CHAPTER ONE

1.0 INTRODUCTION

An estimate of about 80% (4000 million) of the world population rely chiefly

on traditional medicine, which is largely of plant origin, for their primary health

care needs.( Norman, et al., 1985) However, these valuable medicinal resources

in plants are largely untapped due to the inadequacy of scientific, technical and

commercial facilities in developing countries (Olayiwola, 1993). More recently,

the use of herbal therapy has been on the increase. Evidence from scientific

screening of plant extracts appears to be accumulating gradually but steadily.

Several works on the antidiarrheal, antimalarial and antitrypanosomal activities

of plants-based products have observed this assertion (Abdullahi et al., 2001;

John et al., 2001; Nok 2002; Atawodi et al., 2003). The major factors affecting

the increasing interest in herbal therapy include: rising costs of orthodox

medications, low therapeutic index of synthetic compounds and the growing

incidence of drug resistance among the pathogens especially in developing

countries with very weak economic indices (Onyeyilli and Egwu, 1995; Seed,

2000). Therefore, it is generally assumed that the use of plant-based active

principles will offer an alternative, safe and effective means of prevention and

treatment of diseases through self-medication and physicians counsel. One

major requirement in the selection of herbal medicines for use in health services

is safety. Plants extracts should not only be effective in therapy but also safe for

consumption. Therefore, closely associated with screening of plants extracts for

1
their activities against microorganism or disease conditions is the need to know

their toxic potentials (Bulus, et al., 2011).

Mistletoe (Viscum album) is a hemi-parasitic shrub, which grows on the stems

of other trees. It has stems 30–100 centimeters (12–39 inch) long with

dichotomous branching. The leaves are in opposite pairs, strap-shaped, entire,

leathery textured, 2–8 centimeters (0.79–3.15 inch) long, 0.8–2.5 centimeters

(0.31–0.98 inch) broad and are a yellowish-green in colour. This species is

dioecious and the insect-pollinated flowers are inconspicuous, yellowish-green,

2–3 millimeters (0.079–0.118 inch) diameter. The fruit is a white or yellow

berry containing one (very rarely several) seed embedded in the very sticky,

glutinous fruit pulp. It is commonly found in the crowns of broad-leaved trees,

particularly apple, lime (linden), hawthorn and poplar (Tree News and

Spring/summer 2005).

1.1 AIM OF RESEARCH

To evaluate the acute toxicity of ethanolic, leaf extract of Viscum album in

albino rats.

1.2 JUSTIFICATION

The risk of self-medication without prescribed standard dose may results in

untold side effects which may be acute, subacute or chronic toxicity, it is

therefore of great necessity to evaluate the toxic potential of one the most

2
widely used medicinal plants in Nigeria. Mistletoe (Viscum album) so as to give

a scientific authentication or disapproval to the claim of its non-toxicity

1.3 OBJECTIVES OF RESEARCH

 The objectives of this research are:

 To analyse the phytochemical constituents of the plant,

 To identify the lethal dose of the plant(LD50),

 The effect of the plant on hematological parameters.

3
 CHAPTER TWO

2.0 LITERATURE REVIEW

BACKGROUND INFORMATION

The use of herbs in the treatment and management of various ailments is as old

as man (Khosh and Khosh, 2001; Sofowara, 1982). The majority of Africans

prefers as a first line of medications, the use of herbal medicine (Barbara and

Theiss 1992). The mistletoe leaf extract is said to possess antidiabetic (Obatomi,

et al, 1994), immunomodulatory (Solar., et al., 1998), bacteriostatic (Fulder,

1998) effects. The mistletoe plant is an obligate parasite that depends partly on

its host to obtain water and minerals but can carry out photosynthesis (Griggs,

1991).

Investigations aimed at providing the scientific basis to the hypotensive

property of mistletoe leaf extract have already been provided (Obatomi, et al,

1994; Hajto, etal.,1999; Lavastre, et al., 2002).

It is an evergreen semi-parasite that can grow in most parts of the globe.

Mistletoe can grow on either edible or non-edible trees, while only those that

grow on edible plants are used for medicinal purposes (Evans, 2005). The

growth of Mistletoe on different kinds of plants, are of disease curing

specificity, for example, mistletoe grown on Guava, Kolanuts and Citrus are

specific for curing diseases like cancer, hypertension, nervousness and

4
insomnia, while those grown on cocoa is best used for curing diabetes (Ekhaise

et al., 2010).

2.1 TOXICITY

Lectin viscumin has been isolated from Viscum album, which is toxic.

Viscumin is a cytotoxic protein (ribosome inactivating protein, or RIP) that

binds to galactose residues of cell surface glycoproteins and may be internalised

by endocytosis. Viscumin strongly inhibits protein synthesis by inactivating the

60 S ribosomal subunit. The structure of this protein is very similar to other

RIPs, showing the most resemblance to ricin and abrin (Olsnes., et al., 1982).

Mistletoe giving parenterally has been proven to cause inflammation at the

injection site (Magaret., 1999) some of their side effect include gastrointestinal

symptoms such as nausea, vomiting and diarrhea. Intoxication of mistletoe has

also been reported in children. It is a popular believe that this plant is not toxic

in the treatment of several infirmities. Historically, it has been used to treat

hypertension, epilepsy, exhaustion, anxiety, arthritis, vertigo, and degenerative

inflammation of the joints and even majorly as palliative therapy in cancer

treatment (Margaret., 1999).The above claim has over the years been proven

scientifically by experimentation and the plant has been found to contain

pharmacological principles against several ailments (Okwudiri., 2014).

Therefore the study is design to investigate the acute toxicity of the ethanolic

5
leaf extract of this plant and to educate the general public on the risk of self

medication without prescription.

2.2 TAXONOMIC HIERARCHY

  Kingdom Plantae   
  Subkingdom Viridiplantae   
  Infrakingdom Streptophyta   
  Superdivision Embryophyta   
  Division Tracheophyta   
  Subdivision Spermatophytina   
  Class Magnoliopsida   
  Superorder Santalanae   
  Order Santalales   
  Family Santalaceae   
  Genus Viscum   
Species Viscum album 
 

(NODC Taxonomic code 1990)

2.3 COMMON NAMES AND LOCAL NAMES

All heal, bird lime, birdlime mistletoe, devil’s fuge, European mistletoe, golden

bough, herb de la croix (Fr), lignum crucis, mystyldene, visci albi fructus

(berries), viscid albi herba (leaves), visci albi stipites (stem), and viscum

(Pierce.,1999; Charles., 1992; Bissett., 1994; Flemming., 1998)

6
2.4 GEOGRAPHIC DISTRIBUTION

Mistletoe is the common name for most obligate hemiparasitic plants in the

order Santalales. Mistletoes attach to and penetrate the branches of a tree or

shrub by a structure called the haustorium, through which they absorb water and

nutrients from the host plant. The name mistletoe originally referred to the

species Viscum album (European mistletoe, of the family Santalaceae in the

order Santalales); it was the only species native to Great Britain and much of

Europe. A separate species, Viscum cruciatum, occurs in Southwest Spain and

Southern Portugal, as well as North Africa, Australia and Asia. Over the

centuries, the term has been broadened to include many other species of

parasitic plants with similar habits, found in other parts of the world, that are

classified in different genera and even families-such as the Misodendraceae and

the Loranthaceae. In particular, the Eastern mistletoe native to North America,

Phoradendron leucarpum, belongs to a distinct genus of the Santalaceae

family. The genus Viscum is not native to North America, but Viscum album has

been introduced to California. (USDA, NRCS. 2009)

2.5 PLANT DESCRIPTION

It is a semi-parasitic woody perennial commonly found growing on wide range

of trees such as oaks and other deciduous trees (Charles., 1992; Flemming,

1988). The plant is small, dioecious and shrubby, with oblong evergreen

7
leathery entire leaves, clear dichasial branching and four-part flowers which

form white sticky berries. It has a faint but characteristic odor and a bitter taste

(Bisset., 1994). Viscum album is most commonly seen on old apple, ash, oak

trees, hawthorn trees, and rubber trees; mistletoe from oak trees has traditionally

been the most commonly used (Murray., 1995). It is propagated by birds that eat

the berries and then excrete the seeds, or smear them on branches by wiping the

sticky pulp off their beaks. Under proper conditions, the seeds germinate and

the roots penetrate the branch of the host tree. It is considered a semiparasitic

plant because it synthesizes its own chlorophyll but depends on the host for its

supply of water and minerals. The parts used medicinally are the leaves and

stems. V. album is native to Europe and Asia. Imports originate in Bulgaria,

Turkey, the former Yugoslavia, Albania and the former USSR (Bissett., 1994).

