1
ABSTRACT
The aim of was study to determine the proximate, mineral and vitamin composition of stem bark
of Terminalia superba extract. Stem bark extract of Terminalia superba were washed with distilled
water, dried at room temperature, then pulverized, weighed and used for the study. Then 1kg of
dried pulverized of Stem bark extract of Terminalia superba was poured into a small glass
container and 2 litres of distilled water was added to it and stirred. After 48hrs of soaking, it was
filtered using Whatman filter paper. The filtrate was concentrated using rotary evaporator. This
crude aqueous extract was used for the study. The samples were analyzed for proximate
compositions, mineral and vitamin composition using methods of AOAC. The findings of the
study on proximate analysis indicated presence of Carbohydrate, moisture, ash ,protein, fibre and
fats. The findings revealed carbohydrate has the highest value seconded by moisture. The findings
on the mineral composition of terminalia superba in this study revealed the presence of macro
minerals such as calcium, sodium, zinc, potassium and magnesium, phosphorus, oxalate, iron, zinc
and phylate. The findings on vitamin composition revealed Terminalia superba contained vitamin
A, vitamin C, vitamin E, vitamin B12, vitamin B1, vitamin B3, vitamin K, vitamin B2, vitamin D,
vitamin B6 and vitamin B9 in increasing order ofvitamin B2>vitamin B9>vitamin A>vitamin
B3>vitamin B1> vitamin B6 > vitamin D>vitamin C> vitamin B12. The findings of this study
shows that Terminalia superba has nutritional qualities and has the potential for drug development
based on popular uses and biological studies.
2
CHAPTER ONE
INTRODUCTION
1.1 Background of study
Medicinal plants are the source of many important drugs of the modern world. Plants are important
for pharmacological research and drug development (Newman et al., 2019). Most of plant-derived
medicines have been developed on the basis of traditional knowledge in health care and in many
cases, there is a correlation between the indications of pure substances and those of respective
crude extracts used in traditional medicine (Osho et al., 2021). Herbal preparations constitute
valuable natural resource from which chemicals of potential interest for medicine, agriculture,
industry and other areas can be identified and isolated (Sneader et al., 2017).
Interest in medicinal plants has been reborn. This is due to the fact that many synthetic drugs are
potentially toxic and not free of side effects on the host. Examples include, cisplatin which may
cause hearing problem and numbness; entecavir which may cause diarrhea and dizziness and
zidovudine which may cause constipation, weakness and loss of appetite and, they are also costly
(Sneader et al., 2017). The use of traditional medicines is increasing and getting popularity
throughout the developed and developing world (Jia and Zhang, 2020). Herbal medicines are the
finished labeled medicinal product that contains active ingredients, aerial or underground parts of
the plant or other plant material or combinations (Ritch, 2021).
Close to 80% of the marginal (low income earners) people in developing countries rely on
traditional medicine for their primary health care (Latif et al., 2020). With the increase in
preference and demand, herbal product industry is increasing day by day (Shinwari et al., 2018).
Since many of these herbal products are used orally, carrying out proximate and nutrient analysis
of these products and raw material used therein plays a crucial role in assessing nutritional
3
significance and health effects (Kochhar et al., 2018). As far as herbal drug standardization is
concerned, WHO has also emphasized on the need and importance of determining proximate and
micronutrients analysis. Such herbal formulations must pass through standardization processes
(Ojokoh, 2021). The active ingredients of plants that can provide effective therapeutic potential
can occur in all plant structures but concentration is often higher in one part, such part is preferred.
Examples include roots, flower, fruit, leaves, bark of the stem and seeds (Akinleye et al., 2019).
Terminalia superba belongs to the fruit-bearing family called Combretaceae that
contains lots of nutritional benefits (Chinemezu et al., 2018). Traditionally, Terminalia
superba has been used for the treatment of dysentery and wound dressing (Linke et al., 2020). It
is also used to treat circumcision wounds, leprosy and epilepsy, rickets, infertility, gonorrhea,
edema and respiratory disorders (Olowokudejo et al., 2019) and abortion (Owolabi et al., 2018).
Apart from its traditional uses, scientific investigations have reported its; blood-boosting effect
(Njoku-oji et al., 2020), anti-sickling (Umeokoli et al.,2017; Mpiana et al., 2019) antibacterial
(Oyeleke et al.,2019), anti-abortifacient (Owolabi et al., 2018), immune-stimulatory (Daikwo et
al., 2021), antidiarrhoea (Owolabi, 2017), antioxidant (RamdeTiendrebeogo et al., 2021) and profertility in treating azoospermia (Komolafe et al.,2020). Terminalia superba has also been reported
to possess nutritive properties though there are few documented reports on the vitamin and mineral
content, nutritive and phytochemical composition of this plant.
1.2 Aim of the Study
The aim of this study to determine the proximate, mineral and vitamin composition of stem bark
of Terminalia superba extract.
1.3 Objectives of the study
4
1. To determine the vitamin composition of stem bark of Terminalia superba extract .
2. To determine the mineral composition of stem bark of Terminalia superba extract .
3. To determine the proximate composition of stem bark of Terminalia superba extract .
5
CHAPTER TWO
LITERATURE REVIEW
2.1 Medicinal plants
A medicinal plant is any plant which, in one or more of its organs, contains substances that can be
used for therapeutic purposes or which are precursors for the synthesis of useful drugs (Khattab et
al., 2021). This description makes it possible to distinguish between medicinal plants whose
therapeutic properties and constituents have been established scientifically, and plants that are
regarded as medicinal but which have not yet been subjected to a thorough scientific study
(Akachuku and Fawusi, 2018)
A number of plants have been used in traditional medicine for many years. Some do seem to work
although there may not be sufficient scientific data (double-blind trials, for example) to confirm
their efficacy. Such plants should qualify as medicinal plants. The term ‘crude drugs of natural or
biological origin’ is used by pharmacists and pharmacologists to describe whole plants or parts of
plants which have medicinal properties (Akachuku and Fawusi, 2018)
The use of plants for treating diseases is as old as the human species. Popular observations on the
use and efficacy of medicinal plants significantly contribute to the disclosure of their therapeutic
properties, so that they are frequently prescribed, even if their chemical constituents are not always
completely known. For example, Senna alata is used traditionally in Nigeria to treat bacterial and
fungal infections (Shiba et al., 2018). They also showed varying degrees of antibacterial and
antifungal activities against pathogens.
Medicinal plants contain a wide variety of secondary metabolites or compounds such as tannins
terpernoids, alkaloids, flavonoids; that dictates the therapeutic potency of the plants most
6
especially the antimicrobial activities (Egabik et al., 2020).Similar phytochemical constituents
such as flavonoids and tannins were also revealed to be active against pathogenic bacteria such
as Bacillus cereus, Staphylococcus aurous amongst others (Egabik et al., 2020).The tannins
present in medicinal plants make it useful in production of antiseptic soap which are commonly
used in bathing or cleansing of skin surfaces. It was documented in literature that phytochemicals
can be toxic to filamentous fungi, yeasts and bacteria and also, inhibitory to viral reverse
transcriptase (Nduas et al., 2019).
2.1.2 Terminalia sp.
Terminalia (Combretaceae, Myrtales) is a pantropical genus accommodating about 200 species
(McGaw et al. 2018). About fifty of these are native to Africa and distributed throughout the subsaharan region (Lebrun and Stork 1991). Based on both their functional uses and distribution in
Africa, the most important are Terminalia ivorensis A. Chev. and Teminalia superba Engl. and
Diels. in West and Central Africa and T. prunioides M.A. Lawson and T. sericea Burch : DC in
Southern Africa (Lawes et al. 2018).
2.1.3 Terminalia superba (Botanical Description)
Terminalia superba is a large tree, up to 50 m tall and 5 m in girth, bole cylindrical, long and
straight with large, flat buttresses, 6 m above the soil surface; crown open, generally flattened,
consisting of a few whorled branches, leaves simple, alternate, in tufts at the ends of the branches.
Bark fairly smooth, greying, flaking off in small patches; slash yellow, bark surface smooth and
grey in young trees, but shallowly grooved and with elongated, brownish grey scales, inner bark
soft-fibrous, pale yellow (Kimpouni, 2019 ). Root system frequently fairly shallow, and as the tree
ages the taproot disappears. Buttresses, from which descending roots arise at some distance from
the trunk, then support the tree. Leaves simple, alternate, in tufts at the ends of the branches;
7
deciduous, leaving pronounced scars on twigs when shed. Petiole 3-7 cm long, flattened above,
with a pair of sub-opposite glands below the blade; lamina glabrous, obovate , 6-12 x 2.5-7 cm,
with a short acuminate apex. Nerves 6-8 pairs; secondary reticulation inconspicuous. Inflorescence
a 7-18-cm, laxly flowered spike, peduncle densely pubescent; flowers sessile, small, s greenishwhite; calyx tube saucer shaped, with 5 short triangular lobes. Petals absent. Stamens usually twice
the number of calyx lobes (usually 10), in 2 whorls, glabrous; filaments a little longer than calyx;
intra-staminal disc annular, flattened, 0.3 mm thick; densely woolly pubescent. Fruit a small,
transversely winged, sessile, golden-brown smooth nut, 1.5-2.5 x 4-7 cm (including the wings).
Nut without the wing about 1.5 x 2 cm when mature, usually containing 1 seed. The generic name
comes from the Latin ‘terminalis’ (ending), and refers to the habit of the leaves being crowded at
the ends of the shoots (Burkill, 2018).
2.1.4 Taxonomy:
kingdom:
Plantae
Division:
Mangnoliophyta
Class:
Mangnoliopsida
Order:
Myrtales
Family:
Combretaceae
Genius :
Terminalia
Species :
T. superba
Current name: Terminalia superba
Authority:
Engl. & Diels
2.1.5 Origin and geographic distribution
8
Terminalia superba is a tree of about 30-50 m high. It is a member of the genus Terminalia that
comprises around 100 species distributed in tropical regions of the worldwide (Victor et al, 2020).
In Africa it is found in West and Central Africa, from Guinea Bissau east to DR Congo and south
to Cabinda (Angola) (Kimpouni, 2019) In Nigeria it is Indigenous to Cross River State (Burkill.,
2018)
Figure 1: Teminalia superba Tree (www.ecocrop.fao.org/ecocrop)
2.1.6 Nigerian Vernacular Names
EDO
ẹ̀ghọẹ̀n-nófūá, nófūó: white; referring to the flaking bark
EFIK
àfia étò = white tree
IGALA
uji-oko (H-Hansen)
9
IGBO
èdò (auctt.) èdò ó ̣chá = white edo (Amufu)ojiloko (Nkalagu) ojiroko (Owerri) èdò
ó ̣chá = white edo (Egbema) apaụpaụ tịín (Tiemo)
ISEKIRI
egonni
NUPE
eji
URHOBO
unwon ron
YORUBA
afaa , afara (www.ecocrop.fao.org/ecocrop)
2.1.7 Ecology
Terminalia superba is most common in moist semi-deciduous forest, but can also be found in
evergreen forest. It occurs up to 1000 m altitude. It is most common in disturbed forest. It is found
in regions with an annual rainfall of (1000–) 1400–3000 (–3500) mm and a dry season up to 4
months, and mean annual temperatures of 23–27°C. Terminalia superb prefers well-drained,
fertile, alluvial soils with pH of about 6.0, but it tolerates a wide range of soil types, from sandy to
clayey-loamy and lateritic. It does not tolerate prolonged water logging, but withstands occasional
flooding (Richter and Dallwitz, 2018).
