Deciphering the Antitussive, Expectorant, and Anti-Inflammatory Potentials of ShashamKyeongok-Go and Their Phytochemical Attributes: In Vivo Appraisal in ICR Mice
, Seong-Min Ku 2, Dae-Hwa Jung 3, Sae-Kwang Ku 1,4, Md. Mohibbullah 5, Hae-Jeung Lee 6,* and Jae-Suk Choi 7,*
Round 1
Reviewer 1 Report
The manuscript compares the potential antitussive, expectorant, and anti-inflammatory activities of a herbal recipe (SKOG) by using in vivo ICR mice models. The authors have compared these activities to positive control (in two of the assays) and the constituents of SKOG (AR and KOG). This study itself yields interesting results, and might be of great value to the field. However, I have several serious concerns regarding some information written in the document:
The solubility of theobromine in water is roughly 330 mg/L or 0.33 mg/mL (https://pubchem.ncbi.nlm.nih.gov/compound/Theobromine). The authors claimed to have dissolved it in a stock solution of 5 mg/mL in distilled water – this does not seem to make sense, please clarify and elaborate how this was carried out (Line 164-165).
Surprisingly, a positive control is missing from the expectorant assay (Section 2.4.2).
Statistics:
It is a good practice to the test for homogeneity of variance before the ANOVA, and I acknowledge that. However, were the data evaluated for normality beforehand? If the data is not normal, non-parametric statistics should be used instead.
Furthermore, the Kruskal-Wallis test is a non-parametric method that assumes the data is homoscedastic. Whereas a significant deviation from the Levene’s test tells the authors that the data is not homoscedastic – I suggest authors to consult the expert statistician of their institute to reconsider the statistical methodology appropriate for the study.
Please ensure the appropriate statistics have been carried out so that the results can be interpreted within the right context.
The authors need to make sure all the citations reflect what is claimed in the document. I have identified some inappropriate citations, such as:
Line 55-57: Regarding the number of people suffering asthma - please cite the original report/document published by WHO.
Line 65-66: Please provide evidence that A. triphylla is anti-inflammatory, antitussive, and hepatoprotective. The cited studies did not provide this information.
The study cited in reference 6 is about the elucidation of the structures of triterpenoids isolated from A. triphylla, and the authors did not perform any assessments of anti-inflammatory or antitussive properties of the plant.
In reference 8, the authors reported the potential anti-obesity effect of an A. triphylla extract on in vitro and in vivo mice models, rather than its hepatoprotective properties.
In general, I am concerned that the evidence produced is not sufficient for the claims being made, particularly in the conclusion and abstract – and would be reluctant to see such statements being abused for profit by other entities.
For example: The authors have in stated in line 38-39 that the results were comparable or better than the positive control. First of all, this sentence can be considered as an exaggeration as the positive control were used in much lower doses than the SKOG. Furthermore, the statistics were performed against vehicle control, negative control, AR, and KOG, but not the positive control. Thus, the validity of these claims need to be reconsidered after the authors have reconsidered the statistical analyses being used. And not to mention, there was no positive control in the expectorant assay.
In addition to these concerns, the scientific presentation of the manuscript can be greatly improved. It is recommended for the authors to submit the document to a native English speaking individual or an editing office to improve the flow of the language and remove colloquial mannerisms that are not expected in scientific journals.
Various aspects of the document can also be improved, namely:
Please italicize binomial nomenclatures, e.g. A. triphylla
Please define all abbreviations, e.g. Institute of Cancer Research (ICR), dimethyl sulfoxide (DMSO), and so on.
In addition, it would be nice for the authors to suggest a future direction for research on this herbal recipe.
For example, it is particularly interesting that it seemed SKOG at 100 mg/kg can exert somewhat similar potential biological activities to those of 400 mg/kg of AR or KOG individually. Have the compounds behind the potential synergistic activity been studied?
Line 60-61: Please remove the phrase ‘with no potential side effects’ – we do not know enough if these alternative therapeutics have no side effects.
Line 66-69: Please separate citations [6-13] to indicate which compound is reported in which study.
Line 65-66, 69-71, 74-77, and 546-551: These claimed biological activities are overstated as only data from in vitro and in vivo models are referenced. Unless a clinical evidence is present, it is highly recommended for the authors clarify these claims, for examples by stating that the claims were based on in vitro and in vivo models, or by adding the word ‘potential’.