V. album grows in Europe, northern and western Africa, and southwest and

central Asia and Japan; the Asian plant is a special variety, V. album (L) var.

coloratum (Margaret., 1999).

The magic plant, (known as Afomo in Yoruba and Awuruse in Ibo), the experts

say can act as detoxifying or cleansing agent to fight all type of illnesses. Dr.

Aminu Kazeem Olawale(O.I.M) The Chair, Whomp Int`l Centre for Altenative

Therapy said the most important secret of Mistletoe lies in its ability to cleanse

or purify the blood and the entire body tissues of all manner of accumulated

harmful (toxic)substances.

8
2.6 CHEMICAL CONSTITUENTS

Mistletoe’s lectins are cytotoxic glycoproteins of approximately 10,000

molecular weight; they cause cells to agglutinate (Franz., 1986) and inhibit

protein synthesis on the ribosomal level. The lectins, also known as viscumin or

agglutinin, are dual chain molecules. Chain A inhibits protein synthesis and

chain B activates macrophages and releases lymphokines from lymphocytes.

Both the A and B chains of mistletoe lectin I also inhibit allergen-induced

histamine release from leukocytes and collagen-induced serotonin release from

platelets (Franz., 1986). Lectins are structurally similar to two highly

biologically active toxic proteins, ricin and abrin. The amounts and biological

activity of V. album lectins are dependent on the host tree, manufacturing

process, and time of harvest (Bussing., 1999). Viscotoxin is a 46-amino acid

peptide that damages cell membranes, and it is found only in V. album. A

similar constituent of Phoradendron is phoratoxin, a polypeptide about twice

the weight of viscotoxin; it makes up 0.01% to 0.23% of Phoradendron leaves

and stems (Andersson., 1973). Various polysaccharides are thought to be

involved in mistletoe’s antineoplastic effects. The leaves and stems contain

esterified galacturonan, while the berries contain primarily arabinogalactan

(Jordan., 1986). Alkaloids are nitrogenous compounds that may contribute to

mistletoe’s cytotoxicity (Franz., 1986)

9
2.7 ACUTE TOXICITY STUDIES

Acute toxicity describes the adverse effects of a substance that result either from

a single exposure (MSDS 2006) or from multiple exposures in a short period of

time (usually less than 24 hours). To be described as acute toxicity, the adverse

effects should occur within 14 days of the administration of the substance.

(IUPAC 1997).

The U.S. Environmental Protection Agency (EPA) describes the following

methods for determination of acute toxicity:

1. Animal testing. Animal tests are still used where other laboratory

protocols are not available. These tests are combined with other assays

(lethality, microscopy etc.) to minimize the number of animals sacrificed.

Evaluation of acute toxicity data should include the relationship, if any,

between the exposure of animals to the test substance and the incidence

and severity of all abnormalities, including behavioral and clinical

abnormalities, the reversibility of observed abnormalities, gross lesions,

body weight changes, effects on mortality, and any other toxic effects.

2. Use of data from structurally related substances or mixtures. In order

to minimize the need for animal testing for acute effects, the EPA

encourages the review of existing acute toxicity information on chemical

substances that are structurally related to the agent under investigation. In

certain cases it may be possible to obtain enough information to make

10
 preliminary hazard evaluations that may reduce the need for further

animal testing for acute effects.

3. Chemical properties. If a substance is a strong acid then there is really

no need to do skin and eye tests as a corrosive material such as this will

obviously cause great harm.

4. In vitro testing (test tube experiments). Animal rights activists advocate

such methods whenever possible. While in vitro tools have now become

quite powerful, they will never be able to replace completely the need for

animal studies, particularly for pharmaceutical studies.

5. Limit testing. A single group of animals, typically mice or rats, is given a

large dose of the agent. If no lethality is demonstrated, no further testing

is pursued and the substance is classified in a hazard category according

to the dose used.

2.8 USES OF MISTLETOE

Mistletoe is mainly use for the treatment of cancer in Europe (National Cancer

Institute. 2007). While American mistletoe is toxic, European mistletoe is

considered to have medicinal properties till today. The Drug Digests states that

“for several diseases, European mistletoe has been used to treat a wide variety

of physical and mental conditions. Currently, it is best known as an additional

therapy with other drugs and or radiation for treating cancer”. Some HIV/AIDS

11
Organizations (NGO’s) also claim that it can help restore immune systems

(Hoagy., 2008). Away from superstitious beliefs, mistletoe has been used in

medicine to prove much of its older frame as “all healer”. The white-berried

mistletoe (Viscum album) has been documented as a traditional treatment for

diabetes and high blood pressure. Mistletoe extracts represent the most

unorthodox oncology therapy in Germany (National Cancer Institute. 2007).

Ethnobotanical surveys carried out in Palestine showed the use of this plant

(Viscum album) to treat skin diseases and prostate cancer (Khammash., 2005).

In Nigeria, the Hausa and Fulani tribes of Northern Nigeria use mistletoe in the

treatment of cancers and inflammations. (Abubakar et al., 2007). The African

mistletoe, Loranthus bengwensis L. (Loranthaceae), has been widely used in

Nigeria folk medicine to treat Diabetes mellitus (Ibatomi et al., 1994). Another

type of African mistletoe Tapinanthus dodoneifolius revealed a wide spectrum

of antimicrobial activities against certain multiple drug resistant bacteria and

fungal isolates of farm animals. The inhibition of Bacillus sp. Escherichia coli,

Salmonella sp., Proteus sp., Pseudomonas sp., Agrobacterium tumefaciens,

bacterial sp., known to be associated with either crown gall or gastrointestinal

tract and wound infections by this plant extract gives clue to its ethnomedicinal

usage (Fasanu and Oyedapo 2008). Mistletoe leaves have been reported for

treating cholera, nerves and heart problems (Hoagy., 2008). Mistletoe is also

useful for the treatment of insomnia as it relaxes muscles, calms the nerves,

eases palpitation, migraine, nervousness and pains. It has also been observed to

12
slow down the attack of epilepsy and for treating fibroids. Mistletoe is used to

treat arthritis, rheumatism and gout as it increases the production of urine and

the elimination of toxic waste from the system (Anselm., 2009). Mistletoe

leaves contain choline and acetylcholine. Though these compounds act directly

on the autonomic nervous system, the berries contain alkaloids and toxic

substances and should not be ingested (Dutta., 2005).

ROUTE OF ADMINISTRATION OF MISTLETOE (VISCUM ALBUM)

Administration of substances to laboratory animals is often a critical component

of experimental design. Administered substances may include: infectious

disease agents; various therapeutics, such as vaccinations, antimicrobials,

pharmacologic agents, anesthetics, and analgesics; chemical test agents;

radiocontrast agents; electrolytes and other fluids; and nutritive support.

Because substances may be administered repeatedly to the same animal or to

multiple animals on the same study, the dosing methodology is an important

consideration when planning an experiment and during protocol review by

animal care and use committees and represents an essential opportunity for

refining treatment of research subjects. Specific considerations for delivery of

substances to animals are numerous and include factors such as absorption,

distribution, metabolism and excretion of therapeutic or chemical agents; route,

volume, and frequency of administration; duration of treatment; pH, stability,

homogeneity, and osmolality of the substance to be administered; selection of

13
vehicle or solvent for delivering substances that cannot be administered in a

solid or particulate state; solution preparation, including considerations for

sterility if the substance is being administered parenterally; and dosing

apparatus and animal restraint necessary for specific routes of delivery.

Oral Administration.

Administration of substances directly into the mouth (oral administration),

admixed in diet or other foodstuffs, or by orogastric or nasogastric gavage is

common in laboratory animal medicine and research. The oral route is

economical, convenient and relatively safe, depending on the compound being

administered

Intraperitoneal Administration

Commonly used in rats and mice, it results in a faster absorption into the

vasculature than SC administration and is thus akin to IV administration.

