A New Nortriterpenoid, a Sesquiterpene and Hepatoprotective Lignans Isolated from the Fruit of Schisandra chinensis

A new nortriterpenoid, 19(R)-hydroxyl-wuweizidilactone H (1), and a sesquiterpene, (6R)-β-chamigrenic acid (2), together with one known nortriterpenoid, wuweizidilactone H (3), and three known hepatoprotective lignans, micrantherin A (4), gomisin M2 (5) and schizandrin (6) were isolated from the fruit of Schisandra chinensis. Their structures were elucidated by UV, IR, HRESIMS, NMR spectra and X-ray analysis. Among them, the absolute configuration of 2 was confirmed for the first time. In vitro assays, compounds 4–6 (10 μM) exhibited hepatoprotective activities (survival rate: 44%, 43% and 44%) against damage induced by N-acetyl-p-aminophenol (APAP) in human liver carcinoma (HepG2) cells.


Introduction
Fructus schisandrae (Wuweizi in Chinese), the fruit of Schisandra chinensis (Turcz.) Baill., is a traditional Chinese medicine (TCM), and has officially been used as an astringent tonic for more than two thousand years in China [1][2][3][4]. It is always recorded in the Chinese Pharmacopoeia. S. chinensis grows mainly in China, Japan, Korea and Eastern parts of Russia [1,5,6]. Outside China, the fruit of S. chinensis has had monographs in Japanese (2006), Korean (2002), Russian (1990) and American (1999) Pharmacopoeias [7,8]. A monograph on the fruit of S. chinensis has also been included in European Pharmacopoeia since 2008 [7,8]. The first official, internationally recognized monograph on this raw material has been available since 2007 in the international Pharmacopoeia edited by WHO [7,8]. Many types of compounds have been isolated from S. chinensis, including lignans, nortriterpenes, sesquiterpenes and phenolic acids. Some of them, especially dibenzocyclooctadiene lignans, have diverse liver healing properties [9,10]. Bifendate, an effective hepatoprotective drug used clinically for almost forty years, was derived from dibenzocyclooctadiene lignans of S. chinensis [11,12]. Although a lot of research on the fruit of S. chinensis was reported, it is still an interesting subject, and is a hot topic within medicinal chemistry and drug discovery community, and has been studied increasingly in recent years [13,14].