2.1.8 General Uses
The wood, usually traded as ‘limba’, ‘afara’, ‘ofram’ or ‘fraké’, is valued for interior joinery, door
posts and panels, mouldings, furniture, office-fittings, crates, matches, and particularly for veneer
and plywood. It is suitable for light construction, light flooring, ship building, interior trim, vehicle
bodies, sporting goods, toys, novelties, musical instruments, food containers, vats, turnery,
hardboard, particle board and pulpwood. It is used locally for temporary house construction,
planks, roof shingles, canoes, paddles, coffins, boxes and domestic utensils. It is suitable for paper
making, although the paper is of moderate quality. The wood is also used as firewood and for
10
charcoal production. A yellow dye is present in the bark; it is used traditionally to dye fibres for
matting and basketry. The bark is also used for dyeing textiles blackish. In Côte d’Ivoire
Terminalia superba is occasionally used as a shade tree in cocoa and coffee plantations, and in DR
Congo it is used as shade tree in coffee, cocoa and banana plantations (Kimpouni, 2019).
2.1.7 Ethno-Medicinal Uses
Bark decoctions and macerations are used in traditional medicine to treat wounds, sores,
haemorrhoids, diarrhoea, dysentery, malaria, vomiting, gingivitis, bronchitis, aphthae, swellings
and ovarian troubles, and as an expectorant and anodyne. The leaves serve as diuretic and roots as
laxative (Richter and Dallwitz, 2019). Terminalia superba is generally used in traditional medicine
to treat bacterial, fungal and viral infections. The bark of this plant is used to eradicate intestinal
worms and treat gastrointestinal disorders such as enteritis, abdominal pain, diarrhoea, fever,
headache, conjunctivitis. In the Southwest of Côte d’Ivoire the bark of T. superba, called "tree of
malaria", (Orewa et al 2019). In Cameroon it is locally used in the treatment of various ailments,
including diabetes mellitus, gastroenteritis, female infertility and abdominal pains (Adjanohoun et
al., 1996).
2.1.8 Phytochemical Constituents
The results of the phytochemical screening by (2019) showed the presence of tannins, terpenoids,
alkaloids, flavonoids, cardiac glycosides and reducing sugars, with steroids and anthraquinones
absent in the water extract of the leaves and bark of Ficus capensis. Saponin was present in the
bark but absent in the leaves.
11
Terpenoids, flavonoids, steroids, cardiac glycosides and reducing sugars were present in ethanol
extract of the leaves and bark, while anthraquinones were absent. Alkaloids were present in the
ethanol extracts of the bark but absent in leaves. Adebayo and Adeniyi (2020) reported the
presence of tannins in the bark. Saponins were highest in the leaves, reduced in the stem and least
in the bark. Alkaloids and phenolics were highest in the bark while their quantity was least in the
leaf. Terpenoids and flavonoids were highest in the leaf samples. Owolabi et al. (2019) equally
reported the presence of saponins, cardiac glycosides, tannins and flavanoids with traces of
alkaloids and anthracene derivatives in the stem bark. The qualitative and quantitative
phytochemical analyses of aqueous leaf extract revealed the presence of reducing sugar, saponins,
tannins, flavonoids, soluble carbohydrates, alkaloids, steroids, hydrogen cyanide, glycosides,
terpenoids, fats and oil (Higashi et al., 2018). The following compounds were found from nhexane
and ethyl acetate fractions of ethanol extract of Terminalia superba leaves: 4, 4, 24trimelhylcholesta-8-en-3-B-ol, mixture of campesterol, stigmasterol and β-sitosterol, stigmasterol,
3-Bo`glucoside and 4, 5, 7-trihydroxy flavan-3-ol, xantholoxin, and β-amyrin. Francois et al [28]
reported the presence of carvacrol (65.78%), α-caryophyllene (29.81), caryophyllene oxide (25.70
%), linalool (3.97%), 3-tetradecanone (2.90%), geranylacetone (1.20%), 3,7,11- trimethyl-3hydroxy-6,10-dodecadiene-1-
ylacetate(1.53%),
hexahydrofarnesyl
acetone
(1.21%),
α-
caryophyllene (0.81%), 2-methyl-3- hexyne (0.69%) and scytalone (0.69%).
2.1.9 Pharmacological Uses / Biological Activities Of Terminalia superba
Terminalia superba has been scientifically proven to be biologically useful as drug/medicine
because of its antimicrobial, antibacterial, antifungal, antioxidant and anti-sickling activities.
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2.1.10 Microbial inhibitory activities
Several studies have proven the microbial inhibitory activity of Terminalia superba. Microbial
inhibitory activities of the stem, root and leaf of Terminalia superba against test disease-causing
microorganisms were reported [6]. The bark extracts had the highest inhibitions on Pseudomonas
aeruginosa, Candida albicans and Staphylococcus aureus. While Streptococcus faecalis and
Proteus mirabilis were resistant to many antibiotics (87.5%), they were effectively inhibited by all
concentrations of ethanolic Terminalia superba extract. Oyeleke et al. (2021) reported the
inhibitory effect of the leaves and stem bark of Terminalia superba against Esherichia coli and
Shigella species but no activity against Salmonella typhi.
Ghani et al., (2021)reported the crude extract inhibited S. aureus, Escherichia coli, Bacillus subtilis
and Candida pseudotropicalis at 2 mg/ml but P. aeruginosa and Salmonella typhimorium were not
inhibited at the same concentration. Ujats, (2021) also reported antimicrobial activities of the
methanol extract of Ficus capensis leaf against some clinical pathogenic bacteria namely:
Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Staphylococcus aureus, Serratia
marcescens, Pseudomonas aeruginosa, Micrococcus roseus and Bacillus cereus. The leaf extract
had inhibitory effect on all the test organisms except Pseudomonas aeruginosa (Khan and Ahmed,
2018).
2.1.11Antidiarrheal Activity
The leaf aqueous extract of the plant produced a significant dose-dependent increase in percentage
inhibition of the movement of charcoal meal in the small intestine of the mice P < 0.05. The extract
showed a percentage inhibition of 23.52% 31.41% and 48.95% at a dose of 100 mg/kg, 200 mg/kg
and 400 mg/kg. This is comparable to atropine at a dose of 0.1 mg/kg which showed a percentage
13
inhibition of 44.02%. The aqueous extract also exhibited a dose-dependent increase in the average
onset of stooling in the animals using castor oil model. The onset of stooling in atropine- treated
animals was 19 ± 0 min. The leaf extract exhibited a significant dose-dependent decrease in the
number and weight of stool produced by the mice (Lewis and Rader, 2020).
2.1.2.7 Immune-Boosting Activity
The immune system is subject to modification by substances to either enhance or suppress its
ability to resist invasion by pathogens. Justification of the folkloric use of the plant as an immune
boosting agent has been reported (Anandan et al., 2019).
2.2 Proximates
Proximate Analysis is the partitioning of compounds in a plant material into six categories based
on the chemical properties of the compounds. The six categories are: moisture, ash, crude protein
(or Kjeldahl protein), crude lipid, crude fibre and nitrogen-free extracts (digestible carbohydrates)
(Pandey et al., 2018). Proximate and nutrient analysis of medicinal plants plays a crucial role in
assessing their nutritional significance. As various medicinal plant species are also used as food
along with their medicinal benefits, evaluating their nutritional significance can help to understand
the worth of these plants species (Pandey et al., 2018). Carbohydrates, proteins and fats form the
key portion of the diet, whereas minerals and vitamins form somewhat a minor part. As plants
form main portion of our diet; so their nutritive value is imperative (Jimoh and Oladiji, 2020).
Besides these biochemicals; the moisture, fiber, ash contents and the energy values of individual
vegetable species have also been reported to be important to the human health as well as for soil
quality (McSweeney et al., 2020). Each medicinal plant species has its own nutrient composition
besides having pharmacologically important phytochemicals. These nutrients are essential for the
14
physiological functions of human body. Such nutrients and biochemicals like carbohydrates, fats
and proteins play an important role in satisfying human needs for energy and life processes (Novak
and Haslberger, 2000). Fortunately, chemical composition diversity in plants also includes many
compounds that are beneficial to humans: vitamins, nutrients, antioxidants, anticarcinogens, and
many other compounds with medicinal value (Novak and Haslberger, 2000). Plants are also known
to have high amounts of essential nutrients, vitamins, minerals, fatty acids and fibre (Gafar and
Itodo, 2017).
2.2.1 Moisture
Food is any substance consumed to provide nutritional support for the body. It is usually of plant
or animal origin, and contains essential nutrients, such as carbohydrates, fats, proteins, vitamins,
or minerals (Moses et al., 2013). All foods content solids, water and other chemicals. The
moisture contained in a material comprises all those substances which vaporize on heating and
lead to weight loss of the sample. The weight is determined by a balance and interpreted as the
moisture content. According to this definition, moisture content includes not only water but also
other mass losses such as evaporating organic solvents, alcohols, greases, oils, aromatic
components, as well as decomposition and combustion products (Moses et al., 2013). The
moisture content also called as moisture assays is one of the most important analyses performed
on most of the food products.
Water, the simplest of all constituents of foods, is one of the great concern to producer, consumer
and chemist. The weight of food has little significance unless the water content is taken into
consideration (Kenneth, 2018). The accurate determination of moisture poses many challenges as
the difficulty of separating all the water from the food sample, results in underestimation of
moisture content. Whereas, harsher conditions to remove all moisture from a food may result into
15
decomposition of the product, along with/or a loss in sample mass(Kenneth, 2018). Most of the
methods for the estimation of water in food depends on the loss in weight on heating. An exposure
to the air of the drying oven causes the oxidation of certain oils and other constituents. A weight
gain of such constituents offsets the weight loss due to moisture. To remove this error, the drying
should be performed in vacuum(Belli, 2020). The loss in weight on heating is not entirely because
of water but also due to small amount of volatile substance evident to smell present in foods. Most
of the spices however contain notable quantities of volatile oil that pass off with the water (Belli,
2020).
Moisture content in food can have a significant impact on factors such as the product’s taste,
texture, appearance, shape, weight and shelf life ( Oyeleke et al., 2021). It has implications on
legal and labeling requirements, economically important requirements, the shelf life of the food or
food products, food quality measurements, and food processing operations ( Oyeleke et al., 2021).
2.2.2 Total Ash
Ash refers to the inorganic residue remaining after total incineration of organic matter. The ash
content is an indicator of product quality and the nutritional value of food products (Estes, 2016).
When a high ash figure suggests the presence of an inorganic adulterant, it is advisable to
determine the acid insoluble ash (Estes, 2016).
Dry ashing procedures use a high temperature muffle furnace capable of maintaining temperatures
of between 500 and 600 oC. Water and other volatile materials are vaporized and organic
substances are burned in the presence of the oxygen in air to CO2, H2O and N2(Janzen and Tim,
2012). Most minerals are converted to oxides, sulfates, phosphates, chlorides or silicates. Although
most minerals have fairly low volatility at these high temperatures, some are volatile and may be
16
partially lost, e.g., iron, lead and mercury. If an analysis is being carried out to determine the
concentration of one of these substances then it is advisable to use an alternative ashing method
that uses lower temperatures (Janzen and Tim, 2012).
The food sample is weighed before and after ashing to determine the concentration of ash present.
The ash content can be expressed on either a dry or wet basis:
where MASH refers to the mass of the ashed sample, and MDRY and MASH refer to the original
masses of the dried and wet samples (Priyaet al., 2013).
There are a number of different types of crucible available for ashing food samples, including
quartz, Pyrex, porcelain, steel and platinum. Selection of an appropriate crucible depends on the
sample being analyzed and the furnace temperature used (Rakesh et al, 2012). The most widely
used crucibles are made from porcelain because it is relatively inexpensive to purchase, can be
used up to high temperatures (< 1200oC) and are easy to clean. Porcelain crucibles are resistent to
acids but can be corroded by alkaline samples, and therefore different types of crucible should be
used to analyze this type of sample (Rakesh et al, 2012). In addition, porcelain crucibles are prone
to cracking if they experience rapid temperature changes. A number of dry ashing methods have
17
been officially recognized for the determination of the ash content of various foods (AOAC
Official Methods of Analysis). Typically, a sample is held at 500-600 oC for 24 hours.