Injection of substances into the peritoneal cavity is a common technique in

laboratory rodents but rarely is used in larger mammals and humans.

Intraperitoneal injection is used for small species for which intravenous access

is challenging and it can be used to administer large volumes of fluid safely.

Absorption of material delivered intraperitoneally is typically much slower than

for intravenous injection. Although intraperitoneal delivery is considered a

parenteral route of administration, the pharmacokinetics of substances

14
administered intraperitoneally are more similar to those seen after oral

administration, because the primary route of absorption is into the mesenteric

vessels, which drain into the portal vein and pass through the liver.(Lucas, et

al., 1971). Therefore substances administered intraperitoneally may undergo

hepatic metabolism before reaching the systemic circulation. In addition, a

small amount of intraperitoneal injectate may pass directly across the diaphragm

through small lacunae and into the thoracic lymph (Abu-Hijleh, et at., 1995).

15
 CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 MATERIALS

Plant (leaf extract), Animals (96 white albino rats), Cages.

CHEMICALS/REAGENTS:

 tusks solution

 Hayems solution

 Giemsa stain

 Tween 80

 Absolute ethanol

 Oil immersion

Test tubes, test tube racks, filter paper, cuvette, Petri dish, Micropipette,

Dispensable pipettes, Pasteur pipettes, Neaubauer haemocytometer,

Microhaematocrit centrifuge, Microhaematocrit tube reader, Flasks,

Colorimeter (spectrophotometer), light microscope, High-speed centrifuge,

weighing balance, Mortar and Pestle, Spatula, Graduated cylinder.

16
3.2 METHODS

3.2:1 Plant Collection and Identification: Mistleteo (Viscum album) was

obtained from a rubber tree (Hevea brasiliensis), in Orhionmwon Local

government council of Edo state, Nigeria. The plant was identified and

confirmed at the Herbarium of the department of Botany University of Abuja,

Abuja Nigeria. It was dried under room temperature for days, grinded to powder

form using clean mortal and a pestle.

3.2:2 Preparation of Ethanolic Leaf Extract of Viscum album

The Soxhlet extraction method was used for the extraction of the plant

(Soxhlet., 1879), using a soxhlet extractor machine, 1500g of powered leaf plant

was used. The ethanol extract of the plant were prepared by soaking 20g of the

powder sample in 200ml of ethanol for the same 12h. The extract was then

filtered using filter paper, concentrated and stored in airtight container.

Operation (Soxhlet., 1879).

The solvent was heated to reflux. The solvent vapour travels up a distillation

arm, and floods into the chamber housing the thimble of solid. The condenser

was used to cool the solvent vapour, and allowed to drips back down into the

chamber housing the solid material. The chamber containing the solid material

was slowly filled with warm solvent, to allow the desired compound dissolves

17
in the warm solvent. When the Soxhlet chamber is almost filled, the chamber

was then emptied by the siphon. The solvent was returned to the distillation

flask again for continuous process. The cycle was allowed to repeat many times,

over hours. During each cycle, a portion of the non-volatile compound dissolves

in the solvent. After many cycles the desired compound is then concentrated in

the distillation flask. After extraction, the solvent is removed,

3.2:3 Procedure for phytochemicals (qualitative analysis)

Twenty grams of the extract was used for qualitative phytochemical screening

using the method described by Sofowora (1993).

Test for Tannins

About 0.5g of extract was stirred with about 10mls of distilled water and then

filtered. Few drops of 1% ferric chloride solution were added to 2ml of the

filtrate. The occurrence of a blue–black, green or blue green precipitate

indicates the presence of tannins.

Test for Steroids and Triterpenoids

Salkowski test: Crude extract was mixed with chloroform and a few drops of

conc. H2SO4 were added. It was properly shaken and allowed to stand for some

time. Red colour appeared at the lower layer indicating the presence of steroids

and formation of yellow coloured layer indicating the presence of triterpenoids.

18
Test for saponins

1g of extract was boiled with 5ml of distilled water and filtered. To the filtrate,

about 3ml of distilled water was further added and shaken vigorously for about

5minutes.Frothing which persisted on warming was taken as the presence of

saponins.

Test for Phenols

0.5g of the extract was added to 1% ferric (III) chloride in methanol /water

(1:1).A dirty green precipitate indicates the presence of phenols.

Test for Alkaloids

0.5g of extract was stirred with 5ml of 1% aqueous HCl on water bath and then

filtered.1ml of the filtrate was taken individually into 3 test tubes.To one of the

test-tubes, Mayers Wagner and Drangedoffs reagent were added. The formation

of white precipitate indicates the presence of alkaloids.

Test for Terpenoids

0.5ml of acetic anhydride was mixed with 1ml of sample extract and a few

drops of conc. H2SO4 was added. A bluish green precipitate indicates the

presence of terpenoids.

Test for Resins

19
2ml of extract plus equal volume of acetic anhydride solution, then drops of

conc. H2SO4. The presence of violet colouration is indicative of the presence of

resins.

Test for Balsams.

Three drops of alcoholic FeCl3 was added to 4ml of alcoholic extract which is

then warmed. A dark green colouration is formed.

3.3 Experimental animals

White albino rats (Wistar stock) was obtained from a reputable rat farm in

Gwagwalada, Abuja. The animals were fed on finisher’s mash (Pfizer

Company, Nigeria) and portable water ad libitum throughout the study period.

The weight of the animals were measured just before the commencement of the

experiment.

3.4 Experimental Design for Acute Toxicity Study

The acute toxicity study was conducted using modified Lorke’s method (Lorke,

1983) via both oral and intraperitonial route. The study was conducted in two

phases per route using a total of ninety six rats, which were acclimatized for

seven days. In the first phase, nine rats was divided into 3 groups of three rats

each. Groups 1, 2 and 3 animals was given (10, 100 and 1000 mg/kg b.w.) of

the extract, respectively, and a control group of three rats. In the second phase,

further specific doses (1600, 2900 and 5000mg/kg b.w.) of the extract was

administered to three groups of rat comprising of three rats each (group 4, 5, 6),

and a control group of three rats after no signs of toxicity and death recorded in

20
the first phase of treatment. All animals were monitored frequently on the day

of treatment (24hrs) and surviving animals were monitored daily for 14days for

signs of delayed toxicity. Recovery and weight gain was noted as indications of

having survived the delayed toxicity. At the end of 14 days, all surviving rats

were sacrificed, internal organs were examined macroscopically for signs of

toxicity and were further weighed, and the result tabulated as mean ± standard

error of the means.

3.5 HEAMATOLOGY

Blood samples were collected from the rats using the Orbital sinus as adopted

by Hoff and Rlagt (2000). Complete haematological parameters were analysed

and tabulated as mean ± SEM. The Statistical package SPSS was used to

analyse for differences in means.

3.5.1 EVALUATION OF PACKED CELL VOLUME (PCV)

Haematocrits:

Using micro-haematocrit method, whole blood was collected using Ethylene

diamino tetra-acetic acid (EDTA) sample bottle and separated by centrifugation.

Procedure

A capillary haematocrit tube approximately 7cm in length, with a bore of about

1mm was used.

21
The tubes was filled with blood by capillary action, (to about 75% of its length)

The outside tube was dried carefully with a piece of cotton wool

The opposite.

\]e end of the tube was sealed with a special seal.

The sealed tubes was placed in a special high speed microhaematocrit

centrifuge, with the sealed ends near the outside rim of the centrifuge (indeed to

touch the rim)

Then tighten the microhaematocrit centrifuge cover in place

The centrifuge was then turned on for 5 minutes at the speed of 3000r/min.

The spunned microhaematocrit tube was placed on the “Microhaematocrit tube

reader” and the packed cell volume was read in percentage.

3.5:2 EVALUATION OF HAEMOGLOBIN CONCENTRATION

The haemoglobin concentration was determined using the Cyanmethaemoglobin

method.

Procedure

5ml of cyanmethaemoglobin reagent (diluent) was placed into a chemically

clean cuvette

0.02ml of blood was added into the diluent

22
The tube covered firmly and invert two (2) or three (3) times

The blood/reagent mixture was allowed to stand for about 10 minutes, for

maximum conversion of haemoglobin to cyanhaemoglobin.

