A New Sesquilignan Glucoside from Uraria sinensis

A new sesquilignan glucoside, urariasinoside A (1), together with eight known compounds, including two lignans, a sesquilignan, a dilignan, and four flavonoid derivatives were isolated from the aerial parts of Uraria sinensis. Their structures were determined on the basis of extensive spectroscopic analyses and comparison with literature data. Compound 1 was evaluated for in vitro cytotoxicity activity against HL-60, SMMC-7721, A549, MCF-7, SW480, and BEAS-2B cell lines.


Introduction
The genus Uraria (family Leguminosae), consists of 35 species, distributed throughout the tropical regions of Africa, Asia, and Australia [1].Traditionally, some species of this genus are used as folk medicines for the treatment of gonorrhea, cough, chills and fever [2], or as therapies for swelling, coldness, ulcers and stomachalgia [3].Pharmacological investigations have revealed that the extract of U. lagopoides has anti-inflammatory and analgesic activities [4], U. critina exerts nitric oxide-scavenging OPEN ACCESS and antioxidant effects [5], and U. picta has antimicrobial activity [6].Previous phytochemical studies on this genus reported the presence of isoflavanones, triterpenes, steroids, glycosides, and aromatic components [6][7][8].Uraria sinensis Desv.(Hemsl.), a suffruticose sparingly branched perennial herb, is exclusively distributed in the Hubei, Sichuan, Guizhou, Yunnan, Shanxi, Gansu provinces of China [1].To date, this species has not been investigated.As part of our ongoing search for bioactive natural compounds from the genus Uraria, we report the isolation and structure elucidation of a new sesquilignan glucoside, urariasinoside A (1), together with eight known compounds, including two lignans, a sesquilignan, a dilignan, and four flavonoid derivatives from the aerial parts of U. sinensis (Figure 1), as well as the inhibitory activities against five human cancer cell lines (HL-60, SMMC-7721, A549, MCF-7, and SW480) and a normal cell line (BEAS-2B) of compound 1.
The relative configuration of 1 was elucidated by the NOESY (Figure 3) experiment, together with comparison with data of sesquimarocanol B. Likewise, the NOESY correlations of H-8' with H-7 and H-9, of H-8-with H-7' and H-9', and the absence of the NOESY cross-peak between H-8 and H-8', pointed to the trans orientations for H-8/H-8' as that of sesquimarocanol B. Similarly, the relative stereochemistry of H-7''/H-8'' was supposed to cis configuration according to the correlation between H-7'' and H-8''.
In the other respects, previous reports of cytotoxicity activity of secoisolariciresinol-sesquilignan related structures such as sesquimarocanol B hexaacetate [16], suggest compound 1 might have potential activity as an anticancer agent.Therefore, compound 1 was evaluated for its cytotoxic activities against five human cancer cell lines: HL-60 (human myeloid leukemia), SMMC-7721 (hepatocellular carcinoma), A549 (lung), MCF-7 (breast), and SW480 (colon) by the MTT method [25].DDP (cisplatin) and taxol were used as positive controls.However, the bioassay results revealed that compound 1 has no in vitro cytotoxicity (IC 50 > 40 μM) against any of the five tested cancer cell lines.

