CONTRACEPTION
ANTIFERTILITYEFFICACY OF THE PLART ARISTOIJOCHIA INDICA LIRN OR MOUSE
Anita Pslcrashiand Pranab Pakrasi Reproductive Biology Research Section Indian Institute of Experimental Medicine 4, Raja S.C. Mullick Road, Calcutta - 700032, India
ABSTRACT
Two compounds isolated from the alcoholic extract of the roots of Aristolochia indica Linn were tested on day 6 pregnant mice. One of the compounds, pcoumaric acid showed 10046interceptive activity at the single oral dose of 50 m&kg of body weight. The antifertilitvefficacy of these two compounds is discussed. "
Accepted for publication June 11, 1979
JULY
1979 VOL. 20 NO.
1
49
CONTRACEPTION
INTRODUCTION The crude alcoholic extract of the bitter root of the plant Aristolochia indica Linn showed antifertility activity in mice and hamsters when fed orally post-coitum at single dose (1). The crude alcoholic extra&was purified and two constituent compounde, i.e. Aristololactamglucoside and e-coumaric acid (21, were isolated from the above crude extract and were tested in mice at different days of pregnancy. MATERIALS AND METHODS In-bred Swiss albino fertile female mice were caged with their male counterparts in matiqe,cages in 2 :l ratio. They were kept in good husbandry conditions. Day 1 of pregnancy was marked by identificationof vaginal plug. The test materials were administered orally at the dose level of 50 mg/lcgof body weight at different days of pregnancy. Generally the test materials were administered with l$ gum-acacia and water suspension.Proper vehiclefed controls were maintained. Experiment I In this study Aristololactamglucoside was administered to 10 mice of Group 1 on day 6 of pregnancy. Laparotosny was performed under light ether anaesthesia on day 7 along with their controls. The number of implantation sites were counted in each of the animals. Experiment II This study sought to determine the antifertility effect of the E-coumaric acid. The test compound was fed on day 4 (Group 21, day 6 (Group 31, day 8 (Group 41, day 10 (Group 5), day 14 (Group 6) and day 18 Group 7) of pregnancy. Animals were laparotomised on day 6 IGroup 21, day 7 (Group 31, day IO-11 (Group 4) and day 13 (Group 5) under light ether aneasthesia. Other animals were kept until terms. On laparotomy morphological observations of waries and uterine horns were made in treated animals and compared with that of respective pregnant controls. Histological observations of ovaries and uterine horns were performed in Group 3 and the corresponding control animals, using Bouins fixative and employing haematoxylin and eosin staining technique. The litters of Groups 6 and 7 were examined for any gross abnormalities.
50
JULY 1979VOL.20NO.l
CONTRACEPTION
RESULTS The results of the two experiments (Table I) clearly showed that E-coumaric acid was much more effective than the Aristololactum glucoside. On day 6 of pregnancy,Aristololactum glucoside was 306 effective and phenolic compound was lOC$ effective. Results of Group 2 showed the partial effectiveness at this stage. On laparotomy the uterine horns showed scar marks of implantation sites in 85.71;6and in 14.2916 of cases there were intact implantationsites of normal size in comparison to controls. There was no delayed implantation in these cases. -Coumaric acid showed 100 per cent interceptive activity on day 43 and day 8, but in the latter case, it took approximately 72 hours for its action. The litters produced in Group 6 and Group 7 showed normal size and weight without any sign of deformities. Gestation periods were also normal. On laparotomy,morpholcgicalobservation of ovaries in all the aborted animals showed prominent corpora albicanz whereas in control pregnant animals prominent corpora lutea of pregnancy were observed. The ovaries of the aborted mice appeared reddish aLIdseemed to be engorged with blood. Uterus remained thick, non-contractile,long and empty in appearance. In pregnant animals uteri were progestagenic with intact implantationsites. Histology of the ovary of day 7 pregnant mice showed prominent luteal cells (Pig I). The ovarian histology of the aborted mice showed damage of luteal cells with numerous follicles (Fig 2). DISCUSSION Both compounds, Aristololactumglucoside and p-coumaric acid, isolated from the alcoholic extract of The root of Aristolochia indica Linn showed interceptive activity but the promising results of p-coumaric acid encouraged detailed studies on antiferE.lity.Results of Group 2 animals indicate that at this dose level, the compound is partially effective. The compound has no effect possibly in preimplantationstage because from the morphological observation, it was evident that the compound interrupted pregnancy after blastocysts had been implanted. There was no evidence of delayed implantationbecause the blastocysts which were implanted were normal in size and similar to those of the normal pregnant control. Other observations at different days of pregnancies also corroborated our previous findings with crude extract (1). Ovarian histology revealed that z-coumaric acid may damage the corpora lutea in aborted animals. Effectiveness of the test compound administered in regressing the corpora lutea depends upon the stage of pregnancy. Involvement of prolactin as a luteotropic hormone in early post-implantationstage in mouse (3) is proposed by Mantle and Finn (4) and Bartke (5) established that pituitary prolactin is essential
JULY
1979 VOL. 20 NO. 1
51
CONTRACEPTION
TABLE I. Antifertility activities of the compounds Aristolactumglucoside and e-Goumaric acid
Group+
Mice without implantations/i?o. of animals _.
