Determination of Biogenic Amines in Different Parts of Lycium barbarum L. by HPLC with Precolumn Dansylation
Round 1
Reviewer 1 Report
The manuscript »Determination of Biogenic Amines in Different Parts of Lycium Barbarum L. by HPLC with Precolumn Dansylation« deals with validation of the method and quantification of biogenic amines (BA) in various parts of Lycium barbarum L. .
Use of Dansyl chloride for the derivatisation of BA is a well established method and is in use for almost 30 years. In this regard there is lack of novelty of the results related to the validation of the method in model systems (without the interference of the matrix) within the submitted manuscript. Matrix interference are not sufficiently evaluated (which could represent a novelty).
BA amines were evaluated in different parts of an edible plant (Lycium Barbarum L.). The samples were air dried prior to analysis (no control of experimental variables that could lead to the artifacts-transformation of BA in the sample prior to analysis). From the presented data it is not clear how the average values were determined (what were independent experiments). Presented data in figures and tables are also duplicated.
Overall the submitted manuscript is perceived as a preliminary work that should be considerably improved, prior to the publication.
More specific comments are as follows:
Section 4.3.
No control of sample drying. Polyamines can be oxidized by diamine oxidases during sample preparation. Freeze drying, or freezing prior to homogenization would be more appropriate than “drying in the shade”. At least should be tested, whether such practice results in the transformation of BA.
Samples were cut into “small pieces”. What does it mean-dimensions? Are BA really accessible (for example from the bark), when cut to pieces. Homogenization with ball mill would probably be better in regard to extractability.
All in all, there are six extraction stages with two different solvents, which can have a large effect on final result. BA have different polarity that is not uniform and same as that of internal standard (IS). Was that evaluated? Is such complicated protocol really necessary ?
There is evaporation step to dryness. Some biogenic amines are more volatile than others (for example phenethylamine) and are different from that of internal standard (IS). Was influence of that stage evaluated?
Section 4.6.
How exactly were recoveries determined. Were standard solutions added to reconstituted solutions after the evaporation step or in some earlier steps?
Section 4.7.
What was the number of independent experiments that would allow statistical evaluation? If there were multiple samples, were they from one tree or form multiple trees/shrubs?
Section 2.3
The authors state that there is no interference with other substances in the range of integrated peaks of BA. The statement is based on the figure 1d, where no Dns-Cl was added to the sample. Such experiment however does not exclude interferences that are generated after dansylation. As shown in figure 1c there are three very large peaks just “before” putrescine (4). How the authors are convinced that peak designated as histamine (6) is also not an artifact. This is an important issue as authors point to high HIS content as problematic. Are there any other literature data about high HIS contents in the bark of different shrubs/trees? MS detector, or at least sequentially connected fluorescence detectors could be the solution. Danyslated HIS has very low fluorescence signal in comparison to other BA and quotients of UV/fluorescence signal that correspond to HIS from the standard solutions, could strengthen the assumption that peak corresponds to HIS.
What does MET stand for? SPM (12) is missing in the figure legend (Figure 1). Usage of “unspiked” wit IS/Dns-Cl is not appropriate.
Section 2.4.
Linearity range stated in the text do not correspond to the range shown in Table 1.
Data shown in Table 2 and Figure 2 are duplicated. Table 2 or figure 2 should be omitted.
Section 3.
Authors state that HIS content in food is limited by FDA, EFSA… This limitations are set only for fish products and not generally for other foods. Speaking about toxicity in relation to high HIS content in bark is an exaggeration (also in section 5-Conslusions). Determined values are based on dry weight of bark, which cannot be consumed by such high quantities as fish (limit is set on a FW). Would it be realistic that somebody eat a few hundred grams of bark?
The authors state: “The leaves of L. barbarum can prolong longevity in modern scientific research [31].” The reference is from a year 1980 (modern scientific research?) and is a dictionary. Such strong statement could not be based on such a reference. Statement should be omitted.
The content of SPD in the leaves Lycium Barbarum is actually low (in comparison to many other food, especially when normalized to DW). Exposing the health benefits of SPM from Lycium Barbarum leaves is therefore unrealistic. The authors state (ref 33) that SPD at 30-60 mmol/L (4350 mg/L- 8700 mg/L) have some beneficial effects. How is it possible to prepare such a concentration directly from the leaves where the content is in the range of 10mg/kg DW.
