Evaluation on the Potential for Hepatotoxic Components from Herba Epimedii to Induce Apoptosis in HepG2 Cells and the Analysis of the Influence of Metabolism in Liver Microsomes
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsIn the present manuscript, the authors attempt to clarify possible hepatotoxic mechanisms of several active components of Herba Epimedii. The study utilises flow cytometry and western blotting to assess in vitro apoptosis of HepG2 cells and the expression of key proteins associated with apoptosis, Bax, Bcl-2, caspase-3 and caspase 9. In addition, in vitro metabolism and the ability of these compounds to induce apoptosis were evaluated by LC-MS/MS after incubation with liver microsomal fraction of different species. The results indicate that sagittatoside A, baohuoside I and icaritin can induce hepatotoxicity via apoptosis, but that metabolic transformation can alter this ability.
The manuscript is very well written, and the study is well designed and conducted, but some details need further explanation and discussion.
The main problem is that the authors have used metabolic transformation of flavonoids in vitro, but have not provided literature (or experimental) evidence that none of the analysed constituents of Herba Epimedii have in vivo effects on the expression of enzymes involved in phase I and phase II metabolism. In addition to hepatotoxicity, it is not uncommon for adverse effects of herbal supplements to result from interference with liver xenobiotic metabolism via UDP-glucuronosyltransferase and cytochrome p450 activity. Interactions of herbal supplements with microsomal fraction enzymes and potential interactions between herbs and drugs/xenobiotics as a mechanism for adverse effects are often overlooked.
Questions:
1. Can any of the investigated compounds (sagittatoside A, icariside I, baohuoside I and icaritin) alter the expression of enzymes involved in phase I and II metabolism? If so, how meaningful are the results of in vitro metabolic transformation, especially with repeated in vivo use of herbal preparations?
2. The doses you used to treat the HepG2 cells were based on IC50 values from previous in vitro experiments, but do these concentrations correspond to the doses used in vivo?
3. Are there any studies in humans or animals in which flavonoids and/or their metabolites were measured in the blood (or liver) following oral ingestion of the Epimedii herbal preparations that could be compared with and discussed in relation to the in vitro results presented in the manuscript? Are therapeutic doses of the herbal preparations sufficient to induce apoptosis?
Author Response
Thank you for the comments on our manuscript entitled “The evaluation on potential hepatotoxic components from Herba Epimedii induced apoptosis in HepG2 cells and the analysis of the influence of metabolism with liver microsomes” (ID: molecules-2903130). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have studied comments carefully and have made correction which we hope meet with approval. The main corrections in the paper and the responds to the reviewer’s comments are as flowing:
Point 1: Can any of the investigated compounds (sagittatoside A, icariside I, baohuoside I and icaritin) alter the expression of enzymes involved in phase I and II metabolism? If so, how meaningful are the results of in vitro metabolic transformation, especially with repeated in vivo use of herbal preparations? 

Response 1: About the investigated compounds (sagittatoside A, icariside I, baohuoside I and icaritin) altering the expression of enzymes involved in phase I and II metabolism, we have not found the literatures. In this research, we only focused on the final situation of the investigated compounds by metabolism and analysis the effect of metabolic results on apoptosis in order to provide a reference for conforming the hepatotoxic components from Herba Epimedii. So we have not carried out the research of evaluating the impact of the investigated compounds on metabolic process. However, as the reviewer prompted that, it is not uncommon for adverse effects of herbal supplements to result from interference with liver xenobiotic metabolism via UDP-glucuronosyltransferase and cytochrome p450 activity, which happened to have the same view of our next research plan. In our follow-up study, we will evaluate the influence of sagittatoside A, icariside I, baohuoside I and icaritin on the expression of drug metabolic enzyme. Furthermore, we will make a concrete analysis of the effect of the investigated compounds on CYP450 isoforms and the metabolic changes induced by the component interactions.
Point 2: The doses you used to treat the HepG2 cells were based on IC50 values from previous in vitro experiments, but do these concentrations correspond to the doses used in vivo?
