Available via license: CC BY-NC 3.0
Content may be subject to copyright.
Genotoxicity and acute and subchronic toxicity
studies of a standardized m ethanolic extract of
Ficus deltoidea leaves
Elham Farsi,
I
Armaghan Shafaei,
II
Sook Yee Hor,
I
Mohamed B. Khadeer Ahamed,
I
Mun Fei Yam,
I
Mohd Z.
Asmawi,
I
Zhari Ismail
II
I
Universiti Sains Malaysia, School of Pharmaceutical Sciences, Department of Pharmacology, Pulau Penang/Malaysia.
II
Universiti Sains Malaysia, School of
Pharmaceutical Sciences, Department of Pharmaceutical Chemistry, Pulau Penang, Malaysia.
OBJECTIVE: Ficus deltoidea leaves have been used in traditional medicine in Southeast Asia to treat diabetes,
inflammation, diarrhea, and infections. The present study was conducted to assess the genotoxicity and acute
and subchronic toxicity of a standardized methanol extract of F. deltoidea leaves.
METHODS: Sprague Dawley rats were orally treated with five different single doses of the extract and screened
for signs of toxicity for two weeks after administration. In the subchronic study, three different doses of the
extract were administered for 28 days. Mortality, clinical signs, body weight changes, hematological and
biochemical parameters, gross findings, organ weights, and histological parameters were monitored during the
study. Genotoxicity was assessed using the Ames test with the TA98 and TA100 Salmonella typhimurium strains.
Phytochemical standardization was performed using a colorimeter and high-performance liquid chromato-
graphy. Heavy metal detection was performed using an atomic absorption spectrometer.
RESULTS: The acute toxicity study showed that the LD
50
of the extract was greater than 5000 mg/kg. In the
subchronic toxicity study, there were no significant adverse effects on food consumption, body weight, organ
weights, mortality, clinical chemistry, hematology, gross pathology, or histopathology. However, a dose-
dependent increase in the serum urea level was observed. The Ames test revealed that the extract did not have
any potential to induce gene mutations in S. typhimurium, either in the presence or absence of S9 activation.
Phytochemical analysis of the extract revealed high contents of phenolics, flavonoids, and tannins. High-
performance liquid chromatography analysis revealed high levels of vitexin and isovitexin in the extract, and
the levels of heavy metals were below the toxic levels.
CONCLUSION: The no-observed adverse effect level of F. deltoidea in rats was determined to be 2500 mg/kg.
KEYWORDS: Ficus deltoidea; Oral Toxicity; OECD; Genotoxicity; Isovitexin; Vitexin.
Farsi E, Shafaei A, Hor SY, Ahamed MB, Yam MF, Asmawi MZ, et al. Genotoxicity and acute and subchronic toxicity studies of a standardized
methanolic extract of Ficus deltoidea leaves. Clinics. 2013;68(6):865-875.
Received for publication on January 6, 2013; First review completed on January 26, 2013; Accepted for publication on March 6, 2013
E-mail: elhamfarsi@gmail.com / khadeer.nc@gmail.com
Tel.: 604-653 4962/2146 / 6014-241 5410
& INTRODUCTION
A number of studies have highlighted tremendous
medical concerns through the systematic investigation of
herbal remedies and their adverse effects on the vital organs
of animals and humans (1,2). Anti-vitamins, anti-nutritional
factors, immunomodulators, and heavy metals are among
the potential toxic substances (3,4). Because of the absence of
strict quality control and the complex mixture of the
chemicals present in herbal medicines, there is limited
knowledge available about the chemical compositions of
these medicines and their effects on human physiology. This
lack of data necessitates the thorough evaluation of the
safety of medicinal herbs.
Ficus deltoidea (Moraceae), an epiphytic shrub, is widely
distributed in Southeast Asian countries. In Malaysia, F.
deltoidea is locally known as Mas cotek (5). Traditionally, this
plant has been used in to treat inflammation and relieve
pain. It is used to treat several diseases, including gout, high
blood pressure, pneumonia, diarrhea, and skin infections
(6). In addition, F. deltoidea has been used as an aphrodisiac,
particularly to increase male fertility (7). Decoctions of the
leaves of F. deltoidea have been extensively utilized in folk
medicine to decrease the symptoms of diabetes mellitus,
hyperlipidemia, and hypertension, and herbal healers often
recommend the leaves of both male and female plants as
Copyright ß 2013 CLINICS – This is an Open Access article distributed under
the terms of the Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/3.0/ ) which permits unrestricted non-
commercial use, distribution, and reproduction in any medium, provided the
original work is properly cited.
No potential conflict of interest was reported.
DOI: 10.6061/clinics/2013(06)23
BASIC RESEARCH
865
libido boosters and postpartum treatments to strengthen the
uterus (8). Studies have shown that F. deltoidea leaves
possess antinociceptive, wound-healing, and anti-oxidant
properties (6,9,10). The beneficial effects of F. deltoidea on
hypertension, inflammation, and ulcers, its ability to inhibit
carbohydrate-hydrolyzing enzymes, and its wound-healing,
hepatoprotective, and antinociceptive activities have been
verified (10-13). Despite the prevalent use of this plant as a
food and medicine, the toxicity of F. deltoidea has not been
fully explored. An aqueous extract of F. deltoidea leaves
administered orally at 100 and 300 mg/kg/body weight has
been shown not to cause any hematological or biochemical
changes in rats (14). Although herbal medicines/dietary
supplements are not covered under US-FDA drug-regula-
tory criteria because these products are considered safe,
their safety profiles may not have been adequately
documented. Hence, preclinical acute and subchronic
toxicological evaluations using the Organisation for
Economic Cooperation and Development (OECD) guide-
lines need to be undertaken to establish the safety profiles of
drugs of herbal origin (15).
Few scientific data are available to validate the claims of
folklore regarding the use of F. deltoidea as a remedy to treat
various human ailments or to confirm the safety profile of
repeated exposure to the extract of F. deltoidea leaves. To the
best of our knowledge, there have been no genotoxicological
studies to assess the safety of F. deltoidea. Thus, the present
study was designed to evaluate the safety profile of a
standardized methanol extract of F. deltoidea leaves (MEFL).
Acute and 28-day subchronic oral toxicity tests were
conducted in Sprague Dawley (SD) rats according to the
OECD guidelines, and for the first time, the genotoxicity of
MEFL was investigated using Salmonella typhimurium
strains. In addition, qualitative and quantitative phyto-
chemical analyses were performed colorimetrically. The
quantitation of vitexin and isovitexin in MEFL was
performed using HPLC. The detection of heavy metals in
MEFL was conducted using atomic absorption spectro-
metry.
& MATERIALS AND METHODS
Plant material and preparation of the extract. Leaves of
F. deltoidea were purchased from HERBagus Sdn. Bhd.,
Malaysia. Taxonomical authentication was performed by a
senior botanist, V. Shunmugam, and a voucher specimen
(Ref. No. 11204) was deposited at the herbarium of the
School of Biological Sciences, Universiti Sains Malaysia,
Penang. The leaves of the plant were dried in an oven
(37
˚
C) and powdered mechanically. The extract was
prepared with 100 g of powdered material and 1 L of
methanol using a Soxhlet extractor at 50
˚
C. The methanol
extract (yield, 12% w/w) was filtered and evaporated to
dryness under a vacuum. The residue was then lyophilized
using a freeze drier (Labconco Cooperation, Denmark). The
extract was stored at -80
˚
C until used.
