Chemical and Functional Characterization of Extracts from Leaves and Twigs of Acacia dealbata
, Maria Paula Duarte 1, Elisabete Muchagato Maurício 2, João Brinco 3, José Carlos Quintela 4, Marco Gomes da Silva 5 and Margarida Gonçalves 1,6,*Round 1
Reviewer 1 Report
This paper titled “Bioactive Extracts from Leaves and Twigs of the Portuguese Invasive Acacia dealbata” is interesting. This manuscript could be considered for publication in Processes after Major revising.
My comments are as follow:
- Check the abbreviations, the text format of full text.
- English should be improved.
- The authors should analysis the typical bioactive compounds of the extracts.
- For anti-bacterial effect, the Figure should be supported.
- The discussion section should be improved.
- Authors should further investigate the bioactive compounds and the mechanism of the effect. otherwise, this research is too simple.
Author Response
Comment 1: Check the abbreviations, the text format of full text.
Answer: The abbreviations were reviewed in the manuscript text to ensure homogeneity.
Comment 2: English should be improved.
Answer: The manuscript was reviewed to improve writing style and clarity.
Comment 3: The authors should analysis the typical bioactive compounds of the extracts.
Answer: The non-polar fraction of the extracts was analysed by GC-MS and the contribution of the detected analytes for antioxidant or anti-microabial activities was discussed; analysis of the phenolic fraction by LC-MS-MS will be addressed in another publication.
Comment 4: For anti-bacterial effect, the Figure should be supported.
Answer: The authors did not completely understand the meaning of this comment; figures with the inhibition halos were not included because those images did not add useful information to the discussion..
Comment 5: The discussion section should be improved.
Answer: The Discussion Section was reviewed and improved.
Comment 6: Authors should further investigate the bioactive compounds and the mechanism of the effect. otherwise, this research is too simple.
Answer: Some investigation of the nature of bioactive components present was done by characterizing the non-polar fraction of the extracts that may contain non-phenolic compounds that may influence the antioxidant and antimicrobial activity of the extracts. The characterization of the phenolic component will be addressed in another paper that presents data on the phenolic components of extracts obtained from Acacia leaves, twigs, flowers, bark and wood.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
This manuscript reported Acacia dealbata leaves and twigs extract were used for biological activity. However, this manuscript needs major revisions and the following comments will help to increase the quality of the manuscript before publication.
· Manuscript Title should be revise.
· Line No: 17, “Fresh or air-dried biomass were subject to maceration at room temperature or hot extraction at 60 ºC using 70% aqueous ace- 18 tone, ethanol, or methanol.” write clearly
· Abstract should be more specific based on the findings with recommendation.
· Species or genus names should be followed by scientific terminology in the entire text.
· Line No: 86, why author choose particular temperature range for extraction? Clearly state the reason in the text.
· In the "Introduction" section, general description on the importance of manuscript topic is poor. Therefore, the importance of this work cannot be well recognized from general readers. In order to fix this problem, addition of description on recent development in the field of research topic with citing recent comprehensive papers would be important.
· How is the work different or better than those reported earlier? Author needs to highlight this in the introduction and discussion part.
· Why author not used ethyl acetate for extract?
· Line No: 100-104, why you chose ten different and totally how many sample were collected from each trees? Include in the text.
· Author carefully check the scientific terminology in entire text.
· Why you choose particular bacteria’s and yeast for antimicrobial activity. Why author not checked fungal pathogens?
· Why author choose particular Antioxidant assay? Author must include the reason in the text with appropriate reference citation https://doi.org/10.1016/j.jksus.2022.102029; https://doi.org/10.1155/2022/8575026;
· Author should be include pathogens virulence factors https://doi.org/10.1155/2022/7234586
· Extensive editing of English language and style required
· Need to check consistency in reference the in text
· Author should acknowledge the recommendation of present study in the conclusion section.
Author Response
The authors want to thank for the revision of the manuscript and acknowledge the contribution of the reviewer comments to improve the quality of the manuscript.
