Foliar Application of Salicylic Acid Stimulates Phenolic Compound Accumulation and Antioxidant Potential in Saposhnikovia divaricata Herb
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsDear Author,
This study addresses the ecological crisis caused by excessive root harvesting of Saposhnikovia divaricata by proposing a strategy of using the aboveground parts as an alternative to the roots, which has ecological protection and economic value. The experimental design investigated the effects of foliar application of salicylic acid (SA) on the accumulation of phenolic compounds and antioxidant potential in the aboveground parts of Saposhnikovia divaricata. Three geographical provenances (Siberia, Far East, and Mongolia) were included to evaluate the differences in SA responses due to genetic background. Five SA concentration gradients (0.0 - 2.0 mM) were set, and LC-MS/MS was used to comprehensively analyze the metabolites, identifying 48 compounds (38 of which were newly reported). However, there are still some key issues that need to be revised, as detailed below:
1. Introduction: The description of the ecological crisis of Saposhnikovia divaricata can be appropriately condensed, with a focus on highlighting the research innovation points (such as the lack of research on the aboveground parts and the insufficient analysis of metabolites).
2. Materials and Methods:
(1) It is not clearly stated how cross-contamination was avoided during SA treatment, and there is a lack of isolation measures between different treatment groups in the field experiment.
(2) Line 118-119, "full flowering stage" should be specified with a specific developmental stage, such as the percentage of flowering. All flowers cannot bloom simultaneously, and this period should be explained.
(3) It is suggested to refer to 0.0 mM SA as CK and other concentrations as treatment groups.
3. Results and Discussion:
(1) The "n" values in Table 1 are inconsistent (e.g., n = 15 for the 0.0 mM group and n = 17 - 20 for other groups). The reason should be explained (e.g., whether abnormal samples were excluded?).
(2) The Siberian provenance (Goryachinsk) showed the strongest response to SA (total phenols +91.4%), while the Mongolian provenance (Argalan) only increased by +29.6%. The genetic background (such as differences in key enzyme expression) should be discussed to determine whether it affects the efficiency of phenolic biosynthesis.
(3) The volatile oil content of all varieties decreased with increasing SA (Table 1). Literature should be cited to support the antagonistic relationship between SA and the terpene pathway (such as competition for precursor substances).
(4) The 38 newly discovered compounds by LC-MS (such as acylated flavonoid glycosides) should be related to antioxidant activity. It is suggested to correlate their structures with activity (such as the number of phenolic hydroxyl groups and glycosylation positions).
(5) Please check the "Found in group with SA level" column in Table 2. Some compounds in the 0.0 mM group are marked as "+" (e.g., compounds 3 and 9), but the text states that only 17 compounds were detected in the untreated group (Page 7). Consistency should be verified.
(6) The y-axis unit of Figure 3 should be labeled (e.g., mAU).
4. References:
The year format of Ref. 9, 11, and 15 is bolded. Please modify the format according to the journal requirements in detail.
Date: July 17, 2025
Author Response
Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.
Comments 1: Introduction: The description of the ecological crisis of Saposhnikovia divaricata can be appropriately condensed, with a focus on highlighting the research innovation points (such as the lack of research on the aboveground parts and the insufficient analysis of metabolites).
Response 1: Thank you for pointing this out. Description of problems associated with irrational collection and insufficient study are already included in the text of the manuscript. “Among the species with rapidly shrinking natural habitats owing to unsustainable collection is Saposhnikovia divaricata. Because the plant does not produce creeping or adventitious roots to regenerate new individuals, digging up the roots without following sustainable harvesting practices causes rapid population decline and can lead to local extinction. Such activity has already caused a decline of over 60% in natural populations of S. divaricata in the Baikal region, highlighting the urgent need to explore the possibility of harvesting the above-ground parts while preserving the roots. … Specifically, the roots have been found to contain approximately 200 compounds and exhibit more than 20 different types of biological activities [12]. In contrast, only a few chromones [13], flavonoids, and caffeoylquinic acids [14] have been identified in the herb, and no data are available regarding its biological activity.” This information appears to be sufficient to characterize the relevance of the study.
Comments 2: Materials and Methods: It is not clearly stated how cross-contamination was avoided during SA treatment, and there is a lack of isolation measures between different treatment groups in the field experiment.
Response 2: Thank you for pointing this out. This is a logical observation. Experimental fields for different levels of SA were located at least 50 m apart to avoid cross-contamination and wind transfer of the elicitor. Changes have been made to the text.
Comments 3: Materials and Methods: Line 118-119, "full flowering stage" should be specified with a specific developmental stage, such as the percentage of flowering. All flowers cannot bloom simultaneously, and this period should be explained.
