Paullinia pinnata L. (Sapindaceae) is an African tropical plant whose roots and leaves are used in traditional medicine for many purposes, especially for erectile dysfunction, but its action mechanism is unknown. Some previous chemical investigations have been done on leaves showing the presence of triterpene saponins and polyphenols, including cardiotonic catechol tannins and flavonoids.1–3 However, no pharmacological investigation about this traditional use has been carried out on P. pinnata.
Penile erection is a complex process involving a series of sequential and coordinating events between the central nervous system, mainly at the cortical, hypothalamic and spinal levels, which have connections to the efferent and afferent nerves of the penis that trigger and conduct the neurotransmission signals to the organ; and the vascular system, specifically the smooth muscle in the corpora cavernosa and within the media of the arteries that deliver blood to the penis.4–6 The penis can be considered simply as an extension of the vascular system. The physiology of an erection involves a complex balance of contraction and relaxation processes regulating the contractile activity of smooth muscle associated with penile circulation.7–9 Erection results from relaxation of the smooth muscle in the arterioles and cavernous sinuses of the penis.10 This relaxation permits increased blood flow into the sinuses and, as the sinuses fill, they expand against the tunica albuginea to activate the veno-occlusive mechanisms that limit blood outflow.11 The principal agent causing relaxation of smooth muscle leading to the erectile response is nitric oxide (NO),12,13 although vasoactive intestinal polypeptide14,15 and prostaglandin E may participate as well.16 The reaction leading to NO production is the conversion of L-arginine into citrulline, and in the penis this occurs in the nerve terminals of the corpora cavernosa, involving the neuronal NO synthase (NOS; nNOS or NOS I), and presumably also in the endothelium of the cavernosal cisternae and arteries, involving endothelial NOS (eNOS or NOS III). The primary trigger for erection is the nitrergic neurotransmission through the nonadrenergic noncholinergic nerves catalyzed by the stimulation of nNOS activity via Ca2+ binding to the nNOS-associated calmodulin. This process is in part modulated by Ca2+ fluxes through another nNOS-associated protein, the N-methyl-D-asparate receptor.17 The eNOS activation via sheer stress, Ca2+ flux, and phosphorylation appears to be ancillary and supports NO synthesis in the corpora cavernosa during erection.18,19
The NO liberated from penile nerves and endothelium activates guanylyl cyclase in the trabecular and penile arterial smooth muscle, respectively, causing the elevation of cyclic guanosine monophophate (cGMP) levels that stimulate a cGMP protein (PKG) and the subsequent reduction in cytoplasmic Ca2+. This leads to smooth muscle relaxation and penile erection, counteracting adrenergic and related agents that maintain its tone during the flaccid stage. In turn, cGMP breakdown is catalyzed in the smooth muscle by cGMP-dependent phosphodiesterase (PDE) 5A, the target of the PDE5 inhibitors used orally for the treatment of erectile dysfunction, such as sildenafil (Viagra).
Although the cardiovascular effects and many of the drug–drug interactions of sildenafil have already been addressed,20 there has been much concern about possible interactions of sildenafil with other drugs,21 especially in patients at increased cardiovascular risk. This is because sildenafil-induced increases in cGMP levels can potentiate cGMP-mediated dilator responses to other drugs that activate the NO-cGMP pathway and cause severe systemic hypotension and death. For example, the vasodilator actions of nitrates are potentiated with concomitant use of sildenafil.20,22 Therefore, it is generally accepted that sildenafil should not be used by patients taking nitrates or other NO donors, regardless of their hemodynamic site of action, because of the risk of developing potentially life-threatening hypotension.20
Most of the time the penis is flaccid because of contraction of the smooth muscle of the cavernosal arterioles and sinuses that limits blood flow through the penis. Contraction of cavernosal smooth muscle can be induced by a variety of vasoconstrictor agents that ultimately restrict blood flow entering the penis and blood volume contained in the cavernosal tissue. The adrenergic agonist norepinephrine23,24 and endothelin-1 (ET-1)25 are two vasoconstrictors believed to have important roles in regulating penile blood flow. ET-1 has been suggested to be the principal agent that maintains cavernosal smooth muscle in the contracted state, which prevents erection.26–29 Bell et al30 and Saenz de Tejada et al31 have demonstrated the presence of both ETA and ETB-receptor subtypes in erectile tissue. Dai et al32 reported that ET-1 causes vasoconstriction in the penile circulation of the rat and that an antagonist to the ETA receptor prevents vasoconstriction.
The goal of this study was to complete phytochemical investigation of P. pinnata leaves and roots and to test whether polar extracts of both organs induce endothelium-dependent vasorelaxation and modulate the expression of eNOS and ET-1 in endothelial cells. This approach could provide some explanation for the traditional use of P. pinnata in the treatment of erectile dysfunction.
MATERIALS AND METHODS
The leaves and roots of P. pinnata were collected in July 2002 in Ivory Coast, Africa. Plant samples were identified by Prof Ake Assi, one of the founders of the National Floristic Centre of the University of Abidjan. Voucher specimens were deposited at the Herbarium of the Department of Botany (Herbarium code: LIP), Faculty of Pharmacy, University of Lille 2, France.
