Phytochemical Characterization and Antimicrobial Properties of a Hydroalcoholic Extract of Tristerix corymbosus (L) Kuijt, a Chilean Mistletoe Species Hosted on Salix babylonica (L)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In the article “Phytochemical characterization and antimicrobial properties of a hydroalcoholic extract of Trysterix corymbosus (L) Kuijt, a Chilean mistletoe species hosted on Salix babilonica (L)” author evaluated antimicrobial activity of the hydroalcoholic extract from leaves, flowers and fruits of T. corymbosus collected.  The most important study, including the leaves extract, was further characterized as it presented the higher antimicrobial activity against the Gram-positive S. pyogenes, although it was inactive against E. coli, a Gram-negative bacterium.

 

Following corrections were recommended before publications:

The extraction solvent used is 70% EtOH. What is another solvent used for dilution to make 70% EtOH? The second solvent needs to be mentioned in literature.

At what temperature was 300 mL MeOH added to the water to precipitate out compound (room temperature or 0°C or 60-70 °C)? If MeOH is added at cooled temperature, will get most of the product precipitate out.

In figure 6, the chemical structures of some chemical bonds between atoms are not clearly visible. The redraw the structure and paste.

Also, similar in figure 8, top T. Corymbosus- HSF, the chemical structures are not clearly visible.

Section: 4.6.1: In sample preparation and LC-MS analysis:

which solvent used for preparing sample and after resuspending sample in solvent, do it filtered or direct injected to HPLC.

In reference section: Page numbers, volume, and years were missing. For e.g. Ref. 6. Check all other references and correct all the references by using journal guidelines.

Author Response

Comment 1: The extraction solvent used is 70% EtOH. What is another solvent used for dilution to make 70% EtOH? The second solvent needs to be mentioned in literature.

Response 1: Thank you for this observation. The 70% EtOH extraction solvent was prepared by diluting absolute ethanol with distilled water (ethanol–water, v/v). While water dissolved the HSF up to 150 mg/mL for antimicrobial testing. Hydroalcoholic mixtures of ethanol and water are widely reported in the literature for the extraction of polar and moderate polar phytochemicals, and 70% (v/v) ethanol is one of the most used solvent systems in phytochemical and antimicrobial studies. This information has been clarified in the Materials and Methods section (4.2 Plant Material)

Comment 2: At what temperature was 300 mL MeOH added to the water to precipitate out compound (room temperature or 0°C or 60-70 °C)? If MeOH is added at cooled temperature, will get most of the product precipitate out.

Response 2: Thank you for this comment. The 300 mL of methanol was added at room temperature to the concentrated extract (in mostly water) during the solvent removal process after reducing the volume to around 100 mL. The precipitation was observed under these conditions, without prior cooling. Although cooling may favor precipitation, it was not applied in this protocol. This information has now been clarified and corrected in the Materials and Methods section of the revised manuscript (4.2 Plant Material). In addition, the temperature at which precipitation was performed is included in Figure 2.

Comment 3: In figure 6, the chemical structures of some chemical bonds between atoms are not clearly visible. The redraw the structure and paste.

Response 3: Thank you for your suggestion, size of figures has been increased for clarity.

Comment 4: Also, similar in figure 8, top T. Corymbosus- HSF, the chemical structures are not clearly visible.

Response 4:The figure has been improved, now the chemical structures are much more visible.

Comment 5: Section: 4.6.1: In sample preparation and LC-MS analysis:

which solvent used for preparing sample and after resuspending sample in solvent, do it filtered or direct injected to HPLC.

Response 5: Thank you for this comment. The lyophilized sample ware resuspended in 80% ice-cooled methanol in water (v/v) prior to LC-MS analysis. After resuspension, the samples were filtered using a 45µM pore PVDF filter and injected into the LC-MS system. This information has now been clarified and corrected in Section 4.6.1 (Sample Preparation and LC-MS Analysis) of the revised manuscript.

Comment 6: In reference section: Page numbers, volume, and years were missing. For e.g. Ref. 6. Check all other references and correct all the references by using journal guidelines.

Response 6: Thank you for highlighting this issue. We have carefully checked all references and corrected missing or incomplete information, including publication year, volume number, issue number, and page range, in accordance with the journal guidelines. For example, Reference 6 (Phytochemical Screening and Antimicrobial Activities of Stem, Leaves and Fruit Extracts of Viscum album L.) has been updated to include the correct year (2017), volume (11), issue (3), page numbers (1337–1349), and DOI. All other references have been reviewed and corrected as necessary to ensure consistency and compliance with the journal’s reference format.

