INVESTIGATING THE POSSIBLE MODULATORY EFFECTS OF FIVE DIFFERENT

POLYHERBAL MIXTURES IN HIGH FRUCTOSE-FED, STREPTOZOTOCIN-

INDUCED TYPE 2 DIABETIC MALE WISTAR RATS

A RESEARCH PROPOSAL

Submitted to

Babcock University Health Research Ethics Committee

By

The Department of Biochemistry, College of Health and Medical Sciences, Benjamin.S .Carson

School of Medicine, Babcock University, Ilishan-Remo, Ogun State, Nigeria

MARCH, 2017

1
 CHAPTER ONE

INTRODUCTION

Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia resulting from

defects of insulin action, insulin secretion or both, which ultimately results in resulting in

hyperglycemia (ADA, 2009). It has become one of the most important diseases worldwide,

reaching epidemic proportions. Global estimates predict that the proportion of adult population

with diabetes will increase 69% for the year 2030 (Shaw et al., 2010). The major goals in the

treatment of diabetes is to keep blood glucose concentration or level within acceptable limits

thereby reducing the risk of long term complications. The use of polyherbal mixtures, derived

from medicinal plants and minerals which are used for the treatment of different chronic diseases

like diabetes are becoming accepted, for the treatment of certain metabolic disorders, both in

developing and developed countries due to their natural origin and perceived lower toxicity.

More than three-quarter of the world’s population is gradually diverting to herbal medicine due

to its ability to reduce health issues at a cheaper rate and the trend is increasing globally

(Oreagba et al., 2011).

Polyherbal mixtures continue to expand rapidly across the world with many people now

resorting to these products for treatment of various health challenges, however, as there are not

enough evidence produced by common scientific approaches to answer questions of safety and

efficacy about most of the herbal medicines now in use, it is important that these mixtures be

2
scrutinized for their efficacy and safety through clinical research in herbal mixtures,

development of simple bioassays for biological standardization, pharmacological and

toxicological evaluation as well as the development of various animal models for toxicity and

safety evaluation (Pirart , 2004). It is also important to establish the active components of these

herbal extracts.

Therefore, in the present study, the in vivo modulatory effects of selected poly herbal mixtures

will be investigated for their antiglycemic effects in high-fructose-fed Streptozotocin-induced

diabetic experimental male Wistar rats.

3
OBJECTIVES OF THE STUDY

The objectives of this study are enumerated below;

1. To investigate the phytochemical constituents and active compounds using gas

chromatography/ Mass spectrometry (GC/MS) present in Fijk flusher, Yoyo bitters,

Oroki herbal mixture, Fidson Bitters and Ruzu Bitters (FYOFR) in vitro.

2. To investigate the effects of polyherbal mixtures (FYOFR) on biochemical parameters

such as plasma glucose level, total cholesterol, total triglycerides, high density

lipoprotein-cholesterol (HDL cholesterol), low density lipoprotein cholesterol (LDL

cholesterol), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and

histopathological parameters in high fructose-fed-Streptozotocin-induced diabetes in

male Wistar rats.

3. To investigate the effect of polyherbal mixtures (FYOFR) on oxidative stress biomarkers

and antioxidant status histopathological parameters in high fructose-fed-Streptozotocin-

induced diabetes in male Wistar rats.

4. To investigate the effect of polyherbal mixtures (FYOFR) on pancreas and liver

histopathology in high fructose-fed-Streptozotocin-induced diabetes in male Wistar rats.

4
JUSTIFICATION OF STUDY

Long term hyperglycemic condition is associated with damage and failure of many organs such

as eyes, kidney, nerves, heart, and blood vessels, (Ewing et al; 1993). The aim of many anti

diabetic drugs is to keep the blood sugar at acceptable levels in order to prevent damage of other

organs, however many of these drugs are associated with serious side effects such as

hypoglycemia, flatulence, diarrhea, and weight gain. Also they are often unable to lower

glucose concentrations to within the normal range, or to reinstate a normal pattern of glucose

homeostasis (Senthilvel et al., 2006). Many polyherbal mixtures claim to be able to lower

blood sugar without the added risks of various side effects as seen in the case of synthetic drugs,

however, the lack of standardization coupled with the existence of little or no empirical data to

support the medicinal claims of these mixtures has raised major concerns on the use of the

polyherbal mixtures and although they can be considered safe, some are known to be toxic at

high doses and others may have potentially adverse effect after prolonged use, (Abbas, 2006).

