evaluation of nutritional composition of Pterocarpus santalinoides leaves
CHAPTER ONE
1.0 INTRODUCTION AND LITERATURE
1.1 INTRODUCTION
Plants are important for human existence and they are the major sources of foods and medicine (Pieroni A. 2000). Plant-based foods are important components of traditional diets in many parts of developing countries (Kushi LH, et al., 1995). For instance, plant-based foods are very good sources of protein, fibers, minerals and vitamins (Aremu M.O, et al., 2014), and consumption of these vegetables/fruit can help to prevent diabetes, cancers (Hu FB, et al., 2002) and heart disease like stroke, high blood pressure and accumulation of cholesterol (Etudeko I, 1970). The use of plants in traditional medicine has a long history in the life of a man, and it remains the mainstay of primary health care in most of the developing countries. Plant-based medicines are used by over 60% of the world population; in both developing and developed countries where modern medicines are predominantly used (Mythilypriya I, et al., 2017). In African countries large proportions of the population depends solely on herbal medicines for its primary health care needs.
Medicinal plants play an important role in maintenance of human and animal health. A wide array of diseases including life threatening diseases such as cancer, diabetes, hypertension etc. are being treated successfully with medicinal plants. These plants are widely consumed not only for medicinal purposes but also as food. These role played by plants are due to the presence of phytochemicals and nutrients. Phytochemicals which include flavonoids, saponins, tannins and alkaloids. Play key role in defense against numerous diseases. Major minerals such as Ca, Mg, Cl, Na, S, K, P serve as structural components of tissues, function in cell metabolism and acid-base balance (Smith et al., 1988). Many trace elements play vital roles in prevention and treatment of diseases (Parasad et al., 1993). Vitamins have diverse biochemical role which include antioxidation (Bender et al., 2003), precursor for enzymes cofactors and hormones (Berg et al., 2002).
Pterocarpus santalinoides L’Herit ex DC (Family: Fabaceae-papilnoideae) is a shade-tolerant tree 9-12m tall, with low straggling branches, commonly found along riverine forests in Africa and tropical South America. It is native to Brazil, Cameroon, Ghana, Nigeria and Senegal (Orwa C et al., 2009). Various morphological parts of P. santalinoides are used in traditional medicine, in many African countries, to treat an array of human ailments. Locally known as nturukpa, the fresh leaves of P. santalinoides are consumed locally, in soups, by the Igbos of South East Nigeria and is reputed to be useful in the treatment of diarrhoea and other gastrointestinal disorders (Okafor JC et al., 1996). Also, the fresh-leaf extract combined with leaves of Solanum macrocarpum is used in the management of high blood pressure. Similar uses are known among the Igede tribe in Benue State, Nigeria (Igoli JO et al., 2005 ). The bark of the plant plus the leaf is used in the management of sleeping sickness in Ivory Coast. It is also used as an anti-abortive agent and in the treatment of malaria and other infections such as Staphylococcus aureus and Escherichia coli.
1.2 JUSTIFICATION
The leaves of Pterocarpus santalinoides has been used for the past few decades in the treatment of so many kinds of diseases. Some tribes in the Eastern and Southern Nigeria use the leaf
extracts in the treatment of headaches, pains, fever, convulsions, and respiratory disorders and as antimicrobial agents as has been reported for Sansevieria trifasciata (Ogukwe et al., 2004). Its
antidiarrheal property has also been reported by (Okpo and Ching, 2011), but then, despite its popular use in traditional medicine. There is paucity of information on the nutritional and phytochemical constituents of Pterocarpus santaliniodes . Thus, the need for the present research.
1.3 AIM AND OBJECTIVES
This research work is aimed to evaluate the nutrient composition of Pterocarpus santalinoides leaves.
The objectives are
⦁ To establish proximate levels of protein, fat, fibre, carbohydrate, moisture content and ash in Pterocarpus santalinoides leaves
⦁ To determine the mineral contents (focusing on Fe, Zn, Cu, Mn, Na, K, Ca, Mg, and P) of Pterocarpus santalinoides leaves.
⦁ To ascertain the vitamin composition (Vitamin A, C, and E) of Pterocarpus santalinoides leaves.
⦁ To determine the anti-nutrient content (Terpenoids, Tannin, Phenol, Glycosides and Saponin) of Pterocarpus santalinoides leaves.
1.4 LITERATURE REVIEW
According to Keay (1989), Pterocarpus santalinoides is a specie of pterocarpus in the family papilionoidea. It is commonly called red sandal wood in English, it is known as nturukpa in Igbo, gunduru in Hausa and gbengbe in Yoruba. Pterocarpus santalinoides is a tree growing to about 9-12m tall with a trunk diameter up to 1m with low straggling branches. It has thin bark and flaking in small patches, slash yellowish- white exuding drops of red gum. The leaves are pinnate, 10-20cm long, with 5-9 leaflets that are abruptly acuminate, rounded at the base and glossy with about 8 pairs of prominent main lateral nerves looping away from the margin. The leaf stalk is slender with stout leaflet stalks about 2-5mm long. The flowers are orange-yellow, fragrant in axillary’s racemes and panicles, with finely hairy inflorescence branches. The fruit is light brown glabrous pod, 3.5-6cm across, including the soft fleshy narrow wing which extends about three quarters away round the body. The name pterocarpus is based on the Greek word ‘pteran’ meaning a wing and ‘Karpos’ meaning fruit. The specific epithet ‘santalinoides’ refers to its likeness to Pterocarpus santalinus found is Asia. It is a shade tolerant tree commonly found along riverine forests in Africa.
1.4.1 Description of the study Plant: Pterocarpus santalinoides
Pterocarpus santalinoides is a tree 9-12 m tall, 1 m DBH, with low straggling branches. Bark thin and flaking in small patches, slash yellowish-white exuding drops of red gum. Leaves compound, 5-9 leaflets ovate-elliptic, abruptly acuminate, rounded at the base or slightly cuneate, glabrous, glossy, rather coriaceous with about 8 pairs of prominent main lateral nerves looping away from the margin, leaf stalk slender, glabrous stalk 10-20 cm long, leaflet stalk stout 2-5 mm long. Flowers orange-yellow, fragrant in axillary racemes and panicles, inflorescence branches finely hairy, individual flowers with short stalks. Calyx rather narrowly cup-shaped, petals densely hairy outside, about 7 mm long including the prominent triangular teeth, standard petal about 12 mm long and broad. Fruit a light brown glabrous pod, 3.5-6 cm across including the soft, fleshy narrow wing which extends about three quarters way round the body. Pterocarpus santalinoides is monoecious, flowering from December-March, fruits ripening between March-April. (Orwa et al., 2009) P. santalinoides is a shade tolerant tree commonly found along riverine forests in Africa and tropical South America.
Altitude: 200-500 m
Mean annual temperature: 26 degree Celsius
Mean annual rainfall: 1600 mm
Soil type: Prefers well drained soils
Figure 1.0 Diagrammatic representation of Pterocarpus santalinoides tree
1.4.2 Taxonomy of Pterocarpus santalinoides leaves
Kingdom: Plantae
(unranked): Angiosperms
(unranked): Eudicots
(unranked): Rosids
Order: Fabales
Family: Fabaceae
Subfamily: Faboideae
Tribe: Dalbergieae
Genus: Pterocarpus
Species: P. santalinoides
Binomial Name
Pterocarpus santalinoides
1.4.3 Distribution
Pterocarpus santalinoides is a tree specie in the legume family (biology) (Fabaceae); it is locally known as mututi. It has a remarkable bi-continental distribution, native to tropical western Africa (Benin, Burkina Faso, Cameroon, Gambia, Ghana, Guinea, Guinea-Bissau, Ivory Coast, Liberia, Mali, Nigeria, Senegal, Sierra Leone, Togo) and also to South America (Brazil, Colombia, French Guiana, Guyana, Paraguay, Peru, Suriname, Trinidad and Tobago, and Venezuela).(Prado, 1998)
It grows to 9–12 m tall, with a trunk up to 1 m in diameter and flaky bark. The leaves are pinnate, 10–20 cm long, with 5–9 leaflets. The flowers are orange-yellow, produced in panicles. The fruit is a pod 3.5–6 cm long, with a wing extending three-quarters around the margin.
