1186 Biol. Pharm. Bull. 29(6) 1186—1190 (2006) Vol. 29, No. 6
The Antispasmodic Activity of Buddleja scordioides and Buddleja perfoliata on Isolated Intestinal Preparations
Alma Rosa CORTÉS,* Alba Jady DELGADILLO, Marcela HURTADO, Adriana Miriam DOMÍNGUEZ-RAMÍREZ, José Raúl MEDINA, and Kazuko AOKI Departamento Sistemas Biológicos, Universidad Autónoma Metropolitana-Xochimilco; Calzada del Hueso 1100, Colonia Villa Quietud, México, D.F., México, C.P. 04960. Received October 5, 2005; accepted March 3, 2006
The antispasmodic activity of extracts from the aerial parts of Buddleja scordioides and Buddleja perfoliata (family: Scrophulariaceae) was studied on isolated tissue preparations from rabbit and guinea pig intestine. The chloroformic extract from the plants exhibited a significant relaxation on the spontaneous contraction of isolated rabbit jejunum at concentrations ranging from 1 to 400 mg/ml, and also caused an inhibitory effect on both K and Ca2 induced contractions in the same tissue. The extracts at moderate doses (50 mg/ml) reduced 5-hydroxy- tryptamine (5-HT), acetylcholine and histamine induced contractions on isolated guinea pig ileum. Therefore, B. scordioides and B. perfoliata possess similar relaxant mechanism of action, in view of the fact that both inhibit K induce contraction and act through serotoninic, muscarinic and histaminic receptors. So, these data support the idea that the extracts may interfere either with calcium mobilization from intracellular stores, or with cal- cium interaction with regulatory proteins (e.g., calmodulin), or in other steps in the calcium signaling pathway. This leads us to suggest that the spasmolytic effect of both Buddleja species on smooth muscular contractility are due to the same or similar compounds occurring in these two species, which might be present in similar quanti- ties. Key words Buddleja scordioides; Buddleja perfoliata; spasmolytic; calcium antagonist; smooth muscle relaxant
The genus Buddleja, included in the family Loganiaceae, nic, gallic and oxalic acids.4) In folk medicine, it is used in and previously classified in a family of its own, the Buddle- the treatment for tuberculosis as well as for catarrh, ptyalism, jaceae, is now classified in the family Scrophulariaceae. Na- and headaches.6,7) tive to Asia, Africa, North and South America, Buddleja Despite the extensive use of B. scordioides for gastroin- (also Buddleia), is a genus containing approximately 100 testinal problems such as intestinal spasms, no pharmacolog- species, 50 being distributed in America, of which 16 grow ical studies have been carried out to date, for the chemical in Mexico.1) content relationship, throughout the Buddleja genus, where Buddleja species are widespread and share some remark- the species perfoliata may possibly have similar biological able similarities in their medicinal uses. This may well indi- activity. cate the presence of the same or similar compounds with a In the present paper, we evaluated the antispasmodic activ- particular pharmacological action. A pattern is emerging ity of two Buddleja species, Buddleja scordioides and Bud- about the composition of these compounds; flavonoid and iri- dleja perfoliata, which are widely used in Mexico in tradi- doid glycosides being the major secondary metabolites that tional medicine. have been isolated to date, and which are common through- out the genus.2) MATERIALS AND METHODS Due to the interesting phytochemical and ethnopharmaco- logical observations, the genus Buddleja has been investi- Plant Material and Extraction B. scordioides was col- gated extensively; a valuable review on chemical compo- lected in May from the North of Mexico (Chihuahua State) sition and biological activity has been published by and a fresh plant of B. perfoliata was purchased from the Houghton.2,3) Mexico city local market. The plants were identified by Dr. Buddleja scordioides HBK (KUNTH), Loganiaceae, is com- Jerónimo Reyes from the Botanical Garden, Institute of Biol- monly known as