Bioresource Utilization of Djulis (Chenopodium formosanum) Biomass as Natural Antioxidants

Round 1

Reviewer 1 Report

The authors have improved the manuscript 

Author Response

We appreciate the reviewer comments very much and have revised the manuscript accordingly. The specific changes we have made in the revised manuscript are, as follows:

Point 1: The authors have improved the manuscript

Response 1: Thanks for the positive comments.

Reviewer 2 Report

This study presents the antiradical activity of extracts prepared from hulls and roots of red and yellow djulis plant using several in vitro methods. Several methods for antioxidant activity were employed as well as the determination of total polyphenols, flavonoids and sugar content.

However, some minor changes should be performed prior to publication.

1. I think that before comparing the extracts regarding their components it should be added a sentence explaining the abbreviation used (before the first time you used it not after you used it already for several times). For example, were investigated regarding their total sugar content an ethanolic (RE) and a water extract (RW) of djulis roots… (page 2-line 72)
2. You should not mention “using glucose as calibration standard” in Figure 1 legend as it can be easily observed from the y axis (page 3-line 91-92).
3. You should replace “significant different” with significant difference (page 3-line 97, page 4-line 118, page 5-line 140, page 8-line 206)
4. I don’t understand how the antioxidant effect was doubled from 56.13% to 77.54%, maybe you should change it to “increased” (page 5-line 143).
5. You should remove the articles in the case of “the chlorogenic acid” to “chlorogenic acid”, “the rutin” to “rutin” etc. (page 6-line 168).
6. You should change “to stable” into “stabilize” (page 7-line 192)
7. Figures 7a and b should be place in the same line with a single legend for both (same for figure 8 a and b)

“Figure 7a. DPPH free radical scavenging activities of djulis hull and roots samples and b - EC50 of DPPH free radical scavenging activities of djulis hull and roots samples. Data are expressed as mean ± SD (n=3). Different letters above the bars indicates a significant difference (p<0.05)” (page 9-Line 214-215 and 217-219).

8. I would change “concluded” to “depicted” and “constantly topped” to “presented high amounts of “ (page 10- line 240, line 241)
9. I would change “with” to “from” (page 10-line 247)
10. Also, I would remove the word “the” before introducing, in figure legends, antioxidant activity assays names.
11. All references should be revised and cited uniformly accordingly to the journal conditions mentioned in Instructions for authors.

Author Response

Response to Reviewer 2 Comments

We appreciate the reviewer comments very much and have revised the manuscript accordingly. The specific changes we have made in the revised manuscript are, as follows:

Point 1: I think that before comparing the extracts regarding their components it should be added a sentence explaining the abbreviation used (before the first time you used it not after you used it already for several times). For example, were investigated regarding their total sugar content an ethanolic (RE) and a water extract (RW) of djulis roots… (page 2-line 72)

Response 1: Thank you for the positive comment and evaluation. A sentence explaining the abbreviation used had been added at page-2 line 72. The sentence has been re-written and redraft. All changes are marked in yellow background in the revised manuscript.

Point 2: You should not mention “using glucose as calibration standard” in Figure 1 legend as it can be easily observed from the y axis (page 3-line 91-92).

Response 2: Thanks for the comment. The sentence has been re-written and redraft from “Total sugar content of djulis hull and roots samples by using glucose as a calibration standard” to “Total sugar content of djulis hull and roots samples” in Figure 1. All changes are marked in yellow background in the revised manuscript.

Point 3: You should replace “significant different” with significant difference (page 3-line 97, page 4-line 118, page 5-line 140, page 8-line 206)

Response 3: Thanks for the positive evaluation. The word “significant different” had been replaced with “significant difference” in the revised manuscript. All changes are marked in yellow background in the revised manuscript.

Point 4: I don’t understand how the antioxidant effect was doubled from 56.13% to 77.54%, maybe you should change it to “increased” (page 5-line 143).

Response 4: Thank you for the comment. The sentence, “the scavenging effect was doubled from 56.13% to 77.54%” had been rephrased and redraft to “the scavenging effect was increased from 56.13% to 77.54%”. All changes are marked in yellow background in the revised manuscript.

Point 5: You should remove the articles in the case of “the chlorogenic acid” to “chlorogenic acid”, “the rutin” to “rutin” etc. (page 6-line 168).

Response 5: Thanks for the positive comment. The sentence has been re-written and redraft from “the major constituents of polyphenols in djulis was the rutin, followed by the chlorogenic acid and catechin” to “the major constituents of polyphenols in djulis was rutin, followed by chlorogenic acid and catechin” in the Section 2.5. All changes are marked in yellow background in the revised manuscript.

Point 6: You should change “to stable” into “stabilize” (page 7-line 192)

Response 6: Thank you for the comment. The words “to stable” had been changed into “stabilize” in the Section 2.6. All changes are marked in yellow background in the revised manuscript.

Point 7: Figures 7a and b should be place in the same line with a single legend for both (same for figure 8 a and b)

“Figure 7a. DPPH free radical scavenging activities of djulis hull and roots samples and b - EC50 of DPPH free radical scavenging activities of djulis hull and roots samples. Data are expressed as mean ± SD (n=3). Different letters above the bars indicates a significant difference (p<0.05)” (page 9-Line 214-215 and 217-219).

