EFFECTS OF AQUEOUS EXTRACT OF FERMENTED
SEEDS OF prosopis africana ON OKPEYE ASPARTATE
AMINO TRANSFERASE AND LOW DENSITY
LIPOPROTEIN OF WISTAR ALBINO RATS
BY
ANI PRECIOUS EBERECHI
2015030172643
A PROJECT PRESENTED TO THE DEPARTMENT OF APPLIED
BIOCHEMISTRY, FACULTY OF APPLIED NATURAL
SCIENCES, ENUGU STATE UNIVERSITY OF SCIENCE AND
TECHNOLOGY (ESUT)
IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR
THE AWARD OF BACHELOR OF SCIENCE (B.Sc.) DEGREE IN
APPLIED BIOCHEMISTRY
DECEMBER, 2019
i
ii
DEDICATION
I dedicate this research to God Almighty for his protection and preservation through
the period of this research work.
iii
ACKNOWLEDGEMENTS
The biggest of my gratitude goes to God Almighty for the life and provision throughout
my stay in this institution and during the course of this work.
Sincerely appreciate my supervisor Mrs. AKPATA E.I. for her assistance, guidance and
constructive criticism in the course of this work and during my stay in school. I also
appreciate all those who through their sleepless night, I am who I am today, that is the
Head of Department Dr. Achikanu,C. E. and lecturers of this Department.
I must immensely thank my uncle Mr and Mrs. Gabriel Ogbu and my beloved brother
Mr. Augustine Ani for their support both spiritually, financially and way of advice in
the course of my program in school, I must say thank you and God bless.
iv
TABLE OF CONTENT
Title Page
i
Certification
ii
Dedication
iii
Acknowledgement
iv
Table of Contents
v
List of Plates
viii
List of Tables
ix
Abstract
x
CHAPTER ONE
1.0 Introduction
1
1.1 Prosopis Africana
1
1.2 Aim of study
2
1.3 Objectives
2
CHAPTER TWO
2.0 Literature Review
3
2.1 Scientific Classification of prosopis africana seed
4
2.2 Phytochemical Analysis
6
2.3 Bio-pharmaceutical Potentials of prosopis spp
8
5
2.4 Liver
8
2.5 Meaning of Liver Function Test
9
2.5.1
Functions of the liver
9
2.5.2
Uses of the liver function test
10
2.5.3
Classification of liver function test
10
2.5.4
Limitation of liver function test
10
2.6 Aspartate Aminotransferase
11
2.7 Lipid Profile
12
2.8 Cholesterol
12
2.8.1
12
Low density lipoprotein
2.9 UV spectrophometer
13
2.9.1
14
Principle of ultraviolet spectrophotometer
CHAPTER THREE
3.0 Materials ad method
16
3.1 Materials
16
3.2 Plant materials
16
3.3 Animal Study
16
3.4 Equipment's/ Instrument
16
3.4.1
Equipment's
16
3.4.2
Chemicals and Reagents
17
3.5 Methods
18
3.6 Traditionals Fermentations of the prosopis Africana seeds
18
3.7 Experimental Designs
18
6
3.8 Sample Collection
19
3.9 Biochemicals Parameters
19
3.9.1
Determination of aspartate amino transferase
19
3.10
Lipid profile test
20
CHAPTER FOUR
4.0 Results
21
CHAPTER FIVE
5.0 Discussion
24
5.1 Conclusion
25
Reference
26
Statistical Tables
30
Descriptive
30
Anova
31
Post Hoc Test
32
Homogeneous Subsets
33
Appendix
34
7
LIST OF PLATES
Plate 1; Prosopis africana seeds
4
Plate 2; Fermented Prosopis africana seeds
4
Plate 3; Spectrophotometer
13
Plate 4; Effect of aqueous extract of fermented Prosopis africana seed
on low density lipoprotein level in rats
21
Plate 5; Effect of aqueous extract of fermented Prosopis africana seed
on Aspartate aminotransferase activity in rats
8
25
LIST OF TABLES
Table 1; Phytochemical Analysis
3
9
ABSTRACT
The effect of aqueous extract of fermented seeds of Prosopis africana on lipid profile (Low
Density Lipoprotein) and liver marker (Aspartate Amino transferase) of wistar albino rats were
determined in this study. Standard methods were used in preparing aliquots of the sample used for
the analysis. The result showed a significant (p<0.05) increase in low density lipoprotein
cholesterol levels of animals in group 2 fed with 50mg/kg extract (4.535±0.89), group 3 fed with
100mg/kg extract (4.745±1.12) and group 4 fed with 200mg/kg extract (4.753±0.46) when
compared with the animals in group 1 which are fed wiyh normal saline (3.023±0.67). there was a
significant (p<0.05) difference in the low density lipoprotein cholesterol level of animals in group
5 administered 12.5mg/kg orlistat when compared to the low density lipoprotein levels of animals
in the control group 1. The result showed a significant (p<0.05) decrease in the aspartate amino
transferase (AST) activity of the animals in group 2 fed with 50mg/kg extract (53.000±3.74),
animals in group 3 fed with 100mg/kg extract (54.500±6.40), in group 4 animals fed with
200mg/kg extract (41.750±14.22) when compared with the animals in the control group
(71.500±21.83). The AST activity of group 5 rats administered with 12.5mg/kg of orlistat
decreased significantly (p<0.05) when compared to the animals in the control group 1. From the
result, it shows that incorporation of the fermented seed of Prosopis africana in the human diet
may protect against liver damage. However, the increase in the level of low density lipoprotein of
the rats may be as a result of the oil base of the seeds. Therefore, moderate incorporation of the
fermented seeds of Prosopis africana in diet is advised.
