Purification and Characterization of Fractions Containing Polysaccharides from Talinum triangulare and Their Immunomodulatory Effects

Round 1

Reviewer 1 Report

Comments for: In this manuscript, Yeh et al address "Purification and Characterization of Polysaccharides from 2 Talinum triangulare and Their Immunomodulatory Effects". In this study, authors provide the containing polysaccharides fraction extracted from T. triangulare shown the induction of NO production and TNF expression and the inhibition of Hela cells proliferation. Although some of the observations are of potential interest, there are shortcomings in the design of experiments and in the presentation and interpretation of data that require resolution.

Several concerns are addressed below:

1. It is a major concern that the bioactive fractions (large molecular weight) were through crude extract which beside polysaccharides, contains triterpenoids, polyphenols, proteins and a lot unknown compounds. In the fraction, F2, polysaccharides are only 21%. The experiments did not exclude the effect of other compounds and the osmolality problem due to polysaccharides. The present data cannot fully support the title.
2. The half-life of NO is only 6 seconds in cells. In the materials and methods (line 110, 153), you cannot detect NO concentration by Griess reagent which is for nitrite quantitation. The fig.1A and 3B should be corrected. Same reason, it cannot occur in conditioned medium.
3. In fig 4 legend, "the cell viability using PBS-pretreated CM was designed as 100%". Why the first column, PBS treatment is more than 120%.
4. The rational is not clear by using conditioned medium treated cancer cells. The conditioned mediums are not clearly defined in the legends, are they still contain the extracts before treated or has been wash out? If not, it cannot rule out the effect of the extract.

5. The quantification of Fig1B and statistical analysis (with n and p values) should be clearly presented and compared in the results to interpret them.

Author Response

Response to Reviewer 1 Comments

 

Comments for: In this manuscript, Yeh et al address "Purification and Characterization of Polysaccharides from Talinum triangulare and Their Immunomodulatory Effects". In this study, authors provide the containing polysaccharides fraction extracted from T. triangulare shown the induction of NO production and TNF expression and the inhibition of Hela cells proliferation. Although some of the observations are of potential interest, there are shortcomings in the design of experiments and in the presentation and interpretation of data that require resolution.

Several concerns are addressed below:

Point 1: 1. It is a major concern that the bioactive fractions (large molecular weight) were through crude extract which beside polysaccharides, contains triterpenoids, polyphenols, proteins and a lot unknown compounds. In the fraction, F2, polysaccharides are only 21%. The experiments did not exclude the effect of other compounds and the osmolality problem due to polysaccharides. The present data cannot fully support the title.

 

Response 1: Thank you for your valuable feedback on our manuscript. We have changed the title of this manuscript as “Purification and Characterization of Fractions Containing Polysaccharides from Talinum triangulare and Their Immunomodulatory Effects” to support our present data.

 

Point 2: 2. The half-life of NO is only 6 seconds in cells. In the materials and methods (line 110, 153), you cannot detect NO concentration by Griess reagent which is for nitrite quantitation. The fig.1A and 3B should be corrected. Same reason, it cannot occur in conditioned medium.

 

Response 2: Thank you for your valuable comment. We agree your comment and have followed your suggestion. Changes in this manuscript have been made accordingly in Figs. 1A and 3B and lines 111, 147, 154, 231, 240, 242, 262, 326, 328, 349, 367, 371.

 

Point 3: 3. In fig 4 legend, "the cell viability using PBS-pretreated CM was designed as 100%". Why the first column, PBS treatment is more than 120%.

 

Response 3: Thank you for your valuable comment. We have normalized the values of different groups with the PBS group and corrected mistakes of Fig. 4. Thus, a new Fig. 4 has been uploaded and the results of cell viabilities have been rewritten (line 364-366).

 

Point 4: The rational is not clear by using conditioned medium treated cancer cells. The conditioned mediums are not clearly defined in the legends, are they still contain the extracts before treated or has been wash out? If not, it cannot rule out the effect of the extract.

 

Response 4: Thank for your valuable feedback. We have described how condition media (CMs) obtained in the Materials and Methods (line 108-111, 149-153). CMs were obtained by filtered with 0.22μm filters, so the effects of extracts can’t be ruled out. We have mentioned it in the Results and Discussion (line 254-255, 368-370).

 

Point 5: The quantification of Fig1B and statistical analysis (with n and p values) should be clearly presented and compared in the results to interpret them.

