In Vitro Anticariogenic Activity of Some Indian Medicinal Plants Towards Human Oral Pathogen

Indrni Bhttchry, Klpesh Ishnv*, Jenbhi Chuhn

Ashok and Rita Patel Institute of Integrated Studies and Research in Biotechnology and Allied Sciences (ARIBAS), New Vallabh Vidyanagar-388121, Gujarat, India

Regular article

In Vitro Anticariogenic Activity of Some Indian Medicinal Plants Towards Human Oral Pathogen

Indrani Bhattacharyaa, Kalpesh Ishnavaa*, Jenabhai Chauhana

Ashok and Rita Patel Institute of Integrated Studies and Research in Biotechnology and Allied Sciences (ARIBAS), New Vallabh Vidyanagar-388121, Gujarat, India

Oral diseases continue to be a major health problem worldwide. The increasing failure of chemotherapeutics and antibiotics resistance exhibited by pathogenic micro-organisms has led to the screening of several medicinal plants for their potential anticariogenic activity. In the present study, in vitro anticariogenic potential of the organic solvents of 20 plant leaves was evaluated by using f ve oral pathogen, Lactobacillus acidophilus, Lactobacillus casei, Staphylococcus aureus, Streptococcus mutans and Candida albicans. Agar well diffusion method and minimum inhibitory concentration were used for this purpose. Plant extracts exhibited varying degree of inhibited on growth of oral pathogen between hexane and ethyl acetate extracts is highly potential. Maximum activity show hexane (15mm) and ethyl acetate (16mm) extracts of Piper betle against S. mutans. The MIC of crude extract of ethyl acetate of Syzygium rubicundum against L. acidophilus was 0.12 mg/mL and ethyl acetate extract of Piper betle was 1 mg/mL and 0.25 mg/mL respectively for S. mutans and L. casei. The preliminary phytochemical analysis of crude extract of ethyl acetate of P. betle prsent the Phenolic compound and Alkaloid. Clear zone of inhibition against S. mutans located on TLC plate when bioautography performed using ethyl acetate extract of P. betle. The bioactive compound was separated from the crude ethyl acetate extract by using TLC technique. Further, spectroscopy analysis are requiring for determination of structure of bioactive compound. The f nding may be useful in the development of drugs against oral pathogen.

Anticariogenic activity; Oral Pathogen; Leaves; Medicinal Plants

1 Introduction

The oral cavity is very complex and heterogeneous microbial habitat. Oral diseases continue to be a major health problem worldwide [1]. Tooth decay and periodontal disease are among the most important global oral health problems. Tooth loss, caused by poor periodontal health (which affects up to 20% of the adult population worldwide) can lead to signif cant morbidity and premature death. There are mainly many different types of bacteria involved in tooth decaying process. Such as mutans group of streptococci, S. salvarious, S. mitis, S. milleri, S. oralis, S. sanguis, Lactobacillus casei, Actinomyces viscosus etc [2]. Tooth decay, also known as dental caries or cavity, is a disease where in bacterial processes damage hard tooth structure. These tissues progressively break down, producing dental caries. Two groups of bacteria are responsiblefor initiating caries: Streptococcus mutans and Lactobacillus. If left untreated, the disease can lead to pain, tooth loss, infection, and in severe cases death. Today, caries remains one of the most common diseases throughout the world. The link between oral diseases and the activities of microbial species that form part of the microbiota of the oral cavity is well established. Over 750 species of bacteria inhabit the oral cavity and a number of these are implicated in oral diseases [3]. The development of dental caries involves acidogenic and aciduric Gram-positive bacteria, primarily the mutans streptococci, which metabolize sucrose to organic acids that dissolve the calcium phosphate in teeth, causing decalcification and eventual decay. Dental caries is thus a supragingival condition [2]. Sucrose is the only sugar that S. mutans can use to form this sticky polysaccharide. Conversely, many other sugars such as glucose, fructose, and lactose can be digested by S. mutans, but they produce lactic acid as an end product. It is the combination of plaque and acid that leads to dental decay. Due to the role the S. mutans plays in tooth decay, there have been many attempts to make a vaccine for the organism. So far, such vaccines have not been successful in humans [4].

