ANTIFUNGAL EFFECTIVENESS OF CACAO BEAN SHELLS EXTRACT (Theobroma cocoa L.) ON TRICHOPHYTON RUBRUM GROWTH IN VITRO EFEKTIFITAS ANTIFUNGAL EKSTRAK CANGKANG BIJI KAKAO (Theobroma cocoa L.) TERHADAP PERTUMBUHAN TRICHOPHYTON RUBRUM SECARA IN VITRO

Trichophyton rubrum is the most common dermatophytosis etiology. The antifungal agent has several problems such as fungal resistance and therapy side effects. Cacao (Theobroma cocoa L.) bean shells extract contained flavonoids, alkaloids, and saponins which have an antifungal effect. This study aimed to test the in vitro cacao bean shells extract antifungal (especially: Trichophyton rubrum) effectiveness by agar well diffusion method. This was an experimental study with a post-test only control group design. This study used cacao bean shells extract concentration 25%, 50%, 75%, and 100%, ketokonazol as positive control, and aquadest as negative control. The average inhibition diameter of 25%, 50%, 75% and 100% cacao bean shells extract concentration were 10.65 mm, 18 mm, 26.92 mm, and 37.22 mm, respectively, while the ketoconazole inhibition diameter was 51.52 mm. Data were analyzed using the Kruskal-Wallis test and post hoc with Mann-Whitney. The results showed significant differences between each treatment group (p<0.05). Cacao bean shells extract had an antifungal effect in inhibiting the growth of Tricophyton rubrum invitro.


INTRODUCTION
Dermatophyte infect 10-15% human population at least once in their lives (Pires et al., 2014). 2010 Indonesia's health profile data showed that skin and subcutaneous tissue diseases are third ranked of ten most diseases in outpatients in hospitals throughout Indonesia based on the number of visit (Kemenkes, 2011).
Trichophyton rubrum is the most frequently dermatophytosis found in clinical cases. It could be isolated from various superficial and deeper sites of infection, also systemic dissemination in immunocompromised patients (Pereira et al., 2013). Antifungal agents such as griseofulvin, azole derivatives, and allylamine which are used as therapeutic options have some problems in the treatment of dermatophytosis such as fungal resistance, long duration of treatment and therapeutic side effects. In that situation, additional antifungal therapy are needed (Pereira et al., 2015). the process of producing dry cocoa beans produces waste called cacao rind and pulp, then the processing dried cacao bean into cacao products produces waste called cacao bean shells (Kayaputri et al., 2014). Indonesian cacao fruit production was 686,946 tons in 2018 (Direktorat Jendral Perkebunan (Ditjenbun), 2017), and 3.6% of this cacao fruit was cacao bean shells (Kim, et al., 2014), therefore 24,730 tons cacao bean shells be produced.
The cacao bean waste has a low economic value and so far its been used as animal feed and compost only. Phytochemical study on the extract of cacao bean shells (Theobroma cocoa L.) showed antimicrobial compound alkaloid, flavonoid, and saponin which has antifungal effects.
Gas Chromatography-Mass Spectrometry of cacao bean shells extract study showed that cacao bean shell contain 89.5% alkaloid, in the form of caffeine and theobromine (Kayaputri et al., 2014).
Flavonoids modulate the pathway of fatty acid synthesis, thereby disrupting the formation of fungal cell walls.
The saponin induces H2O2, causes fungal lipid membranes peroxidation and makes the membrane permeability increased, so occur the cell leakage (Jiang et al., 2016 (table 1). These results were consistent with Pelczar and Chan (2008) which stated that the higher concentration of an antifungal material, the antifungal activity also be stronger. Davis and Stout (1971) categorized the strength of antifungal agents based on the diameter of the inhibitory zone. The diameter of inhibitory zone ≤ 5 mm is weak, 5-10 mm is medium, 10-20 mm is strong, and ≥ 20 mm is very strong. This study showed that the antifungal activity of cacao bean shells extract on Trichopyhton rubrum growth was in the strong and very strong category.
Kruskal-Wallis test was used to identify a significant difference between groups. Kruskal-Wallis test indicated that there was a significant difference between groups of study (p= 0,003).
This means that there are differences in the results of inhibitory strength between treatment groups. The differences between groups need to be determined to find out the most effective dose. Mann-Whitney Post Hoc was done, showed significant differences betwen groups. which can be used as antifungals (Kayaputri et al., 2014). Flavonoids inhibit the growth of  (Dheeb, 2015). The underlying mechanism of saponins activity are its ability to form complexes with sterols in membrane cells and cause perturbation of membrane (Trdá et al., 2019). Saponin also induces H2O2, causes peroxidation of fungal lipid membranes and makes cell membrane permeability increased and cell leakage occurs (Jiang et al., 2016).

CONCLUSION
Cacao bean shells extract (Theobroma cocoa L.) had in vitro antifungal effectiveness of Trichophyton rubrum growth inhibition by agar well diffusion method, with strong and very strong category result. There were significant differences between all cacao bean shells extract groups p <0.05.