The study was done in an experimental laboratory setting with an in vitro study design, for a period of three months in 2015 at Central Research Laboratory, MAHER University. The study was approved by the Institutional Ethical Committee, Meenakshi Ammal Denal College. Standard strains of the organisms were obtained from MTCC and ATCC. The organisms ordered were Streptococcusmutans MTCC no. 890, Streptococcus oralis MTCC no 2696 and Lactobacillus acidophilus MTCC no. 10307, Streptococcussanguis ATCC no. 10556, Streptococcus salivarius ATCC no. 13419. The organisms were revived and the sugar limited cultures were maintained at a pH of 7 throughout the stationary phase. The leaves of the two plants Solanum nigrum and Phyllanthus niruri were dried in shade and powdered using a blender. The resulting powder was then stored in air tight wide mouth containers. A 25 mg of herbal powders were dissolved in 75 ml ethyl alcohol (1:3 w/v) and extracted at room temperature. The solution was then placed in an orbital shaker and left for three days. The resulting solution was filtered using a whatman filter paper. The filterate was placed in a rotary evaporater. The powder obtained was weighed and 100 mg of the powder was mixed with 1 ml of DMSO (Dimethyl Sulphoxide) and stored in amber coloured bottles at a temperature of –20°C for further use [4].

The antibacterial susceptibility was tested by agar well diffusion method. Inoculums were prepared and adjusted to 0.5 McFarland turbidity standard guidelines [12]. The inoculum suspension from the nutrient broth was streaked uniformly on TSA-BA and MRS agar. Wells were cut into the lawn cultures with a sterile borer of well of 0.5 cm diameter in the pathogen inoculated media. A 50 µl of each extract were aseptically filled into the well. The plates were placed at room temperature to allow diffusion of extract into the agar for a period of one hour. It was then incubated for 24 hours at 37°C. The results were recorded by measuring the diameter of zone of inhibition at the end of 24-48 hours. A 0.2% chlorhexidine was used as a positive control, while DMSO medium without the herbal extracts was taken as a negative control. After incubation the clear zone around the wells was measured in mm and was recorded as the zone of inhibition. The test was done in triplicates and average values were recorded.

The MIC Determination and MBC determination was done using the broth dilution method [12]. A pure culture of the test organism was grown in the respective broth. A 100 µl of the nutrient broth was mixed with 100 µl of the herbal extract (1:1) in column 1 of the microtitration tray. After the herbal extract has been diluted, 100 µl volume of the diluted herbal extract from column 1 was added to each dilution vessel in column 2 using a multichannel pipette, 100 µl was then removed from column 2 and added to column 3. Thus serial dilution beginning from 100 µl to 50 µl, 25 µl, 12.5µl, 6.25 µl till 3.125 µl was performed. The inoculated, serially diluted herbal agent was then incubated for about 24 hours. The series of dilution vessels were then observed for microbial growth, indicated by turbidity in the bottom of the vessel. The last dilution tube that did not show growth corresponded to the MIC of the antimicrobial agent. The MIC dilution and two other dilutions of the herb were then plated to determine the Colony Forming Units (CFUs). The MBC (Minimum Bactericidal Concentration) value was determined as the lowest concentration at which 99.9% of the bacteria showed lysis [12].

The results were tabulated and since the data obtained was non parametric, Mann-Whitney test was used to compare the zones of inhibition of the two herbs.

The results have been summarised in [Table/Fig-1,2,3,4,5,6,7 and 8]. The herb Phyllanthus niruri Labelled as ‘A’ (9.6 mm-11.6 mm) showed lesser antibacterial inhibition than Solanum nigrum (12.3 mm-14.6 mm) labelled as ‘B’. The results were statistically significant for Streptococcus sanguis, Streptococcus salivarius, Streptococcus oralis and Streptococcus mutans (p-value<0.05). Although for Lactobacillus acidophilus, the inhibitory effect of Solanum nigrum was more than Phyllanthus niruri, the p-value>0.05 indicating that it is not statistically significant [Table/Fig-3]. The MIC and MBC were also found to be least. The MIC for Lactobacillus acidophilus (25 and 50 mg/ml) for both the herbs Solanum nigrum and Phyllanthus niruri respectively, indicating that these herbs were effective antimicrobials even in lower concentrations [Table/Fig-9,10].

