Flavonoids from Achyrocline satureioides with relaxant effects on the smooth muscle of Guinea pig corpus cavernosum.
Ethanol extract of the aerial parts of Achyrocline satureioides (Lam.) DC. (Asteraceae) showed a significant, does dependent, relaxant relaxant /re·lax·ant/ (re-lak´sant)
1. lessening or reducing tension.
2. an agent that so acts.
muscle relaxant effect on the smooth muscle of corpus cavernosum strips, obtained from Guinea pig (65.5 [+ or -] 4.1% of relaxation at the dose of 25.0 mg/ml). Bioassay guided fractionation fractionation /frac·tion·a·tion/ (frak?shun-a´shun)
1. in radiology, division of the total dose of radiation into small doses administered at intervals.
2. of this extract furnished two flavonoids flavonoids,
n.pl common plant pigment compounds that act as antioxidants, enhance the effects of vitamin C, and strengthen connective tissue around capillaries. , quercetin quer·ce·tin
A yellow powdered crystalline compound produced synthetically or occurring as a glycoside in the rind and bark of numerous plants, used medicinally to treat abnormal capillary fragility. Also called meletin. and quercetin 3-methyl ether, with important vasorelaxing effects on the corpus cavernosum strips (79.8 [+ or -] 8.4 and 66.0 [+ or -] 4.8% of relaxation respectively at the dose of 0.075 mg/ml). Two methyl derivatives of quercetin obtained by synthesis, quercetin 3,7,3',4'-tetramethylether and quercetin 3,5,7,3',4'-pentamethylether, showed similar relaxant effects at the dose of 0.075 mg/ml (86.4 [+ or -] 8.5 and 67.31 [+ or -] 1.4% of relaxation respectively). The results show that the ethanol extract of A. satureioides and the assayed compounds exhibit significant vasorelaxing properties. Additionally, it is shown that the number of methyl groups in the quercetin nucleus has no significant influence on the effectiveness of these compounds.
Key words: Achyrocline satureioides, flavonoids, smooth muscle relaxation, corpus cavernosum
Impotence is a serious clinical problem in adult men. The failure of penile penile /pe·nile/ (pe´nil) of or pertaining to the penis.
Of or relating to the penis.
of or pertaining to the penis. erection may be due to impaired relaxation of the smooth muscle of the corpus cavernosum and several studies indicate that the relaxation is mediated by nitric oxide (NO) released from nonadrenergic-noncholinergic nerves and from the sinusoidal sinusoidal /si·nus·oi·dal/ (si?nu-soi´dal)
1. located in a sinusoid or affecting the circulation in the region of a sinusoid.
2. shaped like or pertaining to a sine wave. epithelium (Holmquist et al. 1991; Kim et al. 1991; Rajfer et al. 1992; see also Anderson et al. 1998).
Recently we found that extracts of some Argentinian medicinal plants (Baccharis trimera, Haplopappus rigidus, Huperzia saururus, Maytenus ilicifolia, Satureja parvifolia and Senecio Senecio
a widespread genus of the Asteraceae family. The genus contains more than 1200 species of which at least 25 are known to be poisonous. Some of them are listed here; the toxins are a group of pyrrolizidine alkaloids which cause seneciosis hepatic injury, and the dummy eriophyton) possess vasorelaxing effects (Hnatyszyn et al. 2003). Achyrocline satureioides (Lam.) DC. (Asteraceae), commonly known as "marcela", is native to the Southeast of South America. The aqueous extract of its aerial parts is used as choleretic, hepatoprotective, antispasmodic antispasmodic /an·ti·spas·mod·ic/ (-spaz-mod´ik)
1. preventing or relieving spasms.
2. an agent that so acts.
adj. and against male impotence (Morton, 1975; Toursarkissian, 1980). Other investigations have reported antiinflammatory, analgesic, sedative, hepatoprotective, immunostimulating, antiviral, antibacterial and antioxidant properties (Filot da Silva and Langeloh, 1994; Desmarchelier et al. 1998; Kadarian et al. 2002). Phytochemical phy·to·chem·i·cal
A nonnutritive bioactive plant substance, such as a flavonoid or carotenoid, considered to have a beneficial effect on human health. analyses showed the presence of flavonoids, caffeoyl derivatives, coumarins, essential oil, terpenes, sesquiterpenes and monoterpenes (Lopez et al. 1996), as well as two pectic poly saccharides with immunological activity (Wagner et al. 1984; Puhlmann et al. 1992), and recently a new compound, achyrofuran, with hypoglycemic hypoglycemic /hy·po·gly·ce·mic/ (-gli-sem´ik)
1. pertaining to, characterized by, or causing hypoglycemia.
