Network Pharmacological Analysis of a New Herbal Combination Targeting Hyperlipidemia and Efficacy Validation In Vitro

Round 1

Reviewer 1 Report

The authors used computational design and invitro studies to evaluate the effect of herbs in hyperlipidemia. Well, the western blot results are great but still in-depth investigation using in-vivo models are also required. The In-vitro data just give support to in-silico data. Well there are some issues which need to be rectified. 

1.      First of all, there are a lot of studies available of Zingiber officinale on hyperlipidemia and these studies are deeply investigated studies. I didn’t understand what was the moto of authors? What is the novelty of this study which can atleast attract some readers to read this article?

2.      Section 2.6. The authors didn't well explain the sample preparation area. I have many questions over here? how the water sample was dried in rotary evaporator? What was the temperature set for evaporator? When the extract was dried how it was taken out from evaporator? In which solution it was dissolved and how it was freeze? How the sample was prepared for test? What was the extract quantity for doing in-vitro experiment, because there is a standard for extract, if the extract was very concentrated then may be it gives false results? So all these things need to be properly explained.

3.      Section 3.1. Selection of potential compounds. Did the authors screen all compounds and some were selected and some were neglected for the studies? Because authors didn’t provide any information like that in article? So that’s why the title of this section is little confusing for me.

4.      Conclusion is missing

5.      It would be great if the authors provide color graphs in fig.7 and fig.8

6.      Table 1 caption. The name of the plants should be in italics. 

 

 

Author Response

Reviewer 1

The authors used computational design and invitro studies to evaluate the effect of herbs in hyperlipidemia. Well, the western blot results are great but still in-depth investigation using in-vivo models are also required. The In-vitro data just give support to in-silico data. Well there are some issues which need to be rectified. 

• We appreciate for your sincere and constructive comments. As you indicated, in vitro data presented in the study are preliminary result and further in vivo study is needed to confirm the actual efficacy of the herbal combination. We are clearly aware of the limitations of our study. Though, we insist that the value of the study lies in other aspect.
• We revised our manuscript by taking suggestions given by reviewers. We hope you are satisfied with the revised manuscript.

 

1. First of all, there are a lot of studies available of Zingiber officinale on hyperlipidemia and these studies are deeply investigated studies. I didn’t understand what was the moto of authors? What is the novelty of this study which can at least attract some readers to read this article?

• Thank you for raising important issue. As you wrote, there are already many studies investigated herb prescriptions for treating metabolic disorders.
• However, the novelty of our study is in the report of new herbal combination, which act key role in many herbal prescriptions and exhibit better efficacy than single herb.
• The importance of the herbal combination (consist of ZO, PC, AT) found in several herbal prescriptions is revealed for the first time. Although many traditional practitioners have vague idea of the presence of the herbal combination in several lipid-lowering prescriptions, yet there was no relevant study exist on it.
• In our preceding study, we analyzed the herbal composition of several prescriptions used for hyperlipidemia in Korea. While we analyze herbal compositions of prescriptions in certain category (phlegm- and dampness-solving prescriptions) which is closely related with hyperlipidemia, we found overlapping herbs of ZO, PC and AT. The herbal combination of three herbs was discovered by such endeavors.

2. Section 2.6.The authors didn't well explain the sample preparation area. I have many questions over here? how the water sample was dried in rotary evaporator? What was the temperature set for evaporator? When the extract was dried how it was taken out from evaporator? In which solution it was dissolved and how it was freeze? How the sample was prepared for test? What was the extract quantity for doing in-vitro experiment, because there is a standard for extract, if the extract was very concentrated then may be it gives false results? So all these things need to be properly explained.

• To prepare the extract, dried herbs (100 g) were ground to a powder and extracted with 500 mL in distilled water at 100°C for 15 min. In addition, the mixed sample was blended with equal weights of AT, PC, and ZO (33 g each).
• The hot water extracts were filtered twice through 8 μm-pore-size Whatman filter paper and concentrated into 200 ml or less by rotary evaporation at 100°C for 20 min, and then the concentrated samples were lyophilized by vacuum freeze-dryer (Buchi, Switzerland), and freeze-dried to obtain a powder (yield : AT 2.88%, w/w, PC 2.24%, w/w, ZO 3.2%, w/w and Mix 2.4%, w/w).
• Powder was then eluted with DPBS and filtered through a 0.22 μm syringe filter before cell treatment.

