Articulo Unidad2 Sanchez-Mendoza

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JPP 2008, 60: 229236

2008 The Authors

Received September 8, 2007

Accepted October 4, 2007

DOI 10.1211/jpp.60.2.0012

ISSN 0022-3573

Facultad de Qumica,

Departamento de Farmacia,

Universidad Nacional Autnoma

de Mxico, Mxico D.F., 04510,

Mxico

Mara Elena Snchez-Mendoza,

Andrs Navarrete

Escuela Superior de Medicina,

Instituto Politcnico Nacional.

Plan de San Luis y Daz Mirn,

Colonia Santo Toms,

Delegacin Miguel Hidalgo

11340, Mxico D.F., Mxico

Mara Elena Snchez-Mendoza,

Carlos Castillo-Henkel

Correspondence: A. Navarrete,

Facultad de Qumica,

Departamento de Farmacia,

Universidad Nacional Autnoma

de Mxico, Ciudad Universitaria,

Coyoacn 04510, Mxico D.F.,

Mxico. E-mail:

[email protected]

Ackowledgments and funding:

The authors of this work

acknowledge Dr Jess Arrieta,

(Escuela Superior de Medicina)

and Ernestina Cedillo(Universidad de Chapingo) for

the collection of plant material.

Also thanks go to Dr Benito

Reyes (Universidad de Chapingo)

for the berberine X-ray registers.

This work was supported by

grants of Direccin General de

Asuntos del Personal Acadmico

DGAPA-UNAM IN201506 and

CONACYT (Proyecto C01-018).

M. E. Snchez-Mendoza

acknowledges fellowship from

Beca Institucional and Programa

Institucional de Formacin deInvestigadores (PIFI), IPN, to

carry out her graduate studies.

This work was taken, in part,

from the PhD research work of

M. E. Snchez-Mendoza.

Relaxant action mechanism of berberine

identified as the active principle of Argemone

ochroleuca Sweet in guinea-pig tracheal

smooth muscle

Mara Elena Snchez-Mendoza, Carlos Castillo-Henkel and

Andrs Navarrete

Abstract

In this study we investigated the relaxant effect of the aerial parts of Argemone ochroleuca

(Papaveraceae), which is used in Mexican traditional medicine for the treatment of various respi-

ratory diseases such as cough, bronchitis and asthma. The alkaloid berberine was identified as one

of the active relaxant principles EC50 = 11850391M) in the dichloromethane extract of A.

ochroleuca (EC50= 7803215g mL1 with 9512356% of relaxation). Berberine concentration-

dependently relaxed the carbachol-induced precontractions but not histamine- or KCl-induced

precontraction. The relaxant effect of berberine was unaffected by the presence of propranolol

3M), glibenclamide 10M) or ODQ 10M). However, 25-dideoxyadenosine 10M) blocked the

log concentrationresponse curves of berberine. On the other hand, berberine produced a leftward

shift of the log concentrationresponse curves of isoproterenol, forskolin and nitroprusside. Addi-

tionally, berberine produced a parallel rightward shift of the concentrationresponse curve of carba-

chol in a competitive manner with a pA2 of 3870045. The above results suggest that the relaxant

effect of berberine on tracheal muscle is due to its antagonistic effect on muscarinic acetylcholine

receptors.

Introduction

In Mexican traditional medicine several Argemone species are locally known as Chicaloteand the aerial parts of these species are used in the treatment of respiratory ailments andocular disorders (Argueta & Cano 1994). One of these species, Argemone ochroleucaSweet (Papaveraceae), is used as remedy for cough, bronchitis, asthma, cataract removaland ocular allergy and for its sedative, anticonvulsive, tranquilizing, anti-diabetic andantispasmodic properties (Argueta & Cano 1994). Previous chemical investigations onthis species have revealed the presence of alkaloids, such as sanguinarine, chelery-thrine, protopine, berberine, dihydrosanguinarine, dihydrochelerytrine, -allocryptopine,heleritrine, cheilantifoline, scouletrine, reticuline and coptisine (Haisova & Slavik 1973;Israilov et al 1986; Chelombitko & Nazarova 1988; Takken et al 1993). Recently,Fernandez et al (2005) reported that the flavonoid isoquercitrin, isolated from Argemone

platyceras, inhibits carbachol- and leukotriene-D4-induced contraction in guinea-pigairways. However, there are no additional reports concerning the pharmacological effectsof Argemone species on airways smooth muscle. Therefore, the present study was

undertaken to evaluate the claimed asthma-relieving effect of A. ochroleuca, usingthe guinea-pig isolated tracheal rings as an experimental model, to identify, throughbioassay-guided fractionation, the active principle(s) and also to investigate the trachealrelaxant action mechanism of the main active ingredient, berberine, in leaves and flowers of

