Biological Activities of Methanolic Extract of Aegle marmelos against HN Protein of Newcastle Disease Virus
, Muhammad Umar Ijaz 2, Azhar Rafique 1, Asma Ashraf 1,*, Naheed Bano 3, Nimrah Zafar 1, Faisal Tasleem 4, Romina Alina Marc (Vlaic) 5,*, Oana Lelia Pop 6 and Hanadi Talal Ahmedah 7Round 1
Reviewer 1 Report
- The solvent (methanol) controls are still missing in eggs inoculation experiments How can the authors exclude that the observed antiviral activity is due at least in part to the methanol toxicity against NDV?.
- The authors still perform data interpretation in result section (as an example, see lines 86 – 95).
- FRAP Activity:
How was prepared the Standard curve?
How were the results expressed? as μmol trolox equivalent/g dw? Fig 1B, the ordinate axis usually report this FRAP value (mM Trolox/g d.w., for example).
Please specify the numbers of both technical and biological replicates
- In table 1, table 2 and Figure 1 the statistical analysis of the data should be properly reported, by the mean of letters or asterisks, as you prefer.
- Please, always use µg/mL d.w. ,through the text and in the captions, to report the concentrations values
- Total phenolic contents and total flavonoids contents of the methanolic extract. In Table 1. The values are reported as mean ± SEM values. I can read at least 4 value, so it should be n=4 or maybe n = 12.
- Line 125-126: please report one or more references
- Authors should include the information about the equipments suppliers and location (city and state), in parentheses after the name of the items (of the spectrophotometer, for example)
Author Response
The Editor
Agronomy
Subject: Submission of revised manuscript ID: agronomy-1339430
Dear Sir
It is stated that I want to submit revised article entitled, “In vitro biological evaluation and in silico studies on antiviral efficacy of methanolic extract of Aegle marmelos against HN protein of Newcastle Disease Virus”. We are highly thankful to referees whose comments helped in improving this manuscript. We have revised the entire manuscript for language as well as for proper flow of the information. Mostly these are the reviewers’ observations which are addressed in the point by point rebuttal file and also incorporated the same in the text. Below is response to referee comments:
REVIEWER 1
Review comment: The solvent (methanol) controls are still missing in eggs inoculation experiments. How can the authors exclude that the observed antiviral activity is due at least in part to the methanol toxicity against NDV?
Author’s response: Extracts control are added in result section and pointed by track view.
Review comment: The authors still perform data interpretation in result section (as an example, see lines 86 – 95).
Author’s response: Line 86-95 has been added to discussion section
FRAP Activity:
Review comment: How was prepared the Standard curve?
Author’s response: Linear calibration curves were produced with R2= 0.9998 (Figure 1B) and result was calculated as Trolox equivalent per gram dry sample.
Dilute the trolox standard by adding 0.05 g of the compound to 100 mL ethanol (75%), mix well. Make diluitions of 100, 200, 300, 400, 500 μl individually and take absorbance at 700 nm. (See the methodology of FRAP activity).
Review comment: How were the results expressed? as μmol trolox equivalent/g dw? Fig 1B, the ordinate axis usually report this FRAP value (mM Trolox/g d.w., for example).
Author’s response: Mentioned in respective section
Review comment: Please specify the numbers of both technical and biological replicates
Author’s response: All experiments were done in triplicate
Review comment: In table 1, table 2 and Figure 1 the statistical analysis of the data should be properly reported, by the mean of letters or asterisks, as you prefer.
Author’s response: Statistical letters and asterisks are mentioned
Review comment: Please, always use µg/mL d.w. ,through the text and in the captions, to report the concentrations values
Author’s response: Concentrations has been expressed with (µg/mL d.w) in all text
Review comment: Total phenolic contents and total flavonoids contents of the methanolic extract. In Table 1. The values are reported as mean ± SEM values. I can read at least 4 value, so it should be n=4 or maybe n = 12.
Author’s response: It was typological mistake, the dilutions used are 4.
