CHAPTER ONE INTRODUCTION 1.1 VEGETABLE

A vegetable is any part of a plant that is consumed by humans as food as part of a savoury course or meal. The term "vegetable" is somewhat arbitrary, and largely defined through culinary and cultural tradition. It normally excludes other main types of plant food, fruits, nuts and cereal grains but includes seeds such as pulses. Defining a

"vegetable" is hard because so many different parts of a plant are consumed as food; roots, tubers, bulbs, corms, stems, leaf stems, leaf sheaths, leaves, buds, flowers, fruits and seeds. The broadest definition is the word's use adjectivally to mean "matter of plant origin" to distinguish it from "animal", meaning "matter of animal origin". More specifically, a vegetable may be defined as "any plant, part of which is used for food",a secondary meaning then being "the edible part of such a plant".A more precise definition is "any plant part consumed for food that is not a fruit or seed, but including mature fruits that are eaten as part of a main meal"(Sinha et al;2010, Vainio and Bianchini,

2003)Falling outside these definitions are mushrooms and other edible fungi which, although not parts of plants, are often treated as vegetables.

1.2 DIFFEENT CATEGORIES OF VEGETABLE

1.2.1 BULB VEGETABLES

Bulb vegetables are aromatic vegetables which are used widely to add flavour to casseroles, broths, courts-bouillons and soups. Garlic, chive, spring onion, water chestnut, grey shallot and other varieties of onions and leeks come under the category of bulb vegetables. They derived the name because it is the bulbs that are eaten, not the

1 leaves. Some of the bulb vegetables including garlic are known for their medicinal value and recent studies suggest that some of these help in preventing cancer(Fungi vegetables,

2015).

1.2.2 FRUIT VEGETABLES

Fruit vegetables are those which come under the category of both fruits and vegetables. An example is tomato which is a fruit by scientific means as they are developed from the ovary in the base of a flower as in the case of any other fruit. From the culinary point of view, they are termed as vegetables as they are used in savory other than sweet cooking. Example include; Avocados, Chayote, Okra, Olives, Peppers,

Tomatoes, etc. (Fungi vegetables, 2015)

1.2.3 INFLORESCENT VEGETABLES

Clusters of flowers that are arranged on a stem which is composed of a main branch or complex arrangement of branches are Inflorescent Vegetables. Its flowers, flower buds, stems and leaves are eaten as vegetables. These vegetables have great nutritional value and used often in casseroles, cooked as a side dish or is served raw to accompany salads. They are the perfect option for dieters as they are less in calories and are filling and hearty. Examples include; Artichokes, Broccoli, Cauliflower, Banana flower, etc. (Fungi vegetables, 2015).

1.2.4 LEAF VEGETABLES

2 Leafy vegetables are plant leaves that are eaten as a vegetable, sometimes accompanied by petioles and shoots. The number of species of plants with edible leaves coming up to one thousand, most of the leafy vegetables come from herbaceous plants such as lettuce and spinach. Leafy vegetables have high nutritional value though it is the presence of Vitamin K which makes it a viable option. Rich in vitamins, minerals and disease-fighting phytochemicals and fiber, it is effective for weight loss and helps you keep your hunger in check. It also helps in reducing and blood pressure and slows down the process of absorption of carbohydrates into the bloodstream. Containing plenty of water, it keeps you hydrated and helps in maintaining beautiful skin and hair.

Examples include; Spinach, Arugula, Cabbage, Collards, Lettuce, Sea kale, Endive,

Cress, etc. (Fungi vegetables, 2015)

1.2.5 ROOT VEGETABLES

Root vegetables are either roots or stems of the underground plants that are used as vegetables. They are actually, storage organs that are enlarged to store energy in the form of carbohydrates and among them, starchy root vegetables are of particular prominence. Widely available in tropical regions including West Africa, Central Africa and Oceania. Those vegetables that are included in this group are sweet potatoes, carrots, beets, parsnips, jicama, leeks, and Jerusalem artichokes and beets. As they grow beneath the ground, they are highly nutritious, allowing them to absorb as many minerals and nutrients as possible (Fungi vegetables, 2015).

1.3 MEDICINAL PLANT

3 Medicinal plant is defined as any substance with one or more of its organ containing properties that can be used for therapeutic purposes or which can be used as precursors for the synthesis of various drugs. (Sofowora, 1993) Medicinal plants contain numerous biologically active compounds such as carbohydrates, proteins, enzymes, and oils, minerals, vitamins, alkaloids, quinones, terpenoids, flavonoids, carotenoids, sterols, simple phenolic glycosides, tannins, saponins, polyphenols etc. Traditional medicine refers to health practices, knowledge and beliefs incorporating plants, animals and mineral based medicines, spiritual therapies, manual techniques and exercises, applied singularly or in combination to treat, diagnose and prevent illnesses or maintain well being. Over the years, medicinal plants have been found useful in the treatment and management of various health problems. Traditional medicine is undoubtedly a reliable alternative approach to health care delivery in the metropolis because it is cheap, easily accessible and efficacious. Herbal drugs are invariably single plant extracts of fractions thereof or mixtures of fractions/extracts from different plants. Traditional plant medicines might offer a natural key to treat various human ailments. In recent years, there has been an increasing interest by researchers in the use of naturally occurring biologically active compounds of medicinal value (Ananda Rajagopal et al., 2011). The use of plants for medical purposes dates back to antiquity (Sofowora, 1993). Recent research has focused on natural plants product alternative for disease control in developing countries. The majority of rural dwellers do not have access to modern health care, so they mostly depend on medicinal plant to prevent or eliminate diseases. Medicinal plants are cheaper, more accessible to most of the population in the world. Thus, there is need to encourage the use of medicinal plants as potential sources of new drugs. There has therefore been an

4 upsurge in the interest in herbal remedies in several parts of the world with many of the herbal remedial being incorporated into orthodox medical practice (Daniyan and

Muhammad, 2008).

1.4 NUTRITIONAL FACTS OF BASELLA SP.

Basella or vine spinach is a popular tropical leafy-green vegetable commonly grown as backyard plant in the home gardens. In the true sense, it is different from

English spinach (Spinacea oleracea) in that the plant is a creeping vine, and its leaves feature glossy, broad, deep green, thick, and mucilaginous (Ghafoorunissa, 1996).

