Effect of Garcinia kola Seed Extract on Female Reproductive Functions in Rats
The effects of G. kola seed extract on oestrous
cycle, ovulation, implantation and pregnancy were studied in adult female
rats with the aim of its possible use as female contraceptive. The three
experimental groups were fed with G. kola seed extract (200 mg
kg-1 body weight) (GKSE) and the control received distilled
water once daily. Group 1 was fed with GKSE for three weeks and the estrous
cycle monitored and number of ova released recorded. Group 2 consisted
of pregnant rats and received GKSE from day one of pregnancy. Equal numbers
of the rats were sacrificed on days six, eight and 19th of pregnancy.
The total number of implants, resorption and viable foetuses were weighed,
examined and recorded. Group 3 animals received GKSE daily for three weeks;
half of them were sacrificed after three weeks and the others were allowed
three weeks to recover from the effect of the extract. Histology of the
ovary, uterus and fallopian tubes were done. The results showed that the
oestrous cycle was altered with a significant reduction in the occurrence
of estrous and metestrous phase. Ovulation was partially blocked (70%
blockage). No significant difference between the number and weight of
the implantation sites in both pregnant control rats and the GKSE rats.
No significant difference in both the foetal number and weight on day
19 of pregnancy. GKSE made the uterine endometrial gland inactive, stopped
maturation of the follicles, caused acute inflammation of the tubes but
all the organs showed full recovery after administration of GKSE was stopped.
GKSE may be use as a contraceptive with a possible advantage of reversibility.
Traditionally, in Africa the seed is used for the treatment of bronchitis,
throat infections, antipurgative and antiparasitic (Madubunyi, 1995; Okunji
and Iwu, 1991; Adefile-Ositelu et al., 2004). It is known to posses
anti-atherogenic effects (Adaramoye et al., 2005b) antilipoperoxative
effect (Emerole et al., 2005), aphrodisiac effect (Uko et al.,
2001) and gastroprotective effects (Olaleye and Farombi, 2006). The biflavonoids
complex from Garcinia kola has also been shown to have anti-diabetics,
antihepatotoxic and hypolipidemia effect (Tita et al., 2001; Adaramoye
and Adeyemi, 2006a, b). Kolaviron (a natural antioxidant) was isolated
as a defatted ethanol extract from Garcinia kola seed and it contains
a mixture of three compounds Garcinia bioflavonoid GB-1, GB-2 and G.
kola flavanone. (Farombi et al., 2002; Adaramoye et al.,
2005a). Other constituent of Garcina kola seed include, 1-3, 8-11
benzophenones (Olatunde et al., 2004). Traditionally G. kola
seed is believed to destroy sperm cell and make male infertile and this
has been proven scientifically. The G. kola seed extract has been
found to raise peripheral testosterone levels in plasma and reduce sperm
count without damage to the germinal epithelium; thus the proposed used
as a male contraceptives (Akpantah et al., 2003). Ovulation an
important aspect of fertility is an inflammatory process (Epsey, 1994;
Akpantah et al., 2003). According to Braide (1993) the presence
of flavonoids in G. kola seed confirms its anti-inflammatory property.
Thus, this study was carried out to discover the effect of G. kola
seed on female reproduction and its possible use as female contraceptive.
MATERIALS AND METHODS
Plant material: The Garcinia kola seed was purchased from
the local market in Lagos, Nigeria in 2006 and authenticated by The Botany
Department, University of Lagos.
The outer coats were removed and the seed was cut into pieces and air-dried.
The dried seeds were grounded into fine powder and extraction was done
using 70% alcohol in a soxhlet extract. The yield is then concentrated
into a solid form by evaporation using a rotary evaporator at 40 °C.
The resulting residue was further air-dried. Two gram of the extract was
dissolved in 100 mL of distilled water to give 20 mg mL-1 and
stored under -4 °C until required for use.
Animal grouping: Female pubertal adult rat and 4 pubertal male
were collected from the animal house of the College of Medicine, University
of Lagos. They were acclimatized for 2 weeks in the rat room of Physiology
department under standard condition of temperature and illumination.
