Sea Buckthorn Leaves as a Potential Source of Antioxidant Substances

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

 

Authors are suggested to address the following comments!

Introduction Section:

1). The introduction mentions the increasing costs in the animal production industry and the potential of using agro-industrial by-products as alternative feed resources. Could the authors provide more context or references to justify the focus on sea buckthorn (Hippophae rhamnoides L.) specifically? What makes this plant particularly suitable for addressing the mentioned industry challenges?

 

2). While the introduction highlights the potential health benefits and various applications of sea buckthorn leaves, the literature on their bioactive components is stated to be limited. Can the authors clarify which specific bioactive components and secondary metabolites were prioritized in this study, and why? How does this selection align with the reported health benefits?

 

3). The introduction suggests that sea buckthorn leaves can be used as livestock feed and may positively affect the nutritional quality of meat products. Can the authors elaborate on the specific mechanisms or pathways through which these bioactive components influence animal growth and meat quality? Are there any preliminary studies or findings that support these claims?

 

Clarification of Methodology for Antioxidant Compound Quantification

4). The section provides a detailed methodology for determining various antioxidant compounds, yet it is not clear why specific wavelengths and reagents were chosen for each type of compound. Could the authors elaborate on the rationale behind these selections and how they ensure the accuracy and reliability of the measurements?

 

Consistency and Standardization in Measurement Techniques

5). While the methodologies for different antioxidant compounds (TP, TF, TFL, TA, etc.) are well described, there is a lack of information on how the reproducibility and consistency of these measurements are ensured. Can the authors include more details on any standardization procedures or controls used during these analyses to validate the results?

 

 

 

Results and Discussion

 Mechanisms of Oxidative Stress and Antioxidant Defense:

6). The section provides a thorough explanation of the balance between reactive oxygen species (ROS) and antioxidants in the human/animal organism. It emphasizes the enzymatic and non-enzymatic mechanisms that protect against oxidative stress. However, the explanation could be enhanced by providing more specific examples of these mechanisms and their roles in various physiological contexts.

 

Use of Antioxidant Assays:

7). The study utilizes different antioxidant assays to evaluate the antioxidant potential of sea buckthorn leaves. The choice of assays is well-justified, highlighting the strengths and limitations of each method. Yet, the discussion could benefit from a comparative analysis of how the results from these assays correlate with each other and with antioxidant activity in vivo is lacking.

Variability in Polyphenol Content

8). The data presented in Table 1 show significant variability in total polyphenols (TP) and antioxidant activity (AA) across different years and cultivars. While the influence of environmental factors on TP content is acknowledged, a deeper exploration of specific factors (e.g., soil composition, weather conditions) that might contribute to this variability would add value to the findings.

 

Statistical Analysis and Trends

9). The results indicate significant differences in antioxidant activity depending on the year and cultivar, but some trends were not statistically significant. The discussion notes these trends (e.g., the AA of Leikora cv. in the PCL method), but it would be helpful to elaborate on the potential biological or environmental reasons behind these observed trends, even if they are not statistically significant.

 

Flavonoid and Anthocyanin Content

10). The section detailing the content of total flavonoids (TF) and individual flavonoids, such as anthocyanins, provides important insights into the phytochemical composition of sea buckthorn leaves. However, the impact of these compounds on health outcomes or their specific roles in antioxidant defense is not extensively discussed. Including more information on the biological activities and potential health benefits of these specific flavonoids would enhance the relevance of the findings.

Author Response

Dear Reviewer,

We would like to express our sincere gratitude for your insightful review of our manuscript. Below, we have attached a list of changes made in accordance with your suggestions. In the revised version of the manuscript, we have marked the corrected parts of the text in track change mode.

Yours sincerely,

Wioletta Biel

 

Reviewer:

Comments and Suggestions for Authors

Specific points: Authors are suggested to address the following comments!

Response: Thank you for your thorough and constructive feedback. Thank you for your time and consideration in reviewing our text. We appreciate your time and effort in evaluating our work, and we have carefully considered each of your comments.

