EVALUATION OF HEAVY METAL CONCENTRATION OF GRAINS CONSUMED IN

ENUGU METROPOLIS.

BY
JOSHUA CHIDIEBUBE MIRACLE
2018/250985

DEPARTMENT OF MEDICAL BIOCHEMISTRY

FACULTY OF BASIC MEDICAL SCIENCE
UNIVERSITY OF NIGERIA ENUGU CAMPUS

SEPTEMBER, 2023

I
 TITLE

EVALUATION OF HEAVY METAL CONCENTRATION OF GRAINS CONSUMED

IN ENUGU METROPOLIS.

BY
JOSHUA CHIDIEBUBE MIRACLE
2018/250985

DEPARTMENT OF MEDICAL BIOCHEMISTRY

FACULTY OF BASIC MEDICAL SCIENCE
UNIVERSITY OF NIGERIA ENUGU CAMPUS

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS OF THE AWARD OF

BACHELOR OF BIOCHEMISTRY (B.SC), HONOURS DEGREE IN
BIOCHEMISTRY

SUPERVISOR
DR. OBIANYDO H.O.

SEPTEMBER, 2023
i
 DEDICATION

I would like to dedicate this project work to God Almighty for giving me the strength to start

and complete this project work. I would also like to dedicate this project work to my mum,

Mrs. Chinenye Okonkwo for her financial and moral support through which I was able to

conclude this B.Sc. programme.

ii
 ACKNOWLEDGEMENT

I would like to show gratitude to God almighty for the accomplishment of this project-work

and for preventing accidents during experiments and practicals.

I want to thank God for my mother, Mrs chinenye Okonkwo for her support as this project

work was carried out.

She supported well both Spiritually, financially and emotionally as this project work was

carried out, God bless her.

Nevertheless, I would love to give special thanks to my supervisor Dr Obianydo for

approving such an interesting topic and for his guidance as I carried out the project work.

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 TABLE OF CONTENT

Title i

Dedication ii

Acknowledgement iii

Table of content iv

List of tables vi

Abstract vii

CHAPTER ONE

Introduction 1

1.1 Background to the study 1

1.2 Statement of the problem 2

1.3 Research questions 3

1.3 Aim of research 3

1.4 Specific objectives of research 3

1.6 Research hypothesis 4

1.7 Significance of study 4

1.8 Scope of study 4

CHAPTER TWO

Literature review 5

2.1 Heavy metals a cause of toxicity in grains 5

2.2 Phytochemical in grains 14

2.2.1 Alkaloids 14

2.2.2 Terpenoids 14

2.2.3 Phytosterols 15

iv
2.2.4 Phenols 15

2.2.5 Glycosides 16

2.3 Strategies to mitigate the potential risk of heavy metal toxicity in grains 16

CHAPTER THREE

3.1 Materials and reagents 19

3.1.1 Materials 19

3.1.2 Reagents 20

3.2 Grain samples 20

CHAPTER FOUR

Result and interpretation 24

4.1 Phytochemical analysis and heavy metal concentration of rice. 24

4.2 Phytochemical analysis and heavy metal concentration of corn. 27

CHAPTER FIVE

Discussion, conclusion and recommendation 29

5.1 Discussion 29

5.2 Conclusion 29

5.3 Recommendations for further research 30

Reference

v
 LIST OF TABLES

Table 1: Quantitative Phytochemical analysis of rice obtained from four markets in Enugu

metropolis. 24

Table 2: Flame atomic spectroscopy analysis of heavy metals in rice from four markets in

Enugu metropolis. 26

Table 3: Quantitative Phytochemical analysis of corn obtained from four markets in Enugu

metropolis. 27

Table 4: Flame atomic spectroscopy analysis of heavy metals in corn in from four markets in

Enugu metropolis. 28

vi
 ABSTRACT

Grains are an important source of food nutrition in both humans and animals, toxicity in
grains causes a lot of fatal diseases. This Study is focused on reviewing the toxicity of grains
in markets in Enugu metropolis. This study was a case study that involved Grains (namely
rice and corn), from four markets in Enugu metropolis(namely; Ogbete market, New market,
Ogige market and Mayor market). The grains were analyzed for Heavy metals and
phytochemicals, using a spectrum-lab 23A spectrometer.the spectrometer provide information
about the level of heavy metals and phytochemicals in grains. At the end of the study the
grains(namely; corn and rice) had presence of phytochemicals, the heavy metals
concentrations (particularly lead and cadmium) in rice where above minimum allowable
concentration, while the heavy metal concentrations (particularly mercury and arsenic) were
within the minimum allowable concentration while the heavy metal concentrations of lead,
arsenic, cadmium and mercury were within the minimum allowable concentration.

vii
 CHAPTER ONE

INTRODUCTION

1.1 Background to the study

Grains are the main energy and carbohydrate sources for human nutrition globally.

Governmental and non-governmental authorities recommend whole grains as a healthy food

choice. The role of toxicants in (whole) grains in Enugu metropolis and how to mitigate any

potential risk following their consumption has not been reported.

We found that grains contain significant toxicants, the main ones being; toxic heavy-metals

like arsenic, cadmium, mercury and lead[Mom Tatahmentan,, et al., 2018].