Response 2: It is really true as the Reviewer suggested that the in vitro-in vivo correlation of the doses is very important. The treated doses of the investigated compounds in this manuscript were based on IC50 values and had used for detecting indexes related to the apoptosis, including superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA), reactive oxy gen species (ROS) and mitochondrial membrane potential (MMP) in previous vitro experiments (Zhang, L., Xu, A. L., Yang, S., Zhao, B. S., & Wang, T. (2020). In vitro screening and toxic mechanism exploring of leading components with potential hepatotoxicity of herba epimedii extracts. Toxicology in Vitro, 62, 104660.). To keep the consistency and the continuity of these results of researches in vitro, we still followed these doses. But in fact, we also referred to the doses used in vivo and tried to create the relationship between vivo and vitro. In previous vivo experiments, Herba Epimedii extract at 22.90, 45.79, 68.69, 91.05 mg/kg were administered to SD rats once a day for 28 days to evaluate the toxicity in vivo. Though UPLC-MS/MS analysis, the concentrations of sagittatoside A, icariside I, baohuoside I and icaritin in the extract were determined. By calculation, the doses used in vitro only slightly lower than in vivo. In view of transport in vivo, this difference could be acceptable. And we will perfect the in vitro-in vivo correlation of the doses by the hepatoxic evaluation of the investigated compounds in vivo.
Point 3: Are there any studies in humans or animals in which flavonoids and/or their metabolites were measured in the blood (or liver) following oral ingestion of the Epimedii herbal preparations that could be compared with and discussed in relation to the in vitro results presented in the manuscript? Are therapeutic doses of the herbal preparations sufficient to induce apoptosis?
Response 3: There are some literatures about the metabolism studies of flavonoids in Herba Epimedii in animals. However, most of these literatures were about pharmacodynamic components such as icaritin, epimedin C, epimedin A and epimedin B, related to the potential hepatotoxic components from Herba Epimedii were not much, especially about sagittatoside A. One study found baohuoside I could be detected from 0.5 h, and showed a peak at 1 h with a concentration of ∼15 nM after Epimedium extract ingestion, indicating its possiblemetabolic pathway originally from icariin or other glycosides with more than one sugar moieties, and Icariside I were undetectable at 0.5 h, suggesting that very small amounts of them were directly absorbed due to the less concentrations in the Epimedium extract (Shen P, Wong S P, Li J,et al. Simple and sensitive liquid chromatography-tandem mass spectrometry assay for simultaneous measurement of five Epimedium prenylflavonoids in rat sera [J]. Journal of Chromatography B Analytical Technologies in the Biomedical & Life Sciences, 2009, 877(1-2):71-78.). These results cloud enrich the discussion of the manuscript, so we have supplied some related content in some part according to the Reviewer’s suggestion. As for whether therapeutic doses of the herbal preparations sufficient to induce apoptosis, it is hard to conclude right now because of complex processes in vivo and the interaction of many components. We will find out though follow-up studies in vivo as soon as we can.
After a point-by-point reply to the peer reviewers’ comments and suggestions, I believe the level of this manuscript is raised. Thanks for the reviewer’s valuable opinions again, which mean a great deal to our research.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsAuthors should revise the manuscript as per following suggestion
Major:
- Few lines on 'herb-induced hepatotoxicity or herb-drug interaction' focusing on TCM in general, should be incorporated in Introduction or Discussion section.
- An HPLC-MS/MS chromatogram showing presence of the analyzed hepatoxic constituents in the plant extract might add an interest.
Minor:
- English language needs some attention.
- Some statements are too long and ambiguous that should be restructured i in to two.
Please see the attached manuscript file (.pdf) for highlighted comments, too.
Comments for author File: Comments.pdf
Needs minor polishing.
