Ecological Insights to Track Cytotoxic Compounds among Maytenus ilicifolia Living Individuals and Clones of an Ex Situ Collection
, Denise Medeiros Selegato 1, Ian Castro-Gamboa 1, Luiz Vitor Silva do Sacramento 3 and Maysa Furlan 1,*Round 1
Reviewer 1 Report
Manuscript fit the journal's aims and scopes
Need to improve
-introduction: references and state of the art
-aim of the work: meaning and general novelty also compared to other published papers
-discussion: clearly express the novelty and the findings and the interest to reader expecially related to phytochemistry and natural bioactive compound chemistry
general novelty of the manuscript and general consideration due to further opportunities of research in the same area
some part need to be revised in order to allow the reader to clearly understand the experimental protocol and the plant sampling
Further comments are in the pdf of the manuscript
Comments for author File: 
Comments.pdf
Author Response
TO THE SUGGESTIONS OF REVIEWER NUMBER 1
We appreciate the suggestions that might improve the quality of the communication. Please find bellow all the points of clarification that our revision addressed.
Regarding the suggestions made available on the website decision text:
To bring more clarity to the manuscript authors reviewed the text thoroughly. For instance, the modifications made in the introduction are intended to substantially increase the clarity on the aims of the paper.
A more detailed text is present for both items discussion and introduction including more references. We hope the present version could meet the quality criteria of the reviewer, which has raised helpful questions.
Regarding the suggestions included in PDF file:
1- Suggestion on last version of manuscript line 12:
To re write the following “Biodiversity is key for maintenance of life and source of richness. However, chemodiversity in nature might be exclusively phenotype expression of living organisms. Sesquiterpene Pyridine Alkaloids (SPA) and Quinonemethide Triterpenes (QMT) accumulate in root bark of Celastraceae plants.”
Outcome in this present version (Paragraph starting line 11).
“Biodiversity is key for maintenance of life and source of richness. Nevertheless, concepts such as phenotype expression are also pivotal to understand how the chemical diversity varies in a living organism. Sesquiterpene Pyridine Alkaloids (SPA) and Quinonemethide Triterpenes (QMT) accumulate in root bark of Celastraceae plants. However, despite their known bioactive traits, there is still a lack of evidence regarding their ecology functions. “
2- Suggestion on last version of manuscript line 68
“that is reported for domestic species but some examples also for wild plants need to be added and better explained”
Outcome in this present version (Paragraph starting line 72).
“Among the ecosystem features, the habitat plays a key role in driving the physiology of wild type plant species [11]. Hence, it has been established that biosynthesis pathways are feasible to be triggered due to specific ecological interactions [12], resulting in distinct metabolic profiles furnished for different individuals of the same plant species. Examples of this plasticity have been extensively reported for domestic plants, such as wine grapes (Vitis vinifera: Vitaceae [13]) and apples (Malus domestica: Rosaceae [14]). However, only a few studies on literature address wild plants endemic to neotropics, such as reported for Casearia sylvestris (Salicaceae) [15]”
3- In Line 76 there was the following comment: “too general sentence also needing reference please”.
Outcome in this present version (Paragraph starting line 83).
“In Celastraceae, the production of terpenes shifts among different plant species, tissues and season, as reviewed by Alvarenga and Ferro [19]. For instance, our group has shown that the biosynthesis of triterpene friedelin happens in the leaves but produces the precursors of quinone methide triterpenes, which are synthesized in the roots [20]. These variations raise the hypothesis of time-dependent and age-related chemical behaviour, which have been widely reported in current literature [16] but has not been proved yet within this family.
Most commonly found bioactive metabolites in root bark extracts of Celastraceae species are quinonemethide triterpenes (QMT) and sesquiterpene pyridine alkaloids (SPA). Quinonemethide triterpenes display cytotoxic, anti-ulcerogenic and antioxidant effects, as well as anticancer activity against different human cancer cell lines [21–27]. Similarly, the sesquiterpene pyridine alkaloids (SPA) display insect antifeedant [28], insecticidal [29], cytotoxic [30], immunosuppressive[26], anti-HIV [26], antiprotozoal [31], anti-hepatitis C [32] and antitumor [33] activities.”
4- Suggestion on last version of manuscript line 86 “please some reference on the medicinal traditional uses...”
Outcome in this present version (Paragraph starting line 95)
“In this topic, we aim to expose the relevance of multi-trophic interactions and ontogenesis in determining richness of bioactive classes of compounds among ex situ collections of Maytenus ilicifolia Mart. ex Reissek (Celastraceae). This specie is generally known in South America as “Espinheira-santa” and is the most used Maytenus species in tradition medicine, exhibiting high antiulcerogenic effects of its leaves infusion [34].”
5 – Correction on line 89 done accordingly.
6 – Correction on line 94 done accordingly.
7 – Suggestion on last version of manuscript line 99 .
“more details are needed in collection and storage of plants. how authors decided the sampling how did they selected the same stage of plants? more details in this are needed”
Outcome in this present version (starting line 221)
“Individuals were monitored since their transplant from original habitat, during late 90's up to nowadays. Hence, sexual reproduction, through bird’s dispersion of seeds, and asexual reproduction from branches and root shootings, were determined through simple observation of each piece of the collection. All the plants of the collection maintained in the botanical garden were sampled in the present study. Height description and morphological traits of each individual are available as Table 1 and Figure 2 from the Supplementary Material.
Side shootings of roots were harvested, and their bark was separated from the inner parts of the root. For this harvest, the root was unburied, the bark separated and collected, and the remaining parts buried again. There was no significant injury to the plants in the collection, which are still kept alive. After sampling, the barks were rinsed under water flow to remove soil particles and allowed to dry at controlled temperature of 36 °C for 48 hours.
Samples were harvested in an ex situ collection that comprises ten different specimens of Maytenus ilicifolia, as described in Table 1. A total of 10 individuals kept alive in ex situ conditions at the botanical garden of Pharmaceutical Science School/UNESP (BG-PSS) (GPS S: -21.814885, W: -48.201091) were codified (Table 1).
According the resolution 29 from 2007, all harvest procedures are free of previous allowance from a Department of Environment Ministry of Brazil, the MMA–ICMbio. However, MMA–ICMbio have granted us an allowance for all field activities (49727-2). One botanical voucher was included at in house collection under the code DPP005 and is available for consultation at the Institute of Chemistry from the São Paulo State University-UNESP, Araraquara-SP, Brazil. Furthermore, another voucher was deposited under number HRCB 68663 at the “Herbário Rioclarense” in the São Paulo State University (UNESP), Rio Claro, SP, Brazil.”
