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

Background: Medicinal plants are potential resources for isolating drug candidates. Various plants have been reported to possess pharmacological effects including anti-hepatitis C activities. The current study examined the anti-hepatitis C virus (HCV) activities of Acacia mangium extracts in solvents with various polarities and further evaluated the mechanism of action of the extracts using Western blotting and combination treatment models. Methods: The leaves of A. mangium were extracted in two phases, first in ethanol and then in solvents with different polarities (n-hexane, dichloromethane, and methanol). HCV-infected Huh7it-1 cells were treated with the extracts at concentrations of 0.01, 0.1, 1, 10, 50, and 100 µg/mL. Results: The results revealed the strong anti-HCV activities of the extracts. The 50% inhibition concentrations (IC 50s) of the ethanol, n-hexane, dichloromethane and methanol extracts were of 4.6 ± 0.3, 2.9 ± 0.2, 0.2 ± 0.3, and 2.8 ± 0.2 μg/mL, respectively, and no cytotoxic effect was detected. These extracts displayed stronger effects than the positive control ribavirin. The mode of action of the ethanol extract was evaluated at 30 µg/mL, revealing that the inhibitory effect was stronger on the post-entry step than on the entry step. Western blotting revealed that the extracts decreased NS3 protein expression, indicating that virus replication was suppressed. Further evaluation illustrated that combined treatment with the ethanol extract enhanced the anti-viral activity of simeprevir. Conclusions: These results indicated that A. mangium leaves could represent sources of anti-HCV agents.


Introduction
Hepatitis C virus (HCV) infection is an acute or chronic liver disease.HCV infection has a high prevalence globally, and approximately 71 million people are at risk of liver cirrhosis or hepatocellular carcinoma attributable to chronic infection (Lange et al., 2014).To date, no effective hepatitis C vaccine has been developed because HCV is a commonly mutated virus (Pawlotsky et al., 2015;Pawlotsky Jm et al., 2018).
Hepatitis C treatment has evolved with the availability of direct acting antivirals, which have achieved sustained virological response (SVR) rates exceeding 90% (Segarra-Newnham et al., 2020).However, some low-income countries cannot access those therapies because of their costs, and the combination of interferon-alpha and ribavirin (RBV), which produces an SVR rate of 50%, remains in use.Combination treatment has also been reported to have serious side effects and risks of resistance, making this strategy less effective (Swain et al., 2010).Efforts to develop new agents for HCV are necessary.Further issues to overcome include the development of drugs that can inhibit the virus with fewer side effects and affordable prices for all countries.Therefore, it is necessary to develop affordable, safe, and effective HCV therapies.
Traditional herbal medicine has become a popular treatment, and plants are among the primary components of such medicines.Our previous studies reported medicinal plants possessing anti-HCV activities (Adianti et al., 2014;Wahyuni et al., 2013;Wahyuni et al., 2014).Many medicinal plants have also been reported to inhibit HCV by inhibiting various steps of the HCV life cycle (Hussein et al., 2000;Ravikumar et al., 2011;Wahyuni et al., 2016).
A. mangium contains alkaloids, flavonoids, polyphenols, glycosides, saponins, steroids, tannins, and terpenoids, and their leaves contain phenolic groups including tannins and flavonoids. A. mangium was reported to contain 2,3-cis-3,4,7,8tetrahydroxyflavanone and teracacidin (Barry et al., 2005).A. mangium was reported to possess various bioactivities such as anti-oxidant, antibacterial activities, antifungal activities (Batiha et al., 2022;Mihara et al., 2005;Prayogo et al., 2021).Other species of Acacia, Acacia comfisa and Acacia nilotica were reported to inhibit HCV (Lee et al., 2011;Rehman et al., 2011).This current study evaluated the anti-HCV activity of various extracts of A. mangium leaves.In addition, the cytotoxicity of the extracts was evaluated.The mode of action was additionally assayed to determine the part(s) of the HCV life cycle inhibited by the extracts.Moreover, its mechanism of action was examined by Western blotting and combination treatment with current anti-HCV drug.This study was used simeprevir for combination treatment which known as NS3 protease inhibitor.It is particularly effective against HCV genotypes 1 and 4, making it a valuable treatment option for patients infected with those genotypes (Rice et al., 2014).

