Therapeutic Capabilities of Triterpenes and Triterpenoids in Immune and Inflammatory Processes: A Review

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This article untitled “Isolation and Biological Activity Assessment of Triterpenes and Triterpenoids from Plant Leaves: A Review” is interesting and well written but some questions remain.

 

About the following names: terpenoids, triterpenoids, versus terpene, triterpene, what is the difference? Are they used correctly in the text? In a same sentence, triterpene and triterpenoids are found, why? Please explain.

 

Figure 1, a “typical“ structure is proposed, but the choice of the groups as R1-R4 is not good. Oxydated triterpenes in position 29, 30, 21, 22, 16 etc… can be found easily in saponins, so the authors cannot only choose 4 positions (R1-R4).

 

Some compounds, among all of those in the table, are cited in the text. The authors should add the corresponding structure to better understand the structure activity/relationships. 

Author Response

This article untitled “Isolation and Biological Activity Assessment of Triterpenes and Triterpenoids from Plant Leaves: A Review” is interesting and well written but some questions remain.

We would like to thank the reviewer for their insightful comments.

About the following names: terpenoids, triterpenoids, versus terpene, triterpene, what is the difference? Are they used correctly in the text? In a same sentence, triterpene and triterpenoids are found, why? Please explain.

Terpenes, also referred to as terpenoids, are a class of natural products comprising molecules having the formula (C₅H₈)n for n ≥ 2. Triterpenes are terpenes comprising six isoprene units with the chemical formula C₃₀H₄₈, whereas triterpenoids are triterpenes that contain heteroatoms, typically oxygen. Explanation was also added to the manuscript.

Figure 1, a “typical“ structure is proposed, but the choice of the groups as R1-R4 is not good. Oxydated triterpenes in position 29, 30, 21, 22, 16 etc… can be found easily in saponins, so the authors cannot only choose 4 positions (R1-R4).

Oxidated triterpenes and saponins belong to the triterpenoid group, which differs from the triterpenes.

Some compounds, among all of those in the table, are cited in the text. The authors should add the corresponding structure to better understand the structure activity/relationships.

The compounds listed in the text and tables are too numerous to give their chemical structures. To do so would provide the reader with a large amount of information, which would not serve the purpose of the paper and could tire and confuse the reader.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors present a detailed quasi-systematic review (PRISMA criteria) on various aspects associated with the biosynthesis, natural sources, chemical profile and biological activity (particularly anti-inflammatory activity) of natural triterpenes, triterpenoids and saponins, highlighting the limitations of the study and future actions on the subject. The information collected is current and organized for the intended purpose. However, the following is suggested to further improve the uniqueness and scientific soundness of the manuscript:

·       General. A) English grammar and syntax could if the manuscript is reviewed by a native English speaker or by a formal translation agency. B) Authors should minimize the use of abbreviations (e.g. mOD) or define them correctly the first time they are cited (e.g. Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA), IC50). C) Check again the whole manuscript for academic plagiarism (iThenticate report match: 25%).

·       Title/Abstract. Highlight whenever needed the scientific contribution of this new review article on this topic, by comparing it to preceding ones (e.g. doi: 10.1146/annurev-arplant-050312-120229, 10.1007/s00709-019-01411-0, 10.3390/ph16030386).

·       Introduction/sections. A) Long and too wordy paragraphs are difficult to read. Authors are advised to construct them in a more "effective" way (see: https://purdueglobalwriting.center/how-to-write-an-effective-paragraph/). Do the same from this section onwards.

·       Figures & Tables. A) Figures should be provided with enough resolution (≥300 dpi) and tables should be formatted according to this journal´s guidelines. B) A graphical abstract depicting all relevant information could be invaluable.

·   References. A) Please check once again the references´ format according to this journal´s guidelines.

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Author Response

The authors present a detailed quasi-systematic review (PRISMA criteria) on various aspects associated with the biosynthesis, natural sources, chemical profile and biological activity (particularly anti-inflammatory activity) of natural triterpenes, triterpenoids and saponins, highlighting the limitations of the study and future actions on the subject. The information collected is current and organized for the intended purpose. However, the following is suggested to further improve the uniqueness and scientific soundness of the manuscript:

We would like to thank the reviewer for their valuable comments.

• General. A) English grammar and syntax could if the manuscript is reviewed by a native English speaker or by a formal translation agency.

English grammar and syntax have been improved by a native English speaker, as requested.

1. B) Authors should minimize the use of abbreviations (e.g. mOD) or define them correctly the first time they are cited (e.g. Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA), IC50).

All abbreviations, including recognized cytokines, proteins, dosage effects, and microorganisms, are now fully explained upon first mention to familiarize the readers with the topics.

1. C) Check again the whole manuscript for academic plagiarism (iThenticate report match: 25%).

The authors have tried to reduce the plagiarism rate, as asked. However, the academic editor could decide whether a further reduction in the similarity rate is needed.

• Title/Abstract. Highlight whenever needed the scientific contribution of this new review article on this topic, by comparing it to preceding ones (e.g. doi: 10.1146/annurev-arplant-050312-120229, 10.1007/s00709-019-01411-0, 10.3390/ph16030386).

The novelty of our work was compared to that of the reviewer’s highlighted work. The authors primarily aimed to emphasize extracts abundant in triterpenes and triterpenoids, particularly in studies that involved the isolation of these molecules. This study would also facilitate the comparison of plant leaf extracts with established anti-inflammatory medications. To that end, the manuscript has been substantially revised.

• Introduction/sections. A) Long and too wordy paragraphs are difficult to read. Authors are advised to construct them in a more "effective" way (see: https://purdueglobalwriting.center/how-to-write-an-effective-paragraph/). Do the same from this section onwards.