Today, numerous traditional plant treatments for diabetes have been reported. However, only

small quantities of these have been scientifically scrutinized and have undergone medical

evaluation to assess their therapeutic efficacy. The World Health Organization Expert Committee

on diabetes has recommended that traditional medicinal polyherbal mixtures be further

investigated (WHO, 2005). Thus, this study hopes to verify these claims, hence provide scientific

backing for such claims as the elucidation of such claims could provide a major alternative

therapy for the diabetic condition.

5
 CHAPTER TWO

LITERATURE REVIEW

POLYHERBAL MIXTURES

Herbal medicines that are also called botanical medicines or phytomedicines refer to herbs,

herbal materials, herbal preparations and finished herbal products that contain parts of plants or

other plant materials as active ingredients (WHO, 2008). According to the World Health

Organization (WHO), herbal medicines among the general population are medications prepared

from one or more herbs or plant parts including roots, stem, bark, seeds and leaves (WHO,

2013). There are many different types of polyherbal mixtures such as Yoyo Bitters, Swedish

Bitters, Fijk flusher, Osomo, Alomo, and Oroki amongst others available in Nigeria.

YOYO CLEANSER BITTERS

Yoyo cleanser bitters is a polyherbal mixture produced by Abllat Company Nigeria Limited

located in Lagos, Nigeria. The mixture usually comes in a 200ml bottle which comprises of Aloe

Vera, Acinoss Arvensis, Citrus Aurantofolia, Chenopodium Murale and Cinamomum Aromticum

and was formulated to help scavenge free radicals in the body and help remove harmful toxins in

the body, thereby supporting the immune system and the body’s ability to resist disease. The

supplement herbal product is said to be enriched with water soluble vitamins and minerals such

as vitamins B1, B2, B3, B6 & B12. It also has minerals such as copper, zinc & Iron (Ablatt,

2015).

6
 According to the manufacturers, the mixture helps many systems in the body such as the

Urinary and Excretory system, The Circulatory System, Nervous system.e.t.c. It has been

acclaimed to have several benefits from dissolving existing kidney stones and preventing the

formation of new ones to aiding blood circulation, facilitating blood pressure control as well as

eliminating cholesterol, sugar, triglycerides.e.t.c (Ganong, 2003).

Figure 1: Yoyo Cleanser Bitters ([Link], 2015)

7
FRUCTOSE METABOLISM

Fructose is a simple ketonic monosaccharide found in many plants, where it is often bonded to

glucose to form sucrose, a disaccharide. It is absorbed directly into the bloodstream during

digestion. Pure, dry fructose is a very sweet, white, odorless, crystalline solid and is the most

water-soluble of all the sugars (Vasdev et al., 2004).

Figure 2: D-fructose (Nelson et al., 2013).

There is research indicating that excessive fructose consumption is a cause insulin resistance,

obesity, elevated low density lipoprotein cholesterol and triglyceride, leading to yielding type 2

diabetes and cardiovascular disease (Elliot et al., 2002). A recent epidemiological analysis in

humans also found an association between diabetes prevalence and sugar availability (Vasdev et

al., 2004). Utilization of fructose in the body is reliant on fructose phosphorylating enzymes.

Hexokinases are enzymes that phosphorylate hexose sugars generally, fructose included.