Despite several agents being commercially available, these chemicals can alter oral microbiota and have undesirable side-effects such as vomiting, diarrhea and tooth staining [5]. For example, bacterial resistance to most (if not all) of the antibiotics commonly used to treat oral infections (penicillins and cephalosporins, erythromycin, tetracycline and derivatives and metronidazole) has been documented [6]. Other antibacterial agents used in the prevention and treatment of oral diseases including cetylpyridinium chloride, chlorhexidine, amine f uorides or products containing such agents, are reported to exhibit toxicity, cause staining of teeth or in the case of ethanol (commonly found in mouthwashes) have been linked to oral cancer [7].

Plants are medically important we had screened out many important plants from our natural environment plants are known as medicinal because they contain active substances that cause certain reactions, from relenting to the cure of diseases, on the human organism [8]. Knowledge on medicinal plants sometimes means the only therapeutic resource of some communities and ethnic groups. Thus, due to different chemical components involved against a tooth decayed organism responsible [9, 10]. From these screened plants we got some wonderful results which we can use in our daily life as a ingredient in tooth paste or as a medicine to prevent tooth decaying which is the major problem in the world [11, 12]. In fact there is an overwhelming number of studies on the biological activities of plants and their natural product derivatives [13, 14]. There have been numerous reports of the use of traditional plants and natural products for the treatment of oral microbial pathogens.

Hence, the search for alternative products continues and natural phytochemicals isolated from plants used in traditional medicine are considered as good alternatives to synthetic chemicals [15]. The present study was undertaken to screen 20 selected medicinal plants for their efficacy against different oral pathogen under in vitro conditions. Attempt was also made to characterize bioactive compounds at primary level. These information will may be useful to search for new cost-effective drugs of natural or synthetic origin in future.

2 Materials and Method

2.1 Plant materials

The different plant species were selected based on ethnopharmacological information and collected between December, 2009 to February, 2010 from medicinal plant garden, New Vallabh Vidyanager and surroundings place collected (Table 1). Theleaves of all the healthy and disease free plants were used to test the antibacterial activity. The plant specimens were identified by Dr. Kalpesh Ishnava (Plant Taxonomist) at Ashok and Rita Patel Institute of Integrated Study & Research in Biotechnology and Allied Sciences (ARIBAS), New Vallabh Vidyanagar, Gujarat, India.

Table 1 Details of selected medicinal plants leaves

2.2 A preparation of plant leaves extracts

First of all the leaves of respective plants were thoroughly washed with running tap water, blotted and dried under sunlight. For the purpose of making powder it was grinded in grinder (Maharaja Mixer Ltd). From these, 50 grams of powdered material were soaked in 250 ml of hexane for 24 hours at room temperature under shaking condition (130-140 rpm). The extract was f ltered with the help of Whatman filter paper number-1. The filtrate was collected in petridish and dried at room temperature. The dried extract from petridish was scraped and transferred to eppendorf tube. The residual material from the funnel was dried again and resuspended in 250 ml ethyl acetate for 24 hours at room temperature under shaking condition (130-140 rpm). The extract was filtered and collected in petridish. It was dried at room temperature. Similarly, the residual materials from the funnel are preserved and re-extracted with same volume (250 ml) of methanol and then distilled water respectively. In both the cases, the resultant culture filtrate was air dried at room temperature. The dried extract from petridish was scraped and transferred to eppendorf tube and weight.

2.3 Oral Pathogenic Strains

A group of bacteria known to cause tooth decay were selected and purchased from Microbial Type Culture Collection (MTCC) bank, Chandigarh as a freeze dried pure culture. The bacterial cultures were revived by using MTCC specified selective growth medium and preserved as glycerol stocks. The bacteria responsible for dental caries used the Lactobacillus acidophilus (LA) (MTCC-*447), Lactobacillus casei (LC) (MTCC-1423), Streptococcus mutans (SMU) (MTCC-890) and Staphylococcus aureus (SA) (MTCC-96) and Candida albicans (CA) (MTCC-183) for the study.

2.4 Preparation of Inoculums

Fresh microbial cultures were prepared by streaking loopful of bacterial suspension in to organism specific selective media (Hi-media) and incubated at optimal temperature in order to maintain approximately uniform growth rate of each organism. The bacterial cultures from fresh media was compared with 0.5 McFarland turbidity standard, which is equivalent to approximately 1×108bacterial cell count per ml, was maintained throughout the experimentation.

