Characterization of Alginate from Sargassum duplicatum and the Antioxidant Effect of Alginate–Okra Fruit Extracts Combination for Wound Healing on Diabetic Mice

Round 1

Reviewer 1 Report

The authors try to evaluate the effects of Alginate, okra fruit extract and their combination on the evaluation of wound healing in mice previously made diabetic. The evaluation of wound healing was made on 36 male mice divided into 12 groups including normal control (NC), diabetics control (DC), alginate (DA) and alginate-30 okra (DAO) treatment groups in 3 different times. Mice were wounded to give 1 cm length wounds on the glutea (buttocks).

The authors see that  

a) SEC-MALLS analysis demonstrated  that alginate as single and homogeneous polysaccharide. 

b) The 1H-NMR spectrum showed that the Mannuronate/Guluronate ratio of the used alginate was 0.77.

c) Alginate, okra fruit extract and their combination were classified as moderate and strong antioxidants.

d) The numbers of fibrocytes,  fibroblasts, collagen densities significantly increased from 3 to 7 day.

e) In contrast, wound width, neutrophil, macrophages had significantly decreased at 14 day for both DA and DAO groups.

In conclusion the administration of extracts combination increased the re-epithelization of the wound area and 37 wound healing process on diabetic mice. 

My questions are the following:

a) How was the thickness of epidermis measured ?

b) What were the histological parameters for recognizing and discriminating fibroblasts, fibrocytes, macrophages and neutrophil granulocytes from preparations stained with hematoxylin and eosin?

c) Since the recognition of the cells is not easy I strongly recommend immunohistochemical staining which not only shows general views but specific localizations. Consequently, the data shown relating to the counts must be implemented with other results (i.e. immunohistochemical results) that take into account what has been suggested.

d) How was measured the collagen density?

 

Since this paper presents various doubts from a histological point of view I recommend a maJor revision before considering a possible publication

 

 

 

 

Author Response

The authors try to evaluate the effects of Alginate, okra fruit extract and their combination on the evaluation of wound healing in mice previously made diabetic. The evaluation of wound healing was made on 36 male mice divided into 12 groups including normal control (NC), diabetics control (DC), alginate (DA) and alginate-30 okra (DAO) treatment groups in 3 different times. Mice were wounded to give 1 cm length wounds on the glutea (buttocks).

 

The authors see that 

 

1. a) SEC-MALLS analysis demonstrated that alginate as single and homogeneous polysaccharide.

 

1. b) The 1H-NMR spectrum showed that the Mannuronate/Guluronate ratio of the used alginate was 0.77.

 

1. c) Alginate, okra fruit extract and their combination were classified as moderate and strong antioxidants.

 

1. d) The numbers of fibrocytes, fibroblasts, collagen densities significantly increased from 3 to 7 day.

 

1. e) In contrast, wound width, neutrophil, macrophages had significantly decreased at 14 day for both DA and DAO groups.

 

In conclusion the administration of extracts combination increased the re-epithelization of the wound area and 37 wound healing process on diabetic mice.

Answer 1: The authors thank the reviewer for the in-depth analysis of the submitted manuscript.

 

My questions are the following:

 

1. a) How was the thickness of epidermis measured?

Answer 2: The thickness of epidermis in the histology of skin tissues were measured by using the imageJ software.

 

1. b) What were the histological parameters for recognizing and discriminating fibroblasts, fibrocytes, macrophages and neutrophil granulocytes from preparations stained with hematoxylin and eosin?

Answer 3: The parameters for recognizing and differentiating cells were measured based on Hematoxylin & Eosin stains of the morphological of each cell was observed under a microscope.

 

1. c) Since the recognition of the cells is not easy I strongly recommend immunohistochemical staining which not only shows general views but specific localizations. Consequently, the data shown relating to the counts must be implemented with other results (i.e. immunohistochemical results) that take into account what has been suggested.

Answer 4: We thank the reviewer for the suggestion. The immunochemical staining is indeed an excellent technique for the cells recognition but unfortunately, due to limited resources, we couldn’t apply this technique for the present work. We will try to get the necessary funding for our next manuscript.

 

1. d) How was measured the collagen density?

Answer 5: Determination of collagen density was measured by using the ImageJ software based on the percent of area of ​​the collagen fibers, which has a color gradient set. This feature was detailed in the main text.

 

Since this paper presents various doubts from a histological point of view I recommend a maJor revision before considering a possible publication

Answer 6: We appreciate the constructive comments pointed out by the reviewer. We now hope that the reviewed version of the manuscript is suitable for publication.

