The Effects of Aronia melanocarpa Extract on Testosterone-Induced Benign Prostatic Hyperplasia in Rats, and Quantitative Analysis of Major Constituents Depending on Extract Conditions

Round 1

Reviewer 1 Report

Dear authors,

congratulations a very interesting and well-designed work.
The article, in my opinion, is written correctly and it only needs to clarify a few inaccuracies below.

In the Materials and Methods section, it is necessary to complete the data concerning the statistical tests used to prepare the results.

When using the abbreviation for the first time, please give its full development e.g. IACUC (Institutional Animal Care and Use Committee - I think so)

Please explain and document your choice of the dose levels used for the extracts?

I understand that the animals received a specific dose of extracts not in feed or water but with a stomach probe. Therefore, it would be more appropriate to describe the administration as intragastric or oral gavage by cannula.

Is the DHT kit identification correct? The available MyBiosource catalog does not contain the kit with the given number #38342424.

In discussing the results, basic data on animal weight and feed and water consumption during the experiment are missing, which with the test model used seems to be necessary.

Have the authors considered the possibility of dividing the prostate into parts? The prostate in a rat is not a homogenous organ, like in humans too. Many authors point to differences in the level and expression of hormones (testosterone, DHT) and receptors (AR) in different parts of the prostate in rats (differences can reach several dozen times), which is crucial for the development of BPH and PCa. It looks like the photos in Fig. 1 shows only the ventral lobe of the prostate. Do the results presented in this paper concern only this part of the prostate? If so, this should be indicated in the text. Please explain what was the reason for this choice?

In figure 2 (a and b), please give the result per gram.

The description to Figure 3 (Results were expressed as the mean value (± SD) of animals in each group) does not correspond to the graphical blocks shown. Please change the description.

Table 1 is the missing units.

Did the paper undergo a linguistic correction by an English native speaker?

With best regards

 

Author Response

Dear reviewer,

We thank you for providing the helpful and insightful comments to improve our manuscript. Our point-by-point response to your comments is provided below. The modified parts are marked in Red.

 

1. In the Materials and Methods section, it is necessary to complete the data concerning the statistical tests used to prepare the results.

→ Thank you for your careful review on the manuscript. We added the statistical paragraph in Method section.

 

2. When using the abbreviation for the first time, please give its full development e.g. IACUC (Institutional Animal Care and Use Committee - I think so)

→ Thank you for the comments. We added the full terms of IACUC in the text.

 

3. Please explain and document your choice of the dose levels used for the extracts?

→ Though there is no perfect guide for the choice of dose level in the preclinical study of a food material, we searched previous studies for A. melanocarpa extracts and it was found that the administration dose varied 5~20ml extract (juice)/kg. If 100mg/kg is converted into a liquid, the amount equivalent to about 6.6~6.8ml of extract/kg.

In addition, as mentioned in manuscript, this is the first study to evaluate the effect of A. melanocarpa on BPH in vivo. The dose of 100 mg/kg is the most commonly attempted dose in animal experiments on food, especially in the screening step or in the step of confirming the presence or absence of effectiveness.

 

3. I understand that the animals received a specific dose of extracts not in feed or water but with a stomach probe. Therefore, it would be more appropriate to describe the administration as intragastric or oral gavage by cannula.

→ As the Reviewer suggested, we changed p.o. to oral gavage.

 

4. Is the DHT kit identification correct? The available MyBiosource catalog does not contain the kit with the given number #38342424.

→ We corrected catalog number of the DHT kit to MBS265478.

 

5. In discussing the results, basic data on animal weight and feed and water consumption during the experiment are missing, which with the test model used seems to be necessary.

→ Thank you for your careful review on the manuscript. The reason the data of body weight, feed, and water consumption was not presented in the text is that these data is not closely correlated with this study design, and we found no specific differences in feed volume and water intake between the groups. We provided all animals same water and pellet feed ad libitum, under the SPF condition. We checked body weight once a week for calculate and revise administration volume. Also, PI index was a calibrated value by the individual body weight.