escobilla, butterfly-bush, mato, salvia real ogy, Autonomous National University of Mexico. A voucher and salvilla. This plant is widely used for the treatment of di- of each specimen was retained. The aerial parts of the plants arrhea, stomachache (colic) and gastrointestinal disorders.4) were washed with water, air-dried and milled to a coarse In addition, a verbascoside with antibacterial activity,3) triter- powder. The powders were then extracted twice under reflux penoid saponins and other glycosides,5) have been extracted for 4 h using chloroform. The extracts were then filtered and from this species. The presence of some flavonoids such as dried on a rotary evaporator and stored at 20 °C until re- rutine, quercetin and quercitrin has also been reported.4) quired. Buddleja perfoliata HBK (KUNTH), Loganiaceae, known in The crude extracts and polyvinylpyrrolidone 2500 (PVP) Spanish as salvia de bolita, hierva de manita, salvia chiquita, (1 : 4 w/w), used as a co-solvent, were weighed and then dis- salvia de campo, became officially recognised in the 1930 solved separately in a minimum volume of chloroform and Mexican Pharmacopoeia where it was shown to have antisu- methanol, respectively. They were then quantitatively mixed, dorific activity.2) Vague and limited information regarding the dried under a vacuum and dissolved in a Tyrode solution pH types and quantity of chemical compounds found in this 7.4. Control experiments demonstrated that the solvents used plant is available. This plant also contains essential oil, tan- to dissolve plant extracts did not affect the contractile re-
∗ To whom correspondence should be addressed. e-mail: [email protected] © 2006 Pharmaceutical Society of Japan June 2006 1187 sponses of the isolated tissues at the final bath concentration. sium solution was obtained by an equimolar replacement of Chemicals and Drugs All chemicals used were of NaCl by KCl in the Tyrode solution. A concentration re- reagent grade, purchased from Merck Mexico. Acetylcholine sponse was obtained by the cumulative addition of the ex- (ACh), phentolamine, histamine (HIS), 5-hydroxytryptamine tracts at 7-min intervals after addition of 50 mM KCl. The re- (5-HT) and propranolol were obtained from Sigma Chemical laxation of the jejunum was expressed as a percentage of Company, U.S.A. ACh and HIS were dissolved in a Tyrode control K -induced contractions. Verapamil (3.2 10 9— 7 solution, adjusted to pH 4 using ascorbic acid, and all other 3.2 10 M) was used as a positive control. drugs were prepared in suitable physiological saline solution. To assess whether the spasmolytic activity of Buddleja’s Animals Male New Zealand white rabbits (2—2.5 kg) extracts was via a calcium channel blockade, the jejunum and Dunkin Hartley guinea pigs from both sexes (400— was allowed to stabilize in normal Tyrode solution, and was 600 g) of local breed were used for this study. These were then replaced with a Ca2 -free Tyrode solution for 30min. housed at the Animal House of the Autonomous Metropoli- This solution was replaced with K rich (50 mM) and tan University-Xochimilco of Mexico, maintained in con- Ca2 free Tyrode solution. Following an incubation period of trolled conditions at 23—25 °C, under a 12 h light/dark cycle, 20 min and after the confirmation of no spontaneous contrac- and were given a standard diet and free access to water, how- tions of jejunum, Ca2 was added in a cumulative fashion 4 2 ever, food was withdrawn 24 h prior to experimentation. The (1 10 —3 10 M), every 3 min, to obtain control concen- rabbits were sacrificed by a blow to the nape of the neck fol- tration–response curves of Ca2 . The concentration–response lowed by exsanguination and the guinea pigs euthanized with curves of Ca2 were repeated following a 10 min incubation 10) CO2. Animals were treated according to the guidelines for with the extracts. animal care and use of laboratory animals by official Mexi- Spasmolytic Effect on Guinea Pig Ileum The influence can regulations.8) of the extracts on the contractions induced by 