Study on Chemical Composition and Biological Activity of Psidium guajava Leaf Extracts

Round 1

Reviewer 1 Report

The article "Inhibitory Effects of Psidium guajava Leaf Extract Molecules on Activities of Tyrosinase and Collagenase" presents relatively moderately interesting research but it is very poorly and confusingly written and lacking important information. It has not been written in the proper IMRAD format.
The main weaknesses of the article are as follows:
1. Positive controls are missing for several assays
2. Statistical analysis is lacking altogether
3. Figure and tables captions are lacking important information
4. Discussion is missing. The authors should compare their research with research previously published on that topic.
Without changing those the article can not be, in my view, considered for publication at all.

Other remarks are as follows:
The title says only about anti-collagenase and -tyrosinase activity. But other activities have been investigated as well. The title should reflect that.

Line 73: "2.2. Chromatic analysis", please provide additional details (1-2 sentences) on the principles of the assay.

Line 116 "S. aureus " please change format to italic

All figure and table captions: The figure captions should be self-explanatory. Please add additional details so that the reader may obtain full information without looking the rest of the text (e.g. What do the abbreviations A (30%), B(50%) etc. mean so that the reader may know without looking elsewhere, what concentrations of the extracts were tested)

The authors should decide about the names of the extracts. E.g. in table 7 only A,B,C is mentioned, in Figure 5 it is A(30%), Figure 4 is redundant because it is giving well known information. It should be deleted.

Figure 6: In figures with multiple panels each panel should have its name (e.g. a,b,c) and in the caption should explain what is in panel a, what in panel c etc.

The Table 7 is really hard to understand, and it is completely unnecessary. Not all the values for each concentration should be presented so the table 7 should be deleted altogether. The figure 5 is completely meaningless and should also be deleted. Only table 8 should be kept and commented on.

Statistical analysis is completely lacking. The measurements should be repeated at least 3 times and compared using statistical assays. Such analysis should be reported and  only statistically significant differences should be commented on.

Sections "2.5. Free radical scavenging ability measurement (DPPH method)" and "2.8. Tyrosinase inhibition activity assay ". The rates were not measured. Activity was measured. The word "rate" should be replaced with "assay".

Figure 6: the resolution is really low so the photographs can not be used in this condition. Any conclusion that has been reached using these photographs is not valid. New photos with higher resolution should be added.

Line 115: Wrong title is used (it is not 2.6. Tyrosinase inhibition activity assay)
For all formulas: explanation should be added (e.g. what is Abs(control), what is Abs(test), what is Abs(color)).

All the assays: a positive control (something that has proven activity in the respective assay) should be added and the corresponding activity reported. Currently, the controls for antimicrobial activity and Measurement of ability to remove odor of elderly people are lacking.

Line 150: Is collagenase the enzyme? First the enzyme (line 149) and the collagenase (line 150) is mentioned.

Figures 1,2 and 3 caption "Figure 2. GC-MS chromatograms...": In each figure only one chromatogram (not chromatograms) is presented. Other than that, those three figures may be combined into one 3-panel figure. Names of the tentatively identified compounds should be added either to the figure or to the separate table. That table should be mentioned in the table caption.

Discussion is missing altogether. The authors should compare their research with research previously published on that topic. E.g. has antioxidant activity of some of the detected compounds been previously described, what are the results of the previous research, new references should be added, the meaning and importance of each assay should be added etc.

Conclusion should be short, and some of the items on the list should be incorporated in the Results and discussion section.

Lines 148-151: The sentence "The substrate was 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg (1.2 mg/mL) (Sigma Aldrich, USA). After adding 50 μL of each sample, 75 μL of enzyme, and 125 μL of substrate to 0.4 mg/mL of collagenase (CD130-100MG, Sigma Aldrich, St. Louis, MO, USA), the mixture was reacted for 30 minutes in a water bath at 37 °C. " is not clear.

Line 157-158: The sentence "The experimental group was added with a sample." is not clear.

The whole article is poorly written. Not only English should be improved but the article should be edited by someone (a researcher or a professional editing agency) that has experience in writing scientific articles for international journals.

