Sophora alopecuroides L. is widely distributed in the desert of northwest and north of China. The seeds of this herb have been used as the traditional Chinese ethnic medicine “Buya” in northwest of China to treat acute diarrhea and enteritis [1]. Pharmacological studies show that the plant exhibits a variety of biological activities including anticancer, antivirus, antibacterial, and anti-neuroinflammatory effects [1,2,3]. Modern chemical research is mainly focused on its alkaloids and flavonoids [4, 5]. However, studies of phenolic compounds are rare. This prompted us to further study its chemical compositions. Five phenolic natural products were isolated from S. alopecuroides. On the basis of the spectral data, they were determined as (3S,2R)-methyl 2-(4-hydroxybenzyl)tartrate (1), eucomic acid (2) [6], methyl eucomate (3) [7], piscidic acid (4) [8], and ethyl 2-(4-hydroxybenzyl)tartrate (5) [9]. Among them, compound 1 is a new compound, and compounds 1–3, and 5 were isolated from the seeds of S. alopecuroides for the first time. The cytotoxity of all isolated compounds was tested against two human hepatocellular carcinoma cell lines, HepG2 and Hep3B. All compounds 1–5 exhibited weak anticancer activity against HepG2 and Hep3B cell lines.

Compound 1, (3S,2R)-methyl 2-(4-hydroxybenzyl)tartrate, was isolated as a white powder. The molecular formula C12H14O7 was indicated by EI-MS at m/z 269 [M – H]–, combined with the NMR spectral data. The characteristic maximum absorptions at 205, 225, and 272 nm in the UV spectrum suggested that 1 was an aromatic derivative. Its IR spectrum showed strong absorption bands for a hydroxyl group (3390 cm–1) and benzene ring (1612, 1608 cm–1).

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The 13C NMR spectrum showed the presence of two carbonyl carbons at δ 175.7 and 173.4, six aromatic carbons including one oxygenated aromatic carbon at δ 157.2 and five aromatic carbons at δ 132.5, 115.7, and 127.7, one methoxyl carbon at δ 52.4, one methylene carbon at δ 41.7, one oxygenated methine carbon at δ 76.6 and one quaternary carbon at δ 81.8. The 1H NMR spectrum showed a 1,4-disubstituted phenyl group [δ 7.07 (2H, d, J = 8.5 Hz, H-2′, 6′), 6.65 (2H, d, J = 8.5 Hz, H-3′, 5′)], one isolated methylene [δ 3.13 (1H, d, J = 13.8 Hz, H-7′b), 2.97 (1H, d, J = 13.8 Hz, H-7′a)], and an oxygenated methine at δ 4.57 (1H, s, H-3), similar to the data of piscidic acid (4) [8]. An additional signal in 1 is a methoxy at δ 3.70 (3H, s, H-5). The HSQC and 1H–1H COSY spectrum of 1 provided unambiguous assignments of proton and carbon signals. In the HMBC spectrum, the key HMBC long-range correlations combined with the chemical shifts of these proton and carbon resonances revealed the presence of a 2-substituted tartaric acid moiety in compound 1. In addition, HMBC correlations (Fig. 1) from a methylene at δ 2.97 and 3.13 (H-7′) to the carbonyl carbon at δ 175.7 (C-1), a quaternary carbon at δ 81.6 (C-2), and the aromatic carbons at δ 127.9 (C-1′) and 132.5 (C-2′, 6′) determined that the 4′-hydroxybenzyl moiety was located at C-2. The HMBC correlation from methoxyl at δ 3.70 (4-OMe) to δ 173.4 (C-4) demonstrated that the tartaric acid was esterified at the 4-carboxyl moiety. Thus, 1 was elucidated as methyl 2-(4-hydroxybenzyl)tartrate. The similarities of the positive optical rotations for 1 (\( {\left[\alpha \right]}_D^{25} \) +46.96°, c 0.12, MeOH) and piscidic acid [8] suggest that compound 1 possesses the same absolute configuration. Therefore, the structure of compound 1 was determined as (3S,2R)-methyl 2-(4-hydroxybenzyl)tartrate.

Fig. 1.
figure 1

Key HMBC and 1H–1H COSY correlations for compound 1.

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Compounds 1–5 were evaluated against the hepatocellular carcinoma cell lines HepG2 and Hep3B. Preliminary toxicity towards human cells was investigated using a human embryonic kidney cell line, HEK293 (IC50 > 100 μM). All compounds 1–5 exhibited weak anticancer activity against HepG2 and Hep3B cell lines (Table 1).

