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Effects of Genistein, An Isoflavone Isolated from Genista trident ata, on Isolated Guinea-Pig ileum and Guinea-Pig Heal Myenteric Plexus M.D. Herrera'3. E. Marhuenda', andA. Gibson2 Departamento de Farmacia y Tecnologia Farmaceütica. Facultad de Farmacia. Universidad de Sevilla. Sevilla, Spain 2 King's College London. Pharmacology Department. University of London, London, U.K. Address for correspondence Received: September 30. 1991

The inhibitory action of the major con-

stituent of Genista tridentata L. (Papilionaceae), 4',5,7-trihydroxyisoflavone (genistein), on contractions induced by agonists and electrical field stimulation of

Genistein was isolated according to the published procedures (9) from Genista tridentata L. (Papilionaceae) collected in Huelva (Spain) during the flowering stage of the plant.

Pharmacological method:

smooth muscle was analysed. Genistein inhibited

In vitro experiment

twitches evoked by electrical-stimulation of strips of guinea-pig ileum with an IC50 value of 34 jM. Genistein (34 tM) inhibited contractions of the guinea-pig ileum

The isolated tissue preparations were carried out according to the technique of Magnus (10).

by several agonists in a non-selective, antispasmodic action and had no effect on inhibition of 3H-ACh release from ileal myenteric plexus. Genistein (34.tM) produces an increase in cAMP levels of guinea-pig ileum which resulted in a smooth muscle relaxation which leads us to

think that there must be a blockade of its phosphodiesterase. Key words

Genista tridentata, Papilionaceae, genistein, antispasmodic activity.

Isolated guinea-pig ileum Male albino guinea-pigs (300—350g) were sacrified by a blow to the head and the abdominal cavity was opened. A stretch of the mid-ileum was isolated and divided into 2 segments. Preparations were suspended in an organ bath (2 ml) containing Krebs-bicarbonate solution (118mM NaCI; 4.7mM KCI; 2.5mM CaCl2; 1.0mM MgSO4; 1.0mM KH2PO4; 25mM NaHCO3, and 11 mM glucose) at 37 C and bubbled with 95% 02/5% CO2 under a I g resting load. Isometric contractions were recorded by an HSE force transducer coupled to a GRAPHTEC polygraph rec-

order.

(1C50):

Introduction

It is well known that flavonoids have a range of pharmacological effects. In the search of the literature about spasmolytic activity we have found some investigations carried out with these compounds (1, 2). Genistein (4',5,7-trihydroxyisoflavone) is an isoflavonoid reputed for its estrogenic activity (3) and other actions, i.e.,

antifungal (4), hypolipidemic (5), and an inhthitor of tyrosine-specific protein kinases (6). We have started a study of its antispasmodic activity (7) going deeply into its mechanism of action, as this activity is usually associated with flavonoids.

Effect of genistein on field-stimulated ileum After 30 minutes equilibration period, the preparations

were stimulated electrically [0.1 Hz; 70V; 1 ms puls; generated by a GRASS S48 stimulator (11)]. Once twitches became stable, differ-

ent doses of genistein (6.8 x 106M to 1.0 x 104M, dissolved in

ethanol-water, 2: 1) were added accumulatively to the bath solution. The antispasmodic potency was evaluated by calculating the concentration of genistein required to inhibit twitches by 50 % (IC50) (12). Exposure of control preparations to the same volume of

ethanol-water as used to dissolve genistein did not inhibit contractions by twitches. Effect of genistein on acetyicholine and 6-oxoPGE, induced contractions: After equilibration, concentrationeffect curves of acetylcholine (5 x 109M to 2 x i0 M) and 6-oxoPGEI (1—400 ng/ml) were established and their submaximal concentrations were calculated graphically (13). A single concentration of genistein (34 riM) was added to one ileal segment and its ac-

Pharmacological studies of the total isoflavone fraction from some plants of the genus Pueraria (Papilionaceae) and the pure components: daidzein and daidzin (isoflavones) have demonstrated that they showed a papaverin-like spasmolytic action (8).

tivity was assessed by its ability to prevent the contractions induced by a submaximal concentration of acetylcholine (2 x 10-8 M)

or 6-oxo-PGE1 (100 ng/ml). The responses to genistein were calculated as percent of the control values.

Effect of genistein on cyclic nucleotide levels: Segments of ileum (100—125mg wet weight) were weighed and allowed to equilibrate for 15 minutes. Papaverine (20 M) or

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Materials and Methods

Abstract

Planta Med. 58(1992) 315

Effects of Genistein. An Isoflavone Isolatedfrom Genista tridentata genistein (34MM) or nothing for controls were added (5 mm contact time). The tissues were quickly removed (after 5 mm) and added to 1 ml of 6% trichloroacetic acid (TCA) for 10mm on ice. Tissues were minced with scissors and centrifuged for 3 mm. A little later, TCA was extracted using 5 washes with 2.5 ml ofdiethyl ether saturated with water and the remaining aqueous layer was

choline chloride (Sigma). (methyl-3H)-choline chloride (Amersham. U.K.). Hemicholinium-3 (Sigma). papaverine hydrochloride (Hopkin & Williams). 6-oxo-PGE1 (Hopkin & Williams).