2. an agent that lowers blood glucose levels. activity has been described (Carney et al. 2002). The high amount of flavonoids present in the polar extracts of this plant has been considered to be, at least in part, responsible for many of the reported biological activities (Middleton, 1996).
In the search for bioactive compounds with vasorelaxing properties we tested the ethanol extract of A. satureioides, as well as two active flavonoids isolated from this extract and their tetra- and pentamethyl derivatives, using strips of Guinea pig corpus cavernosum.
Materials and Methods
The aerial parts of A. satureioides were collected in the Province of Buenos Aires, Argentina, and identified by Dr. G. Giberti. A voucher specimen (BAF 2981) has been deposited at the Museo de Farmacobotanica, Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Argentina.
Extraction and isolation
The air-dried powdered plant material (50 g) was extracted twice by maceration mac·er·a·tion
1. Softening by soaking in a liquid.
2. Softening of the tissues after death by autolysis, especially of a stillborn fetus. with ethanol (100 ml each) for 24 h at room temperature. Both extracts were taken to dryness under reduced pressure (yield 12.0% w/w). For bioassay-guided fractionation the crude ethanol extract was subjected to preparative paper chromatography on Whatman 3MM. An ascending run in 25% HOAc yielded two major bands detectable by UV at 366 nm (R[f.sub.1] -0.11, yellow, and R[f.sub.2] -0.33, dark purple). Both bands were cut out and eluted with MeOH. The eluate eluate /el·u·ate/ (el´u-at) the substance separated out by, or the product of, elution or elutriation.
The solution of solvent and dissolved matter resulting from elution. was concentrated and further purified by column chromatography (Sephadex LH 20), which afforded quercetin (Q; 0.02% w/w) and quercetin 3-methyl ether (Q3; 0.07% w/w). The identity of Q and Q3 was confirmed by TLC TLC total lung capacity; thin-layer chromatography.
1. thin-layer chromatography
2. . UV, and MS analyses. Quercetin 3,7,3',4'-tetramethyl ether (Q4) and quercetin 3,5,7,3',4'-pentamethyl ether (Q5) were obtained by methylation methylation,
n a phase-II detoxification pathway in the liver; methyl groups combine with toxins to rid the body of various substances.
(meth´ of quercetin (Sigma Chem. Co., St. Louis, MO, USA) according to the method described by Mabry et al. (1970) and their identity was confirmed by TLC, UV and MS analyses.
The ethanol extract (200 mg) was dissolved with 25% ethanol in water and used at final concentrations of 2.5, 5.0, 10.0 and 25.0 mg/ml. The flavonoids Q, Q3, Q4 and Q5 were dissolved in Krebs' solution (KS) and used at final concentrations of 0.025, 0.050 and 0.075 mg/ml.
Tissue preparation and experimental procedure
Male albino Guinea pigs (450-650 g) were killed by cervical dislocation and exsanguinated. The penis was removed, placed in KS gassed with 95% [O.sub.2] and 5% C[O.sub.2] (37 [degrees]C, pH = 7.4) and dissected. The remaining corpus cavernosum tissue was cut into a helical strip and mounted in an organ-bath chamber with KS. After a 60 min stabilization, L-phenylephrine ([10.sup.-5] M) was added to adjust the maximal contractile tension (see Ballard et al. 1998). Then the testing solution was added at the desired final concentration. Controls were performed using vehicle. The change in isometric force was measured during 5-7 min with a force-displacement transducer Model FT 03 connected to a Grass Instrument polygraph Model 79 (Quincy, MA, USA). Tissue tones were tested by adding acetylcholine (Sigma Chem. Co., St. Louis, MO, USA; [10.sup.-4] M) before and after each experience. The relaxation was expressed as a percentual reduction of the contractile tension induced by L-phenylephrine.
