- Email: [email protected]
The antigonadotropic activity of Lithospermum ruderale I. The lack of steroid-like activity at the receptor level
CONTRACEPTION
IHEANrI~PIcAc!rIvITyoFLI~~~ I. TIiEIXKOFSIXXX~LIEEACIIVPIYAT!IHEEZEPIDE~ WilliamE. Findleyand BarryR. Jacobs Department of Obstetricsand Gynecology, The University of Texas MedicalSchcolat Ecustcn,TexasMedicalCenter,Eoustcn,Texas 77030
Previous work has demznstrated that extracts from the plant Lithospermrm ruderalehave antigonadotrcpic activity. The possibility of steroidal substancesbeim resncnsiblefor this activity was examinedat the receptorlevel.-It w&a foundthat only at very high concentraticnsof crude lit&sperm extract (IS) was progesterone, testosterone, or estradiol displaced fran their respectiveuterine receptors.Because such hich dosesof IS may be toxicand because they are 'lotrequiredfor in viva or _in vitrobio&ivity, it is ooncluded that the antigcnadotr~iGi%pcnents of Lithospe rmum ruderale probably do not act as a steroid.
Extracts fran a variety of plants have been shown to have antigonadotropic activity(1). In 1941,Train-et al. (2)reported that certain Indian tribes of Nevada used an extract of the plant Lithcspernum ruderaleas a contraceptive.The ensuingstudies of this plant involved feeding dried material to mice. This resulted in anestrus(3),reportedly by inhibitingthe productia of luteinizing hormone(LE) (4,5). More recently,extracts of Lithospermumruderale (hereafter referredto as lithosperm), have been sy7)to zreass endogemus gonadotrcpic activity in ccckerels gcnadotropicactivity of excgenouslyadministeredIH' and follicle stimulating hormcne@SE) (8). The actual mechanism of action of lithospermis unkmwn and is therefore being studied in this laboratory.This reportis ccncerned with elucidatingthe mode of actionof the antigcnadotropic coqznents of lithosperm. Many plants are kna+n to contain estrogens and estrcgenicsubstances(9). Such ampounds could account for the antigcmadotropic activities which havebeen observed in lithospermextracts. Consequently this study was undertakento determine if ccqonents of lithcsperm extractare capableof actingas steroids at the receptor level. Accepted for publication December 28, 1979
FEBRUARY
1980VOL.21N0.2
199
CONTRACEPTION
All incubations were performedin TEIG buffer,consisting of 10 z+l Tris-HCl(pH7.4), 1.5 XTM BETA, and 12 r&l thioglycerolwith 10% ruderalewere reduced glycerol. Coarselygroundrootsof Lithospermrm to a finepowder with a mortar and pestle and suspended in TEIG buffer. This suspension was then spun at 14009x 20 minutes to renove particulate matter. Steroid receptorswere prepared as previouslydescribed (10). Bovine uterine tissue was hcmcgenized at 4“C with a Tekmar Tissuemizer in a 4-fold excess (ml/g tissue) of TEX buffer. A 200,OOOgsupernatant(cytceol) was used as the receptorsource. Receptors were added to tubes containing3H-steroidand cold (hanolcgous) steroid,lit-m extract,or buffer alone. After an overnightincubatimat 4"C, 0.5 ml of buffer containingcharcoal/ dextranwas addedto each tube which was then vortex& and centrifuged. The supernatantattaining receptor-bound 3H-steroid was decanted into scintillationvials containing 5 ml Scintiverse (Fisher).Vialswere counted5 minutes in a Packard Tricarb Liquid ScintillationSpectrometer. 3H-Steroidsincludedprogesterone(P), testosterone (T)j and 17Wstradiol (E). Diethylstilbesterol (DES) was used as the H-E ccmpetitor.
