CHAPTER ONE

INTRODUCTION

1.1 Background of the Study

Plants are capable of producing and synthesizing diverse groups of

organic compounds and are divided into two major groups: primary

and secondary metabolites. Primary metabolites (proximates) are

metabolites that are directly involved in normal growth,

development, and reproduction. It usually performs a physiological

function in the organism (i.e. an intrinsic function) (Prins et al.,

2010). Primary metabolites are typically present in many

organisms or cells. It is also referred to as a central metabolite,

which has an even more restricted meaning (present in any

autonomously growing cell or organism). Some common examples

of primary metabolites include: lactic acid, and certain amino acids

(Prins et al., 2010).

Proximates are used in the analysis of biological materials as a

decomposition of a human-consumable good into its major

constituents. They are a good approximation of the contents of

packaged comestible goods and serve as a cost-effective and easy

verification of nutritional panels (Eurofins, 2013). Examples

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proximates are: Carbohydrates, Proteins, Lipid, Fiber, Ash,

Moisture, Fat, vitamins and minerals etc.

Carbohydrates are one of the most important food groups in the

diet. They provide essential elements that the body needs for

instant energy production and various vital functions (Pichon et al.,

2006). Carbohydrates are an important source of energy, forming

around 40% to 80% of our total energy intake. Low-carbohydrate

diets may miss the health advantages – such as increased intake of

dietary fiber afforded by high-quality carbohydrates found in

legumes and pulses, whole grains, fruits, and vegetables

(Seidelmann et al., 2018). Several health organizations recommend

carbohydrates should form the main source of energy over other

food groups for the following reasons: Carbohydrates provide easily

available energy for oxidative metabolism and energy production.

Carbohydrates help maintain a healthy blood sugar level. Unlike fat

and protein, a high carbohydrate intake is not linked to adverse

health effects, providing the carbohydrates are derived form a

variety of sources (Seidelmann et al., 2018).

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Sources of carbohydrates food sources that are rich in

carbohydrates include cereals, root crops or tubers, sugar, pulses

and legumes, vegetables, fruits and milk products. Grains and

vegetables such as rice, wheat, maize, barley, potatoes, yams,

cassava, and sweet potatoes are rich in starch. Nuts, legumes and

high-fibre grain or whole grains contain significant amounts of

dietary fibre, about a third of which is present as hemicellulose. The

most important carbohydrate is glucose, a simple sugar

(monosaccharide) that is metabolized by nearly all known

organisms. Glucose and other carbohydrates are part of a wide

variety of metabolic pathways across species: plants synthesize

carbohydrates from carbon dioxide and water by photosynthesis

storing the absorbed energy internally, often in the form of starch or

lipids. Plant components are consumed by animals and fungi, and

used as fuel for cellular respiration. Oxidation of one gram of

carbohydrate yields approximately 16 kJ (4 kcal) of energy, while

the oxidation of one gram of lipids yields about 38 kJ (9 kcal). The

human body stores between 300 and 500 g of carbohydrates

depending on body weight, with the skeletal muscle contributing to

a large portion of the storage (Maughan, 2013). Energy obtained

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from metabolism (e.g., oxidation of glucose) is usually stored

temporarily within cells in the form of ATP (Mehta, 2013).

Proteins are essential nutrients for the human body (Hermann,

2016). They are one of the building blocks of body tissue and can

also serve as a fuel source. As a fuel, proteins provide as much

energy density as carbohydrates: 4 kcal (17 kJ) per gram; in

contrast, lipids provide 9 kcal (37 kJ) per gram. The most important

aspect and defining characteristic of protein from a nutritional

standpoint is its amino acid composition (IM, 2005). Protein is a

nutrient needed by the human body for growth and maintenance.

Aside from water, proteins are the most abundant kind of molecules

in the body. Protein can be found in all cells of the body and is the

major structural component of all cells in the body, especially

muscle. This also includes body organs, hair and skin. Proteins are

also used in membranes, such as glycoproteins. When broken down

into amino acids, they are used as precursors to nucleic acid, co-

enzymes, hormones, immune response, cellular repair, and other

molecules essential for life. Additionally, protein is needed to form

blood cells (IM, 2005). Meat, dairy, eggs, soy, fish, whole grains, and

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cereals are sources of protein (Steinke, et al., 1992). Examples of

food staples and cereal sources of protein, each with a

concentration greater than 7%, are (in no particular order)

buckwheat, oats, rye, millet, maize (corn), rice, wheat, sorghum,

amaranth, and quinoa (Young and Pellett, 1994). Vegetarian

sources of proteins include legumes, nuts, seeds and fruits.

Vegetarian foods with protein concentrations greater than 7%

include soybeans, lentils, kidney beans, white beans, mung beans,

chickpeas, cowpeas, lima beans, pigeon peas, lupines, wing beans,

almonds, Brazil nuts, cashews, pecans, walnuts, cotton seeds,

pumpkin seeds, hemp seeds, sesame seeds, and sunflower seeds

(IM, 2005).

Lipid is a macrobiomolecule that is soluble in nonpolar solvents

(IUPAC, 1997). Non-polar solvents are typically hydrocarbons used

to dissolve other naturally occurring hydrocarbon lipid molecules

that do not (or do not easily) dissolve in water, including fatty acids,

waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E, and

K), monoglycerides, diglycerides, triglycerides, and phospholipids.

The functions of lipids include storing energy, signaling, and acting

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as structural components of cell membranes (Fahy et al., 2009;

Subramaniam et al., 2011). Lipids have applications in the cosmetic

and food industries as well as in nanotechnology (Mashaghi, et al.,

2013)

Fiber or roughage is the portion of plant-derived food that cannot be

completely broken down by human digestive enzymes (BNF, 2018)

Dietary fibers are diverse in chemical composition, and can be

grouped generally by their solubility, viscosity, and fermentability,

which affect how fibers are processed in the body (LPI, 2018)

Dietary fiber has two main components: soluble fiber and insoluble

fiber, which are components of plant foods, such as legumes, whole

grains and cereals, vegetables, fruits, and nuts or seeds (LPI, 2018;

USDA,NALAS,IM,FNS, 2005). A diet high in regular fiber

consumption is generally associated with supporting health and

lowering the risk of several diseases (LPI, 2018; Veronese et al,

2018). Soluble fiber (fermentable fiber or prebiotic fiber) are fiber

which dissolves in water. They are generally fermented in the colon

into gases and physiologically active by-products, such as short-

chain fatty acids produced in the colon by gut bacteria. Examples

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are beta-glucans (in oats, barley, and mushrooms) and raw guar

gum. Psyllium a soluble, viscous, non-fermented fiber is a bulking

fiber that retains water as it moves through the digestive system,

easing defecation. Soluble fiber is generally viscous and delays

gastric emptying which, in humans, can result in an extended

feeling of fullness (LPI, 2019). Inulin (in chicory root), wheat

dextrin, oligosaccharides, and resistant starches (Keenan et al.,

2015), (in legumes and bananas), are soluble non-viscous fibers

(LPI, 2019). Regular intake of soluble fibers, such as beta-glucans

from oats or barley, has been established to lower blood levels of

LDL cholesterol, a risk factor for cardiovascular diseases (Veronese

et al., 2018). Insoluble fiber which does not dissolve in water that

is inert to digestive enzymes in the upper gastrointestinal tract.

Examples are wheat bran, cellulose, and lignin. Coarsely ground

insoluble fiber triggers the secretion of mucus in the large intestine,

providing bulking. Finely ground insoluble fiber does not have this

effect and can actually have a constipating effect.[2] Some forms of

insoluble fiber, such as resistant starches, can be fermented in the

colon (Lockyer and Nugent, 2017)

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Ash is one of the components in the proximate analysis of biological

materials, consisting mainly of salty, inorganic constituents. Ash

also refers to all non-aqueous, non-gaseous residues that remain

after something burns. It includes metal salts which are important

for processes requiring ions such as Na+ (Sodium), K+ (Potassium),

and Ca2+ (Calcium). It also includes trace minerals which are

required for unique molecules, such as chlorophyll and hemoglobin

(IUPAC, 2006).

Ash content determination is the process of mineralization for pre-

concentration of trace substances prior to a chemical analysis,

(IUPAC, 2006) such as chromatography, or optical analysis, such as

spectroscopy.

Moisture is simply water diffused in a relatively small quantity.

