shradhanand College, University of Delhi, Alipur- 110036, 4Government Dental
College Bangalore Victoria Hospital Bangalore-560027

ABSTRACT
The herbal formulations HMR (herbal mouth rinse) and HTG (Herbal toothpaste gel) were
subjected for clinical study. Wherein 30 healthy subjects were selected and divided in three
groups each group recommended different products using Chlorhexidine as standard, the
paraffin stimulated saliva sample were taken before and after 0h, 2h, 5h and 8h use of
formulations. The microbial growth counts using MSB and Lactobacillus MRS Agar for
Streptococcus and lactobacillus species respectively were evaluated for reduction of microbial
growth after use of herbal formulations. Results indicate herbal toothpaste gel was effective
beyond 8-hour in daily use and herbal rinse was active for 2-5 hours only. Thus it can be
recommended for fluorinated area and for children below 6-year of age.

KEY WORDS: Clinical Study, herbal mouth rinse and Herbal toothpaste gel, Chlorhexidine
mouth rinse.

INTRODUCTION
Dental diseases are recognized as major public health problem through out the world. Human oral cavity provides
all requirements for growth of various microorganisms, resulting in dental caries. Teeth and their supporting
structure the gum (gingival) are subjected to infection by cariogenic bacteria that causes cavity and pyorrhea, which
if left untreated can eventually lead to gingivitis. Recent study suggests that such chronic low-grade localized
infection such as pyorrhea contribute to heart disease and coronary heart disease ( Hujoel et al, 2001) Streptococcus
mutans, Lactobacilli species and Actinomyces species are major cariogenic bacteria (Zhang et al., 2001)
Streptococcus species are main causative bacteria, among these S. mutans found in a greater number follow by
Actinomyces species and lactobacilli species(Lee et al, 1986). Depressive symptoms favor abundant growth of
lactobacilli bacteria, depress subjects are risk of having caries and possible other diseases (Sirpa et al, 1999).

The production of bacteriocin like inhibitory substance “mutacins” by mutans streptococci is transported between
mother and child, which are implicated as the principal initiator microorganism in child dental caries. Maternal
caries increase is a significant factor influencing the caries experience of the children ( Gronroos et al, 1998). The
antibacterial activities of Phyllanthus emblica fruits are reported from ancient times ( Iqbal et al 1998, Rani et al,
2004). The antioxidant activities of this plant are also studied(Grover et al, 1989) Antiproliferative activity of
Phyllanthus emblica is recently reported(Khan et al, 2002)
In our study antimicrobial activity on cariogenic bacteria by methanolic extract of phyllanthus emblica fruits are also
reported. Phyllanthus emblica is a part of “TRIPHALA” in Indian traditional system of medicine has reported a
76.6% reduction of MMP-9 in adult periodontal patient (Abraham et al, 2002). Conventionally chlorhexidine
gluconate (0.2%) mouthwash is available in the market for the treatment of oral infection. Saccharin is used as
sweetening agent in chlorhexidine rinse and gel preparation, Chlorhexidine gluconate is most widely used
anticariogenic agents, which also produced cytotoxicity on gingival(Hidalgo et al, 2001 and Babich , 1995) Use of
Glycyrrhiza glabra as natural sweetening agent, and for anticarcinogenic activity is also reported(Segal, et al, 1985).
Chlorhexidine gluconate has good antibacterial activity against the microorganism responsible for oral infection(
Ullsfoss et al 1994 ).Though chlorhexidine was introduced in 1957 s, as an antiseptic and in 1970 in dental
community, is still considered one of the most effective antiplaque agents in dentistry. However long term use of
chlorhexidine is limited by its disagreeable test and propensity to stain the teeth brown( Addy et al , 1991)

There are many clinical studies in which antimicrobial mouth rinses have been used in relation to plaque inhibition
and plaque growth (Scherer , et al, 1998). However there is clinically relevant evidence to suggest that mouth rinse

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 Aftab Alam et al: Continental J. Pharmacology and Toxicology Research 2: 19 - 26, 2008.

containing active agent are effective against carcinogenic bacteria( Vanka , et al, 2001) Limited information’s are
available regarding to herbal formulation hence this study is designed and carried out with the following aim and
objective. To compare the efficacy of HMR rinse, HTG and chlorhexidine mouth rinse on cariogenic microorganism
(Sreptococcus mutans and lactobacilli species) for daily use of formulations.