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)).
3. Method section: add sentence in cytotoxicity assay section, lines 3-5 of this section.

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% CO 2 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 1x10 5 ) was chosen for anti-HCV assay.The stock was stored at À80°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 (IC 50 ) (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 (CC 50 ) (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 0.25Â, 0.5Â, 1Â, 2Â, and 4ÂIC 50 for monotherapy and combination.All treatments were performed for 48 h incubation.The IC 50 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).

Results
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 IC 50 of 0.2 AE 0.3 μg/mL, whereas the IC 50 s of the extracts ranged 2.8-4.6 μg/mL (Table 1).While the positive control of ribavirin revealed the IC 50 values of 10.4 AE 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).

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).

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 postinfection processes such as virus replication, virus assembly, and virus release.The raw data are available in the Underlying data (Permanasari and Wahyuni, 2022d).
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 IC 50 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).

Discussion
The study found that A. mangium possesses potential anti hepatitis C virus activity through some mechanism.The anti-HCV activities of the A. mangium leaves extracted using solvents of different polarities, namely 96% ethanol, n-hexane, dichloromethane, and methanol was examined.The data illustrated the strong anti-HCV activities of all extracts.Moreover, no toxic effect was observed according to the CC 50 and selectivity index.
Ethanol is commonly used as a solvent in traditional drug development.Therefore, the strong anti-HCV activities of the ethanol extract and the lack of cytotoxicity provide necessary information for developing extracts of A. mangium as an alternative or complementary anti-HCV agent.However, we used various solvents with different polarities to further isolate the active anti-HCV compounds.The result demonstrated that the dichloromethane extract of A. mangium possessed the strongest inhibitory activity.Mode of action analysis was performed as the first screening assay to determine the mechanism of A. mangium extract.
The results illustrated that the ethanol extract more strongly inhibited HCV in the post-entry stage than in the entry stage.
Virus production starts with binding of the virus to the host cell receptor, followed by virus entry and endocytosis.These processes are included in the entry step.The entry of HCV into hepatocytes is mediated by the viral E1 and E2 glycoproteins, which are the surface proteins of viral particles.HCV infection occurs through complex interactions of  viral lipoviral particles with cellular factors, including low-density lipoprotein receptors, glycosaminoglycans, scavenger receptor class B type I, tetraspanin (CD81), claudin-1, and occludin (Dubuisson et al., 2008;Moriishi and Matsuura, 2003).Viral particles enter host cells through clathrin-mediated endocytosis, after which they are sent to the endosome.Meanwhile, the post-entry steps include translation, replication, and assembly.NS3-NS5 form a replication complex that produces new viral genomic RNA.Genomic RNA and HCV core proteins accumulate to form a nucleocapsid, which is excreted through the lumen side of the endoplasmic reticulum.After this excretion, the nucleocapsid can interact with very low-density lipoprotein (VLDL), followed by translocation to the Golgi for maturation.Mature HCV-VLDL complexes are released exocytically via the VLDL secretory pathway.Finally, new cells can be infected by released free HCV particles or by cell-to-cell transmission (Fénéant et al., 2014;Lindenbach and Rice, 2013;Zeisel et al., 2015).
To further clarify the mechanism of the anti-HCV effects of A. mangium extract, Western blotting was performed to evaluate the effect of the extract on NS3 protein.A. mangium extract decreased NS3 protein levels versus the control.NS3 is a non-structural virus protein that plays an important role in replication.It is an attractive target for HCV treatment.
Inhibition of NS3 could result in decreased virus production.Moreover, the anti-HCV activity of the extract was evaluated in combination with the NS3 protein inhibitor simeprevir. A. mangium extract was demonstrated to enhance the inhibitory activity of simeprevir against HCV.This suggested that the extract potentiated the effect of simeprevir on secondary targets of HCV.
Chemical compounds play an important role in anti-HCV activities.Further isolation of the active compounds from A. mangium against HCV is needed.However, it has been reported that Acacia species are rich in polyphenols, flavonoids, alkaloids, saponins, and terpenoids.It was reported compounds in the genus Acacia include epicatechin, quercetin, proacaciaside I, and proacaciaside II (Figure 5).Those compounds were previously demonstrated to exhibit bioactivities such as anti-bacterial, anti-fungal, and anti-parasitic effects, which could contribute to anti-HCV properties (Chew et al., 2011;Rangra et al., 2019).