Fructokinase phosphorylates fructose specifically. In muscle hexokinase phosphorylates fructose

to fructose 6-phosphate, a direct glycolytic intermediate (Nelson et al., 2013). However, the

affinity of hexokinase for fructose is relative low because of the non-specificity of hexokinase

coompared to fructokinase. The major hexokinase found in pancreatic β-cells is glucokinase, this

means fructose can not be adequately metabolized in the pancreas because glucokinase is

specific to glucose and not fructose (Devlin, 2010). Again, the liver expresses majorly

glucokinase which is highly specific to glucose as its substrate. However, there is need for

8
fructokinase in order to deliver fructose into hepatic glycolysis (Harvey et al., 2011). Hepatic

fructokinase phosphorylates fructose to yielding fructose 1-phosphate which is hydrolyzed by a

specific aldolase (Nelson et al., 2013). Humans express three major forms of aldolases; aldolase

A, aldolase B and aldolase C. Aldolase A is expressed in most tissues. Aldolase A catalyses the

hydrolysis of fructose 1, 6-bisphosphate into dihydroxyacetone phosphate and glyceraldehyde 3-

phosphate, this reaction is reversible and is useful for glycolysis and gluconeogenesis (Harvey et

al., 2011). Aldolase B is expressed primarily in the liver. Aldolase B catalyzes the hydrolysis of

both fructose-1, 6-bisphosphate and fructose 1-phosphate. Aldolase C is primarily expressed in

the brain. In the liver, aldolase B can catalyze both fructose-1, 6-bisphosphate and fructose 1-

phosphate as substrates (Michael, 2016). So, when presented with fructose 1-phosphate the

enzyme yields dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. The

dihydroxyacetone phosphate is converted to glceraldehyde 3-phosphate by the action of triose

phosphate isomerase, yielding two molecules of glceraldehyde 3-phosphate which enters

glycolysis (Koolman et al., 2005).

9
Figure 3: Entry of fructose into the glycolytic pathway in hepatocytes, kidney and small

intestine (Michael, 2016).

10
STREPTOZOTOCIN

Streptozotocin (STZ) (2-deoxy-2-(3-methyl-3-nitrosourea)-1-D-glucopyranose) is composed of

nitrosoureas moiety with a methyl group attached at one end and a glucose molecule at the other

end. STZ is a cytotoxic glucose analogue .i.e it is a naturally occurring chemical that is toxic to

the insulin-producing beta cells of the pancreas. It is produced by the soil bacterium

Streptomyces achromogenes, which exhibits broad spectrum of antibacterial properties. The

selective toxicity of STZ to beta cells occurs because of its preferential accumulation in beta

cells through uptake via GLUT2 glucose transporter. Insulin dependent diabetes mellitus can be

induced by either single high dose or multiple low- dose STZ injections (Ganda et al., 1976). At

low dose, STZ induces pancreatic beta-cell apoptosis and at high dose it causes necrosis. STZ is

also used in medicine for various purposes such as treating certain cancers of the islet of

Langerhans and used in medical research to produce animal model for hyperglycemia in a large

dose a also Type 2 Diabetes or Type 2 Diabetes in low doses. STZ can also cause carcinogenesis

and renal, hepatic and muscle myoblast toxicity, these side effects can be avoided by using lower

doses (Gul et al., 2002).

11
MECHANISM OF STREPTOZOTOCIN

Streptozotocin is a glucoasmine-nitrosourea compound. Just like every other alkylating agents in

the nitrosoureas class, it is toxic to cells by causing damage to the DNA. DNA damage induces

activation of poly ADP-ribosylation a process that is more important for the diabetogenicity of

streptozotocin than DNA damage itself (Som et al., 2001). Poly ADP-ribosylation leads to

depletion of cellular NAD+ and ATP, further reduction of the ATP content and subsequent

inhibition of insulin synthesis and secretion. STZ is similar to glucose in structure. Therefore can

be easily mistaken as glucose by the cell and its by pancreatic β-cells via the GLUT 2 transporter

where it causes β-cell death by DNA fragmentation due to the nitrosourea moiety. Three major

pathways associated with cell death are:

 DNA methylation through the formation of carbonium ion (CH 3+) resulting in the

activation of the nuclear enzyme poly ADP-ribose synthetase as part of the cell repair

mechanism and consequently, NAD+ depletion;

 Nitric oxide production

 Free radical generation as hydrogen peroxide ( Reactive Oxygen Species)

