Comparative Analysis of Biological Activity of Artificial and Wild Agarwood

Round 1

Reviewer 1 Report

The manuscript entitled “Evaluation of biological activity of alcohol-soluble extract of artificial agarwood”, authored by Sheng Ma, Penglian Wei, Mengji Qiao, Yunlin Fu, Yingjian Li, and Zhigao Liu, deals with the evaluation of the biological activity of wild and artificial agarwood.  The great limitation of the work concerns the use of really simple methodologies which, in the absence of analytical characterization, are hardly commentable. Aside from that, a number of changes need to be made.

• Please, consider changing the title of the article and making it more attractive.
• Keywords should be words not contained in the title, at most present in the abstract. Their usefulness is to make easier the searching of the article using the common scientific search engines. Since several keywords are already present in the title, and/or repeated several times in the abstract, I strongly advise the authors to replace some of them and add more. As journal guidelines clearly report, a limited number of keywords can be used (maximum 10). Consequently, authors should carefully choose them.
• Authors should write the number report the numerical digits as words if they are not linked to empirical experiments or they are dates. For example, line 27 "twenty-one species"; "twenty countries", etc ...
• In the captions of the figures, the meaning of the acronyms shown in the figure should be clearly indicated also in the caption of each figure.
• Moreover, The word "clearance" should be changed and replaced with a CI₅₀ (50% Inhibitory Concentration) indicating also the measurement unit.
• In materials and methods section, the equation should be written and reported using the Microsoft word tool, as described in the guidelines for authors.
• Concerning the assays evaluating the antioxidant properties of the extracts, It is not clear which is the units of measurement reported in the tables. This problem is also present for enzymatic assays.
• Did the authors use a standard (eg gallic acid) as a control? this aspect is very important, as the results could (and should) also be expressed as mmol of gallic acid equivalent per g of raw material.
• The analytical displayed in the graphs of the revised manuscript lack in standard deviation. From the section of materials and methods it can be deduced that the experiments were carried out in triplicate, however from the data reported it is not possible to understand if the standard deviations of the various experiments were admissible.

Author Response

Responce to Reviewers’ Comments

The manuscript entitled “Evaluation of biological activity of alcohol-soluble extract of artificial agarwood”, authored by Sheng Ma, Penglian Wei, Mengji Qiao, Yunlin Fu, Yingjian Li, and Zhigao Liu, deals with the evaluation of the biological activity of wild and artificial agarwood.

1. The great limitation of the work concerns the use of really simple methodologies which, in the absence of analytical characterization, are hardly commentable. Aside from that, a number of changes need to be made.

Response 1: Thanks for your question. In our previous work, we conducted qualitative and quantitative analysis of the chemical composition of five batches of artificial agarwood by GC-MS, HPLC and UV spectrophotometry, and published a paper (References: Ma S, Fu Y, Li Y, et al. The formation and quality evaluation of agarwood induced by the fungi in Aquilaria sinensis[J]. Industrial Crops and Products, 2021, 173: 114129), in which the sum of the relative contents of sesquiterpenes and 2-(2-phenylethyl)chromones ranged from 90.55 to 98.99%. The chemical composition table determined by GC-MS is at the bottom of this document.

2. Please, consider changing the title of the article and making it more attractive.

Response 2: Thanks for your suggestion. We have changed the title of the manuscript to “Comparative analysis of biological activity of artificial and wild agarwood”.

3. Keywords should be words not contained in the title, at most present in the abstract. Their usefulness is to make easier the searching of the article using the common scientific search engines. Since several keywords are already present in the title, and/or repeated several times in the abstract, I strongly advise the authors to replace some of them and add more. As journal guidelines clearly report, a limited number of keywords can be used (maximum 10). Consequently, authors should carefully choose them.

Response 3: Thanks for the suggestion. We have changed it (line 24 and 25).

4. Authors should write the number report the numerical digits as words if they are not linked to empirical experiments or they are dates. For example, line 27 "twenty-one species"; "twenty countries", etc ...

Response 4: Thanks for your advice, we have changed in the line of 29, 42, 47 and 48.

5. In the captions of the figures, the meaning of the acronyms shown in the figure should be clearly indicated also in the caption of each figure.

Response 5: Thanks for your kind remind, we have changed it (line: 195~198, 235~238, 263~266, 294~297 and 326~329).

6. Moreover, The word "clearance" should be changed and replaced with a CI₅₀(50% Inhibitory Concentration) indicating also the measurement unit.

Response 6: Thank you for your reminder. In the manuscript, we also compared the IC₅₀ values of the samples, and we have replaced it with the IC₅₀ value in line 186, 190, 228, 230, 287, 318, 376 and 428.

7. In materials and methods section, the equation should be written and reported using the Microsoft word tool, as described in the guidelines for authors.

Response 7: Thanks for your kind remind, we have modified it (line 109, 124, 137, 157 and 174).

8. Concerning the assays evaluating the antioxidant properties of the extracts, It is not clear which is the units of measurement reported in the tables. This problem is also present for enzymatic assays.

Response 8: Thank you for your reminder. The unit of IC₅₀ value is mg/mL. Within the design concentration range, the scavenging power and inhibitory power of artificial and wild agarwood concentration on free radicals and enzymes increase with the increase of mass concentration. Therefore, at the maximum mass concentration, we mainly discuss the maximum clearance or inhibition ability of free radicals and enzymes for artificial and wild agarwood, as well as the differences between them. The concentration in the table corresponds to the maximum inhibitory or scavenging capacity of artificial and wild agarwood, which is expressed by the clearance or inhibition rate (%).

