Coleus amboinicus (Lour.) is a medicinal plant containing various bioactive compounds. Endophytes are microorganisms living inside intracellular tissue of plants and known as the source of bioactive compounds. In order to explore the potential of endophyte in producing novel bioactive compounds, this study focused on isolating endophytic fungi from the leaves of C.amboinicus, characterisation and screening their metabolite bioactivity during submerged culture fermentation. Isolation of endophytic fungi from the leaves segment of C.amboinicus was conducted on PDA media and fungus identification was carried out by analyzing its morphology and molecular examination. The production of metabolites was examined using thin layer chromatography and gas chromatography. Identification of the endophytic fungus showed 98.84% similarity with Eutypa linearis. This species was fermented submergedly in PDB medium for 14 days under dark and exposed to light and the fermentation broth was extracted using ethyl acetate. The results showed that exposure to the light did not significantly influence metabolite production. The ethyl acetate extract exhibited antioxidant and cytotoxic activities. Antioxidant activity of this extract as examined by DPPH assay showed IC50 of 105.31 ± 2.11 µg/mL. Cytotoxic activity against Hela cell line was known to be the best among other cell lines (IC50 301.53 ± 11.34 µg/mL) although it was found to be non selective (SI<1). The extract contained five major compounds namely Benzenemethanol, 4-nitro-(CAS) p-Nitrobenzyl alcohol; 2-Pentadecanone (CAS) Pentadecan-2-one; (1R*,6S*,10R*)-5,5-Dimethyl-11,12-dioxatricyclo[8.2.1.0(1,6)] tridecan-10-ol; 9,12-Octadecadienoic acid (Z,Z)-, methyl ester (CAS) Methyl linoleate; and 3-Furanacetic acid, 4-hexyl-2,5-dihydro-2,5-dioxo- (CAS) 2-carboxymethyl-3-N-hexyl-maleic anhydride.
Abstract. Eltivitasari A, Rahmawati, Gemantari BM, Romadhonsyah F, Nurrochmad A, Wahyuono S, Astuti P. 2021. Effect of light exposure on secondary metabolites production of an endophytic fungus Arthrinium rasikravindrae and its antioxidant and anticancer activities. Biodiversitas 22: 3156-3163. Endophytic microorganisms are one of the promising sources in producing bioactive compounds, to be developed for new drug candidates. They are found to have the ability to generate the same compounds as their host plant. Metabolite producing capacity of the endophytes is known to be affected by light exposure during fermentation process. This study focused on an endophytic fungus Arthrinium rasikravindrae isolated from Coleus amboinicus stem to reveal out its metabolite profiles due to light exposure as well as its bioactivity consequences. A. rasikravindrae was cultured on potato dextrose broth medium for 14 days and fermented in dark and exposed to natural light. Metabolite profiling was performed using TLC and GC-MS analysis. The activities were observed using DPPH assay for antioxidant and MTT assay for cytotoxicity potential. The results showed that A. rasikravindrae ethyl acetate extract produced during dark and exposed to light fermentation conditions contained different compounds but there was some which showed similarity with their host plant. Methyl octadec-9-enoate was found in all fermentation conditions as well as in C. amboinicus stem extract. Besides methyl octadec-9-enoate, methyl palmitate was also found present in both A. rasikravindrae extract fermented exposed to light and its host plant. The antioxidant activity of extract generated from dark fermentation condition was better as compared to that exposed to light with IC50 value of 66.36±0.53 vs 556.92±34.37 µg/mL. However, cytotoxic activity screening against several cancer cell lines exhibited opposing results in which extract from light-exposed fermentation resulted in better cytotoxic activity (IC50 value of 291.40 ± 2.34 µg/mL on WiDr, 336.80 ± 5.05 µg/mL on T47D, and 404.73 ± 3.46 µg/mL on Hela cell lines). Extract obtained from dark fermentation condition showed IC50 value of more than 500 µg/mL in all tested cancer cell lines. Preliminary examination on cytotoxic activity against WiDR cells suggested that the extract from light-exposed fermentation might induce cell death through mechanisms involving cell cycle arrest.