DNA METHYLATION

An explanation of the cell death that results from STZ induction is due to the methylating

activity of the methylnitrosourea moiety of STZ especially at the O 6 position of guanine, leading

to DNA damage with resultant necrosis of the pancreatic beta cells, through the depletion of

cellular energy stores (Piepper AA). The resultant activation of polyADP-ribose polymerase

(PARP), in an attempt to repair the damaged DNA, depletes the cellular NAD + and consequently,

12
ATP stores as a result of overstimulation of DNA repair mechanisms. Although STZ also

methylates proteins, this DNA methylation is most responsible for beta cell death, though STZ

methylation of proteins could also contribute to its toxicity to the beta pancreatic cells. STZ

could also react at other sites of DNA such as the ring nitrogen and exocyclic oxygen atoms of

DNA bases, predominantly producing 7-methylguanine, 3-methyladenine which leads to DNA

breaks, activates poly-ADP-ribose polymerase and subsequently depletes NAD + (Stauffacher et

al., 1970).

NITRIC OXIDE PRODUCTION

Another possible mechanism of the diabetogenic action of streptozotocin that results in cell death

has been attributed to its ability to act as nitric oxide (NO) donor in pancreatic cells which

inhibits aconitase activity, leading to DNA alkylation and damage (Friederich et al., 2009).

Streptozotocin has been shown to increase the activity of guanyl cyclase and the formation of

cGMP, which are characteristic actions of NO. β-cells are particularly sensitive to damage by

nitric oxide and free radicals because of their low levels of free radical scavenging enzymes (GA

et al., 1999).

REACTIVE OXYGEN SPECIES

Oxidative stress has recently been shown to be responsible for pancreatic β-cell dysfunction

caused by glucose toxicity in hyperglycemia. Several reaction mechanisms are thought to be

involved in the genesis of oxidative stress in both diabetic patients and diabetic animals and they

include: glucose auto-oxidation, protein glycation, formation of advanced glycation production

13
(Hunt et al., 1990). During these processes reactive oxygen species are produced and cause

tissue damage. STZ treatment causes significant increase in malonaldehyde but decreases

antioxidant enzymes such as: catalase, glutathione peroxidase and superoxide dismutase

activities when compared with control animals in experiments (Matsuoka et al., 1998).

Decreases in antioxidant activities, and simultaneous increases in malonaldehyde (MDA)

activities, indicate the susceptibility of pancreas to STZ’s induction of oxidative stress (Gul et

al., 2002).

14
 CHAPTER THREE

METHODOLOGY

Selection of Poly herbal mixtures

The polyherbal mixtures, Yoyo bitters®, Oroki herbal mixture®, Fijk Flusher®, Ruzu Bitters®

and Fidson bitters® were purchased at XXXXXX, Nigeria.

Reagents and Chemicals

Hexane, Dimethyl Sulphoxide (DMSO), Quarcetin, Aluminium chloride, Potassium acetate,

Ethanol, Gallic acid, Folin Ciocalteu reagent, Sodium bicarbonate, Tannin, Vanillin,

Hydrochloric acid, Methanol, Acetic acid, Aluminium hydroxide, Saponin standard,

Tetraoxosulphate(VI) acid, D-Fructose (Burgoyne, India), Streptozotocin (Sigma Aldrich,

Germany), Glibenclamide (Vapi Care Pharma, India), Metformin (Shine Pharmaceuticals, India),

Carboxyl methylcellulose (Divine Bakers, Nigeria), Sodium Citrate, Citric acid, Aspartate

Transaminase (Randox kit), Alanine amino Transferase (Randox kit), High Density Lipoprotein

(Randox kits), Total Cholesterol (Randox kits), Total Triglycerides (Randox kits). All other

chemicals and reagents to be used will be of analytical grade.

Fractionation/Extraction of Polyherbal mixtures

The fractionation process will be carried out according to the method described by Onyeaghala et

al. (2015) with slight modifications. 2 x 200ml of the each of the bitters will be exhaustively

extracted using n-hexane. The extraction will be carried out in a ratio of 1:1 (Bitters: hexane).

