Icarifil, a Natural Mixture Based on L-Citrulline and L-Carnitine as a Novel Multicomponent Nutraceutical to Modulate ROS and PDE5

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this paper authors studied Icarifil (trademark) effect on proliferation and cell turgor of muscle epithelium cells, as well as anti-oxidant and PDE5 inhibitory activity, in order to prove its efficiency in reducing penile dysfunction. Although efficiency (according to analysed parameters) of product seems clear, there are some concern which should be addressed.

 

More information on Icarifil preparation are needed (particularly information on the choice of diluent). If the chosen diluent carries any potential cytotoxicity, whether the control medium contains an equivalent quantity of this diluent?

 

What is rationale for combining specific mixtures of components and missing for example Ginsenosids?

Methodology for determination of cell turgor should be more clear and authors should cite source for this methodology.

Although authors stated that concentration of Icarifil solution used for experiments was 0,5 mg/ml, final concentrations used in every assay are unclear and should be stated. Stating volume of Icarifil is not reflecting its final concentration in culture as medium volume was changed as well.

The application of statistical tests is not clear. Why the authors used so many different tests.

Authors compare Tadafil 10 with Icarifil+Tadafil 5 in 3 times regime. Do they have results for Tadafil 5 in 3 days regime?

 

Minor corrections:

Table 1: Ginsenoids should be Ginsenosids?

Line 304: whether ..increase of efficiency of PDE5.. should be ..increase of efficiency of PDE5 inhibitor..?

Comments on the Quality of English Language

Minor editing of English language required

Author Response

In this paper authors studied Icarifil (trademark) effect on proliferation and cell turgor of muscle epithelium cells, as well as anti-oxidant and PDE5 inhibitory activity, in order to prove its efficiency in reducing penile dysfunction. Although efficiency (according to analysed parameters) of product seems clear, there are some concern which should be addressed.

More information on Icarifil preparation are needed (particularly information on the choice of diluent). If the chosen diluent carries any potential cytotoxicity, whether the control medium contains an equivalent quantity of this diluent?

What is rationale for combining specific mixtures of components and missing for example Ginsenosids?

We understand the concerns of the referee related to the different ways to combine the single components of the mixture. Based on literature data, we have assembled the final mixture in a proper amount to have an efficient formulation without resulting toxic.

 

To better explain this concept the article has been edited in:

 

“Consequently, in this work, we investigated the possibility to combine various nutraceuticals in a unique combination named Icarifil (L-Citrulline, L-Carnitine, Eruca vesicaria, Panax ginseng, Tribulus terrestris, Turnera diffusa, Taurine, Vitamin E, Zinc). Specifically, we assembled the final mixture in a proper amount to have therapeutical efficacy without resulting toxic”.

 

Methodology for determination of cell turgor should be more clear and authors should cite source for this methodology. Although authors stated that concentration of Icarifil solution used for experiments was 0,5 mg/ml, final concentrations used in every assay are unclear and should be stated. Stating volume of Icarifil is not reflecting its final concentration in culture as medium volume was changed as well.

The authors understand the concerning of the reviewer related to the unclarity of the method. Materials and methods section has been re-edited in order to explain more clearly the methodology adapted from Hempling et al, 1981, State of water and electrolytes in mammalian cells during maturation and differentiation.

The application of statistical tests is not clear. Why the authors used so many different tests.

The authors apologize for the unclearness. The data were analysed by different statistical tests, in order to verify the robustness of the produced data. However, in the manuscript only the ONE WAY ANOVA test was used to compared the data, as no statistically significant variation was visible when the TWO WAY ANOVA was used. Materials and Methods section has been re-edited accordingly.

 

Authors compare Tadafil 10 with Icarifil+Tadafil 5 in 3 times regime. Do they have results for Tadafil 5 in 3 days regime?

The authors apologies for the misunderstanding. The administration of tadalafil requests a posology of 5 or 10 mg as a single dose, no more than once a day. Therefore, in this work, tadalafil 5 (a single dose) in co-administration with Icarifil 3 (times a day) were tested. Figure and manuscript text have been reedited in order to make it clearer for the reader.

 

Minor corrections:

Table 1: Ginsenoids should be Ginsenosids?

Sorry for the typos. The correction has been made.

Line 304: whether ..increase of efficiency of PDE5.. should be ..increase of efficiency of PDE5 inhibitor..?

