Antiprotozoal Activity and Cytotoxicity Screening of Lippia adoensis (Hochst.) Extracts: Growth Inhibition of Plasmodium, Leishmania, and Trypanosoma Parasites
, Mariscal Brice Tchatat Tali 1, Darline Dize 1, Cyrille Armel Njanpa Ngansop 1, Boniface Pone Kamdem 1,* and Fabrice Fekam Boyom 1,2
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript described the inhibitory effect of Lippia adoensis extract on Plasmodium, Leishmania, and Trypanosoma. The experiments were reasonably designed and described with enough details.
I have one critique which is to provide all the individual fluorescence intensity readings and calculated IC50 of each experiment in a supplemental spreadsheet. Table 1 alone is not sufficient. And I suppose that the IC50 presented in Table 1 is mean with SD (or SEM?), which is not labeled anywhere in the manuscript.
Author Response
Response to the reviewer’s comments point by point.
Manuscript ID: joma-3292203
Title: Antiprotozoal activity and cytotoxicity screening of Lippia adoensis (Hochst.) extracts: Growth inhibition of Plasmodium, Leishmania and Trypanosoma parasites
Referee 1 :
The manuscript described the inhibitory effect of Lippia adoensis extract on Plasmodium, Leishmania, and Trypanosoma. The experiments were reasonably designed and described with enough details.
I have one critique which is to provide all the individual fluorescence intensity readings and calculated IC50 of each experiment in a supplemental spreadsheet. Table 1 alone is not sufficient. And I suppose that the IC50 presented in Table 1 is mean with SD (or SEM?), which is not labeled anywhere in the manuscript.
Answer : Thank you very much for the observation of the reviewer. However, all the required information on antiprotozoal tests (antiplasmodial, antitrypanosomal and antileishmanial assays) is already incorporated in the main manuscript.
Nevertheless, raw data on an Excel sheet is also attached for your kind reference.
We request the reviewer for re-evaluation and kind consideration.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsNeglected tropical disease are indeed a problem especially in third world countries and new drugs are needed as the current drugs available are toxic and also facing resistance from the parasites. The initiative to test a plant against the protozoan parasites is a good one. However this work needs to be improved and I have highlighted my concerns in my comments.
Comments for author File: Comments.pdf
Author Response
Response to the reviewer’s comments point by point.
Manuscript ID: joma-3292203
Title: Antiprotozoal activity and cytotoxicity screening of Lippia adoensis (Hochst.) extracts: Growth inhibition of Plasmodium, Leishmania and Trypanosoma parasites
Referee 2 :
Neglected tropical disease are indeed a problem especially in third world countries and new drugs are needed as the current drugs available are toxic and also facing resistance from the parasites. The initiative to test a plant against the protozoan parasites is a good one. However this work needs to be improved and I have highlighted my concerns in my comments.
Summary
The authors tested the hydroethanolic extracts of the leaves and twigs of Lippia adoensis against various parasitic protozoa namely Plasmodium falciparum 3D7 and Dd2, Leishmania donovani and Trypanosoma brucei brucei . They also tested the cytotoxic effects of the extracts using Vero cells. They then did in vitro phytochemical analysis of the extracts as well as identifying molecular clusters using GNPS.
General comments
The paper falls short of publication in many ways however these can be rectified through labwork as well as additional information. The idea of testing the extracts of the plant against the protozoan parasites is good but the execution is not done well. It is important to find the active principle in an extract or at least attempt to especially when you have the assays at your disposal. Simply doing phytochemical analysis and using LCMS based software to try and identify compounds in the extracts does not help in identification of the active principle and simply leads to a speculative discussion. In the introduction the authors also show inconsistencies in the chemical composition of the essential oil of the plant that was tested and this leaves one wondering whether the plant could be used in drug discovery as the difference in chemical composition may lead to inconsistence bioactivities. The merit of the work is the bioassays but unfortunately without attempting to identify the active principle, it simply isn't enough to do an invitro phytochemical analysis as well as identifying molecular clusters.
Answer : Thank you for your suggestions. This work is a preliminary study that mainly aims to validate the traditional use of leaves and twigs of Lippia adoensis in the treatment of certain protozoal infections through in vitro studies. We further evaluated the cytotoxicity of the extracts on Human mammalian cells Vero. The results obtained from in vitro studies substantiated the ethnopharmacological use of Lippia adoensis extracts in treating malaria and other protozoal infections.
