Diversity and Biological Activity of Secondary Metabolites Produced by the Endophytic Fungus Penicillium ochrochlorae

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Overall

The manuscript appears to be in its initial stages and therefore requires considerable attention.

Some observations and suggestions are included in the manuscript.

However, here are some examples.

The manuscript's English language requires a great deal of attention; it appears to have been written with the help of artificial intelligence.

The introduction does not address the concept of metabolic mimicry between plants and fungi. This aspect will help to understand that the 27-nor-3,4-secocycloartane schinortriterpenoid and Penioch A are metabolic constituents of both fungus P. ochrochloron and the host plant K. angustifolia.

In the Results section, Tables 1-4 are missing information, giving the impression that they are "hiding information."

In the References section, 10 references are from 2020 to the present. 55 gives the impression that this is not a novel work. This is one of the many reasons why the introduction needs improvement.

I noticed that some citations are incorrect, particularly in the "Materials and Methods" section.

There is information missing from the "Materials and Methods" section.

In the Results section, Tables 1-4 are missing information, giving the impression that they are "hiding information."

In the References section, 10 references are from 2020, out of 55 to date. It gives the impression that this is not a novel work. This is one of the many reasons why the introduction needs improvement.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The manuscript appears to be in its initial stages and therefore requires considerable attention. The manuscript's English language requires a great deal of attention; it appears to have been written with the help of artificial intelligence.

Author Response

Dear Reviewer,

Thanks a lot for reviewing our paper “Diversity and biological activity of secondary metabolites produced by endophytic fungus Penicillium ochrochlorae (Fermentation-3663865)”. We appreciate your approval of our last manuscript revision. Your comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our research. We have studied the comments carefully and have made revision which emphasis on the text in red font.

Now, we have modified our manuscript point by point according to the comments of reviewer. They mainly include:

1.Comment: Some observations and suggestions are included in the manuscript.

Reply: We have added “Abbreviations” content at the end of the manuscript, including:

NMR    Nuclear Magnetic Resonance

MTT    3-[4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium] bromide

DMSO   Dimethyl Sulfoxide

DPPH   1,1-Diphenyl-2-picrylhydrazyl

DMEM   Dulbecco's Modified Eagle Medium

TLC     Thin-Layer Chromatography

2.Comment: The manuscript's English language requires a great deal of attention; it appears to have been written with the help of artificial intelligence.

Reply: We embellished and modified the language of the text in detail, corrected the grammar problem, and the full text did not use artificial intelligence to assist the writing of the paper. The text content of this paper passed the AI writing rate detection of the journal.

3.Comment: The introduction does not address the concept of metabolic mimicry between plants and fungi. This aspect will help to understand that the 27-nor-3,4-secocycloartane schinortriterpenoid and Penioch A are metabolic constituents of both fungus P. ochrochloron and the host plant K. angustifolia.

Reply: We have added the content description about the metabolism of endophytic fungi to produce host analogues, as well as the modification and transformation of host metabolites in the “Introduction”, please check in the corresponding part of the article.

4.Comment: In the Results section, Tables 1-4 are missing information, giving the impression that they are "hiding information."

Reply: We express our sincere gratitude for your reminder and guidance. In this study, it was not our intention to withhold any information. However, the scope of activity tests conducted on compounds 1-20 was primarily constrained by the limited quantities of the isolated compounds. As evidenced, the weight obtained from the separation of certain compounds was insufficient to facilitate the concurrent execution of three distinct types of biological activity assays across multiple concentration gradients. For instance, compound 3 yielded 5 mg, compound 8 yielded 5.1 mg, compound 11 yielded 4.1 mg, and compound 17 yielded 4.9 mg. Consequently, we undertook comprehensive literature research focusing on the structural characteristics of these compounds and ultimately prioritized the most promising biological activities for testing. This led to the selection of antibacterial activity assays for compounds 1-14, antioxidant activity assays for compounds 15-17, and anti-tumor cytotoxicity assays for compounds 18-20.