Conclusions
A. mangium leaf extracts possess strong anti-HCV activities without toxic effects.The extracts strongly inhibited the post-entry step, decreased NS3 protein levels, and enhanced the anti-HCV activities of simeprevir.These results suggest that A. mangium could be used to develop complementary and alternative treatments for HCV.
Figshare: in vitro activity of A. mangium against HCV.raw data figure 1.docx (It demonstrated the figure of cells with infected cells, the tables which showed number of infected cells and percent inhibition of the three replication of experiments.).https://doi.org/10.6084/m9.figshare.20977714(Permanasari and Wahyuni, 2022b).
Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

Moustafa Sarhan
Zoology Department, Faculty of Science, Al-Azhar University, Assiut, Egypt In the current study, the authors evaluated the anti-HCV activity of various extracts of Acacia mangium leaves.They also determined the cytotoxicity of the extracts.The mode of action experiments revealed the reduction of NS3 protein levels during the post-entry step with some effects on the entry step.Overall, the work is good however there are some concerns: The title needs to be improved to be more indicative of the work, like this ...... "Evaluation of anti-hepatitis C virus of leaves extracts of Acacia mangium" 1.
The graphs in Fig. 1 and Fig. 2 are not so clear.To make them more clear you can use colors for drawing the lines.

2.
In the mode of action experiments, why you did not check the effect of the extract on the cells alone for 2 hours and then add the virus?

3.
Why was ethanol extract selected to check the mode of action?Methanol extract also has a good effect!Dichloromethane has the highest effect and SI! 4.
You may need to use fractionation-guided HPLC to find out the bioactive compound(s) in the leaf extract.You should add to the discussion and conclusion.

Are sufficient details of methods and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate?

○
New efforts also result in the approval of direct-acting antivirals, or DAAs, which aid in the ○ recovery from HCV in three months.Sofosbuvir, Daclatasvir, and other DAAs are quite effective.The major restriction here is the emergence of resistance to these new medications.To validate the title of this article, I would recommend that the authors evaluate the antiviral effects of the plant extracts on HCV-resistant strains.
Please write the species names in italics and correct spelling errors: Acacia nilotica, not 'Acacia nilotoca'.
Since the authors used this drug as a positive control in their tests, actual data on this compound needs to be presented in the introduction as well as its major limitation.

○
Why did the authors confine the study to in vitro systems rather than including an in vivo examination?summaries of the opportunities provided by in vitro systems must be presented here.

Methodology:
Please also include the length of the maceration of plant extracts.

○
Please properly cite Wahyuni, 2013 and include it in the references.But this reference is also old.

○
The Huh7it cell systems must be introduced correctly.What makes them superior to other JFH-1 systems?
○ Two positive controls (ribavirin and simeprevir) were employed independently in this study with no evidence of their mode of action.To minimize confusion, this must be described explicitly.Also, clarify why Sofosbuvir a very popular replication inhibitor has not been used in this assessment.It would have helped to understand the extent of this effect on the replication step.

○
For the suppression of HCV infectivity under the section "Anti-HCV activity," I would propose that the authors specify which viral proteins the primary (human) antibody employed for immunostaining precisely targets, and that they solely refer to prior publications (Permanasari & Wahyuni, 2022).

