Constituents from the Seeds of Sophora Alopecuroides L.

Three new isoflavone glucosides, kudonol A−C (1–3), two new ester derivatives of phenylpropanoid, kudolignan A and B (4–5) and five known compounds, (−)-maackiain (6), neoliquiritin (7), methyl 4-coumarate (8), methyl ferulate (9) and (+)-wikstromol (10), were isolated from an extract of dried seeds of the traditional Chinese medicinal plant Sophora alopecuroides L. Their structures were established by NMR and HRESIMS data analyses. The monosaccharide part’s configuration of isoflavone glucosides was confirmed by acid hydrolysis and analyzed by a JAsco OR-4090 chiral detector, comparing it to standard substance D-glucose. The cytotoxicity effects against HeLa, Hep3B, MCF-7 and H1299 cells were tested by CCK-8 assay.


Introduction
Sophora alopecuroides L. is a traditional Chinese herbal plant, known as "Ku-Dou-Zi" in China, and is a perennial herbaceous plant in the Leguminosae family, which is widely distributed in the deserts of northern China, especially in the Xinjiang and Ningxia provinces as well as the Inner Mongolia Autonomous Region. The seeds of S. alopecuroides have a long history as a traditional Chinese medicine, utilized for the treatment of eczema, acute pharyngolaryngeal infection, sore throat, acute dysentery, and gastrointestinal hemorrhage [1].
Previous phytochemical investigations on S. alopecuroides have led to the isolation of alkaloids, flavonoids, isoflavonoids, and rotenoids [2]. Compounds isolated from the seeds of this plant have been used to treat leukemia [3], trophoblastic tumors and inflammation in traditional Chinese medicine. For example, sophocarpine was found to be effective against tumors, both in cell proliferation and metastasis in liver cancer [4].
During our present exploration for the diterpenoids of S. alopecurioides, five previously undescribed compounds were isolated and identified successfully for the first time ( Figure 1). The structures of the isolated isoflavone glucosides were determined from various spectroscopic data. Finally, the inhibitory effects of some of the isolates against four cancer cell lines were evaluated in vitro using a CCK-8 bioassay.

Results and Discussion
Compound 1 was obtained as a yellow powder and exhibited an ion peak at m/z 591.1512 ([M − H] − , calcd 591.1581) in its HRESIMS data, which indicated a molecular formula of C31H28O12. The indices of hydrogen deficiency (IHDs) of compound 1 was 18. The 13 C NMR data of compound 1 comprised the parent nucleus of an isoflavonoid and a monosaccharide. On the basis of the 1 H-NMR spectroscopic data (Table 1)