Table 1. Cytotoxic Activity of Compounds 1–5 (IC50, μM; mean ± SD, n = 3)
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Experimental

General. All melting points were determined on a Buchi 510 melting point apparatus and are uncorrected. Optical rotations were measured on a PerkinElmer polarimeter 341. IR spectra were determined on a Nicolet Magna FT-IR 750 spectrometer (νmax in cm–1). The NMR spectra were recorded on the Bruker DRX-600 NMR spectrometer for 1H and 13C NMR. The chemical shifts were given on the δ (ppm) scale with tetramethylsilane as internal standard, and the coupling constants (J) were in Hz. The EI-MS data were obtained on a Finnigan-MAT-95 mass spectrometer. Commercial silica gel (Qing Dao Hai Yang Chemical Group Co., 200–300 and 300–400 mesh) was used for column chromatography. Precoated Silical gel plates (Yan Tai Zi Fu Chemical Group Co., G60F-254) were used for analytical TLC.

Plant Material. The seeds of Sophora alopecuroides L. were purchased from the herbal market during May of 2015 at Xining, Qinghai Province, China and identified by one of our authors, Prof. Xinzhou Yang. A voucher specimen (No. SC0217) has been deposited in the Herbarium of South-Central University for Nationalities, Wuhan, China.

Extraction and Isolation. Dried seeds of S. alopecuroides (1.0 kg) were triturated and then extracted sequentially by maceration with 95% EtOH three times (3 L each) at room temperature. The solvent was evaporated under reduced pressure to obtain a crude extract (170 g). The extract was suspended in water and then partitioned successively with petroleum ether (PE), EtOAc, and n-BuOH to afford the PE fraction (12 g), EtOAc fraction (29 g), and n-BuOH fraction (55 g), respectively. The EtOAc fraction (20 g) was subjected to the column chromatography over silica gel eluting with CH2Cl2–MeOH (1:0→50:1→30:1→20:1→10:1→5:1→1:1→0:1, containing 0.1% formic acid) to give five fractions (1–5). Fraction 2 (4.5 g) was chromatographed over silica gel with EtOAc–acetone (100:1→30:1→20:1→10:1→5:1→0:1, containing 0.1% formic acid) to afford four fractions (2.1–2.4). Fractions 2.2 (200 mg) and 2.3 (315 mg) were further purified on a Sephadex LH-20 column, eluting with MeOH (containing 0.1% formic acid) to afford compounds 3 (22 mg) and 4 (17 mg), respectively. Fraction 3 (4.3 g) was subjected to column chromatography over silica gel with EtOAc–acetone (20:1→10:1→8:1→5:1→0:1, containing 0.1% formic acid) to afford three fractions (3.1–3.3). Fraction 3.3 (370 mg) was further purified on a Sephadex LH-20 column eluted with MeOH (containing 0.1% formic acid) and then purified by semipreparative HPLC (H2O–CH3CN, 80%:20% → 30%:70%, 40 min, containing 0.1% formic acid in both mobile phases) to afford 1 (18 mg) and 5 (21 mg), respectively. Fraction 5 (2.2 g) was separated by Sephadex-LH20 eluted with MeOH (containing 0.1% formic acid) to afford five fractions (5.1–5.5). Fraction 5.3 (300 mg) was further purified by semipreparative HPLC (H2O–CH3CN, 70%:30% → 30%:70%, 40 min, containing 0.1% formic acid in both phases) to afford compound 2 (31.3 mg).

(3S,2R)-Methyl 2-(4-hydroxybenzyl)tartrate (1), white powder; \( {\left[\alpha \right]}_D^{25} \) +46.96° (c 0.12, MeOH). UV (MeOH, λmax, nm) (log ε): 205 (1.04), 225 (1.16), 277 (0.38). IR (film, νmax, cm–1): 3390, 2950, 2810, 1731, 1612, 1608, 1515, 1442, 1226, 1118, 840, 782. 1H NMR (600 MHz, CD3OD, δ, ppm, J/Hz): 7.07 (2H, d, J = 8.5, H-2′, 6′), 6.65 (2H, d, J = 8.5, H-3′, 5′), 4.57 (1H, s, H-3), 3.70 (3H, s, 4-OMe), 3.13 (1H, d, J = 13.8, H-7′b), 2.97 (1H, d, J = 13.8, H-7′a). 13C NMR (150 MHz, CD3OD, δ, ppm): 175.7 (C-1), 173.4 (C-4), 157.2 (C-4′), 132.5 (C-2′, 6′), 127.9 (C-1′), 115.7 (C-3′, 5′), 81.6 (C-2), 76.6 (C-3), 52.4 (C-5), 41.7 (C-7′). ESI-MS m/z 269 [M – H]–. HR-ESI-MS m/z 269.0665 [M – H]–.

