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PHYTOCHEMICAL, ANTIMICROBIAL AND ANTIINFLAMMATORY STUDIES
ON CRATEVA RELIGIOSA LEAF EXTRACTS
BY
EZEALISIJI KENNETH MADUABUCHI
PG/M.PHARM/06/41036
BEING A PROJECT REPORT SUBMITTED TO THE DEPARTMENT OF
PHARMACEUTICAL AND MEDICINAL CHEMISTRY, FACULTY OF
PHARMACEUTICAL SCIENCES, UNIVERSITY OF NIGERIA, NSUKKA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
AWARD OF MASTER OF PHARMACY (M.PHARM) DEGREE
DR. AJALI, U. (RESEARCH SUPERVISOR)
DEPARTMENT OF PHARMACEUTICAL AND MEDICINAL
CHEMISTRY, FACULTY OF PHARMACEUTICAL SCIENCES,
UNIVERSITY OF NIGERIA, NSUKKA
FEBRUARY, 2009
CERTIFICATION
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This project report titled “Phytochemical, anti-microbial and anti-
inflammatory studies on Crateva religiosa leaf extracts” is hereby certified as
meeting the requirement for the award of Master of Pharmacy (M.Pharm)
degree in the Department of Pharmaceutical and Medicinal Chemistry,
Faculty of Pharmaceutical Sciences, University of Nigeria, Nsukka.
____________________ ____________________ STUDENT SUPERVISOR
__________________ ____________________ HEAD OF DEPARTMENT EXTERNAL EXAMINER
DEDICATION
To the Almighty God; my wife Mrs. Mary Ann;
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to my son Ikenna
and to my mother Mrs. Rachael Ezealisiji (Adadiora mma)
ACKNOWLEDGEMENT
Words are most inadequate to express my sincere gratitude to the
Almighty God, who provided me with this wonderful opportunity and the
required grace which saw me through this academic exercise. My profound
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gratitude goes to my research supervisor, Dr. Ajali, U. for his immeasurable
support, advice and guidance which helped to see me through this work.
I am ever grateful to Dr. Mba C.J the Head of Department of
Pharmaceutical and Medicinal Chemistry for being there for me during this
work.
My warmest thanks to my friends: Agbo M. and Okonkwo T. for their
support and Mr. Mbaoji E. of Department of Industrial Chemistry for good
and wonderful support during the course of this work.
My gratitude also goes to Mr. and Mrs. Emeka N. and Pharm. Ugoeze
K. for much cherished support and encouragement. My appreciation goes to
my darling wife Mary Ann Nwakaego and my son Ikenna for standing by me
all through the programme also my brothers and sisters and also to my cousin
Onyeka E. for his prayers.
Ezealisiji, Kenneth Maduabuchi
FEBRUARY, 2009
TABLE OF CONTENTS
Title page- - - - - - - - - - - i
Certification- - - - - - - - - - ii
Dedication- - - - - - - - - - -
iii
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Acknowledgement- - - - - - - - -
iv
Table of contents- - - - - - - - - - v
List of Tables- - - - - - - - - -
viii
List of Figures- - - - - - - - - -
ix
Abstract- - - - - - - - - - - x
CHAPTER ONE: GENERAL INTRODUCTION
1.1 Introduction- - - - - - - - - - 1
1.2 Crateva religiosa- - - - - - - - - 2
1.3 Otitis Media- - - - - - - - - - 3
1.3.1 Etiology- - - - - - - - - - 3
1.3.2 Pathophysiology- - - - - - - - - 4
1.3.3 Acute Otitis Media- - - - - - - - 4
1.3.4 Use of Anti-inflammatory Agents in the Treatment of
Otitis Media- - - - - - - - - 5
1.4 Origin of Antibiotics- - - - - - - - - 5
1.4.1 Classification and Mechanism of Action of Antibiotics- - - 5
1.5 Inflammation- - - - - - - - - - 6
1.5.1 Acute Inflammation- - - - - - - - 7
1.5.2 Chronic Inflammation- - - - - - - - 7
1.5.3 Pathophysiology of Inflammation- - - - - - 8
1.5.4 Cellular Events in Inflammation- - - - - - 8
1.5.5 Mediators of Inflammation- - - - - - - 8
1.5.6 Cell-derived Mediators- - - - - - - - 8
1.6 Anti-inflammatory Agents- - - - - - - 9
1.6.1 Steroidal Anti-inflammatory Agents- - - - - 9
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1.6.2 Non-steroidal Anti-inflammatory Agents- - - - -
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1.6.3 Anti-inflammatory Herbs- - - - - - - -
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1.7 The Aim and Objectives of the Study- - - - - -
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CHAPTER TWO: EXPERIMENTAL
2.1 Materials and Methods- - - - - - - -
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2.1.1 Apparatus and Instruments (Equipment)- - - - -
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2.1.2 Reagents and Solvents- - - - - - - -
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2.2 Plant Material- - - - - - - - - -
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2.2.2 Micro-organisms- - - - - - - - -
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2.3 Extraction- - - - - - - - - -
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2.4 Thin Layer Chromatography of Aqueous Methanol Soluble Fraction -
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2.4.1 Trial Analytical TLC- - - - - - - -
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2.4.2 Thin Layer Chromatography of the Diethylether Extract- - -
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2.5 Preparation of Stock Sample Solution in DMSO- - -
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2.6 Antimicrobial Test on Aqueous Methanol and Diethyl ether Extracts-
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2.7 Determination of Minimum Inhibitory Concentration (MIC)- -
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2.8 Media- - - - - - - - - - -
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2.8.1 Nutrient Agar- - - - - - - - -
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2.9 Preparative TLC of Extracts - - - - - - -
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2.10 Isolation of TLC Bands - - - - - - -
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2.11 Antimicrobial Screening of the TLC Bands - - - - -
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2.11.1 Microbiological Test on Ear Swab a Patient with Otitis Media- -
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2.12 Phytochemical Test on All the Extracts- - - - -
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2.13 UV Spectral Determination- - - - - - -
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2.14 Acute Toxicity (LD50) Test- - - - - - -
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2.14.1 Egg Albumin-induced Edema in Rats- - - - -
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2.15 Statistical Analysis- - - - - - - - -
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CHAPTER THREE: RESULTS
3.1 Extraction Yield- - - - - - - - -
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3.2 Phytochemical Analysis- - - - - - - -
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3.3 Chromatographic Separation- - - - - - -
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3.3.1 Chromatographic Fractionation of the Methanol Extract- - -
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3.4 Phytochemical Results of the TLC Fractions- - - - -
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3.5 The Rf Values of TLC Bands - - - - - - -
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3.6 Antimicrobial Activity- - - - - - - -
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3.6.1 Anti-microbial Screening of the Extracts- - - - -
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3.6.2 Anti-microbial Screening of the TLC Fractions- - - -
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3.6.3 Minimum Inhibitory Concentration (MIC) of Standard Drug
and TLC Fractions- - - - - - - -
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3.7 Result of Acute Toxicity Test- - - - - - -