Author Response
Thank you for the comments on our manuscript entitled “The evaluation on potential hepatotoxic components from Herba Epimedii induced apoptosis in HepG2 cells and the analysis of the influence of metabolism with liver microsomes” (ID: molecules-2903130). The reviewer made a lots of meticulous comments and suggestions, which are all valuable and very helpful for revising and improving our paper. We have studied comments carefully and have made correction which we hope meet with approval. We very appreciate the reviewer’s help. The main corrections in the paper and the responds to the reviewer’s comments are as flowing:
Point 1: Few lines on 'herb-induced hepatotoxicity or herb-drug interaction' focusing on TCM in general, should be incorporated in Introduction or Discussion section. 

Response 1: We accept the reviewer’s advice and have added these contents in the appropriate section.
Point 2: An HPLC-MS/MS chromatogram showing presence of the analyzed hepatoxic constituents in the plant extract might add an interest.
Response 2: It is really true as the reviewer suggested that the HPLC-MS/MS chromatogram could add an interest. We have showed the HPLC-MS/MS chromatogram of the Herba Epimedii extract in a previous paper (Zhang, L., Xu, A. L., Yang, S., Zhao, B. S., & Wang, T. (2020). In vitro screening and toxic mechanism exploring of leading components with potential hepatotoxicity of herba epimedii extracts. Toxicology in Vitro, 62, 104660.), and the chromatogram of this article is respectively only about each investigated compound. So in view of avoiding repeat and saving space, we only present the detected data and think the chromatogram may be not necessary.
Point 3: English language needs some attention.
Response 3: We are very sorry for failing to show a required ability of words in English language. For improving the written expression, we have seek some professional help from American Journal Experts (a language polishing company) and polished the manuscript language.
Point 4: Some statements are too long and ambiguous that should be restructured in to two.
Response 4: We are very sorry for failing to express some sentence very well. We have rewritten the corresponding part according to the reviewer’s notes and try our best to make the expression meaning more rigorous and accurate.
Point 5: Please see the attached manuscript file (.pdf) for highlighted comments, too.
Response 5: We have studied the highlighted comments attached in the file carefully and have made the appropriate correction earnestly.
After a point-by-point reply to the peer reviewers’ comments, as the first author, I deeply realize the deficiency of writing English manuscript and precise describing. Through studying the reviewers’ suggestions, I believe the level of this manuscript is raised. We will avoid repeating any of these problems in the future and more scientifically explain the results of our researches. Thanks for the reviewer’s valuable opinions again, which mean a great deal to our research.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsComments to the Authors
The article examines the impact of sagittatoside A, icariside I, baohuoside I, and icaritin on the apoptosis of HepG2 cells, using the expression of key proteins associated with this process as a measure of hepatotoxic mechanism. The study employed flow cytometry and Western blotting to evaluate the flavonoids' ability to induce apoptosis without or after incubation with human liver microsomes. Furthermore, the HPLC-MS/MS detection method was used to analyze the in vitro metabolism of flavonoids with liver microsomes from various species. The remaining percentage of compounds over time and their half-life were subsequently calculated.
There are some issues to be pointed out:
Minor issues:
1. Abstract: “After metabolic incubation, sagittatoside A and icaritin could still induce apoptosis due to less metabolic elimination, while icariside I showed the ability to induce apoptosis, and baohuoside I lost this ability, probably due to metabolic transformation.” (lines 23-26) – The meaning of the sentence is unclear.
2. Introduction: “To examine the toxicity of icariin, epimedin A, epimedin B, epimedin C, sagittatoside A, sagittatoside B, 2”-O-rhamnosylicariside II and baohuoside I, zebrafish embryos were exposed to these compounds to simulate the liver.” (lines 45-47) – Further explanation is needed of the process by which the embryos simulate the liver.
3. The section of Methods needs to be completed:
- “… Bcl-2 (1:2000, ab196495), Bax 116 (1:1000, ab32503) …” – These are probably antibodies – anti-Bcl-2 IgG – and so on.
- “Millipore, Bedford, MA” (line 121) – should be as follows – Millipore, Bedford, MA, USA and etc.
- “… cell pellets were washed three times with PBS, centrifuged at 2000 rpm …” (lines 139-140) – probably washed by centrifugation?
- What was the composition of “lysis buffer” (line 155)?