Line 77-81: Please consider rewriting the hypothesis in a clearer, less lengthy manner.
Line 109-112: The instrumentations of the UPLC system can be described further. E.g. the autosampler, column oven, degasser, etc.
Line 118-121: The source of these standards needs to be clarified. Were they standards purchased/donated or prepared in house? In case of the latter, the purification/synthesis method should be included in the manuscript.
Line 137-138: A reference is required for the UPLC separation method used.
Alternatively, if the method was developed by the authors for this study, please provide a validation of the analytical method based on an appropriate guideline (e.g. Guidelines for Single Laboratory Validation of Chemical Methods for Dietary Supplements and Botanicals by AOAC, FDA, or ICH)
Line 164-165: Please include the reference which suggests theobromine should be used as a positive control in the antitussive assay. Theobromine was not used as a positive control in reference 21 nor 22.
Section 2.3.3: Similar to relative ear weight in section 2.5.5. Changes in relative lung weight could have been measured to ensure that the results of the analyses were not attributed to difference in lung weight due to the treatments.
Line 300-305: The unit of the concentration of the compounds need to be elaborated further - What is the mg/kg written here? Is it mg/kg FW or DW sample? Or is it based on oral administration?
Figure 1: The figure has a very poor resolution – it is not clear at all what information is displayed. It is suggested to expand the size and increase the resolution to make it more legible and include it as supplementary materials if neccessary.
Table 3: DEXA is missing from the results, and somehow TB is there. Is this a typo?
Line 534-551: This paragraph merely a repetition of what have been written in the introduction and method sections.
Line 597-598: This sentence is in contradiction with line 468-469.
Author Response
Reviewer #1
The manuscript compares the potential antitussive, expectorant, and anti-inflammatory activities of a herbal recipe (SKOG) by using in vivo ICR mice models. The authors have compared these activities to positive control (in two of the assays) and the constituents of SKOG (AR and KOG). This study itself yields interesting results, and might be of great value to the field. However, I have several serious concerns regarding some information written in the document:
- The solubility of theobromine in water is roughly 330 mg/L or 0.33 mg/mL (https://pubchem.ncbi.nlm.nih.gov/compound/Theobromine). The authors claimed to have dissolved it in a stock solution of 5 mg/mL in distilled water – this does not seem to make sense, please clarify and elaborate how this was carried out (Line 164-165).
Response: Yes, your point is correct. We used TB solution in the form of a suspension. We have changed our statement to “TB was suspended and prepared in distilled water to the working concentration of 5 mg/ml.” in the revised manuscript (Line: 1391-1392).
- Surprisingly, a positive control is missing from the expectorant assay (Section 2.4.2).
Response: We have already mentioned a positive control [AM (250 mg/kg administered mice; line 209)] in the expectorant assay section in the submitted manuscript (Line: 1553).
Statistics:
- It is a good practice to the test for homogeneity of variance before the ANOVA, and I acknowledge that. However, were the data evaluated for normality beforehand? If the data is not normal, non-parametric statistics should be used instead. Furthermore, the Kruskal-Wallis test is a non-parametric method that assumes the data is homoscedastic. Whereas a significant deviation from the Levene’s test tells the authors that the data is not homoscedastic – I suggest authors to consult the expert statistician of their institute to reconsider the statistical methodology appropriate for the study. Please ensure the appropriate statistics have been carried out so that the results can be interpreted within the right context.
Response: In authors, Hae-Jeung Lee is a specialist in statistics. According to her check, the statistic analysis in this study were revised as you pointed out.
- The authors need to make sure all the citations reflect what is claimed in the document. I have identified some inappropriate citations, such as:
Line 55-57: Regarding the number of people suffering asthma - please cite the original report/document published by WHO.
Response: We have corrected to “It represents global public health problems which are likely to be enormously higher, more than 339 million people currently suffer from asthma [3].”
[3] Global, regional, and national incidence, prevalence, and years lived with disability for 328 diseases and injuries for 195 countries, 1990–2016: a systematic analysis for the Global Burden of Disease Study 2016. Lancet 2017, 390, 1211–1259.
