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Article

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

by
Chiara Amante
1,
Chiara De Soricellis
1,
Gianni Luccheo
2,
Anna Di Vernieri
2,
Luigi Luccheo
2,
Giovanni Falcone
1 and
Pasquale Del Gaudio
1,*
1
Department of Pharmacy, University of Salerno, Via Giovanni Paolo II, 132, 84084 Fisciano, Italy
2
Anvest Health S.r.l., Via Rosario Livatino, 84083 Castel San Giorgio, Italy
*
Author to whom correspondence should be addressed.
Appl. Sci. 2023, 13(22), 12358; https://doi.org/10.3390/app132212358
Submission received: 11 September 2023 / Revised: 2 November 2023 / Accepted: 7 November 2023 / Published: 15 November 2023
(This article belongs to the Section Food Science and Technology)
Browse Figures
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Abstract

:
Erectile dysfunction (ED) is a pathology that affects both an individual’s life and their partner’s, representing an increasing health concern. Currently, phosphodiesterase type 5 inhibitors (PDE5i) remain the first-line therapy for erectile dysfunction, and no other drugs are approved. However, the high cost of these drugs and related adverse events limit their use. Since the concept of nutraceuticals has been extended from health promotion to the treatment of diseases, in this work, a novel mixture of different natural substances named Icarifil (L-Citrulline, L-Carnitine, Eruca vesicaria, Panax ginseng, Tribulus terrestris, Turnera diffusa, Taurine, Vitamin E, Zinc) was studied to verify its ability to increase the proliferation of muscle epithelium cells and cell turgor, as well as, due to containing antioxidants, its ability to reduce the amount of ROS that are mediators in the pathophysiological process of ED. Moreover, Icarifil showed efficacy in inhibiting PDE5 levels higher than 65% compared to the control and 35% compared to the mixture of L-Citrulline and L-Carnitine, the major components of the formulation. In association with the low dosage of a phosphodiesterase (PDE) inhibitor (tadalafil), the inhibitory capacity of tadalafil was significantly increased and comparable to high-dosage drug response, promoting the possibility of reducing the daily dosage of the drug and consequently its adverse effects.

