Possibilities of Using Phyto-Preparations to Increase the Adaptive Capabilities of the Organism of Test Animals in Swimming

Round 1

Reviewer 1 Report

The paper details the protective effects of three test substances on heart and liver in response to cold water immersion. The foundation of the study is interesting, but as a whole the paper lacks detail and clarity. The signficance of the study and the rationale behind the study design are not made clear, nor are the test substances ever sufficiently described.

The abstract has more in-depth information on methodology than is strictly needed for an abstract, and could do with a lot more background information, such as significance of the study and some brief description or detail on the three test substances. The introduction likewise requires more depth. It is much too short and vague, and would be imrpoved with the addition of an explanation of the signficance of the study, where this study fits into previous research, and detail around the choice and background of the three test substances, time points, and analysed tissues and enzymes. The methods would also be improved with more detail, specifically in relation to animal husbandry. The results are somewhat confusing to follow, comparing 5 groups across 4 different timepoints. Perhaps it would be clearer as graphs, rather than, or in addition to, tables? The discussion is much better in terms of the level of detail, but is still not very cohesive. Rather than summarising the results and then seperately detailing evidence from previous research, it would be better to interweave the two. This would help clarify how this work fits into the broader picture.

Specific points:

[Lines 3 and 23] Can you explain what you mean by “the organism of test animals”?

[Line 23] It would be more useful to state the strain of rat, rather than just “outbred”.

[Lines 38, 117 and 141] What is meant by “intact level”?

[Lines 51-52] More references are needed to provide evidence of “numerous research”.

[Line 53] "the performance" of what?

[Line 54] "permanent exposure" of what and to what? Are you talking about organisms that live in cold water year-round?

[Lines 56-57] This sentence needs a reference.

[Line 63] What strain?

[Lines 64-66] Specific details of animal housing are needed.

[Line 67] What is the significance of the cold water? If the aim of the study was to investigate responses to cold-induced stress, why was a cold room not enough? If this was done to emulate previous studies, please provide a reference to them in a rationale behind the study design.

[Line 88] By "some animals", do you mean the remaining 5 of each group, or was there an additional time point?

[Line 96] What were the animals anaesthetised and euthanised with?

[All tables] What is the signficiance of the divide symbols? Please confirm if the numbers in brackets are 95% confidence intervals.

[Line 125] What do you mean by "multidirectional"? Do you mean that of the control and tretament group, one increased and the other decreased? Why can you not specify which is which?

[Line 144] That one of the test substances is synthetic should be mentioned and discussed before.

[Lines 146-147] Is this referring to the two non-synthetic substances? How did they differ? Again, be specific.

[Line 151-153] This is discussion, not a result.

[Line 155] Why are changes in ALT and AST described individually for the acute and subacute timepoints, but as a ratio for the chronic? The ratio is mentioned in the discussion of the subacute measure.

[Line 166] What, specifically, are you referring to by "its"?

[Lines 182-190] This is just a repeat of the results (lines 151-155).

[Line 200] The first sentence needs to be expanded upon.

[Line 231] It should be made clear much earlier on that the GBBH inhibitor is a reference group for the other two test substances.

[Line 2353-236] This as the aim for the paper should have been explicitly stated in both the abstract and introduction.

Author Response

Comments and Suggestions for Authors

The paper details the protective effects of three test substances on heart and liver in response to cold water immersion. The foundation of the study is interesting, but as a whole the paper lacks detail and clarity. The signficance of the study and the rationale behind the study design are not made clear, nor are the test substances ever sufficiently described.

The abstract has more in-depth information on methodology than is strictly needed for an abstract, and could do with a lot more background information, such as significance of the study and some brief description or detail on the three test substances. The introduction likewise requires more depth. It is much too short and vague, and would be imrpoved with the addition of an explanation of the signficance of the study, where this study fits into previous research, and detail around the choice and background of the three test substances, time points, and analysed tissues and enzymes. The methods would also be improved with more detail, specifically in relation to animal husbandry. The results are somewhat confusing to follow, comparing 5 groups across 4 different timepoints. Perhaps it would be clearer as graphs, rather than, or in addition to, tables? The discussion is much better in terms of the level of detail, but is still not very cohesive. Rather than summarising the results and then seperately detailing evidence from previous research, it would be better to interweave the two. This would help clarify how this work fits into the broader picture.

