Energy dispersive x-ray microanalysis, which was attached to variable pressure scanning electron microscopy (VPSEM), was used in this study for elemental analysis. EDX-VPSEM enables unprocessed samples to be viewed and examined. In this study, point and identification (ID) method was used which means specific part of the sample was looked over and the mineral contents were identified. Six replicates of fresh and unprocessed Cola nitida seed were observed for their surface morphological structure by using scanning electron microscope (SEM). The seed had to be cut into a number of 1 cm3 slices and was mounted onto the aluminum stub with a carbon double-sided tape. Samples were then viewed under the EDX-VPSEM (LEO 1455 Variable Pressure-EDX) at various magnifications. The specimen chamber pressure and spot size were varied to obtain the optimum condition. The specimen chambers pressure were 25 to 35 Pascal and the spots size were 2, 1.5 and 1.3 mm2. Together with the image, elemental analysis using EDX attached to the SEM had been carried out by acquiring three elemental spectrums from each sample (Nooraini and Fauziah, 2003). Calibration of EDX was done using cobalt standard.
Preparation of 5% Cola Nut Aqueous Extract
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The aqueous extract of Cola nut (Cn) was prepared freshly every 2 days from modified green tea extraction protocol, according to Conney et al. (1992). Fifty grams of fresh Cola nut were ground in 500 ml of distilled water and filtered. The nuts were re-extracted with 500 ml of distilled water to obtain 5% w/v of water extract (50 g nut/ Lit). Then it was used to obtain the 1 and 2.5% w/v of Cola nut water extract. The 1, 2.5, and 5% w/v Cola nut water extracts were stored at -20 °C till used.
Figure 3.1. Cola nut water extract preparation flow chart.
Obtained estimations were statistically analyzed to investigate the variation between different groups by ANOVA with post-hoc comparisons. Furthermore, Descriptive and Homogeneity of variance test in addition to the Tukey test (as a post-hoc test) was applied in this study. P-value less than 0.05 were considered as significant difference between study groups. The statistical analysis was done using SPSS software, version 16.
About 0.1 ml of the 200 mg Diethylnitrosamine (DEN)/ kg bodyweight of the rat solution was needed to inject to each rat having weight between 150-200 g. 1.0 ml DEN was dissolved in 2.33 ml corn oil (Mazola) to achieve the concentration of 200 mg DEN/ kg bodyweight of the rat.
1.0 g 2-Acetylaminofluorene (AAF) was dissolved into 50.0 ml acetone. 1.5 ml of this solution was then dropped to 150 g rat chow to obtain the final concentration at 0.02% (w/w) AAF in the diet. After that, the acetone was dried in vacuum at 15 mmHg for an hour.
In this study, the semi-thin slides from TEM method were used (Figure ..). Semi-thin sectioning was conducted by ultramicrotome and using glass knifes. The 1 µm thick sections were placed onto glass slide and stained with Toluidine Blue. The slides were dried on hot plate and then washed with water. When the slides were completely dried, they were mounted with cover slip and a drop of DPX (gum). The slides had been kept at room temperature for a few days before they were examined under the light microscope.
The lesion scoring was conducted according to the modified method of Stevens et al. (2002). In this method three characteristic were considered: necrosis of hepatocytes, the presence of inflammatory cells (mainly lymphocytes), and the presence of fibrosis. These features evaluated and classified according to the scoring system illustrated in table 2. The scoring of inflammation, necrosis, and fibrosis was done for each liver section under the light microscope. Because the semi-thin slides were used for lesion scoring, the whole area of section was examined and scored. Lesion scores were expressed in mean ± SD. The severity of necrosis, inflammation, and fibrosis was based on the morphological changes of cells.
Grade of inflammation or necrosis
Mild necrosis of periportal hepatocytes
Moderate necrosis of periportal hepatocytes
Severe necrosis of periportal hepatocytes
Always on Time
Marked to Standard
Inflammation but no necrosis
Focal necrotic cells with Councilman bodies
Severe focal cell damage
Necrosis of liver cells bridges between portal tracts
Stage of fibrosis
Enlarged portal tracts
Fibrosis of periportal area
Fibrosis in septa but no distortion of liver architecture
Fibrosis with regenerative nodules (cirrhosis)
Table .. Staging and grading of liver rats during hepatocarcinogenesis.
