Fetal bovine serum, 1640 medium and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Gibco-BRL (Grand Island, NY, USA). G418 was obtained from Invitrogen Life Technologies (Carlsbad, CA, USA). D-GalN, t-BHP, TAA, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), insulin, hydrocortisone and silybin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Lamivudine was purchased from GlaxoSmithKline (China) Investment Co., Ltd. (Beijing, China). HBV DNA PCR-fluorescence quantitation kit and enzyme-immunoassay (EIA) kits for the detection of hepatitis B surface antigen (HBsAg) and hepatitis B envelope antigen (HBeAg) were provided by Shanghai Kehua Bio-engineering Co., Ltd (Shanghai, China). A plasmid mini preparation kit was purchased from Axygen Biosciences (Union City, CA, USA). The viral DNA kit was obtained from Omega Bio-Tek, Inc. (Norcross, GA, USA). SYBR^® Premix Ex Taq™ II, Taq DNA polymerase, PMD-18T vector kit, DNA marker and conventional PCR reagents were obtained from Takara Biotechnology (Dalian) Co., Ltd. (Dalian, China). All other reagents were of the highest commercial grade available.

The whole herb of T. mongolicum Hand.-Mazz. was purchased from Haozhou herbal medicine market (Anhui, China) and verified by Professor Liurong Chen, College of Pharmaceutical Sciences, Zhejiang University (Hangzhou, China). The voucher specimen (TM-200405) of the plant was deposited at the herbarium of the College of Pharmaceutical Sciences, Zhejiang University. The dried whole herb of T. mongolicum (10 kg) was extracted three times with 95% ethanol. The extract was combined and concentrated under reduced pressure to generate a dark tar-like mass, which was dissolved in hot water. This solution was treated with 5% sodium carbonate, followed by three extractions with chloroform to remove lipophilic constituents. The remaining aqueous extract was further partitioned with ethyl acetate following acidification to pH 2 using 1N hydrochloric acid. The ethyl acetate fraction was washed with water to pH 7 and condensed under reduced pressure (0.06 Mpa) to afford a dark-brown powder (160 g), which was termed TME for further use.

The total phenolic content of TME was determined colorimetrically using ferric chloride-potassium ferricyanide reagent by a modified colorimetric method (13). Absorbance was measured at 763 nm with a UV-1600 Spectrophotometer (Beijing Rayleigh Analytical Instrument Co., Beijing, China). The total phenolic content of TME was calculated using the standard curve of caffeic acid. The total flavonoid content in TME was determined using sodium nitrite-aluminum nitrate-sodium hydroxide reagent by colorimetric assay (14). Absorbance was measured at 500 nm and the total flavonoid content of TME was calculated using the standard curve of rutin. Chemical analyses were repeated three times.

TME was analyzed using a HPLC system (Waters 2695, Waters, Milford, MA, USA) and a symmetry^® C18 (5 μm, 4.6 × 150 mm) column was used (Waters). The mobile phase consisted of two eluents: 0.1% acetic acid (B) and methanol (A). To achieve separation, the flow rate was set at 0.8 ml/min and 5 μl sample was injected. The following gradient elution method was adopted: Initially, 90% B and 10% A maintained for 10 min followed by 55 min linear change to 0% B and 100% A. The detection wavelength was set at 280 nm with a column temperature of 30°C.

TME (100 g) was dissolved in 70% methanol. The solute was applied to a polyamide column and washed with water followed by 30, 50, 70 and 100% of methanol. The separated fractions were repeatedly chromatographed on silica gel, reversed phase silica gel (RP-18) and Sephadex LH-20. The 30 and 50% methanol fractions afforded compound 1 (35 mg) and compound 2 (100 mg), respectively. Their structures were elucidated by spectroscopy (Varian INOVA 400 MHz Nuclear Magnetic Resonance Spectrometer; Varian, Palo Alto, CA, USA) and compared with literature data (15,16). The standard solutions of compound 1 and compound 2 were prepared in the same mobile phase for HPLC analysis.

The experimental protocol was approved by the Animal Ethics Committee of Zhejiang Province (Zhejiang, China), in accordance with the international standard on the care and use of experimental animals. Rat hepatocytes were isolated from three-day-old Sprague-Dawley (Zhejiang Experimental Animal Center, Hangzhou, China) rats according to the method of Anil Kumar et al (17). The isolated hepatocytes were suspended in 1640 medium and subsequently transferred to 96-well culture plates at a density of ~1.0×10⁵ cells/ml. Following hepatocyte attachment to the plate, cells were treated with hepatotoxic agents and analyzed in the following assays.

