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 Table of Contents  
ORIGINAL RESEARCH
Year : 2021  |  Volume : 13  |  Issue : 3  |  Page : 281-287

Efficacy of red betel leaf (Piper crocatum) against Candida albicans over acrylic denture surface: An in vitro study


Department of Prosthodontics, Faculty of Dentistry, Padjadjaran University, Bandung, Indonesia

Date of Submission27-Dec-2020
Date of Decision17-Jan-2021
Date of Acceptance03-Mar-2021
Date of Web Publication18-Jun-2021

Correspondence Address:
Ista Meidarlina
DDS, Department of Prosthodontics, Faculty of Dentistry, Padjadjaran University, Jalan Sekeloa Selatan 1, Bandung, Jawa Barat.
Indonesia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jioh.jioh_359_20

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  Abstract 

Aims and Objectives: The aim of this article is to assess the efficacy of red betel leaf against Candida albicans over acrylic denture surface. Materials and Methods: This research used an experimental design. The efficacy of red betel leaf was assessed through minimum inhibitory concentration (MIC) and immersion tests. The results of MIC test using microdilution method were determined using turbidimetric and spectrophotometric methods. The immersion test used 30 sterilized identical heat-cured acrylic resin plates (10×10×2 mm), five each for six groups contaminated by Candida albicans and immersed in different denture cleansers (red betel leaf extract with concentrations of 10%, 20%, 30%, and 40%, alkaline peroxide denture cleanser “fitty dent”® as positive control, and sterile distilled water as negative control). The number of microorganisms was calculated and analyzed using analysis of variance followed by post hoc test. Results: MIC of red betel leaf extract was 2.34% (turbidimetric method) and 0.29% (spectrophotometric method). The immersion test showed that the statistical analysis of the six groups was significant (P<0.01). The more the concentration of red betel leaf extracts (10%, 20%, 30%, and 40%), the lesser the number of colonies of C. albicans (48, 8.6, 4.4, and 4.2 ×103 CFU/mL); meanwhile, the reduction value increased to 62.09%, 93.21%, 96.53%, and 96.68%. There were no significant differences between 30% and 40% red betel leaf extract concentration (P = 0.89). The highest number of colonies was in negative control (126.6 × 103 CFU/mL) and the smallest was in positive control (1.3×103 CFU/mL). Conclusion: Red betel leaf had efficacy against C. albicans over acrylic denture surface at 30% concentration.

Keywords: Candida albicans, Denture Cleanser, Denture Prosthesis, Red Betel Leaf Extract


How to cite this article:
Meidarlina I, Damayanti L, Rikmasari R. Efficacy of red betel leaf (Piper crocatum) against Candida albicans over acrylic denture surface: An in vitro study. J Int Oral Health 2021;13:281-7

How to cite this URL:
Meidarlina I, Damayanti L, Rikmasari R. Efficacy of red betel leaf (Piper crocatum) against Candida albicans over acrylic denture surface: An in vitro study. J Int Oral Health [serial online] 2021 [cited 2021 Jul 30];13:281-7. Available from: https://www.jioh.org/text.asp?2021/13/3/281/318457


  Introduction Top


Denture prosthesis is used as a rehabilitation tool in cases of tooth loss to restore normal stomatognathic function. The material that is widely used as denture base is heat-cured acrylic resin. The anatomic surface is an ideal place for attachment of Candida albicans.[1],[2]Candida albicans is known as an opportunistic microorganism in oral environment that can be pathogen in individuals whose immune system decreases, by producing toxic hydrolytic enzymes and causing denture stomatitis.[3] The presence of C. albicans in the oral cavity of healthy humans with teeth ranges from 20% to 50%,[4] which can increase to 60–100% in individuals who lose teeth and use dentures due to changes in the microbial flora of their oral cavity.[5]

Denture stomatitis can be prevented by adequate denture hygiene.[3],[6],[7] Research by Lee et al.[7] revealed that combined cleaning techniques (mechanical and chemical) were the most effective, but the majority of denture users are elderly with decreased motor function, so the best method for this group is chemical cleaning.[8]

Commercial denture cleansers that are widely available in the market are generally made of synthetic chemicals such as alkaline peroxide, sodium hypochlorite, and chlorhexidine gluconate.[7] Herbal-based denture cleansers have not been widely available in the market, even though many medicinal plants grow around us.[9],[10],[11] This study carried out to assess the efficacy of red betel leaf against C. albicans over acrylic denture surface as a herbal-based denture cleanser.


