Evaluation of Antioxidant and Antimicrobial Efficacy of Camellia Sinensis and Alstonia Scholaris Extracts on Streptococcus Mutans and Lactobacillus Acidophilus-An in Vitro Study

Context Dental caries is showing an upward trend in India and there is a need to explore innovative strategies to prevent the disease. Literature evaluating antibacterial activity of Camellia sinensis and Alstonia scholaris plant extracts on Streptococcus mutans and Lactobacillus acidophilus is practically non-existent. To assess the minimum inhibitory concentration (MIC) and antimicrobial efficacy of Camellia sinensis and Alstonia scholaris on S. mutans and L. acidophilus. This was an in vitro study carried over a period of three months. The leaves of Camellia sinensis and Alstonia scholaris were collected, and crushed to obtain coarse powder. Plant extraction was performed using Soxhelet appartus. Antioxidant assay was performed for both the plant extracts against DPPH radical using Spectrophotometer at 517nm. Inhibition percentage was calculated through absorbance value measured from spectrophotometer. Antimicrobial activity of both the plant extracts against Microbial Type Culture Collection strains of Streptococcus mutans and Lactobacillus acidophilus was assessed using Agar well diffusion method. 0.2% Chlorhexidine was used as positive control and ethanol as negative control. The experiment was performed in triplicates. Mean inhibition zone in each set of experiment was computed using three readings after accounting for well diameter. One Way Analysis of Variance (ANOVA), Tukey’s post hoc test and independent sample‘t’ test were performed to compare the mean inhibition zone. The plant extracts were effective against Streptococcus mutans and Lactobacillus acidophilus. Camellia sinensis at 4% concentration produced a mean inhibition zone of 30.3± 3.9 mm against Streptococcus mutans and 23.8± 2.2 mm against Lactobacillus acidophilus. Alstonia scholaris at 10% concentration produced a mean inhibition zone of 21.6± 2.8 mm against Streptococcus mutans and 24.1± 1.6 mm against Lactobacillus acidophilus. Camellia sinensis and Alstonia scholaris have significant antioxidant and antimicrobial property against Streptococcus mutans and Lactobacillus acidophilus.

essential medicine. 3 In Japan, there is a tradition to drink green tea after every meal to cleanse their mouth. 4 Green tea is enriched with proteins, phenolic compounds, flavonoids and minerals. 3 Many studies have reported its antibiotic, anti inflammatory, anti oxidative, antifungal, anti diabetic, anti viral, anti mutagenic properties. [5][6][7][8][9][10][11] The limited literature suggests that green tea interferes with caries formation at every step and inhibit the process. 11 Similarly, Alstonia scholaris has also reported potent anti microbial activity in ancient era. 12,13,14,15 Alstonia scholaris belongs to the family of Apocynaceae. The plant is called as Datyuni and Chatiun in Hindi, Devil tree in English, Doddapala in kannada and Saptaparna in Sanskrit. Traditionally, the plant is used as analgesic, immunomodulant in liver disorders, kidney problems, skin disorders, respiratory disorders, urinogenital disorders, Central Nervous System disorder, cardiac disorders and gastro-intestinal disorders. The plant possesses antimicrobial, antioxidant, anticancer, analgesic, antiinflammatory, anti fertility, and anti inflammatory activity. It contains various phytochemicals like alkaloids, phlobatanins, phenolics, steroids, saponins, flavonoids and tannins. 16 Streptococcus mutans (S. mutans) is one among the leading micro organisms responsible for dental caries. 1 It initiates the dental caries and Lactobacillus acidophilus (L. acidophilus) is further responsible for its progression. 17 The literature available on the antimicrobial efficacy of Camellia sinensis and Alstonia scholaris plant extracts on these oral microorganisms is scanty. Hence, this research was undertaken to systematically assess the minimum inhibitory concentration and antimicrobial efficacy of Camellia sinensis extract and Alstonia scholaris on S. mutans and L. acidophilus.

Materials and Method study design and setting
This was an in vitro study conducted at Division of Biotechnology and Bioinformtics, Department of Water and Science, Faculty of Life Science, JSS Academy of Higher Education and Research over a period of three months. Study protocol was approved by the Institutions ethics committee (IEC).

Plant material
The leaves of Camellia sinensis and Alstonia scholaris plants were collected from in and around Mysuru after authentication by a taxonomist. The leaves were rinsed with water and shade dried over a period of three-four weeks at room temperature. The dried leaves were powdered using domestic blender and mixer grinder to obtain fine powder. Thereafter, the powder was filled in airtight plastic bottles and stored in refrigerator at 4 0 C until further use.