Dried and powdered leaves (59 g) and roots (110 g) were extracted with CH2Cl2 (3×500 mL) and MeOH (3×500 mL) at room temperature for 24 h. Combined MeOH extracts were filtered and reduced in vacuo yielding 2.4 g (leaf) and 17.0 g (roots) of dried extracts. Thin-layer chromatography (TLC) analyses were carried out on precoated silica gel 60 F254 and cellulose sheets (Merck). Polyphenolic groups were detected by UV fluorescence and spraying with 1% 2-aminoethyl diphenylborinate (flavonoids) and 2% 4-(dimethylamino) cinnamaldehyde/HCl (tannins), and they were quantified by the means of spectrocolorimetric assay according to the European Pharmacopoeia protocols33 on a Hitachi U-1100 spectrophotometer.
Only leaf and root MeOH extracts that contain major polar compounds were used for the pharmacological tests to be in accordance with traditional protocols used in African medicine. The MeOH residues were dissolved in appropriate solutions according to the experiments to prepare different concentrations. All solvents and chemical reagents were purchased from Carlo Erba (Rodano MI, Italy) and Sigma-Aldrich (St Louis, MO).
Arterial Relaxation Study
All experimental procedures used in this study were carried out according to the guidelines of the Nara Medical University Animal Welfare Committee, adhering to the terms of the Declaration of Helsinki. Male Wistar rats (5–8 weeks old) were purchased from Charles River Laboratory (L'arbreles, France). Animals were anesthetized with sodium thiopental (5 mg/kg, intravenous) and heparinized (2000 IU). The vasoreactivity of isolated thoracic aorta of male Wistar rats was analyzed as previously described.34 Briefly, the thoracic aorta was quickly removed, cleaned of all adipose and connective tissues, and cut into rings of 3-mm length. The rings were placed in bath chambers (40 mL) for isolated organs containing modified Krebs salt solution of the following composition (mmol/L): NaCl 130, CaCl2 1.6, MgSO4 1.2, KH2PO4 1.2, KCl 4.7, NaHCO3 14.9, glucose 5.5, which was maintained at 37°C, pH 7.4, and bubbled with 95% O2 and 5% CO2. The rings were mounted on stainless hooks in chambers (Radnoti Glass Technology, Monrovia, CA) under passive tension gradually adjusted to 2 g resting tension and then connected to force transducers. Changes in isometric force were recorded during the 90 min of the equilibration period followed by the contractile response to a depolarizing concentration of KCl (70 mmol/L) to measure maximal contraction to KCl in each ring. The contractile response of aorta rings was detected by adding cumulative concentrations of phenylephrine (PE; 10−9–3.10−5 mmol/L) and is expressed as the percentage of the maximal response to KCl.
After washout, rings were incubated with N G-nitro-L-arginine methyl ester (L-NAME; 10 μmol/L) for 30 min, whereas control arterial rings received no treatment. The rings were then contracted with 10 μmol/L PE and cumulative concentration-response curves for leaf and root methanolic extracts of P. pinnata (0.0125–1.2 g/L) were constructed. The effects of each drug concentration were measured 6–10 min after the responses became steady. The relaxation response was analyzed as a percentage decrease from the maximal contraction induced by PE.
Bovine aortic endothelial cells (BAEC) were isolated as previously described35 and cultured in Dubelco's modified Eagle's medium (DMEM) supplemented with 10% newborn calf serum, 600 ng/mL glutamine, and 100 U/mL penicillin. BAEC were used at passages 4 to 8. BAEC were then subcultured in 6-well plates. Subconfluent cells were treated for 4 h with the presence of extracts at 0.001 to 1 mg/L or without extracts (control: medium with 0.1% DMSO).
RNA Extraction and Analysis
After treatment, total cellular RNA was extracted from cells using Trizol (Life Technologies, France). For quantitative polymerase chain reaction (QPCR), total RNA was reverse transcribed using random hexameric primers and Superscript reverse transcriptase (Life Technologies). cDNAs were quantified by real-time PCR on an MX 4000 (Stratagene, La Jolla, CA) using specific primers for bovine eNOS: 5′-GC-AAC-CAC-ATC-AAG-TAC-GCC-ACC-3′ and 5′-GAT-GCA-GAG-CTC-CGT-GAT-CTC-CAC-3′ for bovine ET-1: 5′-T-GCT-GCT-CTT-CCC-TGA-TGG-3′ and 5′-GGC-ATC-TCT-TCC-TGT-GGA-CTG-TCG-3′ for bovine 36B4: 5′-CAT-GCT-GAA-CAT-CTC-CCC-CTT-CTC-C 3′ and GGG-AAG-GTG-TAA-TCA-GTC-TCC-ACA-G 3′. PCR amplification was performed in a volume of 25 μL containing 100 nmol/L of each primer, 4 mmol/L MgCl2, the Brilliant Quantitative PCR Core Reagent Kit mix as recommended by the manufacturer (Stratagene), and SYBR Green 0.33X (Sigma-Aldrich). The thermal cycling conditions were 95°C for 10 min, followed by 40 cycles of 30 s at 95°C, 55°C, and 72°C. The eNOS and ET-1 mRNA levels were subsequently normalized to internal control 36B4 mRNA.