The cuvette was wiped with cotton wool and then placed in a

spectrophotometer, to read and record the absorbent at 540nm transmission,

haemoglobin concentration is determine from the value of a standard curve.

3.5:3 EVALUATION OF TOTAL ERTHROCYTE COUNTS

Haemocytometer Method

Procedure:

A clean red cell micro-pipette attached to an aspiration rubber tube was used.

Blood sample was drawn carefully by aspiration to the 0.5 mark of the pipette

The excess blood from the start of the pipette was wiped off using cotton wool.

The pipette was plunged into a cuvette containing the diluting fluid (Hayems

solution)

Carefully, the diluting fluid was drawn by aspiration into the pipette, past the

bulb, and to the 101 mark.

The aspiration rubber tube was removed carefully and the pipette was fixed

between the thumb and the index finger and then mixed gently by inversion

23
The diluted blood was discharged into clean haemocytometer counting

chamber, and was allowed several minutes for erthryocytes to settle to a single

layer

A cover-slip was applied and a high objective (x40 magnification) of the

microscope was used to count the erythrocytes

The total number of cells in five squares in the centre of counting chamber was

counted and multiply by 10,000.

3.5:4 EVALUATION OF ERYTHROCYTIC INDICES

From the packed cell volume (PCV), the haemoglobin concentration (Hb) and

the total erythrocyte counts (RBC) values obtained, it was possible to calculate

the erythrocytic indices. The values are necessary for morphological

classification of anaemia using the formula below.

Mean Corpuscular Volume-MCV.

Unit:- in fentolitres (fl)

MCV= PCV x10/RBC= “a”fl

Mean Corpuscular Haemoglobin-MCH

Unit:- pictogram (pg)

MCH = Hbx10/RBC= “b”pg

24
Mean Corpuscular Haemoglobin Concentration –MCHC

Unit:-grams per deciliter (gm/dl)

MCHC=Hbx100/PCV= “c”/dl

3.5.5 TOTAL WHITE BLOOD CELL COUNTS

Haemocytometer Method

Procedure:

A clean white cell micro-pipette attached to an aspiration rubber tube was used.

Blood sample was drawn carefully by aspiration to the 0.5 mark of the pipette

The excess blood from the start of the pipette was wiped off using cotton wool.

The pipette was plunged into a cuvette containing the diluting fluid (tusk

solution)

Carefully, the diluting fluid was drawn by aspiration into the pipette, past the

bulb, and to the 11 mark.

The aspiration rubber tube was removed carefully and the pipette was fixed

between the thumb and the index finger and then mixed gently by inversion

The diluted blood was discharged into clean haemocytometer counting

chamber, and was allowed several minutes for leukocytes to settle.

25
A cover-slip was applied and a high objective ( x40 magnification) of the

microscope was used to count the leukocytes

The total number of cells in sixteen squares (16) within the larger ruled areas in

the corner of counting chamber was counted and multiply by 50

3.5:6 PREPARATION OF A BLOOD FILM FOR EVALUATION OF

DIFFERENCIAL WHITE BLOOD CELL

Procedure:

A cleaned glass slide was place on a flat surface and was held firmly in place

with the forefingers and thumb, along the length of the slide

The blood was gently mixed slowly inverting the vacutainer several times

A small drop of blood was place on one end of the slide with a capillary tube

An improvised spreader (a second slide) was held along its length, between the

thumb and fore fingers of the other hand.

The second slide was gradually moved backwards to have a contact with the

drop of blood at an angle of 45 degrees

The blood was allowed to spread by capillary action along the short edge of the

slide, and immediately push forward quickly and smoothly at an angle of 40

degrees with minimum pressure.

26
Wave the slide in the air to dry and inscribe all necessary identification marks

on the slide

The smear was then fixed immediately in methanol for 3-5 minutes

After which the fixed smear was stained using Giemmsa stain for 20-30 minutes

and wash.

The slides were then examine under the light microscope using oil immersion,

from the edge to the centre and the different types of white cells identified and

counted respectively.

3.6 Statistical Analysis:

The statistical analyses was carried out using statistical package for social

sciences (SPSS computer package). Percentage organ-body weight ratios and

rats’ body weights would be expressed as mean ± SD. Values in all groups

would be compared using the analysis of variance (ANOVA). Statistical

significance was determined using Duncan test at p<0.05 (Murray., 1992).

27
 CHAPTER FOUR

4.0 RESULTS

4.1 Plant Extraction

After extraction, the percentage yield of the extract was calculated from the

initial 1500g of the powdered plant leaves using the expression below.

weight of extract ( g )
Percentage yield= X 100
powdered weight used ( g)

150
¿ X 100
1500

¿ 10

The extraction process gave a yield of 10w/w.

28
4.2 Phytochemical Analysis Result.

Table 1: Shows the Result of the Qualitative Phytochemical Analysis.

The result of the qualitative phytochemical analysis revealed the presence of

tannins, saponins, phenols, steroids, alkaloids, terpenoids, resins and balsams.

S/N PHYTOCHEMICALS STATUS

1 Tannin +
2 Saponin +
3 Phenol +
4 Steroid +
5 Triterpeniod -
6 Alkaloid +
7 Terpeniod +
8 Flavoniod -
9 Volatile oil -
10 Resins +
11 Balsams +
12 Glycosides -
13 Cardiac glycosides -
14 Cardenolides -

29
+= Present, - = Absent

Table 2: Shows the Result of Quantitative Phytochemical Analysis.

phytochemicals Alkaloid Saponin Tannin Phenol

Abs 0.021 0.0028 0.044 0.032
% content 4.20 0.56 8.8 6.4

30
4.3 ACUTE TOXICITY EFFECT OF ETHANOLIC LEAF EXTRACT OF

VISCUM ALBUM ADMINISTERED ORALLY AND INTRAPERITONEAL

TO WHITE ALBINO RATS.

Acute toxicity studies shows the extract administered orally at 10, 100, and

1000mg/kg respectively does not produce death nor signs of toxicity at 24hours

of administration and 14days after administration. At the end of the first phase

no death was recorded and signs of toxicity were not noticed. Therefore, I

proceeded to the second phase, at which one rat died at 2900mg/kg of the

extract administered orally. The lethal dose was then calculated using geometric

mean expression below

LD50 = √ D 0× D100

D0 = highest dose that gives no mortality.

D100 = lowest dose that produced mortality.

The lethal dose of extract administered orally was 1204mg/kg.

The lethal dose of extract administered intraperitoneally was estimated at

1265mg/kg

31
4.4 Effect of Oral Administration of Ethanolic Leaf Extract of Viscum

album on the Body Weight of Rats during Acute Toxicity Experiment.

Table 3 Shows the Effect of Oral Acute Administration of Ethanolic Leaf

Extract of Viscum album in body weight of rats.

Groups/ × Bw × Bw × Bw

Doses Before After gain %Weight gains

Administration Administration

1. 10 mg/kg 161.1 ± 15.13a 176.1 ± 13.53a 15 9.31

2. 100 mg/kg 161.7 ± 15.56a 176.9 ± 19.51a 15.2 9.40

3. 1000 mg/kg 154.6 ± 10.17a 155.8 ± 6.28a 1.2 0.78

4. Control 164.2 ± 18.19a 177.5 ± 30.66a 13.3 8.09

32
4.4 The result of oral administration of ethanolic leaf extract of Viscum album

on body weight during acute toxicity experiment, shows an increase in weight

of rats treated with various doses of extract.

33
4.5 Effect of Oral Administration of Ethanolic Leaf Extract of Viscum

album on the Body Weight of Rats during Delayed Toxicity Experiment.

Table 4 shows the effect of oral delayed administration of ethanolic leaf extract

of Viscum album in body weight of rats.

Groups/ × Bw × Bw × Bw

Doses Before After gain %Weight gains

Administration Administration

1. 10 mg/kg 155.6 ± 14.64a 206.6 ± 14.95a 51 32.8

2. 100 mg/kg 160.5 ± 27.37a 192.9 ± 37.17a 32.4 20.2

3. 1000 mg/kg 155.4 ± 13.11a 178.5 ± 13.07a 23.1 14.9

4. Control 149.0 ± 7.09a 205.9 ± 14.14a 56.9 38.2

34
4.5 The result of oral administration of ethanolic leaf extract of Viscum album

on body weight during delayed toxicity experiment, shows an increase in weight

of rats treated with various doses of extract.

