Sophora flavescens Aiton is a perennial herbaceous plant widely distributed in China, Russia, and India [1, 2]. Recent studies showed that it has antibacterial, antipyrotic, antipyretic, antiarrhythmic, antiasthmatic, antiulcerative, anti-HBV, and antineoplastic effects, and it has been used to treat jaundice, leukorrhea, carbuncles, pyogenic infections of the skin, scabies, enteritis, and dysentery [1, 3]. Previous phytochemical investigations led to the isolation of alkaloids, flavonoids, coumarins, phenylpropanoids, quinones, and triterpenoid saponins from this species [4]. A phytochemical investigation on the constituents of S. flavescens was carried out and led to the isolation of a new quinolizidine alkaloid, 17β-hydroxysophoridine (1), along with nine known quinolizidine alkaloids, matrine (2) [5], sophocarpine (3) [5], sophoridine (4) [5], isomatrine (5) [6], 7,11-dehydromatrine (6) [7], mamanine (7) [8], sophoranol (8) [9], N-oxymatrine (9) [5], and N-oxysophocarpine (10) [5]. The current report describes the isolation, structure elucidation, and cytotoxic evaluation of these compounds.

figure a

Compound 1 was obtained as a yellow oil. Its molecular formula was determined as C15H24N2O2 by positive HR-ESI-MS at m/z 265.1914 [M + H]+ (calcd for C15H25N2O2, 265.1916). Its IR spectrum exhibited absorption bands for the hydroxy (3431 cm–1), carbonyl (1629 cm–1), and quinolizidine moiety (2934, 2859, 2810, and 2749 cm–1). The 1H NMR data showed a characteristic signal at δH 5.40 (1H, d, J =7.2 Hz, H-17) for one oxygenated methine. The 13C and DEPT NMR spectra displayed 15 carbon resonances, including five methines (one oxygenated carbon), nine methylenes, and one carbonyl group. The 1D NMR data of 1 were similar to those of sophoridine (4), except for an additional hydroxy group at C-17 in 1, as deduced from the HMBC correlations of H-17 to C-4 (δC 31.0), C-5 (δC 34.9), C-6 (δC 58.6), and C-11 (δC 54.7), and the correlations between H-17 and H-5 in the 1H–1H COSY experiment. The structure was further confirmed by the correlations in the HMBC experiment (Fig. 1).

Fig. 1
figure 1

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

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The relative stereochemistry of 1 was established through an analysis of coupling constants and a ROESY experiment. The large coupling constant (J =12.6, 8.0 Hz) [4] of H-6 with H-5 and H-7 suggested that these three protons, H-5, H-6, H-7, had β-, α-, and α-orientation, respectively, which was confirmed by the correlations of H-6 with H-4b, H-5 with H-4a, and H-11 with H-8b. In the ROESY spectrum of 1, the correlations of H-5 with H-11 indicated that H-11 was β-oriented, while the correlations of H-6 and H-4b with H-17, suggested that OH-17 has β-orientation.

Compounds 1 and 5–8 were evaluated for their in vitro cytotoxicity against HL-60, SMMC-7721, A-549, MCF-7, and SW480 cancer cell lines using the reported MTT method [10]. However, none of them were active against the above five human cancer cell lines.

Experimental

General. UV spectra were recorded on a Shimadzu UV-2401A spectrophotometer. IR spectra were obtained on a Tensor 27 spectrometer. 1D and 2D NMR spectra were performed on Bruker AM-400, DRX-500, or Avance III-600 spectrometers with TMS as internal standard. HR-ESI-MS were carried out using VG Auto Spec-3000 or API-Qstar-Pulsar instruments. Column chromatography (CC) was performed using silica gel (100–200 and 200–300 mesh, Qingdao Marine Chemical Co. Ltd., Qingdao, People’s Republic of China), and Sephadex LH-20 (Amersham Pharmacia Biotech, Sweden). Semi-preparative HPLC was performed on an Agilent 1100 liquid chromatograph with Zorbax SB-C18 (5 μm, 9.4 ×250 mm). MPLC was performed on a Lisui EZ Purify III system, including pump manager P03, detector modules P02, and fraction collector P01 (Shanghai Lisui Chemical Engineering Co., Ltd., China), and on a column packed with MCI gel (CHP 20P, 75–150 μm; Mitsubishi Chemical Corporation, Japan). Fractions were monitored by TLC, and spots were visualized using Dragendorff’s reagent. Solvents were distilled prior to use.

