1

EZEAGU, CASMIR U

(PG/M.Sc/11/58652)

Nwamarah Uche

Digitally Signed by: Content manager’s Name

DN : CN = Weabmaster’s name

O= University of Nigeria, Nsukka

OU = Innovation Centre

Faculty of Biological Sciences

Department of Biochemistry

ANTIDIABETIC AND ANTIOXIDANT EFFECTS OF BRYOPHYLLUM

PINNATUM IN ALLOXAN-

INDUCED DIABETIC RATS

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CHAPTER ONE

INTRODUCTION

Tropical forests are biologically lavished with diverse ecosystem of plants whose

potential value as natural pharmacy is yet to be discovered (Cohen-Kohler, 2007). The native

people have for centuries used plants as medicine. There is need to stimulate traditional healers.

Traditional medicine involves the use of herbs, animal parts and minerals in prevention and

treatment of diseases, however, herbal medicine is the most widely used of the three. Right from

the beginning, all or parts of all plant species have been used in medicine. Herbal medicine has

been gaining wide acceptance in recent times. One of the reasons for this trend is the high cost of

orthodox medicines which put the intending beneficiaries beyond the reach of many people

particularly the poor countries (Osujih, 1993; Cohen-Kohler, 2007). There has been the

erroneous impression that herbal medicine has fewer side effects (Larrey, 1994; Chan, 2009).

Although many of the claims ascribed to herbal medicine have not been proven scientifically,

some herbs have been extensively studied, thereby warranting their use as alternative to or

complements of orthodox medicines.

A major criticism associated with the use of herbal medicine is the absence of scientific

evaluation of their safety profiles, since many of them have turned out to be toxic (Ernst, 2005;

Yeung et al., 2008). About 80% of the world population is dependent wholly or partially on

plant-based drugs (WHO, 1996). In Nigeria and most developing countries of the world, rural

and urban dwellers, literate or illiterate rely heavily on herbal preparations for treatment of

various diseases despite availability of orthodox medicine (Nwabuisi, 2002).

1.1 Bryophyllum pinnatum

Bryophyllum pinnatum is a perennial herb that grows in the wild and used as a traditional

medicinal as well as ornamental plant in tropical Africa, China, Australia, tropical America and

India. The Genus belongs to the family of Crassulaceae and is cultivated as ornamental house

plant on rocks or in the garden (Kulka, 2006). It is commonly called air plant, Canterbury bell,

cathedral bell, life plant and resurrection plant. It is found in many parts of the world largely

because of it is easy to cultivate. This may also be the reason for its extensive use in herbal

medicine. It is commonly found in the Southern part of Nigeria. The plant is a perennial herb

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with hollow stem. It is 1-1.5 meters high does not have branches. The leaves appear to be the

most valuable part of the plant for medicinal purposes. The lower and uppermost leaves are

simple but the middle ones are usually pinnately compound with 3-5 leaflets, opposite flat and

elliptic blades which are 5-20 cm long and 2-10 cm wide. The margins of the leaves are crenate

and sometimes produce bulbils (Wagner and Sohmer, 1999). The leaves are fleshy dark green

that are distinctly scalloped and trimmed in red. The plant flowers in November-March and fruits

in April. It is astringent, sour in taste, sweet in post digestive effect and has hot potency. It is

well known for its haemostatic and wound healing properties (Kahn et al., 2004). Various

species of B. pinnatum are used medicinally in Indo-China and Philippine islands. It is

naturalized throughout the hot and moist parts of India. The leaves and bark are bitter tonic,

astringent, to the bowels, analgesic, carminative, useful in diarrhea, ulcer and vomiting (Pal et

al., 1999).

Fig. 1: The plant Bryophyllum pinnatum

(Source: Government Reserved Area (GRA) Nsukka )

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1.1.1 Morphology of Bryophyllum pinnatum

Bryophyllum is a rapidly-growing succulent herb. The leaves are thick and fleshy,

simple, or compound in pairs, on reddish stems. Plantlets grow from the notches of the leaf

margins, and they can develop either while still attached to the mother plant, or when detached.

The plant can also reproduce from adventitious shoots from the juvenile leaf bases. The flowers

are in paniculate cymes 20–80 cm long, each one pendant on pedicels 1–2.5 cm in length. Each

flower is about 5 cm long and bell-shaped, greenish or yellowish, reddish by the stems, and very

attractive. The plant can form dense stands in disturbed and dry areas. It is especially invasive in

parts of Hawaii that are of low elevation. In Fiji it is a weed of waste places and cultivated fields,

and along roadsides, naturalized on rocky coasts and slopes, and sometimes in dry forest.

Bryophyllum pinnatum is heterolytic expressing both simple and compound leaf

morphology simultaneously. During the initial growth of B pinatum, the apical meristem or

growth tip of undifferentiated tissue produces juvenile leaves at the base at the base of the plant.

These leaves remain juvenile even as the stem continues to elongate and other leaves are

produced (Wagner and Sohner, 1999). These leaves at the bottom of the plant are old in age

which indicates that they are the first on the plant but still juvenile in terms of their phase of

development. As B. pinnatum continues to grow into adult phase, the apical meristem produces

adult leaves. These leaves are young in age but are adult with regard to their phase of

development. Bryophyllum pinnatum produces simple leaf morphology at its early stage of

development and compound leaf morphology at the adult phase of development (Wagner and

Sohner, 1999).

1.1. 2 Taxonomy of Bryophyllum pinnatum.

Kingdom: Plantae.

Subkingdom: Tracheobionta (vascular plants).

Superdivision: Spermatophyta (seed plants).

Division: Magnoliophyta (flowering plants).

Class: Magnoliopsida (dicotyledons)

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Order: Saxfragales.

Family: Crassulaceae (stone crop family).

Genus: Bryophyllum

Botanical Name: Bryophyllum pinnatum. Synonym: (Kalanchoe pinnatum)

Fig 2: Bryophyllum pinnatum leaf

1.1.3 Geographical distribution of Bryophyllum pinnatum

Bryophyllum pinnatum is the air plant, miracle or life plant is a native to Madagascar. It is

a popular houseplant and has become naturalized in temperate regions of Asia, the pacific and

Caribbean, Australia, Newzealand, West Indies, Macaronesia, Galapagos, Polynesia and Hawaii.

It is widely distributed in Philippines and it is known as katakataka which means astonishing or

remarkable. Much of the reason for the widespread naturalization of this plant can be traced to its

popularity as a garden plant. The group is notable for vegetatively growing small plantlets on the

fringes of the leaves which eventually drop off and root. These plantlets arise from mitosis of

meristematic type tissue in notches in the leaves (Wagner and Sohner, 1999).

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Some other species include:

Kalanchoe adelae ( Bryophyllum adelae)

Kalanchoe beauverdii (Bryophyllum beauverdii)

Kalanchoe bouvetii (Bryophyllum bouvetii)

Kalanchoe campanulata

Kalanchoe fedtschenkoi

Kalanchoe gastonis-bonnieri (Bryophyllum gastonis-bonnieri)

Kalanchoe laxiflora (Bryophyllum crenatum)

Kalanchoe macrochlamys (Bryophyllum macrochlamys)

Kalanchoe manginii (Bryophyllum manginii)

Kalanchoe marnieriana (Bryophyllum marnierianum)

Kalanchoe miniata (Bryophyllum miniatum)

Kalanchoe prolifera (Bryophyllum proliferum)

Kalanchoe pubescens (Bryophyllum aliciae, Bryophyllum pubescens)

Kalanchoe rosei (Bryophyllum rosei)

Kalanchoe schizophylla (Bryophyllum schizophyllum )

Kalanchoe serrata (Bryophyllum lauzac-marchaliae)

Kalanchoe streptantha (Bryophyllum streptanthum)

Kalanchoe suarezensis (Bryophyllum suarezense)

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1.1.3.1 Common names of Bryophyllum pinnatum

Chinese: Lao di sheng gen

Chuukese: Peekoonia

English: Air plant, Canterbury bells, cathedral bells, life plant, Mexican love plant, miracle plant,

mother-of-thousands, resurrection plant

Fijian: Bulatawamudu, thakamana, thakomana

French: Gros pourpier clochette, herbe tortue, kalanchoé penné, mangé-tortue, soudefafe

Hawaiian: ‘Oliwa ku kahakai

I-Kiribati: Te ang

Maori (Cook Islands): ngāngā‘ere toka

Marshallese: Jemata, kibilia

Mokilese: Lamalam

Niuean: Akau tupu he lau, tupu he lau, tupu noa

Pingelapan: Seh lamelan

Samoan: Mimiti, pagi

Spanish: Hoja del aire.

1.1.4 Pharmacological activities of Bryophyllum pinnatum

1.1.4.1 Hepatoprotective and Nephroprotective activities of Bryophyllum pinnatum

The juice of the fresh leaves of Bryophyllum pinnatum is used effectively for the

treatment of jaundice in Bundelkhand region of India. The juice of the leaves was found more

effective than the ethanol extract as evidenced by in-vivo and in-vitro histopathological studies

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for hepatoprotective activity of the plant leaves in folk medicine for jaundice (Yadav and Dixit,

2003).

1.1.4.2 Antimutagenic activity

The plant has potent antihistamine and antiallergic activity (Nassis et al., 1992). The

methanol extract of the leaves has also been reported to have histamine receptor (H1) antagonism

in the ileum, peripheral vasculature and bronchial muscle. The methanol extract also protect

against chemically induced anaphylactic reactions and death by selectively blocking histamine

receptors in the lungs (Nassis et al., 1992; Pal et al., 1999).

1.1.4.3 Antihypertensive activity of Bryophyllum pinnatum

The herb possesses hypotensive activity and this lends credence to the folkloric use of the

herb in the management of hypertension (Ojewole, 2002). The plant is commonly used in the

management of all types and grades of hypertension by some Yorubas of the western Nigeria.

Calcium is the most abundant macroelement present in the plant. Normal extracellular calcium

concentrations are necessary for blood coagulation and for the integrity of intracellular cement

substances (Okaka and Okaka, 2001). The low sodium content of B. pinnatum might be an added

advantage due to the direct relationship of sodium intake with hypertension on human (Dahl,

1972). The effects of aqueous and methanol leaf extracts were examined on arterial blood

pressures and heart rates of normal and spontaneously hypertensive rats using invasive and non-

invasive techniques. Both the extracts produced dose-related significant decreases in arterial

blood pressures and heart rates of anaesthetized normotensive and hypertensive rats. The

hypotensive effects of the extracts were more pronounced in the hypertensive than normotensive

rats (Ojewole, 2002).

1.1.4.4 Analgesic, Anti-inflammatory and wound healing activity of Bryophyllum pinnatum

The high saponin content justifies the use of the plant extracts to stop bleeding and in

treating wounds. Saponin has the property of precipitating and coagulating red blood cells. Some

of the characteristics of saponin include formation of foams in aqueous solution, haemolytic

activity, cholesterol binding properties and bitterness. These properties bestow high medicinal

activities on the extracts of Bryophyllum pinnatum (Khan et al., 2004). Tannins have astringent

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properties, hasten the healing of wounds and inflamed mucous membranes. These perhaps

explain why traditional medicine healers in southern Nigerian often use herb in treating wounds

and burns. Aqueous extract of Bryophyllum pinnatum can demonstrate strong analgesic potency

comparable in time and dose dependent manner to a non steroidal anti-inflammatory drug (Igwe

and Akunyili, 2005).

1.1.5 Indigenous Uses of Bryophyllum pinnatum

The Creoles in America use the slightly roasted leaves in the treatment of cancer,

inflammations and a leaf infusion for fever. The Palikur in India mix the leaf juice with coconut

oil and then rub it on the fore head for migraines and headache. In Mexico and Nicaragua, it is

used to promote menstruation and assist in child birth (Gwehenberger et al., 2004). In Nigeria

and other West African countries, its fleshy leaves are frequently used as a herbal remedy for an

array of human disorders including: boils, wounds, bruises, insect bites, arthritis, rheumatism,

headaches and body pains (Khan et al., 2004). It is employed in the treatment of kidney stones,

gastric ulcers, burns, skin disorders and edema of the legs (Da Silva et al., 1995). Externally, the

pulp of the leaves or the juice is applied on traumatic injuries to arrest the bleeding as it contracts

the minute arterioles and promote the healing without leaving a scar. The herb is highly

recommended in bleeding disorder, piles and menorrhagia (Dalziel, 1937).

1.2 Diabetes Mellitus

The term diabetes mellitus describes a metabolic disorder of multiple aetiology

characterized by chronic hyperglycemia with disturbances of carbohydrate fat and protein

metabolism resulting from defects in insulin secretion, insulin action, or both (Sundaram, 1996).

Translated from ancient Greek, diabetes mellitus means 'honey sweet flow' and this stemmed

from a time in which tasting a patient's urine was still part of the physician's diagnostic

repertoire. The effects of diabetes mellitus include long-term damage, dysfunction and failure of

various organs.

1.2.1 Symptoms of Diabetes Mellitus

Diabetes mellitus may present with characteristic symptoms such as thirst, polyuria,

blurring of vision, and weight loss. In its most severe forms, ketoacidosis or a non-ketotic

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hyperosmolar state may develop and lead to stupor, coma and, in absence of effective treatment,

death. Often, symptoms are not severe, or may be absent, and consequently hyperglycaemia

sufficient to cause pathological and functional changes may be present for a long time before the

diagnosis is made.

The long-term effects of diabetes mellitus include progressive development of the

specific complications of retinopathy with potential blindness, nephropathy that may lead to

renal failure, and/or neuropathy with risk of foot ulcers, amputation, Charcot joints, and features

of autonomic dysfunction, including sexual dysfunction. People with diabetes are at increased

risk of cardiovascular, peripheral vascular and cerebrovascular disease (Bonnefont et al., 2000).

Several pathogenetic processes are involved in the development of diabetes. These include

processes which destroy the beta cells of the pancreas with consequent insulin deficiency, and

others that result in resistance to insulin action. The abnormalities of carbohydrate, fat and

protein metabolism are due to deficient action of insulin on target tissues resulting from

insensitivity or lack of insulin.

Diabetics have been reported to have significantly higher free radical activity as well as

significantly lower concentrations of antioxidants, compared with healthy controls (Sundaram,

1996). In diabetic condition, persistent hyperglycaemia and hyperlipidemia cause increased

production of free radicals especially reactive oxygen species, for all tissues from glucose auto-

oxidation and protein glycosylation (Aragno et al., 1999; Bonnefont et al., 2000). These changes

are of greater magnitude in patients with disease complications than in those without disease

complications. It is therefore possible that supplementing with nutrient foods and herbs that have

antioxidant activity would help prevent diabetic gangrene and organ damage (Sundaram, 1996).

These radicals are generated as by-products of normal cellular metabolism. However, certain

conditions are known to disturb the balance between ROS production and cellular defence

mechanisms. The imbalance can result in cell dysfunction and destruction resulting in tissue

injury. The elevated levels of ROS in diabetics might be due to increased production of free

radicals and or decreased destruction of free radicals by enzymatic catalase, glutathione

peroxidase (GSH-px), and superoxide dismutase (SOD) antioxidants. The levels of these

antioxidant enzymes critically influence the susceptibility of various tissues to oxidative stress

and are associated with the development of complications in diabetes. The enzymes superoxide

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dismutase, glutathione peroxidase and catalase activities contribute to eliminate superoxide

anions, hydroxyl radicals and hydrogen peroxide respectively (Soto et al., 2003).

1.2.2 Blood Glucose Regulation

The homeostatic mechanism which keeps the blood glucose concentration in a

remarkably narrow range is composed of several interacting systems of which hormone

regulation is the most important. There are two types of mutually antagonistic metabolic

hormones that are affecting blood glucose levels as shown in Fig. 3 below: catabolic hormones

such as glucagon, growth hormone e.g pituitary hormone, glucocorticoid e.g. cortisol,

catecholamines e.g. adrenaline, noradrenaline, dopamine which increases blood glucose; while

anabolic hormone (insulin) decreases blood glucose. The human blood sugar level should be

fairly constant at all times and this is made possible by the action of the two antagonistic

hormones, insulin and glucagon. Both insulin and glucagon are secreted by the pancreas, and

thus are referred to as pancreatic endocrine hormones. It is the production of insulin and

glucagon by the pancreas which determines if a patient has diabetes, hypoglycaemia or some

other sugar problem (John and Henry, 2001).

