- Email: [email protected]
Post-coital antifertility activity of Acalypha indica L.
Journal of Ethnopharmacology 67 (1999) 253 – 258 www.elsevier.com/locate/jethpharm
Post-coital antifertility activity of Acalypha indica L. Shivayogi P. Hiremath a,*, K. Rudresh a, Shrishailappa Badami a, Saraswati B. Patil b, Somanath R. Patil b a
Department of Chemistry, Gulbarga Uni6ersity, Gulbarga-585 106, India Department of Zoology, Gulbarga Uni6ersity, Gulbarga-585 106, India
b
Received 1 August 1998; received in revised form 19 October 1998; accepted 2 November 1998
Abstract Four successive solvent extracts of the whole plant Acalypha indica L. (Euphorbiaceae) were tested for post-coital antifertility activity in female albino rats. Of these, the petroleum ether and ethanol extracts were found to be most effective in causing significant anti-implantation activity. The antifertility activity was reversible on withdrawal of the treatment of the extracts. Both the extracts at 600 mg/kg body weight showed estrogenic activity. Histological studies of the uterus were carried out to confirm this estrogenic activity. © 1999 Elsevier Science Ireland Ltd. All rights reserved.
1. Introduction One approach pursued to identify new antifertility agents is the search for their presence in natural sources. Many plant preparations are reported to have fertility regulating properties and a few have been tested for such effects. But so far no single plant is available which can be developed further as a potent antifertility agent. Hence the search continues. Acalypha indica L. (Euphorbiaceae) is a weed widely distributed throughout the plains of India. It has been reported to be useful in treating pneumonia, asthma, rheumatism and several other ailments (Kirtikar and Basu,
* Corresponding author.
1935; Anonymous, 1948; Chopra et al., 1956; Nadakarni and Nadakarni, 1982). This plant was cited as an emmenagogue by Nadakarni and Nadakarni (1982). The leaves of Acalypha grandis have also been reported to possess contraceptive activity (Bourdy and Walter, 1992). Several chemical (Donw and Steyn, 1938; Talapatra et al., 1981; Nohrstedt et al., 1982; Manzoor-i-Khuda et al., 1985; Asolkar et al., 1992) and biological (Caius and Mhaskar, 1923; Hiremath et al., 1993) investigations have been carried out on this plant, but, so far no antifertility testing has been done. Hence, in continuation of our work on antifertility activity of plants (Hiremath and Hanumantha, 1990; Hiremath et al., 1990, 1994, 1996), we were interested in subjecting Acalypha indica L. to antifertility testing in female albino rats.
0378-8741/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 9 8 ) 0 0 2 1 3 - X
254
S.P. Hiremath et al. / Journal of Ethnopharmacology 67 (1999) 253–258
2. Materials and methods The whole plant of Acalypha indica L. was collected from the fields in and around Gulbarga during August and September, 1996 and was authenticated at the Herbarium, Department of Botany, Gulbarga University, Gulbarga (HGUG No.279). The plant was shade dried, powdered and subjected to Soxhlet extraction (500 g) successively and separately with petroleum ether (60 – 80°C, 2 l), chloroform (2 l), ethanol (95%, 2 l) and distilled water (2 l). The extracts were concentrated to dryness in a flash-evaporator under reduced pressure and controlled temperature (50 – 60°C). The petroleum ether and chloroform extracts yielded dark brown solids weighing 20.0 and 32.0 g, respectively. All the extracts were stored in a refrigerator. The extracts were prepared in Tween − 80 (1%), suspended in distilled water and they were administered to the rats at doses of 300 and 600 mg/kg body weight orally by means of an intragastric catheter. Colony-bred female albino rats (Wistar strain), weighing (150–200 g), were maintained under controlled standard animal house conditions with access to food and water ad libitum. Vaginal smears from each rat were monitored daily. Only rats with normal estrous cycles were selected. Anti-implantation activity was determined as described by Khanna and Chaudhury (1968). Rats found in proestrous phase of the cycle were caged with males of proven fertility, in the ratio of 2:1 and examined the following morning for evidence of copulation. Rats exhibiting thick clumps of spermatozoa in their vaginal smears were separated and that day was designated as day 1 of pregnancy and those rats were divided into nine groups containing eight rats in each group. The extracts were administered at 300 and 600 mg/kg body weight orally from days 1 to 7 of pregnancy. Control rats received the vehicle (Tween-80, 1%) only. On day 10, laparotomy was performed under light ether anesthesia and semisterile conditions. The uteri were examined to determine the number of implantation sites. The rats were allowed to recover and deliver after full term. Each pup was weighed and examined for gross defects. The litters were allowed to grow to check post-na-
tal growth and monitor any congenital abnormalities. The petroleum ether and ethanol extracts were found to be the most active of the extracts of Acalypha indica L., hence they were subjected to a detailed investigation for potential estrogenic and antiestrogenic activity. Colony-bred (Wistar strain) immature female albino rats, 21–23 days old, weighing between 35 and 45 g, were bilaterally ovariectimised by dorsolateral approach under light ether anaesthesia and sterile conditions. They were divided into six groups consisting of eight rats each. The first group served as a control end received vehicle only (Tween-80, 1%). The second group received ethinyl estradiol in olive oil, 1 mg/rat per day, subcutaneously. The third and fourth groups received the petroleum ether and ethanol extracts at a dose of 600 mg/kg body weight, respectively. The fifth and sixth groups received, in addition to ethinyl estradiol, a test dose of the petroleum ether and ethanol extracts at 600 mg/kg body weight, respectively. All the above treatments were given for 7 days. On the 8th day, the rats were sacrificed by decapitation, the uteri dissected out and surrounding tissues removed. The uteri were blotted on filter papers and weighed quickly on a sensitive balance and fixed in Bouin’s fluid for 24 h. The tissues were dehydrated and embedded in paraffin. The paraffin sections were cut at 6 mm and stained with haematoxylin–eosin for histological observations. The diameter of the uterus, thickness of the endometrium and the height of the endometrial epithelium were measured in 20 randomly selected sections using an ocular micrometer. Statistical analysis was carried out using Student’s t-test. The results were judged significant if PB 0.05.