Plant extraction
The extraction process of finely ground Camellia sinensis and Alstonia scholaris were carried out using Soxhlet apparatus. "Thimble" was filled with 50 g of ground powder and loaded into the Soxhlet extractor. Subsequently, distillation flask was filled with solvent (ethanol). This cycle was repeated many times to get desired concentrated compound into the distillation flask. A rotary evaporator at 30°C-60°C was used to dry and concentrate the solvent extract under reduced pressure (30 ± 10 mbar) to a syrupy consistency. Finally, extract was dried at room temperature. The weight of the dried mass was recorded for further experimental studies.

anti oxidant assays
The Anti oxidant activity of Camellia sinensis and Alstonia scholaris was measured against 1, 1-diphenyl-2-picrylhydrazyl (DPPH) free radical activity. DPPH is a nitrogen centered free radical which gets reduced to diphenyl picryl hydrazine upon reduction. The color changes from purple to yellow/ colorless upon reduction. 15

dPPh assay
Standard 1ml of ascorbic acid solution (control) was mixed with 3 ml of 0.002% DPPH solution and checked for absorbance using Ultra Violet-Visible Spectrophotometer at 517nm. Similarly, 1ml of Camellia sinensis and Alstonia scholaris was mixed with 0.002% DPPH solution at different concentrations (25,50,75,100,125 and 150 ìg/ml in methanol) and checked for absorbance at 517 nm. The mixtures were kept in dark for 30 min to stabilize them before measuring the absorbance. High free radical-scavenging was indicated by low absorbance of the mixture which in turn reflects high anti oxidant activity.

The percent inhibition was calculated for
Camellia sinensis and Alstonia scholaris as: Where A is Absorbance of control and B is Absorbance of Test. Experiments were conducted in triplicates.

Bacteria
MTCC (Microbial Type Culture Collection) strains of S. mutans (MTCC 890) and L. acidophilus (MTCC 10307) were collected. Bacteria were enlivened at chemical laboratory for further microbiological assay. Bacterial cultures were maintained on Brain Heart Infusion (BHI) agar slants with periodic sub culturing and stored at 4° C.

Antimicrobial efficacy testing
Agar well/ disc diffusion method was used to assess the antimicrobial efficacy of plant extracts (50 µl volume). 0.2% Chlorhexidine as positive control and ethanol as negative control was used. Initially antimicrobial efficacy was checked at varying concentration (1%, 2%, 3%, 4%, 6%, and 10%) for both the plant extracts. A transparent scale was used to measure the diameter of the inhibition zone at three different planes on the undersurface of agar plate. The experiment was further triplicated using the most effective single concentration based on the initial experimentation. Diameter of inhibition zone was computed at three different planes on the undersurface of agar plate. Mean inhibition zone was computed based on results of these experiments. Minimum inhibitory concentration was that minimum concentration of plant extract that inhibited the growth of these microorganisms.

statistical analysis
Data analysis was done using SPSS version 22. Mean diameter of inhibition zone was compared using One Way Analysis of Variance (ANOVA) and Tukey's post hoc test between test, positive and negative control for both the bacteria. Anti microbial efficacy between 4% Camellia sinensis and 10% Alstonia scholaris extracts against S. mutans and L. acidophilus was compared using independent sample't' test. Anti-oxidant capacity of Ascorbic acid (standard), Camellia sinensis and Alstonia scholaris was compared at different concentration using One-Way Analysis of Variance (ANOVA) and Tukey's post hoc test. The statistical significance was fixed at 0.05.

results
The details of plant extracts and bacteria used in the study are denoted in Table 1 and 2 respectively. The result showed potent antibacterial and anti-oxidant activity of Camellia sinensis and Alstonia scholaris extracts against S. mutans and L. acidophilus. Anti microbial activity was based upon the assessment of mean zone of inhibition. Larger mean zone of inhibition indicated higher antimicrobial activity. Similarly, anti-oxidant activity was assessed by recording absorbance value through spectrophotometer. Percentage inhibition of DPPH free radical was calculated through absorbance value. High inhibition percentage indicated high anti oxidant activity.

