Bio-Guided Fractionation of Oil Palm (Elaeis guineensis) Fruit and Interactions of Compounds with First-Line Antituberculosis Drugs against Mycobacterium tuberculosis H37Ra

Round 1

Reviewer 1 Report

The authors report the structure analysis and in vitro antimicrobial assays of nine fatty acids identified from OPF mesocarp. The separation and identification of compounds was carried out by flash chromatography and GC-MS. Four fatty acids showed weak anti-TB activity and their additive effects with first-line TB drugs were also investigated.

The manuscript is, in principle, suitable for publication, but it needs some improvements prior to that.

1. Page 2, Lines 80-81: Activities ranging from 400 ug/mL to 1600 ug/mL, in my opinion, should be considered weak activity.
2. Page 3, Lines 93-96: This long sentence is a bit confusing. 40 compounds were detected by GC-MS? 10 compounds were identified? How? 9 commercially available compounds were screened? Did you obtain the 9 compounds from commercial suppliers or purified them from n-hexane fraction? Please add more details here.
3. Page 3, Lines 96-99: How did you quantify these compounds? Peak area? Triplicates? Please specify.
4. Page 3, Lines 99-100: Why DMSO is in the OPF mesocarp sample? Shouldn’t the solvent be excluded from calculations?
5. Page 3, Lines 100-101: “Due to the fact that OPF is 50% oil, all the active compounds identified were fatty acids.” Did you mean the active components of OPF are all fatty acids? The fact is that only 9 commercially available fatty acids were tested.
6. Figure 1a: I could not see “40 detected compounds”. And please label the 10 “identified” compounds.
7. Figure 1b can be moved to supporting information.
8. Some parts of Tables 2 and 3 were missing.
9. Page 6, Lines 144 – 157:Reformate this paragraph.

 

 

Author Response

"Please see the attachment."

Author Response File: Author Response.pdf

Reviewer 2 Report

I wrote the letter carefully and I compared the manuscripts and I have to say that there was a considerable work done on the manuscript. Although some of my doubts remain regarding the publication of this manuscript in this journal, I have to take the view to publish, as the authors have corrected fundamental errors and significantly supplemented the text. In addition, the topic of using a cheap nutraceutical to treat a serious illness is becoming more and more actual these days.

Author Response

I wrote the letter carefully and I compared the manuscripts and I have to say that there was a considerable work done on the manuscript. Although some of my doubts remain regarding the publication of this manuscript in this journal, I have to take the view to publish, as the authors have corrected fundamental errors and significantly supplemented the text. In addition, the topic of using a cheap nutraceutical to treat a serious illness is becoming more and more actual these days.

 

We thank the reviewer for recognising the improvements of the revised manuscript and appreciating its potential value given the increasing interest in cheap nutraceuticals as tools to manage serious illnesses such as tuberculosis.

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 authors report the structure analysis and in vitro anti-mycobacterial assays , including a synergistic investigation of some fatty acids identified from OPF mesocarp. Two fatty acids showed additive effects with known anti-TB drugs. However, there are some major issues need to be addressed before it can be accepted in the journal or any other journals.

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Major comments.

1. The authors gave a nice introduction of TB and the current challenges in anti-TB drug discovery in the current Introduction section. However, not enough background of existing literature on the oil palm chemical investigations, particularly fatty acids reported from oil palm, known bioactivity of those fatty acid, anti-TB activity, synergistic studies, etc…. It is now not very clear to me, in the whole manuscript, what has been reported before by others, and what is the new findings from the authors. A major revision of the Introduction is needed.
2. Line 42 or in the whole Introduction: “additive effect” needs to be better explained on how compounds with additive effects with known drugs can be used to reduce their required doses and toxicity. I understand how compounds with synergistic effects with known drugs can be useful for anti-TB treatments, but not convinced by compounds with additive effects with known drugs, especially when the compounds (in this manuscript, for example) only showed MIC at 50 ug/mL. How could these compounds reduce the required doses of existing anti-TB drugs?
3. For activity test, the MIC of fractions used in this study are at the level of mg/mL. From my experience, these concentrations are too high even for the extracts. Considering dilutions in the assay, concentrations of the stock might be even higher. Wasn’t there a solubility issue? And this definitely affects the accuracy of the MIC/MBC data. Another concern is samples with such high concentrations would potentially affect the determination of MICs, as a result of colour interference.
4. Table 2: Library matching rates for decanoic acid and tri (2-ethyhexyl) trimelitate were only 87%. Are these values good enough to unambiguously identify compounds? Also, in my opinion, purification and identification of these compounds from OPF mesocarp is a big part of this manuscript. Most of the fatty acids in this study, particularly nonanoic acid, decanoic acid, and dodecanoic acid are all commercially available. In fact, very cheap. The authors need to think about the value/significance of this study, and it needs to be addressed better in the manuscript.
5. Conclusion: “Although the inhibitory effects of these acids against mycobacteria have been described, the potential of these compounds are re-visited using newer methods and expanded to include investigations into their use as anti-TB adjuvants.” What methods did others use and how newer your method was? This statement is not convincing. More evidence needed. “Importantly, we report novel observations of additive interaction of…” This sentence seems OK…, but then I read from the Result part, saying that synergistic effects have been reported previously… So please make it very clear to the audience what is the novel part of this study.

