Chinese Chemical Letters  2017, Vol. 28 Issue (7): 1465-1468   PDF    
Uncinatic acids A-C, three new carboxylated flavonoids from Selaginella uncinata
Rui Liua,b, Hui Zoua,b, Ping-Sheng Xua, Zhen-Xing Zoua, Jing Lib, Fei Chenga, Rui-Huan Liub, Gan Zhoua, Kang-Ping Xub, Gui-Shan Tana,b,*    
a Xiangya Hospital of Central South University, Changsha 410008, China;
b Xiangya School of Pharmaceutical Sciences, Central South University, Changsha 410013, China
Abstract: Three new carboxylated flavonoids, uncinatic acids A-C (1-3), were isolated from the whole herb of Selaginella uncinata. Their structures were established on the basis of extensive NMR analysis including 1D, 2D NMR experiments and HR-ESIMS techniques. All of them share the carboxylation structural characteristic. Compounds 1 and 2 belong to novel naturally occuring furanoflavonoids which is firstly reported in genus Selaginella. Such furanoflavonoids with dicarboxylic acid structrure have never been discovered before. In addition, the isolates were tested for their cytotoxicity against A549 and BGC-823 cell lines in vitro.
Key words: Selaginella uncinata     Selaginella     Uncinatic acids A-C     Carboxylated flavonoids     Furanoflavonoids     Anticancer     Cytotoxicity    
1. Introduction

Selaginella uncinata (also known as "Cuiyuncao" in China), a member of Selaginella genus belonging to the family of Selaginellaceae, is widely distributed in the south part of China and extensively used to treat jaundice, dysentery, edema, and beriberoid diseases [1]. In addition, S. uncinata is also used as a cancer treatment in national minority of China. Previous phytochemical studies on S. uncinata revealed the presence of flavonoids [2-5], biflavonoids [3-7], chromone glycosides [8], and steroidal saponins [9]. Flavonoids is the most widely distributed natural products in nature with extensive pharmacological effects in particular anticancer effect [10-13]. Therefore, people contribute fairly amount of efforts to search for new flavonoids for the treatment of tumor. In our continuing investigation of new bioactive secondary metabolites from this species, three new carboxylatedflavonoids, uncinatic acids A-C (1-3) (Fig. 1) were isolated from S. uncinata. Herein, the isolation and detailed structural elucidation are described, and the cytotoxicity of these compounds are evaluated.

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Fig. 1. Chemical structures of compounds 1–3.

