CHAPTER 3: METHODOLOGY
3.1 Study Design
This research is an experimental study which was conducted to investigate the effectiveness of treatment of Cassia alata as antioxidant effects in cardiovascular system of hyperglycemic rats. The antioxidant activity was tested from the leaf of Cassia alata using its aqueous extract. For this study, 30 Wistar rats weighing between 180 to 200g were used. They were housed in standard cages in a room with a 12 hour light/dark cycle and 50 to 60% relative humidity at a temperature of about 30°C. The animals had fed with standard food and water without limit. This study was conducted in two groups of streptozotocin (STZ) induced diabetic rats in which each group will consist of 15 rats. For the first group of STZ induced diabetic rats had fed with Cassia alata aqueous extract for 20 days meanwhile for the second group of STZ induced diabetic rats had fed with normal saline as a negative control for 20 days. The two groups of STZ induced diabetic had divided into 2 batches. First batch of diabetic rats was consisted of 6 rats for both groups while the second batch was consisted of 9 rats for both groups. All of the rats in two groups had fasted for 12 hour before induced diabetic via STZ injection. After an injection, the rats with level of fasting blood sampling (FBS) above 200 mg/dl will take for further investigation. Henceforth, the treatment was began in which the Cassia alata aqueous extract will administer orally after 48 hour diabetic induction done for every batch of two groups of induced diabetic rats. This process was completed within 20 days. The provision of treatment was done according to the group, i) the group of induced diabetic rats had given Cassia alata aqueous extract at dosage of 2ml/kg rats weight according to the batch

respectively, ii) the negative control group which also induced diabetic rats had given a normal saline at dosage of 2ml/kg rats weight as a negative control. After the period of treatment provision completed within 20 days, the rats were killed with chloroform and the blood was taken to check the level of blood glucose as well as the heart and aorta had dissected out to further investigation on biochemical parameter test which are determination of end product lipid peroxidation by malondialdehyde assay (MDA), total antioxidant scavenging assay, 1,1-diphenyl-2-picrylhydrazyl (DPPH) and antioxidant catalase activity (CAT).

Figure 3.1 shows the schematic diagram of the study design flowchart.
Sampling preparation of Cassia alata leaf

Preparation aqueous extract of Cassia alata leaf

Induced diabetic by injection of streptozotocin (STZ)

Checking blood glucose level after 2 days
15 hyperglycemic rats treat with normal saline for 20 days

15 hyperglycemic rats treat with Cassia alata leaf aqueous extract for 20 days

Rat surgery to obtain heart, aorta and blood

Total antioxidant assay (DPPH free radical scavenging activity)

Checking blood glucose level after treatment
Estimation of antioxidant catalase activity

Determination of lipid peroxidation (MDA assay)

Statistical analysis (Independent t-test)