 

Response 5: We thank your valuable feedback. We didn’t perform statistical analysis of results (images) in Fig. 1B. Only results of Fig. 1A showed statistical analysis (with n and p values). We have corrected this mistake in figure legend of Fig. 1 (line 264-267).

Author Response File: Author Response.docx

Reviewer 2 Report

This manuscript reported the purification, characterization of polysaccharide extracted and isolated from Talinum triangulare. During the extraction and isolation, 3X3 orthogonal array was used to assist the experiment design, as a result, the best process condition was optimized. In addition, the resulting mixture was undergoing the chemical analysis as well as the bioactivity assessment. Overall, the selling point of this manuscript resided in the design of extraction as well as the weak cytotoxicity displayed. The theme matched pretty well with the journal of processes. Therefore, as far as I am concerned, this manuscript can be assigned as major revision.

 

Two major questions and concerns were listed as below.

 

1. For the 3X3 orthogonal array assisted experiment design, the highest yield is 3.1%, while there are several pretty closed values, such as 2.8%, 2.9%. I just wonder if any duplication was made to confirm the significance of different. If the standard deviation is 0.5% or higher, the extraction condition may not affect the final yield at all. To me, whether group 5 in table 2 is the best condition may need to be verified.

 

2. For the bioactivity assay, especially the cytotoxicity study, can we use the IC50 values for the comparison use? In addition, was there any positive control, for example, other known cytotoxic polysaccharide can be used for another comparison use? Without this information, it might be difficult to evaluate the potency of the isolated.

Author Response

Response to Reviewer 2 Comments

 

Comments and Suggestions for Authors

This manuscript reported the purification, characterization of polysaccharide extracted and isolated from Talinum triangulare. During the extraction and isolation, 3X3 orthogonal array was used to assist the experiment design, as a result, the best process condition was optimized. In addition, the resulting mixture was undergoing the chemical analysis as well as the bioactivity assessment. Overall, the selling point of this manuscript resided in the design of extraction as well as the weak cytotoxicity displayed. The theme matched pretty well with the journal of processes. Therefore, as far as I am concerned, this manuscript can be assigned as major revision.

 

Two major questions and concerns were listed as below.

 

Point 1: For the 3X3 orthogonal array assisted experiment design, the highest yield is 3.1%, while there are several pretty closed values, such as 2.8%, 2.9%. I just wonder if any duplication was made to confirm the significance of different. If the standard deviation is 0.5% or higher, the extraction condition may not affect the final yield at all. To me, whether group 5 in table 2 is the best condition may need to be verified.

 

Response 1: Thank for your valuable feedback. We have double checked our raw data of yields of polysaccharides in both of the Tables 1 and 2, and we found that we have done these experiment three times. Therefore, we have presented the values of yields of polysaccharides to the second decimal place as mean ± standard deviation (SD) in both of the Tables 1 and 2 (line 204, 220). From our data, the highest SD is 0.12% for the HRE method and 0.11% for the UAE method. Therefore, the group 5 in Table 2 is still the best condition for extraction of polysaccharides from T. triangulare.

 

Point 2: For the bioactivity assay, especially the cytotoxicity study, can we use the IC50 values for the comparison use? In addition, was there any positive control, for example, other known cytotoxic polysaccharide can be used for another comparison use? Without this information, it might be difficult to evaluate the potency of the isolated.

 

Response 2: Thank for your valuable suggestions. For cytotoxicity assay, we agree your point of view to use the IC50 values for the comparison of bioactivity. However, fractions of LTTP were not directly added into the media of cancer cells, but they treated macrophages to obtain CMs then indirectly inhibited proliferations of cancer cells (sections 2.4.2 and 2.8 in Materials and Methods). We can’t obtain the IC50 values of different fractions of LTTP for cancer cells. Therefore, we compared the cytotoxic effects of different fractions of LTTP through inhibitory effects of CMs for cancer cells in section 3.8 of Results and Discussions.

The positive control of cytotoxic polysaccharide in this study is the lipopolysaccharides (LPS). We used 1 μg/mL LPS as controls in results of Figures 1, 3 and 4. Therefore, we can evaluate the potency of LTTP fractions in section 3.8 of Results and Discussions.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The author has addressed my concerns, and the manuscript can be accepted in present form now.

Author Response

Response to Reviewer 2 Comment

 

Comments and Suggestions for Authors

The author has addressed my concerns, and the manuscript can be accepted in present form now.

Response: Thank for your valuable comment.

Author Response File: Author Response.docx