Author Response

Author's Reply to the Review Report (Reviewer 1)

The article "Inhibitory Effects of Psidium guajava Leaf Extract Molecules on Activities of Tyrosinase and Collagenase" presents relatively moderately interesting research but it is very poorly and confusingly written and lacking important information. It has not been written in the proper IMRAD format.

The main weaknesses of the article are as follows:

1. Positive controls are missing for several assays

2. Statistical analysis is lacking altogether

3. Figure and tables captions are lacking important information

4. Discussion is missing. The authors should compare their research with research previously published on that topic.

Without changing those the article can not be, in my view, considered for publication at all.

Other remarks are as follows:

The title says only about anti-collagenase and -tyrosinase activity. But other activities have been investigated as well. The title should reflect that.

Answer : Thank you. As you mentioned, the title was changed to “Inhibitory effects of Psidium guajava leaf extract molecules on activities of tyrosinase·collagenase and trans-2-onenal removal” to also reflect the activity of odor in the elderly(Page 1, line 2~3).

 Line 73: "2.2. Chromatic analysis", please provide additional details (1-2 sentences) on the principles of the assay.

Answer : Thank you. As you mentioned, 2.2 Added additional details (1-2 sentences) about the analysis principles to Color Analysis(Page 2, line 77~83).

Line 116 "S. aureus " please change format to italic

 Answer : Thank you. As you mentioned, all S. aureus have been changed to italic format(Page 3, 8, line 134, 274).

All figure and table captions: The figure captions should be self-explanatory. Please add additional details so that the reader may obtain full information without looking the rest of the text (e.g. What do the abbreviations A (30%), B(50%) etc. mean so that the reader may know without looking elsewhere, what concentrations of the extracts were tested) The authors should decide about the names of the extracts. E.g. in table 7 only A,B,C is mentioned, in Figure 5 it is A(30%), Figure 4 is redundant because it is giving well known information. It should be deleted.

Answer :  Thank you. As you mentioned, modifications have been made so that readers can understand just by looking at the tables and figures. In addition, Table 7 and Figure 5, which overlap with Figure 4, have all been deleted(Page 5, 6, 7, 8, 9, 10, line 206, 222~223, 256, 270, 281~282, 290~292, 303~304, 314~315).

Figure 6: In figures with multiple panels each panel should have its name (e.g. a,b,c) and in the caption should explain what is in panel a, what in panel c etc.

Answer : Thank you. As you mentioned, (A), (B), and (C) were added to Figure 6, and the contents were explained(Page 8, line 280~282(Figure 2)).

The Table 7 is really hard to understand, and it is completely unnecessary. Not all the values for each concentration should be presented so the table 7 should be deleted altogether. The figure 5 is completely meaningless and should also be deleted. Only table 8 should be kept and commented on.

Answer : Thank you. As you mentioned, Table 7 and Figure 5 have been deleted and explained in Table 8(Page 8, line 270(Table 7))

Statistical analysis is completely lacking. The measurements should be repeated at least 3 times and compared using statistical assays. Such analysis should be reported and  only statistically significant differences should be commented on.

Answer : Thank you. As you mentioned, measurements were repeated three times, and error bars were plotted on bar graphs in Figures 3 to 5 for statistical analysis(Page 9, 10, line 289(Figure 3), 302(Figure 4), 313(Figure 5)). 

Sections "2.5. Free radical scavenging ability measurement (DPPH method)" and "2.8. Tyrosinase inhibition activity assay ". The rates were not measured. Activity was measured. The word "rate" should be replaced with "assay".

Answer : Thank you. As you mentioned, in sections 2.5, 2.8, and 2.9, the rate was changed to assay(Page 3, 4, line 123, 126, 163, 167, 180).

Figure 6: the resolution is really low so the photographs can not be used in this condition. Any conclusion that has been reached using these photographs is not valid. New photos with higher resolution should be added.

Answer : Thank you. As you mentioned, figure 6 was replaced with a higher resolution new photo(Page 8, line 280(Figure 2)).

Line 115: Wrong title is used (it is not 2.6. Tyrosinase inhibition activity assay)

For all formulas: explanation should be added (e.g. what is Abs(control), what is Abs(test), what is Abs(color)).