The introduction section has been revised in a way that readers cannot be hindered by the length of the sentences.

• Figures & Tables. A) Figures should be provided with enough resolution (≥300 dpi) and tables should be formatted according to this journal´s guidelines.

The resolution of the Figures is 300 dpi and all tables and paragraphs are formatted according to the journal’s guidelines.

1. B) A graphical abstract depicting all relevant information could be invaluable.

A graphical abstract has been provided to enhance the readability of the review, as suggested by the reviewer.

• References. A) Please check once again the references´ format according to this journal´s guidelines

References are formatted according to the journal’s guidelines.

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Our manuscript has been checked by a native English-speaking colleague.

Reviewer 3 Report

Comments and Suggestions for Authors

Overall, the manuscript is not clearly written. Information is presented in a somewhat disorganized manner, and the manuscript requires substantial revision before it can be considered for publication. In fact, the work mainly addresses the activity of extracts rather than the activity of individual compounds.

Detailed comments:

The title should be more precise, indicating that the review focuses on the anti-inflammatory activity, specifically the anti-inflammatory effects of extracts rather than isolated triterpenes and triterpenoids alone.

The introduction needs careful revision, as some statements lack precision. For example:

"Bioactive compounds (…) are categorized into several classes, including terpenoids, alkaloids, nitrogen-containing compounds..." – Most alkaloids are, in fact, nitrogen-containing compounds.

"Bioactive compounds are natural secondary metabolites" – This is not always accurate, as primary metabolites can also exhibit bioactive properties.

Additionally, some information is repetitive; see, for example, lines 36–38 and lines 70–73.

Line 76: "Triterpenoids are a class of chemicals that are found in many different places…" – What do the authors mean by "places"?

The difference between triterpenes and triterpenoids should be explained in the introduction for clarity.

2. Methodology: What were the exclusion criteria?

Section 5: Biological Activity of Triterpenes Derived from Leaf Extracts – The idea of this section is unclear. The title suggests it should focus on the activity of isolated triterpene compounds. Instead it describes the various activities (not only anti-inflammatory) of certain plant extracts. It is not even clear if these extracts contain any triterpenes. See Table 1, none of the triterpenes were detected in the extracts. In my opinion, this section should be removed. 

Section 6: The authors interchangeably use the terms "triterpenoids" and "triterpenes." These are not exact synonyms, and the terminology should be standardized. Furthermore, as above, the title suggests this section should discuss the activity of isolated compounds, but the authors frequently describe the activity of extracts in which triterpenes were identified.

Line 365: "Fractionation is a highly effective method for assessing anti-inflammatory activity" – Fractionation is not a method for assessing activity but rather a process of separating compounds.

Table 2 lacks clarity, and often it is unclear which type of anti-inflammatory activity was investigated. For instance, the term "IB50" is ambiguous. Also, the statement "Potent anti-cancer activity in vivo due to anti-inflammatory activity in NF-κB and MAPK-ERK pathways" needs clarification. Additionally, what parameter  the value of IC50 for Jatropha gossypiifolia described?, and so on…. Please critically review the entire table and add necessary information. All abbreviation used should be explained in footnote under the table.

A separate analytical section regarding the isolation of triterpenes and triterpenoids should be added (as suggested by the article title). Some relevant information is included, but it is mixed with biological activity.

Author Response

Overall, the manuscript is not clearly written. Information is presented in a somewhat disorganized manner, and the manuscript requires substantial revision before it can be considered for publication. In fact, the work mainly addresses the activity of extracts rather than the activity of individual compounds.

We would like to thank the reviewer for their valuable comments.

Detailed comments:

The title should be more precise, indicating that the review focuses on the anti-inflammatory activity, specifically the anti-inflammatory effects of extracts rather than isolated triterpenes and triterpenoids alone.

The title has been appropriately modified, as suggested.

The introduction needs careful revision, as some statements lack precision. For example:

"Bioactive compounds (…) are categorized into several classes, including terpenoids, alkaloids, nitrogen-containing compounds..." – Most alkaloids are, in fact, nitrogen-containing compounds.

The sentence is accordingly modified, as requested.

"Bioactive compounds are natural secondary metabolites" – This is not always accurate, as primary metabolites can also exhibit bioactive properties.

The sentence has been revised, as suggested.

Additionally, some information is repetitive; see, for example, lines 36–38 and lines 70–73.

Lines 33-38 refer to the formation of triterpenes, while lines 70-73 refer to the formation of triterpenoids.

Line 76: "Triterpenoids are a class of chemicals that are found in many different places…" – What do the authors mean by "places"?

There was a typographical error, and the word “plants” was mistyped as “places”. We would like to apologize for the inconvenience. The error has been corrected.

The difference between triterpenes and triterpenoids should be explained in the introduction for clarity.

The difference between triterpenes and triterpenoids has been highlighted, as requested.

2. Methodology: What were the exclusion criteria?

It is now clearly stated in the manuscript that several studies were excluded since they did not match the search criteria. For example, studies examining other parts of plants or not conducting inflammation-assessing assays were excluded. Review articles mistakenly classified as research were excluded as well.

Section 5: Biological Activity of Triterpenes Derived from Leaf Extracts – The idea of this section is unclear. The title suggests it should focus on the activity of isolated triterpene compounds. Instead it describes the various activities (not only anti-inflammatory) of certain plant extracts. It is not even clear if these extracts contain any triterpenes. See Table 1, none of the triterpenes were detected in the extracts. In my opinion, this section should be removed. 

This is a comprehensive review in which a simple recording of the existing literature is conducted. Moreover, in the “Limitations” section, there is a thorough discussion of the limitations of these studies. There are also recommendations for new research based on these limitations and how they can be overcome.

