The Chloroform Extracts of Vietnamese Sophora flavescens Ait. Inhibit the Proliferation of HepG2 Cells through Apoptosis Induction

Round 1

Reviewer 1 Report

The manuscript presented herein demonstrated the potential of Sophora Flavescens root extract in proliferation inhibition of HepG2 cells through inducing apoptosis. Experiments are technically sound and the clarity of writing is exemplary. However, a few revisions are required before further consideration.

1. The title is confusing. The title easily leads to the concept that matrine and oxymatrine inhibit the proliferation of HepG2 cells through apoptosis. Reviewer did not see any evidence in the manuscript supporting the idea that matrine and oxymatrine are the acting agents.
2. Following the point above, could we test the cytotoxicity of purified matrine and oxymatrine? As shown in reference 11, prenylated flavanonol from Sophora Flavescens root extract showed ‘moderate inhibition towards those three tumor cell lines at 14.3, 19.2, 31.5 μg/mL, respectively,’ which is similar to what we observed in this manuscript. The cytotoxicity of Sophora Flavescens root extract could originate from prenylated flavanonol instead of matrine and oxymatrine as implied in the title.
3. Line 137, what is the point of 71 cycles of 60 ⁰C?
4. Do we observe other compounds other than matrine and oxymatrine?
5. Could reviewer add the western uncut picture in the supplemental material?

Author Response

Dear Prof. Dr. Takayoshi Kobayashi and Editorial board,

 

We are very grateful to the Editor for your consideration of our manuscript. We would like to thank the Reviewers for careful and thorough reading of manuscript and for the thoughtful comments and constructive suggestions, which help to improve the quality of this manuscript. Each comment has been carefully considered point by point and responded. Responses to the reviewers and changes in the revised manuscript are as follows.

 

Manuscript ID: applsci-1715622

Title: Matrine and Oxymatrine-rich extracts of Vietnamese Sophora flavescens Ait. inhibited the proliferation of HepG2 cells by apoptosis induction

 

Response to Reviewer 1 Comments:

Point 1:

1. The title is confusing. The title easily leads to the concept that matrine and oxymatrine inhibit the proliferation of HepG2 cells through apoptosis. Reviewer did not see any evidence in the manuscript supporting the idea that matrine and oxymatrine are the acting agents.

Response 1. Thank you so much for your advice. The title has been corrected to “The chloroform extracts of Vietnamese Sophora flavescens Ait. inhibit the proliferation of HepG2 cells through apoptosis induction”.

 

Point 2:

1. Following the point above, could we test the cytotoxicity of purified matrine and oxymatrine? As shown in reference 11, prenylated flavanonol from Sophora Flavescens root extract showed ‘moderate inhibition towards those three tumor cell lines at 14.3, 19.2, 31.5 μg/mL, respectively,’ which is similar to what we observed in this manuscript. The cytotoxicity of Sophora Flavescens root extract could originate from prenylated flavanonol instead of matrine and oxymatrine as implied in the title.

Response 2. The previous study showed that prenylated flavanonol or some novel flavonoids isolated from Sophora flavescens had been identified as the potent antitumor activities. Besides that, the other studies have been reported that matrine and oxymatrine from Sophora flavescens also exhibit the proliferative inhibition and induces apoptosis on several cancer cells such as pancreatic cancer cells [1,2], prostate cancer cells, or against other cancer including cervical cancer, ovarian cancer, colorectal cancer [3,4,5]. These studies showed that there is a high potential of matrine and oxymatrine from Sophora flavescens against tumor cells. Thus, this study applied the chloroform extracts which have the highest contents of matrine and oxymatrine to evaluate the hepG2 cells proliferation and apoptosis.

 

