This in vitro study was carried out at School of Dental Sciences, KIMSDU, Karad in November 2015 after due permission from the Institutional Ethics Committee (Ref No: KIMSDU/ICMR/STS/2015). The study duration was six months and was carried out between January and June of 2016.

A pilot study prior to this investigation was carried out to find out the feasibility of the study with the below mentioned extracts. Based on the Alpha type I error set at 0.05 and power of 90%, a sample size of 15 was determined to be adequate for the current study. Finely, powdered form of 50 grams each of pudina (Menthaarvenis), tulsi (Ocimumtenuiflorum) and curry leaves (Murrayakoenigii) were obtained from an Ayurvedic Pharmacy (Hakim ChiChi Pharmacy, Surat, India) without any added preservatives.

The powdered samples were divided into four groups as follows (n=15)

The saliva samples used for this study were collected from the five patient’s mouth diagnosed with at least three active carious lesions after a written informed consent was taken. Whole, stimulated saliva by spitting method was collected by one of the investigators by asking the patient to bite on a standard piece of paraffin wax. The saliva was transferred to a sterile screw capped tube and the test microorganism S. mutans were isolated and subcultured using a specific media, mitis salivarius agar, bacitracin (HiMedia Laboratory Pvt., Ltd., Bombay India).

The extract of herbal products was prepared by dissolving 1 μg of the powder in 1 μL of distilled water as per the protocol given by Prashant GM et al., [1]. Once the extract was prepared, dilutions were prepared to achieve a range of 500% to 5% (V/V). To obtain 500% (V/V), 50 gm of powder was added to 10 mL of distilled water. Minimum Inhibition Concentration (MIC) was then determined using agar well diffusion.

The MIC of the extracts was determined using the method proposed by Thongston C et al., [13]. Fifteen samples (n=15) for each group were prepared and an Agar Well Diffusion Technique was used to determine the MIC for all the groups. The bacterial isolates seeded on Mueller Hinton agar plates were fed with a 0.5 mL volume of each solution using aseptic technique and were incubated at 37°C for 24 hours. The least concentration of extracts showing clear zone of inhibition in millimeters (mm) was measured using vernier calipers (Aerospace Digimatic Vernier Caliper 150 mm/6 inch, India) and was considered as the MIC [Table/Fig-1,2]. Once the MIC was determined; the antimicrobial efficacy was then tested using the modified direct contact test.

[Table/Fig-2]:

Zone of Inhibition for Pudina group depiciting 500%, 250%, 100% (V/V).

The antibacterial property (cfu/mL) of the extracts was determined using the protocol set by Zhang H et al., [11]. A 96 well microtitre plate was held vertically and an area of fixed size on the side wall of the wells was coated with an equal amount (100 μL) of each extract (Group 1, Group 2, Group 3) with an applicator tip. A 10 μL of bacterial suspension {3×10⁸ cfu/mL which contains 3×10⁶ bacteria determined using a spectrophotometer (UV 2550 Shimadzu, Kyoto, Japan)} was carefully placed on the surface of each extract. Bacterial suspensions with 0.2% chlorhexidine served as a control. After incubation in 100% humidity at 37°C for two minutes, 240 μL of Tryptic Soya Broth (TSB) was added to each well. After gently mixing with pipette for one minute, the bacterial suspension from each well was transferred and serially diluted in TSB. The viability of bacteria was appraised by culturing aliquots of 20 μL onto Tryptic Soya Agar (TSA) plate subsequent to 10-fold sequential dilutions. The colony forming units/mL was calculated using the formula cfu/mL=(no. of colonies×dilution factor)/volume of culture plate after 24 hours incubation at 37°C. All the tests were performed three times using the same titre plate to reduce the manual error. Triplicate testing methodology was used.

The significance level (p-value) was set to <0.05. All tests were performed using the software program SPSS for Windows, version 23.0 (IBM, NY, USA). The results were analysed using ANOVA test followed by Tukey post-hoc test for intergroup comparison.

The zone of inhibition for each group (in mm) attained using agar well diffusion of different groups are listed in the [Table/Fig-1] and depicted in [Table/Fig-2]. The mean, standard deviation and standard error for antibacterial property of extracts using modified direct contact test in millimeters is presented in [Table/Fig-3]. The growth of S. mutans was measured and analysed. The data was subjected to ANOVA and Tukey’s post-hoc test.