Effect of Extracts on Peroxisome Proliferator Activated Receptor-α Transcriptional Activity Using Chimeric Protein Constructs
Cos-7 cells were obtained from ATCC (CRL-1651). Cells were transfected in Opti MEM without fetal calf serum (FCS) for 3 h at 37°C, using polyethylenimine, with reporter (pGal5-Tk-pGL3) and expression plasmids for (pGal4-hPPAR-α as described Raspé et al.36 Transfection was stopped by addition of DMEM supplemented with 10% FCS. After 16 h of culture, cells were trypsinized and seeded in 96-well plates, then incubated 16 h in DMEM containing 0.2% FCS with different compounds.
Cells were treated with either vehicle (0.1% vol/vol DMSO), P. pinnata root or leaf extracts (0.01–100 mg/L). Their capacity to induce PPAR-α activity was compared with the efficiency of potent PPAR-α activators fibrate (ciprofibrate, fenofibric acid, wy 14,643, gemfibrozil) at the indicated concentration. (Fenofibric acid was a gift from Dr A. Edgar of Laboratoires Fournier, Daix, France; the other fibrates were purchased from Sigma Chemical, St Louis, MO.) At the end of the experiment, luciferase and the β-galactosidase assays were performed. The results were expressed in RLU/βGal ratio. The amount of hPPAR/βGal bound to coactivators in the presence of the indicated ligands is expressed relative to that measured in presence of ligand wy 14,643 (100%).
In Vitro Scavenging Effects of P. pinnata Extracts
Versus Superoxide Oxygen (O2 .-)
Scavenging effects of P. pinnata leaf and root extracts were carried out on an a cellular generation of
by using the hypoxanthine-xanthine oxidase system.37 This system produces
and the reaction is based on the cytochrome C reduction by
unscavenged by studied extracts. Reduced cytochrome C is quantified spectrophotometrically as its maximum of absorbance is detected at 550 nm. The reaction mixture contained 1 mmol/L EDTA, 0.1 mmol/L hypoxanthine in 50 mmol/L KOH, 20 μmol/L cytochrome C with various concentrations of extracts (50–600 mg/L) in a final volume of 1.5 mL, buffered in KH2PO4-KOH (pH 7.4). The reaction was started by adding 0.1 U of xanthine oxidase. Absorbance was measured against a reference cuvette which did not contain the
The amount of
was calculated using Beer-Lambert law with the extinction coefficient ε550=2.1×102 mol/cm. Percentage of the inhibited
production by extracts was calculated and results were expressed in inhibitory concentration (IC50) values.
Versus Hydrogen Peroxide (H2O2)
H2O2 was determined by means of the peroxidase-dependent oxidation of phenol red.38 Phenol red oxidation by H2O2 leads to a color change in H2O2 basic medium (addition of NaOH) and is detected spectrometrically at 610 nm. Samples contained 100 μL of extract in various concentrations (see above). The total volume was brought to 1 mL by adding the appropriate amount of phosphate-buffered saline (PBS) pH 7.4 and incubated for 15 min at 37°C. Then, the tubes were overlaid with 1 mL of 0.2 mg/mL phenol red dye containing 17 U/mL horseradish peroxidase type II. After 15 min at 37°C, 50 μL of 1 mol/L NaOH was added and absorbances were recorded. Absolute values were derived from standard curves obtained with the reagent H2O2 (1–20 μmol/L). Percentage of inhibited H2O2 amount by extracts was calculated, and results were expressed in IC50 values.
Versus Hypochlorous Acid (HOCl)
HOCl was measured by means of the chlorination of taurine.39 HOCl unscavenged by studied extracts reacts with taurine to give TnCl, which combined with potassium iodide leads to a yellow coloring (oxidation of I− into I2) detected at 350 nm. Sample cuvettes contained 100 μL of 600 μmol/L HOCl, 100 μL of 150 mmol/L taurine, and 100 μL of extracts in various concentrations (see above), for a total volume of 1 mL in PBS at pH 7.4. Assays were terminated by the addition of catalase (50 μg). After the addition of 20 mmol/L of potassium iodide, absorbance was measured against reference cuvettes at 350 nm. The differential spectrum was directly proportional to the amount of HOCl, calculated with the extinction coefficient E350=22.9 mmol/cm. Percentage of inhibited HOCl amount by extracts was calculated, and the results were expressed in IC50 values.
Low-Density Lipoprotein (LDL) Oxidation Inhibition Test
Human LDLs were isolated from freshly drawn blood from healthy, normolipidemic, fasting volunteers. LDLs were isolated by sequential density gradient ultracentrifugation in sodium bromide density solutions in the density range of 1.019 to 1.063 g/mL as previously reported.40 The protein concentration was determined by Peterson's method41 using bovine serum albumin as the standard. Oxidation of LDL was initiated by copper. To eliminate the possible role of copper chelation, oxidation was also induced by water soluble 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH), which generates free radicals, by its spontaneous thermal decomposition.