Reviewer 2 Report

Comments and Suggestions for Authors

Phytochemical characterization and antimicrobial properties of a hydroalcoholic extract of Trysterix corymbosus (L) Kuijt, a Chilean mistletoe species hosted on Salix babilonica (L) by Hidalgo et al. deals in investigating aforementioned herbal species for its antimicrobial potential, this including chemical composition of plant's extract. In general the paper is written nicely, research was conducted in proper manner, and it was an interesting overview to read. I have several suggestions/comments/questions for Authors, and they are as follows:

1. Regarding the title, and the rest of the manuscript- herbal species is S. babylonica, not babilonica.
2. Abstract- Similar to previous case- I believe it is tristerix, and not trysterix (as given in keywords, please check).
3. Introduction- very nicely written, especially the first part. I would have to disagree on the location of Figure 1- this belongs to MM section and not Introduction, as this is a part (plant material) of YOUR research, and just random picture describing the plant and host plant. Please make the required change of location. Together with this, please separate the figure description from rest of the text. 
4. Aim of the study- strongly suggesting, not mandatory-- aim should be a bit shorter, this way resembles more of an abstract- I am of the opinion that it should be shorter, and less detailed.
5. Results- line 163 I believe that call-out should be for table 2? Also regarding the table title- row? of it should be raw? Figure 2 is nicely done, I find it quite useful. Figure, also nicely done, I especially like that growth of microorganisms is semiconfluent as it can be seen in C.albicans. Table 3, it is not necessary to add mg/ml after every number, especially if you have stated units in table itself. Regarding this segment, and the paper in general- I have to say that I really appreciate the fact that the Authors have investigated not only water or ethanol and methanol fraction, as it is done in over 90% of paper, but also the non soluble, lipidic fraction, the chemistry, the activity, and most interestingly mechanism of activity- all the features of a really valuable and useful scientific article. 
6. MM segment - paragraph 454-457 FC method for phenolics was described, yet I found no results of this test in Results segment...? Further on, why do you say flavonoids....? Section 4.4 what was the height of solidified agar in petri plates? I am asking this because I believe that 35 microliters seems as insufficient amount for wells 6 mm in diameters... 
7. Conclusion- the biggest problem with present paper- I invite you to delete the entirety of currently present conclusion, either to move it to discussion, which ever you find better, Conclusion of your paper should be about YOUR research, what was CONCLUDED and by no means it should contain references to other people's work, nor to give vague comments and future ideas. Please change it, completely. 

Looking forward to new, improved version. Kindest regards.

Author Response

Reviewer 2

Comment 1: Regarding the title, and the rest of the manuscript- herbal species is S. babylonica, not babilonica.

Response 1: See below response 2.

Comment 2: Abstract- Similar to previous case- I believe it is tristerix, and not trysterix (as given in keywords, please check).

Response 2: Thank you for pointing out the errors in the species names. The host species name has been corrected throughout the manuscript from S. babilonica to S. babylonica. In the case of mistletoe species, the plant genus was corrected from Trysterix to Tristerix in the abstract, keywords, and throughout the manuscript. All corrections have been duly implemented in the revised version of the text.

Comment 3: Introduction- very nicely written, especially the first part. I would have to disagree on the location of Figure 1- this belongs to MM section and not Introduction, as this is a part (plant material) of YOUR research, and just random picture describing the plant and host plant. Please make the required change of location. Together with this, please separate the figure description from rest of the text. 

Response 3: Thank you for this helpful suggestion. We agree with the reviewer that part of Figure 1 is more appropriately placed in the Materials and Methods section, as it illustrates the plant material and host species used in this study. However, parts of this figure are useful for the introduction. To conciliate these two points of view and to keep the structure of the introduction section, Figure 1 was splited. Now the old Figure 1a is presented as Figure 9 in the Material and methods section (4.1.) indicating the place where samples were taken. Old Figures 1bcdf are now included in a new version of Figure 1. Also, legend has been separated from the main text and presented independently as a figure caption, in accordance with the journal guidelines. In addition, a couple of misspellings were resolved in the foot notes.

Comment 4: Aim of the study- strongly suggesting, not mandatory-- aim should be a bit shorter, this way resembles more of an abstract- I am of the opinion that it should be shorter, and less detailed.

