Three New Phenolic Compounds from Sabia parviflora and Their Hepatoprotective Activity

Three compounds were obtained from Sabia parviflora Wall., and their structures were identified through nuclear magnetic resonance (NMR) spectroscopy, particularly 2-dimensional (2D)-NMR. The molecular masses were determined using quadrupole-time-of-flight-mass spectrometry. Electronic circular dichroism spectra were used to determine the absolute configuration of compound 1. The 3 new compounds were identified as 2,2,7,7-tetramethyl-8S,10-dihydroxy-2,7,8,9-tetrahydro-2H-naphtha[2,3-b]pyran-6-one-10-O-β-D-glucopyranoside, (2Z)-4-(3-carboxy-4-hydroxyphenyl)-2-methylbut-2-enoic acid, and (2Z)-5-(3-carboxy-4-hydroxyphenyl)-5-oxo-2-methylpent-2-enoic acid. The liver protective activities of these compounds were tested by HepG2 and LO2 cell lines, which were induced using free fatty acids.


Introduction
Sabia parviflora Wall., family Sabiaceae, is a plant mainly distributed in southwest China. It is a traditional Chinese herbal medicine that is mainly used for the treatment of "damp heat jaundice, hepatitis and hemostasis." 1,2 Numerous chemical studies have shown that the plant contains triterpenoids, 1,3 flavonoids, 4,5 alkaloids, 1,6 phenolic acids, phenylpropane, and polysaccharides. 7 Previous pharmacological studies have revealed that the plant's compounds possess antiviral, 8 antiinflammatory, 5, 9 antiliver injury, 5,9 and antioxidant 10 activities.
Compound 3, isolated as a yellow oil, had a molecular formula of C 13     C-NMR spectra of compound 3 were similar to those of compound 2, except for subtle differences at C-5 ′ . The signal at δ C 192.1 was used to define the carbonyl group. The HMBC cross peak (δ 7.67/192.1) indicated that the carbonyl carbon was connected to C-1. Furthermore, in the ROESY experiment, the cross peak at δ H 6.00, H-3 ′ /δ H 1.88, H-2 ′ a supports the Z-configuration of the C-2 ′ -C-3 ′ double bond. Therefore, compound 3 was established as (2Z)-5-(3-carboxy-4-hydroxyphenyl)-5-oxo-2-methylpent-2-enoic acid. The NMR data of compounds 2 and 3 are listed in Table 2, and the HMBC spectrum is shown in Figure 2.
Compounds 1-3 were evaluated for their hepatoprotective activities. The results demonstrated that the 3 compounds had no hepatoprotective activity with no significant difference being observed between the cells in the presence or absence of these 3 compounds.

Materials
Analytical methanol and glacial acetic acid were purchased from Sinopharm Chemical Reagent Co., Ltd. Dimethylsulfide-d 6 (DMSO-d 6 ) was used as a solvent to dissolve monomers. All chemicals used in this study were of analytical grade.
Deionized ultrapure water was obtained using a MillQ Millipore Ultrapure Water Purification System (USA).
Methanol and acetonitrile for separation and purification were from Shanghai McLean Company, silica gel (300-400 mesh) for chromatographic separation from Qingdao Haiyang Chemical Group, and DIAION HP-20 macroporous resin from Mitsubishi. The medium-pressure liquid chromatography system was equipped with a Lisui EZ Purify III system, which was from Shanghai Lisui Chemical Engineering. The RP-C18 (25 µm) chromatographic column was purchased from Merck Company. The Ryong C18 column (10 µm, 30 × 250 mm) was equipped with a preparative high performance liquid chromatography (HPLC) system. We also used a Ryong C18 column (5 µm, 20 × 25 0 mm) for further purification. A Cosmosil C18 column (5 µm, 4.6 × 50 mm) was used in the component purity test. A Shimadzu LC-30A HPLC was connected to a TOF™5600 + mass spectrometer. The important NMR data were obtained using a Bruker Avance spectrometer. Palmitic acid (33.3%) and oleic acid (66.6%) were purchased from Sigma.
The 2 cell lines used in this study, HepG2 and LO2, were purchased from American Tissue Culture Collection.

Plant Material
The stems of S parviflora were collected in Ziyun City and identified by Professor Guo-Yue Zhong, Jiangxi from University of Traditional Chinese Medicine. A voucher specimen is deposited in the corresponding author's laboratory, the Center of

Cell Viability Assay
HepG2 and LO2 cells were preserved in Roswell Park Memorial Institute 1640 (RPMI 1640) medium under a humidified atmosphere of 5% CO 2 at 37°C. The medium contained 5% fetal bovine serum, penicillin (100 μm L −1 ), and streptomycin sulfate (100 μm L −1 ). HepG2 and LO2 cells were seeded and inoculated on 96-well plates at a density of 1 × 10 4 cells/well for 24 h. Then, 100 μL of medium containing different concentrations of the 3 compounds was added to each well, except for the blank and model wells. After 4 h, free fatty acids (1 mmol L −1 ) were added to all groups except the blank. After 24 h, the supernatant was discarded and a 10% CCK8 reagent (100 μL/well) was added and incubated for 2 h. In our experiment, a microplate reader was used to measure the absorbance of the sample at 450 nm. Cell viability is presented as a relative percentage of the untreated control. 12,13 Hydrolysis of Compound 1 and GC Analysis As reported in the previous literature, 14   (2 mL × 4). The aqueous layer was neutralized with 2 mol L −1 NaOH and dried to obtain the monosaccharides. Then, in the presence of pyridine (2 mL), the dry powder was added to L-cysteine methyl ester hydrochloride (1.5 mg) and kept at 60°C for 1 h. Next, trimethylsilylimidazole (1.5 mL) was added to the reaction mixture and kept at 60°C for 30 min. The supernatant (4 μL) was directly subjected to GC and compared with a glucose standard. GC was performed using the following experimental conditions: Agilent-7890b ECD detector, Chromatographic column: HP-5 (30 m × 0.25 nm, 0.25 μm), flow: 2 mL min −1 ; split ratio 30:1. temperature: 250°C.

Conclusion
Three new compounds were isolated from S parviflora. These 3 compounds showed no significant activity regarding liver protection. Further research is needed in order to understand other possible biological activities of these compounds.