Antihyperlipidemic and Antioxidant Effects of Phaseolus Vulgaris Linn in Wistar Rats

ANTIHYPERLIPIDEMIC AND ANTIOXIDANT EFFECTS OF PHASEOLUS VULGARIS LINN IN WISTAR RATS

AHANTE EJIROGHENE

PG/MSC/13/66902

A RESEARCH PROJECT SUBMITTED

DEPARTMENT OF PHARMACOLOGY & TOXICOLOGY

FACULTY OF PHARMACEUTICAL SCIENCES

UNIVERSITY OF NIGERIA, NSUKKA

SUPERVISOR: PROF. P.A. AKAH & DR. C.S. NWORU

AUGUST, 2015

ANTIHYPERLIIDEMIC AND ANTIOXIDANT EFFECTS OF PHASEOLUS VULGARIS LINN IN WISTAR RATS

AHANTE EJIROGHENE

PG/MSc./13/66902

A RESEARCH PROJECT SUBMITTED TO DEPARTMENT OF PHARMACOLOGY & TOXICOLOGY

FACULTY OF PHARMACEUTICAL SCIENCES

UNIVERSITY OF NIGERIA, NSUKKA

IN PARTIAL FULFILMENT FOR THE AWARD OF MASTER OF SCIENCE (MSC) IN PHARMACOLOGY & TOXICOLOGY

AUGUST, 2015

iii

CERTIFICATION

This research, antihyperlipidemic and antioxidant effects of Phaseolus vulgaris L. in Wistar rats, has been read and approved as having met the requirements of the Department of

Pharmacology&Toxicology, Faculty of Pharmaceutical Sciences, University of Nigeria, Nsukka, for award of degree of Master of Science in Pharmacology and Toxicology.

......

Ahante Ejiroghene

….………………………. ..……………………... Prof. P.A. Akah Dr. C.S. Nworu (Supervisor I) (Supervisor II)

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DEDICATION

This project is dedicated: To: The Almighty God Now unto The King Eternal, Most Gracious God And Merciful God Be The Honour And Glory Forever And Ever, Amen.

ACKNOWLEDGEMENT

I wish to acknowledge God Almighty who in his infinite mercies granted me the strength and patience, ordering my steps in everything to the successful completion of this project, May His name be glorified.

I wish to express my profound gratitude to my supervisors; Prof P.A. Akah and Dr C.S. Nworu for their guidance, supervision, and candid assistance in making this project a successful one and also for making it achieve an acceptable standard. I also thank all the members of staff of Pharmacology and Toxicology, I pray God continue to increase every one of you in all aspect of your life.

To my parents Mr and Mrs E.M. Ahante and my siblings (Mr Emena, Miss Gare and Barr. Oke) whose love, support, advice and friendship have kept me, I pray that God will shower you with His infinite Favour and abundant Grace in Jesus name Amen.

Finally, my gratitude goes to my wonderful and special friend Osifo Itohan Mercy whose life has inspired me to be a better person in all I do, and also to all my colleagues at the department of Pharmacology and Toxicology for always raising the bar higher and pushing me to work harder, may God reward you all

To everyone who wished me well, may God continue to bless and enrich you in love.

Ahante Ejiroghene

August 2015

Table of Contents Title page ------i

Table of Contents ------vi

Lists of Tables ------ix

Lists of Figures ------x

Abstract ------xi

CHAPTER ONE

1.0 Introduction ------1

1.1Review on Phaseolus vulgaris ------3

1.2Hyperlipidemia ------6

1.2.1Lipoproteins------6

1.2.2 Classification of lipoprotein ------8

1.2.3Lipoproteins metabolism ------10

1.2.4 Classification of hyperlipidemia ------13

1.2.5 Management of hyperlipidemia------18

1.2.5.1 Non-Pharmacological interventions------18

1.2.5.2Pharmacological interventions ------19

1.3 Antioxidants ------26

1.3.1 Classification of antioxidants------27

1.3.2 Types of antioxidants ------28

1.3.3 Oxidative Stress ------30

1.3.4 Antioxidants and Cardiovascular diseases ------30

1.4 Induction of hyperlipidemia------31 vi

vii

1.4.1Carbon Tetrachloride (CCl4) ------32

1.5 Statement of the problem ------33

1.6 Aim of Study ------33

1.7 Objectives of Study ------34

1.8Significance of Study ------34

1.9 Justification of Study------34

CHAPTER TWO

2.0 Materials and Method------35

2.1Experimental animals ------35

2.2 Plant material ------35

2.3 Phytochemical screening------36

2.4 Hplc fingerprinting ------36

2.5Acute toxicity (LD50) ------37

2.6Hypolipidemicstudies ------37

2.6.1Experimental Design ------37

2.6.2Acute Study of the effects of extract and fractions of PVE on lipid profile- 38

2.6.3Sub-acute Study of the effects of extract and fractions of PVE on lipid profile- 38

2.6.4Chronic Study of the effects of extract and fractions of PVE on lipid profile----- 39

2.6.5BiochemicalDetermination of Lipid profile parameters ------40

2.6.5.1 Determination of Total Cholesterol Level (TC) ------40

2.6.5.2 Determination of serum Triglycerides levels (TRIGS) ------42

2.6.5.3 Determination of serum High Density Lipoproteins Cholesterol (HDL-C) --- 42

2.6.5.4 Determination of serum Low Density Lipoproteins Cholesterol (LDL-C) ----- 43

2.6.5.5 Determination of serum Very Low Density Lipoproteins Cholesterol (VLDL-C) -- 44

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viii

2.7Determination of Body weight ------44

2.8 Determination of Atherogenic Index------44

2.9Studies on Antioxidants effects of extract and fractions of PVE ------44

2.9.1DPPH scavenging assay of extract and fractions of PVE------44

2.9.2Nitric oxide scavenging assay of extract and fractions of PVE------45

2.9.3In-vivo Study ------45

2.9.4Estimation of Catalase activity ------45

2.9.5 Estimation of Lipid-peroxidation activity ------46

2.9.6Estimation of Glutathione Peroxidase activity------46

2.10Method of data analyses ------47

CHAPTER THREE

3.0 Results ------48

CHAPTER FOUR

4.0 Discussion, Conclusion and Recommendations ------90

4.1 Discussion ------90

4.2 Conclusion ------94

4.3References ------95

APPENDICES ------108

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List of Tables

Table Page

1 Fredrickson Hyperlipidemia classification ------15

2Phytochemical screening of all fractions of Phaseolus vulgaris L. ------49

3 LD50of Phaseolus vulgaris L. ------50

4 The effects of crude of Phaseolus vulgaris L.on antioxidants enzyme ------88

5 The effects of various extract of Phaseolus vulgaris L. ------89

List of Figures

Figures Pages

1. Showing structure of Phaseolus vulgaris ------3

2. Structure of Lipoproteins------7

3. Showing diagram of lipoproteins metabolism------12

4. Antioxidants and cardiovascular system ------31

5. HPLC analysis studies ------51

6. The effect of crude extract of Phaseolus vulgaris L. on lipid profile

parameters (acute study)------55

7. The effect of crude extract ofPhaseolus vulgaris L. on body weight of Wistar

rats (acute study) ------56

8. The effects of crude extract of Phaseolus vulgaris L. on atherogenic index of

Wistar rats (acute study) ------57

9. The effects of various fractions of Phaseolus vulgaris L. on lipid profile

parametrs of Wistar rats (acute study) ------60

10. The effects of various fractions of Phaseolus vulgaris L. on body weight of

Wistar rats (acute study)------61

11. The effects of various fractions of Phaseolus vulgaris L. on atherogenic index of

Wistar rats (acute study) ------62

12. The effect of crude extract of Phaseolus vulgaris L. on lipid profile parameters

(sub-acute study) ------65

13. The effect of crude extract of Phaseolus vulgaris L. on body weight of Wistar rats

(sub-acute study) ------66

14. The effects of crude extract of Phaseolus vulgaris L. on atherogenic index of

Wistar rats (sub-acute study) ------67

15. The effects ofvarious fractions of Phaseolus vulgaris L. on lipid profile

parameters of Wistar rats (sub-acute study) ------70

16. The effects of various fractions of Phaseolus vulgaris L. on body weight of

Wistar rats (sub-acute study) ------71

17. The effects of various fractions of Phaseolus vulgaris L. on atherogenic index of

Wistar rats (sub-acute study) ------72

18. The effects of high fatty diet on lipid profile parameters ------75

19. The effect of on crude extract of Phaseolus vulgaris L. on lipid profile parameters

(chronic study) ------76

20. The effect of crude extract of Phaseolus vulgaris L. on body weight of Wistar rats

(chronic study)------77

21. The effects of crude extract of Phaseolus vulgaris L. on atherogenic index of

Wistar rats (chronic study) ------78

22. The effects of various fractions of Phaseolus vulgaris L. on lipid profile

paramters of Wistar rats (chronic study) ------81

23. The effects of various fractions of Phaseolus vulgaris L. on body weight of

Wistar rats (chronic study) ------82

24. The effects of various fractions of Phaseolus vulgaris L. on atherogenicindex of

Wistar rats (chronic study) ------83

25. DPPH scavenging activity of various extract of Phaseolus vulgaris L.------85

26. Nitric oxide scavenging activity of various extract of Phaseolus vulgaris L. ------86

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ABSTRACT

In this study, the antihyperlipidemic and antioxidants activities of the extract and fractions of Phaseolus vulgaris L. were evaluated. The crude extract (PVE) of dried pulverized plant material was obtained by maceration in methylene chloride/methanol (1:1) while the solvent fractions were obtained by successive solvent-solvent partition in separating funnel between the crude extract suspended in aqueous medium and solvents of increasing polarity to obtain the n-hexane fraction (PVHF), ethylacetate fraction (PVEF), and butanol fraction (PVBF) in that order.Antihyperlipidemic effects of the extracts and fractions were investigated using acute, sub- acute and chronic models. In all threemodels, treatment with (PVE) caused significantreduction (P<0.05) in the lipid profile parameters with the most significance seen in the sub acute study where total cholesterol was decreased by 38.44%. Triglycerides level was significantly decreased (P<0.05) by 18.18% in the acute study model.Similarly, very low density lipoprotein (VLDL- C)and low density lipoprotein (LDL-C) wasrespectively decreased by 18.18% and 48.73% in the acute antihyperlipidemia model. In contrast, high density lipoprotein (HDL-C) level was significantly (P<0.05) increased in the acute phase by 20.85%.In all study protocols involving the various fraction there were significant increase(P<0.05) in lipid profile. PVEF (400 mg/kg) produced- the most significant reduction in total cholesterol level in the acute study with percentage decrease of 22.10% compared to the control treatment. Triglycerides level was similarly reduced by 21.59% and 15.91% at PVHF (200 mg/kg) and PVEF (200 mg/kg) with the acute study.The sub-acute protocol showed significant decrease at PVBF 200 mg/kg percentage decrease of 17.28%. VLDL-C level for the fraction study showed significant decrease (P<0.05) at PVHF 200 mg/kg and PVEF 200 mg/kg with percentage decrease of 21.59% and 15.91% during the acute protocol, the sub-acute protocol showed significant decrease at PVBF 200 mg/kg percentage decrease of 17.28. LDL-C level for fraction extract study showed dose dependent significance decrease (P<0.05) seen at PVHF 100 mg/kg, PVEF100 mg/kg, and 200 mg/kg with percentage decrease of 47.19%, 41.62% and 53.39% respectively during the acute protocol, Finally in the chronic protocol a significant decrease was seen with PVHF 200mg/kg, PVEF 100 mg/kg, and PVBF 200 mg/kg with percentage decrease of 26.03%, 24.82% and 20.05% respectively. HDL-C level for extract study showed dose dependent significant increase (P<0.05) seen at PVHF 100 mg/kg, PVEF 100 mg/kg and 200 mg/kg with percentage increase of 30.44%, 28.88% and 30.86%. DPPH reduction and nitric oxide scavenging assays were used in the investigation of the extract and fractions for the in vitro antioxidant activities study, the antioxidant activities of the extract and fractions were further determined in vivo in rats. Antioxidant enzymes and factors such as catalase, glutathione peroxidase, and lipid peroxidation activities were measured in carbon tetrachloride-treated rats treated with or without the extract and fractions studies. The highest percentage reduction of DPPH was 80.61% seen with PVEF and PVBF fraction at 400 mg/kg. The highest percentage reduction of nitric oxide was 75.86% seen with PVHF at 200 mg/kg. The in vitro study showed significant increased (P<0.05) scavenging activity with the PVHF and PVEF having scavenging activity comparable with ascorbic acid. In in-vivo antioxidant assay showed that the Lipid peroxidation levels estimated by thiobarbituric acid reaction showed no significant (P>0.05) increase or decrease in the serum MDA of both the treated and untreated group, while in catalase activity estimation showed significant (P<0.05) increase was seen with PVE 100 mg/kg of 71.05%, Glutathione peroxidise activity showed the most significant percentage (P<0.05) increase of 76.19% for PVBF 100 mg/kg.The results of the study showed that the extracts and fractions of Phaseolus vulgaris

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xiii posses anti-hyperlipidemic and antioxidant with the PVEF showing better and more consistent effects with all protocols used in the investigation.

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CHAPTER ONE

1.0. INTRODUCTION

A large volume of scientific research suggests that in situations of oxidative stress, reactive oxygen species (ROS) are generated and a homeostatic environment between anti-oxidant and oxidation is created, which are known to be an important concept for maintaining a healthy

- biological system (Davies, 2000). Reactive oxygen species (ROS) such as superoxide anions (O2

), nitric oxide (NO) and hydroxyl radical (OH-) aids in the inactivation of enzymes and this result in damage to important cellular components which leads to complication such as coronary heart diseases (Gessin et al., 1990).

Disease of coronary origins such as stroke, atherosclerosis, etc., continues to be a leading cause of death in most countries of the world (Davey, 1993).One of the greatest risk factors in the severity and prevalence of coronary heart diseases is disorders of lipid metabolism known as hyperlipidemia (Grundy, 1986). According to reports by the World Health Organization approximately 56% of coronary heart diseases are as a result of hyperlipidemia and this result in about 4.4million deaths each year worldwide (World Health Organization, 2002).

Hyperlipidemia is a disorder of lipoprotein metabolism, including lipoprotein overproduction or deficiency and manifested by elevation of the serum total cholesterol, low-density lipoprotein

(LDL) cholesterol and triglyceride concentrations with a decrease in the high-density lipoprotein

(HDL) cholesterol concentration (Adam, 2005).

Man as always looked for a way to fight and control diseased state with inspiration and aid from his immediate natural environment; this guidance’s have been used for centuries as remedies for human diseases because they contain components of therapeutic value (Nostro et al., 2000). The use of medicinal plants in the management of diseases is common around the world (Aliyu et al.,

2007). Herbal medicine, the study and use of medicinal properties of plants, is an aspect of

modern medicine (World Health Organization, 2008).The use of traditional medicine is recognized as the most viable method of identifying of new medicinal plants species

(Farnsworth, 1966; Ajanahoun et al., 1991).

Medicinal plants are used for their beneficial antioxidant and lipid lowering effects thus reducing the risk of cardiovascular diseases in many countries. HMG-CoA reductase inhibitor that is the statins is the most recent lipid lowering drugs. They are very effective in lowering total and

LDL- cholesterol and have been shown to reduce coronary events and mortality. They have very few side effects and are now usually the drugs of first choice (Neal, 2002). Also, ascorbic acid and tocopherols are widely used anti-oxidants. In recent times antioxidants derived from natural sources mainly plants have been intensively used to prevent oxidative damage because of its advantage over synthetic ones; as they are easily obtained, economical and have slight or negligible effects (Onay-ucar et al., 2006). Although the adverse effect of statins is relatively low, one rare effect called rhabdomyolysis can be very serious (Miller, 2001). Statins and fibrates both used in elevated cholesterol, especially in combination; cerivastatin (Baycol) was withdrawn in 2001 after numerous incidence of rhabdomyolysis (Armitage, 2007). Hence, there is an urgent need to research natural products that would have minimal or no lipid lowering side effects.