 

6. Have the authors considered the possibility of dividing the prostate into parts? The prostate in a rat is not a homogenous organ, like in humans too. Many authors point to differences in the level and expression of hormones (testosterone, DHT) and receptors (AR) in different parts of the prostate in rats (differences can reach several dozen times), which is crucial for the development of BPH and PCa. It looks like the photos in Fig. 1 shows only the ventral lobe of the prostate. Do the results presented in this paper concern only this part of the prostate? If so, this should be indicated in the text. Please explain what was the reason for this choice?

→ Thank you for your careful review on the manuscript. As you pointed out, the ventral lobes are the target of the study. We also collected dorsal lobes at necropsy in the first place, but the absolute/relative weight of the dorsal lobes were not show significant differences between the groups in this study. In our point of view, the weight and size was the most important indicator of BPH, so that we are focused on the ventral lobes. We added more information about these considerations.

 

7. In figure 2 (a and b), please give the result per gram.

→ The amount of prostate tissue used for the measurement of prostate DHT and 5-alpha reductase level was about 50 mg/animal. We followed the user guide of the commercial ELISA kit, and the range of standard calibration curve was within pg. We believe that the actual value provides more accurate information than to convert the unit.

 

8. The description to Figure 3 (Results were expressed as the mean value (± SD) of animals in each group) does not correspond to the graphical blocks shown. Please change the description.

→ We added the mean and SD in the result, and recalculated the values.

 

9. Table 1 is the missing units.

→ Thank you for the comments. The missing Table 1 (serum biochemistry data) and the description on the results are added in the text.

 

10. Did the paper undergo a linguistic correction by an English native speaker?

→ Before submitting the article we received English correction by a colleague. We authors have reviewed and revised the manuscript during this revision.

 

With best regards

Reviewer 2 Report

Nutrients #794225

The manuscript "The Effects of Aronia melanocarpa Extract on Testosterone‐Induced Benign Prostatic Hyperplasia in Rats, and Quantitative Analysis of Major  Constituents Depending on Extract Conditions" presents relevant information about the beneficial effects of A. melanocarpa extracts on testosterone-induced benign prostatic hyperplasia (BPH) in rats. However, some sections of this manuscript can be improved.

 

Corrections are suggested to the authors in order to complete some points, clarify some doubts or correct some mistakes:

 

General considerations:

The reviewer considers that there is not much information on the statistical analysis applied (normality and homogeneity tests included). Furthermore, the Tables do not show statistical analysis. It is recommended to complete these points.

Authors are invited to add more critical discussion, using new references.

In Figures, significant letters should be added.

Please, check the format of the whole text carefully according to the Journal guidelines.

Please, it is recommended to check specifically:

- Abstract is too long. The abstract should be a single paragraph of about 200 words maximum.

- Check the font size in the text and in the Figures and Tables.

 

Specific suggestions:

Introduction

Lines 68-88. More references should be added concerning Aronia melanocarpa bioactivity (i.e. line 20, anti-diabetic, hypolipidemic hepatoprotective, and cardiovascular-protective effects).

Lines 89-91. Authors are recommended to justify deeper the importance of anthocyanins.

Line 92. "... depends on the temperature and solids content". Solids content? could authors clarify this point?

Lines 94-97. These lines could be in material and methods.

Line 95. Could the authors explain why different methods of extraction are used?

Lines 97-99. the objective is not clearly described. In this work, correlations between the major compounds of the extracts and the improvement in BPH are not presented. The effect of each extraction process on the different composition of each extract was also not evaluated. It is recommended rewrite this part.

 

Material & Methods

Line 113. Please, remove the colons.

Line 116. Please, specify, once the extraction is complete, how the extract is preserved.

Line 152. In the section "Results and discussion" there is no comment on "Blood Collection and Biochemical Analysis". What were these analyses done for?

Line 157. The term "triglyceride" is duplicated, please correct.

Lines 181 and 193. It is recommended to place points 2.8 and 2.9 after point 2.2, since all these points refer to the fruit extract.