Ash content represents the total mineral content in foods. Determining the ash content may be
important for several reasons (Igoli et al., 2020). It is a part of proximate analysis for nutritional
evaluation. Ashing is the first step in preparing a food sample for specific elemental analysis.
Because certain foods are high in particular minerals, ash content becomes important. One can
usually expect a constant elemental content from the ash of animal products, but that from plant
sources is variable (Igoli et al., 2020). This helps determine the amount and type of minerals in
food; important because the amount of minerals can determine physiochemical properties of foods,
as well as retard the growth of microorganisms (Hasler, 2020).
2.2.3 Crude Fiber
The crude fibre representing the cell wall material left after boiling with dilute acid and alkali in
the process, is a mixture of cellulose, lignin and pentosans, together with sand, silica and other
mineral matter locked in the tissues and little nitrogenous matter after grinding and defatting,
boiling with sulphuric acid solution, and separation and washing of the insoluble residue (Marc et
al., 2013). This residue is boiled with sodium hydroxide solution, separated, washed, and dried
and the insoluble residue is then weighed. The loss in mass on incineration is also noted.
In crude fibre, a sample is boiled sequentially with dilute acid and then with dilute alkali, and then
sequentially washed with ethanol and diethyl ether, and the residue is subtracted by its ash, and
the result is defined as crude fiber (Munaet al., 2016).Crude fiber is primarily measured to
comprehend indigestible parts in feeds, and is consisted mainly of a part of lignin, pentosan, chitin,
etc., in addition to cellulose (Forster et al, 2000).
These compounds are collectively called as fiber; however, the sum of their individually measured
18
contents is significantly different from the crude fiber content obtained by the method shown
above, and the former is always larger. This is because a part of lignin and hemicellulose is
dissolved during the boiling procedure, and the percentage of dissolution varies depending on the
feed type and thus is not constant (Forster et al, 2000).
Crude fiber clearly corresponds only to feeds of plant origin considering the component
compounds; however, a small amount of it is contained in feeds of animal origin. This is because
organic residue that is not dissolved by acid/alkali boiling is observed in feeds of animal origin,
and the residue is chitin and some of scleroprotein (albuminoid), which are completely different
from so-called crude fiber in content (Jason et al., 2014).
There is a close relationship between the crude fiber content and the nutrition value of the feed,
and generally the higher the crude fiber content is, the lower the nutrition value is (Sykes, 2001).
On the other hand, fiber is an important nutrient for ruminant livestock. Fiber that was previously
in the scope of feed analysis was only crude fiber of general nutrients, which was insufficient for
the evaluation of fiber(Rutherford, 2015). This is because plant fiber is mainly consisted of cell
wall which is comprised of substances such as cellulose, hemicellulose, lignin and pectin, but crude
fiber quantitated in general analysis does not include hemicellulose, etc. To measure fiber as
accurate as possible, detergent analysis using detergents was developed in Europe and the US,
while enzymatic analysis using enzymes was developed in Japan led by the National Institute of
Animal Industry (Hammer et al., 2019).
In detergent analysis, a feed is heated using a neutral detergent and is separated into soluble and
insoluble parts. Organic matter in the insoluble part corresponds to cell wall, and is defined as
neutral detergent fiber (NDF). The “low digestivity” fraction in cell wall is measured by heat
19
treatment with an acidic detergent, and is defined as acid detergent fiber (ADF) (Hammer et al.,
2019).
In enzymatic analysis, starch and protein are degraded by enzymes and is separated into soluble
and insoluble parts. Organic matter in the insoluble part corresponds to cell wall, and is defined as
organic cell wall (OCW) (Kennett, 2018). The “indigestible” fraction after the degradation of
starch and protein is measured by degrading cellulose with enzymes, and is defined as fiber with
low digestivity or organic b fraction (Ob) (Mas, 2013).
2.3.1 Benefits of a high-fiber diet
a)
Normalizes bowel movements. Dietary fiber increases the weight and size of your stool
and softens it. A bulky stool is easier to pass out decreasing the chance of constipation.
Loose of watery stools, fiber may help to solidify the stool because it absorbs water and
adds bulk to stool (Hasler, 2020).
b)
Helps maintain bowel health. A high-fiber diet may lower the risk of developing
hemorrhoids and small pouches in the colon (diverticular disease). Studies have also found
that a high-fiber diet likely lowers the risk of colorectal cancer (Robert et al., 2019). Some
fiber is fermented in the colon. Researchers are looking at how this may play a role in
preventing diseases of the colon (Robert et al., 2019).
c)
Lowers cholesterol levels. Soluble fiber found in beans, oats, flaxseed and oat bran may
help lower total blood cholesterol levels by lowering low-density lipoprotein, or "bad,"
cholesterol levels. Studies also have shown that high-fiber foods may have other hearthealth benefits, such as reducing blood pressure and inflammation (Robert et al., 2019).
d)
Helps control blood sugar levels. In people with diabetes, fiber — particularly soluble
fiber — can slow the absorption of sugar and help improve blood sugar levels. A healthy
20
diet that includes insoluble fiber may also reduce the risk of developing type 2 diabetes
(Robert et al., 2019).
Gut function and dietary fibre: Although small intestine is unable to digest dietary fibre, fibre
helps to ensure good gut function by increasing the physical bulk in the bowel, and thereby
stimulating the intestinal transit. Once the indigestible carbohydrates pass into the large intestine,
some types of fibre such as gums, pectins and oligosaccharides are broken down by the gut microflora
(Belli, 2020). This increases the overall mass in the bowel and has a beneficial effect on the makeup of our gut microflora. It also leads to formation of bacterial waste products, like the short-chain
fatty acids, which are released in the colon with beneficial effects on our health (see our dietary
fibre articles for more information).
2.2.4 Fat
Fats are naturally occurring molecules that are part of our diet. They belong to a larger group of
compounds lipids that also include waxes, sterols (e.g. cholesterol) and fat-soluble vitamins.
However, this distinction is not always clear, and sometimes the term fats also include other lipids,
such as cholesterol (Okwu, 2020).
Dietary fats molecules originate from plants and animals. In plants, they are found in seeds (e.g.
rapeseed, cottonseed, sunflower, peanut, corn and soybean), fruits (e.g. olive, palm fruit and
avocado) and nuts (e.g. walnuts and almonds) (Ashok and Upadhyaya, 2012). Common animal fat
sources are meat, (oily) fish (e.g. salmon, mackerel), eggs and milk. Both plant, or, as often called,
vegetable fats, and animal fats can be consumed as they naturally occur, but also indirectly, for
example in pastry and sauces, where they are used to improve texture and taste (Okwu, 2020).
21
Milk yields many popular animal fat products, such as cheese, butter, and cream. Apart from milk,
animal fat is extracted primarily from rendered tissue fats obtained from livestock animals.
Dietary fats, together with carbohydrates and proteins, are the main source of energy in the diet,
and have a number of other important biological functions. Besides being structural components
of cells and membranes in our bodies (e.g. our brain consists mainly of fats), they are carriers of
fat-soluble vitamins from our diet. Fat metabolites are involved in processes such as neural
development and inflammatory reactions (Petrovska, 2012). When stored, body fat provides
energy when the body requires, it cushions and protects vital organs, and helps to insulate the body
(Petrovska, 2012).
The oils and fats from oilseeds and fruits as well as from animal fatty tissues correspond quite
closely with those extracted by diethyl ether (Mas, 2013). Practically, all the sterols and
phosphorus containing organic compounds notably the lecithins are extracted with the glycerides.
Essential oil and resins are the chief constituents of the ether extract of certain spices. Similarly,
pepper contains nitrogenous ether soluble substance, piperine (alkaloids)(Muna, 2018). Other
solvents viz., chloroform, carbon tetrachloride, carbon disulphide and petroleum distillates of
lower or higher boiling points dissolve fats and oils and can be used but the yield and composition
of the extract differ somewhat with the solvent (Muna, 2018). Free fat can be extracted by the less
polar solvents such as petroleum ether and diethyl ether, whereas the bound fat requires more polar
solvents viz., alcohols for their extraction. The bound fat may be broken down by hydrolysis or
other chemical treatment to yield free fat (Randall et al., 2016). Hence, the amount of extracted
fat found in food products will depend on the method of analysis used.
2.2.5 CrudeProtein
22
Protein are biological polymers composed of amino acids. Amino acids, linked together by peptide
bonds, form a polypeptide chain (O'Rourke, 2018). One or more polypeptide chains twisted into a
3-D shape form a protein. Proteins have complex shapes that include various folds, loops, and
curves. Folding in proteins happens spontaneously. Chemical bonding between portions of the
polypeptide chain aid in holding the protein together and giving it its shape. There are two general
classes of protein molecules: globular proteins and fibrous proteins. Globular proteins are
generally compact, soluble, and spherical in shape. Fibrous proteins are typically elongated and
insoluble. Globular and fibrous proteins may exhibit one or more of four types of protein structure
(O'Rourke, 2018).
All natural foods contain protein, although trace amounts are found in honey and maple sugar.
Crude protein is defined as the value obtained by quantitating nitrogen in a sample by the Kjeldahl
method (in which nitrogen compounds in the sample is degraded by sulfuric acid to become
ammonia, sodium hydroxide is added, steam distillation is conducted under the alkaline conditions,
distilled ammonia is absorbed in acid and measured by titration) and multiplying the result by the
factor 6.25 (6.38 for milk products) (Regalado, 2018). Therefore, crude protein includes ammonia,
etc., that are not of protein origin Generally the nitrogen content of protein is 16 % on average;
thus the inverse number of this (100/16 = 6.25) is used as the factor. However, as the factor is
different between samples (5.83 for flour; 5.95 for rice), the crude protein of some feeds is different
from the pure protein content; crude protein is measured to be excessively small in materials of
milk product origin such as casein, and excessively large in flour and soybean (Wallace et al.,
2015).The quantification of total protein in food and food products can be performed directly or
by determining total nitrogen from conversion of crude protein using a suitable conversion factor
(O'Rourke, 2018).The protein content is calculated from the total nitrogen determined by either
23
Kjeldahl method or Dumas/Pregl-Dimas method. Amides (abundant in young shoots), ammonium
salts, nitrates, lecithin, nucleic acid, purines of tea, coffee, cocoa and meat extracts in addition to
protein contain nitrogen in varying proportions (Regalado, 2018).
Although small, these compounds thus add error to the calculated protein estimate. However, the
protein calculated by factor is a valuable figure, not only because it represents approximately the
true protein present but also because it is an index of the content of other groups (Hian, 2018). The
protein content can also be determined directly by formal titration, UV spectrophotometry, Lowry
method, Dye binding method, IR spectrophotometry, NMR spectroscopy, turbidimetry,
refractometry, etc.
2.2.5.1 Function of proteins
Structure, Repair and Maintenance: Protein is vital for building, maintaining and
repairing body tissue. Hair, skin, eyes, muscles and organs are all made from protein.
Children need more protein per pound of body weight than adults because they are growing
new tissues made from protein (Ojokoh, 2021).
Energy: Protein is a source of energy. Consumption of protein more than normal will
affect the body maintenance and other necessary functions sand so it can be converted for
energy in the body. If it is not needed for energy it will be converted to fat and stored on
the body (Hansch et al., 2019).
Hormones: hormones are protein based. Hormones help control many body functions. For
example, insulin is a protein based hormone that regulates blood sugar.
24
Enzymes: Enzymes are proteins that increase the rate of chemical reactions in the body.