Review comment: Authors should include the information about the equipments suppliers and location (city and state), in parentheses after the name of the items (of the spectrophotometer, for example)
Author’s response: Mentioned (for example; UV-visible spectrophotometer (Biotech Instruments, Winooski, VT, USA)).
REVIEWER 2
Reviewer comment: The manuscript entitled "Biological Activities of methanolic extract of Aegle marmelos against HN protein of Newcastle Disease Virus" is well-structured and studied, however in my opinion it needs to be checked thoroughly in case of any mistypes, grammatical errors, extra spaces, etc. please find my comments and suggestions below:
Author’s response: Grammatical error, extra spaces and extra brackets has been checked and removed.
Abstract
Review comment: L28,29: please add unit for IC50 values
Author’s response: Unit for IC50 values has been added
Introduction
Review comment: L40,43,44,etc., please correct the references, in case of excess brackets without ref.!
Author’s response: Extra brackets has removed
Review comment: L43: "poses" is correct? or possesses?
Author’s response: Corrected
Review comment: L50: please mention the successful bioactive phytochemicals which are being used as anticancer and/or antiviral drugs
Author’s response: Phytochemicals has been added from literature. (See introduction second paragraph).
Review comment: L64: please revise "reatment"
Author’s response: Word is replaced with treatment
Results
Review comment: L73-77: I do not understand the reason of this paragraph, moreover, it has language mistakes e.g. "The richest bio-resources of conventional medicines, modern drugs, nutraceuticals, curative intermediates, and artificial drug chemicals are medicinal plants.", I would recommend to remove this part
Author’s response: Paragraph has been removed according to instructions
Review comment: L82-84: "Total phenolic contents of the extracts were calculated from the regression equation of calibration curve (Y = 0.0108x; R2 = 0.993) and expressed as mg gallic acid equivalents per gram (mg GAE/g) of sample in dry weight (mg/g)." belongs to the methods section not results, the similar cases should also be checked (e.g. L96-98)
Author’s response: L82-84 and L96-98 sentences has been removed and added to methodology of respective part.
Review comment: L105: please revise the heading "2.2.1DPPH antioxidant effects", to for instance "DPPH assay"
Author’s response: Heading has been replaced
Review comment: L108,109: please reword and revise "IC50 for leaves extract of A. marmelos was 63.52±1.48 while fruit extract was 52.06±1.62 (Figure 1A).", add please also
Author’s response: The line has been reword and revised.
Review comment: L251-252: please revise "The HN protein coordinate crystal structure was obtained from."
Author’s response: The line has been revised.
Review comment: Table 7: the chemical structures are very low quality, please replace them with high ones
Author’s response: The structure quality has been improved.
Review comment: Fig. 4: It is too difficult for the readers to see the details, please provide a better quality figure
Author’s response: The figures quality has been improved.
Discussion
Review comment: In my opinion only comparing the data with the previous studies is not sufficient, the rationale of present findings should also be discussed, e.g. discuss why the antioxidants are different between fruits and leaves, any proofs? correlated to the phenolic contents? please describe
Author’s response: Discussion section has been improved with respective of instructions.
References
Review comment: Please check all the references again, some of them are not complete, e.g. 59, 62
Author’s response: References have been checked. Both of 59 and 62 references are the links for GOLD and discovery studio software used for molecular docking was downloaded.
Thank you once again for your valuable comments. I am available if there are any further queries.
Best Regards,
Marc (Vlaic) Romina Alina
Reviewer 2 Report
The manuscript entitled "Biological Activities of methanolic extract of Aegle marmelos against HN protein of Newcastle Disease Virus" is well-structured and studied, however in my opinion it needs to be checked thoroughly in case of any mistypes, grammatical errors, extra spaces, etc. please find my comments and suggestions below:
Abstract
L28,29: please add unit for IC50 values
Introduction
L40,43,44,etc., please correct the references, in case of excess brackets without ref.!
L43: "poses" is correct? or possesses?