1. Introduction

Erectile dysfunction (ED), a common male sexual dysfunction, is defined by the National Institutes of Health as the inability to obtain or keep an erection firm enough to sexually satisfy men and their partners [1]. This pathology affects individuals’ lives and their partners’ rational, emotional, and physical well-being [2]. ED represents an increasing health concern; in fact, it is estimated that more than 300 million men worldwide will be affected by it by 2025, an increase of 150 million compared to 1995 [3]. Numerous pathophysiological pathways, including psychological, neurological, hormonal, vascular, and cavernosal pathways, can affect erectile function [4]. The major risk factor associated with ED is age, and the increasing prevalence of ED is linked to the global aging population [5]. Moreover, ED facilitates the risk of future progressive cardiovascular and cerebrovascular events and all-cause mortality [6,7].
Overall, 80% of ED is caused by endothelial dysfunction related to the nitric oxide/cyclic guanosine 3′5′-monophosphate (NO-cGMP) system in the corpus cavernosum [8]. NO regulates vascular tone and blood flow by activating guanylate cyclase (GC), which converts guanosine triphosphate (GTP) into cyclic guanosine monophosphate (cGMP). cGMP, by reducing the intracellular calcium level, induces muscle vasodilation and consequently the influx of blood into the penis and erection.
Subsequently, the enzyme phosphodiesterase type 5 (PDE5) breaks down the cGMP and allows a return to the detumescence phase. Since the delay of the catabolism of cGMP by PDE5s allows penile tumescence and erection to be prolonged [9], oral PDE type 5 inhibitors (PDE5i) are considered the ejection treatment for ED. However, the high cost of these drugs, the lack of responsiveness in patients with certain comorbidities, and related adverse events such as headache, skin rash, visual disturbances, and dyspepsia limit their use (reduced compliance) [10].
L-Citrulline (CIT), an organic compound and a non-essential amino acid, is synthesized endogenously from the body or via the diet through watermelon [11]. The use of CIT in ED is correlated with its metabolic conversion in Arginine, which is the only physiological substrate for NOS [10]. Scientific evidence has demonstrated that L-arginine administration can restore endothelial vasodilation and improve NO synthesis in animal models and humans with ED. Since in subjects with cardiovascular disorders associated with erectile dysfunction (diabetes, ischemic heart disease, hypercholesterolemia, hypertension), the ability of the endothelium to synthesize NO is impaired (endothelial dysfunction), Arginine, a precursor of NO, can restore this function. However, Arginine, administered orally, shows reduced bioavailability because of a significant intestinal pre-systemic metabolization by arginases. Unlike L-arginine, CIT is not subject to intestinal pre-systemic metabolization [12]. Therefore, oral administration increases, in a dose-dependent manner, the plasma levels of Arginine to a greater extent (about double) compared to direct administration of Arginine [11].
L-Carnitine supplies muscle tissue with energy through the β-oxidation of lipids to produce ATP. It presents antioxidant activity by preserving the endothelial function from oxidative stress. Its role as an anion scavenger in combination with other natural substances or PDE5i was confirmed by different studies [13,14].
Icariin is a natural flavonoid PDE5i extracted from Eruca vesicaria, administered orally and developed to be an effective phytotherapeutic agent for its ability to preserve penile hemodynamics, smooth muscle, endothelial integrity, and neuronal expression of NOS in the penis of diabetic rats [11]. Erucine, an isothiocyanate extracted from Eruca vesicaria, is a slow donor of H2S, which is released into biological substrates, causing myorelaxation and vasodilatory activity of the smooth muscles with consequent filling of the sinusoids of the cavernous bodies and penile erection. Erucine also possesses antioxidant activity which is essential to avoid the inactivation of NO via ROS [15,16].
Panax ginseng is widespread and known in Eastern countries as an aphrodisiac [17]. Ginseng extractions and ginsenosides have been reported to induce vasodilatation of the corpus cavernosum via the NO/cGMP pathway, mediated by the endothelial and neuronal NOS enzymes [18]. Ginsenosides also increase the conversion of L-Arginine into L-Citrulline, stimulating the synthesis of NO.
Tribulus terrestris (TT) contains Protodioscin, a steroidal saponin precursor of androgens, which increases the endogenous synthesis of testosterone and dehydroepiandrosterone. Scientific studies have shown that TT treatment improves performance and drastically increases the serum testosterone level in rats [19]. Moreover, Gauthaman et al. have reported that Protodioscin is involved in the nitric oxide synthase pathway determining relaxation of the corpus cavernosum [20].
Turnera diffusa, commonly known as damiana, is extensively diffused as a tonic, a stimulant, and an aphrodisiac in folk medicine [21]. Its active ingredients, including arbutin, caffeine, and flavonoids, promote sexual function, reduce post-ejaculation latency time, and increase libido.
Taurine, a sulfur-containing amino acid, is one of the most abundant free amino acids in the human body involved in several different pathways: osmotic regulation, immunomodulation, and oxidative stress inhibition [22]. Taurine improves erectile function in diabetic mice, reducing penile fibrosis and endothelial dysfunction (upregulation of the eNOS/cGMP pathway), revealing itself as an alternative therapy for ED [23].
Aging is associated with a decrease in NO synthesis due to the increased levels of superoxide anion O2− in the endothelium and cavernosal smooth muscle. Since Vitamin E (α-tocopherol) is a lipid-soluble antioxidant and oxygen-free radical scavenger, it plays an important role in modulating erectile function by exercising protection against O2− [24].
Also, zinc deficiency may cause ED, and therefore zinc supplementation is commonly included in the diet to improve sexual function [25].
Recently, the concept of nutraceuticals was extended from health promotion to the treatment of diseases, not only for their potential therapeutic effects but also for their safety. Consequently, in this work, we investigated the possibility of combining various nutraceuticals in a unique combination named Icarifil (L-Citrulline, L-Carnitine, Eruca vesicaria, Panax ginseng, Tribulus terrestris, Turnera diffusa, Taurine, Vitamin E, Zinc) to reduce penile dysfunction. Specifically, based on the literature data, the final mixture was assembled using the smallest amount of each component reporting a positive effect on the different pathways involved in erectile dysfunction in order to have a possible therapeutical efficacy without resulting toxicity.