 

We appreciate the constructive comments pointed out by the reviewers. We now hope that the reviewed version of the manuscript is suitable for publication

 

I am looking forward your favourable consideration.

Thank you very much for kind collaboration.

 

With best regards,

Pratiwi Pudjiastuti

Author Response File: Author Response.pdf

Reviewer 2 Report

The authors have tested antioxidants from okra fruit in combination with alginate on wound healing in diabetic mice. The low M/G ratio indicated that the isolated alginate is a good absorber. The isolated flavonoids were classified as strong antioxidants. Application of these compounds on incisional wounds in diabetic mice seemed to have beneficial effects.

This articles demonstrates to possibility to use alginate+flavonoids to limit morbidity in diabetic wounds.

Major points in the article.

1. In general the grammar needs to be checked throughout the paper.
   1. More detail from the mentioned literature is needed in the introduction (and discussion) to explain the observed effects. For example: how does ion exchange result in increased cell regeneration (line 68-70)?
2. Histology
   1. Section 2.9 Different types of cells were discriminated in histology (how many observers, blinded?). This is however not a very specific type of analysis, better use immunohistochemistry by using vimentin, CD68, MPO and collagen as targets. For collagen, Masson trichrome, picrosirius red or collagen hybridizing peptide are also available.
   2. Similarly, wound width is difficult to determine from the supplied images in figure 3. Why not stain for cytokeratins (e.g. panCK or CK15) thereby visualizing keratinocytes/newly formed epidermis and the wound gap.
   3. Results histology (fig 3-9): results cannot be claimed based on images like in figure 3.

Minor points.

1. Missing details in M&M
   1. Section 2.7 How were blood samples taken for glucose analyses and how was glucose measured? Number of animals per time point is 3? Please add this info to table 2. NB blood samples of day 1 were not from euthanized animals?
   2. How were animals divided over the groups? line 179: “The replication of the animal was based on the formula of Federer” is not sufficient. I find 12 groups a bit odd. There were in fact 4 groups with 9 animals per group. At 3 time points, 3 animals per group were euthanized for sample collection.
   3. Wounding: why was chosen for incision and why on the glutea?
2. Although proper statistics have been conducted, please present the data as mean +/- standard deviation. SEM must not be used: the Standard Error of the Mean of the sample is an estimate of how far the sample mean is likely to be from the population mean, whereas the standard deviation (SD) of the sample is the degree to which individuals within the sample differ from the sample mean. SEM is often used to polish the results: SEM values are (much) lower than SD.
3. Restructure Results
   1. Section 3.1 should be moved to materials & methods.
   2. Section 3.2 is superfluous or should be moved to subsections of 3.2.1-3.2.3.
   3. Section 3.3 (is 44 mg/g high or low?) can be fused with 3.4.
   4. Line 258-259 “indicating the potentiality of okra fruit extract in 258 reducing blood glucose level on diabetic mice.” should be moved to discussion.
4. What do the formulas in 3.4 refer to?
5. How does TGA (3.2.2) aid in the research goal of this paper?
6. Results in table 1 were performed in how many replicates, and was this test repeated?
7. Section 3.5 Report the results with a maximum of 2 decimals.
8. Table 2: add “n=3 animals per time point”. Significant differences were not observed?

Author Response

The authors have tested antioxidants from okra fruit in combination with alginate on wound healing in diabetic mice. The low M/G ratio indicated that the isolated alginate is a good absorber. The isolated flavonoids were classified as strong antioxidants. Application of these compounds on incisional wounds in diabetic mice seemed to have beneficial effects.

 

This articles demonstrates to possibility to use alginate+flavonoids to limit morbidity in diabetic wounds.

Answer 1: We thank the reviewer for the analysis of our manuscript.

 

Major points in the article.

 

In general the grammar needs to be checked throughout the paper.

Answer 2: The paper was checked by a native speaker and the grammar improved throughout the manuscript as suggested by the reviewer.

 

More detail from the mentioned literature is needed in the introduction (and discussion) to explain the observed effects. For example: how does ion exchange result in increased cell regeneration (line 68-70)?

Answer 3: We have revised the introduction and discussion adding the suggested references accordingly to the referee’s suggestions.

 

Histology

Section 2.9 Different types of cells were discriminated in histology (how many observers, blinded?). This is however not a very specific type of analysis, better use immunohistochemistry by using vimentin, CD68, MPO and collagen as targets. For collagen, Masson trichrome, picrosirius red or collagen hybridizing peptide are also available.

Similarly, wound width is difficult to determine from the supplied images in figure 3. Why not stain for cytokeratins (e.g. panCK or CK15) thereby visualizing keratinocytes/newly formed epidermis and the wound gap.