2. Results and discussion

Compound 1 was obtained as yellow amorphous powder. The molecular formula was determined as C19H12O9 by HR-ESIMS (negative-ion mode, m/z 383.0415 [M—H]-, calcd. for C19H11O9, 383.0409), corresponding to fourteen degrees of unsaturation in accordance with its 13C NMR spectrum (Table 1) resolving 19 Catom resonances. Its UV maximal absorptions at 291 and 335 nm. Its IR spectrum showed the presence of carbonyl group (1746, 1644 cm-1) and benzene ring (1606, 1555, 1505 cm-1). The 1H NMR spectrum (Table 1) of 1 indicated two hydroxyl singlets [δH 12.12 (s, 5-OH and 10.04 (s, 7-OH)], one 1, 3, 4-trisubstituted benzene ring [δH 8.37 (d, 1H, J = 1.0 Hz, H-2'), 7.71 (d, 1H, J = 8.5 Hz, H-5'), 7.63 (dd, 1H, J = 8.5, 1.0 Hz, H-6')], one 1, 2, 3, 5-tetrasubstituted benzene ring [δH 5.95 (d, 1H, J = 2.0 Hz, H-8), 5.90 (d, 1H, J = 2.0 Hz, H-6)], one Obearing methine group [δH 5.72 (dd, 1H, J = 13.0, 3.0 Hz, H-2)], two aliphatic protons [δH 3.28 (dd, 1H, J = 17.5, 13.0 Hz, H-3α), 2.82 (dd, 1H, J = 17.5, 3.0 Hz, H-3β)]. Additionally, signals for one methylene (δC 45.2) and one methine (δC 79.1) were observed in the 13C NMR spectrum (Table 1 and Fig. S2 in Supporting information) of 1. According to the characteristics of these NMR data, compound 1 should be a dihydroflavone. In the HMBC spectrum (Fig. 2), the long-range correlations of H-2 (δH 5.72) with C-1' (δC 135.2), C-3 (δC 45.2), and H-3 (δH 3.28, 2.82) with C-4 (δC196.4), C-10 (δC 102.2) confirm the dihydroflavone skeleton. The HMBC correlations of 5-OH (δH 12.12) with C-5 (δC 164.0), C-10 (δC 102.2), C-6 (δC 96.4), and H-6 (δH 5.90) with C-7 (δC 167.2), C-5 (δC 164.0), C-10 (δC 102.2) indicated a 5, 7-dihydroxylated pattern. Except for ten degrees of unsaturation of the dihydroflavone skeleton, the structure of 1 still remains four degrees of unsaturation. In addition, the 13C NMR spectrum indicated two carboxyl groups δC 164.3 (8'-COOH) and 160.5 (7'-COOH), two olefinic carbon δC 152.2 (C-8') and 117.6 (C-7') without unsaturated hydrogen suggested the existence of a fully substituted double band. The molecule remains one degree of unsaturation. These data together with the HMBC correlation between H-2' and C-7' indicated a furan ring with two carboxyl subsituents which should be along C-4' (O-bearing) and C-3' of the ring B. Furthermore, the circular dichrosim (CD) spectrum of 1 showed a negative cotton effect at 289 nm and a positive cotton effect at 332 nm, which suggested the configuration of C-2 in 1 was S by comparison with the CD data with that of literature [14]. The completely assignment of 1H and 13C NMR signals are shown in Table 1 based on the HSQC and HMBC spectra analysis. Thus, compound 1 was elucidated as (2S)-5-(5, 7-dihydroxy-4-oxochroman-2-yl)benzofuran-2, 3-dicarboxylic acid, named uncinatic acid A.

Table 1
1H NMR (500 MHz) and 13C NMR (125 MHz) data of compounds 1-3, in DMSO-d6, δ in ppm, J in Hz).

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Fig. 2. The key HMBC correlations of 13.

Compound 2 was isolated as yellowamorphous powder. The HRESIMS showed a [M + H]+ peak at m/z 383.0392 (calcd. 383.0397). In combination with the 13C NMR spectrum (Table 1 and Fig. S6 in Supporting information), the molecular formula of 2 was deduced as C19H10O9, implying fifteen degrees of unsaturation. Comparison of the 1H NMR and 13C NMR data of 2 to those of 1 prompted that the presence of unsaturated bond between C-2 and C-3 in the structure of 2, which means compound 2 should be a flavone rather than a dihydroflavone. It was furtherevidenced by HMBC corellationsof δH 6.96 (s, 1H, H-3) with δC 182.2 (C-4), δC 163.9 (C-2) and δH 8.88 (d, 1H, J = 1.0 Hz, H-2'), 8.17 (dd, 1H, J = 7.5, 1.0 Hz, H-6') with δC 163.9 (C-2). Except that, all remaining 1H NMR and 13C NMR signals of compounds 1 and 2 are similar. Therefore, compound 2 was determined as 5-(5, 7-dihydroxy-4-oxo-4H-chromen-2-yl)benzofuran-2, 3-dicarboxylic acid, named uncinatic acid B.

