Anti-Inflammatory, Antimicrobial, and Vasoconstriction Activities of an Anti-Hemorrhoidal Mixture of Alchemilla vulgaris, Conyza bonariensis, and Nigella sativa: In Vitro and Clinical Evaluations

Round 1

Reviewer 1 Report

After careful revision of the manuscript by Said et al entitled ‘Anti-inflammatory, antimicrobial, and vasoconstriction activities of an antihemorrhoids mixture of Alchemilla vulgaris, Conyza bonariensis, and Nigella sativa: In vitro and clinical evaluations’ considering the interest of the topic for scientific community and the well-presented results, I suggest to revise both the English style and the overall manuscript according to the comments reported below, before possible consideration for publication.

-Abstract: use the full name and Italics for scientific name the first time it appears, then use the abbreviation in italics form (e.g. change ‘E. coli’ by ‘Escherichia coli’): check this in overall manuscript;

-Abstract: change ‘µg/ml’ by ‘µg/mL’: check this in overall manuscript;

-Page 2, introduction: check for the correct grammar in the following sentence: ‘The former one is those that occur inside…’;

-Page 2, introduction: change ‘CB was found to exert antioxidant’ by ‘CB was found to exert antioxidant’;

-Abstract and page 2, introduction: use the plant scientific name and delete the acronym for Nigella sativa, Conyza bonariensis, and Alchemilla vulgaris. In particular, as said before, use use the full name and Italics for scientific name the first time it appears, then use the abbreviation in italics form;

-Page 2 of introduction: modify this sentence: ‘Alchemilla vulgaris: Alchemilla plants (lady's mantle) are used in…’;

-Page 2 of introduction: please modify the last sentence of introduction;

-Page 3, paragraph 2.1: delete the bold for ‘voucher specimen’; moreover, add information on plant extract preparation;

-Page 3, paragraph 2.2. change ‘herbal cream “HPC”’ by ‘herbal HPC cream’;

-Page 3, paragraph 2.3: change ‘g/l’ by ‘g/L’ or ‘µg/ml and ng/ml’ by ‘µg/mL and ng/mL’ or ‘hours’ by ‘h’ or ‘2 X 10⁵cells/ml’ by ‘2x10⁵ cells/mL’ check this in overall text;

-Page 3, paragraph 2.3: change ‘Cells then treated with various concentrations of the HPC’ by ‘Cells were then treated with different HPC concentrations’ adding also the various concentrations used in brackets.

-Page 3, paragraph 2.6: the same comment made for abstract: change ‘Escherichia coli’ by ‘E. coli’; check this in overall text;

-Page 3, paragraph 2.6: check ‘°C’ and verify in overall text;

-Page 4, paragraph 2.7: change ‘O2 and CO2’ by ‘O2 and CO₂’; change ‘minutes’ by ‘min’. Check this in overall text;

-Page 5, paragraph 2.9: change ‘Day’ by ‘day’;

Add a brief conclusion;

-Table 1: check the scientific names form, change ‘gr’ by ‘g’; use small letters for ‘seeds’. Moreover, the percentage is necessary after ‘93.8’ and ‘100’?;

-Check all Figures: ‘µg/mL’ instead of ‘µg/mL’ in Figure 2, 4 and so on. Figure 6: the colours are wrong (where is the blue?); Figure 3: check the word ‘control’.

Author Response

Reviewer 1.

Thank you very much for your suggestions and comments. We have changed our manuscript according to your wishes. Changes are added in the text using Track Changes. 

-Abstract: use the full name and Italics for scientific name the first time it appears, then use the abbreviation in italics form (e.g. change ‘E. coli’ by ‘Escherichia coli’): check this in overall manuscript;

Done

-Abstract: change ‘µg/ml’ by ‘µg/mL’: check this in overall manuscript;

Done

-Page 2, introduction: check for the correct grammar in the following sentence: ‘The former one is those that occur inside…’;

Done

-Page 2, introduction: change ‘CB was found to exert antioxidant’ by ‘CB was found to exert antioxidant’;

Corrected

-Abstract and page 2, introduction: use the plant scientific name and delete the acronym for Nigella sativa, Conyza bonariensis, and Alchemilla vulgaris. In particular, as said before, use use the full name and Italics for scientific name the first time it appears, then use the abbreviation in italics form;

Corrected

-Page 2 of introduction: modify this sentence: ‘Alchemilla vulgaris: Alchemilla plants (lady's mantle) are used in…’;

Done

-Page 2 of introduction: please modify the last sentence of introduction;

Done

-Page 3, paragraph 2.1: delete the bold for ‘voucher specimen’; moreover, add information on plant extract preparation;

Done

-Page 3, paragraph 2.2. change ‘herbal cream “HPC”’ by ‘herbal HPC cream’;

Done

-Page 3, paragraph 2.3: change ‘g/l’ by ‘g/L’ or ‘µg/ml and ng/ml’ by ‘µg/mL and ng/mL’ or ‘hours’ by ‘h’ or ‘2 X 10⁵cells/ml’ by ‘2x10⁵ cells/mL’ check this in overall text;

Done

-Page 3, paragraph 2.3: change ‘Cells then treated with various concentrations of the HPC’ by ‘Cells were then treated with different HPC concentrations’ adding also the various concentrations used in brackets.