Commonly found in the backyard gardens of many south Asian families, it is gaining popularity in some of the tropical and temperate climates of America, Australia and

Europe for its succulent, nutritious greens, and tender stems. Vine spinach belongs to the

Basellaceae family, and has two chief cultivars, Basella alba, which features green-stems and deep-green leaves, and Basella rubra with purplish-stems and deep-green leaves with pink veins (Daniyan and Muhammad, 2008).

5 Fig. 1:0 Morphology of Basella alba plant (A), along with Fruits (B), Bud (C), adaxial (D) and abaxial (E) surfaces of the leaf.

There are many plant species available all over the world which has been used for the multi beneficial activities. India and China are the two major countries that are richer in many of the medicinal plant species. In spite of millions of chemically synthesized drug for a number of diseases; natural products of plant origin has got its own importance and has remained the most important source of new drugs. One such medicinal herb is

Basella

The present study is focused towards compiling the ethanobotanical and scientific importance of above mentioned plant.

1.4.1 TAXONOMY OF THE PLANT Kingdom: Plantae Phylum: Magnoliophyta Class: Magnoliopsida Order: Caryophyllales Family: Basellaceae Genus: Basella Species: alba (green stem)

6 Species: rubra(purplish stem) 1.4.2 VERNACULAR NAMES English: Ceylon spinach, Malabar spinach, Indian spinach, Hindi: lalbachlu , po,

Bengali: Puishak, Oriya: Poi saga, Konkani: Valchibhaji, Kannada: Basalesoppu, Telugu:

Bachhali, Tamil: KodippasaLi, Tulu: Basale, Marathi: Mayalu, Portuguese: Bertalha,

Filipino: Alugbati, Vietnamese: Mồ ngt ơ i, Sinhalese: VelNiviti (Sudu), Sanskrit name:

Upodika, Indian name: Poi (Kumar, 2010).

1.4.3 CULTIVATION DETAILS

Requires a well-drained moisture-retentive soil rich in organic matter and a warm sunny sheltered position, prefers a sandy loam. Tolerates fairly poor soils but does much better in rich soils, Tolerates high rainfall, Tolerates a pH in the range 4.3 to 7. A frost- tender perennial, it is not hardy outdoors in Britain but can be grown as a spring-sown annual. A fast growing plant, capable of producing a crop within 70 days from seed in a warm climate, though it requires a minimum daytime temperature of 15°c if it is to keep growing vigorously so it seldom does well outdoors in Britain, It does tolerate low light levels plus night temperatures occasionally falling below 10°c, and so can do well in a cold greenhouse. Plants do not flower if the length of daylight is more than 13 hours per day. Widely cultivated for its edible leaves in the tropics, there are some named varieties.

It is an excellent hot weather substitute for spinach (Huxley, 1992).

Basella species is a wildly cultivated, cool season vegetable with climbing growth habit. It is a succulent, branched, smooth, twining herbaceous vine, several meters in length. Stems are purplish or green. Leaves are fleshy, ovate or heart-shaped, 5 to 12 cms

7 long, stalked, tapering to a pointed tip with a cordate base. Spikes are axillary, solitary,

5-29 cm

Long. Fruit is fleshy, stalkless, ovoid or spherical, 5-6 mm long, and purple when mature.

Mainly leaves and stems are used for the medicinal purpose (Kumar, 2010).

1.4.4 EDIBLE USE

Edible Parts: Leaves.

Edible Uses: Colouring; Tea.

Leaves and stem tips - raw or cooked. A pleasant mild spinach flavour, the leaves can be used as spinach or added to salads. Do not overcook the leaves or they will become slimy.

The mucilaginous qualities of the plant make it an excellent thickening agent in soups, stews etc where it can be used as a substitute for okra, Abelmoschatus esculentus. A nutritional analysis of the leaves is available. An infusion of the leaves is a tea substitute.

The purplish sap from the fruit is used as a food colouring in pastries and sweets. The colour is enhanced by adding some lemon juice (Huxley, 1992).

1.4.5 COMPOSITION Figures in grams (g) or miligrams (mg) per 100g of food. Leaves (Dry weight) 275 Calories per 100g Water: 0% Protein: 20g; : 3.5g; Carbohydrate: 54g; Fiber: 9g; Ash: 19g; Minerals - Calcium: 3000mg; Phosphorus: 0mg; Iron: 0mg; Magnesium: 0mg; Sodium: 0mg; Potassium: 0mg; Zinc: 0mg; Vitamins - A: 50mg; Thiamine (B1): 0.7mg; Riboflavin (B2): 1.8mg; Niacin: 7.5mg; B6: 0mg; C: 1200mg; 1.4.6 ETHANOBOTANY

8 Basella species has been used for many of its useful product from ancient times.

Nowadays its properties have been utilized for the extraction of some useful material so that it can be used for the beneficial human activities. Some of the uses of this plant parts in the cure of certain problems occurred to humans has been explained here: Daily consumption of Basella species has a positive effect on total-body vitamin A stores in men. The paste of root of red Basella species along with rice washed water is taken in the morning in empty stomach for one month to cure irregular periods by the rural people of

Orissa, India. Leaves of B. alba is used for the treatment of hypertension by Nigerians in

Lagos, and malaria in cameroonian folk medicine. The plant has been reported for its antifungal, anticonvulsant, analgesic, anti-inflammatory and androgenic activities and or the treatment of anemia. The leaves of B. Alba are traditionally used in ayurveda system of medicine to bring sound refreshing sleep when it is applied on head about half an hour before bathing. A paste of the root is applied to swellings and is also used as a rubefacient. Sap is applied to acne eruptions to reduce inflammation (NHMRC, 2006).

Decoction of leaves used for its mild laxative effects. Pulped leaves applied to boils and ulcers to hasten suppuration. Sugared juice of leaves is useful for catarrhal afflictions.

Leaf-juice mixed with butter, is soothing and cooling when applied to burns and scalds.

In Ayurveda, it is used for hemorrhages, skin diseases, sexual weakness, and ulcers and as laxative in children and pregnant women. The plant is febrifuge, its juice is a safe aperient for pregnant women and a decoction has been used to alleviate labour. It is also an astringent and the cooked roots are used in the treatment of diarrhea. The leaf juice is a demulcent, used in cases of dysentery. This plant serves as a Thai traditional vegetable.