LDLs were extensively dialysis against 0.01 mmol/L PBS, pH 7.4, under N2 at 4°C. Then, oxidation was induced at 30°C by adding 20 μL of 16.6 μmol/L CuSO4 or 2 mmol/L AAPH to 160 μL of LDL (125 μg of protein/mL) in the presence or not of 20-μl P. pinnata extracts from 0.030 to 0.125 g/L. During copper or AAPH-induced LDL oxidation, diene-conjugated formation was followed by measuring optical density at 234 nm every 10 min for 8 h at 30°C with a thermostated Spectra Max Plus Molecular Devices spectrophotometer (96 wells, Molecular Devices, Sunnyvale, CA).42 There was a positive relationship between lag phase durations and the concentrations of antioxidant extracts (0.030–0.125 g/L) contained in the LDL mixture. We considered that the drug had 100% activity (ED100) when control lag phase duration doubled, and we defined ED50 as the concentration of the drug that increased this control lag phase by 1.5 times.
Results were expressed as mean±SD when a minimal number of 3 independent experiments were performed in triplicate. The comparison of data between >2 groups was analyzed by ANOVA comparison. Significant differences were then subjected to post hoc analysis by using the Sheffe test. A value of P<0.05 was accepted as statistically significant.
Phytochemical screening of P. pinnata roots and leaves by means of TLC showed that the major chemical substances in methanolic extracts were catechic tannins and flavonoids. TLC analysis of polar extracts demonstrated that these tannins were of a catechic type with a high degree of polymerization. Therefore, their isolation has not been possible by using current analytical methods. Consequently, in this work, only total methanolic extracts were used for pharmacological testing. The quantification of tannins and flavonoids yielded the following proportions: 15% and 0.5% in roots and 3.8% and 3.40% in leaves, respectively (Fig. 1).
The aorta ring strip of rat exhibited a strong contraction after an initial application of 10−5 mmol/L phenylephrine to the organ bath. Root and leaf extract applications (0.0125–1.2 g/L) potentially relaxed the contraction induced by phenylephrine in a concentration-dependent manner, as shown in Figure 2. Whatever the concentration, the importance of the amplitude of vasorelaxation was significant with root extracts, whereas this amplitude began to be significant with 0.05 g/L of leaf extracts. Root and leaf extracts at 1.2 g/L decreased the contraction by 87.19%±3.9% and 97.55%±2.1% (n=6; P<0.001), respectively.
To examine the involvement with endothelium-dependent relaxation via NO activation, a pretreatment for 30 min with L-NAME (a nonselective NO synthesis inhibitor) was carried out. The vasorelaxation was significantly attenuated because 0.0125 g/L for root extracts and 0.025 g/L for leaf extract. Increased concentrations of root and leaf extracts from 0.0125 to 1.2 g/L induced a significantly lower vasorelaxation than it did in the absence of L-NAME. The IC50 with L-NAME are, respectively, 0.8 and 0.36 g/L versus control for root extracts and 0.84 and 0.14 g/L for leaf extracts (Figs. 3 and 4).
To examine the involvement of eNOS and ET-1 expressions in arterial endothelium as a major factor contributing to arterial vasomotricity, treatment of BAEC with P. pinnata extracts was initiated. BAEC were incubated 4 h with 1 ng/mL to 1 μg/mL root and leaf methanolic extracts of P. pinnata. Total mRNA expressed in BAEC was amplified by real-time PCR. Then eNOS and ET-1 mRNA expressions in the cells were quantified by QPCR. As shown in Figure 5, root extract stimulated eNOS mRNA expression in endothelial cells in a concentration-dependent manner. Concentrations of 0.001, 0.01, 0.1, and 1 mg/L significantly stimulated eNOS expression with relative quantities of 1.89±0.23, P<0.05, 1.95±0.22, P<0.05, 2.88±0.33, P<0.01, and 4.5±0.37, P<0.01, respectively, in comparison with controls (1.01±0.15). Root extract inhibited ET-1 mRNA expression in a concentration-dependent manner by reducing the relative ET-1 mRNA quantities of 10.9±0.22, NS, 0.8±0.11, P<0.05, 0.46±0.1, P<0.01, and 0.21±0.1, P<0.01, respectively, compared with controls (1.0±0.2; Fig. 6).
Comparable observations were made with leaf methanolic extract (Fig. 5). Concentrations of 0.001 μg/mL, 0.01 mg/L, 0.1 mg/L, and 1 mg/L of leaf extract significantly stimulated eNOS expression, with relative quantities of 1.36±0.25, P<0.05, 1.65±0.39, P<0.05, 2.58±0.25, P<0.01, and 3.52±0.45, P<0.01, respectively, in comparison with controls (1.02±0.17). Leaf extract inhibited ET-1 mRNA expression in a concentration-dependent manner by reducing the relative ET-1 mRNA quantities of 1.01±0.18, NS, 0.8±0.1, P<0.05, 0.42±0.1, P<0.01, and 0.21±0.11, P<0.01, respectively, in comparison with control (1.0±0.2; Fig. 6).
