You are on page 1of 6

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
b
Department of Zoology, Gulbarga Uni6ersity, Gulbarga-585 106, India

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 1935; Anonymous, 1948; Chopra et al., 1956;


Nadakarni and Nadakarni, 1982). This plant was
One approach pursued to identify new antifer- cited as an emmenagogue by Nadakarni and
tility agents is the search for their presence in Nadakarni (1982). The leaves of Acalypha grandis
natural sources. Many plant preparations are re- have also been reported to possess contraceptive
ported to have fertility regulating properties and a activity (Bourdy and Walter, 1992). Several chem-
few have been tested for such effects. But so far ical (Donw and Steyn, 1938; Talapatra et al.,
no single plant is available which can be devel- 1981; Nohrstedt et al., 1982; Manzoor-i-Khuda et
oped further as a potent antifertility agent. Hence al., 1985; Asolkar et al., 1992) and biological
the search continues. Acalypha indica L. (Euphor- (Caius and Mhaskar, 1923; Hiremath et al., 1993)
biaceae) is a weed widely distributed throughout investigations have been carried out on this plant,
the plains of India. It has been reported to be but, so far no antifertility testing has been done.
useful in treating pneumonia, asthma, rheumatism Hence, in continuation of our work on antifertil-
and several other ailments (Kirtikar and Basu, ity activity of plants (Hiremath and Hanumantha,
1990; Hiremath et al., 1990, 1994, 1996), we were
interested in subjecting Acalypha indica L. to an-
* Corresponding author. tifertility 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 tal growth and monitor any congenital


abnormalities.
The whole plant of Acalypha indica L. was The petroleum ether and ethanol extracts were
collected from the fields in and around Gulbarga found to be the most active of the extracts of
during August and September, 1996 and was au- Acalypha indica L., hence they were subjected to a
thenticated at the Herbarium, Department of detailed investigation for potential estrogenic and
Botany, Gulbarga University, Gulbarga (HGUG antiestrogenic activity. Colony-bred (Wistar
No.279). The plant was shade dried, powdered strain) immature female albino rats, 21–23 days
and subjected to Soxhlet extraction (500 g) succes- old, weighing between 35 and 45 g, were bilater-
sively and separately with petroleum ether (60 – ally ovariectimised by dorsolateral approach un-
80°C, 2 l), chloroform (2 l), ethanol (95%, 2 l) and der light ether anaesthesia and sterile conditions.
distilled water (2 l). The extracts were concen- They were divided into six groups consisting of
trated to dryness in a flash-evaporator under re- eight rats each. The first group served as a control
duced pressure and controlled temperature end received vehicle only (Tween-80, 1%). The
(50 – 60°C). The petroleum ether and chloroform second group received ethinyl estradiol in olive
extracts yielded dark brown solids weighing 20.0 oil, 1 mg/rat per day, subcutaneously. The third
and 32.0 g, respectively. All the extracts were and fourth groups received the petroleum ether
stored in a refrigerator. The extracts were pre- and ethanol extracts at a dose of 600 mg/kg body
pared in Tween − 80 (1%), suspended in distilled weight, respectively. The fifth and sixth groups
water and they were administered to the rats at received, in addition to ethinyl estradiol, a test
doses of 300 and 600 mg/kg body weight orally by dose of the petroleum ether and ethanol extracts
means of an intragastric catheter. at 600 mg/kg body weight, respectively. All the
Colony-bred female albino rats (Wistar strain), above treatments were given for 7 days. On the
weighing (150–200 g), were maintained under 8th day, the rats were sacrificed by decapitation,
controlled standard animal house conditions with the uteri dissected out and surrounding tissues
access to food and water ad libitum. Vaginal removed. The uteri were blotted on filter papers
smears from each rat were monitored daily. Only and weighed quickly on a sensitive balance and
rats with normal estrous cycles were selected. fixed in Bouin’s fluid for 24 h. The tissues were
Anti-implantation activity was determined as de- dehydrated and embedded in paraffin. The
scribed by Khanna and Chaudhury (1968). Rats paraffin sections were cut at 6 mm and stained
found in proestrous phase of the cycle were caged with haematoxylin–eosin for histological observa-
with males of proven fertility, in the ratio of 2:1 tions. The diameter of the uterus, thickness of the
and examined the following morning for evidence endometrium and the height of the endometrial
of copulation. Rats exhibiting thick clumps of epithelium were measured in 20 randomly selected
spermatozoa in their vaginal smears were sepa- sections using an ocular micrometer. Statistical
rated and that day was designated as day 1 of analysis was carried out using Student’s t-test.
pregnancy and those rats were divided into nine The results were judged significant if PB 0.05.
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. 3. Results
Control rats received the vehicle (Tween-80, 1%)
only. On day 10, laparotomy was performed un- Of the four extracts of Acalypha indica L. eval-
der light ether anesthesia and semisterile condi- uated for post-coital antifertility activity, the
tions. The uteri were examined to determine the petroleum ether extract at 300 and 600 mg/kg
number of implantation sites. The rats were al- significantly inhibited pregnancy in three of eight
lowed to recover and deliver after full term. Each rats with a mean number of implants of 5.8759
pup was weighed and examined for gross defects. 1.76 (PB 0.05) and six of eight rats with a mean
The litters were allowed to grow to check post-na- number of implants of 2.1259 1.39 (PB 0.001),
S.P. Hiremath et al. / Journal of Ethnopharmacology 67 (1999) 253–258 255