 

 

I got an impression that the manuscript had been prepared in a hurry, and so many minor “fixes” have been overlooked. I list only the obvious ones, the senior CI should screen the revised copy carefully.

 

Minor comments.

1. (in the title and elsewhere) In the Latin scientific names of organisms, names at the species level and below (species, subspecies, variety) are not capitalized; those at the genus level and above (e.g., genus, tribe, subfamily, family, class, order, division, phylum) are capitalized. For example, “Mycobacterium Tuberculosis” needs to be corrected to “Mycobacterium tuberculosis” even when it’s part of the Title!
2. “Mtb” or “tb” are more commonly used as abbreviation of “Mycobacterium tuberculosis”.
3. Change all “Mycobacterium tuberculosis” or “ tuberculosis” to “Mtb” except for the first one appeared in the manuscript.
4. Line 13: “Natural compounds”, my pet hate. so please use “Natural products” instead.
5. Line 55 and elsewhere “n-hexane” needs to be changed to “n-hexane”.
6. Line 33: Reference 1 needs to be replaced with the latest 2020 WHO TB report, which has been released in October.
7. Line 39: “MDR-TB” is the abbreviation of “multidrug-resistant tuberculosis”.
8. Line 61 and other subtitles are all too long.
9. Line 64: “MABA”, spell out the full name.
10. Table 1 and lines 63-64: “Sequential partition of crude methanolic extract of OPF mesocarp was done using n-hexane, chloroform, ethyl acetate, and butanol, consecutively”, which means for example, chloroform was added to the aqueous crude extract, which had been partitioned by n-hexane already? If this was the case, in Table 1, mass of crude extract for chloroform should be 5-3.621 g, and yield needs to be recalculated.
11. Line 69-71: “only the n-hexane partition was selected for further investigation due to the low yield of the chloroform partition”. It would make more sense if the n-hexane fraction was selected for further investigation because it contained the highest amount of fatty acid or other reasons, not because it had the highest yield. From my perspective, I would rather work on the chloroform fraction as it showed the best activity. The low yield of the chloroform fraction would result in a more potent compound from the fraction!
12. Line 74-75: well without drugs/samples should be called “negative control” and isoniazid is “positive control”! And normally the solvent (DMSO?) should be used for the negative control.
13. Line 76-85 and Figure 1: I didn’t see any value of including the TLC results in this manuscript. Was it used to determine solvent system for flash chromatography? Some information can be included in the Methods if the authors feel they need to, but other than that, I don’t think there is anything worth mentioning in the main text, including Figure 1A.
14. Figure 1B: More information needs to be added to the chromatogram, at least names of each compound corresponding to their peaks. Resolution of the current chromatogram is not good enough.
15. Line 80: “40 compounds were identified, of which 9 compounds were characterised…”. I would like to see the 40 compounds in Figure 1B if possible. Also, this sentence is a bit ambiguous. “40 compounds were identified” means you know the identities of these 40 compounds. I would think the case should be “40 compounds were detected”, otherwise, please provide a list of the 40 compounds.
16. Line 83-84: “Identified compounds amounted to 0.23% of the fraction, while the rest was DMSO”. I couldn’t understand this sentence. Please rewrite.
17. Line 93: “Fraction 8 was found to contain 40 compounds, of which 9 were fully characterised structurally”. This is not true, at least from what have been written in the current manuscript. Nine compounds were identified only by comparison with database. This is not a fully structural characterisation.
18. Section 2.2 and Table 2: Purification of the 9 compounds were not mentioned here or in the Methods. Did the authors obtain pure compounds, otherwise, how did they get the MICs/MBCs of each compound? Please specify in the manuscript.
19. Table 2: CAS No of tri (2-ethyhexyl) trimelitate is “3319-31-1”. Please check.
20. Lines 118-120: Concentrations of the other compound/drug need to be added. For example, MIC of nonanoic acid was reduced from 50 μg/mL to 6.25 μg/mL by how much of streptomycin addition?
21. Lines 129 – 135: This paragraph should be in the main text.
22. Is subtitle 2.4 missing??
23. Lines 136 – 139 and Figure 2a: Bacterial survival rates of the negative control (no drug treatment) decreased greatly from Day 6, and reached to < 50% on Day 8. Is this a normal result? As a result, neither the single drug treatments nor the drug combinations did not kill 100% or 90% of bacteria as there was only less than 50% bacteria survived in the blank control. Please explain in the manuscript.
24. Line 139: “TEM”, spell out the full name.
25. Lines 146 – 154: Please separate descriptions of Figure 2a from Figure 2b.
26. Line 155: It should be “2.5 …”
27. Line 158: a space is missing.
28. Line 176: 80% methanol with 20% WHAT?
29. Line 192 – 193: “100μL of M7H9 broth was added into all wells except for negative controls.” why not added to negative controls?
30. Line 215 – 224: no weights of each fractions are reported.
31. Purification of the compounds is missing from the manuscript. And compounds information, such as spectroscopic data needs to be added.