  Materials and Methods Top


This research used an experimental design. The efficacy of red betel leaf was assessed through the minimum inhibitory concentration (MIC) and immersion tests. The results of MIC test using microdilution method were determined using turbidimetric and spectrophotometric methods. Observations for the turbidimetric method (quantitative analysis based on turbidity measurements) were based on visual appearances. This can lead to bias on results because red betel leaf extract has a dye that can influence the results; therefore, it continued with spectrophotometric calculations. The immersion test used 30 sterilized identical heat-cured acrylic resin plates (10 × 10 × 2 mm), five each for six groups contaminated by C. albicans and immersed in different denture cleansers (red betel leaf extract with concentrations of 10%, 20%, 30%, and 40%, alkaline peroxide denture cleanser “fitty dent”® as positive control, and sterile distilled water as negative control) for 30 min. Determination of the number of samples in this study was done using the Federer formula.[9]Candida albicans colonies were counted by a colony counter in units of CFU/mL and analyzed using analysis of variance (ANOVA) followed by post hoc with independent t-test.

This research was conducted in the integrated laboratory of Padjadjaran University’s Faculty of Dentistry and in the central laboratory of Padjadjaran University, Bandung, Indonesia, from March to September 2020.

Preparation and standardization of C. albicans (ATCC 10231)

Candida albicans derived from American Type Culture Collection [ATCC] 10231 was implanted on the surface of the agar medium by selecting several colonies using a sterile loop wire, incubated at 37°C for 18–24 h. Then it was suspended into a liquid medium, incubated for 18–24 h at 37°C. The turbidity of the C. albicans suspension was standardized to a standard of 0.5 McFarland (1–2×108 CFU/mL) and then diluted with a ratio of 1:20 to become a suspension with a density of 5×105 CFU/mL.

Preparation of red betel leaf extract (Piper crocatum)

Two kilograms of fresh red betel leaves (P. crocatum) picked from red betel plantations in the Garut West Java (coordinates 6º56′49–7º45′00 South latitude and 107º25′8–108º7′30 East longitude, an altitude of 700 m above sea level), Indonesia, are cleaned, chopped, and dried in an oven at 40°C for 4 × 24 h and then mashed into fine powder (simplicia) using a blender (Miyako BL 152 PF AP, Indonesia). The extraction of red betel leaves (P. crocatum) was carried out by the maceration method, which was immersed in a 96% ethanol solution for 1 × 24 h with a 1:4 ratio of ingredients and solvents. The resulting filtrate was filtered with filter paper (Whatman no. 1). The maceration process was repeated for 6 consecutive days. The results of the filtrate from filtering with a filter paper were evaporated by a rotary vacuum evaporator at 40°C, and then the extract was stored in a dark container at 4°C.[12] A total of 10 g of 100% thick red betel leaf extract was dissolved in dimethyl sulfoxide (DMSO) 25% and then diluted with sterile distilled water to obtain concentrations of 10%, 20%, 30%, and 40% with the dilution formula C1.V1=C2.V2 (C1: initial concentration, C2: desired concentration, V1: initial volume, and V2: desired volume).[12]

Preparation of alkaline peroxide denture cleanser “fitty dent”® (positive control)

Dissolve the alkaline peroxide commercial denture cleanser tablet “fitty dent”® (Fittydent Int GMBH A-7423, Pinkafeld, Austria) according to the recommendation on the product packaging. The dissolution process is carried out by placing the effervescent tablet “fitty dent”® (Fittydent Int GMBH A-7423, Pinkafeld, Austria) in 100 mL warm water (55°C) until the tablet looks run out.

Specimen fabrication (preparation of heat-cured acrylic resin plates)

Thirty pieces of modeling wax of the size 10 × 10 × 2 mm were placed into a stainless steel cuvette using a plaster cast. The wax removal procedure is carried out after the plaster cast hardens, and then the mold smeared with separating liquid cold mold seal. The powder and the liquid of heat-cured acrylic resin (QC-20, UK) were mixed according to the manufacturer’s recommendations (polymer: monomer ratio of 3:1 volume) and packed into the mold. A hydraulic press at 1200 psi was used to pack the denture-based resin, subsequently the excess resin was removed, and finally 800 psi was applied for 15 min. Samples were then polymerized by a conventional heat method with metal flasks in a water bath at 70°C for 90 min, followed by 100°C for 30 min. All samples were cooled at room temperature.