1.4.4 Uses of Pterocarpus santalinoides
Keay (1989) reported that Pterocarpus santalinoides provides a wealth of useful products which include:
Food: The leaves are eaten as a vegetable
Fodder: Livestock browse its young shoot and leaves.
Timber: The wood is not hard but termite resistant.
Gum or resin: The cuttings on the stem exude a red gum.
Tannin or dyestuff: The bark contains tannins and dyes used for dyeing.
Medicine: The tree bark is used as stomach ache remedy while the leaf is used in
treatment of diarrhea and high blood pressure.
Erosion control: It is important specie for soil conservation in water catchments areas.
Shade or Shelter: It is a good windbreak around settled areas and farms.
Nitrogen fixation: Pterocarpus santalinoides forms nodules with nitrogenase activity. The
nodules are generally spherical but occasionally elongated.
Soil improver: The leaf litter from Pterocarpus santalinoides on decomposition slowly
releases nitrogen and significantly increases soil exchangeable calcium and magnesium in
the soil.
Ornamental: It is a beautiful tree with good gardening attributes, it’s showy flower, beautiful foliage and form make it a suitable ornamental tree.
Boundary or barrier: Poles from Pterocarpus santalinoides are used for fencing.
1.4.5 NUTRITIONAL VALUE
1.4.5.1 Carbohydrate
Carbohydrates refers to polyhydroxy aldehydes or ketones and their specialized and other compounds that produce them on hydrolysis (Abugre, 2011) and are essential food energy provider among the macronutrients, giving between 40 and 80 percent of total energy intake. Carbohydrate also serves as stored forms of energy as glycogen in liver and muscles. It also provides major source of energy and responsible for breaking-down of fatty acids inhibiting ketosis (Hassan et al., 2006).
Carbohydrates are the major nutrients of fruits and vegetables, with sucrose representing one third of total sugars (Singh et al., 1993). This disaccharide is one of the important parameters for the assessment of the commercial quality of the fruit, since consumers prefer the sweetest fruits.
1.4.5.2 Protein
Crude Proteins are important organic compounds which have high molecular weight found in all living tissues. They are derived from amino acids and may be categorized based on factors such as solubility and shape. Simple proteins contain only amino acids as building blocks whiles conjugated proteins consist of amino acids but in addition, a non- protein or prosthetic group which may be glycoprotein, lipoprotein, chromoprotein (Abugre, 2011). The World Health organization (WHO) recommends a protein intake of 56g of protein a day for a (75kg) man and 48g for a (64kg) woman. The recommendations of the UK Department of Health and Social Security (DHSS) are slightly higher, at about 68g a day for sedentary or moderately active men, and 54g a day for women.
1.4.5.3 Lipid
1.4.5.4 Fiber
Dietary Fiber (DF) plays an important role in decreasing the risks of many disorders such as constipation, diabetes, Cardiovascular Diseases (CVD), diverticulosis and obesity (Bassi et al., 1994). Plant foods are the only sources of Dietary Fiber. All the fractions (cellulose, lignin, hemicellulose, pectins, gums and mucilages) of DF are the major constituents of plant cell wall (Douet et al., 2004). FAO/WHO discussion document on carbohydrates recommended dropping the terms soluble and insoluble fiber (Charro and Barreiro, 1957). The physiological effects of Total Dietary Fiber (TDF), in the forms of insoluble and soluble fractions of foods, have a significant role in human nutrition (Desmaison and Adrian, 1986).
1.4.6 MINERALS
Minerals are chemical constituents used by the body in many ways. Although they yield no energy, they have important roles to play in many activities in the body (Eruvbetine, 2003). Every form of living matter requires these inorganic elements or minerals for their normal life processes (Ozcan, 2003). Minerals may be broadly classified as macro (major) or micro (trace) elements. The macro-minerals include calcium, phosphorus, sodium and chloride, while the micro-elements include iron, copper, cobalt, potassium, magnesium, iodine, zinc, manganese, molybdenum, fluoride, chromium, selenium and sulfur (Eruvbetine, 2003). The macro-minerals are required in amounts greater than 100 mg/dl and the micro-minerals are required in amounts less than 100 mg/dl (Murray et al., 2000). Micronutrient deficiencies are a major public health problem in many developing countries, with infants and pregnant women especially at risk (Batra and Seth, 2002). Infants deserve extra concern because they need adequate micronutrients to maintain normal growth and development (Rush, 2000). The micronutrient deficiencies which are of greatest public health significance are iron deficiency, causing varying degrees of impairment in cognitive performance, lowered work capacity, lowered immunity to infections, pregnancy complications e.g. babies with low birth weight, poor learning capacity and reduced psychomotor skills (Batra and Seth, 2002). Medical reports show that very severe anemia is a direct cause of maternal and child mortality (Chakravarty and Ghosh, 2000). There have been suggestions that more than anything else, lack of adequate information about the composition of varied feed resources in some regions have been the major drawback to their utilization, rather than real shortage (Aletor and Omodara, 1994). For instance, there is very limited information on the mineral elements in some plants used as human food and animal feeds consumed in Nigeria, especially the newly introduced varieties of diets and the lesser known legumes. Some of the earlier information on mineral elements was based on analysis employing less sensitive methods, which may not be reliable.
1.4.6.1 THE BIOCHEMISTRY AND FUNCTIONS OF THE INDIVIDUAL MINERAL ELEMENTS
The basic functions performed by the minerals are: they are structural components of body tissues, are involved in the maintenance of acid-base balance and in the regulation of body fluids, in transport of gases and in muscle contractions (Malhotra, 1998; Murray et al., 2000). For example, high dietary sodium is implicated in cardiovascular and renal disorders. Consequently, high dietary sodium is often discouraged in patients/subjects who suffer from or are prone to hypertension. Also, knowledge of the importance of the mineral elements in plants is essential as the global trend in nutrition and medicine is shifting towards the consumption of plant foods (fruits and vegetables) and medicinal plants (phyto-medicines) respectively, because the plant kingdom is reported to be full of large numbers of beneficial substances to both human and animal health (Soetan, 2008). The symptoms of a mineral deficiency depend on the function and mobility of the element. The metabolism of soil bacteria makes nitrogen available to plants (Roberts, 1985).
1.4.6.2 Macro element composition
1.4.6.3 Sodium
Sodium is the principal cation in extracellular fluids. It regulates plasma volume and acid-base balance, involved in the maintenance of osmotic pressure of the body fluids, preserves normal irritability of muscles and cell permeability, activates nerve and muscle function and involved in Na+/K+-ATPase, maintenance of membrane potentials, transmission of nerve impulses and the absorptive processes of monosaccharides, amino acids, pyrimidines, and bile salts.
1.4.6.4 Potassium
Potassium is the principal cation in intracellular fluid and functions in acid-base balance, regulation of osmotic pressure, conduction of nerve impulse, muscle contraction particularly the cardiac muscle, cell membrane function and Na+/K+-ATPase. Potassium is also required during glycogenesis. It also helps in the transfer of phosphate from ATP to pyruvic acid and probably has a role in many other basic cellular enzymatic reactions. Its metabolism is regulated by aldosterone.
1.4.6.5 Calcium
Calcium is an essential nutrient that plays a vital role in neuromuscular function, many enzyme-mediated processes and blood clotting, as well as providing rigidity to the skeleton by virtue of its phosphate salts. Its non-structural roles require the strict maintenance of ionized calcium concentration in tissue fluids at the expense of the skeleton if necessary and it is therefore the skeleton which is at risk if the supply of calcium falls short of the requirement.
Calcium functions as a constituent of bones and teeth, regulation of nerve and muscle function. In blood coagulation, calcium activates the conversion of prothrombin to thrombin and also takes part in milk clotting. It plays a vital role in enzyme activation. Calcium activates large number of enzymes such as adenosine triphosphatase (ATPase), succinic dehydrogenase, lipase etc. It is also required for membrane permeability, involved in muscle contraction, normal transmission of nerve impulses and in neuromuscular excitability. A reduced extracellular blood calcium increases the irritability of nerve tissue, and very low levels may cause spontaneous discharges of nerve impulses leading to convulsions (Hays and Swenson, 1985; Malhotra, 1998; Murray et al., 2000). Calcium absorption requires calcium-binding proteins and is regulated by vitamin D, sunlight, parathyroid hormone and thyrocalcitonin. Thyrocalcitonin decreases plasma calcium and phosphate levels whereas parathyroid hormone increases them.
