Adsorption/Desorption on Macroporous Resins of Okicamelliaside in the Extract of Camellia nitidissima Chi Leaves
Round 1
Reviewer 1 Report
“Adsorption/Desorption on Macroporous Resins of Okicamelli- 2
aside in the Extract of Camellia nitidissima Chi Leaves”.
Abstract
It is necessary to highlight the novelty of this research work.
Add the standard deviation to the results showed in the Abstract.
Introduction:
The introduction section is very confusing and needs to be completely rewritten.
Line 28-29:
“C. nitidissima has abundant phenolic components, saponins, polysaccharides and other substances, many of which exhibit antioxidant, anti-cancer and anti-hyperglycaemic effects”
This phrase is a bit confusing, please rewrite to avoid the reader thinking that saponins or polysaccharides might exhibit antioxidant, anticancer, and anti-hyperglycaemic effects.
Line 34-38:
“The C. nitidissima leaves not only contain partial bioactive components which are found in C. nitidissima flowers, but also some characteristic components, for example, OCS (Figure 1b), whose structure is elucidated as 3,4-dioxoloellagic acid 4’-O-β-D-glucopyra-noside, is an ellagic acid derivative previously observed in Camellia japonica (C. japonica) and C. nitidissima leaves”.
OCS is present in C. nitidissima leaves and was previously observed in C. nitidissima leaves. Please rewrite it, it's too confusing.
Line 38-41:
“It exhibits excellent anti-allergic activity. Compared with ketotifen fumarate, an antihistamine drug, it is 12,000 times more potent in inhibiting the degranulation of RBL-2H3 cells [6]. It significantly inhibits the vascular hyperpermeability in a passive cutaneous anaphylaxis mouse model [7].”
It is necessary to join the sentences, to facilitate the understanding to the reader
Line 45:
“The content of OCS in C. nitidissima leaves ranges from 0.51 to 1.33%.”
Please add the standard deviation values.
Line 47-48:
“However, studies on the extraction and separation of OCS from C. nitidissima leaves are few.”
Please add the references of these studies.
Line 49-52:
Many studies have been carried out on the extraction and isolation of bioactive compounds from plant tissues, but most of the methods have limitations, such as the use of extraction solvents that are toxic, the high cost of new methods and the inability to recycle consumables, which limits the development of products after the active ingredient [9,10].
The isolation of bioactive compounds from plants is a very old methodology.
Nothing new is shown in figure 1, it is better to delete it.
It is necessary to highlight the novelty of this article, and add a sentence with the main advances achieved with this research work.
Materials and Methods:
Line 67-68
“They were powdered using an 800Y pulveriser (Xichu, Jinhua, China) and filtered through a 60 Tyler mesh sieve. The leaf powder was stored in a foil bag at -20℃ until use.”
Add the company and the characteristics of the equipment used to pulverize the material. The sieving process was manual or mechanical. With a single sieve it is not possible to select the particle size distribution.
Line 71-74
“The powder prepared in 4.1. was mixed with 60% ethanol at a solid-liquid ratio of 1:30 and extracted in a water bath at 80°C for 10 min. in the supernatant ethanol was added to the supernatant to achieve a final concentration of 70%. The mixture was precipitated overnight. The supernatant was then rotary-evaporated and freeze-dried to obtain a crude extract E1.”
The ethanol concentration must be well expressed.
What was the moisture content of the powder? What was measured?.
How was the extraction reactor?. It was stirred mechanically?
What rotary evaporator was used? What was the final concentration of the concentrated liquor?
It is necessary to clarify the extraction process.
Results and Discussion:
Line 187-194:
“Adsorption capacity describes the amount of OCS adsorbed on each gram of resin. Adsorption rate is the percentage of OCS adsorbed by the resin from the extraction [14]. Figure 2a shows that the adsorption capacity of the five resins was in descending order: AB-8 > HPD100 > NKA-9 > D101 > DM130. AB-8 had the highest adsorption capacity and rate of 280.11 μg/g and 82.39% respectively. HPD100, NKA-9 and D101 followed closely, their adsorption capacities ranged from 229.36 to 251.89 μg/g, and adsorption rates were all above 70%. Overall, the adsorption of these four resins was relatively good. The DM130 resin had an adsorption capacity of less than 200 μg/g and were significantly less effective than the remaining four.”
Please add the standard deviation values.
Figure 2. (a).
Upgrade the figure quality.
Standard deviation values need to be added to the results shown throughout the article. Review section 3.2. It is difficult to understand when the authors speak of the pure OCS or of the extract obtained from the leaves of C. nitidissima.
Discussion
The discussion should be based on the results shown, and not on other research papers.
Line 355-356:
“Japanese scholars had used three fillers for the separation of OCS using chromatographic columns, resulting in a lengthy and complicated operation”.
Add reference.
Conclusions
It is necessary to highlight the novelty of this article in the conclusions.
Author Response
Point 1: It is necessary to highlight the novelty of this research work. Add the standard deviation to the results showed in the Abstract.
Response 1: The novelty of this research work has been added to the Abstract and standard deviations have been added to the results showed in the Abstract.
Point 2: “C. nitidissima has abundant phenolic components, saponins, polysaccharides and other substances, many of which exhibit antioxidant, anti-cancer and anti-hyperglycaemic effects”. This phrase is a bit confusing, please rewrite to avoid the reader thinking that saponins or polysaccharides might exhibit antioxidant, anticancer, and anti-hyperglycaemic effects.
Response 2: This phrase has been amended to read ”C. nitidissima has abundant phenolic components, saponins, polysaccharides and other substances. And pharmacological studies have shown that C. nitidissima exhibits antioxidant, anti-cancer and anti-hyperglycaemic effects”.
Point 3: “The C. nitidissima leaves not only contain partial bioactive components which are found in C. nitidissima flowers, but also some characteristic components, for example, OCS (Figure 1b), whose structure is elucidated as 3,4-dioxoloellagic acid 4’-O-β-D-glucopyra-noside, is an ellagic acid derivative previously observed in Camellia japonica (C. japonica) and C. nitidissima leaves”. OCS is present in C. nitidissima leaves and was previously observed in C. nitidissima leaves. Please rewrite it, it's too confusing.
