A new anthraquinone from seed of Cassia obtusifolia

Seeds of Cassia obtusifolia L. are known as homology of medicine and food material, which is a commonly consumed beverage in China. One new compound, 8-hydroxy-1,7-dimethoxy-3-methylanthracene-9,10-dione-2-O-β-d-glucoside (1), together with 11 known compounds, including seven anthraquinones (2–8), was isolated from the seeds. The 2D NMR data of compound 2 are reported for the first time. The structures of the compounds were established on the basis of 1D and 2D NMR, IR and HR-ESI-MS spectra. The cytotoxic activities of all the compounds against five cell lines (LO2, HCT-116, A549, HepG2 and SGC7901) were evaluated by using CCK8 methods. Compounds 1, 3 and 7 show moderate cytotoxicity towards HCT-116 cells compared with oxaliplatin.


Introduction
Cassia obtusifolia Linn., a member of the genus Cassia (Leguminosae), is a well-known traditional Chinese medicinal plant. It belongs to the medically and economically important family Leguminosae (syn. Fabaceae), subfamily Caesalpinioideae (Joshi & Kapoor 2003). Semen Cassiae is the dry ripe seed of C. obtusifolia (Leguminosae). This herb is distributed mainly in China, Korea, India and the west tropical regions (Vadivel et al. 2012). The seeds are reported to have the effects of improving eyesight, alleviating constipation and lowering hypertension and hyperlipidemia (Hao et al. 2001). In previous investigations of this plant, a number of constituents were isolated, including anthraquinones, anthrones, flavonoids and triterpenoids (Guo et al. 1998;Zhang et al. 2009;Sob et al. 2010). Many studies have indicated that anthraquinones have antitumour or antimetastasis effects (Zhang et al. 1998;Lee 2001;Kuo et al. 2004). In previous research, we had isolated cytotoxic anthraquinone glycosides from marine Streptomyces sp. strain (Lu et al. 2012). With the aim of finding new cytotoxic anthraquinone, we investigated the chemical constituents of Semen Cassiae. In our ongoing efforts towards identifying new bioactive components from C. obtusifolia, we have conducted an investigation of the ethanol extract from the seeds of C. obtusifolia and isolated one new anthraquinone (see Figure 1), as well as nine known compounds and evaluated their anticancer activities in several different cancer cell lines. Herein, we describe the experimental details of the separation process as well as information pertaining to the structural elucidation of these compounds based on their spectroscopic properties. In addition, we examined the anti-tumour activities of the anthraquinones.

Results and discussion
2.1. Structural elucidation of the isolated compounds Compound 1 was obtained as an orange amorphous powder. It was positive to Borntrager's reaction, revealing that it was a hydroxyl anthraquinone compound (Wagner & Bladt 1996.) High-resolution mass spectroscopy (HR-MS) analysis of the pseudomolecular peak at m/z 475.1452 [M 2 H] 2 (calculated for 475.1447) in the ESI-MS, in combination with the 1 H and 13 C NMR data (Table S1), supported the molecular formula of C 23 H 24 O 11 and indicated 12 degrees of unsaturation. The 1 H NMR spectrum showed a set of two ortho-coupled aromatic hydrogens at d 7.68 (d, J ¼ 8.4 Hz, H-5) and d 7.40 (d, J ¼ 8.4 Hz, H-6), suggesting a tetrasubstituted aromatic ring B, one singlet aromatic proton at d 7.86 due to H-4 showed the presence of penta-substituted ring A in addition to the singlet for methyl group at d 2.43. Also, 1 H NMR spectrum revealed a signal at d 13.11, assigned to a hydroxyl group H-bonded to a carbonyl. The spectrum also showed a distinct signal for anomeric proton, a hexose anomeric proton resonance at d 5.01 (1 H, d, J ¼ 7.4 Hz).
13 C NMR spectra displayed 12 aromatic carbon signals, 4 of them with oxygen substitution (d 154.3, 153.7, 153.3 and 151.8). The signals at d 188.3 and 180.2 were assigned to the two carbonyl carbons. The 13 C NMR spectrum also reveals two methoxy carbon signals at d 61.4 and 56.2, and one methyl at d 17.6. In addition, assignments of the aglycone carbons were aided by comparison with the reported chemical shifts of emodin 2-O-b-glucoside (Choi et al. 1996). In the HMBC spectrum, the signals at d 2.43 (H-11) had correlations with carbons C-2, C-3 and C-4, indicating that the methyl group was located at C-3. The methoxyl at C-1 and C-7 was confirmed by the HMBC correlation signals (see Figure S1 and Table S1), H-12/C-1 and H-13/ C-7, respectively. Long-range correlation between H-1 0 and C-1 confirmed the position of the aglycone substituent on C-1 of anthraquinone parent skeleton. The spectrum also showed a distinct signal for anomeric proton resonance at d 5.01 (1H, d, J ¼ 7.4 Hz), indicating glucosyl with b-configuration. Based on the above-mentioned data, compound 1 is deduced to be 8hydroxy-1,7-dimethoxy-3-methylanthracene-9,10-dione-2-O-b-D-glucoside.
Compound 2 was obtained as an orange amorphous powder. It was positive to Borntrager's reaction, revealing that it was a hydroxyl anthraquinone compound. HR-MS analysis of the pseudomolecular peak at m/z 491.1354 [M -H] -(calculated for 491.1346) in the ESI-MS, in combination with the 1 H and 13 C NMR data (Table S1), supported the molecular formula of C 23 H 24 O 12 . Based on the detailed analysis of its NMR spectra as well as the comparison of the NMR data with those of compound 1, it was found that they were very similar, except that compound 2 has the substitution of a hydroxyl at C-6 position. The substitution positions of ring B were confirmed by the HMBC spectra (see Figure S1 and Table S1). The aromatic proton at d H 7.18 correlations with the carbon C-6, -7, -10, -8a and -10a, indicating that aromatic proton was located at C-5. The correlations between d 13.25 (-OH) and C-7, -8, -8a confirmed hydroxyl at C-8. The correlation between d H 3.85 (-OCH 3 ) with d 139.8 (C-7) suggested that the methoxyl is substituting at C-7 position. Thus, the position of the last one hydroxyl was unambiguously at C-6. Thus, compound 2 was elucidated as 6,8-dihydroxy-1,7-dimethoxy-3-methylanthracene-9,10-dione-2-O-b-D-glucoside.

