CHAPTER ONE

INTRODUCTION

1.1 Background of the study

Citrus fruits among other uses are sources of food and medicine. Fruits are divided into
climacteric and non-climacteric classes based on their respiration pattern during
ripening. In non-climacteric category are the grapefruit, lemon, orange, melon,
pineapple and the strawberry. In these fruits the respiratory pattern shows slow drift
downwards after detachment from the parent plant while climacteric classes undergo a
distinct ripening phase e.g. bananas, pears and avocados (Alibone, 2000).
Orange, lemon and grape fruit have the same morphology, fruit development and
maturation except in the percentage composition of ascorbic acid and sugars (Alibone,
2000). Ascorbic acid also known as Vitamin C, L-ascorbic acid and the Antiscorbutic
Vitamin, when pure is a white crystalline water-soluble vitamin found especially in
citrus fruits and vegetables. It is synthesized by most organisms from glucose but man
and other primates and various other species must obtain it from their diet (Alibone,
2000).
Ascorbic acid is the most abundant vitamin in orange, lemon and grape fruit (Ralph &
Bender, 2000). The peel is especially rich in ascorbic acid. There is a considerable
variation in the ascorbic acid content of juice of different fruit (Ralph & Bender, 2000).
The citrus species are medium sized tree of the rutacene family. They are evergreen
trees that give fruits of different forms and sizes (from round to oblong) which are full
of fragrance, flavour and juice. Citrus fruits have a rough, robust and bright colour
(from green to yellow) skin or rind known as epicarp which covers the fruits and
protects them from damages (Okwu & Emenike, 2000).

The glands contain the essential oils that the fruit its typical citrus fragrance. The
endocarp is rich in soluble sugars and contains significant amounts of ascorbic acid,
pectin, fibres, different organic acids and potassium salt which gives the fruit its
characteristic citrus flavour (Roger, 2002).
Ascorbic acid is required in the synthesis of collagen in connective tissues,
neurotransmitters, steroid hormones, carnitine, and conversion of cholesterol to bile acid
and enhances iron bio-availability (Robert et al., 2000). Ascorbic acid is a great
antioxidant and helps to protect the body against pollutants (Okwu, 2004). It is also a
biological reducing agent linked to prevention of degenerative disease such as cataracts,
certain cancers and cardiovascular diseases (Gary, 2004). It promotes healthy cell
development, proper calcium absorption, normal tissue growth and repair such as
healing of wounds and burns and strengthening the wall of the capillaries.
Ascorbic acid is needed for healthy gums, to help protect against infection and assisting
with clearing up infection; and is thought to enhance the immune system and help
reduce cholesterol levels and high blood pressure (Robert et al., 2000). A deficiency of
ascorbic acid in the body results in scurvy, a disease characterised by sore, spongy
gums, loose teeth, fragile blood vessels, swollen joints and anaemia (Piff et al., 1998).
A shortage of ascorbic acid may also result in haemorrhages under the skin and a
tendency to bruise easily, poor wound healing, oedema and weakness (Enloe et al.,
1985). Lack of energy, poor digestion, bronchial infection and colds are also indicative
of an under-supply of ascorbic acid (Ronzio & Robert, 1992). Many of the deficiency
symptoms can be explained by a deficiency in the hydroxylation of collagen, resulting in
defective connective tissues (Henry et al., 2004). Ascorbic acid is widely used as
antioxidant in frozen fruits, canned meat, beverages, beer and other food items. It is
used medically as drug and in pharmaceutical industry for the manufacture of drugs rich
in ascorbic acid (Gary, 2004). Ascorbic acid is used in the treatment of curvy and
prickly heat and thus required in the tropics in relatively larger quantities so as to ensure
among other things the normal functioning of the hyperactive sweat glands. Its
application in animal feed is fast growing as it supplements the Vitamin C content of the
feed (Gary, 2004). Apart from the citrus fruits, few other sources of ascorbic acid are
green leafy vegetables, berries, guavas, tomatoes, melons, papayas and white potatoes.
Ascorbic acid content of citrus fruits is never constant but varies with some factors
which include climatic/environmental conditions, maturity state and position on the tree,
handling and storage, ripening stage, specie and variety of the citrus fruit as well as
temperature. It is in the light of this that the quest to have knowledge of the ascorbic
acid content of some selected citrus fruits in South Eastern Nigeria was born with
respect to temperature, ripening stage, specie and time.
1.2 Statement of the problem
The major active ingredient in orange is ascorbic acid which is also known as vitamin C.
This vitamin C helps protects the body system against various diseases by nourishing
the body system. Despite the beneficial aspect of this ascorbic acid, the medium for
transporting and storing orange before processing it to finished products is very poor in
our locality which could affect the level of the ascorbic acid present in orange used for
finished products. Hence, this study seeks answer to quantify the level of ascorbic acid
in orange in our locality.
1.3 Aim and objectives
The aim of this study is quantitative analysis of ascorbic acid in orange.
Its specific objectives are;
i. To extract ascorbic acid in orange
ii. To determine the levels of ascorbic acid in orange
iii. To compare the level of ascorbic acid in orange with the standard
1.4 Significance of the study
This study will help farmers improve the way the transport and store these oranges for
further production. Also, this information will be useful to food processing industries
who utilize orange as their main raw materials for production
1.5 Scope and limitation
This study is limited to quantitation of ascorbic acid in orange. However, due to time
and financial constrain, the GC-MS analysis of the ascorbic acid would not be possible.
 CHAPTER TWO
LITERATURE REVIEW

