Flavonoids from Achyrocline satureioides with relaxant effects on the smooth muscle of Guinea pig corpus cavernosum.Summary
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.
Anderson KE. Waldeck KNL. Persson K (1998) Mediators and mechanisms of relaxant in rabbit urethral muscle. Br J Pharmacol 123: 617-624
Aydin S. Ozbek H, Yilmaz Y, Atilla MK, Bayrakli H, Cetin H (2001) Effects of sildenafil citrate, acetylcholine, and sodium nitroprusside on the relaxation of rabbit cavernosal tissue in vitro. Urology 58: 119-124
Ballard SA, Gungell CJ, Tang K, Turner LA, Price ME, Naylor AM (1998) Effects of sildenafil sildenafil /sil·den·a·fil/ (sil-den´ah-fil?) a phosphodiesterase inhibitor that relaxes the smooth muscle of the penis, facilitating blood flow to the corpus cavernosum; used as the citrate salt to treat erectile dysfunction. on the relaxation of human corpus cavernosum tissue in vitro and on the activities of nucleotide phosphodiesterase phosphodiesterase /phos·pho·di·es·ter·ase/ (-di-es´ter-as) any of a group of enzymes that catalyze the hydrolytic cleavage of an ester linkage in a phosphoric acid compound containing two such ester linkages. isozymes. J Urol 159: 2164-2171
Carney JR, Krenisky JM, Williamson RT, Luo J (2002) Achyrofuran, a New Antihyperglycemic Dibenzofuran from the South American Medicinal Plant Achyrocline satureioides. J Nat Prod 65: 203-205
Chuang AT, Strauss JD, Murphy RA, Steers WD (1998) Sildenafil, a type-5 cGMP phosphodiesterase inhibitor, specifically amplifies endogenous cGMP-dependent relaxation in rabbit corpus cavernosum smooth muscle in vitro. J Urol 160: 257-261
Desmarchelier C, Coussio J, Ciccia G (1998) Antioxidant and free radical scavenging effects in extracts of the medicinal herb Achyrocline satureioides (Lam.) DC. ("marcela"). Braz J Med Biol Res 31: 1163-1170
Duarte J, Perez Vizcaino F, Utrilla P, Jimenez J, Tamargo J, Zarzuelo A (1993) Vasodilatory effects of flavonoids in rat aortic smooth muscle. Structure--activity relationships. Gen Pharmacol 24: 857-862
Duarte J, Perez-Palencia R, Vargas F, Ocete MA, Perez-Vizcaino F, Zarzuelo A, Tamargo, J (2001) Antihypertensive effects of the flavonoid quercetin in spontaneously hypertensive rats. Br J Pharmacol 133: 117-124
Filot Da Silva L, Langeloh A (1994) Comparative study of antispasmodic activity of hydroalcoholic 80% (v/v) extracts of Achyrocline satureioides (Lam.) DC. (Asteraceae) with papaverine papaverine (pəpăv`ərēn), alkaloid found in opium that acts as a muscle relaxant and vasodilator. The drug relaxes the smooth muscle of the larger blood vessels and is used to increase the blood supply to the brain or to the heart, as and atropine atropine (ăt`rəpēn, –pĭn), alkaloid drug derived from belladonna and other plants of the family Solanaceae (nightshade family). on rat isolated jejunum jejunum: see intestine. . Acta Farm Bonaer 13: 35-40
Hnatyszyn O, Moscatelli V, Garcia J, Rondina R, Costa M, Arranz C, Balaszczuk A, Ferraro G, Coussio J (2003) Argentinian plant extracts with relaxant effect on the smooth muscle of the corpus cavernosum of Guinea pig. Phytomedicine (in press)
Holmquist F, Hedlund H, Andersson KE (1991) L-[N.sup.6]-nitroarginine inhibits non-adrenergic, non-cholinergic relaxation of isolated human corpus cavernosum. Acta Physiol Scand 141: 441-442
Kadarian C. Broussalis AM, Mino J, Lopez P, Gorzalczany S, Ferraro G, Acevedo C (2002) Hepatoprotective activity of Achyrocline satureioides (Lam) DC. Pharmacol Res 45: 57-61
Kim N, Azadzoi KM, Goldstein I, Saenz de Tejada I (1991) A nitric oxide-like factor mediates nonadrenergic-noncholinergic neurogenic neurogenic /neu·ro·gen·ic/ (-jen´ik)
1. forming nervous tissue.
2. originating in the nervous system or from a lesion in the nervous system. relaxation of penile corpus cavernosum smooth muscle. J Clin Invest 88: 112-118
Ko WC, Kuo SW, Sheu JR, Lin CH, Tzeng CM, Chen CM (1999) Relaxant effects of quercetin methyl ether derivatives in isolated Guinea pig trachea and their structure-activity relationships. Planta Med 65: 273-275
Laekeman GM, Claeys M, Rwangabo PC, Herman AG, Vlietinck AJ (1986) Cardiovascular effects of 3-Methylquercetin. Planta Med 6: 433-558
Lopez P, Broussalis A, Rodriguez M, Coussio J, Ferraro G (1996) Analisis de muestras comerciales de "marcela" (Achyrocline satureioides). Acta Farm Bonaer 15(4): 243-249
Mabry TJ, Markham KR, Thomas MB (1970) The Systematic Identification of Flavonoids. Springer-Verlag-Berlin-Heildelberg, New York, pp 30-31
Middleton E (1996) Biological properties of plant flavonoids: an overview. Int J Pharmacog 34: 344-348
Morton JF (1975) Current folk remedies of Northern Venezuela. Q J Crude Drug Res 13: 97-121
Perez-Vizcaino F, Ibarra M, Cogolludo AL, Duarte J, Zaragoza-Arnaez F, Moreno L, Lopez-Lopez G, Tamargo J (2002) Endothelium-independent vasodilator vasodilator /vaso·di·la·tor/ (-di-la´ter)
1. causing dilatation of blood vessels.
2. a nerve or agent that does this.
n. effects of the flavonoid quercetin and its methylated metabolites in rat conductance and resistance arteries. J Pharmacol Exper Ther 302(1): 66-72
Puhlmann J, Knaus U, Tubaro L, Schaefer W, Wagner H (1992) Immunologically active metallic ion-containing polysaccharides of Achyrocline satureioides. Phytochemistry phytochemistry,
n the scientific study and classification of the chemical constituents of plants. 31(8): 2617-2621
Rajfer J, Aronson WJ, Bush PA, Dorey FJ, Ignarro IJ (1992) Nitric oxide as a mediator of relaxation of the corpus cavernosum in response to nonadrenergic, noncholinergic neurotransmission. New Eng J Med 326: 90-94
Toursarkissian M (1980) Plantas medicinales de la Argentina: sus nombres botanicos, vulgares, usos y distribucion geografica. Editorial Hemisferio Sur S.A., Buenos Aires, p 25
Wagner H, Proksch A, Riess-Mauer I, Vollmar A, Odenthal S, Stuppner H, Jurcic K, Le Turdu M, Heur YH (1984) Immunostimulant im·mu·no·stim·u·lant
An agent that stimulates the immune system.
n a substance that encourages and sustains the immune system and its responses.
1. action of polysaccharides (heteroglycans) from higher plants. Preliminary communication. Arzneim Forsch 34(6): 659-661
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: firstname.lastname@example.org