3. Results Of the four extracts of Acalypha indica L. evaluated for post-coital antifertility activity, the petroleum ether extract at 300 and 600 mg/kg significantly inhibited pregnancy in three of eight rats with a mean number of implants of 5.8759 1.76 (PB 0.05) and six of eight rats with a mean number of implants of 2.1259 1.39 (PB 0.001),
S.P. Hiremath et al. / Journal of Ethnopharmacology 67 (1999) 253–258
respectively (Table 1). The ethanol extract at doses of 300 and 600 mg/kg body weight also showed anti-implantation activity in three of eight and five of eight rats, with a mean number of implants of 6.00 91.76 (P B 0.05) and 3.25 9 1.60 (P B0.001), respectively. However, both the doses of the chloroform and distilled water extracts were found to be ineffective and the number of implantation sites in these cases were comparable with the control rats. No toxic effects were observed either by gross visual examination or in the weight of animals. After discontinuation of treatment, all the animals were mated. This resulted in pregnancy and delivery of normal litters, indicating that the action of the extracts was reversible. The estrogenic and the antiestrogenic activity of the petroleum ether and ethanol extracts is shown in Tables 2 and 3. Oral administration of the petroleum ether and ethanol extracts at 600 mg/ kg body weight caused a significant increase in uterine weight in immature rats (versus control, P B 0.001). The uterotrophic potency, as shown by the weight of the uterus, is about 37% of that of the ethinyl estradiol in the case of the petroleum ether and 32% of that of ethinyl estradiol in the case of the ethanol extract, respectively. The uterotrophic changes, such as the
diameter of the uterus, (P B 0.001), thickness of the endometrium (PB 0.001) and height of the endometrial epithelium (P B 0.05), were significantly increased when compared with control rats. The uteri of these rats were inflated and full of fluid resembling the proestrous/estrous uterus. The epithelium of the endometrium consisted of spindle-shaped cells with basal nuclei. The stroma consisted of loose and edematous fibroblast-type cells. The treated rats showed open vaginas, while all the control rats had closed vaginas. Examination of the vaginal smears of treated rats revealed predominantly cornified and nucleated epithelial cells. However, their number was less than in ethinyl estradiol-treated rats. It appears that the petroleum ether and ethanol extracts have weak estrogenic activity, but no antiestrogenic activity at 600 mg/kg dose.
4. Discussion In the present study, Acalypha indica L. was tested for its anti-implantation and estrogenic properties. Among the four extracts tested at two different doses, the petroleum ether and ethanol extracts at 600 mg/kg body weight dose were more potent in their anti-implantation activity, as
Table 1 Effect of extracts of Acalypha indica L. on implantation in rats when fed orally from days 1 to 7 of pregnancya Treatment
Dose (mg/kg body weight)
No. of rats having no implantation sites on day 10
Mean number of implants 9 S.E
% of rats having no implantation sites on day 10
Control
–
Nil
11.000 9 0.46
Nil
Petroleum ether extract
300 600
3 6
05.875 9 1.76* 02.125 9 1.39**
37.5 75.0
Chloroform extract
300 600
Nil Nil
10.500 90.29 11.625 90.37
Nil Nil
Ethanol extract
300 600
3 5
06.000 91.76* 03.2509 1.60**
37.5 62.5
Distilled water extract
300 600
Nil Nil
10.0009 0.46 08.375 9 0.70
Nil Nil
a
Each group consisted of eight rats. * PB0.05; when compared with control. ** PB0.001; when compared with control.
255
S.P. Hiremath et al. / Journal of Ethnopharmacology 67 (1999) 253–258
256
Table 2 Estrogenic and anti-estrogenic activity of the petroleum ether and ethanol extracts of Acalypha indica L. Group
Treatment (dose)
Uterine weight (mg/100 g body weight; mean9S.E.)
