Antiprotozoal Potential of Cultivated Geranium macrorrhizum Against Giardia duodenalis, Trichomonas gallinae and Leishmania infantum

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

     This is an interesting and clear-cut work to demonstrate the antiprotozoal capability of extracts from the plant Geranium microrrizae L, through its essential oils (EOs), particularly b-elemenone and germacrone, major constituents of such EOs (with 40-42% of the former and 36-40% of the latter). This was done on the parasites Giardia duodenalis, Leishmania spp. and Trichomonas spp.  However, the work would require revision. The comments and parts to be improved, according to this reviewer, are:

-Regarding the title. Authors used the term “biotechnologically cultivated … G. microrrhizum”, but it could be argued whether such definition, sustained in the use of a VENLO-type greenhouse for the propagation and maintenance of the selected plants along 6 months, could be defined as “biotechnological” nowadays.  Is it actually a biotechnological cultivation the one followed? At Material and Methods, the expected controlled conditions are only defined as “standard environmental conditions”. What does it mean, specifically?  Do these conditions differ significatively of what is done in standard commercially-based cultivars, or this was done in particular conditions, typical of in vitro cultivation, i.e. affecting sunlight, temperature and various plant growth regulators (PGRs), that could favour a better growth, better yields of certain metabolites (like secondary ones found at the EOs) and better protection against virus and fungus. I.e. a recent paper on the in vitro cultivation analysis of another variety of Geranium can be found at M. Harutyunyan et al, 2025, Functional Food Science 5, 364-377. 

      Also, another recent publication on the effects of gamma rays treatment on the in vitro cultivation (and micropropagation) of the same distinct Geranium variety (Pelargonium g.) and production of EOs also appeared recently: O.F. Abou El-Leel et al, 2025, BMC Plant Biology 25, 1447.  It would be wise that authors comment in the revised manuscript that these approaches could be used (including the use of bioreactors), particularly when a large increase in the production yield of a particular metabolite (i.e. germacrone here) is wished.

-Control compounds:   Authors must indicate very earlier that Metronidazole and Amphotericin B served as reference compounds for anti-Giardia /anti-Trichomonas and anti-leishmanial activities, respectively, otherwise non-expert readers will become lost until page 15, in which this indication is found.

-Compounds 1,2,3 and 4.  Authors should indicate more frequently and clearly that the comparative analysis of compounds in this work has been done on compounds 1-4, of which number 1 es b-elemenone, number 2 is germacrone, and numbers 3-4 are synthetic derivatives starting from germacrone … because confusion on their identities could be produced easily.  At this respect, it could be useful to keep the name of b-elemenone as it is in the comparative Table 3 (instead of compound 2), the first site in which such numbering is used, as well as give number 1 to germacrone, and 2,3 to the other derivatives.  It is confusing that number 2 (and not 1) is assigned to germacrone, as it appears in such table 3 as well as in Fig. 7.  Also, it would be useful to include the structure of b-elemenone in such Figure 7.

-At page 10, lines 230-232, such sentence probably it would be better to be eliminated because it doesn’t merit to remind that germacrone exhibited cytotoxicity against canine DH82 macrophages when subsequently it is said that such property can be attributed to the added DMSO at 2%.  At the following sentences, three from lines 233-241, all the asseverations there seems to be related with the reference [30], placed at the end. This is confusing and should be modified to make clearer such relationship.   By the way, at line 235 the term germacrome is followed by a (1).  Should this one termed as (2) instead of, or this is nuclear?  Revise it, please.

-Side effects of germacrone. At the next paragraph, and in fact along the manuscript, the biotech/biomed properties of germacrone are emphasised, but little is said on its proved or potential undesirable side-effects.  Given that there are quite a number of references dealing with this issue, including those that indicate possible damage at liver and other organs/cells, this should be collected and indicated here (i.e. one of 2020, de Tomáš Zárybnický, 2020, at Nutrients 12(6): 1720; and Suyan Liu et al, 2024, at Etnopharmacology).

-Lines at the end of Discussion:  such lines, 319-322, are in fact a general comment applicable to many other scientific studies, useless there, and it would be better to remove it.

Author Response

This is an interesting and clear-cut work to demonstrate the antiprotozoal capability of extracts from the plant Geranium microrrizae L, through its essential oils (EOs), particularly b-elemenone and germacrone, major constituents of such EOs (with 40-42% of the former and 36-40% of the latter). This was done on the parasites Giardia duodenalis, Leishmania spp. and Trichomonas spp.  However, the work would require revision. The comments and parts to be improved, according to this reviewer, are:

Comment: Regarding the title. Authors used the term “biotechnologically cultivated … G. microrrhizum”, but it could be argued whether such definition, sustained in the use of a VENLO-type greenhouse for the propagation and maintenance of the selected plants along 6 months, could be defined as “biotechnological” nowadays.  Is it actually a biotechnological cultivation the one followed?

