Anti-Shigellosis Activity and Mechanisms of Action of Extracts from Diospyros gilletii Stem Bark

Round 1

Reviewer 1 Report

Manuscript ID ddc-2831376

Anti-shigellosis activity and mechanisms of action of extracts from Diospyros gilletii stem bark.

This manuscript describes a study investigating the antibacterial activity of extracts from Diospyros gilletii, a medicinal plant traditionally used for treating diarrheal conditions, against Shigella species, which are responsible for causing shigellosis. Here's a critical review of the provided information:

Relevance of the Study: Shigellosis is a significant health issue, and the increasing resistance of Shigella species to existing therapies emphasizes the need for alternative treatments.

Exploring traditional medicinal plants for potential antibacterial properties aligns with the global search for new therapeutic options.

Methodology: The study employs various solvents for extracting compounds from Diospyros gilletii, providing a comprehensive analysis of potential antibacterial components. The use of a microdilution method for evaluating antibacterial activity and cytotoxicity tests on Vero and Raw cells adds scientific rigor to the study.

Results: The extracts from Diospyros gilletii show promising antibacterial activity against Shigella species without being toxic to normal cells. Time kill kinetics provide insights into the bactericidal and bacteriostatic effects of the extracts.

Mechanistic Insights: The study delves into the mechanisms of action by assessing bacterial membrane permeability and the ability of the extracts to inhibit and eradicate Shigella biofilms. The revealed mechanisms, such as increased membrane permeability and damage, contribute to a better understanding of how Diospyros gilletii extracts act against Shigella.

However, there are some aspect for Improvement:

This manuscript lacks specific details about the experimental setup, concentrations of extracts used, and specific conditions of the tests. Providing this information would enhance the study's reproducibility.

Statistical Analysis: While minimum inhibitory concentration (MIC) values are provided, information about statistical analysis or replicates is missing. Including such details would strengthen the reliability of the results.

Context on Diospyros gilletii: Some additional information about Diospyros gilletii, such as its traditional uses, chemical composition, or known pharmacological properties, could provide a more comprehensive background for readers unfamiliar with the plant.

Peer Review and Validation: The study's credibility could be further enhanced by mentioning if it has undergone peer review and if the results have been validated by other researchers or institutions.

Overall Impression:

The study presents valuable insights into the antibacterial properties of Diospyros gilletii extracts against Shigella species, offering potential alternative treatments for shigellosis. Addressing the suggested improvements would strengthen the study's scientific validity and enhance its contribution to the field.

Author Response

RESPONSE TO THE REVIEWER’S COMMENTS POINT BY POINT

Reviewer 1:

Manuscript ID ddc-2831376

Anti-shigellosis activity and mechanisms of action of extracts from Diospyros gilletii stem bark.

This manuscript describes a study investigating the antibacterial activity of extracts from Diospyros gilletii, a medicinal plant traditionally used for treating diarrheal conditions, against Shigella species, which are responsible for causing shigellosis. Here's a critical review of the provided information:

Relevance of the Study: Shigellosis is a significant health issue, and the increasing resistance of Shigella species to existing therapies emphasizes the need for alternative treatments.

Exploring traditional medicinal plants for potential antibacterial properties aligns with the global search for new therapeutic options.

Methodology: The study employs various solvents for extracting compounds from Diospyros gilletii, providing a comprehensive analysis of potential antibacterial components. The use of a microdilution method for evaluating antibacterial activity and cytotoxicity tests on Vero and Raw cells adds scientific rigor to the study.

Results: The extracts from Diospyros gilletii show promising antibacterial activity against Shigella species without being toxic to normal cells. Time kill kinetics provide insights into the bactericidal and bacteriostatic effects of the extracts.

Mechanistic Insights: The study delves into the mechanisms of action by assessing bacterial membrane permeability and the ability of the extracts to inhibit and eradicate Shigella biofilms. The revealed mechanisms, such as increased membrane permeability and damage, contribute to a better understanding of how Diospyros gilletii extracts act against Shigella.

However, there are some aspect for Improvement:

This manuscript lacks specific details about the experimental setup, concentrations of extracts used, and specific conditions of the tests. Providing this information would enhance the study's reproducibility.

Reply: We agree with the reviewer’s inquisitiveness. The preparation of the extract has now been incorporated in the sub-section 4.4. The range of concentrations used for in vitro assays has now been indicated in the material and methods’ section.

Statistical Analysis: While minimum inhibitory concentration (MIC) values are provided, information about statistical analysis or replicates is missing. Including such details would strengthen the reliability of the results.

Reply: We agree with the reviewer’s apprehension. The missing information has now been incorporated at the appropriate place (Table 1) of the manuscript. The changes have been coloured red with yellow stripes.

Context on Diospyros gilletii: Some additional information about Diospyros gilletii, such as its traditional uses, chemical composition, or known pharmacological properties, could provide a more comprehensive background for readers unfamiliar with the plant.

Reply: Thank you for the remark. Information about the traditional uses, chemical composition, or known pharmacological properties of Diospyros gilletii has now been incorporated in the introductory part of the manuscript. The changes have been coloured red with yellow stripes. In fact, the following statements have been added:

-Traditional uses: Although little is documented on the ethnomedicinal uses of D. gilletti, various organs (leaves, fruits, stem and bark) of other Diospyros species were recorded used to treat malaria, headache, dysentery, fever, and diarrhoea [22, 23]. The decoction of D. mafiensis and D. rotundifolia roots, as well as D. ferrea fruits are used for the traditional treatment of diarrhoea in Mozambique [24], South Africa [25], and India [26], respectively. The leaf and bark decoction of D. mespiliformis is used in the Central African Region to treat malaria, diarrhoea and dysentery, whereas the roots are used as chewing stick for oral hygiene [27, 28, 29]. In Cameroon, Diospyros gilletti is used by the Pygmées Baka people to treat respiratory tract diseases [30].