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3.8 Result of Anti-inflammatory Analysis- - - - - -
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CHAPTER FOUR: DISCUSSION AND CONCLUSION- - -
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References- - - - - - - - - - -
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Appendices- - - - - - - - - - -
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LIST OF TABLES
Table 1: Examples of Some Anti-inflammatory Herbs- - - -
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Table 2: Classes of Phytocompounds present in the methanolic and
diethyl ether extracts and powdered leaves- - - - -
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Table 3: Trial TLC of methanol extract- - - - - -
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Table 4: Result of phytochemical analysis of the TLC bands- - -
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Table 5: The Rf values of the TLC bands- - - - - -
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Table 6: Culture and sensitivity analysis of ear swab of patient
with otitis media- - - - - - - - -
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Table 7: Result of the antimicrobial screening of methanolic and
diethyl ether extracts- - - - - - - -
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Table 8: Result of antimicrobial screening of TLC fractions- - -
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Table 9: Average Minimum Inhibitory Concentration of TLC bands- -
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Table 10: Values of X2, IZD, logarithms of concentration, MIC of TLC
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fractions with significant activities and standard drugs- - -
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Table 11: Result of acute toxicity (LD50) test (methanol extract) - -
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Table 12: Result of acute toxicity (LD50) test (diethyl ether extract)- -
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Table 13: Mean paw volume ± SEM (ml)- - - - - -
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Table 14: Percentage Inhibition of Acute Inflammation of the rat paw-
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LIST OF FIGURES
Fig. 1: Chemical Structures of Some Glucocorticoids- - -
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Fig. 2: Chemical Structures of Non-steroidal
Anti-inflammatory Drugs- - - - - -
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ABSTRACT
Crateva religiosa leaf extract squeezed with hands is used in traditional
medicine in the management of otitis media. In this work, antimicrobial and
anti-inflammatory activities of leaf extract of the plant with diethylether and
methanol were evaluated. 280 g of the leaf powder was extracted sequentially
with diethyl ether and methanol respectively. Trial thin layer chromatographic
studies on the methanol extract using silica gel coated glass plates and
different solvent systems gave (n-hexane:chloroform 1:2) as the best solvent
system which afforded 10 bands. This solvent system was used for preparative
TLC and the 10 bands were scraped and eluted to obtain 10 fractions.
Phytochemical analyses were carried out on the extracts and fractions. The
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extracts were evaluated for antimicrobial and anti-inflammatory activities
using agar diffusion method and egg-albumin-induced rat hind paw oedema
respectively. Gentamycin, chloramphenicol and aspirin were used as
standards. The fractions were only evaluated for antimicrobial activities.
Diethyl ether and methanol extracts gave yields of 25% and 27% respectively,
which were reasonable. Silica gel and n-hexane:chloroform (1:2) gave good
chromatographic resolution of the components for the methanol extract. The
extracts had antimicrobial activities which were comparable with those of the
standard drugs. The methanol extract was a better antimicrobial agent than the
diethyl ether extract. From the chromatographic and phytochemical studies,
the agents responsible for anti-microbial activity of the fractions were mainly
steroidal terpenoids. The diethyl ether extract had anti-inflammatory activity
that was concentration dependent and significant (P<0.001) at 100 mg/kg with
percentage inhibition of oedema value of 65.6. These results justified the use
of the extracts of Crateva religiosa leaf in the management of bacterial and
inflammatory related diseases such as otitis media in ethnomedicine but
further work is recommended for adequate standardization.
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CHAPTER ONE
GENERAL INTRODUCTION
1.1 Introduction
Herbal medicines could also be referred to as indigenous medicines,
phytomedicines or plant medicines. According to the guidelines issued by the
World Health Organization, herbal medicines should be regarded as, finished,
labeled medicinal products that contain an active ingredient aerial or
underground parts or other plant material or combination thereof, whether in
the crude state or as plant preparation (WHO 1991). Plant materials include
juices, gums, fatty oils, essential oils, and any other substances of this nature.
The problems of herbal medicines that require urgent attention are in
the area of processing and handling. The stability of the products and things
like the shelf life, solid dosage forms and liquid preparation are not well
elaborated. There are no dosage specifications (Ezeugwu, 2007).
Unregulated or inappropriate use of herbal medicines and practices can
have negative or dangerous effects. In some cases renal failure has been
reported with the use of herbal medicines, while in other cases there could be
hepatic necrosis. For instance, the herb “Ma Huang” (Ephedra) is traditionally
used in China to treat respiratory congestion. In the United States, the herb
was marketed as a dietary aid, whose over-dosage led to at least a dozen
deaths, heart attacks and strokes (Oliver Berver, 1986).
In Belgium, at least 70 people required renal transplant or dialysis for
interstitial fibrosis of the kidney due to herbal medicine (Oliver Berver, 1986).
Herbal medicines should be properly formulated. Herbal formulation refers to
the process of preparing or manufacturing a herbal dosage form with
ingredients possessing standardized properties specified by Pharmacopoea or
any other official drug compendium.
Seventy countries have national regulation on herbal medicines but the
legislative control of medicinal plants has not evolved around a structured
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model. This is because medicinal products or herbs are defined differently in
different countries and diverse approaches have been adopted with regard to
licensing, dispensing, manufacturing and trading. It is important for
government to formulate national policy and regulation for the proper use of
herbal medicine and its integration into national health care system in line
with the provisions of the WHO strategies on herbal medicines. Regulatory
mechanism must be established to control the safety and quality of products
and of herbal medicine practice.
The presence of medicinally active principle in herbs, have been known
from time immemorial. The ancient Egyptians, the Indians, Chinese and Asia
minor have used herbal medicine in one way or the other. Studies have shown
that these herbal remedies contain one medicinally active principle or the
other. Reserpine was extracted from Ralwolfia spp. Atropine and Hyoscine
were extracted from Atropa belladonna/hyosciamus spp respectively.
