Anti-Obesity Activities of Standardized Ecklonia stolonifera Extract in 3T3-L1 Preadipocytes and High-Fat-Diet-Fed ICR Mice

Round 1

Reviewer 1 Report

In this manuscript, Han et al. evaluated the effectiveness of ESETM in reducing lipid accumulation in 3T3-L1 adipocytes and the anti-obesity effect in mice fed with high-fat diet. The authors further characterized the functions of ESETM in reducing adipogenesis and lipogenesis, in stimulating lipolysis, and in promoting thermogenesis browning in the obese mice model and adipocytes, and observed consistent results with dose dependency of SESTM. 

This work is sound in systematically evaluating the biochemistry effectiveness of ESETM in obesity models for potential future clinical trial of this compound. Therefore, this work meets the requirements of scope by the journal Applied Sciences. The data in this manuscript are straightforward and well documented. A few minor issues need to be addresses before publication in Applied Sciences.

The authors used both ESE and ESETM in the 3T3-L1 adipocytes experiments, but only characterized dieckol and ESE by HPLC in Figure 1. The HPLC of ESETM is also suggested, and the effectiveness of dieckol on adipocytes should be shown to support the indication that dieckol is the key effective component of the extract.

It will be more informative to readers when showing the statistics with p or q value or number of asterisk symbols instead of values with letters.

Author Response

Response to Reviewer 1 Comments

 

Point 1: The authors used both ESE and ESETM in the 3T3-L1 adipocytes experiments, but only characterized dieckol and ESE by HPLC in Figure 1. The HPLC of ESETM is also suggested, and the effectiveness of dieckol on adipocytes should be shown to support the indication that dieckol is the key effective component of the extract.

 

Response 1: We have re-written this part according to the Reviewer’s suggestion. We subsequently added data on the content of dieckol in ESETM by HPLC analysis. and added the result (table 1, figure 1d)

 

Point 2: It will be more informative to readers when showing the statistics with p or q value or number of asterisk symbols instead of values with letters.

 

Response 2: Thanks for the reviewer's suggestion. Letter displays are often used to report results of all pairwise comparisons among treatment means in comparative experiments. In captions to tables and charts using such letter displays, it is crucial to explain properly what the letters mean. As the Reviewer suggested, I have revised the section explaining the meaning of the letters in the paper to provide the reader with more information.

Reviewer 2 Report

Review comments:

 

The manuscript described the inhibitory effect of ESETM on weight gain by the evidence of in vitro and in vivo experiments. The conclusion seems to be convincing.

 

Anyhow, there are some problems that need to be addressed:

 

1. In the name of Ecklonia Stolonifera, “S” should be not capitalized. It is capitalized in several locations of the manuscript, such as title, keywords, etc.
2. Line 84: ESE and ESETM were obtained from Naturalway Co., Ltd. (Pocheon, Korea). There are two authors from Naturalway Co. Ltd, Pocheon, Gyeonggi 11160, Korea. So this is not a commercial product of the company. ESE and ESETM were prepared based on research collaboration, then the verification of the initial material is necessary. Who has identified the material of Ecklonia stolonifera, where is the specimen, and what is the number?
3. Line 85 and 86: Ecklonia stolonifera was immersed in 70% edible ethanol and subjected to circulation extraction at 70°C for 9 h. The boiling point of 70% ethanol is 78.2℃. 70°C is not high enough to boil the solution for circulation extraction.
4. Has the extraction method been optimized? Why 70% ethanol? Since the authors have claimed this is used for the clinical trial. It is necessary to optimize the preparation method.
5. The cytotoxicity of ESETM was evaluated. There are many requirements for the materials for clinical trials including toxicity studies. Mostly in vivo experiments (short-term and long-term) are required.
6. In vivo experiment, n =5. Is it too less for mice experiments? SD value is quite large in this case. The number of each group for the mice experiment is usually more than 6.
7. Line 132: 24°C ± 5°C can be 24 ± 5°C
8. Line 133: 55% ± 5% can be 55 ± 5%
9. Line 155: 5 × 106 cells should be 5 × 10⁶ cells
10. Line 177: CO2 incubator, 2 should be subscript.
11. Line 219 and 220: “A PDA spectrum of the dieckol and ecklonia stolonifera extract is shown in Figure 1a. As a result, by comparing the chromatograms of dieckol and ecklonia stolonifera extract.” The description is not appropriate. PDA spectrum should be UV spectrum. It is not dieckol and Ecklonia stolonifera It should be the UV spectrum of dieckolt and the peak at the same retention time (12.? min) in the profile of ESE. The figure’s resolution is not high enough, this retention time is not readable.
12. Line 219, 220, and 222: “ecklonia stolonifera extract”, can be ESE.
13. “Ecklonia stolonifera extract” appeared many times in the manuscript. Since “Ecklonia stolonifera extract (ESE)” is already mentioned in the introduction, there is no need to repeat the full name again. The same problem exists for ESETM.
14. The resolution of the figures is a little low.
15. In Figure 1c, another dominant peak can be observed in the chromatogram. What is that peak? Since the material was proposed to use for functional food, it will be nice if the majority of the peaks in the profile of the ESETM can be clarified. The content of dieckol is 27 mg/g, which is 2.7%. What are the components in the remaining 97.3%?
16. The content of dieckol (27 mg/g) in ESE is different from the content in the two recent publications from the same group (Jin et al. Cells 2020, 9(4), 871. Jin, et al. J. Funct. Foods 2021, 82, 104511) (23.7 mg/g). But the extraction method is the same and obtained from the same group  (Naturalway Co., Ltd. (Pocheon, Korea). The profile of ESE in Figure 1c is very different from the one in Figure 1 of Jin, et al. J. Funct. Foods 2021, 82, 104511. Therefore, it is very important to clarify the materials.
17. Below Table 1: Each value are the means ± SD of samples (n = 3). What is the “each”?