Answer : Thank you. As you mentioned, 2.6 title has been modified. Additionally, we explained Abs(control), Abs(test), Abs(color), and Abs(sample) of the formula(Page 3, line  128~130, 132)

All the assays: a positive control (something that has proven activity in the respective assay) should be added and the corresponding activity reported. Currently, the controls for antimicrobial activity and Measurement of ability to remove odor of elderly people are lacking.

Answer : Thank you. As you mentioned, results and details of a positive control group have been added(Page 8, 9, line 270, 290, 295, 308). 

Line 150: Is collagenase the enzyme? First the enzyme (line 149) and the collagenase (line 150) is mentioned.

Answer :  Yes. You're right. Collagenase is an enzyme.

Figures 1,2 and 3 caption "Figure 2. GC-MS chromatograms...": In each figure only one chromatogram (not chromatograms) is presented. Other than that, those three figures may be combined into one 3-panel figure. Names of the tentatively identified compounds should be added either to the figure or to the separate table. That table should be mentioned in the table caption.

Answer : Thank you. As you mentioned, figures 1 to 3 have been combined into one and marked as (A), (B), and (C). The names of the identified compounds are listed in Tables 3–5(Page 6, line 221~223).

Discussion is missing altogether. The authors should compare their research with research previously published on that topic. E.g. has antioxidant activity of some of the detected compounds been previously described, what are the results of the previous research, new references should be added, the meaning and importance of each assay should be added etc.

Answer : Thank you. As you mentioned, in the case of guava extract, we were the first to approach it. Previous research results from other extracts were added and explained. Through the results of this study, it was further explained that guava extract is a natural material that has whitening, wrinkle improvement, and deodorizing effects in the elderly through high antioxidant and antibacterial effects, and references were added(Page 6, 7, 11, 12, line  233~235, 264~268, 396~401, 404~409).

Conclusion should be short, and some of the items on the list should be incorporated in the Results and discussion section.

Answer : Thank you. As you mentioned, the conclusion section was shortened and included in the results and discussion sections(Page 7, 10, line 264~268, 319~331).

Lines 148-151: The sentence "The substrate was 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg (1.2 mg/mL) (Sigma Aldrich, USA). After adding 50 μL of each sample, 75 μL of enzyme, and 125 μL of substrate to 0.4 mg/mL of collagenase (CD130-100MG, Sigma Aldrich, St. Louis, MO, USA), the mixture was reacted for 30 minutes in a water bath at 37 °C. " is not clear.

Answer : Thank you. As you mentioned, the sentence you mentioned (Lines 148-51) was not clear, so I edited it to make it clearer(Page 4, line 172~176).

Line 157-158: The sentence "The experimental group was added with a sample." is not clear.

Answer :  Thank you. As you mentioned, the sentence you mentioned (Line 157-58) was not clear, so I edited it to make it clearer(Page 4, line 180~182).

Comments on the Quality of English Language

The whole article is poorly written. Not only English should be improved but the article should be edited by someone (a researcher or a professional editing agency) that has experience in writing scientific articles for international journal

Answer :  Thank you. As you mentioned, edited again in English.

Author Response File: Author Response.pdf

Reviewer 2 Report

In my opinion current version of manuscript has got too little scientific value to be published in Molecules.  Introduction can be improved by adding more information on compounds detected in guava leaves. For these compounds Authors should add evidences on these compounds biological activity. Some sentences on guava fruits from the Introduction should be deleted. There is also a lack of real goal of the presented study. In materials and methods there is lack of TPC, TFC and statistic description.  Why Authors used selected antienzymes activities?  Please compare lines 115 vs. 135 (are these both assays of tyrosinase inhibition?). In my opinion extract yield of water-ethanol solution in range 30/70 to 70/30 is rather incredible. Usually, higher ethanol content increase extract yield (proteins, lipids, etc). I do not see the reason of figure 4 application to this manuscript (description in lines 194-197 does'not dispel my doubt. In general, there is a lack of discussion in this study. Please explain 2,4-dimethyl-1- heptene content in 30 and 70% extract ant a lack of this compound in 50% extract. There are other significant mistakes like incorrect use therms ability vs capacity of antioxidant properties in this study; figure 6 is invisible; clear zones are 3 mm (line 242) or 30 mm (line 299). Paper before further uploading should be generally revised.

In my opinion English language should be corrected.

Author Response

Author's Reply to the Review Report (Reviewer 2)

In my opinion current version of manuscript has got too little scientific value to be published in Molecules.  

