EVALUATION OF THE TOXICITY OF THE

METHANOLIC EXTRACT OF MORINGA

OLEIFERA LEAVES ON MICE

BY

ORIMALADE, TINUADE TITILADE

MATRICULATION NUMBER: 110802095

DECEMBER, 2014

ii

iii

DEDICATION
This project is dedicated to God, the creator of Heaven and Earth and the giver of knowledge who
has been my source of strength all through the years, the Father of our Lord Jesus Christ, to whom,
through whom and for whom are all things now and ever more.

iv

ACKNOWLEDGEMENTS
With a heart full of appreciation, I give thanks to God, the monarch of the universe for sparing my
life and for sound health that He bestowed on me throughout my four years in the Department of
Cell Biology and Genetics, University of Lagos, Akoka.
Words cannot express how grateful I am to my family for their encouragement and unrelenting
effort in supporting my education. For their prayers and priceless aid throughout the period of this
work, I say thank you and may the good God bless you.
I appreciate PROF. JOY OKPUZOR, my project supervisor, whose patience, unparalleled aid and
bits of advice and instructions aided the success and completion of my project.
Special thanks to my friends and my colleagues whose assistance is second to none in the
completion of this work. May God bless you all enormously.
To everyone who assisted in any way during the course of this project, thank you and God bless

TABLE OF CONTENTS
Title page

ii

Certification.

iii

Dedication.

iv

Acknowledgements.

Table of Contents.

vi

List of Tables

ix

List of Figures

Abstract.

xi

CHAPTER ONE
1.0 Introduction and literature review.
1.1 Introduction

1.2 botanical classification of Moringa oleifera

1.2.1 Morphology And Botanical Description Of Moringa oleifera

1.2.2 Origin And Geographic Distribution

1.2.3 Scientifically Proven Uses

1.2.4 Phytochemistry And Crude Nutrient Analysis

1.2.5 Uses Of Moringa oleifera

1.3 Solvent Extraction

1.4 Toxicity

1.4.1 Acute Toxicity Test

1.4.2 Sub Acute Toxicity Test

10

1.5 Biochemical Parameters

10

1.5.1 Cholesterol

10

1.5.2 Triglyceride

10

1.5.3 Aminotransferases

11

1.5.4 Alkaline Phosphatase

11
vi

1.5.5 Albumin

12

1.5.6 Urea

12

1.6 Objectives

12

CHAPTER TWO
Materials And Methods.

13

2.1 Materials

13

2.2 Methods

13

2.2.1 Plant Sample Collection And Identification

13

2.2.2 Extract Preparation

13

2.2.3 Phytochemical Screening

14

2.2.4 Experimental Animals

14

2.2.5 Method Of Dilution Of Extract

14

2.2.6 Experimental Design

14

2.2.7 Method Of Administration

15

2.3 Weekly Body Weight

16

2.4 Acute Toxicity Test

16

2.5 Sub Acute Toxicity Study

16

2.6 Biochemical Parameters Assay

16

2.7 Statistical Analysis

17

CHAPTER THREE
Result
3.0 Percentage Yield of Methanol Extract

18

3.1 Qualitative Phytochemical Analysis

18

3.2 Acute Toxicity

20

3.3 Sub Acute Toxicity

20
vii

3.4 Mice Weight

20

3.5 Biochemical Parameters

21

CHAPTER FOUR
Discussion

25

Conclusion

26

REFERENCES

27

APPENDICES

32

viii

LIST OF TABLES
Pages

Table 1 nutritive value of the leaves of Moringa oleifera per 100 grams of edible portion

Table 2: Dosage administered to mice in each group per body weight

16

Table 3: The percentage yield of methanol extract of Moringa oleifera

19

Table 4: The results of the phytochemical composition of the leaves of Moringa oleifera

20

Table 5: Effects of graded doses of M. oleifera on plasma biochemical parameters of mice

21

ix

LIST OF FIGURES
Pages

Figure 1: various useful part of Moringa oleifera

Figure 2: the effects of graded doses of M. oleifera on plasma biochemical parameters

21

Figure 3: Effect of Moringa oleifera extract on liver enzymes level (ALP, ALT, AST)

22

Figure 4: Effect of Moringa oleifera extract on Lipid profile level

23

Figure 5: Effect of Moringa oleifera extract on Lipoprotein profile level

24

ABSTRACT
Moringa oleifera is grown and used in many countries around the world as a multi -purpose tree
with medicinal, nutritional, therapeutic potential and socio-economic values. Different parts of M.
oleifera have been shown to exhibit wide pharmacological activities. Testing the toxicity and
safety of plant extracts is a key step before further efficacy tests could be performed. This study
is set out to establish the acute and sub acute toxicity the methanol extract of M. oleifera will
have on some biochemical parameters . This laboratory based

study

had Moringa oleifera

leaves harvested , air dried and pulverized. Extraction was done using methanol as solvent.
M.oleifera leaves contain important phytochemicals like carbohydrates, alkaloids, glycosides,
saponins, tannins, flavonoids, resins, protein and acidic components. The control group received
no extract but 400, 800, 1200, 2000 (mgkg-1) were given in a single daily oral dose to five (5)
Albino SCID laboratory BALB/C per cage, (cage I, II , III, IV, V) . On the last day blood was
collected for biochemical analysis. The analysis showed that there was no significant changes in
the Total Cholesterol (Mmol/L), Triglyceride(Mmol/L), Alanine Amino Transferase(mol/L),
Aspartate Amino Transferase(mol/L), Urea(Mmol/L), Alkaline Phosphate(mol/L), Albumin
(Mmol/L) levels when compared with the control. In conclusion, methanol leaf extract of M.
oleifera given orally to mice in a single dose for 28 days had no toxic effects on some biochemical
parameters and could be useful for therapeutic treatment of many diseases.

xi

CHAPTER ONE
INTRODUCTION AND LITERATURE REVIEW
1.1 INTRODUCTION
Medicinal plants have over the years been the lifeline of many indigenes which do have
access to, or cannot afford pharmaceuticals. In recent years the shift from synthetic drugs to
natural products is considered a panacea to wellness that is cutting across all socio-economic
barriers (Anwar et al.,2007). The inhibition to the widespread use of medicinal plants is
gradually being eroded by the inability of orthodox drugs to cure long standing and common
ailments such as malaria, diabetes and hypertension .The coupling agent of acceleration to the
use of medicinal plants, herbal plants, natural products etc., is the emergence of nutraceutical
plants (Chinmoy, 2007), these are natural plant products that link nutritional plants and
medicinal plants. One 21st century plant that has taken center-stage and promises to be the
catalyst towards the achievement of the millennium development goals of reducing poverty,
disease and malnutrition is Moringa oleifera (Eshak and Osman, 2013).
Moringa oleifera (Family: Moringaceae) is a multipurpose tree with significant medicinal
and nutritional, therapeutic potential and socio-economic value. M. oleifera is an angiosperm
plant, the tree itself is rather slender, with drooping branches that grow to approximately 10m
in height, Its native of the Indian subcontinent, It is now cultivated in all tropical and subtropical regions of the world (Ramachandran, 1980).
The enthusiasm for the health benefits of M. oleifera is variably labeled as Miracle Tree, Tree
of Life, the Best Friend, Gods Gift to Man, Savior of the Poor (Kasolo et al., 2010). The
English common names include Moringa, drumstick tree; from the appearance of the long,
slender, triangular seed pods, Horseradish tree; from the taste of the roots which resembles
horseradish, miracle tree: from its capacity to withstand prolonged periods of drought and
Ben oil tree; from the oil derived from the seeds, in Hausa it is known as zogallagandi, in