The idea to carry out a phytochemical analysis was to identify the groups of active principles that can guide us to further detailed chemical studies towards isolation and characterization of the antiprotozoal compounds of Lippia adoensis. As highlighted in the conclusion section, isolation and characterization is ongoing in our laboratory as a continuation to this preliminary research contribution. Limitations regarding detailed chemical analysis of the plant extracts have also been highlighted in a separate section « Limitations and perspectives » right before the conclusion section of the manuscript.
Specific comments
Line 9: The authors used 70 % ethanol for extraction, from my experience 80 % methanol gives the best extraction. Did they try other solvents for extraction and what was the basis of using 70 % ethanol?
Answer : 70 % ethanol was used as the solvent of extraction in this study to mimic the traditional preparation of the plant extract using palm wine.
Line 15: the statement 'As a result' needs to be changed as it gives the impression the GNPS phytochemical screening gave the growth inhibition results.
Answer : The change has now been amended as suggested.
Line 25: Bioassay guided fractionation would be the best approach to identify the active compound(s). Metabolomic studies alone cannot give conclusive results as to what the active principle(s) is(are).
Answer : We agree with the reviewer’s suggestion. As already discussed, the main aim of this work was to validate the traditional use of leaves and twigs of Lippia adoensis in treating certain protozoal infections through in vitro studies. A preliminary phytochemical analysis aided to unveil the chemical functional groups contained in the plant. However, the approach suggested for isolation and characterization of the active principles is fully taken into consideration in our project on the identification of antiprotozoal lead compounds from medicinal plants, including Lippia adoensis.
Liebe 33, 34: 'These diseases claim the lives of a billion people worldwide annually'. This statement is incorrect.
Answer : Thank you. The statement has now been corrected.
Line 36: What do the authors mean by 'Environmental changes'?
Answer : Environmental changes encompass alterations in the environment, driven by both natural processes and human activities, including climate change, pollution, deforestation, and biodiversity loss, with impacts ranging from local to global (McMichael et al., 2008 ; Sorgho et al., 2021).
Line 36, 37: How does eliminating one NTD in each of 50 countries indicate that we are at the midway point of eradicating NTDs in 100 countries by 2030?
Answer : Thank you. In the World Health Organization report published in 2024 « https://www.emro.who.int/media/news/50th-country-recognized-by-who-for-eliminating-a-neglected-tropical-disease-marking-the-halfway-point-towards-the-100-country-target-set-for-2030.html », it is clearly mentioned the following statement « Eliminating one NTD in each of 50 countries signifies a halfway point towards the 2030 goal of eliminating at least one NTD in 100 countries, as 50 is half of 100, marking significant progress in the global fight against these diseases ». Thus, we believe that, the World Health Organization uphold the most trustful information and predicted data regarding the epidemiology of neglected diseases, including certain protozoal diseases.
Line 38: 'This notorious disease', which disease are the authors referring to? You have to write Malaria and not 'This notorious disease' as you have not mentioned malaria in your previous sentence.
Answer : 'This notorious disease' has now been replaced by ’Malaria’
Line 42, 44: Please cite the source of this data before beginning the next sentence.
Answer : A source has now been cited for the statement.
Line 50: I would not begin a sentence with 'Caused'.
Answer : The sentence has now been rewritten.
Line 62: The authors wrote human American trypanosomiasis, this is Chagas disease but in the previous sentences they are referring to human African trypanosomiasis, 'Sleeping sickness'.
Answer : The change has now been amended as suggested.
Line 74: Change revolutionise to revolutionised.
Answer : The change has now been amended as suggested.
Line 78, 79: change the word derivative to derivatives for both diamidine and nitrofuran.
Answer : The change has now been amended as suggested.
Line 82, 83: The statement 'there is interest of natural product-based drug screening over high throughput screening of combinatorial libraries' is debatable as natural products also bring challenges like adherence to Lipinskis rules, intellectual property issues, sustainability, changes in chemical composition due to seasonal changes, just to mention a few.
Answer : Thanks. Challenges regarding sustainability and changes in the plant’s chemical composition, etc. are some limitations in drug discovery campaigns from plants (Laftouhi et al., 2023). In the meantime, accumulated evidence has shown that even more than 50% of the currently available synthetic drugs originated from medicinal plant products (Butler, 2005 ; Ranasinghe et al., 2023), thus justifying the need to search for effective antiparasitic treatments from medicinal plants.
