HEMATOLOGICAL STUDY OF PIPER GUINEENSE ON ADULT FEMALE WISTAR

RAT

1.0 CHAPTER ONE: INTRODUCTION

1.1 BACKGROUND OF THE STUDY

In ancient times and till date, many traditional agricultural societies rely on edible
plants to provide important energy and micronutrients (Grivetti and Ogle, 2000).A
type of such edible plant is the spice called Piper guineense. Herbal medicines
have been used since earliest times to treat illnesses and restore good health and
today, herbalism remains the most widely practised form of medicine worldwide,
(Ali et al 2015). Globally, medicinal plants are very useful for the treatment and
management of diseases or infections. They are mostly particularly useful in
countries, where, due to their low income status, they can hardly afford imported
and expensive conventional medicine, (WHO 2005). According to the World
Health Organization, 1992 report, it is estimated that 80% of people worldwide
rely on herbal medicines for some aspects of their primary health care, (WHO
2003). Plants which are medicinal and used by animals as foods have contributed
immensely to health care, (Ali 2015). These medicinal plants include Piper
guineense, Gnetumgnemon, Congremalatifolium, Azadirachta. Piper guineense is a
plant that has edible leaves and seeds, it is traditionally known as “uziza”, “iyere”
and “masoro” in Igbo, Yoruba and Hausa languages in Nigeria and West African
black pepper. It belongs to the family Piperaceae with over 700 species around the
world mainly in tropical and sub-tropical regions of the world (Olonisakinet al.,
2006) and climbs up to 12m high by its adventitious rootlets (Busia, 2007). The
fresh fruits of this plant are drupe mesocarp with an oval shape and the mature
ones appear black in colour. The plant has been found to have a range of effects
ranging from antioxidant effects (Adegokeet al., 1999), antimicrobial (Davidson,
1997; Ahnet al., 2007), antibacterial (Nwinyiet al., 2009), tocolytic (Williams et
al., 2013), hepatoprotectiveeffects (Nwozoet al., 2012) and increasing uterine
muscle contractions (Udoh, 1999) and therefore could also be included in our daily
diets (Echo et al., 2012). Studies on the dose related effect of aqueous extract of
the seeds of Piper guineense on the livers and kidneys of wistar rats revealed that
there were some pathological differences in the tissues of the organs causing
increasing hepatic and renal toxicities with increasing doses in the animals
(Ebeyeet al., 2007). Other studies on the short and long term oral administration of
the aqueous extract of Piper guineense showed a significant increase in body
weights, values of red and white blood cells, packed cell volumes and mean
corpuscular haemoglobin concentrations which are dependent on the duration of
the treatments (Hassan et al., 2010).

Haematology refers to the study of the constituents of blood and its morphology. It
can also be referred to as the study of the changes in some blood indices due the
effect of some environmental or chemical substances, (Je et al 2001). Hence,
haematological parameters are the features indices, or characteristics relating to
blood. Otherwise they are called indices of blood characterization, which include
haemoglobin, red blood cells, packed cell volume, mean cell volume, mean cell
haemoglobin, mean cell haemoglobin count, white blood cells and platelets. The
study of these parameters is of ecological and physiological interest, (Jiang et al
2001). The use of blood examination as a way of assessing the health status of
animals has been documented, (Johnson, 1998). Haematological assays seldom
provide an ethiological diagnosis but they remain nevertheless indispensable
diagnostic tools to evaluate health and disease conditions in human beings and
animals for monitoring the response and progress of patients to therapeutic regimes
and to offer a prognosis. The routine collection and processing of blood samples
allows the evaluation of haematological responses to diseases, (Kakkaret al.,
1995).

Normal pregnancy is characterized by profound changes in almost every organ and

system to accommodate the demands of fetoplacental unit, (Harrison 2006). In
normal pregnancy, the physiological change in haemoglobin concentration [HGB]
and platelet count during pregnancy are well known phenomena, (Yip 2000). It is
also one of the physiological conditions capable of causing remarkable and
dramatic changes in haematological variables. A pregnancy is influenced by many
factors, some of which include culture, environment, socioeconomic status, and
access to medical care. Many physiological hematological changes occur during
pregnancy due to continuous development of fetus. These changes revert back to
normal after puerperium, (Dennenet al 2011). But, these changes are required to
meet metabolic demands of mother and also ensure adequate oxygen delivery to
fetus, (Salas et al 1993). Depending upon the degree of change in the
hematological profile, the pregnancy outcome may vary, (Akinbamiet al 2013).
Thus, it becomes important to monitor hematological parameters during
pregnancy, thereby improving its outcome. The haematological indices also have
an impact on pregnancy and its outcome, (Yip 2000). The haematological indices
of an individual to a large extent reflect their general health, (Who 2004). Blood is
a special type of connective tissue composed of formed elements in a fluid matrix.
Many of the hematological indices are influenced by many factors like sex,
seasonal variation, lactation, pregnancy health, and nutritional status, (Smith
1993). It is also acknowledged that for comparisons between individuals and with
reference data in a clinical diagnostic situation, it is necessary to consider the
normal variations due to sex, age, and breed in order to increase diagnostic
precision, (James et al 2008). In normal pregnancy the haematological indices of
an individual to a large extent reflect their general health (Who 2004) and many
studies such as (Osonugaet al. 2011) and Shaw et al. 2010) have identified the
haematological indices of the pregnant woman as one of the factors affecting
pregnancy. Anaemia (low haemoglobin) is a widely identified haematological
abnormality, (Centre for Disease Control and Prevention 2005) and it is also
associated with adverse pregnancy outcome, (Garn et al 1981).

1.2 STATEMENT OF THE PROBLEM

According to World Health Organization, one woman dies every minute from a
pregnancy-related complication. The main causes causing mortality are due to
antepartum and postpartum hemorrhage, unsafe abortion, eclampsia, obstructed
labour and infection, (Chandra et al 2012) .Thus, it is important to know variation
in hematological profile during the entire course of pregnancy as well as delivery
such that adverse incidents leading to maternal mortality can be minimized.

Blood is the most easily accessible diagnostic tissue. Variations in haematological

parameters are infuenced by any disease condition which afects the haemopoietic
physiology. Tis is likely to happen with an endemic disease such as malaria that
afects the host homeostasis (Mainaet al 2010).

1.3 AIMS AND OBJECTIVE OF THE STUDY

The aim of this research is to study hematological Study of Piper guineense on

adult female wistar rat

1.3.1 SPECIFIC OBJECTIVES OF THE STUDY

1. To determine the haematological study of experimental rat before

administration of Piper guineense
 2. To determine the blood level ofdifferent stages of pregnancy during
administration of Piper guineense

1.4 RESEARCH QUESTIONON OF THE PIPER GUINEENSE

1. What is the negative and positive effect of Piper guineenseon the blood level
of a pregnancy?
2. Does piper guineense has any effect on the blood level of a pregnant wistar
rat?
1.5 SIGNIFICANCE OF THE STUDY

This study will have academic and clinical significance indecent it will add to the
already existing reservoir of knowledge for research student who may be determine
to explore on this research area and also to the management of pharmaceutical
companies for a clear over view of piper guineense on the values of some major
haematological indices among different stages of pregnancy and new therapeutic
strategy.