Phaseolus vulgaris (family: Fabaceae) is a plant whose leaf, bark, roots and seeds are used for medicinal purposes. It is commonly known as kidney bean, various parts of the plant have been used extensively for the treatment of diabetes mellitus traditionally (Chopra et al., 1958).

Previous studies have reported the hypolipidemic activities of the aqueous extract (Roman-

Ramos et al., 1995), as well as, the anti-inflammatory, antimutagenic, antioxidant, antimicrobial and antioxidant activities of the extract (Jorgeet al.,2013). The present study aims to establish the anti-hyperlipidemic and antioxidants effects of various fractions of Phaseolus vulgaris L. fruit and determine the most active of the fractions. 2

1.1. Review on Phaseolus vulgaris

Figure 1: Showing structure of Phaeolus vulgaris (adapted from Rasbak, 2005)

Phaseolus vulgaris, commonly called the green bean, kidney bean, or common bean, is a herbaceous annual plant in the Fabaceae (legume or bean family) that originated in Central and

South America and is now cultivated in many parts of the world for its beans, it can also be classified as "bush beans" or "pole beans", depending on their style of growth. These include the kidney bean, the navy bean, the pinto bean, and the wax bean (U.S Department of

Agriculture, 2010).

Scientific Classification

Kingdom: Plantae

Phylum: Angiosperms

Order: Fabales

Family: Fabaceae

Genus: Phaseolus

Species: P.vulgaris

The genus Phaseolus vulgaris includes all species of legume seeds normally known as common beans. Archeological investigations showed that common beans originated on the American

Continent, specifically in southern United States, Mexico, Central America, and the northern part of South America (Gepts and Dpbouk, 1991). In the sixteenth century, the species P. vulgaris was brought into Europe from where it was introduced into Africa and has become a very important crop in many regions of the world. Dry common bean is a legume widely consumed

throughout the world and it is recognized as the major source of dietary protein in many African countries (Guzman-Maldonado and Paredes-Lopez, 1998)

Phaseolus vulgaris L. is the most important legume worldwide for direct human consumption.

The crop is consumed principally for its dry (mature) beans, shell beans (seeds at physiologicalmaturity), and green pods. When consumed as seed, it beans constitute an important source of dietary protein (22% of seed weight) that complements cereals for over half a billion people mainly in Latin America (Gepts, 2001).Phaseolus vulgaris L. (Red Kidney

Beans) have a notable place in traditions around the world and in the traditions of many cultures such as its anti-diabetic activity (Campillo 2004; Carai et al.,2009; Mishra et al., 2010). A large variability exists in common bean seeds; color and size are two important quality characteristics for the consumers. Seed size and weight depend on genetic variations, cultivar and environmental conditions (Gonzalez de Mejia et al., 2005). The seed color of beans is determined by the presence and concentration of flavonol glycosides, anthocyanins, and tannins

(Beninger and Hosfield, 2003; Aparicio-Fernandez et al., 2005).

Recently, P. vulgaris is gaining increasing attention as a functional or nutraceutical food, due to its rich variety of phytochemicals with potential health benefits such as proteins, amino acids, complex carbohydrates, dietary fibers, oligosaccharides, phenols, saponins, flavonoids, alkaloids, tannins, among others (Geil and Anderson, 1994; Mishra et al.,2010). Important biological activities have been described for fibers, phenolic compounds, lectins, trypsin inhibitors, and phytic acid from common beans (Queiroz-Monici et al., 2005); antioxidant (Heimler et al.,

2005); anticarcinogenic (Hangen and Bennink, 2002) effects. P. vulgaris seeds have a notable place in the folklore throughout the world and in the traditions of many cultures such as pharmacotherapeutic effects (Hangen and Bennink, 2002; Mishra et al., 2010)

1.2 HYPERLIPIDEMIA

It is the most common form of dyslipidemia (which is an abnormal amount of lipids in the blood). It is an abnormally elevated level of any or all lipidsand/or lipoproteins in the blood

(Dorlands, 2007). It has been ranked as one of the greatest risks factors contributing to the general development and severity of coronary heart diseases (Barney et al., 1968). It has been postulated that in many individuals excess weight gives rise to cardiovascular disease, Type 2 diabetes, hypertension, stroke, osteoarthritis and some forms of cancers (Barney et al., 1968).

The lipoproteins that are elevated in hyperlipidemia are a biochemical assembly that contains both proteins and lipids, bound to the proteins, which allows fats to move through the water inside and outside the cells. Examples include the plasma lipoprotein particles such as high density (HDL) and low density lipoproteins(LDL), which enable fats to be carried in the blood stream, the transmembrane proteins of the mitochondrion and the chloroplast and bacterial lipoproteins. Many enzymes, transporters, structural proteins, antigens, adhesions and toxins are lipoproteins. (Madan et al.,2003).

1.2.1 LIPOPROTEINS

Lipids are transported in the circulation packaged in lipoproteins. Lipoproteinsare complex aggregates of lipids and proteins that render the lipids compatible withthe aqueous environment of body fluids and enable their transport throughout the body to tissues where they are required(Christie, 2014).

Structure of lipoproteins

Lipoproteins are assembled from polar and neutral lipids, as well as specific proteins, which are referred to as apoproteins or apolipoproteins as seen in figure 1. Apolipoproteins are amphiphilic proteins capable of interacting with both lipids and the surrounding aqueous environment of the plasma. The principal lipid components of lipoproteins include the non-polar lipids, triglycerides and cholesteryl esters, and the polar lipids, phospholipids and unesterified cholesterol (Small,

1986).

Figure 2: Showing structure of lipoprotein (adapted from Ginsberg, 2015)

1.2.2 CLASSIFICATION OF LIPOPROTEINS

Lipoproteins are classified according to their density. The lowest density lipoproteins are the chylomicrons followed by the chylomicron remnants, very low density lipoproteins VLDLs, intermediate density lipoproteins IDLs, low density lipoproteins, LDLs, and high density lipoproteins, HDLs (Bryant, 2003).

The lipoproteins densities are related to the relative amounts of lipids to proteins in the complex.

The higher the protein content the higher the density of the lipoprotein(Bryant, 2003). Density is determined largely by the relative concentrations of triacylglycerols, proteins and by the diameters of the broadly spherical particles, which varyfrom about 6000A in CM to 100A or less in the smallest HDL (Christie, 2014).

Chylomicrons

Chlyomicrons are large and have the lowest protein to lipid ratio and hence have thelowest density of all the lipoproteins. Dietary lipids are carried from the intestinal mucosa cells to other tissues by lipoproteins called chylomicrons. Chylomicrons contain phospholipids and proteins on the surface sothat the hydrophilic surfaces are in contact with water. The hydrophobic molecules are enclosed in theinterior (Bryant,2003).

The chylomicrons are transported via the intestinal lymphatic system and enter the blood stream at the left subclavian vein. During circulation throughout the body, triacylglycerols are removed by the peripheral tissues by endothelial-bound lipoprotein lipase with entry of fatty acids into muscle for energy production and adipocytes for storage (Christie, 2014).

Very low density lipoproteins The triacylglycerols of the remnant chylomicrons, together with cholesterol and cholesterol esters, are secreted by the liver into the circulation in the form of VLDL, which contain one molecule of the full-length form of apo B, apo B100 (Christie, 2014). In addition, an appreciable amount of triacylglycerol in VLDL is synthesised in the liver from free fatty acids reaching it from adipose tissue via the plasma in thepost-absorptive and fasted states. In effect, VLDL serves to buffer the plasma free fatty acids released following lipolysis in adipose tissue in excess of the requirements of muscle and liver (Christie, 2014).

Low density lipoproteins

LDL are the main carriers of cholesterol to the adrenals and adipose tissue, where there are receptors requiring apo B100 that are able to take in the LDL by a similar process to that occurring in liver(Christie, 2014). LDLs bind to specific cell receptors located on the plasma membrane of target cells known as LDL receptor. This LDL binding domain has electrostatic interactions with the positively chargedarginine and lysine residues of apo-B100(Bryant, 2003).

Within these tissues, the cholesterol esters are hydrolysed to yield free cholesterol, which is incorporated into the plasma membranes as required(Christie, 2014). Any excess cholesterol is re-esterified by an acyl-CoA-cholesterol acyltransferase for intracellular storage. Other peripheral tissues havemuch lower requirements for cholesterol, but that delivered by the LDL may be helpful in suppressing synthesis of cholesterol de novo within cells. It may also inhibit the expression of lipoprotein receptors (Christie, 2014).

High density lipoproteins

HDL are the most complicated and diverse of the lipoproteins, as they contain many different protein constituents, whose main purpose is to enable secretion of cholesterol from cells,esterification of cholesterol in plasma, transfer of cholesterol to other lipoproteins, and the return of cholesterol from peripheral tissues to the liver for excretion – a process that has been termed ‘reverse cholesterol transportHDLs are secreted by liver and intestinal cells(Christie,

2014).

The nascent HDLs are disked shaped, but they become spherical as they acquire free cholesterol from cell membranes and triacylglycerols from other lipoproteins(Bryant, 2003).The primary function of HDLs is to remove excess cholesterol and carry the excess to the liver to be metabolized into bile salts. This function of cholesterol removal from the tissues underlies the inverse relationship between the plasma concentration of HDLs and the incidence of heart diseases. HDL is commonly called the good cholesterolwhen HDL is actually the transporter of plasma cholesterol back to the liver. HDL contains enzymes that either esterify cholesterol or transfer cholesteryl esters. Lechithin-cholesterol transferase (LCAT) is a peripheral enzyme that circulates with HDL(Bryant, 2003).

1.2.3 LIPOPROTEINS METABOLISM

The handling of lipoproteins in the body is referred to as lipoprotein metabolism. It is divided into two pathways, exogenous and endogenous, depending in large part on whether the lipoproteins in question are composed chiefly of dietary (exogenous) lipids or whether they originated in the liver (endogenous) (Skylight Biotech, 2013)

Exogenous pathway

Over 95% of dietary lipids are triglycerides; the rest are phospholipids, free fatty acids (FFAs), cholesterol (present in foods as esterified cholesterol), and fat-soluble vitamins. Dietary triglycerides are digested in the stomach and duodenum into monoglycerides (MGs) and FFAs by gastric lipase, emulsification from vigorous stomach peristalsis, and pancreatic lipase(Goldberg, 2015).Dietary cholesterol esters are de-esterified into free cholesterol by these same mechanisms. MGs, FFAs, and free cholesterol are then solubilized in the intestine by bile acid micelles, which shuttle them to intestinal villi for absorption. Once absorbed into enterocytes, they are reassembled into TGs and packaged with cholesterol into chylomicrons, the largest lipoproteins(Goldberg, 2015).

As they circulate through the lymphatic vessels, nascent chylomicrons bypass the liver circulation and are drained via the thoracic duct into the bloodstream. The hydrolyzed chylomicrons are now considered chylomicron remnants. The chylomicron remnants continue circulating until they interact via apolipoprotein E with chylomicron remnant receptors, found chiefly in the liver. This interaction causes the endocytosis of the chylomicron remnants, which are subsequently hydrolyzed within lysosomes. Lysosomal hydrolysis releases glycerol and fatty acids into the cell, which can be used for energy or stored for later use (Goldberg, 2015).

Endogenous pathway

In the endogenous pathway, the liver assembles and secretes triglyceride-rich very low-density lipoprotein (VLDL) particles, which transport triglycerides from the liver to peripheral tissues.

After hydrolysis of the triglycerides by LPL, the VLDL particles are reduced to intermediate- density lipoproteins (IDL), which can be taken up by the liver or can be further hydrolyzed to

LDL particles. During this conversion, the particles become depleted of triglycerides but retain considerable amounts of cholesterol (Eisenberg et al., 1973).

LDL circulates and is absorbed by the liver and peripheral cells. Binding of LDL to its target tissue occurs through an interaction between the LDL receptor and apolipoprotein B-100 or E on the LDL particle. Absorption occurs throughendocytosis, and the internalized LDL particles are hydrolyzed within lysosomes, releasing lipids, chiefly cholesterol (Wikipedia, 2015)

Figure 3: Showing diagram of lipoprotein metabolism (Adapted from Chhabra, 2012)

1.2.4 CLASSIFICATION OF HYPERLIPIDEMIA

Hyperlipidemias may basically be classified as either familial (also called primary) caused by specific genetic abnormalities, or acquired (also called secondary) when resulting from another underlying disorder that leads to alterations in plasma lipid and lipoprotein metabolism .Also, hyperlipidemia may be idiopathic, that is, without known cause (Chait and Brunzell,

1990).Hyperlipidemias are also classified according to which types of lipids are elevated, that is hypercholesterolemia, hypertriglyceridemiaor both in combined hyperlipidemia.

Familial (primary)

This is a disease in which the person inherits defective genes for the formation of LDL receptors on the membrane surfaces of the body’s cell (Brown and Goldstein, 1999). In the absence of these receptors, the liver cannot absorb eitherintermediate-density or low densitylipoprotein

(William, 2008). Without this absorption, the cholesterol machinery of the liver cells goes on a rampage, producing new cholesterol; it is no longer responsive to the feedback inhibition of too much plasma cholesterol concentration, the number of VLDLs released by the liver into the plasma increases immensely as results (Adiels et al., 2008).

Familial hyperlipidemias are classified according to the Fredrickson classification, which is based on the pattern of lipoproteins on electrophoresis or ultracentrifugation (Fredrickson and

Less 1965). It does not directly account forHDL, and it does not distinguish among the different genes that may be partially responsible for some of these conditions.This classification can be divided into various groups as shown in Table 1 below. Patients with full blown familial hypercholesterolemia may have blood cholesterol concentrations of 600 – 1000mg/dl, levels that are four to six times normal (American Heart Association, 2009). Many of these people die

before age 20 because of myocardial infarction or other sequel of atherosclerotic blockage of blood vessels throughout the body (Bugger and Abel, 2008).

PHENOTYPE GENERIC ELEVATED ELEVATED LIPID

DESIGNATION LIPOPROTEIN CLASS

CLASS

I Exogenous hyperlipidemia Chylomicrons Triglycerides

IIA Hypercholesterolemia LDL Cholesterol

IIB Combined hyperlipidemia LDL,VLDL Cholesterol, triglycerides

III Remnant hyperlipidemia IDL Triglycerides, Cholesterol

IV Endogenous VLDL Triglycerides

hyperlipidemia

V Mixed hyperlipidemia VLDL, Triglycerides, Cholesterol

Chylomicrons

Table 1: Showing Fredrickson classification of hyperlipidemiaAdapted from (Fredrickson andLess1965;Dorlands2007)

Acquired (secondary)

Acquired hyperlipidemias (also called secondary dyslipoproteinemias) often mimic primary forms of hyperlipidemia and can have similar consequences(Chait and Brunzell, 1990). They may result in increased risk of premature atherosclerosis or, when associated with marked hypertriglyceridemia,may lead to pancreatitis and other complications of the chylomicronemia syndrome (Chait and Brunzell, 1990).

Epdiemology

Worldwide, a third of ischaemic heart disease is attributable to high cholesterol. Overall, 2.6 million deaths (4.5% of total) and 29.7 million disability adjusted life years (DALYS), or 2.0% of total DALYS is estimated to be as a result of raised cholesterol. Raised total cholesterol is a major cause of disease burden in both the developed and developing world as a risk factor for

Ischemic heart disease and stroke (World Health Organization, 2015).

Earlier studies from Nigeria reported that dyslipidemia was rare amongst Nigerians.

Onyemelukwe and Stafford in 1981 suggested that protective cholesterol (HDL-C) was significantly higher in tropical Africa,while Kesterloot et al. in Benin, South South Nigeria in

1989 showed that blacks had a low prevalence of dyslipidemia (Kesteloot et al., 1989).However recent studies show that the prevalence of dyslipidemia in apparently healthy professionals in

Asaba, South South Nigeria, is on the increase. The studies found a very high prevalence of dyslipidemia (60.0%) with low HDL-C being the commonest pattern of dyslipidemia (60%).

Other patterns of dyslipidemia reported were high LDL-C (51%), TC (23%) and TG (5%) levels

(Odenigbo and Oguejiofor, 2008).

There has also been reported that high prevalence of dyslipidemia can be found among individuals of upper social class attending Igbinedion Hospital and Medical Research Centre,

Okada in South-South Nigeria. Most of the subjects (60.4%) had hypercholesterolemia while

22.6% had elevated TG (Agboola-Abu and Onabolu, 2000).

