Review Reports - Changes in Active Components, Antioxidant Activity and Alcohol Dehydrogenase Activity of Penthorum chinense Pursh at Different Harvest Times

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

I consider that the manuscript attempts to establishing optimal harvest times for Penthorum chinense in the Gulin-Luzhou region for flowers, leaves and stems, by correlating developmental stages with phytochemical peaks and antioxidant activity with excellent results. However, some observations are made that could contribute to improve the manuscript.

Title

Line 3. Penthorum chinense should be in italic.

Introduction

Lines 33. Penthorum should be in italic, because it's referring to genus.

Line 34. Check the family where this species is located, because according to POWO (Plants of the World Online) it belongs to the Penthoraceae family.

Lines 60-62. Revise the sentence for a better understanding.

Line 77. There is quite a large space before the beginning of Materials and Methods.

Materials and Methods

Lines 80-86. If we take into account that the compounds present in P. chinense tend to vary with the environment, do you consider that taking the samples in a single year is sufficient to obtain consistent results? Explain. I think that at least the temperatures and rainfall should be mentioned during the collection period.

Lines 80-86. The number of samples taken at each time point should be mentioned.

Avoid starting sentences with numbers.

Results

Line 225. Table 1 should be moved to the next page.

Line 243. Consider use loci in italics.

Line 224. July 5 should also be considered among the days with the highest dry mass.

Line 250. Avoid drawing conclusions in this section and leave it for discussion. Extent to the rest of this chapter.

Line 252. The name of the X-axis is missing from Table 1. The same for Table 2.

Conclusions

Line 432-436. I consider it is important that this sentence is developed within the discussion of the article and not in the conclusions.

Author Response

Detailed response to reviewers

Dear Authors,

Comment 1: Line 3. Penthorum chinense should be in italic.

Response: We revised the Penthorum chinense in Line 3.

Comment 2: Lines 33. Penthorum should be in italic, because it's referring to genus.

Response: We revised the Penthorum chinense in Line 33.

Comment 3: Line 34. Check the family where this species is located, because according to POWO (Plants of the World Online) it belongs to the Penthoraceae family.

Response: We have checked the family and revised it as “Penthoraceae”.

Comment 4: Lines 60-62. Revise the sentence for a better understanding.

Response: We have revised the sentence for the better understanding.

Comment 5: Line 77. There is quite a large space before the beginning of Materials and Methods.

Response: We deleted the space.

Comment 6: Lines 80-86. If we take into account that the compounds present in P. chinense tend to vary with the environment, do you consider that taking the samples in a single year is sufficient to obtain consistent results? Explain. I think that at least the temperatures and rainfall should be mentioned during the collection period.

Response: Thank you for the comment. We have now added temperature and rainfall data for the collection period in the revised manuscript (lines 81–82) to provide environmental context; we agree that multi-year sampling would further strengthen the findings, and we have noted this as a consideration in the Conclusion and Outlook.

Comment 7: Lines 80-86. The number of samples taken at each time point should be mentioned.

Response: We added the weight of samples taken at each time point.

Comment 8: Avoid starting sentences with numbers.

Response: We revised the relevant sentences.

Comment 9: Line 225. Table 1 should be moved to the next page.

Response: Table 1 have been moved to the next page.

Comment 10: Line 243. Consider use loci in italics.

Response: We revised it.

Comment 11: Line 224. July 5 should also be considered among the days with the highest dry mass.

Response: We revised the sentence more clearly.

Comment 12: Line 250. Avoid drawing conclusions in this section and leave it for discussion. Extent to the rest of this chapter.

Response: We deleted the relevant conclusions in this section.

Comment 12: Line 252. The name of the X-axis is missing from Table 1. The same for Table 2.

Response: We added the name of the X-ax in Table 1 and 2.

Comment 13: Line 432-436. I consider it is important that this sentence is developed within the discussion of the article and not in the conclusions.

Response: Thank you for the comment. We agree that these future perspectives are better elaborated in the Discussion. In fact, the influences of environmental factors on compound synthesis and the economic validation of the harvesting strategy have already been discussed in detail in the Discussion section (lines 351-357). The sentence in the Conclusions has been retained only as a brief summary.

