Preparation of Polyphenol-Rich Herbal Beverages from White Willow (Salix alba) Bark with Potential Alzheimer’s Disease Inhibitory Activity In Silico
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThis study entitled “Preparation of polyphenol rich herbal beverages from white willow (Salix alba) bark with potential Alzheimer’s disease inhibitory activity in silico” explores the neuroprotective capabilities of a herbal infusion, focusing on its antioxidative and antidementia properties. Utilizing in vitro and in silico models, suggesting its application as a functional beverage against neurodegenerative diseases. The findings demonstrate the white willow capacity as a moderate AChE and BuChE inhibitor and its significant antioxidative effects, highlighting its therapeutic potential.
The manuscript is well-written and gives a scientific perspective on the field of functional food. However, some minors’ comments should be clarified:
Line 24-25: Should improve the quality of the figure with a better resolution.
Line 96: Please provide additional information regarding the conditions under which the plant was collected.
Line 151: Add formula/eq in this assay
Line 160: Add formula/eq in this assay
Line 230: Maybe authors could improve antioxidant discussion.
Author Response
The authors would like to thank the reviewer for the time they took to read through this manuscript and give us valuable feedback.
Comment 1: Line 24-25: Should improve the quality of the figure with a better resolution.
Response 1: Thanks for pointing this out. We have improved the quality of the figures to 300 dpi and will attach these higher quality images with the manuscript revisions.
Comment 2: Line 96: Please provide additional information regarding the conditions under which the plant was collected.
Response 2: The dried white willow bark (Salix alba) chips were commercially brought from Just ingredients UK, as a result we do not have the information on the conditions under which the plant was collected.
Comment 3: Line 151: Add formula/eq in this assay
Response 3: The calculation of DPPH assay was based on a standard curve (y = 0.3313x, R2 = 0.9998) using ascorbic acid as a standard. This information is not always included in the methodology of manuscripts so we will let the editor decide if it should be included.
Comment 4: Line 160: Add formula/eq in this assay
Response 4: The calculation of FRAP assay was based on a standard curve (y = 0.0021x, R2 = 0.9997) using Trolox as a standard. This information is not always included in the methodology of manuscripts so we will let the editor decide if it should be included.
Comment 5: Line 230: Maybe authors could improve antioxidant discussion.
Response 5: Thanks for your comments. We agree with this comment and have included additional information regarding on the mechanism behind the antioxidant assays to improve antioxidant discussion, as per line 234-237.
“Several assays have been commonly used to examine the antioxidant activities of herbal extracts including DPPH and FRAP [28-30]. The DPPH assay determines the ability of an antioxidant to donate an electron to stable the DPPH free radicals. The ability of an antioxidant to reduce ferric to ferrous ion through redox reaction is estimated in the FRAP assay [31].”
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors describe an interesting study in developing a white willow beverage that may have anti-AD effects.
As a general comment, BEVERAGES is a food-science oriented journal, so that the beverage aspects should be stressed. Is a pH2 product with citric acid still drinkable or marketable as a beverage? This is more acidic than coca cola and bad for the teeth? What would be the consumer advice for best preparation practice of an infusion?
Please also made a remark about the marketability of the beverage. Should this become some medicinal tea product? Or a food supplement? Does the bark need novel food approval in the EU?
The following revisions might be considered:
Line 26: delete fullstop after Salix
Section 2.1: can the plant material be better characterised. What species, variety? Analytical data of the plant material? Authentication of the material?
Section 2.2. and throughout: please add city for all suppliers, not only country
Throughout: add space between number and units
Throughout: consistently use l or L for liter (check guidelines, I am not sure)
Eq 1 and 2: delete, trivial equations (ref 26 is sufficient)
All figures: the resolution is insufficient, improve
Throughout: no comma before et al.
Table 2 and 3: please add extraction solvent to legend (this is for water?)
Section 3.4: the units should be kcal/mol not mol-1
References: carefully check for MDPI style. The journals should be abbreviated, use either doi: or http://doi.org/...
Author Response
The authors would like to thank the reviewer for the time they took to read through this manuscript and give us extreamly valuable feedback.
Comment 1: As a general comment, BEVERAGES is a food-science oriented journal, so that the beverage aspects should be stressed. Is a pH2 product with citric acid still drinkable or marketable as a beverage? This is more acidic than coca cola and bad for the teeth? What would be the consumer advice for best preparation practice of an infusion? Please also made a remark about the marketability of the beverage. Should this become some medicinal tea product? Or a food supplement? Does the bark need novel food approval in the EU?
