CHAPTER ONE

1.0 INTRODUCTION

1.1 Background of the Study

Usefulness of plant resources has a long time effect on both plant and animal (Barnes et

al., 2007). Traditional or herbal medicine still remain a major part of medicine, even

with the evolution of modern medicine, in some cases it is highly preferred to modern

medicine in mitigating against disease. Conversely, little attention has been placed on the

toxicity of by natural products due to the unfounded belief thatdrugs obtained from nature

are devoid of any toxicramifications (Stickel et al., 2000). WHO estimated that 80% of

the world still depend on medicinal plant, and this is largely due to high cost of synthetic

drugs especially with increasing economic challenges in developing countries (Magaji et

al., 2008).

Medicinal plants have been used virtually by all cultures as source of medicine; over

5000 plants are known to be used for medicinal purpose in Africa. Though only few have

been described or studied (Taylor et al., 2001). A medicinal plant is any plant that

possesses therapeutic properties or possesses beneficial pharmacological effects on the

human body (Motaleb M. A., et al., (2011). Medicinal plant parts contain active

ingredients which can be used to synthesize useful drugs (Afolabi F. and Afolabi O. J.

(2013), these parts are also the precursor for chemo-therapeutical semi-synthesis

(Doughari 2012). These parts include leaves, roots, rhizomes, stems, bark, flowers, fruits,

grains, or seeds. In developing countries, the use of traditional medicine has been

normalized as a basis for achieving healthy living (Jordan et al., 2010).

1
 Securinega virosa is one of the great African medicinal plants described as a true “cure

all”, of which all parts are used as remedies, particularly the root. It is a dense, low

branching, many branched shrub, sometimes a small spreading tree up to about 6 m high,

although, more commonly 2 to 3 m, evergreen or deciduous (Neuwinger, 1996). It is

widely distributed throughout tropical Africa, also in India, Malaya, China and Australia

(Dalziel, 1936).

The root is used in many parts of Africa in the treatment of fever, body pain, stomach

ache rheumatism, diarrhea, pneumonia and epilepsy (Neuwinger, 1996). The alcoholic

extract of S. virosa root exhibited antibacterial and antifungal activities (Khan et al.,

1980; Sawhney, 1978). The cytotoxic properties of the alcoholic leaf extract in tumor

cells has been reported (Tatematsu, 1991).

The plant exhibited significant antimalarial activity against Plasmodium falciparum, in

vitro, comparable to quinine used as standard drug (Gbeassor et al., 1989). The aqueous

extract of the roots exhibited hypoglycemic effect (Moshi et al., 2000).

1.2 Statement of Problem

In recent years, there has been an increase in the demand for herbal medicine globally.

There’s an estimated increased in the world population, and this is expected to be more in

regions where traditional medicine remains the main source of primary healthcare (WHO,

2012). Approximately eighty percent of the World population still depends upon the use

of herbal remedies for their health care. Nigeria and many other countries in West Africa

are blessed with severalvarieties of medicinal plants which are of used for various

purposes.This traditional method of treating ailment is transferred from one generation to

2
 the other all over the world. Dependence on plants usage has been attributed to their

affordability, effectiveness, safety, cultural preferences, and ample accessibility at all

times and when it is needed (Oladele et al., 2020).

Globally, traditional healers are using various medicinal plants for the treatment of

COVID-19 (Oladele et al., 2020).African countries such as Madagascar (Malagasy Covid

organic or Tambavy CVO), Nigeria and a host of others attempt to develop medicine to

mitigate the ravaging coronavirus using plant extract, from Artemisia, an anti-malarial

plant that’s grows on the island and 6-Acetylswietenolide, a terpenoid from Khaya

grandifoliola showed significant inhibition against coronavirus 3-chymotrypsin-like

protease (Gyebi et al., 2020) respectively.

Due to this poor quality control, there is a growing concern about the implication of the

use of the medicine. Toxicity testing can reveal some of the risks that may be associated

with the use of herbs, therefore avoiding potential harmful effects when used as

medicine. The test on the safety, quality, and effectiveness of drugs remains an important

aspect of medicine production. Assumptions have been made that the use of herbal drugs

in humans is going on without any noticeable toxic effects (Sushma et al., 2012). These

assumptions are not based on any scientific empirical evidence but on the fact that herbal

medicine is considered natural (Glesler, 1992).

