molecules Article
Vasodilator Activity of Compounds Isolated from Plants Used in Mexican Traditional Medicine Francisco J. Luna-Vázquez 1 ID , César Ibarra-Alvarado 1, *, María del Rayo Camacho-Corona 2, *, Alejandra Rojas-Molina 1 , J. Isela Rojas-Molina 1 , Abraham García 2 and Moustapha Bah 1 1
2
*
Laboratorio de Investigación Química y Farmacológica de Productos Naturales, Facultad de Química, Universidad Autónoma de Querétaro, C.P. 76010 Querétaro, Mexico;
[email protected] (F.J.L.-V.);
[email protected] (A.R.-M.);
[email protected] (J.I.R.-M.);
[email protected] (M.B.) Universidad Autónoma de Nuevo León, Facultad de Ciencias Químicas, Ciudad Universitaria, San Nicolás de los Garza, CP 66451 Nuevo León, Mexico;
[email protected] Correspondence:
[email protected] (C.I.-A.);
[email protected] (M.d.R.C.-C.); Tel.: +52-442-1921200 (ext. 5527) (C.I.-A.); +52-81-8329-4000 (ext. 3414) (M.d.R.C.-C.)
Received: 6 April 2018; Accepted: 12 June 2018; Published: 18 June 2018
Abstract: Arterial hypertension is one of the main risk factors in the development of cardiovascular diseases. Therefore, it is important to look for new drugs to treat hypertension. In this study, we carried out the screening of 19 compounds (triterpenes, diterpenes, sesquiterpenes, lignans, and flavonoids) isolated from 10 plants used in Mexican traditional medicine to determine whether they elicited vascular smooth muscle relaxation and, therefore, could represent novel anti-hypertension drug candidates. The vasorelaxant activity of these compounds was evaluated on the isolated rat aorta assay and the results obtained from this evaluation showed that three compounds induced a significant vasodilatory effect: meso-dihydroguaiaretic acid [half maximal effective concentration (EC50 ), 49.9 ± 11.2 µM; maximum effect (Emax), 99.8 ± 2.7%]; corosolic acid (EC50 , 108.9 ± 6.7 µM; Emax, 96.4 ± 4.2%); and 5,8,40 -trihydroxy-3,7-dimethoxyflavone (EC50 , 122.3 ± 7.6 µM; Emax, 99.5 ± 5.4%). Subsequently, involvement of the NO/cyclic guanosine monophosphate (cGMP) and H2 S/ATP-sensitive potassium channel (KATP ) pathways on the vasodilator activity of these compounds was assessed. The results derived from this analysis showed that the activation of both pathways contributes to the vasorelaxant effect of corosolic acid. On the other hand, the vasodilator effect of meso-dihydroguaiaretic acid and 5,8,40 -trihydroxy-3,7-dimethoxyflavone, partly involves stimulation of the NO/cGMP pathway. However, these compounds also showed an important endothelium-independent vasorelaxant effect, whose mechanism of action remains to be clarified. This study indicates that meso-dihydroguaiaretic acid, corosolic acid, and 5,8,40 -trihydroxy-3,7-dimethoxyflavone could be used as lead compounds for the synthesis of new derivatives with a higher potency to be developed as drugs for the prevention and treatment of cardiovascular diseases. Keywords: corosolic acid; 5,8,40 -trihydroxy-3,7-dimethoxyflavone; meso-dihydroguaiaretic acid hydrogen sulfide; nitric oxide; vasorelaxation
1. Introduction Arterial hypertension is considered a major risk factor in the development of several cardiovascular diseases, such as myocardial infarction and stroke [1–3], which together were responsible for 15 million deaths in 2015 [4]. Although several drugs are currently used in the treatment of high blood pressure, less than a third of the hypertension cases are successfully treated [5–8]. It has been broadly demonstrated that endothelial dysfunction, characterized by a diminished availability of endothelial relaxing factors, significantly contributes to the development of hypertension Molecules 2018, 23, 1474; doi:10.3390/molecules23061474
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and thus, other cardiovascular diseases [9–11]. Particularly, reduced release of the gasotransmitters gasotransmitters NO andimpaired H2S results in an of impaired regulation of the vascular tone NO and H2 S results in an regulation the vascular tone [12–14]. Therefore, the [12–14]. NO/cyclic Therefore, the NO/cyclic guanosine monophosphate (cGMP) and H 2S/ATP-sensitive potassium guanosine monophosphate (cGMP) and H2 S/ATP-sensitive potassium channel (KATP ) pathways are channel (KATP) pathways are valuable targets for innovative treatments in hypertension [12–14]. valuable targets for innovative treatments