Review Reports - Birch Sap Attenuates Inflammatory Cytokines and Improves Skin Parameters in Cellular and Animal Models of Skin Irritation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Based on my review of the entire manuscript, this is a very interesting study and follows good research practices. It would be further strengthened by expanding the discussion to address the limitations of using birch sap in liquid form, particularly regarding its potential impact on shelf life and future applications.

Overall Significance and Contribution
The manuscript presents scientifically sound data on the anti-inflammation and mechanism of birch sap in both cellular and animal models. The findings support its potential application as a naturally derived cosmetic ingredient, particularly for promoting skin comfort and maintaining epidermal barrier integrity. The study addresses a notable gap in the literature by providing experimental data on anti-inflammatory biomarkers using validated analytical assays.
Originality and Relevance to the Field
The topic is original and relevant, as systematic scientific evaluation of birch sap’s bioactivities remains limited. This work contributes meaningful evidence that advances current understanding of birch sap as a functional natural material with potential cosmetic applications.
Methodological Considerations
The methodology is generally robust; however, future work may benefit from additional considerations such as stability testing of liquid birch sap, which could influence its shelf life and practical usability. Expanding dose–response evaluations in humans would further strengthen the biological interpretation.
Results, Tables, and Figures
The tables and figures are clear and effectively support the results. The presentation of anti-inflammatory and previous phytochemical findings is well organized and contributes to the overall readability of the manuscript.
Conclusions
The conclusions are consistent with the evidence presented and appropriately address the main research question without overinterpretation.
References
The reference list is generally appropriate and sufficiently updated; however, it could be further strengthened by including more recent publications within the last 5–10 years.

Author Response

Dear reviewer:

We have addressed the following questions and made relevant changes to the manuscript accordingly. Thank you for your kind opinion.

1. Overall Significance and Contribution
The manuscript presents scientifically sound data on the anti-inflammation and mechanism of birch sap in both cellular and animal models. The findings support its potential application as a naturally derived cosmetic ingredient, particularly for promoting skin comfort and maintaining epidermal barrier integrity. The study addresses a notable gap in the literature by providing experimental data on anti-inflammatory biomarkers using validated analytical assays.

Reply:

Thank you for your kind opinion.

2. Originality and Relevance to the Field
The topic is original and relevant, as systematic scientific evaluation of birch sap’s bioactivities remains limited. This work contributes meaningful evidence that advances current understanding of birch sap as a functional natural material with potential cosmetic applications.

Reply:

Thank you for your kind opinion.

3. Methodological Considerations
The methodology is generally robust; however, future work may benefit from additional considerations such as stability testing of liquid birch sap, which could influence its shelf life and practical usability. Expanding dose–response evaluations in humans would further strengthen the biological interpretation.

Reply:

Thank you for your opinion and valuable suggestions. We agree that stability testing and expanding human dose-response evaluations are crucial for establishing the translational value and reliability of birch sap as a cosmetic ingredient. Accordingly, we have added the issue of liquid birch sap stability into the Discussion section (Limitations), acknowledging the necessity of conducting stability tests to determine the shelf life and impact on practical usability in formulations. We have also refined the Conclusion to more strongly emphasize that future work must include rigorous clinical investigations to fully verify the potential of birch sap as a valuable, well-defined, and safe active agent, which directly addresses the need for expanding dose-response data in humans.

4. Results, Tables, and Figures
The tables and figures are clear and effectively support the results. The presentation of anti-inflammatory and previous phytochemical findings is well organized and contributes to the overall readability of the manuscript.

Reply:

Thank you for your kind opinion.

5. Conclusions
The conclusions are consistent with the evidence presented and appropriately address the main research question without overinterpretation.

Reply:

Thank you for your kind opinion.

6. References
The reference list is generally appropriate and sufficiently updated; however, it could be further strengthened by including more recent publications within the last 5–10 years.

Reply:

Thank you for your valuable suggestion regarding the reference. We agree that incorporating more recent publications strengthens the current relevance of the literature review. We have thoroughly reviewed the reference list and updated it by adding several more recent publications (within the last 5–10 years) to support the key claims and discussions in the revised manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

The findings from these studies suggest that birch-derived material may have some antioxidant, soothing, anti-inflammatory, and—under certain conditions—skin barrier or healing support potential. In a cosmetic context, this may translate into soothing sensitive skin, reducing irritation, protecting against oxidative stress, and possibly gently supporting hydration or the barrier.

