Using HPLC–DAD and GC–MS Analysis Isolation and Identification of Anticandida Compounds from Gui Zhen Cao Herbs (Genus Bidens): An Important Chinese Medicinal Formulation
, Saadia Masood 2, Sobia Nisa 3, Abdul Qayyum 4,*, Muhammad Ishaque 1, Khurram Shahzad 5, Waseem Ahmed 6, Zahid Hussain Shah 7, Hameed Alsamadany 8, Seung-Hwan Yang 9 and Gyuhwa Chung 9,*Round 1
Reviewer 1 Report
The comments are in the file attached
Comments for author File: 
Comments.pdf
Author Response
Dated: 8th September 2021
Dear Editor,
Greetings,
Thank you very much for your time and comments regarding our manuscript (molecules-1360969). Our manuscript “Using HPLC-DAD and GC-MS Analysis Isolation and Identification of Anticandida Compounds from GUI ZHEN CAO Herbs (Genus Bidens): An Important Chinese Medicinal Formulation” has been revised carefully and here we are giving our response to the reviewers’ comments. We have improved the manuscript according to the reviewers’ comments and suggestions. All the revisions can be easily identified from manuscript highlighted with yellow color.
Once again thanks for your co-operation and valuable comments and suggestion. Moreover, the efforts of the reviewer are highly appreciated. We are hoping for pleasant response and further good comments (if any) from your side.
Dr. Abdul Qayyum
Department of Agronomy,
The University of Haripur 22620 Pakistan
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We are thankful to editor and reviewers for timely completion of review process and providing us with valuable feedback.
Response to Reviewer # 1
Dear reviewer, we are grateful to you for your comments and suggestions for the improvement of our research manuscript. We have tried our best to revise the manuscript in light of your comments.
Comment 1: The Introduction is not completely exhaustive, in fact it dwells a lot on the potential use of traditional Chinese medicine and Gui Zen Cao with unusual tables for an introduction, but almost nothing is reported on the importance of the treatment against Candida and above all on which bases the authors decided to evaluate the anticandida effect.
Response: These plants are selected on the basis of their ethnomedicinal importance. As you have suggested I have separately added data (Page No. 2; Paragraph 2) about their uses in skin infections, vaginitis and anticandida activity in introduction and also in table. Highlighted with yellow color.
Comment 2: Furthermore, the authors stated that they validated the traditional use against diarrhea (see L. 303-308) but there is no evidence they have carried out any study on the matter.
Response: Candida species have been often considered but infrequently documented as a credible cause of diarrhea. I have also specified on Page No 17; Paragraph No 2 of discussion). Highlighted with yellow color.
https://pubmed.ncbi.nlm.nih.gov/8645834/
https://pubmed.ncbi.nlm.nih.gov/54741/
Comment 3: Furthermore, the introduction specifies the plants that become part of the Gui Zhen Cao, but not their percentages and, above all, which parts of the plants are used.
Response: In available literature the percentages of these plants are not mentioned. However In Chinese-English Manual of Common-Used Herbs, under guizhencao all plant parts of these three Bidens species are docomented to be used. I have added this in introduction Page No 2; Paragraph No 1). Highlighted with yellow color.
Comment 4: I would suggest adding a paragraph in the materials and methods in which to report which parts of the single plants were used (leaves, stem, root, flower and achenes), the methods of collection, drying and treatment before chemical analysis, and the effect evaluation. Furthermore, I would suggest explaining the methods of preparation of the reproductive parts used for the quantification of Phytochemicals.
Response: Added under heading 2.2 of Materials and Methods (Page No. 5). Highlighted with yellow color.
Comment 5: In the Discussion, authors should first discuss the quantity and quality of the chemical compounds detected and any favorable effects. For example, some substances are known to be used as antioxidants: here the authors could highlight these effects and discuss the amount of substance detected and the beneficial effects reported in the literature.