2.5 Bioassay for Antimicrobial activity

2.5.1 Agar Well Diffusion Method

In the present study, to test antibacterial activity, twenty different plant extracts were used. The antibacterial activity was studied by agar well diffusion method [16]. From the stock, 100 mg of each plant extract were suspended in one milliliter of Dimethyl sulfoxide (DMSO). In order to make agar plates, the petriplates were thoroughly washed using detergent, dried and sterilized in autoclave at 15 lbs pressure for 15 minutes. Approximately 25 ml of sterilized selective medium was poured in to each petridish and solidified at room temperature. The plates were incubated at 37 ºC for sterility checking for overnight. Agar plates were marked and divided in to 4 equal parts, labeled for specif c organism and extract number. A fresh bacterial culture of 100 μl having 108CFU/ml was spread on agar plates with glass spreader. A well of 10 mm diameter punched off at previously marked petriplates in to agar medium with sterile cup borer and then it was f lled with 100 μl of respective plant leaves extract. Plates were placed for 30 min in refrigerator for diffusion of extracts and then incubated at 37 °C (or specif ed temperature) for 24 h or more depending upon the organisms, until appearances of zone of inhibition. The zone of inhibition (excluding well diameter) was measured as a property of antibacterial activity.

Antibiotic, Cefadexin, Erythromycin and Tetracycline (Intas Laboratory Pvt Ltd., Ahmadabad, Gujarat, India) was used as standard at a concentration of 100 μg/ml and 100% DMSO were used as positive control and negative control respectively. Bioassay was performed in duplicate and repeated twice.

2.5.2 Antifungal activity

The plant leave extracts of twenty different plant extracts (Table 2) were screened for antifungal activity by agar well diffusion method [16] with sterile cork borer of size 08.0 mm. The cultures of 48 hours old grown on YEPD (yeast extract peptone dextrose agar medium) were used for inoculation of fungal strain on YEPD plates. An aliquot (0.02 ml) of inoculums was introduced to molten YEPD media and poured in to a petri dish by pour plate technique. After solidification, the appropriate wells were made on agar plate by using cork borer. In agar well diffusion method 0.05 ml of plant leave extracts of twenty different plant extracts were introduced serially after successful completion of one plant analysis. Incubation period of 24-48 h at 30 °C was maintained for observation of antifungal activityof plant leave extracts. The antifungal activity was evaluated by measuring zones of inhibition of fungal growth surrounding the plant extracts. The complete antifungal analysis was carried out under strict aseptic conditions. The zones of inhibition were measured with antibiotic zone scale in mm and the experiment was carried out in triplicates.

2.5.3 Minimum Inhibitory Concentration (MIC) Determination

Minimum inhibitory concentration was evaluated by the two fold serial broth dilution method [17]. Plant extracts showing more than 09 mm inhibition zone were selected for MIC. Selective broth medium was used for dilutions as well as preparing inoculums. The bacterial cell density was maintained uniformly throughout the experimentation at 1×108CFU/ml by comparing with 0.5 McFarland turbidity standards. Plant extract of 40 μl from stock solution (100 mg/mL) was taken in to f rst dilution tube containing 960 μl of selective medium broth and mixed well. From these, 500 μL were transferred to second tubes containing 500 μl broths. This step was repeated nine times and from last tube 500 μl solutions was discarded. The 100 μl of test organisms was added in each tube. The f nal volume of solution in each tube was made up to 0.6 ml. The MIC was tested in the concentration range between 4.0 mg/ml to 0.0031 mg/ml. Tubes were incubated at optimal temperature and time in an incubator. Growth indicator 2,3,5-triphenyl tetrazolium chloride solution (100 μl of 0.1%) was incorporated in each tube to find out the bacterial growth inhibition. Tubes were further incubated for 30 min under dark conditions. Bacterial growth was visualized when colorless 2,3,5-triphenyl tetrazolium chloride was converted into red color formazone in the presence of bacteria. Each assay was repeated thrice by using DMSO and selective medium as control.

2.6 Preliminary phytochemical analysis

Qualitative phytochemical analysis of all the plant leaves extracts selected, based on MIC value was perform as per the methodology of Parekh and Chanda, 2007 [18].

