Evaluation of Antimicrobial Efficacy of Three Commercially Available Herbal Mouthwashes - An In Vitro Comparative Study

Introduction

Oral health is an integral and key part of overall health and wellbeing of an individual. Poor oral hygiene is one of the major causes which lead to impaired oral health. Most of the dental diseases are associated with dental plaque. Hence, plaque control plays an importaant role in ensuring excellent oral health. 

The oral health measures followed should focus on good aesthetics, comfort and function of dentogingival tissues that can be maintained by not only dental professionals but also by patients on their own. Secondly, oral hygiene methods such as tooth brushing, flossing, using interdental brushes may not be effectively carried out by individuals and thus may not be sufficient in controlling plaque.^1,2 Hence the use of antimicrobial therapeutic agents has been proposed as an additional measure to reduce oral bacterial load. There is a continued interest in identifying efficient antiplaque agents that could be used daily by patients. These additional antiplaque agents such as mouthwashes need to possess antimicrobial properties to reduce, control and prevent different kinds of oral diseases. Despite the development of effective antibacterial, chemical based mouthwashes, there is an increasing popularity of natural compounds due to their biocompatibility, low side effects and less chance of developing bacterial resistance, thus paving their way into dentistry.³ Moreover, the use of traditional herbs to treat diseases is known to mankind for thousands of years. As per the WHO data, almost 80% of the world population mainly depend on traditional medicine for their primary healthcare needs.^4,5

For dental application, many commercially available herbal products are marketed in the form of either toothpastes or mouthwashes. Mouthwash is a safe and effective way to deliver the antiseptics which can even reach difficult areas of the oral cavity. Most of the herbal mouthwashes used contain the extracts of Neem, Alovera, Yavani satva, Nagavalli, Gandhapurataila, Pilu, Bibhitaka, Tulsi, Echinacea, Chameli leaves etc due to their potent anti-microbial and anti-inflammatory effect.⁶ Although there are numerous herbal mouthwashes present in the market, results are inconsistent regarding their clinical benefits along with paucity of meta-analytical evidence.

Hence to provide a comprehensive evidence to the clinicians and patients, an effort was made in our study to examine and compare the antimicrobial effectiveness of three different commercially available herbal mouthwashes against four periodontal pathogens namely Streptococcus mutans (S. mutans), Porphyromonas gingivalis (P. gingivalis), Aggregatibacter actinomycetemcomitans (A.a comitans) and Candida albicans (C. albicans).

Materials and Methods

The present in vitro investigation was carried out for a duration of 12 months after obtaining approval from Ethics committee of the institution (IEC02/2018). The antimicrobial efficacy of three commercially available herbal mouthwashes i.e Oramint herbal mouthwash (West-Coast pharmaceutical works Ltd), Hiora mouthwash (Himalaya Drug Company, Bengaluru, India) and Colgate plax fresh tea mouthwash (Colgate – Palmolive India Ltd.) was tested and compared on four different standard bacterial strains i.e Streptococcus mutans, Porphyromonas gingivalis, Candida albicans and Aggregatibacter actinomycetemcomitans. Microbial culturing was further carried out using suitable culture media.

The study involved a key person and a blinded examiner where the key person prepared the microbial strain cultures on petri dishes, filled them with different mouthwashes and coded before handing over to the examiner. Determination of microbial sensitivity was done by ditch plate diffusion method. The examiner measured the diameter of zone of inhibition to check for the antimicrobial sensitivity of the mouthwashes and the whole procedure was repeated four times.

For in vitro testing setup, the inoculum was prepared using a loop and swab, colonies were transferred to the plates. Turbidity of the broth was adjusted to 0.5 McFarland turbidity standard and was vortexed. As an alternative method, with the help of a photometric device, the suspension was standardized.

