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Preliminary investigation of the estrogenic potential of grandiflorenic acid from Montanoa tomentosa
Jownal of Ethnophmnacology, 23 (1933) 329-331 Elsevier Scientific Publishers Ireland Ltd.
Short
329
communication
PRELIMINARY INVESTIGATION OF THE ESTROGENIC POTENTIAL OF GRANDIFLORENIC ACID FROM MOIWWVOA TOMEN!l’OSA EZRA BEJAR* Unidad de Inveetigacion en Medic& Tmd icionul y Desarrollo Argentinu no. 1 CP 62790, Xochitepec, Morelos IMetiol
de Medicamentos,
I.M.S.S.,
(Accepted April 23.1933)
Introduction Grandiflorenic acid (GA) is a diterpene isolated from Montanoa tomentosa ssp. tomentosa Cervantes (Compositae), a well-known abortifacient plant of Mexico (Gallegos, 1983). Several studies have demonstrated the presence of this compound in the “tea” traditionally used by native women (Enriquez et al., 1983) and its inhibitory effect on the contractility of uterine smooth muscle in vitro (Bejar et al., 1984a, 1984b3. The proposed mechanism of action of GA on uterine contractility is a blockade of calcium intake to smooth muscle cells (Bejar et al., 1984b3 which according to Batra and Bengtsson (1978) could be similar to that of the steroids. Gibberellic acid (GA,), a compound biosynthetically formed in plants from the kaurene ring, has been shown to possess estrogenic activity (Gawienowski and Chatterjee, 1980). Therefore, the present investigation was undertaken to assess if GA would also have some estrogenic activity which might explain the abortifacient effect elicited by Montanoa tomentosa in vivo.
C”2
Fig. 1. Grandiflorenic acid. lPferest addre88:Department of Physiology and Pharmacology, School of Pharmacy, University of the Pacific, Stockton CA 95211, U.S.A. 0373-3741/33/$01.40 01933 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
Materials and methods
All injections were administered subcutaneously using 0.1 ml of pure GA dissolved in corn oil. GA was isolated from M. tomentosa as reported elsewhere (Lozoya et al., 1983). Two different assays were carried out. In Group 1, Wistar rats weighing 150 g were ovariectomized under ether anesthesia and left to recover for 20 days. In Group 2, intact Wistar rats weighing 150-200 g and maintained on a 12-h KXOO- 1800 h) light cycle were used after completing two consecutive 5day estrus cycles. The ovariectomized rats of Group 1 were injected once a day with a test drug for 5 days and killed on day 6. The uteri were dissected out at the bifurcation, blotted dry, and weighed immediately on an analytical balance. The uteri were then allowed to dry for 3 days at 135OC and weighed again. The normally cycling intact rats of Group 2 were treated with either GA or vehicle for 10 days and a vaginal smear was analyzed daily at 1100 h. All results were statistically evaluated using Student’s t-test. Resnlts and discussion Wet and dry uterine weights of GA-treated rats were slightly less than those of control rats but this difference was not statistically significant. No evidence of swelling or vascularization was noted for the GA-treated uteri and all uteri (including the controls) appeared blanched and thin on gross observation (Table 1). In the second group, the number of cornified cells was not altered in GAtreated rats relative to controls although a slight but non-significant increase in the length of the estrus cycle was observed in the GA-treated rats (Table 2). The normal 5day cyclic pattern was reestablished 10 days after termination of treatment. Doses of GA used in the first group were 6 times higher than those reported as estrogenic in the case of GA,. For the second group, the doses used were equivalent to those effective in mice (Gawienowski and Chatterjee, 1980). Although slight differences in the uterine response of mice and rats can be expected, it has been shown that the uterine weight method displays similar sensitivity for estradiol in both species (Lauson et al., 1939; TABLE 1 MEAN UTERINE WEIGHT f S.E.M. OF OVARIECTOMIZED WISTAR RATS TREATED WITH 259 rg/kg OF GA DAILY FOR 5 DAYS W = 5 per group) Treatment
