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Endothelium-independent vasorelaxant effect of a Berberis orthobotrys root extract via inhibition of phosphodiesterases in the porcine coronary artery.

Abstract

Background: Berberis orthobotrys Bien ex Aitch. (Berberidaceae) is a plant indigenous of Pakistan that is locally used for the treatment of hypertension.

Hypothesis: This study evaluated the vasoactive properties of a Berberis orthobotrys root extract and its fractions, and investigated the role of the endothelium and the underlying mechanism.

Study design: An aqueous methanolic extract of Berberis orthobotrys roots was prepared and submitted to a multi-step liquid-liquid fractionation with solvents of increasing polarity. Vascular reactivity of the different fractions was assessed using porcine coronary artery rings either with or without endothelium, and in the presence or absence of specific pharmacological tools. The ability of Berberis orthobotrys extracts to affect phosphodiesterase (PDE) activity was evaluated using a radioenzymatic method and purified phosphodiesterases.

Results: The aqueous methanol extract induced similar relaxations in coronary artery rings with and without endothelium, and, amongst the three derived preparations, the butanol fraction (BFBO) was slightly but significantly more effective than the ethyl acetate fraction and the aqueous residue in rings without endothelium. Analysis of the butanol fraction (BFBO) by LC-ELSD-MS indicated the presence of four major isoquinoline alkaloids including berberine. BFBO significantly potentiated the relaxations induced by cyclic GMP- and cyclic AMP-dependent relaxing agonists, and inhibited contractions to KCl, Ca[Cl.sub.2], and U46619 in endothelium denuded rings. In contrast, BFBO did not affect relaxations to endothelium-dependent vasodilators. BFBO concentration-dependently inhibited the cyclic GMP-hydrolyzing activity of basal PDE1, calmodulin-activated PDE1 and PDE5, and of cyclic AMP-hydrolyzing activity of PDE3 and PDE4 with [IC.sub.50] values ranging from 40 to 130[micro]g/ml.

Conclusion: The butanol fraction of the aqueous methanol extract of Berberis orthobotrys roots induced pronounced endothelium-independent relaxations and inhibited contractile responses by acting directly at the vascular smooth muscle in the coronary artery. Moreover, BFBO potentiated relaxations induced by both cyclic GMP- and cyclic AMP-dependent vasodilators most likely due to its ability to inhibit several vascular PDEs, and in particular PDE4 and PDE5.

Keywords:

Berberis orthobotiys

Vasorelaxation

Endothelium

Phosphodiesterases

Introduction

Berberis is a large genus which belongs to the family Berberidaceae and includes over 400 species, such as Berberis aquifolium (Oregon grape), Berberis aristata (Indian barberry), Berberis thunbergii (red barberry), Berberis vulgaris (barberry) and Berberis orthobotrys (named "ishkeen" in Pakistan). This later plant is an indigenous shrub of Pakistan which is used by the people of the Gilgit region for the treatment of hypertension. Chemical studies of Berberis species have led to the identification of berberine, a yellow isoquinoline alkaloid, which has a long history of medicinal use in both Ayurvedic and Chinese medicine (Vuddanda et al., 2010). Several pharmacological properties have been previously reported for berberine, including antibacterial and anti-inflammatory activities, a beneficial effect on dyslipidemia and hyperglycemia, inhibition of cyclooxygenase-2, and improvement of neurological disorders (Kulkarni and Dhir, 2010). Berberine has also been shown to inhibit the generation of reactive oxygen species and the subsequent mitochondrial membrane potential collapse, as well as caspase-3 activation induced by oxLDL in endothelial cells (Hsieh et al., 2007). Moreover, berberine is a potent antineoplastic compound that inhibits cell proliferation through p53dependent G1 arrest and p53-independent G2 arrest in HL-60 cells (Khan et al., 2010). Various clinical trials have evaluated its therapeutic properties in cardiovascular diseases (Derosa et al., 2012). Indeed, berberine treatment was shown to significantly decrease mortality in patients with congestive heart failure (Zeng et al., 2003). In addition, berbamine, another alkaloid extracted from B. vulgaris, has been shown to increase myocardial contractility by increasing myofilament [Ca.sup.2+]-sensitivity via a protein kinase C[epsilon]-dependent signaling pathway (Zhang et al., 2011).