For example, digestive enzymes are involved in the digestion of protein, carbohydrate and
fat (Hansch et al., 2019).
Transportation and Storage of Molecules: Transport proteins bind and carry molecules
within cells and throughout the body. For example, haemoglobin is a protein that transports
oxygen in the blood. Protein is also used to store certain molecules such as the protein
ferritin, which combines with iron for storage in the liver (Hansch et al., 2019).
Antibodies: Protein forms antibodies which identify and help to destroy antigens such as
bacteria and viruses. For example Immunoglobulin G (IgG) (Hansch et al., 2019).
2.2.6 Carbohydrate
Carbohydrates are made of the elements carbon, hydrogen and oxygen. Each carbon atom is
bonded to at least one oxygen atom. All carbohydrates include an aldehyde or ketone group and a
hydroxyl group. Carbohydrates may form long chains that are either straight or branched. But, no
matter the final structure, all complex carbohydrates structure is composed of individual units
called monosaccharides, or single sugars. Each monosaccharide is made of the carbohydrates
formula: CH2On where n is number greater than two.
Physical Properties: Carbohydrates are a varied class of macromolecules and thus have different
physical properties. Glucose, for example, is a crystalline white powder at room temperature. It
has a melting point of 153 to 156 degrees Celsius ( Oyeleke et al., 2021).
25
Starch is a complex carbohydrate and contains many molecules of glucose strung together. As a
result of the increased complexity it has a higher melting point of 256-258 degrees Celsius. At
room temperature starch is also a white crystalline powder ( Oyeleke et al., 2021).
Monosaccharides are connected with glycosidic bonds. This type of bond is found only in
carbohydrates and connects two monosaccharides together with an ether group. Glycosidic bonds
are categorized based on which elements are involved in bonding to the carbon. Most glycosidic
bonds use only carbon, hydrogen and oxygen, but some may use sulfur or nitrogen. For example,
an N-glycosidic bond is a carbon attached to a nitrogen and is common in the addition of
carbohydrates to proteins (Belli, 2020).
Glycosidic bonds are assembled using dehydration synthesis, also known as a condensation
reaction. In this reaction, the hydroxyl group of one carbon atom leaves with an additional
hydrogen atom from the other carbon, creating water. There are two main types of glycosidic
bonds, 1,4-glycosidic bond and the 1,6-glycosidic bond. These two types of bonds are based on
which carbon atoms are being attached in the monosaccharides. The two types of glycosidic bonds
determine the structure of the carbohydrate chain. A 1,4-glycosidic bond creates a linear structure
and the 1,6-glycosidic bond creates a branch structure.
There are also two types of bonds, alpha and beta, depending on the stereochemistry of the bond.
If the bond is formed below the glucose ring the bond is alpha and if the bond is formed above the
ring the bond is beta. This has important implications for humans because the digestive tract is
only able to break down alpha linkages. So, plant based carbohydrates like cellulose that use beta
linkages cannot be broken down and absorbed.
26
2.6.1 Functions of carbohydrates in the body
Carbohydrates are an essential part of diet. Most importantly, they provide the energy for the most
obvious functions of the body, such as moving or thinking, but also for the ‘background’ functions
that most of the time even noticed (Kenneth, 2018). During digestion, carbohydrates that consist
of more than one sugar get broken down into their monosaccharides by digestive enzymes, and
then get directly absorbed causing a glycaemic response. The body uses glucose directly as energy
source in muscle, brain and other cells. Some of the carbohydrates cannot be broken down and
they get either fermented by the gut bacteria or they transit through the gut without being changed.
Interestingly, carbohydrates also play an important role in the structure and functions of cells,
tissues and organs (Kenneth, 2018).
Carbohydrates as energy source and their storage: Carbohydrates broken down to mainly glucose
are the preferred source of energy for the body, as cells in the brain, muscle and all other tissues
directly use monosaccharides for their energy needs (Kenneth, 2018). Depending on the type, a
gram of carbohydrates provides different amounts of energy:

Starches and sugars are the main energy-providing carbohydrates, and supply 4 kilocalories (17
kilojoules) per gram

Polyols provide 2.4 kilocalories (10 kilojoules) (erythritol is not digested at all, and thus gives 0
calories)

Dietary fibre 2 kilocalories (8 kilojoules) (Kenneth, 2018).
Monosaccharides are directly absorbed by the small intestine into the bloodstream, where they are
transported to the cells in need. Several hormones, including insulin and glucagon, are also part of
27
the digestive system. They maintain our blood sugar levels by removing or adding glucose to the
blood stream as needed (Kenneth, 2018).
If not used directly, the body converts glucose to glycogen, a polysaccharide like starch, which is
stored in the liver and the muscles as a readily available source of energy. When needed, for
instance, between meals, at night, during spurs of physical activity, or during short fasting periods,
our body converts glycogen back to glucose to maintain a constant blood sugar level (Kenneth,
2018).
The brain and the red blood cells are especially dependent on glucose as energy source, and can
use other forms of energy from fats in extreme circumstances, like in very extended periods of
starvation. It is for this reason that our blood glucose must be constantly maintained at an optimum
level. Approximately 130 g of glucose are needed per day to cover the energy needs of the adult
brain alone (Kenneth, 2018).
2.3 Vitamins
Vitamins are organic compounds that are necessary for normal growth and maintenance of life. The
body cannot synthesize vitamins; they must be taken in food and food supplements. Adequate intake
is necessary for normal functioning of the body (Tanko et al., 2019). Thirteen essential vitamins
have been isolated and these are divided into 2 categories; Water soluble vitamin and fat soluble
vitamins. The water soluble vitamins include vitamin C and the B group of vitamins. These water
soluble vitamins are not stored by the body and can be readily depleted. The fat soluble vitamins
include vitamin A, D, E and K. They can be stored in the body. Vitamin D sometimes is not regarded
28
as essential because it can be synthesized in the body by the action of ultra violet rays of sun on 7
dehydrocholcalciferol in the skin of humans (Petrovska, 2021).
Carnitine a vitamin like compound very indispensable for survival and health are not strictly
"essential" because the human body has some capacity to produce them from other compounds
(www.nutrition. Org. uk/healthyliving). Specific conditions are known to arise as a consequence of
a dietary deficiency of one or more of the vitamins. These conditions can be avoided if meals are
well planned and carefully prepared (Nnam 2021). Dietitians advice that the practice of warming or
heating vegetable soups every morning should be discouraged as many vitamins are lost in the
process. Excess of some vitamins especially the fat soluble vitamins could be dangerous to health.
In general vegetables are good sources of vitamins. The factors that influence the amount of vitamins
in green leafy vegetables are cultivars, maturity and light (Egbuna, 2018). Green leafy vegetables
are the richest source of thiamin and riboflavin, ascorbic acid and beta carotene (Pro-vitamin A).
Oguntona, (2017) and Egbuna (2018) reported that Niacin and folate are found in reasonable amount
in green leafy vegetables. Other sources of vitamins include meat, eggs poultry, fish, and cereals.
2.3.1 Vitamin E
Vitamin E is a fat-soluble vitamin existing in eight different forms. In humans, α-tocopherol is the
most active form, and is the major powerful membrane bound antioxidant employed by the cell
(Hensley et al., 2017). Pryor (2018) reported that the main function of Vitamin E is to protect against
lipid peroxidation. Also, a number of evidences suggest that α-tocopherol and ascorbic acid function
synergistically in a cyclic-type process (Rahman, 2019). α-tocopherol is converted to an αtocopherol radical by the donation of a labile hydrogen to a lipid or lipid peroxyl radical during
29
antioxidant reaction. The α-tocopherol radical can therefore be reduced to the original α-tocopherol
form by ascorbic acid (Kojo, 2017).
2.3.2 Vitamin C
Vitamin C is also known as ascorbic acid. It is an important and efficient water-soluble antioxidant
and hence functions in aqueous environments of the body (Rahman, 2019). Vitamin C works
synergistically with vitamin E and the carotenoids as well as other antioxidant enzymes. Importantly,
vitamin C functions in the regeneration α-tocopherol from α-tocopherol-radicals in membranes and
lipoproteins (Kojo, 2017). It also raises intracellular glutathione levels thus playing an important
role in protein thiol group protection against oxidation (Naziroglu and Butterworth, 2020).
2.3.3 Vitamin B Complex
A number of research works on Vitamin B1 have reported the role of vitamin B1 in the treatment of
oxidative stress-related diseases(Gibson and Zhang, 2019). Thiamine diphosphate which is the
active form of vitamin B1 functions as a co-factor for several enzymes, which are important in the
biosynthesis of reducing equivalents used in oxidant stress defenses (Singleton and Martin, 2018).
Thiamine deficiency has been reported to promote an increase in oxidative stress and
neurodegeneration (Gibson and Zhang, 2019); however, its effectiveness in treating diseases
associated with free radicals is still unclear (Bonnefont-Rousselot, 2017; Nascimento et al., 2020).
Nicotinamide, the amide form of niacin (vitamin B3) is a known precursor for both nicotinamide
adenine dinucleotide (NAD/NADH), and nicotinamide adenine dinucleotide phosphate (NADP).
These reducing equivalents play an important role in energy metabolism, signal transduction, aging,
cellular injury (Aksoy et al., 2020), and show significant inhibition of oxidative damage induced by
ROS (Feng et al., 2020).
30
Riboflavin (Vitamin B2) is another co-factor, which is converted to flavin dinucleotide. This serves
as a coenzyme for glutathione reductase and other enzymes (Manthey et al., 2020). Low intakes of
Riboflavin have been associated with different diseases including cancer and cardiovascular diseases
and there is evidence that treatment with riboflavin can provide some benefit against diseases
associated with oxidative stress (Bonnefont-Rousselot, 2017).
2.3.4 Vitamin K
Vitamin K is naturally produced by the bacteria in the intestines, and plays an essential role in
normal blood clotting, promoting bone health, and helping to produce proteins for blood, bones,
and kidneys (Eneobong 2018).
Good food sources of vitamin K are green, leafy-vegetables such as turnip greens, spinach,
cauliflower, cabbage and broccoli, and certain vegetables oils including soybean oil, cottonseed
oil, canola oil and olive oil. Animal foods, in general, contain limited amounts of vitamin K.
To help ensure people receive sufficient amounts of vitamin K, an Adequate Intake (AI) has been
established for each age group (Jeruto et al., 2021).
Without sufficient amounts of vitamin K, hemorrhaging can occur. Vitamin K deficiency may
appear in infants or in people who take anticoagulants, such as Coumadin (warfarin), or antibiotic
drugs (Mahesh and Statish, 2019). Newborn babies lack the intestinal bacteria to produce vitamin
K and need a supplement for the first week. Those on anticoagulant drugs (blood thinners) may
become vitamin K deficient, but should not change their vitamin K intake without consulting a
physician. People taking antibiotics may lack vitamin K temporarily because intestinal bacteria are
sometimes killed as a result of long-term use of antibiotics (Rahman, 2019). Also, people with
chronic diarrhea may have problems absorbing sufficient amounts of vitamin K through the
intestine and should consult their physician to determine if supplementation is necessary.
31
Although no Tolerable Upper Intake Level (UL) has been established for vitamin K, excessive
amounts can cause the breakdown of red blood cells and liver damage (Rice-Evans, 2018). People
taking blood-thinning drugs or anticoagulants should moderate their intake of foods with vitamin
K, because excess vitamin K can alter blood clotting times. Large doses of vitamin K are not
advised.
2.3.5 Vitamin D
Vitamin D plays a critical role in the body’s use of calcium and phosphorous. It works by
increasing the amount of calcium absorbed from the small intestine, helping to form and maintain
bones (Omage and Azeke, 2017). Vitamin D benefits the body by playing a role in immunity and
controlling cell growth and may protect against osteoporosis, high blood pressure, cancer, and
other diseases. Children especially need adequate amounts of vitamin D to develop strong bones
and healthy teeth (Schroeter et al., 2019).