The authors described in detail in vitro experiments with HaCaT cells in which cytokine expression after inflammatory stimulation and the activity of signaling pathways (MAPK, NF-κB) were measured, as well as in vivo experiments (on models of irritated or damaged skin) with functional measurements of the skin (moisture, TEWL, barrier, hydration, histology, erythema, blood flow).

The results are presented in a transparent manner, the figures are described in detail, the conclusions are clearly stated, and the discussion clarifies many doubts arising from the wording used by the authors.

However the conclusions may be purely hypothetical. The authors described strong regulation of inflammatory cytokines, MAPK/NF-κB blockade, improvement of the skin barrier and physiological parameters of the skin after irritation (erythema, TEWL, perfusion, histology) — it sounds very ambitious. However, there are currently no published, peer-reviewed studies that would confirm such a broad and consistent effect for birch sap (as opposed to bark extracts). This makes the whole concept plausible.

I believe that if the authors add proper justification for their hypotheses and make minor corrections in the formatting and style of section descriptions and figure captions, the manuscript could be published in the journal Cosmetics.

Here is why I have doubts about certain wording in the conclusions:

Literature reviews and synthetic articles on betulin mention its anti-inflammatory effect — but mainly in the context of macrophages, systemic inflammation, or pain models, not in the context of epidermal skin or keratinocytes. In order to unequivocally confirm that oral birch sap (or in cosmetics) has such strong, broad anti-inflammatory and barrier-restoring effects as those described in the manuscript, further work should be carried out involving standardized preparations with a well-known chemical composition (how much betulin, flavonoids, sugars. A comprehensive set of assessments must be conducted to ensure safety, tolerance, and allergy risk. These include also
a toxicological evaluation and meticulously designed clinical trials in humans (controlled, randomized) to ascertain whether the observed effects in laboratory settings translate into tangible improvements in skin condition.

Indeed, birch sap contains 11 types of fatty acids, 18 types of amino acids, 4 types of vitamins, and 18 types of minerals, as well as nicotinic acid compounds, essential oil, betulinic acid, saponins, cell division elements, growth elements, sulfur and ammonia elements, and pyridoxine. Among them, amino acids, fatty acids, and vitamins play an important role in maintaining skin barrier function, reducing inflammation, moisturizing the skin, healing wounds, and whitening. The rich minerals contained in birch sap are also very valuable in skin care. However, there have been no reports of the clinical use of birch sap in the regeneration of sensitive skin [Shu, X., Zhao, S., Huo, W., Tang, Y., Zou, L., Li, Z., ... & Wang, X. (2023). Clinical study of a spray containing birch juice for repairing sensitive skin. Archives of Dermatological Research, 315(8), 2271-2281].

In a study by Choi et al., the preparation of Betula platyphylla extract was described in detail, along with its yield (10.7%). "Dried B. was purchased from Human Herb (Gyeongbuk, Korea). The roots (100 g) were chopped using a blender with 1 L of 70% ethanol solution at room temperature for 24 hours and then concentrated under vacuum. The resulting extract solution was then filtered, concentrated in a water bath under vacuum, frozen, and freeze-dried to obtain ethanol extracts (yield: 10.7%). Oh, S. R., Um, J. Y., Choi, H. J., Im, C. K., Kim, K. J., Jung, J. W., ... & Kim, S. J. (2012) [Betula platyphylla attenuated mast cell-mediated allergic inflammation in vivo and in vitro. Life sciences, 91(1-2), 20-28].

In this regard, it is known that extracts have a higher concentration of active ingredients than juices. The authors provided the method of juice composition analysis described in the previous study, but a brief description should be included here. The composition of birch juice is more complex than the authors state, not just oligosaccharides alone. Are the described positive effects on mouse skin and HaCaT cells solely due to the presence of fructose, glucose, and fucose? I see no connection between the sugar content measurement and anti-inflammatory effects, TEWL reduction, increased hydration, erythema or normal blood flow. It is known in the pharmacological literature that triterpenoids characteristic of birch, especially betulin (and its derivatives), have widely described anti-inflammatory effects. The mechanism may involve activation of the antioxidant/cytoprotective system (e.g., via the Nrf2 pathway), inhibition of inflammatory factors, and enhanced inhibition of inflammation-related kinases (MAPK, COX-2, iNOS) in cell and animal models.

