Effects of the Germinated Glycine max and Angelica gigas Nakai Mixture on Hepatic Lipid Metabolism and Bone Turnover Balance in Ovariectomized Rats
, Hwal Choi 1, Tae Sung Jung 2 and Jungkee Kwon 1,*
Round 1
Reviewer 1 Report
The language and organization of this manuscript are not acceptable in its present form. The manuscript can be reconsidered after a substantial revision. My individual comment is listed below.
- The period of the experiment four weeks is not sufficient to study the Effects of the Germinated Glycine max and Angelica gigas Nakai Mixture on Hepatic Lipid Metabolism and Bone Turnover Balance in Ovariectomized Rat at least the period of the experiment need eight weeks
- Page 4- line 156 mentions the name, model, and original of the centrifuge, and in line 199
- Page 4-line 156 Convert rpm to xg
- Some abbreviations must mention meaning (PBS, BCA reagent, BSA)
- Line215 adds a reference to statistical analysis
- The title of Table 1 Effects of GG and GAM mixtures on body and some organ weights of the OVX-induced rat model.
- Write under table Values are presented as mean ± SD (n = 3). Mean values within the same row with different superscript letters differ significantly (P< 0.05).
- Duncan's analysis is not mentioned in the methods
- Do you have to determine the amount of phytoestrogen in soybeans and the total isoflavone
- Do you have to determine the amount of Decursin in AG root
- GAM has anti-obesity effects the discussion is very poor please clarify the reason GAM has anti-obesity effects
- Combining the percent ratio of GG and AG not cited in methods
- - All references need a DOI added and need to be updated
Author Response
On behalf of the authors, I would like to thank you for providing us the opportunity to improve our manuscript once again. We appreciated the careful reading of our manuscript as well as commenting and suggesting for better manuscript.
We have carefully rewritten and reorganized our manuscript according to the comments from you.
We hope that you agree with our manuscript that has been not only through revised but also strengthened by tour comments.
Thank you for your kind consideration.
The language and organization of this manuscript are not acceptable in its present form. The manuscript can be reconsidered after a substantial revision.
Response: Thank you for suggesting about language editing again. We attached the certification of language editing from eWorldEditing, Inc.
The period of the experiment four weeks is not sufficient to study the Effects of the Germinated Glycine max and Angelica gigas Nakai Mixture on Hepatic Lipid Metabolism and Bone Turnover Balance in Ovariectomized Rat at least the period of the experiment need eight weeks.
Response: Before proceeding with this experiment, the pilot experiment was conducted after confirming that a period of 4 weeks after OVX can cause symptoms in menopause women. To analyze the pilot experiment’s results, we measured ALP activity which is related with osteoporosis and total cholesterol which is related with body lipid. The results of pilot experiment are attached through the file.
Page4 line 156 mentions the name, model, and original of the centrifuge, and in line 199
Page 4 line 156 Convert rpm to xg
Some abbreviations must mention meaning (PBS, BCA reagent, BCA)
Line215 adds a reference to statistical analysis
The title of Table 1 Effects of GG and GAM mixture on body and some organ weights of the OVX induced rat model. Write under table Values are presented as mean ± SD (n =3). Mean values within the same row with different superscript letter differ significantly (P< 0.05)
Response: We revised the part according to the reviewer's opinion and the reference. Thank you for your careful review.
Duncan’s analysis is not mentioned in the methods.
Response: We analyzed all the results using Tukey analysis method. Duncan is a one-way distribution analysis, and Turkey is also a similar system with one-way distribution analysis, so we conducted statistical analysis using this. Nor is Duncan supported in our statistical system. For this a reason, we did not mention about Ducan’s analysis.
Do you have to determine the amount of phytoestrogen in soybeans and the total isoflavone.
Response: We did not have to determine the amount of phytoestrogen in soybeans and the total isoflavone. Because the aim of this study is measuring the effects of soybeans and Angelica gigas Nakai Mixture as a extraction. For this reason, we did not analyze the amount of phytoestrogen in soybean and the total isoflavone.
Do you have to determine the amount of Decursin in AG root.
Response: We did not have to determine the amount of Decursin in AG root with the same reason why we had not determined the amount of phytoestrogen in soybeans and the total isoflavone.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The objective of present study was to evaluate the effect of germinated Glycine max and Angelica gigas Nakai on hepatic, bone mineralization in a menopause model. The topic of study is very clear and provided enough data but need to address the specific points. Other than this, MS needs extensive English grammar editing for presentation. Based on the thorough revision I suggest this manuscript address some of the original information; however, a substantial revision is required to improve the quality and contents of the article.