respectively (Table 1). The ethanol extract at diameter of the uterus, (P B 0.001), thickness of
doses of 300 and 600 mg/kg body weight also the endometrium (PB 0.001) and height of the
showed anti-implantation activity in three of eight endometrial epithelium (P B 0.05), were signifi-
and five of eight rats, with a mean number of cantly increased when compared with control rats.
implants of 6.00 91.76 (P B 0.05) and 3.25 9 1.60 The uteri of these rats were inflated and full of
(P B0.001), respectively. However, both the doses fluid resembling the proestrous/estrous uterus.
of the chloroform and distilled water extracts The epithelium of the endometrium consisted of
were found to be ineffective and the number of spindle-shaped cells with basal nuclei. The stroma
implantation sites in these cases were comparable consisted of loose and edematous fibroblast-type
with the control rats. cells. The treated rats showed open vaginas, while
No toxic effects were observed either by gross all the control rats had closed vaginas. Examina-
visual examination or in the weight of animals. tion of the vaginal smears of treated rats revealed
After discontinuation of treatment, all the animals predominantly cornified and nucleated epithelial
were mated. This resulted in pregnancy and deliv- cells. However, their number was less than in
ery of normal litters, indicating that the action of ethinyl estradiol-treated rats.
the extracts was reversible. It appears that the petroleum ether and ethanol
The estrogenic and the antiestrogenic activity of extracts have weak estrogenic activity, but no
antiestrogenic activity at 600 mg/kg dose.
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 4. Discussion
uterine weight in immature rats (versus control,
P B 0.001). The uterotrophic potency, as shown In the present study, Acalypha indica L. was
by the weight of the uterus, is about 37% of that tested for its anti-implantation and estrogenic
of the ethinyl estradiol in the case of the properties. Among the four extracts tested at two
petroleum ether and 32% of that of ethinyl estra- different doses, the petroleum ether and ethanol
diol in the case of the ethanol extract, respec- extracts at 600 mg/kg body weight dose were
tively. The uterotrophic changes, such as the 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 No. of rats having no Mean number of % of rats having no
body weight) implantation sites on day 10 implants 9 S.E implantation sites on day 10

Control – Nil 11.000 9 0.46 Nil


Petroleum ether 300 3 05.875 9 1.76* 37.5
extract 600 6 02.125 9 1.39** 75.0
Chloroform 300 Nil 10.500 90.29 Nil
extract 600 Nil 11.625 90.37 Nil
Ethanol extract 300 3 06.000 91.76* 37.5
600 5 03.2509 1.60** 62.5
Distilled water 300 Nil 10.0009 0.46 Nil
extract 600 Nil 08.375 9 0.70 Nil

a
Each group consisted of eight rats.
* PB0.05; when compared with control.
** PB0.001; when compared with control.
256 S.P. Hiremath et al. / Journal of Ethnopharmacology 67 (1999) 253–258

