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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. |
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