3. Section 3.1. Selection of potential compounds. Did the authors screen all compounds and some were selected and some were neglected for the studies? Because authors didn’t provide any information like that in article? So that’s why the title of this section is little confusing for me.

• Thank you for sincere comments on procedures in selecting compounds. We searched the pharmacological properties of herbal compounds in TCMSP with cutoff standards of OB (oral bioavailability, ≥30%) and DL (drug-likeness ≥ 18).
• The OASIS, which is also a pharmacological database of herbal medicine, was also referenced for adding well-known bioactive compounds with plenty of reference papers.
• The detailed process of screening potential compounds is described in ‘2.1.1 Data acquisition of herbs from the online database’.

 

4. Conclusion is missing

- We are pleased to have a comment on significant part of the manuscript.

- We added the summarized conclusion of our study in line 472-479.

- The description in conclusion part is as follows;

“The pharmacological activities of three herbs including ZO, AT, PC against dyslipidemia were estimated via network pharmacological approaches. The herbs po-tentially target several key proteins and pathways critical to lipid metabolism. The effi-cacy and mechanism of the herbal extracts were investigated in hepatic steatosis model in vitro. The mixed herb extract showed stronger potential against hepatic steatosis as compared to single herb extracts. As a result, we suggest that the herbal combination could be a candidate drug for hyperlipidemia which can alternate statin, with significant benefits on modulating lipid metabolism.”

 

5. It would be great if the authors provide color graphs in fig.7 and fig.8

• We authors agreed with your opinion on adding color on the bar charts in fig 7 and 8.
• Therefore, we modified our figure 7 and 8 by your suggestion.

 

6. Table 1 caption. The name of the plants should be in italics. 

• We appreciate your kind comments on our mistake. We corrected it immediately.

Author Response File: Author Response.docx

Reviewer 2 Report

 

Comments to the Author

In the manuscript entitled "Network Pharmacological Analysis of a New Herbal Combination Targeting Hyperlipidemia and Efficacy Validation In Vitro", the authors have undertaken a study to understand the effect of new herbal combination against hyperlipidemia using Network pharmacology method. The topic of the paper is interesting. The experimental design was rational and the results were reliable. I recommend the acceptance of the manuscript after minor revision.

The comments are as follow:

1) Page 3, line number 130, A PPI network with STRING was produced using a query list of target genes. What is the confidence score of the interactions collected from STRING database?

2) Page 3, line number 133, The total PPI 132 network was clustered functionally using MCODE, What are the clustering parameters?

3) Page 12, line number 342, Modify figure 7B blots with labels.

 

4) Throughout the manuscript, typographical and graphical errors need to be corrected. For example : Page 4, line number 183, 4 × 104 cells/well. 

Author Response

In the manuscript entitled "Network Pharmacological Analysis of a New Herbal Combination Targeting Hyperlipidemia and Efficacy Validation In Vitro", the authors have undertaken a study to understand the effect of new herbal combination against hyperlipidemia using Network pharmacology method. The topic of the paper is interesting. The experimental design was rational and the results were reliable. I recommend the acceptance of the manuscript after minor revision.

• We are very pleased to have kind comments on our study. We tried to share the result of research on novel herbal combination with reasonable explanation on mechanisms.
• We assume that the herbal combination consisting of ZO, PC and AT plays key role in modulating lipid metabolism in many herbal prescriptions. We believe this study be valuable for other peer researchers and clinical practitioners.

 

1) Page 3, line number 130, A PPI network with STRING was produced using a query list of target genes. What is the confidence score of the interactions collected from STRING database?

• We appreciate for the comments on confidence score. We used medium confidence. (0.400)
• We wrote confidence score in the revised manuscript in ‘2.3 Protein-protein interaction (PPI) network construction’ part (confidence score = 0.400).

2) Page 3, line number 133, The total PPI 132 network was clustered functionally using MCODE, What are the clustering parameters?

• We used following parameter settings:

Degree cutoff: 2 ,

Cluster finding: haircut,

node score cut off: 0.2

K-core: 2,

Max depth: 100;

These parameters were default setting in MCODE cluster.

• The MCODE app detects densely connected regions in whole protein networks to find molecular complexes, based on vertex weighting by local neighborhood density and outward traversal from a locally dense seed protein to isolate the dense regions according to given parameters. (reference: https://bmcbioinformatics.biomedcentral.com/articles/10.1186/1471-2105-4-2)

 

3) Page 12, line number 342, Modify figure 7B blots with labels.