Nearly all materials contain at least a diminutive volume of

moisture as a component of the molecular makeup. Moisture is a

given in the mass of materials, however the relative percentage is

dynamic and therefore not constant (Phillips, 2017). Moisture has

different effects on different products, influencing the final quality

of the product. Wood pellets, for instance, are made by taking

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remainders of wood and grinding them to make compact pellets,

which are sold as a fuel (Phillips, 2017). They need to have relatively

low water content for combustion efficiency. The more moisture that

is allowed in the pellet, the more smoke that are released when the

pellet is burned (Phillips, 2017). Water transports other nutrients to

cells, carries wastes away, aids digestion and more. It makes up

more than half your weight. Sources: water; juices and other

beverages; soups and many "solid" foods (fruits, vegetables, breads,

etc.). The human body is made up of over 70% water. Our blood is

more than 80%‚ our brain over 75%‚ and the human liver is

amazing 96% water (Phillips, 2017). Water performs the following

functions in the body:

1. Works to keep muscles and skin toned

2. Aids in weight loss

3. Transports oxygen & nutrients to cells

4. Eliminates toxins & waste from the body

5. Regulates body temperature.

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Vitamins are organic molecule (or a set of molecules closely related

chemically, i.e. vitamers) that is an essential micronutrient which

an organism needs in small quantities for the proper functioning of

its metabolism. Vitamins also remains essential nutrients for the

healthy maintenance of the cells, tissues, and organs that make up

a multi-cellular organism; they also enable a multi-cellular life form

to efficiently use chemical energy provided by food it eats, and to

help process the proteins, carbohydrates, and fats required for

cellular respiration (Bender, 2003). Vitamins are classified as either

water-soluble or fat-soluble. In humans there are 13 vitamins: 4

fat-soluble (A, D, E, and K) and 9 water-soluble (8 B vitamins and

vitamin C). Water-soluble vitamins dissolve easily in water and, in

general, are readily excreted from the body, to the degree that

urinary output is a strong predictor of vitamin consumption

(Fukuwatari and Shibata, 2008). Because they are not as readily

stored, more consistent intake is important (Bellows and Moore,

2008). Fat-soluble vitamins are absorbed through the intestinal

tract with the help of lipids (fats). Vitamins A and D can accumulate

in the body, which can result in dangerous hypervitaminosis. Fat-

soluble vitamin deficiency due to malabsorption is of particular

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significance in cystic fibrosis (Maqbool and Stalling, 2008). They

also play important roles in bodily functions such as metabolism,

immunity and digestion.

mineral is a chemical element required as an essential nutrient by

organisms to perform functions necessary for life (Zoroddu et al.,

2019; Berdanier et al.,2013). However, the four major structural

elements in the human body by weight (oxygen, hydrogen, carbon,

and nitrogen), are usually not included in lists of major nutrient

minerals (nitrogen is considered a "mineral" for plants, as it often is

included in fertilizers). These four elements compose about 96% of

the weight of the human body, and major minerals (macrominerals)

and minor minerals (also called trace elements) compose the

remainder. They are needed in the buildup and function of

important biomolecules in the human body. Although, minerals are

not a source of energy in the body but they are necessary for

the maintenance of normal bio-chemical processes in the body

(Zhao et al. 2016). These Minerals are classified into two; macro and

micro (trace) minerals. Macro-minerals are nutritionally important

minerals such as sodium, calcium, phosphorus, magnesium

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and potassium. They are classified as macro because the average

adult daily requirement is greater than 100 mg/day (Akinyeye et al.

2010). Micro elements are essential group of minerals, which are

needed, in small quantity for the day-to-day metabolic processes in

man. Trace minerals include iron, copper, zinc, iodine, manganese

etc. They are regarded, as trace elements because their daily

requirement should be below 100 mg, above which can be toxic to

health. However, the deficiency of any of these trace elements can

lead to serious health challenges (Akinyeye et al. 2010).

Phytochemistry is the study of phytochemicals, which are chemicals

derived from plants. Phytochemists strive to describe the structures

of the large number of secondary metabolites found in plants, the

functions of these compounds in human and plant biology, and the

biosynthesis of these compounds (Hasler, 2014). Plants synthesize

phytochemicals for many reasons, including protecting themselves

against insect attacks and plant diseases. The compounds found in

plants are of many kinds, but most can be grouped into four major

biosynthetic classes: alkaloids, phenylpropanoids, polyketides, and

terpenoids (Hasler, 2014). Phytochemistry can be considered a

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subfield of botany or chemistry. Activities can be led in botanical

gardens or in the wild with the aid of ethnobotany. Phytochemicals

are biologically active, naturally occurring chemical

compounds found in plants, which provide health benefits for

humans as medicinal ingredients and nutrients (Hasler, 2014).

They protect plants from disease and damage, and also

contribute to the plant’s colour, aroma and flavour. In

general, the plant chemicals that protect plants from

environmental hazards such as pollution, stress, drought, UV

exposure and pathogenic attack are called as phytochemicals

(Gibson et al., 1998; Mathai, 2000). Recently, it has been clearly

shown that they also have roles in the protection of human

health, when their dietary intake is significant (Hayer, 2015). Till

date over 4,500 phytochemicals have been reported and are

classified on the basis of their protective functions, and physical

and chemical characteristics, amongst these about 350

phytochemical have been studied in detail (Koche et al., 2010).

Phytochimicals are found in fruits, vegetables, legumes, whole

grains, nuts,seeds, fungi, herbs and spices (Baser and (Buchbauer,

2010). Broccoli, cabbage, carrots, onions, garlic, whole wheat bread,

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tomatoes, grapes, cherries, strawberries, rasp-berries, beans and

soy foods are also the common sources of phytochemicals

(Moorachian, 2000). Phytochemicals accumulate in different parts

of the plants, such as in the root, stem, leaf, flower, fruit and

seed (Costa et al., 1999). Many phytochemicals, particularly

the pigment molecules like anthocyanines and flavonoids,

are often concentrated in the outer layers of the various plant

parts like leaves and fruits of vegetables. Phytochemcials vary

from plant to plant depending upon the variety, climatic

growing conditions (Rao, 2003). These compounds have biological

properties such as antioxidant activity, anti-microbial effect,

modulation of detoxification enzymes, stimulation of the immune

system, decrease of platelet aggregation and modulation of hormone

metabolism and anticancer property (Hamburger and

Hostettmann, 1991). At the same time, Horborne (1999) reported

the anti-nutritional properties of some plant chemicals.

The exact classification of phytochemicals has not been given

so far, because of their diverse forms and structures

classically, the phytochemicals have been classified as primary

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or secondary metabolites, depending on their role in plant

metabolism. Primary metabolites include the common sugars,

amino acids, proteins, purines and pyrimidines of nucleic

acids, chlorophylls etc. Secondary metabolites are the remaining

plant chemicals such as alkaloids, terpenoids, flavonoids,

tannins, lignans, plant steroids, curcumines, saponins, phenolics

, and cardiac glucosides (Hahn, 2015; Ramawat et al., 2009).

Flavonoids are water soluble polyphonelic molecules containing 15

carbon atoms. They are the largest group of plant phenols and also

the most studied one (Dai and Mumper, 2010). They are

polyphenolic compounds that are ubiquitous in nature and occur

as aglycones, glucosides and methylated derivatives. More than

4,000 flavonoids have been recognized, many of which occur in

vegetables, fruits and beverages like tea, coffee and fruit drinks

(Pridham, 2010). Flavonoids appear to have played a major role in

successful medical treatments in ancient times, and their use has

persisted up to now. Most flavonoids occur naturally associated

with sugar in conjugated form and within any one class, may be

characterized as monoglycosidic, diglycosidic etc.

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Phenolic compounds are the largest category of phytochemicals

and are most widely distributed in the plant kingdom

(Walton et al., 2003). Phenolics are hydroxyl group (-OH)

containing classof chemical compounds where the (-OH) group is

bonded directly to an aromatic hydrocarbon group. Phenol

(C6H5OH) is considered the simplest class of this group of

natural compounds. Being a secondary metabolite, they have an

important role as defense compounds. Phenolics exhibit several

properties beneficial to humans and its antioxidant properties

are important in determining their role as protecting agents against

free radical-mediated disease processes. The three most important

groups of dietary phenolics are flavonoids, phenolicacids and

polyphenols.

Tannins (or tannoids) are a class of astringent, polyphenolic

biomolecules that bind to and precipitate proteins and various other

organic compounds including amino acids and alkaloids. Tannin

compounds are widely distributed in many species of plants, where

they play a role in protection from predation (including as

pesticides) and might help in regulating plant growth (Ferrell et

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al .,2006). The astringency from the tannins is what causes the dry

and puckery feeling in the mouth following the consumption of

unripened fruit, red wine or tea (McGee and Harold, 2004). Tannins

are an important ingredient in the process of tanning leather.

Tanbark from oak, mimosa, chestnut and quebracho tree has

traditionally been the primary source of tannery tannin, though

inorganic tanning agents are also in use today and account for 90%

of the world's leather production (Marion and Roy Thomson, 2006).

