Antihypertensive activity of Salvia elegans Vahl. (Lamiaceae): ACE inhibition and angiotensin II antagonism

Journal of Ethnopharmacology 130 (2010) 340–346

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Ethnopharmacological communication

Antihypertensive activity of Salvia elegans Vahl. (Lamiaceae): ACE inhibition and angiotensin II antagonism Enrique Jiménez-Ferrer, Fidel Hernández Badillo, Manases González-Cortazar, Jaime Tortoriello, Maribel Herrera-Ruiz ∗ Centro de Investigación Biomédica del Sur, Instituto Mexicano del Seguro Social (IMSS), Argentina No. 1, CP 62790, Xochitepec, Morelos, Mexico

a r t i c l e

i n f o

Article history: Received 25 February 2010 Received in revised form 1 May 2010 Accepted 9 May 2010 Available online 19 May 2010 Keywords: Salvia elegans Angiotensin II Angiotensin converting enzyme

a b s t r a c t Ethnopharmacological relevance: Salvia elegans Vahl. (Lamiaceae), recognized with the popular name of “mirto” is widely used in Mexico for healing purposes, and also them as antihypertensive treatment. Aim of the study: The high prevalence of this illness and the side effects of antihypertensive drugs conducted us to the evaluation of the Salvia elegans extract on angiotensin II action. Materials and methods: The acute response of blood pressure to angiotensin II administration was measured in mice. We also tested in vitro the inhibitory effect on angiotensin convertase enzyme. Additionally, characterization of the pharmacological effect of the extract fraction was obtained. Results: We obtained dose–response curve for the administration of complete extract and extract fractions. Due to the hydroalcoholic extract (SeHA) treatment blood pressure decreased significantly from systolic dose of 0.75 mg kg−1 (p < 0.05) and even had an antihypertensive effect that was greater than that treatment with losartan. SeHA extract decreased the Emax of the AG II hypertensive effect by about 20% in both systolic and diastolic pressures, treatment with losartan also decreased the same parameter between 6% and 8% for systolic and diastolic pressures, respectively. Fractions SeF8 and SeF8-8 showed similar levels of AG II ED50 for both pressures compared with losartan, these fractions showed major compounds with maximum absorbance peaks at 221, 289 and 330 nm typical of flavonoids. In the inhibition assay the activity of angiotensin converting enzyme (ACE), the extract SeHA showed percentage inhibition (%IACE) of 50.27 ± 5.09% (n = 5). SeBuOH fraction is found to have greater inhibitory capacity of achieving a IACE 78.40 ± 2.24% (n = 5), which was similar to the values obtained in the presence of the SeF8-22 fraction (82.61 ± 1.74%) and lisinopril (87.18 ± 1.16%). The changes in the value of KM suggest that components of the extracts and fractions were recognized by the enzyme’s active site. The main compounds of the fractions SeBuOH, SeF8-22 were by flavonoid and phenyl propanoid types, according to UV absorption spectra of the fractions. Conclusions: The experimental results demonstrated the antihypertensive effect of Salvia elegans and it was due to the AG II antagonism and inhibition of angiotensin converting enzyme. © 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Salvia elegans Vahl. (Lamiaceae) is widely recognized by the name “mirto”, which is used for other species of the same genus (Martínez, 1970). Salvia elegans is a plant utilized in Mexican traditional medicine for treating central nervous system (CNS) disorders, such as anxiety and insomnia (Aguilar et al., 1994). Also, the Pame, an indigenous community living in San Luis Potosí and Queretaro, Mexico, refers the use of “yerba del burro” (Salvia elegans) as a remedy against headache (cephalea) in adults who are

∗ Corresponding author. Tel.: +52 777 361 2155; fax: +52 777 361 2155. E-mail addresses: cibis [email protected], cibis [email protected] (M. Herrera-Ruiz). 0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2010.05.013

under great emotional stress associated with ringing or buzzing in the ears (tinnitus) and dizziness. These symptoms can be related to high blood pressure, despite that, local healers do not recognize the hypertension as an ailment (Biblioteca Digital de la Medicina Tradicional Mexicana). Pharmacological studies indicate that Salvia elegans possesses antidepressant and anxiolytic properties, which have been observed in behavioral models with mice and rats (Herrera-Ruiz et al., 2006; Mora et al., 2006). In Mexico, certain species of the genus Salvia are utilized to treat diseases associated with renal pathologies, such as Salvia laevis Benth., Salvia leucantha Cav., and Salvia elegans Vahl. (Aguilar, 1994). There are no reports that show the pharmacological activity of Salvia elegans on high blood pressure models. However, it has been demonstrated that other species of the genus possess hypotensor activity in animals submitted to different hypertension

