Standardization of Diploid Codonopsis laceolata Root Extract as an Anti-Hyperuricemic Source
and Seung-Sik Cho 1,2,*Round 1
Reviewer 1 Report
In this manuscript, Song et al. have presented the analytical methodology for the chemical standardization of C. lanceolata root extracts. As part of the work, the authors prepared solvent extracts and quantified lobetyolin, an extract ingredient with anti-hyperuricemic properties. For the quantitative determination of the tested biomarker, they developed and validated HPLC method with UV detection.
My doubts concern the identification of lobetyolin during the chromatographic analysis. As it is known, lobetyolin has the same UV spectrum as some other polyacetylenes such as lobetyolinine. These substances may coelute on separation by HPLC. The question arises how lobetyolin was identified and co-elution of such compounds excluded. The retention time and the UV spectrum may be insufficient to reliably identify similar compounds.
The aim of this study was to optimize the extraction process, but in this manuscript there is no detailed description of this procedure.
In the Results and Discussion section the authors presented only their results. There is no discussion part presented.
These are my minor comments and questions about the current manuscript:
Title:
line 2: ‘roof’ should be ‘root’
line 2:‘diploid’ – this term is redundant
Introduction:
line 45-46: there is no reference to the sentence ‘In the previous study, we isolated the XO inhibitor lobetyolin from the extract of C. anceolata root’
line 50: there is no citation
line 59: there is no reference of Seol et al. in References section
line 61-62: It is not true. Qiao et al. presented the content of lobetyolin in C. laceolata
line 72: what do you mean by industrialization?
Materials and Methods:
There is no detailed procedure described for sample preparation and extraction. Please, add it.
Why was the standard (lobetyolin) dissolved in the mobile phase and the extracts in methanol? In what solvent were dillutions of standard made?
Please, provide the source of the standard (lobetyolin) used in HPLC analysis (supplier, country)
Table 1: What was the temperature of the chromatography column? Please, add it.
Results and Discussion
Figure 1: The UV spectrum (?) is of poor quality. It cannot be analyzed There is a discrepancy between the wavelength in Figure 1 (280 nm) and in Table 1 (254 nm). Which one is true?
line 154: Lobetyolin was also determined in the water extract. Please, correct it in the Legend.
Conclusions:
line 157: What do you mean ‘chemical profile’?
References:
Numerous mistakes in the References. Please, check them and correct in a proper format.
Author Response
Response to the Reviewers’ Comments-1
In this manuscript, Song et al. have presented the analytical methodology for the chemical standardization of C. lanceolata root extracts. As part of the work, the authors prepared solvent extracts and quantified lobetyolin, an extract ingredient with anti-hyperuricemic properties. For the quantitative determination of the tested biomarker, they developed and validated HPLC method with UV detection.
My doubts concern the identification of lobetyolin during the chromatographic analysis. As it is known, lobetyolin has the same UV spectrum as some other polyacetylenes such as lobetyolinine. These substances may coelute on separation by HPLC. The question arises how lobetyolin was identified and co-elution of such compounds excluded. The retention time and the UV spectrum may be insufficient to reliably identify similar compounds.
The aim of this study was to optimize the extraction process, but in this manuscript there is no detailed description of this procedure.
In the Results and Discussion section the authors presented only their results. There is no discussion part presented.
Response: First of all, we thank the reviewer for the insightful comment. We fulfilled the request and actively utilized the ms form specified by the editor.
This report is about the standardization of C. lanceolata root extracts, as an anti-hyperuricemic source, and establishment of an analysis method. As described in the introduction, we previously reported the anti-hyperuricemic efficacy of lobetyolin.
In addition, C. lanceolata root extracts was successful in clinical trials for hypersensitivity immune response in Korea in 2018(IRB no 2017-09, Semyung univesity Korean hospital).
The clinical efficacy of C. lanceolata root extracts was additionally described in the introduction (2page, highlighted with red color).
We reported additional anti- hyperuricemic efficacy in 2020. Therefore, prior to the development of functional food of C. lanceolata root, an official report on the analysis method for quality control was required and submitted to this journal. Therefore, the application of the results of this study was limited to the quality control of C. lanceolata.
When comparing substances with spectra of similar substances to lobetyolin, as pointed out by the reviewer, it is the case that two or more plant extracts are mixed. In this manuscript, we reported on the validity of the analytical method for quality analysis of lobetyolin in C. lanceolata extract.
For the development of functional materials in Korea, we performed limited solvent selection and analysis feasibility studies for specific materials.