High-performance liquid chromatography (HP LC)
Chemicals. HPLC-grade methanol and formic acid
(Merck Chemicals, Germany) were used for the HPLC
analysis. Two standards, vitexin and isovitexin
(ChromaDex, USA), were used for the HPLC analysis.
HPLC analysis. The HPLC analysis of MEFL to determine
the vitexin and isovitexin contents was performed according
to the methodology of Fu et al. (16). This analysis was
performed using an Agilent Technologies Series 1100
system equipped with a degasser, an autosampler, a
column heater, a quaternary pump, and a UV detector. A
reversed-phase Nucleosil C18 column (250 mm64.6 mm,
5 mm) was maintained at 25
˚
C, and a 10 ml volume of injected
sample was eluted using an isocratic mobile phase composed
of methanol:water:formic acid (33:66.37:0.67 v/v/v) at a flow
rate of 1 ml/min. The separation time was 30 min. The
detection wavelength was 330 nm. Standard calibration
curves were established by plotting the peak areas against
different concentrations. The reference standards for vitexin
and isovitexin were used to determine the retention times of
these compounds and to spiked with the samples. The
external standard method was used to quantify the bioactive
markers in the sample of the extract.
Preparation of samples and standard solutions for
HPLC analysis
A 100 mg portion of the methanol extract of F. deltoidea
was dissolved in 25 ml of methanol and sonicated for 10-
15 min. The contents were transferred to a 25 ml volumetric
flask, and the volume was brought up to 25 ml. All samples
were filtered through a 0.45 mm filter (Whatman). Similarly,
the reference compounds were weighed (approximately
5 mg), each dissolved in 5 ml of methanol, and then filtered
through a 0.45 mm filter (Whatman). The stock solution was
used to prepare further dilutions. The samples were kept in
a refrigerator at -20
˚
C prior to analysis.
Phytochemical screening and heavy metal analysis
The total contents of protein, polysaccharides, glycosapo-
nins, phenolics, flavonoids, and tannins in MEFL were
estimated colorimetrically (17,18). The total phenolic content
was determined using the Folin-Ciocalteu reagent with
gallic acid as a standard, and the results were expressed as
mg of gallic acid equivalents. The total flavonoid content
was determined using the AlCl
3
colorimetric method with
quercetin (QTN) as the standard, and the results were
expressed as mg of QTN equivalents. The amount of total
condensed tannins was expressed as (+)-catechin equiva-
lents (CT, mg (+) catechin/g sample). The levels of lead (Pb),
cadmium (Cd), arsenic (As), and mercury (Hg) in MEFL
were determined using an atomic absorption spectrometer
(Perkin Elmer, AAnalyst 800, Canada) according to the
standard method of the British Pharmacopoeia 2008 (19).
Analysis of antimutagenic effects
The antimutagenic effects of MEFL at different concentra-
tions (15.625 to 500 mg/well) were tested using the
Salmonella typhimurium strains TA98 and TA100 for frame-
shift and base-pair substitution mutagenesis, respectively,
with (indirect effect) and without (direct effect) metabolic
activation. S. typhimurium TA100, TA98, TA1535, and
TA1537 are the most commonly used strains for bacterial
mutation assays within the pharmaceutical industry (20). 2-
Nitrofluorene (2-NF) and 2-anthramine (2-AA, Chemtron,
Singapore) were used as the indirect-acting mutagens in the
metabolic activation system, and sodium azide phosphate
(Chemtron, Singapore) was used as a direct-acting mutagen
for TA98 or TA100. The broth (Oxoid, Malaysia) and
reagents were prepared according to the method of Maron
and Ames (21), and a preincubation mutagenicity test was
Safety evaluation of Ficus deltoidea
Farsi E et al.
CLINICS 2013;68(6):865-875
866
performed (22). Moltox rat liver LS-9 (S9 mix, Chemtron,
Singapore) was added in the indirect antimutagenic effect
test to activate the metabolism of the mutagen. Incubated
TA98 or TA100 cells (1610
8
cells in 0.1 ml), the extract
(100 ml), and the mutagen (10 ml) were mixed in a sterile test
tube with a cap (12675 mm). Sodium phosphate buffer
(0.5 ml, 0.1 M, pH 7.4) was added to the direct mutagen–
containing tubes, and 0.5 ml of 10% S9 mix was added to the
indirect mutagen–containing tubes. After preincubation at
37
˚
C in a shaking water bath for 30 min, 2 ml of top agar
containing 10% histidine/biotin solution was added and
then spread on a minimal glucose agar plate. After the
plates had been incubated at 37
˚
C for 48 h, the His+
revertant colonies were counted, and the percent inhibition
induced by the extract treatment was calculated.
Experimental animals
SD rats of either sex (8 weeks of age) were obtained from
the animal house of the School of Pharmaceutical Sciences,
Universiti Sains Malaysia. The animals were housed under
standard environmental conditions (temperature, 25
˚
C;
humidity, 51%¡10%) with a 12-h light–dark cycle and
were provided a standard pellet diet (Gold Coin Holdings
Sdn Bhd) and water ad libitum. The study was approved by
the Animal Ethics Committee of Universiti Sains Malaysia,
Penang, Malaysia [Protocol No: USM/Animal Ethics
Approval/044/(58)].
Acute toxicity study in rats
Healthy adult female SD rats (200-225 g) were used in the
acute toxicity study. The study was conducted according to
the OECD guidelines for chemicals using a fixed-dose
procedure (23). One group of rats was dosed by oral gavage
with a single limit dose of 5,000 mg/kg MEFL dissolved in
0.5% carboxymethyl cellulose (CMC), and 0.5% CMC alone
was administered to another group as a control. After this
single administration, the animals were observed for signs
of possible toxicity every hour for the first six hours and
then every day for 14 days. All animals were weighed daily
and monitored for any signs of toxicity and for mortality for
up to 14 days. Food and water consumption were recorded
daily. The rats were observed visually to identify the
following: changes in the skin, fur, eyes, and mucous
membranes; effects on the respiratory system, circulatory
system, autonomic nervous system, and central nervous
system; and changes in somatomotor activity and beha-
vioral patterns. The animals were euthanized on the last day
of experiment, and the LD
50
values were estimated.
Subchronic toxicity study in rats
A subchronic repeated dose (28 days) study in rats was
conducted according to the OECD testing guidelines (24).
SD rats of both sexes were randomly distributed to four
groups of six animals each. MEFL prepared in 0.5% CMC
was orally administered daily for 28 days in single doses of
750 mg/kg (group I), 1250 mg/kg (group II), or 2500 mg/
kg (group III). The control rats (group IV) received only
vehicle (0.5% CMC). The body weight was recorded on days
0, 7, 14, and 28. Along with food and water consumption,
signs of toxicity and mortality were also recorded daily
throughout the study period. At the end of the experiment,
all rats were anesthetized by carbon dioxide inhalation, and
blood samples were collected via cardiac puncture into non-
heparinized and EDTA-containing tubes for biochemical
and hematological analyses. After blood collection, the
animals were sacrificed by cervical dislocation, and their
organs were isolated to assess histopathological changes.
