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Energy diffusing x-ray microanalysis, which was attached to variable force per unit area scanning negatron microscopy ( VPSEM ) , was used in this survey for elemental analysis. EDX-VPSEM enables unprocessed samples to be viewed and examined. In this survey, point and designation ( ID ) method was used which means specific portion of the sample was looked over and the mineral contents were identified. Six replicates of fresh and unrefined Cola nitida seed were observed for their surface morphological construction by utilizing scanning negatron microscope ( SEM ) . The seed had to be cut into a figure of 1 cm3 pieces and was mounted onto the aluminium stub with a C double-sided tape. Samples were so viewed under the EDX-VPSEM ( LEO 1455 Variable Pressure-EDX ) at assorted magnifications. The specimen chamber force per unit area and topographic point size were varied to obtain the optimal status. The specimen Chamberss force per unit area were 25 to 35 Pascal and the musca volitanss size were 2, 1.5 and 1.3 mm2. Together with the image, elemental analysis utilizing EDX attached to the SEM had been carried out by geting three elemental spectrums from each sample ( Nooraini and Fauziah, 2003 ) . Calibration of EDX was done utilizing cobalt criterion.

Preparation of 5 % Cola Nut Aqueous Extract

The aqueous infusion of Cola nut ( Cn ) was prepared newly every 2 yearss from modified green tea extraction protocol, harmonizing to Conney et Al. ( 1992 ) . Fifty gm of fresh Cola nut were ground in 500 milliliter of distilled H2O and filtered. The nuts were re-extracted with 500 milliliter of distilled H2O to obtain 5 % w/v of H2O infusion ( 50 g nut/ Lit ) . Then it was used to obtain the 1 and 2.5 % w/v of Cola nut H2O infusion. The 1, 2.5, and 5 % w/v Cola nut H2O infusions were stored at -20 & A ; deg ; C boulder clay used.

Figure 3.1. Cola nut H2O extract readying flow chart.

Datas Analysis

Obtained appraisals were statistically analyzed to look into the fluctuation between different groups by ANOVA with post-hoc comparings. Furthermore, Descriptive and Homogeneity of discrepancy trial in add-on to the Tukey trial ( as a post-hoc trial ) was applied in this survey. P-value less than 0.05 were considered as important difference between survey groups. The statistical analysis was done utilizing SPSS package, version 16.

Diethylnitrosamine Preparation

About 0.1 milliliter of the 200 milligrams Diethylnitrosamine ( DEN ) / kg bodyweight of the rat solution was needed to shoot to each rat holding weight between 150-200 g. 1.0 milliliter DEN was dissolved in 2.33 milliliters maize oil ( Mazola ) to accomplish the concentration of 200 milligrams DEN/ kg bodyweight of the rat.

2-Acetylaminofluorene Preparation

1.0 g 2-Acetylaminofluorene ( AAF ) was dissolved into 50.0 milliliter propanone. 1.5 milliliter of this solution was so dropped to 150 g rat Zhou to obtain the concluding concentration at 0.02 % ( w/w ) AAF in the diet. After that, the propanone was dried in vacuity at 15 mmHg for an hr.


Light Microscopy

In this survey, the semi-thin slides from TEM method were used ( Figure.. ) . Semi-thin sectioning was conducted by ultramicrotome and utilizing glass knifes. The 1 µm thick subdivisions were placed onto glass slide and stained with Toluidine Blue. The slides were dried on hot home base and so washed with H2O. When the slides were wholly dried, they were mounted with screen faux pas and a bead of DPX ( gum ) . The slides had been kept at room temperature for a few yearss before they were examined under the light microscope.

Lesion Scoring

The lesion marking was conducted harmonizing to the modified method of Stevens et Al. ( 2002 ) . In this method three feature were considered: mortification of hepatocytes, the presence of inflammatory cells ( chiefly lymphocytes ) , and the presence of fibrosis. These characteristics evaluated and classified harmonizing to the marking system illustrated in table 2. The marking of redness, mortification, and fibrosis was done for each liver subdivision under the light microscope. Because the semi-thin slides were used for lesion marking, the whole country of subdivision was examined and scored. Lesion tonss were expressed in average ± SD. The badness of mortification, redness, and fibrosis was based on the morphological alterations of cells.

Grade of redness or mortification









Portal redness

Mild mortification of periportal hepatocytes

Moderate mortification of periportal hepatocytes

Severe mortification of periportal hepatocytes









Inflammation but no mortification

Focal necrotic cells with Councilman organic structures

Severe focal cell harm

Necrosis of liver cells bridges between portal piece of lands

Phase of fibrosis

Phase mark







Enlarged portal piece of lands

Fibrosis of periportal country

Fibrosis in septa but no deformation of liver architecture

Fibrosis with regenerative nodules ( cirrhosis )

Table.. Staging and scaling of liver rats during hepatocarcinogenesis.