In addition, the authors did not describe the method of obtaining the juice or the content of components in birch juice, only the ratio of oligosaccharides 3:5:8. – line 183 [18].

In the aforementioned study [Shu, X., Zhao, S., Huo, W., Tang, Y., Zou, L., Li, Z., ... & Wang, X. (2023). Clinical study of a spray containing birch juice for repairing sensitive skin. Archives of Dermatological Research, 315(8), 2271-2281], the inclusion criteria were as follows: age between 18 and 60; good health; positive lactic acid stimulation test result and skin discomfort during seasonal changes in previous years - In the case of the clinical study mentioned, the effect concerned sensitive/hyper-reactive skin, not chemically or allergically irritated skin (as in the inflammatory model described). This is an important difference: “sensitive skin”/“skin with active inflammation/barrier damage + inflammation.” Can this be compared?

Minor corrections:

- References should be cited in the following style: References should be numbered in order of appearance and indicated by a numeral or numerals in square brackets—e.g., [1] or [2,3], or [4–6]. See the end of the document for further details on references. In the paper, the numbers are before the full stop.

- Individual sections should be marked:

3.1. Subsection

3.1.1. Subsubsection

- Figure 1 – line 16 – it is difficult to interpret because the authors have included too much information. It may be worth highlighting the basic physiological measurements in a different color.

- Section names should not be in the form of sentences (3.4, 3.5, 3.7 – the number is repeated, it should be 3.8 in line 281) – lines 217-218, 235-236, 266, 281.

- Figures 3, 4, 5 – descriptions should not be in the form of sentences.

Author Response

Dear reviewer:

We have addressed following questions and revised the manuscript accordingly.

Dear Authors,

Here is why I have doubts about certain wording in the conclusions:

Literature reviews and synthetic articles on betulin mention its anti-inflammatory effect — but mainly in the context of macrophages, systemic inflammation, or pain models, not in the context of epidermal skin or keratinocytes. In order to unequivocally confirm that oral birch sap (or in cosmetics) has such strong, broad anti-inflammatory and barrier-restoring effects as those described in the manuscript, further work should be carried out involving standardized preparations with a well-known chemical composition (how much betulin, flavonoids, sugars. A comprehensive set of assessments must be conducted to ensure safety, tolerance, and allergy risk. These include also a toxicological evaluation and meticulously designed clinical trials in humans (controlled, randomized) to ascertain whether the observed effects in laboratory settings translate into tangible improvements in skin condition.

Reply:

Thank you for your highly insightful and supportive comments. We agree that the broad anti-inflammatory and barrier-restoring effects we observed, while promising, necessitate robust justification and further translational research. We have addressed this by thoroughly revising the Discussion section to justify our hypotheses by linking the multi-component nature of birch sap (e.g. polysaccharides, triterpenoids, trace minerals) to established mechanisms of skin modulation, which collectively account for the robust, multi-target effects observed in vitro and in vivo. We have also significantly strengthened the Limitations and Conclusion sections to explicitly call for: standardized preparations with known chemical compositions, a comprehensive set of assessments to ensure safety and tolerance, thorough toxicological evaluation, and rigorously designed clinical trials to translate our laboratory findings into tangible improvements in human skin condition.

In addition, the authors did not describe the method of obtaining the juice or the content of components in birch juice, only the ratio of oligosaccharides 3:5:8. – line 183 [18].