Section 6: The authors interchangeably use the terms "triterpenoids" and "triterpenes." These are not exact synonyms, and the terminology should be standardized. Furthermore, as above, the title suggests this section should discuss the activity of isolated compounds, but the authors frequently describe the activity of extracts in which triterpenes were identified.

The authors erroneously employed the term triterpenes instead of triterpenoids in section 6, likely due to negligence. The issue has been corrected following an accurate recommendation from the reviewer. Concerning extracts containing triterpenoids, the Limitation section indicates that many studies have not isolated the particular molecules, attributing the anti-inflammatory effect to the cumulative presence of components in each extract.

Line 365: "Fractionation is a highly effective method for assessing anti-inflammatory activity" – Fractionation is not a method for assessing activity but rather a process of separating compounds.

The sentence has been accordingly modified, as asked.

Table 2 lacks clarity, and often it is unclear which type of anti-inflammatory activity was investigated. For instance, the term "IB50" is ambiguous. Also, the statement "Potent anti-cancer activity in vivo due to anti-inflammatory activity in NF-κB and MAPK-ERK pathways" needs clarification. Additionally, what parameter  the value of IC50 for Jatropha gossypiifolia described?, and so on…. Please critically review the entire table and add necessary information. All abbreviation used should be explained in footnote under the table.

It is now highlighted that IB₅₀ value referred to protein denaturation assay. Regarding the anti-cancer and anti-inflammatory correlation as per the study involving Osmanthus fragrans var. aurantiacus, the manuscript has been revised appropriately to ensure the readers understand the effect of the isolated compounds maslinic and corosolic acids. In both HCT116 and HT29 cell lines, the phosphorylation levels of p65, Iκ-Bα, and ERK were considerably reduced by the ethyl acetate fraction at a concentration of 100 μg/mL, while the protein expression levels were unaffected. However, in the HT29 cells, the phosphorylation of Iκ-Bα was primarily reduced at a lower concentration of 50 μg/mL. In addition, since COX-2 is a downstream target of NF-κB and ERK1/2 that mediates inflammation-related carcinogenic pathways, the leaf ethyl acetate fraction reduced COX-2 expression in colorectal cancer cells. Moreover, the greater anti-inflammation potency of corosolic acid against maslinic acid was also highlighted. Finally, in most cases, the authors omitted the term “NO production” when referring to IC₅₀ values, which is now corrected. The Table has been further revised accordingly with the appropriate abbreviations explained in the table footer.

A separate analytical section regarding the isolation of triterpenes and triterpenoids should be added (as suggested by the article title). Some relevant information is included, but it is mixed with biological activity.

An analytical section involving extraction and isolation techniques has been inserted in the manuscript to provide a clearer picture of our study to the readers.

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript “Isolation and Biological Activity Assessment of Triterpenes and Triterpenoids from Plant Leaves: A Review” [compounds-3295172] written by Martha Mantiniotou, Vassilis Athanasiadis, Dimitrios Kalompatsios, Eleni Bozinou and Stavros I. Lalas is a review article about triterpene(oids) isolated from plant leaves. The title does not provide any further restriction on this broad topic, but the authors primarily refer to immune responses and anti-inflammatory activity, which are influenced by correlation between triterpenes and triterpenoids.

The reviewer has expertise in the field of organic chemistry, molecular structure determination and molecular interactions and hence refers to the manuscript from this point of view.

The authors examine this topic comprehensively and focus on the current developments of the last few years, in which the fields of immune responses and anti-inflammatory activity have developed significantly. The review begins with some basic information on triterpenes and triterpenoids, which are, however, inadequately presented from a chemical perspective. The focus on immune responses and anti-inflammatory activity is addressed intensively. However, this part of the manuscript lacks a comprehensible structure. The information from different publications seems to be simply strung together. No comprehensible structure emerges about the relationships between natural sources (plant species), molecular structures and resulting immunochemical effects.

The manuscript therefore contains a lot of information from corresponding original works. However, it does not provide a comprehensible overview of the topic chosen by the authors. The present work is hence of little interest in the fields Organic Chemistry and Phytochemistry. It is of some value in Pharmaceutical Chemistry and Immunochemistry. Therefore, the topic is of some interest and the compiled excerpts from the original works are also fundamentally worthy of publication. However, the inappropriate presentation of (bio)chemical principles, the confusing connected presentation of original and the lack of critical consideration of y context mean that the manuscript is currently not in a publishable form.

The reviewer therefore recommends that the manuscript be rejected and that it be allowed for resubmission to “compounds” after a fundamental revision. To this end, the authors should carefully consider the comments listed below.

Comments:

1) The title of the manuscript should better reflect the content of the work.

2) The presentation of the chemical principles and the biosynthesis should be thoroughly revised. It would be sensible to involve a chemist with in-depth knowledge of terpenes and terpene biosynthesis.
 - The graphical representation of the structures in Fig. 1 is unusual for a chemist. For triterpenes, position 3 is usually positioned at the bottom right of the structure. Stereochemistry should be considered in all structures.
- The biosynthesis of squalene epoxide is the same for all triterpenes. It is crucial to consider the following steps (cyclization) when discussing different triterpenes. However, this is not new knowledge; it has been known for decades.
- The presentation in the texts (introduction and biosynthetic pathways) is confusing from a chemical point of view. A lot of basic information is given, but without going into the context. For example, the sentence: "Typically, this necessitates elevated temperature and pH levels, the utilization of expensive reagents and the introduction of protective groups to the reactive centers of the molecule [11–14]." does not do justice to the chemistry of triterpenes that has been carried out over more than a century. The authors should therefore revise the entire paragraphs and work more intensively on the points that are important for their future work. Irrelevant details should be omitted.

