Potent Effect of Phlorotannins Derived from Sargassum linifolium as Antioxidant and Antidiabetic in a Streptozotocin-Induced Diabetic Rats Model
, Ragaa A. Hamouda 2,3, Mai Abdel Naby 4, Tarek M. Mohamed 4, Turki M. Al-Shaikh 2 and Abeer Khamis 4
Michael T. Simonich
Round 1
Reviewer 1 Report
The manuscript presents a study of the effects of Sargassum sp. phlorotannins on the maintenance of a diabetic state in rats. The overall finding is that the phlorotannins are potent antai-diabetics. The subject matter is timely of should be of high interest across multiple fields of research.
Unfortunately, the manuscript suffers considerably from a lack of English language proficiency and no apparent attempt was made by the authors to have the manuscript professionally edited for English proficiency prior to submission. This is truly unfortunate as numerous such services are available online and are relatively inexpensive. This should be done before the manuscript can be accepted for publication.
Additional Comments:
1) Figure 2 is labeled as a UV spectrum which is not possible since the data it contains are from an FT-IR analysis.
2) There are two tables called Table 1.
3) The graphs in Figs 3 & 4 should not have the measured data points connected by lines as the intervening values were not measured. However, the curve formed in Fig 3 by connecting the data points would strongly suggest that a linear model (y=mx+b) was not the best choice for regression analysis of those data.
4) The last figure with the histological pictures has no number or label and is thus difficult to evaluate.
Author Response
Dear Editor, Applied Sciences Journal
Thank you very much for the message to let us to that our manuscript needs some revisions to be made before publication. We think that the comments are of great value to improve the quality of our manuscript. Now we have carefully revised the manuscript according to the comments and we have answered the questions of the reviewers . Answers to all reviewers' questions were done point by point and marked by red colour. Also, all corrections within the manuscript text have been made in red. We try to follow the recommendations of referees and we hope the revised manuscript is now suitable for publication and are looking forward to receiving good news from your journal. Thank you again.
Prof. Saly Gheda (Corresponding author)
Review 1
The manuscript presents a study of the effects of Sargassum sp. phlorotannins on the maintenance of a diabetic state in rats. The overall finding is that the phlorotannins are potent anti-diabetics. The subject matter is timely of should be of high interest across multiple fields of research.
Unfortunately, the manuscript suffers considerably from a lack of English language proficiency and no apparent attempt was made by the authors to have the manuscript professionally edited for English proficiency prior to submission.
Response: English language checked and improved
This is truly unfortunate as numerous such services are available online and are relatively inexpensive. This should be done before the manuscript can be accepted for publication.
Additional Comments:
- Figure 2 is labeled as a UV spectrum which is not possible since the data it contains are from an FT-IR analysis.
Response: Done, the figure2 legend corrected
- There are two tables called Table 1.
Response: Done, corrected
- The graphs in Figs 3 & 4 should not have the measured data points connected by lines as the intervening values were not measured. However, the curve formed in Fig 3 by connecting the data points would strongly suggest that a linear model(y=mx+b) was not the best choice for regression analysis of those data.
Response: Done, we choise lorgarithmic regression, which is a more appropriate method for modeling non-linear relationships.
4) The last figure with the histological pictures has no number or label and is thus difficult to evaluate.
Response: Done, corrected
Author Response File: 
Author Response.docx
Reviewer 2 Report
This manuscript describes the antioxidant and antidiabetic activity of the phlorotanins from Sargassum linifolium, both in vitro and in vivo. The manuscript is interesting, many experiments have been conducted, as detailed is the description of the preparation of the fraction and chemical analysis. I would like to ask the authors in which solvent the extract obtained is solubilized, to perform the assays shown in the manuscript. The high concentrations used for the experiment with cells undoubtedly demonstrate the lack of cytotoxicity; however, I would like to ask the authors how it is possible to use such high concentrations. What is the maximum solubility of the extract in your solvent? In the event that the solvent used in cell medium was organic, it is difficult to think that using the same volume of solvent in control, its toxicity remains low. In this regard, I ask the authors to replace the figure of the vitality expressed as a percentage, with one that reports the absorbance values to 630 nm and in which the controls of the cells in the presence and in the absence of the solvent are reported. In the manuscript clearly indicate the origin of the normal cells used. The authors should also explain the choice of the dose of extract used in the experiments in vivo. Is there a correspondence between the dose used and the one used in cells? In this manuscript, only the antidiabetic activity in vivo in the rats has been demonstrated.Therefore, every possible reference to an antidiabetic activity of the extract in other species must be used with great caution. In the title, in the abstract (Lane 38) and in the text it must be said that in this manuscript the extract used is effective in rats.