Specific:
1. Chemical composition of GG and AG is not given. Provide the chemical composition of supplements and feed also. It is also possible that feed ingredients might affect the parameters. Additionally, the treatments (alcohol/heat) could also degrade the compounds.
2. Why authors selected the small age rat (6 weeks) not adult? Some of the rats might have stunt growth during growing phase.
3. Why estrogen hormone was not measured as a direct indicator in response to supplements?
Author Response
On behalf of the authors, I would like to thank you for providing us the opportunity to improve our manuscript once again. We appreciated the careful reading of our manuscript as well as commenting and suggesting for better manuscript.
We hope that you agree with our manuscript that has been not only through revised but also strengthened by your comments.
Thank you for your kind consideration
Chemical composition of GG and AG is not given. Provide the chemical composition of supplements and feed also. It is also possible that feed ingredients might affect the parameters. Additionally, the treatments (alcohol/heat) could also degrade the compounds.
Response: We recognized and agreed with that it is possible that feed ingredients might affects the parameter. And the chmical compositions which are effective to symptoms are important. However, we did not have to determine the amount of chemical composition of GG and AG. Because the aim of this study is measuring the effects of soybeans and Angelica gigas Nakai Mixture as extractions not only chemical composition for functional food.
Why authors selected the small age rat (6 weeks) not adult? Some of the rats might have stunt growth during growing phase.
Response: We conducted the experiment referring to this paper (Yousefzadeh, Nasibeh, et al. "Ovariectomized rat model of osteoporosis: a practical guide." EXCLI journal 19 (2020): 89.). We selected 6 weeks-old rats. Because when under 6months-old rats have subjected to OVX, they have similar symptoms with postmenopausal women such as low response of cortical bone and response to intervention/treatment.
Why estrogen hormone was not measured as a direct indicator in response to supplements?
Response: The aim of this study is evaluating the preventive effects on postmenopausal symptoms by mixing Glycine max and Angelica gigas Nakai with reducing the amount of phytoestrogen for estrogen-sensitive women. Also mixing Angelica gigas Nakai with Glycine max keeping the same concentration, the absolute amount of Glycine max will decrease. This means GAM group’s estrogen hormone concentration are lower than GG group. For these reasons we did not measure estrogen hormone and estrogen receptors activities.
Reviewer 3 Report
The work demonstrated the potential of Glycine max and Angelica gigas for development as a beneficial ingredient for food to improve menopausal symptoms using ovariectomized Rats for the experiment. The animal experiment was approved and conducted according to the guidelines for animal experiments in the protocol.
The research result merits the work to be considered for publication because the findings demonstrated that combining GG and AG at a 7:3 ratio had anti-obesity, anti-inflammation, and anti-osteoporotic effects in an OVX-induced rat model. In similitude, a detailed understanding of anti-osteoporotic effects in pre-osteoblast MC3T3-E1 cells showed that GAM stimulated mineralization and in RANKL-induced osteoclast differentiation from RAW264.7 cells showed that GAM suppressed osteoclastogenesis.
However, the following observations are recommended for consideration
· Of course, seeds germinate hence “Germinated” in the title makes it seem Glycine max and Angelica gigas Nakai are not of plant seed source but of something else. Kindly revise the title to “Effects of Glycine max and Angelica gigas Nakai Mixture on Hepatic Lipid Metabolism and Bone Turnover Balance in Ovariectomized Rats”
· Also revise “germinated” in line 15 and Line 93. What is the difference between sprouted soybean and the germinated soybean embryo in Line 93. The term germination seems unclear. Authors are requested to specify the germination stage for clarity throughout the article if they want to go by “germinated soybean” or authors are recommended to certainly clarify.
· Define LDL and HDL in the abstract and revise the Line 189-190
· Cite a reference for line 53-54
· kindly revise Line 71-72 statement for clarity. At what germination stage is soybean found of increased nutritional value and bioactive compounds.
· In the materials and method section kindly specify the extract thus powder extract or liquid extract.
· Line 234 is a repetition of methodology in section 2.9. omit the repeated statement
· Line 253, 230, 233, 266, 279, 294, 312, 353, 412, 418, 454, 466, and other sections throughout the article has the wording “we” omit the “we” and rewrite the statement to make the work more scientific.
