Concentration-Dependent Rheological and Sensory Effects of Walnut Leaf Extract in Cosmetic Emulsion Creams

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript is of interest for cosmetic formulators: understanding how the addition of an active ingredient affects the texture and application properties of a cosmetic formulation is of great practical relevance. The combined use of rheological characterization and sensory analysis provides a clear and comprehensive picture of the structural and applicative properties of emulsions and gives indications on how these properties can be modified following reformulation or introduction of new ingredients, even at low concentrations.

However, before the paper can be considered for publication, several important revisions and improvements are needed to ensure the scientific soundness of the work and to provide a more complete and accurate discussion of the topic 

 

The manuscript is of interest for cosmetic formulators: understanding how the addition of an active ingredient affects the texture and application properties of a cosmetic formulation is of great practical relevance. The combined use of rheological characterization and sensory analysis provides a clear and comprehensive picture of the structural and applicative properties of emulsions and gives indications on how these properties can be modified following reformulation or introduction of new ingredients, even at low concentrations.

However, before the paper can be considered for publication, several important revisions and improvements are needed to ensure the scientific soundness of the work and to provide a more complete and accurate discussion of the topic.

 

• Lines 42–44: In the abstract conclusion, the statement “Walnut leaf extract acts as a concentration-dependent rheological modifier, strengthening the emulsion network at low levels but inducing softening and rapid recovery at higher concentrations” is not entirely appropriate. The extract should not be described as a “rheological modifier”, since this term refers to ingredients that are specifically added to a formulation with the purpose of improving and controlling its rheological, structural, suspending, and stability properties. In this case, it would be more accurate to state that the active ingredient has a significant impact on the rheological properties of the emulsion (a behaviour that is quite common for many plant extracts and active molecules) rather than classifying it as a “rheology modifier”.

 

• Line 204-205: The selected shear rate range is very limited. It would have been more appropriate to start from a lower initial shear rate (e.g., 0.001 or 0.0001 s⁻¹) to properly visualize the Newtonian plateau (if present) and to determine the viscosity at rest, which is directly related to the sample’s consistency before application and product pick-up.

Moreover, the manuscript refers to both zero-shear viscosity (η₀) and infinite-shear viscosity (η∞), but these values do not appear to have been calculated using a mathematical model such as the Carreau–Yasuda model. It is not sufficient to consider the viscosity at 0.1 s⁻¹ as the zero-shear viscosity, since at that shear rate the sample is already in motion and partially deformed, and therefore does not represent the stationary viscosity. Similarly, the viscosity at 1000 s⁻¹ cannot be assumed to correspond to the infinite-shear viscosity, which should be obtained from model fitting of the complete flow curve. In addition, measurements at very high shear rates tend to be less accurate and should be interpreted with caution.

 

• Line 295-306 and Table 4: A previously said, the parameter reported here is not η₀, but simply the viscosity measured at 0.1 s⁻¹. Referring to this value as zero-shear viscosity is not scientifically correct. Please fit the flow curve with an appropriate constitutive model (e.g., Carreau–Yasuda or Cross) to estimate both η₀ and η∞ or simply rename the reported parameters as “viscosity at 0.1 s-1” and “viscosity at 1000 s-1” throughout the text, figures, and tables, and avoid the symbols η₀ and η∞.

 

• Line 315 – 316: The authors state that “This suggests that certain classes of polyphenolic compounds, common to many plant extracts, can act as internal modifiers of the cream's microstructure.” To substantiate this hypothesis and verify whether the walnut leaf extract truly interacts with the emulsifying system and alters the formulation’s microstructure, it would be useful to perform optical microscopy analysis. This could highlight morphological changes, such as variations in the droplet size and distribution of the dispersed phase, thereby providing direct evidence of the extract’s impact on the emulsion microstructure.

 

• Line 318-319: The statement “formulation F2, which, despite its intermediate η₀, exhibited the lowest infinite-shear viscosity (η∞)” should be reconsidered. Considering the standard deviations, this difference does not appear to be statistically significant. Moreover, viscosity values measured at high shear rates are generally less reliable and should be interpreted with caution.