Preliminary experiments revealedthat lithcepermextract (IS) did not displaceP, T, or E at a dose of 60 ug (0.3 mg equivalents/ml) or less. The resultsdepictedin Figure1 indicate that scareinhibition of 3H-steroid bindingdid occur at a IS dose of 600 ug (3.0 nrg equivalents/ml). Whileonly 16-17%of the specificcounts bound of E and T were displacedby the 600 ug dose of IS, 52% of the P was displaced. A aznpetivebirdingassay was thereforeperformedwith P. The results(Figure2) indicatethat P is approximately290,000 times as potentas the crudeLS cn a weight basis. It should also be noted that,at the high concentrations of crude Is, the nonspecificcounts remainingin the supernatant were increasedup to 4.5-fold (Figure 3); that is theseconcentrations of LS were sufficientto inhibit the actual5H-P adsorptionto the charcoal used to separate the free steroidfran the receptor-bound steroidin the supernatant.
200
FEBRUARY
1980 VOL. 21 NO. 2
CONTRACEPTION
0
100 200 ng E
60
0
100
60
200
100
ng P
600 119 LS
ng T
0
600 ug LS
200
600
60
lJg
LS
Effect of litbosperm extract an the binding of Figure 1. 3H-steroids to the appropriate uterine cytcsol receptors. To tubes containing 3H-estradiol 3H-testosterone (E) , (T) r 3H-progesterone (P) was tied either buffer alone (O), litho&: doses extract (Is), or a large excess of cold hormone at the indicated. uterine Steroid receptors fran a 200,ooog W=ol After preparaticm were then added. overnight incubation dextran-coated charcoal was vortexed with zccntents of each tube & adsorb the free steroid. Following a 1400g centrifugation, the receptor-bound %-I-steroid in the supernatant was counted. Mean 2 standard deviation, n = 2.
FEBRUARY
1980 VOL. 21 NO. 2
201
CONTRACEPTION
100.
L
,
I
w P
0.51
ug u
5
10
I
I
2
5
20
50
I
10
20
100 200
50
I
100 200
500 1000 2000
Effect of increasingdases of progesterone (P) or lithosperm extract(Is) on the binding of 3H-P to bovine uterine were the same as those of cytceolP receptors.The assayccnditicns
Figure 2.
&an
+ standard deviation contained'within
points,n = 2.
H’ ‘Ii........ L I.
1
250
I
500
I
I
1000
2000
ug IA
Inhibitianof 3H-P adsorpticntocharchoalty Figure3. high ccncentrations of lithosperm.Same exteriment as Figure2 except that an equalvolumeof bufferwas substituted for the receptor preparation addedto each tube. Dottedline: baselineunadsorbed cpn raining in supernatant in the absence of lithospermextract. Mean f standard deviation, containedwithinpoints,n = 2.
202
FEBRUARY 1980VOL.21N0.2
CONTRACEPTION
DISUJSSION The antigonadotropic activityof lithosperm has been attributed,at leastin part,to lithcepermic acid (11,12). This polyphenolicacid has a molecularweightof 538 and represents2-3%of the dry weight of the LS (13). If litt-cspermic acid was the sole factor inhibitingP bindingin Figure2, then it is 3400-5100fold lesspotentthanP on a molarbasis. In the assays reported herein, the 3H-P not bound to the receptorsis adsorbedby dextran-coated charcoalwhich is then removed by centrifugaticn. The resultingsupernatant should thereforecontain only 3H-P boundto receptors.Hcwever, the data sti in Figure 3 indicatethat at LS doses of 25Oug or more, there is sufficient materialin solutionto directly inhibit the adsorptionof 3H-P by the charcoal. If the LS curve in Figure 2 is corrected for this artifact,litbpermic acid remains2900-4300fold lesspotent than P. This overall lack of potency (2900-5100 fold) indicates that lithospermicacid is not binding to intracellularprogesterone receptors. The bindingof 3H-Pwas inhibitedby crude lithospermextracts at doses increasing fran 250 ug (417 ug/ml incubationvolume). Such