Fresh weight (mg)
Dry weight (mg)
Corn oil vehicle
94.2 f 36.1
12.9
f 4.9
GA
76.4 f 15.4
11.5
f 5.3
331 TABLE 2 MEAN LENGTH OF ESTRUS CYCLE (* S.E.M.) OF INTACT RATS TREATED WITH GA (36 &kg, FOR 10 DAYS W = 8 per group) Treatment
Length (days)
Corn oil vehicle
5.40 f 0.55
GA
6.38 f 1.55
Astwood, 1938). The ratios of the potency of estrogenic activity between stilbestrol and the natural estrogens are also considered similar (Sondern and Sealey, 1940). In the present study, there were no evidences of estrogenic activity for GA on the rat at the doses tested. However, a less potent estrogenic effect by GA cannot be discarded. References A&wood, E.B. (1938) A six-hour assay for the quantitative determination of estrogen. Endocrinology 23,25-31. Batra, S. and Bengtsson, B. (1978) Effects of diethylstilboestrol and ovarian steroids on the contractile responses and calcium movements in rat uterine smooth muscle. Journal of Physislogy lLoxd& 276,329-342. &jar, E., Enriquez, R.G. and Lozoya, X. (1984a) The in vitro effect of grandiflorenic acid and zoapatle aqueous crude extract upon spontaneous contractility of the rat uterus during oestrus cycle. Journal of Ethnopharmacology 11, 87-97. Bejar. E., Lozoya, X., Enriquez. R.G. and Escobar, L. (1984b) Comparative effect of zoapatle (Montunou tomentosa) products and on verapamil on the in vitro uterine contractility of rat. Archives de Investigti Medica Mexico) 15,223- 235. Dorfman, R.I. (1954) Bioassay of steroid hormones. Physiological Reviews 34, 138-166. Enriquez, R.G., Escobar, L.I., Romero, M.L., Chavez, M.A. and Lozoya, X. (1983) Determination of grandiflorenic (kauradienoic) acid in organic and aqueous extracts of Montanoo tomentosa (zoapatle) by reversed phase high performance liquid chromatography. Journal of Chromatography 256.297-361. Gallegos, A.J. (1983) The zoapatle. I: a traditional remedy from Mexico emerges to modern times. Contraception 27.211-225. Gawienowski, A.M. and Chatterjee, D. (1980) Effect of prostaglandin inhibitor on the uterotrophic response of estradiol and gibberellic acid. Life Sciences 27, 1393- 1396. Lauson, H.D., Heller, C.G., Golden, J.B. and Sevringhaus. E.L. (1939) The immature rat uterus in the assay of estrogenic substances and a comparison of estradiol, estrone and estriol. Endocrinology 24,35- 44. Lozoya, X., Enriquez, R.G., Bejar, E., Estrada, A.V., Giron, H. and Gallegos, A.J. (1983) The zoapatle. V: The effect of kauradienoic acid upon uterine contractility. Contraception 27, 267 -279. Sondern, C.N. and Sealey, J.L. (1940) The comparative estrogenic potency of diethyl stilbestrol, estrone, estradiol and estriol. Enakrin&gy 27.670-672.
Short
329
communication
PRELIMINARY INVESTIGATION OF THE ESTROGENIC POTENTIAL OF GRANDIFLORENIC ACID FROM MOIWWVOA TOMEN!l’OSA EZRA BEJAR* Unidad de Inveetigacion en Medic& Tmd icionul y Desarrollo Argentinu no. 1 CP 62790, Xochitepec, Morelos IMetiol
de Medicamentos,
I.M.S.S.,
(Accepted April 23.1933)
Introduction Grandiflorenic acid (GA) is a diterpene isolated from Montanoa tomentosa ssp. tomentosa Cervantes (Compositae), a well-known abortifacient plant of Mexico (Gallegos, 1983). Several studies have demonstrated the presence of this compound in the “tea” traditionally used by native women (Enriquez et al., 1983) and its inhibitory effect on the contractility of uterine smooth muscle in vitro (Bejar et al., 1984a, 1984b3. The proposed mechanism of action of GA on uterine contractility is a blockade of calcium intake to smooth muscle cells (Bejar et al., 1984b3 which according to Batra and Bengtsson (1978) could be similar to that of the steroids. Gibberellic acid (GA,), a compound biosynthetically formed in plants from the kaurene ring, has been shown to possess estrogenic activity (Gawienowski and Chatterjee, 1980). Therefore, the present investigation was undertaken to assess if GA would also have some estrogenic activity which might explain the abortifacient effect elicited by Montanoa tomentosa in vivo.