-Chemical composition: As a matter of fact, the chemistry of Diospyros gilletii and other Diospyros species were studied and reported by several authors [14, 15, 16, 17, 18]. Thanks to the research by Tameye [17], which detailed the extraction and isolation of pure compounds in various parts (leaves, stem bark, and twigs) of Diospyros gilletii that led to the identification of seven coumarins [11-O-(E)-cinnamoylnorbergenin (1) (leaves); 4-O-p-hydroxybenzoylnorbergenin (2); 4-O-galloylnorbergenin (3) and norbergenin (4) (leaves, stem bark and twigs); per-acetylated derivative of 4-O-p-hydroxybenzoylnorbergenin (5) and per-allylated derivative of 4-O-p-hydroxybenzoylnorbergenin (6) (stem bark); and 11-O-p-hydroxybenzoylnorbergenin (7) (twigs)] (Figure 1S, supplementary material); three pentacyclic triterpene acids [ursolic acid (8) and ursolic acid 28-allyle (9) (leaves); and corosolic acid (10) (leaves and twigs)]; and  five pentacyclic triterpenoids [lupeol (11) (leaves, stem bark and twigs); betulin (12) (leaves and twigs); betulinic acid (13) (leaves and stem bark); oleanolic acid (14) (leaves) and rotundic acid (15) (twigs)] (Figure 2S, supplementary material). Compounds 8, 10, 11, 13 and 14 were also identified in Diospyros fragrans by the same research group [18]. Furthermore, previous studies by Mallavadhani et al. [19] have shown that Diospyros plants afford an incredible amount of naphthoquinones, which can be used as a baseline for taxonomic studies of this plant. As summarized and discussed in Maridass’s research paper [20], the chemistry of Diospyros plants has led to the identification of numerous classes of compounds, such as flavonoids, terpenoids, fatty acids, coumarins, naphthoquinones, among others. More recently, Ribeiro et al. [21] documented the existence of triterpenoids, naphthoquinone, fatty acids, flavonoids, phenols, monoterpenoids, and diterpenoids and others in seventeen Diospyros species [15, 16, 21].

-Pharmacological properties: A few authors revealed that extract and compounds from D. gilletti possess antioxidant and antibacterial activities, without cytotoxicity on KB-3-1 (human epithelial carcinoma cells) and HT-29 (human colorectal adenocarcinoma cells with epithelial morphology) cell lines [14, 17].

Peer Review and Validation: The study's credibility could be further enhanced by mentioning if it has undergone peer review and if the results have been validated by other researchers or institutions.

Reply: Thank you very much for the suggestion.

Overall Impression:

The study presents valuable insights into the antibacterial properties of Diospyros gilletii extracts against Shigella species, offering potential alternative treatments for shigellosis. Addressing the suggested improvements would strengthen the study's scientific validity and enhance its contribution to the field.

Reply: We thank your valuable time and efforts to review our manuscript. Your comments and suggestions have significantly improved the quality of our manuscript.

We request the reviewer for re-evaluation and kind consideration.

References:

14. Tameye, N.S.J.; Akak, C.M.; Happi, G.M.; Frese, M.; Stammler, H.-G.; Neumann, B.; Lenta, B.N.; Sewald, N.; Nkengfack, A.E. Antioxidant norbergenin derivatives from the leaves of Diospyros gilletii De Wild (Ebenaceae). Phytochem. Lett. 2020, 36, 63-67.
15. Akak, C.M.; Djama, C.M.; Nkengfack, A.E.; Tu, P.-F.; Lei, L.-D. New coumarin glycosides from the leaves of Diospyros crassiflora (Hiern). Fitoterapia 2010, 81 (7), 873-877.
16. Akak, C.M.; Nkengfack, A.E.; Tu, P.F. Norbergenin derivatives from Diospyros crassiflora (Ebenaceae). Nat. Prod. Commun. 2013, 8 (11) 1575-1578.
17. Tameye, J.N.S. Phytochemical studies of Diospyros gilletii De Wild and Diospyros fragrans Gürke (Ebenaceae), chemical transformations and antibacterial, antioxidant and cytotoxic activities of extracts and isolated compounds. PhD Thesis Dissertation. University of Yaounde 1, Faculty of science, Department of Chemistry. 2022, 289 Pages. https://savoirs.cames.online/jspui/handle/20.500.12177/11202
18. Tameye, N.S.J.; Akak, C.M.; Tabekoueng, G.B.; Mkounga, P.; Bitchagno, G.T.M.; Lenta, B.N.; Sewald, N.; Nkengfack, A.E. 2022. Chemical constituents from Diospyros fragrans Gürke (Ebenaceae). Biochem. Syst. Ecol. 2022, p. 104373. https://doi.org/10.1016/j.bse.2021.104373
19. Mallavadhani, U.V.; Panda, A.K.; Rao, Y.R. Pharmacology and chemotaxonomy of Diospyros. Phytochemistry 1998, 49 (4) 901-951. doi: 10.1016/s0031-9422(97)01020-0.
20. Maridass, M. Phytochemicals from genus Diospyros (L.) and their biological activities. Ethnobot. Leafl. 2008, 12, 231-244.
21. Ribeiro A, Serrano R, da Silva IBM, Gomes ET, Pinto JF, Silva O. The genus Diospyros: A review of novel insights into the biological activity and species of Mozambican flora. Plants (Basel). 2023 Jul 31;12(15):2833. doi: 10.3390/plants12152833.
22. Ragasa C., Puno M., Sengson J., Shen , Rideout J. et Raga D. (2009). Bioactive triterpenes from Diospyros blancoi. Natural Product Research, 23(13), 1252-1258.
23. Rauf, A.; Uddin, G.; Patel, S.; Khan, A.; Halim, S.; Bawazeer, S.; Ahmad, K.; Muhammad, N.; Mubarak, M. 2017. Diospyros, an under-utilized, multi-purpose plant genus: A review. Pharmacother. 2017, 91, 714–730.
24. Mmongoyo J.A., Nair M.G., Linz J.E., Wu F., Mugula J.K., Dissanayake A.A., Zhang C., Day D.M., Wee J.M., Strasburg G.M. Bioactive compounds in Diospyros mafiensis roots inhibit growth, sporulation and aflatoxin production by Aspergillus flavus and Aspergillus parasiticus. World Mycotoxin. J. 2017, 10, 237-248.
25. Kaikabo A.A., Suleiman M.M., Samuel B.B., Eloff J.N. Antibacterial activity of eleven South African plants use in treatment of diarrhoea in folkloric medicine. Afr. J. Tradit. Complement. Altern. Med. 2009, 6, 315-316.
26. Kadavul K., Dixit A.K. Ethnomedicinal studies of the woody species of Kalrayan & Shervarayan hills, Eastern Chats, Tamil Nadu. Indian J. Tradit. Knowl. 2009, 8, 592–597.
27. Adzu B., Amos S., Muazzam I., Inyang U.S., Gamaniel K.S. Neuropharmacological screening of Diospyros mespiliformis in mice. J. Ethnopharmacol. 2002a, 83, 139-143.
28. Adzu B., Amos S., Dzarma S., Muazzam I., Gamaniel K.S. Pharmacological evidence favouring the folkloric use of Diospyros mespiliformis Hochst in the relief of pain and fever. J. Ethnopharmacol. 2002b, 82, 191-195.
29. Belemtougri R.G., Constantin B., Cognard C., Raymond G., Sawadogo L. Effects of two medicinal plants Psidium guajava (Myrtaceae) and Diospyros mespiliformis L. (Ebenaceae) leaf extracts on rat skeletal muscle cells in primary culture. J. Zhejiang Univ. Sci. B. 2006, 7, 56-63.
30. Brisson, R. « Mythologie des Pygmées Baka (Sud-Cameroun): mythologie et contes. » Peeters Publishers, 1999, 416 p.

