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medical bio-chemistry project material sample

OXIDATIVE STRESS LEVEL IN FEMALES WITH HEART DISEASES USING VITAMIN A, C AND E AS DETERMINANTS

OXIDATIVE STRESS LEVEL IN FEMALES WITH HEART DISEASES USING VITAMIN A, C AND E AS DETERMINANTS

INTRAOCULAR PRESSURE MARKERS IN MALARIAL INFECTED MICE RECEIVING PHYLLANTHUS AMARUS TREATMENT

 

 

                                                   ABSTRACT

Heart disease is associated with elevated oxidative stress via increased generation of reactive oxygen species (ROS), and decline in antioxidant defences. Increased oxidative stress is thought to play a role in the development of cardiovascular diseases. The present study was carried out to see the levels of vitamin C, vitamin E and total antioxidant (AO) in hypertensive female patients with heart disease. Twenty-two patients (all women) with history of Hypertension from outpatient clinic unit of the State Central Hospital, Benin City, Edo State, Nigeria where studied. Eight control subjects (all women) with no history of hypertension and heart diseases were studied. The raw group data of their age, weight, height, blood pressure and pulse rate of the subjects were obtained.  They were selected on the basis of general physical examination Serum level of vitamin A, C and E were obtained using documented method. Serum levels of vitamin A,C, and E were 380.24±68.13 U/L and 135.69±21.32 U/L, 1.23±0.13 mg/dl and 1.20±0.09 mg/dl, 136.26±9.72 U/L  and 185.41±1.84 U/L in experimental and control. Vitamin A shows significant increase with experimental when compared with control, but Vitamin C shows mild increase when experimental group was compared with control group, but did not attain significant at (p<0.05) and Vitamin E shows moderate significant decrease when experiment group compared with control group at (p<0.05). This study reveals a significant reduction in serum vitamin E level of hypertensive patients as compared to the controls with the mean vitamin C level showing no significant difference. In this research, the scientific data do not justify the use of antioxidant vitamin supplements for CVD risk reduction.

 

 

 

 

 

LIST OF TABLES

Table 4.1: shows the effect of hypertension

on the blood pressure, enzyme and non

enzyme antioxidants in hypertensive

female patients          –                –       –       –       –       –  62

TABLE OF CONTENTS

PAGE

Cover page        –       –       –       –       –       —      –       –       -i

Title page  –       –       –       –       –       –       –       –  –    –       -ii

Certification      –       –       –       –       –       –       –       –       -iii

Dedication         –       –       –       –       –       –       –       – –     -iv

Acknowledgements    –       –       –       –       –       –  –    –       -v

Abstract    –       –       –       –       –       –       –       –  –    –       -vi

List of tables and figure     –       –       –       –       –       –  –    -vii

Table of content         –       –       –       –       –       –       –  –    -vii

 

CHAPTER ONE

1.1    INTRODUCTION  –     –       –       –       –       –  –    –       –1

1.2    Aims and Objectives          –       –       –       –  –    –       -5

1.3    Scope of study  –       –       –       –       –  –    –       –       -6

1.4    Significance of study:         –       –       –       –  –    –       -6

 