3) The authors should create a "common thread" in the composition of the original papers. Effects, molecular structures and biological sources should not be listed, but rather related to each other. This can also be done in a more drawn-out organization of the tables, whereby the bioactivities should be given priority. However, the relationships mentioned above should also be taken into account.

4) Based on the revision in 3), the text should be structured much more intensively. This should be done with appropriate chapters and subheadings.

5) The relationships identified by comparing the results from the original studies should then be highlighted more clearly in a conclusion. Currently, only a (previously known) basic effectiveness of the plant extracts is mentioned here.

Author Response

The manuscript “Isolation and Biological Activity Assessment of Triterpenes and Triterpenoids from Plant Leaves: A Review” [compounds-3295172] written by Martha Mantiniotou, Vassilis Athanasiadis, Dimitrios Kalompatsios, Eleni Bozinou and Stavros I. Lalas is a review article about triterpene(oids) isolated from plant leaves. The title does not provide any further restriction on this broad topic, but the authors primarily refer to immune responses and anti-inflammatory activity, which are influenced by correlation between triterpenes and triterpenoids.

The reviewer has expertise in the field of organic chemistry, molecular structure determination and molecular interactions and hence refers to the manuscript from this point of view.

The authors examine this topic comprehensively and focus on the current developments of the last few years, in which the fields of immune responses and anti-inflammatory activity have developed significantly. The review begins with some basic information on triterpenes and triterpenoids, which are, however, inadequately presented from a chemical perspective. The focus on immune responses and anti-inflammatory activity is addressed intensively. However, this part of the manuscript lacks a comprehensible structure. The information from different publications seems to be simply strung together. No comprehensible structure emerges about the relationships between natural sources (plant species), molecular structures and resulting immunochemical effects.

The manuscript therefore contains a lot of information from corresponding original works. However, it does not provide a comprehensible overview of the topic chosen by the authors. The present work is hence of little interest in the fields Organic Chemistry and Phytochemistry. It is of some value in Pharmaceutical Chemistry and Immunochemistry. Therefore, the topic is of some interest and the compiled excerpts from the original works are also fundamentally worthy of publication. However, the inappropriate presentation of (bio)chemical principles, the confusing connected presentation of original and the lack of critical consideration of y context mean that the manuscript is currently not in a publishable form.

The reviewer therefore recommends that the manuscript be rejected and that it be allowed for resubmission to “compounds” after a fundamental revision. To this end, the authors should carefully consider the comments listed below.

We would like to thank the reviewer’s responses with the point of enhancing our manuscript from an Organic Chemistry aspect.

Comments:

1) The title of the manuscript should better reflect the content of the work.

The title has been substantially revised, as the reviewer suggested.

2) The presentation of the chemical principles and the biosynthesis should be thoroughly revised. It would be sensible to involve a chemist with in-depth knowledge of terpenes and terpene biosynthesis. - The graphical representation of the structures in Fig. 1 is unusual for a chemist. For triterpenes, position 3 is usually positioned at the bottom right of the structure. Stereochemistry should be considered in all structures.- The biosynthesis of squalene epoxide is the same for all triterpenes. It is crucial to consider the following steps (cyclization) when discussing different triterpenes. However, this is not new knowledge; it has been known for decades. - The presentation in the texts (introduction and biosynthetic pathways) is confusing from a chemical point of view. A lot of basic information is given, but without going into the context. For example, the sentence: "Typically, this necessitates elevated temperature and pH levels, the utilization of expensive reagents and the introduction of protective groups to the reactive centers of the molecule [11–14]." does not do justice to the chemistry of triterpenes that has been carried out over more than a century. The authors should therefore revise the entire paragraphs and work more intensively on the points that are important for their future work. Irrelevant details should be omitted.

Despite being established concepts for several decades, the authors opted to incorporate the biosynthesis of triterpenes and triterpenoids in their work, enabling readers to comprehend the progression "from farm to tablet" regarding pharmaceuticals. Consequently, the procedures for extraction and isolation of the molecules have been incorporated, while the primary focus of the study is on the anti-inflammatory activity of these extracts. To that end, Figure 1 gives a concept of biosynthetic compounds, but has been revised with a specific focus on highlighting the chirality of all presented molecules. However, some irrelevant information about triterpene biosynthesis has been removed. Regarding the specific sentence “Typically, this necessitates elevated…”, the authors wanted to emphasize the need to extract plant-based molecules.

3) The authors should create a "common thread" in the composition of the original papers. Effects, molecular structures and biological sources should not be listed, but rather related to each other. This can also be done in a more drawn-out organization of the tables, whereby the bioactivities should be given priority. However, the relationships mentioned above should also be taken into account.

The authors have substantially revised the Tables, highlighting the bioactivities from the plant leaf extracts compared to positive control drugs (where possible).

4) Based on the revision in 3), the text should be structured much more intensively. This should be done with appropriate chapters and subheadings.

The authors agree on establishing a new individual section and subsection including information about the analytical section to emphasize the significance of extraction and separation of these chemicals. The two sections addressing the anti-inflammatory effects of terpenes and triterpenes have been incorporated as subsections of section 5.

5) The relationships identified by comparing the results from the original studies should then be highlighted more clearly in a conclusion. Currently, only a (previously known) basic effectiveness of the plant extracts is mentioned here.

The conclusions of several studies have been revised in order to emphasize the important findings of each study. It should be noted that several studies involved the discovery of novel compounds, which is now highlighted in the manuscript. However, it is frequently noted that in some studies, the compounds were not identified, and in others, the required tests for anti-inflammatory properties were not conducted.

Reviewer 5 Report

Comments and Suggestions for Authors

It is a systematic review, prepared according to the PRISMA guidelines, based eventually on 41 out of 200 records identified. The flowchart for literature search and exclusion of records is presented. I find it useful for other researchers.

Limitations of the field of research are indicated. Conclusions and future perspectives are appropriate.