Vaginal cornification
I
Control
071.98 902.64
Nil
II
Ethinyl estradiol (1 mg/rat per day)
330.34 9 17.59*
+++a
III
Petroleum ether extract (600 mg/kg)
123.799 07.28*
+ to ++a
IV
Ethanol extract (600 mg/kg)
108.52 909.94*
+ to ++a
V
Ethinyl estradiol (1 mg/rat per day)+petroleum ether extract (600 mg/kg)
258.13 9 05.44
*,**
+++a
VI
Ethinyl estradiol (1 mg/rat per day)+ethanol extract (600 mg/kg)
402.62 9 12.81*,**
+++a
a +, nucleated epithelial cells; ++, nucleated and cornified cells; +++, cornified cells. * PB0.001; when compared with control. ** PB0.01; when compared with ethinyl estradiol.
75 and 62.5% of the rats failed to show any implantation sites, respectively. However, the chloroform and distilled water extracts were inactive, as the number of implantation sites in these cases were comparable with the control rats. The loss of implantation caused by the petroleum ether and ethanol extracts may be due to antizygotic, blastocytotoxic or anti-implantation activity as described by Hafez (1970). The petroleum ether and the ethanol extracts also exhibited estrogenic activity as shown by the significant increase in uterine weight, diameter of the uterus, thickness of endometrium, height of the endometrial epithelium and vaginal epithelial cornification in immature rats. It is well known that for implantation exact equilibrium of estrogen and progesterone is essential, and any disturbance in the level of these hormones may cause infertility (Psychoyos, 1966). The compound of hormonal values usually disturbs the hormonal milieu in the uterus and provokes an infertility effect. In this study, the histological evidence of the uterus treated with petroleum ether and ethanol extracts clearly supports an unfavourable uterine milieu. Therefore, the anti-implantation activity may be due to estrogenic activity, causing the expulsion of ova from the tube, disrupting the luteotrophic activity of the blastocyst (Pincus, 1965; Anderson, 1972).
It is interesting to note that they possess around 35% of the estrogenic efficacy of ethinyl estradiol and thus may reduce some of the unwanted side effects of estrogens. Simultaneous administration of ethinyl estradiol and petroleum ether extract caused a highly significant increase in the uterine weight when compared with control (P B0.001). But, the degree of uterotrophic potency was less than that produced by ethinyl estradiol (P B 0.01), when compared with standard ethinyl estradiol. It also caused a highly significant increase in uterine diameter, thickness of the endometrium end height of the endometrial epithelium (versus control, PB 0.001). The simultaneous administration of ethinyl estradiol and the ethanol extract also caused a highly significant increase in the uterine weight (versus control, PB 0.001). However, the extent of uterotrophic response was greater than that produced by ethinyl estradiol alone (P B 0.01). These observations have also been confirmed when the uterotrophic changes, such as the diameter of the uterus, thickness of the endometrium and height of the endometrial epithelium, were compared with the control and the standard ethinyl estradiol treatments. It appears that the petroleum ether and the ethanol extracts have weak estrogenic activity when given alone, but the petroleum ether extract
S.P. Hiremath et al. / Journal of Ethnopharmacology 67 (1999) 253–258
257
Table 3 Histological changes in the uterus and endometrium after treatment with petroleum ether and ethanol extract of Acalypha indica L. Treatment (dose)
Diameter of uterus (mm9 S.E.)
Thickness of endometrium (mm9S.E.)
Height of endometrial epithelium (mm9 S.E.)
Control Ethinyl estradiol (1 mg/rat per day)
338.999 3.30 812.519 4.27**
52.35 9 2.10 240.71 92.19**
19.59 92.25 46.03 9 2.60*
Petroleum ether extract (600 mg/kg)
625.069 1.44**
85.06 9 2.23**
32.5291.19*
Ethanol extract (600 mg/kg)
512.069 2.15**
78.39 9 1.59**
29.58 91.01*
**,****
288.44 95.41
54.38 91.86**,***
249.10 91.08**,****
50.35 90.69**,***
Ethinyl estradiol (1 mg/rat per day)+ petroleum ether extract (600 mg/kg)
919.899 3.00
Ethinyl estradiol (1 mg/rat per day)+ethanol extract (600 mg/kg)
859.369 1.12**,****
**,****
* PB0.05; when compared with control. ** PB0.001; when compared with control. *** PB0.05; when compared with ethinyl estradiol. **** PB0.001; when compared with ethinyl estradiol.
has shown anti-estrogenic activity when given along with standard ethinyl estradiol. However, the ethanol extract did not show any anti-estrogenic activity when given along with ethinyl estradiol at the tested dose. The phytochemical studies reported on Acalypha indica L. revealed the presence of several sterols in the petroleum ether extract (Talapatra et al., 1981). A flavone kaempferol (Asolkar et al., 1992) has also been reported from the ethanolic extract. In our phytochemical studies of this plant two flavonoids, chrysin and galangin (Hiremath et al., 1998), have been isolated from the ethanol extract. The preliminary investigations of the antifertility studies revealed that the two newly isolated flavonoids have promising antifertility activity. Several sterols (Hall and Fraser, 1983) and flavonoids (Pincus, 1965; Psychoyos, 1966; Khanna and Chaudhury, 1968; Hafez, 1970; Anderson, 1972) have been reported to possess antifertility activity. Therefore, the antiimplantation activity of the extracts of Acalypha indica L. might be due to the presence of such compounds.