Response: Added on Page No 9 of Results, Paragraph No 1. Highlighted with yellow color.
Comment 6: In L. 336-341 it is highlighted that dehydroabietic acid, present in a multitude of plants, was detected for the first time in the genus Bidens: well, this characteristic could be better exploited assuming also other uses supported by the bibliography. As for the anti-candidacy effect, I would suggest to the authors to enrich the topic through a more in-depth bibliographic study.
Response: This is the first report of dehydroabietic acid from genus bidens. However I have added uses of dehydroabietic acid isolated from other plant species. For anticandida activity, studies has been performed on antifungal activity of this compound which I have added in discussion section (Page No 18; Paragraph No 4) but anticandida effect of dehydroabietic acid has not been explored before. Highlighted with yellow color.
Comment 7: In the Conclusions, in addition to summarizing the findings, it is advisable to talk about future study perspectives: for example, evaluation of the toxicity of isolated substances through cytotoxicity experiments on normal cell cultures, use of two or more substances to evaluate the possible synergistic effect, etc.
Response: Future perspectives are added in conclusion (Page 18). Highlighted with yellow color.
Minor Remarks:
Comment: L. 43 and L. 47: there is a need of references.
Response: Reference added. See Page 2; 1st Paragraph. Highlighted with yellow color. In case of Line 43 references were added with in Table 1.
Comment: L. 53: replace the comma with a full stop.
Response: Replaced.
Comment: L. 74: add “flower” at F and G points.
Response: Added. Page No. 5. Highlighted with yellow color.
Comment: L. 105: something is missing between “with” and “of”. Will you check?
Response: Corrected, Page No. 6. Highlighted with yellow color.
Comment: L. 228: a full stop and “The” are missing.
Response: “Full stop” and “The” added. Page No. 9. Highlighted with yellow color.
Comment: L. 230: please, specify which are the reproductive parts.
Response: Added. Page No. 9; 3rd Paragraph. Highlighted with yellow color.
Comment: L. 241: delete “is”: it is better past tense. Please, change throughout the manuscript.
Response: Changed throughout the manuscript and highlighted with yellow color.
Comment: L. 303-305: please, add reference.
Response: Reference added. Page No. 17. Highlighted with yellow color.
Comment: L. 308: how did you validate the traditional use? (See general comments)
Response: Explained under discussion section. 2nd Paragraph; Page No. 17. Highlighted with yellow color.
Comment: L. 319: add a reference.
Response: Reference added. Page No. 17. Highlighted with yellow color.
Comment: L. 325-326: “anicandida” should be “anticandida”.
Response: Changed. See 2nd Paragraph; Page 18. Highlighted with yellow color.
Comment: L. 343: the authors say: “various studies”, but one reference is reported.
Response: More references are added. See 5th Paragraph. Highlighted with yellow color.
Comment: L. 344: “destroys” should be “destroy”.
Response: Modified as “cause reduction in cell size and disruption in”. See 5th Paragraph; Page 18. Highlighted with yellow color.
Comment: L. 345: “Previous studies”: only one reference is reported.
Response: More references are added. See 5th Paragraph. Highlighted with yellow color.
Reviewer 2 Report
The article deals with the analysis, isolation and identification of antifungal substances from the three components of traditional Chinese medicine Gui Zhen Cao, Biden Pilosa, B. biternata and B. bipinnata. The plant material was analyzed by simple methods for the content of a number of groups of substances (phenolic, tannins, alkaloids, terpenoids, Saponins, oxalate and lipids). In in vitro tests, the antimicrobial effects of various extracts from these plants were tested against several species of Candida. The selected extract was separated by TLC chromatography and its components tested for antimicrobial activity. Two substances identified by mass spectrometry have been declared the major antimicrobial components.
The manuscript contains the results of a large number of experiments with a not always clear goal, the article is quite confusing. Some of the antimicrobial tests mentioned in the results are not adequately described in the methodology.