extractswere dark brm in appearance, beingsanewhatopagueabove the 1000 ug dose. These high ccncentraticnsof crude material do not allowfor an accurateevaluationof receptor binding. As discussed above,therewas actuallysufficient materialin solution to directly interferwith the adsorption of 3H-P to the dextran-ccatedcharcoal of LS which interferedwith receptor (Figure3). The concentrations bindingwere also much higher (>>33 fold) than those required to Likewise, inhibitLEF-stimulated gonadotropin releasein vitro (15)when the serum concentrationsof IS are X&iiZZd fran the doses reportedwith in vivo experiments(4,5,6),antigonadotripic activity is observedwith zlmely smallconcentrations of Is. The aboveobservations suggestthat the inhibitionobserved with the largeIs concentraticns was not due to the specificbindingof sane Is ccsponentto the cytcsolP receptors.Rather, the LSwas apparently exertinga sore general inhibitioncn receptor birding, perhaps by limitingthe availability of eitherthe 3H-P or the receptor binding sites. Futureexperiments will be conductedwith partially purified extractin order to minimizeany nonspecific effectsdue to the large excessof crudematerial. AlthoughP bindingwas the rrvlst sensitiveto LS inhibition, E and T bindingwere also inhibitedslightlyby the 600 ug dose of IS (Fiure 1). Sincemore than 91% of the 3H-E was displzed by DES, the 9H-E bindingdid not involvesteroidbindingglobulins(TE8G)but rather the intracellular receptors(16,17).Uterineestrogen receptors,as were used in this study,are quite specific and sensitive for detecting
FEBRUARY
1980VOL.21N0.2
203
CONTRACEPTION
estrogensincluding phytoestrogens (14). Hckvever,as discussed above for P, the inhibition of the binding of both 3H-E and 3H-T by the high dcse of I.5probablydid not involvespecificbindingof IS to the E receptorsand T receptors, respectively. In sumnary, these preliminaryexperimentssuggest that, unlike other plant-derivedantigcnadotropic substances,lithosperm,or at leastlithospermic acid, exerts its antigonadotropic effect without actingas a steroid. Studiesdesignedto further elucidateboth the chemicalnatureof the amponents-of lithospermand their mode of acticnare currentlyin progressand will be the subject of future reports.
The authorswish to expresstheirappreciation to Dr. Frank Zeller, IndianaUniversity, for kindlysupplyingthe lithospermroots used in this study. REFERENCES
1. Brondegaard, VJ. 23:167-172, 1973.
Contraceptive plant drugs.
Planta Med
2. Train,P, Henrichs,JBandArcher,WA. Medicinaluses of plants by Indiantribesof Nevada. Contributions towarda flora of Nevada. No. 33, Divisionof Plantl%cploraticn and Introducticn.Bureau of Plant Industry, US Dept of Agriculture, Washington, D.C., 1941, p. 102. 3. Cranston,EM. The effectof Lithosperxum ruderale cn the estrous cycleof mice. J PharmacolExp Ther 83:130-142, 1945. 4.
Drasher,ML. Furtherobservations QI the inhibition of luteinizing hormoneby lithcsperm.Endocrinology 47:399-413, 1950.
5. Noble,I&, Plunkett,ER and Graham,KCB. Directhormcne inhibition by extractsof Lithospermnnruderale. J Endccrinol10:212-227, 1954. 6. Breneman,WB and Zeller, FJ. Lithcsperm inhibitim of anterior pituitaryhormones.BiochemBiophysBes Camn 65:1047-1053, 1975. 7. Zeller,FJ, Breneman,WB, Carmack, M, and Kelly, CJ. Biphasic effectof extractsof Lithospermum ruderalecn the uptake of by testesand thyroids of cockerels. Biochem Biophys F&s & 67:1234-1241, 1975. 8. Breneman,WB, Zeller,FJ, Carmack,M, Kelley,CJ. In vivo inhibitionof gonadotropins and thyrotropin in the chick-b extracts of Lithcspermum ruderale.Gen Canp Endocrinol28:24-32,1976.