Clinically, it will inform the pregnancy women on the possible risks or gain
accruable in the consumption of piper guineense, specifically the role of variation
in white blood cell (WBC) count, haemoglobin, packed cell volume (PCV),
granulocytes, lymphocyte, and platelet in the diagnosis of complications or
challenges during pregnancy. In a more general term, this study will be beneficial
to health facilities both (Local, State and at Federal Level), Ministry of Health at
all levels, as well as the general public.

1.6 JUSTIFICATION OF THE STUDY

This study will help to show the effect of known concentration and the level of full
blood count of Piper guineense on different stages of pregnancy in female albino
wistar rats. The choice of female albino wistar rats for this study is apt as
according to the research carried out by Nwachukwuet al. (2010) on the uses of
some medicinal plants, they found out that Piper guineense ‘Uziza’ is suitable for
treating Infertility in women. According to Okoye and Ebeledike (2013) the leaves
and fruits of Piper guineense are used in preparing food for the pregnant and
lactating mothers, because people believed that they promote good health.
Biomedical Research Association fact sheet, animals make good research subjects
in that animals are biologically similar to humans. In fact, mice and rats share more
than 98% DNA with humans; therefore, animals are susceptible to many of the
same health problems as humans. Animals have a shorter life cycle than humans
and as a result, they can be studied throughout their whole life span or across
several generations. In addition, the environment around animals (diet,
temperature, lighting), can be easily controlled which would be difficult to do with
humans. Thus the decision to use the albino wistar rats in the investigation of the
effect of hematological Study of Piper guineense on the different stages of
pregnancy in female is justified

1.7 SCOPE OF THE STUDY

This study is limited only to biochemical analysis.

2.0 CHAPTER TWO: LITERATURE REVIEW

2.1 PIPER GUINEENSE

The use of plant medicine dates back to ancient times, the nearly all year round
availability and the relatively cheaper cost of medicinal plants in sub Saharan
Africa makes them more attractive as therapeutic agents when compared to the
orthodox medicine, (Agbor&Ngogany, (2005), Ebeyee, et al 2007) The use of
herbal medicine is on the increase globally, (World Health Organization (1996),
hence the increased interest on research on herbal formulations and preparations.
(Tilburt, &Kaptchuk, (2008). Some of these plants are used as condiments and
ingredients in food thus referred to as local spices. One of such plants used as a
local spice in Nigeria is Piper guineense traditionally called „Uziza‟ amongst the
Igbo tribe, „Iyere‟ amongst the Yoruba tribe, „Etinkene‟ amongst the Efik and
Ibibio tribe and „Ebe-ahinhiakpoke‟ amongst the Edo tribe. The leaves and seeds
of Piper guineense were initially consumed in the Southern part of Nigeria but
overtime its consumption has spread across the entire country even into
neighboring countries like Benin republic, Ghana, Liberia, Cameroun amongst
others (Okwute, (1992). Also, its incorporation in intercontinental dishes and its
high demand by Nigerians in foreign countries has promoted its use in countries far
and wide. An assessment of the effect of Piper guineense consumption on various
human organs is essential to ascertain merits and detect demerits if any in order to
avert health conditions that may arise overtime due to continual use. From time
past and present, extracts and medicines from plants have made immense
contributions to the overall health and wellbeing of human beings (Anyanwu and
Nwosu, 2014). From time immemorial, the use of plants for the treatment of
diseases has been widely accepted due to their healing properties. According to
World Health Organization, many microorganisms are developing resistance to
several drugs which is posing severe threat to the general public health hence
requires actions across all sectors to curb this menace. Cyperusesculentus (tiger
nut) tuber oil meal-based diet prevents the accumulation of toxic material in the
body (Bamishaiyeet al., 2009). As a result of this, there is a growing interest in
plant’s usage for medicinal purposes due to the presence of several antibacterial
compounds present in them. Kareem et al. (2010) agrees that medicines developed
from plants are comparably safer than their synthetic counterparts thus rendering
enormous therapeutic benefits at an economical treatment rate. P. guineense
‘Uziza’ is one of such beneficial plants. The antimicrobial ability of plant extracts
and oils has established a platform for the processing and transformation of these
plant products into pharmaceuticals, preservatives and natural medicine. The use
of plants for the treatment of diseases has been widely accepted due to their
healing properties. According to World Health Organization, many
microorganisms are developing resistance to several drugs which is posing severe
threat to the general public health hence requires actions across all sectors to curb
this menace. As a result of this, there is a growing interest in plant’s usage for
medicinal purposes due to the presence of several antibacterial compounds present
in them. Kareem et al. (2010) agree that medicines developed from plants are
comparably safer than their synthetic counterparts thus rendering enormous
therapeutic benefits at an economical treatment rate.

Piper guineense ‘Uziza’ is of the family of Piperaceae. It is a West African species

of Piper; the spice derived from its dried fruit is known as West African pepper,
Ashanti pepper, Benin pepper, false cubeb, Guinea cubeb, uziza pepper or
(ambiguously) "Guinea pepper", and called locally kale, kukauabe, masoro, sasema
and sorowisa. It is a close relative of cubeb pepper and a relative of black pepper
and long pepper. Unlike cubeb pepper, which is large and spherical in shape,
Ashanti peppers are prolate spheroids, smaller and smoother than Cubeb pepper in
appearance and generally bear a reddish tinge. The stalks of Ashanti pepper berries
are also distinctly curved whilst those of cubeb pepper are completely straight
(Katzer, 2015). The plants that provide Ashanti pepper are vines that can grow up
to 20m in length, climbing up boles of trees by means of adventitious roots. These
are n ative to topical regions of Central and Western Africa and are semi-cultivated
in countries such as Nigeria where the leaves (known as uziza) are used as a
flavoring for stews and in making of soup. Research shows that Ashanti peppers
have preservative and anti-oxidant properties. In a comparison study of three
native West African peppers on the preservation of catfish, Ashanti peppers were
discovered to be the most effective as reported by Kiin-Kabariet al. (2011).

2.2 SCIENTIFIC CLASSIFICATION OF PIPER GUINEENSE

Kingdom: Plantae

Division: Magnoliophyta

Class: Magnoliopsida

Order: Piperales
Family: Piperaceae

Genus: Piper

Species: guineense

(Source: Schumach. and Thonn, 2005).

