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Zoapatle XII. In vitro effect of kaurenoic acid isolated From Montanoa frutescens and two derivatives upon human spermatozoa
Journal of Ethnopharmacology, Elsevier Scientific
18 (1986) Publishers Ireland Ltd.
89-94
89
ZOAPATLE XII. IN VITRO EFFECT OF KAURENOIC ACID ISOLATED FROM MONTANOA FRUTESCENS AND TWO DERIVATIVES UPON HUMAN SPERMATOZOA
A. VALENCIA, A, WENS, H. PONCE-MONTE& N, PEDRdN, L. QUIJANOa, J. CALDER6Na, F. G6MEZa and T. RIbSa
AJ. GALLEGOS,
Divisi6n de Biologia Molecular, Unidad de Investigacibn Biomkdica, Centro MMico National, Inetituto Mexican0 de1 Seguro Social, Apartado Postal 73-032, Mkxico, D.F. 03020 and bInstituto de Quimica, Univereidad Autbnoma de Mbxico, Ciudad Universitaria, Mkico, D.F. 04510 (Mexico) (Accepted
August 12,1986)
Summary Zoapatle aqueous crude extract (ZACE) from Montanoa tomentosa has been used as an oral contraceptive in traditional Mexican medicine for centuries. Kaurenoic acid, as isolated from Montanoa frutescent, and the methyl esters of 15-hydroxydihydro-kaurenoic acid and 15-ketodihydrokaurenoic acid were tested in vitro on human sperm motility and viability. The estimated EDs0 concentrations immobilization were 374,126 and 58 pg/ml, respectively, using 15 X lo6 sperms/500 ~1. Kaurenoic acid and both of its derivatives displayed only weak to negligible capacity for killing human sperms.
Introduction It is known that several compounds of vegetable origin possess a spermicidal effect in vitro (Stolsenberg, 1974; Setty et al., 1976). Despite the great diversity of chemical structures, most have adverse reactions that prevent their use (Farnsworth and Waller, 1982). There are several recent reports describing the various biological activities associated with the use of zoapatle aqueous crude extract (ZACE) (Senties and Amayo, 1964; Landgren et al., 1979; Ponce-Monter et al., 1983). Its use in traditional medicine is as a contraceptive agent with clear uterotonic and luteolytic effects in women when administered orally (Landgren et al., 1979; Gallegos and Cortes, 1977; Gallegos, 1985). Only recently the in vitro sperm inhibitory capacity of ZACE was noted when the extract was prepared from Montunoa frutescent. When ZACE was prepared from its traditional source, Montanoa tomentosa, the effect was not present (Wens et al., 1985). The 0378-8741/$02.45 0 1986 Elsevier Scientific Published and Printed in Ireland
Publishers Ireland Ltd.
90
present study reports the in vitro assay of one compound from Montanoa frutescens (kaurenoic acid) and two semi-synthetic derivatives (the methyl esters of 15-hydroxydihydrokaurenoic acid and 15-ketodihydro-kaurenoic acid) upon the viability and motility of human spermatozoa and compares these effects with a clinically used tertiary amine (verapamil). Materials and methods Isolation of kaurenoic acid and preparation of derivatives Montanoa frutescens Cerv. (Compositae, Heliantheae) (Funk, 1980) was collected from the state of Morelos (near Mexico City). Three kilograms of authentic leaves were extracted with hexane and the resultant extract chromatographed through a silica gel column, eluted with benzene/ethyl acetate mixtures. From the polar fractions, kaurenoic acid (compound 1) and its 15~~ isovalerate were isolated. The latter compound was treated with an ether solution of diazomethane, obtaining the methyl ester derivative. Hydrolysis was performed with KOH to produce the alcohol. Further hydrogeneration with PtOz afforded the methyl ester of 15a-hydroxydihydro kaurenoic acid (compound 2). Subsequent Jane’s oxidation resulted in the methyl ester of 15-keto-dihydro-kaurenoic acid (compound 3). Appropriate physical and spectroscopic data were obtained from all three compounds after purification. Structural formulas are shown in Fig. 1. Semen collection