The primary food sources of vitamin D are milk and other dairy products fortified with vitamin D.
Vitamin D is also found in oily fish (e.g., herring, salmon and sardines) as well as in cod liver oil.
In addition to the vitamin D provided by food, we obtain vitamin D through our skin which
produces vitamin D in response to sunlight (Schroeter et al., 2019).
The Recommended Dietary Allowance (RDA) for vitamin D appears as micrograms (mcg) of
cholecalciferol (vitamin D3). From 12 months to age fifty, the RDA is set at 15 mcg. Twenty mcg
of cholecalciferol equals 800 International Units (IU), which is the recommendation for
maintenance of healthy bone for adults over fifty. Table 1 lists additional recommendations for
various life stages (Jeruto et al., 2021).
Exposure to ultraviolet light is necessary for the body to produce the active form of vitamin D.
Ten to fifteen minutes of sunlight without sunscreen on the hands, arms and face, twice a week is
32
sufficient to receive enough vitamin D. This can easily be obtained in the time spent riding a bike
to work or taking a short walk with arms and legs exposed. In order to reduce the risk for skin
cancer one should apply sunscreen with an SPF of 15 or more, if time in the sun exceeds 10 to 15
minutes (Chukwujeku et al., 2020).
Symptoms of vitamin D deficiency in growing children include rickets (long, soft bowed legs) and
flattening of the back of the skull. Vitamin D deficiency in adults may result in osteomalacia
(muscle and bone weakness), and osteoporosis (loss of bone mass). Vitamin D deficiency has been
associated with increased risk of common cancers, autoimmune diseases, hypertension, and
infectious disease. Research shows that vitamin D insufficiency affects almost 50% of the
population worldwide; an estimated 1 billion people (Ramos-Moraleset al., 2017). The rising rate
of deficiency has been linked to a reduction in outdoor activity and an increase in the use of
sunscreen among children and adults. In addition, those who live in inner cities, wear clothing that
covers most of the skin, or live in northern climates where little sun is seen in the winter are also
prone to vitamin D deficiency. Since most foods have very low vitamin D levels (unless they are
enriched) a deficiency may be more likely to develop without adequate exposure to sunlight.
Adding fortified foods to the diet such as milk, and for adults including a supplement, are effective
at ensuring adequate vitamin D intake and preventing low vitamin D levels. In the absence of
adequate sun exposure, at least 800 to 1,000 IU of vitamin D3 may be needed to reach the
circulating level required to maximize vitamin D’s benefits (Singh, 2018).
2.4 Mineral
Trace element are also known as micronutrient and are found only in minute quantity in the body
yet they are vitally important. The quantity in which they are found is so small, that they can only
be detected by spectrographic method. The following are considered as micronutrient: copper,
zinc, iron and cobalt (Skjevdal, 2017).
33
2.4.1 Copper
Copper is not poisonous in its metallic state but some of its salts are poisonous, especially the most
common salts of copper are the Sulphate or the blue vitriol and the sub-acetate or Verdigris
(Remeuf and Lenoir 2013). Copper Sulphate is a crystalline salt with blue colour and metallic taste
in a small dose of 0.5 g it acts as an emetic but in large doses, as an irritant poison produces gastric
and intestinal irritation. Copper subacetate is a blue green salt formed by the action of vegetable
acids on copper cooking vessels which are not properly lined and which have been used for
cooking and storage for a long time (Skjevdal, 2017). Copper compounds of Arsenic include
Scheele’s green (Copper Arsenite), Paris green or emerald green (Copper Aceto-arsenite) etc.
Copper is a powerful inhibitor of enzymes. Poisoning effect of copper will commence within 15
to 30 minutes.
Copper is a reddish brown nonferrous mineral which has been used for thousands of years by many
cultures (Haenlein and Wendorff, 2006). The name for the metal comes from Kyprios, the Ancient
Greek name for Cyprus, an island which had highly productive copper mines in the Ancient world.
Its atomic number is 29, placing it among the transition metals. The metal is highly conductive of
both electricity and heat, and many of its uses take advantage of this quality. Copper can be found
in numerous electronics and in wiring. It is also used to make cooking pots. This metal is also
relatively corrosion resistant. For this reason, it is often mixed with other metals to form alloys
such as bronze and brass (Haenlein, 2014). The metal is closely related with silver and gold, with
many properties being shared among these metals. Modern life has a number of applications for
34
copper, ranging from coins to pigments, and demand for the metal remains high, especially in
industrialized nations. Many consumers interact with it in various forms on a daily basis.
Copper, vitally is important to root metabolism, help form compounds and protein, amino acid and
a host organic compounds. It act as a catalyst or part of the enzyme system, it help to produce dry
matter through stimulation of growth, prevent development of chlorosis, resetting and dieback
(Sudith and Grey, 2006).
An excess of copper result in degeneration of the liver. It causes blood in urine and poor utilization
of nitrogen.
Copper is needed for the formation of bone, hemoglobin and red blood cells, copper also promotes
healthy nerves, a healthy immune system and collagen formation (Sudith and Grey, 2006). Copper
work in balance with zinc and vitamin c along with manganese, magnesium and iodine copper
plays an important role in memory and brain function. Nuts, molasses and oats contain copper but
liver is the best easily assimilated source. This complexing is of great important in maintaining
adequate copper in solution for plant use (Johe et al., 2011).
In the human body, most of the copper, Cu, is present as Cu+ (cuprous) and oxidized Cu2+ (cupric)
compounds. Copper is therefore an intermediary for electron transfer in redox reactions. The
oxidation states, Cu3+ and Cu4+, are uncommon (Kondyli et al., 2011). The average daily intake
of copper is between 0.5 and 1.5 mg, coming mainly from seeds, grains, shellfish, nuts, beans and
liver. The current recommended dietary intake in the USA is 0.9 mg/day (Carles et al., 2017).
Copper is mainly absorbed in the duodenum and proximal jejunum, with a little bit of absorption
occurring in the stomach and the distal portion of the small intestine (Jacobus et al., 2010). The
human copper transport protein 1 (hCTR1), located at the level of enterocytes, transfers the ion
following the reduction of dietary Cu2+ into Cu+. In hepatocytes, copper binds to metallothioneins
35
(MTs), to reduced glutathione (GSH) or to one of the copper chaperones regulating the traffic of
intracellular copper (CCS: chaperone for superoxide dismutase 1 SOD1, which is the sole cytosolic
cuproenzyme; COX17: chaperone for cytochrome C oxygenase; ATOX1 antioxidant-1: chaperone
for the ATPases, ATP7A and ATP7B). The group of transmembrane copper transporters includes
CTR1, ATP7A and ATP7B. ATP7A (expressed in the placenta, gut and nervous system) and
ATP7B (expressed in the hepatocytes, where it exports copper into the bile and provides copper
to nascent ceruloplasmin, and in the nervous system) are linked to the enzyme, tyrosinase, and the
ceruloplasmin, respectively (Whitney et al., 2011).
Acquired copper deficiency is mainly attributable to nutritional deficiency, and may be seen in
mal- nourished low-birth-weight infants, newborns, and small infants. Copper deficiency has also
been reported to develop after gastrointestinal surgery, intractable diarrhea, and prolonged
parenteral or enteral nutrition. However, since copper supplementation of intravenous and enteral
nutritional formulas was made mandatory, the incidence of copper deficiency has decreased
dramatically (Boulanger et al., 2008). An acquired copper excess state has been described in cases
of Indian childhood cirrhosis, non-Indian childhood cirrhosis, excessive copper intake, and
parenteral bolus administration of copper for the treatment of copper deficiency.
1. Bone lesions in copper deficiency states: Rachitic-like or scorbutic-like changes
(enlargement of the epiphyseal area and changes in the margin) are observed in the bones
of extremities. They may be accompanied by osteoporosis and occipital horn formation
after adolescence (Brozos et al., 2014). These are attributable to functional impairment of
copper requiring enzymes, such as ascorbate oxidase and lysyloxidase, associated with
copper deficiency.
36
2. Hair abnormalities: Change of hair texture, namely, kinky-hair, may be observed in
children with Menkes disease. Hair changes are, however, considered rare in cases with
secondary copper deficiency (Brozos et al., 2014). On the other hand, the possibility of
changes in the hair should be borne in mind in cases of prolonged copper deficiency. The
copper content of the hair and nail is decreased in cases of copper deficiency (Brozos et
al., 2014).
3. Vascular lesions: Menkes disease is characterized by tortuosity and winding of arteries
and increased capillary fragility (Lidon et al., 2014). Caution must be exercised to avoid
prolonged copper deficiency in humans, since this may lead to abnormal vascular tortuosity
and increased capillary fragility.
4. Central nervous system disorder and convulsion: Reports of central nervous system
disorder and convulsion associated with secondary copper deficiency are rare, but they are
characteristic features of Menkes disease (Brozos et al., 2011). Progressive Menkes disease
can be fatal. Prolonged copper deficiency may cause degeneration of the cerebrum and
cerebellum (numerous copper- requiring enzymes are present in the brain, such as
dopamine-hydroxylase and cytochrome c oxidase), associated with slowing of mentation
and muscular rigidity, as well as hemorrhagic changes due to increased capillary (Lidon et
2.4.2 Zinc
Zinc is an essential component of many enzyme in the dehydrogenase, proteinase and peptidase
groups, it is a minue catalyst (Mcintosh et al., 2014). Zinc help to make acetic acid in the root to
prevent rotting, it Is used to control blight and allow dead twigs on trees to shed off.
Zinc contribute to test weight, increased corn ear, size, promote cornsilking, hastens maturity,
chlorophyll formation, enzyme functions and regulates plant growth.
37
Zinc is absolutely essential for production of sperm. Zinc is well known for it’s need in animal
nutrient and most commercial livestock producer supplement animal feed with these minerals. For
that reason, manures from commercial livestock operation are very good sources of zink. It also
increases the need for vitamin A. (Foth, 2009).
The importance of zinc in biological systems was recognized as early as 1869 by Raulin during
studies on Aspergillusniger (Scheiber et al., 2014). Although the importance of zinc in animals
had been established. Since then, the importance of zinc in human metabolism and growth in both
health and disease has become well established (Rouault et al., 2011). It is currently known that
over 200 zinc metalloenzymes exist in the human body. Of these, many e.g., carbonic anhydrase,
alkaline phosphatase, carboxypeptidase A, B etc., perform a variety of vital functions. However,
zinc deficiency does not exert its effects through deficient function of these enzymes alone. Zinc
also performs a vital biological role in maintenance of biomembranes and is also considered
essential for DNA replication, transcription and translation. Other important roles attributed to zinc
include maintenance of adequate immune function and brain development (Gardner and Hogue,
2015). Our knowledge on the scope and contribution of zinc nutrition to paediatric physiology has
widened considerably since the initial limitation to six clinical ‘syndromes’ and zinc is now
considered crucial to the maintenance of satisfactory growth in childhood. Zinc deficiency has
been shown to effect the function of human growth hormone by modulating with the function of
the polypeptide hormone-receptor ‘zinc sandwich. This could provide a mechanism to explain the
close relationship between alteration in zinc nutrition and plasma insulin-like growth factors
(Kondyli et al., 2011). A growth limiting mild zinc deficiency state has been described in young
boys with short stature. Although initial studies of zinc supplementation of the diet failed to show
any significantly, greater effect on growth and appetite, other studies of zinc supplementation have
38
shown to significantly improve the linear growth and weight gain of preschool children with
stunting (Kondyli et al., 2011).
2.4.3 Calcium
Calcium is an essential bulk mineral. It makes up bones and teeth and is essential for the
transmission of information of the right calcium balance for the maintenance of health cannot be
overestimated (Bland, 2009).