The rats were fed with the rat pallets and had access to drinking water
ad libitum. The average weights of the animals used were 145-170
g. The female rats that have undergone two successive four days cycle
were divided into three experimental groups.
Estrous and ovulation study: Five animals received 200 mg kg-1
b.wt. of GKSE once daily for three weeks. Another 5 animals receive equal
volume of distilled water daily for three weeks and serve as the control.
The pattern of the estrous cycle was studied in all animals by taking
the vaginal smear. At the end of three weeks, each animal was sacrificed
on the morning of estrous by cervical dislocation. The fimbriated part
of the oviduct was dissected out, suspended in normal saline and placed
on a microscopic slide to count the number of ova in the oviduct.
Implantation and pregnancy study: Thirty animals in this group
were mated on the afternoon of proestrous and the male remain in the cage
until the morning of estrous. The presence of spermatozoa (determined
by microscopic examination of the vaginal smear) the next morning indicated
conception and day one of pregnancy (Oderinde et al., 2002). The
pregnant rats were divided into two groups of 18 rats each. Rats in group
1 receive 200 mg kg-1 b.wt. of GKSE orally from day four of
pregnancy while rats in group 2 were given equal volume of distilled water
starting from day four of pregnancy serving as the control. Localized
changes in endometrial vascular permeability, indicative of implantation
were assessed on days six and eight of pregnancy. Six rats each from each
group were randomly selected and given 0.3 mL of 0.5% Evans blue dye via
the tail vein. They were killed 15 min later and the uteri were separated
from fat and connective tissues and opened to ascertain the implantation
sites as described by Bolarinwa and Olaleye (1997). Where present, the
dye sites were carefully dissected out with a scalpel blade and weighed
to the nearest 0.01 mg on a mettler balance. The corned out sites were
counted and the number of uterine dye sites per rat recorded. The remaining
12 rats were sacrificed on day 19 of pregnancy, foetuses were removed
from the pregnant rats by ventral laboratory and fetal weight, number
and the resorption sites were determined.
Histological study: Twenty four rats were divided into two groups.
The 12 rats in group 1 received 200 mg kg-1 b.wt. of GKSE for
three weeks. The remaining 12 rats serve as control and received equal
volume of distilled water. At the end of 3 weeks, six rats from group
three were sacrificed by cervical dislocation. The remaining 12 rats from
both groups were allowed another 3 weeks for recovery before they were
sacrificed. The fallopian tube, ovary and uterus were removed from each
animal, weighed and processed for histological study. The weights of all
the animals were monitored and recorded through-out the 3 weeks of study.
Histological processing: The organs were cut in slabs of about
0.5 cm thick transversely and fixed in Bouin`s fluid for a day after which
it was transferred to 70% alcohol for dehydration. After 6 h it was transferred
to 90% alcohol and left overnight. From 90% to 3 changes of absolute alcohol
for 1 h each, then into chloroform for about 10 h and later transferred
into fresh chloroform for about 30 min. The tissues were placed in two
changes of molten paraffin wax for 20 min each in an oven at 57 °C.
They were placed vertically in molten paraffin wax inside a metal mould
and left overnight to cool and solidify. They were later trimmed and mounted
on wooden blocks. Serial sections were cut using rotary microtome at 5
microns. Sections were floated on a water bath to spread out and later
picked into albumenised slides and dried on a hot plate at 52 °C.
Slides were put in staining rack and placed in staining wells containing
xylene to dewax; absolute alcohol (2 changes); 70% alcohol and then to
water for 5 min after which they were stained with Haematoxylin (Hx) for
3 min. Excess Hx was washed off with water and differentiated with 1%
acid alcohol. Sections were rinsed in running tap water and left for 5
min for blueing. Sections were stained with 1% eosin and washed off with
water. They were dehydrated with 70 and 90% absolute alcohol and cleared
in xylene to remove all traces of water. Mountant (a drop) was placed
on the surface of slide and covered with a 22 by 22 mm cover slip.
