Royal Jelly Mitigates Cognitive Decline and Anxiety in Female Mice: A Promising Natural Neuroprotective Solution for Alzheimer’s Disease

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript presents an experimental study combining phytochemical characterization, antioxidant assays, behavioral tests, and histopathological evaluation in mouse model of Alzheimer’s disease. The topic is relevant and timely, within the scope of Compounds. The iThenticate similarity score is also OK. However, it also requires some revisions.

Detailed comments:

Lines 39-42, this should be supported by proper reference, i.e. https://doi.org/10.56782/pps.421

Lines 91–96: The origin, batch-to-batch variability, and quality control of the royal jelly sample should be described in more detail. Was only a single batch used in the study?

Lines 118–123: The preliminary phytochemical screening relies on classical colorimetric tests, which are largely qualitative and nonspecific. The limitations of these methods should be acknowledged.

Line 68, this statement should be supported by reference, i.e. https://doi.org/10.56782/pps.273

Lines 145–155: There is confusion in the description of total sugar content, particularly in the final sentence where results are again expressed as “gallic acid equivalents,” which is incorrect. Please revise as gallic acid is not a sugar. It is either a typo or a methodological mistake.

Lines 181–190 and 435: Antioxidant activity is described as “moderate”, but the reported inhibition percentages are below 7%, which is very low. This should be addressed, and the biological relevance of these antioxidant results should be discussed more critically.

Lines 323–325: Conducting multiple behavioral tests on the same day may introduce stress-related confounders. Please justify this experimental design choice.

Figure 1, there is really no need to present those ticks for peaks with wavenumbers lower than 500 cm-1.

Figures 2 and subsequent as well, the explanations for “a”, “b”, etc. should be added in the captions.

Lines 527–530: The forced swim test results are difficult to interpret in the context of Alzheimer’s disease. Reduced immobility may reflect altered motor activity rather than antidepressant-like effects. This limitation should be discussed.

Line 548–556: Histopathological observations are described qualitatively. Quantitative analysis would significantly strengthen the conclusions.

 

Author Response

To Reviewer #1 comments:

This manuscript presents an experimental study combining phytochemical characterization, antioxidant assays, behavioral tests, and histopathological evaluation in mouse model of Alzheimer’s disease. The topic is relevant and timely, within the scope of Compounds. The iThenticate similarity score is also OK. However, it also requires some revisions.

Authors’ response: Firstly, we thank you for evaluating our manuscript and acknowledging its scientific relevance. We appreciate your positive feedback and have carefully considered your points as follows:

1- Reviewer comment: Lines 39-42, this should be supported by proper reference, i.e. https://doi.org/10.56782/pps.421

Authors’ response: Thank you for this observation. We agree that a suitable reference was needed and have included it as recommended (Line 42).

2- Reviewer comment: Lines 91–96: The origin, batch-to-batch variability, and quality control of the royal jelly sample should be described in more detail. Was only a single batch used in the study?

Authors’ Response:  Thank you for emphasizing the importance of batch details and quality control. The royal jelly, sourced from BALPARMAK in Istanbul, Turkey, was a single batch, so inter-lot variability was not addressed. The Folin-Ciocalteu assay, suitable for all batches, was used for quality control. This information was added to the Materials and Methods section (Lines 101 and 102).

3- Reviewer comment: Lines 118–123: The preliminary phytochemical screening relies on classical colorimetric tests, which are largely qualitative and nonspecific. The limitations of these methods should be acknowledged.

Authors’ Response: We agree with the reviewer that the classic colorimetric tests upon which our study relies are indeed primarily qualitative and nonspecific. However, these methods provide a rapid and effective preliminary assessment of the presence of various secondary metabolites, which is essential for early-stage exploratory research.

