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Vasodilation and radical-scavenging activity of imperatorin and selected coumarinic and flavonoid compounds from genus Casimiroa.

ABSTRACT

Hypertension is a very widespread condition which is not strictly considered as an illness but if not countered, progressively causes damage to all tissues and loss in their functionality. For this reason the find of new antihypertensive agents is prominent and medicinal plants and their derivatives are valuable forthe purpose. The genus Casimiroa (Rutaceae) includes plants from Central America and Mexico; among these, Casimiroa edulis Have et Lex. and Casimiroa pubescens Ramirez are the most relevant species, even for their medicinal uses. The decoction of leaves and seeds is traditionally taken as a tea mainly to lower blood pressure. The object of this research was the study of vascular activity of coumarinic and flavonoid compounds isolated from seeds of Casimiroa spp. in comparison with Casimiroa edulis and Casimiroa pubescens extracts. The phenolic compounds isolated from Casimiroa were herniarin (Her), imperatorin (Imp), 8-geranyloxypsoralen (GOP) and 5,6,2',3',4'- pentamethoxyflavone (PMF). All these compounds induced vasorelaxation on rat arterial tissues although with different effectiveness. To study the cellular mechanisms of the vasorelaxation exhibited by imperatorin, we used selective inhibitors of different receptors and enzymes, such as atropine, pyrilamine, nifedipine, L- NAME and DETC. In a further step of this research, we evaluated the radical-scavenging activity of Casimiroa extracts and isolated compounds by means of DPPH assay. In general, we observed that the scavenging activities increased in a concentration-dependent manner for all substances. The phenolic compounds highlight a synergism of vasodilation and antioxidant activity which may be very useful in the management of cardiovascular diseases. Among the evaluated compounds, imperatorin shows a significant vasorelaxant activity even higher than acetylcholine and similar to nitrite, and also useful antiradical capabilities. All these properties suggest its possible role against hypertension and vasculopathies, even if in vivo studies are needed to determine the actual applications.

Keywords:

Casimiroa seed extracts

Imperatorin

Vasodilation

Radical-scavenging activity

Coumarins

Flavones

Introduction

Hypertension is a global health issue that causes cardiovascular illnesses and disabilities in about 1 in 3 adults worldwide. Further, findings from serial surveys show an increasing prevalence of hypertension in developing countries, possibly caused by urbanization, population ageing, changes to dietary habits and social stress (Ibrahim and Damasceno, 2012). Although the benefits of treating hypertension have been known since the 1960s, inadequate control remains frequent in many countries. Overall, the antihypertensive treatments for preserving cardiovascular function are of high interest in medicine. Traditional medicines propose various plants linked to this specific area. In Traditional Mexican Medicine, the genus Casimiroa is used as anxiolytic and antihypertensive remedy (Randolph Major, 1958; Sarris et al., 2013). In Mexico and in countries with subtropical climate Casimiroa edulis fruits are available in local markets for food use as a dessert, while leaf and seed extracts are readily available for traditional medicine. The use of Casimiroa is also described in the African continent, particularly as Egyptian folk medicine. In Europe and Italy the genus Casimiroa is little-known. However, the Italian Ministry of Health has included the seeds of Casimiroa edulis in the list of herbs not permitted in food supplements (G.U. n. 57 of 09/03/2007, Annex 1). Of course, the fruits are admitted.

