Antibacterial Effects of Chewing Stick Extracts on Streptococcus Mutans

Background: Streptococcus mutans is considered as a major microorganism causing tooth decay, affecting individuals globally. Different types of chewing sticks possess anti cariogenic properties, traditionally been consumed in maintaining dental hygiene. Usage of these sticks can be economical in developing and rural regions where dental caries is a major health concern. Objective: The current study aims to evaluate antibacterial action of different type of chewing sticks extract on Streptococcus mutans. Methods: Aqueous extracts of Azadiracta indica (Neem), Melia azedarach (Bakain), Mangifera indica (Mango), Salvadora persica (Peelu), Terminalia chebula (Harhar), Dalbeigia sissoo (Tali) and Juglans regia (Akhrot) plants which have justified folk use were prepared in Punjab University College of Pharmacy, Punjab University. Next, in vitro antimicrobial activity was studied by broth dilution method in Post Graduate Medical Institute, Lahore. Moreover, minimum inhibitory concentration (MIC) as well as the minimum bactericidal concentration (MBC) were quantitatively evaluated on basis of turbidity index. Results: There is a significant effect of aqueous extracts of plants on the bacterial inhibition. MIC and MBC values were in the range of 6.125 to 100 mg/mL against S. mutans. The notable effect occurred with aqueous extracts of Azadiracta indica showing MIC and MBC values as 6.25mg/mL and 12.5 mg/mL, respectively. Alternatively, Salvadora persica demonstrated 8.33mg/mL and 16.66 mg/mL values of these parameters Conclusions: Each plant studied exhibited moderate to high antibacterial activity against tested bacterial strain. However, Azadirachta indica and Salvadora persica aqueous extracts showed promising effect against Streptococcus mutans

to the tooth decalcification.Global burden of disease (GBD) report that 60 to 90% of school going children anti-plaque and anti-fungal properties.World Health Organization (WHO) suggested the practice of chewing stick in 1986 as well as 2000 and concluded that additional work is needed to assess its effectiveness against 2,6 dental microorganism.In line with the above, different parts of plants have been tested for antimicrobial activity 6,7 globally.A study conducted in Pakistan in 2014 concluded that Miswak taken from the root of the peelu tree exhibited antimicrobial activity against all the common 2 oral pathogens.Nevertheless, some knowledge gaps do exist.The current study aims to evaluate antibacterial action of different type of chewing sticks extract on Streptococcus mutans.
Variety of in vitro methods have been used to evaluate the antibacterial effect, of chewing sticks including the agar diffusion, Petri dish bioassay, and the use of Similarly in a study, Streptococcus mutans gave an inhibitory zone of 17.33 mm at a concentration of 5 400μg/ml to alcohlic extract of Salvadora persic .
Similarly in a study, 400 mg/mL of aqueous and methanolic extracts of Salvadora persic was the most effective on all strains using the agar dilution and minimum inhibitory concentration methods.The methanol extract exhibited a stronger antibacterial activity against Gramnegative (3.3-13.6 mm) than Gram-positive (1.8-8.3 7 mm) bacteria.Keeping the above into consideration and lack of evaluation by broth dilution method in previous studies, we aimed to evaluate the antimicrobial potential of natural herbs used by rural people in Pakistan like Azadiracta indica (Neem), Melia azedarach (Bakain), Mangifera indica (Mango), Salvadora persica (Peelu), Terminalia chebula (Harhar), Dalbeigia sissoo (Tali) and Juglans regia (Akhrot) by broth dilution method so that safe and economical technique can be discovered to limit caries in emerging nations.