MATERIALS AND METHODS

Plants materials selection and extraction
Fruits of Phyllanthus emblica Linn and roots of Glycyrrhiza glabra Linn were collected from local market in
Bangalore and authenticated by Shri Gajendra Rao, Survey Officer, Regional Research Institute (Ay.). 200 gram of
drugs were defatted by petroleum ether and then subjected to soxhlet extraction for 6-hours with methanol. The
solvent was removed using rotary evaporator to get a dry residue. These extracts were stored in airtight container at
4 0C.

Formulations
Mouth rinse (Vanka , et al, 2001 and Aftab 2007)
HMR contains Phyllanthus emblica extract as active constituent, Glycyrrhiza glabra active as well as sweetening
agent, peppermint oil as flavor, and the concentration of Phyllanthus emblica and Glycyrrhiza glabra are not less
than 20mg/ml, pH-7

Tooth gel. ( Vanka , et al, 2001,Aftab , 2007 )

HTG contains Phyllanthus emblica extract as active constituent, Glycyrrhiza glabra active as well as sweetening
agent, Carbopol as gelling agent, Sorbitol as humectants, calcium carbonate as abrasive, xanthin gum as thickening
agent, peppermint oil as flavor, and the concentration of Phyllanthus emblica and Glycyrrhiza glabra are not less
than 20mg/g. pH-7
In vitro evaluation of herbal toothpaste gel and herbal mouth rinse of Phyllanthus emblica and Glycyrrhiza
glabra extracts almost equivalent to commercial formulations (Noveon, 1911)

Clinical evaluations
To compare the efficacy of HT-gel and Herbal mouth rinse with chlorhexidine mouth rinse on cariogenic
microorganism (Sreptococcus mutans and lactobacilli species) for duration of inhibition activity during 12 h.

Subject Selection: 30 male subjects of 18-25 years age were selected from Al-Ameen College of Pharmacy
Bangalore, Karnataka, India. After taking consent, the selected subjects were medically healthy with no systemic
disease, free from diabetics and did not have use any antibiotic or antiseptic mouthwash during the last two weeks.
Smokers were not included in this group (Aftab et al, 2008)

Procedure
Grouping of subjects (Almas ,et al, 2004)
All Subjects were divided into 3- groups with 10 subjects in each group

Group 1 – HMR: Ten subjects were asked to rinse their mouth with 15ml of “HMR” for 5 min. A 2ml of stimulated
saliva was collected before and after 0-h, 2h, 5h, and 8-h, rinsing with HM Rinse.

Group 2 – Chlorhexidine gluconate (0.2%): Ten subjects were asked to rinse with 15ml of Chlorhexidine gluconate
(0.2%) for 5 min. A 2ml of stimulated saliva was collected before and after 0-h, 2h, 4h, 8-h, and 12-h rinsing with
Chlorhexidine.

Group 3 –HTG: Ten subjects were asked to apply 1g of HT-gel thoroughly in oral cavity for 6 min. A 2ml of
stimulated saliva was collected before and after 0-h, 2h, 4h, 8-h, and 12-h application of HT-gel.

Collection of saliva
Midmorning stimulated salivary sample were collected from the subjects, followed by a small piece of paraffin wax
(1cm Long) and asked to chew for a period of 30 second swallowing only saliva but not the paraffin. There after the
subjects were asked to continue chewing the wax and the saliva was collected at two-minute interval for total period
of 6 minutes. 2ml of saliva was collected in to a sterile glass cup. This process is repeated for 2h, 4h, and 8h. The
dietary factor was not included in this study.

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 Aftab Alam et al: Continental J. Pharmacology and Toxicology Research 2: 19 - 26, 2008.