Schizophyllum commune (fam Schizophyllaceae) is an endophytic fungus isolated from a medicinal plant Coleus amboinicus (Lour.) Spreng. This study was aimed to identify metabolite profiles, characterize bioactivities (antioxidant and cytotoxic), and analyze metabolite production of the fungus due to light exposure and initial fungal culture age. Fungal cultures aged 7, 14, and 21 days old on PDA were incubated in Potato Dextrose Broth (PDB) fermentation medium for 2 weeks either in full light or dark condition. Metabolite profiles were analyzed using thin-layer chromatography (TLC) and gas chromatography-mass spectrometry (GC-MS). The 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays were used to determine antioxidant and cytotoxic activity. The results showed that light exposure and different age of fungal cultures affected metabolite production based on TLC analysis. Phytochemical screening of ethyl acetate extract of fermentation indicated the presence of flavonoids, terpenoids, steroids, phenols, and alkaloids. There are five major compounds detected by GC-MS. Bioactive screening showed that this extract had DPPH scavenging activity (IC50 = 109.19 ± 0.77 µg/mL) and cytotoxic activities, the best cytotoxic activity was on HeLa cell (IC50 = 492.23 ± 24,43 µg/mL) with selectivity index (SI) value is 2.33.
As the enzyme responsible for the uric acid formation, Xanthine oxidase was considered to be a therapeutic target for hyperuricemic treatment. This study was carried out to assess the potential of caryophyllene, and its derivates usually present in the natural product to inhibit Xanthine oxidase. The molecular docking using Autodock Tool and Biovia Discovery Studio was conducted to visualize the molecular interaction and to reveal the structure-activity relationship of those compounds. The results showed that the derivates of caryophyllene showed a higher affinity to Xanthine Oxide than Allopurinol. Among those all, caryophyllene oxide has the most stable bonding to xanthine oxide. Structure-activity relationship analysis showed that the chemical properties of the compound affected the affinity and molecular interaction to the enzyme as the target site in this study. The number of double bonds, substituents position, conformational structure related to steric hindrance, and the presence of lactone ring were assumed to influence the xanthine oxide inhibitory activity of caryophyllene derivates.
Organic Cation Transporter 1 (OCT1) is the primary transporter of metformin mainly located in hepatocytes, which plays an important role in metformin action to inhibit gluconeogenesis so as to reduce blood glucose. Genetic polymorphism of SLC22A1 that encodes OCT1, one of which is rs622342, has been widely reported and proven to decrease the antidiabetic effect of metformin. This study aimed to design primers and to obtain an optimum condition for polymerase chain reaction (PCR) process that can detect the genetic polymorphism of SLC22A1 rs622342. Primers were computationally designed in primer 3 webpage and analyzed with Primer BLAST and Oligo Analyzer. Optimization of PCR condition was conducted for temperatures of denaturation, annealing, and elongation as well as for the number of cycles in PCR process. Sensitivity test was performed on PCR condition using a variety of volumes and DNA template qualities undergoing multiple freeze-thaw cycles. The obtained pair, forward primer (5’- CAG AGA GAA TCA GTG AGC TGT G-3’) and reverse primer (5’- CCC AGG CTG GTC TTT TTA AG-3’), was proven to be capable of amplifying DNA sequence containing SNPs in rs622342 at 96°C denaturation, 60°C annealing, and 72°C elongation temperatures with a 30-cycle iteration. Such PCR condition could amplify DNA with 0.2 µL of template volume and 7 freeze-thaw cycles. Therefore, in addition to the selected primer pairs and PCR condition to analyze SNPs in rs622342, this study also recommends that the volume of DNA template having undergone multiple freeze-thawing be increased if the amplicon PCR products are unqualified.
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