Phytopharmacology 2012, 2(1) 170-178 Antihypertensive effect of Lepechinia caulescens extract on spontaneously hypertensive rats Samuel Estrada-Soto1,*, Gabriel Navarrete-Vázquez1, Ismael Léon-Rivera2, María Yolanda Rios2, Berenice Aguilar-Guadarrama2, Patricia Castillo-España3, Rolffy Ortiz-Andrade4, Francisco Aguirre-Crespo5 1 Facultad de Farmacia, 2Centro de Investigaciones Químicas and 3Centro de Investigación en Biotecnología, Universidad Autónoma del Estado de Morelos, Avenida Universidad 1001, Colonia Chamilpa, 62209, Cuernavaca, Morelos, México. 4 Facultad de Química, Universidad Autónoma de Yucatán, Mérida, Calle 421 No. 41 x 26 y 28 Col. Industrial, C.P. 97150 Mérida Yucatán 97150, México. 5 División Ciencias de la Salud, Universidad de Quintana Roo, Boulevard Bahía s/n esq. Ignacio Comonfort, Col. del Bosque, 77019, Chetumal, Quintana Roo, México. *Corresponding Author: enoch@uaem.mx Received: 13 February 2012, Revised: 17 February 2012 Accepted: 18 February 2012 Abstract The aim of the present study was to examine the antihypertensive effect of methanolic extract from Lepechinia caulescens (MELc) and to determine the thoracic aorta reactivity after long-term treatment with MELc. Results showed that MELc at 38 and 120 mg/Kg induced a significant decrease of heart rate (HR), systolic blood pressure (SBP) and diastolic blood pressure (DBP) in comparison with control and similar than captopril (30 mg/Kg). Also, MELc (120 mg/Kg) induced a long-term antihypertensive activity when still down SBP and DBP from fifth day until the end of experiment. Vascular reactivity of vessels from extract-treated animals was improved when were stimulated with carbachol and sodium nitroprusside. However, treatment with noradrenaline enhanced contractile response on these preparations. In conclusion, MELc produced significant antihypertensive and bradycardic effects that may be related with an activation of NO/cGMP pathway. Keywords: Antihypertensive agent; Lamiaceae; Lepechinia caulescens; Ursolic acid; Oleanolic acid; SHR rats ; Lepechinia caulescens; heart rate; systolic blood pressure; diastolic blood pressure; Vascular reactivity; NO; cGMP pathway; cyclic guanosine monophosphate; Nitric oxide; tilianin; naringenin; discretamine; astragaloside IV; galangin; epigallocatechin gallate; Carbamylcholine; noradrenaline; ursolic acid; captopril; nitroprusside; eNOs; Mexico; ursolic acid; oleanolic acid; reactive oxygen species; ROS; terpinen-4-ol, salvigenin; spathulenol Introduction The World Health Organization published in 2007 its Guidelines for Assessment and Management of Cardiovascular Risk, where reports an estimated of 58 million deaths globally in 2005, from them 30% was produced by Cardiovascular diseases (CVD) (WHO, 2011). CVD are most prevalent causes of death in Western population so far. Moreover, hyp- 170 © 2012 Inforesights Publishing UK Estrada-Soto et al. Figure 1. Chemical structure of Ursolic Acid (Left) and Spathulenol (Right). ertension is one of the most prevalent causes of CVD by impaired vascular relaxation process due to appearance of endothelial dysfunction and oxidative stress (Endemann and Schiffrin, 2004). Therapeutic strategies to combat the consequent damage to the vascular endothelium are generally aimed at modulating the molecular and biochemical mechanisms underlying this dysfunction (Navarrete-Vázquez et al., 2010). One approach to treat the affected endothelium involves improving endothelium-dependent vasodilatation, which is mediated by augmenting the influence of endothelial protective factors (prostacyclin and nitric oxide). Therefore, there are a lot of compounds with NO-release stimulation properties that produce significant relaxant effect in vessels, such as tilianin, naringenin, discretamine, astragaloside IV, galangin, epigallocatechin gallate, inter alia (Hernández-Abreu et al., 2009; Morello et al., 2006; Sánchez-Salgado et al., 2010; Silva et al., 2009; Kim et al., 2007; Zhang et al., 2007). In this context, it is well known that the study of medicinal plant species had allowed the isolation of several agents used as leads for the development of new therapeutic drugs (Gurib-Fakim, 2006). Although there is available a low-cost therapy, the Mexican folk medicine policies promote the use of medicinal plants for the treatment of different diseases, and some herbal medicines are real choices for