Mechanochemical-Assisted Extraction and Pharmacological Study of Triterpenoids from Antrodia Camphorata

Round 1

Reviewer 1 Report

Figures should be improved or moved to an Annex, as supplementary material.

Please change the format of lin3s 181-186

An scheme or Figure of the Ball Mill or Mechanochemical apparatus should be included

Tables 1 and 2 should be improved (Format)

Author Response

Dear Reviewer:

Thank you for your comments concerning our manuscript entitled “Mechanochemical-assisted Extraction and Pharmacological study of Triterpenoids from Antrodia Camphorata” (applsci-601807).

Those comments are all valuable and very helpful for revising andimproving 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: Figures should be improved or moved to an Annex, as supplementary material.

Response 1：It is really true as Reviewer suggested that figures should be moved to an Annex in most cases, however, Applied Sciences recommends authours insert pictures into manuscripts, and we also uploaded a ZIP archive containing all the figures and tables.

Point 2: Please change the format of lin3s 181-186.

Response 2: We are very sorry for our negligence of mistake about format, we have corrected the format of these lines.

Point 3: An scheme or Figure of the Ball Mill or Mechanochemical apparatus should be included.

Response 3: As reviewer suggested that we have made a scheme of the Ball Mill apparatus, the figure is in the revised version of manuscript.

Point 4: Tables 1 and 2 should be improved (Format).

Response 3: We are very sorry for our negligence of mistake about format, we have revised the spacing and typesetting of these tables.

Please see the attachment to find the revised version of manuscript .

We would like to express our great appreciation to you for comments on our paper.

Thank you and best regards.

Yours sincerely,

Rongjun He

Corresponding author:

Rongjun He

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments to Authors:

Please explain why antcin A was the only triterpenoid that showed a higher yield using ethanol hot reflux extraction method (EHRE). In Fig 1. More than one peak has higher intensity in EHRE compared to please clarify if any of these peaks corresponds to triterpenoids compound? Please explain if triterpenoids compounds beside the other compounds may have positive or negative effect on biological properties of Antrodia Camphorata extract. “Compound 2 and 3 were the R, S configuration of Antcin K”, as mass spectrometry couldn’t in fact differentiate the R, S configuration of Antcin K but it require 2D-NMR analysis. The manuscript is missing the discussion of some parts. Please rewrite the results and discussion sections. Organic solvents were used in both methods (EHRE & MAEM) during the extraction oftriterpenoids from Antrodia Camphorata. So please highlight this fact when re-organizing the manuscript and concluding your findings. Antrodia Camphorata should be italic throughout the manuscript. Authors would check the style of writing references in accordance to journal instruction. Fig 2 is unclear, the clarity and resolution should be adjusted. The authors could be benefited for the following references for support the methodology section: 

Zhu, P., Fu, X., Li, J., Tse, A. K. W., Guo, H., Yin, C., ... & Hossen, M. J. (2018). Antrodiacamphoratamycelia exert anti-liver cancer effects and inhibit STAT3 signaling in vitro and in vivo. Frontiers in pharmacology, 9, 1449. Thulin, M., Moore, A. J., El-Seedi, H., Larsson, A., Christin, P. A., & Edwards, E. J. (2016). Phylogeny and generic delimitation in Molluginaceae, new pigment data in Caryophyllales, and the new family Corbichoniaceae. Taxon, 65(4), 775-793.

Author Response

Dear Reviewer:

Thank you for your comments concerning our manuscript entitled “Mechanochemical-assisted Extraction and Pharmacological study of Triterpenoids from Antrodia Camphorata” (applsci-601807).

Those comments are all valuable and very helpful for revising andimproving 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: Please explain why antcin A was the only triterpenoid that showed a higher yield using ethanol hot reflux extraction method (EHRE).

Response 1：

①. In the discussion section, We have noted that according to the result, the components with medium polarity were much more easily extracted by MAEM.

②. In terms of molecular structure, Antcin K, C and H have hydroxyl groups but Antcin A does not, we tend to think that comparing to Antcin A, long-term high temperature treatment will have a negative impact on these triterpenoids. 