35
4.6 Effect of Intraperitoneal Administration of Ethanolic Leaf Extract of

Viscum album on the Body Weight of Rats during Acute Toxicity

Experiment.

Table 5 shows the effect of intraperitoneal acute administration of ethanolic

leaf extract of Viscum album in body weight of rats.

Groups/ × Bw × Bw × Bw

Doses Before After gain %Weight gains

Administration Administration

1. 10 mg/kg 161.6 ± 21.01a 164.1 ± 15.92a 2.5 1.55

2. 100 mg/kg 165.7 ± 17.03a 164.1 ± 15.90a -1.6 -0.97

3. 1000 mg/kg 138.5 ± 6.40a 136.6 ± 12.37a -1.9 -1.37

4. Control 148.8 ± 14.62a 145.9 ± 3.95a -2.9 -1.95

36
4.6 The result of intraperitoneal administration of ethanolic leaf extract of

Viscum album on body weight during acute toxicity experiment, shows an

increase in weight of rats treated with 10mg/kg dose and decrease in 100,

1000mg/kg doses of extract, with the control showing decrease in body weight.

37
4.7 Effect of Intraperitoneal Administration of Ethanolic Leaf Extract of

Viscum album on the Body Weight of Rats during Delayed Toxicity

Experiment.

Table 6 shows the effect of intraperitoneal delayed administration of ethanolic

leaf extract of Viscum album in body weight of rats.

Groups/ × Bw × Bw × Bw

Doses Before After gain %Weight gains

Administration Administration

1. 10 mg/kg 166.4 ± 11.20a 208.5 ± 18.96a 42.1 25.3

2. 100 mg/kg 136.4 ± 18.34a 182.7 ± 22.76a 46.3 33.9

3. 1000 mg/kg 150.5 ± 11.56a 189.0 ± 10.57a 38.5 25.6

4. Control 140.1 ± 4.57a 180.8 ± 4.84a 40.7 29.0

38
4.7 The result of intraperitoneal administration of ethanolic leaf extract of

Viscum album on body weight during delayed toxicity experiment, shows an

increase in weight of rats treated with various doses of extract.

39
4.8 Effect of Oral Administration of Ethanolic Leaf Extract of Viscum

album on the Body Weight of Rats during Second Phase Delayed Toxicity

Experiment.

Table 7 shows the effect of oral delayed administration of ethanolic leaf extract

of Viscum album in body weight of rats.

Groups/ × Bw × Bw × Bw

Doses Before After gain %Weight gains

Administration Administration

1. 1600 mg/kg 179.7 ± 15.70a 194.0 ± 17.95a 14.3 7.96

2. 2900 mg/kg 176.9 ± 17.71a 186.7 ± 19.05a 9.8 5.54

3. 5000 mg/kg 166.6 ± 13.12a 189.0 ± 4.61a 22.4 13.4

4. Control 170.4 ± 6.76a 175.1 ± 20.79a 4.7 2.76

40
4.8 The result of oral administration of ethanolic leaf extract of Viscum album

on body weight during second phase delayed toxicity experiment, shows an

increase in weight of rats treated with various doses of extract.

41
4.9 Effect of Intraperitoneal Administration of Ethanolic Leaf Extract of

Viscum album on the Body Weight of Rats during Second Phase Acute

Toxicity Experiment.

Table 8 shows the effect of intraperitoneal acute administration of ethanolic

leaf extract of Viscum album in body weight of rats.

Groups/ × Bw × Bw × Bw

Doses Before After gain %Weight gains

Administration Administration

1. 1600 mg/kg 183.3 ± 19.05a 179.7 ± 17.45a -3.6 -1.96

2. 2900 mg/kg 178.0 ± 7.50a -

3. 5000 mg/kg 183.7 ± 9.12a -

4. Control 177.4 ± 7.35a 176.0 ± 14.10a -1.4 -0.79

42
4.9 The result of intraperitoneal administration of ethanolic leaf extract of

Viscum album on body weight during acute toxicity experiment, shows a

decrease in weight of rats treated with 1600mg/kg and also a deccrese in body

weight of the control group.

43
4.10 Effect of Intraperitoneal Administration of Ethanolic Leaf Extract of

Viscum album on the Body Weight of Rats during Second Phase of Delayed

Toxicity Experiment.

Table 9 shows the effect of intraperitoneal delayed administration of ethanolic

leaf extract of Viscum album in body weight of rats.

Groups/ × Bw × Bw × Bw

Doses Before After gain %Weight gains

Administration Administration

1. 1600 mg/kg 182.0 ± 24.44a -

2. 2900 mg/kg 196.3 ± 11.09a -

3. 5000 mg/kg 187.7 ± 21.41a -

4. Control 159.3 ± 8.09a 190.0 ± 11.93a 30.7 19.3

44
4.10 The result of intraperitoneal administration of ethanolic leaf extract of

Viscum album on body weight during delayed toxicity experiment, shows an

increase in weight of rats of the control group.

45
 4.11 Signs of Acute/Delayed Toxicity of Viscum album Leaf Extract in white

Albino Rats during Oral and Intraperitoneal Administration Route.

Table 10 Oral Administration

Signs 10mg/kg bw 100mmg/kg 1000mg/kg Control

bw bw
Change in behavior Normal Normal Normal Normal

Difficulty in breathing Absent Absent Absent Absent

Stretching of hind limb Absent Absent Absent Absent

Persistent cage scratching Absent Absent Absent Absent

Recumbency Absent Absent Absent Absent

Cold to touch Absent Absent Absent Absent

Feed intake Normal Normal Normal Normal

Stool consistency Normal Normal Normal Normal

Death Absent Absent Absent Absent

46
 Table 11 Oral Administration.

Signs 1600mg/kg 2900mg/kg 5000mg/kg Control

bw bw bw
Change in behavior Present Present Present Present

Difficulty in breathing Absent Absent Absent Absent

Stretching of hind limb Absent Absent Absent Absent

Persistent cage scratching Absent Absent Absent Absent

Recumbency Present Present Present Present

Cold to touch Absent Absent Absent Absent

Feed intake Reduced Reduced Reduced Reduced

Stool consistency Normal Normal Watering Normal

Death Absent Present Absent Absent

47
 Table 12 Intraperitoneal Administration

Signs 10mg/kg bw 100mmg/kg 1000mg/kg Control

bw bw
Change in behavior Absent Absent Absent Absent

Difficulty in breathing Absent Absent Absent Absent

Stretching of hind limb Absent Absent Absent Absent

Persistent cage scratching Absent Absent Absent Absent

Recumbency Absent Absent Absent Absent

Cold to touch Absent Absent Absent Absent

Feed intake Normal Normal Normal Normal

Stool consistency Normal Normal Normal Normal

Death Absent Absent Absent Absent

48
 Table 13 Intraperitoneal administration

Signs 1600mg/kg 2900mmg/kg 5000mg/kg Control

bw bw bw
Change in behavior Present Present Present Present

Difficulty in breathing Absent Absent Absent Absent

Stretching of hind limb Absent Absent Absent Absent

Persistent cage scratching Absent Absent Absent Absent

Recumbency Present Present Present Present

Cold to touch Absent Absent Absent Absent

Feed intake Reduced Reduced Reduced Reduced

Stool consistency Normal Normal Normal Normal

Death Present Present Present Present

49
4.12 Effect of Oral Administration of Ethanolic Leaf Extract of Viscum

album on Haematology during Phase (1) Acute Toxicity Experiment.

