Influence of Harvesting Stage on Phytochemical Composition, Antioxidant, and Antidiabetic Activity of Immature Ceratonia siliqua L. Pulp from Béni Mellal-Khénifra Region, Morocco: In Silico, In Vitro, and In Vivo Approaches

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Researchers examined the amounts of total sugars, polyphenols, flavonoids, and tannins in immature carob pulp from the Moroccan region of Béni-Mellal-Khnéfra in order to assess the pulp's potential as an antioxidant and antidiabetic. I have the following suggestions:

 

1.      The authors list previous studies that addressed the same subject in the Introduction, but they should highlight how their work varies from those studies and the novelty it represents.

2.      Page 3, Line 116: Were the voucher specimens verified and placed in the Herbarium to validate the analysed plant material?

3.      Page 8, Line 359 – How do you account for the nearly ten-fold decrease in total sugar content in your work when compared to Benchikh et al. (2016) research?

4.      Page 8, Line 361 - what does the abbreviation DW stand for? Were the values the authors compared to the literature expressed in the same way?

5.      Page 9, Line 380 - The authors should not merely quote the values that other researchers found in their papers when discussing the results; instead, they should attempt to explain the significant differences in their results when compared with other studies (for example Page 9, Line 390).

6.      Page 11, Line 460 - similar to what is mentioned under point 5.

7.      Table 3 – What is carob jus in the title? Also, the subscript should provide clarification regarding the abbreviation used to express the content of phenolic components.

8.      Page 13, Line 506 - the results of the content of phenolic components should be compared with similar researches and discussed, e.g. Amira Richane, Ben Mansour Rim, Megdiche wided, Ksouri Riadh, Attia Khaoula, Moujahed Nizar, Ben Ismail Hanen, Variability of phenolic compounds and antioxidant activities of ten Ceratonia siliqua L. provenances, Biochemical Systematics and Ecology, Volume 104, 2022, 104486, ISSN 0305-1978, https://doi.org/10.1016/j.bse.2022.104486...

9.      Page 22, Line 764 - Which value of binding energy indicate high binding affinity (below what binding energy values can one speak of high binding affinity)?

10.  Figure 5 - Figure and text in the figure is vague. Please increase the resolution of the figure (it is impossible to see which interactions are occurring).

Author Response

Reviewer 1

Researchers examined the amounts of total sugars, polyphenols, flavonoids, and tannins in immature carob pulp from the Moroccan region of Béni-Mellal-Khnéfra in order to assess the pulp's potential as an antioxidant and antidiabetic. I have the following suggestions:

Thank you for your thorough review and valuable suggestions regarding our study on total sugars, polyphenols, flavonoids, and tannins in immature carob pulp from Béni-Mellal-Khénifra. We appreciate your efforts to enhance the manuscript's quality and are committed to addressing your feedback to improve our work.

Comment 1: The authors list previous studies that addressed the same subject in the Introduction, but they should highlight how their work varies from those studies and the novelty it represents.

Response 1: The authors are grateful for the suggestion to highlight the originality of their work in comparison to previous studies. 

With regard to existing research, which was conducted in Algeria, Cyprus, and Tunisia, it primarily focused on comparing the levels of bioactive compounds across the three main stages of carob maturation (immature, pre-mature, and mature).

However, our study introduces several distinct aspects:

1. To our knowledge, this is the first study specifically examining the variation in bioactive compound content over five periods during the immature stage of fresh carob pulp.
2. The polyphenols were identified at the stage where their concentrations were highest, thus enabling the determination of the types of phenolic compounds most abundant during this stage.
3. The antidiabetic properties of immature carob pulp extracts have been evaluated using a combination of in vitro, in vivo and in silico methods, thereby providing a more comprehensive assessment of their potential.
4. Our research is the first of its kind conducted in the Béni-Mellal-Khénifra region, and more broadly, in Morocco. This adds a new dimension to the existing literature by exploring the bioactive properties of carob in a different environmental and geographical context.

We hope these clarifications address your comment effectively and underscore the originality and significance of our study.

 

Comment 2: Page 3, Line 116: Were the voucher specimens verified and placed in the Herbarium to validate the analysed plant material?

Response 2: The authors thank you and appreciate your insightful comment regarding the plant material validation. We would like to confirm that the identification of the plant was carried out by a professional botanist, Professor Fennane Mohammed, from the Scientific Institute in Rabat, Morocco. A reference specimen of the plant has been deposited in the plant section of the herbarium of Mohammed Premier University in Oujda, Morocco, under reference number HUMPOM74. This guarantees that the plant material used in our study has been correctly verified and authenticated.