Line 98: This is rather unexpected. Can the authors provide a possible explanation as to why linalool was not found in the wild type plant.
Answer : Thank you. The absence of linalool in the wild type plant might result in the genetic variability, whereby certain genes, which are silent in the wild plant type might undergo mutations in mutant plant type to produce a number of secondary metabolites, including linalool. Indeed, mutations can disrupt normal gene function, potentially leading to the production of secondary metabolites not found in the wild type. This can occur through activation of previously silent genes, increased expression of existing genes, or the creation of new pathways (Erb and Kliebenstein, 2020).
Line 100- 110:The variability in the chemical composition of the essential oil raises questions as to whether this plant can be a reliable source of active compounds.
Answer : Thank you. Plant secondary metabolites, such as flavonoids, alkaloids, tannins, among others, which are well known to exhibit antiprotozoal effects (Nardella et al., 2018, Ungogo et al., 2020 ; Chepkirui et al., 2021) are not generally expected in the plant’s essential oil, Thus, secondary metabolites of Lippia adoensis can not be restricted only to those reported thus far in its essential oil.
Line 128-131: Investigating the antiparasitic effects was a good idea, but the authors should have followed up by assaying the fraction by partitioning with n-hexane/ heptane, chloroform, ethylacetate and n-butanol. Partitioning and testing the fractions would have given an indication as to which compounds may be responsible for the activity.
Answer : Thank you. As already discussed, this work is a preliminary study that mainly aims to validate the traditional use of leaves and twigs of Lippia adoensis in the treatment of certain parasitic infections through in vitro studies. We further assessed the cytotoxicity of the extracts on Human mammalian cells Vero. The results obtained from in vitro studies substantiated the ethnopharmacological use of Lippia adoensis extracts in treating certain protozoal infections. Nevertheless, the observation of the reviewer is worthwhile and is fully taken into consideration in our plant drug discovery campaigns.
Line 165: Where was the human blood obtained?
Answer : The blood was collected from group O+ individuals (healthy volunteers).
Line 172: P. falciparum in italics.
Answer : « P. falciparum » has now been italicized as suggested.
Line 181: Why were the tests done in duplicate not triplicate, with duplicate it is not easy to identify an outlier if you have only two readings.
Answer : Thanks. Although triplicates generally offer larger sample size and a more robust statistical analysis and outlier detection, other authors (Julio et al., 2007 ; Green and Lee, 2023) have argued that duplicate testing is also effective at identifying and mitigating irregular laboratory errors, and is best suited for assays predisposed to such error, where costs are minimal.
Line 223: why did the authors use varying concentrations of the positive control. Normally you use a concentration where you expect 100 % mortality of your parasite. Why use a range of concentrations?
Answer : We believe that, to know about the concentration where 100 % mortality is expected, it is suitable to vary the concentrations so as to easily locate it.
Line 246: Trypanosoma brucei in italics.
Answer : The change has now been amended as suggested.
Line 260: The authors should have done triplicates.
Answer : Thanks. Although triplicates generally offer larger sample size and a more robust statistical analysis and outlier detection, other authors (Julio et al., 2007 ; Green and Lee, 2023) have argued that duplicate testing is also effective at identifying and mitigating irregular laboratory errors, and is best suited for assays predisposed to such error, where costs are minimal.
Line 322: the in vitro phyto chemical screening does not really add much value to the paper as it confirms the presence of compounds that occur in plants in general.
Answer : We agree with that. However, this work is a preliminary study that mainly aims to validate the traditional use of leaves and twigs of Lippia adoensis in the treatment of certain parasitic infections through in vitro studies. We further evaluated the cytotoxicity of the extracts on Human mammalian cells Vero. The results obtained from in vitro studies substantiated the ethnopharmacological use of Lippia adoensis extracts in treating malaria and other protozoal infections. The type of plant secondary metabolites indicated in this work will guide us or other researchers to develop/select appropriate methods for compound isolation and characterization.
Line 325: On what basis do the authors think carbohydrates could be responsible for the antiparasitic activity?
Answer : Growing evidence has shown the antiparasitic effects of carbohydrate compounds/derivatives (Kang et al., 2015 ; Vasconcelos and Pomin, 2018 ; Singh and Tripathi, 2023).