2.1 Citrus sinensis

Citrus × aurantium f. aurantium (Citrus × sinensis), a hybrid between pomelo (Citrus

maxima) and mandarin (Citrus reticulata), also known as the sweet oranges, is a
commonly cultivated species of orange that includes Valencia oranges, blood
oranges and navel oranges.

The orange fruit is an important agricultural product, used for both the juicy fruit pulp
and the aromatic peel (rind). Orange blossoms (the flowers) are used in several different
ways, as are the leaves and wood of the tree.

The genus Citrus L., which belongs to the Rutaceae family consists of different forms
such as trees, shrubs, and herbs in the world (Sicari et al., 2018) . The citrus genus is the
most cultivated and traded variety in the world as a garden plant and one of the most
important commercial fruit crops grown on all continents of the world (Wang et al.,
2019). Various species of Citrus are C. limon (lemon), C. medica (citron), C. aurantium
(sour orange), C. paradise (grapefruit), C. reticulata (mandarin, tangerine), C.
clementina (clementine) and C. sinensis (sweet orange). Amongst them, C. sinensis
(Orange) is extensively grown for its fruits, which have food and medicinal
properties (Wu et al., 2021).
C. sinensis probably originated from South East Asia and were cultivated in China by
2500 BCE (Saini et al., 2022). It is one of the most commonly employed ingredients
in Traditional Chinese Medicine with preparations derived from peels, young fruit,
mature fruit, flower, and other tissues. All of these parts are processed to prepare food
items in China and are popularly used in Chinese prescriptions due to the presence of
important phytochemicals such as flavonoids, vitamins, and phenolic acids confirming
that food and medicine came from the same source (Murat, 2021).
About 48 million tonnes of C. sinensis (Orange) were produced worldwide in January
2021–2022 with China, Brazil, the European Union, Mexico, and United States being
the top producers. Several nations are using oranges primarily for juice extraction,
which accounts for 1.7 million tonnes of orange production by January 2021–2022 (Juan
et al., 2016). Global C. sinensis production for 2022–2023 is projected to be 5% lower
at 47.5 million tonnes. This is because of decreased production in US and EU. The
decreased production decreases fruit consumption and processing worldwide (Pineda et
al., 2022).
Thirst-quenching fruit attracts all age groups with its multifold health benefits, mainly
consumed fresh or in juice. C. sinensis fruit is rich in vitamins, minerals, and dietary
fibers which are essential for normal growth and development of healthy life. Starting
with the pulp, seeds, leaves, and skin of the fruit, oranges have various advantages. One
noteworthy example is that a healthy, antibiotic-free product with no hazardous residues
for humans can be produced by employing the proper amounts of orange waste and by-
products as a supplement in the poultry diet (Nicolosi et al., 2000). C. sinensis essential
oil in clinical studies showed reduced anxiety levels in patients of dental treatment as
well as in healthy volunteers submitted to anxiogenic situations (Zaker-Esteghamati &
Abdelfattah, 2018). Recent studies indicate that C. sinensis supplementation may be
useful as a prophylactic agent against SARS-CoV-2 infection and complementary
treatment for COVID-infected patients (Mannucci, 2018). These few examples had
proved the worth of the nutritional benefits of C. sinensis in everyday life.