2.7 TLC-Bioautography

Out of 20 plants leaves extracts tested for anticariogenic activity, only one showing maximum growth inhibition against S. mutans (MTCC-890), L. casei (MTCC-1423) and S. aureus (MTCC-96) was selected and used for bioautography. By using capillaries 10 μl of aqueous extract of Piper betle leaves extract (100mg/mL stock solution) was spotted on to 0.25 mm thick precoated silica gel 60 F254 plate (Merck, Germany). The band length was 2 mm thick. After air drying the TLC plate was run using pre-standardized solvent system, Toluene: ethyl acetate: methanol (3:2:1). The chromatogram was observed under UV illumination and used for bioautography. Organisms specific agar medium, seeded with specif c bacteria Streptococcus mutans, Lactobacillus casei and Staphylococcus aureus was overlaid on to the silica gel plate loaded with sample and incubated at 37 °C for 24 h. On the next day, the plate was flooded with 2,3,5-triphenyl tetrazolium chloride (0.1%) to visualize growth inhibition. The area of inhibition zone was appeared as transparent against reddish background (lawn of living bacteria).

3 Results and Discussion

There has been revival of great interest in medicinally important plants. This is because of increased awareness of the limitations of ability synthetic pharmaceutical products to control major diseases. The present study has both significance and relevance. Plant synthesizes large number of secondary metabolites; they may serve as futurereservoir of novel drugs and therapeutic agents. Many of these metabolites have been showed to be active against oral pathogens [13, 19].

In the present study, the antimicrobial sensitivity assay of plant extracts against tooth decaying organisms was carried out. The leaves of 20 plants were sequentially extracted using hexane, ethyl acetate, methanol and distilled water and then used for antimicrobial assay. The result of sensitivity of tooth decaying organisms (SMU, LC, SA, LA, CA) was assessed by visualizing the presence or absence of inhibition zone and measuring the zone of diameter. The results are summarized as under Table 2:

Table 2 Antibacterial activities of different plant leave extract against oral pathogen and their zone of inhibition (in mm)

3.1 Streptococcus mutans

Hexanolic extract of Piper betle (15 mm) and Saraca asoca (8 mm) showed the highest activity whereas Ficus benghalensi, Cassia fistula, Nerium indicum showed lowest activity against this bacteria. Rest of the 11 plants doesn’t show any activity against these bacteria (Table 2). Ethyl acetate extracted Piper betle (16 mm) and Mangifera indica (12 mm) showed the highest activity against these bacteria (Table 2). S. asoca, B. montanum, A. marmelos and A. sativa these extracts showed lowest activity against S. mutans. Rest of the 5 plants doesn’t show any activity against S. mutans. M. indica (12 mm) and M. hexandra (11 mm) showed the highest activity (Table 2) whereas, E. divarticata and A. marmelos showed lowest activity against S. mutans when methanolic fraction was used. Twelve plants were found inactive against this bacterium. Aqueous extract of A. digitata (7 mm) and E. divaricata (6 mm) showed the highest activity against this organism (Table 2). A. marmelos showed the lowest activity against this organism and rest of the 15 plants doesn’t show any activity against this organism. Comparatively, less activity was found when aqueous extract of A. digitata, E. divaricata, C. congesta, A. sativa and A. marmelos used.

3.2 Lactobacillus acidophilus

Hexanolic extract of S. rubicundum (13 mm) and A. marmelos (12 mm) presented the highest activity (Table 2) whereas, P.rubra, M. hexandra and piper betle presented lowest activity against this organism. Twelve plants don’t show any activity against L. acidophillus. In ethyl acetate, S. rubicundum (13 mm) and E. divaricata (10 mm) presented highest activity. S. asoca and P. rubra, M. hexandra extracts showed the lowest activity against this organism (Table 2). 12 plants were found to be inactive against this bacterium. Eight plants extracted with methanol are active with maximum zone of inhibition in M. indica (10 mm) and M. hexandra (9 mm) against L. acidophilus. E. divarticata and A. marmelos showed the moderate activity against this organism (Table 2) and rest of the 12 plants doesn’t show any activity against L. acidophilus. Aqueous extracts of F. virens (9 mm) and M.indica (7 mm) showed the highest activity against L. acidiphilus. A. marmelos, S. rubicundum, M. hexandra showed the lowest activity against this organism (Table 2). Whereas, 15 plants doesn’t show any activity against this organism.