- The description of PAGE, which precedes Western blotting, is missing (line 156) – Authors should briefly describe a protocol used.
- The dilution of antibodies is not stated (lines 159-160).
- Describe “gel imagery system” (line 163) – manufacturer, SW used ….
- What is meant by “Negative controls (incubation without substrate) were prepared in the same manner” (lines 179-180)?
- What is “gm” in the equation (3)?
4. Results:
- Paragraphs 3.1 and 3.3 should be combined; there is no reason to separate them.
- “Icariside I did not cause a change in the apoptosis rate (2.39±0.34%) at the tested concentration. The apoptosis rate of the cells treated with baohuoside I was 3.93±0.20% (P < 0.05). The apoptosis rate of the cells treated with icaritin was 25.93±0.66% (P < 0.01). Overall, these results suggested that sagittatoside A, baohuoside I and icaritin induced HepG2 cell apoptosis, which was mainly consistent with the results from the early apoptosis assessment.” (lines 238-243) – How to understand this paragraph with conflicting information?
- “Values are presented as the mean ± standard deviation of triplicate experiments” (line 251 and 280) is in contradiction with: “All data are expressed as the mean ± standard error of the mean (SEM)” in the Methods (lines 227-228) – One way or the other?
- Typos: “sagittatoside An increased” (lines 264-265).
5. Discussion is well written but some statements need to be clarified:
“… it was proposed that the values from beagle dog microsomes were more similar to those from human microsomes, and when comparing the two kinds of rodents, the values from SD rats were closer to those from humans.” (lines 461-462) – This expression is unclear.
Major issues:
1. The UDP-glucuronosyltransferase, like CYPs, is located in the endoplasmic reticulum. Please comment on how to distinguish the impact of Phase I (CYPs) and Phase II (UDP-GT) enzymes when NADPH is used to support CYP-mediated metabolism in both kits. One may expect another experiment with “Phase II” system without NADPH. Has the experiment been done?
2. After oral administration, flavonoid glycosides, when are not degraded in the stomach due to acidic conditions, may be cleaved into saccharides and aglycones in the small or large intestines by human and/or bacterial glycosidases. This means that it is mainly aglycones that reach the liver via the enterohepatic circulation. Therefore, examining the metabolic fate of flavonoid glycosides in microsomes and HepG2 cells may not be appropriate in terms of the potential human risks associated with these compounds. An explanation is needed: How does it fit in with the research objectives?
3. The experiments that provided data on the elimination curves of Phase I and II metabolism were conducted in the presence of NADPH and UDP-GA, respectively. However, control experiments without these cofactors were not conducted. Therefore, it is impossible to rule out the involvement of other microsomal enzymes and processes in the depletion of flavonoids from the reaction mixture. Conclusions should be rephrased accordingly.
4. The metabolism of flavonoids by microsomes (membrane fraction of endoplasmic reticulum prepared from liver), expressed as their depletion from the reaction mixture, is a significant part of the biotransformation of these xenobiotics. However, this is not the only important aspect of the biotransformation of these xenobiotics. Essential biotransformation reactions, specifically Phase II, occur in the cytoplasm of hepatocytes. Additionally, the metabolism of ingested flavonoids via enterocytes in the intestines and possibly microbiota should not be disregarded. Calculation of 'predicted liver clearance in vivo (CLint(liver, in vitro))' is not useful unless the flavonoids are administered intravenously, which is not the case. Please comment on this issue.
5. “The influence of metabolism on the apoptosis ability of these compounds could be summarized as follows: by metabolism through oral Herba Epimedii, some sagittatoside A remained in its original form because less metabolic elimination occurred” (lines 449-452) – As mentioned above, when administered orally, flavonoid glycoside(s) may be cleaved in the stomach and/or in the intestines by glycosidases and potentialy by gut microflora. Additionally, the metabolism mediated by enterocytes in the intestines must be considered. Therefore, the use of 'Phase I and II metabolic stability kits' cannot provide a complete picture. Any limitations of this approach should be discussed in the appropriate section.
