Opuntia ficus-indica L. Mill Residues—Properties and Application Possibilities in Food Supplements

Round 1

Reviewer 1 Report

The article presents an assessment of quality parameters (Mass uniformity, thickness and diameter; Tablet breaking force; Friability; Disintegration; Scanning electron microscopy (SEM); Functional properties: Lipid and water holding capacity; Color measurement; Antioxidant properties) for tablet formulations developed by mixing two commercial excipients microcrystalline cellulose (M) and α-lactose-monohydrate (L) and cactus pear residues. The results were compared with the tolerance limits established by United States Pharmacopoeia Commission.

The experimental data were obtained in a well-organised work. The manuscript is well written, and contains the key-elements according to the journal requirements. Material and Methods is excellent written, every step is enough detailed to repeat the measurements in same conditions.

However, the manuscript do not consider the determination of some trace elements in Dietary Supplements (at least in the introduction Section). See for reference:

 

Senila, M., Cadar, O., Senila L., Hoaghia, A., Miu, I. 2019, Mercury determination in natural zeolites by thermal decomposition atomic absorption spectrometry: Method validation in compliance with requirements for use as dietary supplements, Molecules, 2019, 24, 4023.

Author Response

Response to Reviewer 1 Comments

Point 1: The article presents an assessment of quality parameters (Mass uniformity, thickness and diameter; Tablet breaking force; Friability; Disintegration; Scanning electron microscopy (SEM); Functional properties: Lipid and water holding capacity; Color measurement; Antioxidant properties) for tablet formulations developed by mixing two commercial excipients microcrystalline cellulose (M) and α-lactose-monohydrate (L) and cactus pear residues. The results were compared with the tolerance limits established by United States Pharmacopoeia Commission.

The experimental data were obtained in a well-organised work. The manuscript is well written, and contains the key-elements according to the journal requirements. Material and Methods is excellent written, every step is enough detailed to repeat the measurements in same conditions.

However, the manuscript do not consider the determination of some trace elements in Dietary Supplements (at least in the introduction Section). See for reference:

Senila, M., Cadar, O., Senila L., Hoaghia, A., Miu, I. 2019, Mercury determination in natural zeolites by thermal decomposition atomic absorption spectrometry: Method validation in compliance with requirements for use as dietary supplements, Molecules, 2019, 24, 4023.

 

Response 1:

 It is important to mention that this work only reports the main characteristics of the tablets, due to the nature of the base sample, which is rich in fiber and antioxidant compounds (Barba et al., 2017). About the evaluation of trace elements, it is contemplated to perform the analysis of the best formulation, for a future publication.

Barba, F. J., Putnik, P., Kovačević, D. B., Poojary, M. M., Roohinejad, S., Lorenzo, J. M., & Koubaa, M. (2017). Impact of conventional and non-conventional processing on prickly pear (Opuntia spp.) and their derived products: From preservation of beverages to valorization of by-products.

According to the recommendations, some trace elements that have been reported were added as a part of the introduction in lines 64-66 as follows: 

The non-edible portion constituted as peel, comprise two fractions mesocarp and pericarp, which are rich in antioxidants, soluble sugars and dietary fiber, as well as trace elements such as calcium, magnesium, potassium and manganese (El Kossori et al., 1998).

 

El Kossori, R. L., Villaume, C., El Boustani, E., Sauvaire, Y., & Méjean, L. (1998). Composition of pulp, skin and seeds of prickly pears fruit (Opuntia ficus indica sp.).

Author Response File: Author Response.docx

Reviewer 2 Report

Article: Applied Sciences-779244

Title: Opuntia ficus-indica L. Mill residues as a Potential Dietary Supplement

 

The manuscript from Manzur-Valdespino et al. gives an insight into the physical-chemical properties of tablets prepared from Opuntia ficus-indica L. Mill residues. The manuscript could be of interest for the readership of the journal. With some exceptions, the experiments have been well conducted and the results are clearly showed. However major revisions are required. The manuscript is not very easy to read and should be revised by English native.