5.Comment: In the References section, 10 references are from 2020 to the present. 55 gives the impression that this is not a novel work. This is one of the many reasons why the introduction needs improvement.

Reply: Following your suggestion, we have reviewed and updated the references throughout the entire text, incorporating numerous recent publications. Kindly examine the revised “Reference” list.

6.Comment: I noticed that some citations are incorrect, particularly in the "Materials and Methods" section. There is information missing from the "Materials and Methods" section.

Reply: Under your supervision, we have rectified the incorrect references in the Materials and Methods section, provided comprehensive details regarding the model information of the instruments and equipment, and included the separation process and detailed information for all compounds. Kindly review these revisions in the revised “Materials and Methods” section.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

In this manuscript, Hu et al. reported the assessment of the secondary metabolites produced by Penicillium ochrochlorae. While 19 out of the 20 compounds they isolated have been identified before, they revealed a new metabolite, penioch A (20). They also evaluated the biological activities of the isolated compounds. Overall, this manuscript indeed showed the large potential of Penicillium ochrochlorae to produce diverse natural products and thus is suitable for publication in Fermentation. However, the authors should address my following comments prior to publication.

 

Major comments:

• The structural characterization of compound 20 needs to be clearer. First, the m/z value of 20 is way off from its theoretical value, leading to around 733 ppm error. The authors should double check the ESI-MS analysis.
• Second, it is uncommon to see that carbon signals have splitting patterns as shown in Table 1. I assume the authors did not exclude C-H coupling during the NMR analysis. But the 13C NMR spectrum in the SI does not clearly show the splitting patterns. Please clarify this.

 

Minot comments:

• Line 14, “polyketones” should be “polyketides”.
• Line 22, “1-17” should be bold.
• Line 24, a space is missing between “74.9” and “to”.
• Line 35, the first sentence of Introduction needs a reference.
• Line 75, “Hydroxyisosclerone” should be “hydroxyisosclerone”
• Line 83, “of 1 showed” should be “of 20 showed”
• Line 94, “triterpenoid-nigranoic acid (3)” should be “triterpenoid-nigranoic acid (18)”
• Line 96, it is better to number the carbons in 18 since they are discussed in the text.
• Line 116, “1-14” should be bold.

Author Response

Dear Reviewer,

Thanks a lot for reviewing our paper “Diversity and biological activity of secondary metabolites produced by endophytic fungus Penicillium ochrochlorae (Fermentation-3663865)”. We appreciate your approval of our last manuscript revision. Your comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our research. We have studied the comments carefully and have made revision which emphasis on the text in red font.

Now, we have modified our manuscript point by point according to the comments of reviewer. They mainly include:

 

1.Major comments:

The structural characterization of compound 20 needs to be clearer. First, the m/z value of 20 is way off from its theoretical value, leading to around 733 ppm error. The authors should double check the ESI-MS analysis.

Second, it is uncommon to see that carbon signals have splitting patterns as shown in Table 1. I assume the authors did not exclude C-H coupling during the NMR analysis. But the 13C NMR spectrum in the SI does not clearly show the splitting patterns. Please clarify this.

Reply: Prompted by your reminder, we conducted a thorough re-evaluation of the mass spectrometry and high-resolution mass spectrometry data of compound 20. This review revealed a typing error in our manuscript reported theoretical value for compound 20. Based on the test results, the HR-ESI-MS analysis of compound 20 indicated an m/z value of 479.3493 [M+Na]⁺, which is consistent with the calculated value for C₃₀H₄₈O₃Na of 479.3496. The corrected high-resolution mass spectrometry data have been uploaded to the revised supplementary materials for detailed reference.