1. There are some articles confirming that these extracts can increase physical performance:

About Ligusticum wallichii:

• Wei Z, Dong C, Guan L, Wang Y, Huang J, Wen X. A metabolic exploration of the protective effect of Ligusticum wallichiion IL-1β-injured mouse chondrocytes. Chin Med. 2020 Feb 1;15:12. doi: 10.1186/s13020-020-0295-0. PMID: 32025239; PMCID: PMC6995652.
• Shao Z, Wu P, Wang X, Jin M, Liu S, Ma X, Shi H. Tetramethylpyrazine Protects Against Early Brain Injury and Inhibits the PERK/Akt Pathway in a Rat Model of Subarachnoid Hemorrhage. Neurochem Res. 2018 Aug;43(8):1650-1659. doi: 10.1007/s11064-018-2581-0. Epub 2018 Jun 27. PMID: 29951731.
• Song XG, Zhou W, Chen C, Wang SM, Liang SW. [Study on material base of Ligusticum wallichii for treating brain ischemia and its molecular mechanism based on molecular docking]. Zhongguo Zhong Yao Za Zhi. 2015 Jun;40(11):2195-8. Chinese. PMID: 26552180.
• Bo Jiang, MD, PhD, Chao Huang, MD, PhD, Xiang-Fan Chen, MD, Li-Juan Tong, MSc, Wei Zhang, MD, Tetramethylpyrazine Produces Antidepressant-Like Effects in Mice Through Promotion of BDNF Signaling Pathway, International Journal of Neuropsychopharmacology, Volume 18, Issue 8, June 2015, pyv010, https://doi.org/10.1093/ijnp/pyv010
• Yang B, Li H, Qiao Y, Zhou Q, Chen S, Yin D, He H, He M. Tetramethylpyrazine Attenuates the Endotheliotoxicity and the Mitochondrial Dysfunction by Doxorubicin via14-3-3γ/Bcl-2. Oxid Med Cell Longev. 2019 Dec 3;2019:5820415. doi: 10.1155/2019/5820415. PMID: 31885804; PMCID: PMC6914960.
• Wang J, Liu N, Zhang F. Tetramethylpyrazine Protects Oxidative Stability and Gelation Property of Rabbit Myofibrillar Proteins. Food Sci Anim Resour. 2019 Aug;39(4):623-631. doi: 10.5851/kosfa.2019.e52. Epub 2019 Aug 31. PMID: 31508592; PMCID: PMC6728824.
• Li X, Wang Q, Ren Y, Wang X, Cheng H, Yang H, Wang B. Tetramethylpyrazine protects retinal ganglion cells against H2O2‑induced damage via the microRNA‑182/mitochondrial pathway. Int J Mol Med. 2019 Aug;44(2):503-512. doi: 10.3892/ijmm.2019.4214. Epub 2019 May 29. PMID: 31173163; PMCID: PMC6605642.
• Chen H, Cao J, Zhu Z, Zhang G, Shan L, Yu P, Wang Y, Sun Y, Zhang Z. A Novel Tetramethylpyrazine Derivative Protects Against Glutamate-Induced Cytotoxicity Through PGC1α/Nrf2 and PI3K/Akt Signaling Pathways. Front Neurosci. 2018 Aug 15;12:567. doi: 10.3389/fnins.2018.00567. PMID: 30158850; PMCID: PMC6104130.

 

About Eleutherococcus senticosus: 