Piper guineense is of the family of Piperaceae and contains over 700 species all
over the world. It is a perennial woody climber that grows up to 10m or more in
height. It has alternate and simple leaves with a petiole 2-5cm long. It is a plant of
the wet tropics that requires heavy and well distributed rainfall and temperature.
The ideal soil for production is a well-drained alluvium with high humus content.
It can also be grown in red laterite soil (Okoye and Ebeledike, 2013). According to
Dada et al. (2013), Piper guineensis ‘Uziza’ is a local spice that comprises of
dillapiol, 5-8% of piperine, elemicine, 10% of myristicine and safrole and these
chemicals exhibit bactericidal and antimicrobial effects on certain microorganisms.
These effects have been associated with the presence of phytonutrients such as
flavonoids, alkaloids, glycosides, essential oils, tannins, saponins, peptides and
phenols in this spectacular plant. According to the research carried out by
Nwachukwuet al. (2010), on the uses of some medicinal plants, they found out that
Piper guineense ‘Uziza’ is suitable for treating Infertility in women and Low sperm
count in men.
A B

Fig 1 Essence of African by aditmicrosys 2021

A. Piper guineense ripe seed and leaf

B. Piper guineense unripe seed
C. Piper guineense dry seed

2.3 PLANT DISTRIBUTION OF P. GUINEENSE

The plant is native to tropical regions of central& Western Africa and is semi-
cultivated in countries like Nigeria where it is found commonly in the southern part
(Dalziel, 1955). It grows in evergreen rainforest, forest edges, usually in wet
places, gallery forest along rocky rivers of an elevation of 750-1,650m. There are
more than 700 species of this plant throughout tropical and subtropical regions of
the world (Anyanwu, and Nwosu, (2014). This plant has so many uses including
culinary, medicinal, cosmetic and insecticidal uses (Martins (2013), Dalziel, 1955).

2.4 BOTANICAL DESCRIPTION OF PIPER GUINEENSE

Piper guineense is a perennial climbing herb that is found growing mostly in

tropical regions such as Africa and other tropical zones of the world (Oyemitanet
al., 2015). The plant belong to the genus Piper of the Piperaceae family and from
genus piper. It is a West African spice plant commonly called Ashanti pepper
(Katzer and Gernot (2015). It is often cultivated for its fruits, leaves and roots, by
local farmers who earn a living by selling it to traditional healers and the populace.
It is an herb which could grow up to 20m tall, climbing by means of its
adventitious roots (Besonget al., 2016). It has various parts which are utilized for
different medicinal purposes. It is known as Uziza in Igbo and Iyere in Yoruba.
Other common names are Benin pepper, Guinea pepper and false cubeb, and
Ashanti pepper (Besong et al., 2016, Katzer and Gernot (2015). Spices generally
are parts of various plants cultivated for their aromatic pungent or otherwise
desirable substances. They consist of rhizomes, bulbs, flower bud, fruit, seed, and
leaves. They usually are categorized into tiny wild fruits, nuts, herbs, and leafy
vegetables. It consists of black berry fruits, seeds, leaves, rhizomes and flower
buds (Besonget al., 2016). The flowers which produces the fruits are greenish
yellow, the fruits when riped are reddish brown and occur in clusters and then
produces the seed. The fruit is small and round in shape and has a hot pungent taste
when chewed. The fruits are sold in markets as flavouring agents (Freieslebenet
al., 2015). The leaves are green and oval in shape and are used to prepare soup and
decoctions for the treatment of various ailments, West Africa. It has Piper has
numerous species which have been reported to have varying chemical constituents
based on their diverse nature (Besonget al., 2016, Oyemitanet al., 2015). Species
found in the same geographical region could have variations in their chemical
constituents. It is a vegetable plant that is used to enhance the taste of food. The
plants that provide Ashanti pepper are vines that grow up to 20m tall climbing up
bole of trees by means of adventitious roots. It is a perennial plant that is
characterized by heart-shaped leaves and oval, petiole, alternate, 12cm long. The
leaves which have a peppery taste, are pale greenish color when fresh and darker
green when frozen or dried. The inflorescence is a pedicel flower spike between 3
and 6cm long and the peduncle 5mm long. Flowers are greenish yellow and
arranged in a spiral along the spine (Agoha, 1974). The fruits of Piper guineense
occur in clusters, small, reddish or reddish brown when ripe and black when dry
(Okwute, 1992). The fruit is a drupe mesocarp or fleshy, oval, 5mm in diameter.
Medicinal plants are accessible medicinal resources used frequently by a large
population of people for the treatment and prevention of diseases. Ethno-medicinal
practice either encourages or discourages the use of certain natural remedies by
pregnant women and breast feeding mothers in many cultures. In Nigeria and some
other cultures the seeds of P. guineense are used as spices in diets by breast feeding
mothers to enhance lactation. The seeds of the plant are also used traditionally to
expel the placenta after childbirth. The plant is however believed by some to be
unsafe in pregnancy but there is no scientific evidence of this
2.5 PHYTOCHEMISTRY

The different parts of the plant have been characterized and their chemical
composition determined. They are used as therapeutic agents in minor ailments
(Okwu, 2001). Phytochemicals are not vitamins or minerals but are bioactive
compound found in plant foods that work with nutrient and dietary fibers to protect
against disease (Pal, and Verma, 2013). The presence of phytochemicals like
alkaloids in both the leaves and seed extracts of Piper guineense signified the
possession of medicinal properties within the plant. The flavonoids possess
antioxidant, anti-inflammatory, anti-tumor, anti-allergic and antiplatelet properties
(Okoye, and Ebeledike, (2013). They are also found to have cholesterol lowering
ability (Manta et al 2014). Alkaloids which are natural products present in Piper
guineense are made up of heterocyclic nitrogen that has anti-malarial,
antihypertensive, antiarrhythmic and anticancer properties (Heikenset al 1995).
Alkaloids are being used as CNS stimulant, powerful pain relievers, topical
anesthetic in ophthalmology among others (Ashok, and Upadhyaya, (2012).
Tannins are compounds with proline-rich proteins that help to inhibit the
absorption of iron when present in the gastrointestinal lumen thus reducing the
bioavailability of iron due to the presence of compounds that help in the treatment
of diseases like enteritis, gastritis, and esophagitis. Plants that contain tannins as
their primary component are astringent, thus very beneficial for the management of
diarrhea, dysentery, inflammation of the mucous membrane (Echo, et al 2012).
Saponins have anti-carcinogenic properties (Adesokan, and Akanji, (2010). and
may also play an important role in antimalarial activity of plants (Trease and Evans
2002). Piper guineense also contains cardiac glycosides in a significant amount and
cardiac glycosides are useful in the management of diseases associated with the
heart (Dada et al 2002). Piper guineense also contains dillapiol, 5-8% of piperine,
elemicine, 10% of myristicine and safrole and these chemicals exhibit bactericidal
and antimicrobial effects on some micro-organisms (KlinKabariet al 2011). Piper
guineense like other members of the piper family contains 5-8% of the chemical
“piperine” which gives them their “heat”. They also contain large amounts of beta-
carophyllene which is being investigated as an anti-inflammatory agent (Isaac,
(2012).

2.6 PHYTOCHEMICAL COMPOSITION OF THE LEAF OF PIPER

GUINEENSE

Class of Phytocompound Inference

Alkaloids +++

Flavonoids +++

Saponins ++

Tannin +++

Resins +

Acidic compound -

Steroids -

Terpenoids -

Essential oil ++

(Source: Okoye and Ebeledike, 2013)

Key

+++ = Present in high concentration

++ = Present in moderate concentration

+ = slightly or sparingly present

- = Absent.