The liver, kidneys, adrenal glands, lungs, brain, spleen,
heart, testes, ovaries, uterus, thymus, and gut were excised,
weighed using an analytical lab balance (Mettler-Toledo
AX-204, Japan), and examined macroscopically. These
organs were then finally fixed in 10% buffered neutral
formalin for histopathological examination.
Hematological and biochemical analyses
The following hematological parameters were analyzed
using an automatic hematology analyzer (Sysmex-XT-1800
Germany): red blood cells (RBCs), white blood cells (WBCs),
neutrophils, lymphocytes, eosinophils, monocytes, baso-
phils, hemoglobin concentration (Hb), hematocrit (Ht),
mean corpuscular volume (MCV), mean corpuscular hemo-
globin (MCH), mean corpuscular hemoglobin concentration
(MCHC), and platelet count (Plt).
The following serum biochemical parameters were
measured using a biochemistry autoanalyzer (Olympus
640 Japan): alkaline phosphatase (ALP), aspartate amino-
transferase (AST), alanine aminotransferase (ALT), lactate
dehydrogenase, creatine phosphokinase, total protein, total
albumin, albumin/globulin ratio, phosphorus, calcium,
sodium, potassium, chloride, and total and conjugated
bilirubin.
Histopathological analysis
For the histopathological analysis, three randomly
selected rats in each experimental group were euthanized,
and the organs listed above were harvested and fixed in
10% buffered neutral formalin for 48 hours and then in
bovine solution for 6 hours. The fixed organs were
processed for paraffin embedding. Sections (5 mm thick)
were cut using a microtome, processed using an alcohol-
xylene series, and stained with hematoxylin and eosin (25).
Statistical analysis
The statistical analysis was performed using the Statistical
Package for the Social Sciences (SPSS 16.0 package). The
data are given as the mean¡S.E., and the analysis was
performed using one-way analysis of variance (ANOVA).
Significant differences between the control and treatment
groups were identified using Dunnett’s test. p- values of
,0.05 and 0.01 were considered significant.
& RESULTS
HPLC analysis of MEFL. The HPLC chromatogram of the
pure standards (Figure 1A) illustrated their R
f
values
and allowed the corresponding peaks in the MEFL
chromatogram to be identified (Figure 1B). Vitexin
accounted for 18.76%¡1.12% of the dry weight of MEFL,
and isovitexin accounted for 9.68%¡1.18%. The results of
this study are similar to those of previous studies suggesting
that the flavone C-glycosides vitexin, and isovitexin are the
major chemical constituents of MEFL along with other
flavonoids (5). The chemical structures of the biomarkers
used in this study are given in Figure 1. Good linearity and
retention times and method validation using five-point
calibration curves were obtained for all replicates. The
quantitative results for the bioactive markers (% dry weight)
are illustrated in Figure 1C. The concentrations in the
CLINICS 2013;68(6):865-875 Safety evaluation of Ficus deltoidea
Farsi E et al.
867
Safety evaluation of Ficus deltoidea
Farsi E et al.
CLINICS 2013;68(6):865-875
868
samples were estimated based on the calibration curves for
vitexin and isovitexin over the range of 5 to 200 mg/ml. The
quantitative percentages of the dry weights of the standards
were calculated using the formulas Y = 23.90X - 65.44
(R2 = 0.9992) and Y = 28.305X - 28.245 (R2 = 0.9982),
respectively, where Y is the peak area for the analyte and
X is the concentration of the analyte (mg/ml).
Phytochemical screen ing and heavy metal analysis
of MEFL
The results of the quantitative analysis of the total
contents of proteins, polysaccharides, glycosaponins, flavo-
noids, phenolics, and tannins present in MEFL are graphi-
cally depicted in Figure 1D. The results revealed that the
levels of heavy metals such as cadmium (detec-
ted = 0.07 ppm, specification # 0.1), mercury (not detected),
and arsenic (detected = 0.4 ppm, Specification #0.4) in
MEFL were below toxic levels (26). In contrast, lead had a
level slightly higher (0.76 ppm) than the permitted limit
(0.7 ppm).
Bacterial reverse mutation te st
The Ames test was used to analyze the anti-mutagenic
potential of MEFL. In this study, S. typhimurium strains
TA98 and TA100 were used to measure the induction of
frameshift and base-pair mutations, respectively. Mutagens
make bacteria histidine independent, and thus, the mutated
bacteria can form colonies on histidine-deficient medium.
The mutagens used were either direct acting (NaN
3
and 2-
nitrofluorene) or required microsomal activation (2-AA).
Adding antimutagenic agents considerably reduces the
reverse mutation effects of mutagens.
The antimutagenic effects of MEFL were tested in S.
typhimurium strains TA98 and TA100, both in the presence
and absence of the S9 mix. The cytotoxicity of MEFL in
S. typhimurium was preliminarily investigated in tests
performed with TA100 using the plate pre-incubation
method with or without the addition of the S9 mix. MEFL
did not cause any decrease in the number of histidine
+
revertant colonies compared with the negative control
values obtained for the tester stains. Because MEFL
exhibited no toxicity toward the tester strains, a concentra-
tion of 500 mg per plate was set as the upper limit of
the concentration range tested. The test of the antimutagenic
activity of MEFL was performed both in the absence of
the S9 mix, in which NaN
3
and 2-nitrofluorene were used
as standard direct mutagens, and in the presence of the S9
mix, in which 2-AA was used as a standard indirect
mutagen.
In both assays, no genotoxicity was noted at the tested
concentrations. In the plate incorporation assay performed
without rat liver S9 metabolic activation (Table 1), no
biologically or statistically significant increase in the
number of revertants was observed with the S. typhimurium
TA98 or TA100 strain following treatment with MEFL at
levels of 15.62 to 500 mg/well. In the pre-incubation test
(Table 1), the assay with metabolic activation using the rat
liver S9 fraction indicated that there was no statistically
significant increase in the number of revertants for the S.
typhimurium TA90 and TA100 strains. MEFL at concentra-
tions up to 500 mg per plate did not increase the number of
Table 1 - Inhibitory effects of MEFL on direct mutagenicity induced by 2-nitrofluorene (NF) in TA98 cells or sodium azide
phosphate (SA) in TA100 cells without the S9 mix.