Igbo it is called okweoyibo, and the common name in Yoruba is ewe-igbale. (Palwal et
al.,2011).
M. oleifera is an edible plant, it may be used as forage for livestock, a wide variety of
nutritional and medicinal virtues have been attributed to its roots, bark, leaves, flowers and
fruits (kurmar et al., 2010). Different parts of M. Oleifera (leaves, pods, seeds, bark and
flowers) have been shown to exhibit wide pharmacological activities and used in Ayurvedic
medicine (Dangi et al., 2002). A recurring explanation for the therapeutic actions of M.
oleifera medication is the relatively high antioxidant activity of its leaves (verma et al.,
2009).
As the search to discover new therapeutic agents and dietary supplements derived from plants
offers many advantages, determining the toxicity and safety of plant extracts is crucial to
assure the safety and is a key step before further efficacy tests could be performed,
Interestingly a toxic substance might elicit pharmacological effects at a lower non-toxic dose.
Hence toxicity tests in animals play key role in determining the safety of medicinal plants
that are found to contain bioactive compounds (Reddy et al.,2013).
1.2 BOTANICAL CLASSIFICATION OF MORINGA OLEIFERA
Kingdom:

Plantae

Division:

Magnoliophyta

Class:

Magnoliopsida

Order:

Violes

Family:

Moringaceae

Genus:

Moringa

Species:

Oleifera

(Mishra et al., 2011).

Moringa oleifera (synonym: Moringapterygosperma) is the most widely cultivated species of
the genus Moringa, which is the only genus in the family Moringaceae, Moringa; derived
2

from the Sinhalese name morunga is the sole genus in the flowering plant family
moringaceae with 12 deciduous tree species In addition to M. Oleifera, which is a diploid
species with 28 chromosomes, and the most common species, several other species of
Moringa have proven to be useful sources of food, fiber, medicinals, and other products.
These include M. Concanensis, M. Drouhardii, M. Longituba, M. Ovalifolia, M. Peregrina,
and M. Stenopetala (Roloff et al., 2009).

1.2.1

MORPHOLOGY

AND

BOTANICAL

DESCRIPTION

OF

MORINGA

OLEIFERA
Moringa oleifera is a small, fast-growing evergreen or deciduous tree that usually grows as
high As 9 m, with a soft and white wood and corky and gummy bark, Roots have the taste of
horseradish (Mishra et al., 2011), Leaves are longitudinally cracked leaves, 30-75 cm long
main axis and its branch jointed, glandular at joints, leaflets are glabrous and entire, the
leaflets are finely hairy, green and almost hairless on the upper surface, paler and hairless
beneath, with red-tinged mid-veins, with entire (not toothed) margins, and are rounded or
blunt-pointed at the apex and short-pointed at the base, the twigs are finely hairy and green
(Roloff et al., 2009). Flowers are produced throughout the year, in loose axillary panicles up
to 15 cm long; individual flower stalks up to 12 mm long and very slender; 5 pale green
sepals 12 mm long, finely hairy, 5 white petals, unequal, a little longer than the sepals; 5
stamens with anthers, 5 without style slender, flowers have very sweet smelling (Palwal et
al.,2011).

Plate 1: showing various useful part of Moringa oleifera. (a) tree, (b) root, (c) leaves (d)
flowers (e) pod(fruit), (f) seeds.
Source: (Paliwal et al.,2011).

1.2.2 ORIGIN AND GEOGRAPHIC DISTRIBUTION

Drumstick tree is indigenous to the Himalayan foothills of South Asia from northeastern
Pakistan (33 N, 73 E) to northern West Bengal State in India and north eastern Bangladesh
where it is commonly found from sea level to 1,400 m on recent alluvial land or near
riverbeds and streams (Ramachandran, 1980). It grows at elevations from sea level to 1400
m. It is cultivated and has become naturalized in other parts of Pakistan, India, and Nepal, as
well as in Afghanistan, Bangladesh, Sri Lanka, Southeast Asia, West Asia, the Arabian
peninsula, East and West Africa, throughout the West Indies and southern Florida, in Central
and South America from Mexico to Peru, as well as in Brazil and Paraguay (Roloff et
al.,2009).
4

1.2.3 SCIENTIFICALLY PROVEN USES

The leaves have been shown to exhibit anti hypertensive (Dangi et al., 2002),
hypocholesterolaemic (Ghasi et al., 2000), antiulcer (Pal et al., 2006) and wound healing
properties (Rathi et al., 2006). Leaf, fruit and seed extracts of M. Oleifera protect against
oxidative DNA damage by scavenging free radicals (Bharali et al., 2003). The roots are
mildly diuretic, contain anti fertility activity (Shukla et al., 1988) and are used as a stimulant
in paralytic, epilepsy and hysteria conditions. The bark is regarded as an antiscorbic,
antifungal antitubercular (Bhatnagar et al., 1961) and antiurolithiatic activity (Jameel et al.,
2010). Bark exudes reddish gum used in the treatment of diarrhoea and known to have
filaricidal activity. Bark has been shown to decrease the cardiotoxicity and lipid peroxidation
by stimulating endogenous antioxidant enzymes (Mahendra et al., 2010).
Renitta et al. (2009), reported antimicrobial activity from the ethanolic extract of leaves,
Seeds and flowers of

Moringa oleifera against microorganisms like

Escherichia coli,

Klebsiella, Pneumoniae, Enterobacterspp, Proteus mirabilis, Pseudomonas aeroginosa,

Salmonella typhi , Staphylococcus aureus, Streptococcus and Candida albicans.
S.G. Mahajan et al.(2007), reported anti-arthritic activity of ethanolic extract of seeds of
Moringa oleifera lam. in adjuvant-induced arthritis in adult female wistar rats.
M. Bajpai et al. (2005), reported antioxidant activity from the leaves of Moringa oleifera, the
Antioxidant property was found due to presence of kaempferol.
Costa-Lotufo et al. (2005), reported anticancer activity of Moringa oleifera. The extracts
were tested for cytotoxicity by using the brine shrimp lethality assay, sea urchin eggs assay,
hemolysis assay and MTT assay using tumor cell lines.
Ganguly and Guha (2008), evaluated ethanolic extract of Moringa oleifera leaves in

alteration of brain monoamines (norepinephrine, dopamine and serotonin) & EEG wave
pattern in Alzheimer's disease in rats. Treatment with Moringa oleifera leaf extract restores
the monoamine levels of brain regions to near control levels.