Tannins comprise a large group of natural products widely

distributed in the plant kingdom. They have a great structural

diversity, but are usually divided into two basic groups: the

hydrolyzable type and the condensed type. Hydrolyzable tannins

include the commonly occurring gallic acid and the ellagic acid.

Hydrolyzable tannins are readily degraded into smaller molecules

and are found in many plant species but particularly in high

concentrations in nutgalls growing on Rhussemialata(Chinese and

Korean gallotannins) and Quercusinfectoria (Turkish and Chinese

gallotannins), the seedpods of Caesalpiniaspinosa (Tara tannins),

and the fruits of Terminaliachebula. They react with proteins to

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produce the typical tanning effect of leather (Muller and Harvey,

2009). Medicinally, this is important for treatment of inflamed or

ulcerated tissues. They also contribute most of the astringent

property that is noted when drinking tannin containing beverages.

The condensed tannins, also known as proanthocyanidins, are

much more resistant to decomposition and merely yield polymers or

precipitates when acidified. The basic monomers of condensed

tannins are epicatechin and catechin (identical except for

orientation of the molecules) (Muller and Harvey, 2009)

These are then extended by the successive addition of similar

phenol units to produce polymers (polyphenols) (Subhuti.,2003).

Although both types of tannin have been used to treat diseases in

traditional medicine, the hydrolyzable tannins have long been

considered official medicinal agents in Europe and North America.

They have been included in many pharmacopoeias. In the older

editions in particular, they are specifically referred to as tannic acid

and were recommended for the treatment of inflammation and

ulceration, including topical application for skin diseases and

internally for intestinal ulceration and diarrhea. The condensed

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tannins also have important medicinal roles as stable and potent

antioxidants.

Alkaloids are natural products that contain heterocyclic nitrogen

atoms and are always basic in character. The name of alkaloids

derives from the ‘alkaline’ nature and it was used to describe any

nitrogen-containing base (Muller-Harvey, 1999). Alkaloids occur in

many different species in numerous genera and families of vascular

plants as well as in certain species of fungi. It has been estimated

that some fifteen percent or more of all vascular plants contain

alkaloids. A number of amines produced by animals possess

physical and chemical properties rather similar to those of

alkaloids. By traditions and conventions, these animals’ amines are

generally not considered as alkaloids. The occurrence of alkaloids in

different plant organs and tissues and their relationship to aspects

of the physiology of the plants are interesting part of alkaloids.

There are various classes of alkaloids according to the heterocyclic

ring system they contain are listed below. Pyrrolidine alkaloids-

These contain pyrrolidine (tetrahy-dropyrrole) ring system. For

example, hygrine found in leaves of Erythroxylumspp. &

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Leonotisspp. Pyridine alkaloids. These have piperidine

(hexahydropyridine) ring system. For example, coniine, piperine

and isopelletierine. Pyrrolidinepyridine alkaloids. These contain the

heterocyclic ring system with pyrrolidi-nepyridine. For example

myosmine, nicotine alkaloid found in tobacco

(Nicotianatabaccum).Pyridine-piperidine alkaloids. This family of

alkaloids contains a pyridine ring system joined to a

piperidine ring system. For example, anabasine alkaloid from

Anabasis aphyllan. Quinoline alkaloids-These have the basic

heterocyclic ring system quinoline. For example, quinine

Koche et al. 2010 occurs in the bark of cinchona tree.

Isoquinoline alkaloids-They contain heterocyclic rig system

isoquinoline. For example, opium alkaloids like narcotine,

papaverine, morphine,codeine, and heroine. Alkaloids are

significant for the survival of plant because they ensure their

protection against micro-organisms (antibacterial and antifungal

activities), insects and herbivores (feeding deterrents) and also

against other plants by means of allelopathy (Molineux et al.,

1996). The use of alkaloids containing plants as dyes,

spices, drugs or poisons can be traced back almost to the

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beginning of civilization. Alkaloids have many pharmacological

activities including anti-hypertensive effects (many indole

alkaloids), anti-arrhythmic effect (quini-dine, spareien), anti-

malarial activity (quinine), and anti-cancer actions

(dimericindoles, vincristine, vinblastine). These are just a few

examples illustrating the great economic importance of this

group of plant constituents. Some alkaloids have stimulant

property as caffeine and nicotine, morphine are used as the

analgesic and quinine as the antimalarial drug (Wink et al.,

1998).

Terpenoids comprises of natural products which have been derived

from five-carbon isoprene units. Most of the terpenoids have

multi cyclic structures that differ from one another by

their functional groups and basic carbon skeletons. These

types of natural lipids can be found in every class of living

things and therefore considered as the largest group of naturally

occurring secondary metabolites (Elbein et al., 1999). Many of

these are commercially interesting because of their use as

flavours and fragrances in foods and cosmetics (Horborne and

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Tomas Barberan,1991). Terpenes are widespread in nature,

mainly in plants as constituents of essential oils. Their building

block is the hydrocarbon isoprene, CH2=C(CH3)-CH=CH2.

Hemiterpenoids consists of a single isoprene unit. The only

hemiterpene is the isoprene itself, but oxygen-containing

derivatives of isoprene such as isovalericacid and prenol are

also classified as hemiterpenoids. Monoterpenoids have two

isoprene units. Monoterpenes may be of two types linear (acyclic)

or rings containing e.g. Geranyl pyrophosphate, Eucalyptol, Limon-

ene, Citral, Camphor and Pinene. Sesquiterpenes have three

isoprene units e.g. Artemisinin, Bisabolol and Fernesol, cyclic

compounds, such as Eudesmol found in Eucalyptus oil.

Saponins are steroid or triterpenoid glycosides that are present in

many feedstuffs. They have a bitter taste, can form foams in

aqueous solutions and haemolyse red blood cells. They are known

to depress growth performance in both poultry and swine. Their

antinutritional properties seem related to their ability to form

complexes with sterols, in particular those in membranes of animal

cells (Puupponen et al., 2008). This appears to result in increased

22
permeability of the intestinal mucosa. Poultry, compared to other

monogastrics, are more sensitive to saponins. Significant saponin

levels are present in alfalfa meal with minor levels in other legumes

such as soya beans, rapeseed and various varieties of peas (Niba,

2003). In general saponins are of minor concern in monogastric

animals because they are present at only law levels in common

feedstuffs.

The term saponin is derived from (Saponariavaccaria Quillajasapon

aria), a plant, which abounds in saponins and was once used as

soap. Saponins therefore possess ‘soaplike’ behaviour in water, i.e.

they produce foam. On hydrolysis, an aglycone is produced, which

is called sapogenin. There are two types of sapogenin: steroidal and

triterpenoidal. Usually, the sugar is attached at C-3 in saponins,

because in most sapogenins there is a hydroxyl group at C-3.

Quillajasaponariais known to contain toxic glycosides quillajic acid

and the sapogeninsenegin. Quillajic acid is strenutatory and

senegin is toxic. Senegin is also present in Polygala senega.

Saponins are regarded as high molecular weight compounds in

which, a sugar molecule is combined with triterpene or steroid

23
aglycone. There are two major groups of saponins and these

include: steroid saponins and triterpenesaponins. Saponins are

soluble in water and insoluble in ether, and like glycosides on

hydrolysis, they give aglycones. Saponins are extremely poisonous,

as they cause heamolysis of blood and are known to cause cattle

poisoning (Kay, 2007). They possess a bitter and acrid taste,

besides causing irritation to mucous membranes. They are mostly

amorphous in nature, soluble in alcohol and water, but insoluble in

non-polar organic solvents like benzene and n-hexane.

Saponins are also important therapeutically as they are shown to

have hypolipidemic and anticancer activity. Saponins are also

necessary for activity of cardiac glycosides. The two major types of

steroidal sapogenin are diosgenin and hecogenin. Steroidal

saponins are used in the commercial production of sex hormones

for clinical use. For example, progesterone is derived from

diosgenin. The most abundant starting material for the synthesis of

progesterone is diosgenin isolated from Dioscorea species, formerly

supplied from Mexico, and now from China (Sarker and Nahar,

2007). Other steroidal hormones, e.g. cortisone and hydrocortisone,

24
can be prepared from the starting material hecogenin, which can be

isolated from Sisal leaves found extensively in East Africa (Sarker

and Nahar, 2007).