Response 3: This phrase has been amended to read ”OCS (Figure 1b) is present in C. nitidissima leaves, and its structure was elucidated as 3,4-dioxoloellagic acid 4’-O-β-D-glucopyranoside”.
Point 4: “It exhibits excellent anti-allergic activity. Compared with ketotifen fumarate, an antihistamine drug, it is 12,000 times more potent in inhibiting the degranulation of RBL-2H3 cells [6]. It significantly inhibits the vascular hyperpermeability in a passive cutaneous anaphylaxis mouse model [7]”. It is necessary to join the sentences, to facilitate the understanding to the reader.
Response 4: This phrase has been amended to read “OCS showed 12,000 times higher anti-degranulation activity than that of the anti-histaminic drug, ketotifen fumarate, tested on RBL-2H3 cells [6], and it significantly inhibited the vascular hyperpermeability in a passive cutaneous anaphylaxis mouse model [7]”.
Point 5: “The content of OCS in C. nitidissima leaves ranges from 0.51 to 1.33%”. Please add the standard deviation values.
Response 5: This phrase has been amended to read “The content of OCS in C. nitidissima leaves ranges from 0.51 (± 0.04) % to 1.33 (± 0.06) %”.
Point 6: “However, studies on the extraction and separation of OCS from C. nitidissima leaves are few”. Please add the references of these studies.
Response 6: “Inhibitory Effects of an Ellagic Acid Glucoside, Okicamelliaside, on Antigen-Mediated Degranulation in Rat Basophilic Leukemia RBL-2H3 Cells and Passive Cutaneous Anaphylaxis Reaction in Mice.” and “Screening, Preparation and Investigation of the Antitumor Activity of Punch Camptothecin in Camellia nitidissima Chi Leaves” two references have been added. This phrase has been amended to read “However, studies on the green separation of OCS from C. nitidissima leaves are few [6,8]”.
Point 7: “Many studies have been carried out on the extraction and isolation of bioactive compounds from plant tissues, but most of the methods have limitations, such as the use of extraction solvents that are toxic, the high cost of new methods and the inability to recycle consumables, which limits the development of products after the active ingredient [9,10]”. The isolation of bioactive compounds from plants is a very old methodology.
Response 7: This phrase has been amended to read “Macroporous resins, as a recyclable and environmentally friendly material [10], are characterised by fast adsorption rates [11] and good adsorption effects. They have been widely used in food and medicine industry [12]”.
Point 8: Nothing new is shown in figure 1, it is better to delete it.
Response 8: Figure 1 was intended to show the reader the plant form of C. nitidissima leaves and the molecular structure of the OCS as a supplementary illustrative element, so it has not been deleted.
Point 9: It is necessary to highlight the novelty of this article, and add a sentence with the main advances achieved with this research work.
Response 9: The novelty of this article has been added to the article. And a sentence with the main advances achieved with this research work “In this study, the adsorption/desorption of OCS on different resins were compared, and the separation condition with AB-8 resin was optimized to obtain a green OCS extract with high OCS content” has been added to the Introduction.
Point 10: “They were powdered using an 800Y pulveriser (Xichu, Jinhua, China) and filtered through a 60 Tyler mesh sieve. The leaf powder was stored in a foil bag at -20℃ until use”. Add the company and the characteristics of the equipment used to pulverize the material. The sieving process was manual or mechanical. With a single sieve it is not possible to select the particle size distribution..
Response 10: This phrase has been amended to read “They were powdered using a mechanical pulveriser (Xichu 800Y, Jinhua, China). The powder (moisture content of 8.87 (±0.10) %) is then manually shaken through a 60 mesh sieve to remove oversized particles, ensuring that the powder is well homogenised and in full contact with the extraction solution for full extraction. The leaf powder was stored in a foil bag at -20℃ until use”. The equipment company used to pulverize the material is Xichu. The sieving process was manual. The purpose of sieving with a sieve is to remove large particles and to ensure the uniformity of the powder.
Point 11: “The powder prepared in 4.1. was mixed with 60% ethanol at a solid-liquid ratio of 1:30 and extracted in a water bath at 80°C for 10 min. in the supernatant ethanol was added to the supernatant to achieve a final concentration of 70%. The mixture was precipitated overnight. The supernatant was then rotary-evaporated and freeze-dried to obtain a crude extract E1”.
The ethanol concentration must be well expressed.
What was the moisture content of the powder? What was measured?
How was the extraction reactor? It was stirred mechanically?
What rotary evaporator was used?
What was the final concentration of the concentrated liquor?
It is necessary to clarify the extraction process.
Response 11: This phrase has been amended to read “1.00 g of dried leaves powder and 30 mL of ethanol: water (60:40 v/v) were mixed in a beaker and the beaker was placed in an electric thermostatic water bath (Jinghong DK-S26, Shanghai, China) at 80°C for 10 min to extraction. After completion of the water bath, the extracts were centrifuged (Eppendorf 5810R, Hamburg, Germany) at 8000 rpm for 10 min at 20°C. The anhydrous ethanol was added to the supernatant to reach a final concentration of 70%. The mixture was precipitated overnight. The supernatant was then concentrated in a rotary evaporator (BUCHI Labortechnik AG V-850 vacuum controller & R-215 rotavapor, Switzerland) to remove all the ethanol from the solution and then freeze-dried to give the final crude extract E1. E1 is powdered and yellow-brown in colour”.
The ethanol concentration is expressed as “ethanol: water (60:40 v/v)”.
The moisture content of the powder was 8.87 (±0.10) %, this piece is supplemented in 2.1. In addition, the morphology and the colour of the E1have been added accordingly in 2.2.
The extraction reactor was an electric thermostatic water bath, the brand of this machine is jinghong, from Shanghai, China, model DK-S26. This machine is not a stirring type, it is a thermostatic water bath.
