Clinical Assessment of a Pharmaceutical Polyphenol Fruit Extract Gel to Inhibit Dental Plaque

Oral bacteria (e.g. Porphyromonas gingivalis) are associated and potentially co-causative in several medical conditions including Alzheimer’s disease and increased cardiovascular risk. Naturally occurring polyphenols are a devel-oping class of antimicrobial pharmaceutical agents. The use of plant-based polyphenol compounds is an attractive area of medical and dental research due to their high safety factor from their presence in vegetables and fruits. This preliminary study evaluated polyphenols found in berries to reduce bacterial dental plaque accumulation. The study was an in vivo single blind randomized controlled trial of seven subjects with a novel tooth gel using validated measurements. Results showed a significant reduction in plaque accumulation (p = 0.005) and a non-significant trend to reduce gingivitis (p = 0.08). The bioactive gel showed no local or systemic adverse effects and was well tolerated by subjects.


Introduction
The oral cavity contains an impressive and diverse array of microorganisms of bacteria, viruses, and fungal elements. In addition, the human dietary intake may contain multiple environmental toxins from their incorporation into the food chain. Direct breathing of polluted air from smog and industrial chemical exposures adds to an altered oral microbiome. While most oral bacteria are Pharmacology & Pharmacy commensal, one anaerobic type is Porphyromonas gingivalis (P. gingivalis) and forms part of the "red complex" that is responsible for destructive periodontal disease. There is an accumulating body of evidence from animal models and human cadaver studies that P. gingivalis is a pathogen of interest in the causation of Alzheimer's disease. This bacterium secretes proteases that cleave amyloid and tau causing the formation of β-amyloid proteins and neurofibrillary tangles [1]. P. gingivalis is an opportunistic bacteria that inhabit periodontal pockets. The pockets are initially established by local inflammation and calcification of supragingival dental plaque. The reduction of dental plaque would be a logical and feasible first step approach to prevent habitation of P. gingivalis.
From a dental perspective, there have been extensive epidemiological, clinical and bacteriological studies that have long demonstrated the direct link between the accumulation of dental plaque, gingivitis, periodontitis and caries [2] [3] [4] [5]. Current global oral health techniques of plaque removal employ tooth brushing with a dentifrice and the use of interdental devices including dental floss, toothpicks and interdental brushes [6]. A key research goal for clinical translation has been to assess the nature of dental plaque for disruption of the bacterial biofilm via chemical means [2]. To achieve this disruptive mechanism many dentifrices now contain synthetic pharmaceutical agents such as chlorhexidine, metal fluoride complexes (stannous fluoride) and triclosan. These common chemical additives have been shown to reduce plaque accumulation with chlorhexidine being the most effective [6] [7]. Current estimates suggest that less than one-third of the population of developed countries can be expected to practice adequate mechanical plaque removal [8]. In developed countries, there is an increased awareness for the aging population to have a decline in oral health and increased caries due to the physical limitations of personal oral cleansing habits. Therefore, it can be argued that supplementation of oral hygiene practices with a chemotherapeutic agent would be beneficial in limiting the oral disease burden to hard and soft dental tissues [7].
Chlorhexidine whilst effective at plaque control has unwanted side effects of the staining of the teeth, propensity to calculus formation and altered taste [9] [10] [11].
Increasingly, herbal compounds have been studied for their efficacy in inhibiting the formation of dental plaque by their antibacterial effect on periopathogenic and cariogenic bacteria [12]. Herbal compounds have been shown to have antimicrobial properties, increase salivary pH buffering capacity, reduce inflammation and assist in wound healing [13]. Berries are a particular area of interest within the herbal research domain as they have numerous bioactives termed polyphenols that have demonstrated anti-inflammatory activities. Studies of these naturally occurring polyphenols have found these chemicals exert an inhibitory effect on microorganisms commonly found in dental plaque that is a precursor to periodontitis, periodontal pocket formation and dentinal caries [14] [15].
Quercetin is one such polyphenol that has an inhibitory effect on periodontal  [16]. Recently raspberry leaf, containing quercetin and kaempferol polyphenols, has been shown to significantly reduce pain and reverse disease progression in the mucosal autoimmune disease of oral lichen planus in vivo [17].
The current aim of this pilot study was to assess the efficacy of a novel herbal topical gel formulation comprising of a concentrated extract obtained from several professional herbal grade berry sources that contain quercetin. Herbal extracts containing quercetin include raspberry leaf, juniper berries and blueberry fruit extract. Specifically, the study was to quantify the accumulation of dental plaque and its corresponding gingival health in vivo using the herbal extract in a tooth gel. An additional aim was to evaluate if the test polyphenol formulation caused any unwanted oral esthetic changes similar to the detrimental cosmetic consequences of chlorhexidine that have significant tooth enamel staining characteristics. A final aim was to evaluate any adverse local or systemic effects.

