Surgical Research Laboratory, institute of Medical Sciences, Banaras Hindu University, Varanasi - 221 005 lndia
EFFECTS OF HIBISCUS ROSA SZNENSIS ON PREGNANCY OF RATS By S. D. KHOLKUTE and K. N. UDUPA
The total bknzkne extract of H i 6 s c u s r o s a s i n e n s i s (China rose plant) flowers showed potent, orally effective antifertility activity in albino rats when administered from day 1 to 10 of pregnancy. This activity o f the extract was found to depend upon the dose and duration of treatment. The extract was not found t o affect the tubual transport of the zygote as well as blastocyst per se from implanting. However, as studied by pontamine blue reaction and administration of extract during implantation, it seems that, at least in 70°/o of the females, the antifertility activity of the drug was mediated via interruption in implantation. The results. obtained by delayed implantation technique further suggest that the extract, in ony way or other, alters the delicate estrogen-progesterone balance. The pregnancy maintenance by higher dose of oestrone in extract treated females further lend support to the curtailnzent of estrogen. However, invoking ,,anti-implantation factor" alone, is not responsible enough to achieve full contraception and the drug causes obsorption of foetus.
Hibiscus rosa sinensis (China rose plant) is a common ornamental plant cultivated widely throughout India. The flower portion of this' plant is attributed to possess antifertility property in ancient Ayurvedic literatures. In folklores of Kerala, this plant is advocated for its contraceptive action. Apart from this, the flower portion has been mentioned for its alleged antifertility properties by various authors (KRITIKARand BASU, 1935, NADKARNI and NADKARNI, 1954, CHOPRAand CHOPRA, 1955, CASEY, 1960, CHAUDHURY, 1966). BATTA and SANTHAKUMARI (1971) showed the presence of the post-coital antifertility activity in various extracts of flowers which was confirmed by studies of KHOLKUTE and UDUPA(1974). In the present communication, an attempt has been made to pinpoint the probable modus operandi of this plant utilising various experimental models like, administration of drug during various embryonic stages of pregnancy, pontamine blue reaction and delayed implantation technique.
I
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Abstract
322
Kholkute and Udupa
Planta medica Vol. 29 1976
Materials and Methods
Antifertility Effects T h e antifertility activity of the extract was screened by the method of KHANNAand CHAUDHURY (1968) with modification in the duration of treatment. The test screens the post-coital antifertility effects and involves treating female rats following mating and then inspecting the uteri. The absence of implantation is used as the objective of the test. Some of the animals received the extract from day 1 to 10 of pregnancy, some received from day 1 t o 6 and in other groups of animals, the extract was administered from day 4 t o 6 and day 1 to 3 of pregnancy; in three different dose levels 100 n~g/kg; 150 mglkg and 250 mglkg. T o the animals in control groups sterile group nut oil was similarly intubated. The duration of the treatment was selected depending upon the chronological events in embryonic development of the rat. O n day 11 of pregnancy, all the animals were laparotomised by midventral incision under light ether anaesthesia. T h e number of implantation sites in both the uterine horns and corpora lutea in ovaries were recorded. Any animal with atleast one normal foetus was considered to be pregnant. The presence of placental scars or reabsorbed foetus was taken as sign of termination of pregnancy. After the opcration, the animals were sutured as usual and were returned to their respective cages. The treatment was withdrawn from day 10 of pregnancy and the animals were allowed to deliver. After parturition, if it occured, the number of litters delivered were counted and examined macroscopically for gross teratogenic effects. Effect of Exogenous Estrogen on pregnancy in rats treated with total Benzene extract o f H.rosa sinensis In other experiment, the effect of estrogen on extract treated pregnant females was studied. T h e total benzene extracts was administered in the dose of 250 mg/kg from day 1 to 10 of pregnancy. From day 4 to 6 of pregnancy the extract treated animals received daily subcutaneous injection of 1pg. oestrone (Merck, Germany) in 0.1 ml of sterile ground nut oil. Appropriate controls receiving sterile ground oil were maintained. O n day 11 postcoitum, all the animals were autopsied. The number of implantation sites in utero and corpora lutea in ovaries were recorded. At autopsy, the embryonic swellings were cut, blotted on filter paper and weighed to nearest 0.1 mg. on Single Pan Electric Balance. T h e average weight was calculated. Effect of Total Benzene extract administration of Pontamine Blue reaction in pregnant rats Twenty mated females were equally divided in 2 groups on day 1 post-coitum. In the treated group of animals, total benzene extract was orally administered in the dose of 250 mg/kg from day 4 to 6 of pregnancy. T o the animals in control group sterile ground nut oil was similarly intubated.