A. ochroleuca.


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230 Mara Elena Snchez-Mendoza et al

Materials and Methods

Drugs

Acetylcholine chloride, histamine dihydrochloride, carbacholchloride, dl-propranolol hydrochloride, -isoproterenolhydrochloride, forskolin, nitroprusside, glibenclamide,

ODQ (1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one), 2 5-dideoxyadenosine and ipratropium bromide were purchasedfrom Sigma Chemical Co. (St Louis, MO). The other reagentsused were of analytical grade. Glibenclamide, ODQ andforskolin were dissolved in dimethyl sulfoxide (DMSO)and diluted with water. The other drugs were dissolved indistilled water. The extracts and berberine were suspended indistilled water with traces of Tween 80. The final concentra-tion of DMSO or Tween 80 was less than 0.1% and did notsignificantly affect the trachea response.

Plant material

Argemone ochroleuca Sweet aerial parts were collected inTexcoco, Estado de Mexico, Mexico during June of 2005. Thespecimens were identified by botanists from the UniversidadAutnoma Chapingo Herbarium and a voucher sample wasdeposited (No. 17559).

Extraction and isolation

The plant aerial part, including branch, leaves and flowers butnot seeds, was air-dried at room temperature under shadow.

After grinding, 5 kg of plant was extracted at room temper-ature with hexane (3 30L, 48h each), then with CH2Cl2(330 L, 48 h each) and finally with MeOH (330 L, 48 heach); evaporation of the solvents in vacuum gave 85g,33g and 95g of syrupy residues, respectively. The crudeextracts were tested for activity at different concentrations177316 g mL1 in the trachea ring assay. The CH2Cl2extract showed the most relaxant activity. This extract (32 g)was subjected to percolation over silica gel (0.0630.200mmmesh, 200g) using a step gradient of hexane (1.5L; F1),hexaneCH2Cl2 (1:1, 1.5 L; F2), CH2Cl2 (1.5 L; F3), EtOAc(1.5 L; F4) and MeOH (1.5 L; F5) (Figure 1). These fractions

were tested for activity 177316 g mL

1 in the trachealring assay. Fraction F5 was the most active and showed amajor yellow component in TLC. Ten grams of F5 weretaken for a subsequent fractionation on a silica gel column(100g) using CH2Cl2 F

1 EtOAc F2, EtOAcMeOH(95:5, F3), EtOAcMeOH (4:1, F4) and finally MeOH F5as eluents (Figure 1). The fraction F4 (4.7g) was the mostactive fraction and gave a yellowish powder, which was puri-fied from MeOHCH2Cl2 to afford a yellow solid (700 mg,0.014%), melting point 142C (with decomposition). Thissolid was identified as berberine (Figure 2) by IR, NMR andmass spectrometric analysis (Blask et al 1988).

Berberine: IR (KBr): max =3434, 1572, 1505, 1386 cm1.FAB+MS m/z= 336 100; 1H NMR (DMSO-d6): = 980(1H, s, H-8), 8.84 (1H, s, H-13), 8.14 (1H, d, J= 93 Hz, H-11), 7.98 (1H, d, J= 93 Hz, H-12), 7.74 (s, H-1), 7.05 (s, H-4), 6.12 (2H, s, OCH2O), 4.87 (2H, t, J= 55 Hz, H-6), 4.05(3H, s, CH3O), 4.03 (3H, s, CH3O), 3.18 (2H, t, J= 55Hz,

Berberine (700mg, 0.014%)

Hexane extract(85g)

Percolation Residue

F1

1.4g

F2

2.6g

Plant(5kg)

Residue

CH2Cl2 extract(33g)

MeOH extract(95g)

Residue

F3

2.2g

F4

14.5g

F5

10.1g

F10.37g

F21.4g

F32.0g

F44.7g

F51.5g

Yellowish powder

Crystallization

Figure 1 Bioassay-guided fractionation ofArgemone ochroleuca.

