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The Relaxant Activity of Safranal in Isolated Rat Aortas Is Mediated Predominantly Via an Endothelium-Independent Mechanism - Vasodilatory Mechanism of Safranal

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The Relaxant Activity of Safranal in Isolated Rat Aortas Is Mediated Predominantly Via an Endothelium-Independent Mechanism - Vasodilatory Mechanism of Safranal ISSN 2093-6966 [Print], ISSN 2234-6856 [Online] Journal of Pharmacopuncture 2016;19[4]:329-335 DOI: https://doi.org/10.3831/KPI.2016.19.034 Original article The Relaxant Activity of Safranal in Isolated Rat Aortas is Mediated Predominantly via an Endothelium-Independent Mechanism - Vasodilatory mechanism of safranal - Bibi Marjan Razavi1, Mojtaba Alipoor Amanloo2, Mohsen Imenshahidi3, Hossein Hosseinzadeh3* 1Targeted Drug Delivery Research Center, Department of Pharmacodynamics and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran 2 School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran 3 Pharmaceutical Research Center, Department of Pharmacodynamy and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran Key Words Results: Safranal induced relaxation in endotheli- um-intact aortic rings precontracted by using PE or aorta ring, indomethacin, L-NAME, saffron, KCl in a concentration-dependent manner, with a vasodilatory effect maximum relaxation of more than 100%. The relaxant activity of safranal was not eliminated by incubating Abstract the aortic rings with L-NAME (EC50 = 0.29 vs. EC50 = 0.43) or with indomethacin (EC50 = 0.29 vs. EC50 = 0.35), Objectives: Safranal is a pharmacologically active com- where EC50 is the half maximal effective concentration. ponent of saffron and is responsible for the unique aro- Also, the vasodilatory activity of safranal was not mod- ma of saffron. The hypotensive effect of safranal has ified by endothelial removal. been shown in previous studies. This study evaluates the mechanism for the vasodilatory effects induced by Conclusion: This study indicated that relaxant activity safranal on isolated rat aortas. of safranal is mediated predominantly through an en- dothelium-independent mechanism. Methods: To study the vasodilatory effects of safranal (0.2, 0.4 and 0.8 mM), we contracted isolated rat tho- racic aorta rings by using 10-6-M phenylephrine (PE) or 1. Introduction 80-mM KCl. Dimethyl sulfoxide (DMSO) was used as a control. The vasodilatory effect of safranal was also eval- The endothelium produces a variety of substances uated both on intact and denuded endothelium aortic that play important roles in the regulation of circula- rings. Furthermore, to study the role of nitric oxide and tion and vascular wall homeostasis. Among those sub- prostacyclin in the relaxation induced by safranal, we stances, vasodilatory factors, such as nitric oxide (NO), incubated the aortic rings by using L-NAME (10-6 M) or prostacyclin (PGI2) and endothelium-derived hyper- indomethacin (10-5 M), each for 20 minutes. polarizing factor (EDHF), or vasoconstrictive factors, such as thromboxane (TXA2) and endothelin-1 (ET- 1), have critical roles in the control of blood-vessel- Received: Aug 03, 2016 Reviewed: Aug 19, 2016 Accepted: Aug 30, 2016 wall homeostasis [1]. Evidence exists for abnormal This is an Open-Access article distributed under the terms of the Creative Commons *Corresponding Author Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) Hossein Hosseinzadeh. Pharmaceutical Research Center, Department of Pharmacody- which permits unrestricted noncommercial use, distribution, and reproduction in any namics and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, medium, provided the original work is properly cited. Mashhad, Iran. Tel: +98-513-881-9042 Fax: +98-513-882-3251 This paper meets the requirements of KS X ISO 9706, ISO 9706-1994 and ANSI/NISO E-mail: [email protected] Z39.48-1992 (Permanence of Paper). ⓒ 2016 Korean Pharmacopuncture Institute http://www.journal.ac 330 http://www.journal.ac Journal of Pharmacopuncture 2016;19(4):329-335 endothelium-dependent relaxation in hypertension [2], were carried out in accordance with the requirements es- which is a highly prevalent cardiovascular disease and is tablished by the Ethics Committee, Mashhad University regarded as an important risk factor for developing oth- of Medical Sciences (Verification number: 87124; date of er diseases, such as endothelial dysfunction, metabolic approval: Nov 9, 2009). syndrome, diabetes, renal dysfunction, congestive heart Safranal was obtained from Fluka (Switzerland). Phe- failure, coronary artery disease, and stroke [3]. Although nylephrine (PE) hydrochloride, acetylcholine chloride many antihypertensive drugs are available, some adverse (ACh), indomethacin, and N-nitro-L-arginine methylester effects have been reported following their use [4]. There- (L-NAME) were purchased from Sigma-Aldrich (Germa- fore, natural plants and their active