Diagnosis

In an effort to introduce the term “metabolic syndrome” into clinical practice for hyperlipidemia, several organizations, including the World Health Organization, National Cholesterol Education

Program, and International Diabetes Foundation, has proposed criteria for its diagnosis. These criteria which are also required by the American Heart Association proposed that at least three of the following criteria for the diagnosis of the metabolic syndrome:

• Waist circumference of at least 40 inches (102 cm) in men or 35 inches (89 cm) in

women, measured at the top of the iliac crest at the end of a normal expiration

• Triglyceride level of at least 150 mg per dL (1.70 mmol per L), or receiving

pharmacologic therapy for elevated triglyceride levels

• HDL cholesterol level of less than 40 mg per dL (1.05 mmol per L) in men or less

than 50 mg per dL (1.30 mmol per L) in women, or receiving pharmacologic therapy

for reduced HDL cholesterol levels

• Systolic blood pressure of at least 130 mm Hg or diastolic blood pressure of at least

85 mm Hg, or receiving pharmacologic therapy for hypertension

• Fasting glucose level of at least 100 mg per dL (5.6 mmol per L), or receiving

pharmacologic therapy for elevated fasting glucose levels.(CARRIE 2006)

Signs and symptoms

There are usually no symptoms of hyperlipidemia in the early years. Uncommonly, hyperlipidemia can manifest with yellowish nodules of fat in the skin beneath eyes, elbows and knees, and in tendons; sometimes a large spleen and liver occur, or whitish rings around the eye's iris occur. Longstanding elevated cholesterol is typically associated with cardiovascular disease and therefore, can lead to heart attack, stroke and/or peripheral vascular disease(American Heart

Association, 2015)

1.2.5 MANAGEMENT OF HYPERLIPIDEMIA

Hyperlipidemia is a common risk factor for the development of cardiovascular disease. The

Adult Treatment Panel III (ATP III) of the National Cholesterol Education Program has for the past decade recommended non-pharmacologic treatment as initial therapy in most patients with hyperlipidemia (National Cholesterol Education Program, 2001),before incorporation of pharmacological treatment, if and when necessary.

1.2.5.1 NON-PHARMACOLOGICAL INTERVENTION

This approach to management of hyperlipidemia can be termed therapeutic life style changes and in based on panel's review of the available evidence in 1999 that concluded that diet and exercise can have a beneficial effect on serum levels of total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, and triglycerides (Robert, 2010).

The TLC diet recommendations include obtaining 25 to 35 percent of daily calories from fats, and restricting saturated fats to less than 7 percent of total calories and cholesterol to less than

200 mg per day. However, physicians and patients are often unsure of how much change in blood lipid levels can be expected when the TLC diet is prescribed, and wonder which lifestyle

changes have the greatest effects (Kelly, 2010). A large number of dietary factors may influence lipid levels. These include modification of nutritional components, consumption of specific foods, use of food additives and supplements, and major dietary approaches (Robert, 2010).

1.2.5.2 PHARMACOLOGICAL INTERVENTIONS

Clinical trials of hyperlipidemia therapy should address outcomes that matter most to patients, such as morbidity, mortality, quality of life, and cost, rather than stressing disease-oriented evidence, such as the ability to reduce cholesterol levels (Robert, 2010).Antihyperlipidemic drug therapy is used to lower serum levels of cholesterol and triglycerides. The primary health care provider may use one drug or, in some instances, more than one antihyperlipidemic drug for those with poor response to therapy with a single drug. Three types of antihyperlipidemic drugs are currently in use, as well as one miscellaneous antihyperlipidemic drug. The various types of drugs used to treat hyperlipidemia are:

• Bile acid sequestrants

• HMG-CoA reductase inhibitors

• Fibric acid derivatives

• Niacin

The target LDL level for treatment is less that 130 mg/dL. If the response to drug treatment is adequate, lipid levels are monitored every 4 months. If the response is inadequate, another drug or a combination of two drugs is used. Antihyperlipidemic drugs decrease cholesterol and triglyceride levels in several ways. Although the end result is a lower lipid blood level, each has a slightly different action.

HMG-COA REDUCTASE INHIBITORS

HMG-CoA reductase (3-hydroxy-3-methyl-glutaryl-CoA reductase) isthe rate- controlling enzyme of the mevalonate pathway, the metabolic pathway that produces cholesterol and other isoprenoids. This group of antihyperlipidemic agents inhibits the first committed enzymatic step of cholesterol synthesis, and they are the first line and more effective treatment for patients with elevated LDL cholesterol (Harvey et al., 2009). Normally in mammalian cells this enzyme is suppressed by cholesterol derived from the internalization and degradation of low density lipoprotein (LDL) via the LDL receptor as well as oxidized species of cholesterol.

Competitive inhibitors of the reductase induce the expression of LDL receptors in the liver, which in turn increases the catabolism of plasma LDL and lowers the plasma concentration of cholesterol, an important determinant of atherosclerosis (Enterz Gene)

Drugs that inhibit HMG-CoA reductase, known collectively as HMG-CoA reductase inhibitors (or "statins"), are used to lower serum cholesterol as a means of reducing the risk forcardiovascular disease (Farmer, 1998). These drugs include lovastatin, atorvastatin, pravastatin, simvastatin, pitavastatin, rosuvastatin and fluvastatin (John Hopkins, 2004). Red yeast rice extract, one of the fungal sources from which the statins were discovered, contains several naturally occurring cholesterol-lowering molecules known as monacolins (Lin et al.,2008).

Mechanisms of action

They are analogs of HMG, the precursor of cholesterol; hence they compete effectively to inhibit

HMG-CoA reductase, the rate limiting step in cholesterol synthesis. By inhibiting the de novo cholesterol synthesis they deplete the intracellular supply of cholesterol (Harvey et al., 2009).

Depletion of intracellular cholesterol causes the cell to increase the number of specific cell

surface LDL receptors that can bind and internalize circulating LDLs. Thus the end result is a reduction in plasma cholesterol, both lowered cholesterol synthesis and by increased catabolism of LDL. It can also increase plasma HDL level in some patients (Harvey et al., 2009).Few adverse effects have been associated with the statins which are mainly related to liver and muscle functions. They are contraindicated during pregnancy and in nursing mothers and may also increase the level of warfarin if given concomitantly (Harvey et al., 2009).

FIBRATES

The fibrates are a class of amphipathic carboxylic acids. They are used for a range of metabolic disorders, mainlyhypercholesterolemia(high cholesterol), and are therefore hypolipidemic agents. The magnitude of lipid changes depends, however, on the patient’s pretreatment lipoprotein status(Tikkanen, 1992). They include benzafibrates, ciprofibrates, gemfibrozil, fenofibrates and clofibrates.

Mechanism of action

The primary mode of action of the fibrates is via activation of the nuclear transcription factor

PPARα, predominantly expressed in tissues that metabolise fatty acids, such as the liver, kidney, heart and muscle (Chapman, 2003, Fazio and Linton, 2004). Fibrates may also interact with other

PPAR receptors to varying degrees (Chapman, 2003).On activation by binding of the fibrate,

PPARα binds as heterodimers with RXR, which subsequently recognizes and binds to specific

PPARαresponse elements leading to modulation of expression of the target genes.

Fibrates should be avoided in patients and alcoholics who are rediposed to hypertriglyceridaemia and are at risk of rhabdomyolysis. Myositis can also occur; although unusual it can be severe

(Rang et al., 2003). It should not be taken together with coumarin and its safety in regnant and

lactating mothers has not been established. It should also be avoided in patients with severe hepatic and renal dysfunction(Harvey et al., 2009).

BILE ACID BINDING RESINS

Bile acid sequestrants such as cholestryramine, colestipol and colesevelam are medications for lowering LDL cholesterol in conjunction with diet modification (Ogbru and Marks, 2015)

Mechanisms of action

They are anion-exchange resins that binds negatively charged bile acid and bile salts in the small intestine. The resins bile complex is excreted in the feaces, thus preventing the bile acids from returning to the liver by the entrerohepatic circulation (Harvey et al., 2009).

Lowering the bile acid concentration causes hepatocytes to increase conversion of cholesterol to bile acids, resulting in a replenished supply of these compounds, which are essential components of the bile. Consequently, the intracellular cholesterol concentration decrease, which activates an increased hepatic uptake of cholesterol-concentrating LDL particles, leading to a fall in plasma

LDL (Harvey et al., 2009).In some patients, a modest rise in plasma HDL levels is also observed. The final outcome of this sequence of events is a decreased total plasma cholesterol concentration. Systematic toxicity is low, since resins are not absorbed but gastrointestinal symptoms of nausea, abdominal bloating. They can also interfere with the absorption of fat- soluble vitamins, and of drugs such as chlorothiazide, digoxin and warfarin (Rang et al., 2005).

NIACIN (NICOTINIC ACID)

Niacin can reduce LDL levels by 10 to 20 percent and is the most effective agent for increasing

HDL levels. Niacin can be used in combination with statins, and a fixed dose combination of lovastatin and long acting niacin is available (Harvey et al., 2009).

Niacin (also known as vitamin B3 or nicotinic acid) is an organic compound, this colorless, water-soluble solid is a derivative of pyridine, with a carboxyl group at the 3-position. Nicotinic acid and niacinamide are convertible to each other with steady world demand rising from 8,500 tonnes per year in the 1980s to 40,000 in recent years (Cantarella et al., 2011).

Mechanisms of action

Niacin therapeutic effect is mostly through its binding to G protein coupled receptors, niacin receptor 1 (NIACR1) and niacin receptor 2 (NIACR2), that are highly expressed inadipose tissue, spleen, immune cells and keratinocytes but not in other expected organs such as liver, kidney, heart or intestine (Soga et al., 2003; Gass, 2003).

NIACR1 and NIACR2 inhibit cyclic adenosine monophosphate (cAMP) production and thus fat breakdown in adipose tissue and free fatty acids available for liver to produce triglycerides and very-low-density lipoproteins (VLDL) and consequently low-density lipoprotein (LDL) or "bad" cholesterol (Gille et al., 2008.,Wanders and Judd 2011). Decrease in free fatty acids also suppress hepatic expression of apolipoprotein C3(APOC3) and PPARγ coactivator-1b (PGC-1b) thus increase VLDL turn over and reduce its production (Hernandez et al., 2010)It also inhibits diacylglycerol acyltransferase-2 (important hepatic TG synthesis).

The mechanism behind increasing HDL is not totally understood but it seems to be done in various ways. Niacin increases apolipoprotein A1 levels due to anti catabolic effects resulting in higher reverse cholesterol transport. It also inhibits HDL hepatic uptake, down-regulating production of the cholesterol ester transfer protein (CETP) gene (Villineset al., 2012). Finally, it stimulates the ABCA1 transporter in monocytes and macrophages and up-regulates peroxisome proliferator-activated receptor γ results in reverse cholesterol transport (Rubic et al., 2004)

The most common adverse effects of niacin therapy are an intense cutaneous flush and pruritus.

Administration of aspirin prior to taking niacin decreases the flush. Niacin inhibits tubular secretion of uric acid and thus, predisposes to hyperuricemian and gout. Impaired glucose tolerance and hepatotoxicity have been reported (Harvey et al., 2009).

CHOLESTEROL ABSORPTION INHIBITORS

Cholesterol absorption inhibitors are a class of compounds that prevents the uptake of cholesterol from the small intestine into the circulatory system (Patrick et al., 2002, Dujovne et al., 2002). An example is ezetimibe

Mechanisms of action

They selectively inhibit instestinal absorption of dietary and biliary cholesterol in the small intestine, leading to decrease in the delivery of intestinal cholesterol to the liver. This causes a reduction of hepatic cholesterol stores and an increase in clearance of cholesterol from the blood

(Harvey et al., 2009).Ezetimibe is rapidly and extensively conjugated by uridine diphosphate- glucuronosyltransferase in the intestine and is then quickly circulated to the liver and returned to the intestines via the bile; with only minimal urinary excretion (Zhu et al., 2000).The estimated effective half-life of ezetimibe in humans is 24 h (Zhu et al., 1999). Accumulation within the epithelial cell of the small intestine, high extraction from the portal venous blood, and rapid enterohepatic recycling appear to cause localization of ezetimibe within the intestinal lumen, with a small associated systemic exposure (Van Heek et al.,2000).Ezetimbe has no clinically meaningful effect on the plasma concentrations of the fat soluble vitamins. Patients with moderate to severe hepatic insufficiency should not be treated with ezetimble (Finkel et al.,

2009).

OTHER THERAPY

Other substances used as hypolipidemic therapy are probucol and fish oil. Probucol lowers the concentration in the plasmaof both LDLand HDL. Its place in therapy has not been defined. It has distinctive properties that could be either desirable (e.g. antioxidant properties) or the reverse

(e.g. lowering HDL and prolonging cardiac action potential). Its mechanisms of action are not understood and it should be avoided in patients with a long Q-T interval (Rang et al., 2005).

Fish oil (Omega-3)reduces plasma triglycerides concentration but increase cholesterol. Plasma triglycerides concentrations are less strongly associated with coronary artery disease than is cholesterol and an effect of fish oil on cardiac morbidity or mortality is unproven. The mechanisms of action are also unknown. Although there is epidemiological evidence that eating fish regularly does reduce ischemic heart disease. Fish oil is contraindicated in patients with type

IIa hyperlipoproteinaemia because of the increase in LDL-cholesterol that it causes (Rang et al.,

2005).

COMBINATION DRUG THERAPY

Combination regimens should be considered for use in patients who fail to meet target values and are compliant with their current therapy. Patients with moderate to severe hypercholesterolemia generally have more difficulty reaching their goals, although considerable reductions in LDL do occur (Safeer and Lacivita, 2000). If further reductions in LDL are required, and the benefits outweigh the risks, a statin in conjunction with niacin is an effective combination.More patients reach their lipid goals with a statin and niacin than with any other combination regimen(Safeer and Lacivita, 2000). Myopathy and rhabdomyolysis are serious concerns when statins are combined with fibrates or niacin. It is important for physicians to

educate their patients about the adverse effects of these medications and instruct them to report muscle pain or tenderness immediately.

Compliance with bile acid sequestrants is a problem. Approximately one third of the patients for whom bile acid sequestrants are prescribed will not take the full dosage because of constipation or poor palatability.Combination therapies that include a bile acid sequestrant have lower success rates than other combinations.The statins have the best compliance or maintenance rates, followed by niacin, gemfibrozil and bile acid sequestrants (Schectman and Hiatt., 1996). Despite the use of single or combined drug therapy to treat hypercholesterolemia, only 50 percent of treated patients reach the lipid goals outlined in the NCEP recommendations (Schectmanand

Hiatt, 1996). Combination therapy increases the likelihood of reaching target values, but poor compliance is a variable that can foil even the most aggressive therapeutic interventions (Safeer and Lacivita, 2000).

1.3 ANTIOXIDANTS

Antioxidants are substances protect cells from the damaged caused by unstable molecules known as free radicals. Antioxidants interact with and stabilize free radicals and may prevent some of the damaged free radicals might otherwise cause. Free radical damaged may lead to cancer.

Examples of antioxidants include beta-carotene, lycopene, vitamins C, E, A and other substances

(Sies, 1997).They may are molecules that neutralize the effect of free radicals that damage living cells (Hennekens and Cheung, 1994).An antioxidant is a molecule that inhibits the oxidation of other molecules. Oxidation is a chemical reaction involving the loss of electrons or an increase in oxidation state.

Oxidation reactions can produce free radicals. In turn, these radicals can start chain reactions.

When the chain reaction occurs in a cell, it can cause damage or death to the cell. Antioxidants

terminate these chain reactions by removing free radical intermediates, and inhibit other oxidation reactions. They do this by being oxidized themselves, so antioxidants are often reducing agents such as thiols, ascorbic acid, or polyphenols (Sies, 1997). Antioxidants are widely used in dietary supplements and have been investigated for the prevention of diseases such as cancer, coronary heart disease and even altitude sickness (Bazllie et al., 2009). Although oxidation reactions are crucial for life, they can also be damaging; plants and animals maintain complex systems of multiple types of antioxidants, such as glutathione, vitamin C, vitamin A, and vitamin E as well as enzymes such as catalase, superoxide dismutase and various peroxidases. Insufficient levels of antioxidants or inhibition of the antioxidant enzymes, cause oxidative stress and may damage or kill cells. Antioxidants are abundant in nature and are presents in vegetables and fruits, they can also be found in other foods such as meats, poultry, nuts and fish (Borek, 1991).