Author Response
Thank you for the comments on our manuscript entitled “The evaluation on potential hepatotoxic components from Herba Epimedii induced apoptosis in HepG2 cells and the analysis of the influence of metabolism with liver microsomes” (ID: molecules-2903130). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have studied comments carefully and have made correction which we hope meet with approval. The main corrections in the paper and the responds to the reviewer’s comments are as flowing:
Point 1: Abstract: “After metabolic incubation, sagittatoside A and icaritin could still induce apoptosis due to less metabolic elimination, while icariside I showed the ability to induce apoptosis, and baohuoside I lost this ability, probably due to metabolic transformation.” (lines 23-26) – The meaning of the sentence is unclear.

Response 1: We are very sorry for failing to express this sentence very well. So, we have rewritten this sentence like “ After metabolic incubation, sagittatoside A and icaritin metabolism mixture could still induce apoptosis due to less metabolic elimination, while icariside I and baohuoside I metabolism mixture respectively got and lost the ability to induce apoptosis, probably due to quick metabolism and metabolic transformation.”
Point 2: Introduction: “To examine the toxicity of icariin, epimedin A, epimedin B, epimedin C, sagittatoside A, sagittatoside B, 2”-O-rhamnosylicariside II and baohuoside I, zebrafish embryos were exposed to these compounds to simulate the liver.” (lines 45-47) – Further explanation is needed of the process by which the embryos simulate the liver.
Response 2: As Reviewer suggested that, we have added the process of using zebrafish embryos to simulate the liver and supplied the related reference. The content has been rewritten like “Zebrafish embryos has hepatocytes from 2 days after fertilization and exhibited concordance in the metabolic pathway compared to the traditional models (McGrath et al., 2008; Driessen et al., 2015).”
Point 3: The section of Methods needs to be completed:
- “… Bcl-2 (1:2000, ab196495), Bax 116 (1:1000, ab32503) …” – These are probably antibodies – anti-Bcl-2 IgG – and so on.
- “Millipore, Bedford, MA” (line 121) – should be as follows – Millipore, Bedford, MA, USA and etc.
- “… cell pellets were washed three times with PBS, centrifuged at 2000 rpm …” (lines 139-140) – probably washed by centrifugation?
- What was the composition of “lysis buffer” (line 155)?
- The description of PAGE, which precedes Western blotting, is missing (line 156) – Authors should briefly describe a protocol used.
- The dilution of antibodies is not stated (lines 159-160).
- Describe “gel imagery system” (line 163) – manufacturer, SW used ….
- What is meant by “Negative controls (incubation without substrate) were prepared in the same manner” (lines 179-180)?
- What is “gm” in the equation (3)?
Response 3: We have re-written the part of Methods according to the Reviewer’s suggestion.
“… Bcl-2 (1:2000, ab196495), Bax 116 (1:1000, ab32503) …” was rewritten as “anti-Bcl-2 (1:2000, ab196495), anti-Bax (1:1000, ab32503)” according to the instructions.
“Millipore, Bedford, MA” was rewritten as“Millipore, Bedford, MA, United States”.
“… cell pellets were washed three times with PBS, centrifuged at 2000 rpm …”was rewritten as“Briefly, the collected cell pellets were washed three times by centrifugation at 2000 rpm and 4 °C for 5 min with PBS and resuspended in binding buffer..
The composition of “lysis buffer” according to the instructions were 25 mM Tris•HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS.
We have added description of PAGE like “Then, to destroy the 3-dimensional protein structure, the proteins in the loading buffer were heated at 95 °C for 5 min. An equal amount of protein from each sample was separated by SDS-PAGE on 10% SDS–polyacrylamide gels.”
We have added description of the dilution of antibodies like “the membrane was incubated in blocking buffer (5% nonfat dairy milk in TBST) for 2 h and probed with various antibodies (1:1000) overnight at 4 °C. The membrane was then probed with a secondary antibody (1:5000) for 2 h at room temperature”.