C14

02

01 C2

C1

C3C4

C4A

C13B

C13A

C5

C6

06

C17

05

07B

07A

N7

C8

C9

03

04

C15

C10C11

C16

C12

C13

C12A

C8A

Figure 2 Perspective drawing of the X-ray structure of berberinebicarbonate.


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Smooth muscle relaxant effect of berberine from Argemone ochroleuca 231

H-5). 13C NMR (DMSO-d6): = 1507 (C-10), 150.1 (C-3),148.0 (C-2), 145.7 (C-8), 143.9 (C-9), 137.8 (C-13a), 133.3(C-12a), 131.0 (C-4a), 127.0 (C-11), 123.9 (C-12), 121.7 (C-8a), 120.7 (C-13b), 120.4 (C-13), 108.7 (C-4), 105.7 (C-1),102.2 OCH2O 622 C10-OCH3 573 C9-OCH3 55.6(C-6), 26.6 (C-5). These data corresponded with those in theliterature (Blask et al 1988).

Unequivocal identification of berberine structure, asberberine bicarbonate C21H19NO7 in this plant, was deter-mined by X-ray diffraction analysis (Figure 2). X-ray datawere collected on an Enraf-Nonius CAD4 with a wave-length of 0.71073 (molybdenum) using a yellow crystalgrown from CH2Cl2MeOH. The structure was solved andrefined by using SHELXL97. Data collection was conductedat 293K on a monoclinic crystal, P21/n a= 7034018 ,b= 157602 c= 162952 = 10018512o V=177796 3 Z = 4 R1 = 49% wR2 = 115% GOF =0998.

Animals and guinea-pig trachea preparation

Male guinea-pigs, 300450 g, bred under conventional condi-tions and fed with standard diet (Purina Pellets) and drinkingwater, were used. Procedures involving animals and theircare were conducted in conformity with the Mexican Offi-cial Norm for Animal Care and Handing (NOM-062-ZOO-1999, Especificaciones Tcnicas para la Produccin, Cuidadoy Uso de Animales de Laboratorio) and in compliancewith international rules on the care and use of laboratoryanimals.

The guinea-pigs were euthanized by intraperitoneal injec-tion of an overdose of sodium pentobarbital 95mg kg1.The chest was opened to obtain the trachea, which wastransferred to a dish containing warm Krebs solution.After removal of excess connective tissue and fat thetrachea was divided into eight small rings of about 2-mmlength containing two or three cartilaginous segments. Eachtracheal ring was hung between two hooks inserted into thelumen, and placed in a 10-mL organ bath containing Krebssolution with composition (in mm): NaCl 118, KCl 4.7,NaH2PO4 12 MgSO47H2O 12 CaCl22H2O 25 NaHCO325 and glucose 11.1. This solution was maintained at 37C

and bubbled with 5% CO295% O2. Isometric tensionwas recorded through an eight-channel Biopack Systempolygraph MP100 via a Grass FT 03E force transducer. Thedata were digitalized and analysed by mean of software fordata acquisition (Acknowledge 3.7.3). Tissues were placedunder a resting tension of 1.5 g and allowed to stabilize for60 min and they were washed with fresh Krebs solution at15-min intervals before starting the experiments. After astabilization period the tracheal rings were contracted withacetylcholine chloride 30 m twice at 30-min intervals andwashed after stimulation with fresh Krebs solution. Thirtyminutes after the tissues were contracted with carbachol

chloride 3 m, cumulative additions of crude extracts(17.7, 31.6, 56.2, 100, 177 or 316 g mL1), fractionsor reference drugs were made to the bath to yield therequired tracheal relaxant effects and allowed to reach asteady state at each concentration. The relaxant potencies ofcrude extracts or reference drugs were expressed as EC50

(half-maximal effective concentration). In another set ofexperiments the tissues were precontracted with carbachol3 m, histamine 30 m or KCl (30mm) and cumulativeconcentrationresponse curves for dichloromethane extractor berberine were constructed.