Calcium is part of bones and teeth. In addition, it plays a role in neuromuscular excitability
(decreases it), good function of the conducting myocardial system, heart and muscle contractility,
intracellular information transmission and blood coagulation ability (Torres-Hernandez et al.,
2008). Osteoporosis and osteomalacia are the most common manifestations of calcium deficiency;
a less common but proved disorder attributable to Ca deficiency is hypertension. Based on newly
acquired epidemiological data, implication of Ca deficiency in other disorders is currently being
discussed (Wallace et al., 2005). The recommended Ca daily intake for adults’ ranges between
700 and 1000 mg. Some population groups may need a higher intake.
Calcium deficiency is a condition in which the body has an inadequate amount of calcium. Calcium
is a mineral that is essential for many aspects of health, including the health of bones and teeth,
and a normal heart rhythm (Cornell and Pallansch, 2008). This mineral is also required for muscle
contractions and relaxation, nerve and hormone function, and blood pressure regulation. Calcium
must be ingested daily and absorbed effectively in order to maintain optimal health. Most people
can get enough calcium by eating a variety of foods rich in calcium. Foods that naturally contain
calcium include milk and other dairy products; green, leafy vegetables; seafood, nuts, and dried
beans. Calcium is also added to orange juice, breakfast cereals, breads, and other fortified food
products. High dietary calcium intake is necessary for infants, children and adolescents in order to
39
promote bone growth and formation. Pregnant women also have higher calcium needs, because it
is required for the normal development of fetal bones. In addition, women who have reached
menopause need to ensure an adequate amount of calcium intake to reduce the risk of osteoporosis
(Cornell and Pallansch, 2008).
2.4.4 Magnesium
Magnesium is referred to as a macromineral, which means that our food must provide us with
hundreds of milligrams of magnesium everyday. Inside our bodies,magnesium is found mostly in
our bones (60-65 %), but also inour muscles (25 %) and in other cell type and body fluids.
Magnesium is sometimes regarded as smoothie mineral, since it has the ability to relax our
muscles. Our nerves also depend upon magnesium to avoid becoming overexcited and this aspect
of magnesium links this mineral to maintenance of healthy blood pressure (Meiners and Derise,
2009).
The metabolic role of magnesium include:
1. Bone Formation: About two thirds of all magnesium in our body is found in our bones.
Bone magnesium has two very different role to play in our health. Some of the magnesium
in our bones helps to give them their physical structure. This magnesium is part of the bone
crystal lattice and is found in this bone scaffolding together with the minerals. Other
amount of magnesium, however, are found on the surface of the bone. This surface
magnesium does not appear to be involved in the bones structure, instead acts as a storage
site for magnesium which the body can draw upon in time of poor dietary supply. (Touyz,
2009).
40
2. Verve and Muscle Relaxation: Magnesium help regulate the body nerve and muscle tone.
Magnesium serves as a chemical gate blocker as long as there is enough magnesium around
(Meiners and Derise, 2009). This gate blocking by magnesium helps keep the nerve
relaxed. If our diet provide us with too little magnesium, this gate blocking can fail and the
nerve cell can become over activated when some nerve cell are activated, they can send
too many messages to the muscles and cause the muscle to over contract. This chain of
events helps explain how magnesium deficiency can trigger muscle tension soreness and
muscle fatigue.
3. Presence of an Enzymes: Enzymes are special proteins that help trigger chemical
reactions. Over 300 different enzyme in the body require magnesium in order to function.
Magnesium is involved in the metabolism of protein, carbohydrates and fats. It help genes
function properly, our cardiovascular system, digestive system, nervous system, muscles,
kidneys, liver and brains all rely on magnesium for their metablic function (Meiners and
Derise, 2009).
2.4.5 Phosphorus
Phosphorus is the second most abundant essential mineral in the human body after calcium. It not
only plays a role in numerous biologic processes, including energy metabolism and bone
mineralization, but also provides the structural framework for DNA and RNA (Cheaters et al.,
2013). It is synthesized through various biochemical pathways such as glycolysis and beta
oxidation. As a part of signal transduction, phosphate is used in cyclic adenosine monophosphate
(AMP) and products of deoxyribonucleoside diphosphates like dADP, dCDP, dGDP, and dUDP
(O’Dell et al., 2016). Approximately 80% to 90% of phosphorus is present in the bones and teeth
in the form of hydroxyapatite (Ca10 (PO4)6(OH)2) (Cheaters et al.,2013). The remainder is
41
present in extracellular fluid (ECF), soft tissues and erythrocytes. Serum and plasma contain only
a small fraction of total body phosphorus in the form of inorganic phosphate, lipid phosphorus,
and phosphoric ester phosphorus. Thus, changes in serum phosphate levels do not necessarily
reflect the body’s total store of phosphorus (Cheaters et al., 2013).
A normal diet provides approximately 20 mg/kg/day of phosphorus of which 16 mg/kg/day is
absorbed in the small intestine (predominantly in the jejunum) by both para-cellular and intracellular processes. The intra-cellular process is mediated via Sodium- Phosphate co-transport
present on villi of small intestine (Chase et al., 2014). The paracellular pathway is a concentration
gradient-dependent, passive transport system. Increase in dietary phosphorus leads to an increase
in phosphate absorption with little evidence of an upper limit or saturation of absorption process
(O’Dell et al., 2016). Three mg/kg/day of phosphorus is secreted into the intestine via pancreatic
and intestinal secretions, giving a net phosphorus absorption of around 13 mg/kg/day. Once
absorbed, phosphate/phosphorus enters the ECF and circulation where it is taken up by bones,
teeth, and other soft tissues via the action of sodium phosphate co-transporters. Three sodiumphosphate co- transporters have been described: NaPi-I, NaPi-II (a, b, and c) and NaPi-III. NaPiIIb is localized in the small intestine whereas NaPi-IIa and NaPi-IIc are found in the kidneys, with
the former responsible for at least 85% of renal phosphate absorption through intracellular
processes (Cheaters et al., 2013). The rate of reabsorption and mineralization is important in
determining the serum phosphorus concentration. Approximately 3 mg/kg/day of phosphorus is
exchanged between mineralized bones and the ECF.
Phosphate is filtered freely across the glomerulus-about 13 mg/ kg/day in a normal adult-and 60%
to 70 % of filtered phosphate is reabsorbed in the proximal tubule (with 10 % to 15% reabsorbed
in the distal tubule) (Bettger and O’Dell, 2012). This transport is mediated via a sodium-gradient
42
dependent process and sodium-phosphate co-transporters (type I and type IIa/IIc) on the luminal
brush border membrane of the proximal tubules (Wagner, 2016). Seven mg/kg/day of phosphorus
is excreted through the feces. Alterations in the expression of co-transporters on the luminal brush
border membrane and microvilli of the small intestine determine the rate of phosphate
reabsorption.
2.4.6 Manganese
Manganese (Mn) is an essential micronutrient in most organisms. In plants, it participates in the
structure of photosynthetic proteins and enzymes. Its deficit is dangerous for chloroplasts because
it affects the water splitting system of photosystem II (PSII), which provides the necessary
electrons for photosynthesis (Buchanan, et al., 2012). However, its excess seems also to be
particularly damaging
to
the photosynthetic apparatus. Thus, Mn has two roles in the plant
metabolic processes: as an essential micronutrient and as a toxic element when it is in excess
(Ducic and Polle, 2015). Mn toxicity is favored in acid soils with decreasing pH, the amount of
exchangeable manganese – mainly Mn2+ form – increases in the soil solution. This Mn form is
available forplants and can be readily transported into the root cells and translocated to the shoots,
where it is finally accumulated (Marschner, 2015). In contrast, other forms of Mn predominate at
higher pH values, such as Mn (III) and Mn (IV), which are not available and cannot be accumulated
in plants (Rengel, 2012).
There is some evidence that human diseases such as amylotrophic lateral sclerosis, acromegaly
and epilepsy are associated with low tissue levels of manganese and that the manganese intake of
many people is below the estimated safe, adequate dietary intake (Saric and Lucchini, 2011).
Reported deficiency symptoms include ataxia, fainting, hearing loss, weakness in tendons and
ligaments and, possibly, type 2 diabetes mellitus (since low levels of manganese reduce insulin
43
production and impair glucose metabolism). Manganese deficiency might also develop from
failure to absorb the metal, which normally takes place in the small intestine via a carrier-mediated
mechanism (Cowan et al., 2012). Manganese and iron compete for sites of absorption in the gut,
while fibre, phytates, calcium, phosphorus and excessive intake of magnesium may also interfere
with manganese absorption.
Absorption of ingested manganese is generally low but appears to be relatively higher in infants
than in adults. Bioavailability of manganese from different food types is variable, but is generally
low, due to poor solubility of manganese salts. Once absorbed, Mn in the hepatic portal blood
binds to albumin and alpha-2 macroglobulin. A small proportion of Mn in the systemic circulation
is bound to transferrin (Zouni et al., 2011).
Use of manganese supplementation to treat fatigue, nervousness and irritability (possibly by
enhancing brain enzyme activity) and poor memory (by inducing SOD and protecting brain tissue)
was originally reported by Carl Pfeiffer in his book “Mental and Elemental Nutrients” (Pfeiffer,
2014). Pfeiffer suggested that manganese, along with zinc, will help decrease copper levels by
both decreasing absorption and increasing urinary losses. He claimed that copper, in physiological
but higher than normal amounts, can cause psychological problems and even schizophrenia and
that this reflects an underlying tissue manganese deficiency.
2.4.7 Sodium
Sodium Is a metallic element with a symbol Na, the same group with Li, K, Rb, Cs is widespread
in nature in the form of salts (nitrates, carbonates, chlorides), atomic number 11 and atomic weight
22,9898, sodium is the sixth most abundant element in The Earth crust, which contains 2,83% of
sodium in all its forms. Na is, after chloride, the second most abundant element dissolved in
seawater (Lidon et al., 2014). The most important sodium salts found in nature are sodium chloride
44
(halite or rock salt), sodium carbonate (trona or soda) sodium borate (borax), sodium nitrate and
sodium sulfate. Sodium salts are found in seawater (1,05 %), salty lakes, alkaline lakes and mineral
spring water. Is an essential element for all animal life (including human) and for some plant
species (kumar et al., 2009). In animals, sodium ions are used in opposition to potassium ions, to
allow the organism to build up an electrostatic charge on cell membranes and thus allow
transmission of nerve impulse when the charge is allowed to dissipate by a moving wave of voltage
change. Sodium is thus classified as a “dietary inorganic macro-mineral” for animals.
Sodium is a compound of many foodstuff, for instance of common salt, it’s is necessary for humans
to maintain the balance of the physical fluid system, is also required e physical fluids system for
nerve and muscle functioning (Giebisch et al., 2015). Too much sodium can damage our kidneys
and increase the chances of high blood pressure. The amount of sodium a person consumes each
day varies from individual to individual and from culture to culture, some people get as little as
2g\day, some as much as 20 g (Pendias and Pendias, 2012). Sodium essential, but contriversely
surrounds the amount required. Contact of sodium with water, including perspiration causes the
formation of sodiumContact to the skin may cause itching, tingling, thermal and caustic burns and
permanent damage. Contact with eye may result in permanent damage and loss of sight.
2.4.8 Iron
Rich source of dietary iron include milk, red meat, beans, fish, leaf vegetable and fortified bread.
Iron in low amount is found in molasses. Although some studies suggested that heme/hemoglobin
from red meat has effects which may increase the likelihood of colorectal cancer. Iron in milk is
more easily absorbed than iron in vegetable (Lidon et al., 2014).
45
Iron distribution is heavily regulated in mammals, partly because iron have a high potential for
biological toxicity (Nanami et al., 2005).