For example, the results of chemical analyze of polyphenols, alkaloids, etc. are presented. (table 3) in vegetative parts and reproductive parts, antimicrobial tests give results for five different parts of plants (Fig. 5).
Of the 49 fractions of TLC / HPLC (Fig. 6) chromatography, two were selected as the components with the main antimicrobial activity, although at least two others appear to be comparable to "component 2". Do the authors have any literature information on the antimicrobial efficacy of the two substances (linoleic acid, dehydroabietic acid)?
I recommend reworking / supplementing the manuscript so that it is more clear, the aim, design and description of the experiments are more obvious and, if possible, the chemical composition of the extracts used for in vitro antimicrobial tests is supplemented. At the same time, authors should consider more carefully the discussion and evaluation of experimental results.
Author Response
Dated: 8th September 2021
Dear Editor,
Greetings,
Thank you very much for your time and comments regarding our manuscript (molecules-1360969). Our manuscript “Using HPLC-DAD and GC-MS Analysis Isolation and Identification of Anticandida Compounds from GUI ZHEN CAO Herbs (Genus Bidens): An Important Chinese Medicinal Formulation” has been revised carefully and here we are giving our response to the reviewers’ comments. We have improved the manuscript according to the reviewers’ comments and suggestions. All the revisions can be easily identified from manuscript highlighted with yellow color.
Once again thanks for your co-operation and valuable comments and suggestion. Moreover, the efforts of the reviewer are highly appreciated. We are hoping for pleasant response and further good comments (if any) from your side.
Dr. Abdul Qayyum
Department of Agronomy
The University of Haripur 22620 Pakistan
***************************************************************************
We are thankful to editor and reviewers for timely completion of review process and providing us with valuable feedback.
Response to Reviewer # 2:
Comment: The manuscript contains the results of a large number of experiments with a not always clear goal, the article is quite confusing. Some of the antimicrobial tests mentioned in the results are not adequately described in the methodology.
For example, the results of chemical analyze of polyphenols, alkaloids, etc. are presented. (Table 3) in vegetative parts and reproductive parts, antimicrobial tests give results for five different parts of plants (Fig. 5).
Response: In the present study quantitative phytochemical analysis has been carried out on its re-productive and vegetative parts in order to identify the class of phytochemicals responsible for its traditional uses. Whereas antimicrobial tests of five different parts were conducted target the compound responsible for anticandida activity.
Meanwhile I have tried to clear the goals of present article (Page No. 2; Last 4 Lines of 2nd Paragraph). Highlighted with yellow color.
Comment: Of the 49 fractions of TLC / HPLC (Fig. 6) chromatography, two were selected as the components with the main antimicrobial activity, although at least two others appear to be comparable to "component 2".
Response: The other two peaks that are appeared to be comparable with component 2 were appeared to be impure after isolation that’s why they were not tested further.
Comment: Do the authors have any literature information on the antimicrobial efficacy of the two substances (linoleic acid, dehydroabietic acid)?
Response: The available literature on antimicrobial activity of linoleic acid and dehydroabietic acid is added in discussion (2nd Last paragraph before conclusion; Page No. 18). Highlighted with yellow color.
Comment: I recommend reworking / supplementing the manuscript so that it is more clear, the aim, design and description of the experiments are more obvious and, if possible, the chemical composition of the extracts used for in vitro antimicrobial tests is supplemented. At the same time, authors should consider more carefully the discussion and evaluation of experimental results.
Response: Reworking has been done and more discussion is added (Highlighted with yellow color). Chemical composition of extract for antimicrobial tests is not known; in fact present study is conducted to identify the chemical compounds in the extract.
Round 2
Reviewer 2 Report
Dear authors,
The modifications and additions made reflect my suggestions and I have no further comments on the manuscript. I wish you a good citation score and success in your further research.
Author Response
Dear Reviewer
Thanks for accepting the paper in as such form. Thanks
Dr. Abdul Qayyum