 

Comment 3: Page 8, Line 359 – How do you account for the nearly ten-fold decrease in total sugar content in your work when compared to Benchikh et al. (2016) research?

Response 2: We greatly appreciate your comment regarding the nearly tenfold decrease in total sugar content when compared to the study by Benchikh et al. (2016). After careful analysis, we believe the following factors may explain this discrepancy:

1. Methodology:
   While both studies utilized the same method for quantifying total sugars (Dubois et al., 1956), the operational conditions differed. Benchikh et al. (2016) analyzed lyophilized carob pod powder, whereas our analysis was conducted on fresh carob pulp. The use of lyophilized versus fresh material can significantly affect sugar concentration, as water content in fresh pulp may dilute the measured sugar levels.

 

1. Sample origin:

Benchikh et al. (2016) collected carob samples from Algeria, while our samples were obtained from Morocco. Regional variations such as climate, soil composition, and agricultural practices can have a substantial impact on the phytochemical composition of carob pods, including sugar content.

1. Study period:

The samples in Benchikh et al.'s study were collected in 2012, while ours were gathered between April and June 2022. Environmental conditions, such as temperature, rainfall, and seasonal variations across the different years, could also have influenced the biochemical composition of the carob pods.

1. Comparison context:

We initially chose to compare our results with those of Benchikh et al. (2016) because, to our knowledge, it is the only study that quantifies total sugars in immature carob pods. Our intent was to provide a reference point and illustrate potential variations between the studies. However, as you correctly pointed out in your comment Number 4, it is not appropriate to compare results expressed using different units (fresh weight vs. dry weight), as this could lead to misleading interpretations. Therefore, we have decided to remove this comparison from the discussion of our results.

We hope this explanation clarifies the observed differences and demonstrates our rationale for revising this section. We sincerely appreciate the opportunity to address your concerns and thank you for your valuable input.

 

Comment 4: Page 8, Line 361 - what does the abbreviation DW stand for? Were the values the authors compared to the literature expressed in the same way?

Response 4: The authors appreciate your comment regarding the abbreviation "DW" and the comparison of our results with the literature.

    The abbreviation "DW" stands for "Dry Weight", and we acknowledge that this was not clearly defined in the manuscript.

    Regarding the comparison with the literature, we would like to clarify that our results were expressed on a fresh weight basis, whereas the values reported in the literature, particularly for total sugars, were expressed on a dry weight (DW) basis. The study by Benchikh et al. (2016), which we used as a reference, is the only one we found that quantifies total sugars in immature carob pods. However, while this difference in the basis of expression (fresh weight vs. dry weight) may introduce ambiguity and make the comparison less relevant, we acknowledge that this comparison does not provide an accurate reference for the total sugar content during the immature stage of fresh carob pulp development. For this reason, we have decided to remove this part of the comparison.

We hope this clarification addresses your concern, and we will make the necessary adjustments in the manuscript.

Comment 5: Page 9, Line 380 - The authors should not merely quote the values that other researchers found in their papers when discussing the results; instead, they should attempt to explain the significant differences in their results when compared with other studies (for example Page 9, Line 390).

Response 5: We thank you for drawing our attention to the need to explain the significant discrepancies between our results. We confirm that we will further enrich the manuscript by providing additional justifications for the observed differences.

The authors would like to inform you that the direct comparisons from the literature have been removed, as previously outlined in our initial response.

We hope these revisions address your concerns and enhance the clarity of our discussion.

Comment 6: Page 11, Line 460 - similar to what is mentioned under point 5.

Response 6: The authors are thankful for this important remark. We have considered the suggestion to further explain the significant differences observed between our results. Accordingly, we have enriched the discussion on antioxidant activity by adding additional references and elaborating on the potential factors that could account for these discrepancies. These additions provide a clearer understanding of the results obtained in our study.

 

Comment 7: Table 3 – What is carob jus in the title? Also, the subscript should provide clarification regarding the abbreviation used to express the content of phenolic components.

Response 7: Thank you for pointing out the problem with Table 3's title and the need to clarify the abbreviation used for phenolic compounds.