• We are grateful for your precious suggestion on the labels of the figure.
• As per your request, we inserted labels on the figure 7B for convenience of the readers.

 

4) Throughout the manuscript, typographical and graphical errors need to be corrected. For example : Page 4, line number 183, 4 × 104 cells/well. 

• Thank you for pointing out the mistake. We revised the point correctly.
• Moreover, we checked for minor errors of spacing, typo errors throughout the manuscript and corrected it.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors answerd some of my questions. But for my point 2 the authors didnt answer my questions properly. They just re-written the sentences from Manuscript. My main point is "WHAT WAS THE QUANTITY OF THE EXTRACT WHEN SELECTED FOR IN-VITRO TEST? THERE IS A STANDARD FOR CRUDE EXTRACTS DOING IN-VITRO or IN-VIVO experiememts. If the authors didnt properly mention this. It means all the results are totally wrong. if the quantity of extract cross the standard then the results are counted as false results. So this is the critical thing authors should respond. So i still stay on major revision.

Author Response

Reviewer comments:

The authors answerd some of my questions.

But for my point 2 the authors didnt answer my questions properly.

They just re-written the sentences from Manuscript.

My main point is "WHAT WAS THE QUANTITY OF THE EXTRACT WHEN SELECTED FOR IN-VITRO TEST? THERE IS A STANDARD FOR CRUDE EXTRACTS DOING IN-VITRO or IN-VIVO experiememts.

If the authors didnt properly mention this.

It means all the results are totally wrong.

if the quantity of extract cross the standard then the results are counted as false results.

So this is the critical thing authors should respond. So i still stay on major revision.

 

Response to reviewer

Dear reviewer,

• We appreciate your sincere comments raised on our manuscript. We feel grateful that we had another chance to explain.
• However, we authors could not fully understand the true meaning of reviewer question. We tried to respond every comment based on what we understand. We really hope that our answers will address the issues raised by the reviewer.

 

1. About “What was the quantity of the extract when selected for in-vitro test.”

• If you mean the amount of dried herbal extract, we already mentioned it in response to reviewer by presenting the extraction yield (AT 2.88%, PC 2.24%, ZO 3.20%, Mix 2.40%). This means we procured 2.88g, 2.24g, 3.20g, 2.40g of each AT, PC, ZO, and Mix powder as we started extraction with 100g of herbs.
• If you are speculating that we used several more herbs (except AT, PC, ZO and Mix) on investigating the drug efficacy, it is not true. We didn’t test other herbal extracts. As we mentioned in our manuscript, these three herbs and mixture were the main interests.
• So it means that we did not included/excluded herbs by selecting them with any intention or standard. Only the ZO, PC, AT were chosen to be investigated. 
• Dried extracts were easily soluble to water; therefore we used DPBS as eluent to prepare stock solution to make final concentration of 10 mg/ml. No precipitation was observed in stock solution.
• As we already conducted studies with several other herbs using their extracts, we think we have proper knowledge and experience on preparing herbal extractions. We are certain that no problem was observed in extraction processes.

1. About “THERE IS A STANDARD FOR CRUDE EXTRACTS DOING IN-VITRO or IN-VIVO experiememts”. Also, you mentioned as “if the quantity of extract cross the standard then the results are counted as false results”.

• What “standard” are you exactly saying about? We authors yet heard of any specific standard or criteria applied in extraction process as figures to determine the quality of extract.
• The presented extraction yield (2.24 - 3.20%) is within the normal range as compared to other study.
• If you talking about the content of each herb comprising herbal prescription (composition of herbs in prescriptions), it depends on the herbal prescriptions. However, we didn’t use any herbal prescription, but used three herbs and the mixture of three herbs in this study.
• In other cases, when you evaluate our research, you may have wondered which of the various compounds of herbal extracts we chose to study. However, we didn’t use any bioactive compounds for test in this study, neither.

 

• In conclusion, we insist that the samples were appropriately prepared. To be Honest, we are much embarrassed to have critical comments on herbal preparation part, since we used the method for a long time, and it is generally used by researchers.
• If we misunderstood your intention, please specify your question. We will answer it with pleasure. Or if we missed important facts/issues related with this, please enlighten us.

Many thanks,

 

Round 3

Reviewer 1 Report

The authors addressed all my questions and i am satisfied with it.