MiC of Camellia sinensis and Alstonia scholaris extracts against S. mutans and L. acidophilus
Camellia sinensis did not demonstrate any inhibitory activity against S. mutans and L. acidophilus at concentrations 1%, 2% and 3%. Alstonia scholaris did not demonstrate any inhibition against S. mutans and L. acidophilus at 1%, 2% 3%, 4% and 6%. Hence, Minimum Inhibitory Concentration for Camellia sinensis against S. mutans and L. acidophilus was considered to be 4% while for Alstonia scholaris, it was 10%.
In the initial experiment, Camellia sinensis produced a mean inhibition zone of 27.5± 0.7mm against S. mutans and 26.0± 1.4mm against L. acidophilus at 4% concentration which was significantly lower when compared with mean Zone of Inhibition produced by 0.2% Chlorhexidine against S. mutans (32.5±0.7mm) and L. acidophilus (34.0±1.4mm) (p < 0.05, Table  3). Mean zone of inhibition significantly increased with increasing concentration of Camellia sinensis against S. mutans (p < 0.05, Table 3). However, there was no significant difference in the mean zone of inhibition produced by Camellia sinensis at 4% and 6% concentrations on these bacteria (p = 0.38, Table 3). Mean zone of inhibition significantly decreased with increasing concentration of Camellia sinensis against L. acidophilus (p < 0.05, Table 3).Here also, the difference between 4% and 6% was not statistically significant (p > 0.41, Table  3).
Alstonia scholaris at 10% concentration produced a mean inhibition zone of 23.0± 2.8mm and 25.5± 2.1mm against S. mutans and L. acidophilus respectively which was significantly lower than that produced by 0.2% chlorhexidine (p < 0.05, Table 3).
Mean inhibition zone of Camellia sinensis at 10% concentration against S. mutans was significantly higher (37.50± 3.54 mm) than that produced by 10% Alstonia scholaris (23.00± 2.83 mm). However, the difference in mean inhibition zone between 10% Camellia sinensis and 0.2% chlorhexidine was not statistically significant.
Antioxidant activity of Camellia sinensis and Alstonia scholaris in comparison with standard Ascorbic acid The mean Inhibition percentage was found to be significant significantly higher for Camellia sinensis and Alstonia scholaris in comparison with Ascorbic acid at all the levels of concentration (25% 50%, 75%, 100%, 125% and 150%) (p < 0.05, Table 7). The mean Inhibition percentage increased with increasing concentrations of plant extracts. Multiple pair wise comparison between different concentrations of each plant extracts   Table 7).

