Isolation and characterization of bioactive metabolites from two ethno pharmacologically important Zingiberaceae plants of Bangladesh and their endophytic fungi

Abstract

The work explained in this thesis elaborates the isolation and identification of endophytic fungi from two ethnopharmacologically significant Zingiberaceae plants of Bangladesh, namely Curcuma longa L. and Zingiber officinale Rosc. The term “ethnophamacologically” means that these kinds of plants are traditionally used by specific groups of people for medicine purpose from very ancient period. A detail description was given for chemical screening of the crude extracts of the plants as well as their endophytic fungi. Isolation of secondary metabolites was done from the extracts of plants and their endophytic fungi by a combination of repeated column chromatography and preparative thin layer chromatography (PTLC). Structure clarification of pure compounds was made by their spectral data (GC-MS, 1D and 2D NMR). Finally bioassays of different extracts and isolated pure compounds were performed to observe antioxidant, antimicrobial and cytotoxic effect. The antioxidant activity was measured by the DPPH free radical scavenging method and antimicrobial activity was performed by the disc diffusion method. The cytotoxic activity of various extracts was completed by brine shrimp lethality bioassay. But the pure isolated compounds’ cytotoxic activity was analyzed on a variety of cell line cultures, for example, Lung cancer cell line A-549, kidney fibroblast cell line BHK-21, human cervical carcinoma cell line (HeLa). A network pharmacology-based approach in combination with molecular docking analysis was also used to identify a compound’s multiple targets that may have linked to its pharmacological action. A total of seven endophytic fungi were isolated from C. longa and, a total of five endophytic fungi were isolated from Z. officinale. All the endophytes were segregated from diverse parts of the plants (petiole, leaf, root, bark). Through morphological and molecular analysis it was revealed that among twelve endophytic fungi, eight are Fusarium species, three are Clonostacys species, and one is cladosporium species. All crude extracts contain flavonoids, coumarins, anthrocyanins, isocoumarins or their derivatives types of compounds which was revealed through chemical screening by thin layer chromatography (TLC). The crude methanolic extract of C. longa showed significant antioxidant (IC50 value 19.85 μg/ml) and cytotoxic activity (LC50 value1.81 μg/ml) compared to their standards. Its vaccuum liquid chromatography (VLC) fractions also showed significant antioxidant and cytotoxic activity. In case of Z. officinale, the plant methanolic crude extracts showed very significant antioxidant activity (IC50 value 2.08 viii μg/ml) and mild to moderate antibacterial activity (zone of inhibition 7-10 mm). Its fractions obtained through the modified Kupchan partition method also showed good antioxidant and antibacterial activity. The obtained results prove the phytochemical as well as pharmacological significance of these plants. A total of 27 metabolites and 21 metabolites were discovered through GC-MS analysis of crude methanolic extracts of C. longa and Z. officinale respectively. Most of them are bioactive constituents. Two compounds have been isolated from the plant C. longa, which were characterized as 4-hydroxy-3-methoxy cinnamic acid (CL-1) and methyl ferulate (CL-4). Among seven endophytic fungi isolated from this plant, CLRE-3 (Clonostachys rosea) was selected for large-scale cultivation based on chemical and biological investigation. From its ethyl acetate extract four compounds were isolated and characterized as Bassiatin (CLE-7), Nectriafurone (CLE-14), 8-O-methyl nectiafurone (CLE-11) and 8-O-methyl bostrycoidin (CLE-18)). Bassiatin, 8-O-methyl nectiafurone and 8-O-methyl bostrycoidin were obtained from this endophytic fungus for the first time. Among five endophytic fungi isolated from Z. officinale ZOLE-2 (Cladosporium cladosporoides) was selected for large-scale cultivation by observing its chemical and biological analysis. From its ethyl acetate extract two compounds were isolated and characterized as ergosterol (ZOE-1) and 3, 5, dihydroxy-ergosta-7, 22-diene-6-one (ZOE-6). Following biological investigation it was revealed that almost all isolated compounds were bioactive. CLE-18(2) and ZOE-6 indicated mild to moderate antibacterial activity against E. coli, P. aeruginosa and S. aureus bacteria with a zone of inhibition of 10 to 16 mm. CL-4 and CLE-7 showed moderate antibacterial activity against E. coli bacteria with a zone of inhibition of 10 mm. CL-1 showed noteworthy antioxidant activity with IC50 value 4.77 μg/ml in comparison with standards ASA (IC50 value 9.01 μg/ml) and BHA (IC50 value 11.42 μg/ml). It also exhibited cytotoxic activity against A-549 cancer cell line which was done by the trypan blue exclusion method. CLE-14 showed cytotoxic activity against BHK-21 cell line with IC50 value of 663.4μg/ml compared to standard doxorubicin (IC50 value of 327.1 μg/ml). It did not exhibit any activity against the HeLa cell line. CLE-7 also displayed cytotoxic activity against the A-549 cancer cell line. A computational study was done against Bassiatin (CLE-7) and lung cancer. Thus MMP9, PTGS2 and ERBB2 were unveiled as the top three common targets between the disease and compound. CLE-7 targets these proteins with fairly good binding score compared to ix standards based on molecular docking analysis. Thus it can be considered as a potential candidate for lung cancer treatment. The isolation of endophytic fungi and their chemical constituents from these plants have been revealed for the first time in Bangladesh through this investigation. From this study it is clear that these plants and their endophytic fungi would be a good resource of reference in the scientific world because of possessing various types of bioactive compounds. This analysis meets up the overall aims and objectives of the study for isolation and characterization of bioactive metabolites which can be used as a scaffold for innovative medicinal chemistry amendment to build up new drugs.