Asmah Rahmat, Fahmida Parveen
Saib, and Nurul Amira Buslima
Derpartment of Nutrition and Dietetics,
Faculty of Medicine and Health Sciences, University Patra Malaysia, 43400
Serdang, Selangor, Malaysia.
Asstract – Hepatocellular carcinoma
(HCC) is the fifth most common malignancy in the world. It is much prevalent in
many of the developing countries than in the industrialized world. The effect
of Ficus Benjamina extract and Pereskia Saecnarosa extract was studied
on the level of micro and macro minerals in normal and induced liver cancer
rats. The extracts of Ficus Benjamina
and Pereskia Saecnarosa were prepared
and were given to the rats through ad
libitum. Forty-four male rats from the species Sprague Dawley were used as a model. They were acclimatized for
duration of one week and further divided into 6 groups at random. The 6 groups
were: control, cancer, normal rats supplemented with Ficus Benjamina, cancer rats supplemented with Ficus Benjamina, normal rats supplemented with Pereskia Seacnarosa, cancer rats supplemented with Pereskia Seacnarosa. Twelve weeks later,
the blood of each rat was taken for the analysis of calcium, iron and zinc and
body weight was taken before and after the treatment. The very obvious result
was the highest weight gained by the normal rat supplemented with Ficus Benjamina extract. However, the
weight gain was not significant to the control group but it was significant to
the cancerous group. In all the 6 groups irrespective of their treament, the
level of minerals was almost the same. From the statistical analysis, it was
found that there was a significant relationship in the level of calcium between
the cancerous rats and the cancerous rats given Pereskia Saecnarosa (p<0.05). Moreover, a significant
relationship in the level of iron was found between the cancerous rats and the
cancerous rats supplemented with Pereskia
Seacnarosa (p<0.05). However the cancerous rats supplemented with Pereskia Seacnarosa showed a lower level of ferrum in their blood serum.
Furthermore a significant relationship was found in the level of zinc between
the control rats and normal rats supplemented with Pereskia Seacnarosa (p<0.05).
In conclusion, there is no significant relationship between the effect of Ficus Benjamina and Pereskia Seacnarosa extract and the level of calcium, iron and
zinc.
Keyword
– Ficus Benjamina, Pereskia Seacnarosa, hepatocarcinogenesis,
micro and macro minerals.
I. INTRODUCTION
Since ancient time,
plants leave have been used an exemplary sources of medicine. Ayurveda and
other Indian literature have mentioned the use of plants in the treatment of
various ailments [1]. Indeed, most of the new applications of plant secondary
metabolites and their derivatives over the haft century have been applied towards
combating cancer [2]. Since ancient times, more than 3,000 plant species were
reported to be used in the treament of cancer [3]. Since the year 1980’s more
than 60% of cancer approved drugs developed are of natural products origin [4]
and 25% are directly derived from plants [5]. Plants products are regarded as
potential chemopreventive agents act to block reverse or prevent the
developement of invasive cancers [6]. Recently, tropical and subtropical plants
are perceived to produce a large variety of phytochemicals or secondary
metabolites and possess a wide range of cancer preventive. Lots of importance
is given to cancer as cancer is one of the leading diseases of new era and it
is also one of the greatest killers. In Malaysia, it is one of the leading
causes of morbidity and mortabity [7].
Antioxidants are
defined as compounds that inhibit the oxidation of other molecules by
inhibiting the initiation the propagation of oxidizing chain reactions [8]. are
two basis of antioxidants, synthetic and natural. Synthetic antioxidants are
compounds with phenolic structures of vatious degrees of alkyl substitution,
whereas natural antioxidants can be phenolic compounds (tocopherols, flavonoids
and phenolic acid). Nitrogen compounds alkaloids, chlorophyll derivatives,
amino acids and amines) or carotenoids as well as ascorbic acid [9].
Many of natural
antioxidants, especially flavonoids, exhibit a wide range of biological
effects, including antibacterial, antiviral, anti-inflammatory, antiallergic,
anti-thrombotic and vasodilatory actions. Antioxidants activity is the
fundametal property important for life. Many of the biological functions, such
as antimutagenicity, anti-carcinogenocity and antiaging, among others,
originate from this property [10]. The antioxidant activity of several plant
materials has cerently been reported.
