Determination of the Antioxidant and Antiproliferative Properties of Pomegranate Peel Extract Obtained by Ultrasound on HCT-116 Colorectal Cancer Cell Line

Round 1

Reviewer 1 Report (New Reviewer)

Overall, the quality of the submitted manuscript is good. The authors used different techniques to evaluate antioxidant and antiproliferative properties of pomegranate peel extract. The experimental design is carefully crafted and the manuscript provided is suitable for publication in the Molecules journal. However, the described phytochemicals are very preliminary. Therefore, it is recommended that the authors use High Performance Liquid Chromatography (HPLC) coupled to UV detectors or a mass spectrometer, thus allowing the identification of compounds present in the extract. The article is solid, concise and well written. This article is acceptable for publication with minor revisions.

Author Response

Thank you for your kind comments. Regarding the identification of the compounds present in extracts, we must restart the extraction in order to realize the HPLC, which requires time. However, further studies are being conducted in which we will be performing extraction, LCMS, anti-inflammatory and antibacterial tests on pomegranate peel extracts.

 

Author Response File: Author Response.docx

Reviewer 2 Report (New Reviewer)

1. The introduction and discussion should be focused more on the observations and novelty of this study and supported with related references.

2. Equations must be written with standard and one uniform.

3. Qualitative images for cytotoxic assays are required.

4. Statistical analysis paragraph must be added to the materials and methods section.

5. Many typos and mistakes were observed. Thus, English language editing is required.

6. More concluding remarks must be also added. 

Author Response

• The introduction and discussion should be focused more on the observations and novelty of this study and supported with related references
• Introduction and discussion were updated and more references were introduced.
• Equations must be written with standard and one uniform.
• Equation are written in standard and one uniform
• Qualitative images for cytotoxic assays are required.
• Qualitative images for cytotoxic effects were added.
• Statistical analysis paragraph must be added to the materials and methods section.
• Statistical analysis paragraph was added in materials and methods section
• Many typos and mistakes were observed. Thus, English language editing is required.
• The mistakes were corrected.
• More concluding remarks must be also added.
• More concluding remarks were added

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report (New Reviewer)

Accept

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 manuscript submitted for review is, in my opinion, of poor quality. Also, the presented topic and the obtained results do not show originality. The manuscript lacks modern methods for the chemical characterization of the obtained extracts, which is difficult to relate to the demonstrated biological activities. No statistical study. I do not recommend it for publication.

Author Response

Statistical study was performed. Unfortunately, we could not apply more methods for the chemical characterization of the extracts obtained at the Lebanese University due to the economic crisis that affected our country, Lebanon. We look forward to expanding our research in our further studies. May we ask you to take this into consideration.

Author Response File: Author Response.docx

Reviewer 2 Report

[I] The title of the manuscript should be rearranged to be more adequate.

“Determination of the antioxidant and antiproliferative properties of ultrasound obtained extracts from pomegranate peel on colorectal cancer HTC-116 cells”. Or something like this, because term “anticancer” is not properly used in this article. Authors determined antiproliferative activity of the extracts in the in vitro experiments (not anticancer activity in vivo).

[II] The extracts were characterized by some reactions for groups of structure and for “anticancer” activity extracts should be well characterized by HPLC or HPLC-MS methods to identify single compounds. As we know that extracts often exerts better effect than single isolated compounds analysis of the extract is ok, however these experiments on cell line are not connected directly with in vivo activity.

In the manuscript we have found term anticancer (see line; 490, 518,title of the section 2.8, and title of the manuscript) – it must be replaced by antiproliferative.

[III] The botanical name of the species used in the study must be given (Punica granatum L.) and there are some varieties used as fruit found in the market (it should be specified if any).

[IV] Previously published work covered the topic of the ultrasound assisted extraction of polyphenols from Pomegranate peel (see ref 7 and 8). As we see in this research (ref. 7 and 8) extraction was optimized and the parameters such as; extraction power, extraction time, extraction solvent used e.g. water, methanol, ethanol were applied, and concentrations of ethanol were tested (from 10 to 90 %), so what was the idea to compare maceration with ultrasound extraction, if many research was done proving the better effectiveness of the extraction assisted by ultrasound, and it is nothing new.

