Flavonoids from Morus alba L. Leaves: Optimization of Extraction by Response Surface Methodology and Comprehensive Evaluation of Their Antioxidant, Antimicrobial, and Inhibition of α-Amylase Activities through Analytical Hierarchy Process
Round 1
Reviewer 1 Report
The aim of the present paper is not very clear and should be rewritten.
The Response Surface Methodology was done by choosing ethanol as solvent. However, other solvents were also used without explaining the reason for using them. Why ethanol was the most appropriate?
Many abbreviations were used, without explanation. For instance, MIC stands for what??
Extensive English editing and revision is required throughout the manuscript.
Author Response
Response to Reviewer 1 Comments
Point 1: The aim of the present paper is not very clear and should be rewritten.
Response 1: The aim of this paper in abstract has been rewritten as blow: Abstract: To explore the flavonoids from Morus alba L. leaves (MLF), the process of extracting was to optimized by response surface methodology and the antimicrobial and antioxidant activities were evaluated in vitro. The yield of flavonoids reached to 50.52 mg g-1 under the optimized extraction conditions (i.e., extraction temperature, 70.85℃; solvent concentration, 39.30%; extraction time, 120.18 min; and liquid/solid ratio, 34.60:1). The total flavonoids were extracted in organic solvent with various polarities petroleum ether (MLFp), ethyl acetate (MLFe), and n-butanol (MLFb). In vitro, the four MLF samples exhibited good antioxidant activities for scavenging of 2, 2’-azinobis-(3-ethylbenz-thiazoline-6-sulphonate) radical, 1, 1-diphenyl-2-picrylhydrazyl radical, and total reducing power. Regarding antimicrobial efficacy, the MLF samples suppressed the development of Staphylococcus aureus, Bacillus subtilis, and Bacillus pumilus. The MLF samples inhibitedα-amylase activity to a certain extent. The analytical hierarchy process (AHP) was used to evaluate comprehensively the bioactivities of MLF samples. AHP results revelead that the bioactivity comprehensive score (78.83 μg mL-1) of MLFe was the optimal among the four MLF samples. MLF also exhibited nonhemolytic property. All bioactivities suggested the potential of MLFe as a candidate resource in the food and drug industries.
Point 2: The Response Surface Methodology was done by choosing ethanol as solvent. However, other solvents were also used without explaining the reason for using them. Why ethanol was the most appropriate?
Response 2: The ethanol was employed as solvent mainly based on the following two reasons. Firstly, flavonoids were better soluble in ethanol, and ethanol was cheaper and environmentally friendly. Secondly, ethanol is the preferred solvent for total flavonoids extraction in many references.
a) Yang, L., Cao, Y.-L., Jiang, J.-G., Lin, Q.-S., Chen, J., & Zhu, L. (2010). Response surface optimization of ultrasound-assisted flavonoids extraction from the flower ofCitrus aurantiumL. var.amaraEngl. Journal of Separation Science, 33, 1349–1355. doi:10.1002/jssc.200900776
b) Huang, W., Xue, A., Niu, H., Jia, Z., & Wang, J. (2009). Optimised ultrasonic-assisted extraction of flavonoids from Folium eucommiae and evaluation of antioxidant activity in multi-test systems in vitro. Food Chemistry, 114(3), 1147–1154. doi:10.1016/j.foodchem.2008.10.079
c) Zhang, G., He, L., & Hu, M. (2011). Optimized ultrasonic-assisted extraction of flavonoids from Prunella vulgaris L. and evaluation of antioxidant activities in vitro. Innovative Food Science & Emerging Technologies, 12(1), 18–25. doi:10.1016/j.ifset.2010.12.003
In this study, solvents such as petroleum ether, ethyl acetate and n-butanol were also used because of their different polarity. Ethanol extracts are extracted from the above solvents, allowing the components in the total flavonoids to be distributed in different solvents according to polarity. This is also a common preparation method for the study of the activity of different polar flavonoids.
