Seasonal Biochemical Variations in Mediterranean Halophytes and Salt-Tolerant Plants: Targeting Sustainable Innovations in Ruminant Health

Comments 1: This manuscript provides insights into seasonal biochemical variations in Mediterranean halophytes and salt-tolerant plants: Sustainable Innovations in Ruminant Health. The aim is to evaluate the effects of seasonality on biochemical properties, including proximate composition, minerals, antioxidant properties, and phenolic composition, of the aerial organs of halophytes and salt-tolerant species, with a view to their future exploitation in ruminant production as novel nutraceuticals or phytotherapeutics.

The work is generally relevant. Reviewing the cited literature, I see a considerable number of review articles, and the results shown in this manuscript are aligned with research published over the last 5 years.

The topic of this research has several published articles, but the species in this study are different; the information they provide is important for enriching the topic.

(...)

The conclusions of the work are consistent with the evidence presented, and it is clear that knowledge of these studied species maximizes their potential for ruminant nutrition and that they are excellent nutraceuticals and phytotherapeutics.

The references are appropriate; it is worth mentioning that 50% are from the last 5 years, making the manuscript a very up-to-date document on the subject.

I have no relevant comments (...)

Response 1: We would like to greatly acknowledge the reviewer’s comments on our article.

Comments 2: (…) It would have been interesting to be able to analyze the amino acid content in addition to proteins and lipids. In the case of species subjected to salt stress, they have high amino acid contents, for example, proline, which is also an important marker of stress in plants.

Response 2: Thank you for your valuable suggestion. We agree that this is a relevant topic that can be addressed in future work, thereof we have now pinpointed this subject in the Discussion section (lines 405-409).

Comments 3: (…) Furthermore, the parameters used to select tolerant species are not clear (lines 100-101). (…)

Response 3: From an initial list of salt-tolerant plants that we were able to identify in the Algarve Coast in previous projects, we selected these nine species based on the combined knowledge on their 1) traditional veterinary uses, systematically reviewed in [10], 2) phytochemical content reported in the literature as well as 3) their abundance in the wild setting in 2017/2018, targeting their use as nutraceuticals or phytotherapeutical products, with a particular focus on gastrointestinal nematodes infections. We have previously summarized this, however, in attempt to clarify this point we have improved it as suggested (lines 98-103).

Comments 4: (...) I only believe that the letters in Figure 3 are too small; increasing their size would allow for better reading.

Response 4: As recommended, we have increased the letters size and quality of all figures and legends across the document.

Comments 1: Why was the extract evaporated and dissolved in DMSO? What was the purpose and why could an acetone extract, which is typical for determining polyphenols, not be used?

Response 1: Thank you for your pertinent questions. Firstly, extracts were dried to accurately determine their mass, then, stock and working solutions were prepared by dissolving the dried extracts in DMSO. Considering that the extracts consist of a complex mixture of various compounds, DMSO was chosen as it is widely used for its ability to dissolve polar and non-polar metabolites, being chemically stable and compatible with diverse biological and chemical assays (e.g., in vitro cell culture techniques, antioxidant methods). Regarding the employed extraction solvents, the 80% acetone water mixture was previously shown as best to extract polyphenols from different halophyte species, yielding increased antioxidant results, such as L. monopetalum, I. crithmoides and Crithmum maritimum ^1,2. We have added this point to more accurately justify this choice, in lines 346-348. Moreover, extracts prepared with acetone/water mixtures are often reported as exhibiting enhanced anthelmintic activity^3,4, which aligns with our previously published findings on the anthelmintic value of these salt-tolerant species extracts^5,6.

1.      Trabelsi, N. et al. 2010. LWT - Food Sci. Technol., 43, 632-639. https://doi.org/10.1016/j.lwt.2009.11.003

2.      Jallali I et al. 2014. Food Chem, 145:1031-8. https://doi.org/10.1016/j.lwt.2009.11.003

3.      Barrau, E. et al. 2005. Parasitology, 131, 531. https://doi.org/10.1017/S0031182005008024

4.      Vargas-Magaña, J. J. et al. 2014. Vet. Parasitol., 206, 322–327. https://doi.org/10.1016/j.vetpar.2014.10.008

5.      Oliveira, M. et al., 2021. Sci Rep, 11, 24303. https://doi.org/10.1038/s41598-021-03472-9

6.      Oliveira, M. et al., 2022. Front Plant Sci, 13, https://doi.org/0.3389/fpls.2022.934644

Comments 2: The methodology for determining antiradical properties should be described in detail.