1.2.4 PHYTOCHEMISTRY AND CRUDE NUTRIENT ANALYSIS

Scientific analysis has shown and confirmed that M. Oleifera leaves are indeed a powerhouse
of essential macro- and micro-nutrients, the leaves are believed to contain approximately 46
types of antioxidants, 90 nutrients, 18 amino acids (among which 8 are the essential ones), It
is on record that, a gram of Moringa oleifera leaves contain more vitamin C than oranges,
more vitamin A than carrots, more calcium than milk, more potassium than banana, more
iron than spinach (Richardson, 2009).

Table 1: The nutritive value of the leaves of Moringa oleifera per 100 grams of edible
portion

Carotene (Vit. A)

18.9 mg

Thiamin (B1)

2.64 mg

Riboflavin (B2)

20.5 mg

Niacin (B3)

8.2 mg

Vitamin C

17.3 mg

Calcium

2,003 mg

Calories

205 cal

Carbohydrates

38.2 g

Copper

0.57 mg

Fat

2.3 g

Fiber

19.2 g

Iron

28.2 mg

Magnesium

368 mg

Phosphorus

204 mg

Potassium

1,324 mg

Protein

27.1g

Zinc

3.29 mg

Source: Fuglie,1999.
Phytochemical studies on Moringa oleifera by Ndong et al. (2007), revealed major
polyphenols such as quercetin glucosides, rutin, kaempferol glycosides and chlorogenic acids
in Moringa oleifera powder by HPLC analysis. Singh et al. (2009). reported presence of
gallic acid, chlorogenic acid, ellagic acid, ferulic acid, kaempferol, quercetin and vanillin
from the aqueous extracts of leaves, fruits and seeds of Moringa oleifera.

Verma et al. (2009) reported presence of phenolic acids like gallic acid, chlorogenic acid,
ellagic acid, ferulic acid and flavonoids like kaempferol, quercetin and rutin from the leaves
of Moringa oleifera.
Sashidhara et al. (2009), from the roots of Moringa oleifera isolated and characterized
Aurantiamide acetate 4 and 1, 3-dibenzyl urea.
1.2.5 USES OF MORINGA OLEIFERA
The wood of M. Oleifera is little used outside of its native range except as a fuel wood and
occasionally for light construction (Palwal et al., 2011 ). The corky bark yields a coarse fiber,
which is utilized in making mats, paper, and cordage. The stem exudes a mucilaginous gum
that is used in leather tanning and calico printing (Ramachandran et al., 1980). In many parts
of its range, the leaves and twigs are used as fodder for cattle, sheep, goats, and camels .The
flowers are a good source of pollen for honey bees, the tree is mainly valued for its edible,
tender pods, which have a taste very similar to asparagus, these are eaten as a nutritious
vegetable, either cooked or pickled, the seeds contain 19 to 47 percent oil, known
commercially as ben oil; it is similar to olive oil and is used for human consumption, and in
cosmetics and soaps (Rolloff et al., 2009).
The dried, powdered, seeds have been used both in crude form and following extraction of
their active principle in petroleum ether through a hot percolation process, as an effective
and low-cost coagulant for removing turbidity and reducing bacterial and viral contamination
from drinking water in rural communities (Mandloi et al.,2004).
1.3 SOLVENT EXTRACTION
Considerable effort has been made by researchers to find efficient extraction methods in
order to get high efficiency and efficacy, efficiency refers to the yield of extraction, where as
efficacy refers to the potency (magnitude of bioactivity / the capacity to produce an effect) of
the extract, for isolation of biological components, extraction from plant is one of the more
sustainable approaches (Jadhav et al., 2009). It should be noted that choice of appropriate
8

solvent is of essential importance along with application of a compatible extraction method,

solvent extraction is the most popular method of extraction (Gupta et al., 2012). For the
extraction of therapeutically desired active constituents various solvents such as water,
ethanol, chloroform, ethyl acetate, methanol, etc. are commonly used, sometimes mixtures of
solvents are also used to get better extraction efficiency, Methanol proved most suitable
solvent for extraction of flavonoids (Ramanuj et al., 2012).
In particular, methanol has been generally found to be more efficient in extraction of lower
molecular weight polyphenols, In preparing anthocyanin-rich phenolic extracts from plant
materials, an acidified organic solvent, most commonly methanol is used, methanol is more
commonly used than ethanol to elute non-tannin compounds(Dai and Mumper, 2010).

1.4 TOXICITY
Toxicity referred to as the degree to which a substance can damage an organism and toxicity
is measured by varying methods viz. Changes in cell morphology, cell viability, metabolic
activity, and oxidative stress. (Asharani et al.,2009).
Toxicity testing is performed to assess the safety or hazards presented by substances such as
industrial chemicals, consumer products, and pharmaceuticals, many of the current toxicity
test methods include the use of laboratory animals (e.g., mice, rats, rabbits) (Bhardwaj et al.,
2012).

1.4.1 ACUTE TOXICITY TEST

Acute toxicity refers to those adverse effects occurring following oral or dermal
administration of a single dose of a substance, or multiple doses given within 24 hours, acute
toxicity tests are generally the first tests conducted, they provide data on the relative toxicity
likely to arise from a single or brief exposure (Bhardwaj et al., 2012).

Acute toxicity is involved in estimation of lethal dose 50 (LD50) the dose which has proved
to be lethal (causing death) to 50% of the tested group of animals, determination of acute oral
toxicity is usually an initial screening step in the assessment and evaluation of the toxic
characteristics of all compounds. (Akhila et al., 2007).

1.4.2 SUB ACUTE TOXICITY TEST

Sub acute toxicity tests are employed to determine what side effects will arise from
Repeated administration of a drug, detailed clinical observations and pathology examinations
are conducted (Bhardwaj et.al, 2012).

1.5 BIOCHEMICAL PARAMETERS

1.5.1 CHOLESTEROL
Cholesterol is a soft, fat-like substance found in the bloodstream and in all your bodys cells
Cholesterol is the precursor of cholesterol ester, bile acids and steroid hormone, Cholesterol
synthesis is primarily dependent on hepatocyte metabolism but may occur in any tissue,. the
dietary cholesterol ester is utilized almost completely in the liver, and losses are in the form
of bile acids and free cholesterol and its derivatives in bile (Howard et al., 2009).

1.5.2 TRIGLYCERIDE
Triglycerides are the most common type of fat in your body, they come from food, and your
body also makes them, high levels of blood triglycerides are often found in people who have
high cholesterol levels, heart problems, are overweight or have diabetes (Howard et al.,
2009).