Statistics All values were expressed as mean SEM. The

evaporated (14—17).

data were statistically analyzed according to Student's t-test.

attached, was gently separated from the underlying circular muscle (18). The dissected muscle strips were attached via cotton threads to a retaining rod at one end — with a transducer at the other end, and mounted in 2 ml organ baths containing Krebsbicarbonate solution with choline cholride (1pM) at 37°C and bubbled with 95% 02/5 %C02 to maintain the pH at 7.4.

Longitudinal muscle-myenteric plexus pre-

parations (30—50mg) were suspended under a resting tension of 1 g and allowed to equilibrate for 45 mm with several changes of solution. The preparations were stimulated electrically (0.2 Hz;

70V; 0.5 ms puls, generated by a GRASS 48 stimulator) and the isometric contractions of the longitudinal muscle were recorded by an HSE force transducer. Tissues were stimulated to obtain a stable baseline of isometric contractions. During the final 20 mm of

stimulation. lOpCi 3!I-choline (methyl 311-choline chloride, 81.9 Ci/mmol) was added to the bath solution (19). After switching

off the stimulation, the tissues were washed three times every 10 mm during one hour with Krebs-bicarbonate solution containingcholine chloride(1 pM) and Hemicholinium-3 (10pM) (20).

Triplicate 0.5 ml aliquots of each bathing solution were collected 10 mm after the last wash and, again, from

each preparation at 20, 30, and 40mm. All preparations were then stimulated for 10mm, and a final set of triplicate aliquots was collected.

Results and Discussion

In our preliminary study on the antispasmodic activity of the flavonic extract of Genista tndentata L. and of an isoflavone, genistemn, isolated from it, we noticed that genistein significantly modifies the curves

formed by barium chloride and histamine, by working directly on the smooth muscle like a non-specific relaxant.

Thus we have continued our studies on its possible mechanism of action.

Genistein reduces the responses of guinea-pig ileum to electrical field stimulation (Fig. 1). The IC50 value of genistein (50% inhibitory concentration) is 34 pM and it was calculated graphically.

The submaximal contractions induced by ACh (20 nM) and 6-oxo-PGE1 (100 ng/ml) were inhibited by 52.3 6.02% (p < 0.005) and 70.8 3.7% (p < 0.001),

respectively, thus confirming the non-specific antispasmodic action (Fig. 2). Genistein (34 pM) has no effect on 3H-ACh release to the synaptic cleft. This action was expressed as the % release increase (Fig. 3) and the results obtained with genistein are very different from those obtained with the noradrenaline pattern.

Genistein (34pM) and noradrenaline (10pM) were added to the Krebs solution 5mm before the last 10mm of stimulation. Aliquots were transferred to scintillation counting vials containing 5 ml LIQUISCINT and the radioactivity was de-

In the experiments on cyclic nucleotide levels of guinea-pig ileum, genistein (34 pM) as well as

termined by a Beckman LS 6800 liquid scintillation spectrometer.

phosphodiesterase. This increase resulted in a smooth muscle relaxation and, perhaps, it was the cause of the antispasmodic activity. On the other hand, the selected

The amount of radioactivity released during the last 10mm of stimulation was determined by subtracting the counts collected in the previous period 121).

Chemicals

papaverine (20 pM) produce an increase in the cAMP level, which leads us to think that there must be a blockade of its

concentrations did not modify significantly the cGMP levels (Table 1).

The following drugs and chemicals were used: (—)-arterenol bitartrate (Sigma), acetyicholine chloride (Sigma), cyclic AMP assay kit and cyclic GMP RIA kit (Amersham. U.K.).

Fig. 1 Effect of genistein on twitch responses of guinea-pig ileum elicited by electrical field stimulation.

0.6 g

M)

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Effect of genistein on 311-acetylcholine release: Male albino guinea-pigs weighing 400—450 g were decapited: the small intestines were removed with about 10cm of terminal ileum being discarded. The intestine was cut into segments 10cm in length, and the longitudinal muscle, with the myenteric plexus

M. D. Herrera eta!.

316 Planta Med. 58(1992)

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Fig. 22 Effect Effect of conFig. of genistein genistein on on acetylcholine acetyicholine and and 6-oxo-PGE1 6-oxo-PGE1 induced nduced COfl tractions. tractions. 16

'

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Fig, 3 Effect of genistein on 3H-acetylcholine release from guinea-pig Veal myenteric plexus.

Table 1 Effect of genistein and papaverine on cyclic nucleotide levels.

Control Genistein (34DM) Papaverine (20DM)

cAMP (pmol cAMP/mg tissue

cGMP (pmol cGMP/mg tissue)

0.1824 0.0253 0.2839 0.0230

0.0416 0.0068

0.3030 0.0229

0.0533 0.0040 (n.s.) 0.0605 0.0061 (n.s.)

p < 0.025; n.s. not significant.

Acknowledgements This work was supported by a grant awarded by the Spanish Ministry of Education and Sciences.

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