AIP Conference Proceedings 2193, 030015 (2019); https://doi.org/10.1063/1.5139352 2193, 030015
© 2019 Author(s).
Explore type and concentration of Indonesian propolis wax in inhibiting Candida albicans: In vitro study anti- candidiasis
Cite as: AIP Conference Proceedings 2193, 030015 (2019); https://doi.org/10.1063/1.5139352 Published Online: 10 December 2019
Robiatul Adawiyah, Muhamad Sahlan, Sri A. Soekanto, et al.
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Explore Type and Concentration of Indonesian propolis wax in inhibiting Candida albicans: in vitro study Anti-
candidiasis
Robiatul Adawiyah
1,2,9, Muhamad Sahlan
3,9, Sri A. Soekanto
4,9, Diah K. Pratami
5, Faiqueen D.S.F. Adnan
6, Silvana Saputri
6, Redita N. Putri
6, Siti Farida
7,8,9,a)1Parasitology Department, Faculty of Medicine, Universitas Indonesia, Jakarta, 10430, Indonesia
2Parasitology Clinical Program, Faculty of Medicine, Universitas Indonesia, Jakarta, 10430, Indonesia
3Department of Chemical Engineering, Majoring Technology Bioprocess, Faculty of Engineering, Universitas Indonesia, Depok, West Java, 16424, Indonesia
4Faculty of Dentistry, Universitas Indonesia, Jakarta, 10430, Indonesia
5Lab of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Pancasila University, Jakarta, 12640, Indonesia
6Undergraduate program, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia
7Department of Medical Pharmacy, Faculty of Medicine, Universitas Indonesia, Jakarta, 10430, Indonesia
8Drug DevelopmentCluster, Indonesia Medical Education and Research Institute, Universitas Indonesia, Jakarta, 10430, Indonesia
9Research Center for Biomedical Engineering, Faculty of Engineering, Universitas Indonesia, Depok, West Java, 16425, Indonesia
Corresponding author: a)[email protected]
Abstract. Candida sp. is the most aetiology of superficial and systemic fungal infection in human. The rising number of Candida albicans resistant as antifungal has drawn attention to a new alternative treatment’s finding. Natural substances like propolis become a preferred option due to the expectation of having fewer side effects. Propolis as one substance products of bees, containing polyphenols and flavonoids was already known to have anti-inflammatory, anti-viral, antioxidant, and antimicrobial activities. Propolis wax from Sulawesi, Indonesia has never been tested on Candida albicans. The aim of this study is to determine the best concentration of propolis, which effective against Candida albicans. This study was conducted at the Parasitology Laboratory, Faculty of Medicine, Universitas Indonesia. Candida albicans on Mueller-Hinton agars were tested to a disk diffusion that contains three types of Propolis wax (regular, coral, mixed) with different concentrations, 1%, 5%, and 7%.
The result indicated that 5% and 7% were the effective concentration for every type of propolis wax and no difference between the type of Propolis wax (Kruskal Wallis and Mann Whitney). Propolis wax at concentration 5% and 7% could be an alternative substance in overcoming candidiasis infections.
Keywords: propolis, concentration, candida
INTRODUCTION
Mycoses are divided into superficial and systemic / invasive infection. Candida sp. is the most aetiology of the mycoses [1]. Fifteen Candida sp. known as a cause human disease, but >90% of invasive disease and superficial candidiasis are caused by the 5 most common pathogens, C. albicans, C. tropicalis, C. parapsilosis, C. glabrata, and C. krusei [2]. Candidiasis infect both immunocompetent and immunocompromised individuals. But, for systemic candidiasis, the majority is on immunocompromised patients, such as neutropenia, hematological malignancy, organ transplant recipients, the patient receives chemotherapy and long-term of broad-spectrum antibiotics patients [3].
However, for the superficial candidiasis the risk factors are Diabetes Mellitus and pregnancy [2]. The most clinical manifestation of superficial candidiasis is vaginal candidiasis [4]. The drugs for candidiasis treatment currently are
Propolis wax is one of the substances products by bees containing polyphenols and flavonoids have already known to have anti-inflammatory, anti-viral, antioxidant, and antimicrobial activities [6,7]. Study in Brazil has shown that green propolis origin from Brazil could inhibit the growth of C. Albicans, which resistant to nystatin [8]. However, propolis from different origins has a different composition and potential [9]. Indonesia is a tropical country with great potential for propolis because propolis is available in large quantity. Some studies about propolis of Tetragonula sp.
(origin of Sulawesi island, Indonesia) has conducted, there were isolation and characterization of the propolis (Sahlan, 2018) and also phytochemical profile-antioxidant activity of it (Pratami, 2018) [10,11]. The aim of this research is exploring the type and effective concentration of Indonesian propolis wax in inhibiting Candida sp. by in vitro study in order to find an alternative drug for candidiasis.
MATERIALS AND METHODS Extraction of Propolis Tetragonula sp.