10
CHAPTER ONE
1.0 INTRODUCTION
1.1 Prosopis africana
prosopis africana also known as african mesquite or iron tree is the only species of prosopis that
is indigeneous to tropical africa. Its tree could be between 4-20m long. This tree is characterized
by a deep, fast growing tap root. prosopis africana (pa) is mostly found growing in the savanna
regions of western African. (keay et al., 1964). it has different names by the various ethnic groups
in Nigerian. prosopis africana is the only species found in the savanna, especially in senegal and
Nigeria. Because this species is not cultivated, it is often referred to as wild endangered but edible
(Ola-adams et al., 1993), as a lost crop or as a lesser crop (Okafor et al., 1993) prosopis africana
has vast social, economic, cultural, medicinal and agricultural values. It is widely used and
consumed in the entire country and beyond. It is very popular for its seeds, highly priced food
condiment or seasoning, rich in protein, fatty acids and other vital nutrients and minerals
(Ayanwuyi et al., 1993). High consumption rate and the potential use of prosopis africana as a
good source of foreign earnings for Nigeria, the traditional method of post-harvest processing
provides a poor quality product with low nutrient content. This has limited its utilization both
locally and internationally. In Nigeria, there is scarcely large scale producer of prosopis africana
that use machinery for its storage, handling or processing. Many small scale producers’ carry out
these operations manually. The genus prosopis accommodates 44 species, of which 40 are native
to north and south Americas 28 species of this genus comes from Africa. Chemical compounds in
prosopis spp. Change certain physiological processes in the human body. Besides medicinal
applications, different mesquite species have other uses. Since its wood is extremely hard and
durable, and is of appealing colouration, it is used for making furniture's and parquet flooring. Its
wood is also used for construction, as firewood, or for charcoal production (Mwangi et al., 2008).
11
woodern chips provide much for gardening (Pasiecznik et al., 2001). a beverage, known as “anapa”,
is produced by mixing mesquite pods in water after being fermented, it produces the alcoholic
beverage “chichi” (Vilela et al., 2009). owing to its high carbohydrate level, mesquite wood can
also be used to produce bioethanol.
1.2 AIM OF STUDY
The aim of this study is to determine the possible protective effect of aqueous extract of fermented
prosopis africana seed on low density lipoprotein and Aspartate amino transferase.
1.3 OBJECTIVES
 To determine the effect of aqueous extract of fermented prosopis africana seeds on lipid
profile (LDL)
 To determine the effect of aqueous extract of fermented prosopis africana seeds on liver
marker (AST).
12
CHAPTER TWO
2.0 LITERATURE REVIEW
prosopis africana (Guill., Perrott. and Rich.) Taub. (Leguminosae, sub-family Mimosoideae) is
the only native prosopis in Africa. It has a natural distribution from Senegal to Ethiopia in the
north, from Guinea to Cameroon in the south, and from Uganda to Egypt in the east; but it has
disappeared from extensive parts of its range due to over-exploitation, such as excessive cutting
of stems and branches resulting in limited natural regeneration (Pasiecznik et al., 2004). In West
Africa, it extends throughout the Sudanian and Guinean ecozones in the southern part of its range
and into the Sahelian ecozone in the northern part of its range. It does not tolerate habitually dry
sites, preferring 600–1500 mm annual rainfall. Trees produce a deep taproot, grow slowly but can
attain a height of 20 m in natural stands, and can be coppiced for successive harvests. Trees in
natural stands generally have an erect form, but they may be multi-stemmed due to forking in the
lower trunk. The breeding system has not been studied, but it is assumed to be primarily
outcrossing, as reported for other species in the genus (Bessega et al., 2000; Dhillon, et al., 2003).
Prosopis species are insect pollinated (Toro, 2002), but the guild of pollinators of prosopis africana
has not been investigated. Seeds are naturally dispersed by browsing animals, such as camels,
cattle and goats at the end of the dry season (Tybirk, 1991), and perhaps also by humans who
collect the pods to feed to their animals and collect cow dung to fertilize their fields. Seed dispersal
distances could vary, therefore, from relatively short to relatively long distances, and this would
affect the population genetic structure (Hamrick et al., 1992). There has been only one published
study of genetic variation in prosopis africana: signifificant differences were observed in height
among four Nigerian provenances in a nursery test (Akinnagbe et al., 2007). prosopis africana is
very important for farming and pastoralist communities in the West African Sahel (Agboola, 2004).
The wood is moderately dense (basic density = 687 kg/m3) at one site in Burkina Faso and very
13
resistant to termites and fungi (Ge´ rardin et al., 2004), making it useful for construction poles and
planks, mortars, pestles, and handles for farm implements. It is also highly valued for firewood
and charcoal (Pasiecznik et al., 2004), and is the preferred species by many blacksmiths in the
region. Its leaves, succulent branches and pods provide fodder for cattle and goats, which is
essential during the 9-month long dry season. In some areas, people use the fermented seeds as a
food condiment (Barminas et al., 1998; Aremu et al., 2006; Kalinganire et al., 2007), so there is a
thriving market for the seeds in these areas. The leaves, branches, bark and roots are used for
several traditional medicines (Arbonnier, 2002; Kalinganire et al., 2007). In addition, trees fix
atmospheric nitrogen (Halliday, 1984) and, therefore, can improve soil fertility in the traditional
parkland agroforestry system. Because it has many uses for farming and pastoralist communities,
there is intensive extraction pressure on prosopis africana in parkland agroforests and statecontrolled forests in the West African Sahel. Excessive branch lopping and pod harvesting, for
example, have seriously reduced the natural regeneration in some areas. This, together with the
fact that few communities protect and manage natural regeneration, has dramatically reduced the
abundance of P. africana in many areas. In addition, farmers and pastoralists state that many trees
are dying due to increasingly hotter, drier conditions in the region (ICRAF-IFAD, 2006).