3.3 Lactobacillus casei

Growth of this organism was inhibited by hexanolic extract of Piper betle (9 mm) (Table 1). Low 4 mm inhibition was found when F. benghalensis extract was used (Table 2). Rest of the 18 plants doesn’t show any activity against this organism. Ethyl acetate extract of Piper betle (13 mm) and M. indica (12 mm) showed the highest inhibition on the growth of L. casei (Table 2). S. asoca, F. virens and C. fistula extract exhibited variable growth inhibition pattern against this organism (Table 2). Whereas, 15 plants found to be inactive against L. casei oral bacteria. When extracted with methanol M. indica (10 mm) and C. f stula (9 mm) showed the highest activity against this organism (Table 2). S. asoca and Piper betle comparatively low activity was found when extract was used. Sixteen plants don’t show any activity against this organism. Aqueous extract of only two plants were found to inhibit L. casei at low level C. f stula (4 mm) and F. virens (3 mm) (Table 2). No activity was found in rest of the 18 plants.

3.4 Staphylococcus aureus

Hexanolic extract of Piper betle (8 mm) showed the highest activity whereas D. indica (6 mm) exhibit moderate activity (Table 2). Remaining 18 plants doesn’t show any activityagainst this organism. In ethyl acetate extracts only 4 plants found to be inhibitory to S. aureus. They are Piper betle (14 mm), M. indica (8 mm), D. indica (8 mm) and N. indica (4 mm) (Table 2). Rest of the 16 plants found to be inactive against this organism. In methanolic extract 5 plants showed activity against S. aureus .This includes M. indica (8 mm), M. hexandra (8 mm), S. asoca (8 mm), F. virens (7 mm) and P. betle (7 mm) (Table 2). Fifteen plants don’t show any activity against this organism. Distilled water extract of 2 plants P. betle (6 mm) and F. virens (5mm) showed the moderate activity against this organism (Table 2). Eighteen plants are totally inactive against this organism.

3.5 Candida albicans

Only one plant found to be active against this fungal organism. The antifungal substances are extractable in ethyl acetate and hexane of P. betle and showed zone of inhibition of 14 mm and 12 mm respectively (Table 2). None of the solvents found to be good for the extraction of compound from the 19 plants.

3.6 Determination of minimum inhibitory concentrations (MIC)

The minimum inhibitory concentrations (MIC) of selected palnt extract (hexane, ethyl acetate, methanol, distilled water) against all five organisms (SMU, LA, LC, SA, CA) was determine usually by using two fold serial dilution method and 2, 3, 5-Tri phenyl tetrazolium chloride. The result is summarized in Figure 1. The MIC of crude extract of ethyl acetate of S. rubicundum against LA was 0.12 mg/mL (Figure 1), lower than that of the hexane extract 2 mg/mL (Figure 1). The MIC value M. hexandra methanolic extract is 2 mg/mL and 1 mg/mL against SMU and SA respectively (Figure 1). Some plant when extracted with hexane exhibited MIC value 4 mg/mL against SMU. The MIC of M. indica ethyl extract for SMU, LC and SA are 1 mg/mL, 0.5 mg/mL respectively (Figure 1). The MIC value methanolic extract some plant was 1 mg/mL and, 2 mg/mL respectively for SMU and LC (Figure 1). The Hexane extract of A. marmelos was 4 mg/mL against LA (Figure 1). D. indica ethyl acetate extract exhibited 4 mg/mL MIC value against SA (Figure 1). The MIC value of .N. indicum methanolic extract was 4 mg/mL against LA (Figure 1). The MIC of ethyl acetate extract of P. betle was 1 mg/mL and 0.25 mg/mL respectively for SMU and LC (Figure 1), the hexanolic extract of some plant gives MIC value of 1 mg/mL, 2 mg/mL and 1 mg/mL respectively SMU, LC and SA (Figure 1).

Fig. 1 The MIC (mg/mL) of selected plant leaves extract against oral pathogens

3.7 Phytochemical analysis

The phytochemical test was carried out for all plant extracts selected for MIC determination and Table 3. The methanolic extract of M. hexandra shows a positive result for tannins, saponins, phenolic compound and alkaloid, thus these compounds may be present in the extract and hexane extract shows positive result for alkaloid only, thus alkaloid may be present in the extract (Table 3). The hexanolic extract of S. rubicundum found positive for alkaloid whereas steroids and alkaloid both are found in case of ethyl acetate extract (Table 3). Distilled water extract of some plant shows positive result for tannins, phenolic compound and alkaloid, thus these compounds may be present in the extract (Table 3). The ethyl acetate extract of M. indica positive for tannins and alkaloids, whereas methanolic extract shows positive result for tannins, saponins, steroids, phenolic compound and alkaloid (Table 3). The hexane extract and ethyl acetate extract of P. betle found positive for tannins, saponins, cardiac glycosides, terpenoids, phenolic compound and alkaloids (Table 3). The ethyl acetate extract of D. indica spositive for alkaloid. The methanol extract of N. indicum found positive for saponins, terpenoids, phenolic compounds and alkaloids (Table 3). Finally the hexane extract of A. marmelos found positive for terpenoids, phenolic compound and alkaloids, thus these compounds may be present in the extract (Table 3).

