Lichens as a Natural Source of Compounds Active on Microorganisms of Human Health Interest

Round 1

Reviewer 1 Report

The communication paper entitled “Lichens as a natural source of compounds active on microorganisms of human health interest” described lichen extraction by acetone and cyclohexane. The extracts evaluated the antimicrobial activity before separating each fraction for structure elucidation by LC-MS/MS. It can be accepted for publication in Appl. Sci. after the major revision. The comment is as follows.

1. In the introduction, the authors should make it concise by focusing on the natural compounds from lichen and reducing the content of each pathogen.

2. Line 74, give the full name of AMR.

3. Line 124, did the authors record the lichen appearance such as macroscopic and microscopic characteristics?  Lichen identification is important to know the origin or source of natural products. How do you preserve the lichen for further experiments?

4. Line 138, the stability of the extracts was evaluated. Give the detail of the extracts whether they were in liquid (solvent) or dried state because it influenced their stabilities.

5. In the result, provide the figure of spots on TLC to show compound separation.

6. Lines 177-178, provide the physical appearance of dried LC1 and LC2 extracts and the definition of extraction rate.

7. Table 1 showed the result of the inhibition zone and stability of LC1 and LC2.

7.1 It had one data set. The authors must separately present the activity and stability of two extracts.

7.2 Give the full name and unit of IZ and add the SD data.

7.3 The inhibition zone against B. subtilis was 16 mm, and the activity on Day 1 was “++”. Based on the definition of IZ more than 15 mm, it was “+++”. Please recheck the result.

8. Lines 212 and 215, specify the tested pathogen in the antimicrobial activity.

9. Line 215, LC2 fractions had no antimicrobial activity. It might be from the dilution effect because the samples were not concentrated. Similarly, the amount or concentration of purified compound from LC1 fractions was not stated for antimicrobial assay. The authors should provide the data on the amount or concentration of LC1 and LC2 fractions and the negative control used in this experiment.

10. Line 220, the interesting peaks of LC1 and LC2 extracts were found at the retention time of 6, 10, and 11 min. There are some questions related to this statement and Table 2.

10.1 What fractionations gave interesting peaks? and What compound did each peak represent?

10.2 How to calculate the % yield in Table 2? or Was it the total yield from those three peaks?

10.3 The figure of the three interesting peaks should be shown.

11. More discussion must be given such as

11.1 Different solvents used to extract the lichen affect the compound separation and antimicrobial activity

11.2 Yield of the main metabolites in LC1 and LC2

11.3 How the main metabolites inhibit the pathogens, especially C.albican.

11.4 The limitation of this study

Author Response

Please  see the attachment

Author Response File: Author Response.docx

Reviewer 2 Report

Reviewer’s comments to the authors

Manuscript title: Lichens as a natural source of compounds active on microorganisms of human health interest

The following are my comments and critique that should be addressed before acceptance for publication:

Major comments:

The authors strongly encouraged clarifying the identified strains, I have doubts about whether the implementation of the work has led to meaningful results.

Minor comments:

# As a non-native speaker, I found the manuscript easy to read and understand. However, there are some grammatical errors and, in some instances, the phrasing needs to improve.

# Abstract should support quantitative values

# Define abbreviations upon the first appearance in the text, especially scientific names

# The authors should provide the extraction methods of Lichens in detail

# Dilution? specify

# Why used distilled water instead of DMSO for fractions dilution? Clarify, the authors used solvent or DMSO as negative control? How much concentration of DMSO if so?

# It’s strange to check the antimicrobial activity for 30 days. Is there

 Any changes in media?

# How much concentration for the tested extracts on each disk

# When did the author collect the Lichens?  Do the potential active compounds of Lichens extracts change by season?  I hope to add a description of it.

# How did the authors identify the structures of the ingredients of the Lichens extracts?  Were they identified by the retention time of HPLC/LC-MS?  I suggest showing the HPLC data of the standard samples to the authors.

# Lake of statistical data (data analysis) in the methodology part in addition to results

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Thank you for your revision. I have some suggestions as follows.

1. Add the MS database used to search for chemical identification, and their confidence scores.

2. Do a statistical analysis to confirm that the extracts in LC1 and LC2 are stable within 30 days (Table 1). Also, give the detail of the analysis in the method.

3. Give more discussion related to higher extraction yield in acetone (Table 2).

Author Response

Please see the attachment.

Author Response File: Author Response.doc