Author Response
Dear Editor, Applied Sciences Journal
Thank you very much for the message to let us to that our manuscript needs some revisions to be made before publication. We think that the comments are of great value to improve the quality of our manuscript. Now we have carefully revised the manuscript according to the comments and we have answered the questions of the reviewers . Answers to all reviewers' questions were done point by point and marked by red colour. Also, all corrections within the manuscript text have been made in red. We try to follow the recommendations of referees and we hope the revised manuscript is now suitable for publication and are looking forward to receiving good news from your journal. Thank you again.
Prof. Saly Gheda (Corresponding author)
Review2
Which solvent the extract obtained is solublizeÙŠ extract obtained is solublizediiiiiiiiiiiiiiiiiiiiii?
Response: Phlorotannins were extracted from brown seaweeds according to the method of Koivikko et al., (2007) the end result were supernatant of phlorotannis in water which were dried by rotary evaporator and stored at 4 oC until used.
What is the maximum solubility of the extract in your solvent?
Response: Phlorotannins are water soluble and this is also confirmed by Jennings&Steinbery (1997)
The authors should also explain the choice of the dose of extract used in the experiments in vivo.
Response: Diabetic rat were treated with phlorotannis from S. linifolium extract (60 mg/kg body weight) following the reference of (Jung et al., 2014), one rat with weight 200 gram take about 12 mg of phlorotannis soluble in 1 ml of water
In the manuscript clearly indicate the origin of the normal cells used
Response: The normal cell was Human prostatic stromal myofibroblast normal cell line
(WPMY-1). This cell was obtained from Vecsera company,Cairo, Egypt
Is there a correspondence between the dose used and the one used in cells? In this manuscript
Response: Cytotoxicity of phlorotannis extracts was performed by MTT assay , Seaweed extracts ranging from 250 to 1000 µg/ml dissolved in water, but in vivo study extract (60 mg/kg body weight) because in vivo there are million of cell in the body of rat.
In the title, in the abstract (Lane 38) and in the text it must be said that in this manuscript the extract used is effective in rats.
Done, title changed to: Potent effect of phlorotannins derived from Sargassum linifolium as antioxidant and antidiabetic in a streptozotocin-induced diabetes rat model
Done, this sentence added to the abstract:
Response: The results in the present study recommended using phlorotannins derived from Sargassum linifolium as an antioxidant, anti-diabetic, in vivo study. It could be used in developing medicinal preparations for treating diabetes and its related symptoms.
Author Response File: Author Response.docx
Reviewer 3 Report
The work described in this manuscript has little novelty because many studies have been done to extract and characterize the phlorotannins from brown algae. Many studies about the extraction, characterization and toxicity/safety of phlorotannin extracts can be found in citation 8. The studies of antioxidant activities and anti-diabetic activities of phlorotannins extracted from different brown algae have also been studied as shown in the literature cited, particularly, a reader can find all information from references [3, 4, 15-17]. The methods used for this study were all reported too. The only difference of this study from others is the species of brown algae.
The justification of the need of this study is not provided in the Introduction.
Section 2.4: provide the type and strain of rats used in the study, and the company (name and location) from which the rats were purchased. Describe how rats were housed after receiving and before the experiment.
Total 87 references were cited in this is a research paper, which is too many. Please cut to about 40 by removing the irrelevant references.
Significant improvement in English writing is needed.
