Antibacterial Assay of the Whole Plant Ethanolic Extract of Amaranthus viridis , Aerva sanguinolenta and Cynodon dactylon against Streptococcus mutans and Lactobacillus acidophillus – An in vitro Microbiological Study

Background: Dental caries, a multifactorial, microbial disease of calcified portion of tooth, can be controlled by controlling the microbial factor either by synthetically derived or naturally derived antibacterial components. Aim: Antibacterial assay of three whole plant ethanolic extracts against two cariogenic bacteria to find out a plant extract which could be used as therapeutically effective method of controlling dental caries by controlling its microbial factor. Materials and Methods: Antimicrobial activity of 1000 µg/ml, 750 µg/ml, 500 µg/ml and 250 µg /ml concentration of Amaranthus viridis (A1), Aerva sanguinolanta (A2), Cynodon dac-tylon (A3) ethanolic whole plant extracts were measured by agar disc diffusion and broth microdilution method against Streptococcus mutans , and Lactobacillus acidophilus , 5% DMSO (negative control) and 30% Vancomycin (positive control). Result: The 1000 µg/ml (100%), ethanolic extracts of (A2) showed greatest inhibition zone (mean-11.860 mm) against Streptococcus mutans and (mean= 9.730 mm) against Lactobacillus acidophillus which were more than negative control but less than positive control. (A2) exhibited the lowest MIC of 0.02050 µg/ml and 0.02050 µg/ml against Streptococcus mutans and Lactobacillus acidophillus respectively. Conclusion: Aerva sanguinolenta whole plant ethanolic extract can be used to inhibit growth of cariogenic bacteria, thereby can be used for controlling microbial factor of dental caries in human being.


INTRODUCTION
Prevalence of dental caries has increased in last few decades. It is a microbial disease of calcified portion of tooth where innumerous cariogenic microorganisms like Lactobacillus acidophilus, Streptococcus mutans etc. Favour the adherence and accumulation of plaque biofilm by metabolizing sucrose into sticky glucan and degrading the dietary carbohydrates to produce lactic acid leading to localized demineralization and eventually the formation of dental caries. [1,2] Lactobacilli have been found in high numbers in both superficial and deep caries. [3] Streptococcus mutans are found in dental plaque and are cariogenic in animal models. [4] In 1924 Clarke isolated the organisms from human carious lesions Table 1. [5] Few studies have demonstrated antimicrobial activity against selected oral pathogens from natural plant sources. [6][7][8] Only 1% of plant source has been evaluated till date for research, thus lot more to be investigated. Amaranthus viridis (Baun note), [9][10][11][12] Aerva sanguinolenta (Lal bishalyakarani) [13][14][15] and Cynodon dactylon (Durba grass) [16][17][18] are widely and easily grown, all season, hardy plant with wild cultivation status. Table  2. The three medicinal plants are with many therapeutic virtues including antibacterial activity and abundantly grows without extra effort in the North Gangetic plane of North 24 Parganas, West Bengal, India.
The different Types of Antibacterial susceptibility testing's for plant extracts are Diffusion methods, Thin-layer chromatography (TLC), Dilution method, Time-kill test, ATP bioluminescence assay, Flow cytofluorometric method. [19] The diffusion and bioautographic methods are described as qualitative techniques since these methods give an idea only about the presence or absence of substances with antimicrobial activity. On the other hand, dilution methods are considered as quantitative assays since they determine the minimal inhibitory concentration. [20] The present study evaluated antibacterial assay of the whole plant ethanolic extract of Amaranthus viri-Pharmacognosy Research, Vol 13, Issue 4, Oct-Dec, 2021

MATERIALS AND METHODS
Requisite ethical clearance (GNIDSR/IEC/18-18) and permission to undertake the study was obtained from the respective committee.
Collection, authentication and pre-treatment of plant sample

Preparation of plant extract
The coarse powder of (A1), (A2) and (A3) were subjected to hot continuous extraction with ethanol ( Mercks) respectively by Soxhlet apparatus (Borosil) to get an wet extract which was concentrated by distillation using rotaryvaccum evaporator (RE100PR0MFGD silicogex/ USA Takashi ) to obtain a dry extracts which were stored in sterilized glass beaker in a refrigerator at 4 degree centigrade. Preparation of plant extracts were performed in the Guru Nanak Institute of pharmaceutical Science and Technology, Kolkata.           samples were added to the first few wells of the microplate starting with a concentration of 100mg/ml of extracts, 5mg/ml of the positive control was taken and then two-fold serially diluted down the wells. The assay was repeated twice with two replicates per assay Figure 1. The lowest     concentration where no visible turbidity was produced after a total incubation period of 48 hr was regarded as final MIC.