2.1 SCIENTIFIC CLASSIFICATION OF PROSOPIS AFRICANA SEED
KINGDOM
Planta
Sub Kingdom
Tracheobionta
Super Division
Spermatophyta
Division
Magnoliophyta
Class
Magnoliopsida
14
Sub Class
Rosidae
Order
Fabales
Family
Fabaceae
Source:
(Achi and Okereka, 1999)
15
2.2 Phytochemical Analysis
Table 1: Phytochemical composition of seed and pod extract of Prosopis africana (Ajiboyeet et al,
2013)
S/N
Test
Seed Extract
Pod extract
1.
Phlobatanmin
+
+
2.
Flavonoid
+
-
3.
Cyanoglycoside
-
+
4.
Tannin
+
+
5.
Saponin
+
+
6.
Steroid
+
+
7.
Alkaloid
+
+
Keys:
Present = +
Absent = -
16
Plate 1: Prosopis africana Seed
Plate 2: Fermented Prosopis Africana Seeds (Okpeye)
17
2.3 BIOPHARMACEUTICAL POTENTIALS OF PROSOPIS SPP.
All parts of prosopis spp. Are traditionally used by indigenous people for curing various ailments
(khejra et al., 2001). water extracts of leaves and bark are traditionally used to cure mouth and
throat infections as well as bronchitis and ulcers,internal diseases including parasites and urinary
diseases and skin parasitic infections as well as dermatitis (Pasieczik et al., 1999). leaf smoke is
traditionally used to cure eye infections and extracts are recommended for use against snakebite
and scorpion sting (Pimental et al., 1960).
Alkaloids, flavonoids, terpenes and phenolic compound are the most important bio-active
substances of prosopis spp. Terpenes are used as insecticides and their pharmacological properties
include antibacterial, antifungal, antihelminthic, antimalarial and molluscicidal activities (Gurih
fakim et al., 2006). Phenolic compounds from mesquite show anti-inflammatory, anti-tumor, antiHIV, anti- infective, vasodilatory, antinulcerogenice analgesic, and immune stimulant activities
(Stefanavie et al., 2015). Flavonoid have attracted interest recently, due to the discovery of their
pharmacological activities (Lou et al., 2014). Alkaloids from mesquite are applied as analgesics
and anti-malaria agents. Alkaloids of prosopis speciealso demonstrate a broad spectrum of antifual activities against fungi such as fusarium, drechslera and alternaria.The tree contributes to
nutrient recycling and prevention of soil erosion.
2.4 LIVER
Liver is a self-regenerating organ that plays important roles in the body. It functions not only in
metabolism and removal of exogenous toxins and therapeutic agents responsible for metabolic
derangement but also in the biochemical regulation of fats, carbohydrates, amino acids, protein,
blood coagulation and immune-osculation function (Ram et al.,1999). Due to its ability to
regenerate, even a moderate cell injury is not reflected by measurable changes in its metabolic
18
function. However, damage caused by lipid peroxidation on the membrane of the hepatocytes
allows the leakage of some cytosine enzymes of the liver into the blood stream (Plaa et al., 1982).
2.5 MEANING OF LIVER FUNCTION TESTS
Liver function tests (LFTs or LFs), also referred to as a hepatic panel, are groups of blood tests
that provide information about the state of a patient's liver (Mary, 2009). These tests include
prothrombin time (PT/INR), aPTT, albumin, bilirubin (direct and indirect), and others. The liver
transaminases aspartate transaminase (AST or SGOT) and alanine transaminase (ALT or SGPT)
are useful biomarkers of liver injury in a patient with some degree of intact liver function (Johnston
DE, 1999). Most liver diseases cause only mild symptoms initially, but these diseases must be
detected early. Hepatic (liver) involvement in some diseases can be of crucial importance. This
testing is performed on a patient's blood sample. Some tests are associated with functionality (e.g.,
albumin), some with cellular integrity (e.g., transaminase), and some with conditions linked to the
biliary tract (gamma-glutamyl transferase and alkaline phosphatase).
2.5.1
FUNCTIONS OF THE LIVER (LIPID METABOLISM)
The liver carries out the following major functions in lipid metabolism:
 It facilitates the digestion and absorption of lipids by the production contains cholesterol and
bile salts synthesized within the liver denovoor from uptake of lipoprotein cholesterol.
 The liver has active enzymes system for synthesizing and oxidizing fatty acids and for
synthesizing triacylglycerol’s phospholipids.
 It converts fatty acids to ketone bodies (ketogenic).
 It plays an integral part in the synthesis and metabolism of plasma lipoprotein (Murray et al.,
2003).
19
2.5.2
Uses of Liver Function Tests
the various uses of liver function tests include:
 Screening: They are non-invasive yet sensitive screening modality for liver dyfunction.
 Pattern of Disease: they are helpful to recognize the pattern of liver disease. Like being helpful
in differentiating between acute viral hepatitis and various cholestasis disorders and chronic
liver diseases. (CLD).
 Assess severity: they are helpful to assess the severity and predict the outcome of certain
disease like primary biliary cirrhosis.
 Follow up: they are helpful in the follow up of certain liver diseases and also helpful in
evaluating response to therapy like autoimmune hepatitis.
2.5.3
CLASSIFICATION OF LIVER FUNCTION TESTS
 Test of the livers capacity to transport organic aurous and to metabolize drugs (serum,
bilirubin, urine bilirubin and urine urobilirubin).