Antifertility activity in $3
30
1 Control
3/10 o/7
0
2
G/7
85.71
3
25/25
100
4
7/7
100
5
O/7
6+
-/a
7+
-/a
Control
o/10
0 0 0 0
* Animals of each group received the test material at the single dose level of 50 mg/kg. + Animals of groups 6 and 7 were not laparatomized.
52
JULY
1979 VOL. 20 NO. 1
CONTRACEPTION
Fig I. Histological picture of the day 7 pregnant mouse showing prominent corpora lutea of pregnancy. (ii & E, x 80)
Fig
2. Histological picture of the aborted mouse showing damage of luteal cells. (H &E, x 100)
JULY 1979VOL.20NO.l
53
CONTRACEPTION
for the maintenance of pregnancy up to day 9. We also observed that our compound is 100 per cent effective at the early postimplantationalstage and furthermore our unpublished observation shows that exogenous prolactin can counteract the effect of the compound. From the above facts,it may not be unwise to envisage the idea of antiprolactinaction of the compound by which it produces interception.It has no action when placenta takes charge of the fetus or pregnancy becomes independent of maternal pituitary. The comptid also has no effect on parturition (5) or gestation period when administered in later stages of pregnancy.
ACKNOWIEDGEMENT Authors are thankful to the Director of this Institute for granting senior fellowship to P.Pakrasi and to Dr. B. Achari for the isolation of compounds. REFERENCES 1.
Pakrashi, A. and Pakrasi, P.L. : Interceptiveand abortifacient activity of Aristolochia indica Linn and possible mode of action. Indian J E-1 15 : 428 (1977)
2.
Pakrashi, S.C., Ghosh Dastidar, P.P., Bose, S., and Achari, B : New Phenanthrene derivatives from Aristolochia indica. Phytochemistry 16 : 1103 (1977).
3.
Choudhury, J.B. and Greenwald, G.S. : Luteotropic complex of the mouse. Anat Ret 163 : 373 (1969b)
4.
Mantle, P.G. and Finn, C.A. : Investigationson the mode of action of Dr6-Methyl-8 Cyanomethylergoline in suppressing regnancy in the mouse. J. Reprod. Fert, 24 : 441 ?1971) Bartke, A. : The maintenance of gestation and the initiation of lactation in the mouse in the absence of pituitary prolactin. J Reprod Pert 27 : 121 (1971)
5.
54
JULY
1979 VOL. 20 NO. 1
ANTIFERTILITYEFFICACY OF THE PLART ARISTOIJOCHIA INDICA LIRN OR MOUSE
Anita Pslcrashiand Pranab Pakrasi Reproductive Biology Research Section Indian Institute of Experimental Medicine 4, Raja S.C. Mullick Road, Calcutta - 700032, India
ABSTRACT
Two compounds isolated from the alcoholic extract of the roots of Aristolochia indica Linn were tested on day 6 pregnant mice. One of the compounds, pcoumaric acid showed 10046interceptive activity at the single oral dose of 50 m&kg of body weight. The antifertilitvefficacy of these two compounds is discussed. "
Accepted for publication June 11, 1979
JULY
1979 VOL. 20 NO.
1
49
CONTRACEPTION
INTRODUCTION The crude alcoholic extract of the bitter root of the plant Aristolochia indica Linn showed antifertility activity in mice and hamsters when fed orally post-coitum at single dose (1). The crude alcoholic extra&was purified and two constituent compounde, i.e. Aristololactamglucoside and e-coumaric acid (21, were isolated from the above crude extract and were tested in mice at different days of pregnancy. MATERIALS AND METHODS In-bred Swiss albino fertile female mice were caged with their male counterparts in matiqe,cages in 2 :l ratio. They were kept in good husbandry conditions. Day 1 of pregnancy was marked by identificationof vaginal plug. The test materials were administered orally at the dose level of 50 mg/lcgof body weight at different days of pregnancy. Generally the test materials were administered with l$ gum-acacia and water suspension.Proper vehiclefed controls were maintained. Experiment I In this study Aristololactamglucoside was administered to 10 mice of Group 1 on day 6 of pregnancy. Laparotosny was performed under light ether anaesthesia on day 7 along with their controls. The number of implantation sites were counted in each of the animals. Experiment II This study sought to determine the antifertility effect of the E-coumaric acid. The test compound was fed on day 4 (Group 21, day 6 (Group 31, day 8 (Group 41, day 10 (Group 5), day 14 (Group 6) and day 18 Group 7) of pregnancy. Animals were laparotomised on day 6 IGroup 21, day 7 (Group 31, day IO-11 (Group 4) and day 13 (Group 5) under light ether aneasthesia. Other animals were kept until terms. On laparotomy morphological observations of waries and uterine horns were made in treated animals and compared with that of respective pregnant controls. Histological observations of ovaries and uterine horns were performed in Group 3 and the corresponding control animals, using Bouins fixative and employing haematoxylin and eosin staining technique. The litters of Groups 6 and 7 were examined for any gross abnormalities.