References

1. Tianyou Liu, Yan Song, Hua Chen, Shangha Pan, Xueying Sun, Matrine Inhibits Proliferation and Induces Apoptosis of Pancreatic Cancer Cells in Vitro and in Vivo, Biological and Pharmaceutical Bulletin, 2010, Volume 33, Issue 10, Pages 1740-1745.
2. Wu, C., Huang, W., Guo, Y., Xia, P., Sun, X., Pan, X., & Hu, W. (2015). Oxymatrine inhibits the proliferation of prostate cancer cells in vitro and in vivo. Molecular Medicine Reports, 11, 4129-4134.
3. Zhou YJ, Guo YJ, Yang XL, Ou ZL. Anti-Cervical Cancer Role of Matrine, Oxymatrine and Sophora Flavescens Alkaloid Gels and its Mechanism. J Cancer. 2018;9(8):1357-1364.
4. Zhang, X., Hou, G., Liu, A. et al. Matrine inhibits the development and progression of ovarian cancer by repressing cancer associated phosphorylation signaling pathways. Cell Death Dis 10, 770 (2019).
5. Li X, Sun J, Xu Q, Duan W, Yang L, Wu X, Lu G, Zhang L, Zheng Y. Oxymatrine Inhibits Colorectal Cancer Metastasis via Attenuating PKM2-Mediated Aerobic Glycolysis. Cancer Manag Res. 2020;12:9503-9513

 

Point 3:

1. Line 137, what is the point of 71 cycles of 60 ^oC?

Response 3. This is the process of Melting curve analysis which can be performed with real-time PCR detection, as followed:

The Start temperature: 60.0^oC

            The End temperature: 95.0^oC

            Hold time:         15 s

            Temperature Increment: 0.5 ^oC

            Estimated Total time: 30m 07s

            Number of cycles: 71

 

Point 4:

1. Do we observe other compounds other than matrine and oxymatrine?

Response 4. We would like to present the other compounds from SFA extract as followed:

The analysis was performed on an HPLC 1200 system (Agilent Technologies, USA), equipped with a binary solvent delivery apparatus, an auto-sampler, coupled to a Bruker micrO-TOF QII (Bruker, Germany) system. The mobile phase consisted of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in methanol). The analytical sample (injection volume: 5 µL) was separated in an ACE C18 150 x 4,6 mm, 3,5 µm (150 mm× 4.6 mm × 3.5 µm particle size; UK) at the flow rate of 0.5 mL. min-1 and the column temperature of 40⁰C. The gradient program was set as follows: 10 % solvent B was maintained initially for 1 min, followed by a gradual increase to 100% over 20 min, and then maintained at 100% solvent B for 10 min. The MS data were collected in the range 100–1000 m/z under positive ion modes. The desolvation gas (nitrogen) was set to 10 L.min-1 at a temperature of 300⁰C. The Nebulizer gas (nitrogen) was set to 35 psi, and the source temperature was 300⁰C. The capillary voltage was set to 3500 V. Data were collected in the centroid mode. Accurate masses and elemental compositions were calculated using the Data Analysis software (Bruker, Germany) incorporated in the instrument.

 

Mass error in parts per million (ppm) = (m/z_exact – m/z_accurate) * 10⁶/ m/z_exact

 

Identification of tentative compounds to EA extracts of Sophora flavescens Ait. on the basis of LC-MS results.

No.	Tentative Identifications	tR (min)	Measured Mass (m/z) [M + H]+	Elemental	m/z error
				Composition	(ppm)
1	Formononetin	16.9	269.0804	C16H12O4	-3.8
2	Kurarinol	17.1	457.2246	C26H32O7	4.2
3	7,4'-Dihydroxy-5-methoxy-8(γ,γ-dimethylallyl)-flavanone	17.8	355.1554	C21H22O5	2.3
4	Trifolirhizin	17.9	447.1293	C22H22O10	0.3
5	3’,7-Dihydroxy-4’-methoxy isoflavone	18.1	285.0756	C16H12O5	-2.6
6	Kushenol I/N	18.3	455.2065	C26H30O7	-1.1
7	Kuraridine	18.6	439.2122	C26H30O6	0.2
8	Sophoraisoflavanone A	18.7	371.1485	C21H22O6	-2.7
9	Xanthohumol	19.1	355.1534	C21H22O5	-3.3
10	Kushenol C	19.6	439.2122	C26H30O6	0.2
11	isoxanthohumol	19.8	355.1534	C21H22O5	-3.3
12	2’_Methoxykurarinone	20.1	453.2275	C27H32O6	-0.5
13	Sophoraflavanone G	20.1	425.1945	C25H28O6	-4.6
14	Isokurarinone	20.7	439.2142	C26H30O6	4.8
15	Kushenol A	21.1	409.2031	C25H28O5	3.8
16	Kushenol D	21.4	453.2275	C27H32O6	-0.5
17	Kusamol R	22	423.218	C26H30O5	1.9
18	Kurarinone	22.1	439.2132	C26H30O6	2.5

 

Point 5:

1. Could reviewer add the western uncut picture in the supplemental material?

Response 5. As follows the comment of the reviewer, we have carried out again the western blot analysis to get the better bands in X-ray film. We also re-do the relative expression analysis of Bax and Bcl-2 with fixing 1 as the reference.

