The study was carried out after obtaining clearance from Yenepoya University ethics committee, Mangaluru. Study duration was of one year conducted in 2014-15. Dry twigs of Salvadora Persica were purchased from authorized Ayurvedic medical shop in Mangaluru, Karnataka. The twigs were then powdered using household electric blender. Fifty grams of the plant powder was loaded in the thimble of Soxhlet apparatus. It was fitted with appropriate size round bottom flask with 250 ml absolute ethanol, and upper part was fitted with condenser for alcoholic extract and with water in round bottom flask for water extract. The filtrates were concentrated using a rotary evaporator. The extracts were transferred into clean vials and stored at 4⁰C till further use. The yield from 50 grams of Miswak twigs was seven grams.

The aqueous and alcoholic extracts were dissolved in Dimethyl Sulfoxide (DMSO) to prepare different concentrations i.e., 200μg/ml, and 400μg/ml and used for microbial analysis.

Collection & isolation of test organisms

Test organisms were collected from patients who visited the Yenopoya dental clinic, Mangalore for treatment. Informed consent was taken from the patients after explaining the study details. Cariogenic organisms were collected by scraping soft caries from carious cavities of affected teeth using excavator and periodontal pathogens from periodontal pockets using paper points. After collection the paper points were dropped into 20 ml of Brain-Heart Infusion (BHI) broth which was used as transport media.

The collected samples were placed in an anaerobic chamber or incubator. After 48 hours of incubation, they were plated on variety of selective and nonselective media such as sheep blood agar, chocolate agar and SDA, manually under strict aseptic conditions in a laminar air flow chamber. Following a period of 24 hours of aerobic/anaerobic incubation, the culture plates were inspected for the colonies. Each colony was identified by colony morphology, gram staining, catalase test, pigment production and aerotolerance test, etc.

The isolates were preserved in BHI broth and RCM broth with serial subcultures every 72 hours for the entire study period. The purity of the broth cultures of each microorganism was confirmed by microscopic examination of gram-stained smears, cultivation of the organisms on sheep blood agar medium and used as inoculum for antibacterial assay. Cariogenic organisms such as Streptococcus mutans, Streptococcus mitis and Lactobacilli and periodontal pathogens such as Peptostreptococcus and Prevotella intermedia as well as Candida albicans were studied.

The pure cultures of organisms were emulsified in BHI broth which was then incubated at 37°C for 24 hours. The suspensions were diluted by adding sterile BHI broth to match/obtain a turbidity equivalent to Mc Farland 0.5 standard 5X10⁵CFU/ml. These suspensions were used as inoculum for testing the effect of crude extracts by agar diffusion method and to determine the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the extract by broth dilution method [6].

The antibacterial screening was carried out using agar diffusion method described by Lino and Deogracious with slight modifications [7]. The freshly prepared inoculum was swabbed all over the surface of the Muller Hinton Agar plate using sterile cotton swab. Five wells of 6mm diameter were bored in the medium with the help of sterile cork-borer having 6mm diameter and were labelled properly and fifty micro-litres of different concentrations (200μg/ml and 400μg/ml) of plant extract and same volume of positive and negative control was filled in the wells with the help of micropipette. Chlorhexidine was used as positive control. Distilled water was used as control for aqueous extract and DMSO. Plates with lid closed were left for some time till the extract diffused in the medium. Plates were incubated at 37°C for 24 hour. Zone of inhibition was measured using scale [7].

Various dilutions of the extracts which had antibacterial activity in the previous assay were taken in sterile test tubes to determine MIC. Freshly prepared nutrient broth was used as diluents. Crude extract was diluted by two fold serial dilution method. Nutrient broth in a test tube was considered as control. Except the control tube, 50μl of the standard culture inoculums was added to each test tube. The tubes were incubated at 37°C for 24 hours and then examined for growth. Growth was confirmed by observing turbidity. One ml of bacterial culture was pipetted from the mixture obtained in the determination of MIC tubes which did not show any growth and subcultured onto Muller Hinton Agar and incubated at 37^oC for 24 hour. The concentration at which there was no single colony of bacteria after incubation was taken as MIC [7].