L50: please mention the successful bioactive phytochemicals which are being used as anticancer and/or antiviral drugs
L64: please revise "reatment"
Results
L73-77: I do not understand the reason of this paragraph, moreover, it has language mistakes e.g. "The richest bio-resources of conventional medicines, modern drugs, nutraceuticals, curative intermediates, and artificial drug chemicals are medicinal plants.", I would recommend to remove this part
L82-84: "Total phenolic contents of the extracts were calculated from the regression equation of calibration curve (Y = 0.0108x; R2 = 0.993) and expressed as mg gallic acid equivalents per gram (mg GAE/g) of sample in dry weight (mg/g)." belongs to the methods section not results, the similar cases should also be checked (e.g. L96-98)
L105: please revise the heading "2.2.1DPPH antioxidant effects", to for instance "DPPH assay"
L108,109: please reword and revise "IC50 for leaves extract of A. marmelos was 63.52±1.48 whilefruit extract was 52.06±1.62 (Figure 1A).", add please also
L251-252: please revise "The HN protein coordinate crystal structure was obtained from.."
Table 7: the chemical structures are very low quality, please replace them with high ones
Fig. 4: It is too difficult for the readers to see the details, please provide a better quality figure
Discussion
In my opinion only comparing the data with the previous studies is not sufficient, the rationale of present findings should also be discussed, e.g. discuss why the antioxidants are different between fruits and leaves, any proofs? correlated to the phenolic contents? please describe
References
Please check all the references again, some of them are not complete, e.g. 59, 62
Good luck!
Author Response
The Editor
Agronomy
Subject: Submission of revised manuscript ID: agronomy-1339430
Dear Sir
It is stated that I want to submit revised article entitled, “In vitro biological evaluation and in silico studies on antiviral efficacy of methanolic extract of Aegle marmelos against HN protein of Newcastle Disease Virus”. We are highly thankful to referees whose comments helped in improving this manuscript. We have revised the entire manuscript for language as well as for proper flow of the information. Mostly these are the reviewers’ observations which are addressed in the point by point rebuttal file and also incorporated the same in the text. Below is response to referee comments:
REVIEWER 1
Review comment: The solvent (methanol) controls are still missing in eggs inoculation experiments. How can the authors exclude that the observed antiviral activity is due at least in part to the methanol toxicity against NDV?
Author’s response: Extracts control are added in result section and pointed by track view.
Review comment: The authors still perform data interpretation in result section (as an example, see lines 86 – 95).
Author’s response: Line 86-95 has been added to discussion section
FRAP Activity:
Review comment: How was prepared the Standard curve?
Author’s response: Linear calibration curves were produced with R2= 0.9998 (Figure 1B) and result was calculated as Trolox equivalent per gram dry sample.
Dilute the trolox standard by adding 0.05 g of the compound to 100 mL ethanol (75%), mix well. Make diluitions of 100, 200, 300, 400, 500 μl individually and take absorbance at 700 nm. (See the methodology of FRAP activity).
Review comment: How were the results expressed? as μmol trolox equivalent/g dw? Fig 1B, the ordinate axis usually report this FRAP value (mM Trolox/g d.w., for example).
Author’s response: Mentioned in respective section
Review comment: Please specify the numbers of both technical and biological replicates
Author’s response: All experiments were done in triplicate
Review comment: In table 1, table 2 and Figure 1 the statistical analysis of the data should be properly reported, by the mean of letters or asterisks, as you prefer.
Author’s response: Statistical letters and asterisks are mentioned
Review comment: Please, always use µg/mL d.w. ,through the text and in the captions, to report the concentrations values
Author’s response: Concentrations has been expressed with (µg/mL d.w) in all text
Review comment: Total phenolic contents and total flavonoids contents of the methanolic extract. In Table 1. The values are reported as mean ± SEM values. I can read at least 4 value, so it should be n=4 or maybe n = 12.
Author’s response: It was typological mistake, the dilutions used are 4.
Review comment: Authors should include the information about the equipments suppliers and location (city and state), in parentheses after the name of the items (of the spectrophotometer, for example)
Author’s response: Mentioned (for example; UV-visible spectrophotometer (Biotech Instruments, Winooski, VT, USA)).