Antimicrobial activity
Using an automatic micropipette with sterile plastic tips 1.5ml of saliva was transferred into the ependroff tubes and
agitated for 30 sec on vortex mixture. The salivary samples were diluted in 0.05M Phosphate buffer (pH-7.0) to the
dilution of 10-2 - 10-3( Larmas , 1992 and Adamkova ,et al, 2004). For the cultivation of mutant streptococcus
mutans and lactobacilli species, 50 µL of the dilution was incubated on MSB agar media and Lactobacillus MRS
Agar media (Kohler B,et al, 1979 and Westergren ,et al, 1978 ). The agar Plate was incubated anaerobically in
anaerobic jar (5%Co2 and 95% N2) at 37oC for 48 h.. Mutant streptococci and lactobacilli colonies are identified on
the agar plate by its typical colonial morphology. The number of mutans streptococci and lactobacilli colony
forming unit (CFU) per mal of saliva were estimated and scored as below (Kulkarni VV, et al, 2003 and Pai MR, et
al 2004)

Score Streptococcus mutans Lactobacilli species

Score-1 < 104 CFU/ml <103 CFU/ml

Score-2 104- 105 CFU/ml 103-104 CFU/ml
Score-3 105-106 CFU/ml 104-105 CFU/ml
Score-4 > 106 CFU/ml >105 CFU/ml

Statistical analyses were carried out compare the percentage difference inhibition of carcinogenic bacteria and
average bacterial growth before and after use of formulations. The percentage difference of salivary bacteria was
given in Table -1 (S. mutans) and Table-2 (Lactobacilli sp.) growth and graph show the average bacterial growth
before and after 0h, 2h, 5h, and 8h were shown in graph 1 (S. mutans) and graph 2 (lactobacilli species).

RESULTS
Clinical study
In present study comparative clinical study of herbal formulation and marketed formulation were carried in
Government dental college. The study was designed to analysis the effectiveness of formulation in 8-hour use. For
the study 30 subjects were divided in three groups i.e. Herbal rinse groups were 10 subjects, Chlorhexidine groups
were 10 subjects and Herbal tooth gel groups were 10 subjects. Each subject was previously given demonstration
before collecting the saliva 2ml paraffin stimulated saliva was collected before and after 0 hour, 2 hours, 5 hours and
8 hours use of formulation. The antimicrobial activity was evaluated using MSB agar for S. mutans and
Lactobacillus MRS Agar for Lactobacillus species and the bacterial colony was counted using colony counter after
48-hour incubation in 10% CO2 incubator. The percentage difference of Streptococcus mutans after 0h, 2h, 5h and
8h are reported in Table No 1 and percentage difference of Lactobacilli species after use of 0h, 2h, 5h and 8h are
reported in Table No 2. The graph shows the average microbial growth in saliva before and after use of
formulations. Figure No 1, indicate the average S. mutans in saliva before and after 0h, 2h, 5h and 8h and Figure No.
2, indicate the average lactobacillus species before and after 0h, 2h, 5h and 8h use of formulations.

DISCUSSION
In present study the main aim was to prepare an effective formulation for human use, and maintain proper oral
hygiene upto 8 hours. The selected formulations were comparatively evaluating in-vivo in a study carried out at
Government Dental College Bangalore. The midmorning paraffin stimulated saliva was taken before and after
immediate (0h), 2h, 5h and 8h use of formulation. The microbial count of each salivary extract of herbal rinse groups
indicated after use, 90% inhibition of SM and lactobacilli species and after 5h, 10% reduction of SM and
lactobacilli species indicate herbal mouth rinse (HM rinse) was effective for 5 hour only. The microbial count of
each salivary extract of CHx rinse groups indicated after use 100% reduction of SM and lactobacilli species and
after 8h, 20% reduction of SM and 0.00% reduction of Lactobacilli species, concluded that CHx mouth rinse was
effective for 5 to 8 hours. Herbal toothpaste gel (HT-gel) groups were indicate after application 100% reduction of
SM and lactobacilli species and after 8h, 70 % reduction of SM and 60% reduction of Lactobacilli species,
concluded that HT-gel was effective for greater than 8 hours. In present study herbal formulation do not contain any
fluoride ingredient so it can be used in fluorosis condition and area where water fluoride level is high and also
recommended for children below 6 years.

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 Aftab Alam et al: Continental J. Pharmacology and Toxicology Research 2: 19 - 26, 2008.

CONCLUSION
The clinical study of formulation indicates HT-gel (1gram) was active for 12-hours in maintaining oral hygiene
condition while CHX (Std-10ml) was active for 5-8 hours and Herbal mouth rinse (10ml) was active for 2-5 hour.