Structural Analysis
In this study, the CH2Cl2 fraction of the 75% EtOH extract of the fruit of S. chinensis yielded one new nortriterpenoid, 19(R)-hydroxyl-wuweizidilactone H (1), a sesquiterpene, (6R)-β-chamigrenic acid (2) (the absolute configuration of which was confirmed for the first time), one known nortriterpenoid, wuweizidilactone H (3), and three known hepatoprotective lignans, micrantherin A (4), gomisin M2 (5) and schizandrin (6) ( Figure 1). The structures of these compounds were elucidated on the basis of spectroscopic or X-ray analysis. Compound 1, obtained as white crystal (MeOH), m.p. > 200 °C, had a molecular formula of C28H36O11 derived from its HRESIMS spectrum at m/z 571.2156 [M + Na] + , and showed 11 degrees of unsaturation. The IR spectrum of 1 showed the presence of hydroxyl groups (3404 cm −1 ), a carbonyl group (1767 cm −1 ), and a double bond (1668 cm −1 ). The 1 H-and 13 C-NMR spectra displayed that 1 contained 28 carbons, including four methyl groups (one secondary and three tertiary carbons), five methylenes (five aliphatic carbons), ten methines (four oxygenated, one olefinic, and five aliphatic carbons), and nine quaternary carbons (two carbonyls, six oxygenated, and one olefinic carbon). These observations suggest that 1 has a highly oxygenated nortriterpenoid with a schisanartane skeleton that has eight rings, one double bond, and two carbonyl groups matching the observed degrees (11) of unsaturation. The analysis of NMR data of 1 and HRESIMS revealed that the structure of 1 was similar to that of wuweizidilactone H (3) [16], except for an extra hydroxyl group at C-19.  Compound 1, obtained as white crystal (MeOH), m.p. > 200 • C, had a molecular formula of C 28 H 36 O 11 derived from its HRESIMS spectrum at m/z 571.2156 [M + Na] + , and showed 11 degrees of unsaturation. The IR spectrum of 1 showed the presence of hydroxyl groups (3404 cm −1 ), a carbonyl group (1767 cm −1 ), and a double bond (1668 cm −1 ). The 1 H-and 13 C-NMR spectra displayed that 1 contained 28 carbons, including four methyl groups (one secondary and three tertiary carbons), five methylenes (five aliphatic carbons), ten methines (four oxygenated, one olefinic, and five aliphatic carbons), and nine quaternary carbons (two carbonyls, six oxygenated, and one olefinic carbon). These observations suggest that 1 has a highly oxygenated nortriterpenoid with a schisanartane skeleton that has eight rings, one double bond, and two carbonyl groups matching the observed degrees (11) of unsaturation. The analysis of NMR data of 1 and HRESIMS revealed that the structure of 1 was similar to that of wuweizidilactone H (3) [16], except for an extra hydroxyl group at C-19. The HMBC cross-peaks of H-19 (δ H 4.22)/C-9 (δ C 82.4) and 19-OH (δ H 5.56)/C-10 (δ C 101.5) determined this hydroxyl group's position is at C-19. The positions of the other three hydroxyl groups were confirmed by the HMBC correlations of 7-OH (δ H 4.61)/C-6 (δ C 31.7), 9-OH (δ H 4.62)/C-11 (δ C 35.2) and 12-OH (δ H 4.41)/C-11 (δ C 35.2) ( Figure 2). The strong NOE correlations of 19-OH/9-OH/12-OH determined the β-orientation of the H-19 ( Figure 2). HRESIMS, UV, IR, 1D and 2D NMR spectra data see Supplementary data.  The structure of 1 was completely determined by single crystal X-ray diffraction analysis ( Figure 3). Accordingly, the structure of 1 was elucidated as 19(R)-hydroxyl-wuweizidilactone H ( Figure 1). Compound 2, obtained as white crystals (MeOH), m.p. > 200 °C, showed a molecular ion peak in the HRESIMS spectrum at m/z 233.1542 [M − H] − , which was consistent with a molecular formula of C15H22O2 and indicative of five degrees of unsaturation. The data of 1 H-, 13 C-NMR and HMBC correlations ( Figure 2) confirmed the planar structure of 2 is the same as β-chamigrenic acid [20]. The complete absolute configuration of C-6 in 2 was determined by single crystal X-ray diffraction analysis ( Figure 3). Accordingly, the structure of 2 was elucidated as (6R)-β-chamigrenic acid ( Figure 1). HRESIMS, UV, IR, 1D and 2D NMR spectra data see Supplementary data.
Though the structure of (6S)-β-chamigrenic acid had been shown in published papers, the absolute configuration of C-6 had not been confirmed by any data [20]. In addition, Scifinder recorded the structure of (6R)-β-chamigrenic acid (CAS 103089-10-7) from a Japanese patent [21], but actually, only the structure of (6S)-β-chamigrenic acid was shown in this patent [21], which was also not validated by any supporting data.
The  The structure of 1 was completely determined by single crystal X-ray diffraction analysis ( Figure 3). Accordingly, the structure of 1 was elucidated as 19(R)-hydroxyl-wuweizidilactone H ( Figure 1).  Figure 2) confirmed the planar structure of 2 is the same as β-chamigrenic acid [20]. The complete absolute configuration of C-6 in 2 was determined by single crystal X-ray diffraction analysis ( Figure 3). Accordingly, the structure of 2 was elucidated as (6R)-β-chamigrenic acid ( Figure 1). HRESIMS, UV, IR, 1D and 2D NMR spectra data see Supplementary data.
Though the structure of (6S)-β-chamigrenic acid had been shown in published papers, the absolute configuration of C-6 had not been confirmed by any data [20]. In addition, Scifinder recorded the structure of (6R)-β-chamigrenic acid (CAS 103089-10-7) from a Japanese patent [21], but actually, only the structure of (6S)-β-chamigrenic acid was shown in this patent [21], which was also not validated by any supporting data.
The  The structure of 1 was completely determined by single crystal X-ray diffraction analysis ( Figure 3). Accordingly, the structure of 1 was elucidated as 19(R)-hydroxyl-wuweizidilactone H (Figure 1).  (Figure 2) confirmed the planar structure of 2 is the same as β-chamigrenic acid [20]. The complete absolute configuration of C-6 in 2 was determined by single crystal X-ray diffraction analysis ( Figure 3). Accordingly, the structure of 2 was elucidated as (6R)-β-chamigrenic acid (Figure 1). HRESIMS, UV, IR, 1D and 2D NMR spectra data see Supplementary data.
Though the structure of (6S)-β-chamigrenic acid had been shown in published papers, the absolute configuration of C-6 had not been confirmed by any data [20]. In addition, Scifinder recorded the structure of (6R)-β-chamigrenic acid (CAS 103089-10-7) from a Japanese patent [21], but actually, only the structure of (6S)-β-chamigrenic acid was shown in this patent [21], which was also not validated by any supporting data.
The The structure of 1 was completely determined by single crystal X-ray diffraction analysis ( Figure 3). Accordingly, the structure of 1 was elucidated as 19(R)-hydroxyl-wuweizidilactone H (Figure 1).  The structure of 1 was completely determined by single crystal X-ray diffraction analysis ( Figure 3). Accordingly, the structure of 1 was elucidated as 19(R)-hydroxyl-wuweizidilactone H (Figure 1).   (Figure 2) confirmed the planar structure of 2 is the same as β-chamigrenic acid [20]. The complete absolute configuration of C-6 in 2 was determined by single crystal X-ray diffraction analysis ( Figure 3). Accordingly, the structure of 2 was elucidated as (6R)-β-chamigrenic acid ( Figure 1). HRESIMS, UV, IR, 1D and 2D NMR spectra data see Supplementary data.
Though the structure of (6S)-β-chamigrenic acid had been shown in published papers, the absolute configuration of C-6 had not been confirmed by any data [20]. In addition, Scifinder recorded the structure of (6R)-β-chamigrenic acid (CAS 103089-10-7) from a Japanese patent [21], but actually, only the structure of (6S)-β-chamigrenic acid was shown in this patent [21], which was also not validated by any supporting data.
The    (Figure 2) confirmed the planar structure of 2 is the same as β-chamigrenic acid [20]. The complete absolute configuration of C-6 in 2 was determined by single crystal X-ray diffraction analysis ( Figure 3). Accordingly, the structure of 2 was elucidated as (6R)-β-chamigrenic acid (Figure 1). HRESIMS, UV, IR, 1D and 2D NMR spectra data see Supplementary data.
Though the structure of (6S)-β-chamigrenic acid had been shown in published papers, the absolute configuration of C-6 had not been confirmed by any data [20]. In addition, Scifinder recorded the structure of (6R)-β-chamigrenic acid (CAS 103089-10-7) from a Japanese patent [21], but actually, only the structure of (6S)-β-chamigrenic acid was shown in this patent [21], which was also not validated by any supporting data.
The CHCl 3 )} [21]. Only one chiral carbon was in the structure of β-chamigrenic acid. Thus, it was concluded that the complete reported absolute configuration of β-chamigrenic acid should be R (but not S), which is the same as that of 2.