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H . rosa sinensis flowers were collected from University campus, dried in shade and powdered. T h e powdered material was then extracted with benzene by soxhlation. T h e extractive thus obtained was further concentrated by vacuum distillation under reduced pressure and controlled temperature (56-60° C) for 'administration to test animals the benzene extract was dissolved in sterile ground nut oil and intubated by soft rubber chatheter tube. Inbred stain of female albino rats, 3 t o 5 months old and weighing between 130 to 170 gms were selected for experiments. Colony bred adult fertile males were used for mating. T h e animals were maintained under uniform environment. Standard laboratory diet (Anidiet, Poona, India) and tap water were supplied ad libidum.
Effects of Hibiscus on Pregnancy of Rats
323
Effect of Total Benzene extract on delayed lrnplantation in rats T h e method used for inducing implantation after a delay of 6 days was essentially similar t o the one described by COCHRANE and MEYER(1957). Mated females were bilaterally ovariectomised on day 3 of pregnancy. O n the same day the females were subcutaneously injected with 4 mg. Progesterone (Luteocyclin, Ciba) in 0.2 rnl sterile oil, until they were sacrificed. The animals were laparotomised on day 8 of pregnancy to ensure that there was no implantation. After this, the animals received daily subcutaneous injections of 0.1 pg. Oestrone (in 0.1 ml sterile oil) from day 8 to 12. The animals were divided into following groups: (1) Delayed implanted group (above treatment only). (2) Delayed implanted group to which sterile ground nut oil was intubated from day 3 to 12 of pregnancy, this group served as control. (3) Delayed implanted group t o which 250 mglkg total Benzene extract was administered from day 3 t o 12. (4) Animals receiving 250 nlglkg total Benzene extract from day 3 to 7 of pregnancy in addition to progesterone and oestrone. (5) Delayed implanted group receiving 250 mglkg of extract from day 8 to 12 of pregnancy. All the animals were sacrificed on day 13 post-coitum and the number of implantation sites in utero were recorded. All the datas were stastistically analysed by students 't' test.
Results Antifertility Effect T h e effects of total benzene extract of H. rosa sinensis flowers on pregnancy in rats is shown in Table I, when administered in the dose of 100 mglkg from day 1 to 10 of pregnancy in 50% of the animals, the pregnancy was inhibited as evident by absence of implantation sites in utero (Group A); while a t the dose af 150 mg/kg, out of 1 0 females in 7 animals pregnancy was not observed. The number of implants in this groups was significantly reduced when compared to the control (Group B; P < 0.05). However, the number of corpora lutea in ovaries were comparable to the number of implantation sites in utero (P > 0.1; Table I). Maximum antifertility activity was observed after the administration of the extract in the dose of 250 mglkg from day 1 t o 10 of pregnancy. Out of 10 treated animals, only 1was pregnant. Furthermore, the animal failed to deliver any litter (Group C ) . Similar results were obtained, when this treatment was repeated (Group D).
Downloaded by: National University of Singapore. Copyrighted material.
O n the morning of day 6 of pregnancy, all the rats were injected intravenously with 0.5 ml of Pontamine blue dye (2.5% in normal saline). Fifteen minutes after the injection, the animals were laparotomised. T h e number of blue spots in utero were recorded. After the operation the animals were sutured and were returned to their respeaivc cages. O n day 11 post-coitum, the animals were sacrificed by decapitation and the number of implantation sites in utero and corpora lutea in ovaries were recorded.
