Development of Cosmetic Formulations Containing Olive Extract and Spirulina sp.: Stability and Clinical Efficacy Studies

Round 1

Reviewer 1 Report

I have read the manuscript of D’Angelo Costa et al based on the development of stable cosmetic formulations with suitable physical-mechanical properties and pleasant sensory properties to obtain cosmetic formulations containing olive extract ans Spirulina sp with natural antioxidants to reduce the oxidative stress caused by solar radiation and pollution. The study is well structured, the methods used are pertinent and the results are plausible. The sudy and the characterization of the formulations developed it is well done and my suggestion is to publish this work after Major revisions, see below.

1. The material and methods (section 2) should be corrected and divided into subchapters to improve understanding. Add 2.1 Materials, section. The authors should describe all the raw materials, Please add company, city and country. Remove characteristics from table 1.
2. Elaboration of emulsions. The authors should describe more correctly all the equipment used in section 2 in all manuscript. Please add: Commercial brand, model. And company, city and country in brackets. For example Stirrer. Describe temperatue, add stirring times,……

3.      In all the manuscript the vehicle formulation I suppose it refers to the formulation F7. It is confusing, please use nomenclature with F.

4. Why the texture profiles were evaluated only in the F6 and F7 formulations? Why not F5? Explain it.
5. Why is the rotation speed  from 0 to 50 and 50 to 0 rpm described in section 2.1.2 and instead in the graphs presented in Figure 2 it is from 0 to 100 and 100 to 0 rpm, and Figure 3 it is from 0 to 200 and 200 to 0 rpm? Why are the rheograms in Figure 2 different from those in Figure 3 in terms of shear rate? Explain it
6. Please use rpm or 1/s, not mixed
7. I would appreciate the authors viscosity graphs and a table with the viscosity values ​​of the samples obtained. If the authors do not have space in the manuscript, add it to the supplementary material.
8. If olive extract and spirulina sp have antioxidant properties, how come the authors have not tested formulations without BHT and SMBS? Explain it.

9.      The particle size distribution has a significant influence on several fundamental properties of the final emulsion: stability, viscosity, optical properties, mechanical strength. Use DLS and/or other techniques to determine Particle size.

10.  Do the authors really consider that the addition of 0,1% of SMBS causes a decrease in viscosity? Describes in detail this process, give references and explain it. Also add the viscosity values ​​and/or viscosities curves where these changes can be seen.

11.  Why the authors did not test sample F7 in terms of rheological parameters, pH, organoleptics characteristics in order to compare it with the other prepared samples (F1-F6)?

12.  In all manuscript the formulation containing the association of olive extract with spirulina sp is mentioned many times (fig.4-5-6). Looking at table 1, the formulations that contain olive extract with spirulina sp are F5 and F6. Which is Which is F5 or F6? It is very confusing, please use nomenclature with F in the manuscript.

13.  Conclusions: specify which is the most suitable and stable developed formulation (F1-6) . It's confusing.

14.  Change 3. Results for 4. Results

15. In my opinion chapter 3. Casuistic and methods should be 2.X Casuistics and methods.

16.  Improve resolution figures, especially rheology figures.

17.  Change minutes for min in  all manuscript (4).

Author Response

Reviewer 1: Comments and Suggestions for Authors

I have read the manuscript of D’Angelo Costa et al based on the development of stable cosmetic formulations with suitable physical-mechanical properties and pleasant sensory properties to obtain cosmetic formulations containing olive extract ans Spirulina sp with natural antioxidants to reduce the oxidative stress caused by solar radiation and pollution. The study is well structured, the methods used are pertinent and the results are plausible. The sudy and the characterization of the formulations developed is well done and my suggestion is to publish this work after Major revisions, see below.

The authors are deeply grateful for the comments and attention.

1. The material and methods (section 2) should be corrected and divided into subchapters to improve understanding. Add 2.1 Materials, section. The authors should describe all the raw materials, Please add company, city and country. Remove characteristics from table 1.

