Comparisons of the Anti-Inflammatory Activity of Dendropanax morbifera LEV Leaf Extract Contents Based on the Collection Season and Concentration of Ethanol as an Extraction Solvent

Round 1

Reviewer 1 Report

This study compared the anti-inflammatory activity of 10-year-old Dendropanax 14 morbifera LEV (DM) leaf extracts. They provided evidence that 30% ethanol extract harvested in  May and 60% ethanol extracts collected in August and November displayed the highest inhibitions 24 of nitrite, PGE2, and inflammatory cytokines. Furthermore, the 60% ethanol extract harvested in August suppressed activation of the NF-κB and MAPK signaling pathways. Although it is an interesting finding, it is very preliminary and lacks the depth of understanding of the extracts. For example, which component(s) or gradient(s) of the extract is involved in the activation of NF-κB and MAPK signaling pathways

 

Author Response

Thank you for your insights, we supplemented the discussion about limitation (Line: 302-305). Since this study is a preliminary step, active compounds from DM extracts are not expiscated. Instead, it was estimated which components contribute to suppress inflammation by comparing the amount of chief composition and anti-inflammatory activity.

Author Response File: Author Response.docx

Reviewer 2 Report

Kim et al. compared the anti-inflammatory effect of different Dendropanax morbifera leaf extracts on LPS-induced RAW264.7 macrophage-like cells. They found that 30% ethanol extracts harvested in May and 60% ethanol extracts collected in August and November displayed the highest inhibitions of nitrite, PGE2, IL-6, IL-1β and TNFa, iNOS and COX-2 mRNA levels and reduced the activation of NFκB and MAPK pathways. In addition, they found that the amount of potent compounds depends on the concentration of ethanol in the extraction process. Thus, they concluded that ethanol extracts of Dendropanax morbifera leafs have the anti-inflammatory potential.

While the study might be interesting for experts in the field, the data is not always described in a comprehensive way and/or the conclusions are not always supported by the data. I have some suggestions to improve the quality of the manucript.  

 

Major comments:

1. English language editing is needed.
2. Color coding in the figures might be misleading: same colors are used for the concentration series and for different extracts of DM, sometimes in the same figure. Please, adapt color coding to avoid confusion.
3. Please, indicate whether „triplicate determinations“ in line 160 refer to biological replicates = independent experiments or technical replicates = independent wells of same experiment. Also specify the number of independent experiments in each figure legend.
4. * = statistical significance: compared to what? Needs to be stated in each figure legend.
5. Why did the authors used only one concentration for qPCR and not the same as for nitrite and PGE2 profiling? The authors may want to provide this data.
6. E30 extracts increase PGE2 levels at lower concentration. The authors may discuss why.
7. The paragraph „3.2. Inhibitory effect of DM extracts on proinflammatory cytokine production“ is only insufficiently described. In addition, the statement „All of the treated DM extracts (50 μg/mL) showed significant inhibitory activity for proinflammatory cytokines production“ in line 194-195 needs careful consideration: in some treatments the levels of the proinflammatory cytokines are even significantly increased. Why do lower concentrations of several extracts induce upregulation of PGE2, IL-1beta, IL-6 and TNFalpha, but not nitrite or iNOS and COX-2? Please, describe accordingly and discuss in the manuscript.
8. The first sentence of the paragraph 3.3. „Based on the results of mRNA expression, markers of the NF-κB pathway were identified only for one group with the highest anti-inflammatory activity by season, namely 30% ethanol extract in 206 May and 60% ethanol extracts in August and November (Figure 4).“ is highly confusing (line 205-206). Data in Fig. 4 seem to be data from protein analysis (Western Blot mentioned in the methods) and not mRNA expression. Indicate in the figure legend which extract was used and which concentration? If additional mRNA expression data were used to select the conditions, please show these data at least in the supplement. Moreover, from the figure legend it is not clear what is represented in the graphs (quantification of the blots in a?). Please indicate how many independent experiments were used to quantify the WB data.
9. The authors often write that NFkB is „secreted“ by macrophages. Usually NFkB is sequesterd in the cytosol and translocates to the nucelus to regulate gene expression. The author should carefully proof read their manuscript.
10. What does the correlation between the inflammation-related factors and major active components tells about the quality of DM extracts (Fig. 5)? This is not clear from paragraph 3.5. Usually, r² of approximately 0.5 is considered as poor correlation. Could the authors please state more clearly why there is a correlation and what this means. Moreover, the authors state that nitrite levels were negatively correleted, iNOS mRNA expression was positively correletad, but COX-2 mRNA expression was „only“ correlated. The difference is currenty not clear in the manuscript.
11. As stated earlier, the statement „All of the extracts showed lower inflammatory responses in RAW264.7 cells stimulated with LPS than those in LPS-treated control cells.“ is not reflected by the data, at least not for all concentrations (line 254-256 vs. Fig. 2b, Fig. 3). Here, the authors should be more precise.
12. The discussion part is rather a summary of the results than a disussion. The authors should adapt their manuscript. It is not yet clear what are the new findings and why these are important.
13. The authors conclude that the extractions May/30, Aug/60 and Nov/60 have the most potent anti-inflammatory effects and correlate those to the amount of a- and β-amyrin. Interestingly, the extraction May/30 contains much lower amounts of these terpenoids. Can the authors explain this discrepancy and demonstrate or at least speculate why this extraction exerts its anti-inflammatory effect?
14. Why is effect of extraction so different on nitrite and PGE2? Fig. 2 a vs. b