Reviewer 2 Report

Comments and Suggestions for Authors

Although this manuscript provides some interesting scientific results, several deficiencies should be addressed.

Please clearly state the purpose of this paper.

Please clearly state which components of this plant are important. Please clearly state the basis for your judgment that the quality has improved. Do the differences in components shown here between the flowering stage and the early stages of fruiting affect the expected effects?

What are the characteristic components of this plant? Most of the components being studied appear to be known compounds.

There are multiple reports in the introduction, but please explain how this plant is actually used. Which part of the plant is used?

It is clear that the components vary depending on the growth stage. It is also clear that various factors affect plant growth. As this is an international journal, please write in a way that can be understood and used by researchers worldwide. Papers that are limited to a specific region will not be referenced by other researchers.

Author Response

Detailed response to reviewers

Reviewer #2:

Although this manuscript provides some interesting scientific results, several deficiencies should be addressed.

Comment 1: Please clearly state the purpose of this paper.

Response: We have revised the abstract and introduction to clearly state the purpose of this study: to establish a time-resolved harvesting standard for Penthorum chinense by integrating growth phenology, phytochemical accumulation dynamics, and bioactivity across six developmental stages.

Comment 2: Please clearly state which components of this plant are important. Please clearly state the basis for your judgment that the quality has improved. Do the differences in components shown here between the flowering stage and the early stages of fruiting affect the expected effects?

Response: Thank you for these important questions. The important components of Penthorum chinense are polyphenolic compounds, which are the main bioactive constituents. Therefore, total phenolics, flavonoids, proanthocyanidins, and tannins, which are well-documented as the major bioactive constituents responsible for its hepatoprotective and antioxidant effects. Among these, pinocembrin-7-O-glucoside, PGHG, and thonningianin A are present at relatively high contents. Furthermore, our ongoing animal study has identified thonningianin A as a key hepatoprotective component. Therefore, total phenolic content serves as the most important indicator for quality evaluation.

Quality improvement was assessed based on simultaneous increases in bioactive component contents and antioxidant activity. Higher levels of these components at specific harvest times indicate superior medicinal material quality.

The optimal harvest times differ by plant part: flowers at full blooming stage (July 20) and leaves/stems at early fruiting stage (August 18) . Harvesting at these times maximizes the accumulation of bioactive components, thereby potentially enhancing the expected hepatoprotective and antioxidant effects. Harvesting at suboptimal stages may result in lower bioactive content and reduced efficacy.

Comment 3: What are the characteristic components of this plant? Most of the components being studied appear to be known compounds.

Response: Thank you for the comment. The characteristic components of Penthorum chinense Pursh were identified by high-resolution mass spectrometry in our preliminary study. Based on these results and literature reports, the major bioactive constituents were selected for quantitative analysis. Although these compounds are known, they represent the characteristic and active components of this plant, and their simultaneous quantification is essential for quality control.

Comment 4: There are multiple reports in the introduction, but please explain how this plant is actually used. Which part of the plant is used?

Response: Thank you for this comment. In response, we have now added the relevant section in the Introduction.

Comment 5: It is clear that the components vary depending on the growth stage. It is also clear that various factors affect plant growth. As this is an international journal, please write in a way that can be understood and used by researchers worldwide. Papers that are limited to a specific region will not be referenced by other researchers.

Response: We thank the reviewer for this insightful comment. We agree that growth stage and environmental factors can influence the composition of plant materials, and that a broader perspective is essential for an international readership. Accordingly, we have revised the manuscript to present our findings in a more generalizable context, avoiding regionally restricted interpretations. We believe these revisions enhance the applicability and accessibility of our work for researchers worldwide.

Reviewer 3 Report

Comments and Suggestions for Authors

The introduction section should be supplemented with information justifying the choice of ADH as a parameter in determining the optimal harvesting time.
Testing ADH activity must be justified, as long as the maintaining of enzymatic activity is closely linked to the method applied for plant conditioning, namely at low temperatures. In this work, drying was indeed done by freeze-drying, but if drying is done with hot air (as is done in most cases) the enzyme is denatured and the activity testing is no longer justified.
The abstract provides synthetically information regarding the samples, the methodology and the obtained results. However, the exact dates corresponding to the determined optimal harvesting time should not be mentioned in the abstract because they are closely related to the local pedoclimatic conditions in that year and in the abstract the name of the phenological stage is enough.
Specific comments:
- A little more attention to the writing would increase the value of the work. The substances indices are sometimes written with a subscript, sometimes not (lines 109, 117, 118);
- NaNO₂-Al(NO₃)₃-NaOH is the correct formula in the line 116.