The material and methods have been corrected and the raw materials were described in a separate table, as suggested.

This information was added to the item 2.1. Materials, as shown below:

The raw materials used in the formulations are described in Table I.

Table I - Raw materials.

1. Elaboration of emulsions. The authors should describe more correctly all the equipment used in section 2 in all manuscript. Please add: Commercial brand, model. And company, city and country in brackets. For example Stirrer. Describe temperatue, add stirring times,……

The equipment has been described in a separate table, as suggested.

This information was added to the item 2.1. Materials, as shown below:

“The equipment used is  described in Table II.”

Table II: Equipaments.

1. In all the manuscript the vehicle formulation I suppose refers to the formulation F7. It is confusing, please use nomenclature with F. Ok, we used the nomenclature F.
2. Why the texture profiles were evaluated only in the F6 and F7 formulations? Why not F5? Explain it.

The texture profile and sensory evaluation were only performed on formulations F6 and F7 because they were the formulations chosen to be evaluated in the clinical test. Work of shear and spreadability are correlatable attributes [1] and are important to evaluate to ensure adherence and use by the research participants during the clinical test. This information was added to the item 2.1.3.”Texture Profile”, as shown below:

“The texture profile was evaluated in the formulations F6 and F7, which were chosen to be evaluated in the clinical study.”

[1] Calixto, L.S.; Infante, V.H.P.; Maia Campos, P.M.B.G. Design and Characterization of Topical Formulations: Correlations Between Instrumental and Sensorial Measurements. AAPS PharmSciTech. 2018 19, 1512-1519. https://doi.org/10.1208/s12249-018-0960-0.

1. Why is the rotation speed  from 0 to 50 and 50 to 0 rpm described in section 2.1.2 and instead in the graphs presented in Figure 2 it is from 0 to 100 and 100 to 0 rpm, and Figure 3 it is from 0 to 200 and 200 to 0 rpm? Why are the rheograms in Figure 2 different from those in Figure 3 in terms of shear rate? Explain it

The rotation speed of 0 to 50 rpm described in the item 2.2.1.1. “Rheological Behaviour”  refers to the rotation speed of the spindle during the analysis. It is a measure that we define in the RheocalcT® 1.2.19 software program before starting the analysis. Figures 2 and 3 show the rheograms resulting from the rheological analyses. These rheograms are set on the x-axis as the measured shear rate (1/s) and on the y-axis as shear stress. They are therefore different measurements and cannot be interchanged. This nomenclature standard has been used for some years by our research group [1, 2, 3].

The rheograms in Figure 2 and 3 have different shear rates because they were taken at different times. Figure 2 was the step to check the influence of SMBS in the presence of the active substances under study and Figure 3 was another step to check the rheological parameters of the formulations that would be tested in the clinical trial. As they were carried out on different days and with different formulations, there was a variation in this parameter. However, if you look at the shear stress parameter, it doesn't change. This variable happens because the shear rate is determined by the rotational speed of the spindle, the size and shape of the spindle, the size and shape of the container used and therefore, the distance between the container wall and the spindle surface [4].

[1] Calixto, L.S.; Infante, V.H.P.; Maia Campos, P.M.B.G. Design and Characterization of Topical Formulations: Correlations Between Instrumental and Sensorial Measurements. AAPS PharmSciTech. 2018 19, 1512-1519. https://doi.org/10.1208/s12249-018-0960-0.

[2] César, F.C.S.; Maia Campos, P.M.B.G.  Influence of vegetable oils in the rheology, texture profile and sensory properties of cosmetic formulations based on organogel. Int J Cosmet Sci 2020 42, 494-500. https://doi.org/10.1111/ics.12654

[3] D'Angelo Costa, G.M.; Maia Campos, P.M.B.G. Development of Photoprotective Formulations: Influence of Formulation Composition on the SPF and Mechanical Properties. AAPS PharmSciTec. 2023 24, 97. https://doi.org/10.1208/s12249-023-02558-z.