Line 353: Experiments should be performed in triplicates
Answer : Thanks. Although triplicates generally offer larger sample size and a more robust statistical analysis and outlier detection, other authors (Julio et al., 2007 ; Green and Lee, 2023) have argued that duplicate testing is also effective at identifying and mitigating irregular laboratory errors, and is best suited for assays predisposed to such error, where costs are minimal.
Line 360: Table 1, may the authors be consistent on the number of decimal points. Also give a numerical figure for the CC50 of vero cells rather than saying it is greater than 100 as this figure is used in calculating the SI.
Answer : Thank you. The numerical figure of CC50s of Lippia adoensis extracts on vero cells has now been incorporated in table 1. Values of selectivity indices (SI) have also been calculated and incorporated in table 1.
Line 366, 369: In line 366 the authors refer to values of 10.008 and 97.467 as relevant. In line 369 the authors refer to 29.48 and 26.96 as moderate, yet these lie in between 10 and 96. Should they also not be relevant?
Answer : The concerned sentences have now been rewritten.
Line 385: Shouldn't the molecular networking have confirmed the presence of the compounds known to be abundant in the essential oil of Lippia adoensis?
Answer : Thank you. As already discussed, the genetic variability among plant species might induce significant changes in the plant’s chemical composition.
Line418: 10.008 micrograms per ml is referred to as significant. In line 420 and 421, 97.467, 29.48 and 26.96 micrograms per ml are referred to as moderate. What criteria did the authors use to determine what moderate and significant activity is?
Answer : The sentence has now been rewritten. The changes have been coloured red with yellow stripes across the main manuscript.
Line 422-443: This part of the discussion is very speculative and the activity of the compounds mentioned cannot be linked to the activity of the extracts as the compounds were not identified in the extract.
Answer : GNPS was used to predict potential compounds present in Lippia adoensis extracts. This is just a prediction, thus the observation of the reviewer is correct. The compounds predicted using GNPS may or may not be those responsible for the observed activity. Additional chemical studies are warranted to support such predictions. We have also mentioned the importance of isolation and characterization of Lippia adoensis compounds in a separate section « Limitations and perspectives » right before the conclusion section.
Line 443-445: The authors should have attempted to isolate the compound linalool through column chromatography in order to test this claim.
Answer : Thank you for the observation of the reviewer. As already discussed, this work is a preliminary study that mainly aims to validate the traditional use of leaves and twigs of Lippia adoensis in the treatment of certain parasitic infections through in vitro studies. We further evaluated the cytotoxicity of the extracts on Human mammalian cells Vero. The results obtained from in vitro studies substantiated the ethnopharmacological use of Lippia adoensis extracts in treating malaria and other protozoal infections. Nevertheless, the observation of the reviewer is worthwhile and is fully taken into consideration in our plant drug discovery campaigns.
Line 452-460: The LC-MS feature based detection and molecular networking flow on GNPS did not give any valuable information pertaining to the activity of the extract and what the authors state here is speculative.
Answer : Obviously with predicted results, discussion will mostly be speculative, thus requiring additional studies that will help to support the claims. The reviewer is absolutely correct.
Line 462: The non toxicity on vero cells is not an indication that the extracts are safe to use as this is just one cell line. Further tests would be needed to make this justification that the plant is safe to use. The authors did however mention the need for further testing.
Answer : The reviewer is right, additional studies on other cell lines are desired to support the safe use of this plant. Additional tests regarding cytotoxicity of Lippia adoensis extracts have also been incorporated in a separate section « Limitations and perspectives » right before the conclusion section.
Line 484: The authors cannot conclude that the compounds identified using GNPS were responsible for the antiparasitic effects.
Answer : GNPS was used to predict the potential compounds present in Lippia adoensis extracts. This is just a prediction, thus the reviewer is correct. Additional chemical studies are warranted to support such predictions.
Line 485: The authors state that the IC50 values are 'significantly low', how do they come up with the criterion to determine whether activity is high, low, significant, insignificant, moderate etc?
Answer : The sentence has now been rewritten as suggested.
There is no supplementary data attached. The raw data used to calculate the IC50 and CC50 values has to be presented in the supplementary data.