Plant Material. The roots of Sophora flavescens were bought from the Kunming Chrysanthemum Village medicine market, Yunnan, China, in March 2011 and was identified by Prof. X. Cheng at Kunming Institute of Botany, Chinese Academy of Sciences. A voucher specimen (No. 20110320s01) has been deposited in the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences.

Extraction and Isolation. The dried roots (20 kg) were extracted with MeOH for three times at room temperature successively to afford a black residue (3 kg) after evaporation in vacuo. The residue was dissolved in warm water, acidified with 0.5% HCl to pH 2, and extracted with ethyl acetate. The aqueous layer was neutralized with 10% ammonia/water to pH 9 and extracted with CHCl3 once again and concentrated in vacuo to obtain the total alkaloids (300 g). It was subjected to MPLC (MCI gel) using a stepwise gradient (MeOH–H2O, 40:60 to 100:0) to obtain three fractions, A–C. Fraction A (100 g) was subjected to silica gel CC (200–300 mesh) and eluted with a gradient of petroleum ether–acetone (9:1 to 6:4) to give four further subfractions, A1–A4. Subfraction A1 was further purified by column chromatography on silica gel with petroleum ether–EtOAc (9:1) to afford 2 (1 g) and 3 (900 mg). Subfraction A2 was subjected to semi-preparative HPLC (MeOH–H2O, 11:89) to yield 4 (15 mg) and 5 (12 mg). Subfraction A3 was loaded on a column of silica gel and eluted with CHCl3–acetone–diethylamine (8:2:0.05), and then further purified by Sephadex LH-20 (MeOH), yielding 6 (13 mg) and 7 (7 mg). Fraction B (30 g) was subjected to silica gel CC with CHCl3–MeOH (9:1), followed by semi-preparative HPLC (MeOH–H2O, 10:90) to afford 1 (70 mg) and 8 (600 mg). Fraction C (150 g) was re-separated by silica gel CC (100–200 mesh) using petroleum ether–acetone–diethylamine (5:3.5:0.5 to 5:4:1) to give 9 (57 g) and 10 (17 g).

Compound 1. Yellow oil. [α] 17.8D  − 39.76∘ (c 0.218, MeOH). UV (MeOH, λmax, nm) (log ε): 421 (1.73), 204 (3.27). IR (KBr, νmax, cm–1): 3431, 2934, 2859, 2810, 2749, 1729, 1629, 1463, 1453, 1383, 1333, 1264, 1221, 1111, 1043. For 1H and 13C NMR spectral data, see Table 1. ESI-MS m/z 265 [M + H]+; HR-ESI-MS at m/z 265.1914 [M + H]+ (calcd for C15H25N2O2, 265.1916).

Table 1 1H (500 MHz) and 13C NMR (125 MHz) Spectra of 1 (CD3OD, δ, ppm, J/Hz)
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Matrine (2). White needles (petroleum ether). ESI-MS m/z 249 [M + H]+. 1H NMR (400 MHz, CD3OD, δ, ppm, J/Hz): 4.30 (1H, dd, J =12.7, 4.1, H-17a), 3.81 (1H, m, H-11), 3.06 (1H, t, J =12.7, H-17b), 2.85 (2H, m, H-2a, H-10a), 2.34–2.03 (7H, m), 1.95–1.45 (14H, m). 13C NMR (100 MHz, CD3OD, δ, ppm): 172.9 (C-15), 63.0 (C-6), 58.3 (C-2), 58.2 (C-10), 54.7 (C-11), 44.5 (C-17), 42.8 (C-7), 36.8 (C-5), 33.4 (C-14), 28.7 (C-12), 28.0 (C-4), 27.2 (C-8), 22.1 (C-3), 21.6 (C-9), 19.6 (C-13).