[4] BROOKFIELD AMETEK DV1 Digital Viscometer Operating Instructions Manual No. M14-023-A0416.

1. Please use rpm or 1/s, not mixed

Please note that rpm cannot be exchanged for 1/s and vice versa, as they are different units of measurement. The rpm used in the item 2.2.1.1. "Rheological Behaviour '' refers to the rotational speed of the spindle and the 1/s refers to the shear rate of the rheograms. This nomenclature has been used by our research group, as described in the follow references:

[1] Calixto, L.S.; Infante, V.H.P.; Maia Campos, P.M.B.G. Design and Characterization of Topical Formulations: Correlations Between Instrumental and Sensorial Measurements. AAPS PharmSciTech. 2018 19, 1512-1519. https://doi.org/10.1208/s12249-018-0960-0.

[2] César, F.C.S.; Maia Campos, P.M.B.G.  Influence of vegetable oils in the rheology, texture profile and sensory properties of cosmetic formulations based on organogel. Int J Cosmet Sci 2020 42, 494-500. https://doi.org/10.1111/ics.12654

[3] D'Angelo Costa, G.M.; Maia Campos, P.M.B.G. Development of Photoprotective Formulations: Influence of Formulation Composition on the SPF and Mechanical Properties. AAPS PharmSciTec. 2023 24, 97. https://doi.org/10.1208/s12249-023-02558-z.

1. I would appreciate the authors viscosity graphs and a table with the viscosity values ​​of the samples obtained. If the authors do not have space in the manuscript, add it to the supplementary material.

The table with the viscosity values will be added to the supplementary material, as suggested. The tables as shown below:

Table I: Minimum apparent viscosity of formulations with or without Sodium Metabisulfite (SMBS) in the presence of olive extract alone or in association with Spirulina sp. when stored at room temperature for (T0), 7 (T7), 14 (T14), 28 (T28), 56 (T56) and 84 (T84) days. F1 = formulation without SMBS with 0.5 %w/w olive extract at room temperature; F2= formulation with SMBS with 0.5 %w/w olive extract at room temperature; F3= formulation without SMBS with 0. 2 %w/w of olive extract at room temperature; F4= formulation with SMBS with 0.2 %w/w of olive extract at room temperature; F5= formulation with SMBS with 0. 5 %w/w olive extract and 0.1%w/w Spirulina sp. at room temperature; F6= SMBS formulation with 0.2 %w/w olive extract and 0.1%w/w Spirulina sp. at room temperature.

Table II: Minimum apparent viscosity of the vehicle formulation (F7) and containing the association of olive extract with Spirulina sp. (F6) when stored at room temperature, 37°C and at 45°C for (T0), 7 (T7), 14 (T14), 28 (T28), 56 (T56) and 84 (T84) days.

1. If olive extract and spirulina sp have antioxidant properties, how come the authors have not tested formulations without BHT and SMBS? Explain it.

Spirulina sp. is a microalgae that has been studied by our research group since 2014 [1] and through previous studies the concentration of 0.1% was defined as stable and viable for incorporation into cosmetic formulations. As a result, we focused our research only on the ideal concentration of olive extract in the formulation. During the formula development process, it was observed that the olive extract needed an antioxidant to stabilize it. The choice to use SMBS was because it is widely used to stabilize vitamin C, which has a similar antioxidant profile to olive extract [2]. We therefore focused on evaluating the olive extract in the formulation with and without SMBS at different concentrations, which were formulations F1 to F4.

As the aim of the study was to investigate the association between olive extract and Spirulina sp., the formulations containing this association were made with and without the addition of SMBS. However, during development it was observed that continuing the studies with the formulation without the addition of SMBS would not be feasible, for reasons of stability and test optimization. We therefore evaluated formulations F5 and F6, as the concentration of the olive extract had not yet been defined.

Regarding the question about testing without BHT in the formulation, we didn't do it because it wasn't the focus of the study. BHT was added to the formulation to prevent oxidation of the sunflower oil lipid [3, 4]. Furthermore, this combination of sunflower oil and BHT at the concentration used has already been studied by our group by César and Maia Campos 2020 [5].