Samples were then deflasked, and excess resin was sequentially removed with a low-speed carbide bur. Both the resin surfaces were not finished and polished as a simulation of the anatomical surface of the denture base [Figure 1]. Finally, all samples were immersed in sterile distilled water at room temperature for over 48 h to eliminate any residual monomers. In the last step, the sample was sterilized with an autoclave (Sturdy, SA-3-MA) at 121°C for 20 min.[7],[13]
Figure 1: Acrylic plate

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Contamination of specimens

Assuming the conditions in the mouth, all samples of sterilized acrylic resin plates were immersed in sterile artificial saliva for 1 h. After that, each sample of the heat-cured acrylic resin plate was inserted into a  Petri dish More Details containing C. albicans suspension according to MacFarland standards (1×108 CFU/mL) and incubated for 24 h at 37C.[8],[14]

MIC test

The test of MIC of red betel leaf extract against C. albicans was done using microdilution method. Ninety-six-well microplates (Greiner, Germany) were prepared with the following format: Sabouraud dextrose broth (SDB CM 0147, Oxoid Ltd, UK) media added with red betel leaf extract, SDB media added with DMSO solvent, SDB media added with red betel leaf extract and C. albicans, SDB media added with DMSO solvent and C. albicans. Then, add 100 µL of liquid media (SDB) into the microplate, add 100 µL of red betel leaf extract into the first well of the microplate and then carry out gradual dilutions, and add 10 µL of C. albicans suspension into the microplate. After incubation at 37°C for 16–20 h, the data using turbidimetric method and spectrophotometric method are calculated and processed.

Immersion testing

The immersion test used 30 sterilized identical heat-cured acrylic resin plates without polishing on both the surfaces with a size of 10 × 10 × 2 mm, five each for six groups, contaminated by C. albicans, and immersed in different denture cleansers (red betel leaf extract with concentrations of 10%, 20%, 30%, and 40%, alkaline peroxide denture cleanser “fitty dent”® as positive control, and sterile distilled water as negative control) for 30 min [Figure 2]A and B.
Figure 2: A. Immersing plates in alkaline peroxide and sterilized distilled water. B. Immersing plates in alkaline peroxide and sterilized distilled water

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Tube 1: Five contaminated samples of heat-cured acrylic resin plates were immersed in a sterilized test tube (Pyrex, Corning Inc.) containing alkaline peroxide denture cleanser “fitty dent”® (Fittydent Int GMBH A-7423 Pinkafeld, Austria) for 30 min (positive control).

Tube 2: Five contaminated samples of heat-cured acrylic resin plates were immersed in a sterile test tube (Pyrex, Corning Inc.) containing 40% red betel leaf extract for 30 min.

Tube 3: Contaminated samples of heat-cured acrylic resin plates were immersed in a sterilized test tube (Pyrex, Corning Inc.) containing 30% red betel leaf extract for 30 min.

Tube 4: Contaminated samples of heat-cured acrylic resin plates were immersed in a sterilized test tube (Pyrex, Corning Inc.) containing 20% red betel leaf extract for 30 min.

Tube 5: Contaminated samples of heat-cured acrylic resin plates were immersed in a sterilized test tube (Pyrex, Corning Inc.) containing 10% red betel leaf extract for 30 min.

Tube 6: Contaminated samples of heat-cured acrylic resin plates were immersed in a sterilized test tube containing sterilized distillated water (negative control) for 30 min.

The test plates were removed from the test tube by placing them in 10 mL of 0.9% NaCl and vibrated with a vortex (Eppendorf, Germany) for 30–60 s (dilution of 100) to release C. albicans adhered to the acrylic resin plate.[8],[14] The next procedure was multilevel dilutions 101, 102, and 103. As much as 1 mL of each dilution was cultured on Sabouraud dextrose agar (CM 0041, Oxoid Ltd, UK) and then incubated for 48 h at 37°C.[8],[14]Candida albicans colonies were counted by a colony counter in units of CFU/mL.

Statistical analysis

Microsoft Excel 2016 was used for data entry and SPSS version 12 (SPSS Inc., Chicago, IL, USA) for data analysis. Descriptive statistics such as mean, standard deviation, and range for quantitative variables and frequency and percentage for categorical variables were calculated. One-way ANOVA was performed and continued with post hoc analysis (two-tailed P-values for pairwise independent group t-test) to determine which group of red betel leaf has the best antifungal potency.