and preparation Human volunteers with 48 h of sexual abstinence were the source of semen. Samples were collected in polyethylene beakers. Prior to sperm examination, semen was allowed to liquefy for up to 60 min at room temperature (23 _+2°C). Microscopic semen analysis was performed and only those samples considered normal according to established parameters (Freund and Peterson, 1976) were used for the experiments. The samples were centrifuged for 10 min at 900 X g at room temperature. The pellet was resuspended in Bigger’s Whitten and Wittingham media (BWW) (Biggers et al., 1971) and washed only once using this media. Final concentration of sperm was 60 X 10” spermatozoa/ml. Experimental procedure Each drug was dissolved in 95% ethanol at a concentration of 10 mg/ml. From this solution, 2,4,8 and 16 ~1 were taken and placed in BWW media at pH 7.35 for a final volume of 250 ~1. To each solution, 250 ~1 containing 15 X lo6 of human sperms were added and incubated at 37°C. After 60 s, two aliquots of 10 ~1 each, were taken and assessment of motility and viability of human spermatozoa was made. Percent of motility was evaluated
91
asD 1
coon
keto
. 100
kawenoic
acid
1 200 jJg/ml
. 300 CONCENTRATION
# 400
Fig. 1. Effect of kaurenoic acid and two derivatives on human sperm motility. Incubation was performed in Bigger’s Whitten and Whittingham media pH 7.35 at 37°C for 60 s. Each point represents the average * S.D. of six different experiments Verapamil (O----A)).
under phase-contrast microscopy as described by Zaneveld and Polakoski (1977). Sperm survival was estimated by the method described by Eliasson and Treichl (1971). Linear regression analysis was used to analyse the data. R43SUltS
The effects of (l), (2), (3), and verapamil upon the motility of human spermatozoa are shown in Fig. 1. After 60 s of incubation, the ketokaurenoic acid derivative (3) at a concentration of 320 pg/ml was capable of 100% inhibition of human spermatozoa motility. The hydroxy-kaurenoic acid derivative (2) was less potent since the same concentration was only capable of inhibiting sperm motility by 64%. The least active of the three compounds was kaurenoic acid (1) since (at the same concentration) inhibition of sperm motility was only 51% and not significantly different from the vehicle control. The keto derivative (3) was 5.7 times more potent than verapamil for inhibiting motility of human spermatozoa (Table 1).
92
TABLE I ED,, (gg/ml) FOR INHIBITING MOTILITY OF HUMAN SPERMATOZOA SYSTEM CONTAINING 15 x lo6 SPERMS
IN A TEST
Linear regression analysis was used to analyse the data.
Kaurenoic acid Keto derivative Hydroxy derivative Verapamil -
ED,, (95% confidence limits)
r
Slope
374 58 126 332
0.997 0.993 0.998 0.997
-..____ 0.19 1.27 0.24 0.22
(371-377) (47-69) (123-129) (329-335)
The hydroxy derivative (2) was 2.6 times more potent than verapamil while kaurenoic acid (1) was estimated to be only 0.89. In the assessment of sperm viability, kaurenoic acid and its derivatives as well as verapamil were found to be relatively inactive. Figure 2 shows that the presence of 160 pg of either of .the four compounds in the presence 100 -
90 Kaurenoic
acid
Keto - Kaurenoic Hydroxy-
acld
Kaureneic
acid
Verapamil
o/o
20 -
10 -
100
200
ai/rn!
CONCENTRATION
Fig. 2. The effect of verapamil, kaurenoic acid and two derivatives on human sperm viability. Each point represents average of six different experiments ?r S.D.
93
of 15 X lo6 sperms was capable of decreasing sperm viability only about 10%. At 320 pg/ml, hydroxy-kaurenoic acid appeared to be the most effective spermicide (33%) with kaurenoic acid being somewhat less effective (21%) and keto-kaurenoic acid being least effective (10%). With this incubation time, much higher concentrations would be necessary in order to show useful spermicidal activity.