 

We hope that our corrections could meet your requirements,

 

Thank you so much

 

 

Author Response File: Author Response.docx

Reviewer 2 Report

Dear Editor,

The manuscript Matrine and Oxymatrine-rich extracts of Vietnamese Sophora flavescens Ait. inhibited the proliferation of HepG2 cells by apoptosis induction interestingly reports the anti-proliferative and pro-apoptotic effects of chloroform extract of Matrine and Oxymatrine-rich extract of Sophora flavescens Ait (SFA) on the hepatocarcinoma cell line HepG2. Hallmarks of apoptosis were analyzed in SFA chloroform extract-treated HepG2 cells, including cell shrinkage, cell cycle arrest, nuclear fragmentation and upregulation of pro-apoptotic Bax accompanied by downregulation of anti-apoptotic Bcl2 mitochondrial proteins.

I have found the article justified for this journal based on application-orientated research journal devoted to reporting advances with originality and novelty, in the science and technology of biological research.

The outcome of research is interesting; however, the manuscript cannot be considered for publication as it stands. The study contains number of scientific flaws in terms of accuracy of information, data presentation/interpretation and in the clarity.

 

1.      First, in the Title, I would suggest to change “by apoptosis induction” with “through (or via) apoptosis induction”. The term through or via would be more appropriate when it comes to a biochemical-mediated process such as apoptosis.

 

2.      In the Abstract, the authors should replace “CHCl3” with methanol and should mention the use of other solvents referring to “highest matrine and oxymatrine contents” (compared to?). Hence, chloroform should be added to “SFA extract” in order to distinguish this extract from other solvent extracts.

The sentence (l23-24: “Cell cycle analysis… was increased”) should be improved.

The authors should clarify the “morphological changes in HepG2 nuclear”. It is too vague in the Abstract.

(l28-29): “down-regulation of HepG2” and “inducer against HepG2”, replace “of’ and “against” with “in”.

3.      In the Introduction, the authors should write a paragraph about Apoptosis: Key term mentions in the Title and that would clarify the purpose of the apoptosis-related assays.

Correction on “viable therapies” and clarification requested on “Herbal medicine has been characterized to respond to”.

 

4.      In the Materials and Methods section, the authors should define the chemical compound while mentioning EtOH:H2O and MeOH 5%NH3. Throughout the full section, the authors should use the name of the chemical compound only (to avoid mixing chemical compound and its name, such as in l69-71). Same requirement regarding the Table 1. Full name of each compound should be mentioned as well.

The full terms of abbreviations (i.e. HPLC, HRESIMS) are required.

(l118) status should be added to “apoptosis analysis”.

Missing information regarding the City/State of most of the suppliers. To be completed.

Complete State for Abcam-related information and mention it only once. Delete “USA” from Abcam in the repeated ones.

5.      In the Results section, the first paragraph (l161-163) must be removed.

-          The subtitles do not exactly reflect the described results. For instance, “HepG2 cell morphology” is too restrictive as cell density, an indicator of cell proliferation, is also mentioned. Same remark for 3.3 HepG2 cell proliferation, section in which cell cycle progression, apoptosis, and Bax/Bcl-2 protein expression are described. Thus, the authors are advised to rename the subtitles appropriately.

To conclude cycle progression of HepG2 cells in a time-dependent manner, the authors should verify whether there is a significant difference between the relevant effects. The authors should clarify this point. Same remark for apoptosis-related bar graph.

Better scatter plots should be shown because less analyzed cells are displayed in the scatter plots corresponding to the treated cells, compared to higher cell number analyzed in the scatter plot corresponding to the untreated cells. In addition, the percentage of cells should be mentioned in each quadrant and the experimental conditions should be clarified in the figure legend as well.

Higher magnification of the pointed (by the arrows) nuclear morphology should be also included.

Regarding the Figure 5, the authors should replace the Western blot images with better ones showing the entire bands. The related bar graph is confusing with Gapdh data. Hence, the authors must remove the data corresponding to Gapdh analysis and re-do relative expression analysis of Bax and Bcl-2, fixing 1 as the reference and corresponding to the basal expression level of the target protein.