We want to emphasize that, although these classical colorimetric tests have limitations, they were chosen for their simplicity and for providing preliminary indications of the chemical composition of the royal jelly extract. However, we intend to conduct further studies to deepen our knowledge of the bioactive compounds present in royal jelly extract, using quantitative and more specific approaches, such as chromatography or mass spectrometry, in future investigations. We added this information in the discussion section (Lines 719-724). 

By incorporating this discussion into the revised manuscript, we aim to clarify our analytical approach and highlight the value of these tests as part of an initial study on the bioactive compounds of royal jelly extract.

4- Reviewer comment: Line 68, this statement should be supported by reference, i.e. https://doi.org/10.56782/pps.273

Authors’ Response: Thank you very much for your valuable observation. We agree with the reviewer that these lines need an appropriate reference and, following their recommendation, we have included the aforementioned reference (Line 77).

5- Reviewer comment: Lines 145–155: There is confusroion in the description of total sugar content, particularly in the final sentence where results are again expressed as “gallic acid equivalents,” which is incorrect. Please revise as gallic acid is not a sugar. It is either a typo or a methodological mistake.

Authors’ Response: We are very grateful that you pointed out our methodological error and apologized for it. We confirm that a typo occurred in the description of the total sugar content. The results should indeed be expressed in "maltose equivalents" and not in "gallic acid equivalents". As the reviewer noted, we corrected the description accordingly (Line 166).

6- Reviewer comment: Lines 181–190 and 435: Antioxidant activity is described as “moderate”, but the reported inhibition percentages are below 7%, which is very low. This should be addressed, and the biological relevance of these antioxidant results should be discussed more critically.

Authors’ Response: We chose the term "moderate antioxidant activity" because it reflects not only our results relative to standard references in the field, but also the broader context of antioxidant activity. Although an inhibition percentage below 7% is considered low compared to potent antioxidants, this may still indicate significant antioxidant capacity in the context of royal jelly (El-Guendouz et al., 2020), especially given its regular consumption (Alu’datt et al.,2025; Viuda-Martos et al., 2025). Moreover, its antioxidant effects are biologically relevant in Alzheimer's disease. A previous study demonstrated that royal jelly reduced lipid peroxidation and increased total antioxidant capacity in the hippocampus of Wistar rats administered cerebroventricular beta-amyloid (Raoufi et al., 2023). Another research showed that royal jelly decreased malondialdehyde levels in the brains and plasma of APP/PS1 mice (You et al., 2019). This neuroprotective potential reduced oxidative stress-induced neurotoxicity and neuronal death, thereby improving cognitive deficits in these experimental models (Raoufi et al., 2023; You et al., 2019). Highlighting the essential role of the royal jelly antioxidant actions in Alzheimer's disease.

As requested by the reviewer, we included this text in the discussion section (Lines 725-736).

References:

El-Guendouz et al., 2020. doi: 10.1177/1934578X20908080

El-Guendouz et al., 2020. doi: 10.1080/00218839.2020.1744241

Alu’datt et al., 2025. doi: 10.1080/10408398.2024.2418892

Viuda-Martos et al., 2025. doi: 10.1007/978-3-031-89049-9_10

Raoufi et al., 2023. doi: 10.1007/s11011-023-01168-9

You et al., 2019. doi: 10.3389/fnagi.2018.00428

7- Reviewer comment: Lines 323–325: Conducting multiple behavioral tests on the same day may introduce stress-related confounders. Please justify this experimental design choice.

Authors’ Response: All behavioral tests were conducted on the same day to minimize interindividual variability that may arise when assessments are performed on different days. This approach ensured that environmental conditions, including noise, lighting, and other external factors, remained constant throughout the experimental procedures, thereby enhancing the reliability and reproducibility of the results. Moreover, performing the tests on separate days could lead to habituation or desensitization of the animals to the behavioral challenges, potentially confounding the outcomes. By administering the tests consecutively, behavioral responses were assessed under comparable activation states, allowing for a more accurate comparison between experimental groups.