Remarks:

Please define all acronyms on their first use.

Lines 202-209: This fragment is not sufficiently explained. What type of membrane stability is involved? The experiments evaluated thermal stability of the erythrocyte membrane (at 56oC), a parameter easily measured but of doubtful biological significance. Increased thermal stability of the membrane does not imply inhibition of the interaction of inflammatory agents with these regions; perhaps the authors mean a different study but if so, it should be cited.

Hyaluronidase inhibition assay is reported as a measure of anti-inflammatory activity. Why the role of hyaluronidase in the induction of imflammation was not mentioned before, when discussing inflammation?

Lines 270/271: “can protect mouse albumin, total protein, and malondialdehyde …”, rather: “protect levels of….”, it should not “protect MDA”

Lines 315/316: “volume of edema decreased from 5.56 to 2.59 mm”, volume is not expressed in mm

Lines 340/341: “were isolated through silica gel chromatography”, please be more astute. Indeed, the abstract of Ref. 67 states that “The compounds were isolated by silica gel chromatography” but when you read the paper it becomes clear that the compounds were isolated by column chromatography andtheir purity was checked by silica gel chromatography

Lines 401/402” Fractions of dichloromethane (~85%), ethyl acetate (~86%), and n-butanol (~83%) indicate”, rather: “Fractions of dichloromethane.. etc extracts”

Line 463: “80% of methanol”, rather: „80% methanol”

Lines 589-591: “compound 2 had the greatest inhibition on phosphorylation of ERK 1/2 at 0.5 relative density, whereas the control had ~1.2”, the sentence is not too understandable

Lines 673/674: “the highest concentration of extract (i.e., 40 μM)”, how is it possible to express extract concentration in µM?   

Line 691: “M. albicans”, please in italics

Line 834: “ranging from 30−90 oC”, perhaps „in the range of 30-90oC”. 90oC also for ethyl acetate? Its boiling temperature is 77oC

Line 855: “iNOS expression from 1 to ~0.2 iNOS/actin”, please be more precise (density units?)

Line 900: “Camellia”, please in italics

Table 2: „From literature” is not the best description of the anti-inflammatory effect

Author Response

It is a systematic review, prepared according to the PRISMA guidelines, based eventually on 41 out of 200 records identified. The flowchart for literature search and exclusion of records is presented. I find it useful for other researchers.

Limitations of the field of research are indicated. Conclusions and future perspectives are appropriate.

We would like to thank the reviewer for their valuable comments.

Remarks:

Please define all acronyms on their first use.

All acronyms have now been defined upon their first mention, as recommended by the reviewer.

Lines 202-209: This fragment is not sufficiently explained. What type of membrane stability is involved? The experiments evaluated thermal stability of the erythrocyte membrane (at 56oC), a parameter easily measured but of doubtful biological significance. Increased thermal stability of the membrane does not imply inhibition of the interaction of inflammatory agents with these regions; perhaps the authors mean a different study but if so, it should be cited.

The membrane stability assay was conducted at 56 °C for 30 min, as now indicated in the manuscript. It is also stated that the stability of the membrane after inflammation affects the serum proteins and prevents them from leaking into the tissues during the increased permeability. The mechanism of a plant extract's anti-inflammatory activity was measured by its ability to inhibit hypotonicity impact by membrane stabilization of the bovine red blood cell. In addition, a citation supporting this evidence was added.

Hyaluronidase inhibition assay is reported as a measure of anti-inflammatory activity. Why the role of hyaluronidase in the induction of imflammation was not mentioned before, when discussing inflammation?

The significant role of hyaluronidase is now discussed in detail in the specific research study.

Lines 270/271: “can protect mouse albumin, total protein, and malondialdehyde …”, rather: “protect levels of….”, it should not “protect MDA”

The sentence is corrected, as requested.

Lines 315/316: “volume of edema decreased from 5.56 to 2.59 mm”, volume is not expressed in mm

The word “volume” is replaced by “thickness”, as used in the original paper.

Lines 340/341: “were isolated through silica gel chromatography”, please be more astute. Indeed, the abstract of Ref. 67 states that “The compounds were isolated by silica gel chromatography” but when you read the paper it becomes clear that the compounds were isolated by column chromatography and their purity was checked by silica gel chromatography

The sentence has been written more carefully, as requested.

Lines 401/402” Fractions of dichloromethane (~85%), ethyl acetate (~86%), and n-butanol (~83%) indicate”, rather: “Fractions of dichloromethane.. etc extracts”

The percentages are removed, as suggested.

Line 463: “80% of methanol”, rather: „80% methanol”

The sentence has been corrected, as requested.

Lines 589-591: “compound 2 had the greatest inhibition on phosphorylation of ERK 1/2 at 0.5 relative density, whereas the control had ~1.2”, the sentence is not too understandable

The specific assay revealed a ~50% inhibition of ERK 1/2 in LPS-stimulated RAW 264.7 cells, since the relative density of the specific compound was used at a concentration of 10 μM was measured at ~0.5 relative density of pERK/ERK when compared to ~1.2 of the untreated cells using Western blot analysis. This specification is now included in the manuscript.

Lines 673/674: “the highest concentration of extract (i.e., 40 μM)”, how is it possible to express extract concentration in µM?

It is now clarified that the specific concentration refers to the previously mentioned compounds (i.e., 7, 8, 10, and 11).

Line 691: “M. albicans”, please in italics

1. albicans has been turned into italics, as asked.

Line 834: “ranging from 30−90 oC”, perhaps „in the range of 30-90oC”. 90oC also for ethyl acetate? Its boiling temperature is 77oC

It is now indicated in the manuscript that the authors used an accelerated solvent extraction (ASE) in which high pressures (i.e., 1460 psi) and temperatures are applied to the sample.