1.4.6.6 Magnesium
Magnesium is widely distributed in plant and animal foods, and geochemical and other environmental variables rarely have a major influence on its content in foods. Most green vegetables, legume seeds, beans, and nuts are rich in magnesium, as are some shellfish, spices, and soya flour, all of which usually contain more than 500mg/kg fresh weight. Although most unrefined cereal grains are reasonable sources, many highly-refined flours, tubers, fruits, fungi, and most oils and fats contribute little dietary magnesium (<100mg/kg fresh weight) (Koivistoinen P, 1980). Corn flour, cassava and sago flour, and polished rice flour have extremely low magnesium contents. (Paul A.A, 1978).
1.4.6.7 Phosporus
Phosphorus is located in every cell of the body and is vitally concerned with many metabolic processes, including those involving the buffers in body fluids (Hays and Swenson, 1985). It functions as a constituent of bones, teeth, adenosine triphosphate (ATP), phosphorylated metabolic intermediates and nucleic acids. It serves buffering action, that is, phosphate buffers, functions in the formation of high energy compounds, that is, adenosine triphosphate (ATP) and is involved in the synthesis of phospholipids and phosphoproteins. Practically, every form of energy exchange inside living cells involve the forming or breaking of high-energy bonds that link oxides of phosphorus to carbon or to carbon-nitrogen com-pounds (Hays and Swenson, 1985; Malhotra, 1998; Murray et al., 2000). Vitamin D is probably involved in the control of phosphorus absorption and serum levels are regulated by kidney reabsorption. Phosphorus is an essential macronutrient for plants and one of the three nutrients generally added to soils in fertilizers because of its vital role of energy transfer in living organisms and in plants. Adequate phosphorus availability stimulates early growth and hastens maturity in plants (Sharma et al., 2008). Phosphorus is also needed for soil fertility. In plants, as grasses mature, phosphorus is transferred to the grain. Also, the phosphorus content of the plant is influenced markedly by the availability of phosphorus in the soil. As a result of this, low-quality pastures devoid of legumes and range plants tend to be naturally low in phosphorus, as the forage matures and the seeds fall; characteristically, the range soil is also deficient in phosphorus (Merck, 1986). A large percentage (60-80%) of the total phosphorus of cereal grains and oil seeds exists organically bound as phytic acid. Phytic acid, the hexaphosphoric acid ester of inositol, is present in cereal and legume seeds primarily as the Ca-Mg salt called phytin. The organically bound phosphorus, phytin phosphorus, is largely unavailable to monogastric animals, whereas ruminants can utilize it relatively very well. This species difference is explained by the presence of the enzyme phytase from rumen microorganisms, which hydrolyzes the organically bound phosphorus and renders it available for absorption. This partly, coupled with the slower growth rate of ruminants, accounts for the rather large difference in phosphorus requirements of ruminant and non ruminant animals (Hays and Swenson, 1985). Decrease in serum phosphorus is found in rickets, hyperparathyroidism, De Toni-Fanconi Syndrome. Deficiency disease or symptoms in children causes rickets and in adults, it causes osteomalacia. Increase in serum phosphorus is found in chronic nephritis and hypoparathyroidism. Toxicity disease or symptoms include low serum Ca2+ : P ratio. It may also lead to bone loss (Malhotra, 1998; Murray et al., 2000). Sources of phosphorus include phosphate food additives, green leafy vegetables and fruits, especially banana.
1.4.7 Trace element composition
1.4.7.1 Iron
Iron plays different important roles in the body including the formation of myoglobin and haemoglobin. Most of the iron in the body (65%-75%) forms part of the haemoglobin, a red blood cells component responsible for carrying oxygen from the heart across the body (Darshan, 2010). Myoglobin, on the other hand, transports oxygen to muscle cells (Darshan, 2010). In addition, iron is an important component in the energy producing reactions that takes place in the body. Iron occurs either as non-heme or heme iron, with the former found in both plant and animal tissues while the latter is only found in animal tissues products (Darshan, 2010). The heme iron from animals is highly bioavailable with the absorption rate being between 25 and 35 % as compared to that of heme iron, which only has an absorption rate of 3%. Unfortunately, the non-heme iron is the dominant in the diet among poor people from developing countries, including Kenya. Iron deficiency could range from mild depletion of body iron stores with no health or functional effects, to anemia related iron deficiency which has an impact on the functioning of various organs within the body (Darshan, 2010). The World Health Organization acknowledges iron deficiency anemia as being among the most infamous nutrient deficiencies across the globe, which is contributed towards by various reasons, including low consumption of iron in the diet, impaired absorption of iron, or high losses of blood (Darshan, 2010). Several groups including pregnant women, children, women who have reached puberty, adolescents, older adults, and athletes are more exposed to iron deficiency. Some of the foods that are high in iron include; legumes, whole grains, green leafy vegetables, meats and eggs (Darshan, 2010).
1.4.7.2 Copper
Copper is a constituent of enzymes like amine oxidase, catalase, peroxidase, ascorbic acid oxidase, cytochrome oxidase, plasma monoamine oxidase, erythrocuprin (ceruloplasmin), lactase, uricase, tyrosinase, cytosolic superoxide dismutase e.t.c. and it plays a role in iron absorption (Chandra, 1990). Cu is an essential micro-nutrient necessary for the hematologic and neurologic systems (Tan et al., 2006). It is necessary for the growth and formation of bone, formation of myelin sheaths in the nervous systems, helps in the incorporation of iron in hemoglobin, assists in the absorption of iron from the gastrointestinal tract (GIT) and in the transfer of iron from tissues to the plasma (Murray et al., 2000). It is transported by albumin; bound to ceruloplasmin. Ceruloplasmin has oxidase activity and thereby facilitates the incorporation of ferric iron into transferrin. The copper-containing protein in red blood cells (RBC) is erythrocuperin, in liver, it is hepatocuperin and in brain, it is cerebrocuperin. In general, Cu is poorly absorbed, and under normal conditions >90% of the ingested copper appears in the faeces. Most of the faecal copper is unabsorbed dietary copper, but some of it comes from the bile, which is the major pathway of Cu excretion. Biliary obstruction increases the excretion of copper through the kidney and intestinal wall (Hays and Swenson, 1985). Increased levels of copper are seen in acute infections and in chronic conditions such as cirrhosis, rheumatoid arthritis and in post-operative stages. Increased level is also found in malnutrition (Malhotra, 1998). Clinical disorders associated with Cu deficiencies include anemia, bone disorders, neonatal ataxia, depigmentation and abnormal growth of hair, fur or wool, impaired growth and reproductive performance, heart failure and gastrointestinal disturbances. The incidence of these disorders varies widely among animal species. Cu deficiency has also been associated with cardiac hypertrophy and sudden cardiac failure. (Gardea Torresdey et al. 1990) reported that carboxyl groups found on the cell walls of dead algal biomass are potentially responsible for copper binding. Toxicity disease or symptoms are rare and is secondary to Wilson’s disease (Murray et al., 2000). In Wilson’s disease, a large amount of copper is deposited in liver, brain, etc. Total copper content in the plasma and ceruloplasmin-bound copper content decreases and there is an increased excretion of copper in the urine. Sometimes, Cu may be deposited in the renal tubules giving rise to renal tubular degeneration and this is manifested as glycosuria and amino aciduria (Malhotra, 1998). Excess dietary Cu causes an accumulation of Cu in the liver with a decrease in blood haemoglobin concentration and packed cell volume. Liver function is adversely affected in copper poisoning. Jaundice results from erythrocyte hemolysis and this may lead to death unless treatment is started. Deficiency disease or symptom include anemia (hypochromic, microcytic). Sources include liver, whole grains, molasses, legumes, nuts, shell fish and other sea foods.