A recent study has shown that B. orthobotrys strongly decreased blood pressure in both normotensive and hypertensive rats (Alamgeer et al., 2013). Since previous studies have shown that extracts from plants used traditionally as antihypertensive medicine such Parkia biglobosa, cause potent endothelium-dependent relaxations involving NO of isolated arteries (Tokoudagba et al., 2010), the aim of the present study was to assess the potential of the B. orthobotrys extracts to affect vascular tone, to clarify the role of the endothelium and the underlying mechanism.

Materials and methods

Plant material and isolation

The roots of B. orthobotrys were collected from Shikiyote, Gilgit, Pakistan, in June 2011 and were identified by Dr. Shair Wali Khan, Assistant Professor of Botany, Karakuram International University, Pakistan. The voucher specimen No (BO-15-12) has been deposited in the Herbarium of the Faculty of Pharmacy, University of Sargodha, Pakistan. An aqueous methanol extract from B. orthobotrys was prepared by three successive macerations of 2 kg of roots in 5 l of a water-methanol mixture (70:30) for 72 h at room temperature followed by evaporation giving a drug extract ratio of 5:1. Thereafter, the methanol extract (100 g) was mixed with water (500 ml) and partitioned with the same volume of ethyl acetate (500 ml) for three successive times. The decantation and evaporation of the collected ethyl acetate layer provided 13 g of the ethyl acetate fraction of B. orthobotrys (EFBO). The remaining aqueous layer was further extracted with butanol (500 ml) for three successive times. The butanol layer was collected and evaporated providing 26.1 g of the butanolic fraction of B. orthobotrys (BFBO). The remaining aqueous layer was evaporated and provided 60.9 g of the aqueous residue of B. orthobotrys (ARBO). Each fraction was analyzed by thin-layer chromatography (TLC; Merck, Germany) eluted with n-butanol-acetic acid-water (4:1:1, v/v/v) and the presence of alkaloids was revealed using the Dragendorff's reagent at 366 nm. All extracts were stored at 4[degrees]C before being used.

Chemicals and drugs

Levcromakalim, atrial natriuretic peptide (ANP) and ethylene glycol-bis(2-aminoethylether)-N,N,N',N' -tetraacetic acid (EGTA) were purchased from Tocris Bioscience (Bristol, UK). U46619 was supplied by Cayman Chemical (Ann Arbor, MI, USA), charybdotoxin (synthetic) and apamin by Latoxan (Valence, France), and bradykinin, sodium nitroprusside (SNP), forskolin, 1-EBIO, calcium ionophore A23187, N-[omega]-nitro-L-arginine (L-NA), isoproterenol hydrochloride, berberine hydrochloride by Sigma-Aldrich (Saint-Quentin Fallavier, France). All chemicals were of analytical grade.

HPLC profiles and quantification of berberine by LC/UV

Analytical HPLC analyses of the extracts fractions ARBO, BFBO and EFBO, and of the standards berberine and berbamine were performed on a LC-20 AD instrument system (Shimadzu) equipped with a SPD-M20A PDA detector, an evaporative light scattering detector (ELSD) serie 3300 (Alltech) and a LCMS-8030 detector (Shimadzu). For the ELSD, the N2 flow was set at 2.5 1/min, and evaporation temperature was 60[degrees]C. The mobile phase consisted of 0.1% formic acid (solvent A) and MeOH + 0.1% formic acid (solvent B), and the flow was set to 0.4ml/min. Separations were performed on an Uptisphere[R] Strategy C18-2 column (3.0 x 150 mm, 3 [micro]m, Interchim) which was thermostatted at 40[degrees]C. For quantification of berberine in extracts, stock solutions extracts ARBO, BFBO and EFBO were prepared at concentrations of 8.1, 9.9 and 12.1 mg/ml, respectively. Each extract stock solution was further diluted two and four fold. For the standard of berberine, a stock solution of 1.01 mg/ml was prepared, from which five serial dilutions ranging from 202 to 25 [micro]g/ml were obtained. For each sample, 10.0 [micro]1 was injected. The following gradient was used: 10% B isocratic for 4 min, gradient 4-20 min to 30% B, 20-30 min 30% B isocratic, 30-31 min to 100% B, 31-35 min 100% B isocratic, and 35-36 min to 10% B. The HPLC-UV-ELSD-MS chromatograms are shown in Fig. 1.

