Effect of 0.2% Betel Nut Mouth Rinse on Flow Rate, pH, Bacterial Parameters of Saliva: A Clinical Trial

Introduction

Dental caries is the most prevalent dental disease affecting humans. Although the prevalence of dental caries is significantly reduced, it is still considered a major problem. The etiology and pathogenesis of dental caries is said to be multifactorial.¹

Saliva is a complex body fluid and is essential in maintaining the oral health.² Saliva serves to facilitate This work is licensed under a Creative Commons Attribution-NonCommercial 4.0. the inherent symbiosis existing between the indigenous oral microbiota and their host, exerting an influence on the regional pH via its inherent damping capabilities, and thereby lending support to the development of resilient and heterogeneous microbial assemblages.³ Saliva assumes a significant function in the process of remineralization by bestowing dental tissues with calcium, phosphate, and fluoride.⁴

Salivary flow is an important factor since it favours the cleansing of bacterial substrate, protects the oral surfaces and helps in controlling the development of caries. A reduced salivary flow results in a cascade of events where the biofilm accumulates and matures, resulting in acidification of the micro environment and the selection of acidogenic bacteria. Salivary flow rate may be considered as the most optimal clinical indicator of the protective qualities of saliva, given that other salivary parameters such as pH and buffer capacity are contingent on it.⁵

Betel nut/Areca nut (Areca catechu) of the family Arecaceae, claims to possess anti-ulcer, anti-aging, hypo-lipidemic, anti-diabetic properties and claims to enhance digestion and metabolism.⁶ Betel nut having a sialogogue effect and anti-bacterial property against several bacteria helps in reducing dental caries.⁷ However, chewing of betel nut is attributed to many adverse effects.⁸ Therefore in the present study, betel nut has been used as an oral rinse solution.

It has been reported that mouthwashes are one of the effective and safest delivery systems. These mouthwashes have the ability to inhibit the adhesion of bacteria, colonization, and metabolic activity which eventually affects the growth of bacteria.⁹ Many studies have been conducted based on habitual chewing and ingestion of areca nut, but use of betel nut as a mouth rinse has not been explored. Hence, the purpose of this study was to evaluate and compare the effect on salivary parameters before and after the use of betel nut mouth rinse.

Materials and Methods

Study Setting: KVG Dental College and Hospital, Kurunjibagh, Sullia, Dakshina Kannada.

Study Design: A clinical trial

Study source: Betel nut powder was obtained from raw betel nuts procured from local venders in Sullia taluk.

Preparation of mouth rinse

A 0.2% Betel nut mouth rinse was freshly prepared by dissolving two grams of Betel nut powder in 100 mL of double distilled water under strict aseptic laboratory conditions (Figure 1).

Procedure

Informed consent from the participating students was obtained before the study. This clinical trial was registered with CTRI bearing the number REF/ 2023/06/069367. This study was approved by the Institutional ethics committee, KVGDCH, bearing the reference number IECKVGDCH/UG/01/2021-2022.

The research was undertaken for a period of 30 days, during which the principal investigator carried out various steps of the study protocol. In the first visit, the investigator provided a brief introduction about the study, necessary demographic data was collected from the participants and baseline data was obtained, which included salivary pH, salivary flow rate, and total bacterial count. Baseline salivary sample from each subject was collected and evaluated on day 1. The subjects were instructed not to consume anything, except water, two hours prior to the collection of saliva. The participants were asked to hold their saliva in the mouth for one minute. Subsequently, the participants were instructed to expectorate saliva into sterile eppendorf tubes for 1-5 minutes. Collected samples were then sent to laboratory for baseline data evaluation of salivary pH, salivary flow rate, and total bacterial count.

Subsequently, a volume of five milliliters of the experimental solution was dispensed into sterile, disposable receptacles and administered to the participants. The subjects were instructed to perform a daily oral rinse for a duration of one minute for 15 days. At the end of 15 days, the post rinse salivary samples were collected as described previously for baseline. The salivary specimens were then expeditiously transferred to a vial that was filled with a transport medium and sent for analysis. Subsequently, use of the study product was ceased after 15 days. After a washout period of 15 days, final evaluation was done (Figure 2).

Statistical Analysis

The statistical analysis was done using SPSS software version 27. Descriptive analysis was performed for proportion of gender using Chi Square test and mean age. Pair wise comparison of pH, salivary flow rate and bacterial count between the two time periods was done utilizing the paired t-test. P value <0.05 was considered statistically significant.

Results

The study included 30 healthy adults aged between 18-25 years (CTRI No. REF/2023/06/069367). Data was collected on specially designed Proforma containing four parts. Part A included socio-demographic details. Part B recorded clinical and microbiological parameters at baseline including pH, flow rate and bacterial colony counts. Part C and Part D included details of the intervention and recorded clinical and microbiological parameters at the follow up.

