Phytochemical profile by LC-MS/MS analysis and evaluation of antioxidant, antidiabetic, anti-Alzheimer, and anticancer activity of Onobrychis argyrea leaf extracts

Introduction

Studies on different species of Onobrychis, a member of the Fabaceae family, have revealed a number of biological activities with potential applications in alternative medicine. The phytochemical content of the plant Onobrychis argyrea subsp. argyrea and some of the important biological activities linked to its metabolism are quite limited. Therefore, this study was the first to determine the content of secondary metabolites in this species and to elucidate important biological activities related to metabolism.

Methods

LC-MS/MS was used for the quantitative analysis of bioactive compounds in the aerial part of O. argyrea extracts. The antioxidant properties of the extracts were assessed based on their ability to scavenge DPPH radicals and reduce iron ions. The anti-diabetic effect of the extracts was tested by the inhibition of α-amylase and α-glycosidase enzymes, and the anti-Alzheimer's ability was tested by the inhibition of the enzymes AChE and BChE. The cytotoxic effects of the extracts were tested on four different cancer lines, namely A-549, HT-29, MCF-7 and MDA-MB 453 using the XTT assay. The mechanism of the anti-cancer effect was determined by means of flow cytometry analysis on HT-29 cells.

Results

Twenty-nine phytochemicals were identified via the LC-MS/MS analysis in the plant extracts. The most abundant phytochemicals in the extracts were quinic acid, isoquercitrin, epicatechin, and routine, respectively. Antioxidant analysis showed that among all extracts, methanol extract (ME) was most effective in scavenging DPPH (IC₅₀: 23.93 ± 0.96 µg/mL) and in reducing iron ions (53.24 ± 2.14 mgTEAC/g). Enzyme activity studies demonstrated that ME is a potent inhibitor of α-glycosidase (IC₅₀: 15.06±0.64 µg/mL), AChE (IC₅₀: 24.75±0.76 µg/mL), and BChE (IC₅₀: 3.36±0.14 µg/mL). XTT assay results disclosed the strongest anti-proliferative activity of ME against the human colorectal adenocarcinoma HT-29 cell line with an IC₅₀ value of 33.35±0.92 µg/mL. Flow cytometry analysis revealed that ME caused mitochondrial membrane damage in HT-29 cells (**p<0.01) and subsequently induced apoptosis (**p<0.01) by activating the caspase pathway (**p<0.001).

Conclusion

O. argyrea, rich in bioactive compounds, showed remarkable antioxidant, antidiabetic, Alzheimer's, and anticancer properties in vitro, demonstrating that this plant contains secondary metabolites that may be potential drug candidates for the treatment of metabolic diseases.