The phytochemical analysis showed the present of alkaloids, flavonoids, saponins,

tannin, resins and essential oil. The presence of alkaloids signified the possession
of medicinal properties within the leaves. The flavonoids possess antioxidant
activity and equally antiinflammatory and antiviral infection activities etc.
Flavonoid was said to also have the ability to lower cholesterol level (Okoye and
Ebeledike, 2013). Also, related studies revealed that flavonoids apart from their
antioxidant protective effects, inhibits the initiation, promotion and progression of
tumors (Okwu, 2004).

2.7 NUTRITIONAL VALUE

Piper guineense have nutritional and non-nutritional factors which are responsible
for its aroma, flavor, and preservative properties (Nwankwoet al 2014). The
proximate analysis reveals that the plant contains crude protein, fat, carbohydrate,
vitamins and minerals (Nwankwoet al 2014). The essential oil content is high in
the range of 0.1 to 5% (Adesokan, and Akanji, 2010) while the peroxide value and
free fatty acid contents are generally low (Adesokan, and Akanji, 2010).
Nwankwoet al. 2014 and Igileet al 2013) also studied Uziza Leaf and found that in
addition to the above mentioned, it also contains a high amount of ash. This
implies high mineral content such as calcium, zinc, magnesium, copper and
potassium in the vegetable (Idris, et al 2011). The crude fiber content of Uziza leaf
was also found to be high, so consumption of this leaf could aid digestion,
absorption of water from the body, bulky stool and prevent constipation (Udousoro
and Ekanem 2013). The fat content of Uziza Leaf was found to be low so could be
employed as part of the weight loss regimen. Also, the protein content of this plant
makes it a good source of plant protein. The study also showed that P.guineense
has a high carbohydrate content, so its consumption could provide the body with
the energy needed for daily activities (Okonkwo and Ogu 2014). (Okonkwo and
Ogu 2014, Bell, 1987), also reported that Piper guineense contains vitamin C in
considerable amount and this aids the good health of teeth and gums and also
promotes healing. He reported that Piper guineense contains vitamin A and traces
of vitamin B1 and B2 which all are important for general good health. It also
contains vitamin E which plays a significant role as an antioxidant (Chibuzor, and
Assumpta, 2014).

2.8 TRADITIONAL MEDICINE IN WEST AFRICA OF PIPER

GUINEENSE

West Africa is a region that is richly endowed with medicinal plants which are
used for the treatment of infectious diseases (Oguntibeju, 2018). The population
which are mostly poor income earners uses medicinal plants such as Piper
guineense and Xylopiaaethiopica for the treatment of various diseases. The region
owing to its numerous medicinal plants is a good source of a diversity of bioactive
compounds which could be potential lead compounds for drug discovery
(Olorunnisola, et al.,2013). According to World Health organization (WHO),
greater number of people in the developing countries like in West Africa rely on
medicinal plants for the treatment of infectious diseases. The population often
patronize traditional medical practitioners (TMP) and herb sellers for their primary
health care needs. These TMPs collects medicinal plants which are easily
accessible from the wild or from their farms to prepare decoctions and various
herbal formulations for the treatment of diseases. Medicinal plants growing in
tropical rainforest zone are rich sources of bioactive compounds and as such the
ethnobotanical information of such plants are needed for an effective use as an
antimicrobial lead compounds. In West Africa, the importance of traditional
medicine cannot be overemphasized because of lack of modern facilities in the
rural areas (Ode et al., 2011).

2.9 TRADITIONAL AND MEDICINAL USES OF PIPER GUINEENSE OF

PIPER GUINEENSE

Piper guineense is useful in traditional medicine and its nutritive and medicinal
potentials have been outlined in various pharmaceutical studies (Obodozieet al.,
2010; Uhegbuet al., 2015; Ekundayoet al., 1988; Ene-Obonget al., 2018). The
fruits, seeds and leaves are often prepared in alcohol or as decoctions together with
other herbal formulations, and used in the treatment of various diseases (Besonget
al., 2016; Freieslebenet al., 2015). Ethno-pharmacologically, the roots, leaves,
fruits and seeds of Piper guineense are relevant herbal products in African
traditional medicine, most importantly in West Africa where it is administered to
nursing mothers to stimulate breast milk production and to aid the contraction of
the uterus after child birth (Okigbo and Igwe, 2007). The fruits and seed extracts
from this plant have been reported to be an important ingredient in the preparation
of Niprisan herbal formulation used to treat sickle-cell anaemia (Freieslebenet al.,
2015; Obodozieet al., 2010). Decoctions from the fruits and seeds are used in the
treatment of venereal diseases, rheumatism, gastrointestinal diseases and
respiratory diseases (Udoh, 1999). Piper guineense extracts are used in the
treatment of mental disorder and fever, and have been found to possess sedative
and muscle relaxant properties (Oyemitanet al., 2015). Extracts from Piper
guineense are used as aphrodisiac and it has been reported that Yaji soup, which is
often eaten as an aphrodisiac in most West African countries, is prepared from the
fruits of Piper guineense (Ibrahim et al., 2010; Asaseet al., 2012). Infusions and
decoctions from the fruits of P. guineense are administered orally to treat
bronchitis, cough, and intestinal diseases (Nwozoet al., 2017). Several studies have
shown that extracts of Piper guineense could lower lipid peroxidation thereby
preventing inflammation and oxidative damage and are also used in the treatment
of dysentery (Ogunniran, 2009; Nwozoet al., 2017). The leaves, fruits and whole
plant parts of P. guineense are used to prepare herbal formulation for the treatment
of asthma and its related symptoms (Gbekleyet al., 2017). A previous research
conducted on the ethnomedicinal uses of African medicinal plants has reported that
the leave extracts Piper guineense are used in the treatment of sexually transmitted
diseases (Ajibesinet al., 2011). The fruits and leaves of Piper guineense are ground
and soaked in alcohol with other herbs to prepare concoctions used for the
treatment of epilepsy, convulsion and malaria (Abilaet al., 1993; Umohet al.,
2013). Previous research has shown that Piper guineense extracts are useful in the
treatment of fertility disorder (Mbongueet al., 2005) and have been used to
stimulate sexual behavior in an adult male rat (Kamtchouinget al., 2002). It is also
used as a food preservative and as fragrance in perfume and cosmetic industries
(Nwozoet al., 2017).