treatment of hypertension (Aguilar et al., 1994; Monroy-Ortiz and Castillo-España, 2007). Consequently, Lepechinia caulescens (Ortega) Epling (Lamiaceae) has been used in traditional medicine of Morelos State, Mexico, for the treatment of diabetes, hypertension and related diseases (Monroy-Ortiz and CastilloEspaña, 2007). However, it has not been widely studied to probe its medicinal uses. Some preliminary pharmacological investigations show that methanolic extract of L. caulescens (MELc) and one of its metabolites, ursolic acid (UA) (Figure 1), exert vasorelaxant action on rat aorta rings (Aguirre-Crespo et al., 2005). Furthermore, it was established that their vasorelaxant mechanism is through nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway (Aguirre-Crespo et al., 2006). In addition, other reports showed that this extract was able to induce a spasmolytic effect on spontaneously contraction of rat ileum strips through calcium channel blockade and NO/cGMP pathway (Estrada-Soto et al., 2007). In this context, the aim of current work was to evaluate the short-term in vivo cardiovascular effects of MELc on spontaneously hypertensive rats (SHR). Additionally, long-term cardiovascular effect of MELc was evaluated on same model and vascular reactivity of rat aorta preparations was determined. © 2012 Inforesights Publishing UK 171 Phytopharmacology 2012, 2(1) 170-178 Materials and methods Chemicals and drugs Carbamylcholine (carbachol), noradrenaline HCl (NA), ursolic acid (UA), captopril and sodium nitroprusside (SNP) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Other reagents were analytical grade from local sources. To carry out the experiments, extracts were dissolved in isotonic salt solution (SS). Plant material L. caulescens was collected in February 2004. Briefly, plant material was obtained from its natural habitat (19°05´35.78” N; 98°56´41.99” W; 2 876 m; 2005Google Earth) and was collected and identified by Dr. P. Castillo-España. A voucher specimen (20386) has been deposited at ‘‘Centro de Educación Ambiental e Investigación Sierra de Huatla’’ HUMOHerbarium, Cuernavaca, Morelos, Mexico. Preparation of extracts MELc was obtained as previously described (Aguirre-Crespo et al., 2005). Briefly, air-dried plant material (100 g of aerial parts) was ground into powder and extracted exhaustively by maceration at room temperature with methanol (MeOH; 1 L), which yielded 12.2 g of extract (12.2 %). For in vivo experiments, MELc was dissolved in Tween 80 (2%), brought to the chosen volume with sterile isotonic saline solution (vehicle) and sonicated just before use. Animals Male spontaneously hypertensive rats [SHR: 250-300 g; 408.5 ± 4.1 bpm for hearth rate (HR); 152.6 ± 2.4 and 122.1 ± 1.9 mmHg for systolic blood pressure (SBP) and diastolic blood pressure (DBP), respectively] were used for this study. Animals were maintained under standard laboratory conditions with free access to food and water. All animal procedures were carried out in accordance with our Federal Regulations for Animal Experimentation and Care (Ministry of Agriculture, NOM-062-ZOO-1999, Mexico) and were approved by the Institutional Animal Care and Use Committee. Antihypertensive effect of MELc Acute determination SHR rats were used in the experiments. All experiments were carried out using six animals per group. Doses used were 38.4 and 120 mg/Kg for MELc and 30 mg/Kg for captopril (used as antihypertensive reference drug, suspended in 0.05% of Tween 80 in SS); test samples were administered by orally intragastric route. Control rats received vehicle (SS) at the same volume (0.5ml/100g). SBP, DBP and HR were measured at 0, 2, 4 and 6 hrs after treatment using a non-invasive tail-cuff method (®Letica, PanLab, Barcelona, Spain). 