• Sumiyoshi M, Kimura Y. Effects of Eleutherococcus senticosusCortex on Recovery from the Forced Swimming Test and Fatty Acid β-Oxidation in the Liver and Skeletal Muscle of mice. Nat Prod J. 2016 Mar;6(1):49-55. doi: 10.2174/2210315506999151207145020. PMID: 28553575; PMCID: PMC5427804.
• Gaire B, Lim D. Antidepressant effects of Radix et Caulis Acanthopanacis Santicosi extracts on rat models with depression in terms of immobile behavior. J Tradit Chin Med. 2014 Jun;34(3):317-23. doi: 10.1016/s0254-6272(14)60096-0. PMID: 24992759.
• Ptaszyńska AA, Załuski D. Extracts from Eleutherococcus senticosus(Rupr. et Maxim.) Maxim. Roots: A New Hope Against Honeybee Death Caused by Nosemosis. Molecules. 2020 Sep 28;25(19):4452. doi: 10.3390/molecules25194452. PMID: 32998304; PMCID: PMC7582972.
• Miyauchi-Wakuda S, Kagota S, Maruyama-Fumoto K, Shiokawa Y, Yamada S, Shinozuka K. Acanthopanax senticosus Root Extract Exerts Dual Action on Mouse Ileal Smooth Muscle Function, Leading to Modulation of Gastrointestinal Motility. Biol Pharm Bull. 2020;43(5):817-822. doi: 10.1248/bpb.b19-01027. PMID: 32378558.
• Huang HJ, Huang CY, Lee M, Lin JY, Hsieh-Li HM. Puerariae Radix Prevents Anxiety and Cognitive Deficits in Mice Under Oligomeric Aβ-Induced Stress. Am J Chin Med. 2019;47(7):1459-1481. doi: 10.1142/S0192415X19500757. PMID: 31752523.
• Panossian A, Seo EJ, Efferth T. Novel molecular mechanisms for the adaptogenic effects of herbal extracts on isolated brain cells using systems biology. Phytomedicine. 2018 Nov 15;50:257-284. doi: 10.1016/j.phymed.2018.09.204. Epub 2018 Sep 20. PMID: 30466987.

 

Specific points:

[Lines 3 and 23] Can you explain what you mean by “the organism of test animals”? – „the organism of test animals” is the animals on which the experiments were conducted.

 [Line 23] It would be more useful to state the strain of rat, rather than just “outbred”. – the rats were mongrel.

 [Lines 38, 117 and 141] What is meant by “intact level”? – pre-intervention level

[Lines 51-52] More references are needed to provide evidence of “numerous research”. –

• Bierens JJ, Lunetta P, Tipton M, Warner DS. Physiology Of Drowning: A Review. Physiology (Bethesda). 2016 Mar;31(2):147-66. doi: 10.1152/physiol.00002.2015. PMID: 26889019.
• BAKER MA, HORVATH SM. INFLUENCE OF WATER TEMPERATURE ON OXYGEN UPTAKE BY SWIMMING RATS. J Appl Physiol. 1964 Nov;19:1215-8. doi: 10.1152/jappl.1964.19.6.1215. PMID: 14232327.
• Boujezza H, Sghaier A, Ben Rejeb M, Gargouri I, Latiri I, Ben Saad H. Effects of cold water immersion on aerobic capacity and muscle strength of young footballers. Tunis Med. 2018 Feb;96(2):107-112. PMID: 30324975.

 [Line 53] "the performance" of what? – According to some studies, exposure to cold water on the skin of athletes can increase their performance by stimulating the processes of lipolysis.

[Line 54] "permanent exposure" of what and to what? Are you talking about organisms that live in cold water year-round? – The permanent exposure of temperature leads to certain metabolic changes caused by hormonal deviations.

[Lines 56-57] This sentence needs a reference.
Effects of Acute Cold Stress After Long-Term Cold Stimulation on Antioxidant Status, Heat Shock Proteins, Inflammation and Immune Cytokines in Broiler Heart.

Wei H, Zhang R, Su Y, Bi Y, Li X, Zhang X, Li J, Bao J.Front Physiol. 2018 Nov 13;9:1589. doi: 10.3389/fphys.2018.01589. eCollection 2018.

 

Effects of prior cold stimulation on inflammatory and immune regulation in ileum of cold-stressed broilers.

Su Y, Zhang X, Xin H, Li S, Li J, Zhang R, Li X, Li J, Bao J.Poult Sci. 2018 Dec 1;97(12):4228-4237. doi: 10.3382/ps/pey308.

 

[Line 63] What strain? - The rats were mongrel.

 [Lines 64-66] Specific details of animal housing are needed. - We used mongrel male rats obtained from the Stolbovaya nursery of the Russian Academy of Sciences

 [Line 67] What is the significance of the cold water? If the aim of the study was to investigate responses to cold-induced stress, why was a cold room not enough? If this was done to emulate previous studies, please provide a reference to them in a rationale behind the study design.

4 С water temperature is the standard conditions for cold swimming.

A single administration of Neurotropin reduced the elongated immobility time in the forced swimming test of rats exposed to repeated cold stress.

Nasu T, Kubo A, Queme LF, Mizumura K.Behav Pharmacol. 2019 Oct;30(7):547-554. doi: 10.1097/FBP.0000000000000488.