Acknowledgements The authors are thankful to Professor Malcolm
Hooper, University of Sunderland, Sunderland, UK for useful discussions, and to M/s. Wyeth Laboratories Ltd., Bombay, for supplying a free sample of ethinyl estradiol. One of the authors (KR) is thankful to AICTE, New Delhi, for financial assistance. References Anderson, L.L., 1972. Biology of Mammalian Fertilization and Implantation. Thomas, Springfield, IL. Anonymous, 1948. Wealth of India: Raw materials, vol. l. CSIR, New Delhi. Asolkar, L.V., Kakkar, K.K., Chakre, O.J., 1992. Glossory of Indian Medicinal Plants with Active Principles Part I. Publications and Information Directorate (CSIR), New Delhi. Bourdy, G., Walter, A., 1992. Maternity and medicinal plants in Vanuatu. I. The cycle of reproduction. Journal of Ethnopharmacology 37, 179 – 196. Caius, J.F., Mhaskar, K.S., 1923. The correlation between chemical composition and anthelmintics and their therapeutic values in connection with the Hookworm. Indian Journal of Medical Research 11, 103 – 109. Chopra, R.N., Nayar, S.L., Chopra, I.C., 1956. Glossory of Indian Medical Plants. CSIR, New Delhi. Donw, G., Steyn, J.S., 1938. The presence of hydrocyanic acid in stock feeds and other plants. African Veterinary Medical Association 9, 60 – 64. Hafez, E.S.E., 1970. Reproduction and Breeding, Techniques for Laboratory Animals. Lea and Febiger, Philadelphia, PA.
258
S.P. Hiremath et al. / Journal of Ethnopharmacology 67 (1999) 253–258
Hall, P.E., Fraser, I.S., 1983. Advances in Human Fertility and Reproductive Endocrinology, vol. 2. Raven Press, New York. Hiremath, S.P., Hanumantha Rao, S., 1990. Antifertility efficacy of the plant Striga lutea (Scrophulariaceae). Contraception 42, 467 – 477. Hiremath, S.P., Hanumantha Rao, S., Jain, P.K., Jaya, Y., Sembulingam, K., 1990. Antifertility activity of Striga lutea— Part I. Indian Journal of Physiology and Pharmacology 34, 23 – 25. Hiremath, S.P., Shrishailappa, B., Swamy, H.K.S., Biradar, J.S., 1993. Antimicrobial activity of various extracts of Acalypha indica (Euphorbiaceae). Indian Journal of Microbiology 33, 75– 77. Hiremath, S.P., Shrishailappa, B., Swamy, H.K.S., Patil, S.B., Londonkar, R.L., 1994. Antifertility activity of Striga orabanchioides. Biological and Pharmaceutical Bulletin 17, 1029 – 1031. Hiremath, S.P., Swamy, H.K.S., Shrishailappa, B., Patil, S.B., Londonkar, R.L., 1996. Postcoital antifertility activity of the plant Striga densiflora (Scrophulariaceae) on female albino rats. International Journal of Pharmacognosy 34, 48–52. Hiremath, S.P., Rudresh, K., Shrishailappa B., 1998. Flavonoids of Acalypha indica L. Indian Journal of Heterocyclic Chemistry (communicated).
.
Khanna, U., Chaudhury, R.R., 1968. Antifertility screening of plants — Part I, Investigation of Butea monosperma (Lam) Kutze. Indian Journal of Medical Research 56, 1575 – 1579. Kirtikar, K.R., Basu, B.D., 1935. Indian Medicinal Plants. Lalit Mohan Basu, Allahabad. Manzoor-i-Khuda, M., Choudury, S.A., Reza, T., Choudhury, A.K., 1985. Chemical investigation of Acalypha indica, Linn. Part-I: Chemical constituents of the roots and leaves. Bangaladesh Journal of Scientific and Industrial Research 20, 171 – 175. Nadakarni, K.M., Nadakarni, A.M., 1982. The Indian Medicinal Plants, vol. II. Popular Prakashan, Bombay. Nohrstedt, A., Kant, J.D., Wray, V., 1982. Acalyphin, a cyanogenic glucoside from Acalypha indica. Phytochemistry 21, 101 – 105. Pincus, G., 1965. Control of Fertility. Academic Press, New York. Psychoyos, A., 1966. Recent Research on Egg Implantation, CIBA Foundation Study Group. Talapatra, B., Shyamprasad, G., Talapatra, S.K., 1981. Acalyphamide, a new amide and other chemical constituents of Acalypha indica, Linn. Indian Journal of Chemistry 20B, 974 – 977 and Refs. cited therein.