Direct
TA98 TA100
Concentration
(mg/well)b
Number of revertants
per plate¡SD % inhibition of mutation
Number of revertants
per plate¡SD
% inhibition of
mutation
15.625 294¡16 29 216¡19 27
31.25 322¡8 18 247¡615
62.5 302¡12 26 196¡14 34
125 228¡25 56 238¡819
250 196¡19 69 172¡11 43
500 163¡782133¡31 58
SR 119¡21 19¡5¡8
Sodium azide (0.5) ---- 288+7
2-Nitrofluorene 366¡41 ---
Indirect
15.625 126¡6 6 271¡47
31.25 121¡912279¡16 3
62.5 112¡13 22 267¡13 9
125 86¡24 49 224¡11 31
250 94¡541197¡23 46
500 79¡7 57 164¡863
SR 39¡593¡2
2-anthramine (0.1) 132¡17 284¡6
Values are the mean
¡S.E.M.
a Without the S9 mix.
bn=3.
c With the S9 mix, n = 3.
Figure 1 - HPLC chromatograms of MEFL and mixed standards of vitexin and isovitexin with detection at 330 nm. A) HPLC
chromatogram of the standards (vitexin and isovitexin). B) HPLC chromatogram of MEFL highlighting the peaks corresponding to the
standards at their respective R
f
values. C) The contents of vitexin and isovitexin (% dry weight) present in the fractions of MEFL. D)
Graphical representation of the phytochemical contents of MEFL. All values are expressed as the mean¡S.E.M. (n = 6).
CLINICS 2013;68(6):865-875 Safety evaluation of Ficus deltoidea
Farsi E et al.
869
his
+
revertant colonies over the negative control (Table 1).
The results therefore indicated that MEFL was not muta-
genic in the S. typhimurium mutagenicity assay.
Acute toxicity study
The acute toxicity study was performed according to
OECD guideline 420, which specifies a limit test dose of
5000 mg/kg. No treatment-related mortality was observed
at 5000 mg/kg, and throughout the 14-day observation
period, there were no significant changes in behavior, such
as apathy, hyperactivity, or morbidity, in any of the animals.
No abnormal changes in body weight, respiration rate, or
heart rate attributable to the treatment were noted. Ilyanie
et al. (27) reported that no overt signs of acute toxicity or
death were observed in mice and rats treated with a
methanol extract of F. deltoidea up to the dose of 6400 mg/
kg. In the present study, MEFL was found to be safe at a
dose of 5000 mg/kg, and therefore, the LD
50
value for oral
toxicity was considered to be greater than 5000 mg/kg.
Subchronic toxicity study
Effects of 28 days of oral administration of MEFL on
general behavior and hematological and biochemical para-
meters in rats.
MEFL at doses of 750, 1250, and 2500 mg/kg adminis-
tered orally every 24 hours for 28 days did not result in any
Figure 2 - Body weight changes of male (A) and female (B) SD rats during the 28-day toxicological assessment. The vehicle, 0.5% CMC
(10 ml/kg/day), was administered to rats in the vehicle group. No significant differences were detected between the treated (750, 1250,
2500 mg/kg) and control (vehicle 10 ml/kg) groups. All values are expressed as the mean¡S.E.M. (n = 5). Representative microscopic
findings (C) for the heart, kidneys, liver, lungs, and spleen of SD rats treated orally with 750, 1250, or 2500 mg/kg MEFL or the vehicle
for 28 days.
Safety evaluation of Ficus deltoidea
Farsi E et al.
CLINICS 2013;68(6):865-875
870
mortality in the tested animals. No signs of observable
toxicity were detected during the entire experimental
period. The body weight gains in the treated groups were
different from that in the control group, but the differences
were not significant (Figures 2A and 2B). There were no
differences in general behavior or food and water consump-
tion between the treated groups of rats and the control
group (data not shown). The effects of subchronic treatment
on the hematological parameters are presented in Table 2.
None of the parameters except the mean corpuscular
hemoglobin (MCH) and packed cell volume (PCV) in
female rats treated with 1250 mg/kg MEFL and the
percentage of lymphocytes in female rats treated with
2500 mg/k showed a significant difference with respect to
the untreated group. The changes in MCH and PCV were
not dose dependent because they were only observed in the
group treated with 1250 mg/kg, not in the group treated
with the higher dose.
The biochemical profiles of the treated and control groups
are shown in Table 3. The oral administration of MEFL for
up to 28 days did not cause significant changes in total
protein, albumin, globulin, the albumin/globulin ratio, total
bilirubin, alkaline phosphatase, AST, ALT, ALP, gamma
glutamyl transferase, potassium, sodium, chloride, creati-
nine, or uric acid. However, a dose-dependent increase in
the level of serum urea was observed in male rats. In a
similar subchronic toxicity study (27), it was observed that
the methanolic extract of F. deltoidea leaves at a dose of
200 mg/kg did not cause any abnormal changes as reflected
by the liver and renal function tests, whereas in the present
study, the higher doses (1250 and 2500 mg/kg) induced
significant changes in the serum urea level.
All the tested hematological parameters, including
hemoglobin, total blood count, total white blood cells,
neutrophils, lymphocytes, eosinophils, monocytes, baso-
phils, packed cell volume, mean corpuscular volume, mean
corpuscular Hb, mean corpuscular Hb concentration, and
platelet count, were within the normal range.
Effects of 28 days of oral treatment with MEFL on
histopathological parameters in rats
The results of the histopathological studies provided
evidence supporting the findings of the biochemical
analysis. No histopathological abnormalities were detected
in the heart, liver, spleen, kidneys, or lungs of the control
group. Histopathological sections of heart, liver, spleen,
kidneys, and lungs are shown in Figure 2C. No lesions or
pathological changes related to treatment with MEFL were
observed in the organs of the animals from the treatment
groups, except in the lungs, where there was evidence of
mild inflammation. Nevertheless, the treatment-related
results were very similar to those for the control group.
Effects of 28 days of oral treatment with MEFL on
the organ weights of the rats
The weights of the organs of the control and treated rats
are shown in Table 4. There were no significant differences
in the organ weights between the treated groups and the
control group.
Table 2 - Effects of the subchronic oral administration of MEFL on hematological parameters in SD rats.
