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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 areavailable regarding to herbal formulation hence this study is designed and carried out with the following aim andobjective. 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 METHODSPlants materials selection and extractionFruits of Phyllanthus emblica Linn and roots of Glycyrrhiza glabra Linn were collected from local market inBangalore and authenticated by Shri Gajendra Rao, Survey Officer, Regional Research Institute (Ay.). 200 gram ofdrugs were defatted by petroleum ether and then subjected to soxhlet extraction for 6-hours with methanol. Thesolvent was removed using rotary evaporator to get a dry residue. These extracts were stored in airtight container at4 0 C.FormulationsMouth rinse (Vanka , et al, 2001 and Aftab 2007)HMR contains Phyllanthus emblica extract as active constituent, Glycyrrhiza glabra active as well as sweeteningagent, peppermint oil as flavor, and the concentration of Phyllanthus emblica and Glycyrrhiza glabra are not lessthan 20mg/ml, pH-7Tooth gel. ( Vanka , et al, 2001,Aftab , 2007 )HTG contains Phyllanthus emblica extract as active constituent, Glycyrrhiza glabra active as well as sweeteningagent, Carbopol as gelling agent, Sorbitol as humectants, calcium carbonate as abrasive, xanthin gum as thickeningagent, peppermint oil as flavor, and the concentration of Phyllanthus emblica and Glycyrrhiza glabra are not lessthan 20mg/g. pH-7In vitro evaluation of herbal toothpaste gel and herbal mouth rinse of Phyllanthus emblica and Glycyrrhizaglabra extracts almost equivalent to commercial formulations (Noveon, 1911)Clinical evaluationsTo compare the efficacy of HT-gel and Herbal mouth rinse with chlorhexidine mouth rinse on cariogenicmicroorganism (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 PharmacyBangalore, Karnataka, India. After taking consent, the selected subjects were medically healthy with no systemicdisease, 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)ProcedureGrouping of subjects (Almas ,et al, 2004)All Subjects were divided into 3- groups with 10 subjects in each groupGroup 1 – HMR: Ten subjects were asked to rinse their mouth with 15ml of “HMR” for 5 min. A 2ml of stimulatedsaliva 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 withChlorhexidine.Group 3 –HTG: Ten subjects were asked to apply 1g of HT-gel thoroughly in oral cavity for 6 min. A 2ml ofstimulated saliva was collected before and after 0-h, 2h, 4h, 8-h, and 12-h application of HT-gel.Collection of salivaMidmorning 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 thesubjects were asked to continue chewing the wax and the saliva was collected at two-minute interval for total periodof 6 minutes. 2ml of saliva was collected in to a sterile glass cup. This process is repeated for 2h, 4h, and 8h. Thedietary factor was not included in this study.2
Aftab Alam et al: Continental J. Pharmacology and Toxicology Research 2: 19 - 26, 2008.Antimicrobial activityUsing an automatic micropipette with sterile plastic tips 1.5ml of saliva was transferred into the ependroff tubes andagitated for 30 sec on vortex mixture. The salivary samples were diluted in 0.05M Phosphate buffer (pH-7.0) to thedilution of 10 -2 - 10 -3 ( Larmas , 1992 and Adamkova ,et al, 2004). For the cultivation of mutant streptococcusmutans and lactobacilli species, 50 µL of the dilution was incubated on MSB agar media and Lactobacillus MRSAgar media (Kohler B,et al, 1979 and Westergren ,et al, 1978 ). The agar Plate was incubated anaerobically inanaerobic jar (5%Co 2 and 95% N 2 ) at 37 o C for 48 h.. Mutant streptococci and lactobacilli colonies are identified onthe agar plate by its typical colonial morphology. The number of mutans streptococci and lactobacilli colonyforming unit (CFU) per mal of saliva were estimated and scored as below (Kulkarni VV, et al, 2003 and Pai MR, etal 2004)ScoreScore-1Score-2Score-3Score-4Streptococcus mutans< 104 CFU/ml104- 105 CFU/ml105-106 CFU/ml> 106 CFU/mlLactobacilli species105 CFU/mlStatistical analyses were carried out compare the percentage difference inhibition of carcinogenic bacteria andaverage bacterial growth before and after use of formulations. The percentage difference of salivary bacteria wasgiven in Table -1 (S. mutans) and Table-2 (Lactobacilli sp.) growth and graph show the average bacterial growthbefore and after 0h, 2h, 5h, and 8h were shown in graph 1 (S. mutans) and graph 2 (lactobacilli species).RESULTSClinical studyIn present study comparative clinical study of herbal formulation and marketed formulation were carried inGovernment dental college. The study was designed to analysis the effectiveness of formulation in 8-hour use. Forthe study 30 subjects were divided in three groups i.e. Herbal rinse groups were 10 subjects, Chlorhexidine groupswere 10 subjects and Herbal tooth gel groups were 10 subjects. Each subject was previously given demonstrationbefore collecting the saliva 2ml paraffin stimulated saliva was collected before and after 0 hour, 2 hours, 5 hours and8 hours use of formulation. The antimicrobial activity was evaluated using MSB agar for S. mutans andLactobacillus MRS Agar for Lactobacillus species and the bacterial colony was counted using colony counter after48-hour incubation in 10% CO 2 incubator. The percentage difference of Streptococcus mutans after 0h, 2h, 5h and8h are reported in Table No 1 and percentage difference of Lactobacilli species after use of 0h, 2h, 5h and 8h arereported in Table No 2. The graph shows the average microbial growth in saliva before and after use offormulations. 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.DISCUSSIONIn present study the main aim was to prepare an effective formulation for human use, and maintain proper oralhygiene upto 8 hours. The selected formulations were comparatively evaluating in-vivo in a study carried out atGovernment Dental College Bangalore. The midmorning paraffin stimulated saliva was taken before and afterimmediate (0h), 2h, 5h and 8h use of formulation. The microbial count of each salivary extract of herbal rinse groupsindicated after use, 90% inhibition of SM and lactobacilli species and after 5h, 10% reduction of SM andlactobacilli species indicate herbal mouth rinse (HM rinse) was effective for 5 hour only. The microbial count ofeach salivary extract of CH x rinse groups indicated after use 100% reduction of SM and lactobacilli species andafter 8h, 20% reduction of SM and 0.00% reduction of Lactobacilli species, concluded that CHx mouth rinse waseffective for 5 to 8 hours. Herbal toothpaste gel (HT-gel) groups were indicate after application 100% reduction ofSM 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 anyfluoride ingredient so it can be used in fluorosis condition and area where water fluoride level is high and alsorecommended for children below 6 years.3
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