REVIEWER 2
Reviewer comment: The manuscript entitled "Biological Activities of methanolic extract of Aegle marmelos against HN protein of Newcastle Disease Virus" is well-structured and studied, however in my opinion it needs to be checked thoroughly in case of any mistypes, grammatical errors, extra spaces, etc. please find my comments and suggestions below:
Author’s response: Grammatical error, extra spaces and extra brackets has been checked and removed.
Abstract
Review comment: L28,29: please add unit for IC50 values
Author’s response: Unit for IC50 values has been added
Introduction
Review comment: L40,43,44,etc., please correct the references, in case of excess brackets without ref.!
Author’s response: Extra brackets has removed
Review comment: L43: "poses" is correct? or possesses?
Author’s response: Corrected
Review comment: L50: please mention the successful bioactive phytochemicals which are being used as anticancer and/or antiviral drugs
Author’s response: Phytochemicals has been added from literature. (See introduction second paragraph).
Review comment: L64: please revise "reatment"
Author’s response: Word is replaced with treatment
Results
Review comment: L73-77: I do not understand the reason of this paragraph, moreover, it has language mistakes e.g. "The richest bio-resources of conventional medicines, modern drugs, nutraceuticals, curative intermediates, and artificial drug chemicals are medicinal plants.", I would recommend to remove this part
Author’s response: Paragraph has been removed according to instructions
Review comment: L82-84: "Total phenolic contents of the extracts were calculated from the regression equation of calibration curve (Y = 0.0108x; R2 = 0.993) and expressed as mg gallic acid equivalents per gram (mg GAE/g) of sample in dry weight (mg/g)." belongs to the methods section not results, the similar cases should also be checked (e.g. L96-98)
Author’s response: L82-84 and L96-98 sentences has been removed and added to methodology of respective part.
Review comment: L105: please revise the heading "2.2.1DPPH antioxidant effects", to for instance "DPPH assay"
Author’s response: Heading has been replaced
Review comment: L108,109: please reword and revise "IC50 for leaves extract of A. marmelos was 63.52±1.48 while fruit extract was 52.06±1.62 (Figure 1A).", add please also
Author’s response: The line has been reword and revised.
Review comment: L251-252: please revise "The HN protein coordinate crystal structure was obtained from."
Author’s response: The line has been revised.
Review comment: Table 7: the chemical structures are very low quality, please replace them with high ones
Author’s response: The structure quality has been improved.
Review comment: Fig. 4: It is too difficult for the readers to see the details, please provide a better quality figure
Author’s response: The figures quality has been improved.
Discussion
Review comment: In my opinion only comparing the data with the previous studies is not sufficient, the rationale of present findings should also be discussed, e.g. discuss why the antioxidants are different between fruits and leaves, any proofs? correlated to the phenolic contents? please describe
Author’s response: Discussion section has been improved with respective of instructions.
References
Review comment: Please check all the references again, some of them are not complete, e.g. 59, 62
Author’s response: References have been checked. Both of 59 and 62 references are the links for GOLD and discovery studio software used for molecular docking was downloaded.
Thank you once again for your valuable comments. I am available if there are any further queries.
Best Regards,
Marc (Vlaic) Romina Alina
Round 2
Reviewer 1 Report
This version of the manuscript is ameliorated and the authors answered almost all of my questions.
I still wonder how the authors can distinguish between the in ovo anti NDV activity of their extracts and the in ovo anti NDV activity of the diluent (methanol) of their extracts, without a proper diluent control experiment and without a strong evidence of a dose-response activity of their extracts.
Best whishes
Line 113 “number” shoulb be “letter”, I suppose
Author Response
The Editor
Agronomy
Subject: Submission of revised manuscript ID: agronomy-1339430
Dear Sir,
It is stated that I want to submit minor revisions of article entitled, “In vitro biological evaluation and in silico studies on antiviral efficacy of methanolic extract of Aegle marmelos against HN protein of Newcastle Disease Virus”. I am highly thankful to referees whose comments helped in improving this manuscript. Below is response to referee comments:
Reviewer 1
Reviewer’s comment: This version of the manuscript is ameliorated and the authors answered almost all of my questions. I still wonder how the authors can distinguish between the in ovo anti NDV activity of their extracts and the in ovo anti NDV activity of the diluent (methanol) of their extracts, without a proper diluent control experiment and without a strong evidence of a dose-response activity of their extracts.
