BIOCHEMICAL EFFECT OF ETHANOL SEED EXTRACT OF GARCINIA KOLA ON THE TRIACYLGLYCEROL (TAG) CONCENTRATION IN ALLOXAN-INDUCED DIABETIC RATS

 

CHAPTER ONE

INTRODUCTION AND LITERATURE REVIEW

BACKGROUND OF STUDY

Plants are important in our everyday existence. They provide our foods, produce the oxygen we breathe, and serve as raw materials for many industrial products such as clothes, foot wears and so many others. Plants also provide raw materials for our buildings and in the manufacture of biofuels, dyes, perfumes, pesticides and drugs.

Historically, plants have always provided a source of inspiration for novel drug compounds as plant derived medicines have made large contributions to human health and well being. Medicinal plants which produce and accumulate constituents have medical properties (Adedeji et al., 2006). The use of plants in traditional medical practice has a long drawn history, and remains the mainstay of primary health care in most of the third world. Traditional medicines are used by about 60% of the world population; in both developing and developed countries where modern medicines are predominantly used (Mythilypriya et al., 2007). While an estimated 60-80% Africa’s population depends solely on herbal remedies for its primary health care needs. In diversity, plants are thought to be between 250,000 to 400,000 species spread across all continents from the Antarctic to the Arctic. They thrive in all environments from the flooded planes to the deserts, and from those who live on the seas and oceans to others that thrive on fresh water and ponds.

For classification and easy identifications, plants were divided into different taxonomical groups known as kingdoms; these are further streamlined into phylum, class, order, family genus and species. Within the family of the Clusiacea is found an amazing plant called the bitter kola (Garcinia kola).

Garcinia kola (Heckel) is an angiospermae, belonging to the family Guttiferae and is known in commerce as bitter cola. On chewing, G. kola seed has a bitter astringent and resinous taste, somewhat resembling that of raw coffee, followed by a slight sweetness. Bitter cola is a highly valued ingredient in African ethno medicine because of its varied and numerous uses which are social and medicinal; thus making the plant an essential ingredient in folk medicine. Medicinal plants such as G. kola are believed to be an important source of new chemical substances with potential therapeutic benefits (Eisner, 1990).

Bitter Kola (Garcinia Kola) is popular in Southern Nigeria. The plant is extensively used in herbal medicine and as food. It prevails as a multipurpose tree crop in the home gardens of Southern Nigeria.

Traditionally, the plant is used as a natural antimicrobial. Other medicinal properties of the plant include its usage in the treatment of skin infections in Liberia and Congo Democratic Republic. The powdered bark of the plant is applied to malignant tumors, cancers etc. The plants latex is taken internally for gonorrhea and externally to seal new wounds and prevent sepsis.

LITERATURE REVIEW

World Health Organization (1999) estimated that there were 135 million people in the world with diabetes and that this would rise to 380 million by 2025, this report also highlighted the fact that low and middle income countries will bear the brunt of the increase with Africa contributing significantly to this rise (King et al., 1998). In Nigeria, World Health Organization has disclosed that more than 1.71 million citizens above 15 years are diabetic, 70, 000 children under 15 years develop insulin dependent diabetes each year, if nothing is done, diabetes sufferers will grow to about 484 million by  2030 (Winifred, 2008). Diabetes mellitus and other numerous pathological events such as atherosclerosis and inflammatory processes are associated with the generation of Reactive Oxygen Species (ROS) and consequently the induction of several chain reactions among them, lipid peroxidation (Grober, 2010). Evidence suggests that oxidative cellular injury caused by free radicals contributes to the complications of diabetes mellitus (Baimbolkar and Sainani, 1995). Some of these radicals are extremely reactive and therefore interact with some vital macromolecules including lipids, nucleic acids and protein (Nia et al., 2003).

Before the introduction of insulin in 1922, the treatment of diabetes mellitus relied heavily on the use of traditional plant therapies (Gray and Flatt, 1999). In Africa and beyond, application of traditional medical practices to the treatment of diabetes is quite popular; several plant species have been used for this purpose (Nwaegerue et al., 2007). Active components of these plants are now being investigated and their extract are developed into drugs and little or no negative effects as contraindications, one of such plants is Garcinia kola (Adedeji et al., 2006).