 

Major:

1) The data showed in the tables need to be carefully revised, taking into consideration the decimal figures of errors. For example, in Table 1 “120.7 ± 8.0” should be changed into “121 ± 8”, “162.4 ± 28.9” into “162 ± 29”, “163.6 ± 28.9” into “164 ± 29”, “96.4 ± 13.1” into “96 ± 13”, and so on. Please check also Table 2 and Table 3 for similar modifications.

2) The DPPH and FRAP assays must be validated by the use of appropriate reference standards (Trolox or Ascorbic acid for DPPH, BHT for FRAP) with known antioxidant activity. These data should be included in Table 3.

 

Minor:

Pag 2, line 61: Please change “fiber dietary” with “dietary fiber”.

Pag 6, lines 245-249: The sentence should be re-written in a clearer way.

Author Response

Response to Reviewer 2 Comments

The manuscript from Manzur-Valdespino et al. gives an insight into the physical-chemical properties of tablets prepared from Opuntia ficus-indica L. Mill residues. The manuscript could be of interest for the readership of the journal. With some exceptions, the experiments have been well conducted and the results are clearly showed. However major revisions are required. The manuscript is not very easy to read and should be revised by English native.

Point 1:

1) The data showed in the tables need to be carefully revised, taking into consideration the decimal figures of errors. For example, in Table 1 “120.7 ± 8.0” should be changed into “121 ± 8”, “162.4 ± 28.9” into “162 ± 29”, “163.6 ± 28.9” into “164 ± 29”, “96.4 ± 13.1” into “96 ± 13”, and so on. Please check also Table 2 and Table 3 for similar modifications.

Response 1:

The suggested changes by the reviewer were made

Point 2:

The DPPH and FRAP assays must be validated by the use of appropriate reference standards (Trolox or Ascorbic acid for DPPH, BHT for FRAP) with known antioxidant activity. These data should be included in Table 3.

Response 2:

Data of reference standards has been included in table 3. As well as concentrations of calibration curves for ABTS, DPPH and FRAP assays were also added in the material and methods section.

Point 3:

Minor:

Pag 2, line 61: Please change “fiber dietary” with “dietary fiber”.

Response 3:

The terms has been changed.

Point 4:

Pag 6, lines 245-249: The sentence should be re-written in a clearer way.

Response 4:

The sentence has been re-written in lines 259-264 as follows:

In this context, the GL tablet presented a greater resistance to breakage, less friability and a longer disintegration time compared to the other excipients. These differences can be explained by the influence of the different excipients analyzed, their physical and chemical properties of each one, given that several researchers have reported that the excipients are a considerable critical factor for the development of new pharmaceutical formulations with greater pharmacological functionality (disintegration, dissolution, release and bioavailability of the drug) (Pishnamazi et al., 2019; Van Den Mooter 2012).

 

Pishnamazi, M.; Iqbal, J.; Shirazian, S.; Walker, G. M. and Collins, M.N. Effect of lignin on the release rate of acetylsalicylic acid tablets

Van Den Mooter, G. The use of amorphous solid dispersions: A formulation strategy to overcome poor solubility and dissolution rate, Drug Discovery Today

 

Author Response File: Author Response.docx

Reviewer 3 Report

In subjected paper, the Authors presented studies upon the evaluation quality parameters, functional and antioxidant properties in tablet formulations developed from cactus pear residues.

Authors presented this issue in a clear way by describing it correctly in a chapter “Materials and methods”. They also presented and discussed those results in chapters “Results” and “Discussion”.

Minor note:

Introduction: line 44-45 “It does not belong to the category of food, neither to the category of  medicine product [3].” - This sentence is incorrect - dietary supplements belong to food - in accordance with legal regulations. This sentence should be corrected.

Line 67 “Nowadays, tablets have a greater demand by consumers than other forms of medications [20],” - This is debatable - it depends on age. For example, children and the elderly prefer liquid dietary supplements. Some people have trouble swallowing tablets.