5-HT 7 5 9 6 Isolated Tissue Preparations The spasmolytic activities (1 10 —1 10 M), Ach (1 10 —1 10 M) and HIS 8 6 of B. scordioides and B. perfoliata were investigated using (1 10 —3 10 M), was studied in preparations pre-incu- isolated tissue preparations from rabbit jejunum and guinea bated for 10 min with each extract. pig ileum. Statistical Analysis The results obtained from at least Several segments of jejunum were rapidly removed four different animals are presented as mean S.E.M. Two through a midline abdominal incision. The tissue was sample comparisons were made by a two-tail unpaired Stu- cleaned and placed directly in a 10-ml organ chamber and dent’s t-test. Statistical significance was assumed at p levels was allowed to equilibrate for 30 min in Tyrode solution, pH less than 0.05. The concentrations to produce 50% of the
7.4, and was aerated continuously with a gas mixture of 95% maximal responses (EC50) were calculated by a nonlinear O2–5% CO2, and maintained at 37 °C. An initial tension of curve fitting procedure using Sigma Plot version 8.0. 0.5 g was applied.9) The terminal portion of the guinea pig ileum, the 10 cm RESULTS portion nearest the caecum being discarded, was cleaned and transferred to the Tyrode solution. Segments of ileum Chloroformic extracts from the aerial parts of B. scor- (0.5 cm) were placed directly in a 10-ml organ chamber. The dioides and B. perfoliata caused both a concentration-de- preparations were given an initial load of 1 g and were equili- pendent antispasmodic effect in the spontaneous contraction brated for 1—2 h before the experiments were initiated with of rabbit jejunum, at a concentration of 1—400 mg/ml, which a 15 min washout interval. was similar (respect to inhibition profile) to that caused by The composition of the Tyrode solution (mM) was: NaCl verapamil at a concentration range of 50 to 280 nM, however,
(136.9), KCl (2.7), CaCl2 (1.8), MgCl2 (1.0), NaHCO3 in isolation, neither the resting tension of the tissue nor the (11.9), NaH2PO4 (0.4) and glucose (5.5), pH 7.4. spontaneous contraction frequency was altered (Fig. 1). The experiments were performed on three or four seg- The effect of B. scordioides and B. perfoliata extracts on ments of jejunum or ileum taken from each of four or five spontaneous contraction were not significantly different animals. Segments that did not show spontaneous activity (p 0.05), concentrations that caused 50% inhibition of the were discarded. spontaneous contractions (EC50) were 62.8 11.8 and The force of contraction was recorded simultaneously on a 41.35 5.6 mg/ml, respectively (Fig. 2a). The spasmolytic ef- 4-channel Narco-Biosystem physiograph, equipped with an fect was reversible and the spontaneous activity returned to isometric F-60 force transducer which had been previously normal after washing the preparation. calibrated. Pre-incubation of the tissues with phentolamine and pro- Relaxing Activity on Isolated Rabbit Jejunum The ac- pranolol had no effect on the extract response. However, a tivity of the extracts was tested in isolated spontaneously high concentration of K is known to cause smooth muscle contracting rabbit jejunum, in the absence and presence of contractions through the opening of voltage dependent slow several agents including non-selective a and b-adrenergic Ca2 channels, thus allowing the influx of extracellular Ca2 , 6 antagonists: phentolamine (2 10 M) and propranolol causing a contractile effect. When tested in isolated rabbit je- 6 (1 10 M). Verapamil (50—280nM) was used as a positive junum, the extract of B. scordioides and B. perfoliata, within control. The inhibition of contractions in the test material the same concentration range that inhibited the spontaneous was assessed as a percentage of the basal spontaneous con- contraction, caused a spasmolytic effect on the tissue pre- tractions in the rabbit jejunum. treated with high K (50 mM), which was similar to that of The spasmolytic effect was studied using potassium depo- verapamil. The concentration–response