10

1.5.3 AMINOTRANSFERASES
Aspartate amino transferase (AST, also sometimes termed SGOT) and Alanine amino
transferase (ALT, also sometimes termed SGPT) are normally intracellular enzymes with
mitochondrial and cytoplasmic forms, their names reflect their role in catalyzing chemical
reactions in which amino groups of Alanine and Aspartic acid are transferred to the alphaketo group of ketoglutaric acid, particularly during gluconeogenesis, ALT and AST are
widely distributed in cells throughout the body and are found in liver, heart, skeletal muscle,
kidney, brain, and pancreas, Alt is exclusively cytoplasmic and is found primarily in the liver
and kidney, with only minute amounts in heart and skeletal muscle (Krier and Aijaz 2009).
In adults, AST and ALT activities are significantly higher in males than in females, AST
activity is slightly higher than that of ALT, with the pattern reversing by age 15 in males but
persisting till age 20 in females, In adults, AST activity tends to be lower than that of ALT
until approximately age 60, when they become roughly equal, Liver disease is the most
important cause of increased ALT activity and a common cause of increased AST activity
(Dufour, 2000).

1.5.4 ALKALINE PHOSPHATASE

AP is involved in phosphate ester hydrolysis, although its exact catalytic function is
unknown, Liver and bone AP are the most abundant isoenzyme forms found in the serum, AP
is involved in metabolite transport across cell membranes, is found in decreasing order of
abundance, in placenta, ileal mucosa, kidney, bone, and liver (Dufour, 2000). Bone, liver,
and kidney alkaline phosphatase share a common protein structure they differ in carbohydrate
content, the half-life of the liver isoenzyme is three days, cholestasis stimulates synthesis of
ALP by hepatocytes; bile salts, detergents or other surface-active agents facilitate release of
ALP from cell membranes (Krier and Aijaz 2009).

11

1.5.5 ALBUMIN
Albumin is the most abundant plasma protein produced by hepatocytes, up to 10 g of albumin
is normally produced and secreted each day by the liver, in advanced liver disease, Plasma
and serum albumin level can be used to assess hepatic synthetic function, however, the halflife of plasma albumin is 20 days, which significantly reduces its utility for real-time
assessment of hepatic synthetic function in acute liver disease (Dufour, 2000).

1.5.6 UREA
Urea is the final degradation product of protein and amino acid metabolism, in protein
catabolism the proteins are broken down to amino acids and deaminated, the ammonia
formed in this process is synthesized to urea in the liver, this is the most important catabolic
pathway for eliminating excess nitrogen in the human body, Urea is primarily produced in the
liver and secreted by the kidneys, Urea is the major end product of protein catabolism in
animals, it is the primary vehicle for removal of toxic ammonia from the body, Urea
determination is very useful for the medical clinician to assess kidney function of patients
(Krier and Aijaz 2009).

1.6 OBJECTIVES OF STUDY

The aqueous M. oleifera leaf extract has been widely studied, the objective of the current
study is
i. To determine the safety of M. oleifera methanolic leaf extract by performing, acute and
sub acute (28days repeated dose) oral toxicity studies in Albino SCID laboratory BALB/C
substrain mice.
ii. To identify a dose causing major adverse effects and an estimation of the minimum dose
causing lethality (LD50).

12

CHAPTER TWO
MATERIALS AND METHODS
2.1 MATERIALS
Labconco Freeze dryer, Leadman 02 automated analyser, Leadman standardized reagent kits
Toledo XS204 analytical balance and.Eurosonic ES 480R refrigerator.

2.2 METHODS
2.2.1

PLANT SAMPLE COLLECTION AND IDENTIFICATION

This laboratory based study had Moringa oleifera leaf harvested in Lekki Lagos Nigeria, A
voucher specimen was deposited in the herbarium with register number LUH 6202 for
future reference.

2.2.2 EXTRACT PREPARATION

Leaves were shade dried for one week. They were kept away from high temperatures and
direct sun light to avoid denaturation of active compounds. Dried leaves were pulverized to
coarse powder. Extraction was done using 80% methanol as solvent.
Pulverized leaf sample of M. Oleifera (346g) was weighed into a bottle container; using the
weighing balance and soaked (in 1:4 ratio) in aqueous methanol (water: methanol, 20:80 v/v)
and was allowed to stand for five days. It was filtered using a Buckner funnel and whatman
filter paper no. 1 into a conical flask (500ml).
Determination of Percentage Yield
% Yield = Weight of dried extract 100
____________________________
Weight of pulverized leaves

13

2.2.3 PHYTOCHEMICAL SCREENING

Phytochemical tests were carried out on the methanolic leaf extract of Moringa oleifera using
The procedure outlined by Trease and Evans (1989).
In general, test for the presence or absence of phytochemical compounds using the above
method involved the addition of an appropriate chemical agent to the methanolic leaf extract
of the plant in a test tubes and shaking vigorously or lightly as the case may be. Gentle heat
may sometimes be required as detailed in Appendix.1

2.2.4 EXPERIMENTAL ANIMALS

Thirty male Albino SCID laboratory BALB/C substrain mice of about 12-18 weeks weighing
20 2.0 g were obtained from the animal house of College of medicine, University of Lagos.
Mice were acclimated in the experimental room to controlled laboratory conditions for two
weeks before beginning the experiment. The mice were housed in separate polypropylene
cages in experimental room. All the animals were maintained at 24 2C and the relative
humidity set at 30-70% with a 12:12-hours light-dark cycle maintained on a regular standard
vital pellet feed and distilled water

2.2.5 METHOD OF DILUTION OF EXTRACT

The extract; 0.35g (350mg) was weighed using an electronic weighing balance. It was
transferred into a beaker containing 1ml of normal saline with a spatula. The concentration of
the stock solution was 350 mg ml-1. It was then stored in a clean glass bottle with cover in a
refrigerator.

2.2.6 EXPERIMENTAL DESIGN

Mice were weighed and grouped into six (5) cages: I, II , III, IV,V with each group having a
close range of weights, Group 1 served as control. Each cage was marked to differentiate
14

them. Their weight range was used to calculate the volume of drugs to be administered to
each animal.
()

Volume = Group dose (mg)

1000
_____________________________
Concentration (mg ml-1 ) of stock

STOCK CONC = 1 ml of normal saline = 350mg

Table 2: Dosage administered to mice in each group per body weight

Weight(g) 400mg kg-1

350mg ml-1

800mg kg-1

350mg ml-1

1200mg kg-1

15

0.017

12

0.034

18

20

0.023

16

0.046

25

10

0.029

20

0.057

350mg ml-1

2000mg kg-1

350mg ml-1

0.051

30

0.086

24

0.069

40

0.114

30

0.086

50

0.143

2.2.7 METHOD OF ADMINISTRATION

When administering the extract, a Canula was fitted onto the nozzle of a 1ml syringe thereby
enhancing easy administration. Each animal was picked up with hand by using the thumb and
index finger to hold the loose skin of the back of the neck and then lifting the animal allowing
the back to rest on your palm then place the tail between the small finger and the ring finger,
this is how to restrain the animal. The appropriate volume of each drug or extract was
delivered directly into the oesophagus with the aid of a Canula, the Canula was used to
prevent the influx of drug into the tracheal tube.