Two plant extracts were
used in this research, namedly Ficus
Benjaminaextract and Pereskia
Seacnarosa extract. According to Wagner [11]. Ficus Benjamina is the plant from the family Moracacca (Mullberry
family) that are native to large area including India, Southern China,
SouthEast Asia,Malaysia, Philippines, Northern Australia, and the island of
South Pacific [12]. Whereas, Pereskia
Saecnarosa is categorized as a primitive cactus because of its leaves and
one of an edible herb in Malaysia, though it is not very common. It is known as
“jarum tujuh bilah” among Malaysian and usually they are made into juice after
boiled and drank it every morning for the purpose of health practice and body
refreshing.
In the Ficus species,
there have been some studies carried out on Ficus
elastica as in possessing inflammatory properties and on Ficus citrifolia
as a therapeutic agent against cancer therapy [13]. However, so far, there has
been no study carried out of Ficus Benjamina
as in possessing any anti-cancerous properties. As for Pereskia Saecnarosa is concerned, there has been few researchers
carried out to see its potential in cancer therephy. The leaves of Pereskia Saecnarosa is believed to
possess a dietary component in reducing the severe level in the incidence of
hepato-carcinogenesis.
II.
MATERIALS AND METHODS
2-acetylaminofluorene,
diethylnitrosamine, acetone, rat basal diet, ether, centrifuge apparatus,
spectra A-40 atomic absorption spectrometer, calcium hollow cathode lamp, iron
hollow cathode lamp, zinc hollowcathode lamp.
III.
METHODOLOGY
A.
Sample Preparation and Extraction
The leaves of Ficus Benjamina and Pereskia Saecnarosa were randomly plucked from University
agricultural Park, University Putra Malaysia. The leaves were cleaned, weighed and
further grounded into powder form. A volume of 2000ml of distilled water was
added to the ground leaves to make a concentration of 5.0% (w/v). The solution
were then filtered and given to the group of rats which was concerned. The same
procedures were done to prepare Perseskia
Saecnarosa extract.
B.
Cancer Inducer Preparation
Diethylnitrosamine
(DEN) inducer is prepared by dissolving 1.0 ml DEN in 3.33 ml corn oil, which
is equivalent to
200mg DEN /kg body weight of the rats. About 0.2 ml of the solution was
injected to each rat. Besides that, 1.0g of 2-acetylaminofluorene (AAF) is
dissolved into 50.0ml acetone. 1.5 ml of this solution is then dropped to 150g
rat chow to obtain the final cincentration at 0.02% (w/w) AAF in the diet. The
acetone is then dried in vacuum at 15mmHg for an hour.
C.
Induction of Hepatocarcinogenesis
The cancerous groups
were induced with hepato-carcinogenesis using the method of [14]. which can
induce liver cancer is injected into the rats intraperitoneally with necrogenic
doses of 200 mg/kg body weight to initiate hepatocarcinogenesis. Following a
two weeks period, as described by method of Cameroon [15]. the rats were then
fed with 0.02% (w/w) AAF treated rat chow for another 2 weeks without partial
hecpatectomy to promote hepato-carcinogenesis.
Group
|
Treatment
|
|||
Normal control
(N)
|
Basal diet +water
|
|||
Cancer induced
(C)
|
Basal diet + water
|
AAF + water
|
Basal diet + water
|
|
Normal + FB
(NF)
|
Basal diet + water
|
Basal diet + FB
|
||
Cancer + FB
(CF)
|
Basal diet + water
|
AAF + FB
|
Basal diet + FB
|
|
Normal + PS
(NP)
|
Basal diet + water
|
Basal diet + PS
|
||
Cancer + PS
(CP)
|
Basal diet + water
|
AAF + PS
|
Basal diet + PS
|
|
Week 0 2 4 12
DEN ↑
Fig 1. Protocol to study the effect of Ficus Benjamina and Pereskia
Saecnarosa extract during rat hepatocarcinogenesis.
D.
Animal Management
In the research, a
total of 44 male rats from the species of Sprague Dawley were used as a model.