Below are detailed comments:
Line 21: 1. change to "in a streptozotocin-induced diabetes rat model" , 2. change : described" to "characterized"
line 29: change "the same in" to "the same as in"
Line 30-32: Grammar error.
line 33: change to "There were"
line 36-37: change "improve" to "improved", "reverse" to reversed"
line 52: change "unpleasant" to "undesirable"
Line 61: change to "some algae". use lower case.
line 98: change to 25mg/mL.
Line 103-106: Correct grammar error.
line 122: change to "fasting"
Line 138-139: What were the samples? how they were taken
Line 141-142: how the pancreatic, intestine and liver tissues were removed from rats? Were animals sacrificed before tissue removal and what methods were used to sacrifice the animals?
Section 3.3: The quantification of phlorotannins content by DMBA assay was not described in the Material and Method section.
Line 227-228: grammar error. use highest or maximum, but not both.
Figure 3 and 4: the relationship between Inhibition% and Extract conc is obviously non-linear, but the author did linear regression.
Figure 4: ABTE* is a free radical. The increase of ABTS* activity with extract concentration indicates the stimulation of oxidative activity, not inhibition of free radical activity. Please check your data and redo the graph.
Line 247: check the unit of concentration
Line 274-277: Improve English writing.
Table 6: Adjust the position of the title of Table 6
Line 381: S. linifolium: Make it Italic throughout the manuscript.
line 386: change to “which were administrated”
Line 294--397: confusing sentence.
line 490: delete: against disorder
line 492-494: grammar error.
line 495: change to "lowered glucose, insulin, cholesterol,"
line 497: change to "did not affect kidney functions represented by urea and creatinine levels"
Author Response
Dear Editor, Applied Sciences Journal
Thank you very much for the message to let us to that our manuscript needs some revisions to be made before publication. We think that the comments are of great value to improve the quality of our manuscript. Now we have carefully revised the manuscript according to the comments and we have answered the questions of the reviewers . Answers to all reviewers' questions were done point by point and marked by red colour. Also, all corrections within the manuscript text have been made in red. We try to follow the recommendations of referees and we hope the revised manuscript is now suitable for publication and are looking forward to receiving good news from your journal. Thank you again.
Prof. Saly Gheda (Corresponding author)
Review 3
The work described in this manuscript has little novelty because many studies have been done to extract and characterize the phlorotannins from brown algae
. Many studies about the extraction, characterization and toxicity/safety of phlorotannin extracts can be found in citation 8.
The studies of antioxidant activities and anti-diabetic activities of phlorotannins extracted from different brown algae have also been studied as shown in the literature cited, particularly, a reader can find all information from references [3, 4, 15-17].
The methods used for this study were all reported too.
The only difference of this study from others is the species of brown algae.
Response: Done, in introduction we wrote the following references, and rearrangement the references,
The phlorotannins recovered from brown algae varied depending on the species, region, and thallus zone [14]. Phlorotannins varied quantitative and qualitatively according to seasons [15] and also the quantity and characterization of phlorotannins differ from species to species, and also in the same species according to location so it must be know the characterization and quantity of phlorotannins in each species and in the same species in different locations
The justification for the need for this study is not provided in the introduction
Response: This paragraph added to the introduction part: Numerous studies dealt with the role of of phlorotannins extracted from Sargassum as an antidiabetic, but studies reported role of phlorotanins from Sargassum linifolium species which collected from a region of Gulf Suez, Sinia is lacking. the study also extend to investigate phlorotanins effects on all biochemical and histological parameters of STZ-induced diabetic rats
Section 2.4: provide the type and strain of rats used in the study, and the company (name and location) from which the rats were purchased. Describe how rats were housed after receiving and before the experiment.
Response: Forty white albino males Wistar rats weighing 200-240 g were bought from the Faculty of Agriculture, Center for Experimentation and Agricultural Research, Zagazig University, Egypt. Rats were kept in cages (four to six rats per cage), in a well-ventilated environment, at an appropriate temperature, with a 12 hour light/dark cycle, and fed a pellet-concentrated diet. Rats were given two weeks to acclimatise to the laboratory environment prior to experimentation.