Treatment
a
Control MEFL (mg/kg)
0 mg/kg 750 1250 2500
Male rats
Hemoglobin g/l 145.25¡2.21 140.8¡0.20 141.16¡3.55 140.40¡2.70
Total Red Blood Cells 10
12
/l 8.56¡0.24 8.11¡0.14 8.31¡0.24 8.55¡0.31
Total White Blood Cells 10
9
/l 7.25¡4.81 6.97¡1.16 4.45¡1.69 5.96¡2.05
Neutrophils % 33.25¡6.99 35.50¡2.42 38.25¡6.99 37.20¡6.01
Lymphocytes % 58.50¡6.10 56.67¡2.78 58.50¡6.13 57.20¡5.11
Eosinophils % 2.75¡0.50 3.00¡0.37 3.50¡0.15 2.55¡0.25
Monocytes % 6.00¡20.10 5.00¡0.48 6.33¡2.50 4.80¡1.40
Basophils % 0.00¡0.00 0.00¡0.00 0.00¡0.00 0.00¡0.00
Packed Cell Volume % 45.00 ¡0.81 44.65¡0.99 41.00¡0.86 43.80¡0.99
Mean Corpuscular Volume fl 54.50¡0.57 55.17¡0.65 56.83¡0.40 57.80¡0.80*
Mean Corpuscular Hb pg 17.66¡0.50 18.37¡0.19 18.33¡0.5 18.60¡0.80*
Mean Corpuscular Hb Conc g/l 328.75¡6.65 326.9¡0.5 325.66¡7.99 321.00¡4.47
Platelet Count 10
9
/l 851.25¡146.64 775.5¡83.7 678.5¡125.27* 738.20¡108.88
Female rats
Hemoglobin g/l 149.66¡4.35 141.20¡0.83 143.50¡5.42 140.50¡3.32
Total Red Blood Cells 10
12
/l 8.18¡0.7 7.81¡0.20 8.14¡0.40 8.04¡0.50
Total White Blood Cells 10
9
/l 15.40¡2.02 15.49¡0.75 15.51¡3.80 15.53¡1.02
Neutrophils % 19.56¡3.28 23.16¡2.47 28.66¡2.83 23.40¡2.08
Lymphocytes % 75.50¡3.39 67.17¡2.03 65.33¡9.10 63.80¡4.10*
Eosinophils % 3.80¡0.07 3.00¡0.51 5.80¡0.50 3.80¡0.07
Monocytes % 6.16¡0.31 6.3.00¡1.53 6.80¡0.48 6.70¡0.45
Basophils % 0.00¡0.00 0.00¡0.00 0.00¡0.00 0.00¡0.00
Packed Cell Volume % 48.16¡0.30 45.25¡0.33 45.00¡0.53* 44.00¡0.18*
Mean Corpuscular Volume fl 59.00¡0.17 57.67¡0.42 56.00¡0.14* 57.40¡0.19
Mean Corpuscular Hb pg 18.50¡0.54 18.40¡0.14 17.66¡0.51* 18.20¡0.44
Mean Corpuscular Hb Conc g/l 310.16¡5.49 313.0¡4.30 315.83¡4.26 313.00¡2.54
Platelet Count 10
9
/l 980.16¡25.49 860.3¡24.4 977.50¡20.26 772.60¡21.50
Values are the mean ¡S.E.M.,
a
n=6.
*
p,0.05.
CLINICS 2013;68(6):865-875 Safety evaluation of Ficus deltoidea
Farsi E et al.
871
& DISCUSSION
Despite the popularity of medicinal plants, few scientific
studies have been undertaken to determine the safety of
traditional medicinal herbs. To determine the safety of
medicines and plant products intended for human con-
sumption, systematic toxicological studies must be per-
formed using various experimental models to predict the
toxicity and to set criteria for selecting a safe dose in
humans. Most often, toxicity in animals and humans
manifests in the form of adverse hematological, gastro-
intestinal or cardiovascular effects, and certain adverse
health effects are correlated with structural rearrangements
of the genome caused by different types of DNA damage.
The evaluation of the adverse effects of single and repeated
dosing in experimental animals and the study of mutageni-
city using mutant strains of bacteria may be more relevant
in determining the overall toxicity of plant preparations.
The pharmacological properties of F. deltoidea are widely
known. Despite the widespread use of F. deltoidea in
traditional medicine, there are insufficient data regarding
its toxicity. Therefore, the objective of the present study
was to assess the oral toxicity and genotoxicity of MEFL
in rodents and mutant strains of S. typhimurium, respec-
tively. In the acute toxicity assay, oral treatment with
MEFL was well tolerated. A dose of 5000 mg/kg MEFL
administered to female rats did not cause signs of toxicity,
changes in behavior, or mortality. Any substance with an
LD
50
between 5000 and 15,000 mg/kg is considered non-
toxic (28). Thus, in the present study, MEFL could be
characterized as non-toxic because the LD
50
for this extract
was found to be greater than 5000 mg/kg. Although the
LD
50
does not predict the lethal dose in humans, it provides
a guide for choosing a dose for use in subchronic studies.
The daily administration of the lower dose in the toxicity
study provides some indication of the long-term toxicity of
MEFL. The results of the subchronic (28-day) toxicity study
of MEFL demonstrated that there was no mortality and no
change in the normal behavior or general condition of the
treated rats. These results indicate that MEFL is safe even at
the highest studied dose (2500 mg/kg). In the MEFL-treated
animals, the body weight gain was not significantly
different from that of the control group, suggesting that
MEFL did not alter food intake through appetite suppres-
sion. The weights of the major organs did not significantly
differ from those of the control group. This result implies
that MEFL is non-toxic to these organs, even after 28 days of
exposure.
Treatment with MEFL did not alter the hematological
profile. Significant differences (p,0.05) were found in the
lymphocyte count, MCV, and PVC in female animals
treated with 1250 and 2500 mg/kg MEFL. Because no
Table 3 - Effects of the subchronic oral administration of MEFL on biochemical parameters in SD rats.
a
Treatment
Control MEFL (mg/kg)
0 mg/kg 750 1250 2500
Male rats
Total Protein g/l 74.50¡1.88 76.33¡1.09 68.66¡2.6 69.6¡1.82
Albumin g/l 31.75¡1.58 33.33¡0.95 33.33¡0.95 32.33¡1.58
Globulin g/l 38.17¡0.87 36.00¡0.63 35.67¡1.02 36.33¡1.50
Albumin/Globulin Ratio 0.75¡0.05 0.93¡0.03 0.93¡0.03 0.84¡0.05
Total Bilirubin mmol/l ,2 ,2 ,3 ,2
Alkaline Phosphatase U/l 406.25¡58.74 382.50¡83.11 334.83¡68.08 351.0¡69.54
Alanine Aminotransferase U/l 62.50¡10.37 77.00¡12.56 71.66¡17.42 69.80¡14.88
Aspartate Aminotransferase U/l 248.00¡10.42 241.50¡12.45 239.50¡8.45 252.40¡9.82
Gamma Glutamyl Transferase U/l ,3 ,3 ,3 ,3
Urea mmol/l 6.07¡0.35 6.18¡0.83* 7.12¡0.27* 7.50¡0.39**
Potassium mmol/l 6.30¡ 0.06 5.08¡0.05 5.90
¡0.03 6.26¡0.02
Sodium mmol/l 141.50¡0.31 139.17¡0.40 139.5¡0.40 140.80¡0.48
Chloride mmol/l 98.75¡5.49 100.83 ¡4.31 101.00¡6.63 101.25¡ 2.10
Creatinine mmol/l 30.50¡1.72 30.00¡1.99 31.16¡ 1.16 26.20¡1.27
Uric Acid mmol/l 0.17¡0.02 0.16¡18.35 0.14¡0.05 0.13¡0.04
Female rats
Total Protein g/l 78.50¡1.23 77.83¡1.66 76.20¡2.48 71.23¡2.89
Albumin g/l 33.83¡0.98 35.00¡0.68 34.00¡1.39 33.17¡0.60
Globulin g/l 47.66¡0.79 44.20¡2.94 44.20¡2.94 43.2¡1.12
Albumin/Globulin Ratio 0.65¡0.10 0.77¡0.03 0.74¡0.02 0.64¡0.05
Total Bilirubin mmol/l ,2 ,2 ,2 ,2
Alkaline Phosphatase IU/l 334.33¡5.05 415.83¡4.44 339.80¡11.50 416.8¡21.25
Alanine Aminotransferase U/L 102.33¡2.67 118.50¡3.29 101.83¡4.45 111.33¡2.17
Aspartate Aminotransferase U/L 266.33¡ 13.94 266.83¡4.09 267.17¡14.86 270.6¡15.19
Gamma Glutamyl Transferase U/L ,3 ,3 ,3 ,3
Urea mmol/l 7.16¡0.35 8.30¡0.26* 10.30¡0.28** 7.16¡0.17
Potassium mmol/l 4.48¡0.10 4.42¡0.07 4.57¡0.08 4.33¡0.11
Sodium mmol/l 138.83¡1.72 143.33¡0.95 136.33¡1.00 134.83¡1.65
Chloride mmol/l 101.83¡1.45 99.60¡1.67 100.00¡0.52 98.00¡1.06
Creatinine mmol/l 29.50¡2.24 26.33¡1.50 27.20¡2.68 23.20¡2.38
Uric Acid mmol/l 0.20¡0.05 0.20¡54.09 0.19¡0.05 0.20¡0.09
Values are the mean ¡S.E.M., n = 6.