15

2.3 WEEKLY BODY WEIGHT

The body weight of each rat was assessed using a sensitive balance during the acclimatization
period, once before commencement of dosing, once weekly during the dosing period and
once on the day of sacrifice.

Percentage difference in weight of mice =

final weightinitial weight

initial weight

100

2.4 ACUTE TOXICITY TEST

The acute oral toxicity of the crude Methanolic extracts of M. oleifera was evaluated in mice
using the procedures described by Adedapo et al.(2009). Mice fasted for 8 hours and were
divided into four dosage groups(400, 800, 1200, 2000 mg kg-1) with 5 mice each. After the
dose has been administered, the mice were then allowed free access to food and water and
observed over a period of 48hours for signs of acute toxicity. The number of deaths within
this period of time was recorded.

2.5 SUB ACUTE TOXICITY STUDY

Repeated dose toxicity study was evaluated in mice using the procedures described by Farah
et al.(2013) .The animals were divided into four dosage groups (400, 800, 1200, 2000 mg
kg- 1) of 5 mice each. Mortality, body weights, food and water consumption as well as
observation for general toxicity signs of the animals were evaluated daily for 28 days.

2.6 BIOCHEMICAL PARAMETERS ASSAY

Blood samples were collected for biochemical analysis on the 29th day after mice were
fasted overnight. The animals were sacrificed by cervical dislocation 24 hours after the last
treatment. Blood was collected by occular puncture The blood plasma was obtained by
centrifuging the heparinized blood at 4100g for 10 min at 4C, An automatic blood enzyme
16

analyzer

(LEADMAN)

Cholesterol(Mmol/L),

was

used

for

the

Triglyceride(Mmol/L),

following

Alanine

determinations:

Total

Amino

Transferase(mol/L),

Aspartate Amino Transferase(mol/L), Urea(Mmol/L), Alkaline

Phosphate(mol/L),

Albumin (mol/L). The parameters were determined as described in the LEADMAN

standard operating procedures (2012). Biochemical parameters are expressed in international
units (SI).

2.7 STATISTICAL ANALYSIS

Results were expressed as mean + standard deviation (S.D). Where applicable data were
analyzed using Microsoft Excel 2007 to obtain group means and standard deviations (SD)
and graphs for comparison between the control and test groups.

17

CHAPTER THREE
RESULTS
3.0 PERCENTAGE YIELD OF METHANOL EXTRACT
The percentage yield of the extract was 4.97%. as shown in table 3
Table 3: The percentage yield of methanol extract of Moringa oleifera.
Extract (g)

Percentage (%)

17.2g

4.97

Percentage yield= 17.2/346 100

18

3.1 QUALITATIVE PHYTOCHEMICAL ANALYSIS

Phytochemical analysis of leave extract of Moringa oleifera showed in abundance the
presence of Alkaloids, flavonoids, resins and Protein .Glycoside, tannins and acid component
are present significantly, while saponins was present in little amount, Fats and oil, Steroids
and Terprenoids were absent (Table 4).
Table 4: Phytochemical composition of the leaves of Moringa oleifera.
Constituent

Relative abundance

Alkaloids

++++

Glycosides

++

Reducing sugar

Carbohydrate

+++

Saponins

Tannins

++

Flavonoids

+++

Test for acidic components

++

Protein

+++

Fats and oil

Steroids

Terprenoids

Resins

+++

Key:
- Means absent
+ Means present in little amount
++ Means moderately present
+++ Means present in large amount
19

3.2 ACUTE TOXICITY

No mortality and clinical signs were observed in mice treated with Moringa oleifera
methanolic leaf extract at 2000 mg kg-1 b.wt during the 48 hours observation period

3.3 SUB ACUTE TOXICITY

No mortality or clinical signs were observed in mice administered orally with Moringa
oleifera methanolic leaf extract at 400, 800, 1200 and 2000 mg kg-1 B.wt for a period of 28
days. General behavior of the mice was found to be normal throughout the study period.

3.4 CHANGEES IN THE WEIGHT OF MICE.

No significant variation was observed in the mean body weight of mice treated with Moringa
oleifera methanolic leaf extract at different doses compared with control, as shown in Fig.2
However gain in the body weight was observed in all the groups compared to their initial
weights on day 1 (Table 5).
Table 5: Effects of graded doses of M. oleifera on body weight of mice

GROUPS

Dosage

% difference in

(400mgkg-1)

weight

GROUP 1

10.4

GROUP 2

400

7.85

GROUP 3

800

6.38

GROUP 4

1200

3.04

GROUP 5

2000

3.14

20

25
24

Weight of mice (g)

23

GROUP 1

22

GROUP 2

21

GROUP 3
GROUP 4

20

GROUP 5

19
18

DAY 0

DAY 7

DAY 14

DAY 21

DAY 28

Time (Days)

Figure 2: The effect of graded doses of M. oleifera on body weight of mice

21

3.5 BIOCHEMICAL PARAMETERS

No significant difference was observed in all the biochemical parameters of the test groups
treated with Moringa oleifera methanolic leaf extract at 400, 800, 1200 and 2000 mg kg-1
B.wt compared to control. All the parameters were found to be within the normal range.

Liver enzymes levels (mol/l)

120
100
80
ALT

60

AST
ALP

40
20
0

GROUP 1

GROUP 2

GROUP 3

GROUP 4

GROUP 5

Dosage Groups

Figure 3: Effect of moringa oleifera extract on liver enzymes (ALP, ALT, AST) of mice
Methanolic extract of Moringa oleifera showed no significant effect on the plasma alkaline
phosphatase (ALP) enzyme , alanine amino transferase(ALT) enzyme and aspartate amino
transferase (AST) enzyme concentration; after
(Fig. 3).

22

daily administration for 28 days on mice

Methanolic extract of Moringa oleifera leaf extract showed no significant effect on the total
Cholesterol and Triglyceride concentration after daily administration to the mice for 28 days
(Fig. 4).
5

Lipids profile levels (mmol/l)

4.5
4
3.5
3
TOTAL CHOLESTEROL

2.5

TRIGLYCERIDE

2
1.5
1
0.5
0

GROUP 1 GROUP 2 GROUP 3 GROUP 4 GROUP 5

Dosage Groups

Figure 4: Effect of Moringa oleifera extract on Lipid profile on mice

23

Fig. 5 shows that daily administration of M.oleifera to mice for 28 days presented no
significant change in the levels of Albumin and Urea

60

Lipoproteins levels (mmol/l)

50
40
ALBUMIN

30

UREA
20
10
0

GROUP 1

GROUP 2

GROUP 3

GROUP 4

GROUP 5

Dosage Groups

Figure 5: Effect of Moringa oleifera extract on Albumin and Urea levels of mice
.