Line 197. Please, It is recommended to specify that samples are referred to Aronia fruit.

Line 202. It would be interesting to specify which compounds are aimed to be detected by HPLC.

It is recommended to include a paragraph with the statistical design of the experiments, specifying the statistical tests used for data analysis.

 

Results and Discussion

Line 214. In the text "... (T2) produced the greatest reduction in the mean prostate weight and PI..." however no significant differences were shown for absolute weight. Please, rewrite.

Line 217. Please, remove  "with statistical significance", it is redundant.

Line 220. Could authors explain why there was no significant therapeutic effect after administration of saw palmetto? Is there any work that shows these same results?

Line 251. Although all four extracts showed a tendency to decrease DTH and 5AR, only T1 showed significant differences...authors should take into account the significant differences.

Line 256. Comparisons between PC and Aronia extracts have not been properly shown. In fact, figure 2 shows that they reach similar values, so there is no data to consider that Aronia extracts have a greater effect than PC. Please rewrite.

Line 282. T1 and T2 remarkably reduce the mRNA expression of AR, but these reductions were significant? In figure 3a, no asterisk was shown. When increases or decreases of a parameter are shown without significant differences, it is recommended to indicate this. Please, rewrite.

Lines 285-288 . It's not clear... It seems to be a confusion between PSA and PCNA in the text. Figure 3b corresponds to the PSA or PCNA? Please, clarify this point.

Lines 293-295. In the case of AR and PSA (fig 3a and 3c, respectively) there was not significant differences, thus, authors should avoid strongly asserting these results.

Authors are invited to comment the expression of AR, PSA and PCNA with the treatment T4.

Line 350.  In the text, it seems that methyl 3-O-caffeoylquinic acid has been found, however, according to Table 2, it was not detected. Please, rewrite.

Line 354. Although the authors argue " the contents of all compounds were highest in T1 than the other extracts (T2~T4) ", however, significant differences were not found? Please, rewrite.

Line 355. It is recommended to rewrite this sentence in order to explain better its meaning "The remarkable difference in the contents of components were found in cyanidin glycosides."

It is recommended to discuss the effect of the different extraction processes on the quantity and quality of phenolic compounds.

 

Conclusion

The conclusion should be rewritten in accordance with the above recommendations of the reviewer.

Line 372. Significantly higher? Could the authors clarify where the statistical analyses of these results are reflected?

Line 376. It is recommended not to use terms such as "inhibit" when the treatments did not reach levels similar to controls.

The conclusions have been expressed too specific. Authors are invited to be more generalist.

 

Tables and Figures.

Figure 1, 2, 3. The text explaining the composition of each treatment could be shortened or removed, because it is described in material and methods.

Figure 4. In the text, no reference is made about the chemical structure of compounds isolated from Aronia, how figure 4 contributes to the work?

 

Author Response

Reviewer 2

Dear reviewer,

We thank you for providing the helpful and insightful comments to improve our manuscript. Our point-by-point response to your comments is provided below. The modified parts are marked in Red.

 

1. The reviewer considers that there is not much information on the statistical analysis applied (normality and homogeneity tests included). Furthermore, the Tables do not show statistical analysis. It is recommended to complete these points. In Figures, significant letters should be added.

→ Due to the considerably large standard deviation of data in in vivo experiments, the sufficient statistical significance was not obtained. ^#p value vs. the sham group, and ^*p value vs. the VC group are expressed in Figure 1-3. In Table 1 and 2, the relative composition or content of compounds are expressed without statistical analysis because this is to compare the values ​​between the groups, not to set standards and to compare them.

 

2. Please, check the format of the whole text carefully according to the Journal guidelines.

→ Thank you for the comments. The whole manuscript has been carefully modified according to the Journal guidelines.

 

3. Please, it is recommended to check specifically:

- Abstract is too long. The abstract should be a single paragraph of about 200 words maximum.

- Check the font size in the text and in the Figures and Tables.

→ As the reviewer’s comments, the abstract has been shortened concisely. The font size in text, Figures and Tables are checked and corrected according to the Journal guidelines.