Response:

Thank you for pointing this out. We acknowledge that the use of the term “biotechnologically cultivated” in the title was incorrect. The plants were propagated and maintained in a VENLO-type greenhouse under controlled conditions, but no biotechnological techniques were applied. We have corrected the title accordingly to avoid any misunderstanding.

Comment: At Material and Methods, the expected controlled conditions are only defined as “standard environmental conditions”. What does it mean, specifically?  Do these conditions differ significatively of what is done in standard commercially-based cultivars, or this was done in particular conditions, typical of in vitro cultivation, i.e. affecting sunlight, temperature and various plant growth regulators (PGRs), that could favour a better growth, better yields of certain metabolites (like secondary ones found at the EOs) and better protection against virus and fungus. I.e. a recent paper on the in vitro cultivation analysis of another variety of Geranium can be found at M. Harutyunyan et al, 2025, Functional Food Science 5, 364-377. 

Response

Thank you for your comment. We agree that the previous description was too general. We have revised the Materials and Methods section to specify the controlled greenhouse conditions, including temperature, humidity, and irrigation practices, and clarified that no in vitro techniques or plant growth regulators were used. This information has now been added to the Materials and Methods section for clarity.

Comment: Also, another recent publication on the effects of gamma rays treatment on the in vitro cultivation (and micropropagation) of the same distinct Geranium variety (Pelargonium g.) and production of EOs also appeared recently: O.F. Abou El-Leel et al, 2025, BMC Plant Biology 25, 1447.  It would be wise that authors comment in the revised manuscript that these approaches could be used (including the use of bioreactors), particularly when a large increase in the production yield of a particular metabolite (i.e. germacrone here) is wished.

Response

Thank you for your suggestion. We have incorporated the recommended changes in the Discussion, adding a paragraph that comments on the recent study by Abou El-Leel et al. (2025) and highlighting the potential use of gamma irradiation and bioreactor-based approaches to enhance germacrone production.

Comment: Control compounds:   Authors must indicate very earlier that Metronidazole and Amphotericin B served as reference compounds for anti-Giardia /anti-Trichomonas and anti-leishmanial activities, respectively, otherwise non-expert readers will become lost until page 15, in which this indication is found.

Response

The following sentence has been added at page 3, lines 107-109: “Metronidazole was used as the reference compound for anti-Giardia and anti-Trichomonas activity, while Amphotericin B served as the reference for anti-Leishmania activity.”

Comment: Compounds 1,2,3 and 4.  Authors should indicate more frequently and clearly that the comparative analysis of compounds in this work has been done on compounds 1-4, of which number 1 es b-elemenone, number 2 is germacrone, and numbers 3-4 are synthetic derivatives starting from germacrone … because confusion on their identities could be produced easily.  At this respect, it could be useful to keep the name of b-elemenone as it is in the comparative Table 3 (instead of compound 2), the first site in which such numbering is used, as well as give number 1 to germacrone, and 2,3 to the other derivatives.  It is confusing that number 2 (and not 1) is assigned to germacrone, as it appears in such table 3 as well as in Fig. 7.  Also, it would be useful to include the structure of b-elemenone in such Figure 7.

Response

We appreciate the reviewer’s comment and understand the concern regarding potential confusion about compound numbering. To clarify, the names of germacrone and β-elemenone have been included in Table 3 and Figure to improve readability. However, the numbering was assigned based on the order of isolation and chemical transformation: β-elemenone was the first compound obtained according to its retention index, followed by germacrone, and then compounds 3 and 4, which are synthetic derivatives originating from germacrone. For this reason, we believe that maintaining this numbering is the most appropriate approach. Additionally, we have included the chemical structure of β-elemenone in Figure 7 to further reduce any ambiguity.

Comment: At page 10, lines 230-232, such sentence probably it would be better to be eliminated because it doesn’t merit to remind that germacrone exhibited cytotoxicity against canine DH82 macrophages when subsequently it is said that such property can be attributed to the added DMSO at 2%.  At the following sentences, three from lines 233-241, all the asseverations there seems to be related with the reference [30], placed at the end. This is confusing and should be modified to make clearer such relationship. By the way, at line 235 the term germacrone is followed by a (1).  Should this one termed as (2) instead of, or this is nuclear?  Revise it, please.

Response

The sentence on page 10, lines 230–232, has been removed, and lines 238–241 have been relocated to follow previous reference 29 to establish a clearer relationship with the cited work. Additionally, the compound name “germacrone” was incorrectly followed by the number 1 at line 235 and the number has been deleted.

Comment: Side effects of germacrone. At the next paragraph, and in fact along the manuscript, the biotech/biomed properties of germacrone are emphasised, but little is said on its proved or potential undesirable side-effects.  Given that there are quite a number of references dealing with this issue, including those that indicate possible damage at liver and other organs/cells, this should be collected and indicated here (i.e. one of 2020, de Tomáš Zárybnický, 2020, at Nutrients 12(6): 1720; and Suyan Liu et al, 2024, at Etnopharmacology).