 

6.      Despite the numerous results, the Discussion is too empty in explanation and in suggestion of possible molecular insights. These phytochemicals, particularly matrine, have been well studied as potential anticancer agents. Numerous studied have demonstrated and pinpointed the dysregulated mechanisms induced by the phytochemical, explaining its antiproliferative effect.

Therefore, the authors must work this Discussion section.

 

7.      There are too many grammatical errors. The authors should correct their manuscript with suitable English Writer during resubmission.

 

I hope that my comments will contribute to the improvement of the manuscript and will help you make a decision on the quality of this manuscript.

Faithfully yours,

Comments for author File: Comments.pdf

Author Response

Dear Prof. Dr. Takayoshi Kobayashi and Editorial board,

 

We are very grateful to the Editor for your consideration of our manuscript. We would like to thank the Reviewers for careful and thorough reading of manuscript and for the thoughtful comments and constructive suggestions, which help to improve the quality of this manuscript. Each comment has been carefully considered point by point and responded. Responses to the reviewers and changes in the revised manuscript are as follows.

 

Manuscript ID: applsci-1715622

Title: Matrine and Oxymatrine-rich extracts of Vietnamese Sophora flavescens Ait. inhibited the proliferation of HepG2 cells by apoptosis induction

 

Response to Reviewer 2 Comments:

Point 1:

1. First, in the Title, I would suggest to change “by apoptosis induction” with “through (or via) apoptosis induction”. The term through or viawould be more appropriate when it comes to a biochemical-mediated process such as apoptosis

Response 1. Thank you so much for your comment, the title has been corrected to “The chloroform extracts of Vietnamese Sophora flavescens Ait. inhibit the proliferation of HepG2 cells through apoptosis induction”.

Point 2:

2. In the Abstract, the authors should replace “CHCl3” with methanol and should mention the use of other solvents referring to “highest matrine and oxymatrine contents” (compared to?). Hence, chloroform should be added to “SFA extract” in order to distinguish this extract from other solvent extracts.

Response 2.1. The abstract was corrected and rewritten. We also add the comparation of “highest matrine and oxymatrine contents” from CHCl3 extract, EtOH extract and Ethyl acetate extract. “SFA extract” was replaced by “SFA-CHCl3 extract”.

The sentence (l23-24: “Cell cycle analysis… was increased”) should be improved.

Response 2.2. This sentence was rewritten.

The authors should clarify the “morphological changes in HepG2 nuclear”. It is too vague in the Abstract.

Response 2.3. The morphological changes in HepG2 nuclear were demonstrated in this abstract.

(l28-29): “down-regulation of HepG2” and “inducer against HepG2”, replace “of’ and “against” with “in”.

Response 2.4. The “of’ and “against” were replaced by “in” in these phrases.

Point 3:

3. In the Introduction, the authors should write a paragraph about Apoptosis: Key term mentions in the Title and that would clarify the purpose of the apoptosis-related assays.

Response 3.1. A paragraph about apoptosis was added to the Introduction.

Correction on “viable therapies” and clarification requested on “Herbal medicine has been characterized to respond to”.

 Response 3.2. The “viable therapies” was corrected to “therapies”. The sentences “Herbal medicine has been characterized to respond to the tumor formation, growth, and spread. It's been employed to treat HCC symptoms and the side effects of cancer treatment” were removed.

Point 4:

4. In the Materials and Methods section, the authors should define the chemical compound while mentioning EtOH:H2O and MeOH 5%NH3. Throughout the full section, the authors should use the name of the chemical compound only (to avoid mixing chemical compound and its name, such as in l69-71). Same requirement regarding the Table 1. Full name of each compound should be mentioned as well.

Response 4.1. We have corrected “EtOH:H2O” to “ethanol–water mixture” and “MeOH 5%NH3” to “5% ammonia solution in methanol”. We also corrected the name of the chemical compound in the manuscript.

The full terms of abbreviations (i.e. HPLC, HRESIMS) are required.

Response 4.2. The full terms of abbreviations were added to the Abbreviation section.

(l118) status should be added to “apoptosis analysis”.

Response 4.3. The SFA-CHCl3 extract treatments of HepG2 cells by time-dependent manner was added to flow cytometry analysis to evaluate the apoptosis and cycle progression of these cells.