Compound 3 was obtained as yellow amorphous powder. The HR-ESIMS exhibited a pseudo-molecular-ion peak at m/z 313.0361 ([M—H]-; calcd. 313.0354), corresponding to the molecular formula C16H10O7. The UV spectrum of 3 indicated the absorption at 222, 270 and 325 nm, attributed to a conjugated aromatic system. The IR spectrum revealed the presence of hydroxyl (3400 cm-1), conjugated carbonyl (1659, 1651 cm-1) and aromatic ring (1605 and 1486 cm-1). The 1H NMR spectrum (Table 1) exhibited signals for two hydroxyl groups [δH 12.90 (s, 1H, 5-OH) and 10.92 (s, 1H, 7-OH)] one 1, 3, 4-trisubstituted aromatic ring [δH 8.40 (d, 1H, J = 2.0 Hz, H-2'), 8.20 (dd, 1H, J = 8.5, 2.0 Hz, H-6'), 7.12 (d, 1H, J = 8.5 Hz, H-5')], one 1, 2, 3, 5-tetrasubstituted benzene ring [δH 6.50 (d, 1H, J = 2.0 Hz, H-8), 6.21 (d, 1H, J = 2.0 Hz, H-6)] and one singlets [δH 6.95 (1H, s, H-3)]. The 13C NMR spectrum, together with HSQC spectrum, revealed 16 C-atom signals including two C=O groups, six CH groups, and eight quaternary C-atoms (Table 1 and Figs. S10, S11 in Supporting information). On the basis of these NMR characteristics, compound 3 should be an apigenin derivative. Besides the fifteen carbons of apigenin skeleton, the remaining carbonyl was observed in 13C NMR spectrum at δC 171.4. The unambiguous linkage sites were determined by HMBC spectrum (Fig. 2). In the HMBC spectrum, the correlations of H-2' (δH 8.40) with C-7' (δC 171.9), C-4' (δC 164.3), C-2 (δC 163.0), and H-5' (δH 7.12) with C-3' (δC 114.6), C-1' (δC 112.0) suggested the carbonyl was attached to C-3'. Thus, compound 3 was elucidated as 5-(5, 7-dihydroxy-4-oxo-4H-chromen-2-yl)-2-hydroxybenzoic acid, named uncinatic acid C.

Compounds 1-3 were tested for their inhibitory effects on the lung cell line A549 and gastric cancer cell line BGC-823. All compounds were tested using non-cytotoxic concentrations with the positive control 5-fluorouracil (5-Fu). Compounds 1 and 2 presented potent anticancer activities with IC50 values of 20.51 ±1.21 and 44.89 ± 6.70 aginst A549 cells, 26.14 ± 3.49 and 72.27 ± 5.14μmol/L against BGC-823 cells (Table 2). No significant cytotoxic activity of compound 3 were observed under all tested concentrations. Uncinatic acid (1) is the most effective among the isolates against both of cell lines.

Table 2
Inhibitory effects of compounds 1-3 on A549 and BGC-823Cells (n = 3).

3. Conclusion

Since Banks et al. [15] reported the unique genome of Selaginella and predicted that Selaginella has potential to synthesize a repertoire of novel secondary metabolites, studies about the secondary metabolites of Selaginella has a bloom. The present study reported three novel carboxylated flavonoids from S. uncinata. New compounds 1 and 2 are furanoflavonoids, with very unique structures from natural sources. Compounds 1 and 2 also display effective anticancer activities against A549 and BGC-823 cells. This study firstly revealed S. uncinata may be a potential candidate for anticancer including lung cancer and gastric cancer and compounds 1 and 2 play an important role in this effect.

4. Experimental 4.1. General

Optical rotations were measured on a WZZ-2S polarimeter. UV spectra were obtained on a UV-2450 spectrometer (SHIMADZU, Kyoto, Japan). IR spectra were obtained using KBr disks on an AVATAR 360FT-IR spectrophotometer (Nicolet Instrument Corporation, Madison, WI, USA). HRESIMS were recorded on a Micromass Zabspec (Micromass UK Ltd, Manchester, UK) HRMS spectrometer. NMR spectra, including 1H and 13C NMR, HSQC and HMBC experiments, were recorded on a Bruker AV-500 MHz spectrometer (Bruker, Karlsruhe, Germany) with tetramethylsilane (TMS) as the internal standard. CD spectra were measured on an Olis DSM 20 CD spectrophotometer (Borgart, GA, USA). Macroporous resin D101 (AnHui SanXin Resin Technology Co. Ltd., AnHui, China) and Polyamide (100-200 mesh; China National Medicine Corporation Ltd., Shanghai, China), Sephadex LH-20 (TOYOPEARL TOSOH, Tokyo, Japan) were used for column chromatography (CC) and silica gel GF-254 (Qingdao Marine Chemical Factory, Qingdao, China) was used for Thin Layer Chromatography (TLC). Pre-HPLC experiments were conducted using an Agilent HPLC system and carried on preparative YMC Pack ODS-A (3 μm, 150 mm × 10 mm, YMC Co., Ltd, Kyoto, Japan).