1.3 Justification of the Study

The primary aim of toxicological assessment of any herbal medicine is to identify

unfavorable effects and to determine the level of exposure that could result in harm or

damage to the body system of the consumer. Also, a very important aspect of this study

3
on toxicity is to determine toxic plant extract or constituents in the different stages of

development of drugs from plant sources. Knowledge of this will help in the proper

removal of harmful toxicants and modification of other beneficial constituents for safer

consumption (Gamaniel, 2000).One of the basic criteria set by WHO for the use of herbs

as medicines is that they shouldbe shown to be non-toxic (WHO, 2011). Although the use

of ethnomedicine iswidespread in Africa, most of the plants have not been thoroughly

investigated for theirtoxicities (Sowemimo et al., 2007). Besides studies on the quality

and efficacy of herbalmedicines, it is necessary to ensure the safety of a product and this

entails toxicity. Toxicity testing of herbal drugs has also been found to have a lot of

benefits (OECD, 2008). Notably, it is easy to identify the toxic effects and thus determine

the limitof exposure levels especially to sensitive populations. Once these toxicants are

known theymay be discarded or modified via dosage adjustment, chemical group, or

structural

adjustments (Obidike and Salawu, 2013).

According to WHO criteria set for the use of herbal medicine, it must be certified non-

toxic (WHO, 2012). The use of traditional medicine is widespread in Africa, and most of

the plants used have not been thoroughly investigated for their toxicities (Sowemimo et

al., 2007). Besides studies on the quality and efficacy of herbalmedicines, it is necessary

to ensure the safety of a product and this entails toxicitytesting. Toxicity testing of herbal

drugs has also been found to have a lot of benefits(OECD, 2008). Particularly, it is easy

to identify toxic effects and to determine the level of exposure required by a sensitive

population. Once the toxicants are ascertained, they may be discarded or modified

through dosage adjustment or structural adjustment (Obidike and Salawu, 2013).Plants

4
 parts such as leaves, stem, bark, and root bark extracts of Securinega virosa have been

used in the management of many disorders in Nigeria (Magaji et al., 2014; Tanko et al.,

2008). There is still lean information on the toxicity profile of the leaf extract of

Securinega virosa. Hence, the toxicity data are therefore required to predict the safety

and effect of continuous exposure to the plant, because of its widespread use.

Aim of the Study

This study aims to compare the effect of methanol and acqeous leave extract of

Securinega virosa in Albino rat.

1.5 Objectives of the study

1. To evaluate the effect of methanol and aqueous leaf extract of Securinega virosa on

some

hematological parameters in albino rats.

2. To assess the histological changes on some vital organs of albino rats exposed to

methanol and aqueous extract of Securinegavirosa.

5
 CHAPTER TWO

2.0 LITERATURE REVIEW

Medicinal plants naturally synthesize and accumulate some secondary metabolites, like

alkaloids, sterols, terpenes, flavonoids, saponins, glycosides, cyanogenic, tannins, resins,

lactones, volatile oils as well as others (Motaleb et al., 2011) The part of plants used

include; leaves, roots, rhizomes, stems, barks, flowers, fruits, grains, or seeds and these

contain chemical components which are used for control and treatment of diseases.

Araujo et al.,(2012) stated that medicinal plants have been used as a means of curing or

preventing diseases known as "phytotherapy" in all regions of the world, with regional

variations due to the influence of cultural characteristics of the population, as well as its

flora, soil, and climate. According to Motaleb et al., (2011) researchers have found that

people in different parts of the world tend to use the same or similar plants for treating

the same illness. WHO estimated that 80% of the people globally rely on herbal

medicines (Motaleb et al.,2011, Adoum, 2016). Anokwuruet al., 2011) partially for their

primary health care (Borde et al., 2014, Merina et al.,2012, Deshmukh, & Sakarkar,

2011, Mbhele N. et al., 2015, Amri, E. 2014). Medicinal plants have assessed their

therapeutic efficacy and toxicology, or safety of use evaluated, among other properties,

are scientifically approved to be used by people in their basic needs, due to their ease of

access, low cost, and compatibility with cultural traditions (Ramos et al., 2012).

