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PROJECT TOPIC AND MATERIAL ON PHYTOCHEMICAL AND BIOLOGICAL STUDIES OF ETHANOL EXTRACT OF CHROZOPHORA SENEGALENSIS

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ABSTRACT

The ethanol extract of root of Chrozophora senegalensis use in ethanomedicine to treat
diarrhea, rheumatism, taeniasis, stomach ache, inflammation and veneral diseases was subjected
to Phytochemical and Biological studies. Phytochemical analysis was carried out using standard
phytochemical procedures which revealed the presence of flavonoids, tannins, alkaloids,
saponins. Chromatographic separation of the ethylacetate fraction using silica gel led to the
isolation of three compounds, identified as 5-hydroxy – 2- (4-hydroxyphenol) – 4 – chromenone
– 7- glucose (Apigenin – 7- O- glucoside); 5,7 – dihydroxy-2- (4 hydroxyphenyl) – 4 –
chromenone – 3 – glucose (Aromadendrin -3-O-glucoside) and 2 – (3,4 – dihydroxy phenyl) –
3,5,7 – trihydroxy 4 H – chromen 4 – one (Quercetin) using spectrocopic analysis. The mean
number of writhing in acetic acid induced-writhing in mice is 18.8 with normal saline while at
dose of 600, 300 and 150mg/kg of ethanol extract has 1.3,1.7 and 3.7 respectively. The standard
drug (ketoprofen, 10mg/kg) has 0.6 mean number of writhing.The Anti-infammatory studies
showed that at 135min, the mean volume of paw oedema is 0.43 with normal saline which is
control while the standard which is ketoprofen (10mg/kg) has 0.29. The ethanol extract at dose
of 600, 300 and 150mg/kg has mean volume of 0.26,0.30 and 0.31 respectively.The extract in
analgesic and anti-inflammatory studies showed statistically significant activities when
compared to control at p less than 0.05 limits. Biological studies conducted on the ethyl acetate
include: acute toxicity using Lorke method and was found to be 2154.1 mg/kg. Analgesic using
acetic acid induced writhing, anti-inflammation using carrageenan induced paw oedema test in
rats. Antimicrobial was conducted using Agar well diffusion and tube dilution technique with
Escherichia coli, Staphylococcus aureaus, Pseudomonas aeruginosa, Bacillus subtilis, Candida
albica and Trichopyton mentaagrophyte as the test organisms with inhibition zones ranging
from 15-22mm at 20mg/ml while at concentration of 50mg/ml gave range of 19-25 mm. Ethyl
viii
acetate extract (20mg/ml) produce zones of inhibition which ranges from 15-22 mm but using
50mg/ml the zone of inhibition ranges between 19-25 mm. The n-butanol fraction also has
activity against the microorganisms tested at 20mg/ml with zone of inhibition between 15-23
mm and at 50mg/ml the range was found to be between 16-26mm. The MIC of ethanol extract,
ethylacetate and n-butanol soluble fraction ranges from 0.06 to 0.25mg/ml while the MBC
ranges from 0.25 to 1.0mg/ml. This study showed that the plant has analgesic, anti-inflammatory
and antimicrobial activity. The study validate the ethnomedicinal claim of the plant

TABLE OF CONTENTS

Title Page
Title page i
Declaration ii
Certification iii
Acknowledgement iv
Abstract v
Table of content vii
List of Tables. xi
List of Figures xiii
Abbreviations xiv