Missing information regarding the City/State of most of the suppliers. To be completed.

Response 4.4. The City/State of the suppliers were added to the reagents in Materials and Methods.

Complete State for Abcam-related information and mention it only once. Delete “USA” from Abcam in the repeated ones.

Response 4.5. The Abcam-related information was corrected.

Point 5:

5. In the Results section, the first paragraph (l161-163) must be removed.

Response 5.1. This paragraph was removed.

-          The subtitles do not exactly reflect the described results. For instance, “HepG2 cell morphology” is too restrictive as cell density, an indicator of cell proliferation, is also mentioned. Same remark for 3.3 HepG2 cell proliferation, section in which cell cycle progression, apoptosis, and Bax/Bcl-2 protein expression are described. Thus, the authors are advised to rename the subtitles appropriately.

Response 5.2. The subtitles were corrected in the Result section.

To conclude cycle progression of HepG2 cells in a time-dependent manner, the authors should verify whether there is a significant difference between the relevant effects. The authors should clarify this point. Same remark for apoptosis-related bar graph.

Response 5.3. We have presented the significant difference between groups in G0/G1 phase, S phase, and G2/M phase of Figure 3. We also added the percentage of HepG2 cells in each quadrant of apoptosis scatter plots by flow cytometry. The old Figure 4E was removed.

Better scatter plots should be shown because less analyzed cells are displayed in the scatter plots corresponding to the treated cells, compared to higher cell number analyzed in the scatter plot corresponding to the untreated cells. In addition, the percentage of cells should be mentioned in each quadrant and the experimental conditions should be clarified in the figure legend as well.

Response 5.4. For apoptosis analysis, the cell number from SFA-CHCl3 extract treatment groups were 4×10⁴ cells to 5 ×10⁴ cells which were lower than control group (6 ×10⁴ cells). These reduced cell numbers in SFA-CHCl3 extract treatment caused by apoptosis induction which lead to cell death.

Higher magnification of the pointed (by the arrows) nuclear morphology should be also included.

Response 5.5. The Figures of nuclear morphology were adjusted in Figure 4E.

Regarding the Figure 5, the authors should replace the Western blot images with better ones showing the entire bands. The related bar graph is confusing with Gapdh data. Hence, the authors must remove the data corresponding to Gapdh analysis and re-do relative expression analysis of Bax and Bcl-2, fixing 1 as the reference and corresponding to the basal expression level of the target protein.

Response 5.6. We have carried out again the western blot analysis to get a better band in X-ray film exposure. We also re-do the relative expression analysis of Bax and Bcl-2 with fixing 1 as the reference.

Point 6:

6. Despite the numerous results, the Discussion is too empty in explanation and in suggestion of possible molecular insights. These phytochemicals, particularly matrine, have been well studied as potential anticancer agents. Numerous studied have demonstrated and pinpointed the dysregulated mechanisms induced by the phytochemical, explaining its antiproliferative effect.

Therefore, the authors must work this Discussion section.

Response 6.1. We have discussed more the potentials of matrine,  oxymatrine, and the evaluation of the combination of these compounds in hepG2 cell proliferation.

 Point 7:

7. There are too many grammatical errors. The authors should correct their manuscript with suitable English Writer during resubmission.

Response 7.1. We have checked and corrected the grammatical errors in our manuscript.

We hope that our corrections could meet your requirements,

 

Thank you so much

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

This manuscript has been improved after the revision and should be considered for publication. 

Author Response

Dear Prof. Dr. Takayoshi Kobayashi and Editorial board,

We are grateful for your consideration of our manuscript. We would like to thank the Reviewers for careful and thorough reading of the manuscript and for the thoughtful comments and constructive suggestions, which help to improve the quality of this manuscript. Each comment has been carefully considered point by point and responded. Responses to the reviewers and changes in the revised manuscript are as follows.

 

Manuscript ID: applsci-1715622

Title: Matrine and Oxymatrine-rich extracts of Vietnamese Sophora flavescens Ait. inhibited the proliferation of HepG2 cells by apoptosis induction

 

Response to Reviewer 1 Comments:

 

- This manuscript has been improved after the revision and should be considered for publication. 

Response. Thank you so much for your comments, which improve the quality of our manuscript.