Response 7: Thanks for the positive evaluation. Figures 7a and b, and Figures 8a and b had been placed in the same line with a single legend for both. All changes are marked in yellow background in the revised manuscript.

Point 8: I would change “concluded” to “depicted” and “constantly topped” to “presented high amounts of “(page 10- line 240, line 241)

Response 8: We appreciate for the correction mentioned. The sentence has been re-written and redraft from “Concluded from the antioxidant activities analysis, ethanolic extracted red colored dujulis hull (RE), and water extracted red colored djulis hull (RW) constantly topped for the total sugar content, total phenolic content, total flavonoid content, and the antioxidant activities tests” to “Depicted from the antioxidant activities analysis, ethanolic extracted red colored dujulis hull (RE), and water extracted red colored djulis hull (RW) presented high amounts of the total sugar content, total phenolic content, total flavonoid content, and the antioxidant activities tests” in the Section 2.8. All changes are marked in yellow background in the revised manuscript.

Point 9: I would change “with” to “from” (page 10-line 247)

Response 9: Thanks for the comment. The word “with” had been replaced with “from” in the Section 2.8. All changes are marked in yellow background in the revised manuscript.

Point 10: Also, I would remove the word “the” before introducing, in figure legends, antioxidant activity assays names.

Response 10: Thanks for the positive appraisal. The word “the” before introducing, in figure legends, antioxidant activity assays names had been removed. Furthermore, some grammatical errors, verb tense, singular/plural, and improper words had been modified and revised. All changes are marked in yellow background in the revised manuscript.

Point 11: All references should be revised and cited uniformly accordingly to the journal conditions mentioned in Instructions for authors.

Response 11: Thank you very much for the positive comment and suggestions. All references were revised and cited uniformly accordingly to the journal conditions mentioned in Instructions for authors.

Author Response File: Author Response.docx

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

The study presents the antioxidant activity of six extracts either ethanolic or water from hulls or roots of red and yellow djulis plant determined using several in vitro methods such as DPPH, ABTS, FRAP, CUPRAC and DMPD as well as investigations regarding their total polyphenols, flavonoid and sugar content.

The paper presents novelty considering that in the literature were compared before only some in vitro method and in this study were applied the most well-known radical scavenger activity methods however the article requires some revisions that need to be addressed.

1) Page 1-line 2-3

    I would suggest the title to be changed into ”In vitro assays of antioxidant activity of Djulis extracts (Chenopodium formosanum) from biomass as potential natural antioxidants” in order to better describe the content of the paper and emphasize the several methods used for radical scavenger activity determination.

2) Page 2-Abstract (line 30-33)

I would not mention the abbreviation of methods name in the abstract, considering that afterwards it is a section dedicated denoted abbreviations.

3) Page 1-Keywords (line 39-40)

“Key words” should be change into “Keywords”.

4) Page 3-Introduction 1 (line 66-67)

I would suggest rephrasing the paragraph:” Recently, djulis has attracted many researchers interests because of its intense color, functional and nutritional composition, as well as for its bioactive components”.

5) Page 4-Introduction (line 73-76)

I would suggest rephrasing the sentence, from the one presented in the manuscript you cannot understand that it is about reducing the occurring of different types of cancer.

6) Page 4-Introduction (line 83-88)

I would recommend that if you identified several studies regarding djulis hull antioxidant activity to mention the results that they obtained and what methods were used for the determination and cite their research properly. Otherwise, from your sentence it is understood that there was no novelty of your research and defy the scope of publishing original articles.

7) Page 5-2.1 Total sugar content (line 101-103)

It should be rephrased as “Total sugar content of djulis hull and roots ethanolic and water extracts was presented in Figure 1. The highest amount of total sugar was for RE…”

Also, in the material and methods you should present the calibration curve used (y=ax+b, R²=…) for quantification and in text mentioned as mg SUGAR NAME/ g extract or g plant depending on your data. This way is easier for anyone reading your article to be able to compare the results.

8) Page 6 – Figure 1 (Line 121)

I would think that in the graph should be mentioned also the sugar used as calibration standard mg sugar/ g extract or g D.W. etc.

Also, I would suggest to recheck your results used to be represented as graph because I find it difficult to understand how the ethanolic extracts total sugar content is higher than in the case of water extracts taking into account the fact the sugars solubility in water is significantly higher. Also, these results are consistent with other studies?

9) Page 7– (Line 130-141)

I saw that you compared your results with a study reported by Sun et al., 2019. It would be nice to also present their results in comparison with the ones from your manuscript and also if there are consistent with your results, or higher or lower amounts.

10) Page 8-Total flavonoid content (Line 146-150)

I would suggest that the instead of mg/g units to explain better as mg QE/ g extract or g DW because there are important differences between units, and it is important to be understood clearly. (QE-quercetin equivalent). Use the modified units in the graph (Fig. 3) also.