172 © 2012 Inforesights Publishing UK Estrada-Soto et al. Sub-acute experimental model SHR rats were allotted into two groups, untreated control (SS) and MELc group. All experiments were carried out using six animals per group. Treated MELc extract group received 120 mg/kg body weight/day for 10 days. Every day, SBP, DBP and HR were measured before administration of samples test. In vitro experiments At the end of sub-acute experiment (MELc and SS groups), thoracic aortic rings were obtained and prepared from SHR rats. The animals were sacrificed by cervical dislocation. Tissue segments were dissected-out, cleaned and placed in organ baths containing warmed (37°C) and oxygenated (O2:CO2, 19:1) Krebs solution (10 mL; composition mM: NaCl, 118; KCl, 4.7; CaCl2, 2.5; MgSO4, 1.2; KH2PO4, 1.2; NaHCO3, 25.0; EDTA, 0.026; glucose, 11.1, pH 7.4). Changes in tension were recorded by Grass-FT03 force transducers (Astromed, West Warwick, RI, USA), connected to a MP100 analyzer (®BIOPAC Instruments, Santa Barbara, CA, USA) as previously described (Aguirre-Crespo et al., 2006). All tissues (3-5 mm) were mounted by stainless steel hooks under an optimal tension of 3 g in organ baths with Krebs solution. After equilibration (30 min), rings were contracted with NA (0.1 µM) and washed every 30 min for 2 hrs. The absence of endothelium was confirmed by the lack of a relaxing response to carbachol (1 µM). Thus, cumulative-response curves to carbachol (0.1 nM to 1 µM, endothelium-intact rings), SNP (1 nM to 0.32 µM, endothelium-denuded rings) and NA (0.1 nM to 0.32 µM, endothelium-denuded rings) were recorded for each ring. The effects of agents were determined by comparing the muscular tone of the contraction before and after addition of the test materials. Muscular tone was calculated from the tracings, using Acknowledge software (BIOPAC system). Data analysis Results are expressed as the mean of six experiments ± S.E.M. ConcentrationResponse Curves (CRC) were plotted and fitted by specific software (ORIGIN® 8.0). The time-course curves represent either number of measurements of HR, SBP and DBP versus time (h.). The statistical significance (p<0.05) of differences between means was assessed by an analysis of variance (ANOVA). Results MELc (120 mg/Kg) showed important reduction of HR than SS group (Figure 2a) but was less potent compared to captopril treated group (p<0.05). However, basal values of HR were recovered at 24 h after administration (data not showed). In addition, MELc at 38 and 120 mg/Kg also induced significantly reduction of SBP (Figure 2b) and DBP (Fig. 2c) compar-ed with control group (vehicle). Moreover, it was more potent than positive control group (p<0.05). In fact, both doses of MELc kept down SBP and DBP levels after 24 hrs (data not showed). It is important to mention that doses of 38 mg/Kg of MELc were selected in order to compare the effect of the test sample with the dose used for captopril at 30 mg/kg (50 and 100 mg/Kg are therapeutic doses used for the treatment of hypertension), also 120 mg/Kg of MELc were established using a median logarithm above ratio based on first dose. © 2012 Inforesights Publishing UK 173 Phytopharmacology 2012, 2(1) 170-178 Figure 2. Effect of intragastric administration of MELc at doses of 38.4 and 120 mg/Kg and captopril (30 mg/Kg) on (a) heart rate, (b) systolic blood pressure and (c) diastolic blood pressure in SHR (n=6; *p< 0.01, MELc vs SS). Orally sub-acute treatment with MELc (120 mg/Kg, 10 days) can regulate the rise of diastolic and systolic blood pressures in SHR without changes on HR (Figure 3). After 6 days of treatment, MELc showed a significant reduction on SBP and DBP than control group (112.63.3 and 90.63.6 vs. 131.34.0 and 107.82.5 mm Hg, for SBP and DBP, respectively). After evaluation of sub-acute effect of MELc in SHR, thoracic aortas were dissected out from both experimental groups and concentration-response curves for sodium nitroprusside (SNP), carbachol and noradrenaline (NA) were constructed. SNP and carbachol relaxed curves (Figure 4a and 4b) were shifted to the left on aortic rings obtained from rats treated with MELc (CI50: 6.3  0.03 nM vs. 0.398  0.1 µM for NPS; and 3.98  0,2 nM vs. 0.398  0.1 M for carbachol) compared to the control group, respectively. Maximum effect of SNPinduced relaxation was unaffected in MELc group (Emax: 99.10.7 vs. 97.012.3 % of relaxation); however, the maximal relaxation induced by carbachol in the extract group was 174 © 2012 Inforesights Publishing UK Estrada-Soto et al. Figure 3. Sub-acute antihypertensive effect of MELc (120 mg/Kg) in SHR (n=6; **p < 0.01, SS vs. MELc). Figure 4. Concentration-Response Curves to noradrenaline (NA), carbachol and sodium nitroprusside (SNP) in control conditions (SS) and treated with MELc (120 mg/Kg/day, 10 days), in SHR. (n= 4; **p < 0.01, SS vs. MELc). © 2012 Inforesights Publishing UK 175 Phytopharmacology 2012, 2(1) 170-178 approximately 20% more than control group (94.72 ± 1.89 vs. 75.86 ± 5.93 %). Finally, there