Author Response File: Author Response.pdf

Reviewer 2 Report

Dear Editor,

The original manuscript entitled “Anti-shigellosis activity and mechanisms of action of extracts from Diospyros gilletii stem bark” is well-written, structured and developed by Talla et al. in suitable English with a clear structure. They evaluated the antibacterial activity of D. gilletii extracts against some Shigella species. They found growth inhibitory activities of D. gilletii extracts against S. boydii, S. flexneri and S. sonnei. This study is practical and interesting; however, there are some points of view which should be addressed and implemented as described below:

-        My main concern is the components of the extract. Did the authors analyse the extract? What are the main compounds in the extract? Which one can be considered the most effective antibacterial compound? Which compounds are mainly present in the extract? Please describe more the chemical composition and the profile of functional compounds in the extract comprehensively and compare it with other natural extracts with similar chemical profiles regarding the antibacterial activity in the discussion section. Please add the table of contents for the extract. Please determine which compound can be mainly regarded as the responsible component for each antibacterial activity evaluation in the discussion section. All sections must be revised accordingly.

-        Please illustrate the results of the biofilm evaluation by showing the microplates and the formed biofilms accordingly. 

Author Response

RESPONSE TO THE REVIEWER’S COMMENTS POINT BY POINT

Reviewer 2:

Dear Editor,

The original manuscript entitled “Anti-shigellosis activity and mechanisms of action of extracts from Diospyros gilletii stem bark” is well-written, structured and developed by Talla et al. in suitable English with a clear structure. They evaluated the antibacterial activity of D. gilletii extracts against some Shigella species. They found growth inhibitory activities of D. gilletii extracts against S. boydii, S. flexneri and S. sonnei. This study is practical and interesting; however, there are some points of view which should be addressed and implemented as described below:

Reply: We thank you for your optimistic view toward our manuscript.

-        My main concern is the components of the extract. Did the authors analyse the extract? What are the main compounds in the extract? Which one can be considered the most effective antibacterial compound? Which compounds are mainly present in the extract? Please describe more the chemical composition and the profile of functional compounds in the extract comprehensively and compare it with other natural extracts with similar chemical profiles regarding the antibacterial activity in the discussion section. Please add the table of contents for the extract. Please determine which compound can be mainly regarded as the responsible component for each antibacterial activity evaluation in the discussion section. All sections must be revised accordingly.

Reply: Thank you for the remark. As suggested, we have now revised and discussed the observed antibacterial activity in respect of the compounds of Diospyros gilletii. A brief overview of chemical composition of Diospyros gilletii has also been incorporated in the introductory part of the manuscript. The changes have been coloured red with yellow stripes within the main text.