The inducer for insulin secretion is high blood glucose. Although, there is always low

level of insulin secreted by the pancreas, the amount secreted into the blood increases as the

blood glucose rises. Similarly, as blood glucose falls, the amount of insulin by the beta cells

decreases. Insulin has an effect on number of cells, including muscle, red blood cells and fat

cells. In response to insulin, these cells absorb glucose out of the blood, having the net effect of

lowering the high blood glucose levels into the normal range (John and Henry, 2001). Glucagon

is secreted by the alpha cells of the pancreas in the same pattern as insulin but in opposing

biochemical mechanism. If the blood glucose is high, no glucagon will be secreted. When blood

glucose has decreased such as during period of fasting, more glucagon will be secreted. The

effect of glucagon is to facilitate the release of glucose from the liver cells into the blood stream,

with the net effect of increasing blood glucose. Glucagon also facilitates gluconeogenesis.

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Fig. 3. Glucose regulation

1.3 Classification of diabetes

1.3.1 Type 1 diabetes mellitus

This form of diabetes is called insulin-dependent diabetes, Type 1 diabetes, or juvenile-

onset diabetes.Type 1 diabetes results from autoimmune mediated destruction of the beta cells of

the pancreas (Zimmet et al., 1994). The rate of destruction of beta cells is quite variable, being

rapid in some individuals and slow in others (Zimmet et al., 1994). The rapidly progressive form

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is commonly observed in children, but also may occur in adults (Humphery et al., 1998). The

slowly progressive form generally occurs in adults and is sometimes referred to as latent

autoimmune diabetes in adults (LADA). Some patients, particularly children and adolescents,

may present with ketoacidosis as the first manifestation of the disease (Japan and Pittsburgh

Childhood Diabetes Research Groups, 1985). Others have modest fasting hyperglycaemia that

can rapidly change to severe hyperglycaemia and/or ketoacidosis in the presence of infection or

other stress. Still others, particularly adults, may retain residual beta-cell function, sufficient to

prevent ketoacidosis, for many years (Zimmet, 1995).

Individuals with this form of Type 1 diabetes often become dependent on insulin for

survival and are at risk for ketoacidosis (Willins et al., 1996). At this stage of the disease, there is

little or no insulin secretion as manifested by low or undetectable levels of plasma C-peptide

(Hother-Nielsen et al., 1988). Markers of immune destruction, like islet cell autoantibodies,

and/or autoantibodies to insulin, and autoantibodies to glutamic acid decarboxylase (GAD) are

present in 85-90 % of individuals with Type 1 diabetes mellitus when fasting diabetic

hyperglycaemia is initially detected (Verge et al., 1996). The peak incidence of this form of Type

1 diabetes occurs in childhood and adolescence, but the onset may occur at any age, ranging

from childhood to the ninth decade of life (Molbak et al., 1994). There is a genetic predisposition

to autoimmune destruction of beta cells. It is also related to environmental factors that are still

poorly defined. Although, patients are usually not obese when they present with this type of

diabetes, the presence of obesity is not incompatible with the diagnosis. These patients may also

have other autoimmune disorders such as Graves' disease, Hashimoto's thyroiditis, and Addison's

disease (Betterle et al., 1983).

1.3.2 Type 2 diabetes mellitus

Diabetes mellitus of this type is regarded as non-insulin-dependent diabetes, or adult-

onset diabetes. It is a term used for individuals who have relative (rather than absolute) insulin

deficiency (Lillioja et al., 1993). People with this type of diabetes are frequently resistant to the

action of insulin (DeFronzo et al., 1997; Lillioja et al., 1993). These individuals do not need

insulin treatment to survive. This form of diabetes is frequently undiagnosed for many years

because the hyperglycaemia is often not severe enough to provoke noticeable symptoms of

diabetes (Mooy, et al., 1995; Harris, 1993). Nevertheless, such patients are at increased risk of

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developing macrovascular and microvascular complications (Mooy et al., 1995; Harris, 1993).

There are probably several different mechanisms which result in this form of diabetes, and it is

likely that the number of people in this category will decrease in the future as identification of

specific pathogenetic processes and genetic defects permits better differentiation and a more

definitive classification with movement into "Other types".

Although, the specific aetiologies of this form of diabetes are not known, by definition

autoimmune destruction of the pancreas does not occur and patients do not have other known

specific causes of diabetes. The majority of patients with this form of diabetes are obese; obesity

itself causes or aggravates insulin resistance (Campbell and Carlson, 1993; Bogardus et al.,

1985). Many of those who are not obese by traditional weight criteria may have an increased

percentage of body fat distributed predominantly in the abdominal region (Kissebah et al., 1982).

Ketoacidosis is infrequent in this type of diabetes; when seen it usually arises in association with

the stress of another illness such as infection (Banerji et al., 1994; Umpierrez et al., 1995).

Whereas patients with this form of diabetes may have insulin levels that appear normal or

elevated, the high blood glucose levels in these diabetic patients would be expected to result in

even higher insulin values had their beta-cell function been normal (Polonsky et al., 1996). Thus,

insulin secretion is defective and insufficient to compensate for the insulin resistance. On the

other hand, some individuals have essentially normal insulin action, but markedly impaired

insulin secretion. Insulin sensitivity may be increased by weight reduction, increased physical

activity, and/or pharmacological treatment of hyperglycaemia but is not restored to normal

(Simonson et al., 1984; Wing et al., 1994). The risk of developing Type 2 diabetes increases

with age, obesity, and lack of physical activity (Zimmet, 1992). It occurs more frequently in

women with prior GDM and in individuals with hypertension or dyslipidaemia. Its frequency

varies in different racial/ethnic subgroups (Valle et al., 1997). It is often associated with strong

familial, likely genetic predisposition. However, the genetics of this form of diabetes are

complex and not clearly defined. Some patients who present with a clinical picture consistent

with Type 2 diabetes have autoantibodies similar to those found in Type 1 diabetes, and may

masquerade as Type 2 diabetes if antibody determinations are not made. Patients who are non-

obese or who have relatives with Type 1 diabetes and who are of Northern European origin may

be suspected of having late onset Type 1 diabetes.

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1.3.2.1 Gestational diabetes

Gestational diabetes is carbohydrate intolerance resulting in hyperglycaemia of variable

severity with onset or first recognition during pregnancy (Kissebah et al., 1982). It does not

exclude the possibility that the glucose intolerance may antedate pregnancy but has been

previously unrecognized. The definition applies irrespective of whether or not insulin is used for

treatment or the condition persists after pregnancy (Kissebah et al., 1982). Women who become

pregnant and who are known to have diabetes mellitus which antedates pregnancy do not have

gestational diabetes but have "diabetes mellitus and pregnancy" and should be treated

accordingly before, during, and after the pregnancy. In the early part of pregnancy (e.g. first

trimester and first half of second trimester) fasting and postprandial glucose concentrations are

normally lower than in normal, non-pregnant women (Polonsky et al., 1996). Elevated fasting or

postprandial plasma glucose levels at this time in pregnancy may well reflect the presence of

diabetes which has antedated pregnancy, but criteria for designating abnormally high glucose

concentrations at this time have not yet been established (Polonsky et al., 1996). The occurrence

of higher than usual plasma glucose levels at this time in pregnancy mandates careful

management and may be an indication for carrying out an OGTT. Nevertheless, normal glucose

tolerance in the early part of pregnancy does not itself establish that gestational diabetes may not

develop later. Individuals at high risk for gestational diabetes include older women, those with

previous history of glucose intolerance, those with a history of large for gestational age babies,

women from certain high-risk ethnic groups, and any pregnant woman who has elevated fasting,

or casual, blood glucose levels. It may be appropriate to screen pregnant women belonging to

high-risk populations during the first trimester of pregnancy in order to detect previously

undiagnosed diabetes mellitus. Formal systematic testing for gestational diabetes is usually done

between 24 and 28 weeks of gestation (Valle et al., 1997).

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1.3.2.2 Genetic defects of beta cell function

Several forms of the diabetic state may be associated with monogenic defects in beta-cell

function which are frequently characterized by onset of mild hyperglycaemia at an early age

(generally before age 25 years). They are usually inherited in an autosomal dominant pattern

(Byrne et al., 1996). Patients with these forms of diabetes, formerly referred to as maturity-onset

diabetes of the young (MODY), have impaired insulin secretion with minimal or no defect in

insulin action (Byrne et al., 1996; Clement et al., 1996). Abnormalities at three genetic loci on

different chromosomes have now been characterized. The most common form is associated with

mutations on chromosome 12 in a hepatic nuclear transcription factor referred to as HNF1alpha

(Yamagata et al., 1996). A second form is associated with mutations in the glucokinase gene on

chromosome 7p (Froguel et al., 1996; Vionnet et al., 1992).

Glucokinase converts glucose to glucose-6-phosphate, the metabolism of which in turn

stimulates insulin secretion by the beta cell. Thus, glucokinase serves as the "glucose sensor" for

the beta cell. Because of defects in the glucokinase gene, increased levels of glucose are

necessary to elicit normal levels of insulin secretion. A third form is associated with a mutation

in the HNF4alpha gene on chromosome 20q (Yamagata et al., 1996). HNF4alpha is a

transcription factor which is involved in the regulation of the expression of HNF1alpha. A fourth

variant has recently been ascribed to mutations in another transcription factor gene, IPF-1, which

in its homozygous form leads to total pancreatic agenesis (Stoffers et al., 1997). Specific genetic

defects in other individuals who have a similar clinical presentation are currently being defined.

Point mutations in mitochondrial DNA have been found to be associated with diabetes mellitus

and deafness (Walker and Turnbull, 1997). The most common mutation occurs at position 3243

in the tRNA leucine gene, leading to an A to G substitution.

An identical lesion occurs in the MELAS syndrome (mitochondrial myopathy,

encephalopathy, lactic acidosis, and stroke-like syndrome); however, diabetes is not part of this

syndrome, suggesting for unknown reasons different phenotypic expressions of this genetic

lesion (Johns, 1995). Genetic abnormalities that result in the inability to convert proinsulin to

insulin have been identified in a few families. Such traits are usually inherited in an autosomal

dominant pattern (Gruppuso et al., 1984; Robbins et al., 1984) and the resultant carbohydrate

intolerance is mild. Similarly, mutant insulin molecules with impaired receptor binding have

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been identified in a few families. These are also associated with autosomal inheritance and either

normal or only mildly impaired carbohydrate metabolism (Haneda et al., 1984; Sanz et al.,

1986).

1.3.2.3 Role of Insulin in Lipid Metabolism

Fat cells respond to insulin within minutes of its administration (Clement et al., 1996).

There will be reduction in the release of fatty acids from fat stores. At the same time there will be

increase in the synthesis of triacylglycerol to increase fat esterification and storage. So the

overall effect is decrease in fatty acids in blood. Insulin inhibits an enzyme called ‘lipase’ in fat

cells. Lipase function is to breakdown fat and release free fatty acids in to the blood. Once its

activity is inhibited by insulin, it no longer releases fatty acids from stored fat. Insulin also

increases the activity of another enzyme called ‘lipoprotein lipase’ in blood vessel endothelial

cells. The function of lipoprotein lipase is to synthesize triacylglycerol using free fatty acids. So

fatty acid levels are decreased. In diabetes there is deficiency of insulin (Campbell and Calson,

1993). The chylomicrons and VLDL (both are types of lipoproteins in blood that carry

triglycerides from intestinal food and liver respectively) are released into blood waiting for the

enzyme lipoprotein lipase to act. Due to insulin deficiency which is needed by it, the enzyme

cannot function to its full extent. This increases triglycerides in blood causing

‘Hypertriglyceridemia’, a type of high cholesterol state. The above mechanism also operates in

type 2 diabetes. In addition there will be some other mechanisms are responsible for

hypertriglyceridemia in type 2 diabetes. In type 2 diabetes, there is insulin resistance state which

is not an ideal condition for the enzyme lipoprotein lipase. And also the type 2 diabetics are

obese, where there will be excess VLDL, related to obesity. VLDL is not metabolized

completely increasing the level of Intermediate density lipoproteins in the blood. (IDL carries

very high cholesterol in them) (Campbell and Calson, 1993).

1.4 Alloxan

Alloxan (2, 4, 5, 6- tetraoxypyrimidine; 2, 4, 5, 6- pyrimidinetetrone) is an oxygenated

pyrimidine derivative. It is present as alloxan monohydrate in aqueous solution (Lenzen, 2008).

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1.4.1 Biological effect of alloxan.

Alloxan is a toxic analogue of glucose that selectively destroys insulin- producing beta

cells of the pancreas when administered to rodents and many other animals (Lenzen, 2008). This

causes insulin dependent diabetes mellitus (alloxan diabetes) with characteristics similar to type

1diabetes in those rats. Alloxan is selectively toxic to insulin-producing beta cells because it

preferentially accumulates in beta cells through uptake via the GLUT2 glucose transporter.

Alloxan in the presence of intracellular thiols, generates reactive oxygen species (ROS) in a

cyclic reaction with its reduction product, dialuric acid. The beta cell toxic action of alloxan is

initiated by free radicals formed in the redox reaction. Alloxan and the product of its reduction

establish a redox cycle with the formation of superoxide radicals (Mrozikiewicz et al., 1994).

These radicals undergo dismutation to hydrogen peroxide. Consequently, highly reactive

hydroxyl radicals are formed by the fenton reaction as shown below:

Fe2+

+ H2O2 →Fe3+

+ .OH + OH-,

Fe3+

+ H2O2 → Fe2+

+ .OOH + H+ .

The action of reactive oxygen species with a simultaneous massive increase in cytosolic

calcium concentration causes rapid destruction of beta cells. A study suggests that alloxan does

not cause diabetes in humans (Lenzen, 2008). Other studies show some correlation between

alloxan plasma levels and type 1 diabetes in children (Mrozikiewicz et al., 1994).

Fig. 4: Alloxan structure

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1.5 Antioxidants

An antioxidant is a molecule that inhibits the oxidation of other molecules (Helmut,

1997). Oxidation is a chemical reaction that transfers electrons or oxygen from a substance to an

oxidizing agent (Helmut, 1997). 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 further oxidation reactions. They do this by being oxidized themselves,

so antioxidants are often reducing agents such as thiols, ascorbic acid, or polyphenols (Helmut,

1997). Although initial studies suggested that antioxidant supplements might promote health,

later large clinical trials with a limited number of antioxidants detect no benefit and even

suggested that excess supplementation with certain putative antioxidants may be harmful

(Prabhat et al., 1995; Bjelakovic et al., 2007).

Antioxidants are classified into two broad divisions, depending on whether they are

soluble in water (hydrophilic) or in lipids (hydrophobic). In general, water-soluble antioxidants

react with oxidants in the cell cytosol and the blood plasma, while lipid-soluble antioxidants

protect cell membranes from lipid peroxidation (Helmut, 1997). These compounds may be

synthesized in the body or obtained from the diet. The different antioxidants are present at a wide

range of concentrations in body fluids and tissues, with some such as glutathione or ubiquinone

mostly present within cells, while others such as uric acid are more evenly distributed. Some

antioxidants are only found in a few organisms and these compounds can be important in

pathogens and can be virulence factors (Miller and Britigan, 1997). The relative importance and

interactions between these different antioxidants is a very complex question, with the various

metabolites and enzyme systems having synergistic and interdependent effects on one another

(Chaudiere and Ferrari-Iliou, 1999).

The action of one antioxidant may therefore depend on the proper function of other

members of the antioxidant system. The amount of protection provided by any one antioxidant

will also depend on its concentration, its reactivity towards the particular reactive oxygen species

being considered, and the status of the antioxidants with which it interacts. Some compounds

contribute to antioxidant defense by chelating transition metals and preventing them from

catalyzing the production of free radicals in the cell. Particularly important is the ability to

sequester iron, which is the function of iron-binding proteins such as transferrin and

20

Seleniferritin. Selenium and zinc are commonly referred to as antioxidant nutrients, but these

chemical elements have no antioxidant action themselves and are instead required for the activity

of some antioxidant enzymes (Miller and Britigan, 1997).