[1]  Corauce D, Camargo FB Jr, Maia Campos PMBG, inventors. Universidade de Sao Paulo (USP) and Ouro Fino Participações e Empreedimentos S/A, assignee. Spirulina containing cosmetic composition and cosmetic treatment method. US20140023676A1. January 23, 2014.

[2] Maia, A.M.; Baby, A.R.; Pinto, C.A.; Yasaka, W.J.; Suenaga, E.; Kaneko, T.M.; Velasco, M.V. Influence of sodium metabisulfite and glutathione on the stability of vitamin C in O/W emulsion and extemporaneous aqueous gel. Int J Pharm. 2006 28, 130-5. https://doi.org/10.1016/j.ijpharm.2006.05.038.

[3]  Sanhueza, J.; Nieto, S.; Valenzuela, A. Thermal Stability of Some Commercial Synthetic Antioxidants. JAOCS 2000 77, 933-936. https://doi.org/10.1007/s11746-000-0147-9

[4] Abdalla, A. E.; Roozen, J. P. Effect of plant extracts on the oxidative stability of sunflower oil and emulsion. Food Chemistry 1999 64, 323-329. https://doi.org/10.1016/S0308-8146(98)00112-5.

[5] César, F.C.S.; Maia Campos, P.M.B.G.  Influence of vegetable oils in the rheology, texture profile and sensory properties of cosmetic formulations based on organogel. Int J Cosmet Sci 2020 42, 494-500. https://doi.org/10.1111/ics.12654

1. The particle size distribution has a significant influence on several fundamental properties of the final emulsion: stability, viscosity, optical properties, mechanical strength. Use DLS and/or other techniques to determine Particle size.

We appreciate the suggestion to carry out the DLS experiment. It will be considered in the future studies of our research group.

Considering that the samples of this study have already been discarded, it would not be possible to carry out this experiment under the ideal conditions for comparison.

1. Do the authors really consider that the addition of 0,1% of SMBS causes a decrease in viscosity? Describe in detail this process, give references and explain it. Also add the viscosity values ​​and/or viscosities curves where these changes can be seen.

Yes. There was a numerical and visible decrease in the viscosity of the formulations when added with SMBS. As we can see in the table below, the minimum apparent viscosity values of the F2, F4, F5 and F6 formulations are different from the formulations F1 e F3.

We believe that this is due to the interaction of SMBS with the polymeric structure of the formulation. It is known that SMBS is a reducing agent and participates in polymerization reactions [1]. The formulation developed has organogel as a viscosity agent. An organogel is a semi-solid structure composed of an oil phase structured with a polymer [2]. Organogels have advantages such as low cost, increased chemical and physical stability of formulations, improved rheological properties and increased delivery profile [2]. The use of vegetable oil as the oil phase can soften its structure of organogel, as it impacts on gelling [2]. As the organogel uses sunflower oil in its composition and more sunflower oil was added to the formulation, the polymer structure may have been softened and the addition of a reducing agent such as SMBS at a low concentration (0.1%) may have affected the polymerization process. As a result, there may have been a loosening of the π-π stacking bonds in this structure, causing the viscosity to decrease. It is important to note that this decrease in viscosity did not affect the stability results or cause phase separation. Furthermore, this decrease in viscosity made the formulation more fluid and spreadable, which is an expected characteristic in cosmetic formulations to ensure greater adherence to the use of the cosmetic.

Table I: Minimum apparent viscosity of formulations with or without Sodium Metabisulfite (SMBS) in the presence of olive extract alone or in association with Spirulina sp. when stored at room temperature for (T0), 7 (T7), 14 (T14), 28 (T28), 56 (T56) and 84 (T84) days. F1 = formulation without SMBS with 0.5 %w/w olive extract at room temperature; F2= formulation with SMBS with 0.5 %w/w olive extract at room temperature; F3= formulation without SMBS with 0. 2 %w/w of olive extract at room temperature; F4= formulation with SMBS with 0.2 %w/w of olive extract at room temperature; F5= formulation with SMBS with 0. 5 %w/w olive extract and 0.1%w/w Spirulina sp. at room temperature; F6= SMBS formulation with 0.2 %w/w olive extract and 0.1%w/w Spirulina sp. at room temperature.

