Two new triterpenoids from Gypsophila oldhamiana

Abstract Two new triterpenoids (1–2) were isolated and elucidated from the roots of Gypsophila oldhamiana, together with four known triterpenoids (3–6). Their structures were identified to be 3β-hydroxyolean-13(18)-ene-23, 28-dioic acid (1), 3β, 12α-dihydroxy-23-carboxyolean-28, 13β-olide (2), 3β, 16α-dihydroxy-23-oxoolean-13(18)-en-28-oic acid (3), gypsogenin (4), quillaic acid (5) and gypsogenic acid (6) by spectral methods. All compounds were tested for their cytotoxicities against human tumour cell lines (lung cancer H460 and gastric cancer SGC-7901) and for their antiangiogenic effects using a zebra fish model. All compounds showed interesting antiangiogenic activities and the significant cytotoxicities against H460.


Introduction
Gypsophila oldhamiana Miq. known as 'xia cao' in China belongs to the Caryophyllaceae family. It is distributed in the north regions of China. Its roots have been used as a traditional Chinese medicine to treat fever, consumptive disease, infantile malnutrition and so on (The Chinese Medicine Dictionary 1977). The roots of Gypsophila species are an especially rich source of triterpenoid saponins (Hostettmann & Marston 1995;Yotova et al. 2012) and have some alkaloids (Zhang et al. 2015). The triterpenoid saponins from Gypsophila species were reported to have cytotoxic activities (Bai et al. 2007;Arslan et al. 2012;Arslan et al. 2013), α-glucosidase inhibition activities (Yao et al. 2010) and so on. The aglycone of the above-mentioned compounds are gypsogenin, gypsogenic acid and quillaic acid (Böttger & Melzig 2011), as well as vaccaric acid (Luo & Kong 2006) and segetalic acid (Luo et al. 2008). In previous studies, we found that G. oldhamiana is also a rich source of triterpenoids (Bai et al. 2007;Wu et al. 2011). As part of a continuing effort to discover secondary metabolites from local medicinal plants, the major constituents of the cytotoxic fraction from the extract of G. oldhamiana were investigated. Herein, in this study, we report the isolation and structure elucidation of two new triterpenoids (1-2), together with four known compounds (3-6) ( Figure 1). Additionally, the compounds 1-6 were also investigated for their cytotoxic activities against human tumour cell lines (lung cancer H460 and gastric cancer SGC-7901) and for their antiangiogenic effects using a zebra fish model.

Results and discussion
The methanol extract of the air-dried roots of G. oldhamiana was fractionated into petroleum ether (60-90 °C) fraction, etoAc fraction, n-BuoH fraction and water fraction. Then, the above four fractions were tested for their cytotoxic activities against human tumour cell lines (lung cancer H460), and the IC 50 values of the petroleum ether (60-90 °C) fraction, the etoAc fraction, the n-BuoH fraction and the water fraction were 196.45, 12.87, 50.46 and 33.22 μg/mL, respectively. The cytotoxic activity of the etoAc extract was the highest. So, the portion etoAc was subjected to successive column chromatography to give compounds 1-6.
Compound 2 was isolated as white solid powder. Its molecular formula was determined to be C 30 H 46 o 6 . Hr-eSI-MS spectrum of 2 measured in the negative ion mode gave a quasi-molecular ion peak at m/z: 501. . The 13 C NMr and DePT spectra revealed the presence of six methyl carbons, ten methylene carbons, five methine carbons and nine quaternary carbons. The 13 C NMr spectra further demonstrated the presence of two carboxyl carbon at δ 179.7 (C-23) and δ 177.4 (C-28), three oxymethine carbons δ 74.6 (C-3), 68.5 (C-12) and 90.0 (C-13) and six methyl carbons (δ 11.1, 16.0, 17.1, 20.3, 21.9, 32.1). From the above data, we found the signals in NMr spectra arising from rings B, C, D and e of 2 were very similar to 3β, 12α-dihydroxyolean-28, 13β-olide (Ali et al. 2002;García-Granados et al. 2004; Csuk & Siewert 2011) except for the signals from the ring A, a carboxyl group at C-23 position, in 2 instead of those due to a methyl group at C-23 position. In the HMBC spectrum, correlation signals from δ 4.06 (H-12) to δ 22.7 (C-11), δ 43.5 (C-14), δ 52.4 (C-17) and δ 90.0 (C-13) and from H-12 and δ 3.15 (H-18, dd, J = 11 Hz, 4 Hz) to C-13 were exhibited, which confirmed the location of a hydroxyl and a hydroxyl lactone groups at C-12 and C-13. Furthermore, according to the signals of the Hr-eSI-MS and the Ir, we concluded that compound 2 was the γ-lactone (13 → 28). The HMBC cross-peaks of δ 4.70 (H-3) to δ 26.9 (C-2), 53.7 (C-4), 179.7 (C-23) and 11.1 (C-24) confirmed the location of the hydroxyl group at C-3. Additional correlations between H-3 and H-5 and between H-12 and H-26 were also detected in the NoeSY map. These correlations enable to confirm that H-3 was α-oriented and H-12 was β-oriented. The presence of a carboxyl group at C-23 position was also confirmed by the correlations between H-25 and H-24, H-26 in the NoeSY map. From the above data, the structure of 2 was deduced to be 3β, 12α-dihydroxy-23-carboxyolean-28, 13β-olide.
The cytotoxic activities of 1-6 against the growth of human tumour cell lines (lung cancer H460 and gastric cancer SGC-7901) were evaluated in vitro, using cisplatin as a positive control. All of compounds displayed cytotoxic activities against the human tumour cell lines.
The antiangiogenic activities of compounds 1-6 were tested using a zebra fish model, in terms of the inhibition on the growth of intersegmental vessels, with PTK787 as a positive control. Accordingly, all compounds showed interesting antiangiogenic activities. At a 5 μg/mL concentration, compound 1 showed 100% inhibition on the growth of intersegmental vessels, as good as PTK787.