1.6 The Oxidative Challenge in Biology

A paradox in metabolism is that, while the vast majority of complex life on Earth requires

oxygen for its existence, oxygen is a highly reactive molecule that damages living organisms by

producing reactive oxygen species (Davies, 1995). Consequently, organisms contain a complex

network of antioxidant metabolites and enzymes that work together to prevent oxidative damage

to cellular components such as DNA, proteins and lipids. In general, antioxidant systems either

prevent these reactive species from being formed, or remove them before they can damage vital

components of the cell (Davies, 1995). However, reactive oxygen species also have useful

cellular functions, such as redox signaling. Thus, the function of antioxidant systems is not to

remove oxidants entirely, but instead to keep them at an optimum level (Rhee, 2006).

The reactive oxygen species produced in cells include hydrogen peroxide (H2O2),

hypochlorous acid (HClO), and free radicals such as the hydroxyl radical (·OH) and the

superoxide anion (O2−) (Valko et al., 2007). The hydroxyl radical is particularly unstable and

will react rapidly and non-specifically with most biological molecules. This species is produced

from hydrogen peroxide in metal-catalyzed redox reactions such as the Fenton reaction (Stohs

and Bagchi, 1995). These oxidants can damage cells by starting chemical chain reactions such

as lipid peroxidation, or by oxidizing DNA or proteins. Damage to DNA can cause mutations

and possibly cancer, if not reversed by DNA repair mechanisms (Nakabeppu et al., 2006) while

damage to proteins causes enzyme inhibition, denaturation and protein degradation (Stadtman,

1992).

The use of oxygen as part of the process for generating metabolic energy produces

reactive oxygen species (Raha and Robinson, 2000). In this process, the superoxide anion is

produced as a by-product of several steps in the electron transport chain. Particularly important

is the reduction of coenzyme Q in complex III, since a highly reactive free radical is formed as

an intermediate (Q·−). This unstable intermediate can lead to electron "leakage", when electrons

jump directly to oxygen and form the superoxide anion, instead of moving through the normal

series of well-controlled reactions of the electron transport chain. Peroxide is also produced

21

from the oxidation of reduced flavoproteins, such as complex I. However, although these

enzymes can produce oxidants, the relative importance of the electron transfer chain to other

processes that generate peroxide is unclear (Seaver and Imlay, 2004). In plants, algae, and

cyanobacteria, reactive oxygen species are also produced during photosynthesis,( Demmig-

Adams, and Adams, 2002) particularly under conditions of high light intensity (Krieger-

Liszkay, 2004).This effect is partly offset by the involvement of carotenoids in photoinhibition,

which involves these antioxidants reacting with over-reduced forms of the photosynthetic

reaction centres to prevent the production of reactive oxygen species ( Kerfeld, 2004).

1.6.1 Pro-oxidant activities

Antioxidants that are reducing agents can also act as pro-oxidants. For example, vitamin

C has antioxidant activity when it reduces oxidizing substances such as hydrogen peroxide

(Duarte and Lunec , 2005). However, it will also reduce metal ions that generate free radicals

through the Fenton reaction (Carr and Frei, 1999; Stohs and Bagchi, 1995).

2 Fe3+ + Ascorbate → 2 Fe2+ + Dehydroascorbate

2 Fe2+ + 2 H2O2 → 2 Fe3+ + 2 OH· + 2 OH−

The relative importance of the antioxidant and pro-oxidant activities of antioxidants are

an area of current research, but vitamin C, which exerts its effects as a vitamin by oxidizing

polypeptides, appears to have a mostly antioxidant action in the human body (Carr and Frei,

1999; Valko et al., 2005). However, less data is available for other dietary antioxidants, such as

vitamin E (Schneider, 2005). or the polyphenols ( Halliwell, 2008 ; Ristow and Zarse, 2010).

Likewise, the pathogenesis of diseases involving hyperuricemia likely involves uric acid's direct

and indirect pro-oxidant properties. That is, paradoxically, agents which are normally considered

antioxidants can act as conditional pro-oxidants and actually increase oxidative stress. Besides

ascorbate, medically important conditional pro-oxidants include uric acid and sulfhydryl amino

acids such as homocysteine. Typically, this involves some transition-series metal such as copper

or iron as catalyst. The potential role of the pro-oxidant role of uric acid in (e.g.) atherosclerosis

and ischemic stroke is considered above. Another example is the postulated role of homocysteine

in atherosclerosis (Valko et al., 2005).

22

1.6.2 Oxidative stress

Oxidative stress represents an imbalance between the systemic manifestation of reactive

oxygen species and a biological system's ability to readily detoxify the reactive intermediates or

to repair the resulting damage (Valko et al., 2005). Disturbances in the normal redox state of

cells can cause toxic effects through the production of peroxides and free radicals that damage all

components of the cell, including proteins, lipids, and DNA. Further, some reactive oxidative

species act as cellular messengers in redox signaling. Thus, oxidative stress can cause disruptions

in normal mechanisms of cellular signaling. In man, oxidative stress is thought to be involved in

the development of many diseases or may exacerbate their symptoms. These include cancer,

Parkinson's disease, Alzheimer's disease (Valko et al., 2007), atherosclerosis, heart failure,

(Singh et al., 1995) myocardial infarction (Ramond et al., 2011) Schizophrenia; (Boskovic et

al., 2011), fragile X syndrome, Sickle Cell Disease (Amer et al., 2006) and chronic fatigue

syndrome (Gwen et al., 2005). However, reactive oxygen species can be beneficial, as they are

used by the immune system as a way to attack and kill pathogens. Short-term oxidative stress

may also be important in prevention of aging by induction of a process named mitohormesis

(Gems and Partridge, 2008). Chemically, oxidative stress is associated with increased production

of oxidizing species or a significant decrease in the effectiveness of antioxidant defenses, such as

glutathione (Schafer and Buettner, 2001). The effects of oxidative stress depend upon the size of

these changes, with a cell being able to overcome small perturbations and regain its original

state. However, more severe oxidative stress can cause cell death and even moderate oxidation

can trigger apoptosis, while more intense stresses may cause necrosis (Lennon et al., 1991).

Production of reactive oxygen species is a particularly destructive aspect of oxidative

stress. Such species include free radicals and peroxides. Some of the less reactive of these

species (such as superoxide) can be converted by oxidoreduction reactions with transition metals

or other redox cycling compounds (including quinones) into more aggressive radical species that

can cause extensive cellular damage. The major portion of long term effects is inflicted by

damage on DNA (Evans and Cooke, 2004).Most of these oxygen-derived species are produced at

a low level by normal aerobic metabolism. Normal cellular defense mechanisms destroy most of

these. Likewise, any damage to cells is constantly repaired. However, under the severe levels of

oxidative stress that cause necrosis, the damage causes ATP depletion, preventing controlled

apoptotic death and causing the cell to simply fall apart (Lee and Shacter, 1999).

23

1.6.3 Lipid Peroxidation and Free Radical

Lipid peroxidation refers to the oxidative degradation of lipids. It is the process in which

free radicals "steal" electrons from the lipids in cell membranes, resulting in cell damage. This

process proceeds by a free radical chain reaction mechanism. It most often affects

polyunsaturated fatty acids, because they contain multiple double bonds in between which lie

methylene -CH2- groups that possess especially reactive hydrogen. Peroxidation (auto-oxidation)

is responsible not only for deterioration of food (rancidity) but also for damage to tissue in vivo,

where it may be a source of cancer, inflammatory diseases, atheriosclerosis and aging (Murray et

al., 2003). The deleterious effects are considered to be caused by free radicals (ROO; RO; OH·)

produced during peroxide formation from fatty acids containing interrupted double bonds. As

with any radical reaction, lipid peroxidation is a chain reaction providing continuous supply of

the free radicals that initiate further peroxidation (Halliwell et al., 1999). The reaction consists

of three major steps: initiation, propagation, and termination (Halliwell et al., 1999).

1.6.3.1 Initiation

Initiation is the step in which a fatty acid radical is produced. The most notable initiators

in living cells are reactive oxygen species (ROS), such as OH· and HOO, which combines with a

hydrogen atom to form water and a fatty acid radical (Halliwell et al., 1999).

1.6.3.2 Propagation

The fatty acid radical is not a very stable molecule, so it reacts readily with molecular

oxygen, thereby creating a peroxyl-fatty acid radical. This too is an unstable species that reacts

with another free fatty acid, producing a different fatty acid radical and lipid peroxide, or cyclic

peroxide if it had reacted with itself. This cycle continues, as the new fatty acid radical reacts in

the same way (Halliwell et al., 1999).

1.6.3.3 Termination

When a radical reacts with a non-radical, it always produces another radical, which is

why the process is called a "chain reaction mechanism." The radical reaction stops when two

radicals react and produce a non-radical species. This happens only when the concentration of

radical species is high enough for there to be a high probability of collision of two radicals.

24

Living organisms have evolved different molecules that speed up termination by catching free

radicals and, therefore, protecting the cell membrane. One important such antioxidant is vitamin

E. Other anti-oxidants made within the body include the enzymes superoxide dismutase,

catalase, and peroxidase (Halliwell et al., 1999).

Fig. 5: Mechanism of lipid peroxidation.

25

1.7 Aim and Objectives of the study

1.7.1 Aim of the study

This study aims to investigate the anti-diabetic and anti-oxidant effect of the ethanol

extract of Bryophyllum pinnatum on alloxan-induced diabetic rats.

1.7.2 Research Objectives

This work is designed to achieve the following specific objectives:

(i) To determine the median lethal toxicity (LD50) of the extracts

(ii) To determine the effect of the extract on blood glucose concentration of the rats

(iii) To determine the effect of the extract on lipid profile Total cholesterol, HDL, LDL and

TAG of the rats

(iv) To determine the effect of the extract on the pro- and anti-oxidant potentials of the rats.

(v) To determine the effect of the extract on haematological parameters of the rats.

26

CHAPTER TWO

MATERIALS AND METHODS

2.1 Materials

2.1.1 Equipment/ Apparatus Manufacturer

One touch glucometer Lifescan, USA

PCV tubes Pyrex England

Haematocrit centrifuge Vickas Ltd England

PCV reader Pyrex England

Pasteur pipette Pyrex England

Colorimeter E1 Scientific CO. India

Syringes Mono-ject China

Measuring cylinder Pyrex England

Weighing balance Vickas Ltd England

Refrigerator Haier thermocool China

Beaker Pyrex England

Centrifuge Vickas Ltd England

Micropipette Perfect USA

2.1.2 Reagents/ Chemicals Manufacturer

Alloxan Sigma Aldrich Germany

Glacial acetic acid May and bakers England

Sodium carbonate May and baker England

Sodium chloride BDH England

Trichloroacetic acid Merck Darmstadt Germany

Thiobarbituric acid May and bakers England

Sodium Dodesyl sulphate BDH England

2,4-dinitrophenyl hydrazine Merck Darmstadt Germany

Thiourea solution Randox USA

Cupric sulphate solution Merck Darmstadt Germany

Sulphuric acid BDH England

27

Potassium dichromate Sigma Aldrich Germany

Combined colour reagent QCA Spain

Molisch’s reagent QCA Spain

Ethanol BDH E ngland

Dragenddroff’s reagent Randox USA

Wagner’s reagent Randox USA

Picric acid solution Merck Darmstadt Germany

Ammonia solution BDH E ngland

Fehling’s solution A and B Teco USA

Potassium hydroxide solution Merck Darmstadt Germany

Olive oil Solive oil Nigeria

Ferric chloride Merck Darmstadt Germany

Lead acetate solution Merck Darmstadt Germany

Ethyl acetate BDH England

Aluminium chloride solution BDH England

Chloroform BDH England

1% thiobarbituric acid May and bakers England

Sodium hydroxide May and bakers England

Trition-X Teco USA

Drabkin’s solution Teco USA

Cumen hydroxide QCA Spain

2.1.3 Plant Material

Fresh leaves of Bryophullum pinnatum were collected from GRA in Nsukka LGA of

Enugu State, Nigeria. The leaves were identified by Mr Alfred Ozioko of Bioresources

Development and Conservation Programme (BDCP), Nsukka, Nigeria.

2.1.4 Experimental Animals

A total of twenty-one (21) male albino rats with an average weight of 170±16g were

obtained from the Animal House of Department of Zoology, University of Nigeria, Nsukka. The

animals were acclimatized to laboratory conditions for one week under a standard condition with

28

12 hour light and dark cycles and maintained on a regular feed and water ad libitum. The mice

used for LD50 were 8-10 weeks old with average weight of 16±1.4g

2.2 Methods

2.2.1 Preparation of Extract

Fresh leaves of Bryophyllum pinnatum were plucked and washed with distilled water.

The leaves were spread on a mat in a well ventilated room with regular turning to avoid

decaying. The leaves were shade dried for four weeks after which they were pulverized. One

thousand, one hundred and twenty-five grams (1125g) of dry leaves were coarsely ground. The

ground leaves were soaked for 48 hours in 95% absolute ethanol with occasional shaking after

which they were filtered using muslin sieve. The filtrate was later evaporated to dryness using

rotary evaporator at 60˚C.

2.2.2 Determination of LD50 of the Extract

The Median lethal dose (LD50) of the ethanol extract was determined by the method of

(Lorke, 1983). Six groups of three adult albino-mice each weighing between 13-20g were used

for this experiment. The experiment was divided into two phases. Phase one comprises groups

1-3 and they were administered with 10, 100 and 1000 mg/kg body weight of extract respectively

while phase two comprises groups 4-6 and were administered 1600, 2900 and 5000 mg/kg body

weight of the extract respectively. The extract was dissolved in 3% tween 80 and made up to the

required volume with distilled water and administered orally to the mice according to their

groups. The mice were observed for 24 hours and the number of deaths was recorded. The LD50

was calculated using geometric mean as the square root of the product of the lowest lethal dose

and the highest non-lethal dose.

LD50=√XY

Where X= Highest dose without death,

Y= lowest dose with death.

29

2.2.3 Experimental Design

In this experiment, the rats were divided into seven groups of three rats each. Group 1

was administered 1ml of 3% tween 80 solution which represented the vehicle. The treatment

were carried out using (per oral) orogastric tube (gavage).

Group 1: Normal rats were Non diabetic (negative control), no treatment was given.

Group 2: Positive control, Diabetic rats without treatment.

Group 3: Diabetic rats treated with standard drug (2.5 mg/kg b.w of glibenclamide.

Group 4: Diabetic rats treated with 200 mg/kg body weight of extract

Group 5: Diabetic rats treated with 400 mg/kg body weight of extract.

Group 6: Non diabetic rats treated with 200 mg/kg body weight of extract.

Group 7: Non diabetic rats treated with 400 mg/kg body weight of extract.

2.2.4 Diabetes Mellitus Induction

On the seventh day of acclimatization, the rats were starved overnight prior to induction

of diabetes mellitus with a view to enabling the alloxan to penetrate the vital organs of the rats.

Diabetes was induced by intraperitoneal injection of a single dose of 150mg/kg body weight of

1% alloxan monohydrate dissolved in freshly prepared normal saline. After twenty-four hours,

all the rats that have blood glucose level of 200mg/dl and above were considered diabetic and

were selected for the experiment.

2.2.5 Preparation of reagents for phytochemical analysis

5% (w/v) ferric chloride solution

A quantity, 5.0g of ferric chloride was dissolved in 100ml of distilled water.

Ammonium solution

A known volume, 187.5ml of the stock concentrated ammonium solution was dissolved in

32.5ml of distilled water.

45% (v/v) ethanol

Absolute ethanol, (45ml) was mixed with 55ml of distilled water.

Aluminium chloride solution

Aluminium chloride, (0.5g) was dissolved in 100ml of distilled water.

30

Wagner’s reagent

A quantity, (2.0g) of iodine crystals and 3.0g of potassium iodide were dissolved in 40ml

of distilled water and then made up to 100ml with distilled water.

Mayer’s reagent

A known weight, 13.5g of mercuric chloride was dissolved in 50ml of distilled water.

Also, 5.0g of potassium iodide was dissolved in 20ml of distilled water. The two solutions were

mixed and the volume was made up to 100ml with distilled water.

Dilute sulphuric acid

A known volume, 10.9ml of concentrated sulphuric acid was mixed with 5.0ml of

distilled water and made up to 100ml.

Lead sub-acetate solution

A quantity, 45ml of 15%lead acetate (15g of lead acetate in 100ml of distilled water) was

dissolved in 20ml of absolute ethanol and made up to 100ml with distilled water.