In the present study, we compared the in vitro vasodilation and antioxidant activity of Casimiroa edulis (CE) and Casimiroa pubescens (CP) seed extracts with their isolated coumarin and flavone derivatives, as herniarin (Her), imperatorin (Imp), 8-geranyloxypsoralen (GOP), and 5,6,2',3',4'-pentamethoxyflavone (PMF) (Fig. 1). Further, the cellular mechanism of vasodilatation was investigated by the use of standard compounds known for their vascular activity, such as acetylcholine and nitrite, an endothelium-dependent and endothelium-independent vasodilator, respectively (Joannides et al., 2006). Previous studies evidenced that Casimiroa spp. extracts cause relaxant effects on rat aorta and caudal arteries mainly by activation of [M.sub.3]-muscarinic receptor through the cGMP-dependent NO pathway (Froldi et al., 2011).Taking into account this knowledge, we determined the influence of selective inhibitors such as atropine, pyrilamine, nifedipine, L-[N.sup.G]- nitro-arginine methyl ester (L-NAME), and diethyldithiocarbamate (DETC) on the in vitro dilation of imperatorin, as a reference compound isolated from Casimiroa seeds. In addition to the vascular dilation activity, we also studied the scavenging activity of CE, CP and single isolated compounds. For this, we also determined the Total Phenolic Content (TPC), the Total Flavonoid Content (TFC) for both extracts, and carried out for all substances the 1,1-diphenyl-2- picrylhydrazyl (DPPFI) scavenging assay.

Materials and methods

Casimiroa samples

Seeds of Casimiroa edulis and Casimiroa pubescens were collected in the wild and their voucher specimens were deposited at the Plerbarium of Facultad de Ciencias UNAM. Casimiroa pubescens was collected between Vithe and Cardonalito Towns in Ixmiquilpan County, Flidalgo (voucher number FCME-84835-2). Casimiroa edulis was collected in the surroundings of Comala County, Colima (voucher number FCME-84837-2).

Preparation of the Casimiroa extracts

Dried and milled seeds of each selected species were extracted with methanol for 24 h (3 times). The three extracts were gathered and, after evaporation of the solvent at low pressure, the total dry weight was recorded: the yields for Casimiroa edulis (CE) and Casimiroa pubescens (CP) were 4.1 [+ or -] 2.3% and 8.1 [+ or -] 2.1% w/w, respectively. For each extract the furocoumarin imperatorin was quantitatively characterized by FIPLC-DAD (Fig. 3). Previously, imperatorin has been identified in Casimiroa edulis leaf extract (Awaad et al., 2012). Imperatorin in CE and CP was 0.39 [+ or -] 0.05% and 0.73 [+ or -] 0.09% w/w, respectively.

Isolation of Casimiroa components

Melting points were taken on a Fieser-Jones apparatus and are uncorrected. IR spectra were obtained on a Perkin-Elmer mod. 337. The samples were dissolved in chloroform (CH[Cl.sub.3]). The Nuclear Magnetic Resonance spectra were generated on a Varian XL-300 spectrometer, using deuteron chloroform (CD[Cl.sub.3]) as solvent and TMS as reference. Known compounds were identified by comparison of the spectroscopic ([sup.1]H and [sup.13]C NMR) and physical (mp, [[alpha]]D) data with those of literature.

8-Ceranyloxypsoralen

From the fractions eluted with 8:2 hexane/ethyl acetate, 8geranyloxypsoralen was isolated (Fig. 1). The identification of this coumarin was achieved by analysis of its [sup.1]H NMR and [sup.13]C (COSY, F1ETCOR) spectra and in literature (Row et al., 2006).

Hemiarin

Chromatography of extracts was performed using an open column packed with silica (MN-Kiesegel G). Elution was performed with a mixture of hexane-ethyl acetate with ascending polarity. 53 fractions of 125 ml each were collected. Fractions with a similar profile in thin plate chromatography were gathered. From the fractions eluted with 9:1 hexane/ethyl acetate, herniarin (862.3 mg) was isolated. The comparison of spectroscopic data with those published in literature allowed the identification (Petrulova-Poracka et al., 2013).

Imperatorin

An aliquot (33.22 g) of extract was taken up in 21.7 g of celite and placed in the top of a column packed with silica (MN-Kiesegel G). The chromatographic column was eluted with solvent mixtures of increasing polarity: 60 fractions of 150 ml were collected and those that showed similar profiles in thin layer chromatography were gathered. From the fractions eluted with 6:4 hexane/ethyl acetate, imperatorin was isolated. The comparison of its spectral data with the data published in literature allowed the identification of this coumarin (Garcia-Argaez et al., 2000).

