Isolation and Antiprotozoal Effects of Two Sesquiterpene Lactones from Ptilostemon chamaepeuce subsp. cyprius (Asteraceae)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Building upon the recent report on the antileishmanial activity of the ethanolic leaf extract from Ptilostemon chamaepeuce subsp. cyprius (Pcc, Asteraceae), the authors have now isolated its major sesquiterpene lactone, deacylcynaropicrin, along with a minor derivative, 13-hydroxy-11β,13-dihydro-deacylcynaropicrin. The main compound was evaluated for antileishmanial activity against both promastigote and amastigote forms of Leishmania infantum. In addition, both sesquiterpene lactones were tested against other protozoan pathogens, including L. donovani, Trypanosoma brucei rhodesiense, and Plasmodium falciparum. Cynaropicrin, which bears a hydroxymethacrylate ester moiety absent in its deacyl analogues, demonstrated substantially higher potency than both deacylcynaropicrin and its hydroxylated derivative against all tested parasites, as well as against the T. brucei pteridine reductase enzyme (TbPTR1). The results indicate that the ester functional group in cynaropicrin plays a key role in enhancing its antiprotozoal activity. However, cynaropicrin also exhibited significant cytotoxicity toward mammalian cell lines (L6 and J774A.1), limiting its potential as a drug development candidate. Despite this, the findings provide valuable insight into the structure–activity relationships (SAR) of this class of compounds, highlighting functional group contributions to antiparasitic potency and cytotoxicity. Overall, this study is well-designed and executed, and the results make a valuable contribution to the ongoing search for new compounds active against neglected tropical diseases. Therefore, I recommend acceptance of the manuscript after minor revisions, as outlined below:

1. Figure 1 shows that four fractions were obtained through chromatographic fractionation of the crude extract; however, sesquiterpene lactones were detected only in two of them. Please clarify the composition of fractions 3 and 4.
2. Provide information on the purity of the isolated compounds (1–3).
3. Table 1 should be reformatted or optimized to improve the clarity and readability of the presented results.
4. The tested compounds exhibited notable in vitro toxicity. This aspect should be discussed in the manuscript, ideally by comparing the observed effects with those of chemically related compounds.
5. Please clarify why compound 2 was not evaluated against Leishmania infantum.

Author Response

Reviewer 1

Building upon the recent report on the antileishmanial activity of the ethanolic leaf extract from Ptilostemon chamaepeuce subsp. cyprius (Pcc, Asteraceae), the authors have now isolated its major sesquiterpene lactone, deacylcynaropicrin, along with a minor derivative, 13-hydroxy-11β,13-dihydro-deacylcynaropicrin. The main compound was evaluated for antileishmanial activity against both promastigote and amastigote forms of Leishmania infantum. In addition, both sesquiterpene lactones were tested against other protozoan pathogens, including L. donovani, Trypanosoma brucei rhodesiense, and Plasmodium falciparum. Cynaropicrin, which bears a hydroxymethacrylate ester moiety absent in its deacyl analogues, demonstrated substantially higher potency than both deacylcynaropicrin and its hydroxylated derivative against all tested parasites, as well as against the T. brucei pteridine reductase enzyme (TbPTR1). The results indicate that the ester functional group in cynaropicrin plays a key role in enhancing its antiprotozoal activity. However, cynaropicrin also exhibited significant cytotoxicity toward mammalian cell lines (L6 and J774A.1), limiting its potential as a drug development candidate. Despite this, the findings provide valuable insight into the structure–activity relationships (SAR) of this class of compounds, highlighting functional group contributions to antiparasitic potency and cytotoxicity. Overall, this study is well-designed and executed, and the results make a valuable contribution to the ongoing search for new compounds active against neglected tropical diseases. Therefore, I recommend acceptance of the manuscript after minor revisions, as outlined below:

1. Figure 1 shows that four fractions were obtained through chromatographic fractionation of the crude extract; however, sesquiterpene lactones were detected only in two of them. Please clarify the composition of fractions 3 and 4.