The authors apologize for the typos reported in the manuscript. The text has been corrected.

Reviewer 2 Report

Comments and Suggestions for Authors

In this article, Amante et al. have evaluated the effect of a herbal formulation and suggest it to be a beneficial nutraceutical to modulate ROS and inhibit PDE5. However, the exact methodology used for every measurement is questionable and hence the claims cannot be supported by the data presented.

See specific comments below:

1.       Measurement of cell proliferation: In Fig 1, 100 ul Icarafil-treated cells show ~ 45% proliferation, while in Fig 2 they show about 55% proliferation. This likely represents the error in treatment/ measurement. The whole data must be used for comparing with the appropriate control (combined L-citrulline and L-carnitine treatment).  

2.       Measurement of Cell Turgor: It is mentioned that the cells were cultured in 24-well plates and “After 24 h, the wells emptied of the medium were weighed and compared with their weight before treatment. The weight obtained was then normalized concerning the number of cells present in the well” (lines 155 and 156). No reference is provided for this methodology used. How can one measure the weights of individual wells of a 24-well plate, accurately enough to identify changes in cell mass? How did the authors make sure that the result is not because of residual and inter-cellular liquids?

3.       Phosphodiesterase 5 (PDE5) inhibition: An ELISA was used to measure PDE5 “inhibition”. The source of the kit used for this measurement is not specified. ELISA would generally measure the protein amounts and not activity. Hence, the ELISA results should be presented not as “inhibition” but as a reduction in protein levels.

4.       ROS measurements: It is not clear from lines 180-183, how exactly was this measurement performed- was the dye added along with the treatment (line 181) or added afterwards (line 183)? Also, if the cells were harvested before the dye labeling, how was this done (scraping/ trypsinization)? How was the data normalized? In figure 7, why is there a “control” for each individual treatment?

5.       Statistical tests: On lines 190-192, it is mentioned “The statistical test used was the Two-Way ANOVA followed by Tukey's post-test, or the One-WAY ANOVA followed by the Bonferroni test as a post-test, or the Mann-Whitney test where appropriate.” It is not clear from the figure legends, which analysis was used for which figure.    

Author Response

In this article, Amante et al. have evaluated the effect of a herbal formulation and suggest it to be a beneficial nutraceutical to modulate ROS and inhibit PDE5. However, the exact methodology used for every measurement is questionable and hence the claims cannot be supported by the data presented.

See specific comments below:

1. Measurement of cell proliferation: In Fig 1, 100 ul Icarafil-treated cells show ~ 45% proliferation, while in Fig 2 they show about 55% proliferation. This likely represents the error in treatment/ measurement. The whole data must be used for comparing with the appropriate control (combined L-citrulline and L-carnitine treatment).  

Concerning the first consideration, since the cells used were different, is possible to have different results in terms of proliferation. Regarding the second point of view, to better understand the activity of the formulation, in second test (conduced on murine penile muscle epithelium cells) different combination of the components present in Icarifil were tested.

To better explain this concept, test has been modified in:

“Since good activity was already highlighted at the smallest quantity tested, to avoid toxicity, 100 µL of Icarifil was used to quantify the proliferation of the murine penile muscle epithelium cells. To better understand which of the components present in Icarifil had greater activity, different combinations of it were tested”.

2. Measurement of Cell Turgor: It is mentioned that the cells were cultured in 24-well plates and “After 24 h, the wells emptied of the medium were weighed and compared with their weight before treatment. The weight obtained was then normalized concerning the number of cells present in the well” (lines 155 and 156). No reference is provided for this methodology used. How can one measure the weights of individual wells of a 24-well plate, accurately enough to identify changes in cell mass? How did the authors make sure that the result is not because of residual and inter-cellular liquids?

The authors understand the concerning of the reviewer related to the unclarity of the method. Materials and methods section has been re-edited in order to explain more clearly the methodology adapted from Hempling et al, 1981, State of water and electrolytes in mammalian cells during maturation and differentiation.

3. Phosphodiesterase 5 (PDE5) inhibition: An ELISA was used to measure PDE5 “inhibition”. The source of the kit used for this measurement is not specified. ELISA would generally measure the protein amounts and not activity. Hence, the ELISA results should be presented not as “inhibition” but as a reduction in protein levels.