disCussion
Dental caries is demonstrating an upward trend in India and other developing countries. Treatment of dental caries is quite expensive and not a realistic option for developing countries such as India. Although, chlorhexidine is considered as a gold standard in preventing dental plaque formation, it causes some minor side effects on long term use like discoloration of teeth, taste alteration, oral/ mucosal ulceration and parotid swelling. 1,2,5 The need to evolve innovative strategies to prevent dental caries has resulted in multiple studies which have evaluated the antimicrobial efficacy of plant extracts on dental caries microorganisms in the recent past. [18][19][20][21] Camellia sinensis and Alstonia scholaris plants were known for their medicinal properties since centuries. 10 Lack of sufficient literature evaluating antimicrobial efficacy of these plant extracts on dental caries bacteria led us to undertake this in vitro study which assessed anti oxidant and antimicrobial activity of Camellia sinensis and Alstonia scholaris extracts on Streptococcus mutans and Lactobacillus acidophilus.
Camellia sinensis did not demonstrate any inhibitory activity against S. mutans and L. acidophilus at initial concentration (1%, 2% and 3%). Alstonia scholaris did not demonstrate any inhibiotry activity against S. mutans and L. acidophilus at initial concentration of 1%, 2%, 3%, 4% and 6%. This may be due to lack of sufficient concentration of phytochemical constituents in plant extracts for inhibiting the bacteria. Based on these results, MIC of Camellia sinensis and Alstonia scholaris against S. mutans and L. acidophilus was considered to be 4% and 10% respectively. Inhibitory activity of Camellia sinensis is attributed to the presence of various phytochemicals. It has been reported in the literature that Camellia sinensis has the potential to inhibit dextran and levan produced from sucrose by acting upon S. mutans. 7 It has been found to even interfere with the process of bacterial attachment to tooth enamel. 11 Plant is said to contain anti-microbial and anti-oxidant activity due to the presence of polyphenols and catechins. 10 Similarly, some studies have reported anti-microbial, phyto chemical and antioxidative property of different parts viz. leaves flowers, bark, root and latex of Alstonia scholaris. 15,16,22,23 The available literature over assessment of anti-microbial activity of Alstonia scholaris against S. mutans and L. acidophilus is limited. Araghizadeh A et al assessed the antimicrobial activity of Camellia sinensis against S. mutans at a concentration starting from 1.56mg/ ml to 50 mg/ml. There was no inhibition of bacteria at a concentration of 1.56mg/ ml. However, a mean zone of inhibition of 7.5±2.76 mm was formed at a concentration of 3.12 mg/ml which increased to 36.3±1.08 mm at a concentration of 50mg/ ml. 5 A review study by Reygaert W C reported that MIC of Camellia sinensis against S. mutans to vary from 2.58-3.98 mg/ ml. 24 The mean zone of inhibition for S. mutans increased with increasing concentration of Camellia sinensis. On the other hand, the  25,26,27,28 Anita P et al to assessed anti-microbial efficacy of Camellia sinensis against S. mutans and L. acidophilus. Study reported significant increase in mean zone of inhibition on increasing concentration of Camellia sinensis against S. mutans from 10.00 ± 0.01mm at 100 µg concentration to 12.66 ± 0.58mm at 200µg and finally 18.33± 0.58mm at 300µg concentration. They found a statistically significant increase in mean zone of inhibition with increasing concentration of Camellia sinensis against L. acidophilus from 8.33± 0.58mm at 100µg concentration to 10.00± 0.01mm at 200µg concentration and finally 12.67± 0.58mm at 300µg concentration. 2 These results were similar to the results of present study with regard to S. mutans while contradictory with respect to L. acidophilus. Tahir A and Moeen R also found anti-microbial efficacy of Camellia sinensis against S. mutans and L. acidophilus to increase with increasing concentration. 11 Khan M R et al reported Alstonia scholaris to inhibit the growth of S. mutans similar to the results of our study. 12, Although, literature indicating antibacterial activity of Alstonia scholaris against gram positive and gram negative bacteria are available, 19,25,26 studies reporting antimicrobial activity of Alstonia scholaris against S. mutans and L. acidophilus are practically non-existent. Hence, We could not compare the results with other studies.
Anita P et al. reported 0.2% Chlorhexidine to exhibit a higher mean zone of inhibition against S. mutans and L. acidophilus in comparison with Camellia sinensis at 300µg concentration. 2 our findings were similar to the results of this study and others. 1,2,6 Chlorhexidine is considered the gold standard antiplaque agent and it was found in our study as well that it has the potential to inhibit these microorganisms. However, in view of its side effects on long term use, mouth rinses made of these extracts could be considered as potential alternates with further research.
Camellia sinensis at 4% demonstrated higher mean zone of inhibition against S. mutans than against L. acidophilus similar to the findings of a study by Tahir A and Moeen R who found Camellia sinensis at concentrations of 5000, 10000, 15000 and 20000 mg/ml to have greater mean zone of inhibition against S. mutans (19, 24, 34 and 35 mm ) than against L. acidophilus (15, 20, 29 and 33mm). 11 This finding was also similar to study conducted by Anita P et al. 2 Alstonia scholaris at 10% demonstrated a higher mean zone of inhibition against L. acidophilus than S. mutans. Although, the extract was found to be very effective against L. acidophilus, we could not compare this finding vowing to non-availability of studies on the efficacy of this extract on dental caries bacteria.
Mean inhibition percentage of DPPH radical increased with increasing concentration of Ascorbic acid, Camellia sinensis and Alstonia scholaris. The increase in mean inhibition percentage was found to be significant for all three samples. However, Alstonia scholaris showed a significant high inhibition percentage than standard Ascorbic acid and Camellia sinensis at all concentrations except at 25% where Camellia sinensis showed a higher mean inhibition percentage. Higher mean inhibition percentage indicated high antioxidant activity. This illustrates that Camellia sinensis and Alstonia scholaris have enough potent antioxidant activity to scavenge free radicals. Various studies have reported antioxidant and antibacterial property of Alstonia scholaris. 15,16,25,26 Similar results were reported by James J et al, Ramachandra Y L et al and Jain D P et al. 18,27,28 Ramachandra Y et al found an increase in mean inhibition percentage from 26.37% to 72.82% with increase in Alstonia scholaris concentration of 200µg/ml to 1000 µg/ml. 18 Jain D P et al reported a significant increase in inhibition percentage of DPPH radical from 10.82 ± 2.2 to 81.13 ± 2.6 with increase in concentration of Camellia sinensis from 10 mg/ ml to 180 mg/ ml. 27 Our results were similar to the results of all these studies. novelty The study evaluated the MIC, antibacterial activity of two potential plant extracts against two most disease causing pathogens in oral environment besides evaluating their anti-oxidant capacity. Evaluation of antimicrobial activity of Alstonia scholaris against Streptococcus mutans and Lactobacilli acidophilus was the first of its kind study. limitation Phytochemical constituents of plant extracts vary depending upon their geographical location, brewing time, fermentation, etc. A qualitative and quantitative assay of phytochemical constituents could have validated the results of this study which could not be undertaken. We assessed antimicrobial activity of these extracts against Streptococcus mutans and Lactobacilli acidophilus while oral cavity has other pathogenic microorganisms as well.
Hence, more such researches can be performed to confirm anti-oxidant and antimicrobial property of Camellia sinensis and Alstonia scholaris against other oral bacteria.