1.3.1 CLASSIFICATION OF ANTIOXIDANTS

Antioxidants are grouped into two namely, primary or natural antioxidants and secondary or synthethic antioxidants (Hamid et al., 2010).

Primary or natural antioxidants

They are the chain breaking antioxidants which react with lipid radicals and convert them into more stable products. Antioxidants of this group are mainly phenolic in structures and include the following (Hurrell, 2003):

(1) Antioxidants mineral – these are co factor of antioxidants enzymes. Their absence will definitely affect metabolism of many macromolecules such as carbohydrates. Examples include selenium, copper, iron, zinc and manganese

(2) Antioxidants vitamin – it is needed for most body metabolic functions. They include vitamin

C, vitamin E, vitamin B.

(3) Phytochemicals- These are phenolic compounds that are neither vitamins nor minerals. These include flavonoids, catechin, carotenoids, betacarotene, and lycopene e.t.c.

Secondary or synthethic antioxidants

These are phenolic compounds that perform the function of capturing free radicals and stopping the chain reactions, the compound include (Hurrell, 2003)

i. Butylated hydroxyl anisole (BHA).

ii. Butylated hydroxyrotoluene (BHT) iii. Propyl gallate (PG) and metal chelating agents (EDTA) iv. Tertiary butyl hydroquinone (TBHQ)

v. Nordihydro guaretic acid (NDGA)

1.3.2 TYPES OF ANTIOXIDANTS

Ascorbic acid

Ascorbic acid or vitamin C is a monosaccharide antioxidant found both in animals and plants. As one of the enzymes needed to make ascorbic acid has been lost by mutation during human evolution, it must be obtained from diet and is a vitamin. Most other animals are able to produce this compound in their bodies and do not require it in their diets. In cells, it is maintained in its reduced form by reaction with isomerase and glutaredoxins. Ascorbic acid is a reducing agent

and can reduce and thereby neutralize reactive oxygen species such as hydrogen peroxide

(Ortega, 2006; Antioxidants and Cancer Prevention, 2007).

Glutathione

Glutathione is a cysteine- containing peptide found in most forms of aerobic life. It is not required in the diet and is instead synthesized in cells from its constitutent amino acids.

Glutathione has antioxidant properties since the thiol group in its cysteine moiety is a reducing agent and can be reversibly oxidized and reduced. In cells Glutathione is maintained in the reduced form by the enzyme Glutathione reductase and is turn reduces other metabolites and enzymes systems, such as ascorbates in Glutathione-ascorbate cycle, Glutathione peroxidases and glutaredoxins, as well as reacting directly with oxidants (Meister and Anderson, 1983). Due to its high redox state, Glutathione is one of the most important cellular antioxidants.

Tocopherols

Vitamin E is the collective name for a set of eight related tocopherols, which are fat soluble vitamins with antioxidants properties. Of these, tocopherol has been most studied as it has the highest bioavailability; with the body preferentially absorb and metabolizing this form (Herrera and Barbas, 2001).

It has been claimed that the α-tocopherol form is the most important lipid soluble antioxidant and that it protects membranes from oxidation by reacting with lipid radicals produced in the lipid peroxidation chain reaction.

Antioxidants system in our body

The body has developed several endogenous antioxidant systems to deal with the production of

ROI. These systems can be divided into enzymatic and non-enzymatic groups.The enzymatic antioxidants system include Catalase, which convert H2O and O2-, and glutathione peroxidase, which reduces H2O2 and superoxide dismutase (SOD), which catalyses the conversion of O2-O to

H2O2 and H2O.The non-enzymatic antioxidants include lipid soluble vitamins, vitamin E and vitamin A or provitamin A (beta-carotene) and water soluble vitamin C. (Amitom, 2001)

1.3.3 OXIDATIVE STRESS

Inflammation is an important protective response to cellular injury, which destroy and remove the injurious agent and injured tissue, thereby promoting tissue repair. When this crucial and normally beneficial response occurs in an uncontrolled led manner, the result is excessive cellular damage which causes chronic inflammation and destruction of normal tissues by enhancing oxidative stress (Rahman, 1996). The excessive release of free radical species in an inflammatory condition may lead to damage of living cells and tissue in the body and can be referred to as “Oxidation Stress”.

However, inflammation is an important protective response to cellular injury and defense mechanism to destroy invasive pathogens and remove damaged tissues. It can be said that inflammation are sites of increased free radical production (Frevel, 2006). In the acute inflammatory response, the production of this entire battery of free radicals is an important mechanism. However, the problem begins when the inflammatory process persist and becomes chronic, because the inherent high level of oxidative stress turns against the healthy body tissue.

1.3.4 ANTIOXIDANTS AND CARDIOVASCULAR DISEASES

The oxidative modification of circulating lipoproteins by free radicals, particularly low-density lipoproteins (LDL), is important for the development of atherosclerosis(Steinberg et al., 1989).

Smaller, denser LDL particles, which are known to be a risk factor for cardiovascular diseasemay promote atherogenesis for several reasons(Rajman et al., 1994, Rajman et al., 1996).

These modified LDL particles do not bind readily to the endogenous LDL receptor and are therefore not cleared from the circulation by this mechanism. They penetrate the intima more easily, are more readily oxidized, possibly because they contain less antioxidant protection, and are taken up by the macrophage scavenger receptors, accelerating foam cell formation (figure 2).

This early histoogical feature leads to the development of atherosclerotic plaques (Steinberg et al., 1989, Rajman et al., 1994)

Figure 4:Antioxidants and cardiovascular disease (adapted from Nutall and Martin 1999)

1.4 INDUCTION OF HYPERLIPIDEMIA

In experimental studies, hyperlipidemia is usually produced by dietary manipulation or genetic manipulations. However, it also occurs as a toxic response to certain detergents such as Triton. It is a non-anionic detergent of polymeric (Okazazi et al., 1990) structure that has been

successfully used in several studies to induce hypercholesterolemia (Kourounakis et al.,2002;

Okunevich and Ryzhenkov 2002; Horak et al., 2002).

Several studies have shown that systematic administration of Triton WR1339 (non-ionic detergent) to fasted rats causes an elevation in the plasma lipid levels (Moss and Dajani, 1971).

Initially, there is a twofold or three fold increases in the lipid levels over the control value, 24h after administration of triton (Devi and Sharma, 2004). The increase in the plasma lipids is thought to be either due to increased hepatic synthesis of cholesterol or a physical alteration in

VLDL by triton resulting in their removal from the blood. Drugs interfering with cholesterol synthesis were shown to be active in phase one, while drugs interfering with cholesterol excretion and metabolism were found to be active in Phase I (Devi and Sharma 2004).

A single parenteral administration of Triton to adult rats produces a hyperlipidemia in which cholesterol, triglycerides and phospholipids. The majority of experimental evidence supports the concept that Triton physically alters very low density lipoproteins, rendering them refractive to the action of lipolytic enzymes of blood and tissue. This prevents or delays their removal from blood and secondarily stimulates the synthesis of lipid, enhancing the hyperlipidemia.Garattini et al. and Paoletti suggestedthe use of Triton-induced hyperlipidemiaas an approach to screen for or to differentiatethe mechanism of action of hypolipidemicdrugs. Triton WR-1339 has been widely used to block clearance of triglyceride-rich lipoproteins to induce acute hyperlipidemia in several animals (Schurr et al., 1972). This model is widely used for a number of different aims (Ye-Yun et al., 2005) particularly, in rats it has been used for screening natural or chemical hypolipidemic drugs (Harbowy and Balentine 1996) Schurr et al demonstrated that a parenteral administration of a dose of TritonWR-1339 to 56 adult rats induced hyperlipidemia.

1.4.1 Carbon tetrachloride (CCL4)

Carbon tetrachloride is a non-flammable colorless liquid with a heavy, sweet odour. Before

1970, carbon tetrachloride was widely used as a clearing fluid in homes and industry. CCL4 is still used to manufacture propellants and other industrial chemicals. Carbon tetrachloride is said to evaporate quickly and is heavier than water.Carbon tetrachloride is known to affect almost every functional site of animal tissues, often due to their bioaccumulation and poor excretion

(Okoko, 2009).

Carbon tetrachloride is rapidly absorbed from the gastrointestinal tract through the hepatic portal vein to the hepatocytes where it undergoes drug biotransformation or metabolism. Hepatic disease remains a global health problem. However, an appreciable amount of hepatic protective ethnomedical drug used in the treatment of serious liver disorders remains unresolved. Hence, the search for new drugs is still on-going (Dash et al., 2007).

1.5 STATEMENT OF THE PROBLEM

LDL- cholesterol and have been shown to reduce coronary events and mortality. They have very few side effects and are now usually the drugs of first choice (Neal, 2002).

Although the adverse effect of statins is relatively low, one rare effect called rhabdomyolysis can be very serious (Miller 2001). Statins and fibrates both used in elevated cholesterol, especially in combination; cerivastatin (Baycol) was withdrawn in 2001 after numerous incidence of rhabdomyolysis (Armitage, 2007). There is an urgent need to investigate and develop natural products that have profound lipid lowering effects with minimal side effects.

1.6 AIM OF STUDY

The aim of the study is to evaluate the antihyperlipidemic effects and antioxidants effects of methylene chloride: methanol (50:50) extracts and n-hexane, ethylacetate and butanol fractions of the fruits ofPhaseolus vulgaris L. in Wistar rats.

1.7 OBJECTIVES

i) To evaluate the antihyperlipidemic effects of various fractions of Phaseolus vulgaris L. in

in Wistar rats.

ii) To evaluate the antioxidant effect of various fractions of Phaseolus vulgaris L. using in

vitro and in vivo studies.

iii) To determine which fractions of the extracts have the best lipid lowering effects and

antioxidant properties.

1.8 SIGNIFICANCE OF STUDY

This study is significant as the need has arisen for the search of plants with lipid lowering effects due to the numerous adverse effects and limitation of current therapies. There is also a need for drugs with antioxidant activities which will be beneficial in the prevention of cardiovascular diseases.

1.9 JUSTIFICATION

The use of fresh fruits of Phaseolus vulgaris Lis very popular amongst traditional herbal practitioners in Orba community of Enugu State of Nigeria, as a “potent medicine for heart

diseases”. It has been reported that the benefits of Phaseolus vulgaris L. in the treatment of cardiovascular ailments is related to its hypolipidemic properties (Roman-Ramos et al., 1995).

These facts motivated the present studies in which the antioxidants and lipid lowering effects of the various extracts and solvents fractions of the plant were evaluated in vivo and in vitro.

CHAPTER TWO

MATERIALS AND METHODS

2.1 EXPERIMENTAL ANIMALS

Adult Swiss albino mice weighing (20-25g) and Wistar albino male rats weighing (120 - 200g) were procured from laboratory animal facility of the Department of Pharmacology and

Toxicology, University of Nigeria, Nsukka. Animals were housed in steel cages under standard conditions and fed with standard pellets and water ad libitum. The animals were allowed to acclimatize for two weeks, prior to the commencement of the study.

2.2 PLANT MATERIAL

Fresh fruit of Phaseolus vulgaris L. were obtained from Obollo-Afor in Nsukka Local

Government Area Enugu State, Nigeria and authenticated by Mr. A. Ozioko, a plant taxonomist with Bio-resources Development and Conservation Programme (BDCP) Center, Nsukka, Enugu

State, Nigeria.

The plant materials were sun dried and then pulverized using the laboratory grinding machine at the Department of Crop Science, University of Nigeria, Nsukka. 1kg of the powdered extract

was dissolved in 2.5 L of a mixture of methanol and methylene chloride in the ratio 1:1 to obtain the crude extract (PVE). A portion of the extract was suspended in distilled water and fractions where made by adding solvents with increasing polarity successively i.e. n-hexane (PVHF), ethylacetate (PVEF) and n-butanol (PVBF). The layers were separated accordingly and the fractions were dried.

2.3 PHYTOCHEMICAL SCREENING

Chemical tests for the phytochemistry of Phaseolus vulgaris were carried out using the standard procedures as described by Trease and Evans (1989).

2.4 HPLC FINGERPRINTING

2.4.1 ANALYTICAL HPLC

Pump: P 580A LPG Dionex

Autosampler: ASI-100T (injection volume = 20 μL) Dionex

Detector: UVD 340S (Photodiode array detector) Dionex

Column oven: STH 585 Dionex

Column: Eurospher 100-C18, [5 μm; 125 mm × 4 mm] Knauer

Pre-column: Vertex column, Eurospher 100-5 C18 [5-4 mm] Knauer

Software: Chromeleon (V. 6.30)

2.4.2 PREPARATION OF SAMPLES

About 2 mg of the PVE was reconstituted with 2 mL of HPLC grade methanol. The mixture was sonicated for 10 min and thereafter centrifuged at 3000 rpm for 5 min. 100 μL of the dissolved samples was transferred into HPLC vial containing 500 μL of HPLC grade methanol.

2.5.3 HPLC-DAD Analysis

HPLC analysis was carried on the sample with a Dionex P580 HPLC system coupled to a photodiode array detector (UVD340S, Dionex Softron GmbH, Germering, Germany). Detection was at 235, 254, 280 and 340 nm. The separation column (125 × 4 mm; length × internal diameter) was prefilled with Eurospher-10 C18 (Knauer, Germany), and a lineargradient of nanopure water (adjusted to pH 2 by addition of formic acid) and methanol was used as eluent.

The compounds were detected by comparing the retention times and uv spectral with inbuilt library. Identification was based on library hit similarity of >99%

2.5 ACUTE TOXICITY (LD50):

The acute toxicity (LD50) study was carried on the crude extract of Phaleosus vulgaris L. using

Locke method of (1983). Animals (mice) of either sex were used. Organization of Economic

Corporation and Development’s (OECD) guidelines (OECD, 2000), was used for dosage selection.

In phase I, the mice were placed in three groups (n = 3). Groups 2, 3 and 4 received 10mg/kg,

100mg/kg and 1000mg/kg of the extracts respectively. The animals were observed and the number of death recorded after 24 h.

In phase II, four groups (n = 3) of mice were used. Doses are selected based on the lethality in

Phase I. Since there was no death in Phase 1, higher doses were selected (1600, 2500, 3900, and

5000mg/kg), and the animals were observed and the number of death recorded after 24 h.

The LD50 was calculated using the geometric mean of the highest non-lethal dose and the least toxic dose.

50 = √ℎℎ ℎ ×

2.6 HYPOLIPIDEMIC STUDIES

2.6.1 EXPERIMENTAL DESIGN

Acute, sub-acute and chronic studies to determine the lipid lowering effects of the extract and fractions Phaseolus vulgaris L. were carried out in this study.

2.6.2 ACUTE STUDY OF THE EFFECTS OF EXTRACT AND FRACTIONS OF PVE

ON LIPID PROFILE

In the experimental procedure involving the crude extract (PVE), twenty-five rats were randomly divided into five groups of five rats. Group 1 rats served as the positive control and were treated with 10 mg/kg of atorvastatin, Group 2 (untreated group), Group 3, 4, 5 where treated with the

PVE at 100mg/kg, 200mg/kg and 400mg/kg.

In second phase of this procedure involving fractions of PVE, thirty-five rats were used for the study randomly divided into seven groups of five rats each. Group 1 rats served as the positive control and were treated with 10 mg/kg of atrorvastatin, Group 2 (untreated group), while group

3 and 4 were treated with PVHF fraction at 100mg/kg and 200 mg/kg with group 5 and 6 administered the PVEF fraction at 100mg/kg and 200 mg/kg and group 7 and 8 treated with

PVBF fraction at 100mg/kg and 200mg/kg respectively.

Each group were given various doses of the test drugs, an hour later intra-peritoneal injection of

200mg/kg of Triton WR-1339 was administered to each rats (Khana et al., 2002, Banerjee et al.,

2006., Gajbhiye et al., 2008, Rajasekaran et al., 2013 ). Twenty-four hours after treatment, blood was drawn from the retro-orbital plexus of the rats and centrifuged at 4000 r.p.m. for 10 min, after which the serum was collected and biochemical analysis was done using randox lipid profile kit.