components need to ny). The dimethyl sulfoxide (DMSO) (a solvent of safranal) be considered as possible new antihypertensive drugs that and other chemicals used in this study were obtained from can be used with safety, efficacy, and cultural acceptability Merck (Germany). and that may have fewer side effects [5]. Rats were killed by using intraperitoneal (IP) injection Saffron (Crocus sativus L.) is a plant belonging to the Iri- of ketamine/xylazine. Then, the thorax was opened, after daceae family and has been used from ancient times as a which the thoracic aorta was quickly removed, cleaned herbal medicine, spice, food colorant, and flavoring agent from adherent connective tissues, and cut into rings (4 - 5 [6]. Saffron and its active components have valuable phar- mm in length). Special care was taken to ensure that en- macological properties, including anticonvulsant [7], anti- dothelial damage did not occur during tissue preparation. depressant [8-10], anti-inflammatory and antinociceptive Two stainless-steel stirrups were passed through the lu- [11], antioxidant [12], antitumor [13, 14], and hypotensive men of each ring. One stirrup was connected to an isomet- [15, 16] properties. Furthermore, saffron extract or its ac- ric force transducer (POWERLAB, AD Instrument, Austral- tive components have been shown to have protective ef- ia) to measure the tension in the vessels. The rings were fects against some toxic agents, including diazinon and placed in a 25-mL organ chamber (Organ Bath, 4 chan- acrylamide, in the brain, liver, and cardiovascular system nels, AD Instrument, Australia) containing Krebs solution of rats [17]. Research has identified more than 150 chemi- gassed with 95% O2/5% CO2 and maintained at 37°C. The cals in saffron stigma [18], and the pharmacological effects composition of the Krebs solution was as follows: 118.0- of saffron are related to its three major components: cro- mM NaCl, 4.7-mM KCl, 1.2-mM NaH2PO4, 1.2-mM MgSO4, cins, which are considered as coloring agents [19], glyco- 25.0-mM NaHCO3, 11.1-mM glucose, and 2.5-mM CaCl2 side picrocrocin, which is responsible for saffron’s bitter (pH = 7.4) [30]. Rings were placed under a resting tension taste, and safranal, a monoterpene aldehyde, which is re- of 2 g, which had been determined in preliminary studies sponsible for saffron’s unique aroma [13, 20]. to be optimal, and allowed to equilibrate for 1 hour. During Safranal has been shown to possess various pharmacolog- this equilibration period, the physiological salt solution ical activities, including antioxidant [21], anticonvulsant was replaced every 15 minutes. The Labchart 7.3 (AD-In- [22], anti-anxiety and antidepressant [8], antitussive [23], struments) software was used for this study. antinociceptive [24], and anti-ischemia [25] activities. Sa- For the study of the vasodilatory effects of safranal, the franal has also been shown to be able to improve impaired thoracic aorta rings isolated from rats were contracted memory and learning ability in rats [26]. The cardiovascu- by using 10-6-M PE or 80-mM KCl in two separate exper- lar protective effects of safranal have been demonstrated iments. When the vasoconstriction curves for the rings in several studies. Safranal decreased myocardial con- reached the plateau phase of maximum tension, safranal tractility through a calcium channel-blocking property in (0.2, 0.4 and 0.8 mM) was added, and the tensions were guinea pigs [27]. In addition, safranal protected the cardio- recorded. The vasodilatory effect of safranal was expressed vascular system against isoproterenol-induced myocardi- as a percentage of relaxation to maximum constriction in- al infarction in Wistar rats [28]. Moreover, the hypotensive duced by 10-6-M PE or 80-mM KCl. DMSO was used as a effects of safranal have been demonstrated in anaesthe- control. The vasodilatory effect of safranal on constriction tized normotensive and hypertensive rats [15]. In another induced by PE in rat thoracic aortas was also evaluated for study, safranal reduced the mean systolic blood pressure both intact endothelium rings and denuded endothelium in rats treated chronically with desoxycorticosterone ace- rings. To confirm that the endothelial layer had been re- tate (DOCA) salt [29]. Regarding the hypotensive effect of moved, we tested with a single dose of acetylcholine (10-6 safranal, the aims of this study are to determine whether M) after phenylephrine (10-6 M) in denuded vessels. The safranal exhibits vasodilatory effects on isolated rat aortas maximum relaxation induced by acetylcholine was less and to evaluate the mechanism for the vasodilatory effects, than 30%. if any, induced by safranal in isolated rat aortas. To examine whether the safranal induced vasorelaxation was mediated by the NO, we incubated the aortic rings in L-NAME (10-5 M) for 20 minutes, after which the thorac- 2. Material and Methods
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