9. Did the authors use a standard (eg gallic acid) as a control? this aspect is very important, as the results could (and should) also be expressed as mmol of gallic acid equivalent per g of raw material.

Response 9: Thanks for your suggestion. Ascorbic acid is usually used for this antioxidant because of its strong antioxidant capacity (Reference: Scalzo R L. Organic acids influence on DPPH scavenging by ascorbic acid[J]. Food Chemistry, 2008, 107(1): 40-43.). We choose it as a control.

10. The analytical displayed in the graphs of the revised manuscript lack in standard deviation. From the section of materials and methods it can be deduced that the experiments were carried out in triplicate, however from the data reported it is not possible to understand if the standard deviations of the various experiments were admissible.

Response 10: Thanks for your question. There is standard deviation analysis in the figure, that is, the error bar representation, but some of the errors are small and the point on the curve is slightly larger, the part is not so obvious on the figure. And we have added in the title of each table with “Each value represents the mean ± SD (n = 3); SD, standard deviation”.

Table：Components and relative contents of 5 batches of artificial agarwood (C12 and C18 represented agarwood produced by A. crassna after 12 and 18 months of inoculation, respectively; A6-A18 represented agarwood produced by A. sinensis after 6, 12 and 18 months of inoculation, respectively).

Author Response File: Author Response.docx

Reviewer 2 Report

Dear Authors.

Manuscript entitled: Evaluation of biological activity of alcohol-soluble extract of artificial agarwood, have been reviewed.

Five different methods were used to compare biological activity of artificial and natural agarwood.

The introduction of the article provided sufficient background and included  relevant references. Material & Method are described precisely and potentionally reproductible.

Results are also described very preciselly, but the the discussion and results in one chapter seems to be not homogenous.

I would recommend to separate into two different chapters.

Few overtypings have been found as well as recommendation for the improving of titles in Tab4-6.

Line 81: dried. after drying = dried. After drying

Line 88: the sieved powder was taken, to which 250 mL of 95% ethanol was added = English correction required.

Table 4 – change the Title. (The same with Table 5 and Table 6)

When the sample concentration was 3.5 mg/mL, the total reducing powers of all batches 263 (five) and controls. = The total reducing powers of all five batches and controls.  The information about sample concentration is not necessary because it is a part of Table 4

Line 266: dots placed in O2.- and NO. are in wrong position – please correct!

It would be better if the authors separate Results and Discussion as a two chapters!

Lines 266 – 292 – as a part of discussion is not logically placed. Please separate the discussion and connect to the exact part – when authors presents DPPH activity – place discussion directly after this results, that reader can compare immediately…

OR – provide separate chapter DISCUSSION, when authors can summarize discuss their results with published ones.

Lines 285 – 292 presents summarizations which should be at the end of the Discussion.. not in the middle of the manuscript.. OR it should be as a part of conclusion.

The same with lines 318 – 347 and 383-406

best wishes

Author Response

Responce to Reviewers’ Comments

Manuscript entitled: Evaluation of biological activity of alcohol-soluble extract of artificial agarwood, have been reviewed.

Five different methods were used to compare biological activity of artificial and natural agarwood.

The introduction of the article provided sufficient background and included relevant references. Material & Method are described precisely and potentionally reproductible.

Results are also described very preciselly, but the the discussion and results in one chapter seems to be not homogenous.

I would recommend to separate into two different chapters.

Few overtypings have been found as well as recommendation for the improving of titles in Tab4-6.

1. Line 81: dried. after drying = dried. After drying

Response 1: Thank you for your correction. We have deleted "after drying" in the manuscript.

2. Line 88: the sieved powder was taken, to which 250 mL of 95% ethanol was added = English correction required.

Response 2: Thank you for your suggestion, we have changed "to which 250 mL of 95% ethanol was added" to "and 250 mL of 95% ethanol was added".

3. Table 4-change the Title. (The same with Table 5 and Table 6)

When the sample concentration was 3.5 mg/mL, the total reducing powers of all batches 263 (five) and controls. = The total reducing powers of all five batches and controls. The information about sample concentration is not necessary because it is a part of Table 4

Response 3: Thank you for your advice, we have modified the titles of Table 4~6, and correction as follow:

Table 4. The total reducing powers of all five batches and controls (line 280).

Table 5. The inhibition rates of acetylcholinesterase activity in all five batches and controls (line 310).

Table 6. The inhibition rates of α-glucosidase activity in all five batches and controls (line 350).

4. Line 266: dots placed in O2.- and NO. are in wrong position – please correct!

Response 4: Thank you for your suggestion. We have corrected it to “O₂^.-, OH^., and NO^.” (line 355).

5. It would be better if the authors separate Results and Discussion as a two chapters!

Lines 266-292 as a part of discussion is not logically placed. Please separate the discussion and connect to the exact part when authors presents DPPH activity place discussion directly after this results, that reader can compare immediately. Or provide separate chapter DISCUSSION, when authors can summarize discuss their results with published ones. Lines 285-292 presents summarizations which should be at the end of the Discussion not in the middle of the manuscript. Or it should be as a part of conclusion.The same with lines 318 -347 and 383-406.

Response 5: Thank you very much for your good advice. We have separated “Results and Discussion” into two chapters.

Author Response File: Author Response.docx