CHAPTER TWO

2.0    LITERATURE REVIEW       –       –       –       – –     –       – 7

2.1    HEART DISEASE       –       –       –                —      –       – 7

2.2    TYPES OF HEART DISEASE       —      –  –    –       –       – 8

2.2.2 Hypertensive heart disease         —      –       – –     –       -8

2.2.3 Heart failure    – –       –       –       –       –  –    –       -8

2.2.4 Cor pulmonale or pulmonary heart disease         —      -9

2.2.5 Valvular heart  disease      –       –       –       —      —      9

2.2.6 Cerebrovascular disease    –       –       –       –  –    —      -9

2.2.7 Congenital heart disease   –       –       –       —      -10

2.3    Epidemiology of Cardiovascular Disease     –       –  –    -10

2.4    Risk factors       –       –       –       –       –  –    —      —      -11

2.5    OXIDATIVE STRESS –       –       –       –  –    —      -13

2.6    Physiological Sources of Reactive Oxidant

Species in Cells         –       –       –       –       –       —-  13

2.6.1 Mitochondrial respiration as a source

of reactive oxidant  species in cells    –       –       –  –    -14

2.6.2 NADH/NADPH oxidase system as a source of

reactive oxidant species in the cell    –       –       –  –    -17

2.6.3 Xanthine oxido-reductase system as a source of

reactive oxidant species in the cell     –       –      –         –  –    -20

2.6.4 NOS uncoupling as a source of reactive

oxidant species in the cell. Uncoupled NO –     —          21

2.7    Reactive Oxidant Species Formation and

Cardiovascular Disease     –       –       –       –      –         —       21

2.7.1 Oxidative stress and endothelial

Dysfunction in aterosclerosis     –       –       –     ­-          –  –    -24

2.7.2 Oxidative stress and hypertension      –       —  –   —      31

2.7.3 Oxidative stress and cardiovascular ischemia –   —      -33

2.7.4 Oxidative stress and heart failure    – –       –  –    -35

2.7.5 Oxidative stress and postoperative arrhythmias –  –    -39

2.8    Antioxidants and Cardiovascular Disease   –       -39

2.8.1 Antioxidants     –       –                –       –  –    —      —      -40

2.8.2 The Use of Antioxidants     –       –       –       –  –    —      -42

2.8.3 Dietary Intervention and Risk of

Cardiovascular Disease     –       –       –       –       –       –  –    -42

2.8.4 Antioxidants and Cardiovascular Risk        —  –   —      -45

2.8.5 Vitamin C and Cardiovascular Disease       —  –   —      -48

2.8.6 Vitamin E and Cardiovascular Disease       –       —      -51

CHAPTER THREE

3.0    MATERIALS AND METHOD        –       –       –       —      -56

3.1    MATERIALS      –       –       –       –       –  –    —      —      -56

3.1.1 Instruments      –       –       –       –       –       –  –    —      -56

3.1.2 Apparatus and glass wares        –       –       –  –    —      -56

3.1.3 Reagents   –       –       —      –       –       –  –    —      —      -57

3.1.4 Specimen –       –                –       –       –  –    —      -57

3.1.5 Blood Serum     –       –       –       —      –  –    —      —      -57

3.2    METHODS         –       –       –       –                –  –    —      -57

3.2.1 Study group      –       –       –       –         –     —      —      -57

3.2.2 Clinical assessment  –       –       –                —      —      -58

3.2.3 Sample collection and preservation    –       –       –  –    —58

3.3    SAMPLE ANALYSIS   –       –       –       –  –    —      —      -58

3.3.1 Serum vitamin E estimation     –       –       –  –    —      -58

3.3.2 Serum  Vitamin A estimation     –       –       –  –    —      -60

3.3.3 Serum Vitamin C estimation      –       –       –  –    —      -60

3.4    STATISTICAL ANALYSIS    –       –       –  –    —      —      -61

CHAPTER FOUR

4.0    RESULTS —      –       –       –       –      –       –                -62

 CHAPTER FIVE

5.0 DISCUSSION       –       –       –       –       –       –       –  –    — 64

5.1 Conclusion –       –       –       –       –       –       –       –   –      67

References

 

 

                                CHAPTER ONE

1.1     INTRODUCTION

Heart disease(cardiovascular disease), defined as coronary artery disease, hypertensive heart disease, congestive heart failure, peripheral vascular disease, and atherosclerosis including cerebral artery disease and strokes, is the leading cause of death in the United States and disability in the  world today, (Thom, 1989). In the United States, the heart disease death toll is nearly one million each year, and in 2002 the estimated cost of heart disease treatment was $326.6 billion, (Shekelle et al., 2003). To provide early prognosis and better therapies for preventing and curing these diseases, an understanding of the basic pathophysiologic mechanisms of heart disease is essential. Growing evidence indicates that oxdant stress production of reactive oxygen species (ROS) and other free radicals under pathophysiologic conditions is integral in the development of cardiovascular diseases (CVD).

Free radicals are molecules containing one or more unpaired electrons in atomic or molecular orbital, (Gutteridge et al., 2000). Reactive free radicals play a crucial part in different physiological processes ranging from cell signaling, inflammation and the immune defense, (Elahi et al., 2006). There is increasing evidence that abnormal production of free radicals lead to increased stress on cellular structures and causes changes in molecular pathways that underpins the pathogenesis of several important human diseases, including heart disease, neurological disease and cancer and in the process of physiological ageing, (Pacher 2008; Vassalle et al., 2008). One of the major contributors of oxidative stress is the reactive oxygen species (ROS) family of molecules. These include free radicals such as superoxide anion (O2-), hydroxyl radical (HO-), lipid radicals (ROO-) and nitric oxide (NO). Other reactive oxygen species, hydrogen peroxide (H2O2), peroxynitrite (ONOO-) and hypochlorous acid (HOCl), although are not free radicals but they have oxidizing effects that contribute to oxidative stress. ROS has been implicated in cell damage; necrosis and cell apoptosis due to its direct oxidizing effects on macromolecules such as lipids, proteins and DNA, (Izakovic et al., 2006). Production of one free radical can lead to further formation of radicals via sequential chain reactions, (Cronin et al., 2005).