③. After ball milling treatment, the raw materials are crushed more thoroughly. After mechanochemical reaction, Antcin K, C and H will be more easily extracted.

Point 2: In Fig 1. More than one peak has higher intensity in EHRE compared to (MAEM?) please clarify if any of these peaks corresponds to triterpenoids compound?

Response 2：

①. In Fig. 1b (EHRE), some peaks indeed have higher intensity compared to Fig 1a (MAEM), but this is for a reason that in Fig 1a (MAEM), intensity of peaks 2 and 3 has been greatly improved while the concentrations of the two samples for HPLC are the same, both are 2mg/mL, when at the same concentration, the concentration of one compound increases, the concentration of other comounds will decrease relatively, so some of the peaks in Fig 1a (MAEM) seem lower than those in Fig 1b (EHRE), mainly due to the significant increase in the concentration of peaks 2 and 3 in Fig 1a (MAEM).

②. We also analyzed Fig 1a (MAEM) and Fig 1b (EHRE) by LC-MS. However, due to the space limitation, we only put the mass spectrometry analysis pictures of MAEM. The results show that the marked peaks in Fig 1b (EHRE) correspond to those in Fig 1a (MAEM). 

③. By comparing the LC-MS results of the samples we used with the mass-charge ratios (M/Z) of these triterpenoids in the references, we can determine the corresponding triterpenoids of these peaks. The following references play an important role in proving our results[1][2][3].

Huang, H.; Hsu, J. L.; Yu, C. C.; Chang, L. C.; Chen, C. R.; Huang, T. C.; Chang, C. I.   Qualitative and quantitative analysis of seven signature components in the fruiting body of Antrodia cinnamomea by HPLC—ESI—MS/MS . Acta Chromatogr. 2015, 28, 387-402. Wei-De, W. , Pin-Shern, C. , Omar, H. A. , Arafa, E. A. , Hung-Wei, P. , & Jingyueh, J. , et al. Antrodia cinnamomeaboosts the anti-tumor activity of sorafenib in xenograft models of human hepatocellular carcinoma. Sci Rep, 2018 , (1), 12914. Hsiu-Man, L. , Chun-Hung, C. , Chia-Chang, C. , Wan-Lin, C. , Charng-Cherng, C. , & Robert, P. . . Comparison of the apoptotic effects of supercritical fluid, extracts of Antrodia Cinnamomea mycelia on hepatocellular carcinoma cells.Molecules, 2014, 19(7), 9033-9050.

Point 3: Please explain if triterpenoids compounds beside the other compounds may have positive or negative effect on biological properties of Antrodia Camphorata extract.

Response 3：From the point of view of traditional Chinese medicine theory, besides triterpenoids, there are other substances with physiological activity. Through other studies, it is found that there may also be synergistic or antagonistic effects, in Jiyang Su’s research[4], the combination of Ganoderma lucidum spore polysaccharide and paclitaxel has better effect on inhibiting tumor. Similar effects may exist between triterpenoids or other components. These are also the further research we need to do in the future.

Su, J.Y.; Li, D.; Chen, Q.J.; Li, M.X.; Su, L.; Luo, T.; Liang, D.L.; Lai, G.X.; Shuai, O.; Jiao, C.W.;     Wu, Q.P.; Xie, Y.Z.; Zhou, X.X. Anti-breast Cancer Enhancement of a Polysaccharide From Spore of Ganoderma lucidum With Paclitaxel: Suppression on Tumor Metabolism With Gut Microbiota Reshaping. Front Microbiol. 2018, 9, 3099.

Point 4: “Compound 2 and 3 were the R, S configuration of Antcin K”, as mass spectrometry couldn’t in fact differentiate the R, S configuration of Antcin K but it require 2D-NMR analysis.