x
CHAPTER ONE: INTRODUCTION
1.0 Introduction 1 1.1 Statement of Research problem 11 1.2 Justification of Research 11 1.3 Aim 12
1.4 Objective 12
1.5 Hypothesis 13
CHAPTER TWO:
2.0 Literature Review of the plant 14
2.1 Ethnomedicinal uses of Chrozophora senegalensis 14
2.2 Phytochemistry and Pharmacological activities of Chrozophora senegalensis 15
CHAPTER THREE: 17
3.0 Materials and Methods 17
3.1 Materials 17
3.1.1 Chemicals and equipments 17
3.1.2 Identification, collection and preparation of plantmaterial 17
3.1.3 Standard drugs and sample used 17
3.1.4 Animal used 17
3.1.5 Microorganisms 18
3.2 Methods 18
xi
3.2.1 Extraction procedure 18
3.2.2 Preliminary Phytochemical screening 18
3.2.3 Thin layer Chromatography (TLC) 21
3.2.4 Column Chromatography (CC) 22
3.2.5 Spectroscopic Analysis of Isolated Compounds 24
3.3 Pharmacological Studies 24
3.3.1 Acute toxicity studies (LD50) in mice 24
3.3.2 Analgesic studies of ethanol extract 24
3.3.3 Effect of the ethanol extract on carrageenan induced oedema in rats 25
3.3.4 Determination of zone of inhibition 26
CHAPTER FOUR:
4.0 Result 27
4.1 Yield Of Crude extract and fraction 27
4.2 Result of Phytochemical Screening 29
4.3 Column chromatography of ethyl acetate fraction 30
4.4 TLC of isolated compound 31
4.5 Spectral Analysis of compound A4 32
4.5.1 UV spectroscopy of compound A4 31
4.5.2 Proton NMR of compound A4 32
4.5.3 13CNMR of compound A4 33
4.5.4 Mass spectrum of A4 34
xii
4.6 Spectral Analysis of Compound B1 36
4.6.1 UV Spectroscopy of compound B1 35
4.6.2 proton NMR of compound B1 36
4.6.3 13CNMR of compound B1 37
4.6.4 Mass spectroscopy of compound B1 38
4.7 Spectral Analysis of Compound C1 40
4.7.1 Proton NMR of compound C1 39
4.7.2 Mass spectra of compound C1 40
4.8 Pharmacological Studies 42
4.8.1 Acute Toxicity Studies of Ethanol extract of Chrozophora senegalensis root in mice 41
4.8.2 Analgesic Study of Ethanol Extract of Chrozophora senegalensis root 41
4.8.3 Anti-inflammatory Study of Ethanol Extract of Chrozophora senegalensis root 42
4.8.4 Anti microbial Study of Ethanol Extract, Ethyl acetate and n-butanol fractions 44
CHAPTER FIVE;
5.0 Discussion 47 CHAPTER SIX:
6.0 SUMMARY 52
6.1 CONCLUSION AND RECOMMENDATION 58 REFERENCES 58