The fruit provides dark violet color for food colorant. Basella mucilage has been used in

9 Thai traditional medicine as topical application for irritant, bruise, ringworm and laboring. Stem and leaves are used as mild laxative, diuretic and antipyretic. In India, it has been used for antipruritis and burn, and has been used in Bangladesh for acne and freckle treatment. The Ayurvedic treatment in India has been used B. Alba leaves and stem for anticancer such as melanoma, leukemia and oral cancer. Root and leaves has been used for the removal of after birth, stomach pains and increase milk production.

Basella alba is administered orally for the treatment of anal prolapsed or hernia. Ground leaves of Basella alba are rubbed on the human hand to introduce the whole preparation into the animal vagina every morning for the treatment of sterility. The leaf juice is used in Nepal to treat dysentery, catarrh and applied externally to treat boils. The mucilaginous qualities of the plant make it an excellent thickening agent in soups, stews, etc. The purplish sap from fruits is used as a colouring agent in pasteries and sweets. Basella alba has been used for the treatment of Anemia in women, coughs, cold (leaf with stem), and cold related infections. Maceration is taken orally for infertility, pelvic inflammatory disease, orchitis, epididymytis, threatened abortion, spurious labour. Leaves are used in constipation, poultice for sores, urticaria and gonorrhea. It is also used in poultice local swellings, intestinal complaints etc. (Yasmin et al.,2009) The mucilaginous liquid obtained from the leaves and tender stalks of plants is popular remedy for headaches.

(Jadhav et al., 2011).

CHAPTER TWO 2.0 LITERATURE REVIEW 2.1 Biological lipids are a chemically diverse group of compounds, the common and deflning feature of which is their insolubility in water. The biological functions of the

10 lipids are as diverse as their chemistry. Fats and oils are the principal stored forms of

energy in many organisms (Paulose, et all.,1996). Phospholipids and sterols are major

structural elements of biological membranes. Other Iipids, although present in relatively

small quantities, play crucial roles as enzyrne cofactors,electron carriers, Iight-absorbing

pigments, hydrophobic anchors for proteins, "chaperones" to help membrane proteins

fold, emulsifying agents in the digestive tract, hormones, and intracellular messengers

( folch et all., 1957).

S/N CATEGORY CATEGORY EXAMPLES CODE Fatty acids FA Oleate, stearoyl-CoA, palmitoylcarnitine 1 2 Glycerolipids GL Di- and triacylglycerols

3 Glycerophospholipids , phosphatidylserine, GP phosphatidylethanolamine

4 Sphingolipids SP Sphingomyelin, ganglioside GM2

5 Sterol lipids ST Cholesterol, progesterone, bile acids

6 Prenol lipids PR Farnesol, geraniol, retinol, ubiquinone

7 Saccharolipids SL Lipopolysaccharide

8 Polyketides PK Tet racycline. aflatoxin

Table 2.0: Eight major categories of biological lipids

2.2 PHOSPHOLIPIDS Phospholipids are a class of lipids that are a major component of all cell membranes

as they can form bilayers. Most phospholipids contain a diglyceride, a phosphate

group, and a simple organic molecule such as ; one exception to this rule is

11 sphingomyelin, which is derived from sphingosine instead of glycerol. The first phospholipid identified in 1847 as such in biological tissues was , or phosphatidylcholine, in the egg yolk of chickens by Theodore Nicolas Gobley, a French chemist and pharmacist. The structure of the phospholipid molecule generally consists of hydrophobic tails and a hydrophilic head. Biological membranes in eukaryotes also contain another class of lipid, sterol, interspersed among the phospholipids and together they provide membrane fluidity and mechanical strength. Purified phospholipids are produced commercially and have found applications in nanotechnology and materials science (Mashaghi et al., 2013).

CLASSES OF PHOSPHOLIPIDS

There are five general classes of phospholipids. These are phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and phosphatidylsphingomyelin. The special characteristics of the different classes of phospholipids depend upon which element is attached,at the head i.e., whether serine, choline, inositol or ethanolamine is attached, although it is now beginning to be realized that the activity of these phospholipids may also be influenced by which fatty acids make up the tails.

2.2.1 PHOSPHATIDYLCHOLINE

Phosphatidylcholine is an excellent source of choline, a B vitamin.

Phosphatidylcholine is the major component of cell membranes and is critical for brain

12 and liver function. In the brain, phosphatidylcholine is the precursor to acetylcholine. In digestion, phosphatidylcholine is part of the bile complex that emulsifies fats to facilitate absorption. Technically, phosphatidylcholine is lecithin, although the term is now used more frequently for the mixed phospholipid byproducts of seed oil refining.

2.2.2 PHOSPHATIDYLETHANOLAMINE

Phosphatidylethanolamine is usually the second most abundant phospholipid in animal and plant lipids and is a key building block of cell membrane bilayers. It can be transformed into phosphatidylcholine, but it nevertheless has its own roles in the cell.

This phospholipid aids in the assembly of membrane proteins and without it they may not function properly. It may be needed to orient enzymes correctly in the inner membrane.

2.2.3 PHOSPHATIDYLINOSITOL

Phosphatidylinositol is present in all tissues and cell types. It is especially abundant in brain tissue, where it can make up as much as 10% of the phospholipids.There is usually less phosphatidylinositol in tissues than there is phosphatidylcholine, phosphatidylethanolamine or phosphatidylserine, which is to say that it is a minor phospholipid constituent of cell membranes. In cell membranes, it is usually located on the inner side. Phosphatidylinositol is the primary source of the arachidonic acid required for biosynthesis of eicosanoids, including prostaglandins.

Derivatives of this phospholipid serve as messenger molecules with the nervous system.

13 2.2.4 PHOSPHATIDYLSERINE (PS)

Phosphatidylserine has an affinity for the proteins found within the cellular membrane matrix. PS is most concentrated in the cells of the brain and nerves. PS promotes the stability and the integrity of the cellular membrane and it promotes the ability of cells to maintain that internal balance known as homeostasis.