2. Materials and Methods

L-Citrulline, L-Carnitine, Eruca vesicaria (Icariina 5%), Panax ginseng (Ginsenosids 80%), Tribulus terrestris, Turnera diffusa (damiana), Taurine, Vitamin E, and Zinc were kindly donated by Anvest Health s.r.l. (Castel San Giorgio, Salerno, Italy). Fetal bovine serum (FBS) that was qualified and heat-inactivated was acquired from Gibco (Thermo Fischer Scientific, Milan, Italy), and Dulbecco’s Modified Eagle’s Medium (DMEM) was purchased from Euroclone (Euroclone, Milan, Italy). Carboxyfluorescein succinimidyl ester was acquired from Biolegend, SanDiego, CA, USA) and 2′, 7′-dichlorofluorescein-diacetate (DCFH-DA), a fluorescent probe, from Sigma (St. Louis, MO, USA).

2.1. Preparation of Icarifil

The physical mixture (Icarifil, registration number 018623914) was prepared by mixing appropriate amounts of ingredients (as reported in Table 1) in a mortar using the geometric dilution technique.

2.2. Cell Proliferation Studies of Human Muscular Epithelium and Murine Penile Muscle Epithelium

Human muscle epithelial cells and murine penile muscle epithelium cells (SAP1) were isolated from the culture medium and resuspended in PBS to obtain a final suspension of 1 × 106 cells/mL and 1 × 107 cells/mL, respectively. Icarifil was dissolved in PBS under magnetic stirring to obtain a stock solution of 0.5 mg/mL. After that, cells were stained with 5 μM of carboxyfluorescein succinimidyl ester and incubated at room temperature for 10 min. Cells were seeded in 96-well plates (5 × 105 cells/well) with 150 μL of the medium before the initiation of proliferation assays. The epithelial cells were then treated with different amounts (100, 200, and 300 μL) of the 0.5 mg/mL solution, while penile muscle epithelium cells were treated with 100 μL of the 0.5 mg/mL solution and different combinations of the single components of Icarifil (in quantities equivalent to those present in the mixture). In both cases, the culture medium and culture medium with PBS were used as controls. The treated cells were incubated for 48 h, subsequently harvested, and the percentage of proliferating cells was determined via flow cytometry.

2.3. Determination of Induced Human Muscular Epithelium Cell Turgor

Human muscle epithelial cells were isolated from the culture medium and resuspended in PBS to obtain a final suspension of 1 × 107 cells/mL. After that, they were plated in 24-well plates (2.5 × 104 cells/well) with 500 μL of the medium. The cell turgor was correlated with the increase in weight of the treated cell by using an method adapted from Hempling et al. [26]. The epithelial cells were then treated with a solution composed of 50% of Icarifil and 50% of the culture medium and compared with the medium alone used as a control. Specifically, 200, 300, and 450 μL of Icarifil (0.5 mg/mL) were diluted 1:1 with medium and tested. After 24 h, the medium was carefully removed from each well and the plate was weighed on a precision balance (XPR204S, Mettler Toledo, Columbus, OH, USA) and compared with its weight before treatment. The weight obtained was then normalized in relation to the number of cells contained in each plate at the end of the experiment. The normalized weight increases of the mixture-treated cells were compared with those of untreated cells to evaluate the increases in membrane permeability and cell turgor. All the experiments were conducted in triplicate.

2.4. Measurement of Phosphodiesterase Type 5 (PDE5) Protein and Transcript Levels

Studies of the effects of the complete mixture on phosphodiesterase type 5 were conducted via ELISA assay, using a commercial ELISA kit (BD OptEIA, BD Biosciences, CA) following the manufacturer’s instructions. To evaluate the expression of PDE5 in treated and untreated samples, real-time PCR was performed using 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 by Zhang et al. [27]. The studies were performed using both human muscle epithelial cells and mouse penile muscle epithelial cells. After their culture in DMEM medium supplemented with 10% fetal bovine serum, the cells were treated with single components of Icarifil (0.5 mg/mL), different combinations of the components in amounts equal to those present in Icarifil, and with Icarifil. The cells were also treated with the inhibitor of the phosphodiesterase V, tadalafil (2,3,6,7,12,12a-exahydropyrazino[1′,2′:1,6]pyrido[3,4-b]indole-1,4-dione substituted in position 2 by methyl and in position 6 by the 1,3-benzodioxol-5-yl group) alone or in co-treatment with Icarifil (0.5 mg/mL). Cells were homogenized in a 0.01 M of PBS solution (pH 7.4) and subsequently centrifuged (3000 rpm, 4 °C, 20 min). The supernatant was used to quantify the PDE5 levels through immunoenzymatic analysis. The experiments were carried out sixfold.