a) Sultan, A., Bahang, Aisa, H. A., & Eshbakova, K. A. (2008). Flavonoids from Dracocephalum moldavica. Chemistry of Natural Compounds, 44(3), 366–367. doi:10.1007/s10600-008-9065-4
b) Kang, W.-Y., Li, C.-F., & Liu, Y.-X. (2009). Antioxidant phenolic compounds and flavonoids of Mitragyna rotundifolia (Roxb.) Kuntze in vitro. Medicinal Chemistry Research, 19(9), 1222–1232. doi:10.1007/s00044-009-9265-x
c) Toor, R. H., Tasadduq, R., Adhikari, A., Chaudhary, M. I., Lian, J. B., Stein, J. L., … Shakoori, A. R. (2018). Ethyl acetate and n-butanol fraction of Cissus quadrangularis promotes the mineralization potential of murine pre-osteoblast cell line MC3T3-E1 (sub-clone 4). Journal of Cellular Physiology. doi:10.1002/jcp.27707
And the contents ratio of flavonoids in petroleum ether (MLFp), ethyl acetate (MLFe), and n-butanol (MLFb) was 7.33: 62: 28.67.
Point 3: Many abbreviations were used, without explanation. For instance, MIC stands for what??
Response 3: According your suggestion, all abbreviations had been explained in the first time that appears in the revised manuscripts.
Point 4: Extensive English editing and revision is required throughout the manuscript.
Response 4: We are sorry to trouble you for our terrible English, and according to our reviewer’s suggestion, we have used a language editing service to improving the language in the manuscript.
Reviewer 2 Report
Comments and Suggestions for Authors:
In this paper, the extraction conditions of flavonoids from dried mature leaves of Morus alba were optimized. However, due to some inaccuracies and not fully exhausted discussion, this manuscript should be improved before it could be considered for publication.
1. The optimal extraction conditions were identified as follow: extraction temperature of 70.85℃, solvent concentration of 39.30%, extraction time of 120.18 min, and liquid/solid ratio of 34.6:1, the actual yield of MLF was 50.52 mg g-1. Does the authors used these conditions for extract to confirm the predicted values? There is no relevant experimental data in the Materials and Methods section.
2. From 279 to 291, this paragraph introduces the activity of various flavonoids and it is not suitable for discussion. It is better to move to the introduction section.
3. The authors suggested the multi-bioactivities of MLFe were related to flavonoids. It is necessary to measure the amount of flavonoids in petroleum ether (MLFp), ethyl acetate (MLFe), and n-butanol (MLFb) to confirm the components distribution. Usually, flavonoid glycosides were also found in butanol layer after liquid-liquid partition.
4. Line 21,147-148, 153…Degree Celsius symbol (℃) can be inserted in the word file, please do not use“。+ C”.
5. Line 40, please add reference for “The flavonoids contained the active hydrogen …and eliminate the toxicity”.
6. Line 271, solid/liquid ratio 1 : 14 “g mL-1”, the ratio will not have units.
7. Line 331, the spell of “flavoniods” à flavonoids
8. The manuscript is not carefully written; a great number of typographical and punctuation errors are present throughout the text.
I hope that my comment is useful for the improvement of the article.
Author Response
Response to Reviewer 2 Comments
Point 1: The optimal extraction conditions were identified as follow: extraction temperature of 70.85℃, solvent concentration of 39.30%, extraction time of 120.18 min, and liquid/solid ratio of 34.6:1, the actual yield of MLF was 50.52 mg g-1. Does the authors used these conditions for extract to confirm the predicted values? There is no relevant experimental data in the Materials and Methods section.
Response 1: We have added the statement of “After optimization, the adequacy of the model equation was validated by comparing the experimental data with the predicted results of the optimized model.” in the Materials and Methods section 4.2. And the confirmation of the predicted values by these conditions for extract was exhibited in section 2.3. Verification of predictive model.
Point 2: From 279 to 291, this paragraph introduces the activity of various flavonoids and it is not suitable for discussion. It is better to move to the introduction section.
Response 2: The introduction of the activities of various flavonoids was used to explain the multiple-bioactivities of MLF. So we decided to stay the same as it was, after careful consideration.
Point 3: The authors suggested the multi-bioactivities of MLFe were related to flavonoids. It is necessary to measure the amount of flavonoids in petroleum ether (MLFp), ethyl acetate (MLFe), and n-butanol (MLFb) to confirm the components distribution. Usually, flavonoid glycosides were also found in butanol layer after liquid-liquid partition.