 

Minor comments:

15. The supplementary figures are not mentioned in the main text. Please refer to these figures in the manuscript and state what is shown and why this is important?
16. Information about LPS induction is scarce. I kindly ask the authors to include information about when, how long and the density of cell cultures for induction.
17. The legends of the axes in multiple figures is distorted/warped. The authors may want to correct this.
18. qPCR method needs to be described in more detail. The authors may easily consider the MIQE guidelines.
19. WB method: which antibodies were used? (primary, secondary, company, order number, concentration)
20. 2 is shown at two pages. Although a figure legend might be diplayed at a separate page, the figure itself should fit to one page.
21. In the abstract the authors write that „ethanol extracts of DM leaves may have the potential to enhance inflammatory responses“, which is somehow controversial to the anti-inflammatory effects of DM extracts.

 

 

 

Author Response

1. English language editing is needed.

: Thank you for your suggestion. But the manuscript had been edited by a professional English language Editing company (Editage, Republic of Korea).

2. Color coding in the figures might be misleading: same colors are used for the concentration series and for different extracts of DM, sometimes in the same figure. Please, adapt color coding to avoid confusion.

: Thank you for providing these insights. Their color was revised.

3. Please, indicate whether „triplicate determinations“ in line 160 refer to biological replicates = independent experiments or technical replicates = independent wells of same experiment. Also specify the number of independent experiments in each figure legend.

: That indicates “independent wells of same experiment” in most experiments, but Western blot analysis was carried out independent experiments. I expressed in each figure legend.

4. * = statistical significance: compared to what? Needs to be stated in each figure legend.

: Thank you for providing these insights. All data were compared with LPS-treat group. I indicated mean in the figure legend.

5. Why did the authors used only one concentration for qPCR and not the same as for nitrite and PGE2 profiling? The authors may want to provide this data.

: The inhibitory effect of DM extracts with three concentrations (12.5, 25, 50 μg/mL) on nitrite and PGE₂ was revealed through cells supernatant. Thus, for double check, qPCR was carried out only 50 μg/mL. The oxidation of L-arginine was catalyzed by nitric oxide synthase (NOS), releasing unstable and reactive NO. Among the isoforms of NOS, iNOS is activated by inflammatory stimulation and concerned with production of NO. A well-known inflammatory response in macrophages starts by combining LPS and TLR, which brings about a cascade of kinases. COX has two isoenzyme forms, COX-1 and COX-2. COX-1 appears in various cells and mediates the physiological activity, whereas COX-2, known as an inducible enzyme, is found in immune cells treated with LPS and inflammatory cytokines. Previous studies described that COX-2 is accountable for the synthesis of the prostanoids, which are relevant in critical and chronic inflammatory conditions. Thus, the hyperaction of COX-2 can provoke production of the PGE₂ (Line: 282-290). Therefore, tendency of production may differ.