Inoculation of agar plate was done within 15 minutes following the adjustment of inoculum to McFarland 0.5 turbidity standard. Sterile cotton swab was dipped into the inoculum and rotated against the wall of the tube above the liquid to remove excess inoculum. Entire surface of the agar plate was swabbed three times while the plates were rotated approximately 60° between streaking to ensure an even distribution. Hitting the sides of petri plate, aerosol creation was avoided carefully. Inoculated plates were rested for a minimum of three minutes to a maximum of 15 minutes before making wells.

Addition of compound to the plate

Twelve petri dishes were prepared and a narrow well of five millimetres diameter was made in every dishes with a punch. Five such wells were prepared on each dish. With the help of micropipette, 75µL, 50µL, 25µL, 10µL and 5µL of full concentration of three mouthwashes were added in each of the microbial strain petri dishes.

Incubation

Plates were incubated in less than 15 minutes of compound application. Plates were inverted and stacked not more than five high and were incubated for 18-24 hours at 37ºC in an incubator. For facultative anaerobes and anaerobic microorganism, the plates were incubated in a CO₂ jar at 37ºC.

Reading the plates

Plates were read after the growth was nearly confluent. Diameter of zone of inhibition was measured to nearest whole millimetre by holding the measuring scale. After measuring the zone of inhibition, the petri dishes were decoded and the results were obtained.

Statistical analysis

The data analysis was performed using SPSS (IBM version 23) software. Descriptive statistics included mean and standard deviation. Differential statistics included one way ANOVA for comparison between groups (inter group comparison), followed by post-hoc Tukey’s test for multiple comparisons. The statistical significance level was set at a ‘p’ value of 0.05 at 95% confidence interval.

Results

The cultured strains were tested using ditch plate method. On testing the antibacterial efficacy of the three herbal mouthwashes against different standard pathogenic bacterial strains, better efficacy was observed with the increase in concentration of mouthwashes. Irrespective of the herbal formulations used, the maximum inhibition was observed at the concentration of 75µL/mL for all the tested products (Table 1).  

At 75 microliter/mL concentration, Oramint mouthwash showed the highest mean zone of inhibition with a mean diameter of inhibition of 28.2500 mm for S. mutans, 19.7500 mm for P. gingivalis and 21.0000 mm for A.a. comitans. Candida albicans showed high sensitivity towards Hiora mouthwash with a mean zone of inhibition of 13.2500 mm. The mean zone of inhibition of Colgate plax against Candida albicans was found to be 13.0000 mm. Statistical significant difference was observed on comparison of the mouthwashes tested. But the difference of mean zones of inhibition of Hiora and Colgate plax against Candida albicans was not statistically significant. (Table 2 and Figure 1).

Discussion

With the growing resistance of pathogens towards antibiotics, increasing prevalence of oral diseases and with the after effects of chemical based mouthwashes, herbal extracts are gaining wide popularity in oral hygiene products with encouraging results. Herbal medicines contain natural phytochemicals that have demonstrated potent antimicrobial, anti-inflammatory, and antioxidant properties.^7,8 They have been proven to be beneficial in reduction of plaque as well as gingivitis.^9,10 Numerous herbal based mouthwashes are available as over the counter products claiming to have greater antibacterial activity. However, the results regarding their clinical benefits are inconsistent in the literature.^11-13 Hence, in the present in vitro study, we tested the antimicrobial efficacy of three commonly available mouthwashes with herbal ingredients against the known oral pathogens like Streptococcus mutans, Candida albicans, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans using ditch plate diffusion method.