Garcinia kola can be used as an antidiabetic agent, it could exert a beneficial effect in the diabetics by enhancing insulin secretion or improving the mimicking of insulin secretion action (Gray and Flatt, 1999), the plant phytochemical constituents include dimeric flavoid, biflavoid, xanthone and benzophenones (Alaba, 2007). Hydroxycitric acid is the principal acid of the fruit, this acid was shown to be a potent inhibitor of ATP-dependent citrate lyase which catalyses the cleavage of citrate to oxaloacetate and Acetyl-CoA  (Manhendran  et al., 2000). Acetyl-CoA is used by Acetyl-CoA carboxylase, the regulatory enzyme of lipogenesis in the Liver (Vance and Vance, 1996). Constituents of Garcinia kola was responsible for its use as an antidote for piosion and its effectiveness in the reduction of blood sugar with corresponding pharmacological actions against allergies. The properties of Garcinia kola makes it a stimulant and as a herbal remedy has been documented (Cowan, 2010).

Garcinia kola: Distribution

Garcinia kola (Bitter kola) is a type of kola found in all parts of Nigeria.

Scientific classification of Garcinia kola

Its biological/binomial name is Garcinia kola Heckel and belongs to the species of flowering plant in the Clusiaceae or Guttiferae family. Its natural habitat is subtropical or tropical moist lowland forests. This fruit is classified scientifically as

Kingdom                         Plantae

Angiosperm

Eudicots

Rosids

Order                              Malpighiales

Family                            Clusiaceae

Genus                            Garcinia

Species                          Garcinia kola

Wikipedia (2013)

Bitter kola is a popular agricultural produce available in large quantity in West Africa particularly in Nigeria and it is a tree that grows in rainforests. It has been identified as a potent antibiotic which could be effective in the treatment of many diseases. The fruit, seed, nuts and bark of the plant have been used for centuries in folk medicine to treat ailments from coughs to fever. It is used extensively in the preparation of herbal drugs either as stimulant supplement or as herbal remedies as well. However, bitter kola is considered as an effective agricultural produce in the treatment of cough, liver disease, diarrhea, seeds are used for bronchitis, throat infections, tuberculosis and other bacterial infections (Iwu et al., 1990; Braide, 1991). It is also chewed as an aphrodisiac. When food is suspected to be infected by bacteria, it can be chewed to prevent the development of any infection or poisonous reactions. It contains chemical compounds that will help the breakdown of glycogen in the liver and has other medicinal use which accounts for its longevity property in man. It is considered an anti- poison due to its role in detoxifying of the system.

Ecology of the plant Garcinia kola

Garcinia kola is a dicotyledonous plant found in moist rain forests and swamps and grows as a medium sized tree up to a height of about 12m high. It is cultivated through the seedlings or with cuttings. It grows more easily using the cuttings. The bitter kola plant is found in countries across west and central Africa and it is distributed by man around the towns and villages of such countries like; Nigeria, Ghana, Cameroon, Sierra Leone, Togo, Congo Democratic Republic, Angola, Liberia, Gambia etc. Across the places where it grows it is known by various names such as bitter kola, male kola (English name), orogbo (Yoruba), Aku ilu (Igbo) and Namijin goro( Hausa). It is also known as false kola  mainly  due  to  the  absence  of  stimulants  which characterizes the kola nut seeds. It is also known as male kola due to the reported aphrodisiac properties of Garcinia kola.

Health benefits of Garcinia kola

1. Improving lung function: Garcinia kola has been used for centuries to treat chest cold in traditional medicine. In a study, tests were performed on mice which reports that Garcinia kola improved respiratory function after 28 days of use of Garcinia kola extract. The study showed that Garcinia kola works by dilating the alveoli ducts and sacs in the lungs by improving the strength of the fibers in the lung tissue. This beneficial lung property are attributed to its high antioxidant content.
2. Reduce eye pressure: an increase in eye pressure can lead to glaucoma according to a report from the New York Times health guide. Researchers at LASUTH tested the effectiveness of eye drops that contain 0.5% extract of Garcinia kola. The results of their study showed that the optamolic solution contained in the Garcinia significantly reduced eye pressure when use twice a day.
3. Reduces arthritis: Osteoarthritis is the most common form of arthritis characterized by pain, inflammation and limited movement in the joints. According to the national institute of health, causes of osteoarthritis can include joint injury, obesity and aging. Since many arthritis patients prefer natural herbal medicine to other pain relievers and medications,

researchers at Obafemi Awolowo University tested the effects of Garcinia kola against arthritis. The result concluded that Garcinia kola significantly reduced the inflammation and pain while increasing the joint movement in patients that had osteoarthritis symptoms. It is believed that Garcinia kola works well as a result of its antioxidant properties. The side effects reported from the Kola use in this study were weight loss, increase in sex drive and extended sleep which may be considered helpful to meet their individual and marital demands.