curves show exactly larized for adding K (50 mM) to the tissues; the high potas- the same pattern for both Buddleja extracts, with no signifi- 1188 Vol. 29, No. 6 cant difference in the EC50 values of 27.6 3.1 mg/ml and 27.3 4.6 mg/ml, respectively and with the 400 mg/ml bath concentration response to KCl completely abolished by the extracts (Fig. 2b). Throughout these experiments, there was no significant change in the K responses of the preparations treated with an equivalent volume of the vehicle. A contractile response was obtained by the addition of cal- 4 2 cium (1 10 —3 10 M) to a previously decalcified rabbit jejunum. Under the same conditions, but in presence of the extracts of B. scordioides and B. perfoliata a decrease in the contractile response was observed, producing a shift in the Ca2 curves (Fig. 3), with maximum inhibition at 50 mg/ml of the extracts reaching 55.8 5.1% and 62.6 4.9%, respec- tively. Serotonin (5-HT), Ach and HIS caused a rapid con- traction of guinea pig ileum, reaching the maxima within 30 s of contact. Similarly, both extracts of B. scordioides and B. perfoliata at a concentration of 50 mg/ml reduced the tis- sue response to these drugs without any significant difference between them (p 0.05). The spasmolytic effect of the ex- tracts in the preparations contracted with 5-HT was more pronounced that those contracted with HIS or Ach (Fig. 4). The concentration–response curves to 5-HT, in the pres- ence of 50 mg/ml of B. scordioides and B. perfoliata extracts,
Fig. 2. Concentration–Response Curves of the Inhibitory Effect of (a) B. scordioides and B. perfoliata Extracts on Spontaneous Contractions and (b) Both Extracts and Verapamil on the K -Induced Contraction in Isolated Rabbit Jejunum Values shown are mean S.E.M., n 5.
Fig. 1. Comparative Tracing of the Effect of Chloroformic Extracts of B. scordioides, B. perfoliata and Verapamil on Spontaneous Contraction of Rabbit Jejunum Fig. 3. Effect of B. scordioides and B. perfoliata (50 mg/ml) on Cumula- tive Concentration–Response Curve to CaCl2 in Isolated Rabbit Jejunum Values shown are mean S.E.M., n 4.
Fig. 4. Effect B. scordioides and B. perfoliata (50 mg/ml) on Tension Development to (a) 5-HT, (b) Ach and (c) HIS in Isolated Guinea Pig Ileum Values shown are mean S.E.M., n 5. June 2006 1189 shifted to the right, and the maximal height of contractions cium transport across membranes, or blocking inositol decreased to 40.7 1.4% and 35.7 5.1%, respectively (Fig. triphosphate (IP3) mediated Ca2 release from internal 4a). stores. At a same concentration of both extracts (50 mg/ml) the In the following, an attempt to summarize the current concentration–response curves to Ach and HIS also de- knowledge about IP3 participation is presented. Ach interacts creased. The reduction of the maximum response with re- with muscarinic receptors on ileum smooth muscle cell spect to the acetylcholine control was 57.2 4.4% and membranes and thereby increases the activity of membrane 54.3 3.5% (Fig. 4b), and 72.8 6.1% and 64.8 2.6% with bound phospholipase-C enzymes and the generation of inosi- respect to the HIS control (Fig. 4c). tol triphosphate, which on being released into the cytoplasm, When the tissues were pre-incubated with 100 mg/ml of B. will interact with receptors on intracellular Ca2 store sites scordioides and B. perfoliata (data not shown), the responses and cause the release of calcium.13) of HIS decreased to 52.9 3.4% and 38.7 6.3%, respec- In addition, histamine-induced contractions generated by tively. H1 receptor activation, locate postsynaptically to produce de- polarization and tonic contractions of smooth muscle.14) The DISCUSSION binding of HIS to a H1 receptor in smooth muscle results in the opening of receptor operated channels, thereby allowing The results from the present study indicate that both crude sodium influx, which causes a depolarization of the cell chloroformic extracts of B. scordioides and B. perfoliata had membrane. This depolarization opens voltage dependent cal- a relaxant