1.4.7.3 Zinc
Zinc is an essential component of a large number (>300) of enzymes participating in the synthesis and degradation of carbohydrates, lipids, proteins, and nucleic acids as well as in the metabolism of other micronutrients. Zinc stabilizes the molecular structure of cellular components and membranes and in this way contributes to the maintenance of cell and organ integrity. Furthermore, zinc has an essential role in polynucleotide transcription and thus in the process of genetic expression. Its involvement in such fundamental activities probably accounts for the essentiality of zinc for all life forms. Zinc plays a central role in the immune system, affecting a number of aspects of cellular and humoral immunity. Shankar and Prasad have reviewed the role of zinc in immunity extensively (Shankar AH. Et al., 1998). Lean red meat, whole-grain cereals, pulses, and legumes provide the highest concentrations of zinc: concentrations in such foods are generally in the range of 25–50mg/kg (380–760mmol/kg) raw weight. Processed cereals with low extraction rates, polished rice, and chicken, pork or meat with high fat content have a moderate zinc content, typically between 10 and 25mg/kg (150–380 mmol/kg). Fish, roots and tubers, green leafy vegetables, and fruits are only modest sources of zinc, having concentrations <10mg/kg (<150mmol/kg) (Sandström B. 1989). Saturated fats and
oils, sugar, and alcohol have very low zinc contents.
1.4.7.54 Manganese (Mn)
Manganese is a cofactor of hydrolase, decarboxylase, and transferase enzymes (Murray et al., 2000). It is involved in glycoprotein and proteoglycan synthesis and is a component of mitochondrial superoxide dismutase. Manganese is a co-factor in phosphohydrolases and phosphotransferases involved in the synthesis of proteoglycans in cartilage. Mn is a part of enzymes involved in urea formation, pyruvate metabolism and the galactotransferase of connective tissue biosynthesis (Chandra, 1990). Mn activates several important enzyme systems and in this capacity it is required for the synthesis of acid mucopolysaccharides, such as chondroitin sulphate, to form the matrices of bones and egg shells. Consequently skeletal deformities and defects in shell quality occur when the manganese intake is inadequate (Gordon, 1977). The fact that Mn is concentrated in the mitochondria has led to the suggestion that, in vivo, manganese is involved in the partial regulation of oxidative phosphorylation. Absorption of Mn is inhibited by the presence of excessive amounts of calcium and phosphorus in the diet. The absorption and retention of manganese from foods low in iron, such as milk, are relatively high. If milk is supplemented with iron, the percentage of manganese absorbed is reduced (Gruden, 1977). Increased absorption of manganese has been reported during pregnancy in sows (Kirchgessneret al., 1981) and with coccidiosis infection in chickens (Southern and Baker, 1983). Mn deficiency has been demonstrated in several animal species including laboratory animals, pigs, poultry, and possibly in cattle. Its severity depends greatly on the degree and duration of the deficiency and on the maturity of the animal (Hays and Swenson, 1985). Manganese deficiency presents with the following signs; in pigs, lameness, enlarged hock joints, and shortened legs, in cattle, leg deformities with over knuckling, in chicks, poultries and ducklings, perosis or slipped tendon; and in chick embryos, nutritional chondrodystrophy. In laboratory animals, effects of deficiency include deformities of bone, poor growth, impaired reproduction, egg shell formation, and blood clotting. Some of these defects are related to the role of the manganese ion as the most effective activator of glycosyl transferase enzymes in the synthesis of mucopolysaccharides and glycoproteins (Leach, 1974). In other species, congenital defects in embryonic bone development result from Mn deficiency. Birds are much more susceptible to manganese deficiency than mammals because their requirements for this element are considerably higher and this is attributable partly to relatively poor absorption from the intestine (Gordon, 1977). Deficiency disease or symptoms is unknown in humans. Mn overexposure reportedly may have an adverse effect on central nervous system (CNS) function and mood (Tan et al., 2006). Toxicity disease or symptoms by inhalation poisoning produces psychotic symptoms and Parkinsonism. Corn is extremely low in manganese (4-12 ppm) and so animals fed high-corn diets especially if supplemented with animal by-products, which are also low in manganese content, may receive inadequate amounts. The high requirement of poultry and the low levels of Mn in many of the ingredients of poultry diets make Mn supplementation highly important (Hays and Swenson, 1985). Sources include whole grains, tea, legumes, nuts and seeds.
1.4.8 Vitamins
1.4.8.1 Vitamin A
Vitamin A (retinol) is an essential nutrient needed in small amounts by humans for the normal functioning of the visual system; growth and development; and maintenance of epithelial cellular integrity, immune function, and reproduction. These dietary needs for vitamin A are normally provided for as preformed retinol (mainly as retinyl ester) and provitamin A carotenoids. essential foods, which may include sugar, cereals, condiments, fats, and oils (Rodriguez-Amaya DB, 1997). Provitamin A carotenoids are found in green leafy vegetables (e.g. spinach, amaranth, and young leaves from various sources), yellow vegetables (e.g. pumpkins, squash, and carrots), and yellow and orange non-citrus fruits (e.g. mangoes, apricots, and papayas). Red palm oil produced in several countries worldwide is especially rich in provitamin A (Booth SL, et al., 1992). Some other indigenous plants also may be unusually rich sources of provitamin A. Such examples are the palm fruit known in Brazil as burití, found in areas along the Amazon River (as well as elsewhere in Latin America), and the fruit known as gac in Viet Nam, which is used to colour rice, particularly on ceremonial occasions. Foods containing provitamin A carotenoids tend to have less biologically available vitamin A but are more affordable than animal products. It is mainly for this reason that carotenoids provide most of the vitamin A activity in the diets of economically deprived population. Although vitamin A status cannot be assessed from dietary intake alone, intake assessment can provide evidence of risk of an inadequate status. However, quantitative collection of dietary information is fraught with measurement problems. These problems arise both from obtaining representative quantitative dietary histories from individuals, communities, or both, and from interpreting these data while accounting for differences in bioavailability, preparation losses, and variations in food composition data among population groups (Rodriguez-Amaya DB, 1997). This is especially difficult in populations consuming most of their dietary vitamin A from provitamin carotenoid sources. Simplified guidelines have been developed recently in an effort to improve the collection of reliable dietary intake information from individuals and communities (Parker RS et al., 1999).
1.4.8.2 Vitamin C
Vitamin C is present in many 'fresh' foods, especially fruits and leafy vegetables. It acts primarily as a water-soluble reductant in vivo, although it can become a pro-oxidant in the presence of oxygen and ferrous ions or copper (Basu T.K et al., 1996). It is essential as the cofactor for some mixed function oxidase enzymes, notably those involved in the synthesis of hydroxylysyl and hydroxyprolyl residues in nascent collagen. It also has other redox functions, many of which are not yet fully understood. Human beings, higher primates, and some other species cannot synthesize vitamin C, and therefore require a dietary source. Vitamin C is essentially nontoxic but there remains controversy about risk: benefit ratios of large daily intakes in milligram (Maiani G, et al., 1993).
1.4.8.3 Vitamin E
Vitamin E occurs in plasma as a variety of tocopherols, of which the alpha- and gamma isomers are usually the major ones. Different foods vary in the tocopherol (and tocol) isomers which they contain: z-tocopherol is biologically the most active isomer in mammals. Vitamin E circulates in the lipoproteins and chylomicrons, and the molar ratio to cholesterol is a good index of vitamin E status. Unlike vitamin A, there is no strong homeostatic control of the amount in the blood. Blood levels rise progressively with increasing intakes (Howard L.J, 1990). Vitamin E is a major lipid-soluble antioxidant and membrane component, and helps to protect tissues against oxygen-derived free radical attack, especially protecting vulnerable oxidizable polyunsaturated fatty acids. Although a deficiency disease-syndrome is well-established for some animal species, clinical deficiency occurs rarely in humans. Vitamin E is generally considered to be non-toxic, even at high intakes (Goss-Sampson M.A, et al 1989)
1.5 ANTI-NUTRITIONAL FACTORS
Anti-nutritional factors are a chemical compounds synthesized in natural food and / or feedstuffs by the normal metabolism of species. These anti-nutritional factors are also known as ‘secondary metabolites’ in plants and they have been shown to be highly biologically active (Habtamu and Nigussie, 2014). Anti-nutritional factors (ANF) are compounds which reduce the nutrient utilization and/or food intake of plants or plant products used as human foods or animal feeds and they play a vital role in determining the use of plants for humans and animals (Soetan K. O. and Oyewole , 2009). The toxicity due to the consumption of various forages is very common among the farm animals. The anti-nutritional factors present in the forages are mainly responsible for this (Smitha- Patelet al., 2013). Anti-nutritional factors may be divided into two major categories. They are:
⦁ Proteins (such as lectins and protease inhibitors) which are sensitive to normal processing temperatures.