- Line 65-66: Please provide evidence that A. triphylla is anti-inflammatory, antitussive, and hepatoprotective. The cited studies did not provide this information. The study cited in reference 6 is about the elucidation of the structures of triterpenoids isolated from A. triphylla, and the authors did not perform any assessments of anti-inflammatory or antitussive properties of the plant. In reference 8, the authors reported the potential anti-obesity effect of an A. triphylla extract on in vitro and in vivomice models, rather than its hepatoprotective properties.
Response: We have added new references to provide evidence that A. triphylla shows anti-inflammatory, antitussive, and hepatoprotective potentials.
Hu, J.R.; Jung, C.J.; Ku, S.M.; Jung, D.H.; Ku, S.K.; Choi, J.S. Antitussive, expectorant, and anti-inflammatory effects of Adenophorae Radix powder in ICR mice. Journal of Ethnopharmacology2019, 239, 111915.
Gum, S.I.; Lee, D.U.; Cho, M.K. Protective effects of water extracts composed of Adenophora triphylla var. japonica Hara on the acetaminophen-induced hepatotoxicity. Korean Journal of Food Science and Technology2007, 39(6), 688-693.
- In general, I am concerned that the evidence produced is not sufficient for the claims being made, particularly in the conclusion and abstract. And I would be reluctant to see such statements being abused for profit by other entities. For example: The authors have in stated in line 38-39 that the results were comparable or better than the positive control. First of all, this sentence can be considered as an exaggeration as the positive control were used in much lower doses than the SKOG. Furthermore, the statistics were performed against vehicle control, negative control, AR, and KOG, but not the positive control. Thus, the validity of these claims need to be reconsidered after the authors have reconsidered the statistical analyses being used. And not to mention, there was no positive control in the expectorant assay.
Response: We have deleted “Moreover, the results were comparable or even better to positive control used in the respective study.” in the revised manuscript (Line:38).
- In addition to these concerns, the scientific presentation of the manuscript can be greatly improved. It is recommended for the authors to submit the document to a native English speaking individual or an editing office to improve the flow of the language and remove colloquial mannerisms that are not expected in scientific journals.
Response: We have revised the manuscript by a native English speaking individual.
Various aspects of the document can also be improved, namely:
- Please italicize binomial nomenclatures, e.g. A. triphylla
Response: We have correct to italicize binomial nomenclatures throughout the manuscript.
- Please define all abbreviations, e.g. Institute of Cancer Research (ICR), dimethyl sulfoxide (DMSO), and so on.
Response: We have defined all abbreviations including ICR, DMSO, ANOVA, and SPSS (Line: 1370, 1333, 1636, and 1641).
- In addition, it would be nice for the authors to suggest a future direction for research on this herbal recipe. For example, it is particularly interesting that it seemed SKOG at 100 mg/kg can exert somewhat similar potential biological activities to those of 400 mg/kg of AR or KOG individually. Have the compounds behind the potential synergistic activity been studied?
Response: We are also very interested in the potential synergistic activity of SKOG, and we intend to study compounds that exert such synergistic effects through further researches (Line: 2359-2361).
- Line 60-61: Please remove the phrase ‘with no potential side effects’ – we do not know enough if these alternative therapeutics have no side effects.
Response: We have removed the phrase ‘with no potential side effects’ (Line: 60-61).
- Line 66-69: Please separate citations [6-13] to indicate which compound is reported in which study.
Response: We have separated citations [6-13] to indicate individual compound(s) reported in A. triphylla (Line: 72-74).
- Line 65-66, 69-71, 74-77, and 546-551: These claimed biological activities are overstated as only data from in vitro and in vivomodels are referenced. Unless a clinical evidence is present, it is highly recommended for the authors clarify these claims, for examples by stating that the claims were based on in vitro and in vivo models, or by adding the word ‘potential’.
Response: We have added ‘in vitro’, ‘in vivo’, or ‘potential’ in the sentences (Line: 70, 75-77, 82, 1839).
- Line 77-81: Please consider rewriting the hypothesis in a clearer, less lengthy manner.
Response: We have rewritten the hypothesis in a clearer, less lengthy manner (Line: 83-85).
- Line 109-112: The instrumentations of the UPLC system can be described further. E.g. the autosampler, column oven, degasser, etc.