Response 3: Thank you for pointing this out. Yes, that's a fair point. Full flowering stage was defined as >80 percentage of flowering. Changes have been made to the text.
Comments 4: Materials and Methods: It is suggested to refer to 0.0 mM SA as CK and other concentrations as treatment groups.
Response 4: Thank you for pointing this out. We partly understand this remark and mention that treatment level 0 is the control group. “Control plants were sprayed with water containing 0.1% MeOH only (0.0 mM SA).” – lines 120-121. But we would like to achieve uniformity in the designation of groups, so we chose this description option. To be fair, this decision may be changed if the editor/s feels that your formatting method is better.
Comments 5: Results and Discussion: The "n" values in Table 1 are inconsistent (e.g., n = 15 for the 0.0 mM group and n = 17 - 20 for other groups). The reason should be explained (e.g., whether abnormal samples were excluded?).
Response 5: Thank you for pointing this out. Yes, indeed, the number of samples in the groups was different, which is due to their selection before analysis. Section 2.2 indicates that the total number of samples in a single group was 40. Some of the plants were rejected due to inappropriate size, color, and stage of development. This is not a laboratory experiment, but a field experiment, so the likelihood of the emergence of individuals that do not meet the requirements of the experiment increases. To avoid additional questions, this information can be excluded from the manuscript (we wanted to present the most truthful version of the results).
Comments 6: Results and Discussion: The Siberian provenance (Goryachinsk) showed the strongest response to SA (total phenols +91.4%), while the Mongolian provenance (Argalan) only increased by +29.6%. The genetic background (such as differences in key enzyme expression) should be discussed to determine whether it affects the efficiency of phenolic biosynthesis.
Response 6: Thank you for pointing this out. That's right, these studies are already being conducted at the present time. We study differences in the activity of key enzymes involved in the biosynthesis of phenolic and terpene compounds. It is not possible to fit this information into the framework of this manuscript. This was not intended to be the purpose of this work.
Comments 7: Results and Discussion: The volatile oil content of all varieties decreased with increasing SA (Table 1). Literature should be cited to support the antagonistic relationship between SA and the terpene pathway (such as competition for precursor substances).
Response 7: Thank you for pointing this out. This statement already has experimental confirmation, but as stated earlier, this is the topic of a separate message. The manuscript focuses on the possibility of using the herb as an alternative source of bioactive compounds, so including information on biosynthesis would not be entirely appropriate in our opinion.
Comments 8: Results and Discussion: The 38 newly discovered compounds by LC-MS (such as acylated flavonoid glycosides) should be related to antioxidant activity. It is suggested to correlate their structures with activity (such as the number of phenolic hydroxyl groups and glycosylation positions).
Response 8: Thank you for pointing this out. You are absolutely right; the structure of the identified compounds influences antioxidant activity. This was confirmed by data from studies with pure substances, as well as by the results of HPLC with pre-column sample derivatization with a DPPH radical (like in our previous works https://doi.org/10.3390/molecules181114105, https://doi.org/10.3390/molecules191118296, https://doi.org/10.3390/molecules22010016, etc.). This study was combined with the results of the study of the structure of new flavonoids and submitted to the publisher.
Comments 9: Results and Discussion: Please check the "Found in group with SA level" column in Table 2. Some compounds in the 0.0 mM group are marked as "+" (e.g., compounds 3 and 9), but the text states that only 17 compounds were detected in the untreated group (Page 7). Consistency should be verified.
Response 9: Thank you for pointing this out. (facepalm) Of course, this is an incredible mistake due to carelessness. The authors were unable to correctly count the 16 crosses. Thank you very much for your attention (shame on us). Changes have been made to the text.
Comments 10: Results and Discussion: The y-axis unit of Figure 3 should be labeled (e.g., mAU).
Response 10: Thank you for pointing this out. Changes have been made to the figure.
Comments 11: References: The year format of Ref. 9, 11, and 15 is bolded. Please modify the format according to the journal requirements in detail.
Response 11: Thank you for pointing this out. Changes have been made to the reference section.
Reviewer 2 Report
Comments and Suggestions for AuthorsCompliments on an excellent study.
There are only a minor issues to be solved.
Line 17: Please rephrase to provide a logical claim. In its current form, it says that even 0.0 mM positively influenced phenolic production.
Line 27-29: 'This could potentially reduce 'the use of S. divaricata roots by partially substituting them in medical applications, thereby helping to preserve natural populations of this valuable plant.'. No, please rephrase. SA application will not potentially reduce the use itself, but the overexploitation of natural populations.