Wens et al. (1985) have reported that ZACE prepared from M. frutescens is capable of instant immobilization of human spermatozoa. Results of the present study suggest that kaurenoic acid may be partially responsible for the immobilization effect. An interesting effect observed in the present study is that while kaurenoic acid and both of its laboratory-made derivatives have an immobilizing effect, they have only weak or negligible capacity for killing human sperms. A concentration as high as 160 pg in the test system decreased the number of live sperms only 10%. Doses required for immobilization of 50% of human sperms ranged from 58 to 374 pg/ml for the three test compounds using 60 s of exposure. The amount of substance required in our study -was approximately 225 times less than the doses reported by Waller et al. (1980) utilizing gossypol in an in vitro system with unwashed human sperms. In general terms, the active ingredients currently used in intravaginal contraceptives (surfactants such as nonoxynol) fall in the millimolar concentration range. These preliminary observations with kaurenoic acid and its derivatives may be useful in stimulating the synthesis of analogs with possible application in the development of new intravaginal contraceptive agents. References Biggers, J.D., Whitten, W.K. and Whittingham, D.G. (1971) The culture of mouse embryos in vitro. In: J.C. Daniel (Ed,), Methods in Mammalian Embryology, W.H. Freeman and Co., San Francisco, pp. 86-116. Eliasson, R. and Treichl, B. (1971) Supravital staining human spermatozoa. Fertility and Sterility 22, 134-137. Farnsworth, N.R. and WaIIer, D.P. (1982) Current status of plant products reported to inhibit sperm. Research Frontiers in Fertility Regulation 2, l-16. Freund, M. and Peterson, R.N. (1976) Semen evaluation and fertility. In: E.S.E. Hafez (Ed.), The Human Semen and Fertility Regulation in Men, C.V. Mosby, St. Louis, p. 344. Funk, V.A. (1980) Systematics of Montanoa Cerv. (Compositae, Heliantheae), Ohio State University via University Microfilms, Ann Arbor, Michigan. Gallegos, A.J. and Cortes, G.V. (1977) Composition and methods for fertility control. U.S. Patent 4006227. GaBegos, A.J. (1985) Zoapatle VI: Revisited. Contraception 31, 487-497. Landgren, B.M., Aedo, A.R., Hagenfeld, K. and DiczfaIusy, E. (1979) Clinical effects of orally administered effects of Montanoa tomentosa in early human pregnancy, American Journal of Obstetrics and Gynecology 135, 480-484.
94
Ponce-Monter, H., Giron, H., Lozoya, X., Enriquez, R., Bejar, E., Estrada, A.V. and Gallegos, A.J. (1983) Zoapatle III. Biological and uterotonic properties of aqueous plant extract. Contraception 27, 239-253. Quijano, L., Calderon, J.S., Gbmez, F. and Rfos, T. (1979) Montafrusin, a new germacrolide from Montanoa frutescens. Phytochemistry 18, 843-845. Senties, G.L. and Amayo, R. (1964) Efecto de1 Chihuapahtli sobre el dtero human0 gravido. Gaceta M6dica 4,343-350. Setty, B.S., Kamoj, V.P., Garg, H.S. and Khanna, N.M. (1976) Spermicidal potential of saponins isolated from indian medicinal plants. Contraception 14, 571-579. Stolsenberg, S.J. (1974) Spermicidal actions of extracts and compounds from Phytolacca odecandra. Contraception 10, 135-143. Waller, D.F., Zaneveld, L.J. and Fong, H.H. (1980) In oitro spermicidal activity of gossypol. Contraception 22, 183-187. Wens, A., Valencia, A., Pedron, N., Ponce-Monter, H., Guzmln, A. and Gallegos, A.J. (1985) Zoapatle IX. In vitro effect of Montanoa tomentosa and Montanoa frutescens upon human sperm and red cells. Contraception 31, 523-531. Zaneveld, LJ. and Polakoski, K.L. (1977) Collection and physical examination of the ejaculate. In: E.S.E. Hafez, (Ed.), Techniques of Human Andrology, North Holland Publishing Co. Amsterdam, p. 147.
