Bioassay Guided Extraction, Isolation and Characterization of Phytoconstituents from Datura Innoxia Mill.

Abstract

Natural products have been used to treat human disease since the dawn of medicine. The present study aimed to isolate and characterize bioactive lead compounds from an underexplored medicinal folklore D. innoxia Mill. Total 12 extracts from each of the leaf, stem and fruit part were prepared by sonication aided maceration as the extraction technique. The extracts were subjected to a range of phytochemical and in vitro biological assays in order to optimize most operative plant part and extraction solvent for preparative extraction. Phytochemical analysis included standard colorimetric assays to determine phenolic and flavonoid contents whereas, RP-HPLC was performed to quantify specific polyphenolic compounds. MTT assay was used to determine antipromastigote activity against L. tropica while disc diffusion assay was used to establish antibacterial, antifungal as well as protein kinase inhibitory spectrum. Starch-iodine chromogenic assay elucidated the α-amylase inhibitory potential whereas, brine shrimp lethality, MTT and SRB assays were employed to determine cytotoxic potential of the subject plant. Highest amount of gallic acid equivalent phenolic and quercetin equivalent flavonoid contents were obtained in the distilled water and ethyl acetate-ethanol extracts of leaf i.e. 29.91 ± 0.12 and 15.68 ± 0.18 mg/mg dry weight (DW), respectively. RP-HPLC detected significant amounts of catechin, caffiec acid, apigenin and rutin ranging from 0.16–5.41 μg/mg DW. Highest DPPH radical scavenging activity (IC50 16.14 μg/ml) was recorded in the ethyl acetate-acetone stem extract. Maximum total antioxidant capacity and reducing power potential were obtained in the aqueous leaf and ethyl acetate stem extracts i.e. 46.98 ± 0.24 and 15.35 ± 0.61 mg ascorbic acid equivalent/g DW respectively. Ethanol-chloroform leaf extract manifested a noteworthy antileishmanial activity (IC50 3.98 ± 0.12 μg/ml). A significant antimicrobial activity was exhibited by leaf extracts against Micrococcus luteus and n-hexane fruit extract against Aspergillus niger (MIC 3.70 and 12.5 μg/ml, respectively). Moderate inhibition of α-amylase enzyme activity was observed in all the three plant parts whereas, ethyl acetate and methanol-chloroform extracts of leaf exhibited conspicuous protein kinase inhibitory activity with 22 mm bald phenotype. Significant cytotoxicity against brine shrimps (IC50 85.94 ± 0.16 μg/ml), THP-1 (IC50 3.49 ± 0.17 μg/ml) and Hep G2 (6.54 ± 0.10 μg/ml) cell lines was manifested by the methanol-chloroform leaf extract, n-hexane fruit extract and chloroform leaf extract, respectively. In view of the aforementioned results, leaf and fruit parts were selected for preparative Abstract x extraction with ethyl acetate-methanol (1:1) and chloroform as their extraction solvents, respectively. The preparative extracts of leaf (DCL) and fruit (DCF) were then partitioned using solid phase extraction and the resulting fractions were biologically evaluated to prospect fraction hits for lead development. In case of leaf fractions, moderately polar and polar fractions were found to be potential candidates for the isolation of antileishmanial compounds whereas polar fractions exhibited profound protein kinase inhibitory and cytotoxic activities. In case of fruit fractions nonpolar fractions were identified as potential hits for cytotoxic leads isolation. Total three compounds (CL-1, CL-3 and CF-5) were isolated using normal phase vacuum and medium pressure column chromatography as the isolation technique. Compound CL-3 demonstrated noteworthy antileishmanial activity (IC50 8.34 ± 1.21 μg/ml) and cytotoxic potential against MCF-7 (IC50 4.3 ± 0.93 μg/ml), LU-1 (6.9 ± 1.3 μg/ml) and PC3 (0.01 ± 0.001 μg/ml) cancer cell lines. In protein kinase inhibition assay, maximum bald growth inhibition zone of 11 ± 2.21 mm was formed around the CL-1 loaded disc whereas, CF-5 demonstrated remarkable cancer chemopreventive activity through inhibition of NFκB and NO production with IC50 1.1 ± 0.9 and 3.3 ± 0.6 μg/ml, respectively. Crystallography and NMR spectroscopy characterized the structure of CL-1, CL-3 and CF-5 as β-sitosterol, isowithametelin and (4a, 4b, 6b, 8b, 10b, 14a) 7, 10 dimethyl dinoroleanan-12 en-3-one (new terpenoid), respectively. In principle, the results of the current study endorses D. innoxia as a substantial source of bioactive lead compounds