Isolation and Identification of Two Potent Phytotoxic Substances from Afzelia xylocarpa for Controlling Weeds
Round 1
Reviewer 1 Report
I have read this research with great interest and I am happy that the methodology has been taken care of very well. The manuscript is very clear and the motivations, goals and objectives to be achieved are well defined. The application of the research results can have great relevance in agriculture and towards environmental sustainability. The literature search was carried out very carefully and the basic structure is therefore robust. I have no negative notes to add and the manocrit can be published as is.
Author Response
Please see the attachment.
Author Response File: 
Author Response.docx
Reviewer 2 Report
I found the manuscript interesting. But I feel there are some alterations need prior to publication:
- Some improvement on English is necessary. For example, what to the authors mean (lane 96) with: “An aliquot concentration (0.6mL)….”
- I suggest the authors to rephrase the objective written in the introduction: the manuscript describes the phytotoxic activity of A. xylocarpa leaves, and not of A. xylocarpa.
- Please add units to table 1 (shoot length)
- Please add the controls to figure 1 and 2.
- Xx scale on figures 4 and 5 is not linear as it should be.
Author Response
Please see the attachment
Author Response File: Author Response.docx
Reviewer 3 Report
In this work, the authors test the effect of an extract and two molecules (vanillic and trans-ferulic acid) isolated from Afzelia xylocarpa leaf on model plants for a possible application as a green herbicide.
Beyond the formal improvements needed along with the manuscript, in the light of the field application of these substances remain a basic concern: how to justify the use of Afzelia xylocarpa extracts to respect/protect Nature when Afzelia xylocarpa is itself an endangered species (https://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.976.2993&rep=rep1&type=pdf; https://doi.org/10.1111/j.1755-0998.2008.02334.x; https://www.publish.csiro.au/pc/PC100101). In addition, other plant species can produce high quantities of the two phenolic compounds here isolated and tested (e.g. Angelica sinensis), as well as it is already known as the inhibitory effect produced by phenolics (just before the eighties). So, is of utmost importance that the authors explain their study design, the importance of this research, and why it is necessary to use this extract and not another one or perform further studies as stated in conclusions.
English revision is needed
Below additional notes.
Keywords help a searcher choose the most relevant research article to read.. Keyword search results are directly related to the quality of the keywords and this can significantly enhance the frequency of citations it receives, increasing your credibility in your field. Short single/general word should be avoided because your paper would be lost in a sea of papers falling in different study field, moreover use words already present in the title it is a pity…because you are losing an opportunity to increase your visibility
The majority of keywords chosen are already in the title, then, I suggest you change them.
The introduction misses the weeds problem, suggested by the title but not mentioned in this section. I mean, weeds are the reason for the use of controlling chemicals and the search for green solutions..
In general, the section of Material and methods should be organized/ shortened to avoid repeats in descriptions..
L19 two substances:
L43-44 the sentence should be reshaped in the light of the article cited: They possess higher oxygen- and nitrogen-rich molecules with relatively few so called ‘heavy atoms’, a halogen substitute, and are characterized by the absence of ‘unnatural’ rings. As a matter of fact, allelopathic is not a synonym of phytotoxic and all of these chemical features enable them (allelopathic substances) to be more easily degraded than the synthesis herbicides. As it is the sentence does not reflect the meaning of the author cited.
L50 target plant – the weeds you missed in this introduction
L54 Fabaceae, the third largest family of flowering plant in terms of species richness…some substances they produce are not only phytotoxic but should be toxic (https://core.ac.uk/download/pdf/82490389.pdf) or have an adverse effect on human health as vicine /convicine produced by fava beans
L65-67 Among the isolated molecules there are also phenolics (e.g. vanillic and ferulic acid) as reported by Phuong [you cited at 33]; some of them have been already reported as germination inhibitors since 1978 (https://doi.org/10.1023/A:1006269716762; https://doi.org/10.1007/BF01014693; https://doi.org/10.1007/BF00989499)
L68-69 “..may exhibit phytotoxic activity with phytotoxic substances, which could be used in biological weed management” this sentence should be simplified to avoid repetitions…cancel with phytotoxic substances
L70-71 the phytotoxic potential of A. xylocarpa has not been investigated or has not yet investigated.
