Antihypertensive and vasorelaxant effects of dihydrospinochalcone-a isolated from Lonchocarpus xuul lundell by no production: Computational and ex vivo approaches.
Keywords: Anti hypertensive Di hydrospinochalcone-A Lonchocarpus xuul Docking lsocordoin Nitric oxide Vasorelaxant
Current work was conducted to evaluate the vasorelaxant effect of dihydrospinochalcone-A (1) and iso-cordoin (2), compounds type chalcone isolated from Lonchocarpus xuul, an endemic tree of the Yucatan Peninsula, Mexico. Compounds 1 and 2 were found to induce significant relaxant effect in a concentration-dependent manner on aortic rat rings pre-contracted with noradrenaline (NA, 0.1 [mu]M). Compound 1 was the most active and its effect was endothelium-dependent (Emax = 79.67% and [EC.sub.50] = 21.46 [mu]M with endothelium and Emax = 23.58% and [EC.sub.50] = 91.8 [mu]M without endothelium, respectively). The functional mechanism of action for 1 was elucidated. Pre-incubation with L-NAME (unspecific nitric oxide synthase inhibitor), indomethacin (unspecific COX inhibitor), ODQ (soluble guanylyl cyclase inhibitor), atropine (cholinergic receptor antagonist), TEA (unspecific potassium channel blocker) reduced relaxations induced by 1. Oral administration of 50 mg/kg of compound 1 exhibited significant decrease in diastolic and systolic blood pressure in SHR rats. The heart rate was not modified. Compound 1 was docked with a crystal structure of eNOS. Dihydrospinochalcone-A showed calculated affinity with eNOS in the Cl binding pockets, near the catalytic site; Trp449, Trp447 and His373 through aromatic and [pi]--[pi] interactions, also His463 and Arg367 are the residues that make hydrogen bonds with the carbonyl and hydroxyl groups.
In conclusion, dihydrospinochalcone-A induces a significant antihypertensive effect due to its direct vasorelaxant action on rat aorta rings, through NO/sCG/PKG pathway and potassium channel opening.
[c]2013 Elsevier GmbH. All rights reserved.
Flavonoids are a group of naturally occurring polyphenolic compounds that are found in fruits, vegetables, nuts, seeds, herbs, spices, stems, flowers, as well as tea and red wine. They are prominent components of citrus fruits and other food sources and are consumed regularly with the human diet, with over 8000 individual compounds known (Middleton et al. 2000). Flavonoids are usually subdivided according to their substituents into flavones, flavanols, flavanones, flavanonols, anthocyanidins and chalcones (Gonzalez-Castejon and Rodriguez-Casado 2011). Flavonoids are potent antioxidants, and therefore one of the main interests in the compounds has involved protection against cardiovascular and other diseases. Antioxidation is, however, only one of the many mechanisms through which flavonoids could exert their actions. Many studies have suggested that flavonoids exhibit wide pharmacological activities, including antiallergenic, antiviral, anti-inflammatory, antidiabetic, cardiovascular diseases protection, vasodilators, among others (Perez-Vizcaino and Duarte 2010; Duarte et al. 2001; Harborne and Williams 2000).
Chalcones, or 1,3-diary1-2-propen-1 -ones, consist of open-chain flavonoids in which the two aromatic rings are joined by a three-carbon [alpha], [beta]-unsaturated (or saturated) carbonyl system. A vast number of naturally occurring chalcones are polyhydroxylated in the aryl rings. The free-radical quenching properties of the phenolic groups present in many chalcones have raised interest in using the compounds or chalcone rich plant extracts as drugs or food preservatives. Chalcones have been reported to possess many useful properties, including anti-inflammatory, antimicrobial, antifungal, antioxidant, cytotoxic, antitumor and anticancer activities (Nowakowska 2007).
Hypertension is a cardiovascular disease with the most epidemiological impact in the world, and also represents a major risk factor for developing other diseases as endothelial dysfunction, metabolic syndrome, diabetes, renal dysfunction, congestive heart failure, coronary artery disease, and stroke (Ogihara et al. 2005). Currently, there are antihypertensive drugs used to control arterial blood pressure classified as vasodilators, sympathicolytic drugs, calcium-channel blockers, drugs acting on renin-angiotensin system and diuretics (Staffileno 2005). Despite this, pharmaceutical companies have shown that natural products still represent an extremely valuable source for production of new chemical entities for the treatment of untreated diseases, since they represent privileged structures selected by evolutionary mechanisms over a period of millions of years (Smith and Ashiya 2007; Koehn and Carter 2005; Harvey 2007). In addition, the World Health Organization (WHO) estimates that about 65-80% of the world's population in developing countries due to the poverty and lack of access to modern medicine; depend essentially on plants for their primary health care (Smith and Ashiya 2007; Koehn and Carter 2005; Harvey 2007).