 

Cold water swimming is a more appropriate stress model.

The changes of nociception and the signal molecules expression in the dorsal root ganglia and the spinal cord after cold water swimming stress in mice.

Feng JH, Sim SM, Park JS, Hong JS, Suh HW.Korean J Physiol Pharmacol. 2021 May 1;25(3):207-216. doi: 10.4196/kjpp.2021.25.3.207.

 

Modulation of corticosterone and changes of signal molecules in the HPA axis after cold water swimming stress.

Feng JH, Sim SM, Park JS, Hong JS, Suh H.Anim Cells Syst (Seoul). 2021 Feb 23;25(1):37-45. doi: 10.1080/19768354.2021.1890211.

The room temperature was not changed, it had standard vivarium temperature (18-21 C).

[Line 88] By "some animals", do you mean the remaining 5 of each group, or was there an additional time point? – The animals were also slaughtered before the start of cold exposure to determine the initial level of enzyme activity.

 [Line 96] What were the animals anaesthetised and euthanised with? – were decapitated under ether anesthesia, in compliance with the rules of euthanasia, according to Helsinki Declaration on the Humane Treatment of Animals

[All tables] What is the signficiance of the divide symbols? Please confirm if the numbers in brackets are 95% confidence intervals. Confirm

[Line 125] What do you mean by "multidirectional"? Do you mean that of the control and tretament group, one increased and the other decreased? Why can you not specify which is which? –

“Multidirectional” means that each drug caused its own changes.

 [Line 144] That one of the test substances is synthetic should be mentioned and discussed before.

Using meldonium to improve the adaptation of patients with cardiovascular disease to the effects of heat and correction of associated oxidative stress].

Smirnova MD, Svirida ON, Vitsenia MV, Kuzmina AE, Lankin VZ, Tikhaze AK, Konovalova GN, Ageev FT.Kardiologiia. 2014;54(7):53-9. doi: 10.18565/cardio.2014.7.53-59.

 

[Mildronate effects on oxidant stress in type 2 diabetic patients with diabetic peripheral (sensomotor) neuropathy].

Statsenko ME, Poletaeva LV, Turkina SV, Apukhtin AF, Dudchenko GP.Ter Arkh. 2008;80(10):27-30.

 

[Lines 146-147] Is this referring to the two non-synthetic substances? How did they differ? Again, be specific.

 [Line 151-153] This is discussion, not a result. –

 [Line 155] Why are changes in ALT and AST described individually for the acute and subacute timepoints, but as a ratio for the chronic? The ratio is mentioned in the discussion of the subacute measure.

 [Line 166] What, specifically, are you referring to by "its"? –

At the same time, against the background of the enzyme gamma-butyrobetaine hydroxylase inhibitor use, such changes did not occur, which makes it possible to interpret action of the drug as cardio-protective.

 [Lines 182-190] This is just a repeat of the results (lines 151-155).

[Line 200] The first sentence needs to be expanded upon. – this is the subtitle.

[Line 231] It should be made clear much earlier on that the GBBH inhibitor is a reference group for the other two test substances. –

[Line 235-236] This as the aim for the paper should have been explicitly stated in both the abstract and introduction. – The aim was added.

 

Reviewer 2 Report

The manuscript presented semms to describe the effects of various adaptogens on some parameters in rats forced to swim in cold water. However, in my opinion, the manuscript should not be published in its present form. My comments and suggestions are as follows:

1. The introduction is too short and has not provided some important information about the adaptogens used. Moreover, it is not explained here or in the Materials and methods section why the authors chose meldonium, eleutherococcus and Ligusticum Wallichii extract.
2. The purpose of the study is not precisely stated.
3. Materials and Methods:
   1. Row 71: 5 or 7 days a week?
   2. "Injected per os" - how was it administered to the rats - orally or intragastrically?
   3. Lines 88-89: Were these animals given meldonium etc. prior to decapitation? How many animals were used to determine initial enzyme activity levels?
   4. What about the basic parameters obtained during animal experiments i.e. body weight and body gain, water and feed intake?
   5. The weight of the organs should be given. Why did the authors choose only heart and liver?
   6. What about blood analysis? Were basic hematological parameters determined?
   7. ALT and ASP are not antioxidant enzymes.
   8. Why did the authors not determine ALT and AST in serum/plasma? It is very difficult to know whether an increase in the activity of these enzymes is indicative of cell destruction (especially when determined in homogenates). Serum/plasma activities are more indicative of cell destruction.
   9. Why did the authors choose to measure only GPO and SOD? What about catalase activity, TBARS or MDA concentration, GSH concentration in homogenates or TOS/TAS in serum? Only two enzymes in only two organs seems a bit inadequate.
4. Why did the Authors not provide baseline levels of the measured parameters (mean +- standard deviation or SEM)? Since the Authors sacrificed animals to obtain these data, they should include them, especially since the rest of the results are presented as % of baseline levels. Their absence makes all the results unreliable.
   Moreover, the results in the tables should also be presented as mean +- standard deviation or SEM.
5. In the discussion section, the authors do not discuss the results obtained and their significance, rather they repeat their results. This chapter should be rethought and rewritten.
6. Line 244: Is this correct?