Reply: No STLs were detected in these fractions. Other types of constituents were not within the focus of this study. The text in Mat&Met section 2.3 was modified to express this: “Only fractions Fr.1 and Fr.2 contained STLs according to LC/MS analyses. They were both separately separated…”

 

1. Provide information on the purity of the isolated compounds (1–3).

Reply: The following statement was added at the end of section 2.3: “The purity of the isolated STLs was >90% in all cases as estimated from the ¹H-NMR spectra (Figures S1 (1), S7 (2) and S13 (3), Supplementary materials).” Figure S13 was newly added for this purpose.

 

1. Table 1 should be reformatted or optimized to improve the clarity and readability of the presented results.

Reply: The superscript numerals^1-7 indicating footnotes were replaced by letters^a-g to avoid confusion.

Furthermore, the table entries were arranged in a clearer manner without line breaks, which makes the table more easily readable.

 

1. The tested compounds exhibited notable in vitro toxicity. This aspect should be discussed in the manuscript, ideally by comparing the observed effects with those of chemically related compounds.

Reply: In fact, only compound 3 exhibits notable cytotoxicity against mammalian cells. This fact is already duly mentioned (paragraph after Table 1: “Due to its comparatively high toxicity, it may not be a desirable candidate for in vivo testing of antileishmanial activity”). A comparison with other STLs may not be so helpful and would unnecessarily distract from the important message. There are indeed many examples of STLs with even higher toxicity, but also less toxic ones, but these are not under study.

 

1. Please clarify why compound 2 was not evaluated against Leishmania infantum.

Reply: This was due to the small amounts isolated. This is already mentioned in the sentence: “The minor STL 2 of Pcc, due to its small isolated quantity, could only be tested against Ldo, Tbr, and Pfc (Table 1).” (Paragraph between Figure 4 and Table 1).

We thank the reviewer for the time and effort to help us improve our manuscript.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

1. The authors highlight that compound 2, lacking α,β-unsaturated carbonyl groups, is more active against P. falciparum than compound 1 (IC₅₀ 29 vs 92 µmol/L). Expand the discussion to address possible mechanisms. This currently stands as an interesting but unexplained observation.
2. Figures 3 and 4 present dose–response curves with SEM, but the number of biological replicates vs. technical replicates is not entirely clear, beyond stating “triplicate and at least two independent runs.” Add explicit N values for biological replicates for each data set in figure legends or methods.
3. The study evaluates L. infantum in two stages but only uses axenic amastigotes for L. donovani and bloodstream forms for T. brucei. Add a brief rationale explaining the chosen models (e.g., availability, standardization at Swiss TPH, comparability with earlier publications).
4. Cynaropicrin exhibits cytotoxicity more potent than its antiparasitic activity (e.g., CC₅₀ L6 = 1.29 µmol/L vs IC₅₀ Lin = 23 µmol/L). Elaborate on how these toxicity levels compare to clinically used drugs, and discuss whether semi-synthetic modification strategies could improve selectivity.
5. Compound 2 is only briefly characterized and tested in a limited manner due to yield limitations. Add a few sentences in the discussion addressing: whether future work could target semi-synthetic production from cynaropicrin or compound 1; and its potential relevance despite low abundance in the plant.
6. Conslusions are missing.

Author Response

Reviewer 2

1. The authors highlight that compound 2, lacking α,β-unsaturated carbonyl groups, is more active against P. falciparum than compound 1 (IC₅₀ 29 vs 92 µmol/L). Expand the discussion to address possible mechanisms. This currently stands as an interesting but unexplained observation.

Reply: The possible mechanism is covalent modification of vital biological nucleophiles, most likely SH groups. The respective sentence has been extended; “…(i.e. “enone” structures able to interact by covalently modifying and irreversibly inhibiting biological nucleophiles such as enzymes, transcription factors etc.; see e.g. [2-4]….”).

 

2. Figures 3 and 4 present dose–response curves with SEM, but the number of biological replicates vs. technical replicates is not entirely clear, beyond stating “triplicate and at least two independent runs.” Add explicit N values for biological replicates for each data set in figure legends or methods.

Reply: The n values have been added in the figure caption.

 

3. The study evaluates L. infantum in two stages but only uses axenic amastigotes for L. donovani and bloodstream forms for T. brucei. Add a brief rationale explaining the chosen models (e.g., availability, standardization at Swiss TPH, comparability with earlier publications).

