Cytotoxicity Activity and Reproductive Profiles of Male Rats Treated with Methanolic extracts of Ficus deltoidea
S.A. Sharipah Ruzaina,
M.S. Nurul Hidayah
Ficus deltoidea is a shrub belongs to Moraceae family. The Malaysian traditional medicinal practitioners use the F. deltoidea leaves to treat poor circulation after giving birth and to reduce blood glucose level and blood pressure. In this study, methanolic extract of F. deltoidea fruit and leaf were tested on human leukemic HL 60 cell line for their cytotoxicity activity and its effects on reproductive system of male rats. The methanol extracts was employed to assess its effects on body weight, testes, epididymis, sperm count, sperm viability and sperm morphology. Both methanolic leaf and fruit extracts showed significant cytotoxic activity against HL-60 cell line with IC50 value of 11.4 and 13.6 μg mL-1, respectively. The administration of the F. deltoidea leaf extract did not significantly alter the body weight of male rats. Testes and epididymis weight, sperm count, sperm viability and the number of normal sperm morphology were significantly decreased when treated with leaf extract. Meanwhile, rats treated with extract from stem showed significant increase in epididymis weight, sperm count and sperm viability. However, there is a reduction in testes weight and the number of normal sperm morphology of male rats. Cytotoxic activity against HL 60 cell line of the leaf methanolic extract was more potent than fruit methanolic extract. There was a significant effect on testes and epididymis weight, sperm count, sperm viability and the number of normal sperm morphology. However, the mechanism of action of F. deltoidea extracts still remains unclear and an intensive research should be needed in the future.
to cite this article:
J.S. Norrizah, A. Norizan, S.A. Sharipah Ruzaina, D. Dzulsuhaimi and M.S. Nurul Hidayah, 2012. Cytotoxicity Activity and Reproductive Profiles of Male Rats Treated with Methanolic extracts of Ficus deltoidea. Research Journal of Medicinal Plants, 6: 197-202.
Received: June 24, 2011;
Accepted: September 16, 2011;
Published: November 02, 2011
In Malaysia, F. deltoidea family Moraceae is commonly known as mas cotek,
serapat angina, telinga beruk, ara terbang and kangkalibang. The aerial parts
of F. deltoidea are highly commercialized. The traditional medicinal
practitioners used the decoction of the leaves to treat various diseases. Various
parts of the plant can be used to treat poor blood circulation after giving
birth (Zaki, 2005). Medicinally, the fruit and leaf can
be used to recover arthritis and to reduce blood glucose and blood pressure
(Aris et al., 2009). There were few studies have
been done on the identification of phytochemicals and biological activities
of this plant. It was reported that petroleum ether, chloroform, methanol and
water extracts of F. deltoidea leaves contain saponin, amino acid, flavonoids
and terpenoids (Shafaei et al., 2011). It was
also found that extracts of the plant showed more than 90% inhibition when tested
with Ferric Thiocyanate Method (FTC) and thiobarbituric acid method (TBA) and
good radical scavenging activity with DPPH method. Previous finding has reported
the blood glucose lowering effect of F. deltoidea (Aminudin
et al., 2007; Adam et al., 2007).
Another study on the antidiabetic mechanism of F. deltoidea has revealed
that methanolic extract of this plant stimulated insulin secretion from BRIN
BD11 cell lines by 1-enhance both basal and insulin-stimulated glucose 3 folds
as compared to 1.1 mM glucose alone (Hamid et al.,
2008). According to Adam et al. (2009) F.
deltoidea extracts have the ability to enhance the basal and insulin-stimulated
glucose uptake into liver cell and possess either insulin-mimetic activity or
insulin-sensitizing activity or combination of both activities. It was also
found that extracts from F. deltoidea possess α-glucosidase inhibitory
activity in vitro (Adam et al., 2010).
