Comparative evaluation of the efficacy of a herbal mouthwash and chlorhexidine mouthwash on select periodontal pathogens: An in vitro and ex vivo study.

BACKGROUND: Several herbal mouthwash and herbal extracts have been tested in vitro and in vivo in search of a suitable adjunct to mechanical therapy for long-term use. In this study, we aimed to look at the antimicrobial effect of the herbal mouthwash and chlorhexidine (CHX) mouthwash on select organisms in in vitro test and an ex vivo model.
MATERIALS AND METHODS: The antimicrobial effects were determined against standard strains of bacteria that are involved in different stages of periodontal diseases. The in vitro tests included determination of minimum inhibitory concentration (MIC) using broth dilution and agar diffusion. In the ex vivo part of the study supragingival dental plaque were obtained from 20 periodontally healthy adult volunteers. Descriptive analysis was done for the entire quantitative and qualitative variable recorded.
RESULTS: The MIC by broth dilution method found no statistically significant difference between the mouthwashes. The agar dilution method showed CHX was more effective as compared to the herbal mouthwash against standard strains of Streptococcus mutans, Streptococcus sanguinis, and Aggregatibacter actinomycetemcomitans. However, no difference was observed between the mouthwashes for Porphyromonas, Pseudomonas aeruginosa, and Fusobacterium nucleatum. The ex vivo results conclude that none of the selected mouthwashes were statistically significantly different from each other.
CONCLUSION: In the present study, CHX showed higher levels of antimicrobial action than the herbal mouthwash against bacterial species. The results reinforce the earlier findings that the in vitro testing is sensitive to methods and due diligence is needed when extrapolating the data for further use. However, long-term use and in vivo effectiveness against the periopathogens need to be tested in well-planned clinical trials.

INTRODUCTION

Ideally, it is required that any antimicrobial/antiseptic agent used should able to modify the oral environment by being specifically effective against pathogens without altering the normal flora.[1] There are several types of mouthwash available in the market today worldwide. Many of these mouthwashes have not been tested adequately, and the information is lacking as to when and how to use these agents for maximum benefit.[2] Chlorhexidine (CHX) digluconate has been the agent of choice as an antiplaque agent when compared to others and is considered as the gold standard.[3] However, due to its side effects, its acceptance by patients can be limited, especially when a longer period of use is recommended.[4]

Mouthrinses are primarily known to affect the supragingival and marginal plaque, and its effect on the subgingival plaque is limited, because of the tight gingival contact with the tooth in healthy individuals.[5]

Several herbal mouthwash and herbal extracts have been tested in vitro and in vivo in search of a suitable adjunct to mechanical therapy for long-term use.[6789] In the present study, one such herbal mouthwash was selected which has proved to be promising antimicrobial agent in the past studies. The herbal mouthwash chosen in this study contains Pilu (Salvadora persica) Bibhitaka (Terminalia bellirica), Nagavalli (Piper betle), essential oils, namely, Gandhapura taila (Gaultheria fragrantissima), Ela (Elettaria cardamomum), flavoring agents Peppermint satva (Mentha), and Yavani satva (Trachyspermum ammi). S. persica also known as Miswak have shown anti-plaque properties.[10] T. bellirica is known to have antimicrobial, antioxidant, and antibiofilm activity,[1112] whereas Piper beetle and E. cardamomum have antimicrobial properties.[131415]

In the present study, a panel of standard strains of bacterial Streptococcus mutans, Streptococcus sanguinis which are important as initial colonizers and vital in dental caries were selected.[16] Also included in the panel were important periodontal pathogens such as Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis, also known as a keystone pathogen,[17] Fusobacterium nucleatum which is considered to be a bridging organism,[18] and Pseudomonas aeruginosa which is most commonly colonized on the dorsum of the tongue.[19] In this study, we aimed to look at the antimicrobial effect of the herbal mouthwash and CHX mouthwash on these select organisms in two different in vitro tests and an ex vivo model.

MATERIALS AND METHODS

The current study included two in vitro tests to determine the minimum inhibitory concentration (MIC) using broth and agar dilution technique along with the ex vivo testing of the antimicrobial action on the supragingival plaque from healthy individuals. The protocol for the research project was approved by the ethics committee of the institution. The antimicrobial effects were determined against standard strains of bacteria which are involved in different stages of periodontal diseases. These bacterial strains include aerobic bacteria P. aeruginosa (ATCC 25619), facultative anaerobic bacteria including S. mutans (ATCC 25175), S. sanguinis (ATCC 10556), A. actinomycetemcomitans (ATCC 43718), and obligate anaerobic organism F. nucleatum (ATCC 25586) and P. gingivalis (ATCC 33277).