Reply:

Thank you for your detailed and insightful analysis of our study's context, composition data, and the strength of our mechanistic conclusions. We recognize the crucial distinction between highly concentrated birch extracts and the crude birch sap used in our study. The birch sap was directly acquired from Betula platyphylla, subsequently frozen, and air-shipped from Hokkaido, Japan to Taiwan, as detailed in the manuscript. Birch sap is natural water-based solutions containing mostly sugars, minerals and organic acids [1,2]. Sugars, including monosaccharides and polysaccharides, are major component of birch sap [1,3–5]. The determination of monosaccharides composition of polysaccharides may be crucial in understanding its bioactivities. The analysis protocol was as previous report published by Huang et al. [6]. Polysaccharides are also reported to have anti-oxidative and anti-inflammatory effects [7–10]. Prebiotic oligosaccharides are also reported to improve skin hydration, prevent photo-aging and regulate gut-skin axis and immune response [11]. Plant-derived polysaccharides have beneficial effects on anti-aging, wound healing and skin barrier recovery through mechanisms of scavenging reactive oxygen species, regulating telomerase activity, modulating gut-skin axis and improving skin tight junction function [12–14]. Oral supplementation of polysaccharides may have beneficial effects on skin conditions and reduce skin inflammation [15,16]. In addition to polysaccharides, minerals, such as strontium, selenium, magnesium, and calcium also play a role in combating oxidative stress and promote skin barrier recovery [17,18]. Triterpenoids such as betulin or its derivatives betulinic acids, while abundant in birch bark, are lipophilic and may only be present in trace amount in water-based birch sap [19,20]. We came to the conclusion that multi-component nature of birch sap (including its polysaccharides, mineral, and organic acid profile) yields the robust, synergistic anti-inflammatory and barrier-supportive effects observed across all our in vitro and in vivo models. We have addressed the effects of these constituents and their known mechanisms in the revised Discussion section to provide the necessary justification for our broad findings, and we explicitly mention the unrefined nature of the sap in the Limitation section.

Reply:

We appreciate the reviewer's insightful comment regarding the difference between our DNCB-induced inflammatory animal model and the sensitive skin context of the referenced clinical study [17]. We agree that these two conditions represent different points on the spectrum of barrier dysfunction and inflammation. However, our study is highly relevant to establishing the cosmetic potential of birch sap for sensitive skin for the following reasons:

Robustness of Mechanistic Action: Our research demonstrates that birch sap exerts a broad inhibitory effect on the core signaling pathways that drive inflammation in the skin. Specifically, we showed that birch sap significantly suppresses the phosphorylation of MAPK and NF-κB. Elevated activity of these pathways is a hallmark of skin irritation and inflammation [21]. By targeting these canonical inflammatory pathways simultaneously, birch sap demonstrates a robust, multi-target activity.
Consistency in Physiological Outcomes: Regardless of the trigger, both conditions suffer from impaired epidermal integrity and dysregulated inflammatory responses [21–23]. Crucially, the physiological improvements observed in our DNCB model are consistent with the clinical observations in sensitive skin [17]. We observed a normalization of skin barrier function. The clinical study similarly reported increased stratum corneum hydration and reduced TEWL. We demonstrated that birch sap normalizes the inflammation-induced cutaneous blood flow. The clinical study also reported diminished erythema and skin perfusion.

Our findings provide preclinical evidence by confirming that birch sap can not only improve minor discomfort but also effectively attenuate the cascade of inflammation and reverse the severe barrier compromise induced by a strong chemical challenge. In conclusion, the efficacy demonstrated in our robust DNCB model substantiates the mechanism and confirms the therapeutic potential for the broader application of promoting skin comfort and maintaining epidermal health, as seen in the sensitive skin population.

Minor corrections:

Reply:

We have made revisions accordingly.

- Individual sections should be marked:

3.1. Subsection

3.1.1. Subsubsection

Reply:

We have made revisions accordingly.

- Figure 1 – line 16 – it is difficult to interpret because the authors have included too much information. It may be worth highlighting the basic physiological measurements in a different color.

Reply:

We appreciate the suggestion to highlight the measurement days. In the original figure, the DNCB administration days were already marked with a background color. To avoid visual redundancy and potential confusion, especially since physiological measurements may be conducted on the same day as the DNCB challenge (Day 1), we chose to use a more distinct marker to represent physiological measurements. We have therefore highlighted the measurement time points using red star signs in the revised figure, which we believe offers superior clarity and visibility to the reader

- Section names should not be in the form of sentences (3.4, 3.5, 3.7 – the number is repeated, it should be 3.8 in line 281) – lines 217-218, 235-236, 266, 281.

- Figures 3, 4, 5 – descriptions should not be in the form of sentences.

Reply:

We have made revisions accordingly.

References:

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