2.1.1 Description

The genus Citrus L., which belongs to the Rutaceae family consists of different forms
such as trees, shrubs, and herbs in the world (Bellavite et al., 20220). The citrus genus is
the most cultivated and traded variety in the world as a garden plant and one of the most
important commercial fruit crops grown on all continents of the world (Chand et al.,
2020). Various species of Citrus are C. limon (lemon), C. medica (citron), C. aurantium
(sour orange), C. paradise (grapefruit), C. reticulata (mandarin, tangerine), C.
clementina (clementine) and C. sinensis (sweet orange). Amongst them, C. sinensis
(Orange) is extensively grown for its fruits, which have food and medicinal
properties (Bellavite, 2020).
C. sinensis probably originated from South East Asia and were cultivated in China by
2500 BCE. It is one of the most commonly employed ingredients in Traditional Chinese
Medicine with preparations derived from peels, young fruit, mature fruit, flower, and
other tissues. All of these parts are processed to prepare food items in China and are
popularly used in Chinese prescriptions due to the presence of
important phytochemicals such as flavonoids, vitamins, and phenolic acids confirming
that food and medicine came from the same source (Bellavite, 2020).
About 48 million tonnes of C. sinensis (Orange) were produced worldwide in January
2021–2022 with China, Brazil, the European Union, Mexico, and United States being
the top producers. Several nations are using oranges primarily for juice extraction,
which accounts for 1.7 million tonnes of orange production by January 2021–
2022 (Torre et al., 2019). Global C. sinensis production for 2022–2023 is projected to be
5% lower at 47.5 million tonnes. This is because of decreased production in US and EU.
The decreased production decreases fruit consumption and processing worldwide (Jose
et al., 2017).
Thirst-quenching fruit attracts all age groups with its multifold health benefits, mainly
consumed fresh or in juice. C. sinensis fruit is rich in vitamins, minerals, and dietary
fibers which are essential for normal growth and development of healthy life. Starting
with the pulp, seeds, leaves, and skin of the fruit, oranges have various advantages. One
noteworthy example is that a healthy, antibiotic-free product with no hazardous residues
for humans can be produced by employing the proper amounts of orange waste and by-
products as a supplement in the poultry diet (Jose et al., 2017).
C. sinensis essential oil in clinical studies showed reduced anxiety levels in patients of
dental treatment as well as in healthy volunteers submitted to anxiogenic situations (Jose
et al., 2017).
Recent studies indicate that C. sinensis supplementation may be useful as a prophylactic
agent against SARS-CoV-2 infection and complementary treatment for COVID-infected
patients (Jose et al., 2017).
These few examples had proved the worth of the nutritional benefits of C. sinensis in
everyday life.
Scientific classification
Kingdom: Plantae
Clade: Angiosperms
Clade: Eudicots
Clade: Rosids
Order: Sapindales
Family: Rutaceae
Genus: Citrus
Species: C. sinensis

Uses

 The orange blossom, which is the state flower of Florida,[2] is highly fragrant and
traditionally associated with good fortune. It has long been popular in bridal
bouquets and head wreaths.
 Orange blossom essence is an important component in the making of perfume.
 Orange blossom petals can also be made into a delicately citrus-scented counterpart
to rosewater, known as "orange blossom water" or "orange flower water". It is a
common ingredient in French and Middle Eastern cuisines, especially in desserts
and baked goods. In some Middle Eastern countries, drops of orange flower water
are added to disguise the unpleasant taste of hard water drawn from wells or stored
in qullahs (traditional Egyptian water pitchers made of porous clay). In the United
States, orange flower water is used to make orange blossom scones and
marshmallows.
 In Spain, fallen blossoms are dried and used to make orange tea.
 Orange blossom honey (or citrus honey) is obtained by putting beehives in the citrus
groves while trees bloom. By this method, bees also pollinate seeded citrus varieties.
This type of honey has an orangey taste and is highly prized.[citation needed]

2.1.2 Traditional/ ethnomedicinal uses

C. sinensis is consumed all over the world as an excellent source of vitamin C, which is
a powerful natural antioxidant that builds the body's immune system. It has been used
traditionally to treat various ailments like constipation, cramps, colic,
diarrhea, bronchitis, tuberculosis, cough, cold, obesity, menstrual disorder, angina,
hypertension, anxiety, depression and stress, asthma, vomiting, fever, hiccoughs and
for indigestion ((Jose et al., 2017). In Chinese Traditional medicine, the orange has a
long history as a cooling agent for coughs, colds, and respiratory disorders. It is used as
a traditional symbol of good luck in China.

2.1.3 Applications

C. sinensis is rich in many valuable phytoconstituents and are widely used for their
beneficial effects. Orange essential oils have many applications in different sectors. In
the food industry, oil is used as an antifungal agent to avoid crop and food deterioration
during the transportation stage. In the cosmetic and cleaning sectors, oils are commonly
applied due to their aromatic properties. In the Pharmaceutical industry, oils find a place
for their preventive properties against some diseases like cancer, diabetes, antiviral, and
inflammations. In the chemical industry, the orange peel can be used to produce
methane, after the essential oil is extracted (Jose et al., 2017).