In Ref 7 Authors optimized solvents such as; water, methanol, ethanol, acetone (each at 50, 60, 70, 80 90  and 100 % of the concentration), and amount of the powdered substance extracted by 100 mL of the solvent (it was 2, 3, 5, 6, 8 and 10 g/100 mL), and also extraction temperature.

In Ref 8 Authors optimized power control (20, 60 and 100% and the extraction time 5, 10, 15 minutes, yield values and DPPH, FRAP and IC50 for ROS scavenging ability.

Authors said – see line 422-423; “…the conditions under which ultrasonic extraction must take place were optimized” – only extraction power; 10, 50 and 100 W was tried.

And in the 524-526; “This study give an idea of the efficiency of ultrasound assisted extraction ….need to be to optimize…temperature, solvent, time, …” – this idea was perfectly presented in many references – see examples below.

Some recent references should be used and cited, e.g.;

1)            Zivkovic J. et al. “Optimization of ultrasound-assisted extraction of polyphenolic compounds from pomegranate peel using response surface methodology.” Separation and Purification Technology, 2018; 194, 40-47.  – the UAE with ethanol (10 – 90 %) and the time of extraction  was optimized, TPC calculated, and many more.

2)            Garcia P. et al. “Recovery of bioactive compounds from pomegranate peel (Punica granatum L.) using pressurized liquid extraction” – it will be helpful to construct some discussion about results.

3)            Mo Y., et al. “Pomegranate peel as a source of bioactive compounds – a mini review on their physiological functions.” – Frontiers in nutrition, 2022, doi:10.3389/fnut.2022.887113.

[V] In reviewed manuscript the Discussion is not properly done. Authors should compare their own results with previous studies and highlighted what is different, better, etc. We have no information about previous results.

[VI] “polyphenols and flavonoids” - - flavonoids are polyphenols also. The reported in previously published studies in pomegranate peel groups of polyphenols are; tannins, phenolic acids and flavonoids, anthocyanidins (sometimes considered as the group of the latter). TPC is the method of estimation of the total polyphenols (on basis of their orto-diphenolic groups) so flavonoids are counted in this method also. See this problem line 472, 475,

[VII] Figures 3 and 5 should be corrected because the OY axis scale should start with 0. What Authors present on these Figures suggest big differences in TPC and TFC between extracts, however it does not the big difference at all.

[VIII] The extraction time was the same in maceration and in ultrasound extraction (it was in the each case 35 minutes – why? – nothing about optimization of the time of extraction) – so Authors have not the background to say; (see e.g. lines; 227, 424, 510) that the ultrasonication afforded the biggest yield “in the less time” because the time was in each extraction 35 minutes (not more  and not less). From the other hand 35 minutes for maceration (even with stirring) is a very short time.

[IX] The expensiveness of the UAE is not big as compared with e.g. Soxhlet extraction, however there are methods more effective and in the biggest scale unexpensive and eco-friendly (e.g. supercritical fluid extraction SFE, frequently used to obtain big amount of the plant extracts).                                                

Line; 419; “alternative solvents” – example? Alternative to which solvents?

[X] When the methods are described Authors should use the past time, because it was already done.

[XI] Plant systematic names should be written in italics.- see references; 6,8,10,11,12.

[XII] line 399 citation needed.

[XIII] – Authors used term “denaturation” – it is process depending on several factors in which proteins lose their tertiary, and secondary structure. So in case of polyphenols maybe better is to use “degradation”.

[XIV] In each case when quantitative data are presented, standard deviation should be given (statistical analysis is required). The same in case of calibration curve equations; for gallic acid and rutin (rutoside); the SD of the intercept and the slope of calibration curve are required.

In other hand when equation is presented there is no need to present the calibration curve as a Figure (so Fig 2 and Fig 4 are redundant).

[XV] Line 143; is “rutin curve” should be “rutin calibration curve”

[XVI] In the section Materials and Methods subsection “Reagents and Chemicals” should be inserted, where producers and purity of the reagents and standards should be listed.

[XVII] Line28; “…viability of cells drops…” should be “viability of cells decreased…”

[XVIII] Line 37; should be “…deaths worldwide as the result of CRC [1].”