To evaluate the participation of ATP-sensitive potassiumchannel and -adrenergic receptors, glibenclamide 10 m,

propranolol 3 m, or their vehicles, were incubated for5 min, after the carbachol 3 m-precontracted tissue reacheda steady state, then berberine was added at different concen-trations (26, 47, 84, 151, 268 476 m). Similarly, 2 5-dideoxyadenosine 10 m or ODQ 10 m were added5min before the addition of berberine (26, 47, 84, 151,268, 476 m), or its vehicle, after carbachol 3 m-inducedprecontraction reached a steady state. To observe the effectof berberine (151, 268, 476 m) on the relaxant responsesof isoproterenol (1010 to 316 106m), forskolin (108

to 106 m) and nitroprusside (1010 to 105m) to carbachol3 m-induced precontractions, trachea rings were incubatedwith berberine after the precontraction reached a steady statefor 5 min before the first addition of relaxant drug.

To determine the antagonistic effects of berberineagainst the contractions elicited by carbachol, cumula-tive concentrationresponse curves for carbachol (1010 to102m) were constructed in the absence or in the pres-ence of different concentrations of the berberine, as follows:after the tissues were incubated with different concentra-tions of berberine bicarbonate (151, 268, 476m), iprat-ropium bromide (177103 316103 562103 1102 m, used as reference drug) or their vehicles (distilled

water with Tween 80 in traces, for berberine and distilledwater for ipratropium) for 15 min, carbachol was cumula-tively added into normal Krebs solution. The antagonisticpotentials of berberine and ipratropium were expressed aspA2 values.

Data analysis

The EC50 (for relaxant effect and antagonistic effect experi-ments) values were calculated by linear regression (Tallarida2000). All the values are shown as mean s.e.m. of atleast six experiments. The differences among these values

were statistically calculated by one-way analysis of vari-ance, and then determined by Dunnetts t-test (Montgomery1991). P < 005 was considered statistically significant. ThepA2 value was calculated according to the Schild equation:log A/A1 = log B logKB, where A is the EC50 ofcarbachol in absence of the antagonist, A is the EC50 ofcarbachol in the presence of antagonist, KB is the affinityconstant for the antagonist and B is the concentration (m) ofthe antagonist. The value of pA2 is the abscissa to the originwhen A/A = 2 (Tallarida & Murray 1987).

Results

Hexane, dichloromethane and methanol extracts obtainedfrom aerial parts without seeds of Argemone ochroleuca,concentration-dependently P < 005 relaxed the carbachol3 m-induced precontractions (Figure 3); the values of


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maximal relaxation were 5913 486 9512 356 and3187 376% respectively. The dichloromethane extractwas also the most potent relaxant extract EC50 = 7803215 g mL1. This extract showed a more potent relaxanteffect on the carbachol 3 m-induced precontrations thanwhen the precontractions were elicited by 30 m histamine(386 54% of relaxation), but it was unable to relax theguinea-pig trachealis muscle when it was precontracted with30 mm KCl. These results encouraged us to perform thebio-guided fractionation of the dichloromethane extract of

Argemone ochroleuca. The scheme of fractionation and iden-tification of the tracheal smooth muscle relaxing compoundfrom this extract is showed in Figure 1. After percola-tion of dichloromethane extract with different solvents, theactivity was found in two fractions: one obtained with EtOAc(F4, Figure 1; EC50 = 1439 73 g mL1) and the otherwith methanol (F5, Figure 1; EC50 = 110258 g mL1).The methanol fraction was used for further fractionation

using open column chromatography. The fraction eluted withEtOAcMeOH (4:1), obtained as a yellowish semi-solid,showed the highest activity (F4, Figure 1; EC50 = 5600415 g mL1). The major component of this mixture wasprecipitated from MeOHCH2Cl2 (1:1) as a yellow solid

Extract log (g mL1)

1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6

Contraction(%)

20

0

20

40

60

80

100

120

Figure 3 The relaxant effects of hexane (), dichloromethane () andmethanol () extracts of A. ochroleuca (177316 g mL1) or control() on carbachol 3 m-induced precontraction in guinea-pig trachea.Each point value represent the mean s.e.m., n = 6. P < 005, vsrespective control at the same time; Dunnetts t-test after analysis ofvariance test.