Steroids are biological active organic compounds with four rings

arranged in a specific molecular configuration. Steroids have two

principal biological functions: as important components of cell

membranes which alter membrane fluidity; and as signaling

molecules. Hundreds of steroids are found in plants, animals and

fungi. All steroids are manufactured in cells from the sterols

lanosterol (opisthokonts) or cycloartenol (plants). Lanosterol and

cycloartenol are derived from the cyclization of the triterpene

squalene (Lanosterol biosynthesis, 2006). Steroids are also referred

to as ‘cardiac glycosides’ are one of the most naturally occurring

plant phyto-constituents that have found therapeutic applications

as arrow poisons or cardiac drugs. The cardiac glycosides are

basically steroids with an inherent ability to afford a very specific

and powerful action mainly on the cardiac muscle when

administered through injection into man or animal. Steroids

(anabolic steroids) have been observed to promote nitrogen

25
retention in osteoporosis and in animals with wasting illness

(Maurya et al., 2008).

Cardiac glycosides are a class of organic compounds that increase

the output force of the heart and decrease its rate of contractions by

acting on the cellular sodium-potassium ATPase pump (Patel,

2016). Their beneficial medical uses are as treatments for

congestive heart failure and cardiac arrhythmias; however, their

relative toxicity prevents them from being widely used (Ambrosy et

al., 2014). Most commonly found as secondary metabolites in

several plants such as foxglove plants, these compounds

nevertheless have a diverse range of biochemical effects regarding

cardiac cell function and have also been suggested for use in cancer

treatment (Riganti et al., 20110). Cardiac glycosides can be more

specifically categorized based on the plant they are derived from, as

in the following list. For example, cardenolides have been primarily

derived from the foxglove plants Digitalis purpurea and Digitalis

lanata, while bufadienolides have been derived from the venom of

the cane toad Bufo marinus, from which they receive the “bufo”

portion of their name (Watanabe, et al., 2003).

26
1.2. Statement of the Problem

A. cathartica is claimed to have a lot of economic value such as

medicinal, and pesticidal values. These claims have not been clearly

justified. Hence the study on assessment of the chemical

constituents of the leaves of allamanda cathartica will significantly

justify the phytochemical analysis of allamanda cathartica uses as

herbs and in alternative and environmentally friendly means to

tackle healt issues.

1.3. Objective of the Study

The objective of this study is to assess the chemical constituents of

the leaves of Allamand cathartica

1.4. Significance of the Study

At the successful completion of this work, the chemical constituents

of the leaves of allamanda cathartica will be exposed with the

medicinal values of these plants which will be beneficial in the

following groups:

1. Therapeutic industry: In traditional systems of medicine,

different parts (leaves, stem, flower, root, and even whole

27
 plant) of Allamand acathartica Linn (Golden trumpet wine)

showed its efficacy in treatment of different diseases.

2. Farmers: farmers would benefits from the outcome of the

research because the chemical constituents of the plants

showed pesticidal properties, which means it can be used in

pest control. It can also be used as a feed for livestocks

3. Industry: Industries like pharmaceutical would greatly

benefits from the study because the extract from this natural

plant can be used in production of antidotes, antiviral drugs,

anti-inflammatory drugs and anti-malaria drugs etc; chemical

industry would also benefits from the study because the plant

can be used in production of hydrocarbons.

4. The researcher: It would be a great benefit to researcher

because at the successful completion of this work, the

researcher would be exposed to a wide knowledge of the plant

and its great value in the society.

1.4 Scope of the Study:

The scope of this study was the Assessment of the chemical

constituents of the leaves of Allamanda cathartica.

28
1.5 Research Questions:

1. What are the proximate assessed from the leaves of Allamanda

cathartica?

2. What are the percentage compositions of proximate assessed

from the leaves of Allamanda cathartica?

3. What are Phytochemicals assessed from the leaves of

Allamanda cathartica?

4. What are the percentage compositions of Phytochemicals

assessed from the leaves of Allamanda cathartica?

CHAPTER TWO

LITERATURE REVIEW

2.1. Review of Study Plant

29
Allamanda is a genus of flowering plants in the family Apocynaceae.

They are native to the Americas, where they are distributed

from Mexico to Argentina. Some species are familiar as ornamental

plants cultivated for their large, colorful flowers (Fakhrudin et al.,

2013). The members of this family are found throughout the world,

but they are more commonly met with in the tropical regions. There

is a great variation in the habit of the plants of this family. They

may be herbs, erect or twining shrubs or trees. The leaves are

simple, petiolate, usually opposite decussate. In rare cases the

leaves are alternate or even whorled (e.g., in Nerium odorum,

Alstonia, etc.) Usually the leaves are exstipulate and very rarely they

may be stipulate. Usually the inflorescence is of cymose type. It is

very rarely solitary as in Vinca. In Carissa, the flowers are found to

be arranged in corymbose cymes. In Plumeria, the flowers are

arranged in terminal cymes. In Alstonia, the flowers are found to be

arranged in umbellate branched panicled cymes. In Rauvolfia, the

flowers are arranged in umbellate or corymbose cymes. The flowers

are pedicellate, bracteate, bracteolate, hermaphrodite,

actinomorphic, regular, sometimes slightly, zygomorphic, complete,

hypogynous and pentamerous. In rare cases the flowers are

30
tetramerous with reduction to two in the pistil. Usually it consists

of five sepals, gamosepalous. The calyx is generally divided almost

to the base. The aestivation is quincuncial. Usually the corolla

consists of five petals, gamopetalous. It is generally salver or funnel

shaped. The corolla tube usually possesses hairy appendage or

scales. The aestivation is contorted. It consists of five stamens

alternating with the petals. The stamens are situated on the tube or

the throat of the corolla (i.e., epipetalous). The filaments are short;

anthers introrse, polyandrous or connate and often adhere to the

stigma. The antherlobes are sometimes empty at their base and

prolonged into spines. It consists of two carpels. The carpels may be

free (apocarpous) or connate (syncarpous); superior, sometimes

partly inferior as in Plumeria. The style is simple and the stigma is

thick and often bilobed. Rarely the number of carpels exceeds, i.e.,

3 to 5. Usually a nectar secreting disc is situated beneath the

gynoecium. In syncarpous gynoecium, the ovary may be unilocular

with parietal placentation or it may be bilocular with axile

placentation. In the case of separate ovaries the placentation is

marginal. The ovary is superior or half-inferior. Numerous ovules

are found to be situated on parietal placentas or in two chambered

31
ovaries on marginal walls. In the case of free ovaries, the fruit is a

pair of follicles. Sometimes the fruits of separate ovaries are fleshy

and indehiscent, or may be one seeded, e.g., Cameraria. In the case

of syncarpous ovary, usually the fruit is indehiscent, fleshy and

berry-like, e.g., in Landolphia. In Cerbera, it may be a drupe. This

fruit is coconut like and distributed by means of water currents. In

certain genera, possessing syncarpous ovaries a two-valved capsule

is found, e.g. In Aspidosperma and Allamanda. In dry fruits the

seeds are generally winged, e.g. In Plumeria. Sometimes the seed

bears a tuft of hairs at the base, e.g. In Kickxia, and sometimes at

both ends, e.g., In Strophanthus. The embryo is straight, with or

without endosperm.

Economic Importance Apocynaceae

The family is of little economic value. Some plants are grown as

ornamentals, while some possess medicinal properties.

1. Alstonia scholaris; This is a small tree grown as an

ornamental. Its wood is quite light and used for carvings. In

Myanmar, the black boards are prepared from its wood. The bark

possesses medicinal properties, which is used for diarrhoea and

32
dysentery. The wood of Alstonia scholaris has been recommended

for the manufacture of pencils, as it is suitable in nature and the

tree grows rapidly and is easy to cultivate (Tonanont, 2004).

2. Beaumontia grandiflom: It is a climbing shrub, usually grown

as an ornamental for its large, white fragrant flowers. It is a small

genus of evergreen woody vines in the Apocynaceae.

3. Beaumontia jerdoniana: This is also grown as an ornamental.

4. Anodendron paniculatum: It is a small genus of evergreen

woody plant in the Apocynaceae.

5. Carissa carandas: is a species of flowering shrub in the family

Apocynaceae. It produces berry-sized fruits that are commonly used

as a condiment in Indian pickles and spices. It is a hardy, drought-

tolerant plant that thrives well in a wide range of soils. Common

names in English include Bengal currant, Christ's thorn, Khare

CP(2007) carandas plum and karanda

Important genera in the family Apocynacea include:

Allamanda, Alstonia, Amsonia, Angadenia,

Apocynum, Araujia, Asclepias, Calotropis,

33
Carissa,Amsonia,Catharanthus, Cryptostegia, Carissa, Pentalinon,

Plumeria, Mandevilla,Metastelma, Nerium, Thevetia, Vinca.

Allamanda is a genus of flowering plants in the family Apocynaceae.