2.1 DEVELOPMENT OF MEDICINAL PLANTS

The development of medicinal has a long history among humans dating back to the

Sumerian and the Akadian civilization in the 3rd millennium (Doughari 2012). The first

6
 documentation of medicinal plant was done way back in 1500BC in a document titled

“use of medicinal plants, animal and human anatomy” (Djordjevic, 2017). The first

botanical garden in Athens founded by Theophrastus (371-286 BC) along with his

students where over 500 medicinal plants were described, afterward other scientists

emerged, and by 10century, thousands of medicinal plants and their properties have been

described. Further research by other scientists results in the first isolation of alkaloids.

2.2 METHODOLOGICAL EXTRACTION FOR TOXICOLOGICAL

INVESTIGATION

Extraction is an important step in the itinerary of phytochemical processing for the

discovery of bioactive constituents from plant materials. Extraction of medicinal plants is

a process of separating active plant materials or secondary metabolites such as alkaloids,

flavonoids, terpenes, saponins, steroids, and glycosides from inert or inactive material

using an appropriate solvent and standard extraction procedure (Abubakar and Hague

2020). Selection of a suitable extraction technique is also important for the

standardization of herbal products as it is utilized in the removal of desirable soluble

constituents, leaving out those not required with the aid of the solvents. Solvents

commonly used in extraction of medicinal plants are polar solvent (e.g., water, alcohols),

intermediate polar (e.g., acetone, dichloromethane), and nonpolar (e.g., n-hexane, ether,

chloroform) (Abubakar and Hague 2020).

The choice of solvent depends on the type of plant, part of plant to be extracted, nature of

the bioactive compounds, and the availability of solvent. In general, polar solvents such

as water, methanol, and ethanol are used in extraction of polar compound, whereas

7
 nonpolar solvents such as hexane and dichloromethane are used in extraction of nonpolar

compounds (Pandey and Tripathi, 2014, Altemimi et al., 2017).

2.2.1 Aqueous extract preparation

This method of preparation is known as decoctiona. This is a process that involves

continuous hot extraction using specified volume of water as a solvent. A dried, grinded,

and powdered plant material is placed into a clean container. Water is then poured and

stirred. Heat is then applied throughout the process to hasten the extraction. (Ingle et

al.,2017, Majekodunmi 2015). The process is lasted for a short duration usually about

15min. The ratio of solvent to crude drug is usually 4:1 or 16:1. It is used for extraction

of water soluble and heat stable plant material (e.g. leaves and stems) (Pandey and

Tripathi 2014).

Water is a polar solvent used in the laboratory for extraction and preparation of aqueous
solution. it is cheap, nontoxic and nonflammable and highly polar up to 1000 polarity.
Using water in preparation of an aqueous solution requires high temperature or exposure
to heat for absolute concentration of the extract. Though it is affordable and readily
available, it is known to inhibit bacterial and mold growth; and it may result in the
hydrolysis (Das et al., 2010, Tiwari et al., 2011).

2.2.2 Methanoic extract preparation

This method of preparation is known as infusion, this process involve maceration. The

drug material is grinded into fine powder, and then placed inside a clean container. The

extraction solvent (methanol or alcohol) hot or cold is then poured on top of the plant

material, soaked, and kept for a short period of time. (Ingle et al., 2017, Majekodunmi

2015). This method is suitable for extraction bioactive constituents that are readily

soluble. In addition, it is an appropriate method for preparation of fresh extract before

8
 use. The solvent to sample ratio is usually 4:1 or 16:1 depending on the intended use

(Azwanida 2015).

Methanol as a main constituent of this extract, is miscible with water add could extract

polar secondary metabolites, It is self-preservative at a concentration above 20%. It is

nontoxic at low concentration, and as small amount of heat is required for concentrating

the extract (Das et al., 2010, Tiwari et al., 2011).

2.3 LIVER ENZYMES

Liver enzymes are enzymes which catalyzes some important biochemical reactions in the

liver, they include the Aminotransferase (Aspartate Aminotransferase and Alanine

Aminotransferase Gammaglutamlytransferase) and the phosphatase (Alkaline

Phosphatase) (Thapa and Walia, 2007).

The classification of bilirubin into direct and indirect bilirubin are based on the original

van der Bergh method of measuring bilirubin. Bilirubin is altered by exposure to light so

serum and plasma samples must be kept in dark before measurements are made. When

the liver functions.