Comments for author File: Comments.pdf

Author Response

Detailed response to reviewers

Reviewer #3:

The timing of plant harvest is very important in terms of productivity, but also quality. To capitalize on plants with therapeutic potential, it is necessary to analyze the moment when the accumulation of bioactive compounds is maximum. The paper reports the research conducted to establish the optimal harvesting time of Penthorum chinense Pursh, a plant used in traditional Chinese medicine for its hepatoprotective effects. Six harvesting dates corresponding to the phenological stages were tested analyzing total phenols, flavonoids, proanthocyanidins, tannins, antioxidant capacity and alcohol dehydrogenase (ADH) activity. The study may be of interest to growers and processors of plants that are used for their potentially curative properties. For the scientific community, it can constitute a basis for the development of more in-depth studies that take into account other determining factors for selecting the optimal harvesting time (soil composition, climatic conditions, cultivation technique, use of fertilizers and plant protection products).

Comment 1: The introduction section should be supplemented with information justifying the choice of ADH as a parameter in determining the optimal harvesting time.

Response: Thank you for this suggestion. We have added a brief justification for selecting ADH as a parameter in the Introduction, noting its relevance to liver function and the traditional hepatoprotective use of P. chinense. The change has been highlighted in the manuscript.

Comment 2: The investigation methodology is adequate to the set objectives. ADH is important for plant survival and when extracted from vegetal matrix may show therapeutic properties for human health. Testing ADH activity must be justified, as long as the maintaining of enzymatic activity is closely linked to the method applied for plant conditioning, namely at low temperatures. In this work, drying was indeed done by freeze-drying, but if drying is done with hot air (as is done in most cases) the enzyme is denatured and the activity testing is no longer justified.

Response: Thank you for your comment. We wish to clarify that our study measures ADH-activating activity of extracts from Penthorum chinense, not the endogenous ADH activity within the plant material. In this in vitro assay, plant extracts are added to an ADH enzyme system to evaluate their activating effects as an indicator of hepatoprotective potential. Therefore, the key concern is the stability of bioactive compounds (e.g., flavonoids), not the preservation of endogenous plant ADH. Freeze-drying was used to protect these thermosensitive constituents, but the assay rationale does not depend on maintaining native enzyme activity in the plant.

Comment 3: The analytical methods were chosen appropriately. The abstract provides synthetically information regarding the samples, the methodology and the obtained results. However, the exact dates corresponding to the determined optimal harvesting time should not be mentioned in the abstract because they are closely related to the local pedoclimatic conditions in that year and in the abstract the name of the phenological stage is enough.

Response: Thank you for this suggestion. We agree that specific harvest dates are tied to local conditions and may limit generalizability. Accordingly, we have revised the abstract to retain only the phenological stages (full blooming stage and early fruiting stage) as indicators of optimal harvest time, removing the exact dates. The changes have been highlighted in the manuscript.

Comment 4: A little more attention to the writing would increase the value of the work. The substances indices are sometimes written with a subscript, sometimes not (lines 109, 117, 118).

Response: Thank you for suggestion and we carefully revised the article.

Comment 5: NaNO₂-Al(NO₃)₃-NaOH is the correct formula in the line 116.

Response: We revised the NaNO₂-Al(NO₃)₃-NaOH.

Reviewer 4 Report

Comments and Suggestions for Authors

The aim of the study - to determine the optimal harvest times for the flowers, leaves, and stems of Penthorum chinense based on analyses of their biologically active components, antioxidant activity, and alcohol dehydrogenase activity, using PCA to interpret the data - was achieved. The study was well-designed and carried out, and the conclusions were presented appropriately.