in hypertension [12–14]. In Mexico, plants are an important element of traditional medicine, and many of them are In Mexico, plants are an important element of traditional medicine, and many of them are considered part of Mexican cultural heritage from prehispanic and colonial times [15,16]. considered part of Mexican cultural heritage from prehispanic and colonial times [15,16]. Nevertheless, Nevertheless, relatively few systematic scientific studies have been conducted to fully characterize relatively few systematic scientific studies have been conducted to fully characterize the chemical the chemical composition and pharmacological activities of Mexican medicinal plants. composition and pharmacological activities of Mexican medicinal plants. In the present study, we carried out a pharmacological screening of 19 secondary metabolites In the present study, we in carried outtraditional a pharmacological screening 19 secondary metabolites isolated from 10 plants used Mexican medicine in order to of detect leads that could be isolated from 10 plants used in Mexican traditional medicine in order to detect leads could developed as potential drug candidates to treat hypertension. The plant sources of thethat tested becompouds developedwere: as potential drug candidates to treat hypertension. plant sources of the tested Amphipterygium adstringens (Schltdl.) Standl. The (cuachalalate), Celaenodendron compouds were: Amphipterygium adstringens (Schltdl.) Standl. (cuachalalate), Celaenodendron mexicanum mexicanum Standl. (palo prieto), Crataegus gracilior J.B. Phipps (tejocote), Croton alamosanus Rose (vara Standl. (palo prieto), Crataegus gracilior J.B. Phipps (tejocote), Croton alamosanus Rose (vara blanca), Croton glabellus L. (cascarillo), Galphimia glauca Cav. (árnica roja), Jatropha neopauciflora blanca), Pax Croton (cascarillo), Galphimia glauca (árnica roja), Jatropha neopauciflora (sangre (sangreglabellus de grado),L.Larrea tridentata (Sessé and Moc. Cav. ex DC.) Coville (gobernadora), Perezia adnataPax A. Gray de(pipitzá grado), Larrea (Sessé and Moc. deexzorrillo). DC.) Coville (gobernadora), Perezia A. Gray huac), andtridentata Teloxys graveolens (epazote These plants are widely usedadnata in Mexican traditional medicine to treat several illnesses, such as gastric ulcers, stomach ache, gastroenteritis, (pipitzáhuac), and Teloxys graveolens (epazote de zorrillo). These plants are widely used in Mexican diarrhea, dysentery, sores and infections of the such oral cavity, skinulcers, ulcers [17–21], disorders and traditional medicine to treat several illnesses, as gastric stomachrenal ache, gastroenteritis, kidney stones (Larreasores tridentata) [17], cough,ofasthma, tachycardia andulcers to improve coronary blood flowand diarrhea, dysentery, and infections the oral cavity, skin [17–21], renal disorders (Crataegus gracilior) and [17], cancer (Croton alamosanus) [23]. and to improve coronary blood flow kidney stones (Larrea[20,22], tridentata) cough, asthma, tachycardia The compounds evaluated were previously purified (Crataegus gracilior) [20,22], and cancer (Croton alamosanus) [23].by our research group and our collaborators [18,19,24–26] and include: triterpenes: 3-α-hydroxymasticadienonic acid (1), 3α-our The compounds evaluated were (a) previously purified by our research group and hydroxytirucalla-7,22Z-dien-26-oic acid (2), corosolic acid (3), galphin A (4), galphin B (5), collaborators [18,19,24–26] and include: (a) triterpenes: 3-α-hydroxymasticadienonic acid (1), galphimidin (6), and 3β-trans-p-coumaroyl-oxy-16-β-hydroxy-20(29)-lupene (7); (b) sterols: 3α-hydroxytirucalla-7,22Z-dien-26-oic acid (2), corosolic acid (3), galphin A (4), galphin B (5), β−sitosteryl β−D-glucopyranoside (8); (c) diterpenes: (3R,4R,6S)-p-menth-1-eno-3,6-diol (9), 6,7galphimidin (6), and 3β-trans-p-coumaroyl-oxy-16-β-hydroxy-20(29)-lupene (7); (b) sterols: β-sitosteryl diacetylaustro inulin (10), and 6-O-acetylaustro inulin (11); (d) sesquiterpenes: perezone (12), and β-D-glucopyranoside (8); (c) diterpenes: (3R,4R,6S)-p-menth-1-eno-3,6-diol (9), 6,7-diacetylaustro pipitzol (13); (e) lignans: 3′-demethoxy-6-O-demethyl-isoguaiacin (14) and meso-dihydroguaiaretic inulin (10), and 6-O-acetylaustro inulin (11); (d) sesquiterpenes: perezone (12), and pipitzol (13); acid (15); 0and (f) flavonoids: 5,4′-dihydroxy-3,7,8-trimethoxyflavone (16), 5-hydroxy-3,7,4′(e)trimethoxyflavone lignans: 3 -demethoxy-6-O-demethyl-isoguaiacin (14) and (18), meso-dihydroguaiaretic (15);1).and (17), 5,8,4′-trihydroxy-3,7-dimethoxyflavone and pinostrobin (19)acid (Figure 0 -dihydroxy-3,7,8-trimethoxyflavone (16), 5-hydroxy-3,7,40 -trimethoxyflavone (17), (f)Although flavonoids: 5,4 it has been previously demonstrated that some of these compounds possess biological 0 -trihydroxy-3,7-dimethoxyflavone (18), and pinostrobin (19) (Figure 1). Although it has been 5,8,4 activities, such as antibacterial and antifungal [21,25,27], antiprotozoal [18,24,28,29], antipreviously demonstrated that some of these compounds biological activities, as antibacterial inflammatory [30], anti-diabetic [31,32], and anticancer possess [23], at present, there are no such studies aimed at and antifungal [21,25,27], antiprotozoal [18,24,28,29], anti-inflammatory [30], anti-diabetic [31,32], and assessing their vasodilatory activity. anticancer [23], present, there are no studies aimed at assessing their compounds vasodilatoryelicited activity.vascular In the firstatphase of this study, we assessed whether the selected In themuscle first phase of this study, we isolated assessed rat whether selected compounds smooth smooth relaxation in the aorta.theThereafter, the most elicited potentvascular vasodilator compounds, which also displayed the highest maximum effects (Emax) ≥which 96%],also muscle relaxation in the isolated rat aorta. Thereafter, the most potent[maximum vasodilatoreffect compounds, were evaluated to determine if the NO/cGMP and H 2S/KATP channel pathways involved in their if displayed the highest maximum effects [maximum effect (Emax) ≥ 96%], were were evaluated to determine mechanism of and action. the NO/cGMP H2 S/KATP channel pathways were involved in their mechanism of action.
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Figure 1. Cont.
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4 R=H 5 R=OAc
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16 R1= OMe R2= OH 17 R1= H R2= OMe 18 R1= OH R2= OH
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Figure 1. Chemical structures of studied compounds. Figure 1. Chemical structures of studied compounds.
2. Results 2. Results The results from the pharmacological evaluation of the selected compounds indicated that 3-αThe results from the acid pharmacological evaluation of the selected compounds indicated that hydroxymasticadienonic (1), corosolic acid (3), galphimidin (6), meso-dihydroguaiaretic acid 3-α-hydroxymasticadienonic acid (1), corosolic acid (3), galphimidin (6), meso-dihydroguaiaretic (15), 5,8,4′-trihydroxy-3,7-dimethoxyflavone (18), and pinostrobin (19) elicited a significant 0 -trihydroxy-3,7-dimethoxyflavone (18), and pinostrobin (19) elicited a significant acid (15), 5,8,4All vasodilation. these six secondary metabolites exhibited a maximum vasodilator effect up to 96%, vasodilation. All these metabolites exhibited a maximum vasodilator up to which was higher thansix thatsecondary of Acetylcholine (ACh), used as the positive control (Tableeffect 1). meso96%, which was higher than that of Acetylcholine (ACh), used as the positive control (Table Dihydroguaiaretic acid (15) exhibited a comparable potency to that of Ach, while corosolic acid (3) 1). meso-Dihydroguaiaretic acid (15) exhibited a(18) comparable potency to that of Ach, while corosolic acid and 5,8,4′-trihydroxy-3,7-dimethoxyflavone turned out to be approximately two-fold less potent 0 -trihydroxy-3,7-dimethoxyflavone (18) turned out to be approximately two-fold less (3)than andthe 5,8,4 positive control (Figure 2 and Table 1). Based on these results, involvement of the NO/cGMP and H 2S/Kthe ATP channel in the 2mechanisms underlying vasorelaxant action of mesopotent than positivepathways control (Figure and Table 1). Based onthe these results, involvement of the dihydroguaiaretic acid (15) and 5,8,4′-trihydroxy-3,7-dimethoxyflavone (18), both isolated from NO/cGMP and H2 S/KATP channel pathways in the mechanisms underlying the vasorelaxant action of 0 -trihydroxy-3,7-dimethoxyflavone Larrea tridentata, and corosolic purified from Crataegus gracilior, was analyzed. meso-dihydroguaiaretic acid (15)acid and(3)5,8,4 (18), both isolated from Larrea tridentata, and corosolic acid (3) purified from Crataegus gracilior, was analyzed.