2.5. Measurement of Reactive Oxygen Species (ROS) Levels in Murine Penile Muscle Epithelium Cells

Modulation of the intracellular level of ROS by citral in murine penile muscle epithelium cells (SAP1) was measured using the 2′, 7′-dichlorofluorescein-diacetate (DCFH-DA) fluorescent probe. Cells were plated (1 × 106 cells/mL) in 60 mm culture dishes and incubated with 10 μM DCFH-DA for 30 min at 37 °C. SAP1 cells were pretreated with Icarifil or with different combinations of the single components in amounts equivalent to those present in Icarifil for 30 min before treatment for 2.5 h with citral to induce oxidative stress.
At the end of the treatment, the cells were rinsed, harvested, and centrifuged at 1000 rpm for 5 min. The fluorescence signal was measured using the flow cytometer detected in the FL1-A channel.

2.6. Statistic Analysis

Statistical analysis was performed using GraphPad Prism 9.04 (San Diego, CA, USA). The statistical test used was the One-WAY ANOVA followed by the Bonferroni test as a post-test, or the Mann–Whitney test when appropriate. The p values are indicated in the individual graphs. The results are expressed as means ± SD. Data were statistically analyzed using an unpaired Student’s t-test. A p-value of <0.05 was considered statistically significant.

3. Results and Discussion

3.1. Cell Proliferation

In order to determine the biochemical mechanisms by which our formulation could be used for ED, the current in vitro studies were conducted.
The capacity of the formulation to improve cell proliferation was evaluated using human muscle epithelial cells as a preliminary test. As can be seen from Figure 1, Icarifil was capable of positively and significantly stimulating cell proliferation. Specifically, different amounts of a 0.5 mg/mL solution were tested. It was found that 100 µL of the solution was able to increase cell proliferation by about 22% compared to the control, while 200 µL of the solution was able to increase the degree of proliferation up to 29%; however, the further increase in the amount of the dose of Icarifil did not lead to a significant change in its effect.
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. As shown in Figure 2, Icarifil was able to increase cell proliferation by about 43% compared to the control, whereas various combinations of the components used, although they still showed a positive action on cell proliferation, never achieved an effect above 29%. Different works have reported that the combination of various nutraceuticals provides results superior those compared to single agents, probably due to the synergic effect between the components in the mixture [13,14].

3.2. Human Muscular Epithelium Cell Turgor

Figure 3 shows the normalized weight gain of human muscle epithelial cells treated with different dosages of a solution consisting of Icarifil (0.5 mg/mL) and the culture medium in a 1:1 ratio. In order to determine the effectiveness of Icarifil in increasing human muscular epithelium cell turgor, a non-invasive technique for measuring rapid changes in cell water volume was used [26]. The direct relationship between weight increase and treatment of Icarifil was interpreted as a result of a change in membrane permeability and cell turgor. Specifically, Figure 3 exhibits how the addition of Icarifil solution was able to increase permeability and salt/nutrient retention from the treated cells by about 10 and 15% compared to the control when using 400 µL or 600 µL, respectively, of the total solution of Icarifil and medium. At 900 µL, the activity reached a plateau, probably due to internal osmotic pressure effects.