Effect of Extracts on PPAR-α Transcriptional Activity
To avoid interference with endogenous nuclear receptors, we used 2 chimeras comprising the DNA-binding domain of the yeast transcription factor Gal4 fused to the ligand binding domain of human PPAR-α and a reporter vector containing 5 copies of the Gal4 response element cloned in front of the herpes simplex thymidine kinase promoter and the luciferase reporter gene. The advantage of this assay is that the background is low because the chimeras and the Gal4 transcription factor are not expressed in nontransfected target cells. When cotransfected with appropriate reporter vector in Cos-7 cells, the Gal4-hPPAR-α chimera was activated by the reference fibrates in a concentration-dependent manner. The effects of fibrates were concentration dependent. A significant activation was already noticed at a concentration as low as10−7 mmol/L (ciprofibrate and fenofibric acid) and at 10−5 mmol/L (wy 14,643, gemfibrozil). Data obtained with root and leaf extracts were pooled to be sufficiently numerous to be statistically tested in comparison with controls. It was not technically possible to test the effect of root and leaf methanolic extracts over 0.1 g/L. This dose tends to increase by 15% PPAR-α activity in comparison with 10−5 mmol/L WY (100% activity) in the system described by Raspé et al36 without reaching the level of significance (P=0.06). Figure 7 shows that strong PPAR-α activators (ciprofibrate, fenofibric acid) start to induce PPAR-α transcriptional activation at 10−7 mmol/L, whereas this effect was not observed at the same dose with wy 14,643 or gemfibrozil.
ROS Scavenging Activities
Scavenging of O2 .-, H2O2, and HOCl
Results expressed in IC50 values (with 95% confidence intervals) are found in Table 1. Both extracts scavenged all tested ROS, but root extract activities were stronger than leaf extracts. The best activities are obtained for the scavenging of H2O2 and HOCl for root methanolic extracts. Moreover, for the 2 extracts,
value is more important.
is not stable and difficult to scavenge, which is probably caused by its rapid reaction rate.
Inhibition of LDL Oxidation
The capacity of leaf and root methanolic extracts to reduce or inhibit copper or AAPH-initiated LDL oxidation was tested and the kinetic of LDL oxidation led us to calculate the ED50. To eliminate the possible role of copper chelation, oxidation was induced by water-soluble AAPH, which generates free radicals by its spontaneous thermal decomposition. Only the free radical scavenging mechanism contributes toward the inhibitory action of these compounds. This study confirms that extracts inhibit copper-induced LDL oxidation and shows that they highly reduce AAPH-induced LDL oxidation. The values of ED50 for leaves and roots were 18 and 68 mg/L for copper and 22 and 30 mg/L for AAPH, respectively (Table 2).
In the presence of the tested molecules there was a strong correlation in lag phase duration between these two oxidative procedures of LDL. Therefore, these molecules are capable of scavenging free radicals and of inhibiting LDL peroxidation.
This study shows that P. pinnata root and leaf polar extracts induce vasorelaxation of PE precontracted Wistar rat aortas. The inhibition of NO synthesis with L-NAME attenuated the vasorelaxation induced by the extracts, suggesting that the relaxation was partly mediated through the NO-dependent pathway. These extracts have strong antioxidant properties and they increase eNOS gene expression in BAEC while they decrease ET-1 gene expression in these cells. Furthermore they tend to induce PPAR-α activation.
P. pinnata roots and leaves are chiefly composed of polyphenols such as tannins and flavonoids, with content varying from 0.5% to 15% in methanolic extracts (see Results). The polyphenol content of P. pinnata is a great criterion for assessing the probable biological effect of its extracts. Indeed, polyphenols possess a multitude of biological activities including their protective effect versus free radicals.43–45 It is well documented that polyphenols induce endothelium-dependent vasorelaxation46 and alter the bioactivity of eNOS and ET-1 in an opposing manner because they increase eNOS activity46 and decrease ET-1 expression.47
It has recently been demonstrated that red wine polyphenol-induced, endothelium-dependent NO-mediated relaxation is caused by the redox-sensitive PI3-kinase/Akt-dependent phosphorylation of eNOS in artery, resulting in an increased formation of NO.48 Furthermore, it has been reported that red wine polyphenols induce vasorelaxation not only by increasing eNOS activity but also by protecting NO degradation through their antioxidant properties.46
NO signaling is mainly mediated by the guanylate cyclase/cyclic guanylate monophosphate pathway. The effects of this second messenger system are limited by enzymatic degradation through PDEs. Oral PDE-5 inhibitors (eg, sildenafil) are approved for the treatment of erectile dysfunction. PDEs.49 It has recently been reported that polyphenols from red grapes inhibit human cGMP-specific PDE-5.50 This result suggests that P. pinnata root and leaf polar extract-induced vasorelaxation may be sustained by smooth muscle PDE inhibition by their polyphenol content.
Red wine polyphenols inhibit ET-1 gene expression in endothelial cells.47,51 This action was associated with modifications in phosphotyrosine staining, indicating that the active components of red wine cause specific modifications in tyrosine kinase signaling.51 Furthermore, we have recently reported that natural phenolic compounds other than red wine polyphenols also inhibit ET-1 gene expression and ET-1 secretion in BAEC.52
Today, there is no identified mechanistic link between the capacity of natural polyphenols to simultaneously increase eNOS activity and decrease ET-1 synthesis; furthermore the existence of a mechanistic link is only putative. Nevertheless, it could be suggested that PPAR-α activation could be a significant link between eNOS and ET-1. Numerous facts suggest this hypothesis: PPARs are targets for natural polyphenols,53 and our study shows that the highest tested dose of root and leaf extracts tend to slightly enhance the transcriptional activity of PPAR-α similarly as low doses of potent PPAR-α activators (Gemfibrosil, WY, ciprofibrate and fenofibric acid).