Total 87 references were cited in this is a research paper, which is too many. Please cut to about 40 by removing the irrelevant references.
Response: Its two difficult to reduce the references because we have many experiments, introduction plus material and methods have references contain 37 references and discussion 40
Significant improvement in English writing is needed.
Response: Done, English writing improved and checked with languge grammer
Below are detailed comments:
Line 21: 1. change to "in a streptozotocin-induced diabetes rat model" , 2. change : described" to "characterized"
Response: Done, changed
line 29: change "the same in" to "the same as in"
Response: Done, changed
Line 30-32: Grammar error.
Response:Done, Corrected
line 33: change to "There were"
Response:Done, changed
line 36-37: change "improve" to "improved", "reverse" to reversed"
Response:Done, changed
line 52: change "unpleasant" to "undesirable"
Response:Done, changed
Line 61: change to "some algae". use lower case.
Response:Done, changed
line 98: change to 25mg/mL.
Response:Done, changed
Line 103-106: Correct grammar error.
Response
Corrected
line 122: change to "fasting"
Response
Corrected
Line 138-139: What were the samples? how they were taken
Response
Added
Blood samples were separated from all experimental rats, after overnight fasting, from the ocular vein
Line 141-142: how the pancreatic, intestine and liver tissues were removed from rats? Were animals sacrificed before tissue removal and what methods were used to sacrifice the animals?
Response :Added
At the end of the experiment, rats being given 300 mg/kg of sodium pentobarbital intraperitoneally as anaesthetic. and pancreatic tissues were collected and fixed with 10% formalin.
Section 3.3: The quantification of phlorotannins content by DMBA assay was not described in the Material and Method section.
Response
Added in details
The phlorotannin was also characterized by Dimethoxy benzaldehyde assay (DMBA) as a method estimated by Stern et al. [21], Stock solutions of DMBA (2 g/100 ml glacial acetic acid), 16.0 ml concentrated hydrochloric acid per 100.0 ml glacial acetic acid were prepared and kept at room temperature before starting the assay The working reagent must be made fresh. Ten mg of algal extract was dissolved in 1ml methanol and different volume of extract stock (0-20µl) was taken in test tubes, methanol was added to make total volume 20µl in each test tube. 10 µl of N, N dimethylformamide (DMF) was added to each tested sample, to precipitate protein. HCl reagent 1.25 ml was mixed with 1.25 ml of DMBA reagent, and added to each tested sample. Tested sample was placed in 30°C water bath and was covered, after 60 min the absorbance was recorded at 510 nm. The total phlorotannis content was determined using the linear equation based on the calibration curve of phloroglucinol as stander.
Line 227-228: grammar error. use highest or maximum, but not both.
Response
Corrected
Figure 3 and 4: the relationship between Inhibition% and Extract conc is obviously non-linear, but the author did linear regression.
Response:Done, Thank you for bringing this to our attention. We have updated our analysis for figs. 3 and 4 to use logarithmic regression, which is a more appropriate method for modeling non –linear relationships
Figure 4: ABTE* is a free radical. The increase of ABTS* activity with extract concentration indicates the stimulation of oxidative activity, not inhibition of free radical activity. Please check your data and redo the graph.
Response:Done, the ABTS inhibition % were corrected in curve
Line 247: check the unit of concentration
Response
Done, corrected
Line 274-277: Improve English writing.
Response
Deleted
Table 6: Adjust the position of the title of Table 6
Response
Adjusted
Line 381: S. linifolium: Make it Italic throughout the manuscript.
Response
Done, now italic in all manuscript
line 386: change to “which were administrated”
Response
Changed
Line 294--397: confusing sentence.
Response
Deleted
line 490: delete: against disorder
Response
Deleted
line 492-494: grammar error.