2.3 FATTY ACIDS

Fatty acids (FA) are class of compounds containing long hydrocarbon chain and a terminal carboxylic group. They are building block of saponifiable lipids, only traces occur in free (unesterified) form in cells and tissues.

All fatty acids possess a long non-polar hydrocarbon chain which may be saturated or may contain one or more double bonds Fatty acids with two or more double bonds are called polyunsaturated fatty acids.

Polyunsaturated fatty acids are better in human nutrition than saturated fatty acid and they are derived from fat and oils. The properties of fatty acid and of lipids are markedly dependent on their degree of saturation. Unsaturated fatty acids have a lower melting point than the saturated fatty acid of the same chain length.

Examples of saturated fatty acids;

CH3 (CH2)10COOH Lauric acid

CH3 (CH2)12COOH Myristic acid

CH3 (CH2)14COOH Palmitic acid

CH3 (CH2)16COOH stearic acid

Examples of unsaturated fatty acids;

CH3 (CH2)5CH=CH(CH2)7COOH Palmitoleic acid

14 CH3 (CH2)7CH=CH(CH2)7COOH Oleic acid

CH3 (CH2)4CH=CH CH2CH=CH(CH2)7COOH Linoleic acid (LA).

CH3CH2CH=CHOH2CH= CH CH2CH=CH(CH2)7COOH Linolenic acid (-LA).

CH3 (CH2)4CH=CH(CH2)4(CH2)2COOH Arachidonic acid (AA).

2.3.1 PHYSIOLOGICAL ROLES OF FATTY ACIDS

Fatty acids have four physiological roles, namely;

1. Fatty acids derivatives serve as hormones and intracellular messengers.

2. Many proteins are modified by the covalent attachment of fatty acids which

targets them to membrane locations.

3. Fatty acids are fuel micro molecules.

4. Fatty acids are fuel molecules. They are stored as triacyglycerol which are

uncharged esters of glycerol also called neutral fats or triglycerol.

2.3.2 DIETARY FATS AND FATTY ACIDS

Dietary fat includes all the lipids in plant and animal tissues that are eaten as food.

The most common fats (solid) or oils (liquid) are glycerolipids. Fatty acids constitute the main components of these lipid entities and are required in human nutrition as a source of energy, and for metabolic and structural activities. The most common dietary fatty acids have been subdivided into three broad classes according to the degree of unsaturation; saturated fatty acids (SFA) have no double bonds, monounsaturated fatty acids (MUFA) have one double bond and polyunsaturated fatty acids (PUFA) have two or more double bonds. In general, these fatty acids have an even number of carbon atoms and have unbranched structures. The double bonds of naturally occurring unsaturated fatty acids

15 are very often of the cis orientation. A cis configuration means that the hydrogen atoms attached to the double bonds are on the same side. If the hydrogen atoms are on opposite sides, the configuration is termed trans.

2.3.3 SATURATED FATTY ACIDS

The Saturated Fatty Acids (SFA) has the general formula R-COOH. They are further classified into four subclasses according to their chain length: short, medium, long and very long. There are various definitions used in the literature for the SFA sub-classes.

The Expert Consultation recognized that there is a need for universal definitions and recommends the following definitions for the SFA sub-classes.

• Short-chain fatty acids: Fatty acids with from three to seven carbon atoms.

• Medium-chain fatty acids: Fatty acids with from eight to thirteen carbon atoms.

• Long-chain fatty acids: Fatty acids with from fourteen to twenty carbon atoms.

• Very-long-chain fatty acids: Fatty acids with twenty one or more carbon atoms.

16 Table 2.2: Common saturated fatty acids in food fats and oil

TRIVIAL NAME SYSTEMATIC ABBREVIATION TYPICAL SOURCES NAME Butyric butanoic C4:0 dairy fat

caproic hexanoic C6:0 dairy fat

caprylic octanoic C8:0 dairy fat, coconut and palm kernel oils capric decanoic C10:0 dairy fat, coconut and palm kernel oils lauric dodecanoic C12:0 coconut and palm kernel oils myristic tetradecanoic C14:0 dairy fat, coconut and palm kernel oils palmitic hexadecanoic C16:0 most fats and oils

stearic octadecanoic C18:0 most fats and oils

arachidic eicosanoic C20:0 peanut oil

behenic docosanoic C22:0 peanut oil

lignoceric tetracosanoic C24:0 peanut oil

2.3.4 UNSATURATED FATTY ACIDS

The unsaturated fatty acids are also further classified into three sub-groups according their chain lengths. Various definitions have also been used in the literature for the sub-classes of unsaturated fatty acids, but no universally accepted definitions exist.

Therefore, the Expert Consultation recommends the following definitions.

• Short-chain unsaturated fatty acids: Fatty acids with nineteen (19) or fewer carbon atoms.

17 • Long-chain unsaturated fatty acids: Fatty acids with twenty (20) to twenty four (24) carbon atoms.

• Very-long-chain unsaturated fatty acids: Fatty acids with twenty five (25) or more carbon atoms.

2.3.5 MONOUNSATURATED FATTY ACIDS

More than one hundred cis-MUFA occur in nature, but most are very rare compounds. Oleic acid (OA) is the most common MUFA and it is present in considerable quantities in both animal and plant sources.

S/N COMMON SYSTEMATIC DELTA TYPICAL SOURCES NAME NAME ABBREVIATION 1. palmitoleic cis-9- 16:1Δ9c (9c-16:1) marine oils, macadamia oil, hexadecenoic most animal

2. oleic cis-9- 18:1Δ9c (9c-18:1) all fats and oils, especially olive octadecenoic (OA) oil, canola oil and high-oleic sunflower and safflower oil

3. cis-vaccenic cis-11- 18:1Δ11c (11c-18:1) most vegetable oils octadecenoic 4. gadoleic cis-9-eicosenoic 20:1Δ9c (9c-20:1) marine oils

cis-11- 20:1Δ11c (11c-20:1) marine oils eicosenoic 5. erucic acid cis-13- 22:1Δ13c (13c-22:1) mustard seed oil, high erucic docosenoic rapeseed oil

6. nervonic cis-15- 24:1Δ15c (15c-24:1) marine oils tetracosenoic Table 2.3: Some common cis-monounsaturated fatty acids in fats and oils

18 2.3.5 POLYUNSATURATED FATTY ACIDS Natural polyunsaturated fatty acids (PUFA) with methylene-interrupted double bonds and all of cis configuration can be divided into 12 families, ranging from double bonds located at the n-1 position to the n-12 position (Gunstone, 1999). The most important families, in terms of extent of occurrence and human health and nutrition, are the n-6 and n-3 families.). linolenic acid (ALA) is the parent fatty acid of the n-3 family.