○
I believe that at least one mean comparison test should be used to detect the statistical difference between IC50 values.
○ What about the isobologram curves now that combination experiments have been completed?Before concluding the Simeprevir relationship, it would have been great to know whether this combination is synergistic or antagonistic.

Results & Discussion
According to my interpretation of Table 1, the dichloromethane extract has the lowest IC50 value and the highest selectivity index (SI), and therefore should be used for further analysis.I'm curious why the authors chose to focus on the ethanol extract instead.Please stick to facts.

○
Based on this result, I think the phytochemical characterization of the dichloromethane ○ extract by HPLC/UV is necessary.Kindly support these findings with in silico studies of Acacia mangium compounds with the HCV NS3/4A protease.

○
The information in Figure 1  The effect of this extract on NS3/4A activity is also required

Conclusion
Before concluding that A. mangium extracts might be exploited to produce supplementary and alternative therapies for HCVmore in vivo research is needed to evaluate the anti-HCV potential and assess the toxicity.

Is the work clearly and accurately presented and does it cite the current literature? Partly
Is the study design appropriate and is the work technically sound?Yes

Are sufficient details of methods and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate?Partly Are all the source data underlying the results available to ensure full reproducibility?Yes

Are the conclusions drawn adequately supported by the results? Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Pharmacology, Hepatology and Virology

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have
Once-daily dosing: Simeprevir is typically administered as a once-daily oral medication, which improves convenience and patient adherence to the treatment regimen.
Weaknesses of Simeprevir: Resistance development: The long-term use of Simeprevir, especially as a monotherapy, can lead to the development of viral resistance, potentially reducing its effectiveness.
Limited efficacy in certain populations: Simeprevir may not be as effective in certain patient populations, such as those with liver cirrhosis or prior treatment failure.
Drug interactions: Simeprevir has the potential for drug interactions, particularly with certain medications metabolized by cytochrome P450 enzymes, which may require dosage adjustments or careful monitoring.
Why did the authors confine the study to in vitro systems rather than including an in vivo examination?summaries of the opportunities provided by in vitro systems must be presented here.

Answer:
The study of in vitro systems refers to conducting experiments or investigations in a controlled laboratory environment outside of a living organism.In vitro studies involve using cells, tissues, or organs cultured in a laboratory setting, often in artificial conditions that mimic aspects of the natural environment.And for HCV the in vivo model was very limited Methodology: Please also include the length of the maceration of plant extracts.

Answer:
The maceration process has been described Please properly cite Wahyuni, 2013 and include it in the references.But this reference is also old.
Answer: The appropriate method was described very well in Wahyuni 2013.And the method was used the same until now.
The Huh7it cell systems must be introduced correctly.What makes them superior to other JFH-1 systems?
Answer: The Huh7 cell system, is a widely used in vitro cell culture model for studying the hepatitis C virus (HCV).It is derived from the Huh-7 human hepatoma cell line and has been extensively employed for HCV research, including the study of the JFH-1 strain of the virus.Two positive controls (ribavirin and simeprevir) were employed independently in this study with no evidence of their mode of action.To minimize confusion, this must be described explicitly.Also, clarify why Sofosbuvir a very popular replication inhibitor has not been used in this assessment.It would have helped to understand the extent of this effect on the replication step.
Answer: We used ribavirin and simeprevir that still used as anti-hcv drug in some area in developing countries due to limited others DAAs For the suppression of HCV infectivity under the section "Anti-HCV activity," I would propose that the authors specify which viral proteins the primary (human) antibody employed for immunostaining precisely targets, and that they solely refer to prior publications (Permanasari & Wahyuni, 2022).
Answer: We used the serum of a patient with a positive HCV genotype 2a, which did not have a specific viral protein target.
I believe that at least one mean comparison test should be used to detect the statistical difference between IC 50 values.
Answer: We complete it with Student's t-test: The t-test is a parametric statistical test that compares the means of two groups.Since we have two sets of IC 50 values What about the isobologram curves now that combination experiments have been completed?Before concluding the Simeprevir relationship, it would have been great to know whether this combination is synergistic or antagonistic.