From a logistical perspective, conducting all tests on the same day also optimized time and resource management, facilitating more efficient experimental execution without compromising scientific rigor. Notably, behavioral responses are influenced by prior experiences and by the animals' physiological or emotional state. Synchronizing the behavioral assessments enabled a more precise evaluation of treatment-induced effects within a defined temporal window following administration. In addition, assessing multiple behaviors within a short timeframe allowed the identification of potential interactions or cumulative effects of the treatments on the same day, providing deeper insight into the effects of royal jelly extract.

Although it is recognized that exposure to multiple behavioral tests may induce stress and potentially influence outcomes, this methodological decision was made to balance experimental validity, efficiency, and scientific robustness. To mitigate stress-related effects, all procedures followed protocols approved by the institutional ethics committee, and tests were conducted in a calm and controlled environment. Furthermore, each animal was allowed to rest for approximately 5 minutes between test phases and nearly 10 minutes between tests, thereby reducing stress and supporting optimal behavioral performance.

8- Reviewer comment: Figure 1, there is really no need to present those ticks for peaks with wavenumbers lower than 500 cm-1.

Authors’ Response: We thank the reviewer for this pertinent observation and for the opportunity for us to explain our choice.

We agree with the reviewer's observation. However, the inclusion of peaks with wavenumbers below 500 cm⁻¹ is scientifically justified for several reasons. Signals in this low-wavenumber region may provide complementary information regarding the chemical structures of the compounds present, contributing to a more comprehensive understanding of molecular interactions and physicochemical properties within the samples. In spectroscopic analyses, it is common practice to present the full spectral range, including regions of lower apparent significance, to provide a complete overview of the data. This approach facilitates comparison with previously published studies and allows for the identification of subtle spectral features that may be relevant under specific analytical or experimental conditions.

Furthermore, bands observed below 500 cm⁻¹ may be associated with interaction phenomena, such as weak intermolecular forces or low-energy vibrational or electronic transitions, which, although less prominent, may carry meaningful implications for interpreting the results. Therefore, while some spectral features in this region may appear redundant at first glance, their inclusion adds contextual value to the figure and supports a holistic interpretation of the spectroscopic data. Maintaining these features in the graphical representation enhances the completeness and interpretability of the analysis, particularly for future comparative or mechanistic studies.

9- Reviewer comment: Figures 2 and subsequent as well, the explanations for “a”, “b”, etc. should be added in the captions.

Authors’ Response: We sincerely appreciate the reviewer's observation.

In the graphs of each of the figures in the paper, bars with the same letters do not show a statistical difference in themselves. Different letters indicate a statistical difference compared with the other groups. These analyses were performed using SPSS software.

As recommended by the reviewer, we included this explanation in all figure legends.

10- Reviewer comment: Lines 527–530: The forced swim test results are difficult to interpret in the context of Alzheimer’s disease. Reduced immobility may reflect altered motor activity rather than antidepressant-like effects. This limitation should be discussed.

Authors’ Response: We acknowledge that interpreting the results of the Forced Swim Test (FST) in the context of Alzheimer's Disease (AD) can be challenging, but its use in our study is justified. The FST is a widely recognized method for assessing anxiety and depression-like behaviors in animal models, allowing consistent comparisons with another AD study (Torres-Lista and Giménez-Llort, 2014). While reduced immobility can indicate changes in motor activity, it often reflects an active response to treatment, particularly relevant to our investigation of royal jelly's extract neuroprotective effects. Moreover, we complemented the FST with other assessments, such as the Morris Water Maze, to provide a holistic view of cognitive and behavioral impacts. The test also helps elucidate neurobiological mechanisms in AD by showing how treatment may modulate pathways associated with depression and anxiety, which have direct clinical implications for AD patients and contribute to the development of natural therapies.

As requested by the reviewer, we included this text in the discussion section (Lines 788-797).