Sophocarpine (3). White needles (petroleum ether). ESI-MS m/z 247 [M + H]+. 1H NMR (400 MHz, CD3OD, δ, ppm, J/Hz): 6.62 (1H, m, H-13), 5.80 (1H, dt, J =9.8, 1.9, H-14), 4.05 (1H, dd, J =12.9, 4.7, H-17a), 3.96 (1H, m, H-11), 3.14 (1H, t, J =12.9, H-17b), 2.82 (2H, m, H-2a, H-10a), 2 60 (1H, m, H-6), 2.21–1.47 (14H, m). 13C NMR (100 MHz, CD3OD, δ, ppm): 167.7 (C-15), 140.9 (C-13), 124.1 (C-14), 64.7 (C-6), 58.3 (C-10), 58.3 (C-2), 52.9 (C-11), 43.1 (C-17), 42.8 (C-7), 35.9 (C-5), 28.7 (C-4), 28.3 (C-12), 27.3 (C-8), 22.0 (C-3), 21.6 (C-9).

Sophoridine (4). White needles (MeOH). ESI-MS m/z 249 [M + H]+. 1H NMR (400 MHz, CD3OD, δ, ppm, J/Hz): 3.40–3.30 (3H, m, H-11, H2-17), 2.87 (2H, m, H-2a, 10a), 2.30 (3H, m, H2-14, H-6), 2.14 (2H, m, H-2b, 10b), 2.03 (2H, m, H-3a, 5), 1.86 (2H, m, H-4a, 12a), 1.60–0.90 (10H, m). 13C NMR (100 MHz, CD3OD, δ, ppm): 172.5 (C-15), 63.5 (C-6), 57.5 (C-2), 56.6 (C-11), 50.6 (C-10), 49.6 (C-17), 41.6 (C-7), 32.9 (C-14), 31.3 (C-5), 30.5 (C-12), 28.6 (C-4), 23.5 (C-3), 22.8 (C-8), 22.7 (C-9), 19.4 (C-13).

Isomatrine (5). Colorless prisms (MeOH). ESI-MS m/z 249 [M + H]+. 1H NMR (400 MHz, CD3OD, δ, ppm, J/Hz): 3.92 (1H, m, H-11), 3.72 (1H, dd, J =9.7, 3.9, H-17a), 3.53 (1H, d, J =9.7, H-17b). 13C NMR (100 MHz, CD3OD, δ, ppm): 174.0 (C-15), 62.8 (C-6), 56.1 (C-2), 52.9 (C-11), 52.1 (C-10), 43.0 (C-17), 38.3 (C-7), 33.4 (C-14), 29.9 (C-5), 26.6 (C-12), 25.3 (C-4), 20.3 (C-3), 19.7 (C-8), 19.2 (C-9), 17.6 (C-13).

7,11-Dehydromatrine (6). Colorless prisms (acetone). ESI-MS m/z 247 [M + H]+. 1H NMR (400 MHz, CD3OD, δ, ppm): 4.24 (1H, m, H-17a), 3.31 (1H, m, H-17b). 13C NMR (100 MHz, CD3OD, δ, ppm): 171.6 (C-15), 136.4 (C-11), 109.1 (C-7), 62.7 (C-6), 58.0 (C-10), 56.2 (C-2), 41.9 (C-17), 33.5 (C-14), 33.2 (C-5), 29.6 (C-12), 27.6 (C-4), 27.6 (C-8), 25.4 (C-9), 22.3 (C-3), 20.5 (C-13).

Mamanine (7). Colorless oil. ESI-MS m/z 263 [M + H]+. 1H NMR (400 MHz, CD3OD, δ, ppm, J/Hz): 7.53 (1H, dd, J =6.5, 9.0, H-4), 6.39 (1H, t, J =8.7, H-3), 6.25 (1H, d, J =6.8, H-5), 3.59 (2H, m, H2-17). 13C NMR (100 MHz, CD3OD, δ, ppm): 166.4 (C-2), 152.5 (C-6), 143.8 (C-4), 118.3 (C-3), 105.1 (C-5), 64.8 (C-10), 63.7 (C-17), 61.1 (C-16), 57.5 (C-14), 44.6 (C-9), 40.4 (C-7), 34.6 (C-11), 30.3 (C-8), 26.4 (C-13), 25.3 (C-12).