8 – Suggestion on last version of manuscript line 126
To reformulate “The major part of individuals has also displayed the content of both QMT metabolites. Enzymatic activity of CYP450, particularly from clans 85 and 71, yield reactions on ring E of triterpenes in many domestic plants [30]. They might be involved in modifications among these metabolites, which is a matter still unresolved in terms of biosynthetic pathways studies”
Outcome in this present version (Paragraph starting line 125)
“In many domestic plants, the enzymatic activity of CYP450 during the biosynthesis of QMT yields reactions on the ring E, mostly on positions 19, 20, 21 and 22 [35]. There is a suggestive involvement of this enzymes in the functionalization of QMT, however it remains an unresolved matter for biosynthetic pathways studies.”
9 – correction on line 135 and 137 done accordingly.
10 – Suggestion on last version of manuscript line 143 and 145 to include reference for NMR data on the literature.
Outcome in this present version (Paragraph starting line 131)
“Results from the 1HNMR spectra of all samples showed similar chemical shifts between maytenin (1) and pristimerin (2), with major differences on the aromatic signals from ring A and B, the presence of a singlet from the methoxyl hydrogens of pristimerin at d3.48 (s, 3H, H-29) and the significant shift in the methyl group at H-27. NMR spectra of M. ilicifolia raw extract (CDCl3, 600 MHz) showed five maytenin (1) signals at d6.28 (d, J 7.2 Hz, 1H, H-7), d6.46 (d, J 0.8 Hz, 1H, H-1), d6.97 (dd, J 7.2 and 0.8 Hz, 1H, H-6), d1.38 (s, 3H, H-25) and d2,14 (s, 3H, C-23); and six pristimerin (2) signals at d6.31 (d, J 7.1 Hz, 1H, H-7), d6.48 (d, J 0.8 Hz, 1H, H-1), d6.95 (dd, J 7.1 and 0.8 Hz, 1H, H-6), d2,16 (s, 3H, C-23), d1.36 (s, 3H, H-25) and d0.47 (s, 3H, H-27), with major peaks highly convoluted in between d0-2.5. Confirmatory analysis by Heteronuclear Multiple Bond Correlation (1H-13CHMBC) and Heteronuclear Single Quantum Coherence (1H-13CHSQC) have also been assessed for both metabolites and are displayed in Supplementary Materials as Table 3. Data from literature, including data generated by our own research group, was used for comparison [36,37].”
11 – Suggestion on last version of manuscript line 174 was to “need to be better explained by the hypothesis”
Outcome in this present version (Paragraph starting line 178)
“These results above mentioned show that maytenin and pristimerin productions are not depended on the plants age, as observed in the clustering patterns. These novel data, herein firstly presented, disproves the hypothesis that QMT biosynthesis are dependent to the plant's age. Hence, ontogenetic shifts on biosynthesis enzymes, such as CYP450, are unlikely to play roles in phenotypical plasticity in M. ilicifolia. Furthermore, the clustering was also not dependent on the reproduction of the plant (clones/individuals). This finding, in particular, might reinforce the hypothesis over gene regulation of the biosynthesis pathways. Currently literature on Plant Sciences have reported that triterpenes production on Bupleurum falcatum L. (Apiaceae) can be epigenetically altered, once β-amirin synthase production can be overstimulated via jasmonate treatment [38].”
12- Suggestion on last version of manuscript line 174 was that “some part of the description allowing readers to understand the experimental details need to be reported in the results section in order to appreciate the choices of the sampling and the differences in the individuals. a couple of simple sentences about this are needed in the comment of results”
Outcome in the present version is at section 3.1.
13- Question on line 241 of the previous version of the manuscript if “sample is the extract after solvent removal?”. Yes, it is.
Outcome in the present version is at section 3.2.
14- Suggestion on line 261 “indication on the statistical evaluation are needed”
Outcome in this present version (Paragraph starting lines 281 and 206)
“HCA is an agglomerative clustering analysis in which the measurement of distance between pairs creates a hierarchy between samples. In this procedure, most similar points are grouped together, creating a dendogram plot that clusters samples based on their similarity.
For HCA experiments, samples I-X were clustered, in triplicate, based on the QMTs and SPA production measured by the semi-quantitative HPLC-DAD and 1H NMR data. For this analysis, the similarity was calculated using Euclidean distance between the samples total integrated area. Ward was chosen as the optimum linkage method due to the fewer susceptibility to outlier effects. All data were statistically analyzed using algorithms created in MATLAB software R2017a® (MathWorks, USA) at a 95% confidence level.”
“Hierarchical clustering analysis (HCA) has been consistently efficient in the analysis of natural and multivariate chemical data [42–44]. For M. ilicifolia, this unsupervised chemometric analysis grouped the samples that shared a similar metabolic production using both HPLC-DAD and 1HNMR data. Some important advantages of this clustering analysis are the possibility to apply high-dimensional data typical from ‘omics' studies, the possibility of both quantitative and qualitative evaluation and the detection of pleiotropic effects that might influence the chemical variation.”
Updated References:
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4. Joly, C.A.; Metzger, J.P.; Tabarelli, M. Experiences from the Brazilian Atlantic Forest: ecological findings and conservation initiatives. New Phytol. 2014, 204, 459–473.
5. Joly, C.A.; Rodrigues, R.R.; Metzger, J.P.; Haddad, C.F.B.; Verdade, L.M.; Oliveira, M.C.; Bolzani, V.S. Biodiversity conservation research, training, and policy in São Paulo. Science (80). 2010, 328, 1358–1359.
6. Carnevale Neto, F.; Pilon, A.C.; Selegato, D.M.; Freire, R.T.; Gu, H.; Raftery, D.; Lopes, N.P.; Castro-Gamboa, I. Dereplication of natural products using GC-TOF mass spectrometry: Improved metabolite identification by spectral deconvolution ratio analysis. Front. Mol. Biosci. 2016, 3.
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10. Mayrhofer, S.; Teuber, M.; Zimmer, I.; Louis, S.; Fischbach, R.J. Diurnal and Seasonal Variation of Isoprene Biosynthesis - Related Genes in Grey Poplar Leaves. Plant Physiol 2005, 139, 474–484.
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14. Nieuwenhuizen, N.J.; Green, S.A.; Chen, X.; Bailleul, E.J.D.; Matich, A.J.; Wang, M.Y.; Atkinson, R.G. Functional genomics reveals that a compact terpene synthase gene family can account for terpene volatile production in apple. Plant Physiol 2013, 161, 787–804.
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Author Response File: Author Response.pdf
Reviewer 2 Report
Unfortunately it is still hard to evaluate this manuscript. I do not have access to supplementary material and can not check essential contents.
Table 2 is not mentioned in the text – is it misnumbering?
