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Thank you for addressing the last requests!
[# PeerJ Staff Note - this decision was reviewed and approved by Vladimir Uversky, a PeerJ Section Editor covering this Section #]
The authors of this manuscript have carefully answered the questions raised in the previous review. Some experiments were repeated or supplemented, and several obvious errors in the writing and data were corrected. However, there are still a few minor issues in this article that need to be noted and modified.
1. The author does not have a good understanding of the Questions 1. They tried to convert the amount of PGE2 to percent (%), and indicate as COX-2 activities (Figures 2B and 2C). Obviously, this is not appropriate. The problem with this part is the inconsistency between figures and texts. The author claimed in MATERIALS AND METHODS that the cells were treated with LPS and each herb for 6 h (simultaneous treatment) or LPS for 6 h and thereafter with each herb for 1 h (sequential treatment). Then, exogenous arachidonic acid (10 µM) for 30 min (Lines 118-121). This is consistent with the experimental method described in the annotation(Lines 507-511), But it is completely inconsistent with the “Time schedule of treatment” shown in Figure 3A.
2. I still don't see any image results of Western blots were quantified by densitometry using image software. Of course, this is just a suggestion.
3. A large number of WB tests were repeated and positive controls were added. But the stripes of WB should not be spliced( in Figure 6C).
As you will see, our reviewers found your manuscript valuable. There are, nonetheless, many instances in your paper where the description in the text does not agree with the schemes (e.g. Fig.3A disagrees with lines 96-97, etc.etc.etc.) units are in error (e.g. Table 1, in the 100ug/ml column units should by nM instead of uM) and so forth. Please address all of those inconsistencies.
I agree with reviewer #2 regarding the absence of evidence that the effect of shokyo or kankyo relies on inhibition of PLA2.
This manuscript dose an excellent job demonstrating significant effect of Shokyo and Kankyo on prostaglandin E2 production. The article is well written, treats an actual problem. Also, it has focus on importance of traditional Asian medicine.
The experiment has designed very well, with appropriate statistical analysis.
The findings are valid and interesting and it has focused on role of traditional Japanese medicine and also spread organic and easier way for more usage of traditional anti-inflammatory drugs.
Japanese traditional medicine uses most of the Chinese therapies including acupuncture and moxibustion, but Kampō in its present-day sense is primarily concerned with the study of herbs. Kambo formulas were introduced from China and it is fully integrated into the modern health care system in Japan. Kampo can be an ideal choice of therapy.
I think this article excellent and is an important link between Shokyo and Kankyo and their water-extracted fractions of ginger with mechanisms of action via the inhibition of cPLA2 activity. This is an interesting topic of research. I personally enjoyed the role of gingerols and shogaols in the reduction of LPS-induced PGE2 production. And a new significant role of these extracts for inflammatory diseases.
Some specific questions are as follows:
1. Lines 96 and 97: The method of measuring cyclooxygenase (COX)-2 activity is inconsistent with Figure 3A.
2．Line 160: Authors claimed that Shokyo and kankyo (both 100 µg/ml) strongly reduced LPS-induced PGE2 production (Figure 2B), and the effects of kankyo were stronger than those of kankyo. The expression is ambiguous and inconsistent with it in the section “ABSTRACT”.
3. The dosage of 6-shogaol (Lines 215-223) is inconsistent with Figure 4A. There are some confusion and mistakes of its units.
4. Some experimental results are delineated vaguely. For example, the descriptions of cell viability on line 151, Western Blot analysis of ERK phosphorylation in “RESULTS” (lines 215 to 223) and in “DISCUSSION” (line 239) are all confusing.
5. It was absent of direct evidences to prove that shokyo and kankyo inhibited cPLA 2 activity. The conclusion about COX-2 activity (lines 174 to 184), one of the main point of this article, was obtained through an indirect experiment. It is better to have more direct experimental evidence here, for example, enzyme activity testing.