Over the past decade, our research team has undertaken extensive investigations into triterpenoids within Schisandraceae plants, successfully isolating and characterizing the skeletal properties and structural features of numerous such compounds [1-5]. Under your expert guidance, we meticulously re-evaluated the 1D-NMR and 2D-NMR spectra of compound 20, including the HSQC, HMBC, and ¹H-¹HCOSY spectra, to elucidate the coupling relationships between compound C-H. Additionally, we examined a substantial number of spectra from compounds analogous to compound 20, thereby affirming the accuracy of its structural analysis. The observed splitting patterns in the carbon spectrum of compound 20, as noted in the original manuscript, may have resulted from insufficient purity during material separation, a phenomenon documented in certain scholarly publications. Consequently, in the revised supplementary materials, we have provided an updated and clearer NMR spectrum, with signal values for both carbon and hydrogen spectra automatically annotated using advanced analysis software. We anticipate that this enhancement will facilitate a more intuitive comprehension of the compound's spectral information.

Reference:

1. Qin, D.; Wang, L.; Han, M. Wang, J.Q.; Song, H.C.; Yan X.; Duan, X.X.; Dong, J.Y. Effects of an endophytic fungus Umbelopsis dimorphaon the secondary metabolites of host-Plant Kadsura angustifolia. Front. Microbiol. 2018, 9, 2845.
2. D.; Shen, W.Y.; Wang, J.Q.; Han, M.J.; Chai, F.N.; Duan, X.X.; Guo, J.L.; Gao, T.C.; Zuo, S.H.; Dong, J.Y. Enhanced production of unusual triterpenoids from Kadsura angustifoliafermented by a symbiont endophytic fungus, Penicillium sp. SWUKD4.1850. Phytochemistry. 2019,158, 56-66.
3. Qin, D.; Shen, W.Y.; Gao, T.C.; Zuo, S.H.; Song, H.C.; Xu, J.R.; Yu, B.H.; Peng, Y.J.; Guo, J.L.; Tang, W.W.; Dong, J.Y. Kadanguslactones A-E, further oxygenated terpenoids from Kadsura angustifoliafermented by a symbiotic endophytic fungus, Penicillium ochrochloron1850. Phytochemistry. 2020, 174, 112335.
4. Han, M.J.;Qin, D., Ye, T.T.; Yan,X.; Wang, J.Q.; Duan,X.X.; Dong, J. An endophytic fungus from Trichoderma harzianum SWUKD3.1610 that produces nigranoic acid and its analogues. Natural Product Research, 2018, 1-9.
5. Wang,L.; Qin, ; Zhang, K.; Huang, Q.; Liu, S.; Han, M.J.; Dong, J.Y. Metabolites from the co-culture of nigranoic acid and Umbelopsis dimorpha SWUKD3.1410, an endophytic fungus from Kadsura angustifolia. Natural Product Research, 2017, 31, 1414-1421.

 

2.Minot comments:

Line 14, “polyketones” should be “polyketides”.

Line 22, “1-17” should be bold.

Line 24, a space is missing between “74.9” and “to”.

Line 35, the first sentence of Introduction needs a reference.

Line 75, “Hydroxyisosclerone” should be “hydroxyisosclerone”

Line 83, “of 1 showed” should be “of 20 showed”

Line 94, “triterpenoid-nigranoic acid (3)” should be “triterpenoid-nigranoic acid (18)”

Line 96, it is better to number the carbons in 18 since they are discussed in the text.

Line 116, “1-14” should be bold.

Reply: Thank you for your carefully correction again. All the above minor errors and grammar errors have been modified in the corresponding parts of the text. Please check the red font of the corresponding part of the text.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

fermentation-3663865-peer-review

Overall

The manuscript appears to be in its second stages and therefore requires a great deal of attention.

Some observations and suggestions are included in the manuscript.

However, here are some examples.

The manuscript's English language requires a great deal of attention; it appears to have been written with the help of artificial intelligence.

The introduction improved, now it is robust.  

However, there is an inconsistency in the bioactive effect of the extract's components. The antimicrobial, cytotoxic, and antioxidant activity seems like a mere afterthought; it lacks scientific support or rigor. The authors could reinforce their research purpose, both in the introduction and in the discussion.

This is an opportunity to establish consistency between the manuscript title, purpose, results, and discussion.