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Table 1. Values of half maximal effective concentration (EC50) and maximum effect (Emax) of positive control (Acetilcholine, ACh) and test compounds. Table 1. Values of half maximal effective concentration (EC50 ) and maximum effect (Emax) of positive EC50 (μM) Emax (%) control Plant/Compound (Acetilcholine, ACh) and test compounds. acetilcholine (ACh) 58.8 ± 8.9 69.5 ± 5.7 Amphypterygium adstringens Plant/Compound EC50 (µM) Emax (%) 3-α-hydroxymasticadienonic acid (1) 206.1 ± 11.6 98.2 ± 3.1 acetilcholine (ACh) 58.8 ± 8.9 69.5 ± 5.7 Celaenodendron mexicanum Amphypterygium adstringens 3α-hydroxytirucalla-7,22Z-dien-26-oic acid (2) 331.3 ± 42.1 6.1 ± 3.1 3-α-hydroxymasticadienonic acid (1) 206.1 ± 11.6 99.5 ±98.2 Crataegus gracilior Celaenodendron mexicanum Corosolic acid (3) 108.9 ± 6.7 4.2 ± 6.1 3α-hydroxytirucalla-7,22Z-dien-26-oic acid (2) 331.3 ± 42.1 96.4 ±99.5 Croton alamosanus Crataegus gracilior Corosolic acid (3) 108.9 ± 6.7 80.5 ±96.4 5-hydroxy-3,7,4′-trimethoxyflavone (17) 377.1 ± 37.1 3.7 ± 4.2 Croton alamosanus (3R,4R,6S)-p-menth-1-ene-3,6-diol (9) 1622.8 ± 73.8 99.5 ± 8.3 0 -trimethoxyflavone (17) 5-hydroxy-3,7,4 377.1 ± 37.1 80.5 ± 3.7 Croton glabellus (3R,4R,6S)-p-menth-1-ene-3,6-diol (9) 1622.8 ± 73.8 99.5 6-O-acetylaustro inulin (10) 413.5 ± 22.4 47.1 ± 2.8 ± 8.3 Croton glabellus 6,7-diacetylaustro inulin (11) 261.0 ± 9.1 99.5 ± 3.8 6-O-acetylaustro inulin (10) 413.5 ± 22.4 47.1 ± 2.8 Galphimia glauca 6,7-diacetylaustro inulin (11) 261.0 ± 9.1 99.5 ± 3.8 Galphin 592.3 ± 21.7 99.5 ± 9.1 Galphimia glauca A (4) 1030.7 23.2± 9.1 GalphinGalphin A (4) B (5) 592.3±±39.4 21.7 99.5 ±99.5 (6) 145.9 ± 9.2 5.3 ± 23.2 GalphinGalphimidin B (5) 1030.7 ± 39.4 99.5 ±99.5 Jatropha Galphimidin (6)neopauciflora 145.9 ± 9.2 99.5 ± 5.3 Jatropha3β-trans-p-coumaroyl-oxy-16-β-hydroxy-20(29)-lupene neopauciflora (7) 63.2 ± 5.8 27.5 ± 1.9 3β-trans-p-coumaroyl-oxy-16-β-hydroxy-20(29)-lupene (7) 63.2±±19.7 5.8 β-sitosteryl β-D-glucopyranoside (8) 314.7 71.2 ±27.5 5.3 ± 1.9 β-sitosteryl β-tridentata D -glucopyranoside (8) 314.7 ± 19.7 71.2 ± 5.3 Larrea Larrea tridentata meso-dihydroguaiaretic acid (15) 49.9 ± 11.2 99.8 ±2.7 meso-dihydroguaiaretic acid (15) 49.9 ± 11.2 99.8 ± 2.7 5,4′-dihydroxy-3,7,8-trimethoxyflavone (16) 587.8 ± 33.4 80.5 ± 5.6 5,40 -dihydroxy-3,7,8-trimethoxyflavone (16) 587.8 ± 33.4 80.5 ± 5.6 5,8,4′-trihydroxy-3,7-dimethoxyflavone (18) 122.3 ± 7.6 99.5 ± 5.4 5,8,40 -trihydroxy-3,7-dimethoxyflavone (18) 122.3 ± 7.6 99.5 ± 5.4 3′-demethoxy-6-O-demethyl-isoguaiacin (14) 604.5 ± 60.1 ± 7.3 ± 7.3 0 3 -demethoxy-6-O-demethyl-isoguaiacin (14) 604.5 ± 60.1 91.70 91.70 Perezia adnata Perezia adnata perezone (12) 524.5 ± 32.5 2.2 ± 2.2 perezone (12) 524.5 ± 32.5 99.5 ±99.5 (13) 249.4 ± 10.2 2.4 ± 2.4 pipitzolpipitzol (13) 249.4 ± 10.2 59.8 ±59.8 Teloxys graveolens Teloxys graveolens pinostrobin (19) 234.9 ± 9.9 99.5 ±99.5 pinostrobin (19) 234.9 ± 9.9 4.8 ± 4.8