Table 3 Phytochemical analysis of crude leaves extract of selected plants

3.8 Bioautography

The hexane and methanolic extract of P. betle was selected for the bioautography as it gives largest zone of inhibition against most of the selected organisms. The bioautography was done against all organisms. After addition 2,3,5-triphenyl tetrazolium chloride (0.1%) the entire plate changes to reddish colour which leads to clear visualization of zone. From that zone the band is identified which responsible for inhibition of the growth of the organism. Clear zone of inhibition against SMU located on TLC plate when bioautography performed using ethyl acetate of P. betle. Clear zone of inhibition was found in case of SMU, LC, SA when used hexanolic extract of P. betle.

3.9 Analytical TLC

In order to find out active principle present in P. betle hexane extract and ethyl acetate extract, TLC solvent system was standardized (toluene: ethyl acetate: methanol-3:2:1) and used for subsequent analysis. The bioactive compound was separated from the crude hexane and ethyl acetate extract by using TLC technique (Rfvalue: 0.66, 0.68, 0.72, 0.77, 0.83, 0.88). The resultant chromatogram was used for bioautography against all 4 organisms. The UV analysis of TLC plate run from P. betle hexane and ethyl acetate extract showed no fluorescence at 245 nm and blue fluorescence at 365 nm respectively. The presences of bands also confirm by iodine vapour. Development of TLC plate using cardiac glycosides, alkaloids, saponins and terpenoids specific reagent may indicate presence of cardiac glycosides, alkaloids, saponins and terpenoids .respectively. Further, spectroscopy and chromatographic analysis are requiring for determination of structure of bioactive compound.

There has been revival of great interest in medicinally important plants. This is because of increased awareness of the limitations of the synthetic pharmaceutical products to control major diseases. Plant synthesizes large number of secondary metabolites they may serve as future reservoir of novel drugs and therapeutic agents. Many phytochemicals have been shown to be active against oral pathogens [13, 19].

Nalina and Rahim (2006) studied effect of crude aqueous extract of leaves of Piper betle on virulence activity of S. mutans [20]. Though large number of researchers studied effect of plant extract in different parts of the world. There is meager information on the effect of leafy plants on tooth decaying organisms. Natural products have been used to prevent oral diseases, especially plaquerelated diseases, such as dental caries [21].

Seghal et al., (2005) studied inhibitory effect of various extract of Calotropis procera against Candida albicans and found that there is signif cant inhibition in the growth of C. candida [22].

Most of the selected organisms are sensitive to hexanolic and ethyl acetate extracts of Peper betle. Therefore, it is subjected for MIC determination and phytochemical characterization. The f nding may be useful in the development of drugs against dental caries.

Antibiotic which are in commercial use have few well defined target on bacteria and fungi. Disruptions of cell wall, inhibition of DNA replication or protein synthesis are some of the common mechanism for antibiotic activity. Since the target of these antibiotics is few well def ned, there is a rapid evolution of bacterial drugs. Moreover, there is increasing resistance to available antimicrobials. This bacterial mechanism is widely present in the bacterial system and became world health problem.

4 Conclusion

Plant extracts have great potential as antimicrobial compounds against microorganisms drugs and therapeutic agents against oral pathogens. Thus, they can be used in the treatment of different infectious diseases. Due to ever increasing problem of dental caries, the present study on antimicrobial screening of medicinal plants is both, signif cant and relevant, since plant synthesizes array of secondary metabolites they may served as future reservoir of novel drug. The good inhibitory potential of hexanolic and ethyl acetate extracts of Peper betle plants against Candida albicans and panel of cariogenic bacteria will be useful in the future development of effective formulations of drugs for the control of human oral pathogens.

Acknowledgements

Authors are thankful to Charutar Vidya Mandal(CVM), Vallabh Vidyanagar and Director of Ashok and Rita Patel Institute of Integrated Studies and Research in Biotechnology and Allied Sciences (ARIBAS), New Vallabh Vidyanagar, Gujarat, India for providing necessary support for research and laboratory facility.

