In vitro study comparison of purple leaf extract as denture cleanser at different concentration towards S. mutans growth on flexible denture

Open a cess: www.balimedicaljournal.org and ojs.unud.ac.id/index.php/bmj In vitro study comparison of purple leaf extract as denture cleanser at different concentration towards S. mutans growth on flexible denture


INTRODUCTION
Tooth loss is a common thing in society due to accidents, illnesses, or natural aging processes. If not treated immediately, it will produce anatomical, physiological, and functional changes that can even lead to psychological trauma. The use of dentures can help restore phonetic or speech functions, mastication, aesthetic or beauty functions, and preserve oral tissues. The polyamide thermoplastic resin (nylon) is a flexible denture base with distinctive physical and aesthetic properties. This denture has excellent flexibility and stability and can be made thinner with specific thicknesses. So, it is flexible, lightweight, and not easily broken. 1 It is essential to prevent denture damage and minimize the formation of bacterial colonies or candida on the denture's surface. They can cause dental and oral problems such as halitosis, caries, stomatitis, periodontal disease, inflammation of the palatal mucosa, and denture stomatitis. 2 The causes of denture stomatitis include C. albicans, bacterial infections, allergies, psychological factors, lack of denture hygiene, salivary flow, and nutrients. 3 Colonization of bacteria and fungi causes patients' salivary pH to be more acidic and a trigger factor of denture stomatitis. The acidic condition is due to carbohydrate fermentation by C. albicans and S. mutans. 4, 5 Denture cleansing can be done mechanically with a soft toothbrush or by chemical means using a disinfectant. Denture cleansers should preferably be bactericidal and fungicidal, easy to use, and compatible with all denture materials. Ideal denture cleansers should have the following characteristics: nontoxic, easily removed, leaving no irritant, irritating, or dissolving organic and inorganic materials contained in the denture, does not damage denture materials, is stable in storage, and is preferable of a bactericide and fungicide. 6 The use of plant materials as an alternative ingredient of denture cleanser is expected to provide benefits.
The material is easy to obtain, cheap, and relatively safer than the chemical-based. 7 Purple leaf or Graptophylum pictum is one of the plants that are often used as traditional medicine, such as hemorrhoid drug. 8 Chemical content of purple leaves consists of alkaloids, flavonoids, tannins, alkaloids, steroids, and saponins. Flavonoids are the largest group of phenol compounds, simple monocyclic phenols, phenylpropanoids, and phenolic quinones. 9 The general properties of phenol can increase cell permeability and precipitate proteins. 10 Flavonoids can inhibit microorganisms because of their ability to form complex compounds with proteins and are antiviral. 9 Research in medicine shows that purple leaves are useful for healing hemorrhoids, anti-bacterial, analgesic, and anti-inflammatory. Research conducted in dentistry shows that 40% purple leaf extract has the highest anti-fungal power to the growth of C. albicans on denture acrylic resin plate. 3 So, it is essential to do research that aims to compare purple leaf extract's effectiveness as denture cleanser at concentration: 1.25%; 2.5%; 10%, and 40%, towards S. mutans growth on flexible denture plates.

Methods
This study was using laboratory experimental research design in-vitro with posttest-only control group design. The local ethic commission has approved this study. This study compared the effectiveness of purple leaf extract at different concentrations: 1.25%; 2.5%; 10% and 40%, fittident® and aquadest as denture cleanser towards the growth of S. mutans on flexible denture plates, all group repeated three times.

Bacteria and reagents
This study used S. mutans ATCC 35668 from the microbiology laboratory, Faculty of Medicine, Universitas Udayana. The S. mutans has incubated for 24 hours at 37 0 C with suspension according to 0.5 Mc. Farland standard (3x10 6 CFU/ml), on the surface of the thermoplastic nylon plate size 10x10x2 mm. Mueller Hinton agar was used for the disc diffusion method.

Preparation of purple leaves extract
Graptophyllum Pictum or purple leaves is a traditional medicine act as anti-bacterial, analgesic, and anti-inflammatory for healing hemorrhoids. The extracting process consists of extracting and evaporating processes. Purple leaves as much as 100 grams are dried and ground and then extracted using 70% ethanol for 3 hours. The liquid extract was concentrated with a Vacuum Rotary Evaporator to obtain 100% purple leaf extract. The extract is then weighed with an analytical balance. We diluted 1.25 grams of purple leaf extract plus distilled water up to 100 ml to make 1.25% extract concentration. Making a solution of purple leaf extract of 2.5%, 10% and 40% was carried out the same method with the weight of purple leaf extract 2.5 g, 10 g, and 40 g.