The three mouthwashes selected for the current study included fresh tea extract mouthwash (Colgate plax), Hiora mouthwash (Himalaya) and Oramint herbal mouthwash (Healthvit). Oramint mouthwash contains a group of ingredients that had been widely used in every Indian household for many centuries as a part of traditional medicinal system. In our study, when compared to other tested herbal mouthwashes, Oramint mouthwash showed significant (p <0.05) higher antibacterial activity against all the tested pathogens except C. albicans. Oramint mouthwash contains herbal ingredients like Neem, Babool, Tulsi etc which could explain its superior antibacterial activity. Various studies in the past literature reported antibacterial effects of Neem and Babool. Neem extract functions by inhibiting bacterial growth, adhesion and thus prevents plaque formation.¹⁴ Higher antibacterial activity of Neem compared to chlorhexidine was reported in previous studies. The same study also reported on the antimicrobial activity of Babool and suggested it to be a potent ingredient for antimicrobial oral rinses.¹⁵ Tulsi has been traditionally used in treating plethora of human diseases. Tulsi extract has been demonstrated to be beneficial in managing periodontitis and highly effective against periodontal pathogens.^16,17

Hiora herbal mouthwash contains combination of natural herbs like Bibhitaki (Terminalia bellirica), Nagavalli (Piper betle), Pilu (Salvadora persica), Peppermint satva, Yavani satva, Gandhapura taila, Ela, Piper betle. These herbs are known to exhibit antimicrobial, anti- inflammatory, antioxidant and immunity booster properties.^6,18 Betel leaf extract has been found to be effective against both gram positive and gram negative organisms.¹⁹

Meswak (Salvadora persica) is also known as toothbrush tree. Miswark extract exhibits potent antibacterial and antifungal effects. The results of the current study found Hiora mouthwash to be highly effective against C. albicans compared to the other mouth rinses. The presence of meswak in Hiora mouthwash could be responsible for its beneficial effects. Previous data also supports the possible antifungal activity of miswak.²⁰

The green tea extract mouthwash tested in the current study showed effectiveness against standard strains of bacteria. Green tea is known to contain catechin which are potent antibacterial agents.²¹ A recent systematic review also suggests a possible positive effect of green tea in treating periodontitis.²² Previously, multiple studies have claimed that oral formulations that contain herbal medicinal ingredients can inhibit the growth of periopathogens and reduce dental plaque as well as inflammation of gingiva.^3,23 The findings of our study also supports this assertion wherein all the three tested herbal mouthwashes were found to be effective in inhibiting the growth of cultured strains of the pathogens with some variation in their effectiveness against each test organism. Among the three herbal mouthwashes, Oramint mouthwash showed significant results against the tested periopathogens. Whereas, Hiora herbal mouthwash and Colgate plax fresh tea extract mouthwash showed good antimicrobial activity against C. albicans. Although Hiora mouthwash produced a greater level of inhibition against C. albicans, its antifungal activity was comparable to Colgate plax with the difference being not statistically significant.

In our study, the experimental procedure was repeated four times to obtain reliable results which could be an added advantage. However, as it was an in vitro investigation, there could be a room for variation in outcomes. Hence these changes should be kept in mind while applying the same in clinical situations.

Conclusion

Herbal products are generally considered to be safe with low toxicity coupled with low cost, making it a sought after treatment modality among general population. Within the limitations of the current study, it can be concluded that all the three commercially available herbal mouthwashes showed varying degree of effectiveness against the tested organisms. Among the tested mouthwashes, Oramint mouthwash outshined due to its overall antimicrobial efficacy. However, this study was done under in vitro setting. Hence further randomized clinical trials regarding the same are required to support the claim before attempting to generalize these results.

Nevertheless, this in vitro method can be effectively used as a screening technique to check the antimicrobial efficacy of herbal mouthwashes before their in vivo application. However, there is a need to carry out future research focusing on testing and comparing the substantivity of various herbal mouthwashes.

Clinical significance

The herbal mouthwashes tested in our study demonstrated varying degree of antimicrobial activity against the periopathogens. Hence, they can be used as additional agents along with the standard mechanical oral hygiene procedures.

Conflicting Interest

None

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Acknowledgement

We would like to thank the Dr. Kishore Bhat, Head, Central Research Laboratory, Maratha Mandal’s NGH Institute of Dental sciences, Belgaum for guiding the study and Dr. Tripti MDS, Public Health Dentistry for carrying out the statistical analysis for the study.