Antispasmodic and Antimicrobial Diterpenic Acids from Viguiera hypargyrea Roots Alejandro Zamilpa1,2, Jaime Tortoriello2, Victor Navarro2, Guillermo Delgado3, Laura Alvarez1
Abstract
Letter
Two spasmolytic diterpene acids, ent-beyer-15-en-19-oic acid and ent-kaur-16-en-19-oic acid, have been isolated from the roots of Viguiera hypargyrea by bioassay-guided fractionation. Beyerenoic acid was also active against Staphylococcus aureus, Enterococcus feacalis and Candida albicans.
Viguiera hypargyrea Blake (Asteraceae) is a perennial herb growing in Northern MØxico and it is commonly known with the local name of ªplateadaº [1]. Underground parts of this plant have been used in Mexican traditional medicine for the treatment of gastrointestinal disorders [2]. Previous phytochemical study on the aerial parts resulted in the isolation of ent-kaurene diterpene acids and sesquiterpene lactones [3], some of which showed potent cytotoxic activity [4]. However, the chemical constituents of the roots have not been isolated. In this work different extracts obtained from roots of V. hypargyrea were submitted to a pharmacological evaluation by using the gastrointestinal transit in mice, the electrically induced contractions of guinea-pig ileum and antimicrobial assays, as pharmacological models that could make evident a spasmolytic, antidiarrhoeic and/or anti-infection activity. Afterwards, in order to identify the chemical bioactive compounds, a bioguided chromatographic separation was developed. This procedure was monitored by the isolated guinea-pig ileum model. When tested with the in vitro spasmolytic assay, the methanolic and ethyl acetate extracts of V. hypargyrea roots did not show any notorious effect, while the n-hexane extract induced a 68.67 % of inhibition of the contraction induced electrically (Table 1). Bioassay-guided fractionation of the active n-hexane extract led to the isolation of an active fraction (78.13 % of inhibition). Further separation afforded two active compounds. Compound 1 was identified as ent-kaur-16-en-19-oic acid by comparison with authentic sample isolated from V. hypargyrea aerial parts [3]; extensive Affiliation: 1 Centro de Investigaciones Químicas, Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos, MØxico ´ 2 Centro de Investigación BiomØdica del Sur, Instituto Mexicano del Seguro Social, Xochitepec, Morelos, MØxico ´ 3 Instituto de Química, Universidad Nacional Autónoma de MØxico, Circuito Exterior, Ciudad Universitaria, Coyoacµn, MØxico, D. F., MØxico Correspondence: Dra. Laura Alvarez ´ Centro de Investigaciones Químicas ´ Universidad Autónoma del Estado de Morelos ´ Av. Universidad 1001, Col. Chamilpa, Cuernavaca 62210 Morelos ´ Mexico ´ E-Mail:
[email protected] ´ Fax: +52-73-297997 Received: March 23, 2001 ´ Accepted: October 28, 2001 Bibliography: Planta Med 2002; 68: 281±283 ´ Georg Thieme Verlag Stuttgart ´ New York ´ ISSN 0032-0943
281
Letter
analysis of 13C-, 1H- and DEPT-NMR data indicated 2 and 3 to be ent-beyer-15-en-19-oic acid and ent-kaur-9(11),16-dien-19-oic acid respectively. Beyerenoic acid (2) showed an inhibition of contraction of 63.54 %, while kaurenoic acid (1) produced an inhibition of 8.04 % at the dose of 10 mg/ml. Moreover, the effect of beyerenoic acid was concentration dependent and showed a ED50 of 4.9 mg/ml. Chromatographic analyses of the ethyl acetate extract showed that this was mainly composed by the same diterpenic mixture found in the n-hexane extract, together with oleanolic acid (4) and the triterpene saponin glucopyranosyl oleanolate (5) which have been previously isolated from V. decurrens [5]. Altough the EtOAc extract displayed the highest antimicrobial activity, evaluation of the major constituents of this extract (1, 2, 4 and 5; Table 2) showed that only beyerenoic acid (2) inhibited the growth of Staphylococcus aureus and Enterococcus feacalis reaching an MIC of 0.012 mg/ml (Table 2).