2.10 BIOLOGICAL ACTIVITIES OF PIPER GUINEENSE

2.10.1 Antifungal Activities of Piper Guineense

The effect of Piper guineense extracts on various fungal pathogens has been
reported (Dada et al., 2013; Dzoyemet al., 2014; Abialaet al., 2015). Piper
guineense is effective against pathogenic fungal strains such as
Fusariumoxysporum, Fusariumsolani, Macrophominaphaseolina,
Fusariumverticillioides and Botryodiplodiatheobromae which are responsible for
rots in watermelon fruits and other vegetable fruits (Abialaet al., 2015). Piper
guineense extracts are often used as botanical fungicides by local farmers because
of high cost associated with synthetic fungicides. Evaluation of the antifungal
potentials of Piper guineense extracts along with other Cameroonian spices,
revealed that it is effective against Candida albicans, Candida parapsilosis,
Candida tropicalis, Candida krusei, Candida lusitaniae, Cryptococcus neoformans,
Candida guilliermondii and Candida glabrata (Dzoyemet al., 2014). However the
cold water maceration and hot water decoctions of the plant extracts do not have
activity against Candida albicans (Okigbo and Igwe, 2007). The activity of Piper
guineense on Candida albicans demonstrates that its extracts could be effectively
utilized in treatment of infections caused by opportunists Candida strains in
antifungal therapy. The antifungal efficacy of the fruit and leaf extracts of Piper
guineense on various Candida strains (Candida albicans, Candida tropicalis,
Candida parapsilosis, and Candida glabrata) supports the use of the plant in the
treatment of toilet infections and sexually transmitted diseases. Piper guineense
extracts were used as a natural insecticide to control maize weevil,
Sitophiluszeamais (Asawalam, 2006).

Furthermore, Dada et al., 2013, reported that ethanolic and hexane extracts of
Piper guineense inhibited the growth of Aspergillusflavus and Aspergillusniger.
The result shows that Piper guineense has interesting antifungal properties and can
be used as a good therapeutic agent in the discovery of a new antifungal drug.
Research has shown that fractions, natural compounds and extracts from Piper
guineense can be explored as antifungal agents in the prevention of skin infection
(Nganeet al., 2003). The efficacy of these fractions and extracts on panel of
organisms such as Microsporumgypseum, Trichophytonmentagrophytes,
Trichophytonrubrum, Aspergillusflavus, Scopulariopsisbrevicaulis, and
Cryptococcus neoformans shows that they can be effectively used to combat
diseases and infections caused by these filamentous fungi.

2.10.2 Antibacterial Activities of Piper Guineense

Bacterial infections often result to death if not well treated, and Piper guineense
has been widely reported to exhibit antibacterial properties (Okekeet al., 2001;
Konninget al., 2004; Anyanwu and Nwosu, 2014). Extracts and fractions from
various parts of this plant have antibacterial activity against Gram-positive and
Gram-negative bacterial strains (Tekwuet al., 2012; Dada et al., 2013). It has been
observed that the extraction solvent and method of extraction play a role in the
inhibitory activity of the extracts on the bacteria, and previous research have
shown that hexane, methanol and ethanol extracts are more effective than the water
fractions (Dada et al., 2013, Konninget al., 2004). However, essential oils from the
fruits of Piper guineense did not have activity against Escherichia coli, Salmonella
typhi, Klebsiella sp., and Pseudomonas aeruginosa (Olonisakinet al., 2006). Piper
guineense is effective against Mycobacterium tuberculosis which is a threat to
human life (Tekwuet al., 2012). The ethanol extracts of Piper guineense has been
reported to be effective against Bacillus subtilis, Escherichia coli, Staphylococcus
aureus, Salmonella typhi, Klebsiellapneumoniae, and Proteus vulgaris (Okekeet
al., 2001). The antibacterial evaluation of the plant extracts on Streptococcus
faecalis did not show remarkable activity (Okigbo and Igwe, 2007). The
antibacterial activities of Piper guineense on Acinetobacter spp., Bacillus cereus,
Escherichia coli, Salmonella spp., Shigelladysenteriae, Staphylococcus aureus by
Dada et al., 2013, revealed that bioactive compounds from the plant are good
antibacterial agent and could be a lead to the discovery of a new antibacterial drug.
Previous research has shown that ethanol extracts of Piper guineense has
remarkable activity against E. coli (Anyanwu and Nwosu, 2014), and these
researchers observed that the extracts inhibited the growth of B. subtilis, E. coli,
and S. aureus with the least activity observed in Piper aeruginosa.

2.10.3 Antimicrobial Activity

Anyanwu and Nwosu (2014). studied the antimicrobial activity of aqueous and
ethanolic extracts of Piper guineense leaves on some bacterial and fungal
organisms namely Staph.aureus, E.coli, P. aeroginosa, C. albicans using agar Well
diffusion method and minimum inhibitory concentration. The result of this study
showed that the leaf extracts of Piper guinnense inhibited the growth of all the
microbial isolate tested. This implies that the leaf extract has antimicrobial action.
The extracts had a broad spectrum activity as their actions were independent of
gram reaction. The higher antimicrobial activity of the extracts was observed
against E.coli followed by Staph. aureus. The antimicrobial effect of this plant can
be attributed to the phytochemicals present in it, they are rich in flavonoids,
tannins, saponins and alkaloids which have been found to have antimicrobial
properties. The study showed that the aqueous extract of Piper guineense has less
antimicrobial activity than the ethanolic extract against the isolates. This may be
due to insolubility of the active compounds in water or the presence of inhibitors of
antimicrobial components (Nwinyi, et al 2009).
2.10.4 Antioxidant effect

Omodamiro and Ekeleme 2013, (Etimet al 2013), studied the antioxidant activity
of Piper guineense. The result showed that the leaves of this plant exhibited free
radical scavenging effects. This could be attributed to the presence of phenolic
compounds in the plant which is a major group of compounds that act as primary
antioxidants or free radical scavengers. In another study, the seed extracts of Piper
guineense was found to rapidly scavenge nitric oxide in vitro at different intervals
(Ngane, et al 2003).

2.10.5 Aphrodisiac property

An aphrodisiac is a substance that when consumed, increases sexual behavior

(Kamthouinget al 2002). The activity of aqueous extract of Piper guineense on the
sexual behavior of male rats was studied by Kamtchouinget al.2002, using such
criteria as penile erection, copulating behavior and orientation activities towards
themselves and the female rats (anogenital sniffing, mounting). The rats received
122.5 mg/kg of the aqueous extract for 8 days and the results showed that this plant
extracts stimulated male sexual behavior, it significantly increased penile erection
and frequency of intromission and ejaculation. The plant extract was also found to
increase the orientation of males towards females by increasing mounting and
anogenital behavior. So this study showed that Piper guineense has aphrodisiac
property (Kamtchouinget al.2002).

2.10.6AMIDES AND ALKALOIDS IN PIPER GUINEENSE

Amides and alkaloids from Piper guineense and other Piper species are called
piperamides and these piperamide compounds have gained recognition in recent
years for their strong efficacy as antibacterial, antifungal, anti-inflammatory, and
antitumor agents in drug discovery (Adesinaet al., 2003; Nageswariet al., 2018;
Bezerraet al., 2005; Scott et al, 2005). Piperine, a potent alkaloid which has been
reported to have antimicrobial activity is contained in substantive amount in P.
guineense (Adesina et al., 2003, Scott et al., 2005). Other piperamide alkaloids
found in Piper guineense include piperylin, 4, 5dihyropiperlonguminin,
piperlonguminin , and 4, 5-dihydropiperine (Scott et al., 2005). The chemical
structure of the piperamide compounds found in Piper guineense are found in
publication (II).