A weakness of the study is the absence in the Introduction of general information regarding the world-wide distribution of the studied plant and literature data covering the results of previous studies on the biologically active components found in the plant used in traditional folk medicine. Furthermore, what is the current application of Penthorum chinense - which prompted the undertaking of such a comprehensive study as presented in this work.

In the title of the paper, the name of the plant under study (Penthorum chinense) should be written in italics.

The paper uses a large number of abbreviations, the meanings of which must be explained where the abbreviation first appears. This applies to the following abbreviations: GAE, DW, TFC, RE, TPAC, OD, CE, TTC, TAE, DPPH, VE, TE, ABTS, FRAP, ADH, PGHG, KMO, ADH, PAL, C4H, 4CL, MdANS, MdUFGT, MYB, CHS, DFR, ANS.

Please specify the composition of the reagents: line 107 – Folin-Ciocalteu reagent, line 137 – Folin-Denis reagent.

Line 116: The combination NaNO₂-aluminum(NO)₃)₃-NaOH should be written as NaNO₂-Al(NO₃)₃-NaOH

Is it necessary to mention the name of the laboratory equipment manufacturer (BioTek Instruments, Inc., USA) as many as six times?

The text indicates that 14 metabolites were quantitatively analyzed (line 260), whereas Table 2 presents data for only 12 metabolites. Please clarify this issue.

The paper cites 59 references related to the research topic. A large portion of these works were published in recent years. For two references (refs. 10 and 31), the journal title is missing, while for refs. 6, 9, 10, 23, 35, and 47, the journal volume is missing.

Author Response

Detailed response to reviewers

Reviewer #4:

Comment 1: A weakness of the study is the absence in the Introduction of general information regarding the world-wide distribution of the studied plant and literature data covering the results of previous studies on the biologically active components found in the plant used in traditional folk medicine. Furthermore, what is the current application of Penthorum chinense - which prompted the undertaking of such a comprehensive study as presented in this work.

Response: We have expanded the Introduction to include global distribution, previously reported bioactive components, and current applications of P. chinense, thereby strengthening the study rationale (see Introduction section).

Comment 2: In the title of the paper, the name of the plant under study (Penthorum chinense) should be written in italics.

Response: We revised the title (Penthorum chinense) in italics.

Comment 3: The paper uses a large number of abbreviations, the meanings of which must be explained where the abbreviation first appears. This applies to the following abbreviations: GAE, DW, TFC, RE, TPAC, OD, CE, TTC, TAE, DPPH, VE, TE, ABTS, FRAP, ADH, PGHG, KMO, ADH, PAL, C4H, 4CL, MdANS, MdUFGT, MYB, CHS, DFR, ANS.

Response: We have now defined all abbreviations (GAE, DW, TFC, RE, TPAC, OD, CE, TTC, TAE, DPPH, VE, TE, ABTS, FRAP, ADH, PGHG, KMO, PAL, C4H, 4CL, MdANS, MdUFGT, MYB, CHS, DFR, ANS) at their first occurrence in the manuscript.

Comment 4: Please specify the composition of the reagents: line 107 – Folin-Ciocalteu reagent, line 137 – Folin-Denis reagent.

Response: We thank the reviewer for the careful reading and the opportunity to clarify the details of the reagents. Regarding line 107 (Folin-Ciocalteu reagent) and line 137 (Folin-Denis reagent), we have now specified their compositions in the revised manuscript as follows:

Folin-Ciocalteu reagent: This reagent is a commercial mixture composed of sodium tungstate, sodium molybdate, phosphoric acid, hydrochloric acid, and lithium sulfate. It is widely used for the determination of total phenolic content, as it reacts with both phenolic and non-phenolic reducing substances.

Folin-Denis reagent: This reagent is a classic formulation consisting of sodium tungstate, phosphoric acid, and phosphomolybdic acid (or sodium molybdate in some formulations). It is specifically employed for the quantification of tannins and phenolic compounds.

Comment 5: Line 116: The combination NaNO₂-aluminum(NO)₃)₃-NaOH should be written as NaNO₂-Al(NO₃)₃-NaOH.

Response: We revised the relevant words.

Comment 6: Is it necessary to mention the name of the laboratory equipment manufacturer (BioTek Instruments, Inc., USA) as many as six times?