References:

Torres-Lista and Giménez-Llort, 2014. doi: 10.1016/j.beproc.2014.05.001

11- Reviewer comment: Line 548–556: Histopathological observations are described qualitatively. Quantitative analysis would significantly strengthen the conclusions.

Authors’ Response: Thank you for your insightful feedback.

We value the suggestion for quantitative analysis and plan to implement it in our future research to enhance our findings. We believe that a combined approach will not only strengthen our conclusions but also contribute to the broader understanding of the effects of royal jelly on neurodegenerative processes associated with Alzheimer's disease. However, in the present study, we focused on whether royal jelly extract would have beneficial effects in this Alzheimer's model in females. Therefore, the histological analyses aimed to identify potential neuroprotective effects of the extract, so that, in ongoing investigations, we can examine the mechanisms underlying these beneficial effects and conduct an in-depth study of brain histology using other stains and immunofluorescence.

 

We sincerely appreciate the reviewer's valuable comments, which have improved our paper. We have carefully responded to each comment and hope the paper will be suitable for publication in Compounds.

Best Regards,

Graziele Freitas de Bem

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript investigated the chemical composition of royal jelly extract and its effects on neurobehavioral changes in a female AD model.  It revealed that the extract was rich in chemical composition such as phenols and flavonoids, exhibiting antioxidant activity, and significantly improving cognitive deficits, neurodegeneration, and anxiety behaviors in AD model mice. These findings provide a new perspective for the application of royal jelly and the development of preventive strategies for AD.

1. Please reconsider the use of "Flavonoids" in the title. The data in the manuscript do not sufficiently demonstrate that flavonoids are the primary active components in royal jelly, as other types of substances such as polyphenols and polysaccharides are also present in high amounts. The major limitation is that there is no data supporting the relationship between specific components and the observed bioactivity.
2. It is recommended to replace "phytochemical" with "chemical" throughout the manuscript, given that the constituents under investigation are animal derived.
3. Line 54, please supplement with specific details regarding the reported "beneficial effects of royal jelly in Alzheimer's models", and explicitly state how this manuscript's findings differ from those earlier reports.
4. While the manuscript highlights gender disparity in AD prevalence and employs a female mouse model, does royal jelly potentially have different effects in male or mice? Please provide some supplementary information based on the literatures.
5. It is recommended to add detailed reaction conditions (e.g., temperature and time for heating) in Table 1.
6. Materials and Methods section, please provide the concentrations of all experimental samples and reaction reagents, such as the concentration of the extract/reagent referenced in Line 132.
7. Sections 2.3.3 and 2.3.4, why was "calibration" chosen as the reference instead of glucose?
8. Line 15, "gallic acid" is incorrect.
9. Line 221, what is the relationship between "34 female Naval Medical Research Institute mice" and “n = 25” in line 23.
10. The experimental design of this manuscript primarily evaluates the preventive effect of royal jelly in Alzheimer's disease (AD). Why was an assessment of its therapeutic effect not conducted, and what considerations informed this decision?
11. Figure 1, please label the positions of the key absorption peaks emphasized in the manuscript. Furthermore, please validate the accuracy of the 1700 cm⁻¹ in Line 414 and check the presence of an absorption peak near ~600 cm⁻¹ in Figure 1 as presented in Table 5.
12. Table 6, please explain the meaning of "inhibition percentage".
13. Section 3.6, please provide a concise explanation of the main evidence for “absence of any signs of toxicity”.
14. Please check the data of the ALZ-STD group in Figures 4 and 5, which appears to be inconsistent with the description in the manuscript.
15. Please check the correspondence between the description in section 3.8.1.2 and the data in Figure 1.

Author Response

To Reviewer #2 comments:

The manuscript investigated the chemical composition of royal jelly extract and its effects on neurobehavioral changes in a female AD model.  It revealed that the extract was rich in chemical composition such as phenols and flavonoids, exhibiting antioxidant activity, and significantly improving cognitive deficits, neurodegeneration, and anxiety behaviors in AD model mice. These findings provide a new perspective for the application of royal jelly and the development of preventive strategies for AD.