They were housed in a seperate cage in the animal house at the Faculty of
Medicine and Health Sciences of University Putra Malaysia with woodchip bedding
in a well ventilated room with equal periods of daylight and darkness with temperature
32 +/- 20C. Hygienic conditions are maintained by twice-weekly
changes of the woodchip beds and daily changes of water and treatment. Each
rats initially weighing between 150 to 200 g (7-8 weeks old) were acclimatized
for at least 1 week before use, on basal diet and water ad
libitum. All rats are randomly distributed into 6 group comprising groups
of normal control (N), cancer-induced control (C), normal with 0.1ml of Ficus Benjanina (NF), cancer with 0,1ml
of Ficus Benjanina, (CF),
E. Experiment Termination
All rats are killed
after 12 weeks periods of experimental protocol. The rats were starved for 24
hours before being sacrificed. At the termination of the experiment, tha
animals are weighed and immediately before death, blood is collected by
venipuncture and placed into EDTA tubes. The samples were centrifuged at 3,000
rpm with bench centrifuge for 10 minutes and the plasma was collected to
perform analysis of marco minerals.
F. Macro Minerals Analysis
The atomic absorption
spectroscopy (AAS) was used to determine the following mineral level: calcium,
iron and zinc in the blood plasma of the 44 male rats after their respective
treatment. A SpectrAA-40 atomic absorption spectrometer, PSC-56 programmable
sample changer, Epson LX-80 printer and calcium, ferum, and zinc hollow cathode
lamps from Varian were used in the procedure. The main instrument parameters
for the analysis were as Table 1.
Table 1: Parameters for the selected macro minerals analysis
Parameters
|
Zinc
|
Ferum
|
Calcium
|
Wavelength
(nm)
|
213.9
|
248.3
|
422.7
|
Stil
width (nm)
|
1.0
|
0.2
|
0.5
|
Lamp
current (mA)
|
4
|
8
|
3
|
Burner
height (nm)
|
10
|
10
|
4
|
Acetylene
flow (L/min)
|
2.00
|
2.50
|
2.50
|
Air
flow (L/min)
|
13.5
|
13.5
|
13.5
|
Standard
|
0.1 - 0.5
|
0.1 - 0.5
|
4.0 – 20.0
|
concentration
(µg/ml)
|
As for reagents
concerned, the water used were doubly distilled and deionized. All the reagents
used for the preparation of standard solutions and anti-interference reagents
were ultra pure grade. The standard solutions were prepared in glass bottles,
previously washed with diluted nitric acid and rinsed with deionized water. One
milliliter of serum was placed in a test tube diluted to 10 milliliter with
deionized water. The test tube was placed in the auto sampler carousel after
mild shaking. The measurement was automatically carried out and the results
were the everage of two replicated according to the standard addition
calibration method. The final result was presented as unit of mg/100g sample.
G. Data Analysis
All data were presented
as mean ± standard deviation of duplicates and statistically analyzed using
Statistical Package for Social Sciences (SPSS) version 19.0. One-way analysis
of variance (ANOVA0 was used to compare means of different groups. A
significant difference was considered at the level of p< 0.05.
IV.
RESULTS AND DISCUSSION
A.
Comparison of Body Weight
The average final
weights gained of the rats were shown in Table 2. The table clearly shown that
the group of normal rats supplemented with Ficus
Benjamina extract was the highest weight gained among all groups. Actually
the group of normal rats supplemented with Ficus
Benjamina extract has gained weight significantly (p<0.05) compared with
the DEN/AFF treated group, but the weight gain is not significant to the other
groups. The small sample size may be an explanation of this effect. The
presence of some bioactive compounds in Ficus
Benjamina might have lead to the weight gained, where it might have
increased the appetite of the rats.
Table
2: Effect of Ficus Benjamina extract, Pereskia
Saecnarosa extract and DEN/AAF on the final body weight gained
Group
|
Body weight gained (g)
|
Normal control (N)
|
178.94
± 8.40
|
Cancer induced (C)
|
149.26
± 4.56a
|
Normal – FB (NF)
|
213.28
± 19.12a
|
Cancer – FB (CF)
|
176.74
± 4.10
|
Normal – PS (NP)
|
180.81
± 8.46
|
Cancer – PS (CP)
|
150.28
± 14.55b
|
Value are the mean ± standard deviation
|
|
Letter a : significantly different from the control group
(p<0.05)
|
|
Letter b : significantly different from the control cancer
(p<0.05)
|
|
There was also a
significant reduction on weight in the DEN/AAF treated group to the control
group. The cancerous group had lost a significant among of weight. The
significant reduction in body weight by the cancerous rats might be because of
low food consumption and hence low of weight. The reduction in body weight by
the cancerous rats showed that the effect of DEN/AAF has been met. A research
cattied out by Hosaka. [16] also showed the same pattern in weight gained.