324
Kholkute and Udupa
Planta medica Vol. 29 1976
Table I Antifertility effects of tota1,Benzene extract of H. rosn sinensis on pregnancy in rats. (Mean Group
Control A
B C D
Dose (mdkg)
Duration No. Pregn. No. Implants. (days) No. treated. Pregn. Animals.
100 150 25 0 250
No. of Corpora lutea
Litters delivered.
5 SD)
Antifertility (%)
10.7 k 1.9 7.1 k 1.3 4.9 k 0.8 Nil Nil
50 70 100 100
E Control
F G Control H
* P < 0.05,in all cases P > 0.1.
" P < 0.05when compared to No. of Implants in utero. In contrast to this (Group E), the administration of extract a t the dose of 250 mg/kg from day 1 to 3 of pregnancy, no significant effect on the pregnancy was observed. After 150 mg/kg extract treatment from day 1 t o 6 inclusive, in 60% of animals pregnancy was terminated. Furthermore, a significant reduction in number of implants was observed (Group E, P < 0.05). At the dose of 250 mg/kg (Group F) for the same duration, in 80% of females, pregnancy was terminated. .Administration in the dose of 250 mglkg from day 4 t o 6 inclusive, terminated pregnancy in 70% of females (Group G ) and significantly reduced the number of implants (P < 0.05). N o side effects were observed. T h e animals delivered between day 22 t o 24 as the control. T h e litters delivered were normal. Effect of Exogenous Estrogen Administration of total benzene extract (250 mg/kg) from day 1 to 10 caused loss of pregnancy in 9 out of 1 0 animals when examined on day 11 (Table 11, Group B). T h e animal which was pregnant, showed 5 implantation sites and a marked reduction in the average weight of implant was observed (P 0.1 (A) P < 0.05 (B)
10
13.9
11.9 2 2.2
Effectof Pontamine Blue Reaction All the 10 females in the control group showed blue spots on day 6 (Table 111). However, in treated group, only 3 animals showed blue spots which was significantly reduced in number (P
EFFECTS OF HIBISCUS ROSA SZNENSIS ON PREGNANCY OF RATS By S. D. KHOLKUTE and K. N. UDUPA
The total bknzkne extract of H i 6 s c u s r o s a s i n e n s i s (China rose plant) flowers showed potent, orally effective antifertility activity in albino rats when administered from day 1 to 10 of pregnancy. This activity o f the extract was found to depend upon the dose and duration of treatment. The extract was not found t o affect the tubual transport of the zygote as well as blastocyst per se from implanting. However, as studied by pontamine blue reaction and administration of extract during implantation, it seems that, at least in 70°/o of the females, the antifertility activity of the drug was mediated via interruption in implantation. The results. obtained by delayed implantation technique further suggest that the extract, in ony way or other, alters the delicate estrogen-progesterone balance. The pregnancy maintenance by higher dose of oestrone in extract treated females further lend support to the curtailnzent of estrogen. However, invoking ,,anti-implantation factor" alone, is not responsible enough to achieve full contraception and the drug causes obsorption of foetus.
Hibiscus rosa sinensis (China rose plant) is a common ornamental plant cultivated widely throughout India. The flower portion of this' plant is attributed to possess antifertility property in ancient Ayurvedic literatures. In folklores of Kerala, this plant is advocated for its contraceptive action. Apart from this, the flower portion has been mentioned for its alleged antifertility properties by various authors (KRITIKARand BASU, 1935, NADKARNI and NADKARNI, 1954, CHOPRAand CHOPRA, 1955, CASEY, 1960, CHAUDHURY, 1966). BATTA and SANTHAKUMARI (1971) showed the presence of the post-coital antifertility activity in various extracts of flowers which was confirmed by studies of KHOLKUTE and UDUPA(1974). In the present communication, an attempt has been made to pinpoint the probable modus operandi of this plant utilising various experimental models like, administration of drug during various embryonic stages of pregnancy, pontamine blue reaction and delayed implantation technique.
I
Downloaded by: National University of Singapore. Copyrighted material.
