Isolation, Structural Elucidation of Three New Triterpenoids from the Stems and Leaves of Schisandra chinensis (Turcz) Baill.

Schisandra chinensis (Turcz) Baill. is sufficiently well known as a medicinal plant worldwide, which modern research shows has many pharmacological activities such as hepatoprotective, anti-inflammatory effect, potent anti-HIV-1 activity, anti-tumor effect, and activity on the central nervous system. With considerable chemical investigation, three new triterpenoids (1–3), together with four known triterpenoids were isolated from the S. chinensis (Turcz) Baill. Their structures were elucidated by 1D- and 2D-NMR spectroscopic analyses, single-crystal X-ray diffraction and high-resolution mass spectroscopy, which were identified as Schisanlactone I (1), Schinalactone D, (2), Schisanlactone J, (3) Kadsuphilactone B (4), Schisanlactone C (5), Schisphendilactone B (6), and Schinchinenlactone A (7). The cytotoxicity of those compounds (1–7) was tested against Hep-G2 cell lines, but no apparent antitumor activity was observed at 50 µg/mL using MTT method.


Introduction
Schisandra chinensis (Turcz) Baill., Chinese magnolia vine, is widely distributed in China, the most eastern parts of Russia, Japan, Korea, USA, Europe, and all over the world [1]. The plant of S. chinensis (Turcz) Baill. can be found in the Chinese Pharmacopoeia, Russian Pharmacopoeia, Japanese Pharmacopoeia, Korean Pharmacopoeia, American Pharmacopoeia, and the International Pharmacopoeia [2]. According to various authors, the genus Schisandra includes from 20 to 30 species, and the major chemical composition of S. chinensis (Turcz) Baill. is lignans and triterpenes [2,3]. The seeds and fruits have been used to treat various diseases such as cough insomnia and arthritis [4]. Modern research shows it has many pharmacological activities such as hepatoprotective, anti-inflammatory effect, potent anti-HIV-1 activity, anti-tumor effect and activity on the central nervous system [5][6][7][8][9]. Most of phytochemical studies have concentrated on the analysis of fruits, and a great majority of them investigated lignans, but our studies of vegetative parts of stems and leaves are scarce. As a part of our effort to search for novel triterpene from S. chinensis (Turcz) Baill., we report here the isolation and structure determination of the new triterpenoids: Schisanlactone I (1), Schinalactone D (2), and Schisanlactone J (3), in addition to four known triterpenoids: Kadsuphilactone B (4), Schisanlactone C (5), Schisphendilactone B (6) and Schinchinenlactone A (7) (Figure 1). Kadsuphilactone B (4), Schisanlactone C (5), Schisphendilactone B (6) and Schinchinenlactone A (7) (Figure 1).

Structure Elucidation
The structures of the new compounds (1-3) were elucidated on the basis of extensive spectroscopic analyses, including a series of 1D-and 2D-NMR experiments ( 1 H-1 H COSY, HSQC and HMBC), single-crystal X-ray diffraction, IR spectrum, and mass spectrometry data. The known compounds (4-7) were identified by comparison of their experimental spectral data with the literature data [10][11][12].
Compound 1 was obtained as white amorphous powder with the molecular formula determined to be C32H50O5 according to the molecular ion peak [M + H] + at m/z 515.3737 (calculated for 515.3758) observed in HR-ESI-MS and NMR spectroscopic data. Analysis of NMR data (Table 1) indicated that compound 1 highly resembled Schisanlactone H [13]. The only difference was the replacement of the ethoxycarbonyl group in 1, which could be deduced by the HMBC correlations between H-31 (δH 4.11, q, J = 7.13 Hz) and methyl group (δC 14.57), ester group (δC 177.05), otherwise 1