Introduction can be improved by adding more information on compounds detected in guava leaves. 

For these compounds Authors should add evidences on these compounds biological activity. Some sentences on guava fruits from the Introduction should be deleted. 

Answer : Thank you. As you mentioned, Information on compounds detected in guava leaves and evidence of biological activity were added to the introduction, and the sentence about guava fruits was deleted(Page 2, line 51~57).

There is also a lack of real goal of the presented study. In materials and methods there is lack of TPC, TFC and statistic description.  

Answer : Thank you. As you mentioned, TPC, TFC and measurements were repeated three times, and error bars were plotted on bar graphs in Figures 3 to 5 for statistical analysis (Page 7, 9, 10, lines 247~249, 288(Figure 3), 301(Figure 4), 312(Figure 5)).

Why Authors used selected antienzymes activities?  Please compare lines 115 vs. 135 (are these both assays of tyrosinase inhibition?). In my opinion extract yield of water-ethanol solution in range 30/70 to 70/30 is rather incredible. Usually, higher ethanol content increase extract yield (proteins, lipids, etc). I do not see the reason of figure 4 application to this manuscript (description in lines 194-197 does'not dispel my doubt. In general, there is a lack of discussion in this study. 

Answer : Thank you. As you mentioned, line 115 is an antibacterial analysis and line 135 is an analysis of tyrosinase inhibition. 2.6 Title modified. Additionally, the yield is not for proteins or lipids, but for the entire extract, and as the ethanol content increases, the yield of the entire extract decreases. Figure 4 was deleted because it is general information(Page 3, line 132).

Please explain 2,4-dimethyl- heptene content in 30 and 70% extract ant a lack of this compound in 50% extract. There are other significant mistakes like incorrect use therms ability vs capacity of antioxidant properties in this study; 

Answer :  Thank you. As you mentioned, in the case of GC analysis, if a specific component is detected in large quantities for the extraction conditions, other components appear relatively lacking. Efficacy also varies accordingly. Compared to previous research results, ingredients extracted from guava leaves have shown high antioxidant and antibacterial effects, and have been shown to have whitening, wrinkle improvement, and odor removal effects. Related references have been added(Page 6, 11, line 233~235, 396~401).

figure 6 is invisible; clear zones are 3 mm (line 242) or 30 mm (line 299). Paper before further uploading should be generally revised.

Answer : Thank you. As you mentioned, figure 6 was replaced with a higher resolution new photo(Page 8, line 280(Figure 2))

Comments on the Quality of English Language

In my opinion English language should be corrected.

Answer : Thank you. As you mentioned, edited again in English.

Author Response File: Author Response.pdf

Reviewer 3 Report

The study is thorough and presents significant findings on the use of Psidium guajava leaf extract as a natural material with antioxidant, antibacterial, and enzyme-inhibitory properties. However, there are some main concerns that the authors should address.

1. Were any toxicity or side effects observed or anticipated with the use of guajava leaf extract in high concentrations?

2. For the GC-MS, how did you figure out the known compounds? did you also purify them or you compared them to the standard compounds?

3. From the GC-MS, I didn’t see significant differences between 30%, 50%, and 70% extractions. How can you compare their activities?

4. How many times did you repeat the results of the yield of different extracts?

5. Figure 4, please give the name of each compound and double-check the structures.

6. Figure 5 needs to be improved.

7. Please clarify what is the control group.

8. The study focuses on S. aureus but could be expanded to include a broader range of bacteria to better assess the extract's antibacterial spectrum.

Author Response

Author's Reply to the Review Report (Reviewer 3)

The study is thorough and presents significant findings on the use of Psidium guajava leaf extract as a natural material with antioxidant, antibacterial, and enzyme-inhibitory properties. However, there are some main concerns that the authors should address.

1. Were any toxicity or side effects observed or anticipated with the use of guajava leaf extract in high concentrations?

Answer : Thank you for your good comments. Of course it is expected. We will conduct MTT’assay toxicity analysis in the next additional study.

2. For the GC-MS, how did you figure out the known compounds? did you also purify them or you compared them to the standard compounds?