     As a matter of fact, the chemistry of Diospyros gilletii and other Diospyros species has been studied and reported by several authors (Tamaye et al., 2020, https://doi.org/10.1016/j.phytol.2020.01.012; Tamaye et al., 2022, https://doi.org/10.1016/j.bse.2021.104373; Akak et al., 2010; https://doi.org/10.1016/j.fitote.2010.05.011; Akak et al., 2013, https://pubmed.ncbi.nlm.nih.gov/24427945/; etc.). Thanks to the PhD research work by Tamaye (2022) (https://savoirs.cames.online/jspui/handle/20.500.12177/11202) that detailed the extraction and isolation of pure compounds in various parts (leaves, stem bark, and twigs) of Diospyros gilletii along with another Diospyros species namely Diospyros fragrans (leaves and roots). As a result, Diospyros gilletii afforded eleven pure compounds from the leaves [4-O-p-hydroxybenzoylnorbergenin (1), 11-O-(E)-cinnamoylnorbergenin (2); 4-O-galloylnorbergenin (3); norbergenin (4); ursolic acid (5); corosolic acid (6); lupeol (7); botulin (8); betulinic acid (9); oleanolic acid (10) and ursolic acid 28-allyle (11)), eight compounds from the stem bark [4-O-p-hydroxybenzoylnorbergenin (1); 4-O-galloylnorbergenin (3); norbergenin (4); lupeol (7); botulin (8); betulinic acid (9); per-acetylated derivative of 4-O-p-hydroxybenzoylnorbergenin (12); and per-allylated derivative of 4-O-p-hydroxybenzoylnorbergenin (13)], and twelve compounds from the twigs, among which only eight could be characterized [4-O-p-hydroxybenzoylnorbergenin (1); 4-O-galloylnorbergenin (3); 11-O-p-hydroxybenzoylnorbergenin (14); norbergenin (4); corosolic acid (6); rotundic acid (15); lupeol (7); and betulinic acid (9)]. Compounds 5, 6, 7, 9 and 10 were also identified in Diospyros fragrans by the same research group (Tameye et al., 2022; https://doi.org/10.1016/j.bse.2021.104373). Furthermore, previous studies (Mallavadhani et al., 1998, doi: 10.1016/s0031-9422(97)01020-0) have shown that Diospyros plant species are known to produce an incredible amount of naphtoquinones, which could be considered as a chemical marker for a taxonomic study (Mallavadhani et al., 1998). As summarized and discussed in a review article by Maridass (2009), the chemistry of Diospyros plants has led to the identification of numerous classes of compounds, such as flavonoids, terpenoids, fatty acids, coumarins, naphtoquinones, among others (Maridass, 2009, https://core.ac.uk/download/pdf/60544644.pdf).  In a recently published paper (Ribero et al., 2023), a detailed phytochemical composition of plants from the genus Diospyrus was discussed. Thus, triterpenoids, naphthoquinone, fatty acids, flavonoids, phenols, monoterpenoids, diterpenoids, etc. were found to be the most prominent compounds in seventeen Diospyros species (Ribeiro et al., 2023, doi: 10.3390/plants12152833). Therefore, there is no denying that Diospyros gilletii and other Diospyros species contain a myriad of secondary metabolites that were identified and reported thus far.

      In this study, the observed antibacterial activity might be attributed to the presence of numerous metabolites [phenolic acids (betulinic acid, corosolic acid, ursolic acid and quercitol), coumarins (norbergenin, 4-O-galloylnorbergenin), and the cholesterol derivative β-sitosterol] found in Diospyros gilletii stem bark (Tamaye et al., 2020). As already discussed, Diospyros gilletii contain a number of bergenin and norbergenin derivatives, which are well known to possess antibacterial activity (Liu et al., 2018; Tamaye, 2022; Tamaye et al., 2022). In a recently published review article by Salimo et al. (2023), the antibacterial activity of bergenin and its derivatives were reported in 15 in vitro studies and 4 in vivo experiments (Salimo et al., 2023). Noteworthy, bergenin and its congeners participate in several mechanisms, such as lipid peroxidation inhibitory activity, free radical scavenging activity (Kour et al., 2019), and initiating apoptosis and cell cycle arrest in the G0/G1 phase (Mehta et al., 2022). Moreover, these compounds are able to easily cross the complex and multilayer lipopolysaccharide of bacteria cell walls, thereby causing lysis and cell death (Nyemb et al., 20018). Thus, it is not unreasonable to speculate that these compounds might have contributed to the anti-shigellosis activity.

Overall, Diospyros gilletii contain a myriad of secondary metabolites, including terpenes and terpenoids, coumarins (Figure 1S & 2S; see supplementary material), naphtoquinones, among others; however, a detailed chemistry study of this plant using latest generation (more sophisticated) techniques (HPTLC, UPLC, LC-MS, etc.) is warranted to depict the antibacterial potential hits and their pharmacokinetic profile. 

-        Please illustrate the results of the biofilm evaluation by showing the microplates and the formed biofilms accordingly.

Reply: We acknowledge that. However, all relevant information is already available in the text.

We request the reviewer for re-evaluation and kind consideration.

References

-Akak, C.M.; Djama, C.M.; Nkengfack, A.E.; Tu, P.-F.; Lei, L.-D. New coumarin glycosides from the leaves of Diospyros crassiflora (Hiern). Fitoterapia 2010, 81 (7), 873-877.

-Akak, C.M.; Nkengfack, A.E.; Tu, P.F. Norbergenin derivatives from Diospyros crassiflora (Ebenaceae). Nat Prod Commun. 2013, 8 (11) 1575-1578.

-Kour, H.; Raina, R.; Verma, P.K.; Pankaj, N.K.; Singh, S.P. Phytochemical ingredients and pharmacological properties of Bergenia ciliata. J. Vet. Pharmacol. Toxicol. 2019, 18, 1–10.

-Liu, B.; Wang, M.; Wang, X. Phytochemical analysis and antibacterial activity of methanolic extract of Bergenia purpurascens against common respiratory infection causing bacterial species in vitro and in neonatal rats. Microb. Pathog. 2018, 117, 315-319.

-Mallavadhani, U.V.; Panda, A.K.; Rao, Y.R. Pharmacology and chemotaxonomy of Diospyros. Phytochemistry. 1998, 49 (4) 901-951. doi: 10.1016/s0031-9422(97)01020-0.

-Maridass, M. Phytochemicals from genus Diospyros (L.) and their biological activities. Ethnobotanical Leaflets 2008, 12, 231-244.

-Mehta, S.; Kadian, V.; Dala, S.; Dalal, P.; Kumar, S.; Garg, M.; Rao, R. A fresh look on bergenin: vision of its novel drug delivery systems and pharmacological activities. Future Pharmacol. 2022, 2 (1), 64-91.

-Nyemb, J.N.; Djankou, M.T.; Talla, E.; Tchinda, A.T.; Ngoudjou, D.T.; Iqbai, J.; Mbafor, J.T. Antimicrobial, α-glucosidase and alkaline phosphatase inhibitory activities of bergenin, the major constituent of Cissus populnea roots. Med. Chem. 2018, 8, 426-430.