Morphine were obtained from opium puppy while penicillin was extracted
from the mould Penicillium notatum.
Advanced science has led us into the principle of structural
modification of the active agent such that any of these naturally occurring
agents can be modified structurally to give us newer analogues with better
potency, efficacy and less side effects. This is the reason why up till this day
the pharmaceutical chemist is still much interested in the search of “leads”
from herbs and natural products.
1.2 Crateva religiosa
Genus: Crateva
Botanical name: Crateva religiosa
Family: capparidaecceae
Specie: religiosa
Local name: ‘Akpu nta’
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Crateva religiosa is a small plant or shrub which grow wild in the
Easter and Western Nigeria and Ghana. It is indigenous to Eastern Nigeria. It
grows and measures up to 1.5 m tall, has trifoliate leaves with ash-green stem
which has a profound lenticel visible to the naked eye. The trifoliate leaves are
lathery and a cross section of the leaf shows prominent secondary metabolites
under the microscope.
The leaves of Crateva religiosa are used locally by the native of
Eastern Nigeria, especially the Okigwe region as medicament for the
treatment of ear ache or exudative ear infection.
The leaves when squeezed produce the leaf extract, two or four drops of
this leaf extract are dropped into the affected ear, though it is painful, it
produces total cure within four to five days of treatment.
Little or no work has been done on Crateva religiosa.
1.3 Otitis Media
Otitis media is an ear infection. Three out of four children experienced
otitis media by the time they are 3 years old (Berman, 1995). There are two
main types. The first type is called acute otitis media (AOM). This means that
parts of the ear are infected and swollen. It also means that fluid and mucus
are trapped inside the ear. Acute otitis media can be painful.
The second type is called otitis media with effusion (fluid) or OME.
This means, fluid and mucus stay trapped in the ear after the infection is over.
Otitis media with effusion makes it harder for the ear to fight new infections.
The fluid can also affect hearing.
1.3.1 Etiology
Otitis media usually happen when viruses and/or bacteria get inside the
ear and cause infection. It often happens as a result of another illness, such as
cold. It is harder for children to fight illness than it is for adults, so children
develop ear infections more often (Del Mar et al., 1997). Some researchers
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believe that other factors, such as being around cigarette smoke, can
contribute to ear infection.
Streptococcus pneumoniae spp and Staphylococcus aureus have been
implicated in acute otitis media. Also Haemophilus influenza and Morexalla
catarralis are involved.
1.3.2 Pathophysiology
When the ears are infected, the eustachian tubes become inflamed and
swollen. The adenoids can also become infected.
Swollen and inflamed eustachian tubes often get clogged with fluid and
mucus from a cold. If the fluids plug the openings of the eustachian tubes, air
and fluids get trapped inside the ear. These tubes are smaller and straighter in
children than in adults.
This makes it harder for fluids to drain out of the ear and is one reason
that children are more susceptible.
Also the adenoids are located in the throat, near the eustachian tubes;
adenoids can become infected and swollen. They can also block the openings
of the Eustachian tubes, trapping air and fluid.
1.3.3 Acute Otitis Media: Treatment in an Era of Increasing
Antibiotic Resistance
Antibiotic resistance is increasing among the pathogens that commonly
cause acute otitis media. This development may merit changes in the
traditional antibiotic treatment of acute otitis media. Amoxicillin remains the
antibiotic of first choice, although a higher dosage (80 mg per kg per day)
may be indicated to ensure eradication of resistant Streptococcus pneumonia.
Also, gentamycin and chloramphenicol could be used in the treatment. Oral
cefuroxime or amoxicillin- clavulanate and intramuscular ceftriaxone are
suggested second choice drugs for treatment failure. Compliance with
antibiotic regimens is enhanced by selecting agents that require less frequent
dosing (such as one or two times daily).
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1.3.4 Use of Anti-inflammatory Agents in the Treatment of Otitis Media
Since this condition, otitis media involve painful swelling of the
adenoids, management with anti inflammatory agent could be beneficial.
The anti inflammatory drug of choice is ibuprofen, though acetaminophen
(paracetamol) could also be useful.
These set of drugs tend to reduce the rate of inflammation and hence the
pain associated with otitis media.
1.4 Origin of Antibiotic
Historical aspects: The concept that substance derived from one living
organism may kill another organism (antibiosis) is almost as old as the science
of meteorology. The Chinese were aware, over 2500 years ago of therapeutic
property of moldy curd of soybeans applied to carbuncle and boils; they now
used this as standard treatment in such disorder.
Definition: Antibiotics are chemical substances produced by various species
of microorganisms that suppress the growth of other micro-organisms and
may eventually destroy them. They differ markedly in physical, chemical and
pharmacological properties.
1.4.1 Classification and Mechanism of Action
Antibiotics are classified based on chemical structure and mechanism of
action.
(i) Agents that inhibit synthesis of active enzymes or that disrupt
bacterial cell walls to cause loss of viability and cell lysis; these include
the penicillins and cephalosporins
O ═ C N CH COOH
Penicillin G
CH2 C NH CH CH C
O ││
S CH3
CH3
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(ii) Agents that act directly on the cell membrane of the micro organism
affecting permeability and leading to leakage of intercellular compound
e.g. nystatin and detergents.
(iii) Agents that affect the function of bacterial ribosomes to cause
reversible inhibition of protein synthesis, they are bacteriostatic in
nature
E.g. chloramphenicol
(iv) Agents that bind to the 30s ribosomal subunit and alter protein synthesis
which eventually lead to cell death.
Eg aminoglycosides (gentamycin)
(v) Agents that affect nucleic acid metabolism such as rifampicin.
(vi) The antimetabolites including Trimethoprim and sulphonamides
(vii) The nucleic acid analogues such as vidarabine and acyclovir
1.5 Inflammation
Inflammation is caused by a variety of stimuli including physical
damage, UV irradiation, microbial invasion and immune reactions. The
classical key features of inflammation are redness, warmth, swelling and pain.
Inflammation cascades can lead to the development of diseases such as
chronic asthma, rheumatoid arthritis, multiple sclerosis, inflammatory bowel
disease and psoriasis. Many of these diseases are debilitating and are
becoming increasingly common in our aging society.