Reply: The reason is that L. don axenic amastigotes are the standard assay at Swiss TPH and tests with intracellular amastigotes are only conducted in case of compounds showing high activity.

We added a short statement explaining this in the Mat&Met section 2.5.3.

 

4. Cynaropicrin exhibits cytotoxicity more potent than its antiparasitic activity (e.g., CC₅₀ L6 = 1.29 µmol/L vs IC₅₀ Lin = 23 µmol/L). Elaborate on how these toxicity levels compare to clinically used drugs, and discuss whether semi-synthetic modification strategies could improve selectivity.

Reply: The selectivity being strongly inverse (i.e. SI <<1), cynaropicrin in unaltered form cannot be considered useful and a comparison with used drugs would just distract from this fact. However, we have extended the last sentence in the paragraph after Table 1: “Due to its comparatively high toxicity, it may not be a desirable candidate for in vivo testing of antileishmanial activity and further development; this problem might, however be overcome by syn-thetic modifications, but this would have to be the subject of extensive further re-search.” We hope the reviewer is satisfied with that.

 

5. Compound 2 is only briefly characterized and tested in a limited manner due to yield limitations. Add a few sentences in the discussion addressing: whether future work could target semi-synthetic production from cynaropicrin or compound 1; and its potential relevance despite low abundance in the plant.

Reply: Even though the activity level is rather low, we added the statement: “In view of this unexpected activity, it might be an interesting topic in future studies to investigate the antiplasmodial effect of this compound more closely. In spite of its small yield obtainable from the plant material, it could be accessible semi-synthetically from 1 or 3.”

 

6. Conslusions are missing.

Reply: According to the journal’s instructions for authors, conlusions are optional: “The structure should include an Abstract, Keywords, Introduction, Materials and Methods, Results, Discussion, and Conclusions (optional) sections.“. In fact, we had decided to do without a separate “Conclusions” section since the essential conclusions were already mentioned in the Results & Discussion. However, since this was also requested by the editor, and in spite of inevitably adding a certain element of redundance to the manuscript, we have appended a separate “Conclusions” section.

We thank the reviewer for the time and effort to help us improve our manuscript.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript reports the isolation of deacylcynaropicrin and a minor derivative from Ptilostemon chamaepeuce subsp. cyprius, along with their antiprotozoal evaluation against several parasites. The authors also compare these compounds with cynaropicrin from artichoke. While the work provides valuable structure-activity relationship insights for sesquiterpene lactones, several issues require attention before publication.