Statistical analysis
The Statistical software IBM SPSS statistics 20.0 (IBM Corporation, Armonk, NY, USA) was used for the analyses of the data and Microsoft word and Excel were used to generate graphs, tables etc. Results from the study were analysed for statistical significance using two way Anova and Tukey's test for multiple comparison. P<0.05 was considered statistically significant and P<0.001 was considered highly statistically significant.
In the present study also the four concentrations (1000 µgm/ml), (750 µgm/ml), (500 µgm/ml ) and (250 µgm/ml) of ethanolic whole plant extracts of Amaranthus viridis (A1), Aerva sanguinolenta (A2) and Cynodon dactylon (A3) showed mean inhibition zone ranging from 9.050mm -6.250mm, 11.860mm -7.470mm and 8.780mm -6.610mm respectively against Streptococcus mutans and showed mean inhibition zone ranging from 7.860mm-6.250mm, 9.730mm -6.610mm and 8.780mm -6.610mm respectively against Lactobacillus acidophillus (Table  1). Similar to present study many other studies revealed the positive in vitro antibacterial effect of Amaranthus viridis, [9][10][11][12] Aerva sanguinolenta [13] and Cynodon dactylon [16][17][18] plant extracts respectively against different gram positive and gram negative bacteria. Phytochemical screening of A1, A2, A3 in a previous study by present authors revealed presence of phytoconstituents in A1-Alkaloids and Flavanoids, A2-Flavanoids, A3-Alkaloids, Flavonoid, Carbohydrate, Steroid, Protein, Cardiac glycoside. [33] The greatest antimicrobial activity of A2 could be attributed to it's flavanoid content which has antibacterial activity. [34] Tagousop C N et al. (2018) in an in vitro study revealed the antibacterial activities of flavonoid glycosides obtained from G. glandulosum were in some cases equal to, or higher than those of ciprofloxacin. Flavanoid acted by disrupting the membrane permeability leading to leakage of cellular components and eventually cell death of bacteria. [35] The antimicrobial activity of a plant extract is considered to be highly active if the MIC < 100 μg/mL. [36] Thus in the present study Aerva sanguinolenta (A2) possess highly active antibacterial activity as it's MIC is mean of 0.02050 µg/ml for both Streptococcus mutans and Lactobacillus acidophillus and is lowest among the three extracts. Determination of MIC would aid in dose determination of plant extract to be used for prevention of demineralization of human enamel. In an another part of study by present authors it was revealed that human enamel sample exposed to 2ml of 0.2% solution of Aerva sanguinolenta [one tenth of MIC of Aerva sanguinolenta (A2)] for 2 min could inhibit biofilm induced demineralization of enamel in a closed batch culture technique utilizing three types of biofilm setup that is S. mutans mono species, -10307) as shown in Figure 12. Antibacterial effect of Aerva sanguinolanta (A2) is more than negative control (5% DMSO) but much less than positive control [Vancomycin disc (Himedia )] which showed mean inhibition zone of 18.200 mm and 36.710 mm respectively against Streptococcus mutans and Lactobacillus acidophillus respectively Figure 3, 6.

Result of Antimicrobial assay by broth micro dilution method
The lower is the value of MIC of plant extract, greater is the antibacterial activity of the plant extract against a specific bacteria. MIC of Aerva sanguinolenta (A2) with a mean of 0.02050 µg/ml for Streptococcus mutans and 0.02050 µg/ml for Lactobacillus acidophillus is lowest among the three extracts with a significant variance (by Anova, F=191916.464 and P= <0.001**) and (by Anova, F=16241744.64 and P= <0.001**) respectively Figure 16 and 17.

DISCUSSION
The three plant samples were chosen as they are abundantly and easily grown, hardy, all season plant of North Gangetic Plane of West Bengal, India with known ethno medicinal and experimental antibacterial activity.  L. acidophilus mono species and combined S. mutans and L. acidophilus dual species. [37] CONCLUSION Qualitative and quantitative antibacterial assay revealed that antibacterial activity of Aerva sanguinolenta (A2) is more than Amaranthus viridis (A1) and Cynodon dactylon (A3) and negative control (5% Dimethyl sulfoxide) but less than positive control [Vancomycin (30 µcg)] against both Streptococcus mutans and Lactobacillus acidophilus. Antibacterial effect of Aerva sanguinolenta (A2) is more against Streptococcus mutans than Lactobacillus acidophilus.