6. E30 extracts increase PGE2 levels at lower concentration. The authors may discuss why.

: Thank you for your points. Sometimes, inflammation reaction was increased in low concentration (12.5 μg/mL). Unlike in humans, inflammation could be excessively induced in an in vitro study, because plants contain various phytochemicals and interact with each other. Furthermore, this study was performed using cell viability data and anti-inflammatory effects were induced at a high concentration (50 μg/mL). Although identification of the principal bioactive components is based on simple principles, discovering active compounds is difficult if multiple metabolic pathways are involved in plants. Thus, further research should be conducted to determine what causes increased inflammation in the low concentration sample group compared to the LPS-treated group (Line: 261-269).

7. The paragraph „3.2. Inhibitory effect of DM extracts on proinflammatory cytokine production“ is only insufficiently described. In addition, the statement „All of the treated DM extracts (50 μg/mL) showed significant inhibitory activity for proinflammatory cytokines production“ in line 194-195 needs careful consideration: in some treatments the levels of the proinflammatory cytokines are even significantly increased. Why do lower concentrations of several extracts induce upregulation of PGE2, IL-1beta, IL-6 and TNFalpha, but not nitrite or iNOS and COX-2? Please, describe accordingly and discuss in the manuscript.

:  It is the same as the response to the previous point. Sometimes, inflammation reaction was increased in low concentration (12.5 μg/mL). Unlike in humans, inflammation could be excessively induced in an in vitro study, because plants contain various phytochemicals and interact with each other. Furthermore, this study was performed using cell viability data and anti-inflammatory effects were induced at a high concentration (50 μg/mL). Although identification of the principal bioactive components is based on simple principles, discovering active compounds is difficult if multiple metabolic pathways are involved in plants. Thus, further research should be conducted to determine what causes increased inflammation in the low concentration sample group compared to the LPS-treated group (Line: 261-269).

8. The first sentence of the paragraph 3.3. „Based on the results of mRNA expression, markers of the NF-κB pathway were identified only for one group with the highest anti-inflammatory activity by season, namely 30% ethanol extract in 206 May and 60% ethanol extracts in August and November (Figure 4).“ is highly confusing (line 205-206). Data in Fig. 4 seem to be data from protein analysis (Western Blot mentioned in the methods) and not mRNA expression. Indicate in the figure legend which extract was used and which concentration? If additional mRNA expression data were used to select the conditions, please show these data at least in the supplement. Moreover, from the figure legend it is not clear what is represented in the graphs (quantification of the blots in a?). Please indicate how many independent experiments were used to quantify the WB data.

: Thank you for your insight, based on mRNA expression of iNOS and COX-2 results, The effects on the NF-κB signaling pathway were demonstrated using the extracts showing the highest anti-inflammatory activities (Figure 4), namely 30% ethanol extract in May and 60% ethanol extracts in August and November (Figure 2c, d). I modified the paragraph (Line: 205-207).

9. The authors often write that NFkB is „secreted“ by macrophages. Usually NFkB is sequesterd in the cytosol and translocates to the nucelus to regulate gene expression. The author should carefully proof read their manuscript.

: Thank you for your point, we modified it (Line: 271).

10. What does the correlation between the inflammation-related factors and major active components tells about the quality of DM extracts (Fig. 5)? This is not clear from paragraph 3.5. Usually, r2 of approximately 0.5 is considered as poor correlation. Could the authors please state more clearly why there is a correlation and what this means. Moreover, the authors state that nitrite levels were negatively correleted, iNOS mRNA expression was positively correletad, but COX-2 mRNA expression was „only“ correlated. The difference is currenty not clear in the manuscript.