The results obtained were statistically analysed using Kruskal-Wallis test and Mann-Whitney U test. All statistical analysis was done using SPSS software version 15.0. The level of significance was set at p < 0.05.

With two different concentrations of water extract (200μg/ml and 400μg/ml), the tested cariogenic bacteria showed no zone of inhibition except for Streptococcus mutans at 400μg/ml which gave an inhibitory zone of 13mm. Periodontal pathogens, when subjected to the same concentrations of water extracts, both Prevotella Intermedia and Peptostreptococcus, gave a zone of inhibition of 6mm and 6.67mm and 12.67mm and 10.67mm at concentration of 200 and 400μg/ml respectively. Only the higher concentration showed an inhibitory zone of 6mm against Candida albicans while 200μg/ml did not show any effect.

When the same organisms were subjected to alcoholic extracts of similar concentrations, significant zone of inhibition was noted particularly in 400μg/ml concentration. The cariogenic bacteria Lactobacillus acidophillus, Streptococcus mutans and Streptococcus mitis gave an inhibitory zone of 10.67 mm, 17.33 mm and 8.67 mm, respectively at a concentration of 400μg/ml. The periodontal pathogens namely Prevotella and Peptostreptococcus showed an inhibitory zone of 11.33 mm and 10 mm, respectively at concentration of 200μg/ml and 16.67 mm and 16.63 mm at concentration 400μg/ml. Candida also showed an inhibitory zone of 10.67 mm at 400μg/ml and 6.33 mm at 200μg/ml. Statistical analysis of the results showed a significant increase in effect from 200μg/ml to 400μg/ml in both water and alcoholic extracts (p-value < 0.05). Similarly alcoholic extract showed a significantly higher effect compared to water extract (p-value < 0.05) [Table/Fig-1].

When the effect on different pathogens was compared, it was noted that both extracts had significant effect on periodontal pathogens compared to others [Table/Fig-1]. The MIC and MBC recorded is shown in [Table/Fig-2].

Plants are important sources of therapeutic medicines and the tribal and ethnic communities rely a lot on these natural products for their healthcare needs. According to WHO, herbal medicines serve the health needs of about 80% of the world population, especially for millions of people in the rural areas of developing countries [8]. The beneficial medicinal effects of plant materials including the antibacterial activity typically result from the secondary products present in the plant although, it is usually not attributed to a single compound but a combination of the metabolites [9]. Various chemicals in Miswak extracts, such as sodium chloride, potassium chloride, salvadourea, Salvadoraine, saponins, tannins, vitamin C, silica, and resin, cyanogenic or lignan glycosides, alkaloids, terpenoids, oleic, linoleic, and stearic acids may be responsible for the antimicrobial and cleaning effects of Miswak [4].

Though the exact mechanism by which the active components of the plant materials contribute to antibacterial activity is not known, the antimicrobial effect may be through one of the mechanisms such as inhibition of cell wall synthesis, damage to cell membrane, inhibition of nucleic acid synthesis, inhibition of protein synthesis etc. Useful antimicrobial phytochemicals can be divided into several categories like phenolics, polyphenols, flavones, flavonoids, flavonols, quinones, tannins, coumarins, terpenoids, essential oils, alkaloids, lectins and polypeptides etc., [10]. Phenolic compounds exert antimicrobial property by causing enzyme inhibition, probably through interaction with sulfhydryl groups [11]. Quinones probably act on adhesins, cell wall polypeptides and membrane-bound enzymes. They also deprive microorganisms of substrates [11]. Flavones, flavonoids, and flavonols complex with bacterial proteins and cell walls and exhibit antimicrobial activity. Tannins form a layer over enamel by their astringent effect, thus providing protection against dental caries. Coumarins act by stimulating macrophages. Terpenoids and essential oils act against bacteria, fungi, viruses and protozoa by causing membrane disruption [10]. Alkaloids also exhibit antimicrobial properties [12]. Lectins and polypeptides act possibly through formation of ion channels in the microbial membrane [13,14] or competitive inhibition of adhesion of microbial proteins to host polysaccharide receptors [10]. Other compounds that have antimicrobial properties are polyamines [15], isothiocyanates [16,17] thiosulfinates [18], and glucosides [19,20].