Table 14: Acute (oral) Administration

Haematological Group 4 Group 1 Group 2 Group 3

parameters control 10mg/kg bw 100mg/kg bw 1000mg/kg bw

PCV ( %) 37.33 ± 3.17 37.33 ± 2.96 36.67 ± 0.33 31.33 ± 3.28a

Hb (g/dL) 12.40 ± 1.01 12.43 ± 0.97 12.50 ± 0.10 10.43 ± 1.10a
RBC (×106/µL) 9.17 ± 1.90 9.20 ± 1.33 4.98 ± 1.23a 5.80 ± 2.02a
MCV (fl) 46.60 ± 14.16 35.17 ± 3.49a 35.17 ± 3.49a 89.13 ± 50.48a
MCH (pg) 15.47 ± 4.69 14.40 ± 3.19 29.16 ± 8.47a 29.73 ± 16.83a
MCHC (g/dL) 33.23 ± 0.03 33.30 ± 0.00 33.20 ± 0.00 33.27 ± 0.03
Total (WBC×103/µL) 13.00 ± 0.87 7.98 ± 2.47a 10.43 ± 2.19a 7.57 ± 0.73a
Neutrophils (%) 15.67 ± 2.33 24.00 ± 4.35a 28.33 ± 6.93a 42.00 ± 12.49a
Lymphocytes (%) 84.00 ± 2.08 75.33 ± 4.63a 71.00 ± 6.65a 56.67 ± 12.81a
Monocytes (%) 1 1 1 2
Basophils (%) - 1 1 -
Eosinophils (%) - - - -

n = 3,
Results expressed as; mean ± SEM
Superscript ‘a’ indicate significance at ( P < 0.05 ).

50
Result of the effect of ethanolic leaf extract of Viscum album administered

orally on heamatologic parameters shows significant reduction in the value of

Red blood cells at 100mg/kg and 1000mg/kg respectively, slight reduction in

the packed cell volume and heamoglobin value at 1000mg/kg, a decrease in

total white blood cells count, though, there was an increase in neutrophil count

and a decrease in lymphocytes count in the doses at ( P < 0.05 ), when compared

to the control as shown in table 4.7 during the first phase of the oral acute

toxicity study.

51
4.13 Effect of Oral Administration of Ethanolic Leaf Extract of Viscum

album on Haematology during phase (1) Delayed Toxicity Experiment.

Table 15: Delayed (oral) Administration

Haematological Group 4 Group 1 Group 2 Group 3

parameters control 10mg/kg bw 100mg/kg bw 1000mg/kg bw

PCV ( %) 46.67 ± 0.88 40.33 ± 1.16a 41.67 ± 0.33a 39.00 ± 1.00a

Hb (g/dL) 15.53 ± 0.29 12.40 ± 1.15a 13.77 ± 0.13a 10.96 ± 0.67a
RBC (×106/µL) 6.63 ± 0.38b 5.83 ± 1.72 9.27 ± 1.58a 4.53 ± 0.54
MCV (fl) 73.77 ± 3.37 79.53 ± 14.97a 47.43 ± 7.49a 88.17 ± 9.05a
MCH (pg) 24.67 ± 1.211 24.33 ± 6.08 15.80 ± 2.48a 25.33 ± 4.92
MCHC (g/dL) 33.27 ± 0.33 30.77 ± 2.48a 33.23 ± 0.33 28.27 ± 2.47a
Total (WBC×103/µL) 12.57 ± 2.85 7.97 ± 1.67a 11.97 ± 1.48 12.90 ± 3.70
Neutrophils (%) 18.33 ± 0.88 27.40 ± 2.65a 30.77 ± 4.04a 19.00 ± 6.65a
Lymphocytes (%) 77.67 ± 2.18 71.00 ± 2.08a 67.67 ± 5.23a 75.33 ± 7.86a
Monocytes (%) 4 3 5.6 5.6
Basophils (%) - 1 - -
Eosinophils (%) - - - -

n = 3,
Results expressed as; mean ± SEM
Superscript ‘a’ indicate significance at ( P < 0.05 ).

52
Result of the effect of ethanolic leaf extract of Viscum album administered

orally on heamatologic parameters shows significant reduction in the packed

cell volume in all doses and a slight reduction in heamoglobin value in all the

doses, but an increase in the value of Red blood cells at 100mg/kg. there was a

decrease in total white blood cells count at 10mg/kg, though, there was an

increase in neutrophils count in all doses and a decrease in lymphocytes count

in the doses at ( P < 0.05 ), when compared to the control as shown in table 4.8

during the first phase of the oral delayed toxicity study.

53
4.14 Effect of Oral Administration of Ethanolic Leaf Extract of Viscum

album on haematology during phase (2) Acute Toxicity Experiment.

Table 16: Acute (oral) Administration

Haematological Group 4 Group 1 Group 2 Group 3

parameters control 1600mg/kg bw 2900mg/kg bw 5000mg/kg bw

PCV ( %) 40.00 ± 3.05 41.00 ± 2.08 27.00 ± 13.50a 44.67 ± 1.86a

Hb (g/dL) 13.30 ± 3.05 13.63 ± 0.71 8.97 ± 4.48a 14.73 ± 0.98
RBC (×106/µL) 6.07 ± 0.48 11.83 ± 1.06a 7.90 ± 4.19 10.33 ± 2.18a
MCV (fl) 66.00 ± 2.40 35.10 ± 2.96a 23.70 ± 12.51a 46.20 ± 7.15a
MCH (pg) 21.00 ± 2.40 11.67 ± 0.98a 7.86 ± 4.15a 15.23 ± 2.44a
MCHC (g/dL) 33.20 ± 2.40 33.20 ± 0.05 22.10 ± 11.05a 33.26 ± 0.12
Total (WBC×103/µL) 10.73 ± 2.88 13.60 ± 2.00a 9.86 ± 5.04 15.46 ± 2.63a
Neutrophils (%) 31.00 ± 5.68 20.33 ± 8a. 8.36 ± 4.90a 21.33 ± 3.33a
Lymphocytes (%) 68.67 ± 5.92 78.33 ± 8.66a 40.36 ± 23.38a 77.00 ± 3.51a
Monocytes (%) - 2 31.5 1.6
Basophils (%) - - - -
Eosinophils (%) - - - -

n = 3,

Results expressed as; mean ± SEM

Superscript ‘a’ indicate significance at ( P < 0.05 ).

54
Result of the effect of ethanolic leaf extract of Viscum album administered

orally on heamatologic parameters shows a significant reduction in the packed

cell volume at 2900mg/kg dose, and increase at 5000mg/kg dose, and a

reduction in heamoglobin value at 2900mg/kg dose, but an increase in the value

of Red blood cells in all the doses. There was a decrease in total white blood

cells count at 1600mg/kg and 5000mg/kg doses, though, there was a decrease in

neutrophil count in all doses and increase in lymphocytes count at 1600mg/kg

and 5000mg/kg, monocytes count was also high at 2900mg/kg, at (P < 0.05 ),

when compared to the control as shown in table 4.9 during the second phase of

the oral acute toxicity study.

55
4.15 Effect of Oral Administration of Ethanolic Leaf Extract of Viscum

album on haematology during phase (2) Delayed Toxicity Experiment.

Table 17: Delayed (oral) Administration

Haematological Group 4 Group 1 Group 2 Group 3

parameters control 1600mg/kg bw 2900mg/kg bw 5000mg/kg bw

PCV ( %) 43.00 ± 2.08 45.33 ± 2.85a 42.00 ± 1.00 41.67 ± 7.86

Hb (g/dL) 14.30 ± 0.68 15.10 ± 0.95a 13.97 ± 0.33 13.86 ± 0.80
RBC (×106/µL) 6.23 ± 1.87 8.23 ± 2.05a 13.17 ± 1.21a 10.13 ± 1.90a
MCV (fl) 86.90 ± 32.50 66.50 ± 23.73a 32.77 ± 3.67a 45.63 ± 12.41a
MCH (pg) 28.83 ± 10.76 22.10 ± 7.91a 10.80 ± 1.24a 15.17 ± 4.14a
MCHC (g/dL) 33.20 ± 0.05 33.26 ± 0.01 33.20 ± 0.00 33.23 ± 0.03
Total (WBC×103/µL) 12.20 ± 1.15 16.33 ± 0.98a 16.10 ± 2.00a 11.20 ± 3.63
Neutrophils (%) 25.66 ± 12.78 24.33 ± 1.20a 26.00 ± 4.58a 18.00 ± 5.56a
Lymphocytes (%) 73.00 ± 13.11 74.67 ± 1.66 71.67 ± 4.80 81.00 ± 5.68a
Monocytes (%) 1 2 2 1
Basophils (%) - - - -
Eosinophils (%) - - - -

n = 3,
Results expressed as; mean ± SEM
Superscript ‘a’ indicate significance at ( P < 0.05 ).