Response 4: Thank you for this valuable suggestion. We agree that the aim of the study was overly detailed and resembled an abstract. Therefore, the aim has been revised, shortened, and reformulated to be more concise and focused, in accordance with the style and guidelines of Antibiotics. The revised aim is now clearly stated in the Introduction of the manuscript.

The last paragraph now reads as follows:

In this work, we studied the phytochemical composition and antimicrobial activity of a hydroalcoholic extract of Tristerix corymbosus collected in central Chile.

Comment 5: Results- line 163 I believe that call-out should be for table 2? Also regarding the table title- row? of it should be raw? Figure 2 is nicely done, I find it quite useful. Figure, also nicely done, I especially like that growth of microorganisms is semiconfluent as it can be seen in C.albicans. Table 3, it is not necessary to add mg/ml after every number, especially if you have stated units in table itself. Regarding this segment, and the paper in general- I have to say that I really appreciate the fact that the Authors have investigated not only water or ethanol and methanol fraction, as it is done in over 90% of paper, but also the non soluble, lipidic fraction, the chemistry, the activity, and most interestingly mechanism of activity- all the features of a really valuable and useful scientific article.

Response 5:

• We have corrected the citation for Table 1 to Table 2.
• We thank you for your comments on Figure 2, we have made modest improvements by adding the dissolvent to test for antimicrobial activity, by suggestion of reviewer 4. Also, temperature of MeOH precipitation was added.
• We have deleted mg/ml from tables 3 and 4
• We appreciate your kind comments on our work

 

Comment 6: MM segment - paragraph 454-457 FC method for phenolics was described, yet I found no results of this test in Results segment...? Further on, why do you say flavonoids....? Section 4.4 what was the height of solidified agar in petri plates? I am asking this because I believe that 35 microliters seems as insufficient amount for wells 6 mm in diameters... 

Response 6: We apologize for including phenolic compounds quantification in M&M section. This part and the mention of flavonoids were part of an old version of this manuscript, before receiving HPLC-MS/MS results. At that point, the manuscript was a little bit biased by the typical polyphenolic composition reported by others. After that, lipidic surfactants become the main active compounds in the analyzed fractions, while phenolic compounds and flavonoids lose protagonism. The mention of phenolic compounds quantification and flavonoids was eliminated.

Regarding the volume of the formed well in the agar. In the plates we are using (90 mm plastic plates), the height of the solidified agar is around 4 mm. With that in mind, a maximum volume of around 90 µL would be possible. In practice, a meniscus is formed that would facilitate overflowing the agar surface. In our hands, volumes over 40-45 µL risk losing sample by overloading (depending on the vehicle used); therefore, we have obtained consistent results by using a safe volume of 35 µL.

Comment 7: Conclusion- the biggest problem with present paper- I invite you to delete the entirety of currently present conclusion, either to move it to discussion, which ever you find better, Conclusion of your paper should be about YOUR research, what was CONCLUDED and by no means it should contain references to other people's work, nor to give vague comments and future ideas. Please change it, completely. 

Response 7: Thank you for this important and constructive comment. We agree with the reviewer that the previous version of the Conclusion section was overly broad and included background information and references to other studies. The Conclusion has been completely rewritten to focus exclusively on the main findings and outcomes of the present research, without citing external literature or discussing future perspectives. Any contextual or comparative elements have been moved to the Discussion section where appropriate. The revised Conclusion now strictly summarizes what was concluded from our experimental results, in accordance with the journal guidelines.

New Conclusions

In this study, a hydroalcoholic extract of Tristerix corymbosus was prepared, fractionated, and evaluated for its phytochemical composition and antimicrobial activity. The hydrosoluble fraction obtained after methanol-induced precipitation exhibited the strongest antimicrobial effects, particularly against Gram-positive bacteria, including standard and clinical isolates of Staphylococcus aureus.

Phytochemical screening and LC–MS analysis revealed that this active fraction was predominantly composed of lipidic compounds, including glycerolipids, glycerophospholipids, lysophosphatidylcholines, and fatty acid amides. Functional assays demonstrated that the antimicrobial activity of this fraction was associated with disruption of bacterial membrane integrity.

Overall, these results demonstrate that T. corymbosus contains bioactive lipid-rich fractions with significant antimicrobial activity, supporting its relevance as a source of natural antimicrobial compounds.