Comment 1: Manuscript Title should be revised.
Answer: The manuscript title was revised. The title proposed was: “Chemical and Functional Characterization of Extracts from Leaves and Twigs of Acacia dealbata.”
Comment 2: Line No: 17, “Fresh or air-dried biomass were subject to maceration at room temperature or hot extraction at 60 ºC using 70% aqueous acetone, ethanol, or methanol.” write clearly.
Answer: Line nº 17 was rephrased as follows: “Fresh and air-dried biomass were extracted by maceration at room temperature and by hot extraction at 60°C using aqueous solutions of acetone, ethanol, and methanol”.
Comment 3: Abstract should be more specific based on the findings with recommendation.
Answer: The abstract was reviewed to better present the main results obtained in the work and for overall organization of the text.
Comment 4: Species or genus names should be followed by scientific terminology in the entire text.
Answer: The manuscript was corrected accordingly.
Comment 5: Line No: 86, why author choose particular temperature range for extraction? Clearly state the reason in the text.
Answer: The text in line Line No: 86 is not related to the extraction temperature, but the authors supposed that the reviewer was referring to line 186. To clearly state the choice of the extraction temperature the following phrase was included in lines 184 to 186: “Hot extraction was performed at 60 ºC for 1 h, for both leaves and twigs, to evaluate the effect of temperature in extract yield and properties. Higher temperatures were not selected to avoid thermal degradation of the extracts”.
Comment 6: In the "Introduction" section, general description on the importance of manuscript topic is poor. Therefore, the importance of this work cannot be well recognized from general readers. In order to fix this problem, addition of description on recent development in the field of research topic with citing recent comprehensive papers would be important.
Answer: The following text was modified and included in the Introduction Section to better clarify contributions from literature and the novelty of the present work:
“Extraction of value-added components from Acacia spp. has been focused mainly on the bark, flower, wood, and leaves as reviewed by Correia et al. [15]. Concerning the leaves fraction, antioxidant and/or antimicrobial activities have been determined for A. farnesiana [16], A. karroo [17–20], A. longifolia [21], A. pycnantha [22,23], A. saligna [24–27],. The antioxidant and antimicrobial activities of A. dealbata leaves were evaluated by Borges et al. [28], for acetonic and ethanolic extracts of fresh A. dealbata leaves, obtained with different extraction methods. Ethanolic extracts of dried A. dealbata leaves were also found to have antimicrobial activity against the food poisoning agent Bacillus cereus [29].
Research on extraction of functional components from twigs of Acacia spp. is scarce. Extracts of A. nilotica twigs were characterized for antimicrobial activity against oral pathogens [30–33], while extracts of A. pennata twigs were described as having some potential application in the prevention of Alzheimer´s disease [34].
The extraction methods explored so far included solid-liquid extraction for a pre-determined period under agitation at room temperature (1 h [28], 8 h [33], or 2 days [29,32]) ultrasound-assisted extraction [28], Soxhlet [28,34], microwave-assisted extraction [28], cold percolation [30], and supercritical fluid extraction [31,35]. Most of these methods are energy-consuming and/or involve high investment costs and may promote thermal degradation of the extract components [36].
This study aimed to investigate the production of bioactive extracts from leaves and twigs of A. dealbata, using maceration at room temperature, a method with low energy requirements and easy to implement on an industrial scale. Extraction with different aqueous solvents (acetone, ethanol, and methanol) was applied to fresh and dried biomass, collected in two different locations in order to evaluate the influence of solvent polarity, biomass water content, and geographic origin on the characteristics of the extracts, namely their yield, composition, and properties. To the best of our knowledge, this is also the first study on the production and characterization of bioactive extracts from A. dealbata twigs. The extracts were characterized for mass yield, total phenolic content, total flavonoid content, total proanthocyanidins content, in vitro antioxidant activity tests, and antimicrobial activity against several bacteria and yeasts. To identify non-polar components that might contribute to the antioxidant or antimicrobial properties of the extracts, a representative set of leaves and twigs ex-tracts was fractionated and characterized by GC-TOFMS”.