Response: Thank you for your suggestion and deleted the needless equipment manufacturer.

Comment 7: The text indicates that 14 metabolites were quantitatively analyzed (line 260), whereas Table 2 presents data for only 12 metabolites. Please clarify this issue.

Response: We apologize for the inconsistencies. Line 260 has been corrected from “14” to “18” metabolites. Table 2 originally displayed only 12 metabolites due to page width limitations; in the revised manuscript, the table has been reformatted (landscape layout) to present all 18 quantified metabolites completely.

Comment 8: The paper cites 59 references related to the research topic. A large portion of these works were published in recent years. For two references (refs. 10 and 31), the journal title is missing, while for refs. 6, 9, 10, 23, 35, and 47, the journal volume is missing.

Response: We have carefully revised the references as suggested. The missing journal titles for refs. 10 and 31, as well as the missing volume numbers for refs. 6, 9, 10, 23, 35, and 47, have all been added. Thank you for pointing out these omissions.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Comment 1: Please clearly state the purpose of this paper.

>>This paper emphasizes the importance of the quantity of chemical components. On the other hand, when the total amount of components is important, the overall yield should be compared without separating it into stems, leaves, and flowers.

>> The comparison of chemical component amounts is based on dry weight, but this should differ when considering total yield. For example, since leaves yield more than flowers, even if the chemical component amount of flowers is high in dry weight, the total chemical component amount of leaves should be higher. Also, each chemical component is distributed relatively uniformly. It seems unnecessary to divide plants into flowers, leaves, and stems.

Comment 3: What are the characteristic components of this plant? Most of the components being studied appear to be known compounds.

>> If all the components are known compounds, what is the purpose of evaluating this plant as a medicinal plant? Does this plant contain more of the target chemical component compared to other plants? Please clearly state your purpose.

Comment 4: There are multiple reports in the introduction, but please explain how this plant is actually used. Which part of the plant is used?

Response: Thank you for this comment. In response, we have now added the relevant section in the Introduction.

>> Please explain the added parts.

>> Please clearly indicate the corrections using red or another color.

Author Response

Detailed response to reviewers

Comment 1: This paper emphasizes the importance of the quantity of chemical components. On the other hand, when the total amount of components is important, the overall yield should be compared without separating it into stems, leaves, and flowers.

Response: We thank the reviewer for this thoughtful comment, which highlights a critical question regarding the rationale of our experimental design. We appreciate the opportunity to clarify our logic.

The reviewer correctly notes that if the sole objective were to maximize the total chemical yield per plant, a whole-plant analysis would be sufficient. However, the aim of our study is not merely to identify the harvest time that yields the highest total chemical content, but rather to establish a practical, field-applicable harvesting strategy that balances multiple real-world considerations.

Our rationale for separately analyzing stems, leaves, and flowers is threefold:

Alignment with commercial practice.In actual production, Penthorum chinenseis harvested and sold by separate plant parts—stems, leaves, and flowers—because these parts have substantially different market values. Leaves and flowers command higher prices due to their richer bioactive compound profiles, while stems are less valuable. Therefore, a whole-plant analysis would not directly inform the harvesting decisions made by producers, who already separate parts in practice.
Enabling a time‑segmented, organ‑specific harvest strategy.A fundamental challenge in field production is that stems, leaves, and flowers grow together on the same plant, making it impractical to harvest only one part at a single time point without affecting the yield and quality of the remaining parts. By monitoring the accumulation dynamics of each organ across six developmental stages, we can identify the optimal harvest window for each part and propose a sequential harvest schedule(e.g., harvest flowers at early flowering, followed by leaves, then stems) that aligns with natural phenological progression. This approach allows producers to maximize economic returns while maintaining practical field management.
Providing mechanistic insight.Separating the analysis by organ enables us to understand whythe overall quality changes over time—specifically, which organ contributes most to the overall chemical profile at each developmental stage. This mechanistic understanding is essential for developing scientifically grounded harvesting guidelines that can be adapted to different production conditions.

In summary, while whole-plant analysis would address the question of “when is total yield highest,” our organ‑specific approach addresses the more practical and nuanced question of “how to schedule harvests of different parts to maximize both economic value and medicinal quality under real‑world production constraints.” We have clarified this rationale in the revised manuscript (Introduction section).