Response 1: Thanks for your insightful comments and questions. This is the first study looking at the preparation of willow infusions/ beverages, as a result we looked at the preparation of a model willow beverage to get an understanding of the impact of solvent, temperature and pH on its production. Before being sold (and marketed) as a beverage further work would need to be done on optimisation of the beverage for dental health or for other health applications. Although it should be noted that while low pH infusions may be too acidic to consume alone, they may be used as beverage ingredients. Before being sold as a beverage further work would also need to be completed on the regulatory status of willow in the EU and we have included these considerations in the conclusion (Lines 655-662). This work will provide the basis for future studies focusing on the points mentioned.
Comment 2: Line 26: delete full stop after Salix
Response 2: We deleted the full stop after Salix as requested in line 26.
Comment 3: Section 2.1: can the plant material be better characterised. What species, variety? Analytical data of the plant material? Authentication of the material?
Response 3: Thanks for your comments. The willow bark is a commercial product that we purchased from Just ingredients UK. The information regarding on the plant materials provided from the supplier shows it is white willow bark (Salix alba) chips and originally from eastern Europe.
Comment 9: Section 2.2. and throughout: please add city for all suppliers, not only country
Response 9: Thanks for your comments. We have added city information of all chemicals and reagents, in lines 109 – 112.
Comment 10: Throughout: add space between number and units
Response 10: We have added space between number and units throughout the manuscript as requested. This is highlighted in red in the attached document.
Comment 11: Throughout: consistently use l or L for liter (check guidelines, I am not sure)
Response 11: We have changed every l to L as requested.
Comment 12: Eq 1 and 2: delete, trivial equations (ref 26 is sufficient)
Response 12: Thanks for your comments. We have deleted Eq 1 and 2.
Comment 13: All figures: the resolution is insufficient, improve
Response 13: Thanks for your comments. We have improved the resolution of all figures as requested and original figures will be uploaded with the revised manuscript.
Comment 14: Throughout: no comma before et al.
Response 14: Thanks for your comments. We have made changes as requested and they are highlighted in red in the attached manuscript.
Comment 15: Table 2 and 3: please add extraction solvent to legend (this is for water?)
Response 15: Thanks for your comments. We have added extraction solvent into the legend of Table 2 and 3, as per line 323 and 400.
Comment 16: Section 3.4: the units should be kcal/mol not mol-1
Response 16: We have made changes as requested and they are highlighted in red in the attached manuscript.
Comment 17: References: carefully check for MDPI style. The journals should be abbreviated, use either doi: or http://doi.org/...
Response 17: Thanks for your comments. We have updated the reference list to ensure it meets the requirement for MDPI journals.
Author Response File: 
Author Response.docx
Reviewer 3 Report
Comments and Suggestions for AuthorsDear Authors,
Firstly, I would like to congratulate you on an attempt to discuss such an important topic and to present the findings. I sincerely hope that these comments/suggestions assist you in the improvements of the manuscript.
Comments;
1. Abstract will require adjustment following the addressing of all comments.
a. Image used in the abstract should be of better quality.
2. Introduction section
a. It would be also beneficial if authors can state that current medication (rather than drugs) for the management of Alzheimer’s disease are ineffective.
3. Methods
a. Can authors provide information on how long was the powder stored for? (light/away from light)
b. In places there is to much for the unnecessary information included. For example, Duran glass bottle (Line 111). There is no need for this amount of detail.
c. What is meant by target temperature? Please elaborate
d. How was the initial experimental procedure set (in regards the choice of solvent ration and temperature). Did authors consider using Box-Behnken method?
e. For HPLC analysis, did authors consider using internal standard for accurate determination of the concentration.
f.
4. Results and Discussion
a. This section is presented well. In figure 2, can authors ensure that same sale is used on Y axis
b. In places there is a repetition of the included information such as, use of data in figures and in text is same. Choose one or the other.
c. Through the discussion section, it is important that authors emphasize the in silico nature of this study rather than findings being seen as the AD management outcome.
5. Conclusion
a. Can authors also provide clear indication of what are the limitations of this project.
6. References
a. Please update the reference list following the journal guidelines. This includes the appropriate use of references in text and in the reference list.
b. Please ensure that all references are consistent and that there is no duplication
c. Please ensure that all references use or full name or abbreviation of the included journals.
