AIP Conference Proceedings 2344, 040013 (2021); https://doi.org/10.1063/5.0047205 2344, 040013
© 2021 Author(s).
The effectivity of Indonesian propolis from Tetragonula biroi bee as an antifungal
agent for Candida sp. and Cryptococcus neoformans
Cite as: AIP Conference Proceedings 2344, 040013 (2021); https://doi.org/10.1063/5.0047205 Published Online: 23 March 2021
Robiatul Adawiyah, Alifa Husnia AlHaq, Davita Kristabel Mandala, Muhammad Sahlan, Diah Kartika Pratami, and Siti Farida
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The Effectivity of Indonesian Propolis from Tetragonula biroi Bee as an Antifungal Agent for Candida sp. and
Cryptococcus neoformans
Robiatul Adawiyah
1,2,3,a), Alifa Husnia AlHaq
4,b), Davita Kristabel Mandala
5,c), Muhammad Sahlan
3,5,d), Diah Kartika Pratami
3,6,e), Siti Farida
3,7,f)1Department of Parasitology, Faculty of Medicine, Universitas Indonesia, Jakarta, 10430, Indonesia
2Study Program of Clinical Parasitology, Faculty of Medicine, Universitas Indonesia, Jakarta, 10430, Indonesia
3Research Center for Biomedical Engineering, Faculty of Engineering, Universitas Indonesia, Depok, 16424, West Java, Indonesia3
4Undergraduate programm, Faculty of Medicine, Universitas Indonesia, Jakarta, 10430, Indonesia
5Department of Chemical Engineering, Faculty of Engineering, Universitas Indonesia, Depok, 16424, West Java, Indonesia.
6Lab of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Pancasila University, Jakarta, 12640, Indonesia.
7Department of Pharmacy, Faculty of Medicine, Universitas Indonesia, Jakarta, 10430, Indonesia
a)corresponding author: [email protected]
b)[email protected], c)[email protected], d)[email protected],
e)[email protected], f)[email protected]
Abstract: In recent years, there has been an increase in the incidence of fungal infections accompanied by a decrease in susceptibility to fluconazole as one of the drug choices for fungal infections. So that it needs to be considered the existence of an alternative drug that is effective and also expected to have a minimal side effect, one of which is Propolis. This research was done to determine the effectivity of Indonesian Propolis from Tetragonula biroi bee on the growth of Candida sp. and Cryptococcus neoformans. An experimental study to test the susceptibility of Propolis using disc diffusion technique performed on six types of fungi, e.g. Candida albicans, Candida glabrata, Candida parapsilosis, Candida krusei, Candida tropicalis, dan Cryptococcus neoformans. The samples tested were Indonesian propolis emulsion from Tetragonula biroi bee with a concentration of 10 mg/ml, 50 mg/ml, and 70 mg/ml. The sample selection based on the result of “Exploration of Antifungal Potential of Indonesian Propolis from Tetragonula biroi Bee“ from the Faculty of Engineering, University of Indonesia. Indonesian Propolis emulsion from Tetragonula biroi bee can inhibit the growth of Candida sp., especially Candida glabrata and Candida krusei, and also Cryptococcus neoformans with an optimum concentration of 10 mg/ml. The lower concentration than 10 mg/ml of Indonesian Propolis emulsion from Tetragonula biroi bee could be considered to provide more optimal results.
Keywords: fungal infection, Indonesian, Propolis, susceptibility test
INTRODUCTION
The last three decades, the incidence of fungal infections has increased, superficial and systemic infections respectively. Some factors that play a role in increasing the incidence are immunosuppressive therapy, invasive surgical procedures, and the use of broad-spectrum antibiotics. The most common cause of fungal infections is
the Candida sp., especially Candida albicans, Candida glabrata, Candida parapsilosis, Candida krusei and Candida tropicalis [1]. Besides Candida sp., Cryptococcus neoformansis the other pathogen for humans [2].