18 (1986) Publishers Ireland Ltd.
89-94
89
ZOAPATLE XII. IN VITRO EFFECT OF KAURENOIC ACID ISOLATED FROM MONTANOA FRUTESCENS AND TWO DERIVATIVES UPON HUMAN SPERMATOZOA
A. VALENCIA, A, WENS, H. PONCE-MONTE& N, PEDRdN, L. QUIJANOa, J. CALDER6Na, F. G6MEZa and T. RIbSa
AJ. GALLEGOS,
Divisi6n de Biologia Molecular, Unidad de Investigacibn Biomkdica, Centro MMico National, Inetituto Mexican0 de1 Seguro Social, Apartado Postal 73-032, Mkxico, D.F. 03020 and bInstituto de Quimica, Univereidad Autbnoma de Mbxico, Ciudad Universitaria, Mkico, D.F. 04510 (Mexico) (Accepted
August 12,1986)
Summary Zoapatle aqueous crude extract (ZACE) from Montanoa tomentosa has been used as an oral contraceptive in traditional Mexican medicine for centuries. Kaurenoic acid, as isolated from Montanoa frutescent, and the methyl esters of 15-hydroxydihydro-kaurenoic acid and 15-ketodihydrokaurenoic acid were tested in vitro on human sperm motility and viability. The estimated EDs0 concentrations immobilization were 374,126 and 58 pg/ml, respectively, using 15 X lo6 sperms/500 ~1. Kaurenoic acid and both of its derivatives displayed only weak to negligible capacity for killing human sperms.
Introduction It is known that several compounds of vegetable origin possess a spermicidal effect in vitro (Stolsenberg, 1974; Setty et al., 1976). Despite the great diversity of chemical structures, most have adverse reactions that prevent their use (Farnsworth and Waller, 1982). There are several recent reports describing the various biological activities associated with the use of zoapatle aqueous crude extract (ZACE) (Senties and Amayo, 1964; Landgren et al., 1979; Ponce-Monter et al., 1983). Its use in traditional medicine is as a contraceptive agent with clear uterotonic and luteolytic effects in women when administered orally (Landgren et al., 1979; Gallegos and Cortes, 1977; Gallegos, 1985). Only recently the in vitro sperm inhibitory capacity of ZACE was noted when the extract was prepared from Montunoa frutescent. When ZACE was prepared from its traditional source, Montanoa tomentosa, the effect was not present (Wens et al., 1985). The 0378-8741/$02.45 0 1986 Elsevier Scientific Published and Printed in Ireland
Publishers Ireland Ltd.
90
present study reports the in vitro assay of one compound from Montanoa frutescens (kaurenoic acid) and two semi-synthetic derivatives (the methyl esters of 15-hydroxydihydrokaurenoic acid and 15-ketodihydro-kaurenoic acid) upon the viability and motility of human spermatozoa and compares these effects with a clinically used tertiary amine (verapamil). Materials and methods Isolation of kaurenoic acid and preparation of derivatives Montanoa frutescens Cerv. (Compositae, Heliantheae) (Funk, 1980) was collected from the state of Morelos (near Mexico City). Three kilograms of authentic leaves were extracted with hexane and the resultant extract chromatographed through a silica gel column, eluted with benzene/ethyl acetate mixtures. From the polar fractions, kaurenoic acid (compound 1) and its 15~~ isovalerate were isolated. The latter compound was treated with an ether solution of diazomethane, obtaining the methyl ester derivative. Hydrolysis was performed with KOH to produce the alcohol. Further hydrogeneration with PtOz afforded the methyl ester of 15a-hydroxydihydro kaurenoic acid (compound 2). Subsequent Jane’s oxidation resulted in the methyl ester of 15-keto-dihydro-kaurenoic acid (compound 3). Appropriate physical and spectroscopic data were obtained from all three compounds after purification. Structural formulas are shown in Fig. 1. Semen collection and preparation Human volunteers with 48 h of sexual abstinence were the source of semen. Samples were collected