Out of these, rheumatoid arthritis and osteo-arthritis are the major
inflammatory diseases affecting people worldwide. Rheumatoid arthritis is an
inflammatory condition that usually affects multiple joints. It affects 0.3-10%
of the general population and is more prevalent among women in developed
─CH CH─NH─C─CHCl2
O ││
OH │
CH2OH │
O2N─
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countries. Osteo-arthritis which is characterized by loss of joint cartilage that
leads to pain and loss of function primarily in the knees and hips, affects 9.6%
of men and women aged 60 years. Increasing life expectancy and aging
populations are expected to make osteo-arthritis the fourth leading cause of
disability by the year 2020 (Labenti et al., 1992). The plant-based medicine
initially dispensed in the form of crude drugs such as tinctures, teas, poultices,
powders and other herbal formulations, now serve as the basis of novel drug
discovery (Gautam and Jackak, 2007).
The bark and the leaves of Cinnamomum species (family Lauraceae)
are commonly used as spices in home kitchens and their distilled essential oils
or synthetic analogs are used as flavoring agents.
1.5.1 Acute Inflammation
This is initial response to tissue injury, which is mediated by the release
of autocoids and usually precedes the development of immune response. The
degree and time of inflammatory response depends on the extent of damage
caused on the tissue (Katzung et al., 1998). Generally, acute inflammation is
a reversible process (Djukanovic et al., 1990). However, there may be serious
problems when organ function is compromised, for example, in meningitis,
hepatitis and asthma. The inflammatory reactions also usually soon subside
and the inflammation is unlikely to cause permanent damage if treated
promptly.
1.5.2 Chronic Inflammation
Inflammation termed chronic may start with relatively rapid onset or
slow insidious and even in unnoticed manner and tends to persist for several
weeks, months or years. It is a vague and indefinite terminology and result
when the injuring agent persists in the lesion. Usually, in such a situation, the
host tissue responds in a manner that is not sufficient to overcome completely
the continuing effect of the injuring agent. Chronic inflammation may arise in
various organs following acute inflammation. It is of longer duration and
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associated histologically with the presence of lymphocytes and macrophages,
the proliferation of blood vessels, fibrosin and tissue necrosis (Cotran et al.,
1999). Chronic inflammation is characterized by infilteration with
mononuclear cell, which include macrophages, lymphocytes and plasma cells.
1.5.3 Pathophysiology of Inflammation
The complex sequence of events that characterize inflammatory
reaction can be broadly categorized into vascular and cellular processes.
These events involved in inflammation are separated into three distinct stages:
vaso-constriction, vasodilation and vascular permeability. These occur in
response to the initial trauma, a neurologic response results in
vasoconstriction of blood vessels leading to the injured tissue with the
resultant decrease in blood flow (Cotran et al., 1999), into the area, this is
followed gradually by vasodilation and vascular permeability.
1.5.4 Cellular Events
Cellular events in response to inflammatory stimuli involve the
recruitment of leucocytes and tissue macrophages to sites of injury. Activation
of macrophages by products of infection and inflammation results in a rapid
enlargement of the cells. The enlarged cells start their phagocytic actions
minutes after initiation of inflammation (Guyton and Hall, 2000). This is
followed by leucocyte migration towards the site of injury.
1.5.5 Mediators of Inflammation
The events involved in the inflammatory response are mediated by
biologically active molecules broadly classified into cell-derived mediators
and plasma proteases.
1.5.6 Cell-derived Mediators
These include: histamine, serotonin, lysosomal enzymes,
prostaglandins, leukotrenes, platelets activation factors (PAF), reactive
oxygen species, nitric oxide and cytokines. These mediators are derived from
cells such as neutrophils, monocytes/macrophages, platelets, mast cells, etc.
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Some of them such as histamine, serotonins and lysosomal enzymes are
preformed and released on situation while others are newly synthesized upon
induction by appropriate inflammatory stimuli.
1.6 Anti-inflammatory Agent
Anti-inflammatory effect refers to the property of a substance or treatment
that reduces inflammation. Anti-inflammatory drugs make up about half of
analgesics, remedying pain by reducing inflammation.
1.6.1 Steroidal anti-inflammatory agents
Many steroids, specially glucocorticoids, reduce inflammation or
swellilng by binding to cortisol receptors. These drugs are often referred to as
corticosteroids.
The Corticosteroids
Interest in the chemistry and biological properties of these steroids was
aroused because of the discovery of the anti-inflammatory activity of
cortisone, and its therapeutic usefulness in the treatment of rheumatoid
arthritis and other disease where the symptoms can be attributed to
inflammatory reactions. They are also good for allergic manifestation.
Examples of glucocorticoids include: cortisol, prednisone and prednisolone
C O
O
O
CH2OH
/////OH
Cortisol (Natural)
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These analogues are more potent antirheumatic and anti-allergic agent
than the parent compounds and produce fewer undesirable side effects.
1.6.2 Non-steroidal Anti-inflammatory Agents
Non-steroidal anti-inflammatory drugs (NSAIDS) alleviate pain by
counteracting the cyclo-oxygenase (COX) enzyme. On its own COX enzyme
synthesizes prostaglandins, creating inflammation. The NSAIDS prevent the
prostaglandins from being synthesized, thus reducing or eliminating the pain.
Some common examples of NSAIDS are: aspirin, ibuprofen and naproxen.
The newer specific COX-inhibitors although probably sharing a similar mode
of action are not classified together with the traditional NSAIDS.
In addition to medicinal drugs, many herbs have anti-inflammatory
qualities, including hyssop, ginger, Turmeric, Arrica montara which contain
helenalin, a sesquiterpene lactone, and willow bark, which contains salicylic
acid, a substance related to the active ingredient in aspirin. Cannabichromene,
one of the many cannabinoids present in the cannabis plant, has been shown to
reduce inflammation. On the other hand, there are analgesics which are
Prednisone
C O
O
O
CH2OH
/////OH
Prednisolone
C O
O
O
CH2OH
/////OH
Fig 1: Chemical Structures of Some Glucocorticoids
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commonly associated with anti-inflammatory drugs but which have no anti-
inflammatory effects. An example is paracetamol, called acetaminophen in the
US and sold under the brand name Tylenol.
1.6.3 Anti-inflammatory Herbs
Among the best-known herbs with anti-inflammatory properties are
Matricaria recutita, Curcuma longa, Zingiber officinale, Glycyrrhza glabra,
Salix abba, and Amica Montana. Some commercially available formulae are
listed as shown in table 1
(http://www.who.int/mediacentre/factsheets/Fs134/en/.).
