Anti-Inflammatory Effect of Three Isolated Compounds of Physalis alkekengi var. franchetii (PAF) in Lipopolysaccharide-Activated RAW 264.7 Cells
Round 1
Reviewer 1 Report
Comments to authors:
- please define the abbreviations the first time they appear on the text starting from the abstract for example UV, HR-ESI-Ms
- Please correct scientific names to italic formatting
- please correct the font and the format of the measuring units
- Please follow the template and author's instructions for the manuscript preparation. it is obvious the article was prepared for other journal and even the font is not consistent
- as there is a section for materials please list all materials used in this section there
- why do the authors use only RAW246.7 cell lines please justify
- Please include the proton NMR for compounds 1-3
- It is very difficult to read the labeling of the axis on the figures please improve image quality
- there should be a western blot analysis for such a study
- Authors should provide an ELISA assay for cytokines under evaluation in this study
- Authors should highlight the novelty of this approach. the naturally isolated compounds from 1-3 have been widely reported and many kinds of literature have discussed their therapeutic potential. I believe the manuscript lacks the novelty of new work
- The English language requires major revision, many typos, and grammatical mistakes
Author Response
Response to comments from Referee # 1
(Reviewer 1)
1.please define the abbreviations the first time they appear on the text starting from the abstract for example UV, HR-ESI-Ms
Response: As reviewer suggested, we have defined the abbreviation (see also, Lines 19-20).
2. Please correct scientific names to italic formatting
Response: We have corrected scientific names (see also, Line 17,24,37,47).
3.please correct the font and the format of the measuring units.
Response: We have corrected the font and format in the current revision (see also, 111, 115, 116).
4. Please follow the template and author's instructions for the manuscript preparation. it is obvious the article was prepared for other journal and even the font is not consistent
Response: We corrected format in a revised version of the manuscript according to Journal’s author's instructions (see also, Lines 180-191).
5. as there is a section for materials please list all materials used in this section there
Response: We have included list of all materials in the Methods section (see also, Lines 70-79)
6. why do the authors use only RAW246.7 cell lines please justify
Response: We added justification using RAW246.7 cell lines in Materials and Methods (See also, lines 119-122)
The RAW264.7 murine macrophage cell line has been used extensively to carry out in vitro screens for anti-inflammatory candidate agents. The cell line response is considered to reflect the potential human de novo response. Therefore, we used this cell line to evaluate the isolated three compounds from PAF for bioactivity and to predict their potential effect in vivo or on primary cells. (Reference; Anti-inflammatory effects of methanol extract of Antrodia cinnamomea mycelia both in vitro and in vivo.Wen CL, Chang CC, Huang SS, Kuo CL, Hsu SL, Deng JS, Huang GJ.J Ethnopharmacol. 2011 Sep 1;137(1):575-84.)
7. Please include the proton NMR for compounds 1-3
Response: As reviewer suggested, we included the NMR spectrum (See also, Lines 97-101)
1H NMR (200MHz, CDCl3) δ ppm: 12.13 (1H, s, OH), 7.92 (d, J = 2.0 Hz, H-2'), 7.85 (1H, dd, J = 8.8. 2.0 Hz, H-6'), 7.02 (1H, d, J = 2.0 Hz, H-5'), 6.72 (1H, d, J = 2.0 Hz, H-8), 6.33 (1H, d, J = 2.0 Hz, H-6), 3.94 (3H, s, OCH3-3'), 3.93 (3H, s, OCH3-7).
13C NMR (50MHz, CDCl3) δ ppm: 176.8 (C-4), 166.8 (C-7), 162.0 (C-5), 157.8 (C-8a), 149.9 (C-4'), 148.4 (C-3'), 147.3 (C-2), 137.1 (C-3), 123.6 (C-1'), 123.0 (C-6'), 116.2 (C-5'), 112.2 (C-2'), 104.9 (C-4a), 98.5 (C-6), 92.9 (C-8), 56.5 (OCH3-3', OCH3-7).
8. It is very difficult to read the labeling of the axis on the figures please improve image quality
Response: we have changed the labeling of all figures to improved image quality in the current revision.
9.there should be a western blot analysis for such a study.
10.Authors should provide an ELISA assay for cytokines under evaluation in this study
Response (9-10): Reviewer thought that additional experiments including Western blot and ELISA should be performed. We appreciate the reviewer’s helpful advice. Although it would be worthy to obtain the more convincing evidence of anti-inflammation effect of PAF with Western blot and ELISA data, it was unfortunately difficult to perform this study immediately because we could only obtain a very small quantity of isolated samples from PAF and it was very hard to get enough sample and to perform experiments such as Western blot, ELISA or Immuno histochemistry with a tiny sample. It should be noted that our current results clearly demonstrate that three isolated compounds from physalis alkengi var. franchetii can suppress inflammatory responses in LPS stimulated macrophage with traditional RT-PCR and qPCR assay as qualitative and quantitative technique, providing more reliable and reproducible quantification of IL-1β and TNF-α mRNA. We will examine protein levels of TNF-α and IL-1β with Western blot and ELISA experiments in the future.
11. Authors should highlight the novelty of this approach. the naturally isolated compounds from 1-3 have been widely reported and many kinds of literature have discussed their therapeutic potential. I believe the manuscript lacks the novelty of new work
Response: We addressed this point and highlighted the novelty of our study as mentioned in Introduction and discussion part of the present revised version in line xx.
“In order to examine their potential anti-inflammatory effects of bioactive compounds from P. alkekengi var. francheti. (PAF), we have isolated three compounds from PAF, and identified as 3,7-dimethylquercetin, physalin B and isophysalin B. For the first time, we reported that the known flavonoid, 3,7-dimethylquercetin was isolated as constituents of PAFP. Moreover, 3,7-dimethylquercetin has never been isolated previously from the genus Physalis. We further investigated phytochemical and pharmacological activity of three compounds in the present study. For the first time we have shown that three isolated compounds from PAF can suppress inflammatory responses in LPS stimulated macrophage with RT-PCR and qPCT assay.
12. The English language requires major revision, many typos, and grammatical mistakes
Response: we have corrected several minor errors and made some minor edits in order to approve readability.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The authors addressed my requested point in this new revision. Therefore, I am now able to recommend this manuscript for publication in current issues in molecular biology.
Author Response
We really appreciate your work.
Round 2
Reviewer 1 Report
No further comments to the authors
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
The authors addressed some of the requested points in this new revision. However, the most important issue was not addressed! The qPCR data for IL-1β upon 100 µg/mL physalin B treatment (Figure 2D) is in direct opposition to the RT-PCR result shown in Figure 2C, wherein the RT-PCR result there is no change in IL-1β upon 100 µg/mL physalin B treatment and with qPCR, there is a strong decrease in mRNA expression.
The authors deem it not important to clarify this confusion in the IL-1β gene expression upon treatment with 100 µg/mL physalin B. Also as written by the authors (lines 156-160): “However, there was no significant difference of the mRNA level of IL-1β after treatment of physalin B among groups. As shown in Figure 2D, treatment of RAW 264.7 cells with LPS alone resulted in significant increases in TNF-α and IL-1β mRNA expression as compared to the control group (P < 0.001). However, there was a significant difference of the mRNA level of IL-1β and TNF-α after treatment of physalin B (100 μg/ml) compared to the LPS group (P < 0.001).” Therefore, I am unable to recommend this manuscript for publication in current issues in molecular biology.
Reviewer 2 Report
No comments to the authors.
