Comparative Insights into the Pharmacological Significance of Selected Pakistani Datura Species

Abstract

Medicine derived from plant sources have been used by human populations for their healthcare needs since antiquity. Phytopharmaceuticals developed from traditional medicine can provide effective therapeutic alternatives to conventional medications. The current study was designed to provide comparative insights into the pharmacological significance of two Datura species i.e., DS and DI (Datura stramonium Linn. and Datura innoxia Mill.). Initially, 32 extracts of root (R), stem (S), fruit (F) and leaf part (L) of both species were procured by successive extraction in solvents of increasing polarity i.e., n-hexane (NH), ethyl acetate (EA), methanol (M) and distilled water (Dw) using ultra-sonication assisted maceration process. The extracts were then examined through phytochemical and in vitro biological assays to determine the bioactive plant parts and most effective solvents for preparative scale extraction in latter phase of the study. Phenolic and flavonoid contents were estimated through colorimetric assays. The EA extracts of both species were the most proficient in terms of their phenolic and flavonoid contents, followed by NH and M extracts. The same trend was observed in antioxidant assays including total antioxidant capacity (TAC) and total reducing power (TRP) of the crude extracts. RP-HPLC analysis was done to detect and quantify selected polyphenols. Presence of pharmacologically significant polyphenolic compounds i.e., gallic acid, rutin, apigenin, myricetin, kaempferol and catechin was confirmed in EA and M extracts of DS and DI plants. EA leaf and fruit extracts of DS (DSL-EA and DSF-EA) while EA leaf and stem extracts of DI (DIL-EA and DIS-EA) were found to be most abundant in selected polyphenols. The preliminary in vitro cytotoxicity and anticancer potential was investigated using brine shrimp lethality and protein kinase inhibition assay respectively. Significant cytotoxicity was observed against brine shrimp larvae, 75% of DS extracts resulted in LC50 values of < 25 μg/ml while LC50 values < 23 μg/ml were recorded for 75% of DI extracts. In protein kinase inhibition assay, the EA leaf extract of both species showed significant results with DIL-EA and DSL-EA resulting in highest bald phenotype zones i.e., 19 and 12.50 mm respectively. Preliminary investigations revealed the noteworthy bioactivity of medium to low polarity extracts of both species, leaf part showed comparatively stronger bioactivity in these experiments and Dw extracts were excluded from further screening. The anticancer properties of remaining extracts (NH, EA and M) of both species were investigated by determining their cytotoxicity against PC-3, DRSML QAU xi MDA-MB 231 and MCF-7 cancer cell lines. DIL-EA was the most potent extract with IC50 < 3 µg/ml against each cancer cell line while among DS extracts, DSR-EA and DSL-EA displayed slight cytotoxicity. The extracts were then further shortlisted, and NH and EA leaf extracts of both species (DSL-NH, DSL-EA, DIL-NH and DIL-EA) were selected for the next phase of the study. The safety profile of these extracts was established via MTT assay against isolated human lymphocytes and rat macrophages and none of the extracts revealed any significant cytotoxicity against normal cells. DSL-EA at 20 µg/ml showed significant (72.65±1.98%) nitric oxide (NO) scavenging potential followed by DIL-EA (41.72±1.33%). The in vivo anti-inflammatory (acute carrageenan induced paw edema and croton oil induced anal edema), anti-depressant (tail suspension test) and analgesic potential (hot plate method) of these extracts was also investigated in Balb/c mice. DSL-EA and DIL-EA exhibited significant anti inflammatory activity and mild antidepressant and analgesic action. These results helped in selection of two most potent extracts for chronic in vivo studies, i.e., EA extracts of the leaf part of DS and DI (DSL-EA and DIL-EA). These two extracts were extensively examined in benzene induced leukemia and carbon tetrachloride (CCL4) induced liver damage in Sprague Dawley rats through a series of hematological, biochemical, histological and immunohistochemistry studies. Significant alleviative effects of DSL-EA led to the selection of this extract for preparative scale extraction and isolation of bioactive compounds. Three compounds, stigmasterol (compound a), ferulic acid (compound b) stigmasterol-D-glucoside (compound c) were isolated from DSL-EA using normal phase column chromatography. The structure of these compounds was confirmed using 1D and 2D nuclear magnetic resonance (NMR) techniques. The compounds selected for in vivo evaluation were stigmasterol-D glucoside (C1) and ferulic acid (C2). The antileukemic and anti-inflammatory potential of C1 and C2 was extensively investigated in Balb/c mice. Marked improvement in hematological, biochemical, histological and regulation of molecular markers of cancer and inflammation was observed in C1 and C2 treated groups. The extensive evaluation of the pharmacological potential of DS and DI in our study has helped in the scientific validation of their ethnomedicinal claims. Both species, particularly DS, proved to be a very prominent source of anticancer and anti-inflammatory agents.