ACKNOWLEDGEMENTS.
We would like to thank Prof. B.G Shivananda, Principal Al-Ammen College of Pharmacy, Dr. Salma Khanam
HOD, Dept. of Pharmacognosy Al-Ammen College of Pharmacy Bangalore Karnataka India, for providing such
good environment for this projects. Dr. Jaye Prasad N Shethy Principal Gov. Dental College Bangalore Karnataka
India, provided us with additional data for his dual-buffering system and also provide facilities for achievement of
target.

Table –1: Percentage differences of S. mutans after 0h, 2h, 5h and 8h use of formulations

Group Time 4 3 2 1 0 Total

intervals (n%) (n%) (n%) (n%) (n%) (n%)
HMR 0 hour - 1 5 3 1 10
(10%) (50%) (30%) (10%) (100%)

2hour - - 2 6 2 10
(20%) (60%) (20%) (100%)

5hour - - - 1 9 10
(10%) (90%) (100%)
8 hour - - - - 10 10
(100%) (100%)
CHx Rinse 0 hour - 4 5 1 0 10
(40%) (50%) (10%) (0.00%) (100%)

2 hour - 1 5 3 1 10
(10%) (10%) (30%) (10%) (100%)

5 hour - - 3 4 3 10
(30%) (40%) (30%) (100%)
8 hour - - - 2 8 10
(20%) (80%) (100%)

HTG 0 hour 2 5 3 0 0 10
(20%) (50%) (30%) (0.00%) (0.00%) (100%)

2 hour - 4 5 1 0 10
(40%) (50%) (10%) (0.00%) (100%)

5 hour - - 5 4 1 10
(50%) (40%) (10%) (100%)
8 hour - - 1 6 3 10
(10%) (60%) (30%) (100%)

CHx= Chlorhexidine gluconate (0.2%), HMR= Herbal mouth rinse, HTG= Herbal toothpaste gel.

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 Aftab Alam et al: Continental J. Pharmacology and Toxicology Research 2: 19 - 26, 2008.

Table –2: Percentage differences of lactobacillus species after 0h, 2h, 5h and 8h use of selected formulation.

Group Time 4 3 2 1 0 Total

intervals (n%) (n%) (n%) (n%) (n%) (n%)
HMR
0 hour - - 2 7 1 10
(20%) (70%) (10%) (100%)

2hour - - - 8 2 10
(80%) (20%) (100%)

5hour - - - 1 9 10
(10%) (90%) (100%)

8 hour - - - - 10 10
(100%) (100%)
CHx Rinse
0 hour - - 5 5 0 10
(50%) (50%) (0.00%) (100%)

2 hour - - 4 5 1 10
(40%) (50%) (10%) (100%)

5 hour - - - 5 5 10
(50%) (50%) (100%)

8 hour - - - - 10 10
(100%) (100%)
HTG
0 hour - 2 5 3 0 10
(20%) (50%) (30%) (0.00%) (100%)

2 hour - - 6 4 0 10
(60%) (40%) (0.00%) (100%)

5 hour - - 2 7 1 10
(20%) (80%) (10%) (100%)

8 hour - - - 6 4 10
(60%) (40%) (100%)

CHx= Chlorhexidine gluconate (0.2%), HMR= Herbal mouth rinse, HTG= Herbal toothpaste gel

23
 Aftab Alam et al: Continental J. Pharmacology and Toxicology Research 2: 19 - 26, 2008.

Figure 1: Average reduction of Streptococcus mutans before and after 0h, 2h, 5h and 8h use of formulations.

Reduction of Streptococcus mutans

4
Average S. mutans in

3.5
3 Before use
2.5 0h
saliva

2 2h
1.5 5h
1 8h
0.5
0
HMR CHx MR HTPG
Formula tions

Figure 2: Average reduction of Lactobacillus species before and after 0h, 2h, 5h and 8h use of formulations.

Reduction of Lactobacillus species

ill 2.5
i
c
a 2
b a Before
o v
li 1.5
tc use
a
s
la n 1 0h
e i
g . 0.5
a p
s
r
e
v 0
A HMR CHx MR HTPG
Formulations

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Received for Publication: 21/08/2008

Accepted for Publication: 28/08/2008

Corresponding Author
Suman Yadav
Department of Chemistry, Swami Shradhanand College, University of Delhi, Alipur- 110036, India.

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