⦁ Other substances which are stable or resistant to these temperatures and which include, among many others, polyphenolic compounds (mainly condensed tannins), non-protein amino acids and galactomannan gums (Osagie,1998). The major ones includes: toxic amino acids, saponins, cyanogenic glycosides, tannins, phytic acid, gossypol, oxalates, goitrogens, lectins (phytohaemagglutinins), protease inhibitors, chlorogenic acid and amylase inhibitors (Akandeet al., 2010). More often than not, a single plant may contain two or more toxic compounds, generally drawn from the two categories, which add to the difficulties of detoxification. According to Aletor (1993), there are several anti-nutritional factors that are very significant in plants used for human foods and animal feeds and some most common ones with their mechanism of toxicity and impact on animal health and productivity are discussed hereunder
1.5.1 Tannins
Tannin is an astringent, bitter plant polyphenolic compound that either binds or precipitates proteins and various other organic compounds including amino acids and alkaloids. Tannins are the most widely occurring antinutritional factors found in plants. These compounds are present in numerous tree and shrub foliages, seeds and agro-industrial by-products (Dube et al., 2004). Tannins have a property of binding to protein to form reversible and irreversible complexes due to the existence of a number of phenolic hydroxyl groups (Patra and Saxena, 2010). Tannins are water soluble phenolic compounds with a molecular weight greater than 500 and hydrolysable tannins and condensed tannins are two different groups of these compounds (Smitha Patel. et al., 2013). The two types differ in their nutritional and toxic effects. The condensed tannins have more profound digestibility-reducing effect than hydrolysable tannins, whereas, the latter may cause varied toxic manifestations due to hydrolysis in rumen (Akande et al., 2010). Tannins are heat stable and they decreased protein digestibility in animals and humans, probably by either making protein partially unavailable or inhibiting digestive enzymes and increasing fecal nitrogen. Tannins are known to be present in food products and to inhibit the activities of trypsin, chemotrypsin, amylase and lipase, decrease the protein quality of foods and interfere with dietary iron absorption. Tannins are known to be responsible for decreased feed intake, growth rate, feed efficiency and protein digestibility in experimental animals. If tannin concentration in the diet becomes too high, microbial enzyme activities including cellulose and intestinal digestion may be depressed. Tannins also form insoluble complexes with proteins and the tannin-protein complexes may be responsible for the anti-nutritional effects of tannin containing foods (Habtamu and Nigussie, 2014).
1.5.2 Saponins
Saponins are secondary compounds that are generally known as non-volatile, surface active which are widely distributed in nature, occurring primarily in the plant kingdom. They are structurally diverse molecules and consist of non polar aglycones coupled with one or more monosaccharide moieties. This combination of polar and non-polar structural elements in their molecules explains their soap-like behavior in aqueous solutions. The structural complexity of saponins results in a number of physical, chemical, and biological properties, which include sweetness and bitterness, foaming and emulsifying , pharmacological and medicinal, haemolytic properties, as well as antimicrobial, insecticidal activities (Habtamu and Ngusse, 2014). Saponins reduce the uptake of certain nutrients including glucose and cholesterol at the gut through intra-lumenal physicochemical interaction. Hence, it has been reported to have hypo cholesterolemic effects (Umaru et al., 2007). Saponins are among several plant compounds which have beneficial effects. Among the various biological effects of saponins are antibacterial and antiprotozoal (Avato et al., 2006).
1.5.3 Glycosides
Glycosides in general, are defined as the condensation products of sugars (including polysaccharides) with a host of different varieties of organic hydroxy (occasionally thiol)
compounds (invariably monohydrate in character), in such a manner that the hemiacetal
entity of the carbohydrate must essentially take part in the condensation. Glycosides are
colorless, crystalline carbon, hydrogen and oxygen-containing (some contain nitrogen and
sulfur) water-soluble phytoconstituents, found in the cell sap. Chemically, glycosides contain a carbohydrate (glucose) and a non-carbohydrate part (aglycone or genin) (Kar, 2007; Firn, 2010). Alcohol, glycerol or phenol represents aglycones. Glycosides are neutral in reaction and can be readily hydrolyzed into its components with ferments or mineral acids.Glycosides are classified on the basis of type of sugar component, chemical nature of aglycone or pharmacological action. The rather older or trivial names of glycosides usually has a suffix ‘in’ and the names essentially included the source of the glycoside.
1.5.4 Terpenoids
Terpenoids are compounds synthesized from five carbon isoprene units mainly isopentenyl pyrophosphate and its isomer dimethylallyl pyrophosphate by terpene synthases. Terpenoids have antioxidant properties and also interact with most regulatory proteins. Plant extracts have been used both traditionally and in modern medicine in the treatment of cancer and inflammatory
diseases. Terpenes are used as inhibitors of NF-kB in modern medicine (Piero N.M et al., 2015). NF-kB system is a cytoplasmic sensor that responds to various internal and external signals like genotoxic stress and hypoxia as well as disturbances in the immune system. NF-kB also plays a major role in the development of cellular resistance against apoptosis and anti-apoptotic signaling. Most terpenes in plants occur as terpene derivatives (terpenoids). Sesquiterpenoids are the main tepernes and are known to have NF-kB signaling inhibitory effect while triterpenoids and diterpenoids are also believed to have several potent inhibitors of NF-kB signaling system (Mertens-Talcott SU et a., 2003). Terpenoids also improve the skin tone, increases the concentration of antioxidants in wounds, and restore inflammed tissues by increasing blood supply (Aggarwal B.B et al., 2006). Terpenoids also improve lung function (Mujoo K et al., 2001). The leaves and seeds of S. spectabilis are used in the treatment of diabetes due to the presence phytochemicals including terpenoid. Terpenoids have shown to reduce diastolic
blood pressure and lower the sugar level in blood in hypertensive and diabetic patients respectively (Grace M.H et al., 2013).
1.5.5 Phenols
Phenolics, phenols or polyphenolics (or polyphenol extracts) are chemical components that
occur ubiquitously as natural colour pigments responsible for the colour of fruits of plants.
Phenolics in plants are mostly synthesized from phenylalanine via the action of phenylalanine ammonia lyase (PAL). They are very important to plants and have multiple functions. The most important role may be in plant defence against pathogens and herbivore predators, and thus are applied in the control of human pathogenic infections. They are classified into (i) phenolic acids and (ii) flavonoid polyphenolics (flavonones, flavones, xanthones and catechins) and (iii) non-flavonoid polyphenolies. Caffeic acid is regarded as the most common of phenolic compounds distributed in the plant flora followed by chlorogenic acid known to cause allergic dermatitis among humans (Kar, 2007). Phenolics essentially represent a host of natural antioxidants, used as nutraceuticals, and found in apples, green-tea, and red-wine for their enormous ability to combat cancer and are also thought to prevent heart ailments to an appreciable degree and sometimes are anti-inflammatory agents. Other examples include flavones, rutin, naringin, hesperidin e.t.c
CHAPTER TWO
2.0 MATERIALS AND METHOD
2.1 REAGENTS
Reagents used in the study
Reagent Specification Company/Country
Xylene Analar England
Ethanol BDH Chemicals England
Phosphotungstate reagent Analar England
Petroleum ether BDH chemical England
FeCl3 BDH Chemical England
KOH solution BDH Chemicals England
Distilled water Pure SSU lab
Vitamin A standard BDH chemical Tokyo, japan
Vitamin C standard BDH chemical Tokyo, Japan
Vitamin E standard BDH chemical Tokyo, japan
H3PO4 Analar England
2.2 EQUIPMENT/APPARATUS
Table 2.2; Equipment and apparatus used in the study
Materials/Apparatus Specification Company/Country Name
Weighing balance PC-4400/Metal Tokyo/Japan
Beaker Glass England
Conical Flask Glass Pyrex, England
Measuring cylinder Glass Pyrex, England
Spectrophotometer AE-350 ERMAInc./Tokyo, Japan
Water bath GD100 Grant Instruments (Cambridge) Ltd./England
Centrifuge Machine 800D/Metal
Pipette Robber England
Test tube Glass Pyrex England
UV lamp China
Muffle furnace Lento Furnace Gallenkamp USA
2.3 Plant Collection and Identification
Fresh leaves of Pterocarpus santalinoides were collected from Gwandu local government in Kebbi State Nigeria, in the month of July, 2018. The plant voucher number is 516 A, from the family of Fabaceae. It was taxonomically identified and authenticated by a botanist at the Department of Biological Science, Kebbi State University of Science and Technology, Aliero.