15
The mixture of bitters and hexane will be put into a separating funnel, shaken well together and

allowed to stand for about 45 minutes to ensure complete extraction of the non-polar components

of the bitters. The two resultant fractions (Polar and Non-polar fractions) will be collected

separately, concentrated at 40°C under reduced pressure using a rotary evaporator, dried in a hot

air oven at 40°C and stored in a refrigerator until further use. Each of these fractions as well as

the whole polyherbal mixture (concentrated at 40°C under reduced pressure using a rotary

evaporator) will be analyzed via Gas chromatography-Mass Spectrometry as well as via

phytochemical analytic methods as the case may be.

Gas Chromatography- Mass Spectrometry (GC/MS) Analysis of the Polar and Non- Polar

Fractions of the various Polyherbal Mixtures

GC-MS analyses of the polar and non-polar fractions of the various bitters will be carried out at

the Basic Research Department of the National Research Institute for Chemical Technology,

Zaria, Kaduna using a QP2010 PLUS GCMS. Initial temperature of 80°C will be held for 1min

and gradually increased to a final temperature of 280°C at a rate of 10°C/min. 1µl of the various

fractions of the various bitters will be injected. The temperature of the heater will be set at

250°C, pressure at 108.0kPa, carrier gas (Helium, 99.999% purity, flow rate= 1.58ml/min,

average velocity=46.3cmsec-1). The constituent compounds will be identified by comparing their

retention times and mass weights with those of known compounds obtained by GC as well as

mass spectra stored in the library of the National Institute of Science and Technology.

16
Quantification of the Phytochemical Components of Various Bitters

The various bitters will be analyzed for their polyphenol, flavonoid, tannin, saponin and alkaloid

constituents quantitatively using standard procedures.

Determination of Total Polyphenol Content

The total polyphenol content of the polar and non polar fractions of the various bitters will be

estimated using the Folin Ciocalteu method as described by Obaseki et al. (2016).

Determination of Total Flavonoid Content

The total flavonoid content of the polar and non polar fractions of the various bitters will be

determined by the aluminium chloride colorimetric method as described by Soladoye &

Chukwuma. (2012).

Determination of Tannin Concentration

The Tannin content of the various bitters will be determined by the modified vanillin-HCl

methanol method as described by Noha et al. (2011).

17
Determination of Total Saponin Content

Total Saponin content of the various bitters will be determined by the vanillin-sulphuric acid

colorimetric method as described by Makkar et al. (2007) and modified by Senguthuvan et al.

(2014).

Determination of Alkaloid Content

The total alkaloid content of the bitters will be determined via gravimetric method as described

by Asika et al. (2016).

ANIMAL STUDIES

Experimental Animals

Fifty four (54) male albino rats of the Wistar strain of weights 120-180g will be purchased from

Babcock University animal house, Ilishan Remo, Ogun State. They will be kept in aerated plastic

cages at ambient temperature and humidity with a 12-hour light-dark schedule at the Babcock

University Animal House and acclimatized for two weeks. They will be placed on a rat pelleted

diet and water ad libitum. An ethical approval will be obtained from the Babcock University

Health Research Ethics Committee (BUHREC). All the animal experiments and protocol will

conform to the guidelines of National Institute of Health (NIH, 2000) for laboratory animal care

and use.

18
Induction of Diabetes and Treatment Plan

54 rats will be divided into nine groups of 6 rats per group. All the rats except those in the

Positive control will be induced with diabetes type 2 with 20% fructose in drinking water ad

libitum for two weeks followed by single intraperitoneal injection of Streptozotocin (40mg/kg

body weight) dissolved in citrate buffer with a pH 4.4 (Wilson & Islam, 2012). All treatment

will be administered via the oral route for a period of 10 days. The clinical drugs i.e. Metformin

and Glibenclamide will be administered at therapeutic dose whereas the various bitters will be

administered according to the manufacturer’s prescription for adult humans.

Experimental Design

Group 1: 0.5% Carboxy methyl cellulose (1ml/kg) body weight only.

Group 2: Fructose (20%w/v) + Streptozotocin (40mg/kg) + 0.5% Carboxy methyl cellulose

(1ml/kg) body weight only.