The brand name of the rotary evaporator is BUCHI from Switzerland and it consists of a vacuum controller (AG V-850) and a rotary evaporator (R-215), which operate together.
The final concentration of the concentrated liquor should be ethanol free.
The extraction process has been supplemented in 2.2.
Point 12: “Adsorption capacity describes the amount of OCS adsorbed on each gram of resin. Adsorption rate is the percentage of OCS adsorbed by the resin from the extraction [14]. Figure 2a shows that the adsorption capacity of the five resins was in descending order: AB-8 > HPD100 > NKA-9 > D101 > DM130. AB-8 had the highest adsorption capacity and rate of 280.11 μg/g and 82.39% respectively. HPD100, NKA-9 and D101 followed closely, their adsorption capacities ranged from 229.36 to 251.89 μg/g, and adsorption rates were all above 70%. Overall, the adsorption of these four resins was relatively good. The DM130 resin had an adsorption capacity of less than 200 μg/g and were significantly less effective than the remaining four.”
Please add the standard deviation values.
Response 12: The standard deviations have been added to the article and this sentence has been amended to read ” AB-8 had the highest adsorption capacity and rate of 2.80 (± 0.14) mg/g and 82.39 (± 4.16) %, respectively. HPD100, NKA-9 and D101 followed closely, their adsorption capacities ranged 2.29-2.52 mg/g, and adsorption rates were all above 70%. Overall, the adsorption of these four resins was relatively good. The DM130 resin had an adsorption capacity of less than 2.00 mg and were significantly less effective than the remaining four”.
Point 13: Upgrade the figure2a quality.
Response 13: Figures 2, 5 and 6 have all been added with error lines. Due to the layout of the article, the images are small and the original images are available in the zip archive. If there is a problem with pixelation please ask the editor to contact me promptly.
Point 14: Standard deviation values need to be added to the results shown throughout the article. Review section 3.2. It is difficult to understand when the authors speak of the pure OCS or of the extract obtained from the leaves of C. nitidissima.
Response 14: The standard deviations have been added to the article. The content of 3.2 has been changed to include a detailed description of the method of making further extracts.
Point 15: The discussion should be based on the results shown, and not on other research papers.
Response 15: The discussion has been changed. And the content of other research papers has been deleted.
Point 16: “Japanese scholars had used three fillers for the separation of OCS using chromatographic columns, resulting in a lengthy and complicated operation”. Add reference.
Response 16: A reference “Okicamelliaside, an Extraordinarily Potent Anti-Degranulation Glucoside” has been added. This phrase has been amended to read “In addition to this, compared to the Japanese scholars who used three fillers to separate OCS, toxic reagents were also used [5]”.
Point 17: It is necessary to highlight the novelty of this article in the conclusions.
Response 17: The discussion has been changed. This study highlights the use of AB-8 resin for the separation of OCS as an effective technique on the basis of the adsorption/desorption of OCS on the resin. And the method proposed in this paper is green and provides a theoretical basis for the application of OCS in food and medicine.
In addition, the data in this chapter has been checked and found to have a multiplier error, so the incorrect data and units have been corrected, and the corresponding pictures have been changed, so please let the editor contact me if you have any other questions.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The manuscript "Adsorption/Desorption on Macroporous Resins of Okicamelli-aside in the Extract of Camellia nitidissima Chi Leaves," authored by Hanyu Zheng, Ying Gao, Jianyong Zhang, Xin Meng, Qizhen Du, and Junfeng Yin, described adsorption and desorption studies. According to the results, appropriate resin selection as well as adsorption and desorption conditions can result in high polyphenol transfer. In this study, the authors show that, ideally, OCS should be adsorbing onto resin at room temperature in order to promote multilayer adsorption. In order to minimize the repulsive forces opposing adsorption as well as ensure complete adsorption, an appropriate adsorption time and sample concentration need to be chosen.
Some issues must be clarified before the manuscript is published.
1. The abstract of the paper is too long the authors must reduce it. Abstract has to be reframed with clear outline of the work.
2. Novelty of work is missing, the authors must shows this.
3. What is the type of the adsorption in this study?
4. Why is the choice of the method employed in this study? Proper citation for this study should be introduce
5. The introduction section is poorly written that lacks motivation and purpose of this study. To put the study in its state of the art, the authors must update the Introduction section, by recent works found in the literature. Introduction section should be revised. Some news references which should be added: DOI :10.1039/d0ra03581e, DOI :10.1016/j.jmrt.2019.11.045.
6. Correct all typos throughout the manuscript.
7. The quality of all figures must be improved.
8. Thoroughly go through the manuscript and check for grammar and spelling corrections & space between two sentences, and articles use may be incorrect, etc.. And also check the manuscript formats
9. Check the reference format.
10. Conclusion should be modifies with important findings.
11. Units are missing at some places and include them at proper places
Once you address all of the above-mentioned comments, I will gladly review your manuscript again.
Comments for author File: 
Comments.pdf
Author Response
Point 1: The abstract of the paper is too long the authors must reduce it. Abstract has to be reframed with clear outline of the work.
Response 1: The abstract has been reworked in the article to outline the work for this study.
Point 2: Novelty of work is missing, the authors must shows this.
Response 2: Novelty of work has been added in the article. This study highlights the use of AB-8 resin for the separation of OCS as an effective technique on the basis of the adsorption/desorption of OCS on the resin. And the method proposed in this study is green and simple , providing a theoretical basis for the application of OCS in food and medicine.
Point 3: What is the type of the adsorption in this study?
Response 3: The correlation coefficients of AB-8 resin for both models were close to 1 (R2 > 0.99), indicating that both models were good predictors of the kinetics of OCS adsorp-tion by AB-8 macroporous resin. For the other four resins, the pseudo-second-order model seems to be a better fit. This suggests that the adsorption process of OCS on the resins may be a chemisorption process.
Point 4: Why is the choice of the method employed in this study? Proper citation for this study should be introduce.
