Abstract
Plant extracts and phytochemicals may prevent chronic diseases via activation of adaptive cellular stress response pathways including induction of antioxidant and phase II detoxifying enzymes. The regulatory regions of these inducible genes encode the electrophile-response element (EpRE). This study tested the EpRE induction ability of Maerua subcordata (fruit, leaf, root, seed) methanol extracts and selected candidate constituents thereof, identified by liquid chromatography coupled with multistage mass spectroscopy, employing an EpRE luciferase reporter gene assay using hepa-1c1c7 mouse hepatoma cells. A parallel Cytotox CALUX assay using human osteosarcoma U2OS cells was used to monitor any non-specific changes in luciferase activity or cytotoxicity. Results showed that fruit, root, and seed extracts were non-cytotoxic up to a concentration of 30 gram dry weight per litre but the leaf extract exhibited some cytotoxicity and that the leaf (despite some cytotoxicity), fruit, and seed extracts showed strong induction of EpRE mediated gene expression while induction by the root extract was minimal. Selected candidates included glucosinolates, isothiocyanates, and some biogenic amines. Subsequent studies showed that methyl-, ethyl-, isopropyl-, isobutyl- isothiocyanates, and sec-butyl thiocyanate as well as glucobrassicin induced concentration (1–100 μM) dependent EpRE-mediated gene expression while the biogenic amines stachydrine and trigonelline acted as inhibitors of EpRE-mediated gene expression at 100 μM. The identification of glucolepidiin, glucobrassicin, glucocapparin, stachydrine, and trigonelline in all extracts was confirmed using standards and based on multiple reaction monitoring; yet, glucobrassicin level in the root extract was negligible. In conclusion, this study provided a first report on EpRE mediated gene expression effects of M. subcordata; and despite detection of different glucosinolates in all extracts, those containing glucobrassicin particularly displayed high EpRE induction. Because EpRE inducers are cytoprotective and potential chemopreventive agents while inhibitors are suggested adjuvants of chemotherapy, results of this study imply that process manipulation of this plant may result in herbal preparations that may be used as chemopreventive agents or adjuvants of chemotherapies.
Highlights
Compelling scientific evidences revealed an inverse relationship between consumption of plant foods rich in some phytochemicals and the risk of several chronic diseases [1,2,3,4,5]
While a multitude of cellular targets are affected by phytochemicals, upregulation of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway could be a key factor in the cytoprotective properties of phytochemicals by means of which they promote cellular adaptation [1, 6,7,8,9]
Arecaidine hydrochloride was purchased from Alfa Aesar (Karlsruhe, Germany); N-acetylagmatine from Cayman Chemicals-Europe (Sanbio Uden, the Netherlands); sinigrin potassium salt and glucolepidiin potassium salt were from Extrasynthese (Genay Cedex, France); Effects of Maerua subcordata on electrophile-response element (EpRE)-mediated gene expression in vitro glucobrassicin potassium salt, stachydrine hydrochloride, and trigonelline hydrochloride were from PhytoLab (Vestenbergsgreuth, Germany); agmatine sulfate, dimethyl sulfoxide (DMSO), glucosinolate hydrolysis products, pipecolic acid, tert-butylhydroquinone, Viscozyme L, and resazurin sodium salt were from Sigma–Aldrich (Schnelldorf, Germany/ Zwijndrecht, The Netherlands)
Summary
Compelling scientific evidences revealed an inverse relationship between consumption of plant foods rich in some phytochemicals and the risk of several chronic diseases [1,2,3,4,5]. The basic protective mechanism has not been clearly explained yet, one suggested mechanism is that phytochemicals activate adaptive cellular stress response pathways. In the presence of Nrf activators (oxidative stress, some chemicals including phytochemicals), the Nrf dissociates from Keap and translocates in to the nucleus where it assembles to a cis-acting regulatory sequences called antioxidant response element (ARE) or electrophile-response element (EpRE), located in the promoter region of genes encoding various antioxidant and phase 2 detoxifying enzymes and resulting in their expression [8,10]. Several reactive cysteine residues in Keap function as sensors of cellular redox changes and oxidation/covalent modification of some of these critical cysteine thiols in Keap would hamper its role in Nrf degradation thereby stabilizing Nrf and facilitating its nuclear accumulation [8, 10,11,12]. Nrf inhibitors have been suggested as adjuvant therapies to sensitize cancers with high expression of Nrf2 [13] or as treatment options of chemo- and radio-resistant forms of cancer [14]
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