We acknowledge that ‘carob juice’ in the title was a typographical error. The table's correct title will be updated as follows:

‘Table 3. HPLC-UV MS/MS phenolic profile of methanolic extracts of immature carob pulp (MEICP) from different locations (TG, TM, and AW).

In addition, the abbreviations used to express the content of phenolic components are clearly defined and clarified in the revised manuscript (below Table 3).

We thank you for your attention to these details and will make the necessary corrections.

 

Comment 8: Page 13, Line 506 - the results of the content of phenolic components should be compared with similar researches and discussed, e.g. Amira Richane, Ben Mansour Rim, Megdiche wided, Ksouri Riadh, Attia Khaoula, Moujahed Nizar, Ben Ismail Hanen, Variability of phenolic compounds and antioxidant activities of ten Ceratonia siliqua L. provenances, Biochemical Systematics and Ecology, Volume 104, 2022, 104486, ISSN 0305-1978, https://doi.org/10.1016/j.bse.2022.104486...

Response 8: The authors thank you for this suggestion. We have carefully read the recommended article (Richane et al., 2022) and applied a similar approach used in their discussion to compare and interpret our results. We have also cited this article as a reference to further enrich our discussion. As highlighted in our responses to comments #3 and #4, there are no studies directly comparable to ours; however, we have made our best efforts to compare our results with those obtained for other fresh fruits.

 

Comment 9: Page 22, Line 764 - Which value of binding energy indicate high binding affinity (below what binding energy values can one speak of high binding affinity)?

Response 9: Thank you for your question. Regarding binding affinity, we compare the affinity of the molecules studied with that of acarbose, a drug used as a reference for the enzyme. If a molecule exhibits a higher affinity than acarbose, it indicates that it has a strong affinity.

 

Comment 10: Figure 5 - Figure and text in the figure is vague. Please increase the resolution of the figure (it is impossible to see which interactions are occurring).

Response 10: Thank you for your observation. For Figure 5, the interactions are clear in the Word document. I believe the quality of the figure decreased after exporting to PDF format. We have sent this figure separately in a Word document to ensure that the results and text in the figure are clear.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This is a fairly comprehensive manuscript about the evolution of content of phytoactive compounds in carob at different stages of plant maturity. Some preliminary tests showed also promise for further studies testing the antidiabetic activities of some othese phytocompounds.

The manuscript is also well tailored to the topic of the special issue it was submitted to.

Nevertheless, there is room for further improvement of the manuscript. Here are my comments and suggestions on a line-by-line basis:

Line 7: Not sure about this, but shouldn't the last name "chigr" be spelled with a capital C?

Lines 41-45: Switch all verbs to past tense to be consistent with the rest of the abstract

Line 49: cross out the second full stop on this line

Section 1: The whole introduction is just one large paragraph. Please split it into several shorter paragraphs based on the topics discussed in these separate paragraphs

Line 132: Provide technical details about the centrifuge: type, manufacturer, place and country, including g force because rpm's depend of centrifuge g force

Add an additional subsection to Materials & Methods about all chemicals/reagents used in all protocols, listing all of them with their vendor and purity of each (or at least reagent grade). For example include sulphuric acid, phenol, sodium carbonate, Folin-Ciocalteu reagent, AlCl3, gallic acid, catechin, vanillin, methanol, ethanol, conc. HCl, DPPH, TPTZ, FeCl3, ABTS, potassium persulfate etc. (all chemicals must be included in this subsection)

Line 140: add also place and country for manufacturer of spectrophotometer

Line 160: 99% concentrated hydrochloric acid is an impossible concentration. About 35-37% is the maximum attainable in practice. Please reconsider your claim!

Line 162: What causes the absorbance at 500 nm in this case? How was the quantification performed? Using what standard to produce the standard curve and taking readings against what?

Line 169: Is the 1/1000 ratio volumetric? Please clarify

Line 184: Only in the case of ABTS assay you mention triplicate measurements performed. In the case of all other techniques only one measurement was done? If so, this is unacceptable.

Line 188: Again, what kind of ratio? Volumetric?

Line 194: Overly complicated phrasing. Simpler to just state: "Polyphenols were extracted in methanol."

Line 212: List the nine standards by their name and mention the source from where you obtained them.

Line 224 and throughout the manuscript as this issue is recurring: symbol for millilitre should be mL (with a capital L)

Line 230: unripe (misspelled, need an "e" at the end)

Line 324: 40 x 40 x 40 what? Units are not mentioned

Line 341: The sentence "The analyses were conducted in duplicate to get three results" makes no sense. Please clarify or correct!