Results and Discussion
Compound 1 was obtained as a yellow powder and exhibited an ion peak at m/z 591.1512 ([M − H] − , calcd 591.1581) in its HRESIMS data, which indicated a molecular formula of C 31 H 28 O 12 . The indices of hydrogen deficiency (IHDs) of compound 1 was 18. The 13 C NMR data of compound 1 comprised the parent nucleus of an isoflavonoid and a monosaccharide. On the basis of the 1 H-NMR spectroscopic data (Table 1) performed on compound 1 afforded D-glucose, which was determined using a JAsco OR-4090 detector with D-glucose standard sample. Then, a parahydroxy cinnamic acid was deduced in the structure from the signals at δ H 7.52 (2H, d, J = 8.4 Hz) and 6.79 (2H, d, J = 8.4 Hz) and a pair of trans-ene hydrogen signals at δ H 7.56 (1H, d, J = 15.6 Hz, 7"'-H) and 6.39 (1H, d, J = 15.6 Hz, 8"'-H) in the 1 H NMR spectrum as well as a carbonyl carbon signal at δ C 166.3 in the 13 C NMR spectrum. The analysis of the spectroscopic data indicated that compound 1 was similar to the known compound calycosin-7-O-D-glucopyranoside [5], and the difference mainly lay in the parahydroxy cinnamic acid group. The exact structure of compound 1 was established by 2D-NMR. In the HMBC spectrum, a methoxide signal at δ H 3.80 had a long-range correlation with δ C 147.6(C-4 ), which indicated a methoxy group connected on the C ring of this isoflavone. A long-range correlation was observed between δ H 5.18 (H-1 ) and δ C 161.2 (C-7), which located the D-glucose at C-7 of the isoflavone, and the anomeric proton at δ H 5.18 (d, J = 7.2 Hz) also indicated the presence of a β glycosidic bond. The parahydroxy cinnamic acid group was posited to the C-6 of D-glucose by the HMBC correlations of δ H 4.45 (H-6")/δ C 166.3 (C-9"'). Based on the abovementioned data, the structure of compound 1 was elucidated as Calycosin-7-O-β-D-(6"-hydroxy cinnamate)-glucopyranoside and named as kudonol A.  signal at δ C 168.1 in the 13 C NMR spectrum. Additionally, a long-range correlation was observed from δ H 4.46 (H-6") to δ C 168.1 (C-9"'), which located the cinnamic acid at C-6 of the glucose. Compared with compound 1, the structure of compound 2 has a hydroxy group instead of the methoxy group present in the B ring of the isoflavone parent nucleus. Consequently, the structure of compound 2 was elucidated to be 7,3 ,4 -trihydroxyisoflavone-7-O-β-D-(6"-cinnamic acid)-glucopyranoside and named as kudonol B.
Compound 3 had the molecular formula C 28 H 24 O 12 according to the HRESIMS data from its quasi-molecular ion peak at m/z 553.1336 ([M + H] + , calcd 553.1268). On the basis of spectroscopic data, compound 3 was deduced to be an isoflavone glycoside, which was similar to compound 1; the main difference was that cinnamic acid was replaced by a 2-hydroxy benzoyl group, as well as a hydroxy group instead of the methoxy at C-4 . The hydrolysis experiment on compound 3 gave a monosaccharide, which was identified to be of the D-glucopyranosyl group by the corresponding standard substances. Through the analysis of spectral data, a salicylic acid group was deduced from δ C 113.5, 162.8, 118.4, 137.0, 120.4, 131.2 and 171.0 in the 13 C NMR spectrum, and that was attached to the C-6 position of the glucose with the carboxyl of itself, which was indicated from the long-correlation between δ H 4.81 (H-6") and δ C 171.0 (C-7"'). Consequently, the structure of compound 3 was elucidated to be 7,3 ,4 -trihydroxyisoflavone-7-O-β-d-(6"-salicylic acid)-glucopyranoside and was named as kudonol C.
Compound 4 was obtained as a pale yellow powder and exhibited an ion peak at m/z 455.1673 ([M + Na] + , calcd 455.1682) in its HRESIMS data, which indicated a molecular formula of C 23 H 28 O 8 . The 1 H NMR spectrum of compound 4 (see Table 2) showed six aromatic proton signals at δ H 7.16 (1H, d,  Table 2) showed 23 carbon signals. Aside from the carbon signal of the ethoxy unit and the three methoxy groups, the remaining 18 carbon signals included a carbonyl signal at δ C 169.1 (C-9), double ring olefin carbon signals at δ C 116.8 (C-8) and 146.1 (C-9), 12 aromatic carbons, and three aliphatic carbons at 83.7 (C-7 ), 84.7 (C-8 ) and 62.3 (C-9 ). The HMBC correlations of H-7 at δ H 7.61 with C-2, of H-8 at δ H 6.40 with C-6 and C-9, and of H-7 at δ H 4.40 with C-2 , C-5 , C-6 and C-8 confirmed the presence of two phenyl propanoid units. In the HMBC spectrum, the correlation of H-8 at δ H 4.54 with C-4 at δ C 151.8 suggested that compound 4 was an 8 -O-4 system neolignan. The methoxy group was determined to be at C-3, C-3 and C-7 , based on the HMBC correlation of the methoxy groups at δ H 3.83 with C-3 at δ C 151.8 and at δ H 3.82, with C-3 at δ C 148.9 and δ H 3.26, and with C-7 at δ C 83.7. Compound 5, a pale yellow powder, gave the same molecular formula through HRESIMS, the spectroscopic data indicated that compound 5 was similar to compound 4, except for the difference of their absolute configuration. Regarding the configurations of compounds 4 and 5, the difference in chemical shifts of both H-9 protons is the parameter that must be used to establish the relative configuration around the chiral centers H-7 and H-8 [6,7], in compound 4 both H-9 protons seem to be isochronous (δ H 3.86), whereas there is a difference of 0.20 ppm (δ H 3.71 and 3.51) in the chemical shift of these protons in compound 5, thus confirming that 4 and 5 have erythro and threo relative configurations, respectively, namely these are an erythro/threo pair. Thus, the structure of compound 4 was determined to be erythro-4 ,9 -dihydroxy-3,3 ,7 -trimethoxy-9-ethyoxyl-8−4-oxyneolignan and was named as kudolignan A (4), and 5 was determined to be threo-4 ,9 -dihydroxy-3,3 ,7 -trimethoxy-9-ethyoxyl-8−4-oxyneolignan named as kudolignan B (5). The other known compounds were identified as (−)-maackiain (6) [8], neoliquiritin (7) [9], methyl 4-coumarate (8) [10], methyl ferulate (9) [11] and (+)-Wikstromol (10) [12] from the comparison of their NMR data with that reported in the literature.
Compounds 1-6 were tested for their cytotoxicity against four human cancer cell lines (HeLa, Hep3B, MCF-7 and H1299) and one normal human liver cell line (LO2) (see Table 3). Experimental results show that all of the compounds showed no cytotoxicity to the LO2 cell line, compound 2 showed moderate inhibition of cell proliferation against several cancer cell lines, and compound 1 showed moderate inhibition of cell proliferation against Hep3B cells. On the basis of structural analysis, the activity of inhibiting the compounds' cytotoxicity against tumor cells was probably due to the trans-cinnamic acid moiety.

General Experimental Procedures
NMR spectra were run in DMSO-d 6 and CD 3 OD on a Varian Mercury NMR spectrometer. Analytical HPLC data were collected on an Agilent 1260 infinity II instrument (Thermo Scientific dionex). The UV spectra were measured by an Agilent 1260 infinity II UV-vis spectrophotometer in methanol. Preparative HPLC was performed by a Saipuruisi MH-LC 52 instrument with an Elite UV2300 detector and an YMC C18 column (250 × 20 mm, 5 µm). The HRESIMS data were obtained using an Agilent 1290 series 6540 UHD accurate mass Q-TOF mass spectrometer using direction injection. The sugar configurations were determined by a JAsco OR-4090 chiral detector. Column chromatographic separations were carried out on silica gel H-60 (Qingdao Marine Chemical Group Corporation, Qingdao, China), Polyamide (Shanghai Yiyan biology technology Co. Ltd., Zhengzhou, China) and Sephadex LH-20 (Shanghai Yi-He biological technology Co. Ltd., Shanghai, China). The Acetonitrile and Methanol used were of chromatographic grade, and were purchased from Fisher in America. All other solvents used of chemical grade (Da-Mao Chemical Co. Ltd., Tianjin, China).

Plant Materials
The dry seeds of S. alopecuroides L. were collected from Alxa League in Inner Mongolia Autonomous Region and identified by Prof. Liu Yong, Jiang-Xi University of Chinese Medicine. The specimens were deposited in the specimen room of traditional Chinese Medicine in Shenyang Pharmaceutical University (SPU-2014-0714-06).