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models. For example, administration of the Salvia miltiorrhiza aqueous extract to rats with renovascular high blood pressure in the Goldblatt model (renin–angiotensin–aldosterone system [RAAS]dependent) causes diminution of systolic blood pressure without modifying renin levels in plasma; in addition, the extract possesses an inhibitory effect on the angiotensin converting enzyme (ACE) (Kang et al., 2002). When Salvia miltiorrhiza was administered, it prevented left ventricular hypertrophy, and significantly inhibited the ventricular mass index (VMI) by collagen compositions in left ventricle, probably through the inhibition of the cardiac aldosterone action (Han et al., 2002). It was also able to inhibit structural remodeling in rats with pulmonary hypertension by suppression of heme oxygenase-1 and nitric oxide synthase (eNOS) (Chen et al., 2003). Another species comprises Salvia scutellarioides, which is widely employed in traditional Colombian medicine for the treatment of high blood pressure and as a diuretic (Ramírez et al., 2006). Administration of 2 g/kg of the aqueous extract of this species in rats showed diminution of diastolic blood pressure (induced with G-nitro-l-arginine-methyl ester [l-NAME]) measured by a non-invasive method. This effect lasted only 2 weeks after administration, without modifying systolic blood pressure and without provoking histological changes in l-NAME damage-related heart and kidney (Ramírez et al., 2006). Recognizing the use of Salvia elegans in traditional medicine to treat symptoms associated with high blood pressure, in addition to the pharmacological antecedents of the Salvia genus as a hypotensor, the objective of the present work was to evaluate the antihypertensive capacity of Salvia elegans extracts and fractions in a hypertension model induced by acute administration of angiotensin II (AG II) as well as the ability to inhibit ACE in an in vitro model. 2. Materials and methods 2.1. Plant material and extract preparation The aerial parts (flowers, leaves, and stems) were collected from the state of Puebla, southwest of Mexico. Plant material was identified by Abigail Aguilar-Contreras, M.Sc., the IMSSM Herbarium Director (located in National Medical Center, Mexico City). Voucher specimens were stored at this site for future reference (IMSSM-14588). The collected plant was dried at environmental temperature under conditions of darkness for 2 weeks. The material was ground (in a Pulovez electric mill) to obtain particles of <4 mm. We performed extraction of a 60% ethanol solution at 50 ◦ C for 2 h. Later, the extract was filtered on Whatman #1 filter paper, retaining the plant material to carry out a new extraction under the same conditions and with a new solvent. The extract obtained was concentrated in a rotary evaporator under low pressure conditions. The extraction yield was quantified, and the material obtained was denominated by hydroalcoholic extract (SeHA). 2.2. Fractioning of the organic extract The SeHA obtained was concentrated in a rotary evaporator, and the solvent residue was eliminated by freeze drying. We performed SeHA liquid–liquid separation with n-butanol/water and obtained two extracts (organic and aqueous), which were concentrated and evaporated by freeze drying. The organic phase was the active fraction (SeBuOH, 44.4 g), which was submitted to separation through percolation with silica gel (70:230 mesh) with hexane:ethyl acetate:methanol, and 83 fractions were obtained. Fractioning follow-up was conducted by means of thin-layer chromatography (TLC), the fractions were grouped into 25 sub-fractions

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according to the similarity of their components, and these were denominated SeF1–SeF25. Of these, the SeF8 fraction (5.3 g) presented antihypertensive activity; thus, it was submitted to chromatographic separation in a silica gel column (170 g) with a chloroform:methanol gradient, obtaining 86 fractions. These fractions were grouped according to chemical similarity in 29 sub-fractions, to which the key SeF81–SeF8-29 was assigned. The fractions SeF8-8, -22, and -28 were active in the pharmacological test and TLC analysis was used in order to obtain evidence of the constituents responsible for the biological activity, specifically for flavonoids according to the reaction with 2-aminoethyl diphenylborinate and by ultraviolet (UV) light spectrophotometry. The fractions were analyzed under high performance liquid chromatography (HPLC) with a Lichrospher 100 RP-18 (12.5-cm) column, with an A = TFA 1.1% and a B = CH3 CN:H2 O 1:1 solvent system with a discontinuous gradient of concentration (Table 1) and a constant 1-ml/min flow. A (%)