Cytotoxicity induced by TME treatment was detected by MTT assay as follows: Hepatocytes were cultured in 1640 medium in the presence of 1–100 μg/ml TME for 48 h and subsequently 10 μl MTT (5 mg/ml) was added to the cells in each well. Following culturing for 4 h, the medium was removed and the blue formazan crystals that had formed were dissolved in dimethylsulfoxide. The absorbency of formazan generated from MTT was measured at 570 nm using an ELX 800 universal microplate reader (BioTek Instruments, Winooski, VT, USA). Cell survival was defined as the quantity of formazan produced relative to that of the untreated control cells.

The hepatocytes were incubated for a further 48 h in fresh culture medium containing 1–100 μg/ml TME after the cells had been incubated for 8 h with 20 mM D-GalN, 2 h with 8 mM TAA and 1.5 h with 6.5 mM t-BHP, respectively. Silibin was used as a reference drug. Hepatocyte injury was assessed according to cell viability. Cell viability was expressed according to protection rate (%) and proliferation index.

Duck hepatocytes were isolated from 20-day-old embryonated, unhatched, duck eggs laid by DHBV-infected ducks according to a modified method (18). Cells were maintained at 1.0×10⁵ cells/ml in 1640-medium containing 2 mM glutamine, 1 μg/ml insulin, 7.5 μg/ml hydrocortisone, 10% (v/v) fetal bovine serum, 100 U/ml penicillin and 100 U/ml streptomycin at 37°C in a 5% CO₂ atmosphere. Congenital DHBV infection was confirmed by PCR assay on the viral DNA obtained from the allantoic fluid of duck embryos using DHBV-specific primers. The forward primer was 5′-AAC CAT TGA AGC AAT CAC TAG AC-3′ and the reverse primer was 5′-ATC TAT GGT GGC TGC TCG AAC TA-3′.

Cytotoxicity induced by TME treatment was measured using the MTT method as follows: Infected duck hepatocytes were cultured in 1640-medium with a concentration of 1.0×10⁵ cells/ml in the presence of 1–100 μg/ml TME for six days and subsequently the MTT assay was performed as described previously. Cytotoxicity was described according to the quantity of formazan produced relative to that produced by the untreated control cells.

In order to measure the DHBV DNA levels, the infected duck hepatocytes were treated with varying concentrations of TME and the medium with the test compound was replaced every three days. On the third and sixth days, the DHBV DNA level in the replaced culture supernatants was detected with a SYBR-green dye-based quantitative PCR assay as follows: DHBV DNA was extracted and amplified with a Bio-Rad iQ5 Real Time PCR system (Bio-Rad Laboratories Inc., Irvine, CA, USA). The thermal program comprised of an initial denaturation at 94°C for 10 min followed by 40 amplification cycles at 94°C for 30 sec, 55°C for 30 sec, and then 72°C for 45 sec. The forward primer was 5′-AGC TGG CCT AAT CGG ATT AC-3′ and the reverse primer was 5′-TGT CCG TCA GAT ACA GCA AG-3′. SYBR^® Premix Ex Taq™ II was used to amplify and detect the DNA level during the reaction. A plasmid containing the DHBV genome was used to generate the standard curve for quantifying DHBV levels. Lamivudine was used as the reference drug.

HepG2.2.15 cells were provided by the State Key Laboratory for the Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University (Hangzhou, China). The cells were maintained in DMEM containing 2 mM glutamine, 10% (v/v) heat-inactivated fetal bovine serum and 380 μg/ml of G418 at 37°C (95% humidity, 5% CO₂). The cytotoxicity of TME in HepG2.2.15 cells was analyzed by the MTT assay. The cells were seeded in a 96-well plate at a concentration of 1.0×10⁵ cells/ml. Varying concentrations of TME were applied to culture wells in triplicate. Following six days of incubation, the MTT assay was performed as described previously. Cytotoxicity was described according to the quantity of formazan produced, compared with that produced by the untreated cultures.