Response 4: The introduction incorporates the motivation and purpose of the study. Okicamelliaside from C. nitidissima leaves can be used in thera-peutic drugs or nutritional foods. The separation of the active molecules using macroporous resins is the most basic method. However, which resin is the best for separating OCS and the underlying mechanism for its superiority remains unclear. In this study, the adsorption/desorption of OCS on different resins were compared, and the separation condition with AB-8 resin was optimized to obtain a green OCS extract with high OCS content.
Point 5: The introduction section is poorly written that lacks motivation and purpose of this study. To put the study in its state of the art, the authors must update the Introduction section, by recent works found in the literature. Introduction section should be revised. Some news references which should be added: DOI :10.1039/d0ra03581e, DOI :10.1016/j.jmrt.2019.11.045.
Response 5: The introduction section has been revised to add the motivation and purpose of the study. “Combined Statistical Physics Models and DFT Theory to Study the Adsorption Process of Paprika Dye on TiO2 for Dye Sensitized Solar Cells” and “A Microscopic and Macroscopic Investigation of the Adsorption of N719 Dye on ZnONanopowders (ZNP) and ZnONanorods (ZNR) for Dye Sensitized Solar Cells Using Statistical Physics Treatment and DFT Simulation” two references have been added.
Point 6: Correct all typos throughout the manuscript.
Response 6: All typos throughout the manuscript have been corrected.
Point 7: The quality of all figures must be improved.
Response 7: Figures 2, 5 and 6 have all been added with error lines. Due to the layout of the article, the images are small and the original images are available in the zip archive. If there is a problem with pixelation please ask the editor to contact me promptly.
Point 8: Thoroughly go through the manuscript and check for grammar and spelling corrections & space between two sentences, and articles use may be incorrect, etc. And also check the manuscript formats.
Response 8: Corrected incorrect grammar and spelling & spaces between two sentences. Incorrect use of articles, formatting of manuscripts have been corrected.
Point 9: Check the reference format.
Response 9: The format of the references has been corrected in accordance with journal requirements.
Point 10: Conclusion should be modifies with important findings.
Response 10: The conclusion has been modified in line with the key elements of this study.
Point 11: Units are missing at some places and include them at proper places.
Response 11: The units have been modified to put it in the right place.
In addition, the data in this chapter has been checked and found to have a multiplier error, so the incorrect data and units have been corrected, and the corresponding pictures have been changed, so please let the editor contact me if you have any other questions.
Author Response File: Author Response.docx
Reviewer 3 Report
This work investigated the performance of Adsorption/Desorption on Macroporous Resins of Okicamelliaside in the Extract of Camellia nitidissima Chi Leaves. The authors did much work. However, some issues need to be minor revised before considering acceptance.
1. What was the pH of the solution at equilibrium for the different resins studied?
2. Can the resins be recycled multiple times?
3. The novelty of this research should be well presented in the Introduction.
4. It is suggested that the author briefly explain this work's enlightenment to other researchers.
5. In terms of duplications. I checked and found that the repetition rate of your manuscript is still 40%. To further process your manuscript, could you please check and revise according to the updated duplication report.
Comments for author File: Comments.pdf
Author Response
Point 1: What was the pH of the solution at equilibrium for the different resins studied?
Response 1: The pH of the resins at equilibrium is added in 2.5.1 of the article as "the pH of HPD100, D101, DM130, AB-8 and NKA-9 resins at equilibrium are 8.33 (± 0.13), 7.45 (± 0.67), 9.14 (± 0.17), 7.35 (± 0.34) and 8.80 (± 0.33), respectively”.
Point 2: Can the resins be recycled multiple times?
Response 2: Yes, the resin can be recycled multiple times.
Point 3: The novelty of this research should be well presented in the Introduction.
Response 3: The Introduction has been changed. This study highlights the use of AB-8 resin for the separation of OCS as an effective technique on the basis of the adsorption/desorption of OCS on the resin. And the method proposed in this study is green and simple , providing a theoretical basis for the application of OCS in food and medicine.
Point 4: It is suggested that the author briefly explain this work's enlightenment to other researchers.
Response 4: The enlightenment of this study has been added to the article. Okicamelliaside from C. nitidissima leaves can be used in thera-peutic drugs or nutritional foods. The separation of the active molecules using macroporous resins is the most basic method. However, which resin is the best for separating OCS and the underlying mechanism for its superiority remains unclear. In this study, the adsorption/desorption of OCS on different resins were compared, and the separation condition with AB-8 resin was optimized to obtain a green OCS extract with high OCS content.
Point 5: In terms of duplications. I checked and found that the repetition rate of your manuscript is still 40%. To further process your manuscript, could you please check and revise according to the updated duplication report.
Response 5: Sentences with a high repetition rate in the inspection report were corrected, but the methods used in this study were similar to those used in the study in the references, resulting in a high repetition rate in the methods section.
In addition, the data in this chapter has been checked and found to have a multiplier error, so the incorrect data and units have been corrected, and the corresponding pictures have been changed, so please let the editor contact me if you have any other questions.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
Point 1: It is necessary to highlight the novelty of this research work. Add the standard deviation to the results showed in the Abstract.
Response 1: The novelty of this research work has been added to the Abstract and standard deviations have been added to the results showed in the Abstract.
Response 1:
No standard deviations were added.
Point 2: “C. nitidissima has abundant phenolic components, saponins, polysaccharides and other substances, many of which exhibit antioxidant, anti-cancer and anti-hyperglycaemic effects”. This phrase is a bit confusing, please rewrite to avoid the reader thinking that saponins or polysaccharides might exhibit antioxidant, anticancer, and anti-hyperglycaemic effects.
Response 2: This phrase has been amended to read ”C. nitidissima has abundant phenolic components, saponins, polysaccharides and other substances. And pharmacological studies have shown that C. nitidissima exhibits antioxidant, anti-cancer and anti-hyperglycaemic effects”.