B (%)

Time (min)

83 83 70 70 20 20 83 83

17 17 30 30 80 80 17 17

0 7 8 10 11 13 14 15

2.3. Animals ICR albino mice weighing 30–36 g were used (Harlan, México, D.F.). All animals were housed eight per cage and were maintained under laboratory conditions at 25 ◦ C, with a normal 12 h:12 h light/dark schedule (lights on at 7:00 a.m.) and free access to water and food (pellets, Harlan rodent lab diet). The mice were allowed 3 weeks to adapt to the laboratory environment prior to experiments. Experiments were carried out between 8:00 a.m. and 12:00 p.m. All studies were conducted in accordance with official Mexican norm NOM-062-ZOO-1999 (technical specifications for production, care, and use of laboratory animals). Minimum number of animals and minimum duration of observation required to obtain consistent data were employed. 2.4. Evaluation of the antihypertensive activity of Salvia elegans First, we constructed a dose–response curve of the effects produced by intravenous (i.v.) administration of AG II (angiotensin II, Sigma–Aldrich) (0.5, 0.75, 1.0, 1.5, and 2.0 ␮g kg−1 ). Each mouse was anesthetized with sodium pentobarbital (55 mg kg−1 , Pfizer) (50 mg kg−1 i.p.); the animals were placed under these conditions in an immobilizer and connected to a non-invasive blood pressure measurement equipment (LE 5002 Storage Pressure Meter, Biopac Systems MP 150). The basal blood pressure was measured (eight records on average) for systolic and diastolic pressures. An increase of this parameter was provoked by administration of the corresponding dose of AG II i.v. In order to evaluate the effect produced by Salvia elegans on blood pressure in mice, 15 groups of eight animals each were treated as follows: the negative control group received 100 ␮l of saline solution by oral pathway (o.p.); positive control groups were treated with 10 mg kg−1 o.p. of losartan (an angiotensin type 1 [AT1] receptor agonist, Merck) in 100 ␮l of vehicle and for treatments with Salvia elegans, the dose was chosen from the traditional use, 3 g of a decoction from this plant (5% extraction yield) provides an estimated dose of 1.5–2.1 mg kg−1 in humans. Then, when comparing the pharmacokinetics of humans with respect to that of a

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Table 1 Parameters of the pharmacologic activity of extract from Salvia elegans. Compound

AG II SeHA Losartan SeF8 SeF8-8

Systolic blood pressure

Diastolic blood pressure −1

Emax (mmHg)

ED50 (␮g kg

204.10 169.40 180.46 153.09 119.83

2.75 10.85 2.79 2.40 2.85

mouse, a dose of 20 mg kg−1 was shown for animal model (ReaganShaw et al., 2008). With this basis, a dose of 10 mg kg−1 o.p. of SeHA or fractions was used. Finally, the active fractions were SeF-8 and SeF8-8. After 1 h of all treatments, the animals were administered with corresponding doses of AG II i.v. (0.5, 0.75, 1.0, 1.5, and 2.0 ␮g kg−1 ), and the systolic and diastolic blood pressures were measured. 2.5. Evaluation of angiotensin converting enzyme (ACE) inhibition The enzymatic activity assay of ACE in vitro was carried out quantifying the hydrolysis of N-[3-(2-Furyl)acryloyl]-Phe-Gly-Gly (FAPGG, Sigma–Aldrich) by ACE. Briefly, a final volume of 730 ␮l of which 530 ␮l corresponds to the substrate solution de FAPGG (3 mM in reaction buffer) and 200 ␮l to the reaction buffer (HEPES 25 mM, NaCl 300 mM, pH 8.4) was incubated during 3 min at 37 ◦ C. The reaction was started by adding 20 ␮l of ACE solution (0.05 U/ml) to the test reaction, the samples were incubated during 60 min, and the reaction was stopped by adding 80 ␮l of 5% trifluoroacetic acid solution and the samples were centrifuged at 10,000 rpm for 5 min at room temperature. In the bioassay of ACE inhibition, 200 ␮l of buffer reaction was substituted by the same volume of extract SeHA (100 mg/ml), SeBuOH (100 mg/ml), SeF8-22 (0.5 mg/ml), and SeF828 (0.25 mg/ml) and the therapeutic drug lisinopril (200 ␮g/ml) was used as a reference ACE inhibitor. For FAPGG kinetic hydrolysis determination in the presence of the inhibitors, solutions of different concentrations of FAPGG (0.05, 0.1, 0.2, 0.3, 0.6, 0.8, 1.0, 2.0, 3.0, and 4.0 mM) dissolved in HEPES reaction buffer were prepared, and 530 ␮l of each solution was added to 200 ␮l of the inhibitor in order to adjust the inhibitor concentration at 200 ␮g/ml. The enzymatic activity was calculated by quantifying the decrease of FAPGG concentration by recording the decrease absorbance at 345 nm using a reversed phase HPLC (GRACE® Altima HP C18 HL column [53 mm × 7 mm, 3 ␮m]) with isocratic solvent system consisting of acetonitrile 1.1% TFA in water (75:25, v/v) at a flow rate of 1.5 ml/min. FAPGG displayed a retention time of 8.67 min; the enzyme inhibition was calculated by comparing the enzymatic activity with, and without, inhibitor using the following equation: %IACE = I × 100; where I = 1 − a and a = activity with inhibitor/activity without inhibitor (Segel, 1975). 2.6. Kinetic calculations The kinetic parameters were calculated by adjusting curves to the Michaelis–Menten equation: vo = (Vmax × [S])/(KM + [S]). The inhibition type and the inhibitory constants were calculated from the double reciprocal plot, using the equation: mi = m(1 + [I]/Ki ), where mi is the slope of lineal plot from inhibited reaction, m the slope of lineal plot from reaction without inhibitor, [I] the concentration of inhibitor, and Ki is the inhibitory constant.