Response 2: Thank you for your comment. The individual results concerning C. mariscus seasonal samples are identified in the manuscript as published elsewhere¹ (Fig 2-4; 7). Having this in mind, and since the methodologies performed dealing with the phenolic content and antioxidant properties of the other eight species extracts are the same and were done in parallel, we decided that we would not be exhaustively detailing them herein, as it would be redundant, and thereof, we remit the readers to the respective reference of the published work.

¹ Oliveira, M. et al. 2021. Plants, 10, 556, https://doi.org/10.3390/plants10030556 

Comments 3: The captions on the drawings are illegible.

Response 3: Thank you for pinpointing that. As suggested, we have increased the letters size and quality of all figures and legends across the document.

Comments 4: Have the indicated uses (e.g. nutraceuticals, feed) been confirmed by toxicological and pharmacological studies, or are they based solely on the content of bioactive ingredients?

Response 4: In this study, we have not addressed either the toxicological or pharmacological studies of these plants biomass and/or extracts, thereof, the prospected uses are based on their content of bioactive ingredients. However, we did have in consideration some aspects on the choice of the salt-tolerant and halophyte species to work with. For example, H. italicum aerial parts are traditionally used as forage to stimulate rumination in bovines and to improve milk quality in sheep^1,2 and in vitro investigations of its decoctions and infusions did not reveal toxicity³; I. crithmoides is an edible halophyte, widely appreciated in Mediterranean gastronomy and there is report of field observations of goats grazing on this plant along the Lebanese coast⁴; Plantago sp., including P. coronopus, and Medicago sp. are widely used as fodders for livestock⁵, M. sativa (alfalfa) being one of the most used resources; L. monopetalum has been pointed out as a potential non-conventional fodder resource for ruminant animals along North African countries and P. lentiscus is a tannin-rich fodder resource ⁶. C. mariscus, L. monopetalum, I. crithmoides and P. lentiscus 70% acetone extracts were generally safe when tested in vitro against different cell lines, and only C. soldanella displayed significant toxicity⁷. Still, we agree that, particularly for nutraceutical/phytotherapeutic applications, further investigations should not neglect the toxicological and pharmacological aspects of the future developed products.

1.      Uncini Manganelli et al. (2001). J. Ethnopharmacol., 78, 171- 191. https://doi.org/10.1016/s0378-8741(01)00341-5

2.      Guarrera P. M. (2005). Fitoterapia, 76(1), 1–25. https://doi.org/10.1016/j.fitote.2004.09.006

3.      Zurayk, R.A., & Baalbaki, R. (1996). Arid Soil Research and Rehabilitation, 10(3), 213-223. https://doi.org/10.1080/15324989609381436

4.      Pereira, C. G. et al. 2017. J. Pharm. Biomed. Anal., 145, 593–603. https://doi.org/10.1016/j.jpba.2017.07.007

5.      Oliveira, M. et al. 2021. J Ethnopharmacol, 267, 113464, https://doi.org/10.1016/j.jep.2020.113464

6.      Silanikove et al., 1996. Small Rum Res. 21, 195-201. https://doi.org/10.1016/0921-4488(95)00833-0

7.      Lopes, A. et al. (2016). Ind. Crop. Prod., 94, 299–307. https://doi.org/10.1016/j.indcrop.2016.08.040

4. Response to Comments on the Quality of English Language

Point 1: The English could be improved to more clearly express the research.

Response 1: As suggested, we have improved the English along the document.

Comments 1:

Abstract

1. All plant species in the manuscript should be italicized, except the taxonomic authorities.

Introduction

1. The hyphen should be removed from the word "salinization". Line 39.

2. Many words have a hyphen that was not removed when submitting the final version of the manuscript. Please review the entire manuscript and remove these hyphens. In the introduction, some were highlighted in blue.

3. All plant species in the manuscript should be italicized, except the taxonomic authorities.

Response 1: We sincerely appreciate the reviewer’s comments. It appears that the previously submitted manuscript experienced formatting issues, which we have carefully addressed and made the necessary corrections throughout the revised submission.

Comments 2:

Materials and Methods

1. Please review all plant species names and check them against The Plant List or Tropicos databases.

2. In the chemical compound AlCl3, the number 3 should be used as a subscript. Please correct this. Line 138

3. The number -1 should be used as a superscript. Line 146. This error is repeated several times throughout the manuscript.

4. ¿The P-value was P=0.05 or P≤0.05? Line 184.

Response 2: Thank you for your valuable comments and suggestions, which were all addressed in the revised manuscript, and justified below.

1.     We have reviewed and checked all plant species names, modified them according to the NCBI Taxonomy Browser, and referenced it accordingly ¹ (lines 91-198)

2-3. As previously stated, we have corrected the formatting issues including the subscript number in AlCl₃ (line 149) as well as for the sub- and superscript letters or numbers throughout the manuscript.