(700 mg, 0.014%), whose melting point began with decom-position at 142C, and it was identified as berberine bicar-bonate (Figure 2) by comparison of its spectral data (IR, 1HNMR, 13C NMR and MS) with the literature data (Blasket al 1988) and by X-ray diffraction analysis. This purecompound concentration-dependently relaxed the carbachol-induced precontractions, but it was unable to relax theguinea-pig trachealis muscle when it was precontracted with30 m histamine (25% relaxation only; P > 005 vs control)or 30mm KCl. The EC50 value for berberine bicarbonatewas 11850391 m 468155 g mL1 while the EC50value for ipratropium bromide, used as reference drug, was00032000015 m.

The relaxant effect of berberine was not affected bypretreatment with glibenclamide 10 m, an ATP-sensitivepotassium-channel blocker. Also, the -adrenoceptor antag-onist propranolol at 3 m did not antagonize the relaxanteffect of berberine, a concentration that significantly blocked

the effect of isoproterenol (316 1010 to 106m) to relaxcarbachol-induced precontractions. In the same form ODQ10 m, a soluble guanylate cyclase inhibitor, blockedthe effect of nitroprusside (1010 to 105m), but did notaffect the log concentrationresponse curves of berberine.However, 2 5-dideoxyadenosine 10 m, an adenylatecyclase inhibitor, blocked the log concentrationresponsecurves of berberine, in the same manner that it blockedthe effect of forskolin (1 108 to 177 106 m) to relaxcarbachol-induced precontractions (Table 1).

On the other hand, berberine significantly reduced the

EC50 values of isoproterenol, forskolin and nitroprus-side (Table 2).In other experiments, berberine produced a parallel

rightward shift of the concentrationresponse curve ofcarbachol EC50 = 010 0014 m in a competitivemanner at concentration of 151 m EC50 = 034 0099 m 268 m EC50 = 218 0388 m and 476 m(EC50 = 559 136 m; Figure 4A), with a pA2 of387 0045. Ipratropium bromide also produced a parallelrightward shift of the concentration response curve ofcarbachol (Figure 4B), but this displacement was morepotent than berberine, showing a pA2 of 905 0057

(Figure 5).

Table 1 EC50 m values of berberine and forskolin against carbachol3 m-induced precontractions in guinea-pig tracheal smooth muscle inthe absence and presence of 2,5-dideoxyadenosine

2,5-Dideoxyadenosine

m

Berberine

bicarbonate EC50

m

Forskolin EC50

m

0 11850391 0262004510 194887605 09470121

Values are presented as the means s.e.m., n= 6 (where n is the numberof experiments). P < 005, vs respective control; Students no-pairedt-test.


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Table 2 EC50 m values of isoproterenol, forskolin and nitroprusside against carbachol 3 m-inducedprecontractions in guinea-pig trachealis in the absence and presence of berberine bicarbonate

Berberine (m) Isoproterenol (m) Forskolin (m) Nitroprusside (m)

0 006400069 02190018 08160149151 0008100036 003800038 001400043

268 0002700003 001700082 0008600022

476 0000900002 0011700028 0001900007

Values are presented as the means s.e.m., n= 6 (where n is the number of experiments). P < 005 versusrespective control; Dunnetts t-test after analysis of variance test.

Carbachol log (M)

10 8 6 4 2

Contraction(%)

20

0

20

40

60

80

100

120

Carbachol log (M)

10 8 6 4 2 0

Contraction(%)

20

0

20

40

60

80

100

120A B

Figure 4 The inhibitory effects of berberine bicarbonate (A: 151 m

(), 268 m

(), 476 m

()) or ipratropium bromide (B: 177103

m

(), 316103 m (), 562103 m (), 1102 m ()) on the contractile response of carbachol () on guinea-pig trachea. Each point valuerepresents the mean s.e.m. of 59 experiments.