1. Table 1 has significant formatting problems with superscript references appearing in the middle of IC₅₀ values (e.g., "4.92±0.71³" where "3" appears to be a reference). The table layout is confusing and makes it difficult to discern which values are from the current study versus literature. Supplementary Tables S2 and S3 have several formatting issues with excessive merged cells and redundant headers that obscure the actual data. It is recommended to restructure these tables for clarity.
2. The TbPTR1 inhibition assay was only performed at a single concentration for compounds 1 and 2, which is insufficient for proper characterization. For compound 1, the authors estimate an IC₅₀ of "about 35-40 μmol/L" based on 53% inhibition at 38 μmol/L, but this extrapolation lacks proper dose-response data. It is suggested to perform full dose-response curves for at least compound 1 to determine accurate IC₅₀
3. Throughout the manuscript, compounds are referred by multiple identifiers (numbers, names, abbreviations). For example, compound 1 is variously called "deacylcynaropicrin (1)", "the main constituent", "the main STL", and sometimes just "1". A consistent naming convention should be established and followed throughout.
4. The authors note that compound 2 (lacking reactive α, β-unsaturated carbonyl groups) shows better activity against falciparum than compound 1 (which has these groups), contradicting their own statement that "vigorous biological and antiprotozoal activity of STLs usually being associated with the presence of reactive α, β-unsaturated carbonyl groups. (Lines 272-273)". It is suggested to explain this contradiction through deeper explanation or additional experiments.
5. Cytotoxicity was evaluated using different cell lines (L6 rat skeletal myoblasts and J774A.1 murine macrophages), making direct comparisons challenging. The authors should justify this choice and discuss how differences between these cell lines might affect interpretation of selectivity indices.
6. The manuscript states that cynaropicrin was "retested against infantum for direct comparison," but fails to explain why previous data was insufficient or how the current testing conditions differ from prior studies. This methodological detail is essential for evaluating the validity of the comparisons.
7. Due to limited quantities, compound 2 was only tested against a subset of parasites. While understandable, the authors should acknowledge how this limitation affects the comprehensiveness of their structure-activity conclusions. If possible, additional biological replicates for compound 2 against falciparum would strengthen the finding about its activity.
8. The materials and methods section contains redundant descriptions, particularly regarding extraction procedures. Streamlining this section would improve readability without sacrificing essential methodological details.
9. While this is the first report of these compounds in chamaepeuce subsp. cyprius, the antiprotozoal activities of cynaropicrin have been extensively documented. The manuscript should more clearly describe what new insights this study provides beyond confirming known structure-activity relationships.
10. The authors note that cynaropicrin's high cytotoxicity limits its therapeutic potential but don't sufficiently discuss alternative approaches (e.g., structural modifications to reduce toxicity while maintaining activity, delivery systems to improve selectivity). These discussions would strengthen the manuscript's impact.
11. The preliminary enzyme inhibition results lack statistical analysis or error measurements, making it difficult to assess the reliability of the reported inhibition percentages.
12. There are several inconsistencies, e.g. “In Vitro” (not italicized in Line 145) and “In Vitro” (italicized in Line183). It is recommended to read the whole manuscript carefully and remove all such inconsistencies.
13. Lines 192-194: Authors stated, “For compounds 1 and 2, single-concentration assays were performed at a fixed concentration of 10 μg/mL, corresponding to 38 and 36 μmol/L, respectively.” The described statement is confusing. It is suggested to rewrite all such statements to circumvent the confusion.

Author Response

Reviewer 3

This manuscript reports the isolation of deacylcynaropicrin and a minor derivative from Ptilostemon chamaepeuce subsp. cyprius, along with their antiprotozoal evaluation against several parasites. The authors also compare these compounds with cynaropicrin from artichoke. While the work provides valuable structure-activity relationship insights for sesquiterpene lactones, several issues require attention before publication.

1. Table 1 has significant formatting problems with superscript references appearing in the middle of IC₅₀ values (e.g., "4.92±0.71³" where "3" appears to be a reference). The table layout is confusing and makes it difficult to discern which values are from the current study versus literature. Supplementary Tables S2 and S3 have several formatting issues with excessive merged cells and redundant headers that obscure the actual data. It is recommended to restructure these tables for clarity.

Reply: The content of Table 1 is indeed complex. Following the reviewer’s suggestion, we tried to improve the readability and comprehensibility: The superscript numerals^1-7 indicating footnotes were replaced by letters^a-g to avoid confusion. Furthermore, the table entries were arranged in a clearer manner, without line breaks between IC₅₀ values and their deviations, which makes the table more easily readable.

We have also cleared up Tables S2 and S3 and hope they no longer obscure the data.

 

2. The TbPTR1 inhibition assay was only performed at a single concentration for compounds 1 and 2, which is insufficient for proper characterization. For compound 1, the authors estimate an IC₅₀ of "about 35-40 μmol/L" based on 53% inhibition at 38 μmol/L, but this extrapolation lacks proper dose-response data. It is suggested to perform full dose-response curves for at least compound 1 to determine accurate IC₅₀

Reply: The compounds’ activity was indeed tested only once in the single-concentration assay since this is our usual procedure. We only determine IC₅₀ data in case of more active compounds, since the measurements, especially the production of the recombinant enzyme, are rather time consuming and expensive. However, according to our experience with this assay which shows quite small deviations (see our previous work Possart et al., DOI 10.3390/molecules27010149 and 10.3390/molecules28227526 with data of various IC₅₀ determinations in the Supplementary Materials), we consider it legitimate to estimate an approximate IC₅₀ range from one measured data point in this case since this measured % inhibition value is very close to 50%. We have clearly stated that this is only an estimate. We have modified the statement to: “…so that a rough estimate of the IC₅₀ would be about 35-40 µmol/L” and hope the reviewer will allow us to keep this statement without performing a full IC₅₀ determination. We do not perform this assay on an “every day basis”, so that we would have to produce new recombinant enzyme first, which is an expensive and time-consuming procedure and certainly not possible to do within the time frame of a manuscript revision.