Post-coital antifertility activity of Acalypha indica L. Shivayogi P. Hiremath a,*, K. Rudresh a, Shrishailappa Badami a, Saraswati B. Patil b, Somanath R. Patil b a
Department of Chemistry, Gulbarga Uni6ersity, Gulbarga-585 106, India Department of Zoology, Gulbarga Uni6ersity, Gulbarga-585 106, India
b
Received 1 August 1998; received in revised form 19 October 1998; accepted 2 November 1998
Abstract Four successive solvent extracts of the whole plant Acalypha indica L. (Euphorbiaceae) were tested for post-coital antifertility activity in female albino rats. Of these, the petroleum ether and ethanol extracts were found to be most effective in causing significant anti-implantation activity. The antifertility activity was reversible on withdrawal of the treatment of the extracts. Both the extracts at 600 mg/kg body weight showed estrogenic activity. Histological studies of the uterus were carried out to confirm this estrogenic activity. © 1999 Elsevier Science Ireland Ltd. All rights reserved.
1. Introduction One approach pursued to identify new antifertility agents is the search for their presence in natural sources. Many plant preparations are reported to have fertility regulating properties and a few have been tested for such effects. But so far no single plant is available which can be developed further as a potent antifertility agent. Hence the search continues. Acalypha indica L. (Euphorbiaceae) is a weed widely distributed throughout the plains of India. It has been reported to be useful in treating pneumonia, asthma, rheumatism and several other ailments (Kirtikar and Basu,
* Corresponding author.
1935; Anonymous, 1948; Chopra et al., 1956; Nadakarni and Nadakarni, 1982). This plant was cited as an emmenagogue by Nadakarni and Nadakarni (1982). The leaves of Acalypha grandis have also been reported to possess contraceptive activity (Bourdy and Walter, 1992). Several chemical (Donw and Steyn, 1938; Talapatra et al., 1981; Nohrstedt et al., 1982; Manzoor-i-Khuda et al., 1985; Asolkar et al., 1992) and biological (Caius and Mhaskar, 1923; Hiremath et al., 1993) investigations have been carried out on this plant, but, so far no antifertility testing has been done. Hence, in continuation of our work on antifertility activity of plants (Hiremath and Hanumantha, 1990; Hiremath et al., 1990, 1994, 1996), we were interested in subjecting Acalypha indica L. to antifertility testing in female albino rats.
0378-8741/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 9 8 ) 0 0 2 1 3 - X
254
S.P. Hiremath et al. / Journal of Ethnopharmacology 67 (1999) 253–258
2. Materials and methods The whole plant of Acalypha indica L. was collected from the fields in and around Gulbarga during August and September, 1996 and was authenticated at the Herbarium, Department of Botany, Gulbarga University, Gulbarga (HGUG No.279). The plant was shade dried, powdered and subjected to Soxhlet extraction (500 g) successively and separately with petroleum ether (60 – 80°C, 2 l), chloroform (2 l), ethanol (95%, 2 l) and distilled water (2 l). The extracts were concentrated to dryness in a flash-evaporator under reduced pressure and controlled temperature (50 – 60°C). The petroleum ether and chloroform extracts yielded dark brown solids weighing 20.0 and 32.0 g, respectively. All the extracts were stored in a refrigerator. The extracts were prepared in Tween − 80 (1%), suspended in distilled water and they were administered to the rats at doses of 300 and 600 mg/kg body weight orally by means of an intragastric catheter. Colony-bred female albino rats (Wistar strain), weighing (150–200 g), were maintained under controlled standard animal house conditions with access to food and water ad libitum. Vaginal smears from each rat were monitored daily. Only rats with normal estrous cycles were selected. Anti-implantation activity was determined as described by Khanna and Chaudhury (1968). Rats found in proestrous phase of the cycle were caged with males of proven fertility, in the ratio of 2:1 and examined the following morning for evidence of copulation. Rats exhibiting thick clumps of spermatozoa in their vaginal smears were separated and that day was designated as day 1 of pregnancy and those rats were divided into nine groups containing eight rats in each group. The extracts were administered at 300 and 600 mg/kg body weight orally from days 1 to 7 of pregnancy. Control rats received the vehicle (Tween-80, 1%) only. On day 10, laparotomy was performed under light ether anesthesia and semisterile conditions. The uteri were examined to determine the number of implantation sites. The rats were allowed to recover and deliver after full term. Each pup was weighed and examined for gross defects. The litters were allowed to grow to check post-na-
tal growth and monitor any congenital abnormalities. The petroleum ether and ethanol extracts were found to be the most active of the extracts of Acalypha indica L., hence they were subjected to a detailed investigation for potential estrogenic and antiestrogenic activity. Colony-bred (Wistar strain) immature female albino rats, 21–23 days old, weighing between 35 and 45 g, were bilaterally ovariectimised by dorsolateral approach under light ether anaesthesia and sterile conditions. They were divided into six groups consisting of eight rats each. The first group served as a control end received vehicle only (Tween-80, 1%). The second group received ethinyl estradiol in olive oil, 1 mg/rat per day, subcutaneously. The third and fourth groups received the petroleum ether and ethanol extracts at a dose of 600 mg/kg body weight, respectively. The fifth and sixth groups received, in addition to ethinyl estradiol, a test dose of the petroleum ether and ethanol extracts at 600 mg/kg body weight, respectively. All the above treatments were given for 7 days. On the 8th day, the rats were sacrificed by decapitation, the uteri dissected out and surrounding tissues removed. The uteri were blotted on filter papers and weighed quickly on a sensitive balance and fixed in Bouin’s fluid for 24 h. The tissues were dehydrated and embedded in paraffin. The paraffin sections were cut at 6 mm and stained with haematoxylin–eosin for histological observations. The diameter of the uterus, thickness of the endometrium and the height of the endometrial epithelium were measured in 20 randomly selected sections using an ocular micrometer. Statistical analysis was carried out using Student’s t-test. The results were judged significant if PB 0.05.
