Effect of oleic, lauric and myristic acids on phenylephrine-induced contractions of isolated rat vas deferens

Transcription

1 Indian Journal of Experimental Biology Vol. 49, September 2011, pp Effect of oleic, lauric and myristic acids on phenylephrine-induced contractions of isolated rat vas deferens ML Arruzazabala, Y Pérez, Y Ravelo, V Molina*, D Carbajal, R Mas & E Rodríguez Centre of Natural Products, National Centre for Scientific Research 25 th Ave, 158 st., P.O 6880, Cubanacán, Havana City, Cuba Received 24 February 2011; revised 24 June 2011 D-004, a lipid extract of Roystonea regia fruits that contains oleic, lauric and myristic acids as major components inhibits α1-adrenoreceptors-mediated contractile responses in isolated rat vas deferens and prostate trips; no study has demonstrated a similar effect for oleic, lauric or myristic acids individually. Therefore, the effects of D-004 (250 µg/ml), oleic (100 µg/ml), lauric (50 µg/ml) or myristic (25 µg/ml) acids and their combined effects on phenylephrine (PHE: mol/l) induced contractions has been studied. No treatment changed the basal tone of the preparations, but all inhibited PHE-induced contractions. D-004 produced the highest inhibition, followed by lauric acid, which was more effective than myristic and oleic acids against PHE-induced contractions of control group. D-004 and the mixture of the three acids produced similar inhibitions. Keywords: D-004; Oleic, lauric and myristic acids; Phenylefrine induced contractions, Vas deferens Benign prostatic hyperplasia (BPH), a common disease in the aging men, is a histological condition that consists in an hyperplastic process leading to a measurable growth of glandular-epithelial and stromal/muscle prostatic tissue, which may contribute to the lower urinary tract symptoms (LUTS) common in men with BPH 1, 2. Hormonal factors, like the androgenic hormones testosterone (T) and dihydrotestosterone (DHT), play a crucial role in the etiology of BPH 3, while growth factors and estrogens may contribute to this condition as well. LUTS, however, may or may not be linked with prostate enlargement and bladder outlet obstruction 2. Indeed, urinary outlet obstruction in BPH does not result from the urethral compression produced by the hypertrophied prostate only, but from the enhanced stimulation of α 1 -adrenoceptors (AR) of both urethral and prostatic smooth muscle 2,4,5, being reported an increased number of α 1 -AR in the prostate of patients with BPH 6. Therefore,α 1 -AR-blockers are among the first-line pharmacological options to treat BPH, mainly effective in relieving LUTS in these patients 7. Plant extracts, like saw palmetto lipid extracts (SPLE), are widely used to treat BPH and related *Correspondent author Telephone: [537] Fax: [537] vivian.molina@cnic.edu.cu LUTS Although some trials have failed to find differences versus placebo 11,12, most studies have supported that saw palmetto is effective to treat BPH/LUTS, with an efficacy comparable to that of finasteride and tamsulosin 13,14. Multiple, rather than a single mechanism, contribute to the efficacy of SPLE in BPH, like the inhibition of prostate 5 α-reductase activity and the antagonism of α 1 -AR in vitro and in vivo 18,19. In addition, major components of SPLE, like lauric, oleic, myristic acids, have been shown to produce similar effects, since lauric acid is a potent inhibitor of 5 α-reductase activity, oleic and myristic acids being also able to inhibit the enzyme activity 15,20. A recent study also demonstrated that like SPLE, lauric, oleic, myristic and linoleic acids also inhibited the specific binding of radioligands to α 1 AR, receptors 21. D-004 is a lipid extract of the royal palm (Roystonea regia) fruits that contains a mixture of free fatty acids wherein oleic, lauric, palmitic and myristic acids are major components. D-004 has been shown to inhibit competitively rat prostate 5αreductase activity in vitro 22, and orally given prevented T-induced, but not DHT-induced, prostate hyperplasia (PH) in rodents As SPLE, D-004 also antagonizes α 1 -AR-mediated responses in both in vitro and in vivo conditions. In vitro, D-004 inhibited markedly and dose-dependently