3.3. Reduction of Phosphodiesterase Type 5 (PDE5) Protein and Transcript Levels

To determine whether the formulation affects the expression of the PDE5 enzyme, human muscle epithelial cells were incubated with Icarifil and L-Citrulline in the same amount present in the mixture. Figure 4 reports the variations in PDE5 protein level and transcriptional levels of epithelial cells after treatment. Icarifil (0.5 mg/mL) was able to reduce the level of human PDE5 in a significantly higher manner than the vehicle used as a control, and L-Citrulline was dosed in an equivalent amount present in the mixture. Specifically, Icarifil showed efficacy in reducing PDE5 protein levels higher than L-Citrulline by 22% and 45% compared to the control. This difference further increased when transcriptional levels of PDE5 were evaluated, where the total mixture was more effective than L-Citrulline alone at levels of about 40%. It is clear from these data that when L-Citrulline is used in combination with the other component in a unique formulation, the efficacy on the PDE5 enzyme is more evident than when it is used by itself [28].
To verify whether these results translated into mouse penile muscle cells, the cells were incubated with Icarifil or components comprising the formulation. The results showed that Icarifil (0.5 mg/mL) was able to reduce the activity of phosphodiesterase type 5 significantly more effectively than the control and the mixture of L-Citrulline and L-Carnitine alone or in combination with Eruca vesicaria, Turnera diffusa, and Tribulus Terrestris, dosed in an equivalent ratio to that present in the mixture (Figure 5A). Specifically, the total mixture showed efficacy in reducing PDE5 levels higher than 65% compared to the control and 35% compared to the mixture of L-Citrulline and L-Carnitine, the major components of Icarifil. This difference was also evident in the relative transcriptional levels of PDE5 (Figure 5B), where Icarifil was substantially more effective than the control (about 57%) and the mixture consisting of L-Citrulline and L-Carnitine (about 22%). These results could be explained by the synergic effect between the components of the multicomponent mixture, demonstrating that this complex mixture could reduce PDE5 levels more effectively than the single components.
After assessing the ability of Icarifil to reduce the activity of PDE5 enzyme more than the single extract, its activity was compared with a well-known PDE5 inhibitor. Therefore, mouse penile muscle cells were treated with an aqueous solution of tadalafil alone (10 µmol/L and 5 µmol/L) and in co-treatment with Icarifil (0.5 mg/mL). As can be seen in both panels of Figure 6, the capacity of tadalafil to reduce PDE5 protein and transcriptional levels at both tested concentrations showed significantly superior results compared to the control and Icarifil, where the highest concentration (10 µmol/L) was able to return an inhibitory response more than double that of the lowest concentration (5 µmol/L) and more than four times higher than Icarifil at 0.5 mg/mL. However, it is interesting to note that, when Icarifil was co-administered with 5 µmol/L tadalafil, a synergistic effect on PDE5 was exhibited. In fact, the administration of Icarifil three times a day in association with tadalafil administered once a day was able to reduce the activity of the PDE5 enzyme, as shown in Figure 6. In fact, this association led to activity being registered when tadalafil was administered at a concentration of 10 µmol/L. The presence of Icariina in Icarifil located near the entrance of the PDE5 active binding site modulates the affinity of this enzyme for its ligand [29]. Moreover, Citrulline and Carnitine administered together with PDE5 inhibitors are able to increase the efficacy of inhibitors on PDE5 [13,30].

3.4. Modulation of the Intracellular Level of ROS

Reactive oxygen species (ROS) act as one of the important mediators in the pathophysiological process of ED. Studies have shown that increased ROS reduces NO production or bioavailability, leading to impaired endothelial function and erectile function [31]. Therefore, 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. This effect was greatest in the case of Icarifil, capable of counteracting the formation of ROS by about 70% compared to the control, whereas the individual mixtures, also due to the quantity of the various antioxidant agents present, proved capable of reducing the levels of ROS at the intracellular level by a maximum of 58%, as in the case of the mixture composed of L-Citrulline, L-Carnitine, and Eruca vesicaria. However, the mixture presented better activity thanks to other nutraceuticals, Vitamin E, Taurine, and Zn, which, acting as an antioxidant, may have suppressed testis oxidant enzyme activity and testosterone synthesis, blocking oxidative stress [32]. Hence, it seems plausible that a new formulation containing antioxidant elements could have a beneficial effect on ED, reducing the risk of ROS-mediated damage.

4. Conclusions

Phosphodiesterase type 5 inhibitors (PDE5i) are the first-choice class of drugs in therapy for erectile dysfunction. However, their high cost and side effects led to searches for other alternatives. This study has demonstrated the possibility of using a mixture of natural substances (L-Citrulline, L-Carnitine, Eruca vesicaria, Panax ginseng, Tribulus terrestris, Turnera diffusa, Taurine, Vitamin E, Zinc) to modulate ROS and PDE5 levels properly while increasing muscular cell proliferation and cell turgor. Such results show how this specific mixture, Icarifil, could be considered a safe alternative or an adjuvant to PDE5i drugs, even if additional clinical studies are necessary to evaluate a dose, bioavailability, efficacy, and safety.