Materials used

For this study, we used an herbal mouthwash (HiOra [HiOra Mouthwash Regular-Manufactured by the Himalaya Drug Company, Bengaluru, India.]). Each gram of HiOra mouthwash containing: Pilu (S. persica, 5.0 mg), Bibhitaka (T. bellirica, 10.0 mg), Nagavalli (Piper beetle, 10.0 mg) (b) Powders: Peppermint satva (Mentha spp 1.6 mg), Yavani satva (T. ammi 0.4 mg) and Oils: Gandhapura Taila (G. fragrantissima 1.2 mg) and Ela (E. cardamomum 0.2 mg).

CHX mouthwash: Commercially available nonalcoholic 0.12% CHX mouthwash (Hexidine® 60 ml ICPA Health Products Ltd, Mumbai, India.)

For the in vitro testing, bacterial strains were maintained on blood agar media, mitis salivarius agar, and brain–heart infusion agar as appropriate for that species. Media and supplements were obtained and prepared in accordance with a manufacturer's instructions (HiMedia Laboratories Pvt Ltd., Mumbai, India).

In vitro tests

First, for the broth dilution assays, test strains were inoculated into tubes containing serial dilutions of antimicrobial agents (i.e., 100, 50, 25, 12.5, 6.25, 3.12, 1.6, 0.8, 0.4, and 0.2 μl/ml) in brain–heart infusion broth and incubated at 35°C for 48–72 h. The MIC was defined as the highest dilution of the agent that inhibited bacterial growth, as determined by lack of turbidity.

Second, for agar method, all microorganisms were maintained on tryptic soy agar supplemented with 5% defibrinated sheep blood 5.0 μg/ml hemin and 0.5 μg/ml vitamin K1.[20] Later, serial dilutions (20–200 μg/ml) of test mouthwash were added 1% by volume (i.e., 300 μl) to 30 ml of media individually. In each case, agar was autoclaved and then equilibrated at 45°C before the addition of mouthwash. These agars were poured under sterile conditions and utilized subsequently. Bacterial suspensions were inoculated onto test media and incubated as appropriate for that species. The MIC was defined as the lowest concentration of test agent that inhibited bacterial growth, either no bacterial colonies or only a few small colonies.

Ex vivo testing

Finally, in the ex vivo part of the study, supragingival dental plaque was obtained from 20 periodontally healthy adult volunteers (13 males and 7 females) after obtaining their informed consent. For these participants above 18 years of age and in good general health, diagnosed as periodontally healthy having probing depth ≤3 mm on clinical examination were included. Each participant had at least 20 natural teeth, with no orthodontic treatment or fixed or removable prosthesis. Potential individuals were excluded if with relevant medical conditions or diseases, smokers, or under antibiotic, anti-inflammatory, or corticosteroid therapy in the last 2 months. Participants with a history of any use of mouth rinse in the last month and oral prophylaxis in the last 3 months were also excluded from this study.

Similar to the previously described procedure, a dentist completed the oral examination for all participants and provided them with commercially available nonfluoride dentifrice and a soft-bristled toothbrush to be used for 1 week before their scheduled visit for dental plaque sampling. All study participants were asked to refrain from oral hygiene for a minimum of 12 h before plaque collection.[2021] A total of six teeth were randomly selected, air-dried and isolated with cotton rolls. Supragingival plaque samples were collected from the buccal, lingual, and interproximal surfaces of the teeth, pooled in a plastic vial containing reduced transport fluid, and sent to laboratory for processing. Individual samples were dispersed onto 30 ml blood agar media, kanamycin blood agar media, brain–heart agar media, anaerobic agar media, and mitis salivarius agar media containing 1% by volume of 100% of each mouthwash.[2021] Anaerobic cultures were incubated in an anaerobic environment (95% nitrogen, 10% hydrogen, and 5% carbon dioxide) at 35°C–37°C. After 5–7 days, total colony count was performed using a counting grid.

Descriptive analysis was done for the entire quantitative and qualitative variable recorded.

Data were analyzed by two-way analysis of variance and Kruskal–Wallis (95% confidence level). Pair-wise comparison was performed using Mann–Whitney U-test. Dose effects were examined by Tukey's multiple post hoc tests. All analysis was performed using SPSS 11.0 version (SPSS Inc. Released 2001 SPSS for Windows, Version 11.0. Chicago, IL).