In vivo survey

The process of rat hepatocarcinogenesis, which was used in this survey, was modified from the Solt and Farber ( 1976 ) protocol. Harmonizing to modified method, rats did non undergo the partial hepatectomy ( selective force per unit area ) phase. In this survey rats indiscriminately distributed into 11 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 survey protocol was shown in Figure 2.1.


Fifty five male Sprague Dawley rats ( Rattus norwegicus ) , 150-200 g ( 6-8 hebdomads old ) , were acclimatized for at least 1 hebdomad before usage, on basal diet and H2O. Rats were housed separately in metabolic coops with woodchip bedclothes in a good ventilated room with equal periods of daytime and darkness with temperature 32 ± 2 & A ; deg ; C. Hygienic conditions were maintained by twice-weekly alterations of the woodchip beds and day-to-day alterations of H2O bottles.

At the beginning of the survey, rat ‘s organic structure weight was taken, and so rats were divided indiscriminately into 11 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 organic structure weight ) Diethylnitrosamine ( DEN ) dissolve in maize oil to originate hepatocarcinogenesis followed by a recovery period of 2 hebdomads on basal diet. After that, to advance hepatocarcinogenesis, rats were fed with 0.02 % w/w AAF-mixed rat Zhou for another 2 hebdomads. The rats in N, NCn1, NCn2.5, and NCn5 groups were induced by neither DEN nor AAF, but they were gotten a individual intraperitoneal injection with maize oil to move as normal rats.


The rats in N and C groups had free entree to H2O. Each rat in NCn1, NCn2.5, NCn5, CCn1, CCn2.5, and CCn5 groups were severally given about 50 milliliters of 1, 2.5, 5, 1, 2.5, and 5 % w/v Cola nut infusion ( Cn ) daily utilizing H2O bottles. The Cola nut H2O infusion was given as a replacement for H2O to rats. While each rat in GL0.001, GL0.0025, and GL0.005 groups were given about 50 milliliters of 0.001, 0.0025, and 0.005 % w/v GL assorted with H2O daily, as an option to H2O. Except the two hebdomads period, from the 2nd hebdomad to the 4th hebdomad, in C, CCn1, CCn2.5, CCn5, GL0.001, GL0.0025, and GL0.005 groups ; all rats were given radical diet. Body weights were recorded every two hebdomads during the experience.

Post intervention

After 11 months from the beginning of the survey, rats were sacrificed. All rats were starved for 24 hours before being sacrificed. At the expiration of the experiment, the rats were weighed and complete necropsies were perfumed after the rats had been sacrificed by beheading under trichloromethane anaesthesia. The liver of each rat were washed in ice-cold 0.9 % NaCl solution every bit shortly as possible and their weight were recorded. A portion of liver tissues of each rat were sliced in 1 centimeter midst and were fixed in 10 % buffered formol to implant in paraffin blocks and procedure for TUNEL Assay. Besides a figure of 1 mm3 liver pieces were cut from each rat. They were fixed in 4 % Glutaraldehyde for 12 to 24 hours at 4 & A ; deg ; C to undergo the transmittal negatron microscopy. The left over tissues were kept in -80 & A ; deg ; C for Real clip quantitative RT-PCR.

DEN = Diethylnitrosamine ; 200 mg/ kilogram organic structure weight, intraperitoneal injection

AAF = 2-Acetylaminofluorene ; 0.02 % w/w, assorted with rat Zhou

Cn = Cola nut ; 1, 2.5, and 5 % w/v H2O infusion

GL = Glycyrrhizin ; 1, 2.5, and 5 mg/ 100 milliliter H2O

Figure 2.1. Modified survey protocol of Solt and Farber ( 1976 ) to analyze the consequence of Cola nut H2O infusion on rat ‘s liver during hepatocarcinogenesis.

Real-time Quantitative Reverse Transcriptase Polymerase Chain Reaction

RNA Extraction

The entire RNA was purified utilizing the Qiagen RNeasy® Mini Kit. Following collection 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 & A ; deg ; C instantly. RNA was isolated harmonizing to the Qiagen RNeasy® Mini Handbook demonstrated in Figure. In this survey, tissues were disrupted utilizing rotor-stator homogenizer. ?-Mercaptoethanol ( ?-ME ) was added to Buffer RLT before usage by adding 10 ?l ?-ME per 1 milliliter Buffer RLT. Buffer RPE is supplied as a dressed ore. Before utilizing for the first clip, four volumes of ethanol 100 % ( Merck ) were added to obtain a on the job solution. The concentration of gathered RNA was evaluated by NanoDrop spectrophotometer. RNA was kept at -80 & A ; deg ; C to utilize in Real-time RT-PCR at the same twenty-four hours of isolating.