Group 3: Fructose (20%w/v) + Streptozotocin (40mg/kg) + Glibenclamide (0.071mg/kg) body

weight only.

Group 4: Fructose (20%w/v) + Streptozotocin (40mg/kg) + Metformin (7.14mg/kg) body weight

only.

Group 5: Fructose (20%w/v) + Streptozotocin (40mg/kg) + Fijk Flusher® (0.2ml/kg) body weight

only.

Group 6: Fructose (20%w/v) + Streptozotocin (40mg/kg) + Yoyo Bitters® (0.42ml/kg) body

weight only.

19
Group 7: Fructose (20%w/v) + Streptozotocin (40mg/kg) + Oroki Herbal Mixture® (0.86ml/kg)

body weight only.

.Group 8: Fructose (20%w/v) + Streptozotocin (40mg/kg) + Fidson Bitters® (0.43ml/kg) body

weight only.

.Group 9: Fructose (20%w/v) + Streptozotocin (40mg/kg) + Ruzu Bitters® (0.43ml/kg) body

weight only.

SACRIFICING OF ANIMALS

Rats will be made to fast overnight following the last administration and will be euthanized by

cervical dislocation before being sacrificed. Blood will be collected by cardiac puncture into

different sample tubes for biochemical investigations including liver function tests (AST and

ALT determination) and lipid profiles. Plasma as well as serum as the case may be will be

separated after centrifugation at 4,200 rpm at room temperature for 5 minutes. The liver and

pancreas will be carefully excised, cleared of adhering tissues, and weighed. Weight will be

recorded in grams and expressed as g/kg body weight. A small portion of the excised liver and

pancreas will be fixed in 10% formaldehyde and subsequently prepared for histology. The

remaining portion of the excised liver and pancreas will be weighed and homogenized in four

volumes of 100 mM of phosphate buffer (pH 7.4). The plasma, serum and liver and pancreas

homogenate obtained from each animal will be then analyzed to assess selected biochemical

parameters.

20
PHYSIOLOGICAL MEASUREMENT

The body weights of the rats will be measured on Day 1, 4, 7 and 10 of treatment as well as upon

final fructose feeding.

BIOCHEMICAL PARAMETERS OF INTEREST

Blood Glucose Estimation

Blood samples from the experimental animals will be analyzed for estimation of blood glucose

levels using an Accu-Check active Glucometer. This will be done prior to start of dietary

manipulation, at the end of two weeks of dietary manipulation, after Streptozotocin injection (to

confirm diabetes) and on days 1, 4, 7 and 10 of treatment.

Principle

When blood is dropped on the yellow squared spot on the test strips inserted inside the

Glucometer, glucose in the blood reacts with the chemical reagent on the test strip. The

glucometer test strip is based on double sequential enzyme reaction in which an enzyme, glucose

oxidase (GOD) converts glucose to hydrogen peroxide and glucuronic acid while peroxidase

oxidizes the dye in the test strip to produce a color. The blood glucose level in mg/dl will be

displayed on the screen after 20 seconds. The reactions involved in this process are shown

below;

glucose oxidase
Glucose + O2 Glucose + H2O2

peroxidase
H2O2 + dye oxidized dye + H2O2 + H2O

21
Procedure

The rats will be fasted overnight (16 h) and blood samples will be obtained from rats by gently

nipping the tail with a lancelet, and then gently squeeze the tail to let out 2 to 3 drops of fresh

venous whole blood on a glucometer strip properly inserted in a glucometer. The glucose level in

mg/dl will be displayed on the digital display screen of the glucometer and will be recorded.

Liver Function Tests

Standard procedures will be used in the assay for liver enzymes such as plasma alanine

aminotransferase, aspartate aminotransferase and alkaline phosphatase.

Determination of Plasma Alanine Amino Transferase (ALT)

Assay of alanine aminotransferase (ALT) will be carried out using the procedure provided by the

Randox kit manufacturer and following the principle described by Reitman and Frankel (1957).