Authors’ response: Firstly, we would like to express our sincere gratitude for your willingness to evaluate this manuscript and to recognize its scientific relevance. We truly appreciate your positive evaluation and have carefully considered your point-to-point, as follows:

1- Reviewer comment: Please reconsider the use of "Flavonoids" in the title. The data in the manuscript do not sufficiently demonstrate that flavonoids are the primary active components in royal jelly, as other types of substances such as polyphenols and polysaccharides are also present in high amounts. The major limitation is that there is no data supporting the relationship between specific components and the observed bioactivity.

Authors’ Response: We are very grateful for your comment highlighting the need to reconsider the use of the flavonoids in the title of the manuscript. We agree with the reviewer that we need to deepen our studies to better highlight the potential of flavonoids for the beneficial effects observed. Furthermore, extensive research has demonstrated the importance of the synergistic effects of bioactive compounds on neuroprotection. Therefore, we followed the reviewer's recommendation and modified the manuscript title as follows:

Royal jelly mitigates cognitive decline and anxiety in female mice: a promising natural neuroprotective solution for Alzheimer's disease.

2- Reviewer comment: It is recommended to replace "phytochemical" with "chemical" throughout the manuscript, given that the constituents under investigation are animal derived.

Authors’ Response: Thank you very much for your valuable observation. We agree with the reviewer and replace "phytochemical" with "chemical" throughout the manuscript.

3- Reviewer comment: Line 54, please supplement with specific details regarding the reported "beneficial effects of royal jelly in Alzheimer's models", and explicitly state how this manuscript's findings differ from those earlier reports.

Authors’ Response:  We greatly appreciate your feedback and the importance you place on supplementing the text with a detailed description of the effects of royal jelly on AD models and in highlighting the novelty of our study. As recommended by the reviewer, we supplemented the text as follows (Lines 55-65):

Regarding the effects of royal jelly on Alzheimer's disease models, previous studies demonstrated that its natural product reduced beta-amyloid levels (Pan et al., 2018; You et al., 2019) and neurodegeneration (Pan et al., 2018; You et al., 2019; Silva et al., 2020) while increasing antioxidant capacity (Pan et al., 2018; Raoufi et al., 2023; Silva et al., 2020) and neuronal metabolic activity (Pan et al., 2018). Moreover, royal jelly elevated hippocampal BDNF levels (You et al., 2019). Finally, also improved learning and memory impairment (You et al., 2019; Raoufi et al., 2023; Silva et al., 2020). These data highlight its pharmacological potential in AD. We found one article in the literature that investigated the effects of royal jelly in females, which used APP/PS1 transgenic mice; the other studies used only male mice. Therefore, the beneficial effects in females and the mechanisms underlying these actions still require further investigation. Moreover, this study will investigate freeze-dried royal jelly. This technique improves the stability of the extract, a breakthrough that could enhance its effectiveness while preserving bioactive compounds.

References:

Pan et al., 2018. doi: 10.3389/fnagi.2018.00050

You et al., 2019. doi: 10.3389/fnagi.2018.00428

Raoufi et al., 2023. doi: 10.1007/s11011-023-01168-9

Silva et al., 2020. doi: 10.1016/j.heliyon.2020.e03281

4- Reviewer comment: While the manuscript highlights gender disparity in AD prevalence and employs a female mouse model, does royal jelly potentially have different effects in male or mice? Please provide some supplementary information based on the literatures.

Authors’ Response:  We thank the reviewer for this relevant observation regarding the use of the female mouse model and the potential sex-dependent effects of royal jelly.

