Preparation of plant materials
Four edible Thai plants, C. longa, C. formosum, M. charantia and M. oleifera were purchased from a local market in Bangkok. The plants were dried under shade for 3 weeks and grounded into powder and stored at 4°C until further use. The powder was then extracted by two different solvent systems, the first being a 80% hydroalcoholic solvent (80% ethanol and 20% distilled water) and the second, a 50 mM Tris-HCl buffer (pH 7.5). For hydroalcoholic extraction, samples were shaken in 80% hydroalcoholic solvent for 16 h at room temperature. Then, the solutions were filtered, and then the filtrate was re-suspended and shaken in ethanol for 48 h. All extracts were pooled, re-filtered and concentrated using a rotary evaporator and then lyophilized. Prior to testing, the lyophilized extracts were dissolved in distilled water to produce 1 g/L of plant extracts. The extract yield (%, w/w) was determined from all hydroalcoholic extracts by using the formula:
(Additional file1: Table S1).
For buffer extraction, the samples were incubated in buffer for 48 h at 4°C in the dark, filtered, and then centrifuged at high speed for 30 min at 4°C. The supernatant of each sample was collected and dialysed in Tris-HCl for 48 h, then total protein was determined using the Bradford assay, using BSA as a protein standard. The total protein of each buffer extract is presented in Additional file1: Table S1.
Cell culture and transfection
Both COS-7 and HepG2 (ATCC, gifts from The Centre of Excellence Clinical Virology and Molecular Biology Research, Chulalongkorn University) were cultured in DMEM (Gibco, Invitrogen) and supplemented with 10% (v/v) heat inactivated fetal bovine serum (Gibco, Invitrogen) and 1%(v/v) antimicotic antibiotic (Gibco, Invitrogen). All cell lines were cultured in 75 cm3 sterile tissue culture flasks at 37°C under a 5% CO2 atmosphere. For quantitative real time-PCR, HepG2 cells were seeded on 24-well plates with approximately 1 x 105 cells in each well. Seeded cells were treated with either 30 μg/mL of hydroalcoholic crude extracts or buffer extracts containing 0.3 μg total protein and then transfected with 1 μg of the DNA expression plasmid of HBV genome (pHBV48, a gift from M-H Lin, National Taiwan University) and using the Lipofectamine™ 2000 in triplicate. This plasmid has been reported to express full-length HBV and is able to produce HBV particles in cells. For the positive control, cells were transfected with 1 μg of pHBV48 without addition of crude extracts. The ratio between Lipofectamine™ 2000 (μL) and DNA (μg) was 3:1. After 5 days of incubation, cells were subjects to DNA extraction and real-time analysis.
Cell viability assay (MTT assay)
COS-7 and HepG2 cells were seeded on 96-well plates with a cell density of approximately 1.5x 103 cells per 150 μL of media in each well. After 24 h of incubation, cells were added with 0 (solvent only), 50, 150 and 300 μg/mL of hydroalcoholic extracts. For buffer extracts, cells were incubated with 0 (buffer only), 0.5, 1, 1.5 and 2 μg of total protein extracts from each plant. A cell-free control was also included to exclude any false positive results from the assay. Each sample was performed in quadruplicate and cells were incubated for 3, 5 and 7 days. After incubation, the culture medium was removed from each well by aspiration and 4 μg of MTT (Invitrogen) was added into each well. After 3 h of incubation, DMSO (Amresco) was added to dissolve the purple formazan of MTT. The absorbance was then measured by a microplate reader at a wavelength of 570 nm. The cell viability (%) was calculated using the formula:
Quantitative real time- PCR analysis of HBV cccDNA
Total DNAs were isolated from each well using a genomic DNA Extraction mini kit (RBC Bioscience) according to the manufacturer’s instruction. HBV cccDNA was amplified and quantified by real-time PCR assay using specific primers: HBV_CCC_F1 (5′- actcttggactccagcaatg-3′) and HBV_CCC_R1 (5′-ctttatacgggtcaatgtcca-3′) with SYBR-green. These pair of primers was designed to match the region corresponding to the gap and incomplete region in the partially double- stranded HBV DNA. Therefore, they specifically amplify DNA fragments from HBV cccDNA but not from genomic DNA. Negative control (no DNA template) was included to determine contamination whereas the positive control (transfected cells with 1 μg of pHBV48) was performed to yield quantitative information. Each sample was performed in triplicate. A result was indicated in terms of a relative quantitation by the comparative threshold (delta-delta Ct) method, (2-ΔΔCt). In this study, the reference gene was beta-globin. The target gene was the cccDNA of transfected HBV and the calibrator was cells transfected with only the HBV plasmid.
Student’s t-test was used for comparing data between control cells (COS-7) and human liver cancer (HepG2) cells in the viability assay. It also used to determine significant differences between the positive control (transfected cells with pHBV48 without addition of crude extracts) and treated cells with crude extracts in the real-time analysis. A statistic t was calculated using the formula:
and are means of % cell viability of COS-7 cells and HepG2 cells respectively; Sp is the sample standard deviation (uncertainty value); S1
2 is COS-7 sample variance; S2
2 is HepG2 sample variance; n1 is number of COS-7 sample and n2 is number of HepG2 sample. The t distribution was used with the degree of freedom (df) = n1 + n2 -2. A p-value was determined from the probability table. A P value < 0.05 indicated the presence of a statistically significant difference.