2.6.3 SUB-ACUTE STUDY OF THE EFFECTS OF EXTRACT AND FRACTIONS OF

PVE ON LIPID PROFILE

PVE at 100mg/kg, 200mg/kg and 400mg/kg.

In experimental procedure involving fractions, thirty-five rats were randomly divided into seven groups of five rats each. Group 1 rats served as the positive control and were treated with 10 mg/kg of atrorvastatin, Group 2 (untreated group), while group 3 and 4 were treated with PVHF fraction at 100mg/kg and 200 mg/kg with group 5 and 6 administered the PVEF fraction at

100mg/kg and 200 mg/kg and group 7 and 8 treated with PVBF fraction at 100mg/kg and

200mg/kg respectively.

Hyperlipidemia was induced using Triton WR-1339 at 100mg/kg to all rats in each groups, after

72h of triton administration, the test extract was administered for 7days, on the 8th day the animals where sacrificed with blood collected and centrifuged at 4000 r.p.m for 10 min, after which the serum was collected and biochemical analysis (Mohan et al., 2013)

2.6.4 CHRONIC STUDY OF THE EFFECTS OF EXTRACT AND FRACTIONS OF

PVE ON LIPID PROFILE

PVE at 100mg/kg, 200mg/kg and 400mg/kg respectively.

100mg/kg and 200 mg/kg and group 7 and 8 treated with PVBF fraction at 100mg/kg and

200mg/kg respectively.

Three rats were drawn at random from each group and analysis where done to determine the baseline value of lipid profile parameters. Each group was given a high fatty diet throughout the experimental duration (4weeks), after the first week the same three rats from each groups were tested to determine the level of induction of hyperlipidemia.

After satisfactory hyperlipidemic state where achieved all rats were given various doses of the test drugs for three weeks after which they were sacrificed with blood collected and centrifuged at 4000 r.p.m for 10 min, after which the serum was collected and biochemical analysis done.

The high fat diet comprised of the chow enriched with high calorie and 1%cholesterol. High fat diet induced hyperlipidemia is one of the common method to induce hyperlipidemia (Chandratre et al., 2011).

2.6.5 BIOCHEMICAL DETERMINATION OF LIPID PROFILE PARAMETERS

Lipid profile parameters were determined using biochemical kit from randox laboratories United

Kingdom.

2.6.5.1 DETERMINATION OF TOTAL CHOLESTEROL LEVEL

PRINCIPLE: The cholesterol is determined after enzymatic hydrolysis and oxidation. The indicator quinoneimine is formed from hydrogen peroxide and 4-aminoantipyrine in the presence of phenol and peroxidase (Trinder, 1969).

Cholesterol ester + H2O cholesterol esterase cholesterol + fatty acids

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

2H2O2 + Phenol + 4-Aminoanitipyrine peroxidase Quinoeimine + 4H2O

PROCEDURE: pipette into the respective tubes the following as shown below

Blank Standard Sample

Distilled H2O 10 - -

Standard - 10 ul -

Sample - - 10ul

Reagent R1 1000 ul 1000 ul 1000 ul

The following tubes were mixed and incubated for 10 min at 250C. The absorbance of the sample

(Asample) and standard (Astandard) was measured against the blank within 60 min

Calculation:

Conc. of cholesterol in sample= . x Concentration of standard (mg/dl) .

2.6.5.2 DETERMINATION OF TRIGLYCERIDES LEVEL

PRINCIPLE: The triglycerides were determined after enzymatic hydrolysis with lipases. The indicator was a quinoneimine formed from hydrogen peroxide, 4-aminophenazone and 4- chlorophenol under the catalytic influence of peroxidase (Tietz 1990).

Triglycerides + H2O lipase Glycerol + Fatty acids

Glycerol + ATP glycerol kinase (GK) Glycerol-3-phosphate + ADP

Glycerol-3-phosphate + O2 Dihydoxyacetone + phosphate + H2O2

2H2O2 + 4-aminophenazone + 4-chlorophenol peroxidase Quinoeimine + HCL + 4H2O

PROCEDURE: pipette into the respective tubes the following as shown below

Blank Standard Sample

Sample - - 10 ul

Standard (CAL) - 10 ul -

Reagent R1 1000 ul 1000 ul 1000 ul

The following tubes were mixed and incubated for 10 min at 250C . The absorbance of the sample (Asample) and standard (Astandard) were measured against the blank within 60 min.

Calculation:

Triglycerides concentration = . x Concentration of standard (mg/dl) .

2.6.5.3 DETERMINATION OF HIGH DENSITY LEVEL CHOLESTEROL

Principle: Low density lipoproteins (LDL and VLDL) and chylomicrons fractions were precipitated quantitatively by the addition of phosphotungstic acid in the presence of magnesium ions. After centrifuging, the cholesterol concentration in the HDL fractions, which remained in the supernatant, was determined.

1: Precipitation:

Standard Sample

Sample - 200 ul

Standard (CAL) 200 ul -

Diluted precipitate 500 ul 500 ul

(RI)

These were mixed and allowed to stand for 10min at room temperature, and were centrifuged at

4000r.p.m for 10 min. After this, clear supernatant were separated within two hours to determine the cholesterol content by CHOD-PAP method.

2. Cholesterol CHOD-PAP Assay

Blank Standard Sample

Sample - - 100 ul

Standard (CAL) - 100 ul -

Distilled H2O 100 ul - -

Reagent R1 1000 ul 1000 ul 1000ul

The following tubes were mixed and incubated for 10 min at 250C. The absorbance of the sample

(Asample) and standard (Astandard) were measured against the blank within 60 min.

Calculation:

HDL-Cholesterol concentration = . x Concentration of standard (mg/dl) .

2.6.5.4 DETERMINATION OF LOW DENSITY LEVEL CHOLESTEROL

Low density lipoproteins cholesterol was determined using Friedwald and Less 1972 principle, which stated that low density lipoproteinscould be calculated from the value of total cholesterol,

HDL cholesterol and triglycerides using the following formula

LDL = TC - HDL - TG/5.0 (mg/dL)

2.6.5.5 DETERMINATION OF VERY LOW DENSITY LEVEL CHOLESTEROL

Very low density lipoproteins cholesterol was determined using Friedwald and Less 1972 principle, which stated that very low densitylipoproteins could be calculated from the value of triglycerides, the following Formula TRIGS/5.5

2.7 DETERMINATION OF BODY WEIGHT

Pre and post body weight of Wistar rats were determined by weighing the rats using an electronic weighing balance.

2.8 DETERMINATION OF ATHEROGENIC INDEX

Atherogenic index was determined by using the Formula log (TC/HDLC), is a useful predictor in determining the risk of cardiovascular diseases (Nwagha et al.,2010)

2.9 STUDIES ON ANTI-OXIDANT EFFECTS OF EXTRACT AND FRACTIONS OF

PVE

Anti-oxidant activities were determined by in-vivo and in-vitro studies. In the in-vitro studies free radical scavenging assay was determine using DPPH radical scavenging activity and nitric oxide scavenging activity while in-vitro activities were determine by measuring tissue estimates of catalase, glutathione-peroxidase and lipid peroxidation activities.

2.9.1 DPPH scavenging activity of extract and fractions of PVE

The free radical scavenging activity of the test substances were determined by measuring, the change in absorbance of DPPH radicals at 517nm (Blois, 1958). The sample extract (0.2 mL) was diluted with methanol and 1.8 mL of DPPH solution (0.1 mM) was added. After 30 min, the absorbance is measured at 517 nm. The percentage of DPPH scavenging activity was determined as follows, DPPH Radical Scavenging Activity (%) = [(A0−A1)/A0] where A0 is the absorbance of control and A1 was the absorbance of sample.

2.9.2 Nitric oxide scavenging activity of extract and fractions of PVE

Nitric oxide scavenging activity was measured spectrophotometrically by using Greiss reaction

(Marcocci et al., 1994). Extract, prepared in ethanol, was added to different test-tubes in varying concentrations (25mg- 400mg). Sodium nitroprusside (5mM) in phosphate buffer was added to each test tube to make volume up to 1.5ml. The solutions were incubated at 25ºC for 30 min.

Thereafter, 1.5ml of Griess reagent (1% sulphanilamide, 0.1%naphthylethylenediamine dichloride and 2% phosphoric acid) was added to each test tube. The absorbance was measured, immediately, at 546 nm and percentage of scavenging activity was measured with reference to ascorbic acid as standard.

2.9.3 In-vivo study

Fifty five rats were randomly divided into eleven groups of five rats each; Group 1 rats served as the positive control and were treated with ascorbic acid. Group 2 (untreated group), Group 3, 4, 5

where treated with the crude extract at 100mg/kg, 200mg/kg and 400mg/kg respectively while group 6 and 7 where treated with n-hexane fraction at 100mg/kg and 200mg/kg with group 8 and

9 administered the ethylacetate fraction at 100mg/kg and 200mg/kg and group 10 and 11 treated with butanol fraction at 100mg/kg and 200mg/kg respectively.

Test and control group animals were intoxicated with CCl4 (1.25ml) for 2 days followed by administration of test and control substances for 7days. After the treatments, the animals were starved overnight and sacrificed under mild chloroform anesthesia. Blood and liver tissues were harvested and use for further analysis (Omonhinmin and Agbara, 2013)

2.9.4 Estimation of catalase activity

Principle: The ultra violet absorption of hydrogen peroxide can easily be measured at 240nm.

On the decomposition of hydrogen peroxide with catalase the absorption decreases with time and from this decreases catalase activity can be measured.

Procedure: Phosphate buffer (2.5 ml) was pipette into a test tube along with 2ml of hydrogen peroxide and 0.5ml of sample. To 1 ml portion of the reaction was added 2ml of dichromate acetic acid reagent. The absorbance at 240 nm was determined into 4 places at a minute interval

(Aebi, 1983). Catalase activity was calculated using the following equation:

Catalase concentration = (0.23 × log Abs 1/Abs 2) ÷ 0.00693.

2.9.5 Estimation of lipid peroxidation

Principle: Malondialdehyde (MDA) reacts with thiobarbituric acid to form a red or pink colored complex which in acid solution, absorbs maximally at 532nm.

MDA + 2TBA MDA: TBA adduct + H2O

Procedure: Lipid peroxidation was determined spectrophotometrically by measuring the level of the lipid peroxidation product, malondialdehyde (MDA). A volume of 0.1 mL of liver tissue

homogenate samples were treated separately with 2 mL of TBA-TCA-HCl reagent (1:1:1 ratio,

0.37% TBA, 0.25 N HCl, 10% TCA) and incubated at 95oC for 40mins and cool in water. 0.1ml of 20% SDS (sodium dodecyl sulphate) was added and the absorbance at 532nm was determined against a blank (Wallin B et al., 1993).

2.9.6 Estimation of Glutathione peroxidase

Principle: Gluthatione peroxidase catalyses the oxidation of gluthatione (GSH) by cumene hydroperoxide. In the presence of Gluthatione Reductase (GR) and NADPH, the oxidized

Glutathione reductase (GSSG) is immediately converted to the reduced form with a concomitant oxidation of NADPH to NADP+. The decrease in absorbance at 340nm is measured in a spectrophotometer.

Procedure: Glutathione peroxidase (GPx) activity was determined spectrophotometrically

(Agergaard and Jensen, 1982). A volume of 0.1 ml of liver tissue homogenate samples was added to 3mls of phosphate buffer solution, 0.05ml of gluaiacol and 0.03ml of hydrogen peroxide. The absorbance was taken at 436 nm for 2 min at 30secs interval.

2.10 METHOD OF DATA ANALYSES

Significant difference between control and experimental groups were obtained by one way

ANOVA using SPSS version 16 Dunnet post hoc. All data obtained were expressed as

Mean±SEM (standard error of mean). Graphical representation was done using Graph pad Prism.

P-values <0.05 were considered significant.

CHAPTER THREE

RESULTS

3.1 FRACTIONATION

After extraction, the crude extract yielded a residue of 58.6g (11.72% w/w) while the various fraction yields are as follows n-hexane 4.85g (8.27% w/w), ethylacetate 6.72g (11.47% w/w) and butanol 4.87g (8.31% w/w).

3.2 PHYTOCHEMICAL SCREENING

The phytochemical screening results revealed that all the extract showed positive reactions for the presence of tannins, saponins and flavonoids while moderate amounts of carbohydrates was seen with all the extract, and trace amount of steroids was found in all the extract. The presence

of trace amount of alkaloids was observed with the extract and fractions, except the butanol fraction, while terpenoids was seen in the butanol and the crude extract, Resin was not observed in extracts and fractions (Table 2).

Table 2: Phytochemical screening of all fractions of Phaseolus vulgaris L.

Phytochemicals Crude n-hexane fraction Ethylacetate Butanol fraction

Extract fraction

Tannins +++ ++ +++ ++

Saponins +++ +++ +++ +++

Flavonoids ++ +++ +++ ++

Steroids + + + +

Alkaloids + + + -

Terpernoids + - - +

Carbohydrates + ++ ++ +

Resins - - - -

Proteins - - - -

Reducing ++ ++ + +

Sugar

+ = activity present - = no activity present

3.3 ACUTE TOXICITY (LD50)

In acute toxicity study, oral administration of PVE did not cause lethality or signs of intoxication in the mice even at doses up to 5000 mg/kg (Table 3). Therefore the oral LD50 of the extracts in mice is greater than 5000 mg extract/kg body weight (Lorke, 1983).

Table 3: LD50 of Crude extract of Phaseolus vulgaris L.

Phase 1 Treatment No. of Death

Mice

10 mg/kg 0/3

100 mg/kg 0/3

1000 mg/kg 0/3

Phase 2 Treatment No. of Death

Mice

1600 mg/kg 0/3

2900 mg/kg 0/3

3500 mg/kg 0/3

5000 mg/kg 0/3

3.4 HPLC-FINGERPRINTING STUDIES

The qualitative HPLC-fingering and comparison with various database revealed the presence of various compounds such as derivatives of catechin 4’-methylcatechin and 3’,4’-dimethylcatechin

(Figure 5)

Figure 5: HPLC-profile studies Identified compounds: Methyl derivatives of cathechin 1 and 2

NB: Identification was based on HPLC-DAD library hit. The peaks which were not identified either did not show any library hit.

3.4.1 STRUCTURE OF COMPOUNDS IDENTIFIED

HO O

OH 4’-Methylcathechin (1)

HO O

OH 3',4'-Dimethylcathechin (2)

3.5 ANTI-HYPERLIPIDEMIC STUDY OF EXTRACT AND FRACTIONS OF PVE

3.5.1 ACUTE STUDY

An acute study to determine the effects of the extract and fractions of Phaseolus vulgaris L. on lipid profile parameters, body weight and atherogenic index was carried out and the results shown in Figure 5 - 10 below.

3.5.1.1 ACUTE STUDY OF PVE EXTRACTS

In this hyperlipidemic studies, administration of the extract caused a significant

(P<0.05)decrease in the levels of total cholesterol, triglycerides, LDL-C and VLDL-C while there was a significant (P<0.05)increase in HDL-C blood level. The blood levels of total cholesterol showed a significant decrease from 164.52 in the untreated group to 141.48, 131.04,

132.12 in the groups treated with PVE (100 mg/kg, 200 mg/kg, 400 mg/kg) representing a decrease of 14.00%, 20.35% and 19.69% respectively when compared to the untreated groups with the PVE 200 mg/kg and 400mg/kg showing a significant decrease and the PVE 100 mg/kg showing non-significant (P>0.05) decrease in total cholesterol level. Similarly, triglyceride levels was decreased from 158.40in the untreated group to 148.32, 139.68, and 129.60 in the groups treated with PVE (100, 200, and 400 mg/kg) representing a decrease of 6.36%, 11.82%, and 18.18%, respectively when compared to the untreated group with the PVE 400mg/kg showing a significant decrease (P<0.05) and the PVE 100 mg/kg and 200 mg/kg showing non- significant decrease (P>0.05) in triglycerides level. The VLDL-C levels also showed a similar decrease as the triglycerides level between the untreated and the treated with decrease from

31.68 in the untreated group to 29.66, 27.94, and 25.99 in the groups treated with PVE (100, 200, and 400 mg/kg) representing a decrease of 6.38%, 11.81%, and 17.96%, respectively when compared to the untreated group with the PVE 400mg/kg showing a significant decrease

(P<0.05) and the PVE 100 mg/kg and 200 mg/kg showing non-significant decrease (P>0.05) in

VLDL-C level.

