Hepatoprotective constituents from halophyte plant, Limonium tetragonum (Thunb.) Bullock, attenuating HSC-T6 proliferation and RAW264.7 activation

Halophytes are naturally salt-tolerant plants growing in high salinity soil. In the course of searching for hepatoprotective halophyte plants growing near southern and western seashores in Korea, it was found that the methanolic extract of Limonium tetragonum (Thunb.) Bullock (Plumbaginaceae) leaves showed the potent antiproliferative activity in murine hapetic stellate cell line, HSC-T6 cells. Due to the lack of information on hepatoprotective activities of L. tetragonum, our work was aimed at investigating antifibrotic activities of L. tetragonum in HSC-T6 cells and the relevant effects in hepatocytes and macrophage cells. Also, the bioactive constituents contributing to hepatoprotective effects of L. tetragonum were identified. Culturing HSC-T6 cells on uncoated plastic plates is known to cause spontaneous activation, leading to myoblastic phenotype, mimicking the process seen in vivo. The treatment of the activated HSC-T6 with the extract significantly attenuated cellular proliferation in dose-dependent manners for 48h incubation. Also, excessive production and deposition of collagen in the activated HSC-T6 cells was effectively reduced. To rule out the non selectivity of the sample, the cytotoxicity in primary cultured rat hepatocyte was examined. The hepatocytes were intact up to 48h in response to the treatment of all compounds at the concentration up to 100 µg/ml. A proinflammatory cytokine, TNF-α produced in lipopolysaccharide (100 ng/ml)-stimulated RAW264.7 macrophage cells was also inhibited by the pre-treatment of the compounds. To elucidate bioactive constituents from L. tetragonum leaves, the methanolic extract of L. tetragonum was suspended in water and successively partitioned with n-Hexane, CHCl₃, EtOAc and n-BuOH. The EtOAc soluble fraction which showed the most potent antiproliferative activity in HSC-T6 cells was further subjected to repeated column chromatography to elucidate six phenolic compounds including five flavonoids.

Keywords: Limonium tetragonum, Antifibrotic, Hepatic Stellate Cells, constituents