Acacia mangium: A promising plant for isolating anti-hepatitis C virus agents

Materials

A. mangium leaves were obtained from an area in Mojokerto Regency, Indonesia. The plant was verified by an expert botanist from Materia Medica Indonesia, East Java (see the Underlying data (Wahyuni, 2022)).

The materials used in the bioassays were as follows: Huh7it-1 cells (Apriyanto et al., 2016); adapted variant of HCV strain (JHF1a) (Yu et al., 2010); Dulbecco’s Modified Eagle’s Medium (DMEM, GIBCO Invitrogen) supplemented with 10% of fetal bovine serum (FBS, GIBCO Invitrogen), 150 μg/mL kanamycin (Sigma-Aldrich), and non-essential amino acids (NEAAs, GIBCO Invitrogen); Dulbecco’s phosphate-buffered saline (GIBCO Invitrogen); formaldehyde (HCHO, Sigma-Aldrich); trypsin-EDTA (Sigma-Aldrich); 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTT, Sigma-Aldrich); bovine serum albumin (BSA, Biowest); Triton X-100 Sigma-Aldrich); 3,3′-diaminobenzidine (DAB, Thermo Fisher Scientific); anti-HCV human antibody and HRP-conjugated goat anti-human Ig antibody (Thermo Fisher Scientific); RIPA buffer; polyacrylamide gel and polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA); β-actin antibody (MBL, Nagoya, Japan); and a chemiluminescence detection system (Bio-Rad; GE Healthcare, UK).

Preparation of A. mangium leaf extracts

A. mangium leaves (2 kg) were dried, ground into powder, and further extracted with two kind of extraction procesess. First, 200 g of powder was extracted by maceration process with a total of 2 liters of 96% ethanol and another 200 g was successively extracted with 2 liters of n-hexane, dichloromethane, and methanol. Specifically, 10 mg of the extract powder were dissolved in 100 μL of dimethyl sulfoxide to obtain 100,000 μg/mL stock solution (Wahyuni et al., 2013).

Cell and virus culture

Huh7it-1 cells were cultured in DMEM supplemented with 10% FBS, 150 μg/mL kanamycin, and NEAAs in 5% CO₂ at 37°C and maintained for bioassay purposes. Cells were incubated at 37 °C for 2 days. Cells which showed more than 80% confluence were used for further bioactivity assay. The detailed protocol for cell passage is available at https://dx.doi.org/10.17504/protocols.io.n92ldpbd7l5b/v1.

Virus stock was obtained by propagating HCV in Huh7it-1 cells. Culture supernatants at day 3, 5 and 7 after virus infection were collected. Virus titers were calculated by titration assay (wahyuni, 2013). In brief the virus harvested was diluted on x5, x25, x125, x625, and x3125 then put onto Huh7it-1 cells and incubated for 4 hours, the remaining virus was removed and refed with new medium for further incubation for 2 days. The infected cells were stained with DAB staining reagent and further calculated. The number of viruses represented as titer virus. The high titer virus stock (higher than 1x10⁵) was chosen for anti-HCV assay. The stock was stored at −80°C until use (Wahyuni et al., 2018).

Anti-HCV activity

An anti-HCV assay was conducted using HCV-infected Huh7it-1 cells. Various concentrations of the extracts were mixed with virus solution (multiplicity of infection of 0.1) and inoculated into the cells at a final concentration of 0.01, 0.1, 1, 10, 50, or 100 μg/mL. The cultures were incubated for 48 h with 2 h of virus inoculation and further incubation for 46 hours at 37°C. The viral levels of the supernatants were examined by titration assay. Culture cell supernatant was collected, diluted 10x with medium and inoculated to the Huh7it cells. This was incubated for 2 days and the infected cells were calculated after the immunostaining process. The inhibitory effect of the extracts were calculated compared to the untreated control. The 50% inhibitory activity was conducted by SPSS software version 25 (Wahyuni et al., 2019; Wahyuni et al., 2018). Ribavirin was used as the positive control.