5,6,2',3',4'-Pentamethoxyflavone

Fractionation of the ethyl acetate extract (48.9 g) of dried and milled seeds of Casimiroa pubescens (1173g) was carried out by means of vacuum liquid chromatography (silica gel, step gradient of hexane in ethyl acetate). Fractions eluted with 7:3 mixtures yielded 82.3 mg of 5,6,2',3',4'-pentamethoxyflavone. The compound was identified by comparison of its [sup.1]H and [sup.13]C NMR spectra with the data reported in literature (Garcia-Argaez et al., 2005). The identification was achieved by using a combination of m.p., UV, El-mass, [sup.1]H NMR and [sup.13]C NMR spectroscopy.

Pharmacological studies

Caudal arterial tissues

The present study conforms to the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996). The protocols were approved by the Research Ethics Committee of Padova University. Six-month old male rats (Wistar, 300-400 g) were anaesthetised through inhalation of methoxyflurane before sacrifice, hence the caudal arteries were removed. The arteries were cleaned of adhering tissue and arterial rings, 3 mm long, were placed in a tissue bath under a resting tension of 19.6mN. The tissue bath was filled with Krebs-Ringer solution at 37[degrees]C, with the following composition (mM): NaCl 118.3, KCl 4.7, Ca[Cl.sub.2]-2[H.sub.2]O 2.5, MgS[O.sub.4]-7[H.sub.2]O 1.2, K[H.sub.2]P[O.sub.4] 1.2, NaHC[O.sub.3] 25, D-glucose 11.1, and was bubbled with 95% [O.sub.2] and 5% C[O.sub.2] (p[O.sub.2] = 345 [+ or -] 8mmHg), pH = 7.2. Tissues were left to rest for 45 min before viability was assessed with standard start procedures. The endothelial integrity was assessed by the relaxation caused by 1 [micro]M acetylcholine in 10 [micro]M phenylephrine precontracted tissues. The isometric tension was recorded by a high-sensitivity transducer (Ugo Basile, type DYO Comerio, Italy) connected to a PC-based Acqknowledge acquisition system (BIOPAC Systems, Inc., 42 Aero Camino Santa Barbara, CA 93117).

Determination of total phenolic content (TPC) and total flavonoid content (TFC)

The TPC was determined using the Folin-Ciocalteu reagent (Singleton and Rossi, 1965; Ainsworth and Gillespie, 2007). The TPC of each extract is expressed as milligrams of gallic acid equivalents (GAE) per gram of extract.

The total flavonoid content (TFC) of Casimiroa extracts was estimated by aluminium chloride (Al[Cl.sub.3]) colorimetric method (Petry et al., 2001), and TFC was expressed as milligrams of quercetin equivalents (QE) per gram of extract.

Scavenging DPPH radicals

The free radical-scavenging activity of samples was measured by 1,1 -diphenyl-2-picryl-hydrazyl (DPPH) according to the method described by Brand-Williams (1995). The scavenging effect was estimated based on the percentage of DPPH radical scavenged as the following equation:

Percentage effect(E%)= ([[Abs.sub.control] - [Abs.sub.sample]]/[Abs.sub.control]) x 100

Different sample concentrations were used in order to obtain antiradical curves for calculating the EC50 values, which were used to compare the radical-scavenging activity.

High performance liquid chromatography (HPLC) analysis

The chromatographic fingerprinting of Casimiroa spp. extracts was obtained performing an HPLC analysis of each methanolic seed extract, recording the UV spectra of the single peak (Diode Array Detector). The characterization was performed by a previously reported method (Froldi et al., 2011), with some modifications for the determination of imperatorin, which for the first time was identified in Casimiroa seed extracts. Chromatographic analyses were carried out by the use of HPLC system (Waters Corporation, USA) equipped with Waters 1525 Binary HPLC pump and Waters 2998 Photodiode Array Detector. The separation was performed on a XTerra RP 18 column (4.6 x 15 mm 3.5 [micro]m. Waters, USA) by a linear methanol increasing concentration gradient in the mobile phase from 50 to 90% (v/v) in 50 min, maintaining the flow rate at 1 ml/min (Solution A; acetic acid 2% and Solution B: methanol) and peaks were detected at 254 nm. The sample extract was filtered through 0.2 [micro]m membrane filters, and then 10 [micro]l aliquots from the filtrate were injected into the HPLC system. Identification of compounds was performed on the basis of the retention time, co-injections, and spectral matching with imperatorin standard. For the preparation of the calibration curves, standard stock solutions of imperatorin were prepared in methanol, filtered through 0.22 [micro]m filters (Millipore), and appropriately diluted (0.01-100 [micro]g/ml) to obtain the desired concentrations in the quantification range. The calibration graphs were plotted after linear regression of the peak areas vs. concentrations. For the quantification of marker compound by HPLC, a five-point calibration curve was generated by the least squares method.