Preparation of Denture Cleansers
The flexible denture plates were sterilized using an autoclave with temperature 121 o C for 15 minutes. Then, it immersed in sterile saliva for 1 hour, then rinsed with PBS and inserted into test tube containing TSB (trypticase soy broth) media containing S. mutans suspension, then incubated for 24 hours at 37 o C. After that, the plates were inserted into a closed test tube, each containing a purple leaf extract of 1.25%, 2.5%, 10%, and 40% concentrations, and sterile aquades (control). The immersion period used is 5 minutes. Furthermore, the plate was rinsed with PBS and then inserted into test tube containing TSB media. All test tubes were vibrated on vortex for 1 minute to release S. mutans attached to plate. S. mutants were grown on Nutrient Broth -MHB agar then incubated for 24 hours at 37 o C, then counting bacterial colonies using colony counter.

Statistical Analysis
Data were analyzed using the One-Way Anova test with 95% confidence level (α = 0,05%). LSD (Least Significant Different) was then tested to determine the significance of differences in the treatment group.

RESULT
The mean of S. mutans colonies on flexible denture base plate in each Graptophylum pictum extract concentration group can be seen in Table 1. It was showed that 2,5% Graptophyllum pictum extract had the highest mean of colonies growth (43.3333), while the lowest mean of colonies growth was in 10% concentration (21.0000).
The result of the One-Way ANOVA test ( Table  2) showed that there was a significant difference (p <0.05) of bacterial colony count between some extract concentration groups. Table 3 showed that the LSD-multi comparisons test results on the Post Hoc analysis showed a significant difference between the extract concentrations of 1.25% to 10% and the 2.5% extract to 10% and 40%. All extract concentrations have significant differences in suppressing bacterial growth when compared to the control group. Extracts with 10% concentration

ORIGINAL ARTICLE
were considered to have the most optimum effectiveness. The extracts at these concentrations were significantly different with 1.25% and 2.5% concentration but did not differ significantly with the extracts with 40% concentrations.
The qualitative phytochemical analysis (Table  4) showed that the Graptophylum pictum plant extract contains a mixture of phytochemicals such as Flavonoid, Tannin and Saponin. In vitro, antioxidant activity of Graptophylum pictum extract were determined using spectrophotometric methods. In Table 5 the Graptophylum pictum extract showed flavonoid content (4340,30 mg/100 QE wb), tannin content (1104,62 mg/100g TAE wb) and total phenolic content (1082,25 mg/100g GAE wb). However, the Graptophylum pictum exhibited very weak antioxidant capacities (3038,41 mg/L GAEAC) with the IC 50% value is 212,77 mg/ml.   . Noted: * The mean difference is significant at the 0.05 level work using protein denaturation, thereby increasing the permeability of cell membranes. Denaturation of proteins causes a disruption in cell formation that alters the composition of protein components. The function of the disrupted cell membrane may cause increased cell permeability, resulting in cellular damage. 3 Denaturation of proteins can damage cells irreversibly and irreparably. Tanin is also thought to have the ability to inhibit growth or kill S. mutans by collapsing and precipitating proteins from the solution by forming an insoluble compound. 13 Based on this, purple leaf extract's effectiveness is lower in suppressing bacterial growth at concentrations below 10%, possibly due to lower levels of flavonoids and tannins. Still, at concentrations of 40%, their effectiveness has no significant difference with a 10% concentration of the possibility because the levels of flavonoids and tannins have reached optimal ability. However, the Graptophylum pictum exhibited very weak antioxidant capacities, with the IC 50% value is 212,77 mg/ml.

CONCLUSION
All extract concentrations effectively suppress the growth of bacteria, but which has the optimum effect is an extract with a concentration of 10%.

DISCUSSION
In the early formation of salivary-pellicles, grampositive bacteria, Streptococcus sp. became the first bacteria to attach to the denture base and form a colony. S. mutans, which have extracellular polysaccharide (PSE) that other bacteria do not own, the substrate becomes the path for bacteria and other fungi attached to the denture base. The colonization of bacteria and fungi will proliferate into plaque leading to denture stomatitis. 11 The denture base material also affects the ability of S. mutans bacteria attachment on the denture base. Whereas, based on the results of the research on removable denture base ingredients shows that the number of S. mutans bacteria on the nylon thermoplastics plate are less than the heat-cured acrylic resins. This study showed that the highest colonies' growth was in 2,5% Graptophyllum pictum extract concentration, while the lowest colonies' growth average was in 10% concentration. This study showed that the Purple leaf or Graptophylum pictum plant extract contains a mixture of phytochemicals as Flavonoid, Tannin, and Saponin. Purple leaf extract contains antibacterial flavonoids, capable of inhibiting the growth of S. mutans by forming complex compounds against extracellular proteins that interfere with the integrity of bacterial cell membranes. 12 Flavonoids