2.3.1 Alanine Aminotransferase(ALT):

This is an enzyme that helps to process proteins. An enzyme is a protein that helps to

speed up chemical reactions. Various enzymes occur in the cells in the body. Large

amounts of ALT occur in liver cells. When the liver is injured or inflamed, the blood

level of ALT usually rises.

9
2.3.2 Aspartate aminotransferase(AST):

This is another enzyme usually found inside liver cells. When a blood test detects high

levels of this enzyme in the blood it usually means the liver is injured in some way.

However, AST can also be released if heart or skeletal muscle is damaged. For this

reason ALT is usually considered to be more specifically related to liver problems.

2.3.3 Alkaline Phosphatase (ALP)

This enzyme occurs mainly in liver cells next to bile ducts, and in bone. The blood level

is raised in some types of liver and bone disease.

2.3.4 Liver Disease

Common liver diseases are:Acute Hepatic Porphyria, Alagille Syndrome, Autoimmune

hepatitis, Biliary Atresia,Budd-Chiari Syndrome, Cirrhosis, other medical problems can

also causes it. The damage to the liver usually can’t be reversed.

2.3.5 Liver function Test and its Parameters

Liver has to perform different kinds of biochemical, synthetic and excretory functions, so

no single biochemical test can detect the global functions of liver. All laboratories usually

employ a battery of tests for initial detection and management of liver diseases and these

tests are frequently termed “Liver function tests” (ThapaandWalia, 2007). Although they

are of little value in assessing the liver function parameters.In spite of receiving a lot of

criticism for this terminology, the phrase ‘Liver function tests’ is firmly entrenched in the

medical lexicon. It might be argued that ‘Liver injury tests’ would be a more appropriate

terminology (Thapa and Walia, 2007). Moreover, the clinical history and physical

examination play important role to interpret the functions (Rosen and Keefe, 2000). The

10
 role of specific disease markers, radiological imaging and liver biopsy cannot be

underestimated (Daniel and Marshall, 1999).

2.4 Effect of aqueous extract of Securinega virosaroots on

Bodyweight of Australian white albino rabbits

A study by Mainen et al, (2000) on the effect of aqueous extract of Securinega virosa,

found that the extract caused a dose-dependent decrease in the bodyweight of the albino rat,

at a dose of 1.0g/kg of body weight, had a reduced effect compared to a dose of 0.4g/kg

body weight, suggesting that the effect of extract peak at a dose of 0.4 and 1.0g/kg body

weight

Blood glucose

The extract showed a significant effect on the blood glucose of the albino rat, but this was

observed only during hyperglycemia. It was observed that, once the glucose level reaches a

fasting level, the extract did not affect, this indicates the inability of the extract to cause

hypoglycemia. This was tested in both fed and fasted albino rabbits, though it suggested that

the extract may be acting an excursion of the postprandial blood glucose, the mechanism of

its action remain unknown and requires more research.

2.5 Anti-Ulcerogenic Potential of Aqueous Extract of Securinega virosa Leaf

Gastric ulcer contributes majorly to morbidity and mortality in Nigeria and many other

third-world countries (Agbakwuru et al., 2006). The aggressive secretion of gastric juice

contents (HCl and pepsin) and the inhibition of blood supply, mucus, prostaglandins

syntheses in the stomach (Oloyede et al., 2015) contribute to its etiology. The secretion

which is a physiological event aggravates the ulcer through cell damage and capillary

11
 destruction and inhibited wound healing (Tarnawski and Halter, 1995). A study by Salawu

et al (2019) found that the presence of flavonoids and tannins both improve mucus secretion

and also prevent ulcer development respectively, by improving vase-constricting effects

(Aguwa and Nwako, 1988), suggesting that the plant extract can reduce indomethacin-

induced gastric ulcer. A macroscopic examination of the body part showed no structural

difference in the gastric mucosa between the treated and untreated-induced group with no

sign of ulceration, which suggests an ulcer healing effect of cimetidine. The gastric mucosa

of the groups treated with a varied dose of the extract appeared somewhat whitish.