We have demonstrated that PPAR-α activation inhibits ET-1 gene expression in endothelial cells,51,54,55 and these data have been extensively confirmed.46,53 In our previous study54 we showed that 10−7 mmol/L wy14,643 inhibited endothelin secretion through the PPAR-α transrepression pathway, whereas in the present study wy14,643 did not induce PPAR-α transcriptional activity at this low dose. These data suggest that the capacity of PPAR=α to induce transcriptional activity is achieved at a highest level of PPAR-α agonist than its capacity to induce transrepression (or that our test to measure the capacity of PPAR-α agonists to induce a transcriptional activity is not very sensitive). Under this condition it cannot be precluded to suggest that the slight tendency of P. pinnata root and leaf extracts to induce a PPAR-α transcriptional activity in our system would not be sufficient to induce a PPAR-α transrepression activity in endothelial cells and the resulting inhibition of ET-1 secretion. It has been shown that PPAR-α agonists increase NOS expression in vascular endothelial cells mainly through mechanisms of stabilizing eNOS mRNA.56
In our study, animals were not pretreated by P pinnata root and leaf extracts before assessing ex vivo aortic vasorelaxation; therefore, eNOS and ET-1 gene expression may not have been significantly modified in the aortic endothelium when vasorelaxation was tested because the endothelium had not been exposed long enough to P pinnata extracts to notably alter the mRNA machinery. Alternatively, it could be suggested that
- The in vitro duration of contact between the aortic endothelium and the polyphenols contained in the extracts was sufficient to induce endothelium-dependent NO-mediated relaxation, the redox-sensitive PI3-kinase/Akt-dependent phosphorylation of eNOS, and the resulting increase in NO formation.48
- P. pinnata root and leaf polar extracts–induced vasorelaxation may be caused by the putative capacity of their polyphenol content to inhibit smooth muscle PDE-5 activity.49
- We have shown in this study that leaf root and leaf polyphenol extracts of P pinnata are potent antioxidants in different systems. They inhibit LDL oxidation and reduce oxidation mediated by
- , H2O2, and HOCl. Therefore, these extracts could protect NO against its oxidative degradation, increasing its life and its concentration around the smooth muscle cells, and increasing the endothelium-dependent vasorelaxation. The capacity of root extract to inhibit
- -mediated oxidation is higher than the capacity of leaf extract to inhibit oxidation by this free radical (EC50: 165±19 vs 324±24 mg/L/ROS respectively). The role of tannins+flavonoids is likely to be important in the antioxidant effect of these extracts because there is an inverse relationship between EC50 and the tannin+flavonoid contents of root and leaf extracts (15.5% and 7.2%, respectively). These data support the hypothesis that both extracts improved endothelium-dependent vasorelaxation by inhibiting
- -induced NO oxidation because lower doses of root extracts than leaf extracts were necessary to induce endothelium-dependent vasorelaxation.
P. pinnata root and leaf extracts increase endothelium-dependent relaxation through the NO pathway. It could be suggested that (1) the expression of eNOS and ET-1 genes was not modified in our experimental system testing vasorelaxation because of the short-term contact of arterial endothelium with P. pinnata root and leaf extracts; (2) some molecules' content in P. pinnata root and leaf extracts could increase PPAR-α activation and the capacity of P. pinnata extracts to increase eNOS and decrease ET-1 mRNA levels in endothelial cell cultures could be mediated partly by a slight PPAR-α activation and the resulting capacity of PPAR-α to mediate trans- repression of specific genes; (3) P. pinnata could induce endothelium-dependent vasorelaxation through the NO pathway by increasing the redox-sensitive PI3-kinase/Akt-dependent phosphorylation of eNOS in artery48 and/or by inhibiting the smooth muscle cell cGMP-specific phosphodiesterase-5.50
It is more than likely in our study that the capacity of P. pinnata leaf and root extracts to induce endothelium-dependent vasorelaxation through the NO pathway depends on their capacity to inhibit NO oxidation and therefore on their properties to increase NO half-life and bioactivity.
It is more than likely that the capacity of P pinnata to induce endothelium-dependent vasorelaxation through the NO pathway as well as inhibit ET-1 synthesis could constitute pharmacological mechanisms explaining its efficacy in traditional African medicine treatment of impotence.
1. Abourashed EA, Toyang NJ, Choinski J, et al. Two new flavone glycosides from Paullinia pinnata
. J Nat Prod. 1999;62:1179–1181.
2. Kerharo J, Adam JG. La Pharmacopée Sénégalaise Traditionnelle. Paris: Vigot; 1974.
3. Bowden K. Isolation from Paullinia pinnata
of material with action on the isolated frog heart. Br J Pharmacol. 1962;18:173–174.
4. Allard J, Giuliano F. Central nervous system agents in the treatment of erectile dysfunction: how do they work? Curr Urol Rep. 2001;2:488–494.
5. Andersson KE. Erectile physiological and pathophysiological pathways involved in erectile dysfunction. J Urol. 2003;170:S6–S13.