[XIX] Line 144; should be ; “The calibration curve was prepared using rutin standard.”

Author Response

 [I] The title of the manuscript should be rearranged to be more adequate.

“Determination of the antioxidant and antiproliferative properties of ultrasound obtained extracts from pomegranate peel on colorectal cancer HTC-116 cells”. Or something like this, because term “anticancer” is not properly used in this article. Authors determined antiproliferative activity of the extracts in the in vitro experiments (not anticancer activity in vivo).

- The manuscript’s title was changed from “Determination of anticancer and antioxidant activities of pomegranate on colorectal cancer by ultrasonic extraction techniques” to “Determination of the antioxidant and antiproliferative properties of pomegranate peel extract obtained by ultrasound on HTC-116 colorectal cancer cell line”.

 

[II] The extracts were characterized by some reactions for groups of structure and for “anticancer” activity extracts should be well characterized by HPLC or HPLC-MS methods to identify single compounds. As we know that extracts often exerts better effect than single isolated compounds analysis of the extract is ok, however these experiments on cell line are not connected directly with in vivo activity.

In the manuscript we have found term anticancer (see line; 490, 518,title of the section 2.8, and title of the manuscript) – it must be replaced by antiproliferative.

- The term “anticancer” was replaced with “antiproliferative”.

[III] The botanical name of the species used in the study must be given (Punica granatum L.) and there are some varieties used as fruit found in the market (it should be specified if any).

- The botanical name of the pomegranate (Punica granatum L.) was given in the line 50.

[IV] Previously published work covered the topic of the ultrasound assisted extraction of polyphenols from Pomegranate peel (see ref 7 and 8). As we see in this research (ref. 7 and 8) extraction was optimized and the parameters such as; extraction power, extraction time, extraction solvent used e.g. water, methanol, ethanol were applied, and concentrations of ethanol were tested (from 10 to 90 %), so what was the idea to compare maceration with ultrasound extraction, if many research was done proving the better effectiveness of the extraction assisted by ultrasound, and it is nothing new.

In Ref 7 Authors optimized solvents such as; water, methanol, ethanol, acetone (each at 50, 60, 70, 80 90  and 100 % of the concentration), and amount of the powdered substance extracted by 100 mL of the solvent (it was 2, 3, 5, 6, 8 and 10 g/100 mL), and also extraction temperature.

In Ref 8 Authors optimized power control (20, 60 and 100% and the extraction time 5, 10, 15 minutes, yield values and DPPH, FRAP and IC50 for ROS scavenging ability.

Authors said – see line 422-423; “…the conditions under which ultrasonic extraction must take place were optimized” – only extraction power; 10, 50 and 100 W was tried.

And in the 524-526; “This study give an idea of the efficiency of ultrasound assisted extraction ….need to be to optimize…temperature, solvent, time, …” – this idea was perfectly presented in many references – see examples below.

Some recent references should be used and cited, e.g.;

1)         Zivkovic J. et al. “Optimization of ultrasound-assisted extraction of polyphenolic compounds from pomegranate peel using response surface methodology.” Separation and Purification Technology, 2018; 194, 40-47.  – the UAE with ethanol (10 – 90 %) and the time of extraction  was optimized, TPC calculated, and many more.

2)         Garcia P. et al. “Recovery of bioactive compounds from pomegranate peel (Punica granatum L.) using pressurized liquid extraction” – it will be helpful to construct some discussion about results.

3)         Mo Y., et al. “Pomegranate peel as a source of bioactive compounds – a mini review on their physiological functions.” – Frontiers in nutrition, 2022, doi:10.3389/fnut.2022.887113.

- The idea “This study give an idea of the efficiency of ultrasound assisted extraction ….need to be to optimize…temperature, solvent, time, …” was removed. The suggested references were included, thank you.

[V] In reviewed manuscript the Discussion is not properly done. Authors should compare their own results with previous studies and highlighted what is different, better, etc. We have no information about previous results.

- Previous studies were included in the discussion.

