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Assessment of antimicrobial activity of different concentrations of Tinospora cordifolia against Streptococcus mutans : An in vitro study

Background: The antimicrobial property of Tinospora cordifolia has been tested against a variety of microorganisms in the literature. The present study aimed to assess the antimicrobial activity of different concentrations of commercially available T. cordifolia powder against Streptococcus mutans . Materials and Methods: An in vitro study was undertaken in which extract of T. cordifolia was obtained using 100% ethanol by maceration. Seven different concentrations were prepared and tested against S. mutans in brain–heart infusion agar medium. Plates were incubated aerobically at 37°C for 48 h, and zone of inhibition was measured using Vernier caliper. 0.2% chlorhexidine and dimethylformamide were used as positive and negative controls respectively. The data were analysed by descriptive analytic tests. Results: The maximum antibacterial activity of T. cordifolia was observed with a volume of 40 μ l at 2% concentration with a zone of inhibition of 19 mm. A 30 μ l volume of 0.2% chlorhexidine showed a zone of inhibition of 28 mm, and no zone of inhibition was observed with dimethylformamide. Conclusion: Tinospora exhibited antimicrobial activity against S. mutans . However, it needs to be confirmed further with in vivo studies.


INTRODUCTION
The use of plants for medicinal purposes is as old as human civilization itself. Medicinal plants have been used for curing diseases in different traditional systems of medicine such as Ayurveda, Siddha, European, Tibetan, and Unani. [1] Herbal medicine is still the mainstay of treatment in about 75%-80% of people in many developing countries for their primary health care because of better cultural acceptability and compatibility with the human body and fewer side effects. [2] One such immensely valuable plant regarding its constituents and pharmacology is Tinospora cordifolia of the Menispermaceae family, commonly called as Guduchi in Sanskrit. [3] It is a deciduous climbing shrub with small greenish flowers, having enormous medicinal value in all its parts such as leaves, stem, and also the root. [4] It is a Rasayana (rejuvenator) and anti-aging medicine in Ayurveda, used to improve the immune system and the body resistance against infections. [5] It has also been found that Tinospora has antispasmodic, [6] antipyretic, [7] anti-inflammatory, [8] anticomplementary, [9] and immunomodulatory activities. [10] In addition to it, Tinospora has been found to exhibit antidiabetic, [11] hepatoprotective, [12] anticancer, [13] and antioxidant properties [14] as well. It has been listed as an insecticide, an antifungal agent, and an antibacterial agent. [15,16] Antimicrobial features have been found in its root, stem, and leaf extracts on pathogenic microorganisms. [17] Ethanolic and aqueous extracts of T. cordifolia have been successfully tested against various bacteria such as Staphylococcus epidermidis, Escherichia coli, Aspergillus niger, Candida albicans, and Staphylococcus aureus. [18,19] Unfortunately, no much research has been done to explore its antimicrobial properties against oral organisms. Hence, the present in vitro study was undertaken to assess the antimicrobial properties of Tinospora cordifolia against Streptococcus mutans, the most common organism associated with dental plaque and caries.

Study design
An in vitro study was designed to assess the antimicrobial activity of different concentrations of commercially available Tinospora powder against S. mutans. The study was approved by the Institutional Ethical Committee (SRDCHRC/146/11).

Preparation of Tinospora cordifolia extract
Tinospora powder was first sieved to obtain uniform sized particles. 300 g of finely sieved powder of T. cordifolia was macerated with 1 L of 100% ethanol. It was then subjected to filtration with Whatman filter paper (No. 1) to obtain a clear filtrate. This filtrate was reduced at 80°C temperature in a water bath for 48 h to get a solid residue of T. cordifolia extract. From 300 g of Tinospora powder dissolved in 1 L of ethanol, 1.6 g of residue (extract) was obtained.

Preparation of seven different concentrations of Tinospora cordifolia extract
One gram of extract was dissolved in 10 ml of dimethylformamide to obtain a 10% concentration of extract. One milliliter of the extract was transferred to a sterilized test tube and labeled as 10%. The remaining 9 ml of the extract was diluted further with dimethylformamide to obtain seven different concentrations (2%, 3%, 4%, 5%, 6%, 7%, and 8%). In this study, 0.2% chlorhexidine was used as positive control, and dimethylformamide was used as negative control. The method for obtaining different concentrations was adopted from a study by Agarwal et al. [2] Bacterial sample Pure strains of S. mutans (MTCC 890) obtained from Microbial Type Culture Collection and Gene Bank, Chandigarh, were used in the present study.

Culture media and methods
Glass petri dishes and brain-heart infusion agar were used for culture and to determine the zones of inhibition. 20 ml of freshly prepared, autoclaved, brain-heart infusion agar media was transferred to each petri dish. Four circular wells to accommodate four different volumes (10 μl, 20 μl, 30 μl, and 40 μl) of the T. cordifolia extract were cut in the agar plates. Nine such petri dishes were prepared and labeled, seven of the seven different concentrations of Tinospora extract, one for the positive control (chlorhexidine), and one for the negative control (dimethylformamide). The sterile environment around the Petri dishes was maintained using split lamps. S. mutans (MTCC 890) strains were mixed with saline and swabbed to each agar plate using sterile wooden tongue blades. The plates were then incubated aerobically at 37°C for 48 h. After 48 h, the zone of inhibition was measured using a Vernier caliper. Data were tabulated and expressed as descriptive as this observational study did not require any inferential analysis.