RESULTS

In vitro results

The broth dilution procedure was utilized to compare the effects of the herbal mouthwash and CHX on a set of standard laboratory strains [Figure 1]. CHX against S. mutans was sensitive at 6.25 μg/ml, while the herbal mouthwash was sensitive at 12.5 μg/ml. MIC of the herbal mouthwash (25 μg/ml) showed less inhibition than CHX (12.5 μg/ml) for S. sanguinis. CHX also showed higher MIC value of 6.25 μg/ml when compared to 25 μg/ml that of the herbal mouthwash for A. actinomycetemcomitans. Against P. gingivalis and P. aeruginosa MIC value of 50 μg/ml and 12.5 μg/ml was observed for the herbal mouthwash and CHX, respectively. For F. nucleatum, the MICs of CHX (1.6 μg/ml) were lower than the herbal mouthwash (25 μg/ml). One hundred percent of the selected bacterial strains were susceptible to CHX in the range of 50–25 μg/ml, whereas the same for the herbal mouthwash was 66.66%. At a concentration of 25–12.5 μg/ml, CHX was susceptible for 83% of the bacterial strains, whereas for the herbal mouthwash, 33.33% bacteria's were sensitive. Finally, at a concentration of 12.5–6.25 μg/ml of the herbal mouthwash, all the selected bacteria's were resistant, whereas 50% bacterial strains were found to be sensitive to CHX. The MIC by broth dilution method concludes that there was no statistically significant difference observed between the selected groups of mouthwashes [Figure 2].

Figure 1

Comparison of the effects of herbal mouthwash and chlorhexidine on a set of standard laboratory strains using broth dilution method

Figure 2

Percentage of susceptible bacteria with herbal mouthwash and chlorhexidine by broth dilution method

The agar dilution method determined the inhibitory capacity of each mouthwash for each organism. According to Figure 3, CHX was more effective as compared to the herbal mouthwash against standard strains of S. mutans, S. sanguinis, and A. actinomycetemcomitans. The mean CFU of S. mutans by CHX was 0.51 ± 0.73 which is significantly less as compared to the herbal mouthwash (1.92 ± 0.13). For S. sanguinis, CHX again showed significantly lesser mean CFU (0.26 ± 0.58) when compared to the herbal mouthwash (2.17 ± 0.08). For A. actinomycetemcomitans, a significant value by CHX (0.59 ± 0.81) was observed against herbal mouthwash (1.88 ± 0.28). Herbal mouthwash showed a mean CFU value of (0.10 ± 0.21) while CHX showed no CFU with P. gingivalis. For P. aeruginosa, CHX displayed a lesser mean CFU (0.54 ± 0.52) as compared to (0.74 ± 0.26) of herbal mouthwash. Finally, for F. nucleatum, herbal mouthwash showed less effectiveness that is (1.49 ± 0.14) as compared to CHX (0.99 ± 0.90). There was no statistically significant difference observed between CHX and herbal mouthwash against P. gingivalis, P. aeruginosa, and F. nucleatum.

Figure 3

Mean colony-forming units of the bacteria with herbal mouthwash and chlorhexidine by in vitro agar dilution method

Ex vivo results

Comparison of the two mouthwashes with mean log CFU counts of all organisms on specific plates demonstrated that CHX gave the least mean CFU (0.50 ± 0.18) followed by the herbal mouthwash (1.77 ± 0.16) [Figure 4].

Figure 4

Mean colony-forming units of the bacteria from dental plaque with herbal mouthwash and chlorhexidine by ex vivo method

Similarly, a pair-wise comparison was done using Tukey's multiple post hoc tests and a P < 0.05 was considered statistically significant. The ex vivo results conclude that none of the selected mouthwashes were statistically significantly different from each other. CHX by far was found to be most effective although the herbal mouthwash demonstrates some effectiveness against the select bacteria.

DISCUSSION

Dental plaque has long been considered to be the predominant etiological agent in developing caries, gingivitis, and periodontal disease.[22] Adequate plaque control is very vital to prevent the occurrence of the abovementioned disease. This control of plaque can be achieved by mechanical, chemical, or by a combination of the two. Mouthwash which is a chemical plaque control technique should be used along with mechanical hygiene.[23] However, in certain individuals with a disability or with lack of skill and motivation, mouth rinsing is an easier alternative to supragingival plaque control.

Mouthwash has been recommended being a regular adjunct along with mechanical therapy to maintain oral health.[24] Among the mouthwashes, CHX is considered to be the gold standard and is part of the part of the periodontal treatment regimen.[25] However, CHX is known to have various side effects ranging from minor effects such as alteration in patient taste sensation and staining of teeth to certain less common effects such as mucosal erosion or parotid swelling.[262728] Considering the adverse effects of the use of CHX, its use for long-term therapy has been limited or not actively recommended.[29]

Several mouthwashes without the similar negative effects as CHX have been tried for long-term therapy, but none has been successful in providing similar anti-plaque and anti-gingivitis effect as the latter one. In recent time, herbal mouthwashes have gained attention for their antimicrobial properties, but none has been able to match that of CHX.[30]

In the present study, HiOra® a herbal mouthwash was tested in vitro against a set of specific aerobic bacteria P. aeruginosa, facultative anaerobic bacteria including S. mutans, S. sanguinis, A. actinomycetemcomitans, and obligate anaerobic organism F. nucleatum and P. gingivalis that contribute to periodontal diseases.

