SECONDARY METABOLITES OF PESTALOTIOPSIS MICROSPORA, FUSARIUM SOLANI AND DIAPORTHE LITHOCARPUS, ENDOPHYTIC FUNGI ISOLATED FROM INDONESIAN ARTOCARPUS AND THEIR BIOACTIVITIES

• Main Author: Riga
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/45315
• Institution: Institut Teknologi Bandung

Artocarpus is one of the important plant genus in family Moraceae which has been widely used as the traditional medicine to treatment inflammation, malaria, fever and diarrhea. Phytochemical studies of Artocarpus reported that the main secondary metabolites are prenylated phenolic compounds including flavonoids, stilbenes and 2-arylbenzofuranes with important biological activities such as cytotoxic, antimicrobial, antimalarial and antioxidants, that make Artocarpus becomes one of the interesting natural sources in bioavtive compound discovery. Further exploration of bioactive compounds from Artocarpus can also be conducted by advanced technologies such as developing culture of its endophytic fungi. Endophytic fungi are microorganisms living within tissues in plant organ, such as in roots, stems, leaves, tubers, fruits and flowers, without causing any visible manifestation of disease to the host plants. Endophytic fungi have been reported to synthesize secondary metabolites with various structures and bioactivities. However, there are no reports regarding the chemical investigation of endophytic fungi derived from Artocarpus. In this research, the chemical constituents of endophytic fungi isolated from Artocarpus were investigated. The purpose of this research was isolation and identification of endophytic fungi from A. heterophyllus and A. champeden and then isolation, identification and bioactivity evaluation of secondary metabolites obtained from endophytic fungi. Endophytic fungi were characterized based on analysis of the ITS region on the rDNA. Endophytic fungi were cultivated on PDB media and incubated at 28 0C for their optimum cultivated time. Then, the mycelia and media were separated using Buchner funnel. The mycelia were extracted with MeOH (three times) and the media were extracted with EtOAc (three times) to obtain methanol and ethyl acetate extract. Fractionation and purification of secondary metabolites from the extracts of endophytic fungi were conducted using various chromatographic techniques including vacuum liquid, column and radial chromatography. The structures of the isolated compounds were determined by means of spectroscopic data, NMR 1D (1H and 13C), NMR 2D (HSQC, HMBC, NOESY and COSY) and MS. Secondary metabolites obtained from endophytic fungi were evaluated for their cytotoxicity against murine leukemia P-388 cells according to MTT assay method expressed in IC50 value. The isolated compounds were also examined for their antimicrobial activities against five bacteria (Escherichia coli, Bacillus subtilis, Pseudomonas fluorescens, Micrococcus luteus, Saccharomyces cerevisiae and Sporobolomyces salminocolor) and a fungus (Sporobolomyces salminocolor) following agar dilution method expressed in inhibition zone. Two endophytic fungi identified as Pestalotiopsis microspora and Diaporthe lithocarpus have been isolated from the tissues of A. heterophyllus and Fusarium solani has been isolated from A. champeden. All endophytic fungi were reported for the first time from Artocarpus. In this research, twenty-two compounds have been isolated from the endophytic fungi where three of them are new compounds and two other compounds are firstly reported from endophytic fungi. Among twenty-two compounds, eight compounds have been obtained from P. microspora, five compounds from F. solani and nine compounds from D. lithocarpus. The eight compounds isolated from P. microspora were a new lactone, (+)-acetylpestalotine (1), three known lactones ((-)-pestalotine (2), (6S,7S,8R)-hydroxypestalotine (3) and necpiron D (5)), a lignan (+)-pinoresinol (4) and three sesquiterpene derivatives (+)-cydonicacid (6), (+)-cydowicacid (7) and (+)-dendocarbin L (8). Moreover, the five other compounds obtained from F. solani were a new alkaloid fusarindopyrrolidine (12), a known 2-azaanthraquinone alkaloid 7-desmethylskorpinone (13) and three skitalon and isokleron derivatives (3,4,8-trihydroxy-1(2H)-naphtalenone (9), 8-metoxynaphtalen-1-ol (10) and 1,8-dimethoxynaphtalena (11)). Additionally, the nine other compounds yielded from D. lithocarpus consisted of a new compound diaporthindoicacid (14), a known tricotesen macrolide derivative mirotesin C (15), two anthrquinone derivatives (1,2,8-trihydroxyianthraquinone (16) and emodin (17)), two coumarin derivatives (scopoletin (18) and coumarin (19)), two phenyl ethanol derivatives (2-phenylethanol (20) and tyrosol (21)) and arbutin (22). Discovery the new compounds (1,12 and 14) from each endophytic fungus in this research showed that endophytic fungus is a potencial source in searching the new secondary metabolites. The cytotoxic properties of isolated compounds against murine leukemia P-388 cells showed that the most compounds isolated from P. microspora were active and moderate (IC50 6.37-33.17 ?M). Among eight compounds from P. microspora, two compounds (5 and 7) were active against murine leukemia P-388 cells (IC50 6.37 and 7.30 ?M) and compounds 1-4 and 6 were moderate with IC50 in the range of 10.10-33.17 ?M. All compounds (9, 10 and 11) from EtOAc extract of F. solani tested against murine leukemia P-388 cells were not active, whereas compounds (12 and 13) from MeOH extract were active with IC50 values of 6.03 and 7.65 ?M, respectively. Furthermore, two compounds (15 and 17) obtained from D. lithocarpus were active against murine leukemia P-388 cell (IC50 values of 0.63 and 1.52 ?M), a compound (22) was moderate and six other compounds (14, 16, 18-21) were not active. The analysis of structure-activity relationship suggested that the existence of the hidroxyl group at C-9 and C-8 in lactone derivatives (2, 3 and 5) can increase the activity. The presence of the cyclic ring in bisabolane-type sesquiterpenes (6 and 7) can enhance the cytotoxic activity. Meanwhile, the presence of a methyl group at C-6 and a hidroxyl group at C-3 in anthraquinone derivatives (16 and 17) is the significant factor to increase the cytotoxicity. The antimicrobial activities of isolated compounds showed that tricotesen macrolide derivative, mirotesin C (15), was most active against six microbes with inhibition zone values of 14.17-16.00 mm. The bisabolane-type sesquiterpene cydowicacid (7) exhibited anti microbial activies against B. subtilis dan M. luteus, E. coli, P. fluorescens and S. salminocolor. Two anthraquinone derivatives, 1,2,8-trihydroxyianthraquinone (16) and emodin (17), were also active to inhibit the growth of microbes. Compound 16 was active against E. coli, B. subtilis and S. cerevisiae, while compound 17 was active against E. coli, B. subtilis, P. fluorescens, M. luteus and S. cerevisiae with the values of inhibition zone in the range of 11.67-14.67 mm. A new compound (+)-acethylpestalotine (1) also showed antimicrobial activity against B. subtilis with an inhibition zone value of 12.17 ± 0.29 mm. Meanwhile, the other compounds were incative as antimicrobial agent. Results of this research showed that compounds 7, 15 and 17 could be used as the candidate of lead compounds for cancer and anti microbes. Compounds 7, 15 and 17 were isolated from endophytic fungi derived from A. heterophyllus. The results of this study indicated that endophytic fungi can produce the new compounds and can also synthesize the same secondary metabolites with the host plant Artocarpus. Furthermore, the invention of all compounds from endophytic fungi isolated from Artocarpus can enhance the phytochemical information including the diversity of structures and skeletones.