Essential oil is widely used in the preparation of toilet soaps, perfumes, cosmetics, and
other home care products, personal hygiene products, or as a green solvent It is one of
the popular oils in aroma therapy including massage, inhalations, and baths since ancient
times. In the textile, plastics, and paint industries, oil is used as a masking agent in order
to cover up undesirable odors. Oil is extensively used in pharmaceutical formulations
for its flavoring properties. Recently, food and packaging industries are using the oil for
its natural antimicrobial properties as consumer concerns increase and to ensure safe
foodstuff (Qiao et al., 2008).
Phenolic compounds: Several scientists proved that regular consumption of phenolic
compounds present in orange reduces the risk of disease and is also beneficial for human
health. Phenolic compound plays a noteworthy role to combat the incidence of diseases
such as cancer, heart disease, hepatic injury, and neurodegenerative disorders. They are
useful against many other health issues such as osteoarthritis and ocular disorders and
act as phytoestrogen and anti-thyroidal compounds. These compounds have strong
antioxidant and free radical scavenging activity and may have practical applications in
the food industry by replacing the chemical antioxidant with this natural
antioxidant (Qiao et al., 2008).
Pectin is one of the compounds found everywhere in the peel part mainly in flavedo
(epicarp), albedo (mesocarp), and edible portion (endocarp). They are mostly found in
higher plants’ cell walls and are called complex polysaccharides compounds. Pectin is
produced industrially and is widely used as a thickening, coating, and encapsulating
material due to its stabilizing, gelling, and thickening characteristics in jam and
confectionery products. Due to its film-forming capacity, pectin may be used as a
packaging material to improve the sensory attributes of some foods and for their shelf
life (Perez-Cacho et al., 2007).

2.1.4 Chemical composition

The chemical composition of C. sinensis varies with cultivars, year of harvest and
postharvest storage conditions, planting area, fruit variety, part of the fruit, climate, and
degree of maturation. Despite of this fruit is rich in many important phytoconstituents
present in various parts of C. sinensis viz leaves, seed, flower, juice, and peels. Different
scientists studied and reported several types of chemical compounds which include the
groups like volatile oil, flavonoids (e.g., flavanones, flavanone glycosides, and
polymethoxylated flavones), carbohydrates, coumarins, peptides, fatty acids, steroids,
alkanes, hydroxyamides, carotenoids, carbamates, vitamins and alkylamines.
The primary and secondary metabolome of C. sinensis is chemically diverse. Numerous
structural classes and thousands of unique compounds have been identified and
described (Boukroufa et al., 2015). These studies’ findings undeniably showed that
oranges are a good source of chemicals that could be used as functional additives in
human health products and potential medication candidates.
Plate 1: Unripe orange
 Plate 2: Ripe orange