Author Response

Response to Reviewer 2 Comments

 

Point 1: In the name of Ecklonia Stolonifera, “S” should be not capitalized. It is capitalized in several locations of the manuscript, such as title, keywords, etc.

 

Response 1: We are very sorry for our incorrect writing, and We have made corrections according to the Reviewer’s comments.

 

Point 2: Line 84: ESE and ESETM were obtained from Naturalway Co., Ltd. (Pocheon, Korea). There are two authors from Naturalway Co. Ltd, Pocheon, Gyeonggi 11160, Korea. So this is not a commercial product of the company. ESE and ESETM were prepared based on research collaboration, then the verification of the initial material is necessary. Who has identified the material of Ecklonia stolonifera, where is the specimen, and what is the number?

 

Response 2: In this study, ESE and ESETM were obtained from the Naturalway Co. Ltd, Pocheon, Gyeonggi 11160, Korea, and ESE is the product produced after the raw material standardization. Currently, Ecklonia stolonifera extract is registered as a functional raw material for health functional food (May help to liver health) approved by the Korea Ministry of Food and Drug Safety. It is a material with proven functionality and safety (Registration date is 2018-03-26, No. 2004001510493). Our study demonstrated that the extract has dual functionality by validating its anti-obesity activity of the extract.

 

Point 3: Line 85 and 86: Ecklonia stolonifera was immersed in 70% edible ethanol and subjected to circulation extraction at 70°C for 9 h. The boiling point of 70% ethanol is 78.2℃. 70°C is not high enough to boil the solution for circulation extraction.

 

Response 3: We agree that The boiling point of 70% ethanol is 78.2℃. However, The evaporation temperature of alcohol is 31.5°C, so even at 70°C, which does not reach the boiling point, efficient circular extraction can be performed.

 

Point 4: Has the extraction method been optimized? Why 70% ethanol? Since the authors have claimed this is used for the clinical trial. It is necessary to optimize the preparation method.

 

Response 4: Thanks for your comment. Ecklonia stolonifera extract used this article is registered as a functional raw material for health functional food (May help to liver health) approved by the Korea Ministry of Food and Drug Safety. In addition, the company has optimized and standardized the production process for extracts. ESETM is also prepared from standardized ESE.

 

Point 5: The cytotoxicity of ESETM was evaluated. There are many requirements for the materials for clinical trials including toxicity studies. Mostly in vivo experiments (short-term and long-term) are required.

 

Response 5: We agree with you that comment. However, ecklonia stolonifera extract is registered as a functional raw material for health functional food (May help to liver health) approved by the Korea Ministry of Food and Drug Safety (Registration date is 2018-03-26, No. 2004001510493). Therefore, the safety of ESE has been proved.