Author Response

Comments and Suggestions for Authors

The manuscript presented semms to describe the effects of various adaptogens on some parameters in rats forced to swim in cold water. However, in my opinion, the manuscript should not be published in its present form. My comments and suggestions are as follows:

1. The introduction is too short and has not provided some important information about the adaptogens used. Moreover, it is not explained here or in the Materials and methods section why the authors chose meldonium, eleutherococcus and Ligusticum Wallichii extract.

 

A similar answer was in the first review

 

1. The purpose of the study is not precisely stated. – The purpose of the work was to study the phyto-preparations potential to increase the adaptive capabilities of the body of experimental animals during swimming.

 

1. Materials and Methods:
1. Row 71: 5 or 7 days a week? – 7 days a week
2. "Injected per os" - how was it administered to the rats - orally or intragastrically? - orraly
3. Lines 88-89: Were these animals given meldonium etc. prior to decapitation? How many animals were used to determine initial enzyme activity levels? These animals were given meldonium etc. prior to decapitation. After cold exposure, 5 rats were randomly selected from each group.
4. What about the basic parameters obtained during animal experiments i.e. body weight and body gain, water and feed intake? Rats weighed 350 - 400 g. Animals were fed dry food and drink ad libitum.

 

1. The weight of the organs should be given. Why did the authors choose only heart and liver?

 

It is easier to perform a biochemical analysis with the heart and liver due to the high weight.

 

1. What about blood analysis? Were basic hematological parameters determined?

No, they were not.

1. ALT and ASP are not antioxidant enzymes.

They are just enzymes.

1. Why did the authors not determine ALT and AST in serum/plasma? It is very difficult to know whether an increase in the activity of these enzymes is indicative of cell destruction (especially when determined in homogenates). Serum/plasma activities are more indicative of cell destruction.

We had reagents only for determination ALT and AST in homogenates.

1. Why did the authors choose to measure only GPO and SOD? What about catalase activity, TBARS or MDA concentration, GSH concentration in homogenates or TOS/TAS in serum? Only two enzymes in only two organs seems a bit inadequate.

We had reagents only for determination only for determination GPO and SOD.

 

 

1. Why did the Authors not provide baseline levels of the measured parameters (mean +- standard deviation or SEM)? Since the Authors sacrificed animals to obtain these data, they should include them, especially since the rest of the results are presented as % of baseline levels. Their absence makes all the results unreliable.
   Moreover, the results in the tables should also be presented as mean +- standard deviation or SEM.

 

Another reviewer agreed that there should be a median (interquartile span) – I also agree with him – because I am not sure about the normal law of the distribution of parameters.

 

1. In the discussion section, the authors do not discuss the results obtained and their significance, rather they repeat their results. This chapter should be rethought and rewritten.

 

1. Line 244: Is this correct? incorrect

 

Round 2

Reviewer 2 Report

The Authors did not answer many crucial issues, and hardly improved manuscript, so, in my opinion the manuscript in the present form should be rejected. Moreover, the Authors did not included the answers to the reviewer in the revised form of the manuscript.

1. The introduction still lacks the information about the used adaptogens and it is not known, why Authors choose specifically Ligusticum Wallichii extract and extract of eleutherococcus. According to the “Instructions of Authors”:
   “The introduction should briefly place the study in a broad context and highlight why it is important. It should define the purpose of the work and its significance, including specific hypotheses being tested. The current state of the research field should be reviewed carefully and key publications cited. Please highlight controversial and diverging hypotheses when necessary. Finally, briefly mention the main aim of the work and highlight the main conclusions. Keep the introduction comprehensible to scientists working outside the topic of the paper.”