[Table/Fig-4]:

Antibacterial zones of inhibition against Streptococcus sanguis on TSA-BA. NC-Negative control, B-Solanum nigrum (14.3 mm), PC-Positive control, A-Phyllanthus niruri (11.6 mm)

[Table/Fig-5]:

Antibacterial zones of inhibition against Streptococcus salivarius on TSA. NC-Negative control, B-Solanum nigrum (14.3 mm), PC-Positive control, A-Phyllanthus niruri (9.6 mm)

[Table/Fig-6]:

Antibacterial zones of inhibition against Streptococcus oralis on TSA-BA. NC- Negative control, B-Solanum nigrum (14.3 mm), PC- Positive control. A-Phyllanthus niruri (11.6 mm)

[Table/Fig-7]:

Antibacterial zones of inhibition against Streptococcus mutans on TSA- BA. NC- Negative control, B-Solanum nigrum (14.6 mm), PC- Positive control, A- Phyllanthus niruri (11.3 mm)

[Table/Fig-8]:

Antibacterial zones of inhibition against Lactobacillus acidophilus on MRS agar. NC-Negative control, B-Solanum nigrum (12.3 mm), PC- Positive control, A-Phyllanthus niruri (11 mm)

In the Indian subcontinent herbal medicines are widely used and very often are the only treatment option for more than 70 percent of the population, particularly in rural areas. Over the years the potential chemotherapeutic property of herbal drug and its ability to counteract the antibiotic resistance has led to numerous innovation using herbal molecules [13]. Phyllanthus niruri and Solanum nigrum are indigenous herbs which have been used in India for treatment of various ailments [9].

The present in vitro study showed that the herbs Solanum nigrum and Phyllanthus niruri have an inhibitory effect on the growth of cariogenic organisms Streptococcus mutans, Streptococcussanguis, Streptococcus salivarius, Streptococcus oralis and Lactobacillus acidophilus which were cultured in their respective mediums. Any agent showing a zone of inhibition of more than 3 mm is considered to be an effective antimicrobial [12]. The result of our study showed both the herbs Solanum nigrum and Phyllanthus niruri had antimicrobial inhibitory zones ranging from 9.6-14.6 mm [Table/Fig-1,2], indicating that both the herbs canrestrict the growth of cariogenic organisms. Further when the zones of inhibition of the two herbs Solanum nigrum and Phyllanthus niruri were compared using Mann-Whitney test, the antibacterial activity of Solanum nigrum was found to be more than Phyllanthus niruri. In the Mann-Whitney test the results were statistically significant for Streptococcus sanguis (p<0.043), Streptococcus salivarius (p<0.043), Streptococcusoralis (p<0.043) and Streptococcus mutans (p<0.043) [Table/Fig-3]. Thus we can infer from the study that the herb Phyllanthus niruri and Solanum nigrum have inherent antimicrobial property. The MIC values of the herbs was lowest for Lactobacillus acidophilus (A=50 mg/mm, B=25 mg/ml) and Streptococcus oralis (B=50 mg/ ml) [Table/Fig-9,10] than for the other three organisms taken in the study, indicating that the herbs are potent antimicrobials on these two oral pathogens even at very low concentration.