It also has 18 carbon atoms, but three double bonds. In contrast to LA, the first double bond in ALA is 3 carbon atoms from the methyl end of the fatty acid chain, and hence the n-3 name. Arachidonic acid (AA) is the most important n-6 PUFA of all the n-6 fatty acids because it is the primary precursor for the n-6 derived eicosanoids. AA is present at low levels in meat, eggs, fish, algae and other aquatic plants (Wood et al., 2008; Ackman,

2008a). Eiocosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are the most important n-3 fatty acids in human nutrition.

19 2.4 COMMON SOURCE OF N-6 POLYUNSATURATED FATTY ACIDS COMMON NAME SYSTEMATIC N MINUS TYPICAL SOURCES S/N NAME ABBREVIATION Cis-9,cis-12- 1. Linoleic acid octadecadienoic 18:2n-6 Most vegetable oils. acid [LA] Evening primrose, borage 2. Cis-6,cis-9,cis12- 18:3n-6 and blackcurrant seed -linolenic acid octadecatrienoic [GLA] oils acid Dihomo -linolenic Cis-8,cis-11,cis- Very minor component in 3. acid 14-eicosatrienoic 20:3n-6 animal tissues. acid [DHGLA] 4. Arachidonic acid Cis-5,cis-8,cis- 20:4n-6 Animal fats, 11,cis-14- eicosatrienoic acid [AA] liver,egg,lipids,fish. Cis-7,cis-10,cis- Very minor component in 5. Docosatetraenoic 13,cis-16- 22:4n-6 animal tissues acid docosatetraenoic acid Cis-4,cis-7,cis- Very minor component in 6. Docosapentaenoic 10,cis-13,cis-16- 22:5n-6 animal tissues acid docosapentaenoic acid

20 TABLE 2.4: common sources of n-6 polyunsaturated fatty acid (White, 2008).

S/N COMMON SYSTEMATIC N MINUS TYPICAL NAME NAME ABBREVIATION SOURCES Cis-9,cis-12,cis-15- Flaxseed oil, perilia 1. -linolenic acid octadecatrienoic acid 18:3n-3 oil, canola oil, [ALA] soybean oil. Stearldonic acid Cis-6,cis-9,cis-12,cis- Fish oils, 15-octadecatetraenoic 18:4n-3 genetically 2. acid [SDA] enhanced soybean oil, blackcurrant seed oil, hemp oil. Cis-8,cis-11,cis- 3. Eicosapentaenoic 14,cis-17- 20:4n-3 Very minor acid eicosapentaenoic acid component in animal tissues. Cis-5,cis-8,cis-11,cis- Fish, especially only 14,cis-17- 20:5n-3 fish (salmon, eicosapentaenoic acid [EPA] herring, anchovy, smelt & mackerel. Docosapentaenoic Cis-7,cis-10,cis- Fish, especially only 4. acid 13,cis-16,cis-19- 22:5n-3 fish (salmon, docosapentaenoic [n-3 DPA] herring, anchovy, acid smelt & mackerel Cis-4,cis-7,cis-10,cis- Fish, especially only 5. Docosahexaenoic 13,cis-16,cis-19- 22:6n-3 fish (salmon,

21 acid docosahexaenoic acid [DHA] herring, anchovy, smelt & mackerel

TABLE 2.5 : common sources of n-3 polyunsaturated fatty acid (White, 2008).

CHAPTER THREE

3.0 MATERIALS AND METHOD 3.1 COLLECTION AND PREPARATION OF SAMPLE The sample was collected from school main campus farm, Iworoko-Ekiti,

Irepodun-Ifelodun local government area of Ekiti state and Ado-Ekiti. It was properly sorted, washed, dried and milled into powdered form and kept in an air tight plastic sample bottle prior to analysis.

3.2 LIPIDS CONCENTRATION DETERMINATION

The lipids concentration for the samples was carried out by the following modified AOAC (2005) official method for the fatty acids methyl ether, sterol and total crude fat analysis, while the modified method of Raheja et all (1973) was employed in the phospholipids analysis. the analysis of the extracted fatty acids, steroids and phospholipids was carried out using the gas chromatography method.

3.3 TOTAL CRUDE FAT DETERMINATION

The crude fat analysis was carried out by the following ester extraction method of

AOAC 92.30 (A, 2006). The reagents and apparatus employed includes: petroleum ether,

Condenser, Soxhlet extraction unit, Oven, 250ml capacity boiling flask, Weighing balance, Thimble, Heating mantle, No 4-filter paper and glass wool.

22 3.3.1 PROCEDURE

250ml capacity in the oven at extracting flask was dried in the oven at 105 oC, transferred to the desiccators to cool to the laboratory temperature and the weight of the flask was measured. 0.25g of the sample was weighed into the labeled porous thimble.

200ml of the petroleum ether was measured and added to the dried 250ml capacity flask.

The covered porous thimble with the sample was placed in the condenser of the soxhlet extractor for 5hrs. The porous thimble was removed with care and the petroleum ether in the top container tube was collected for recycling for reuse. The extraction flask was removed from the heating mantle arrangement when it was almost free of petroleum ether. The extraction flask with the oil was oven dried at 105 oC for one hour. The flask containing the dried oil was cooled in the desiccators and the weight of the cooled flask with the dried oil was measured.

3.4 FATTY ACID METHYL ESTER DETERMINATION

Fatty acid profile-saturated, mono- and polyunsaturated analysis was carried out but following the modified AOAC 965.49 and AOAC 966.06 official method.

3.4.1 PROCEDURE

50mg of the extracted fat content of the sample was saponified (esterified) for five minutes at 95 oC with 3.4ml of the 0.5M KOH in dry methanol. The mixture was neutralized by using 0.7M HCL. 3ml of the 14%boron trifluoride in methanol was added.