 

• Line 337-338: “The frequency sweep tests (Figure 2) revealed that the storage modulus (G′) was higher than the loss modulus (G″) across the entire frequency range for all samples”. However, Figure 2 clearly shows that for sample F2, G′ and G″ values are comparable over several frequency intervals. If this is not due to measurement error, such behaviour may indicate a loss of elasticity and possible structural instability of the emulsion.

Have any mechanical stress centrifugation tests been performed to assess the mechanical stability of the prototypes and validate the conclusions about the structural robustness of the formulations.

 

• Table 5: Please specify the frequency values at which G′, G″, and tan δ listed were taken.

The standard deviations reported for formulations F1 and F2 are unusually high. Such variability may indicate sample heterogeneity or instability within the systems. Can you give an explanation?

Additionally, a discrepancy is observed between Figure 2 and Table 5: in the frequency sweep graph, F3 shows higher G′ values compared to the other formulations, but this trend is not reflected in Table 5: considering the reported standard deviations, G′ for F3 is never significantly higher than that of F2.

 

• Line 472-474: The authors correctly state that “the addition of some extracts may alter the viscosity or textural profile of the formulation: it can increase or decrease viscosity, emulsion stability, droplet size.”

To further support this statement, I recommend performing a Temperature sweep rheological test to evaluate the influence of temperature on G′ and G″ and obtain predictive information on the thermal sensitivity and long-term stability.

 

• Line 549-551: The authors state that “The correlation analysis between rheological and sensory data demonstrated that…” However, it is not clear whether and how the correlation analysis was performed. Please report in the text statistical details, such as correlation coefficients, significance levels (p-values), or the type of test applied (e.g., Pearson or Spearman correlation). Without these data, it is difficult to assess the robustness and reliability of the claimed relationships between rheological and sensory parameters.

 

• The authors often refer to the polyphenolic content of the walnut leaf extract, but no quali-quantitative composition data are provided, nor are the results of its chemical characterization discussed. A more detailed discussion linking the chemical composition of the extract to its interactions with the emulsified system would significantly enhance the understanding of the observed rheological behaviour.

 

 

Author Response

Reviewer 1

The manuscript is of interest for cosmetic formulators: understanding how the addition of an active ingredient affects the texture and application properties of a cosmetic formulation is of great practical relevance. The combined use of rheological characterization and sensory analysis provides a clear and comprehensive picture of the structural and applicative properties of emulsions and gives indications on how these properties can be modified following reformulation or introduction of new ingredients, even at low concentrations.

However, before the paper can be considered for publication, several important revisions and improvements are needed to ensure the scientific soundness of the work and to provide a more complete and accurate discussion of the topic.

 

Comments 1. Lines 42–44: In the abstract conclusion, the statement “Walnut leaf extract acts as a concentration-dependent rheological modifier, strengthening the emulsion network at low levels but inducing softening and rapid recovery at higher concentrations” is not entirely appropriate. The extract should not be described as a “rheological modifier”, since this term refers to ingredients that are specifically added to a formulation with the purpose of improving and controlling its rheological, structural, suspending, and stability properties. In this case, it would be more accurate to state that the active ingredient has a significant impact on the rheological properties of the emulsion (a behaviour that is quite common for many plant extracts and active molecules) rather than classifying it as a “rheology modifier”.

Response 1. Thank you for this observation. We agree with the reviewer that the term “rheological modifier” is not appropriate in this context, as the walnut leaf extract was not added to control the rheological properties of the formulation. Accordingly, we have revised the sentence in the abstract and throughout the manuscript. The text now states that the extract “affects rheological behavior” rather than describing it as a “rheological modifier.” All changes are highlighted in red in the revised manuscript.