Whole grains usually contain more toxicants than refined products [Papanikolaou, et al.,

2017].

However, whole grains also provide more nutrients that may reduce the impact of these

toxicants. Strict regulatory thresholds aim to minimize the risk of toxicants to public health.

The risk posed by toxins from whole grains does not outweigh the known nutritional benefits

of whole grain consumption. Globally, there are over 50,000 edible plants. Just three of these

(rice, maize, millet and oat) provide about 60 per cent of the world's food energy intake[fao,

et al., 2018].

Grains have a long history of use by humans, dating back to prehistoric times. Admittedly,

pancakes and bread were still a long way off during the Middle Stone Age; however,

evidence suggests that some humans in Africa at that time (i.e., 105,000 years ago) did

indeed eat grain-based snacks [Mercader, et al.,2019].

1
Nowadays, grains are food staples and represent the primary source of carbohydrates even in

Enugu metropolis, there are many industrial sites in Enugu metropolis and this has resulted in

the pollution of farm lands where grain crops are cultivated.

1.2 Statement of the problem

Along with being our main energy and carbohydrate sources, grains also naturally contain

toxicants and, as a food category, represent one of the main dietary sources of food borne-

contaminants[Frank Thielecke, et al., 2018]

The definition of a toxicant is “any substance not intentionally added to food which is present

in such food as a result of the production, manufacture, processing, preparation, treatment,

packing, packaging, transport or holding of such food or as a result of environmental

contamination[Brussels, et al., 1993]

The potential sources for the contamination of grains are mostly environmentally based and

include air, dust, soil, water, insects, rodents, birds, animals, humans, storage and shipping

containers, and handling and processing equipment[Frank Thielecke,, et al., 2018]

Most heavy metals act as contaminants or toxicants of grains ranging from lead to cadmium

to arsenic by [Van der Kamp et al., 2014] Grains are generally consumed by the masses; it

could be assumed that a good population of Enugu state resident would be vulnerable to

toxicity of heavy metals. This work seeks to review the level of toxicity in certain grains in

markets in Enugu metropolis and specify the possible effects of accumulation of these heavy

metals [Tchounwou, et al., 2012].

Toxic metals primarily originating from industrialization have polluted the environment,

including food and caused adverse human health effects through food chains. Exposure to

2
low and high levels of arsenic(As), lead (Pb) and cadmium(Cd) can cause cancerous and non-

cancerous effects [Boffetta, et al., 1993].

Toxicity of heavy metals has been attributed to skin, lung, kidney and bladder cancers [chen

q.y., et al 2019] while cadmium and lead toxicity specifically has been linked to prostrate

cancers [chen, et al.,2019].

Hence the interest for the investigation.

1.3 Research questions

The research questions of this research study include;

i. What are the risks associated with consumption of grains from markets in Enugu

metropolis?

ii. What is the level of toxicity in grains from Enugu metropolis?

iii. How can one mitigate this risks and toxicity if they are in existence?

1.3 Aim of research

The aim of this project is to ascertain the level of heavy metals in concentration of grains

consumed in Enugu metropolis.

1.4 Specific objectives of research

The objectives of this research includes;

I. To determine the concentration of heavy metals (arsenic, lead, mercury and cadmium)

in some grains consumed in Enugu metropolis.

3
 II. To compare the heavy metal level in the grains with the Food and Agriculture

Organization/World Health Organization permissible limit.

III. To determine the concentration of phytochemicals contained in this grains.

1.6 Research hypothesis

It is hypothesized that;

 Grains in Enugu metropolis contain a high level of toxicity.

The null hypothesis states that;

 Grains in Enugu metropolis do not contain a any level of toxicity

1.7 Significance of study

The significance of this study includes;

 To confirm the level of heavy metals in grains in Markets in Enugu metropolis

 To Evaluate how to mitigate risks associated with contamination of heavy metals

in grains.

1.8 Scope of study

The scope of this study is restricted to grains from four market in Enugu metropolis namely;

Ogbete market, New market, Mayor market and Ogige market.

4
 CHAPTER TWO

LITERATURE REVIEW

2.1 Heavy metals a cause of toxicity in grains

Heavy metals are found naturally in the earth. Arsenic, cadmium, lead, and mercury are toxic

elements that are omnipresent at low concentrations in the environment [Clemens et

al.,2016]. They can enter plant, animal, and human tissues via inhalation, diet, and manual

handling. Toxic heavy metals such as cadmium, mercury, lead and arsenic can not only

compete with minerals such as calcium, magnesium or iron for absorption but can also bind

to vital cellular components, such as structural proteins, enzymes, and nucleic acids, where

they can interfere with their functioning [Andrade et al .,2017].

Heavy metals are a major cause of the toxicity of grains even in Enugu metropolis. Chronic

exposure to heavy metals can lead to wide-ranging health problems. For example, arsenic

affects the skin, lungs, brain, kidneys, liver, metabolic system, cardiovascular system,

immune system, and endocrine system. Cadmium impacts the bones, kidneys, liver, lungs,

testes, brain, immune system, and cardiovascular system [Tchounwou et al., 2012].