As demonstrated in the previous question, royal jelly has beneficial effects on memory and learning deficits, as well as on beta-amyloid deposition and neurodegeneration, in male experimental models mimicking Alzheimer's disease (Pan et al., 2018 ; You et al., 2019; Raoufi et al., 2023; Silva et al., 2020). The only study we found that investigates the effects of royal jelly in males and females in these experimental models did not separate the sexes (You et al., 2019), making it impossible to examine sex differences. The literature lacks information about sex differences regarding royal jelly effects in Alzheimer's disease, with investigations mainly focused on males, highlighting the importance of studying females. However, hormonal differences between the sexes, including variations in estrogen and testosterone levels, may affect both susceptibility to disease and response to therapeutic interventions. Regarding this, a previous study demonstrated that royal jelly has estrogenic activity (Mishima et al., 2005), as its fatty acids can modulate estrogen receptors (Moutsatsou et al., 2010; Suzuki et al., 2008), thereby improving menopausal symptoms. Moreover, a randomized placebo-controlled clinical trial showed that eight weeks of royal jelly administration alleviated menopausal symptoms (Sharif and Darsareh, 2019). This royal jelly property has an essential role in AD since estrogen deficiency after menopause is associated with autonomic nervous changes, leading to memory impairment and increased susceptibility to AD (Pan et al., 2019).

As requested by the reviewer, we included this text in the discussion section (Lines 739-750).

References:

Pan et al., 2018. doi: 10.3389/fnagi.2018.00050

You et al., 2019. doi: 10.3389/fnagi.2018.00428

Raoufi et al., 2023. doi: 10.1007/s11011-023-01168-9

Silva et al., 2020. doi: 10.1016/j.heliyon.2020.e03281

Mishima et al., 2005. doi: 10.1016/j.jep.2005.04.012

Moutsatsou et al., 2010. doi: 10.1371/journal.pone.0015594

Suzuki et al., 2008. doi: 10.1093/ecam/nem036

Sharif and Darsareh, 2019. doi: 10.1016/j.ctcp.2019.08.006

Pan et al., 2019. doi: 10.3390/molecules24061149

5- Reviewer comment: It is recommended to add detailed reaction conditions (e.g., temperature and time for heating) in Table 1.

Authors’ Response:  Thank you very much for your valuable observation.

As recommended by the reviewer, we added detailed reaction conditions in the table 1 legend as follows:

For the Gallic/Catechin tannin test, the mixture of 10 ml of Stiasny extract and reagents was heated in a water bath at 90 °C for 15 minutes, and the sugar test was performed at 40-60 °C until it boiled. All other tests took place at room temperature.

6- Reviewer comment: Materials and Methods section, please provide the concentrations of all experimental samples and reaction reagents, such as the concentration of the extract/reagent referenced in Line 132.

Authors’ Response: In our experimental protocols, the extract used in the study is a 1% solution diluted in water. For the polyphenol assay, the Folin-Ciocalteu reagent is diluted 1/10 in distilled water, sodium carbonate is 7.5%, and gallic acid is 1%. For the flavonoid assay, aluminum nitrate is 0.1%, ammonium acetate is 7.7%, and quercetin is 1%. For the total sugar assay, we used 5% phenol with concentrated sulfuric acid, and maltose was also 1%. Finally, for the reducing sugar assay, the DNS reagent is prepared with 3.5% dinitrosalicylic acid, potassium-sodium tartrate, and sodium hydroxide, containing 1% maltose.

As recommended by the reviewer, we added this information to the Materials and Methods section.

7- Reviewer comment: Sections 2.3.3 and 2.3.4, why was "calibration" chosen as the reference instead of glucose?

Authors’ Response:  The term "calibration" refers to the method of creating standard curves that allow quantification of sugar concentrations in samples. Maltose was chosen as the gold standard for several reasons.

Maltose, being a disaccharide, exhibits analytical and behavioral properties in tests that are comparable to those of the sugars present in royal jelly, especially in the context of the method used for the determination of total sugars and reducing sugars. Using maltose, we can observe a reaction similar to that of the sugars present in our sample, thus providing reliable, consistent results.