 

Comments 2. Line 204-205: The selected shear rate range is very limited. It would have been more appropriate to start from a lower initial shear rate (e.g., 0.001 or 0.0001 s⁻¹) to properly visualize the Newtonian plateau (if present) and to determine the viscosity at rest, which is directly related to the sample’s consistency before application and product pick-up. Moreover, the manuscript refers to both zero-shear viscosity (η₀) and infinite-shear viscosity (η∞), but these values do not appear to have been calculated using a mathematical model such as the Carreau–Yasuda model. It is not sufficient to consider the viscosity at 0.1 s⁻¹ as the zero-shear viscosity, since at that shear rate the sample is already in motion and partially deformed, and therefore does not represent the stationary viscosity. Similarly, the viscosity at 1000 s⁻¹ cannot be assumed to correspond to the infinite-shear viscosity, which should be obtained from model fitting of the complete flow curve. In addition, measurements at very high shear rates tend to be less accurate and should be interpreted with caution.

Response 2. We thank the reviewer for this valuable and constructive comment. In accordance with the suggestion, we have completely revised the methodology for flow curve measurement and the corresponding interpretation of rheological parameters.

First, the shear rate range has been extended to 0.001–1000 s⁻¹. This modification is implemented in Section 2.9.3 (Flow Curve Test) of the revised manuscript. We agree that the previously used range was not sufficient to cover the potential Newtonian plateau and this limitation has now been removed.

Second, instead of approximating the zero-shear viscosity (η₀) and infinite-shear viscosity (η∞) from individual experimental points, the revised manuscript now presents the values ​​of η₀ and η∞ obtained by mathematically fitting the model. Following the reviewer's recommendation, the full viscosity curves were fitted using the Cross, Caro-Yasuda and Caro models, while the Ostwald (power law) model was used to extract the parameters K and n. The model showing the highest correlation coefficient (R²) was selected for interpretation. These changes are reflected in sections 2.9.3 and 3.2.2, as well as in the revised Table 7.

Third, as the reviewer correctly pointed out, the very low shear plateau behavior is not clearly observed in all formulations. Accordingly, we now explicitly state that η₀ estimates for formulations with poorly defined low shear plateaus (especially F1 and F3) are treated as unreliable, and instead, the experimental viscosity at 0.001 s⁻¹ is used as a robust indicator of near-steady state behavior. This clarification is provided in section 3.2.2 of the revised manuscript.

 

Comments 3. Line 295-306 and Table 4: A previously said, the parameter reported here is not η₀, but simply the viscosity measured at 0.1 s⁻¹. Referring to this value as zero-shear viscosity is not scientifically correct. Please fit the flow curve with an appropriate constitutive model (e.g., Carreau–Yasuda or Cross) to estimate both η₀ and η∞ or simply rename the reported parameters as “viscosity at 0.1 s-1” and “viscosity at 1000 s-1” throughout the text, figures, and tables, and avoid the symbols η₀ and η∞.

Response 3. We thank the Reviewer for the comment. Following your recommendation, we have implemented all the corrections described in our previous response.

 

Comments 4. Line 315 – 316: The authors state that “This suggests that certain classes of polyphenolic compounds, common to many plant extracts, can act as internal modifiers of the cream's microstructure.” To substantiate this hypothesis and verify whether the walnut leaf extract truly interacts with the emulsifying system and alters the formulation’s microstructure, it would be useful to perform optical microscopy analysis. This could highlight morphological changes, such as variations in the droplet size and distribution of the dispersed phase, thereby providing direct evidence of the extract’s impact on the emulsion microstructure.

Response 4. We thank the reviewer for this valuable suggestion. We performed optical microscopy analysis to verify whether walnut leaf extract affects the microstructure of the emulsion. The corresponding methodology, micrographs, and an expanded discussion of the observed morphological changes have been added to the revised manuscript.

 

Comments 5. Line 318-319: The statement “formulation F2, which, despite its intermediate η₀, exhibited the lowest infinite-shear viscosity (η∞)” should be reconsidered. Considering the standard deviations, this difference does not appear to be statistically significant. Moreover, viscosity values measured at high shear rates are generally less reliable and should be interpreted with caution.

Response 5. We thank the reviewer for this comment. The statement referring to formulation F2 as having the lowest infinite shear viscosity (η∞) was removed during the revision of the rheological analysis. As suggested, the interpretation of the high shear viscosity value has been corrected and updated accordingly, and the current version of the manuscript no longer contains this sentence.