Response: We have added “Waters ACQUITYT SM (Sample Manager) Autosampler, Waters ACQUITYT CH-A Column Oven (20 oC)” (Line: 1227-1237).
- Line 118-121: The source of these standards needs to be clarified. Were they standards purchased/donated or prepared in house? In case of the latter, the purification/synthesis method should be included in the manuscript.
Response: We have added the sentence” The standard preparations of this experiment were from the Sigma-Aldrich (St. Louise, MO, USA) or Extrasynthese (GenayCedex, France)” (Line: 1246-1248).
- Line 137-138: A reference is required for the UPLC separation method used. Alternatively, if the method was developed by the authors for this study, please provide a validation of the analytical method based on an appropriate guideline (e.g. Guidelines for Single Laboratory Validation of Chemical Methods for Dietary Supplements and Botanicals by AOAC, FDA, or ICH).
Response: We have added to “the UPLC separation method were followed the method of the Korean Pharmaceutical Manufacturing Plant (2019-11) [23]. In the PDA analysis, lupeol, acteoside, catalposide, and 5H2F were analyzed at a wavelength of 280 nm, whereas lobetyolin and syringaldehyde were analyzed at 310 and 254 nm, respectively [24]” in revised manuscript (Line: 1231 and 1237]
- Line 164-165: Please include the reference which suggests theobromine should be used as a positive control in the antitussive assay. Theobromine was not used as a positive control in reference 21 nor 22.
Response: We have added new reference [Line: 90 and 2442].
[23] Hu, J.R.; Jung, C.J.; Ku, S.M.; Jung, D.H.; Ku, S.K.; Choi, J.S. Antitussive, expectorant, and anti-inflammatory effects of Adenophorae Radix powder in ICR mice. Journal of Ethnopharmacology 2019, 239, 111915.
- Section 2.3.3: Similar to relative ear weight in section 2.5.5. Changes in relative lung weight could have been measured to ensure that the results of the analyses were not attributed to difference in lung weight due to the treatments.
Response: Your point is good. Unfortunately, we have missed this in this study, but we will consider it in further researches.
- Line 300-305: The unit of the concentration of the compounds need to be elaborated further - What is the mg/kg written here? Is it mg/kg FW or DW sample? Or is it based on oral administration?
Response: Yes, it is considered as mg/kg of FW. In response to your comment, we have added (FW) in the revised manuscript [Line: 1858, 1859, 1861].
- Figure 1: The figure has a very poor resolution – it is not clear at all what information is displayed. It is suggested to expand the size and increase the resolution to make it more legible and include it as supplementary materials if neccessary.
Response: Figure 1 is a picture taken from a computer screen, and as the picture size was reduced, the resolution decreased. We transferred this picture to appendix section as Figure A1.
- Table 3: DEXA is missing from the results, and somehow TB is there. Is this a typo?
Response: Yes. We have corrected to ‘DEXA 1 mg/kg’ from ‘TB 50 mg/kg’ in Table 3 [Line: 2124].
- Line 534-551: This paragraph merely a repetition of what have been written in the introduction and method sections.
Response: We have deleted this paragraph.
- Line 597-598: This sentence is in contradiction with line 468-469.
Response: This is an error. In response to your comment, we have revised to Among treatments, SKOG 400 and 200 mg/kg exhibited clear decreases of ear redness and edema as compared with those mice received AR and KOG doses at 400 mg/kg, respectively and SKOG 100 mg/kg showed similar xylene-induced ear redness and edema gross signs as compared with those of AR and KOG 400 mg/kg, respectively. In addition, SKOG 400 mg/kg showed favorable decreases of ear redness and edema gross signs as comparable to those of DEXA 1 mg/kg, but AR and KOG 400 mg/kg, SKOG 200 and 100 mg/kg showed slighter inhibitory effects on the xylene-induced ear redness and edema as compared to those of DEXA 1 mg/kg, in the current gross observation. (Figure A3) (Lines: 1949-1954).
Thank you for your valuable comments on our manuscript. We have modified our manuscript according to your suggestions and we sincerely ask that the revised manuscript be re-examined and considered for publication.
Reviewer 2 Report
Actually it is very interesting scientific work as well as we need in this moment like scientific research containing novel therapeutic agent for respiratory system due to the huge need for these agent due to COVID-19
-All the Latin names must be in Italic manner.