If the results are similar to other studies, I would like to see in the manuscript also their results: for example: “are similar to the results reported by x and y in the range of a-b mg QE/g DW”.

11) Page 10-Trolox equivalent antioxidant capacity (Line 168-189)

It would be best if you were able to express all extracts antioxidant activity (TEAC) as Trolox equivalents (using a calibration curve using Trolox), this way it would be easier to compare your results with others that used a slightly different procedure. Also, take into account that TEAC is a method that has a linearity domain (at low concentration) and afterward no matter how much you increase extract concentration the ABTS+ ^.  will be completely degraded, easily seen by the fact that the solution it is no longer colored. Therefore, you should maybe rewrite the discussion of results obtained for your extracts.

12) Page 11-Ferric reducing antioxidant power (Line 192-211)

It would be best to express the FRAP activity of extracts as ascorbic acid equivalents.

13) Page 13-CUPRAC (Line 218-234)

It would be best to express the CUPRAC activity of extracts as ascorbic acid equivalents and in the discussion section to compare the results obtained by several methods for antioxidant activity determination.

14) Page 15-DPPH (Fig. 7).

I would suggest that in the legend the extracts be put in the same order as the curves to be easier to see the results. Also, you could express your results as ascorbic acid equivalents (maybe create a Table with all of the antioxidant activity results expressed as the same standard substance and then compare them). Figure 7 and 8 should become A and B.

15)Page 16-18-DMPD (Fig. 9-Fig 10)

You could express your results as ascorbic acid equivalents. Figure 9 and 10 should become A and B.

16) Page 19 – Conclusions (Line 311)

This section should be improved with adding data expressed as ascorbic acid…for example: RE and RW extracts exhibited high amount of total sugar (ranging from… to…)…etc.

17) Page 20-Experimental should be named Materials and Methods

The authors didn’t present which materials they used.

General mentions:

1. All references should be cited according to the recommendation from the journal. In the text, reference numbers should be placed in square brackets [ ], and placed before the punctuation; for example [1], [1–3] or [1,3]. Also, references should be numbered accordingly to the place they appear in text [1], [2], [3] etc.

For example:

Journal Articles:
1. Author 1, A.B.; Author 2, C.D. Title of the article. Abbreviated Journal Name Year, Volume, page range.

“Abderrahim, F.; Huanatico, E., Segura, R.; Arribas, S.; Gonzalez, M. C.; Condezo-Hoyos, L. Physical features, phenolic compounds, betalains and total antioxidant capacity of colored quinoa seeds (Chenopodium quinoa Willd.) from Peruvian Altiplano. Food Chem. 2015, 183, 83-90.”

And in text: “Djulis (Chenopodium formosanum Koidz.) …. to Taiwan (Bhargava et al., 2006)” should be changed to “Djulis (Chenopodium formosanum Koidz.) …. to Taiwan [1]”.

2.The article does not respect the journal format: Front mater: 1. Introduction; 2. Materials and methods; 3. Results; 4. Discussion (3 and 4 could be combined) 5. Conclusions and Back matter: Acknowledgements, Author contribution (see Instructions for Authors from the journal page), Conflict of interests.

3. You should only mention the abbreviation of a method name the first time that you used it and afterwards used it. It is highly unnecessary to mention it every time.
4. To improve the discussion or the introduction section you can refer to the following research articles:

 Lin, Y.; Lin, Y.; Chan, S.; Chun, Y.; Lin, Y.; Kan, K.; Yu, C. Anti Cutaneous Aging Effect of Red Djulis (Chenopodium formosanum) Extract on Gene Expression of Human Dermal Fibroblast. Preprints 2019, 2019090028. doi: 10.20944/preprints201909.0028.v3

Lee, C.-W., Chen, H.-J., Xie, G.-R., & Shih, C.-K. (2019). Djulis (Chenopodium Formosanum) Prevents Colon Carcinogenesis via Regulating Antioxidative and Apoptotic Pathways in Rats. Nutrients, 11(9), 2168. doi:10.3390/nu11092168 

Chyau, C.-C., Chu, C.-C., Chen, S.-Y., & Duh, P.-D. (2018). The Inhibitory Effects of Djulis (Chenopodium formosanum) and Its Bioactive Compounds on Adipogenesis in 3T3-L1 Adipocytes. Molecules, 23(7), 1780. doi:10.3390/molecules23071780 

5. Also, I think that the whole manuscript should be revised concerning English corrections.

Author Response

Response to Reviewer 1 Comments

We appreciate the reviewer comments very much and have revised the manuscript accordingly. The specific changes we have made in the revised manuscript are, as follows:

1) Page 1-line 2-3

I would suggest the title to be changed into “In vitro assays of antioxidant activity of Djulis extracts (Chenopodium formosanum) from biomass as potential natural antioxidants” in order to better describe the content of the paper and emphasize the several methods used for radical scavenger activity determination.

