Comprehensive Characterization of Lignans from Forsythia viridissima by UHPLC-ESI-QTOF-MS, and Their NO Inhibitory Effects on RAW 264.7 Cells

Round 1

Reviewer 1 Report

L17 number agreement; replace “have” with “has”

L18 insert a connecting sentence about the study motivation between first and second sentence of the abstract. Is it known that lignans show anti-inflammatory activity?

L24 comment on the difference between lignin and lignin; compound 3 and 4 are lignans, aren’t they?

L22: introduce shortly why you investigate NO inhibitory effects

L25: introduce the abbreviations LPS and iNOS

L27-28: the last sentence is a week statement about the meaning of the study. Pls make the importance more clear.

Add a figure illustrating the chemical structures of the detected compounds

L60: delete “The”

L61: replace suspended with diluted

L65 number agreement “were”/was

L68: Rephrase e.g. “The lignans present in F. viridissima were identified by calculation of the molecular formula from exact mass and MSe spectra with the characteristic mass fragmentation patterns.”

L71 number agreement “its”/their

L73 replace fragmentation with fragment ions

L75 number agreement “molecular ions”

Always place m/z before the value of the m/z throughout the manuscript. Delete all “of” between m/z and their values

L77 pls explain the labeling of the chroms in the legend of fig s1. Moreover, the order of the sup figures seems awkward and should follow their appearance as reference in the manuscript.

All supp figures are labeled with “S” after the number, all references with “S” before the number, pls adjust

L78: Rephrase: ”The characteristic MS fragment of m/z 137 of matairesinol was also observed (Figure S7) in agreement with [16, 17].

L 84: do you refer to figure 1 here?

L86 rephrase: “The daughter ions of both the compounds also exhibited diagnostic ions at m/z 137 and 151.

Reduce size of the upper panel in Fig 1 to match the lower panel. Make the two panels visually more clear. Improve the legend: explain A and B, label the different panels etc. For better comprehension, highlight the two regions in the molecule where ring opening is suggested in the lower panel- according to your belief, does this happen in the gas phase or in solution? Moreover, you suggest a twofold charged species here?? And finally, was the suggested fragmentation confirmed or is this just a proposal of yours? I would assume, other cleavages would also be possible?

L95-96: Rephrase “In contrast, the observed fragmentation at m/z of 247 could occur due to the unsaturation of the position 7 and 8 followed by a rearrangement resulting in loss of the substituted benzene moiety (Figure 2) [16, 17].”

Fig 2: upper panel can be reduced in size and, anyway, is redundant with Fig 1. Lower panel first structure contains overlapping elements, pls improve. The proposed mechanism contains several H migration, what makes you think that the rearrangements are not simultaneously? Pls better illustrate the proposed mechanism using arrows to highlight the purpose of the figure

L102 number agreement “molecular ions”

L104 number agreement “spectra…were obtained”

Table 1: legend: “Compounds identified by molecular formulas calculated from the accurate mass and MSe fragments of UHPLC-ESI-QTOF-MS analyses.” Consider using “MSe” instead MS fragments for consistency

L109: number agreement “roots exert”

L111 explain selection of the cell line

L118 6,7 and 8 also show inhibition in a dose-dependent manner? Just not linear?

Fig3: shift graphs for inactive compounds to the supps. The legend is too short; e.g. explain the stars and color-coding of the bars and the numbers above the graph; axis labeling could be a bit more suggestive

Add a reasonable conclusion

L139 add a paragraph summarizing all used chemicals and suppliers

L144 “subfractions”

L149 you sure you used w/v not v/v; moreover, either of the two is written in italics

L154 0.0 L/h is “off”; missing e in “cone”; two modes- does that mean you´measured each sample twice?

L155 really 600.0 L/h? (check precision for given number of digits)

L157 delete “The”, missing e in encephaline, add lock mass in pos mode

L167 detail how the compounds were applied, did you change the medium? If not, how did you achieve homogenous distribution?

L172 comment on how valid the quantity of nitrite works as a proxy for the discussed NO; in fact, the whole manuscript never mentions that you in fact used a nitrite assay; how you ensure that we see the effect of NO not nitrite?