The blood LDL-C levels showed a dose dependent significant (P<0.05) decrease from 92.68 in the untreated group to 63.58, 47.88, and 47.52 in the group treated with PVE (100, 200, and 400 mg/kg) representing a percentage decrease of 31.40%, 48.39% and 48.73% respectively. The

HDL-C blood levels shows a dose dependent significant increase (P<0.05) from 40.32 in the untreated group to 48.24, 51.48 and 58.68 in the group treated with PVE (100, 200, and 400 mg/kg) representing a percentage increase of 19.64%, 20.02% and 20.85%.

In the study to determine the effects of the extracts on body weight there was no significant increase seen when the untreated group is compared to the treated groups, also in the atherogenic index there was a dose dependent significant (P<0.05) decrease from 0.60 in the untreated group to 0.49, 0.43, 0.35 in the group treated with PVE (100, 200, and 400 mg/kg)with the PVE 200 mg/kg and 400mg/kg showing a significant decrease and the PVE 100 mg/kg showing non- significant decrease in atherogenic index.

Fig 6: The effects of crude extract of Phaseolus vulgaris L. on lipid profile parameters

Fig 7: The effects of crude extract of Phaseolus vulgaris L. on body weight of Wistar rats

Fig 8: The effects of crude extract of Phaseolus vulgaris L. on atherogenic index of Wistar

rats

3.5.1.2 ACUTE STUDY OF FRACTIONS OF PVE

In hyperlipidemic studies involving various fractions, administration of the fractions caused a significant (P<0.05)decrease in the levels of total cholesterol, triglycerides, LDL-C and VLDL-C while there was a significant (P<0.05)increased in HDL-C blood level. The blood levels of total cholesterol showed a dose related significant decreased from 164.52 in the untreated group to

153.72, 132.48 in the group treated with PVHF (100 mg/kg and 200mg/kg), and to 140.40,

128.16 in the group treated with PVEF while the group treated with PVBF showed an increase

(166.28 mg/kg) in the 100 mg/kg group and a decrease (151.52) in the 200 mg/kg group. This represents a significant decrease of 19.47% for PVHF at 200 mg/kg and 22.10 for PVEF at 200 mg/kg. Similarly, triglyceride levels was decreased from 158.40in the untreated group to 154.80,

124.20 in the group treated with PVHF (100 mg/kg and 200mg/kg), and to 148.32, 133.20 in the group treated with PVEF while the group treated with PVBF showed a decrease of 145.08,

127.08 in the 100 mg/kg group and the 200 mg/kg group. This represents a significant decrease of 21.59% for PVHF at 200 mg/kg, 15.91% for PVEF at 200 mg/kg and 19.77% for PVBF at

200 mg/kg. The VLDL-C levels also showed a similar relationship between the untreated and the treated as the triglycerides level with decrease from 31.68 in the untreated group untreated group to 30.93, 24.84 in the group treated with PVHF (100 mg/kg and 200mg/kg), and to 29.66, 26.64 in the group treated with PVEF while the group treated with PVBF showed a decrease of 29.02,

25.42 in the 100 mg/kg group and the 200 mg/kg group. This represents a significant decrease of

21.59% for PVHF at 200 mg/kg, 15.91% for PVEF at 200 mg/kg and 19.76% for PVBF at 200 mg/kg.

The blood LDL-C levels showed a decrease from 92.68 in the untreated group to 85.32, 48.96 in the group treated with PVHF (100 mg/kg and 200mg/kg), and to 54.10, 43.20 in the group treated with PVEF while the group treated with PVBF showed a decrease of 97.66, 87.22 in the

100 mg/kg group and the 200 mg/kg group. This represents a significant decrease (P<0.05) of

47.19% for PVHF at 200 mg/kg and 41.62% for PVEF at 100 mg/kg and 53.39% for PVEF at

200 mg/kg. The blood HDL-C levels showed a increase from 40.32 in the untreated group to

37.44, 57.96 in the group treated with PVHF (100 mg/kg and 200mg/kg), and to 56.52, 58.32 in the group treated with PVEF while the group treated with PVBF showed a slight decrease of

39.60, 38.52 in the 100 mg/kg group and the 200 mg/kg group. This represents a significant increase (P<0.05) of 30.44% for PVHF at 200 mg/kg and 28.66% for PVEF at 100 mg/kg and

30.86% for PVEF at 200 mg/kg.

In the study to determine the effects of the fractions on body weight there was no significant increase (P>0.05) seen when the untreated group is compared to the treated groups, also in the atherogenic index there was a change from 0.60 in the untreated group to 0.62, 0.33 in the group treated with PVHF (100 mg/kg and 200mg/kg), and to 0.42, 0.36 in the group treated with

PVEF while the group treated with PVBF showed a decrease of 0.57, 0.53 in the 100 mg/kg group and the 200 mg/kg group respectively. This represent a significant decrease (P<0.05) in the PVHF 200 mg/kg, PVEF 100 mg/kg and 200 mg/kg.

Fig 9: The effects of various fractions of Phaseolus vulgaris L. on lipid profile parameters

on Wistar rats

Fig 10: The effects of various fractions of Phaseolus vulgaris L. on body weight of Wistar

rats

Fig 11: The effects of various fractions of Phaseolus vulgaris L. on atherogenic index of Wistar rats

3.5.2 SUB-ACUTE STUDY

A sub-acute study to determine the effects of various extract of Phaseolus vulgaris L. on lipid profile parameters, body weight and atherogenic index was carried out and the results shown in

Figure 11-16 below.

3.5.2.1 SUB-ACUTE STUDY OF PVE EXTRACTS

In this hyperlipidemic studies, administration of the extract caused a significant

(P<0.05)decrease in the levels of total cholesterol, triglycerides, and VLDL-C while there was a non-significant decrease (P>0.05) in LDL-C levels with HDL-C levels showing a non- significant (P>0.05)increased in HDL-C blood level. The blood levels of total cholesterol showed a significant decreased from 129.24 in the untreated group to 122.76, 119.88, 114.84 in the groups treated with PVE (100 mg/kg, 200 mg/kg, 400 mg/kg) representing a decrease of

5.01%, 7.26% and 38.44% respectively when compared to the untreated groups with the PVE

200 mg/kg and 400mg/kg showing a significant decrease and the PVE 100 mg/kg showing non- significant (P>0.05) decrease in total cholesterol level. Similarly, triglyceride levels was decreased from 100.08 in the untreated group to 92.52, 91.44, and 87.12 in the groups treated with PVE (100, 200, and 400 mg/kg) representing a decrease of 7.55%, 8.63%, and 12.95%, respectively when compared to the untreated group with the PVE 200 mg/kg and PVE 400mg/kg showing a significant decrease (P<0.05) and the PVE 100 mg/kg showing non-significant decrease (P>0.05) in triglycerides level. The VLDL-C levels also showed a similar relationship between the untreated and the treated as the triglycerides level with decrease from 20.02 in the untreated group to 18.50, 18.28, and 17.42 in the groups treated with PVE (100, 200, and 400 mg/kg) representing a decrease of 7.59%, 8.69%, and 12.99%, respectively when compared to the untreated group with the PVE 200 mg/kg and PVE 400mg/kg showing a significant decrease

(P<0.05) and the PVE 100 mg/kg showing non-significant decrease (P>0.05) in VLDL-C level.

The blood LDL-C levels showed a non-significant (P>0.05) decrease from 60.98 in the untreated group to 53.64, 45.99, and 48.96 in the group treated with PVE (100, 200, and 400 mg/kg) representing a decrease in 12.04%, 24.58% and 19.71%. The HDL-C blood levels shows a non- significant increase (P>0.05) from 48.24 in the untreated group to 50.76, 52.56, 54.00 in the group treated with PVE (100, 200, and 400 mg/kg) representing a decrease in 19.64%, 20.02% and 20.85%.

Fig 12: The effects of crude extract of Phaseolus vulgaris L. on lipid profile parameters

Fig 13: The effects of crude extract of Phaseolus vulgrais L. on body weight of wistar rats

Fig 14: The effects of crude extract of Phaseolus vulgaris L. on atherogenic index of Wistar

rats

3.5.2.2 SUB-ACUTE STUDY OF FRACTIONS OF PVE

In this hyperlipidemic studies, administration of the extract caused a significant

(P<0.05)decrease in the levels of total cholesterol, triglycerides, and VLDL-C while there was a non-significant decrease (P>0.05) in LDL-C levels with HDL-C levels showing a non- significant (P>0.05)increased in HDL-C blood level. The blood levels of total cholesterol showed a decreased from 129.24 in the untreated group to 125.92, 117.72 in the group treated with PVHF (100 mg/kg and 200mg/kg), and to 120.06, 112.68 in the group treated with PVEF while the group treated with PVBF showed an decrease to 121.32 and 114.48 in 100 mg/kg group and 200 mg/kg group respectively. This represents a significant decrease (P<0.05) of

12.81% for PVEF at 200 mg/kg and 14.76% for PVBF at 200 mg/kg with the remaining group showing a non-significant decrease (P>0.05). Similarly, triglyceride levels was decreased from

100.08in the untreated group to 98.64, 93.60 in the group treated with PVHF (100 mg/kg and

200mg/kg), and to 98.28, 99.00 in the group treated with PVEF while the group treated with

PVBF showed a decrease of 95.76, 82.80 in the 100 mg/kg group and the 200 mg/kg group. This represents a significant decrease (P<0.05) of 17.28% for PVBF at 200 mg/kg,with the remaining group showing a non-significant decrease (P>0.05). The VLDL-C levels also showed a similar relationship between the untreated and the treated as the triglycerides level with decrease from

20.02 in the untreated group untreated group to 19.73, 19.30 in the group treated with PVHF

(100 mg/kg and 200mg/kg), and to 19.66, 19.80 in the group treated with PVEF while the group treated with PVBF showed a decrease of 19.15, 16.56 in the 100 mg/kg group and the 200 mg/kg group. This represents a significant decrease (P<0.05) of 17.28% for PVBF at 200 mg/kg,with the remaining group showing a non-significant decrease (P>0.05).

The blood LDL-C levels showed a decrease from 60.98 to 56.88, 43.34 in the group treated with

PVHF (100 mg/kg and 200mg/kg), and to 51.70, 40.39 in the group treated with PVEF while the group treated with PVBF 100 mg/kg showed an increase to 62.47 while the group treated with

PVBF 200 mg/kg showed a decrease to 39.60. There was no-significant decrease (P>0.05) in

LDL-C in all treated group. The blood HDL-C levels showed a decrease from 48.24 in the untreated group to 47.88 in the group treated with PVHF 100 mg/kg and an increase to 51.12 in the group treated with PVHF 200 mg/kg, in the group treated with PVEF there was an increase to

49.32, 55.44 in PVEF 100 mg/kg and 200mg/kg respectively, in the group treated with PVBF

100 mg/kg there was a decrease to 45.72 and an increase to 54.22 in the group treated with

PVBF 200 mg/kg. There was no significant decrease (P>0.05) in HDL-C in all treated group.

In the study to determine the effects of the fractions on body weight there was no significant increase seen when the untreated group is compared to the treated groups, also in the atherogenic index there was a no significant (P>0.05) decrease seen when the untreated group when compared to all treated group.

Fig 15: The effects of various fractions of Phaseolus vulgaris L. on lipid profile parameters of Wistar rats

Fig 16:The effects of various fractions of Phaseolus vulgaris L. on body weight of Wistar rats

Fig 17: The effects of various fractions of Phaseolus vulgaris L. on atherogenic index of Wistar rats

3.5.3 CHRONIC STUDY

A chronic study to determine the effects of various extract of Phaseolus vulgaris L. on lipid profile parameters, body weight and atherogenic index was carried out and the results shown in

Figure 17-23 below.

3.5.3.1 CHRONIC STUDY OF PVE EXTRACTS

In this hyperlipidemic studies, administration of the extract caused a significant

(P<0.05)decrease in the levels of total cholesterol, triglycerides, LDL-C and VLDL-C while there was a significant (P<0.05)increased in HDL-C blood level. The blood levels of total cholesterol showed a decreased from 173.08 in the untreated group to 161.64, 156.60, 151.92 in the groups treated with PVE (100 mg/kg, 200 mg/kg, 400 mg/kg) representing a decrease of

6.61%, 9.52% and 12.22% respectively when compared to the untreated groups with the PVE

200 mg/kg and 400mg/kg showing a significant decrease (P<0.05) and the PVE 100 mg/kg showing non-significant (P>0.05) decrease in total cholesterol level. Similarly, triglyceride levels was decreased from 138.60in the untreated group to 137.52, 133.20, and 127.44 in the groups treated with PVE (100, 200, and 400 mg/kg) representing a decrease of 0.78%, 3.89%, and 8.05%, respectively when compared to the untreated group with the PVE 400mg/kg showing a significant decrease (P<0.05) and the PVE 100 mg/kg and 200 mg/kg showing non-significant decrease (P>0.05) in triglycerides level. The VLDL-C levels also showed a similar relationship between the untreated and the treated as the triglycerides level with decrease from 27.72 in the untreated group to 27.50, 26.64, and 25.49 in the groups treated with PVE (100, 200, and 400 mg/kg) representing a decrease of 0.79%, 3.90%, and 8.05%, respectively when compared to the untreated group with the PVE 400mg/kg showing a significant decrease (P<0.05) and the PVE

100 mg/kg and 200 mg/kg showing non-significant decrease (P>0.05) in VLDL-C level.

The blood LDL-C levels showed a dose dependent significant (P<0.05) decrease from 87.12 in the untreated group to 67.18, 62.28, and 52.12 in the group treated with PVE (100, 200, and 400 mg/kg) representing a decrease in 22.89%, 28.51% and 40.17% with all group showing significance. The HDL-C blood levels shows a dose dependent significant increase (P<0.05) from 58.32 in the untreated group to 66.96, 67.68 and 83.52 in the group treated with PVE (100,

200, and 400 mg/kg) representing a decrease in 12.90%, 13.83% and 30.17% with the PVE 400 mg/kg showing significance.

In the study to determine the effects of the extracts on body weight the percentage increase of the untreated group 6.71% when compared to the treated PVE 100 mg/kg (38.06), PVE 200 mg/kg

(16.97%) and PVE 400 mg/kg (14.50%) there was a significant decrease (P<0.05) seen in the

PVE 400 mg/kg group, also in the atherogenic index there was a dose dependent significant

(P<0.05) decrease from 0.38 in the untreated group to 0.32, 0.30, 0.26 in the group treated with

PVE (100, 200, and 400 mg/kg)with the PVE 200 mg/kg and 400mg/kg showing a significant decrease (P<0.05) and the PVE 100 mg/kg showing non-significant decrease (P>0.05) in atherogenic index.

