Hop (Humulus lupulus L.) Specialized Metabolites: Extraction, Purification, Characterization in Different Plant Parts and In Vitro Evaluation of Anti-Oomycete Activities against Phytophthora infestans

Round 1

Reviewer 1 Report

The article brings valuable information about the in-vitro antifungal activity of the hop extracts prepared from the different hop organs against Phytophthora infestans. Most of the extracts are made from hop by-products. Interestingly the most promising results were obtained for the leaf extract. Unfortunately, the possible active compounds in leaf extract were not identified. The research topic is very actual in light of the current pressure to reduce pesticide use and attempt to follow the circular economy. The results will definitely be of interest to other researchers. The introduction of the article is well structured. On the other hand, the part materials, methods, and results need improvement. The information in the methodology can sometimes lead to misleading conclusions (see below). The result part could be shortened, and I would definitely recommend that the authors present the results from the same design experiment in the same way. Also, merging some tables and omitting some pictures would improve the clarity of the results.

In conclusion, I do recommend the manuscript for acceptance after major revisions. The comments are attached.

Comments for author File: Comments.pdf

Author Response

Dear reviewer,

Thank you for your relevant comments.

Please see our reply in the attached file.

Best regards,

Céline Rivière

Author Response File: Author Response.pdf

Reviewer 2 Report

In recent times, scientific research of hop (Humulus lupulus L.) in the world has shown that the substances xanthohumol and humulones have a significant antiviral effect. This was confirmed against the dangerous virus HIV-1, which causes AIDS in humans. It is also interesting to find that the chemical components of hops generally had higher antimicrobial activity than other substances isolated from plants such as thymol, menthol, nerol, eucalyptol, cinnamon and clove silica.

However, the most fundamental scientific studies have shown the inhibitory effects of hop secondary metabolites against certain types of cancer cells. The most interesting, studied and promising of these for potential use as a chemopreventive agent or drug is clearly xanthohumol.

Recently, there have been studies showing the biopesticide potential of selected hop components. Their antimicrobial activities against plant phytopathogens, including bacteria and fungi, have been demonstrated.

On the other hand, agro-ecological knowledges about different types of cultivation interventions and influences with their evaluation on biomass production, metabolite production and its composition are very important.

The present study of authors is the current research to study the qualitative and quantitative characteristics of essential oil of hop cones as well as extracts from four organs (cones, leaves, stems, and rhizomes) and evaluating the in vitro anti-oomycete activity against the pseudo-fungus Phytophthora infestans. The activity of the main compounds of hop, xanthohumol, and mix of α-acids and α,β-acids was determined and compared to copper sulfate as a reference active substance. Abstract section consist needs more information about the importance of using biofungicides in regard to plant or animal protection. The introduction of manuscript is appropriate to describe, that hop, as a medicinal plant, which include sedative, antispasmodic, estrogenic, antioxidant, antiproliferative and anti-inflammatory bioactivities, special antimicrobial properties (antibacterial and antiviral).

Material and Methods are described adequately. I appreciate a description of preparation of extracts, sub-extracts, and purified compounds of hop and analytic-chemical analyses. For the purpose of seeking alternatives to conventional treatments to manage diseases caused by Phytophthora infestans, were presented the anti-oomycete potential of essential oil of hop cones as well as crude hydroethanolic extracts and apolar sub-extracts of different parts of hop plant. I agree with the authors that there is a need for next evaluations of this research fields. The literature citations are adequate enough and finally the manuscript does not contain any irrelevant information’s.

In regard to my opinion the contents of the manuscript in line with policy of the journal Agronomy MDPI, the text has been prepared according to the format and style of journal including the body of the manuscript, page size and referencing. The manuscript should be accepted in the form after its small correction in regard to my notes and recommendation.

Author Response

Dear reviewer,

Thank you for your relevant comments.

Please see our reply in the attached file.

Best regards,

Céline Rivière

Author Response File: Author Response.pdf

Reviewer 3 Report

The thematic of the work is quite interesting. Despite the extraction procedures of hop phytochemicals have been well covered in the literature, the application of its extracts in antifungal activity is a great topic. The manuscript must be improved prior to its acceptance. There are several errors in grammar and the presentation of the results. The speaking on the manuscript could be changed from the first person: instead of "our work", "we", it could be used an impersonal and direct speaking such as "the present work", "the results showed". English must be reviewed in all text. All paragraphs are very extensive; and the reading gets lost in all the long sentences of the manuscript. The authors tend to repeat several information along the text.