282
active fraction (F-1, 15.0 g) eluted with n-hexane-ethyl acetate (97: 3 v/v, 6900 ml) which was composed of a diterpenic acid mixture. This mixture (500 mg) was further purified by column chromatography on silica gel (30.0 g) impregnated with 50 % silver nitrate solution [7], using mixtures of n-hexane-CH2Cl2 resulting in the isolation of 40 mg of 1 (4 : 1 v/v, 245 ml), 220 mg of 2 (7: 3 v/v, 45 ml) and 3 mg of 3 (0 : 100 v/v, 40 ml). The order of elution was 1 (Rf = 0.65), 2 (Rf = 0.45) and 3 (Rf = 0.40) observed on silica gel plates impregnated with 50 % AgNO3 and eluted with n-hexane-ethyl acetate (4 : 1 v/v). Compound 1 {0.02 % yield, m. p. 165 ± 167 8C; [a]D25: ± 44.88 (c 0.1, CHCl3)} was identified as ent-kaur-16-en-19-oic acid by comparison (IR, TLC) with an authentic sample. Identification of 2 {0.14 % yield, m. p. 185 ± 186 8C [a]D25: + 0.548 (c 0.18, CHCl3)} and 3 {0.002 % yield, m. p. 158 ± 159 8C, [a]D25: + 18.38 (c 0.12, CHCl3)}was based on com-
Table 1
Evaluation of the gastrointestinal transit in the mice assay showed that there were no differences with any of the extracts when they were compared with vehicle (5 % Tween 80 solution), only loperamide, drug used as reference, significantly decreased gastrointestinal tract propulsion (29.3 %) at the doses of 2.0 mg/ kg. It was demonstrated that the n-hexane extract possesses an excellent spasmolytic effect without modifying significantly the gastrointestinal transit of mice. This interesting activity could be attributed to beyerenoic acid (2), which is the major constituent of the active extract. This compound could also be held responsible for the antimicrobial activity displayed by the n-hexane and EtOAc extracts. These results support the traditional use of V. hypargyrea roots for the treatment of gastrointestinal disorders in Mexico.
Substance
a
Roots of V. hypargyrea (4.5 kg) were milled and extracted successively with n-hexane, ethyl acetate and methanol for three times each, at room temperature to yield, after solvent evaporation under vacuum, 120 g of n-hexane extract, 178 g of ethyl acetate extract and 300 g of methanol extract. The active n-hexane extract was fractionated by vacuum liquid chromatography [6] over silica gel (300 g) using n-hexane-ethyl acetate mixtures to yield an
Inhibition of ileum contraction (%)
25
60.67 6.3
Ethyl acetate extract
25
10.56 2.1
Methanol extract
25
2.16 2.6
F-1
25
78.13 3.9
Beyerenoic acid (2)
10
63.54 6.1a
Beyerenoic acid (2)
5.0
52.5 3.0
Beyerenoic acid (2)
3.0
21.37 3.0
Kaurenoic acid (1)
10
Papaverine
10
8.04 2.5a 82.8 2.1
p < 0.0001 in the Student's ªtº test.
Minimal inhibitory concentration exhibited by Viguiera hypargyrea extracts and constituents 2, 4 and 5
Substance
MIC values (mg/ml) S.a
E.c
E.f
P.m
C.a
n-Hexane extract
2.5
>10
2.5
>10
2.5
Ethyl acetate extract
1.25
>10
1.25
10
1.25
Methanol extract Beyerenoic acid (2)
>10 0.012
>10 >0.4
>10 0.012
Oleanolic acid (4)
>0.4
>0.4
>0.4
Glucopyranosyl oleanolate (5)
>0.4
>0.4
>0.4
Gentamicin (0.002 ± 0.0128 mg/ml) Nystatin
0.008
0.016
0.016
>10
>10
>0.4
>0.4
>0.4
>0.4
>0.4
>0.4
±
±
±
0.02
S.a. (Staphylococcus aureus) ATCC2913, C.a. (Candida albicans) ATCC10231, E.c. (Escherichia coli) ATCC25922, P.m. (Proteus mirabilis) ATCC43071 and E.f. (Enterococcus feacalis) ATCC29212.
Letter ¼ Planta Med 2002; 68: 281 ± 283
The roots from V. hypargyrea Blake were collected near to Durango City, on September 15th of 1997 and identified by Dr. Robert Bye. A botanical sample was prepared and deposited for reference at the National Herbarium of Mexico (MEXU) with the code number MEXU961417.
Concentration (mg/ml)
n-Hexane extract
Table 2
Materials and Methods
Inhibition of electrically induced contractions of guinea-pig ileum by the tested substances
parison of mass, 1H-, 13C-, and DEPT-NMR data with literature values [8], [9]. Copies of the original spectra are available from the author of correspondence. Ethyl acetate extract was applied to a silica gel column (500 g) eluting with an n-hexane-EtOAc (9 : 1, 8 : 2, 1 : 1; v/v, 8 l for each eluent) and an EtOAc-MeOH gradient to afford diterpene mixture of 1 ± 3, (2.45 g, 1.3 % yield) on elution with n-hexane-EtOAc (9 : 1 v/v), compound 4 {420 mg, 0.23 % yield; [a]D25: + 37.88 (c 0.19, CHCl3)} on elution with n-hexane-EtOAc (4 : 1 v/v) and compound 5 {(264 mg, 0.14 % yield; [a]D25: + 31.08 (c 0.14, MeOH)} on elution with EtOAc-MeOH (9 : 1 v/v). These compounds were identified by direct comparison with authentic samples.