The herbal extracts used in this mouthwash such as S. persica, T. bellerica, P. betle, G. fragrantissima, E. cardamomum, and Mentha spp. have been known to have antimicrobial activity on some of the common oral pathogens.[111431] To the best of our knowledge from the data available, the activity of these herbal extracts on common periodontal pathogens is lacking.

There have been reports of the herbal mouthwash showing positive beneficial effects on the clinical parameters.[32] In another study, the ex vivo effect of the herbal mouthwash has been seen on organisms from supragingival dental plaque in healthy and chronic periodontitis patients.[6] However, in this study, we aimed to look at the panel of organisms which are important in both the supra and subgingival dental plaque and play vital role in plaque formation and disease.

One of the variables examined in the present study was the effect of the mouthwashes on the MIC after exposure to different media. Earlier studies have shown that exogenous protein present in the media can influence the antimicrobial activity of the antibacterial compounds.[20] About 20% addition of horse serum in the broth media has shown to increase the MIC for teicoplanin but not for vancomycin.[33]

In our study, MIC by broth technique showed the highest sensitivity with CHX mouthwash for all the selected bacterial strain followed by the herbal mouthwash, but the difference was not statistically significant. However, when agar was used to test the MIC the herbal mouthwash had a significantly lesser effect as compared to CHX against S. mutans, S. sanguinis, A. actinomycetemcomitans, and F. nucleatum. For P. gingivalis and P. aeruginosa, CHX was more effective than the herbal mouthwash, but the difference was not significant. These findings reinforce the earlier findings that variation in the media can affect the MIC values of a compound and that MIC values are method dependent.[20] It may be that the constituents of the agar media could have influenced some of the antimicrobial properties of the herbal mouthwash. The presence of exogenous proteins in the media or the ability of media components to reduce the antimicrobial activity or the biding of the mouthwash components to protein in the media can influence the antimicrobial efficacy.[34]

Dental biofilm is a polymicrobial heterogenic community which provides several advantages to the microorganism to thrive with help of metabolic and phenotypic flexibility.[35] Certain bacteria undergo genetic transformation by acquiring select genes in their genomes which render them as new phenotype in the biofilm community.[36] In such situations, in vitro testing of antimicrobial using standard strains may not suffice and cannot be extrapolated to in vivo conditions. Considering this, we employed ex vivo antimicrobial testing of dental plaque samples from an oral cavity of humans similar to the studies in the past.[213738] The dental plaque obtained represents the polymicrobial community similar to the dental plaque in clinical samples. The entire spectrum of cultivable polymicrobial species from the plaque samples was tested against the CHX and herbal mouthwash similar to the previous study by Srinivasan et al.[21] Both the mouthwashes demonstrated effectiveness without any significant difference. The results of our study were similar to the findings of Bhat et al. who compared the efficacy of CHX and the herbal mouthwash on the supragingival dental plaque which was initially cultured on blood agar plate.[6] In another study, Mahajan et al. evaluated the efficacy of CHX, herbal mouthwash, pomegranate extract, tulsi, clove, and neem on supragingival plaque.[7] Their findings were similar to the present study with no significant difference between CHX and the herbal mouthwash, in their abilities to inhibit bacteria from dental plaque. It has been considered that the removal of plaque during the sampling process disturbs the three-dimensional structures of the plaque which can alter the quantification of antimicrobial effectiveness.[37] In this aspect, the ex vivo technique employed by us involved minimal manipulation of the dental plaque before the antimicrobial testing of the mouthwash, which is different from previous studies involving alteration of plaque by initial culture or by utilizing selective species from the plaque.[67]

CONCLUSION

In the present study, CHX showed higher levels of antimicrobial action against the select bacterial species. However, the herbal mouthwash too was effective in these bacterial species in both in vitro and ex vivo testing. The results reinforce the earlier findings that the in vitro testing is sensitive to methods and due diligence is needed when extrapolating the data for further use with in vivo studies. The results of the present study do confirm with the earlier studies that the herbal mouthwash can be used as an adjunct to oral mechanical cleaning.[67] However, long-term use and in vivo effectiveness against the periopathogens need to be tested in well-planned clinical trials.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.