Line 855: “iNOS expression from 1 to ~0.2 iNOS/actin”, please be more precise (density units?)

The iNOS expression was calculated in relative band intensity of iNOS/actin through Western blot assay and is now clearly stated in the manuscript.

Line 900: “Camellia”, please in italics

Camelia has been turned into italics, as asked.

Table 2: „From literature” is not the best description of the anti-inflammatory effect

The specific term has been replaced by “n.a.”, meaning not applicable which is also indicated in each Table footnote.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

Some improvements have been made; however, the manuscript still requires revision.

Title: „Isolation and Potential Anti-inflammatory and Biological Activity Assessment of Triterpenes and Triterpenoids from Plant Leaves: A Review” - The title is not appropriate. The current version of the title suggests that the manuscript describes isolated compounds. The expression "leaf extracts containing triterpenes and triterpenoids" is more relevant than "triterpenes and triterpenoids from plant leaves," Additionally, "and biological activity" is unnecessary because anti-inflammatory action is a type of biological activity. Alternatively, you could rephrase it to include "and other biological activities." Remove the word "isolation" from the title, or add an accurate description of the methods used for the isolation of triterpenes/triterpenoids (see my comment below).

My previous comment: Section 5: Biological Activity of Triterpenes Derived from Leaf Extracts – The idea of this section is unclear. The title suggests it should focus on the activity of isolated triterpene compounds….

I do not understand the Authors' response, so I reiterate my comment: the purpose of this section remains unclear. For example, why did the Authors include the study by Mancarz et al.? In the mentioned paper, no triterpenes were determined. In turn, Banni and Jayaraj did not assess any anti-inflammatory activity (or, in fact, any activity at all).

Additionally, the Authors included studies where the presence of triterpenes was identified using a non-specific colorimetric test. Such tests indicate the presence of triterpenes in most extracts. Please provide  information about this in the table (or as a footnote) instead of simply using "n.a."—this will allow readers to better assess the reliability of this investigation.

Table 1: What does the IC50 value for Liquidambar styraciflua refer to?

Moreover, the titles of sections 5.1 and 5.2 should be revised to better reflect their content. Instead of using the phrase "Triterpenes Derived from Leaf Extracts," which suggests the text focuses on isolated compounds, the phrase "Leaf Extract Containing Triterpenes" should be used for better clarity.

4.1 Section: The Authors added a new section as I suggested, guided by the title of the paper (Isolation and …); however, the Authors should focus on the extraction of plant material specifically in the context of isolating triterpenes/triterpenoids. Currently, this section is just a general description of the overall procedure. Moreover, some sentences are misleading or even incorrect. See example below:

„Solvent extraction through maceration, distillation process, and Soxhlet extraction are all procedures that are based on the same principle. It should be noted that several green techniques could also be employed for that reason. These techniques include accelerated solvent extraction, microwave-assisted extraction, ultrasound-assisted extraction (…)” - distillation is not a method of extracting plant material. Accelerated solvent extraction, microwave-assisted extraction, and ultrasound-assisted extraction are not necessarily 'green' techniques, it depends on solvents used. They just can enhance the efficiency of the extraction process.

The Authors should critically revise this section and supplement it with specific techniques used for the isolation of triterpenes. Alternatively, if the word "isolation" is removed from the title of the paper, this section could be omitted.

Author Response

Some improvements have been made; however, the manuscript still requires revision.

Title: „Isolation and Potential Anti-inflammatory and Biological Activity Assessment of Triterpenes and Triterpenoids from Plant Leaves: A Review” - The title is not appropriate. The current version of the title suggests that the manuscript describes isolated compounds. The expression "leaf extracts containing triterpenes and triterpenoids" is more relevant than "triterpenes and triterpenoids from plant leaves," Additionally, "and biological activity" is unnecessary because anti-inflammatory action is a type of biological activity. Alternatively, you could rephrase it to include "and other biological activities." Remove the word "isolation" from the title, or add an accurate description of the methods used for the isolation of triterpenes/triterpenoids (see my comment below).

We would like to acknowledge the reviewer’s insistence regarding the enhancement of the readability of our manuscript. The title has been changed. A new title ‘’Therapeutic Capabilities of Triterpenes and Triterpenoids in Immune and Inflammatory Processes: A Review’’ focuses more on the specific therapeutic capabilities of triterpenes and triterpenoids, particularly in immune and inflammatory processes. It aligns closely with the main theme of our review paper and might appeal to readers interested in the medicinal and therapeutic aspects of these compounds.

My previous comment: Section 5: Biological Activity of Triterpenes Derived from Leaf Extracts – The idea of this section is unclear. The title suggests it should focus on the activity of isolated triterpene compounds….

Section 5 title has been changed, as recommended by the reviewer in another comment below.

I do not understand the Authors' response, so I reiterate my comment: the purpose of this section remains unclear. For example, why did the Authors include the study by Mancarz et al.? In the mentioned paper, no triterpenes were determined. In turn, Banni and Jayaraj did not assess any anti-inflammatory activity (or, in fact, any activity at all).

The study from Mancarz et al. was included in the study since the authors involved anti-inflammatory assay (i.e., hyaluronidase inhibition) which is also highlighted in both the Discussion section and Table 1. However, the triterpene identification was done through phytochemical analysis, a major drawback that is now included in the manuscript. Regarding Sida cordata (whose leaf extracts were investigated by Banni and Jayaraj), this plant is known for its anti-inflammatory properties. The authors highlighted the need for anti-inflammatory assays which would support this evidence. The same applies to the studies of Chipenzi et al., Gavrilova et al., Ng et al., Kosanam and Papusula, and Bezruk et al. Hence, the specific studies have been removed from the review since they do not match the criteria stated in the Methodology section. Changes were also made in the PRISMA Figure.

