Inhibitory effects of crude extracts from some edible Thai plants against replication of hepatitis B virus and human liver cancer cells

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.

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)[17] 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[17]. 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.

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[18]. 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:

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[19]. 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[20] 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:

X ¯ andX ¯ 2 are means of % cell viability of COS-7 cells and HepG2 cells respectively; S_p 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[20]. A P value < 0.05 indicated the presence of a statistically significant difference.

To examine whether extracts from tested edible Thai plants could inhibit the viability of liver cancer cells, an optimisation of the MTT assay was performed on the non-cancerous cell (COS-7) for evaluating: (i) the appropriate concentration of crude extract needed such that there was no cytotoxic effects on the normal cells, (ii) the amount of MTT compound needed and (iii) the duration of effects. We used hot water extracts of C. longa as our analytical control because its cytotoxicity and anti-HBV activity were previously reported on the liver-cancer cells[9]. Subsequently, COS-7 cells were treated with various concentrations of C. longa hot water extract (100-900 μg/mL) and different amounts of MTT (2-8 μg) and then the MTT assay was performed after 3, 5 and 7 days post-incubation. A high variation of cell viability was observed when cells were treated with 8 μg of MTT while using 2 μg of MTT produced an unclear pattern of dose dependent effects at any time setting (Figure1). At day 5, the cytotoxicity effect of C. longa extract was moderately elevated in a dose dependent manner with 4 μg of MTT and the concentrations of extracts at higher than 300 μg/mL drastically affected cell viability (less than 65% of cell viability, Figure1B). At day 7, the inhibitory effect was absent and most of the cells were found to be unhealthy when viewed under a microscope leading to relatively high variation of data (Figure1C). Therefore, the appropriate amount of MTT was 4 μg and the optimal concentration of C. longa hot water extract that had no effect on the normal cells was less than 300 μg/mL. The best inhibitory effect was observed at day 5. These optimal conditions were used when analysing the inhibitory effects of all crude extracts on COS-7 and HepG2 cells. The data analysed at day 5 were shown as bar graphs in Figure2 (see supplemental information for the actual numbers of percentage of cell viability, Additional file1: Table S2).

From Figure2A, our data indicated that 50 and 150 μg/mL of hot water extracts from the bulbs of C. longa could specifically affect cell viability of HepG2. However, Kim et al. (2009) reported that 500 μg/mL of C. longa hot water extract had no cytotoxicity effect on the same kind of cell. This variation of concentration may be due to the different conditions used for the MTT assay. Interestingly, crude extract of C. longa by Tris-HCl buffer did not inhibit cell viability on HepG2 cells (Figure2B). This result may suggest that proteins are not involved in the inhibitory effects. Nonetheless, higher amount of total protein may be needed to confirm the MTT result of C. longa buffer extract. Curcumin is the product obtained by solvent extraction of C. longa and is known to have anti-liver cancer activity[21]. However, our result showed that cytotoxicity effect of 80% hydro-alcoholic C. longa crude extract on HepG2 cells had no significant differences from the COS-7 cells (Figure2C). Therefore, the higher concentration of C. longa 80% ethanol extraction may be required to have an effect on the cells.

For the buffer extracts, the results indicated that 0.5 μg of total protein extraction from leaves of C. formosum significantly decreased cell viability of the HepG2 cells without affecting cell viability of COS-7 cells (Figure2B: p < 0.001). A similar observation was found for 2 μg of total protein extraction from the leaves of M. charantia and the fruits of M. oleifera (Figure2B: p < 0.01 and 0.001 respectively). However, higher amounts of protein extracts from the leaves of C. formosum seemed to have a greater cytotoxic effect on normal cells than the HepG2 cells. Up to now, there has not been a report on biological active peptides or hepatoprotective proteins from the extraction of C. formosum. Therefore, it is very interesting to examine novel peptides or a proteomic profile of C. formosum buffer extract. In addition, the crude extract of C. formosum leaves by 80% ethanol displayed a significant difference of inhibitory effect between the HepG2 and COS-7 cells (Figure2C: p < 0.001). This effect is likely due to chlorogenic acid, dicaffeoylquinic acid and ferulic acid derivatives that are previously reported to play important roles in the anticancer activity of C. formosum[11]. Notably, crude extracts by 80% ethanol from the fruits and leaves of M. charantia and M. oleifera did not significantly inhibit cell viability of HepG2 cells (Figure2C). Therefore, the MTT results (Figure2) suggested that the crude extracts of C. formosum are shown to have the best cytotoxicity effect against liver cancer cells whereas hydroalcoholic extracts of M. oleifera (fruit and leaf) and M. charantia (fruit and leaf) do not contain compounds that act specifically against the anti-liver cancer activity.

To study the effect of crude extracts on the replication of HBV, the level of HBV transcription template (cccDNA) was determined using quantitative real-time PCR. This experiment included C. longa hot water extract as the analytical control and we found that the extract significantly inhibited cccDNA of HBV with 85% inhibition (data not shown). This result was consistent with the previous report indicating that C. longa hot water extract has anti-HBV activity[9]. In addition, almost all the buffer extracts (0.3 μg of total protein) significantly decreased the level of HBV cccDNA in transiently transfected HepG2 cells with the DNA expression plasmid of HBV genome (Figure3). The best inhibition from the buffer extractions was observed in C. longa (bulb), C. formosum (leaf) and M.oleifera (leaf) with about 80% inhibition whereas M. oleifera (fruit) showed to have a moderate inhibitory effect with about 50% inhibition. Notably, buffer extracts from the fruits and leaves of M. Charantia were found to drastically reduce gene expression of the reference gene (beta-globin) when compared with untreated cells, thus we did not analyse their effects on the level of HBV cccDNA (Figure3). This is the first report on the anti-HBV activity of crude extracts by the buffer method from C. longa (bulb), C. formosum (leaf) and M.oleifera (leaf). Further experiments will be carried out to investigate any potential anti-HBV compounds from these plant extracts including their mechanism inside the cells.

Furthermore, our results suggest that 30 μg/mL aqueous ethanol extracts of M. olifera (fruit) and C. longa (bulb) drastically decreased the level of HBV cccDNA with greater than 85% inhibition. The 30 μg/mL hydroalcoholic extracts of C. formosum (leaf) and M. Charantia (leaf) also showed a moderate inhibition of the level of HBV cccDNA with 50% inhibition. Hence, our results suggested that crude extracts from 80% ethanol of C. longa (bulb), C. formosum (leaf), M. olifera (fruit) and M. Charantia (leaf) contain compounds that could inhibit HBV replication. On the other hand, 30 μg/mL ethanol extracts of M. Charantia (fruit) and M. oleifera (leaf) had no effect on the level of HBV cccDNA (Figure3). Therefore, the M. oleifera (leaf) ethanol extracts have antiviral activities against herpes simplex virus type 1 (HSV-1)[15] and Sindbis virus[22], but not HBV.