Answer:
We have completed with the diagram which showed synergetic effect by Combination Index (CI) analysis through compusyn software.The Combination Index is a quantitative measure used to evaluate drug interactions in combination therapies.It is calculated based on the dose-response curves of individual drugs and the combination.The CI value assesses whether the observed effect of the combination is synergistic (CI < 1), additive (CI = 1), or antagonistic (CI > 1).

Results & Discussion
According to my interpretation of Table 1, the dichloromethane extract has the lowest IC50 value and the highest selectivity index (SI), and therefore should be used for further analysis.I'm curious why the authors chose to focus on the ethanol extract instead.Please stick to facts.

Answer:
The author was more explored in ethanol extract because of the potency in developing extanol extract as herbal drug product.
Based on this result, I think the phytochemical characterization of the dichloromethane extract by HPLC/UV is necessary.
Kindly support these findings with in silico studies of Acacia mangium compounds with the HCV NS3/4A protease. 1.
"The current study examined the anti-hepatitis C virus (HCV) activities of Acacia mangium extracts in solvents with various polarities and further evaluated the mechanism of action of the extracts on the protein virus and combination treatment models."-Are you evaluating effects on combination treatment models too or on protein virus?I do not think the models are also dependent variables.

2.
"Anti-hepatitis C virus activities was conducted by in vitro culture cells..." -Please replace "by" with "using".Because culture cells cannot conduct a test.Also, better to say cell culture than "culture cell".

3.
"Further examined its NS3 protein inhibition was evaluated by western blotting assay..." -This whole statement is grammatically incorrect.Take out "examined" and replace "by" with "using".

4.
What is ribavirin?Any brief details on it and why was it used as a control? 5.
"The mode of action of the ethanol extract was evaluated at 30 µg/mL, revealing that the inhibitory effect was stronger on the post-entry step than on the entry step."-what does entry and post-entry steps mean in this context and why study kinetics at a higher concentration than the 4.6 ug/ml for ethanol extract? 6.
Why use ribavirin as a positive control and also use simeprevir in combination treatment?Comparing simeprevir-based treatment with the control will not make sense.7. "...The current study examined the anti-hepatitis C virus (HCV) activities of Acacia mangium extracts in solvents with various polarities..." -Please indicate here early enough that you used the leaves of the plant.This was not mentioned until your conclusion and it should not be so.

Methods
Why extract compounds from 200g of leaf powder?What informed this figure?1.
At what temperature was the extraction done? 2.
For how long was the maceration done? 3.
For your MTT assay, why did you not rinse your cells and take out media and add DMSO before incubating again for colour development for spectral reading?DMSO is added to MTT assays at the end of the experiment to dissolve the formazan crystals that were created during the experiment.If DMSO is not added and the formazan crystals are not dissolved, this would cause significant changes in the absorbance spectrum due to the sodium bicarbonate that would still be there.

Cytotoxic effect result
How sure are you that you have removed interference from cell culture media and that your result is not as a result of potential interference? 1.

Other concerns
What compounds from the plant leaf specifically are responsible for the results you have gotten? 1.
Figure 5 throws light on 5 bioactive compounds.Are the observations as a result of all of these compounds or some of them or just one of them? 2.
Does it mean that the results are only due to these five compounds or there may be other compounds beyond these five that may give the results you have?