Dragendorff’s reagent

Bismuth carbonate (0.85g) was dissolved in 100ml of glacial acetic acid and 40ml of

distilled water to give solution A. Another solution called solution B was prepared by dissolving

8.0g of potassium iodide in 20ml of distilled water. Both solutions were mixed to give a stock

solution.

Molisch reagent

A known weight 1.0g of α-naphtol was dissolved in 100ml of absolute ethanol.

2% (v/v) Hydrochloric acid

A known volume, 2.0ml of concentrated hydrochloric acid was diluted with distilled

water and made up to 100ml.

1% (w/v) picric acid

A quantity, 1.0g of picric acid was dissolved in 100ml of distilled water.

2.2.6 Phytochemical Analysis of the Fresh and Dry Leaf Extact of B. pinnatum

The preliminary analysis of the phytochemical composition of the ethanol extract was

carried out according to the method of (Harborne, 1984) which involved testing for the presence

of the following plant constituents: alkaloids, flavonoids, carbohydrates, glycosides, proteins,

saponins, steroidal aglycone, tannins, oil, acids, resins and terpenoids.

31

2.2.6.1 Test for the presence of alkaloids

The ethanol extract (0.2g) of B. pinnatum leaf was boiled with 5ml of 2% hydrochloric

acid on a water bath. The mixture was filtered using Whatman No 1 filter paper. One mililitre of

the filtrate was treated with 2 drops of Dragendroff’s reagent (bistmuth potassium iodide

solution). A red precipitate indicates the presence of alkaloids (Harborne, 1984).

2.2.6.2 Test for the presence of glycosides

The ethanol extract (2g) of B. pinnatum leaf was mixed with 30ml of water. The mixture

was heated in a water bath at 60˚C for 5 minutes and filtered. A mixture of Fehling’s solution A

and B of equal volumes (0.2ml each) was added to 5ml of the filtrate until it turned alkaline

when tested with litmus paper. It was then boiled in water bath for 3 minutes at 60˚C and

observed.

2.2.6.3 Molisch test

One gramme (1g) of the extract was boiled with 2ml of distilled water and filtered. To the

filtrate, few drops of alpha naphtol solution (Molisch’s reagent) were poured down the side of

the test tube to form a lower layer.

2.2.6.4 Test for acid compounds

The extract (0.1g) was placed in a dry test tube and 10 ml of distilled water was added.

The mixture was warmed over water bath at 60˚C and later cooled. A strip of water wetted

neutral litmus paper was dipped into the filtrate and observation was made.

2.2.6.5 Test for the presence of steroids.

To a mixture of 10 ml of lead acetate solution (90%w/v) and 20 ml of aqueous ethanol

(50%) was added 1g of the extract in a 200ml conical flask. The mixture was placed on a boiling

water bath for 2 minutes, cooled and filtered. The filtrate was washed twice with 15ml of

chloroform. A 5ml aliquot of the extract was evaporated to dryness in a water bath. To the

residue, 2ml of 3,5-dinitrobenzoic acid solution (in 2% ethanol) and 1ml of 1N sodium

hydroxide solution were added.

32

2.2.6.6 Test for the presence of flavonoids

The extract (0.2g) was dissolved in 10ml of ethyl acetate and heated in boiling water for

3minutes. The mixture was filtered using whatman No1 filter paper and the filtrate was used for

the test. 4ml of the filtrate was shaken with 1% (1ml) of aluminium chloride solution and

observed.

2.2.6.7 Test for Resin

The extract, 0.2g was re-extracted with 15ml of 95% ethanol. The alcohol extract was

then poured into 20ml of distilled water in a beaker and observed.

2.2.6.8 Test for the Presence of Tannins

The extract, (2g) were boiled in 5ml of 45% ethanol for 5 minutes. The mixture was

cooled and filtered. 1ml of the filtrate was added 3 drops of lead acetate solution

2.2.6.9 Test for the Presence of Saponins

The extract, 0.1g was boiled in 6ml of distilled water for 5 minutes. The mixture was

filtered while still hot. The filtrate was used for the following tests:

Emulsion Test

Olive oil 2 drops were added to 1.0ml of the filtrate. The mixture was shaken and

observed.

Frothing Test

The filtrate, 1 ml was mixed with 4ml of distilled water and then shaken vigorously and

observed.

2.2.7 Quantitative Phytochemical Analysis of Fresh and Dry Leaf Extract

2.2.7.1 Steroid Determination

This was determined by the method described by Okeke and Elekwa (2003). A known

weight, 2g of each sample was dispersed in 100ml of distilled water and homogenized in a

laboratory blender. The homogenate was filtered and the filtrate was eluted with normal

ammonium hydroxide solution (pH 9). A volume of 2ml of the elute was put in a test tube and

mixed with 2ml of chloroform. A known quantity, 3ml of ice-cold acetic acid anhydride was

33

added to the mixture in a flask and 2 drops of conc H2SO4 were cautiously added. Standard sterol

solution was prepared and treated as described above. The absorbances of standard and prepared

sample were measured in a spectrophotometer at 420nm.

2.2.7.2 Saponin Determination

Each sample, (1g) was weighed and macerated with 10ml of petroleum ether and

decanted into a beaker. Petroleum ether, (10ml) was decanted into the beaker, mixed with the

filtrate and evaporated to dryness; this was followed by addition of 6ml of ethanol. A fraction of

the resultant mixture 2ml was pipetted into a test tube and this was followed by addition of 2ml

of colour reagent. Absorbance was measured at 550nm after 30 minutes of standing.

2.2.7.3 Flavonoid Determination

This was determined according to the method of Harborne (1998). A portion (5g) of the

sample was refluxed in 50ml of 2M HCl solution for 30 minutes. It was allowed to cool and then

filtered with Whatman No1 filter paper. A measured volume of the extract was treated with equal

volume of ethyl acetate starting with a drop. The solution was filtered into a weighed crucible.

The filtrate was heated to dryness in an oven at 60˚C. The dried crucible was weighed again and

the difference in the weight gave the quantity of flavonoid in the sample.

2.2.7.4 Determination of Reducing Sugar

One gramme of each sample was weighed and macerated with 20ml of distilled water,

boiled for 5minutes and cooled. 1ml of phosphomolybdic acid and 7ml of distilled water were

added and absorbance was measured at 420nm.

2.2.7.5 Alkaloid Determination

The determination of alkaloid was described by Harborne (1998). A quantity (5g) of the

sample was weighed into a 250ml beaker and 200ml of 10% acetic acid in ethanol was added,

covered and allowed to stand for 2 hours. This was filtered and the extract was concentrated on a

water bath to one-quarter of its original volume. Concentrated ammonium hydroxide was added

drop-wise to the extract to obtain the precipitate which was collected and washed with dilute

34

ammonium hydroxide and then filtered. The residue is the alkaloid which was dried and

weighed.

2.2.7.6 Terpenoid Determination

One gramme of the sample was macerated into 50ml of ethanol. The solution was

filtered. The filtrate (2.5ml) was pipetted into a test tube followed by 2.5ml of 5% aqueous

phosphmolibdic acid solution, 2.5ml conc H2SO4 was gradually added, mixed and allowed to

stand for 30minutes. The solution was made up to 12.5ml with ethanol and absorbance was taken

at 700nm.

2.2.7.7 Glycoside Determination

The sample (1g) was added into a test tube and 2.5ml of 15% ethanol was added and

filtered. A known quantity (2.5ml) of chloroform was added and shaken vigorously; the lower

layer was then collected and evaporated to dryness after which 3ml of glacial acetic acid was

added followed by 0.1ml of 5% ferric chloride, 0.25ml of concentrated hydrogen sulphide (H2S).

The setup was shaken and put in the dark for 2hours and absorbance was read at 530nm.

2.2.7.8 Tannin Determination

The method of Swain (1979) was used for the determination of the tannin content of B

pinnatum. A quantity, 0.2g of finely ground sample was measured into a 50ml beaker. A known

volume (20ml) of 50% methanol was added and covered with paraffin and placed in a water bath

at 77-80˚C for 1hr and stirred with a glass rod to prevent bumping. The extract was filtered using

a double layer of Whatman No1 filter paper into a 50ml volumetric flask. Known volumes of

20ml of distilled water, 2.5ml of Folin-Denis reagent and 10ml of 17% Na2CO3 were added and

mixed properly. The mixture was made up to mark with distilled water and allowed to stand for

20 minutes when a bluish colouration developed. Standard tannic acid solutions of range 0-

10ppm were treated similarly as 1ml of sample above. The absorbances of tannic acid standard

solution as well as samples were read after colour development at 760nm. The tannin content

was calculated using the formula:

Tannin (%) = Absorbance of sample ×Average gradient×Dilution factor

Weight of sample × 10000

35

2.2.7.9 Cyanide Determination

The extract, (1g) was macerated with 50ml of distilled water and allowed to stand for 24

hours and filtered. The filtrate (1ml) was pipetted into a test tube and 4ml of alkaline picrate

solution was added, boiled for 5minutes, cooled and absorbance was measured at 490nm.

2.2.7.10 Soluble Carbohydrate Determination

The sample (1g) was macerated with 50ml of distilled water and filtered. Also 1ml of the

filtrate was pipetted into a test tube and 2ml of saturated picric acid was added. Absorbance was

measured at 530nm.

2.2.8 Anti-Diabetic Evaluation

The rats were divided into seven groups of three rats each group. Group one contained

negative control rats which were given 1ml of 3% tween 80 solution. They were administered

1ml of 3% tween 80 solution. Group two were untreated diabetic rats. The extract was

administered daily to group 4-7 for twenty-one days while 2.5 mg/bw of glibenclamide standard

drug was administered to group 3. Blood glucose concentration was measured on weekly interval

to ascertain the hypoglycaemic effect of the extract on the rats being treated. This was done by

slightly cutting the tail vein with a sharp scissors. Blood samples were collected by nipping and

smearing the tail on the indicated portion of glucometer strip until it was fully soaked by blood.

The strip was inserted into the Accu-check glucometer and blood glucose concentration was read

within few seconds. On day 22 the rats were fasted overnight and blood samples collected

through ocular puncture into EDTA bottle for haematological analysis. The blood samples for

lipid profile determination were collected into a plain container without anticoagulant and the

serum obtained. The rats were anaesthetized with chloroform after the collection of blood

samples.

2.2.9 Determination of Haematological Parameters of the Rats

2.2.9.1 Determination of Packed Cell Volume

This was determined by using standard haematological procedure as described by Ochei and

Kolhakar (2005).

Principle: When whole blood is subjected to a centrifugal force for maximum

36

RBC parking, the space occupied by the RBC is measured and expressed as percentage of the

whole blood volume.

Method: Using microhaematocrit method, a well-mixed anticoagulant whole blood was allowed

to enter capillary haematocritn tubes until they are they are 2/3 filled with blood. Blood filling

was done for each tube. One end of each tube was sealed with plastic seal and placed in the

medial groove of the centrifuge, head exactly opposite each other, with sealed end away from the

centre of the centrifuge. All tubes were spun for 5minutes at 3000 revolution per minute (rpm).

The tubes were removed when as soon as the centrifuge had stopped spinning.

Calculation: PCV was obtained for each tube using microhaematocrit-reader by measuring the

height of the RBC column and expressing it as a ratio of the height of the total blood column.

PCV (%) = Height of cell column x 100

Height of total blood column

2.2.9.2 Determination of Haemoglobin (Hb) Concentration

The concentration of haemoglobin was determined by standard haematological procedure

as described by Ochei and Kolhakar (2005).

Principle: Whole blood is added to drabkin’s reagent: a solution containing KCN and

K3Fe(CN)6. KCN converts Hb-Fe²+ (ferrous) to Hb-Fe³+ (ferric) state to form methaemoglobin

complex which combines with KCN to form a stable pigment cyanmethaemoglobin complex.

The colour intensity of this mixture is measured in a spectrophotometer at a wavelength of

540nm (or using a yellow-green filter). The optical density (OD) of the solution is proportional

to the haemoglobin concentration. All forms of Hb (Hb-C, Hb-O, etc) except Hb-S are measured

with this cyanmet-method.

Method: Exactly 5.0ml of Drabkin’s solution was pipetted into two test tubes and a well mixed

sample sample of EDTA blood (0.02ml) was pipetted into these tubes, rinsing the pipette five

times with the reagent, until all the blood has been removed from the pipette. The solutions were

well mixed and allowed to stand at 25˚C for 10 minutes in order to allow the formation of cyan-

methaemoglobin. The mixtures were transferred into cuvettes and read in a spectrophotometer at

a wavelength of 540nm. The drabkin’s reagent in tube 1 was used to blank the machine (setting

the percentage transmittance at 100%). The readings were recorded and transferred into a pre-

calibrated chat and the actual Hb values in g/dl were determined.

37

Calculation: Hb in g/dl = Absorbance of test x conc. Of standard (in mg/dl)

Absorbance of standard

2.2.9.3 Determination of Red Blood Cell (RBC) Counts

Red blood cell (RBC) count was determined using the method of Ochei and Kolhartkar,

(2005).

Principle: When whole blood is diluted with an isotonic fluid, it prevents lysis and facilitates

counting of the red cells. Some isotonic solutions in use include Haymen’s solution, Gower’s

solution or 0.85%NaCl solution.

Method: using the thoma (manual counting) method, anticoagulated blood was drawn up to the

0.5ml mark in the RBC count pipette and diluted to a 101 mark with RBC diluting fluid (1:200

dilution). Dilution was repeated with the replicate tube counting chamber was cleaned; both

pipettes were shaken three times , counting chamber filled (first expelling the first 4 drops of the

mixture, allowing approximately three minutes for RBCs to settle. Red cells were counted using

the counting steps as follows.

1. The filled counting chamber was carefully placed on the microscope stage.

2. Using low power (x10 objective) the large centre square was placed in the middle of the field

of vision and the entire large square was carefully examined for even distribution of RBCs.

3. The high-dry was carefully changed, moving the counting chamber so that the small upper

left corner square (this square is further sub-divided into 16 even smaller squares) is completely

in the field of vision.

4. All the RBCs were counted in the squares, also counting the cells on the two of the margins

but excluding those lying on the other sides.

Calculation: The RBCs (in mm³) =cells counted x correction for volume x correction for

dilution

= RBCs counted in 5 small squares × 200 × 1.0/0.2 (or 50)

=number of RBCs counted in five squares × 104.

2.2.9.4 Determination of Total White Blood Cell (WBC) Count

Total white blood cell (WBC) count was determined using the method of Ochei and

Kolhartkar (2005).

38

Principle: When whole blood is mixed with weak acid solution, it dilutes the blood and

haemolyes the RBCs, enabling the WBCs to be counted.

Method: Manual WBC counting method was used as follows:

Dilution of Blood:

i. The blood specimen was mixed appropriately for one minute; using aspirator and WBC

pipette, blood was drawn to the 0.5 mark in the pipette.

ii. Blood was removed from the outside of the pipette with clean gauze.

iii. Holding the pipette almost vertically, the tip was placed into the counting diluting fluid to

draw it slowly. While gently rotating the pipette, to ensure proper mixing, the diluting fluid was

aspired until it reached 11 mark.

iv. The pipette was placed in a horizontal position and firmly holding the index finger of the

either hand over the opening in the tip of the pipette, aspirator was detached from other end of

the pipette. This is 1:20 dilution.

v. Having completed the dilution of blood, the counting chamber and cover glass were cleaned

with a lint free cloth.

Filling the counting chamber: Approximately, 0.02ml of well mixed EDTA blood sample was

added to 0.38ml of diluted fluid, dispensed into a small container. One of the grids of the

counting chamber was filled with re-mix of the diluted blood sample using a Pasteur pipette

taking care not to overfill the area. The filled area was left undisturbed for two minutes to allow

the WBCs to settle, after which the underside of the chamber was dried and placed on a

microscope stage.

Counting the white blood cells: Using x 10 objective lens with the condenser iris closed

sufficiently to give good contrast, the ruling of the chamber and the white cells were focused

until the cells appeared as small black dots. The cells in the four large squares of the chamber

were then squarely counted.