Furthermore, the body
weight gained of the DEN/AAF group also differs significantly (p<0.05) with
the DEN/AAF group supplemented with the Pereskia
Saecnarosa extract group Cancerous rats supplemented with Pereskia Seac-narosa extract have higher
body weight gained Cancerous rats do not have appetite but Pereskia Saecnarosa extract might have increased the appetite of
the rats, resulting in a weight gained.
B. Plasma Mineral Analysis Using Atomic Absorption Spectroscopy (AAS)
From the result shown
in Table 3, all the plant extracts did not influence the level of minerals in
the rat’s plasma. In all the 6 groups irrespective of their treatment, the
levels of minerals were almost the same. This may be because the rats have been
consuming the plant extract for a too short period of time. For a herb to be
effective, it must be consumed over a long period of time.
Table
3. Selected mineral level in mg/100g of sample in each group
Group
|
Calcium
|
Iron
|
Zinc
|
Normal control
(N)
|
1.66±
0.475
|
0.58
± 0.292
|
0.010
± 0.387
|
Cancer induced
(C)
|
1.89
± 0.203
|
0.71
± 0.029
|
0.63
± 0.335
|
Normal - FB
(NF)
|
1.44
± 0.575
|
0.07
± 0.026
|
0.64
± 0.391
|
Cancer - FB
(CF)
|
1.88
± 0142
|
0.06
± 0.028
|
0.79
± 0.441
|
Normal - PS
(NP)
|
1.43
± 0.648
|
0.08
± 0.028
|
0.83
± 0.416b
|
Cancer - PS
(CP)
|
1.48
±0.503a
|
0.07
± 0.017a
|
0.10
± 0.068
|
Values are the mean = standard deviation.
Letter a: significantly different from the control group
(p<0.05)
Letter b: significantly different from the control cancer
(p<0.05)
From the statistical
analysis, there was a significant relationship in the level of calcium between
the cancerous rats and the cancerous rats given Pereskia Saecnarosa. It is know that calcium can prevent
precancerous cells from becoming cancerous [17]. But vitamin D is essential for
the absorption and metabolism of calcium. If calcium is taken but the level of
vitamin D is too low, the calcium will not be absorbed and will result in high
level of calcium in the blood.
Moreover, a significant
relationship in the level of iron was found between the cancerous rats and the
cancerous rats supplemented with Pereskia
Saecnarosa. However, the cancerous rats supplemented with Pereskia Saecnarosa showed a low level
of iron in their blood plasma. There might be substances in Pereskia Saecnarosa which inhibit the
absorption of iron. Iron plays an important role in the prevention of cancer
but should not be taken in excess due to the initiation of production of
radicals by iron complexes. According to Charles, [18], excess stores of iron
can increase the risk of cancer in man and probably women as well. Furthermore,
a significant relationship was found in the level of zinc between the control
rats and normal rats supplemented with Pereskia
Saecnarosa.
In a research carried
out by undergraduate student from the Faculty of Medicine and Health Sciences, University
Putra Malaysia, the level of 4 minerals were analyzed from the leaves of the
plant Ficus Benjamina. The minerals
of interest were selenium, calcium, copper and iron. The result showed that
calcium (9.12mg/100g) was of the highest amount, followed by iron
(0.64mg/100g), selenium (0.46mg/100g) and copper (0.06mg/100g). The nutrients
that were present in Ficus Benjamina
leaves have a great potential to be used as a source of anticancer medicine.
V.
CONCLUSIONS
Results of the body
weight gain by the normal group of rats supplemented with Ficus Benjamina and by the normal group of rats supplemented with Pereskia Saecnarosa showed that both
plants extract have the potential to increase appetite. Moreover, the low body
weight of cancerous rats indicates that the effect of Diethylnitrosamine and
2-acetyl-aminofluorene have been successful. As for the levels of minerals were
concerned, there were almost no variation among the groups. This may due to the
short period of taking the plant’s extract.
ACKNOWLEDGMENT
A warm appreciation
dedicated to the Department of Nutrition and Dietetics, University Putra
Malaysia for the support, help and contribution in this research project.
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