Thứ Tư, 24 tháng 7, 2013

Comparing the Effect of Ficus Benjamina Extract and Pereskia Saecnarosa Extract on the Level of Micro and macro minerals in Normal and Induced Liver Cancer Rats





Comparing the Effect of Ficus Benjamina Extract and Pereskia Saecnarosa Extract on the Level of Micro and macro minerals in Normal and Induced Liver Cancer Rats

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.


              AsstractHepatocellular 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), normal with 0.1ml of Perseskia Saecnarosa (NP), and cancer with 0,1ml of Perseskia Saecnarosa (CP). A total of 5-8 rats are allocated for each group. The rats in normal control group had only a basal diet and water throughout the experiment. For the groups of treatment and cancer – induced + treatment, about 0.1ml samples will be given every day using force feeding. For two weeks period, groups of cancer-induced are feed with a treated AAF chow pellet in their diet as substitude to basal diet in the 3rd week of experiment following two weeks of DEN injection to promote hepatocarcinogenesis. On the 5th week, feeding of treated AAF chow pellet is terminated and the rats were then given back basal diet until the end of the experiment. 
     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.


Nguồn: V.Van Toi ct al (Eds.) 
4th International Conference on Biomedical Engineering in VietNam, 
IFMBE Proceedings 40. Pp. 208 -212. 2013
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