Statistical analysis: Values were expressed as Mean ±
SEM. The statistical analysis was carried out using students`t-test. The
level of significance for all experiment was p≤0.05. Simple percentages
were used to present the alteration in the different phases of estrous
All the dams survived and no death was recorded through-out the study.
GKSE had no significant effect on the weight of the animals studied (Table
Estrous and ovulation studies: The estrous pattern was also significantly
altered following administration of GKSE. The occurrence of estrous reduced
drastically from 20% in control to 7.5% in GKSE. Metestrous also reduces
from 44.8% in control to 31.3% in GKSE and Proestrous and diestrous increases
from 20.9 and 14.3% in control to 26.5 and 34.7% in GKSE (Table
The number of ova in the oviduct was significantly reduced (p<0.05)
in the GKSE treated rats when compared with the control (10.80 ±
2.6 for the control and 3.2 ± 0.8 for GKSE). Thus GKSE produces
70.4% blockage in ovulation.
Implantation and pregnancy studies: There is no significant difference
between the number of the implantation site and the weight of the implantation
sites in both pregnant control rats and the GKSE treated rats (Table
The observations on foetal condition on day 19 of pregnancy are shown
in Table 4. Observation of the uterus of the GKSE treated
and control rats showed no significant difference in both the foetal number
Histological studies: The histological section through the fallopian
tube of the GKSE treated rats showed acute inflammation of the tubes which
was absent in the control groups of rats (Fig. 1a,
b). The various follicular stages are visible in the sections through
the ovary of the control and recovery rats (Fig. 2a, c)
with no developing follicles but corporal albican seen in the G. kola
treated rats (Fig. 2b). The uterus of the G.
kola treated rats showed an inactive endometrial gland (Fig. 3b). The uterus showed
full recovery after administration of GKSE was stopped (Fig.
1b, 2c, 3b).
||Effect of normal saline (0.9%) and Garcinia kola
seed extract (200 mg kg-1 b.wt.) on weekly body weight
|NS: Means not significant (p>0.05), *Growth rate
per week = Weight by week 3-Weight by week 1
||Effect of administration of 200 mg kg-1 Garcinia
kola seed extract on the phases of estrous cycle
||Effect of normal saline (0.9%) and Garcinia kola
seed extract (200 mg kg-1 b.wt.) on implantation in
rat (days 6 and 8 of pregnancy)
|Photomicrographs of the ovary from
the GKSE treated (a) and recovery (b) rats
Sections showing histology of ovary with no developing follicles,
old degenerate luteum
Sections showing histology of the ovary at various follicular stages,
Grafian follicles and numerous Corpus luteum and congestion
of the stroma
|Photomicrographs of the fallopian
tube from the control (a), G. kola treated (b) and recovery
Section showing normal histology of the fallopian tube except for
edema and congestion in the control rats
Sections showing acute inflammation of the fallopian tube in GKSE
Section showing histology of fallopian tube in the recovery rats with
unremarkable feature except for oedema and congestion
|Photomicrographs of the uterus from
the G. kola treated (a) and recovery (b) rats
Sections through the uterus of the GKSE treated rats showing inactive
Sections showing histology of the uterus, endometrium at secretory
phase in the recovery rats
||Effect of normal saline (0.9%) and Garcinia kola
seed extract (200 mg kg-1 b.wt.) on pregnancy at day
19 of gestation
GKSE has no significant effect on the weight of growth rate of the rats
similar result was reported by Akpantah et al. (2003). But this
is contrary to the report of Braide and Grill (1990) who reported retarded
growth in a group of rats fed with 10% w/w dry powder seed of Garcinia
kola for six weeks. The difference in observed weight may be due to
the mode of administration (in their study, G. kola seed powder
was mixed with the rat`s feed) or the presence of tannin which as explained
by them, causes malabsorption and thus nutritional deficit. But in this
study the process of extraction would have reduce the effectiveness of
tannins in the extract used.