Line 111: „The amount yielded is recorded at Table 1” while table 1 is situated on page 10 and is entitled “Table 1. Maytenus ilicifolia individuals kept alive in ex situ conditions at the botanical garden of Pharmaceutical Science School/UNESP (BG-PSS) (GPS S: -21.814885, W: -48.201091).“
Some other mistakes are highlighted in the pdf file.
Due to the inability to evaluate the manuscript, I recommend its rejection in its current form.
Comments for author File: Comments.pdf
Author Response
TO THE SUGGESTIONS OF REVIEWER NUMBER 2.
We appreciate the suggestions that might improve the quality of the communication. Please find bellow all the points of clarification that our revision addressed.
Regarding the suggestions made available on the website decision text:
The supplementary material is available at the Journal’s platform. Moreover, all numbering from Figures and Tables were corrected accordingly.
Regarding the suggestions included in PDF file:
1 - Suggestion on last version of manuscript lines 112, 114 and 286:
The supplementary material is available at the Journal’s platform compress3ed in “.zip” file.
2 - Correction on line 145 done accordingly.
Updated References:
1. Lovelock, J. The Vanishing Face of Gaia; Basic Books, 2009; ISBN 978-0-465-01549-8.
2. Crutzen, P.J.; Stoermer, E.F. The “Anthropocene.” Glob. Chang. Newsl. 2000, 41, 17–18.
3. Pavarini, D.P.; Da Silva, D.B.; Carollo, C.A.; Portella, A.P.F.; Latansio-Aidar, S.R.; Cavalin, P.O.; Oliveira, V.C.; Rosado, B.H.P.; Aidar, M.P.M.; Bolzani, V.S.; et al. Application of MALDI-MS analysis of Rainforest chemodiversity: A keystone for biodiversity conservation and sustainable use. J. Mass Spectrom. 2012, 47, 1482–1485.
4. Joly, C.A.; Metzger, J.P.; Tabarelli, M. Experiences from the Brazilian Atlantic Forest: ecological findings and conservation initiatives. New Phytol. 2014, 204, 459–473.
5. Joly, C.A.; Rodrigues, R.R.; Metzger, J.P.; Haddad, C.F.B.; Verdade, L.M.; Oliveira, M.C.; Bolzani, V.S. Biodiversity conservation research, training, and policy in São Paulo. Science (80). 2010, 328, 1358–1359.
6. Carnevale Neto, F.; Pilon, A.C.; Selegato, D.M.; Freire, R.T.; Gu, H.; Raftery, D.; Lopes, N.P.; Castro-Gamboa, I. Dereplication of natural products using GC-TOF mass spectrometry: Improved metabolite identification by spectral deconvolution ratio analysis. Front. Mol. Biosci. 2016, 3.
7. FAPESP. Knowledge and sustainable use of Brazilian biodiversity: Biota-FAPESP Program/The State of São Paulo Research Foundation. São Paulo; Prol Editora Gráfica Ltda., 2008.
8. Pilon, A.C.; Valli, M.; Dametto, A.C.; Pinto, M.E.F.; Freire, R.T.; Castro-Gamboa, I.; Andricopulo, A.D.; Bolzani, V.S. NuBBEDB: An updated database to uncover chemical and biological information from Brazilian biodiversity. Sci. Rep. 2017, 7, 1–12.
9. Valli, M.; Dos Santos, R.N.; Figueira, L.D.; Nakajima, C.H.; Castro-Gamboa, I.; Andricopulo, A.D.; Bolzani, V.S. Development of a natural products database from the biodiversity of Brazil. J. Nat. Prod. 2013, 76, 439–444.
10. Mayrhofer, S.; Teuber, M.; Zimmer, I.; Louis, S.; Fischbach, R.J. Diurnal and Seasonal Variation of Isoprene Biosynthesis - Related Genes in Grey Poplar Leaves. Plant Physiol 2005, 139, 474–484.
11. Gershenzon, J.; Dudareva, N. The function of terpene natural products in the natural world. Nat Chem Bio 2007, 3, 408–414.
12. Inderjit, W.D.A.; Karban, R.; Callaway, R.M. The ecosystem and evolutionary contexts of allelopathy. Tr. Ecol Evol 2011, 26, 655–662.
13. Martin, D.M.; Aubourg, S.; Schouwey, M.B.; Daviet, L.; Schalk, M.; Toub, O.; Steven, T.L.; Bohlmann, J. Functional annotation, genome organization and phylogeny of the grapevine (Vitis vinifera) terpene synthase gene family based on genome assembly, FLcDNA cloning, and enzyme assays. BMC Plant Biol. 2010, 10, 226–248.
14. Nieuwenhuizen, N.J.; Green, S.A.; Chen, X.; Bailleul, E.J.D.; Matich, A.J.; Wang, M.Y.; Atkinson, R.G. Functional genomics reveals that a compact terpene synthase gene family can account for terpene volatile production in apple. Plant Physiol 2013, 161, 787–804.
15. Bueno, P.C.P.; Pereira, F.M. V; Torres, R.B.; Cavalheiro, A.J. Development of a comprehensive method for analyzing clerodane-type diterpenes and phenolic compounds from Casearia sylvestris Swartz (Salicaceae) based on ultra high performance liquid chromatography combined with chemometric tools. J. Sep. Sci. 2015, 38, 1–21.
16. Pavarini, D.P.; Pavarini, S.P.; Niehues, M.; Lopes, N.P. Exogenous influences on plant secondary metabolite levels. Anim Feed Sci Technol 2012, 176, 5–16.
17. Muntendam, R.; Happyana, N.; Erkelens, C.; Bruining, F.; O, K. Time dependent metabolomics and transcriptional analysis of cannabinoid biosynthesis in Cannabis sativa var. Bedrobinol and Bediol grown under standardized condition and with genetic homogeneity. Online Int J Med Plant Res 2012, 1, 31–40.
18. Happyana, N.; Kayser, O. Monitoring Metabolite Profiles of Cannabis sativa L. Trichomes during Flowering Period Using 1H NMR-Based Metabolomics and Real-Time PCR. Planta Med 2016, 82, 1217–23.
19. Alvarenga, N.; Ferro, E.A. Bioactive Triterpenes and Related Compounds from Celastraceae. Stud. Nat. Prod. Chem. 2006, 33, 239–307.
20. Souza-Moreira, T.M.; Alves, T.B.; Pinheiro, K.A.; Felippe, L.G.; De Lima, G.M.A.; Watanabe, T.F.; Barbosa, C.C.; Santos, V.A.F.F.M.; Lopes, N.P.; Valentini, S.R.; et al. Friedelin Synthase from Maytenus ilicifolia: Leucine 482 Plays an Essential Role in the Production of the Most Rearranged Pentacyclic Triterpene. Sci. Rep. 2016, 6, 1–13.