*
p,0.05,
**
p,0.01.
Safety evaluation of Ficus deltoidea
Farsi E et al.
CLINICS 2013;68(6):865-875
872
corresponding changes were observed in the other para-
meters, the significant changes in the MCV and PCV may
be attribute d to differences in the volumes of the col lected
blood samples. The number of lymphocytes was signifi-
cantly (p, 0.05) reduced in female rats treated with the dose
of 2500 mg/kg, indicating that the defense mechanisms are
likely altered at this dose in female rats. However , the
differential leukoc yte coun ts for eosinophils and mono-
cytes remained within the reference value range (29),
which strongly suggests that there is no relation to
treatment with MEFL. Almost all biochemical paramete rs
analyzed remai ned within the reference levels for the
species (29). However, a dose- dependent increase in the
serum urea level was observed in male rats; this increase
could be related to renal overload. As an in crease in the
plasma level of urea is indicative of renal overlo ad, acute
renal failure or an increase in protein catabolism (30) . A
previous subchronic study (27) foun d that the oral
administration of the extract at a lower dose (200 mg/kg )
did not induce abnormal changes in the serum urea level.
This result suggests that high doses of the extract may
contribute to renal overload.
When the plasma membranes of liver cells are damaged, a
variety of enzymes located in the cytosol are released into
the bloodstream. The levels of these enzymes in the serum
are quantitative measures of the extent and type of
hepatocellular damage. The lack of alteration in the liver
parameters (alkaline phosphatase, aspartate transaminase,
alanine transaminase, lactate dehydrogenase, creatine phos-
phokinase, total protein, albumin/globulin ratio, and
bilirubin) showed that the administration of MEFL for 28
days is not toxic to the liver. Furthermore, the results
showed that the indicators of kidney function (creatinine,
uric acid, phosphorus, calcium, sodium, potassium, and
chloride) remained unaffected. Thus, it is reasonable to
assume that the subchronic administration of MEFL did not
cause any damage to the liver or the kidneys.
These results were confirmed by the histopathological
examination of selected organs (heart, liver, lungs, spleen,
and kidneys) harvested from treated and control animals.
This analysis revealed normal architecture for all vital
organs. In the liver parenchyma of animals treated with
MEFL at doses up to 2500 mg/kg, normal-sized cells with a
centrally located euchromatic nucleus and a very prominent
nucleolus were observed. The hepatic vascular distribution
was homogeneous when compared with that of the control
group (Figure 2C), with a normal hepatic portal triad. All
vital organs studied had a normal histological architecture
Table 4 - Effects of the subchronic oral administration of MEFL on organ weights in SD rats.
Organ weight g
a
Treatment
Control MEFL (mg/kg)
0 mg/kg 750 1250 2500
Male rats
Brain 0.49¡0.02 0.51¡0.04 0.51¡0.03 0.51¡0.02
Heart 0.98¡0.06 0.82¡0.01 0.79¡0.04* 0.88¡0.04
Liver 8.96¡0.75 8.11¡0.22 8.11¡0.13 8.76¡0.07
Thymus 0.27¡0.10 0.23¡0.03 0.23¡0.01 0.27¡0.02
Spleen 0.21¡0.02 0.21¡0.03 0.27¡0.02 0.29¡0.02
Kidney (right) 0.37¡0.01 0.20¡0.01 0.24¡0.1 0.32¡0.01
Kidney (left) 0.38¡0.01 0.30¡0.01 0.25¡0.01 0.34¡0.01
Adrenal Gland (right) 0.03¡0.00 0.02¡0.00 0.02¡0.00 0.03¡0.00
Adrenal Gland (left) 0.03¡0.00 0.03¡0.00 0.03¡0.00 0.03¡0.00
Lungs 1.41¡0.02 1.44¡0.02 1.21¡0.02 1.34¡0.03
Testis (right) 0.55¡0.02 0.55¡0.02 0.54¡0.00 0.53
¡0.02
Testis (left) 0.56¡0.01 0.52¡0.02 0.56¡0.01 0.54¡0.02
Stomach 3.78¡0.50 4.24¡0.21 3.64¡0.17 3.75¡0.20
Stomach (empty) 1.32¡0.01 1.513¡0.01 1.28¡0.02 1.38¡0.01
Gut 11.50¡0.47 12.52¡0.51 11.59 ¡.43 13.43 ¡.53*
Gut (empty) 6.72¡0.24 7.97¡0.20 6.93¡0.19 8.63 ¡.24*
Female rats
Brain 0.48¡0.03 0.49¡0.03 0.50¡0.02 0.48¡0.03
Heart 0.70¡0.01 0.70¡0.01 0.72¡0.01 0.70¡0.01
Liver 7.28¡0.55 7.23¡0.22 7.56¡0.19 7.65¡0.21
Thymus 0.27¡0.02 0.23¡0.01 0.24¡0.01 0.20¡0.01
Spleen 0.20¡0.02 0.21¡0.02 0.21¡ 0.02 0.23¡0.02
Kidney (right) 0.39¡0.01 0.31¡0.01 0.39¡0.01 0.34¡0.01
Kidney (left) 0.29¡0.02 0.30¡0.01 0.29¡0.01 0.28¡0.01
Adrenal Gland (right) 0.03¡0.001 0.03¡ 0.002 0.03¡0.001 0.03¡0.001
Adrenal Gland (left) 0.03¡0.002 0.03¡0.002 0.03¡0.002 0.03¡0.002
Lungs 1.87¡0.02 1.59¡0.05 1.69¡0.03 1.85¡0.02
Ovary (right) 0.06¡0.01 0.06¡0.002 0.06¡0.004 0.05¡0.006
Ovary (left) 0.06¡0.02 0.05¡0.04 0.05¡0.03 0.05¡0.02
Uterus 0.19¡0.01 0.19¡0.01 0.19¡0.01 0.19¡0.05
Stomach 3.85¡0.32 3.34¡0.17 3.28¡0.17 2.72¡0.26*
Stomach (empty) 1.29¡0.07 1.33¡0.03 1.37¡0.03 1.33¡0.03
Gut 10.17¡0.48 11.15¡0.41 11.50¡0.17 10.46¡0.26
Gut (empty) 5.99¡0.18 4.64¡0.20 5.30¡0.031 6.44¡0.03
Values are the mean ¡
S.E.M.,
a
n=6.