In vivo study
The procedure of rat hepatocarcinogenesis, which was used in this study, was modified from the Solt and Farber (1976) protocol. According to modified method, rats did not undergo the partial hepatectomy (selective pressure) stage. In this study rats randomly distributed into eleven groups: Normal Control group (N), Normal + Cn 1% w/v (NCn1), Normal + Cn 2.5% w/v (NCn2.5), Normal + Cn 5% w/v (NCn5), Cancer Control group (C), Cancer + Cn 1% w/v (CCn1), Cancer + Cn 2.5% w/v (CCn2.5), Cancer + Cn 5% w/v (CCn5), Cancer + GL 0.001% w/v (GL0.001), Cancer + GL 0.0025 % w/v (GL0.0025), and Cancer + GL 0.005% w/v (GL0.005). Animal study protocol was shown in Figure 2.1.
Fifty five male Sprague Dawley rats (Rattus norwegicus), 150-200 g (6-8 weeks old), were acclimatized for at least 1 week before use, on basal diet and water. Rats were housed individually in metabolic cages with woodchip bedding in a well ventilated room with equal periods of daylight and darkness with temperature 32 ± 2 °C. Hygienic conditions were maintained by twice-weekly changes of the woodchip beds and daily changes of water bottles.
At the beginning of the study, rat's body weight was taken, and then rats were divided randomly into eleven groups (N, NCn1, NCn2.5, NCn5, C, CCn1, CCn2.5, CCn5, GL0.001, GL0.0025, and GL0.005) which each group contained 5 rats. Cancer were induced into C, CCn1, CCn2.5, CCn5, GL0.001, GL0.0025, and GL0.005 groups by intraperitoneal injection of 200 (mg/kg body weight) Diethylnitrosamine (DEN) dissolve in corn oil to initiate hepatocarcinogenesis followed by a recovery period of 2 weeks on basal diet. After that, to promote hepatocarcinogenesis, rats were fed with 0.02% w/w AAF-mixed rat chow for another 2 weeks. The rats in N, NCn1, NCn2.5, and NCn5 groups were induced by neither DEN nor AAF, but they were gotten a single intraperitoneal injection with corn oil to act as normal rats.
The rats in N and C groups had free access to water. Each rat in NCn1, NCn2.5, NCn5, CCn1, CCn2.5, and CCn5 groups were respectively given about 50 ml of 1, 2.5, 5, 1, 2.5, and 5% w/v Cola nut extract (Cn) daily using water bottles. The Cola nut water extract was given as a substitute for water to rats. While each rat in GL0.001, GL0.0025, and GL0.005 groups were given about 50 ml of 0.001, 0.0025, and 0.005% w/v GL mixed with water daily, as an alternative to water. Except the two weeks period, from the second week to the fourth week, in C, CCn1, CCn2.5, CCn5, GL0.001, GL0.0025, and GL0.005 groups; all rats were given basal diet. Body weights were recorded every two weeks during the experience.
After eleven months from the beginning of the study, rats were sacrificed. All rats were starved for 24 hours before being sacrificed. At the termination of the experiment, the rats were weighed and complete autopsies were perfumed after the rats had been sacrificed by decapitation under chloroform anesthesia. The liver of each rat were washed in ice-cold 0.9% NaCl solution as soon as possible and their weight were recorded. A part of liver tissues of each rat were sliced in 1 cm thick and were fixed in 10% buffered formalin to embed in paraffin blocks and process for TUNEL Assay. Also a number of 1 mm3 liver slices were cut from each rat. They were fixed in 4% Glutaraldehyde for 12 to 24 hours at 4 °C to undergo the transmission electron microscopy. The left over tissues were kept in -80 °C for Real time quantitative RT-PCR.
DEN = Diethylnitrosamine; 200 mg/ kg body weight, intraperitoneal injection
AAF = 2-Acetylaminofluorene; 0.02% w/w, mixed with rat chow
Cn = Cola nut; 1, 2.5, and 5% w/v water extract
GL = Glycyrrhizin; 1, 2.5, and 5 mg/ 100 ml water
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Figure 2.1. Modified study protocol of Solt and Farber (1976) to study the effect of Cola nut water extract on rat's liver during hepatocarcinogenesis.