3.
Please, do consider English Language Editing to avoid some errors.4.
Thank you for the opportunity.

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility?Partly

Are the conclusions drawn adequately supported by the results? Partly
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: 1. Plant bioactive compounds extraction, purification, identification, characterization, bioactivity assessment, bioavailability and potential industrial applications (e.g.bioactive compound-based nanoparticles, nano-emulsions, etc) 2. Food Science and Engineering.3. Physical processing and treatment of biomass I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

At what temperature was the extraction done?
Answer: The extraction was done in the room temperature.11.For how long was the maceration done?Answer: The maceration was done overnight in three times.
12. For your MTT assay, why did you not rinse your cells and take out media and add DMSO before incubating again for colour development for spectral reading?DMSO is added to MTT assays at the end of the experiment to dissolve the formazan crystals that were created during the experiment.If DMSO is not added and the formazan crystals are not dissolved, this would cause significant changes in the absorbance spectrum due to the sodium bicarbonate that would still be there.

Answer:
The MTT assay was conducted as you was mentioned.We have revised in the manuscript.
After 2 days incubation, the medium was discarded, rinse with medium then 150 mikroliter of 10% of MTT reagent was added and incubate for 4 hours.After incubation time removed the MTT and DMSO was added to dissolve the formazon.
13. How sure are you that you have removed interference from cell culture media and that your result is not as a result of potential interference?Answer: The cytotoxic assay was done following our previous method and replication was also conducted to ensure the valid result.
Other concerns 14.What compounds from the plant leaf specifically are responsible for the results you have gotten?Answer: Our study have not deermine the single compounds with responsible to the activities.Following to the references epicatechin, quercetin, proacaciaside I, and proacaciaside II were reported as isolated compounds of Acacia genus that may contribute in their activities.
15. Figure 5 throws light on 5 bioactive compounds.Are the observations as a result of all of these compounds or some of them or just one of them?Answer: The compounds in Figure 5 were known as isolated compounds of Acacia genus that may give contribution in its activities.
16. Does it mean that the results are only due to these five compounds or there may be other compounds beyond these five that may give the results you have?Answer: Our studies have not determined the specific compounds with contribute for its anti-HCV activities.There are strill many unidentified compounds from this plants.

Ziwen Wang
Tianjin Normal University, Tianjin, China Current study evaluated the anti-HCV activity of various extracts of A. mangium.In addition, the cytotoxicity of the extracts was evaluated.The mode of action was additionally assayed to determine the part(s) of the HCV life cycle inhibited by the extracts.Moreover, its mechanism of action was examined by Western blotting and combination treatment with current anti-HCV drug.The work is important for the development of novel antivirals, and the results are interesting.I recommended it to be indexed after minor revision.
For Abstract: Parts Background and Methods need to be reorganized, and the content does not match the requirements.Part Background only needs to introduce the research background.Part Methods does not need to write the specific operation process.
For Introduction: The research progress on the biological activity of Acacia mangium extract should be increased.The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias • You can publish traditional articles, null/negative results, case reports, data notes and more • The peer review process is transparent and collaborative • Your article is indexed in PubMed after passing peer review • Dedicated customer support at every stage • For pre-submission enquiries, contact research@f1000.com

Figure 1 .
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.

Figure 2 .
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.

Figure 3 .
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 AE SEM of three independent experiments.

Figure 4 .
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.

Table 1 :
Extracts from different solvents show different activities because the chemical components in them have changed.Can you detect which components have changed by liquid chromatography?In addition, have you ever tested the biological activity of different batches of extracts from the same solvent?Is the work clearly and accurately presented and does it cite the current literature?Yes Is the study design appropriate and is the work technically sound?

Table 2 .
Anti-Hepatitis C virus activities of Acacia mangium in single and combination with simeprevir.
*Data represent the mean AE SD of three independent experiments.
and Table1is almost the same.I would advise the authors to concentrate solely on Table1because it includes IC50, CC50, and SI values, which are more relevant.BecauseIC50 is a result of a dose-dependent impact, showing it directly explains it and eliminates the need for Fig.1.Fig 5: Honestly, I don't see the importance of these compounds since you have not isolated all these from your plant.In my opinion, you must isolate these compounds and test their effect on replication.What about the effects of these extracts on other non-structural proteins including the RdRp which is a key enzyme in the replication step?