 

Comments 6. Line 337-338: “The frequency sweep tests (Figure 2) revealed that the storage modulus (G′) was higher than the loss modulus (G″) across the entire frequency range for all samples”. However, Figure 2 clearly shows that for sample F2, G′ and G″ values are comparable over several frequency intervals. If this is not due to measurement error, such behaviour may indicate a loss of elasticity and possible structural instability of the emulsion.

Response 6. We thank the reviewer for this observation. The sentence has been revised, and the updated text now reflects that the F2 formulation shows frequency intervals where G′ and G″ are comparable. This behavior is not due to measurement error, but corresponds to a reduced elastic contribution in F2, which is also consistent with its higher value of tan δ. Figure 2 and the accompanying discussion have been corrected accordingly.

 

Comments 7. Have any mechanical stress centrifugation tests been performed to assess the mechanical stability of the prototypes and validate the conclusions about the structural robustness of the formulations.

Response 7. We appreciate the reviewer's question. Mechanical stress tests, such as centrifugation, were not performed in this study, as the structural robustness of the formulations was assessed through multiple complementary rheological measurements (amplitude gap, frequency gap, flow curves, and thixotropy), which together provide a reliable assessment of mechanical stability. Given the consistent viscoelastic behavior in these tests and the absence of any signs of structural failure, additional centrifugation testing was deemed unnecessary for the scope of this work.

 

Comments 8. Table 5: Please specify the frequency values at which G′, G″, and tan δ listed were taken.

Response 8. We thank the reviewer for his comment. The frequency at which G′, G″ and tan δ were extracted is now listed in the table legend. All viscoelastic parameters in Table 5 are taken at 10 rad/s.

 

Comments 9. The standard deviations reported for formulations F1 and F2 are unusually high. Such variability may indicate sample heterogeneity or instability within the systems. Can you give an explanation?

Response 9. We thank the reviewer for this observation. The larger standard deviations reported for formulations F1 and F2 are now addressed in the revised manuscript in the Results and Discussion section 3.2.3. Viscoelastic Properties and Structural Stability

 

Comments 10. Additionally, a discrepancy is observed between Figure 2 and Table 5: in the frequency sweep graph, F3 shows higher G′ values compared to the other formulations, but this trend is not reflected in Table 5: considering the reported standard deviations, G′ for F3 is never significantly higher than that of F2.

Response 10. We thank the Reviewer for pointing out this inconsistency. During the revision of the manuscript, we re-checked the raw data and identified that Figure 2 and Table 5 were originally derived from measurements taken at different time points. Both the figure and the table have now been updated using the same dataset, and the trend observed in the frequency sweep is now fully consistent with the statistical results presented in the table. We have clarified this in the revised version of the manuscript.

 

Comments 11. Line 472-474: The authors correctly state that “the addition of some extracts may alter the viscosity or textural profile of the formulation: it can increase or decrease viscosity, emulsion stability, droplet size.” To further support this statement, I recommend performing a Temperature sweep rheological test to evaluate the influence of temperature on G′ and G″ and obtain predictive information on the thermal sensitivity and long-term stability.

Response 11. We thank the reviewer for a helpful suggestion. Although a temperature change test was not performed, a literature review found that emulsions stabilized with Olivem-type emulsifiers maintain their structural integrity under moderate thermal variations. A clarification on thermal robustness has been added to section 3.2.3 of the revised manuscript.

 

Comments 12. Line 549-551: The authors state that “The correlation analysis between rheological and sensory data demonstrated that…” However, it is not clear whether and how the correlation analysis was performed. Please report in the text statistical details, such as correlation coefficients, significance levels (p-values), or the type of test applied (e.g., Pearson or Spearman correlation). Without these data, it is difficult to assess the robustness and reliability of the claimed relationships between rheological and sensory parameters.

Response 12. We thank the Reviewer for this observation. In the revised manuscript, we have removed the phrase “correlation analysis” because no formal statistical correlation test was performed. Instead, the text has been rephrased to describe the qualitative agreement between sensory impressions and rheological trends, without implying statistical testing. This correction eliminates any ambiguity regarding the type of analysis conducted.