To determine HBV antigen and HBV DNA levels, HepG2.2.15 cells were treated with various concentrations of TME and the medium with test compound was replaced every three days. On the third day, the replaced medium was assayed for HBsAg and HBeAg. On the sixth day, the replaced medium was measured for HBsAg, HBeAg and HBV DNA. Lamivudine served as a positive control. The HBsAg and HBeAg levels in the replaced culture supernatants were determined by using HBsAg and HBeAg EIA kits, respectively. The results were measured at 450 nm by a MULTISKAN MK3 multi-well microplate reader (Thermo Fisher Scientific Inc., Waltham, MA, USA).

HBV DNA levels in replaced culture supernatants were detected with a HBV DNA PCR-fluorescence quantitation kit. HBV DNA was extracted and amplified with Bio-Rad iQ5 Real Time PCR system (Bio-Rad, Hercules, CA, USA). The thermal program comprised of an initial denaturation at 94°C for 2 min followed by 40 amplification cycles each of two steps: 95°C for 5 sec and 60°C for 30 sec. The forward primer was 5′-CCG TCT GTG CCT TCT CAT CTG-3′, the reverse primer was 5′-AGT CCA AGA GTA CTC TTA TAG AAG ACC TT-3′ and the Taqman probe was FAM-CCG TGT GCA CTT CGC TTC ACC TCT GC. A plasmid containing the full-length insert of the HBV genome was used to prepare the standard curve.

Experimental results were expressed as the mean ± standard deviation and subjected to a one-way analysis of variance and Student’s t-test. P<0.05 was considered to indicate a statistically significant difference.

The structures of compounds 1 and 2 were identified as caffeic acid and luteolin7-O-β-D-glucopyranoside, respectively (Fig. 1). The results of quantification indicated that the total phenolic acid content of TME was 36.27%, using caffeic acid as the control standard, and the total flavonoid was 49.90% with rutin as the control. HPLC analysis demonstrated that caffeic acid and luteolin-7-O-β-D-glucopyranoside were the major phenolic and flavonoid components in the extract and their absolute contents amounted to 0.6 and 1.0%, respectively (Fig. 2).

The cytotoxicity of TME toward neonatal rat primary hepatocytes was tested. The result showed that TME, at concentrations of 1–100 μg/ml was almost nontoxic to the cells. Cytotoxicity was induced in neonatal rat primary hepatocytes by exposure to 20 mM D-GalN, 8 mM TAA and 6.5 mM t-BHP, respectively. Subsequently, the cells were treated with TME. As shown in Table I, TME concentrations between 1 and 100 μg/ml improved cell viability in a dose-dependent manner. Silibin, used as the reference drug, induced a similar, but weaker, effect, than that of TME at the same concentration.

The cytotoxicity test indicated that TME was nontoxic to primary duck fetal hepatocytes at concentrations between 1–100 μg/ml. The effect of TME on the DHBV DNA level is shown in Table II. Following cell treatment with TME for three days, TME at concentrations between 50–100 μg/ml significantly reduced DHBV DNA levels in culture supernatants compared with vehicle. On the sixth day, TME markedly inhibited the DHBV DNA replication at a concentration of 1–100 μg/ml. Lamivudine, used as the standard drug, also induced a similar effect.

Cellular toxicity tests revealed that TME was harmless to HepG2.2.15 cells at concentrations between 1–100 μg/ml. Expressions of HBsAg and HBeAg in culture supernatants were assayed after the cells had been incubated with TME for 3 days (Table III). The results indicated that TME had significant inhibitory effects on HBsAg expression at concentrations between 10–100 μg/ml and the HBeAg expression at concentrations between 50–100 μg/ml. TME produced the maximum inhibition rates, on HBsAg and HBeAg expression at a concentration of 100 μg/ml, of 59.47 and 66.37%, respectively. The levels of HBsAg, HBeAg and HBV DNA in culture supernatants were measured after the cells had been treated with TME for 6 days (Table IV). At concentrations between 10–100 μg/ml, TME significantly inhibited HBsAg expression. At concentrations between 25–100 μg/ml, TME significantly reduced HBeAg levels. At a concentration of 100 μg/ml, TME produced the maximum inhibition rates of 91.39 and 91.72% on HBsAg and HBeAg expression, respectively. Furthermore, TME markedly inhibited HBV DNA replication at 25–100 μg/ml.