Real-time Quantitative Reverse Transcriptase Polymerase Chain Reaction
The total RNA was purified using the Qiagen RNeasy® Mini Kit. Following collecting of sacrificed rat's liver, the liver of each rat were washed in ice-cold 0.9% NaCl solution and the tissues were kept in -80 °C immediately. RNA was isolated according to the Qiagen RNeasy® Mini Handbook demonstrated in Figure . In this study, tissues were disrupted using rotor-stator homogenizer. β-Mercaptoethanol (β-ME) was added to Buffer RLT before use by adding 10 μl β-ME per 1 ml Buffer RLT. Buffer RPE is supplied as a concentrate. Before using for the first time, four volumes of ethanol 100% (Merck) were added to obtain a working solution. The concentration of collected RNA was evaluated by NanoDrop spectrophotometer. RNA was kept at -80 °C to use in Real-Time RT-PCR at the same day of isolating.
Figure . Total RNA purification according to Qiagen RNeasy® Mini Handbook.
Primer pairs for AFP and ALB genes were designed using World Wide Web tools. The Primer3 version 0.4.0 (http://frodo.wi.mit.edu/primer3/) and the Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) were used in this study. The picked primers were performed a BLAST search to compare primer query sequences with database of sequences and evaluate the uniqueness of the query. Rattus norvegicus alpha-fetoprotein (Afp), mRNA (NCBI Reference Sequence: NM_012493.1), and Rattus norvegicus albumin (Alb), mRNA (NCBI Reference Sequence: NM_134326.2) were used as the reference mRNA sequences (Appendix A). Since fluorescence from SYBR Green I increases strongly upon binding of the dye to any double-stranded DNA, it is particularly important to minimize nonspecific primer annealing by careful primer design. For the highest efficiency in real-time RT-PCR using SYBR Green I, targets should ideally be 60-200 bp in length.
Real-time Quantitative RT-PCR
Real-time Quantitative RT-PCR was conducted using the QuantiFast™ SYBR® Green RT-PCR kit (Qiagen). Use of 2x QuantiFast SYBR Green RT-PCR Master Mix together with QuantiFast RT Mix allows both reverse transcription and PCR to take place in a single tube, that all reagents required for both reactions were added at the beginning.
2x QuantiFast SYBR Green RT-PCR Master Mix, QuantiFast RT Mix, diluted primers, and RNase-free water were stored at -20 °C. Real-time Quantitative RT-PCR was carried out following the manufacturer's procedure as below:
2x QuantiFast SYBR Green RT-PCR Master Mix, template RNA, primers, and RNase-free water were thawed. The individual solutions were mixed and placed on ice. QuantiFast RT Mix should be taken from -20 °C immediately before use, always kept on ice, and returned to storage at -20 °C immediately after use.
Reaction mix was prepared according to Table 1. The reaction mix was thoroughly mixed and appropriate volumes were dispensed into PCR vessels or plates.
2x QuantiFast SYBR Green RT-PCR Master Mix
QuantiFast RT Mix
Template RNA (added at step 4)
Total reaction volume
Template RNA (≤100 ng/reaction) was added to the individual PCR vessels or wells containing the reaction mix.
Real-time cycler (Rotor-Gene 6000, Corbett) were programmed (Table 2). The PCR vessels or plates were placed in the real-time cycler and then the program was run.
PCR initial activation step
Number of cycles 35-40
Melting curve analysis of the RT-PCR products was performed to verify their specificity and identity.
Transmission Electron Microscopy
Transmission electron microscopy was conducted according to the Hayat (1986) procedure. The liver tissues from rats were sliced into a number of 1 mm3 pieces and put into separate vials. As a primary fixation, the tissues were fixed in 4% Glutaraldehyde for 12 to 24 hours at 4 °C. The tissues were then washed with 0.1 M Sodium Cacodylate buffer 3 times of 10 minute each. Later they were post-fixed in 1% Osmium Tetroxide for 2 hours at 4°C and washed again with 0.1 M sodium cacodylate buffer for 3 changes of 10 minutes each. The samples were dehydrated in a series of ascending acetone dilution as in (Table 2). The samples were then infiltrated with resin and acetone mixture as in (Table 2).