Answer : Thank you for your good comments. GC-MS analysis allows for easy qualitative analysis of ingredients without purification or comparison with standard compounds. Therefore, we did not compare tablets or standard compounds. However, the compounds were identified by comparing previous research results. In the next additional study, we plan to conduct quantitative analysis using LC-MS (standard compound). In addition, references to related research results have been added(Page 6, 7, 11, 12, line 233~235, 264~268, 396~401, 404~409)

3. From the GC-MS, I didn’t see significant differences between 30%, 50%, and 70% extractions. How can you compare their activities?

Answer : This can be confirmed by the area peak on the GC-MS graph. According to Lambert beer's law, as the concentration increases, the absorbance (area) peak appears higher and the content of the compound increases. The areas were compared by combining Figures 1 to 3(Page 6, line 221)

4. How many times did you repeat the results of the yield of different extracts?

Answer : The yield was confirmed by repeating it three times. Description added in 2.1(Page 2, line 74~75).

5. Figure 4, please give the name of each compound and double-check the structures.

Answer : Figure 4 was deleted because it is general information.

6. Figure 5 needs to be improved.

Answer : Thank you. Duplicate Table 7 and Figure 5 have been deleted and explained in Table 8(Page 8, line 270(Table 7))

7. Please clarify what is the control group.

Answer : Thank you. As you mentioned, results and details of a positive control group have been added(Page 8, 9,  line 270, 290, 295, 308).                                 

 8. The study focuses on S. aureus but could be expanded to include a broader range of bacteria to better assess the extract's antibacterial spectrum.

Answer :  Thank you for your good comments. In the next additional study, we will conduct research that includes not only S. aureus, but also a wider range of bacteria such as E. coli, Pseudomonas aeruginosa, and Candida.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The article "Inhibitory Effects of Psidium guajava Leaf Extract Molecules on Activities of Tyrosinase and Collagenase" presents relatively moderately interesting research but it is very poorly and confusingly written and lacking important information. It has not been written in the proper IMRAD format.
The main weaknesses of the article are as follows:
1. Positive controls are missing for several assays: a positive control (something that has proven activity in the respective assay) should be added and the corresponding activity reported. Currently, the controls for antimicrobial activity and Measurement of ability to remove odor of elderly people are lacking.
2. Statistical analysis is lacking altogether
3. Discussion is missing altogether. The authors should compare their research with research previously published on that topic. E.g. has antioxidant activity of some of the detected compounds been previously described, what are the results of the previous research, new references should be added, the meaning and importance of each assay should be added etc.
Without changing those the article can not be, in my view, considered for publication at all.
A minor problem:  In several places, e.g. sections "2.5. Free radical scavenging ability measurement (DPPH method)" and "2.8. Tyrosinase inhibition activity assay " but elsewhere too:  The rates were not measured. Activity was measured. The word "rate" should be replaced with "assay".

Author Response

1. Positive controls are missing for several assays: a positive control (something that has proven activity in the respective assay) should be added and the corresponding activity reported. Currently, the controls for antimicrobial activity and Measurement of ability to remove odor of elderly people are lacking.

Answer : Thank you for your good comments. The results of a positive control group with proven activity were added to each analysis(Table 7, Figure 2-5) . In particular, in the elderly odor removal analysis, we added control of antibacterial activity(Figure 2) and measurement results of odor removal ability(Figure 3).(Page 8, Line 281(Tabe 7), 285-286, 293(Figure 2), Page 9, Line 305(Figure 3), 323(Figue 4), Page 10, Line 337(Figure 5)).

2. Statistical analysis is lacking altogether

Answer : Thank you for your good comments. We added statistical analysis.(Page 5, Line 205(Table 1), Page 8, 297-303, Page 9, Line 312-320, Page 10, Line 328-336) .

3. Discussion is missing altogether. The authors should compare their research with research previously published on that topic. E.g. has antioxidant activity of some of the detected compounds been previously described, what are the results of the previous research, new references should be added, the meaning and importance of each assay should be added etc.

Answer : Thank you for your good comments. This study was compared with previously published studies. Among the contents of previous studies, we compared and analyzed whether they had antioxidant activity compared to the compounds detected in this study, and the meaning and importance of each analysis method were additionally explained.(Page 2, Line 52-64, Page 6, Line 237-245, Page 8 Line 276-279, 300-303, Page 9, 317-320, Page 10, 333-335].