3. Results Of the four extracts of Acalypha indica L. evaluated for post-coital antifertility activity, the petroleum ether extract at 300 and 600 mg/kg significantly inhibited pregnancy in three of eight rats with a mean number of implants of 5.8759 1.76 (PB 0.05) and six of eight rats with a mean number of implants of 2.1259 1.39 (PB 0.001),
S.P. Hiremath et al. / Journal of Ethnopharmacology 67 (1999) 253–258
respectively (Table 1). The ethanol extract at doses of 300 and 600 mg/kg body weight also showed anti-implantation activity in three of eight and five of eight rats, with a mean number of implants of 6.00 91.76 (P B 0.05) and 3.25 9 1.60 (P B0.001), respectively. However, both the doses of the chloroform and distilled water extracts were found to be ineffective and the number of implantation sites in these cases were comparable with the control rats. No toxic effects were observed either by gross visual examination or in the weight of animals. After discontinuation of treatment, all the animals were mated. This resulted in pregnancy and delivery of normal litters, indicating that the action of the extracts was reversible. The estrogenic and the antiestrogenic activity of the petroleum ether and ethanol extracts is shown in Tables 2 and 3. Oral administration of the petroleum ether and ethanol extracts at 600 mg/ kg body weight caused a significant increase in uterine weight in immature rats (versus control, P B 0.001). The uterotrophic potency, as shown by the weight of the uterus, is about 37% of that of the ethinyl estradiol in the case of the petroleum ether and 32% of that of ethinyl estradiol in the case of the ethanol extract, respectively. The uterotrophic changes, such as the
diameter of the uterus, (P B 0.001), thickness of the endometrium (PB 0.001) and height of the endometrial epithelium (P B 0.05), were significantly increased when compared with control rats. The uteri of these rats were inflated and full of fluid resembling the proestrous/estrous uterus. The epithelium of the endometrium consisted of spindle-shaped cells with basal nuclei. The stroma consisted of loose and edematous fibroblast-type cells. The treated rats showed open vaginas, while all the control rats had closed vaginas. Examination of the vaginal smears of treated rats revealed predominantly cornified and nucleated epithelial cells. However, their number was less than in ethinyl estradiol-treated rats. It appears that the petroleum ether and ethanol extracts have weak estrogenic activity, but no antiestrogenic activity at 600 mg/kg dose.
4. Discussion In the present study, Acalypha indica L. was tested for its anti-implantation and estrogenic properties. Among the four extracts tested at two different doses, the petroleum ether and ethanol extracts at 600 mg/kg body weight dose were more potent in their anti-implantation activity, as
Table 1 Effect of extracts of Acalypha indica L. on implantation in rats when fed orally from days 1 to 7 of pregnancya Treatment
Dose (mg/kg body weight)
No. of rats having no implantation sites on day 10
Mean number of implants 9 S.E
% of rats having no implantation sites on day 10
Control
–
Nil
11.000 9 0.46
Nil
Petroleum ether extract
300 600
3 6
05.875 9 1.76* 02.125 9 1.39**
37.5 75.0
Chloroform extract
300 600
Nil Nil
10.500 90.29 11.625 90.37
Nil Nil
Ethanol extract
300 600
3 5
06.000 91.76* 03.2509 1.60**
37.5 62.5
Distilled water extract
300 600
Nil Nil
10.0009 0.46 08.375 9 0.70
Nil Nil
a
Each group consisted of eight rats. * PB0.05; when compared with control. ** PB0.001; when compared with control.
255
S.P. Hiremath et al. / Journal of Ethnopharmacology 67 (1999) 253–258
256
Table 2 Estrogenic and anti-estrogenic activity of the petroleum ether and ethanol extracts of Acalypha indica L. Group
Treatment (dose)
Uterine weight (mg/100 g body weight; mean9S.E.)