Response 1: Thank you for the positive comment and evaluation. After referring to related literature (Brahim et al., 2013; Fidrianny et al., 2015; Pandey et al., 2008; Zhu et al., 2019), we conserved the title as “Bioresource Utilization of Djulis (Chenopodium formosanum) Biomass as Natural Antioxidants” to deliverance and present the main focus of this study: fully utilization of the djulis hulls and roots biomass as eco-friendly antioxidant additives for goods or commodities of bio-products, which will not harmful to the environment

2) Page 2-Abstract (line 30-33)

I would not mention the abbreviation of methods name in the abstract, considering that afterwards it is a section dedicated denoted abbreviations.

Response 2: Thank you for the comment. The sentence has been re-written and redraft. All changes are marked in yellow background in the revised manuscript.

3) Page 1-Keywords (line 39-40)

“Key words” should be change into “Keywords”.

Response 3: The word “Key words” had changed into “Keywords”. The changes are marked in yellow background in the revised manuscript.

4) Page 3-Introduction 1 (line 66-67)

I would suggest rephrasing the paragraph: “Recently, djulis has attracted many researchers interests because of its intense color, functional and nutritional composition, as well as for its bioactive components”.

Response 4: Thank you for the comment and positive evaluation. The sentence has been re-written and redraft from “In recent years, djulis has fascinated by many investigators because of its gorgeous color, functional and nutritional composition, and bioactive constituents” to “Recently, djulis has attracted many researchers interests because of its intense color, functional and nutritional composition, as well as for its bioactive components” in the Section of Introduction. All changes are marked in yellow background in the revised manuscript.

5) Page 4-Introduction (line 73-76)

I would suggest rephrasing the sentence, from the one presented in the manuscript you cannot understand that it is about reducing the occurring of different types of cancer.

Response 5: Thanks for the positive evaluation. The sentence had been re-written and redraft at the Section of Introduction. All changes are marked in yellow background in the revised manuscript.

6) Page 4-Introduction (line 83-88)

I would recommend that if you identified several studies regarding djulis hull antioxidant activity to mention the results that they obtained and what methods were used for the determination and cite their research properly. Otherwise, from your sentence it is understood that there was no novelty of your research and defy the scope of publishing original articles.

Response 6: Thanks for the comment. Several novel studies regarding the antioxidant activity, the results that they obtained, and methods were used for the determination of djulis hull had been mentioned in the Introduction section. All changes are marked in yellow background in the revised manuscript.

7) Page 5-2.1 Total sugar content (line 101-103)

It should be rephrased as “Total sugar content of djulis hull and roots ethanolic and water extracts was presented in Figure 1. The highest amount of total sugar was for RE…”

Also, in the material and methods you should present the calibration curve used (y=ax+b, R2=…) for quantification and in text mentioned as mg SUGAR NAME/ g extract or g plant depending on your data. This way is easier for anyone reading your article to be able to compare the results.

Response 7: Thank you for the comment. The sentence had been rephrased from “Total sugar content of djulis hull and roots which extracted by ethanol and water was showed in Figure 1. RE with 794.7±4.18 mg/g was top for the total sugar content as compared with the other samples.” to “Total sugar content of djulis hull and roots ethanolic and water extracts was presented in Figure 1. The highest amount of total sugar was for RE…”. Furthermore, the calibration curve used (y=0.1522x–0.17, R²=0.99) for quantification was mentioned in the section 4.3. Total sugar content was expressed as mg GLU g^-1 D.W. All changes are marked in yellow background in the revised manuscript.

8) Page 6 – Figure 1 (Line 121)

I would think that in the graph should be mentioned also the sugar used as calibration standard mg sugar/ g extract or g D.W. etc.

Also, I would suggest to recheck your results used to be represented as graph because I find it difficult to understand how the ethanolic extracts total sugar content is higher than in the case of water extracts taking into account the fact the sugars solubility in water is significantly higher. Also, these results are consistent with other studies?

Response 8: In the graph of Figure 1, the sugar used as calibration standard (mg GLU g^-1 D.W.) was mentioned. In the meantime, the betacyanin pattern of djulis (Chenopodium fromosanum), a native cereal plant in Taiwan, was reported by Tsai et al. (2010). Betalains are water-soluble nitrogen-containing vacuolar pigments, consisting of the red to red-violet betacyanins and the yellow-orange betaxanthins. They are immonium conjugates of betalamic acid with cyclo-Dopa (cyclo-3,4-dihydroxyphenylalanine) and amino compounds (amino acids, amines, or derivatives), respectively. Examples are betanin (betanidin-5-O-β-glucoside) of red beet and indicaxanthin (betalamic acid linked to proline) of cactus pear, the first structurally characterized betalains. Glycosylation and acylation result in a diversity of betacyanin structures. The betanidin aglycone is usually linked with glucose, occasionally with glucuronic acid, sophorose, rhamnose, and apiose, at the C-5 or C-6 position. Further modification occurs by aliphatic or aromatic acid esterification of the sugar moiety. Hence, we hypothesis that alcohol extraction may increase the isolation of the biological active compounds which link by glycoside linkage. Causing the ethanolic extracts total sugar content is higher than in the case of water extracts taking into account the fact the sugars solubility in water is significantly higher.

9) Page 7– (Line 130-141)

I saw that you compared your results with a study reported by Sun et al., 2019. It would be nice to also present their results in comparison with the ones from your manuscript and also if there are consistent with your results, or higher or lower amounts.