Calculation; the number of white cells per litre of blood was calculated as follows:

The total number of white cells counted was divided by 2

The number obtained was then divided by 10

The result was then multiplied by 109

39

2.2.9.5 Assay for Lipid Peroxidation

Lipid peroxidation was determined spectrophtometrically by measuring the level of lipid

peroxidation product, malondialdehyde (MDA) as described by Wallin et al. (1993).

Principle:

Malondialdehyde (MDA) reacts with thiobarbituric acid to form red or pink coloured

complex which absorbs maximally at 532nm.

MDA+ 2 TBA -------> MDA: TBA adduct + H20

Reagent preparation

i. 1.0% Thiobarbituric acid (TBA): A quantity, 1.0g of thiobarbituric acid was dissolved in

83ml of distilled water on warming and after dissolution the volume was made up to 100ml with

distilled water.

ii. 25% Trichloroacetic acid (TCA): A quantity, 12.5g of trichloroacetic acid was dissolved in

distilled water and made up to 50ml in volumetric flask with distilled water.

iii. Normal saline solution (NaCl) : A quantity, 0.9g of NaCl was dissolved in 10ml of distilled

water and made up to 100ml with distilled water.

Procedure

To 0.1ml of plasma in a test tube was added 0.45 ml of normal saline and mixed

thoroughly before adding 0.5ml of 25% trichloroacetic acid and 0.5ml of 1% thiobarbituric acid.

The same volume of trichloroacetic acid, and saline was added to the blank. Distilled water

(o.1ml) was added to the blank instead of plasma. Then the mixture was heated in a water bath at

95˚C for 40 mins. Turbidity was removed by centrifugation. The mixture was allowed to cool

before reading the absorbance of the clear supernatant at 532nm against reagent blank at 600 nm.

Thiobarbituric acid reacting substances were quantified as lipid peroxidation products by

referring to the standard curve of MDA concentration.

2.2.9.6 Assay of Catalase Activity

Catalase activity was assayed by the method described by Abei (1983).

40

Principle

The ultraviolet absorption of hydrogen peroxide can be easily measured at 240nm. On the

decomposition of hydrogen peroxide with catalase, the absorption decreases with time and from

this decrease, catalase activity can be measured.

Reagent

a. Phosphate buffer, pH 7.0

b. 0.2M H2O2

c. 5% potassium dichromate

d. Glacial acetic acid

Procedure

Phosphate buffer (2.5ml) was pipetted into a test tube followed by 2ml of hydrogen

peroxide and 0.5ml of serum. To 1ml of portion of the reaction mixture was added 2ml of

bichromate acetic acid reagent. Absorbance was determined at 240nm at every 15 seconds

interval into four places for one minute.

Catalase activity was calculated using the equation below:

Catalytic activity (unit/L) = 0.23 × logA1/A2

0.00693

Where A1 = A 240 at t = 0.

A2=A 240 at t = 15 seconds.

0.23 and 0.00693 are constants.

2.2.9.7 Determination of High Density Lipoprotein (HDL) Concentration

The concentration of high density lipoprotein (HDL) was determined according to the

method of Albers et al. (1978).

Procedure:

A set of clean test tubes (1ml) was set up and 0.3ml of the serum was pipetted into each

of them. One drop of the precipitant solution (dextran sulphate and magnesium acetate) was

added to the serum in each of the test tube, mixed and allowed to stand for 15mins at 25˚C. It

was then centrifuged at 3000 revolutions per minute (rpm) for 10 minutes. A volume of 1ml each

of cholesterol esterase, cholesterol oxidase, peroxidase, buffer (pH 6.8), phenol, 3,5-

41

dichlorophenol, 4-amino antipyrine was added to another set of test tubes labeled standard, blank

and sample test (labeled according to group number). Also 1ml of the supernatant of the

centrifuged samples was added to each of the labeled test tubes. The entire solution was mixed

thoroughly and allowed to stand for 10 minutes at 25˚C. The absorbance of the sample and the

standard against the sample blank was read at a wavelength of 500nm. The HDL cholesterol

content of each sample in mg/dl was calculated using the formula:

Absorbance of the sample × concentration of the standard

Absorbance of the standard

2.2.9.8 Determination of Serum Low Density Lipoprotein Concentration

The concentration of low density lipoprotein (LDL) was determined according to the

method of Assman et al. (1984).

Procedure

The serum (0.2ml) was pipetted into sets of clean labeled test tubes. Three drops of

precipitate solution (polyvinyl sulphate, sodium EDTA, polyethylene glycol, monomethyl ether)

were addedto the serum in the test tubes, mixed properly and allowed to stand for 10minutes at

25˚C. Thereafter the mixture was centrifuged at 3000rpm for 10 minutes. A volume of 1ml each

of cholesterolesterase, cholesterol oxidase, peroxidase, buffer (pH 6.8), phenol, 3,5-

dichlorophenol, 4-amino antipyrine was added to another sets of test tubes labeled standard,

blank and sample test (labeled according to group numbers). Also 0.1ml of the supernatant

sample was added appropriately to the labeled tubes. The entire solution was mixed thoroughly

and allowed to stand for 10 minutes at 25˚C, after which 0.1ml of the standard (equivalent to

200mg/dl of cholesterol) was added to the test tube labeled standard, mixed thoroughly and

allowed to stand for 10 minutes at 25˚C. the absorbance of the sample and the standard against

the sample blank was read at a wavelength of 505nm. The cholesterol content of the supernatant

was calculated using the formula:

Absorbance of the sample × concentration of the standard

Absorbance of the standard.

The LDL-cholesterol concentration was calculated using the relationship:

42

LDL (mg/dl) = Total cholesterol ˗ 1.5 × supernatant cholesterol (Assman et al., 1984).

2.2.9.9 Determination of Serum Total Cholesterol Concentration

Total cholesterol concentration was determined according to the method of Allain et al.

(1974).

Procedure

The serum (0.2ml) was pipetted into sets of clean labeled test tubes, 3 drops of precipitant

solution (polyvinyl sulphate, sodium EDTA, poly ethylene glycol monomethyl ester) was added

to the serum in the test tube, mixed properly and allowed to stand at 25˚C. The mixture was

centrifuged at 3000rpm for 10 minutes. A volume of 1ml each of cholesterol esterase, cholesterol

oxidase, peroxidase, pipes buffer (pH 6.8), phenol, 3,5-dichlorophenol, 4-amino antipyrine was

added to another set of test tubes labeled standard , blank and sample test labeled according to

group numbers. Also 0.1ml of the supernatant derived from the centrifugation of the precipitant

sample mixture was added appropriately to the test tubes. The entire media was mixed

thoroughly and allowed to stand for 10 minutes at 25˚C after which 0.1ml of the standard

(equivalent to 200mg/dl cholesterol) was added to the test tube labeled standard, mixed well and

allowed to stand for 10 minutes at 25˚C. the absorbance of the sample and the standard against

the sample blank was read at a wavelength of 505nm. The cholesterol content of the supernatant

was calculated using the formula:

Absorbance of the sample × concentration of the standard

Absorbance of the standard

2.2.9.10 Determination of Triacylglycerol (TAG) Concentration

Triacylglycerol concentration was determined by the method of Bucalo and David

(1973).

Procedure

A known volume, 1ml each of 4-chlorophenol, 4-amino antipyrine, ATP, MgCl2, glycerol

kinase, glycerol-3-phosphate oxidase, peroxidase and lipase was pipetted into a set of test tubes

labeled standard, blank and sample test. Also, 1ml of the supernatant derived from the

43

centrifugation of precipitate sample mixture was added appropriately to the labelled test tubes.

The entire media were mixed thoroughly and allowed to stand for 10 minutes at 25˚C, after

which 0.1ml of the standard solution was added to the test tube labelled standard, mixed

thoroughly and was allowed to stand for 10 minutes at 25˚C. The absorbance of the sample and

the standard against the reagent blank was read at a wavelength of 500nm.

The triacylglycerol content of each of the supernatants was calculated using the formula:

Absorbance of the sample × concentration of the standard

Absorbance of the standard

2.3 Statistical Analysis

Statistical analysis was determined using one way analysis of variance (ANOVA)

together with post Hoc test (multiple comparisons) i.e. LSD using statistical package for social

sciences. Data obtained from the test groups (mean of triple determination ± SD) were compared

with their respective controls and the differences were considered significant at p<0.05.

44

CHAPTER THREE

RESULTS

3.1 Percentage Yield of Ethanol Extract of Bryophyllum pinnatum Leaves

Table 1 shows a weight of 500g of dried sample of Bryophyllum pinnatum with a

percentage yield and weight of the extract as 4% and 20g respectively.

Table 1: Percentage yield of the extract of Bryophyllum pinnatum leaves

Weight of dried sample (g) Percentage yield of extract (%) Weight of extract (g)

500.00 4.00 20.00

45

3.2 Qualitative Phytochemical Screening of the Ethanol Extract of Bryophyllum

pinnatum

Table 2 shows that both dry and fresh samples of the leaves contain alkaloids, flavonoids,

steroids, glycosides, terpenoids, carbohydrates, tannins. There is no significant increase in the

phytochemical composition of the fresh samples as compared to the dry samples of the plant.

Table 2: Qualitative phytochemical constituents of the fresh and dry samples of B pinnatum

Phytochemicals Fresh Samples Dry Samples

Alkaloids ++ +++

Flavonoids +++ +++

Steroids + +

Glycosides ++ ++

Terpenoids + +

Carbohydrates + +

Tannins ++ ++

Saponin ++ ++

Hydrogen cynide + +

Reducing sugar ++ ++

Key: + = slightly present ++ = moderately present +++ = highly present --- = not detected

46

3.3 Quantitative Phytochemical Constituents of Both Fresh and Dry samples of

Bryophyllum pinnatum

Table 3 shows mean of triple determination of the phyto-constituents of the fresh and dry

samples of Bryophyllum pinnatum. The antioxidant property of the plant extract could be

attributed to the high presence of flavonoids, tannins, glycosides and alkaloids (Ververidis et al.,

2007). All the edible plants that possess anti-oxidant properties contain high level of flavonoids

and phenolic compounds. Consequently, the lower concentration of Hydrogen cyanide is

observable evidence of the low toxicity of the plant. Also, the fresh samples have higher

concentration of reducing sugar, saponins and alkaloids than the dry samples. This is due the

difference in moisture content between the fresh and the dry samples.

Table 3: Quantitative phytochemical constituent of fresh and dry samples.

Phytochemical constituents Fresh samples (mean of 3 Dry samples (mean of 3 det-

of B pinnatum (mg/100g) determinations ±SD) erminations ±SD)

Flavonoids

Glycosides

Carbohydrates

Hydrogen cyanide

Reducing sugar

Saponins

Steroids

Tannins

Terpenoids

Alkaloids

3.25±0.03

3.40±0.25

0.79±0.01

0.10±0.00

500±25

1.7±0.50

0.27±0.01

2.25±0.02

0.51±0.01

2.90±0.10

3.26±0.02

3.5±0.20

0.79±0.00

0.09±0.00

350±25

1.1±0.15

0.27±0.00

2.26±0.02

0.51±0.00

3.25±0.10

47

3.4 Acute Toxicity (LD50) Test of Bryophyllum pinnatum Leaf Extract

The acute toxicity test (LD50) of the ethanol extract was indeterminable as there

was no death recorded and no obvious toxicological signs at a dose of 5g/kg body weight.

The acute toxicity test of the extract indicated that that the plant is not toxic and is an

indication that the leaves are safe for human and animal consumption.

Table 4: Phase: I and II of the acute toxicity (LD50) test of Bryophyllum pinnatum leaf

extract.

Dosage (mg/kg body weight) Mortality

Phase I

Group 1 10 0/3

Group 2 100 0/3

Group 3 1000 0/3

Phase II

Group 4 1600 0/3

Group5 2900 0/3

Group6 5000 0/3

48

3.5 Effect of Ethanol Extract of Bryophyllum pinnatum on Glucose

Concentration of Allooxan Induced Diabetic Rats

Fig. 6 shows the effect of ethanol extract of Bryophyllum pinnatum on glucose

concentration of alloxan-induced diabetic rats. Significant reduction (p<0.05) was

observed in the glucose concentration of groups 4 and 5 diabetic rats treated with 200

mg/kg and 400 mg/kg body weight of the extract respectively compared with the glucose

concentration of the diabetic untreated rats in group 2. There was also a significant

reduction (p<0.05) in the glucose concentration of diabetic rats in group 3 treated with

glibenclamide standard drug compared with the glucose concentration of the diabetic

untreated rats in group 2.

49

Group 1: Normal rats (negative control), no treatment was given. Group 2: Positive control, Diabetic rats without treatment. Group 3: Diabetic rats treated with standard drug (2.5 mg/kg b.w of glibenclamide. Group 4: Diabetic rats treated with 200 mg/kg body weight of extract. Group 5: Diabetic rats treated with 400 mg/kg body weight of extract. Group 6: Non diabetic rats treated with 200 mg/kg body weight of extract. Group 7: Non diabetic rats treated with 400 mg/kg body weight of extract.

50

3.6 Effect of Ethanol Extract of Bryophyllum pinnatum on Packed Cell Volume (PCV)

Level of Alloxan Induced Diabetic Rats

Fig. 7 shows non-significant increase (p>0.05) in packed cell volume (PCV) of the rats in

the test groups (groups 4, 5, 6 and7) compared with the PCV of the diabetic untreated rats (group

2). On the other hand, non-significant decrease (p>0.05) was observed in the PCV level of test

rats compared with the PCV of diabetic rats (group 3 treated with standard drug. Neither

significant (p>0.05) increase nor decrease was observed in the PCV of rats in the test groups

compared with the PCV of normal control rats in group 1.

51

Group 1: Normal rats (negative control), no treatment was given. Group 2: Positive control, Diabetic rats without treatment. Group 3: Diabetic rats treated with standard drug (2.5 mg/kg b.w of glibenclamide. Group 4: Diabetic rats treated with 200 mg/kg body weight of extract. Group 5: Diabetic rats treated with 400 mg/kg body weight of extract. Group 6: Non diabetic rats treated with 200 mg/kg body weight of extract. Group 7: Non diabetic rats treated with 400 mg/kg body weight of extract.

52

3.7 Effect of Ethanol Extract of Bryophyllum pinnatum on Catalase Activity of

Alloxan Induced Diabetic Rats

Fig. 8 shows the effect of Bryophyllum pinnatum extract on catalase activity of

alloxan induced diabetic rats. Significant increase was observed (p<0.05) in the catalase

activity of groups 4 and 5 treated with 200 and 400 mg/kg b.w of the extract respectively

compared with the group 2 diabetic untreated rats. The catalase activity of group 3 also

significantly (p<0.05) increased compared with group 2 diabetic untreated group 2. There

was no significant increase (p>0.05) in the catalase activity of the diabetic treated groups

(group 3, 4, and 5) compared with the normal control group 1.

53

Group 1: Normal rats (negative control), no treatment was given. Group 2: Positive control, Diabetic rats without treatment. Group 3: Diabetic rats treated with standard drug (2.5 mg/kg b.w of glibenclamide. Group 4: Diabetic rats treated with 200 mg/kg body weight of extract. Group 5: Diabetic rats treated with 400 mg/kg body weight of extract. Group 6: Non diabetic rats treated with 200 mg/kg body weight of extract. Group 7: Non diabetic rats treated with 400 mg/kg body weight of extract.

54

3.8 Effect of Ethanol Extract of Bryophyllum pinnatum on Malondialdehyde

Concentration (MDA) of Alloxan Induced Diabetic Rats

Fig. 9 shows the effect of ethanol extract of Bryophyllum pinnatum on

malondialdehyde concentration of alloxan induced diabetic rats. The level of

malondialdehyde in the tissue is an indication of membrane lipid peroxidation. The

ethanol extract of Bryophyllum pinnatum was able to significantly (p<0.05) reduce the

malondialdehyde level of groups 4 and 5 respectively treated with 200 and 400mg/kg

b.w of the extract compared with group 2 diabetic untreated rats. Significant reduction

(p<0.05) was also observed in the malondialdehyde level of groups 6 and 7 normal rats

respectively treated with 200 and 400 mg/kg b.w of the extract compared with diabetic

untreated group 2.

