Halophytes as Medicinal Plants against Human Infectious Diseases

Round 1

Reviewer 1 Report

The authors propose a manuscript titled “Halophytes as medicinal plants against human infectious diseases”. The article give original information and is well written. on interesting topic on Halophytes that for many decades was used as entirely empirical, with no data of the bioactive compounds underlying the different applications. Many of these plants have a high nutritional, nutraceutical and pharmacological value and may represent added value to the emerging activity of saline agriculture and targeted modification of the rhizosphere with plant-growth promoting bacteria are being attempted as a tool to modulate the plant metabolome and enhance the expression of interesting metabolites. The authors in the review proposed highlight the potential of halophytes as a valuable, and still unexplored, source on antimicrobial compounds for clinical applications.

The manuscript deserve of some informations to be able published on international audience.

Introduction

·      Lines 29-30. Please specify. ….Approximately 3000 species in the world? or in a specific terriotry?;

·      Lines 30-31. Please complete in the suggested way (see your figure 2). “Halophytes have an extremely broad distribution, occurring over a wide range mostly in costal and wetlandshabitats [Perrino et al. 2021],….”;

·      Lines 39-42. Insufficient adjustment leads to osmotic and oxidative stress, ion toxicity and nutrient deficiency [choose reference]. High concentrations of NaCl in soil reduce water availability to the roots, the water potential of leaves and will ultimately limit nutrient uptake [choose reference];

·      Lines 51-54. Ethnobotanical literature provides evidence that because of their phytochemical richness, halophytic medicinal plants have long been used to treat various infectious diseases [choose reference], particularly in developing countries, where traditional medicine remains the first approach to minor ailments (10, Abenavoli et al. 2021).

Add reference:

Ø Perrino, E.V.; Magazzini, P.; Musarella, C.M. Management of grazing “buffalo” to preserve habitats by Directive 92/43 EEC in a wetland protected area of the Mediterranean coast: Palude Frattarolo, Apulia, Italy. Euro-Mediterranean Journal for Environmental Integration2021, 6, 32. doi: 10.1007/s41207-020-00235-2

Ø Abenavoli, L.; Milanovic, M.; Procopio, A.C. et al. Ancient wheats: beneficial effects on insulin resistance. Minerva Medica 2021 12(5), 641-50. doi: 10.23736/S0026-4806.20.06873-1.

2. Halophytes in traditional medicine and ethnopharmacology

Figure and table are clear. I give some suggestions

·      Line 74. Many halophytes have been used for centuries in TM [choose references] instead “Innumerous halophytes have been used for centuries in TM”;

·      Lines 104-105. Please report the scientific name in the complete way when the species is cited for the first time in the manuscript. Please check whole document in this way. 

ü Ziziphus spina-christi (Mill.) Georgi

ü Calotropis procera (Aiton) Dryand

ü Datura stramonium ….

ü Withania somnifera …

ü …..

3. Halophytes as a source of antimicrobial compounds and 4. 4. Current limitations and future prospects

Figures and tables are clear. Remember only my suggestion on the correct way to reporting the scientific name of plants.

·      Lines 300-301. Many bioactive metabolites are expressed by plants in response to stress or induced by microbial symbionts, and environmental conditions [choose reference as Perrino et al. 2021] colonizing plant tissues.

Perrino, E.V.; Valerio, F.; Jallali, S.; Trani, A.; Mezzapesa, G.N. Ecological and Biological Properties of Satureja cuneifolia Ten. and Thymus spinulosus Ten.: Two Wild Officinal Species of Conservation Concern in Apulia (Italy). A Preliminary Survey. Plants 2021, 10, 1952. doi: 10.3390/plants10091952.

Author Response

Thank you for your corrections and suggestions. We have made all the suggested alterations in the article text. 

Please see below, in blue,  a point-by-point response to the reviewers’ comments and concerns. All page numbers refer to the revised manuscript file with tracked changes. Please let us know if you would like more information to be included in the manuscript.

Introduction

• Lines 29-30. Please specify. ….Approximately 3000 species in the world? or in a specific terriotry?;

Line       30: …. approximately 3000 species in the world are….

• Lines 30-31. Please complete in the suggested way (see your figure 2). “Halophytes have an extremely broad distribution, occurring over a wide range mostlyin costal and wetlands habitats [Perrino et al. 2021],….”;

Line       32 ……over a wide range mostly in costal and wetlands….