2.10.7 Effect on Fertility

Memuduet al. 2015, studied the effect of crude extracts of dry fruits of Piper
guineense on male fertility parameters using adult Sprague dawley rats. 200mg/kg
of the extract was given to 2 groups of rats for four weeks and eight weeks
respectively. The result showed that the extract improved male reproductive
functions. It improved sperm motility, sperm function, testicular spermatogenesis
and weight and this could be attributed to its androgenic and aphrodisiac
properties. In essence Piper guineense should be recommended as a form of
treatment for male fertility problems especially those associated with hormonal
secretion. In another study, the effect of ethanol extract of Piper guineense was
investigated in mice. Findings revealed that the extract has a negative effect on
mice reproduction (Ekanem, et al 2017).

2.10.8 Antiparasitic effect

In this study, the effect of methanol extract of the seeds of Piper guineense on
some parasites was investigated and found to be active against goldfish
(Carassiusauratusauratus, Pisces cyprinidae) monogenean parasites (Ekanem, et al
2004)

2.10.9 Effect on Hematological parameters

Uhegbuet al.2015) investigated the effect of aqueous extract of P.guineense on
some hematological parameters of albino Wistarrats.The result showed that
intraperitoneal injection of Piper guineense seed extracts at a dose of 10mg/kg and
20mg/kg to the 2 groups respectively for 21 days significantly increased the
haemoglobin level, white blood cell and red blood cell counts of the animals. The
increase in white blood and red blood cell count may be attributed to the protein
content of the seed extract (Uhegbuet al.2015). Plant seeds are rich in
phytochemicals, vitamins and minerals, hence, may be the cause of increased
production of white and red blood cells in the animals (Joan and Michiho, (2013).

2.10.10 Sedative and anxiolytic effects

The aromatherapeutic potential of P.guineense essential oil was investigated in

mice via inhalation administration and the result showed significant sedative and
potent anxiolytic activities (Eweteet al 1996).

2.10.11 Insecticidal effect

Investigations with an ethanolic extract from five Nigerian plants showed that
extract of Piper guineense and C.odoratashow the best potential for development as
botanical insecticides (Sara et al 2015)

2.10.12 Pharmaco-enhancer effect

Piper guineense has been shown to inhibit specific liver cytochrome enzymes
(Soladoye et al 2010).

2.10.13 Anti-tumor effect

In this work, Piper guineense and other plant species were investigated for its anti-
tumor effect. Results showed that quite a number of plant parts including Piper
guineense seeds were found to be efficient in the management of cancer (Saba, and
Tomori, (2007).

2.10.14 contractile effect

The effect of ethanolic extraction of West African black pepper (Piper guineense)
on contractility of gastrointestinal smooth muscles of Guinea pig was carried out.
The results showed a significant contraction of GIT smooth muscles through the
cholinergic and histaminergic receptors (Nwachi, and Igbinobaro, 2012).

2.10.15 Antihyperlipidemic effect

Piper guineense extract was investigated for its hypolipidemic effect. The results
showed that it decreased the plasma cholesterol and triglycerides level in treated
rats (Gabriel et al 2012).

2.10.16 Anti-atherogenic effect

In a study aimed at evaluating the anti-atherosclerotic activities of Piper guineense

on atherogenic diet fed hamsters. The results suggested that Piper guineense had
significant antioxidant and antiatherogenic effects against atherogenic diet
intoxication (Nwozoet al 2012).

2.10.17 Hepatoprotective effect

Nwozoet al. 2012 studied the hepatoprotective effect of aqueous extract of Piper
guineense on ethanol-induced liver toxicity in male albino rats, in this experiment
chronic administration of ethanol to the animals for 21 days enhanced lipid
peroxidation with reduced levels of glutathione as well as reduced activity of
superoxide dismutase (SOD) and glutathione –S- transferase, serum triglyceride
levels, alanine transaminase (ALT), and aspartate transaminase (AST) activities
were also elevated but these was attenuated by co-administration of the extract of
Piper guineense at a dose of 100 or 200mg/kg. Administration of this plant during
ethanol exposure inhibited hepatic lipid peroxidation and ameliorated SOD and
AST activities and also restored GSH levels significantly. This study showed that
aqueous extract of Piper guineense has potent antioxidants which have
hepatoprotective properties.

2.10.18 Larvicidal property

A laboratory evaluation of ethanolic extracts of Piper guineese (seeds and leaves)

on mosquito larvae carried out by Ihemanmaetal, et al 2012) showed that the leaf
and seed extract of Piper guineense have Larvicidal effects at high concentration
on mosquito larvae. Piper guineense seeds and leaves have larvicidal effects on
mosquitoes with 90% and 80% mortality at 100mg/ml respectively. This could be
attributed to the phytochemicals present in the plant extracts. The more
concentrated the plant extract is, the more the larvicidal effect. Hence, this plant
could be utilized in the production of botanical larvicides which could be used as a
substitute for the synthetic ones as they are readily available, affordable, eco-
friendly and biodegradable (Ihemanmaetal, et al 2012).

2.11 HEMATOLOGICAL CHANGES IN PREGNANCY

Pregnancy is associated with various physiological changes, which tend to affect

most of the body system and some of these begin immediately after conception
continuing through delivery to the postpartum period in order to accommodate
both the maternal and fetal needs. The hematologic system adapts to make
provision for fetal hematopoiesis, ensuring adequate blood supply to the enlarged
uterus and its content thereby protecting both mother and fetus against the effects
of impaired venous return in both the supine and erect positions in addition to
safeguarding against bleeding at delivery. The maternal blood volume at term is
about 50% above the non-pregnant level in normal pregnant women, averaging
about 100ml/kg and the presence of a fetus is not needed for the hematological
changes as an increase in blood volume have been seen in women with
hydratidiform mole, (Pritchard 1965, Jansenet al 2005).

2.11.1 Major hematological changes seen can be broadly categorized under:

1) Physiologic Anemia

2) Leukocyte and Immunological function

3) Mild Thrombocytopenia

4) Coagulation and fibrinolysis

2.12 PHYSIOLOGIC ANEMIA

This is also referred to as dilutional anemia and it results from an increased plasma
volume to red blood cell ratio. It is detected between the late second and third
trimester with peak at 30-34weeks GA (Assali and Brinkman 1972). Its absence
has been associated with an increased tendency for stillbirths while its presence
conveys benefits related to decreased blood viscosity and reduced resistances to
blood flow culminating in increased perfusion of the placenta (Stephansson et al
2000). Maternal hemoglobin level tends to increase by the third day postpartum
and there is a diuresis-induced resolution of pregnancy-induced anemia by the 6th
week postpartum. The average hemoglobin level is about 12.5g/dl at term and
according to the World Health Organization (WHO), anemia is hemoglobin level
less than 11g/dl. It is classified as severe, when less than 7g/dl and hemoglobin
level less than 4g/dl is very severe necessitating urgent treatment to prevent the
development of congestive heart failure (World Health Organization (2001). The
United States Center for Disease Control (CDC) denotes anemia as hemoglobin
less than 10.5g/ dl in the 2nd trimester and less than 11g/dl in the 1st and 3rd
trimesters ((1998) Recommendations to prevent and control iron deficiency in the
United States). The frequent use of iron supplementation in pregnancy has been
associated with a reduction in maternal anemia at delivery and only about 6% of
pregnant women on Iron supplementation should present with a hemoglobin level
less than 11 g/dl (American College of Obstetricians and Gynecologists 2008). The
postpartum hematocrit is usually similar to the prelabor hematocrit by the 7th day
postpartum in the absence of excess blood loss or disease states such as
preeclampsia (Pritchard et al 1962).