In material an methods include the response of the first review.

In the Results section, Tables 1-4 are missing information, giving the impression that they are "hiding information."

In the References section, 19 references are from 2019 to the present. 67 gives the impression that this is not a novel work. This is one of the many reasons why the title, introduction, results, discussion and conclusion needs to be improved.

In the Results section, Tables 1-4 are missing information, giving the impression that they are "hiding information." However, there is an inconsistency in the bioactive effect of the extract's components. The antimicrobial, cytotoxic, and antioxidant activity seems like a mere afterthought; it lacks scientific support or rigor. The authors could reinforce their research purpose, both in the introduction and in the discussion.

This is an opportunity to establish consistency between the manuscript title, purpose, results, and discussion.

Data confirming the absolute configuration of compound 20 are needed.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

fermentation-3663865-peer-review

Overall

The manuscript appears to be in its second stages and therefore requires a great deal of attention.

Some observations and suggestions are included in the manuscript.

However, here are some examples.

The manuscript's English language requires a great deal of attention; it appears to have been written with the help of artificial intelligence.

The introduction improved, now it is robust.  

However, there is an inconsistency in the bioactive effect of the extract's components. The antimicrobial, cytotoxic, and antioxidant activity seems like a mere afterthought; it lacks scientific support or rigor. The authors could reinforce their research purpose, both in the introduction and in the discussion.

This is an opportunity to establish consistency between the manuscript title, purpose, results, and discussion.

In material an methods include the response of the first review.

In the Results section, Tables 1-4 are missing information, giving the impression that they are "hiding information."

In the References section, 19 references are from 2019 to the present 67 gives the impression that this is not a novel work. This is one of the many reasons why the title, introduction, results, discussion and conclusion needs to be improved.

In the Results section, Tables 1-4 are missing information, giving the impression that they are "hiding information." However, there is an inconsistency in the bioactive effect of the extract's components. The antimicrobial, cytotoxic, and antioxidant activity seems like a mere afterthought; it lacks scientific support or rigor. The authors could reinforce their research purpose, both in the introduction and in the discussion.

This is an opportunity to establish consistency between the manuscript title, purpose, results, and discussion.

Data confirming the absolute configuration of compound 20 are needed.

Author Response

Dear Reviewer,

Thank you very much for taking the time to review our manuscript “Diversity and biological activity of secondary metabolites produced by endophytic fungus Penicillium ochrochlorae (Fermentation-3663865)”. Your comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our further research. Please find the detailed responses below and the corresponding revisions highlighted red track changes in the re-submitted files. Now, we have modified our manuscript point by point according to your comments of reviewer. They mainly include:

Comment 1.Some observations and suggestions are included in the manuscript.

Response 1: Thank you very much for your careful review of the manuscript. We have carefully checked every issue you raised in the file you uploaded and made modifications in the corresponding positions in the revised manuscript.

 Comment 2.The manuscript's English language requires a great deal of attention; it appears to have been written with the help of artificial intelligence.

Response 2:Thank you again for your reminder. We invited peer experts to polish and modify the language of the manuscript, and conducted another AI writing rate check. The check results showed that the AI assisted writing rate was below 10%, which meets the academic paper publication requirements of the journal.

Comment 3.However, there is an inconsistency in the bioactive effect of the extract's components. The antimicrobial, cytotoxic, and antioxidant activity seems like a mere afterthought; it lacks scientific support or rigor. The authors could reinforce their research purpose, both in the introduction and in the discussion.This is an opportunity to establish consistency between the manuscript title, purpose, results, and discussion.

Response 3:Thank you for pointing this out. We agree with this comment. Therefore, we consulted a large amount of literature and rewrote the abstract, introduction, result and discussion sections of the manuscript as much as possible, and established the consistency between the manuscript title, purpose, results, and discussion. Please check the corresponding sections in the text for the modifications mentioned above.

Comment 4. In the Results section, Tables 1-4 are missing information, giving the impression that they are "hiding information."