Discussion

In this study, we found strong evidence that Argemoneochroleuca elicits a relaxant effect in tracheal smooth muscle.The relaxant EC50 value for the dichloromethane extract ofaerial parts of this plant was significantly lower than the

EC50 values obtained for the methanol and hexane extracts(Figure 3). This suggested that the dichloromethane extractcontains active relaxant compounds. These results provideexperimental support for the use of this plant as a remedyfor asthma in Mexican traditional medicine (Argueta & Cano1994). In addition, from the bioassay-guided fractionation

of the dichloromethane extract, berberine bicarbonate wasisolated as one of the active principles responsible for therelaxant effect.

Berberine has been reported before in Argemoneochroleuca and other Argemone species (Haisova &Slavik 1973; Israilov et al 1986; Chelombitko & Nazarova

1988; Takken et al 1993). Berberine has been reported tohave several pharmacological actions, including anti-tumour(Iizuka etal 2003), anxiolytic (Peng etal 2004), anal-gesic (Yesilada & Kpeli 2002), in-vitro antioxidant poten-tial (Shirwaikar et al 2006), acetylcholinesterase inhibition(Ingkaninan et al 2006) and anti-diarrhoeal activity. Further-


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3.2

0.0 0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

0.5

1.0

1.5

2.0

2.5

3.0

3.3 3.4 3.5 3.6 3.7 3.8 3.9

Berberine bicarbonate log (M)

Log((A/A)1)

Log((A/A)1)

AB

a=18.410

b=4.745

pA2=3.870.045

a=17.291

b=1.910

pA2=9.050.057

7.8 8.0 8.2 8.4 8.6 8.8

Ipratropium log (M)

Figure 5 Schild plot for berberine bicarbonate (A) or ipratropium bromide (B).

more, berberine has cardiovascular effects (Lau et al 2001)and in isolated vascular preparations causes relaxation (Koetal 2000).

The effect of berberine on the isolated guinea-pig trachealismuscle has been studied by Abdel-Haq et al (2000) and itwas found that the berberine relaxant effect is not antago-nized either by the -blocker timolol or by xanthine aminecongener, an adenosine antagonist. In our study, the cumu-lative concentrationrelaxing response curve of berberine

to carbachol-induced precontraction was not modified bypropranolol 3 106m, a non selective -adrenoceptorblocker, suggesting that its relaxant effect is not through theactivation of -adrenoceptors. These results are in agree-ment with those obtained by Abdel-Haq et al (2000). Despiteberberine potentiating isoproterenol relaxation (Table 2),it is not possible to consider that berberine acts on -adrenoceptors, because it has been reported that the relaxanteffect of berberine is not antagonized by timolol (Abdel-Haq etal 2000) and propranolol (this study), two very wellknown -adrenoceptor antagonists. Hence the synergisticeffect should be due to other mechanisms.

It is known that the opening of K+ATP channels mediatesthe relaxation of isolated guinea-pig trachea (Thirstrup et al1997). To investigate whether K+ATP channels are involvedin the guinea-pig trachea relaxation response to berberine,the preparation was pretreated with glibenclamide, a K+ATP-channel blocker; however, glibenclamide did not affect its

relaxant response, suggesting that berberines relaxant effectis not through the opening of ATP-sensitive potassiumchannels. Several NO-releasing compounds, such as sodiumnitroprusside and endogenous NO, activate soluble guanylatecyclase (sGC) that increases cyclic GMP (cGMP) and relaxesairway smooth muscle (Hall 2000). Berberine produced aparallel leftward shift of the log concentrationresponsecurves of nitroprusside to carbachol 3 m-induced precon-tractions of the trachealis, and significantly reduced the EC50

values (Table 2). However the tracheal relaxant effect wasnot inhibited by pretreatment with the sGC inhibitor ODQ.These results suggest that berberine induced relaxation of theguinea-pig trachea by a mechanism of action that is unliketo the activation of sGC/cGMP pathways, although it hasbeen suggested that endothelial NO is likely different to beinvolved in the berberine vasodilating activity (Chiou et al1998; Ko et al 2000).

The involvement of the activation of adenylatecyclase/cyclic AMP (AC/cAMP) pathways in therelaxant action induced by berberine was drawn from the factthat berberine produced a significant reduction of the EC50

of forskolin (Table 2), an activator of adenylate cyclase(Seamon et al 1983). Furthermore, in the presence of theadenylate cyclase inhibitor 2 5-dideoxyadenosine (Sabouniet al 1991), the relaxant effect of berberine was reducesignificantly (Table 1). This experiment suggested that therelaxant effect of berberine is in part via the stimulation


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of adenylate cyclase. However, more experimental data arenecessary to confirm this postulation.