Quantification of berberine in the extracts was done by LC/UV at 345 nm and revealed the presence of 5.4%, 15% and 1.6% berberine in Berberis fractions ARBO, BFBO and EFBO respectively.

Vascular reactivity studies

Pig hearts were collected from the local slaughterhouse (Copvial, Holtzheim) and vascular reactivity was assessed as indicated previously (Ndiaye et al., 2003). Briefly, left circumflex coronary arteries were excised, cleaned of loose connective tissue and flushed with PBS without calcium to remove remaining blood. Rings of porcine coronary arteries (4-5 mm in length) were then suspended in organ baths containing oxygenated (95% [O.sub.2], 5% C[O.sub.2]) Krebs bicarbonate solution (composition in mM: NaCl 119, KCl 4.7, K[H.sub.2]P[O.sub.4] 1.18, MgS04 1.18, Ca[Cl.sub.2] 1.25, NaHC[O.sub.3] 25 and D-glucose 11, pH 7.4, 37[degrees]C) for the determination of changes in isometric tension (basal tension 5 g). The integrity of the endothelium was checked with bradykinin (0.3 [micro]M). For the assessment of the vasorelaxant properties of the extracts, rings were contracted (about 80% of the maximal contraction) with U46619 (a thromboxane [A.sub.2] receptor agonist) before construction of a concentration-relaxation curve to an extract. For assessment of the inhibition of contractile responses, rings were exposed to BFBO for 30 min before construction of a concentration-contraction curve either to KCl, U46619 or Ca[Cl.sub.2] in the presence of 40 mM KCl. In some experiments, rings were exposed to a low concentration of BFBO (10[micro]g/ml) for 30 min before contraction to U46619 and the subsequent construction of a concentration-dependent relaxation curve to an agonist.

Determination of PDE activity

Vascular cyclic nucleotide phosphodiesterases (PDE1, PDE3, PDE4 and PDE5) were purified from smooth muscle of bovine aorta (Lugnier et al., 1986). The cyclic nucleotide hydrolytic activity assay was carried out using a radioenzymatic method as previously described (Keravis et al., 2005) at a substrate concentration of either 1 [micro]M cyclic AMP or 1 [micro]M cyclic GMP in presence of 10.000 cpm [[sup.3]H]-cyclic AMP or [[sup.3]H]-cyclic GMP as a tracer. PDE1 was assessed in presence of cyclic GMP, in basal state (1 mM EGTA) and in a calmodulin-activated state (18 nM calmodulin with 10 [micro]M Ca[Cl.sub.2]). PDE3 and PDE4 were assessed in presence of cyclic AMP and 1 mM EGTA. Since PDE3 and PDE4 were partially cross-contaminated during their chromatographical separation, determination of specific PDE4 activity was performed in the presence of an excess of cyclic GMP to inhibit PDE3 activity. Reciprocally the determination of specific PDE3 activity was carried out in the presence of 50 [micro]M rolipram to selectively inhibit PDE4. PDE5 activity was measured with 1 [micro]M cyclic GMP in the presence of 1 mM of EGTA. The concentration of compounds that produced 50% inhibition of substrate hydrolysis ([IC.sub.50]) was calculated by non-linear regression analysis of concentration-response curves performed with different concentrations of BFBO dissolved in dimethyl sulfoxide (DMSO). Results are provided as mean [+ or -] S.E.M of 3 different [IC.sub.50] determinations on each purified vascular PDEs according to Lugnier et al., (1986).

Statistical analysis

Data are presented as mean [+ or -] S.E.M. of n different experiments. The [IC.sub.50] value corresponds to the concentration of the product inhibiting 50% of the response. Mean values are compared using analysis of variance followed by the Bonferroni post-hoc test (comparison of selected pairs), using GraphPad Prism 5 (GraphPad Software, Inc., San Diego, CA, USA). The difference was considered to be significant for a P value less than 0.05.