Response 4: We have supplemented the information and biological activity data of all compounds in Tables 1-4, improving the statistical scientificity of the relevant data. Please check the changes in the newly uploaded data.

Comment 5. In the References section, 19 references are from 2019 to the present. 67 gives the impression that this is not a novel work. This is one of the many reasons why the title, introduction, results, discussion and conclusion needs to be improved.

Response 5: Thank you very much for your reminder. We have updated the references as much as possible to ensure that 60% of them are research results after 2020. In addition, this will be a great reminder and help for our future paper writing.

Comment 6. Data confirming the absolute configuration of compound 20 are needed. 

Response 6: In recent years, our research team has undertaken extensive investigations into triterpenoids within Schisandraceae plants, successfully isolating and characterizing the skeletal properties and structural features of numerous triterpenes analogues from nigranoic acid [1-7]. The structures and absolute configurations of these compounds were elucidated by detailed spectroscopic analyses, including nuclear magnetic resonance (NMR) spectra, electronic circular dichroism (ECD) calculations, and single-crystal X-ray diffraction. The structure and absolute configuration of nigranoic acid (18) were determined by Sun et al. in 1996 [8]. Structurally, compound 20 is a degradation product of the 27th carboxyl carbon of compound 18, and the absence of the 27th carboxyl group does not affect the absolute configuration change of the side chain of compound 18. Therefore, theoretically speaking, the absolute configuration of compound 18 and compound 20 are consistent. In addition, due to the limited separation weight of compound 20 and the supplement to biological activity test, there was not enough weight to supplementary testing of ECD or X-ray diffraction analysis after completing various NMR analyses and activity tests. Therefore, in the results section, we only clarified the relative configuration of compound 20. We hope this response will satisfy you and look forward to your understanding of this fact.

Reference:

1. Qin, D.; Wang, L.; Han, M. Wang, J.Q.; Song, H.C.; Yan X.; Duan, X.X.; Dong, J.Y. Effects of an endophytic fungus Umbelopsis dimorphaon the secondary metabolites of host-Plant Kadsura angustifolia. Front. Microbiol. 2018, 9, 2845.
2. Qin, D.; Shen, W.Y.; Wang, J.Q.; Han, M.J.; Chai, F.N.; Duan, X.X.; Guo, J.L.; Gao, T.C.; Zuo, S.H.; Dong, J.Y. Enhanced production of unusual triterpenoids from Kadsura angustifoliafermented by a symbiont endophytic fungus, Penicilliumsp. SWUKD4.1850. Phytochemistry. 2019,158, 56-66.
3. Qin, D.; Shen, W.Y.; Gao, T.C.; Zuo, S.H.; Song, H.C.; Xu, J.R.; Yu, B.H.; Peng, Y.J.; Guo, J.L.; Tang, W.W.; Dong, J.Y. Kadanguslactones A-E, further oxygenated terpenoids from Kadsura angustifoliafermented by a symbiotic endophytic fungus, Penicillium ochrochloron1850. Phytochemistry. 2020, 174, 112335.
4. Han, M.J.; Qin, D., Ye, T.T.; Yan, X.; Wang, J.Q.; Duan, X.X.; Dong, J.Y. An endophytic fungus from Trichoderma harzianum SWUKD3.1610 that produces nigranoic acid and its analogues. Natural Product Research, 2018, 1-9.
5. Wang, L.; Qin, D; Zhang, K.; Huang, Q.; Liu, S.; Han, M.J.; Dong, J.Y. Metabolites from the co-culture of nigranoic acid and Umbelopsis dimorpha SWUKD3.1410, an endophytic fungus from Kadsura angustifolia. Natural Product Research, 2017, 31, 1414-1421.
6. Dong, J.Y., Chen, Y.G., Song, H.C., He, Y.P., Li, L., Zhong, Y.P., 2007a. Hydroxylation of nigranoic acid to 6β-hydroxynigranoic acid by Caryospora carllicarpa YMF1.01026. Chin. Chem. Lett. 18, 165-167.
7. Dong, J.Y., Chen, Y.G., Song, H.C., Zhu, Y.H., Zhou, Y.P., Li, L., 2007b. Hydroxylation ofthe triterpenoid nigranoic acid by the fungus Gliocladium roseum00133. Chem. Biodivers. 4, 112-117.
8. Sun, H.D.; Qiu, S.X.; Lin, L.Z.; Wang, Z.Y.; Lin, Z.W.; Pengsuparp, T. 1996. Nigranoic acid, a triterpenoid from Schisandra sphaerandra that inhibits HIV-1 reverse transcriptase. J. Nat. Prod. 59, 525-527.