  Results Top


The test results of MIC of red betel leaf extract against C. albicans using microdilution method were determined by turbidimetric and spectrophotometric methods. Observations through the turbidimetric method (quantitative analysis based on turbidity measurements) showed that the level of turbidity was visually visible at a concentration of 2.34%, so it was estimated that the inhibition value is at a concentration of 2.34%. Red betel extract has a dye which can lead to bias and can influence the results; therefore, it continued with spectrophotometric calculations. Calculations showed that the spectrophotometric absorbance values of red betel leaf extract concentrations ranging from the smallest to the concentration of 0.15% were homogeneous in the amount of 0.7 mg/mL. Absorbance values showed a decline of 0.29% which was 0.45 mg/mL, thereby concluding that the inhibition began to appear at a concentration of 0.29%.

The immersion test showed the effects of different denture cleansers against C. albicans over heat-cured acrylic surface [Table 1]. The colony counter calculations showed that the fungal colonies in negative control (sterile distilled water) and red betel leaf extract with concentrations of 10%, 20%, 30%, and 40% were only seen after three stratified dilutions (103), while in alkaline peroxide denture cleanser “fitty dent”® (Fittydent Int GMBH A-7423 Pinkafeld, Austria) as positive control, colonies were already seen at the first dilution. The statistical analysis of the six groups was significant (P < 0.01). The more the concentration of red betel leaf extracts (which increased to 10%, 20%, 30%, and 40%), the lesser the number of colonies of C. albicans (which decreased to 48, 8.6, 4.4, and 4.2×103 CFU/mL). Meanwhile, the reduction value increased to 62.09%, 93.21%, 96.53%, and 96.68%. There were no significant differences between 30% and 40% of red betel leaf extract concentration (P = 0.89) [Table 2]. The highest number of colonies was in negative control as much as 126.6×103 CFU/mL with zero reduction value, and the smallest number was in positive control as much as 1.3 × 101 CFU/mL. The reduction value shows the highest number (98.87%).
Table 1: Growth of microorganisms on resin specimens after different immersions in denture cleanser (one factor ANOVA)

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Table 2: Advanced test (post hoc) with independent t-test

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  Discussion Top


Removable denture prostheses made of heat-cured acrylic material are widely used as a rehabilitation tool in cases of tooth loss because this material has the following advantages: non-toxic if the ratio of materials and manipulations is according to the manufacturer’s recommendations, does not irritate, does not dissolve in oral fluids, aesthetically pleasing, easy to manipulate, easy to repair, and small change in dimensions. Dentures made of heat-cured acrylic resin also have disadvantages. The disadvantages are porosity and easy absorption of fluids, so that they can affect plaque adhesion especially on the anatomical surface of the denture.[1],[2],[8] Porosity in the base of heat-cured acrylic resin dentures can occur due to trapping of air during the mixing process, contraction of monomers during the polymerization process, the mixing process of the polymer-monomers that is lacking, evaporation of monomers associated with exothermic reactions and the presence of monomer residues, improper processing temperature, molding forming process, and inadequate cuvette compression process.[15] The surface characteristics of the denture base also contributed to the increase in C. albicans colonies.[16] The anatomical surface of the denture is the part of the surface that is not polished, the surface is rough and irregular, adapting the anatomy of the patient’s mucosa, so that if the oral cleaning and denture cleaning are inadequate, it will facilitate the accumulation of plaque on that surface.[3]

There are various types of denture cleansers available in the market, including those based on chemicals such as alkaline peroxide, hydrogen hypochlorite, and chlorhexidine gluconate. Research on herbal-based denture cleansers has begun to be widely carried out, including green betel leaf, robusta coffee bean paste, cinnamon bark, and basil leaf juice.[1],[7],[14],[17],[18]

In this study, we observed the MIC of red betel leaf extract against C. albicans and assessed the efficacy of red betel leaf against C. albicans over acrylic denture surface by the immersion test. Three different denture cleansers were presented. They were herb base (red betel leaf extract with concentrations of 10%, 20%, 30%, and 40%), chemical base (alkaline peroxide denture cleanser “fitty dent”®) as positive control, and sterilized distillate water as negative control. All of them differed in their efficacy against C. albicans over acrylic denture surface.