Tables 1 & 2: The number of decimals reported varies greatly, which is inconsistent; however, decimals in the errors in this case are insignificant because the mean values are in the order of thousands or hundreds

Table 3: What does "AIR" stand for? Explain any acronyms used! Also, "5-O-" is written in a separate column, really off from the rest of the name it is part of.

Table 7: There is no need for capitalization of k in "kcal" (little k stands for kilo-)

Line 878: Data availability statement is missing

Bibliography not formatted consistently:

>some journal names are abbreviated, but others are not

>page numbers or article numbers must be provided for all references (some are missing right now)

 

Comments on the Quality of English Language

Some tense inconsistencies within the same paragraph and a few misspellings, but otherwise English is okay

Author Response

Reviewer 2

This is a fairly comprehensive manuscript about the evolution of content of phytoactive compounds in carob at different stages of plant maturity. Some preliminary tests showed also promise for further studies testing the antidiabetic activities of some othese phytocompounds.

The manuscript is also well tailored to the topic of the special issue it was submitted to.

Nevertheless, there is room for further improvement of the manuscript. Here are my comments and suggestions on a line-by-line basis:

Thank you for your detailed and insightful feedback on the manuscript.

The authors also appreciate your constructive criticism and suggestions for improvement. We will carefully review your comments on a line-by-line basis and address each point to improve the manuscript.

Thank you again for your thorough review and for helping us to strengthen our work.

 

Comment 1: Line 7: Not sure about this, but shouldn't the last name "chigr" be spelled with a capital C?

Response 1: Thank you for your suggestion. The last name "Chigr" has been corrected to start with a capital "C" as recommended.

Comment 2: Lines 41-45: Switch all verbs to past tense to be consistent with the rest of the abstract

Response 2: Thank you for your observation. The verbs in lines 41-45 have been switched to past tense to ensure consistency with the rest of the abstract.

Comment 3: Line 49: cross out the second full stop on this line

Response 3: Thank you for pointing this out. The second full stop on line 49 has been removed as requested.

Comment 4: Section 1: The whole introduction is just one large paragraph. Please split it into several shorter paragraphs based on the topics discussed in these separate paragraphs

Response 4: Thank you for your suggestion. The introduction has been revised and split into several shorter paragraphs, each focusing on distinct topics discussed within the section.

Comment 5: Line 132: Provide technical details about the centrifuge: type, manufacturer, place and country, including g force because rpm's depend of centrifuge g force

Response 5: Thank you for your observation. We have provided the technical details of the centrifuge as requested. The centrifuge used was a Sigma 2-16P, manufactured by Sigma Laborzentrifugen GmbH, located at An der Unteren Söse 50, 37520 Osterode am Harz, Germany. The g-force (RCF) at 4500 RPM is 2150 × g.

Comment 6:  Add an additional subsection to Materials & Methods about all chemicals/reagents used in all protocols, listing all of them with their vendor and purity of each (or at least reagent grade). For example, include sulphuric acid, phenol, sodium carbonate, Folin-Ciocalteu reagent, AlCl3, gallic acid, catechin, vanillin, methanol, ethanol, conc. HCl, DPPH, TPTZ, FeCl3, ABTS, potassium persulfate etc. (all chemicals must be included in this subsection)

Response 6: Thank you for your valuable suggestion. The authors have incorporated a new subsection into the Materials and Methods section, in which they provide comprehensive details regarding the chemicals and reagents utilized in the protocols. As per your request, we have provided a list of the chemicals used in the study, along with the vendors from whom they were procured and the purities of the chemicals.

Comment 7: Line 140: add also place and country for manufacturer of spectrophotometer

Response 7: Thank you for your suggestion. The additional details have been included as follows: The spectrophotometer used is a Shimadzu UV-2401 PC UV spectrophotometer, serial number A 10834232128CS, manufactured by Suzhou Instruments Manufacturing, Suzhou, Jiangsu, China.

Comment 8: Line 160: 99% concentrated hydrochloric acid is an impossible concentration. About 35-37% is the maximum attainable in practice. Please reconsider your claim!

Response 8: The authors would like to express their gratitude for bringing this omission to their attention. They acknowledge the error and have amended the concentration of hydrochloric acid in the revised manuscript to 37%.

Comment 9: Line 162: What causes the absorbance at 500 nm in this case? How was the quantification performed? Using what standard to produce the standard curve and taking readings against what?