CHCOOH
CH3
(CH3)2CHCH2
Ibuprofen
CHCOOH
CH3
CH3O
Naproxen
CH2COH
O CH3O
N CH3
C
O
Cl
Indomethacin
OCOCH3
COOH
Aspirin
Fig. 2: Chemical Structures of Non-steroidal Anti-inflammatory Drugs
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Table 1: Examples of Some Anti-inflammatory Herbs
Herb Name Preparation
Arnica Arnica flowers, liquid extract, Arnica flowers oil.
Bromelain Maetizyme
Echinacea Echinacea-C
German chamomile Matricaria recutita liquid extract
Ginger Ginger liquid extract
Licorice Glycyrrhiza glabra fluid extract
Tumeric Tumeric liquid extract
Witch Hazel Witch Hazel liquid extract
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1.7 The Aim and Objectives of Study
It is very important to provide readily available, cheap and affordable
drugs to everyone who is in need of medication at the right time.
The aim and objectives of this research work include:
(a) To investigate the antimicrobial and anti-inflammatory activity of the
plant leaf extract of Crateva religiosa.
(b) To establish the phytochemical constituents responsible for the
observed activity via activity guided study.
(c) And finally to semi-characterise the resultant constituents.
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CHAPTER TWO
EXPERIMENTAL
2.1 Materials and Methods
2.1.1 Equipment (Apparatus and Instruments)
The instruments used included soxhlet extractor, rotary-vacuum
evaporator type 349/2 (Buchi, Germany), water bath, test tubes, conical flasks,
measuring cylinder, beakers, pipettes, funnels, filter paper, KG.D-7470 mass
balance (Sauter, Germany), silica gel, spreader, chromatoplates, petri dishes,
cultures of test organisms, sterile cork borers, inoculating loop, incubator,
autoclave, indelible marker, UV2102 PC Spectrophotometer (UNICO,
Germany), Plethysmometer (Thomas Wiley, Germany), syringes.
2.1.2 Reagents and Solvents
The reagents were sourced commercially, and included methanol,
ethylacetate, diethylether, n-hexane, and chloroform (Sigma Aldrich,
Germany) while acetic acid, dimethylsulphoxide (DMSO), methyl-ethyl-
ketone (MEK) (BDH, Germany) and tetraoxosulphate (vi) acid, (by May and
Baker Ltd, Dageha, England), distilled water. All solvents were of analytical
grade.
Other reagents used included: Acetic anhydride, Acetone, Ethanol, Sodium
Hydroxide, Hydrochloric acid, Mayer’s reagent, Wagner’s reagent,
Dragendoff’s and Hager’s reagents, Molisch’s reagent, Million’s reagent,
nutrient agar, Fresh egg albumin, Tween 80, Aspirin (Emzor ®), Diclofenac
(Hovid ®).
2.2 Plant Material
The fresh leaves of Crateva religiosa were harvested from Okigwe, Imo
State of Nigeria in February, 2008. They were authenticated by Mr. Ozioko of
Bioresource Development and Conservation Programme (BDCP) Centre, No.
23 Aku Road, Nsukka. The leaves were dried at room temperature in the
27
laboratory, pulverized to coarse powder with a mechanical grinder and stored
in sealed transparent glass container.
2.2.1 Animals
Adult Swiss albino mice (15-45 g) and rats (110-179 g) of either sex
were obtained from the animal house of the Faculty of Veterinary Medicine,
University of Nigeria, Nsukka. The animals were housed in plastic cages
(mice) and stainless steel cages (rats) under standard conditions and fed with
pellet animal feed and potable water ad libitum. They were acclimatized in the
laboratory for 7days before the start of the experiments.
2.2.3 Micro-organisms
The micro-organisms used were clinical isolates maintained in the
Pharmaceutical Microbial Laboratory of the Department of Pharmaceutics,
University of Nigeria, Nsukka. They incuded: Bacillus subtilis,
Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae,
Salmonella typhi, Escherichia coli and Candida albicans.
2.3 Extraction
280 g of dry powdered leaves of Crateva religiosa were extracted
sequentially on different quantities of plant material with diethylether and
95% methanol using soxhlet extractor.
The extracts were concentrated in vacuo using rotary evaporator to
obtain a deep brown reddish sticky solid for methanol and dark-green solid for
diethylether.
2.4 Thin Layer Chromatography of the Aqueous Methanol Extract
2.4.1 Trial Analytical TLC
Adsorbents used: fluorescent silica gel pre-coated plate. 20cm x 20cm.
(polygram® SIL G/UV254)
Procedure
A solution of the aqueous methanol extract was streaked on two
different locations using capillary a tube and the plate was allowed to dry. The
28
plate was immersed in a chromatographic tank containing n-hexane and
chloroform (1:2 v/v). The plate was developed and when the solvent front
reached a predetermined distance, the development was stopped and the plate
removed from the tank, dried and the bands visualized with either iodine or
ultraviolet light. Other solvent systems were equally tried. The best system
was however, n-hexane; chloroform 1:2 (v/v).
2.4.2 Thin Layer Chromatography of the Diethylether Extract
The above procedure was repeated for the diethyl ether extract for
which the best solvent was n-hexane: chloroform (2:1 v/v).
2.5 Preparation of Stock Sample Solution in DMSO, (CH3)2 S0
(i) 20 ml of DMSO was added and used to dissolve 50 g each of the
extracts and the required volume made up with water to obtain 10 mg/ml.
Other concentrations were obtained through serial dilutions.
(ii) Standard Drugs:
Weight of Drug Equivalent to 20 mg of each of the standard drugs was
dissolved in 2 ml of DMSO, providing a concentration of 10 mg/ml of
solution.
2.6 Antimicrobial Test of the Aqueous Methanol and Diethyl ether
Extracts
10 mg/ml of the extract was prepared using dimethylsulphoxide
(DMSO). The solutions were used directly without further dilution. The
method employed was agar diffusion
Procedure: A 0.1 ml of each test organism prepared by culture and re-
isolation, was transferred to its corresponding plates previously labeled. One
bottle of molten agar was opened and poured into the plate and properly
mixed to ensure a homogenous mixture. These were aseptically done to avoid
contamination. They were allowed to cool before cutting holes using sterile
cork–borer provided, after dividing the plate on the back of the bottom plate
using a permanent-marker.
29
Using the sterile pipette with the tip attached, two drops were allowed
into its corresponding hole previously cut in the agar plate and allowed 30
minutes pre–diffusion time after which the plates were incubated at 370C for
bacteria and 250C for fungi for 48 hours. The same procedure was repeated for
the control drugs.