Njoroge AD et al., showed that the in vitro antimicrobial efficacy of Phyllanthus niruri extracted in water, methanol and dichloromethane were bacteriolytic on various pathogenic bacteria (Bacillus pumilus, Staphylococcus aureus, Bacillus subtilis, Micrococcus luteus, Escherichia coli and Klebsiella pneumonia) and on fungus Candida albicans. The antibacterial action observed against the pathogens Bacillus pumilus with methanolic and dichloromethane extracts exhibited wider zones of inhibition measuring 18-20mm [14]. Obiagwu IN et al., did a similar study using different concentrations of ethanolic and aqueous extracts of Phyllanthus niruri on Escherchiacoli, Staphylococcus aureus, Salmonella typhi, Pseudomonas aeruginosa and Klebsiella aerogenes. Pseudomonas aeruginosa was found to be most susceptible among the ethanolic extracts [11]. The MIC obtained for Escherchia coli was 50 mg/ml which is similar to the MIC value obtained for Lactobacillus acidophilus in our study. The antimicrobial activity was more for the methanolic extract than for aqueous extract. Since organic solvents are better than aqueous extract proved through previous studies, probably due to the fact that active components readily dissolve in solvent compared to water, so in our study we used ethanolic extract of the leaves of Solanum nigrum and Phyllanthus niruri.

The antibacterial effect of dried powdered Solanum nigrum fruits which were extracted in different organic solvents (Petroleum ether, chloroform, Dichloromethane, ethyl acetate, acetone, methanol) and water were evaluated against the pathogens Micrococcusluteus, Staphylococcus aureus, Salmonella typhi, Eschericha coli and fungus Candida albicans. Herbs which were extracted by using more polar solvents like methanol (7-14 mm zone of inhibition) had significant antimicrobial activity compared to those extracted with less polar solvents (3.6-9.8 mm) [15]. A similar study was done by Kasa P et al., who investigated the ethanolic and methanolic extracts of Solanum nigrum against pathogenic bacteria like Bacillussubtilis, Escherichia coli, Klebsiella pneumonia and Pseudomonas aeruginosa. The whole plant, stem and berries were individually extracted with the organic solvent and evaluated. Whole plant extract showed potential antibacterial activity than stem and berries [4]. Sridhar TM et al., evaluated the solvent extracts of seeds, leaf and roots of Solanum nigrum using various organic solvents like ethanol, methanol, ethyl acetate, diethyl ether, chloroform and hexane. Among the extracts, ethyl acetate extracts obtained from the seeds of Solanum nigrum were shown to exhibit significant activity against enteric pathogens Proteus vulgaris, Klebsiella and Bacillus subtilis [16].

Yogananth N et al., studied the antibacterial effect of Solanumnigrum against eight pathogenic organisms namely Enterococcus faecalis, Escherchia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Shigella flexneri, Salmonella typhi, Staphylococcus aureus and Vibrio cholera. The ethanolic extract was having significant antibacterial activity against all pathogens excluding Staphylococcus aureus and Pseudomonas aeruginosa [17]. Our study was the first such attempt to study the effect of these herbal leaf extracts of Solanum nigrum and Phllanthus niruri on caries causing pathogens.

Thus the present study confirms that the herbs Solanum nigrum and Phyllanthus niruri are potential anticaries agents. Numerous plants have been used to extract biomolecules and have been incorporated in toothpastes for use in oral care therapy [18-20]. The anticariogenic potential of the herbs in our current study, once proven through research can be incorporated into products for oral care.

The limitation of this research include- it was not performed with various types of extract at different concentration, and studied only the alcoholic extracts of the herbs. This was an in vitro study, since the in vivo environment is unique, we cannot anticipate the same results when the herb is used in vivo settings. The present study used only the leaves of the herbs; the other parts of the plant like the seeds, stem, and fruits should also be explored for their antibacterial potential. The functional activity of the herbs Solanumnigrum and Phyllanthus niruri should be interpreted by cell culture studies as well and the molecular mechanisms of action should be elucidated.

Hence, we can conclude from the present study that Solanumnigrum and Phyllanthus niruri are potent antibacterial agents against Streptococcus mutans, Streptococcus sanguis, Streptococcus salivarius, Streptococcus mitis, and Lactobacillus acidophilus. When the activity of the two herbs were compared Solanumnigrum showed greater activity than Phyllanthus niruri. This in vitro study showed that the herbs have substantial anticariogenic potential, further research can lead to isolation of the key molecules responsible for the antibacterial action.

Conflict of interest: No conflict of interest has been reported for the same.