55

Group 1: Normal rats (negative control), no treatment was given. Group 2: Positive control, Diabetic rats without treatment. Group 3: Diabetic rats treated with standard drug (2.5 mg/kg b.w of glibenclamide. Group 4: Diabetic rats treated with 200 mg/kg body weight of extract. Group 5: Diabetic rats treated with 400 mg/kg body weight of extract. Group 6: Non diabetic rats treated with 200 mg/kg body weight of extract. Group 7: Non diabetic rats treated with 400 mg/kg body weight of extract.

56

3.9 Effect of Ethanol Extract of Bryophyllum pinnatum on Haemoglobin

Concentration of Alloxan Induced Diabetic Rats

Fig. 10 shows the effect of the extract on haemoglobin concentration of alloxan

induced diabetic rats. The extract was able to significantly (p<0.05) increase the

haemoglobin concentration of the treated groups 4, 5, 6 and 7 compared with the diabetic

untreated group 2. The reduction in haemoglobin concentration of group 2 might be due

to glycosylation of the red cells which prevented the possibility of binding of the haem

molecule. There is no significant increase between groups 6 and 7 normal rats

respectively treated with 200 and 400 mg/kg b.w of the extract compared with group 1

normal control.

57

Group 1: Normal rats (negative control), no treatment was given. Group 2: Positive control, Diabetic rats without treatment. Group 3: Diabetic rats treated with standard drug (2.5 mg/kg b.w of glibenclamide. Group 4: Diabetic rats treated with 200 mg/kg body weight of extract. Group 5: Diabetic rats treated with 400 mg/kg body weight of extract. Group 6: Non diabetic rats treated with 200 mg/kg body weight of extract. Group 7: Non diabetic rats treated with 400 mg/kg body weight of extract.

58

3.10 Effect of Ethanol Extract of Bryophyllum pinnatum on Low Density

Lipoprotein Concentration of Alloxan-Induced Diabetic Rats

Fig. 11 shows the effect of ethanol extract of Brophyllum pinnatum on Low

Density Lipoprotein (LDL). Significant reduction (p<0.05) was observed in LDL

concentration of groups 4 and 5 treated rats compared with group 2 untreated rats. No

significant decrease (p>0.05) in LDL concentration was observed in groups 6 and 7

normal rats respectively treated with 200 and 400 mg/kg b.w of the extract compared

with group 1 normal control. LDL concentration also significantly (p<0.05) decreased in

group 3 treated with standard drug compared with group 2 diabetic untreated.

59

Group 1: Normal rats (negative control), no treatment was given. Group 2: Positive control, Diabetic rats without treatment. Group 3: Diabetic rats treated with standard drug (2.5 mg/kg b.w of glibenclamide. Group 4: Diabetic rats treated with 200 mg/kg body weight of extract. Group 5: Diabetic rats treated with 400 mg/kg body weight of extract. Group 6: Non diabetic rats treated with 200 mg/kg body weight of extract. Group 7: Non diabetic rats treated with 400 mg/kg body weight of extract.

60

3.11 Effect of Ethanol Extract of Bryophyllum pinnatum on Red Blood Cell of

Alloxan Induced Diabetic Rats

Fig. 12 shows the effect of ethanol extract of Bryophyllum pinnatum on the red

blood cell concentration of alloxan induced diabetic rats. There was no significant

increase (p>0.05) in the red blood cell concentration of the treated groups (groups 4 and

5) compared with diabetic untreated group 2. Significant increase (p<0.05) was observed

in group 3 treated with standard drug compared with group 2 untreated rats. There was no

significant increase (p>0.05) in groups 6 and 7 compared with group 1 normal control.

61

Group 1: Normal rats (negative control), no treatment was given. Group 2: Positive control, Diabetic rats without treatment. Group 3: Diabetic rats treated with standard drug (2.5 mg/kg b.w of glibenclamide. Group 4: Diabetic rats treated with 200 mg/kg body weight of extract. Group 5: Diabetic rats treated with 400 mg/kg body weight of extract. Group 6: Non diabetic rats treated with 200 mg/kg body weight of extract. Group 7: Non diabetic rats treated with 400 mg/kg body weight of extract.

62

3.12 Effect of Ethanol Extract of Bryophyllum pinnatum on High Density

Lipoprotein of Alloxan Induced Diabetic Rats

Fig. 13 shows the effect of ethanol extract of Bryophyllum pinnatum on high

density lipoprotein. High density lipoprotein concentration significantly (p<0.05)

increased in the test groups (groups 4, 5, 6 and 7) compared with group 2 untreated rats.

HDL also increased significantly (p<0.05) in group 3 compared with group 2. No

significant increase (p>0.05) in HDL was observed in groups 3, 4, 5, 6 and 7 compared

with group 1 normal control.

63

Group 1: Normal rats (negative control), no treatment was given. Group 2: Positive control, Diabetic rats without treatment. Group 3: Diabetic rats treated with standard drug (2.5 mg/kg b.w of glibenclamide. Group 4: Diabetic rats treated with 200 mg/kg body weight of extract. Group 5: Diabetic rats treated with 400 mg/kg body weight of extract. Group 6: Non diabetic rats treated with 200 mg/kg body weight of extract. Group 7: Non diabetic rats treated with 400 mg/kg body weight of extract.

64

3.13 Effect of ethanol extract of B pinnatum on Triacylglycerol level of alloxan

induced diabetic rats.

Fig. 14 shows the leaf effect on triacylglycerol level of the rats. No significant decrease

(p>0.05) was observed in triacylglycerol level in group 4 treated with 200 mg/kg b.w of

the extract compared with group 2. Significant reduction in triacylglycerol level (p<0.05)

was observed in the standard control group (group 3) compared with group 2 untreated

rats. No significant reduction was observed in the treated groups (groups 5, 6 and 7)

compared with group 3 treated with standard drug.

65

Group 1: Normal rats (negative control), no treatment was given. Group 2: Positive control, Diabetic rats without treatment. Group 3: Diabetic rats treated with standard drug (2.5 mg/kg b.w of glibenclamide. Group 4: Diabetic rats treated with 200 mg/kg body weight of extract. Group 5: Diabetic rats treated with 400 mg/kg body weight of extract. Group 6: Non diabetic rats treated with 200 mg/kg body weight of extract. Group 7: Non diabetic rats treated with 400 mg/kg body weight of extract.

66

3.14 Effect of Ethanol Extract of Bryophyllum pinnatum on Total cholesterol Level of

Alloxan Induced Diabetic Rats

Fig. 15 shows the effect of Bryophyllum pinnatum leaf extract on total cholesterol level

of alloxan-induced diabetic rats. The extract was able to significantly (p<0.05) lower the total

cholesterol level of the diabetic treated groups (groups 4 and 5) compared with untreated

group 2. No significant decrease (p>0.05) was observed in the diabetic treated groups

(groups 4 and 5) compared with non-diabetic treated group 6 and 7. There was no significant

decrease (p>0.05) in the total cholesterol level of the standard control group (group 3)

compared with the normal control group.

67

Group 1: Normal rats (negative control), no treatment was given. Group 2: Positive control, Diabetic rats without treatment. Group 3: Diabetic rats treated with standard drug (2.5 mg/kg b.w of glibenclamide. Group 4: Diabetic rats treated with 200 mg/kg body weight of extract. Group 5: Diabetic rats treated with 400 mg/kg body weight of extract. Group 6: Non diabetic rats treated with 200 mg/kg body weight of extract. Group 7: Non diabetic rats treated with 400 mg/kg body weight of extract.

68

3.15 Effect of Ethanol Extract of Bryophyllum pinnatum on Total White Blood Cell Level of

Alloxan Induced Diabetic Rats

Fig. 16 shows the effect of the extract of Bryophyllum pinnatum on total white blood cell level of

alloxan induced diabetic rats. The extract significantly (p<0.05) reduced the number of white blood cell

count in those diabetic groups (groups 4 and 5) treated with the extract compared with group 2untreated

rats. The standard control group treated with glibenclamide also decreased significantly (p<0.05)

compared with group 2 diabetic untreated.

69

Group 1: Normal rats (negative control), no treatment was given. Group 2: Positive control, Diabetic rats without treatment. Group 3: Diabetic rats treated with standard drug (2.5 mg/kg b.w of glibenclamide. Group 4: Diabetic rats treated with 200 mg/kg body weight of extract. Group 5: Diabetic rats treated with 400 mg/kg body weight of extract. Group 6: Non diabetic rats treated with 200 mg/kg body weight of extract. Group 7: Non diabetic rats treated with 400 mg/kg body weight of extract.

70

CHAPTER FOUR

DISCUSSION

4.1 Discussion

This study is a report of the effects of ethanol extract of Bryophyllum pinnatum on

alloxan-induced diabetic rats. Diabetes mellitus is a chronic disorder caused by partial or

complete insulin deficiency, which produces inadequate glucose metabolism and leads to acute

and chronic complications. Premature and extensive arteriosclerosis involving renal, peripheral

and cardiovascular vessels remain the major complications of diabetes mellitus

(Thirunavukkarasu et al., 2003). Currently, the management of diabetes mainly involves

lowering of blood glucose level by the use of biguanides, sulphonylureas, diphenyalanine

derivatives, thiazolidinediones and glucosidase inhibitors in addition to insulin. However, due to

some side effects, the efficacies of these compounds are debatable. There is therefore a demand

for new compounds for the management of diabetes (Thirunavukkarasu et al., 2003). Hence,

plants have been suggested as a rich, yet unexplored, source of potentially useful antidiabetic

active ingredients. However, only a few have been subjected to detailed scientific investigation

due to lack of mechanism-based available in-vitro assays (Saxena and Vikram, 2004).

The phytochemical composition of the ethanol extracts of the leaves of Bryothyllum

pinnatum indicated the abundance of many secondary metabolites including flavonoids,

alkaloids, tannins, saponins, glycosides, etc. From the results, flavonoids, glycosides, alkaloids,

and tannins were found in higher amounts in both dry and fresh samples while steroids, hydrogen

cyanides and carbohydrates were found in very low concentration in both samples. Also,

reducing sugar, saponins, terpenoids and glycosides were moderately high in both samples.

There is no significant difference (p>0.05) between the quantitative phytochemical constituents

of the dry and fresh samples. The flavonoids, saponins and alkaloids are said to have medicinal

properties in animal (Living stone et al., 1997). However, in high concentrations, these

secondary metabolites are toxic and may impair body metabolism (Wieslaw et al., 1999). The

results of the phytochemical composition of Bryophyllum pinnatum is in consistent with the

finding of Puri and Baral, (1998) and consequently suggested that the plant extract might be

responsible for reducing the blood glucose concentration in alloxan-induced diabetic rats.

71

The acute toxicity (LD50) test of ethanol leaf extract indicated that the plant leaves were

not toxic. The result showed that no casualty was recorded at a dose as high as 5000 mg/kg body

weight. This is an indication that the leaves are relatively safe for human and animal

consumptions. The results of the effect of different doses of the extract showed that the extract

was active in reducing the blood glucose concentration in normal rats but cannot induce

hypoglycaemia. This reveals an indication that the plant extract could be use in the treatment of

other metabolic disease without any side effect on the normal glucose metabolism. The

antihyperglycaemic effect of Bryophyllum pinnatum leaf extract on alloxan–induced diabetic rats

showed that the extract significantly (p< 0. 05) reduced the glucose level of the test rats in a

dose-dependent manner. It further revealed that 2.5 mg/kg b.w of glibenclamide (standard drug)

significantly (p< 0.05) reduced the blood glucose concentration compared with the blood

glucose level of group 4 and 5 rats treated with graded doses of the extract. Administration of

Bryophyllum pinnatum leaf extract led to significant reduction (p<0.05) in blood glucose level,

which is consistent with the report of (Latha and Pari, 2003) and Badole et al. (2006) which

showed that graded doses of the extract were responsible for lowering of the blood glucose level

in diabetic subjects.

Diabetic rats were observed to have significant increases in plasma lipid concentrations

which are responsible for several cardiovascular disorders (Alarcon- Aguilair et al., 2002). The

high lipid levels observed in diabetic rats might be due to the increased mobilization of free fatty

acids from peripheral deports and could also be attributed to lipolysis (Nikkhila and Kekki,

1973). The levels of serum lipids are usually elevated in diabetes thereby damaging the insulin-

secreting cells of the pancreas (Best and Tylor, 1989). Since the pancreas of the alloxan-induced

diabetic rat was partially destroyed, the possible mechanisms by which the blood glucose

concentrations of the groups treated by the extract might be reduced is through potentiation of

pancreatic secretion of insulin from the intact beta cells of the islets of langerhans (Suba et al.,

2004) coupled with extra pancreatic mechanisms like glycogenolysis and enhanced glycogenesis

by the liver and or enhanced transport of blood glucose to peripheral tissues. Bryophyllum

pinnatum leaf extract was investigated act to by stimulating the secretion of insulin from the beta

cells by a mechanism similar to that of oral hypoglycemic agents by depolarization of islets

membrane which consequently alters ion influx or affecting reports responsible for insulin

secretagogues. This was in consistent with the findings of Trivedi et al. (2004). The results were

72

comparable with that of glibenclamide, which acts by stimulation of insulin release (Hardy and

McNuthy, 1997) thus further confirming that the extract lowers the blood glucose level by

pancreatic regulation. The anti-diabetic effect of the extract can also be attributed to the presence

of flavonoids. It was reported that flavonoids constitute the active biological principles of most

medicinal plants with hypoglycamic and anti-diabetic activities. Flavonoids facilitate the

incorporation of fatty acids into triglycerides in the liver and adipose tissue (Best and Tylor,

1989). It is well known that LDL plays an important role in arteriosclerosis and Results show

that oral administration of ethanol extract of Bryophyllum pinnatum for 21 days significantly

(p<0.05) reduced LDL concentration and hyperglycaemia. Polonged diabetes mellitus is a risk

factor for coronary heart diseases. This abnormally high level of serum lipids is mainly due to

the uninhibited actions of lipoic enzymes on the fat depot mainly due to lack of insulin (Ei-soud

et al., 2007). Under normal condition insulin activates the enzyme lipoprotein lipase, which

hydrolyses triglycerides. However in diabetic state, lipoprotein lipase is not activated due to

insulin deficiency resulting in hypertriglyceridemia. Alteration of serum lipid profile is known to

occur in diabetes and this is likely to increase the risk of coronary heart diseases. A significant

reduction (p<0.05) in serum lipids particularly the LDL, and triacylglycerol achieved through the

administration of the extract was considered as being beneficial for the long term prognosis of

the disease (Chattopadhay and Bandyopadhay 2005). The ethanol extract of Bryophyllum

pinnatum leaves produced significant beneficial effects in the lipid profile in the treated diabetic

rats, significantly reducing (p<0.05) total cholesterol, low density lipoprotein and triacylglycerol

whereas high density lipoprotein was significantly increased (p<0.05) compared with untreated

diabetic rats.

The decrease in cholesterol and LDL levels achieved by the administration of

Bryophyllum pinnatum leaf extract indicates a possible protection against hypercholesterolemia

with its complications. Insulin deficiency may also be responsible for dyslipidaemia because

insulin has an inhibitory action on 3-hydroxy-3-methylglutaryl coenzyme A (HMG–COA)

reductase, a key rate limiting enzyme responsible for the metabolism of cholesterol rich LDL

particles.

Lipid peroxidation is one of the characteristic features of chronic diabetes (Satheesh and

Pari 2004). In this study, a significant increase (p<0.05) in the concentration of malondiadehyde

(MDA), the product of lipid peroxidation was observed in the diabetic rats but a significant

73

reduction (p<0.05) of MDA was observed in the diabetic rats treated with Bryophyllum pinnatum

ethanol leaf extract. The treatment of diabetic rats with Bryophyllum pinnatum extract could have

likely regenerated the β-cells of the pancreas and potentiation of insulin secretion from the

surviving β-cells. The increase in insulin secretion and the consequent decrease in blood glucose

level may lead to inhibition of lipid peroxidation and control lipolytic enzymes (Odetola et al.,

2006). The beta cells of the pancreas are susceptible to damage caused by oxygen free radicals

since the antioxidant defense system is weak under diabetic condition (Gorray et al., 1993).