21. Carvalho, P.R.F.; Silva, D.H.S.; Bolzani, V.S.; Furlan, M. Antioxidant quinonemethide triterpenes from Salacia campestris. Chem Biodivers 2005, 2, 367–372.
22. Jeller, A.H.; Silva, D.H.S.; Lião, L.M.; Bolzani, V.S.; Furlan, M. Antioxidant phenolic and quinonemethide triterpenes from Cheiloclinium cognatum. Phytochem 2004, 65, 1977–1982.
23. dos Santos, V.A.F.F.M.; Santos, D.P.; Castro-Gamboa, I.; Zanoni, M.V.B.; Furlan, M. Evaluation of Antioxidant Capacity and Synergistic Associations of Quinonemethide Triterpenes and Phenolic Substances from Maytenus ilicifolia (Celastraceae). Molecules 2010, 15, 6956–6973.
24. Costa, P.M.; Ferreira, P.M.; Bolzani, V.S.; Furlan, M.; dos Santos, V.A.F.F.M.; Corsino, J.; de Moraes, M.O.; Costa-Lotufo, L. V; Montenegro, R.C.; Pessoa, C. Antiproliferative activity of pristimerin isolated from Maytenus ilicifolia (Celastraceae) in human HL-60 cells. Toxicol Vitr. 2008, 22, 854–863.
25. Paz, T.A.; dos Santos, V.A.F.F.M.; Inácio, M.C.; Pina, E.S.; Pereira, M.A.S.; Furlan, M. Production of the Quinone-Methide Triterpene Maytenin by In Vitro Adventitious Roots of Peritassa campestris (Cambess.) A.C.Sm. (Celastraceae) and Rapid Detection and Identification by APCI-IT-MS/MS. Biomed Res Int 2013, 2013, 485837.
26. Queiroga, C.L.; Silva, G.F.; Dias, P.C.; Possenti, A.; Carvalho, J.E. Evaluation of the antiulcerogenic activity of friedelan-3β-ol and friedelin isolated from Maytenus ilicifolia (Celastraceae). J Ethnopharmacol 2000, 72, 465–468.
27. Souza-Formigoni, M.L.; Oliveira, M.G.; Monteiro, M.G.; da Silveira-Filho, N.G.; Braz, S.; Carlini, E.A. Antiulcerogenic effects of two Maytenus species in laboratory animals. J Ethnopharmacol 1991, 34, 21–27.
28. Liu, J.K.; Jia, Z.J.; Wu, D.G.; Zhou, J.; Wang, Q.G. Insect antifeeding agents: sesquiterpene alkaloids from Celastrus angulatus. Phytochemistry 1990, 29, 2503−2506.
29. Núñez, M.J.; Guadaño, A.; Jimenez, I.A.; Ravelo, A.G.; Gonzalez-Coloma, A.; Bazzocchi, I.L.́ Insecticidal Sesquiterpene Pyridine Alkaloids from Maytenus chiapensis. J. Nat. Prod. 2004, 67, 14–18.
30. Whitson, E.L.; Mala, S.M.V.D.; Veltri, C.A.; Bugni, T.S.; Silva, E.D.; Ireland, C.M. Oppositines A and B, Sesquiterpene Pyridine Alkaloids from a Sri Lankan Pleurostylia opposita. J. Nat. Prod. 2006, 69, 1833−1835.
31. dos Santos, V.A.F.F.M.; Regasini, L.O.; Nogueira, C.R.; Passerini, G.D.; Martinez, I.; Bolzani, V.S.; Graminha, M.A.S.; Cicarelli, R.M.B.; Furlan, M. Antiprotozoal Sesquiterpene Pyridine Alkaloids from Maytenus ilicifolia. J Nat Prod 2012, 75, 991–995.
32. Jardim, A.C.G.; Igloi, Z.; Shimizu, J.F.; Santos, V.A.; Felippe, L.G.; Mazzeu, B.F.; Amako, Y.; Furlan, M.; Harris, M.; Rahal, P. Natural compounds isolated from Brazilian plants are potent inhibitors of hepatitis C virus replication in vitro. Antivir. Res 2014, 115, 39–47.
33. Gonzalez, A.G.; Tincusi, B.M.; Bazzocchi, I.L.; Tokuda, H.; Nishino, H.; Konoshima, T.; Jimenez, I.A.; Ravelo, A.G. Anti-tumor promoting effects of sesquiterpenes from Maytenus cuzcoina (Celastraceae). Bioorg. Med. Chem 2000, 8, 1773−1778.
34. Jorge, R.M.; Leite, J.P. V; Oliveira, A.B.; Tagliati, C.A. Evaluation of antinociceptive, anti-inflammatory and antiulcerogenic activities of Maytenus ilicifolia. J. Ethnopharmacol. 2004, 94, 93–100.
35. Field, B.; Osbourn, A.E. Metabolic diversification - independent assembly of operon-like gene clusters in different plants. Sci 2008, 320, 543–547.
36. Corsino, J.; Bolzani, V.S.; Pereira, A.M.S.; França, S.C.; Furlan, M. Bioactive sesquiterpene pyridine alkaloids from Maytenus aquifolium. Phytochemistry 1998, 48, 137–140.
37. dos Santos, V.A.F.F.M. Metabolomic, biological and proteomic aspects of Maytenus ilicifolia and Salacia campestris (Celastraceae), 2010.
38. Kim, Y.S.; Cho, J.H.; Park, S.; Han, J.Y.; Back, K.; Choi, Y.E. Gene regulation patterns in triterpene biosynthetic pathway driven by overexpression of squalene synthase and methyl jasmonate elicitation in Bupleurum falcatum. Planta 2011, 233, 343–355.
39. Achnine, L.; Huhman, D. V; Farag, M.A.; Sumner, L.W.; Blount, J.W.; Dixon, R.A. Genomics-based selection and functional characterization of triterpene glycosyltransferases from the model legume Medicago truncatula. Plant J 41AD, 875–887.
40. Estevam, C.S.; Cavalcanti, A.M.; Cambui, E.V.F.; Araújo Neto, V.; Leopoldo, P.T.G.; Fernandes, R.P.M.; Araujo, B.S.; Porfírio, Z.; Sant’Ana, A.E.G. Phytochemistry and microbiological assay of the bark extracts of Maytenus rigida Mart. (Celastraceae). Braz J Farm. 2009, 19, 299–303.
41. Monks, T.J.; Jones, D.C. The metabolism and toxicity of quinones, quinonimines, quinone methides, and quinone-thioethers. Curr Drug Metab 2002, 3, 425–438.
42. Depke, T.; Franke, R.; Brönstrup, M. Clustering of MS2 spectra using unsupervised methods to aid the identification of secondary metabolites from Pseudomonas aeruginosa. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 2017, 1071, 19–28.