Figure. Entire RNA purification harmonizing to Qiagen RNeasy® Mini Handbook.

Primer Design

Primer braces for AFP and ALB cistrons were designed utilizing World Wide Web tools. The Primer3 version 0.4.0 ( hypertext transfer protocol: // ) and the Primer-BLAST ( hypertext transfer protocol: // ) were used in this survey. The picked primers were performed a BLAST hunt to compare primer question sequences with database of sequences and measure the singularity of the question. Rattus norvegicus alpha-fetoprotein ( Afp ) , mRNA ( NCBI Reference Sequence: NM_012493.1 ) , and Rattus norvegicus albumen ( Alb ) , mRNA ( NCBI Reference Sequence: NM_134326.2 ) were used as the mention messenger RNA sequences ( Appendix A ) . Since fluorescence from SYBR Green I increases strongly upon binding of the dye to any double-stranded Deoxyribonucleic acid, it is peculiarly of import to minimise nonspecific primer tempering by careful primer design. For the highest efficiency in real-time RT-PCR utilizing SYBR Green I, marks should ideally be 60-200 bp in length.

Real-time Quantitative RT-PCR

Real-time Quantitative RT-PCR was conducted utilizing 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 rearward written text and PCR to take topographic point in a individual tubing, 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 H2O were stored at -20 & A ; deg ; C. Real-time Quantitative RT-PCR was carried out following the maker ‘s process as below:

2x QuantiFast SYBR Green RT-PCR Master Mix, templet RNA, primers, and RNase-free H2O were thawed. The single solutions were assorted and placed on ice. QuantiFast RT Mix should be taken from -20 & A ; deg ; C instantly before usage, ever kept on ice, and returned to storage at -20 & A ; deg ; C instantly after usage.

Reaction mix was prepared harmonizing to Table 1. The reaction mix was exhaustively assorted and appropriate volumes were dispensed into PCR vass or home bases.



Concluding concentration

2x QuantiFast SYBR Green RT-PCR Master Mix

12.5 ?l


Primer A


1 ?M

Primer B


1 ?M

QuantiFast RT Mix

0.25 ?l

Template RNA ( added at measure 4 )


?100 ng/reaction

RNase-free H2O


Entire reaction volume

25 ?l

Template RNA ( ?100 ng/reaction ) was added to the single PCR vass or Wellss incorporating the reaction mix.

Real-time cycler ( Rotor-Gene 6000, Corbett ) were programmed ( Table 2 ) . The PCR vass or home bases were placed in the real-time cycler and so the plan was run.




Reverse written text

10 min

50 & A ; deg ; C

PCR initial activation measure

5 min

95 & A ; deg ; C

Two-step cycling


10 s

95 & A ; deg ; C

Combined annealing/extension

30 s

61 & A ; deg ; C

Number of rhythms 35-40

Melting curve analysis of the RT-PCR merchandises was performed to verify their specificity and individuality.

Transmission Electron Microscopy

Transmission negatron microscopy was conducted harmonizing to the Hayat ( 1986 ) process. The liver tissues from rats were sliced into a figure of 1 mm3 pieces and set into separate phials. As a primary arrested development, the tissues were fixed in 4 % Glutaraldehyde for 12 to 24 hours at 4 & A ; deg ; C. The tissues were so washed with 0.1 M Sodium Cacodylate buffer 3 times of 10 infinitesimal each. Subsequently they were post-fixed in 1 % Osmium Tetroxide for 2 hours at 4 & A ; deg ; C and washed once more with 0.1 M Na cacodylate buffer for 3 alterations of 10 proceedingss each. The samples were dehydrated in a series of go uping acetone dilution as in ( Table 2 ) . The samples were so infiltrated with rosin and propanone mixture as in ( Table 2 ) .

Table 2. Dehydration and Infiltration







35 %

50 %

75 %

95 %

100 %

10 min

10 min

10 min

10 min

15 min ( 3changes )



100 % rosin

100 % rosin

1 Hour

2 Hourss


2 Hourss

The specimens were placed into beam capsules and filled up with the rosin for implanting, and so they were polymerized in an oven at 60 & A ; deg ; C for 24-48 hours. Semi-thin sectioning was carried out by ultramicrotome and utilizing glass knifes. The 1 µm thick subdivisions were placed onto glass slide and stained with Toluidine Blue. The interested portion of the specimen was spotted and selected by detecting the slides under the light microscope for ultrathin sectioning. The Toluidine Blue-stained slides were kept to be used in histological surveies and lesion marking.