Grain is a commonly consumed food throughout the world [NLM, et al., 2018]. Arsenic,

Lead and Cadmium are among the toxic metals that pose serious health effects in humans,

exposure to these metals through diet is of concern especially among rice consumers.

Human Exposure to these toxic metals can occur through occupation, air, pollution or diet.

Dietary exposure is the most common route through which this toxic metal enter the human

body [Denng f. et al., 2019].

5
The grains may contain toxic metals which the human body could be exposed through

ingestion [Williams p.n, et al ., 2007].

2.2 Cadmium

Cadmium is a toxic trace element found as an environmental contaminant, both through

natural occurrence and from industrial and agricultural sources. Foods are the main source of

cadmium exposure, second to smoking. Wheat and rice are major cadmium contributors to

the diet. Cadmium is stored in the endosperm, the source of white flour. Grain genotypes vary

and can influence the level of grain cadmium accumulation [Clemens et al., 2016].

Cadmium like Arsenic has also be found to accumulate more in rice than other

grains[mehargaai, et al., 2013], brown rice contains more cadmium than white rice likely

because the grain contains an outer layer which accumulates metals more easily[Sun G., et

al.,2008].

2.2.1 Mechanism of action

Cadmium has an effect in apoptosis and cell differentiation(Rami, et al., 2014) ,Cadmium in

low concentration attaches itself to the human mitochondria and inhibit both oxidative

phosphorylation and cellular respiration(Patrick et al., 2003)

Cadmium leads to DNA strand breaks, DNA- protein cross links in cell lines, cadmium

inhibits the activity of antioxidant enzymes, such as superoxide dismutase, and copper/zinc-

dismutase(Filipic et al., 2012)

Cadmium can modulate the cellular level of Ca 2+ and the activities of caspases and nitrogen-

activated protein kinases (MRPKs) in the cells, in which these processes cause apoptosis

indirectly(Brama M et al., 2012)

6
Cadmium can induce ROS production and result in oxidative stress. This mechanism may

express the role of cadmium in organ toxicity, carcinogenicity and apoptotic cell

death(Caspian et al., 2017)

2.2.2 Effects and symptoms of cadmium toxicity

 Cadmium bone and Itai-itai disease: Several studies mentioned cadmium can affect

the skeletal system, exposure to cadmium caused skeletal demineralization, whereby

it may directly interact with bone cells, diminish mineralization, also inhibit

procollagen C-proteinases and collagen production(Staessen JA , et al., 2010)

 Renal damage in cadmium toxicity:Cadmium predominantly accumulates in kidney

and liver, but it can be found in other tissues such as bone and placenta. It has been

reported that occupational and environmental exposures to cadmium have implicated

renal dysfunction(Jarup L. et al., 2002)

 Cadmium and reproductive system:Several previous studies found that cadmium

has the potential to affect reproduction and development in several mammalian

species, and recent studies have also confirmed these findings(Thompson J, et al.,

2008)

 Cadmium and cardiovascular system: In vitro studies have indicated the

involvement of cadmium in endothelial dysfunction as well as carotid intima-media

thickness (IMT). Moreover, the formation of atherosclerotic plaques were promoted

in vivo(Fagerberg B, et al 2012)

 Cadmium and other systems: The acute central and peripheral neurotoxicity of

cadmium has been recently reported (Ismail SM et al., 2015) Cadmium may also

induce cellular damage and lipid peroxidation in brain. Its effect on monoaminoxidase

7
 (MAO) is responsible for oxidative deamination of monoamine

neurotransmitters(Ismail SM et al., 2015)

2.3 Arsenic

Generally both white and brown rice have high Concentrations of arsenic than other

grains[NLM, et al.,2018]

Arsenic is a ubiquitous element, which is introduced to the environment from both natural

and anthropogenic sources. The toxicity of arsenic compounds strongly depends on their

chemical forms—inorganic arsenic is considered to be more toxic than the organic form. All

plants can absorb some arsenic, but rice, cultivated in flooded conditions, can absorb much

more than other grains. On average, a 30-fold increase in arsenic has been reported for rice in

flooded as opposite to non-flooded conditions [Clemens et al., 2016].The soil in the fields,

when covered with water, creates conditions that allow arsenic to be converted to more

readily absorbable forms, although the actual amounts eaten in the diet will depend on type of

rice, growing, processing and cooking conditions.

Arsenic accumulates most in the outer layer of rice, which is the reason that whole grain rice,

with its bran intact, can have up to 80% more arsenic than white rice. It is also safe to eat by

infants and young children, who have higher intakes of arsenic relative to their body size than

older children and adults.

Moreover rice for example is a staple food that contributes toxic inorganic arsenic to humans

through diet[Schoof R.a, et al., 1999], brown rice accumulates more arsenic than white rice

this is likely because brown rice has a germ layer that retains more in organic arsenic [NLM,

et al., 2018]

8
2.3.1 Mechanism of action

Arsenic poisoning is a global health issue affecting millions of people worldwide through

environmental and occupational exposure, as well as intentional suicide and homicide

attempts. Although arsenic homicides commonly receive media publicity, the primary source

of arsenic toxicity to the general population is by contaminated water, soil and food products.