General experimental procedures
The1D and 2D NMr spectra were obtained on a Bruker Avance 400 spectrometer ( 1 H: 400 MHz, 13 C: 100 MHz) with TMS as internal standard in C 5 D 5 N. Chemical shifts were given in values of ppm and coupling constants in Hertz. Ir (KBr) spectra were recorded by Thermo Nicolet Nexus 670 FT-Ir. Hr-eSI-MS were obtained on a Bruker Daltonics Inc. APeXII spectrometer. optical rotations were obtained in the solvents specified with a JASCo P-1020 polarimeter (JASCo, Tokyo, Japan). Preparative HPLC was conducted using a KNAuer K-501 HPLC pump and uV Detector K-2501 with a Agilent-C 18 column (21.2 × 150 mm, 7 μm). Analytical HPLC was recorded using Waters 2695 with a Waters-C 18 column (4.6 × 250 mm, 5 μm). Silica gel (200-300 mesh, Qingdao Marine Chemical Industry Factory, Qingdao, China), and Sephadex LH-20 (25-100 μm, Pharmacia Fine Chemical Co., Ltd. uppsala, Sweden) were used for column chromatography. TLC was performed on precoated silica gel plates (Qingdao Haiyang Chemical Co.), using the solvent systems CHCl 3 -MeoH (9:1), and detection was obtained by 10% sulphuric acid/etoH, followed by heating at 100 °C for triterpenoids.

Plant material
The roots of G. oldhamiana were collected from Laoshan Mountain in Qingdao, Shandong, China, in August 2000. They were identified by Prof Li-Li Sun in Shandong Academy of Chinese Medicine. A voucher specimen (No. S200008) was stored in the Institute of Materia Medica, Shandong Academy of Medical Sciences, Jinan, Shandong Province China.

Compound 1
White

Cytotoxicity assays
The procedure for the cytotoxic activity was performed according to the MTT method (Mosmann, 1983;Gerlier & Thomasset 1986). In this study, human tumour cell lines (lung cancer H460 and gastric cancer SGC-7901) were used. In brief, the human tumour cells were incubated in rPMI 1640 medium (Gibco Co., uSA) containing 10% foetal bovine serum supplemented with 100 units/mL penicillin and 100 μg/mL streptomycin in a 96-well plate at 37 °C for 24 h, while suspended cells were seeded with an initial density of 1 × 105 cells mL −1 . Then, the compounds were added and cells were cultured for 48 h at 37 °C, with cisplatinum as positive controls. Last, 10 μL of 5 mg/mL MTT (Sigma, St. Louis, Mo, uSA) in phosphate-buffered saline was added to each well, and the tumour cells were incubated for another 4 h at 37 °C in carbon dioxide incubator. The supernatant was collected from each well, and 200 μL of DMSo was added. The absorbance of the produced formazan was measured at 560 nm in a microplate reader. The inhibition (%) was calculated, and the IC 50 value defined as the concentration of sample necessary to inhibit the growth to 50% of the control was calculated by a modified Karber formula.

Antiangiogenesis assay
The compounds 1-6 dissolved by 100% DMSo were prepared. These solutions were diluted in sterile salt water (5 mM NaCl, 0.17 mM KCl, 0.4 mM CaCl 2 , 0.16 mM MgSo 4 ) to obtain final solutions of various concentrations in 0.1% DMSo. Aliquots were placed into 96-well culture plate. Then, zebra fish embryos at 24 hpf (hours post-fertilisation) were transferred into 96-well culture plate. PTK787 was used as the positive control. All embryos were incubated at 28.5 °C. After 48 h of treatment, the intersegmental vessels of embryos were visualised with green fluorescent protein labelling and endogenous alkaline phosphatase staining. The antiangiogenic activities of compounds were calculated from the inhibition ratio of angiogenesis (Murphey & Zon 2006).

Conclusion
The structures of the two new triterpenoids and four known compounds were established by 1D and 2D NMr techniques, other spectroscopic evidence and comparison with the literature. Compounds 1-6 showed the significant cytotoxicities against H460 and interesting antiangiogenic activities. Compounds 4 and 5 also presented the strong cytotoxicities against SGC-7901. At a 10.29 μM concentration, compound 1 showed 100% inhibition on the growth of intersegmental vessels, as good as PTK787. Further investigations are ongoing in our laboratory.

Supplementary material
Supplementary material relating to this article is available online, alongside Tables S1-S3 and Figures S1-S20.