 

 

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Authors deal with very interesting topic: cheap nutraceutical can be used to treat very dangerous and resistant bacteria of current world. Authors use very easy procedures to obtain extracts but with quite discutable results - when they obtain similar results for non-polar water/aqua extract and polar n-hexane or chloroform extracts..

There was described antimicrobial effect of the extracts from Elaeis Guineensis several times, e.g. (leaf extract) in Chong, K & Zakaria, Zuraini & Sasidharan, Sreenivasan & Kalnisha, P & Lachimanan, Yoga & Ramanathan, Surash. (2007). Antimicrobial Activity of Elaeis Guineensis Leaf. Pharmacologyonline. 3.), but the relation to Mycobacterium seems interesting to me. Synergistic effect shown in the graph (between antibiotic and extract) seems very promising.

On the other hand, other authors also mention that threre is a pool of antioxidant phenolic compounds in the extracts, such as gallic acid or para-hydroxybenzoate. Did authors do any detection of such compounds? I suggest to the authors to screen these extracts also on these phenolic cmps that can also act antibacterial (gallic acid).

 

Author Response

Please see attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors describe the discovery of additive effects of several fatty acids with first-line anti-Tb drugs against Mtb. While this work is important and of broad interest, I do not recommend this article for publication in Molecules for the following reasons:

1. The anti-mycobacterium potential of fatty acids, and their synergistic effects with anti-TB drugs have been reported before by other authors. Even through the authors argued that they have expanded the activity investigation with different fatty acids, my opinion is that this work does not demonstrate enough new results from what have been discovered before.
2. The quality of this work is also relatively low. Only a small portion of compounds detected from OPF were identified, and were identified by GC-MS only. Except for a flash chromatography, no further purification has been conducted. And the fatty acids obtained from commercial suppliers were further evaluated for biological potential in terms of anti-TB activity. Neither fractions, or the compounds (individually or in combination with anti-TB drugs) showed good enough activity.

 

Other questions and issues are listed below.

1. Line 27: “50” in “LC50” needs to be in lower case;
2. Line 27: “…positing this compound as a novel anti-TB adjuvant candidate” is not true. None of the compounds in this manuscript should be considered novel, including their activity.
3. Line 34: Since reference 1 has been updated to the 2020 WHO TB report, the data here needs to be updated from 2018 to 2019 as well.
4. Table 1: how was yield of the “aqueous” phase calculated as “93.17”?? This does not seem right. In fact, the whole table, except for MICs of each fraction, could be deleted or moved to experimental section.
5. The authors answered my previous comment regarding to selection of hexane fraction as “the yield difference between n-hexane (3.621g) and chloroform (0.106g) was 34-fold, where as the difference in MIC was only 2-fold. Given the time required for extraction and challenges of obtaining sufficient amounts from chloroform partition for further testing, we made the decision to only pursue n-hexane partition.”. As a natural product chemist, I totally understand people conducting further work on any of their active fraction, which is totally fine. However, I’m not convinced by the explanation they provided in the cover letter. Firstly, the yield difference between n-hexane (3.621g) and chloroform (0.106g) was 34-fold, and the chloroform fraction showed higher activity with this low yield. This means the minor compounds in the chloroform fraction would be much more active than your major compounds in the high yield hexane fraction. Secondly, I don’t see much difference in “time required for extraction and challenges of obtaining sufficient amounts from chloroform partition for further testing” from what have been described in the manuscript for working on hexane fraction. This is a minor comment. Just to clarify.
6. Figure 1: both A and B are in very poor quality, not visible at all.

Author Response

Please see the attachment. Thank you.

Author Response File: Author Response.docx