Response 2: Now it's more understandable
Point 3: “The C. nitidissima leaves not only contain partial bioactive components which are found in C. nitidissima flowers, but also some characteristic components, for example, OCS (Figure 1b), whose structure is elucidated as 3,4-dioxoloellagic acid 4’-O-β-D-glucopyra-noside, is an ellagic acid derivative previously observed in Camellia japonica (C. japonica) and C. nitidissima leaves”. OCS is present in C. nitidissima leaves and was previously observed in C. nitidissima leaves. Please rewrite it, it's too confusing.
Response 3: This phrase has been amended to read ”OCS (Figure 1b) is present in C. nitidissima leaves, and its structure was elucidated as 3,4-dioxoloellagic acid 4’-O-β-D-glucopyranoside”.
Response 3: OK
Point 4: “It exhibits excellent anti-allergic activity. Compared with ketotifen fumarate, an antihistamine drug, it is 12,000 times more potent in inhibiting the degranulation of RBL-2H3 cells [6]. It significantly inhibits the vascular hyperpermeability in a passive cutaneous anaphylaxis mouse model [7]”. It is necessary to join the sentences, to facilitate the understanding to the reader.
Response 4: This phrase has been amended to read “OCS showed 12,000 times higher anti-degranulation activity than that of the anti-histaminic drug, ketotifen fumarate, tested on RBL-2H3 cells [6], and it significantly inhibited the vascular hyperpermeability in a passive cutaneous anaphylaxis mouse model [7]”.
Response 4: OK
Point 5: “The content of OCS in C. nitidissima leaves ranges from 0.51 to 1.33%”. Please add the standard deviation values.
Response 5: This phrase has been amended to read “The content of OCS in C. nitidissima leaves ranges from 0.51 (± 0.04) % to 1.33 (± 0.06) %”.
Response 5: OK
Point 6: “However, studies on the extraction and separation of OCS from C. nitidissima leaves are few”. Please add the references of these studies.
Response 6: “Inhibitory Effects of an Ellagic Acid Glucoside, Okicamelliaside, on Antigen-Mediated Degranulation in Rat Basophilic Leukemia RBL-2H3 Cells and Passive Cutaneous Anaphylaxis Reaction in Mice.” and “Screening, Preparation and Investigation of the Antitumor Activity of Punch Camptothecin in Camellia nitidissima Chi Leaves” two references have been added. This phrase has been amended to read “However, studies on the green separation of OCS from C. nitidissima leaves are few [6,8]”.
Response 6: OK
Point 7: “Many studies have been carried out on the extraction and isolation of bioactive compounds from plant tissues, but most of the methods have limitations, such as the use of extraction solvents that are toxic, the high cost of new methods and the inability to recycle consumables, which limits the development of products after the active ingredient [9,10]”. The isolation of bioactive compounds from plants is a very old methodology.
Response 7: This phrase has been amended to read “Macroporous resins, as a recyclable and environmentally friendly material [10], are characterised by fast adsorption rates [11] and good adsorption effects. They have been widely used in food and medicine industry [12]”.
Response 7: OK
Point 8: Nothing new is shown in figure 1, it is better to delete it.
Response 8: Figure 1 was intended to show the reader the plant form of C. nitidissima leaves and the molecular structure of the OCS as a supplementary illustrative element, so it has not been deleted.
Point 9: It is necessary to highlight the novelty of this article, and add a sentence with the main advances achieved with this research work.
Response 9: The novelty of this article has been added to the article. And a sentence with the main advances achieved with this research work “In this study, the adsorption/desorption of OCS on different resins were compared, and the separation condition with AB-8 resin was optimized to obtain a green OCS extract with high OCS content” has been added to the Introduction.
Response 9: It is necessary to highlight the novelty of this article, which justifies its publication.
Point 10: “They were powdered using an 800Y pulveriser (Xichu, Jinhua, China) and filtered through a 60 Tyler mesh sieve. The leaf powder was stored in a foil bag at -20℃ until use”. Add the company and the characteristics of the equipment used to pulverize the material. The sieving process was manual or mechanical. With a single sieve it is not possible to select the particle size distribution..
Response 10: This phrase has been amended to read “They were powdered using a mechanical pulveriser (Xichu 800Y, Jinhua, China). The powder (moisture content of 8.87 (±0.10) %) is then manually shaken through a 60 mesh sieve to remove oversized particles, ensuring that the powder is well homogenised and in full contact with the extraction solution for full extraction. The leaf powder was stored in a foil bag at -20℃ until use”. The equipment company used to pulverize the material is Xichu. The sieving process was manual. The purpose of sieving with a sieve is to remove large particles and to ensure the uniformity of the powder.
Response 10:OK
Point 11: “The powder prepared in 4.1. was mixed with 60% ethanol at a solid-liquid ratio of 1:30 and extracted in a water bath at 80°C for 10 min. in the supernatant ethanol was added to the supernatant to achieve a final concentration of 70%. The mixture was precipitated overnight. The supernatant was then rotary-evaporated and freeze-dried to obtain a crude extract E1”.
The ethanol concentration must be well expressed.
What was the moisture content of the powder? What was measured?
How was the extraction reactor? It was stirred mechanically?
What rotary evaporator was used?
What was the final concentration of the concentrated liquor?
It is necessary to clarify the extraction process.
Response 11: This phrase has been amended to read “1.00 g of dried leaves powder and 30 mL of ethanol: water (60:40 v/v) were mixed in a beaker and the beaker was placed in an electric thermostatic water bath (Jinghong DK-S26, Shanghai, China) at 80°C for 10 min to extraction. After completion of the water bath, the extracts were centrifuged (Eppendorf 5810R, Hamburg, Germany) at 8000 rpm for 10 min at 20°C. The anhydrous ethanol was added to the supernatant to reach a final concentration of 70%. The mixture was precipitated overnight. The supernatant was then concentrated in a rotary evaporator (BUCHI Labortechnik AG V-850 vacuum controller & R-215 rotavapor, Switzerland) to remove all the ethanol from the solution and then freeze-dried to give the final crude extract E1. E1 is powdered and yellow-brown in colour”.
The ethanol concentration is expressed as “ethanol: water (60:40 v/v)”.