Chemicals

Imperatorin (9-[(3-Methyl-2-buten-l-yl)oxy]-7h-furo[3,2-g] [1 ]benzopyran-7-one) standard, DPPH (2,2-diphenyl-l-picrylhydrazyl) radical, DMSO (Dimethyl sulfoxide), methanol and all other chromasolv[R] reagents were purchased from Sigma-Aldrich, Milano, Italy. The purity of all substances was >98%.

Statistical analysis

Each experiment was performed at least six times and the results are presented as the Mean [+ or -] S.E.M. The relaxing response was expressed in terms of percent decrease of the maximal contraction caused by 0.5 [micro]M phenylephrine (Phe). The [IC.sub.50] value was defined as the concentration of substance that reduced by 50% the maximum contraction elicited by phenylephrine and calculated from the concentration-response curve, which was analyzed by nonlinear regression (curve fit) using GraphPad Prism[R] programme (GraphPad Software, San Diego, CA, USA). The dilatation induced by agonists was quantified by use of the [EC.sub.50], also expressed as pD2 (-log[EC.sub.50]). The statistical analysis were performed by the GraphPad Prism[R] programme, using the five-parameter analysis for the [EC.sub.50] estimation of antioxidant activity (Chen et al., 2013). The statistical comparisons between treatment and control data were performed by ANOVA followed by Bonferroni t test; a value of p < 0.05 was considered statistically significant.

Results

To study the antihypertensive action of Casimiroa spp., we determined the vascular activity of methanolic extracts of seeds from Casimiroa edulis (CE) and Casimiroa pubescens (CP) in comparison with standard vasodilators such as nitrite and acetylcholine, respectively endothelium-independent and endothelium-dependent agents, in phenylephrine-precontracted caudal arterial tissues (Fig. 2). Both extracts (20 [micro]g/ml) showed a significant dilatation of arterial tissue, higher for CP (84.0 [+ or -] 3.4%) in comparison to CE (40.0 [+ or -] 1.6); further, the effects of both Casimiroa extracts were always higher than the relaxation induced by 10 [micro]M acetylcholine (17.0 [+ or -] 1.9%). Further, we characterized the Casimiroa extracts using HPLC-DAD apparatus determining the imperatorin amount in each extract (Fig. 3). Its concentration was equal to 0.73 [+ or -] 0.09% w/w in CP, and 0.39 [+ or -] 0.05% w/w in CE. Moreover, we determined the total phenolic content and the total flavonoid content both in CP and CE (Table 1). The extracts showed a larger amount in phenolic compounds, much higher than the presence in flavonoids.

In order to have a deeper knowledge of the vasodilatation of extracts and to search for newer dilators, we studied the activity of isolated compounds such as herniarin (Her), imperatorin (Imp), 8-geranyloxypsoralen (GOP), and 5,6,2',3',4'pentamethoxyflavone (PMF). These compounds at micromolar concentrations started to reduce arterial tension, reaching the plateau of effect in about twenty min; the relaxation was stable and promptly reversible after washout. Imperatorin tested from 0.3 to 300 [micro]M induced a concentration-dependent vasorelaxation on phenylephrine-precontracted arterial tissues (Fig. 4A); the EC50 was 4.2 [micro]M (pD2 = 5.37 [+ or -] 0.16). Also GOP, Hern, and PMF caused similar relaxation but with different potency (Fig. 4C). The potency order was: PMF>Imp>Hern>GOP, moreover all showed higher activity than acetylcholine, the physiological endothelium-dependent vasodilator (Fig. 4B and C).