204
FEBRUARY 1980 VOL. 21 NO. 2
CONTRACEPTION
9.
Farnsworth, MH, Bingel,As, Cordell,GA, Crane,FA, and Fang, HHS. Pontential valueof plantsas sourcesof new antifertilityagents II. J PharmSci 64:717-754, 1975.
10. Jacobs,B and SmithR. Evidencefor a receptor-likeprotein for progesterone in bovineovariancytosol. Endccrinolcgy.In press. 11. Gassner,FK, Hopwocd,ML, Jachle,W, Johnson, G, and Sunderwirth, SG. Antifertility activityof an oxidized polyphenolicacid from Lithospermum ruderale.Proc Sot Exp Biol Mad 114:20-25, 1963. 12. Wagner,H, Horhaxmer, L, and Frank,V. Lithcspermsaure, das antihormonale Wirkprinzipvcn Lycopus europaeus L. (Wolfsfusz)und SynphytumofficinateL. (Bienwell) . Arzneim-Forsch20:705-713, 1970. 13. Kelly,CJ, Mahajan,JR, Brooks,LC, Neubert,IA, Breneman,WH, and acidsof Lithcsperxnnn ruderale Dougl. ex Carmack,M. Polyphenolic Lehm. (Boraginaceae). 1. Isolaticnand structuredeterminationof lithospermic acid. J Grg Chem 40:1804-1815, 1975. bindingto sheep 14. Shutt,DA and Cox, HI. Steroidand phyto-estrogen uterinereceptors in vitro. J Endccr 52:299-310, 1972. -15. Findley,W. The inhibition of LRF-induced gcnadotropinrelease -in vitroby lithosperm.In preparaticm. 16. Murphy,B.E. Bit-dingof testosteroneand estradiol in plasma. Can. J. Biochem.46:299-302, 1968. Unique 17. Soloff, M.S., Creange, J.E., arKI Potts, G-0. estrogen-binding properties of rat pregnancyplasma. Endocrinology 88:427-432, 1971.
FEBRUARY
1980VOL.21N0.2
205
IHEANrI~PIcAc!rIvITyoFLI~~~ I. TIiEIXKOFSIXXX~LIEEACIIVPIYAT!IHEEZEPIDE~ WilliamE. Findleyand BarryR. Jacobs Department of Obstetricsand Gynecology, The University of Texas MedicalSchcolat Ecustcn,TexasMedicalCenter,Eoustcn,Texas 77030
Previous work has demznstrated that extracts from the plant Lithospermrm ruderalehave antigonadotrcpic activity. The possibility of steroidal substancesbeim resncnsiblefor this activity was examinedat the receptorlevel.-It w&a foundthat only at very high concentraticnsof crude lit&sperm extract (IS) was progesterone, testosterone, or estradiol displaced fran their respectiveuterine receptors.Because such hich dosesof IS may be toxicand because they are 'lotrequiredfor in viva or _in vitrobio&ivity, it is ooncluded that the antigcnadotr~iGi%pcnents of Lithospe rmum ruderale probably do not act as a steroid.