Results and discussion: line 272-274 “In this sense, the tablets of the green variety have a fiber content of 17.1 g/100g and the tablets 273 of the red variety, 10.9 g/100g, which would cover 68 and 43% respectively of the recommended daily 274 intake for adults [50].” - This sentence needs to be corrected. Rather, no one will consume 100 g tablets a day. Please refer to the daily portion of consumption. What daily portion may be recommended? (in supplements we are not talking about doses but about portions of consumption - doses are in the case of drugs)

I would also like to add that the advisor is not a native speaker  and because of that I suggest that before having it published, language expert should read it.

Author Response

Response to Reviewer 3 Comments

 

In subjected paper, the Authors presented studies upon the evaluation quality parameters, functional and antioxidant properties in tablet formulations developed from cactus pear residues.

Authors presented this issue in a clear way by describing it correctly in a chapter “Materials and methods”. They also presented and discussed those results in chapters “Results” and “Discussion”.

 

Point 1:

Introduction: line 44-45 “It does not belong to the category of food, neither to the category of  medicine product [3].” - This sentence is incorrect - dietary supplements belong to food - in accordance with legal regulations. This sentence should be corrected.

 

Response 1:

The sentence has been corrected in line 45-46 as follows:

These supplements are regulated by the Food and Drug Administration (FDA, 2019) as food and not as drugs (FDA, 2019).

 

U.S. Food & Drug Administration. Diettary Supplements.

 

Point 2:

Line 67 “Nowadays, tablets have a greater demand by consumers than other forms of medications [20],” - This is debatable - it depends on age. For example, children and the elderly prefer liquid dietary supplements. Some people have trouble swallowing tablets.

 

Response 2:

The reviewer is in all reason therefore the sentence has been changed in line 74-76 as follows:

The drugs or food supplements are introduced into the body through the mouth and are absorbed in the digestive tract, between other dosage forms as liquids, emulsions or powders, the use of tablets is a convenient and easy way of administration (Allen and Ansell, 2013).

 

Allen, L., & Ansel, H. C. (2013). Ansel's pharmaceutical dosage forms and drug delivery systems. Lippincott Williams & Wilkins.

 

Point 3:

Results and discussion: line 272-274 “In this sense, the tablets of the green variety have a fiber content of 17.1 g/100g and the tablets 273 of the red variety, 10.9 g/100g, which would cover 68 and 43% respectively of the recommended daily 274 intake for adults [50].” - This sentence needs to be corrected. Rather, no one will consume 100 g tablets a day. Please refer to the daily portion of consumption. What daily portion may be recommended? (in supplements we are not talking about doses but about portions of consumption - doses are in the case of drugs)

 

Response 2:

The reviewer is in all reason, therefore the sentence has been corrected in line 287-296 as follows:

In this sense, each tablet with green residues have a fiber content of 0.24 g and 0.15 g to red residues aproximately. According to the recommended daily intake of fiber for adults (25-30g/day) (Cho, 2001), in the present study, the daily portion of consumption would be of 6 tablets/day, therefore, the tablets would cover 5.7% (green tablets) and 2.6% (red tablets). Although the content of fiber is low, the impact over the physiological benefits and in the food industry could be important. According with Cardenas et al. (1997), the fiber in the cactus pear fruit is characterized with the presence of a high insoluble fietary fiber content and mucilages with high molecular weight which interacts in an entangled network, able to hydrate and increase the viscosity of the liquid phase in the food system. Therefore, the physicochemical properties of these samples could be affected in presence of water.

Cho, S. S. Handbook of Dietary Fiber.

Cardenas, A., Higuera-Ciapara, I., & Goycoolea, F. Rheology and aggregation of cactus (Opuntia ficus indica) mucilage in solution.

 

Point 4:

I would also like to add that the advisor is not a native speaker and because of that I suggest that before having it published, language expert should read it.

 

Response 4:

The manuscript has been reviewed and all observations made by a native speaker have been added in the manuscript track changes.

Author Response File: Author Response.docx

Reviewer 4 Report

This is an interesting manuscript which evaluates the potential use of powder from Opuntia ficus-indica (L.) Mill residues (mesocarp and pericarp) of cactus pear fruit for the food supplement industry in development of tablet formulations obtained by mixing with two commercial excipients (microcrystalline cellulose and lactose). Opuntia ficus-indica residues is a product that has been recognized for its biological activities, i.a. it is relatively rich in natural antioxidants.