2.13 PLASMA VOLUME CHANGES

The total plasma volume at term is 4700-5200mls due to a gain of about 1100-
1600mls and an average of 10-15% increase in plasma volume by 6-12 weeks
gestation with a progressive rapid rise till the 34th week (Pritchard 1965, Lund and
Donovan1967). The result is a plasma volume that is about 30-50% above that of
the non-pregnant, which later regresses to about 10-15% above that of the non-
pregnant by the 3rd week postpartum and is at normal non-pregnant level by the
6th weekPossible reasons that have been espoused for the increased plasma volume
include a response to the under-filled vascular system resulting from systemic
vasodilatation, reduced systemic vascular resistance and blood pressure as well as
increased cardiac output associated with sodium and water retention.

2.14 RED BLOOD CELL CHANGES

The erythrocyte volume is about 250-450mls higher at term in women on iron

supplements compared to those not supplemented and about 20-30% higher than
that in the non-pregnant state (Pritchard 1965). A 15-20% increase in red blood
cell mass is seen in non-iron supplemented pregnant women bearing in mind that
1ml of red blood cell mass has about 1.1mg of elemental iron (Hytten and Lind
1973). A progressive increase occurs from 8-10 weeks gestation till the end of
pregnancy but the average red blood cell life span is slightly reduced (Pritchard
1965). The average mean corpuscular volume is about 4 fl higher in healthy
pregnant women but decreases to an average of 80-84 fl in those not on Iron
supplements (Whittaker et al 1996). Erythropoietin levels are 50% higher due to
the higher metabolic oxygen requirement and this accounts for the moderate bone
marrow erythroid hyperplasia and elevated reticulocyte count. There is also an
increased transportation of oxygen across the placenta due to the combination of a
reduced maternal RBCs oxygen affinity from an elevated 2,3 Diphosphoglycerate
and a low maternal pCO2 (Madsen and Ditzel1984).

2.15 PHYSIOLOGIC EFFECTS OF HYPERVOLEMIA AND ANEMIA

A reduced resistance to flow from the decreased blood viscosity results in an

increased placental perfusion and reduced cardiac work. The increase in total blood
volume at term, which averages about 47% (+/- 15) above the non-pregnant serves
as a reserve against normal blood loss at delivery and peripartum hemorrhage
(Zeeman et al 2009). Blood loss at delivery range from 500mls at normal singleton
vaginal delivery, 1000mls at twin delivery and cesarean section to 1500mls at
cesarean hysterectomy (Pritchard 1965). The increased cardiac output to the
placenta leads to an increased supply of nutrients to the fetus and those to the
kidney and skin encourages excretion of maternal and fetal waste products and
control of maternal temperature while a reduction in large cerebral blood flow has
also been reported (Zeeman et al 2003). A lack of the hypervolemic changes and
physiologic hemodilution has been shown to be associated with an increased risk
of stillbirths especially small for gestational age associated stillbirths (Stephansson
et al 2000).

2.16 PREGNANCY REQUIREMENTS

The normal total body iron content in a non-pregnant woman is about 2g, which is
about half that of men while the iron stores are only about 300mg (Pritchard and
Mason1964). Iron supplementation is needed for the replacement of the
approximately 1000mg needed during pregnancy, about 500mg of which is
expended in the development of the maternal red blood cell mass, while the fetus
and placenta utilize about 300mg. Approximately 200mg of iron is excreted via the
skin, urine and gastrointestinal tract. It is absorbed in the duodenum in the ferrous
form and about 30mg of daily elemental Fe is needed as prophylaxis and more may
be required depending on the degree of maternal anemia. An average of 3-4mg of
iron needs to be absorbed per day during pregnancy although this is non-uniform
as about 6-7mg is needed daily in the late second to third trimester (Pritchard and
Scott 1970). Iron supplementation is usually given as 325mg of oral tablets, in the
form of ferrous sulfate with 65mg of elemental iron, ferrous gluconate with 35mg
and ferrous fumarate with 107mg of elemental Iron. In addition 400-800mcg of
Folic acid is recommended daily during pregnancy for the increased red blood cell
production while about 50-100mcg is needed pre-pregnancy.

2.17 CHRONIC SEVERE ANEMIA AND CONSEQUENCES

Possible sequelae of inadequate Iron stores and folate deficiency derived from
reduced intake and chronic hemolytic states, such as malaria and sickle cell disease
are as listed in box 1 while box 2 encompasses standard laboratory tests.

Box 1
A. Spontaneous abortion

B. Low birth weight infants

C. Prematurity

D. Reduced amniotic fluid volume

E. Fetal cerebral vasodilation

F. Non Reassuring Fetal heart tracing

G. Increased neonatal mortality and H. Increased maternal mortality

Box 2

Standard laboratory Evaluation

a) Complete Blood count

b) Reticulocyte count

c) Peripheral smear review

d) Serum Iron level

e) Total Iron-binding capacity

f) Serum Ferritin level and

g) Hemoglobin electrophoresis, if applicable

In areas where iron deficiency is prevalent, offspring’s have demonstrated a

positive correlation between prenatal iron/folic acid supplementation and
intellectual functioning but fetal red blood cell production is not impaired even in
the presence of severe maternal iron deficiency anemia (Christian et al 2010,
Gambling et al 2011).
2.18 LEUKOCYTE AND IMMUNOLOGICAL FUNCTION

A pregnancy related leukocytosis with an increase in neutrophils has been seen

from the second month of pregnancy with an upward trend observed as pregnancy
advances (Pramanik et al 2007).

