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Article
Peer-Review Record

Legal Cannabis sativa L. Dried Inflorescences: Cannabinoids Content and Cytotoxic Activity against Human HepG2 Cell Line

Appl. Sci. 2023, 13(8), 4960; https://doi.org/10.3390/app13084960
by Maria Assunta Acquavia 1, Carmen Tesoro 1, Raffaella Pascale 2, Angela Ostuni 1, Ilenia Matera 1, Giuliana Bianco 1, Laura Scrano 3, Sabino A. Bufo 1,4, Rosanna Ciriello 1, Angela Di Capua 1,* and Filomena Lelario 1,*
Reviewer 1: Anonymous
Reviewer 2:
Reviewer 3: Anonymous
Appl. Sci. 2023, 13(8), 4960; https://doi.org/10.3390/app13084960
Submission received: 9 March 2023 / Revised: 1 April 2023 / Accepted: 13 April 2023 / Published: 14 April 2023

Round 1

Reviewer 1 Report

reject

Comments for author File: Comments.pdf

Author Response

The introduction is long.

Thank you for your comment. However, we didn’t receive any suggestion on which part should be shortened or reformulate. For this reason, we decided to not make this change, as we think that all the information reported in the introduction are useful to explain the aim of our work.

 

What was the percentage of similarity resulting from the plagiarism of the text of the article?

Thank you for your question. We double checked the percentage of similarity by using the software Grammarly, and results reported less than 10%.

 

The full name of each of these items should be listed below the table.

Thank you for your suggestion. We apologize if our original caption did not show names of each compound listed in the table. We added them for clarification in the caption of Table 1. (line 153-154)

 

There is no title of materials and methods. Is the materials and methods section after the findings?

Thank you for your question. We agree with the reviewer, and we will format our paper according to the template proposed by Applied Science if it will be accepted for publication.

Reviewer 2 Report

Acquavia et al report on the cannabinoid content of 34 commercially available Cannabis sativa L. dried inflorescences. They furthermore test 3 of these for cytotoxic activity against a human cancer-derived cell line HepG2. The study is presented in a clear and informative way. I think this study could be of interest and I suggest a few experimental and presentation improvements.

 

Experimental comments.

Three Cannabis sativa L extracts were choses for IC50 quantitation against the HepG2 cell line. As far as I can tell, authors want to establish if Cannabis sativa L extracts could be useful as an anti-cancer agent. I believe for such an experiment to be informative the cytotoxic test requires additional crucial controls. 1. Further extracts need to be included, which correspond to a different THC/CBD ratio – such that conclusions could be drawn as to the effect of THC/CBD ratio. 2. Extracts need to be tested against at least one additional cell line which is derived from normal tissues. To show that cancer-derived line(s) are more sensitive than normal cells to the toxic effect of the Cannabis sativa L extracts. 3. It is possible that the cytotoxic activity associated with the Cannabis sativa L extracts is unrelated to THC and CBD content but rather to further active ingredients in the Cannabis sativa L extracts. Authors could use paired comparisons with extract which have the lowest CBD and TCH concentrations (preferentially at similar THC/CBD ratios) and correlate IC50s to extract which have the highest CBD and TCH concentrations and thus be able to conclude a causal relationship.

 

Presentation comments.

1. Authors could present their spectral results more visually. For example, by generating mirror plots, where database and experimentally derived spectra are compared on the same graph.

2. Authors need to clarify if extract or CBD/THC concentrations are used on Figure 2)

3. Could authors please clarify paragraph lines 223-231, specifically in regard to the statements about THC/CBD ratio.

Author Response

Acquavia et al report on the cannabinoid content of 34 commercially available Cannabis sativa L. dried inflorescences. They furthermore test 3 of these for cytotoxic activity against a human cancer-derived cell line HepG2. The study is presented in a clear and informative way. I think this study could be of interest and I suggest a few experimental and presentation improvements.

 

Experimental comments

 

Three Cannabis sativa L extracts were chosen for IC50 quantitation against the HepG2 cell line. As far as I can tell, authors want to establish if Cannabis sativa L extracts could be useful as an anti-cancer agent. I believe for such an experiment to be informative the cytotoxic test requires additional crucial controls.

 

1.Further extracts need to be included, which correspond to a different THC/CBD ratio – such that conclusions could be drawn as to the effect of THC/CBD ratio.

  1. Extracts need to be tested against at least one additional cell line which is derived from normal tissues. To show that cancer-derived line(s) are more sensitive than normal cells to the toxic effect of the Cannabis sativa L extracts.
  2. It is possible that the cytotoxic activity associated with the Cannabis sativa L extracts is unrelated to THC and CBD content but rather to further active ingredients in the Cannabis sativa L extracts. Authors could use paired comparisons with extract which have the lowest CBD and TCH concentrations (preferentially at similar THC/CBD ratios) and correlate IC50s to extract which have the highest CBD and TCH concentrations and thus be able to conclude a causal relationship.