The mixture was heated for five minutes at temperature of 90 oC to achieve complete methylation process. The fatty acid methyl esters were thrice extracted from the mixture with redistilled n-hexane. The content was concentrated to 1ml for gas chromatography

23 analysis and 1µl was injected port of GC. The gas chromatography GC condition for the

analysis of fatty acid methyl esters are as follows;

GC HP 6890 powered with HP Chemstation Rev.A.09.01 (1206) Software Injection Split Ratio 20:1 Carrier Gas Nitrogen Inlet temperature 250 oC Column type HP INNOwax Column Dimension 30m x0.25mm x 0.25µm Oven program Initial Temperature @ 60 oC First Ramping @10 oC /min for 20mins Maintained for 2mins Second Ramping @ 15 oC /min for 4mins Maintained for 8mins Detector FID Detector temperature 320 oC Hydrogen pressure 22psi Compressed Air 35psi

3.5 STEROLS DETERMINATION Sterols and cholesterol analysis were carried out by following the modified

AOAC 970.51 official methods

3.5.1 PROCEDURE

The aliquots of the extracted fat were added to the screw-crapped test tubes. The

samples was saponified at 95 oC for 10mins by using 3ml of 10ml% KOH in ethanol, to

which 0.20ml of benzene had been added to ensure miscibility. Deionized water 3ml was

added and 2ml of hexane was used in extracting the non-saponified materials (sterols

e.t.c.). Three extracyions, each with 2ml of hexane were carried out for one hour, 30mins,

and 30mins respectively, to achieve complete extraction of the sterols. The hexane was

24 concentrated to 1ml in the vial for gas chromatography analysis and 1µl was injected into

the injection port of GC. The GC conditions for the analysis of sterols are as follows:

GC HP 890 powered with HP Chemstation Rev.A.09.01 (1206) Software Injection Split Ratio 20:1 Carrier Gas Nitrogen Inlet temperature 250 oC Column type HP INNOwax Column Dimension 30m x0.25mm x 0.25µm Oven program Initial Temperature @ 60 oC First Ramping @10 oC /min for 20mins Maintained for 4mins Second Ramping @ 15 oC /min for 4mins Maintained for 10mins Detector FDI Detector temperature 320 oC Hydrogen pressure 22psi Compressed Air 35psi

3.6 PHOSPHOLIPIDS DETERMINATION

Modified method of Rehaja et al., (1973) was employed in the analysis of the

extracted oil phospholipids content determination.

3.6.1 PROCEDURE

0.01g of the extracted fat was added to the tubes. To ensure complete dryness of

the oil for phospholipids analysis, the solvent was completely removed by passing stream

of Nitrogen gas on the oil. 0.40ml of chloroform was added to the content of the tube and

it was followed by the addition of 0.10ml of the chromo-genic solution. The content of

the tube was heated at temperature of 100 oC in the water bath for about 1mins 20secs.

The content was allowed to cool to the laboratory temperature and 5ml of the hexane gas

was added and the tube with this content shock several times. The solvent and the

aqueous layers were allowed to be separated. The hexane layer was recovered and

25 allowed to be concentrated to 1.0ml for gas chromatography analysis using pulse frame

photometry detector. The GC conditions for the analysis for the phospholipids are as

follows:

GC HP6890 powered with HP Chemstation Rev.A.09.01 (1206) Software Injection Split Ratio 20:1 Carrier Gas Nitrogen Inlet temperature 250 oC Column type HP INNOwax Column Dimension 30m x0.25mm x 0.25µm Oven program Initial Temperature @ 50 oC First Ramping @10 oC /min for 20mins Maintained for 4mins Second Ramping @ 15 oC /min for 4mins Maintained for 5mins Detector PFPD Detector temperature 320oC Hydrogen pressure 22psi Compressed Air 35psi

3.7 CORRELATION COEFFICIENT OF STANDARD USE IN LIPIDS

DETERMINATION

The correlation coefficient is a statistical indices that shows the quality assurance

of the calibration curves performed. It ranges between 1 for high positive correlation to -1

for high negative correlation, with 0 indicating a purely random relationship. The value of

the correlation coefficient must be above 0.95 for it to be acceptable. Any value less than

this should be rejected.

26 CHAPTER FOUR

4.0 RESULT AND DISCUSSION

4.1 CRUDE FAT (%)

Crude fat refers to the crude mixture of fat soluble material present in a sample.

The food function soluble in polar solvent is generally reported crude fat or crude lipid

27 and may be fractioned into several groups on the basis of their chemical and physical properties of their hydrolysis products.

The result obtained for the crude fat percentage of Basella alba and Basella rubra are represented in Table 4.1

TABLE 4.1 Levels of Crude Fat (%) in Basella alba and Basella rubra

B.RUBR parameter B.ALBA A MEAN S/D CV% Crude Fat 3.38 4.27 3.83 0.629 16.5 Total Fat 2.70 3.42 3.06 0.503 16.5 E (kJ/100g) 100 126 113 18.6 16.5

B.Alba= Basella alba B. Rubra= Basella rubra S.D= Standard Deviation CV%= Coefficient of Variation E= Enegy (kJ/100g) The crude fat composition of Basella alba and Basella rubra as indicated in Table 4.1 above include 3.38 and 4.27 with mean and standard deviation of 3.83 and 0.63 respectively. Out of the two samples, result showed that Basella rubra has the highest crude fat content compare to Basella alba and with the value of 3.42. Consequently, Basella rubra is a better source of crude fat than Basella alba. Fat gives energy to the body system than twice of the energy protein and carbohydrate gives (Osborne and Vogt, 1998).