Best regards

 

Reviewer 2 Report

Dear Editor,

The manuscript Matrine and Oxymatrine-rich extracts of Vietnamese Sophora flavescens Ait. inhibited the proliferation of HepG2 cells by apoptosis induction has been considerably improved. Overall, I am satisfied with the answers and with most of the changes. However, there are still minor corrections to be made:

-          Typo mistake on “has showed”, “could inhibits”, “each wells”, “2 ug/mL Hoechst”, from 24h to 74h” to be corrected.

 

-          “by time-dependent manner” is not well used throughout the manuscript. Better to use these following terms: time course, over the time or effect in a time-dependent manner. Therefore, “HepG2 cell proliferation by time-dependent manner”, “Cycle progression of HepG2 cells by time-dependent manner”, “The apoptosis analysis of HepG2 cells by time-dependent manner” must be rewritten.

 

-          In Figure 3, it is indicated that the percentage of HepG2 cells in S phase significantly increased in 48 h “(**P < 0.01, 48 h group vs. other groups)”. However, according to Fig 3B there is no significant difference between 48 h and 72 h. Therefore, “vs. other groups” must be clarified and corrected.

 

-          Addition of the molecular weight along the Western blot images.

 

-          In the Discussion and the Conclusion, keep indicating SFA-CHCl3 when it comes to the current study.

 

-          There are too many typo mistakes in the Supplementary data that must be corrected as well.

 

After making the requested corrections, the manuscript can be considered for publication.

I hope that my comments will help you make your final decision.

Faithfully yours,

Comments for author File: Comments.pdf

Author Response

Dear Prof. Dr. Takayoshi Kobayashi and Editorial board,

We are grateful for your consideration of our manuscript. We would like to thank the Reviewers for careful and thorough reading of manuscript and for the thoughtful comments and constructive suggestions, which help to improve the quality of this manuscript. Each comment has been carefully considered point by point and responded. Responses to the reviewers and changes in the revised manuscript are as follows.

Manuscript ID: applsci-1715622

Title: Matrine and Oxymatrine-rich extracts of Vietnamese Sophora flavescens Ait. inhibited the proliferation of HepG2 cells by apoptosis induction

 

Response to Reviewer 2 Comments:

-          Typo mistake on “has showed”, “could inhibits”, “each wells”, “2 ug/mL Hoechst”, from 24h to 74h” to be corrected.

Response 1. Thank you so much for your comment, we have corrected these mistakes as followed:

Page 3, line 4: “has showed” was corrected to “showed”

Page 3, line 6: “could inhibits” was corrected to “could inhibit”

Page 5, in method “2.4. Cell density determination”: “each wells” was corrected to “each well”.

Page 6, in method “2.6. Nuclear staining”: “2 ug/mL Hoechst” was corrected to “2 µg/mL Hoechst”

Page 10, in the legend of Figure 3: “from 24h to 74h” was corrected to “from 24h to 72h”. 

-          “by time-dependent manner” is not well used throughout the manuscript. Better to use these following terms: time course, over the time or effect in a time-dependent manner. Therefore, “HepG2 cell proliferation by time-dependent manner”, “Cycle progression of HepG2 cells by time-dependent manner”, “The apoptosis analysis of HepG2 cells by time-dependent manner” must be rewritten.

Response 2. “by time-dependent manner” was corrected by “over the time” throughout the manuscript.

-          In Figure 3, it is indicated that the percentage of HepG2 cells in S phase significantly increased in 48 h “(**P < 0.01, 48 h group vs. other groups)”. However, according to Fig 3B there is no significant difference between 48 h and 72 h. Therefore, “vs. other groups” must be clarified and corrected.

Response 3.  Thank you so much for your comment. This is corrected to “(**P < 0.01, 48h group vs. control group and 24h group”

-          Addition of the molecular weight along the Western blot images.

Response 4. The molecular weight was added to the Western blot images in Figure 5.

-          In the Discussion and the Conclusion, keep indicating SFA-CHCl3 when it comes to the current study.

Response 5. “SFA-CHCl3 extract” was used to replace “SFA extract” in the Discussion and the Conclusion and throughout the manuscript.

-          There are too many typo mistakes in the Supplementary data that must be corrected as well.

Response 6. The typo mistakes in the Supplementary data were corrected.

 

We hope that our corrections could meet your requirements,

Thank you so much.