Response
Corrected
line 495: change to "lowered glucose, insulin, cholesterol,"
Response
Changed
line 497: change to "did not affect kidney functions represented by urea and creatinine levels"
Response
Changed
Author Response File: Author Response.docx
Reviewer 4 Report
Extensive language editing is needed
The first sentence of the abstract can be removed and put in the introduction supported with references. to enrich the introduction the following refs can be used:
https://www.mdpi.com/797270
https://www.sciencedirect.com/science/article/pii/S1319562X20306823
Name the normal cell line used for the in vitro cytotoxicity,
what was the confluency of the cells used.
Author should not rely on only UV and FTIR for the identification of compound, Mass spectroscopy is required for it.
the results p[resented for the cytoxicity is not convincing as it is strating from 80%. provide the raw data or revise the graph.
How does the author justify the 10% cell death after using a plant extract ?
What is the discovery of this study?
Author Response
Dear Editor, Applied Sciences Journal
Thank you very much for the message to let us to that our manuscript needs some revisions to be made before publication. We think that the comments are of great value to improve the quality of our manuscript. Now we have carefully revised the manuscript according to the comments and we have answered the questions of the reviewers . Answers to all reviewers' questions were done point by point and marked by red colour. Also, all corrections within the manuscript text have been made in red. We try to follow the recommendations of referees and we hope the revised manuscript is now suitable for publication and are looking forward to receiving good news from your journal. Thank you again.
Prof. Saly Gheda (Corresponding author)
Review 4
Extensive language editing is needed
Response
Language checked and improved
The first sentence of the abstract can be removed and put in the introduction supported with references. to enrich the introduction the following refs can be used:
https://www.mdpi.com/797270
https://www.sciencedirect.com/science/article/pii/S1319562X20306823
Response
Added
Name the normal cell line used for the in vitro cytotoxicity,
Response
Done , the name was added: Human prostatic stromal myofibroblast cell line
what was the confluency of the cells used.
Response
The cells used to discover the effects of phlorotannins extracted from S. linifolium brown alga on the WPMY1 normal cell line, to prove that the phlorotannins was safe to use
Author should not rely on only UV and FTIR for the identification of compound, Mass spectroscopy is required for it.
Response
We used UPLC-MS/MS analysis in previously published work by Kamal et al., [45], which confirmed the presence of Carmalol derivatives, Dihydroxy pentafuhalol, Trihydroxy Hexafuhalol, Hydroxy tetrafuhalol, and Phlorotannis-7 phloroglucinol.
the results presented for the cytoxicity is not convincing as it is strating from 80%. provide the raw data or revise the graph.
Response
We used for treatments low concentrations of phlorotannins. Cytotoxicity of phlorotannis extracts was performed by MTT assay , algal extracts ranging from 250 to 1000 µg/ml dissolved in water and I treatments rats by dose of 60 mg/kg body weight
How does the author justify the 10% cell death after using a plant extract ?
Response
We used for treatments low concentrations of phlorotannins, we start the test in vitro250 µg /mL to 1000 µg /mL, and more than 90% of the cells were still alive at all concentrations and the rats treated by dose of 60 mg/kg body weight following the reference of (Jung et al., 2014)
What is the discovery of this study?
Response
Numerous studies dealt with the role of of Phlorotannins extracted from Sargassum as an antidiabetic, but studies reported role of phlorotanins from Sargassum linifolium species which collected from a region of Gulf Suez, Sinia is lacking. Also the study reported the role of phlorotanins as antioxidant, antidiabetic in vitro and in vivo and the study also extend to investigate all biochemical and histological parameters of STZ-induced diabetic rats. Total phlorotannins extracted from alga was 10.87 mg/ gm dry weight and considered best amount . All these measurements gave an indication of the possibility of using this new species of Sargassum in the treatment of diabetes and pharmaceutical industry.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
The manuscript is now in good shape for publication. No further comments.
Author Response
no comments have been received
Reviewer 2 Report
I thank the authors for answering my questions
Author Response
no comments have been received
Reviewer 3 Report
The readability and scientific soundness of the revised manuscript is significantly improved. Most of my comments are addressed properly. however, there are a few new issues to be addressed before the manuscript can be published.
