Hepatoprotective phenylethanoid glycosides from Cirsium setosum

Abstract Two new phenylethanoid glycosides, namely β-D-glucopyranoside, 1″-O-(7S)-7-(3-methoxyl-4-hydroxyphenyl)-7-methoxyethyl-3″-α-L-rhamnopyranosyl-4″-[(8E)-7-(3-methoxyl-4-hydroxyphenyl)-8-propenoate] (1) and β-D-glucopyranoside, 1″-O-(7S)-7-(3-methoxyl-4-hydroxyphenyl)-7-methoxyethyl-3″-α-L-rhamnopyranosyl-4″-[(8E)-7-(4-hydroxyphenyl)-8-propenoate] (2), together with six phenylethanoid glycosides were isolated from Cirsium setosum. Their structures were elucidated by their spectroscopic data and references. Compounds 2, 4, 5, 7 and 8 (10 μM) exhibited moderate hepatoprotective activities. Compounds (3–8) were obtained from this plant for the first time.


General procedures
The melting points were recorded on a XT5B microscopic melting point apparatus (Beijing Tech electro-optical instrument factory, China) which were uncorrected. The optical rotations were measured on a Perkin-Elmer 241 polarimeter at 20 °C. The uV spectra were measured with an Australia GBC uV-916 spectrophotometer, and a Nicolet 5700 FT-IR spectrometer was used for scanning IR spectroscopy using KBr pellets. HR-ESI-MS data were measured using a Q-Trap LC/MS/MS (Turbo Ionspray Source) spectrometer. 1D and 2D NMR spectra were recorded on Bruker-400 with TMS as internal standard. Reversed-phase HPLC was performed using Agilent 1200 series with a DIKMA (4.6 × 250 mm) analytical column packed with C18 (5 μm). Column chromatography was performed on Sephadex LH-20 (Amersham Pharmacia, Sweden), silica gel H, Qingdao,China). TLC was performed on precoated silica gel GF254 plates, and the spots were visualised under uV light (254 or 356 nm) or by spraying with 10% H 2 So 4 in 95% EtoH followed by heating.

Plant material
The whole plant of C. setosum was collected from Xixia Country, Henan Province, China in october 2013. A voucher specimen (No. NNu-201310) has been deposited in Nanyang Normal university. The plant material was dried, finely powdered and used for the successive extraction.

Extraction and isolation
The whole air-dried and powdered plant of C. setosum (10.0 kg) was extracted three times with 95% EtoH (15 L × 3) heating under reflux to give 1.2 kg of crude extract (Almeida et al. 2011). The combined extracts were successively partitioned with petroleum ether, ethyl acetate and n-butanol to yield three fractions: petroleum ether soluble fraction (104.5 g), ethyl acetate soluble fraction (188.4 g) and n-butanol soluble fraction (268.2 g). According to the results of bioassay-guided investigation of C. setosum, we found that the n-butanol soluble fraction showed potential hepatoprotective activity.

Acid hydrolysis of compounds 1-8
Compounds 1-8 (5 mg each) were treated in 5% HCl (0.5 mL) and heated at 90 °C for 2 h, respectively (Chang & Case 2005). After cooling, each reaction mixture was extracted with EtoAc, and the aqueous layer was neutralised with 0.1 M NaoH. As a result, glucose and rhamnose were obtained from compound (1-4), which were detected by thin layer chromatography (TLC) with authentic sugars. Similarly, we obtained glucose from compounds (5-8). The type of glucose and rhamnose were identified by TLC method with authentic sugars (Ma et al. 2014).

Hepatoprotective assay
The compounds (1-8) were evaluated for their hepatoprotective activities against D-galactosamine-induced toxicity in HL-7702 cells using a MTT colorimetric method. The HL-7702 cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 3% foetal calf serum, 100 units/mL penicillin and 100 units/mL streptomycin in 5% Co 2 and incubated at 37 °C, which were placed in a 96-well microplate and precultured for 24 h (Hsiao et al. 2013). The cultured cells were measured for cytotoxic effects which exposed to 40 mM D-galactosamine after 24 h. At last, the medium was replaced for the serum-free medium (0.5 mg/mL MTT) for 3.5-h incubation. The medium was removed and added DMSo (150 μL/well) into the microplate, and the formazan crystals were redissolved. The optical density (oD) was measured by a microplate reader at a wavelength of 492 nm, and the inhibition was calculated as inhibition (%) = [(oD (sample) − oD (control) )/(oD (normal) − oD (control) )] × 100 (Liu et al. 2012).

Statistical analysis
We had evaluated the pharmacological activities of compounds (1-8) with the bicyclol (hepatoprotective activity drug) as the positive control for their hepatoprotective activities against D-galactosamine-induced toxicity in HL-7702 cells. The results of hepatoprotective activities were given in Table S2. The percentage of inhibition of compounds 2, 4, 5, 7 and 8 (10 μM) were calculated using the following formula: inhibition (%) = [(oD (sample) − oD (control) ) /(oD (normal) − oD (control) )] × 100, respectively. Moreover, all the values were expressed as means ± SD of three experiments. The significance of unpaired observations between normal or control and tested samples was determined by Student's t-test (Ma et al. 2014). The differences were considered significant at p < 0.05. Consequently, compounds 2, 4, 5, 7 and 8 (10 μM) showed moderate hepatoprotective activities.