: We modified the content (Line: 238-245). The correlation was carried out to assess which components contribute greatly to suppress inflammation, I supplemented that in manuscript (Line: 309-311). 

11. As stated earlier, the statement „All of the extracts showed lower inflammatory responses in RAW264.7 cells stimulated with LPS than those in LPS-treated control cells.“ is not reflected by the data, at least not for all concentrations (line 254-256 vs. Fig. 2b, Fig. 3). Here, the authors should be more precise.

: We modified this point of concern (Line: 257-259).

12. The discussion part is rather a summary of the results than a disussion. The authors should adapt their manuscript. It is not yet clear what are the new findings and why these are important.

: We revised the discussion point (Line: 323-327, 330-335).

13. The authors conclude that the extractions May/30, Aug/60 and Nov/60 have the most potent anti-inflammatory effects and correlate those to the amount of a- and β-amyrin. Interestingly, the extraction May/30 contains much lower amounts of these terpenoids. Can the authors explain this discrepancy and demonstrate or at least speculate why this extraction exerts its anti-inflammatory effect?

: We expect it to affect unknown compounds, so I added it (Line: 327-329).

14. Why is effect of extraction so different on nitrite and PGE2? Fig. 2 a vs. b

: Nitrite and PGE2 are influenced by iNOS and COX-2, respectively. The oxidation of L-arginine was catalyzed by nitric oxide synthase (NOS), releasing unstable and reactive NO. Among the isoforms of NOS, iNOS is activated by inflammatory stimulation and concerned with production of NO. A well-known inflammatory response in macrophages starts by combining LPS and TLR, which brings about a cascade of kinases. COX has two isoenzyme forms, COX-1 and COX-2. COX-1 appears in various cells and mediates the physiological activity, whereas COX-2, known as an inducible enzyme, is found in immune cells treated with LPS and inflammatory cytokines. Previous studies described that COX-2 is accountable for the synthesis of the prostanoids, which are relevant in critical and chronic inflammatory conditions. Thus, the hyperaction of COX-2 can provoke production of the PGE₂ (Line: 282-290). Therefore, tendency of production may differ.

15. The supplementary figures are not mentioned in the main text. Please refer to these figures in the manuscript and state what is shown and why this is important?

: We mentioned the supplementary material in line 244-245. The figure 5 and supplementary materials showed the correlation analysis between all compounds and inflammatory cytokines. Most importantly, α-amyrin and β-amyrin showed a significant difference in decreasing inflammatory cytokines among these compounds of DM. Therefore, this result provides that they serve as inflammatory inhibitor (Line: 309-311).

16. Information about LPS induction is scarce. I kindly ask the authors to include information about when, how long and the density of cell cultures for induction.

: We added the density of cell and LPS concentration (Line: 93-95). RAW264.7 cells were seeded at 1×106 cells/well in a 96-well plate. After 24 h, the cells were incubated for 12 h in 5% CO2 atmosphere at 37 °C with varying concentrations of DM extracts and treated with LPS (2 μg/mL).

 

17. The legends of the axes in multiple figures is distorted/warped. The authors may want to correct this.

: Thank you for your insight, we modified the axes of legends by adjusting font size.

18. qPCR method needs to be described in more detail. The authors may easily consider the MIQE guidelines.

: We supplemented more detail (Line: 102 and 104-108). To isolate RNA, the cells were lysed using TRIzol reagent (1 mL) (Life Technologies, Rockville, MD, USA) following the manufacturer’s protocol. After adding chloroform (0.5 mL) for 5 min, the supernatants were mixed with isopropanol (0.5 mL). cDNA was synthesized from RNA using Transcriptor cDNA Synthesis Kit (Roche Molecular Systems, Inc., Pleasanton, CA, USA). The Universal Probe Library (UPL) method was used to quantify the expression of iNOS, COX-2, and GAPDH in the cells using a Light Cycler 96 system (Hoffmann La Roche, Basel, Switzerland). The PCR thermal cycling was as follows: initial denaturing at 95 °C for 600 s, 85 cycles at 95 °C for 10 s, and 60 °C for 30 s. Relative mRNA expression levels were quantified using the comparative 2−∆∆Cq method and normalized to the amount of GAPDH.