Viral titration and immunostaining

Huh7it-1 cells were incubated with serial dilutions of the supernatant for 48 h. The cells were fixed with formaldehyde, stabilized with triton and subjected to immunostaining with primary antibody (human serum) and secondary antibodies (HRP-conjugated goat anti-human). The detailed immunostaining protocol is available in the Underlying data (Permanasari and Wahyuni, 2022a). DAB staining was performed to visualize the infected cells. The percent inhibitory effect was calculated by comparing the reduction of infected cells to the control (Wahyuni et al., 2018).

Mode of action analysis

The mode of action assay was performed to examine whether A. mangium extract affected the entry or post-entry step of the HCV life cycle. Three parallel experiments were performed. First, the extract was only added to the cells during viral inoculation. Second, the extract was only added to the cells after viral inoculation. Third, the cells were treated with the extract both during and after inoculation. After 48 h of incubation, all supernatants were collected to examine their viral levels by titration assay. Viral supernatant was diluted with medium and inoculated to the Huh7it cells. Infected cells were calculated to further determine the percentage inhibitory against HCV. Anti-HCV activity was expressed by 50% inhibition concentration (IC₅₀) (Hafid et al., 2017).

Cytotoxicity assay

The MTT (3-(4,5- Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay was used to measure cytotoxicity. Huh7it-1 cells were incubated with various concentrations of the extracts for 48 h. After incubation, 10% MTT was added to the cultures for 4 h. The remaining reagent was discarded and DMSO was asses in each wells to dissolved the formazon formation. Absorbance was measured at 560 and 750 nm to calculate the percentage cell viability relative to the control. SPSS software version 25 probit analysis was used to calculate the 50% cytotoxic concentration (CC₅₀) (Wahyuni et al., 2018). The protocol of the MTT assay is available at http://dx.doi.org/10.17504/protocols.io.6qpvr4x5pgmk/v1.

Immunoblotting assay

Huh7it-1 cells were treated with mixtures of the extracts (10 or 50 μg/mL) and HCV. After incubation for 2 days at 37 °C, the cells were collected, lysed, and protein levels were determined using a BCA assay kit (Thermo Fisher Scientific). Equal amounts of proteins were subjected to SDS–polyacrylamide gel electrophoresis followed by transfer to a polyvinylidene difluoride membrane. Samples were run in transfer buffer at 0.3 A for 30 minutes followed by processing to SDS running buffer at 0.1 A for 25 minutes. The membrane was applied into a blocking buffer of skim milk and reacted to antibodies. The primary antibody was an HCV NS3 mouse monoclonal antibody (clone H23; Abcam, Cambridge, MA, USA), and the secondary antibody was HRP-conjugated goat anti-mouse immunoglobulin. β-actin (MBL, Nagoya, Japan) served as the internal control (Permanasari et al., 2021; Widyawaruyanti et al., 2021). Membranes were incubated at room temperature for 1 hour in each antibody. NS3 protein expression was detected using an enhanced chemiluminescence detection system (GE Healthcare, Buckinghamshire).

Anti-HCV activity of the combination of A. mangium extract and simeprevir

Combination treatment was performed by adding equal volumes of A. mangium extract and simeprevir (Toronto Research Chemical, Canada). Simeprevir was added at 0.25×, 0.5×, 1×, 2×, and 4×IC₅₀ for monotherapy and combination. All treatments were performed for 48 h incubation. The IC₅₀ of simeprevir when used in combination with A. mangium extract and monotherapy were calculated and compared using the SPPS probit assay version 25 (Wahyuni et al., 2020).

In vitro activity of A. mangium against HCV

All A. mangium leaf extracts strongly inhibited HCV in a dose dependent manner (Figure 1). Inhibition concentrations of 50% of all extracts were calculated by probit analysis. Dichloromethane extract displayed the strongest effects, with an IC₅₀ of 0.2 ± 0.3 μg/mL, whereas the IC₅₀s of the extracts ranged 2.8–4.6 μg/mL (Table 1). While the positive control of ribavirin revealed the IC₅₀ values of 10.4 ± 0.2 μg/mL. All of the extracts were demonstrated to possess stronger activity compared to the positive control of ribavirin. The raw data are available in the Underlying data (Permanasari and Wahyuni, 2022b).