43. Selegato, D.M.; Freire, R.T.; Tannús, A.; Castro-Gamboa, I. New Dereplication Method Applied to NMR-Based Metabolomics on Different. J. Braz. Chem. Soc. 2016, 0027, 1–11.
44. Shawky, E. Multivariate analyses of NP-TLC chromatographic retention data for grouping of structurally-related plant secondary metabolites. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 2016, 1029–1030, 10–15.
45. Nosengo, N. New tricks for old drugs. Nat 2016, 534, 314–316.
46. Peng, B.; Xu, L.; Cao, F.; Wei, T.; Yang, C.; Uzan, G.; Zhang, D. HSP90 inhibitor, celastrol, arrests human monocytic leukemia cell U937 at G0/G1 in thiol-containing agents reversible way. Mol Cancer 2010, 9, 79–92.
Author Response File: Author Response.pdf
Reviewer 3 Report
Daniel Petinatti Pavarini et al reported the ecological insights of cytotoxic compounds, sesquiterpene pyridine alkaloids (SPA) and quinonemethide triterpenes (QMT), in Maytenus ilicifolia living individuals and clones of ex situ collection. The topic is interest, but more references are needed to support the experimental design.
Why choose quinonemethide triterpenes (QMT) and sesquiterpene pyridine alkaloids (SPA) for classification? Do they have any ecological function?
Reference is needed to support the selection of chemometric method, such as Hierarchical Clustering Analysis (HCA), for group classification based on the concentration of individual component or a few compounds.
Figure 5 does not have any scientific value.
Author Response
To the suggestions of reviewer number 3.
We appreciate the suggestions that might improve the quality of the communication. Please find bellow all the points of clarification that our revision addressed.
Regarding the suggestions made available on the website decision text:
1 - Daniel Petinatti Pavarini et al reported the ecological insights of cytotoxic compounds, sesquiterpene pyridine alkaloids (SPA) and quinonemethide triterpenes (QMT), in Maytenus ilicifolia living individuals and clones of ex situ collection. The topic is interest, but more references are needed to support the experimental design. Why choose quinonemethide triterpenes (QMT) and sesquiterpene pyridine alkaloids (SPA) for classification? Do they have any ecological function?
Response: In fact, the ecology functions of QMT and SPA in Celastraceae are still unclear. However saponins from Celastraceae and, other species are reported to as bioactive as we displayed in the Line 198: ” the extensive presence of QMT and low abundance of SPA suggests a constitutive accumulation of QMTs, similar to the accumulation of sapogenins in Medicago truncatula roots [39]. It has been already reported that Maytenus rigida root bark extracts display antimicrobial saponins [40].
Our group has successfully determined bioactive traits of these two types of compounds using in vitro and in vivo essays (new references [21-25] [31-32]) in last two decades. One of our interests for the present work was to better understand time-dependent phenotypical plasticity of bioactive metabolites, and to do so we aimed the terpenes produced in the root bark of Maytenus ilicifolia, which are majorly QMT and SPA. We explored this approach in the new version of this manuscript in the line 83: “In Celastraceae, the production of terpenes shifts among different plant species, tissues and season, as reviewed by Alvarenga and Ferro [19]. For instance, our group has shown that the biosynthesis of triterpene friedelin happens in the leaves but produces the precursors of quinone methide triterpenes, which are synthesized in the roots [20]. These variations raise the hypothesis of time-dependent and age-related chemical behaviour, which have been widely reported in current literature [16] but has not been proved yet within this family.”
Hence, in a nutshell, we target QMT and SPA in such a manner we could produce original research to understand time dependent effects on bioactive terpenes production in Maytenus ilicifolia.
2 - Reference is needed to support the selection of chemometric method, such as Hierarchical Clustering Analysis (HCA), for group classification based on the concentration of individual component or a few compounds.
Outcome in this present version (Paragraph starting lines 283 and 308)
“HCA is an agglomerative clustering analysis in which the measurement of distance between pairs creates a hierarchy between samples. In this procedure, most similar points are grouped together, creating a dendogram plot that clusters samples based on their similarity.
For HCA experiments, samples I-X were clustered, in triplicate, based on the QMTs and SPA production measured by the semi-quantitative HPLC-DAD and 1H NMR data. For this analysis, the similarity was calculated using Euclidean distance between the samples total integrated area. Ward was chosen as the optimum linkage method due to the fewer susceptibility to outlier effects. All data were statistically analyzed using algorithms created in MATLAB software R2017a® (MathWorks, USA) at a 95% confidence level.”
“Hierarchical clustering analysis (HCA) has been consistently efficient in the analysis of natural and multivariate chemical data [27–29]. For M. ilicifolia, this unsupervised chemometric analysis grouped the samples that shared a similar metabolic production using both HPLC-DAD and 1HNMR data. Some important advantages of this clustering analysis are the possibility to apply high-dimensional data typical from ‘omics' studies, the possibility of both quantitative and qualitative evaluation and the detection of pleiotropic effects that might influence the chemical variation.”
3 - Figure 5 does not have any scientific value:
Correction done accordingly. Figure 5 was transferred to the Supplementary Materials.
Updated References:
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2. Crutzen, P.J.; Stoermer, E.F. The “Anthropocene.” Glob. Chang. Newsl. 2000, 41, 17–18.
3. Pavarini, D.P.; Da Silva, D.B.; Carollo, C.A.; Portella, A.P.F.; Latansio-Aidar, S.R.; Cavalin, P.O.; Oliveira, V.C.; Rosado, B.H.P.; Aidar, M.P.M.; Bolzani, V.S.; et al. Application of MALDI-MS analysis of Rainforest chemodiversity: A keystone for biodiversity conservation and sustainable use. J. Mass Spectrom. 2012, 47, 1482–1485.
4. Joly, C.A.; Metzger, J.P.; Tabarelli, M. Experiences from the Brazilian Atlantic Forest: ecological findings and conservation initiatives. New Phytol. 2014, 204, 459–473.
5. Joly, C.A.; Rodrigues, R.R.; Metzger, J.P.; Haddad, C.F.B.; Verdade, L.M.; Oliveira, M.C.; Bolzani, V.S. Biodiversity conservation research, training, and policy in São Paulo. Science (80). 2010, 328, 1358–1359.
6. Carnevale Neto, F.; Pilon, A.C.; Selegato, D.M.; Freire, R.T.; Gu, H.; Raftery, D.; Lopes, N.P.; Castro-Gamboa, I. Dereplication of natural products using GC-TOF mass spectrometry: Improved metabolite identification by spectral deconvolution ratio analysis. Front. Mol. Biosci. 2016, 3.
7. FAPESP. Knowledge and sustainable use of Brazilian biodiversity: Biota-FAPESP Program/The State of São Paulo Research Foundation. São Paulo; Prol Editora Gráfica Ltda., 2008.