In another step we determined the activity of nitrite (Fig. 5), that is a potent and universal vasodilator which mimics the activity of organic nitrates known for their use in angina pectoris treatment (Nossaman et al., 2010). The dilation caused by these compounds is a typical endothelium-independent vasorelaxation (Ignarro, 1989). In this study, the maximal vasorelaxant activity of natural compounds and nitrate was quantitatively equal, but the potency of PMF, Imp and Her was about 100 times higher than that of nitrite, which has an EC50 of 190.6 [micro]M (pD2 = 3.72 [+ or -] 0.09).

Furthermore, to investigate the mechanisms involved in vasodilation we chose to deeply study Imp as a reference compound among those isolated from Casimiroa extracts. In order to achieve this aim, we used selective inhibitors at different receptors such as atropine, a muscarinic antagonist (van Zwieten and Doods, 1995), and pyrilamine, a selective [H.sub.1] histamine antagonist (Jones and Kearns, 2011). Neither atropine (1 [micro]M) nor pyrilamine (1 [micro]M) changed the effects of Imp (Fig. 6). Furthermore, 50 nM nifedipine, an L-type calcium channel antagonist (Godfraind, 1987), was ineffective vs. the dilation of Imp (Fig. 6). To study the role of nitric oxide, we examined the effect of the NO synthase inhibitor L-NAME, 10 [micro]M incubated for 30 min, which also left unchanged the activity of imperatorin (Fig. 6). Finally, we studied the effect of 5mM diethyldithiocarbamate, a superoxide dismutase (SOD) inhibitor (Heikkila et al., 1976), which unexpectedly increased the dilatation induced by Imp (Fig. 6).

To deeply study the activity of Casimiroa extracts and phytoconstituents we determined their antioxidant capacity using the DPPH assay. Both CE and CP elicited a concentration-dependent antioxidant activity, starting from 20 p-g/ml and reaching an effect of 72.0 [+ or -] 3.2% and 58.4 [+ or -] 2.5% respectively, at the concentration of 600p,g/ml (Fig. 7). Among the isolated compounds Imp and GOP revealed higher antioxidant activity; unexpectedly, the latter exerts an evident activity reaching the 91.9 [+ or -] 0.6% at 5.0 mM (Fig. 7), while the coumarin Hern showed a very low antioxidant profile with an effect of 11.5 [+ or -] 1.4% at 5.0 mM (Fig. 7). At the same concentration imperatorin showed an action of 73.8 [+ or -] 1.5%, with a [IC.sub.50] of 2.25 (1.68-3.02) mM. The antioxidant potency order based on estimated [EC.sub.50] was: geranyloxypsoralen> imperatorin > pentamethoxyflavone > herniarin. The ascorbic acid, which was used as a reference antioxidant, always showed a higher DPPH scavenging effect; at the concentration of 50 [micro]M ascorbic acid showed a radical-scavenging activity of 96.4 [+ or -] 0.2% (Fig. 7).

Discussion

The experimental data clearly show the vasodilation capacity of specific coumarin and flavone derivatives: imperatorin, 5,6,2',3',4'pentamethoxyflavone, herniarin and 8-geranyloxypsoralen, compared with acetylcholine and nitrite, well-known reference compounds in pharmacology as endothelium-dependent and independent vasodilators, respectively. All the phytoconstituents show a significant and quantifiable capacity in decreasing the smooth muscle tension of rat caudal artery, which is a valuable experimental model of resistance vessels (Jantschak et al., 2010). Further, this research corroborates previous data showing vasorelaxation by various Casimiroa spp. extracts (Froldi et al., 2011); in the present experiments, the methanolic seed extracts from Casimiroa edulis and Casimiroa pubescens exhibit vasorelaxant activity comparable to nitrite and even higher than acetylcholine. In this framework, we can observe the consistency among the activity of single chemical constituents and the plant extracts.