· Line 318 is a repetition of line 42-43 kindly revise it for clarity
· Line 332 stated “GAM did not have cytotoxic effects on MC3T3-E1 cells” however section 4 lacks a clear mechanism to the line 332 statement. Authors are recommended to elaborate on key mechanisms that brought about such findings.
· Revise Line 384-392 with key mechanisms that led to the reported result.
· Line 420-421 is a repeated statement found in line 38-41. Kindly revise by avoiding the repetition of contextual statements. Similar repetitions are observed in other sections of the manuscript. Kindly read thoroughly and revised such sections accordingly.
Comments for author File: Comments.pdf
Author Response
On behalf of the authors, I would like to thank you for providing us the opportunity to improve our manuscript once again. We appreciated the careful reading of our manuscript as well as commenting and suggesting for better manuscript.
We hope that you agree with our manuscript that has been not only through revised but also strengthened by your comments.
Thank you for your kind consideration
Seeds germinate hence “Germinated” in the title makes it seem Glycine max and Angelica gigas Nakai are not of plant seed source but of something else. Kindly revise the title to “Effects of Glycine max and Angelica gigas Nakai Mixture on Hepatic Lipid Metabolism and Bone Turnover Balance in Ovariectomized Rats
Response: The reason we use “Germinated” in the title is that germination process makes composition changes in soybean. Specifically, increased phytoestrogen which has the preventive effects on postmenopausal symptoms in soybean by germination was confirmed other studies. Unfortunately, the reason mentioned above we could not revise the title of article.
Also revise “germinated” in line 15 and Line 93. What is the difference between sprouted soybean and the germinated soybean embryo in Line 93. The term germination seems unclear. Authors are requested to specify the germination stage for clarity throughout the article if they want to go by “germinated soybean” or authors are recommended to certainly clarify.
Response: We revised the part according to the reviewer's opinion and the reference. Thank you for your careful review.
Define LDL and HDL in the abstract and revise the Line 189-190
Response: We revised the part according to the reviewer's opinion and the reference. Thank you for your careful review.
Cite a reference for line 53-54
Response: We revised the part according to the reviewer's opinion and the reference. Thank you for your careful review.
kindly revise Line 71-72 statement for clarity. At what germination stage is soybean found of increased nutritional value and bioactive compounds.
Response: We revised the part according to the reviewer's opinion and the reference. Thank you for your careful review. Germination can be divided 5 stage imbibtion, respiration, effect of light on seed germination, mobilization of reserves, development of embryo axis into seedling. The third stage ‘effect of light on seed germination’ affect to the compound’s changing during the germination. The amounts of light and time can affect the amount of compound in the soybean. Our answer is referring with ‘Germination and Fermentation of Soybeans: Two Healthy Steps to Release Angiotensin Converting Enzyme Inhibitory Activity Compounds. (2019)’.
In the materials and method section kindly specify the extract thus powder extract or liquid extract.
Response: We extracted the samples as liquid after that dried as powder form. And we revised the part according to the reviewer's opinion and the reference. Thank you for your careful review.
Line 234 is a repetition of methodology in section 2.9. omit the repeated statement
Response: We revised the part according to the reviewer's opinion and the reference. Thank you for your careful review.
Line 253, 230, 233, 266, 279, 294, 312, 353, 412, 418, 454, 466, and other sections throughout the article has the wording “we” omit the “we” and rewrite the statement to make the work more scientific.
Response: We revised the part according to the reviewer's opinion and the reference. Thank you for your careful review.
Line 318 is a repetition of line 42-43 kindly revise it for clarity.
Response: We revised the part according to the reviewer's opinion and the reference. Thank you for your careful review.
Line 420-421 is a repeated statement found in line 38-41. Kindly revise by avoiding the repetition of contextual statements. Similar repetitions are observed in other sections of the manuscript. Kindly read thoroughly and revised such sections accordingly.
Response: We revised the part according to the reviewer's opinion and the reference. Thank you for your careful review.
Reviewer 4 Report
This study represents an interesting vision of a combined mixture of ingredients with a powerful biological effect as a functional food. The results obtained are very beneficial by demonstrating that the mixture (GAM) decreases postmenopausal inflammation, fat accumulation, liver damage and bone absorption.
11) Line 454: The authors indicate a reference (#47) that does not appear in the References section. Are the authors referring to reference #46?