The moisture content of the powder was 8.87 (±0.10) %, this piece is supplemented in 2.1. In addition, the morphology and the colour of the E1have been added accordingly in 2.2.
The extraction reactor was an electric thermostatic water bath, the brand of this machine is jinghong, from Shanghai, China, model DK-S26. This machine is not a stirring type, it is a thermostatic water bath.
The brand name of the rotary evaporator is BUCHI from Switzerland and it consists of a vacuum controller (AG V-850) and a rotary evaporator (R-215), which operate together.
The final concentration of the concentrated liquor should be ethanol free.
The extraction process has been supplemented in 2.2.
Response 11: “The final concentration of the concentrated liquor should be ethanol free.” What was the concentration of the solution obtained after the concentration process (grams of solid extract/grams of solution)?
Point 12: “Adsorption capacity describes the amount of OCS adsorbed on each gram of resin. Adsorption rate is the percentage of OCS adsorbed by the resin from the extraction [14]. Figure 2a shows that the adsorption capacity of the five resins was in descending order: AB-8 > HPD100 > NKA-9 > D101 > DM130. AB-8 had the highest adsorption capacity and rate of 280.11 μg/g and 82.39% respectively. HPD100, NKA-9 and D101 followed closely, their adsorption capacities ranged from 229.36 to 251.89 μg/g, and adsorption rates were all above 70%. Overall, the adsorption of these four resins was relatively good. The DM130 resin had an adsorption capacity of less than 200 μg/g and were significantly less effective than the remaining four.”
Please add the standard deviation values.
Response 12: The standard deviations have been added to the article and this sentence has been amended to read ” AB-8 had the highest adsorption capacity and rate of 2.80 (± 0.14) mg/g and 82.39 (± 4.16) %, respectively. HPD100, NKA-9 and D101 followed closely, their adsorption capacities ranged 2.29-2.52 mg/g, and adsorption rates were all above 70%. Overall, the adsorption of these four resins was relatively good. The DM130 resin had an adsorption capacity of less than 2.00 mg and were significantly less effective than the remaining four”.
Response 12: OK
Point 13: Upgrade the figure2a quality.
Response 13: Figures 2, 5 and 6 have all been added with error lines. Due to the layout of the article, the images are small and the original images are available in the zip archive. If there is a problem with pixelation please ask the editor to contact me promptly.
Response 13: OK
Point 14: Standard deviation values need to be added to the results shown throughout the article. Review section 3.2. It is difficult to understand when the authors speak of the pure OCS or of the extract obtained from the leaves of C. nitidissima.
Response 14: The standard deviations have been added to the article. The content of 3.2 has been changed to include a detailed description of the method of making further extracts.
Response 14: OK
Point 15: The discussion should be based on the results shown, and not on other research papers.
Response 15: The discussion has been changed. And the content of other research papers has been deleted.
Response 15: The discussion could be improved.
Point 16: “Japanese scholars had used three fillers for the separation of OCS using chromatographic columns, resulting in a lengthy and complicated operation”. Add reference.
Response 16: A reference “Okicamelliaside, an Extraordinarily Potent Anti-Degranulation Glucoside” has been added. This phrase has been amended to read “In addition to this, compared to the Japanese scholars who used three fillers to separate OCS, toxic reagents were also used [5]”.
Response 16: OK
Point 17: It is necessary to highlight the novelty of this article in the conclusions.
Response 17: The discussion has been changed. This study highlights the use of AB-8 resin for the separation of OCS as an effective technique on the basis of the adsorption/desorption of OCS on the resin. And the method proposed in this paper is green and provides a theoretical basis for the application of OCS in food and medicine.
In addition, the data in this chapter has been checked and found to have a multiplier error, so the incorrect data and units have been corrected, and the corresponding pictures have been changed, so please let the editor contact me if you have any other questions.
Response 17: The conclusion could be improved.
Author Response
Point 1: It is necessary to highlight the novelty of this research work. Add the standard deviation to the results showed in the Abstract.
Response 1: The novelty of this research work has been added to the Abstract and standard deviations have been added to the results showed in the Abstract.
Response 1: No standard deviations were added.
Point 2: “C. nitidissima has abundant phenolic components, saponins, polysaccharides and other substances, many of which exhibit antioxidant, anti-cancer and anti-hyperglycaemic effects”. This phrase is a bit confusing, please rewrite to avoid the reader thinking that saponins or polysaccharides might exhibit antioxidant, anticancer, and anti-hyperglycaemic effects.
Response 2: This phrase has been amended to read ”C. nitidissima has abundant phenolic components, saponins, polysaccharides and other substances. And pharmacological studies have shown that C. nitidissima exhibits antioxidant, anti-cancer and anti-hyperglycaemic effects”.
Response 2: Now it's more understandable.
Point 3: “The C. nitidissima leaves not only contain partial bioactive components which are found in C. nitidissima flowers, but also some characteristic components, for example, OCS (Figure 1b), whose structure is elucidated as 3,4-dioxoloellagic acid 4’-O-β-D-glucopyra-noside, is an ellagic acid derivative previously observed in Camellia japonica (C. japonica) and C. nitidissima leaves”. OCS is present in C. nitidissima leaves and was previously observed in C. nitidissima leaves. Please rewrite it, it's too confusing.
Response 3: This phrase has been amended to read ”OCS (Figure 1b) is present in C. nitidissima leaves, and its structure was elucidated as 3,4-dioxoloellagic acid 4’-O-β-D-glucopyranoside”.
Response 3: OK
Point 4: “It exhibits excellent anti-allergic activity. Compared with ketotifen fumarate, an antihistamine drug, it is 12,000 times more potent in inhibiting the degranulation of RBL-2H3 cells [6]. It significantly inhibits the vascular hyperpermeability in a passive cutaneous anaphylaxis mouse model [7]”. It is necessary to join the sentences, to facilitate the understanding to the reader.
Response 4: This phrase has been amended to read “OCS showed 12,000 times higher anti-degranulation activity than that of the anti-histaminic drug, ketotifen fumarate, tested on RBL-2H3 cells [6], and it significantly inhibited the vascular hyperpermeability in a passive cutaneous anaphylaxis mouse model [7]”.