Table 2. Dehydration and Infiltration
15 min (3changes)
The specimens were placed into beam capsules and filled up with the resin for embedding, and then they were polymerized in an oven at 60 °C for 24-48 hours. Semi-thin sectioning was carried out by ultramicrotome and using glass knifes. The 1 µm thick sections were placed onto glass slide and stained with Toluidine Blue. The interested part of the specimen was spotted and selected by observing the slides under the light microscope for ultrathin sectioning. The Toluidine Blue-stained slides were kept to be used in histological studies and lesion scoring.
Ultrathin sectioning was carried out on interested area. For ultrathin sectioning, the diamond knife is recommended. Then, the silver/gold sections were picked up with a grid. Grids were dried using filter papers. The dried grids were stained with Uranyl Acetate for 10 minute and washed with 50% filtered alcohol. After that, they were stained with Lead for 10 minutes and washed with double distilled water. Following each step the grids were dried with filter papers. The specimens were then examined under transmission electron microscope (LEO).
Figure Liver tissue Transmission Electron Microscopy Method. Semi-thin slides was prepared and examined for histological studies and lesion scoring.
The DeadEnd™ Fluorometric TUNEL System (Promega) kit was used in this study. The DeadEnd Fluorometric TUNEL System detects the fragmented DNA of apoptotic cells by catalytically incorporating fluorescein-12-dUTP at the 3´-OH ends of the DNA using the enzyme Terminal Deoxynucleotidyl Transferase (recombinant), which forms a polymeric tail using the principle of the TUNEL (TdT-mediated dUTP Nick-End Labeling) assay.
The liver tissues from each rat were sliced in 1 cm thick pieces and they were fixed in 10% formalin for a few days. Then the tissue samples were embedded in paraffin blocks and were cut using microtome in 4 to 5 micron. Pasted on slide samples were dried on a hot plate at 50-55 °C for 30 minutes. The slide could be kept at room temperature before they were processed further.
DeadEnd™ Fluorometric TUNEL System was conducted as stated by the manufacturer's instruction (Promega), which have been illustrated in Figure . . The fluorescein-12-dUTP labeled DNA could be visualized directly by fluorescence microscopy. Observing and analyzing the apoptotic cells under the fluorescence microscope were immediately done once preparing of the slides had been finished. Samples were viewed under a fluorescence microscope using a standard fluorescein filter set to view the green fluorescence of fluorescein at 520 ± 20nm; view red fluorescence of propidium iodide at >620nm and blue DAPI at 460nm. All the slides were examined. Five spots were randomly selected at 20X magnification to conduct the systematic scoring method. On each spot, green stained cells were counted as a TUNEL-positive apoptosis cells.
Pretreatment of Paraffin-Embedded Tissue
Wash slides twice in xylene, 5 minutes each wash
Immerse in 100% ethanol for 5 minutes
Wash slides in decreasing concentrations of ethanol (100%, 95%, 85%, 70%, 50%), 3 minutes each wash
Immerse in 0.85% NaCl for 5 minutes
Immerse in PBS for 5 minutes
Immerse slides in 4% formaldehyde in PBS for 15 minutes
Immerse slides twice in PBS, 5 minutes each time
Add 100μl of a 20μg/ml Proteinase K solution. Incubate at room temperature for 8-10 minutes.
Immerse slides in PBS for 5 minutes
Immerse slides in 4% formaldehyde in PBS for 5 minutes
Immerse slides in PBS for 5 minutes
Add 100μl Equilibration Buffer, Equilibrate at room temperature for 5-10 minutes
Add 50μl of TdT reaction mix to the tissue on an area, no larger than 5 square centimeters. Do not allow tissue to dry completely. Cover slides with Plastic Coverslips to ensure even distribution of the mix. Incubate slides for 60 minutes at 37°C in a humidified chamber; avoid exposure to light from this step forward.
Remove Plastic Coverslips, immerse slides in 2X SSC for 15 minutes
Immerse slides three times in PBS, 5 minutes each time
Immerse slides in 40ml of propidium iodide solution freshly diluted to 1μg/ml in PBS for 15 minutes at room temperature in the dark in a Coplin jar
Wash the samples by
Immerse slides three times in deionized water for
5 minutes at room temperature
Drain off excess water from the slides and wipe the area surrounding the tissue with tissue paper
Add one drop of Anti-Fade solution to the area containing the treated tissue and mount slides using glass coverslips. Seal the edges and let dry for 5-10 minutes.
Observe Under Fluorescent Microscope
Table . Preparing slides method according to DeadEnd™ Fluorometric TUNEL System (Promega).