Eucomic acid (2), white powder. 1H NMR (600 MHz, CD3OD, δ, ppm, J/Hz): 7.08 (2H, d, J = 8.4, H-2′, 6′), 6.69 (2H, d, J = 8.4, H-3′, 5′), 2.97 (1H, d, J = 16.3, H-3b), 2.96 (1H, d, J = 13.3, H-7′a), 2.88 (1H, d, J = 13.3, H-7′b), 2.58 (1H, d, J = 16.3, H-3a). 13C NMR (150 MHz, CD3OD, δ, ppm): 175.3 (C-1), 173.0 (C-4), 156.9 (C-4′), 132.0 (C-2′, 6′), 127.4 (C-1′), 115.2 (C-3′, 5′), 76.4 (C-2), 44.7 (C-3), 43.1 (C-7′). ESI-MS m/z 239 [M – H]–.

Methyl eucomate (3), white powder. 1H NMR (600 MHz, CD3OD, δ, ppm, J/Hz): 7.08 (2H, d, J = 8.4, H-2′, 6′), 6.69 (2H, d, J = 8.4, H-3′, 5′), 3.66 (3H, s, H-5), 2.99 (1H, d, J = 15.8, H-3b), 2.97 (1H, d, J = 13.7, H-7′a), 2.88 (1H, d, J = 13.3, H-7′b), 2.61 (1H, d, J = 15.8, H-3a). 13C NMR (150 MHz, CD3OD, δ, ppm): 177.3 (C-1), 172.5 (C-4), 157.4 (C-4′), 132.6 (C-2′, 6′), 127.5 (C-1′), 115.8 (C-3′, 5′), 76.9 (C-2), 52.4 (C-5), 45.5 (C-3), 43.8 (C-7′). ESI-MS m/z 253 [M – H]–.

Piscidic acid (4), white solid. 1H NMR (600 MHz, CD3OD, δ, ppm, J/Hz): 6.97 (2H, d, J = 8.5, H-2′, 6′), 6.59 (2H, d, J = 8.5, H-3′, 5′), 4.29 (1H, s, H-3), 2.96 (1H, d, J = 13.8, H-7′b), 2.80 (1H, d, J = 13.8, H-7′a). 13C NMR (150 MHz, CD3OD, δ, ppm): 174.3 (C-1), 173.3 (C-4), 156.2 (C-4′), 131.6 (C-2′, 6′), 126.9 (C-1′), 115.0 (C-3′, 5′), 80.2 (C-2), 75.3 (C-3), 41.3 (C-7′). ESI-MS m/z 255 [M – H]–.

Ethyl 2-(4-hydroxybenzyl)tartrate (5), colorless solid. 1H NMR (600 MHz, CD3OD, δ, ppm, J/Hz): 7.07 (2H, d, J = 8.5, H-2′, 6′), 6.69 (2H, d, J = 8.5, H-3′, 5′), 4.55 (1H, s, H-3), 4.19 (2H, q, J = 7.1, H-5), 3.15 (1H, d, J = 13.8, H-7′b), 2.99 (1H, d, J = 13.8, H-7′a), 1.26 (3H, t, J = 7.1, H-6). 13C NMR (150 MHz, CD3OD, δ, ppm): 175.6 (C-1), 173.3 (C-4), 157.1 (C-4′), 132.3 (C-2′, 6′), 127.7 (C-1′), 115.6 (C-3′, 5′), 81.5 (C-2), 76.3 (C-3), 62.3 (C-5), 41.3 (C-7′), 14.2 (C-6). ESI-MS m/z 283 [M – H]–.

Cytotoxity Assay. Human hepatocellular carcinoma cell lines HepG2 and Hep3B and human embryonic kidney cell line HEK293 were purchased from the American Type Culture Collection (ATCC; Rockville, MD, USA). The cytotoxic assay was performed as previously described [10,11,12]. Briefly, three cell lines suspended in RPMI 1640 containing 10% fetal bovine serum (FBS) were seeded at 1 × 104 cells (100 μL) per well in a flat 96-well plate and incubated in a humidified atmosphere of 5% CO2 in air at 37°C. After 24 h, the medium containing different concentrations of compounds 1–5 was added to incubate cells for 24 h, and 0.1% DMSO was used as solvent control. After that, the cells were fixed with EtOH–H2O (95:5), stained with crystal violet solution, and lysed with a solution of 0.1 N HCl in MeOH. The absorbance in control and drug-treated wells was measured in an automated microplate reader (Bio-Rad 550) at 550 nm.