Similarly, the gastric mucosa of the groups treated with 35 mg kg-1 extract and 70 mg kg-1

extract shows no sign of ulceration, an indication of ulcer healing effects of aqueous extract

of S. virosa leaf. But at a higher dose of up to 140mg kg-1 showed ulceration with severe

injury.

2.6 Anti-diabetic properties ofSecurinega virosa leaf extract

Diabetes is one of the oldest known diseases of the man whose devastating effect is

increasing by the day and severity almost at an epidemic level. It is a disease of disordered

metabolism of carbohydrates, protein, and fat which is caused by the complete or relative

insufficiency of insulin secretion and /or insulin action (Balkau et al.,2000). A study to test

the hypoglycemic effect of methanol extract of S. virosa leaves in streptozocin-induced

diabetes Winstar rat by Tanko et al (2008) found that the phytochemical constituent of the

leave extract includes the presence of reducing sugars, cardiac glycosides, resin, tannins,

saponins, glycosides, flavonoids, glycerin carbohydrate anthraquine, and steroids. The

toxicity of the extract was noticed after 2-4hours after administration of the extract. This

was followed by a decreased locomotion and sensitivity to touch in the induced animal.

12
 This suggests that the lethal dose of the extract is 1264.9mg/kg of body weight. The effect

of three doses (100, 300, and 600mg/kg) of methanol extract of S. virosa leaves, insulin, and

the control group in streptozocin-induced diabetic Winstar rats did not show any significant

change in the blood glucose levels when compared to the untreated control after 2 hours of

observation. However, there was a significant decrease in blood glucose after 4, 8, and

24hours after administration. The least dose of 100mg/kg was found to be the most effective

than the other two doses of 300 and 600mg/kg body weight. These, therefore, suggest that

an S. virosa leaf contains anti-diabetic properties, which promote glucose uptake and

metabolism or inhibiting hepatic gluconeogenesis.

2.7 Sedative properties ofSecurinega virosa root bark extract

According to the World Health Report (WHO, 2001), approximately 450 million people

suffer from a mental or behavioral disorder, yet only a small minority of them receive

even the most basic treatment This amounts to 12.3% of the global burden of disease and

is speculated to keep rising to over 15% by 2021( Reynolds, 2003). Insomnia is a

common complaint of inadequate sleep affecting 15-40% of the world population, which

complicates several disorders, and of which less than 15% receive appropriate treatment

(Jiang et al., 2007). The use of chemical drugs in the management of the condition using

benzodiazepines and non-benzodiazepine drugs are associated with untoward effects such

as daytime fatigue, cognitive impairment, and physical dependence. This has necessitated

the patronage of herbal remedies with claims of lower side effects (Magaji et al., 2015).

A study by Magaji et al (2015) on the sedative properties of S.virosa was able to isolate

bergenin from the root of S. virosa and it sleep-inducing properties, it is said to also have

been isolated from the leaves of S.virosa (Sanogo et al., 2009). The bergenin is said to

13
 also possess antioxidant (Takahashi et. al., 2003), anti-inflammatory (Nazir et al., 2007),

antiretroviral (Piacente et al., 1996), antiarrythmic (Pu et al., 2002), and hepatoprotective

(Lim et al., 2000) properties. The study found that bergenin behaved similarly to the n-

butanol fraction from which it was isolated by decreasing the onset of sleep without

significantly affecting the total sleep duration, suggesting that it may contribute to the

sleep-inducing property of the extract since other phytochemicals such as flavonoids,

saponins, and alkaloids found in the extract and fraction have been reported to have

sleep-modulating properties.

2.8 Anti-sickling properties ofSecurinega virosa leaf extract

Sickle cell disease is a potentially devastating condition that is caused by an autosomal

recessive inherited hemoglobinpath, which results in the hallmark clinical sequale vas

occlusion. It is seen worldwide but occurs mostly in Africa and less commonly in those

of Mediterranean, Latino, and East Indian. It is estimated that 16% of the population in

Africa has sickle hemoglobinopathy which is the highest proportion worldwide. The

Americas and the East Mediterranean region represent the next highest proportion of

sickle cell hemoglobinopathy as delineated by the World Health Organization

(Angastiniotis and Modelle, 1998). Despite a variety of antisickling agents, there is still a

paucity of antisickling medicines. This is because of the potential toxicities associated

with most of these agents. hydroxyurea, a classical example that possesses antisickling

activity causes bone marrow suppression which greatly limits its use (Strouse et al.,

2008). A study by Abere et al., (2014) on the antisickling properties of S.virosa leaf

extract found that there was a significant difference effect in the inhibition of sodium

metabisulphite induced sickling at different concentrations (100, 300, and 500 mg/ml).