Regarding the natural populations, what I see missing in the introduction and the discussion section is the mention of how to preserve those natural populations. Are there any conventional propagation methods, some conservation programs, or restoration activities? Are there any protocols for its rapid multiplication?
The Materials and methods are very well explained and detailed. What I see missing here is an explanation regarding the usage of reference analytical standards for each of the detected compounds. Did you purchase a standard for each of them? If not, did you use some kind of re-calculation to quantify the compounds and then express the micrograms per milliliter?
Results are very clear and comprehensible. Compliments on that.
In the discussion section, after line 423, please add a broader paragraph regarding the application of your results. Please have in mind a very broad audience of the Horticulturae Journal.
Please provide some practical guidelines for producers. How can they benefit from your results? Is it economically viable, having in mind the price of SA?
Otherwise, the paper is excellent.
Author Response
Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.
Comments 1: Line 17: Please rephrase to provide a logical claim. In its current form, it says that even 0.0 mM positively influenced phenolic production.
Response 1: Thank you for pointing this out. We agree with this comment. Original version – “A field study showed that foliar application of SA at concentrations ranging from 0.0 to 2.0 mM on one-year-old S. divaricata positively influenced the total phenolic content in the herb”. Corrected version – “A field study showed that foliar application of SA on one-year-old S. divaricata positively influenced the total phenolic content in the herb”. You are absolutely right. The original version leads to an incorrect interpretation of the meaning.
Comments 2: Line 27-29: 'This could potentially reduce 'the use of S. divaricata roots by partially substituting them in medical applications, thereby helping to preserve natural populations of this valuable plant.'. No, please rephrase. SA application will not potentially reduce the use itself, but the overexploitation of natural populations.
Response 2: Thank you for pointing this out. We agree with this comment. Original version – “This could potentially reduce the use of S. divaricata roots by partially substituting them in medical applications, thereby helping to preserve natural populations of this valuable plant.” Corrected version – “This could potentially reduce the overexploitation of natural populations of S. divaricata helping to preserve this valuable plant.” You are absolutely right. The original version leads to an incorrect interpretation of the meaning.
Comments 3: Regarding the natural populations, what I see missing in the introduction and the discussion section is the mention of how to preserve those natural populations. Are there any conventional propagation methods, some conservation programs, or restoration activities? Are there any protocols for its rapid multiplication?
Response 3: Thank you for pointing this out. In our opinion, this would make sense if we were talking about plants for which conservation measures have already been developed to preserve natural populations. So, to speak, so as not to be unfounded and to talk about real and effective ways of preservation. There are no effective methods for S. divaricata conservation yet, and work on studying the extent of damage to natural populations has only just begun. Therefore, it is not possible to provide such information or even speculate on possible solutions to the problem.
Comments 4: The Materials and methods are very well explained and detailed. What I see missing here is an explanation regarding the usage of reference analytical standards for each of the detected compounds. Did you purchase a standard for each of them? If not, did you use some kind of re-calculation to quantify the compounds and then express the micrograms per milliliter?
Response 4: Thank you for pointing this out. We agree with this comment. I can’t imagine how this information was left out of the materials. Two tables were added to Supplementary materials – Table S1. Reference standards used for HPLC profiling and quantification; Table S2. Regression equations, correlation coefficients, standard deviation, limits of detection, limits of quantification and linear ranges for reference standards.
Comments 5: In the discussion section, after line 423, please add a broader paragraph regarding the application of your results. Please have in mind a very broad audience of the Horticulturae Journal.
Response 5: Thank you for pointing this out. As for practical recommendations, one should be careful, as large-scale practical results in this area is needed. All we can say is that “The results of this study have the potential for the practical application both in medical plant agriculture and in rational natural management. Producers who are already cultivating S. divaricata may be useful to consider about using salicylic acid elicitation on their plantations to improve the quality of their crops and increase the economic benefit of a higher quality harvest. This would not only increase the economic benefit from the higher quality of the marketed crop, but could theoretically reduce the pressure on natural populations as a result of more goods being available on the market. Preliminary field trials are needed to confirm these findings, but the known effectiveness of salicylic acid in improving crop quality may help convince farmers to use it.” But in our opinion, it is still too early to draw even such a conclusion.
Comments 6: Please provide some practical guidelines for producers. How can they benefit from your results? Is it economically viable, having in mind the price of SA?