Author’s response: Neutral red method of Koch et al. (2008) was used for evaluating maximum nontoxic concentration (MNTC) of the extracts. Neutral red is a dye for staining living cells. Viable cells will take up the dye and incorporate it into the lysosomes. The cells were seeded into 24-well plates. The medium was removed after 24 h of incubation at 37°C and then 100 ml of fresh DMEM containing different dilutions of the sterile extracts of fruit and leaves (separately) (15, 30, 60, 90, 120 and 150 μg/ml were added and incubated again. After 48 h, the extracts were aspirated, and 0.2 ml of the neutral red solution (40 μg/ml) was added to wells and incubated for 1 h at 37°C. After removing the neutral red, rinsing with 0.5 ml acetic acid buffer and shaking for 15 min, absorbance was measured at 550 nm. The control wells contained extract free medium. Mean absorbance of the cell control wells was assigned 100% viability. On the basis of observations, no toxicity was observed at 120 µg/mL, which is taken as MNTC. For further investigation, we have used 120 µg/mL and below concentrations for antiviral activity.
Reviewer’s comment: Line 113 “number” should be “letter”, I suppose
Author’s response: Word has been replaced.
Reviewer 2
Reviewer’s comment: Unfortunately I can not see any changes in order to improve the quality of structures in Table 7, Figure 4,
moreover it is better to highlight whole the revised parts, in that way the revisions can be tracked easier.
Author’s response: Structures in table 7 and figure 4 has been improved.
Best regards!
Reviewer 2 Report
Unfortunately I can not see any changes in order to improve the quality of structures in Table 7, Figure 4,
moreover it is better to highlight whole the revised parts, in that way the revisions can be tracked easier.
Author Response
The Editor
Agronomy
Subject: Submission of revised manuscript ID: agronomy-1339430
Dear Sir,
It is stated that I want to submit minor revisions of article entitled, “In vitro biological evaluation and in silico studies on antiviral efficacy of methanolic extract of Aegle marmelos against HN protein of Newcastle Disease Virus”. I am highly thankful to referees whose comments helped in improving this manuscript. Below is response to referee comments:
Reviewer 1
Reviewer’s comment: This version of the manuscript is ameliorated and the authors answered almost all of my questions. I still wonder how the authors can distinguish between the in ovo anti NDV activity of their extracts and the in ovo anti NDV activity of the diluent (methanol) of their extracts, without a proper diluent control experiment and without a strong evidence of a dose-response activity of their extracts.
Author’s response: Neutral red method of Koch et al. (2008) was used for evaluating maximum nontoxic concentration (MNTC) of the extracts. Neutral red is a dye for staining living cells. Viable cells will take up the dye and incorporate it into the lysosomes. The cells were seeded into 24-well plates. The medium was removed after 24 h of incubation at 37°C and then 100 ml of fresh DMEM containing different dilutions of the sterile extracts of fruit and leaves (separately) (15, 30, 60, 90, 120 and 150 μg/ml were added and incubated again. After 48 h, the extracts were aspirated, and 0.2 ml of the neutral red solution (40 μg/ml) was added to wells and incubated for 1 h at 37°C. After removing the neutral red, rinsing with 0.5 ml acetic acid buffer and shaking for 15 min, absorbance was measured at 550 nm. The control wells contained extract free medium. Mean absorbance of the cell control wells was assigned 100% viability. On the basis of observations, no toxicity was observed at 120 µg/mL, which is taken as MNTC. For further investigation, we have used 120 µg/mL and below concentrations for antiviral activity.
Reviewer’s comment: Line 113 “number” should be “letter”, I suppose
Author’s response: Word has been replaced.
Reviewer 2
Reviewer’s comment: Unfortunately I can not see any changes in order to improve the quality of structures in Table 7, Figure 4,
moreover it is better to highlight whole the revised parts, in that way the revisions can be tracked easier.