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; Vaginal


mean9S.E.) 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 258.13 9 05.44 *,**
+++a
extract (600 mg/kg)
VI Ethinyl estradiol (1 mg/rat per day)+ethanol extract 402.62 9 12.81*,** +++a
(600 mg/kg)

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 It is interesting to note that they possess around
implantation sites, respectively. However, the 35% of the estrogenic efficacy of ethinyl estradiol
chloroform and distilled water extracts were inac- and thus may reduce some of the unwanted side
tive, as the number of implantation sites in these effects of estrogens.
cases were comparable with the control rats. Simultaneous administration of ethinyl estra-
The loss of implantation caused by the diol and petroleum ether extract caused a highly
petroleum ether and ethanol extracts may be due significant increase in the uterine weight when
to antizygotic, blastocytotoxic or anti-implanta- compared with control (P B0.001). But, the de-
tion activity as described by Hafez (1970). gree of uterotrophic potency was less than that
The petroleum ether and the ethanol extracts produced by ethinyl estradiol (P B 0.01), when
also exhibited estrogenic activity as shown by the compared with standard ethinyl estradiol. It also
significant increase in uterine weight, diameter of caused a highly significant increase in uterine di-
the uterus, thickness of endometrium, height of ameter, thickness of the endometrium end height
the endometrial epithelium and vaginal epithelial of the endometrial epithelium (versus control,
cornification in immature rats. PB 0.001). The simultaneous administration of
It is well known that for implantation exact ethinyl estradiol and the ethanol extract also
equilibrium of estrogen and progesterone is essen- caused a highly significant increase in the uterine
tial, and any disturbance in the level of these weight (versus control, PB 0.001). However, the
hormones may cause infertility (Psychoyos, 1966). extent of uterotrophic response was greater than
The compound of hormonal values usually dis- that produced by ethinyl estradiol alone (P B
turbs the hormonal milieu in the uterus and pro- 0.01). These observations have also been confi-
vokes an infertility effect. In this study, the rmed when the uterotrophic changes, such as the
histological evidence of the uterus treated with diameter of the uterus, thickness of the en-
petroleum ether and ethanol extracts clearly sup- dometrium and height of the endometrial epithe-
ports an unfavourable uterine milieu. Therefore, lium, were compared with the control and the
the anti-implantation activity may be due to es- standard ethinyl estradiol treatments.
trogenic activity, causing the expulsion of ova It appears that the petroleum ether and the
from the tube, disrupting the luteotrophic activity ethanol extracts have weak estrogenic activity
of the blastocyst (Pincus, 1965; Anderson, 1972). 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 Thickness of Height of endometrial


(mm9 S.E.) endometrium (mm9S.E.) epithelium (mm9 S.E.)

Control 338.999 3.30 52.35 9 2.10 19.59 92.25


Ethinyl estradiol (1 mg/rat per day) 812.519 4.27** 240.71 92.19** 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*
Ethinyl estradiol (1 mg/rat per day)+ 919.899 3.00 **,****
288.44 95.41 **,****
54.38 91.86**,***
petroleum ether extract (600 mg/kg)
Ethinyl estradiol (1 mg/rat per day)+ethanol 859.369 1.12**,**** 249.10 91.08**,**** 50.35 90.69**,***
extract (600 mg/kg)