2. What was the rationale for an use of meldonium? This should be explained also.
3. About the animals decapitated for the determination of initial levels:
   The Authors state that “Some animals were also slaughtered before the start of cold exposure to determine the initial level of enzyme activity.” But in the Authors’ answer: “These animals were given meldonium etc. prior to decapitation. After cold exposure, 5 rats were randomly selected from each group.”
   So did the animals were decapitated before or after the cold exposure?
   Why the Authors still did not provide the initial levels of enzymes activity, especially when they decapitated 20 animals, so 1/5 of all animals in this experiment?! Moreover, they state that “The activity of enzymes AST, ALT, GPO was expressed in mmol/min g of tissue, and the activity of SOD was expressed in μmol / min g of tissue”. This really make all the results not reliable at all.

4. “What about the basic parameters obtained during animal experiments i.e. body weight and body gain, water and feed intake? Rats weighed 350 - 400 g. Animals were fed dry food and drink ad libitum.”
   This is not an answer to that question. In the animal experiments there is a need to check the health status of the animals, and one of basics parameters are body weight, body gain, water and feed intake through the experiment, and they should be provided.

5. The weight of the organs should be given. Did any differences in the organ weight were observed?
6. What was the rationale of determining ALT and AST in tissues? Is there any connection with the activity of these enzymes and adaptive capabilities?
7. There is no information that the results are presented as medians and what are the numbers in the brackets. The Authors did explain it only in the answers to other Reviewer.
8. The discussion section was not revised at all. In my opinion this section is still the repetition of their results and did not provide any significance of the obtained results, so it not acceptable.

 

Author Response

Dear Reviewer,

Thank you very much for your time and valuable comments, which all have been considered and incorporated. The detailed list of responses is given below. We hope that the modifications and explanation will be acceptable for you.

Yours sincerely,

Rydzik, corresponding author

 

 

1. The introduction still lacks the information about the used adaptogens and it is not known, why Authors choose specifically Ligusticum Wallichii extract and extract of eleutherococcus. According to the “Instructions of Authors”:
   “The introduction should briefly place the study in a broad context and highlight why it is important.It should define the purpose of the work and its significance, including specific hypotheses being tested. The current state of the research field should be reviewed carefully and key publications cited. Please highlight controversial and diverging hypotheses when necessary. Finally, briefly mention the main aim of the work and highlight the main conclusions. Keep the introduction comprehensible to scientists working outside the topic of the paper.”

 

Answer: The most commonly used phytopreparations are ligusticum wallichii¹, Eleutherococcus² and others³. There are publications that meldonium is a stress protector, especially in cold swimming conditions⁴. At the same time, the research results are very contradictory. In particular, there was a publication about the absence of adaptogenic effects in Eleutherococcus⁵. Some authors call meldonium the "gold standard" for stimulating physical performance in swimming⁶.

 

 

2. What was the rationale for an use of meldonium? This should be explained also.

 

Answer: described before

 

3. About the animals decapitated for the determination of initial levels:
   The Authors state that “Some animals were also slaughtered before the start of cold exposure to determine the initial level of enzyme activity.” But in the Authors’ answer: “These animals were given meldonium etc. prior to decapitation. After cold exposure, 5 rats were randomly selected from each group.”

So did the animals were decapitated before or after the cold exposure?
Why the Authors still did not provide the initial levels of enzymes activity, especially when they decapitated 20 animals, so 1/5 of all animals in this experiment?! Moreover, they state that “The activity of enzymes AST, ALT, GPO was expressed in mmol/min g of tissue, and the activity of SOD was expressed in μmol / min g of tissue”. This really make all the results not reliable at all.

 

 

Answer: We slaughtered the animals after the cold exposure. Also, 5 randomly selected animals from each group were slaughtered before cold exposure to determine the initial activity of the enzymes. Added a table with the initial activity of the enzymes.

 

 

4. “What about the basic parameters obtained during animal experiments i.e. body weight and body gain, water and feed intake? Rats weighed 350 - 400 g. Animals were fed dry food and drink ad libitum.”
   This is not an answer to that question. In the animal experiments there is a need to check the health status of the animals, and one of basics parameters are body weight, body gain, water and feed intake through the experiment, and they should be provided.