We revised the manuscript faithfully following the reviewer's advice.
We would be greatly appreciated if the reviewer gave their understanding for the derivation of the results of this manuscript.
These are my minor comments and questions about the current manuscript:
Title:
line 2: ‘roof’ should be ‘root’
Response: Thank you for your kind advice. we have changed typos
line 2:‘diploid’ – this term is redundant
Response: Thank you for your kind advice. The term diploid was used because the supplier produced as diploid and extracted it. Our raw materials are not harvested from the wild.
Introduction:
line 45-46: there is no reference to the sentence ‘In the previous study, we isolated the XO inhibitor lobetyolin from the extract of C. anceolata root’
Response: Thank you for your kind advice. We added the reference.
line 50: there is no citation
Response: Thank you for your kind advice. We added the reference.
line 59: there is no reference of Seol et al. in References section
Response: Thank you for your kind advice. We added the reference.
line 61-62: It is not true. Qiao et al. presented the content of lobetyolin in C. laceolata
Response: Thank you for your kind advice. We added the missing report and reference.
line 72: what do you mean by industrialization?
Response: Thank you for your kind advice. We change the in terms of development as functional food source
Materials and Methods:
There is no detailed procedure described for sample preparation and extraction. Please, add it.
Why was the standard (lobetyolin) dissolved in the mobile phase and the extracts in methanol? In what solvent were dillutions of standard made?
Please, provide the source of the standard (lobetyolin) used in HPLC analysis (supplier, country)
Response: According to the reviewer’s advice, we have corrected typos and described the source of standards.
Table 1: What was the temperature of the chromatography column? Please, add it.
Response: According to the reviewer’s advice, we added temperature in Table 1
Results and Discussion
Figure 1: The UV spectrum (?) is of poor quality. It cannot be analyzed There is a discrepancy between the wavelength in Figure 1 (280 nm) and in Table 1 (254 nm). Which one is true?
Response: According to the reviewers advice, we have corrected typos in table 1
line 154: Lobetyolin was also determined in the water extract. Please, correct it in the Legend.
Response: According to the reviewers advice, we have corrected typos in table 5
Conclusions:
line 157: What do you mean ‘chemical profile’?
Response: According to the reviewer’s advice, we have corrected typos as lobetyolin contents
References:
Numerous mistakes in the References. Please, check them and correct in a proper format.
Response: Thank you for your kind advice. We checked and corrected the ref as MDPI styles.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The subject of the publication is interesting, but the content of the work does not correspond to the title of the publication. The authors mainly present the results of the determination of lobetyolin frm C. lanceolata by HPLC. They don't show how they got to the chromatographic assay conditions. How they optimized the procedure. Much of the work is validation, which is not a major topic and is rather a routine activity. However, the extraction method is also treated superficially. Two solvents (water, ethanol) under some conditions (without details how these conditions were selected). Work from a scientific point of view very poor.
Author Response
Response to the Reviewers’ Comments-2
The subject of the publication is interesting, but the content of the work does not correspond to the title of the publication. The authors mainly present the results of the determination of lobetyolin frm C. lanceolata by HPLC. They don't show how they got to the chromatographic assay conditions. How they optimized the procedure. Much of the work is validation, which is not a major topic and is rather a routine activity. However, the extraction method is also treated superficially. Two solvents (water, ethanol) under some conditions (without details how these conditions were selected). Work from a scientific point of view very poor.
Response: First of all, we thank the reviewer for the insightful comment. We fulfilled the request and actively utilized the ms form specified by the editor.
This report is about the standardization of C. lanceolata root extracts, as an anti-hyperuricemic source, and establishment of an analysis method. As described in the introduction, we previously reported the anti-hyperuricemic efficacy of lobetyolin.
In addition, C. lanceolata root extracts was successful in clinical trials for hypersensitivity immune response in Korea in 2018(IRB no 2017-09, Semyung univesity Korean hospital).
The clinical efficacy of C. lanceolata root extracts was additionally described in the introduction (2page, highlighted with red color).
We reported additional anti- hyperuricemic efficacy in 2020. Therefore, prior to the development of functional food of C. lanceolata root, an official report on the analysis method for quality control was required and submitted to this journal. Therefore, the application of the results of this study was limited to the quality control of C. lanceolata.
The method of deriving the analysis method and whether water/ethanol is selected are briefly described in Materials and Methods (3page, highlighted with red color). Our manuscript was submitted in the form of Note. In the note, we have summarized only the important results we need.