6. In addition, the Western blot results (Figures 3 and 4) showed that neither the herbs extraction nor 6-shogaol altered LPS-induced expression of COX-2, cPLA2 or annexin 1, and there was also no significant inhibitory activity on LPS-induced phosphorylation of NF-κB p65 and ERK. The experiment lacked corresponding positive control. Besides, the relative gray value should be calculated.
7. It is not enough just to evaluate p65 phosphorylation level for researching the role of shokyo and kankyo on NF-κB signal pathway, and some complementary experiments will make the results more convincing, for example, some detection about the phosphorylation of I-κB or p65 transportation to the nucleus.
The literature and background introduction are quite sufficient.
There are many obvious errors, confusing statements in this manuscript and the experimental design needs to be improved.
The manuscript "Effects of shokyo (Zingiberis Rhizoma) and kankyo (Zingiberis Processum Rhizoma) on prostaglandin E2 production in lipopolysaccharide-treated mouse macrophage RAW264.7 cells” was professionally represented in unambiguous clear English language throughout the manuscript with structure aligned with PerrJ standard.
Additionally, raw data have been supplied with high quality relevant figures. The introduction elaborated the context with a need for further consideration of certain studies and reviews of aqueous ginger and its effect of PGE2 levels in-vitro and in-vivo which will be thoroughly mentioned later on.
Toshiaki Ara et al represented a manuscript aligned with the PeerJ scope with consistently defined research questions. All the methodologies are well described and the data is analysed in a satisfactory modern manner.
Robust data with well discussed findings. Nevertheless, The metabolomic profiling of shokyo and kankyo materials is to be provided if not declared in the certificated of analysis provided by Tsumura & Co and the COA provided by the commercial vendor is to be added to the supplementary section.
A) The anti-inflammatory mechanisms of the ginger armoury of phytochemicals and either aqueous or alcoholic extracts were comprehensively vetted in the recent review (Ameliorative and protective effects of ginger and its main constituents against natural, chemical and radiation-induced toxicities: A comprehensive review, Food and Chemical Toxicology, 2018 https://doi.org/10.1016/j.fct.2018.10.048) for both the in-vitro and the in-vivo studies. This review should have been considered and utilised in the introductory section to draw the need for more studies on the aqueous ginger extract to be discussed briefly. Furthermore, some studies elaborated in the aforementioned review are to be considered in the discussion and/or the introduction sections for examples:
- The inhibition of induced cytokines in HaCaT cells and Mice by aqueous ginger extract (Zingiber officinale protects HaCaT cells and C57BL/6 mice from ultraviolet B-induced inflammation J. Med. Food, 13 (3) (2010), pp. 673-680).
- The declined expression of IFN-γ, IL-6 and iNOS with inhibition of the NF-k B activation in LPS challenged mice by aqueous ginger extract (Y.Y. Choi, M.H. Kim, J. Hong, S.-H. Kim, W.M. YangDried ginger (zingiber officinalis) inhibits inflammation in a lipopolysaccharide-induced mouse model Evid. Based Complement Alternat. Med., 2013 (2013), p. 914563).
- The orally administered Cuttlebone complex (CBC) including fresh ginger roots demonstrated a protective potentiality against indomethathin-induced gastric ulcer in rats via reducing the gastric ulcerous lesions and significant increment of the indomethacin-declined PGE2 levels in the stomach in a dose dependent manner (M.-Y. Chien, Y.-T. Lin, F.-C. Peng, H.-J. Lee, J.-M. Chang, C.-M.Yang, C.-H. Chen, Gastroprotective potential against indomethacin and safety assessment of the homology of medicine and food formula cuttlebone complex Food Funct., 6 (8) (2015), pp. 2803-2812).
B) Discuss the ginger up-regulatory effect on PGE2 in the stomach and its down-regulatory effect in macrophages and inflammation site.
C) Global metabolomic profiling of shokyo and kankyo used materials for robustness and repeatability and QC purposes in any further studies.
D) A section on the limitations of the study and ideas for future research could be included.
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