Answer : Thank you very much for your observation. However, all the required information on antiprotozoal tests (antiplasmodial, antitrypanosomal and antileishmanial assays) is already incorporated in the main manuscript.
Nevertheless, raw data on an Excel sheet is also attached for your kind reference.
References
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-Erb M, Kliebenstein DJ. Plant Secondary Metabolites as Defenses, Regulators, and Primary Metabolites: The Blurred Functional Trichotomy. Plant Physiol. 2020 Sep;184(1):39-52. doi: 10.1104/pp.20.00433.
-Green AD, Lee GR. An appraisal of the practice of duplicate testing for the detection of irregular analytical errors. Clin Chem Lab Med. 2023 Nov 10;62(4):627-634. doi: 10.1515/cclm-2022-0605.
-Julio M. Singer, Antonio C. Pedroso-de-Lima, Nelson I. Tanaka, Verónica A. González-López, To triplicate or not to triplicate? Chemometrics and Intelligent Laboratory Systems 86 (2007) 82–85.
-Kang HK, Seo CH, Park Y. The effects of marine carbohydrates and glycosylated compounds on human health. Int J Mol Sci. 2015 Mar 16;16(3):6018-6056.
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-McMichael AJ, Friel S, Nyong A, Corvalan C. Global environmental change and health: impacts, inequalities, and the health sector. BMJ. 2008 Jan 26;336(7637):191-194.
-Nardella, F., Gallé, JB., Bourjot, M., Weniger, B., Vonthron-Sénécheau, C. (2018). Antileishmanial and Antitrypanosomal Activities of Flavonoids. In: Mérillon, JM., Riviere, C. (eds) Natural Antimicrobial Agents. Sustainable Development and Biodiversity, vol 19. Springer, Cham. https://doi.org/10.1007/978-3-319-67045-4_7
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-Singh K, Tripathi RP. Carbohydrate derivatives fight against malaria parasite as anti-plasmodial agents. Carbohydr Res. 2023 Sep;531:108887. doi: 10.1016/j.carres.2023.108887.
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-Ungogo MA, Ebiloma GU, Ichoron N, Igoli JO, de Koning HP, Balogun EO. A Review of the Antimalarial, Antitrypanosomal, and Antileishmanial Activities of Natural Compounds Isolated From Nigerian Flora. Front Chem. 2020 Dec 23;8:617448. doi: 10.3389/fchem.2020.617448.
-Vasconcelos AA, Pomin VH. Marine carbohydrate-based compounds with medicinal properties. Mar Drugs. 2018 Jul 9;16(7):233. doi: 10.3390/md16070233.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThe World Health Organization (WHO) classified malaria, leishmaniasis and African trypanosomiasis as the triad parasitic diseases that are more prevalent in tropical regions, especially in Sub-Saharan Africa. These diseases claim the lives of a billion people worldwide annually.
In 2022, there were an estimated 249 million malaria cases and 608,000 malaria deaths worldwide. And regarding Leishmaniasis, it affects mostly the poorest regions of the globe and annually there are an estimated 1 million new cases. In addition, Human African trypanosomiasis (HAT, sleeping sickness) menaces millions of people.
Currently, there are several challenges to be faced such as drug resistance, the toxic effects of these therapies, and consequently the discovery of effective antiprotozoal agents including those developed from medicinal plants. Lippia adoensis is an example of medicinal plants used for the traditional treatment of malaria and many other diseases
The authors pretend to explore the antiplasmodial, antileishmanial, and antitrypanosomal activities of Lippia adoensis extracts. Crude extracts from L. adoensis inhibited growth of P. falciparum (3D7) (IC50 =10.00-97.46 μg/mL), P. falciparum (Dd2) (IC50=26.96-29.48 μg/mL), L. donovani (IC50=22.87-10.52 μg/mL), and T. brucei brucei (IC50=2.30-55.06 μg/mL).
These authors suggest the antiparasitic effects of L. adoensis extracts, and their results seem to support the use of this plant in the traditional treatment of malaria conditions.
The article is quite interesting. However, some minor comments regarding the content should be clarified.
1/ Line 34: Why did the authors consider Malaria as a Neglected Disease?
2/ Lines 246-267: Please, explain the selection of Trypanosoma brucei brucei? Why not Trypanosoma brucei rhodesiense and T. brucei gambiense (Human African tripanosomiasis)?
