Chemical and Biological Investigation on Zanthoxylum rhetsa, Micromelum minutum and their Endophyte Isolates

Abstract

Zanthoxylum rhetsa (Roxb.) DC and Micromelum minutum (G. Forst.) Wight & Arn belonging to the family Rutaceae have been investigated in this research to isolate secondary metabolites and evaluate bioactivities. In addition, five endophytic fungi were isolated from Z. rhetsa and identified at the genus level. Phytochemical investigation of the two plants yielded twenty four compounds which were identified by extensive NMR (1H NMR, 13C NMR, DEPT-135, 1H-1H COSY, HSQC, HMBC and NOSEY) experiments and ESI mass spectrometry. The root bark of Zanthoxylum rhetsa afforded twenty one chemical compounds viz. 2,11-didemethoxy-vepridimerine A, N-methylatanine, 3-dimethylallyl-4,8-dimethoxy-1-methyl-2-quinolone, zanthodioline, 8-methoxy-N-methylflindersine, skimmianine, canthin-6-one, arnottianamide, 8-O-demethylchelerythrine, chelerythrine, 7-methoxynitidine, oxynitidine, xanthyletin, pluviatilol, (+)-piperitol-γ-γ-dimethylallylether, lupeol, β-sitosterol, stigmasterol, methyl oleate, methyl stearate and stearic acid. Among them 2,11-didemethoxy-vepridimerine A is a new dimeric prenylated quinolone alkaloid. 7-methoxynitidine also appeared as a new natural compound containing a benzophenanthredine nucleus, however, it was synthesized previously. N-methylatanine and 3-dimethylallyl-4,8-dimethoxy-1-methyl-2-quinolone are 2-quinolone alkaloids and isolated for the first time from the genus Zanthoxylum. The pyranoquinoline alkaloid, zanthodioline, two benzophenanthridine alkaloids, 8-O-demethylchelerythrine and oxynitidine and the lignan, pluviatilol are reported for the first time from Z. rhetsa. Phytochemical investigations of the methanolic extract of the whole plant of Micromelum minutum provided two coumarins namely micromelin and murrangatin as well as one sterol, stigmast-4-en-3-one, the latter being reported for the first time from M. minutum. To evaluate bioactivities, different fractions of the crude methanol extracts of the two plants, obtained by partitioning, and two pure compounds, isolated in the present study, were screened for antioxidant, cytotoxicity, thrombolytic and antimicrobial activity. In vitro antioxidant activity was assessed by DPPH radical scavenging method using butylated hydroxytoluene as the standard. Among the fractions tested, the aqueous and chloroform soluble fractions of Z. rhetsa exhibited significant antioxidant activities with IC50 values of 55.25±2.78 and 96.00±4.13 μg/ml respectively as compared to the standard 29.23±5.38 μg/ml. Among the different fractions of M. minutum tested, pet ether and chloroform soluble fractions showed prominent antioxidant activities with IC50 value of 49.46 and 67.53 μg/ml compared to the standard 31.02 μg/ml. 8-methoxy-N-methylflindersine showed good antioxidant activity with IC50 value of 71.18±1.74 μg/ml. In the brine shrimp lethality bioassay, the aqueous fraction of Z. rhetsa showed very promising cytotoxicity with LC50 value of 0.46 μg/ml as compared to the standard vincristine sulphate (LC50 0.43 μg/ml). The pet ether and chloroform fractions of M. minutum showed good brine shrimp larvicidal activity with LC50 values of 1.15 and 1.50 μg/ml respectively as compared to vincristine sulphate (LC50 0.27 μg/ml). In the thrombolytic assay, the aqueous fraction of Z. rhetsa again showed the highest activity when compared to other fractions tested with clot lysis 50.5%±3.3% and the standard streptokinase showed 70.6%±7.9% lysis. Zanthodioline and 8-methoxy-N-methylflindersine showed mild thrombolytic activities of 32.0%±9.0% and 17.5%±1.6%. In vitro antimicrobial activity was determined against six gram-positive and nine gram-negative bacteria by the standardized disc diffusion method using kanamycin as the reference drug. Carbon tetrachloride, crude methanolic extract and aqueous fraction of Z. rhetsa demonstrated the highest antimicrobial activity against Vibrio cholera, Bacillus subtilis and Klebsiella pneumonia respectively with zone of inhibition of 18.67±3.21, 14.33±1.53 and 14.0±3.61mm as compared to the standard 24.67±0.58, 19.33±2.08 and 25.33±1.53 mm. 8-methoxy-N-methylflindersine showed poor antimicrobial activity against Bacillus subtilis, Sarcina lutea, Staphylococcus aureus. Pet ether soluble fraction of M. minutum showed good antimicrobial activity against Staphylococcus aureus and Escherichia coli with zone of inhibition of 19.0 mm and 17.0 mm as compared to the standard kanamycin 22.0 mm and 24.0 mm respectively. A total of five endophytic fungi were isolated from the plant Z. rhetsa and were identified morphologically up to the genus level on the basis of macroscopic and microscopic characteristics as Colletotricum sp., Fusarium sp., Colletotricum sp., Clonostachys sp. and Mucor sp. Among of the fungal extract, the extract of Clonostachys sp. showed good brineshrimp larvicidal activity with LC50 value of 0.94 μg/ml as compared to the standard vincristine sulphate (LC50 0.48 μg/ml). Finally, the molecular docking and molecular dynamics (MD) simulations was performed on thirteen compounds isolated in this study to determine their binding affinities against the main protease (Mpro) of SARS-CoV-2 by using AutoDock Vina and the YASARA software package respectively. Among the pure compounds, 2,11-didemethoxy-vepridimerine A, N-methylatanine, 3-dimethylallyl-4,8-dimethoxy-1-methyl-2-quinolone, showed the binding affinity of -8.5, -6.3 and -6.1 Kcal/mol respectively as compared to the standard remdesivir, which showed the binding affinity of -7.8 Kcal/mol.