2.7 Determination of Minimum Inhibition Concentration (MIC)
From stock solution of extracts and standard drugs, two fold serial
dilutions of different concentrations were prepared and the organism seeded
into the petri dishes prepared in duplicate. After gelling, holes were cut in the
agar and marked. The holes were made to contain 2 drops. A pre–diffusion
time of 30 minutes was allowed and incubated at 370C for 24 hours.
0.1 mg/ml of standard antibiotics chloramphenicol and gentamycin
were used. Their MIC were determined and recorded.
2.8 Media
2.8.1 Nutrient Agar
The nutrient agar was prepared by suspending 28 g of the powder in 1
liter of distilled H2O and allowed to soak for 10 minutes. It was sterilized at
1210C (15 Ib/sq in) for 15 minutes.
2.9 Preparative TLC of Extracts
Preparation and Activation
Chromatographic plates each measuring 20 cm by 20 cm by 0.05 cm
thickness were prepared by mixing silica gel with distilled water in the ratio of
1:2 (w/v). The slurry was then poured into a Uniplan spreader which was set
at a thickness of 0.5 mm. The plates were then dried at room temperature and
activated at 1000C for one hour before use.
Spotting, Development and Detection
The aqueous methanol extract was dissolved in aqueous methanol and
subsequently, a streak of spots was made horizontally 5 cm up the plate.
30
The spots were allowed to dry and reloaded three more times. The plate
was dipped into a chromatographic tank of appropriate size containing n-
hexane, chloroform mixture (1:2 v/v). After development, the plates were
removed from the tank, air –dried and appropriately viewed under UV light.
Ten bands were revealed.
2.10 Isolation of TLC Bands
From the developed plates, the bands were carefully and separately
scraped and each fraction eluted with methanol.
Procedure
The scrapped bands were separately soaked in enough methanol and the
contents exhaustively washed. The mixtures were filtered and the filtrate
concentrated under vacuum. These solutions were stored to be used for
antimicrobial and phytochemical tests as well as UV determinations.
2.11 Anti-microbial Screening of the TLC Fractions
An aliquot of each extract was evaporated to dryness and re-dissolved
in an appropriate volume of DMSO, to produce a 10 mg/ml of solution. 10
mg/ml solutions of gentamycin and chloramphenicol were used as positive
control as described in table 8.
The inhibition zone diameters, IZDs were measured, recorded and the
MIC was calculated from them.
2.11.1 Microbiological Studies on an Ear Swab of a Patient with Otitis
Media with Effusion
Procedure
Using a sterile swab stick, a purulent effusion sample was collected
from a patient with otitis media. This sample was sent to a private
microbiology laboratory for culture and identification test. The result of the
culture and sensitivity were recorded in table 6.
31
2.12 Phytochemical Test on All the Extracts
Phytochemical tests were performed on the extracts and fractions for
the following classes of compounds as outlined by Trease and Evans (1983)
and Harbourne (1988).
Glycosides - The Hydrolysis Test
0.1 g of each extract was boiled in 5 ml of dilute HCl for 15 minutes
and filtered. The filtrate was subjected to the Fehlings test after neutralization
with 20% aqueous solution of NaOH. A dense brick red precipitate would
indicate the presence of glycoside.
Saponins
Extraction: A little quantity of each extract was boiled with 5 ml of distilled
water for 5 minutes.
The mixture was filtered, allowed to cool and the filtrate used for the
following test.
(a) Frothing Test: 1 ml of the filtrate was diluted with 2 ml of distilled
water and shaken. A stable froth indicates the presence of saponins.
(b) Emulsion Test: Another aliquot of the filtrate was mixed with 2 ml of
water and 1 drop of olive oil and shaken. An emulsion indicates the presence
of saponins.
Carbohydrates
Extraction: About 0.1 g of each extract was shaken with wate, boiled and
filtered, filtrate was divided into portions.
(a) General Test- Molisch’s Test: To the filtrate, a few drops of Molisch’s
reagent were added. Concentrated H2SO4 was then poured down the test tube.
A purple ring at the interface of the two layers indicated the presence of
carbohydrates.
(b) Specific Test for Free Reducing Sugars- Reduction of (Fehling’s
Test): To 1 ml of the filtrate was added equal volumes of Fehling’s
32
solution A and B and boiled on a water–bath. The presence of a reducing
sugar is shown by the presence of brick red precipitate.
Test for Tannins and Flavonoids
Extraction: 0.1 g of each extract was boiled with 6 ml of distilled water for 3
minutes on water –bath. The mixture was filtered and the resulting filtrate
divided into two portions.
i. Ferric Chloride Test – 1 ml of filtrate was diluted with distilled water
(1:4) and a few drops of FeCl3 solution added. A blue or green coloured
precipitate would indicate the presence of phenolic nucleus.
a. Specific Test for Flavonoids – Shinoda Test: 0.5 g of powdered
sample was boiled in ethanol for 5 minutes and filtered. To the filtrate was
added four pieces of magnesium filings, followed by a few drops of
concentrated HCl. A pink or red colour would indicate the presence of
flavonoids.
b. Specific Test for Tannins (Albumin Test): To aqueous extracts of the
sample were added about equal volume of egg albumin. A precipitate will
indicate the presence of tannins.
Terpenoids and Steroids – Salkowski Test: 1 g of powdered sample was
dissolved in chloroform and filtered. To the filtrate was added 5 ml of Conc.
H2S04 to form a lower layer. A reddish brown colour at the interface would
indicate the presence of terpene.
Liebermann – Burchard Test: Portion of powdered sample was agitated in
acetic acid. Then a mixture of chilled acetic anhydride and Conc. H2S04 (19:1)
was poured down the side of the test tube. The formation of a greenish blue
colour in the chloroform layer would indicate the presence of
steroids/triterpenes
Alkaloids
0.1 g of the dried material/fraction isolate was boiled for minutes with 5
ml of 2NHCL on a water bath. The mixture was filtered and to 1 ml portions
33
of the filtrate was added 2 drops of Dragendorff’s reagent, Wagner’s or
Mayer’s reagent.
Formation of a reddish-brown precipitate, yellow precipitate and a
cream precipitate respectively indicates a positive result.
Proteins
a. Millions Test: To a small portion of the sample was added a few
milliliters of Million’s reagent, a white precipitate, which changed to brick red
on boiling would indicate the presence of proteins.
b. Biuret Test: To the sample, was added a few drops of CuSO4 solution,
a violet precipitate would indicate proteins.