4.2. Plant material

The air-dried whole herbs of S. uncinata were collected from Huaihua, Hunan Province, China, and identified by Prof. Zheng-Ji Li (Xiamen University, Xiamen, China). A voucher specimen (No. 20090710) was deposited in Xiangya School of Pharmaceutical Sciences, Central South University.

4.3. Extraction and isolation

The air-dried whole herbs of S. uncinata (20 kg) were extracted two times with 70% aq. EtOH to obtain the extract (2.5 kg). Then, the extract was subjected to macroporous resin (D101) column chromatography (CC) with a gradient elution using EtOH/H2O (0:100 → 40:60 →70:30 → 95:5) to yield four portions (the water portion, 40% EtOH portion, 70% EtOH portion and 95% EtOH portion). The 95% EtOH portion (80 g) was separated on a silica gel column, eluted with a system of CHCl3/MeOH gradient elution (100:0 to 0:100) to afford 10 fractions (A-J). Fraction F (CHCl3-MeOH, 90:10) was chromatographed on a Sephadex LH-20 gel CC and eluted with MeOH (isocratic elution) to obtain 35 subfractions, based on TLC analyses (CHCl3/MeOH 10:1). Sub-fraction F18-20 was further purified by semi-preparative HPLC (Acetonitrile/H2O 35:65, 3 ml/min) to obtain compound 3 (15.3 mg, tR 22.7 min). Subfraction F23-25 was purified by semi-preparative HPLC (Acetonitrile/H2O 26:74, 3 ml/min) obtain compounds 1 (10.6 mg, tR 13.0 min) and 2 (7.0 mg, tR 16.6 min).

Uncinatic acid A (1). Amorphous yellow powder. UV (MeOH): 291, 335. (KBr, cm-1): 1746, 1644, 1606, 1555, 1505. 1H NMR and 13C NMR: see Table 1. HR-ESIMS: 383.0415 ([M-H]-, calcd. for C19H11O9-, 383.0409).

Uncinatic acid B (2). Amorphous yellow powder. UV (MeOH): 268, 328. 1H NMR and 13C NMR: see Table 1. HRESIMS: 383.0392 ([M + H]+, calcd. for C19H11O9+, 383.0397).

Uncinatic acid C (3). Amorphous yellow powder. UV (MeOH): 222, 270, 325. IR (KBr, cm-1): 3400, 1659, 1651, 1605, 1486 cm-1. 1H NMR and 13C NMR: see Table 1. HR-ESIMS: 313.0361 ([M-H]-, calcd. for C16H9O7-, 313.0354).

4.4. Activity assay

Human lung cancer A549 cells and gastric cancer BGC-823 cells were purchased from the Chinese Academy Cell Bank (Shanghai, China), and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Thermo Scientific, USA), 100 IU/mL penicillin G and 100 μg/mL streptomycin (Invitrogen Life Technologies, USA) in a CO2 incubator (5% CO2) at 37 ℃. Trypsin and dimethyl sulfoxide (DMSO) were purchased from Amersco Co. (USA).

All compounds were screened for cytotoxicity against BGC-823 and A-549 human cancer cell lines using the MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) method. The cells were cultured in DMEM mediumwith 10% fetal bovine serum (HyClone) at 37 ℃ in a humidified atmosphere supplied with 5% CO2.100 μL of adherent cells was seeded into each well of a 96-well cell culture plate and allowed to adhere for 12 h before drug addition. Each tumor cells were exposed to the test compounds at various concentrations in triplicate for 48 h with 5-fluorouracil (5-Fu) as a positive control. Absorbance was determined by a microplate spectrophotometer at 490 nm.

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (No. 31370370), Key Project of Science and Technology of Hunan Province, China (Nos. 2014SK2002 and 2013SK5077), Key Project of Application Technology Research and Development of Haikou (No. 2015-039) andSocial Development and Technology Specific Program of Hainan Province (No. SF201419).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cclet.2017.03.004.

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