Please reduce the use of the word phytotoxic because you used this word 7 times in a few lines (L67-71). I mean, simplify the aim of this work avoiding redundant repetitions.
L73 and their activity tested on a few model plants at laboratory conditions.
L76 Plant material and receptor plant species. The receptor concept belongs to the pharmacological and biochemical field with studies performed at molecular/cellular scale. In this case, the plants are used as models (some because characterized by high responsiveness and others to represent the target species to be controlled in crops). Then, “receptor plant species” is inappropriate even if used by others. Please change the chapter header to Plant material and target plant species/ or model plants. In the light of the previous clarification, it is necessary to amend the term “receptor” along with the whole manuscript.
Section 2.3
In general, this section should be simplified avoiding repetitions (e.g. filter paper)
L94 to asses not determine
L94-95 A.xylocarpa is mentioned twice in the same sentence please modify the sentence
L95 change D.W. to dry weight (DW) all acronyms should be reported for the first time with their meaning into parenthesis. Please report along with the text DW and not D.W:
L97 containing a filter paper sheet;
L97-98 After the solvent evaporation, the filter paper sheets were soaked/moistened with…
L101 please explain why you used germinated seeds
L102 change “All treatments” to “All plates”
L104 six replicates
L105 results were expressed as percentage ratio using the following formula:
L106 not treatment length but “treated length”
L107 identification of the active compounds. You cannot say phytotoxic because you have not yet proved this effect on plants.
L109 in vacuo is Latin, so it should be in italics
L111-112 did you tested the effect of the different fractions on L.sativum only?
In order to identify the most active fraction, the cress assay was used (reference)…” as reported above” is not clear because you give mention four species to test the extract and now only one.
L114-115 Both the aqueous and ethyl acetate fractions were examined for phytotoxic activity at concentrations of 1, 3, 10, and 30 mg D.W. equivalent A. xylocarpa extract/mL This sentence is already present at lines 94-95
L126 The most active fraction (F2) ….
L130 chemical structures were assessed.
L132 as I told before “phytotoxic” cannot be used until all results are shown…so until that moment “biological activity” fits better. Please correct this item along with the manuscript.
L146 3mM in which volume?
Table 2 the letters of statistical analysis should be reported as superscript character
Figure 3. the vanillic acid and trans-ferulic acid molecules are not necessary and even a duplicate of those reportesìd in fig 4 and fig 5.
Figure 4 E has low readability, I suggest discarding the molecular structure (the substance tested is reported in the caption) to allow figure enlargement; the same should be done for Figure 5.
L272 xylocarpa, an a was lost, the same at L278
L273 remove [] as at L279
L274 & L280 The bars on each experiment show mean ± SE with three replications change to “ reported values are expressed as mean ± SE of three replicates”
Figure 6 to identify the treatment on the x-ax use letters e.g VA, tFA, VA+tFA; increase the size of 6C
L314-315 This study evidenced the phytotoxic effect of A. xylocarpa extract on the model species, and identify in vanillic and trans ferulic acid as the major bioactive molecules
Author Response
Please see the attachment
Author Response File: Author Response.docx
Round 2
Reviewer 3 Report
Dear Authors,
below I reported the basic concern I have, your response, and the pasted text from the Introduction. After that, you can find my comments.
#Comment 1: Beyond the formal improvements needed along with the manuscript, in the light of the field application of these substances remain a basic concern: how to justify the use of Afzelia xylocarpa extracts to respect/protect Nature when Afzelia xylocarpa is itself an endangered species (https://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.976.2993&rep=rep1&type=pdf;https://doi.org/10.1111/j.1755-0998.2008.02334.x; https://www.publish.csiro.au/pc/PC100101).In addition, other plant species can produce high quantities of the two phenolic compounds here isolated and tested (e.g. Angelica sinensis), as well as it is already known as the inhibitory effect produced by phenolics (just before the eighties). So, is of utmost importance that the authors explain their study design, the importance of this research, and why it is necessary to use this extract and not another one or perform further studies as stated in conclusions.
Response: We are grateful for your valuable suggestions. We accepted and agreed with the reviewer's comments. Therefore, we have explained the importance of the results of this research that could be useful in further studies. Those sentences were inserted in the introduction section (L77-82).