“Negative controls (incubation without substrate) were prepared in the same manner” means there is no the investigated components in the incubation mixture of the negative controls.
The “gm” in the equation (3) is the unit to descript the quality of microsomes in liver (Li, Z., Zhao, S., Yuan, Y., Zhang, L., Song, Z., Tian, X., & Zhang, X. (2019). In vitro metabolic profiles of motolimod by using liquid chromatography tandem mass spectrometry: Metabolic stability, metabolite characterization and species comparison. Journal of Pharmaceutical and Biomedical Analysis, 167, 90–99.).
Point 4: Results:
- Paragraphs 3.1 and 3.3 should be combined; there is no reason to separate them.
- “Icariside I did not cause a change in the apoptosis rate (2.39±0.34%) at the tested concentration. The apoptosis rate of the cells treated with baohuoside I was 3.93±0.20% (P < 0.05). The apoptosis rate of the cells treated with icaritin was 25.93±0.66% (P < 0.01). Overall, these results suggested that sagittatoside A, baohuoside I and icaritin induced HepG2 cell apoptosis, which was mainly consistent with the results from the early apoptosis assessment.” (lines 238-243) – How to understand this paragraph with conflicting information?
- “Values are presented as the mean ± standard deviation of triplicate experiments” (line 251 and 280) is in contradiction with: “All data are expressed as the mean ± standard error of the mean (SEM)” in the Methods (lines 227-228) – One way or the other?
- Typos: “sagittatoside An increased” (lines 264-265).
Response 4: We have re-written the part of Results according to the Reviewer’s suggestion.
Paragraphs 3.1 and 3.3 were separated in order to present the studies in a logical order.
For the “conflicting information” (lines 238-243), we have explained in the discussion. The phenomenon probably induced by metabolic transformation.
We are very sorry there are some clerical errors in describing the content of Statistical analysis. We have corrected them as “All data are expressed as the mean ± standard deviation of the mean” (lines 227-228).
We feel ashamed for these clerical errors and have corrected the typos.
We are very sorry there are some clerical errors in describing the content of Figure2. We have corrected them.
Point 5: “… it was proposed that the values from beagle dog microsomes were more similar to those from human microsomes, and when comparing the two kinds of rodents, the values from SD rats were closer to those from humans.” (lines 461-462) – This expression is unclear.
Response 5: Considering the reviewer’s suggestion, we have rewritten the part like “it was proposed that the values from beagle dog microsomes were more similar to those from human microsomes. Comparing the metabolic parameters of the investigated components between in two kinds of rodents’ microsomes, the values from SD rats were closer to those from humans”
Point 6: The UDP-glucuronosyltransferase, like CYPs, is located in the endoplasmic reticulum. Please comment on how to distinguish the impact of Phase I (CYPs) and Phase II (UDP-GT) enzymes when NADPH is used to support CYP-mediated metabolism in both kits. One may expect another experiment with “Phase II” system without NADPH. Has the experiment been done?
Response 6: It is really true as the reviewer suggested that one may expect another experiment with “Phase II” system without NADPH. But according to our understanding, the most of commercial Phase I and II metabolic stability kits contain NADPH used to activate metabolic enzyme. Distinguishing the impact of Phase I and Phase II enzymes in vitro mainly only depend on the kinds of metabolic enzyme. And the process in vivo as the reviewer mentioned is more continuous and complex. In our follow-up study, we will evaluate the metabolic stability in vivo of sagittatoside A, icariside I, baohuoside I and icaritin, pay more attention to the influence of the whole metabolic system.
Point 7: After oral administration, flavonoid glycosides, when are not degraded in the stomach due to acidic conditions, may be cleaved into saccharides and aglycones in the small or large intestines by human and/or bacterial glycosidases. This means that it is mainly aglycones that reach the liver via the enterohepatic circulation. Therefore, examining the metabolic fate of flavonoid glycosides in microsomes and HepG2 cells may not be appropriate in terms of the potential human risks associated with these compounds. An explanation is needed: How does it fit in with the research objectives?
