Four sesquiterpenoids from the vegetable raw material Schisandra chinensis (Turcz.) Baill.: leaves and stems

Abstract Four sesquiterpenoids A-D (1–4) were isolated from the ethanol extracts of the leaves and stems from Schisandra chinensis. Their structures and absolute configurations were elucidated by a combination of NMR, MS and ECD. Compounds 1–4 (10 μM) exhibited moderate hepatoprotective activities against APAP-induced LO2 cell damage with increasing cell viability by 18%, 17%, 16%, and 19% compared to the model group (bicyclol, 26%) at 10 μM, respectively. All the compounds displayed no cytotoxic activity against five human cell lines, which also suggested the safety of leaves and stems of S. chinensis as an edible vegetable in a degree. Graphical Abstract


Introduction
Schisandra chinensis (Turcz.) Baill., a deciduous woody liana belonging to the Schisandraceae family, is widely distributed in northeastern China, Japan and Korea (Xue et al. 2015). Simultaneously, the woody stems and leaves of S. chinensis are utilized as a folk herbal medicine for the treatment of asthma, influenza, frostbite and gastrointestinal dysfunction in northeastern China (Shao 1995). In addition, the leaves of S. chinensis are edible as a vegetable to enhance the immune function in Northeast China. It's reported that triterpenoids, lignans, and polysaccharides have been isolated from the woody stems and leaves of S. chinensis (Zheng et al. 2014;Yang et al. 2019;Zhao et al. 2020). Several studies have shown that the woody stems and leaves of S. chinensis possess anti-aging, anti-viral (Song et al. 2013) and antifeedant  activities. In order to further characterize the chemical constituents and explore their hepatoprotective activity, our teams used several chromatographic technologies to isolate four sesquiterpenoids (1-4) from the ethyl acetate extracts of the woody stems and leaves of S. chinensis (Figure 1). We report herein the isolation, structural elucidation and hepatoprotective activity assay of these compounds.
The stereochemistry of 1 was determined on the basis of the NOESY and circular dichroism. The NOESY correlation ( Figure S2) of H 3 -13 with H-7, H-4 established H-3, H-4, CH ¼ CH-6 and H 3 -13 were cofacial orientations and randomly assigned as b-oriented. Also, the coupling constant between H-3 and H-4 was 6.6 Hz, indicating that H-3 and H-4 were on the same side. The absolute configuration of the vicinal diol moiety in 1 was determined by ECD spectra of its complex with Mo 2 (OAc) 4 solution (Snatzke's method) (Frelek et al. 1999;Yan et al. 2014). According to the helicity rule relating the sign of the Cotton effect of the diagnostic O-C-C-O moiety (Tang et al. 2009), the negative Cotton effect at 310-340 nm in the ECD spectrum indicated a 5 R configuration, on the contrary, which is 5S configuration. Compound 1 was confirmed by ECD spectroscopy to have a negative Cotton effect ( Figure S1-S2). Therefore, the absolute configuration of C-5 was determined as R. In addition, the absolute configuration was determined by experimental and calculated ECD and assigned as (3S,4R,5R,6R)-absolute configuration ( Figure S3). Therefore, the structure of compound 1 was determined to be schiterpenoid A.
Compound 2, a colorless oil, was found to have a molecular formula of C 15 H 20 O 6 as shown by its positive HRESIMS ion peak at m/z 319.1152 [M þ Na] þ (calcd for C 15 H 20 NaO 6 , 319.1158). The 1 H NMR spectrum of 2 (Table S1)  . The 13 C NMR spectrum displayed six nonprotonated carbons including an olefinic (d C 152.1, C-9), two carbonyls (d C 170.1, C-15 and 180.9, C-11), a quaternary (d C 53.6, C-1), and two oxygenated quaternary (d C 89.8, C-5 and 82.9, C-6) carbons together with nine protonated carbons. The large vicinal coupling constant (15.6 Hz) between H-7 (d H 6.51) and H-8 (d H 6.69) suggested that the olefin moiety was in the E configuration. According to the above features, the NMR spectroscopic data of 2 were very similar to those of 8 0 -oxo-6-hydroxy-dihydrophaseic acid (Bai et al. 2012), except for the absence of resonance for an oxygen-bearing isolated methylene group and the presence of methyl group at d H 2.34 (3H, d, J ¼ 0.6 Hz), which indicated that a hydroxymethyl group at C-10 in 8 0 -oxo-6-hydroxy-dihydrophaseic acid was replaced by a methyl group in 2. It was further confirmed by HMBC correlations between the methyl proton at d H 2.34 and the olefinic carbons at d C 152.1 (C-9), 139.2 (C-8) and 122.4 (C-14). The relative configuration of 2 was deduced from a NOESY experiment, in which the correlations between H-14 and H-10 verified the conjugated diene was 8E,14Z-configuration (Bai et al. 2012). The correlation of H-3 with H 3 -13, H 3 -12; H-7 with H 3 -13, H 3 -12 established HO-3, HO-6 were cofacial orientations and randomly assigned as a-oriented, contrarily, CH 3 -12 and CH 3 -13 were assigned as b-oriented. ECD calculation succeeded in establishing the absolute configuration of compound 2 as (1S,3R,5R,6S)absolute configuration ( Figure S3). Therefore, the structure of compound 2 was determined to be schiterpenoid B  1H, m, H-9), and two olefinic protons at d H 5.94 (1H, dd, J ¼ 15.6, 6.6 Hz, H-7) and 5.77 (1H, dd, J ¼ 15.6, 1.2 Hz, H-8). The 13 C NMR spectrum displayed six nonprotonated carbons including a quaternary (d C 40.8, C-1), two oxygenated quaternary (d C 80.6, C-6 and 76.4, C-5) carbons and ten protonated carbons. According to the above features, the NMR spectroscopic data of 3 were very similar to those of tectoionol B (Francisco et al. 2008), the main differences included the following three aspects. Firstly, the large vicinal coupling constant (15.6 Hz) between H-7 (d H 5.94) and H-8 (d H 5.77) suggested that it had a set of trans-ene bonds in this structure (Sun et al. 2007). Secondly, a hydrogen of methylene group at C-4 in tectoionol B was replaced by a hydroxy group in 3. Even more remarkably, a double bond at C-5/C-6 in tectoionol B has been formed a ternary oxygen ring through oxidation dehydration reaction. This was further confirmed by HMBC correlations between the oxymethine proton at d H 3.58 and the proximal carbons at d C 80.6 (C-6), 76.4 (C-5), 66.6 (C-2) and 40.1 (C-3). In the NOESY spectrum, the correlations of H-2 with H-4, H 3 -12; H-7 with H-4, H 3 -13 established HO-2, HO-4 were cofacial orientations and randomly assigned as a-oriented, CH 3 -12 and CH 3 -13 were assigned as b-oriented. The absolute configuration was determined by experimental and calculated ECD and assigned as (2S,4R,5R,6S)-absolute configuration ( Figure S3). Therefore, the structure of compound 3 was determined to be schiterpenoid C.

