Anti-Bacterial and Anti-Fungal Activities from Macaranga bancana Leaves Extract

Macaranga bancana is one of the Indonesian medicinal plants that is empirically used to treat infectious disease. The aim of this study was to evaluate anti-bacterial and antifungal activities of M. bancana against human pathogenic microbes, i.e., Escherichia coli, Staphylacoccus aureus, and Candida albicans. The dried leaves of M. bancana were extracted using cold extraction method with n-hexane, dichloromethane, ethylacetate, methanol, and ethanol solvents. The evaluation of anti-microbial activity from these extracts at the concentration of 1000 μg/ml was conducted using microdilution method. Amoxicillin, cephadroxil, and ketoconazole were used as positive controls. All extracts showed 100% inhibitory activity against S. aureus, with the exception of ethanol extract which was 72.8%. Nevertheless, methanol and ethanol extracts showed 100% inhibitory activity against E. coli, while the lowest activity was shown by n-hexane extract ( 45.2%). All extracts exhibited 100% inhibitory activity against C. albicans. In conclusion, leaves extract of M. bancana is a potential source of anti-bacterial and anti-fungal agents.


Introduction
The irrational use of antibiotics cause many pathogenic microbes to adapt to their environment and become resistant to synthetic antibiotic drugs.This has encouraged the development of novel antimicrobial substances derived from natural products. 1M. bancana plant, a member of Euphorbiaceae family, is widely found in Indragiri Hulu, Province of Riau, Indonesia.Talak Mamak tribe from Indragiri Hulu empirically use the leaves and fruits of this plant to treat diarrhea.Previous studies showed that various plants from the same genus, such as M. gigantea, M. pruinosa, M. tanarius and M. triloba had strong antimicrobial activity against S. aureus. 2,3Salah et al reported that M. monandra possessed anti-fungal activity. 4Nevertheless, the antimicrobial activity from M. bancana had not been previously reported.
Therefore, this study was conducted to evaluate the anti-bacterial and anti-fungal activities of M. bancana against human pathogenic microbes, i.e., E. coli, S. aureus, and C. albicans.

Extract preparation
M. bancana leaves were collected from Indragiri Hulu, Province of Riau, in March 2018.Plant determination was conducted at Department of Biology, Faculty of Mathematics and Natural Sciences, University of Riau.The leaves were cleaned, air-dried, and powdered.Subsequently, 20 g of the leaves powder were cold extracted using n-hexane, dichloromethane (DCM), ethyl acetate (EtOAc), methanol and ethanol for 3 x 24 hours, respectively.The filtrate was collected and concentrated using rotary evaporator.

Anti-microbial activity
The anti-microbial activities of the extracts against S. aureus, E. coli ATCC 35218, and C. albicans ATCC 10231 were conducted using microdilution method. 5The extracts were dissolved in dimethyl sulfoxide (DMSO) in the concentration of 1000 μg/ ml.Amoxsan®, cefadroxil and ketokenazole at 1000 μg/ml were used as positive control, while DMSO was used as negative control.Precultures of the tested microorganisms were made by inoculating 25 ml of nutrient broth medium for bacteria (WP medium for fungus) and incubated for 18 hours at 37 0 C for bacteria (4 days at room temperature for fungus).Subsequently, the cell density was adjusted to 107 CFU/ml. 6Microbial suspension of 20 μl was distributed in each well containing extract, nutrient broth, and resazurin.The plate was incubated at 37 0 C for 24 h for the bacteria or 4 days for the fungus and the optical density of each was measured by using microplate reader.The experiment was run in three replicates.

Data analysis
The inhibition activity was calculated using the formula: % growth = x 100 % inhibition = 100 -% growth

Results and Discussion
In this study, anti-bacterial and anti-fungal activities of M. bancana leaves extracts were evaluated against three pathogenic microbes (S. aureus, E. Coli, and C. albicans) using microdilution method.This method was used because it has several advantages, e.g., can be used to analyzed several different samples at one time, requires small amount of samples, and has high sensitivity. 7 this assay, the resazurin was added as microbial growth indicator.The addition of resazurin in the assay can predict the presence of visual anti-microbial activity (qualitative) through colour change.A compound which has anti-microbial activity changes from pink to blue, while those which does not have activity or weak activity stays pink.This reaction is resulted from the activity of oxyreductase in the microbe that converts blue resazurin into pink resofurin. 5This has long been explained by the presence of an outer membrane permeability barrier in Gram-negative bacteria, which limits the access of the antimicrobial agents to their targets in the bacterial cells.Furthermore, all extracts showed high inhibition activity against C. albicans with 100% inhibition, similar with ketoconazole as positive control.
Strong anti-fungal activity of the extract is caused by the activity of compounds that prevent the synthesis of ergosterol by inhibiting the P450 enzyme.This will cause the depletion of ergosterol, which can lead to a change in membrane permeability and cell membrane damage. 9Nevertheless, the further investigation is needed to investigate the mode of action of the extracts towards the fungal.

Conclusion
Leaves extract of M. bancana is a potential source of anti-bacterial and anti-fungal agents.