The results certainly provide useful information as they show quality parameters as well as functional and antioxidant properties in tablet formulations which can be useful for application in food supplement industry.

However, in my opinion, the manuscript requires minor adjustments and additions before publication. Please, see my comments and suggestions below.

1. I suggest revising the title to the following “Opuntia ficus-indica (L.) Mill residues – properties and application possibilities in food supplements”

2. Potential toxicants in the food chain are derived from natural or industrial sources. My main point concerns the discussion section, and the need for a review of information on the health risk of exposure to the potential contaminants in this product released from the cactus pear residues, e.g.  heavy metals, residual pesticides, and natural toxins, such as those produced by plants (phytotoxins). This is key to ensure safety and high-quality of dietary supplements. 

3. Finally, please add strengths and limitations of the study.

 

Author Response

Response to Reviewer 4 Comments

This is an interesting manuscript which evaluates the potential use of powder from Opuntia ficus-indica (L.) Mill residues (mesocarp and pericarp) of cactus pear fruit for the food supplement industry in development of tablet formulations obtained by mixing with two commercial excipients (microcrystalline cellulose and lactose). Opuntia ficus-indica residues is a product that has been recognized for its biological activities, i.a. it is relatively rich in natural antioxidants.

The results certainly provide useful information as they show quality parameters as well as functional and antioxidant properties in tablet formulations which can be useful for application in food supplement industry.

However, in my opinion, the manuscript requires minor adjustments and additions before publication. Please, see my comments and suggestions below.

Point 1:

I suggest revising the title to the following “Opuntia ficus-indica (L.) Mill residues – properties and application possibilities in food supplements”

Response 1:

The title has been changed as suggested by the reviewer

Point 2:

Potential toxicants in the food chain are derived from natural or industrial sources. My main point concerns the discussion section, and the need for a review of information on the health risk of exposure to the potential contaminants in this product released from the cactus pear residues, e.g. heavy metals, residual pesticides, and natural toxins, such as those produced by plants (phytotoxins). This is key to ensure safety and high-quality of dietary supplements.

Response 2:

The content of heavy metals or residual pesticides, are very low in the cactus pear and the sentences have been included in the discussion section in lines 371-376 as follows: 

On the other hand, studies have been carried out regarding the safety of the consumption of cactus pear fruit, for example the presence of pesticide residues (malathion, chlorpyrifos, permethrin, diazinon, dimethoate, spinosad and abamectin) and heavy metals (copper, chromium) arsenic, cadmium, lead and selenium) have been demonstrated,  whose levels are in amounts less than the maximum limits of toxic residues established by the North American Free Trade Agreement [Aldama-Aguilera et al., 2008; Motese et al., 2018] indicating that these phytotoxic elements does not trigger toxicological health risks.

 

However, it is a very important point to perform in vivo studies to complete the information with studies about the presence of contaminants in the residues of cactus pear in order to provide a safe product for human consumption.

 

Aldama Aguilera, C., Cibrián-Tovar, J.; Anaya-Rosales, S.; Pérez, L.; Guadalupaseso, M.; et al. Límites máximos de residuos e intervalos de seguridad de plaguicidas en tuna, Opuntia ficus-indica.

Mottese, A. F.; Naccari, C.; Vadalà, R.; Bua, G. D.; Bartolomeo, G.; Rando, R.  et al. Traceability of Opuntia ficus‐indica L. Miller by ICP‐MS multi‐element profile and chemometric approach

 

And in the conclusion section (line 382-384) was added as follows: 

Further research about phytotoxicity is needed sine there are no reports about that, as well as in vivo model studies, in order to evaluate the health effects of a dietary supplement developed from cactus pear by-products.

Point 3:

Finally, please add strengths and limitations of the study.