 

4. Lines 68-88. More references should be added concerning Aronia melanocarpa bioactivity (i.e. line 20, anti-diabetic, hypolipidemic hepatoprotective, and cardiovascular-protective effects).

→ As the reviewer suggested, the biological activities of A. melanocarpa are described and references are added.

 

5. Line 92. "... depends on the temperature and solids content". Solids content? could authors clarify this point?

→ Solid content means ‘brix’ of carrot juice. The sentence was revised.

 

6. Lines 89-91. Authors are recommended to justify deeper the importance of anthocyanins.

→ According to the Reviewer’s comment, the features and importance of anthocyanins are additionally described and references are added.

 

7. Lines 97-99. the objective is not clearly described. In this work, correlations between the major compounds of the extracts and the improvement in BPH are not presented. The effect of each extraction process on the different composition of each extract was also not evaluated. It is recommended rewrite this part.

→ Thank you for the comments. The Introduction has been revised all over to clarify the objective of research.

 

8. Line 95. Could the authors explain why different methods of extraction are used? Authors are invited to add more critical discussion, using new references.

→ The reason for setting the extraction conditions was additionally described in relation to the characteristics and stability of anthocyanins in Introduction.

 

9. It is recommended to discuss the effect of the different extraction processes on the quantity and quality of phenolic compounds.

→ According to the Reviewer’s comment, Result 3.5 was revised.

10. Lines 94-97. These lines could be in material and methods.

→ As the reviewer suggested, the sentence in line 94-97 was modified.

 

11. Line 113. Please, remove the colons.

→ The colons is deleted.

 

12. 12. Line 157. The term "triglyceride" is duplicated, please correct.

→ The duplicated term was deleted.

 

13. Lines 181 and 193. It is recommended to place points 2.8 and 2.9 after point 2.2, since all these points refer to the fruit extract.

→ Thank you for your careful review on the manuscript. The order of subsection in Method and Materials was modified.

 

14. Line 197. Please, It is recommended to specify that samples are referred to Aronia fruit.

→ “The sample” was changed to “Aronia extract prepared using ASE”.

 

15. Line 116. Please, specify, once the extraction is complete, how the extract is preserved.

→ Thank you for the comments. The preparation method of the extract obtained by ASE for in vivo and chemical study was additionally described.

 

16. Line 152. In the section "Results and discussion" there is no comment on "Blood Collection and Biochemical Analysis". What were these analyses done for?

→ Thank you for the comments. The missing Table 1 (serum biochemistry data) and the description on the results are added in the text.

 

17. Line 202. It would be interesting to specify which compounds are aimed to be detected by HPLC.

→ Thank you for the comments. Target compounds in HPLC and LC/MS analysis were presented in Results section instead.

 

18. It is recommended to include a paragraph with the statistical design of the experiments, specifying the statistical tests used for data analysis.

→ Thank you for your careful review on the manuscript. We added the statistical paragraph in Method section.

 

19. Line 214. In the text "... (T2) produced the greatest reduction in the mean prostate weight and PI..." however no significant differences were shown for absolute weight. Please, rewrite.

→ The sentence has been revised.

 

20. Line 217. Please, remove "with statistical significance", it is redundant.

→ “with statistical significance" is removed in the sentence.

 

21. Line 220. Could authors explain why there was no significant therapeutic effect after administration of saw palmetto? Is there any work that shows these same results?

Line 256. Comparisons between PC and Aronia extracts have not been properly shown. In fact, figure 2 shows that they reach similar values, so there is no data to consider that Aronia extracts have a greater effect than PC. Please rewrite.

→ As the Reviewer suggested, the description on the use of Saw palmetto (PC) and the results has been added. Also, the expression that “Aronia extract was more potent than positive control” has been removed.

 

22. Line 251. Although all four extracts showed a tendency to decrease DTH and 5AR, only T1 showed significant differences...authors should take into account the significant differences.

→ As the Reviewer suggested, the description of result 3.2 has been revised.