Response 4：It can be defined that the specific configuration of R or S configuration of Antcin K need further 2D-NMR analysis to draw a conclusion. In this manuscript, we have obtained compound 2 and 3 which can only be charaterized by the mixture of R-Antcin K and S-Antcin K, the focus of this paper is the effects of different pretreatment methods on the pharmacological activity of the mixture of Antrodia Camphorata extract, so there is no futher study to determine the specific configutation of Antcin K. However, we will revise this sentence to be: Compound 2 and 3 belonged to Antcin K with different R and S configurations.

Point 5: The manuscript is missing the discussion of some parts. Please rewrite the results and discussion sections.

Response 5：We have made correction according to the Review’s suggestions. And we have done our best to revise it. All the red marks in the article have been revised. If we still fail to meet your requirements, we will be very grateful to you for pointing out for us what specific areas need to be revised and what discussions need to be supplemented.

Point 6: Organic solvents were used in both methods (EHRE & MAEM) during the extraction of triterpenoids from Antrodia Camphorata. So please highlight this fact when re-organizing the manuscript and concluding your findings.

Response 6：We are very sorry for our statement mistakes, the correct statement should be: avoid the extensive use of organic reagents. We will make the appropriate revision when re-organizing the manuscript.

Point 7: Antrodia Camphorata should be italic throughout the manuscript. Authors would check the style of writing references in accordance to journal instruction.

Response 7：We have checked the whole manuscript carefully and changed all the Antrodia Camphorata into italic. We have checked the style of writing references in accordance to journal instruction.

Point 8: Fig 2 is unclear, the clarity and resolution should be adjusted.

Response 8：We are very sorry for our negligence, we have replaced all those unclear pictures with clearer ones.

Point 9: The authors could be benefited for the following references for support the methodology section:

Zhu, P., Fu, X., Li, J., Tse, A. K. W., Guo, H., Yin, C., ... & Hossen, M. J. (2018). Antrodia camphorata mycelia exert anti-liver cancer effects and inhibit STAT3 signaling in vitro and in vivo. Frontiers in pharmacology, 9, 1449. 

Thulin, M., Moore, A. J., El-Seedi, H., Larsson, A., Christin, P. A., & Edwards, E. J. (2016). Phylogeny and generic delimitation in Molluginaceae, new pigment data in Caryophyllales, and the new family Corbichoniaceae. Taxon, 65(4), 775-793. 

Response 9：These two references are very valuable. We have read these two references carefully. They are very helpful to us, and we have refered these two references in this manuscript in “3.2. Chemical analysis of compounds by LC-MS” section and “3.4. Anticancer activity of TAEM and TAEE” section.

Please see the attachment to find the revised version of manuscript .

We would like to express our great appreciation to you for comments on our paper.

Thank you and best regards.

Yours sincerely,

Rongjun He

Corresponding author:

Rongjun He

Round 2

Reviewer 2 Report

In fig 3: Compounds 2 & 3 were R-Antcin K and S-Antcin K. Distinguishing between R- and S- form is unclear . Authors could double check and correct the structure.

Author Response

Dear Reviewer:

Thank you for your comments concerning our manuscript entitled “Mechanochemical-assisted Extraction and Pharmacological study of Triterpenoids from Antrodia Camphorata” (applsci-601807).

According to the comment, we have revised the structure of Compound 2 and 3 in Fig 3.Please see the attachment to get the revised manuscript. The 25th carbon of Antcin K is chiral carbon atom. It is determined that the R and S configuration of Antcin K mainly depends on the spatial position of the carboxyl groups connected on the carbon. Referring to the connection mode of the 25th carbon and carboxyl group in the reference[1] (see Fig. 2 of the reference specifically), we use wave lines instead of straight lines to indicate that the structure of Antcin K is R or S configuration.

1.  Chen, C. Y. , Chien, S. C. , Tsao, N. W. , Lai, C. S. , Wang, Y. Y. , & Hsiao, W. W. , et al. Metabolite profiling and comparison of bioactivity in antrodia cinnamomea and antrodia salmonea fruiting bodies. Planta Medica, 2015，82, 244-249.

We would like to express our great appreciation to you for comments on our paper.

Thank you and best regards.

Yours sincerely,

Rongjun He

Corresponding author:

Rongjun He

Author Response File: Author Response.pdf