When used as chewing sticks, in addition to these biologically active chemicals, the beneficial effect of Miswak could be because of mechanical cleansing effect of fibrous component. The substantial amount of silica detected in S. persica ashes contribute to Miswak’s mechanical action in plaque removal [21]. However, there is no sufficient literature regarding the mechanical cleansing efficacy of Miswak, as compared to the manual or electric tooth brushes. Current studies available frequently lack specific details concerning the time, duration, and frequency of their use which prevents meaningful assessment of the mechanical cleaning effect of Miswak upon oral health. The increase in saliva production when the chewing stick is left in the mouth for some time could also be responsible for promoting better cleansing and thus maintenance of oral health [3].

While comparing the efficacy of different extraction methods, the alcoholic extract of Salvadora Persica, showed better inhibitory zones compared with aqueous extracts. This observation is in accordance with the findings of Maria et al., and Doughari et al., who reported a strong antibacterial effect of alcoholic extract of tested plants in comparison to aqueous extract [7,9]. The differences in the activities of alcoholic and water extracts may be due to varying degrees of solubility of the active constituents in these two solvents. From this observation we can infer that alcohol is relatively a more efficient solvent than water for the extraction of bioactive compounds of Salvadora Persica than water. Difference in potency and diversity of compounds extracted by using organic solvents and water has also been reported previously [9]. However, in contrast to our results, one study done in Iraq has shown that the aqueous extract was more potent than the alcoholic form in inhibiting microorganisms [4]. In a study conducted by Almas K it was seen that even the alcoholic extracts of Salvadora persica (obtained from Pakistan) had no inhibitory effect on Streptococcus mutans, Staphylococcus aureus and Candia albicans [5]. He has attributed this to the fact that it took him almost one month to test the antimicrobial activity of extracts, so the extract was not fresh, at the time of experiment which could have caused the loss of antimicrobial activity.

In the present study, the results showed that the alcoholic extracts of Salvadoraa persica at a concentration of 400μg/ml was more effective against the anaerobic bacteria (lactobacilli, Peptostreptococcus) than the facultative aerobes (streptococci). Anaerobic bacteria are inherently more sensitive to antimicrobial substances than the aerobes; this could be the reason behind above observation.

Numerous extrinsic and intrinsic parameters influence the inhibition produced by the plant extracts against particular organism. Variable diffusability of plant extract in agar medium may also affect zone of inhibition. The antibacterial property may not demonstrate as zone of inhibition in proportion to its efficacy if there is decreased diffusability [9]. Therefore, MIC and MBC values, the lowest concentration of antibacterial substance required to produce a sterile culture has also been computed here. Demonstration of antibacterial activity against the test bacteria is an indication that alternative antibiotic substances in these plants could be used for the development of newer antibacterial agents in future. The observed low MIC and MBC values against these bacteria suggest that the plant extracts has the potential to treat ailments arising from the bacterial pathogens effectively.

It is possible that these plant materials can also be contributing to oral health through efficient free radical scavenging activity and antioxidant capacity [7].

With respect to plant materials, the beneficial antimicrobial effects could be related to the active chemical constituents It was beyond the scope of our study to isolate the active principle. Further studies are needed to bring forth the details of the active components in Salvadora persica, mechanism of action of these chemicals as well as their potentials in combination with other plant extracts.

Our study has confirmed the antimicrobial beneficial effects of alcoholic extracts of Salvadora persica. However, with respect to this plant, an alternative mechanism of synergistic effects of various natural substances when used in combination or when used along with other antibiotics should be considered as an area of future research.