Three new feruloyl glucuronopyranosyl glycerols from Eriocaulon buergerianum with their anti-fibrotic effects on hepatic stellate cells

Abstract Three new feruloyl glucuronopyranosyl glycerols, 1-O-α-D-(2′-feruloyl) glucuronopyranosyl glycerol (1), 1-O-α-D-(3′-feruloyl) glucuronopyranosyl glycerol (2), and 1-O-α-D-(4′-feruloyl) glucuronopyranosyl glycerol (3) were isolated and purified from the capitulum of Eriocaulon buergerianum Koern. Their structures were identified by spectroscopic and chemical methods. Molecular docking study showed that 1 is a potential inhibitor of TGF-β1 receptor. Bioassay results revealed that 1 exhibited strong inhibitory activity against the TGF-β-induced expression of α-smooth muscle actin (α-SMA) and fibronectin in human hepatic stellate LX-2 cells. The results in this study indicated that the new feruloyl glucuronopyranosyl glycerol 1 has preventive and therapeutic potentials against hepatic fibrosis. Graphical Abstract


Introduction
Liver fibrosis is a pathology result of wound-healing process due to overexpression of extracellular matrix (ECM) proteins produced by a variety of pathogenic factors, such as viral hepatitis, alcoholic toxicity, non-alcoholic steatohepatitis, fatty liver, autoimmune, and chronic persistent drug-induced liver injury (Tsuchida and Friedman 2017;Friedman 2003). In the liver, hepatic stellate cells (HSCs) play an important role in the progress of fibrosis, and which are considered as the main ECM source in fibrotic liver (Kumar et al. 2017). Following hepatic injury, continuous accumulation of ECM extremely rich in collagen I and III leads to scar deposition and liver fibrosis ). In addition, with the development of hepatic fibrosis, the excessive generation of a-smooth muscle actin (a-SMA) is noteworthy indicator of liver fibrosis (Caviglia et al. 2018). When HSCs are activated, transdifferentiated into the myofibroblast-like cell type, secreting platelet-derived growth factor (PDGF) and transforming growth factor (TGF-b) can accelerate the progression of hepatic fibrosis (Xu et al. 2012). Thus, inhibition the activation of HSCs has been considered to be a good therapeutic strategy in drug development for hepatic fibrosis .
Natural compounds derived from herbal medicines have played an irreplaceable role in combating liver fibrosis, because of their high activity and low risk of side effects. Some natural products have revealed anti-hepatic fibrosis effects by targeting HSCs activation, such as ferulic acid, glycyrrhetinic acid, salvianolic acid, curcumin and oxymatrine (Mu et al. 2018;Xu et al. 2015;Chan et al. 2020).
Eriocaulon buergerianum Koern. is a medicinal herb widely distributed in China, which was first recorded in Kaibao Materia Medica of the Song Dynasty (Jin et al. 2016). The dried capitulum with peduncle of E. buergerianum are used as Chinese herbal medicine Gu-Jing-Cao for treating Liver-Fire ascending, opacity of the cornea, swelling and pain of eyes (Chen and Feng 2012). Up to now, Gu-Jing-Cao has been used in the treatment of liver and eye diseases in China (Zhou et al. 2011). Previous studies have demonstrated that the predominant constituents of Eriocaulon genus plants are flavonoids (flavonols, flavones, isoflavones and xanthones) and naphthopyranones (Qiao et al. 2012;Fang et al. 2008;Ho and Chen 2002), with varied biological activities including antioxidant (Yuan et al. 2010), antitumor (Xu et al. 2013), neuroprotection (Wang et al. 2011), antibacterium (Fang et al. 2008) and anti-diabetes (Zhu and Ye 2010). However, no studies have been reported on the anti-hepatic fibrosis effect of E. buergerianum. Herein, we report the isolation and structure elucidation of three new feruloyl glucuronopyranosyl glycerols, 1-O-a-D-(2 0 -feruloyl) glucuronopyranosyl glycerol (1), 1-O-a-D-(3 0 -feruloyl) glucuronopyranosyl glycerol (2), and 1-O-a-D-(4 0 -feruloyl) glucuronopyranosyl glycerol (3) from water extract of E. buergerianum (Figure 1). The compounds 1-3 were evaluated as potential TGF-b type I (TGF-b1) receptor inhibitors by molecular docking, and further bioassay results revealed that the 1 inhibited TGF-b-induced expression of a-SMA and fibronectin in human hepatic stellate LX-2 cells.
By using the chemical method of acid hydrolysis (Yamada et al. 2010), the absolute configuration of the glucuronic acid moiety of 1, 2 and 3 was finally determined to be D-glucuronic acid (t R ¼ 16.8 min, its derivative of L-cysteine methyl ester) by comparing its retention time with those of the authentic samples of D-glucuronic acid (t R ¼ 16.8 min, its derivative of L-cysteine methyl ester) and L-glucuronic acid (t R ¼ 17.3 min, the derivative of D-cysteine methyl ester and D-glucuronic acid) (Supporting material Figure S34).
We also searched all the published papers from 1955 to 2022 in PubMed, and we found that all the reported natural glucuronide compounds (more than 240) isolated from plants, with the absolute configuration, are in D form (e.g. _ Zuchowski et al. 2014;Magid et al. 2006).