Done

-Page 3, paragraph 2.6: the same comment made for abstract: change ‘Escherichia coli’ by ‘E. coli’; check this in overall text;

Done

-Page 3, paragraph 2.6: check ‘°C’ and verify in overall text;

Done

-Page 4, paragraph 2.7: change ‘O2 and CO2’ by ‘O2 and CO₂’; change ‘minutes’ by ‘min’. Check this in overall text;

Done

-Page 5, paragraph 2.9: change ‘Day’ by ‘day’;

Done

Add a brief conclusion;

We separated the conclusions from the Discussion and added them at the end of the Discussion as a new section

-Table 1: check the scientific names form, change ‘gr’ by ‘g’; use small letters for ‘seeds’. Moreover, the percentage is necessary after ‘93.8’ and ‘100’?;

Done

-Check all Figures: ‘µg/mL’ instead of ‘µg/mL’ in Figure 2, 4 and so on. Figure 6: the colours are wrong (where is the blue?); Figure 3: check the word ‘control’.

Corrected

Reviewer 2 Report

Congratulations on the work done in order to obtain the results. Unfortunately, without some fundamental experiments these results are not useful. First of all chemical characterization of the plant material is needed. Likewise, if you would like to improve manuscript my suggestion would be to use microdilution instead of disc diffusion assay.

Author Response

Corrected

Reviewer 2

Comments and Suggestions for Authors

Congratulations on the work done in order to obtain the results. Unfortunately, without some fundamental experiments these results are not useful. First of all chemical characterization of the plant material is needed. Likewise, if you would like to improve manuscript my suggestion would be to use microdilution instead of disc diffusion assay.

Thank you very much for your suggestions and comments. However, since the chemical characterizations of the used three plants are already done and published by other research groups, there is no need to repeat these experiments. We referred to these publications in the Discussion.

In addition, due the correct Covid-19 pandemic and continuous lockdowns as well as restricted financial resources we are unable to carry out new experiments. We have submitted a research proposal on evaluation of synergistic effects of the main active compounds of NS, CB, and AV in the treatment of haemorrhoids. The results will be published in a new paper. 

The present paper contains a large amount of data with 12 Figures and Tables.

Reviewer 3 Report

In this manuscrip is described the preparation of an extract of three plants and its formulation; in addition, in vitro, in vivo and clinical studies were performed concerning its use in hemorrhoids treatment. Some problems in the manuscript can be detected:

a) English problems can be detected – examples: “

 HPC showed a dose-dependent antibacterial activity against E. coli (60% of the in-hibition observed with the Ampicillin at 5mg/disc), and a significant vasoconstriction effects on intestinal vein rings (40% increase compared to phenylephrine).”; “In a RDBPC with 77 hemorrhoi-dal disease (HD), (patients, ages 19-61 years, the median grade of hemorrhoids, 2.0) we determined the anti-hemorrhoid efficacy and safety of HPC.” (abstract); “Current conventional treatment of …  … ice baths.” (introduction), others. Therefore all manuscript must be checked and corrections performed.

b) abbreviations:

-some were defined in abstract and not used here

-must be defined in first use after abstract (in introduction,..); in addition, and as example, in introduction we can find AV, but later the name “Alchemilla vulgaris” also appeared

Therefore all manuscript must be checked and corrections performed.

c) justification of the study and other relevant introduction points – in introduction, it is needed a higher clarification on the presently used therapeutical approached for hemorrhoid; in addition, the basis for the use of this extract from these plants must be developed and clarified

d) experimental part problems:

-despite being previously described, the extract preparation should be presented in higher detail; in addition, at least a partial chemical characterization must be performed and included in the manuscript; moreover, the technological characterization of the cream should be partially performed and the data included in the manuscript

-why LDH (and not MTT) assay in cell and what was the basis for the selection of bacterium in antimicrobial studies? LD50 in cytotox. assays?

-more details are needed in in vivo and clinical studies; for example, in in vivo studies, what was the quantity of cream used and the time of exposition?