 

Answer: в ходе эксперимента вес животных не изменился. Added to the text

 

From our point of view, the weight of the animals did not change due to their sufficient maturity (12 weeks).

 

5. The weight of the organs should be given. Did any differences in the organ weight were observed?

 

Answer: There were no differences in the weight of organs isolated from animals of different groups. Added to the text.

 

 

6. What was the rationale of determining ALT and AST in tissues? Is there any connection with the activity of these enzymes and adaptive capabilities?

 

Answer:

In a number of studies, these enzymes are used to study the adaptive capabilities of rats in swimming conditions . The use of these enzymes is due to the fact that intense physical activity can stimulate cytolysis .

 

The text was added to the article in the discussion section

 

7. There is no information that the results are presented as medians and what are the numbers in the brackets. The Authors did explain it only in the answers to other Reviewer.

 

Answer:

Confidence intervals were determined on basis of calculating odds ratios (OR) with p <0.05.

It has already been added to the text

 

8. The discussion section was not revised at all. In my opinion this section is still the repetition of their results and did not provide any significance of the obtained results, so it not acceptable.

 

The section has been edited and expanded

Round 3

Reviewer 2 Report

Authors did improve the manuscript, but there are still some flaws, which prevent, in my opinion, accepting it.

• reader still do not know, why Ligusticum Wallichii extract and extract of eleutherococcus were chosen in this study.
• new table was prepared with the initial activities of studied enzymes, but activity of SOD is expressed there as mmol/min g (umol/min g in the text) and the names of the groups are different form the rest tables.
• the activity of GPO in the liver in control group is 3-fold higher than in the rest groups. Therefore it is impossible to compare this result with other groups. Calculations based on table 1 and 2 are presented below:
  
  

  	From table 2	Calculated from table 1
  Control group	84,62%	519 mmol/min g
  0,9% NaCl solution	87,43%	166 mmol/min g
  Inhibitor of the enzyme gamma-butyrobetaine hy-droxylase	112,90%	245 mmol/min g
  Wallich ligusticum extract	225,86%	524 mmol/min g
  Eleutherococcus extract	78,05%	188 mmol/min g

  So comparing the percentage in the case of hepatic GPO is not correct.
• There is still not know, in what form the results are presented (mean, median?). In the new table 1 also this information is missing.
• The Authors stated in some places that "numerous studies" confirm something, but there is only one reference after this kind of statement.
• They explanation about ALT and AST is not sufficient, because the Authors cited work, in which the serum level of these enzymes were determined.

 

Author Response

Authors did improve the manuscript, but there are still some flaws, which prevent, in my opinion, accepting it.

Answer:

1. reader still do not know, why Ligusticum Wallichii extract and extract of eleutherococcus were chosen in this study.

Answer:  We have added a description of these extracts and their properties and references in the introduction

 

2. new table was prepared with the initial activities of studied enzymes, but activity of SOD is expressed there as mmol/min g (umol/min g in the text) and the names of the groups are different form the rest tables.

 

Answer: Yes, it is the techical problem –we corrected in table, it is μmol/min g (red color)

The names of gropus also corrected

3. the activity of GPO in the liver in control group is 3-fold higher than in the rest groups. Therefore it is impossible to compare this result with other groups. Calculations based on table 1 and 2 are presented below:

Answer: It the techical error. We corrected. The result is 161. We market by red color

 

	From table 2	Calculated from table 1
Control group	84,62%	519 mmol/min g
0,9% NaCl solution	87,43%	166 mmol/min g
Inhibitor of the enzyme gamma-butyrobetaine hy-droxylase	112,90%	245 mmol/min g
Wallich ligusticum extract	225,86%	524 mmol/min g
Eleutherococcus extract	78,05%	188 mmol/min g

1. So comparing the percentage in the case of hepatic GPO is not correct.

Answer: Please see the answer up, it was the techical error

 

2. There is still not know, in what form the results are presented (mean, median?). In the new table 1 also this information is missing.

Answer: We add this information to the tables

 

3. The Authors stated in some places that "numerous studies" confirm something, but there is only one reference after this kind of statement.

Answer: We added literature

 

4. They explanation about ALT and AST is not sufficient, because the Authors cited work, in which the serum level of these enzymes were determined.

Answer: literature is added

 

 

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.