24

CHAPTER FOUR
DISCUSSION AND CONCLUSION
There has been a shift from the use of synthetic drugs to natural products as this is considered
a panacea to wellness and it cuts across all socio-economic barriers (Anwar et al.,2007).
The methanolic leaf extract of M. oleifera containing significant amount of phytochemicals
indicates that the plant may have preventive and curative properties. Flavonoids have been
implicated in the management of diverse diseases. A recurring explanation for the therapeutic
actions of M. oleifera medication is the relatively high flavonoid activity of its leaves (verma
et al., 2009).
Mice used in the study gained weight which may be as result of high nutritive value of M.
oleifera. Previous research had shown that Moringa oleifera leaves contain more vitamin C
than oranges, more vitamin A than carrots, more calcium than milk, more potassium than
banana, more iron than spinach (Richardson, 2009).
It is interesting to note however that mice in the controlled group gained more weight than
mice in the experimental groups. Weight gained for the experimental mice however
decreased with increase in dose. This is in accordance with the study conducted by Adedapo
et al., (2009). This may have implication when searching for medicinal plants with active
compounds that could reduce weight. It is likely that the bioactive ingredient in the plant
reduces the rate of metabolism of fats in the treated mice and is more efficient at a higher
dose than lower dose.
The median lethal dose of was found to be >2000 mg kg-1 b.wt. This indicates that at 2000mg
kg-1 dose, the methanolic leaf extract of M. oleifera is safe for consumption and for medical
use, at doses above this level however it is unclear how the animal may react as reported by
Adedapo et al. (2009) who evaluated the safety of aqueous extract of M. oleifera leaves of at
2000mg kg-1 in rats and observed no toxic changes.

25

The non toxic nature of the leaf extract is indicated by minimal differences in the
biochemical parameters. Mice in the entire group did not have any significant increase in
Total cholesterol, Triglyceride, alkaline phosphatase (ALP), alanine aminotransferase (ALT)
and aspartate aminotransferase (AST), when compared with control, this suggest that M.
oleifera methanolic leaf extract may modulate liver enzymes levels and lipids profile and this
report shows a correlation with that of Reddy et al.(2013). He reported that M. oleifera stem
bark extract did not alter Liver enzyme profile in Swiss albino mice. Adedapo et al. (2009),
reported a significant increase in alkaline phosphatase having administered Moringa oleifera
aqueous leaf extract to the rats using a low dose of 1600mg kg-1 and Ezekwe and Ugwu
(2013), reported no significant increase observed in ALP and ALT at different groups when
compared with the control but in AST, there was a significant increase in the values of all the
groups. Similarly there was no significant increase in albumin and urea levels when
compared with control, non toxic nature of the leaf extract is also indicated by these
biochemical parameters which did not alter significantly compared to control mice, this is in
consonance with the study conducted by Reddy et al.,(2013).

CONCLUSION
The results presented in this paper has shown that a single oral dose of M. oleifera methanol
leaf extracts of 400, 800, 1200, 2000 (mg kg-1 b-w) was unable to induce mortality or toxic
effects in mice. Similarly, sub acute toxicity test in mice dosed 400, 800, 1200, 2000 (mg kg1

b-w) during 28 days demonstrated that the extract is innocuous and has no toxic effects on

biochemical parameters of Albino SCID laboratory BALB/C substrain mice.

Therefore the area of cultivation of the plant may play an important role in the outcome of
the results from such studies.

26

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32

APPENDICES

A.1: DETERMINATION OF PHYTOCHEMICALS

The phytochemical constituent of the methanol extract was carried out based on the
procedures outlined by Trease and Evans (1983)
PREPARATION OF REAGENTS FOR PHYTOCHEMICAL ANALYSIS 5% (w/v)
Ferric chloride solution: 5.0 g of ferric chloride was dissolved in 100 ml of distilled water.
Ammonium solution: 187.5 ml of the stock concentrated ammonium solution was diluted in
31.25 ml of distilled water and then made up to 500 ml with distilled water.
45% (v/v) ethanol: 45 ml of absolute ethanol was mixed with 55 ml of distilled water.
Aluminium chloride solution: 0.5 g of aluminium chloride was dissolved in 100 ml of
distilled water.
Dilute sulphuric acid: 10.9 ml of concentrated sulphuric acid was mixed with 5.0 ml of
distilled water and made up to 100 ml.
Lead sub-acetate solution: 45 ml of 15% lead acetate (i.e. 15.0 g of lead acetate in 100 ml
of distilled water) was dissolved in 20 ml of absolute ethanol and made up to 100 ml with
distilled water.
Wagners reagent: 2.0 g of iodine crystals and 3.0 g of potassium iodide were dissolved in a
40 ml of distilled water and then made up to 100 ml (with distilled water).
Mayers reagent: 13.5 g of mercuric chloride was dissolved in 50 ml of distilled water.
Also, 5.0 g of potassium iodide was dissolved in 20 ml of distilled water. The two solutions
were mixed and the volume made up to 100 ml with distilled water.
Dragendorffs reagent: 0.85g of bismuth carbonate was dissolved in 100 ml of glacial
acetic acid and 40ml of distilled water to give solution A. Another solution called solution B
was prepared by dissolving 8.0 g of potassium iodide in 20 ml of distilled water. Both
solutions were mixed to give a stock solution.
Molisch reagent: 1.0 g of -naphthol was dissolved in 100ml of absolute ethanol. 2% (v/v)
Hydrochloric acid 2.0 ml of concentrated hydrochloric acid was diluted with some distilled
water and made up to 100 ml.
1% (w/v) Picric acid: 1.0 g of picric acid was dissolved in 100 ml of distilled water.

QUALITATIVE PHYTOCHEMICAL ANALYSIS OF M.OLEIFERA

33

TEST FOR ALKALOIDS

0.2g of the sample was boiled with 5 ml of 2% HCl on a steam bath. The mixture was
filtered and 1ml portion of the filtrate was treated with 2 drops of the following reagents
(i) Dragendorffs reagent: An orange precipitate indicates the presence of alkaloids.
(ii) Mayers reagent: A creamy-white precipitate indicates the presence of alkaloids.
(iii) Wagners reagent: A reddish-brown precipitate indicates the presence of alkaloids.
(iv) Picric acid (1%): A yellow precipitate indicates the presence of alkaloids.