Arsenic (As) is a nearly tasteless odorless toxic metalloid element that is found ubiquitously

in the environment. Arsenic comes in four common valence states: As(o), As(III), As(V) and

Arsine gas and three common forms: inorganic salt, organic salt, and gaseous form (Olsen V,

et al., 2004)

Although regional distribution appears to be the dominant epidemiologic factor affecting

arsenic toxicity, there is evidence to believe that the manifestation of arsenic toxicity differs

between men and women. Some studies indicate that men are more affected than women by

arsenic-related skin conditions(Ferraris D, et al., 2016)

2.3.2 Effects and symptoms of toxicity

Acute

Gastrointestinal: Gastroenteritis is the most common symptom seen with acute toxicity.

Doses under 5mg results in nausea, vomiting, diarrhea, and abdominal pain. Symptoms begin

minutes to hours after ingestion and typically resolve within 12 hours, but symptoms may

persist for days after exposure. The classic description of diarrhea is as "rice water" or

cholera-like diarrhea that may be bloody. Arsenic has effects on the gastrointestinal mucosa

leading to vasodilation and sloughing of mucosal tissue which can lead to vesicle damage.

9
Cardiovascular: Hypotension is another hallmark characteristic of arsenic poisoning,

secondary to dehydration and volume loss. Reports exist of EKG abnormalities have been

noted hours to days after exposure including QTc prolongation, QRS lengthening, non-

specific ST changes, T wave flattening, and additional cases of torsades de pointes.

Pulmonary: Cough, dyspnea, chest pain may be seen, more commonly with inhalation. Non-

cardiogenic pulmonary edema can present from increased capillary permeability.

Renal: Proteinuria, hematuria and acute renal failure have been reported hours to days after

exposure.

Neurologic: Classically, neurologic symptoms occur in the subacute phase, weeks after

exposure; however, there are reports of neurologic symptoms occurring hours after

exposure including headache, delirium, encephalopathy, and seizures (Matthew k, et al.,

2022)

Subacute (1 to 3 weeks):

Neurologic: Reversible sensorimotor polyneuropathy is a well-documented symptom of

arsenic toxicity, typically seen one to three weeks after acute exposure, but it may occur

insidiously with chronic exposure. Dysesthesias predominately occur in a stocking-and-glove

distribution, starting with the soles of the feet and ascending. In severe toxicity, clinicians

have noted a loss of deep tendon reflexes, vibratory sensation, temperature sensation,

weakness and gait disturbances. Additionally, there are reports of headaches, confusion,

memory loss, seizures, and delirium.

Hematology: A wide variety of hematologic abnormalities exist, including leukopenia,

thrombocytopenia, basophilic stippling and anemia ranging from normochromic, normocytic

to hypochromic, microcytic as well as hemolytic anemia. (Matthew k, et al., 2022)

10
Chronic:

Dermatologic: Multiple dermatologic signs have been documented, prominently diffuse or

spotted hyperpigmentation, described as having a "raindrop appearance" which may be

preceded by hypopigmentation occurs six months to three years after exposure. Reynolds-

Aldrich-Mees lines or more classically described Mees lines occur in approximately 5% of

patients. Mees’ lines show a 1 to 2mm wide transverse white bands across the fingernails

presents 4 to 6 weeks after exposure. Other dermatologic signs include palmer-planter

hyperkeratosis, eczematoid lesions, warts, and alopecia.

Arsenic exposure has links to basal cell carcinoma, as well as squamous cell carcinoma

secondary to hyperkeratosis. Skin cancer in arsenic exposure has presented in sunless

exposure on palms, soles, and abdomen(Abernathy Co, et al., 1992)

2.4 Lead

Lead is also a ubiquitous element, found naturally in the earth’s crust at an average level of

10 mg/kg [Hershikoc, et al ., 2005].

There does not appear to be large differences in the location of lead stored in different

compartments of the grain. There is no known safe blood lead level, but chronic exposure to

lead of the above levels can seriously harm particularly a child’s health.

2.4.1 Mechanism of action

11
Lead toxicity is an important environmental disease and its effects on the human body are

devastating. There is almost no function in the human body which is not affected by lead

toxicity (Anjum Zara, et al 2015)

Lead is a highly poisonous metal affecting almost every organ in the body. Of all the organs,

the nervous system is the mostly affected target in lead toxicity, both in children and adults.

The toxicity in children is however of a greater impact than in adults. This is because their

tissues, internal as well as external, are softer than in adults. Long-term exposure of adults

can result in decreased performance in some tests of cognitive performance that measure

functions of the nervous system. Infants and young children are especially sensitive to even

low levels of lead, which may contribute to behavioural problems, learning deficits and

lowered IQ (Rubin & Strayer, 2008). Long-time exposure to lead has been reported to cause

anaemia, along with an increase in blood pressure, and that mainly in old and middle aged

people. Severe damage to the brain and kidneys, both in adults and children, were found to be

linked to exposure to heavy lead levels resulting in death. In pregnant women, high exposure

to lead may cause miscarriage. Chronic lead exposure was found to reduce fertility in males

(Sokol & Berman,1991)

2.4.2 Effects of toxicity and symptoms

i. Pregnant women who have elevated blood lead levels are at a risk of premature birth

or of babies with a low birth weight. The foetus may be adversely affected at blood

lead concentrations well below 25 μg per deciliter (Bellinget et al., 1987)

ii. Lead disrupts the maintenance of the cell membrane, red blood cells with a damaged

membrane become more fragile, which results in anaemia (White et al.,2007).