Garcinia kola seed have been reported to have anti-inflammatory
(Madubunyi, 1995). In the past, anti-inflammatory drugs have been employed
in blocking ovulation (Gaytan et al., 2002), thus this explains
the observed reduction in number of egg released and the occurrence of
the estrous phase in the G. kola treated rats.
Phytochemical analysis has shown that Garcinia kola contains flavonoids
and some cardiac glycosidesespecially cardenolides (Cotterih et al.,
1978). Studies have shown that flavonoids such as apigenin are a major
component of Garcinia kola seed (Iwu and Igboko, 1982). Flavonoids will
inhibit cyclo-oxygenase enzymes (Liang et al., 1999). It has been
revealed that all traditional non-steroid anti-inflammatory drugs produce
most of their effects by blocking COX-2 (Stand, 2000). Mice with COX-2
deficiency suffer defective reproductive functions as COX-2 plays an important
role in follicular rupture (Lim et al., 1997). Thus GKSE may be
blocking ovulation as reported in this study by suppressing the activity
or formation of COX-2 thereby preventing follicular rupture.
Estrous phase is the period of vagina cornification and the drastic reduction
its occurrence following GKSE treatment may be due to the presence of
apigenin an effective inhibitor of the enzyme aromatase. Aromatase is
important in estrogen synthesis i.e., in the conversion of androgen to
estrogens (Jeong et al., 1999).
GKSE significantly reduces the occurrence of estrous while producing
70% blockage of ovulation without damage to the reproductive tissues (ovary,
uterus and fallopian tubes). Thus it may be use as a contraceptive having
the advantage of reversibility.
Adaramoye, O.A., E.O. Farombi, E.O. Adeyemi and G.O. Emerole, 2005.
Comparative study on the antioxidant properties of flavonoids of Garcinia Kola
seeds. Pak. J. Med. Sci., 21: 331-339.Direct Link |
Adaramoye, O.A., V.O. Nwaneri, K.C. Anyanwu, E.O. Farombi and G.O. Emerole, 2005.
Possible anti-atherogenic effect of kolaviron (a Garcinia kola
seed extract) in hypercholesterolaemic rats. Clin. Exp. Pharm. Physiol., 32: 40-46.CrossRef | PubMed |
Adaramoye, O.A. and E.O. Adeyemi, 2006.
Hypoglycaemic and hypolipidaemic effects of fractions from kolaviron, a biflavonoid complex from Garcinia kola
in streptozotocin-induced diabetes mellitus rats. J. Pharm. Pharmacol., 58: 121-128.PubMed |
Adaramoye, O.A. and E.O. Adeyemi, 2006.
Hepatoprotection of d-galactosamine-induced toxicity in mice by purified fractions from Garcinia kola
seeds. Basic Clin. Pharmacol. Toxicol., 98: 135-141.CrossRef | PubMed |
Adefile-Ositelu, A.O., A.K. Adefule, B.O. Oosa and P.C. Onyenefa, 2004.
Antifungal activity of Garcinia kola
nut extract as an ocular bacterial isolates in Lagos. Nig. Q. J. Hosp. Med., 14: 112-114.
Akpantah, A.O., A.A. Oremosu, M.O. Ajala, C.C. Noronha and A.O. Okanlawon, 2003.
The effect of crude extract of Garcinia kola
seed on the histology and hormonal milieu of male Sprague-Dawley rats' reproductive organs. Niger. J. Health Biomed. Sci., 2: 40-46.CrossRef | Direct Link |
Bolarinwa, A.F. and S.B. Olaleye, 1997.
Blastocyst implantation: Effect of thyroidectomy and thyroxine replacement in the rat. Afr. J. Med. Med. Sci., 26: 135-137.Direct Link |
Braide, V.P. and V. Grill, 1990.
Histological alterations by a diet containing seeds of Garcinia kola
. Effect on liver, kidney and intestine in the rat. Gegesbarug Morphol. Jahrb., 136: 95-101.PubMed | Direct Link |
Braide, V.P., 1993.
Anti-inflammatory effect of Kolaviron, bio-flavonoid extract of Garcinia kola
. Fitoterapia, 64: 433-436.