Results histology (fig 3-9): results cannot be claimed based on images like in figure 3.

Answer 4: We thank the reviewer for the suggestion. The immunochemical staining is indeed an excellent technique for the cells recognition but unfortunately, due to limited resources, we couldn’t apply this technique for the present work. We will try to get the necessary funding for our next manuscript.

 

Minor points.

 

Missing details in M&M

Section 2.7 How were blood samples taken for glucose analyses and how was glucose measured? Number of animals per time point is 3? Please add this info to table 2. NB blood samples of day 1 were not from euthanized animals?

Answer 5: Analyses of blood samples taken for glucose were conducted from veins on the tail area of ​​mice and measured using a glucometer (test stripe). Yes. The number of animals per time point is 3. No. NC blood samples of day 1 were not from euthanized animals.

 

How were animals divided over the groups? line 179: “The replication of the animal was based on the formula of Federer” is not sufficient. I find 12 groups a bit odd. There were in fact 4 groups with 9 animals per group. At 3 time points, 3 animals per group were euthanized for sample collection.

Answer 6: Actually there were 12 groups (NC₃, NC₇, NC₁₄, DC₃, DC₇, DC₁₄, DA₃, DA₇, DA₁₄, DAO₃, DAO₇, DAO₁₄), because were used 3 different times, so for NC group there were 3 groups (NC₃, NC₇, NC₁₄), and so on.

 

Wounding: why was chosen for incision and why on the glutea?

Answer 7: Because the incisional wound can be performed reintegration of the separated tissue section, so the wound gap can be observed. Based on some literature, diabetic mouse models were applied to dorsal (glutea) wounds to form chronic wounds.

 

Although proper statistics have been conducted, please present the data as mean +/- standard deviation. SEM must not be used: the Standard Error of the Mean of the sample is an estimate of how far the sample mean is likely to be from the population mean, whereas the standard deviation (SD) of the sample is the degree to which individuals within the sample differ from the sample mean. SEM is often used to polish the results: SEM values are (much) lower than SD.

Answer 8: We have now presented data as a mean +/- standard deviation (SD) on the results of the analysis.

 

Restructure Results

Section 3.1 should be moved to materials & methods.

Answer 9: Agreed. I have been moved section 3.1 to material and methods.

 

Section 3.2 is superfluous or should be moved to subsections of 3.2.1-3.2.3.

Answer 10: Agreed. I have been moved section 3.2 to subsection.

 

Section 3.3 (is 44 mg/g high or low?) can be fused with 3.4.

Answer 11: Total flavonoid yields of okra fruit extracts tend to be high compared to some literature. I have been fused section 3.2 with 3.4.

 

Line 258-259 “indicating the potentiality of okra fruit extract in 258 reducing blood glucose level on diabetic mice.” should be moved to discussion.

Answer 12: Agreed. I have been move the statement to discussion.

 

What do the formulas in 3.4 refer to?

Answer 13: We refer to the formula in section 2.6 about the antioxidant assay.

 

How does TGA (3.2.2) aid in the research goal of this paper?

Answer 14: TGA analysis was carried out in order to provide the readers with a complete characterization of the used alginate.

 

Results in table 1 were performed in how many replicates, and was this test repeated?

Answer 15: 3 replicates of each sample. Yes. This test was repeated 3 times.

 

Section 3.5 Report the results with a maximum of 2 decimals.

Answer 16: I have changed and corrected this.

 

Table 2: add “n=3 animals per time point”. Significant differences were not observed?

Answer 17: I have added the statement on the Table 2. Significant different were observed and marked with a letter notation of each groups on Table 2.

 

We appreciate the constructive comments pointed out by the reviewers. We now hope that the reviewed version of the manuscript is suitable for publication

 

I am looking forward your favourable consideration.

Thank you very much for kind collaboration.

 

With best regards,

 

Pratiwi Pudjiastuti

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Undoubtedly the authors have improved their work but the problems related to cellular identification remain and unfortunately seem not to be overcome. On the other hand, the cells present must be identified without a shadow of a doubt to justify the subsequent counts. The photographs shown do not clarify doubts.

These are my suggestions:

a) Devote a chapter to the identification of the cells or the histological parameters considered: eg Neutrophils were recognized because ....., Fibroblasts were recognized because ..... and so on.

b) Use tables and not graphs where arbitrary units are taken into account. This method should be described in the materials and methods describing them precisely. In the literature there are methods that use intensity scales with appropriate indices (0, 1, 2, 3)

c) Table the counts considered as mean and standard deviation

Author Response

Please see attachment.

Author Response File: Author Response.docx