They are native to the Americas, where they are distributed from

Mexico to Argentina. Some species are familiar as ornamental

plants cultivated for their large, colorful flowers. Most species

produce yellow flowers; A. blanchetii bears pink (de Souza-Silva,

2009). Plants of the genus are evergreen trees, shrubs, or vines

(Fakhrudin et al., 2013). They contain a white latex. The leaves are

opposite or arranged in whorls of up to 5. The blades are generally

oval and smooth-edged, and some are leathery or lightly hairy. The

inflorescence is a compound cyme. The flower has five lobed sepals

and a bell- or funnel-shaped corolla of five petals, yellow in most

species. The fruit is a schizocarp containing two to four seed

(Fakhrudin et al., 2013).

Some species in the genus Allamanda includes; Allamanda

angustifolia, Allamanda blanchetii Allamanda calcicola, Allamanda

cathartica,Allamanda doniana,Allamanda laevis,Allamanda

34
martii,Allamanda nobilis, Allamanda oenotherifolia, Allamanda

polyantha, Allamanda puberula, Allamanda schottii, Allamanda

setulosa. Allamanda thevetifolia. Allamanda weberbaueri etc.

The species is a shrub or woody vine, clambering or sometimes

twining, much branched, 2-8 metres in length, with abundant

milky latex. Stems grayish, cylindrical, glabrous or puberulous.

Leaves in whorls of 3 or 4; blades 8-13 × 1.5-3.5 cm, oblong,

elliptical, coriaceous, the apex acuminate, the base acute, the

margins undulate and revolute; upper surface glabrous, dark green,

shiny, with a prominent mid-vein; lower surface yellowish green,

with the mid-vein thickened; petioles 5-10 mm long; stipules

transformed into 4 small intrapetiolar glands (Acevedo-Rodríguez,

2013). Flowers arranged in axillary cymes. Calyx greenish, 5

lanceolate sepals, 12-18 mm long; corolla infundibuliform, yellow,

the tube 7-9 cm long, the limb approximately 8 cm in diameter,

with five rounded, revolute lobes. Capsules ellipsoid, covered with

nusmerous spines, infrequent; seeds numerous, oval, compressed,

1.2-1.5 cm long, with a discolorous, wing-like margin (Acevedo-

Rodríguez, 2013).

35
Economic importance of Allamanda cathartica

In some tropical countries, plant parts such as leaves, flower, root,

and stem are used in traditional medicine for giving protection to

human from ancient time (Acevedo-Rodriguez, 2005). Different

ethnopharmacological reports suggest that the roots are used

against jaundice, malaria, and an enlarged spleen. The flowers act

as a laxative, and also used as an antibiotic against Staphylococcus

spp. In Trinidad, it is also used for treating malaria and jaundice

(Ghosh et al., 2018). In Guiana, the latex is employed for colic and

also used as purgative in Southeast Asia. Leaves are used against

cough and headaches. Allamanda cathartica flower extracts show

potential anti-inflammatory and anti-oxidant activities. Plumieride,

the primary compound of flower extracts has potential anti-

inflammatory activities. It has also been recorded as a medicinal

plant for the medication of various conditions such as feverish

infections like dysentery gonorrhea, and hepatitis. The leaf extracts

have visible anti-fertility potency in male, membrane stabilizing

activity and anti-microbial property against multiple drug-resistant

36
pathogens. The milky sap is also accepted for its anti-bacterial and

possibly anti-cancer activities.

Lots of works has been carried out in the areas of proximate

analysis of plant materials.

Okwuonu et al., (2017) researched on proximate constituents of

Aspilia africana (Wild sunflower) flowers, they made a remark that

proximate analysis is an analysis carried out scientifically to

partition the right amounts of both nutrients and non-nutrients

(hazardous substances) within an organic material into categories

based on common chemical properties. It is employed by many

researchers for the purpose of studying things like animal feed, bio-

fuels and coal. On the other hand, food, supplements and plants

products for human consumption are tested to ascertain their

nutrient levels, found out that the analysis of the dry crude flowers

of Aspilia Africana have 8.47% moisture content, 17.53% Protein,

2.37% fat, 9.17% crude ash, 9.10% crude fibre and 53.4%

carbohydrate. These results are comparable to the findings of

Uduak and Umana, (2013) who reported moisture content of 15.7%,

protein content of 7.87%, fat content of 3.86%, ash content of

37
6.10%, crude fibre of 12.30% and carbohydrate content of 75.97%

in the stem of Aspilia africana.

Moses et al., (2019) researched Proximate, mineral and

phytochemical compositions of Vernonia amygdalina leaves grown

under hot and humid tropical conditions and found out higher

crude protein, dry matter and ether extract contents than the air-

dried samples. The air-dried samples, however, had higher ash

content, than the oven-dried samples. That notwithstanding, the

crude fibre content did not vary significantly. Dry matter content of

the leaves was quite high, ranging between 87.40 to 89.82%.It was

realized that the drying methods applied, had significant (p<0.05)

effects on the proximate composition of the leaves.

Etejere et al (2017) worked on proximate composition of

Chromolaena odorata and discovered that the proximate

composition showed percentage moisture, ash, fibre, fat, protein

and carbohydrate which ranged from 2.22 ± 0.71 –9.09 ± 0.72%,

6.00 ± 0.30 –10.00 ± 0.05%, 0.03 ± 0.01 –0.04 ± 0.01%, 10.00 ±

0.93 –50.00 ± 1.36%, 2.50 ± 0.49 –10.81 ± 1.57% and 19.07 ± 0.5 –

65.70 ± 1.39% respectively. Carbohydrate was highest with mean

38
value of 44.65 ± 20.5% while crude fibre was least with mean value

of 0.03 ± 0.01%. C. odorata most importantly its leaf and root,

contained bioactive substances that could be explored for

pharmaceutical purposes.

Nweze and Nwafor (2014) researched on proximate and Mineral

Composition of Leaf Extracts of Moringa oleifera Lam and obtained

that carbohydrate(57.01%),protein(18.92%),fats(2.74%),fibre(9.31%),

moisture(4.09%)and ash(7.95%). The results from this study prove

the extensive use of the leaves of this plant in ethnomedicine and its

potentials in drug formulation.

Otache and Kparobo,(2017). Worked on proximate and Mineral

composition of leaves of Azadirachta indica and discovered that

Azadirachta indica contains (12.10 to 14.30) %, (3.88 to 4.03) %,

(1.22to 4.04) %, (2.89 to 3.18) % and (9.25 to 10.86) % for Moisture,

Ash, Protein, Fat and Fibre respectively.

Rita and Dutta, (2016) researched on proximate analysis of Papaya

(Carica papaya) Leaves and found the leaves to be rich in proteins

and ash. Dry matter content was also very high. Crude fat and

Crude Fibre content were found to be very low. Leaves contained

39
Dry Matter 89.60%, Crude Protein 13.1%, Crude Fat 3.5%, Crude

Fibre 1.95%, Total Ash 18.3%, NFE 63.1%, Acid Insoluble Ash4.4%.

Offor, (2015), researched on proximate analyses of Psidium Guajava

Leaves and come out with the result that proved proximate

composition (%) of moisture, ash, fat, fibre, protein and

carbohydrate recorded 10.74 ± 0.08, 4.35± 0.21, 1.37 ± 0.36, 10.37

± 0.05, 18.64 ± 0.05 and 54.53± 0.25 respectively.

Idoko et al., (2014), worked on proximate analysis and mineral

composition of some leafy vegetables consumed in Nigeria and

found the proximate analysis results showed that the moisture, ash,

crude lipid, crude fibre, crude protein and carbohydrate ranged as

followed; 10.20 to 15.17%, 6.65 to 28.33%, 1.44 to 11.29%, 0.52 to

2.33% 2.24 to 35.21% and 22.66 to 62.80% respectively.

Usunobun et al., (2014) researched on proximate composition of

annona muricata leaves and The result of the proximate composition

showed that the leaves contained 88.99%dry matter, 11.01%

moisture, 25% crude protein, 14.96%ash, 22.20% crude fiber, 21.22

% fat and 16.62% carbohydrate contents.

40
Nkoli and Amako (2020) worked on proximate and mineral

compositions of scent Leaf (Ocimum gratissimum) and Bitter Leaf

(Vernonia amygdalina) Leaves and the results of proximate analysis

revealed the presence of moisture (12.28 0.02% and 10.010.01%)

protein (35.370.11% and 22.200.02%) and total ash (6.000.20%

and 5.750.10%) each in V.amygdalina and O.gratisimum

respectively

Lots of works have also been carried out in the areas of

phytochemical analysis of plant materials. Nkoli and Amako (2020)

conducted a research on phytochemical and mineral compositions

of scent Leaf (Ocimum gratissimum) and Bitter Leaf (Vernonia

amygdalina) Leaves and investigated that Ocimum gratissimum and

Vernonia amygdalina revealed the presence of saponin, tannin,

flavonoids, steroids, terpenoids and alkaloids. O.gratissimum also

contained phenols but lacked anthroquinones while V. amygdalinav

contained both anthroquinones and phenols. However, the

quantitative phytochemical analysis revealed the presence of all the

secondary metabolites in varied quantities in both leaves extracts.