12
 iii. Lead is also speculated to alter the permeability of blood vessels and collagen

synthesis (Needlemann, 2004).

iv. Lead interferes with the activity of an essential enzyme called delta-aminolevulinic

acid dehydratase, or ALAD, which is important in the biosynthesis of heme, the

cofactor found in haemoglobin causing anaemia (Patrick et al., 2006).

2.5 Mercury

The levels of mercury in the earth’s crust are usually around 0.02 mg/kg. Like arsenic, this

element occurs in organic and inorganic forms. The organic form is considered most toxic.

2.5.1 Mechanism of action

Mercury is more volatile as it is heated and is readily absorbed when it is aerosolized by

vacuuming. Pulmonary, central nervous system (CNS), and renal toxicity are common with

elemental mercury. The gastrointestinal tract primarily absorbs inorganic mercury salts, and

secondarily they are absorbed through intact skin. The salts mainly cause renal and

gastrointestinal (GI) toxicity. Organic mercury is also primarily absorbed by the GI tract and

secondarily through intact skin. Organic mercury toxicity typically leads to neurologic

symptoms which are characteristically delayed(Newman RK, et al., 2022)

2.5.2 Effects of toxicity and symptoms

i. Primary absorption of elemental mercury will cause shortness of breath, cough, fever,

nausea, vomiting, diarrhea, headache, metallic taste, salivation, and visual

disturbance. Severe exposure may lead to respiratory distress and failure. Ingested

elemental mercury can be metabolized to inorganic mercury salts and can also

develop those symptoms (Rusyniak DE, et al 2010)

13
 ii. Acute ingestion of organic salts typically will cause a metallic taste and a graying of

the oral mucosa. The most common findings of significant ingestion include

abdominal pain, hemorrhagic gastroenteritis, acute tubular necrosis, and

shock(Rusyniak DE, et al 2010)

2.6 Phytochemical in grains

Phytochemicals are a group of chemical compounds that occur naturally in plants,

conferring colour, flavour, aroma and texture. (Ramona et al., 2017). Phytochemicals can be

classified into major groups based on their chemical structure and this major groups are:

alkaloids, sulphur-containing phytochemicals, terpenoids and polyphenols(Ramona et al.,

2017).

2.6.1 Alkaloids

Alkaloids are bases which are nitrogenous in nature, Alkaloids have a wide range of

pharmacological activities. including anti-malaria (e.g. quinine), anti-asthma

(e.g. ephedrine), anti-cancer (Kittakoop P, et al.,2014)

2.6.2 Effects of alkaloids

Alkaloids are among the most important and best-known secondary metabolites,, i.e. biogenic

substances not directly involved in the normal growth, development, or reproduction of the

organism. Instead, they generally mediate ecological interaction, which may produce a

selective advantage for the organism by increasing its survivability or fecundity. In some

cases their function, if any, remains unclear (Aniszewski, et al., 2007)

2.6.3 Terpenoids

14
Terpenoids, also known as isoprenoids are the most numerous and structurally diverse

natural products found in many plants (Rama R.M, et al., 2019).

They are the largest class of plant secondary metabolites, representing about 60% of

known natural products, many terpenoids have substantial pharmacological bioactivity and

are therefore of interest to medicinal chemists (Ashour M, et al., 2010).

Terpenoids can also be classified according to the type and number of cyclic structures they

contain: linear, acyclic, monocyclic, bicyclic, tricyclic, tetracyclic, pentacyclic, or

macrocyclic (Ludwiczuk A, et al.,2017).

2.6.4 Effects of terpenoids

Terpenoids are known to induce toxic effects in humans and animals, terpenoids also have

local irritant effects and thus are capable of causing gastrointestinal disorder(Armelle T, et al.,

2014)

2.7 Phytosterols

Phytosterols are phytosteroids , similar to cholesterol that serve as structural components of

biological membranes of plants(Moreau, et al., 2018).Phytosterols are a compound found in

many plant foods.

2.7.1 Effects of phytosterols

Some studies suggest a role for phytosterols in cancer treatment and prevention(Khan Z, et

al., 2022)

Side effects, if any, tend to be mild and may include constipation, nausea, upset stomach,

heartburn, flatulence, and the discoloration of stools(Nccih et al., 2019).

15
2.7.2 Phenols

Phenols are the largest group of Phytochemicals, they also include flavonoids and tannins,

more than 5000 flavonoids have been identified(Harbone, et al., 2000).

2.7.3 Effects of phenols

Phenols have health benefits which include quenching of oxygen free radicals, the protection

and regeneration of dietary antioxidants (e.g. vitamin E) and the chelation of pro-oxidant

metals (Terao, 2009).They possess health b properties by stopping chronic diseases,

including cardiovascular diseases (CVDs), cancer, diabetes, osteoporosis and certain vision

diseases (Wahle et al., 2010).

2.7.4 Glycosides

Glycosides play numerous important roles in living organisms. Many plants store chemicals

in the form of inactive glycosides(Marco, et al., 2007).