in polyethylene beakers. Prior to sperm examination, semen was allowed to liquefy for up to 60 min at room temperature (23 _+2°C). Microscopic semen analysis was performed and only those samples considered normal according to established parameters (Freund and Peterson, 1976) were used for the experiments. The samples were centrifuged for 10 min at 900 X g at room temperature. The pellet was resuspended in Bigger’s Whitten and Wittingham media (BWW) (Biggers et al., 1971) and washed only once using this media. Final concentration of sperm was 60 X 10” spermatozoa/ml. Experimental procedure Each drug was dissolved in 95% ethanol at a concentration of 10 mg/ml. From this solution, 2,4,8 and 16 ~1 were taken and placed in BWW media at pH 7.35 for a final volume of 250 ~1. To each solution, 250 ~1 containing 15 X lo6 of human sperms were added and incubated at 37°C. After 60 s, two aliquots of 10 ~1 each, were taken and assessment of motility and viability of human spermatozoa was made. Percent of motility was evaluated
91
asD 1
coon
keto
. 100
kawenoic
acid
1 200 jJg/ml
. 300 CONCENTRATION
# 400
Fig. 1. Effect of kaurenoic acid and two derivatives on human sperm motility. Incubation was performed in Bigger’s Whitten and Whittingham media pH 7.35 at 37°C for 60 s. Each point represents the average * S.D. of six different experiments Verapamil (O----A)).
under phase-contrast microscopy as described by Zaneveld and Polakoski (1977). Sperm survival was estimated by the method described by Eliasson and Treichl (1971). Linear regression analysis was used to analyse the data. R43SUltS
The effects of (l), (2), (3), and verapamil upon the motility of human spermatozoa are shown in Fig. 1. After 60 s of incubation, the ketokaurenoic acid derivative (3) at a concentration of 320 pg/ml was capable of 100% inhibition of human spermatozoa motility. The hydroxy-kaurenoic acid derivative (2) was less potent since the same concentration was only capable of inhibiting sperm motility by 64%. The least active of the three compounds was kaurenoic acid (1) since (at the same concentration) inhibition of sperm motility was only 51% and not significantly different from the vehicle control. The keto derivative (3) was 5.7 times more potent than verapamil for inhibiting motility of human spermatozoa (Table 1).
92
TABLE I ED,, (gg/ml) FOR INHIBITING MOTILITY OF HUMAN SPERMATOZOA SYSTEM CONTAINING 15 x lo6 SPERMS
IN A TEST
Linear regression analysis was used to analyse the data.
Kaurenoic acid Keto derivative Hydroxy derivative Verapamil -
ED,, (95% confidence limits)
r
Slope
374 58 126 332
0.997 0.993 0.998 0.997
-..____ 0.19 1.27 0.24 0.22
(371-377) (47-69) (123-129) (329-335)
The hydroxy derivative (2) was 2.6 times more potent than verapamil while kaurenoic acid (1) was estimated to be only 0.89. In the assessment of sperm viability, kaurenoic acid and its derivatives as well as verapamil were found to be relatively inactive. Figure 2 shows that the presence of 160 pg of either of .the four compounds in the presence 100 -
90 Kaurenoic
acid
Keto - Kaurenoic Hydroxy-
acld
Kaureneic
acid
Verapamil
o/o
20 -
10 -
100
200
ai/rn!
CONCENTRATION
Fig. 2. The effect of verapamil, kaurenoic acid and two derivatives on human sperm viability. Each point represents average of six different experiments ?r S.D.
93
of 15 X lo6 sperms was capable of decreasing sperm viability only about 10%. At 320 pg/ml, hydroxy-kaurenoic acid appeared to be the most effective spermicide (33%) with kaurenoic acid being somewhat less effective (21%) and keto-kaurenoic acid being least effective (10%). With this incubation time, much higher concentrations would be necessary in order to show useful spermicidal activity.