Author Response File: Author Response.docx

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript has improved significantly and can now be properly reviewed.

As this draft is currently written, this is the first version of a manuscript. However, the manuscript has already undergone three revisions.
Now, my request to the authors is: Provide evidence showing that the purified metabolites of P. ochrochloron are not metabolites obtained from wheat bran.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

No comments

Author Response

Dear Reviewer,

Thank you very much for taking the time to review our manuscript. Your comments are all valuable and very helpful for revising and improving our paper. Please find the detailed responses below and the corresponding revisions highlighted red track changes in the re-submitted files. Now, we have modified our manuscript point by point according to your comments of reviewer. They mainly include:

 

Comment 1.Provide evidence showing that the purified metabolites of P. ochrochloron are not metabolites obtained from wheat bran.

 

Response 1: Thank you very much for the reviewer's question.We provide the following explanation regarding the study of secondary metabolites of P. ochrochloron:

• Firstly, a similar question was also raised by the reviewers in our previous research [1]. In the study of secondary metabolism of endophytic fungi Umbelopsis dimorpha 1410, we analyzed the chemical composition of the co-fermented products of Umbelopsis dimorpha SWUKD3.1410 with different metabolic substrates using HPLC (Figure 1), confirming that the co-fermented products of strain Umbelopsis dimorpha with host materials and wheat bran are produced by strain biotransformation and self metabolism, rather than the chemical composition of wheat bran. In Figure 1d, we can see that the sterilized stored wheat bran extract has almost no metabolic products, which is significantly different from the analysis results of other treatment groups.

 

Figure 1. Effects of an endophytic fungus Umbelopsis dimorpha on the secondary metabolites of host-plant Kadsura angustifolia [1].

• Secondly, we have published three studies on the secondary metabolites of the research object of this article, ochrochloron. In the first and second studies, P. ochrochloronco cultured with host plant materials and obtained a large amount of biotransformation triterpenoids (Figure 2 and Figure 3) [2-3]. In the third study, we isolated a large amount of resorcilic acid lactones from the liquid fermentation metabolites of P. ochrochloron (Figure 4) [4]. In this study, 20 compounds were produced through the metabolism of P. ochrochloron using wheat bran as a solid fermentation substrate, including included polyketides (1-14), lignans (15-17) and schitriterpenoid/norschitriterpenoid (18-20). Based on the structural similarity, synthetic pathways, and existing research results of the compounds, compounds 1-20 should all be metabolites of the strain itself.

 

Figure 2. Metabolites produced by co-cultivation of Penicillium sp. SWUKD4.1850 and host plant Kadsura angustifolia [2].

 

Figure 3. Metabolites produced by co-cultivation of P.ochrochloron SWUKD4.1850 and host plant Kadsura angustifolia [3].

 

Figure 4. Metabolites from liquid fermentation (PDB) of P.ochrochloron [4].

• Finally, through extensive literature search, we can see that many secondary metabolites of fungi have been analyzed and studied, mainly through two methods: solid fermentation and liquid fermentation. Wheat bran is the most commonly used substrate for solid fermentation and is widely used in fields such as natural medicinal chemistry and food. As is well known, the main chemical components of wheat bran are ergosterol, polysaccharides, and phenolic acids. Therefore, based on the structural characteristics of metabolites obtained in this article and existing research on the strain, we believe that compounds 1-20 are secondary metabolites produced by ochrochloronusing wheat bran metabolism, rather than wheat bran itself.

