ISOLATION, OPTIMIZATION AND CHARACTERIZATION OF SECONDARY METABOLITES FROM SOIL BORNE FUNGI SCRELOTIUM AND ASPERGILLUS SPECIES

Abstract

In the present dissertation, our research findings on the production of bioactive secondary metabolites by soil borne fungi are discussed. The application of microorganisms for the welfare of human beings is the main goal of biotechnology. In this study, two phytopathogenic fungi, Sclerotium rolfsii and Aspergillus flavus were isolated from soil samples collected from Malakand Division, Khyber Pakhtunkhwa, Pakistan. Growth parameters (nutrient media, temperature, pH, incubation period, and static/shaking intervals) were optimized for achieving maximum production of bioactive secondary metabolites. Five nutrient media were used for fungal growth. S. rolfsii produced maximum amounts of metabolites in Czapek yeast broth media (CYB), whereas maximum formation of metabolites by A. flavus was found in potato dextrose media (PDB). Furthermore, the crude secondary metabolites in ethyl acetate (EtOAc) and n-hexane extract obtained from each medium were screened for their activities against different pathogenic bacteria. The EtOAc and n-hexane fractions obtained from the CYB medium were more active against pathogenic bacteria as compared to the crude metabolites obtained from the other media in the case with S. rolfsii, whereas PDB was the media, in which A. flavus yielded the largest quantities of secondary metabolites, showed significant results against pathogenic bacteria. EtOAc and n-hexane fractions of both fungi were screened for their in vitro effects, including antifungal, phytotoxic, and insecticidal properties and brine shrimp lethality. The in vivo activities of the metabolites, such as acute toxicity, analgesic, and sedative action, were also studied. The results showed that the ethyl acetate (EtOAc) fraction was more active as compared to the n-hexane fraction due to the presence of polar constituents. The results obtained from the examinations of the biological activities indicated that both fungi produced bioactive secondary metabolites which have antimicrobial, phytotoxic, insecticidal, cytotoxic, analgesic, and sedative effects. The EtOAc fraction of S. rolfsii and A. flavus was significantly more active against carbonic anhydrase with IC50 values of 45.40 ±0.75 and 59.89 ±1.65, respectively. Similarly, the n-hexane fraction of both fungi also showed significant results against carbonic anhydrase (62.5 and 63% at 0.2 mg/mL, respectively) with IC50 values (45.40 ±0.75 and 61.3 ±1.75, correspondingly). The EtOAc and n-hexane fraction of both fungi exerted insignificant influence against urease. In short, the isolated crude metabolites exhibited remarkable inhibition activity against carbonic anhydrase. The bioactive EtOAc fraction of both fungi were further subjected to column chromatography (cc), which yielded one new and five known compounds. Their purity was confirmed by thin-layer chromatography. The structures of the isolated compounds were elucidated by using various modern spectroscopic analyses, including 1H-NMR, 13C-NMR, HMBC, and EI-MS spectra. One new and four known secondary metabolites were isolated from S. rolfsii and one known compound was isolated from A. flavus. The bioactive compounds isolated from S. rolfsii were: cinnamic acid (1), chlorogenic acid (2), Screlotiumol (3), o-coumaric acid (4), and gallic acid (5), whereas kojic acid (6) was isolated from A. flavus. Two compounds were obtained in high quantity, chlorogenic acid (2) and screlotiumol (3), and assessed for their effects on the reversion of multidrug resistant (MDR) mediated by P-glycoprotein (P-gp). In cancer cell lines, the multidrug resistant P-glycoprotein is a target for chemotherapeutic drugs. Both tested compounds showed an excellent MDR reversing impact against the mouse T-lymphoma cell line in a dose-dependent manner. Furthermore, compounds (2 and 3) were subject to molecular docking. Optimal effects of molecular docking were obtained by compounds (2 and 3) as compared to the standard treatment. Therefore, the preliminary results obtained in the present investigation indicate that these compounds could be used in the selection of potential targets for the treatment of cancer.