Ultrathin segmenting was carried out on interested country. For ultrathin sectioning, the diamond knife is recommended. Then, the silver/gold subdivisions were picked up with a grid. Grids were dried utilizing filter documents. The dried grids were stained with Uranyl Acetate for 10 minute and washed with 50 % filtered intoxicant. After that, they were stained with Lead for 10 proceedingss and washed with dual distilled H2O. Following each measure the grids were dried with filter documents. The specimens were so examined under transmittal negatron microscope ( LEO ) .

Figure Liver tissue Transmission Electron Microscopy Method. Semi-thin slides was prepared and examined for histological surveies and lesion marking.


The DeadEnd™ Fluorometric TUNEL System ( Promega ) kit was used in this survey. The DeadEnd Fluorometric TUNEL System detects the disconnected Deoxyribonucleic acid of apoptotic cells by catalytically integrating fluorescein-12-dUTP at the 3?-OH terminals of the Deoxyribonucleic acid utilizing the enzyme Terminal Deoxynucleotidyl Transferase ( recombinant ) , which forms a polymeric tail utilizing the rule of the TUNEL ( TdT-mediated dUTP Nick-End Labeling ) assay.

The liver tissues from each rat were sliced in 1 centimeter midst pieces and they were fixed in 10 % formol for a few yearss. Then the tissue samples were embedded in paraffin blocks and were cut utilizing microtome in 4 to 5 micrometer. Pasted on slide samples were dried on a hot home base at 50-55 & A ; deg ; C for 30 proceedingss. The slide could be kept at room temperature before they were processed farther.

DeadEnd™ Fluorometric TUNEL System was conducted as stated by the maker ‘s direction ( Promega ) , which have been illustrated in Figure. . The fluorescein-12-dUTP labelled Deoxyribonucleic acid could be visualized straight by fluorescence microscopy. Observing and analysing the apoptotic cells under the fluorescence microscope were instantly done one time preparing of the slides had been finished. Samples were viewed under a fluorescence microscope utilizing a standard fluorescent dye filter set to see the green fluorescence of fluorescent dye at 520 ± 20nm ; position red fluorescence of propidium iodide at & A ; gt ; 620nm and bluish DAPI at 460nm. All the slides were examined. Five musca volitanss were indiscriminately selected at 20X magnification to carry on the systematic marking method. On each topographic point, green stained cells were counted as a TUNEL-positive programmed cell death cells.


Pretreatment of Paraffin-Embedded Tissue


Remove Paraffin

Wash slides twice in xylene, 5 proceedingss each wash



Immerse in 100 % ethyl alcohol for 5 proceedingss



Wash slides in diminishing concentrations of ethyl alcohol ( 100 % , 95 % , 85 % , 70 % , 50 % ) , 3 proceedingss each wash



Immerse in 0.85 % NaCl for 5 proceedingss



Immerse in PBS for 5 proceedingss


Apoptosis Detection



Immerse slides in 4 % methanal in PBS for 15 proceedingss



Immerse slides twice in PBS, 5 proceedingss each clip



Add 100?l of a 20?g/ml Proteinase K solution. Incubate at room temperature for 8-10 proceedingss.



Immerse slides in PBS for 5 proceedingss


Repeat Fix

Immerse slides in 4 % methanal in PBS for 5 proceedingss



Immerse slides in PBS for 5 proceedingss



Add 100?l Equilibration Buffer, Equilibrate at room temperature for 5-10 proceedingss



Add 50?l of TdT reaction mix to the tissue on an country, no larger than 5 square centimetres. Do non let tissue to dry wholly. Cover slides with Plastic Coverslips to guarantee even distribution of the mix. Incubate slides for 60 proceedingss at 37 & A ; deg ; C in a humidified chamber ; avoid exposure to visible radiation from this measure frontward.


Stop Chemical reaction

Remove Plastic Coverslips, immerse slides in 2X SSC for 15 proceedingss



Immerse slides three times in PBS, 5 proceedingss each clip



Immerse slides in 40ml of propidium iodide solution newly diluted to 1?g/ml in PBS for 15 proceedingss at room temperature in the dark in a Coplin jar


Wash the samples by

Immerse slides three times in deionized H2O for

5 proceedingss at room temperature


Drain off

Drain off extra H2O from the slides and pass over the country environing the tissue with tissue paper


Saddle horse

Add one bead of Anti-Fade solution to the country incorporating the treated tissue and saddle horse slides utilizing glass coverslips. Seal the borders and allow dry for 5-10 proceedingss.

Observe Under Fluorescent Microscope

Table. Fixing slides method harmonizing to DeadEnd™ Fluorometric TUNEL System ( Promega ) .

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