Neutrophils range between 5-12,000/μl with up to 15,000/μl seen in the third

trimester and during labor and the puerperium; levels of 25,000/μl or more have
been attained (Taylor et al 1981). Leucocyte levels have been demonstrated to
correlate with cervical dilation and labor progress and should resolve by the 6th
day postpartum, if associated with pregnancy (Acker et al 1985). Lymphocytes and
monocytes do not demonstrate a significant change while eosinophils and
basophils might either be slightly decreased in number or remain the same.
Complement factors C3 and C4 are noted to be increased in the 2nd and 3rd
trimesters while C-reactive protein (CRP) level, a serum acute phase reactant has
also been found to be elevated during pregnancy with further increase noted during
labor (Richaniet al 2005, Anderson et al 2013). In order for the maternal body to
accept the fetal graft, there is various changes in the immunological function,
which generally leads to a decreased cell mediated immunity and an increased
humoral or antibody-mediated immunity (Redmanet al2014). The suppression of
T-helper 1 and T-cytotoxic 1 cells leads to decreased Interleukin -2, Tumor
necrosis factor, interferon gamma and alpha. Up-regulation of T-helper 2 and T-
cytotoxic 2 cell function do lead to an increased Interleukin 4, 6 and 13
(Michimataet al 2003). There is an increased ratio of CD8 to CD4 T-lymphocytes
and an increased peak level of Immunoglobulin A and G has also been reported
(Luppiet al 2002). The spleen is about 50% enlarged by term relative to its size in
the first trimester in a normal pregnancy and about 68% larger than its size in non-
pregnant controls,(Maymonet al 2007, Gayeret al 2012).

2.19 PLATELETS

Pregnancy is a relatively hypercoagulable state with an increased platelet activity

and consumption (Valeraet al 2010). This combined with the hemodilution state
leads to a mean platelet count that is slightly lower than that in the non-pregnant
state (Matthewset al 1990). An increased platelet production can be inferred from
the increase in circulating platelets width and volume. There is also an increase in
thromboxane A2 with an increased tendency for platelets aggregation in
pregnancy, (Hayashiet al 2002).

2.20 GESTATIONAL THROMBOCYTOPENIA

It is usually asymptomatic or mild, tends to present without a prior history and has
been described in about 5% of pregnancies. A platelet level between 70,000 and
150,000/mm3 was described by Burrows and Kelton in about 8% of pregnancies
but mostly resolves by 4 weeks postpartum (Burrows and Kelton 1993, Burrows
and Kelton 1988).

2.21 COAGULATION AND FIBRINOLYSIS

Pregnancy is regarded as a procoagulant state. The coagulation cascade is in an

activated state with about 20-200% increase in fibrinogen and factors II, VII, VIII,
X and XII while the level of factors XI and XIII decrease (Hellgren2003, Johnson
1997). Fibrinogen level increases from about 300mg/dl pre-pregnancy to as much
as 600mg/dl at term, averaging about 450mg/dl (Baker and Cunningham 1999).
There is an increased Von Willie brand factor, increased fibrinolytic inhibitors
such as plasminogen activator inhibitor type 1 and 2 (PAI-1 & 2) and thrombin
cleavage products (KU et al 2003, Hui et al 2012). An increased resistance to
activated protein C has been seen, while the anticoagulant protein S and
fibrinolysis are reduced. The free protein S antigen level is used for screening for
protein S in pregnancy(American College of Obstetricians and Gynecologists
Women’s Health Care Physicians 2010). Factors V, IX, antithrombin and protein C
are relatively unchanged while thrombin level progressively increases throughout
pregnancy (Hellgren2003, Goodwinet al 2002,McLeanet al 2012). The whole
blood clotting time and bleeding time are unchanged but there is about 20%
decrease in the prothrombin and plasma thromboplastin times. D-Dimer and the
erythrocyte sedimentation rate are elevated in pregnancy and this limits their
diagnostic usefulness during pregnancy. The hitherto D-dimer concentration
threshold of 0.50 mg/L cannot be used to rule out venous thromboembolism (VTE)
in the third trimester (Klineet al 2005). Overall,the coagulation profile change
indicates a substantially increased production in relation to the physiological
increase in plasma volume and tends to prevent postpartum hemorrhage due to
thrombus formation in addition to the myometrial contraction (Kennyet al 2014).
Adverse effects include an increased risk of venous thromboembolism (VTE) in
pregnancy and the postpartum period with VTE occurring in 0.7/1000 women. It is
about 3-4x higher in the puerperium than in the non-pregnant thereby making close
monitoring and the institution of preventive measures as well as early treatment
especially important in women with inherited thrombophilia (McColl et al 1997).
Persistent hypercoagulation has been demonstrated during the first 3 weeks of
delivery; however, pregnancy related hypercoagulable state should resolve by 6-8
weeks postpartum (Sahaet al 2009)
3.0 CHAPTER THREE: MATERIALS AND METHOD

3.1 MATERIALS

1. 35 Adult male wistar rat

2. Aqueous leaf extract of Piper guineense
3. Wired-guazed cage
4. 2ml Needle and Syringe
5. Cannula
6. EDTC
7. Capillary tube
8. Digital Weighing Balance
9. Distilled Water
10.Dissection Kits

3.3 PLANT (STEM BARK) MATERIALS

The dry seed of piper guineense will be obtained from ogbete main market and the
research will be carried out in the animal house of ESUT College of Medicine
Parklane, Enugu state, Nigeria.

3.4 PLANT EXTRACT PREPARATION

The dry seed of piper guineense will be obtained from ogbete main market and
will be milled into fine powder. The powder sample will be weighed and about
1000g of the sample will be soaked in 500ml of distilled water for 48hrs. After the
extraction, the sample will be filtered through whatman paper No 1 filter paper to
obtain a pure filtrate. The filtrate will be allowed to evaporate at 40 oc for 3hrs.
After which the residue will be stored in a refrigerator at 4 oc . The dosage will be
prepared fresh on the day of the experiment prior to administration to the rats by
oral administration.

3.5 EXPERIMENTAL ANIMALS

 A total of thirty- five rats weighing 150-200g, will be procured from

laboratory house of ESUT College of medicine.
 The rat will be housed under standard laboratory conditions of light,
temperature and humidity. The animal will be given free access to food and
tap water.

 The rats will be randomly grouped into five (5) experimental groups of
seven (7) each. Five female (5) and two male (2).

Group 1 – Normal control

Group 2 - first trimester

Group 3 – Second trimester

Group 4 – third trimester

Group 5 - delivery

3. 8 EXPERIMENTAL DESIGN

A total of twenty five rat weighing 150-250g, twenty five female and ten male. The
blood will be draw from the saphenous vein. The female and male rats will be mate
(5:2) and the presence of copulatory plug in female rats confirms pregnancy and
indicates gestation day (GD) 1. Pregnant animals will be separated and grouped
into five (5 animals each). Extract will dissolved in normal saline and group 2 will
be given extract for the first trimester of the extract, while the 3rd group received
extract for second trimester. Group 4 will be give extract for the third
trimesterPiper guineense of 100g (Umohetal.,2013). Group 5 will the
delivery, at the end of each stage, the blood will be drawn with capillary tubes
through saphenous vein. Body weights of pregnant animals will be recorded on
group1, group 2 and group 3.
Group Dosage duration
Number of Induction/extract
rats
Group 1 5 Food and water nil
Group 2 5 extract only 13mg First trimester
Group 3 5 extract only 20.7mg Second
trimester
Group 4 5 extract only 28mg Third
trimester
Group 5 5 extract only 31.8mg delivery