Methods
This was cross-sectional study, which was conducted in the Department of Microbiology, Post Graduate Medical Institute and Punjab University College of Pharmacy, Punjab University through non-probability convenient sampling technique.
Standard reference strain of Streptococcus mutan ATCC 25175 American Type Culture Collection, USA was used in this study.
The fresh chewing sticks of medicinal plant with appropriate diameter 0.5-1cm and length 15-20cm were collected from Jinnah bagh (Lawrence garden), Lahore.Peelu (Salvadora persica) was collected from Lalsuhanra, Bahawalpur, Dried chewing sticks of un appropriate diameter and length were excluded from study Permission was taken from management of Bagh e Jinnah , Lahore and Lalsuhanara Park for sample collection and authenticated by botanist, Government College, Lahore.Sample of the collected plants were deposited as a specimen (voucher No: GC.Herb Bot 1160) in the herbarium, Botany Department, Government College, Lahore, Pakistan.Ethical permission for the study was obtained from Post graduate Medical Institute, Lahore.
Samples were dried and ground into coarse powder.Extracts of the powdered plant material were prepared by maceration method in Punjab University College 5 of Pharmacy, Punjab University.For this purpose; 100 grams of powdered plant material were added in one liter distilled water, pH 7.0 in a container.Mixture was filtered under UV light lamp through Whatmans filter paper after 24 hours.This process was repeated three times to obtain concentrated extract.The extracts were concentrated in vacuum at 35°C, inoculated a sterile nutrient agar slant with the filtered extract to check the sterility of the extracts and frozen it 9 at -80ºC .The frozen extracts were dried at -44°C under vacuum 0.2 mbar over 3 days using a freeze drier (Christ Lyophilizer).The extracts were kept in screw glass capped bottles and stored in refrigerator till use.Stock solutions of the plant extract were prepared by suspending 600mg of extract in 3ml of distilled water to get 200mg/ml concentration.These stock solutions 8 were stored at -20 °C.
Liofilchem stick containing freeze-dried form of strain (Streptococcus mutans) was obtained.Blood agar and Nutrient agar were prepared in Microbiology Laboratory (PGMI).The stick was opened and sub cultured on blood agar and nutrient agar.For 24 hours, at 37 °C plates remained incubated; afterwards bacterial growth obtai-ned was checked by gram staining and catalase reaction.
Using aseptic technique, at least four to five well-isolated colonies were selected from the nutrient agar plate.The top of each colony was touched with a loop, and inoculated into 5 ml of Brain Heart Infusion Broth.After incubation at 35°C, turbidity was checked against the 0.5 McFarland turbidity standard.Turbidity was adjusted to 0.5 McFarland standards by either diluting aseptically with Brain Heart Infusion Broth or further incubation.The inoculums prepared contained approximately 1-2×108 CFU/ml.Tube dilution technique was performed to evaluate In 9 vitro antimicrobial susceptibility testing.We added 1.0 ml of the culture suspension to all broth dilution tubes.In the first tube, 2ml of plant extract solution was added.From tube number 2-9, 1ml of sterile brain heart infusion was added 1.0 ml of the extract in tube 1 was nd shifted to the 2 tube from 1st tube.Process was continued till tube number 8. From 8th tube 1.0 ml was thrown away.Last tube having1ml of sterile broth served as a control. 1 ml of the culture suspension was added to all of tubes.Subsequent concentration of plant extract was one-half of original concentration in each tube.In this way dilutions of each plant extract were made i.e. 200 mg/ml, 100 mg/ml, 50 mg/ml, 25, 12.5, 6.25, 3.125, and 1.56 mg/ml.Tubes were incubated at 35 °C for 16-20 hours.To prevent drying out, the tubes were sealed 9 with tight fitting screw caps.
Three tubes were included in each batch as control: one tube having distilled water and Streptococcus mutans; second tube having brain heart infusion (add in material) broth and third tube with brain heart infusion broth and Streptococcus mutans.The MIC was read as the lowest concentration of the extract that completely inhibited the growth (no turbidity).MIC broth tube without visible growth was used to check the MBC.From the broth dilution tubes without visible growth, standardized loop containing approximately 108 CFU/ml of organism were sub-cultured onto appropriately labeled blood agar plates.The plates were then incubated at 37°C for 24 hour.Following overnight incubation, MBC plates were evaluated for the presence 9 or absence of colony growth.MIC and MBC were repeated thrice to confirm result reliability.

Figure 1: Minimum Inhibitory Concentration of different Chewing Sticks Extracts
All the tests were performed in triplicate and data were presented as mean deviation.The collected information was analysed by SPSS using one way analysis of variance and compared using Turkey's honestly significant difference (HSD) at 5% level of significance.To meet the normality assumption of the ANOVA, the  data were transformed by square root transformation.