Wens et al. (1985) have reported that ZACE prepared from M. frutescens is capable of instant immobilization of human spermatozoa. Results of the present study suggest that kaurenoic acid may be partially responsible for the immobilization effect. An interesting effect observed in the present study is that while kaurenoic acid and both of its laboratory-made derivatives have an immobilizing effect, they have only weak or negligible capacity for killing human sperms. A concentration as high as 160 pg in the test system decreased the number of live sperms only 10%. Doses required for immobilization of 50% of human sperms ranged from 58 to 374 pg/ml for the three test compounds using 60 s of exposure. The amount of substance required in our study -was approximately 225 times less than the doses reported by Waller et al. (1980) utilizing gossypol in an in vitro system with unwashed human sperms. In general terms, the active ingredients currently used in intravaginal contraceptives (surfactants such as nonoxynol) fall in the millimolar concentration range. These preliminary observations with kaurenoic acid and its derivatives may be useful in stimulating the synthesis of analogs with possible application in the development of new intravaginal contraceptive agents. References Biggers, J.D., Whitten, W.K. and Whittingham, D.G. (1971) The culture of mouse embryos in vitro. In: J.C. Daniel (Ed,), Methods in Mammalian Embryology, W.H. Freeman and Co., San Francisco, pp. 86-116. Eliasson, R. and Treichl, B. (1971) Supravital staining human spermatozoa. Fertility and Sterility 22, 134-137. Farnsworth, N.R. and WaIIer, D.P. (1982) Current status of plant products reported to inhibit sperm. Research Frontiers in Fertility Regulation 2, l-16. Freund, M. and Peterson, R.N. (1976) Semen evaluation and fertility. In: E.S.E. Hafez (Ed.), The Human Semen and Fertility Regulation in Men, C.V. Mosby, St. Louis, p. 344. Funk, V.A. (1980) Systematics of Montanoa Cerv. (Compositae, Heliantheae), Ohio State University via University Microfilms, Ann Arbor, Michigan. Gallegos, A.J. and Cortes, G.V. (1977) Composition and methods for fertility control. U.S. Patent 4006227. GaBegos, A.J. (1985) Zoapatle VI: Revisited. Contraception 31, 487-497. Landgren, B.M., Aedo, A.R., Hagenfeld, K. and DiczfaIusy, E. (1979) Clinical effects of orally administered effects of Montanoa tomentosa in early human pregnancy, American Journal of Obstetrics and Gynecology 135, 480-484.
94
Ponce-Monter, H., Giron, H., Lozoya, X., Enriquez, R., Bejar, E., Estrada, A.V. and Gallegos, A.J. (1983) Zoapatle III. Biological and uterotonic properties of aqueous plant extract. Contraception 27, 239-253. Quijano, L., Calderon, J.S., Gbmez, F. and Rfos, T. (1979) Montafrusin, a new germacrolide from Montanoa frutescens. Phytochemistry 18, 843-845. Senties, G.L. and Amayo, R. (1964) Efecto de1 Chihuapahtli sobre el dtero human0 gravido. Gaceta M6dica 4,343-350. Setty, B.S., Kamoj, V.P., Garg, H.S. and Khanna, N.M. (1976) Spermicidal potential of saponins isolated from indian medicinal plants. Contraception 14, 571-579. Stolsenberg, S.J. (1974) Spermicidal actions of extracts and compounds from Phytolacca odecandra. Contraception 10, 135-143. Waller, D.F., Zaneveld, L.J. and Fong, H.H. (1980) In oitro spermicidal activity of gossypol. Contraception 22, 183-187. Wens, A., Valencia, A., Pedron, N., Ponce-Monter, H., Guzmln, A. and Gallegos, A.J. (1985) Zoapatle IX. In vitro effect of Montanoa tomentosa and Montanoa frutescens upon human sperm and red cells. Contraception 31, 523-531. Zaneveld, LJ. and Polakoski, K.L. (1977) Collection and physical examination of the ejaculate. In: E.S.E. Hafez, (Ed.), Techniques of Human Andrology, North Holland Publishing Co. Amsterdam, p. 147.