TEST FOR FLAVONOIDS 0.2 g of the sample was heated with 10 ml ethyl acetate in
boiling water for 3 minutes. The mixture was filtered and filtrate was used for the following
tests.
(i) Ammonium test: 4 ml of the filtrate was shaken with 1 ml of dilute ammonium solution
to obtain two layers. The layers were allowed to separate. A yellow precipitate in the
ammonium layer indicates the presence of flavonoids.
(ii) Aluminium chloride test: 4 ml of the filtrate was shaken with 1 ml of 1% aluminium
chloride solution and observed for light yellow colouration that indicates the presence of
flavonoids.

TEST FOR GLYCOSIDES About 2.0 g of the sample was mixed with 30 ml of distilled
water and heated in a water bath for 5minutes. The mixture was filtered and the filtrate used
for the following tests.
(i) 0.3 ml of Fehlings solutions A and B was added to 5 ml of the filtrate until it turned
alkaline (tested with litmus paper) and heated on a water bath for 2 minutes. A brick-red
precipitate indicates the presence of glycosides.
(ii) About 15 ml of dilute sulphuric acid was used instead of distilled water; the above
process was repeated and filtered. 0.3 ml of Fehlings solutions A and B was added to 5 ml of
the filtrate until it turned alkaline (tested with litmus paper) and heated on a water bath for 2
minutes. A brick-red precipitate indicates the presence of glycosides.

TEST FOR PROTEINS About 5 ml of distilled water was added to 0.1 g of the sample. The
mixture was left to stand for 3 hours and then filtered. To 2 ml portion of the filtrate was

34

added 0.1 ml of Millons reagent. The mixture was shaken and kept for observation. A yellow
precipitate indicates the presence.
TEST FOR CARBOHYDRATES: About 0.1 g of the sample was shaken vigorously with
water and filtered. To the aqueous filtrate was added few drops of Molisch reagent followed
by vigorous shaking again. Then, 1 ml of concentrated sulphuric acid was carefully added
down the side of the test tube to form a layer below the aqueous solution. A brown ring at the
interface indicates the presence of carbohydrates.
TEST FOR REDUCING SUGARS: 0.1 g of the sample was shaken vigorously with 5 ml
of distilled water and filtered. To the filtrate was added equal volumes of Fehlings solutions
A and B and shaken vigorously. A brick-red precipitate indicates the presence of reducing
sugars
TEST FOR SAPONINS: About 0.1 g of the sample was boiled with 5 ml of distilled water
for 5 minutes. The mixture was filtered while still hot. The filtrate was used for the following
tests.
(i) Emulsion test: 1ml of the filtrate was added to two drops of olive oil. The mixture was
shaken and observed for the formation of emulsion.
(ii) Frothing test: 1 ml of the filtrate was diluted with 4 ml of distilled water. The mixture
was shaken vigorously and then observed on standing for a stable froth.

TEST FOR TANNINS: 2 g of the sample was boiled with 5 ml of 45% ethanol for 5
minutes. The mixture was cooled and then filtered and the filtrate was treated with the
following solutions.
(i) Lead sub acetate solution: To 1 ml of the filtrate was added 3 drops of lead sub acetate
solution. A gelatinous precipitate indicates the presence of tannins.
(ii) Bromine water: To 1 ml of the filtrate was added 0.5 ml of bromine water and then
observed for a pale brown precipitate.
(iii) Ferric chloride solution: 1ml of the filtrate was diluted with distilled water and then 2
drops of ferric chloride solution was added. A transient greenish to black colour indicates the
presence of tannins.
TEST FOR OILS: 0.1 g of the sample was pressed between filter papers and the papers
observed. Translucency of the filter paper indicates the presence of oils.
TEST FOR RESINS: (i) Precipitate test: 0.2 g of the sample was extracted with 15 ml of
96% ethanol. The alcoholic extract was poured into 20 ml of distilled water in a beaker. A
precipitate occurring indicates the presence of resins. (ii) Colour test: : 0.12 g of the sample
35

was extracted with chloroform and concentrated to dryness. The residue was redissolved in 3
ml of acetone, and 3 ml of concentrated HCl added and heated in a water bath for 30 minutes.
A pink color which changes to magenta indicates the presence of resins.
TEST FOR TERPENOIDS AND STEROIDS: About 9 ml of ethanol was added to 1 g of
the sample and refluxed for a few minutes and filtered. The filtrate was concentrated to 2.5
ml on a boiling water bath and 5 ml of hot water was added. The mixture was allowed to
stand for 1hour and the waxy matter filtered off. The filtrate was extracted with 2.5 ml of
chloroform using a separating funnel. To 0.5 ml of the chloroform extract in a test tube was
carefully added 1 ml of concentrated sulphuric acid to form a lower layer. A reddish-brown
interface shows the presence of steroids. Another 0.5 ml aliquot of the chloroform extract
was evaporated to dryness on a water bath and heated with 3 ml of concentrated sulphuric
acid for 10 minutes on water. A grey colour indicates the presence of terpenoids

A.2:BIOCHEMICAL ANALYSIS DATA

TOTAL
CHOLESTEROL(mmol/l)
GROUP1
1
2
3
4
5

GROUP 2

4.4
4.6
4.6
4.7
4.5
0.114018
4.56

SD
MEAN

GROUP1
1
2
3
4
5

GROUP1
1

4.2
4.6
4.6
4
4.2
0.268328
4.32

GROUP 2
1.2
1.4
1.2
1.3
1.3
0.083666
1.28

SD
MEAN

GROUP 3

1.1
1
1
1
1
0.044721
1.02

4.5
4.6
4.5
4.7
4.7
0.1
4.6

TRIGLYCERIDE(mmol/l)
GROUP 3
GROUP 4 GROUP 5
2.2
2.2
2.1
2.1
2.2
2
2.3
2
2.3
2
2.1
2.1
2
2.3
2.1
0.130384 0.114018 0.109545
2.12
2.16
2.12

ALANINE AMINO
TRANSFERASE(mol/l)
GROUP 2
GROUP 3
40.5
40.1
36

GROUP 4 GROUP 5
4.1
4.4
4.6
4.8
4.5
4.2
4.8
4.1
4.5
4.3
0.254951 0.270185
4.5
4.36

GROUP 4 GROUP 5
40.5
40.2
40.8

2
3
4
5

41.2
42.8
40.9
39.7
1.143241
41.02

SD
MEAN

ASPARTATE AMINO
TRANSFERASE(mol/l)
GROUP1
1
2
3
4
5

GROUP 2

38
41.9
40
40.5
41
1.454991
40.28

SD
MEAN

GROUP1
1
2
3
4
5

1
2
3
4
5

100.5
100.2
100.9
100.6
100.4
0.258844
100.52

11.6
12.5
11.9
12
12.3
0.350714
12.06

GROUP 3

41.5
39.9
39.5

44
43.7
41.9
37

GROUP 4 GROUP 5
40.1
40.2
39.7
39.9
37.8
39.8
40.8
40.5
38.8
39
1.167476 0.563028
39.44
39.88

ALKALINE
PHOSPHATE(mol/l)
GROUP 3
GROUP 4 GROUP 5
109.5
111.2
113.1
109
108.9
109.9
110.2
110.5
110.7
110
112.2
111
111.4
107.9
106.9
0.90111
1.7358 2.247665
110.02
110.14
110.32