Response 9: Thank you for the comment. The results with a study reported by Sun et al., 2019 was presented in comparison with the ones from the manuscript. All changes are marked in yellow background in the revised manuscript.

10) Page 8-Total flavonoid content (Line 146-150)

I would suggest that the instead of mg/g units to explain better as mg QE/ g extract or g DW because there are important differences between units, and it is important to be understood clearly. (QE-quercetin equivalent). Use the modified units in the graph (Fig. 3) also.

If the results are similar to other studies, I would like to see in the manuscript also their results: for example: “are similar to the results reported by x and y in the range of a-b mg QE/g DW”.

Response 10: Thanks for the positive appraisal.  Total flavonoid content was expressed as mg QE g^-1 D.W. Moreover, the results with a study reported by Sun et al., 2019 was presented in comparison with the ones from the manuscript. All changes are marked in yellow background in the revised manuscript.

11) Page 10-Trolox equivalent antioxidant capacity (Line 168-189)

It would be best if you were able to express all extracts antioxidant activity (TEAC) as Trolox equivalents (using a calibration curve using Trolox), this way it would be easier to compare your results with others that used a slightly different procedure. Also, take into account that TEAC is a method that has a linearity domain (at low concentration) and afterward no matter how much you increase extract concentration the ABTS+ .  will be completely degraded, easily seen by the fact that the solution it is no longer colored. Therefore, you should maybe rewrite the discussion of results obtained for your extracts.

Response 11: Thank you for the positive comment. After referring to related literature (Babu et al., 2013; Karadag et al., 2009; Magalhaes et al., 2008; Moukett et al., 2015; Schlesier et al., 2002; Shahidi and Zhong, 2015), we conserved with TEAC quantification and antioxidant capacity expression.

12) Page 11-Ferric reducing antioxidant power (Line 192-211)

It would be best to express the FRAP activity of extracts as ascorbic acid equivalents.

Response 12: Thank you for the positive comment. After referring to related literature (Babu et al., 2013; Karadag et al., 2009; Magalhaes et al., 2008; Moukett et al., 2015; Schlesier et al., 2002; Shahidi and Zhong, 2015), we maintained with FRAP quantification and antioxidant capacity expression.

13) Page 13-CUPRAC (Line 218-234)

It would be best to express the CUPRAC activity of extracts as ascorbic acid equivalents and in the discussion section to compare the results obtained by several methods for antioxidant activity determination.

Response 13: Thank you for the positive comment. After referring to related literature (Babu et al., 2013; Karadag et al., 2009; Magalhaes et al., 2008; Moukett et al., 2015; Schlesier et al., 2002; Shahidi and Zhong, 2015; Sharma et al., 2011), we conserved with CUPRAC quantification and antioxidant capacity expression.

14) Page 15-DPPH (Fig. 7).

I would suggest that in the legend the extracts be put in the same order as the curves to be easier to see the results. Also, you could express your results as ascorbic acid equivalents (maybe create a Table with all of the antioxidant activity results expressed as the same standard substance and then compare them). Figure 7 and 8 should become A and B.

Response 14: Thank you for the positive comment. After referring to related literature (Babu et al., 2013; Karadag et al., 2009; Magalhaes et al., 2008; Moukett et al., 2015; Schlesier et al., 2002; Shahidi and Zhong, 2015; Sharma et al., 2011), we conserved with DPPH quantification and antioxidant capacity expression. Furthermore, Figure 7 and 8 had become Figure 7A and Figure 7B.

15)Page 16-18-DMPD (Fig. 9-Fig 10)

You could express your results as ascorbic acid equivalents. Figure 9 and 10 should become A and B.

Response 15: Thank you for the positive comment. After referring to related literature (Babu et al., 2013; Karadag et al., 2009; Magalhaes et al., 2008; Moukett et al., 2015; Schlesier et al., 2002; Shahidi and Zhong, 2015; Sharma et al., 2011), we conserved with DMPD quantification and antioxidant capacity expression. Moreover, Figure 9 and 9 had become Figure 8A and Figure 8B.

16) Page 19 – Conclusions (Line 311)

This section should be improved with adding data expressed as ascorbic acid…for example: RE and RW extracts exhibited high amount of total sugar (ranging from… to…)…etc.

Response 16: Thanks for the comment. The conclusion had been re-write. All changes are marked in yellow background in the revised manuscript.

17) Page 20-Experimental should be named Materials and Methods

The authors didn’t present which materials they used.

Response 17: Thank you for the positive evaluation. The Experimental had been named as Materials and Methods. In the section of Materials and Methods, the materials used, treatments, and preparation had been present. All changes are marked in yellow background in the revised manuscript.

References

Babu, D.; Gurumurthy, P.; Borra, S.; Cherian, K. M.; Antioxidant and free radical scavenging activity of triphala determined by using different in vitro models. J Med Plants Res., 2013, 7, 2898-2905.