14
The fractions of the crude extract of S. virosa inhibited sodium metalsulphite-induced

sickling of HbSS red blood cells to various degrees. The inhibitory activity of S. virosa

could be due to the presence of bioactive compounds. Phenolic compounds which are

present in S. virosa have been reported to possess antioxidant activity.

15
 CHAPTER THREE

3.0 MATERIALS AND METHOD

3.1 MASERIALS

Fresh leaves of Securinega virosawere collected from several communities within

Plateau state. The plant will be identified by a local herbalist from the area. The

taxonomy of Securinega virosaplant was authenticated by a botanist. The leaves were

washed off sand and particles, dried under a shade, and then reduced to fine powdery

form with a blender.

3.2 Methanol leave extract preparation

The powder were prepared by weighing 660g of the powder using analytical balance and

soaked in absolute methanol for 4 days with frequent agitation at room temperature

(dilution factor of 1:5). The extract was filtered with Whitman paper No.1 and the residue

of fine powder were re-soaked with a fresh portion of methanol twice for four days each

time at room temperature. The filtrate were concentrated under reduced pressure in a

vacuum at 45˚C and evaporated to dryness on a rotary evaporator.

3.3 Aques leave extract preparation

Equal mass of the dried powder were used. The dried powder were soaked in 500ml

distilled water for 30mins followed by boiling in water bath for 30min. the extract were

allowed to cool, filtered and then freeze-dried at -200C until used.

3.4 Laboratory animal acquisition and maintenance

20 albino rats weighing 100g and more were used. These animals were bred and housed

in the Animal Facility of the National Veterinary Research Institute (NVRI), Vom,

16
 Plateau State. The animals were separated in five groups of 4 and kept for 14 days for

acclimatization in a cage lined with wood shavings, maintained at room temperature with

adequate ventilation, and an environment naturally illuminated with 12hours of light and

12hours of darkness. They were fed on a standard diet (vital feed in pellets) and allowed

access to clean drinking water ad libitum.

Extract administration were done in the following sequence:

Group one- normal control = 4 albino rats (5ml)

Group two- 1st dose (Methanol) = 4 albino rats (4ml)

Group three- 2nd dose (Methanol) = 4 albino rats (2ml)

Group four- 1st dose (Aqueous) = 4 albino rats (4ml)

Group five- 2nd dose (Aqueous) = 4 albino rats (2ml)

3.5 Toxicological studies

Lorke’s (1983) method will be used with modifications (rats instead of mice will be used)

to test the safety of the leave extract which proceeded in 5 phases. The animals will be

deprived of food for 6-8h before administration of the extract. In phase 1, the first three

groups of five rats (n=4) will be used. S. virosa aqueous extract will be administered

orally in the same procedure will be carried out on the second group of five albino rat

(n=4), but methanol leave extract will be administered orally to the second group. The

treated albino rats will be observed for 3hours post-administration for signs of toxicity.

17
3.6 Hematological and Liver Function estimation

Using syringe and an EDTA bottle blood sample was collected and haematological

parameters of red blood cell count (RBC), haemoglobin (Hb), packed cell volume (PCV),

white blood cell count (WBC), mean cell volume (MCV), will be analyzed according to

the standard techniques described by Baker et al. (2015) and Cheesbrough (2005).And to

carry out the (ALT,ALS ALP) Liver Function Test.

3.7 Statistical analysis

The result was analyzed using a two-way Analysis of Variance (i.e. ANOVA). A P-value

of <0.05 was considered significant. Significant differences between control and

experimental was assessed by the least significant difference (LSD) and student’s t-test.

18
 CHAPTER FOUR

4.0 RESULT

Figure 1 shows variation in hematological parameters between the control albino rats and

the other group administered with the extract (i.e. Methanoic and Aqueous extract). There

was a significant increase in the lymphocyte, Hemoglobin (hereafter Hb), and mean

corpuscular volume(hereafter MCV) between the control group and the group

administered with the different amount of both the Methanoic and aqueous extract

(p<0.05), while the White blood cell (hereafter WBC), Packed cell volume (hereafter

PCV) and Red blood cell (hereafter RBC) showed no significant different between the

three groups and between the quantity of doses administered (p>0.05).