[1] Abreu, C. M. R.; Mendonça, P. V.; Serra, A. C.; Popov, A. V.; Matyjaszewski, K.; Guliashvili, T.; Coelho, J. F. J. Inorganic Sulfites: Efficient Reducing Agents and Supplemental Activators for Atom Transfer Radical Polymerization. ACS Macro Lett. 2012, 1, 11, 1308–1311  https://doi.org/10.1021/mz300458x.

[2] Martinez, R.M.; Rosado, C.; Velasco, M.V.R.; Lannes, S.C.S.; Baby, A.R. Main features and applications of organogels in cosmetics. Int J Cosmet Sci. 2019 41, 109-117. https://doi.org/10.1111/ics.12519.

1. Why the authors did not test sample F7 in terms of rheological parameters, pH, organoleptics characteristics in order to compare it with the other prepared samples (F1-F6)?

Sample F7 was tested in terms of rheological parameters, pH and organoleptic characteristics. The results are shown in Figure 3. I believe there was confusion because the nomenclature of the formulations was not standardized in the manuscript. However, this nomenclature has now been standardized.

The results of rheological parameters for F1 - F6 are shown in Figure 2. The results were separated because the aim of Figure 2 was to compare the presence and absence of SMBS in the presence of the active substances. Figure 3 aims to provide more detail on the stability of F6 and F7, which are the formulations that were tested in the clinical trial.

1. In all manuscript the formulation containing the association of olive extract with spirulina sp is mentioned many times (fig.4-5-6). Looking at table 1, the formulations that contain olive extract with spirulina sp are F5 and F6. Which is Which is F5 or F6? It is very confusing, please use nomenclature with F in the manuscript.

 The nomenclature with F has been changed in the manuscript, as suggested.

1. Conclusions: specify which is the most suitable and stable developed formulation (F1-6) . It's confusing.

The specification of the chosen formulation F6 was added in the conclusion, as suggested. The conclusion is shown below:

“The cosmetic formulation containing olive extract and Spirulina sp. in combination (F6) was stable and presented good sensory properties and spreadability.

The proposed formulation (F6) showed a hydrating effect and improved the skin barrier after 12 weeks of daily application.

An improvement of morphological and structural characteristics of the skin was observed only in the group that applied the formulation containing the association of antioxidants for 12 weeks by improvement of stratum corneum and stratum granulosum quality scores due to an increase in stratum granulosum interkeratinocyte reflectance and  brightness of stratum corneum. .

Finally, the proposed formulation (F6) was stable and effective in protecting the skin and reducing skin changes related to photoaging.”

1. Change 3. Results for 4. Results Ok

15. In my opinion chapter 3. Casuistic and methods should be 2.X Casuistics and methods.

The change in the numbering of the Casuistic and methods item was made, as suggested.

16. Improve resolution figures, especially rheology figures.

The resolution figures were improved, as suggested.

17. Change minutes for min in all manuscript (4).

Minutes were replaced by min throughout the manuscript, as requested.

Reviewer 2 Report

At what temperature (the article states that it was cold) was the emulsion made?

Pattern 1, please standardize the font, size and coloring.

Were test subjects advised to use a specific amount of emulsion?

Why was the sensory evaluation performed only for preparations F6 and F7?

How were the probands trained?

In my opinion the language is correct

Author Response

Reviewer 2:  Comments and Suggestions for Authors

At what temperature (the article states that it was cold) was the emulsion made?

The authors are deeply grateful for the comments and attention.

The cold emulsification process was done at room temperature (25ºC). This information was added to the item 2.2.1. Studied Formulations, as shown below.

“The studied formulations were made by a cold process at room temperature (25ºC) due to the presence of an organogel that has the property of cold emulsification.”