50
JULY 1979VOL.20NO.l
CONTRACEPTION
RESULTS The results of the two experiments (Table I) clearly showed that E-coumaric acid was much more effective than the Aristololactum glucoside. On day 6 of pregnancy,Aristololactum glucoside was 306 effective and phenolic compound was lOC$ effective. Results of Group 2 showed the partial effectiveness at this stage. On laparotomy the uterine horns showed scar marks of implantation sites in 85.71;6and in 14.2916 of cases there were intact implantationsites of normal size in comparison to controls. There was no delayed implantation in these cases. -Coumaric acid showed 100 per cent interceptive activity on day 43 and day 8, but in the latter case, it took approximately 72 hours for its action. The litters produced in Group 6 and Group 7 showed normal size and weight without any sign of deformities. Gestation periods were also normal. On laparotomy,morpholcgicalobservation of ovaries in all the aborted animals showed prominent corpora albicanz whereas in control pregnant animals prominent corpora lutea of pregnancy were observed. The ovaries of the aborted mice appeared reddish aLIdseemed to be engorged with blood. Uterus remained thick, non-contractile,long and empty in appearance. In pregnant animals uteri were progestagenic with intact implantationsites. Histology of the ovary of day 7 pregnant mice showed prominent luteal cells (Pig I). The ovarian histology of the aborted mice showed damage of luteal cells with numerous follicles (Fig 2). DISCUSSION Both compounds, Aristololactumglucoside and p-coumaric acid, isolated from the alcoholic extract of The root of Aristolochia indica Linn showed interceptive activity but the promising results of p-coumaric acid encouraged detailed studies on antiferE.lity.Results of Group 2 animals indicate that at this dose level, the compound is partially effective. The compound has no effect possibly in preimplantationstage because from the morphological observation, it was evident that the compound interrupted pregnancy after blastocysts had been implanted. There was no evidence of delayed implantationbecause the blastocysts which were implanted were normal in size and similar to those of the normal pregnant control. Other observations at different days of pregnancies also corroborated our previous findings with crude extract (1). Ovarian histology revealed that z-coumaric acid may damage the corpora lutea in aborted animals. Effectiveness of the test compound administered in regressing the corpora lutea depends upon the stage of pregnancy. Involvement of prolactin as a luteotropic hormone in early post-implantationstage in mouse (3) is proposed by Mantle and Finn (4) and Bartke (5) established that pituitary prolactin is essential
JULY
1979 VOL. 20 NO. 1
51
CONTRACEPTION
TABLE I. Antifertility activities of the compounds Aristolactumglucoside and e-Goumaric acid
Group+
Mice without implantations/i?o. of animals _.
Antifertility activity in $3
30
1 Control
3/10 o/7
0
2
G/7
85.71
3
25/25
100
4
7/7
100
5
O/7
6+
-/a
7+
-/a
Control
o/10
0 0 0 0
* Animals of each group received the test material at the single dose level of 50 mg/kg. + Animals of groups 6 and 7 were not laparatomized.
52
JULY
1979 VOL. 20 NO. 1
CONTRACEPTION
Fig I. Histological picture of the day 7 pregnant mouse showing prominent corpora lutea of pregnancy. (ii & E, x 80)
Fig
2. Histological picture of the aborted mouse showing damage of luteal cells. (H &E, x 100)
JULY 1979VOL.20NO.l
53
CONTRACEPTION
for the maintenance of pregnancy up to day 9. We also observed that our compound is 100 per cent effective at the early postimplantationalstage and furthermore our unpublished observation shows that exogenous prolactin can counteract the effect of the compound. From the above facts,it may not be unwise to envisage the idea of antiprolactinaction of the compound by which it produces interception.It has no action when placenta takes charge of the fetus or pregnancy becomes independent of maternal pituitary. The comptid also has no effect on parturition (5) or gestation period when administered in later stages of pregnancy.
ACKNOWIEDGEMENT Authors are thankful to the Director of this Institute for granting senior fellowship to P.Pakrasi and to Dr. B. Achari for the isolation of compounds. REFERENCES 1.
Pakrashi, A. and Pakrasi, P.L. : Interceptiveand abortifacient activity of Aristolochia indica Linn and possible mode of action. Indian J E-1 15 : 428 (1977)
2.
Pakrashi, S.C., Ghosh Dastidar, P.P., Bose, S., and Achari, B : New Phenanthrene derivatives from Aristolochia indica. Phytochemistry 16 : 1103 (1977).
3.
Choudhury, J.B. and Greenwald, G.S. : Luteotropic complex of the mouse. Anat Ret 163 : 373 (1969b)
4.
Mantle, P.G. and Finn, C.A. : Investigationson the mode of action of Dr6-Methyl-8 Cyanomethylergoline in suppressing regnancy in the mouse. J. Reprod. Fert, 24 : 441 ?1971) Bartke, A. : The maintenance of gestation and the initiation of lactation in the mouse in the absence of pituitary prolactin. J Reprod Pert 27 : 121 (1971)
5.
54
JULY
1979 VOL. 20 NO. 1
