Artemlavanins A and B from Artemisia lavandulaefolia and Their Cytotoxicity Against Hepatic Stellate Cell Line LX2

Abstract Two new sesquiterpenoids, artemlavanins A (1) and B (3), together with fifteen known compounds (2 and 4−17) were isolated from the EtOH extract of Artemisia lavandulaefolia. The structures of new compounds were elucidated by extensive spectroscopic analyses (HRESIMS, 1D and 2D NMR) and ECD calculations. Compound 1 was a sesquiterpenoid lactone possessing a rearranged eudesmane skeleton; compounds 2–5, 6–8, 9 and 10–12 belonged to the eudesmane, guaiane, oppositane and farnesane sesquiterpenoids, respectively; compounds 13–17 were the phenyl derivatives with a 4-hydroxyacetophenone moiety. Twelve compounds (1–3, 5–7, 10–12, 14, 15 and 17) displayed cytotoxicity against hepatic stellate cell line LX2 (HSC-LX2) with IC50 values ranging from 35.1 to 370.3 μM. Compounds 2, 7, 10–12 and 17 exhibited the stronger cytotoxicity than silybin (IC50, 169.6 μM) with IC50 values of 82.1, 35.1, 95.0, 83.8, 81.6 and 90.1 μM. Compound 7 as the most active one showed significant inhibition on the deposition of human collagen type I (Col I), human hyaluronic acid (HA) and human laminin (HL) with IC50 values of 10.7, 24.5 and 13.3 μM. Graphic Abstract Electronic supplementary material The online version of this article (10.1007/s13659-020-00254-0) contains supplementary material, which is available to authorized users.


Introduction
Hepatic fibrosis is characterized by the abnormal accumulation of extracellular matrix (ECM), which results from the liver diseases such as viral hepatitis and alcoholic or nonalcoholic steatohepatitis. The global prevalence of alcohol-use disorders, diabetes and hepatitis B virus were approximately 75 million, 422 million and 257 million people [1], thus increasing the risks for liver cirrhosis or cancer and leading to a significant burden for liver diseases and medical costs. Although over 20 candidate molecules for hepatic fibrosis involving the inhibition of inflammatory response, ECM production and HSCs activation have achieved positive progress in recent years, most of which are being assessed in clinical trials and none of them have been commercialized as antifibrotic drugs [2][3][4][5]. Therefore, the discovery of antifibrotic agents with high efficacy and low side effect are urgently required. Recently, a review of 60 natural products with antihepatic fibrosis activity has been reported, indicating alkaloids, polysaccharides, flavonoids, polypeptides, terpenoids and polyphenols as the active ingredients [6].

Plant Materials
The

Extraction and Isolation
The air-dried aerial parts of Artemisia lavandulaefolia (20 kg) were powdered and extracted with 95% EtOH for two times (2 × 100 L × 4 day) at room temperature. The combined extract was concentrated under reduced pressure to yield the residue, which was suspended in H 2 O and subsequently partitioned with EtOAc. The EtOAc fraction (814 g) was subjected to silica gel column chromatography (CC) using EtOAc-petroleum ether (PE) gradient (0:100

Cytotoxicity Assay
The cytotoxicity of compounds was determined using the MTT method. Briefly, HSC-LX2 were cultured in 96-well plates at a density of 1 × 10 4 cells/well in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37 ℃ with 5% CO 2 . After overnight incubation, cells were treated with compounds at different concentrations for 48 h. Subsequently, the culture medium was exchanged by 100 μL of MTT reagent (1 mg/mL). After co-incubation for 4 h at 37 °C, the solution was removed and 100 μL of DMSO was added to dissolve the formazan crystals. The absorbance was recorded on a microplate reader at 490 nm. All the experiments were carried out in triplicate. The inhibitory ratios were calculated as [A (control) −A (sample) ]/A (control) × 100%, and IC 50 values were calculated using GraphPad Prism 5 (Graph-Pad Software, San Diego, CA, USA).

Col I, HA and HL Secretion Assay
HSC-LX2 were seeded at 8 × 10 4 cells/well in 24-well plates overnight and then treated with the compounds at different concentrations. After 72 h incubation, Col I, HA and HL levels of culture media were collected and determined using the commercial ELISA kits according to the manufacturer's instructions.