Author Response
Authors are grateful to reviewer’s valuable comments concerning our manuscript entitled ‘Determination of Biogenic Amines in Different Parts of Lycium Barbarum L. by HPLC with Precolumn Dansylation’ (ID: molecules-1090722, Molecules). Those comments are all valuable and very helpful for revising and improving our manuscript, as well as the important guiding significance to our researches. We have studied comments carefully and have made correction which we hope meet with approval. Revised portion are marked in red in the manuscript. The main corrections in the paper and the responds to the reviewer’s comments are in attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The authors describe a modified method published in many other papers to analyze biogenic amines in different parts of a plant, concretely of Lycium barbarum L.
The abstract and the title include all the matters studied in the paper, and the introduction justifies the research done, and the objectives have been clearly stated. Moreover, the Material and methods section has been properly explained.
Some suggestions or questions about the manuscript:
Different acronyms have been used. What is the meaning of MET? ACN is acetonitrile? The inclusion of a list of acronyms is suggested.
Results
Figure 2 could be deleted. Similar Information is showed in Table 2.
Discussion
Discussion about Histamine is not adequate. Histamine is indeed regulated in different countries. However, generally, histamine content is regulated for fish products, especially from the Scombridae family. In the European Union (Italy and Finland are included) is regulated fresh and ripened fish and fish sauce. On the other hand, the bark of this plant is consumed directly or is an ingredient of drugs, infusions…then, probably the histamine content will considerably diminish?
Speculation about the function of SPD and SPM is commented, e.g., in the text is written " the authors speculate the high content of SPD in the leaves is crucial for L. barbarum on anti-aging, and it can, to a certain extent, contribute to the anti-oxidative and anti-aging effect of the leaves “, this sentence it not admissible of a scientific point of view. Data obtained no supported this speculation.
Do the authors consider that the content of SPM in young leaves is high? This paragraph “The highest content of SPM in the young leaves can, to a certain extent, contribute to the anti-oxidative and anti-aging effect of the leaves” is speculative.
Author Response
We would like to thank you for giving us a chance to revise our manuscript entitled “Determination of Biogenic Amines in Different Parts of Lycium Barbarum L. by HPLC with Precolumn Dansylation” (ID: molecules-1090722, Molecules). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have studied your comments carefully and have made revision which marked in red in the manuscript. The main corrections in the paper and the responds to the your comments are in the attachment.
Author Response File: Author Response.pdf
Reviewer 3 Report
Yun Ai et al. reports interesting findings regarding the content of biogenic amines in Lycium barbarum L.
Overall the text is well written, but it can be improved in the Discussion section. The results are presented clearly and consistently support the Authors’ conclusions but should be revised as their numerical format is concerned. The procedure and instrumental method used to determine the amines is not a novelty, but this can also be regarded as a point of strength of the study since the Authors can undoubtedly claim that the method used is well consolidated to determine amines in biological matrices. Maybe the Authors can provide some more references to the use of Dns-Cl for derivatization of amines before HPLC-UV analysis.
Some flaws of the manuscript are listed below to help the authors improve their already brilliant presentation.
Section 2.2 “An addition of irritating ammonia etc..” I do not understand the need for the adjective “irritating”. I think that it is out of context here.
Section 2.3
The authors claim that “There was no overlap between each standard amine.” However, looking at the chromatograms presented, it does not seem to be so. The peaks of chromatograms in Figures 1b and 1c in the cluster at about 20 min seem not baseline separated. It is probably a matter of low quality of the illustrations. It is suggested to provide pictures of better quality and more readable.
In Figure 1, one trace is missing, i.e., illustrating a blank of Dns-Cl reacted without matrix, to document the selectivity of the separation concerning by-products of the derivatization reactant.
Section 2.4
“Good linearity” should be reworded as “Good fitting with the linear model for the response (R2 etc.).”
The term “reproducibility” should be corrected with “intermediate precision,” which the authors demonstrated by assaying the inter-day precision.
Tables 1 and 2, and first line of page 5. The authors did not define what is “MET”.
Table 1: Recovery, is it % mean ±RSD or % mean ±SD? Please check.