 Test that detect injury to hepatocytes (serum enzymes test) – amino transferases, alkaline
phosphatase, glut amyl trans-peptidase, 5-nucleatidase, leucine aminopeptidase.
 Test of the livers biosynthetic capacity-serum protein, albumin, prealbum, serum
ceruloplasmin, procollagen III peptide, a lantitrypsin, a fetoprotein, prothrombimtime (Thapa
et al., 2007).
2.5.4
LIMITATION OF LIVER FUNCTION TEST (LFT)
Lack sensitivity: The LFT may be normal in certain liver disease like cirrhosis, non-cirrhotic portal
fibrosis, congenitail hepatic fibrosis, etc.
20
Lack specificity: They lack specificity and are not specific for any particular disease. Serum
albumin may be decreased in chronic disease and also in nephrotic syndrome. Aminotransferases
may be raised in cardiac disease and hepatic disease. (McClatchey K et al., 2002).
Except for serum bile acids the LFT are not specific for liver disease and all the parameters may
be elevated for pathological processes outside the liver. (Anciaux ml et al., 1986).
2.6 ASPARTATE AMINOTRANSFERASE (AST)
The levels of aspartate aminotransferase (AST) are enzymes found mainly in the liver, but also
found in red blood cells, heart cells, muscles tissue and other organs, such as the pancreas and the
kidneys. AST formerly is called serum glutamine oxaloacetie transaminase (GOT). AST levels are
a valuable aid primarily in the diagnosis of liver disease. Although not specific for liver disease it
can be used in combination with other enzymes to monitor the cause of various liver disorder. The
normal concentrations in the blood are from 5 to 40 UI-1 for AST. However, when body tissue or
organ such as the liver or heart is diseased or damaged additional AST is released into the blood
stream, causing levels of the enzymes to rise. Therefore, the amount of AST in the blood is directly
related to the extent of the tissue damage. After severe damage, AST levels rise from 10 to 20
times and greater than normal. On the other hand, the ratio of AST to ALT (AST/ALT) sometimes
can help determine whether the liver or their organ has been damaged (Hafkenscheid et al., 1979).
The assay of AST Activity based on the following enzymes reaction. L-aspartate + 2-ketoglutarate
oxaloacetate +
GOT
L-glutamate. (Wang et al., 2002). Due to the clinical importance of
AST/GOT in monitoring patients with liver diseases, AST/GOT detection have been reseeded by
a number of scientists all over the world as well as International Federation of Clinical Chemistry
(IFCC) and the scanclinallian Committee on Enzymes (SCE) (Hanson et al., 1964).
21
2.7
LIPID PROFILE
Lipid profile or lipid panel is a panel of blood tests that serves as an initial broad medical screening
tool for abnormalities in lipids, such as cholesterol and triglycerides. The results of this test can
identify certain genetic disease and can determine approximate risks for cardio-vascular disease,
certain forms of pancreatitis, and other disease. Lipid panals are commonly ordered as part of a
physical exam, along with other panels such as the complete blood count (CBC) and basic
metabolic panel (BMP) (Murray et al., 2003). Liver plays an essential role in lipid metabolism,
several stages of lipid synthesis and transportation. It has been well documented that chronic liver
dyfunction might interfere with lipid metabolism in vivo and could changes plasma lipid and
lipoprotein patterns (Miller. 1990).
2.8 CHOLESTEROL
Cholesterol is a waxy substance that is present in the blood plasma and in all animal tissues.
Chemically, cholesterol is an organic compound belonging to the steroid family, its molecular
formula is C27H46O. Cholesterol is essential to life, it is a primary component of the membrane
that surrounds each cell and it is the starting material or an intermediate compound from which the
body synthesizes bile acids, steroid hormones and vitamin D. cholesterol circulates in the blood
stream and is synthesized by the liver and several other organs. Cholesterol is insoluble in the
blood. It must be attached to certain protein complexes called lipoproteins in order to be
transported through the blood stream (Abell et al., 1952).
2.8.1
Low Density Lipoprotein (LDL)
Lipoprotein molecules enable the transportation of lipids (fats), such as cholesterol phospholipids
and triglycerides within the water around the cells (extracellular fluid) including the blood stream.
Studies have shown that higher levels of type B LDL particles (as opposed to type A LDL particles)
22
are associated with health. Problems including cardiovascular disease. Although the nick name is
overly simplistic and thus misleading LDL cholesterol molecules are often informally called bad
cholesterol because they can transport their content of many fat molecules into artery walls attract
macrophages and thus drive atherosclerosis. In contrast, high density lipoprotein cholesterol
molecules are frequently referred to as good cholesterol or healthy cholesterol, because they
remove fat molecules from macrophages in the wall of arteries (Murray et al., 2003).
Niacin (B3), lowers LDL by selectivity inhibiting hepatie diacyglycerol acyltransferase 2, reducing
triglyceride synthesis and very low density lipoprotein secretion through a receptor. HM74
(Meyers et al., 2004) and HM74A or GPR109A (Soudijn et al., 2007). LDL particles appear
harmless until they are within the blood vessel walls and oxidized by free radicals, it is postulated
that ingesting anti-oxidants and minimizing free radical exposure may reduce LDL’s contribution
to atherosclerosis, though results are not conclusive (Teissedre et al., 1996).
2.9 UV SPECTROPHOTOMETER: The ultraviolet-visible spectrophotometer measures the
intensity of light passing through the water sample I.e. absorbance, in relation to its concentration,
working basically on Beer’s Lambert law and measures within the wavelength range of 200-800nm.