Extracts fran a variety of plants have been shown to have antigonadotropic activity(1). In 1941,Train-et al. (2)reported that certain Indian tribes of Nevada used an extract of the plant Lithcspernum ruderaleas a contraceptive.The ensuingstudies of this plant involved feeding dried material to mice. This resulted in anestrus(3),reportedly by inhibitingthe productia of luteinizing hormone(LE) (4,5). More recently,extracts of Lithospermumruderale (hereafter referredto as lithosperm), have been sy7)to zreass endogemus gonadotrcpic activity in ccckerels gcnadotropicactivity of excgenouslyadministeredIH' and follicle stimulating hormcne@SE) (8). The actual mechanism of action of lithospermis unkmwn and is therefore being studied in this laboratory.This reportis ccncerned with elucidatingthe mode of actionof the antigcnadotropic coqznents of lithosperm. Many plants are kna+n to contain estrogens and estrcgenicsubstances(9). Such ampounds could account for the antigcmadotropic activities which havebeen observed in lithospermextracts. Consequently this study was undertakento determine if ccqonents of lithcsperm extractare capableof actingas steroids at the receptor level. Accepted for publication December 28, 1979
FEBRUARY
1980VOL.21N0.2
199
CONTRACEPTION
All incubations were performedin TEIG buffer,consisting of 10 z+l Tris-HCl(pH7.4), 1.5 XTM BETA, and 12 r&l thioglycerolwith 10% ruderalewere reduced glycerol. Coarselygroundrootsof Lithospermrm to a finepowder with a mortar and pestle and suspended in TEIG buffer. This suspension was then spun at 14009x 20 minutes to renove particulate matter. Steroid receptorswere prepared as previouslydescribed (10). Bovine uterine tissue was hcmcgenized at 4“C with a Tekmar Tissuemizer in a 4-fold excess (ml/g tissue) of TEX buffer. A 200,OOOgsupernatant(cytceol) was used as the receptorsource. Receptors were added to tubes containing3H-steroidand cold (hanolcgous) steroid,lit-m extract,or buffer alone. After an overnightincubatimat 4"C, 0.5 ml of buffer containingcharcoal/ dextranwas addedto each tube which was then vortex& and centrifuged. The supernatantattaining receptor-bound 3H-steroid was decanted into scintillationvials containing 5 ml Scintiverse (Fisher).Vialswere counted5 minutes in a Packard Tricarb Liquid ScintillationSpectrometer. 3H-Steroidsincludedprogesterone(P), testosterone (T)j and 17Wstradiol (E). Diethylstilbesterol (DES) was used as the H-E ccmpetitor.
Preliminary experiments revealedthat lithcepermextract (IS) did not displaceP, T, or E at a dose of 60 ug (0.3 mg equivalents/ml) or less. The resultsdepictedin Figure1 indicate that scareinhibition of 3H-steroid bindingdid occur at a IS dose of 600 ug (3.0 nrg equivalents/ml). Whileonly 16-17%of the specificcounts bound of E and T were displacedby the 600 ug dose of IS, 52% of the P was displaced. A aznpetivebirdingassay was thereforeperformedwith P. The results(Figure2) indicatethat P is approximately290,000 times as potentas the crudeLS cn a weight basis. It should also be noted that,at the high concentrations of crude Is, the nonspecificcounts remainingin the supernatant were increasedup to 4.5-fold (Figure 3); that is theseconcentrations of LS were sufficientto inhibit the actual5H-P adsorptionto the charcoal used to separate the free steroidfran the receptor-bound steroidin the supernatant.
200
FEBRUARY
1980 VOL. 21 NO. 2
CONTRACEPTION
0
100 200 ng E
60
0
100
60
200
100
ng P
600 119 LS
ng T
0
600 ug LS
200
600
60
lJg
LS
Effect of litbosperm extract an the binding of Figure 1. 3H-steroids to the appropriate uterine cytcsol receptors. To tubes containing 3H-estradiol 3H-testosterone (E) , (T) r 3H-progesterone (P) was tied either buffer alone (O), litho&: doses extract (Is), or a large excess of cold hormone at the indicated. uterine Steroid receptors fran a 200,ooog W=ol After preparaticm were then added. overnight incubation dextran-coated charcoal was vortexed with zccntents of each tube & adsorb the free steroid. Following a 1400g centrifugation, the receptor-bound %-I-steroid in the supernatant was counted. Mean 2 standard deviation, n = 2.
FEBRUARY
1980 VOL. 21 NO. 2
201
CONTRACEPTION
100.
L
,
I
w P
0.51
ug u
5
10
I
I
2
5
20
50
I
10
20
100 200
50
I
100 200
500 1000 2000
Effect of increasingdases of progesterone (P) or lithosperm extract(Is) on the binding of 3H-P to bovine uterine were the same as those of cytceolP receptors.The assayccnditicns
Figure 2.
