A New Tetrahydrofuran Lignan Diglycoside from Viola tianshanica Maxim

A new lignan glycoside, tianshanoside A (1), together with a known phenylpropanoid glycoside, syringin (2) and two known lignan glycosides, picraquassioside C (3), and aketrilignoside B (4), were isolated from the whole plant of Viola tianshanica Maxim. The structure of the new compound was elucidated by extensive NMR (1H, 13C, COSY, HSQC, HMBC and ROESY) and high resolution mass spectrometry analysis. The three lignans 1, 3, and 4 did not exhibit significant cytotoxicity against human gastric cancer Ags cells or HepG2 liver cancer cells. This is the first report of the isolation of a lignan skeleton from the genus Viola L.


Introduction
Viola tianshanica Maxim (Violaceae) is a perennial herb widely distributed in Central Asia, especially in the Xinjiang Uygur Autonomous Region (XUAR) of China [1]. The whole herb, including the roots, have been used in traditional Uygur medicines as an antifebrile and detoxicating agent for the treatment of fever, headache, pharyngalgia and acute pyogenic infections such as boils, furuncles and carbuncles [2]. Modern pharmacological studies have demonstrated that the extracts of V. tianshanica have anti-inflammatory [3], anti-bacterial [4] and anti-oxidative [5] activities. It is used in XUAR as a substitute for Viola yedoensis. Very little is known about the phytochemical composition of V. tianshanica

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with the exception of several flavonol-O-glycosides [6] and cyclotides [7] that have been reported. As part of our continued investigation of the constituents of V. yedoensis and its substitutes [8,9], a phytochemical study of the ethanolic extract of the herb was thus carried out. Herein, we report the isolation and structural elucidation of a new lignan glycoside, tianshanoside A (1), along with three known glycosidic lignan derivatives, syringin (2) [10], picraquassioside C (3) [11], and aketrilignoside B (4) [12] (Figure 1), from V. tianshanica. Compounds 1, 3, and 4 were evaluated for their cytotoxicity against the human gastric cancer (Ags) and liver cancer HepG2 cell lines.

Results and Discussion
Repeated column chromatography of the ethyl acetate fraction of the ethanolic extract of V. tianshanica led to the isolation of a new tetrahydrofuran lignan diglycoside (1). Compound 1 was obtained as a white amorphous powder and possessed the molecular formula of C 32 H 44 O 16 as evidenced by a HRESIMS peak at m/z 707.2531 [M + Na] + in combination with its 1 H-NMR, 13 C-NMR and DEPT spectra. Maximum absorption bands at 230 and 280 nm in the UV spectrum suggested the existence of conjugated groups in 1. The IR spectrum exhibited absorption bands for free hydroxyl groups (3387 cm −1 ) and benzene rings (1637 and 1513 cm −1 ). Detailed analysis of 1 H-NMR (Table 1) and HSQC spectra showed that 1 contained two ABX spin systems assignable to two 1,3,4-trisubstituted benzene rings at δ H 6.64 (1H, d, J = 8.0 Hz, H-5), 6.68 (1H, brs, H-6), and 6.82 (1H, brs, H-2); and 6.69 (1H, brs, H-5′), 6.62 (1H, dd, J = 8.0, 1.4 Hz, H-6′), and 6.73 (1H, d, J = 1.4 Hz, H-2′). The 1 H-NMR chemical shifts observed for these two benzene systems together with the presence of two oxygenated methyl proton signals at δ H 3.72 (6H, s) suggested the presence of two guaiacyl (3-methoxy-4-hydroxyphenyl) groups in this compound [13]. The 13 C-NMR assignments corroborated the 1 H-NMR assignments for both aromatic rings. In addition, the 1 H-NMR spectrum of 1 also established a C(7)-O-C(9′) tetrahydrofuran skeleton [14], similar to that of 4 [12]. The proton signals at δ  The 1 H-NMR spectrum of 1 also gave signals for two glucose moieties (Table 1). In the HSQC spectrum, two anomeric protons at δ  Table 1). All the information mentioned above substantiates the identity of 1 as a tetrahydrofuran lignan diglucoside [15,16].
The HMBC correlations between H-7 and C-1, C-2, and C-6 of the benzene ring indicated the linkage of C-7 and this aromatic ring. Similarly, C-7′ was connected to C-1′ of another aromatic ring as evidenced by the HMBC correlations between H-7′ and C-1′, C-2′ and C-6′ ( Figure 2). The attachment of the inner glucose residue to C-9 was suggested by the HMBC correlation of the anomeric proton at δ H 4.32 (d, J = 7.7 Hz) with the signal at δ H 66.7 (C-9). The 1→2 interglycosidic linkage with the second glucose moiety was deduced from the HMBC correlation between H-1′′′ (δ H 4.43, d, J = 7.8 Hz) and C-2′′ at δc 81.9. The assignment of the 1→2 interglycosidic linkage of the two glucoses has also been confirmed by comparison with the 13 C NMR spectral data of the reference [17].
The known compounds 2-4 were identified as syringin (2) [10], picraquassioside C (3) [11], and aketrilignoside B (4) [12] (Figure 1), respectively, by comparison of their NMR and MS data with those reported. All these compounds were reported for the first time from V. tianshanica. Compounds 1, 3, and 4 were tested in vitro for their cytotoxicities against two human tumor cell lines, gastric cancer Ags and liver cancer HepG2 cells, using the CCK-8 assay. None of the tested compounds showed any significant cytotoxic activities at concentrations up to 0.2 mM.

Plant Material
The whole plant of V. tianshanica was collected from the Xinjiang Uygur Autonomous Region, China, in June 2012, and authenticated by Dr. Zhihong Cheng. A voucher specimen (NO. 201206TSJC) is deposited at the Herbarium of Materia Medica, School of Pharmacy, Fudan University, Shanghai, China.

Cytotoxicity Evaluation
Two human cancer cell lines, the gastric cancer Ags cells and HepG2 liver cancer cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The Ags cell line was cultured in a Ham's/F12 medium (Hyclone, Logan, UT, USA), supplemented with 10% fetal bovine serum (Gibco). The HepG2 cell line was cultured in DMEM medium (Hyclone), supplemented with 10% fetal bovine serum (Gibco). Cells were maintained at 37 °C in a 95% air-5% CO 2 atmosphere. The cytotoxicity of the compounds was determined in vitro using the Cell Counting Kit-8 (CCK-8) assay kit (Dojindo Laboratories, Tokyo, Japan), according to the manufacturer′s instructions. The sample compounds were dissolved in DMSO, and then further diluted. Cells were plated at a density of 1.0 × 10 5 per well in a 96-well microplate and incubated at 37 °C for 24 h. The cells were treated with various concentrations of test compounds (the final concentrations of the compounds were 0, 25, 75, 100, 150, 200 μM/mL) in quadruplicate for 72 h. Then, 20 μL of the solution of CCK-8 was added into each well, and the plates were further incubated for an additional 1 h. The absorbance was measured at 450 nm with a Varioskan Flash microplate reader (Thermo Scientific, Waltham, MA, USA).

Conclusions
Three lignan glycosides including a new one, tianshanoside A, and a phenylpropanoid glycoside were isolated for the first time from the dried whole plant of V. tianshanica. This is the first report of lignans from Viola species. Three lignan glycosides exhibited no significant inhibitory activities against two human tumor cell lines, gastric cancer Ags or liver cancer HepG2.