In addition, although glucose is a typical sugar in other studies, maltose has shown better sensitivity and accuracy in the methods we used. This is particularly relevant in calibration procedures with sulfuric acid and dinitrosalicylic acid, where the maltose reaction is better documented and validated for these types of analyses.

In summary, the choice of maltose as the reference standard is based on its chemical relevance to the compounds present in our sample and its proven effectiveness in the analytical methods used (Wang et al., 2010). As recommended by the reviewer, we added this information to the Materials and Methods section (Lines 167-171).

Reference:

Wang et al., 2010. doi: 10.1111/j.1750-3841.2009.01504.x

8- Reviewer comment: Line 15, "gallic acid" is incorrect.

Authors’ Response: We are very grateful that you pointed out our methodological error and apologized for it. We confirm that a typo occurred in the description of the total sugar content. The results should indeed be expressed in "maltose equivalents" and not in "gallic acid equivalents". As the reviewer noted, we corrected the description accordingly (Line 166).

9- Reviewer comment: Line 221, what is the relationship between "34 female Naval Medical Research Institute mice" and “n = 25” in line 23.

Authors’ Response:  We thank the reviewer for this relevant observation.

The total number of mice mentioned, 34, includes all mice used, including the 9 mice dedicated to the toxicity test. The remaining 25 are the number needed to assess neuroprotective activity in our experiment. We divided these 25 mice into 5 groups of 5 each, enabling a systematic, controlled evaluation of the neuroprotective effects of royal jelly.

As recommended by the reviewer, we added this information to the Materials and Methods section (Lines 239 and 240).

10- Reviewer comment: The experimental design of this manuscript primarily evaluates the preventive effect of royal jelly in Alzheimer's disease (AD). Why was an assessment of its therapeutic effect not conducted, and what considerations informed this decision?

Authors’ Response:  The main objective of our study was to explore the preventive potential of royal jelly in modulating risk factors associated with Alzheimer's disease. Protecting brain health before the onset of disease is essential, as treating AD requires an accurate understanding of its stage of progression. Evaluating the preventive effect enables us to act early, before symptoms appear. In addition, there are still many questions regarding the pathophysiology of AD, making treatment challenging. Once damaged, neuronal cells are difficult to treat or regenerate. Conducting studies on the therapeutic effect would require models already affected by the disease, which would introduce additional complexities into the analysis of results and complicate the determination of disease stage.

For these reasons, we have chosen to focus our efforts on the preventive effect. However, we recognize the importance of exploring therapeutic effects in future studies, which may offer a more comprehensive perspective on royal jelly's potential in Alzheimer's disease.

11- Reviewer comment: Figure 1, please label the positions of the key absorption peaks emphasized in the manuscript. Furthermore, please validate the accuracy of the 1700 cm⁻¹ in Line 414 and check the presence of an absorption peak near ~600 cm⁻¹ in Figure 1 as presented in Table 5.

Authors’ Response:  Thank you very much for your valuable observation.

As recommended by the reviewer, we reformulated Figure 1 and substituted it in the paper as follows:

12- Reviewer comment: Table 6, please explain the meaning of "inhibition percentage".

Authors’ Response:  Thank you very much for your valuable observation.

The "inhibition percentage" in Table 6 refers to the antioxidant effectiveness of royal jelly extract, as measured by the DPPH and ABTS methods, which quantify its ability to neutralize free radicals responsible for oxidative stress, highlighting its potential role in neuroprotection. The IC50 of 0.36 mg/ml for DPPH indicates the concentration required to reduce the radical's intensity by 50%, corresponding to 6.52% inhibition, suggesting moderate efficacy compared to the reference of 0.065 mg/ml. Similarly, for ABTS, the IC50 of 0.38 mg/ml and the 5.94% inhibition also indicate an antioxidant capacity lower than the reference of 0.26 mg/ml. These results highlight the importance of further exploring the potential synergistic effects of royal jelly's compounds for health applications.