• Lines 39-42. Insufficient adjustment leads to osmotic and oxidative stress, ion toxicity and nutrient deficiency [choose reference].

Line 40-41….. Insufficient adjustment leads to osmotic and oxidative stress, ion toxicity and nutrient deficiency [4].

        [4] Flowers, T.J.; Munns, R.; Colmer, T.D. Sodium chloride toxicity and the cellular basis of salt tolerance in halophytes. Annals of botany 2015, 115, 419-431.

 High concentrations of NaCl in soil reduce water availability to the roots, the water potential of leaves and will ultimately limit nutrient uptake [choose reference];

Line 41-43….High concentrations of NaCl in soil reduce water availability to the roots, the water potential of leaves and will ultimately limit nutrient uptake [8].

[8]Flowers, T.J.; Colmer, T.D. Salinity tolerance in halophytes. New Phytologist 2008, 945-963.

• Lines 51-54. Ethnobotanical literature provides evidence that because of their phytochemical richness, halophytic medicinal plants have long been used to treat various infectious diseases [choose reference], particularly in developing countries, where traditional medicine remains the first approach to minor ailments (10, Abenavoli et al. 2021).

Line 52-55…. Ethnobotanical literature provides evidence that because of their phytochemical richness, halophytic medicinal plants have long been used to treat various infectious diseases, particularly in developing countries, where traditional medicine remains the first approach to minor ailments [12,13].

[12] Ksouri, R.; Ksouri, W.M.; Jallali, I.; Debez, A.; Magné, C.; Hiroko, I.; Abdelly, C. Medicinal halophytes: potent source of health promoting biomolecules with medical, nutraceutical and food applications. Critical reviews in biotechnology 2012, 32, 289-326.

[13] Abenavoli, L.; Milanovic, M.; Procopio, A.C.; Spampinato, G.; Maruca, G.; Perrino, E.V.; Mannino, G.C.; Fagoonee, S.; Luzza, F.; Musarella, C.M. Ancient wheats: beneficial effects on insulin resistance. Minerva Medica 2020, 112, 641-650.

Add reference:

Ø Perrino, E.V.; Magazzini, P.; Musarella, C.M. Management of grazing “buffalo” to preserve habitats by Directive 92/43 EEC in a wetland protected area of the Mediterranean coast: Palude Frattarolo, Apulia, Italy. Euro-Mediterranean Journal for Environmental Integration2021, 6, 32. doi: 10.1007/s41207-020-00235-2

Line 33….reference added [2]

Add reference:

Ø Abenavoli, L.; Milanovic, M.; Procopio, A.C. et al. Ancient wheats: beneficial effects on insulin resistance. Minerva Medica 2021 12(5), 641-50. doi: 10.23736/S0026-4806.20.06873-1.

 Line 55….reference added [13]

2. Halophytes in traditional medicine and ethnopharmacology

Figure and table are clear. I give some suggestions

• Line 74. Manyhalophytes have been used for centuries in TM [choose references] instead “Innumerous halophytes have been used for centuries in TM”;
• Lines 104-105. Please report the scientific name in the complete way when the species is cited for the first time in the manuscript. Please check whole document in this way. 

ü Ziziphus spina-christi (Mill.) Georgi

ü Calotropis procera (Aiton) Dryand

ü Datura stramonium ….

ü Withania somnifera …

ü …..

The scientific names were reported in the complete way as suggested.

3. Halophytes as a source of antimicrobial compounds and 4. 4. Current limitations and future prospects

Figures and tables are clear. Remember only my suggestion on the correct way to reporting the scientific name of plants.

Thank you for your suggestion, however to no overload the tables, we suggest that the authors of the species/subspecies should not be included.

Lines 300-301. Many bioactive metabolites are expressed by plants in response to stress or induced by microbial symbionts, and environmental conditions [choose reference as Perrino et al. 2021] colonizing plant tissues.

Line 360….reference added [116]

Perrino, E.V.; Valerio, F.; Jallali, S.; Trani, A.; Mezzapesa, G.N. Ecological and Biological Properties of Satureja cuneifolia Ten. and Thymus spinulosus Ten.: Two Wild Officinal Species of Conservation Concern in Apulia (Italy). A Preliminary Survey. Plants 2021, 10, 1952. doi: 10.3390/plants10091952.

Reviewer 2 Report

The review concerns the problems of the use of halophytic plants for human medical purposes.