Response

Thank you for your observation. The reference regarding germacrone’s inhibition of CYP2B6 was already included in the manuscript ([35]). To address your suggestion, we have now added information from the second recommended source (Zárybnický et al., Nutrients 2020), highlighting that germacrone can induce oxidative stress and dysregulate cholesterol and lipid metabolism in hepatocyte-like cells, which suggests a potential risk of hepatotoxicity.

Comment: Lines at the end of Discussion:  such lines, 319-322, are in fact a general comment applicable to many other scientific studies, useless there, and it would be better to remove it.

Response

We apologize for the mistake, the last paragraph of discussion (lines 319-322) has been deleted.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This is a fine manuscript addressing an interesting and relevant topic in the evaluation of the antiparasitic activity of Geranium macrorrhizum. The overall presentation is generally clear, and the work has potential. However, some methodological and reporting issues currently limit the biological conclusions. I therefore provided major and minor comments below, including recommendations for a couple additional experiments, which should be addressed before the manuscript can be considered suitable for publication.

Major comments

- The Abstract states “potent, broad-spectrum activity” (Page 1, Lines 32-34) of essential oils, but several essential oil IC₅₀ values are reported as beyond the highest tested concentration, indicating limited activity under the tested conditions. Please revise the Abstract/Discussion to accurately reflect the spectrum and potency supported by the data and highlight germacrone appropriately.

- The manuscript would benefit from a stronger framing of the three parasitic diseases addressed, particularly in the Introduction (or early in the Discussion). At present, the motivation for focusing on these infections is not fully developed before transitioning to natural products as alternatives. I suggest adding a dedicated paragraph highlighting the clinical importance of the target parasites and the limitations of current therapies beyond resistance. For example, avian trichomoniasis caused by T. gallinae can represent a serious ecological threat, with the potential to even decline bird populations. In addition, nitroimidazoles face broader constraints, including regulatory restrictions in food-producing animals… metronidazole has been prohibited for use in animals destined for human consumption in many countries due to carcinogenicity concerns (https://doi.org/10.1038/s41598-025-10668-w). Providing this context would strengthen the rationale and translational relevance of the study.

- In the Methods section, SI is defined as SI = IC₅₀/CC₅₀, but in Table 3 authors define SI as CC₅₀/IC₅₀, and the reported SI values appear consistent with that latter definition. Please correct the SI definition throughout the manuscript, and verify that all SI values are correctly calculated.

- I strongly recommend Table 2 to be reformulated. Listing all identified compounds, along with their retention time (or retention index, as appropriate) and relative abundance (%). In addition, summarize compounds by chemical class (mono/sesquiterpenes, oxygenated vs non oxygenated) and report the percentage contribution of each class.

- The authors state that DMSO was kept at <1% final concentration in all assays. However, Table 4 includes DMSO 2% as a control. It is of concern that DMSO alone produced toxicity similar to germacrone in DH82 cells. This raises a major issue that some reported effects (including apparent antiparasitic activity and/or host cell toxicity) could be driven partly by the vehicle. Please, standardize and clearly report the final DMSO concentration for each assay and ensure that all treatment groups are compared against the matched DMSO control.

- In virtue of these results, additional experiments including DMSO controls are highly recommended for the experiments of the effect of germacrone on parasites morphology and related cell morphometric parameters.

- A recurring issue in the manuscript is the choice of concentrations used for downstream experiments relative to the activity observed in the initial screening. For example, germacrone IC₅₀ values for extracellular protozoa fall in the 37-55 µg/mL range (depending on the parasitic organism). Yet higher concentrations were used in the intracellular assay (50-100 µg/mL) and were reported to kill host cells. Please justify the concentrations selected for mechanistic/morphological and intracellular assays. For the morphological experiments even the time of exposure is higher when compared to the initial 24 h in the screenings. The ideal point would be the inclusion of experiments at concentrations anchored to biological endpoints (0.5×/1×/2× IC₅₀, and concentrations that remain non-cytotoxic to host cells).

- The morphometric analysis is based on 30 photographed trophozoites, captured via a mobile phone through the microscope. This approach is really clever and can be acceptable, but only if the workflow is transparent and controlled. Authors are recommended to clarify: 1) how fields/parasites were selected (randomization, avoidance of selection bias); 2) whether the evaluator was blinded; 3) whether the 30 measurements are from multiple independent experiments (biological replicates) or a single run.

- Some interpretations in the Discussion (for example, the conserved impact on cytoskeletal dynamics) goes beyond what can be concluded from morphometric analysis alone. Please tone down mechanistic statements unless supported by direct mechanistic assays.

- The Methods section should include details regarding essential oil extraction to ensure reproducibility. Please report: 1) the amount of botanical material used to obtain each essential oil (including whether fresh or dried material was used and any relevant pre-processing); 2) the hydrodistillation conditions, particularly the distillation time; and 3) how the oils were handled and stored after extraction (drying agent if used, storage temperature, protection from light, and duration of storage prior to analysis/biological assays). These details are essential because yield and composition can be affected by extraction and storage conditions.

