Three new compounds isolated from the whole plants of Salsola collina pall

Abstract One new alkaloid, 6, 7-dimethoxyisoquinoline-N-oxide (1), one new benzofuran derivative, 3,7-dimethyl-6-acetyl-8-benzofuranol (2) and one new lignan, salsolains A (3), along with seven known compounds (4–10), were isolated from the whole plant of Salsola collina Pall. Their structures were elucidated by extensive analysis of spectroscopic data (IR, UV, HR-ESI-MS, 1 D and 2 D NMR), and their absolute configurations were determined by the X-ray crystallography and ECD calculation. The activities of compounds 1–10 against inflammatory cytokines IL-6 and TNF-α levels on LPS-induced RAW 264.7 macrophages were assessed, especially, compound 5 (50 μM) exhibited the most significant anti-inflammatory activity with the secretion levels of IL-6 and TNF-α at 3.87% and 4.03%, respectively. Graphical Abstract


Introduction
Salsola collina Pall, belonging to the Salsola genus in Chenopodiaceae family, is widely distributed in the northeast of China. The whole plant is traditionally utilized for the treatment of burns, rabies bites and high blood pressure (Kuang et al. 1979). A metabolite profiling driven analysis of other two Salsola plants revealed that the Salsola genus contained various classes of phytochemical components, which showed potential important activities (Rasheed et al. 2013). While previous phytochemical investigations on the title plant have led to the isolation of alkaloids, lignans flavonoids and carbohydrates, etc. (Syrchina et al. 1989;Zhao and Ding 2004;Xiang et al. 2007). Some of these alkaloids and lignans showed diverse bioactivities, including antibacterial (Li and Zuo 2010;, anti-viral (Wu et al. 2016;Xu et al. 2019). and anti-inflammatory activities (Li et al. 2013;Zhang et al. 2017;Fang et al. 2018;Wu et al. 2018). As part of our continuing endeavors to search for significant bioactive constituents from the plants, our group have carried out the current experimental study of the extracts from the Salsola collina Pall. As a result, one new alkaloid (1), one new benzofuran derivative 3,7-dimethyl-6-acetyl-8-benzofuranol (2) and one new lignan, salsolains A (3), together with seven known compounds (4-10) (Figure 1), were isolated and identified. Herein, we reported the isolation, structural elucidation, as well as the anti-inflammatory activities of these compounds.
Pro-inflammatory cytokines such as TNF-a, IL-1b, IL-6, IFN-c are the key mediators for the pathogenesis of osteoarthritis, inflammation is a physiological response of the body, mediated by inflammatory or immune cells to against harmful injury (Beg 2002). Hence, the inhibition of proinflammatory cytokines will be an important strategy for the treatment of these inflammatory conditions (Huang et al. 2015). Suppression of TNF-a and IL-6 production in macrophages is a key anti-inflammatory approach and may provide a strategy for drug development. In this investigation, we further evaluated the anti-inflammatory activities of the isolated compounds 1-10 using exposure to LPS-induced IL-6 and TNF-a production in the mouse macrophage RAW264.7 cell line. As shown in (Table S2), compounds 1-10 exhibited the secretion levels of IL-6 ranging from 3.87% to 87.92%, and the secretion levels of TNF-a ranging from 4.03% to 60.81%. Among of them, compound 5 showed the most significant activity with the secretion levels of IL-6 and TNF-a at 3.87% and 4.03%, respectively.

General experimental procedures
HRESIMS data were carried out using an Agilent 6210 ESI/TOF mass spectrometer (Massachusetts, USA). 1 D and 2 D NMR spectra were obtained on a Bruker AV-400 spectrometer with TMS as internal standard. Preparative HPLC were performed using a COSMOSIL C18 preparative column (5 lm, 20 Â 250 mm). TLC analysis was performed using silica gel GF 254 plates (Yantai Chemical Industry Research Institute, Yantai, China). Optical rotations were acquired by a Jasco P-1020 digital polarimeter (JASCO, Tokyo, Japan). UV spectra were measured by a JASCO V-550 UV/VIS spectrophotometer (JASCO, Tokyo, Japan). A JASCO FT/IR-480 plus FT-IR spectrometer was used for scanning the IR spectra with KBr pellets (JASCO, Tokyo, Japan). All chemical reagents were purchased from Tianjin Damao Chemical Company (Tianjin, P. R. China).
3.4. X-ray diffraction analysis X-ray crystallographic analysis of 2 was performed on an Agilent Gemini S Ultra CCD diffractometer with Cu Ka radiation.

Cell viability assay
RAW 264.7 cells were purchased from American Type Culture Collection. Cells were seeded in a 96-well plate at the density of 5 Â 10 4 cells/mL for 24 h and then cells were treated with compounds 1-10 for 24 h. The mixture was then removed and each well of the plates was incubated with 30 ml of MTT (5 mg/mL) at 37 C for 4 h. After complete removal of the supernatant, DMSO (200 lL/well) was added into the plates and to dissolve the formazan produced in the cells. The absorbance was recorded by a microplate reader at 570 nm. As a result, compounds 1-10 at the concentration of 50 mM had no obvious cytotoxic activities on RAW 264.7 cells after 48 h treatment.

Assay of enzyme-linked immunosorbent assay (ELISA)
To quantify the level of proinflammatory cytokines, RAW 264.7 cells were placed in a 24-well plate at a density of 2 Â 10 5 /mL for 24 h. Then, cells were pretreated with 50 lM of isolated compounds 1-10 for 12 h before stimulation with LPS for another 12 h. The level of IL-6 and TNF-a in cell supernatants were quantify with ELISA kits according to the manufacturer's protocols. In this assay, data were expressed as mean ± SD. Statistical significance was considered when P value < 0.05.

Conclusion
In summary, a phytochemical study was carried out on Salsola collina Pall, one new alkaloid (1), one benzofuran derivative (2) and one new lignan (3), as well as seven known compounds (4-10) were isolated from this plant. Their structures were determined by detailed spectroscopic analysis, X-ray crystallography and ECD calculation. The anti-inflammatory activities of 1-10 on LPS-stimulated RAW 264.7 cells were measured ( Figure S6), and compound 5 exhibited stronger anti-inflammatory effect than that of positive control resveratrol.