 

Comments 13. The authors often refer to the polyphenolic content of the walnut leaf extract, but no quali-quantitative composition data are provided, nor are the results of its chemical characterization discussed. A more detailed discussion linking the chemical composition of the extract to its interactions with the emulsified system would significantly enhance the understanding of the observed rheological behaviour.

Response 13. We thank the reviewer for this valuable comment. In response, we have expanded the manuscript by adding qualitative and quantitative phytochemical characterization of walnut leaf extracts (sections 2.3–2.6 and 3.1).

 

Reviewer 2 Report

Comments and Suggestions for Authors

The study addresses an interesting topic, exploring the formulation and sensory/rheological characterization of Juglans regia-based emulsions. However, some methodological aspects should be clarified to improve reproducibility and scientific robustness.

1. The Materials and Methods section does not clearly describe the human study design. Please specify how the 20 volunteers were selected (sex, skin type, inclusion/exclusion criteria) and whether all tested all formulations (F1-F4).

2. Consider reporting any observations on tolerability or adverse effects, as this information is relevant from a dermatological standpoint.

3. The correlation between rheological parameters and sensory perception could be better discussed, as it may reveal meaningful structure-function relationships.

4. The statistical approach should be reconsidered for sensory data (non-parametric tests may be more appropriate).

5. The discussion would benefit from citing recent works that highlight innovation in cosmetic research and evaluation, such as:

   • Di Guardo et al. Artificial Intelligence in Cosmetic Formulation: Predictive Modeling for Safety, Tolerability, and Regulatory Perspectives. Cosmetics 2025;12(4):157. DOI: 10.3390/cosmetics12040157

   • Michelini et al. Non-Invasive Imaging for the Evaluation of a New Oral Supplement in Skin Aging: A Case-Controlled Study. Skin Res Technol 2025. DOI: 10.1111/srt.70171

Overall, this is a technically sound study that would gain clinical and translational depth by refining methodological details and contextualizing findings within current dermatological research trends.

Author Response

Reviewer 2

The study addresses an interesting topic, exploring the formulation and sensory/rheological characterization of Juglans regia-based emulsions. However, some methodological aspects should be clarified to improve reproducibility and scientific robustness.

 

Comments 1. The Materials and Methods section does not clearly describe the human study design. Please specify how the 20 volunteers were selected (sex, skin type, inclusion/exclusion criteria) and whether all tested all formulations (F1-F4).

Response 1. We appreciate your comment. The Materials and Methods section describing the sensory analysis design has been updated according to your suggestions.

 

Comments 2. Consider reporting any observations on tolerability or adverse effects, as this information is relevant from a dermatological standpoint.

Response 2. We appreciate the reviewer’s valuable remark. A sentence has now been added to the Sensory Evaluation section to report the tolerability observations. Specifically, we clarified that no adverse skin reactions (e.g., redness, irritation, itching, burning, or discomfort) were observed during or after product application, indicating that all formulations were well tolerated under the test conditions.

 

Comments 3. The correlation between rheological parameters and sensory perception could be better discussed, as it may reveal meaningful structure-function relationships.

Response 3. We thank the reviewer for this comment. In response, the discussion in Section 3.5 has been revised and expanded to more clearly describe how sensory perceptions match the rheological behavior of formulations. The updated text now highlights key structure-function relationships without introducing additional analysis, ensuring that the interpretation remains consistent with the data.

 

Comments 4. The statistical approach should be reconsidered for sensory data (non-parametric tests may be more appropriate).

Response 4. We appreciate your comment. However, we have already applied non-parametric tests for the analysis of the sensory characteristics of the formulations - the Chi-square test, the Friedman test, and the sum of ranks with multiple sample comparisons using Fisher’s LSD test. High rank sums correspond to a high level of perception for each sensory attribute. Differences were considered statistically significant if p < 0.05 and highly significant if p < 0.01. It is presented in section 3.4. We overlooked the inclusion of the statistical analysis description in the Statistical Analysis section. The correction has now been made. The statistical analysis and results presentation are in accordance with the other studies evaluating sensory attributes of the creams.