3/ Lines 360-361: The SI value of the extracts of Lippia adoensis leaves against T. brucei brucei was >43.47. What might be expected when treating T. brucei rhodesiense or T. brucei gambiense? This discussion should be included in the text.
Author Response
Response to the reviewer’s comments point by point.
Manuscript ID: joma-3292203
Title: Antiprotozoal activity and cytotoxicity screening of Lippia adoensis (Hochst.) extracts: Growth inhibition of Plasmodium, Leishmania and Trypanosoma parasites
Referee 3
The World Health Organization (WHO) classified malaria, leishmaniasis and African trypanosomiasis as the triad parasitic diseases that are more prevalent in tropical regions, especially in Sub-Saharan Africa. These diseases claim the lives of a billion people worldwide annually.
In 2022, there were an estimated 249 million malaria cases and 608,000 malaria deaths worldwide. And regarding Leishmaniasis, it affects mostly the poorest regions of the globe and annually there are an estimated 1 million new cases. In addition, Human African trypanosomiasis (HAT, sleeping sickness) menaces millions of people.
Currently, there are several challenges to be faced such as drug resistance, the toxic effects of these therapies, and consequently the discovery of effective antiprotozoal agents including those developed from medicinal plants. Lippia adoensis is an example of medicinal plants used for the traditional treatment of malaria and many other diseases
The authors pretend to explore the antiplasmodial, antileishmanial, and antitrypanosomal activities of Lippia adoensis extracts. Crude extracts from L. adoensis inhibited growth of P. falciparum (3D7) (IC50 =10.00-97.46 μg/mL), P. falciparum (Dd2) (IC50=26.96-29.48 μg/mL), L. donovani (IC50=22.87-10.52 μg/mL), and T. brucei brucei (IC50=2.30-55.06 μg/mL).
These authors suggest the antiparasitic effects of L. adoensis extracts, and their results seem to support the use of this plant in the traditional treatment of malaria conditions.
The article is quite interesting. However, some minor comments regarding the content should be clarified.
1/ Line 34: Why did the authors consider Malaria as a Neglected Disease?
Answer: Malaria is considered a neglected disease, despite being preventable and curable, because it disproportionately affects the poor and lacks adequate resources and global attention, leading to a cycle of poverty and limited access to treatment and prevention (Carlton et al., 2011; Hotez et al., 2020).
2/ Lines 246-267: Please, explain the selection of Trypanosoma brucei brucei? Why not Trypanosoma brucei rhodesiense and T. brucei gambiense (Human African tripanosomiasis)?
Answer: Trypanosoma brucei brucei is used as a model organism to study human African trypanosomiasis (HAT), and related parasitic diseases, due to its well-characterized life cycle, ease of study in a laboratory setting, and the insights it provides into parasite-host interactions (Shaw and Roditi, 2023). This pathogen (Trypanosoma brucei brucei) is susceptible to trypanosome lytic factors (TLFs) in human serum, making it a good model for studying innate immunity and parasite-host interactions, while T. b. gambiense and T. b. rhodesiense are resistant to these TLFs (Tesoriero et al., 2018).
3/ Lines 360-361: The SI value of the extracts of Lippia adoensis leaves against T. brucei brucei was >43.47. What might be expected when treating T. brucei rhodesiense or T. brucei gambiense? This discussion should be included in the text.
Answer: Although Trypanosoma brucei brucei is a preferred model organism to study human African trypanosomiasis, previous in vitro antitrypanosomal reports using T. brucei rhodesiense or T. brucei gambiense in culture revealed that there is no difference in activity with tests on T. brucei brucei (Balber et al., 1985; Brun et al., 1989; Likeufack et al., 2006; Merschjohann and Steverding, 2006).
References
-Balber AE, Gonias SL, Pizzo SV. Trypanosoma brucei gambiense: growth in vitro of bloodstream forms inhibited by dichlorodiammineplatinum (II) compounds and hypolipidemic drugs. Exp Parasitol. 1985 ; 59 (1) 74-80.
-Brun R, Baeriswyl S, Kunz C. In vitro drug sensitivity of Trypanosoma gambiense isolates. Acta Trop. 1989 Oct;46(5-6) 369-376.
-Carlton JM, Sina BJ, Adams JH. Why is Plasmodium vivax a neglected tropical disease? PLoS Negl Trop Dis. 2011; 5 (6) e1160. doi: 10.1371/journal.pntd.0001160.