Results

Analysis of B. orthobotrys by LC-ELSD-UV-MS

Four samples were obtained by maceration of B. orthobotrys roots: the aqueous methanol crude extract, the ethyl acetate fraction (EFBO), the butanol fraction (BFBO), and the aqueous residue (ARBO). Analysis of the most active fraction (BFBO) by LC-ELSDUV-MS showed the presence four major compounds 1 to 4. Compound 3 was identified as berberine, by co-chromatography with an authentic reference, and by the molecular ion [[M].sup.+] observed at m/z 336.0 for the quaternary alkaloid. Compounds 2 and 4 displayed similar UV spectra as berberine 3 (U[V.sub.max]: 225, 275 and 345 nm), and their structures were also assigned to protoberberine alkaloids. The MS spectrum of 2 showed a molecular ion [[M].sup.+] at m/z 338.0, while compound 4 displayed a molecular ion [[M + H].sup.+] at m/z 352.0. Peak 1 showed a pseudomolecular ion [[M + H].sup.+] at m/z 609.2, and a fragment ion at m/z 305.0. The UV spectrum was similar to that of berbamine, and compound 1 was thus a bisbenzylisoquinoline alkaloid with a MW of 608.2.

Quantification by LC/UV at 345 nm revealed the presence of 15.0% berberine in the most active fraction BFBO. The other two fractions ARBO and EFBO contained 5.4% and 1.6% berberine respectively.

B. orthobotrys root extracts and its fractions cause endothelium-independent relaxations

The ability of a B. orthobotrys root aqueous methanol extract and its fractions to affect vascular tone was assessed using porcine coronary artery rings contracted submaximally with the thromboxane [A.sub.2] receptor agonist U46619. The aqueous methanol extract of B. orthobotrys caused similar concentration-dependent relaxations in rings with and without endothelium, with [IC.sub.50] values of 25.45 [+ or -] 5.03 and 19.07 [+ or -] 4.30 [micro]g/ml, respectively (Fig. 2A). These findings indicate that the extract is acting preferentially at the vascular smooth muscle. Among the different fractions obtained from the aqueous methanol extract of B. orthobotrys, the butanol fraction (BFBO) was slightly but significantly more potent that the other fractions in rings without endothelium (Fig. 2B). Indeed, BFBO at concentrations of 30 and 100 [micro]g/ml caused significantly greater relaxations compared with the ethyl acetate fraction and the aqueous fraction (Fig. 2B). The [IC.sub.50] values were 37.50 [+ or -]5.59, 96.30 [+ or -]7.70 and 71.89 [+ or -] 13.20 [micro]g/ml for BFBO, the ethyl acetate fraction and the aqueous fraction, respectively. The maximal relaxation of BFBO was achieved at 300 [micro]g/ml and amounted to 94.98 [+ or -] 1.69% (Fig. 2). Since the butanol fraction was the most active one, all further investigations to characterize the mechanism underlying the vasoactive properties of B. orthobotrys were performed with this fraction.

BFBO does not affect relaxations induced by eNOS activators and potassium channels openers in coronary artery rings

The endothelium-dependent relaxing agonists bradykinin and A23187 caused concentration-dependent relaxations in coronary artery rings with endothelium, which were not affected by BFBO at a concentration of 10[micro]g/ml, which caused less than 25% relaxation (Figs. 3A and B). Similarly, BFBO did not significantly affect the relaxation to levcromakalim (an activator of KATP) and 1-EBIO (an activator of I[K.sub.Ca]) in rings without endothelium (Figs. 4A and B). These findings indicate that BFBO does not affect the endothelium-mediated NO-dependent relaxation, and potassium channel-induced relaxation of the vascular smooth muscle.

BFBO potentiates endothelium-independent relaxations induced by cyclic CMP- and cyclic AMP-elevating agents

As vascular tone is finely regulated by cyclic nucleotides levels in the vascular smooth muscle, we have investigated the effect of BFBO on relaxations induced by agents elevating either cyclic GMP or cyclic AMP. BFBO at a concentration of 10 [micro]g/ml significantly potentiated the relaxation in response to cyclic GMP-elevating vasodilators such as atrial natriuretic peptide and SNP in rings without endothelium (Figs. 5A, B). The [IC.sub.50] values were 14.2 [+ or -]2.0 and 5.0[+ or -]0.8nM for atrial natriuretic peptide, and 117.5 [+ or -]5.2 and 33.7 [+ or -]4.1 [micro]M for SNP, in the absence and presence of BFBO, respectively (Figs. 5A, B). Similarly, BFBO (10 [micro]g/ml) significantly shifted to the left the concentration-relaxation curves to cyclic AMP-elevating vasodilators forskolin and isoproterenol in rings without endothelium (Figs. 5C, D). The [IC.sub.50] values were 445.2 [+ or -]32.1 and 208.3 [+ or -] 15.3 nM for forskolin, and 147.4 [+ or -]6.7 and 29.5 [+ or -] 3.5 nM for isoproterenol, in the absence and presence of BFBO, respectively (Figs. 5C, D). These results indicate that BFBO significantly potentiates relaxations induced by cyclic nucleotide-elevating agents, suggesting that BFBO might stimulate the formation of cyclic GMP and/or cyclic AMP, and/or possibly also, decrease their degradation by inhibiting PDEs.