[1] Petersen PE, Bourgeois D, Ogawa H, et al. The global burden of oral diseases and risks to oral health. Bull World Health Organ, 2005, 83: 661-669.

[2] Loesche W. Dental caries and periodontitis: contrasting two infections that have medical implications. Infect Dis Clin Nth Am, 2007, 21: 471-502.

[3] Jenkinson HF, Lamont RJ. Oral microbial communities in sickness and in health. Trends Microbiol, 2005, 13: 89-95.

[4] Klein JP, Scholler M. Recent advance in the development of a Streptococcuc mutans vaccine. Journal of Epidemiology, 1998, 4: 419-425.

[5] Park KM, You JS, Lee HY, et al. Antibacterial agent from the root bark of Morus alba against oral pathogens. J Ethnopharmacol, 2003, 84: 81-85.

[6] Bidault P, Chandad F, Grenier D. Risk of bacterial resistance associated with systemic antibiotic therapy in periodontology. J Can Dent Assoc, 2007, 73: 721-5.

[7] Rodrigues F, Lehmann M, Amaral VS, et al. Genotoxicity of three mouthwash products, Cepacol, Periogard, and Plax, in the Drosophila wing-spot test. Environ Mol Mutagen, 2007, 48: 644-649.

[8] Cowan MM. Plant products as antimicrobial agents. Clin Microbiol Re., 1999, 12: 564-82.

[9] Ishnava K, Chauhan J, Barad M. Anticariogenic and phytochemical evaluation of Eucalyptus globules Labill. Saudi J Biological Sci., 2013, 20: 69-74.

[10] Barad M, Ishnava K, Chauhan J. Anticariogenic activity and phytochemical studies of crude extracts from some Indian plant leaves. J Intercultural Ethanoparmacol, 2014, 3: 85-90.

[11] Chitme HR, Chandra R, Kaushik S. Studies on antidiarrheal activity of calotropis gigantea R. Br. in experimental animals. J Pharm Pharmaceut Sci, 2003, 7: 70-75.

[12] Kim HS. Do not put too much value on conventional medicines. J Ethnopharmacol, 2005, 100: 37-39.

[13] Hebber SS, Harsha VH, Hegde GR, et al. Ethanomedicine of Dharwad district in Karnataka, India-plant in oral healthcare. J ethnopharmacol, 2004, 94: 261.

[14] Pai MR, Acharya LD, Udupa N. Evalution of antiplaque activity of Azadivacta indica leaf extract gel-a 6 week clinical study. J Ethanopharmocol, 2004, 90: 99-103.

[15] Prabu GR, Gnanamani A, Sadulla S. Guaijaverin - a plant flavonoid as potential antiplaque agent against Streptococcus mutans. J Appl Microbiol, 2006, 101: 487-495.

[16] Perez C, Paul M, Bezique P. An Antibiotic assay by the agar well diffusion method. Alta Biomed, 1990, 15: 113.

[17] Chattopadhyay D, Maiti K, Kundu AP, et al. Antimicrobial activity of Alstonia macrophylla: folklore of bay island. Journal of Ethnopharmacology, 2001, 77: 49-55.

[18] Parekh J, Chanda SV. In vitro antimicrobial activity and phytochemical analysis of some Indian medicinal plants. Turk. J Biotechnol, 2008, 31: 53-58.

[19] Parimala Devi B, Ramasubramaniaraja R. Dental caries and medicinal plants - An overview. Journal of pharmacy research, 2009, 2: 1669-1675.

[20] NalinaT, Rahim ZHA. Effect of Piper betle L. extract on the virulence activity of Streptococcus mutans-in vitro studies. Pakistan Journal of Biological science, 2006, 9: 1470-1475.

[21] Koo H, Pearson SK, Scott-Anne K. Effect of apigenin and t-farnesol on glucosytransferase activity and caries development ub rats. Oral microbial immunol, 2002, 17: 337-343.

[22] Sehgal R, Arya S, Kumar VL. Inhibitory effect of extract of latex of Calopropis procera against Candida albican.. A prelimary study. Indian Journal of pharmacology, 2005, 17: 334-335.

* Author to whom correspondence should be addressed. Address: Ashok and Rita Patel Institute of Integrated Studies and Research in Biotechnology and Allied Sciences (ARIBAS), New Vallabh Vidyanagar-388 121, Gujarat, India; Email: ishnavakb203@yahoo. com

Received: 2015-10-11 Accepted: 2015-12-21