Alkaloid and Nitrogenated Compounds from Different Sections of Coryphantha macromeris Plants and Callus Cultures

Reviewer comment

Authors response

Line 42 – 43. The discussion of PAL enzyme could be irrelevant as the research focused on characterization of compounds profile instead of elucidation of biosynthetic pathways.

The information related to PAL enzyme was deleted from the introduction section.

Line 119. Minor edits on English – improper use of italics.

Corrected

Line 236, 253, 287 and throughout the text. Lack of detailed explanation on how the compounds are “assigned”. Suggest specifying compound identification method under section “Materials and Methods”. Currently only compound detection method has been specified in the section.

The compound in line 236 was assigned as N-Methyltyramine. The description is provided based on spectrometric evidence existing in the literature. The cited manuscripts present spectrometric evidence that we were able to find within the mass spectrum (fragments at m/z: 121.0651 and 103.0546). Additionally, we analyzed the fragmentation pattern of the proposed compound using ChemDraw and the experimental obtained mass spectral information. Based on this, we assigned the identity of the detected metabolite. In the reviewed version, we included the fragmentation steps in brackets. Regarding the compounds from lines 253 and 287, the identity was assigned using the same procedure.

Table 1. There are no MSn ions shown for the “unknown” compounds in the table. Specify if they are not detected or not reported.

In the reviewed version, we included the fragment ions for unknown compounds. Additionally, we included the molecular formula of the given metabolites.

We also realized about typo-mistakes and corrected them within Table 1.

Table 1. Regarding “tentative identification” of the compounds – it’s unclear how the compounds have been identified except proposal based on ion fragmentation pattern. A full justification of the compound identity should be demonstrated by inclusion of authentic chemical standards, which should then validate retention time and fragmentation of the compounds.

In this job, the untargeted metabolite analysis was performed. Based on our experience in LC-MS, retention time cannot always be employed to assign peaks since retention time modifications may occur when chromatographic matrices interact with highly complex mixtures. In this regard, for natural product discovery and untargeted metabolite analysis, High-Resolution Mass Spectrometry (HRMS) approaches represent a powerful tool to assign candidates in complex biological samples. By analyzing fractions with a particular m/z value or molecular mass and by assigning candidates using HRMS, an accuracy below 5 ppm can be achieved. Hence, we use the term "tentatively identified" or "assigned".

In addition, some compounds previously reported for other cacti species are cited, which support the occurrence of these metabolites on closely related plant species. In this regard, one of the manuscript's contributions is describing the assigned molecules' fragmentation pattern.

Authentical chemical standards are not available now for us; in addition, authentical standards are usually employed in targeted metabolite profiling; in this job, we performed for the first time the untargeted analysis of alkaloids and nitrogenated compounds in C. macromeris plants and callus cultures. Based on this, we propose to include "Untargeted profiling of…" in the title.

Table 1. Multiple compounds, e.g., salsoline, toluic acid, etc., are associated with more than one retention time - need to justify whether those are due to occurrence of isomers, etc. Suggest show comparison to authentic standards.

The word “isomer” was included in table 1.

Line 387. Suggest use word "detected" instead of "identified". Current data suggests that multiple compounds are associated with more than one retention time, which needs justification and further characterization of the detected compounds. Identification of the compounds should be supported by comparison to authentic chemical standards.

The word “identified” was modified by “detected” in line 387.

Reviewer comment

Authors response

Abstract: Ok

Introduction: L43-46 Of which type of metabolite in particular?

This section was deleted by reviewer recommendation.

L58-60: Please specify the active alkaloids from Lophophora diffusa (Peyote) 

corrected

L72-77: Why are these finding significant unless they identified the active alkaloids of medical/ industrial uses? and why mescaline, hordenine etc are important?

The provided information form part of the immediate evidence on cactus alkaloids. The main importance of the described information is due to the psychotropic potential of different species. In our experience, C. macromeris is popularly known to have psychotropic potential; nevertheless, no alkaloids with psychotropic properties were detected in our investigation.

General: Be careful with second mentioning of scientific names and unit used are in different format. 

The information was reviewed and modified.

2.3 L149 Why dark? and how is it important?

By using dark conditions, we aimed to avoid photodegradation and light-mediated modification of metabolites.

L155-156: Not sure why freeze drying is necessary here?

The information was corrected. The freeze-drying process was performed after concentration under reduced pressure.

2.4 is very in detail, I suggest reducing the word and may be add citations.

We propose to include the full information for research article.

I have not objecting the use of UHPLC MS/MS, however to be considered as the full research article, I would expect to see different parameters measured in the experiment. i.e., biomass of the callus, yield of the extract. For chemical analyses, may be add total alkaloid contents as to justify if different material sources produces different amount of alkaloids. Interim of Chemical identification, different tool should be also used i.e., NMR if available.  Considering the present amount of work, I would suggest therefore, the first report after the fine revision of the MS.

The revision of MS was performed. Some modifications were included in the manuscript. Currently, no NMR is available for us.

In the reviewed version of the manuscript, in lines 125-126, we included the expression “…callus tissue analyzed after 9 weeks of culture, when the biomass production reached its maximum yield, as reported previously for C. macromeris callus cultures…” to specify and demonstrate that the biomass production and its kinetic behavior was reported previously

Reviewer comment

Authors response

The authors have assessed the production of metabolites from an endangered species using plants grown under different conditions. The study is well planned and executed. However, it would have been interesting if multiple genotypes were taken to compare, whether alkaloids with recognized psychotropic properties reported previously are produced only in certain physiological conditions in particular genotypes. Overall an interesting work with clear outline and execution. 

Different genotypes and different cacti species are being considered for future investigations; at this time, we aimed to evaluate the alkaloid and nitrogenated metabolites modifications according to the culture system. Additionally, we proposed the fragmentation pattern of newly detected compounds for cacti species and demonstrated that no alkaloids with psychotropic properties are present in C. macromeris.

Reviewer comment

Authors response

Suggest the authors to explicitly specify compound identification method in "materials and methods", i.e., have an individual subsection with title "compound identification" and list the use of previous literature as spectrometric reference for each compound identified.

It would be helpful to include "untargeted metabolomics" in subtitles under "materials and methods" to justify analysis method without authentic standards.

The word “Untargeted metabolomic” was included instead the word “phytochemical” within the manuscript.

An individual subsection entitled “2.4.1 Metabolite identification using fragmentation pattern analysis“ was incorporated, as well as its description in the M&M section. The description is provided as follows: “For metabolite identity assignment, an analysis of the existing spectrometric evidences in the literature and the fragmentation pattern analysis of molecules were carried out. Compound structure´ search was performed using databases such as METLIN, isoMETLIN, The Human Metabolome Database (HMDB), Scopus, ScienceDirect, NCBI, ChemSpider, MassBank, and PubChem. Meanwhile, the chemical structure drawing was performed using ChemDraw professional 15.0 software. The resulting data was then compared with the obtained experimental spectrometric information. The identification of metabolites relied on their elemental composition and precise mass calculation, utilizing a defined mass tolerance window below 6 ppm.”