-Hotez PJ, Aksoy S, Brindley PJ, Kamhawi S. What constitutes a neglected tropical disease? PLoS Negl Trop Dis. 2020, 14(1):e0008001. doi: 10.1371/journal.pntd.0008001.
-Likeufack AC, Brun R, Fomena A, Truc P. Comparison of the in vitro drug sensitivity of Trypanosoma brucei gambiense strains from West and Central Africa isolated in the periods 1960-1995 and 1999-2004. Acta Trop. 2006 Nov;100(1-2) 11-16.
-Merschjohann K, Steverding D. In vitro growth inhibition of bloodstream forms of Trypanosoma brucei and Trypanosoma congolense by iron chelators. Kinetoplastid Biol Dis. 2006; 5,3. doi: 10.1186/1475-9292-5-3.
-Shaw S, Roditi I. The sweet and sour sides of trypanosome social motility. Trends Parasitol. 2023; 39(4): 242-250.
-Tesoriero C, Xu YZ, Mumba Ngoyi D, Bentivoglio M. Neural damage in experimental Trypanosoma brucei gambiense infection: The suprachiasmatic nucleus. Front Neuroanat. 2018; 12:6. doi: 10.3389/fnana.2018.00006.
Author Response File: Author Response.pdf
Reviewer 4 Report
Comments and Suggestions for AuthorsThis article examines the possibility of the antiparasitic effects of L. adoensis extracts against Plasmodium, Leishmania and Trypanosoma parasites. The authors exhibit that the crude extracts of L. adoensis exhibit low, medium and high in some cases antiparasitic effect while they have also showed that these extracts had propable no cytotoxic effect to mammalian VERO cells.
The article is of some interest for the readers as the there is a high demand and need of new antiparasitic compounds especially of natural product origin.
However, before considering publication in JOMA journal, i propose some major and some minor corrections of the article below:
Introduction:
Sentence line 36: In which neglected disease is the sentence referring?
Line 38: Malaria disease must be mentioned before, otherwise it does not make any sense. Is this sentence related with the above sentence. If yes please write it clearer, otherwise please rewrite both sentences.
Materials and methods:
Paragraph 2.2.1.b
a) Please mention how the measurement is correlated with the viability of the parasites (as it is also mentioned with the resazurin method)
b) Please add some representative giemsa images before and after the treatment
Paragraph 2.2.2
Antileishmanial activity is better represented by in vitro intracellular amastigotes' assay instead of just checking only against the promastigote form as the intracellular amastigotes are the form of the parasite in the mammalian host. Please check also the in vitro intracellular amastigotes' activity of the extracts.
Paragraph 2.3.4 . b
Please rephrase the sentence in lines 332-334
Why the experiments are performed only 2 times? It is commonloy used to perform such assays in triplicates for 3 times minimum unless there is a major restriction
Table 1:
Please decide how many decimals you are going to use in all values of the table.
Results:
Paragraph 3.1.1
Line 366: "relevant and low, respectively"
Figure 2: please add the image with better resolution
Discussion:
Line 420: "...revealed low IC50..." instead of moderate
Please keep in mind that an accepted S.I for an antiparasitic compound is >5 or >10
Conclusion:
Line 485: "The significant low IC50..." is not applied to all assays, there is also moderate IC50s and must be mentioned
Throughout the text:
Put in italics throughout the text, in the figure legends, in the paragraph titles and in references: "in vitro", "L. donovani", "T. brucei" , "et al." and all the names of the parasites and plants
L. donovani throughout the text instead of L. Donovani
There are no names and affiliations of the aticle's authors, thus there is no way to check if there is excess self-citation in the references.
Author Response
Response to the reviewer’s comments point by point.
Manuscript ID: joma-3292203
Title: Antiprotozoal activity and cytotoxicity screening of Lippia adoensis (Hochst.) extracts: Growth inhibition of Plasmodium, Leishmania and Trypanosoma parasites
Referee 4
This article examines the possibility of the antiparasitic effects of L. adoensis extracts against Plasmodium, Leishmania and Trypanosoma parasites. The authors exhibit that the crude extracts of L. adoensis exhibit low, medium and high in some cases antiparasitic effect while they have also showed that these extracts had propable no cytotoxic effect to mammalian VERO cells.