Hepatoprotective Activities of 4-6
To assess the biological activities of these three compounds, a human liver carcinoma cell (HepG2) injury model induced by N-acetyl-p-aminophenol (APAP) was adopted. Bicyclol, a hepatoprotective drug in clinic, was used as a positive control. As shown in Table 1, compounds 4-6 at a concentration of 10 µM showed moderate hepatoprotective activities.  (6) is one of the main lignans [8]. The good hepatoprotective effect of schizandrin (6) in vivo and in vitro has been studied before [22][23][24]. However, the hepatoprotective effect of micrantherin A (4) and gomisin M 2 (5) has not been reported yet. From our experiment in vitro, micrantherin A (4) and gomisin M 2 (5) showed a similar hepatoprotective effect as that of schizandrin (6), which suggested they also may be promising compounds for development of functional food materials beneficial to liver protection. The damaged HepG2 cell used in the hepatoprotective experiment model has been published in many papers [25][26][27], which suggests that this bioassay method is reliable.

Plant Material
The cultivated fruit of Schisandra chinensis was collected from Ji'an County, Tonghua City, Jilin

Hepatoprotective Effects of Compounds on Damaged HepG2 Cells Induced by APAP
The hepatoprotective effects of compounds 4-6 against damage induced by APAP in human liver carcinoma (HepG2) cells were determined by the MTT colorimetric assay as previously described [26,28]. Each cell suspension of 2 × 10 4 cells in 200 µL of RPMI 1640 containing fetal calf serum (10%), penicillin (100 U/mL), and streptomycin (100 µg/mL) was placed in a 96-well microplate and pre-cultured for 24 h at 37 • C under a 5% CO 2 atmosphere. Fresh medium (100 µL) containing bicyclol and test samples were added, and the cells were cultured for 1 h. Then, the cultured cells were exposed to 25 mM DL-galactosamine for 24 h. Then, 100 µL of 0.5 mg/mL MTT was added to each well after the withdrawal of the culture medium and incubated for an additional 4 h. The resulting formazan was dissolved in 150 µL of DMSO after aspiration of the culture medium. The optical density (OD) of the formazan solution was measured on a microplate reader at 492 nm.
The dibenzocyclooctadiene lignans are the main constituents in S. chinensis, and have attracted a lot of attention and been studied extensively since the 1970s [29][30][31]. Varied bioactivities of dibenzocyclooctadiene lignans, such as hepatic protection, anti-hepatitis B virus, anti-inflammation and cytotoxic activity have been reported previously [3,5,32,33]. However, no more than 20 nortriterpenoids have been found in S. chinensis since 2007, and only cytotoxic activity was reported [28]. Thus, the more nortriterpenoids and their related bioactivities need to be further studied extensively.