-Tameye, J.N.S. Phytochemical studies of Diospyros gilletii De Wild and Diospyros fragrans Gürke (Ebenaceae), chemical transformations and antibacterial, antioxidant and cytotoxic activities of extracts and isolated compounds. PhD Thesis Dissertation. University of Yaounde 1, Faculty of science, Department of Chemistry. 2022, 289 Pages. https://savoirs.cames.online/jspui/handle/20.500.12177/11202

-Tameye, N.S.J.; Akak, C.M.; Happi, G.M.; Frese, M.; Stammler, H.-G.; Neumann B.; Lenta, B.N.; Sewald, N.; Nkengfack, A.E. Antioxidant norbergenin derivatives from the leaves of Diospyros gilletii De Wild (Ebenaceae). Phytochemistry Letters 2020, 36, 63-67. https://doi.org/10.1016/j.phytol.2020.01.012

-Tameye, N.S.J.; Akak, C.M.; Tabekoueng, G.B.; Mkounga, P.; Bitchagno, G.T.M.; Lenta, B.N.; Sewald, N.; Nkengfack, A.E. 2022. Chemical constituents from Diospyros fragrans Gürke (Ebenaceae). Biochem. Syst. Ecol. 2022, p. 104373. https://doi.org/10.1016/j.bse.2021.104373

-Ribeiro A, Serrano R, da Silva IBM, Gomes ET, Pinto JF, Silva O. The genus Diospyros: A Review of Novel Insights into the Biological Activity and Species of Mozambican Flora. Plants (Basel). 2023, 12 (15) 2833. doi: 10.3390/plants12152833.

-Salimo, Z.M.; Yakubu, M.N.; da Silva, E.L.; de Almeida, A.C.G.; Chaves, Y.O.; Costa, E.V.; da Silva, F.M.A.; Tavares, J.F.; Monteiro, W.M.; de Melo, G.C.; Koolen, H.H.F. Chemistry and pharmacology of bergenin or its derivatives: A promising molecule. Biomolecules 2023, 13 (3) 403. doi: 10.3390/biom13030403.

Author Response File: Author Response.pdf

Reviewer 3 Report

This manuscript by Talla et al. studies the anti-shigellosis activity and potential mechanisms of action of extracts from D. gilletii stem bark. The introductions, methods, discussion, and conclusion sections are well described.  Several tests were carried out to evaluate the alcoholic extracts' potential. The results are very promising. The authors included good standards, the data ,and MIC values are well presented. This study helped to validate that this plant can be successfully used for treatment of diarrhea caused by Shigella species. 

Minor revisions. 

1. add a picture of the plant itself.

2. Typos. add a space between the units and the degree symbol (e.g., 37 ºC). This appears several times

Author Response

RESPONSE TO THE REVIEWER’S COMMENTS POINT BY POINT

Reviewer 3:

This manuscript by Talla et al. studies the anti-shigellosis activity and potential mechanisms of action of extracts from D. gilletii stem bark. The introductions, methods, discussion, and conclusion sections are well described.  Several tests were carried out to evaluate the alcoholic extracts' potential. The results are very promising. The authors included good standards, the data ,and MIC values are well presented. This study helped to validate that this plant can be successfully used for treatment of diarrhea caused by Shigella species. 

Reply: We thank you for the optimistic view of the reviewer toward our manuscript.

Minor revisions. 

1. add a picture of the plant itself.

Reply: We have now added a picture (Figure 8) of the studied plant in our manuscript.

2. Typos. add a space between the units and the degree symbol (e.g., 37 ºC). This appears several times

Reply: We agree with the reviewer’s suggestion. We have now added a space between the units and the degree symbol throughout the manuscript.

We request the reviewer for re-evaluation and kind consideration.

Author Response File: Author Response.pdf

Reviewer 4 Report

Overall the manuscript is ok. The author has investigated the antibacterial activity of Diospyros gilletii extracts against a number of Shigella species, including, Shigella flexneri, Shigella boydii, Shigella dysenteriae, and Shigella sonnei. The mechanistic basis of the antibacterial efficacy of D. gilletii extracts is also evaluated through biofilm inhibition and eradication, and membrane permeabilization assays.

The introduction part is ok. But in the methodology, the author should make it as "Materials and Methods", instead of writing separately.

The author needs to make some necessary changes, the author should remove the preparation of extracts and preparation of solutions part from the antibacterial activity heading and make it as a separate heading as 4.1.

What is "BEI resources", explain fully not in abbreviation form.

The author has mentioned that they have used "animals" in their study, and they need to provide the ethical committee approval details.

The table and figure numbering are not proper, the author has started the manuscript with Table 2, and Figure 6, why so?

Extensive English language editing and typographical corrections are required throughout the manuscript.

Extensive English language editing and typographical corrections are required throughout the manuscript.

Author Response

RESPONSE TO THE REVIEWER’S COMMENTS POINT BY POINT

Reviewer 4:

Overall the manuscript is ok. The author has investigated the antibacterial activity of Diospyros gilletii extracts against a number of Shigella species, including, Shigella flexneri, Shigella boydii, Shigella dysenteriae, and Shigella sonnei. The mechanistic basis of the antibacterial efficacy of D. gilletii extracts is also evaluated through biofilm inhibition and eradication, and membrane permeabilization assays.

Reply: We thank you for your positive view of the reviewer toward our manuscript.

The introduction part is ok. But in the methodology, the author should make it as "Materials and Methods", instead of writing separately.

The author needs to make some necessary changes, the author should remove the preparation of extracts and preparation of solutions part from the antibacterial activity heading and make it as a separate heading as 4.1.

Reply: Thank you. The changes have been amended as suggested. The preparation of extracts and preparation of solutions part have now been separated from antibacterial activity heading and rewritten under a separate heading. The changes have been colored red with yellow stripes in the main text.

What is "BEI resources", explain fully not in abbreviation form.