We sincerely thank the reviewer for pushing us to articulate this logic more clearly, which has substantially strengthened the manuscript.

Comment 2: The comparison of chemical component amounts is based on dry weight, but this should differ when considering total yield. For example, since leaves yield more than flowers, even if the chemical component amount of flowers is high in dry weight, the total chemical component amount of leaves should be higher. Also, each chemical component is distributed relatively uniformly. It seems unnecessary to divide plants into flowers, leaves, and stems.

Response: We thank the reviewer for these comments.

Dry weight content vs. total yield.This study focuses on establishing a quality-basedharvest standard. In medicinal materials, bioactive content per dry weight is the core indicator of quality and market value. We acknowledge that total yield is important for maximizing output, which we have noted as a limitation for future study (Discussion, Lines 359-363).
Uniform distribution of components.Our data show that bioactive components are not uniformly distributedacross plant parts—flowers and leaves contain significantly higher levels than stems. Organ-specific analysis is therefore necessary to provide precise harvest guidance.
Necessity of organ-specific analysis.In practice, stems, leaves, and flowers are harvested and sold separately due to different market values. Separate analysis enables a time‑segmented harvest strategy, allowing each part to be harvested at its optimal quality.

We appreciate the reviewer's comments, which have helped clarify the contributions and limitations of this study.

Comment 3: If all the components are known compounds, what is the purpose of evaluating this plant as a medicinal plant? Does this plant contain more of the target chemical component compared to other plants? Please clearly state your purpose.

Response:

We thank the reviewer for this critical question. The purpose of this study is not to discover new compounds, but to establish a scientifically validated harvest standard for Penthorum chinense, a plant with well-documented traditional use but lacking systematic quality control guidelines.Our rationale is as follows:

Known compounds ≠ established harvest standards.Although the bioactive components of P. chinenseare known, their accumulation dynamics across developmental stages have not been systematically characterized. Without this knowledge, producers lack evidence-based guidance on when to harvest to obtain material with optimal quality. This study fills that gap.
Higher content is not the point.We are not claiming that P. chinensecontains higher levels of these compounds than other plants. Rather, we are addressing a specific practical need: for this particular plant, which is already cultivated and marketed, what is the best harvest time to ensure consistent quality? The value of our study lies in optimizing the production process for an existing medicinal plant, not in comparing it to other species.
Quality control requires quantification.Even for known compounds, simultaneous quantification is essential for establishing quality control standards. This study provides the foundational data needed to develop such standards for P. chinense.

In summary, our purpose is to provide a scientific basis for harvest timing to ensure consistent quality of P. chinense products, addressing a real-world problem faced by producers despite the fact that the compounds are already known.

Comment 4: There are multiple reports in the introduction, but please explain how this plant is actually used. Which part of the plant is used? Thank you for this comment. In response, we have now added the relevant section in the Introduction. Please explain the added parts.

Response: We thank the reviewer for the follow-up question. We added the following sentences to the Introduction to clarify the actual usage and the specific plant parts used: “Penthorum chinense is traditionally used as a whole plant for hepatoprotective purposes. In contemporary practice, the aerial parts—including stems, leaves, and flowers—are harvested and utilized, with leaves and flowers commanding higher market value due to their richer bioactive component profiles.”

We added this after the existing sentence describing the traditional use of aboveground parts. This addition specifies that flowers are also utilized in contemporary practice and highlights the economic distinction among plant parts, which aligns with the rationale for our organ-specific analysis.

Comment 5: Please clearly indicate the corrections using red or another color.

Response: We thank the reviewer for this comment. We have revised the manuscript to present our findings in a more generalizable context for an international readership. The specific revisions are as follows, and we have highlighted them in yellow in the revised manuscript:

In the Introduction: We removed regionally specific statements (e.g., references to Gulin County only) and broadened the description to emphasize that Penthorum chinense is cultivated in multiple regions across China and East Asia.
Throughout the manuscript: We replaced regionally specific terms such as “in Gulin” with more general terms such as “in major production areas” or “under typical cultivation conditions.”

We believe these revisions make the findings more accessible and relevant to researchers worldwide.