 

23. Line 282. T1 and T2 remarkably reduce the mRNA expression of AR, but these reductions were significant? In figure 3a, no asterisk was shown. When increases or decreases of a parameter are shown without significant differences, it is recommended to indicate this. Please, rewrite.

→ In PCR experiments, the standard deviation of the VC group was considerably large. Though we observed the tendency to decrease in AR and PSA expressions in A. melanocarpa-treated group, the statistical significance was not obtained. The sentence was modified, and this consideration is added in the text.

 

24. Lines 285-288 . It's not clear... It seems to be a confusion between PSA and PCNA in the text. Figure 3b corresponds to the PSA or PCNA? Please, clarify this point.

→ Figure 3a refers to PCNA and Figure 3b refers to PSA expression. The sentence was corrected.

 

25. Lines 293-295. In the case of AR and PSA (fig 3a and 3c, respectively) there was not significant differences, thus, authors should avoid strongly asserting these results.

→ Thank you for the comments. As the reviewer suggested, we clearly stated that statistical significance was not obtained on AR and PSA result, and described the “trendency” of mRNA expression change based on the average value of each group. Also, for data where statistical significance was not confirmed, the expression 'significant' was deleted.

 

26. Authors are invited to comment the expression of AR, PSA and PCNA with the treatment T4.

→ The description on T3 and T4-treated group was added.

 

27. Line 350. In the text, it seems that methyl 3-O-caffeoylquinic acid has been found, however, according to Table 2, it was not detected. Please, rewrite.

→ Through chromatographic separation, we isolated four compounds including 1-(3, 4-dihydroxycinnamoyl cyclopenta-2, 3-dilo), methyl 3-O-caffeoylquinic acid and cyanidin glycosides and cyanidin-3-xyloside from A. melanocarpa extract. Using these compounds as standard compounds, we performed quantititive experiments using HPLC. As the reviewer pointed out, methyl 3-O-caffeoylquinic acid was not dectected. To avoid a confusion, the sentence was modified.

28. Line 354. Although the authors argue " the contents of all compounds were highest in T1 than the other extracts (T2~T4) ", however, significant differences were not found? Please, rewrite. Line 355. It is recommended to rewrite this sentence in order to explain better its meaning "The remarkable difference in the contents of components were found in cyanidin glycosides."

→ Thank you for your careful review on the manuscript. The remarkable difference in the contents of components between Aronia extracts T1~T4 was cyanidin glycosides. The contents of cyanidin-3-glucoside and cyanidin-3-xylose were highest in T1. As suggested, the sentences was revised.

 

29. The conclusion should be rewritten in accordance with the above recommendations of the reviewer. Line 372. Significantly higher? Could the authors clarify where the statistical analyses of these results are reflected? Line 376. It is recommended not to use terms such as "inhibit" when the treatments did not reach levels similar to controls.

→ The expressions such as ‘significantly’ or ‘strongly’ was deleted where the inhibition/increase rate is not accurately presented. Accordingly, Conclusion section was revised based on comments the reviewer pointed out.

 

30. The conclusions have been expressed too specific. Authors are invited to be more generalist.

→ Thank you for the comments. Instead detailing the conclusion, we complemented and further discussed the results in “Results and Discussion” section.

 

31. Figure 1, 2, 3. The text explaining the composition of each treatment could be shortened or removed, because it is described in material and methods.

→ As the Reviewer suggested, the sentences explaining each treatment are removed. Instead, the sentence “The preparation and administration of T1~T4 are described in Materials and Methods” was added in Figure legends 1~3.

 

32. Figure 4. In the text, no reference is made about the chemical structure of compounds isolated from Aronia, how figure 4 contributes to the work?

→ Four compounds used in HPLC experiment (not LC/MS) was isolated by chromatographic separation. These were used as standard compounds for the quantitation of the content in Aronia extracts, T1~T4. To avoid the confusion, the figure legend 4 was removed in the text. Instead, the structures of each target compounds in LC/MS and HPLC was included in Table 2 and Table 3, respectively.

 

Wigh Best Regards,