Minor comments

- The authors should perform a careful, full manuscript revision to correct multiple formatting and typographical inconsistencies. In several places, scientific terms that should be italicized (genus/species names) are not, and in other instances units/notation appear incorrectly formatted (superscripts in density units and related expressions). Please standardized throughout the text.

- Please delete the last paragraph of discussion, which appears to be the instructions from the journal template.

- (Line 35, page 5) (Line 142, page): please replace “exposition” for “exposure”.

- Tables/figures: Please include in all tables/figure legends the controls, solvent percentages, and what “n” represents (biological vs technical replicates), as well as the statistical test used for that group of data (and the values related to the asterisks).

- (Pages 9-10, Lines 207 - 214): There seems to be some confusion between botanical family and genus... Pelargonium is not the same as Geranium, but both belong to the same Geraniaceae family.

Author Response

This is a fine manuscript addressing an interesting and relevant topic in the evaluation of the antiparasitic activity of Geranium macrorrhizum. The overall presentation is generally clear, and the work has potential. However, some methodological and reporting issues currently limit the biological conclusions. I therefore provided major and minor comments below, including recommendations for a couple additional experiments, which should be addressed before the manuscript can be considered suitable for publication.

Major comments

Comment: The Abstract states “potent, broad-spectrum activity” (Page 1, Lines 32-34) of essential oils, but several essential oil IC₅₀ values are reported as beyond the highest tested concentration, indicating limited activity under the tested conditions. Please revise the Abstract/Discussion to accurately reflect the spectrum and potency supported by the data and highlight germacrone appropriately.

Response

Considering that de IC₅₀ of EOAP and EOF are between 67-120 μg/mL for L.  infantum and T. gallinae, we have rewritten the sentence (page 1, lines 32-34) as follows: “The results demonstrated strong activity of essential oils against Trichomonas gallinae, and Leishmania infantum, indicating the relevance of lipophilic compounds….” As essential oils are complex mixtures of compounds several authors consider this IC₅₀ values as strong activity.

Comment: The manuscript would benefit from a stronger framing of the three parasitic diseases addressed, particularly in the Introduction (or early in the Discussion). At present, the motivation for focusing on these infections is not fully developed before transitioning to natural products as alternatives. I suggest adding a dedicated paragraph highlighting the clinical importance of the target parasites and the limitations of current therapies beyond resistance. For example, avian trichomoniasis caused by T. gallinae can represent a serious ecological threat, with the potential to even decline bird populations. In addition, nitroimidazoles face broader constraints, including regulatory restrictions in food-producing animals… metronidazole has been prohibited for use in animals destined for human consumption in many countries due to carcinogenicity concerns (https://doi.org/10.1038/s41598-025-10668-w). Providing this context would strengthen the rationale and translational relevance of the study.

Response

The following paragraph has been added to the Introduction: “Protozoan parasites encompass a diverse range of eukaryotic organisms, some of which are significant contributors to globally neglected human diseases (Menezes & Tasca, 2023). These diseases pose a substantial public health challenge, affecting millions of individuals worldwide. Control and prevention strategies are increasingly undermined by the development of resistance to commonly used chemotherapeutic agents (Pramanik et al., 2019). Among the protozoan parasites of medical and veterinary importance, Giardia duodenalis, Leishmania spp., and Trichomonas spp. stand out due to their widespread impact on humans, mammals and birds.

Leishmania spp. cause leishmaniasis, a group of diseases transmitted through the bite of infected female phlebotomine sandflies. More than one billion people currently live in areas endemic for leishmaniasis and are at risk of infection (World Health Organization (WHO), 2024). Treatment primarily relies on pharmacological therapies; however, many available drugs (pentavalent antimonials, liposomal amphotericin B, and pentamidine) are associated with high toxicity, limited efficacy, complex administration protocols, and significant costs. Furthermore, resistant strains have emerged, complicating disease management (Sasidharan & Saudagar, 2021). G. duodenalis is an extracellular enteric protozoan responsible for widespread diarrheal diseases, with over 300 million cases reported annually, particularly in low-income and developing regions (Cernikova et al., 2018). Despite the availability of treatments, the increasing resistance of Giardia to nitroimidazole-based therapies, such as metronidazole (MTZ), poses a growing concern (Argüello-García et al., 2020; Menezes & Tasca, 2023). In birds, Trichomonas gallinae is a flagellated oropharyngeal parasite that causes granulomas and starvation, significantly impacting avian populations. While Columbiformes serve as the primary reservoirs, other domestic and wild bird species are also susceptible (Amin et al., 2014). The current treatment for T. gallinae infections also relies on nitroimidazoles, yet no preventive treatments have been approved in the EU, and therapy failures linked to resistant strains have been documented (European Commission, 1995).

There is an urgent need for alternative treatments against Leishmania spp., G. duodenalis, and Trichomonas spp.”