Vaginal cornification
I
Control
071.98 902.64
Nil
II
Ethinyl estradiol (1 mg/rat per day)
330.34 9 17.59*
+++a
III
Petroleum ether extract (600 mg/kg)
123.799 07.28*
+ to ++a
IV
Ethanol extract (600 mg/kg)
108.52 909.94*
+ to ++a
V
Ethinyl estradiol (1 mg/rat per day)+petroleum ether extract (600 mg/kg)
258.13 9 05.44
*,**
+++a
VI
Ethinyl estradiol (1 mg/rat per day)+ethanol extract (600 mg/kg)
402.62 9 12.81*,**
+++a
a +, nucleated epithelial cells; ++, nucleated and cornified cells; +++, cornified cells. * PB0.001; when compared with control. ** PB0.01; when compared with ethinyl estradiol.
75 and 62.5% of the rats failed to show any implantation sites, respectively. However, the chloroform and distilled water extracts were inactive, as the number of implantation sites in these cases were comparable with the control rats. The loss of implantation caused by the petroleum ether and ethanol extracts may be due to antizygotic, blastocytotoxic or anti-implantation activity as described by Hafez (1970). The petroleum ether and the ethanol extracts also exhibited estrogenic activity as shown by the significant increase in uterine weight, diameter of the uterus, thickness of endometrium, height of the endometrial epithelium and vaginal epithelial cornification in immature rats. It is well known that for implantation exact equilibrium of estrogen and progesterone is essential, and any disturbance in the level of these hormones may cause infertility (Psychoyos, 1966). The compound of hormonal values usually disturbs the hormonal milieu in the uterus and provokes an infertility effect. In this study, the histological evidence of the uterus treated with petroleum ether and ethanol extracts clearly supports an unfavourable uterine milieu. Therefore, the anti-implantation activity may be due to estrogenic activity, causing the expulsion of ova from the tube, disrupting the luteotrophic activity of the blastocyst (Pincus, 1965; Anderson, 1972).
It is interesting to note that they possess around 35% of the estrogenic efficacy of ethinyl estradiol and thus may reduce some of the unwanted side effects of estrogens. Simultaneous administration of ethinyl estradiol and petroleum ether extract caused a highly significant increase in the uterine weight when compared with control (P B0.001). But, the degree of uterotrophic potency was less than that produced by ethinyl estradiol (P B 0.01), when compared with standard ethinyl estradiol. It also caused a highly significant increase in uterine diameter, thickness of the endometrium end height of the endometrial epithelium (versus control, PB 0.001). The simultaneous administration of ethinyl estradiol and the ethanol extract also caused a highly significant increase in the uterine weight (versus control, PB 0.001). However, the extent of uterotrophic response was greater than that produced by ethinyl estradiol alone (P B 0.01). These observations have also been confirmed when the uterotrophic changes, such as the diameter of the uterus, thickness of the endometrium and height of the endometrial epithelium, were compared with the control and the standard ethinyl estradiol treatments. It appears that the petroleum ether and the ethanol extracts have weak estrogenic activity when given alone, but the petroleum ether extract
S.P. Hiremath et al. / Journal of Ethnopharmacology 67 (1999) 253–258
257
Table 3 Histological changes in the uterus and endometrium after treatment with petroleum ether and ethanol extract of Acalypha indica L. Treatment (dose)
Diameter of uterus (mm9 S.E.)
Thickness of endometrium (mm9S.E.)
Height of endometrial epithelium (mm9 S.E.)
Control Ethinyl estradiol (1 mg/rat per day)
338.999 3.30 812.519 4.27**
52.35 9 2.10 240.71 92.19**
19.59 92.25 46.03 9 2.60*
Petroleum ether extract (600 mg/kg)
625.069 1.44**
85.06 9 2.23**
32.5291.19*
Ethanol extract (600 mg/kg)
512.069 2.15**
78.39 9 1.59**
29.58 91.01*
**,****
288.44 95.41
54.38 91.86**,***
249.10 91.08**,****
50.35 90.69**,***
Ethinyl estradiol (1 mg/rat per day)+ petroleum ether extract (600 mg/kg)
919.899 3.00
Ethinyl estradiol (1 mg/rat per day)+ethanol extract (600 mg/kg)
859.369 1.12**,****
**,****
* PB0.05; when compared with control. ** PB0.001; when compared with control. *** PB0.05; when compared with ethinyl estradiol. **** PB0.001; when compared with ethinyl estradiol.
has shown anti-estrogenic activity when given along with standard ethinyl estradiol. However, the ethanol extract did not show any anti-estrogenic activity when given along with ethinyl estradiol at the tested dose. The phytochemical studies reported on Acalypha indica L. revealed the presence of several sterols in the petroleum ether extract (Talapatra et al., 1981). A flavone kaempferol (Asolkar et al., 1992) has also been reported from the ethanolic extract. In our phytochemical studies of this plant two flavonoids, chrysin and galangin (Hiremath et al., 1998), have been isolated from the ethanol extract. The preliminary investigations of the antifertility studies revealed that the two newly isolated flavonoids have promising antifertility activity. Several sterols (Hall and Fraser, 1983) and flavonoids (Pincus, 1965; Psychoyos, 1966; Khanna and Chaudhury, 1968; Hafez, 1970; Anderson, 1972) have been reported to possess antifertility activity. Therefore, the antiimplantation activity of the extracts of Acalypha indica L. might be due to the presence of such compounds.