Brahim, H.; Akkal, S.; Christine, B.; Laouer, H.; Dijoux-Franca, M.-G., Evaluation of antioxidant activity, free radical scavenging and cuprac of two compounds isolated from Scorzonera undulata ssp deliciosa. Adv. Environ. Biol., 2013, 7, 591-594.

Fidrianny, I.; Budiana, W.; Ruslan, K.; Antioxidant activities of various extracts from ardisia sp leaves using dpph and cuprac assays and correlation with total flavonoid, phenolic, carotenoid content. Int. J. Pharmacogn. Phytochem., 2015, 7.

Karadag, A.; Ozcelik, B.; Saner, S.; Review of Methods to Determine Antioxidant Capacities. Food Anal. Meth., 2009, 2, 41-60.

Magalhaes, L. M.; Segundo, M. A.; Reis, S.; Lima, J. L.; Methodological aspects about in vitro evaluation of antioxidant properties. Analytica chimica acta., 2008, 613 (1), 1-19.

Moukette, B. M.; Pieme, C. A.; Njimou, J. R.; Biapa, C. P. N.; Marco, B.; Ngogang, J. Y.; In vitro antioxidant properties, free radicals scavenging activities of extracts and polyphenol composition of a non-timber forest product used as spice: Monodora myristica. Biol Res., 2015, 48 (1), 15-15.

Pandey, U.; Pandey, J.; Enhanced production of biomass, pigments and antioxidant capacity of a nutritionally important cyanobacterium Nostochopsis lobatus. Bioresour. Technol., 2008, 99 (10), 4520-4523.

Shahidi, F.; Zhong, Y.; Measurement of antioxidant activity. J. Funct. Food., 2015, 18, 757-781.

Sharma, U. S.; Kumar, A.; In vitro antioxidant activity of Rubus ellipticus fruits. J Adv Pharm Technol Res., 2011, 2 (1), 47-50.

Tsai, P. J.; Sheu, C. H.; Wu, P. H.; Sun, Y. F.; Thermal and pH stability of betacyanin pigment of Djulis (Chenopodium formosanum) in Taiwan and their relation to antioxidant activity. J Agric Food Chem., 2010, 58 (2), 1020-5.

Zhu, M.; Huang, Y.; Wang, Y.; Shi, T.; Zhang, L.; Chen, Y.; Xie, M.; Comparison of (poly)phenolic compounds and antioxidant properties of pomace extracts from kiwi and grape juice. Food Chem., 2019, 271, 425-432.

General mentions:

All references should be cited according to the recommendation from the journal. In the text, reference numbers should be placed in square brackets [ ], and placed before the punctuation; for example [1], [1–3] or [1,3]. Also, references should be numbered accordingly to the place they appear in text [1], [2], [3] etc.

For example:

Journal Articles:

1. Author 1, A.B.; Author 2, C.D. Title of the article. Abbreviated Journal Name Year, Volume, page range.

“Abderrahim, F.; Huanatico, E., Segura, R.; Arribas, S.; Gonzalez, M. C.; Condezo-Hoyos, L. Physical features, phenolic compounds, betalains and total antioxidant capacity of colored quinoa seeds (Chenopodium quinoa Willd.) from Peruvian Altiplano. Food Chem. 2015, 183, 83-90.”

And in text: “Djulis (Chenopodium formosanum Koidz.) …. to Taiwan (Bhargava et al., 2006)” should be changed to “Djulis (Chenopodium formosanum Koidz.) …. to Taiwan [1]”.

Response 1: Thank you for the positive evaluation. The format of the references had been cited, re-write and re-correct according to the Author guideline which recommended from the journal.

2.The article does not respect the journal format: Front mater: 1. Introduction; 2. Materials and methods; 3. Results; 4. Discussion (3 and 4 could be combined) 5. Conclusions and Back matter: Acknowledgements, Author contribution (see Instructions for Authors from the journal page), Conflict of interests.

Response 2: Thank you for the comment. The format of the article had been re-write and re-correct according to the Author guideline which recommended from the journal.

3. You should only mention the abbreviation of a method name the first time that you used it and afterwards used it. It is highly unnecessary to mention it every time.

Response 3: Thanks for the positive comment. Appropriate abbreviation used in the study had been re-write and re-correct. All changes are marked in yellow background in the revised manuscript.

4. To improve the discussion or the introduction section you can refer to the following research articles:

 Lin, Y.; Lin, Y.; Chan, S.; Chun, Y.; Lin, Y.; Kan, K.; Yu, C. Anti Cutaneous Aging Effect of Red Djulis (Chenopodium formosanum) Extract on Gene Expression of Human Dermal Fibroblast. Preprints 2019, 2019090028. doi: 10.20944/preprints201909.0028.v3

Lee, C.-W., Chen, H.-J., Xie, G.-R., & Shih, C.-K. (2019). Djulis (Chenopodium Formosanum) Prevents Colon Carcinogenesis via Regulating Antioxidative and Apoptotic Pathways in Rats. Nutrients, 11(9), 2168. doi:10.3390/nu11092168

Chyau, C.-C., Chu, C.-C., Chen, S.-Y., & Duh, P.-D. (2018). The Inhibitory Effects of Djulis (Chenopodium formosanum) and Its Bioactive Compounds on Adipogenesis in 3T3-L1 Adipocytes. Molecules, 23(7), 1780. doi:10.3390/molecules23071780

Response 4: Thank you for the positive suggestion. Appropriate references had been added. All changes are marked in yellow background in the revised manuscript.