Materials
The concentrated polyphenol berry extract was prepared in a suitable, easy to dispense gel formulation to replicate commercial toothpaste gels used by the

Data Measurements
The study was conducted as a single blinded, placebo controlled, randomized trial. Prior to the commencement of the study each subject had all teeth undergo cleaning and prophylaxis and confirmed by erythrosine plaque disclosing tablet (CareDent, Sydney, Australia). Clinically validated measurements and photographic images were recorded at day 0 (baseline) and at day 5 (termination of the study). Photographic images were used to visually confirm the plaque accumulation in subjects using the erythrosine plaque disclosing method at baseline and day 5. Images were captured with a high resolution digital Canon SLR 550D camera with flash using intraoral mirrors.
For each gel application the subjects were to rinse with water and then apply the gel formulation to the buccal, mesial and distal surfaces of the posterior teeth

Subjects
The study recruited seven volunteers (three female and four males) ranging from 26 to 62 years (mean 42.9, SD 16.6) from a private dental clinic setting. Subjects were in good oral health with no evidence of dental pathology such as enamel decalcification, active dental caries, gingival inflammation or periodontal disease. The inclusion criteria specified eligible participants having no more than one missing tooth per side. Participants with crowned teeth were included in the study because the Silness and Löe PI measures plaque at the gingival one-third

Statistics
Statistical methods employed student's paired t-test with a level of significance p < 0.05.

Results
No

Discussion
This study showed very promising results by day 5 to reduce plaque from the polyphenol gel. Plaque is the major etiological factor of dental caries, gingivitis         [27] [28]. However, one limitation of the PI and GBI in its current form as used in this study was that it does not correct for missing teeth that resulted in a reduction of three reading points for the PI and two reading points for the GBI for Herbal medicine including dental use of plant extracts has cultural and economic significance in many third world countries. First-world countries are now recognizing the benefits of researching natural compounds containing polyphenol bioactive compounds. Several of these compounds such as curcumin derived from turmeric have anti-cancer properties and are undergoing intense scrutiny in hospital cancer research trials. Quercetin is also receiving considerable interest as it has demonstrated multimodal inhibitory effects in several key pathways in cancer progression [29]. This was tested in the oral environment where the quercetin-containing herb raspberry leaf reversed disease progression in the precancerous lesions of oral lichen planus [16]. It is probable that further research will identify other dental applications of herbal and natural compounds.
From a dental health management perspective, increased plaque particularly on exposed root surfaces can trigger rapid and widespread tooth decay. This area is specifically vulnerable as it leads to problematic restorative dentistry where root caries mechanically undermines the functional load-bearing capacity of the tooth with a resulting crown fracture. In addition, pulpal tissue is quickly involved from root caries leading to non-vitality and periapical infection. Several "at-risk" groups of the population are vulnerable to rampant caries and its devastating outcomes, for example, aged care residents and the elderly have declining dexterity and mental aptitude to perform adequate oral health care. This is exacerbated by the multiple medications used in aged care where the side effect is a reduction in salivary flow rate and its anti-cariogenic pH buffering capacity. Other patients who have received head and neck radiotherapy for cancer management suffer a similar dental crisis [30]. Maintaining optimal oral health with polyphenols would be helpful for bis-phosphonate patients where dental extractions can cause major medical complications of infection, osteomyelitis, osteonecrosis and life-threatening cellulitis.
The inhibition of dental plaque can delay or prevent periodontal pocket formation and potentially the subsequent presence and pathogenic disease states attributed to P. gingivalis. This bacterium is a strong risk factor for Alzheimer's disease (AD) that now affects over 24 million people in the world. Over the last 20 years there have 160 drugs trialed to treat AD but all have failed USA Food and Drug Administration clearance due to safety issues or lack of efficacy. Prevention of AD by P. gingivalis inhibition is a logical step forward, particularly if safe and efficacious naturally sourced antimicrobial compounds are available.
This preliminary investigation used limited subject numbers but was conducted in a randomized, placebo-controlled manner. The early results are highly encouraging to perform a follow-up trial of a larger number of subjects focusing on a group with high plaque accumulation and caries incidence. An advantage of conducting research on the oral cavity is the ability to use blinded place-