Higher levels of lipid peroxides and lower level of catalase activities indicate oxidative

stress. The levels of antioxidant defense system are always diminishing in alloxan-induced

diabetic rats (Prince and Menon, 1998) which correlates with (Fig. 8) group 2 diabetic untreated

rats. Calatase is a haemoprotein which catalyses the reduction of hydrogen peroxides and

protects the tissues from damage from reactive hydroxyl radicals. A reduction in catalase activity

results in a number of superoxide anion radicals and hydrogen peroxides. It was observed that

the treatment of alloxan-induced diabetic rats with the extract of Bryophyllum pinnatum leaf

extract significantly increased (P< 0.05) the catalase activity of the treated rats when compared

with the untreated rats (Fig. 8).

The red blood cell count showed no significant increase (p > 0.05) in the level of red

blood cell of diabetic rats treated with graded doses of the extract compared with the group 2

diabetic untreated rats. This might suggest that the leaf of Bryophyllum pinnatum cannot be used

as a blood booster. However, there were significant increase (p<0.05) in the normal rats treated

with the extract when compared with untreated rats.

Packed cell volume (PCV) can be used as a screening tool for anaemia and can also

indicate the degree of fluid loss during dehydration. The treatment of alloxan-induced diabetic

rats with the leaf extract increased the PCV levels of the treated rats but the increase was non

significant (p> 0.05).

Haemoglobin is an oxygen carrying pigment in the red cells. Treatment of the diabetic

rats with graded doses of the extract significantly (p< 0.05) increased the haemoglobin

concentration of the diabetic rats when compared with the diabetic untreated rats. The reduction

in haemoglobin level of the diabetic but untreated rats might be due to glycosylation of the red

blood cells which prevent its frequent interaction with the haem components (Hanis et al., 1984).

74

The total white blood cell count determines the body’s ability to fight infections. The

treatment of alloxan-induced diabetic rats with the ethanol extract of Bryophyllum pinnatum

significantly reduced (P< 0.05) the total white cell count of treated rats as shown in figure 16

when compared with the diabetic untreated rats. The elevation of the total white blood cells in

the diabetic untreated rats is an indication of inflammation, acute infection or tissue damage.

4.2 Conclusion

The result of the present study has shown that the ethanol extract of Bryophyllum

pinnatum had hypoglycemic effect and the 200 and 400 mg/kg body weight of the extract

possessed efficacy that compared effectively with the reference drug (glibenclamide). The

extract equally had significant decrease (p<0.05) on lipid profile such as low density lipoprotein,

triacylglycerol and total cholesterol and significant increase (p<0.05) in high density lipoprotein

cholesterol implying that the extract could be utilized in management of heart-related diseases.

Catalase activity also increased significantly (p<0.05) while malondialdehyde concentration

reduced significantly (p<0.05) in the test groups treated with graded doses of the extract thus

justifying its use in the treatment and management of diabetes mellitus and its related

complications at the traditional level among the people of the south Western and south Eastern

Nigeria and many other countries of the world.

4.3 Suggestions for Further Studies

These are based on my findings from the literature and not a follow up from my work.

(a) Neuropharmacological effects of aqueous leaf extract of Bryophyllum pinnatum in

experimental animals.

(b) Antiulcer activity of methanol extract of Bryophyllum pinnatum in rats.

(c) Effect of Bryophyllum pinnatum versus fenoterol on uterine contractility.

(d) Analgesic effects of aqueous extract of Bryophyllum pinnatum in experimental animals.

(e) Neurosedative and muscle relactant activities of aqueous extract of Bryophyllum pinnatum in

experimental animals.

75

REFERENCES

Abei, H.E. (1983). Catalase. In: Methods of Enzymatic Analysis. 3rd edn. pp 273-285.

Alarcon-Aguilair, F.J., Campos-Sepulveda, A.E., Xolalopa-Molina, S., Hernadez-Galicia, E. and Roman-Ramos, R. (2002). Hypoglycaemic activity of Ibervillea sonorare root in healthy and diabetic rats. Pharmacological Biology, 40:570-575.

Albers, J.J., Warnick, G.R., and Cheung, M.C. (1978). Dertermination of High Density Lipoprptein. Lipids., 13:926-932.

Allain, C.C., Poon, L.S., Chan, C.S.G., Richmond,W. and Fu, P.C. (1974). Enzymatic determination of serum cholesterol. Clinical chemistry, 20: 470-475.

Amer, J., Ghoti, H., Rachmilewitz, E., Koren, A., Levin, C. and Fibach, E. (2006). "Red blood cells, platelets and polymorphonuclear neutrophils of patients with sickle cell disease exhibit oxidative stress that can be ameliorated by antioxidants". British Journal of

Haematology, 132 (1): 108–113.

Aragno, M., Tamagn, E., Gato, V., Parola, S.Danni, O. and Boccuzzi, G (1999). Dehydroepiandrosterone protects tissues of streptozotocin-treated rats against oxidative stress. Free Radical Biology Medicine, 26 (11/12): 1467-147 4 Assman, G., Jab, H.U., and Hormert, U. (1984). LDL- Cholesterol determination in blood following precipitation of LDL with polyvinyl sulphate. Clinical Chemistry Acta., 140:77-84 Badole, S., Patel, N., Bodhankar, S., Jain, B. and Bhardwaj, S. (2006). Antihyperglycaemic activity of aqueous leaf extract of Cocculus hirsutus in alloxan induced diabetic mice. Indian Journal of Pharmacology, 38: 49-53.

Banerji, M. A., Chaiken, R. I., Huey, H., Tuomi, T., Norin, A. J. and MacKay, I. R. (1994).GAD antibody negative NIDDM in adult black subjects with diabetic ketoacidosis andincreased frequency of human leukocyte antigen DR3 and DR4: flatbush diabetes. Diabetes, 43: 741-45.

Berges, R.R., Windeler, J., Trampisch, H.J. and Senge, T.H. (1995). Randomized, placebo- controlled, double-blind clinical trial of beta-sitosterol in patients with benign prostatic hyperplacia. Lancet, 345: 1529-1532.

76

Best, C.H and Tylor, N.B. (1989). Biological Effect of Insulin. In: Williams, W. (Editors): Physiological Basis of Medical Practice. Cambridge University Press, London pp 878- 890. Betterle, C., Zanette, F., Pedini, B., Presotto, F., Rapp, L.B and Monsciotti, C.M. (1983). Clinical and subclinical organ-specific autoimmune manifestations in type 1 (insulin- dependent) diabetic patients and their first-degree relatives. Diabetologia, 26: 431-36. Bjelakovic, G., Nikolova, D., Gluud, L.L., Simonetti, R.G and Glud, C. (2007). "Mortality in randomized trials of antioxidant supplements for primary and secondary prevention: systematic review and meta-analysis". Journal of American Medical Association, 297 (8): 842–857. Bogardus, C., Lillioja S., Mott, D. M., Hollenbeck, C. and Reaven, G. (1985). Relationship between degree of obesity and in vivo insulin action in man. American Journal of

Physiology, 248: 286- 291. Bonnefont, R.D., Jaudon, M.C., Bastard, J.P. and Delattre, J. (2000). Consequences of the diabetic status on the oxidant/antioxidant balance. Diabetes Metabolism, 26: 163-176. Boskovic, M., Vovk, T., Kores Plesnicar, B. and Grabnar, I. (2011). "Oxidative stress in Schizophrenia". Current Neuropharmacology, 9 (2): 301–312. Bucalo, G., and David, H. (1973). Quantitative determination of serum triacylglycerol by use of enzymes. Clinical chemistry, 19: 476-482. Byrne, M.M, Sturis, J., Menzel, S., Yamagata, K., Fajans, S.S. and Dronsfield, M.J. (1996). Altered insulin secretory response to glucose in diabetic and nondiabetic subjects with mutation in the diabetes susceptibility gene MODY 3 on chromosome 20. Diabetes, 45: 1503-1510. Campbell, P.J. and Carlson, M.G. (1993). Impact of obesity on insulin action in NIDDM. Diabetes. 42: 405-410.

Carr, A. and Frei, B. (1999). "Does vitamin C act as a pro-oxidant under physiological conditions?". Journal of the Federation of American Society for Experimental Biology13 (9): 1007–1024. Chan, T.Y.K. (2009). Potential risks associated with the use of herbal anti-obesity products .Drug Safe, 32: 453-456. Chattopadhay, R.F. and Bandyopadhay, M. (2005). Effect of Azadirachta indica leaf extract on serum lipid profile changes in normal and streptozotocin induced diabetic rats. African

Journal of Biomadical Research, 8: 101-104.

77

Chaudiere, J. and Ferrari-Iliou, R. (1999). "Intracellular Antioxidants: From chemical to biochemical mechanisms". Food and Chemical Toxicology, 37 (9–10): 949–962. Clement, K., Pueyo, M.E., Vaxillaire, M., Rakotoambinina, B., Thuillier, F. and Passa, P. (1996). Assessment of insulin sensitivity in glucokinase-deficient subjects. Diabetologia, 39: 82- 90. Cohen-Kohler, J.C. (2007). The morally uncomfortableglobal drug gap. Clinical Pharmacology and Therapeutics.,84:610-614. Dahl, L.K. (1972). Salt and Hypertension. American Journal of Clinical Nutrition, 25: 231-238.

Dalziel, J.M. (1937). The Useful Plants of West Tropical Africa. London. Crown Agents for the Colonies. 28-29. Demmig-Adams and Adams, W. (2002). Antioxidants in photosynthesis and human nutrition. Science., 298 (5601): 2149-2153. Da-Silva, S.A., Costa, S.S., Mendonca. S.C., Silva, E.M., Moraes, V.L. and Rossi Bergmann, B. (1995). Therapeutic effect of oral Kalanchoe pinnata leaf extract in murine leishmaniasis. Acta Tropica, 60 (3): 201-210. Davies, K.J. (1995). "Oxidative stress: The paradox of aerobic life". Biochemical Society

Symposia, 61: 1–31. De-Fronzo, R.A., Bonadonna, R.C. and Ferrannini, E.(1997). Pathogenesis of NIDDM. In: Alberti K.G.M.M, Zimmet P, DeFronzo R.A, Eds. International Textbook of Diabetes

Mellitus, 2nd Edn.: John Wiley, 1997, Chichester. pp 635-712. Duarte, T.L and Lunec, J. (2005). "Review: When is an antioxidant not an antioxidant? A review of novel actions and reactions of vitamin C". Free Radical Research, 39 (7): 671–86. Ei-soud, N.H., Khalil, M.Y., Oraby, F.S. and Farrag, A.R. (2007). Antidiabetic effects of fenugreek alkaloid extract in streptozotocin induced hypoglycaemic rats. Journal of

Applied Sciences., 3:1073-1083.

Ernst, E. (2005). The efficacy of herbal medicine-anoverview. Fundamental Clinical Pharmacology, 19: 405-409. Evans, M.D and Cooke, M.S (2004). "Factors contributing to the outcome of oxidative damage to nucleic acids". Bioessays, 26 (5): 533–542.

78

Froguel, P., Vaxillaire, M., Sun, F., Velho, G., Zouali, H. and Butel, M.O. (1992). Close linkage of glucokinase locus on chromosome 7p to early-onset non-insulin-dependent diabetes. Nature, 356: 162-164.

Gems, D., and Partridge, L. (2008). "Stress-response hormesis and aging: "that which does not kill us makes us stronger" Cell Metabolism, 7 (3): 200–203. Gomes, A., Vedasiromoni, J.R., Das, M. and Ganguly, D.K. (1995). Antihyperglycaemic effect

of black tea (Cemellia sinensis) in rat. Journal of Ethnopharmacology,27:243-75

Gorray, K.C., Sorresso, D., Moak, S.A., Maimon, J. Greenwald, R. and Schneider, B.S. (1993).

“Comparison of superoxide dismutase activities in isolated rats and guinea pig islets of

langerhans”, Hormone and Metabolic Research, 25 (12): 649-650.

Gruppuso, P.A., Gorden, P., Kahn, C.R., Cornblath, M., Zeller, W.P. and Schwartz, R. (1984) Familial hyperproinsulinemia due to a proposed defect in conversion of proinsulin to insulin. New England Journal of Medicine, 311: 629-634. Gwehenberger, B., Rist, L., Huch, R. and Von-Mandach, U. (2004). Effect of Bryophyllum

pinnatum versus fenoterol on uterine contractility. European Journal of Obstet

Gynecology and Reproductive Biology, 113 (2): 164-171. Gwen, K., Vance, A., Margaret, M., Alexande,r H., Christine, U. and Jill, J. F. (2005). "Oxidative stress levels are raised in chronic fatigue syndrome and are associated with clinical symptoms". Free Radical Biology and Medicine, 39 (5): 584–589. Halliwell, B. (2008). "Are polyphenols antioxidants or pro-oxidants? What do we learn from cell culture and in vivo studies?" Archives of Biochemistry and Biophysics, 476 (2): 107–112. Halliwell, B., Gutteridge, J.M. and Gregory, T. Q. (1999). The mechanism of initiation of lipid peroxidation. Evidence against a requirement for an iron (II)-iron(III) complex. Biochemistry Journal, 258:617-620. Haneda, M., Polonsky, K.S., Bergenstal, R.M., Jaspan, J.B., Shoelson, S.E. and Blix, P.M. (1984) Familial hyperinsulinemia due to a structurally abnormal insulin. Definition of an emerging new clinical syndrome. New England Journal of Medicine, 310: 1288-1294.

Hanis, C.L., Agullar, L., Tulloch, B., Garcia, C and Schull, W.J. (1984). Glycosylated hemoglobin determination from capillary blood samples: utility in an epidemiologic

survey of diabetes. American Journal of Epidemiology, 119: 159–66.

Harborne, J.B. (1984). Textbook of Phytochemical Method: A Guide to Modern Technique of Plant Analysis,2nd Edn; Chapman and Hall Ltd, London. pp 99-105.

79

Harborne, J.B. (1998). Phytochemical methods. A Guide to Modern Technology of Plant Analysis,3rd Edn.Chapman and Hall, New York.pp88-185. Hardy, K.J. and McNutty, S.J. (1997). Oral hypoglycaemic agents. Medical Digest., 23: 5-9. Harris, M.I. (1993). Undiagnosed NIDDM; Clinical and public health issues. Diabetes Care, 16: 642-652. Helmut (1997). "Oxidative stress: Oxidants and antioxidants". Experimental physiology 82 (2): 291–295. Hother-Nielsen, O., Faber, O., Sorensen, N.S. and Beck-Nielsen, H. (1988). Classification of newly diagnosed diabetic patients as insulin-requiring or non-insulin-requiring based on clinical and biochemical variables. Diabetes Care, 11: 531-537.

Humphrey, A.R.G., McCarty, D.J., Mackay, I.R., Rowley, M.J., Dwyer, T. and Zimmet, P. (1998). Autoantibodies to glutamic acid decarboxylase and phenotypic features associated with early insulin treatment in individuals with adult-onset diabetes mellitus. Diabetic Med, 15: 113-119. Igwe, S.A. and Akunyili, D.N. (2005). Analgesic effects of Aqueous extracts of the leaves of Bryophyllum pinnatum. Pharmaceutical Biology, 43 (8): 658-661. Japan and Pittsburgh Childhood Diabetes Research Groups (1985). Coma at onset of young insulin-dependent diabetes in Japan: the result of a nationwide survey. Diabetes, 34: 1241-1246. John, B. and Henry, M.D. (2001). Clinical diagnosis and management laboratory methods. Clinical Chemistry; 20 (4): 570-575. Johns, D.R. (1995). Mitochondrial DNA and disease. New England Journal of Medicine, 333: 638-644. Kerfeld, C. (2004). "Water-soluble carotenoid proteins of cyanobacteria". Archives of

Biochemistry and Biophysics, 430 (1): 2–9. Khan, M., Patil, P. A. and Shobba, J.C. (2004). Influence of Bryophyllum pinnatum leaf extract on wound healing in albino rats. Journal of Natural Remedies, 4: 41-46. Kissebah, A.H., Vydelingum, N., Murray, R., Evans, D.F., Hartz, A.J. and Kalkhoff, R.K. (1982). Relationship of body fat distribution to metabolic complications of obesity. Journal of Clinical Endocrinology and Metabolism, 54: 254-60. Krieger-Liszkay, A. (2004). "Singlet oxygen production in photosynthesis". Journal of

Experimental Botany, 56 (411): 337–346.

80

Kulka, R.G. (2006). Cytokinin inhibits epiphyllous plantlet development on leaves of Bryophyllum pinnatum. Marnierianum Experimental Botany, 17 (77): 122-125. Larrey, D., (1994). Liver involvement in the course of phytotherapy. Presse Medicale, 23: 691-

693.