19. WB method: which antibodies were used? (primary, secondary, company, order number, concentration)

: We added more information (Line: 120-125 and 126). The primary antibodies against nuclear factor kappa B (NF-κB) (#8242, Cell signaling), phosphorylated NF-κB (p-NF-κB) (#3033, Cell signaling, Danvers, MA, US), IκB kinase α (IKKα) (#2682, Cell Signaling), phosphorylated IKKα/β (#2697, Cell signaling), c-Jun N-terminal kinase (JNK) (#9592, Cell Signaling), phosphorylated JNK (p-JNK) (#4668, Cell Signaling), p38 MAPK (#8690, Cell Signaling), and phosphorylated p38 MAPK (p38 MAPK) (#9215, Cell Signaling) and secondary antibody were obtained from Cell signaling.

20. 2 is shown at two pages. Although a figure legend might be diplayed at a separate page, the figure itself should fit to one page.

: We modified site of figure 2.

21. In the abstract the authors write that „ethanol extracts of DM leaves may have the potential to enhance inflammatory responses“, which is somehow controversial to the anti-inflammatory effects of DM extracts.

: Thank you for your point. We modified that these results suggest that ethanol extracts of DM leaves may have the potential to regulate inflammatory responses (Line: 28).

 

Author Response File: Author Response.docx

Reviewer 3 Report

The “Comparisons of anti-inflammatory activity and Dendropanax morbifera LEV leaf extract contents based on the collection season and concentration of ethanol as an extraction solvent” is a well-written and well-thought manuscript.  Authors are already aware of the shortcoming of the manuscript in the form of lacking systematic study of the roles of active compounds and detailed mechanisms on induction of related phenotypic responses from DM extracts. Although further experiments is required to explain each DM extract components role on induction of inflammatory or anti-inflammatory response, current format of manuscript is acceptable for publication in the current format.

Author Response

Thank you for your review.

Round 2

Reviewer 1 Report

The reviewer's comments are not addressed.

Author Response

Thank you for your first comment. We reflected that.

Reviewer 2 Report

I would like to thank the authors for adressing my points to improve the quality of their work. While the manuscript has been significantly improved, I have few comments that have not been addressed.

1. I understand that the manuscript has been edited by a professional English language editing company, but there are sentences that are not grammatically correct, to name a few:

Line 26: The amyrin and polyphenol compounds highly depended on the amount of ethanol used during each season.

Line 36: In particular, chronic low-grade inflammation the principal cause of various chronic diseases, such as cardiovascular disease, type 2 diabetes mellitus, metabolic syndrome, non-alcoholic fatty liver disease and various cancers

Line 162: Before quantification proinflammatory compounds, the cytotoxicity of RAW264.7 cells treated with various DM extracts was tested

Figure legends: All data were compared with the LPS-treat group

Thus, careful proof reading/some english editing of the manuscript is still needed.

 

2. The authors expanded their discussion, but they did not consider to describe their data accordingly in the results section.

Line 192: "The treated DM extracts (50 μg/mL) showed significant inhibitory activity for proinflammatory cytokines production." This is simply not true for all 50 μg/mL treatments: W100/August shows increase in IL-6, E60/May + W100/August + E30/November show increase in IL-1beta, W100/May + E60/May + E30/November show increase in TNF-alpha. In addition, the extracts were not treated, but were used to treat the cells. Thus, the term „treated DM extracts“ appears to be misleading (related to english editing in comment 1).