2 ARRUZAZABALA et al.: OLEIC, LAURIC & MYRISTIC ACIDS ON CONTRACTILE RESPONSES 685 norepinephrine (NE) and phenylephrine (PHE)- induced contractions in isolated preparations of rat vas deferens and prostate trips, respectively 27, 28. Also, oral treatment with D-004 to rats significantly reduced the histological features of PH and the urodynamic changes (void volume reduction) induced with PHE 29,30, and modestly, but significantly, attenuated the NE-induced hypertensive effects 28. To our knowledge, however, no work had demonstrated that weather any of the major components of D-004 can inhibit α 1 -AR-mediated responses. The present study investigated whether the major components (oleic, lauric and myristic acids) of D-004 were able to inhibit PHE-induced contractions in isolated rat vas deferens. Animals Young adult male SD rats of 9 weeks old, weighing g, were purchased from the National Centre for Laboratory Animals Production (CENPALAB, Havana, Cuba). Animals were adapted to laboratory conditions (temperature 25 ± 3 C, 60 ± 5% RH and 12:12 h L:D cycle) for 7 days. Food (rodent chow obtained from CENPALAB) and water were provided ad libitum. Animals handle were maintained in accordance with the Cuban Regulations for the use of laboratory animals and ethical principles for animal management. An independent ethical board approved the use of the animals in the study. Chemicals D-004 (batch ) was obtained from the Chemistry Department of the Centre of Natural Products (Havana City, Cuba). The batch had the following composition (percentages of the relative weight of each acid with respect to that of the raw material): caprilic (C 8: 0; 0.4%), capric (C 10:0 ; 0.7%), lauric (C 12:0 ; 23.9%), myristic (C 14:0 ;10.9%), palmitic (C 16:0 ; 10.4%), palmitoleic (C 16:1 ; 0.1%), stearic (C 18:0 ; 2.5%), oleic (C 18:1 ; 32.9%), linoleic (C 18:2 ; 9.1%), and linolenic (C 18:3 ; 0.1%), which was assessed with a validated gas chromatography method. Batch purity (total content of these acids within the raw material) was 91.7%. D-004 and oleic, lauric and myristic acids (Sigma Chemical CO, St Louis, USA) were suspended in a 2% Tween 65/H 2 O vehicle, and suspensions were prepared immediately before use. Phenylephrine (PHE) was obtained from QUIMEFA, Havana Cuty, Cuba. In vitro effects on PHE-induced vas deferens contractions The animals were anaesthetized with ether. The vas deferens were dissected free from extraneous tissue and mounted in organ baths containing Tyrode solution and bubbled with 5% CO 2 and 95% O Experiment 1 In this experiment, the effects of adding D-004 (250 µg/ml), oleic (100 µg/ml), lauric (50 µg/ml) or myristic acid (25 µg/ml) were observed. The respective concentrations of each acid were equivalent to those present in the added amount (250 µg/ml) of D-004. In brief, following an equilibration period of 30 min, a control set of experiments were conducted. Rat vas deferens preparations were suspended in organ bath containing tyrode solution with Tween 65/H 2 O (control) and contractions were induced with successive accumulative concentrations of PHE ( mol/l). Then, suspensions of D-004, oleic or lauric or myristic acid at the concentrations referred above were added to bath solution in independent experiments, and 20 min after the contractile responses to PHE were recorded. Experiment 2 In experiment 2, the effects of D-004 (250 µg/ml) and of combined effect of oleic (100 µg/ml), lauric (50 µg/ml) and myristic acid (25 µg/ml) on PHE-induced contractions in rat vas deferens was studied. Briefly, after a 30 min equilibration period, independent experiments assessed the effects of tyrode solutions containing Tween 65/H 2 O (control), D-004 (250 µg/ml) or oleic (100 µg/ml) + lauric (50 µg/ml) + myristic acid (25 µg/ml) on rat deferens vas contractions induced with successive accumulative concentrations of PHE ( mol/l). Contractions of the tissues were recorded isotonically using a lever transducer attached to a Nihon Kohden transducer attached to the Nihon Kohden polygraph. Statistical