Response 4: OK
Point 5: “The content of OCS in C. nitidissima leaves ranges from 0.51 to 1.33%”. Please add the standard deviation values.
Response 5: This phrase has been amended to read “The content of OCS in C. nitidissima leaves ranges from 0.51 (± 0.04) % to 1.33 (± 0.06) %”.
Response 5: OK
Point 6: “However, studies on the extraction and separation of OCS from C. nitidissima leaves are few”. Please add the references of these studies.
Response 6: “Inhibitory Effects of an Ellagic Acid Glucoside, Okicamelliaside, on Antigen-Mediated Degranulation in Rat Basophilic Leukemia RBL-2H3 Cells and Passive Cutaneous Anaphylaxis Reaction in Mice.” and “Screening, Preparation and Investigation of the Antitumor Activity of Punch Camptothecin in Camellia nitidissima Chi Leaves” two references have been added. This phrase has been amended to read “However, studies on the green separation of OCS from C. nitidissima leaves are few [6,8]”.
Response 6: OK
Point 7: “Many studies have been carried out on the extraction and isolation of bioactive compounds from plant tissues, but most of the methods have limitations, such as the use of extraction solvents that are toxic, the high cost of new methods and the inability to recycle consumables, which limits the development of products after the active ingredient [9,10]”. The isolation of bioactive compounds from plants is a very old methodology.
Response 7: This phrase has been amended to read “Macroporous resins, as a recyclable and environmentally friendly material [10], are characterised by fast adsorption rates [11] and good adsorption effects. They have been widely used in food and medicine industry [12]”.
Response 7: OK
Point 8: Nothing new is shown in figure 1, it is better to delete it.
Response 8: Figure 1 was intended to show the reader the plant form of C. nitidissima leaves and the molecular structure of the OCS as a supplementary illustrative element, so it has not been deleted.
Point 9: It is necessary to highlight the novelty of this article, and add a sentence with the main advances achieved with this research work.
Response 9: The novelty of this article has been added to the article. And a sentence with the main advances achieved with this research work “In this study, the adsorption/desorption of OCS on different resins were compared, and the separation condition with AB-8 resin was optimized to obtain a green OCS extract with high OCS content” has been added to the Introduction.
Response 9: It is necessary to highlight the novelty of this article, which justifies its publication.
Point 10: “They were powdered using an 800Y pulveriser (Xichu, Jinhua, China) and filtered through a 60 Tyler mesh sieve. The leaf powder was stored in a foil bag at -20℃ until use”. Add the company and the characteristics of the equipment used to pulverize the material. The sieving process was manual or mechanical. With a single sieve it is not possible to select the particle size distribution..
Response 10: This phrase has been amended to read “They were powdered using a mechanical pulveriser (Xichu 800Y, Jinhua, China). The powder (moisture content of 8.87 (±0.10) %) is then manually shaken through a 60 mesh sieve to remove oversized particles, ensuring that the powder is well homogenised and in full contact with the extraction solution for full extraction. The leaf powder was stored in a foil bag at -20℃ until use”. The equipment company used to pulverize the material is Xichu. The sieving process was manual. The purpose of sieving with a sieve is to remove large particles and to ensure the uniformity of the powder.
Response 10: OK
Point 11: “The powder prepared in 4.1. was mixed with 60% ethanol at a solid-liquid ratio of 1:30 and extracted in a water bath at 80°C for 10 min. in the supernatant ethanol was added to the supernatant to achieve a final concentration of 70%. The mixture was precipitated overnight. The supernatant was then rotary-evaporated and freeze-dried to obtain a crude extract E1”.
The ethanol concentration must be well expressed.
What was the moisture content of the powder? What was measured?
How was the extraction reactor? It was stirred mechanically?
What rotary evaporator was used?
What was the final concentration of the concentrated liquor?
It is necessary to clarify the extraction process.
Response 11: This phrase has been amended to read “1.00 g of dried leaves powder and 30 mL of ethanol: water (60:40 v/v) were mixed in a beaker and the beaker was placed in an electric thermostatic water bath (Jinghong DK-S26, Shanghai, China) at 80°C for 10 min to extraction. After completion of the water bath, the extracts were centrifuged (Eppendorf 5810R, Hamburg, Germany) at 8000 rpm for 10 min at 20°C. The anhydrous ethanol was added to the supernatant to reach a final concentration of 70%. The mixture was precipitated overnight. The supernatant was then concentrated in a rotary evaporator (BUCHI Labortechnik AG V-850 vacuum controller & R-215 rotavapor, Switzerland) to remove all the ethanol from the solution and then freeze-dried to give the final crude extract E1. E1 is powdered and yellow-brown in colour”.
The ethanol concentration is expressed as “ethanol: water (60:40 v/v)”.
The moisture content of the powder was 8.87 (±0.10) %, this piece is supplemented in 2.1. In addition, the morphology and the colour of the E1have been added accordingly in 2.2.
The extraction reactor was an electric thermostatic water bath, the brand of this machine is jinghong, from Shanghai, China, model DK-S26. This machine is not a stirring type, it is a thermostatic water bath.
The brand name of the rotary evaporator is BUCHI from Switzerland and it consists of a vacuum controller (AG V-850) and a rotary evaporator (R-215), which operate together.
The final concentration of the concentrated liquor should be ethanol free.
The extraction process has been supplemented in 2.2.
Response 11: “The final concentration of the concentrated liquor should be ethanol free.” What was the concentration of the solution obtained after the concentration process (grams of solid extract/grams of solution)?
Point 12: “Adsorption capacity describes the amount of OCS adsorbed on each gram of resin. Adsorption rate is the percentage of OCS adsorbed by the resin from the extraction [14]. Figure 2a shows that the adsorption capacity of the five resins was in descending order: AB-8 > HPD100 > NKA-9 > D101 > DM130. AB-8 had the highest adsorption capacity and rate of 280.11 μg/g and 82.39% respectively. HPD100, NKA-9 and D101 followed closely, their adsorption capacities ranged from 229.36 to 251.89 μg/g, and adsorption rates were all above 70%. Overall, the adsorption of these four resins was relatively good. The DM130 resin had an adsorption capacity of less than 200 μg/g and were significantly less effective than the remaining four.”