Response 3:

Strengths and Limitations of the study were added after the conclusions section, in lines 385-390 as follows:

The strengths of the present study consist in the use of an agricultural by-product, through its use in a novel product, which may have beneficial effects on human health, and which also represents an economic benefit for the producers of the fruit. On the other hand, among the limitations it is found that the cactus pear is a seasonal fruit, so the residues must be collected and stored during the harvest season. It is also necessary to subject the product further in vitro tests, in order to evaluate positive or negative effects on human health.

Author Response File: Author Response.docx

Reviewer 5 Report

Review’s reply 

The paper of Manzur-Valdespino et al proposed tablets formulated with Opuntia ficus-indica residues as novel nutritional supplement for dietary supplement industry demonstrating that among formulation tested the Green Opuntia tablet formulation had both good pharmaceutical and functional health proprieties. The authors deal with two of the topics more current and of fast- growing interest for the scientific community and the consumers such as recovery and utilization of byproducts and the development of innovative nutritional supplements. Despite of large use of some components from Opuntia spp (e.g. mucilage) as pharmaceutical excipient, for instance, for the controlled drug delivery few investigations have been performed, in vitro and in vivo, both on the Op extract formulations and their health benefits.

The reviewer believes that this work gives a good contribution to the field and it is well-organized and sufficiently described. Even if the research provided a basis for the potential development and use of products from residues of Opuntia ficus-indica L. Mill the work raises some concerns which will need to be addressed to improve the manuscript's quality.

Therefore, the manuscript can be recommended for publication provided the minor revisions outlined below have been adequately addressed by its authors.

 

REVIEWER COMMENTS.

Introduction (lanes 40-47):

The authors should state in the Introduction that the use of different agro-food by-products, including these from Opuntia, and their derivatives represents a new field of research as there is a growing interest in the development of novel products with different applications as nutraceuticals, medicines, food and cosmetics, also with antioxidant properties useful for preventing or managing of oxidative stress related diseases (aging, cancer, metabolic disorder, hepatic pathologies and so on) as they can influence the cellular oxidative balance. I consider this point interesting, so it would be good for the authors to strengthen this matter with a comment in which they attach importance of using plant extracts and also their residues in various industrial applications.

 As regards, the authors should tackle all these matters in the introduction providing a complete and current state of the research in this field so matching with the Applied Sciences requests and making the aims of authors comprehensible to all scientists

These papers below should be quoted to up-to-date the references:

•  Di Mauro MD, Tomasello B, Giardina RC, Dattilo S, Mazzei V, Sinatra F, Caruso M, D'Antona N, Renis M. Sugar and mineral enriched fraction from olive mill wastewater for promising cosmeceutical application: characterization, in vitro and in vivo studies. Food Funct. 2017 Dec 13;8(12):4713-4722.
• Varzakas T, Zakynthinos G, Verpoort F. Plant Food Residues as a Source of Nutraceuticals and Functional Foods. Foods. 2016;5(4):88. Published 2016 Dec 10. doi:10.3390/foods5040088

Lanes 90-93: The authors should explain how they select the amount of ingredients to make the tablet formulations.

 

Paragraph 2.6.1. Extraction of Antioxidants: the major concern of this manuscript is the determination of antioxidant capacity of formulations based on an aqueous-organic extraction, a chemical approach that doesn't represent the real condition of the gastrointestinal environment. The authors should uphold their choice to carry out this extraction method and discuss how other extraction procedures could influence the total antioxidant power of formulations.

These sentences should check carefully: lanes 214-216 (“could propiciate….; easiest size”); lanes 275-276 (“significant higher values for, in contrast RL..”); lanes 289-287 (lipid blood concentrations metabolism )

Lanes 237 and 247: capital letter after full stop. Check for other typing mistakes

Author Response

Response to Reviewer 5 Comments

The paper of Manzur-Valdespino et al proposed tablets formulated with Opuntia ficus-indica residues as novel nutritional supplement for dietary supplement industry demonstrating that among formulation tested the Green Opuntia tablet formulation had both good pharmaceutical and functional health properties. The authors deal with two of the topics more current and of fast- growing interest for the scientific community and the consumers such as recovery and utilization of byproducts and the development of innovative nutritional supplements. Despite of large use of some components from Opuntia spp (e.g. mucilage) as pharmaceutical excipient, for instance, for the controlled drug delivery few investigations have been performed, in vitro and in vivo, both on the Op extract formulations and their health benefits.