13- Reviewer comment: Section 3.6, please provide a concise explanation of the main evidence for “absence of any signs of toxicity”.

Authors’ Response:  We thank the reviewer for this relevant observation.

The absence of signs of toxicity following the administration of royal jelly at doses of 150 mg/kg, 300 mg/kg, and 1000 mg/kg to female mice for 14 days is justified, as behavioral changes were monitored and clinical signs were assessed to quickly detect any potential adverse effects and toxicity. The absence of mortality and notable behavioral changes, such as agitation or lethargy, indicates that no acute toxicity was induced. These results align with established toxicological standards, which require studies to demonstrate tolerance to the tested doses to validate a product's safety. Thus, the experimental results confirm that royal jelly at these concentrations has a good safety profile, justifying its use for further research into its neuroprotective properties.

As recommended by the reviewer, we added this information to the Results section (Lines 482-488).

14- Reviewer comment: Please check the data of the ALZ-STD group in Figures 4 and 5, which appears to be inconsistent with the description in the manuscript.

Authors’ Response:  We are very grateful that you pointed out our error in the results description and apologized for it. As recommended by the reviewer, we rewrite the result as follows (Lines 523-530 and 540-547):

In the Morris water maze test, the control group (C) showed no statistically significant difference in the time it took the ALZ group to find the platform. However, both groups took less time (p ≤ 0.05) than the ALZ-STD group, which reached the platform more slowly (Figure 4). Notably, mice treated with royal jelly extract at 150 mg/kg (ALZ-D1) and 300 mg/kg (ALZ-D2) improved their performance compared to the ALZ and ALZ-STD groups, showing a highly significant difference compared to the ALZ group (p ≤ 0.01), highlighting the treatment's effectiveness in improving cognitive deficits (Figure 4).

In the Morris water maze test, the control group (C) exhibited the shortest time to reach the platform (P ≤ 0.01 and P ≤ 0.001) compared with the ALZ and ALZ-STD groups, indicating optimal memory function (Figure 5). In contrast, the Alzheimer model mice (ALZ) took significantly longer time (P ≤ 0.01) to reach the platform, indicating cognitive deficits (Figure 5). Mice treated with royal jelly extract at doses of 150 mg/kg (ALZ-D1) and 300 mg/kg (ALZ-D2) also demonstrated improved performance, compared to the ALZ-STD group, and showed a highly significant difference compared to the ALZ group (p ≤ 0.01) (Figure 5).

15- Reviewer comment: Please check the correspondence between the description in section 3.8.1.2 and the data in Figure 1.

Authors’ Response: The results of the FTIR-ATR analysis indicate that royal jelly is rich in secondary metabolites, such as polyphenols and flavonoids, as well as functional groups associated with antioxidant properties. These compounds are known for their ability to neutralize free radicals and modulate biological pathways crucial to neuroprotection. As a result, administering royal jelly to mice improved behavior in tests such as the raised-cross maze, where a reduction in anxiety-related signs was observed.

This behavioral improvement suggests that the metabolites found in royal jelly promote neuronal health and help alleviate cognitive deficits associated with Alzheimer's disease. Thus, the results highlight how the richness of polyphenols and flavonoids not only supports cellular health through antioxidant actions but also positively impacts the behaviors of treated mice, reinforcing the idea that royal jelly can serve as a promising therapeutic solution for neurodegenerative disorders.

As recommended by the reviewer, we have carefully revised the English of the entire text to ensure the paper is suitable for publication in Compounds.

We sincerely appreciate the reviewer's valuable comments, which have improved our paper. We have carefully responded to each comment and hope the paper will be suitable for publication in Compounds.

Best Regards,

Graziele Freitas de Bem 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have revised and improved their work, current version can be accepted.