2.7.5 Effects of glycosides

The most common side-effects of glycosides include dizziness, fatigue, headache, anxiety,

gastrointestinal upset, change in taste and blurred vision.

2.8 Strategies to mitigate the potential risk of heavy metal toxicity in grains

There are obvious sectors along the food production chain that can impact the risk of

contaminants for human health, including production/farming, processing, distribution, and

retail and out-of-home production. Within these sectors there are a myriad of steps, which

are not covered in detail here but will warrant a separate research. An important role in this

simplified approach falls to the appropriate authorities, who provide a regulatory framework

16
for managing any risk. We have identified here that production/farming and

processing/manufacturing are two other crucial sectors that influence the risk of

contamination..

With respect to heavy metals, increases in understanding of the molecular pathways

responsible for the accumulation of arsenic and cadmium have been made, leading to the

development of crop varieties with significantly reduced concentrations of toxic metals in

their edible parts [Clemens et al .,2016]. Other agronomic strategies to reduce the toxic metal

accumulation in grains include liming; the application of organic material to reduce the

bioavailability of metals such as cadmium in the soil; silicon fertilization to saturate the

transport pathway that mediates arsenic accumulation; and the optimal management of

irrigation regimes to control cadmium and arsenic bioavailability [Zhao et al .,2015].

(i) Processing/Manufacturing of grains

Once the grains are harvested, a prime role in the mitigation of the risk of contaminant falls to

the processing/manufacturing steps. Whole grains undergo thorough cleaning and dehulling,

with debranning (as is done for refined grain) applied to a limited extent in order to retain the

nutritional superiority.

Such processing can also affect the nutritional value of cereals in a positive way. For

example, when grains are milled into flour, the intact botanical structures are opened, which

lowers the amount of resistant starch (a type of starch that is not digested in the small

intestine, therefore functions at a dietary fibre) [Bednar et al., 2001]. Current strategies to

influence heavy metal composition typically relate to milling and cooking. For example, the

milling of durum wheat reduced the presence of the elements nickel > arsenic > cadmium >

lead [Cubadda et al., 2003]. There may also be potential positive effects of heat. During

17
thermal processes such as baking and extrusion, phenolic compounds, which have antioxidant

properties, are generated [Ragaee et al., 2014].

(2) Regulations

With regulations in place that set standards for maximum levels of contaminants as a result of

farming practices and the processing of grains, the question remains what the consumer

can do in order to mitigate any potential risk from ingestion of contaminants from whole

grains. Common hygiene standards as well as guidance for the consumer by the means of

“date marks” are useful tools. For example, advising the consumer to adhere to the date mark,

‘use by’, may reduce likelihood of consuming any mould (a certain types of fungus) that may

grow on bread that has been stored inappropriately: In this section, we outline dietary

strategies and highlight the inherent properties within whole grains, which when consumed as

part of a healthy balanced diet, may reduce the risk of contaminants.

(3) Washing Process

Washing of grains like white rice is likely to reduce concentration of toxic metals such as

Lead and cadmium by 57% and 46% respectively [NLM, et al., 2018].

Washing of grains like rice is accompanied by some lose of heavy metals [NLM, et al.,

2018]

18
 CHAPTER THREE

MATERIALS AND METHODS

III.1 Materials

3.1.1 Instruments and Equipments

The instruments and equipment used for this research work includes;

i. Digital weighing balance

ii. Heater

iii. Electronic Blender

iv. Heating block

v. Centrifuge

vi. Test-tubes

vii. Muslin cloth

viii. Spectrophotometer(Spectrum-lab).

ix. Measuring cylinder

x. Containers

xi. Beaker

xii. Test-tube rack

xiii. Pipette

xiv. Chromogen

xv. Temperature controlled water-bath

xvi. Digestion Flask

xvii. Distilled water

19
 3.1.2 REAGENTS/CHEMICALS

The reagents used for this research work are;

i. Tetraoxosulphate(VI) acid (H2SO4)

ii. Phosphomolybdic acid

iii. Vanillin

iv. Ethanol

v. Folin’s reagent

vi. Sodium carbonate (Na2CO3)

vii. Perchloric acid

viii. Alkaline pirate

ix. Potassium acetate

x. Formaldehyde

xi. Aluminium chloride (AlCl3)

xii. Chromogen

xiii. Nitric acid (HNo3)

III.2 METHODS

3.2.1 Sample collection

Two different grain samples Rice (Oryza sativa) and Maize (Zea mays) were collected from

4 major markets in Enugu state; Ogbete market, New market, Mayor market and Ogige

market.

20
 III.3 Sample Extraction

III.3.1 Maceration Extraction Method(used for rice extraction).

The Method used for extraction of rice was the Maceration extraction method, to start the

extraction method, 20g of rice sample, was measured out and soaked in 30ml of ethanol

overnight and crushed to pieces using mortar and pestle, 20ml of ethanol was added and left

for 4hours (for extraction of filtrate). The mixture was then filtered using muslin cloth and

store in a labelled container to obtain a filtrate.