Point 1: Introduction Section:

1. The introduction mentions the increasing costs in the animal production industry and the potential of using agro-industrial by-products as alternative feed resources. Could the authors provide more context or references to justify the focus on sea buckthorn (Hippophae rhamnoides L.) specifically? What makes this plant particularly suitable for addressing the mentioned industry challenges?
2. While the introduction highlights the potential health benefits and various applications of sea buckthorn leaves, the literature on their bioactive components is stated to be limited. Can the authors clarify which specific bioactive components and secondary metabolites were prioritized in this study, and why? How does this selection align with the reported health benefits?

Response: Thank you for your time and consideration in reviewing our text. In revised version of our manuscript, including in the "Inroduction" section we have taken care to emphasize provide more context and references to justify the focus on sea buckthorn in this aim. We have extended the introduction according to your suggestion. We believe that this addition will strengthen our manuscript.

3. The introduction suggests that sea buckthorn leaves can be used as livestock feed and may positively affect the nutritional quality of meat products. Can the authors elaborate on the specific mechanisms or pathways through which these bioactive components influence animal growth and meat quality? Are there any preliminary studies or findings that support these claims?.

Response: Thank you for this suggestion, we have added information about this problems in chapter Introduction.

Point 2: Clarification of Methodology for Antioxidant Compound Quantification. Consistency and Standardization in Measurement Techniques

1.The section provides a detailed methodology for determining various antioxidant compounds, yet it is not clear why specific wavelengths and reagents were chosen for each type of compound. Could the authors elaborate on the rationale behind these selections and how they ensure the accuracy and reliability of the measurements?

2. While the methodologies for different antioxidant compounds (TP, TF, TFL, TA, etc.) are well described, there is a lack of information on how the reproducibility and consistency of these measurements are ensured. Can the authors include more details on any standardization procedures or controls used during these analyses to validate the results?

Response: Thank you for these questions. We appreciate your curiosity in this aspect. The authors determined the analyzes according to the procedures described in the cited methods for each substance. Due to limitations in the text, the descriptions are in an abbreviated version, but for each ingredient the authors have detailed data for the methodologies, including the following data:

DPPH Assay. The antioxidant activity was expressed as a percentage of inhibition of the free DPPH•+ radical by the following formula:

Antioxidant activity (%) = (1- Aprobe/ ADPPH) x 100

Aprobe – absorbance of the test sample, after one hour incubation in the dark

ADPPH – absorbance of the solution DPPH

The result obtained was also expressed in terms of Trolox equivalent. For this purpose, Trolox solutions of various concentrations were prepared to determine a standard curve (Fig. 1):

 

ABTS Assay: Antioxidant activity was expressed as a percentage of inhibition of the free ABTS•+ cation radical by the following formula:

Antioxidant activity (%) = 100 × (A0 - Aw) / A0

A0 – absorbance of initial ABTS•+ solution

Aw – absorbance of the test sample, after 6 minutes incubation in the dark

 

 

 

 

 

The result obtained was also expressed in terms of Trolox equivalent. For this purpose, Trolox solutions of various concentrations were prepared to determine a standard curve (fig.2 ):

Total Polyphenol (TP) Determination: Gallic acid solutions of various concentrations were prepared to determine a standard curve (Fig. 3):

 

 

 

 

 

 

 

Total Flavonoid (TF) Determination: quercetin solutions of various concentrations were prepared to determine a standard curve (Fig. 4):

 

Total Flavonols (TFL) Determination: quercetin solutions of various concentrations were prepared to determine a standard curve (Fig. 5):

Total Anthocyanin (TA) Determination:

Anthocyanin concentrations in mg/g dry matter were calculated using the following formula:

Total anthocyanins (mg/g DM) = [A530 – 0.25 × A657] × TV/ [d_wt × 1000]

where:

A530 – absorbance of samples at a wavelength of 530 nm

A657 – absorbance of samples at a wavelength of 657 nm

TV – total extract volume (ml);

d_wt – dried herb weight (g)

 

The Proanthocyanidins (PAC) Determination: The formula for calculating the percentage of total proanthocyanidins in the dry weight of sea buckthorn leaves was calculated from the following formula:

Total proanthocyanidins (%) = A550 × 78.26 × wr / % DM.

where:

A550 – absorbance of samples at a wavelength of 550 nm

wr – dilution factor

% DM – percentage of dry matter in the tested sample

 

Total Tanins (TT) Determination: solutions of tannic acid of various concentrations were prepared to determine a standard curve (Fig. 6):