CHAPTER ONE

1.0 INTRODUCTION
The term “traditional medicine” refers to ways of protecting and restoring health that
existed before the arrival of modern medicine (WHO,1996). As the term implies, these
approaches to health belong to the traditions of each country, and have been handed down
from generation to generation. In practice, the term “traditional medicine” refers to the
following components: acupuncture, traditional birth attendant, mental healers and herbal
medicine (WHO, 1996). Traditional medicines are relatively inexpensive, locally available
and are usually readily accepted by the local populace. It was reported that about 60 – 85
percent of the population in developing countries depend on traditional or indigenous form
of medicine (African Pharmacopoeia, 1985; Farnsworth, 1988). It is in recognition of these
facts that the WHO has been attempting to incorporate traditional medicine officially into
the health care systems of developing countries.
Traditional medicine is plagued with disadvantages that include lack of scientific proof of
efficacy of its remedies, lack of precise diagnosis of ailments by the traditional practitioners
and lack of precise dosage of their medication that are not standardized (D’Arcy, 1991; WHO
Drug Information, 1995). It is erroneously believed that herbal medicines as natural
products are safe and thus lack toxic effects (D’Arcy, 1991; WHO Drug Information, 1995).
Several reports and reviews have recently shown that these so-called safe herbal
preparations are either toxic or have several side effects (D’Arcy, 1991; WHO Drug
Information, 1995). Plants are regarded as nature’s bio-chemical factory because they serve
as source of various types of chemical compounds. Prominent among these compounds are
18
the plant secondary metabolites which are diverse complex molecules that are valued and
exploited by man for their pharmacological and other properties (Miller, 1973).
Over the years, the World health assembly has adopted a number of resolutions drawing
attention to the fact that most of the populations in various developing countries around
the world depends on traditional medicine for primary health care, that the work force
represented by practitioners of traditional medicine is a potentially important resource for
the delivery of health care and that medicinal plants are of great importance to the health
of individuals and communities.(WHO,1996)
Through its traditional medicine programmed, the World Health Organization (WHO)
supports, member States in their efforts to formulate national policies on traditional
medicine, to study the potential usefulness of traditional medicine including evaluation for
practices and examination of the safety and efficacy of remedies, to upgrade the knowledge
of traditional and modern health practitioners as well as to educate and inform the general
public about proven traditional health practices (WHO 1996), it is working closely with 19
collaborating centers in ten countries (Belgium, China Democratic People Republic of Korea,
Italy, and Japan, Romania, Sudan, United State of America and Vietnam).
A genuine interest in various traditional practices now exist among practitioners of modern
medicine and growing number of practitioners of traditional indigenous or alternative
system are beginning to accept and use some of the modern technology (WHO,1996). This
will help foster teamwork among all categories of health workers within the framework of
primary health care. The reason for the inclusion of traditional healers in primary health
care are manifold; the healers know the socio cultural background of the people; they are
highly respected and experienced in their work, economic considerations; the distance to be
19
covered in some countries; the strength of traditional belief the shortage of health
professionals, particularly in rural areas to name just a few. (WHO, 1996).
Herbal medicine is assuming greater importance in the primary health care of individuals
and communities in many developing countries and there has been an increase in
international trade in herbal medicines. However, in most countries the herbal medicines
market is not adequately regulated, and the products are therefore unregistered and often
not controlled by regulatory bodies. The establishment of regulation and registration
procedures has become a major concern in both developed and developing countries
(WHO, 1996).
Traditional medicine has maintained its popularity in a number of Asian countries, such as
China India, Japan and Pakistan. In China for example traditional medicines (herbal
preparations) account for 30% to 50% of the total medicinal consumptions. In 1993, the
total sales of herbal medicines amounted to more than US $ 2.5 billion. In Japan from 1974
to 1989, there was a 15- fold increase in Kampoh (Chinese method) medicinal preparations
in comparison with only 2.6 -fold increase in the sales of main stream pharmaceutical
products. The Japanese per capital consumption of herbal medicine appears to be the
highest in the world (WHO 1996).
There has also been a growing interest in traditional and alternative system of medicine in
many developed countries. One- third of American adult have used alternative treatment
and 60% of the public in the Netherlands and Belgium, and 74% in the United Kingdom are
in favor of complementary medicine being available within the framework of the National
Health Service. A survey among Member States of the European Union in 1991 identified
about 1400 herbal drugs used in the European Economic Community (WHO 1996).
20
Unregulated or inappropriate use of traditional medicines and practices can have negative
or dangerous effects. For instance, the herb Ephedra is traditionally used in China to treat
respiratory congestion. In the United States, the herb was marked as dietary aid, whose
over dosage led to at least a dozen of deaths, due to heart attacks and strokes. In addition
to patient safety issues, there is the risk that growing herbal market and its great
commercials benefit might pose a threat to biodiversity through the over harvesting of the
raw material for herbal medicines and other natural health care products. These practices, if
not controlled, may lead to the extinction of endangered species and the destruction of
natural habitats and resources (WHO, 2003).
Plants are indispensible sources of medicine since time immemorial. Studies on natural
product are aimed to determine medicinal values of plants by exploration of existing
scientific knowledge, traditional uses and discovery of potential chemotherapeutic agents.
Phytochemicals are used as templates for lead optimization programs, which are intended
to make safe and effective drugs (Balunas and Kingbour , 2005)
Plant secondary metabolites can be classified under groups of chemical compound such as
