Comparative Action of Blue Food Colorants (Genipin, Patent Blue V, and Brilliant Blue FCF); Their Effect on Oxidative Stress in Human Plasma and Blood Platelets In Vitro

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article written by Beata Olas et al present comparative action of blue food colorants their effect on oxidative stress in .human plasma and blood platelets in vitro. Understanding the mechanisms of action of food colorants on the human body is crucial. We need to understand both the positive and negative aspects of their effects due to their frequent use in food colouring. The authors selected three popular blue food colorants for the study (one natural colorant – genipin, and two synthetic colorants – brilliant blue FCF and patent blue V). In my opinion, the topic taken up by the authors is important, however manuscript can be accepted for publication after major revision. The authors should revise the manuscript according to the following comments:

1. The choice of these three food colorants suggests to readers that they belong to the same class of organic compounds. However, genipin is an oxabicyclo[4.3.0]nona-4,8-diene derivative, and brilliant blue and patent blue are triarylmethane derivatives. Therefore, authors should provide chemical structures of these compounds.
2. In the Introduction section, the authors should also explain that, unlike the other two colorants, genipin is not a natural blue pigment. The blue color appears as a result of genipin reacting with amino acids contained in proteins.
3. Do the authors in their studies observe the formation of a blue colour when adding genipin to the plasma or after adding H2O2/Fe2+ to the system?
4. Please comment further on Figure 1A regarding plasma lipid peroxidation.
5. Does genipin inhibit the oxidation of ABTS?
6. Due to the different properties of genipin, i.e. the ability to cross-linking and react with amino acids/proteins, it is possible that the genipin-protein complex is responsible for the antioxidant properties of genipin.

Author Response

The article written by Beata Olas et al present comparative action of blue food colorants their effect on oxidative stress in .human plasma and blood platelets in vitro. Understanding the mechanisms of action of food colorants on the human body is crucial. We need to understand both the positive and negative aspects of their effects due to their frequent use in food colouring. The authors selected three popular blue food colorants for the study (one natural colorant – genipin, and two synthetic colorants – brilliant blue FCF and patent blue V). In my opinion, the topic taken up by the authors is important, however manuscript can be accepted for publication after major revision. The authors should revise the manuscript according to the following comments:

Response: Thank you for your helpful comments. All of them have been taken into consideration when revising the manuscript.

 

1. The choice of these three food colorants suggests to readers that they belong to the same class of organic compounds. However, genipin is an oxabicyclo[4.3.0]nona-4,8-diene derivative, and brilliant blue and patent blue are triarylmethane derivatives. Therefore, authors should provide chemical structures of these compounds.

Response: We have added these chemical structures on Figure 1.

2. In the Introduction section, the authors should also explain that, unlike the other two colorants, genipin is not a natural blue pigment. The blue color appears as a result of genipin reacting with amino acids contained in proteins.

Response: We have added more information about it:  “The hydrolysis of geniposide and gardenoside by the β-glucosidase results in the production of genipin (obtained from G. jasminosis and G. americana), a water-soluble iridoid monoterpenoid whose maximum ab-sorbance (496 nm) does not change with the pH of the environment. Genipin can be reacted with amino acids such as glycine, lysine or phenylalanine to obtain the blue dye Gardenia Blue. In addition, plant-derived bioactive compounds, including genipin offer multi-targeted property (antibacterial, anti-inflammatory, antithrombotic, antioxidant, and other) and low cytotoxicity. Recent reviews indicate a growing interest in genipin, which acts as a precursor of blue pigments and a natural crosslinking agent for food and biomaterial related applications [Olas et al., 2020 and 2021; Brys et al., 2022].”

3. Do the authors in their studies observe the formation of a blue colour when adding genipin to the plasma or after adding H2O2/Fe2+ to the system?

Response: Genipin, patent blue V, and brilliant blue FCF were dissolved in water. The final concentrations of tested blue colorants in human plasma, and blood platelet samples were 1, 2, 10, 20, and 200 μM. We have not observed the formation of a blue colour when adding tested blue colorants (at used concentrations) to the plasma or blood platelets.

4. Please comment further on Figure 1A regarding plasma lipid peroxidation.

Response: We have added short information about it: “As demonstrated in Figure 2A (old Fig. 1A), two tested blue colorants: genipin, and brilliant blue FCF (at four used concentrations: 2, 10, 20, and 200 µM) reduced plasma lipid peroxidation induced by H2O2/Fe2+.”

5. Does genipin inhibit the oxidation of ABTS?

Response: We have added more information about it. Here, we have noted that genipin (at two used concentrations: 10, and 200 µM) induces statistically significant changes in the level of Trolox in human plasma treated with H2O2/Fe2+ (Fig. 6). We have also noted that all used blue colorants (1 – 200 µM) do not change the level of parameters of oxidative stress, including ABTS in plasma and blood platelets without H2O2/Fe2+ (data not presented).

6. Due to the different properties of genipin, i.e. the ability to cross-linking and react with amino acids/proteins, it is possible that the genipin-protein complex is responsible for the antioxidant properties of genipin.