Figure 3.1: Study Design Flowchart

3.2 Materials
Streptozotocin (STZ), 1,1,3,3-tetramethoxypropane (TMOP), 1,1-diphenyl-2-picrylhydrazyl (DPPH), Ascorbic acid, Thiobarbituric acid (TBA), Thiochloroacetic acid (TCA) was obtained by Sigma Chemical Company, St Louise, USA. Copper Sulphate Pentahydrate (CuSO4.5H2O), Folin-phenol reagent, Hydrochloric acid (HCl) was obtained from Ajax Chemical, Auburn, Australia; Phosphate buffer, Potassium chloride (KCl), Sodium carbonate (Na2CO3), Sodium citrate and Sodium hydroxide (NAOH) was obtained from ICN Biomedicals, Inc., Ohio, USA; Sodium potassium tartarate (KNaC4H4O6) were obtained from Merck, Darmstadt, Germany; Sucrose, albumin serum bovin (BSA) was obtained from Applichem, Darmstadt, Germany; Chloroform, Citric acid and Methanol was obtained from BDH Laboratory Supplies, Poole, England.
3.3 Apparatus and Laboratory Equipments
Centrifuge (MR 22i Joan, France), dissection set (Draper UK), glucometer (US, Roche Diagnostics), frozen ice box (VX350 Joan, France), spectrophotometer (Zuzi Model 4201/20), vortexs (Stuart Scientific, UK), pH meter (MP 220 Mttller-Toledo, Switzerland), homogenizer (HumanLab Instrument Co, South Korea), magnetic stirrer (IKA C-MAC HS7), syringe 1ml (Terumo, Japan), weighing scale (Shimadzu Corporation, Japan), water bath (BU-420 Yih Der, Taiwan), Whatman No. 1 filter paper (Kubota Corporation, Japan), Waring Blender (Waring Product Division Torrington City, USA).
3.4 Preparation of Materials and Chemical Reagent
3.4.1 50mg/ml of Streptozotocin Solution
The preparation of STZ solution to induced diabetic was done by diluted 200mg of STZ into 4ml of normal saline 0.9%.
3.4.2 Materials of Organ Homogenate
3.4.2.1 KCI-Sucrose Buffer Solution
0.1M phosphate buffer pH 7.5 was prepared by diluted with 500ml of distilled water. 85.6g of sucrose 0.25M and 11.5g of KCI 0.154M were diluted into the phosphate buffer solution until evenly. Distilled water was added to make a final volume of solution as much as 1 L.
3.4.3 Materials of Protein Concentration
3.4.3.1 Copper Sulphate Pentahydrate Solution (CuSO4.5H2O)
0.2625g of CuSO4.5H2O was added into distilled water and was diluted to makes the final volume of solution as much as 25ml.
3.4.3.2 Sodium Hydroxide Solution
37.5g of NaOH was added into distilled water and was diluted until reach a final volume of 100ml.
3.4.3.3 Biuret Reagent
25ml of CuSO4.5H2O solution was mixed with 100ml of NaOH solution. The mixtures were stirred until evenly.
3.4.3.4 Natrium Chloride Solution
0.8766g of NaCI was dissolved into distilled water until reach a final volume of 100ml. The solution was stirred until evenly by using magnetic stirrer.
3.4.3.5 Solution of Standard Protein 100µg/ml
0.01g of BSA was dissolved into 100ml of 0.15M NaCI solution. The mixtures were stored at 4°C and were used as a stock solution.
3.4.4 Preparation of Materials for Estimation of Total Antioxidant Activity (DPPH Free Radical Scavenging Activity)
3.4.4.1 Methanolic Solution of 1, 1-diphenyl-2-picrylhydrazyl (DPPH)
0.05g of DPPH was dissolved into 100ml of methanol. The solution was stirred until evenly.
3.4.4.2 Standard Solution of Ascorbic Acid
1.7613g of ascorbic acid was diluted with 10ml of distilled water. The solution was stirred until evenly.
3.4.5 Preparation of Materials for Estimation of Antioxidant Catalase Activity.
3.4.5.1 Dichromate Acetic Acid Reagent
1ml of potassium dichromate was diluted with 4ml of acetic acid and the mixtures were let to dissolve evenly.
3.4.6 Materials for Determination of Lipid Peroxidation Activity
3.4.6.1 Solution of Acid Hydrochloric 0.1N
1ml of HCI was diluted with 100ml of distilled water. The solution was stirred until evenly.
3.4.6.2 Thiochloroacetic Acid Solution
20g of TCA was dissolved into 100ml of distilled water. The solution was stirred until evently.
3.4.6.3 Thiobarbituric Acid Solution
670mg of TBA was dissolved into 100ml of distilled water and the solution was stirred until evenly.