Previously, other authors have reported on vasorelaxant activity of some phenolic compounds (Xu et al., 2007; Lin et al., 2010; Dong et al., 2011). Quercetin (3,3',4,5,7-pentahydroxyflavone) at 10 [micro]M and resveratrol (3,4',5-trihydroxystilbene) at 30 [micro]M caused relaxation of phenylephrine-precontracted aorta, reaching the maximum effect (100% inhibition) at the concentration of 100-300 [micro]M (Ke Chen and Pace-Asciak, 1996). Generally, most of the researches on vascular activity of phenolic compounds have been performed in rat aorta, in a concentration range from 10 to 300 [micro]M (Campos-Toimil et al., 2002; Ajay et al., 2003; Chen et al., 2004); while to now, few data are published on coumarin and furocoumarin and none on the 5,6,2',3',4'-pentamethoxyflavone. About imperatorin, Zhang et al. (2011), determined on rat aortic rings a pD2 of 4.8 [+ or -] 0.07, which is lower than that found in the present research (pD2 = 5.37 [+ or -] 0.16), the diversity may depend on the different arterial tissue and the type of precontracture. Effectively, in the present research, imperatorin showed higher potency, starting to be active from 0.3 [micro]M. In this context, we focused the attention on the activity of imperatorin, which for the first time was isolated here from the seeds of Casimiroa edulis and Casimiroa pubescens, although recently imperatorin has been identified in the leaves of Casimiroa edulis (Awaad et al., 2012). Also the furocoumarin has been reported in various medicinal plants, such as Cnidium monnieri (Sun et al., 1990; Chiou et al., 2001), Heracleum rapula (Liu et al., 2006), Angelica spp. (Widelski et al., 2009; Marumoto and Miyazawa, 2010; Kumar et al., 2013), Clausena lansium (Adebajo et al., 2009), and also in edible vegetables such as parsnip, parsley, and fennel. Few researchers already studied the imperatorin activity on vascular tissues (He et al., 2007; Nie et al., 2009; Zhang et al., 2010; Li et al., 2011). He et al. (2007) found a vasorelaxation in both rat mesenteric and human omental arteries, but only at concentrations higher than 10 [micro]M. According to them, the action of imperatorin depends on a reduction of calcium availability, from both extracellular and intracellular sources. Afterwards, other authors showed that the vasorelaxation by imperatorin is not related to store-operated [Ca.sup.2+] entry (SOCE), but more similar to that of nifedipine (Zhang et al., 2010, 2011). In the present study, the 1,4-dihydropyridine [Ca.sup.2+] channel antagonist nifedipine did not inhibit the vasorelaxation of imperatorin, rather slightly increased this one, suggesting none interaction between nifedipine and imperatorin intracellular mechanisms. The last data disagree with Zhang et al. (2011) who showed the hypotensive effects of imperatorin in hypertensive rats (SHRs), also suggesting for the furocoumarin an activity of L-type calcium channel antagonism. The present data show that imperatorin dilatation is independent either from muscarinic and histamine receptors, or from endothelial nitric oxide (NO) synthesis, in agreement with previous literature data (He et al., 2007; Zhang et al., 2010). Recently, authors suggested a relative selectivity of imperatorin as inhibitor within the PDE4 family, mainly for PDE4A and PDE4B (Ivey et al., 2008; Pantel et al., 2011); this may be a mechanism responsible for the vasorelaxant effect induced by imperatorin; further work will be needed to establish this possibility. DETC is a metal ion-chelating agent known as inhibitor of SODs (Heikkila et al., 1976), and also attenuate the LPS-stimulated induction of NOS in vascular smooth muscle (Loegering et al., 1995). The inhibitor DETC increased the vasodilation of 10-30 [micro]M imperatorin, suggesting a role of the radical [O.sub.2.sup.-] in the regulation of arterial tone. Thus we think to determine the scavenging activity of Casimiroa extracts and single constituents to study if there is a correlation between vasodilation and antioxidant capacity. For this we used the DPPH assay which is the best, easiest and widely used method for testing preliminary free radical-scavenging activity of a compound or a plant extract (Moon and Shibamoto, 2009). Among the compounds, geranyloxypsoralen followed by imperatorin showed the stronger antioxidant activities, whereas herniarin up to 5 mM showed none activity. Plants produce an extraordinary diversity of phenolic metabolites, which may be considered as effective antioxidant compounds thanks to their structural arrangement; for this reason, they are excellent oxygen radical scavengers because the electron reduction potential of the phenolic radical is lower than the electron reduction potential of oxygen radicals. Few authors have been investigated on the possible relationship among antioxidant activity, vasodilation capacity, and phenolic content of plant extracts having different conclusions (Burns et al., 2000). In our experiments, the vasodilation and the antioxidant activity are not correlated; indeed between the two extracts CE caused a minor dilatation but higher antioxidant activity, as opposed to CP. On the other hand, 500 [micro]M imperatorin shows a significant antioxidant activity of 22.7 [+ or -] 1.1%, while 8-geranyloxypsoralen has similar activity at lower concentration (100 [micro]M). Thus imperatorin exhibits the higher vasodilation with minor radical-scavenging activity, vice versa for 8-geranyloxypsoralen.