 

Thank you for your comments. We appreciate the reviewer’s insightful suggestion and agree that it would be interesting and useful to consider the anti-cancer activity for these extracts. We are planning to continue our studies by using samples with different THC/CBD ratio and by conducting experiments on normal cells as well. Unfortunately, plants with high content in THC are not legally available and it requires special permission.

Such an analysis is beyond the scope of our paper, which aims only to determine cannabinoids’ content using a validate analytical technique and to show some preliminary results regarding the efficacy of plant extracts versus pure compound. For this reason, we compared extract with similar ratio in THC/CBD and we only evaluated the citotoxicity, without drawing conclusion about the anticancer activity.

 

Presentation comments

1.Authors could present their spectral results more visually. For example, by generating mirror plots, where database and experimentally derived spectra are compared on the same graph.

Thank you for your comment. We have now included a new Figure (Figure 2) in Section 2.3 (line 220) where we show the chromatograms and the UV spectra for comparison between extracts and pure compounds.

 

  1. Authors need to clarify if extract or CBD/THC concentrations are used on Figure 2)

Thank you for your comments. In figure 2 (now figure 3) we reported the extract concentration.

 

  1. Could authors please clarify paragraph lines 223-231, specifically in regard to the statements about THC/CBD ratio.

Thank you for pointing out it. In the introduction, we mentioned the importance of CBD/THC ratio to categorize Cannabis sativa for medicinal purposes as they interact differently with endocannabinoids system (line 81-82). To better clarify this point, we included the following sentence “due to the different interaction with cannabinoid receptor CBD 1 and 2” at line 224.

 

Reviewer 3 Report

This study reports the results of “Legal Cannabis sativa L. dried inflorescences: Cannabinoids content and cytotoxic activity against human HepG2 cell line”. The manuscript is well prepared and the subject is interesting. In my opinion, the manuscript is in a position to be accepted for publication after minor revision. Here are some comments on the manuscript:

 

General remarks:

-The authors investigate 34 samples of Cannabis sativa, which is interesting, but they should justify why they choose just three samples for the study of their effect on viability of hepatoblastoma HepG2 cells.

-The section 4.5 must be revised especially the part of the preparation of plant extracts. (Authors should precise if they test phytocannabinoids extracts or just ethanolic extracts of inflorescence part)

-The authors should add in the results section the HPLC-UV chromatographic profile of all studied Cannabis sativa samples

Author Response

This study reports the results of “Legal Cannabis sativa L. dried inflorescences: Cannabinoids content and cytotoxic activity against human HepG2 cell line”. The manuscript is well prepared and the subject is interesting. In my opinion, the manuscript is in a position to be accepted for publication after minor revision. Here are some comments on the manuscript:

 

General remarks:

-The authors investigate 34 samples of Cannabis sativa, which is interesting, but they should justify why they choose just three samples for the study of their effect on viability of hepatoblastoma HepG2 cells.

Thank you for pointing out it. We agree with this comment as we didn’t report in this section what we mentioned in line 97. The three samples were selected because the 9:1 ratio CBD:THC is optimal to treat inflammation and pain, while with ratio below or equal to 1:6, CBD is protective against THC effects. We added the following sentence at line 277 “The three samples reported a THC:CBD ratios of 1:9, optimal for inflammation and pain treatment.”

 

 

 

-The section 4.5 must be revised especially the part of the preparation of plant extracts. (Authors should precise if they test phytocannabinoids extracts or just ethanolic extracts of inflorescence part)

Thank you for pointing out this. We agree and we revised out text adding the following sentence in section 4.2 “For vitality assays, samples were extracted with Ethanol (EtOH)”( line 354).

 

-The authors should add in the results section the HPLC-UV chromatographic profile of all studied Cannabis sativa samples.

Thank you for your comment. We decided to report only the HPLC-UV chromatographic profile related to the three samples (Sample 1,5 and 20) that have been selected for the biological evaluation. The new Figure is Figure 2 and it has been added in Section 2.3 (line 220).

 

 

Round 2

Reviewer 2 Report

I believe the manuscript by Acquavia et al has been improved and I thank authors for taking into consideration my suggestions. However, authors considered only my minor suggestions for improvement to data presentation. I am still concerned that a reader might be confused about the overall conclusions, as presented in the manuscript. I recommend authors state clearly that due to legal or other limitations (time, scope) they can only present preliminary results concerning the investigated compounds/C.s extracts. specifically no strong conclusions about THC/CBD ratios and their effects on cells should be drawn and it must be stated clearly that relevant effects must be independently tested in a control manner in future experiments.

 

Author Response

Thank you for your comment. To clarify our preliminary results, al line 335 we changed the phrase "The results from the MTT assay showed" with "The results from the MTT assay suggested" and we added the following sentence (line 340): However, further studies should be carried out to test extracts of Cannabis sativa L with different THC:CBD ratios on tumor cells and on cell line derived from normal tissues.

 

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