Others [the evaluation of individual flavonols (rutozide and quercetin) determination, the evaluation of individual anthocyanidins determination, chelerythrine determination]: Initially, standard solutions were prepared for rutozide, quercetin, delphinidin, peonidin, cyanidin, chelerythrine to be determined, at a concentration of 1 mg/ml. The standard solutions and test samples were prepared as extracts using acidified methanol (mixture of methanol and hydrochloric acid 25%, 4:1, v/v). The determinations were performed on 10×10 cm aluminium plates (aluminium oxide 60 F254, type E, Merck, Darmstadt, Germany), dedicated to TLC analyses. The plates were activated with methanol prior to use. They were then dried at 60°C for 1 hour. Samples and standard on thus prepared stationary phase were spotted with 1 or 2 µL glass capillaries using a Vomaticator Linomat (CAMAG, Muttenz, Switzerland), at a distance of 10 mm from the end of the plate, 10 mm from the edge, and 10 mm distance between bands. Standards were applied first to determine the standard curve (1, 2, 3 and 4 µL), followed by the prepared extracts (2 µL each). The plates thus prepared were transferred to an automated chamber (CAMAG ADC, Muttenz, Switzerland) to develop the chromatograms. The mobile phase in the chamber consisted of a chloroform/methanol/acetone/ammonia mixture (10:22:53:0.2, v/v/v/v) and the separation of the samples took 40 min. in glass chromatographic chambers (17.5 x 16 x 8.2 cm, Sigma-Aldrich, Germany). After drying in the dark, the plates were scanned using a Skaner TLC Scanner 3 (CAMAG, Muttenz, Switzerland). The results obtained were calculated using Visual Cats 1.3.4 software. The results of all secondary metabolites were expressed in µM /g DM.

If the reviewer and editor will, so the authors will attach the above information to the manuscript or to Supplementary Materials.

Point 3. Results and Discussion

1. The section provides a thorough explanation of the balance between reactive oxygen species (ROS) and antioxidants in the human/animal organism. It emphasizes the enzymatic and non-enzymatic mechanisms that protect against oxidative stress. However, the explanation could be enhanced by providing more specific examples of these mechanisms and their roles in various physiological contexts.

Response: Thank you for this comment, however, due to the fact that the publication does not concern research on the mechanisms of quenching free radicals, the authors decided to remove this fragment in the R&D chapter of the manuscript.

 

Point 4. Use of Antioxidant Assays: The study utilizes different antioxidant assays to evaluate the antioxidant potential of sea buckthorn leaves. The choice of assays is well-justified, highlighting the strengths and limitations of each method. Yet, the discussion could benefit from a comparative analysis of how the results from these assays correlate with each other and with antioxidant activity in vivo is lacking.

Response: An important analysis concerning AA SBT is the PCL analysis, which appears significantly less frequently in scientific studies, limiting the possibility of evaluating it against others. Table 4 presents Person's correlation coefficients, which confirmed the method's particularly high correlation with ABTS (0.930) and moderate correlation with DPPH (0.576). Of course, the variation in correlation coefficients was expected, as the ABTS and DPPH methods differ significantly. This is reflected in the paragraph beginning with line 446. If there is a need to expand this section, we will, of course, do so.

 

Point 5: Variability in Polyphenol Content: The data presented in Table 1 show significant variability in total polyphenols (TP) and antioxidant activity (AA) across different years and cultivars. While the influence of environmental factors on TP content is acknowledged, a deeper exploration of specific factors (e.g., soil composition, weather conditions) that might contribute to this variability would add value to the findings.

Response: As shown in the study, habitat conditions modified SBT quality. Sea buckthorn is a perennial plant, and therefore grows on a permanent site, which makes it possible to essentially ignore the effect of soil on SBT quality in such an experiment. The soil on which the experiment was conducted is a leached brown soil (Haplic Cambisol), (WRB 2015). At the Ap level, it shows a granulometric composition of loamy sand with a slightly acidic pH. The analysis of the soil minerals showed high levels of phosphorus and moderate levels of magnesium and potassium. Since the soil is not an important source of variability from an experimental point of view, this information was omitted from the study. Of course, this information can be supplemented. The second element of the habitat is climatic conditions, obviously variable in years. These were not discussed, as it was considered that a three-year study period does not allow a reliable assessment of the effect of temperature and precipitation on the quality of SBT, but only allows confirmation of the variability of the raw material in years. It should be emphasized that the primary purpose of the study was not to determine the relationship between active ingredient contents and habitat conditions.