Sophoranol (8). White crystals (MeOH). ESI-MS m/z 265 [M + H]+. 1H NMR (500 MHz, CD3OD, δ, ppm, J/Hz): 4.48 (1H, d, J =14.5, Ha-17), 3.77 (1H, m, H-11), 3.32 (1H, d, J =14.5, H-17b), 3.16–3.02 (2H, m, H-2a, 10a). 13C NMR (125 MHz, CD3OD, δ, ppm): 173.5 (C-15), 68.5 (C-6), 67.0 (C-5), 57.0 (C-10), 56.3 (C-2), 53.4 (C-11), 46.0 (C-17), 36.7 (C-7), 35.8 (C-4), 33.4 (C-14), 28.0 (C-8), 24.5 (C-12), 21.1 (C-3), 21.1 (C-9), 19.5 (C-13).

Oxymatrine (9). Colorless prisms (acetone). ESI-MS m/z 265 [M + H]+. 1H NMR (400 MHz, CD3OD, δ, ppm, J/Hz): 4.87 (1H, dd, J =11.7, H-11), 4.32 (1H, dd, J =12.2, 4.5, H-17a), 4.02 (1H, t, J =12.2, H-17b), 3.20 (5H, m), 2.55 (2H, m, H-3a, 9a), 2.38 (1H, m, H-14a), 2.25 (2H, m, H-14b, 12a), 2.10 (1H, m, H-8a), 1.58–1.87 (9H, m), 1.35 (1H, m, H-12b). 13C NMR (100 MHz, CD3OD, δ, ppm): 172.8 (C-15), 69.7 (C-10), 69.3 (C-2), 67.9 (C-6), 54.9 (C-11), 43.5 (C-7), 43.2 (C-17), 35.6 (C-5), 33.6 (C-14), 29.3 (C-12), 26.7 (C-4), 25.1 (C-8), 19.6 (C-13), 18.2 (C-3), 18.1 (C-9).

Oxysophocarpine (10). Colorless prisms (acetone). ESI-MS m/z 263 [M + H]+. 1H NMR (400 MHz, CD3OD, δ, ppm, J/Hz): 6.68 (1H, m, H-13), 5.84 (1H, dt, J =12.1, 1.9, H-14), 4.77 (1H, m, H-11), 3.94 (2H, m, H2-17), 3.31 (3H, m, H-6, 2a, 10a), 3.05 (2H, m, H-2b, 10b), 2.75 (1H, m, H-12a), 2.56 (2H, m, H-3a, 9a), 2.01 (2H, m, H-5, 12b), 1.97 (2H, m, H-7, 8a), 1.58–1.86 (5H, m). 13C NMR (100 MHz, CD3OD, δ, ppm): 168.8 (C-15), 141.2 (C-13), 124.7 (C-14), 69.5 (C-10), 69.2 (C-2), 67.3 (C-6), 53.4 (C 11), 43.7 (C-17), 41.6 (C-7), 34.0 (C-5), 30.7 (C-12), 26.8 (C-4), 25.2 (C-8), 18.2 (C-3), 18.1 (C-9).

Cytotoxicity Assay. The methyl thiazolyl tetrazolium assay (MTT assay) was used to determine cell viability. The following human tumor cell lines were used: HL-60 (human myeloid leukemia cell line), SMMC-7721 (human hepatocarcinoma cell line), A-549 (lung cancer cell line), MCF-7 (breast cancer cell line), and SW480 (human colon carcinoma). The cancer cells were all seeded in a 96-well plate at a density of 5000 to 10000 cells per well in 100 μL of medium. The test compounds and the positive control, cisplatin, were added to appropriate wells and the cells were incubated for 48 h (37°C, 5% CO2). Then MTT (100 μg) solution was added into the assay plates. After shaking for 10 s, the plates were returned to the incubator and kept for 4 h, after which the cells were lysed with 100 μL 20% sodium dodecyl sulfate (SDS)-50% DMF after removal of 100 μL medium. The optical density of the lysate was measured at 490 nm in a 96-well microtiter plate reader (Bio-Rad 680, USA). The IC50 value of each compound was calculated by the Reed and Muench method [11].