Response 7: After taking a lot of thought, we agree with the reviewer’s point and realize that flavonoid glycosides could be changed in absorption process before being metabolized by liver. This research was designed for investigating whether the potential hepatotoxic components in Herba Epimedii sieved out in vitro could have a chance to develop their hepatotoxicity in vivo and could be the real hepatotoxic material base. As mentioned in the manuscript, this research lays the foundation in some extent for evaluating the potential human risks of Herba Epimedii in vivo. Meanwhile, some literatures have reported the investigated component could be found in the liver following oral ingestion (Shen P, Wong S P, Li J,et al. Simple and sensitive liquid chromatography-tandem mass spectrometry assay for simultaneous measurement of five Epimedium prenylflavonoids in rat sera [J]. Journal of Chromatography B Analytical Technologies in the Biomedical & Life Sciences, 2009, 877(1-2):71-78.). There are some shortcomings in the present study, while we will perfect the in vitro-in vivo correlation by systematically studying the fate in vivo of the investigated compounds.
Point 8: The experiments that provided data on the elimination curves of Phase I and II metabolism were conducted in the presence of NADPH and UDP-GA, respectively. However, control experiments without these cofactors were not conducted. Therefore, it is impossible to rule out the involvement of other microsomal enzymes and processes in the depletion of flavonoids from the reaction mixture. Conclusions should be rephrased accordingly.
Response 8: As Reviewer suggested that, we have rephrased the part of conclusions and realized the existing study method have the limitation.
Point 9: The metabolism of flavonoids by microsomes (membrane fraction of endoplasmic reticulum prepared from liver), expressed as their depletion from the reaction mixture, is a significant part of the biotransformation of these xenobiotics. However, this is not the only important aspect of the biotransformation of these xenobiotics. Essential biotransformation reactions, specifically Phase II, occur in the cytoplasm of hepatocytes. Additionally, the metabolism of ingested flavonoids via enterocytes in the intestines and possibly microbiota should not be disregarded. Calculation of predicted liver clearance in vivo (CLint(liver, in vitro)) is not useful unless the flavonoids are administered intravenously, which is not the case. Please comment on this issue.
Response 9: According to the reviewer’s advices, we have totally realized the difference between the metabolism in vitro and in vivo. We will prove the completely metabolic process of the potential hepatotoxic components from Herba Epimedii and find out more information about their hepatotoxicity though follow-up studies in vivo as soon as we can. As for calculation of predicted liver clearance in vivo (CLint(liver, in vitro)) in this manuscript, although it can’t guide directly metabolism in vivo, we try to compare transversely the metabolic characteristics of the investigated components within the scope of the study and based on the same calculation method.
Point 10: “The influence of metabolism on the apoptosis ability of these compounds could be summarized as follows: by metabolism through oral Herba Epimedii, some sagittatoside A remained in its original form because less metabolic elimination occurred” (lines 449-452) – As mentioned above, when administered orally, flavonoid glycoside(s) may be cleaved in the stomach and/or in the intestines by glycosidases and potentialy by gut microflora. Additionally, the metabolism mediated by enterocytes in the intestines must be considered. Therefore, the use of 'Phase I and II metabolic stability kits' cannot provide a complete picture. Any limitations of this approach should be discussed in the appropriate section.
Response 10: As reviewer suggested that, we have realized the limitation of liver microsomes simulating liver metabolism again and added the related content in the appropriate section.
After a point-by-point reply to the peer reviewers’ comments, as the first author, I deeply realize the deficiency of writing English manuscript and precise describing. Through studying the reviewers’ suggestions, I am greatly inspired and believe the level of this manuscript is raised by revising. We appreciate the reviewers’ work examining draft which i mean a great deal to our research, and we will adopt the reviewers’ suggestions in the follow-up studies. Thanks for the reviewer’s valuable opinions again.
Author Response File: Author Response.pdf
Round 2
Reviewer 3 Report
Comments and Suggestions for AuthorsThe authors addressed all referees' comments in the revised version.The manuscript has been improved to warrant publication in Molecules.