Please add the standard deviation values.
Response 12: The standard deviations have been added to the article and this sentence has been amended to read ” AB-8 had the highest adsorption capacity and rate of 2.80 (± 0.14) mg/g and 82.39 (± 4.16) %, respectively. HPD100, NKA-9 and D101 followed closely, their adsorption capacities ranged 2.29-2.52 mg/g, and adsorption rates were all above 70%. Overall, the adsorption of these four resins was relatively good. The DM130 resin had an adsorption capacity of less than 2.00 mg and were significantly less effective than the remaining four”.
Response 12: OK
Point 13: Upgrade the figure2a quality.
Response 13: Figures 2, 5 and 6 have all been added with error lines. Due to the layout of the article, the images are small and the original images are available in the zip archive. If there is a problem with pixelation please ask the editor to contact me promptly.
Response 13: OK
Point 14: Standard deviation values need to be added to the results shown throughout the article. Review section 3.2. It is difficult to understand when the authors speak of the pure OCS or of the extract obtained from the leaves of C. nitidissima.
Response 14: The standard deviations have been added to the article. The content of 3.2 has been changed to include a detailed description of the method of making further extracts.
Response 14: OK
Point 15: The discussion should be based on the results shown, and not on other research papers.
Response 15: The discussion has been changed. And the content of other research papers has been deleted.
Response 15: The discussion could be improved.
Point 16: “Japanese scholars had used three fillers for the separation of OCS using chromatographic columns, resulting in a lengthy and complicated operation”. Add reference.
Response 16: A reference “Okicamelliaside, an Extraordinarily Potent Anti-Degranulation Glucoside” has been added. This phrase has been amended to read “In addition to this, compared to the Japanese scholars who used three fillers to separate OCS, toxic reagents were also used [5]”.
Response 16: OK
Point 17: It is necessary to highlight the novelty of this article in the conclusions.
Response 17: The discussion has been changed. This study highlights the use of AB-8 resin for the separation of OCS as an effective technique on the basis of the adsorption/desorption of OCS on the resin. And the method proposed in this paper is green and provides a theoretical basis for the application of OCS in food and medicine.
Response 17: The discussion could be improved.
In addition, the data in this chapter has been checked and found to have a multiplier error, so the incorrect data and units have been corrected, and the corresponding pictures have been changed, so please let the editor contact me if you have any other questions.
==================================
Second Revision Comments and Responses
Point 1: No standard deviations were added in the Abstract.
Response 1: Standard deviations have been added to the results showed in the Abstract. This phrase has been amended to read “The separation yielded purified extract with OCS of 290.82 (± 2.17) mg/g which was 6.0 times more than the crude extract (E1, 48.51 (± 0.56) mg/g of OCS)”. The other figures in the Abstract do not relate to the labelling of standard deviations, so the rest remain as they are. Standard deviations have also been added to all references to these two figures in the article.
Point 2: It is necessary to highlight the novelty of this article, which justifies its publication.
Response 2: (1) The subject of this study is relatively new: OCS was first identified in Camellia japonica in 2010, and it is only in the last 5 years that it has become known to be present and more abundant in C. nitidissima leaf. The available literature on the purification of OCS from C. nitidissima leaves is very limited and the method of purification is still somewhat flawed, which may be one of the reasons for the current low utilisation of C. nitidissima leaves. We therefore conducted a study on this in the hope of obtaining a more efficient and convenient method.
(2) The assay method in this study is relatively new: compared to previous HPLC methods for determining OCS content, this study uses QE for a more rapid and qualitatively accurate determination.
(3) Innovation in separation methods is not the main aim of our study. Our main aim was to elaborate on the rationale for the selection of AB-8 and to systematically investigate the various adsorption/desorption properties of AB-8 for OCS on the basis of previous selective separations, on the basis of which we tried to identify the optimal conditions.
Point 3: “The final concentration of the concentrated liquor should be ethanol free.” What was the concentration of the solution obtained after the concentration process (grams of solid extract/grams of solution)?
Response 3: This phrase in 2.2 has been amended to read “The supernatant was then concentrated in a rotary evaporator (BUCHI Labortechnik AG V-850 vacuum controller & R-215 rotavapor, Switzerland) until all the ethanol in the solution was removed and the rotary evaporation ended. The concentrate was then freeze-dried to give the final crude extract E1”. The point at which rotary evaporation stops is when all the ethanol in the solution has been evaporated.
We did not measure the concentration of the solution obtained after the concentration process as the water also evaporates during each rotary evaporation process, so the concentration of the solution after each concentration is not consistent. The concentrated solution is the intermediate product, the final powder (E1) obtained by lyophilising it is the extract we want, and the OCS content in E1 is what needs to be measured in this study.
Point 4: The discussion could be improved.
Response 4: The discussion has been amended to read “In this study, the adsorption/desorption characteristics of OCS on five resins were investigated based on the adsorption/desorption equilibrium and adsorption kinetics. AB-8 resin was found to be an effective resin to adsorb OCS, which followed pseudo-first-order kinetic model (R2 > 0.99). On this basis the optimum adsorption/desorption conditions were further determined, it was found that the optimum adsorption time and sample concentration were 30 min and 2.5 mg/mL respectively, while the optimum desorption was achieved by using 2.1 times the column volume of 60% ethanol solution. The separation by using AB-8 resin could increase OCS content of the extract from 48.51 (± 0.56) mg/g to 290.82 (± 2.17) mg/g. This has the potential to prepare green OCS extract with high content from the leaves of C. nitidissima for health products, providing some theoretical basis for the application of OCS in food and pharmaceuticals. It likewise suggests new ideas for the utilization of C. nitidissima leaves”.
Author Response File: Author Response.docx