56
Result of the effect of ethanolic leaf extract of Viscum album administered

orally on heamatologic parameters shows a slight increase in the packed cell

volume and heamoglobin value at 1600mg/kg dose, and significant increase in

the value of Red blood cells in all the doses. there was an increase in total white

blood cells count at 1600mg/kg and 2900mg/kg doses respectively, though,

there was a reduction in neutrophil count at 5000mg/kg, with an increase in

lymphocytes count at 5000mg/kg dose, at (P < 0.05), when compared to the

control as shown in table 4.10 during the second phase of the oral delayed

toxicity study.

57
4.16 Effect of Intraperitoneal Administration of Ethanolic Leaf Extract of
Viscum album on Haematology during phase (1) acute Toxicity
Experiment.
Table 18: Acute (Intraperitoneal) Administration

Haematological Group 4 Group 1 Group 2 Group 3

parameters control 10mg/kg bw 100mg/kg bw 1000mg/kg bw

PCV ( %) 46.33 ± 1.76 47.33 ± 1.76 43.00 ± 1.53a 45.33 ± 1.20

Hb (g/dL) 15.40 ± 0.59 15.73 ± 0.59 14.30 ± 0.51a 15.07 ± 0.39
RBC (×106/µL) 10.23 ± 4.15 7.43 ± 0.95a 10.60 ± 1.39 12.07 ± 3.59a
MCV (fl) 66.80 ± 28.79 66.27 ± 9.27 42.03 ± 5.72a 44.80 ± 12.59a
MCH (pg) 22.23 ± 9.57 22.0 ± 3.35 14.00 ± 1.91a 14.87 ± 4.18a
MCHC (g/dL) 33.26 ± 0.33 33.23 ± 0.33 33.27 ± 0.33 33.27 ± 0.33
Total (WBC×103/µL) 8.00 ± 2.80 15.10 ± 2.30a 9.88 ± 3.03 a 6.43 ± 1.48a
Neutrophils (%) 20.67 ± 1.76 37.33 ± 12.23a 26.00 ± 3.21a 34.67 ± 12.33a
Lymphocytes (%) 79.00 ± 1.53 62.33 ± 12.77a 71.33 ± 3.17a 65.33 ± 12.33a
Monocytes (%) 1 2 2.3 -
Basophils (%) 1 - 1 -
Eosinophils (%) - - - -

n = 3,
Results expressed as; mean ± SEM
Superscript ‘a’ indicate significance at ( P < 0.05 ).

58
Result of the effect of ethanolic leaf extract of Viscum album administered

intraperitoneally on heamatologic parameters shows a significant reduction in

the packed cell volume and a slight reduction in heamoglobin value at

100mg/kg the doses, and a reduction and increase in the value of Red blood

cells at 10mg/kg and 1000mg/kg doses respectively. There was an increase in

total white blood cells count at 10mg/kg and 100mg/kg, and a reduction at

1000mg/kg dose, though, there was an increase in neutrophil count in all doses

and a decrease in lymphocytes count in all the doses at (P < 0.05), when

compared to the control as shown in table 4.11 during the first phase of the

acute intraperitoneal toxicity study.

59
4.17 Effect of Intraperitoneal Administration of Ethanolic Leaf Extract of
Viscum album on haematology during phase (1) Delayed Toxicity
Experiment.
Table 19: Delayed (Intraperitoneal) Administration

Haematological Group 4 Group 1 Group 2 Group 3

parameters control 10mg/kg bw 100mg/kg bw 1000mg/kg bw

PCV ( %) 36.00 ± 3.21 41.33 ± 2.96a 43.67 ± 2.40a 35.00 ± 1.52

Hb (g/dL) 11.97 ± 1.05 13.73 ± 0.98a 14.53 ± 0.79a 11.63 ± 0.52

RBC (×106/µL) 8.16 ± 1.23 6.00 ± 1.47a 8.00 ± 2.85 9.83 ± 0.95a

MCV (fl) 45.23 ± 3.97 65.03 ± 12.59a 66.46 ± 16.96a 36.63 ± 5.17a
MCH (pg) 15.03 ± 1.32 21.40 ± 4.30a 22.13 ± 5.61a 12.20 ± 1.72a

MCHC (g/dL) 33.27 ± 0.33 33.23 ± 0.33 33.23 ± 0.33 33.20 ± 0.57

Total (WBC×103/µL) 13.50 ± 2.40 11.43 ± 1.43a 9.40 ± 1.81a 7.93 ± 1.58a
Neutrophils (%) 17.00 ± 3.00 24.00 ± 0.58a 20.00 ± 1.53a 25.67 ± 6.49a
Lymphocytes (%) 82.33 ± 2.85 74.33 ± 1.33a 78.00 ± 1.53a 73..67 ± 7.12a

Monocytes (%) 1 1.5 2.5 2

Basophils (%) - - - -
Eosinophils (%) - - - -

n = 3,

Results expressed as; mean ± SEM

Superscript ‘a’ indicate significance at ( P < 0.05 ).

60
Result of the effect of ethanolic leaf extract of Viscum album administered

intraperitoneally on heamatologic parameters shows significant increase in the

packed cell volume and heamoglobin value at 100mg/kg the doses, and a

reduction and increase in the value of Red blood cells at 10mg/kg and

1000mg/kg doses respectively. There was a reduction in total white blood cells

count in all the doses, though, there was an increase in neutrophil count in all

doses and a reduction in lymphocytes count in all the doses at (P < 0.05), when

compared to the control as shown in table 4.12 during the first phase of the

delayed intraperitoneal toxicity study.

61
4.18 Effect of Intraperitoneal Administration of Ethanolic Leaf Extract of
Viscum album on Haematology during phase (2) Acute Toxicity
Experiment.
Table 20: Acute (Intraperitoneal) Administration

Haematological Group 4 Group 1 Group 2 Group 3

parameters control 1600mg/kg bw 2900mg/kg bw 5000mg/kg bw

PCV ( %) 38.33 ± 1.76 47.33 ± 1.86a - -

Hb (g/dL) 12.86 ± 0.47 15.76 ± 1.86a - -
RBC (×106/µL) 8.45 ± 0.47 14.97 ± 0.79a - -
MCV (fl) 45.43 ± 3.50 31.90 ± 3.08a - -
MCH (pg) 15.23 ± 1.05 10.57 ± 1.02a - -
MCHC (g/dL) 33.53 ± 0.33 33.26 ± 0.03 - -

Total (WBC×103/µL) 6.56 ± 1.02 5.77 ± 1.20 - -

Neutrophils (%) 48.00 ± 1.52 65.66 ± 6.56a - -
Lymphocytes (%) 51.67 ± 1.33 33.33 ± 6.17a - -
Monocytes (%) 1 1.5 - -
Basophils (%) - - - -
Eosinophils (%) - - - -

n = 3,
Results expressed as; mean ± SEM
Superscript ‘a’ indicate significance at ( P < 0.05 ).

62
Result of the effect of ethanolic leaf extract of Viscum album administered

intraperitoneally on heamatologic parameters shows significant increase in the

packed cell volume, heamoglobin value, Red blood cells count at1600mg/kg

dose, and a slight reduction in total white blood cells count at 1600mg/kg dose,

though, there was an increase in neutrophil count and a reduction in

lymphocytes count at ( P < 0.05 ), when compared to the control as shown in

table 4.13 during the second phase of the acute intraperitoneal toxicity study.

63
4.19 Effect of intraperitoneal Administration of Ethanolic Leaf Extract of

Viscum album on Haematology during phase (2) Delayed Toxicity

Experiment.

Table 21: Delayed (Intraperitoneal) Administration

Haematological Group 4 Group 1 Group 2 Group 3

parameters control 1600mg/kg bw 2900mg/kg bw 5000mg/kg bw

PCV ( %) 30.33 ± 1.45 13.33 ± 0.00a - -

Hb (g/dL) 10.10 ± 0.49 4.43 ± 0.00a -

-
RBC (×106/µL) 8.10 ± 0.40 2.96 ± 0.00a - -

1. MCV (fl) 37.50 ± 1.93 4.97 ± 0.00a - -

MCH (pg) 12.43 ± 1.10 4.97 ± 0.00a - -

MCHC (g/dL) 33.23 ± 0.03 11.06 ± 0.00a - -

Total (WBC×103/µL) 10.53 ± 0.49 3.83 ± 0.00a - -

Neutrophils (%) 31.67 ± 3.17 7.66 ± 0.00a - -

Lymphocytes (%) 68.33 ± 3.17 25.00 ± 0.00a -

-
Monocytes (%) - 2 - -
Basophils (%) - - - -
Eosinophils (%) - - - -

n = 3,
Results expressed as; mean ± SEM
Superscript ‘a’ indicate significance at ( P < 0.05 ).