In this context, current work has the aim of to determine the vasorelaxant action of dihydrospinochalcone-A (1) and isocordoin (2) (Fig. 1) isolated from Lonchocarpus xuul (Escalantc-Erosa et al. 2012; Yarn-Puc and Pena-Rodriguez 2009), an endemic tree from the Yucatan Peninsula. Mexico. Also, the underlying mode of action and the antihypertensive effect of 1 were determined.
Materials and methods
Chemicals and drugs
Noradrenaline bitartrate (NA), atropine, N-nitro-L-arginine methyl ester (L-NAME), indomethacin, carbamylcholine chloride (carbachol), 1-H-[1,2,4]-oxadiazolo-[4,3a]-quinoxalin-1-one (ODQ), nifedipine, tetraethylammonium chloride (TEA), were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). All other reagents were analytical grade from local sources. Dihydrospinochalcone-A (1) and isocordoin (2), chalcone-type compounds, were isolated from hexanic extract of Lonchocarpus xuul as previously described (Escalante-Erosa et al. 2012; Yam-Puc and Pena-Rodriguez 2009).
Wistar and SHR rats were provided by F.E.S. Iztacala animal facilities, from Universidad Nacional Autonoma de Mexico. Animals (200-250g) were housed in groups of six (n = 6) under laboratory conditions (12 h light/dark cycle, 25 [+ or -]2[degrees]C and 45-65% of humidity). Before experimentation, all animals were fasted for 16 hours with water ad libitum. All animal procedures were conducted in accordance with the Mexican Federal Regulations for Animal Experimentation and Care (SAGARPA, NOM-062-Z00-1999, Mexico), and approved by the Institutional Animal Care and Use Committee (UNAM) based on US National Institute of Health publication (No. 85-23, revised 1985).
Rat aorta ring assay: functional and mechanistic approaches
Experimental evaluation was conducted using a modified protocol (Hernandez-Abreu et al. 2009). Briefly, rats about 250-300 g of body weight were sacrificed by cervical dislocation. Thoracic aorta was isolated, cleaned of fat and connective tissue and, finally, cut in 4-5 mm length rings. Some aortic rings were denuded of endothelium layer by gentle mechanical procedure. Arterial segments were held by stainless steel hooks into 10 mL organ baths, containing warmed (37[degrees]C), and oxygenated ([0.sub.2]/[CO.sub.2], 19:1) Krebs solution. It was used a basal tension of 3 g for these tissues, and changes in basal tension were recorded by Grass-FT03 force transducers (Astromed, West Warwick, RI, USA), connected to a MP100 analyzer (Biopac Instruments, Santa Barbara, CA, USA). Sixty min after stabilizing the preparation, rings were stimulated with noradrenaline (NA, 0.1 [micro]M) during 10 min and washed out to remove stimulant agent. Subsequently, this procedure was repeated three times every 30 min. Finally, absence of endothelium layer was confirmed by the lack of a relaxing response induced by carbachol (1 [micro]M ) stimuli after last contraction. Then, vascular tissue was contracted by NA and, after that, test samples (pure compounds or positive control) were added to bath in quarter-log cumulative concentrations (evaluation period). The relaxant effect was determined by comparison among maximum vascular contraction before and after addition of samples.
To determine the possible vasorelaxant mechanism exerted by test sample we used cholinergic antagonist, potassium channel blocker and enzymatic inhibitors in endothelium signaling pathways throughout rat aorta ring assay. For this protocol, 15 min before last NA stimulation, we pre-incubated aorta rings with L-NAME (10 [micro]M, unspecific nitric oxide synthase inhibitor), indomethacin (10 [micro]M, unspecific COX inhibitor), ODQ (soluble guanylyl cyclase inhibitor), atropine (muscarinic cholinergic receptor antagonist) and TEA (unspecific potassium channel blocker) in according being the case.
In vivo experiments
Antihypertensive activity study of compound 1 was conducted in SHR rats. Animals were allotted into two groups (six animals each): Control rats (CR, group 1) and treated rats (Compound 1, group 2). Treated group received a single intragastric dose of compound 1 (50 mg/kg). Measurements (blood pressure and heart rate) were recorded before and after the treatment of test compound at 0, 1, 3, 5 and 7 h by a tail cuff method using a LE 5001 automatic blood pressure computer (PanLab[TM], Harvard Apparatus, Spain). Percent decrease in heart rate (HR), systolic blood pressure (SBP), and diastolic blood pressure (DBP) were calculated.