Figure 2. Concentration-response Concentration-responsecurves curvesofof the the vasodilator vasodilator effect effect of of corosolic corosolic acid acid (3), (3), mesomesodihydroguaiaretic acid (15), and 5,8,4′-trihydroxy-3,7-dimethoxyflavone 5,8,40 -trihydroxy-3,7-dimethoxyflavone (18). Acetylcholine was used as a positive control.
2.1. Participation of the Endothelium in the Vasorelaxant Response of Compounds 3, 15, and 18 Figure 3 shows the concentration-response curves (CRCs) for corosolic acid (3), mesodihydroguaiaretic acid (15), and 5,8,4′-trihydroxy-3,7-dimethoxyflavone (18) in the presence and Molecules 2018, 23, 1474 6 of 17 absence of endothelium. The vasorelaxation induced by corosolic acid (3) was almost completely blocked in the absence of endothelium, which suggested that endothelial factors mediate its vasodilatory effect. On the other 2.1. Participation of the Endothelium in the Vasorelaxant Response of Compounds 3, 15, and 18 hand, endothelial denudation caused a significant rightward shift of the CRC to mesoFigure 3 shows acid the concentration-response curves (CRCs) for corosolic acid (3), the mesodihydroguaiaretic (15) and 5,8,4′-trihydroxy-3,7-dimethoxyflavone (18), indicating contribution of both and dependent pathways (18) in their mechanism dihydroguaiaretic acid endothelium-independent (15), and 5,8,40 -trihydroxy-3,7-dimethoxyflavone in the presence of and action. absence of endothelium.
Figure Concentration-response curves thethe vasodilator effecteffect of (a)ofcorosolic acid (3),acid (b) mesoFigure 3. 3.Concentration-response curvesofof vasodilator (a) corosolic (3), (b) dihydroguaiaretic acid (15), andand (c) 5,8,4′-trihydroxy-3,7-dimethoxyflavone (18) in(18) the in presence and meso-dihydroguaiaretic acid (15), (c) 5,8,40 -trihydroxy-3,7-dimethoxyflavone the presence absence (E-) of endothelium. A typical trace in which meso-dihydroguaiaretic acid (15) inhibited Pheand absence (E-) of endothelium. A typical trace in which meso-dihydroguaiaretic acid (15) inhibited induced contraction in endothelium-intact (d), and aorta aorta (e). Analysis by an unpaired t-test Phe-induced contraction in endothelium-intact (d), -denuded and -denuded (e). Analysis by an unpaired was made to test for differences between EC50s of each compound in the presence and absence of t-test was made to test for differences between EC50 s of each compound in the presence and absence of endothelium (3, p < 0.0001; 15, p < 0.0001; 18, p = 0.0093). endothelium (3, p < 0.0001; 15, p < 0.0001; 18, p = 0.0093).