Comment: In the Methods section, SI is defined as SI = IC₅₀/CC₅₀, but in Table 3 authors define SI as CC₅₀/IC₅₀, and the reported SI values appear consistent with that latter definition. Please correct the SI definition throughout the manuscript, and verify that all SI values are correctly calculated.

Response

The correct definition of the Selectivity Index (SI) is CC₅₀/IC₅₀, and it has been changed in the Methods section. Also, SI have been checked, and all SI values are correctly calculated

Comment: - I strongly recommend Table 2 to be reformulated. Listing all identified compounds, along with their retention time (or retention index, as appropriate) and relative abundance (%). In addition, summarize compounds by chemical class (mono/sesquiterpenes, oxygenated vs non oxygenated) and report the percentage contribution of each class.

Response

Thank you for your suggestion. Table 2 has been reformulated as recommended. It now includes all identified compounds with their retention time (or retention index, as appropriate) and relative abundance (%). Additionally, the compounds have been summarized by chemical class (mono- and sesquiterpenes, oxygenated vs. non-oxygenated), and the percentage contribution of each class is reported.

Comment: The authors state that DMSO was kept at <1% final concentration in all assays. However, Table 4 includes DMSO 2% as a control. It is of concern that DMSO alone produced toxicity similar to germacrone in DH82 cells. This raises a major issue that some reported effects (including apparent antiparasitic activity and/or host cell toxicity) could be driven partly by the vehicle. Please, standardize and clearly report the final DMSO concentration for each assay and ensure that all treatment groups are compared against the matched DMSO control.

Response

Thank you for pointing this out. We have carefully reviewed all calculations and confirmed that DMSO was kept at a maximum final concentration of 1% in all experiments, including those performed with canine macrophages. The mention of “DMSO 2%” at Table 4 was a typographical error and has now been corrected to reflect the actual concentration used (1%). We have perfomed another experiment to confirm the cytotoxic effects of DMSO 1% and redone Figure 2.

Comment: In virtue of these results, additional experiments including DMSO controls are highly recommended for the experiments of the effect of germacrone on parasites morphology and related cell morphometric parameters.

Response

We appreciate the reviewer’s suggestion. The morphology experiments were performed using 1% DMSO as a control, and this detail has now been explicitly included in the methodology section

Comment: A recurring issue in the manuscript is the choice of concentrations used for downstream experiments relative to the activity observed in the initial screening. For example, germacrone IC₅₀ values for extracellular protozoa fall in the 37-55 µg/mL range (depending on the parasitic organism). Yet higher concentrations were used in the intracellular assay (50-100 µg/mL) and were reported to kill host cells. Please justify the concentrations selected for mechanistic/morphological and intracellular assays. For the morphological experiments even the time of exposure is higher when compared to the initial 24 h in the screenings. The ideal point would be the inclusion of experiments at concentrations anchored to biological endpoints (0.5×/1×/2× IC₅₀, and concentrations that remain non-cytotoxic to host cells).

Response

We employ concentrations of 100, 50 and 10 µg/ml for most of the compounds tested as antiparasitic at the laboratory, as a standard procedure, since most of the most interesting compounds are active at this range. For this reason, 100, 50 and 10 µg/ml were employed. We could eliminate 100 µg/ml, since it does not add information, but we prefer to keep the data of the whole experiment. Besides, to calculate IC₅₀ values high, low and intermediate doses are required. On the other hand, our morphological analysis was designed as a qualitative, cross‑species comparison to document stabilized phenotypes, and for that reason it was conducted at a single time point (48 h) and a fixed germacrone concentration across Giardia duodenalis, Trichomonas gallinae, and Leishmania infantum. We have employed 2x IC50 endpoint to assure biological effect on parasites. At 100 ug/ml, germacrone was non-toxic for Vero cells and for that reason we used this concentration. Besides, we have clarified in the Methods and figure legends the exact concentration (germacrone, metronidazole and Amphotericin B) and exposure time used for each parasite.

Comment: The morphometric analysis is based on 30 photographed trophozoites, captured via a mobile phone through the microscope. This approach is really clever and can be acceptable, but only if the workflow is transparent and controlled. Authors are recommended to clarify: 1) how fields/parasites were selected (randomization, avoidance of selection bias); 2) whether the evaluator was blinded; 3) whether the 30 measurements are from multiple independent experiments (biological replicates) or a single run.

Response

Thank you for your suggestions. We have expanded the Methods section to describe morphometric workflow in detail. Specifically, trophozoites were selected by consecutive sampling during a predefined zig-zag scan, applying objective eligibility criteria (intact, well-focused, non-overlapping) and excluding overlapping/partially visible cells to minimize selection bias. We also state that formal blinding was not implemented. Finally, we have clarified the replication structure: experiments were performed as independent biological experiments with technical quadruplicates except for Giardia duodenalis, that was set up in one well per condition/time point due to Lab-Tek chamber format. For each condition/time point wells were pooled prior to centrifugation to obtain sufficient biomass and a representative pellet for smear preparation, resulting in one stained smear per condition.