steroids, alkaloids, flavonoids, saponins, and tannins etc, many of which have been used by
man as drugs, dyes and many other purposes since prehistoric times. Some of these
secondary metabolites have been known to have a prominent effect on living cells. Some
have important therapeutic properties and have been utilized in the treatment and cure of
ailments. Examples of such therapeutically active plant constitutes are the Cinchona
alkaloids, cardiac glycosides from Digitalis species, Veratrum alkaloids and the anticancer
agents (Vincristine and Vinblastine) from Catharanthus roseue. Plants containing
therapeutically active constituents are known as medicinal plants. It has been recorded that
21
about 25 percent of the active components of currently prescribed medicines were first
identified in higher plants (Balandrin et al., 1985; Farnsworth, 1988).
There has been an increasing interest worldwide on therapeutic values of natural products.
Nature provides mankind with vast therapeutic flora with a wide variety of medicinal
potential. The revival of interest in plant derived drugs is mainly due to the current
widespread belief that “green medicine” is safe and more dependable than the costly
synthetic drugs many of which have adverse side effects. The need of the hour is to screen a
number of medicinal plants for promising biological activity. (Parekh and Chand. 2006)
Theories have been proposed to explain the acquisition of knowledge of medicinal plants.
One such theory states that man deliberately select specific plant materials for the
treatment of his ailments by rationalizing, while another theory states that the knowledge
of medicinal plants was gained by accident. However, it is known that early man could have
gain some scientific knowledge by watching the effect produced by medicinal plants on
domestic animals, on themselves and on their relatives. These explain why most hunters in
Africa and other parts of the world are known to be custodians of some effective traditional
herbal recipes (Sofowara,1982)
Medicinal plants constitute an effective resource for both traditional and modern
medicines,
and herbal medicine has been shown to have genuine utility. In Nigeria, many plants are
used
in traditional medicine as antimicrobial agents but only few are documented. Plants based
system of traditional medicine has continued to play an essential role in health care in
many
22
cultures. The increased use of plant derived products as alternatives to orthodox or
synthetic
drugs and increasing awareness of beneficial effects of natural products has resulted in
increased interest in alternative therapies. Extracts from plants have been utilised for their
antifungal, antiviral and antibacterial activities globally.(Odeh and Tor-Anyin. 2014).
Medicinal plants are the oldest known health care products. Their importance is still
growing although it varies depending on the ethnological, medical and historical background
of each country. Medicinal plants are also important for pharmacological research and drug
development, not only when plant constituents are used directly as therapeutic agents but
also when they are used as basic materials for the synthesis of drugs or as models for
pharmacologically active compounds.(WHO, 1996)
Legislative controls in respect of medicinal plants have not evolved around a structured
control model. There are different ways in which countries defined medicinal plant or herbs.
As a result different approaches have been adopted with regard to licensing, dispensing,
manufacturing and trading in order to ensure the safety, quality, and efficacy of medicinal
plant preparations.(WHO,1996)
WHO has compiled a list of medicinal plants which are widely used in primary health care, in
July 1996, a WHO scientific group involving 100 experts from various countries around the
world adopted the list which includes 28 monographs of 28 medicinal plants originally
prepared by the WHO collaborating centre for Traditional Medicine in Chicago, Illinois,
United States of America (WHO,1996).
23
Medicinal plants with anti-inflammatory properties are considerably employed in the
traditional treatment of several disorders of inflammation. The inflammatory response
involves a complex array of enzyme activation, mediator release, fluid extravasations, cell
migration, tissue breakdown and repair (Vane and Booting, 1987) which are aimed at host
defense and usually activated in most disease conditions. These different reactions in the
inflammatory response cascade are therapeutic targets which anti-inflammatory agents
including medicinal plants interfere with to suppress exacerbated inflammatory responses
usually invoked in such disorders as rheumatoid arthritis, in infection or injury. Inhibition of
the synthesis of pro-inflammatory prostaglandins is one of such therapeutic targets to which
some of the potent anti-inflammatory agents of clinical relevance (e.g. NSAIDs) owe their
activity. Several anti-inflammatory medicinal plants have also demonstrated the ability to
inhibit the synthesis of prostaglandins (Vane and Booting, 1987).
Phytochemical and pharmacological screenings are two important procedures used in the
study of plant constituents and their therapeutic or pharmacological effects. It is the
presence of bio-active plant secondary metabolites in plant that confers medicinal
properties; therefore that study of the properties of these compounds is very important in
establishing or confirming the claims of these plants (Farnsworth, 1966)
Phytochemcial screening is the application of simple chemical test to determine or detect
the presence of accumulated natural plant product or secondary metabolites of plant such
as alkaloids, tannins, steroids/terpenes, glycosides, flavonoids, carbohydrates, resin, gum
and volatile substance in the plant extract. These metabolites are responsible for
therapeutic properties of the pharmacologically active compounds which are referred to as
“active constituents” (Harbone, 1973).
24
One of the major set backs often encountered with drugs of plant source is that, most of the
active compounds occur in minute quantity which requires tedious extraction procedures. In
order to overcome this limitation the synthetic chemist takes the approach of determining
the structure activity relationship of the isolated, biologically active constituents to
synthesize simpler analogues, some of which might be more active and less toxic than the
natural products (Trease and Evans, 1983). A good example is the anti malarial drug quinine
(I), obtained from cinchona bark and its synthetic analogue, Chloroquine. Structural
modification of pure bioactive molecule of plant origin . morphine (II) isolated from
Papavera somniferum) has presented a major breakthrough (Trease and Evans, 1983).