Fig 18: The effects of high fatty diet on lipid profile parameters

Fig 19: The effects of crude extract of Phaseolus vulgaris L. on lipid profile parameters of

Wistar rats

Fig 20: The effects of crude extract of Phaseolus vulgaris L. on body weight of Wistarrats

Fig 21: The effects of crude extract of Phaseolus vulgaris L. on atherogenic index of Wistar

rats

3.5.1.2 CHRONIC STUDY WITH FRACTIONS

In chronic anti-hyperlipidemic studies involving various fractions, administration of the fractions caused a significant (P<0.05)decrease in the levels of total cholesterol, triglycerides, LDL-C and

VLDL-C while there was a significant (P<0.05)increased in HDL-C blood level. The blood levels of total cholesterol showed a dose related significant decreased from 173.08 in the untreated group to 165.96, 162.72 in the group treated with PVHF (100 mg/kg and 200mg/kg), and to 153.76, 158.04 in the group treated with PVEF with the group treated with PVBF 100 mg/kg decreasing to 161.28 mg/kg and PVBF 200 mg/kg decreasing to 159.72. This represents a significant decrease of 11.16% for PVEF at 100 mg/kg, 8.69% for PVEF 200 mg/kg and 6.82% for PVBF at 100 mg/kg and 7.72% for PVBF at 200 mg/kg. The triglyceride levels was increased from 138.60in the untreated group to 147.92 in the group treated with PVHF 100 mg/kg and decreases at PVHF 200 mg/kg to 131.04, while there was decreases to 136.08 and 124.92 in the group treated with PVEF 100 mg/kg and PVEF 200 mg/kg, while the group treated with PVBF showed a decrease to 136.68, 124.56 in the 100 mg/kg group and the 200 mg/kg group. This represents a significant decrease of 9.87% for PVEF at 200 mg/kg and 10.13% for PVBF at 200 mg/kg. The VLDL-C levels also showed a similar relationship between the untreated and the treated as the triglycerides level with increase from 27.72 in the untreated group to 29.58 in the group treated with PVHF 100 mg/kg and decreases at PVHF 200 mg/kg to 26.64, while there was decreases to 27.22 and 24.95 in the group treated with PVEF 100 mg/kg and PVEF 200 mg/kg, while the group treated with PVBF showed a decrease to 27.34, 24.91 in the 100 mg/kg group and the 200 mg/kg group. This represents a significant decrease of 9.92% for PVEF at 200 mg/kg and 10.14% for PVBF at 200 mg/kg.

The blood LDL-C levels showed a decrease from 87.12 in the untreated group to 76.30, 64.44 in the group treated with PVHF (100 mg/kg and 200mg/kg), and to 65.50, 79.62 in the group treated with PVEF while the group treated with PVBF showed a decrease of 77.42, 69.65 in the

100 mg/kg group and the 200 mg/kg group. This represents a significant decrease (P<0.05) of

26.03% for PVHF at 200 mg/kg, 24.82% for PVEF at 100 mg/kg and 20.05% for PVBF at 200 mg/kg. The blood HDL-C levels showed a increase from 58.32 in the untreated group to 60.08,

68.04 in the group treated with PVHF (100 mg/kg and 200mg/kg), and to 61.60 in the group treated with PVEF 100 mg/kg while there was a decrease to 53.64 in the group treated with

PVEF 200 mg/kg, there was also a decrease to 56.52 in the group treated with PVBF 100 mg/kg and an increase to 65.16 in the group treated with PVBF 200 mg/kg. This represented a significant increase (P<0.05) of 14.29% for PVHF at 200 mg/kg and 10.50% for PVBF at 100 mg/kg 200 mg/kg.

In the study to determine the effects of the extracts on body weight, the percentage increase of the untreated group 6.71% was compared to PVHF 100 mg/kg (17.06%), PVHF 200 mg/kg

(12.96%), PVEF 100 mg/kg (11.20%), PVEF 200 mg/kg (11.50%), PVBF 100 mg/kg (12.38%) and PVBF 200 mg/kg (10.00%) respectively, with all groups showing significant percentage decrease except PVHF 100 mg/kg. Also in the atherogenic index there was a change from 0.38 in the untreated group to 0.39, 0.28 in the group treated with PVHF (100 mg/kg and 200mg/kg), and to 0.35, 0.37 in the group treated with PVEF while the group treated with PVBF showed a decrease of 0.38, 0.28 in the 100 mg/kg group and the 200 mg/kg group respectively. This represented a significant decrease (P<0.05) in the PVHF 200 mg/kg, PVBF 200 mg/kg respectively.

Fig 22: The effects of various fractions of Phaseolus vulgaris L. on lipid profile parameters of Wistar rats

Fig 23: The effects of various fractions of Phaseolus vulgaris L. on body weight of Wistar rats

Fig 24: The effects of various fractions of Phaseolus vulgaris L. on atherogenic index of Wistar rats

3.6 ANTI-OXIDANT STUDIES

Anti-oxidant activities were determined by in-vivo and in-vitro studies. In the in-vitro studies free radical scavenging assay was determined using DPPH radical scavenging activity and nitric oxide scavenging activity while in-vitro activities was determined by measuring tissue estimates of catalase, glutathione-peroxidase and lipid peroxidation activities with results represented using Figure 24 and 25; Table 4 and 5

3.6.1 DPPH SCAVENGING ASSAY

The highest percentage assay of DPPH are 80.61% seen with ethylacetate and butanol fraction at

400 mg/kg while the highest percentage for the crude extract was 78.57% at 400 mg/kg and

75.51% also at 400 mg/kg for hexane fraction. The ethylacetate and butanol showed a similar percentage increase as ascorbic acid (85.71%) (Figure 24).

3.6.2 NITRIC OXIDE SCAVENGING ASSAY

The highest percentage assay of nitric oxide was 75.86% seen with hexane fractions at 200 mg/kg while the ethylacetate fractions showed highest percentage activity at 400 mg/kg

(70.69%) with the butanol fraction at 400 mg/kg have the highest percentage (67.24%) for the crude extract the highest percentage (74.13%) was obtained at 50 mg/kg while ascorbic acid had the highest percentage activity occurring at 200 mg/kg (77.59%) (Figure 25).

Fig 25: DPPH scavenging activity of various extract of Phaseolus vulgaris l.

Fig 26:Nitric oxide scavenging assay of various extract of Phaseolus vulgaris L.

3.6.3 IN-VIVO ANTIOXIDANT ASSAY

In in-vivo antioxidant assay showed that the Lipid peroxidation levels estimated by thiobarbituric acid reaction showed no significant increase or decrease in the serum MDA of both the treated and untreated group (Table 4 and 5).The in vitro study showed significant increase (P<0.05) scavenging activity with the PVHF and PVEF having scavenging activity comparable with ascorbic acid. In in-vivo antioxidant assay showed that the Lipid peroxidation levels estimated by thiobarbituric acid reaction showed no significant (P>0.05) increase or decrease in the serum

MDA of both the treated and untreated group, while in catalase activity estimation significant

(P<0.05) increase was seen with PVE 100 mg/kg of 71.05%, Glutathione peroxidise activity showed a significant percentage (P<0.05) increase of 68%, 71.43%, 64.60%, 66.67%, 76.19% and 66.94% for PVHF 100 mg/kg, PVHF 200 mg/kg, PVEF 100 mg/kg, PVEF 200 mg/kg,

PVBF 100 mg/kg and PVBF 200 mg/kg respectively.

Table 4: Effects of crude extract of Phaseolus vulgaris L. on antioxidant enzymes (IU/L)

CATALASE GPX LIPID

PEROXIDATION

NG 0.65± 0.41 0.40±0.04 1.40±0.10

Ascorbic acid 2.32±0.73* 0.99±0.15* 2.18±0.45

PVE 100 mg/kg 2.09±1.68* 0.43±0.04 2.44±0.57

PVE 200 mg/kg 1.16±0.14 0.34±0.02 1.55±0.12

PVE 400 mg/kg 0.85±0.12 0.51±0.28 0.53±0.16

All values expressed as Mean ± SEM, where n=5, all data were analyzed by using one way

ANOVA followed by Dunnet’s test. p<0.05 was taken to be significant

TABLE 5: EFFECTS OF VARIOUS EXTRACT OF PHASEOLUS VULGARIS L. ON

ANTIOXIDANT ENZYMES (IU/L)

CATALASE GPX LIPIDPEROXIDATION

NG 0.65± 0.41 0.40±0.04 1.40±0.10

PVHF 100 mg/kg 0.68±0.16 1.25±0.12* 1.57±0.11

PVHF 200 mg/kg 0.82±0.27 1.40±0.21* 1.54±0.42

PVEF 100 mg/kg 0.62±0.09 1.13±0.21* 1.03±0.36

PVEF 200 mg/kg 0.40±0.01 1.20±0.09* 1.48±0.06

PVBF 100 mg/kg 0.75±0.17 1.68±0.06* 1.25±0.22

PVBF 200 mg/kg 0.89±0.22 1.21±0.17* 0.52±0.10

All values expressed as Mean ± SEM, where n=5, all data were analyzed by using one way

ANOVA followed by Dunnet’s test. p<0.05 was taken to be significant

CHAPTER FOUR

DISCUSSION AND CONCLUSION

4.1 DISCUSSION

This study evaluated the antihyperlipidemic and antioxidant properties of extract and various fractions of Phaseolus vulgaris L.In this study, hyperlipidemic animals received extract and various fractions of Phaseolus vulgaris L. for different models of experimental studies while an in-vivo and in-vitro antioxidant studies were carried out using various fractions of Phaseolus vulgaris L.

There was no death recorded during both phase of acute toxicity study at all doses up to 5000 mg/kg. Theabsence of death observed showed that the LD50 of the extract was greater than 5000 mg/kg body weight, indicating their relative safety. The phytochemical screening results revealed that both extract and fractions showed positive reactions for the presence of tannins, saponins and flavonoids while moderate amounts of carbohydrates was seen with all the extract.

There was a link between flavonoids and atherosclerosis, this was based partially on evidence that some flavonoids possessed antioxidant properties and have been shown to be potent inhibitors of LDL oxidation invitro (Frankel et al., 1993). Flavonoids had also been shown to inhibit platelet aggregation and adhesion (Gryglewski et al., 1987) which might be another way they lower the risk of heart disease, Iso-flavones in soy foods have been reported to lower plasma cholesterol and also to possess estrogen like effects (Dwyer et al., 1994).

The qualitative HPLC-fingering and comparison with various database revealed the presence of various compounds such as derivatives of catechin 4’-methylcatechin and 3’,4’- dimethylcatechin. Catechin a plant secondary metabolite belongs to the group of flavan-3-ols (or

simply flavanols), part of the chemical family of flavonoids (Brown and Goldstein, 1986). High concentrations of catechin which could be found in red wine, black grapes broad beans etc. and its consumption had been associated with a variety of beneficial effects including increased plasma antioxidant activity (ability of plasma to scavenge free radicals, fat oxidation and resistance of LDL to oxidation (Brown et al.,1981).

In all models of hyperlipidemic study, there was a significant decrease in serum levels of lipid profile parameters when compared with the untreated groups. In the acute study models, the decrease in serum triglycerides, total cholesterol, LDL-C and VLDL-Cwas in order of PVEF >

PVHF > PVE > PVBF with the PVBF showing a non-significant decrease, in sub-acute study serum TG, TC and VLDL-C showed a decrease in this order PVEF >PVBF > PVE > PVHF with the PVHF showing a non-significant decrease, while all the fractions showed a non-significant decrease in LDL-C and an increase in HDL-C level. Finally in the chronic study there was a decrease in serum TG, TC, LDL-C and VLDL-C in these order PVEF > PVBF > PVE > PVHF levels. The results of this present hyperlipidemic study is consistent with the findings of Roman-

Ramos et al.,1995 that reported that administration of Phaseolus vulgaris L. resulted in hypolipidemic effects.

The administration of triton and high fatty diet resulted in increase in cholesterol and triglycerides level, this was due to an apparent increased of LDL secretion by the liver, catabolism of LDL and a strong reduction in VLDL (Otway and Robinson, 1967), elevated levels of these lipoproteins plays a crucial role in atherosclerotic lesions that progresses from fatty streaks to ulcerated plaque. HDL-C transports cholesterol from peripheral tissues to the liver thereby reducing the amount stores in tissue anddecreasing the likelihood of getting

atherosclerotic plagues, HDL-Cholesterol is considered to have anti-atherogenic properties, since there is negative correlation between HDL-cholesterol and risk of cardiovascular disease (Eder,

1982).

In all models there were a significant decrease in total cholesterol, triglycerides, low density lipoprotein and very low density lipoprotein with increase in high density lipoproteins, this suggest some interferences in the catabolism and increase rate of transport of cholesterol by

HDL-C.

The animals treated with various Phaseolus vulgaris L. fractions, was seen to show a non- significant difference in the percentage body weight gain in the acute and sub-acute models while there was a significant decrease (P<0.05) in the chronic study, since the feeding patterns of the animals were normal, this might suggest the extract is unlikely to cause obesity (Obi et al.,

2012) and that long term administration can be effective in managing obesity. .

Atherogenic index of plasma (AIP), a logarithmically transformed ratio of molar concentrations of triglycerides to HDL-cholesterol, it is an indices used for the diagnosis and prognosis of cardiovascular diseases. There is a strong correlation of AIP with lipoprotein particle size and this might explain its high predictive value (Dobiasova, 2006). Results from this study suggests that Phaseolus vulgaris L. might havesignificant activity against cardiovascular as seen with the atherogenic indices study especially when used for a long period of time as seen with the chronic study.

The anti-oxidant activities were determined by in-vivo and in-vitro studies. In the in-vitro studies free radical scavenging assay was determined using DPPH radical scavenging activity and nitric oxide scavenging activity while in-vivo activities was determined by measuring tissue estimates of catalase, glutathione-peroxidase and lipid peroxidation activities.

Enzymes such as catalase, MDA, glutathione peroxidases are amongst several other compounds/enzymes in which there primary expression is an indication of the body defense mechanism (Rajesh & Latha 2004; Paramavsivan 2012 et al., 2012). In this study Lipid peroxidation levels estimated by thiobarbituric acid reaction showed no significant increase or decrease in the serum MDA of both the treated and untreated group, while there was an increase in catalase activities, catalase is an anti-oxidant enzyme that is presence almost everywhere, it catalyzes the degradation of hydrogen peroxides, which is a reactive oxygen species (ROS) which is a toxic product of both pathogenic ROS production and normal aerobic metabolism

(Kohen and Nyska, 2002). The increase in catalase activities revealed that the Phaseolus vulgaris

L. has anti-oxidant activities there was also a non-significant increase in glutathione peroxidase activity was observed with extracts treated with the crude extract when compared with the untreated groups, while there was a significant increase for other groups.

The in-vitro antioxidant activity was assayed using DPPH and nitric oxide, DPPH which is stable nitrogen centered free radical exhibits discoloration when it undergoes reduction from a compound. Substances which are able to perform this reaction can be considered as antioxidants, results of this study shows that all test extract and fractions showed significant percentage reduction of DPPH molecules with PVEF and PVBF showing the highest rate at 80.61%. In the study involving nitric oxide, all test extract and fraction showed significant nitrite inhibition with the PVHF showing the highest inhibition at 75.86% sustained level of nitric oxide production leads to vascular collapse and septic shock and its toxicity increases greatly when it reacts with superoxide radical to form the highly reactive peroxy nitrate anion. Antioxidants inhibit formation of nitrite by directly competing with oxygen in the reaction with nitric oxide. Free radicals causes an imbalance in homeostatic environment between antioxidants and oxidants in

the body (Tiwari, 2001), thus leading to oxidative stress that is been linked to the advent of coronary heart diseases (Doughan et al.,2008), Hence the ability of the extract and fractions of

Phaseolus vulgaris to inhibit the formation of free radical is indicative of an antioxidant effects.

The present study showed that Phaseolus vulgaris L. has antioxidant activities albeit not at a significant level which is supported by works of Cadador-Martinez et al., 2002 but which showed significant antioxidant activity as seen with the in-vitro scavenging assay.

Antioxidant therapy may inhibit atheroscelosis which has hyperlipidemia has a risk factors and thereby preventing clinical complications of disease. Hence Phaseolus vulgaris L. can be exploited as an alternative anti-hyperlipidemic and antioxidant therapeutic agent or adjuvant in existing therapy for the treatment of hyperlipidemia. The present study showed that Phaseolus vulgaris L. has antioxidant activities albeit not at a significant level which is supported by works of Cadador-Martinez et al., 2002 but which showed significant antioxidant activity as seen with the in-vitro scavenging assay.

4.2 CONCLUSION

Results of these study shows that the various extract of Phaseolus vulgaris L. has antihyperlipidemic and antioxidant effects with the ethylacetate fractions showing substantially significant activity in both short terms, sub-chronic and chronic models of hyperlipidemia. The results also showed consistent free radical scavenging activity in both in-vivo and in-vitro antioxidant models.Further studies are encouraged in elucidating the exact mechanism of action of the ethylacetate fractions of Phaseolus vulgaris L.,in possessing antihyperlipidemic and antioxidant properties in albino-Wistar rats.