8. Pilon, A.C.; Valli, M.; Dametto, A.C.; Pinto, M.E.F.; Freire, R.T.; Castro-Gamboa, I.; Andricopulo, A.D.; Bolzani, V.S. NuBBEDB: An updated database to uncover chemical and biological information from Brazilian biodiversity. Sci. Rep. 2017, 7, 1–12.
9. Valli, M.; Dos Santos, R.N.; Figueira, L.D.; Nakajima, C.H.; Castro-Gamboa, I.; Andricopulo, A.D.; Bolzani, V.S. Development of a natural products database from the biodiversity of Brazil. J. Nat. Prod. 2013, 76, 439–444.
10. Mayrhofer, S.; Teuber, M.; Zimmer, I.; Louis, S.; Fischbach, R.J. Diurnal and Seasonal Variation of Isoprene Biosynthesis - Related Genes in Grey Poplar Leaves. Plant Physiol 2005, 139, 474–484.
11. Gershenzon, J.; Dudareva, N. The function of terpene natural products in the natural world. Nat Chem Bio 2007, 3, 408–414.
12. Inderjit, W.D.A.; Karban, R.; Callaway, R.M. The ecosystem and evolutionary contexts of allelopathy. Tr. Ecol Evol 2011, 26, 655–662.
13. Martin, D.M.; Aubourg, S.; Schouwey, M.B.; Daviet, L.; Schalk, M.; Toub, O.; Steven, T.L.; Bohlmann, J. Functional annotation, genome organization and phylogeny of the grapevine (Vitis vinifera) terpene synthase gene family based on genome assembly, FLcDNA cloning, and enzyme assays. BMC Plant Biol. 2010, 10, 226–248.
14. Nieuwenhuizen, N.J.; Green, S.A.; Chen, X.; Bailleul, E.J.D.; Matich, A.J.; Wang, M.Y.; Atkinson, R.G. Functional genomics reveals that a compact terpene synthase gene family can account for terpene volatile production in apple. Plant Physiol 2013, 161, 787–804.
15. Bueno, P.C.P.; Pereira, F.M. V; Torres, R.B.; Cavalheiro, A.J. Development of a comprehensive method for analyzing clerodane-type diterpenes and phenolic compounds from Casearia sylvestris Swartz (Salicaceae) based on ultra high performance liquid chromatography combined with chemometric tools. J. Sep. Sci. 2015, 38, 1–21.
16. Pavarini, D.P.; Pavarini, S.P.; Niehues, M.; Lopes, N.P. Exogenous influences on plant secondary metabolite levels. Anim Feed Sci Technol 2012, 176, 5–16.
17. Muntendam, R.; Happyana, N.; Erkelens, C.; Bruining, F.; O, K. Time dependent metabolomics and transcriptional analysis of cannabinoid biosynthesis in Cannabis sativa var. Bedrobinol and Bediol grown under standardized condition and with genetic homogeneity. Online Int J Med Plant Res 2012, 1, 31–40.
18. Happyana, N.; Kayser, O. Monitoring Metabolite Profiles of Cannabis sativa L. Trichomes during Flowering Period Using 1H NMR-Based Metabolomics and Real-Time PCR. Planta Med 2016, 82, 1217–23.
19. Alvarenga, N.; Ferro, E.A. Bioactive Triterpenes and Related Compounds from Celastraceae. Stud. Nat. Prod. Chem. 2006, 33, 239–307.
20. Souza-Moreira, T.M.; Alves, T.B.; Pinheiro, K.A.; Felippe, L.G.; De Lima, G.M.A.; Watanabe, T.F.; Barbosa, C.C.; Santos, V.A.F.F.M.; Lopes, N.P.; Valentini, S.R.; et al. Friedelin Synthase from Maytenus ilicifolia: Leucine 482 Plays an Essential Role in the Production of the Most Rearranged Pentacyclic Triterpene. Sci. Rep. 2016, 6, 1–13.
21. Carvalho, P.R.F.; Silva, D.H.S.; Bolzani, V.S.; Furlan, M. Antioxidant quinonemethide triterpenes from Salacia campestris. Chem Biodivers 2005, 2, 367–372.
22. Jeller, A.H.; Silva, D.H.S.; Lião, L.M.; Bolzani, V.S.; Furlan, M. Antioxidant phenolic and quinonemethide triterpenes from Cheiloclinium cognatum. Phytochem 2004, 65, 1977–1982.
23. dos Santos, V.A.F.F.M.; Santos, D.P.; Castro-Gamboa, I.; Zanoni, M.V.B.; Furlan, M. Evaluation of Antioxidant Capacity and Synergistic Associations of Quinonemethide Triterpenes and Phenolic Substances from Maytenus ilicifolia (Celastraceae). Molecules 2010, 15, 6956–6973.
24. Costa, P.M.; Ferreira, P.M.; Bolzani, V.S.; Furlan, M.; dos Santos, V.A.F.F.M.; Corsino, J.; de Moraes, M.O.; Costa-Lotufo, L. V; Montenegro, R.C.; Pessoa, C. Antiproliferative activity of pristimerin isolated from Maytenus ilicifolia (Celastraceae) in human HL-60 cells. Toxicol Vitr. 2008, 22, 854–863.
25. Paz, T.A.; dos Santos, V.A.F.F.M.; Inácio, M.C.; Pina, E.S.; Pereira, M.A.S.; Furlan, M. Production of the Quinone-Methide Triterpene Maytenin by In Vitro Adventitious Roots of Peritassa campestris (Cambess.) A.C.Sm. (Celastraceae) and Rapid Detection and Identification by APCI-IT-MS/MS. Biomed Res Int 2013, 2013, 485837.
26. Queiroga, C.L.; Silva, G.F.; Dias, P.C.; Possenti, A.; Carvalho, J.E. Evaluation of the antiulcerogenic activity of friedelan-3β-ol and friedelin isolated from Maytenus ilicifolia (Celastraceae). J Ethnopharmacol 2000, 72, 465–468.
27. Souza-Formigoni, M.L.; Oliveira, M.G.; Monteiro, M.G.; da Silveira-Filho, N.G.; Braz, S.; Carlini, E.A. Antiulcerogenic effects of two Maytenus species in laboratory animals. J Ethnopharmacol 1991, 34, 21–27.
28. Liu, J.K.; Jia, Z.J.; Wu, D.G.; Zhou, J.; Wang, Q.G. Insect antifeeding agents: sesquiterpene alkaloids from Celastrus angulatus. Phytochemistry 1990, 29, 2503−2506.
29. Núñez, M.J.; Guadaño, A.; Jimenez, I.A.; Ravelo, A.G.; Gonzalez-Coloma, A.; Bazzocchi, I.L.́ Insecticidal Sesquiterpene Pyridine Alkaloids from Maytenus chiapensis. J. Nat. Prod. 2004, 67, 14–18.