Antifungal options for fungal infected patients are amphotericin B and azole groups, such as fluconazole, itraconazole, and voriconazole [3]. In the amount of decade, fluconazole was used increasingly, but have been many reports also about sensitivity decreased to fluconazole in several species of Candida sp. and Cryptococcus neoformans [3]. There is a report that amphotericin B, itraconazole, and voriconazole, often cause specific side effects and easily interact with other drugs [1]. So it is necessary to find out of alternative drugs that have the potential for antifungal activity with minimal side effects [3].
Propolis is already known to have antifungal properties, namely flavonoid compounds. Each type of Propolis has different content depending on the type of bee producer and its environment [3]. Indonesia has a diversity of bee species and vegetation as a source of resin so that Indonesia has a broad diversity of sources of Propolis [4]. Among the species of bees, Tetragonula biroi is the most of the Propolisbee collector [5]. Therefore, the researchers wanted to find out the effectiveness of Indonesian Propolis from Tetragonulabiroias an antifungal for Candida sp.
and Cryptococcus neoformans and also finding out the concentration of this Propolis in inhibiting the growth of Candida sp. and Cryptococcus neoformans in vitro.
MATERIALS AND METHODS
This research is an experimental study using disc diffusion techniques. This research was conducted at the Parasitology Laboratory of the Faculty of Medicine (FMUI) Universitas Indonesia in the July-November 2018 period with an ethical number from FMUI: 810/UN2.F1/Etik/2018. We used Indonesian Propolis from Tetragonula biroi bee with concentrations of 10 mg / ml, 50 mg / ml, and 70 mg / ml. The Propolis is the result of extracting from Sahlan et al. [6]. The study was conducted on 6 types of wild type fungi from Fungal Culture Collection Centre (FCCC)- Parasitology FMUI. They were Candida albicans, Candida glabrata, Candida parapsilosis, Candida krusei, Candida tropicalis and Cryptococcus neoformans. The test was carried out 3 times. As a positive control, we used a 25 μg fluconazole disk and a negative control used Propolis extraction solvent.
Sensitivity Test with Diffusion Method
The Candida sp. and Cryptococcus neoformans colonies were taken using disposable ose and put into tubes containing sterile aquades. Suspension of Candida sp. and Cryptococcus neoformans were taken using a sterile cotton swab and applied over the surface of Mueller Hinton agar. The plain disc was inserted into the propolis emulsion and waited for 5 minutes until Propolis was absorbed into the plain disc. Prepare fluconazole discs. Discs containing Propolis were placed on the Mueller Hinton agar surface, the same treatment for fluconazole discs. One petri dish for one type of fungi. They were repeated three times. After that, the Petri dishes were put into an incubator at 37°C.
Inhibition zone readings after 48 hours of incubation. Inhibited zones formed were measured using callipers.
RESULTS AND DISCUSSION
The inhibition zone is measured using a calliper with the results in millimeters (mm). The average MIC result showed in Fig. 1. Based on this result, the propolis extract was effective for Candida glabrata and Cryptococcus neoformans colonies.
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FIGURE 1. The average of MIC propolis to each fungi
Normality test from the data shows abnormal results, so the next is Kruskal Wallis nonparametric test. The following are the results of the Kruskal Wallis statistical results in the form of p (asymp. Sig) of each fungus. (Table 1)
TABLE 1. The Kruskal Wallis statistical of MIC results
Fungi P (asymp. sig)
Candida albicans 0,046
Candida krusei 0,155
Candida tropicalis 0,406
Candida parapsilosis 0,081
Candida glabrata 0,050
Cryptococcus neoformans 0,045
The Kruskal Wallis statistical test result showed that there were three significant data (p <0.05). There are Candida albicans, Candida glabrata and Cryptococcus neoformans. For these groups, we used further test, the Mann-Whitney- U test to find out which Propolis group has a significant difference. The Mann-Whitney-U test showed no significant difference between the two treatment groups so that the increase in concentration did not give significant results.