Oils
The sample was rubbed on a filter paper. The presence of a translucent
patch would confirm the presence of oils.
Resins
Dried sample was dissolved in acetic anhydride and one drop of
concentrated H2S04 was added. A purple or violet colour would indicate the
presence of resins.
2.13 UV Spectral Determination
The ten separated TLC fractions were separately dissolved in methanol
and scanned with UNICO –UV2102PC spectrophotometer. These were run
using methanol and blank.
2.14 Acute Toxicity (LD50) Test
The oral acute toxicity test (LD50) of the crude methanol extract and
diethylether extract were determined according to the method described by
Lorke (1983). Albino mice (15–45 g) of either sex were used. The methanol
and diethylether extract were suspended in 10% tween 80 respectively and
administered orally at doses of 10, 100, and 1000 mg/kg to three groups of
mice (n=3) respectively the animals were observed for 24 hours. Based on the
result obtained in these initial tests, doses of 1000, 1600, 2900 and 5000
34
mg/kg for each extract were administered to four different mice. The LD50
was calculated as the geometric mean of the lowest dose that killed a mouse
and the highest dose that showed no death.
2.14.1 Egg Albumin-induced Edema in Rats
The rat paw edema method of Winter et al., (1962) was used. Increase
in the right hind paw volume (Bani et al., 2000) induced by injection of fresh
albumin into the subplanter tissue and was used as a measure of acute
inflammation. Adult Swiss albino rats (100–250 g) of both sexes were divided
into group of three rats. Each group (n=5) received two doses of 50 or 100
mg/kg of the extract in 10% v/v tween 80 administered orally. Control
animals received p.o aspirin and diclofenac 100 mg/kg respectively. One hour
latter, inflammation was induced by injection of 0.1 ml of undiluted fresh egg
albumin into the subplantar of the right hind paw of rats. The volume of the
paw was measured by water displacement using the plethysmometer at 1, 2, 3
and 4 hours respectively after egg albumin injection. Edema formation was
assessed in terms of the difference in the zero time paw volume of the injected
paw and its volume at the different times after egg albumin injection. For each
dose of the extract, percentage inhibition of edema was calculated using the
relation.
Percentage Inhibition (%) of Edema = [1-(a-x)/(b-y)] X 100
Where
(a) = mean paw volume of treated animals after egg albumin injection
(x) = mean paw volume of treated animals before egg albumin
injection
(b) = mean paw volume of control animals after egg albumin injection
(y) = mean paw volume of control animal before egg albumin
injection
35
2.15 Statistical Analysis
The data were analysed statistically and reported as mean ± standard
deviation and were compared using student t-test and regarded as significant
at P < 0.001
36
CHAPTER THREE
RESULTS
3.1 Extraction Yield
280.00 g of pulverized Crateva religiosa leaves gave 70.00 g of
methanolic extract which is a deep brown redish sticky material. The
percentage yield of the extract was calculated as follows:
Methanol extract
And the diethyl ether extract gave a dark green yield of 27.00% (w/w), since
the extraction was separately done.
3.2 Phytochemical Analysis
Results of the tests on extracts of Crateva religiosa dried powdered
leaves and the extracts are presented in table 2.
3.3 Chromatographic Separation
3.3.1 Chromatographic Fractionation of the Methanolic Extract
The results of the trial TLC experiments are shown in Table 3, listing
the solvent system and the number of bands observed when viewed under UV
light.
3.4 Phytochemical Results on the TLC Fractions
The chemical classes of constituents present in the bands are listed in
table 4.
3.5 The Rf Value of TLC Bands
The Rf values of the ten spots viewed under UV light are as shown in
Table 5.
37
3.6 Antimicrobial Activity
The result of the microbiological analysis of the ear swab of a patient
with otitis media yielded growth of Staph. aureus with the sensitivity shown
in Table 6.
3.6.1 Antimicrobial Screening of the Extracts
Table 7 below shows the susceptibility of tests organisms to extracts and
standard drugs.
3.6.2 Antimicrobial Screening of the TLC Fractions
The susceptibility of test organisms to standard drugs and TLC fraction
bands from the aqueous methanol extract with the Inhibition Zone Diameter, IZD
(mm) is shown in Table 8.
3.6.3 Minimum Inhibitory Concentration (MIC) of Standard Drugs and
TLC Fractions
Table 9 below shows the average minimum inhibitory concentration of
TLC fraction bands and standard drugs.
3.7 Result of Acute Toxicity Test
The acute toxicity (LD50) test of aqueous methanol extract and
diethylether extract for both stage one and two are as shown in Tables 11 and
12 respectively.
3.8 Result of Anti-inflammatory Analysis
The results of mean paw volume and percentage inhibition of acute inflammation
of the rat paw are shown in tables 13 and 14 respectively.
38
Table 2: Classes of phytocompounds present in the methanolic and
diethyl ether extracts and powdered leaves RELATIVE ABUNDANCE
Phytochemicals Diethyl ether Extract Plant powder Aqueous Methanol
extract
1 Carbohydrate _ +++ ++
2 Reducing sugar _ _ _
3 Alkaloids + ++ ++
4 Glycoside _ _ _
5 Saponins _ _ _
6 Tannins _ ++ ++
7 Flavonoids + + ++
8 Resins ++ ++ +
9 Proteins ++ ++ ++
10 Oil ++ ++ ++
11 Steroids + + +
12 Terpenoids + + ++
Key – =Absent
+ =Low in concentration
++= moderate concentration
+++= High concentration
39
Table 3: Trial TLC of Aqueous Methanol Extract
Solvent System No. of Spots Colour of Bands
n-hexane/methanol (1:1) 2 Yellow, green
n-hexane/chloroform (1:2) and
5 drops of acetic acid
5 Pale yellow, green, yellow,
yellowish green and green
n-hexane/methanol (2:1) 4 Pale green, yellowish green,
green and dark green.
n-hexane/methanol (2:1) and 5
drops of acetic acid
3 Yellow, green and light
green
Chloroform/methanol (9:1) Not resolved -
n-hexane/chloroform (1:2) 10 As shown in table 3
40
Table 4: Result of Phytochemical Analysis of the TLC Fractions of the
Aqueous Methanol Extract (Solvent System: n-hexane/chloroform 1:2) Phytochemicals Bands
10 9 8 7 6 5 4 3 2 1
1 Flavoniod _ _ _ _ _ _ _ _ + +++
2 Alkaloid _ _ _ _ _ _ _ ++++ ++++ ++++
3 Resins _ + _ _ _ _ _ _ + +
4 Steroids + ++ + + + + + _ +++ +++
5 Terpenoids + ++ + + + + + _ +++ +++
6 Fats & Oil + + + + + + + _ + +
7 Reducing sugar _ _ _ _ _ _ _ _ _ _
8 Tannins + + _ _ + _ _ ++ ++ ++
9 Saponins _ _ _ _ _ _ _ _ _ _
10 Acidic compounds _ _ _ _ _ _ _ _ _ _
11 Glycosides _ _ _ _ _ _ _ _ _ _
12 Carbohydrate. _ _ _ _ _ _ _ + ++ ++
Key
- = Not Present
+ = Present in small concentration
++ = Present in moderately high concentration
+++ = Present in very high concentration
++++ = Abundantly present.