C”2
Fig. 1. Grandiflorenic acid. lPferest addre88:Department of Physiology and Pharmacology, School of Pharmacy, University of the Pacific, Stockton CA 95211, U.S.A. 0373-3741/33/$01.40 01933 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
Materials and methods
All injections were administered subcutaneously using 0.1 ml of pure GA dissolved in corn oil. GA was isolated from M. tomentosa as reported elsewhere (Lozoya et al., 1983). Two different assays were carried out. In Group 1, Wistar rats weighing 150 g were ovariectomized under ether anesthesia and left to recover for 20 days. In Group 2, intact Wistar rats weighing 150-200 g and maintained on a 12-h KXOO- 1800 h) light cycle were used after completing two consecutive 5day estrus cycles. The ovariectomized rats of Group 1 were injected once a day with a test drug for 5 days and killed on day 6. The uteri were dissected out at the bifurcation, blotted dry, and weighed immediately on an analytical balance. The uteri were then allowed to dry for 3 days at 135OC and weighed again. The normally cycling intact rats of Group 2 were treated with either GA or vehicle for 10 days and a vaginal smear was analyzed daily at 1100 h. All results were statistically evaluated using Student’s t-test. Resnlts and discussion Wet and dry uterine weights of GA-treated rats were slightly less than those of control rats but this difference was not statistically significant. No evidence of swelling or vascularization was noted for the GA-treated uteri and all uteri (including the controls) appeared blanched and thin on gross observation (Table 1). In the second group, the number of cornified cells was not altered in GAtreated rats relative to controls although a slight but non-significant increase in the length of the estrus cycle was observed in the GA-treated rats (Table 2). The normal 5day cyclic pattern was reestablished 10 days after termination of treatment. Doses of GA used in the first group were 6 times higher than those reported as estrogenic in the case of GA,. For the second group, the doses used were equivalent to those effective in mice (Gawienowski and Chatterjee, 1980). Although slight differences in the uterine response of mice and rats can be expected, it has been shown that the uterine weight method displays similar sensitivity for estradiol in both species (Lauson et al., 1939; TABLE 1 MEAN UTERINE WEIGHT f S.E.M. OF OVARIECTOMIZED WISTAR RATS TREATED WITH 259 rg/kg OF GA DAILY FOR 5 DAYS W = 5 per group) Treatment
Fresh weight (mg)
Dry weight (mg)
Corn oil vehicle
94.2 f 36.1
12.9
f 4.9
GA
76.4 f 15.4
11.5
f 5.3
331 TABLE 2 MEAN LENGTH OF ESTRUS CYCLE (* S.E.M.) OF INTACT RATS TREATED WITH GA (36 &kg, FOR 10 DAYS W = 8 per group) Treatment
Length (days)
Corn oil vehicle
5.40 f 0.55
GA
6.38 f 1.55
Astwood, 1938). The ratios of the potency of estrogenic activity between stilbestrol and the natural estrogens are also considered similar (Sondern and Sealey, 1940). In the present study, there were no evidences of estrogenic activity for GA on the rat at the doses tested. However, a less potent estrogenic effect by GA cannot be discarded. References A&wood, E.B. (1938) A six-hour assay for the quantitative determination of estrogen. Endocrinology 23,25-31. Batra, S. and Bengtsson, B. (1978) Effects of diethylstilboestrol and ovarian steroids on the contractile responses and calcium movements in rat uterine smooth muscle. Journal of Physislogy lLoxd& 276,329-342. &jar, E., Enriquez, R.G. and Lozoya, X. (1984a) The in vitro effect of grandiflorenic acid and zoapatle aqueous crude extract upon spontaneous contractility of the rat uterus during oestrus cycle. Journal of Ethnopharmacology 11, 87-97. Bejar. E., Lozoya, X., Enriquez. R.G. and Escobar, L. (1984b) Comparative effect of zoapatle (Montunou tomentosa) products and on verapamil on the in vitro uterine contractility of rat. Archives de Investigti Medica Mexico) 15,223- 235. Dorfman, R.I. (1954) Bioassay of steroid hormones. Physiological Reviews 34, 138-166. Enriquez, R.G., Escobar, L.I., Romero, M.L., Chavez, M.A. and Lozoya, X. (1983) Determination of grandiflorenic (kauradienoic) acid in organic and aqueous extracts of Montanoo tomentosa (zoapatle) by reversed phase high performance liquid chromatography. Journal of Chromatography 256.297-361. Gallegos, A.J. (1983) The zoapatle. I: a traditional remedy from Mexico emerges to modern times. Contraception 27.211-225. Gawienowski, A.M. and Chatterjee, D. (1980) Effect of prostaglandin inhibitor on the uterotrophic response of estradiol and gibberellic acid. Life Sciences 27, 1393- 1396. Lauson, H.D., Heller, C.G., Golden, J.B. and Sevringhaus. E.L. (1939) The immature rat uterus in the assay of estrogenic substances and a comparison of estradiol, estrone and estriol. Endocrinology 24,35- 44. Lozoya, X., Enriquez, R.G., Bejar, E., Estrada, A.V., Giron, H. and Gallegos, A.J. (1983) The zoapatle. V: The effect of kauradienoic acid upon uterine contractility. Contraception 27, 267 -279. Sondern, C.N. and Sealey, J.L. (1940) The comparative estrogenic potency of diethyl stilbestrol, estrone, estradiol and estriol. Enakrin&gy 27.670-672.