* 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 Hooper, University of Sunderland, Sunderland,
along with standard ethinyl estradiol. However, the UK for useful discussions, and to M/s. Wyeth
ethanol extract did not show any anti-estrogenic Laboratories Ltd., Bombay, for supplying a free
activity when given along with ethinyl estradiol at sample of ethinyl estradiol. One of the authors
the tested dose. (KR) is thankful to AICTE, New Delhi, for finan-
The phytochemical studies reported on Acalypha cial assistance.
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 References
been reported from the ethanolic extract. In our
Anderson, L.L., 1972. Biology of Mammalian Fertilization and
phytochemical studies of this plant two flavonoids, Implantation. Thomas, Springfield, IL.
chrysin and galangin (Hiremath et al., 1998), have Anonymous, 1948. Wealth of India: Raw materials, vol. l. CSIR,
been isolated from the ethanol extract. The prelim- New Delhi.
inary investigations of the antifertility studies re- Asolkar, L.V., Kakkar, K.K., Chakre, O.J., 1992. Glossory of
vealed that the two newly isolated flavonoids have Indian Medicinal Plants with Active Principles Part I.
Publications and Information Directorate (CSIR), New
promising antifertility activity. Several sterols (Hall Delhi.
and Fraser, 1983) and flavonoids (Pincus, 1965; Bourdy, G., Walter, A., 1992. Maternity and medicinal plants
Psychoyos, 1966; Khanna and Chaudhury, 1968; in Vanuatu. I. The cycle of reproduction. Journal of
Hafez, 1970; Anderson, 1972) have been reported Ethnopharmacology 37, 179 – 196.
Caius, J.F., Mhaskar, K.S., 1923. The correlation between
to possess antifertility activity. Therefore, the anti-
chemical composition and anthelmintics and their therapeu-
implantation activity of the extracts of Acalypha tic values in connection with the Hookworm. Indian Journal
indica L. might be due to the presence of such of Medical Research 11, 103 – 109.
compounds. 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
Acknowledgements Association 9, 60 – 64.
Hafez, E.S.E., 1970. Reproduction and Breeding, Techniques
The authors are thankful to Professor Malcolm 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 Khanna, U., Chaudhury, R.R., 1968. Antifertility screening
Reproductive Endocrinology, vol. 2. Raven Press, New of plants — Part I, Investigation of Butea monosperma
York. (Lam) Kutze. Indian Journal of Medical Research 56,
Hiremath, S.P., Hanumantha Rao, S., 1990. Antifertility effi- 1575 – 1579.
cacy of the plant Striga lutea (Scrophulariaceae). Contracep- Kirtikar, K.R., Basu, B.D., 1935. Indian Medicinal Plants.
tion 42, 467 – 477. Lalit Mohan Basu, Allahabad.
Hiremath, S.P., Hanumantha Rao, S., Jain, P.K., Jaya, Y., Manzoor-i-Khuda, M., Choudury, S.A., Reza, T., Choud-
Sembulingam, K., 1990. Antifertility activity of Striga hury, A.K., 1985. Chemical investigation of Acalypha in-
lutea— Part I. Indian Journal of Physiology and Pharmacol- dica, Linn. Part-I: Chemical constituents of the roots and
ogy 34, 23 – 25. leaves. Bangaladesh Journal of Scientific and Industrial
Hiremath, S.P., Shrishailappa, B., Swamy, H.K.S., Biradar, J.S.,
Research 20, 171 – 175.
1993. Antimicrobial activity of various extracts of Acalypha
Nadakarni, K.M., Nadakarni, A.M., 1982. The Indian
indica (Euphorbiaceae). Indian Journal of Microbiology 33,
Medicinal Plants, vol. II. Popular Prakashan, Bombay.
75– 77.
Nohrstedt, A., Kant, J.D., Wray, V., 1982. Acalyphin, a
Hiremath, S.P., Shrishailappa, B., Swamy, H.K.S., Patil, S.B.,
Londonkar, R.L., 1994. Antifertility activity of Striga ora- cyanogenic glucoside from Acalypha indica. Phytochem-
banchioides. Biological and Pharmaceutical Bulletin 17, istry 21, 101 – 105.
1029 – 1031. Pincus, G., 1965. Control of Fertility. Academic Press, New
Hiremath, S.P., Swamy, H.K.S., Shrishailappa, B., Patil, S.B., York.
Londonkar, R.L., 1996. Postcoital antifertility activity of the Psychoyos, A., 1966. Recent Research on Egg Implantation,
plant Striga densiflora (Scrophulariaceae) on female albino CIBA Foundation Study Group.
rats. International Journal of Pharmacognosy 34, 48–52. Talapatra, B., Shyamprasad, G., Talapatra, S.K., 1981. Aca-
Hiremath, S.P., Rudresh, K., Shrishailappa B., 1998. lyphamide, a new amide and other chemical constituents
Flavonoids of Acalypha indica L. Indian Journal of Hetero- of Acalypha indica, Linn. Indian Journal of Chemistry
cyclic Chemistry (communicated). 20B, 974 – 977 and Refs. cited therein.