3.5 Procedures
3.5.1 Sampling of Plant Material
The leaf of Cassia alata plant was collected at Dengkil for aqueous extraction procedures. Firstly, the freshly collecting plant was washed and rinsed thoroughly with a distilled water to removed any dirt and unwanted particles and was dried at temperature of 40°C for about seven days to make sure the plant of Cassia alata will truly dry (until reaching a constant weight). The plant was not dried under the sun because to protect and do not lose some of the biochemical constituents of the plant. Afterwards, the plant materials of Cassia alata were pounded into smaller particles by using mortar and pestle and then were grounded to fine powder by using an electric blender. After that, the powder samples of Cassia alata leaves were kept in cellphone bags or air-tight container and will be stored in laboratory freezer at temperature of 4°C until being use.
3.5.2 Preparation of Plant Extraction
For aqueous extraction procedure, 200g of Cassia alata powder was mixed with sterile distilled water in two litre beakers. The mixture was boiled by using hot plate for one hour and thirty minutes and then mixed well using magnetic stirrer for obtaining the maximum dissolution. Then, the mixture was left to cooling to 40°C and the suspension was filtered through a cheese cloth. To get more refined filtration, the liquid was filtered again by using Whatman no.1 filter paper. In order to obtain a sterile pure aqueous extract stock of Cassia alata, the resulting filtrate was left to evaporate until final volume of 400ml is reached. The sterile pure aqueous extraction stocks of Cassia alata were stored in the laboratory refrigerator until being used.
3.5.3 Preparation of Induction of Diabetes

Before the induction of STZ, the rats were fasting overnight for 12 hours. Then, STZ solution was injected through tail vein of the rats after 10 minutes of STZ preparation. The level of blood glucose or fasting blood sugar (FBS) was measured after 48 hour by using glucometer. The rats with level of FBS above 200 mg/dl were taken for further investigation.

3.5.4 Preparation of Organ Homogenate
The preparations of organ homogenate were determined according to the method of Noori et al. (2009). The organs, which are heart and aorta were taken out first from the -80°C temperature to let it warm for easily removing them from tube. The organs were cut of 0.5g and were diluted with 4ml of KC1 buffer. After that, the organs were homogenized by using tissue homogenator until creamy formed produced. The organs were then centrifuge at 600g for 60 minutes. The supernatants formed were taken and kept in freezer at temperature of -80°C until being used.
3.5.5 Determination of Protein Concentration in Heart and Aorta
Protein assay was determined according to the method of Itzhaki and Gill (1964). For standard curve of protein, 0.1ml of sample of tissue homogenate was diluted 100 times with 9.9ml of KCI buffer. Then, 1ml of distilled water, standard BSA and tissue homogenate was added into test tube of blank, standard and sample. Next, 4ml of biuret reagent was added into the tubes and was mixed well by vortex mixer. After that, the mixtures were let to stand for 20 minutes in room temperature. The value of absorbance for each tube was measured against a blank at 540nm and each assay was repeated three times (triplicate). The concentration of protein was determined directly from a standard curve of bovine serum albumin at range of 20µg/ml to 100µg/ml. The equation was used to determine the concentration of protein in tissue homogenate (mg/ml).
Protein of tissue homogenate = Protein (µg/ml) x 10-3 x dilution factor (mg/ml)

3.5.6 Determination of Lipid Peroxidation Activity by Malondialdehyde (MDA) Assay
The lipid peroxidation activity was determined by the formation of malondialdehyde (MDA)-thiobarbituric acid reactive substrates (TBARS) adduct according to the method of Ledwozyw et al. (1986). The production of malondialdehyde serves as an index of lipid peroxidation and give a pink coloured reaction once react with thiobarbituric acid (TBA) when having a maximum absorption at 535nm. The MDA standard curve was freshly prepared by diluting the second stock solution of MDA. For the first stock solution, 4.0mM stock MDA was prepared by hydrolyzing 9.6µl (8.812mg) of 1,1,3,3-tetramethoxypropane in 10ml of 0.1N HCI in 100°C for 15 minutes. Then, standard solution was prepared by 100µl of stock solution was diluted in 10ml of KCI buffer which corresponds to 40µM or 40nmoles/ml MDA. The second stock was prepared into five different concentrations (0 to 2.0 nmol/ml) by diluting with KCI buffer. 0.1ml of organ supernatant was prepared into test tubes. Then, 0.4ml of distilled water was added follow by the addition of 2.5ml of TCA. The mixtures were left at room temperature for 15 minutes. After that, 1.5ml of TBA was added into the mixtures and vortex them for 15 seconds. Next, the tubes containing the mixtures were placed in water bath for boiling at 100°C for 15 minutes until a pale pink coloured obtained. After cooling, 4ml of n-butanol was added and then vortex them for 15 seconds. Then, the mixtures were centrifuged at 3000rpm for 10 minutes. The values of absorbance were then measured by using spectrophotometer at 532nm and each assay was done three times (triplicate). The lipid peroxidation activity was calculated by using the formula.
Lipid peroxidation activity MDA (nmol/ml) x diluting factor (nmol/mg protein) = Protein (mg/ml)