The halophytes live in salt media and remove the excess of the salt by its deposition in special intracellular compartments. In order to compensate the decrease of osmotic pressure they accumulate small organic molecules, such as olygosaccharides, polyols, amino acids, betaines et so on. This is one of the reasons of accumulation of natural products – preferably secondary metabolites (phenolic compounds, terpenoids and other) possessing wide spectrum of biological activities. The authors highlight the potential of halophytes as an important but still unexplored source on antimicrobial compounds for clinical use. The authors review an empirical use of halophytes in traditional medicine and the most relevant plant species and metabolites with antiviral, antifungal and antibacterial activities. They also discuss the current limitations in medicinal use of halophyte products and future prospects.

The article is a popular review that may be useful as an introduction into the problem. It is well written, the illustrations including figures and tables are adequate. Nevertheless, I should recommend several improvements:

The new figure with examples of most characteristic biologically active metabolites of halophytes should be created.

 In the last section, it should be necessary to note that mass-spectrometry metabolomics should be based not only on GLC/MS methods but also on HPLC/MS in order to cover not only volatile compounds but also non-volatile metabolites.

I        It also should be clearly noted that the metabolomic investigation should be only first stage of characterization of perspective halophyte species but the next stage – isolation of individual compounds, their structure identification using NMR technologies or even elucidation of structure of new natural products followed by bioassay.

My general recommendation: the manuscript may be published after minor revision.

Author Response

Thank you for your corrections and suggestions. We have made all the suggested alterations in the article text.

Please see below, in blue,  a point-by-point response to the reviewers’ comments and concerns. All page numbers refer to the revised manuscript file with tracked changes. Please let us know if you would like more information to be included in the manuscript:

The new figure with examples of most characteristic biologically active metabolites of halophytes should be created.

A new figure with the most characteristic biologically active metabolites found in halophytes was created (Figure 4. Most common bioactive compounds produced by halophyte plants. lines 360-362).

In the last section, it should be necessary to note that mass-spectrometry metabolomics should be based not only on GLC/MS methods but also on HPLC/MS in order to cover not only volatile compounds but also non-volatile metabolites.

We do agree with the reviewer. "The complexity and diverse chemical properties of plant metabolites demands a combined use of analytical platforms to increase the detection coverage of these compounds in biological samples. To detect volatile and non-volatile metabolites it is essential to use GC and HPLC coupled with mass spectrometry, as well as other techniques, such as UPLC or NMR to ensure that all or at least the majority of compounds are separated, detected, identified, quantified and characterized. Each method should also cover several extraction solvents to ensure the detection of both polar and non-polar compounds". These remarks were included in the last section of the article (lines 345-352).

It also should be clearly noted that the metabolomic investigation should be only first stage of characterization of perspective halophyte species but the next stage – isolation of individual compounds, their structure identification using NMR technologies or even elucidation of structure of new natural products followed by bioassay.

The isolation and chemical characterization of each compound, by NMR technologies and testing them in bioassays is indeed essential to move towards the assessment of the biological activity of each compound. These remarks were included in the last section of the article (lines  352-354 ).

Reviewer 3 Report

In the review of the manuscript titled: Halophytes as medicinal plants against human infectious diseases. The authors have provided a good description and the methodology is also fine. I would like to see this review publish but after some minor modifications as follow;

 
1. What are the main advantages of the mass-spectrometry-based plant metabolomics approach?

2.    The authors stated that there are several studies that establish the link between the empiric use of these plants to treat infections with the photochemistry evidence of the compounds that underlie antimicrobial effects. The authors should provide a comparison in detail.

3.    The authors are requested to briefly discuss methodological approaches for in vitro tests.

4.    The authors should briefly discuss different approaches towards the general objective of plant-derived effective anti-microbial compounds.

Comments for author File: Comments.docx

Author Response

Thank you for your corrections and suggestions. We have made all the suggested alterations in the article text.

Please see below, in blue,  a point-by-point response to the reviewers’ comments and concerns. All page numbers refer to the revised manuscript file with tracked changes. Please let us know if you would like more information to be included in the manuscript:

What are the main advantages of the mass-spectrometry-based plant metabolomics approach?