4. Medicinal significance: Garcinia kola is used in many tropical countries to fight infectious disease such as Acquired Immune Deficiency syndrome (AIDS) and the Ebola virus. It has shown to posses anti-inflammatory, anti-microbial and anti-viral properties.
5. Cold remedy: the kola is often used to treat the symptoms of cold. It is suggested in particular for coughs of sneezing
6. Impotence: Garcinia kola is believed to cure impotence. Frequent intake/chewing of bitter kola have been implied to boost males’ performance in bed.
7. Knee osteoarthritis: kola has been successfully used to treat patients from knee osteoarthritis. It reduces pain and swelling thereby aiding in improved movement.
8. Immunity: kola is well known for its anti-inflammatory and antioxidant properties. It is used to prevent infections and viruses especially of the immune system.
9. Hops substitute: Bitter kola is used as a substitute for hops in breweries. It is especially useful in the prevention of beer spoilage.
10. Weight loss: kola is a natural hunger depressant and also increases the urge to drink more water.

Plate 1: African Garcinia kola (Bitter kola)

DIABETES MELLITUS

Diabetes mellitus, or simply diabetes, is a group of metabolic disorder in which blood glucose levels become too high as the body produces little or no insulin (a hormone secreted by the pancreas that aids in the utilization of glucose for energy) or cannot use insulin properly. This high blood sugar produces the classical symptoms of polyuria (frequent urination), polydipsia (increased thirst), and polyphagia (increased hunger) (Dolores, 2011).

The word diabetes comes from Latin diabētēs, which in turn comes from Ancient Greek (diabētēs) which literally means “a passer through; a siphon.” (Oxford dictionary, 2011). Ancient Greek physician Aretaeus of Cappadocia (1^st century CE) used that word, with the intended meaning “excessive discharge of urine”, as the name for the disease.(John, 2011). The word mellitus comes from the classical Latin word mellītus, meaning “mellite” (i.e. sweetened with honey; honey-sweet).

Globally, as of 2010, an estimated 285 million people had diabetes, with type 2 making up about 90% of the cases.[4] Its incidence is increasing rapidly, and by 2030, this number is estimated to almost double (Wild et al., 2004). Diabetes mellitus occurs throughout the world, but is more common (especially type 2) in the more developed countries. The greatest increase in prevalence is, however, expected to occur in Asia and Africa, where most patients will probably be found by 2030 (Wild et al., 2004). The increase in incidence in developing countries follows the trend of urbanization and lifestyle changes, perhaps most importantly a “Western-style” diet. This has suggested an environmental (i.e., dietary) effect, but there is little understanding of the mechanism(s) at present, though there is much speculation, some of it most compellingly presented (Wild et al., 2004).

Diabetes mellitus is a chronic disease, for which there is no known cure except in very specific situations. Management concentrates on keeping blood sugar levels as close to normal (“euglycemia”) as possible, without causing hypoglycemia. So therefore, there are roles for patient education (also partner or caretakers), dietetic support, and sensible exercise, with the goal of keeping both short-term and long-term blood glucose levels within acceptable bounds. In addition, given the associated higher risks of cardiovascular disease, lifestyle modifications are recommended to control blood pressure (Adler et al., 2000)

Types of diabetes

Diabetes mellitus is classified into four broad categories: type 1, type 2, gestational diabetes, and “other specific types” (Dolores, 2011).

Type 1 diabetes

Type 1 diabetes mellitus is characterized by loss of the insulin-producing beta cells of the islets of Langerhans in the pancreas, leading to insulin deficiency. This type can be further classified as immune-mediated or idiopathic. The majority of type 1 diabetes is of the immune-mediated nature, in which beta cell loss is a T-cell-mediated autoimmune attack (Rother, 2007). There is no known preventive measure against type 1 diabetes, which causes approximately 10% of diabetes mellitus cases in North America and Europe. Most affected people are otherwise healthy and of a healthy weight when onset occurs. Sensitivity and responsiveness to insulin are usually normal, especially in the early stages. Type 1 diabetes can affect children or adults, but was traditionally termed “juvenile diabetes” because a majority of these diabetes cases were in children.

Type 2 diabetes

Type 2 diabetes mellitus is characterized by insulin resistance, which may be combined with relatively reduced insulin secretion (Dolores, 2011). The defective responsiveness of body tissues to insulin is believed to involve the insulin receptor. However, the specific defects are not known. Diabetes mellitus cases due to a known defect are classified separately. Type 2 diabetes is the most common type.

In the early stage of type 2, the predominant abnormality is reduced insulin sensitivity. At this stage, hyperglycemia can be reversed by a variety of measures and medications that improve insulin sensitivity or reduce glucose production by the liver.