&an
+ standard deviation contained'within
points,n = 2.
H’ ‘Ii........ L I.
1
250
I
500
I
I
1000
2000
ug IA
Inhibitianof 3H-P adsorpticntocharchoalty Figure3. high ccncentrations of lithosperm.Same exteriment as Figure2 except that an equalvolumeof bufferwas substituted for the receptor preparation addedto each tube. Dottedline: baselineunadsorbed cpn raining in supernatant in the absence of lithospermextract. Mean f standard deviation, containedwithinpoints,n = 2.
202
FEBRUARY 1980VOL.21N0.2
CONTRACEPTION
DISUJSSION The antigonadotropic activityof lithosperm has been attributed,at leastin part,to lithcepermic acid (11,12). This polyphenolicacid has a molecularweightof 538 and represents2-3%of the dry weight of the LS (13). If litt-cspermic acid was the sole factor inhibitingP bindingin Figure2, then it is 3400-5100fold lesspotentthanP on a molarbasis. In the assays reported herein, the 3H-P not bound to the receptorsis adsorbedby dextran-coated charcoalwhich is then removed by centrifugaticn. The resultingsupernatant should thereforecontain only 3H-P boundto receptors.Hcwever, the data sti in Figure 3 indicatethat at LS doses of 25Oug or more, there is sufficient materialin solutionto directly inhibit the adsorptionof 3H-P by the charcoal. If the LS curve in Figure 2 is corrected for this artifact,litbpermic acid remains2900-4300fold lesspotent than P. This overall lack of potency (2900-5100 fold) indicates that lithospermicacid is not binding to intracellularprogesterone receptors. The bindingof 3H-Pwas inhibitedby crude lithospermextracts at doses increasing fran 250 ug (417 ug/ml incubationvolume). Such extractswere dark brm in appearance, beingsanewhatopagueabove the 1000 ug dose. These high ccncentraticnsof crude material do not allowfor an accurateevaluationof receptor binding. As discussed above,therewas actuallysufficient materialin solution to directly interferwith the adsorption of 3H-P to the dextran-ccatedcharcoal of LS which interferedwith receptor (Figure3). The concentrations bindingwere also much higher (>>33 fold) than those required to Likewise, inhibitLEF-stimulated gonadotropin releasein vitro (15)when the serum concentrationsof IS are X&iiZZd fran the doses reportedwith in vivo experiments(4,5,6),antigonadotripic activity is observedwith zlmely smallconcentrations of Is. The aboveobservations suggestthat the inhibitionobserved with the largeIs concentraticns was not due to the specificbindingof sane Is ccsponentto the cytcsolP receptors.Rather, the LSwas apparently exertinga sore general inhibitioncn receptor birding, perhaps by limitingthe availability of eitherthe 3H-P or the receptor binding sites. Futureexperiments will be conductedwith partially purified extractin order to minimizeany nonspecific effectsdue to the large excessof crudematerial. AlthoughP bindingwas the rrvlst sensitiveto LS inhibition, E and T bindingwere also inhibitedslightlyby the 600 ug dose of IS (Fiure 1). Sincemore than 91% of the 3H-E was displzed by DES, the 9H-E bindingdid not involvesteroidbindingglobulins(TE8G)but rather the intracellular receptors(16,17).Uterineestrogen receptors,as were used in this study,are quite specific and sensitive for detecting
FEBRUARY
1980VOL.21N0.2
203
CONTRACEPTION
estrogensincluding phytoestrogens (14). Hckvever,as discussed above for P, the inhibition of the binding of both 3H-E and 3H-T by the high dcse of I.5probablydid not involvespecificbindingof IS to the E receptorsand T receptors, respectively. In sumnary, these preliminaryexperimentssuggest that, unlike other plant-derivedantigcnadotropic substances,lithosperm,or at leastlithospermic acid, exerts its antigonadotropic effect without actingas a steroid. Studiesdesignedto further elucidateboth the chemicalnatureof the amponents-of lithospermand their mode of acticnare currentlyin progressand will be the subject of future reports.