Reply: we agree with the reviewer’s suggestion. "BEI resources" has now been fully explained in the main text as follows: Biodefense and Emerging Infections Research Resources Repository.

The author has mentioned that they have used "animals" in their study, and they need to provide the ethical committee approval details.

Reply: In this study, the antibacterial and cytotoxic activities, as well as the antibacterial mechanism of action of Diospyros gilletti extracts were performed in vitro.

The table and figure numbering are not proper, the author has started the manuscript with Table 2, and Figure 6, why so?

Reply: The numbering has now been corrected as suggested.

Extensive English language editing and typographical corrections are required throughout the manuscript.

Reply: We have now proofread the manuscript and performed English editing. However, we would be ready to accept any other changes if required.

We request the reviewer for re-evaluation and kind consideration.

Author Response File: Author Response.pdf

Reviewer 5 Report

The work presented to me for review concerns a very interesting topic, arousing interest especially in the African countries where its authors come from. The work is based on a very good assumptions, is done at a good level, and the discussion of the results is well conducted, but the way of its presentation raises serious concerns. Therefore, let me point out the most important of my observations related to the way this work was edited. All these errors and defects are so important that they absolutely require correction before the work will be accepted for publication. Some of them are only subjective observations of the reviewer, but many others are classic editorial errors. And so, in the order they appear in the text, they are:

- Part 1. "Introduction" devotes a lot of space to schigellosis and says very little about the plant used for research.

- Another comment probably results from the imperfection of the template for entering the text of the work. Either way, there is inconsistent page numbering, which makes it much more difficult to pinpoint the location of necessary corrections.

- Since the tested plant is not widely known, it would be good to include a photo of it, as well as a detailed photo of the bark of the stems, because in the literature we can find descriptions of the activity of extracts also obtained from other parts of this plant.

- On line 102 there is information about the extraction efficiency and it is not known what it concerns. Only in lines 395-401 do we find the appropriate explanation. This requires appropriate extension at an earlier stage of the description, so that already in line 102 it is known what extracts and what yields we are talking about.

- This is where the most important remark appears, showing very little care in preparing the text. The presented work lacks Table 1. (numbering starts from 2.) and lack Figures 1-5. (here numbering starts from 6.). And it is not known whether it is just incorrect numbering or whether some important part of the text has been omitted.

- Explanations of abbreviations below the Tables are probably presented in alphabetical order. In my opinion, better readability would be achieved if they were explained in the order they appeared in the text of the Table (or in the text of the work).

- In Figures 6, 7, 8. I suggest keeping the same scale proportions for all charts, which will greatly facilitate their quick and effective mutual comparison.

- On line 174, the word "Figure 1." Appears but I think this is about "Figure 8". The above error confirms the above-mentioned total mess in the numbering of all the presented Figures.

- During the final editing of the text, efforts should be made to eliminate large blank spaces on some pages.

- The use of different nomenclature for extracts in the description of Figure 12 compared to the nomenclature used elsewhere is unclear.

- The initial 13 lines of the "Discussion" chapter are more typical of the “Introduction” and are practically almost a repetition of what the reader could already find in this introductory part.

- Throughout all the text of the work, the term "hydroethanol" is used many times and only in line 396 it is clarified. In my opinion, an explanation of what the authors mean by "hydroethanol" should have been provided much earlier.

- Line 308 contains information about phenolic acids. However, the exemplary substances listed there are not phenolic acids, but are mostly triterpene acids.

- The calculation formulas included in Chapter 4 require graphic unification.

- Lines 534-535 use the abbreviations “MBIC” and “MBEC”. What is unclear to me is the presence of the letter "B" in them. In such situations, the usual form of notation is MIC and MEC.

- There are relatively few, but significant, editorial errors in the work. Therefore, the text still requires another, significant correction in this respect.

And that's all of my detailed comments. This work, as I mentioned at the beginning, is interesting and therefore, after the authors have made all the suggested corrections, I recommend accepting it for publication without re-evaluation. However, if the authors discuss the validity of the comments presented, a new review of the corrected work will be required.

Author Response

RESPONSE TO THE REVIEWER’S COMMENTS POINT BY POINT

Reviewer 5:

The work presented to me for review concerns a very interesting topic, arousing interest especially in the African countries where its authors come from. The work is based on a very good assumptions, is done at a good level, and the discussion of the results is well conducted, but the way of its presentation raises serious concerns. Therefore, let me point out the most important of my observations related to the way this work was edited. All these errors and defects are so important that they absolutely require correction before the work will be accepted for publication. Some of them are only subjective observations of the reviewer, but many others are classic editorial errors. And so, in the order they appear in the text, they are:

- Part 1. "Introduction" devotes a lot of space to schigellosis and says very little about the plant used for research.

Answer: Thank you for your positive view toward our manuscript. In the introduction part, we have now presented more details regarding the chemistry and pharmacology of the studied plant. The changes have been coloured red with yellow stripes. In fact, the following statements have now been added:

-Chemical composition: As a matter of fact, the chemistry of Diospyros gilletii and other Diospyros species were studied and reported by several authors [14, 15, 16, 17, 18]. Thanks to the research by Tameye [17], which detailed the extraction and isolation of pure compounds in various parts (leaves, stem bark, and twigs) of Diospyros gilletii that led to the identification of seven coumarins [11-O-(E)-cinnamoylnorbergenin (1) (leaves); 4-O-p-hydroxybenzoylnorbergenin (2); 4-O-galloylnorbergenin (3) and norbergenin (4) (leaves, stem bark and twigs); per-acetylated derivative of 4-O-p-hydroxybenzoylnorbergenin (5) and per-allylated derivative of 4-O-p-hydroxybenzoylnorbergenin (6) (stem bark); and 11-O-p-hydroxybenzoylnorbergenin (7) (twigs)] (Figure 1S, supplementary material); three pentacyclic triterpene acids [ursolic acid (8) and ursolic acid 28-allyle (9) (leaves); and corosolic acid (10) (leaves and twigs)]; and  five pentacyclic triterpenoids [lupeol (11) (leaves, stem bark and twigs); betulin (12) (leaves and twigs); betulinic acid (13) (leaves and stem bark); oleanolic acid (14) (leaves) and rotundic acid (15) (twigs)] (Figure 2S, supplementary material). Compounds 8, 10, 11, 13 and 14 were also identified in Diospyros fragrans by the same research group [18]. Furthermore, previous studies by Mallavadhani et al. [19] have shown that Diospyros plants afford an incredible amount of naphthoquinones, which can be used as a baseline for taxonomic studies of this plant. As summarized and discussed in Maridass’s research paper [20], the chemistry of Diospyros plants has led to the identification of numerous classes of compounds, such as flavonoids, terpenoids, fatty acids, coumarins, naphthoquinones, among others. More recently, Ribeiro et al. [21] documented the existence of triterpenoids, naphthoquinone, fatty acids, flavonoids, phenols, monoterpenoids, and diterpenoids and others in seventeen Diospyros species [15, 16, 21].

-Pharmacological properties: A few authors revealed that extract and compounds from D. gilletti possess antioxidant and antibacterial activities, without cytotoxicity on KB-3-1 (human epithelial carcinoma cells) and HT-29 (human colorectal adenocarcinoma cells with epithelial morphology) cell lines [14, 17].

-Traditional uses: Although little is documented on the ethnomedicinal uses of D. gilletti, various organs (leaves, fruits, stem and bark) of other Diospyros species were recorded used to treat malaria, headache, dysentery, fever, and diarrhoea [22, 23]. The decoction of D. mafiensis and D. rotundifolia roots, as well as D. ferrea fruits are used for the traditional treatment of diarrhoea in Mozambique [24], South Africa [25], and India [26], respectively. The leaf and bark decoction of D. mespiliformis is used in the Central African Region to treat malaria, diarrhoea and dysentery, whereas the roots are used as chewing stick for oral hygiene [27, 28, 29]. In Cameroon, Diospyros gilletti is used by the Pygmées Baka people to treat respiratory tract diseases [30].

- Another comment probably results from the imperfection of the template for entering the text of the work. Either way, there is inconsistent page numbering, which makes it much more difficult to pinpoint the location of necessary corrections.

- Since the tested plant is not widely known, it would be good to include a photo of it, as well as a detailed photo of the bark of the stems, because in the literature we can find descriptions of the activity of extracts also obtained from other parts of this plant.

Answer: Thank you for the remark. A picture (Figure 8) of the plant has now been incorporated in the material and methods’ section.

- On line 102 there is information about the extraction efficiency and it is not known what it concerns. Only in lines 395-401 do we find the appropriate explanation. This requires appropriate extension at an earlier stage of the description, so that already in line 102 it is known what extracts and what yields we are talking about.

Answer: As suggested, we have now rewritten the concerned part (preparation of extracts) of the material ad methods’ section. The changes have been coloured red with yellow stripes.

- This is where the most important remark appears, showing very l ittle care in preparing the text. The presented work lacks Table 1. (numbering starts from 2.) and lack Figures 1-5. (here numbering starts from 6.). And it is not known whether it is just incorrect numbering or whether some important part of the text has been omitted.

Answer: We agree with the reviewer’s inquisitiveness. We have now incorporated the right numbering in the main text. The changes have been coloured red with yellow stripes.

- Explanations of abbreviations below the Tables are probably presented in alphabetical order. In my opinion, better readability would be achieved if they were explained in the order they appeared in the text of the Table (or in the text of the work).

Answer: The changes have been amended as suggested.

- In Figures 6, 7, 8. I suggest keeping the same scale proportions for all charts, which will greatly facilitate their quick and effective mutual comparison.

Answer: We have now provided a right numbering for the presented figures. The changes have been coloured red with yellow stripes across the manuscript.

- On line 174, the word "Figure 1." Appears but I think this is about "Figure 8". The above error confirms the above-mentioned total mess in the numbering of all the presented Figures.

Answer: We agree with the reviewer’s apprehension. We have now provided a right numbering for the presented figures. The changes have been coloured red with yellow stripes across the manuscript.

- During the final editing of the text, efforts should be made to eliminate large blank spaces on some pages.

Answer: The changes have been amended as recommended.

- The use of different nomenclature for extracts in the description of Figure 12 compared to the nomenclature used elsewhere is unclear.

Answer: We agree with the reviewer’s apprehension. The nomenclature of the extracts has now been corrected in the concerned Figures (6 and 7).

- The initial 13 lines of the "Discussion" chapter are more typical of the “Introduction” and are practically almost a repetition of what the reader could already find in this introductory part.

Answer: We agree with the reviewer’s remark. The initial 13 lines of the discussion section have now been rewritten to avoid repetition with statements embodied in the introductory part.

- Throughout all the text of the work, the term "hydroethanol" is used many times and only in line 396 it is clarified. In my opinion, an explanation of what the authors mean by "hydroethanol" should have been provided much earlier.

Answer: The change has been amended as suggested. The term “hydroethanol” has now been clarified at its first appearance in ten text.

- Line 308 contains information about phenolic acids. However, the exemplary substances listed there are not phenolic acids, but are mostly triterpene acids.

Answer: We agree with the reviewer’s suggestion. The changes have been amended as recommended.

- The calculation formulas included in Chapter 4 require graphic unification.

Answer: The change has now been amended as recommended. Graphic unification has now been applied to the formulas.

- Lines 534-535 use the abbreviations “MBIC” and “MBEC”. What is unclear to me is the presence of the letter "B" in them. In such situations, the usual form of notation is MIC and MEC.

Answer: We agree with the reviewer’s proposition. The change has now been amended as recommended.

- There are relatively few, but significant, editorial errors in the work. Therefore, the text still requires another, significant correction in this respect.