Response: We have added more information about it (the chapter of Discussion): “Gorczyca et al. [2014] have characterized biological properties of genipin cross-linked porous chitosan-collagen-gelatin scaffolds using chitosan-CO2 solution. They have observed good antioxidant properties (ABTS assay) and especially very low in vitro cytotoxicity against fibroblasts. In the other experiment, genipin-crosslinked gelatin/chitosan-based functional films incorporated with rosemary essential oil and quercetin were used, but crosslinking by genipin did not significantly affect the antioxidant performance of the film. On the other hand, the addition of the functional fillers (quercetin and rosemary essential oil) significantly increased the antioxidant activity of the film [Roy and Rhim, 2022].

Recently, results of Angi et al. [2025] have demonstrated that genipin derivative – mito-genipin also modulates oxidative stress in macrophages. For example, mito-genipin inhibited ROS production. Other genipin derivatives (0.01 µM) also effectively attenuated glutamate-induced oxidative damage (by inhibiting ROS over-accumulation and reducing TBARS content) in HT22 cells [Luo et al., 2023].”

 

Reviewer 2 Report

Comments and Suggestions for Authors

From the very beginning, I would like to note that this work is a pioneering one. There is no direct data on the antioxidant potential of genipin, brilliant blue FCF, and proprietary blue V in blood cells treated with the Ho₂/Fe2⁺ system in available sources. Direct data on the ability of genipin to protect blood elements from oxidative stress caused by the H₂o₂/Fe2⁺ system has not been found in available sources. However, there are examples where its pro-oxidant activity is described. Thus, in my opinion, the authors managed to select the specific conditions for this dye to have the opposite effect. Thus, it is obvious that the research should be continued at the proper level and the research results will be useful both in fundamental biochemistry and in medicine. I would like to note the high experimental level, references to the methods used in the calculation, relevant articles and a high level compared to the literature data. However, there are a couple of comments. The work can be published after a little revision.

1. It is necessary to formulate more clearly the purpose of the study, why blue food dyes were chosen for the study.
2. How do the authors explain that the statistical error p in some cases is greater than 0.05? Is it possible to believe these results?
3. There are a lot of statements in the Discussion section that are not supported by references. Links should be added in 1 paragraph after sentences – 1,2,4; 2 paragraph after sentence 1; 3 paragraph after sentence 1.

Author Response

From the very beginning, I would like to note that this work is a pioneering one. There is no direct data on the antioxidant potential of genipin, brilliant blue FCF, and proprietary blue V in blood cells treated with the Ho₂/Fe2⁺ system in available sources. Direct data on the ability of genipin to protect blood elements from oxidative stress caused by the H₂o₂/Fe2⁺ system has not been found in available sources. However, there are examples where its pro-oxidant activity is described. Thus, in my opinion, the authors managed to select the specific conditions for this dye to have the opposite effect. Thus, it is obvious that the research should be continued at the proper level and the research results will be useful both in fundamental biochemistry and in medicine. I would like to note the high experimental level, references to the methods used in the calculation, relevant articles and a high level compared to the literature data. However, there are a couple of comments. The work can be published after a little revision.

Response: Thank you for your helpful comments. All of them have been taken into consideration when revising the manuscript.

1. It is necessary to formulate more clearly the purpose of the study, why blue food dyes were chosen for the study.

Response: We have added short information about it (the chapter of Introduction): “Various chemical compounds presented in foods may modulate the level of oxidative stress. However, the influence of natural and synthetic blue colorants on this process in various elements of human blood is not well known. In addition, phytochemicals found in plants have demonstrated significant potential in the treatment and prevention of diseases associated with oxidative stress.”.

2. How do the authors explain that the statistical error p in some cases is greater than 0.05? Is it possible to believe these results?

Response: Statistical analyses were conducted using Statistica software version 10 (StatSoft). Data normality was evaluated using normal probability plots, while variance homogeneity was assessed by the Brown-Forsythe test. Differences among and between experimental groups were analyzed using one-way analysis of variance (ANOVA), followed by Duncan’s multiple comparison test. For clarity, only statistically significant differences between the tested compounds and the control or positive control groups were indicated.

Human whole blood was drawn from volunteers at “Diagnostyka” blood collection center (Lodz, Poland). All volunteers (aged 21-25; n=6 (3 men, and 3 women)) were healthy and did not smoke. The donors did not drink alcohol or take medicine (including antiplatelet drugs, for example aspirin and its derivatives) for two weeks before blood collection. However, these biological materials, especially human blood platelets are often heterogenic. On the other hand, our earlier papers and other authors [Kołodziejczyk-Czepas et al., Bijak et al., and others] used these biological materials and statistical methods.

3. There are a lot of statements in the Discussion section that are not supported by references. Links should be added in 1 paragraph after sentences – 1,2,4; 2 paragraph after sentence 1; 3 paragraph after sentence 1.

Response: We have corrected it.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors took my previous comments into account and significantly improved their article. I now believe that the article can be accepted for publication in its current form.