The Propolis was extracted followed Pratami (2018) method and was done in Lab of Research Center for Biomedical Engineering, ILRC Building, Universitas Indonesia [11]. Based on the location of the formation, propolis is distinguished over 2 types, i.e. smooth propolis (inside honeycomb) and rough propolis (outside of the honeycomb).
The compounds of rough and smooth propolis may be different which will have an impact on the activity of each.
Antifungal activity possessed by propolis can be used to treat fluor albus. We used three types of propolis included smooth propolis (from inside honeycomb), rough propolis (from outside of the honeycomb) and mixed propolis with three different concentrations (1%, 5%, and 7%), respectively.
Sample of Candida albicans
We used the Candida albicans sample from the stock culture of the Mycology Laboratory, Parasitology Department, Faculty of Medicine, Universitas Indonesia. We had already identified it by Chromogenic Agar Media and Rice cream test. We subcultured it all on Sabouraud Dextrose Agar and used it on a minimum age of 24 hours.
Preparation of Disc Diffusion Test
The susceptibility test was done by the disc diffusion method. The preparation was done to obtain standard turbidity, McFarland 0.5 which is equally a concentration of 1.5 x 108/ ml cell density. The fungi were then transferred to its medium using the counter strike method on the Muller-Hinton medium in the petri dish, and we made them triplo for each (A, B, C). We used nystatin 100 Unit as positive control and ethanol 98% as a negative control. Blank discs were soaked in extract ethanol propolis with three different concentrations, 1%, 5%, and also 7% (%w/v) in 5 minutes, then leave it at room temperature for 3-5 seconds before putting it on to fungal colonies. The petri dish was incubated for 48 hours at room temperature, and it was then measured the diameter of inhibition zone by Vernier caliper. All methods have been approved by the Ethics Commission of the Faculty of Medicine, Universitas Indonesia.
RESULTS
All results of three types of propolis showed less inhibition than nystatin, but mostly propolis has better inhibition than ethanol 96% (Table 1). The inhibition result for 1% concentration propolis showed that smooth and rough type of propolis has better than mixed type. But in 5% and 7% concentration showed different results, the mixed type propolis is better than smooth and rough propolis type. The difference inhibition zone of Candida albicans' growth between mixed propolis at concentration 5% and positive control Nystatin 100 Unit shown in Figure 1.
A statistical analysis by Kruskal Wallis showed there was a significant difference between the three types of propolis. Analysis for between different concentration with Mann-Whitney-U showed that there is the difference between 1% and 5% concentration (p<0.05) but no difference between 5% and 7% concentration (p>0.05).
TABLE 1. The diameter of inhibition zone of three types and three different concentrations of propolis to Candida albicans
Concentration
Zone of inhibition (mm) mean ± SD Smooth
propolis Rough
propolis Mixed
propolis Negative control
(Ethanol) Positive control (Nystatin) 1% 11.5 ± 1,50 10.50 ± 0.50 6.6 ± 1.52 10.00 ± 2.64 27.6 ± 0.58 5% 11.3 ± 1,52 10.10 ± 1.01 11.6 ± 0.57 10.60 ± 0.58 27.1 ± 0.50
7% 10.3 ± 0.57 11.53 ± 1.51 12.1 ± 01 10.06 ± 0.57 27.3 ± 0.57
FIGURE 1. The difference inhibition zone of Candida albicans's growth between mixed propolis at concentration 5% and Nystatin 100 Unit.
DISCUSSION
The all type propolis have an inhibition zone of C. albicans growth, it is understandable because this propolis has the phenolic compound 5.109% (mixed type), 4.943% (rough type), and 4.256% (smooth type) of propolis [12].
Although this phenolic compound is lower than Argentine Propolis, 36% conducted by Gonzales (2003) and Malaysian Propolis, 29.1 % conducted by Shehu (2016) [13,14]. Besides that, this propolis also has the flavonoid 16.38% (smooth type), 8.08% (rough type), and 12.38% (mixed type), which higher than Taiwanese Propolis (range from 2.82 to 3.07%), Brazil (3.26%), China (range from 5.37-7.73%) and Romanian Propolis (range from 4.24 to 19.07%) [12,15,16]. The statistical result with Kruskall-Wallis and Mann-Whitney-U showed that there is a significant difference between 1% and 5% concentration, but no significant difference between 5% and 7% concentration. It means we could use 5% concentration for the next step research.
The limitation of this research is some inhibition zone could not be read (interpretation). Although the inhibition zone of propolis is less than nystatin it is higher than the negative control (ethanol 96%). It means the propolis could inhibit C. albicans growth. This result is a big opportunity for Indonesian propolis as future antifungal drugs for candidiasis.
ACKNOWLEDGMENTS
We are very grateful to Prof. Retno Wahyuningsih for candidiasis discussion, and also Dra. Ridhawati for helping fund arrangement. The funding of this research is by Directorate of Research and Community Service, Universitas Indonesia [grant numbers 120/SP2H/PTNBH/DRPM/2018].
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