General experimental procedures
Optical rotations were measured with a JASCO P2000 automatic polarimeter. UV spectra were recorded on a JASCO V-650 UV spectrophotometer. 1 D and 2 D NMR spectra were recorded on a Bruker Avance 600 spectrometer with solvent peaks as references. HRESIMS data were obtained with an Agilent 1290 Infinity liquid chromatography system and an Agilent 6540 UHD Accurate-Mass Q-TOF mass spectrometer. High-performance liquid chromatography (HPLC) data were recorded on an Agilent 1260 instrument equipped with a photo-diode array (PDA) and a YMC C18 column (250 Â 4.6 mm, 5 lm). Preparative HPLC was a performed on Sanotac instrument China with a UV detector and a YMC C18 column (250 Â 20 nm, 5 lm). Column chromatographic separations were carried out with silica gel Qingdao Marine Chemical Group Corporation,Qingdao,China),SEP,Beijing,China) and ODS (50 lm, YMC, Kyoto, Japan). TLC was conducted with glass precoated with silica gel GF254 (Yantai Chemical Industrial Institute, Yantai, China). Chromatographic grade methanol and acetonitrile were purchased from Fisher. All other solvents were of chemical grade (Da Mao Chemical Co. Ltd., Tianjin, China).

Mo 2 (OAc) 4 induced electronic circular dichroism
According to the published procedure (Gao et al. 2021), about 1:1.2 mixture of diol-to-Mo 2 (OAc) 4 for compound 1 was prepared at the concentration of 1.0 mg/mL. Soon after mixing, the first ECD spectrum was recorded immediately, and its evolution was monitored until stationary (about 15 min after mixing). The inherent ECD of the diol was subtracted. The diagnostic band at around 310 nm in the induced ECD spectra were correlated to the absolute configuration of 1,2-diol unit.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by the National Key Research and Development Program of China (2017YFC1701200), Leading talents of science and technology innovation in Liaoning Province (XLYC1902101). In addition, the collection of medicinal materials was and assisted by the staff from Liaoning Juyuan Biotechnology Co. Ltd. Thanks for the above organizations and individuals.