The reviewer believes that this work gives a good contribution to the field and it is well-organized and sufficiently described. Even if the research provided a basis for the potential development and use of products from residues of Opuntia ficus-indica L. Mill the work raises some concerns which will need to be addressed to improve the manuscript's quality.

Therefore, the manuscript can be recommended for publication provided the minor revisions outlined below have been adequately addressed by its authors.

 

Point 1:

Introduction (lanes 40-47):

The authors should state in the Introduction that the use of different agro-food by-products, including these from Opuntia, and their derivatives represents a new field of research as there is a growing interest in the development of novel products with different applications as nutraceuticals, medicines, food and cosmetics, also with antioxidant properties useful for preventing or managing of oxidative stress related diseases (aging, cancer, metabolic disorder, hepatic pathologies and so on) as they can influence the cellular oxidative balance. I consider this point interesting, so it would be good for the authors to strengthen this matter with a comment in which they attach importance of using plant extracts and also their residues in various industrial applications.

Response 1:

A sentence about the use of different agro-food by-products were already included in the manuscript in lines  71- 73 as follows:

Different products have been developed with cactus pear residues, such as yogurts, snacks or margarine (Hernández-Carranza et al., 2019; Namir et al., 2017; Chougui et al., 2015). Some other fruit wastes have been used for the creation of dietary supplements or products for the pharmaceutical industry, including pineapple, banana or orange peels (Huang and Chou, 2011; Bansal et al., 2014; Srivastava et al.,2011).

Hernández-Carranza, P.; Jattar-Santiago K. Y.; Avila-Sosa, R.; Pérez-Xochipa, I.; Guerrero-Beltrán, J. A.; Ochoa-Velasco, C. E.; and Ruiz-López I. Antioxidant fortification of yogurt with red cactus pear peel and its mucilage.

Namir, M.; Elzahar, K.; Ramadan, M. F.; and Allaf, K. Cactus pear peel snacks prepared by instant pressure drop texturing: Effect of process variables on bioactive compounds and functional properties.

Chougui, N.; Djerroud, N.; Naraoui. F; Hadjal, S; Aliane, K; Zeroual, B. Physicochemical properties and storage stability of margarine containing Opuntia ficus-indica peel extract as antioxidant.

Huang Y L, Chow C J FYJ. Preparation and physicochemical properties of fiber-rich fraction from pineapple peels as a potential ingredient.

Bansal. J.; Malviya, R.; Malaviya. T.; Bhardwaj. V.; Sharma, P. Evaluation of Banana peel Pectin as excipient in solid Oral Dosage Form.

Srivastava, P.; Malviya, R. Extraction, characterization and evaluation of orange peel waste derived pectin as a pharmaceutical excipient.

And a sentence about different applications was included in line 50-55 as follows:

However, several studies have reported that the use of different agro-food by products such as fruit and vegetable residues, including Opuntia ficus-indica L. Mill residues, possess bioactive compounds with a wide spectrum of pharmacological properties such as neuroprotective, antidiabetic, antioxidant, anti-inflammatory, hepatoprotective and anticancer activities, which are used as raw material for the development of nutraceuticals, medicines, food and cosmetics useful to prevent or control diseases (Di Mauro, et. al. 2017, Varzakas, et al. 2016).

Di Mauro MD, Tomasello B, Giardina RC, Dattilo S, Mazzei V, Sinatra F, Caruso M, D'Antona N, Renis M. Sugar and mineral enriched fraction from olive mill wastewater for promising cosmeceutical application: characterization, in vitro and in vivo studies.

Varzakas T, Zakynthinos G, Verpoort F. Plant Food Residues as a Source of Nutraceuticals and Functional Foods. Foods.