Response 6: Thank you for pointing this out. As for the development of practical guidelines for producers, we understand the practical importance of such recommendations, but in this case, we are faced with some difficult moments. Firstly, despite the positive results for which we are confident, it is necessary to understand that practical recommendations must be based on more practical experience. Secondly, it is very reckless to issue a guideline without its approval on the scale of a large region or even a country. If these studies are supported by big money, you will subsequently see proven recommendations in the open press. As for the cost-effectiveness of using salicylic acid, the following can be said. The average cost of 1 kg of salicylic acid suitable for use in agriculture is about $10. This sample will be sufficient to prepare approximately 7150 L of 1 mM solution. Considering that the average consumption of the solution is about 60 ml per plant, the resulting volume will be sufficient to treat about 120,000 plants. It is effective to grow no more than 2 plants per 1 m2 (due to the size of their above-ground part, which takes up a large space), which corresponds to 6 hectares. It turns out that the processing costs (of course, excluding the costs of mechanized labor and equipment depreciation) will amount to about $10. We understand that these calculations are very approximate and the actual situation may differ slightly, but we are confident that the costs will not increase more than 2-3 times (which even in this case will be an insignificant amount).
Reviewer 3 Report
Comments and Suggestions for AuthorsAuthors of the manuscript "Foliar Application of Salicylic Acid Stimulates Phenolic Compound Accumulation and Antioxidant Potential in Saposhnikovia divaricata Herb" presented a study on the effect of foliar spraying with salicylic acid on the accumulation of polyphenolic compounds in Saposhnikovia divaricate. The article raises an important issue related to the protection of biodiversity of wild plants with high medical potential. In general, the research material is well described, but before official publication it requires some correction and additions, especially when it comes to methodology and discussion. Details of the proposed changes are included in the attached file.
Comments for author File: 
Comments.pdf
Author Response
Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.
Comments 1: Section 2.2. Where and when were the seedlings planted and in what conditions did they grow?
Response 1: Thank you for pointing this out. Seedlings were planted on the territory of Experimental Plantation Site No. 07-7j (Mukhorshibir, Republic of Buryatia, Russia; 51°02’46.4’’ N, 107°46’52.7’’E, 830 m a.s.l.) without fertilizer application, with water supplied by an automatic drip irrigation system GWB3240 (ELGO, Caesarea, Israel). Text was corrected.
Comments 2: Section 2.3. Briefly outline the analytical procedure and literature for analysis.
Response 2: Thank you for pointing this out. But I don't quite understand how I should refer to the manual in case of Total Phenolic Content Assay Kit (Zen-Bio, Inc., Durham, UK; cat. No AOX-17) used https://www.zen-bio.com/pdf/cell-manuals/ZBM0126.pdf and Polysaccharide Content Assay Kit (Profacgen, Shirley, NY, USA; cat. No CASQS-K067M) used https://www.profacgen.com/polysaccharide-content-assay-kit-phenol-sulfuric-acid-method-1823.htm. Regarding the method of essential oil analysis, should I attach the diagram of the apparatus? (see fig. below).
Comments 3: Section 2.4. What device was used for vacuum drying?
Response 3: Thank you for pointing this out. Extracts were dried under vacuum using CombiDancer II vacuum evaporator (Hettich AG, Bäch, Swit-zerland) until completely dry. Text was corrected.
Comments 4: Section 2.5. Provide reference substances
Response 4: Thank you for pointing this out. Two tables were added to Supplementary materials – Table S1. Reference standards used for HPLC profiling and quantification; Table S2. Regression equations, correlation coefficients, standard deviation, limits of detection, limits of quantification and linear ranges for reference standards.
Comments 5: Section 2.6. Briefly describe the methodology and equipment used.
Response 5: Thank you for pointing this out. Text was corrected.
Comments 6: Section 2.7. What statistical calculation program was used?
Response 6: Thank you for pointing this out. Text was corrected.
Comments 7: In the discussion, the authors should address the cause of changes in polyphenolic compounds. Although the paper is not biochemical, it should include mention of the metabolic causes of such changes.
Response 7: Thank you for pointing this out. We understand the reviewer's request, but we have an opinion on this matter. The main problem with such reasoning without providing experimental evidence is its incorrectness. It is traditionally believed that the use of elicitors affects the entire chemical composition of the plant and usually leads to an increase in the level of all compounds. Well, at least that is the impression one gets when reading most research articles. In our case, during the start studies it was found that phenolic compounds actually react by increasing their concentrations, while essential oils decrease and polysaccharides show virtually no reaction. Is it possible to approach the explanation of the response of phenolic compounds to the elicitor only based on the previous experience of other researchers? Of course not, since the plant is not quite a typical representative of the Apiaceae family and probably has another biochemical feature under the influence of salicylic acid. We would not like to spread a false opinion that will be published in the Horticulturae journal and may influence the views of other researchers.
Author Response File: Author Response.pdf