Response 9: The absorbance at 500 nm in our study is attributed to the condensed tannins present in the sample, which form a colored complex when reacting with the specific reagents employed in the assay. This wavelength is frequently utilized in spectrophotometric techniques for the quantification of condensed tannins, as it correlates with the absorption peak of these complexes.

The quantification was conducted using a spectrophotometer, with the absorbance measured at 500 nm against a blank consisting of methanol. The use of a blank allowed for the elimination of any potential interference from solvents or reagents.

In order to establish a reliable quantification, a standard curve was generated using catechin as the reference compound. A series of catechin solutions at known concentrations were employed to create the calibration curve, resulting in a high degree of linearity with a correlation coefficient of R² = 0.989. The results were expressed as milligrams of catechin equivalents (CE) per 100 grams of FM, based on the catechin calibration curve. This approach ensures accurate and reproducible quantification of the condensed tannins in the sample.

Comment 10: Line 169: Is the 1/1000 ratio volumetric? Please clarify

Response 10: Thank you for your question. The 1/1000 ratio is indeed volumetric. Specifically, this ratio indicates that the juice extract was diluted 1,000-fold with distilled water. This means that one part of the original juice extract was combined with 999 parts of distilled water, resulting in the final diluted solution. This volumetric approach ensures a precise and consistent dilution factor, which is critical for accurate analysis.

Comment 11: Line 184: Only in the case of ABTS assay you mention triplicate measurements performed. In the case of all other techniques only one measurement was done? If so, this is unacceptable.

Response 11: The authors would like to express their gratitude to the individual who brought this problem to their attention. All analyses, including those not explicitly mentioned as being performed in triplicate, were conducted in triplicate to ensure the accuracy and reliability of the results.
It should be noted that some protocols lack the requisite information, which constitutes an error. To ensure the consistency and rigor of all reported data, the manuscript will be amended to explicitly state that all measurements were performed in triplicate, including those not explicitly mentioned. This information is also included in the tables and figures' titles in the results section.
We would like to express our gratitude for bringing this to our attention. We appreciate your understanding and will make the necessary amendments to the manuscript.

 

Comment 12: Line 188: Again, what kind of ratio? Volumetric?

Response 12: Yes, the 1/1000 ratio is volumetric. We have clarified this detail in the revised manuscript to avoid any confusion.

 

Comment 13: Line 194: Overly complicated phrasing. Simpler to just state: "Polyphenols were extracted in methanol."

Response 13: Thank you for your valuable input. We agree that a simpler formulation is more effective. The manuscript will be revised to state: "Polyphenols were extracted using methanol."

Comment 14: Line 212: List the nine standards by their name and mention the source from where you obtained them.

Response 14: Thank you for your valuable suggestion. We provide the names of the nine standards used in our analysis, along with their respective purity levels where applicable:

1. P-Coumaric acid
2. Naringin
3. Rutin hydrate
4. 4-Hydroxybenzhydrazide
5. Gallic acid
6. Caffeic acid
7. Chlorogenic acid hemihydrate
8. Ferulic acid
9. Catechin

All standards were obtained from Sigma-Aldrich (Darmstadt, Germany).

Comment 15: Line 224 and throughout the manuscript as this issue is recurring: symbol for millilitre should be mL (with a capital L)

Response 15: Thank you for bringing this to our attention. We acknowledge the oversight and will correct the symbol for millilitre throughout the manuscript to ensure consistency, using mL with a capital L. We appreciate your attention to detail and will make these corrections in the updated version of the manuscript.

 

Comment 16: Line 230: unripe (misspelled, need an "e" at the end)

Response 16: Thank you for pointing out the spelling error. We will correct "unrip" to "unripe" by adding the missing "e" at the end in the manuscript. We appreciate your attention to detail.

Comment 17: Line 324: 40 x 40 x 40 what? Units are not mentioned

Response 17: Thank you for your question. The unit of the values "40" corresponds to the number of points in the x, y, and z dimensions, representing the number of divisions in each direction of the grid. In other words, the space is divided into 40 segments in the x, y, and z directions.

Comment 18: Line 341: The sentence "The analyses were conducted in duplicate to get three results" makes no sense. Please clarify or correct!

Response 18: Thank you for pointing this out. The sentence was, in fact, erroneous. The sentence will be revised to accurately reflect the fact that the analyses were conducted in triplicate in order to obtain three results. This amendment will be incorporated into the revised manuscript to ensure clarity and accuracy.