Also, I think that the whole manuscript should be revised concerning English corrections.

Response 5: Thank you very much for the comment. Some grammatical errors, verb tense, singular/plural, and improper words had been modified and revised. Moreover, this paper has been edited by a native English speaker prior to submission.

Author Response File: Author Response.pdf

Reviewer 2 Report

The present paper is interesting because it reports the antiradical effects of Chenopodium formosanum. Authors demonstrated the different biological properties and composition of the several plant extracts they produced. However, some modifications are required:

a) in the title, the name of the species should be written NOT in capital

b) the patronymic of the species should be reported in the text

c) english form should be revised by a mother tonuge Professor

d) statistics in absent, are these results significant?

e) the authors should better compare their results wiht those reported in literature, even if made on other plants, to give them more value and emphasis. I suggest to make some comments and compare their data with those presented in the following recent works: Adv. Environ. Biol, 2013, 7, 591-594; Pharmaceutical biology, 2017, 55(1), 324-329; Medicinal Chemistry Research, 2019, 28(5): 754–767; Journal of Chromatography B, 2015 995, 101-106; Plant Biosystems, 2019, 153(6), 843-852; IJPPR, 2015, 7(4), 859-865; 

Author Response

Response to Reviewer 2 Comments

We appreciate the reviewer comments very much and have revised the manuscript accordingly. The specific changes we have made in the revised manuscript are, as follows:

1) in the title, the name of the species should be written NOT in capital

Response 1: We appreciate for the correction mentioned. The name of the species in the title had been re-write. The changes are marked in yellow background in the revised manuscript.

2) the patronymic of the species should be reported in the text

Response 2: The patronymic or taxonomy tree of the species was reported in the section of Introduction. All changes are marked in yellow background in the revised manuscript.

3) English form should be revised by a mother tongue Professor

Response 3: Thank you very much for the comment. Some grammatical errors, verb tense, singular/plural, and improper words had been modified and revised. Moreover, this paper has been edited by a native English speaker prior to submission.

4) statistics in absent, are these results significant?

Response 4: Thanks for the positive evaluation. The Section of Statistical analysis had been added in the Materials and Methods. The statistical analysis results had also presented on the Section of Results and Discussion, and in the legend of all figures. All changes are marked in yellow background in the revised manuscript.

5) the authors should better compare their results with those reported in literature, even if made on other plants, to give them more value and emphasis. I suggest to make some comments and compare their data with those presented in the following recent works:

Adv. Environ. Biol, 2013, 7, 591-594; Pharmaceutical biology, 2017, 55(1), 324-329; Medicinal Chemistry Research, 2019, 28(5): 754–767; Journal of Chromatography B, 2015 995, 101-106; Plant Biosystems, 2019, 153(6), 843-852; IJPPR, 2015, 7(4), 859-865;

Response 5: Thank you very much for the positive comment and suggestions. The results of the study had been re-written and redraft with the appropriate discussion based on the observed results in the study. Furthermore, the results have been compared with appropriate recent works. All changes are marked in yellow background in the revised manuscript.

Author Response File: Author Response.pdf

Reviewer 3 Report

Dear authors,

I read the manuscript with interest; however I think you have to improve introduction, discussion and the references. 

In my opinion it is very important report that food by-products are rich in valuable compounds such as sugars and polyphenolic compounds that can find useful application in cosmetic and nutraceutical fields

Di Mauro et al., Antioxidants 2019, 8(10), 462; https://doi.org/10.3390/antiox8100462

-line 29 were determined

-line 30 was assessed

-Please delete Bhargava et al., 2006 and add the reference Lee et al., Nutrients, 2019 doi.org/10.3390/nu11092168

-lines 62-64 Please delete this sentence 

-lines 64-65 please modify as follow: Djulis is subdivided into two kinds by color: kapulapula (yellow-colored djulis) and odidile (red-colored djulis). 

-lines 66 please modify as follow Recently, djulis has attracted the attention of many researchers

-lines 80 please add the reference Fava et al., Clean, 2017 doi.org/10.1002/clen.201600042

-line 102 please check was showed

-line 118 please discuss that these sugars can find useful application in cosmetic field (Di Mauro et al., Food Funct. 2017, 8(12):4713-4722. doi: 10.1039/c7fo01363a.)

-lines 139-141 please modify as follow: All these compounds, well-known for their healthy effects, due to their antioxidant, anti-inflammatory, anticancer and antimicrobial properties, are nowadays recognized as valuable molecules in nutraceutical field and in such a context,  djulis represents a challenging bioresource.

-line 141 delete the reference Bandonienė and Murkovic, 2002

-line 161 delete the reference (it is very old!)