On the other hand, where is reference #36 in the text?
Line 266: Is reference #45 in that paragraph correct?
22) Lines 155 - 156: "After 4 weeks of administration, blood was collected from the abdominal aorta vein".
Authors should correct and place the descending (or abdominal) aortic artery. Unless the authors collected from the inferior vena cava.
33) Lines 217 - 219: Delete the following paragraph: "This section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn."
44) Lines 242 - 243: "The serum HDL concentration decreased by 2.0% in the OVX group, compared with the Sham group (Figure 1D) and increased by 15.3% and 39.2% in the GAM50 and GAM100 groups, respectively, compared with the OVX group".
But that decrease is not significant, nor is it significant with the increase in the GAM50 group.
55) Figure 1: In the study of the lipid profile, the greatest advantage of the GAM100 combination is observed in HDL levels, however, it is not observed as effective in regulating total cholesterol and triglycerides compared to an OVX group (although the benefits are evident compared to LDL and for HDL).
66) In the same way, GAM100 is very effective against inflammatory mediators, but do the authors have any suspicions as to why the highest dose (GAM100) seems less effective than the half (GAM50)?
77) Figure 3: What is the reason for higher levels of inflammatory mediators in OVX rats treated with GAM25 compared to the OVX group?
Comments for author File: Comments.pdf
Author Response
On behalf of the authors, I would like to thank you for providing us the opportunity to improve our manuscript once again. We appreciated the careful reading of our manuscript as well as commenting and suggesting for better manuscript.
We hope that you agree with our manuscript that has been not only through revised but also strengthened by your comments.
Thank you for your kind consideration.
Line 454: The authors indicate a reference (#47) that does not appear in the References section. Are the authors referring the reference #46?
Response: We revised the part according to the reviewer's opinion and the reference. Thank you for your careful review.
On the other hand, where is reference #36 in the text?
Response: We revised the part according to the reviewer's opinion and the reference. Thank you for your careful review.
Line 266: is reference #45 in that paragraph, correct?
Response: We revised the part according to the reviewer's opinion and the reference. Thank you for your careful review.
Lines 155 - 156: "After 4 weeks of administration, blood was collected from the abdominal aorta vein".
Authors should correct and place the descending (or abdominal) aortic artery. Unless the authors collected form the inferior vena cava.
Response: We checked it again but mentioned correct place that blood was collected.
Line 217 – 219: Delete the following paragraph: “This section may be divied by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn.
Response: We revised the part according to the reviewer's opinion and the reference. Thank you for your careful review.
Line 242-234. “The serum HDL concentration decreased by 2.0% in the OVX group, compared with the Sham group (Figure 1D) and increased by 15.3% and 39.2% in the GAM50 and GAM 100 groups, respectively, compared with the OVX group.”
But that decrease is not significant, nor is it significant with the increased in the GAM50 group.
Figure 1 : In the study of the lipid profile, the greatest advantage of the GAM100 combination is observed in HDL levels, however, it is not observed as effective in regulating total cholesterol and triglycerides compared to an OVX group (although the benefits are evident compared to LDL and for HDL.
In the same way, GAM100 is very effective against inflammatory mediators, but do the authors have any suspicions as to why the highest dose (GAM100) seems less effective than the half (GAM50)?
Response: When evaluate the protective effects of liver damage and anti-inflammatory effects on OVX rats, GAM100 group is the most effective. While the improvement effects of lipid profile on OVX rats, in GAM100 group deteriorate compared with GAM50 group. For this reason we considered that the dose of GAM50 is more effective than GAM100 dose on the overall point.
Figure 3: What is the reason for higher levels of inflammatory mediators in OVX treated with GAM25 compared to the OVX group?
Response: We administrated all the group and gave same stress to the rats. One of possibility of the reason is the concentration of GAM25 is low to activate anti-inflammation effect on the OVX-induced rats. On the other hand, the sugar content in the mixture induced inflammatory mediators. The other possibility that GAM25 group’s inflammatory mediators’ levels higher than OVX group, it is expected to be the result of different reactivity for each individual to the allergic reaction to our sample.
Round 2
Reviewer 1 Report
no comment
Reviewer 2 Report
The authors have made substantial revision in response to my comments. Also authors made the extensive English editing in revised manuscript. Based on current observation, the manuscript is acceptable in journal.
Reviewer 4 Report
I appreciate the authors who have taken their time to answer all the questions, as well as to improve the manuscript.