9.5
9.2
9.9
10.1
10.5
0.507937
9.84
ALBUMIN (g/l)
GROUP 2

GROUP1

39.9
40.1
39
40
37.2
1.221884
39.24

UREA(mmol/l)
GROUP 3

GROUP 2
7.2
7
6.8
7.5
6.7
0.320936
7.04

SD
MEAN

GROUP 3

GROUP 2

GROUP1

40.1
40
40.3
39.5
40.7
40.4
39.9
40.1
40.3
40.5
40.2
40.7
0.424264 0.270185 0.234521
40.1
40.24
40.5

36
39
37.8
38.2
37
1.148913
37.6

86.4
84.4
88.7
86.1
86.3
1.531992
86.38

SD
MEAN

1
2
3

39.5
39
38.9
38
0.777817
39.1

GROUP 4 GROUP 5
14.3
16.5
14
15.9
13.9
15.6
14.7
16.4
13.9
16.1
0.343511 0.367423
14.16
16.1

GROUP 4 GROUP 5
51.8
53.1
54.5
50.5
52.5
55.5
49.9
53.2
54.9

4
5

40.2
40.9
0.8
40.4

SD
MEAN

45.1
42.4
1.283355
43.42

50.1
53.5
54.5
48
52.9
56
1.368576 0.371484 0.657267
50.06
53.04
55.08

A.3: WEIGHTS OF MICE IN EACH GROUP

DAY 0

WEIGHT BEFORE TREATMENT

GROUPS

CONTROL

TREATMENT
1

AVG

S.D

NO TREATMENT

20.02

20.9

20.7

20.4

20.8

400mg/kg

800mg/kg

1200mg/kg

2000mg/kg

21.5
21.9
22.5
23.5

20.9
21.6
22.7
23.05

21
22.03
22.9
23.5

21.1
22.1
23.02
23.2

21.1
21.7
23
23

20.564

0.3570434

20.7

21.1

20.982

0.1913635

21.12

0.2280351

21.866

0.212791

22.824

0.2210882

23.25

0.2397916

.
DAY 7
GROUPS
CONTROL

TREATMENT
1

NO TREATMENT

21.2

21.0

20.91

AVG

S.D

400mg/kg

21

21.9

21.5

21.7

21.9

21.6

0.3741657

3 800mg/kg

22

22.2

22.3

23

22.5

22.4

0.3807887

1200mg/kg

23

22.9

23.2

23.3

23.1

23.1

0.1581139

2000mg/kg

23.2

23.4

23.7

23.6

23.09

23.398

0.2579147

22.0

22.2

21.2

21.9

21.84

0.3781534

DAY 14
GROUPS
CONTROL

TREATMENT

AVG

S.D

NO TREATMENT

21.9

400mg/kg

22.2

21.3

21.9

21.7

22

21.82

0.3420526

800mg/kg

22.5

22.53

23.07

22.2

22.4

22.54

0.3231873

1200mg/kg

23.05

23.4

23.3

23.2

23.4

23.27

0.148324

2000mg/kg

23.6

23.75

23.8

23.1

23.3

23.51

0.3008322

DAY 21
GROUPS
CONTROL

TREATMENT
1

NO TREATMENT

400mg/kg

800mg/kg

1200mg/kg

2000mg/kg

22.9

22.3

22.9

22.0

22.1

22.5
22.7
23.1
24.1

22.6
23
23.2
23.9

22.4
23.3
23.5
23.6

22.6
22.9
23.3
24

22.9
23.5
23.8
23.7

23.0

22.5

22.8

22.7

22.5

AVG

S.D
22.44

0.4335897

22.6

0.1870829

23.08

0.3193744

23.38

0.2774887

23.86

0.2073644

DAY 28
GROUPS
CONTROL

TREATMENT
1

NO TREATMENT

38

AVG

S.D
22.7

0.212132

400mg/kg

22

23.2

22.7

22.9

23.1

22.78

0.4764452

800mg/kg

23.2

23.7

23.1

23.2

23.1

23.26

0.250998

1200mg/kg

24

23.2

23.3

23.5

23.6

23.52

0.3114482

2000mg/kg

24

23.8

24.1

23.8

24.2

23.98

0.1788854

A.4: PERCENTAGE DIFFERENCE IN WEIGHT OF MICE

Percentage difference in weight of mice=
Group 1 =

22.720.564
20.564

final weightinitial weight

100 = 10.4%

initial weight

22.7821.12
Group 2 =
100 =7.85%
21.12

Group 3 =

23.2621.866

Group 4 =

23.5222.824

Group 5 =

23.9823.25

21.866

22.824
23.25

100= 6.38%

100 =3.04%

100 =3.14%

A.5: ADMINISTERED DOSAGE PER WEIGHT

()

Volume = Group dose(mg)

1000
_____________________________
Concentration In mg/ml of stock

a. group dose = 400mg

if weight = 15g
1000g = 1kg
1000g = 400mg
39

100

15g = ?mg
400mg15g
__________
1000g

= 6mg

6mg
________ =0.017ml
350mg/ml

20g = ?mg
400mg20 g
__________ = 8mg
1000g
8mg
________ =0.023ml
350mg/ml

25g = ?mg
400mg25 g
__________ = 10mg
1000g
10mg
________ =0.029ml
350mg/ml

b. group dose =800mg

15g = ?mg
800mg15 g
__________ = 12mg
1000g
12mg
________ =0.034ml
350mg/ml

20g = ?mg
800mg20 g
__________ = 16mg
40

1000g
16mg
________ =0.046ml
350mg/ml

25g = ?mg
800mg25 g
__________ = 20mg
1000g
20mg
________ =0.057ml
350mg/ml
c. group dosage= 1200mg
15g = ?mg
1200mg15 g
__________ = 18mg
1000g
18mg
________ =0.051ml
350mg/ml

20g = ?mg
1200mg20 g
__________ = 24mg
1000g
24mg
________ =0.069ml
350mg/ml

25g = ?mg
1200mg25g
__________ = 30mg
1000g

30mg
________ =0.086ml
350mg/ml

41

d. group dose= 2000mg

15g = ?mg
2000mg15 g
__________ = 30mg
1000g
30mg
________ =0.086ml
350mg/ml
20g = ?mg
2000mg20 g
__________ = 40mg
1000g
40mg
________ =0.0114ml
350mg/ml

25g = ?mg
2000mg25 g
__________ = 50mg
1000g
50mg
________ =0.143ml
350mg/ml

42