)

Emax (mmHg)

ED50 (␮g kg−1 )

137.05 112.61 128.30 90.86 80.31

2.09 7.21 2.87 2.93 2.30

2.7. Statistical analysis Statistical analysis was performed with an SPSS 11.0 software program and based on analysis of variance (ANOVA) followed by the Bonferroni test. A significant difference was established with respect to the control group, when the p value was <0.05.

3. Results 3.1. Evaluation of Salvia elegans antihypertensive activity I.v. administration of AG II caused an immediate increase of systolic and diastolic blood pressures. This effect was maintained on average for a period of 250 s; thus, recordings of pressure were taken during the first 180 s. The increase induced by AG II of both systolic and diastolic blood pressures was dose-dependent and significantly different (p < 0.05) when data were compared from group of animals that received only the vehicle (0.0 ␮g kg−1 of AG II), such difference was evident since 0.5 ␮g kg−1 of AG II (Fig. 1). Losartan (10 mg kg−1 ) diminished the elevation of diastolic blood pressure induced by AG II; the data show that this effect was significantly different from negative control group at AG II doses of 0.75, 1.0, and 2.0 ␮g kg−1 (p < 0.05) (Fig. 1b). Additionally, it caused diminution of systolic blood pressure at different AG II doses; however, this activity was only significantly different in the group of animals administered 2.0 ␮g kg−1 (p < 0.05) (Fig. 1a). On the other hand, o.p. administration of SeHA (10 mg kg−1 ) induced a significant decrease (p < 0.05) of diastolic blood pressure at all of the tested doses of this hormone (Fig. 1a). The data indicated that SeHA importantly and significantly diminished systolic blood pressure from doses of 0.75 ␮g kg−1 (p < 0.05) (Fig. 1b). The effect produced by the SeHA extract on both diastolic and systolic blood pressures was greater than that observed with losartan, even at the 2.0 ␮g kg−1 dose of AG II. These two groups were statistically different from each other (p < 0.05). From construction of the AG II dose–response curve, we obtained AG II-associated pharmacodynamic parameters, as well as interactions with treatments with SeHA extract, fraction SeF8, fraction SeF8-8, and losartan (Table 1). Treatment with the SeHA extract diminished the Emax hypertensor effect of AG II by approximately 20% in both systolic and diastolic blood pressures. Treatment with losartan also decreased the Emax value of AG II measured as a pressor effect of between 6% and 8% of systolic and diastolic pressures, respectively (Table 1). The fractions exerted important effects on systolic and diastolic blood pressures in such as way that the greatest decrease of Emax was reached with the administration of fractions SeF8 and SeF88 (Table 1). With regard to ED50 values of AG II for both blood pressures, these values were similar to the values obtained after treatment with losartan, as well as with SeF8 and SeF8-8 fractions. When the SeHA extract was administered, we observed an increase in the ED50 value.