The mean age of the patient was 22.9 years, and 80% of the study subjects were males. The qualitative analysis of saliva before and after the study showed significant difference in pH while no significant difference was found in flow rates. Significant reduction in bacterial counts was observed after the mouth rinse was used for the stipulated period of 15 days (Table 1).

The results obtained in our study indicated that using 0.2% betel nut as a mouth rinse has potential to reduce the oral bacterial flora significantly. Although there was a difference in flow rate before and after the intervention, the values obtained were not significant enough to attribute it to mouth rinsing.

Discussion

Dental caries is a multifactorial disease caused by the role of major interrelated factors, including host (saliva and teeth), agent (microorganism), substrate or diet containing sugar and time. S. mutans, one of the major microorganisms that inhabit the oral cavity forms dental plaque by synthesizing water insoluble glucan from sucrose.¹⁰ The present study consisted of 30 healthy adults of age ranging between 18-25 years. Data was collected on specially designed Proforma wherein Part A included socio-demographic details, while Part B recorded clinical and microbiological parameters including salivary pH, salivary flow rate and total bacterial count at baseline. Part C included details of the intervention with 0.2% Betel nut mouth rinse. Part D recorded clinical and microbiological parameters following the intervention.

Betel nut/ areca nut of the family Arecaceae has antioxidant, antibacterial, anti-inflammatory and analgesic, anti-depressant, anti-allergic properties. The main components of areca nut are polyphenols including flavonoids and tannins (up to 29.8%), fat polysaccharides, and proteins. Besides these, the nuts contain alkaloids arecoline (0.1-0.7%) and others in trace amounts, such as arecadine, guvacoline, and guvacine.

De Miranda and colleagues discovered that the hydrolysable tannic acid present in areca nut is responsible for its antibacterial effects. They also observed that the controlled intraoral exposure to the nut can effectively inhibit bacterial growth within the oral cavity,¹¹ since prolonged exposure of betel nut is attributed to many adverse effects.⁸ Arecoline, the main areca alkaloid of the betel nut is reported to have cytotoxic, genotoxic and mutagenic effects, strongly correlating to the incidence of oral submucosal fibrosis, leukoplakia and oral cancer.⁶ Therefore, in the present study, betel nut in the form of a mouth rinse was used to ensure controlled intraoral exposure.

The microbiological parameter in our study in terms of bacterial count showed significant reduction from 93500±16355 CFU to 72333±31037 CFU following intervention with mouth rinse, which is in accordance with Keshava Bhat Sarpangala et al. who reported that procyanidines of areca nut are responsible for the antibacterial properties against the primary cariogenic bacteria, Streptococcus mutans.¹²

In the present study, significant difference was seen in terms of pH change, pre and post the usage of mouth rinse ( i.e. 5.16±0.59 to 5.52±0.65) that can be explained according to the study by Irene et al., who stated that the process of chewing itself stimulates copious amounts of saliva in the mouth and the added slaked lime in the betel quid increases pH in the oral cavity.¹⁰ The main limitation of this study was that there was no control group which could have improved the level of evidence. Secondly, a convenient sample was used to select the study participants instead of a randomized sample and also the smaller sample size is a drawback for the study. This should be addressed in future research. Nevertheless, the strength of the study was that, this was the first study of its kind where 0.2% Betel nut extract was used as a daily mouth rinsing agent and only young adults were chosen as the study subjects. There is a definite scope for follow up in the study to assess the long term impact of mouth rinsing.

Arecoline hydrobromide, is a stronger stimulant to the salivary glands and a more energetic laxative. Even though several studies reported increased salivary flow rate with the use of betel nut, our study did not show significant difference in terms of flow rate, which could be due to the fact that betel nut was used in the form of a mouth rinse rather than a chewable form.

Betel nut extract could be used as a mouth rinse to reduce the concentration of oral bacteria. Karnataka being the largest producer of betel nut contributing to approximately 47% of India’s annual output, also taking leverage of widespread acceptance by local communities, areca nut mouth rinse should be explored and researched through further studies for its potential overall oral health benefits. Limitations of the study includes small sample size and also the taste of the mouth-rinse can be further modified like other flavored, commercially available mouth-rinses and can be tested on larger population.

Conclusion

The results of this study conclusively demonstrated that 0.2% Betel nut extract when used as a mouth rinse reduced the concentration of oral bacteria and hence can contribute towards cultivation of a constructive influence on the entirety of an individual's oral health well-being. Funding Source Funded by RGUHS undergraduate research grant 2021- 22

Conflict of Interests

Nil