Saponin was the highest among the phytochemicals determined in

41
both leaves extracts with the value 5.71±0.12mg/g in V. amygdalina

and 3.52±0.01 mg/g in O. gratissimum, this is followed by

terpenoids with the value 5.64 ± 0.11 mg/g in V. amygdalina and

3.40±0.11 mg/g in O. gratissimum, tannin 4.90 ± 0.23 mg/g in V.

amygdalina and 2.48 ± 0.03 mg/g in O. gratissimum, flavonoids

4.60±0.01mg/g in V. amygdalina and 2.00±0.03 mg/gin O.

gratissimum, alkaloids 3.16±0.16mg/g in V.amygdalina and

2.00±0.02mg/g in O. gratissimum, steroids 0.50 ±0.02mg/g in V.

amygdalina and 0.48±0.02 mg/g in O.gratissimum, phenol and

anthroquinines were higher in O.gratissimum than in V.amygdalina.

Okwuonu et al., (2017) researched on phytochemical and proximate

constituents of Aspilia africana (Wild sunflower) flowers and the

results of the phytochemicals obtained revealed several

therapeutically relevant phytochemicals such as; tannins, saponins,

cardiac glycosides, anthraquinones, flavonoids, alkaloids,

carotenoids, steroids, anthocyanins, phlobatannins, phenolics and

terpenoids in varied quantities in its crude state, most of which are

retained though in smaller quantities in its aqueous extract.

42
Moses et al., (2019) researched on proximate, mineral and

phytochemical compositions of Vernonia amygdalina leaves and the

phytochemicals present in the leaves include Flavonoids, Tannins,

Saponins, Phenols and Terpenoids, and these could serve as

therapeutic purposes in livestock species. The leaves however, had

no alkaloids and cardiac glycosides, unlike similar plant species in

Nigeria, thus, its use as livestock feed would pose no health threats

to animals.

Usunobun et al., (2014) researched on phytochemical composition

of annona muricata leaves also detected phytochemicals in the

ethanolic leaf extracts such as; flavonoids, alkaloids, cardiac

glycoside, tannins, triterpenoid, saponin and reducing sugar. The

findings indicate that Annona muricata leave is a potential source of

highly nutritious feed stuff and phytomedicine. They are of

nutritional, clinical and veterinary relevance considering the diverse

ethnopharmacological uses of the plant in different parts of the

world.

Idoko et al., (2014), conducted a research on phytochemical,

proximates analysis and mineral composition of some leafy

43
vegetables consumed in Nigeria and found the phytochemical

screening results also showed the presence of tannin, alkaloids and

carbohydrate in all the leafy vegetables.

Rita and Dutta, (2016) researched on phytochemicals and

proximate analysis of Papaya (Carica papaya) Leaves and

investigated that the Leaves pawpaw contained tannin and saponin

in the plant extracts tested are 2.65% and 3.57mg/ml respectively.

Offor, (2015), researched on phytochemicals and proximate

analyses of Psidium guajava leaves and the quantitative analyses

showed the concentrations (mg/100g) of the phytochemical

constituents as flavonoids (0.40 ± 0.02), tannins (0.55 ± 0.01),

glycosides (3.76 ± 0.08), alkaloids (0.04 ± 0.00), saponins (3.01 ±

0.01), phenols (0.28 ± 0.00) and steroids (0.09 ±0.00).

Otache and Kparobo, (2017) worked on phytochemicals, proximates

and Mineral composition of leaves of Azadirachta indica and

discovered that Azadirachta indica have phytochemical composition

reveals the ranges of values; (0.53 to 1.12) %, (0.92 to 1.94) %, (4.53

to 6.23) %, (1.85 to 3.96) % and (0.18 to 1.21) % for Tannin,

Saponin, Flavonoid, Alkaloid and Hydrogen Cyanide respectively.

44
The result from this study reveals that the plant can be used for

medicinal purposes as its heavy metal composition falls within

acceptable level recommended by WHO, hence will pose no harmful

effect to human health when administered.

Nweze and Nwafor (2014) researched on phytochemicals,

proximates and mineral composition of leaf extracts of Moringa

oleifera Lam and obtained the qualitative phytochemical

constituents of both leaf extracts of Moringa oleifera showed

presence of all the tested phytochemicals (flavonoids,

anthraquinone, alkaloids, saponins, steroids, terpenoids, cardiac

glycosides, anthocyanin, tannins and carotenoids) with water

extracting more of the phytochemicals.

Etejere et al., (2017) assessed phytochamicals, proximates

composition of Chromolaena odorata and discovered the listed

phytochemicals; alkaloids, saponins, cardiac glycosides, flavonoids,

tannins, phlobatannins, steroids and terpenoids in the plant parts

with the exception of phlobatannin in the stem. The leaf recorded

highest quantity of the phytochemicals (34.08 ± 3.75 %) followed by

the root (30.80± 1.89 %) and the stem (19.70 ± 0.59%). Saponins

45
occurred in highest amount with mean value of 17.67 ± 5.68 most

importantly in root while phenol had the lowest mean value of 0.81

± 0.42.

With the review of all the different leaves of the plants, it proved to

note that most plants contains active metabolites and proximate.

CHAPTER THREE

MATERIALS AND METHODS

46
3.1. Collection of Plant Material

The leaves of Allamanda cathartica (Fig. 1) were collected from

Science Laboratory Technology Botanical Garden, Federal

Polytechnic Oko, Orumba North Local Government Area, Anambra

State, Nigeria.

Fig 3.1: Leaves of A. cathartica

3.2. Methods

Preparation of Necessary Reagents

47
Sodium hydroxide, is industrially produced using the electrolytic

chloralkali process, in which electrolysis of aqueous sodium

chloride solution gives chlorine gas and sodium hydroxide. NaOH is

obtained as a 50% solution in water, and then dried to give solid

sodium hydroxide flakes or pellets.

2 NaCl + 2 H2O → 2 NaOH + Cl2 + H2

Hydrochloric acid, is prepared by dissolving gaseous hydrogen

chloride in water. Because of the corrosive nature of the acid,

ceramic, glass, or sometimes tantalum apparatus is commonly

used.

Hydrogen chloride, Muriatic acid is prepared by warming NaCl

crystals with concentrated H2SO4 (Sulphuric acid).

NaCl+H2SO4→NaHSO4+HCl

Usually, most of the hydrogen chloride/hydrochloric acid that is

formed is a co-product of some other chemical reactions. HCl is also

formed by the chlorination of hydrocarbons.

Preparation of Extracts

48
After collection and identification of the plant leaves, the leaves

were dried on a laboratory bench for 7days and later grounded into

a fine powder using clean mortar and pestle. The powdered

materials were weighed accurately and 20g were macerated in

400ml of 70% ethanol for 72 hours (3 days) in a maceration tank at

room temperature. The plant materials were then filtered and the

filterate obtained was concentrated in water bath at 400C which

yield a semi-solid extract that was then used for proximate and

phytochemical screening.

3.3. Proximate Determination

The determination of Lipid, crude fibre, protein, mineral, protein,

vitamin, ash and carbohydrate will be carried out by methods of

AOAC (2016).

Lipid Determination

For the determination of lipid content, the cold extraction method

was used. Following this procedure, 50g of powder sample were

introduced in volumetric flasks containing 300 mL of a mixture of

chloroform/methanol (200 mL/100 mL) and mixed for 20 min using

49
an agitator. The mixture will be filtered under N2 and the residue

re-extracted in 200 mL of the same solvent and filtered. The

extracts was then mixed and allowed to separate after the addition

of 0.2 mL of NaCl solution 0.7 g/100 mL. The Lipid in the lower

phase solution was flushed out and recovered by rotary evaporation

at 50˚C under liquid Nitrogen. The flasks containing the extracted

oil were weighed and the difference in weight expressed as

percentage oil content.

Lipid % = [Sample weight (g) x [(chloroform layer+amount lost)

(ml)*/3ml] x100

Crude fibre Determination

The organic residue left after sequential extraction of powdered

leaves samples with diethyl ether was used to determine the crude

fibre. The fat-free material was transferred into a beaker and 200ml

of pre-heated1.25%H2SO4 was added and the solution was gently

boiled for about 30mins, constant volume of acid was maintained

by the addition of hot water. The buckner flask funnel fitted with

whatman filter was pre-heated by pouring hot water into the

funnel.

