Development of HPLC Method for Quantification of Sinigrin from Raphanus sativus Roots and Evaluation of Its Anticancer Potential

Abstract

Sinigrin, a precursor of allyl isothiocyanate, present in the Raphanus sativus exhibits diverse biological activities, and has an immense role against cancer proliferation. Therefore, the objective of this study was to quantify the sinigrin in the R. sativus roots using developed and validated RP-HPLC method and further evaluated its’ anticancer activity. To achieve the objective, the roots of R. sativus were lyophilized to obtain a stable powder, which were extracted and passed through an ion-exchange column to obtain sinigrin-rich fraction. The RP-HPLC method using C18 analytical column was used for chromatographic separation and quantification of sinigrin in the prepared fraction, which was attained using the mobile phase consisting of 20 mM tetrabutylammonium: acetonitrile (80:20%, v/v at pH 7.0) at a flow rate of 0.5 mL/min. The chromatographic peak for sinigrin was showed at 3.592 min for pure sinigrin, where a good linearity was achieved within the concentration range of 50 to 800 µg/mL (R2 > 0.99), with an excellent accuracy (−1.37% and −1.29%) and precision (1.43% and 0.94%), for intra and inter-day, respectively. Finally, the MTT assay was performed for the sinigrin-rich fraction using three different human cancer cell lines, viz. prostate cancer (DU-145), colon adenocarcinoma (HCT-15), and melanoma (A-375). The cell-based assays were extended to conduct apoptotic and caspase-3 activities, to determine the mechanism of action of sinigrin in the treatment of cancer. MTT assay showed IC50 values of 15.88, 21.42, and 24.58 µg/mL for DU-145, HCT-15, and A-375 cell lines, respectively. Increased cellular apoptosis and caspase-3 expression were observed with sinigrin-rich fraction, indicating significant increase in overexpression of caspase-3 in DU-145 cells. In conclusion, a simple, sensitive, fast, and accurate RP-HPLC method was developed for the estimation of sinigrin in the prepared fraction. The data observed here indicate that sinigrin can be beneficial in treating prostate cancer possibly by inducing apoptosis.

Phytochemical identification and in silico study of ethanolic extract of white cabbage as a phosphodiesterase 1B inhibitor

“…WC was shown to contain sinigrin (2-propenyl glucosinolate) (65), which is a precursor of allyl isothiocyanate. This compound has diverse biological activities (66), including anticancer, anti-inflammatory, antibacterial, antifungal, antioxidant, and wound healing effects (67). In line with this study, the presence of sinigrin in WCE was previously reported by Dighe and Charegaonkar (68) and Amir et al (69).…”

Red Kale (Brassica oleracea L. ssp. acephala L. var. sabellica) Induces Apoptosis in Human Colorectal Cancer Cells In Vitro

“…Among the compounds identified in red kale subjected to prior in vitro digestion, sinigrin and spirostanol type saponins, usambaresine and hesperitin are known for their biological activity. Sinigrin is a compound that belongs to the glucosinolates, present in various plants of the Brassicaceae family, with well-documented anticancer activity [15,[21][22][23][24][25][26]. Studies have shown its effect on changes in gene expression in cancer cells.…”

“…Moreover, by preventing the activation of the NLRP3 inflammasome or the NF-B/MAPK pathways in macrophages, sinigrin reduces the generation of inflammatory mediators [23]. Sinigrin also induce overexpression of caspase-3 in DU-145 s cells and increase apoptosis [24].…”