effect on rabbit jejunum and guinea pig ileum, and cium channels and Ca2 enters the cell, which induces the re- reversibly blocked spasms induced by specific and unspecific lease of calcium from the sarcoplasmic reticulum. The cy- stimuli. This effect was attained at relatively low concentra- tosolic calcium thus binds to calmodulin, which results in tions and may be related to the symptomatic relief of acute contraction,15) but calcium release from IP3 sensitive stores abdominal pain associated with some gastric disturbances, is negatively regulated by binding of calmodulin to the IP3 obtained with medicinal usage of these plants commonly receptor. practiced in Mexico. The inhibitory effect was probably due Finally, the 5-HT contraction is mediated by the release of to different components acting separately or together. Appar- Ach from the cholinergic neurone and activation of sero- ently, relaxation was not due to postsynaptic stimulation of toninergic receptors on the smooth muscles of the ileum. 5- adrenoreceptors since pre-incubation of the tissues with HT has been described as participating in the regulation of phentolamine and propranolol had no effect on the extract re- intestinal peristalsis in different animals, and produces a con- sponse. tractile effect mediated by Ca2 via 5-HT2 receptors (attenu- At identical bath concentrations, the chloroformic extracts ated by simultaneous generation of cAMP via 5-HT4 recep- of B. scordioides and B. perfoliata demonstrated an in- tors) which may alter or modulate the intestinal physiology.16) hibitory effect on the contraction of ileum induced by 5-HT, Therefore, the effects observed in both Buddleja species to Ach and HIS (Fig. 4). This suggests that both extracts have these three agonists seemed to be Ca2 -dependent and in- no selective inhibitory effect on contractions induced by volves, at least in part, the mobilization of Ca2 from inositol these spasmogens. triphosphate (IP3) sensitive intracellular stores. It has been accepted that the contraction of smooth muscle Consequently, it can be postulated that the therapeutic ac- is dependent on an increase in the concentration of cytosolic tivity exhibited by Buddlejas must be due to the combined Ca2 , and the sensitivity of the contractile elements to Ca2 effects of several chemical constituents present in the plants, in response to changes in the cell. There is evidence that such as triterpene saponins and triterpenic glycosides iso- there is a significant variation in the degree of participation lated from B. scordioides,5) and different flavonoids, such as of extra- and intracellular Ca2 in smooth muscle contrac- quercetin which exhibits a spasmolytic effect through block- tion.11) ade of calcium channels.4,17) In this regard, studies with triter- The increase in intracellular Ca2 is due to either influx pene saponin and triterpenic glycosides obtained from differ- via voltage dependent Ca2 channels (VDCs) or the release ent medicinal plants, have shown spasmolytic activity on iso- from intracellular stores in the sarcoplasmic reticulum. Peri- lated tissues such as guinea pig bronchus and ileum. Specifi- odic depolarization and repolarization regulate the sponta- cally, Saikogenin A, a triterpenoid saponin, one of the com- neous movements of the intestine and at the height of depo- pounds reported for B. scordioides,5) is related to plants from larization, the action potential appears as a rapid influx of the genus Bupleurum. Besides its observed anti-inflamma- Ca2 via VDCs.12) Organic calcium channel antagonists, tory effect,18) it has also been reported that Saikogenin A can such as verapamil inhibit more markedly the entry of calcium suppress asthmatic bronchoconstriction in sensitized guinea through the VDCs. pigs by antagonism of HIS action.19) Similarly, triterpenic In order to confirm the interaction of Buddlejas extracts glycosides, could also be participating in the antispasmodic with voltage dependent Ca2 channels, the tissue was pre- effect.20) treated with high