[VI] “polyphenols and flavonoids” - - flavonoids are polyphenols also. The reported in previously published studies in pomegranate peel groups of polyphenols are; tannins, phenolic acids and flavonoids, anthocyanidins (sometimes considered as the group of the latter). TPC is the method of estimation of the total polyphenols (on basis of their orto-diphenolic groups) so flavonoids are counted in this method also. See this problem line 472, 475,

- This problem was corrected.

[VII] Figures 3 and 5 should be corrected because the OY axis scale should start with 0. What Authors present on these Figures suggest big differences in TPC and TFC between extracts, however it does not the big difference at all.

- Figures 3 and 5 were corrected, the OY axis scale start with 0.

[VIII] The extraction time was the same in maceration and in ultrasound extraction (it was in the each case 35 minutes – why? – nothing about optimization of the time of extraction) – so Authors have not the background to say; (see e.g. lines; 227, 424, 510) that the ultrasonication afforded the biggest yield “in the less time” because the time was in each extraction 35 minutes (not more  and not less). From the other hand 35 minutes for maceration (even with stirring) is a very short time.

- The aim of this study was to optimize the power of ultrasonic extraction that should be applied to realize the extraction of pomegranate peel and to prove that ultrasound can be an effective method during a short period of time, unlike the maceration. That’s why we fixed the time parameter at 35 minutes which is considered a very short time especially for maceration that gave low yield, while ultrasonic extraction gave better yield during the same period of time. Let's say we have to produce 5g of extract using the maceration technique, we will surely need a much longer period of time than 35 minutes, while more than 5g were produced by UAE in 35 min hence the idea that “the ultrasonication afforded the biggest yield in the less time”.

[IX] The expensiveness of the UAE is not big as compared with e.g. Soxhlet extraction, however there are methods more effective and in the biggest scale unexpensive and eco-friendly (e.g. supercritical fluid extraction SFE, frequently used to obtain big amount of the plant extracts).                                                

Line; 419; “alternative solvents” – example? Alternative to which solvents?

- Solvents Alternative to the petroleum solvents (e.g. ethanol, methanol…)

[X] When the methods are described Authors should use the past time, because it was already done.

- The methods were described in the past tence.

[XI] Plant systematic names should be written in italics.- see references; 6,8,10,11,12.

- Plant systematic names were written in italics in the cited references.

[XII] line 399 citation needed.

- The citation was added.

[XIII] – Authors used term “denaturation” – it is process depending on several factors in which proteins lose their tertiary, and secondary structure. So in case of polyphenols maybe better is to use “degradation”.

- The term “denaturation” was replaced by “degradation”.

[XIV] In each case when quantitative data are presented, standard deviation should be given (statistical analysis is required). The same in case of calibration curve equations; for gallic acid and rutin (rutoside); the SD of the intercept and the slope of calibration curve are required.

 In other hand when equation is presented there is no need to present the calibration curve as a Figure (so Fig 2 and Fig 4 are redundant).

- Statistical analysis was performed. Figure 2 and 4 were removed.

[XV] Line 143; is “rutin curve” should be “rutin calibration curve”

- The term “calibration” was added.

[XVI] In the section Materials and Methods subsection “Reagents and Chemicals” should be inserted, where producers and purity of the reagents and standards should be listed.

- The subsection “Reagents and Chemicals” was inserted in the material and methods section.

[XVII] Line28; “…viability of cells drops…” should be “viability of cells decreased…”

- The term “drops” was replaced by “decreased”.

[XVIII] Line 37; should be “…deaths worldwide as the result of CRC [1].”

- The expression “…deaths worldwide as the result of CRC [1]” was added.

[XIX] Line 144; should be ; “The calibration curve was prepared using rutin standard.”

- The expression “A standard curve was prepared using rutin as the reference molecule” was replaced by “The calibration curve was prepared using rutin standard.”

 

 

Author Response File: Author Response.docx

Reviewer 3 Report

I am very grateful you for the invitation to review the manuscript processes-2147618 by Habchi and coauthors “Determination of anticancer and antioxidant activities of pomegranate on colorectal cancer by ultrasonic extraction techniques”.

 

Comments:

1. “Yet these treatments are linked to 46 many serious side effects, which can be avoided by their substitution with natural therapeutic agents" What side effects? Please list all the effects.