19
 180

160

140

120
Concentration

100 WBC(L)
PCV (%)
80 Lymphocytes (%)
Hb(g/dL)
60 MCV(FL)
RBC(%)
40

20

0
Group A Group B Group C Group D Group E

Experimental Group

Figure 1: The determination of Hematological parameters

Figure 2 is a bar graph showing the difference in the measure of AST, ALT and ALP between
the different groups of albino rats. ALP was seen to be significantly higher in the experimental
test groups which were administered with the Methanoic and Aqueous extract, compared to the
control group, those administered with the Methanoic extract had a slightly higher ALP
concentration compared to the test group administered the aqueous extract. ALT concentration
was not significantly different across the test groups, with the aqueous group showing the least
concentration of ALT, between the test group comparison, the aqueous test group had a lower
ALT concentration, though not statistically significant (P>0.05). AST concentration was
significantly different across the test group, Methanoic test group had a significantly higher
concentration of AST compared to the aqueous test group (p<0.05), but was not significantly
different from the control test group (p>0.05)

20
 120

100

80
Concentration

60
AST
ALT
40 ALP

20

0
Group A Group B Group C

Experimental Group

Figure 2: The determination of effect of both Methanoic and Aqueous extract on the Liver
of Albino rat

21
 CHAPTER FIVE

5.0 DISCUSSION, CONCLUSION AND RECOMMENDATION

5.1 Determination of effect of Securinega virosa Methanoic and Aqueous leaf extract on

some hematological parameters.

This study showed that the Methanoic and aqueous extracts of Securinega virosa caused a

significant increase in the level of lymphocyte, Hb, and MCV, while WBC, PCV and

RBC remained relatively unchanged between the two extract groups. Lymphocyte plays a

significant role in the boosting of immune system as seen in the study. The increase in

lymphocyte concentration both by the Methanoic and aqueous with increased dosage,

suggest that Securinega virosa is capable of boosting the immune system in an organism,

Likewise, RBC count was seen to slightly reduce across the group significantly in the

aqueous group (2ml), at which level can result in anemia. Anemia is the reduction in the

erythrocyte in the circulating blood. This could be by excessive red blood cell

destruction, RBC loss or decreased RBC production. These therefore suggest that

aqueous extract of Securinega virosa may be poisonous to the organism. Similarly, there

was a gradual decrease in the WBC level with decreased dosage of both Methanoic and

aqueous extract. This suggests that at a higher dose of the extracts there might be a

trigger in the WBC in the blood sufficient to initiate defense in the body of the host

organism by engulfing and destroying the invadingmicroorganisms (Paul, 1993; Swenson

and Reece, 1993;Adedapoet al., 2005). And there was no obvious difference in how two

extract was responded to by the rats.

22
 The Hb contents was observed to increase significantly which could have resulted in the

increased synthesis of Hb which causes an increase in the oxygen content in the blood

which is supplied to different tissues. This was responsible for the sustained RBC level

that was observed. There was no significant difference in the way the responded to the

two extracts, as Hb level tend to increase with increase in dosage. Similar pattern was

observed in the PCV level, which is also a measure of the erythrocyte level in the blood.

5.2. Effect of Methanoic and Aqueous leaf extract of Securinega virosa on Liver function

in albino rat

Hepatic enzyme markers (ALT, ALP and AST) are formedin the liver and are good

indicators of liver damage and alsoused to measure the hepatic necrosis.The results

further showed that the Securinega virosa leaf extracts increases the hepatic enzyme

markers (ALP),Significant increase in the level of serum ALP by theadministration of

Securinega virosa leave extracts, indicated the damage of liver cells at early stages but if

theextract is used for long period of time it may lead tocholestasis or hyperbilirubin

anemia. There was a significant elevation in AST in the treatmentgroup (Methanoic)

relative to the control and Aqueous group. This is suggestive of the

occurrence of general cellular damage (Cavanaugh 2003, Kabubii et al., 2015).