Comment: Some interpretations in the Discussion (for example, the conserved impact on cytoskeletal dynamics) goes beyond what can be concluded from morphometric analysis alone. Please tone down mechanistic statements unless supported by direct mechanistic assays.

Response

Thank you for your comment. We agree that some mechanistic interpretations in the Discussion were speculative. We have revised these statements to focus on observed morphological changes without inferring underlying mechanisms. Thus, we have deleted the following part of the sentence “suggesting a conserved impact on cytoskeletal dynamics”.

Comment: The Methods section should include details regarding essential oil extraction to ensure reproducibility. Please report: 1) the amount of botanical material used to obtain each essential oil (including whether fresh or dried material was used and any relevant pre-processing); 2) the hydrodistillation conditions, particularly the distillation time; and 3) how the oils were handled and stored after extraction (drying agent if used, storage temperature, protection from light, and duration of storage prior to analysis/biological assays). These details are essential because yield and composition can be affected by extraction and storage conditions.

Response

Thank you for your comment. We have now added the requested details to the Methods section to ensure reproducibility. Specifically, a 100 g sample of Geranium macrorrhizum aerial parts was hydrodistilled in a Clevenger-type apparatus for 2 hours. The resulting essential oil was dried over anhydrous ammonium sulfate, filtered, and stored at 4 °C for subsequent use. These details clarify the amount and type of botanical material, the hydrodistillation conditions, and the post-extraction handling and storage.

Minor comments

Comment:  The authors should perform a careful, full manuscript revision to correct multiple formatting and typographical inconsistencies. In several places, scientific terms that should be italicized (genus/species names) are not, and in other instances units/notation appear incorrectly formatted (superscripts in density units and related expressions). Please standardized throughout the text.

Response

The manuscript has been checked an al the typographic inconsistencies and other errors have been corrected.

Comment: Please delete the last paragraph of discussion, which appears to be the instructions from the journal template.

Response

The paragraph has been deleted from discussion.

Comment: (Line 35, page 5) (Line 142, page): please replace “exposition” for “exposure”.

Response

Exposition has been replaced for exposure on lines 135 and 142 (pages 5 and 6).

Comment: Tables/figures: Please include in all tables/figure legends the controls, solvent percentages, and what “n” represents (biological vs technical replicates), as well as the statistical test used for that group of data (and the values related to the asterisks).

Response

Thank you for your suggestion. We have followed your recommendations and updated all table and figure legends to include the controls, solvent percentages, the meaning of “n” (biological vs. technical replicates), and the statistical test used, along with the explanation of the asterisks.

Comment: (Pages 9-10, Lines 207 - 214): There seems to be some confusion between botanical family and genus... Pelargonium is not the same as Geranium, but both belong to the same Geraniaceae family.

Response

The paragraph regarding Pelargonium spp. has been deleted.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

 

The present article: Antiprotozoal Potential of Biotechnologically Cultivated Geranium macrorrhizum presents the first comprehensive evaluation of the antiprotozoal activity of Geranium macrorrhizum cultivated biotechnologically under controlled greenhouse conditions. Essential oils were extracted and chemically characterized, then tested in vitro against Giardia duodenalis, Trichomonas gallinae, and Leishmania infantum. The oils showed potent broad-spectrum antiparasitic activity with low cytotoxicity. Germacrone was identified as the main bioactive compound, exhibiting strong and selective effects and outperforming related analogues. Microscopic analyses revealed parasite-specific morphological alterations distinct from those caused by conventional drugs. Overall, the findings highlight G. macrorrhizum as a sustainable and promising source of natural antiprotozoal agents. However, several adjustments and corrections are necessary, as described below:

 

1 – Title: Modify the title by including the protozoan species used in the study;

2 – Introduction, lines 46-49: The importance of each parasite needs to be explained in more detail individually. A brief description was provided, but summarizing all three together makes the discussion too superficial;

3 - Introduction: Remember to follow the rules of botanical and zoological nomenclature. For Giardia duodenalis, Leishmania spp., and Trichomonas spp Neumann, 1899, include the describer and the year of description, and for plants, include the species name. This should be done throughout the text for other species mentioned, always adhering to the proper rules of botanical and zoological nomenclature;

4 - Introduction, lines 62-70: It is advisable to include information on the common names of the plant species used in this study;

5 – Results, Table 1: Include the R² value;

6 - Results, Table 2: Chemical composition of G. macrorrhizum EOs: Include all identified compounds along with their respective retention indices. An additional retention index from the literature (e.g., Wiley) should also be provided, as well as a column indicating the percentage of each compound;

7 - Results, Table 3: Include the R² value;

8 - Results, Figure 2:  The title “DH82 × germacrone” is not concise, as there is no direct comparison or confrontation; rather, the effect of germacrone on the cells is being evaluated. Please revise the graph title accordingly;

9 - Results, Figure 3:  Please include a scale bar in the figures, indicating the corresponding size value;

10 - Results, Figures 4, 5 and 6: It is necessary to include in the figure legend which statistical test was used and the meaning of the asterisk;

11 – Discussion: It should be mentioned in the discussion that the selectivity index between parasites and host cells must be at least 20, based on the following references:

*Evans, S.M.; Casartelli, A.; Herreros, E.; Minnick, D.T.; Day, C.; George, E.; Westmoreland, C. Development of a High Throughput in Vitro Toxicity Screen Predictive of High Acute in Vivo Toxic Potential. Toxicol. In Vitro 2001, 15, 579–584

*Nwaka, S.; Hudson, A. Innovative Lead Discovery Strategies for Tropical Diseases. Nat. Rev. Drug Discov. 2006, 5, 941–955.