2.2. Participation of the NO/cGMP and H2S/KATP Channel Pathways in the Vasodilator Response of The vasorelaxation induced by corosolic acid (3) was almost completely blocked in the absence of Compounds 3, 15, and 18
endothelium, which suggested that endothelial factors mediate its vasodilatory effect. On the other The effect of NG-nitro-l-arginine methyl ester (L-NAME, 100 µ M), an inhibitor of endothelial hand, endothelial denudation caused a significant rightward shift of the CRC to meso-dihydroguaiaretic nitric oxide synthase, on the vasorelaxation induced by compound 3 closely resembled that which acid (15) and 5,8,40 -trihydroxy-3,7-dimethoxyflavone (18), indicating the contribution of both occurred in the absence of endothelium, clearly supporting that activation of the NO/cGMP pathway endothelium-independent dependent in their Regarding mechanismmeso-dihydroguaiaretic of action. importantly contributes and to corosolic acidpathways (3)-vasodilation. acid (15)-induced vasodilation, it was significantly reduced (p = 0.006), but not completely blocked, in the
2.2. Participation of the NO/cGMP and H2 S/KATP Channel Pathways in the Vasodilator Response of Compounds 3, 15, and 18
The effect of NG -nitro-l-arginine methyl ester (L-NAME, 100 µM), an inhibitor of endothelial nitric oxide synthase, on the vasorelaxation induced by compound 3 closely resembled that which occurred in the absence of endothelium, clearly supporting that activation of the NO/cGMP pathway importantly contributes to corosolic acid (3)-vasodilation. Regarding meso-dihydroguaiaretic acid (15)-induced vasodilation, it was significantly reduced (p = 0.006), but not completely blocked, in the
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presence of L-NAME, indicating that the effect of this lignan partly depends on stimulation of the presence presence of of L-NAME, L-NAME, indicating indicating that that the the effect effect of of this this lignan lignan partly partly depends depends on on stimulation stimulation of of the the NO/cGMP pathway. On the other hand, the inhibitionof ofendothelial endothelialnitric nitric oxide synthase (eNOS) NO/cGMP pathway. On the other hand, the inhibition oxide synthase (eNOS) NO/cGMP pathway. On the other hand, the inhibition of endothelial nitric oxide synthase (eNOS) with L-NAME produced a slight rightward shift of the CRC of of 5,8,40 -trihydroxy-3,7-dimethoxyflavone with with L-NAME L-NAME produced produced aa slight slight rightward rightward shift shift of the the CRC CRC of of 5,8,4′-trihydroxy-3,75,8,4′-trihydroxy-3,7(18)dimethoxyflavone (p = 0.99) (Figure(18) 4). (p dimethoxyflavone (18) (p == 0.99) 0.99) (Figure (Figure 4). 4).
Figure Vasodilatory effect (3), (b) meso-dihydroguaiaretic acid Figure 4. 4. of (a) (a) corosolic corosolicacid acid (3), meso-dihydroguaiaretic acid (15), and Figure 4.Vasodilatory Vasodilatoryeffect effect of (a) corosolic acid (3), (b)(b) meso-dihydroguaiaretic acid (15), (15), and and (c) (c) 0 5,8,4′-trihydroxy-3,7-dimethoxyflavone (18) in the absence (control) and presence of L-NAME (100 µµM). 5,8,4′-trihydroxy-3,7-dimethoxyflavone (18) in thein absence (control)(control) and presence L-NAMEof (100 M). (c) 5,8,4 -trihydroxy-3,7-dimethoxyflavone (18) the absence and ofpresence L-NAME by analysis variance the curves each compound Analysis byone-way one-way analysisof of variance (ANOVA) wasmade madebetween between thebetween curvesof ofthe each compound (100Analysis µM). Analysis by one-way analysis of(ANOVA) variance was (ANOVA) was made curves of each GG-nitro-l-arginine in the absence and presence of N methyl ester (L-NAME) followed by aa post G in the absence and presence of N -nitro-l-arginine methyl ester (L-NAME) followed byfollowed post hoc hoc compound in the absence and presence of N -nitro-l-arginine methyl ester (L-NAME) by a Tukey’s test pp