Ultraviolet-visible spectroscopy is considered an important tool in analytical chemistry. In fact,
this is one of the most commonly used techniques in clinical as well as chemical laboratories. This
tool is used for the quantitative analysis and identification of chemicals. However, its main use is
for the quantitative determination of different organic and inorganic compounds in solution.
23
Plate 3: Spectrophotometer
2.9.1
PRINCIPLE OF ULTRA VIOLET (UV) SPECTROPHOTOMETER
Basically, spectroscopy is related to the interaction of light with matter. As light is absorbed by
matter, the result is an increase in the energy content of the atoms or molecules. The absorption of
visible light or ultraviolet light by a chemical compound will produce a distinct spectrum.
When ultraviolet radiations are absorbed, this result to the excitation of the electrons from the
ground state towards a higher energy state. The theory revolving this concept states that the energy
from the absorbed ultraviolet radiation is actually equal to the energy difference between the higher
energy state and the ground state.
UV spectroscopy follows the Beer-Lambert Law. This law states that whenever a beam of
monochromatic light is passed through a solution with an absorbing substance, the decreasing rate
of the radiation intensity along with the thickness of the absorbing solution is actually proportional
to the concentration of the solution and the incident radiation.
24
This law is expressed through this equation:
A = log (10/1) = ECI
25
CHAPTER THREE
3.0 MATERIALS AND METHOD
3.1 MATERIALS
3.2 PLANT MATERIALS
The samples prosopis african seeds were collected from Enugu State, Nigeria. The plant materials
were identified authenticated by a botanist in the department of Botany, University of Nigeria
Nsukka.
3.3 ANIMALS STUDY
Twenty-Four (24) adult wister albino rats were used for this study. The animals were obtained
from the animal house of biological sciences, university of Nigeria Nsukka. They were housed in
metal steel cages and acclimatised in the laboratoey for seven days before the experiments. They
were given free access to water and fed with grower mash (Niger feeds, Nigeria) bought from the
local market.
3.4 EQUIPMENT/ INSTRUMENT
3.4.1
EQUIPMENTS
MANUFACTURER
 Centrifuge
Vickas ltd, England
 Colorimeter
El Scientific co.india
 Electron Microscope
Vickas ltd, England
 Oven
Gallenkamp, England
 Pasteur Pipette
Pysex, England
26
 Refrigerator
thermocool, England
 Spectropotometer
Jenway, Uk
 Water bath
Gallenkamp, England
 Cage
Neulex
 Weighing balance
Vickas ltd, England
3.4.2
Manufacturer
Chemicals and Reagents
 Phosphate buffer
AAT Bioquent, UK
 L- aspartate
BDH, England
 2,4-dinitrophenylhydrazine
G.louis
 Serum
 Phosphotungstic acid
Merek Darmstadt, Germany
 α- oxogluterate
 Sodium hydroxide
may and bakers, England
 Anticoagulate (EDTA)
Randox USA
 Magnesium Chloride
Suffolete England
27
3.5 METHODS
3.6 TRADITIONAL FERMENTATION OF THE Prosopis african SEEDS
The seeds of prosopis african were boiled for up to 6 hours and allowed to cool to room
temperature. The seed coats were removed by pressing finger-tips. This coats were later decanted
along with the washing water, leaving the clean seed cotyledons. The clean cotyledons were boiled
for another 1-2 hours, it helped the seeds to become soft with reduced bitterness for easy
fermentation. The cotyledon was later dried through the sieve and wrapped with paw-paw leaf.
The wrapped cotyledons were put in clean bowels covered with the same leafs for a period of four
days (for fermentation to take place) or during natural fermentation. The resultant product which
was brown in color, was Okpehe, a strong smelling mass of sticky cotyledons. The fermented
cotyledons were covered by a whitish mucilaginous film produced during fermentation (Ogunshe
et al, 2007). the fermented seeds were ground into a motar into a smooth paste. The okpehe was
made into balls of 3-5cm diameter arranged into trays and dried for 1-2days in the sun. The product
became black after sun drying.
3.7 EXPERIMENTAL DESIGN
Twenty-four (24) adult wister albino rats were used for this study the animals were maintained
under hygienic condition, with feed and water available adlibitum for seven (7) days before onset
of the experiment. After acclimatization, the animals were randomly divided into five groups of
four (4) rats each. The route of administration was via oral routine with the aid of oral intubation
tube. The groups and doses administered are summarized below.
Group 1: Received 5mg/kg normal saline
Group 2: Received 50mg/kg of fermented prosopis africana seeds aqueous extract.
28
Group 3: Received 100mg/kg of fermented prosopis africana seeds aqueous extract.
Group 4: Received 200mg/kg of fermented prosopis africana seeds aqueous extract.
Group 5: Received 12.5mg/kg of orlistat
3.8
SAMPLE COLLECTION
Blood samples were collected and analyzed on the twenty-one (21) day of the experiment. Blood
was collected into sample bottles through rectobulba plexus in the eye and put into non-heparinzed
sample bottles to obtained serum for the determination of some biochemical parameters.
3.9 BIOCHEMICAL PARAMETERS
3.9.1
Liver function test of rats treated with aqueous extract of fermented prosopis africana
seeds. Live function test Aspartate Amino Transferase (AST) was determined using the method of
(Reitman et al., 1957).
3.9.1
Determination of Aspartate Amino Transferase (AST)
Principle: AST is measured by monitoring the concentration of oxaloacetate hydrazone formed
with 2, 4-dinitrophenylhydrazine. The colour intensity is measured against the blank at 546nm.