64
Result of the effect of ethanolic leaf extract of Viscum album administered

intraperitoneally on heamatologic parameters shows a significant reduction in

the packed cell volume, heamoglobin value, Red blood cells count, total white

blood cells count, neutrophils count, lymphocytes count at 1600mg/kg dose, at (

P < 0.05 ), when compared to the control as shown in table 4.14 during the

second phase of the delayed intraperitoneal toxicity study.

65
 CHAPTER FIVE

5.0 DISCUSSION, CONCLUSION, AND RECOMMENDATIONS

5.1 Discussion

In this study, the ethanolic leaf extract of mistletoe (Viscum album) from a host

plant rubber tree (Hevea brasiliensis) gave a total yield of 10%w/w using

soxhlet apparatus. This was lower than the value obtained in similar extraction

method reported by Ofem et al., (2007) which gave a yield of 31%w/w from

host plant citrus. The differences in percentage yield could result from

difference in host plant.

The result of the qualitative phytochemical analysis revealed the presence of

tannins, saponins, phenols, steroids, alkaloids, terpenoids, resins and balsams,

which is in accordance to the findings of (Deeni and Sadiq 2002). The

quantitative analysis revealed the percentage composition of saponin (0.56%),

alkaloid (4.20%), phenol (6.4%) and tannins (8.8%) respectively. From the

above result, tannins was presence in highest quantity followed by phenol and

alkaloid respectively. From the acute toxicity study of ethanolic leaf extract of

Mistletoe (Viscum album) administered orally and intraperitoneally, the lethal

dose (LD50) of extract administered orally was estimated at 1204mg/kg and

1265mg/kg respectively. These values were higher than that as reported by

66
Ofem et al., (2007), with estimated LD50 at 420.70mg/kg through

intraperitoneal administration in rats.

The result of oral administration of ethanolic leaf extract of Mistletoe (Viscum

album) on mean body weight during acute toxicity experiment, showed an

increase in body weight of rats treated with (10,100,1000mg/kg) doses of

extract, while the results of intraperitoneal administration of ethanolic leaf

extract of mistletoe (Viscum album) on mean body weight during acute toxicity

experiment, showed an increase in weight of rats treated with 10mg/kg dose and

decrease in 100, 1000mg/kg doses of extract.

The result of oral administration of ethanolic leaf extract of Mistletoe (Viscum

album on body weight during second phase acute toxicity experiment, showed

an increase in body weight of rats treated with various doses of extract, the body

weight during second phase acute toxicity experiment also showed an increase

in body weight of rats treated with various doses of extract, in the

intraperitoneal administration of ethanolic leaf extract of mistletoe (Viscum

album) on body weight during acute toxicity experiment, showed a decrease in

weight of rats treated with 1600mg/kg and also a decrease in body weight of the

control group. The reduction in weight could be as a result of feed intake

because in the control group there was also reduction in weight.

Hematological parameters are useful indices that can be employed to assess the

toxic potentials of plant extracts in living systems (Sunmonu and Oloyede,

67
2010). The results of the effect of ethanolic leaf extract of Viscum album

administered orally on heamatologic parameters shows a reduction in the value

of Red blood cells at 100mg/kg and 1000mg/kg respectively, slight reduction in

the packed cell volume and heamoglobin value at 1000mg/kg, a decrease in

total white blood cells count at 10mg/kg, though, there was an increase in

neutrophil count and a decrease in lymphocytes count in all the doses (10, 100,

1000mg/kg) when compared to the control group during the first phase of the

oral acute toxicity study. The reduction in red blood cell, haemoglobin

concentration, packed cell volume could be as a result of the damage of red

blood cell caused by haemolysis.

Result of the effect of ethanolic leaf extract of Viscum album administered

orally on heamatologic parameters showed a reduction in the packed cell

volume at 2900mg/kg dose, and increase at 5000mg/kg dose respectively.

Though there was a reduction in heamoglobin value at 2900mg/kg, and increase

in the value of Red blood cells in all the doses (1600, 2900, 5000mg/kg). There

was a decrease in total white blood cells count at 1600mg/kg and 5000mg/kg

doses, and a decrease in neutrophil count in all doses(1600, 2900, 5000mg/kg),

an increase in lymphocytes count at 1600mg/kg and 5000mg/kg respectively.

monocytes count was also high at 2900mg/kg, when compared to the control

during the second phase of the oral acute toxicity study. In the same second

phase of oral acute toxicity study, there was slight increase in the packed cell

68
volume and heamoglobin concentration at 1600mg/kg dose, and increase in the

value of Red blood cells in all the doses. there was an increase in total white

blood cells count at 1600mg/kg and 2900mg/kg doses respectively, though, a

reduction in neutrophil count and lymphocytes count at 5000mg/kg dose.

During the first phase of the acute intraperitoneal toxicity study, a reduction in

the packed cell volume and a slight reduction in heamoglobin value at

100mg/kg, and red blood cell at 10mg/kg and an increase in the value of Red

blood cells at 1000mg/kg. An increase in total white blood cells count at

10mg/kg and 100mg/kg, and a reduction at 1000mg/kg dose, though, there was

an increase in neutrophil count in all doses and a decrease in lymphocytes count

in all the doses when compared to the control. In the intraperitoneal

administration, there was an increase in the packed cell volume and

heamoglobin value at 100mg/kg, and a reduction and increase in the value of

Red blood cells at 10mg/kg and 1000mg/kg doses respectively. There was a

reduction in total white blood cells count in all the doses (10, 100, 1000mg/kg),

though, there was an increase in neutrophil count in all doses and a reduction in

lymphocytes count in all the doses when compared to the control.

At second phase acute intraperitoneal administration, there was an increase in

the packed cell volume, heamoglobin value, Red blood cells count

at1600mg/kg, and a slight reduction in total white blood cells count at

1600mg/kg dose, though, there was an increase in neutrophil count and a

69
reduction in lymphocytes count, but at acute intraperitoneal administration,

there was a significant reduction in the packed cell volume, heamoglobin value,

Red blood cells count, total white blood cells count, neutrophils count,

lymphocytes count at 1600mg/kg dose, when compared to the control.

This drastic reduction in heamatological parameters maybe due to the effect of

the phytochemical constituent of the extract which could have led to a massive

destruction of Red blood cells causing haemolysis. The observed decrease in

packed cell volume (PCV) is believed to be as a result of the decreased red

blood cells (RBC). Decrease in haematological indices in exposed animals

indicates destruction of erythrocyte, the oxygen-carrying capacity of the blood

and amount of oxygen delivered to the tissues could be affected following the

extract administration since Red blood cells (RBC) and Haemoglobin (Hb) are

very important in transferring respiratory gases (Dede et al,. 2002; De Gruchy.,

1976). The crucial role of white blood cells (WBC) in defending the body

against infection and tissue damage. This supports a reports that mistletoe and

some commonly prescribed medicinal plants contains agents that stimulate the

production of leucocytes (Al- Mamary., 2002; Imoru et al., 2005).

Administration of the mistletoe extract appears to exhibit stimulatory effect on

the effectors cells of the immune system (Al- Mamary., 2002; Imoru et al.,

2005).

70
5.2 Conclusion

It is therefore suggested that the use of this extract especially at high doses

(1600, 2900, 5000mg/kg) can affect the blood system. It is also advisable that

the use of this extract in herbal medicine should be with caution to avoid the

risk of anemia upon treatment.

Also, the increase in the total white blood cells (WBC) count observed, suggests

that the plant extract contain agents that could stimulate the production of

leucocytes, also the decrease in total white blood cells observed might also

suggest that the plant extract contain agent that suppress the immune system as

well.

5.3 Recommendation

This work is opened to researchers for further investigations on the suppressant

effect of the plants extracts on hematopoietic system. Further study should be

carried out on sub-chronic toxicity.

71
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