Additionally, the Authors included studies where the presence of triterpenes was identified using a non-specific colorimetric test. Such tests indicate the presence of triterpenes in most extracts. Please provide  information about this in the table (or as a footnote) instead of simply using "n.a."—this will allow readers to better assess the reliability of this investigation.

Substantial changes are made in Table 1. The use of non-specific colorimetric techniques is highlighted instead of the term “n.a.”.

Table 1: What does the IC50 value for Liquidambar styraciflua refer to?

It is now mentioned that this IC₅₀ value refers to hyaluronidase inhibition in both the Discussion section and Table 1.

Moreover, the titles of sections 5.1 and 5.2 should be revised to better reflect their content. Instead of using the phrase "Triterpenes Derived from Leaf Extracts," which suggests the text focuses on isolated compounds, the phrase "Leaf Extract Containing Triterpenes" should be used for better clarity.

The specific sections have been revised accordingly, as suggested by the reviewer.

4.1 Section: The Authors added a new section as I suggested, guided by the title of the paper (Isolation and …); however, the Authors should focus on the extraction of plant material specifically in the context of isolating triterpenes/triterpenoids. Currently, this section is just a general description of the overall procedure. Moreover, some sentences are misleading or even incorrect. See example below:

„Solvent extraction through maceration, distillation process, and Soxhlet extraction are all procedures that are based on the same principle. It should be noted that several green techniques could also be employed for that reason. These techniques include accelerated solvent extraction, microwave-assisted extraction, ultrasound-assisted extraction (…)” - distillation is not a method of extracting plant material. Accelerated solvent extraction, microwave-assisted extraction, and ultrasound-assisted extraction are not necessarily 'green' techniques, it depends on solvents used. They just can enhance the efficiency of the extraction process.

The authors agree that solvent choice highly impacts the “greenness” of a method. However, it is highlighted in the manuscript that exhaustive extraction cycles performed guarantee the effectiveness of the extractions. Some techniques requiring higher temperatures or mechanical pressures demand a lot of energy for the release of the chosen chemical. Bioactive compounds and essential oils can be extracted from plants using hydrodistillation, an ancient extraction method. This technique along with several conventional techniques. It should be mentioned that for this reason other green methods could also be used. "Green" extraction techniques seek to reduce negative effects on the environment and the usage of hazardous agents and solvents while extracting substances of interest from their natural sources. These methods provide several advantages over conventional extraction methods, including a quicker extraction time, less volume of toxic organic solvents, a higher extraction yield, and the capacity to automate the process for more repeatability using less energy. Regarding the isolation of the specific compounds, the absence of this technique in the majority of the studies renders it redundant. Consequently, section 4 has been revised accordingly.

The Authors should critically revise this section and supplement it with specific techniques used for the isolation of triterpenes. Alternatively, if the word "isolation" is removed from the title of the paper, this section could be omitted.

The term “isolation” has been removed from the title along with its corresponding section, as suggested by the reviewer.

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript “Isolation and Biological Activity Assessment of Triterpenes and Triterpenoids from Plant Leaves: A Review” [compounds-3295172-V2] written by Martha Mantiniotou, Vassilis Athanasiadis, Dimitrios Kalompatsios, Eleni Bozinou and Stavros I. Lalas has been revised by the authors. A list with answers of the authors to all reviewer’s comments, point by point, is added to the new submission. The reviewer is grateful to the authors for taking to heart the previous comments, also from other reviewers and for addressing several concerns.

However, the changes made by the authors only address the suggestions listed by the reviewer to a limited extent. The entire presentation of the chemical principles and the biosynthesis still contains a collection of various facts about triterpene(oid)s. These are still compiled in such a way that scientifically incorrect conclusions and impressions arise. The reviewer therefore encourages the authors again to revise this part of the manuscript. [Figure 1 is just one example. Here, the IUPAC rules should be followed when presenting the structural formulas. See: https://iupac.qmul.ac.uk/ and especially https://iupac.qmul.ac.uk/steroid/3S01.html ] It is certainly sensible to involve colleagues who have fundamental expertise in chemistry and biochemistry in this entire revision in order to be able to present the representations more pointedly from a chemical point of view.

The reviewer also sees little improvement in the structure and clarity of the rest of the manuscript. In his view, facts are still strung together. This only provides a very limited overview that shows relationships . Since the reviewer sees only an insufficient improvement in quality, he cannot deviate from his original assessment. The reviewer therefore further recommends that the manuscript be rejected and that it be allowed for resubmission to “compounds” after a fundamental revision.

Author Response

The manuscript “Isolation and Biological Activity Assessment of Triterpenes and Triterpenoids from Plant Leaves: A Review” [compounds-3295172-V2] written by Martha Mantiniotou, Vassilis Athanasiadis, Dimitrios Kalompatsios, Eleni Bozinou and Stavros I. Lalas has been revised by the authors. A list with answers of the authors to all reviewer’s comments, point by point, is added to the new submission. The reviewer is grateful to the authors for taking to heart the previous comments, also from other reviewers and for addressing several concerns.

We would like to acknowledge the reviewer’s insistence regarding the enhancement of the readability of our manuscript.

However, the changes made by the authors only address the suggestions listed by the reviewer to a limited extent. The entire presentation of the chemical principles and the biosynthesis still contains a collection of various facts about triterpene(oid)s. These are still compiled in such a way that scientifically incorrect conclusions and impressions arise. The reviewer therefore encourages the authors again to revise this part of the manuscript. [Figure 1 is just one example. Here, the IUPAC rules should be followed when presenting the structural formulas. See: https://iupac.qmul.ac.uk/ and especially https://iupac.qmul.ac.uk/steroid/3S01.html ] It is certainly sensible to involve colleagues who have fundamental expertise in chemistry and biochemistry in this entire revision in order to be able to present the representations more pointedly from a chemical point of view.