Method: The blank and sample test tubes were set up in duplicates. A volume, 0.1ml of serum was
pipetted into the sample tubes and 0.5ml of reagent 1 was pipette into both sample and blank tubes.
The solutions were thoroughly mixed and incubated for exactly 30 minutes at 37 0C ml and pH 7.4.
0.5ml of Reagent 2 containing 2, 4-dinitrophenylhydrazine was added into all the test tubes
followed by 0.1ml of sample into the blank tubes. The tubes were mixed thoroughly and incubated
for exactly 20 minutes at 25 0 C and 5.0ml of sodium hydroxide solution was then added to each
tube and mixed. The absorbance was read against the blank after 5 minutes at 546nm. (Reitman, S,
et al., 1957).
29
3.10
lipid profile test (LDL) of rats treated with aqueous extract of fermented prosopis
africana seeds.
3.10.1 Determination of low density lipoprotein (LDL)
the method of (Kameswara et al., 1952) was used
Principle:
LDL-C can be determined as the difference between total cholesterol and the cholesterol content
of the supernatant after precipitation of the LDL fraction by polyvinyl sulphate (PVS) in the
presence of polyethyleneglycol monomethyl ether. (Bargmenyer. H, 1985).
Procedure:
The serum samples were kept at 2-80C. The precipitant solution (0.1ml) was added to 0.2ml of the
serum sample and mixed thoroughly and allowed to stand for 15 min. This was centrifuged at
2,000 x g for 15 min. The cholesterol concentration in the supernatant was determined. The
concentration of the serum total cholesterol as described by Kameswara et al. (1999) was used.
Calculation:
LDL-C (mmol/L) = Total Cholesterol (mmol/L) – 1.5 x Supernatant Cholesterol (mmol/L).
3.11 Statistical Analysis:
The results were expressed as mean+ standard deviation (+ SD). The data obtained were subjected
to one-way analysis of variance (ANOVA). The statistical analysis was done using the statistical
package for social sciences (SPSS), version 23. significant differences were observed at ≤ 0.05
30
CHAPTER FOUR
4.0 RESULTS
Table 4.1: Effect of the aqueous extract of fermented seed of Prosopis africana on LDL level and
AST activity of Wistar albino rats.
GROUPS
LDL
AST
Group 1 (Normal Saline)
3.023± 0.67a
71.500 ± 21.83c
Group 2 ( 50mg/kg extract)
4.535±0.89b
Group 3 ( 100mg/kg
4.745±1.12b
extract)
Group 4 ( 200mg/kg extract)
4.753±0.46b
Group 5 (12.5mg/kg Orlistat)
4.955±0.67b
53.000 ± 3.74b,c
54.500 ± 6.40b,c
41.750 ± 14.22a,b
30.500 ± 1.73a
Means of different alphabets as superscript within each column are significantly (P<0.05) different
and vice versa.
31
Figure 5
Figure 1; Effect of the aqueous extract of fermented seed of Prosopis africana on low
density Lipoprotein level in rats.
Keys:
Group 1 = Normal Saline Control
Group 2 = 50mg/kg of extract
Group 3 = 100mg/kg of extract
Group 4 = 200mg/kg of extract
Group 5 = 12.5mg/kg of Orlistat
32
Figure 2; Effect of the aqueous extract of fermented seed of Prosopis africana on Aspartate
aminotransferase activity in rats.
Keys:
Group 1 = Normal Saline Control
Group 2 = 50mg/kg of extract
Group 3 = 100mg/kg of extract
Group 4 = 200mg/kg of extract
Group 5 = 12.5mg/kg of Orlistat
CHAPTER FIVE
5.0 DISCUSSION
The liver plays a central role in transforming and clearing chemicals and is susceptible to the toxicity
from these agents (friedman et al., 2003). The liver is the vital organ of paramount importance
involved in the maintenance of metabolic function and detoxification from the exogenous and
endogenous challenges such as xenobiotics, drugs, viral infection and chronic alcoholism. If during
all such exposure to the above mentioned challenges the natural protective mechanisms of the liver
are overpowered or compromised, the result is hepatic injury.
Fermented foods play an important social-economic role in developing countries as well as making
a major contribution to the protein requirements of natural population (Achi, 2005).
In this assessment of liver damage by the assay of enzyme activity such as AST activity are largely
used (Dobbs et al., 2003). membrane damage releases the enzyme into circulation; therefore, it can
be measured in serum. AST is predominantly found in mitochondria of hepatocytes. Increase in the
activity of above enzyme would indicate liver toxicity.
Fig 1 shows a significant (p< 0.05) increase in low density lipoprotein cholesterol levels of animals
in group 2 fed with 50mg/kg extract (4.535±0.89), group 3 fed with 100mg/kg extract (4.745± 1.12)
and group 4 fed with (4.753±0.46) when compared with the animals in group 1 which are fed with
normal saline (3.023±0.67). There was a significant (p< 0.05) difference in the low density
lipoprotein cholesterol level of animals in group 5 administered 12.5mg/kg orlistat when compared
to the low density lipoprotein levels of the animals in the control group 1.
Fig 2 shows a significant (p< 0.05) decrease in the aspartate aminotransferase (AST) activity of the
animals in group 2 fed with 50mg/kg extract (53.000±3.74), animals in group 3 fed with 100mg/kg
extract (54.500±6.40), in group 4 animals fed with 200mg/kg extract (41.750±14.22) when
compared with the animals in the control group (71.500±21.83). The AST activity of group 5 rats
administered with 12.5mg/kg of orlistat decreased significant (p< 0.05) when compared to the
animals in the control group.