In the form of extracts from health functional foods in Korea, water/ethanol solvents are usually used.
This is because traditionally, plant extracts are hot water extracts or extracts using alcohol to improve health functions or for therapeutic purposes. In addition, if other organic solvents are used as extraction solvents, there is a risk of toxicity and residue, thus water and ethanol were used to extract the plant materials. For the development of functional materials in Korea, we performed limited solvent selection and analysis feasibility studies for specific materials (2page, highlighted with red color).
We revised the manuscript faithfully following the reviewer's advice.
We would be greatly appreciated if the reviewer gave their understanding for the derivation of the results of revised manuscript.
Author Response File: Author Response.docx
Reviewer 3 Report
English language must be reviewed
The title does not reflect the arcticle content: nothing to do with ant-hyperuricemic activity. No information about this activity is given or discussed
Results do not support the conclusions: it's only an (adequate) method validation which does not imply the proper pharmacological effect of the species
Authors should include a figure or description of the chemical structure of lobetylin, as well as the obtained chromatograms for the different plant extracts
Purity and characteristics for lobetyolin as a standard compound are lacking (supplier...)
Table 5 should express the units in which lobetyolin content is measured
Comments for author File: 
Comments.pdf
Author Response
Response to the Reviewers’ Comments-3
Response: First of all, we thank the reviewer for the insightful comment. We fulfilled the request and actively utilized the ms form specified by the editor.
- The title does not reflect the arcticle content: nothing to do with ant-hyperuricemic activity. No information about this activity is given or discussed. Results do not support the conclusions: it's only an (adequate) method validation which does not imply the proper pharmacological effect of the species
Response: We thank the reviewer for the insightful points.
This report is about the standardization of C. lanceolata root extracts, as an anti-hyperuricemic source, and establishment of an analysis method. As described in the introduction, we previously reported the anti-hyperuricemic efficacy of lobetyolin (please see page 2)
In addition, C. lanceolata root extracts was successful in clinical trials for hypersensitivity immune response in Korea in 2018(IRB no 2017-09, Semyung univesity Korean hospital). The clinical efficacy of C. lanceolata root extracts was additionally described in the introduction (page 2, highlighted with red color).
We reported additional anti- hyperuricemic efficacy in 2020. Therefore, prior to the development of functional food of C. lanceolata root, an official report on the analysis method for quality control was required and submitted to this journal. Therefore, the application of the results of this study was limited to the quality control of C. lanceolata.
- Authors should include a figure or description of the chemical structure of lobetylin, as well as the obtained chromatograms for the different plant extracts. Purity and characteristics for lobetyolin as a standard compound are lacking (supplier...)
Response: According to the reviewer’s advice, we have corrected typos and described the source of standards (page 3, 2.3 highlighted with red color). Structure of lobetyolin was inserted to Figure 1.
The biological function of lobetyolin was described in the introduction(page 2, with red color).
Our manuscript was submitted in the form of Note. In the note, we have summarized only the important results we need.
- Table 5 should express the units in which lobetyolin content is measured
Response: According to the reviewer’s advice, we corrected the terms of contents.
- We checked the pdf file and corrected all the points that reviewers pointed out.
- We revised the manuscript faithfully following the reviewer's advice. However, we could not include all chromatograms for each extract. Recently, it is difficult to recover previous experimental data by replacing the equipment (HPLC) we have. Through the revised Figure 1 and other analytical validity data, it is expected that the reader will have no difficulty in understanding our results.
We would be greatly appreciated if the reviewer gave their understanding for the derivation of the results of revised manuscript.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
The authors did not respond satisfactorily to my question: how co-elution of compounds like lobetyolinine (with the same UV specra as tested lobetyolin) were excluded in C. lanceolata extract. The retention time and the UV spectrum may be insufficient to reliably identify similar compounds.
I accept the remaining responses to the review and the corrections made to the text.
Author Response
Response: First of all, we thank the reviewer for the insightful comment. As in Reference 20, Qiao et al. reported the quantitative results of lobetyolin among Codonopsis species. According to the additive data of this study, lobetyolonin and lobetyol, which are lobetyollin analogues, have similar UV spectra but have different retention times. Therefore, there is no need to consider about overlapping detection with an derivatives of lobetyolin.
Author Response File: Author Response.docx
Reviewer 2 Report
After receiving the response to the previous review and re-review, I accept the work for publication in Processes.
Author Response
We are very thanksful to the reviewers positive responses to our manuscript and approval for publication.