Gestational diabetes

Gestational diabetes mellitus (GDM) resembles type 2 diabetes in several respects, involving a combination of relatively inadequate insulin secretion and responsiveness. It occurs in about 2–5% of all pregnancies and may improve or disappear after delivery. Gestational diabetes is fully treatable, but requires careful medical supervision throughout the pregnancy. About 20–50% of affected women develop type 2 diabetes later in life.

Though it may be transient, untreated gestational diabetes can damage the health of the fetus or mother. Risks to the baby include macrosomia (high birth weight), congenital cardiac and central nervous system anomalies, and skeletal muscle malformations. Increased fetal insulin may inhibit fetal surfactant production and cause respiratory distress syndrome. Hyperbilirubinemia may result from red blood cell destruction. In severe cases, perinatal death may occur, most commonly as a result of poor placental perfusion due to vascular impairment. Labor induction may be indicated with decreased placental function. A Caesarean section may be performed if there is marked fetal distress or an increased risk of injury associated with macrosomia, such as shoulder dystocia.

Other types

Prediabetes indicates a condition that occurs when a person’s blood glucose levels are higher than normal but not high enough for a diagnosis of type 2 DM. Many people destined to develop type 2 DM spend many years in a state of prediabetes which has been termed “America’s largest healthcare epidemic.” (Handelsman, 1998).

Latent autoimmune diabetes of adults (LADA) is a condition in which type 1 DM develops in adults. Adults with LADA are frequently initially misdiagnosed as having type 2 DM, based on age rather than etiology.

Some cases of diabetes are caused by the body’s tissue receptors not responding to insulin (even when insulin levels are normal, which is what separates it from type 2 diabetes); this form is very uncommon. Genetic mutations (autosomal or mitochondrial) can lead to defects in beta cell function. Abnormal insulin action may also have been genetically determined in some cases. Any disease that causes extensive damage to the pancreas may lead to diabetes (for example, chronic pancreatitis and cystic fibrosis). Diseases associated with excessive secretion of insulin-antagonistic hormones can cause diabetes (which is typically resolved once the hormone excess is removed). Many drugs impair insulin secretion and some toxins damage pancreatic beta cells (WHO, 1999).

LIPID PEROXIDATION

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. Lipid peroxides are unstable and decompose to form a complex series of compounds including reactive carbonyl compounds. It most often affects polyunsaturated fatty acids, because they contain multiple double bonds in between which lie methylene bridges (-CH₂-) that possess especially reactive hydrogens. Polyunsaturated fatty acid peroxides generate malondialdehyde (MDA) and 4-hydroxyalkenals (HAE) upon decomposition, and the measurement of MDA and HAE has been used as an indicator of lipid peroxidation. As with any radical reaction, the reaction consists of three major steps: initiation, propagation, and termination.

Malondialdehyde (MDA) is one of many low molecular weight end-products of lipid hydoperoxide decomposition and is the most often measured as an index of lipid peroxidation.3 However, the use a MDA as a marker for lipid peroxidation is controversial. MDA can be formed during eicosanoid metabolism and the analytical methods for measuring MDA are prone to artifactual errors.

Certain diagnostic tests are available for the quantification of the end-products of lipid peroxidation, to be specific, malondialdehyde (MDA) (Marnett, 1999).

Test principle

The MDA assay is based on the reaction of MDA with thiobarbituric acid (TBA); forming an MDA-TBA2 adduct that absorbs strongly at 532 nm.

Butylated hydroxytoluene (BHT) and EDTA are added to the sample and reaction mixture to minimize oxidation of lipids that contribute artifactually during sample processing and the TBA reaction.5,6 The temperature of the reaction mixture has also been reduced to minimize the decomposition of lipid hydroperoxides. Because much of the MDA is protein bound, mostly as a Schiff base, the pH of the reaction has been optimized to facilitate hydrolysis of the MDA. Additionally, the reaction mixture is subjected to derivative spectrophotometric analysis that resolves the problem of the variable and nonlinear baseline observed when attempting to measure the A₅₃₂ absorbance in various biological samples

ANTIOXIDANT ENZYMES

Several enzyme systems catalyze reactions to neutralize free radicals and reactive oxygen species. These enzymes are generally referred to as antioxidant enzymes and they include:

• superoxide dismutase (SOD)
• glutathione peroxidase (GPX)
• glutathione reductase
• catalases (CAT)

These form the body’s endogenous defense mechanisms to help protect against free radical-induced cell damage. The antioxidant enzymes – glutathione peroxidase, catalase, and superoxide dismutase – metabolize oxidative toxic intermediates.

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