Anti-hepatitis B virus active secoiridoids from Swertia kouitchensis

Three new secoiridoids, swertiakoulactone (1) and swertiakosides A and B (2 and 3), were isolated from Swertia kouitchensis. Their structures were elucidated by comprehensive spectroscopic analyses including MS, IR, 1D and 2D NMR data. By the anti-hepatitis B virus (HBV) assay on Hep G 2.2.15 cells line in vitro, compound 1 showed moderate activities inhibiting the HBsAg secretion (IC50 = 1.10 mM, SI = 4.39) and HBV DNA replication (IC50 = 1.16 mM, SI = 4.12).


Introduction
Swertia kouitchensis (Gentianaceae), is a perennial herb mainly distributed in Yunnan, Sichuan, and Guizhou provinces of China, 1 which was widely used as a fork medicine to treat jaundice, indigestion and sore throat. 2 Its congener plant S. mileensis, well-known as Qing-Ye-Dan in Chinese, has been documented in Chinese Pharmacopoeia (1977-2005 editions) for treatment of viral hepatitis. 3 Our previous investigation on S. mileensis resulted in a series of novel anti-HBV active secoiridoid dimers with C 18 and C 20 skeletons (swerilactones A and B 4 , C and D 5 ) and secoiridoid trimers with C 29 skeleton (swerilactones H-K) 6 , as well as three unusual aromatic lactones (swerilactones E-G) 7 and three new secoiridoid glycoside dimers (swerilactosides A-C) 8 . Presently, xanthones, triterpenoids and sterols except for iridoids have been reported from S. kouitchensis. 9,10 As an on-going search for anti-HBV active compounds from natural resources, our investigation on S. kouitchensis led to the isolation of three new compounds, including one secoiridoid, swertiakoulactone (1), and two secoiridoid glycosides, swertiakosides A and B (2 and 3). Herein, we report the isolation, structural elucidation, and anti-HBV properties of compounds 1-3.
Compounds 1-3 were evaluated for their anti-HBV activities, namely inhibiting the secretion of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg), as well as HBV DNA replication in Hep G 2.2.15 cells, as reported previously (tenofovir was used as the positive control). 6 The results showed that compound 1 exhibited moderate activity inhibiting the secretion of HBsAg with an IC 50 value of 1.10 mM (SI = 4.39) and HBV DNA replication with an IC 50 value of 1.16 mM (SI = 4.12). However, compounds 2 and 3 showed no anti-HBV activity and cytotoxicity at the highest tested concentration (2.86 mM and 1.73 mM, respectively).

Experimental Section
General Experimental Procedures. In Vitro Anti-HBV Assay. The anti-HBV procedure was performed according to our previous report. 8 Briefly, compounds 1-3 were evaluated in the Hep G 2.2.15 cell line, which was stably transfected (Lipofectamine 2000 reagent; Invitrogen; Carlsbad, CA, USA) with the HBV genome. The toxicity of the compounds was assayed by using a modified MTT (GIBCO Invitrogen, Carlsbad, CA, USA) method. DMSO (Gibco; solvent control) alone was added to each culture as a solvent control. All the compounds evaluated were dissolved in DMSO for the assays of anti-HBV activity and cytotoxicity. The concentration of DMSO in the culture was kept below 2.5 μL mL -1 to ensure that the growth of the cells was not affected.
Cell Line and Cell Culture. The widely used Hep G 2.2.15 cell line was applied for the assay of anti-HBV activities. In this study, Hep G 2.2.15 cells established from a hepatoma cell line Hep G 2 (ATCC, Manassas, VA, USA) were cultured in RPMI-1640 (GIBCO) medium supplemented with 10% fetal calf serum (GIBCO), 100 μg mL -1 G148 (GIBCO), 100 IU/mL penicillin (GIBCO), and 100 IU/mL streptomycin (GIBCO). All cultures were maintained at 37C in a moist atmosphere containing 5% CO 2 .
Analysis of Secreted HBV Antigens. The sub-toxic concentration of the identified compounds was measured with a serial dilution in 96-well microplates in which cells were seeded at a density of 3×10 4 cells/mL and cultured at 37 C, 5% CO 2 for 12 days. After incubation, the cells and supernatants were collected. The levels of HBsAg and HBeAg in the supernatants were assayed with an ELISA (AutoBio Diagnostics Co., Ltd., Beijing, P. R. of China) method. The absorbance (A) of each well was measured at 490 nm using a microplate reader (Model 680, Bio-Rad, Inc., Hercules, CA, USA).
Determination of HBV DNA Replication. HepG 2.2.15 cells were seeded in 24 well culture plates at a density of 5 × 10 5 cells per well. Every 2 days, medium was changed. After 6 days, compounds were added to the cell cultures, and fresh medium was fed every other day for another 6 days. Cells were collected and total DNA was isolated by using TIANamp Gemomic DNA Kit (TIANGEN, Biotech Co., Ltd, China) following the manufacturer's instructions. For detection of HBV DNA, a real-time PCR assay was used.
Cytotoxicity Assay. The toxicity of compounds 1-3 was assayed by a modified MTT method. In brief, the samples were prepared at different concentrations. After Hep G 2.2.15 cells had been seeded in a 96-well microplate for 4 h, the samples (20 mL) were placed in each well and incubated for 3 days at 37C; then, 0.1 mL MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide, 400 mg mL -1 ] was added for 4 h. After removal of MTT medium, DMSO (100 mL/well) was added into the microplate for 10 min. The formazan crystals were dissolved, and the absorbance was measured on a microplate reader at 490 nm.