The paper provides a synopsis of the biochemical production of compounds within the extensive group of triterpenes. This facilitates the reader's understanding of the biosynthesis of these chemicals in plant tissues. However, a comprehensive examination of the effects of these terpenes would exceed the scope of the literature review. The authors attempted to enhance the readability by implementing some revisions throughout the manuscript, including the Introduction and Discussion sections, and also correcting Figure 1 (including new carbon numbering and ring letters).

The reviewer also sees little improvement in the structure and clarity of the rest of the manuscript. In his view, facts are still strung together. This only provides a very limited overview that shows relationships . Since the reviewer sees only an insufficient improvement in quality, he cannot deviate from his original assessment. The reviewer therefore further recommends that the manuscript be rejected and that it be allowed for resubmission to “compounds” after a fundamental revision.

The authors made a substantial revision in both the Introduction and Discussion sections. The latter section is further divided into several subsections to enhance the readability of the manuscript. The authors believe that the aforementioned corrections would facilitate the approval of the review for publication.

Round 3

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript now entitled “Therapeutic Capabilities of Triterpenes and Triterpenoids in Immune and Inflammatory Processes: A Review” [compounds-3295172-V3] written by Martha Mantiniotou, Vassilis Athanasiadis, Dimitrios Kalompatsios, Eleni Bozinou and Stavros I. Lalas has been revised by the authors. A list with answers from the authors to all reviewer’s comments, point by point, is added to the new submission. The reviewer is grateful to the authors for taking to heart the previous comments, also from other reviewers, and for addressing several concerns.

The reviewer understands the arguments of the authors, who see the focus of the manuscript as being on "Isolation and Biological Activity Assessment of Triterpenes and Triterpenoids". The reviewer also finds the synopsis regarding the biochemical production of compounds to be very helpful in principle. However, the content presented in this part of the manuscript still does not meet the standard for the presentation of chemical and biochemical relationships. The reviewer can understand that the scientific focus of the authors is not on this (chemical) area. However, the aims of the journals (compounds) chosen by the authors clearly have a chemical/molecular focus. The authors should therefore be aware that a large part of the readership has in-depth knowledge of chemical and biochemical details. Any inaccuracies in the synopsis will therefore certainly be noticed and thus unnecessarily diminish the value of the manuscript.

However, the reviewer has not yet been able to get through to the authors with suggestions for changes to the manuscript. Therefore, further discussion as part of a review process does not appear to be very expedient. As the other parts in the manuscript appear to make sense to the reviewer (even if the expertise is not in this area), the recommendation is changed to “major revision”. The reviewer therefore (again) recommends that the authors consult an experienced natural product chemist and revise the manuscript! The points raised in the first two reviews of the reviewer should be taken into consideration.

In order to avoid unduly prolonging the review process with further rounds of review, the reviewer recommends obtaining further expertise from a chemically oriented reviewer for further reviews of the manuscript.

Author Response

The manuscript now entitled “Therapeutic Capabilities of Triterpenes and Triterpenoids in Immune and Inflammatory Processes: A Review” [compounds-3295172-V3] written by Martha Mantiniotou, Vassilis Athanasiadis, Dimitrios Kalompatsios, Eleni Bozinou and Stavros I. Lalas has been revised by the authors. A list with answers from the authors to all reviewer’s comments, point by point, is added to the new submission. The reviewer is grateful to the authors for taking to heart the previous comments, also from other reviewers, and for addressing several concerns.

The reviewer understands the arguments of the authors, who see the focus of the manuscript as being on "Isolation and Biological Activity Assessment of Triterpenes and Triterpenoids". The reviewer also finds the synopsis regarding the biochemical production of compounds to be very helpful in principle. However, the content presented in this part of the manuscript still does not meet the standard for the presentation of chemical and biochemical relationships. The reviewer can understand that the scientific focus of the authors is not on this (chemical) area. However, the aims of the journals (compounds) chosen by the authors clearly have a chemical/molecular focus. The authors should therefore be aware that a large part of the readership has in-depth knowledge of chemical and biochemical details. Any inaccuracies in the synopsis will therefore certainly be noticed and thus unnecessarily diminish the value of the manuscript.

However, the reviewer has not yet been able to get through to the authors with suggestions for changes to the manuscript. Therefore, further discussion as part of a review process does not appear to be very expedient. As the other parts in the manuscript appear to make sense to the reviewer (even if the expertise is not in this area), the recommendation is changed to “major revision”. The reviewer therefore (again) recommends that the authors consult an experienced natural product chemist and revise the manuscript! The points raised in the first two reviews of the reviewer should be taken into consideration.

In order to avoid unduly prolonging the review process with further rounds of review, the reviewer recommends obtaining further expertise from a chemically oriented reviewer for further reviews of the manuscript.

 

We would like to thank the Reviewer for their detailed comments and valuable feedback.

We want to inform you that two of the authors of this manuscript hold degrees in Chemistry, one has a degree in Biology, and the other two have extensive experience in the chemistry of natural compounds. We have made substantial revisions to both the Introduction and Discussion sections to facilitate the reader's understanding of the biosynthesis of triterpenes in plant tissues, regardless of their background. We would like to reiterate that a comprehensive examination of the effects of these terpenes would exceed the scope of our literature review.

However, we understand the reviewer's concerns and have further modified the presentation of the various compounds in Figures 1 and 2 to align with those presented on the PubChem website. We would be happy to incorporate further changes to our manuscript if the reviewer provides specific suggestions. It is challenging for us to make more extensive changes based on unspecific general comments.

Thank you once again for your constructive feedback.