 

3. Throughout the manuscript, compounds are referred by multiple identifiers (numbers, names, abbreviations). For example, compound 1 is variously called "deacylcynaropicrin (1)", "the main constituent", "the main STL", and sometimes just "1". A consistent naming convention should be established and followed throughout.

Reply: It is rather common in phytochemical literature, to use compound numbers and names within a text, sometimes both, sometimes only one. Since it is necessary multiple times throughout a text, sometimes even in the same sentence, to mention a particular compound, it should be allowed to use paraphrases (i.e. compound 1 = deacylcynaropicrin (1) = 1 = the main STL of this plant …) in order to avoid stereotypic repetition of the same terms over and over. This is more a matter of style than a factual inadequacy. We hope the reviewer will accept our opinion.

 

4. The authors note that compound 2 (lacking reactive α, β-unsaturated carbonyl groups) shows better activity against falciparum than compound 1 (which has these groups), contradicting their own statement that "vigorous biological and antiprotozoal activity of STLs usually being associated with the presence of reactive α, β-unsaturated carbonyl groups. (Lines 272-273)". It is suggested to explain this contradiction through deeper explanation or additional experiments.

Reply: We do not contradict our own statement since the mentioned sentence continues: “…it appears unusual that 2, devoid of such moieties, was about three times more active against Pfc than its “enone” parent STL, 1 (IC50 = 29 vs. 92 µmol/L, respectively).” On grounds of the present data and on the backdrop of many previous observations, we can only note that it is indeed unusual to observe this. Any further discussion on possible reasons (except the implicit “the activity is obviously not due to an “enone” here”) would be mere speculation which we would rather avoid. It is not possible within the scope of this manuscript review to conduct further experiments elucidating the full mechanism of action of this compound against plasmodia. We hope the reviewer can accept that.

 

5. Cytotoxicity was evaluated using different cell lines (L6 rat skeletal myoblasts and J774A.1 murine macrophages), making direct comparisons challenging. The authors should justify this choice and discuss how differences between these cell lines might affect interpretation of selectivity indices.

Reply: The reason why different mammalian cell lines were used in the present study is that the J774A.1 cells serve as the host cells for L. infantum and hence available for cytotoxicity assessment in the Nikosia lab, whereas the L6 cells are the standard cells for cytotoxicity assessment in the Swiss TPH lab. Hence both were used, not so much for the purpose of comparisons among each other but in both cases as a rough estimate for selectivity against the parasite. It should not be forgotten that selectivity indices are never more than rough estimates for selectivity since it is a well known fact that different cells (even from the same organism) often show dramatic differences in sensitivity against compounds. However, comparisons are certainly possible between the values where tests with both lines were performed: Compound 1 is rather untoxic against both lines (IC₅₀ > 50 µM) whereas compound 3 is much more toxic against both (IC₅₀ ≤ 2 µM). The relatively high toxicity of 3 is already part of our discussion.

 

6. The manuscript states that cynaropicrin was "retested against infantum for direct comparison," but fails to explain why previous data was insufficient or how the current testing conditions differ from prior studies. This methodological detail is essential for evaluating the validity of the comparisons.

Reply: The previous data was not insufficient but determined almost 10 years ago in another laboratory under different conditions (reference cited), particularly for the amastigotes with a different host cell line (murine fibroblasts) so it was important to retest the compound to make sure that a direct comparison was legitimate. This should be understandable without further details. The reviewer will agree when comparing the results, that these were virtually identical, i.e. the data determined in the present study (23 ± 1 and 10 ± 0) are well within the deviation range of the previous one (31± 14 and 7 ± 4, respectively). One reason why we think it is good to report the results of both determinations is that a reader (or reviewer) otherwise might ask whether the old data for compound 3 of 2017 would compare well with those of compounds 1 and 2 determined for the present communication. To satisfy the reviewer, the respective sentence has been slightly modified to explain the necessity of this reinvestigation; “..retested against Lin in a different laboratory under different conditions [4],”

 

7. Due to limited quantities, compound 2 was only tested against a subset of parasites. While understandable, the authors should acknowledge how this limitation affects the comprehensiveness of their structure-activity conclusions. If possible, additional biological replicates for compound 2 against falciparum would strengthen the finding about its activity.