Reviewer 3 Report

Comments and Suggestions for Authors

In this paper, we explore the phytochemical composition and antibacterial activity of the water alcohol extract of the Chilean mistletoe plant Tristrix corymbosus. Three components were obtained through 70% ethanol extraction and methanol precipitation separation, among which the brown powder component (HSF) showed strong antibacterial activity, especially against Gram positive bacteria (such as S. aureus, S. pyogenes) and yeast (C. albicans). HPLC-MS analysis showed that the component is rich in lipid compounds (such as phospholipids, glycerides, etc.), suggesting that it exerts antibacterial effects by disrupting the cell membrane structure. The study also validated the inhibitory ability of the extract against multidrug-resistant clinical strains.

1. The author are required to verify the spelling and formatting of the article to ensure it meets this journal's submission guidelines.
2. In the legend descriptions labeled (a-f) for Figure 1, "row" ought to be corrected to "raw," and "three" should be corrected to "tree."
3. Section 4.2 ends with 'Finaly' instead of 'Finally'
4. The summary needs to be simplified. It is recommended to briefly describe the extraction method, key results (such as mic value) and main conclusions in the summary.
5. Some sentences are complicated in structure, which affects readability. It is suggested to simplify them.
6. The initial letter of "inhibition" in Figure 3 is capitalized. In the Materials and Methods section, it directly skips to "4.8" after "4.6.2", with the "4.7" chapter missing; Sections 2.2 and 4.2 redundantly describe the preparation process of the extract.
7. Core abbreviations (e.g., HSF, PG, PI, PA) that make their first appearance in the main text are not provided with their full forms and are merely listed in the "Abbreviations" section at the end of the document; moreover, there is inconsistent usage of certain abbreviations, such as the interchangeable use of "HS extract" and "HSF".
8. Some references are missing page numbers (e.g., Reference 6: "Phytochemical Screening and Antimicrobial Activities of Stem, Leaves and Fruit Extracts of Viscum Album L. Journal of Pure and Applied Microbiology 2018"), and the DOI formats of some references are inconsistent (some are labeled as "doi:10.xxx", while others lack such labeling). Check whether the information of each article is complete (author, title, journal name, year, volume number, page number, DOI number), and whether the journal name uses standard abbreviations.
9. The key parameters (temperature, time, shaker speed) of extraction and fractionation process shall be listed in detail in the method section.

Author Response

Comment 1: The author are required to verify the spelling and formatting of the article to ensure it meets this journal's submission guidelines.

Response 1: We have made all possible to meet journal standards

Comment 2: In the legend descriptions labeled (a-f) for Figure 1, "row" ought to be corrected to "raw," and "three" should be corrected to "tree."

Response 2: Thank you for pointing out these typographical or misspelling errors. The terms “row” and “three” in the Figure 1 legend have been corrected to “raw” and “tree,” respectively. These corrections have been duly implemented in the revised manuscript.

Comment 3: Section 4.2 ends with 'Finaly' instead of 'Finally'

Response 3: Thank you for pointing out these typographical errors. The terms “finaly” has been changed with “finally”. These corrections have been duly implemented in the revised manuscript

 

Comment 4: The summary needs to be simplified. It is recommended to briefly describe the extraction method, key results (such as mic value) and main conclusions in the summary.

Response 4: Thanks for raising the point. Abstract was shortened, keeping the kay information.

Comment 5: Some sentences are complicated in structure, which affects readability. It is suggested to simplify them.

Response 5: Several sentences have been improved though the text, even entire paragraphs have been revised. We hope this revised version rich the quality of publication in Antibiotics.

Comment 6: The initial letter of "inhibition" in Figure 3 is capitalized. In the Materials and Methods section, it directly skips to "4.8" after "4.6.2", with the "4.7" chapter missing; Sections 2.2 and 4.2 redundantly describe the preparation process of the extract.

Response 6: Thanks for your comments and corrections. Inhibition in Figure 3 was corrected.

Comment 7: Previous section 4.8. is now called 4.7 to keep consistent.

Response 7: We really appreciate your comments about sections 2.2 and 4.2. Although section 2.2 is somehow redundant with section 4.2, we kept the original structure of section 2.2 with some modification trying to preserve most technical details for section 4.2.

Comment 8: Core abbreviations (e.g., HSF, PG, PI, PA) that make their first appearance in the main text are not provided with their full forms and are merely listed in the "Abbreviations" section at the end of the document; moreover, there is inconsistent usage of certain abbreviations, such as the interchangeable use of "HS extract" and "HSF".