Tables 1 and 2, and throughout the entire text. Numerical results expressed as mean ±SD should be presented considering the rules for rounding numbers and considering the uncertainty of measurement computed after the experiments. To put it simply, I suggest considering the S.D. as an estimate of the total uncertainty of the measurement. Therefore, the rounding can be made based on the first significant figure of the S.D. and applying the basic rules for rounding (see, for example, Agut et al., Journal of Pharmaceutical and Biomedical Analysis 41 (2006) 442–448; Caballero and Harris, Journal of Chemical Education (1998) 75:996). So inconsistent results such as 89.1±4.87 in Tables 1 (but really in both Tables 1 and 2, and in all the text) can be represented more consistently by selecting the appropriate number of digits consistent with the total measurement uncertainty observed.
Table 2. Please clarify the meaning of the comparisons and the p-values. Based on the somewhat confusing discussion written in Section 3, one must speculate that, e.g., 23.67±2.03** mg/kg for SPM in Young leaves (see also previous comment regarding the selection of the appropriate number of significant digits) is a result significantly different (greater) than the other results in the same column for SPM? However, then what are the other p-values in the same column representing? Furthermore, if the comparison is to be made by rows (e.g., the content of SPM compared with that of the other analytes in Young Leaves), then are the p-values evidencing that 12.94±1.81* is a number different (smaller) than 23.67±2.03** at the 95 or 99% probability level?
Section 3. This section should be rewritten, considering the comments above regarding the presentation of the results and clarifying the meaning of the data collected. Also, all information regarding the toxicity of HIS, the effects of the leaves of L. barbarum, SPD, and SPM should be presented in the introduction and not in the Disucssion. The Discussion section should include comparing the data found with other results previously documented and current limitations imposed by regulatory agencies (like the U.S. FDA, as the authors correctly wrote).
Section 4.6. I suggest the following changes in the first sentence.
“The analytical method was validated in terms of selectivity, linearity, LOD, LOQ, precision, and accuracy.”
Author Response
We would like to thank you for giving us a chance to revise our manuscript entitled “Determination of Biogenic Amines in Different Parts of Lycium Barbarum L. by HPLC with Precolumn Dansylation” (ID: molecules-1090722, Molecules). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have studied your comments carefully and have made revision which marked in red in the manuscript. The main corrections in the paper and the responds to the your comments are in the attachment.
Round 2
Reviewer 1 Report
Analytical part of the manuscript is improved.
Authors included results of LC-MS analysis to strengthen the analytical part.
There is nevertheless still problematic interpretation of spermidine importance as antiaging agent in Lycium Barbarum. Statements were added into abstract and at the end of Results and discussion sections (most exposed part of the manuscript).
Abstract:
Compared to those in other parts of L. barbarum, the spermidine content in the young leaves was the highest which might be the one reason of L. barbarum leaves with the anti-oxidative and the anti-aging effect.
Results and discussion:
The leaves of L. barbarum can prolong longevity, enhance exercise endurance and im-proving body anti-oxidation in senile mice [45,46]. In recent years, it is found that SPD potently extendes the lifespan of cells and inhibits oxidative stress in ageing mice[5, 6]. Our previous study suggests that SPD could possibly be one of the key constituents with anti-aging effects of Chinese Tonic-herbs [40]. Therefore, the SPD containing might be the one reason of L. barbarum leaves with the anti-oxidative and the anti-aging effect.
The truth is that the highest spermidine content found in the young leaves is in the range of 15 mg/kg (Table 3, based on dry weight).
When these values are compared to ordinary food (Polyamines in foods: development of a food database ; https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3022763/ ) they are actually lower than in majority of ordinary food. If the data form this database would be normalized to dry weight, it would be even found that content in Lycium Barbarum is actually very low. The above statements in the manuscript should be therefore omitted (unjustified advertising).
There are many typing errors in the revised text. Thorough English editing is recommended.
Author Response
Dear reviewer,
We would like to thank you for giving us a chance to revise our manuscript entitled “Determination of Biogenic Amines in Different Parts of Lycium Barbarum L. by HPLC with Precolumn Dansylation” (ID: molecules-1090722, Molecules). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have studied your comments carefully and have made revision which marked in blue in the manuscript. The main corrections in the paper and the responds to your comments are as flowing:
1. There is nevertheless still problematic interpretation of spermidine importance as antiaging agent in Lycium Barbarum. Statements were added into abstract and at the end of Results and discussion sections (most exposed part of the manuscript).