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Fig. 1. Dose–response curve of i.v. administration of AG II, with hydroalcoholic extract (SeHA), fraction (SeF8), and sub-fraction (SeF8-8) from Salvia elegans and losartan. (a) Systolic pressure and (b) diastolic pressure. , AG II; , losartan; , SeF8; , SeHA; 䊉, SeF8-8. & p < 0.05, data were compared with the lowest doses of AG II; *p < 0.05, data were compared with the vehicle group, ANOVA followed by post hoc Bonferroni test (mean ± standard deviation [SD]).

3.2. Thin-layer chromatography analysis The SeF8 fraction presents a less complex composition than that of the hydroalcoholic (HA) extract. In this extract, was observed a mixture of median polarity compounds, and continuing with the next stage of fractionation, was obtained a SeF8-8 fraction of greatest purity with median polarity compounds. On the analysis of these latter compounds, with regard to their UV light absorbance spectra and their UV absorbance spectra after separation by HPLC, we determined maximum absorbance peaks of 221, 289, and 330 nm (Fig. 2); these are typical of flavonoids, which are the two main components of this fraction. Among the components of fractions SeBuOH, SeF8-22, and SeF8-28, the main compounds were of the flavonoid- and phenyl propanoid types, given that these showed to be positive with the 2aminoethyl diphenylborinate-specific reagent for flavonoid. HPLC chromatographic-peak absorption spectrum analysis of the main compounds in the fractions of greatest purity was as follows: fraction SeF8-22 presented a main compound that had maximum absorbance at 221, 289, and 330 nm, which is typical of flavonoids,

and the SeF8-28 fraction presented two chromatographic peaks, one with absorption spectra indicating that it was a chlorogenic acid-related compound with peak maximum absorbance of 240, 298 and 325 nm. The compounds in question presented maximum absorbance of 220, 298 and 330 nm (Fig. 3). 3.3. Evaluation of angiotensin converting enzyme (ACE) inhibition In the ACE-activity inhibition evaluation model, kinetic behavior followed the model of Michaelis–Menten, allowing for the determination of values of KM = 0.57 mM and Vmax = 4.41 ␮M/min. Enzyme activity in the presence of the SeHA extract demonstrated an ACE inhibition percentage (%IACE) of 50.27 ± 5.09% (n = 5); the SeBuOH fraction exhibited a greater inhibitor capacity, achieving an %IACE of 78.40 ± 2.24% (n = 5), which was similar to values obtained in the presence of fraction SeF8-22 and lisinopril, 82.61 ± 1.74% and 87.18 ± 1.16% (n = 5), respectively, although at a different concentration. We observed that the SeF8-28 fraction had less ACE inhibition (%IACE of 33.64 ± 6.89% (n = 5)), although the concentra-

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Fig. 2. HPLC chromatograms and UV/vis absorption spectra of SeF8-8 fraction indicate the presence of compounds flavanone type (Lin and Harnly, 2007). The peak with TR = 12.029 min corresponding to 208.6, 289.9 and 330.3 nm; peak with TR = 12.600 min corresponding to 221.6, 289.9 and 330.3 nm.

tion was one-half of that utilized with the SeF8-22 fraction. The kinetic parameters indicate to us that the least complex Salvia elegans extracts and fractions presented modifications in the value of KM , which in these cases is considered as KMapp (the apparent Michaelis–Menten constant); this allows us to suppose that the components present in the extracts and fractions were recognized by the enzyme’s active site. The values determined for the Ki , inhibition constant, which evaluates inhibition in terms of concentration, presented a considerable decrease in the Ki value. The SeF8-22 fraction was that which presented the least value (Ki = 0.04 mg/ml), this was even lower than that of SeF8-28 (Ki = 0.18 mg/ml) (Table 2). 4. Discussion Systemic high blood pressure is a multifactorial disease, in which alteration of the RAAS plays a central role in the control of this disorder. Therefore, drugs acting as antagonists or inhibitors of different mechanisms associated with RAAS system are effective for the treatment of hypertension (Atlas, 2007). The biological activity of Salvia elegans showed in this work is consistent with the results reported for antihypertensive activity of the species from genus Salvia (Kang et al., 2002; Han et al., 2002) and with the ethnomedical antecedents (Martínez,