Author Contributions

Conceptualization, P.D.G.; methodology, L.L. and G.F.; validation, C.A.; investigation, C.A. and C.D.S.; writing—original draft preparation, G.L. and P.D.G.; review and editing, L.L. and A.D.V. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Conflicts of Interest

Authors Gianni Luccheo, Anna Di Vernieri, and Luigi Luccheo were employed by the company Anvest Health S.r.l. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Figure 1. Dose-dependent effect of Icarifil (100, 200, and 300 µL solution prepared at 0.5 mg/mL) on the proliferative activity of human muscle epithelial cells compared with culture medium and culture medium + Icarifil solvent, used as controls. Data represent mean  ±  SD, significance ** p < 0.05, using the One-WAY ANOVA test followed by Bonferroni test as a post-test.
Figure 1. Dose-dependent effect of Icarifil (100, 200, and 300 µL solution prepared at 0.5 mg/mL) on the proliferative activity of human muscle epithelial cells compared with culture medium and culture medium + Icarifil solvent, used as controls. Data represent mean  ±  SD, significance ** p < 0.05, using the One-WAY ANOVA test followed by Bonferroni test as a post-test.
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Figure 2. Effect on the proliferative activity of murine penile muscle cells of Icarifil (100 μL of 0.5 mg/mL) and different mixtures of components, dosed in quantities equivalent to those present in the formulation compared to the medium and medium with 100 μL of PBS added used as controls. Data represent mean  ±  SD with significance, ** p < 0.05, using the One-WAY ANOVA test followed by Bonferroni test as a post-test.
Figure 2. Effect on the proliferative activity of murine penile muscle cells of Icarifil (100 μL of 0.5 mg/mL) and different mixtures of components, dosed in quantities equivalent to those present in the formulation compared to the medium and medium with 100 μL of PBS added used as controls. Data represent mean  ±  SD with significance, ** p < 0.05, using the One-WAY ANOVA test followed by Bonferroni test as a post-test.
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Figure 3. Dose-dependent effect of 400, 600, and 900 µL of a solution consisting of Icarifil and culture medium in 1:1 ratio on weight gain of muscle epithelial cells compared with the culture medium used as a control. Data represent mean ± SD with significance ** p < 0.05, *** p < 0.01 using the One-WAY ANOVA test followed by Bonferroni test as a post-test.
Figure 3. Dose-dependent effect of 400, 600, and 900 µL of a solution consisting of Icarifil and culture medium in 1:1 ratio on weight gain of muscle epithelial cells compared with the culture medium used as a control. Data represent mean ± SD with significance ** p < 0.05, *** p < 0.01 using the One-WAY ANOVA test followed by Bonferroni test as a post-test.
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Figure 4. Treatment effect of Icarifil (0.5 mg/mL) on human phosphodiesterase type 5 activity testing human muscle epithelial cells compared with the action of L-Citrulline alone (equivalent amount present in the formulation). PDE5 protein level variation (A) and PDE5 relative transcriptional levels (B) The medium was used as a control. Data represent mean  ±  SD, significance ** p < 0.05, using the One-WAY ANOVA test followed by Bonferroni test as a post-test.
Figure 4. Treatment effect of Icarifil (0.5 mg/mL) on human phosphodiesterase type 5 activity testing human muscle epithelial cells compared with the action of L-Citrulline alone (equivalent amount present in the formulation). PDE5 protein level variation (A) and PDE5 relative transcriptional levels (B) The medium was used as a control. Data represent mean  ±  SD, significance ** p < 0.05, using the One-WAY ANOVA test followed by Bonferroni test as a post-test.
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Figure 5. Treatment of Icarifil (0.5 mg/mL) on the activity of phosphodiesterase type 5 on murine penile muscle cells in comparison with the action of different mixtures of the components, in an amount equivalent to that present in Icarifil. (Panel (A)) PDE5 protein level variation and (Panel (B)) PDE5 relative transcriptional levels. The vehicle alone was used as a control. Data represent mean  ±  SD, significance ** p < 0.05, using the One-WAY ANOVA test followed by Bonferroni test as a post-test.
Figure 5. Treatment of Icarifil (0.5 mg/mL) on the activity of phosphodiesterase type 5 on murine penile muscle cells in comparison with the action of different mixtures of the components, in an amount equivalent to that present in Icarifil. (Panel (A)) PDE5 protein level variation and (Panel (B)) PDE5 relative transcriptional levels. The vehicle alone was used as a control. Data represent mean  ±  SD, significance ** p < 0.05, using the One-WAY ANOVA test followed by Bonferroni test as a post-test.
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Figure 6. Effect of Icarifil on phosphodiesterase type 5 protein level (Panel (A)) phosphodiesterase type 5 transcription level (Panel (B)) on murine penile muscle cells compared with the action of tadalafil at different concentrations and with co-treatment of tadalafil and Icarifil. The vehicle alone was used as control. Data represent mean  ±  SD, significance ** p < 0.05, using the One-WAY ANOVA test followed by Bonferroni test as a post-test.
Figure 6. Effect of Icarifil on phosphodiesterase type 5 protein level (Panel (A)) phosphodiesterase type 5 transcription level (Panel (B)) on murine penile muscle cells compared with the action of tadalafil at different concentrations and with co-treatment of tadalafil and Icarifil. The vehicle alone was used as control. Data represent mean  ±  SD, significance ** p < 0.05, using the One-WAY ANOVA test followed by Bonferroni test as a post-test.
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Figure 7. Flow cytometric analysis of intracellular ROS induced by citral (100 µM) on murine penile muscle cells after treatment with the complete mixture (0.5 mg/mL) and different mixtures of components, dosed in amounts equivalent to those present in the formulation in comparison with the culture medium used as a control. Data represent mean  ±  SD, significance ** p < 0.05, *** p < 0.01 using the One-WAY ANOVA test followed by Bonferroni test as a post-test.
Figure 7. Flow cytometric analysis of intracellular ROS induced by citral (100 µM) on murine penile muscle cells after treatment with the complete mixture (0.5 mg/mL) and different mixtures of components, dosed in amounts equivalent to those present in the formulation in comparison with the culture medium used as a control. Data represent mean  ±  SD, significance ** p < 0.05, *** p < 0.01 using the One-WAY ANOVA test followed by Bonferroni test as a post-test.
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Table 1. Amount of ingredients in the physical mixture (Icarifil).
Table 1. Amount of ingredients in the physical mixture (Icarifil).
SampleAmount (mg)
L-Citrulline1500
L-Carnitine500
Eruca vesicaria (Icariina 5%)200
Panax ginseng (Ginsenosids 80%)150
Tribulus terrestris100
Turnera diffusa (damiana)100
Taurine50
Vitamin E50
Zinc15
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MDPI and ACS Style

