Identification of Hepatoprotective Constituents in Limonium tetragonum and Development of Simultaneous Analysis Method using High‑performance Liquid Chromatography

Background: Limonium tetragonum, a naturally salt‑tolerant halophyte, has been studied recently and is of much interest to researchers due to its potent antioxidant and hepatoprotective activities. Objective: In the present study, we attempted to elucidate bioactive compounds from ethyl acetate (EtOAc) soluble fraction of L. tetragonum extract. Furthermore, the simultaneous analysis method of bioactive EtOAc fraction of L. tetragonum has been developed using high‑performance liquid chromatography (HPLC). Materials and Methods: Thirteen compounds have been successfully isolated from EtOAc fraction of L. tetragonum, and the structures of 1–13 were elucidated by extensive one‑dimensional and two‑dimensional spectroscopic methods including 1H‑NMR, 13C‑NMR, 1H‑1H COSY, heteronuclear single quantum coherence, heteronuclear multiple bond correlation, and nuclear Overhauser effect spectroscopy. Hepatoprotection of the isolated compounds against liver fibrosis was evaluated by measuring inhibition on hepatic stellate cells (HSCs) undergoing proliferation. Results: Compounds 1–13 were identified as gallincin (1), apigenin‑3‑O‑β‑D‑galactopyranoside (2), quercetin (3), quercetin‑3‑O‑β‑D‑galactopyranoside (4), (−)‑epigallocatechin (5), (−)‑epigallocatechin‑3‑gallate (6), (−)‑epigallocatechin‑3‑(3’’‑O‑methyl) gallate (7), myricetin‑3‑O‑β‑D‑galactopyranoside (8), myricetin‑3‑O‑(6’’‑O‑g alloyl)‑β‑D‑galactopyranoside (9), myricetin‑3‑O‑α‑L‑rhamnopyranoside (10), myricetin‑3‑O‑(2’’‑O‑galloyl)‑α‑L‑rhamnopyranoside (11), myricetin‑3‑O‑(3’’‑O ‑galloyl)‑α‑L‑rhamnopyranoside (12), and myricetin‑3‑O‑α‑L‑arabinopyranos ide (13), respectively. All compounds except for 4, 8, and 10 are reported for the first time from this plant. Conclusion: Myricetin glycosides which possess galloyl substituent (9, 11, and 12) showed most potent inhibitory effects on the proliferation of HSCs.