was no significant difference in the maximal contraction to NA between two groups (Figure 4c; Emax: 1.7 ± 0.2 vs. 1.9 ± 0.1 g), but the concentration-response curve was significantly shifted to the left (IC50: 19.95 ± 0.1 vs. 39.81 ± 0.05 M). Discussion Lepechinia caulescens is an herb used in traditional medicine of Mexico for the treatment of Diabetes mellitus, diarrhea and high blood pressure, through daily consumption of oral beverages or teas (Monroy-Ortiz and Castillo-España, 2007). Previous studies carried out at our laboratory have demonstrated that MELc and ursolic acid have a significantly vasorelaxant effect in endothelium-intact rat aorta rings, through release of NO to vascular smooth muscle (Aguirre-Crespo et al., 2005; 2006). Thus, in order to associate the vasorelaxant effect of MELc with the possible cardiovascular effect in hypertensive animals, we employed SHR model to determine its pharmacological effect in acute (6 hours) and subacute (10 days) test. In this context, MELc showed acute cardiovascular effect in a preliminary dose- dependent manner, and this effect is in agreement with previous in vitro results. Moreover, MELc was capable to control hypertension from 6th day of treatment to 10th, which allows us to probe its efficacy as antihypertensive agent. Taken it together, results suggest that MELc induced its antihypertensive effect by vasodilator properties through an activation of NO pathway (Aguirre-Crespo et al., 2005). Also, the exploration of the reactivity of aortic rings from both groups suggested that continuous administration of MELc could induce an augment in the efficiency of systems implicated in relaxation derived from endothelial and smooth muscle cells. So, it was reported that aqueous extract of Salvia miltiorrhiza (Lamiaceae) and UA increased eNOs expression in EA.hy 926 cells from native human umbilical vein endothelial cells (HUVEC). In addition, they enhanced the bioactive NO and cGMP production reduced NADPH oxidase subunit Nox4 expression and suppressed the production of reactive oxygen species (ROS) in human endothelial cells (SteinkampFenske et al., 2007a; Steinkamp-Fenske et al., 2007b). Thus, MELc might change blood pressure by the modification of expression of the enzyms related with NO/cGMP pathway and by a possible restoration of the functional activity of endothelium and smooth muscle cells. Finally, displacement of concentration-response curves to carbachol and SNP in aortic rings from animals treated with the extracts could be related with UA or oleanolic acid (OA) content in MELc by possible eNOS upregulation, enhancement of bioactive NO production and/or by reduction of the oxidative stress on endothelium cells from SHR, by down regulating of Nox4 expression (Steinkamp-Fenske et al., 2007a; Steinkamp-Fenske et al., 2007b). UA is one of the main active components of L.caulescens that induce vasorelaxation in a concentration- and endothelium-dependent manner (Aguirre-Crespo et al., 2006). However, some other metabolites have been isolated from L. caulescens and were reported as possible smooth muscle relaxant agents as terpinen-4-ol, salvigenin and spathulenol (Figure 1) (Lahlou et al., 2002; Perez-Hernandez et al., 2007; Uydeş-Doğan et al., 2005). Thus, we cannot discard their possible participation in cardiovascular MELc’s properties. However, early studies reported that triterpenoids such as oleanolic acid, erythrodiol, maslinic acid or uvaol produced a vasorelaxant activity in aortic rat rings of Wistar and SHR rats (RodriguezRodriguez et al., 2004; 2006). So, amelioration in blood pressure of SHR rats could be related with the presence of triterpenoids in plant material of L. caulescens. Some reports showed that these molecules (60 mg/Kg, p.w. 6 weeks) exert antihyperlipidemic and antioxidant 176 © 2012 Inforesights Publishing UK Estrada-Soto et al. effects in Dalh salt-sensitive rats (Somova et al., 2003), and supports the idea that sub-acute treatment could be involved in prevention of hypertension. Another research work indicated that UA and OA protect to isoproterenol-induced myocardial ischemia (Senthil et al., 2007). Further experiments are necessary in order to corroborate or discard the important participation of NO system in anti-hypertensive effect induced by MELc and participation of UA and OA. 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