Post-treatment of the aqueous extracts of the leaf and fruits of F. deltoidea
(50 mg kg-1) reduced significantly the external glucose load. The
leaf and fruit aqueous extracts showed non-toxic effect in the brine shrimp
Until now, cytotoxicity study on F. deltoidea plant extracts was very
limited. The cytotoxicity of compounds and crude plant extracts can be tested
using various bioassay such as brine shrimp lethality bioassay (Uddin
et al., 2003; Ahmad et al., 2003),
XTT assay (Aisha et al., 2011) and MTT assay
(Endrini et al., 2007). The present study has
been done to evaluate the cytotoxicity effect of F. deltoidea extract
on human leukemic HL 60 cell lines. In addition, the methanol extract of F.
deltoidea leaf and stem was employed to assess its effects on male rat reproductive
MATERIALS AND METHODS
Plant materials: Leaf, fruit and stem of F. deltoidea were collected in 2010 from forest near Lata Kinjang, Tapah Perak, West Malaysia. The herbarium voucher specimen was identified by Shamsul Khamis, Institute of Bioscience, Universiti Putra Malaysia.
Preparation of methanolic extract: The leaf, stem and fruit were air dried and then ground to powdery form. The ground leaf, stem and fruit of F. deltoidea were extracted with methanol. The extracts were evaporated to dryness under reduced pressure. The process was repeated three times to give dark brown crude methanolic extracts.
Culture of cells: The human leukemic HL-60 cell lines were obtained
from Institute of Bioscience, Universiti Putra Malaysia. Cells (from monolayer
subcultures) were trypsinized and resuspended in complete growth medium (CGM)
(10 mL). Cells were maintained in 25 cm2 flasks (Nunc, Denmark) with
10 mL of CGM in a CO2 incubator at 37°C until confluence was
achieved. Medium supplemented with 10% (w/v) FBS (Fetal bovine serum) and 1%
penicillin/streptomycin mixture was aspirated from MCF-7 cells grown to about
90% confluence. Cells were washed with PBS (Phosphate buffer solution), trypsinized,
counted with a haemocytometer and diluted with RPMI-1640 to 5H105
MTT assay: Cytotoxicity or antiproliferative activity was determined
using MTT assay (Mosmann, 1983).
Animals: Sprague-Dawley male rats (180-250 g) were obtained from the
Animals House, Institute for Medical Research (IMR). The animals were kept in
wire mesh cages, acclimated to laboratory conditions (12 D: 12 L; 25+1°C)
and had access to food and water ad libitum. Each animal was certified
fertile by isolated mating technique before inclusion in the study.
Experimental design: A total of 12 male rats divided into three equal
groups and treated as follow: group 1 (n = 12) were administered with 0.1 mL
distilled water and served as control meanwhile group 2 and 3 administered with
50 mg kg-1 b.w day-1 of the F. deltoidea leaf and
stem methanol extracts, respectively for three weeks. During the experiment,
body weight was recorded every week. At the end of the treatment period, rats
were sacrificed by cervical dislocation. The testis and epididymis were excised
from the animals, cleared of adherent tissues and weight.
Sperm analysis: The sperm analysis consisted in the determination of
sperm concentration, progressive motility and morphology. The sperm count was
performed as previously described Raji et al. (2006).
Statistical analysis: All data were expressed as Mean±SEM. Differences between control and treated animals were analyzed by Students t-test using (SPSS) for Windows. The p values of less than 0.05 were considered statistically significant.
RESULTS AND DISCUSSION
In cytotoxicity study, MTT assay was used to analyze the cytotoxic effect of
methanolic F. deltoidea extract on human leukemic HL-60 cell lines. Yellow
MTT enters the cells and it will be reduced to a colored, insoluble formazon
in the mitochondria of the living cells. The cells were then solubilised with
an organic solvent DMSO (Mosmann, 1983). The results
of cytotoxic activity of leaf and fruits of F. deltoidea methanolic extracts
against HL-60 cell line are summarized in Table 1. The IC50
value of the positive control, goniothalamine against HL-60 cell was 2.4 20
μg mL-1. Fruit extract produced a dose-dependent decline in
cell viability with IC50 (inhibitor concentration required for 50%
inhibition of cell viability) of 13.6 μg mL-1 as determined
by MTT assay after 4 h exposure. The leaf extract moderately inhibited the growth
of human leukemic HL-60 cell lines with an IC50 value of 11.4 μg
mL-1. The leaf extract showed better inhibition compared to the fruit
extract. The crude extract of a plant is considered cytotoxic against the treated
cells if IC50 value obtained is less than 20 μg mL-1
(Geran et al., 1972). Thus, the F. deltoidea
fruit and leaf extracts were shown to possess the bioactive compounds that need
to be studied further. It was found that microscopic evaluation of the dead
HL-60 treated with F. deltoidea fruit and leaf extracts exhibited cellular
DNA fragmentation. This suggested that the mechanism of action responsible for
the death of HL-60 cell lines may be caused by apoptosis. However, further evaluation
on apoptotic cell death is necessary which can be done by TUNEL method (terminal
deoxynucleotidyl transferase (TdT)-biotin nick end-labeling) (Khorshid
et al., 2011; Ali et al., 2011).