2. Only one paper in the literature describes the antibacterial, anti-inflammatory and anticancer properties of pomegranate? It would be of great relevance for the authors to indicate more studies that demonstrate these effects throughout the paragraph, as it is also shown that the metabolites have other advantages in the treatment of diseases. So I suggest indicating more papers that have studied these positive effects.

3. Which primary and secondary metabolites are present in the chemical composition of pomegranate? Authors should include this information in the introduction.

4. Why was ethanol chosen as the solvent? The authors should justify the choice, since a wide variety of green solvents have been studied today.

5. The authors should mention why the 50 W power was higher than the 10 and 100 W power, since the 100 W power would normally be higher.

6. Are there other studies that extract the phenolic compounds from pomegranate using other extraction methods? The authors should search the literature to prove that the ultrasound method is superior.

7. Won't the obtained extracts be purified ? It is well known that products destined for the pharmaceutical industry require a high degree of purification. Please describe this.

Comments for author File: Comments.docx

Author Response

1. “Yet these treatments are linked to many serious side effects, which can be avoided by their substitution with natural therapeutic agents" What side effects? Please list all the effects.

- The side effects caused by therapeutic agents are numerous; we mentioned some of them (line 47-48).

2. Only one paper in the literature describes the antibacterial, anti-inflammatory and anticancer properties of pomegranate? It would be of great relevance for the authors to indicate more studies that demonstrate these effects throughout the paragraph, as it is also shown that the metabolites have other advantages in the treatment of diseases. So I suggest indicating more papers that have studied these positive effects.

- More papers (references 5, 6 and 7) were added demonstrating the antibacterial, anti-inflammatory and anticancer pomegranate properties at line 52.

3. Which primary and secondary metabolites are present in the chemical composition of pomegranate? Authors should include this information in the introduction.

- The chemical composition of pomegranate peel was cited in the introduction (line 54- 60).

4. Why was ethanol chosen as the solvent? The authors should justify the choice, since a wide variety of green solvents have been studied today.

- Several studies (e.g. reference 13) showed that ethanol/water mixture is the most appropriate solvent for the aqueous extraction of phenolic compounds from pomegranate. Moreover, ethanol is used as a medicinal solvent due to its low toxicity and non-polar capabilities.

5. The authors should mention why the 50 W power was higher than the 10 and 100 W power, since the 100 W power would normally be higher.

- We mentioned that high power can cause the degradation of the molecules, that’s why the 50 W power gave richest extract in active compound with higher effects than 100 W (line 482-488).

6. Are there other studies that extract the phenolic compounds from pomegranate using other extraction methods? The authors should search the literature to prove that the ultrasound method is superior.

- We included references proving that ultrasound assisted extraction is more beneficial than traditional extraction methods (reference 26, 27)

7. Won't the obtained extracts be purified ? It is well known that products destined for the pharmaceutical industry require a high degree of purification. Please describe this

- We fully agree that extracts should be purified before being used in pharmaceutical industry, but unfortunately we were not able to do it in this study at the Lebanese university because of the economic crisis that has affected our country, Lebanon. Hopefully, we will be purifying our extracts in our next studies.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

 

I stand by my opinion that the manuscript represents little originality. Assisted extraction techniques are not a new topic and have been addressed many times by other authors. Also, the biological properties of pomegranates are well known in terms of anti-cancer properties. It does not recommend the work for publication.

 

Author Response

This manuscript is part of a PhD study conducted at the Lebanese University. The aim of this study is to characterize the biological properties of the local ecotype of pomegranate grown in Lebanon using ultrasound, knowing that the chemical composition differs within the same species depending on the ecotype or cultivar, growing region, climate, maturity, cultural practice and storage, which can affect biological properties.

Other parameters are being studied such as solvents, extraction time, temperature and power to optimize ultrasonic extraction on Lebanese pomegranate peel.

Hopefully we will be applying more methods for the chemical characterization of the extracts. Unfortunately we were not able to do it in this first study because of the economic crisis that affected our country for the past three years.

Reviewer 2 Report

Necessary corrections have been done.

Author Response

The manuscript underwent English revisions. Thank you for your suggestions and comments that helped us to improve our article.