6. Russell S, Nehra A. The physiology of erectile dysfunction. Herz. 2003;28:277–283.
7. Andersson KE. Pharmacology of lower urinary tract smooth muscles and penile erectile tissues. Pharmacol Rev. 1993;45:253–308.
8. Andersson KE, Wagner G. Physiology of penile erection. Physiol Rev. 1995;75:191–236.
9. Lerner SE, Melman A, Christ GJ. A review of erectile dysfunction: new insights and more questions. J Urol. 1993;149:1246–1255.
10. Christ GJ, Richards S, Winkler A. Integrative erectile biology: the role of signal transduction and cell-to-cell communication in coordinating corporal smooth muscle tone and penile erection. Int J Impot Res. 1997;9:69–84.
11. Christ GJ. The penis as a vascular organ. The importance of corporal smooth muscle tone in the control of erection. Urol Clin North Am. 1995;22:727–745.
12. Burnett AL. Role of nitric oxide in the physiology of erection. Biol Reprod. 1995;52:485–489.
13. Melis MR, Argiolas A. Role of central nitric oxide in the control of penile erection and yawning. Prog Neuropsychopharmacol Biol Psychiatry. 1997;21:899–922.
14. Adaikan PG, Kottegoda SR, Ratnam SS. Is vasoactive intestinal polypeptide the principal transmitter involved in human penile erection? J Urol. 1986;135:638–640.
15. Suh JK, Mun KH, Cho CK, et al. Effect of vasoactive intestinal peptide and acetylcholine on penile erection in the rat in vivo. Int J Impot Res. 1995;7:111–118.
16. Andersson KE, Stief CG. Neurotransmission and the contraction and relaxation of penile erectile tissues. World J Urol. 1997;15:14–20.
17. González-Cadavid NF, Ryndin I, Vernet D, et al. Presence of NMDA receptor subunits in the male lower urogenital tract. J. Androl. 2000;21:566–578.
18. Hurt KJ, Musicki B, Palese MA, et al. Akt-dependent phosphorylation of endothelial nitric-oxide synthase mediates penile erection. Proc Natl Acad Sci U S A. 2002;99:4061–4066.
19. Musicki B, Palese MA, Crone JK, et al. Phosphorylated endothelial nitric oxide synthase mediates vascular endothelial growth factor-induced penile erection. Biol Reprod. 2004;70:282–289.
20. Cheitlin MD, Hutter AM, Brindis RG, et al. Use of sildenafil (Viagra) in patients with cardiovascular disease. J Am Coll Cardiol. 1999;33:273–282.
21. Sakuma I, Akaishi Y, Tomioka H, et al. Interactions of sildenafil with various coronary vasodilators in isolated porcine coronary artery. Eur J Pharmacol. 2002;437:155–163.
22. Ishikura F, Beppu S, Hamada T, et al. Effects of sildenafil citrate (Viagra) combined with nitrate on the heart. Circulation. 2000;102:2516–2521.
23. Garcia-Reboll L, Mulhall JP, Goldstein I. Drugs for the treatment of impotence. Drugs Aging. 1997;11:140–151.
24. Traish AM, Netsuwan N, Daley J, et al. A heterogeneous population of alpha 1 adrenergic receptors mediates contraction of human corpus cavernosum smooth muscle to norepinephrine. J Urol. 1995;153:222–227.
25. Mills TM, Pollock DM, Lewis RW, et al. Endothelin-1-induced vasoconstriction is inhibited during erection in rats. Am J Physiol Regul Integr Comp Physiol. 2001;281:R476–R483.
26. Ari G, Vardi Y, Hoffman A, et al. Possible role for endothelins in penile erection. Eur J Pharmacol. 1996;307:69–74.
27. Christ GJ, Lerner SE, Kim DC, et al. Endothelin-1 as a putative modulator of erectile dysfunction: I. Characteristics of contraction of isolated corporal tissue strips. J Urol. 1995;153:1998–2003.
28. Holmquist F, Persson K, Garcia-Pascual A, et al. Phospholipase C activation by endothelin-1 and noradrenaline in isolated penile erectile tissue from rabbit. J Urol. 1992;147:1632–1635.
29. Zhao W, Christ GJ. Endothelin-1 as a putative modulator of erectile dysfunction. II. Calcium mobilization in cultured human corporal smooth muscle cells. J Urol. 1995;154:1571–1579.
30. Bell CR, Sullivan ME, Dashwood MR, et al. The density and distribution of endothelin-1 and endothelin receptor subtypes in normal and diabetic rat corpus cavernosum. Br J Urol. 1995;76:203–207.
31. Saenz de Tejada I, Carson MP, de las Morenas A, et al. Endothelin: localization, synthesis, activity, and receptor types in human penile corpus cavernosum. Am J Physiol. 1991;261:H1078–H1085.
32. Dai Y, Pollock DM, Lewis R, et al. Receptor-specific influence of endothelin-1 in the erectile response of the rat. Am J Physiol Regul Integr Comp Physiol. 2000;279:R25–R30.
33. European Pharmacopoeia, Ve
34. Pu Q, Bordet R, Robin E, et al. Low dose of lipopolysaccharides induces a delayed enhanced nitric oxide-mediated relaxation in rat aorta. Eur J Pharmacol. 1999;377(2-3):209–214.