Comment 7: How is the work different or better than those reported earlier? Author needs to highlight this in the introduction and discussion part.
Answer: This comment has been addressed by the modifications of the Introduction Section outlined in the previous answer and by a revision of the Discussion Section to better clarify the new information obtained in this study.
Comment 8: Why author not used ethyl acetate for extract?
Answer: Hydrophilic solvents were selected for their lower environmental impact, lower toxicity and good performance in the extraction of phenolic components.
Comment 9: Line No: 100-104, why you chose ten different and totally how many samples were collected from each trees? Include in the text.
Answer: The choice of ten trees for sampling on each geographical origin had the objective of having a representative sampling of the biomass from that origin in the form of a composite sample from various individual trees. The following sentence was added to lines 109-110: “Samples of around 500g of branches were collected from each tree and combined to obtain a composite sample from that geographical origin”.
Comment 10: Author carefully check the scientific terminology in entire text.
Answer: A full text revision was performed to check the scientific terminology.
Comment 11: Why you choose particular bacteria’s and yeast for antimicrobial activity. Why author not checked fungal pathogens?
Answer: The choice of microorganisms to evaluate the antimicrobial activity of the extracts was made considering those organisms that are more often tested in other literature reports because they are responsible by frequent infections. A focus has been given to microorganisms relevant for skin infections to demonstrate the potential use of the extracts in skin cleaning products or cosmetics.
Comment 12: Why author choose particular Antioxidant assay? Author must include the reason in the text with appropriate reference citation https://doi.org/10.1016/j.jksus.2022.102029; https://doi.org/10.1155/2022/8575026;
Answer: The chosen tests are widely used in the literature for evaluation of antioxidant properties. The two references mentioned in this comment refer to in vivo studies with cell lines or animal models that are not related to the determination of antioxidant activity with “in vitro” tests. The FRAP test was already supported by literature references; the following reference was added as reference for the use of the DPPH test: Blois, M.S. Antioxidant Determinations by the Use of a Stable Free Radical. Nature 1958, 181, 1199–1200, doi:10.1038/1811199a0.
Comment 13: Author should be include pathogens virulence factors https://doi.org/10.1155/2022/7234586
Answer: The pathogens used in antimicrobial tests were pure strains purchased as such; the relevance of the use of plant extracts with antimicrobial activity against antibiotic-resistant strains as referred in the proposed reference was added to the manuscript as reference 71 in line 554.
Comment 14: Extensive editing of English language and style required
Answer: English language and writing style were revised in all the manuscript.
Comment 15: Need to check consistency in reference in the text
Answer: The format of the references was revised in all the manuscript and in the Bibliography Section.
Comment 16: Author should acknowledge the recommendation of present study in the conclusion section.
Answer: The conclusion section was revised to address this comment.
Author Response File: Author Response.pdf
Reviewer 3 Report
The effective use of invasive species to play its medical and economic value is a meaningful thing. Several questions remain in this paper:
1. The research plan is to study the antioxidant and antibacterial extracts of the plant. Why are the antioxidant and antibacterial active ingredients directly positioned in polar components? Antioxidants may be more polar components, but the antibacterial active ingredient is not necessarily a polar component. The extract obtained by selecting 70 % acetone, ethanol and methanol as solvents was mainly composed of polar components, which could not fully investigate the antibacterial activity of the invasive plant.
2. The ability of 70 % ethanol and 70 % methanol to extract polar components is similar, and the components of the extracts are also similar, so the choice of solvents is somewhat repetitive. This can also be seen from the experimental results. There is not much difference in the extraction rate of the above two solvents. Why not choose water as one of the solvents to extract?
3. Line 128 Why are the impregnation time of leaves and twigs different? Are the plant materials used for extraction crushed and to what extent.
4. Lines 122 and 148 serial number 2.3 appear repeatedly. Lines 459 and 491 serial number 3.4 repeats.
5. The components of total phenols, total flavonoids and proanthocyanidins overlap with each other. The method of determining total phenols, total flavonoids and proanthocyanidins in the extract is simple.