Understanding the contribution of free radical stress in the pathogenesis of disease will allow us to study the development of oxidative stress; a condition that occurs due to an imbalance between cellular production of oxidant molecules and the availability of appropriate antioxidants species that defend against them. In physiological conditions, cells would increase activities of antioxidant enzymes and other antioxidant defenses to counteract occurrence of oxidative stress, (Brunzini et al., 2004). These include radical scavengers such as vitamin E, A, beta carotene and vitamin C, Manganese dependent superoxide dismutase such as manganese superoxide dismutase (Mn-SOD), Copper/Zinc superoxide dismutase (Cu/Zn SOD), glutathione peroxidase, glutathione reductae and catalase (CAT). Decreased risk of cardiovascular death has been associated with higher blood levels of vitamin C and E. In addition, vitamin C, vitamin E, and A have demonstrated antioxidant effects, including beneficial effects on oxidation of low-density lipoprotein. There is evidence that these vitamins affect other risk factors for CVD such as hypertension. Vitamin E may also reduce coronary artery blockage by decreasing blood platelet aggregation. Thus, it was reasonable to expect that supplementation with these antioxidants would decrease the risk of developing CVD.  Large numbers of people are taking antioxidants with the expectation that they will prevent disease. As part of a natural defense system, antioxidants can mitigate the activity of free radicals and other oxidative species that have been implicated in the development of heart disease, (Krzanowski, 1991; Duthie et al., 1999). The epidemiologic and observational literature has suggested a beneficial effect of antioxidant-rich foods, as well as specific antioxidants, on the risk of CVD and stroke, (Asplund, 2002; Tribble, 1999). Because oxidative functions also contribute positively to the health of the cell by their participation in energy metabolism, biosynthesis, detoxification, and cellular signaling, a balance is clearly required between the pro-oxidants and the antioxidant defense system to maintain health, (German et al., 2001).

1.2     Aims and Objectives

The aim of this study is to determine the efficacy of three antioxidants, vitamin E, vitamin C, and A, for the prevention and treatment of cardiovascular disease (CVD) or modification of known risk factors for heart diseases in hypertensive female patients

Specifically, the objective of this study is to determine;

  1. The vitamin A level in hypertensive patient with heart disease.
  2. The vitamin C level in hypertensive patient with heart disease.

INTRAOCULAR PRESSURE MARKERS IN MALARIAL INFECTED MICE RECEIVING PHYLLANTHUS AMARUS TREATMENT

INTRAOCULAR PRESSURE MARKERS IN MALARIAL INFECTED MICE RECEIVING PHYLLANTHUS  AMARUS  TREATMENT

ANIMAL PRODUCTION AND HEALTH TECH. PROJECT TOPICS

 

 

 

ABSTRACT

Phyllanthus amarus has been consistently reported as a rich herb having medicinal value and ethnomedical importance. It has been used to eliminate gallstone, malaria and some other aliments, but the effect of its use on changes in ocular glucose, proteins and lipids — analytes that influence intraocular pressure, has not been fully documented. So, in this study, the effect of ethanolic leaf extract of Phyllanthus amarus on the levels of ocular glucose, proteins and lipids was investigated using homogenized ocular tissue of experimental mice. Forty five (45) adult mice weighing between 22-27g were randomly divided into 9 groups  and used for the study. Group 1: normal control (uninfected and untreated mice) Group 2: malaria control (mice infected with plasmodium berghei and untreated) Group 3: parasitized (infected with P. Berghei treated with 100mg/kg P. amarus) Group 4: parasitized (infected with P. Berghei treated with 200mg/kg P. amarus) Group 5: parasitized (infected with P. Berghei treated with 300mg/kg P. amarus)

Group 6: parasitized (infected with P. Berghei treated with 5mg/kg chloroquine)