*
p,0.05.
CLINICS 2013;68(6):865-875 Safety evaluation of Ficus deltoidea
Farsi E et al.
873
except the lungs, which exhibited signs of an inflammatory
state, with the infiltration of lymphocytes accompanied by
enlarged alveolar macrophages in the air spaces for both the
control and treated groups (Figure 2C). These morphologi-
cal changes in the lungs were most likely caused by the
daily oral gavage and not by MEFL itself because these
alterations were also observed in the control group. The
histological studies suggest that there are no obvious
detrimental effects or morphological disturbances caused
by the daily oral administration of MEFL for 28 days, even
at the highest tested dose of 2500 mg/kg.
The results from the genotoxicity assay showed that, even
at a very high concentration (5000 mg per plate), MEFL did
not increase the number of histidine revertant colonies over
the negative control in the tester strains TA100 and TA98,
either in the presence or absence of S9 metabolic activation.
Because the standard mutagens used in this study (2-NF, 2-
AA, sodium azide phosphate) induced a clear positive
response, the above results indicate that MEFL was not
mutagenic in this assay. The absence of mutagenicity for
MEFL in the tested S. typhimurium strains indicates that
MEFL does not affect the structural integrity of DNA. In
addition, no toxic effects associated with heavy metals in
MEFL were expected because the contents of heavy metals
were below the toxic ranges, with the exception of the lead
content. The content of lead in MEFL was slightly higher
than the acceptable limit. A high lead content can impair the
normal functions of the brain and nervous system, and lead
tends to displace vital minerals such as calcium in the body
(31). Nevertheless, the administration of MEFL did not
cause any lead-associated toxicity in rats. Signs or symp-
toms of toxicity manifest only when the level of lead is
above 0.9 or 1 ppm (32). Therefore, the level of lead detected
in MEFL can be considered the safe upper limit.
Phytochemical screening revealed the presence of phe-
nolics, flavonoids, tannins, glycosaponins, and proteins in
MEFL. The HPLC analysis further showed that in addition
to these classes of chemical constituents, MEFL also
contained remarkably high levels of isovitexin and vitexin.
These two compounds are C-glycosyl flavones, which are
known to be a rich source of biologically active antioxidants
(33) and have received much attention recently because of
their diverse pharmacological properties. Studies conducted
to elucidate the mechanisms of protection against mutagens
have found that the presence of phenolic and flavonoid
compounds can suppress the toxicity and genotoxicity of
toxins because phenolic and flavonoid compounds can
readily scavenge free radicals or activate antioxidant
enzyme cascades.
Based on our results, the oral administration of MEFL
appears to be well tolerated by SD rats. MEFL seemed to
have no discernible clinically significant toxic effects on the
nervous system, respiratory system, or other physiological
functions of animals of both sexes after acute and
subchronic administration. MEFL treatment had inconsis-
tent effects on body growth, organ weights, and hematolo-
gical and biochemical parameters, and these effects failed to
be supported by the gross and histopathologic assessments
of the major organs.
The no-observed adverse effect level (NOAEL) for the 28-
day study with MEFL was considered to be over 2500 mg/
kg/day. This finding suggests that adverse health effects
would not be expected at lower levels of daily MEFL
exposure. Additionally, these findings could aid in the
pharmacological evaluation of plant preparations using this
route of administration in in vivo experimental models, and
they provide reasonable and comprehensive preclinical
evidence of the safety of MEFL, which is necessary to
conduct phase I clinical trials on this standardized plant
extract. However, it should be noted that this NOAEL was
derived only from a subchronic study. Because the observed
effects in animal studies alone cannot always be extra-
polated to the effects in humans, clinical studies are
necessary to precisely define the safe human dosage.
MEFL was not mutagenic in the AMES Salmonella/
microsome assay. Furthermore, no heavy metals were
detected in MEFL that could eventually be responsible for
metal toxicity. Altogether, these results indicate that the
mammalian toxicity of F. deltoidea extract is low and that its
use in traditional medicine presents no genotoxic risks to
humans.
To conduct a more reliable safety assessment based on the
acceptable daily intake criteria, data on the long-term
chronic toxicity, reproductive toxicity, and carcinogenicity
of MEFL should also be collected.
The findings reported herein indicate that the acute and
subchronic (28 day) oral administration of MEFL is safe at
the doses (750, 1250, and 2500 mg/kg body weight/day in
SD rats) tested in this study. In summary, the administration
of MEFL for 28 days did not cause death or visible signs of
toxicity in any animals. Moreover, MEFL did not have
mutagenic effects even at extremely high concentrations in
S. typhimurium strains. The HPLC analysis of MEFL
revealed that vitexin and isovitexin were present at high
levels. The heavy metal analysis of MEFL showed the
absence of toxic levels of heavy metals. Cumulatively, these
findings suggest that the standardized methanol extract of
F. deltoidea can be considered devoid of acute and
subchronic toxicity and genotoxicity. These data suggest
that the consumption of F. deltoidea extract poses no threat of
potential health risks. However, the increased level of serum
urea suggests that a chronic administration study is
necessary to evaluate the renal toxicity of F. deltoidea.
& ACKNOWLEDGMENTS
The authors are grateful to the School of Pharmaceutical Sciences,
Universiti Sains Malaysia, for providing financial and technical support.
& AUTHOR CONTRIBUTIONS
Asmawi MZ, Ismail Z, and Khadeer MB designed the study and assisted
Farsi E, Shafaei A, and Hor SY in conducting the study. Khadeer MB and
Yam MF interpreted the biochemical, hematological, and histopathological
data, and Farsi E and Khadeer MB drafted the manuscript. All authors
reviewed the data and read and approved the final version of the
manuscript.
& REFERENCES
1. Ernst E. Harmless herbs? A review of the recent literature. Am J Med.
1998;104(2):170-8.
2. Pak E, Esrason KT, Wu VH. Hepato-to xicity of herbal remedies: An
emerging dilemma. Prog Transplant. 2004;14(2):91-6.
3. Akueshi EU, Sabo AE, Ogugbuaja VO. Micronutrient and trace content
of calyx and bud of Bomboax buonopozenes (P.Beauv.). Bioresearch.
2005;3:43-65.
4. Okafor PN, Okoronkwo CO, Maduagwu EN. Occupational and dietary
exposure of humans to cyanide poisoning from large scale cassava
processing and ingestion of cassava food. Food Chem Toxicol.
2002;40(7):1001-5.
5. Zunoliza A, Hussain K, Zhari I, Rasadah MA, Mazura P, Jamaludin F,
et al. Evaluation of extracts of leaf of three Ficus deltoidea varieties
Safety evaluation of Ficus deltoidea
Farsi E et al.
CLINICS 2013;68(6):865-875
874
for antioxidant activities and secondary metabolites. Phcog Res.