-line 164 please add reference Tenuta et al., Antioxidants 2020, 9(2), 184; https://doi.org/10.3390/antiox9020184

-Please add the abbreviations and information related to statistical analysis in the legend of all figures

-Regarding Method section, add a paragraph about statistical analysis

Author Response

Response to Reviewer 3 Comments

We appreciate the reviewer comments very much and have revised the manuscript accordingly. The specific changes we have made in the revised manuscript are, as follows:

1) line 29 were determined

Response 1: Thank you for the comment and positive evaluation. The sentence has been re-written and redraft. All changes are marked in yellow background in the revised manuscript.

2) line 30 was assessed

Response 2: Thanks for the comment. The sentence has been re-written and redraft. All changes are marked in yellow background in the revised manuscript.

3) Please delete Bhargava et al., 2006 and add the reference Lee et al., Nutrients, 2019 doi.org/10.3390/nu11092168

Response 3: Thank you for the positive suggestion. Appropriate references (Lee et al., Nutrients, 2019 doi.org/10.3390/nu11092168; and Lu et al., Foods, 2019 doi. 10.3390/foods8110551.) had been added. All changes are marked in yellow background in the revised manuscript.

4) lines 62-64 Please delete this sentence

Response 4: Thank you for the comment. The sentence had been deleted.

5) lines 64-65 please modify as follow: Djulis is subdivided into two kinds by color: kapulapula (yellow-colored djulis) and odidile (red-colored djulis).

Response 5: Thanks for the comment. The sentence had been rewritten from “The peoples of Paiwan subdivided the djulis into two kinds by color: kapulapula (yellow-colored djulis) and odidile (red-colored djulis)” to “Djulis is subdivided into two kinds by color: kapulapula (yellow-colored djulis) and odidile (red-colored djulis)”.

6) lines 66 please modify as follow Recently, djulis has attracted the attention of many researchers

Response 6: The sentence had been rephrased and rewritten. All changes are marked in yellow background in the revised manuscript.

7) lines 80 please add the reference Fava et al., Clean, 2017 doi.org/10.1002/clen.201600042

Response 7: Thank you for the positive suggestion. The references (Fava et al., Clean, 2017 doi.org/10.1002/clen.201600042) had been added. All changes are marked in yellow background in the revised manuscript.

8) line 102 please check was showed

Response 8: Thank you for the comment and positive evaluation. The sentence has been re-written and redraft. All changes are marked in yellow background in the revised manuscript.

9) line 118 please discuss that these sugars can find useful application in cosmetic field (Di Mauro et al., Food Funct. 2017, 8(12):4713-4722. doi: 10.1039/c7fo01363a.)

Response 9: Thanks for the positive suggestion. The references (Di Mauro et al., Food Funct. 2017, 8(12):4713-4722. doi: 10.1039/c7fo01363a.) had been added in Section 2.1. All changes are marked in yellow background in the revised manuscript.

10) lines 139-141 please modify as follow: All these compounds, well-known for their healthy effects, due to their antioxidant, anti-inflammatory, anticancer and antimicrobial properties, are nowadays recognized as valuable molecules in nutraceutical field and in such a context, djulis represents a challenging bioresource.

Response 10: Thank you for the positive comment. The sentence had been rewrite from “All these phenolic classes play a vital role towards the physiological functions, including free radical scavenging, anti-mutagenic, anti-carcinogenic and anti-inflammatory effects” to “All these compounds, well-known for their healthy effects, due to their antioxidant, anti-inflammatory, anticancer and antimicrobial properties, are nowadays recognized as valuable molecules in nutraceutical field and in such a context, djulis represents a challenging bioresource”.

11) line 141 delete the reference Bandonienė and Murkovic, 2002

Response 11: Thanks for the positive appraisal.  The study reported by Bandonienė and Murkovic, 2002 was deleted and replaced with the other latest study. All changes are marked in yellow background in the revised manuscript.

12) line 161 delete the reference (it is very old!)

Response 12: Thanks for the positive appraisal.  The results with a latest study reported by Sun et al., 2019 was replaced and presented in comparison with the ones from the manuscript (Section 2.3). All changes are marked in yellow background in the revised manuscript.

13) line 164 please add reference Tenuta et al., Antioxidants 2020, 9(2), 184; https://doi.org/10.3390/antiox9020184

Response 13: Thank you for the positive suggestion. The references (Tenuta et al., Antioxidants 2020, 9(2), 184; https://doi.org/10.3390/antiox9020184) had been added. All changes are marked in yellow background in the revised manuscript.

14) Please add the abbreviations and information related to statistical analysis in the legend of all figures

Response 14: Thanks for the positive evaluation. The Section of Statistical analysis had been added in the Materials and Methods. The statistical analysis results had also presented on the Section of Results and Discussion, and in the legend of all figures. All changes are marked in yellow background in the revised manuscript.

15) Regarding Method section, add a paragraph about statistical analysis

Response 15: Thanks for the positive evaluation. The Section of Statistical analysis had been added in the Materials and Methods. The statistical analysis results had also presented on the study. All changes are marked in yellow background in the revised manuscript.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

I consider that the authors made the correction implied in the previous review and the manuscript can now reach the high standards of Sustainability journal.

I think it should be accepted in present form.