 

 

 

 

Point 6 Statistical Analysis and Trends: The results indicate significant differences in antioxidant activity depending on the year and cultivar, but some trends were not statistically significant. The discussion notes these trends (e.g., the AA of Leikora cv. in the PCL method), but it would be helpful to elaborate on the potential biological or environmental reasons behind these observed trends, even if they are not statistically significant.

Response: The three-year experiment presented in this study does not allow for a clear formulation of environmental influences on the content of biologically active substances present in the research material (SBT leaves) in such small quantities. This was the reason for considering trends in the observed analyses. The need to extend research on the impact of changing environmental conditions on the composition of SBT is evidenced by the large variability of the content of biologically active substances in individual years of research.

Point 7 Flavonoid and Anthocyanin Content: The section detailing the content of total flavonoids (TF) and individual flavonoids, such as anthocyanins, provides important insights into the phytochemical composition of sea buckthorn leaves. However, the impact of these compounds on health outcomes or their specific roles in antioxidant defense is not extensively discussed. Including more information on the biological activities and potential health benefits of these specific flavonoids would enhance the relevance of the findings.

Response: In response to your recommendation, we took into consideration your suggestion to include more information about TF and individual flavonoids content, and we have made the necessary revisions accordingly. We believe that these additions contribute to a more well-rounded and informative introduction. Once again, we thank you for your valuable feedback.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Although there are many data in these paper, the data organization is quite confusing that it is hard to link figures and tables together.

 

1)     Table 1 has data from groups of 2014, 2015, and 2016, as well as groups of Ascola, Habego, Hergo, Leikora, but figure 2 organized data as Ascola2014, Ascola2015, Ascola2016, meaning the respective cultivar and year. I think 1) the Table 1 should have the same organization as figure 2, Ascola2014, Ascola2015, Ascola2016, Habego2014, Habego2015, Habego2016, etc. If so, 2) Figure 1 should be included in Table 1, otherwise each detection target should have a similar figure as Figure 1. 3) Table 2 and Table 3 have the same problem as the Table 1.

2)     Although PCA study was done in Figure 2, there is no cluster pattern in Figure 2, and there is a similar problem in Figure 3. The current results generally mean everything is different from everything, the difference is not clustered in cultivars or in years. If so, what’s the meaning of the current study.

3)     There is no figure for either leaves or any assay, even for the high performance thin layer chromatography (HPTLC). It is hard to believe the all the experiments were done properly. Please add some figures or experimental proves to the study.

Author Response

Dear Reviewer,

We would like to express our sincere gratitude for your insightful review of our manuscript. Below, we have attached a list of changes made in accordance with your suggestions. In the revised version of the manuscript, we have marked the corrected parts of the text in track change mode.

Yours sincerely,

Wioletta Biel

 

Reviewer:

Comments and Suggestions for Authors

Although there are many data in these paper, the data organization is quite confusing that it is hard to link figures and tables together.

Response: Thank you for your time and consideration in reviewing our text.

Specific comments

Point 1: Table 1 has data from groups of 2014, 2015, and 2016, as well as groups of Ascola, Habego, Hergo, Leikora, but figure 2 organized data as Ascola2014, Ascola2015, Ascola2016, meaning the respective cultivar and year. I think 1) the Table 1 should have the same organization as figure 2, Ascola2014, Ascola2015, Ascola2016, Habego2014, Habego2015, Habego2016, etc. If so, 2) Figure 1 should be included in Table 1, otherwise each detection target should have a similar figure as Figure 1. 3) Table 2 and Table 3 have the same problem as the Table 1.

Response: In accordance with the methodology, all quantitative traits were subjected to two-factor analysis of variance. Tables 1, 2 and 3 show the main effects, i.e. the influence of the study factors, separately years and genotype, on the development of STB quality traits. The request to present in the table the mean of interaction of years x variety, as postulated by the Reviewer, was considered, but unfortunately none of the interactions tested in the analysis of variance was statistically significant. Presentation of these data would not add any additional information, but if necessary we will supplement the paper with relevant figures. On the other hand, PCA analysis was performed on the entire dataset, so the points in the figures are described by variety/year, which is a must with this approach.