Answer: We have now proofread the manuscript and performed English editing. However, we would be ready to accept any other changes if required.

And that's all of my detailed comments. This work, as I mentioned at the beginning, is interesting and therefore, after the authors have made all the suggested corrections, I recommend accepting it for publication without re-evaluation. However, if the authors discuss the validity of the comments presented, a new review of the corrected work will be required.

Answer: We thank you for your efforts and time to reviewing our manuscript. The suggested corrections have significantly improve the quality of the manuscript.

We request the reviewer for re-evaluation and kind consideration.

References:

14. Tameye, N.S.J.; Akak, C.M.; Happi, G.M.; Frese, M.; Stammler, H.-G.; Neumann, B.; Lenta, B.N.; Sewald, N.; Nkengfack, A.E. Antioxidant norbergenin derivatives from the leaves of Diospyros gilletii De Wild (Ebenaceae). Phytochem. Lett. 2020, 36, 63-67.
15. Akak, C.M.; Djama, C.M.; Nkengfack, A.E.; Tu, P.-F.; Lei, L.-D. New coumarin glycosides from the leaves of Diospyros crassiflora (Hiern). Fitoterapia 2010, 81 (7), 873-877.
16. Akak, C.M.; Nkengfack, A.E.; Tu, P.F. Norbergenin derivatives from Diospyros crassiflora (Ebenaceae). Nat. Prod. Commun. 2013, 8 (11) 1575-1578.
17. Tameye, J.N.S. Phytochemical studies of Diospyros gilletii De Wild and Diospyros fragrans Gürke (Ebenaceae), chemical transformations and antibacterial, antioxidant and cytotoxic activities of extracts and isolated compounds. PhD Thesis Dissertation. University of Yaounde 1, Faculty of science, Department of Chemistry. 2022, 289 Pages. https://savoirs.cames.online/jspui/handle/20.500.12177/11202
18. Tameye, N.S.J.; Akak, C.M.; Tabekoueng, G.B.; Mkounga, P.; Bitchagno, G.T.M.; Lenta, B.N.; Sewald, N.; Nkengfack, A.E. 2022. Chemical constituents from Diospyros fragrans Gürke (Ebenaceae). Biochem. Syst. Ecol. 2022, p. 104373. https://doi.org/10.1016/j.bse.2021.104373
19. Mallavadhani, U.V.; Panda, A.K.; Rao, Y.R. Pharmacology and chemotaxonomy of Diospyros. Phytochemistry 1998, 49 (4) 901-951. doi: 10.1016/s0031-9422(97)01020-0.
20. Maridass, M. Phytochemicals from genus Diospyros (L.) and their biological activities. Ethnobot. Leafl. 2008, 12, 231-244.
21. Ribeiro A, Serrano R, da Silva IBM, Gomes ET, Pinto JF, Silva O. The genus Diospyros: A review of novel insights into the biological activity and species of Mozambican flora. Plants (Basel). 2023 Jul 31;12(15):2833. doi: 10.3390/plants12152833.
22. Ragasa C., Puno M., Sengson J., Shen , Rideout J. et Raga D. (2009). Bioactive triterpenes from Diospyros blancoi. Natural Product Research, 23(13), 1252-1258.
23. Rauf, A.; Uddin, G.; Patel, S.; Khan, A.; Halim, S.; Bawazeer, S.; Ahmad, K.; Muhammad, N.; Mubarak, M. 2017. Diospyros, an under-utilized, multi-purpose plant genus: A review. Pharmacother. 2017, 91, 714–730.
24. Mmongoyo J.A., Nair M.G., Linz J.E., Wu F., Mugula J.K., Dissanayake A.A., Zhang C., Day D.M., Wee J.M., Strasburg G.M. Bioactive compounds in Diospyros mafiensis roots inhibit growth, sporulation and aflatoxin production by Aspergillus flavus and Aspergillus parasiticus. World Mycotoxin. J. 2017, 10, 237-248.
25. Kaikabo A.A., Suleiman M.M., Samuel B.B., Eloff J.N. Antibacterial activity of eleven South African plants use in treatment of diarrhoea in folkloric medicine. Afr. J. Tradit. Complement. Altern. Med. 2009, 6, 315-316.
26. Kadavul K., Dixit A.K. Ethnomedicinal studies of the woody species of Kalrayan & Shervarayan hills, Eastern Chats, Tamil Nadu. Indian J. Tradit. Knowl. 2009, 8, 592–597.
27. Adzu B., Amos S., Muazzam I., Inyang U.S., Gamaniel K.S. Neuropharmacological screening of Diospyros mespiliformis in mice. J. Ethnopharmacol. 2002a, 83, 139-143.
28. Adzu B., Amos S., Dzarma S., Muazzam I., Gamaniel K.S. Pharmacological evidence favouring the folkloric use of Diospyros mespiliformis Hochst in the relief of pain and fever. J. Ethnopharmacol. 2002b, 82, 191-195.
29. Belemtougri R.G., Constantin B., Cognard C., Raymond G., Sawadogo L. Effects of two medicinal plants Psidium guajava (Myrtaceae) and Diospyros mespiliformis L. (Ebenaceae) leaf extracts on rat skeletal muscle cells in primary culture. J. Zhejiang Univ. Sci. B. 2006, 7, 56-63.
30. Brisson, R. « Mythologie des Pygmées Baka (Sud-Cameroun): mythologie et contes. » Peeters Publishers, 1999, 416 p.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

accept in present form. thank you for te revision

Reviewer 4 Report

The author has revised as suggested.

The author has revised as suggested.

Reviewer 5 Report

The authors positively responded to all my comments included in my earlier review from the first round of evaluation, and they included all the corrections and additions in the work which I suggested them. There were also other corrections suggested by other reviewers, which had an even more positive impact on the current scientific level, orderliness and appearance of the work. Now, after making an expected corrections, I recommend accepting the work for publication in its current form.