2.3.1 Preparation of Plant Extracts
The leaves of the plant were picked, cut into small pieces and dried at room temperature . It was then reduced to powdered form by grinding with mortar and pestle (Harborne, 1984). Extraction of the powdered plant material was performed using reflux extraction method by weighing 50g of the powder plant sample into 500ml of methanol at temperature between 60oC – 70oC. The process was run for seventy two hours and filtered using Whatman’s no. 1 filter paper (Maidstone, UK), afterwards an oven was used to evaporate the solvent and later concentrated in water to obtain the extract. The extract was kept in a well labeled sterile beaker material and stored in the refrigerator until required.
2.3.2 Determination of Proximate Composition of Pterocarpus santalinoides leaves
Leaves Sample
Proximate Composition (Total Moisture Content, Crude Protein, Crude Fat, Crude Fiber, Ash Content, Total Carbohydrate) of the leaves of Pterocarpus santalinoides, The method of Association of Official Analytical Chemistry (AOAC) 1990 was used for general proximate analysis of the plant samples
2.3.3 Determination of Moisture Content.
Principle:
This was based on heating the sample to eliminate all water content in the sample. This was achieved by placing the sample in an oven at 1050C up to 24 hours. High temperature is not needed to avoid decomposition of organic matter.
Procedure:
A clean crucible was dried to a constant weight in an air oven, cooled in a desiccator and weighed (w1). 2g of the sample was placed in the crucible and weighed (w2) and dried in the oven for eight hours. The crucible and its contents were cooled in a desiccator and weighed (w3). The procedure was continued until a constant weight was obtained out of which the percentage moisture was calculated.
Calculation:
% moisture =loss in weight due to drying x 100
Weight of fresh sample
=W2 – W3x100
W2 –W1
Where w1 = weight of empty dish
W2 = weight of fresh sample + empty dish
W3 = weight of dry sample + empty dish
2.3.4 Determination of Total Ash Content
Principle:
The principle was based on the fact that minerals are not destroyed by high temperature. The ash content was determined from the loss of weight that occurs during igniting at a high temperature of 6000C for 5 hours in a muffle furnace, all the organic matter is burnt off leaving the inorganic substance in the form of ash.
Procedure:
2g of the finely ground sample was weighed (w2) into a previously weighed clean crucible (w1) which had been ignited in the muffle furnace at 6000C for one hour and cooled in a desiccator. The crucible containing the sample was heated in a muffle furnace at 6000C for five hours to burn off all the organic matter after which the crucible was cooled in a desiccator and weighed (w3).
Calculation:
% ash = weight of ash X 100
Weight of sample
= W3 – W1 X 100
W2
Where W1 = weight of empty crucible
W2 = weight of sample
W3 = weight of ashed sample in a crucible
2.3.5 Determination of lipid content
Principle:
This involves a continuous extraction of fat content from the sample using n-hexane in a soxhlet extractor and non-polar component of the sample was easily extracted into organic solvent.
Procedure:
Soxhlet extractor with reflux condenser and a small round bottom flask were mixed and 2g (w2) of the grounded sample was placed in the thimble, the mouth of the porous thimble was covered with clean white cotton in order to distribute the draping n-hexane. The thimble was placed in the extractor and n-hexane was added until it was half in the weighed flask (W1). The flask is then heated for five hours. The thimble was removed with care and the n-hexane in the top container was collected. The extract was removed from the water bath when it is almost free of n-hexane. Finally, the extraction flask containing the oil was weighed (W3) to know the content of the crude lipid.
Calculation:
% crude lipid = weight of lipid extracted x 100
Weight of dried sample
= W3-W1
W2
Where w1 = weight of empty flask
w2 = weight of sample
w3 = weight of flask containing the oil
2.3.6 Determination of Crude Fibre.
Principle:
This was based on the sequential hot digestion with acid and alkaline solution of the defatted sample, followed by thorough washing with boiling water and finally drying off. These ensure the removal of all material.
Procedure
The residue (2g) (W2) obtained from crude lipid extraction was placed in a conical flask; 200ml of distilled water and 20ml of H2SO4 was added and fixed on a heater and boiled for 30 minutes to maintain a constant volume. The sample was filtered in a muslin cloth, rinsed with warm water and spatula was used to scrape the sample into the flask, 20ml of H2SO4and 10% of NaOH was added to the contents. The content was placed 30minutes then filtered with muslin cloth and the sample was rinsed with petroleum ether. It was then allowed to drain and the residue was scraped into a crucible and placed in an oven and was dried for one hour at 1050C and allowed to cooled in a desiccator and weighed (W1). It was then placed in a muffle furnace to ash for two hours at 6000C and allowed to cool in a desiccator and weighed (W3). Percentage fibre was then calculated.
Calculation
% crude fibre = W3-W1 x 100
W2
Where W1 = weight after drying
W2 = weight after ashing
2.3.7 Determination of Crude Protein
Principle
Kjeldahl digestion involves oxidation of organic matter with conc. H2SO4 and a tablet of kjeldahl catalyst. The sulphuric acid converts all form of nitrogen to ammonium sulphate. Subsequent addition of an excess amount of NaOH neutralizes the acid and release ammonia which is distilled into boric acid solution and titrated against HCl to the end point so that the amount of HCl consumed by the ammonia could be calculated.
Procedure
Digestion: 2g of the grounded sample was collected in a clean dry 500ml Kjeldahl flask. One tablet of the mixed catalyst and 20ml of H2SO4 was added. Little amount of distilled water was also added into the flask to digest the organic matter present. The flask was heated in a fume cupboard until clear solution was obtained. The content was cooled and transferred into a volumetric flask.
⦁ Distillation: 10ml of the aliquot was pipette into a Kjeldahl flask and make the volume up to 50ml with distilled water. 20ml of 40% NaOH was added and extract the ammonia out of the sample which was evaporated into 20ml boric acid indicator that was used as the receiver of nitrogen extracted. The ammonia was liberated into the boric acid until the volume was made up to 40ml in the conical flask. The color changes from pink to green.
⦁ Titration: the collected sample with ammonia was then titrated against 0.1N HCl to end point which gave the actual amount of protein in the sample. The color changes from green to pink at the end point and the titre value was recorded.
Calculation
% nitrogen= Tv x N x 0.014 x dilution factor (10ml) x 100
Weight of sample x ml of aliquot
% crude protein = %N x conversion factor (6.25)
Where Tv = titre value
N = normality of acid (0.1N)
Dilution factor = 10
2.3.8 Determination of Carbohydrates (by difference).
Carbohydrates was not determined directly but was obtained by difference as below:
Calculation
% carbohydrate = 100 – (% ash + % protein + % fibre + % lipid).
2.4 Macroelement composition
Determination of Potassium (K)
About 5ml of the sample was pipette into a test tube in duplicate. Then 2mls of colbalnitrite was added, shaken vigorously and allowed to stand for 45 minutes and centrifuged for 15 minutes. The supernatant was drained-off and 2mls of ethanol was added to the residue. The solution was shaken vigorously and centrifuged for another 15 minutes. The supernatant was drained off and 2ml of distilled water was added to the residue. The solution was boiled for 10 minutes with frequent shaking to dissolve the precipitate. About 1ml of 1% choline hydrochloride and 1ml of 2% sodium ferric cyanide was added. Then 2mls of distilled water was also added and the solution was shaken to mix well. The absorbance was taken at 620nm against the blank.(AOAC, 1990).
Determination of Calcium (Ca)
About 1ml of the sample was pipette into a test tube in duplicate. Then 3ml of calcium working reagent was added and absorbance at 512nm was read against the blank. (AOAC, 1990).