The authors appreciate the suggestion. The manuscript has been edited accordingly both in Materials and Methods section specifying the source of the Elisa kit (BD OptEIA, BD Biosciences, CA) and the Results and Discussion sections.

4. ROS measurements: It is not clear from lines 180-183, how exactly was this measurement performed- was the dye added along with the treatment (line 181) or added afterwards (line 183)? Also, if the cells were harvested before the dye labeling, how was this done (scraping/ trypsinization)? How was the data normalized? In figure 7, why is there a “control” for each individual treatment?

 The authors have re-edited the manuscript text in order to clarify the procedure. Moreover, due to the different solubility of the different components of Icarifil, slightly different amount of medium was used to match the equivalent amount present in the mixture. So the authors decided to normalize data to each individual control; statistical analyses was conducted accordingly.

5. Statistical tests: On lines 190-192, it is mentioned “The statistical test used was the Two-Way ANOVA followed by Tukey's post-test, or the One-WAY ANOVA followed by the Bonferroni test as a post-test, or the Mann-Whitney test where appropriate.” It is not clear from the figure legends, which analysis was used for which figure.   

The authors apologize for the unclearness. The data were analysed by different statistical tests, in order to verify the robustness of the produced data. However, in the manuscript only the ONE WAY ANOVA test was used to compared the data, as no statistically significant variation was visible when the TWO WAY ANOVA was used. Materials and Methods section has been re-edited accordingly.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

In revised Manuscript authors responded to some concerns but there are still some issues which should be addressed.

Authors still did not provide information on diluent used for Icarifil preparation, or preparation of its components. Although they added sentence „Specifically, we assembled the final mixture in a proper amount to have therapeutical efficacy without resulting toxic.“, it is not clear how they prove non-toxic effect.

Authors added reference for determination of induced Human Muscular Epithelium Cell Turgor and included some explanation. Still it is unclear how they determined cell number for every well/plate, and whether result is weight/cell (or 10⁷ cells)? How many times authors repeated this experiment?

 

If I understand correctly, authors changed volume of same Icarifil solution (c=0,5mg/ml) to observe its effect on proliferation. What is rationale to increase volume rather than Icarifil concentration (within the same volume). What was volume of medium in control group? Whether volume of different compounds was 100ul in final solution?

Methodology for accessing PDE5 transcript level is missing.

Comments on the Quality of English Language

Minor editing of English language is required.

Author Response

In revised Manuscript authors responded to some concerns but there are still some issues which should be addressed.

Authors still did not provide information on diluent used for Icarifil preparation, or preparation of its components. Although they added sentence „Specifically, we assembled the final mixture in a proper amount to have therapeutical efficacy without resulting toxic.“, it is not clear how they prove non-toxic effect.

We understand the concerns of the referee related to diluent chosen. In the manuscript specific indication was added: “Icarifil was dissolved in PBS under magnetic stirrer to obtain a stock solution of 0.5mg/mL”. Moreover, related to the rational of the produced mixture it has been based on literature data, as now reported in the re-edited manuscript “Specifically, based on literature data the final mixture was assembled using the smallest amount of each component reporting a positive effect on the different pathways in-volved in erectile disfunction in order to have a possible therapeutical efficacy without resulting toxic.”

Authors added reference for determination of induced Human Muscular Epithelium Cell Turgor and included some explanation. Still it is unclear how they determined cell number for every well/plate, and whether result is weight/cell (or 10⁷cells)? How many times authors repeated this experiment?

The results are reported as a percentage related to the increase in weight between the specific sample and its control. Moreover, each sample has been normalized to the number of cells contained in each plate measured at the end of the experiment. All the experiments have been conducted calculating the normalized percentage in weight increase over three plates and reporting its average value. Therefore, the manuscript has modified improving the method.

If I understand correctly, authors changed volume of same Icarifil solution (c=0,5mg/ml) to observe its effect on proliferation. What is rationale to increase volume rather than Icarifil concentration (within the same volume). What was volume of medium in control group? Whether volume of different compounds was 100ul in final solution?

Authors want to evaluate the possibility to doing multiple administration of a single dose rather than tested different concentration. Therefore, with this aim different volumes of icarifil were tested.

Concerning the volume of medium in control group Figure 1 and its caption were modified. In fact, in the figure 1 were reported two control: the only medium and medium with 300 ul of PBS (maximum volume of diluent used for Icarifil), while, in figure 2 the only medium and medium with 100 ul of PBS were reported.