1970; Biblioteca Digital de Plantas Medicinales, 2009; Aguilar, 1994). The experimental design was based on acute high blood pressure of renovascular origin; however, we substituted the liberation of renal ischemia-associated renin designed by Goldblatt (Martínez-Maldonado, 1991) for acute i.v. administration of AG II in ICR mice. The hypertension induced by AG II, was evident at a few seconds of administration of the peptide; the increased effect on blood pressure was maintained until 250 s after AG II administration. The recording of blood pressure was for 3 min; due to this is the period of life of AG II in the bloodstream (Dickinson and Lawrence, 1963). However, there are reports that show that the pressor effect of i.v.-administered AG II can be maintained for time periods up to 120 min (Wienen et al., 1993). The antihypertensive effect of Salvia elegans, both of the HA extract and of the fractions, was more potent than that of losartan. This lesser potency of losartan can be due to the low bioavailability of the drug (Johnston, 1995; Lo et al., 1995) and because the binding of the losartan to AT1 (receptor for AG II) occurs at 24 h after its administration (Fabiani et al., 2000). The antihypertensive activity of the SeHA extract, as well as of fractions SeF8 and SeF8-8, can be due to the presence of ter-

Table 2 Kinetic parameters of the ACE inhibitor activity of extract from Salvia elegans. Fractions

KM a (mM)

Vmax (␮M/min)

Conc. (mg/ml)

Ki (mg/ml)

None SeHA SeBuOH SeF8-28 SeF8-22

0.57 2.64 6.16 1.32 8.77

4.40 5.41 4.36 4.18 4.54

100 100 0.25 0.5

36.46 10.07 0.18 0.04

a b

For inhibition of ACE by inhibitors, KM is defined as KMapp since it is affected by the factor 1 + [I]/Ki . %IACE is expressed as ±SD.

Ki (mM)

0.85

%IACEb 50.27 78.40 33.64 82.61

± ± ± ±

5.09 0.24 6.89 1.74

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Fig. 3. HPLC chromatograms and UV/vis absorption spectra of SeF8-28 fraction. The absorption spectra correspond to phenyl propanoid compound (Lin and Harnly, 2007).

penic acids, such as ursolic and oleanolic acids, these compounds were previously described in Salvia elegans (Marquina et al., 2008), and other terpenes as amyrine type. It was shown that oleanolic acid acts on endothelial nitric oxide synthase (eNOS), provoking vasorelaxation in isolated rat aorta (Rodríguez-Rodríguez et al., 2004, 2008; Rodríguez-Rodriíguez et al., 2006). In addition, it has been proposed that ursolic acid exerts an endothelium-dependent vasorelaxant effect because of the liberation of nitric oxide (NO) and the consequent activation of the soluble cyclase guanylate of vascular smooth muscle cells (Aguirre-Crespo et al., 2006). In the present work, it was shown that the SeHA extract contains flavanone compounds, which are able to inhibit the secretion of endothelin 1 (ET-1, is a vasoconstrictor molecule, causing elevation of blood pressure). On the other hand, the flavanones are able to increase NO production and release (Chiou et al., 2008). Flavones are another group of flavonoids that were identified in this work from Salvia elegans, so the antihypertensive effect of this plant may be due also to these metabolites. For example, the flavones have capacity to activate Ca2+ -dependent K+ conductance, allowing for hyperpolarization followed by the entry of Ca2+ . This process is responsible for the production of NO (Erdogan et al., 2007), which exerts a decrease in vascular smooth muscle contraction, decreasing peripheral resistance to blood flow, thus, lowering blood pressure. In addition, in the present work it was demonstrated that Salvia elegans extracts and fractions have capacity as an ACE inhibitor. First, it was observed that ACE possesses a behavior that follows the Michaelis–Menten kinetic model. Maximum velocity (Vmax ) of