Table 4.2 Levels of Fatty Acids (%) Composition of Basella alba and Basella rubra

B. FATTY ACIDS B. Alba Rubra Mean S/D CV% C6:0 0.00 0.00 0.00 0.00 0.00 C8:0 0.00 0.00 0.00 0.00 0.00 C10:0 0.00 0.00 0.00 0.00 0.00 C12:0 0.00 0.00 0.00 0.00 0.00 C14:0 1.64 1.89 1.77 0.177 10.0 C14.1 ( cis-9) 0.00 0.00 0.00 0.00 0.00

28 C16:0 20 19.2 19.6 0.600 3.07 C16:1 (cis-9) 0.13 0.14 0.135 0.007 5.24 C18:0 4.31 3.73 4.02 0.410 10.2 C18:1 (trans-6) 0.005 0.011 0.008 0.004 53.0 C18:1 (cis-6) 0.18 0.202 0.191 0.02 8.14 C18:1 (trans-9) 0.0004 0.001 0.0007 0.0004 60.6 C18:1 (cis-9) 0.81 0.753 0.782 0.040 5.157 C18:1 (trans-11) 0.00 0.00 0.00 0.00 0.00 C18:2 (cis-9,13) 18.6 19.2 18.9 0.424 2.24 C18:2 (trans-9,12) 0.006 0.013 0.0095 0.005 52.1 C20:0 0.015 0.031 0.023 0.011 49.2 C18:3 (cis-6,9,12) 32.9 37.5 35.2 3.25 9.24 C20:1 (cis-11) 0.056 0.12 0.088 0.045 51.4 C18:3 (cis-9,12,15) 20.9 17.1 19.0 2.68 14.1 C20:2 (cis-11,14) 0.002 0.005 0.003 0.002 54.4 C22:0 0.014 0.029 0.022 0.011 49.3 C20:3 (cis-8,11,14) 0.109 0.091 0.1 0.013 12.7 C22:1 (cis-13) 0.005 0.01 0.0075 0.004 47.1 C20:3 (cis-11,14,17) 0.009 0.019 0.014 0.007 50.5 C20:4 (cis-5,8,11,14) 0.014 0 0.007 0.010 141 C22:2 (cis-13,16) 0.002 0.004 0.003 0.001 47.1 C24:0 0.002 0.004 0.003 0.001 47.1 Total 100 100 100 0.211 0.212 Total SFA 25.98 24.83 25.41 0.813 3.2 MUFA cis 1.187 1.23 1.208 0.030 2.51 trans 0.006 0.013 0.009 0.005 50.8 MUFA TOTAL 1.19 1.242 1.22 0.035 2.87 PUFA n-3 PUFA TOTAL 20.9 17.1 19.0 2.68 14.1 n-6 PUFA TOTAL 51.6 56.8 54.2 3.66 6.75 PUFA TOTAL 72.5 73.93 73.0 0.986 1.35 n-6/n-3 2.5 3.32 2.9 0.600 20.7

B.Alba= Basella alba B. Rubra= Basella rubra S.D= Standard Deviation, CV%= Coefficient of Variation Table 4.2 shows the result for fatty acids content. The saturated fatty acids include steric acid, palmitic acids, lauric acids etc. similarly monounsaturated fatty acids can be categorise to cis and trans monounsaturated fatty acids which include palmitolcic, octadecanonoic acid etc. Also polyunsaturated fatty acids can be classified into n-3 and n-

29 6 polyunsaturated fatty acids which include Arachidonic acid, linoleic acid, alpha- linolenic acid etc.

From Table 4.2, Steric acid was higher in Basella alba than Basella Ruba with the value of 4.31 to 3.73 with the average value of 4.02, in which Basella rubra has the lowest concentration with the value 3.72. Similarly from the table C22:0 has the lowest value ranging from 0.014-0.029 with an average of 0.02. similarly this table shows that there is higher concentration of n-3 PUFA in Basella alba (20.9) compared to the lower value (17.1) that is observe in Basella rubra. Where as, there is lower concentration of n-

6 PUFA observed in Basella alba with the value (51.6) compared to the higher concentration observed for Basella rubra with the value (56.6). The ratio of n-6 and n-3 indicate a higher value for Basella alba with the value (24.7), which is far higher to the value observed for Basella rubra with the value (3.32)

Table 4.3 Differences between the fatty acids composition of Basella alba and Basella rubra

FATTY ACIDS DIFFERENCES C6:0 0.00 C8:0 0.00 C10:0 0.00 C12:0 0.00 C14:0 -0.25 C14.1 ( cis-9) 0.00 C16:0 0.849 C16:1 (cis-9) -0.01 C18:0 0.58 C18:1 (trans-6) -0.006 C18:1 (cis-6) -0.022 C18:1 (trans-9) -0.0006 C18:1 (cis-9) 0.057 C18:1 (trans-11) 0 C18:2 (cis-9,13) -0.6 C18:2 (trans-9,12) -0.007 C20:0 -0.016 C18:3 (cis-6,9,12) -4.6 C20:1 (cis-11) -0.064

30 C18:3 (cis-9,12,15) 3.795 C20:2 (cis-11,14) -0.0025 C22:0 -0.015 C20:3 (cis-8,11,14) 0.018 C22:1 (cis-13) -0.005 C20: (cis-11,14,17) -0.01 C20:4 (cis-5,8,11,14) 0.014 C22:2 (cis-13,16) -0.002 C24:0 -0.002 TOTAL -0.299

These differences above in Table 4.3 show that six of the fatty acids are concentrated in Basella alba while sixteen of the fatty acids are concentrated in Basella rubra. From the result showed above shows that Basella rubra is more concentrated than

Basella alba in fatty acids composition.

Table 4.4 Levels of Phyto-sterol Composition (Mg/100g) of Basella alba and Basella rubra

PHYTO STEROL B.alba B.rubra mean SD CV% CHOLESTEROL 0.0006 0.0031 0.0019 0.0017 93.3 CHOLESTANOL 0.00004 0.0004 0.0002 0.0003 116 ERGOSTEROL 0.0019 0.0027 0.0023 0.0006 24.2 CAMPESTEROL 67.68 103.23 85.5 25.1 29.4 STIG-MASTEROL 8.23 11.43 9.83 2.26 23.0 5AVENASTEROL 52.62 53.48 53.1 0.608 1.146 SITOSTEROL 195.7 274 235.1 55.7 23.7 TOTAL 324.2 442.6 383 83.7 21.8

Table 4.4 above shows the sterol level (mg/100g) of oil of Basella alba and Basella rubra. Sitosterol shows the highest value of 235.09. Therefore, one of sitosterol derivative (β-Sitosterol) has been found to be reducing blood level of cholesterol and hyper-cholesterol. Cholesterol, cholestanol and Ergosterol are only finding in trace in all the two samples. Campesterol, Stigmasterol, Savenasterol are find in all the samples respectively, but varies from sample to sample. Campesterol has the highest value in

Basella rubra (103.23) and the lowest value in Basella alba (67.68), with the average

31 value 85.46. similarly stigmasterol has the lowest value in Basella alba (8.23) and a highest value in Basella rubra (11.43) with the average value of 9.83. Savernasterol in

Basella alba has the lowest value compared to Basella rubra which has the highest value

(53.48). Therefore Basella rubra is a better source of savernasterol of all the two samples. Stigmasterol may be useful in preventing certain Cancer, including overian prostrate, breast and colon cancer (wikipedia.com). They also help to lower cholesterol level, which causes cardiovascular diseases.