3.3.2 Maceration Extraction Method(used for maize)

The extraction method used for extraction of maize was the maceration method, to start the

extraction method, 20g of maize sample was measured out and soaked in 30ml of ethanol

overnight and crushed to pieces using mortar and pestle, 20ml of ethanol was added and left

for 4hours (for extraction of filtrate). The mixture was filtered using muslin cloth and store in

a labelled container to obtain filtrate.

Each filtrate was further centrifuged at 5000rpm for five minutes to obtain a extract. This was

transferred into a container and stored in refrigerator until used for phytochemical analysis.

3.4 Phytochemical analysis

3.4.1 Method used for phytochemical analysis

The method used for phytochemical quantification followed a standard procedure described

by Harbone (1998).

21
3.4.2 Test for glycosides

The prepared extract (0.5 ml) was transferred into a test tube and 0.2 ml of alkaline pirate was

added followed by 2.3 ml of distilled water. The mixture was boiled for 5 minutes, allowed to

cool and absorbance was read at 490 nm against the blank.

3.4.3 Test for saponins

The prepared extract (0.5 ml) was transferred into a test tube containing 1 ml of vanillin

reagent. Then 1.5 ml of 72 % H 2SO4 was added and incubated for 10 minutes at 60 °C in a

temperature-controlled water bath. After cooling in cold water, the absorbance was read at

544 nm against the blank.

3.4.4 Test for alkaloids

The prepared extract (0.5 ml) was added to 1.5 ml of 60 % H 2SO4. After 5 minutes, 1 ml of

0.5 % formaldehyde was introduced into the test mixture and incubated for 2 hours at room

temperature. Absorbance was read at 565 nm against distilled water.

3.4.5 Test for tannins

A measured volume (0.5 ml) of the extract was added to 0.2 ml Folin reagent (1/10 dilution).

Following this, 2.2 ml of distilled water and 0.1 ml of 3.5 % Na 2CO3 was added. After 5

minutes the absorbance at 640 nm against the blank.

3.4.6 Test for flavonoids

A measured volume (0.1 ml) of 10 % AlCl3 was added to 0.5 ml of the crude extract. Then

0.1 ml of 1 M potassium acetate and 2.3 ml of distilled water was added and absorbance was

read at 415 nm after 30 minutes of incubation at room temperature.

22
3.4.7 Test for terpenoids

The extract (0.5 ml) was pipetted into a test tube. 0.5 ml of 5 % phosphomolybdic acid and

1.5 ml of concentrated H2SO4 was added. After incubation for 30 minutes at room

temperature, absorbance was read at 490 nm against the blank.

3.4.8 Test for phenols

The extract (0.5 ml) was transferred into a test tube containing 0.2 ml of 1/10 dilution Folin

reagent. 0.2 ml of 2 % Na2CO3 was added and allowed to stand for 30 minutes at room

temperature. After addition of 2.1 ml of distilled water, the absorbance was read at 640 nm

against the blank.

3.4.9 Test for steroids

A measured volume of the extract, 0.5 ml was pipetted into a test tube containing 1 ml of

chromogen and 1.5 ml of distilled water was added. The mixture was incubated at room

temperature for 30 minutes and absorbance was read at 550 nm against the blank.

3.5 Statistical Analysis

The Statistical analysis software Graph pad prism version 9 was used to analyse data

from this study and the results were expressed as mean + SD.

23
 CHAPTER FOUR
RESULT AND INTERPRETATION

4.1 Phytochemical analysis and heavy metal concentration of rice (Oryza glaberrimaare)

The Quantitative Phytochemical analysis of rice from markets in Enugu

metropolis are showed the concentration levels of active compounds such

as: Glycosides, Saponins, Alkaloids, Tannins, Flavonoids, Terpenoids and

Phenols.

The Phytochemical with the highest concentration in rice is terpenoids

which was ((3977 +1), This is followed by phenols which showed a high

concentrations of (1158+ 119.50), The concentration of Tannins was also

high and was (1001+ 117.10).

Steroids showed the lowest concentration at (96.98+19.07) accompanied

by Saponins which showed a low concentration at (106.4+ 42.75),

followed by Alkaloids which showed low concentrations at (326 + 90.70)

compared to other phytochemical in the rice.

Flavonoids and Glycosides showed average concentrations with flavonoids

being slightly higher than glycosides (see table 1) .

24
Table 1: Quantitative Phytochemical analysis of rice obtained from four markets in

Enugu metropolis.

Parameters Minimum Maximum Mean + SD

Glycosides 636.70 1069 850.70 178.40

Saponins 74.23 168.80 106.40 42.75

Alkaloids 238 422 326 90.70

Tannins 922.50 1175 1001 117.10

Flavonoids 548.60 1112 852.70 231.50

Terpenoids 3010 5485 3977 1081

Phenols 987.70 1244 1158 119.50

Steroids 76.91 113.90 96.98 19.07

25
The mean concentration of heavy metals in rice

samples from four markets in Enugu metropolis

namely; ogbete market, Ogige market, new market and

mayor market were shown in table 2.

The concentration of lead (0.41+ 0.41mg/kg) was

slightly higher than the minimum allowable

concentration. The concentration of cadmium (0.05+

0.02mg/kg) was also higher than the minimum

allowable concentration. The concentration of arsenic

(0.06+0.06mg/kg) and that of mercury

(0.04+0.02mg/kg) were within the minimum allowable

concentration (See Table 2).