The statistical analysis was performed using one way analysis of variance and a value of p <0.05 was considered significant. All data were expressed as mean [+ or -] S.E.M.
Results and Discussion
The ethanol extract of A. satureioides exerted a significant and dose dependent relaxing effect (Table 1). The two active compounds of the ethanol extract were identified as quercetin and quercetin 3-methyl ether and were present at a considerable concentration (0.02 and 0.07% w/w respectively). Since flavonoids have been described to possess relaxant properties (Laekeman et al. 1986; Duarte et al. 1993, 2001; Ko et al. 1999 Perez-Vizcaino et al. 2002), we tested the flavonoids Q, Q3, Q4 and Q5. As shown in Table 2, all tested compounds yielded similar results. As a consequence, it can be concluded that the relaxant activity observed for the flavonoids in our experimental model is independent of the number of methyl groups in the flavonol nucleus. In addition, the results indicate that the relaxant effect of the ethanol extract of A. satureioides is somehow related to the considerable amounts of Q and Q3 present in this extract and give an explanation for the folk medicinal uses of this plant.
On the other hand, in a recent study we assayed sildenafil citrate using the same experimental model described here (Hnatyszyn et al. 2003). The results showed a poor effectiveness (up to 34% of relaxation). probably due to the lack of NO-donor in the system (see also Chuang et al. 1998; Aydin et al. 2001). As a consequence, it seems that the mechanism involved in the relaxation induced by the tested solutions in our study is independent of the presence of a NO-donor. However, the precise mechanism of action by which flavonoids induce the relaxation in the corpus cavernosum strips is still unknown and our data do not give sufficient proof for the use of A. satureioides in male impotence. But, considering the activity observed for the extract and for the isolated flavonoids and taking into account their low toxicity, it seems worthy to continue the search for natural products with a potential application in male impotence.
Table 1. Relaxant effect of the ethanol extract of A. satureioides on L-phenylephrine precontracted corpus cavernosum strips. The controls with vehicle did not show any significant effect (n = 4, *; p > 0.05 vs. 2.5 mg/ml, **; p > 0.05 vs. 5.0 mg/ml). Extract 2.5 5.0 10.0 25.0 (mg/ml) % of relaxation 29.1 48.9 60.2 65.5 [+ or -] 9.3 [+ or -] 6.6* [+ or -] 5.8** [+ or -] 4.1** Table 2. Relaxant effect (% of relaxation) induced by quercetin (Q), quercetin 3-methyl ether (Q3), quercetin 3,7,3',4'-tetramethyl ether (Q4) and quercetin 3,5,7,3',4'-pentamethyl ether (Q5) on L-phenylephrine precontracted corpus cavernosum strips. Acetylcholine ([10.sup.-4] M) was added before and after each measurement to induce maximal relaxation (n = 5, *; p > 0.05 vs. 0.025 mg/ml, **; p > 0.05 vs. 0.050 mg/ml). Compound 0.025 mg/ml 0.050 mg/ml 0.075 mg/ml Q 15.0 [+ or -] 7.1 59.5 [+ or -] 12.2* 79.8 [+ or -] 8.4** Q3 6.5 [+ or -] 4.1 33.3 [+ or -] 11.4* 66.0 [+ or -] 4.8** Q4 9.5 [+ or -] 4.5 47.5 [+ or -] 10.5* 86.4 [+ or -] 8.5** Q5 3.0 [+ or -] 2.2 42.1 [+ or -] 9.0* 67.3 [+ or -] 11.4**
The authors would like to thank UBACYT079 and IQUIMEFA-CONICET for financial support.
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O. Hnatyszyn (1), V. Moscatelli (1), R. Rondina (1), M. Costa (2), C. Arranz (2), A. Balaszczuk (2), J. Coussio (1) and G. Ferraro (1)
(1) Catedra de Farmacognosia, IQUIMEFA (UBA-CONICET)
(2) Catedra de Fisiologia, Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Buenos Aires, Argentina
O. Hnatyszyn, Catedra de Farmacognosia, IQUIMEFA (UBA-CONICET), Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires. Junin 956, (1113) Buenos Aires, Argentina
Tel.: +54-11-4964-8247; Fax: +54-11-4508-3642; e-mail: email@example.com