1. Line 30-32: Different enzyme activities were not expressed in understandable English. change the sentence to "The glucosidase, alpha-amylase, glutathione and catalase levels were 0.11 ± 0.097, 420.5 ± 13, 11.27 ± 3.3 and 8.01±1.31µmol/min/g in G1, and 0.04 ±0.016, 184.75 ± 55.24, 12.78 ± 2.1, and 11.28 ± 1.74 µmol/min/g) in G1 and G4, respectively."
2. Line 160: change "separated" to "withdrawn".
3. Line 164-166: I assume that organ tissues were removed after rats were dead. How did you make sure the animals/rats were dead after anaesthetization? How other organ tissues were removed from the dead animals was not mention.
4.Fig. 3 and Fig. 4: Briefly describe the relationship between antioxidant activity or free radical inhibition and the concentration of phlorotannin extract.
Author Response
Dear Editor, Applied Sciences Journal
Thank you very much for the message to let us to that our manuscript needs few revisions to be made before publication. We think that the comments are of great value to improve the quality of our manuscript. Now we have carefully revised the manuscript according to the comments and we have answered the questions of the reviewer . we hope the revised manuscript is now suitable for publication and are looking forward to receiving good news from your journal. Thank you again.
Prof. Saly Gheda (Corresponding author)
The readability and scientific soundness of the revised manuscript is significantly improved. Most of my comments are addressed properly. however, there are a few new issues to be addressed before the manuscript can be published.
- Line 30-32: Different enzyme activities were not expressed in understandable English. change the sentence to "The glucosidase, alpha-amylase, glutathione and catalase levels were 0.11 ± 0.097, 420.5 ± 13, 11.27 ± 3.3 and 8.01±1.31µmol/min/g in G1, and 0.04 ±0.016, 184.75 ± 55.24, 12.78 ± 2.1, and 11.28 ± 1.74 µmol/min/g) in G1 and G4, respectively."
Response: Done , the sentence changed
- Line 160: change "separated" to "withdrawn".
Response: Done
- Line 164-166: I assume that organ tissues were removed after rats were dead. How did you make sure the animals/rats were dead after anaesthetization? How other organ tissues were removed from the dead animals was not mention.
Response: The method written in details as follows:
At the end of the experimental period, rats were anesthetized by cervical dislocation under sodium pentobarbital (300 mg/kg) intraperitoneally. Blood samples were withdraw from all experimental rats, after overnight fasting, from the ocular vein. Blood samples were centrifuged for 15 min at 3000 rpm to obtain the serum. The clean and clear serum was put into plastic tubes with labels and stored at − 4 °C until used for biochemical assays. After euthanization of rats , small intestine, liver, skeletal muscle, and pancreatic tissue samples were collected.
4.Fig. 3 and Fig. 4: Briefly describe the relationship between antioxidant activity or free radical inhibition and the concentration of phlorotannin extract.
Response:
For Fig.3. the paragraph changed to:
When DPPH solution is mixed with phlorotannis acting as a hydrogen atom donor, a stable non-radical form of DPPH is obtained with the simultaneous change of the violet color to pale yellow. The inhibition could be observed amplified by raising the concentration of extract with the highest reserve of DPPH maximum inhibition with S. linifolium phlorotannins extract at the concentration (50µg/ml), and IC50 was 50.1 µg/ ml (Figure 3). The regression value (R2) is 0.9972,), that means the model is efficient and can accurately predict the response.
For Fig.4. the paragraph changed to:
The ABTS radical cation (ABTS ) is reactive towards most antioxidants including phenolic, during this reaction, phlorotannis donate electrons leading to a disappearance of the blue/green color of this radical. Phlorotannins extracts demonstrated stronger antioxidant activity with ABTS, and the inhibition increased with the extract concentration of S. linifolium. The maximum inhibition with S. linifolium phlorotannins extracts at the concentration (125 µg/ml), and IC50 was (85.4 µg/ ml) Figure 4. The regression value (R2) is 0.9862, that means the model is efficient and can accurately predict the response
Author Response File: Author Response.docx
Reviewer 4 Report
All the comments have been adressed satisfactorily.
Author Response
no comments have been received