Point 2: Although PCA study was done in Figure 2, there is no cluster pattern in Figure 2, and there is a similar problem in Figure 3. The current results generally mean everything is different from everything, the difference is not clustered in cultivars or in years. If so, what’s the meaning of the current study.

Response: The number of 12 points plotted on the first two components of PCA does not form distinct clusters, but nevertheless is distributed in the four quadrants of the Cartesian system. This means that individual leaf samples differ significantly from each other in both AA potential and bioactive component content. The authors note the aggregation of points particularly related to years, but varieties responded differently in years, which is a significant finding. In conclusion, it can be said that the main factor effects are presented in Tables 1 to 3, and the demonstration of the variation of STB composition of the studied varieties in years is presented by PCA analyses. Thus, these were the objectives of the presented study.

 

Point 3: There is no figure for either leaves or any assay, even for the high performance thin layer chromatography (HPTLC). It is hard to believe the all the experiments were done properly. Please add some figures or experimental proves to the study.

Response: We have been working with this research material for many years. We have had several research projects on this plant. The authors of this publication have many years of analytical experience. Our numerous publications are proof of our scientific paper. For example (authors of this manuscript – bold):

• Bośko P., Biel W., Jaroszewska A., Kowalewska R. 2015. The effect of organic farming methods on the chemical composition leaves of sea buckthorn (Hippophae rhamnoides L.). 60th Anniversary of Faculty of Biotechnology and Animal Husbandry International Scientific Conference organised by West Pomeranian University of Technology in Szczecin, 17-18 June 2015; pp. 37-38.
• Bośko P. A., Biel W. 2016. Evaluation of the chemical composition of selected varieties of sea buckthorn (Hippophae rhamnoides L.) leaves. XLV Scientific Session of Group of Animal Nutrition KNZ PAN, Olsztyn, 21-22 June, 2016
• Jaroszewska A., Biel W., Jurgiel-Małecka G., Grajkowski J., Gibczyńska M. 2016. The influence of soil fertility diverse on chemical composition of sea buckthorn (Hippophaë rhamnoides L.) leaves as feed material. Folia Pomer. Univ. Technol. Stetin., Agric., Aliment., Pisc., Zootech. 328(39)3, 97-108. DOI: 10.21005/AAPZ2016.39.3.09.
• Jaroszewska A., Biel W., Stankowski S., Bośko P. Evaluation of the influence of symbiotic mycorrhizal fungi on basic chemical compounds and minerals of sea buckthorn leaves. Journal of Elementology. 21(4), 1029-1041.
• Bośko P., Smetanska I., A., Biel W. 2017. The influence of adding sea buckthorn (Hippophae rhamnoides L.) leaves on the antioxidant activity of fermented beverages. Materiały konferencyjne Nauki Przyrodnicze we Współczesnym Świecie, Szczecin, 2 June, 2017.
• Jaroszewska A., Biel W., Bojanowska-Czajka A., Wierzchnicki R. 2017. Influence of habitat conditions on chemical composition and content of isotopes in sea buckthorn (Hippophae rhamnoides L.) leaves. Acta Sci. Pol. Hortorum Cultus, 16(1), 3–10.
• Biel W., Jendrzejczak E., Jaroszewska A., Witkowicz R., Piątkowska E., Telesiński A. 2017. Nutritional content and antioxidant properties of selected species of Amaranthus L. Italian Journal of Food Science 29(4),728-740.
• Biel W., Jaroszewska A. 2017. The nutritional value of leaves of selected berry species. Scientia Agricola 74 (5), 405-410.
• Jaroszewska A., Biel W. 2017. Chemical composition and antioxidant activity of leaves of mycorrhized sea-buckthorn (Hippophae rhamnoides L.). Chilean Journal of Agricultural Research 77 (2), 155-162.