Comments on the Quality of English LanguageSome minor corrections are required. English in general is good.
Author Response
The authors would like to thank the reviewer for the time they took to read through this manuscript and give us valuable feedback.
Comment 1:
- Abstract will require adjustment following the addressing of all comments.
- Image used in the abstract should be of better quality.
Response 1: Thanks for your comments. The abstract was adjusted in lines 21-24 after addressing all comments.
“Overall, the obtained results indicate that willow bark prepared with hot water and/or with addition of citric acid is rich in bioactive compounds with high antioxidant activity and possible neuroprotective activities in silico, which could serve as valuable ingredients for inclusion in functional beverages.”
We have improved the image quality of the graphical abstract and will uploaded it with the revised manuscript.
Comment 2: Introduction section
It would be also beneficial if authors can state that current medication (rather than drugs) for the management of Alzheimer’s disease are ineffective.
Response 2: Thanks for your comments. We agree with your comments and have made changes in lines 41-44.
“However, the currently used medications for treating AD present some severe side effects, such as hepatotoxicity and SLUGDE syndrome [7]. Current approved medications for AD including cholinesterase inhibitors are symptomatic treatments that can slow down the disease progression, but their effects are limited and individually depended.”
Comment 3: Methods
a.Can authors provide information on how long was the powder stored for? (light/away from light)
Response 3a: The information has been added, as per line 101-103.
“The willow bark powder was put into plastic sample containers and stored away from light at ambient temperature for no longer than 6 months.”
b. In places there is too much for the unnecessary information included. For example, Duran glass bottle (Line 111). There is no need for this amount of detail.
Response 3b: Thanks for your comments. We have made changes and removed unnecessary detail as suggested.
c. What is meant by target temperature? Please elaborate
Response 3c: Thanks for your comments. We agree that the term “target temperature” is ambiguous and we have changed it to extraction temperature in lines 115-116. The extraction temperatures were 25, 40, 60, and 80 °C as described in line 128.
“Willow bark powder (1 g) was placed in a glass bottle that contained 50 mL of solvent and was pre-heated to the extraction temperature.”
d. How was the initial experimental procedure set (in regards the choice of solvent ration and temperature). Did authors consider using Box-Behnken method?
Response 3d: Thanks for your comments. The initial experimental procedure was set-up with reference to previous literature that looked at producing tea infusions. We did not consider using the Box-Behnken method we wanted to monitor the influence of one parameter at a time.
e. For HPLC analysis, did authors consider using internal standard for accurate determination of the concentration.
Response 3e: For HPLC analysis we quantified each compound using an external calibration curve with HPLC grade standards.
Comment 4. Results and Discussion
a. This section is presented well. In figure 2, can authors ensure that same sale is used on Y axis
Response 4a: Thank you for your comments. We chose to vary the scales on the y-axis in this figure as there was huge variation in the concentration of each compound studied and we wanted to highlight the impact of ethanol concentration on each compound.
Comment 4b.
In places there is a repetition of the included information such as, use of data in figures and in text is same. Choose one or the other.
Response 26: Thanks for your comments. We have deleted repetition as requested. The changes made have been highlighted in red in the attached manuscript.
Comment 4c.
Through the discussion section, it is important that authors emphasize the in silico nature of this study rather than findings being seen as the AD management outcome.
Response 4c: Thanks for your comments. We agree that it is important to emphasize the in silico nature of this study and we have made changes as suggested. Line 595, 600, 612, 652.
Comment 5:
5a. Can authors also provide clear indication of what are the limitations of this project.
Response 5a: Thanks for your comments. We have added limitations of this work in lines 655 – 662.
“However, the regulatory status of willow bark for food applications in the European market remains unclear. If a food has not been consumed in the EU to a significant degree before 15 May 1997 it may fall into the novel foods category. Based on the EU novel food catalogue lists, it has not been determined whether willow bark falls into this category, therefore any food containing willow bark may need to be authorised before being placed on the EU market. Thus, it is important to determine the regulatory status of willow bark for manufacturers who are interested in producing willow beverages for the European market.”
Comment 29:
- References
- Please update the reference list following the journal guidelines. This includes the appropriate use of references in text and in the reference list.
- Please ensure that all references are consistent and that there is no duplication
- Please ensure that all references use or full name or abbreviation of the included journals.
Response 6: We have updated the reference list to ensure it meets the requirement for MDPI journals.
Author Response File: Author Response.docx