(I)

(II)
Some of the most useful compounds obtained from plants are of such complex structure
that man is still unable to synthesize them from simpler compounds. And even where such
complex compounds have been synthesized, the synthetic procedures are often so difficult
and expensive that large –scale synthesis for commercial purpose is quite uneconomical.
(Bankole and Ogunkoya, 1977) Man still relies entirely on the natural source for the
commercial supply of some compounds. Typical of such cases are the antibiotic,
penicillin(III) and the anti-hypertensive agent reserpine (IV) Besides being the main source
of certain drugs, chemical compounds extracted from plant sources have also provide very
25
useful intermediates in the synthesis of other commercially important compounds.. For
example, diosgenin (V), obtained from some species of yam, has proved very useful in the
synthesis of sex hormones which help to cure certain diseases.

(III)
(IV)
(V)
Effect of plant extract
Some of the effects elaborated by extracts of plants used in traditional medicine include
antiviral, antitumor, antibacterial, antifungal, analgesic, anti-inflammatory, e.t.c (Sofowora,
1982).
Cyclooxygenase and lipoxygenase play an important role as inflammatory mediators. They
are involved in the release of arachidonic acid, which is a starting point for a general
26
inflammatory response. The exact mechanisms by which flavonoids inhibit these enzymes is
not clear. Quercetin , in particular, inhibit both cyclooxygenase and lipoxygenes activities,
thus diminishing the formation of these inflammatory metabolites (Robak, et al..,1996)
Species of Chrozophora genus (Euphobiaceae family) are distributed in West African and
Asia. These species are monoecious, shrubby herb and annual plants and their leaves, stems
and fruits besides the whole plant have been used in food and traditional medicine for the
treatment of infectious diseases. Many of these species showed high content of protein and
oil with high percentages of fatty acids. The extracts from different parts of the Chrozophora
plants showed the presence of diterpenoids, triterpenoids, flavonoids and chromone
glucosides, Alkaloids, coumarins, chromones xanthones, diterpenoids and phenylpropanoid
glycosides. However, there are many investigations showed this genus have antimicrobial,
anticancer and antioxidant, antiplasmodial, antidiabetes, and anthelmintic property agents,
and phytotoxic activity (Manar, A, et al., 2014).
Importance of flavonoids
Flavonoids are group of polyphenolic compounds, diverse in chemical structure and
characteristics, mainly found in fruits, vegetables and cereals. Therefore, flavonoids are part
of the human diet; over 4000 different flavonoids have been identified within the major
flavonoid classes which include flavonols, flavones, flavanones, catechins, antho-cyanidins,
isoflavones, dihydroflavonols, and chalcones.
Flavonoids are potentially antioxidants, metal chelators and inhibit lipid peroxidation
(Leopoldini et al., 2006).
27
Flavonoids are polyphenolic in nature therefore they can behave as antioxidants. These
properties depend on the number of hydroxyl groups present on the flavonoid structure. It
is said if the number of hydroxyl groups in flavonoids is higher; the antioxidant capacity is
also better (Cao et al., 1997).
Flavonoids are divided into eight different groups: flavonoids (quercetin, myricetin,
kaemferol and rutin) flavonones (taxifolin) flavones (luteolin and apigenin) isoflavones
(daidzein and genistein)Catechins, anthocyanidins, dihydroflanols and chalcones (Lu et al.,
2006). The function of flavonoid in flowers is to provide colors attractive to plant pollinators
(Harbone et al.,1992). In leaves, these compounds are increasingly believed to promoted
physiological survival of the plant, protecting it from, for example, fungal pathogens and U-V
radiation (Harbone et al., 1992).
1.1 Statement of Research problem
Disease related with pain and inflammations are the most important symptoms that bring
the patient to physician. Pain is frequently associated with inflammation. It is an ill-defined,
unpleasant sensation and may be caused by nociceptive or inflammatory agents. There are
several kinds of drugs currently available as anti-inflammatory and anti-nociceptive agents
to relieve pain but unfortunately, the uses of these drugs are not fully satisfactory in all
cases due to their adverse side effects, for example gastric lesions, ulcers and cardiac
abnormalities are caused by non-steroidal anti-inflammatory drugs and opiates (Allura et al.,
2009, Mapphsa et al., 2009). Therefore, development of newer, more powerful and
relatively cheap anti-inflammatory and analgesic drugs with lesser side effects is necessary
for human welfare.
28
1.2 Justification of Research
The plant Chrozophora senegalensis has been used in ethnomedicine for the treatment of
pain and inflammation, therefore this reseach work is desinged to authenticate this
traditional claim
1.3 Aim
The aim of this research work is to isolate some of the bioactive constituents and validate
the ethnomedical claim that Chrozophora senegalensis has Analgesic, Anti-inflammatory
and antimicrobial activities.
1.4 Objectives
The specific objectives are:
Collection and identification of the plant material followed by extraction of the root portion
of the plant using petroleum ether (defatting) than with ethanol.The crude extract will be
fractionated with ethyl acetate and n-Butanol. Preliminary phytochemical screening using
the crude extract and the fractions.Chromatographic separation of the ethyl acetate
fraction, Followed by spectroscopic analysis of the isolated compounds. Analgesic and anti
inflammatory investigation of crude extract and isolated compounds using acetic acid
induced whrithing and paw oedema respectively. Antimicrobial evaluation using clinical
isolates
1.5 Hypothesis
29
H1; The plant Chrozophora senegalensis contain secondary metabolite with Analgesic, Anti
inflammatory and Antimicrobial activity.
HO ; The plant Chrozophora senegalensis does contain secondary metabolite with
Analgesic, Anti-inflammatory and Antimicrobial activity.

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