Isolation and Characterization of Alkaloids from Endophytic Fungi of Argemone mexicana L.: Their Bioactive Potential

Abstract

The present study is aimed to isolate the alkaloid producing endophytic fungi from Argemone mexicana and to investigate their therapeutic potential. Endophytic fungi are symbiotic microorganisms that reside inside the tissues of different parts of the plant. In this study, a total of 42 endophytic fungi were isolated, and 14 isolates were screened for the production of alkaloids. The Dragendroff test confirmed the presence of alkaloids in all crude extracts of the fungal isolates. Further, among all the fungal isolates revealed that AMEF-5 (128.67 ± 0.44 µg/ml), AMEF-9 (100.56 ± 0.36 µg/ml), AMEF-13 (119.78 ± 0.35µg/ml), and AMEF-14 (137.22 ± 0.56 µg/ml) showed the presence of higher alkaloid content. The selected crude extracts were investigated primarily for their bioactive potential. The crude extracts were studied for the preliminary bioassays: antibacterial, antifungal, antioxidant, and anti-inflammatory activities. Out of the 14 isolates, the three extracts, AMEF-5, AMEF-13, and AMEF-14 showed significant anti-inflammatory activity, while AMEF-9 showed maximum antibacterial activity. The above four isolates were identified as Acremonium alternatum (AMEF-5), Purpureocillium lilacinum (AMEF-9), Sarocladium implicatum (AMEF-13, and Cladosporium ramotenellum (AMEF-14) based on the ITS sequences. The obtained sequences were submitted to the NCBI database and accession numbers were obtained as OR759012 for Acremonium alternatum, PQ596420 for Purpureocillium lilacinum, PQ605663 for Sarocladium implicatum, and PQ163911 for Cladosporium ramotenellum. The crude extracts of the isolates AMEF-5, AMEF-9, AMEF-13, and AMEF-14 were purified via column chromatography, and fractions were pooled on the basis of the Rf values obtained from TLC. The pooled fraction were evaluated for their different bioactive potential. Out of the eight fractions of AMEF-5, fraction 3 showed maximum inhibition of lipoxygenase enzyme (IC50 -15.2 ± 0.09 µg/ml), scavenging of the nitric oxide radicals (IC50 -11.38 ± 0.35 µg/ml), protein denaturation (IC50-14.93 ± 0.4 µg/ml), trypsin inhibition (IC50-12.06 ± 0.64 µg/ml), and HRBC stabilization (IC50-11.9 ± 0.22 µg/ml). From the endophytic fungal isolate, AMEF-13, fractions 5 and 6 showed the maximum activity among the other four fractions. Fraction 5 exhibited maximum inhibition of lipoxygenase enzyme (IC50-17.2 ± 0.5 µg/ml), scavenging of the nitric oxide radicals (IC50-11.04 µg/ml), protein denaturation (IC50 7.57 µg/ml), trypsin inhibition (IC50-11.3 µg/ml), and HRBC stabilization (IC50-6.13 µg/ml). Fraction 6 showed the inhibition of lipoxygenase enzyme (IC50-12.1 ± 0.09 µg/ml), scavenging of the nitric oxide radicals (IC50-13.87µg/ml), protein denaturation (IC50-8.32 µg/ml), trypsin inhibition (IC50-13.76 µg/ml), and HRBC stabilization (IC50-7.34 µg/ml). A total of six fractions of AMEF-14 exhibited significant anti-inflammatory activity, but fraction 5 showed the maximum inhibition of lipoxygenase enzyme (IC50-24.3 ± 0.88 µg/ml), scavenging of the nitric oxide radicals (IC50-9.84 µg/ml), protein denaturation (IC50-6.12 µg/ml), trypsin inhibition (IC50-6.95 µg/ml), and HRBC stabilization (IC50-6.21 µg/ml). The preliminary antibacterial activity of the crude extract of the isolate AMEF-9 was more significant than that of the other isolates, and its fractions were further studied for their antimicrobial activity. Out of the 5 fractions of AMEF-9, fraction 3 showed the maximum inhibition against Escherichia coli (IC50-26.98µg/ml), Staphylococcus aureus (IC50-27.12µg/ml), Pseudomonas aeruginosa (IC50-22.49µg/ml) and Bacillus megaterium (IC50-18.32µg/ml). Based on the results of bioactivities, fraction 3 (AMEF-5), fraction 3 (AMEF-9), fractions 5 and 6 (AMEF-13), and fraction 5 (AMEF-14) were characterized, and identified as aconitine, strychnine, brucine, piperine and berberine. Present study results suggest that, the endophytic fungi of Argemone mexicana can be used as a sustainable source to explore the untapped alkaloids.