Author Response

I would like to thank the authors for adressing my points to improve the quality of their work. While the manuscript has been significantly improved, I have few comments that have not been addressed.

1. I understand that the manuscript has been edited by a professional English language editing company, but there are sentences that are not grammatically correct, to name a few:

: Thank you for your points, we revised it as below.

Line 26: The amyrin and polyphenol compounds highly depended on the amount of ethanol used during each season.

→ The contents of amyrin and polyphenol compounds highly dependent on the ethanol concentration used during each season (Line: 26-28).

Line 36: In particular, chronic low-grade inflammation the principal cause of various chronic diseases, such as cardiovascular disease, type 2 diabetes mellitus, metabolic syndrome, non-alcoholic fatty liver disease and various cancers

→ In particular, chronic low-grade inflammation is the principal cause of various chronic diseases such as cardiovascular disease, type 2 diabetes mellitus, metabolic syndrome, non-alcoholic fatty liver disease, and various cancers (Line: 36-38).

Line 162: Before investigation of anti-inflammatory activit, the cytotoxicity of RAW264.7 cells treated with various DM extracts was tested

→ Before the quantification of proinflammatory compounds, the cytotoxicity of RAW264.7 cells treated with various DM extracts was tested (Line: 165-166)

Figure legends: All data were compared with the LPS-treat group

→ Statistical analysis was performed using one-way ANOVA. *: p<0.05 compared to LPS-treated group.

Thus, careful proof reading/some english editing of the manuscript is still needed.

: Thank you for your comment. On the whole, we have corrected the English language once more.

 

2. The authors expanded their discussion, but they did not consider to describe their data accordingly in the results section.

Line 192: "The treated DM extracts (50 μg/mL) showed significant inhibitory activity for proinflammatory cytokines production." This is simply not true for all 50 μg/mL treatments: W100/August shows increase in IL-6, E60/May + W100/August + E30/November show increase in IL-1beta, W100/May + E60/May + E30/November show increase in TNF-alpha. In addition, the extracts were not treated, but were used to treat the cells. Thus, the term „treated DM extracts“ appears to be misleading (related to english editing in comment 1).

: Thank you for your insight. We supplemented that the DM extracts (25 and 50 μg/mL) prepared with hot water and 30% ethanol harvested in May; 30% and 60% harvested in August; all extracts harvested in November showed significant inhibitory activity for the production of the IL-6 (p < 0.05) (Figure 3a). In addition, these extracts also displayed an anti-inflammatory effect at 12.5 μg/mL, except for 60% ethanol extract harvested in November. However, several extracts, including the 12.5 or/and 25 μg/mL of hot water and 60% ethanol harvested in May and hot water extract (all concentrations) harvested in August, showed an increase in IL-6 level. In terms of IL-1β production, 50 μg/mL of DM extracts except for 60% ethanol harvested in May; hot water harvested in August; 30% ethanol harvested in November showed significantly suppressed IL-1β level compared to the LPS-treated group (Figure 3b). The production of IL-1β was higher in hot water (12.5 and 25 μg/mL) and 60% ethanol (all concentrations) extract harvested in May; hot water (all concentrations) and 30% ethanol (12.5 μg/mL) extract harvested in August; hot water (12.5 and 25 μg/mL) and 30% ethanol (all concentrations) extract harvested in November than in LPS-treated cells. The TNF-α level decreased in 50 μg/mL of 30% ethanol extract harvested in May; all extracts harvested in August; hot water and 60% ethanol extract harvested in November (Figure 3c). Partially, some extracts, such as hot water (all concentrations), 30% (12.5 μg/mL), and 60% ethanol (all concentrations) extract harvested in May; hot water (12.5 and 25 μg/mL) and 30% ethanol (12.5 μg/mL) extract harvested in August; hot water (12.5 and 25 μg/mL) and 30% ethanol (all concentrations) extract harvested in November, showed a considerable induction in TNF-α compared to that in the LPS-treated group (Line: 201-219).

Author Response File: Author Response.docx