Cytotoxic activity
The isolated compounds were evaluated for their cytotoxic activity against LO2, HCT-116, A549, HepG2 and SGC7901 cells (Table 1). Many anthroquinone compounds showed moderate cytotoxicity towards HCT-116 and A549 cell lines, compared with the control, emodin and oxaliplatin. In contrast, compounds 9 -12 did not exhibit apparent cytotoxicity even at a sample concentration of 50 mg/mL.
Among the anthroquinone compounds, 1, 3 and 7 showed equal cytotoxicity towards the HCT-116 cell line as compared with oxaliplatin, and their cytotoxicity towards LO2 is lower than the control.
The above-mentioned results indicated that the C-6 hydroxy group might significantly reduce the cytotoxic activity in these compounds. And the difference in the cytotoxicity value between the aglycons and glycosides is not obvious.

Experiment 3.1. General method
Thin layer chromatography was performed on silica gel GF254 (Qingdao Haiyang Chemical Co. Ltd, Qingdao, China) plates. Octadecylsilanised silica gel (ODS; 40-63 mm, YMC, Tokyo, Japan) was used for column chromatography. HPLC was performed on Agilent 1100 couple with Shimpack ODS-C18 (20 £ 250 mm) column. UV-vis absorption spectra were acquired using a Shimadzu UV-1650PC spectrophotometer; NMR spectra were obtained using a Bruker DRX-500 spectrometers using dimethyl sulphoxide-d 6 (DMSO-d 6 ) as solvent with tetramethylsilane as internal reference, the chemical shifts were given in d and coupling constants in Hz. ESI-MS and HR-ESI-MS experiments were recorded on an Agilent 1100 Series MSD Trap mass spectrometer and Mariner ESI-TOF spectrometer, respectively.

Materials and chemicals
Plant material of C. obtusifolia was collected from Fuyang (Anhui, China) in 2011. The plant materials were authenticated by Professor X.J. Wang. The voucher specimen (CO201101) has been deposited at the Nanjing Institute for Comprehensive Utilization of Wild Plants. DMSO-d 6 was obtained from Merck (Darmstadt, Germany). All the other chemicals and solvents used in this study were purchased from Merck and were of analytical grade.

Extraction and isolation
Semen Cassiae (the seeds of C. obtusifolia, 5 kg) was extracted three times with 95% ethanol at ambient temperature for 24 h. The resulting ethanol mixture was then filtered, and the filtrate was collected and concentrated under vacuum at 508C until approximately 90% of the solvent had been evaporated. The ethanol extract (60 g) was isolated by using silica gel chromatography yielding five fractions (Fr. 1 -Fr. 5). Then, Fr. 1 (5 g) was separated by ODS column chromatography affording 3 (60 mg), 4 (88 mg), 5 (23 mg) and 6 (35 mg). Fr. 2.1 (2 g) and Fr. 2.2 (4 g) were obtained from Fr.  3.6. Cytotoxicity and proliferation assay A CCK8 assay was used to measure cell proliferation and viability. Briefly, cells were plated in 96-well plates (1 £ 10 4 cells/well) and routinely cultured for 24 h. Then, cells were treated with various concentrations (50, 10, 2 and 0.4 mg/mL) of compounds 1 -12 or 1% DMSO (vehicle) in DMEM supplemented with 3% FBS. After 44 h of treatment, 10 ml CCK8 solution (2.5 mg/mL) was added to each well, followed by incubation for 4 h at 378C with 5% CO 2 . Absorbance was measured at 450 nm using a microplate reader. The reference compounds emodin and oxaliplatin were treated at the same concentration and condition like compounds 1 -12.
Results were obtained from more than three independent experiments. Statistical analysis of data was performed using one-way analysis of variance followed by Student's t-test with GraphPad PRISM 5 (GraphPad Software Inc., San Diego, CA, USA). Error bars denoted the standard deviation.

Conclusion
In summary, phytochemical analysis of the ethanol extract of Semen Cassiae resulted in the isolation and characterisation of one new compound and together with 10 known compounds, including six anthraquinones. Their structures were elucidated by using chemical and spectroscopic analyses, including 1D, 2D NMR and HR-ESI-MS. Biological studies disclosed that anthraquinone compounds (1, 3 and 7) show equal cytotoxicity towards HCT-116 cells compared with oxaliplatin, and their cytotoxicity towards LO2 is lower than the control.
The results of our chemical investigation further revealed the chemical composition of C. obtusifolia L., and the biological investigation of these compounds also can help us to find new application of traditional Chinese herb.

Supplementary data
Supplementary data associated with this article can be found in the online version. It includes 1D, 2D NMR spectra and data of compound 1.

Disclosure statement
No potential conflict of interest was reported by the authors.