2.2 Antioxidant activity

Over the years, research on antioxidants and free radicals for understanding their role
has grown exponentially. The antioxidant capacity of C. sinensis juice purchased from
the local market was determined by the kinetics of hydrogen peroxide scavenging
method showed k values of 1.2±0.3 × 103 s−1 and 0.4± 0.1 × 103 s−1. H2O2 may be used
as a relative indicator and regulator of antioxidant activity due to the substantial
variance in kinetic constants of H 2O2 elimination in dietary samples (Davis et al., 2016).
In different study, the antioxidant activity of Moro C. sinensis crude juice was
determined by different methods (2,2-diphenyl-1-picrylhydrazyl (DPPH), OH, 2,2′-
azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS+) and Ferric reducing
antioxidant power and the result showed that Moro juice efficiently
scavenges ABTS radical cations reaching up to 64% of quenching corresponding to
14.30 µM using Trolox equivalents (TE) as the reference antioxidant, it also was able to
scavenge DPPH radicals with an antioxidant power corresponding to 14.39± 0.19 µM
TE and eliminated about the 87% of hydroxyl radical generated at 16.40 µM TE. Five
C-glycosyl flavones—lucenin-2, vicenin-2, stellarin-2, lucenin-2–41 -methyl ether, and
scoparin-one 3-hydroxy-3-methylglutaryl glycosyl flavonol, 3-hydroxy-3-
methylglutaryl glycosyl quercetin, and one flavone O-glycoside— the 7-O-
neoesperidoside of chrysoeriol are responsible for the aforementioned antioxidant
effects (Levine et at., 1992). An amperometric biosensor for the quantification of the
scavenging capacity of orange juices displayed values for natural orange juice at
1.01 mM of IC50/µM 10.6± 0.5 while one bought commercially gave values at 2.27,
1.56, 0.91, 2.22 mM of IC50 values 17.0 ± 0.8 µM, 17.2 ± 0.7 µM, 26.4 ± 0.8 µM, and
18.9 ± 0.9 µM, respectively. Substances such as ascorbic
acid (IC50 30.3± 0.9 µM), caffeic acid (no significant results), gallic acid (no significant
results), ferulic acid (no significant results), curcumin (no significant
results), catechol (no significant results), quercetin (no significant results) were used for
control and also for comparing. The orange juice showed the best activity (Levine et at.,
1992). C. sinensis seed extract exhibited antioxidant activity using reducing power and
DPPH radical-scavenging assays, gallic acid (IC50 = 29.5 µM) was used as a control
standard (Levine et at., 1992). The flavonoid content of several methanolic extract
fractions of Navel orange peel (flavedo and albedo of Citrus sinensis) was first analyzed
phytochemically and then evaluated for its antioxidant activity in vitro. The chemical
structures of the fractionated constituents were determined by comparing their retention
times and the obtained UV spectral data with the available bibliographic information
and further confirmed by detailed LC-DAD-MS (ESI+) analysis. The main flavonoid
groups found within the fractions examined were polymethoxylated flavones, O-
glycosylated flavones, C-glycosylated flavones, O-glycosylated flavonols, O-
glycosylated flavanones and phenolic acids along with their ester derivatives. In
addition, the quantitative HPLC analysis confirmed that hesperidin is the
major flavonoid glycoside found in the orange peel. The antioxidant activity of the
orange peel methanolic extract fractions was evaluated by DPPH assay and the
Co(II)/EDTA-induced luminol chemiluminescence assay. Results showed that orange
peel methanolic extracts possess moderate antioxidant activity as compared with the
activity observed for the aglycones, diosmetin (EC50 71.79±13.58 mg
and hesperetin (EC50 29.18 ± 2.80 7 mg). Quercetin (EC50 = 0.06 mg quercetin/mg
DPPH) was used as a positive control and the tested aglycones exhibited a significantly
higher hydroxyl scavenging activity than quercetin (Eipper et al., 1993). Oboh G et al.
investigated the DPPH antiradical action of C. sinensis juice. Results showed that C.
sinensis juice exhibited 84.81% DPPH antiradical effect at 100 µg/mL. The ascorbic
acid (96.36%) was used as positive control showing 96.36% DPPH antiradical effect.
Citrus peel's bound phenolic content demonstrated DPPH scavenging at 1 mg/mL, OH
scavenging at 4 mg/mL, ability to chelate Fe2+ at 0.48 mg/mL, and prevention of
Fe2+induced lipid peroxidation in the pancreas at 142.8 µg/mL (Rumsey et al., 1997).
Acetone-water extract from red oranges (C. sinensis, Torocco) exhibited an intracellular
antioxidant activity of 85% in Caco-2 cells at a concentration of 50 mg/mL. Gallic acid
and vitamin C were employed as positive control. The red-orange extract showed
significant higher antioxidant activity than positive controls (Zetterström, 2009). The
antioxidant activity of methanolic and ethanolic C. sinensis peel extracts demonstrated
significant levels of free radical scavenging activity produced by ABTS, with values of
55.8% and 60.7%, respectively, and DPPH scavenging activity based on its capacity as a
hydrogen donator, with values of 70% and 80%, respectively. Ascorbic acid and water
(20–150 µL) were employed as a control (Marriott, 2020). The scavenging activity
of dichloromethane extract, and diethyl ether, ethyl acetate, and n-butanol fractions
ranged from 3 to 1.1 mg dry extract/mg DPPH. Ethyl acetate fraction exhibited the best
antioxidant activity which is due to the presence of C-glycosylated flavones, O-
glycosylated flavones, polymethoxylated flavones, O-glycosylated flavanones, and
esters of phenolic acids. All these findings clear that C. sinensis is a good source of
antioxidant compounds.

2.3 Vitamin C (Ascorbic acid)

Vitamin C (also known as ascorbic acid and ascorbate) is a water-soluble vitamin found
in citrus and other fruits and vegetables, also sold as a dietary supplement and as
a topical "serum" ingredient to treat melasma (dark pigment spots) and wrinkles on the
face (Belsky et al., 2018). It is used to prevent and treat scurvy (Emadi-Konjin et al.,
2005).

Vitamin C is an essential nutrient involved in the repair of tissue, the formation

of collagen, and the enzymatic production of certain neurotransmitters (Belsky et al.,
2018). It is required for the functioning of several enzymes and is important for immune
system function (Bjelakovic et al., 2012). It also functions as an antioxidant. Most
animals are able to synthesize their own vitamin C. However, apes (including humans)
and monkeys (but not all primates), most bats, some rodents, and certain other animals
must acquire it from dietary sources.

There is some evidence that regular use of supplements may reduce the duration of
the common cold, but it does not appear to prevent infection (Zetterström, 2009). It is
unclear whether supplementation affects the risk of cancer, cardiovascular disease,
or dementia.[16][17] It may be taken by mouth or by injection (Zetterström, 2009).

Vitamin C is generally well tolerated. Large doses may cause gastrointestinal

discomfort, headache, trouble sleeping, and flushing of the skin (Nishikimi & Yagi,
1991). Normal doses are safe during pregnancy.[8] The United States Institute of
Medicine recommends against taking large doses.