35. Gospodarowicz D, Moran J, Braund D, et al. Clonal growth of bovine endothelial cells in tissue culture: fibroblast growth factor as survival agent. Proc Natl Acad Sci U S A. 1976;73:4120–4124.
36. Raspé E, Madsen L, Lefebre AM, et al. Modulation of rat liver apolipoprotein gene expression and serum lipid levels by tetradecylthioacetic acid (TTA) via PPARαactivation. J Lipid Res. 1999;40:2099–2110.
37. Aruoma OL, Halliwell B, Hoey BM, et al. The Antioxidant action of N-acetylcysteine: Its reaction with hydrogen peroxide, hydroxyl radical, superoxide and hypochlorous acid. Free Radical Biol Med. 1989;6:593–597.
38. Pick G, Keisari YA. A simple colorimetric method for the measurement of hydrogen peroxide produced by cells in culture. J Immunol Methods. 1980;38:161–170.
39. Weiss SJ, Klein R, Slvka A, et al. Chlorination of taurine by human neutrophils. J Clin Invest. 1982;70:598–607.
40. Havel RJ, Eder HA, Bragdon JH. The distribution and chemical composition of ultra-centrifugally separated lipoproteins in human serum. J Clin Invest. 1955;34:1345–1353.
41. Peterson GA. Simplification of the protein assay method of Lowry which is more generally applicable. Anal Biochem. 1977;83:346–356.
42. Esterbauer H, Wang G, Phul H. Lipid peroxidation and its role in atherosclerosis. Br Med Bull. 1993;49:566–576.
43. Andriambeloson E, Kleschyov AL, Muller B, et al. Nitric oxide production and endothelium-dependent vasorelaxation induced by wine polyphenols in rats. Br J Pharmacol. 1997;120:1053–1058.
44. Lambert JD, Hong J, Yang GY, et al. Inhibition of carcinogenesis by polyphenols: evidence from laboratory investigations. Am J Clin Nutr. 2005;81:284–291.
45. Vieira O, Escargueil-Blanc I, Meihac O, et al. Effect of dietary phenolic compounds on apoptosis of human endothelial cells induced by oxidized LDL. Br J Pharmacol. 1998;123(3):565–573.
46. Zenebe W, Pechanova O, Andriantsitohaina R. Red wine polyphenols induce vasorelaxation by increased nitric oxide bioactivity. Physiol Res. 2003;52(4):425–432.
47. Corder R, Douthwaite JA, Lees DM, et al. Endothelin-1 synthesis reduced by red wine. Nature. 2001;414(6866):863–864.
48. Ndiaye M, Chataigneau M, Lobysheva I, et al. Red wine polyphenol-induced, endothelium-dependent NO-mediated relaxation is due to the redox-sensitive PI3-kinase/Akt-dependent phosphorylation of endothelial NO-synthase in the isolated porcine coronary artery. FASEB J. 2005;19(3):455–457.
49. Ghofrani HA, Pepke-Zaba J, Barbera JA, et al. Nitric oxide pathway and phosphodiesterase inhibitors in pulmonary arterial hypertension. J Am Coll Cardiol. 2004;43(12):68S–72S.
50. Dell'Agli M, Galli GV, Vrhovsek U, et al. In vitro inhibition of human cGMP-specific phosphodiesterase-5 by polyphenols from red grapes. J Agric Food Chem. 2005;53(6):1960–1965.
51. Khan NQ, Lees DM, Douthwaite JA, et al. Comparison of red wine extract and polyphenol constituents on endothelin-1 synthesis by cultured endothelial cells. Clin Sci (Lond). 2002;103:72S–75S.
52. Martin-Nizard F, Sahpaz S, Kandoussi A, et al. Natural phenylpropanoids inhibit lipoprotein-induced endothelin-1 secretion by endothelial cells. J Pharm Pharmacol. 2004;56(12):1607–1611.
53. Inoue H, Jiang XF, Katayama T, et al. Brain protection by resveratrol and fenofibrate against stroke requires peroxisome proliferator-activated receptor alpha in mice. Neurosci Lett. 2003;352(3):203–206.
54. Delerive P, Martin-Nizard F, Chinetti G, et al. Peroxisome proliferator-activated receptor activators inhibit thrombin-induced endothelin-1 production in human vascular endothelial cells by inhibiting the activator protein-1 signaling pathway. Circ Res. 1999;85(5):394–402.
55. Martin-Nizard F, Furman C, Delerive P, et al. Peroxisome proliferator-activated receptor activators inhibit oxidized low-density lipoprotein-induced endothelin-1 secretion in endothelial cells. J Cardiovasc Pharmacol. 2002;40(6):822–831.
56. Goya K, Sumitani S, Xu X, et al. Peroxisome proliferator-activated receptor alpha agonists increase nitric oxide synthase expression in vascular endothelial cells. Arterioscler Thromb Vasc Biol. 2004;24(4):658–663.
Keywords:© 2006 Lippincott Williams & Wilkins, Inc.
Paullinia pinnata; endothelium; eNOS; endothelin-1; vasodilation; rat aorta