On the other hand, berberine concentration-dependentlyrelaxed the carbachol-induced precontractions but nothistamine- or KCl-induced precontraction. The CH2Cl2extract of A. ochroleuca was also able to relax moreefficiently the carbachol-induced precontractions than

histamine-induced precontraction and it was unable to relaxthe KCl-induced precontractions. These results suggested thatcholinergic receptors are involved in the relaxant effect ofberberine and the extract. Considering the last observation,an additional experiment was designed to investigate theanticholinergic effect of berberine, using as reference drugipratropium, a non-selective muscarinic receptor antagonistwith clinical uses in the treatment of asthma and chronicobstructive pulmonary disease (Eglen et al 1999). In theseexperiments, berberine produced a clear parallel rightwardshift of the concentrationresponse curve of carbachol in acompetitive manner (Figure 4A) with a pA

2of 3870045

(Figure 5A). The antagonistic effect of berberine was consid-erably lower than the antagonistic effect of ipratropium, witha pA2 value of 905 0057 (Figure 5A). These experi-ments provided additional support that the relaxant effectof berberine is through an antagonistic effect on muscarinicreceptors. This experiment is in agreement with the antimus-carinic effect reported for berberine in other tissues (Tsai &Ochillo 1991). It has been established that stimulation ofmuscarinic M3 receptors mediates smooth muscle contractionand although 5080% of muscarinic receptors on airwayssmooth muscle are M2 type receptors, the function of these

receptors is still unclear. Stimulation of M2 muscarinic recep-tors in airways smooth muscle leads to inhibition of activa-tion of adenylate cyclase, which results in an acute loweringof intracellular cAMP levels (Fryer & Jacoby 1998). Otherstudies provide evidence of the contractile role of the M 2receptor when M3 muscarinic receptors are blocked (Ehlert2003; Walker et al 2004). Other reports have suggested thatprolonged stimulation of muscarinic M2 receptors may sensi-tize adenylate cyclase and thus the M2 receptor presenton airway smooth muscle may play two roles: an acuteinhibition of adenylate cyclase, and in the long term, dueto homoeostatic regulation, the upregulation of adenylate

cyclase expression, thus counteracing the action of muscarinicagonists at M3 receptors coupled to phospholipase C (Hall2000; Michal et al 2001; Mistry et al 2005). The resultsobtained in this study provide a key point for the identifica-tion of the subtypes of muscarinic receptor associated withthe action mechanism of the relaxant effect of berberine.

The antagonist muscarinic effect of Argemone platyceras,another Argemone species known also as Chicalote, hasalready been reported (Fernndez et al 2005). However, theflavonoid isoquercitrin was identified as the active principle(Fernndez et al 2005), and that work did not report the pres-ence of berberine.

It has been reported that the in-vivo administration ofberberine induces IL-12 production in macrophages, and ithas been suggested that this response is importantly involvedin the immunotherapy of asthma (Kim et al 2003). Thus, theidentification of berberine as the active relaxant principle in

A. ochroleuca has a relevant role, because this effect can be

related to the traditional use of this medicinal plant by theMexican people in the treatment of asthma.

Finally we cannot discount the presence of other activerelaxant compounds in A. ochroleuca, because in the frac-tionation process we obtained two active fractions (F4 andF5, Figure 1) with a similar level of activity (EC50 = 143973 g mL1 for fraction F4 and EC50= 110258 g mL1

for fraction F5), although these values did not match with theabundant presence of berberine in F5.

Conclusion

In conclusion, this study demonstrated that Argemoneochroleuca produces a clear relaxant effect on guinea-pigtrachealis muscle, where one of the active principles wasidentified as the alkaloid berberine. This compound has theability to produce a weak antagonistic effect on the muscarinicreceptors, and its action mechanism involves the activa-tion of adenylate cyclase/cyclic AMP (AC/cAMP) pathways.

Finally, this study provides experimental support for the tradi-tional use of this medicinal plant in the treatment of asthma.

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