Group 7: uninfected but treated with 100mg/kg P .amarus Group 8: uninfected but treated with 200mg/kg P. amarus Group 9: uninfected but treated with 300mg/kg P. amarus .Each group was treated for

7days and on the 8th day the animals were scarificed under chloroform anaesthesia after an overnight fast. The mice eyes were carefully excised, rinsed in cold normal saline and prepared for the biochemical analysis of glucose, proteins and lipids using standard methods. Results show that Phyllanthus amarus administration (irrespective of dose) did not significantly (p>0.05) alter ocular glucose and protein levels, but increased (p<0.05) lipids (cholesterol and triglycerides) concentrations when compared with control values. The altered ocular lipid homeostasis may have some biochemical implications and clinical significance. This should be validated in further studies.

 

LIST OF TABLES

Table 1: glucose in the serum of p. berghei infected and uninfected mice treated with phyllanthus amarus leaf extract.    ———————————————————        25

Table 2: changes in blood cholesterol and triglyceride induced by plasmodium berghei infected mice    ——————————————————————————–       26

Table 3:  changes in blood albumin and total protein induced by p. berghei infected mice    —————————————————————————————————        27

Cover         ——————————————————————–      i

Title            ——————————————————————–    ii

Certification   —————————————————————-     iii

Dedication      ————————————————————–       iv

Acknowledgement   ——————————————————         v

Abstract         —————————————————————       vi

List of Tables   ————————————————————-     vii

Table of contents   ——————————————————-       viii

CHAPTER ONE

  • INTRODUCTION ———————————————————————–      1

1.1.1 BACKGROUND OF STUDY    ——————————————————-      1

1.2 STATEMENT OF PROBLEM    ——————————————————-        3

1.3 OBJECTIVE OF STUDY    ————————————————————-        4

1.4 SIGNIFICANCE OF STUDY    ——————————————————–        4

1.5 HYPOTHESIS    ————————————————————————–     5

1.6 BRIEF REVIEW OF RELEVANT LITERATURE    ———————————     5

1.7 BIOMAKERS   —————————————————————————-    5

1.8 RETINOPATHY    ————————————————————————   6

1.9 MUSCULAR ODEMA   —————————————————————–    6

1.10 WHAT IS MALARIA?    ———————————————————    7

1.11 CAUSES OF MALARIA       ———————————————————–     8

1.12 EPIDEMIOLOGY OF MALARIA         ———————————————-     8

1.13 GLOBAL AND GEOGRAPHICAL DISTRIBUTION OF MALARIA..   ——        9

1.14TRANSMISSION AND LIFE CYCLE OF PLASMODIUM PARASITE   —–      11

1.15 LIFE CIRCLE OF PLASMODIUM PARASITE   ———————————–    12

CHAPTER TWO   ——————————————————————————    14

  1. 0MATERIALS AND METHOD ——————————————————– 14

2.1 MATERIALS    —————————————————————————–    14

2.2 METHODS    ——————————————————————————–   14

2.2.1 ANIMALS CARE AND HANDLING    ———————————————    14

2.2.2 ANIMAL GROUPING AND INOCULATION WITH PLASMODIUM BERGHEI    —————————————————————————————————–     15

2.2.3 ANIMAL SACRIFICE AND COLLECTION OF SPECIMEN    —————-     16

2.2.4 ANALYSIS OF SPECIMEN   ———————————————————-   16

 

2.3 GLUCOSE ESTIMATION    ———————————————————–    16

2.3.1 REACTION PRINCIPLE    ———————————————————-     16

2.3.2 PROCEDURE    ———————————————————————–     17

 

2.4 TOTAL CHOLESTEROL ESTIMATION    ——————————————   17

2.4.1 PRINCIPLE    ————————————————————————–     17

2.4.2 EQUATION    ————————————————————————–    17

2.4.3 PROCEDURE    ————————————————————————    18

2.4.4 CALCULATION    ———————————————————————   19

 

2.5. TRIGLYCERIDE ESTIMATION    —————————————————    19

2.5.1 PRINCIPLE    ————————————————————————–     19

2.5.2 EQUATION    —————————————————————————   20

2.5.3 PROCEDURE    ———————————————————————–     20

2.5.4 CALCULATION    ——————————————————————–    21

 

2.6 TOTAL PROTEIN ———————————————————————-     21

2.6.1 PRINCIPLE    ————————————————————————–     21

2.6.2 PROCEDURE    ———————————————————————–     21

2.6.3 CALCULATION    ——————————————————————–     22

 