However,since ALT is a more specific biomarker of liver injuryand given the fact that

AST may also be found in othertissues (cardiac, brain and skeletal muscles) these

findingssuggest that the aqueous-methanol Securinega virosa leaf extracts may not

obviously cause toxic manifestations in the liverbut may probably have some toxic effect

on othertissues.

23
 From our observations, the mean AST/ALT ratio of thetreatment group rats was non-

significantly higher thanthat of the control group rats. This result compares wellwith the

work of other authors who studied the effects ofthese parameters post extract

administration (Sagar 2010)

5.3. CONCLUSION

Securinega virosa leaf extracts can serve as a good source of medicinal value in medicine

and health. The extracts did not bring forth any significant difference in hematological

and biochemical parametric effect on the treated rat when compared with the compared

with the control. However there was a major difference in the ALP, MCV, Hb, and

Lymphocyte level compared to the control, but was not different between the two extract

group (Methanoic and Aqueous). The observed effects may possibly be dueto primary or

secondary metabolic products of one orseveral of the phytochemicals present in the

extract.

5.4. RECOMMENDATION

Due to sample size and limitation of time, many other variations between the two extracts

may have been masked; hence, further study with larger sample size and long term

exposure of the rats to the extract would help to further understand the effect of long term

exposure on the albino rats. Similarly, there is a need for further work on thesub-acute

and chronic effects of this extract.

24
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 APPENDIX

Table 1: Showing the effect of Methanoic and Aqueous leaf extract on some hematological
parameters in Albino rat

Group WBC(L) PCV (%) Lymphocytes (%) Hb(g/dL) MCV(FL) RBC(%)

Normal 6.67 48.33 8.12 14.00 64.94 7.09
control
Methanoic 3.80 63.00 81.00 20.80 126.00 5.00
(4ml)
Methanoic 1.80 55.00 61.00 20.20 137.50 4.00
(2ml)
Aqueous 2.20 58.00 80.00 19.10 116.00 6.09
(4ml)
Aqueous 1.10 48.00 65.00 16.10 155.00 3.09
(2ml)
*WBC=white blood cell, PCV=packed cell volume, HB=hemoglobin, MCV=mean cell volume,
RBC= red blood cell, (p<0.05)

Table 2: Showing the effect of Methanoic and Aqueous leaf extract on some Biochemical
properties of the liver in Albino rat.

Experimental group AST ALT ALP

Methanoic 37.20* 16.70 110.00*
Aqueous 5.90 11.00 95.00*
Normal control 22.0 18.20 15.22
*AST=AminoTransferase, ALT= Alamine Transaminase, ALP=Alkaline Phosphatase, (p<0.05)

32
ANOVA TABLE

Table 3: Showing the Hematological parameters

Source of variance Degree of freedom Sum of square Mean square F-ratio

Between treatment V1=K1 SSB=2582.18 MSB=SSB÷K-1 Fcal=MSB
=5-1=4 2582.18÷4=645.55 MSW
Within treatment V2=K(r-1)
5(6-1) = SSW=55581.74 MSW=SSW÷K(r-1) 645.55
5×5=25 55581.74÷25= 2223.2
2223.27 =0.29
Total rk-1
6×5-1=30-1 SST=58163.92
29

Table 4: The determination of effect of both Methanoic and Aqueous extract on Liver of
Albino rats

Source of variance Degree of freedom Sum of square Mean square F-ratio

Between treatment V1=K-1 SSB=1962.44 MSB=SSB/K-1 Fcal=MSB

=3-1=2 1962.44/2 MSW

=981.22

Within treatment V2=K(r-1) SSW=9838.35 MSW=SSW/K(r-1) =981.22

3(3-1) =6 9838.35/6= 1639.73

1639.73 =0.598

Total rk-1 SST=11800.79

3×3-1=8

33
SST = Σx2i – T2
rk

where : T = Total observation

r =Number of rows
k = Number of columns
x = Individual observation
SST = Sum of Square Total

SSB = Σx2j – T2
r rk

where :Tj = Column total

SSW = SST – SSB
Where : SSW = Sum of Square Within

MSB = SSB
K–1
Where : MSB = Mean Square Between

MSW = SSB
K(r – 1)
Where : MSW = Mean Square Within

Fcal = MSB
MSW

34