Comment 19: Tables 1 & 2: The number of decimals reported varies greatly, which is inconsistent; however, decimals in the errors in this case are insignificant because the mean values are in the order of thousands or hundreds

Response 19: Thank you for pointing out the inconsistency in the number of decimals reported in Tables 1 and 2. We will correct this inconsistency by standardizing the decimal places to ensure uniformity. We appreciate your attention to detail and will make the necessary adjustments.

Comment 20: Table 3: What does "AIR" stand for? Explain any acronyms used! Also, "5-O-" is written in a separate column, really off from the rest of the name it is part of.

Response 20: Thank you for your observation. We have corrected the miswritten term "AIR" to "area" in the table. Additionally, we have verified that the name "3-O-p-Coumaroyl-5-O-caffeoylquinic acid" is correctly written. We have also adjusted the formatting to ensure that the elements are properly aligned and readable.

 

Comment 21: Table 7: There is no need for capitalization of k in "kcal" (little k stands for kilo-)

Response 21: Thank you for the observation. We will correct the capitalization in Table 7 to ensure that "kcal" is written with a lowercase "k," as it should be. We appreciate your attention to detail and will make this revision in the updated manuscript.

Comment 22: Line 878: Data availability statement is missing

Response 22: Thank you for noting this. We will add a Data Availability Statement to address the missing information.

Comment 23: Bibliography not formatted consistently:

>some journal names are abbreviated, but others are not

>page numbers or article numbers must be provided for all references (some are missing)

The authors would like to express their gratitude for your comprehensive review and invaluable feedback. We have taken the necessary steps to address all the points you raised regarding the bibliography. In particular, we have ensured that all journal names are now consistently abbreviated and that any missing page numbers or article numbers have been provided. We believe these corrections will enhance the clarity and consistency of the references.

We appreciate your constructive feedback and trust that the revised version meets the required standards.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I have reviewed the revised manuscript, and here are my suggestions:

1.       The numbering of the Figures is incorrect and does not match the text. Figure 2 appears first in the manuscript (Page 14), then Figure 2 again, although it is a different picture (Page 16), followed by Figures 3 (Page 18) and 4 (Page 19), and then again Figures 3 (Page 21) and 4 (Page 22). All pictures should be numbered correctly and adequately mentioned in the text.

2.       Page 4, Line 184, AlCl3 – number 3 should be in the subscript.

3.       Page 4 – Line 197 – g should be separated from the value – this should be corrected throughout the manuscript.

Author Response

I have reviewed the revised manuscript, and here are my suggestions

We would like to sincerely thank you for your valuable comments, which have significantly contributed to improving the quality of our manuscript. We have carefully considered all your suggestions and have corrected the typographical errors and figure numbering issues in the revised version.

We greatly appreciate the time and effort you dedicated to reviewing our work, and we hope that the changes made meet your expectations.

Comment 1:

[The numbering of the Figures is incorrect and does not match the text. Figure 2 appears first in the manuscript (Page 14), then Figure 2 again, although it is a different picture (Page 16), followed by Figures 3 (Page 18) and 4 (Page 19), and then again Figures 3 (Page 21) and 4 (Page 22). All pictures should be numbered correctly and adequately mentioned in the text.]

Response 1:

[We would like to thank the reviewer for pointing out the issue with the incorrect figure numbering and references in the text. We have carefully reviewed and corrected all the figure numbers throughout the manuscript. Each figure is now numbered correctly and cited appropriately in the corresponding sections of the text. These corrections have been highlighted in a different color to make the changes easily identifiable. We believe this revision addresses the issue and improves the clarity of the manuscript.]

Comment 2:

[Page 4, Line 184, AlCl3 – number 3 should be in the subscript.]

Response 2:

[The authors would like to express their gratitude and admiration for your perceptive observation in identifying this issue. The subscript "3" in AlCl3 has been corrected and formatted properly. For clarity, this modification was mentioned in the updated manuscript.]

Comment 3:

[Page 4 – Line 197 – g should be separated from the value – this should be corrected throughout the manuscript.]

Response 3:

[The authors express their gratitude and appreciation for your perceptive observation regarding the formatting inconsistency. They have duly rectified the spacing issue between the numerical values and the unit "g" throughout the manuscript. For convenient reference, these corrections have been highlighted in the revised version.]

Author Response File: Author Response.pdf