 

12 – Materials and Methods, 4.1. Plant material: The voucher number for plant identification and the coordinates of the collection site should be provided;

13 – Materials and Methods, 4.3. Analysis: The description of the GC conditions should be improved. It is tedious for the reader to constantly check the cited reference; please provide a summary instead;

14 – Materials and Methods, 4.4. Compounds Same as described in the previous section.

   

Author Response

The present article: Antiprotozoal Potential of Biotechnologically Cultivated Geranium macrorrhizum presents the first comprehensive evaluation of the antiprotozoal activity of Geranium macrorrhizum cultivated biotechnologically under controlled greenhouse conditions. Essential oils were extracted and chemically characterized, then tested in vitro against Giardia duodenalis, Trichomonas gallinae, and Leishmania infantum. The oils showed potent broad-spectrum antiparasitic activity with low cytotoxicity. Germacrone was identified as the main bioactive compound, exhibiting strong and selective effects and outperforming related analogues. Microscopic analyses revealed parasite-specific morphological alterations distinct from those caused by conventional drugs. Overall, the findings highlight G. macrorrhizum as a sustainable and promising source of natural antiprotozoal agents. However, several adjustments and corrections are necessary, as described below:

Comment: 1 – Title: Modify the title by including the protozoan species used in the study;

Response

The title has been modified as suggested: Antiprotozoal Potential of Biotechnologically Cultivated Geranium macrorrhizum against Giardia duodenalis, Trichomonas gallinae and Leishmania infantum

Comment: 2 – Introduction, lines 46-49: The importance of each parasite needs to be explained in more detail individually. A brief description was provided, but summarizing all three together makes the discussion too superficial;

Response

The following paragraph has been added to the Introduction to explain the importance of each parasite: “Protozoan parasites encompass a diverse range of eukaryotic organisms, some of which are significant contributors to globally neglected human diseases (Menezes & Tasca, 2023). These diseases pose a substantial public health challenge, affecting millions of individuals worldwide. Control and prevention strategies are increasingly undermined by the development of resistance to commonly used chemotherapeutic agents (Pramanik et al., 2019). Among the protozoan parasites of medical and veterinary importance, Giardia duodenalis, Leishmania spp., and Trichomonas spp. stand out due to their widespread impact on humans, mammals and birds.

Leishmania spp. cause leishmaniasis, a group of diseases transmitted through the bite of infected female phlebotomine sandflies. More than one billion people currently live in areas endemic for leishmaniasis and are at risk of infection (World Health Organization (WHO), 2024). Treatment primarily relies on pharmacological therapies; however, many available drugs (pentavalent antimonials, liposomal amphotericin B, and pentamidine) are associated with high toxicity, limited efficacy, complex administration protocols, and significant costs. Furthermore, resistant strains have emerged, complicating disease management (Sasidharan & Saudagar, 2021). G. duodenalis is an extracellular enteric protozoan responsible for widespread diarrheal diseases, with over 300 million cases reported annually, particularly in low-income and developing regions (Cernikova et al., 2018). Despite the availability of treatments, the increasing resistance of Giardia to nitroimidazole-based therapies, such as metronidazole (MTZ), poses a growing concern (Argüello-García et al., 2020; Menezes & Tasca, 2023). In birds, Trichomonas gallinae is a flagellated oropharyngeal parasite that causes granulomas and starvation, significantly impacting avian populations. While Columbiformes serve as the primary reservoirs, other domestic and wild bird species are also susceptible (Amin et al., 2014). The current treatment for T. gallinae infections also relies on nitroimidazoles, yet no preventive treatments have been approved in the EU, and therapy failures linked to resistant strains have been documented (European Commission, 1995).

There is an urgent need for alternative treatments against Leishmania spp., G. duodenalis, and Trichomonas spp.”

Comment: 3 - Introduction: Remember to follow the rules of botanical and zoological nomenclature. For Giardia duodenalis, Leishmania spp., and Trichomonas spp Neumann, 1899, include the describer and the year of description, and for plants, include the species name. This should be done throughout the text for other species mentioned, always adhering to the proper rules of botanical and zoological nomenclature;

Response

Throughout the manuscript, all protozoan and plant species names have been formatted according to the International Codes of Zoological and Botanical Nomenclature, with the author and year indicated at first mention, for example Giardia duodenalis (Stiles, 1902); Trichomonas gallinae (Rivolta, 1878); Leishmania Leishmania infantum (Nicolle, 1908); and Geranium macrorrhizum L., (1753).”