Principle

Alanine amino transferase catalyzes the transfer of L-amino groups from L-alanine to α-oxo-

glutarate, a reaction which produces L-glutamate and pyruvate. In this procedure, the unstable

pyruvate is then complexed with 2, 4-dinitrophenylhydrazine (DNPH) to produce an intensely

coloured hydrazone on the addition of NaOH. This coloured complex absorbs radiation at 530-

550nm. The ALT is measured by monitoring the concentration of pyruvate hydrazone formed

with DNPH. The equation for the reaction is given below:

ALT
α-oxoglutarate + L-alanine L -glutamate + pyruvate

22
Determination of plasma aspartate amino transferase (AST) activity

The activity of AST will be assayed using the procedure provided by the Randox kit

manufacturer and following the principle described by Reitman and Frankel (1957).

Principle

Aspartate amino transferase catalyzes the transfer of L-amino groups from L-aspartate to α-oxo-

glutarate, a reaction that produces L-glutamate and oxaloacetate. In this procedure, the unstable

oxaloacetate is then complexed with 2, 4-dinitrophenylhydrazine (DNPH) to produce an

intensely coloured hydrazone on the addition of NaOH. This coloured complex absorbs radiation

at 530-550nm. The AST activity is measured by monitoring the concentration of oxaloacetate

hydrazone formed with DNPH. The equation for the reaction is given below:

AST
α-ketoglutarate + L-aspartate L-glutamate + Oxaloacetate

Determination of Lipid Profiles

Determination of Serum Total Cholesterol.

Total cholesterol in serum will be estimated using an enzymatic colorimetric assay based on

cholesterol oxidase method following the manufacturer’s instructions with absorbance measured

at 600 nm using a Shimadzu UV-1201 UV/VIS spectrophotometer.

23
Principle

Enzymatic determination of total cholesterol will be performed according to the following

equation (Allain et al., 1974).

cholesterol esterase
Cholesterol ester + H2O Cholesterol + Fatty acids

cholesterol oxidase
Cholesterol + O2 4- Cholesten- 3- one + H2O2

2 H2O2 + Phenol + 4- aminoantipyrene peroxidase Red quinine + 4H2O

Determination of Serum HDL (High Density Lipoprotein) Cholesterol.

Serum HDL will be estimated using an enzymatic colorimetric assay based on the cholesterol

oxidase method after removal of other lipoproteins by precipitation with phosphotungstate-

magnesium, following the manufacturer’s instructions with absorbance measured at 600 nm

using a Shimadzu UV-1201 UV/VIS spectrophotometer.

Principle

The chylomicrons, VLDL and LDL of serum are precipitated by phosphotungstic acid and

magnesium ions. After centrifugation, HDL in the supernatant solution will be measured by

enzymatic method (Gordon et al., 1977).

Determination of Serum LDL (Low Density Lipoprotein) Cholesterol.

Serum LDL cholesterol will be calculated indirectly by the Friedewald’s equation

LDL = Total cholesterol – [HDL + (TGL/5)].

Determination of Serum Triglycerides.

24
Serum triglycerides will be measured using an enzymatic colorimetric assay based on GPO-

DAOS method following the manufacturer’s instructions with absorbance measured at 600 nm

using a Shimadzu UV-1201 UV/VIS spectrophotometer.

Principle

Enzymatic determination of triglycerides will be performed according to the following equation

(Schettler & Nussel, 1975).

glycerol kinase
Triglyceride + H2O Glycerol + Fatty acids

lipoprotein lipase
Glycerol + ATP glycerol-3- phosphate + ADP

glycerol-3-phosphate oxidase
Glycerol-3-PO4 + O2 dihydroxy acetone phosphate+ H2O2

H2O2 + 4- aminoantipyrene + p- chlorophenol peroxidase Red quinoneimine

Estimation of VLDL(Very Low Density Lipoprotein)

Formula: VLDL = triglycerides (mg/dl) (Ahmadi et al., 2008).

Oxidative Stress Markers

Assessment of Lipid Peroxidation (LPO)

Lipid peroxidation will be determined by measuring the formation of thiobarbituric acid reactive

substances (TBARS) according to the method of Varshney and Kale (1990).