Pattern 1, please standardize the font, size and coloring.

The font, size and color were standardized, as suggested.

Were test subjects advised to use a specific amount of emulsion?

The test participants were instructed to use a sufficient amount of product to apply to the entire face with the exception of the eye area. This information was added to item “2.3.1. Clinical study” in the fourth paragraph as text below:

“In addition, the research participants were instructed to use the test products in sufficient quantities to apply to the entire face, with the exception of the eye area.”

Why was the sensory evaluation performed only for preparations F6 and F7?

The sensory evaluation was only performed on formulations F6 and F7 because they were the formulations chosen to be evaluated in the clinical test. This sensory evaluation was important to ensure that the sensory properties were adequate to start the clinical trial, which guarantees adherence and use by the research participants. This information was added to the item 2.3.2. Sensory Analysis as shown below:

“The sensory analysis was performed with the formulations that were chosen to be evaluated in the clinical study (F6 and F7).”

How were the probands trained?

The participants who performed the sensory analysis are people who have knowledge of the sensory parameters evaluated, due to periodic training carried out in our laboratory, as described by Calixto et al. 2018 [1]. 

This reference was added to item  “2.3.2.  Sensory Analysis” as shown below:

“Ten previously trained research participants evaluated the formulations with intensity scales in terms of the parameters of spreadability, stickiness, moisture feeling, skin feel after 5 min and absorption feeling [32].”

[1] Calixto, L.S.; Infante, V.H.P.; Maia Campos, P.M.B.G. Design and Characterization of Topical Formulations: Correlations Between Instrumental and Sensorial Measurements. AAPS PharmSciTech. 2018 19, 1512-1519. https://doi.org/10.1208/s12249-018-0960-0.

Reviewer 3 Report

I have reviewed your manuscript and would like to offer some suggestions for improvement:

1. In the fifth paragraph, there seems to be a highlighted section in yellow. It might be helpful to reconsider this formatting choice to ensure consistency throughout the document.

2. The seventh and eighth paragraphs could be merged to enhance the flow of the text.

3. There appears to be a formatting error in Table I. The word "phases" seems to be misaligned, and the pH value is in a different font. I recommend revisiting the table to correct these issues for clarity and readability.

4. The captions for Figures 3 and 5 seem to be located on separate pages from the corresponding figures. It would be beneficial to ensure that the captions are positioned appropriately to accompany their respective figures for ease of understanding.

Overall, the English is satisfactory.

Author Response

Reviewer 3: Comments and Suggestions for Authors

I have reviewed your manuscript and would like to offer some suggestions for improvement:

In the fifth paragraph, there seems to be a highlighted section in yellow. It might be helpful to reconsider this formatting choice to ensure consistency throughout the document.
            The authors are deeply grateful for the comments and attention.

The section highlighted  in yellow in the fifth paragraph was corrected, as suggested.

The seventh and eighth paragraphs could be merged to enhance the flow of the text.           The seventh and eighth paragraphs were merged, as suggested.

There appears to be a formatting error in Table I. The word "phases" seems to be misaligned, and the pH value is in a different font. I recommend revisiting the table to correct these issues for clarity and readability.

The word "phases" and the font of the pH value were corrected, as suggested.

The captions for Figures 3 and 5 seem to be located on separate pages from the corresponding figures. It would be beneficial to ensure that the captions are positioned appropriately to accompany their respective figures for ease of understanding. The captions for Figures 3 and 5 were positioned on the same page, as the respective figures as suggested.

Round 2

Reviewer 1 Report

Congratulations for the work done. It is very well structured and easy to understand. The introduction of the work is written extensively and includes the latest literature. The results were broadly discussed. I propose to accept this quality paper and publish it in Cosmetics. Just a few comment, When I was referring to clarifying the part of raw materials and equipment, I was referring to introducing it in its corresponding sections in materials and methods , there was no need to do it in table form and I suggested to remove content of table 1 (first version) in the corresponding section of material and methods in form text.