Acknowledgements The authors are thankful to Professor Malcolm
Hooper, University of Sunderland, Sunderland, UK for useful discussions, and to M/s. Wyeth Laboratories Ltd., Bombay, for supplying a free sample of ethinyl estradiol. One of the authors (KR) is thankful to AICTE, New Delhi, for financial assistance. References Anderson, L.L., 1972. Biology of Mammalian Fertilization and Implantation. Thomas, Springfield, IL. Anonymous, 1948. Wealth of India: Raw materials, vol. l. CSIR, New Delhi. Asolkar, L.V., Kakkar, K.K., Chakre, O.J., 1992. Glossory of Indian Medicinal Plants with Active Principles Part I. Publications and Information Directorate (CSIR), New Delhi. Bourdy, G., Walter, A., 1992. Maternity and medicinal plants in Vanuatu. I. The cycle of reproduction. Journal of Ethnopharmacology 37, 179 – 196. Caius, J.F., Mhaskar, K.S., 1923. The correlation between chemical composition and anthelmintics and their therapeutic values in connection with the Hookworm. Indian Journal of Medical Research 11, 103 – 109. Chopra, R.N., Nayar, S.L., Chopra, I.C., 1956. Glossory of Indian Medical Plants. CSIR, New Delhi. Donw, G., Steyn, J.S., 1938. The presence of hydrocyanic acid in stock feeds and other plants. African Veterinary Medical Association 9, 60 – 64. Hafez, E.S.E., 1970. Reproduction and Breeding, Techniques for Laboratory Animals. Lea and Febiger, Philadelphia, PA.
258
S.P. Hiremath et al. / Journal of Ethnopharmacology 67 (1999) 253–258
Hall, P.E., Fraser, I.S., 1983. Advances in Human Fertility and Reproductive Endocrinology, vol. 2. Raven Press, New York. Hiremath, S.P., Hanumantha Rao, S., 1990. Antifertility efficacy of the plant Striga lutea (Scrophulariaceae). Contraception 42, 467 – 477. Hiremath, S.P., Hanumantha Rao, S., Jain, P.K., Jaya, Y., Sembulingam, K., 1990. Antifertility activity of Striga lutea— Part I. Indian Journal of Physiology and Pharmacology 34, 23 – 25. Hiremath, S.P., Shrishailappa, B., Swamy, H.K.S., Biradar, J.S., 1993. Antimicrobial activity of various extracts of Acalypha indica (Euphorbiaceae). Indian Journal of Microbiology 33, 75– 77. Hiremath, S.P., Shrishailappa, B., Swamy, H.K.S., Patil, S.B., Londonkar, R.L., 1994. Antifertility activity of Striga orabanchioides. Biological and Pharmaceutical Bulletin 17, 1029 – 1031. Hiremath, S.P., Swamy, H.K.S., Shrishailappa, B., Patil, S.B., Londonkar, R.L., 1996. Postcoital antifertility activity of the plant Striga densiflora (Scrophulariaceae) on female albino rats. International Journal of Pharmacognosy 34, 48–52. Hiremath, S.P., Rudresh, K., Shrishailappa B., 1998. Flavonoids of Acalypha indica L. Indian Journal of Heterocyclic Chemistry (communicated).
.
Khanna, U., Chaudhury, R.R., 1968. Antifertility screening of plants — Part I, Investigation of Butea monosperma (Lam) Kutze. Indian Journal of Medical Research 56, 1575 – 1579. Kirtikar, K.R., Basu, B.D., 1935. Indian Medicinal Plants. Lalit Mohan Basu, Allahabad. Manzoor-i-Khuda, M., Choudury, S.A., Reza, T., Choudhury, A.K., 1985. Chemical investigation of Acalypha indica, Linn. Part-I: Chemical constituents of the roots and leaves. Bangaladesh Journal of Scientific and Industrial Research 20, 171 – 175. Nadakarni, K.M., Nadakarni, A.M., 1982. The Indian Medicinal Plants, vol. II. Popular Prakashan, Bombay. Nohrstedt, A., Kant, J.D., Wray, V., 1982. Acalyphin, a cyanogenic glucoside from Acalypha indica. Phytochemistry 21, 101 – 105. Pincus, G., 1965. Control of Fertility. Academic Press, New York. Psychoyos, A., 1966. Recent Research on Egg Implantation, CIBA Foundation Study Group. Talapatra, B., Shyamprasad, G., Talapatra, S.K., 1981. Acalyphamide, a new amide and other chemical constituents of Acalypha indica, Linn. Indian Journal of Chemistry 20B, 974 – 977 and Refs. cited therein.