-Correct the aims of your study at the end of introduction section.
-In section: 2.3.1. Animals and husbandry
-Add kindly the humidity levels where the animals were housed as it is an important factor can affect positively or negatively at RS of mice.
-Figure 1. UPLC analysis of identification and quantification of lupeol, syringaldehyde, 5H2F, acteoside, catalposide, and Rg3 ingredients in SKOG, a mixture of AR and KOG. not clear and muted add kindly unmuted one.
-In the discussion section compare kindly your results with other natural products having the same pharmacological effect.
-The MS needs major editing, grammars and typos corrections
Author Response
Reviewer #2
Actually it is very interesting scientific work as well as we need in this moment like scientific research containing novel therapeutic agent for respiratory system due to the huge need for these agent due to COVID-19.
- All the Latin names must be in Italic manner.
Response: In response to your comment, we have correct to italicize binomial nomenclatures.
- Correct the aims of your study at the end of introduction section.
Response: We have corrected the aims of this study at the end of introduction section [Line: 88-92].
- In section: 2.3.1. Animals and husbandry. Add kindly the humidity levels where the animals were housed as it is an important factor can affect positively or negatively at RS of mice.
Response: We have already mentioned like this “relative humidity of 50-55% in a controlled room” (line 146) [Line: 1466].
- Figure 1. UPLC analysis of identification and quantification of lupeol, syringaldehyde, 5H2F, acteoside, catalposide, and Rg3 ingredients in SKOG, a mixture of AR and KOG. not clear and muted add kindly unmuted one.
Response: Figure 1 is a picture taken from a computer screen, and as the picture size was reduced, the resolution decreased. We transferred this picture to the appendix section as Figure A1.
- In the discussion section compare kindly your results with other natural products having the same pharmacological effect.
Response: This manuscript is evaluating the antitussive, expectorant, and anti-inflammatory potentials of ShashamKyeongok-go (SKOG). As we evaluated the three different activities and the status of the organization, the amount of data and the amount of papers being referenced are being quiet large. So, many of our experimental data had to shift to Appendix section. It would be very nice if we could accept your suggestion as much references being added in the manuscript related to other natural products having similar pharmacological effects. I humbly request you to consider the current contents of our manuscript. Thanks for your skillful evaluation on our manuscript.
- The MS needs major editing, grammars and typos corrections
Response: In response to your comment, we have corrected to major editing, grammars and typos corrections (passim).
Thank you for your valuable comments on our manuscript. We have modified our manuscript according to your suggestions and we sincerely ask that the revised manuscript be re-examined and considered for publication.
Round 2
Reviewer 1 Report
I believe the manuscript is improved after the revisions, particularly in the introduction. Thank you for taking the time to make these changes.
I have a few minor comments left regarding the document:
Language:
My last suggestion for this is that the document should be screened one more time by the authors for minor mistakes, such as in line 26 (a ‘which’ is missing after the comma), as well as the reduction of wordy sentences.
I suggest lines 39-40 and 609-611 to be paraphrased or slightly changed so that they are not a copy and paste to each other.
Introduction:
Reference [13] does not talk about mucus production (line 69) – please kindly recheck this.
There is somehow a gap between line 71 and 72
Methods:
Rather than mentioning the sources of the standards at the end of the section (line 123-125), it would be better to list which standards were purchased from purchased from Sigma and which were purchased from Extrsynthese (line 117-119).
Please put reference [23] somewhere in section 2.3.2 as the use of TB as a positive control was based on this study.
Regarding Statistics:
I do not know if my previous comment regarding statistics has been addressed at all, as I have not observed any differences in the methods nor results. If the authors had really evaluated the normality of the data, I think they should mention the test that they used.
Results:
Line 304: There is a typo there with the (FW)
Regarding UPLC Results:
I think my comments regarding the chromatographic picture had not been fully addressed. In case it was not clear enough, what I meant in the previous comment was that I think the readers should know be able to see how well the peaks of these compounds are baseline separated from the other peaks. I believe this will be an important piece of information once such studies get to the stage of finding the compounds responsible for the potential bioactivities observed. Thus, although this is not a critical information for the current manuscript, an illustration of how well the chromatographic separation was carried out in this study will be a huge benefit for future studies. Hence, I strongly suggest the authors to make the UPLC picture in the appendix clearer – this can be performed by for example making each of them on a separate page (with better legibility of the peaks), or if the former is not possible, redrawing the peaks instead of just screen capturing them.