analysis All values of tissues responses were related to maximal response of control group (E/Emax). Comparison between study groups was performed using the Kruskal-Wallis one-way test. Differences between individual groups and control groups were compared using Dunn s test. P < 0.05 was considered significant. Statistical analysis were performed using Pathtox system software (version 4.2.2; Xybion Corp; Cedar Knolls, NJ, USA) Results Experiment 1 The addition of the vehicle, D-004, lauric, oleic or myristic acids did not change the basal tone of the isolated vas deferens, but D-004 and all the three acids inhibited PHE-induced contractions (Table 1). D-004 (250 µg/ml) and lauric acid (50 µg/ml) significantly inhibited the contractions induced with all the concentrations of PHE (

3 686 INDIAN J EXP BIOL, SEPTEMBER mol/l) used in the dose-response curves, meanwhile myristic acid inhibited only the contractions induced with two concentrations of PHE ( and mol/l) and oleic acid only those induced with PHE mol/l. According to the effects on contractions induced with the highest concentration of PHE, the D-004 produced the highest inhibition, followed by lauric acid, while inhibitions with myristic and oleic acids were similar. Experiment 2 The inhibitory effects of the joint addition of the three acids and D-004 on PHE-induced contractions on vas deferens were statistically similar (Table 2). Discussion The results demonstrate that lauric, oleic and myristic acids, major components of D-004, were able to antagonize PHE-induced contractions on isolated rat vas deferens preparations and that the effect of adding together these three acids at the same relative concentrations present in D-004 were the same of those elicited by D-004 (250 µg/ml). In addition to inhibit 5α-reductase in vitro 23 and to prevent testosterone-induced prostate hyperplasia in rodents in vivo 24-27, D-004 markedly and dose dependently inhibited PHE-induced contractions in isolated vas deferens and rat prostate preparations in vitro 27, 28. Although lauric, oleic and myristic acids, major components of D-004, have been identified as bioactive compounds of SPLE that inhibit prostate 5 α-reductase activity 15-17, no study had reported if they can inhibit α 1 -AR-mediated contractile responses. Therefore, in order to know the pharmacologically active constituents of D-004 that contributes to its effects on α 1 -AR-mediated contractions, the present work was undertaken. The present study first confirms the inhibitory effect of D-004 on α 1 -AR-mediated contractions, since the fact that D-004 significantly inhibited PHEinduced contractions of isolated vas deferens is consistent with its effects on nor-epinephrine and PHE-induced contractions in isolated rat vas deferens and prostate preparations 27,28, respectively. In addition, it was observed that the addition of lauric (50 µg/ml), oleic (100 µg/ml) and myristic (25 µg/ml) acids also inhibited significantly PHEinduced contractions of rat vas deferens; lauric acid exhibiting the highest inhibition against PHE-induced contractions of control group, showing that the acids, the major components of D-004, are the active compounds of D-004 responsible of its inhibitory effect. Further, the fact that D-004 (250 µg/ml) and the mixture of the three acids at the same concentrations referred above produced a similar inhibition of the PHE-induced contraction confirms this assumption and practically ruled out the possibility of other(s) component of D-004 involved in this effect. Treatments Table 1 Effect of D-004, OA, LA and MA on PHE-induced contractions (E/Emax, %) of rat vas deferens [Values are mean ± SD] PHE concentrations (mol/l) Control 1.07 ± ± ± ± ± ± ± 0.00 D004 (250 µg/ml) 0 ± ± 0.5 b 3 ± 0.81 c 17 ± 3.43 c 30 ± 6.02 c 41± 7.51 d 42 ± 7.26 d LA (50 µg/ml) 0 ± ± 0.00 a 7.6 ± 2.00 a 20 ± 5.80 b 40 ± b 52 ± b 54 ± be OA (100 µg/ml) 0 ± ± ± ± a 51± ±19.92 e 65± e MA (25 µg/ml) 0 ± ± ± 3.05 a 28 ± 9.36 a 57 ± ± e 72 ± e PHE= phenylephrine, LA=lauric acid, MA=myristic acid, AO=oleic acid, P values: a < 0.05, b < 0.01, c < 0.001, d < ; e < 0.05 as compared with control (a-d) and with D-004 (e) (Dunn test). Treatments Table 2 Effect of D-004 (250 µg/ml) and the mixture of OA (100 µg/ml), LA (50 µg/ml) plus (MA 25 µg/ml) on PHE-induced contractions (E/Emax, %) of rat vas deferens [Values are mean ± SD] PHE concentrations (mol/l) Control 0.8 ± ± ± ± ± ± ± 0.00 D004 0 ± ± ± 3.85 * 22 ± 7.17* 47 ± 12.94* 51± 9.88* 51± 9.88* OA + LA +MA 0 ± ± ± 4.31* 29 ± * 55 ± 13.79* 62 ± 13.19* 62 ± 13.19* PHE= phenylephrine, LA= lauric acid, MA= myristic acid, AO=oleic acid, *P< 0.01 as compared with control (Dunn test)

