Single Cell Plant Model of Equisetum arvense for the Study Antihistamine Effects of Azulene and Sesquiterpene Lactones

Round 1

Reviewer 1 Report

The article concerns the use of single cell of vegetative microspore from spore-bearing plant Equisetum arvense an experimental model for the screening of antihistamine agents. The model substances having antihistamine activity including azulene, sesquiterpene lactones austricine, gaillardine, grosshemine, inulicine and deacetylinulicine as well as sesquiterpene alcohol ledol, on the histamine content in germinating microspores has been determined by fluorescent method. The fluorescent reaction of ortho-phthalic aldehyde and histamine in cells and secretory revealed the ability of tested compounds to reduce the level of this biogenic amine depending on their structure in the following order: grosshemine>azulene>austricine>ledol. Remain substances have the weak activity.

The article is very interesting and useful but have several petty imperfections:

1) Line 78–79. The phrase “At fluorescent histochemical determination of histamine within cells and out o-phthalic aldehyde given from Sigma, were used according to the method described for animal cells [26, 27] and applied for plant cell as well [4, 7, 8]” is not clear. I suppose that “were” should be replaced with “was”.

2) The authors erroneously used the short dashes instead of long ones between the interval values. It should be fixed in all the text.

3) Line 96. Insert the space into “)was”.

4) Lines 116–119. “Azulene (Fluka, Germany) and sesquiterpene lactones produced by VILAR (Moscow) 116 were used in the work. Chemical formulae was shown on Fig.1”. Ledol is listed as lactone because it is presented at the figure but really it doesn’t have any ester link in a cycle. It should be rephrased. The authors know about it but sometimes forget. “was” should be replaced with “were”.

5) The legend to the figure is absent. It should be fixed.

6) Fig 2. There is no a depiction of “h”. It should be fixed.

7) Fig.3. Ledol is not a lactone. The legend should be rephrased.

8) Line 185. Insert short dash between <beta> and hexosaiminidase.

9) Lines 203–204. It is not clear what is similar and what is different in the structures dicussed. The discussion concerning the SAR should be expanded.

10) Line 231 “].Our” should be replaced with “]. Our”.

11) I recommend to assign individual number for each substance, as it was done in the most of chemical articles. The use of the numbers in the discussion instead of full names of substances seems to be more appropriated.

The English also should be polished.

Hence the article is interesting and may be published after minor corrections.

Author Response

Dear Reviewer,

Thank you for useful comments.

1.Most English and technical corrections have been done in text according to remarks 1-10.

2. In remark 9 our comments to QSAR have been included as phrase: “For most sesquiterpene lactones the structure-activity relationship (QSAR) concludes common effects dealt with the gamma-lactone cycle [12], except ledol. The pharmaco-logical understanding of antihistamine activity is primarily related to the effect on specific receptors, but in a broad sense can be mediated by other mechanisms too.”

To our mind, the study of the structure-activity relationship (QSAR) requires appropriate models. However, most computer programs have approximate specificity QSAR determinations today. Besides, comparing the results obtained in a model that uses a plant cell with patterns obtained on the basis of animal systems is at the beginning yet.

3. Remark 11. As for recommendation to exchange names of sesquiterpenes for individual numbers, we tried to do and think that readers will have difficulties in numbers at the explanations of the figures’ legends, results and discussion. It would be better to have names of compounds.

Reviewer 2 Report

Paper titled (Single cell plant model of Equisetum arvense for the study anti-histamine effects of azulene and sesquiterpene lactones) by Roshchina and Konovalov tested a plant model for investigating the antihistamine action of azulene and lactones. The aim of the study is straight forward and design of the experiment is fine. I have the following recommendations:

1-Stat analysis: data should be presented as mean and SD NOT mean and SE

2- Stat analysis: what type of ANOVA was applied? why shy in mentioning the post-hoc test?

3- Authors have to check the normality of distribution of the results by a suitable post hoc test (such as Shapiro-Wilk test or K-S test) before deciding to choose certain ANOVA. If the normality test indicated normal dist of the data, so use one-way ANOVA, if not, use non parametric ANOVA

In all cases choose a suitable post-hoc test

4- Authors should give the source of chemicals, kits and antibodies completely and consistently (code, company, town, state and country) & version for software

5- Figure should be simplified and enlarged to be more clear

6- In figure legends: clarify the type of the presented data and number of variables + stat test applied to these data.

Author Response

Dear Reviewer,

 Thank you for useful comments. 

1. Post - hoc test and one-way ANOVA approach for one factor analysis may be estimated for histochemical fluorescence probes for histamine without sesquiterpene compounds or with their additions. Ho (0 hypothesis) demonstrated that all cells’ responds relate to one general group. Population of Equisetum microspores is a fairly homogeneous, that is why we choose this single cell model. Internal deviations are small. We included SD values’ varieties for histochemical fluorescence probe on histamine in Statistic Section of Methods. Under legends to Figures, the information distracts the attention from the main thing.
2. Figures have been improved by accented lines.
3. Except azulene from Fluka Corporation, other sesquiterpene compounds were received from VILAR Industry, Moscow, by special order.

Round 2

Reviewer 2 Report

In the revised form, auhtors replied that "

1. We included SD values’ varieties for histochemical fluorescence probe on histamine in Statistic Section of Methods. Under legends to Figures, the information distracts the attention from the main thing.
2. Figures have been improved by accented lines.

Howver, I noticed that methods declare results are mean & SE 

Also figure legends were not amended as mentioned

Author Response

We included text with inclusions in red color as first time was. SD are in Methods and under figures in legends. SD did not influence on the significance of results.

Round 3

Reviewer 2 Report

Unfourtnately Authors did not get th emeaning og my last query about using data as Mean&SD 

what authors wrote in methods 'SD were as follows: for Fig.3 2- 8 in variants with salt only and each sesquiterpene, except desacetylinulicine (in this case 10-15); for Fig.5 4-8, except desacetylinulicine (6-10) and for Fig.7 5-9."

Should be deleted & the same for the red parts under figures!!!! delete them

 

Please consult a statistican who can help you present yoour data in figures as mean& SD (NOt as mean &SE)

thanks

Author Response

Dear Dr. Reviewer 2,,

 I have marked in Methods that instead of mean& SEM, in Fig.3, 5, 7 images have included means & SD values. See attached file.

 Regards

 Victoria V.Roshchina