3.5.7 Determination of Total Antioxidant Assay (DPPH Free Radical Scavenging Activity)
The total antioxidant assay (DPPH free radical scavenging activity) of organ homogenates were determined according to the Blois (1985) method. This method was assessed on the basis of the radical scavenging effect of the stable 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical activity. DPPH play a role in accepts an electron or hydrogen radical to become a stable molecule. DPPH in an absolute ethanol appear a deep violet and shows a strong absorption band at 517nm. Ascorbic acid solution was freshly prepared by diluting 1.7613g of ascorbic acid in 10ml of sterile distilled water. Then, the ascorbic acid was subjected to serial dilution up to 1:32. In this method, ascorbic acid was used as the positive control (standard antioxidant). In order to make DPPH methanol solution, 0.05g of DPPH powder was freshly prepared by dissolved it in 100ml of methanol. DPPH methanolic compound were then mixed with serial dilution of organs homogenate as well as ascorbic acid. The mixture then was left for 30 minutes at room temperature in dark condition to allow the reaction to take place. The value of absorbance were then measured at 517nm on a spectrophotometer and each assay was repeated three times (triplicate). The capability to scavenge DPPH radical was calculated by using the following equation.
Antioxidant Activity (mg/ml) = (A control – A sample) A control

*Antioxidant activity was expressed in terms of inhibition of DPPH free radical concentration and was calculated by using the formula:

*A control = 1ml of DPPH methanol solution + 1 ml methanol *A sample = 1ml of DPPH methanol solution + 1ml of organs homogenate

3.5.8 Estimation of Antioxidant Catalase Activity
The activity of antioxidant catalase was assayed by using the method of Sinha (1972). Dichromate in acetic acid was reduced to chromic acetate, when heated in presence of hydrogen peroxide with the formation of per chromic acid as an unstable intermediate. Catalase was allowed to split H2O2 for different periods of time. The reaction was stopped at different time intervals by the addition of dichromate acetic acid mixture and the remaining H2O2 was determined by measuring chromic acetate calorimetrically after heating the reaction mixture.
0.9 ml of phosphate buffer, 0.1 ml of homogenate and 0.4 ml of H2O2 were added. The mixtures were let for 1 minute in order to reaction take place. Next, 2 ml of dichromatic acetic acid reagent was added into the mixtures. The tubes were then kept in a boiling water bath for 10 minutes. After the mixtures were cooled and the green color was developed, the value of absorbance was read at 530 nm. A standard of antioxidant catalase was prepared in the concentration range of 0 to 400 µg/ml. The activity of antioxidant catalase was expressed as µg/ml of tissues homogenate.
3.5.9 Statistical Analysis
Data will be analyzed using the SPSS. Results will be express as mean ±SEM. Statistical analysis will be done using Independent t-test to determine the significant differences among the variable across three tests. If the differences, P < 0.05, it is considered as significant.

3.6 Project Flowchart Figure 3.2 shows the schematic diagram of the Project Flowchart.
Fasting the rats for 12 hours
Preparation of the aqueous extract of Cassia alata leaf
Sample collection of Cassia alata leaf

Induced diabetic by injection of streptozotocin (STZ)
Fed up the hyperglycemic rats with Cassia alata leaf aqueous extract for 20 days

Checking the fasting blood sugar (FBS)

Dissect out the heart and aorta

Killed the hyperglycemic rats with chloroform
Checking the blood glucose level

Measure the end product of lipid peroxidation by malondialdehyde (MDA) assay

Preparation of homogenate process of the heart and aorta

Determination of protein concentration

Analyze the data
Collect the data
Estimation of antioxidant catalase activity
Determination of total antioxidant scavenging assay, 1,1-diphenyl-2-picrylhydrazyl (DPPH)

Figure 3.2: Project Flowchart