Acute Hepatic and Renal Toxicity Assessment of Euphorbia huanchahana (Klotzsch & Garcke) Boissier (Huachangana) in Holtzman Rats
Round 1
Reviewer 1 Report
I what to that the authors for this informative manuscript on the Acute hepatic and renal toxicity of Euphorbia huanchahana (Klotzsch & Garcke) Boissier (huachangana), a medicinal plan extract, in Holtzman rats. The manuscript is simple and easy to follow. In order to improve the scope and readability of the manuscript, the following comments should be considered:
Major comments:
In the introduction, the indication, and potential indications, of the Euphorbia huanchahana (Klotzsch & Garcke) Boissier (huachangana), should be escribe in greater details as it is not clear to this point the purpose of using/studying this drug. Better incorporating this background would help the reader to understand the importance of the study here presented.
The usual use, of the medication, should also be described as rarely medications are only taken once like in the current study. If the treatment is usually used for a period of time, it should be at least mentioned if not tested.
The authors should compare between the response of male and female for the toxicity as they already have the data. If genre differences are identified, it would be very interesting to know for future study. Furthermore, gender-based difference in metabolism are increasingly recognized and documented.
Line 181: “phytochemical analysis did not reveal concentrations of steroids and/or triterpenes (Figure 2).” I don’t see any results presented only on that in the figure 2, only the solubility results are presented. The flow chart is presented in table A2 but not clearly identified as supplemental data? Moreover, there is no methodology and to result formerly presented for this section. This whole section should be reworked.
Figure 3: This graph is very confusing and labelled clearly. Not all the line are identified. I believe this format should be reconsidered to present the data as I was not really able to assess the validity of this section as I was not able to understand it.
Minor comments:
The English should be corrected
Figure 1: Based on international convention it would made more sense that the positive results be in green and the negative in red, not the other way around.
Figure 4: Can a post-hoc analysis be done to determine at which day and the direction on the change are observed. This information should be added to the discussion.
Author Response
Major comments:
In the introduction, the indication, and potential indications, of the Euphorbia huanchahana (Klotzsch & Garcke) Boissier (huachangana), should be escribe in greater details as it is not clear to this point the purpose of using/studying this drug. Better incorporating this background would help the reader to understand the importance of the study here presented.
RESPONSE: We correct the text.
The usual use, of the medication, should also be described as rarely medications are only taken once like in the current study. If the treatment is usually used for a period of time, it should be at least mentioned if not tested.
RESPONSE: We correct the text.
The authors should compare between the response of male and female for the toxicity as they already have the data. If genre differences are identified, it would be very interesting to know for future study. Furthermore, gender-based difference in metabolism are increasingly recognized and documented.
RESPONSE: We added this analysis in the section 3.2.
Line 181: “phytochemical analysis did not reveal concentrations of steroids and/or triterpenes (Figure 2).” I don’t see any results presented only on that in the figure 2, only the solubility results are presented. The flow chart is presented in table A2 but not clearly identified as supplemental data? Moreover, there is no methodology and to result formerly presented for this section. This whole section should be reworked.
RESPONSE: The phytochemical results have been explained, the appendices have been changed from Tables to Figure A1 and A2.
Figure 3: This graph is very confusing and labelled clearly. Not all the line is identified. I believe this format should be reconsidered to present the data as I was not really able to assess the validity of this section as I was not able to understand it.
RESPONSE: A brief description has been included to improve understanding. We consider that the reading by marker is understood with data in percentages.
Minor comments:
The English should be corrected
RESPONSE: We correct the text.
Figure 1: Based on international convention it would made more sense that the positive results be in green and the negative in red, not the other way around.
RESPONSE: We are displaying the positive results in red boxes to highlight these tests. We understand the international context but we think the green result in the graph is missing a bit.
Figure 4: Can a post-hoc analysis be done to determine at which day and the direction on the change are observed. This information should be added to the discussion.
RESPONSE: We added this analysis in the section 3.3.
Author Response File: 
Author Response.docx
Reviewer 2 Report
In this manuscript the authors study the hepatic and renal toxicity of the Euphorbia huanchahana (Klotzsch & Garcke) Boissier (huachangana) in Holtzman rats. The article is interesting but has several aspects that should be clarified.
In my opinion the most important are:
1. The majority of the reference is not in the English language, it is hard to see the previuos researches.
2. Did the author authenticate the plant or plant part by a qualified botanist. A voucher specimen should have been preserved for future reference.
3. In page 3; line no. 100, the author writs "which was macerated with ethanol 70°/7 days", Is it 70°C or what?
4. In page 3; line no. 101, "The product evaporates at", What is the product? Is it the supernatent?
5. In page 3; line no. 101, "The product evaporates at 40°C (104°F)", Why the auhor added a line under the degree symbol?
6. In page 3; line no. 109, "In 1 g of Euphorbia huanchahana (Klotzsch & Garcke) Boissier (huachangana) sample dissolved in 20 mL of ethanol", Please revise this sentenses as you may mean "A weight of 1 g of Euphorbia huanchahana (Klotzsch & Garcke) Boissier (huachangana) sample was dissolved in 20 mL of ethanol.