50
The boiled acid sample mixture was then filtered hot through the

funnel under sufficient suction. The residue will be then washed

several times with boiling water (until the residue will be neutral to

litmus paper) and transferred back into the beaker. Then 200mls of

pre-heated 1.25%NaOH was added and boiled for another 30mins.

Filter under suction and was thoroughly with hot water and twice

with ethanol. The residue was dried at 105 0C for about 4hrs and

weighed. The residue will be transferred into a crucible and placed

in muffle furnace (400-6000C) and ash for 2hrs, then cooled in a

desiccators and weighed %Crude fibre

= (Dry wt of residue before ashing –wt of residue after ashing) x100

wt of sample

Crude Protein Determination

Crude protein of powdered leaf sample was determined by

measuring the nitrogen content of the feed and multiplying it by a

factor of 6.25. This factor was based on the fact that most protein

contains16%nitrogen.Crude protein was determined by kjeldahl

method. The method involved: Digestion, Distillation and Titration.

51
a. Digestion: About 0.5g of the powdered dried leaf of each sample

was weighed into kjeldahl flask and 20mls of concentrated

sulphuric acid was added, 0.1g of copper sulphate, 1g of sodium

sulphate and a speck of selenium tablet was added. After which the

digestion tubes were placed in the rack on the digest or unit. The

sample was digested at 350°C for approximately 2hrs until the

sample showed a clear pale green appearance. The solution was left

until completely cool and will be made to 50 ml mark with distilled

water.

b. Distillation: Steam distillation apparatus was used for

distillation. The distillation apparatus was steamed up and 10ml of

the digest was added into the apparatus via a funnel and allowed to

boil. Then 5ml of sodium hydroxide from the measuring cylinder

was added and distilled into 5ml of 2 %boric acid containing

screened methyl red indicator.

c. Titration: The alkaline ammonium borate formed was titrated

directly with 0.1MHCl.

The titre value which was the volume of acid used was recorded.

The volume of acid used was fitted into the formula

52
%N= 14 xVAx0.1xWx100

1000 x100

VA= volume of acid used, W=weight of sample

% crude protein = % N x6.25

Ash Determination:

According to AOAC, (2016), an empty crucible was accurately

weighed, and then 10ml of sample was weighed in it using sensitive

balance. The sample in crucible was placed in muffle furnace at

550°C for more than 3 hours until white to grey ash was obtained,

then crucible was removed from furnace to a desiccators to cool,

then weighed.

Ash content % = W2 – W1× 100

W3

Where: W1= weight of empty crucible

W2 = weight of crucible with ash.

W3 = weight of sample

53
Carbohydrate Determination

Nitrogen free extract of the powdered dried leaves of each sample

was determined by mathematical calculation. It was obtained by

subtracting the sum of percentages of all the nutrients already

determined from 100.

%NFE=100-(%moisture+% CF+% CP+% EE +% Ash)

NFE represents soluble carbohydrates in the leaves of each sample.

3.4. Phytochemical Determination

The dry crude and aqueous extract of Allamanda cathartica leaves

were tested for the presence of, tannins, alkaloids terpenoids,

saponins, flavonoids, phenol and cardiac glycosides, using standard

procedures for detecting phytocompounds in line with the methods

of Sofowara, (2014).

Qualitative Assessment

Test for Alkaloids

About 2g of well ground plant materials will be put in a test tube

and treated with 1% hydrochloric acid 10ml for 30minutes in a

54
water bath. Then Iml of the filtrate was treated with a few drops of

Mayer’s reagent and a second 1ml portion was treated with

Dragendorff’s reagent. Turbidity or precipitate with either of these

reagents was taken as presence of alkaloids.

Test for Saponins

About 0.5g of plant extract was shaken with water in a test tube.

Frothing which persists on warming was taken as preliminary

evidence for the presence of saponins.

Test for tannins

About 5g of plant extract was sterilized with 10ml of distilled water,

filtered with Whatman filter paper and ferric chloride reagent was

added to the filtrate. Blackish blue precipitate indicated the

presence of hydrolysable tannin while blackish green precipitate

indicated the presence of condensed tannins.

Test for flavonoids

The alcoholic extracts of the plant material was added to a few

pieces of magnesium metal, and concentrated HCl was added. The

55
formation of crimson colouration was taken as evidence for the

presence of flavonoids.

Test for Phenolics

About 0.5g of the powdered dried leaf of sample was boiled with

10ml of distilled water for 5mins and filtered while hot.Then1ml of

ferric chloride solution will be added. Formation of blue- black or

brown colouration indicated the presence of phenol.

Test for Cardiac Glycosides

Few drops of ferric chloride and concentrated sulphuric acid will be

added to a solution of the plant extract in glacial acetic acid. A

reddish brown coloration at the junction of two layers and the

bluish green colour in the upper layer indicated the presence of

glycosides.

Test for Terpenoids

0.5g of the extracted leaf sample was dissolved in 1ml of

chloroform. 1ml of acetic anhydride was added, followed by the

addition of 2ml of concentrated H2SO4. Formation of reddish violet

colouration indicated the presence of terpenoids

56
Quantitative Assesment

Determination of Alkaloids

Five grams of the plant sample was weighed into a 250ml beaker.

Then 200ml of 10% HCL in ethanol was added and covered and

allowed to stand for 4hrs. It was filtered and the extract

concentrated on a water bath to one quarter of the original volume.

Pigments and other unwanted materials was removed by shaking it

with 100ml Chloroform in separating funnel. Distilled water was

added. The lower layer was collected and excess ammonia added to

precipitate the free alkaloid. The whole solution was filtered through

Whatman filter paper No. 42 (125mm). The filtrate was later

evaporated into dryness in an oven and weighed to a constant

weight.

Calculation: % Alkaloids = (W2-W1) / (weight of sample) X 100/1.

W2 =weight of filter paper + Residue after oven drying.

W1 = weight of filter paper

Weight of sample = 5g

57
Saponin Determination

Twenty grams of the sample was put into a conical flask and 100ml

of 20% aqueous ethanol will be added. The Sample was heated over

a hot water bath for 4hrs with continuous stirring at about 55 oC.

The mixture was filtered and residue was again extracted with

another 200ml of 20% ethanol. The combined extracts was reduced

to 40ml over water bath at about 900C. The Concentrate will be

transferred into a 250ml separating funnel and 20ml of diethyl

ether will be added and shaken vigorously. The aqueous layer

(upper layer) will be discarded and the purification process

repeated. Then 60ml of nbutanol will be added (discard the bottom

and recover the upper layer). The combined n-butanol extracts were

washed twice with 10ml of 5% aqueous sodium chloride. The

remaining solution was heated in water bath. After evaporation, the

sample was dried in the oven to dryness, and to a constant weight.

The saponin content was calculated as percentage weight.

Calculation % Saponin = (W2-W1) / (weight of sample) X 100/1.

W2 =weight of crucible + Sample after oven drying.

58
W1 = weight of empty crucible Weight of sample = 10g

Tannin Determination

Half a gram (0.5) of the sample was weighed into 250ml conical

flask and 50ml of distilled water added and then shaken on a

rotating shaker (magnetic stirrer) for 1hr. The mixture was filtered

into a 50ml volumetric flask. The 5ml of the filtrate was pipetted

into 50ml volumetric flask. (5ml of the filtrate was pipetted out into

the test tube mixed with 2ml of 0.1M Fecl 3 in 0.1N HCl and 0.008M

K4Fe(CN)6 (Potassuim ferrocyanide). The absorbance was measured

at 120nm within 10min) For the standard solution, 0.1g of tannic

acid will be dissolved in 100ml of water to form tannic acid solution.

From the tannic acid solution 5ml will be pipetted into another

50ml volumetric flask. A blank sample will be prepared using 5ml

distilled water.

The three prepared solutions will be put in an incubator for 1hr

30mins at 20-30oC. After which the solutions will be made up to the

50ml mark and absorbance of the solutions at 760nm will be

measured using Spectrophotometer.

59
Calculation:

Let absorbance of 5ml of extract be X

Let absorbance of tannic acid solution be Y

Let absorbance of Blank be Z

Tannin in mg/100 = (X-Z)/(Y-Z).

Flavonoids Determination

Total flavonoid content was determined following a method by Firn

(2010). In a 10 ml test tube, 0.3 ml of extract, 3.4 ml of 30%

methanol, 0.15 ml of NaNO2 (0.5 M) and 0.15 ml of AlCl3.6H2O (0.3

M) was mixed.

After 5 min, 1 ml of NaOH (1 M) was added. The solution was mixed

well and the absorbance was measured against the reagent blank at

506 nm. The standard curve for total flavonoids was made using

rutin standard solution (0 to 100 mg/l) under the same procedure

as earlier described. The total flavonoids was expressed as

milligrams of rutin equivalents per gram of dried fraction.