General experimental procedures
HR-ESI-MS were measured by an Agilent G6520 Q-TOF LC-MS spectrometer (Agilent Technologies Inc., Santa Clara, USA). Optical rotations were obtained on an Autopol VI (Rudolph Research Analytical, Hackettstown, NJ, USA). IR spectra were recorded on an IRAffinity-1S Fourier transform infrared spectrophotometer (SHIMADZU, Japan). UV spectra were measured on an Agilent 1260 infinity HPLC equipped with a DAD detector (Agilent Technologies Inc., USA). NMR spectra including 1 H NMR, 13 C NMR and 2 D NMR were recorded on a Bruker Avance III 500 spectrometer (Bruker Corporation, Rheinstetten, Switzerland), with chemical shifts referenced to tetramethylsilane (TMS) as an internal standard. DIAION HP20 (Mitsubishi Chemical Corporation, Japan), Reversed-phase C 18 silica gel (50 lm, YMC Co., Ltd, Kyoto, Japan) and Sephadex LH-20 (Pharmazia, Uppsala, Sweden) were used for column chromatography. 3.6. Anti-fibrotic effects on hepatic stellate cells 3.6.1. Cell culture and treatment Human hepatic stellate cell line LX-2 cells were purchased from Millipore Corporation. The cells were cultured with DMEM supplemented with 2% fetal bovine serum (FBS) (Gibco, Australia), 100 U/mL penicillin, and 100 mg/mL streptomycin, and incubated in 5.0% CO 2 at 37 C. LX-2 cells were plated in 24-well plates at a density of 60,000/well, overnight, starved with DMEM supplemented with 0.5% fetal bovine serum (FBS) for an additional 12 h, and then treated with the compound 1 at different concentrations (1, 10 and 40 lM) for 24 h.

Plant material
3.6.2. Cell viability assay LX-2 cells were plated at 10,000/well in 96-well plates and treated with 1 for 24 h. Cell viability was measured according to the instructions of the CellTiter 96V R AQ ueous Non-Radioactive Cell Proliferation Assay (MTS) kit (Promega).

Western blot analysis
The cells were lysed with SDS loading buffer. The collected protein samples were separated by 10% SDS-PAGE gel. After electrophoresis, proteins were transferred to a polyvinylidene difluoride membrane, blocked with 5% skimmed milk for 1 h at room temperature and incubated with the primary antibodies (GAPDH, CST; a-SMA, Abcam; fibronectin, ABclonal) overnight at 4 C. After incubation with secondary antibodies (1:5000; Jackson ImmunoResearch) for 1 h at room temperature, the blots were visualized using the Omni-ECL Ultrasensitive Chemiluminescence Detection Kit (Epizyme, Shanghai). Western blotting data were analyzed with Image Lab software.
3.6.4. Quantitative real-time PCR Total RNA was isolated from cells using AG RNAex Pro Reagent (Accurate Biotechnology). 1 lg of total RNA was reverse transcribed using ABScript II Reverse Transcriptase (ABclonal). The resulting cDNAs were amplified using 2X Universal SYBR Green Fast qPCR Mix (ABclonal) and a Stratagene Mx3005P instrument (Agilent Technologies). The expression was normalized to that of the control gene GAPDH. PCR primer sequences were as follows: GAPDH, Forward Sequence: GTCTCCTCTGAC TTCAACAGCG, Reverse Sequence: ACCACCCTGTTGCTGTAGCCAA; a-SMA, Forward Sequence: ACTGGGACGACATGGAAAAG, Reverse Sequence: TAGATGGGGACATT GTGGGT; fibronectin, Forward Sequence: GATGCCGATCAGAAGTTTGG, Reverse Sequence: GGTTGTGCAGATCTCCTCGT.