It was included in this new version more information concerning the advantages of using mass-spectrometry detectors in metabolomics:

Lines 347-352…."To detect volatile and non-volatile metabolites it is essential to use GC and HPLC coupled with mass spectrometry, as well as other techniques, such as UPLC or NMR to ensure that all or at least the majority of compounds are separated, detected, identified, quantified and characterized. Each method should also cover several extraction solvents to ensure the detection of both polar and non-polar compounds."

The authors stated that there are several studies that establish the link between the empiric use of these plants to treat infections with the photochemistry evidence of the compounds that underlie antimicrobial effects. The authors should provide a comparison in detail.

Lines 172-184…."Sixty-three species are reported in TM in Rwanda, to treat diarrheal-like symptoms [64]. In fact, the same plants are used to treat different diseases with similar symptomatology – diarrhoea, dysentery, cholera, and gastroenteritis in general, but different aetiology (helminths, bacteria, viruses. Some studies have found that only some plants have anti-microbial properties in in vitro and in vivo studies. And only a few studies have researched the bioactive phytocompounds of some of these plants [64]. This situation is not exclusive to plants with anti-microbial effects. A pharmacological investigation conducted over a pool of plants used in TM to treat cancer patients found only 30% had a significant cytotoxic effect. And only 6 out of 77 bioactive compounds isolated from those plants exhibited beneficial effects with few side effects in clinical trials [65]. Ethnopharmacological and phytochemistry studies are essential for the pharmacological industry to look for and produce new anti-microbial drugs [66]."

64. Gahamanyi, N.; Munyaneza, E.; Dukuzimana, E.; Tuyiringire, N.; Pan, C.-H.; Komba, E.V. Ethnobotany, Ethnopharmacology, and Phytochemistry of Medicinal Plants Used for Treating Human Diarrheal Cases in Rwanda: A Review. Antibiotics 2021, 10, 1231.
65. Merrouni, I.A.; Elachouri, M. Anticancer medicinal plants used by Moroccan people: Ethnobotanical, preclinical, phytochemical and clinical evidence. Journal of Ethnopharmacology 2021, 266, 113435.
66. Neiva, V.d.A.; Ribeiro, M.N.S.; Nascimento, F.R.; Cartágenes, M.d.S.S.; Coutinho-Moraes, D.F.; do Amaral, F.M. Plant species used in giardiasis treatment: ethnopharmacology and in vitro evaluation of anti-Giardia activity. Revista Brasileira de Farmacognosia 2014, 24, 215-224.

The authors are requested to briefly discuss methodological approaches for in vitro tests.

Lines 190-191: …"The development of new antimicrobial, anti-cancer, or anti-inflammatory drugs has been the subject of rising interest. The emergence of various drug-resistant microbes has directed attention to the need for new drugs and evaluating methods [67]."

67. Balouiri, M.; Sadiki, M.; Ibnsouda, S.K. Methods for in vitro evaluating antimicrobial activity: A review. Journal of pharmaceutical analysis 2016, 6, 71-79.

Lines 201-203:…".Other methods, like cytofluorometric and bioluminescent, require specified equipment and are not yet used as standard testing methods. These are quantifiable techniques and provide results within a short time window [67,68]."

67. Balouiri, M.; Sadiki, M.; Ibnsouda, S.K. Methods for in vitro evaluating antimicrobial activity: A review. Journal of pharmaceutical analysis 2016, 6, 71-79.
68. Oppong, J.R. Globalization of communicable diseases. International encyclopedia of human geography 2020, 223.

The authors should briefly discuss different approaches towards the general objective of plant-derived effective anti-microbial compounds.

Lines 333-341: …."The concern around drug-resistant microbes has drawn attention to plant-derived effective antimicrobial compounds. A new life is waiting for plants that have been used for centuries in infectious disease treatment. The detection and quantification of known and even the discovery of new small bioactive molecules produced by plants as secondary metabolites will provide new forms of therapy for numerous infectious and non-infectious illnesses. Appropriate and optimized extraction methods, susceptibility tests, and clinical trials are still required. The prospects of the outcome of further investigations seem promising and may lead to significant advances with the potential discovery of new and effective treatments [114,115]"

114. Savoia, D. Plant-derived antimicrobial compounds: alternatives to antibiotics. Future microbiology 2012, 7, 979-990.
115. Vaou, N.; Stavropoulou, E.; Voidarou, C.; Tsigalou, C.; Bezirtzoglou, E. Towards advances in medicinal plant antimicrobial activity: A review study on challenges and future perspectives. Microorganisms 2021, 9, 2041.