Iron uptake is tightly regulated by the human body which has no regulated physiological means of
excreting iron. Iron plays an important role in biology, forming complex with molecular biology
in hemoglobin and myoglobin, these two compound are common oxygen transport protein in
vertebrate (kumar et al., 2009). Iron is also the metal use at the active site of many important redox
enzyme dealing with circular respiration, oxidation and reduction in plant and animals. Only small
amount of iron is loss daily due to mucosal and skin epithelial cell sloughing, so control of iron
level is mostly by regulating uptake.
46
CHAPTER THREE
MATERIALS AND METHODS
3.1 Materials
3.1.1. Equipment and Instrument Used
All equipment and instrument used in this study were of analytical standard. The major ones
includes; Rotary evaporator (Model 349/2, Corning Ltd, England), Stopper florescence flask
(Pyrex, England), Kjeldaic flask (Pyrex, England), Fume hood (Rugromag), Electronic weighing
balance (Pyrex, England), Crucible (USA), Desiccators (USA), Spectrophotometer (Sharwood
Scientific Ltd, Cambridge UK), Steam bath (Bradford, England), Electric oven (USA), Flame
photometer (Sharwood Scientific Ltd, Cambridge UK), Refrigerator (Haier Thermocol, England),
Elisa with a micro plate rader MF 9602 (Bio trust diagnostic incorporated, USA), Soxlet apparatus
(Bradford, England), Murfle furnace (Bradford, England), Calibrated precision pipettes (USA),
Microtiter plate shaker (USA), Absorbent material (USA), Vortex mixer (USA) and Centrifuge
(Gallenkamp, Germany).
47
3.1.2 Chemicals and Reagents Used
All chemicals and reagents used in this study were of high analytical standard. The chemicals were
sourced from May and Baker, England; BDH, England and Merck, Damstadt, Germany. The
reagents used were commercial kits and products of Randox, QCA, USA and Biosystem Reagents
and Instruments, Spain.
3.1.3 Biological Materials
Terminalia superba is the biological materials used in this study.
Stem bark extract of Terminalia superba was collected in Abakaliki, Ebonyi State
3.2 Methods
3.2.1 Preparation of Plant Extract
Stem bark extract of Terminalia superba were washed with distilled water, dried at room
temperature, then pulverized, weighed and used for the study.
Then 1kg of dried pulverized of Stem bark extract of Terminalia superba was poured into a small
glass container and 2 litres of distilled water was added to it and stirred. After 48hrs of soaking, it
was filtered using Whatman filter paper. The filtrate was concentrated using rotary evaporator.
This crude aqueous extract was used for the study.
3.2 Methods
3.2.1 Proximate Analysis: The samples were analyzed for proximate compositions which include
moisture content, fat/oil, ash, protein, fiber and carbohydrate contents. Official methods of AOAC,
1980 were used in carrying out the proximate analysis.
48
3.2.1.1 Moisture and Volatile Matter (A.O.A.C., 1980)
10g of the sample was transferred into a crucible and put in an oven set at 110OC for three hours.
The dried matter was weighted, and the difference or loss in weight is calculated in percentage as
the estimation of moisture and volatile matter.
The moisture content was calculated as:
% 𝑚𝑜𝑖𝑠𝑡𝑢𝑟𝑒 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 =
𝑂𝑟𝑖𝑔𝑖𝑛𝑎𝑙 𝑊𝑒𝑖𝑔ℎ𝑡 − 𝐷𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡
𝑂𝑟𝑖𝑔𝑖𝑛𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡
𝑋
100
1
3.2.1.2. Fat/Oil Content (AOAC, 1980):
10g of the sample was transferred into a thimble of a soxlet extractor containing 250ml of
petroleum ether in a position and then connected to a condenser. The set up was heated for four
hours and the filtrate was evapourated to remove the solvents and concentrate the fat/oil with a
rotary evaporator, dried and weighed.
The percentage oil content was calculated as:
% 𝐶𝑟𝑢𝑑𝑒 𝑓𝑎𝑡/𝑜𝑖𝑙 =
𝐹𝑖𝑛𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑑𝑟𝑦 𝑓𝑎𝑡/𝑜𝑖𝑙
𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒
𝑋
100
1
3.2.1.3. Crude Protein (MicroKjedahl Method): Protein content was determined according to
AOCS method 5-38 and crude protein calculated using a nitrogen factor (6.25) (IUPAC, 1979).
49
0.1g of the sample was transferred into a Kjedahl flask and 2ml of digestion mixture added. It was
heated until clear. It was made up to 20ml with distilled water, 2ml was transferred into three (3)
test tubes and 0.5ml of Nessler’s reagent added, mixed, and 1ml of 0.1% gum ghatti was added.
The solution was made up to 10ml with water and absorbance taken at 490nm against a blank in 1
in 5 dilutions. The absorbance readings were used to plot a standard curve and the percentage
composition taken.
3.2.1.4 Crude Fiber: The defatted sample was boiled with 200ml of 0.255N sulfuric acid for 1
hour, filtered with suction and the residue was boiled with 200ml of 0.313N sodium hydrozide for
1hour and filtered. The residue was washed with hot water and dried. The dried residue was
weighed into a crucible and ashed in a muffle furnace and the ash weighed.
The percentage crude fiber is calculated as:
% 𝐶𝑟𝑢𝑑𝑒 𝑓𝑖𝑏𝑒𝑟 =
𝑊𝑡 𝑜𝑓 𝑐𝑟𝑢𝑐𝑖𝑏𝑙𝑒 & 𝑎𝑠ℎ 𝑎𝑓𝑡𝑒𝑟 𝑎𝑠ℎ𝑖𝑛𝑔 − 𝑤𝑡 𝑜𝑓 𝑐𝑟𝑢𝑐𝑖𝑏𝑙𝑒
𝑊𝑡. 𝑜𝑓 𝑐𝑟𝑢𝑐𝑖𝑏𝑙𝑒 𝑎𝑛𝑑 𝑠𝑎𝑚𝑝𝑙𝑒 − 𝑤𝑡. 𝑜𝑓 𝑐𝑟𝑢𝑐𝑖𝑏𝑙𝑒
𝑋
100
1
3.2.1.5 Ash Content (AOCS 5-49, 1989): Three clean, dry porcelain crucible was weighed and
2g of the dry sample was transferred into each of the crucible and reweighed. The crucibles
containing the sample were then placed in a muffle furnace at 600OC for three (3) hours.
The ash and the crucibles were cooled in a dessicator and reweighed. The percentage ash content
or the sample was calculated using the formular:
50
% 𝑎𝑠ℎ 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 =
𝑊𝑡 𝑜𝑓 𝑐𝑟𝑢𝑐𝑖𝑏𝑙𝑒 + 𝑎𝑠ℎ 𝑎𝑓𝑡𝑒𝑟 𝑏𝑢𝑟𝑛𝑖𝑛𝑔 − 𝑤𝑡 𝑜𝑓 𝑐𝑟𝑢𝑐𝑖𝑏𝑙𝑒
𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑐𝑟𝑢𝑐𝑖𝑏𝑙𝑒 𝑎𝑛𝑑 𝑠𝑎𝑚𝑝𝑙𝑒
𝑋
100
1
3.2.1.6 Carbohydrate Determination: 1.0g of the ground sample was mixed with 100mls of
distilled water in a flat bottom flask, and retorted for 90 minutes on a hot plate. The mixture was
allowed to cool and 5mls of 1% diastase added, followed by 3mls of toluene. The mixture was
stoppered with cotton wool and allowed to stand overnight. Then the volume was made up to
250ml mark with distilled water, and filtered. 2mls of the filtrate was added in 3 test tubes and
0/14mls of conc. HCI added. The mixture was boiled for 1 hour and then cooled. It was neutralized
with 10% NaOH and the volume made up to 2/5ml mark with distilled water. 1ml of the resulting
mixture was taken into another test tube and 2mls of saturated aqueous picric acid added. Finally,
the absorbance was taken at 330nm.
3.2.2 Determination of Vitamin Compositions
Vitamin and mineral contents were determined using the method of A.O.A.C (1997)
3.2.2.1 Determination of thiamine (vitamin B1) content
(a) Procedure
Exactly 5 ml of the standard and sample were taken in marked test tubes. In each test tube, 5 ml
NH4OH (0.1M) and 0.5 ml 4 –Amino phenol solution were added, mixed well and kept for 5
minutes. Then 10nml of chloroform was introduced into both solutions. The absorbance in a ultraviolet spectrophotometer was read at 420 nm using chloroform as its blank
Conc. (mg) =
𝑆𝐴 ×𝑆𝑡𝑊 ×1 ×10 ×10 ×1 ×𝑆𝑃 ×100 ×1000
𝑆𝑡𝐴 ×100 ×100 ×1 ×𝑊 𝑉 10 ×10
Where SA = Sample absorbance; St = standard weight; Sp = standard purity
StA = standard absorbance; W = weight
51
3.2.2.2 Determination of Riboflavin (Vitamin B2) Content
(a) Procedure
Two grams of the extract was weighed into test tube and was dissolved with 100 ml of distilled
water and 2 ml of glacial acetic acid were added. The solution was boiled for 5 minutes and then
colled. Then, 20 ml of 1.0M sodium hydroxide solution was added and diluted to 350 ml with
distilled water. The solution was filtered and absorbance was read at 444 nm using
spectrophometer (water was used as blank).
Conc. (mg) =
𝐴 ×𝐷𝐹 ×𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑐𝑢𝑣𝑒𝑡𝑡𝑒
𝐸
Where A = Absorbance; E = Extinction coefficient; DF = Dilution factor
3.2.2.3 Determination of Niacin (Vitamin B3) content
(a) Procedure
One gram of the sample was weighed into a test tube and macerated with fifty milliliters of distilled
water and filtered. One milillitre of filtrate was pipette and 6.5 millilitre of distilled water, 0.5
millilitre of ammonium and one milillitre of cyanogens bromide solution (10% w/v) were added
and shaken. One mililitre of sulphanilic buffer (pH 4.5) was added and allowed to stand for five
minutes. Then, 0.5 milillitre of concentrated (Cc1 was added and then diluted with ten milliliters
of distilled water. Absorbance was measured at 430 nm.
3.2.2.4 Determination of Pyridoxine (Vitamin B6)
(a) Procedure
Oen gram of the extract was mixed with twenty milliliters of water for ten minutes and centrifuged
for five minutes. One milliliter of fifty percent sodium acetate, 1 ml of 5% boric acid (in water)
and 1 ml dye (2, 6- di-cholroquinine chorimide) solution were added and mixed. Then, o.2
millilitre of 5.5 percent sodium carbonate was equally added and the absorbance was read at 540
nm.
% vitamins = ×
𝐴𝑏𝑠.𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒−𝐴𝑏𝑠.𝑜𝑓 𝑏𝑎𝑛𝑘 ×𝐷𝐹
𝑆𝑙𝑜𝑝 ×100
52
3.2.2.5 Determination of Cobalamin (Vitamin B12) content
(a) Procedure
Exactly 0.1mg of the extract was added into 25 ml columetric flask and 10ml distilled water was
added to dissolve it. then, 1.25 g of dibasic sodium phosphate, 1.1 g of anhydrous citric acid and
1.0 g of sodium metabisulphate were added. The volume was made up to the mark with water. The
solution was autoclaved at 1210 for 10 minutes and was equally filtered and absorbance recorded
at 530 nm.