BFBO inhibits contractile responses

BFBO significantly inhibited concentration-dependent contractile responses to KC1 in rings without endothelium (Fig. 6A). The maximal contractile response to KC1 (50 mM) was 17.7 [+ or -] 0.4 g, 13.9 [+ or -] 0.5 g, 9.9 [+ or -] 0.8 g, and 2.4 [+ or -] 0.8 g, in the absence (control) and presence of BFBO (10, 30 and 100[micro]g/ml), respectively (Fig. 6A). Similarly, BFBO also strongly inhibited contractile responses to Ca[Cl.sub.2] in the presence of 40 mM KC1 in endothelium denuded coronary artery rings (Fig. 6B). The maximal contraction induced by Ca[Cl.sub.2] (10 mM) was 14.2 [+ or -] 0.6 g, 11.0 [+ or -] 0.6 g, 9.7 [+ or -] 0.6 g, and 4.8 [+ or -] 0.5 g in the absence and presence of BFBO (10, 30 and 100 [micro]g/ml), respectively (Fig. 6B). BFBO also significantly inhibited the contraction induced by U46619 in a concentration-dependent manner. The maximal contraction was 16.9 [+ or -] 3.1 g, 16.2 [+ or -] 1.9 g, 10.1 [+ or -]2.0g and, 3.5 [+ or -] 0.6 g, in the absence (control) and presence of BFBO (10, 30 and 100[micro]g/ml), respectively (Fig. 6C).

BFBO inhibits the activity of vascular cyclic nucleotide phosphodiesterase isoforms

BFBO inhibited different vascular smooth muscle isoforms of PDE in a concentration-dependent manner (Fig. 7). BFBO inhibited PDE1 activity in its basal state ([IC.sub.50] value was 109.8 [+ or -] 6.5 [micro]g/ml), in the calmodulin-activated state (83.9 [+ or -] 5.3 [micro]g/ml), PDE3 activity (130.5 [+ or -]6.1 [micro]g/ml), PDE4 activity (72.0 [+ or -] 9.7 [micro]g/ml), and PDE5 activity (40.4 [+ or -] 2.6 [micro]g/ml; Fig. 7).

Discussion

The present study shows that the butanol fraction BFBO prepared from B. orthobotrys roots inhibited in a concentration-dependent manner contractile responses in rings without endothelium induced either by KC1, Ca[Cl.sub.2] or U46619. Furthermore, BFBO induced similar concentration-dependent relaxations of coronary artery rings with and without endothelium. Interestingly, BFBO potentiated mainly cyclic AMP- and cyclic GMP-mediated vasorelaxation in rings without endothelium, while having little effect on those induced by endothelium-dependent vasorelaxing agonists and by activators of potassium channels at the vascular smooth muscle. The fact that BFBO was slightly but significantly more effective than the ethyl acetate fraction and the aqueous residue indicates that active compounds are present in all fractions and in particular in the butanol fraction.