30. Whitson, E.L.; Mala, S.M.V.D.; Veltri, C.A.; Bugni, T.S.; Silva, E.D.; Ireland, C.M. Oppositines A and B, Sesquiterpene Pyridine Alkaloids from a Sri Lankan Pleurostylia opposita. J. Nat. Prod. 2006, 69, 1833−1835.
31. dos Santos, V.A.F.F.M.; Regasini, L.O.; Nogueira, C.R.; Passerini, G.D.; Martinez, I.; Bolzani, V.S.; Graminha, M.A.S.; Cicarelli, R.M.B.; Furlan, M. Antiprotozoal Sesquiterpene Pyridine Alkaloids from Maytenus ilicifolia. J Nat Prod 2012, 75, 991–995.
32. Jardim, A.C.G.; Igloi, Z.; Shimizu, J.F.; Santos, V.A.; Felippe, L.G.; Mazzeu, B.F.; Amako, Y.; Furlan, M.; Harris, M.; Rahal, P. Natural compounds isolated from Brazilian plants are potent inhibitors of hepatitis C virus replication in vitro. Antivir. Res 2014, 115, 39–47.
33. Gonzalez, A.G.; Tincusi, B.M.; Bazzocchi, I.L.; Tokuda, H.; Nishino, H.; Konoshima, T.; Jimenez, I.A.; Ravelo, A.G. Anti-tumor promoting effects of sesquiterpenes from Maytenus cuzcoina (Celastraceae). Bioorg. Med. Chem 2000, 8, 1773−1778.
34. Jorge, R.M.; Leite, J.P. V; Oliveira, A.B.; Tagliati, C.A. Evaluation of antinociceptive, anti-inflammatory and antiulcerogenic activities of Maytenus ilicifolia. J. Ethnopharmacol. 2004, 94, 93–100.
35. Field, B.; Osbourn, A.E. Metabolic diversification - independent assembly of operon-like gene clusters in different plants. Sci 2008, 320, 543–547.
36. Corsino, J.; Bolzani, V.S.; Pereira, A.M.S.; França, S.C.; Furlan, M. Bioactive sesquiterpene pyridine alkaloids from Maytenus aquifolium. Phytochemistry 1998, 48, 137–140.
37. dos Santos, V.A.F.F.M. Metabolomic, biological and proteomic aspects of Maytenus ilicifolia and Salacia campestris (Celastraceae), 2010.
38. Kim, Y.S.; Cho, J.H.; Park, S.; Han, J.Y.; Back, K.; Choi, Y.E. Gene regulation patterns in triterpene biosynthetic pathway driven by overexpression of squalene synthase and methyl jasmonate elicitation in Bupleurum falcatum. Planta 2011, 233, 343–355.
39. Achnine, L.; Huhman, D. V; Farag, M.A.; Sumner, L.W.; Blount, J.W.; Dixon, R.A. Genomics-based selection and functional characterization of triterpene glycosyltransferases from the model legume Medicago truncatula. Plant J 41AD, 875–887.
40. Estevam, C.S.; Cavalcanti, A.M.; Cambui, E.V.F.; Araújo Neto, V.; Leopoldo, P.T.G.; Fernandes, R.P.M.; Araujo, B.S.; Porfírio, Z.; Sant’Ana, A.E.G. Phytochemistry and microbiological assay of the bark extracts of Maytenus rigida Mart. (Celastraceae). Braz J Farm. 2009, 19, 299–303.
41. Monks, T.J.; Jones, D.C. The metabolism and toxicity of quinones, quinonimines, quinone methides, and quinone-thioethers. Curr Drug Metab 2002, 3, 425–438.
42. Depke, T.; Franke, R.; Brönstrup, M. Clustering of MS2 spectra using unsupervised methods to aid the identification of secondary metabolites from Pseudomonas aeruginosa. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 2017, 1071, 19–28.
43. Selegato, D.M.; Freire, R.T.; Tannús, A.; Castro-Gamboa, I. New Dereplication Method Applied to NMR-Based Metabolomics on Different. J. Braz. Chem. Soc. 2016, 0027, 1–11.
44. Shawky, E. Multivariate analyses of NP-TLC chromatographic retention data for grouping of structurally-related plant secondary metabolites. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 2016, 1029–1030, 10–15.
45. Nosengo, N. New tricks for old drugs. Nat 2016, 534, 314–316.
46. Peng, B.; Xu, L.; Cao, F.; Wei, T.; Yang, C.; Uzan, G.; Zhang, D. HSP90 inhibitor, celastrol, arrests human monocytic leukemia cell U937 at G0/G1 in thiol-containing agents reversible way. Mol Cancer 2010, 9, 79–92.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
The manuscript present several improvement and I appreciate the effort of authors in taking care of referee's comments.
I still believe that for larger audience as well as to have more clear manuscript and more informative
-aim should be clearly expressed.
-key findings and results discussion explaining to readers the usefulness of the obtained data can be addedd.
The sentence in the lines 271-279 that should summarise all the work finidings are in my opinion not sufficiently clear and informative.
I suggest a further revision
Author Response
To the suggestions of reviewer number 1.
We appreciate the suggestions that might improve the quality of the communication.
Please find bellow all the points of clarification that our revision addressed.
Regarding the suggestions made available on the website decision text:
1- Suggestion that “aim should be clearly expressed”
We changed the paragraph that ends the Introduction. The last version is listed bellow:
“In this topic, we aim to expose the relevance of multi-trophic interactions and
ontogenesis in determining richness of bioactive classes of compounds among ex situ
collections of Maytenus ilicifolia Mart. ex Reissek (Celastraceae). This specie is generally known
in South America as “Espinheira-santa” and is the most used Maytenus species in tradition
medicine, exhibiting high antiulcerogenic effects of its leaves infusion [34]. The present
phytochemistry study aimed to determine whether QMT and SPA levels are different among
clones and individuals of M. ilicifolia kept alive in ex situ collection. The relative concentration
of QMT maytenin (1) and pristimerin (2), and the SPA aquifoliunin E1 (3) [Figure 1] were
tracked in raw extracts by HPLC-DAD and 1 HNMR,. Moreover, Hierarchical Clustering
Analysis (HCA) was used to gather groups of individuals according their ability to
accumulate these compounds. Summarily, this communication relies on straightforward
analytical tools to determine how different can the secondary metabolites profile patterns be
within common environmental conditions acting upon individuals.”
2- suggestions that “key findings and results discussion explaining to readers the
usefulness of the obtained data can be addedd.” and “The sentence in the lines 271-279 that
should summarise all the work finidings are in my opinion not sufficiently clear and
informative.”
