ISOLATION, CHARACTERIZATION AND BIOACTIVITY ASSAY OF SECONDARY METABOLITES FROM TALAROMYCES WORTMANII, XYLARIA SP., AND PHOMOPSIS SP., ENDOPHYTIC FUNGI OF INDONESIAN MORUS

• Main Author: HERMAWATI, ELVIRA
• Format: Dissertations
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/21936
• Institution: Institut Teknologi Bandung

Morus (Moraceae) is one of the most economically valuable plants in the silk industry and has been used as traditional medicines since long time ago. Phytochemical studies on Morus plants had showed that this genus is a potential source of bioactive secondary metabolites. Nevertheless, plants are not the only source of secondary metabolites. Tissue cultures and endophytic microorganisms can also be used as alternatives. The first research on the endophytic fungi of the Morus plant has been reported previously from Morus alba. This study has successfully isolated and identified 106 isolates of endophytic fungi from various tissues of M. alba tissues, i.e. roots, leaves, and bark, and then examined them against pathogenic fungi. However, the secondary metabolites on those endophytic fungi have not yet been reported. <br /> <br /> Based on those backgrounds, the objectives of this research were to isolate, to characterize secondary metabolites from endophytic fungi of two species of Indonesian Morus, i.e. Morus cathayana and M. macroura, and to examine the bioactivity of the extracts and isolated compounds against murine leukemia P-388 cells and lung cancer A-549 cells. The single isolate of endophytic fungi was obtained by axenic culture method and identified based on the sequence of molecular rDNA ITS. The endophytic fungi were cultured in a liquid medium for 14 days at 28 °C. The liquid cultured medium was partitioned with EtOAc at room temperature, while the mycelial part was extracted with MeOH. The separation and purification of secondary metabolites have been conducted by using various chromatography techniques, such as liquid vacuum chromatography, gravitational column chromatography, and radial chromatography. The structures of isolated compounds were determined based on spectroscopic data including NMR-1D (1H, 13C, TOCSY1D), NMR-2D (HSQC, HMBC, COSY and NOESY), high resolution mass spectrometry, and X-ray crystallography. The cytotoxic properties were performed against two cancer cells, namely murine leukemia P-388 cells and lung cancer A-549 cells following the MTT method and the activity is expressed in 50% growth inhibitory concentration or IC50. <br /> <br /> This research for the first time has successfully isolated and identified three isolates of endophytic fungi, i.e. Talaromyces wortmanii and Phomopsis sp. from twigs and roots of M. cathayana, and Xylaria sp. from twig of M. macroura. Eighteen compounds have been obtained from those three endophytic fungi including four new compounds. Among eighteen compounds, eight compounds have been isolated Phomopsis sp., two compounds from Talaromyces wortmanii along with eight compounds from Xylaria sp. The eight compounds yielded from EtOAc extract of Phomopsis sp. were three novel naphthoquinone derivatives (epoxyquinophomopsin (1), epoxyquinophomopsin A (2), and epoxyquinophomopsin B (3)), a new sesquiterpene, i.e. a presilphiperfolan derivative (presilphiperfolan-1,12-diol (4)), one benzoquinone derivative (2-hydroxy-5-methoxy-3-methylcyclohexa-2,5-dien-1,4-dion (5)), one phthalide derivative(5,7-dimethoxy-3,6-dimethylphthalide (6)), and two dihydroisocoumarin derivatives (mellein (7) and 8-methoxymellein (8)). Moreover, eight other compounds isolated from EtOAc extract of Xylaria sp. consisted of five cytochalasan derivatives (cytochalasin Q (9), 19,20-epoxycytochalasin Q (10), 18-deoxy-19.20 epoxycytochalasin Q (11), 19,20-epoxycytochalasin C (12), and 19.20-epoxycytochalasin D (13)), as well as three eremophilane derivatives (phaseolinone (14), phomenon (15), and phaseolinone tetrol (16)). In addition, two compounds obtained from MeOH extract of T. wortmanii were one bianthraquinone derivative (skirin (17)) and one azaphilonoid derivative (wortmin (18)). <br /> <br /> Furthermore, the cytotoxic properties of extracts and isolated compounds against P-388 cells showed that the EtOAc extracts of media of Xylaria sp., Phomopsis sp. and T. wortmanii showed IC50 values of 0.14, 8.67 (active) and 62.36 ?g/mL (not active), respectively. Whereas, the MeOH extracts of mycelia of Xylaria sp., Phomopsis sp. and T. wortmanii showed IC50 0.34 (active), >100, >100 ?g/mL (not active), respectively. All of the compounds examined against P-388 cell were active, except compound 8, 17 and 18. In line with the activity of extracts, two naphtoquinone (2 and 3), one presilphiperfolan derivative (4), one benzoquinone derivative and one phtalide derivative (5 and 6) from Phomopsis sp., five cytochalasan derivatives (9-13) and three eremophilane derivatives (14-916) from Xylaria sp. were very active. Even, compound 10, 12 and 13 had IC50 < 0.10 ?g/mL (IC50 < 0.19 ?M). On the other hand, a bianthraquinone derivative and an azaphilonoid derivative (17-18) from MeOH extract T. wortmanii were not active IC50 > 50 ?g/mL (IC50 > 50 ?M) and this result was in accordance with the IC50 of their extract. The analysis of structure activity relationship showed that the presence of a hydroxy group at C-18 and an epoxy group at C-19 and C-20 in cyctochalasan derivatives (9-13) could play an important role in enhancing cytotoxic properties. While, in sesquiterpenes derivatives (14-16), the absence of terminal alkene at C-11 – C-13 in compound 14 and 16 can increase the activity. <br /> <br /> Moreover, the cytotoxicity assay against lung cancer A-549 cells also showed that all the examined compounds were active. The two new compounds (2 and 3) had IC50 values at 12.1 and 6.8 ?M, respectively. The presence of a hydroxy group at C-1 in the compound 3 probably increased the activity of the compound compared to 2. In addition, five cytochalasan compounds were very active with IC50 values of 0.32 - 3.15 ?M. Among cytochalasan derivatives, compounds 10 and 12 were the <br /> <br /> most active. The activity of cytochalasan derivatives could be caused by the presence of a double bond at C-5 and C-6, a hydroxyl group at C-18, and an epoxy at C-19 and C-20. Compound 15 was more active than compound 14, because compound 15 has a terminal alkene at C-11 – C-13. <br /> <br /> According to this research, the secondary metabolites produced by the three endophytic fungi were different from the compounds from the host plant (M. cathayana and M. macroura). The secondary metabolites isolated from Morus are usually stilbenes, 2-arylbenzofurans, xanthones, coumarins, and adduct Diels Alders, while its endophytic fungi in fact dominantly produced terpenoids, quinones, and cytochalasan derivatives. Therefore, the discovery of these compounds can enrich the phytochemistry of endophytic fungi from Indonesian Morus plant.