Antimicrobial activity: Minimal inhibitory concentration (MIC) of extracts and pure compounds was performed by using standard protocols [10]. Assay was performed in duplicate and results were expressed as the lowest concentration that produced complete suppression of colony growth, nystatin and gentamicin were used as positive controls. Ileum studies: Following the intraluminal perfusion model on in vitro guinea pig's ileum described by Lozoya [11], male guinea pigs (600 g) were used. Test samples dissolved in methanol (0.1 %) were added at concentrations of 10, 5 and 3 mg/ml. Assays were performed in triplicate, the contractile response obtained 5 minutes after drug addition was used for comparison with the control. Papaverin was used as positive control. The significance of drug action was assessed using Student's t-test. Gastrointestinal transit in mice: Following the method described by Tortoriello [12], in all groups (ten male, BalbC mice) 5 % tween-80 (Sigma) was used as a vehicule in a total constant volume of 200 ml. Three groups were treated with 500 mg/kg of nhexane, ethyl acetate and methanol extracts. Thirty minutes later, 300 ml of Castor Oil (Pharmacia) dyed with 5 mg/ml of phenol red were administered. After 30 min of treatment, the length traversed by the dye was calculated as a percentage of the total intestine length. Loperamide (2 mg/kg) was used as positive control.
References 1
Letter ¼ Planta Med 2002; 68: 281 ± 283
Blake SF. A revision of genus Viguiera. Contribution Gray Herbarium Harvard University 1918; 54: 11 ± 6 2 Martínez M. Catµlogo de Plantas medicinales de MØxico. Ed. Botas, 1969: 45 3 Alvarez L, Mata R, Delgado G, Romo de Vivar A. Sesquiterpene lactones from Viguiera hypargyrea. Phytochemistry 1985; 24: 2973 ± 6 4 Villarreal ML, Alvarez L, Alonso D, Navarro V, García P, Delgado G. Cytotoxic and antimicrobial screening of selected terpenoids from Mexican Asteraceae species. Journal of Ethnopharmacology 1994; 42: 25 ± 9 5 Marquina S, Maldonado N, Garduæo-Ramírez ML, Aranda E, Villarreal ML, Navarro N, Bye R, Delgado G, Alvarez L. Bioactive oleanolic acid saponins and other constituents from the roots of Viguiera decurrens. Phytochemistry 2001; 56: 93 ± 7
Coll C, Bowden E. The application of vacuum liquid chromatography to the separation of terpene mixtures. Journal of Natural Products 1986; 49: 934 ± 6 7 Enriquez RG, Escobar LI, Romero ML, Chavez MA, Lozoya X. Determination of grandiflorenic acid in organic and aqueous extracts of Montanoa tomentosa (zoapatle) by reversed-phase high-performance liquid chromatography. Journal of Chromatography 1983; 258: 297 ± 301 8 Delgado G, Romo de Vivar A, Cardenas J, Pereda-Miranda R, Huerta E. Ent-beyerene and ent-atisane diterpenes from Viguiera insignis. Phytochemistry 1984; 23: 2285 ± 8 9 Reynolds WT, Enriquez RG, Escobar LI, Lozoya X. Total assignment of 1 H and 13C spectra of kauradien-9(11),16-oic acid with the aid of heteronuclear correlated 2D spectra optimized for geminal and vicinal 13 C-1H coupling constants: or what to do when ªINADEQUATEº is impossible. Canadian Journal of Chemistry 1984; 62: 2421 ± 5 10 Lennette EM, Balows A, Hansler WJ, Shadomy HJ. (Eds) Manual of Clinical Microbiology pp. 1985: 143 ± 53 11 Lozoya X, Becerril G, Martínez M. Intraluminal perfusion model of in vitro guinea pig's ileum as a model of study of the antidiarrheic properties of the guava (Psidium guajava). Archivos de Investigacion Medica (MØxico) 1990; 21: 155 ± 62 12 Tortoriello J and Aguilar-Santamaría L. Evaluation of the calcium-antagonist, antidiarrhoeic and central nervous system activities of Baccharis serraefolia. Journal of Ethnopharmacol. 1996; 53: 157 ± 63
Letter
Animals: Male guinea pigs (600 50 g) and male BalbC mice with 25 to 27 g of body weight were used. All them were obtained from our Animal House and maintained in compliance with the policy on animal care expressed in the Institutional Research Council guidelines.
6
283