Author Response
Answers to the Comments of Reviewer 3
The authors want to thank for the revision of the manuscript and acknowledge the contribution of the reviewer comments to improve the quality of the manuscript.
Comment 1: The research plan is to study the antioxidant and antibacterial extracts of the plant. Why are the antioxidant and antibacterial active ingredients directly positioned in polar components? Antioxidants may be more polar components, but the antibacterial active ingredient is not necessarily a polar component. The extract obtained by selecting 70 % acetone, ethanol and methanol as solvents was mainly composed of polar components, which could not fully investigate the antibacterial activity of the invasive plant.
Answer: The authors agree that non-polar components could also influence the antioxidant and antibacterial properties of the extracts; to better answer this question a set of 7 extracts, representative of different samples and extraction conditions was fractionated, and the non-polar fraction was analysed by GC-TOFMS. Five predominant components (phytol, squalene, a-tocopherol, lupenone, and lupeol) were detected and their contribution to the functional properties of the extracts was discussed. The new data was introduced in the relevant parts of the discussion Section and in Supplementary Materials.
Comment 2: The ability of 70 % ethanol and 70 % methanol to extract polar components is similar, and the components of the extracts are also similar, so the choice of solvents is somewhat repetitive. This can also be seen from the experimental results. There is not much difference in the extraction rate of the above two solvents. Why not choose water as one of the solvents to extract?
Answer: Although aqueous ethanol, methanol and acetone showed a similar behaviour for these samples that is not always the case in plant matrices; moreover, these solvents have different boiling points what may be relevant for extract concentration. Water was not chosen because extraction of phenolic components at room temperature could be limited by the presence of lipophilic components such as waxes. Furthermore, the presence of the organic solvent reduces fermentation and enzymatic degradation processes that could occur during maceration in water at room temperature.
Comment 3: Line 128 Why are the impregnation time of leaves and twigs different? Are the plant materials used for extraction crushed and to what extent.
Answer: The impregnation times of leaves and twigs were decided after a preliminary study. This is explained in the following lines 130-133: “Extraction times were selected for leaves and twigs as the maceration time necessary to achieve constant values of extract yield and extract composition, according to a previous study (Supplementary Materials; Table S2).”
Concerning the preparation of the plant materials, fresh and dried leaves and twigs were manually cut into small pieces (about 1 cm of length) before impregnation with the solvent. This information was now included in the text, in the section “2.1. Plant Material”.
Comment 4: Lines 122 and 148 serial number 2.3 appear repeatedly. Lines 459 and 491 serial number 3.4 repeats.
Answer: The manuscript was corrected.
Comment 5: The components of total phenols, total flavonoids and proanthocyanidins overlap with each other. The method of determining total phenols, total flavonoids and proanthocyanidins in the extract is simple.
Answer: the authors agree, since the first class (total phenols) includes the other two classes (total flavonoids and proanthocyanidins); the characterization of these chemical families within the total phenols group is important because procedures for detailed chemical composition using HPLC always result in some type of discrimination during sample preparation. This approach of characterizing groups of phenolics using specific reactions and colorimetric tests it is often used in the literature and complements the chromatographic for profile discrimination. The LC-MS analysis of the phenolic fraction will be addressed in a different publication that compares the phenolic profile of different Acacia biomass fractions including leaves, twigs, flowers, bark, and wood.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
The author addressed all my comments.
Author Response
The authors thank again your effort and contributions to the revision of the manuscript.
Reviewer 2 Report
The author did not respond to my previous comments (Comment No: 6, 9, 11, 12, 13) and revise carefully with proper evidence. However, still this draft has a few suspicious queries and the author needs to respond to the following comments.
1. As per Silva et al., 2016 report Acacia spp leaf extracts were used in biological applications. Here, why an author repeating the same study?? Is there any novelty behind that included in the text?