A minor problem:  In several places, e.g. sections "2.5. Free radical scavenging ability measurement (DPPH method)" and "2.8. Tyrosinase inhibition activity assay " but elsewhere too:  The rates were not measured. Activity was measured. The word "rate" should be replaced with "assay".

Answer : Thank you for your good comments. All rates have been changed to assay.(Page 3, Line 117, 130, 133, Page 4, Line 170, 174, Page 8, Line 187, Page 9, 323(Figue 4), Page 10, Line 337(Figure 5)).

Author Response File: Author Response.pdf

Reviewer 2 Report

In my opinion - ok

Author Response

In my opinion - ok

Answer : Thank you. Overall, the content and English text have been slightly modified to fit the context (edited in blue).

Author Response File: Author Response.pdf

Reviewer 3 Report

The authors have not made substantial revisions to the manuscript, leaving lots of issues unaddressed. I would not recommend it for publication until it undergoes thorough revision.

1.     Although it is stated that "The quality of 1-docosene was highest at 30, 50, and 70% ethanol," discrepancies in Tables 3-5 and Figure 1 suggest otherwise.

2.     The authors claim the presence of three duplicates, yet this is not reflected in Table 1.

3.     Given the similar contents of the three extracts, as shown in Tables 3-5, the rationale behind only presenting selected peaks raises questions.

4.     While Table 6 indicates that Sample A contains the highest total flavonoid content, Table 7 presents an apparent contradiction by showing Sample A with the poorest antioxidant activity.

5.     The manuscript requires significant improvements in its writing quality.

Writing needs to be improved.

Author Response

1.     Although it is stated that "The quality of 1-docosene was highest at 30, 50, and 70% ethanol," discrepancies in Tables 3-5 and Figure 1 suggest otherwise.

Answer :  Thank you for your good comments. The meaning of the 1-dococene component analysis results for each extract is explained along with Tables 3 to 5 and Figure 1 to maintain consistency in the results(page 6, line 237~245). 

2.     The authors claim the presence of three duplicates, yet this is not reflected in Table 1.

Answer :  Thank you for your good comments. The standard deviation of the yield for three repeated extractions is reflected in Table 1(page 5, line 205(Table 1). 

3.     Given the similar contents of the three extracts, as shown in Tables 3-5, the rationale behind only presenting selected peaks raises questions.

Answer :  Thank you for your good comments. As you mentioned, the extract contains many complex ingredients mixed together. Therefore, considering the similar contents of the three extracts as shown in Table 3-5, the basis for presenting only the selected peaks is that the activities of the sesquiterpene components were compared based on previous research results [reference 29-31]. In reality, unlike single ingredients, complex ingredients may contain the same or greater amounts of active ingredients, but the complex combination of other ingredients may act as a factor in lowering or increasing antioxidant or antibacterial activity(page 6, line 219~221) .

4.     While Table 6 indicates that Sample A contains the highest total flavonoid content, Table 7 presents an apparent contradiction by showing Sample A with the poorest antioxidant activity.

Answer :  Thank you for your good comments. As shown in Table 6, sample A contained the highest total flavonoid content, while Tbale 7 showed the lowest antioxidant activity of sample A. This represents an unexpected result. As shown in Table 5, the flavonoid content of sample C (70% ethanol extract) was low, but it is thought to have high antioxidant activity because there are many other phenolic compounds in addition to flavonoids [Reference 35](page 8, line 276~279). 

5.     The manuscript requires significant improvements in its writing quality.

Answer :  Thank you. Overall, the content and English text have been modified to fit the context(Edited to blue).

Author Response File: Author Response.pdf

Round 3

Reviewer 1 Report

The article "Inhibitory Effects of Psidium guajava Leaf Extract Molecules on Activities of Tyrosinase and Collagenase" is significantly improved. Howver, there are still major obstacles for acceptance, at least in my opinion.

1.
THe article title "Inhibitory Effects of Psidium guajava Leaf Extract Molecules on Activities of Tyrosinase and Collagenase " should be changed so that it does not cover only two assays. Either the other assays should be added to the title or the authors should finf the way to summarize the performed research (a wides example is "Chemical comporition and biological activity of Psidium guajava Extracts" or  "Chemical comporition and cosmetic activity of Psidium guajava Extracts")

2.
"2.6. Antimicrobial activity measurement (disc diffusion method)" and "3.6. Antimicrobial activity measurement (disc diffusion method) results": The positive control should be a substance with a positive antimicrobial activity (e.g. antibiotic).