Red betel leaf extract contains essential oils, flavonoid compounds, alkaloids, tannins, and polyphenols which have antifungal activity.[9],[10] Alkaloids cause damage to fungal cell membranes, flavonoids inhibit fungal cell division or proliferation, tannins have effectiveness in inhibiting the growth of C. albicans, and phenols cause cell membrane lysis and cause fungi to not develop.[9],[19]

Alkaline peroxide denture cleanser “fitty dent”® (Fittydent Int GMBH A-7423 Pinkafeld, Austria) contains sodium perborate, citric acid compounds, sodium bicarbonate, sodium perborate monohydrate, potassium peroxy-monosulfate, and sodium polyphosphate, which function to destroy and oxidize microorganism’s cell walls. When the tablet is dissolved in water, sodium perborate will decompose and form alkaline hydrogen peroxide which functions to oxidize and reduce surface tension resulting in a cleaning action against the denture base.[8],[20]

Sterilized distillated water is sterile water with the formula H2O without other additives. It has pH of 7.0, which is hypotonic, nonpyrogenic, and non-ionic. This fluid is used for irrigation during surgery, to clean the blood at the site of surgery, moisten gauze, for wound irrigation, and soak and wash medical equipment.[21]

The results in this study support a rejection of the null hypothesis for C. albicans removal. The MIC test in this study showed that red betel leaf extract had an inhibitory potency against C. albicans. The immersion test showed the effects of different denture cleansers against C. albicans over heat-cured acrylic surfaces. The more the concentration of red betel leaf extracts, the lesser the number of colonies of C. albicans; meanwhile, the reduction value increased. The highest number of colonies was in negative control with zero reduction value, and the smallest was in positive control (alkaline peroxide), the reduction value of which shows the highest number.

The results in this study were in line with the research of Candrasari et al.,[9] which revealed that the ethanol extract of red betel leaf had an inhibitory potency against the growth of C. albicans ATCC 10231 at a concentration of 10% extract with a diameter of 8 mm. The inhibition zone formed increased with increasing extract concentration. It is also in line with the research of Rezeky et al.[12] that red betel leaf extract had an inhibitory potency against C. albicans at 25% by spectrophotometric calculation. The number of C. albicans colonies decreased along with the increasing concentration of red betel leaf extract. The inhibition value in this study was smaller than the results of the studies of Candrasari et al.[9] and Rezeky et al.[12] The results of this study are also in line with the research conducted by Lee et al.,[7] which stated that the cleaning method by immersing the denture plate in effervescent tablet denture cleanser (alkaline peroxide) was effective in reducing the adherence of C. albicans to the base surface of removable dentures.

There were no significant differences between 30% and 40% of red betel leaf extract concentration (P = 0.89); it showed that the best efficacy of red betel leaf against C. albicans over acrylic denture surface was at 30% concentration as a herbal-based denture cleanser. This research has limitation of only examining the antifungal potency but not examining the antibacterial potency of red betel leaf as denture cleanser. Further detailed researches with comparatively larger sample size, long follow-up, and different kinds of denture prostheses are needed for further in-depth study of efficacy of red betel leaf as herbal-based denture cleanser.

Acknowledgements

We would like to acknowledge Dr. Nina Djustiana, drg, M. Kes, Dean of Faculty of Dentistry, for the support to the study. Extend our thanks to the laboratory assistants Ria Fitria, Reny, Meitasapitri, and Salam Supriatno for contributing to the research. We would also like to thank Drs. H. Bernik Maskun from Faculty of Mathematics and Natural Sciences, Padjadjaran University, Adji Kusumadjati, dr, M. Kes, from Faculty of Medicine, Padjadjaran University, Indonesia for the direction of analyzing data.

Financial support and sponsorship

This study was self-funded by the authors.

Conflict of interest

There are no conflicts of interest.

Author contributions

I. M.: Concept, data analysis, experimental studies, statistical analysis; I. M. and L. D.: design, definition of intellectual content, literature search, data analysis, statistical analysis, manuscript preparation, manuscripts editing, manuscript review. R. R.: design, definition of intellectual content, literature search, manuscript preparation, manuscripts editing, manuscript review. Finally, all authors have approved the manuscript and given consent for publication.

Ethical policy and institutional review board statement

Not applicable as in vitro study.

Patient declaration of consent

Not applicable.

Data availability statement

The data set used in the current study is available. Data are available within the article or its supplementary material.

 
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