3.2.2.6 Determination of Ascorbic Acid (Vitamin C) content
(a) Procedure
Extactly 1 ml of each sample was put in a 25 ml conical flask. Then 10 ml of oxalic acid (0.05 M)
– EDTA (0.02 M) solution was added and the entire mixture left standing for 24 h, to provide the
required reaction time,. After 24 hrs, the sample was filtered through 0.45 um filter paper. Then
2.5 ml of each sample was transferred to a separate 25 ml volumetric brown flask, after which 2.5
ml of the oxalic acid (0.05 M)-EDTA (0.02 M) solution was added, followed by sulphuric each
volumetric brown flask and the volume was made up to 25 ml with distilled water. A known
concentration of Ascorbic acid (0.1% m/v) was used as the standard and the same process was
carried out on the test sample. Absorbance of the test sample (Ax) and of the standard sample (AS)
was read at 760 nm against the black test on a UV/visible spectrophotometer.
The concentration (Cx) of vitamin C (% w/v or mg/ml) in the test sample was calculated using the
formula.
CX =
𝐴𝑥 ×𝐶𝑠
𝐴𝑠
3.2.2.7 Determination of Alpha Tocopherol (Vitamin E) Content
(a) Procedure
Exactly 2.5g of extract was homogenized in a small volume of 0.1N sulphuric acid and the volume
was finally up to 50ml by adding O.1N sulphuric acid slowly, without shaking and the content was
allowed to stand overnight. The content of the flask was shaken vigorously on the next day and
53
filtered through Whatman No 1 filter paper. Aliquots of the filtrate were used for the estimation of
to copherol. The extract, standard and water of 1.5ml were pipette out into three centrifuge tubes
namely test, standard and blank respectively. To all the tubes, 1.5 ml each of ethanol and xylene
were added, stopered, mixed well and centrifuged at 1500×g for 10 minutes. After centrifugation,
the xylene layer was transferred into another tube, taking care not to include any ethanol or protein.
To 1.0 ml of xylene layer, 1.0ml of2, 2-dipyridyl reagent was added, stoppered and mixed. This
reaction mixture was taken in the spectrophotomometric cuvettes and the extinctions of the test
and the standard were read against the blank at 460nm. Then, in turn, beginning with the blank,
0.33ml of ferric chloride solution was added, mixed well and after exactly 15 minutes, the test and
the standard were read against the blank at 520nm.
Tocopherol (ug) =
𝐴520−𝐴460 ×0.29 ×15
𝑆𝑡𝑑 𝐴520
3.2.3 Determination of minerals
3.2.3.1 Atomic absorption spectrophotometry (AAS)
1. Principles of AAS
In atomic absorption spectrophotometry, radiation from an internal light source, emitting
the spectral line is passed through a flame. This radiation corresponds to the energy
required for an electronic transition from the ground state to an excited state. When a
solution is passed as dispersion of fine droplets into a gas/air flame, the element present
and emitted are largely converted to their atomic state. Some of these atoms are excited
and give off radiation at a definite wavelength.
Methodology
The residue remaining was dissolved after the destruction of the organic matter of plant
material (235 digestion or dry ashing method) in hydrochloric acid. A releasing agent was
added to eliminate the interferences by other elements. For calcium and magnesium
addition of releasing agent to sample solution to represent 10 % v/v solution is also needed.
54
An atomic absorption spectrophotometer was set to measure the absorption from hollow
cathode lamp of any of the cations. Using acetylene/air flame. The appropriate wavelength
for each element corresponding to the lamps was selected. The working standard solution
of the cation being measured and adjusted, the controls until steady zero and suitable
maximum readings were obtained. nebulize the intermediate standards and construct a
graph relating galvanometer reading to µg/ml of the cation in all the standard solution.
55
CHAPTER FOUR
RESULT
4.1 Proximate compositions of stem bark of Terminalia superba extract .
The results showed the proximate analysis of the stem bark of Terminalia superba extract which
indicated presence of Carbohydrate, moisture, ash ,protein, fibre and fats. The findings revealed
carbohydrate has the highest value seconded by moisture.
100,00
90,00
80,00
% Composition
70,00
60,00
50,00
Moisture
Fibre
fats
40,00
30,00
Ash
protein
Carbohydrates
20,00
10,00
0,00
56
Figure 4.1 Proximate compositions of stem bark of Terminalia superba extract .
4.2 Mineral composition of stem bark of Terminalia superba extract .
The result below revealed the presence of macro minerals such as calcium, sodium, zinc, potassium
and magnesium, phosphorus, oxalate, iron, zinc and phylate. The findings of the study revealed
that terminalia superba had high quantities of sodium seconded by zinc
57
500,00
a
450,00
400,00
Levels (mg/100g)
350,00
300,00
b
250,00
b
c
200,00
150,00
100,00
50,00
0,00
Calcium
Magnesium
Sodium
Minerals
Figure 4.2 Mineral composition of terminalia superba
Potassium
58
a
3,50
b
3,00
b
Levels (mg/100g
2,50
2,00
1,50
1,00
0,50
0,00
Phosphorus
Zinc
phytate
Minerls
Figure 4.3 Mineral composition of terminalia superba
oxalate
Iron
59
4.3 Vitamin composition of stem bark of Terminalia superba extract .
The figure below revealed the vitamin composition of terminalia superba. The sample contained
vitamin A, vitamin C, vitamin E, vitamin B12, vitamin B1, vitamin B3, vitamin K, vitamin B2,
vitamin D, vitamin B6 and vitamin B9 in increasing order ofvitamin B2>vitamin B9>vitamin
A>vitamin B3>vitamin B1> vitamin B6 > vitamin D>vitamin C> vitamin B12
60
0,60
a
0,50
b
Level of vitamins (mg/100g)
b
A
0,40
C
B1
0,30
B2
c
B3
c
B6
0,20
B12
D
B9
0,10
0,00
A
C
B1
B2
B3
B6
Vitamins
Figure 4.4 Vitamin composition of terminalia superba
B12
D
B9
61
CHAPTER FIVE
DISCUSSION AND RECOMMENDATION
5.1 Discussion
The aim of this study to determine the proximate, mineral and vitamin composition of stem bark
of Terminalia superba extract.
The findings of the study on proximate analysis indicated presence of Carbohydrate, moisture, ash
,protein, fibre and fats. The findings revealed carbohydrate has the highest value seconded by
moisture. Guay et al. (2017) reported a similar findings showing
high concentrations of
carbohydrate (20 %) and moisture (18.5%) in the leaves of Terminalia superba. Asaolu et al
(2009) reported almost the same concentrations of carbohydrate and moisture (11.05% both), ash
(9.01%), crude fibre (4.02%) and fat content (3.51%), in their report on proximate composition of
dry leaves of Terminalia superba.
The carbohydrate content if the extract sjows the plant extract has high energy potential. The
moisture content of in fruits primarily explains the degree of the spoilage. The moisture content
provides for an activity of water soluble enzyme and co enzyme needed for metabolic activities of
the plant (Jeruto et al., 2017). Nutritionally Terminalia superba plant can be beneficial as protein
contain amino acids utilized by the cells of the body to synthesize all the numerous proteins
required for the proper functioning of the cell and also to furnish energy (Jeruto et al., 2017).
From the results also the fibre content in the plant material showed that nutritionally it is of beneficial
effect since it had been reported that food fibre aids absorption of trace elements in the gut and
reduce absorption of cholesterol (Hua, 2019). Crude fibre is also very essential for the digestion of
62
food materials in the food canal of animals (Hasler, 2020). Also from the results Ash in the extract
is generally taken to be a measure of mineral content of original food (Hasler, 2020). The low values
of ash in the fruits of Ficus capiensis may indicate that the sample would be free of foreign matter.
The findings on the mineral composition of terminalia superba in this study revealed the presence
of macro minerals such as calcium, sodium, zinc, potassium and magnesium, phosphorus, oxalate,
iron, zinc and phylate. The findings of the study revealed that terminalia superba had high
quantities of sodium seconded by zinc.
This study was in agreement with the study of Ngozi et al., (2019) on Phytochemical, Proximate
Analysis, Vitamin and Mineral Composition of Aqueous Extract of Terminalia superba leaves in
South Eastern Nigeria. Their study revealed that Terminalia superba contains considerable amount
of Sodium (3.73 ± 0.006), Zinc (2.84 ± 0.005), Iron (1.89 ± 0.004), Calcium (1.86 ± 0.003),
Magnesium (1.92 ± 0.004) and Potassium (0.72 ± 0.006).
The micro minerals present in this plant (iron, zinc and copper) perform various important
functions in humans like the formation of hemoglobin, growth and sexual maturation, facilitating
iron intake, as cofactor for enzymes and so many other functions. Sodium is a macromineral
essential nutrient and is needed by the body in relatively small amounts (provided that substantial
sweating does not occur) to maintain a balance of body fluids and keep muscles and nerves running
smoothly (Jernelöv, 2018). Calcium is required as a component of the human diet, and it is
essential for the full activity of many enzymes, such as nitric oxide synthase, protein phosphatases,
and adenylate kinase. It is also necessary to maintain an optimal bone development (Beto, 2015).
Besides, calcium is also good for growth and maintenance of bones, teeth and muscles (Pravina,
63
Sayaji, & Avinash, 2013). Normal extra cellular calcium concentrations are necessary for blood
coagulation and for the integrity, intracellular cement substances (Mohanty et al., 2019).
Phosphorus is also an important mineral as it has been reported to form the structure of teeth, bones
and cell membranes (Butusov and Jernelöv, 2013). It also acts as a cofactor for many enzymes and
activates the vitamin B complex. Potassium is an essential mineral and electrolyte involved in
heart function, muscle contraction, and water balance. It is necessary for the normal functioning
of all cells. It regulates the heartbeat, ensures proper function of the muscles and nerves, and is
vital for synthesizing protein and metabolizing carbohydrates (Jernelöv, 2018).
The findings on vitamin composition revealed Terminalia superba contained vitamin A, vitamin
C, vitamin E, vitamin B12, vitamin B1, vitamin B3, vitamin K, vitamin B2, vitamin D, vitamin
B6 and vitamin B9 in increasing order ofvitamin B2>vitamin B9>vitamin A>vitamin B3>vitamin
B1> vitamin B6 > vitamin D>vitamin C> vitamin B12. The vitamins content in this study is
equivalent to the study reported on ficus capiensis by Shittu and Alagbe (2020) and Alagbe (2017).
Vitamin A plays a vital role in good sight (vision), support to immune system and inflammatory
systems, cell growth and development, antioxidant activity, promoting proper cell communication
(Tang, 2010). Vitamin B1 involves in the energy production from carbohydrates and fats, its
deficiency in the body could negatively affect the heart as well as the nervous system (Keogh et
al., 2012).
Vitamin B2 promotes iron metabolism and its deficiency also increase the risk of anemia or blood
shortage (Asensi-Fabado and Munne, 2010). Vitamin B3 is essential in production of energy from
dietary proteins, carbohydrates and fats (McDowell, 2010). Functionally B6 is very important
vitamin as it is involved in red blood cell production, carbohydrate metabolism, liver
64
detoxification, brain and nervous system health (Wardlaw et al., 2017). Folates (vitamin B9)
support the cardiovascular system, nervous system and also prevents cardio vascular disease in
human (Hayden and Tyagi, 2014). Vitamin B12 is involved in production of red blood cells and
also prevents the increase in level of homocysteine (Crider et al., 2016). Vitamin C helps to boost
the immune system by scavenging free radicals; its deficiency induces the disease called scurvy.
In case of scurvy, loss of bone strength, lose teeth and bleeding (Wardlaw et al., 2019). Vitamin
D deficiency is associated with many disorders like, osteoporosis, rickets, osteomalacia, loss of
balance, diabetes, rheumatoid arthritis, asthma, depression etc. (Wagner and Greer, 2018). Vitamin
K is important for bone health and blood clotting; its deficiency increases the risk of bone fracture
(Shearer and Newman, 2014).
5.2 Conclusion
The findings of this study shows that Terminalia superba has nutritional qualities and has the
potential for drug development based on popular uses and biological studies.
65
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