Vitamin C was discovered in 1912, isolated in 1928, and in 1933, the first vitamin to
be chemically produced.[18] It is on the World Health Organization's List of Essential
Medicines (Wintergerst et al., 2006). Vitamin C is available as an
inexpensive generic and over-the-counter medication. Partly for its discovery, Albert
Szent-Györgyi and Walter Norman Haworth were awarded the 1937 Nobel
Prizes in Physiology and Medicine and Chemistry, respectively (Wintergerst et al.,
2006). Foods containing vitamin C include citrus
fruits, kiwifruit, guava, broccoli, Brussels sprouts, bell peppers, potatoes,
and strawberries.[13] Prolonged storage or cooking may reduce vitamin C content in
foods.
Vitamin C is an essential nutrient for certain animals including humans. The
term vitamin C encompasses several vitamins that have vitamin C activity in animals.
Ascorbate salts such as sodium ascorbate and calcium ascorbate are used in some
dietary supplements. These release ascorbates upon digestion. Ascorbate and ascorbic
acid are both naturally present in the body, since the forms interconvert according to pH.
Oxidized forms of the molecule such as dehydroascorbic acid are converted back to
ascorbic acid by reducing agents (Bjelakovic et al., 2012).

Vitamin C functions as a cofactor in many enzymatic reactions in animals (including

humans) that mediate a variety of essential biological functions, including wound
healing and collagen synthesis. In humans, vitamin C deficiency leads to impaired
collagen synthesis, contributing to the more severe symptoms of scurvy (Emadi-Konjin
et al., 2005). Another biochemical role of vitamin C is to act as
an antioxidant (a reducing agent) by donating electrons to various enzymatic and non-
enzymatic reactions. Doing so converts vitamin C to an oxidized state – either as semi
dehydroascorbic acid or dehydroascorbic acid. These compounds can be restored to a
reduced state by glutathione and NADPH-dependent enzymatic mechanisms. In plants,
vitamin C is a substrate for ascorbate peroxidase. This enzyme utilizes ascorbate to
neutralize excess hydrogen peroxide (H2O2) by converting it to water (H2O) and oxygen.

2.3.1 Deficiency

Vitamin C blood serum levels are considered saturated at levels > 65

μmol/L (1.1 mg/dL), achieved by consuming amounts which are at, or above,
the Recommended Dietary Allowance, while adequate levels are defined as ≥ 50
μmol/L. Hypovitaminosis of vitamin C is defined as ≤ 23 μmol/L and deficiency occurs
at ≤ 11.4 μmol/L.[29][30] For those 20 years of age or above, data from the U.S. 2003–
04 National Health and Nutrition Examination Survey showed mean and median serum
concentrations of 49.0 and 54.4 μmol/L, respectively. The percent of people reported as
deficient was 7.1% (Nishikimi & Yagi, 1991).

Scurvy is a disease resulting from a deficiency of vitamin C. Without this

vitamin, collagen made by the body is too unstable to perform its function and several
other enzymes in the body do not operate correctly. Scurvy is characterized by spots on
and bleeding under the skin, spongy gums, 'corkscrew' hair growth, and poor wound
healing. The skin lesions are most abundant on the thighs and legs, and a person with the
ailment looks pale, feels depressed, and is partially immobilized. In advanced scurvy
there are open, suppurating wounds, loss of teeth, bone abnormalities and, eventually,
death (Mannucci et al., 2018).

Notable human dietary studies of experimentally induced scurvy were conducted

on conscientious objectors during World War II in Britain and on Iowa state prisoners in
the late 1960s to the 1980s. Men in the prison study developed the first signs of scurvy
about four weeks after starting the vitamin C-free diet, whereas in the earlier British
study, six to eight months were required, possibly due to the pre-loading of this group
with a 70 mg/day supplement for six weeks before the scorbutic diet was fed. Men in
both studies had blood levels of ascorbic acid too low to be accurately measured by the
time they developed signs of scurvy. These studies both reported that all obvious
symptoms of scurvy could be completely reversed by supplementation of only 10 mg a
day.

People in sepsis or septic shock may have micronutrient deficiencies, including low
levels of vitamin C.

2.3.2 Medicinal uses

Vitamin C blood serum levels are considered saturated at levels > 65

μmol/L (1.1 mg/dL), achieved by consuming amounts which are at, or above,
the Recommended Dietary Allowance, while adequate levels are defined as ≥ 50
μmol/L. Hypovitaminosis of vitamin C is defined as ≤ 23 μmol/L and deficiency occurs
at ≤ 11.4 μmol/L (Saini et al., 2022). For those 20 years of age or above, data from the
U.S. 2003–04 National Health and Nutrition Examination Survey showed mean and
median serum concentrations of 49.0 and 54.4 μmol/L, respectively. The percent of
people reported as deficient was 7.1% (Saini et al., 2022).