 

Point 2:

As regards, the authors should tackle all these matters in the introduction providing a complete and current state of the research in this field so matching with the Applied Sciences requests and making the aims of authors comprehensible to all scientists

These papers below should be quoted to up-to-date the references:

Di Mauro MD, Tomasello B, Giardina RC, Dattilo S, Mazzei V, Sinatra F, Caruso M, D'Antona N, Renis M. Sugar and mineral enriched fraction from olive mill wastewater for promising cosmeceutical application: characterization, in vitro and in vivo studies. Food Funct. 2017 Dec 13;8(12):4713-4722.

Varzakas T, Zakynthinos G, Verpoort F. Plant Food Residues as a Source of Nutraceuticals and Functional Foods. Foods. 2016;5(4):88. Published 2016 Dec 10. doi:10.3390/foods5040088

Response 2:

The references have been included in the manuscript. Information about supplement, fiber and antioxidants were already included in the manuscript however, we omit information about some aspects of quality parameters of tablets, therefore, this information has been included in lines 76-79 as follows:

The tablet formulations must observe specific quality parameters that allow to preserve the characteristics of the product without physical changes during transportation and packaging, as well as the evaluation of the dissolution behavior in the human body (Patel and Sun, 2016).

Patel, S.; Sun, C.C. Macroindentation hardness measurement modernization and applications.

 

Point 3:

Lanes 90-93: The authors should explain how they select the amount of ingredients to make the tablet formulations.

Response 3:

The selection of ingredients were according preliminary studies where more excipient was added until it had a complete compaction, (until the tablets were not disassembled, and remained firm). The information has been include in methodology sección in lines 109-110 as follows:

These formulations were established according to preliminary tests where the excipients were added until reaching a complete compaction.

 

Point 4:

Paragraph 2.6.1. Extraction of Antioxidants: the major concern of this manuscript is the determination of antioxidant capacity of formulations based on an aqueous-organic extraction, a chemical approach that doesn't represent the real condition of the gastrointestinal environment. The authors should uphold their choice to carry out this extraction method and discuss how other extraction procedures could influence the total antioxidant power of formulations.

Response 4:

In effect, according with the reviewer the evaluation of antioxidant activity may be underestimate the real antioxidant capacity when used solvents to extract antioxidants without consider physiological conditions. Serrano et al., (2007) had established the difference between a methodology that include physiological conditions and a extraction with solvents. They used an in vitro enzymatic digestion and colonic fermentation to determine the antioxidant capacity. Therefore, we will consider the use of this complete methodology to future studies.

Considering that a significant part of the antioxidants contained in plant foods are not analysed in most antioxidant capacity assays, where the antioxidant extraction is incomplete, we use a methodology carried out according to the validated methodology by Pérez-Jiménez et al. 2008, following an efficient extraction of antioxidants with two extraction cycles performed with aqueous-organic solvents with different polarities (methanol/water and  acetone/water) in order to extract antioxidant compounds with different chemical structures to extraction specific for the  determination of total phenolic content and antioxidant capacity.

Serrano, J., Goñi, I., & Saura-Calixto, F. (2007). Food antioxidant capacity determined by chemical methods may underestimate the physiological antioxidant capacity.

Pérez-Jiménez J, Arranz S, Tabernero M, Díaz- Rubio ME, Serrano J, Goñi I, Saura-Calixto, F. Methodology to determine antioxidant capacity in plant foods, oils and beverages: Extraction, measurement and expression of results.

 

Point 5:

These sentences should check carefully: lanes 214-216 (“could propiciate….; easiest size”);

Response 5:

The sentence has been corrected as “could propitiate” in line 231 and lines 232-233 were rewritten.

 

Point 6:

Lines 275-276 (“significant higher values for, in contrast RL..”);

Response 6:

The sentence has been corrected in lines 297-298 as follows:

Had higher values, in contrast to RL formulation

 

Point 7:

Lines 289-287 (lipid blood concentrations metabolism)

Response 7:

The sentence has been corrected as “lipid blood concentrations” in line 308.

 

Point 8:

Lanes 237 and 247: capital letter after full stop. Check for other typing mistake

Response 8:

The mistakes have been corrected in all manuscript.

 

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The authors addressed all the issues raised previously and the manuscript in its present form merits to be published as is.