Comment: 4 - Introduction, lines 62-70: It is advisable to include information on the common names of the plant species used in this study;

Response

The common names of G. macrorrhizum had been added at lines 62-63 (bigroot geranium, Bulgarian geranium, and rock crane’s-bill).

Comment: 5 – Results, Table 1: Include the R² value;

Response

Thank you for your comment. The R² values have now been included in Table 1 as requested.

Comment: 6 - Results, Table 2: Chemical composition of G. macrorrhizum EOs: Include all identified compounds along with their respective retention indices. An additional retention index from the literature (e.g., Wiley) should also be provided, as well as a column indicating the percentage of each compound;

Response

Table 2 has been updated accordingly. It now includes all identified compounds with their respective retention indices, an additional retention index from the literature, and a column indicating the percentage of each compound.

Comment: 7 - Results, Table 3: Include the R² value;

Response

Thank you for your comment. The R² values have now been included in Table 3 as requested.

Comment: 8 - Results, Figure 2:  The title “DH82 × germacrone” is not concise, as there is no direct comparison or confrontation; rather, the effect of germacrone on the cells is being evaluated. Please revise the graph title accordingly

Response

We thank the reviewer for this observation. The original title has been removed, and the figure caption has been revised to clearly describe the content. The new caption reads: “Figure 2. Cytotoxic effects of germacrone on DH82 canine macrophages, expressed as % cell viability (AmB: amphotericin B).”

Comment: 9- Results, Figure 3:  Please include a scale bar in the figures, indicating the corresponding size value;

Response

Thank you for your comment. We have replaced Figure 3 with a new version that includes a calibrated scale bar, indicating the corresponding size value. This update ensures accurate representation of the morphological observations.

Comment: 10 - Results, Figures 4, 5 and 6: It is necessary to include in the figure legend which statistical test was used and the meaning of the asterisk;

Response

We have updated the legends of Figures 4, 5, and 6 to include the statistical test used and the meaning of the asterisk. The revised legends now specify: *(p < 0.05, Mann–Whitney U test)”.

Comment: 11 – Discussion: It should be mentioned in the discussion that the selectivity index between parasites and host cells must be at least 20, based on the following references:

*Evans, S.M.; Casartelli, A.; Herreros, E.; Minnick, D.T.; Day, C.; George, E.; Westmoreland, C. Development of a High Throughput in Vitro Toxicity Screen Predictive of High Acute in Vivo Toxic Potential. Toxicol. In Vitro 2001, 15, 579–584

*Nwaka, S.; Hudson, A. Innovative Lead Discovery Strategies for Tropical Diseases. Nat. Rev. Drug Discov. 2006, 5, 941–955.

Response

We have revised the Discussion section to include the statement that the selectivity index (SI) between parasites and host cells should be at least 20, as recommended in the cited references (Nwaka & Hudson, 2006). This information has been incorporated to provide proper context for interpreting our results and to align with established criteria for antiparasitic drug development.

Comment: 12 – Materials and Methods, 4.1. Plant material: The voucher number for plant identification and the coordinates of the collection site should be provided;

Response

Pre-selected field-grown plants of G. macrorrhizum cultivar (voucher number R297730), previously reported [13] for superior biomass production, were collected from Ejea de los Caballeros (Spain) and subsequently propagated under greenhouse conditions. The voucher number and collection site coordinates have now been included in the Materials and Methods section.

Comment: 13 – Materials and Methods, 4.3. Analysis: The description of the GC conditions should be improved. It is tedious for the reader to constantly check the cited reference; please provide a summary instead;

Response

We have revised the Materials and Methods section (4.3. Analysis) to include a concise summary of the GC-MS conditions, as requested, so that readers no longer need to refer to the cited reference for these details.

Comment: 14 – Materials and Methods, 4.4. Compounds Same as described in the previous section.

Response

Thank you for your observation. The summary of the cited reference was already included in section 4.4 after figure 7; therefore, we have not modified its content. However, we have moved the paragraph to appear immediately after the reference to improve readability and make the text less tedious for the reader. This adjustment also ensures greater consistency with section 4.3, where similar changes were implemented to provide a clearer and more user-friendly presentation of the methodology.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

     In my first evaluation of this work, after a list I made of specific suggestions for improvement, I wrote: my Overal Recommendation is "Reconsider after major revision" to be certain that most of my recommendations will be followed given that, although minor, they could impact in the quality of the manuscript if not satisfied. However now, after a careful, deep and point per point revision of the manuscript by authors on the present version-v2, I think that the work deserves an Overall Recommendation of "Accept in the present form".

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have adequately addressed the major concerns raised in the previous round of revision, including correcting internal inconsistencies, adding missing methodological details, and providing additional experimental confirmation regarding vehicle effects. They also improved data presentation. In my view, the revised manuscript is now suitable for publication and will be a great contribution to the field.