Structure Elucidation
The structures of the new compounds (1-3) were elucidated on the basis of extensive spectroscopic analyses, including a series of 1D-and 2D-NMR experiments ( 1 H-1 H COSY, HSQC and HMBC), single-crystal X-ray diffraction, IR spectrum, and mass spectrometry data. The known compounds (4-7) were identified by comparison of their experimental spectral data with the literature data [10][11][12].
Compound 3 was obtained as white amorphous powder with the molecular formula determined to be C30H38O5 on the basis of the molecular ion peak [M + H] + at m/z 479.2790 (calculated for 479.2798) observed in HR-ESI-MS and NMR spectroscopic data. Analysis of its NMR data (Table 1) indicated that compound 3 highly resembled Lancilactone A [15]. The main difference was the carbonyl carbon (C-11) in 3 and the hydroxyl replacement group of C-6 in Lancilactone A. In the HMBC spectrum ( Figure 3), the correlations between H-19 (δH 6.72) and the carbonyl group (δC 199.85), between H-12 (δH 2.52 and 2.83) and the carbonyl group (δC 199.85) revealed the carbonyl group was located at C-11, meanwhile C-6 (δC 30.73) of 3 has no hydroxyl replacement group. So, the structure of compound 3 was identified as Schisanlactone J ( Figure 1).   Compound 2 was obtained as white amorphous powder with the molecular formula determined to be C 32 H 50 O 5 on the basis of the molecular ion peak [M + Na] + at m/z 537.3551 (calculated for 537.3556) observed in HR-ESI-MS ( Figure S18) and NMR spectroscopic data (Figures S11-S16). Analysis of its NMR data (Table 1) indicated that compound 2 highly resembled Schinalactone C [14]. The only difference was the replacement of the ethoxycarbonyl group in 2, which could be deduced by the HMBC correlations ( Figure S17) between H-31 (δ H 4.08, q, J = 7.12 Hz), methyl group (δ C 14.58), and ester carbonyl group (δ C 176.31); otherwise, 1 H-1 H COSY correlates ( Figure S17 Analysis of its NMR data (Table 1) indicated that compound 3 highly resembled Lancilactone A [15]. The main difference was the carbonyl carbon (C-11) in 3 and the hydroxyl replacement group of C-6 in Lancilactone A. In the HMBC spectrum (Figure 3), the correlations between H-19 (δ H 6.72) and the carbonyl group (δ C 199.85), between H-12 (δ H 2.52 and 2.83) and the carbonyl group (δ C 199.85) revealed the carbonyl group was located at C-11, meanwhile C-6 (δ C 30.73) of 3 has no hydroxyl replacement group. So, the structure of compound 3 was identified as Schisanlactone J (Figure 1). determined to be (5R, 8S, 10S, 13R, 14S, 17R, 20S, 22R) by X-ray diffraction using Cu Kα radiation with a Flack parameter of 0.04 (9). Thus, the structure of compound 1 was identified as Schisanlactone I (Figure 1). Compound 2 was obtained as white amorphous powder with the molecular formula determined to be C32H50O5 on the basis of the molecular ion peak [M + Na] + at m/z 537.3551 (calculated for 537.3556) observed in HR-ESI-MS and NMR spectroscopic data. Analysis of its NMR data (Table 1) indicated that compound 2 highly resembled Schinalactone C [14]. The only difference was the replacement of the ethoxycarbonyl group in 2, which could be deduced by the HMBC correlations between H-31 (δH 4.08, q, J = 7.12 Hz), methyl group (δC 14.58), and ester carbonyl group (δC 176.31); otherwise, 1 H-1 H COSY correlates H-31 (δH 4.08) with H-32 (δH 1.22). Thus, the structure of compound 2 was identified as Schinalactone D (Figure 1).
Compound 3 was obtained as white amorphous powder with the molecular formula determined to be C30H38O5 on the basis of the molecular ion peak [M + H] + at m/z 479.2790 (calculated for 479.2798) observed in HR-ESI-MS and NMR spectroscopic data. Analysis of its NMR data (Table 1) indicated that compound 3 highly resembled Lancilactone A [15]. The main difference was the carbonyl carbon (C-11) in 3 and the hydroxyl replacement group of C-6 in Lancilactone A. In the HMBC spectrum (Figure 3), the correlations between H-19 (δH 6.72) and the carbonyl group (δC 199.85), between H-12 (δH 2.52 and 2.83) and the carbonyl group (δC 199.85) revealed the carbonyl group was located at C-11, meanwhile C-6 (δC 30.73) of 3 has no hydroxyl replacement group. So, the structure of compound 3 was identified as Schisanlactone J (Figure 1).    The ethyl esters of compounds 1-2 may have been obtained during the extraction with ethanol. The compounds 1-7 were tested for cytotoxic effects against the Hep-G2 human tumor cell lines. Compared with the positive control cisplatin (IC 50 = 3.16 µg/mL), compounds 1-7 displayed no apparent cytotoxicity (IC 50 > 50 µg/mL).

General
Optical rotation was recorded on Autopol IV Automatic Polarimeter (RUDOLPH, Hackettstown, NJ, USA). IR spectra (Figures S10, S19 and S27) were obtained using the IRT racer-100 (SHIMADZU, Kyoto, Japan) with KBr pellets. NMR spectra were recorded on a Bruker DXR-600 instrument (600 MHz for 1 H and 150 MHz for 13 C) with TMS as an internal standard, and the deuterated solvent (CD 3 OD) were used to solubilize the samples. HR-ESI-MS was recorded on a UPLC-Q Exactive MS system (Thermo Fisher, Santa Clara, CA, USA). Silica gel (200-300, 300-400 mesh, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China) was used for the chromatography column. Semi-preparative HPLC was performed on an Agilent Technologies 1260 Infinity II system equipped with a diode array detector and C18 column (250 mm × 9.6 mm, 5 µm, Agilent Technologies, Santa Clara, CA, USA).

Extraction and Isolation
The dried plant material of S. chinensis (Turcz) Baill. (10 Kg) was extracted four times with 95% ethanol (25 L) at room temperature, 48 hours each time. After concentration, the extract was suspended in hot water and then partitioned with petroleum ether and EtOAc to give petroleum ether (A) and EtOAc ether (B, 151 g)

MTT Assay for Measuring Cell Cytotoxicity
Cytotoxicity assay was performed against human Hep-G2 tumor cell lines, which were seeded in 96-well plates and incubated for 24 h. The cells were incubated in DMEM medium. Subsequently, the cells were treated with concentration gradient of compounds 1-7 (cisplatin was employed as positive control) and incubated at 37 • C in 5% CO 2 for 24 h. Then, 10 µL MTT was added to each well and the mixture was incubated for 5 h. The medium was removed and after adding DMSO (150 µL/well) to each plate, the formazan crystals were dissolved and then absorbance was determined with microplate a reader at 490 nm.  Table 1).  Table 1).  1 H-NMR (CD 3 OD, 600 MHz) and 13 C-NMR (CD 3 OD, 150 MHz) (see Table 1).

Conclusions
Three new triterpenoids and four known triterpenoids were isolated from the S. chinensis (Turcz) Baill. The seven compounds were tested for cytotoxic effects against the Hep-G2 human tumor cell lines, which displayed no apparent cytotoxicity.