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APPENDIX

I. The effects of acute study on crude extract of Phaseolus vulgaris L. on lipid profile

parameters on Wistar rats

II. The effects of acute study on various extract of Phaseolus vulgaris L. on lipid profile

parameters on Wistar rats

III. The effects of acute study on crude extract of Phaseolus vulgaris L. on body weight of

Wistar rats

IV. The effects of sub-acute study on various fractions of Phaseolus vulgaris L. on body

weight of Wistar rats

V. The effects of sub-acute study on crude extract of Phaseolus vulgaris L. on atherogenic

index

VI. The effects of sub-acute study on crude extract of Phaseolus vulgaris L. on lipid profile

parameters on Wistar rats

VII. The effects of sub-acute study on various extract of Phaseolus vulgaris L. on lipid profile

parameters on Wistar rats

VIII. The effects of sub-acute study on crude extract of Phaseolus vulgaris L. on body weight

of Wistar rats

IX. The effects of sub-acute study on various fractions of Phaseolus vulgaris L. on body

weight of Wistar rats

X. The effects of sub-acute study on crude extract of Phaseolus vulgaris L. on atherogenic

index

109

XI. The effects of chronic study on crude extract of Phaseolus vulgaris L. on lipid profile

parameters on Wistar rats

XII. The effects of chronic study on various extract of Phaseolus vulgaris L. on lipid profile

parameters on Wistar rats

XIII. The effects of chronic study on crude extract of Phaseolus vulgaris L. on body weight of

Wistar rats

XIV. The effects of chronic study on various fractions of Phaseolus vulgaris L. on body weight

of Wistar rats

XV. The effects of chronic study on crude extract of Phaseolus vulgaris L. on atherogenic

index

XVI. DPPH scavenging activity of various extract of Phaseolus vulgaris L.

XVII. Nitric oxide scavenging activity of various extract of Phaseolus vulgaris L.

XVIII. Effects of crude extract of Phaseolus vulgaris L. on antioxidant enzymes

XIX. Effects of various extract of Phaseolus vulgaris L. on antioxidant enzymes

110

APPENDIX I: The effect of acute study of crude extract of Phaseolus vulgaris l. on

different lipid profile parameters

DOSE TC TRIGS HDL-C LDL-C VLDL- Cholesterol NG 164.52±7.28 158.40±7.90 40.32±3.35 92.68±5.59 31.68±1.58 PVE-1 141.48±9.98 148.32±5.60 48.24±3.57 63.58±9.54* 29.66±1.12 PVE-2 131.04±7.83* 139.68±7.69 51.48±1.85* 47.88±3.18* 27.94±1.54 PVE-4 132.12±5.62* 129.60±5.78* 58.68±2.71* 47.52±7.20* 25.99±1.14* ATOR- 128.88±6.65* 131.04±5.14* 59.40±2.27* 45.79±7.79* 26.21±1.03* 10 All values expressed as Mean ± SEM, where n=5, all data were analyzed by using one way ANOVA followed by Dunnet’s test. p<0.05 was taken to be significant.

DOSE TC TRIGS HDL-C LDL-C VLDL- Cholesterol NG 164.52±7.28 158.40±7.90 40.32±3.35 92.68±5.59 31.68±1.58 PVHF-1 153.72±6.68 154.80±3.90 37.44±3.62 85.32±5.19 30.93±0.79 PVHF-2 132.48±8.06* 124.20±7.83* 57.96±2.98* 48.96±7.45* 24.84±1.56* PVEF-1 140.40±10.92 148.32±5.82 56.52±3.53* 54.10±7.87* 29.66±1.16 PVEF-2 128.16±5.35* 133.20±5.46* 58.32±4.50* 43.20±6.34* 26.64±1.09* PVBF-1 166.28±6.31 145.08±4.09 39.60±3.07 97.66±5.12 29.02±0.82 PVBF-2 151.52±8.72 127.08±5.65* 38.52±4.71 87.22±11.39 25.42±1.13*

APPENDIX II:The effect of acute study ofvarious fractions of Phaseolus vulgaris l.on

different lipid profile parameters

All values expressed as Mean ± SEM, where n=5, all data were analyzed by using one way ANOVA followed by Dunnet’s test. p<0.05 was taken to be significant.

111

DOSE PRE-BODY POST-BODY PERCENTAGE WEIGHT WEIGHT INCREASE (%) ATOR-10 158.00±9.23 159.40±9.08 0.89 NG 158.80±10.38 159.80±10.61 0.63 PVE-1 162.20±6.59 163.60±6.81 0.86 PVE-2 159.60±10.17 160.00±10.06 0.25 PVE-4 156.60±8.50 158.00±8.60 0.89

APPENDIX III: The effect of acute study of crude extract of Phaseolus vulgaris L.on body weight of Wistar rats

All values expressed as Mean ± SEM, where n=5, all data were analyzed by using one way ANOVA followed by Dunnet’s test. p<0.05 was taken to be significant.

DOSE PRE-BODY POST-BODY PERCENTAGE WEIGHT WEIGHT INCREASE (%) PVHF-1 157.60±8.70 158.40±9.19 0.51 PVHF-2 152.80±9.28 153.80±9.52 0.66 PVEF-1 158.00±7.42 158.60±7.79 0.38 PVEF-2 151.60±7.58 151.80±7.69 0.13 PVBF-1 154.40±7.77 155.00±7.57 0.39 PVBF-2 159.00±6.43 159.00±6.02 0

APPENDIX IV: The effect of acute study of various fractions of Phaseolus vulgaris L.on bodyweight of Wistar rats

All values expressed as Mean ± SEM, where n=5, all data were analyzed by using one way ANOVA followed by Dunnet’s test. p<0.05 was taken to be significant.

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APPENDIX V: The effect of acute study of crude extract of Phaseolus vulgaris L.on atherogenic index

ATHEROGENIC INDEX NG 0.60±0.03 PVE-1 0.49±0.04 PVE-2 0.43±0.03* PVE-4 0.35±0.03* ATOR-10 0.34±0.03* All values expressed as Mean ± SEM, where n=5, all data were analyzed by using one way ANOVA followed by Dunnet’s test. p<0.05 was taken to be significant

APPENDIX VI: The effect of acute study various fractions of Phaseolus vulgaris L. on atherogenic index

ATHEROGENIC INDEX NG 0.60±0.03 PVHF-1 0.62±0.04 PVHF-2 0.33±0.04* PVEF-1 0.42±0.02* PVEF-2 0.36±0.03* PVBF-1 0.57±0.03 PVBF-2 0.53±0.08 All values expressed as Mean ± SEM, where n=5, all data were analyzed by using one way ANOVA followed by Dunnet’s test. p<0.05 was taken to be significant.

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APPENDIX VII: The effect of sub-acute study ofcrude extract of Phaseolus vulgaris l.on different lipid profile parameters

TC TRIGS HDL-C LDL-C VLDL-C NG 129.24±2.23 100.08±3.10 48.24±2.51 60.98±3.99 20.02±0.62 PVE-1 122.76±1.05 92.52±2.18 50.76±2.23 53.64±1.79 18.50±0.44 PVE-2 119.88±0.92* 91.44±1.55* 52.56±2.57 45.99±3.20 18.28±0.31* PVE-4 114.84±1.19* 87.12±1.99* 54.00±2.73 48.96±5.88 17.42±0.39* ATOR-10 108.31±2.74 84.24±1.32 56.52±2.51 34.94±5.56 16.85±0.27 All values expressed as Mean ± SEM, where n=5, all data were analyzed by using one way ANOVA followed by Dunnet’s test. p<0.05 was taken to be significant.

APPENDIX VIII: The effect of sub-acute study ofvarious fractions of Phaseolus vulgaris l.on different lipid profile parameters

NG 129.24±2.23 100.08±3.10 48.24±2.51 60.98±3.99 20.02±0.62 PVHF-1 125.92±1.44 98.64±3.86 47.88±6.75 56.88±6.09 19.73±0.77 PVHF-2 117.72±2.46 93.60±3.01 51.12±5.60 43.34±3.91 19.30±0.83 PVEF-1 120.06±6.34 98.28±3.49 49.32±2.88 51.70±9.01 19.66±0.70 PVEF-2 112.68±4.21* 99.00±2.96 55.44±2.08 40.39±6.71 19.80±0.59 PVBF-1 121.32±3.15 95.76±5.07 45.72±2.58 62.47±9.13 19.15±1.01 PVBF-2 114.48±3.49* 82.80±0.81* 54.22±4.53 39.60±5.93 16.56±0.36* All values expressed as Mean ± SEM, where n=5, all data were analyzed by using one way ANOVA followed by Dunnet’s test. p<0.05 was taken to be significant.

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APPENDIX IX: The effect of sub-acute study of crude extract of Phaseolus vulgaris L.on PRE-BODY POST-BODY PERCENTAGE WEIGHT WEIGHT INCREASE (%) NG 125.60±2.34 137.80±2.67 9.71 PVE-1 125.40±3.14 130.00±2.59 3.67 PVE-2 128.80±2.69 134.00±3.51 4.04 PVE-4 129.20±4.49 131.80±4.32 2.01 ATOR-10 129.80±1.86 136.80±1.24 5.39 body weight of Wistar rats

All values expressed as Mean ± SEM, where n=5, all data were analyzed by using one way ANOVA followed by dunnet’s test. p<0.05 was taken to be significant.

APPENDIX X: The effect of sub-acute study of various fractions of Phaseolus vulgaris L.on PRE-BODY POST-BODY PERCENTAGE WEIGHT WEIGHT INCREASE (%) NG 125.60±2.34 137.80±2.67 9.71 PVHF-1 126.80±3.10 132.40±3.70 4.42 PVHF-2 126.60±3.04 133.00±3.01 5.06 PVEF-1 126.80±2.15 132.40±1.89 4.42 PVEF-2 126.20±1.59 134.40±2.20 6.50 PVBF-1 128.80±2.22 134.40±2.04 4.38 PVBF-2 124.40±0.76 130.00±1.61 4.50 body weight of Wistar rats

All values expressed as Mean ± SEM, where n=5, all data were analyzed by using one way ANOVA followed by dunnet’s test. p<0.05 was taken to be significant

APPENDIX XI: The effect of sub-acute study of crude extract of phaseolus vulgaris L.on atherogenic index ATHEROGENIC INDEX NG 0.32±0.03 PVE-1 0.24±0.02 PVE-2 0.24±0.02 PVE-4 0.21±0.03 ATOR-10 0.17±0.03

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All values expressed as Mean ± SEM, where n=5, all data were analyzed by using one way ANOVA followed by Dunnet’s

APPENDIX XII: The effect of sub-acute study of various fractions of Phaseolus vulgaris L.on atherogenic index

ATHEROGENIC INDEX NG 0.32±0.03 PVHF-1 0.33±0.09 PVHF-2 0.27±0.09 PVEF-1 0.30±0.02 PVEF-2 0.25±0.01 PVBF-1 0.32±0.02 PVBF-2 0.24±0.08 All values expressed as Mean ± SEM, where n=5, all data were analyzed by using one wayANOVA followed by dunnet’s

APPENDIX XIII: The effect of chronic study of crude extract of Phaseolus vulgaris l.on different lipid profile parameters

TC TRIGS HDL-C LDL-C VLDL-C

NG 173.08±2.91 138.60±1.80 58.32±1.35 87.12±3.48 27.72±0.36

PVE-1 161.64±4.39 137.52±3.72 66.96±0.67 67.18±4.26* 27.50±0.76

PVE-2 156.60±5.72* 133.20±3.73 67.68±1.94 62.28±4.64* 26.64±0.75

PVE-4 151.92±4.32* 127.44±2.51* 83.52±12.10* 52.12±5.17* 25.49±0.50*

ATOR-10 151.20±2.35* 123.48±1.94* 68.40±2.05 58.10±3.61* 24.70±0.39

All values expressed as Mean ± SEM, where n=5, all data were analyzed by using one way ANOVA followed by dunnet’s

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APPENDIX XIV: The effect of chronic study of various fractions of Phaseolus vulgaris l. on different lipid profile parameters

TC TRIGS HDL-C LDL-C VLDL-C

NG 173.08±2.91 138.60±1.80 58.32±1.35 87.12±3.48 27.72±0.36

PVHF-1 165.96±2.16 147.92±3.86 60.08±1.49 76.30±2.01 29.58±0.77

PVHF-2 162.72±3.04 131.04±3.48 68.04±0.67* 64.44±4.12* 26.64±0.78

PVEF-1 153.76±3.56* 136.08±2.25 61.60±3.36 65.50±5.55* 27.22±0.45

PVEF-2 158.04±2.16* 124.92±1.94* 53.64±0.67 79.62±2.03 24.95±0.39*

PVBF-1 161.28±3.04* 136.68±1.89 56.52±0.92 77.42±3.09 27.34±0.38

PVBF-2 159.72±2.16* 124.56±2.63* 65.16±0.99* 69.65±3.79* 24.91±0.53*

All values expressed as Mean ± SEM, where n=5, all data were analyzed by using one way ANOVA followed by Dunnet’s

APPENDIX XV: The effect of chronic study ofcrude extract of Phaseolus vulgaris l.on body weight

PRE-BODY POST-BODY PERCENTAGE WEIGHT WEIGHT INCREASE (%) NG 146.60±8.22 202.40±11.12 38.06 PVE-1 152.00±9.48 177.80±9.84 16.97 PVE-2 149.00±8.80 170.60±8.69 14.50 PVE-4 146.20±9.36 161.40±8.67* 10.40 ATOR-10 149.00±8.44 159.00±8.39* 6.71 All values expressed as Mean ± SEM, where n=5, all data were analyzed by using one way ANOVA followed by Dunnet’s

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APPENDIX XVI: The effect of chronic study ofvarious extract of Phaseolus vulgaris l.on body weight

PRE-BODY POST-BODY PERCENTAGE WEIGHT WEIGHT INCREASE (%) NG 146.60±8.22 202.40±11.12 38.06 PVHF-1 145.40±9.16 170.20±7.90 17.06 PVHF-2 146.60±8.81 165.60±9.17* 12.96 PVEF-1 151.80±9.68 168.80±10.48 11.20 PVEF-2 147.80±9.07 164.80±6.80* 11.50 PVBF-1 145.40±8.82 163.40±8.17* 12.38 PVBF-2 150.00±6.39 165.00±7.01* 10.00 All values expressed as Mean ± SEM, where n=5, all data were analyzed by using one way ANOVA followed by Dunnet’s

APPENDIX XVII: The effect of chronic study ofcrude ofPhaseolus vulgaris l.on atherogenic index

ATHEROGENIC INDEX NG 0.38±0.01 PVE-1 0.32±0.02 PVE-2 0.30±0.02* PVE-4 0.26±0.03* ATOR-10 0.25±0.01* All values expressed as Mean ± SEM, where n=5, all data were analyzed by using one way ANOVA followed by Dunnet’s

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APPENDIX XVIII: The effect of chronic study ofvarious extractPhaseolus vulgaris l.on atherogenic index

ATHEROGENIC INDEX NG 0.38±0.01 PVHF-1 0.39±0.02 PVHF-2 0.28±0.01* PVEF-1 0.35±0.02 PVEF-2 0.37±0.01 PVBF-1 0.38±0.01 PVBF-2 0.28±0.01* All values expressed as Mean ± SEM, where n=5, all data were analyzed by using one way ANOVA followed by dunnet’s

APPENDIX XIX: DPPH scavenging activity of various extractPhaseolus vulgaris L.

PERCENTAGE (%) Dose (mg/ml) Ascorbic acid PVE PVHF PVEF PVBF 25 40.82 34.69 28.57 31.00 29.59 50 45.92 35.71 35.71 29.59 31.63 100 62.25 51.10 51.62 45.92 52.04 200 75.51 69.39 71.43 64.29 62.25 400 85.71 78.57 75.51 80.61 80.61

APPENDIX XX: Nitric oxide scavenging activity of various extractPhaseolus vulgaris L.

PERCENTAGE (%) Conentration Ascorbic acid PVE PVHF PVEF PVBF (mg/ml) 25 65.52 68.97 62.07 62.06 62.06 50 60.35 74.13 68.97 68.97 60.35 100 25.86 63.79 70.69 67.24 63.79 200 77.59 62.07 75.86 63.79 60.35 400 75.86 56.90 62.07 70.69 67.24

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