 

Point 6: In vivo experiment, n =5. Is it too less for mice experiments? SD value is quite large in this case. The number of each group for the mice experiment is usually more than 6.

 

Response 6: We agree with you that comment. However, our school strictly controls the number of experimental animals in animal experiments. Our school's animal experiment ethics committee does not approve more than 5 experimental animals per group, so we can only apply for the experiment with 5 mice per group.

 

Point 7: Line 132: 24°C ± 5°C can be 24 ± 5°C

 

Response 7: We have made correction according to the Reviewer’s comments.

 

Point 8: Line 133: 55% ± 5% can be 55 ± 5%

 

Response 8: We have made correction according to the Reviewer’s comments.

 

Point 9: Line 155: 5 × 106 cells should be 5 × 10⁶ cells

 

Response 9: We are very sorry for our incorrect writing, and We have made corrections according to the Reviewer’s comments.

 

Point 10: Line 177: CO2 incubator, 2 should be subscript.

 

Response 10: We are very sorry for our incorrect writing, and We have made corrections according to the Reviewer’s comments.

 

Point 11: Line 219 and 220: “A PDA spectrum of the dieckol and ecklonia stolonifera extract is shown in Figure 1a. As a result, by comparing the chromatograms of dieckol and ecklonia stolonifera extract.” The description is not appropriate. PDA spectrum should be UV spectrum. It is not dieckol and Ecklonia stolonifera It should be the UV spectrum of dieckolt and the peak at the same retention time (12.? min) in the profile of ESE. The figure’s resolution is not high enough, this retention time is not readable.

 

Response 11: We have made correction according to the Reviewer’s comments. We subsequently added data on the content of dieckol in ESETM by HPLC analysis. Although the same column brand was used, the peak retention time slightly changed due to the influence of the old and new columns. And we replaced the high-resolution image in Figure 1.

 

Point 12: Line 219, 220, and 222: “ecklonia stolonifera extract”, can be ESE.

 

Response 12: We have made correction according to the Reviewer’s comments.

 

Point 13: “Ecklonia stolonifera extract” appeared many times in the manuscript. Since “Ecklonia stolonifera extract (ESE)” is already mentioned in the introduction, there is no need to repeat the full name again. The same problem exists for ESETM.

 

Response 13: We have made correction according to the Reviewer’s comments.

 

Point 14: The resolution of the figures is a little low.

 

Response 14: We have made correction according to the Reviewer’s comments.

 

Point 15: In Figure 1c, another dominant peak can be observed in the chromatogram. What is that peak? Since the material was proposed to use for functional food, it will be nice if the majority of the peaks in the profile of the ESETM can be clarified. The content of dieckol is 27 mg/g, which is 2.7%. What are the components in the remaining 97.3%?

 

Response 15: We agree with you that comment. However, We could not identify all the substances present in the extract, which would have been too difficult to experiment with. And ecklonia stolonifera extract used in this article is registered as a functional raw material for health functional food approved by the Korean Ministry of Food and Drug Safety. Mover, Dieckol is the main active ingredient of ESE.

 

Point 16: The content of dieckol (27 mg/g) in ESE is different from the content in the two recent publications from the same group (Jin et al. Cells 2020, 9(4), 871. Jin, et al. J. Funct. Foods 2021, 82, 104511) (23.7 mg/g). But the extraction method is the same and obtained from the same group  (Naturalway Co., Ltd. (Pocheon, Korea). The profile of ESE in Figure 1c is very different from the one in Figure 1 of Jin, et al. J. Funct. Foods 2021, 82, 104511. Therefore, it is very important to clarify the materials.

 

Response 16: Thanks for your comment. The ESE in this article and two recent publications were obtained from Naturalway Co., Ltd. (Pocheon, Korea). However, due to different production batches, the dieckol content in ESE will vary. And the dieckol content of 27 mg/g in ESE used in this paper is within 80-120% of the dieckol content of 23.7 mg/g in the standardized index.

 

Point 17: Below Table 1: Each value are the means ± SD of samples (n = 3). What is the “each”?

 

Response 17: We are very sorry for our incorrect writing and it is rectified to "the results are expressed as mean ± SD"

 

Round 2

Reviewer 2 Report

The comments have been properly addressed.