I hope the above answers will satisfy you. Thank you again for your guidance throughout our article, which will be of great help to our future research work.

 

Comment 2.Duplicate citations of references and other minor errors.

Response 2:Thank you very much for your reminder. We have corrected the issue of duplicate citations in the references and revised the minor errors in the details of the article. Please review the newly uploaded manuscript. Thank you again.

 

Reference:

1. Qin, D.; Wang, L.; Han, M. Wang, J.Q.; Song, H.C.; Yan X.; Duan, X.X.; Dong, J.Y. Effects of an endophytic fungus Umbelopsis dimorphaon the secondary metabolites of host-Plant Kadsura angustifolia. Front. Microbiol. 2018, 9, 2845.
2. Qin. D.; Shen, W.Y.; Wang, J.Q.; Han, M.J.; Chai, F.N.; Duan, X.X.; Guo, J.L.; Gao, T.C.; Zuo, S.H.; Dong, J.Y. Enhanced production of unusual triterpenoids from Kadsura angustifoliafermented by a symbiont endophytic fungus, Penicilliumsp. SWUKD4.1850. Phytochemistry. 2019,158, 56-66.
3. Qin, D.; Shen, W.Y.; Gao, T.C.; Zuo, S.H.; Song, H.C.; Xu, J.R.; Yu, B.H.; Peng, Y.J.; Guo, J.L.; Tang, W.W.; Dong, J.Y. Kadanguslactones A-E, further oxygenated terpenoids from Kadsura angustifoliafermented by a symbiotic endophytic fungus, Penicillium ochrochloronSWUKD4.1850. Phytochemistry. 2020, 174, 112335.
4. Song, H.C., Qin, D., Liu, H.Y., Dong, J.Y., You, C., Wang, Y.M. Resorcylic acid lactones produced by an endophytic Penicillium ochrochloronstrain from Kadsura angustifolia. Planta Med. 2020, 86, 1-11.

Author Response File: Author Response.pdf

Round 4

Reviewer 1 Report

Comments and Suggestions for Authors

Please include the arguments from the rebuttal letter in the discussion.
In the supplementary material, include a figure constructed with three chromatographic profiles of crude methanolic extracts of wheat bran, rice, and fungus.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

no comments

Author Response

Dear Reviewer,

Thank you very much for taking the time to review our manuscript. Please find the detailed responses below and the corresponding revisions highlighted red track changes in the re-submitted files. Mainly include:

 

Comment 1. Please include the arguments from the rebuttal letter in the discussion. In the supplementary material, include a figure constructed with three chromatographic profiles of crude methanolic extracts of wheat bran, rice, and fungus.

 

• Response 1: Thank you very much for the reviewer's question.We have supplemented the analysis of the sources of metabolites of ochrochloron in the “Discussion” section of the revised manuscript as suggested by the reviewers. At the same time, thin layer chromatography (TLC) analysis images of methanol crude extracts of wheat bran, rice, and fermented P. ochrochloron were added to the supplementary materials (Figure S2). It can be clearly seen from the images that there are significant differences in the composition of crude extracts of fermented P. ochrochloron (b) and wheat bran (a) as well as rice (c), confirming that compounds 1-20 are secondary metabolites of P. ochrochloron.

 

 

Figure S2. Profiles of thin layer chromatography (TLC) analysis from treated different samples. 

(a) Methanol extract of sterilized wheat bran(1 mL of 1 mg/mL) ;

(b) Methanol crude extract from co-fermentation of ochrochloronwith wheat bran and rice (5 μL of a 1:1 enriched extract) of treated different samples (1 mL of 1 mg/mL);

(c) Methanol extract of sterilized rice (1mL of 1 mg/mL);

(N) Nigranoic acid (1 mL of 0.1 mg/mL)

 

Author Response File: Author Response.docx