26
Table 2: Flame atomic spectroscopy analysis of heavy metals in rice from four
markets in Enugu metropolis.
Parameters Minimum Maximum Mean + SD MAC

Cadmium 0.03 0.08 0.05 0.02 0.05

Arsenic 0.01 0.12 0.06 0.06 0.1

Mercury 0.02 0.07 0.04 0.02 0.05

Lead 0.06 0.85 0.41 0.41 0.1

27
4.2 Phytochemical analysis and Heavy metal concentration of corn (Zea mays).

The results of the quantitative Phytochemical analysis of corn from four

markets in Enugu metropolis namely; ogbete market, mayor market, Ogige

market and new market, were shown in table 3.

The result showed the concentration levels of active compounds such as:

Glycosides, Saponins, Alkaloids, Tanins, Flavonoids, Terpenoids and

Phenols.

The concentration of Phenols (1464+272.1) in corn was highest in the

four markets in Enugu metropolis, followed by the concentration of

Terpenoids (944.5+227.3) which was also high. The concentration of

Alkaloids (685.2+337.7) was also high.

Saponins (116.2+66.71) had the lowest concentration in corn from four

markets Enugu metropolis, followed by the concentration of

Tannins(174.4+116.5) and Steroids(174.9+132.2) which was also in low

concentrations.

The concentration of Flavonoids(242.8+237.5) and Glycosides was of

average concentration from markets in Enugu metropolis(see table 3).

28
Table 3: Quantitative Phytochemical analysis of corn obtained from four markets in

Enugu metropolis.

Parameters Minimum Maximum Mean + SD

Glycosides 177.70 562.70 316.70 169.70

Saponins 58.46 203.10 116.20 66.71

Alkaloids 71 585 685.20 337.70

Tannins 82.50 345 174.4 116.50

Flavonoids 356.30 986.30 242.80 237.50

Terpenoids 754.5 1225 944.50 227.30

Phenols 1278 1868 1464 272.10

Steroids 75.70 356.60 174.90 132.20

29
The table below shows the mean concentration of heavy

metals in corn samples in four markets in Enugu

metropolis namely; ogbete market, mayor market,

Ogige market and new market.

The concentrations of lead, mercury, cadmium amd

arsenic in corn from this four markets in Enugu

metropolis were within the minimum allowable

concentration(see table 4)

30
Table 4: Flame atomic spectroscopy analysis of heavy metals in corn in from four

markets in Enugu metropolis.

Parameters Minimum Maximum Mean + SD Mac

Cadmium 0.01 0.01 0.02 0.01 0.05

Lead 0.03 0.16 0.09 0.1 0.1

Arsenic 0.00 0.03 0.02 0.01 0.05

Mercury 0.04 0.04 0.02 0.01 0.1

31
 CHAPTER FIVE

DISCUSSION, CONCLUSION AND RECOMMENDATION

5.1 Discussion

In this study observed the level of heavy metals in grains in

markets in Enugu metropolis. The literature review was

concentrated on the heavy metal toxicity in grains, the mechanism

of action and the effects of the toxicity and Phytochemicals.

Research was undertaken with Grains particularly rice and corn

from four markets in Enugu metropolis namely; new market,

ogbete market, mayor market and Ogige market.

The results show that the concentrations of lead and cadmium in

rice from this four markets(new market, ogbete market, Ogige

market and mayor market) was higher than the minimum

allowable concentration while the concentration of arsenic and

mercury in rice from this four markets were within minimum

allowable concentration.

32
 During a population health risk assessment on the

consumption of heavy-metal-contaminated food crops,

Owerri, Imo State, it was reported that the concentration of

lead, cadmium, and nickel exceeded the maximum allowable

concentrations for agricultural soil as recommended by the

European Union. Levels of lead, cadmium, and nickel in food

crops were highest in rice(Orisakwe, et al., 2012).

While the concentrations of lead, mercury, cadmium and arsenic

in corn from this four markets in Enugu metropolis was within the

minimum allowable concentration.

There were active Phytochemicals (namely; glycosides,

flavonoids, saponins, alkaloids, tannins, terpenoids, phenols and

steroids) in both high and low concentration in grains and corn

from the four markets( namely Ogige market, ogbete market, new

market and mayor market).

33
5.2 Conclusion

The existence of heavy metals like lead and cadmium above

minimum allowable concentration in rice and existence of active

phytochemicals in grains(particularly; rice and corn) from this

four markets in Enugu metropolis for example could be

dangerous, to mitigate this risks the following actions should be

taken;

i. Regulations should be put in place to ensure that the grains

were adequately processed before being brought to the

market.

ii. Adequate washing of grains before use by final consumer.

iii. Regulations should also be put in place to ensure minimal

preservatives are used in storage of grains

34
5.3 Recommendations for further research

The research work wasn’t carried out on other grains like millet,

wheat, oats because of financial constraints and availability of this

grains in the markets. Further research with this other grains if

accessible will be helpful to determine if there also heavy metals

and active phytochemical in this other grains and show how to

mitigate this risks.

35
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