Determination of Magnesium Content
Five milliliter (5ml) of each sample digest was pipetted into a test tube in duplicate, 1ml of 0.67N sulphuric acid (H2SO4) and 1ml of 0.05% titan yellow was added. Then 1ml of 0.01% gum acacia and 2ml of 10% sodium hydroxide (NaOH) was also added. The solution was mixed and the absorbance was taken at 520nm against the blank (A.O.A.C. 2005)
Determination of Phosphorus Content
The molybdate colorimetric method was used to determine phosphorus. 2 ml of dry ash extract was mixed with equal volume of Vanodo-molybdate colour reagent. The mixture was then diluted with 50 ml distilled water. A standard phosphorus solution was also prepared. The absorbances of the standard and sample were measured at 660nm after fifteen minutes (A.O.A.C. 2005).
2.4 Trace elements
Exactly, 1 g of the sample was weighed and predigested for a short period of time (75°C for 10 minutes) in 69% HNO3 and 30% H2O2 (w/v: 10 ml) and later heated at 120°C. The digested solutions were filtered using Whatman filter paper No. 1 and diluted to 50 ml with deionized water. The concentrations of the micronutrients (Zinc, Iron, Copper, Manganese)in the digested solutions were determined using an atomic absorption spectrophotometer (Kojuncu et al., 2014).
2.5 DETERMINATION OF VITAMINS
2.5.1 Determination of Vitamin A (Rutkowski and Grzegorczyk, 2007).
Principle:
Carotenoids were transported as complexes with lipoprotein. The bonds were broken by the addition of ethanol and the pigments extracted with petroleum ether. Absorbance was determined at 450nm and the concentrations calculated by reference to dichromate standard.
Procedure
Four cleaned dried test tubes were labeled test A and B, standard and blank and the following were pipetted as follows
Reagent Test Standard Blank
Sample A (ml) 1.25 - -
Sample B (ml) 1.25 - -
95% Ethanol (ml) 1.25 - -
Standard - 5 -
P. ether 2.5 - 5
The tubes were shaken thoroughly and centrifuged for 5minutes and the petroleum portion of the tube containing the test solution was pipetted into a cuvette and the absorbance were taken at 450nm against petroleum blank.
Calculation
The value of β-carotene = O.D of test x conc. Of standard
O.D of standard
2.5.2 Determination of vitamin C (Rutkowski and Grzegorczyk, 2007).
Principle:
This was based on color reaction with periodically prepared phosphotungstate reagent and absorbance taken at 700nm.
Procedure:
1ml of the analyzed liquid into the centrifugal test-tube, 1ml of the phosphotungstate (PR) was added and was mixed thoroughly and left in a room temperature for 30minutes. The tube was centrifuged (7000 xg, 10 minutes) and the whole was collected from separated supernatant with a pipette. The supernatant was the test sample for spectrophotometric measurement. The standard was prepared in the same way without centrifugation. The absorbance of the test samples A and of the standard sample A was measured at 700nm against the mixture PR: 50m solution of oxalic acid =1:1 (v/v) was used as the reference sample.
Calculation
Cx =Ax. Cs
As
Where Cs = concentration of the standard solution =56.8μm/L
2.5.3 Determination of vitamin E (Rutkowski and Grzegorczyk, 2007).
Principle:
This was based on the reduction of ferric ions to ferrous ions which form red color with α,α-dipyridyl and the absorbance was measured at 539nm.
Procedure
Four clean test tubes were labeled blank, standard and tests (kenaf and okra) and the following were added as follows:
Reagent blank standard test
Sample K (ml) - - 1.5
Sample O (ml) - - 1.5
Standard (ml) - 1.5 -
Ethanol (ml) 1.5 - -
Xylene (ml) 1.5 1.5 1.5
The contents were mixed and centrifuged for 10 minutes. 1ml of xylene layer was carefully pipetted into another set of clean test tubes. After that 1ml of α,α-dipyridyl reagent was added to all the test tubes as follows:
Reagent Blank Standard Test
Xylene layer (ml) 1.0 1.0 1.0
α,α-dipyridyl (ml) 1.0 1.0 1.0
The test tubes were mixed and the absorbances were taken against blank at 539nm
2.6 TEST FOR PHYTOCHEMICALS
Test for Tannins
About 0.5g of the dried sample was boiled in 20 ml of distilled water in a test tube and then filtered. A few drop of 0.1% ferric chloride was added and observed for brownish green or blue coloration (Tease and Evans, 2005), indicating the presence of tannins.
Test for Saponins
About 2g of the powdered sample was boiled in 20 ml of distilled water in a water bath and filtered. 10 ml of the filtrate was mixed with 5 ml of distilled water and shaken vigorously for a stable persistent froth. The frothing was mixed with 3 drops of olive oil, shaken vigorously, and then observed for the formation of emulation (Sofowora, 1993).
Test for glycosides: To 2ml of plant extract, 1ml of glacial acetic acid and 5% ferric chloride
was added followed by 3drops of concentrated sulphuric acid. Presence of greenish blue colour indicated the presence of glycosides. . (Harborne, 1973),
Test for Phenols: To 1ml of the extract, 2ml of distilled water followed by 5drops of 10%
ferric chloride was added. Formation of blue or green colour indicates the presence of
phenols. (Nweze et al., 2004)
Test for Terpenoids: 5ml of extract of the plant sample was mixed with 2ml of
Chloroform in a test tube and then 3ml of concentrated H2SO4 was carefully added to the mixture to form a layer. An interface with a reddish brown coloration indicates that
terpenoids constituent is present . (Senthilkumar and Reetha, 2009).
CHAPTER THREE
3.0 RESULTS AND DISCUSSION
Pterocarpus santalinoides are widely used as vegetables in the eastern parts of Nigeria, particularly Anambra, Imo, Enugu, Ebonyi and Abia states. There are skeletal literature reports on their nutritional composition. (Mbaella, G.S 1995).However, there are reports on their industrial and pharmaceutical applications (Mbaella, G.S 1995). The result of the present study shows that the plant contain some phytochemicals such as, Tannin, Saponin, phenol and Terpenoids, as shown in Table 1.
Table 1: Qualitative Phytochemical analysis of Pterocarpus santalinoides
Constituents Methanol extract Aqueous extract
Tannin +++ ++
Saponin +++ ++
Glycosides + +
Phenols + ++
Terpenoids ++ ++
Keys = + slight presence, ++ medium presence, +++ heavy presence, - absent.
The results for the proximate composition of Pterocarpus santalinoides leaves are shown below in table 2.
Table 2: Proximate Composition of Pterocarpus santalinoides
Constituent % Yield
Carbohydrate 46.63 + 0.08
Crude protein 13.71 + 0.25
Crude lipid 32.67 + 0.23
Crude fibre 1.33 + 0.29
Moisture 1.17 + 0.76
Ash 4.5 + 0.87
Values are Mean ± SEM of triplicate determinations
.
Below are the elemental composition of Pterocarpus santalinoides leaves in Table 3.0
Table 3: Elemental composition of Pterocarpus santalinoides
Mineral element
Calcium (Ca) 0.812 + 0.06
Sodium (Na) 71.67 + 3.82
Potassium ( K) 2533.3 + 152.75
Phosphorus ( P) 4.69 + 0.03
Magnesium (Mg) 1.05 + 0.09
Manganese 0.1022 + 0.00
Iron (Fe) 1.11 + 0.00
Copper (Cu) 0.23 + 0.00
Zinc (Zn) 0.27 + 0.00
Values are Mean ± SEM of triplicate determinations.
The vitamin constituent of Pterocarpus santalinoides leaves are shown in table 4.0
Table 4: Vitamins constituent of Pterocarpus santalinoides
Vitamins
Vitamin A 47.9 + 1.07
Vitamin C 530.13 + 17.79
Vitamin E 36.39 + 0.39
CHAPTER FOUR
4.0 DISCUSSION, CONCLUSION, AND RECOMMENDATION
4.1 DISCUSSION
Results of phytochemical screening of Pterocarpus santalinoides leaves as shown in Table 1, implies that the plant contain a good number of secondary metabolite such as Tannin, Saponin, Glycosides, phenol and Terpenoids. They were all present in both the methanolic and aqueous extracts. This result is quite similar to what was reported by Okeke and Njoku (2001). The presence of these chemicals lends credence to their pharmacological activities. The phytoconstituents: reported here and elsewhere (Anowi C.F et al., 2012) might be responsible for the ability of these extracts to inhibit the growth of microbes. For instance, tannins are linked with treatment of intestinal disorders, have anti-inflammatory and antibacterial properties and are used for the healing of wounds. Saponins also show anti-inflammatory, anticancer properties and exhibit adverse physiological response in animals (Ndukwe O.K et al., 2013). Terpenoids such as ß-carotene are orange colored and components of carrots, oranges, tomatoes. They liberate vitamin A and have a high antioxidant potential. Phenols and phenolic compounds are known to be toxic to microorganisms (Mason T.L. and Wasserman, 1987).