Obviously, the cholesterol level is very low in the two samples; therefore these samples cannot pose any risk to human in term of high cholesterol level that can cause cardiovascular diseases.

It has been shown that B. Ruba has the highest value of Campesterol

(103.23mg/100g) when compared to B. Alba with the value (67.83mg/100g). From this, one can infer that Basella rubra is highly concentrated in Campesterol than Basella alba.

Table 4.5 Differences between the Level of Sterol in Basella alba and Basella rubra

PHYTO STEROL DIFFERENCES CHOLESTEROL -0.0025 CHOLESTANOL -0.0004 ERGOSTEROL -0.0008 CAMPESTEROL -35.55 STIG-MASTEROL -3.2 5AVENASTEROL -0.86 SITOSTEROL -78.8 TOTAL -118.4 From the Table above, the result of the differences shows that Basella rubra is highly concentrated in all the sterol composition compared to the corresponding Basella alba.

Table 4.6 Levels of Phospholipids Composition (Mg/100g) of Basella alba and

Basella rubra

32 PHOSPHOLIPIDS B.alba B.rubra mean SD CV% PHOSPHATIDYLETHANOLAMINE 463.8 557 511 66.2 13.0 PHOSPHATIDYLCHOLINE 625.8 719.6 672.7 66.3 9.86 PHOSPHATIDYLSERINE 279.7 336.2 308 40.0 13.0 LYSOPHOSPHATIDYLCHOLINE 0.724 1.906 1.315 0.836 63.6 PHOSPHATIDYLINSITOL 308.8 305.7 307.2 2.21 0.718 TOTAL 1678.8 1921 1800 171.2 9.51

Table 4.6 shows various phospholipids level of Basella alba and Basella rubra, of all the various components of phospholipids present in the two samples, Phosphatidylcholine has the highest concentration which varies from sample to sample within the range of

625.8-719.6 mg/100g with an average value of 672.7mg/100g. The second is phosphatidylethanolamine with a value range from 463.8-557.46mg/100g with an average value of 510.63mg/100g. Also phosphotidylinsitol with the value range from

308.8-305.68mg/100g with an average of 307.24mg/100g is third whose concentration is high in the two samples. Others are present lower concentration. But there is an observation that all the phospholipids component has a higher concentration value in

Basella rubra except in phosphatidylinsitol with the value (305.68) to Basella alba which has the highest value (308.8). Phosphatidylcholine is said to be present at high concentration in egg yolks (Ikekoronye and Ngoddy, 1985). Phosphatidylcholine

(Lecithin) are important phospholipids which are commercially useful in the production of chocolate candies, as well in the production of emulsifying agent. It is also the body main emulsifier produced by the liver and released into the small intestine via the gall bladder during digestion (Whitney et all., 1994).

Table 4.7 Differences between the Phospholipids Composition of Basella alba and Basella rubra PHOSPHOLIPIDS DIFFERENCES PHOSPHATIDYLETHANOLAMINE -93.7

33 PHOSPHATIDYLCHOLINE -93.8 PHOSPHATIDYLSERINE -56.6 LYSOPHOSPHATIDYLCHOLINE -1.18 PHOSPHATIDYLINSITOL 3.12 TOTAL -242

Table 4.7 above shows that Basella alba has the lowest concentration of phospholipids content compared to the higher concentration found in Basella rubra

CHAPTER FIVE 5.0 CONCLUSION AND RECOMMENDATION

5.1 CONCLUSION

The work has shown that Basella rubra is highly rich in Crude fat of (4.27%) compare to Basella alba which has the value of (3.38%) in its crude fat composition.

Also the energy generated by Basella alba is lesser (100kJ/100g) than the Basella rubra

34 with the value (126 kJ/100g). Basella alba has the highest value (25.98) while Basella rubra has the lowest value (24.83) of saturated fatty acids.

The mono-unsaturated fatty acid cis composition of Basella alba and Basella rubra is very close in range with the value of (1.19) and (1.23) respectively, but there is lower value obtained for mono-unsaturated fatty acid trans composition for Basella alba and Basella rubra of (0.006) and (0.013).

Basella rubra has the highest composition of n-6 polyunsaturated fatty acids

(total) of (56.8%) and Basella alba with the lowest value of (51.6%) present of polyunsaturated fatty acid. The n-3 value is very high in Basella alba with the value

(20.9%) compared to Basella rubra with the value (19.0). n-3 being the important essential fatty acid is essential vital because it fight cardiovascular diseases.

The sterol value is high in each of the sample under sitosterol with the value

274.5mg/100g in Basella rubra and the lowest value of 195.7mg/100g of Basella alba.

The phospholipids value is also higher in Basella rubra with the value

1921mg/100g with the lowest value of 1678.8mg/100g observed for Basella alba. At a close look, I observed that there is higher concentration of all the phospholipids except for lysophophstidylcholine with a very low value for both species.

5.2 RECOMMENDATION

The result suggested that Basella ubra is a little bit nutritional than Basella alba because of the n-6/n-3 value of 3.32 and 2.5 respectively, similarly the sterol values were slightly higher in Basella rubra and lower in Basella alba with the value 442.6 and

35 324.2mg/100g respectively. Also their phsospholipids value is higher in Basella rubra and also lower in Basella alba with the value 1921 and 1678.8mg/100g respectively.

I hereby recommend that more nutritional work should be carry out on Basella alba and Basella rubra in the area of proximate determination, vitamins and anti- nutritional factors.

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38