The endothelium-independent vasodilators including atrial natriuretic peptide and SNP, forskolin and isoproterenol are well known to induce relaxation respectively by activating the cyclic GMP- and cyclic AMP-dependent relaxing pathways in the vascular smooth muscle (Eckly et al., 1994; Eckly-Michel et al., 1997; Romas et al., 1991; Schoeffter et al., 1987). Indeed, atrial natriuretic peptide is a potent vasorelaxant which activity is mediated by activation of the membrane-bound guanylyl cyclase (Winquist et al., 1984), while SNP, a NO donor, induces vascular smooth muscle relaxation mainly through the activation of the soluble guanylyl cyclase, both pathways leading to the subsequent increase in cyclic GMP levels. The cyclic GMP-dependent relaxing pathway has been suggested to lead to a decrease in the intracellular [Ca.sup.2+] concentration (Eckly-Michel et al., 1997), possibly due to activation of either the sarcoplasmic reticulum [Ca.sup.2+]-ATPase, the plasma membrane [Ca.sup.2+]-ATPase (Cohen et al., 1999), the [Na.sup.+]/[K.sup.+]-ATPase or different potassium channels (Zhao et al., 1997), and possibly also to the reduced opening of L-type [Ca.sup.2+] channels (Ruiz-Velasco et al., 1998), all these mechanisms are known to contribute to decrease the intracellular free calcium concentration.

Similarly, isoproterenol is well known to cause vasorelaxation via activation of [beta]-adrenergic receptors leading to the activation of the cyclic AMP-dependent relaxing pathway (Xu et al., 2006), and forskolin to the direct activation of adenylyl cyclase at the vascular smooth muscle. The cyclic AMP-dependent relaxing pathway is mediated by the activation of protein kinase A promoting the phosphorylation of several proteins involved in inhibition of [Ca.sup.2+] influx across the plasma membrane, inhibition of myosin light chain kinase (Conti and Adelstein, 1981), increase of [Ca.sup.2+] uptake into the sarcoplasmic reticulum and activation of [Ca.sup.2+] efflux from the smooth muscle cells (Scheid and Fay, 1984).

The cyclic nucleotide PDEs, which hydrolyze cyclic AMP and cyclic GMP, play a pivotal role in intracellular signaling cascades (Lugnier, 2006). According to Lorenz and Wells (1983), and Schoeffter et al., (1987), PDE inhibitors potentiate the relaxant effects of isoproterenol, forskolin, and SNP as observed in the present study. In order to evaluate the possibility that BFBO inhibits PDE activity, the direct inhibitory effect of BFBO was determined using purified vascular smooth muscle isoforms of PDEs (Keravis et al., 2005; Lugnier et al., 1986). Vascular smooth muscle has four major types of PDE: PDE1 which mainly hydrolyzes cyclic GMP and is activated by calcium/calmodulin, PDE3 which mainly hydrolyzes cyclic AMP but can also hydrolyze cyclic GMP (since it has a higher affinity for cyclic GMP than for cyclic AMP, and cyclic AMP hydrolysis is competitively inhibited by cyclic GMP), PDE4 which specifically hydrolyzes cyclic AMP, and PDE5 which specifically hydrolyzes cyclic GMP (Lugnier, 2006). BFBO significantly inhibited basal PDE1 and calmodulin-activated PDE1, PDE3, PDE4, and PDE5. It should be noticed that BFBO similarly inhibited basal PDE1 and calmodulin-activated PDE1 supporting the hypothesis that BFBO might inhibit specifically PDE1 by acting at the level of its catalytic site rather than on the calcium/calmodulin-dependent activation process. The present findings provide direct evidence that BFBO is able to inhibit

purified vascular PDEs indicating a direct effect on various PDE isozymes. The relaxant effect of BFBO was not dependent on the presence of endothelium, showing a similar smooth muscle relaxing effect in rings with and without endothelium. A similar effect was also observed in response to non-selective PDEs inhibitors, such as caffeine and theophylline (Lo et al., 2005; Pauvert et al., 2002). Moreover, PDE1 and PDE3 inhibitors have been shown to induce vasorelaxation in an endothelium-independent manner (Noguera et al., 2001) whereas PDE4 and PDE5 inhibitors induced relaxation in an endothelium dependent manner (Komas et al., 1991). In addition, previous studies have shown that increases in the cyclic GMP level cause inhibition of PDE3, thereby, increasing the cyclic AMP level (Eckly and Lugnier, 1994; Lugnier and Komas, 1993). Altogether, the present findings suggest that the inhibition of vascular tone of BFBO is likely to result from an increased cyclic GMP level following inhibition of PDE1 and PDE5, and of the cyclic AMP level subsequent to the inhibition of PDE3 and PDE4.