5.1 CONCLUSION
From the result, it shows that incorporation of the fermented seed of prosopis africana in the human
diet may protect against liver damage. The extract could also say to have potential therapeutie value
in the treatment of some liver disorders. However, the increased in the level of low density
lipoprotein of the rats may be as a result of the oil base of the seeds. Therefore, moderate
incorporation of the fermented seeds of prosopis africana in diet is adviced.
REFERENCE
Abbott R. (2014). “Documenting traditional medical knowledge”. Geneva, Switzerland: WIPO;
Abell, L.L., Levy, B.B., Brodie, B.B. and Kendall, F.E. (1952). A simplified method for the estimation
of total cholesterol in serum and demonstration of its specificity. Journal of Biological
Chemistry,
195: 357-366.
Achi, O.K. (2005). The potential for upgrading traditional fermented foods through biotechnology.
African Journal of Biotechnology, 4(5): 375-380.
Agboola, D.D. (2004). Prosopis africana (Mimosaceae): stems, roots and seeds in the economy of
the savannah areas of Nigeria. Economic Botany. 34–42.
Agboola, D. A. (1995), “Studies on Seed dormancy of selected economic tropical forest tree species”.
Nigerian Journal of Botany 4: 115-126.
Akinnagbe, A., and Oni, O., (2007). Quantitative variations in the growth of progeny seedlings of
Prosopis africana (Guill., Perrott. and Rich.) plus trees in Nigeria.African journal of
Biotechnology. 6: 359–363.
Alabi D.A. (1993). “Parkiabiglobosa: An endangered species. In proceeding of the seminar on lost
crops in Nigeria”. University of Agriculture Abeokuta, Nigeria. Nigeria Journal of Botany.
(265-285).
Amusa T. O., Jimoh S.O., Aridanzi P., and Haruna M.A. (2010). “Ethnobotany and Conservation of
plant resources of kaniji lake national park, Nigeria”. Ethnobotany Research and Application
(18-194).
Arbonnier, M., (2002). Arbres Arbustes et Lianes des Zones Se`ches d’Afrique de l’Ouest. Centre
de Coope´ ration Internationale en Recherche Agronomique pour le De´veloppement
(CIRAD)/Muse´um National d’Histoire Naturelle (MNHN), Montpellier/Paris.
Aremu, M.O., Olonisakin, A., Atolaye, B.O., and Ogbu, C.F. (2006). Some nutritional and functional
studies of Prosopis africana. Electron. Journal of Environmental Agricultural Food
Chemistry. 5: 1640–1648.
Ayanwuyi L. O., Yara A. H., and Abodunde O. M. (2010). “Analgesie and Anti-Inflammatory
effects of methanol stem bark extract of Prosopis Africana”. Pharmaceutical Biological 48:
296-299.
Barminas, J.T., Maina, H.M., and Ali, J., (1998). Nutrient content of Prosopis Africana seeds. Plant
Foods Human Nutrition. 52: 325–328.
Bergmenyer, H. U., (1985). Methods of enzymatic analysis, 3rd edition volume vill. 154-160.
Bessega, C., Ferreyra, L., Julio, N., Montoya, S., Saidman, B., and Vilardi, J.C. (2000). Mating
system parameters in species of genus Prosopis (Leguminosae). Hereditas 132: 19–27.
Campos M.G., Markham K.R. and Proenc da Cunha A. (1997). “Quality assessment of bee-pollens
using flavonoid, phenolic profiles”. Polyph Comm 96:53- 4.
Demonty, I., Ras, R.T., van der Knaap, H.C., Duchateau, G.S., Meijer, L., Zock, P.L., Geleijnse, J.M.
and Trautwein, E.A. (2009). "Continuous dose-response relationship of the LDL-cholesterollowering effect of phytosterol intake". The Journal of Nutrition, 139(2): 271–284.
Dhillon, R.S., Hooda, M.S., Chopra, D., and Arya, S. (2003). Studies on floral biology and breeding
behaviour of Prosopis cineraria (L.) Druce (Khejri). Forrage Res. 29: 71–75.
Diagne O. (1992). “Current development on Prosopis species in Senegal: In Prosopis Species:
Aspects of their values, research and development”. Centre for overseas research and
development, university of Durham, Uk, (47-59).
Dobbs, N.A., Twelves, C.J., Gregory, W., Cruickshanka, C., Richards, M.A. and Rubens, R.D. (2003).
Epirubicin in patients with liver dysfunction. Development and evaluation of a novel dose
modification scheme. European Journal of Cancer, 39: 580-586.
Fasicli I. O., Samani T., Kadiri M., and Agboola D. A. (2000). “Dormancy types and water uptake
in seeds of parkiabiglobosa”. Journal of Natural and Applied Science, 1: (14-20).
Friedman, S.E., Grendell, J.H. and McQuaid, K.R. (2003). Current Diagnosis and Treatment in
Gastroenterology. Lang Medical Books/McGraw-Hill, New York. pp. 664–679.
Ge´ rardin, P., Neya, B., Dumarc¸ay, S., Pe´ trisanns, M., Serraj, M., and Huber, F., (2004).
Contribution of gums to natural durability of Prosopis africana heartwood. Holzforschung
58: 39–44.
Gurib-Fakim A. (2006). “Medicinal plants: tradition of yesterday and drugs of tomorrow”. Mol
Aspects Med 27:1- 93.
Hamrick, J.L., Godt, M.J. and Sherman Broyles, S.L. (1992). Factors influencing levels of genetic
diversity in woody plant species. New for. 6: 95–124.
ICRAF-IFAD, (2006). Renforceme nt des strate´ gies de subsistance a` travers une utilization et une
gestion ame´ liore´es des parcs agroforestiers au Sahel. In: Rapport Annuel du Projet ICRAF
TAG 799, World Agroforestry Centre (ICRAF)/International Fund for Agricultural
Development (IFAD), Bamako/Rome.