1. Ali A, Skedung L, Burleigh S, Lavant E, Ringstad L, Anderson C D, Wahlgren M, Engblom J. Relationship between sensorial and physical characteristics of topical creams: a comparative study on effects of excipients. Int J Pharm. 2022;613:121370.
2. Adejokun DA, Dodou K. Quantitative Sensory Interpretation of Rheological Parameters of a Cream Formulation. Cosmetics. 2020; 7(1):2.

 

Comments 5. The discussion would benefit from citing recent works that highlight innovation in cosmetic research and evaluation, such as:

1. Di Guardo et al. Artificial Intelligence in Cosmetic Formulation: Predictive Modeling for Safety, Tolerability, and Regulatory Perspectives.Cosmetics 2025;12(4):157. DOI: 10.3390/cosmetics12040157

1. Michelini et al. Non-Invasive Imaging for the Evaluation of a New Oral Supplement in Skin Aging: A Case-Controlled Study. Skin Res Technol 2025. DOI: 10.1111/srt.70171

Overall, this is a technically sound study that would gain clinical and translational depth by refining methodological details and contextualizing findings within current dermatological research trends.

 

Response 5. Thank you for this observation. We have inserted the following two references you suggested as references 61 and 65.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I would like to acknowledge the substantial improvements made in the manuscript. The authors have carefully addressed the reviewers’ comments, implemented the suggested changes, and significantly enhanced the clarity and overall quality of the work. In my opinion, the manuscript can now be considered suitable for publication after minor revisions.

- The paragraph currently found in lines 300–314 discusses how the composition of the extract may influence the rheological properties of the emulsion (a point that was strengthened following the previous review). However, this discussion would be more coherent if moved after the description of the rheological characterization. Presenting this interpretation before the rheological data makes the flow harder to follow. I therefore recommend relocating this paragraph to the section immediately following the frequency sweep analysis.

- The term "stability" is used repeatedly throughout the manuscript. However, no experimental evidence of the physical stability is provided (e.g., centrifugation, accelerated aging, or long-term storage tests). Rheology can offer  predictions regarding long-term stability, but it does not provide direct proof. Additionally, the high variability observed in the frequency sweep curves is not typically indicative of good long-term stability. If, instead, the authors refer to "application stability" (i.e., stability during spreading on the skin rather than shelf life, thermal stability, or long-term storage), this interpretation should be made explicit in the text to avoid ambiguity.

Overall, I commend the authors for their efforts in revising the manuscript. After addressing the points above, I believe the paper will be ready for publication.

Author Response

I would like to acknowledge the substantial improvements made in the manuscript. The authors have carefully addressed the reviewers’ comments, implemented the suggested changes, and significantly enhanced the clarity and overall quality of the work. In my opinion, the manuscript can now be considered suitable for publication after minor revisions.

- The paragraph currently found in lines 300–314 discusses how the composition of the extract may influence the rheological properties of the emulsion (a point that was strengthened following the previous review). However, this discussion would be more coherent if moved after the description of the rheological characterization. Presenting this interpretation before the rheological data makes the flow harder to follow. I therefore recommend relocating this paragraph to the section immediately following the frequency sweep analysis.

We thank the reviewer for this suggestion. The suggested change has been implemented. The paragraph previously located at lines 300–314 has been moved to the section immediately following the frequency transition analysis.

- The term "stability" is used repeatedly throughout the manuscript. However, no experimental evidence of the physical stability is provided (e.g., centrifugation, accelerated aging, or long-term storage tests). Rheology can offer  predictions regarding long-term stability, but it does not provide direct proof. Additionally, the high variability observed in the frequency sweep curves is not typically indicative of good long-term stability. If, instead, the authors refer to "application stability" (i.e., stability during spreading on the skin rather than shelf life, thermal stability, or long-term storage), this interpretation should be made explicit in the text to avoid ambiguity.

In accordance with the reviewer's recommendation, all instances where the term stability appeared in a context that could be interpreted as physical stability have been revised.

Overall, I commend the authors for their efforts in revising the manuscript. After addressing the points above, I believe the paper will be ready for publication.