 

Methodology for accessing PDE5 transcript level is missing.

The Materials and Methods section has been updated with the Real-Time PCR methodology used to assess the PDE5 transcription level variation due to the different substances and mixture tested. Namely “To evaluate the expression of PDE5, in treated and untreated samples real-time PCR was performed using a Taqman Low Density Arrays (TLDA, Thermo Fischer Scientific, Milan, Italy). Specific predesigned assays were performed to configure TLDA. PCR primers and probes for PDE5 mRNA were purchased as an Assay-on-Demand (AoD) gene expression product from Applied Biosystems, using the procedure reported in Zhang et al. [27]”

Reviewer 2 Report

Comments and Suggestions for Authors

Some of the important methods have serious flaws which were flagged in the previous review but were not corrected yet.

Major comment

1. Turgor measurement: It is still unclear how the weight of individual cells of 24-well plates could be taken for moisture measurement and assume that all of it is intracellular water. The "Hemling et al." paper that has been cited for this method did not state anywhere that 24-well plates be used, and their weights taken (that was done here). The turgor measurement presented in this paper is fundamentally flawed in this reviewer's opinion and must be removed. 

2. It was asked to specify which statistical analyses were done for which results. This was still not specified and instead a generic statement has been provided on line 196. 

3. For the ROS measurement, the results show that in response to the citral treatment, the ROS levels were lower. It is not clear if the basal ROS levels had any difference. 

Minor comments

 1. For the ROS measurement, it is not specified how long was citral treatment done?

 

Author Response

Some of the important methods have serious flaws which were flagged in the previous review but were not corrected yet.

Major comment

1. Turgor measurement: It is still unclear how the weight of individual cells of 24-well plates could be taken for moisture measurement and assume that all of it is intracellular water. The "Hemling et al." paper that has been cited for this method did not state anywhere that 24-well plates be used, and their weights taken (that was done here). The turgor measurement presented in this paper is fundamentally flawed in this reviewer's opinion and must be removed.

The Authors do not agree with the reviewer. Following the concept expressed in the paper of Hemling et al., an increase in weight normalized over the weight of the control has been used to evaluate the cell turgor. Specifically, each sample has been normalized to the number of cells contained in each plate measured at the end of the experiment and the results are reported as a percentage related to the increase in weight between the specific sample and its control.

2. It was asked to specify which statistical analyses were done for which results. This was still not specified and instead a generic statement has been provided on line 196. 

For each test One-WAY ANOVA followed by the Bonferroni test as a post-test was conducted. In some cases, to have a more statistical significance also the Mann-Whitney test was considered. Therefore, each caption has been modified reporting the test used.

3. For the ROS measurement, the results show that in response to the citraltreatment, the ROS levels were lower. It is not clear if the basalROS levels had any difference. 

The authors apologize for the misunderstanding. The manuscript has been re-edited to clear the methodology. In fact, as now reported in line 327“the intracellular levels of ROS generated by oxidative stress due to the addition of a citral solution to cells were quantified. As shown by the data reported in Figure 7, this induction was significantly reduced when, the cells were pretreated with Icarifil or with different components of it before the treatment of citral” where it is clear to the reader now, that cells were treated with Icarifil and after with citral that induces oxidative stress. Therefore, the results show the response of formulation activity after the induction of citral on ROS levels.

Minor comments

1. For the ROS measurement, it is not specified how long was citral treatment done?

The treatment of citral was conducted for 2.5 hours. This information has been included in the manuscript in the methods section.

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

The revised manuscript has undergone substantial improvements. However, there is a question concerning the revised figures. It appears that authors did not specify to which group the significance is attributed, whether it is the control group or the control+PBS group. The authors have indicated significance within the control+PBS group. Is this mistake?

Comments on the Quality of English Language

Minor editing of English language required

Author Response

It appears that authors did not specify to which group the significance is attributed, whether it is the control group or the control+PBS group. The authors have indicated significance within the control+PBS group. Is this mistake?

The authors apologise for the mistake. The figures have been re-edited in order to properly indicate significance of the data

Reviewer 2 Report

Comments and Suggestions for Authors

I do not agree with the authors' explanation about measuring cell turgor. I do not have any other comments.

Author Response

I do not agree with the authors' explanation about measuring cell turgor. I do not have any other comments.

The authors are sorry the reviewer did not comply with the data and the methodology used to obtain them