In silico docking studies
Discovery Studio, version 3.1 (Accelrys, Inc. San Diego), and Pymol 1.2 were used for visualization. The crystal structure of eNOS was retrieved from the PDB with the accession code 1D00. Docking calculations were conducted with AutoDock, version 4.2. In short, AutoDock performs an automated docking of the ligand with user-specified dihedral flexibility within a protein rigid binding site. The program performs several runs in each clocking experiment. Each run provides one predicted binding mode.
All water molecules and 3-bromo-7-nitroindazole (crystallographic ligand) were removed from the crystallographic structure and all hydrogen atoms were added. For all ligands and proteins, Gasteiger charges were assigned and non-polar hydrogen atoms were merged. All torsions were allowed to rotate during docking. The auxiliary program AutoGrid generated the grid maps. Each grid was centered at the crystallographic coordinates of the crystallographic compound. The grid dimensions were 60A x 60A x 60A with points separated by 0.375 A. The Lamarckian genetic algorithm was applied for the search using default parameters. The number of docking runs was 25. After docking, all solutions were clustered into groups with RMS lower than 2.0 A. The clusters were ranked by the lowest energy representative of each cluster.
Data were expressed as mean [+ or -] S.E.M. and statistical significance was evaluated by using an ANOVA followed by Tukey's test. p values less than 0.05 were considered to denote statistical significance.
Results and discussion
In our current program for the isolation and discovery of natural and synthetic bioactive compounds as potential antihypertensive drugs, we are focusing in the study of natural occurring flavonoids based on their wide spectra of pharmacological activities, mainly as cardioprotective actions (Perez-Vizcaino and Duarte 2010; Dong et al. 2009; Duarte et al. 2001). In this opportunity, it was decided to study prenylated chalcone type flavonoids dihydrospinochalcone-A (1 ) and isocordoin (2), previously isolated from Lonchocarpus xuul (Escalante-Erosa et al. 2012; Yam-Puc and Pena-Rodriguez 2009) for their vasorelaxant and anti hypertensive actions. There exist few studies that show the vasorelaxant activity of chalcones; however, they have shown significant relaxant effect on aortic rings (Duge de Bernonville et al. 2010; Dong et al. 2008, 2010), although there does not exists any report about their mechanism of vasorelaxant action neither any report that shows their antihypertensive effect. In this context, current work is the first attempt to show the underlying mechanism of action and antihypertensive effect of prenylated chalcone 1.
Compunds 1 and 2 showed significant concentration-dependent vasorelaxant effects on the contraction induced by NA (0.1 [micro]M) in rat aorta rings (Fig. 2). The effect of 1 was endothelium-dependent (Emax = 79.67%; [EC.sub.50] = 21.46 [micro]M) and was more active than compound 2, which was endothelium-independent. Both were less potent and efficient than carbachol and nifedipine, used as positive controls (Fig. 2). As mentioned, 1 was the most active; so, we decided to explore its underlying functional mechanism of action. Vasorelaxant effect of chalcone 1 was endothelium-dependent (Fig. 2), suggesting the participation of endothelium-derived relaxing factors (EDRF) such as nitric oxide (NO) and prostacyclin ([PGI.sub.2]), as mediators of this pharmacological effect (Vanhoutte 2001). In current investigation, it was observed that relaxant effect produced by 1 was drastically inhibited when tested in the presence of NAME (10 [mu]M, a nitric oxide synthase inhibitor), ODQ (1 [mu]M, a soluble guanylyl cyclase inhibitor), and atropine (1 [mu]M, an antagonist of muscarinic receptor) (Figs. 3 and 4). Moreover, aorta rings pre-incubated with indomethacin (100 [mu]M, an unspecific cyclooxygenase inhibitor) did not induce any change in the relaxant action of 1 (Fig. 4). As widely described in literature, eNOS and sGC represent key regulatory factors on NO/cGMP system (Schlossmann et al. 2003). Thus, it seems that dihydrospinochalcone-A is involved in a better bioavailability of NO in vessels through activation of eNOS and, subsequently, a stimulation of sGC that increase cGMP synthesis to induce vascular relaxation (Li and Forstermann 2000). This asseveration was confirmed when enzymatic activity of sGC was inhibited with ODQ and vascular relaxation was modified on aorta rings (Fig. 3).
Additionally, in the presence of TEA (5 mM), compound 1 relaxant curve was shifted to the right (Fig. 5), which indicates that an indirect second mechanism of action is involved in relaxant activity related with the [K.sup.+] channel opening. In this context, an increase in cGMP concentration results in the PKG activation, which phosphorylates specific targets, e.g., potassium channels. This leads to their opening with hyperpolarization of membrane and blockade of L-type voltage-gated [Ca.sub.2+] channels (CaV), which causes the reduced cytoplasmic [Ca.sub.2+] concentration and, consequently, vascular smooth muscle relaxation (Hofmann et al. 2000).