RESULTS
A total of seven different concentrations of T. cordifolia extract along with positive and negative control were assessed for antimicrobial activity against S. mutans.
At 2% concentration, a maximum zone of inhibition of 19 mm was seen with a volume of 40 μl followed by 9 mm with 30 μl. On the contrary, no inhibition zone was found with secondary volumes such as 10 μl and 20 μl. At 4% and 5% concentrations, a maximum area of inhibition of 5 mm was observed at a volume of 40 μl while lesser quantities failed to produce any zone of inhibition. At 7% and 8% concentrations, a maximum area of inhibition of 2 mm was observed at a volume of 40 μl while no zone of inhibition was seen with lesser volumes. At 3% and 6% concentrations, no area of inhibition was observed with any of the volumes [ Table 1].
The results with 0.2% chlorhexidine which was used as a positive control showed a maximum zone of inhibition of 28 mm with 30 μl followed by 20 mm with a volume of 40 μl, 8 mm with 20 μl, and the least of 7 mm with 10 μl, respectively [ Table 2].
No zone of inhibition was seen with any of the volumes of dimethylformamide, which was used as a negative control, indicating total lack of antimicrobial activity [ Table 3].

DISCUSSION
The enormous heritage of vast natural, time-tested medicinal resources is worth exploring the possibility of developing efficient, economically viable, and clinically acceptable antimicrobials for human application. One among them is T. cordifolia, an indispensable medicinal plant, referred to in Ayurveda as "Amruth" or the "Nectar of Immortality" in recognition of its ability to impart youthfulness, vitality, and longevity. Preclinical and clinical pharmacological studies affirm the importance of its therapeutic efficacy and hence have placed it as a novel candidate to be used as the primary drug in the treatment of different ailments. [3] Previously, plants such as Tulsi (Ocimum sanctum) and Stevia rebaudiana have been tested for their antimicrobial activity on S. mutans with positive outcomes. [2,20] However, studies regarding antimicrobial properties of Tinospora against oral microorganisms, especially S. mutans, are lacking though studies have evaluated its antimicrobial activity against E. coli, S. aureus, Pseudomonas aeruginosa, and Bacillus subtilis. [21,22] Some studies found that the maximum antimicrobial property was exhibited against S. aureus. [23] In the present study, out of seven different concentrations and volumes tested against S. mutans, maximum inhibition of 19 mm was found with 40 μl at 2% concentration of the extract. Lesser amounts of the same strength failed to elicit the same results. It was also found that the zone of inhibition decreased with the increasing concentration of the extract of Tinospora. Furthermore, a volume of <30 μl of extract in all concentrations except for 2% failed to elicit any zone of inhibition. This indicates that the antimicrobial activity of Tinospora is effective at a lower concentration and higher volume with a maximum antibacterial activity as seen with 40 μl at 2% concentration. However, further studies are required to confirm the same. A study by Vermani et al. on antimicrobial properties of crude Tinospora extract against five dental pathogens showed similar results. The zone of inhibition against S. mutans was 10 mm, 11 mm, 19 mm, and 18 mm with ether, chloroform, methanol, and aqueous extracts of Tinospora, respectively. [24] The antimicrobial property of Tinospora against Streptococcus mutants could be attributed to the secondary metabolites and the phytochemicals present in it such as quinones, polyphenols, alkaloids, flavonoids, tannins, coumarins, terpenoids, lectins, and polypeptides. [25,26] While quinones and flavonoids bind to adhesins form complexes with cell wall and inactivate bacterial enzymes, terpenoids, polyphenols, and tannins cause membrane disruption and form metal ion complexes, thus inactivating the bacteria. [27] In the present study, chlorhexidine was found to be more effective and exhibited better antimicrobial properties against S. mutans with a zone of inhibition of 28 mm, as compared to T. cordifolia extract. However, the well-known side effect of chlorhexidine, i.e., staining of teeth and restorations, alteration of taste sensation, and development of resistant microorganisms, may limit the long-term use of chlorhexidine. [2] On the contrary, Tinospora is abundantly available, easily accessible, economically   feasible, and culturally acceptable and may possess minimal side-effects. Hence, it can be recommended for long-term use to prevent plaque formation by S. mutans and in turn decrease the risk of caries and periodontal disease as plaque is a common etiological factor for both.
In the present study, dimethylformamide was resistant to S. mutans. This could be attributed to its property of being an inert solvent. It was used in the present study to dilute the Tinospora extract and to neutralize the effect of alcohol so that the results could be solely attributed to Tinospora.

CONCLUSION
According to the present study, Tinospora exhibited antimicrobial properties against S. mutans with the maximum activity at 2% concentration. It could be formulated as mouthwash and used to prevent plaque in patients at high risk for caries and gingivitis. However, more research is required to test its antimicrobial properties in the oral environment through in vivo studies and also the adverse effects if any on oral mucosal cells and teeth.

Financial support and sponsorship
Nil.