Discussion:
I would suggest incorporating the comment below somewhere in the discussion so that future research using the paper as a basis for their method can follow the suggestion.
Section 2.3.3: Similar to relative ear weight in section 2.5.5. Changes in relative lung weight could have been measured to ensure that the results of the analyses were not attributed to difference in lung weight due to the treatments.
Lastly, I would suggest bioavailability of the compounds to be assessed in future studies as it is a critical parameter underlying such bioactivities.
Author Response
Reviewer #1
I believe the manuscript is improved after the revisions, particularly in the introduction. Thank you for taking the time to make these changes.
I have a few minor comments left regarding the document:
Language:
My last suggestion for this is that the document should be screened one more time by the authors for minor mistakes, such as in line 26 (a ‘which’ is missing after the comma), as well as the reduction of wordy sentences.
Response: Corrected. (Line: 26-28)
I suggest lines 39-40 and 609-611 to be paraphrased or slightly changed so that they are not a copy and paste to each other.
Response: We have changed this sentence in abstract part. (Line: 38-40)
Introduction:
Reference [13] does not talk about mucus production (line 69) – please kindly recheck this.
Response: We have placed the appropriate reference. (Line: 79)
There is somehow a gap between line 71 and 72
Response: Corrected. (Line: 80)
Methods:
Rather than mentioning the sources of the standards at the end of the section (line 123-125), it would be better to list which standards were purchased from purchased from Sigma and which were purchased from Extrsynthese (line 117-119).
Response: We rewrite the sentence to specify the standards with purchasing company. (Line: 148-152)
Please put reference [23] somewhere in section 2.3.2 as the use of TB as a positive control was based on this study.
Response: We have put the reference in section 2.3.2. (Line: 184)
Regarding Statistics:
I do not know if my previous comment regarding statistics has been addressed at all, as I have not observed any differences in the methods nor results. If the authors had really evaluated the normality of the data, I think they should mention the test that they used.
Response: We have added normality test in the 2.6. Statistical analyses section, whether the data were normally distributed. Thanks to reviewer for valuable suggestion. (Line: 334-335)
Results:
Line 304: There is a typo there with the (FW)
Response: Corrected. (Line: 357)
Regarding UPLC Results:
I think my comments regarding the chromatographic picture had not been fully addressed. In case it was not clear enough, what I meant in the previous comment was that I think the readers should know be able to see how well the peaks of these compounds are baseline separated from the other peaks. I believe this will be an important piece of information once such studies get to the stage of finding the compounds responsible for the potential bioactivities observed. Thus, although this is not a critical information for the current manuscript, an illustration of how well the chromatographic separation was carried out in this study will be a huge benefit for future studies. Hence, I strongly suggest the authors to make the UPLC picture in the appendix clearer – this can be performed by for example making each of them on a separate page (with better legibility of the peaks), or if the former is not possible, redrawing the peaks instead of just screen capturing them.
Response: We have separated individual UPLC chromatogram and enlarged them with higher resolution to make a better legibility of the peaks. (Figure A1)
Discussion:
I would suggest incorporating the comment below somewhere in the discussion so that future research using the paper as a basis for their method can follow the suggestion.
Section 2.3.3: Similar to relative ear weight in section 2.5.5. Changes in relative lung weight could have been measured to ensure that the results of the analyses were not attributed to difference in lung weight due to the treatments.
Response: Yes, we have incorporated your valuable suggestions in the discussion part that we can be considered in future studies. (Line: 668)
Lastly, I would suggest bioavailability of the compounds to be assessed in future studies as it is a critical parameter underlying such bioactivities.
Response: Thank you for valuable suggestion. We will keep in mind this critical parameter in future studies.
Reviewer 2 Report
This work is novel and very interesting and the authors established all the required corrections and suggestions. So I have not anymore comments
Author Response
This work is novel and very interesting and the authors established all the required corrections and suggestions. So I have not anymore comments.
Response: I would like to thank you to valuable reviewer being considered with no further corrections in our revised manuscript.