The ability of Propolis as an antifungal has been reported in many studies [7-17]. In previous studies, it has been proven through phytochemical tests that Indonesian Propolis from Tetragonula biroi bee has a flavonoid (7.83%) and polyphenols (14.72%). LC-MS/MS analysis shows that Propolis contains six antifungal compounds, including ziyuglicoside II, muscanone, dehydroabietic acid, montecristin, plakevulin A and shikimic acid [6].
In this study, there are three fungi showed significant results, especially for Candida albicans, Candida glabrata and Cryptococcus neoformans. The average of the Propolis inhibition concentrations result of Candida albicans is not effective enough to inhibit the growth of Candida albicans in vitro. Our result is different with the report from Tobaldini-Valerio et al., which reported that Propolis had they are Minimum Inhibitory Concentration value of 50% and 90% against Candida albicans on 440 μg/ml [14]. Haghdoost et al., also reported MIC and MFC (Minimum Fungicidal Concentration) values averaging 360 and 1,250 μg/ml [12].
The average of inhibition Propolistest for Candida glabrata showed 13.00 mm, 11.33 mm, and 12.33 mm. While fluconazole as positive control showed no inhibition zone at all (0 mm). This result provides an opportunity for the potential effectiveness of Propolis in inhibiting Candida glabrata [18]. This result is slightly different with Shokri et al. that the average MIC of Propolis was 3.8 mg/ml and at a concentration of 8 mg/ml showed 57.1% of the isolated fungus did not grow again [18]. This result also different from Siqueira et al. that MIC results of Candida glabrata isolated from chronic periodontitis patients are 64 μg/ml [10].
The average inhibition Propolis test for Cryptococcus neoformans showed 10.00 mm, 7.67 mm, and 10.67 mm. In contrast, fluconazole as positive control showed 1.33 mm. This result provides an opportunity for the potential effectiveness of Propolis in inhibiting Cryptococcus neoformans. This result similar to Fernandes et al., which
0 10 20 30 40
C.albicans C. krusei C. tropicalis C. parapsilosis C. glabrata C. neoformans Treatment Propolis extract (mg/ml) 10 Treatment Propolis extract (mg/ml) 50 Treatment Propolis extract (mg/ml) 70 Treatment Positive control Fluconazole
reported Propolis G12 in inhibiting Cryptococcus neoformans ATCC growth, 0.2 mg/ml, respectively [9], and also Thammasit et al., which reported MIC Propolis was 1 mg/ml [9]. The average inhibition Propolis test for Candida krusei and Candida parapsilosis were not significantly different, but the MIC for Candida krusei is much higher than to fluconazole as a positive control [19]. This result is similar to Freires et al., used red Propolis type 3 (South Brazil) and type 13 one (East Brazil) [20].
Propolis concentration was analysis by The Kruskal Wallis and Mann-Whitney-U test. Based on statistical tests comparing the inhibitory power of each Propolis concentration, there were no significant results either between concentration 10 to 50 mg/ml, concentration 10 to 70 mg/ml and concentration 50 to 70 mg/ml for each fungus. This result showed that the increase in concentration does not have a significant effect on inhibition. So we can use the smallest concentration (10 mg/ ml) to minimize the side effects
CONCLUSIONS
There is potential for the effectiveness of Indonesian Propolis from more Tetragonula biroi in inhibiting the growth of Candida glabrata, Cryptococcus neoformans, and Candida krusei in vitro, with an optimum concentration of 10 mg/ ml. It still needs further research on this Propolis, especially on pharmacokinetics and its pharmacodynamic. In vivo testing is also needed to find out more about the effectiveness of Indonesian Propolis from more Tetragonula biroi and its clinical application.
ACKNOWLEDGEMENT
This study was supported by a grant from Universitas Indonesia, PUTI Prosiding 2020, contract no. Nomor: NKB- 892/UN2.RST/HKP.05.00/2020
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