Cotterih, P.J., F. Scheinmenn and I.A. Stenhuise, 1978.
Extractives from guttiferae. Part 34. Kolaflavanone, a new biflavanone from the nuts of Garcinia kola
Heckel. Applications of 13
C nuclear magnetic resonance in elucidation of the structures of flavonoids. J. Chem. Soc. Perkin. Trans., 1: 532-539.
Epsey, L.L., 1994.
Current status of the hypothesis that mammalian ovulation is comparable to an inflammatory reaction. Biol. Reprod., 50: 233-238.CrossRef | Direct Link |
Farombi, E.O., O.O. Akanni and G.O. Emerole, 2002.
Antioxidant and scavenging activities of flavonoid extract (kolaviron) of Garcinia kola
seeds. Pharm. Biol., 40: 107-116.CrossRef | Direct Link |
Gaytan, F., E. Tarradas, C. Morales, C. Bellido and J.E. Sanchez-Criado, 2002.
Morphological evidence for uncontrolled proteolytic activity during the ovulatory process in indomethacin-treated rats. Reproduction, 123: 639-649.CrossRef | Direct Link |
Iwu, M. and O. Igboko, 1982.
Flavonoids of Garcinia kola
seeds. J. Nat. Prod., 45: 650-651.CrossRef | Direct Link |
Jeong, H.J., Y.G. Shin, I.H. Kim and J.M. Pezzuto, 1999.
Inhibition of aromatase activity by favonoids. Arch. Pharm. Res., 22: 309-312.PubMed | Direct Link |
Liang, Y.C., Y.T. Huang, S.H. Tsai, S.Y. Lin-Shiau, C.F. Chen and J.K. Lin, 1999.
Suppression of inducible cyclooxygenase and inducible nitric oxide synthase by apigenin and related flavonoids in mouse macrophages. Carcinogenesis, 20: 1945-1952.CrossRef | Direct Link |
Lim, H., B. Paria, S. Das, J. Dinchuk, R. Langenbach, J. Trzaskos and S. Dey, 1997.
Multiple female reproductive failures in cyclooxygenase-2 deficient mice. Cell, 17: 197-208.PubMed |
Madubunyi, I.I., 1995.
Antimicrobial activities of the constituents of Garcinia kola
seeds. Int. J. Pharm., 33: 232-237.CrossRef | Direct Link |
Oderinde, O., C. Noronha, A. Oremosu, T. Kusemiju and A. Okanlawon, 2002.
Abortifacient properties of aqueos extract of Carica papaya
(Linn) seed on female Sprague-Dawley rat. Nig. Postgrad. Med. J., 9: 95-98.PubMed | Direct Link |
Okunji, C.O. and M.M. Iwu, 1991.
Molluscidal activity of Garcinia kola
biflavanones. Fitoterapia, 62: 74-76.
Olaleye, S.B. and E.O. Farombi, 2006.
Attenuation of indomethacin-and HCl/ethanol-induced oxidative gastric mucosa damage in rats by Kolaviron, a natural biflavonoid of Garcinia kola
seed. Phytother. Res., 20: 14-20.PubMed | Direct Link |
Farombi, O.E., M. Hansen, P. Ravn-Haren and L.O. Dragsted, 2004.
Commonly consumed and naturally occurring dietary substances affect biomarkers of oxidative stress and DNA damage in healthy rats. Food Chem. Toxicol., 42: 1315-1322.CrossRef | PubMed | Direct Link |
Staud, R., 2000.
Comparing COX-2 inhibitors with traditional NSAIDs. Emer. Med., 23: 1-6.
Tita, R.K., P.G. Odeigah, P.U. Agomo and E. Bassey, 2001.
Some Properties of Medicinal Plants used by the Igbos of Nigeria. In: Triats, Tracts and Traces, Wolfgang Kreis (Ed.). Bioline International, Germany, pp: 209-210
Uko, O.J., A. Usman and A.M. Ataja, 2001.
Some biological activities of Garcinia kola
in growing rats. Veterinarski Arhiv., 71: 287-297.Direct Link |