• Biel W., Jaroszewska A., Łysoń E., Telesiński A. 2017. The chemical composition and antioxidant properties of common dandelion leaves compared to sea buckthorn. Canadian Journal of Plant Science. https://doi.org/10.1139/CJPS-2016-0409
• Sytar O., Biel W., Smetanska I., Brestic M. 2018. Bioactive compounds and their biofunctional properties of different buckwheat germplasms for food processing. In book: Buckwheat Germplasm in the World - 1st Edition Edition: 1st Edition. Publisher: Springer Editors: Meiliang Zhou, Ivan Kreft, Galina Suvorova, Yu Tang Sun-Hee Woo. DOI 10.1016/B978-0-12-811006-5.00019-7.
• Jaroszewska A., Biel W., Telesiński A. 2018. Effect of mycorrhization and variety on the chemical composition and antioxidant activity of sea buckthorn berries. Journal of Elementology 23(2):673-684. DOI: 10.5601/jelem.2017.22.3.1434.
• Sytar O, Bośko P, Živčák M, Brestic M, Smetanska I. Bioactive Phytochemicals and Antioxidant Properties of the Grains and Sprouts of Colored Wheat Genotypes. Molecules. 2018; 23(9):2282. https://doi.org/10.3390/molecules23092282
• Witkowicz, R.; Biel, W.; Chłopicka, J.; Galanty, A.; Gleń-Karolczyk, K.; Skrzypek, E.; Krupa, M. Biostimulants and Microorganisms Boost the Nutritional Composition of Buckwheat (Fagopyrum esculentum Moench) Sprouts. Agronomy 2019, 9, 469.
• Biel W. Witkowicz R., Piątkowska E., Podsiadło C. 2019. Proximate Composition, Minerals and Antioxidant Activity of Artichoke Leaf Extracts. Biological Trace Element Research. org/10.1007/s12011-019-01806-3.
• Witkowicz, R., Skrzypek, E., Gleń-Karolczyk, K., Krupa, M., Biel, W., Chłopicka, J., Galanty, A. 2021. Effects of application of plant growth promoters, biological control agents and microbial soil additives on photosynthetic efficiency, canopy vegetation indices and yield of common buckwheat (Fagopyrum esculentum Moench). Biological Agriculture & Horticulture, 1-18.
• Paulina Bośko, Paulina Małkowska, Patrycja Oberska, Andrii Bordakov, Ilona Hrechaniuk, Monika Komperda, Adrianna Kapturska, Wioletta Biel, Katarzyna Michałek. EXPRESSION OF AQUAPORIN 1, 4, 8 AND 9 IN THE LIVER OF RATS FED WITH THE STANDARD DIET AND SUPPLEMENTED WITH DRIED SEA-BUCKTHORN LEAVES (HIPPOPHAE RHAMNOIDES L.). A PILOT STUDY. Pol. J. Natur. Sc., Vol 36(4): 457–468.
• Witkowicz R., Biel W. 2022. A novel method for analyzing mineral ratio profiles of treated buckwheat sprouts (Fagopyrum esculentum Moench). Journal of Food Composition and Analysis. 114. 104800.
• Piątkowska E., Biel W., Witkowicz R., Kępińska-Pacelik J. 2022. Chemical Composition and Antioxidant Activity of Asteraceae Family Plants. Applied Sciences, 12(23), 12293.
• Biel W., Pomietło U., Witkowicz R., Piątkowska E., Kopeć A. 2023. Proximate composition and antioxidant activity of selected morphological parts of herbs. Applied Sciences. 13(3). 1413.

and others.

We have added a figure of the experimental material (from Lipnik (53°41′ N, 14°97′ E) at the Agricultural Experimental Station belonging to the West Pomeranian University of Technology in Szczecin, PL) to the M&M chapter, we can add detailed data from the methods if desired (which is also added in response to other reviewer comments). Some of the research, chemical analyses was carried out during an internship as part of the BAYHOST Program (Bavarian Academic Center for Central, Eastern and Southeastern Europe) by Paulina Bośko in the laboratory of Prof. Iryna Smetanska - University of Applied Sciences Weihenstephan-Triesdorf Steingruberstrasse 2, 91746 Weidenbach, DE (www.hswt.de). The remaining chemical analyses we did in laboratories in West Pomeranian University of Technology in Szczecin and University of Agriculture in Krakow, in Poland.

Below selected photos documentation regarding chemical analysis and plant material to our manuscript (If the reviewer and editor will, so the authors will attach a few selected ones) to the manuscript or to Supplementary Materials.

 

Author Response File: Author Response.pdf