Scurvy is a disease resulting from a deficiency of vitamin C. Without this

vitamin, collagen made by the body is too unstable to perform its function and several
other enzymes in the body do not operate correctly (Saini et al., 2022). Scurvy is
characterized by spots on and bleeding under the skin, spongy gums, 'corkscrew' hair
growth, and poor wound healing. The skin lesions are most abundant on the thighs and
legs, and a person with the ailment looks pale, feels depressed, and is partially
immobilized. In advanced scurvy there are open, suppurating wounds, loss
of teeth, bone abnormalities and, eventually, death (Saini et al., 2022).

Notable human dietary studies of experimentally induced scurvy were conducted

on conscientious objectors during World War II in Britain and on Iowa state prisoners in
the late 1960s to the 1980s. Men in the prison study developed the first signs of scurvy
about four weeks after starting the vitamin C-free diet, whereas in the earlier British
study, six to eight months were required, possibly due to the pre-loading of this group
with a 70 mg/day supplement for six weeks before the scorbutic diet was fed. Men in
both studies had blood levels of ascorbic acid too low to be accurately measured by the
time they developed signs of scurvy. These studies both reported that all obvious
symptoms of scurvy could be completely reversed by supplementation of only 10 mg a
day (Murat & Ramazan, 2021).

People in sepsis or septic shock may have micronutrient deficiencies, including low
levels of vitamin C

Scurvy

The disease scurvy is caused by vitamin C deficiency and can be prevented and treated
with vitamin C-containing foods or dietary supplements. It takes at least a month of little
to no vitamin C before symptoms occur (Wang et al., 2019). Early symptoms are
malaise and lethargy, progressing to shortness of breath, bone pain, bleeding gums,
susceptibility to bruising, poor wound healing, and finally fever, convulsions and
eventual death (Wang et al., 2019). Until quite late in the disease the damage is
reversible, as healthy collagen replaces the defective collagen with vitamin C repletion.
Treatment can be oral supplementation of the vitamin or by intramuscular or intravenous
injection. Scurvy was known to Hippocrates in the classical era. The disease was shown
to be prevented by citrus fruits in an early controlled trial by a Royal
Navy surgeon, James Lind, in 1747, on board HMS Salisbury. From 1796 onward,
lemon juice was issued to all Royal Navy crewmen (Murat & Ramazan, 2021).
Common cold

Research on vitamin C in the common cold has been divided into effects on prevention,
duration, and severity. A Cochrane review which looked at least 200 mg/day concluded
that vitamin C taken on a regular basis was not effective in prevention of the common
cold. Restricting analysis to trials that used at least 1000 mg/day also saw no prevention
benefit. However, taking vitamin C on a regular basis did reduce the average duration by
8% in adults and 14% in children, and also reduced severity of colds (Sicari et al.,
2018). A subset of trials in adults reported that supplementation reduced the incidence of
colds by half in marathon runners, skiers, or soldiers in subarctic conditions (Sicari et
al., 2018). Another subset of trials looked at therapeutic use, meaning that vitamin C
was not started unless the people started to feel the beginnings of a cold. In these,
vitamin C did not affect duration or severity (Sicari et al., 2018). An earlier review
stated that vitamin C did not prevent colds, did reduce duration, did not reduce severity.
The authors of the Cochrane review concluded that:

The failure of vitamin C supplementation to reduce the incidence of colds in the general
population indicates that routine vitamin C supplementation is not justified … Regular
supplementation trials have shown that vitamin C reduces the duration of colds, but this
was not replicated in the few therapeutic trials that have been carried out. Nevertheless,
given the consistent effect of vitamin C on the duration and severity of colds in the
regular supplementation studies, and the low cost and safety, it may be worthwhile for
common cold patients to test on an individual basis whether therapeutic vitamin C is
beneficial for them."

Vitamin C distributes readily in high concentrations into immune cells,

has antimicrobial and natural killer cell activities, promotes lymphocyte proliferation,
and is consumed quickly during infections, effects suggesting a prominent role in
immune system regulation (Wu et al., 2021). The European Food Safety
Authority found a cause and effect relationship exists between the dietary intake of
vitamin C and functioning of a normal immune system in adults and in children under
three years of age (Wu et al., 2021).
Several studies found Vitamin C to have specific antiviral effects in which it inactivates
the RNA or DNA of viruses or in the assembly of the virus.