2.7 ALBUMIN ———————————————————————————–22

2.7.1 PRINCIPLE    ————————————————————————–      22

2.7.2 PROCEDURE    ———————————————————————–      23

2.7.3 CALCULATION    ——————————————————————–     23

2.7.4 STATISTICAL ANALYSIS ————————————————————– 24

CHAPTER THREE    —————————————————————————   25

3.0 RESULTS    ———————————————————————————-   25

CHAPTER FOUR    —————————————————————————–   28

4.0 DICUSSION    ——————————————————————————    28

4.1 CONCLUSION    —————————————————————————– 29

REFERENCES   —————————————————————————30

 

CHAPTER ONE

1.1     INTRODUCTION

1.1.1. BACKGROUND OF STUDY

Nature can be considered as the ultimate chemist, about 80% of the world inhabitants still depend on natural products that have inspired chemists and physicians for years because of their rich structural diversity and complexity considerable advances have been obtained for the understanding of natural product biosynthesis in the recent decades.

Malaria, a mosquito borne infectious disease is endemic in the tropical and sub tropical regions of the world (Ahimanah et al., 2000). It is a deadly disease which lowers life expectancy and a major cause of infant mortality in highly endemic areas (WHO, 2011).

Malaria infection in humans and animals is caused by the Plasmodium. Several species of Plasmodium have the ability to cause malaria in animals, including rodents (mice). These parasites are not direct practical concern to man or his domestic animals. The interest of these parasites is that they are practical model organism in the laboratory for the experimental study of human malaria.

Plasmodium berghei is considered a comparable genetic model to human There is a high degree of genetic conservation this up to 99% (pennachio, 2003) and it is well established that mice also exhibit natural differences in susceptibility to malaria infection (Greenberg et al., 1954). P. berghei is transmitted by Anopheles mosquito and it infects the liver after being injected into the blood stream by the bite of the infected female mosquito. After a few days of development and multiplication, these parasites leave the liver and invade erythrocyte (red blood cells). The multiplication causes Anemia and damage essential organs in the body. P. berghei infection also affects the brain and can cause cerebral complications in laboratory mice.

The plant, phyllanthus schumach (Euphorbiaceac) is commonly known as bhuirali Usually occurs in Asain, Maharashitra, Burma, Nicobar, Islands malesia and America. Phyllanthus amarus schumach is a native to Americans (van Holthoon, 1999). Phyllanthus amarus is small, erect, annual herb that grows 30-40cm in height which is indigenous to the rain forests of the Amazon and other tropical areas throughout the world including America, India and Nigeria. The Spanish name of the plant, chanca piedra, means stone breaker, wind breaker, gulf leaf flower or gala of wind (Celia, A.  et al., 2006).

Phyllanthus amarus is an Ayurvedic system of medicine which is used in the problems of stomach, genitourinary system, liver, kidney, and spleen, it plays an important role in Ayurvedia, an Indian system of medicine and it is used to treat jaundice, gastropathy, diarrhea, dysentery, fevers, scabies, gential, infections, ulcers, and wounds (Patel et al., 2011).

The different plant parts are ethnobotanically used in various diseases and disorders. For example, the leaves are used as expectorant and diaphoretic and the fruits as carminative, laxative, astringent. Diuretic and tonic to the liver. The juice or extract of its thinner roots and young leaves are taken internally to stimulate the kidney. Heyne recorded its uses in the Dutch  Indies (Indonesia) for stomach, aches, gonorrhoea and children cough (Karuna,  et al., 2009).

Research have shown that the plant has demonstrated anti-viral property against hepatitis B virus (Boeira  et al., 2 011), hepatoprotective (Amin,  et al., 2013), anticarcinogenic (Rajeshkumar,  et al., 2002), antimutagenics, anti-nociceptive and anti-inflammatory (Obidike,  et al., 2010), anti-diabetics (Okoli, et al., 2011) and antilipidermic (Khanna,  et al.,  2002) activities.

1.2 STATEMENT OF PROBLEM

Since malaria has been speculated to alter biochemical functions of organs of the body such as brain, liver, heart, and spleen, this research is designed to know if such biochemical alternations also include changes in the intraocular pressure markers in experimental mice. Phyllanthus amarus is a medicinal herb used for the treatment of several diseases including malarial infection. In southern Nigeria the utilization of Phyllanthus amarus is popular but whether it improves or disturbs