||In vitro antiproliferative activity (IC50
μg mL-1) of leaf and fruits of Ficus deltoidea methanolic
extracts against HL-60 cell line
||Effect of Ficus deltoidea on male rat body weight after
|Values are presented as Mean±SEM, n = 12
||Effects of Ficus deltoidea on sperm count, viability
and normal morphology
|Values are presented as Mean±SEM, n = 12
||Weight of testes and epididymis of male rats treated with
leaf and stem extract of Ficus deltoidea
The body weight did not change significantly during 3 weeks of treatment (Table
2). All control rats recorded about 5% weight gain within the period of
the experiment. The body weight of the male rats was reduced to 14.7 and 10.08
g when treated with methanolic leaf and stem extract of F. deltoidea,
There was a significant (p<0.05) decrease in the weight of testes and epididymis in rats treated with methanolic leaf extract of F. deltoidea compared to control (Fig. 1). A significant increase (p<0.05) in epididymis weight was however, recorded in the rats treated with methanolic stem extract of F. deltoidea. Rats from the same group showed insignificant (p<0.05) reduction in testes weight.
A significant (p<0.05) reduction in sperm count was recorded in rats treated with methanolic leaf extract of F. deltoidea. However, a significant (p<0.05) increase in sperm count was recorded in rats treated with methanolic stem extract of F. deltoidea when compared with the control group (Table 3). Percentage live spermatozoa also decreased significantly (p<0.05) in methanolic leaf treated group but significantly increased (p<0.05) in methanolic stem treated group compared to control. The results of sperm morphology showed significant (p<0.05) reduction of normal morphology in methanolic leaf treated group. Similarly, there was a reduction in normal morphology for rats treated with methanolic leaf extract of F. deltoidea but the reduction was not significant (p>0.05).
The decrease in sperm count, sperm viability and normal sperm morphology in
rats treated with methanolic leaf extract of F. deltoidea indicates interference
with testicular spermatogenesis. The decreased in testes and epididymis weight,
the presence of detached head and the impaired sperm motility in rats treated
with F. deltoidea leaf extract suggest that the reproductive organ also
maybe the target for F. deltoidea toxicity. In other research, Quassia
amaras toxicity on male reproductive system leads to the presence of fragile
tails and detached heads, the impaired sperm motility and the decrease in tissue
α-glucosidase (Parveen et al., 2003).
The increase in sperm count indicates that F. deltoidea stem extract
promote the process of spermatogenesis in testes. An increase number of sperm
count may be associated with the increase in serum levels of testosterone. Testosterone
was necessary for the development, growth and normal functioning of the testes
and male accessory reproductive glands (Prins et al.,
1991). The increase in weight of epididymis may also indicate the increase
number of sperm produced by testes as reported earlier by Espinoza-Navarro
and Bustos-Obregon (2005). The number of sperm viability also increased
up to 14.13%, it may be associated with the improvement of epididymal function
of maturation (Sadik et al., 2001).
The results of this study demonstrate that the methanolic leaf extract of F. deltoidea decreased the sperm quality. Thus, it can be suggested that there is the possible exploitation of F. deltoidea extract as an antifertility agent. Interestingly, in contrast, F. deltoidea stem extract improved sperm quality.
In conclusion, the present study demonstrated the cytotoxic activity against human leukemic, HL-60 cell line of the leaf methanolic extract to be more potent than that of the fruit methanolic extract of F. deltoidea. Further study will be conducted to identify the principle and the mechanism by which the F. deltoidea extracts affects reproductive function in rats.
The authors would like thank the Universiti Teknologi MARA, Shah Alam, Malaysia for financing this research project.
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