potassium. Certainly, the contractions in- duced by KCl are dependent on the entry of Ca2 into the CONCLUSIONS cells through voltage-dependent calcium channels, therefore a substance which can inhibit high K -induced contraction The results from this study demonstrate that both crude is, considered to be a Ca2 channel blocker.10) chloroformic extracts of B. scordioides and B. perfoliata had However, it might be also possible that Buddleja could a relaxant effect on rabbit jejunum and guinea pig ileum, and relax the intestinal smooth muscle by interfering with cal- that this activity may be the basis for some of its uses in tra- 1190 Vol. 29, No. 6 ditional medicine. Therefore, the antispasmodic activity tas Medicinales de México II. Composición, usos y actividad found, supports the rationale use, in both folk and traditional biológica,” UNAM, México, 1999. 5) Avila A., Guilhermo J., Romo de Vivar A., Biochem. System. Ecol., 30, medicine of B. scordioides in the treatment of gastrointesti- 1003—1005 (2002). nal cramps, spasms and colic. This activity was also ob- 6) Martínez M., “Las Plantas Medicinales de México,” 5a. ed., Ediciones served with B. perfoliata, which suggests that it could be Botas, México, 1969. used to treat the same afflictions. 7) Linares E., Bye R., Flores B., “Plantas Medicinales de México: Usos y Finally, it can be concluded that both Buddleja species remedios tradicionales,” Instituto de Biología UNAM y Sistemas de Información Geográfica SA de CV, México, 1999. possess similar smooth muscle relaxant mechanism of ac- 8) Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Ali- tion, in view of the fact that both inhibit K induced contrac- mentación, “NOM-062-ZOO-1999. Norma Oficial Mexicana. Especi- tion and they act through serotoninic, muscarinic and hista- ficaciones técnicas para la producción, cuidado y uso de los animales minic receptors. Therefore, these data support the idea that de laboratorio”: http://www.sagarpa.gob.mx/Dgg/NOM/062zoo.pdf , this extracts may interfere either with calcium mobilization 24 February 2006. 9) Cortés A. A. R., Lara Ch. B., Aoki M. K., Pharm. Biol., 42, 24—29 from intracellular stores, or with the calcium interaction with (2004). regulatory proteins (e.g., calmodulin), or at other levels of 10) Gilani A. H., Jabeen Q., Ghayur M. N., Janbaz K. H., Akhtar M. S., J. the calcium signaling pathway as a second messenger, such Ethnopharm., 98, 127—135 (2005). as, by decreasing the contractile machinery sensitivity to cal- 11) Karaki H., Weiss G. B., Life Sci., 42, 111—122 (1988). cium, as discussed previously. 12) Carl A., Lee H. K., Sanders K. M., Am. J. Physiol. Cell Physiol., 271, C9—C34 (1996). This leads us to suggest that the spasmolytic effect of B. 13) Himpens B., Missiaen L., Casteels R., Adv. Organ Biol., 8, 139—180 scordioides and B. perfoliata can be attributed to the same or (2000). similar compounds occurring in both species, and which 14) Zevecz J. H., Yellin T. O., J. Pharmacol. Exp. Ther., 223, 177—182 might be present in similar quantities. (1982). 15) Bolton T. B., Physiol. Rev., 59, 606—718 (1979). 16) Salvador M. T., Murillo M. D., Rodríguez-Yoldi M. C., Alcalde A. I., REFERENCES AND NOTES Mesonero J. E., Rodríguez-Yoldi M. J., Can. J. Physiol. Pharmacol., 78, 359—366 (2000). 1) Ocampo A. G., “Flora del Bajío y de Regiones Adyacentes (115), 17) Gálvez J., Duarte J., Sánchez F., Jiménez J., Zarzuelo A., Phytother. Pátzcuaro,” ed. by Rzedowski J., Calderón G., Instituto de Ecología, Res., 10, 66—69 (1996). AC, México, 2003, pp. 1—35. 18) Cheng J. T., Tsai C. L., Biochem. Pharmacol., 35, 2483—2487 (1986). 2) Houghton P. J., J. Ethnopharm., 11, 293—308 (1984). 19) Park K. H., Park J., Koh D., Lim Y., Phytother. Res., 16, 359—363 3) Hougton P. J., Mensah A. Y., Iessa N., Yong-Hong L., Phytochemistry, (2002). 64, 385—393 (2003). 20) Capasso A., Balderrama L., Sivila S. C., De Tommasi N., Sorrentino 4) Márquez A. C., Lara O. F., Esquivel R. B., Mata E. R., Luna A., “Plan- L., Pizza C., Planta Med., 64, 348—352 (1998).