All the experimental remarks support the traditional use of Casimiroa as hypotensive in Traditional Medicine. Also, the synergism among the compounds of Casimiroa extracts is useful for a greater vasodilation. Furthermore, these extracts show a relevant antioxidant activity that might enhance the cardiovascular benefits noticed in folk medicine.

In addition to in vitro researches, now are also available in vivo preliminary studies, even on the human species, which assess the effects as vasodilators and the antihypertensive activity of plant extracts and single natural phenolic derivatives (Perez-Vizcaino et al., 2009; Zhang et al., 2011, 2012). To evaluate the therapeutic application of any compound, the pharmacokinetics determination is required; imperatorin has been studied in mice after oral administration, showing a good bioavailability (Lii, 2006; Kumar et al., 2013; Lili et al., 2013; Zhao et al., 2013).

Considering all the data together, there are many evidences supporting the usefulness of pivotal trials on imperatorin to determine the pharmacokinetic profile in humans and its effectiveness as an antihypertensive drug. Further, there are strong evidences for the use of medicinal plants as Casimiroa spp. or others containing imperatorin and various phenolic vasodilators in folk medicine.

http://dx.doi.org/10.1016/j.phymed.2013.10.030

ARTICLE INFO

Article history:

Received 2 August 2013

Received in revised form 20 September 2013

Accepted 27 October 2013

Acknowledgments

This work was supported by the Italian Ministry for University and Research (MIUR) 2011 n. 60A04-5521/11.

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R. Bertin (a), Z. Chen (a), M. Martinez-Vazquez (b), A. Garria-Argaez (a), G. Froldi (a), *

(a) Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Largo E. Meneghetti 2, 35131 Padova, Italy

(b) Instituto de Quimica, Univesidad Nacional Autonoma de Mexico, Circuito Exterior, Ciudad Universitaria, Coyoacan, 04510 D.F., Mexico

* Corresponding author. Tel.: +39 0498275092; fax: +39 0498275093.

E-mail address: g.froldi@unipd.it (G. Froldi).

Table 1
The total phenolic content (TPC) and total flavonoid content (TFC)
of methanolic extracts from Casimiroa spp. seeds.

                                                  Total Flavonoid
Plant extracts             Total Phenolic         Content (mg
                           Content (mg gallic     quercetin
                           acid equivalents/g)    equivalents/g)

Casimiroa edulis (CE)      55.89 [+ or -] 5.38    1.87 [+ or -] 0.13
Casimiroa pubescens (CP)   94.95 [+ or -] 3.19    0.59 [+ or -] 0.02

Each value is reported as mean [+ or -] SEM of at least 3 experiments.
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Author:Bertin, R.; Chen, Z.; Martinez-Vazquez, M.; Garria-Argaez, A.; Froldi, G.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Apr 15, 2014
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