Results
The physical properties of aqueous extracts of seven plants, being used as chewing sticks, are given in Table 1.
All extracts were blackish and brown colour.Magnifera indica shows highest yield extract.All the extracts exhibited neutral to acidic pH.Azadiracta indica and Juglans regia extracts were almost neutral Table 1.
The antibacterial activity of different plant extract in terms of MIC and MBC are given in Figure 1and 2, respectively.The results revealed a significant difference among the different extracts tested (F=3.81;df = 8,18; p<0.01).Azadirachta indica and Salvadora persica proved to be the most effective extracts in terms of minimum inhibitory concentrations, (Figure 2).
The MBC was determined by subculturing the test dilution (used in MIC) on to a fresh solid medium and incubated further for 24 hours.The results revealed a significant difference among the different extracts tested (F=3.45;df=8,18; p<0.05).Azadirachta indica and Salvadora persica proved to be the most effective extracts in terms of minimum bactericidal concentrations, Moreover, MBC value of different plant extract is almost two fold higher than corresponding MICs.
There is a significant effect of aqueous extracts of plants on the bacterial inhibition.MIC and MBC values are in the range of 6.125 to 100 mg/mL against S. mutans.The notable effect occurred with aqueous extracts of Azadiracta indica showing MIC and MBC values as 6.25mg/mL and 12.5 mg/mL, respectively.Alternatively, Salvadora persica demonstrated 8.33mg/mL and 16.66 mg/mL values (Figure 1 and 2).

Discussion
The hypothesis of the present study was proved by the results that commonly used miswak in Pakistan possess antimicrobial activity against Streptococcus mutans as their potential anti-plaque effect likely complement mechanical plaque-removing property of chewingsticks.Presence of antibacterial activity in chewing stick extract is consistent with general antibacterial activity reported in the past.
A study was conducted in 2018, and showed very good antibacterial activity of Mangifera indica and Azadirachta indica against Streptococcus mutans.Magnifera indica and Azadiracta indica had the highest antibacterial activity at concentrations of 0.3 mg/ml and 6.25 mg/ml, respectively.Where as in the present study Azadiracta indica showed good antibacterial activity as compared to Magnifera Indica.the difference in the results may be because all plant parts like bark, stem, root, fruit and leave have a various active ingredients that don't have 14 reliable, precise and constant constituents.
In the present study, the MIC of Magnifera Indica aqueous extract was found to be 50mg/ml and MBC was calculated 100mg/ml respectively.Similar results were Overall, this report gives a basis for further in vivo studies and tends to reinforce the use of these extracts as antimicrobial agent in folk medicine.The two plants should be evaluated further in depth to isolate the active components and to clarify their mode of action, hence, their actual effects on denture plaque.However, additional tests, including experimental models and pharmacological applicability, are required before considering these plant extracts as alternative methods in the treatment of oral diseases or for their use in daily oral-care products, gel, rinses, or effervescent tablet preparations, especially for elderly denture wearers.
In vitro susceptibility of chewing sticks extracts was evaluated in present study.In vivo interaction period of the extract with the bacteria in oral cavity is not clear.
Additional work should be conducted including additional microbes: examining the superiority and effectiveness of chewing sticks by in vitro or in vivo tests and associating the incidence of caries amongst chewing stick users and nonusers to elucidate the picture.

Conclusion
Outcomes advocate that chewing sticks can be an effective method for prevention of caries.The aqueous extracts of Azadirachta indica and Salvadora persica proved to be the most effective in terms of minimum inhibitory and minimum bactericidal concentrations against S. mutans.The aqueous extract of Azadiracta indica showed strongest bactericidal activities with MIC and MBC values of 6.125 mg/mL and 12.5 mg/mL, respectively.

IntroductionD
ental caries is among the prevalent dental infection caused by acid producing microorganism leading 1,2 different regions of the World, different plant sticks serve as natural dentifrice with antibacterial, April -June 2023 | Volume 29 | Issue 02 | Page 150

Table 1 :
Physical property of extracts of plants used as chewing sticks

2, 10,11, 12 ,13
They concluded that Neem and Aleo vera gel have anti-15 microbial effect at different concentrations.Pulbutr P et al. also evaluated MIC and MBC of Derris reticulata ethanolic stem extract by broth dilution method against Streptococcus mutans and found it to be effective at low In 2019, a study was conducted to find antimicrobial activity of herbal medicines on Streptococcus Mutans.April -June 2023 | Volume 29 | Issue 02 | Page 151