L176 provide reference for Griess reagent

References: check italic font for the Journal issues throughout the whole list. Check journal abbreviation ref 10

Author Response

We thank the Reviewers for the thoughtful comments. We tried to address those comments and we sincerely hope that the revisions will render this manuscript suitable for publication in Molecules.

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript “Comprehensive characterization of lignans from Forsythia viridissima by UHPLC-ESI-QTOF-MS, and their NO inhibitory effects on RAW 264.7 cells” describes 10 lignans in Forsythia viridissima roots as well as their NO inhibitory effects on RAW 264.7 cells. Actually, all the ten compounds identified were recently reported (Huh et al., DOI: 10.1021/acs.jnatprod.8b00590) as constituents of Forsythia viridissima roots. In addition, there is a lack of information in the experimental section regarding the extraction and fractionation methodology used for the compounds identified or even how they were isolated for anti-inflammatory analysis. Finally, the anti-inflammatory activity of lignans, including of some of those identified in this manuscript is already described in the literature. Therefore, there is a considerably lack of novelty. I suggest reject it.  

Author Response

We thank the Reviewers for the thoughtful comments. We tried to address those comments and we sincerely hope that the revisions will render this manuscript suitable for publication in Molecules.

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Jungmoo Huh et al. analyzed extracts of Forsythia viridissima to identify compounds with anti-inflammatory effects. They identified various lignans and determined their in vitro effects on NO inhibition and iNOS expression. The experiments have been carefully designed and performed.

 

Major compulsory revisions

The authors describe that fruits Forsythia viridissima of have been traditionally used for      inflammatory conditions. Yet they investigated the roots of that plant.      They should give a rational why they investigated the roots and discuss      whether similar or different compounds have been identified in the fruits.

The authors performed elaborate and nice experiments.      However, they do not provide sufficient detailed information to reproduce      them. Please either give more experimental details or provide a reference      where a detailed protocol can be found. Examples:
     Line 60: the extract: please detail ratio of root and solvent,      temperature, extraction time and conditions.
     Line 121: immunoblotting: please detail whether wet or dry blotting was      used, blotting conditions. A section “Immunoblotting” is not present in      the “Materials and Methods” section. What was used as control? Which      antibodies were used (provider/company?), which dilutions of the      antibodies were used? Please revise.
     Line 125: methylene chloride fraction: please detail extraction      conditions. Was the solvent evaporated after extraction before it was used      in cell culture? Was the residue reconstituted for the cell culture experiments?      Which solvent was used for reconstitution?
     Line 144 and following: which software was used for peak analysis?
     Line 167: which solvent was used for the compounds? What was used as      control in the cell viability assays?
     Line 175: which solvent and concentration was used for LPS? What was the      calibration range for the nitrite assay?

The discussion should put the results of the      present study into the context of existing knowledge. So please explain      whether the compounds you identified have been found in other plants /      parts of plants before. Has an anti-inflammatory activity previously been      described for these compounds? If so, in which assay?

 

 

Minor compulsory revisions

The expression in line 47 “Lignan is a      representative secondary metabolite of F.      viridissima.” is not precise. Lignan is not a single compound, lignans      are a group of compounds. Please revise.

Line 52: the authors mention “numerous studies”.      Please specify the type of studies: in      vivo, in vitro, animal,      human studies?

Figure 3: What does the first column in the      nitrite figures represent? Do the columns represent means and standard      deviations? Or standard error? Please provide the number of repetitions of      the experiments (n= ?). The stars obviously indicate statistical significance,      please explain (p< ?). Which statistical test was used? Please add the      topic “Statistical analysis” in the “Materials and Methods” section.

Figure 4: Please provide the number of repetitions of the experiments (n= ?). Please detail in the “Materials and Methods” section how the densitometric analysis was done (software?).

Author Response

We thank the Reviewers for the thoughtful comments. We tried to address those comments and we sincerely hope that the revisions will render this manuscript suitable for publication in Molecules.

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

The authors responded appropriately to the comments and revised their manuscript accordingly. i find the manuscript suitable for publication now.