The analysis of BFBO by LC-ELSD-UV-MS revealed the presence of four main isoquinoline alkaloids. Compound 3 was identified as berberine, while compounds 2 and 4 were assigned as protoberberine alkaloids with MW of 338.0 and 352.1 respectively. Compound 1 was identified as a bisbenzylisoquinoline with a MW of 608.2. Berberine 3 was the most abundant alkaloid in the extract and was present at 15.0% in BFBO. A previous study identified the presence of several other isoquinoline alkaloids in B. orthobotrys roots including pakistanine, 1-O-methylpakistanine, pakistanamine, aromoline, chitraline, kalashine and oxyacanthine (Hussain et al., 1981). Moreover, berberine has been shown previously to decrease blood pressure in vivo and to induce vasorelaxation of rat aortic and mesenteric artery rings (Kang et al., 2002; Ko et al., 2000). Altogether, the findings suggest that B. orthobotrys roots extract is likely to exert beneficial effect on vascular tone, at least in part, through the inhibition of vascular PDEs.

In conclusion, the butanol fraction of B. orthobotrys roots is able to inhibit vascular tone by acting directly at the vascular smooth muscle most likely through activation of the cyclic AMP- and cyclic GMP-dependent relaxing pathways subsequent to the inhibition of different vascular isoforms of PDEs, with a preferential action on PDE5. The active molecules of the B. orthobotrys extract appear to be present to a greater extent in the butanol fraction, which has been shown to contain simple benzylisoquinoline alkaloids such as berberine, and a bisbenzylisoquinoline. The ability of B. orthobotrys root extract to strongly inhibit vascular tone may contribute to explain its potent antihypertensive action in vivo.

http://dx.doi.org/10.1016/j.phymed.2016.04.005

ARTICLE INFO

Article history:

Received 5 January 2015

Revised 4 April 2016

Accepted 5 April 2016

Conflict of interest

The authors have no conflicts in interest to disclose.

Acknowledgments

Authors are thankful to the Higher Education Commission of Pakistan for providing a fellowship to Alamgeer and to Helene Justiniano for PDE assays. This work was supported, in part, by the International Research & Development Program of the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning of Korea (STAR 2015 Hubert Curien Partnership between France and Korea; number 32179WA).

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Alamgeer (a,b), P. Chabert (a), M.S. Akhtar (c), Q, Jabeen (c), J. Delecolle (d), D. Heintz (d), E. Garo (e), M. Hamburger (e), C. Auger (a), C. Lugnier (a), H.-J. Kim (f), M.-H. Oak (f), V.B. Schini-Kerth (a) *

(a) UMR CNRS 7213, Laboratory of Biophotonics and Pharmacology, Faculty of Pharmacy, University of Strasbourg, Illkirch, France

(b) Faculty of Pharmacy, University of Sargodha, Pakistan

(c) Faculty of Pharmacy and Alternative Medicine, Islamia University of Bahawalpur. Pakistan

(d) IBMP, UPR 2357, University of Strasbourg, France

(e) Department of Pharmaceutical Sciences, University of Basel, Switzerland

(f) College of Pharmacy, Mokpo National University, Muan-gun, Jeollanamdo 534-729, Republic of Korea

Abbreviations: ANP, atrial natriuretic peptide; BFBO, butanol fraction of Berberis orthobotrys; Cyclic GMP cyclic guanosine 3',5'-monophosphate; Cyclic AMP, cyclic adenosine 3',5'-monophosphate; eNOS, endothelial nitric oxide synthase; PDE, phosphodiesterase; oxLDL, oxidized low density lipoprotein; EGTA, ethylene glycolbis(2-aminoethylether)-N,N,N,,N,-tetraacetic acid; SNP, sodium nitroprusside; 1EB10, l-ethyl-2-benzimidazolinone; L-NA, N-co-nitro-L-arginine; EFBO, ethyl acetate fraction of Berberis orthobotrys; ARBO, aqueous residue of Berberis orthobotrys; 1-Kc, intermediate-conductance calcium-activated potassium channels.

* Corresponding author. Tel.: +33 3 68 85 41 27. Fax: +33368854313. E-mail address: valerie.schini-kerth@unistra.fr (V.B. Schini-Kerth).

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Title Annotation:Original Article
Author:Alamgeer; Chabert, P.; Akhtar, M.S.; Jabeen, Q.; Delecolle, J.; Heintz, D.; Garo, E.; Hamburger, M.;
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9PAKI
Date:Jul 15, 2016
Words:5551
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