Specific points:

1. Line 18: include the word "hop cone" instead of just "cone";

2. Line 23: Change the term "cone extracts";

3. Line 26: Make a clear state about which insights were highlighted on the work;

4. Line 33: insert "hop cones" and remove "or hops";

5. Line 36: correct the phrase "...by lupulin glands found at the base of the bracts of mature hop cones";

6. Line 38: Standardize the name of bitter acids "α- and β-acids", correct the names over all manuscript;

7. Line 39: Rewrite the sentence, it is long and confuse;

8. Line 42: Instead of hop and its compounds, put "hop constituents". Changes like this can be performed along the manuscript.

9. Like 44: remove "ones";

10. Lines 45-64: This hole paragraph gets all confuse, with the information being given in circles. Rewrite with the sentences getting a understandable sequence. Also, this paragraph is too long, subdivide it according to the information that has been given. My suggestion is to cut it at the line 56, since a novel point is stated up from it.

11. Line 58: replace "which are currently" with "often";

12. Line 63: Remove the last sentence of the paragraph;

13. Line 65: Remove "in this context, we focus here on"; and star the phrase with: Phytophthora infestans (Mont.) is a casual...";

14. Line 77: Starting at "Nevertheless", make it a new paragraph; 

15. Lines 88-02: Rewrite paragraph, turning it more specific. Substitutes "organs" in all manuscript for "parts" or "constituents".

16. Line 102: Add "macerations in the dark", and remove it from the end of the sentence;

17. Line 112: Specify "dw" to the percentages;

18. Line 114: Remove the α in "αβ acids". This error repeats all over the manuscript.

19. The methodologies described in items 2.1.2 and 2.1.3 follow any reference? If yes, put it on the text;

20. Line 156: Correct 40ºC to 40 ºC. Correct it through the manuscript;

21. Items 2.3.3 and 2.3.3: Specify the concentrations tested.

22. Line 246: correct the sentence "observations were done 24 h after germination evaluation..."

23. Separate item 2.3.5 in IC50 and statistical data analysis.

24. Line 285: Rewrite the sentence "... hop parts, a phytochemical investigation was conducted on crude...";

25. Line 301: Substitute "our method" with "the method used" or similar;

26. Line 306: "Form" to "from". Replace the word "organs";

27. Table 3: Specify what is "LOD" in the footnotes;

28. Table 4: Reorganize the table, specifying the titles of the table (growth and germination column). "cones essential oil" seems like two distinct lines lines, remove this column. 

29. Line 353: Substitute the term "modalities";

30. Line 353: Add "For leaf extracts...";

31. Figure 4: Divide it on a table and a figure. The part A of the figure is a table!! Also, there is no need of an separated column at the table from part A, the letters can be put aside from the IC50 values;

32. Figure 5: the same stated for Figure 4;

33. Line 384: Replace the term "organs";

34. Lines 408-410: Rewrite the sentence. When there is no significant difference between values, there is no need to describe it on the text, we consider it as statistically equals. Therefore, the only compounds that presented higher activity than xanthohumol were α-acids. 

35. Item 4: The discussion seems to be repetitive, as it was a resume from the results topic. Present it on a more concise form, removing repeated information. Also, the paragraphs are too extensive! The information keeps being putted on the text with no order;

36. Replace "our study" with "The present study";

37. Line 465: remove sentence;

38. Line 477: Remove "for many years but also";

39. Lines 504-507: Remove the sentence "The comparison....indicators";

40. Line 527: Reference?

41. Line 540: "Even though the main...";

42. Line 543: "...under optical microscope" for each treatment?

43. Line 546: "botanicals" is the right term in this case? Add reference to the sentence.

44. Line 558: Remove "bitter acids and particularly";

45. Line 561: Change "explicate" for "elucidate".

Author Response

Dear reviewer,

Thank you for your relevant comments.

Please see our reply in the attached file.

Best regards,

Céline Rivière

Author Response File: Author Response.pdf