Figure 1. Concentration-dependent inhibition of hepatitis C virus infectivity by the ethanol, n-hexane, dichloromethane, and methanol extracts of Acacia mangium.

Huh7it-1 cells were cultured and inoculated with mixtures of the virus and each extract at various concentrations. Virus inhibition was calculated relative to the untreated control. The data represent the mean of three independent experiments.

Cytotoxic effects of A. mangium extracts

The cytotoxicity assay of the extracts observed no toxic effect in the Huh7it. The percentage of cell viability demonstrated that all extracts possessed cell viability higher than 80% in the concentration of 400 μg/mL. However, n-hexane and dichloromethane extract showed a reduction in the percentage of cell viability at the dose of 400 μg/mL (Figure 2). The raw data are available in the Underlying data (Permanasari and Wahyuni, 2022c).

Figure 2. Percent viability of extracts in Huh7it-1 cells.

Cells were treated with various concentrations of each extract of Acacia mangium. MTT (3-(4,5- Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) reagent was added after 2 days of incubation, and cell viability was examined using a microplate reader at wavelengths of 450 and 630 nm. The percent cell viability was calculated, and the 50% cytotoxic concentrations were determined by SPSS probit analysis. The data represent the mean of three independent experiments.

Mode of action evaluation

The mode of action assay was performed using three series of experiments. The result illustrated that the inhibitory effect was higher on the post-entry step than on the entry step. The percentage virus inhibition of post entry steps revealed more than 20% differences than the entry step (Figure 3). This result suggested that the extract dominantly affected post-infection processes such as virus replication, virus assembly, and virus release. The raw data are available in the Underlying data (Permanasari and Wahyuni, 2022d).

Figure 3. Mode of action analysis of the ethanol extract of Acacia mangium illustrated that HCV inhibition occurred dominantly in the post-entry step.

(A) Cells were cultured with the ethanol extract of A. mangium (30 μg/mL) in three parallel experiments. First, cultured cells were treated with the extract only during inoculation (entry step). Second, cells were treated with the extract only after inoculation (post-entry step). Third, cells were treated with the extract both during and after inoculation. (B) The entry step comprises the processes of viral binding to host receptors, viral entry into the cells and endocytosis, whereas the post-entry comprises translation, replication, assembly, and release. (C) Percent inhibition of the entry step, post-entry step, and both steps. The data represent the mean ± SEM of three independent experiments.

Anti-HCV activity of the combination of A. mangium extract and simeprevir

Evaluation of the combination effect between the extract and simeprevir showed there is an enhancement of the effects of the extract to simeprevir, which is known as an HCV NS3 protein inhibitor. The addition of A. mangium extract increased the anti-HCV effect of simeprevir, as the IC₅₀ of simeprevir when used in combination with A. mangium extract was reduced by 2-fold compared to that of simeprevir alone (Table 2).

Extract of A. mangium inhibited NS3 protein expression

To examine the mechanism of action of the ethanol extract, western blotting analysis was performed. The result demonstrated a reduction of the NS3 protein level due to the extract intervention. Immunoblotting revealed that treatment with A. mangium extract at 10 or 50 μg/mL decreased the NS3 protein expression by 40% and 95%, respectively, versus the control (Figure 4) (Underlying data, Permanasari and Wahyuni, 2022e).

Figure 4. Extract of Acacia mangium reduced NS3 protein levels in a concentration-dependent manner.

Huh7it-1 cells were treated with a mixture of the extract (final concentration, 10 or 50 μg/mL) and virus. The cells were lysed in RIFA buffer, and an equal amount of proteins were separated by SDS–polyacrylamide gel electrophoresis.