4 ARRUZAZABALA et al.: OLEIC, LAURIC & MYRISTIC ACIDS ON CONTRACTILE RESPONSES 687 The addition of D-004 or of oleic, lauric and myristic acids did not affect the basal tone of rat vas deferens, which do not reproduce a sympathomimetic effect of SPLE. Thus, SPLE had been shown to antagonize NE-induced contractions in smooth muscle isolated preparations 31,32, however, Cao et al. 33 reported that its addition to isolated rat prostates strips produced an unexpected contractile baseline effect, not reported before. Considering that D-004 shares some similarities in composition and effects with SPLE, the effects of D-004 and the three acids on vas deferens basal tone was studied. These results suggest that other component(s) of SPLE different from oleic, lauric or myristic acid is (are) involved in this sympathomimetic effect. Since α 1 -AR mediate the contractile response of the prostate, which is responsible for about 50% of the prostatic urethral pressure in BPH patients, a treatment that antagonizes α1-ar mediated responses should be useful to manage BPH/LUTS 4-7. The present results add to the understanding of the active compounds of D-004 that contributes to the antagonism of α 1 -AR blockermediated responses, a key mechanism in its potential benefit to manage BPH/LUTS. Conclusions The results of the present study demonstrate that the major components of D-004 (oleic, lauric and myristic acids) were active to inhibit the contractile responses induced by PHE in rat vas deferens, and that the effect of D-004 was greater than those of the individual acids, but equivalent to that of the sum of these acids. References 1 Nix J & Carson C, Medical management of benign prostatic hypertrophy, Can J Urol, 14 (2007) Roehrborn C G, Pathology of benign prostatic hyperplasia, Int J Impot Res, 20 (2008) S11. 3 Carson C & Rittmaster R, The role of dihydrotestosterone in benign prostatic hyperplasia, Urology, 61 (2003) 2. 4 Schwinn D A & Roehrborn C G. Alpha1-adrenoceptor subtypes and lower urinary tract symptoms, Int J Urol, 15 (2008) Michel M C, Taguchi K & Schafers R S, Alpha 1- adrenoceptor subtypes in the human cardiovascular and urogenital systems, Adv Pharmacol, 42 (1998) Yamada S, Ashizawa N, Ushijima H, Nakayama K, Hayashi E & Honda K, Alpha-1 adrenoceptors in human prostate: characterization and alteration in benign prostatic hypertrophy, J Pharmacol Exp Ther, 242 (1987) Olke M, Hofner K, Berges R R & Jonas U, Drug therapy of benign prostatic hyperplasia syndrome with alpha 1-receptors blockers. Basic principles and clinical results, Urologe A, 41 (2002) Gordon A & Shaughnessy A F. Saw Palmetto for prostate disorders, Am Fam Physician, 67 (2003) Plosker G L & Brodgen RN, Serenoa repens (Permixon). A review of its pharmacology and therapeutic efficacy in benign prostatic hyperplasia, Drugs Aging, 9 (1996) Aliev IuG, Vinarov A, Lokshin KL & Spivak LG, Five-years experience in treating with prostatic hyperplasia patiens with permixon (Serenoa repens Pierre Fabre Medicament), Urologia 1 (2002) Willetts K E, Clements M S & Champion S, Serenoa repens extract for benign prostate hyperplasia: A randomized controlled trial, BJU Int, 92 (2003) Bent S, Kane C, Shinohara K, Nehuhaus J, Hudes E S & Goldberg H, Saw palmetto for benign prostatic hyperplasia, N Engl J Med, 354 (2006) Carraro J C, Raynaud J P, Koch G, Chirsholm G D, Di Silverio F & Teilac P, Comparisons of phytotherapy (Permixon) with finasteride in the treatment of benign prostate hyperplasia: A randomized international study of 1098 patients, Prostate, 29 (1996) Debruyne F, G. Koch., P. Boyle., F.C Da Silva., J.G. Gillenwater & F.C Hamdy. Groupe d etude PERMAL, Comparison of a phytotherapeutic agent (Permixon) with an alpha blocker (Tamsulosin) in the treatment of benign prostatic hyperplasia: A 1 year randomized international study, Prog Urol, 12 (2002) Raynaud J P, Cousse H & Martin P M. Inhibition of type 1 and type 2 5-alpha reductase activity by free fatty acids, active ingredients of Permixon. J Steroid Biochem Mol Biol, 82 (2002) Rhodes L, Primka R L, Berman C, Vergult G, Gabriel M, Pierre-Malice M & Gibelin B, Mechanisms involved in the spasmolytic effect of extracts from Sabal serrulata fruit on smooth muscle, Prostate, 22 (1993) Weisser H, Tunn S, Behnke B & Krieg M L, Effects of the sable serrulata extract IDS 89 and its subfractions on 5-alpha reductase activity in human prostatic hyperplasia, Prostate, 28 (1996) Goepel M, Hecker U, Krege S, Rubben H & Michel M C, Saw palmettos extracts potently and non-competitively inhibit human alpha 1 adrenoceptors in vitro, Prostate, 38 (1999) Goepel M, Dihn L & Mitchell A, Do Saw palmetto extracts block human alpha 1-adrenoceptors subtypes in vivo?, Prostate, 46 (2001) Niederprum H J, Schweikert H U & Zanker K S, Testosterone 5-alpha-reductase inhibition by free fatty acids from Sabal serrulata fruits, Phytomedicine, 1 (1994) Abe M, Ito Y, Oyunzul L, Oki Fujino T & Yamada S, Pharmacological relevant receptor binding characteristics and 5-α reductase inhibitory activity of free fatty acids contained in saw Palmetto extract, Biol Pharm Bull, 32 (2009) Pérez Y, Menéndez R, Más R & González R M, In vitro effect of D-004, a lipid extract of the fruit of the Cuban royal palm (Roystonea regia), on prostate steroid 5α-reductase activity, Curr Ther Res Exptl & Clin, 67 (2006) Arruzazabala M L, Carbajal D, Mas R, Molina V, González V & Rodríguez E, Preventive effect of D-004, a lipid extract from Cuban royal palm (Rostoynea regia) fruits, on prostate

5 688 INDIAN J EXP BIOL, SEPTEMBER 2011 hyperplasia induced with testosterone on intact and castrated rodents, Drugs Exptl Clin Res, 30 (2004) Carbajal D, Arruzazabala M L, Mas R, Molina V, Rodríguez E & González V, Effect of D-004, a lipid extract from Cuban royal palm fruit, on inhibiting prostate hyperplasia induced with testosterone and dihydrotestosterone in rats, Curr Ther Res Exptl Clin, 65 (2004) Carbajal D, Molina V, Mas R & Arruzazabala M L, Therapeutic effect of D-004 a lipid extract from Roystonea regia fruits on prostate hyperplasia induced in rats, Drugs Exp Clin Res, 31 (2005) Noa M, Arruzazabala M L, Carbajal D, Mas R & Molina V, Effect of D-004, a lipid extract from Cuban royal palm fruit, on histological changes of prostate hyperplasia induced with testosterone in rats, Int J Tiss Reac, 27 (2005) Arruzazabala M L, Pérez Y, Molina V, Carbajal D, Ravelo Y & Mas R, Effect of D-004, a lipid extract from royal palm (Roystonea regia) fruits, on phenylephrine-induced contractions of isolated rat prostate, Rev Cubana Farmacia, 43 (2009) Arruzazabala M L, Mas R, Carbajal D & Molina V, Effect of D-004, a lipid extract from the Cuban royal palm fruit, on in vitro and in vivo effects mediated by alpha-adrenoceptors in rats, Drugs R & D, 6 (2005) Arruzazabala M L, Mas R, Molina V, Noa M & Carbajal D, Effect of D-004, a lipid extract from the Cuban royal palm fruit on atypical prostate hyperplasia induced by phenylephrine in rats, Drug R & D, 7 (2006) Arruzazabala M L, Molina V, Más R & Carbajal D, Effects of D-004, tamsulosin and the combined therapy D004 plus tamsulosin on urodynamic changes induced with phenylephrine in rats, Arzn-Forsc Drug Res, 58 (2008) Gutiérrez M, García de Boto M J, Cantabrana B & Hidalgo A, Mechanisms involved in the spasmolytic effect of extracts from Sabal serrulata fruit on smooth muscle, Gen Pharmacol, 27 (1996) Odenthal K P & Rauwald H W, Lipophilic extract from Sabal serrulata inhibits contractions in smooth muscle tissue, Aktuelle Urologie, 27 (1996) Cao N, Haynes J M & Ventura S, Saw palmetto is an indirectly acting sympathomimetic in the rat-isolated prostate gland, Prostate, 66 (2005) 115.