General Procedures
Optical rotations were measured on a Perkin Elmer PE-341LC polarimeter.UV spectra were measured on a Perkin Elmer Lambda 35 spectrometer.CD spectra were recorded on a JASCO J-810 CD spectrometer.IR spectra were recorded on a Bruker Vertex 70 FT-IR microscope instrument (FT-IRmicroscope transmission).NMR spectra were obtained at 400 MHz for 1 H and 100 for 13 C, on Bruker AM-400 MHz spectrometers with solvent peaks being used as references.Two-dimensional HSQC, HMBC, COSY, and NOESY experiments were performed using the pulse sequences provided by Bruker.HRESIMS data were measured using an API QSTAR Pulsar spectrometer.Column chromatography was performed using polyamide (10-30 mesh, Taizhou Luqiao Sijia Biochemical Plastics Factory, Taizhou, China), silica gel (100-200, 200-300 mesh, H, Qingdao Marine Chemical Inc., China), ODS (50 μm, YMC, Kyoto, Japan) and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala Sweden).HPLC separation was performed on an instrument consisting of an Ultimate 3000 controller, an Ultimate 3000 pump, and an Ultimate 3000 UV detector with an YMC (250 × 10 mm, 5 μm) preparative column.GC analysis was performed on an Agilent Technologies 7820A GC instrument, OV-17 column (30 m × 0.32 mm × 0.5 μm, Lanzhou Zhongke Antai Analysis Technology Co. Ltd., Lanzhou, China), hydrogen-flame ionization detector.Enzymatic hydrolysis was treated with cellulase (Shanghai Yuanye Biology & Technology Co. Ltd., Shanghai, China).TLC was carried out on precoated silica gel GF254 plates.Spots were visualized under UV light (254 or 356 nm) or by spraying with 5% H 2 SO 4 in 95% EtOH followed by heating.

Plant Material
The aerial parts of U. sinensis were collected from Shiyan, Hubei Province, China in September 2010 and identified by Dr. Jianping Wang of School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology.The voucher specimen (1009) was deposited in the herbarium of Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology.

Determination of the Absolute Configuration of Sugar Unit in 1
A solution of 1 (1.0 mg), in 2 M aqueous CF 3 COOH (2.0 mL) was heated at 100 °C for 3 h in a water bath.The reaction mixture was diluted in H 2 O (4.0 mL) and extracted with EtOAc (4.0 mL × 3), then the aqueous layer was concentrated to remove CF 3 COOH.The residue was dissolved in pyridine (1.0 mL), to which L-cysteine methyl ester hydrochloride (2.0 mg) in pyridine (1.0 mL) was added.Then, the mixture was kept at 60 °C for 2 h.After the reaction mixture was dried in vacuo, the residue was trimethylsilylated with 1-trimethylsilylimidazole (0.2 mL) at 60 °C for 2 h in a water bath.Finally, the mixture was partitioned between hexane and H 2 O (0.3 mL each) and the hexane extract was analyzed by gas chromatography (GC) under the following conditions: column temperature, 250 °C; injection temperature, 250 °C; carrier N 2 gas; flow rate 1.0 mL/min.In the acid hydrolysate of 1, D-Glucose was confirmed by comparison of the retention times of their derivatives with those of D-glucose and L-glucose derivatives prepared in a similar way, which showed retention times of 13.66 and 14.34 min, respectively.

Enzymatic Hydrolysis of 1
A solution of 1 (2.67 mg) in 0.1 M acetate buffer (pH 4.0, 2.0 mL) was treated with cellulase (3.0 mg) and then the reaction mixture was stirred at 40 °C for 12 h.The reaction mixture was diluted in H 2 O (4.0 mL) and extracted with EtOAc (4.0 mL × 3).After that, the EtOAc extract was further purified by semi-preparative HPLC (50% MeOH in H 2 O, flow rate 1.8 mL/min, wavelength 212 nm) to obtain compounds 1a (1.2 mg, retention time 18.3 min).

MTT Cytotoxicity Assay
Compound 1 was tested against five human cancer cell lines [HL-60 and SMMC-7721, A549 (lung), MCF-7 (breast), and SW480 (colon)] and a normal cell line (BEAS-2B).The antiproliferative assay was performed by the MTT colorimetric method as described previously [25].Briefly, adherent cells were seeded into 96-well tissue culture plates with density of 1 × 10 5 cells/mL.After 12 h, cells were treated with the medium containing different concentrations of test compounds for 48 h.Then, attached cells were incubated with MTT (15 μL, 5 mg/mL, 1 h) and subsequently solubilized in DMSO.The optical density of absorbency at 595 nm was then measured using a microplate reader.Experiments were performed in triplicate, and the values are the averages of three (n = 3) independent experiments.DDP (cisplatin, Sigma, San Francisco, CA, USA) and taxol were used as the positive control.