The results of proximate analysis as shown in Table 2, revealed that in the leaves of Pterocarpus santalinoides the content of Carbohydrate (46.63 + 0.08) is exceptionally high as compared to the other constituent of the plant. The values of lipid (32.67 + 0.23) is higher than that of protein (13.71 + 0.25), the values of Ash content (13.71 + 0.25) happens to be high as compared to that of crude fibre which is (1.33 + 0.29) and the moisture content which is relatively small. These results obtained signifies that Pterocarpus santalinoides are carbohydrate rich leaves due to their relatively high carbohydrate content when compared with the other components of the leaves. The low moisture content of the leaves would hinder the growth of microorganism and storage life would be high (Adeyeye and Ayejugo, 1994). The crude protein content of the leaves of Pterocarpus santalinoides is (13.71%) which is relatively low when compared with cassava leaves (24.88%) piper guinensis (29.78%) and Talinum triangulare (31.00%) as reported by Akindahunsi and Salawu (2005). The values for ash content of Pterocarpus santalinoides (4.5%). The ash content of the leaves is lower than that of some leafy vegetables commonly consumed in Nigeria such as Talinum triangulare. The value of the ether extracts (crude fat) for the leaves of Pterocarpus santalinoides were exceptionally high (32.67%) as compared to 18.0% reported by Okeke and Njoku (2001). This value is high when compared to those of Talinum triangulare 5.90, Amaranthus hybridus (4.80%) (Akindahunsi and Salawu, 2005). Dietary fat functions in the increase of palatability of food by absorbing and retaining flavours. A diet providing 1- 2% of its caloric of energy as fat is said to be sufficient to human beings as excess fat consumption is implicated in certain cardiovascular disorders such as cancer and aging (Antia et al.,2006). The fat content of 32.67 in the dried leaves of P. santalinoides is very low compared to those of calabash seed (43%) (Ekuagbere, 2007) and groundnut (43%) (Apata and Ologhobo, 1994). P. santalinoides has 1.33% crude fibre which is lower when compared to the 2.8% in gourd seed (Ogungbenle, 2006), 4.28% soybean (Akintayo et al. 2002) and 2.53% calabash seed (Ekuagbere, 2007). P. santalinoides are good sources of vegetable protein having a protein content 13.71%. This value was higher than that (3.3%) recorded by the USDA Nutrient Database for Standard Reference (Hall, 1998). Their protein content makes their leaves suitable for consumption, as a necessity for body development. The protein value of P. santalinoides as observed in this study confers on them the advantage as a rich source of vegetable protein over some vegetables such as raw cocoyam leaf (3.4%), cooked cocoyam leaf (2.1%), Amaranthus (6.1%) and Moringa oleifera (4.2%) as reported by Adepoju et al. (2006).The carbohydrate content of P. santalinoides (46.63%) is high and compares well with of Amaranthus hybridus (52.18%) (Akubugwo et al., 2007). Carbohydrates are essential nutrients required for adequate diet. Moisture content is an index of water activity of many foods. The observed value implies that P. santalinoides may have a long shelf life since microorganisms that cause spoilage does not thrive in foods having low moisture content. P. santalinoides have also been recognized as an excellent source of fiber, which is an important consideration for people who suffer from elevated cholesterol levels and in helping to cleanse the colon (Zhao et al., 2007). A number of studies have indicated that components of plants such as dietary fiber have beneficial effects in lowering blood cholesterol levels aside from the decreased intake of saturated fat and cholesterol that occurs with high intakes of plant foods (Ekumankama, 2008). Fibre cleanses the digestive tract, by removing potential carcinogens from the body and prevents the absorption of excess cholesterol (Smith, 1985). Finally fiber binds to cancer-causing chemicals, keeping them away from the cells lining the colon, providing yet another line of protection from colon cancer (Ensminger and Ensminger, 1996).
The results of the elemental composition of Pterocarpus santalinoides as seen in Table 3, shows that the content of Potassium (2533.3 + 152.75) happens to be the highest when compared to other minerals present in the leaves. Potassium assists in muscle contraction and in maintaining fluid and electrolyte balance in body cells (Frossard, 2000). The values for sodium (71.67 + 3.82) is higher than that of Phosphorus which is just (4.69 + 0.03), Iron (1.11 + 0.00), Magnesium (1.05 + 0.09), Calcium (0.812 + 0.06), Zinc (0.27 + 0.00), Copper (0.23 + 0.00), and Manganese 0.1022 + 0.00). Calcium ion regulates a number of physiologic and biochemical processes which includes neuromuscular excitability, blood coagulation, secretary processes, membrane integrity, plasma membrane transport neurotransmitters, bone mineralization and maintenance of healthy teeth (Dutcher and Fiela, 1967; Cheesebrough, 1987). Magnesium forms a part of enzyme activator and also a constituent of bones and teeth (Laestch, 1979; Murray et al., 1990). It also participates in growth metabolism of protein, lipid, carbohydrate and nucleic acid (Harrison and Hoare, 1980; Guthrie, 1989). Manganese is important in growth, reproduction, skeletal structure and nervous system (Riedman, 1976). Manganese deficiency results in depressed reproductive function, abnormalities in the skeletal structures in animals and man. Zinc has been shown to promote wound healing, promote attacks in sickle cell anaemia and control of hereditary diseases (Rafelson et al., 1980). Zinc also plays a role in taste, appetite and growth (Delvin, 1993).
Table 4, shows the results for the analysis of vitamin A, C and E. As seen clearly, the vitamin having the highest concentration is vitamin C (530.13 + 17.79mg/dl), this value is very high as compared to 90mg/dl reported by Okeke and Njoku (2001) on the leaves of this same plant. Vitamin A has a concentration of (47.9 + 1.07mg/dl) and then the least is Vitamin E (36.39 + 0.39mg/dl). The high concentration of vitamin C, is very beneficial, as it serves as a n antioxidant in the body.
Human beings are among the few vertebrates that cannot synthesize vitamin C which is an important antioxidant in the body (David, et al., 2008). Beta carotene is invaluable for the promotion of growth of cells and tissues, resistance to diseases and for delaying the ageing process. It is also important for the maintenance of eye, skin, nails and hair health. The RDA requirement for Beta carotene for a normal healthy, active adult man and non-pregnant woman is 0.3mg/day and 0.27mg/day respectively (FAO, 2001).).Vitamin E is a very potent antioxidant that helps to protect body cells from damage due to reactive oxygen species. It is very important for the formation and normal function of erythrocytes and muscles (Achikanu, C. E., et al., (2013).
4.2 CONCLUSION
From the results obtained so far, It is believed that the results of this study will help to stimulate consumption or utilization of Pterocarpus santalinoides as good sources of nutrients needed for healthy growth. Making them important in combating nutritional deficiencies according to nutritional needs of individuals towards reducing malnutrition cases and enhancing nutrition security.
Generally, the findings of the study have shown that Potassium content is high in this very leaves making it best to supplement with potassium and meeting potassium needs/requirements of the body. sodium, phosphorus and magnesium contents are high, making them of nutritional importance to cover the required levels in the body on consumption. They are rich sources of Vitamin C, A and E. Proximate levels of crude fat, carbohydrate and crude protein are high. They also Contain some phytochemicals like Tannin, Saponin, Terpenoids, Phenol and Glyoside.
It can be concluded therefore, that the leaves of this plant are not only palatable in soup but also very medicinal. All the phytochemicals present in the leaves proved that the plant could serve as anti-inflammatory, anti-oxidant, anti-tumor and anti microbial agent. The nutritional and health benefits of the leaves of this plant have proved the plant to be a potential source of useful drugs and quality food.
4.3 RECOMMENDATION
Looking at this research work, in the quest of exploring the nutritional benefits of Pterocarpus santalinoides, only a few vitamins, minerals and phytochemicals where actually been analysed, limiting our knowledge of it’s overall benefit to man. So, I recommend that further research be carried out to assess the presence of so many other vitamins, minerals and phytochemicals that are not included in this very research.
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