The article is of some interest for the readers as the there is a high demand and need of new antiparasitic compounds especially of natural product origin.
However, before considering publication in JOMA journal, i propose some major and some minor corrections of the article below:
Introduction:
Sentence line 36: In which neglected disease is the sentence referring?
Answer: The sentence has now been rewritten. The changes have been coloured red with yellow stripes.
Line 38: Malaria disease must be mentioned before, otherwise it does not make any sense. Is this sentence related with the above sentence. If yes please write it clearer, otherwise please rewrite both sentences.
Answer: Both the sentences have now been rewritten as suggested. The changes have been coloured red with yellow stripes.
Materials and methods:
Paragraph 2.2.1.b
- a) Please mention how the measurement is correlated with the viability of the parasites (as it is also mentioned with the resazurin method)
Answer: Thank you for the observation. The changes have been amended as suggested.
- b) Please add some representative giemsa images before and after the treatment
Answer: Thank you for the observation. However, all information is already available in the main manuscript.
Paragraph 2.2.2
Antileishmanial activity is better represented by in vitro intracellular amastigotes' assay instead of just checking only against the promastigote form as the intracellular amastigotes are the form of the parasite in the mammalian host. Please check also the in vitro intracellular amastigotes' activity of the extracts.
Answer: We agree with this statement. We have fully considered the suggestion and have incorporated the in vitro intracellular amastigotes' activity of the extracts in the section « Limitations and perspectives» right before the conclusion section.
Paragraph 2.3.4 . b
Please rephrase the sentence in lines 332-334
Answer: The sentence has now been rewritten as suggested. Changes have been coloured red with yellow stripes in the main text.
Why the experiments are performed only 2 times? It is commonloy used to perform such assays in triplicates for 3 times minimum unless there is a major restriction
Answer: Thank you. Although triplicates generally offer larger sample size and a more robust statistical analysis and outlier detection, other authors (Julio et al., 2007 ; Green and Lee, 2023) have argued that duplicate testing is also effective at identifying and mitigating irregular laboratory errors, and is best suited for assays predisposed to such error, where costs are minimal.
Table 1:
Please decide how many decimals you are going to use in all values of the table.
Answer: Thank you. The number of decimals has now been harmonized for all the values in the table.
Results:
Paragraph 3.1.1
Line 366: "relevant and low, respectively"
Answer: The sentence has now been rewritten. The changes have been coloured red with yellow stripes.
Figure 2: please add the image with better resolution
Answer: The resolution of Figure 2 has now been improved as suggested.
Discussion:
Line 420: "...revealed low IC50..." instead of moderate
Answer: We agree with the reviewer’s observation. The sentence has now been rephrased. The changes have been coloured red with yellow stripes.
Please keep in mind that an accepted S.I for an antiparasitic compound is >5 or >10
Answer: We are thankful to the reviewer for this observation.
Conclusion:
Line 485: "The significant low IC50..." is not applied to all assays, there is also moderate IC50s and must be mentioned
Answer: We agree with the reviewer’s inquisitiveness. The sentence has now been rephrased.
Throughout the text:
Put in italics throughout the text, in the figure legends, in the paragraph titles and in references: "in vitro", "L. donovani", "T. brucei" , "et al." and all the names of the parasites and plants L. donovani throughout the text instead of L. Donovani
Answer: Thanks. Scientific names of plants and microorganisms have now been italicized as recommended.
There are no names and affiliations of the aticle's authors, thus there is no way to check if there is excess self-citation in the references.
Answer: Thank you. As per the Journal’s guidelines for manuscript submission, a separate title page with information regarding names and affiliations of the authors is provided during the submission. We believe that the Editorial Team of the Journal may provide a more suitable answer to this query.
We thank the reviewer for invaluable comments and we request for manuscript re-evaluation and kind consideration.
References
-Julio M. Singer, Antonio C. Pedroso-de-Lima, Nelson I. Tanaka, Verónica A. González-López, To triplicate or not to triplicate? Chemometrics and Intelligent Laboratory Systems 86 (2007) 82–85.
-Green AD, Lee GR. An appraisal of the practice of duplicate testing for the detection of irregular analytical errors. Clin Chem Lab Med. 2023 Nov 10;62(4):627-634. doi: 10.1515/cclm-2022-0605.
Author Response File: Author Response.pdf