The authorswish to expresstheirappreciation to Dr. Frank Zeller, IndianaUniversity, for kindlysupplyingthe lithospermroots used in this study. REFERENCES
1. Brondegaard, VJ. 23:167-172, 1973.
Contraceptive plant drugs.
Planta Med
2. Train,P, Henrichs,JBandArcher,WA. Medicinaluses of plants by Indiantribesof Nevada. Contributions towarda flora of Nevada. No. 33, Divisionof Plantl%cploraticn and Introducticn.Bureau of Plant Industry, US Dept of Agriculture, Washington, D.C., 1941, p. 102. 3. Cranston,EM. The effectof Lithosperxum ruderale cn the estrous cycleof mice. J PharmacolExp Ther 83:130-142, 1945. 4.
Drasher,ML. Furtherobservations QI the inhibition of luteinizing hormoneby lithcsperm.Endocrinology 47:399-413, 1950.
5. Noble,I&, Plunkett,ER and Graham,KCB. Directhormcne inhibition by extractsof Lithospermnnruderale. J Endccrinol10:212-227, 1954. 6. Breneman,WB and Zeller, FJ. Lithcsperm inhibitim of anterior pituitaryhormones.BiochemBiophysBes Camn 65:1047-1053, 1975. 7. Zeller,FJ, Breneman,WB, Carmack, M, and Kelly, CJ. Biphasic effectof extractsof Lithospermum ruderalecn the uptake of by testesand thyroids of cockerels. Biochem Biophys F&s & 67:1234-1241, 1975. 8. Breneman,WB, Zeller,FJ, Carmack,M, Kelley,CJ. In vivo inhibitionof gonadotropins and thyrotropin in the chick-b extracts of Lithcspermum ruderale.Gen Canp Endocrinol28:24-32,1976.
204
FEBRUARY 1980 VOL. 21 NO. 2
CONTRACEPTION
9.
Farnsworth, MH, Bingel,As, Cordell,GA, Crane,FA, and Fang, HHS. Pontential valueof plantsas sourcesof new antifertilityagents II. J PharmSci 64:717-754, 1975.
10. Jacobs,B and SmithR. Evidencefor a receptor-likeprotein for progesterone in bovineovariancytosol. Endccrinolcgy.In press. 11. Gassner,FK, Hopwocd,ML, Jachle,W, Johnson, G, and Sunderwirth, SG. Antifertility activityof an oxidized polyphenolicacid from Lithospermum ruderale.Proc Sot Exp Biol Mad 114:20-25, 1963. 12. Wagner,H, Horhaxmer, L, and Frank,V. Lithcspermsaure, das antihormonale Wirkprinzipvcn Lycopus europaeus L. (Wolfsfusz)und SynphytumofficinateL. (Bienwell) . Arzneim-Forsch20:705-713, 1970. 13. Kelly,CJ, Mahajan,JR, Brooks,LC, Neubert,IA, Breneman,WH, and acidsof Lithcsperxnnn ruderale Dougl. ex Carmack,M. Polyphenolic Lehm. (Boraginaceae). 1. Isolaticnand structuredeterminationof lithospermic acid. J Grg Chem 40:1804-1815, 1975. bindingto sheep 14. Shutt,DA and Cox, HI. Steroidand phyto-estrogen uterinereceptors in vitro. J Endccr 52:299-310, 1972. -15. Findley,W. The inhibition of LRF-induced gcnadotropinrelease -in vitroby lithosperm.In preparaticm. 16. Murphy,B.E. Bit-dingof testosteroneand estradiol in plasma. Can. J. Biochem.46:299-302, 1968. Unique 17. Soloff, M.S., Creange, J.E., arKI Potts, G-0. estrogen-binding properties of rat pregnancyplasma. Endocrinology 88:427-432, 1971.
FEBRUARY
1980VOL.21N0.2
205