Amongst the three study groups curry leaves had the lowest mean bacterial count (cfu/mL) [Table/Fig-4]. ANOVA test [Table/Fig-5] revealed a statistically significant difference (p<0.0003) among the groups compared. Tukey’s post-hoc test [Table/Fig-6] was employed for intergroup comparison. This test revealed a statistically significant difference between pudina and curry leaves (p=0.0151), pudina and chlorhexidine (p=0.0002), tulsi and chlorhexidine (p=0.0024) curry leaves and chlorhexidine (p=0.0233) [Table/Fig-6].

[Table/Fig-4]:

Graphical representation of the mean growth of streptococcus mutants.

In recent years, the traditional method of using herbal products in treatment of various diseases has resurfaced and there has been a rise in its use. The reason behind this is the easy availability of herbal products, cost-effectiveness, low toxicity, increased shelf life and lack of microbial resistance. Thus, spices and other herbal products which are normal ingredients of our routine food preparations can provide protection against bacterial pathogens; serving as therapeutic agents containing antibiotics and other medicinal compounds [14,15].

Chlorhexidine (0.2%) was chosen as the control as it is known to have a potent antibacterial effect against S. mutans. But long-term use of chlorhexidine has side effects like staining of teeth, loss of taste and sometimes stenosis of the parotid duct [3]. Unlike it, herbal products are known to have least side effects with an advantage of affordability to all classes of people. Pudina, tulsi and curry leaves are known to have antibacterial properties. But their effect on S. mutans has been assessed using traditional methods with doubtful accuracy. Hence, we have made an attempt to compare the antibacterial properties of pudina, tulsi, curry leaves and chlorhexidine against S. mutans using modified direct contact test for accurate and better evaluation of the antimicrobial efficacy. The powdered herbal products were serially diluted to obtain the MIC against S. mutans. Distilled water was chosen as the solvent instead of methanol to check for the pure antibacterial effect without the properties of methanol overshadowing the results. Bacteriological testing was performed by modified direct contact test as demonstrated by Zhang H et al., [11].

The result of the modified direct contact test showed that the least mean growth of S. mutans was seen with curry leaf, when compared to the control chlorhexidine. The products in their decreasing order of mean growth of S. mutans were chlorhexidine, curry leaves, tulsi and pudina. The anti bacterial properties of curry leaf can be attributed to its unique compounds carbazole derivatives, murrayanol and girinimbine present in the leaves [15-17].

The statistical analysis using ANOVA test (p<0.05) showed significant difference (p=0.0003) between the antibacterial efficacy of the groups against S. mutans. When compared with the control group chlorhexidine (0.2%), curry leaves were the most effective of the three products and this was in accordance to the study done by Chandra Shekar BR et al., [7]. Prabhakar AR and Murlikrishnan NL, conducted a study comparing curry leaves, garlic and tea tree oil against S. mutans and found all three herbal products had a significant antibacterial property against the tested organism [18]. When subjected to the Tukey’s post-hoc test, there was a significant difference between the antimicrobial efficacy of pudina and curry leaves (p=0.0151), pudina and chlorhexidine (p=0.0002), curry leaves and chlorhexidine (p=0.0233); and tulsi and chlorhexidine (p=0.0024). Chaudhary NJ et al., conducted a study comparing the antimicrobial effect of pudina and chlorhexidine and concluded that though pudina was effective against S. mutans; its efficacy was less compared to chlorhexidine [8]. Agarwal P et al., conducted a study evaluating the antimicrobial properties of tulsi and chlorhexidine, results of which concluded chlorhexidine had better action against S. mutans than Tulsi [9]. Thus, the results of this study are in agreement with the previous studies [7-9] where both pudina and tulsi had less efficacy against S. mutans as compared to chlorhexidine (0.2%) and curry leaves were potent against cariogenic bacteria.

This investigation was conducted in vitro with the extracts of tulsi, pudina and curry leaves. The duration of the contact of such extracts with the microorganisms in the oral cavity in-vivo is not clear; hence, further in-vivo studies are required to obtain clinical results comparing these products utilising a multibacterial biofilm.

Under the limitations of this study, curry leaves had better antimicrobial efficacy compared to pudina and tulsi. With the changing trends in dental caries initiation and progression the use of various preventive strategies have been modified and the inclination towards the use of safer, cost-effective herbal products has increased.