L71-82 Based on its value as a traditional medicine and its secondary metabolites, it is possible that this plant may also contain bioactive substances which could be used in biological weed management. Although some of its biological properties have been reported, the suppressive potential of A. xylocarpa leaves has not been investigated. Thus, this study was conducted to determine the phytotoxic activity of A. xylocarpa leaves and to isolate the active substances. These substances were identified, and their activity tested on four model plants at laboratory conditions. It was envisaged that this study would provide to explain the effect of active substances isolated from A. xylocarpa leaves. Moreover, it may indicate the possibility of their future use in applications in terms of weed control. This information could be useful for assessing the interrelations between active substances and action sites in the target plants. That may lead to suitably apply in the fieldwork in the future.
Despite the many tanks profuse, none of the concern raised was neither answered nor mentioned.
Perhaps the authors underestimated its importance, or perhaps I was not clear in explaining the basic concern affecting this research, so I’ll repeat point by point:
It is well known the inhibitory effect produced by phenolics since before the eighties (Einhellig, F.A., Rasmussen, J.A. Synergistic inhibitory effects of vanillic and p-hydroxybenzoic acids on radish and grain sorghum. J Chem Ecol 4, 425–436 (1978). https://doi.org/10.1007/BF00989499; Chandramohan, D., Purushothaman, D. & Kothandaraman, R. Soil phenolics and plant growth inhibition. Plant Soil 39, 303–308 (1973). https://doi.org/10.1007/BF00014797).
It is well known that Afzelia xylocarpa produces phenolics (Phuong, D.L., Thuy, N.T., Long, P.Q. et al. Composition of Fatty Acids, Tocopherols, Sterols, Total Phenolics, and Antioxidant Activity of Seed Oils of Afzelia xylocarpa and Cassia fistula. Chem Nat Compd 55, 242–246 (2019). https://doi.org/10.1007/s10600-019-02659-x). The authors refer to this work mentioning several compounds but missed to cite phenolics listed in table 4 (e.g. vanillic, p-coumaric, and ferulic acid).
The production of phenolics compounds is widely distributed (El-Basyouni, S. Z., Chen, D., Ibrahim, R. K., Neish, A. C., & Towers, G. H. N. (1964). The biosynthesis of hydroxybenzoic acids in higher plants. Phytochemistry, 3(4), 485-492; Levin, D. A. (1971). Plant phenolics: an ecological perspective. The American Naturalist, 105(942), 157-181; Lattanzio, V. (2013). Phenolic Compounds: Introduction 50. Nat. Prod, 1543-1580.)
Afzelia xylocarpa is an endangered species, it becomes for the overexploitation as a timber source and medicinal plant, and should be protected (https://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.976.2993&rep=rep1&type=pdf;https://doi.org/10.1111/j.1755-0998.2008.02334.x; https://www.publish.csiro.au/pc/PC100101; So, T., Theilade, I., & Dell, B. (2010). Conservation and utilization of threatened hardwood species through reforestation? an example of Afzelia xylocarpa (Kruz.) Craib and Dalbergia cochinchinensis Pierre in Cambodia. Pacific Conservation Biology, 16(2), 101-116; Nghia, N. H. (2001, February). Conservation of forest genetic resources in Vietnam with reference to endangered tree species. In Proceedings of the south east Asian moving workshop on conservation, management and utilization of forest genetic resources (Vol. 25).)
Other plant species can produce high quantities of the two phenolic compounds here isolated and tested: Coriander sativum vanillic acid up to 44.09 mg/100 g and ferulic acid up to 23.921 mg/100g; Angelica sinensis total phenolics 30.20 mg/g (file:///C:/Users/Utente/Downloads/foods-09-01221.pdf; https://link.springer.com/content/pdf/10.2478/s11696-013-0485-7.pdf)
In the light of these assumptions here my questions:
- Where is the novelty in isolating phenolics from Afzelia xylocarpa?
- Where is the novelty in proving the inhibitory effect on plants of vanillic and trans-ferulic acid?
- how much is the yield mg/100g of vanillic acid and trans ferulic acid you obtained to justify another large-scale exploitation of an endangered species?
All these points should be argued in the introduction to best explain to readers the value of this research and highlight the scopes.
Regards,
Author Response
Please see the attachment
Author Response File: Author Response.docx