Abstract
322
Kholkute and Udupa
Planta medica Vol. 29 1976
Materials and Methods
Antifertility Effects T h e antifertility activity of the extract was screened by the method of KHANNAand CHAUDHURY (1968) with modification in the duration of treatment. The test screens the post-coital antifertility effects and involves treating female rats following mating and then inspecting the uteri. The absence of implantation is used as the objective of the test. Some of the animals received the extract from day 1 to 10 of pregnancy, some received from day 1 t o 6 and in other groups of animals, the extract was administered from day 4 t o 6 and day 1 to 3 of pregnancy; in three different dose levels 100 n~g/kg; 150 mglkg and 250 mglkg. T o the animals in control groups sterile group nut oil was similarly intubated. The duration of the treatment was selected depending upon the chronological events in embryonic development of the rat. O n day 11 of pregnancy, all the animals were laparotomised by midventral incision under light ether anaesthesia. T h e number of implantation sites in both the uterine horns and corpora lutea in ovaries were recorded. Any animal with atleast one normal foetus was considered to be pregnant. The presence of placental scars or reabsorbed foetus was taken as sign of termination of pregnancy. After the opcration, the animals were sutured as usual and were returned to their respective cages. The treatment was withdrawn from day 10 of pregnancy and the animals were allowed to deliver. After parturition, if it occured, the number of litters delivered were counted and examined macroscopically for gross teratogenic effects. Effect of Exogenous Estrogen on pregnancy in rats treated with total Benzene extract o f H.rosa sinensis In other experiment, the effect of estrogen on extract treated pregnant females was studied. T h e total benzene extracts was administered in the dose of 250 mg/kg from day 1 to 10 of pregnancy. From day 4 to 6 of pregnancy the extract treated animals received daily subcutaneous injection of 1pg. oestrone (Merck, Germany) in 0.1 ml of sterile ground nut oil. Appropriate controls receiving sterile ground oil were maintained. O n day 11 postcoitum, all the animals were autopsied. The number of implantation sites in utero and corpora lutea in ovaries were recorded. At autopsy, the embryonic swellings were cut, blotted on filter paper and weighed to nearest 0.1 mg. on Single Pan Electric Balance. T h e average weight was calculated. Effect of Total Benzene extract administration of Pontamine Blue reaction in pregnant rats Twenty mated females were equally divided in 2 groups on day 1 post-coitum. In the treated group of animals, total benzene extract was orally administered in the dose of 250 mg/kg from day 4 to 6 of pregnancy. T o the animals in control group sterile ground nut oil was similarly intubated.
Downloaded by: National University of Singapore. Copyrighted material.
H . rosa sinensis flowers were collected from University campus, dried in shade and powdered. T h e powdered material was then extracted with benzene by soxhlation. T h e extractive thus obtained was further concentrated by vacuum distillation under reduced pressure and controlled temperature (56-60° C) for 'administration to test animals the benzene extract was dissolved in sterile ground nut oil and intubated by soft rubber chatheter tube. Inbred stain of female albino rats, 3 t o 5 months old and weighing between 130 to 170 gms were selected for experiments. Colony bred adult fertile males were used for mating. T h e animals were maintained under uniform environment. Standard laboratory diet (Anidiet, Poona, India) and tap water were supplied ad libidum.
Effects of Hibiscus on Pregnancy of Rats
323
Effect of Total Benzene extract on delayed lrnplantation in rats T h e method used for inducing implantation after a delay of 6 days was essentially similar t o the one described by COCHRANE and MEYER(1957). Mated females were bilaterally ovariectomised on day 3 of pregnancy. O n the same day the females were subcutaneously injected with 4 mg. Progesterone (Luteocyclin, Ciba) in 0.2 rnl sterile oil, until they were sacrificed. The animals were laparotomised on day 8 of pregnancy to ensure that there was no implantation. After this, the animals received daily subcutaneous injections of 0.1 pg. Oestrone (in 0.1 ml sterile oil) from day 8 to 12. The animals were divided into following groups: (1) Delayed implanted group (above treatment only). (2) Delayed implanted group to which sterile ground nut oil was intubated from day 3 to 12 of pregnancy, this group served as control. (3) Delayed implanted group t o which 250 mglkg total Benzene extract was administered from day 3 t o 12. (4) Animals receiving 250 nlglkg total Benzene extract from day 3 to 7 of pregnancy in addition to progesterone and oestrone. (5) Delayed implanted group receiving 250 mglkg of extract from day 8 to 12 of pregnancy. All the animals were sacrificed on day 13 post-coitum and the number of implantation sites in utero were recorded. All the datas were stastistically analysed by students 't' test.