Since the relaxant effect of 1 was greatly reduced by L-NAME, an established NOS inhibitor, we investigated in silico the putative interaction of compound 1 with eNOS. The aim of the molecular docking tools is to map the possible drug-target interactions to explain the activity shown in order to assist rational drug design. Dihydrospinochalcone-A (1) was docked with eNOS using the program AutoDock 4.2. According to previous studies, the binding pocket of NOS's is large and can be divided into four pockets. The catalytic pocket 'S' is located above the HEME molecule. The 'M' pocket is located between the pocket S and the substrate access channel. The 'C'1' and 'C2' pockets are several angstroms away from catalytic site. Cl and C2 present more different residues among the NOS isoforms (Ji et al. 2003). Therefore, we decided to work with sites Cl and C2, which are conformed by following residues: Trp76, Val106, Leu107, G1n112, Ser248, Gln249, Arg252, A1a268, Asn340, Arg367, His373, Arg374, Trp449, His463, Tyr477, Gln478, Pro479 y Asp48. The grid was centered on the co-crystallized ligand, fitting the cavities between Cl and C2. Chalcone 1 showed calculated affinity with eNOS in the Cl binding pockets, near the catalytic site; through Trp449, Trp447 and His373 aromatic and [phi]--[phi] interactions, and His463 and Arg367 are the residues that make hydrogen bonds with the carbonyl and hydroxyl groups (Fig. 6). Site 'Cl' does not belong to the catalytic region, but instead is an allosteric site, which is possible that modulates the activity of eNOS. Compound 1 binding in Cl site and docking results indicate that this chalcone could interact with the binding pockets that access the catalytic site, and this interaction may activate eNOS. Modeling the ligand-enzyme interaction supports our functional data; even though enzymatic and other in vitro and molecular experiments that further support the direct activation of eNOS by 1 remain to be performed.
Finally, oral administration of 50 mg/kg of compound 1 exhibited significant decrease in diastolic and systolic blood pressure in SHR rats. The heart rate was not modified (Fig. 7). It is important to mention that SHR model is an adequate method for determine antihypertensive activity, since these animals have similar pathophysiological condition as human essential hypertension characterized by high blood pressure, endothelial dysfunction and oxidative stress (Russell and Proctor 2006).
In conclusion, dihydrospinochalcone-A induces a significant antihypertensive effect due to its direct vasorelaxant action on rat aorta rings, through NO/sCG/PKG pathway and potassium channel opening.
This study was financed by a grant from "Apoyo a la Mejora del Perfil Individual del profesorado de tiempo completo (Fondo para la Consolidacion de las Universidades publicas Estatales y con Apoyo Solidario Ejercicio 2009)", Faculty of Pharmacy budgets (2012 and 2013), and grants from CONACyT-CIENCIA BASICA CB-2011-01 (167044). G. Avila-Villareal acknowledges the fellowship awarded by CONACyT to carry out graduate studies.
* Taken in part from Master in Pharmacy thesis of G. Avila-Villarreal.
* Corresponding author. Tel.: +52 777 329 7089: fax: +52 777 329 7089.
E-mail address: firstname.lastname@example.org (S. Estrada-Soto).
0944-7113/$--see front matter [c] 2013 Elsevier GmbH. All rights reserved.
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Gabriela Avila-Villarreal (a), Oswaldo Hernandez-Abreua (a), Sergio Hidalgo-Figueroa (a), Gabriel Navarrete-Vazquez (a), Fabiola Escalante-Erosa (b), Luis M. Pena-Rodriguez (b), Rafael Villalobos-Molina (c), Samuel Estrada-Soto (a), *
(a) Facultad de Farmacia, Universidad Autonoma del Estado de Morelos, Avenida Universidad 1001, Colonia Chamilpa, 62209 Cuernavaca, Morelos, Mexico
(b) Laboratorio de Quimica Organica. Unidad de Biotecnologia, Centro de Investigacion Cientifica de Yucatan, Calle 43, No. 130, Colonia Chuburna de Hidalgo, Merida, Yucatan, Mexico
(c) Unidad de Biomedicina, Facultad de Estudios Superiores lztacala. Universidad Nacional Autanoma de Mexico, Tlalnepantla, Mexico 54090, Mexico
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|Author:||Avila-Villarreal (a), Gabriela; Hernandez-Abreu (a), Oswaldo; Hidalgo-Figueroa (a), Sergio; Navarret|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Nov 15, 2013|
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