2009;4(1):216-23.
6. Hakiman M, Mazziah M. Non enzymatic and enzymatic antioxidant
activities in aqueous extract of different Ficus deltoidea accessions. J Med
Plants Res. 2009;3(3):120-31.
7. Adam Z, Hamid M, Ismail A, Khamis S. Effect of Ficus deltoidea
aqueous extract on blood glucose level in normal and mild diabetic rats.
Malaysian J Health Sci. 2007;5(2):9-16.
8. Sulaiman MR, Hussain MK, Zakaria ZA, Somchit MN, Moin S,
Mohamad AS, et al. Evaluation of the antinociceptive activity of Ficus
deltoidea aqueous extract. Fitoterapia. 2008;79(7-8):557-61, http://dx.doi.
org/10.1016/j.fitote.2008.06.005.
9. Zahra MA, Mahmood AA, Hapipah MA, Suzita MN, Salmah I. Anti-
ulcerogenic activity of aqueous extract of Ficus deltoidea against ethanol-
induced gastric mucosal injury in rats. Res J Med Sci. 2009;3(2):42-6.
10. Abdulla MA, Khaled AA, Faisal MA, Mazin M. Role of Ficus deltoidea
extract in the enhancement of wound healing in experimental rats.
J Biomed Res. 2010;21(3):241-5.
11. Abdullah Z, Hussain K, Ismail Z, Ali RM. Anti-inflammatory activity of
standardised extracts of leaves of three varieties of Ficus deltoidea.
Asian J Pharm Clin Res. 2009;1(3):100-5.
12. Farsi E, Shafaei A, Hor SY, Ahamed MBK, Yam MF, Idress HA, et al.
Correlation between enzymes inhibitory effects and antioxidant activities
of standardized fractions of methanolic extract obtained from Ficus
deltoidea leaves. Afr J Biotechnol. 2011;10(67):15184-94.
13. Adam Z, Hamid M, Ismail A, Khamis S. Effect of Ficus deltoidea extracts
on hepatic basal and insulin-stimulated glucose uptake. J Biol Sci.
2009;9(8):796-803.
14. Fazliana MS, Muhajir H, Hazilawati H, Shafii K, Mazleha M. Effects of
Ficus deltoidea aqueous extract on hematological and biochemical
parameters in rats. Med J Malaysia. 2008;63(Supplement A):103-104.
15. Jadeja RN, Thounaojam MC, Ansarullah Snehal VJ, Mitul DP, Dipak KP,
Sunita PS, et al. Toxicological evaluation and hepatoprotective potential
of Clerodendron glandulosum Coleb leaf extract. Hum Exp Toxicol.
2011;30(1):63-70, http://dx.doi.org/10.1177/0960327110368420.
16. Fu Y, Zu Y, Liu W, Zhang L, Tong M, Efferth T, et al. Determination of
vitexin and isovitexin in pigeonpea using ultrasonic extraction followed
by LC-MS. J Sep Sci. 2008;31(2):268-75, http://dx.doi.org/10.1002/jssc.
200700312.
17. Siddiqui M, Hafizoh S, Ismail Z, Sahib H, Helal M, Abdul Majid
A. Analysis of total proteins, polysaccharides and glycosaponins
contents of orthosiphon stamineus Benth. in spray and freeze dried
methanol: water (1: 1) extract and its contribution to cytotoxic and
antiangiogenic activities. Pharmacogn Res. 2009;1(5):320-26.
18. Ahamed MB, Aisha AF, Nassar ZD, Siddiqui JM, Ismail Z, Omari SM,
et al. Cat’s whiskers tea (Orthosiphon stamineus) extract inhibits growth
of colon tumor in nude mice and angiogenesis in endothelial cells via
suppressing VEGFR phosphorylation. Nutr Cancer. 2012;64(1):89-99,
http://dx.doi.org/10.1080/01635581.2012.630160.
19. British Pharmacopoeia Commission. The Stationary Office, London,
2008.
20. Gatehouse D, Haworth S, Cebula T, Gocke E, Kier L, Matsushima T, et al.
Recommendations for the performance of bacterial mutation assays.
Mutat Res. 1994;312(3):217-33.
21. Maron DM, Ames BN. Revised methods for the Salmonella mutagenicity
test. Mutat Res. 1983;113(3-4):173-215.
22. Mastsushima T, Sugimura T, Nagao M, Yahagi T, Shirai A, Sawamura
M. Factors m odulating mutagenicity in microbial tests. In: Nor-poth KH,
Gamer RC, editors. Short-Term System for Detecting Gzrcinogens.
Springer, Berlin Verlag, 1980;p273-85.
23. OECD: Acute oral toxicity, guideline 420, the OECD guideline for testing
of chemical. 2001, http://dx.doi.org/10.1787/9789264070943-en.
24. OECD: Acute oral toxicity, guideline 407, the OECD guideline for testing
of chemical. 2006, http://dx.doi.org/10.1787/9789264070684-en.
25. Galigher AE, Kayloff EN. Essential of practical microtechnique.
Philadelphia: Lea and Febiger; 1971.
26. European pharmacopoeia. Council of Europe, 2002;p3238-3239.
27. Ilyanie Y, Wong TW, Choo CY. Evaluation of hypoglycemic activity and
toxicity profiles of the leaves of Ficus deltoidea in rodents. J Complement
Integr Med. 2011;8(1), doi: 10.2202 /1553-3840.1469.
28. Zbinden G, Flury-Roversi M. Significance of the LD
50
-test for the
toxicological evaluation of chemical substances. Arch Toxicol.
1981;47(2):77-99, http://dx.doi.org/10.1007/BF00332351.
29. Harkness SE, Wagner JE. Biologia e Clı
´
nica de Coelhos Roedores. 3rd
edition. Livraria Roca, Sa
˜
o Paulo, 1993;p48-55.
30. Adebayo JO, Yakubu MT, Egwim EC, Owoyele VB, Enaibe BU. Effect of
ethanolic extract of Khaya senegalensis on some biochemical parameters
of rat kidney. J Ethnopharmacol. 2003;88(1):69-72, http://dx.doi.org/10.
1016/S0378-8741(03)00193-4.
31. Haider S, Naithani V, Barthwal J, Kakkar P. Heavy metal content
in some therapeutically importa nt medicinal plants. Bull Environ
Contam Toxicol. 2004;72(1):119-27, http://dx.doi.org/10.1007/s00128-
003-0249-0.
32. Blagojevic N, Damjanovic
´
-Vratnica B, Vukasˇinovic
´
-Pesˇic
´
V, Durovic D.
Heavy Metals Content in Leaves and Extracts of Wild-Growing Salvia
Officinalis from Montenegro. J Environ Studies. 2009;18(2):167-73.
33. Osawa T, Katsuzaki H, Hagiwara Y, Hagiwara H, Shibamoto T. A novel
antioxidant isolated from young green barley leaves. J Agric Food Chem.
1992;40(7):1135-8, http://dx.doi.org/10.1021/jf00019a009.
CLINICS 2013;68(6):865-875 Safety evaluation of Ficus deltoidea
Farsi E et al.
875