Response 8: We are very grateful for your detailed corrections. We changed the use of HS extract to HSF. In line 267, LPC and DAG were first mentioned and defined. In line 273 and 274.PG, PI and PA made their first appearance and are defined.

Comment 9: Some references are missing page numbers (e.g., Reference 6: "Phytochemical Screening and Antimicrobial Activities of Stem, Leaves and Fruit Extracts of Viscum Album L. Journal of Pure and Applied Microbiology 2018"), and the DOI formats of some references are inconsistent (some are labeled as "doi:10.xxx", while others lack such labeling). Check whether the information of each article is complete (author, title, journal name, year, volume number, page number, DOI number), and whether the journal name uses standard abbreviations.

Response 9: Thank you for this comment. As indicated above, all references have been carefully reviewed and corrected according to the journal guidelines. Missing information such as page numbers, volume and issue numbers, publication year, and DOI details has been completed, DOI formats have been standardized, and journal names have been adjusted to use standard abbreviations where applicable. These corrections have been implemented throughout the revised manuscript.

Comment 10: The key parameters (temperature, time, shaker speed) of extraction and fractionation process shall be listed in detail in the method section.

Response 10: We apologies for been negligent in this section. Section “4.2. Plant Material extract” was misnamed in the submitted section. Although the parameters of extraction were listed, we have used this instance to improve the information and made some few corrections on it.

Reviewer 4 Report

Comments and Suggestions for Authors

Thank you for submitting this manuscript on the phytochemical and antimicrobial properties of Tristerix corymbosus. The study addresses an interesting ethnopharmacological topic by characterizing a native Chilean plant with potential applications in alternative medicine. The antimicrobial testing against both standard and clinical isolates, combined with HPLC-MS identification of lipidic compounds, provides a solid contribution, particularly in linking the findings to membrane disruption mechanisms and traditional wound-healing uses. The workflow from extraction to bioactivity assays is logical, and the inclusion of qualitative phytochemical screening adds context. However, the manuscript would benefit from revisions. Below, I outline the suggestions for authors:

• Table 1 could include semi-quantitative indicators (e.g., +/++/+++) for positive tests to show relative abundance.
• Table 2 has "row hydroalcoholic extract"—change to "raw" or "crude".
• Figure 2: Clear workflow, but label fractions more precisely (e.g., specify solvents used for resuspension).
• Table 3: Add statistical tests (e.g., ANOVA) if comparing halos/MICs across strains.
• Figure 3: Good representation, but include scale bars or well diameters in the caption.
• Figure 5 chromatograms could label key peaks directly. 
• Figure 7 references are inconsistent (e.g., "Figure 6" in Line 287 and Line 290 should be "Figure 7"). Quantify green/red ratios with means ± SD from replicates; include the calibration curve equation for transparency.

 

Author Response

Comment 1: Table 1 could include semi-quantitative indicators (e.g., +/++/+++) for positive tests to show relative abundance.

Response 1: Thanks for raising that point. Semi-quantitative indicators were added to table 1.

Comment 2: Table 2 has "row hydroalcoholic extract"—change to "raw" or "crude".

Response 2: Thanks for pointing out the error. The misspelling “row” was corrected to “raw”

Comment 3: Figure 2: Clear workflow, but label fractions more precisely (e.g., specify solvents used for resuspension).

Response 3: Thanks for helping to improve the figures. The solvent used (was) indicated both in the schema, in the footnote of figure 2 and clarified in the materials and methods (4.2 Plant Material extract).

Comment 4: Table 3: Add statistical tests (e.g., ANOVA) if comparing halos/MICs across strains.

Response 4: Analysis of variance was performed to values in tables 3 and 4. MIC experiments remained unchanged across replicates, therefore no error is reported.

Comment 5: Figure 3: Good representation, but include scale bars or well diameters in the caption.

Response 5: Scale bars were added in figures 3 and 4.

Comment 6 Figure 5 chromatograms could label key peaks directly.

Response 6: Thanks for the suggestion. The peaks in the spectra were labeled with peak number and name of their predicted molecules.

Comment 7: Figure 7 references are inconsistent (e.g., "Figure 6" in Line 287 and Line 290 should be "Figure 7"). Quantify green/red ratios with means ± SD from replicates; include the calibration curve equation for transparency.

Response 7: Thanks for pointing out our mistake. Figure 7 is now cited correctly.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Authors have answered all the questions and made the suggested changes, I believe that the manuscript is now improved and I will be recommending it for publication. Kindest regards.