Abstract:
Compared to those in other parts of L. barbarum, the spermidine content in the young leaves was the highest which might be the one reason of L. barbarum leaves with the anti-oxidative and the anti-aging effect.
Results and discussion:
The leaves of L. barbarum can prolong longevity, enhance exercise endurance and im-proving body anti-oxidation in senile mice [45,46]. In recent years, it is found that SPD potently extendes the lifespan of cells and inhibits oxidative stress in ageing mice[5, 6]. Our previous study suggests that SPD could possibly be one of the key constituents with anti-aging effects of Chinese Tonic-herbs [40]. Therefore, the SPD containing might be the one reason of L. barbarum leaves with the anti-oxidative and the anti-aging effect.
The truth is that the highest spermidine content found in the young leaves is in the range of 15 mg/kg (Table 3, based on dry weight).
When these values are compared to ordinary food (Polyamines in foods: development of a food database ; https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3022763/ ) they are actually lower than in majority of ordinary food. If the data form this database would be normalized to dry weight, it would be even found that content in Lycium Barbarum is actually very low. The above statements in the manuscript should be therefore omitted (unjustified advertising).
Response: We have deleted the sentence about “spermidine importance as anti-aging agent in Lycium Barbarum”.
Abstract:
In line 24, “Compared to those in other parts of L. barbarum, the spermidine content in the young leaves was the highest which might be the one reason of L. barbarum leaves with the anti-oxidative and the anti-aging effect” has been deleted.
Results and discussion:
In line 323, “The leaves of L. barbarum can prolong longevity, enhance exercise endurance and im-proving body anti-oxidation in senile mice [45,46]. In recent years, it is found that SPD potently extendes the lifespan of cells and inhibits oxidative stress in ageing mice[5, 6]. Our previous study suggests that SPD could possibly be one of the key constituents with anti-aging effects of Chinese Tonic-herbs [40]. Therefore, the SPD containing might be the one reason of L. barbarum leaves with the anti-oxidative and the anti-aging effect.”has been deleted.
Conclusion
In line 434, “Compared to those in other parts of L. barbarum, the SPD content in the young leaves was the highest which might be the one reason of L. barbarum leaves with the anti-oxidative and the anti-aging effect.” has been deleted.
Please see the details in the revised manuscript.
2. There are many typing errors in the revised text. Thorough English editing is recommended.
Response:The English in the revised manuscript has been thoroughly checked and edited.
Please see the details in the revised manuscript.
Once again, thank you very much for your good comments and suggestions.
Reviewer 2 Report
The authors have answered properly the questions or suggestions performed. I would like to make one suggestion in the Introduction section: the Regulation of the European Union about histamine is appliable to countries as Germany, Italy, and Finland. They have not an own regulation about this amine. See this link: https://eur-lex.europa.eu/legal-content/ES/ALL/?uri=CELEX%3A32005R2073Author Response
Dear reviewer,
We would like to thank you for giving us a chance to revise ourmanuscript entitled“Determination of Biogenic Amines in Different Parts of Lycium Barbarum L. by HPLC with Precolumn Dansylation” (ID: molecules-1090722, Molecules). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have studied your comments carefully and have made revision which marked in blue in the manuscript. The main corrections in the paper and the responds to the your comments are as flowing:
The authors have answered properly the questions or suggestions performed. I would like to make one suggestion in the Introduction section: the Regulation of the European Union about histamine is appliable to countries as Germany, Italy, and Finland. They have not an own regulation about this amine. See this link: https://eur-lex.europa.eu/legal-content/ES/ALL/?uri=CELEX%3A32005R2073
Response: The link is about commission regulation (EC) No 2073/2005 of 15 November 2005 on microbiological criteria for foodstuff. We have studied the regulation carefully. 100 mg/kg has been proposed as the permissible limit of histamine by the European Union in the regulation. Therefore, the regulation is also appliable to Germany, Italy, and Finland. According to your valuable suggestion, the limit of histamine has been modified in the revised manuscript.
In line 65, the “European Community, South Africa, Italy, Canada, Finland and” has been modified to “European Union, South Africa, Canada,”.
In line 269-270, the “European Community, South Africa, Italy, Canada, Finland” has been modified to “European Union, South Africa, Canada”.
Please see the details in the revised manuscript.
Special thanks to you for your good comments.