Latha, M and Pari, L. (2003). Antihyperglycaemic effect of Cassia auriculata in experimental diabetic rats and its effects on key metabolic enzymes involved in carbohydrate metabolism. Clinical Experimental Pharmacological Physiology, 30: 38-43. Lee, Y.J. and Shacter, E. (1999). "Oxidative stress inhibits apoptosis in human lymphoma cells". Journal of Biological Chemistry, 274 (28): 19792–19798. Lennon, S.V, Martin, S.J, Cotter, T.G. (1991). "Dose-dependent induction of apoptosis in human tumour cell lines by widely diverging stimuli". Cell Proliferation, 24 (2): 203–14. Lenzen, S. (2008). The mechanisms of alloxan and streptozotocin-induced diabetes. Diabetologia, 51: 216-226. Lillioja. S., Mott, D.M., Spraul, M., Ferraro, R., Foley, J.E. and Ravussin E (1993) . Insulin resistance and insulin secretory dysfunction as precursors of non-insulin-dependent diabetes. Prospective Study of Pima Indians. New England Journal of Medicine, 329: 1988-1992. Livingstone, A.L., Whitehand, L.C. and Kohler, G.O. (1997). Microbiological assay for saponin in alfalfa products. Journal Association of Official Analytical Chemistry., 60:957-958. Lorke, D. (1983). Determination of acute toxicity. Archive of toxicology 53: 275.

Miller, R.A. and Britigan, B.E. (1997). "Role of oxidants in microbial pathophysiology". Clinical

Microbiology Reviews, 10 (1): 1–18. Molbak, A.G., Christau, B., Marner, B., Borch-Johnsen, K. and Nerup J. (1994). Incidence of insulin-dependent diabetes mellitus in age groups over 30 years in Denmark. Diabetic

Medicine, 11: 650-55. 6-226. Mooy, J.M., Grootenhuis, P.A., de Vries, H., Valkenburg, H.A., Bouter, L.M. and Kostense, P.J. (1995). Prevalence and determinants of glucose intolerance in a Dutch population. The Hoorn Study. Diabetes Care, 18: 1270-73. Mrozikiewicz, A., Kielstrokczewska-Mrozikiewivz., Lstrokowicki,Z., Chmara, E., Korzeniowska, K. and Mrozikiewicz,P.M. (1994). Blood levels of children with insulin- dependent diabetes mellitus. Acta Diabetologica, 31: 236-237.

81

Murray, R.K., Granner, D.K., Mayers, P.A. and Rodwell, V.W (2003). Harper’s Biochemistry. 26th Edn McGraw-Hill Companies, New York.,pp 86-91. Nakabeppu, Y. Sakumi, K., Sakamoto, K. Tsuchimoto, D., Tsuzuki, T and Nakatsu, Y. (2006). Mutagenesis and carcinogenesis caused by the oxidation of nucleic acids. Biological

Chemistry., 387(4): 373-379. Nassis, C.Z., Haebisch, E.M. and Giesbrecht, A.M. (1992). Antihistamine activity of Bryophyllum calycinum. Brazilian Journal of Medical and Biological Research, 25: (9) 929-936. Nikkhila, E.A. and Kekki, M. (1973). Plasma triglyceride transport kinetics in diabetes mellitus. Metabolism, 22: 1-22. Nwabuisi, C. (2002). Prophylactic effect of multi-herbal induced mice. East African Journal of

Medicine. 79 (7): 343-346. Ochei, J and Kolhatkar, A. (2005). Medical Laboratory Science: Theory and practice. Tata Mc Graw Hill Publishing Company, New Delhi. pp. 281-283. Odetola, A.A., Akinloye, O., Egunjobi, C., Adekunle, W.A. and Ayoola, A.O. (2006). Possible Antidiabetic and Antihyperglycaemic effects of fermented Parkia biglobosa on alloxan induced diabetic rats. Clinical Experimental Pharmacological Physiology, 33: 808-812. .

Ojewole, J.A.O. (2002). Antihypertension properties of Bryophyllum pinnatum leaf extracts. American Journal of Hypertension,15 (4):A34-A39. Okaka, J.C. and Okaka, A.N.O. (2001). Food composition, spoilage and shelf life extension. Ocjarco Academic Publishers, Enugu, Nigeria. pp 54-56. Okeke, C.U and Elekwa, I. (2003). Phytochemical study of the extract of Gongronema

latifolium Benth. Journal of Health and Visual Sciences, 5 (1): 47-55. Osujih, M. (1993). Exploration of the frontiers of tradomedical practices: Basis for development of alternative medical healthcare services indeveloping countries. Journal of the Royal

Society of Health, 113: 190-194. Pal, S., Sen,T. and Nag, A. T. (1999). Neuropsychopharmacological profile of the methanol fraction of Bryophyllum pinnatum leaf extract. Journal of Pharmacy and Pharmacology. 51: 313-318.

Polonsky, K.S., Sturis, J. and Bell, G.I. (1996) Non-insulin-dependent diabetes mellitus: A genetically programmed failure of the beta cell to compensate for insulin resistance. New

England Journal of Medicine, 334: 777-84.

82

Prabhat, J., Marcus, F., Eva, L., Michael, F. and Salim, Y. (1995). "The antioxidant vitamins and cardiovascular disease: A critical review of epidemiologic and clinical trial data". Annals of Internal Medicine, 123 (11): 860–872.

Prince, S.M and Menon V.P. (2000). Hypoglycaemic and other related actions of Tinospora

cardifolia roots in alloxan induced diabetic rats. Journal of Ethnopharmacology, 70: 401- 406. Puri, D and Baral, N. (1998). Hypoglycaemic effect of Biophytum sensitivum in the alloxan induced diabetic rabbits. Indian Journal of Pharmacological physiology, 42(3): 401-406. Raha, S. and Robinson, B.H. (2000). "Mitochondria, oxygen free radicals, disease and ageing". Trends in Biochemical Sciences, 25 (10): 502–508. Ramond A, Godin-Ribuot D, Ribuot C, Totoson P, Koritchneva I, Cachot S, Levy P, Joyeux- Faure M. (2011). "Oxidative stress mediates cardiac infarction aggravation induced by intermittent hypoxia.". Fundamental and Clinical Pharmacology., 2 (8): 501- 505 Ravenna, L., Di Silverio, F., Russo,M. A., Salvatori, L., Morgante, E., Morrone, S., Cadillo, M.R., Frati, L., Gulino, A. and Petrangeli, E. (1996). Effects of the lipidosterolic extract of Serenoa repens on human prostatic cell lines. Prostate; 29(4): 219-30 Rhee, S.G. (2006). “cell signaling : H2O2, a necessary evil for cell signaling". Science, 312 (5782): 1882–1883. Ristow, M. and Zarse, K. (2010). "How increased oxidative stress promotes longevity and metabolic health: The concept of mitochondrial hormesis (mitohormesis)". Experimental

Gerontology, 45 (6): 410–418. Robbins, D.C., Shoelson, S.E., Rubenstein, A.H. and Tager, H.S. (1984). Familial hyperproinsulinemia: Two cohorts secreting indistinguishable type II intermediates of proinsulin conversion. Journal of Clinical Investigation, 73: 714-719. Sanz, N., Karam, J.H., Horita, S. and Bell, G.I. (1986). Prevalence of insulin-gene mutations in non-insulin-dependent diabetes mellitus. New England Journal of Medicine, 314: 132- 134. Satheesh, M.A. and Pari, L. (2004). Antioxidant effects of Boehavia diffusa in tissues of

alloxan-induced diabetic rats. Indian Journal of Experimental Biology, 42: 669-695. Saxena, A. and Vikram, N.K. (2004). Role of selected Indian plants in the management of type 2 diabetes. Journal of Alternative comptemporary Medicine, 10: 369- 378. Schafer, F.Q and Buettner, G.R. (2001). "Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple". Free Radical Biology and

Medicine, 30 (11): 1191–212.

83

Schneider, C. (2005). "Chemistry and biology of vitamin E". Mol Nutr Food Res, 49 (1): 7–30. Seaver, L. C. and Imlay, J.A. (2004). "Are Respiratory Enzymes the Primary Sources of Intracellular Hydrogen Peroxide?" Journal of Biological Chemistry,279 (47): 48742– 48750. Simonson, D.C., Ferrannini, E., Bevilacqua, S., Smith, D., Barrett, E. and Carlson, R. (1984). Mechanism of improvement in glucose metabolism after chronic glyburide therapy. Diabetes, 33: 838-45. Singh, N., Dhalla, A.K., Seneviratne, C. and Singal, P.K. (1995). "Oxidative stress and heart failure". Molecular and Cellular Biochemistry, 147 (1): 77–81. Slimestad, R., Fossen, T. and Vagen, I.M. (2007). Onions, a source of unique dietary flavonoids. Journal of Agriculture, Food and Chemistry,55(25): 10067-80. Soto, C., Recoba, R., Favari, L., Alvari, C. and Barron, H. (2003). Silymarin increases antioxidant enzymes in alloxan-induced diabetes in rat pancreas. Comparative Biocchemistry and Physiology. 136: 205-212. Stadtman, E. (1992). "Protein oxidation and ageing". Science, 257 (5074): 1220–1224. Stoffers, D.A., Ferrer, J., Clarke, W.L. and Habener, J.F. (1997). Early-onset type-II diabetes mellitus (MODY4) linked to IPF1. Nature Genetics, 117: 138-139. Stohs, S.J and Bagchi, D. (1995). "Oxidative mechanisms in the toxicity of metal ions". Free

Radical Biology and Medicine, 18 (2): 321–336. Suba, V., Murugesan, T., Bhaskara, R.R., Ghosh, L., Pal, M., Mandal, S.C. and Saha, B.P. (2004). Antidiabetic potential of Barleria lupulina extract in rats. Fitoterapia; 75:1-4. Sundaram, R. I. (1996). Antioxidant status and lipid peroxidation in type II diabetes mellitus with and without complications. Clinical Science, 90: 255-260. Swain, T. (1979). Tannins and Lignins. In: Herbivores: Their Interaction with Metabolites. Rosenthal, G.A and Janzen (Eds). Academic Press, New York. pp.203-221. Thirunavukkarasu, V., Anuradha, C.V. and Viswanathan, P. (2003). Protective effect of Fenugreek (Trigonella foenum graecium) seeds in experimental ethanol toxicity. Phytotherapy Research, 17: 737-743. Trivedi, N.A., Mazumda, B., Bhatt, J.D. and Hemavathi, K.G. (2004). Effect of shilajit on blood glucose and lipid profile in alloxan induced diabetic rats. Indian Journal of

Pharmacology, 36: 373-376.

84

Tsushida, T. and Suzuki, M. (1996). Content of flavonol glucosides and some properties of enzymes metabolizing the glucosides in onion. Journal of Japanese Society of Food

Science and Technology; 43:373-6. Umpierrez, G. E., Casals, M. M. C., Gebhardt, S. S. P., Mixon, P. S., Clark, W. S. and Phillips, L. S. (1995). Diabetic ketoacidosis in obese African-Americans. Diabetes, 44: 790-95. Valko, M., Leibfritz, D., Moncol, J., Cronin, M., Mazur, M. and Telser, J. (2007). "Free radicals

and antioxidants in normal physiological functions and human disease". The

International Journal of Biochemistry and Cell Biology, 39 (1): 44–84. Valko M, Morris H. and Cronin, M.T (2005). "Metals, toxicity and oxidative stress". Curr. Med.

Chem. 12 (10): 1161–208. Valko, M., Leibfritz, D., Moncol, J., Cronin, MTD., Mazur, M. and Telser, J. (2007). "Free radicals and antioxidants in normal physiological functions and human disease". International Journal of Biochemistry & Cell Biology 39 (1): 44–84. Valle, T., Tuomilehto, J. and Eriksson, J. (1997). Epidemiology of NIDDM in Europids. In: Alberti K.G.M.M., Zimmet, P. and DeFronzo, R.A, Eds. International Textbook of

Diabetes Mellitus. 2nd Edn. John Wiley, Chichester: pp 125-142. Verge, C.F., Gianani, R., Kawasaki, E., Pietropaolo, M. and Jackson, R.A. (1996). Predicting type I diabetes in first-degree relatives using a combination of insulin, GAD, and ICA512bdc/IA-2 autoantibodies. Diabetes, 45: 926-33. Ververidis, F., Trantas, E., Douglas, C., Vollmer, G., Kretzchmar, G., Pan, O. and Poulos, N. (2007). “Biotechnology of flavonoids and other phenyl propanoid derived natural products part: Chemical diversity, impacts on plant biology and human health”. Biotechnology Journal,2(10): 817-825. Vionnet, N., Stoffel, M., Takeda, J., Yasuda, K., Bell, G.I. and Zouali, H. (1992). Nonsense mutation in the glucokinase gene causes early-onset non-insulin-dependent diabetes. Nature, 356: 721-722. Wagner, W.L. and Sohmer, S.H. (1999). Manual of the Flowering Plants of Hawaii. 1st Edition. University of Hawaii Press, Honolulu. pp: 1948. Walker, M. and Turnbull, D.M. (1997). Mitochondrial related diabetes: a clinical perspective. Diabetes Medicine, 14: 1007-1009.

Wallins, B., Rosegren, B.,Shertzer, H.G. and Caeejo, G. (1993). Lipoprotein oxidation and measurement of thiobarbituric acid reacting substances, formation in a single microtitre plate: its use for evaluation of antioxidants. Analytical Biochemistry, 208:10-15.

85

Wieslaw, O., Marta, J. and Anna, S. (1999). Determination and toxicity of saponins from Amaranthus cruentus seeds. Journal of Agriculture and Food Chemistry, 47: (9) 3685- 3687

Willins, J. A., Scott, R. S., Brown, L. J., Forbes, L.V., Schmidli, R. S. and Zimmet PZ (1996). Islet cell antibodies and antibodies against glutamic acid decarboxylase in newly diagnosed adult-onset diabetes mellitus. Diabetes Research and Clinical Practice. 33: 89-97.

Wing, R.R., Blair, E.H., Bononi, P., Marcus, M.D., Watanabe, R., and Bergman, R.N. (1994) Caloric restriction per se is a significant factor in improvements in glycemic control and insulin sensitivity during weight loss in obese NIDDM patients. Diabetes Care, 17: 30- 36.

World Health Organisation (1996). WHO guidelines for the assessment of herbal medicines, WHO excerpt committee on specification for pharmaceutical preparation. Technical Report series No 863. Geneva.

Yadav,N.P. and Dixit, V.K. (2003). Hepatoprotective activity of leaves of Kalanchoe pinnata. Journal of Ethnopharmacology. 86: 197-202.

Yamagata, K., Furuta, H., Oda, N., Kaisaki, P. J., Menzel, S. and Cox, N.J (1996). Mutations in the hepatocyte nuclear factor-4alpha gene in maturity-onset diabetes of the young (MODY 1). Nature, 384: 458-460.

Yamagata, K., Oda, N., Kaisaki, P.J., Menzel, S., Furuta, H. and Vaxillaire, M. (1996). Mutations in the hepatocyte nuclear factor-1alpha gene in maturity-onset diabetes of the young (MODY 3). Nature, 384: 455-458.

Yamamoto,Y and Gaynor, R.B. (2011). Therapeutic potential of inhibition of the NF-kB pathway in the treatment of inflammation and cancer. Journal of Clinical Investigation; 107 (2): 135.

Yeung, K.S., Gubili, J. and Cassileth, B. (2008). Evidence-based botanical research: Applications and challenges. Hematol. Oncol. Clin. North Am., 22: 661-670. Zimmet, P.Z. (1992). Kelly West Lecture 1991: Challenges in diabetes epidemiology: From West to the rest. Diabetes Care, 15: 232-352.

Zimmet, P.Z. (1995). The pathogenesis and prevention of diabetes in adults. Diabetes Care, 18: 1050-1064.

Zimmet, P.Z., Tuomi, T., Mackay, R., Rowley, M.J., Knowles, W. and Cohen, M. (1994). Latent autoimmune diabetes mellitus in adults (LADA): the role of antibodies to glutamic acid decarboxylase in diagnosis and prediction of insulin dependency. Diabetic

Medicine, 11: 299-303.