2. There are few skin-infecting fungal pathogens available. So, the author must carefully check and report on the text.
3. Still the author not mentioned the virulence factors in the text. Check and carefully revise the draft.
Author Response
Answers to the 2nd round of Comments from Reviewer 2
The authors thank again for your effort and contribution to the revision of this manuscript.
Comments and Suggestions for Authors
The author did not respond to my previous comments (Comment No: 6, 9, 11, 12, 13) and revise carefully with proper evidence. However, still this draft has a few suspicious queries and the author needs to respond to the following comments.
Comment 1. As per Silva et al., 2016 report Acacia spp leaf extracts were used in biological applications. Here, why an author repeating the same study?? Is there any novelty behind that included in the text?
Answer: Silva et al., 2016, characterized the antimicrobial activity of aqueous, ethanolic and methanolic extracts of Acacia and Eucalyptus dry leaves; these authors did not characterize the extracts in terms of dry weight, total phenolics, total flavonoids, total proanthocyanidins, DPPH scavenging activity, FRAP reducing activity or composition of the non-polar fraction of the extracts using GC-MS.
Furthermore, the study by Silva et al., 2016, does not compare extracts obtained from biomass collected at different geographical locations and it doesn’t compare the extracts obtained from dry and fresh biomass.
So, the present manuscript cannot be described as a repetition of the same study because it has a different structure and addresses many variables that are not included in the study by Silva et al., 2016.
Nevertheless, the following sentence was introduced/changed in lines 607-609:
In general, a positive correlation was observed between the antimicrobial activity of extracts from leaves and twigs of A.dealbata, and their total phenolic content, but this association should be confirmed by additional quantitative studies.
Comment 2. There are few skin-infecting fungal pathogens available. So, the author must carefully check and report on the text.
Answer: We tested the activity of the extracts against one skin-infecting fungal pathogen. This information was highlighted in the new paragraph introduced in the result section (lines 584 to 589):
“Candida albicans was included in this study as a model to determine the antifungal activity of the extracts. Despite being a common commensal yeast fungus of the human oral, gastrointestinal, and genital mucosal surfaces and skin, under specific circumstances, such as perturbation of barrier integrity and/or host immune responses, C. albicans cause opportunistic infections that range from superficial infections of the skin to life-threatening systemic infections (Lopes et al, 2022; Mayer et al., 2013)
(Lopes, J. P., & Lionakis, M. S. (2022). Pathogenesis and virulence of Candida albicans. Virulence, 13(1), 89-121, https://doi.org/10.1080/21505594.2021.2019950;
Mayer, F. L., Wilson, D., & Hube, B. (2013). Candida albicans pathogenicity mechanisms. Virulence, 4(2), 119-128. https://doi.org/10.4161/viru.22913).
Comment 3. Still the author did not mention the virulence factors in the text. Check and carefully revise the draft.
Answer: In the discussion section, a paragraph was added to address the importance of microbial virulence factors (lines 517 to 522):
“Natural products from plants could also target microbial virulence factors and thus play an important role to combat microbial infections and overcoming antibiotic resistances (Wu et al, 2019). Microbial virulence factors encompass a wide range of molecules produced by pathogens, such as toxins, enzymes, exopolysaccharides, cell surface structures such as capsules, lipopolysaccharides, glyco- and lipoproteins (Leitão, 2020).
Wu, S. C., Liu, F., Zhu, K., & Shen, J. Z. (2019). Natural products that target virulence factors in antibiotic-resistant Staphylococcus aureus. Journal of agricultural and food chemistry, 67(48), 13195-13211. DOI: 10.1021/acs.jafc.9b05595.
Leitão, J. H. (2020). Microbial virulence factors. International journal of molecular sciences, 21(15), 5320. doi:10.3390/ijms21155320.
Author Response File: Author Response.pdf
Reviewer 3 Report
The authors have made a great effort in addressing all the comments and suggestions, and answered the questions raised in detail.
Author Response
The authors thank again your effort and contributions to the revision of the manuscript.