4. Indeed, we have corrected this to p ≤ 0.05 (line 192).

¹

Comments 3:

Results

1. What is the objective of determining ash in this study? How does it benefit animals?

2. Why do the parameters show the lowest values ​​during dry seasons?

3. All the plant names should be in italics.

4. The figures lack the resolution necessary for publication in a scientific article. This aspect needs to be improved.

5. Why were tannins is not determined in all plants?

Response 3: We deeply appreciate your questions and suggestions, which are addressed below.

1.     Ash provides an estimation of the total mineral and organic matter contents (dry matter – ash = organic matter), thereby adding to the prospection of the overall nutritional value of the biomass. High ash values impact animal feed intake and digestibility, therefore, the use of the plants as nutraceutical plants or forages may be limited in this case scenario. On the other hand, minerals are essential for health, productivity and performance of the animals, at different production levels, which led us to pursue the quantification of the individual elements, targeting the potential exploitation of the biomass as nutraceutical products with enriched mineral content or mineral feed supplements. This topic has been addressed in the Discussion section (lines 421-437).

2.     The overall decrease in CP during the dry seasons is frequent, and closely linked to each plant phenological cycle, but also to the decreased total rainfall and increased drought stress observed in these seasons, which negatively impact plant growth and development. Having this in mind, plants might be allocating their resources to defence instead of growth, through the biosynthesis of other metabolites instead of protein, to cope with the environmental adversities faced during these periods. That is supported by the observed trend in this work of the increased total flavonoid contents in Su/Sp, considering their role in defence against light stress and UV radiation.

3-4. As priorly stated, the formatting issues were resolved, as well as the quality of all figures was improved and added to this revised version.

5.   Indeed, all samples were tested for their tannin content – however, these were only detected and quantified in the samples of those three species (L. monopetalum, P. lentiscus and C. mariscus). To clarify this question, we added ‘’< LOQ’’ to Figure 4, and the respective value for the limit of quantification (LOQ) to the legend of the figure (line 274).

Comments 4:

Discussion

1.     Fundamental questions need to be addressed in more depth. For example, why do nutritional values ​​vary across seasons? What role do antioxidants play in animal nutrition? How could these halophytes be introduced into the field of veterinary medicine and animal nutrition?

Response 4:

Why do nutritional values ​​vary across seasons?

We agree that this fundamental question is of major relevance, but further data would be necessary to more accurately attempt to answer it. In the wild setting, plants are subjected to a myriad of abiotic and biotic factors, which interact simultaneously, influencing primary and secondary metabolisms. It gets even more complex if each plant-related factors are added to the equation (i.e., organs, evolutionary adaptations and ecological requirements) that most probably also contributed to the observed variations. That said, in this work our focus was on when seasonal variations occur and how they impact biotechnological value, by identifying optimal harvesting periods, rather than delving more deeply into why. Nonetheless, we agree that unravelling the ecological features that shape each plant species’ chemical profile is of utmost significance to be explored in future works. In alignment, we have pinpointed this subject in the Conclusions section (lines 519-524).

What role do antioxidants play in animal nutrition?

We believe that this question was previously addressed in the Discussion section, yet as suggested, we did elaborate it aiming at a better understanding of the potentialities of these plants, particularly due to their phenolic contents, in the context of animal nutrition and health (lines 472-482).

How could these halophytes be introduced into the field of veterinary medicine and animal nutrition?

We are grateful for the reviewer’s input. We believe that this question is addressed throughout the Discussion section, either by 1) using them in complementary strategies or as source of feed supplements (lines 415-420), 2) exploiting them as mineral supplements (lines 422-437); 3) as nutraceutical or phytotherapeutical products to be developed, based on their contents of phenolic metabolites of bioactive interest (lines 438-444) or 4) as sources of added-value individual phenolic metabolites (lines 449-471). Moreover, the potentialities of each species and their applications were also highlighted in the Conclusions section (lines 502-518).

Comments 5:

Overall considerations

The entire manuscript should be spell-checked: a large number of hyphenated words are included. Many symbols and numbers should be superscript or subscript. All plant species have spelling errors and should be italicized. All taxonomic authorities should be reviewed. The discussion of the topics mentioned should be further elaborated. The figures presented should be of improved quality.

Response 5:

We are grateful for the reviewer’s meaningful contributions for the improvement of this article. As recommended, we have checked the entire manuscript and resolved the formatting issues, checked plant species taxonomic authorities and spelling and improved all figures’ quality. Lastly, we improved the Discussion section by addressing the topics raised by the reviewer.

4. Response to Comments on the Quality of English Language

Point 1: The English could be improved to more clearly express the research.

Response 1: As suggested, we have improved the English to more clearly state our research.