Amante, C.; De Soricellis, C.; Luccheo, G.; Di Vernieri, A.; Luccheo, L.; Falcone, G.; Del Gaudio, P. Icarifil, a Natural Mixture Based on L-Citrulline and L-Carnitine as a Novel Multicomponent Nutraceutical to Modulate ROS and PDE5. Appl. Sci. 2023, 13, 12358. https://doi.org/10.3390/app132212358

AMA Style

Amante C, De Soricellis C, Luccheo G, Di Vernieri A, Luccheo L, Falcone G, Del Gaudio P. Icarifil, a Natural Mixture Based on L-Citrulline and L-Carnitine as a Novel Multicomponent Nutraceutical to Modulate ROS and PDE5. Applied Sciences. 2023; 13(22):12358. https://doi.org/10.3390/app132212358

Chicago/Turabian Style

Amante, Chiara, Chiara De Soricellis, Gianni Luccheo, Anna Di Vernieri, Luigi Luccheo, Giovanni Falcone, and Pasquale Del Gaudio. 2023. "Icarifil, a Natural Mixture Based on L-Citrulline and L-Carnitine as a Novel Multicomponent Nutraceutical to Modulate ROS and PDE5" Applied Sciences 13, no. 22: 12358. https://doi.org/10.3390/app132212358

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