Results Antifertility Effect T h e effects of total benzene extract of H. rosa sinensis flowers on pregnancy in rats is shown in Table I, when administered in the dose of 100 mglkg from day 1 to 10 of pregnancy in 50% of the animals, the pregnancy was inhibited as evident by absence of implantation sites in utero (Group A); while a t the dose af 150 mg/kg, out of 1 0 females in 7 animals pregnancy was not observed. The number of implants in this groups was significantly reduced when compared to the control (Group B; P < 0.05). However, the number of corpora lutea in ovaries were comparable to the number of implantation sites in utero (P > 0.1; Table I). Maximum antifertility activity was observed after the administration of the extract in the dose of 250 mglkg from day 1 t o 10 of pregnancy. Out of 10 treated animals, only 1was pregnant. Furthermore, the animal failed to deliver any litter (Group C ) . Similar results were obtained, when this treatment was repeated (Group D).
Downloaded by: National University of Singapore. Copyrighted material.
O n the morning of day 6 of pregnancy, all the rats were injected intravenously with 0.5 ml of Pontamine blue dye (2.5% in normal saline). Fifteen minutes after the injection, the animals were laparotomised. T h e number of blue spots in utero were recorded. After the operation the animals were sutured and were returned to their respeaivc cages. O n day 11 post-coitum, the animals were sacrificed by decapitation and the number of implantation sites in utero and corpora lutea in ovaries were recorded.
324
Kholkute and Udupa
Planta medica Vol. 29 1976
Table I Antifertility effects of tota1,Benzene extract of H. rosn sinensis on pregnancy in rats. (Mean Group
Control A
B C D
Dose (mdkg)
Duration No. Pregn. No. Implants. (days) No. treated. Pregn. Animals.
100 150 25 0 250
No. of Corpora lutea
Litters delivered.
5 SD)
Antifertility (%)
10.7 k 1.9 7.1 k 1.3 4.9 k 0.8 Nil Nil
50 70 100 100
E Control
F G Control H
* P < 0.05,in all cases P > 0.1.
" P < 0.05when compared to No. of Implants in utero. In contrast to this (Group E), the administration of extract a t the dose of 250 mg/kg from day 1 to 3 of pregnancy, no significant effect on the pregnancy was observed. After 150 mg/kg extract treatment from day 1 t o 6 inclusive, in 60% of animals pregnancy was terminated. Furthermore, a significant reduction in number of implants was observed (Group E, P < 0.05). At the dose of 250 mg/kg (Group F) for the same duration, in 80% of females, pregnancy was terminated. .Administration in the dose of 250 mglkg from day 4 t o 6 inclusive, terminated pregnancy in 70% of females (Group G ) and significantly reduced the number of implants (P < 0.05). N o side effects were observed. T h e animals delivered between day 22 t o 24 as the control. T h e litters delivered were normal. Effect of Exogenous Estrogen Administration of total benzene extract (250 mg/kg) from day 1 to 10 caused loss of pregnancy in 9 out of 1 0 animals when examined on day 11 (Table 11, Group B). T h e animal which was pregnant, showed 5 implantation sites and a marked reduction in the average weight of implant was observed (P 0.1 (A) P < 0.05 (B)
10
13.9
11.9 2 2.2
Effectof Pontamine Blue Reaction All the 10 females in the control group showed blue spots on day 6 (Table 111). However, in treated group, only 3 animals showed blue spots which was significantly reduced in number (P