7. Table A1 and Table A2 should be scheme 1 and scheme 2.
8. The author should explain the method of determination of the lethal dose (LD50).
9. In page 4; line 148 "After the observation period, the extraction of blood and organs was carried out", Please revise the English language, as the extraction is not suitable here.
10. Section 2.4. Write the sources of the kits used in the various estimations.
11. The HPLC or GC/MS chromatographic analysis of the extract must be performed.
12. In vivo must be Italic.
13. Section 3.1. line 207; the author stated " We found differences in the concentration of TBIL ", Is that different increase or decrease?
14. Section 3.2. " In male rats, the mean concentration of TBIL was 1.07±0.21 mg/dL, DBIL was 0.45±0.07 mg/dL, UBIL was 0.62±0.18 mg/dL, AST was 141.8±34.57 U/L, ALT was 41.16±5.69 U/L, ALP was 298±85.29 U/L, TP was 5.58±0.57 mg/dL, ALB was 3.59±0.31 mg/dL, GLB was 1.94±0.51 mg/dL, and GGT was of 25.44±9.04 mg/dL", These data were already listed in the table, this is unnecessary repetition. Also, in the female results, delete all the repeated data. Instead, author may mention the importance of every parameter to the liver and kidney.
15. Page 6, in the abbreviations of Table 1: " ALT: Aminotransferasa de alanine aminotransferase" check the language, and " ASP: Aspartate aminotransferase", Check the abbreviation.
16. In the same section there was a rat symbol, is it refers to something?
17. In section 3.4. Tracking the weight variation, there is no need to write the weight data in the text, ypu better explain the results.
18. In "Figure 4. D Differences in weight concentration between the control group", English language must be improved allover the manuscript.
19. Page 9, line no. 276, " The liver was evaluated at a dose of 200 mg/kg" Is the dose 2000 or 200 mg/kg?
20. The auther declared the aim as hepatic and renal toxicity, and only evaluated the liver function tests, so, where the data of the kidney function parameters? Kidney function tests must be estimated.
21. In section 4. Discussion; the first paragraph "This preclinical study determined that the extract of Euphorbia huanchahana (Klot-zch & Garcke) Boissier (Huachangana) did not produce acute hepatorenal toxicity at a single dose of 2000 mg/kg. However, we report increases in liver marker concentrations and changes in body weight in a group of Holtzman rats treated with the extract during a 14-day follow-up period." Must be shifted after the second paragraph.
22. In conclusion section, the author declared " Euphorbia huanchahana (Klotzsch & Garcke) Boissier (huachangana) extract did 365 not produce any notable histopathological signs of cell injury or cause any damage during 366 the 14-day follow-up in Holtzman rats", however, the liver function tests and the histopathological examination proved slight toxicity to the liver cells with inflammation!!
Comments for author File: Comments.docx
Author Response
In this manuscript the authors study the hepatic and renal toxicity of the Euphorbia huanchahana (Klotzsch & Garcke) Boissier (huachangana) in Holtzman rats. The article is interesting but has several aspects that should be clarified.
In my opinion the most important are:
- The majority of the reference is not in the English language, it is hard to see the previuos researches.
RESPONSE: We agree with this observation, however, as it is a plant that grows in the Andes of Peru and Bolivia, it is usual for the few available studies to be published in Spanish. That is our interest also with this study, describing the toxicity of Euphorbia huanchahana (Klotzsch & Garcke) Boissier (huachangana) in English.
- Did the author authenticate the plant or plant part by a qualified botanist. A voucher specimen should have been preserved for future reference.
RESPONSE: We included this information in the section 2.2.
- In page 3; line no. 100, the author writs "which was macerated with ethanol 70°/7 days", Is it 70°C or what?
RESPONSE: We correct the text.
- In page 3; line no. 101, "The product evaporates at", What is the product? Is it the supernatent?
RESPONSE: We correct the text.
- In page 3; line no. 101, "The product evaporates at 40°C (104°F)", Why the auhor added a line under the degree symbol?
RESPONSE: We correct the text.
- In page 3; line no. 109, "In 1 g of Euphorbia huanchahana (Klotzsch & Garcke) Boissier (huachangana) sample dissolved in 20 mL of ethanol", Please revise this sentenses as you may mean "A weight of 1 g of Euphorbia huanchahana (Klotzsch & Garcke) Boissier (huachangana) sample was dissolved in 20 mL of ethanol.
RESPONSE: We revised and correct the text.
- Table A1 and Table A2 should be scheme 1 and scheme 2.
RESPONSE: We correct the text.
- The author should explain the method of determination of the lethal dose (LD50). 141-142
RESPONSE: We included this information in the section 2.2.
- In page 4; line 148 "After the observation period, the extraction of blood and organs was carried out", Please revise the English language, as the extraction is not suitable here.
RESPONSE: We correct the text.
- Section 2.4. Write the sources of the kits used in the various estimations.
RESPONSE: We included this information.
- The HPLC or GC/MS chromatographic analysis of the extract must be performed.