Reply: The reviewer is right that further measurements, as is the common nature of all scientific measurement results, would increase the validity of the reported data. However, as the reviewer noticed correctly, it was not possible to perform further tests with compound 2 because it was only obtained in very small quantities. Further tests of the (otherwise quite inactive) compound 2 against Plasmodia might be of interest but will certainly not be conducted with high priority since the level of activity (in spite of being higher than that of 1) is rather low in comparison with other antiplasmodials. However, a short statement in this regard was added (last two sentences before Table 1).

 

8. The materials and methods section contains redundant descriptions, particularly regarding extraction procedures. Streamlining this section would improve readability without sacrificing essential methodological details.

Reply: We have shortened section 2.2 by removing a separate section on “Extraction for the bioassays”. This was possible since the extraction procedure was essentially the same as for the isolation. However, we do not see any further redundancies that could be removed without omitting essential methodological details that might be necessary to allow repetition of the experiments. We would ask the reviewer to specify which parts they would like us to remove.

 

9. While this is the first report of these compounds in chamaepeuce subsp. cyprius, the antiprotozoal activities of cynaropicrin have been extensively documented. The manuscript should more clearly describe what new insights this study provides beyond confirming known structure-activity relationships.

Reply: We think that all essential new insights have already been mentioned. However, following the advice of another reviewer, we added a short “Conclusions” section repeating the essentials. We hope the reviewer will find this suitable.

 

10. The authors note that cynaropicrin's high cytotoxicity limits its therapeutic potential but don't sufficiently discuss alternative approaches (e.g., structural modifications to reduce toxicity while maintaining activity, delivery systems to improve selectivity). These discussions would strengthen the manuscript's impact.

Reply: The statement related to cynaropicrin’s toxicity (end of paragraph after Table 1) was slightly extended to take this into account. It now reads: “Due to its comparatively high toxicity, it may not be a desirable candidate for in vivo testing of antileishmanial activity and further development; this problem might, how-ever be overcome by synthetic modifications, but this would have to be the subject of extensive further research.”

 

11. The preliminary enzyme inhibition results lack statistical analysis or error measurements, making it difficult to assess the reliability of the reported inhibition percentages.

Reply: See above, point 2.

 

12. There are several inconsistencies, e.g. “In Vitro” (not italicized in Line 145) and “In Vitro” (italicized in Line183). It is recommended to read the whole manuscript carefully and remove all such inconsistencies.

Reply: We found one instance of italicized “in vitro” which was corrected so that all instances are now in unitalicized form. However, where the term occurs in headlines, it has to start with capitals “In Vitro” whereas in the normal text it is spelt “in vitro”. We are sure that possible further issues like this may be corrected at the typesetting/proof stage.

 

13. Lines 192-194: Authors stated, “For compounds 1 and 2, single-concentration assays were performed at a fixed concentration of 10 μg/mL, corresponding to 38 and 36 μmol/L, respectively.” The described statement is confusing. It is suggested to rewrite all such statements to circumvent the confusion.

Reply: We do not see any confusion here. 10 µg/mL was the same concentration at which both compounds were tested. It is common to report such concentrations as molar data in µmol/L (previously µM). In the molar dimension, 10 µg/mL corresponds to 0.038 and 0.036 µmol/mL for the two respective compounds since they have different molar mass (262 and 280 g/mol, respectively). Hence 10/262=0.038 µmol/mL and 10/280=0.036 µmol/mL (= 38 and 36 µmol/1000 mL). We hope this explanation is understandable, otherwise please let us know.

We thank the reviewer for the time and effort to help us improve our manuscript.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Accept in present form

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have revised the manuscript according to suggestions; hence, I recommend considering this manuscript for publication.