The currently used chemical mouth rinse, chlorhexidine, was prescribed widely among dental practitioners for its ability to kill bacteria. However, chlorhexidine prolonged use is limited due to its adverse events such as extrinsic tooth staining, taste disturbance, effects on the oral mucosa such as soreness, irritation, mild desquamation, mucosal ulceration, general burning sensation and calculus formation. Thus, an alternative natural active agent is needed to produce mouth rinse with less side effect but offer similar disease prevention effect. This comprehensive four-phase research initiative aims to advance the development and evaluation of a stingless bee honey mouth rinse, particularly focusing on improved formulation and tested in vitro and clinically. In Phase I and II, the mouth rinse development involve the improved formulation by xylitol addition in the formulation with physicochemical assessment such as shelf-life followed by antimicrobial activities against Staphylococcus aureus and cytotoxicity evaluation against oral keratinocytes. Phase III focuses on clinical trial in a pilot study that will be performed on twenty volunteers divided to two groups; control and test group who will be using mouth rinse as adjunctive periodontal therapy agent (TRL 5). Comparative analyses will assess the adjunctive effect of this stingless bee honey mouth rinse in periodontal therapy. A commercially packaged mouth rinse will be designed and produced in collaboration with Bayu Gagah Marketing and will be given to the public as free samples to obtain feedback and validate its further use in the health industry.

INTRODUCTION

This innovative project is aimed to produce natural-based mouth rinse that are safe for users with no side effects, as an alternative to current chemical mouth rinse.

The currently used chemical mouth rinse, chlorhexidine, was prescribed widely among dental practitioners for its ability to kill bacteria. However, our mouth was designed naturally to have normal microbiota living in symbiosis and the change of this normal equilibrium resulted in disease such as reduction in nitrate-reducing bacteria responsible for nitric oxide production. Nitric oxide is one of the molecules involved in blood pressure regulation. Besides this, chlorhexidine was associated with adverse events such as extrinsic tooth staining, taste disturbance, effects on the oral mucosa such as soreness, irritation, mild desquamation, mucosal ulceration, general burning sensation and calculus formation.

On the other hand, there was growing evidence on medicinal properties of stingless bee honey (SBH). SBH has a high concentration of trehalulose, antioxidant, flavonoids, phenolic acids, hydroxyl and aromatics. In vitro, SBH has antioxidant activity that are up to 45% higher than those of Apis mellifera honey and have broad-spectrum antibacterial action against Gram-positive and Gram-negative bacteria as well as fungus. Several studies revealed wound healing and anti-inflammatory capabilities of SBH making it as a promising active agent in mouth rinse formulation to combat oral disease.

General objective:

To develop an anti-inflammatory and natural based mouth rinse from stingless bee honey.

Specific objective:

1. To develop a mouth rinse from mixture of stingless bee honey, xylitol and mint flavor that are stable physicochemically.

2. To investigate the antimicrobial effect and cytotoxicity of SBH mouth rinse.

3. To evaluate the prototype clinical effectiveness in a clinical trial.

   Methodology

   Phase I and II involve laboratory study (Objective 1 and 2). Phase III involve clinical trial (Objective 3).

   i. Study design

   - This is a randomized, single-blinded and placebo-controlled clinical pilot study that will be conducted at Faculty of Dentistry, Universiti Sains Islam Malaysia following ethical approval.

   ii. Participants

   - Sample size was estimated from Sharma et al., 2022 to have 20 samples per arm. Data was analysed using G Power version 3 statistical software. The significance value was set at α value 0.05 with 95% power of the study. From the previous journal, the calculated effect size was 2.2. The calculated sample size was 20 per arm after including a 20% dropout rate.

   - Sixty (twenty per group) participants attending dental clinic at the Faculty of Dentistry, USIM will be invited to participate in the study based on inclusion criteria:

   • 18 to 60 years old patients

   • BPE code 2 and/or below

   • Presence of at least 20 natural teeth

   - These are the exclusion criteria:

   • Diabetes mellitus
   • Smoking
   • Mucosal lesion
   • Allergic to honey
   • Physical or mental disabilities

   iii. Randomization Participants will be randomly allocated to Group A, Group B or Group C based on the order of recruitment. Randomisation will be performed using Microsoft Excel softwate (Microsoft®, USA). Predetermined recruitment numbers (1-30) will be randomly allocated into Groups A, B or C using computer-generated random numbers. Participants' assignment and enrolment in the intervention will be performed by a research assistant.

   iv. Blinding This is a single-blinded study in which each participant and clinicians who will perform data collection will be blinded to the treatment assignment.

   v. Mouth rinse preparation Three different mouth rinse formulations will be prepared;

   • Stingless bee honey (SBH) mouth rinse containing stingless bee honey (Bayu Kelulut®; Bayu Gagah Marketing, Kulim, Malaysia) diluted to a concentration of 20%, xylitol and mint flavor,

   • 0.12% chlorhexidine (CHX) (Oradex, Shah Alam, Malaysia) as gold standard

   • 0.9% saline solution as control.

   vi. Intervention

   Before start of study, scaling and polishing will be performed to remove all calculus and plaque. All participants will be given Colgate® regular toothpaste and a Colgate® soft bristled toothbrush to brush twice daily for washout period (two weeks) and to continue the standardised products throughout the course of study. Standardised brushing technique will be demonstrated by the clinician to ensure they understood the brushing instructions. Participants will be instructed not to use any other oral hygiene aid and abstain from any other mouth rinse than those provided for the study.

   After two weeks of washout period, plaque and bleeding scores will be taken by three calibrated clinicians. After recording the plaque and bleeding scores, scaling and prophylaxis will be performed. A bottle of mouth rinse will be prescribed to the patients, in a sealed bottle.

   - Participants will be instructed to rinse 15ml of mouth rinse twice daily (before breakfast and after dinner) for two minutes, for one month. Group A will receive stingless bee honey mouthrinse, Group B will receive chlorhexidine (Oradex, Malaysia) and Group C will receive 0.9% normal saline solution in a sealed and coded bottle.

   After one month, plaque and bleeding scores will be measured again. Patient's acceptance in terms of taste, halitosis and burning sensation will be measured using visual analogue scale.

   vii. Outcome measures

   - Periodontal parameters such as plaque score and bleeding score will be recorded pre- and post-intervention by calibrated examiners.

   Phase IV: Pre-commercialisation.

Inclusion Criteria:

• 18 to 60 years old patients
• BPE code 2 and/or below
• Presence of at least 20 natural teeth

Exclusion Criteria:

• Diabetes mellitus
• Smoking
• Mucosal lesion
• Allergic to honey
• Physical or mental disabilities

Mouth rinse will be given in a sealed and coded bottle. The assignment for type of mouth rinse will be peeformed by a research assistant who does not perform the treatment and is not responsible in assessing the outcome.