RESPONSE: As we stated in the discussion section, in this study for self-financing reasons we were unable to perform HPLC or GC/MS chromatographic analysis. We understand that this test is important but the results of this study have that limitation (lines 359-368).
- In vivo must be Italic.
RESPONSE: We correct the text.
- Section 3.1. line 207; the author stated " We found differences in the concentration of TBIL ", Is that different increase or decrease?
RESPONSE: We correct the text.
- Section 3.2. " In male rats, the mean concentration of TBIL was 1.07±0.21 mg/dL, DBIL was 0.45±0.07 mg/dL, UBIL was 0.62±0.18 mg/dL, AST was 141.8±34.57 U/L, ALT was 41.16±5.69 U/L, ALP was 298±85.29 U/L, TP was 5.58±0.57 mg/dL, ALB was 3.59±0.31 mg/dL, GLB was 1.94±0.51 mg/dL, and GGT was of 25.44±9.04 mg/dL", These data were already listed in the table, this is unnecessary repetition. Also, in the female results, delete all the repeated data. Instead, author may mention the importance of every parameter to the liver and kidney.
RESPONSE: We include in the text only the information on the hepatic markers of male and female rats so that the reader can already identify the concentrations of these markers in the text. Although data from the tables is repeated, we believe that the text should be independent of the table and thus explain itself. Explanations have been included on what these markers indicate and renal function markers have been included.
- Page 6, in the abbreviations of Table 1: " ALT: Aminotransferasa de alanine aminotransferase" check the language, and " ASP: Aspartate aminotransferase", Check the abbreviation.
RESPONSE: We correct the text.
- In the same section there was a rat symbol, is it refers to something?
RESPONSE: We deleted the rat symbol.
- In section 3.4. Tracking the weight variation, there is no need to write the weight data in the text, ypu better explain the results.
RESPONSE: W In this paragraph we just want to highlight the changes in the weight of rats treated with Euphorbia huanchahana (Klotzsch & Garcke) Boissier (huachangana) We understand that some data are repeated but we believe it is necessary to present both results independently. In addition, the description of the implications of the weight change of the rats is described in the following paragraph:
As shown in Table 3, the body weights of male and female rats were monitored for 3 times, and no significant difference was found between males and females (p>0.05). However, when we compared the overall body weight of the control and experimental groups, we found a difference between the days on which the body weight was assessed (Figure 4).
- In "Figure 4. D Differences in weight concentration between the control group", English language must be improved allover the manuscript.
RESPONSE: We correct the text.
- Page 9, line no. 276, " The liver was evaluated at a dose of 200 mg/kg" Is the dose 2000 or 200 mg/kg?
RESPONSE: We correct the text.
- The auther declared the aim as hepatic and renal toxicity, and only evaluated the liver function tests, so, where the data of the kidney function parameters? Kidney function tests must be estimated.
RESPONSE: We included this results in the 3.3. section.
- In section 4. Discussion; the first paragraph "This preclinical study determined that the extract of Euphorbia huanchahana (Klot-zch& Garcke) Boissier (Huachangana) did not produce acute hepatorenal toxicity at a single dose of 2000 mg/kg. However, we report increases in liver marker concentrations and changes in body weight in a group of Holtzman rats treated with the extract during a 14-day follow-up period." Must be shifted after the second paragraph.
RESPONSE: We correct the text.
- In conclusion section, the author declared "Euphorbia huanchahana (Klotzsch & Garcke) Boissier (huachangana) extract did 365 not produce any notable histopathological signs of cell injury or cause any damage during 366 the 14-day follow-up in Holtzman rats", however, the liver function tests and the histopathological examination proved slight toxicity to the liver cells with inflammation!!
RESPONSE: We correct the text.
Author Response File: Author Response.docx
Reviewer 3 Report
This article, which studied the safety and possible toxic effects of Euphorbia huachahana (Klotzch & Garcke) Boissier (Huachangana), is valuable and of interest to the international scientific readers. The results are outstanding and clear, and the limitations of the study have been mentioned to give the readers a balance view. It is recommended to minor revise before publication. The article has the following questions:
1. Title of Section 1.1. is unnecessary.
2. In Section 3.2, a brief conclusion is needed, similar to Line 260-263 in Section 3.4.
3. Section 3.4 should be numbered 3.3.
Author Response
This article, which studied the safety and possible toxic effects of Euphorbia huachahana (Klotzch & Garcke) Boissier (Huachangana), is valuable and of interest to the international scientific readers. The results are outstanding and clear, and the limitations of the study have been mentioned to give the readers a balance view. It is recommended to minor revise before publication. The article has the following questions:
- Title of Section 1.1. is unnecessary.
RESPONSE: The section was deleted
- In Section 3.2, a brief conclusion is needed, similar to Line 260-263 in Section 3.4.
RESPONSE: We added a sentence explain the results of this section.
- Section 3.4 should be numbered 3.3.
RESPONSE: The section was re numbered.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
The authors commented and corrected on all the queries I had. Answers are clear and provide enhanced scientific validity and rigor. I think the manuscript is ready for publication in its current state.