Johnston DE. (1999). "Special considerations in interpreting liver function tests". Am Fam
Physician. 59 (8): 2223–30. PMID 10221307.
Kameswara, R., Kesavulu, B., Giri, M.M. and Appa Rao, C.H. (1999). Antidiabetic and hypolipidemic
effects of Momordica cymbalaria Hook, fruit powder in alloxan diabetic rats. Journal of
Ethnopharmacology, 67: 103-109
Keay R. W. J., Onochie C. F. A., and Stan Field D. P. I., (1964), Nigeria trees. Federal Department
of Forest Research. Ibadan, Nigeria 11: 16- 187.
Khejra C. D., Khejra, (2001) Vanoshdi Chitravali (Jaributti), Journal of Food and Drug Analysis 1:
269- 70.
Lee, Mary. (2009). Basic Skills in Interpreting Laboratory Data. MD: American Society of HealthSystem Pharmacists. American Journal of Pharmaceutical Education 74(8): 151
Mengel, Mark B.; Schwiebert, L. Peter (2005). Family medicine: ambulatory care & prevention. 4 th
edition in New York by Lange Medical Book/McGraw-Hill. pp: 268–978.
Meyers, C.D., Kamanna, V.S. and Kashyap, M.L. (2004). Niacin therapy in atherosclerosis. Current
Opinion in Lipidology, 15(6): 659–665.
Miller, J.P. (1990). Dyslipoproteinaemia of liver disease. Baillieres Clinical Endocrinology Metabolism,
4(4):807-832
Murray, R.K., Granner, D.K., Mayes, P.A. and Rodwell, V.W. (2003). Harper’s Illustrated
Biochemistry. 26th Edn. McGraw-Hill Companies, Inc. New York. pp. 215-228.
Mwangi E., Swallow B. (2008). “Prosopis Juloflora invesion and rutal livelihoods in the lake
Baringo area of area of kenya. Conserv Soc 2: 130-140.
Okafor J. C., Okolo, H.C., Ejiofor,M.A.N.,Van der, L.J.G.(1994). Strategies for Enhancement of
utilization potential of Edible Woody forest species of South East, Nigerian. Proceedings of
the AETFAT Congress on the Biodiversity of African Plants., Kluwer- Nijhoff Public,
Wageningen, Paris.pp: 684- 695.
Ola-Adams B. A., and H. D. (1993). “Conservation and untilization of endangered edible wild plants
in Nigeria”. In proceeding of the seminar on lost crops of Nigeria, University of Agriculture,
Abeokuta, Nigeria. (33-43).
Otvos, J. (1999). "Measurement of triglyceride-rich lipoproteins by nuclear magnetic resonance
spectroscopy". Clinical Cardiology, 22 (6): II21–1127.
Pasieczrik NM., Felker P., Harries P. J. C., Harsh L. N., Cruzce., Tewarijc and Maldonado L.J.,
(2001). “The Prosopis Julifora-Prosopis pallida complex: A mono-graph. Henry Doubled
Research Association, coventry, UK. pp.172
Pasiecznik N.M., (1999). “Prosopis: pest or providence, weed Tree? European Tropical for
Research Network News 28:12- 4.
Pimental MDL. (1960) P. juliflora (SW)(DC), In: 1 “Simposio Brasilerio
1:330-5.
sobre Algarobera”;
Plaa, G.L. and Hewitt, H.R. (1982). Quantitative evaluation of indices of hepatotoxicity. Fundamental
and Applied Toxicology of the Liver. Raven Press, New York. 6:103-120.
Ram, V.J. and Goel, A. (1999). Past and present scenario of hepatoprotectants. Current Medicinal
Chemistry, 6(3):217-254.
Reitman, S., Frankel, S. (1957). A colorimetric method for the determination of serum glutamic
oxaloacetic and glutamic pyruvic transaminases. American Journal of Clinical Pathology,
28: 56-63
Spore C. T. A., (1993). “The Prosopis Species”. Bulletin of the Technical Centre for Agricultural
and Rural Coorperation (Wagenigen, the Netherlands). Journal of Nigeria Botany 48: 1- 16.
Stefanovic O.D., Tesic J.D. and Comic L. R. (2015). “Melilotus albus and Dorycnium herbaceum
extracts as source of phenolic compounds and their antimicrobial, antibiofilm, and
antioxidant potentials”. Journal of Food Drug Analysis 23:417-24.
Tapia A., Egly Feresin G., Bustos D., Astudillo L., Theoduloz C. and Schmeda-Hirschmann G.
(2000). “Biologically active alkaloids and a free radical scavenger from Prosopis species”.
Journal of Ethnopharmacol 71: 241-6.
Thapa, B.R. and Anuj, W. (2007). Liver function test and their interpretation. Indian Journal of
Peadiatrics, 74(7):663-671.
Vilela A., Bolkovic M.L., Carmanchahi P., Cony M. and de Lamo D. (2009). “Past, present and
potential uses of native flora and wildlife of the Monte Desert Wassnerg”. Journal of Arid
Environment 73: 238-43.
Wang, C.C., Cheng, P.Y., Peng, Y.J., Wu, E.S., Wei, H.P. and Yen, M.H. (2008). Naltrexone protects
against lipopolysaccharide/D-galactosamine-induced hepatitis in mice. Journal of
Pharmacological Science, 108(3):239-247.
Wierman R. and Vieth K. (1983). “Outer pollen wall, an important accumulator site for flavonoids”.
Protoplasma 118:230-3.