FAPGG hydrolysis in the presence of Salvia elegans extracts and fractions was not modified, but KM underwent changes, giving rise to an apparent Michaelis–Menten constant (KMapp ). The kinetic values obtained with the concentrations utilized of each extract or fraction, caused inhibition percentages at an interval that allowed us to visualize the type of inhibition induced by SeHA, SeF8 and SeF8-8 and lisinopril, indicating that all of these provoked a competitive-type ACE inhibition. Therefore, Ki values were greater with respect to the chemical complexity of the samples analyzed; when the sample was less complex, the value of the inhibition constant was less too. However, this kinetic behavior was not maintained for SeF8-28 fractions, which had a greater inhibition constant (Ki = 0.18 mg/ml) than the SeF8-22 fraction (Ki = 0.04 mg/ml). SeF8-28 fractions contain of phenyl propanoid-type derivatives (as revealed by TLC), and the SeF8-22 fraction, that basically contained flavonoids as main compounds, produced a greater inhibitor effect (>4 times greater). There are reports that showed that some flavanones, even when they lack hydroxyl radicals, are able to inhibit ACE (Chen et al., 1992), and it is possible that the flavanone present in Salvia elegans (3-acetoxy-7-methoxyflavone) (Marquina et al., 2008), may be partially responsible for the ACE inhibitor effect of this plant. On the other hand, it may be suggested that the greatest inhibition of the most complex fraction (SeF8-22) chemically, is due to the presence of flavanones. Also, the flavonols present in Salvia elegans could be part of this inhibition activity. These groups of compounds contain hydroxyl radicals in their structure, therefore, they could be more potent this inhibition. It was proposed that these functional

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groups are capable of chelating Zn2+ , an important mechanism in ACE inhibition (Bormann and Melzig, 2000). Despite the importance of control of high blood pressure, it is of greater value to counterattack the disorders generated by chronic RAAS over-activation (cardiac failure, cerebrovascular accident, and renal insufficiency), which is achieved pharmacologically with the use of antagonist drugs for AG II and inhibitors of ACE. These properties were demonstrated for the first time in the Mexican plant Salvia elegans. Therefore, Salvia elegans could be considered as a potent and effective alternative in the treatment for patients suffering from hypertension and vascular damage associated with this disorder. References Aguilar, A., Camacho, J.R., Chino, S., Jacquez-López, M.E., 1994. Herbario Medicinal de Instituto Mexicano del Seguro Social. México, 107 pp. Aguirre-Crespo, F., Vergara-Galicia, J., Villalobos-Molina, R., López-Guerrero, J., Navarrete-Vázquez, G., Estrada-Soto, S., 2006. Ursolic acid mediates the vasorelaxant activity of Lepechinia caulescens via NO release in isolated rat thoracic aorta. Life Science, 791062–791068. Atlas, S.A., 2007. The renin–angiotensin aldosterone system: pathophysiological role and pharmacologic inhibition. Journal Managed Care Pharmacy 13, 9–20. Biblioteca Digital de la Medicina Tradicional Mexicana. Available at: http://www.medicinatradicionalmexicana.unam.mx/index.php (accessed 04.17.10). Bormann, H., Melzig, M.F., 2000. Inhibition of metallopeptidases by flavonoids and related compounds. Pharmazie 55, 29–132. Chen, C.H., Lin, J.Y., Lin, C.N., Hsu, S.Y., 1992. Inhibition of angiotensin-I-converting enzyme by tetrahydroxyxanthones isolated from Tripterospermum lanceolatum. Journal of Natural Products 55, 691–695. Chen, Y., Ruan, Y., Li, L., Chu, Y., Xu, X., Wang, Q., Zhou, X., 2003. Effects of Salvia miltiorrhiza extracts on rat hypoxic pulmonary hypertension, heme oxygenase-1 and nitric oxide synthase. Chinese Medical Journal 116, 757–760. Chiou, C.S., Lin, J.W., Kao, P.F., Liu, J.C., Cheng, T.H., Chan, P., 2008. Effects of hesperidin on cyclic strain-induced endothelin-1 release in human umbilical vein endothelial cells. Clinical and Experimental Pharmacology and Physiology 35, 938–943. Dickinson, C.J., Lawrence, J.R., 1963. A slowly developing pressor response to small concentrations of angiotensin. Its bearing on the pathogenesis of chronic renal hypertension. Lancet 1, 1354–1356. Erdogan, A., Most, A.K., Wienecke, B., Fehsecke, A., Leckband, C., Voss, R., Grebe, M.T., Tillmanns, H., Schaefer, C.A., Kuhlman, R., 2007. Apigenin-induced nitric oxide production involves calcium-activated potassium channels and is responsible for antiangiogenic effects. Journal of Thrombosis and Haemostasis 5, 1774– 1781. Fabiani, M.E., Dinh, D.T., Nassis, L., Casley, D.J., Johnston, C.I., 2000. Comparative in vivo effects of irbesartan and losartan on angiotensin II receptor binding in the rat kidney following oral administration. Clinical Science 99, 331–341.

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