Response 3: The results showed that the components of different polarity in total flavonoids can be extracted by solvents of different polarity. And the contents ratio of flavonoids in petroleum ether (MLFp), ethyl acetate (MLFe), and n-butanol (MLFb) was 7.33: 62: 28.67. The butanol layer contained 28.67% of flavonoids.
Point 4: Line 21,147-148, 153…Degree Celsius symbol (℃) can be inserted in the word file, please do not use“。+ C”.
Response 4: All the degree celsius symbol had been replaced with ℃.
Point 5: Line 40, please add reference for “The flavonoids contained the active hydrogen …and eliminate the toxicity”.
Response 5: The reference of the statement of “The flavonoids contained the active hydrogen …and eliminate the toxicity” was added at the end of the sentence. “Han, R. M.; Zhang, J. P.; Skibsted, L.H. Reaction dynamics of flavonoids and carotenoids as antioxidants. Molecules 2012, 17, 2140-2160.”
Point 6: Line 271, solid/liquid ratio 1 : 14 “g mL-1”, the ratio will not have units.
Response 6: Based on your comments, we have checked the text, and the units of solid/liquid ratio and liquid/solid ratio had been deleted.
Point 7: Line 331, the spell of “flavoniods” à flavonoids
Response 7: The misspellings of “flavoniods” in line 331 had been corrected with “flavonoids”.
Point 8: The manuscript is not carefully written; a great number of typographical and punctuation errors are present throughout the text.
Response 8: We apologize for the typographical and punctuation errors used in the manuscript. We have used a language editing service to improve the language in the revised version.
Reviewer 3 Report
The manuscript entitled "Flavonoids from Morus alba L. leaves: optimization of the extraction by response surface methodology and comprehensive evaluation of their antioxidant, antimicrobial, and inhibition of α-amylase activities by analytical hierarchy process" reports a study about the optimization of the extraction of flavonoids from Morus alba L. leaves (MLF) using experimental design. Additionally, the authors reported some results regarding antioxidant, antimicrobial and α-amylase activities of the extracts and applied analytical hierarchy process. The use of chemometrics tools is useful to extract information of chemical systems, in this case, method of extraction and the biological activity of the extracts. The obtained some optimized conditions of the extraction of MLF that can be useful for further studies of that need to extract flavonoids from this species.
There are a few points to change.
1 - Page 2 - line 82
MLF is an acronym that the meaning must be cited in the first time that appears in the manuscript, independent of the abstract.
2 - Page - line 69 - "RSM" - response surface methodology
Add its meaning please,
3 - Please improve figure 3, page 7-
Some information in the figure is not possible to read,
4 - Conclusions,
Please add more information regarding the results of biological activities, and/or clarify the sentence:
"The good antimicrobial, antioxidant, α-amylase inhibition activities of MLFe suggested its potential usage in food industry"
Author Response
Response to Reviewer 3 Comments
Point 1: Page 2 - line 82 MLF is an acronym that the meaning must be cited in the first time that appears in the manuscript, independent of the abstract.
Response 1: According your suggestion, all abbreviations had been explained in the first time that appears in the revised manuscripts.
Point 2: Page - line 69 - "RSM" - response surface methodology Add its meaning please,
Response 2: The "RSM" in line 69 had been explained with “response surface methodology”.
Point 3: Please improve figure 3, page 7-Some information in the figure is not possible to read,
Response 3: The figure 3 has been improved to read all the information in the figure.
Point 4: Conclusions, Please add more information regarding the results of biological activities, and/or clarify the sentence:"The good antimicrobial, antioxidant, α-amylase inhibition activities of MLFe suggested its potential usage in food industry"
Response 4: The sentence:"The good antimicrobial, antioxidant, α-amylase inhibition activities of MLFe suggested its potential usage in food industry" had been revised and clarified to be “The good antimicrobial, antioxidant, and α-amylase inhibition activity of MLFe suggested its potential usage in the food industry, such as meat preservative, or in the drug industry, such as antibacterial and hypoglycemic agents.”
Round 2
Reviewer 2 Report
Comments and Suggestions for Authors:
Most of the comments have been answered, the manuscript should be accepted for publication in this form.