25
Principle

Under acidic condition, malondialdehyde (MDA) produced from the peroxidation of membrane

fatty acid and food products react with the chromogenic reagent, 2-thiobarbituric acid (TBA), to

yield a pink coloured complex with maximum absorbance at 532 nm and fluorescence at 553 nm.

The pink chromophore is readily extractable into organic solvents such as butanol.

Reagents

1. Trichloroacetic acid (TCA, 30%): TCA (9 g) was dissolved in distilled water and made up to

30 ml with same.

2. Thiobarbituric acid (0.75%): This was prepared by dissolving 0.225 g of thiobarbituric acid

(TBA) in 0.1 M HCl and made up to 30 ml with same.

3. Tris-KCl buffer (0.15 M, pH 7.4): KCl (1.12 g) and 2.36 g of Tris base were dissolved

separately in distilled water and made up to 100 ml with same. The pH was then adjusted to 7.4.

Procedure

An aliquot (0.4 ml) of the organs (liver and pancreas) will be mixed with 1.6 ml of Tris-KCl

buffer to which 0.5 ml of 30% TCA will be added. Then 0.5 ml of 0.75% TBA will be added and

placed in a water bath for 45 minutes at 80 oC. This will be cooled in ice and centrifuged at 3000

g. The clear supernatant will be collected and absorbance measured against a reference blank of

distilled water at 532 nm. The MDA level will be calculated according to the method of Adam-

Vizi and Seregi (1982). Lipid peroxidation in units/mg protein or gram tissue will be computed

with a molar extinction coefficient of 1.56 x 105 M-1cm-1.

26
Antioxidant Markers

Determination of Catalase Activity

Catalase activity will be determined according to the method of Beer & Sizer. (1952).

Principle

Catalase (hydrogen peroxide oxidoreductase) catalyzes the decomposition of H 2O2 to form H2O

and O2. Catalase activity is measured either by decomposition of H 2O2 or by liberation of O2. The

decrease in the absorbance by H2O2 as a function of time is used to follow the catalase-peroxide

reaction. The spectral region for hydrogen peroxide is 210-240 nm. The difference in absorbance

per minute is a measure of catalase activity.

Reagents

1. Dichromate Solution (5%): K2Cr2O7 (5 g) was dissolved in 80 ml of distilled water and made

up to 100 ml with same.

2. Hydrogen peroxide (0.2M): H2O2 (0.67 g) was mixed with distilled water in a 100 ml

volumetric flask and the solution made up to the mark with same.

3. Dichromate/acetic acid: This reagent was prepared by mixing 5% solution of K 2Cr2O7 with

glacial acetic acid (1:3 by volume) and it is stable for about one month.

4. Phosphate buffer (0.01M, pH 7.0): Na 2HPO4.12H2O (3.5814 g) and 1.19 g NaH2PO4.2H2O

dissolved in 900 ml of distilled water. The pH adjusted to 7.0 and distilled water added to make

up to 1 litre.

Procedure

27
2.5 ml of phosphate buffer will be added to 0.1ml of serum and incubated at 25°C for 30 min.

After transferring into a cuvette the absorbance will be measured at 240 nm, 650 µl of hydrogen

peroxide solution will be added to initiate the reaction, the change in absorbance will then be

measured for 3 min (Beer & Sizer, 1952).

Determination of Reduced Glutathione Level (GSH)

The method of (Ellman, 1959) will be followed in estimating the level of reduced glutathione

(GSH).

Determination of Superoxide Dismutase Activity

The level of superoxide dismutase (SOD) activity will be determined by the method of

Marklund. (1974).

STATISTICAL ANALYSIS

Data will be presented as mean ± standard error of the mean (SEM) and will be analyzed using

Statistical Package for Social Sciences (SPSS) software for windows (SPSS, Inc., Chicago,

Illinois, USA). Differences between groups will be determined by one-way analysis of variance,

and posthoc testing will be performed for intergroup comparisons using the least significant

difference (LSD) (Levine, 1991). A p-value <0.05 will be considered significant.

28
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