2.3.3 Side effect

Vitamin C is a water-soluble vitamin, with dietary excesses not absorbed, and excesses
in the blood rapidly excreted in the urine, so it exhibits remarkably low acute toxicity
(Juan et al., 2016). More than two to three grams may cause indigestion, particularly
when taken on an empty stomach. However, taking vitamin C in the form of sodium
ascorbate and calcium ascorbate may minimize this effect (Other symptoms reported for
large doses include nausea, abdominal cramps and diarrhea. These effects are attributed
to the osmotic effect of unabsorbed vitamin C passing through the intestine (Juan et al.,
2016). In theory, high vitamin C intake may cause excessive absorption of iron. A
summary of reviews of supplementation in healthy subjects did not report this problem,
but left as untested the possibility that individuals with
hereditary hemochromatosis might be adversely affected (Nicolosi et al., 2000).

There is a longstanding belief among the mainstream medical community that vitamin C
increases risk of kidney stones (Nicolosi et al., 2000). "Reports of kidney stone
formation associated with excess ascorbic acid intake are limited to individuals with
renal disease (Nicolosi et al., 2000).

Reviews state that "data from epidemiological studies do not support an association
between excess ascorbic acid intake and kidney stone formation in apparently healthy
individuals" although one large, multi-year trial did report a nearly two-fold increase in
kidney stones in men who regularly consumed a vitamin C supplement (Pineda-Lozano
et al., 2022).

CHAPTER THREE

MATERIALS AND METHOD

3.1 Materials

In addition to the standard laboratory equipments and reagents method, the following
reagents and equipments were used. All the reagents used were of analytical grade.
3.1.1 Apparatus

 Conical flask
 Beaker
 Spatula
 Volumetric flask
 Refrigerator

3.1.2 Reagents

 (HPO3)
 H3PO4
 Distilled water
 CUSO4.5H2O
 EDTA/TCA
 CuSO4

3.2 Method

3.2.1 Sample collection

Ripe and unripe citrus fruits of sweet oranges (Citrus sinensis), were harvested from a
located farm in Makurdi, Benue State, Nigeria. 150 matured fruits were used for the
analysis; 30 unripe and 20 ripe. The fruits were stored in cases at room temperature and
were being withdrawn arriving Jalingo. Another set of oranges from the same farm will
be allowed to be transported in regular bags and method of transportation under intense
weather.
3.2.2 Extraction of Fruit Juice
The juice was squeezed out of the fruits manually until most of the juice was collected
in a clean glass beaker and the residual portion of the juice squeezed through layers of
muslin cloth. The extract was then filtered through another clean layer of muslin cloth
to eliminate certain impurities. A portion of the juice was kept in a refrigerator overnight
(18 hours) prior to the analysis, another portion was exposed to intense sunlight for the
same length of time and the third portion was left at ambient temperature.
Ascorbic acid contents of these portions were promptly determined. No further
treatment was given to the extracts before using them immediately for the analysis.

3.2.3 Preparation of Reagent Solutions

A weight of 15 g met phosphoric acid pellets (HPO 3) was dissolved in warm (60˚C)

distilled water using successive portions until the acid completely dissolved and was
transferred to a 500 ml stoppered bottle. The solution was allowed to cool to room
temperature and 40 ml glacial acetic acid was added followed by sufficient distilled
water to make 450 ml. The solution was stored in a refrigerator and was being used each
time for analysis. HPO3 slowly changes to H3PO4 but this solution satisfactorily

remains for 7-10 days when stored in a refrigerator. A weight of 30 g potassium iodide
crystals was dissolved in 100 ml distilled water in a conical flask to give a solution of
30% potassium iodide. The solution was then transferred into a stoppered reagent
bottle. A weight of 2.5 g CUSO4.5H2O was dissolved in a beaker using distilled water.

The solution was transferred to a 100 ml volumetric flask qualitatively by rinsing the
beaker with successive portions of distilled water until it was made up to the 100 ml
mark. The solution became exactly 0.01M copper sulphate (CUSO 4.5H2O) solution. It

was then transferred to a stoppered reagent bottle.

3.2.4 Determination of Ascorbic Acid

This was done using the method of the Association of Vitamin Chemists as described by
Kirk and Sawyer. 5 g of the sample was dispersed in 50 ml of EDTA/TCA solution and
homogenized. The homogenate was filtered using a filter paper and more of the
extractant was used to wash the residue in the filter paper until 100 ml filtrate was
obtained. A 20 ml portion of the filtrate was measured into a conical flask and 10 ml of
30 % potassium iodide solution was added to it, mixed well and then followed by four
drops of 1 % starch solution. The mixture was titrated against 0.01 M CuSO 4 solution

until a blue-black color appeared. A reagent blank was also titrated using 20 ml of
distilled water. The vitamin C content was calculated based on the relationship that 1 ml
CuSO4 = 0.88 mg vitamin C.

Therefore,
Vitamin C mg/100g = 100 x 0.88 x (T-B) x VT
W VA
Where,
W = weight of sample
T = titre value of sample
B = titre value of blank
VT = total extract volume
VA = volume of extract titrated
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