Author’s response: Structures in table 7 and figure 4 has been improved.
Best regards!
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
The research presented in your study has the potential to be published but in an improved form. The language used in the manuscript does not allow me to understand whether the experiments were performed appropriately. Also, the outcome of the experiments and its significance is hard to evaluate. For example, in the methods section, I can not find what solvent (in which concentration) was used for the extraction of antioxidant compounds and whether the controls of pure solvent were used to assess its impact on bacteria tested. Because of the numerous grammar errors, I don't recommend this manuscript be published at this stage. I think, it would be helpful to invite more experienced scientists for the cooperation, that could guide you in reporting your results. I attached a partially corrected manuscript with some remarks.
Comments for author File: 
Comments.pdf
Reviewer 2 Report
The paper “Antiviral efficacy of methanolic extract of Aegle marmelos against HN protein of Newcastle Disease Virus” aims to investigate the phytochemical components, the antioxidant activities, the antibiofilm activity against E. coli and antiviral activity against Newcastle disease virus (NDV) of methanolic extracts of leaves and fruits of Aegle marmelos.
In the leaves and fruit extracts, respectively, TFC were found in the ranges of 13.4 ± 0.18 and 35.07 ± 2.78 (RU mg/g), TPC were found in the ranges of 19.82 ± 1.98 and 58.54 ± 3.72 (GAE mg/g). DPPH and FRAP assay showed that fruit extract of A. marmelos showed significantly higher reducing power than leaves extract. The fruit extract showed an higher antibiofilm activity against E. coli compared to the leaves extract. In ovo assays showed that both leaves and fruits have an antiviral potential at higher concentrations against ND virus. An in silico approach for drug likeness and potential molecular docking of ligand with target proteins was also used.
The authors conclude that their work provides a suggestion that A. marmelos and its phytoconstituents could be used as potential natural treatment for NDV and they claim pharmacological evidence for the presence of active constituents that may be responsible for free radical scavenging, reducing power, antibiofilm potentials and anti NDV activity.
The paper presents, especially in the methodology, serious flaws.
Above all, some controls are missing, i.e. the solvent (methanol) control alone and in presence of E. coli and NDV, at the same final concentrations used for the treatments with the extracts. Both in the biofilm activity assay and in ovo assay the solvent controls (the extracts resuspension solution) is required. Moreover, the authors should state the final concentration (%) of methanol solution applied.
In table 2, whom is “extract control” referred to. Is it the fruits or the leaves extract? Please, consider that you need both of them. And what is the extract control concentration? The highest applied (120 ug/ml)? This does not clearly appear reading the table.
In table 7, there is the same problem. What does the G4 “extract control” represent?
Moreover, the methodology appears confused and approximated, in that it mixes antibacterial assays, antiviral assays, the (spectrophotometric?) quantitation of total phenolics and flavonoids contents and antioxidant assays, but none of this topics are deeply investigated: not the phytochemical profile of the extracts, not their antibacterial activity, not their antiviral activity. In silico tests results should be followed and confirmed by laboratory results, for at least some of the chemicals reported in the tables 3, 4, 5 and 6 and in the figures 3 and 4.
As a result, the manuscript sounds quite disconnected in its parts, with somewhat speculative conclusions.
Results section
The authors should avoid to perform data interpretation or discuss the results in this section.
The paragraph 2.1 Phenolics and Flavonoids quantification incorrectly reports also the IC50 value results. Moreover, it is not clear what organism is referred to; the method to calculate IC50 value should be added in methods.
The subparagraph 2.2.2 Reducing power activity should be FRAP activity
Figure 1A: the colours of the lines seem inverted
Fig 1B, the ordinate axis should report the FRAP value (mM Trolox/g d.w., for example)
Methods section
The procedures from paragraph 4.1 to 4.7 should be better described, including the calibration curves, despite the citation of one or more reference papers. From 4.3 to 4.4 please specify the numbers of both technical and biological replicates
The concentrations of TPC and TFC are reported as µg/mL through all the text. Should be µg of dry weight/mL? In this case, please use µg/mL d.w.