3.
THe names of ALL the positive controls should be added to the Materials and Methods section parts as well.

4.
The authors have made an improvement by adding standard deviation. However, for a proper article statistical comparison between extracts should be performed by using ANOVA with an appropriate post-hoc test, or at least t-test.

5.
Minor suggestions
Table 7, Title: Please change "IC50 for antioxidant capacity of extracts" to "IC50 for DPPH antiradical capacity of extracts"

Author Response

1. The article title "Inhibitory Effects of Psidium guajava Leaf Extract Molecules on Activities of Tyrosinase and Collagenase " should be changed so that it does not cover only two assays. Either the other assays should be added to the title or the authors should find the way to summarize the performed research (a wides example is "Chemical composition and biological activity of Psidium guajava Extracts" or "Chemical composition and cosmetic activity of Psidium guajava Extracts")

Answer: Thank you for your good comments. In reference to the title you suggested, we changed the title to “Study on chemical composition and biological activity of Psidium guajava leaf extracts” to summarize and encompass other studies conducted (Page 1, Line 2~3).

2. "2.6. Antimicrobial activity measurement (disc diffusion method)" and "3.6. Antimicrobial activity measurement (disc diffusion method) results": The positive control should be a substance with a positive antimicrobial activity (e.g., antibiotic).

Answer: Thank you for your good comments. Information on cefotaxime and ampicillin as positive control antibiotics used for antibacterial activity has been added to 2.6 and 3.6 (Page 4, Line 139~140; Page 7, Line 266~268).

3. The names of ALL the positive controls should be added to the Materials and Methods section parts as well.

Answer: Thank you for your good comments. All the positive control group names have been added to the Materials and Methods section parts (Page 3, Line 119~120; Page 4, Line 139~140, 145, 162; Page 5, Line 176).

4. The authors have made an improvement by adding standard deviation. However, for a proper article statistical comparison between extracts should be performed by using ANOVA with an appropriate post-hoc test, or at least t-test.

Answer: Thank you for your good comments. For statistical comparison between extractions, results were added using ANOVA. Most of them showed *p < 0.05, indicating a significant difference between extractions, but the total polyphenol (TPC) and total flavonoid contents (TFC) values in Table 6 did not show a significant difference due to the large standard deviation. (Page 4, Line 177~179; Page 5, Line 195, 203; Page 7, Line 242~244; Page 8, Line 259; Page 8, Line 287; Page 9, Line 300, 313).

5. Minor suggestions Table 7, Title: Please change "IC50 for antioxidant capacity of extracts" to "IC50 for DPPH antiradical capacity of extracts"

Answer: Thank you for your good comments. The title of Table 7 was changed to “IC50 for the DPPH antiradical capacity of the extracts” (Page 7, Line 263).

Author Response File: Author Response.pdf

Reviewer 3 Report

It seems the authors didn’t answer my questions 3 and 4. The authors said “the flavonoid content of the 70% ethanol extract was lower than that of 30% and 50%, the content of other phenolic compounds in addition to flavonoids was high”, however, it is clearly shown in Figure 1, B is more abundant than C, where are the other phenolic compounds? 

Minor editing of English language required

Author Response

It seems the authors didn’t answer my questions 3 and 4. The authors said “the flavonoid content of the 70% ethanol extract was lower than that of 30% and 50%, the content of other phenolic compounds in addition to flavonoids was high”, however, it is clearly shown in Figure 1, B is more abundant than C, where are the other phenolic compounds? 

Answer:  Thank you for your detailed advice. I checked it now. During the editing process, the pictures (B) and (C) changed. I should have looked more closely, sorry for the confusion. Additionally, For statistical comparison between extractions, results were added using ANOVA (Page 4, Line 177~179; Page 5, Line 195, 203, 208; Page 7, Line 242~244; Page 8, Line 259; Page 8, Line 287; Page 9, Line 300, 313).

Minor editing of English language required

Answer: Minor English edits were made (marked in blue).

Author Response File: Author Response.pdf