These were simultaneously addressed by writing a new Paragraph in the Conclusions,
including a couple of new references.
“The dereplication of bioactive metabolites in M. ilicifolia’s raw extracts was successful
due to combination of a few practical jobs: (1) rigorous spectroscopy and chromatographic
data survey of compounds previously investigated in taxonomic related species and (2) the
proper use of analytical tools and channels of detection to narrow information down to the
target group of compounds. One of the major efforts of natural product society around the
globe nowadays is to enable fast identification of targeted compounds in complex mixtures
[47]. One usefulness of this present paper is to became a proof of concept that this two practical
jobs can be adopted for rapid selection of botanical vouchers displaying high contents of active terpenes. This case of success stands alongside others such as using of TOCSY for selection of
polyketide producer’s bacterial strains [48].”
Author Response File: Author Response.pdf
Reviewer 2 Report
Now, when I had an opportunity to read the whole manuscript (including supplementary material) I can write that In my opinion this article shouldn’t be published in Molecules. I believe that for example Chemistry and Biodiversity would be a better forum for its publication. It is not messy anymore, but I do not see any scientific novelty. Statement that the plant clones can produce different quantities of secondary metabolites is not new. NMR data of described compounds are well known (see below). If the manuscript is enriched for example in genetic research (which, maybe, would show some differences) it would be publishable. Moreover, the authors should check whether the plants were healthy (fungal diseases, pests)?, whether the conditions of growing were the same (access to the sun light, irrigation etc.)? Please find the explanation of observed phenomenon.
Hera are my comments to the current content of the manuscript.
The numbering of the figures and tables should be changed for clarity. Now reader have to focus in which table 1 are data – in the manuscript or in the supplementary material. Figure 1 in supplementary material should be changed to figure 5, table 1 in supplementary material should be changed to table 2 etc.
Where is any evidence confirming different than previously described [Endophytic Bacillus megaterium and exogenous stimuli affect the quinonemethide triterpenes production in adventitious roots of Peritassa campestris (Celastraceae) Plant Cell, Tissue and Organ Culture (2017), 131(1), 15-26] configuration at carbon 13 and 14 of Maytenin?
Due to earlier well description of NMR data of compounds 1-3 I believe that there is no any reason to put them in supplementary material, especially that these data are incomplete – lack of some chemical shifts, for example for carbon 21, and many others. Instead of publishing these data the authors should cite proper references and write that the obtained NMR data were consistent with previously described.
Tingenone (Maytenin?) and pristimerin NMR data are described, for example, in SARS-CoV 3CLpro inhibitory effects of quinone-methide triterpenes from Tripterygium regelii, by Ryu YB, Park SJ, Kim YM, Lee JY, Seo WD, Chang JS, Park KH, Rho MC, Lee WS, published in Bioorganic & Medicinal Chemistry Letters Volume 20, Issue 6, 15 March 2010, Pages 1873-1876.
Aquifoline E1 NMR data are described in Bioactive sesquiterpene pyridine alkaloids from Maytenus aquifolium, Phytochemistry, Volume 48, Issue 1, May 1998, Pages 137-140 by J Corsino, V da Silva Bolzani, AM S. Pereira, S C França, M Furlan – in references position 36.
Author Response
To the suggestions of reviewer number 2.
We appreciate the suggestions that might improve the quality of the communication.
Please find bellow all the points of clarification that our revision addressed.
Regarding the suggestions made available on the website decision text
We understand that our present contribution might have caused an impression that
lacks novelty due to the fact we are not dealing with Drug Discovery. Indeed, we are not
bringing new compounds description. However, we are bringing, what is up to our
knowledge, the first time using of cryogenic heteronuclear NMR TOCSY as an analytical tool
to rapidly screen botanical vouchers and identify known valuable compounds among these
vouchers. It is clearly stated in the literature, and now is also cited in our communication
[references 47-48], that such an advancement is highly sought after by Natural Products
Researchers. Our paper can, once published, be a proof of concept for modern NMR
techniques use as a dereplication technique of choice.
About the point raised as “Moreover, the authors should check whether the plants
were healthy (fungal diseases, pests)?, whether the conditions of growing were the same
(access to the sun light, irrigation etc.)? Please find the explanation of observed phenomenon.”
We have monitored the plants since their transplants to the green house, so the health
status is already checked, as stated in the paragraph bellow from the item 3.1. Sampling
“Individuals were monitored since their transplant from original habitat, during late
90's up to nowadays. Hence, sexual reproduction, through bird’s dispersion of seeds, and
asexual reproduction from branches and root shootings, were determined through simple
observation of each piece of the collection. All the plants of the collection maintained in the
botanical garden were sampled in the present study.”
About the point raised in the comment “The numbering of the figures and tables
should be changed for clarity. Now reader have to focus in which table 1 are data – in the
manuscript or in the supplementary material. Figure 1 in supplementary material should be
changed to figure 5, table 1 in supplementary material should be changed to table 2 etc.”
We have strictly followed the instructions and the outcome is the exact numbering
suggested by the reviewer. About the configuration on QMT carbons 13 and 14, we have made a mistake on the
drawing of the structures. Now, thankful to this a well-spotted review we managed to correct
it.
About the NMR data made available in the Supplementary Material, we understand
that the data we used from literature is proper for comparisons of Chemical shifts against our
experimental data. The reference is from Phytochemistry, a gold standard journal in the field
of Natural Products. Furthermore, not every nuclei were listed due to the fact that our paper
prioritize “the proper use of analytical tools and channels of detection to narrow information
down to the target group of compounds” as already discussed in the Conclusions.
Author Response File: Author Response.pdf
Reviewer 3 Report
The authors have made suitable revisions, and the manuscript is now can be published.
Author Response
Thank you for your attention and time.
Author Response File: Author Response.pdf
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
The manuscript molecules-406741 Ecological Insights to track Cytotoxic Compounds among Maytenus iIlicifolia living individuals and clones of ex situ collection
The manuscript suffer of lack of clarity in the aim of the work.
The introduction is not focussed but probably due to the non clear exposition of meaning and significance of the obtained data. Literature search and inclusion of more recent paper on similar theme should be addedd. The authors may include further references related to the importance of biodiviersity also for medicinal plants.
Aim of the work need to be clearly stated. Further weakneess are related to general organization of the work. Why authors selected this particoular specie?
How authors selected the number and type of sampling of the plants?
The manuscript also in my opinion is not properly fitting journal's aims and scopes.
I think that in the present form is not suitable for publication.
Other suggestions are included in the pdf as notes
Comments for author File: Comments.pdf
Reviewer 2 Report
In present form manuscript is messy, unreadable and impossible to evaluate. Figures 2 and 3 were not included to the manuscript. Figures 3 and 4 are not described. Thus in my opinion manuscript should be rejected.