-positive controls are lacking in several experiments (e.g. LDH study) and therefore experiments must be repeated considering this

e) statistics – authors referred the t-Students test, but in figures the asterisks when a ststistical significance occurs are not present and must be included for a better analysis of the results

f) virtually no discussion exists and therefore, authors must complete the manuscript in this section

Author Response

Reviewer 3

Thank you very much for your suggestions and comments. We have changed our manuscript according to your wishes. Changes are added in the text using Track Changes. 

1. a) English problems can be detected – examples: “

 HPC showed a dose-dependent antibacterial activity against E. coli (60% of the in-hibition observed with the Ampicillin at 5mg/disc), and a significant vasoconstriction effects on intestinal vein rings (40% increase compared to phenylephrine).”; “In a RDBPC with 77 hemorrhoi-dal disease (HD), (patients, ages 19-61 years, the median grade of hemorrhoids, 2.0) we determined the anti-hemorrhoid efficacy and safety of HPC.” (abstract); “Current conventional treatment of …  … ice baths.” (introduction), others. Therefore all manuscript must be checked and corrections performed.

Done

1. b) abbreviations:

-some were defined in abstract and not used here

-must be defined in first use after abstract (in introduction,..); in addition, and as example, in introduction we can find AV, but later the name “Alchemilla vulgaris” also appeared

Done

Therefore all manuscript must be checked and corrections performed.

Done

1. c) justification of the study and other relevant introduction points – in introduction, it is needed a higher clarification on the presently used therapeutical approached for hemorrhoid; in addition, the basis for the use of this extract from these plants must be developed and clarified

The Introduction was changed to include the required information

1. d) experimental part problems:

-despite being previously described, the extract preparation should be presented in higher detail;

Done

in addition, at least a partial chemical characterization must be performed and included in the manuscript;

We thank the reviewer for his excellent suggestions. However, we think that since chemical characterization of the used three plants is already done and published by other research groups, there is no need to repeat these experiments. We currently working on evaluation of synergistic effects of the main active compound of NS, CB, and AV in the treatment of haemorrhoids. The results will be published in a new paper.  The present paper contains a large amount of data with 12 Figures and Tables.

moreover the technological characterization of the cream should be partially performed and the data included in the manuscript

We added the required data in a new paragraph

2.2.1 Pharmaceutical Manufacturing Process: Emulsifiers were melted with sunflower seed oil at a temperature of 70 ± 5 °C. Separately, the aqueous phase (water, HPC, preservatives) was heated to the same temperature of 70 ± 5 °C. Then, the oily phase was added to the aqueous phase. The mixture was emulsified using homogenizer at 5000 rpm for 20 – 30 min. The resulted homogenous and white colored cream. In addition, the consistency, appearance, and stability of the cream were examined and approved.

-why LDH (and not MTT) assay in cell and what was the basis for the selection of bacterium in antimicrobial studies?

Both, MTT and LDH assays are widely applied in the assessments of cell viability and cytotoxic effects. We added new Figure with the MTT results (Figure 2B). In addition, we added the MTT assay in the MM and Results

LD50 in cytotox. assays?

LD50 are from published dada. We have changed the sentence to:

These results support the long history of safe use of the three herbs and published data regarding the biosafety of the individual plants. The LD50 values for NS, CB, and AV are 19.8, 17.8, and 17.3 g/Kg weight, respectively [15, 23].

-more details are needed in in vivo and clinical studies; for example, in in vivo studies, what was the quantity of cream used and the time of exposition?

As mentioned in point 2.9

“The patients were asked to apply 2-3 mL of HPC twice daily for 6 days. Each patient came for 5 visits and follow-up; at baseline, at day 2, 4, 6 and an observation endpoint after one month”.

-positive controls are lacking in several experiments (e.g. LDH study) and therefore experiments must be repeated considering this

LDH assay is a widely used cytotoxicity assay and we usually use  0.01 vol% of a cytotoxic surfactant (Triton X-100,Carl Roth GmbH) was used as positive control which results in a very high levels of LDH (430% compared to untreated cells). Adding the positive control to the Figures will reduce the height of the other column and differences between the different treatments. We have added the data of the positive control in text

1. e) statistics – authors referred the t-Students test, but in figures the asterisks when a ststistical significance occurs are not present and must be included for a better analysis of the results

We added the asterisks in the Figures

1. f) virtually no discussion exists and therefore, authors must complete the manuscript in this section

We combined the Results and Discussion in one section under “Results and Discussion” with 37 citations

Round 2

Reviewer 2 Report

It can be accepted

Reviewer 3 Report

Authors improved the manuscript text, which now is more acceptable for publication, in my oppinion.