60
Calculation

Flavonoid content = (RE x V x D x 10-6 x100)/W

RE = Rutin Equivalent (µg/ml)

V = Total volume of sample (ml)

D = Dilution factor

Phenol Determination

The phenolic content in the sample was estimated by Folin-

Ciocalteu’s colorimetric method. About 0.5 ml of the sample extract

was mixed with 2.5 ml of distilled water. To this, 0.5 ml of Folin –

Ciocalteu reagent (1:1) was added and incubated for 3 minutes. To

the tube, 2 ml of 20% Na2CO3 solution was added and the tubes will

be kept in boiling water bath for 1 minute. The tubes was cooled

and the absorbance of reaction mixture was read at 650 nm. A

standard curve was plotted using different concentrations of Gallic

acid (standard, 0-1000 µg/ml).

Total phenolic content was expressed w/w and calculated using the

formula

61
Total phenolic content (% w/w) = (GAE x V x D x 10-6 x 100) / W

GAE = Gallic Acid Equivalent (µg/ml)

V= Total Volume of Sample (ml)

D = Dilution Factor

W = Weight of sample (g)

Cardiac glycosides Determination

A 10% extract of the leaf sample was mixed with 10mL freshly

prepared Baljet's reagent (95mL of 1% picric acid + 5mL of 10%

NaOH). After an hour, the mixture was diluted with 20mL distilled

water and the absorbance was measured at 495 nm by Shimadzu

UV/VIS spectrophotometer model 160A (Kyoto, Japan). Total

glycoside from triple replicates was expressed as mg of the dried

extracts.

62
 CHAPTER FOUR

RESULTS

The results of proximate and phytochemical analysis of A.cathartica

are presented as follows;

4.1 Proximate Analysis

The results of proximate analysis of leaf of (A. cathartica) stated in

Table 1 below indicate that the contain moisture 32.5 ± 0.233%,

lipid 29.76 ± 0.262%, crude protein 10 ± 0.00% and carbohydrate

7.68 ± 0.170%. The leaf show high level of moisture, followed by

lipids, fiber and ash. Moderate in carbohydrate levels finally crude

protein at low levels.

63
Table 1: The result of Proximate analysis of the (A. cathartica).

S/ Proximates Contents % Concentration with SD

1 Moisture 32.5 ± 0.233

2 Protein 3.15 ± 0.00

3 Lipids 29.76 ± 0.262

4 Fiber 13.87 ± 0.189

5 Ash 12.87 ± 0.188

6 Carbohydrate 7.68 ± 0.170

4.2. Phytochemical Analysis

The phytochemical analysis was done in two ways; qualitative and

quantitative.

Qualitative Analysis

64
Table 2 below shows the result of phytochemical analysis

(qualitative) of the leat of (A. cathartica) revealed the presence of

various metabolites such as alkaloid, tannin, flavonoid, saponin,

steroid and phenol in different concentrations.

Table 2: Result of phytochemical screening (qualitative)

S/ Phytochemical Contents Re
sults
N

1 Alkaloid ++

2 Flavonoid +++

3 Cardia glycoside +

4 Saponin ++

5 Phenol +

6 Tannin ++

Key: Presence =+, Absence=-.

Quantitative Analysis

65
Table 3 below reveals the percentage content of phytochemical

analysis (quantitative) of the leaf of (A.cathartica) shows that the leaf

contains alkaloid 6.1 ± 0.1%, tannin 7.42 ± 0.01%, flavonoid 9.5 ±

0.1%, saponin 4.1 ± 0.1%, cardiac 1.56 ± 0.08% and phenol 1.32 ±

0.0024%. The result shows that flavonoid has the higher content in

A. cathartica, tannin, alkaloid and saponin percentage was relatively

high while the least were Phenol and cardiac glycoside.

Table 3: Result of Quantitative Analysis of the leaf of (A. cathartica)

S/ Phytochemical Contents % Concentration with SD

1 Alkaloid 6.1 ± 0.1%,

2 Flavonoid 9.5 ± 0.1%

3 Cardiac glycoside 1.56 ± 0.08%

4 Saponin 4.1 ± 0.1%

5 Phenol 1.322 ± 0.0024%

6 Tannin 7.42 ± 0.01%

66
 CHAPTER FIVE

DISCUSSION

Proximate composition of the leaf of (A. cathartica) revealed that

moisture had percentage concentration of 32.5 ± 0.233 while that of

of Aspilia Africana carried out by Okwuonu et al., (2017) have

8.47% moisture content, 17.53% Protein, 2.37% fat, 9.17% crude

ash, 9.10% crude fibre and 53.4% carbohydrate. which may be due

to environmental factors. The amount lipid in A. cartharcia was high

with the percentage content of 29.76 ± 0.2% compared to that of

A.africana leafs conducted by Uduak and Umana (2013) which had

the percentage concentration of 7.87 ± 0.04% which may be due to

laboratory procedures used to carry out the work. The amount of fat

is adequate for consumption without any health threat. The

percentage concentration of crude protein in A. cathartica was

3.15% while that of A. africana investigated by Uduak and Umana

67
(2013) revealed the percentage crude protein to be 7.87% which

may be due to different procedures used in carrying out the

experiment. Consumption of A. cathartica is necessary as it helps to

defend the body against diseases. The percentage content of ash in

A. cathartica was 23.9 ± 0.25% while that of C.odorata leaves

conducted by Etejer et al. (2017) revealed ash to be 9.09 ± 0.72%,

this may be due to environmental factors. The percentage content of

ash in A.cathartica was 23.9 ± 0.25% while that of A. africana by

Uduak and Umana (2013) contained 6.10 % ash which may be due

to the different locations where the leaf were gotten from. A.

cathartica revealed a content of carbohydrate of 7.68 ± 0.170%

which was very low when compared to that of A. africana leaves

investigated by Okwuonu et al (2017) which revealed that the

percentage content of carbohydrate was 53.4%, this shows that A.

cathartica is not a very good source of carbohydrate and can’t help

enhance the dietary fiber of man.

The phytochemical analysis of A. cathartica revealed that the leafs

of A. cathartica had alkaloid content with the percentage of 6.1 ±

0.1% while that of Ocimun gratissimum leaf investigated by Nkoli

68
and Amako, (2020) had alkaloid content with the percentage of 5.7

± 0.14%, this shows that both plants can be useful to man. The leaf

of A. cathartica contained tannin with percentage of 7.42 ± 0.001%

while that of V. amygdalia (Nigeria) leaf conducted by Nkoli and

Amako, (2020) showed tannin with the percentage of 4.90 ± 0.23%

which shows that both plants are of health benefit. The leaf of A.

cathartica had a flavonoid content with percentage of 9.5 ± 0.1%

while that of A. indica leaves carried out by Otache and

Kparobo(2017) revealed the percentage flavonoid content of 4.53 ±

6.23%, this shows that leaves of A. cathartica. The result from this

study reveals that the plant can be used for medicinal purposes.

The leaves of A. cathartica contained saponin with the percentage of

4.1 ± 0.01 while that of Chromolaena odorata leave extract carried

out by Etejere et al., (2017) revealed that the leaves contained

saponin with the percentage content of 17.67 ± 5.68 this shows that

both plants may have lower the cholesterol level thereby reducing

the risk of heart disease. The percentage concentration of cardiac

glycoside in A. cathartica was 1.56 ± 0.08% while in Chromolaena

odorata conducted by Etejere et al., (2017) it was present with the

69
concentration of 2.05 ± 1.0%, this shows that both plants are

medicinal.

A. cathartica contains phenol with the percentage content of 1.3 ±

0.0024% while that of Psidium guajava leaves investigated by Offor,

(2015), had phenol content with the percentage of (0.28 ± 0.00%

which shows that both plants are beneficial to health.

Conclusion

The results of the proximate analysis of leaf of (A. cathartica)

contained the following proximate; moisture, fat, crude protein, ash,
crude fiber and carbohydrate which revealed the presence of
appreciable amount of nutrients. According to the proximate
screening, of (A. cathartica) possess odour and flower carrying
ability which enhances in medicine. Furthermore, the study shows
that the seeds of country onions are rich in phytochemicals such
as; alkaloid, tannin, flavonoid, saponin, steroid and phenol and that
their utilization should be strongly recommended for good health.

Recommendations

5. Allamanda cathartica contain an appreciable amount of

carbohydrate which can help enhance the dietary fiber of man.

70
 6. The phytochemicals present in country onions can help

increase the effectiveness of vitamin in the body.

7. Daily consumption of A. cathartica can help lower the

cholesterol level to enable man stay healthy.

8. The crude fiber in A. cathartica is good in maintaining a

healthy and regular digestive system.

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