41
Table 5: The Rf value of TLC Bands of Aqueous Methanol Extract
Band R f Value Colours in ordinary light
10 0.92 Intense yellow
9 0.45 Light yellow
8 0.33 Light yellow
7 0.29 Pale green
6 0.22 Intense yellow
5 0.15 Pale green
4 0.13 Pale yellow
3 0.10 Pale yellow
2 0.07 Dark green
1 0.02 Ash green
n-hexane: Chloroform (1:2)
42
Table 6: Sensitivity of Staph. aureus isolated from the ear swab of a
patient with otitis media
Antibiotic used Sensitivity Test
Gentamycin +++
Streptomycin +++
Norbactin +++
Chloramphenicol +++
Ciprofloxacin +++
Floxapen −
Methanol Extract +++
+ = Sensitive, − = Resistant
43
Table 7: Result of the Antimicrobial Screening of Aqueous Methanolic and
Diethyl ether Extracts of Crateva religiosa Leaf (10mg/ml) Solution Extracts /standard
drugs (10 mg/ml)
Inhibition zone diameters (mm)
Bacillus
subtilis
Staph.
aureus
Klebsiella
pneumoniae
Escherichia
coli
Pseudomonas
aeruginosa
Salmonella
typhi
Candida
albican
Aqueous Methanolic
leaf extract
16 18 + + 14 + +
Diethyl ether extract + + + + + + +
Gentamycin 18 22 14 + + + +
Chloramphenicol 25 28 20 + + 28 +
+ indicate growth without inhibition (Lack of Sensitivity).
44
Table 8: Result of antimicrobial screening of TLC Fractions (aqueous
methanolic extract)
Inhibitions zone diameter, IZD (mm)
Bands/standard
drugs
B. subtilis
(10mg/ml)
S.
aureus
K.
pneumonia
P.
aeruginosa
E.
coli
S. typhi C.
albicans
10 15 ± 1.0 30 ± 1.0 + + + + +
9 + + + + + + +
8 + + + + + + +
7 14 ± 1.0 + + 12 ± 1.0 + + +
6 + + + + + + +
5 + + + + + + +
4 + + + + + + +
3 + + + + + + +
2 + + + + + + +
1 + + + + + + +
Gentamycin 18 ± 1.0 22 ± 1.0 14 ± 1.0 + + + +
Chloramphenicol 25 ± 1.0 28 ± 1.0 20 ± 1.0 + + 28 ± 1.0 +
Values are mean IZD ± standard deviation of triplicate determinations.
+ indicate growth without inhibitions (lack of sensitivity)
45
Table 9: Average minimum inhibitory concentration (mg/ml) of TLC
bands and standard drugs
Minimum Inhibitory Concentration (mg/ml)
Bands /drugs B. subtilis S. aureus K.
pneumonia
P.
aerugiosa E.
coli
S. typhi C.
albicans
10 1.06 ± 0.10 0.70 ± 0.17 _ _ _ _ _
7 1.47 ± 0.17 _ _ 1.47 ± 0.07 _ _ _
Gentamycin 0.67 ± 0.02 0.94 ± 0.11 0.70 ± 0.17 _ _ _ _
Chloramphenicol 1.21 ± 0.04 0.98 ± -0.10 0.70 ± 0.17 _ _ 0.98 ±0.10 _
MIC ± S. E. M
46
Table 10: Values of X2, IZD, logarithms of concentration, MIC of TLC
fractions with significant activities and standard drugs Band /Drugs Conc.
(mg/ml)
Log
Conc.
IZD X2 MIC
(mg/ml)
10 B. Subtilis 5.000
2.500
1.250
0.625
0.70
0.40
0.10
_0.20
15
14
10
_
12.25
9.00
1.00
_
1.0551
S. aureus 5.000
2.500
1.250
0.625
0.70
0.40
0.10
_0.20
12
11
10
_
4.00
2.25
1.00
_
0.6955
7 B. Subtilis 5.000
2.500
1.250
0.625
0.70
0.40
0.10
_0.20
12
10
8
_
4.00
1.00
0.00
_
1.4668
P. aeruginosa 5.000
2.500
1.250
0.625
0.70
0.40
0.10
_0.20
10.
9
8
_
1.00
0.25
0.00
_
1.4668
Gentamycin B. Subtilis 5.000
2.500
1.250
0.625
0.70
0.40
0.10
_0.20
14
12
11
10
9.00
4.00
2.25
_
1.6643
S. aureus 5.000
2.500
1.250
0.625
0.70
0.40
0.10
_0.20
16
12
11
9
16.00
4.00
2.25
0.25
0.9373
K. pneumonia 5.000
2.500
1.250
0.625
0.70
0.40
0.10
_0.20
12
11
10
8
4.00
2.25
1.00
0.00
0.6955
47
n= 6 * P < 0 .05. Compared to control inhibitions (%) was calculated relative
to the negative control.
Choramphenicol B. subtilis 5.000
2.500
1.250
0.625
0.70
0.40
0.10
_0.20
114
12
10
9
9.00
4.00
1.00
0.25
1.2139
S. aureus 5.000
2.500
1.250
0.625
0.70
0.40
0.10
_0.20
15
12
9
8.5
12.25
4.00
0.25
0.06
0.9770
K. pneumonia 5.000
2.500
1.250
0.625
0.70
0.40
0.10
_0.20
12
11
10
8
4.00
2.25
1.00
0.00
0.6955
S. typhi 5.000
2.500
1.250
0.625
0.70
0.40
0.10
_0.20
15
12
9
8.5
12.25
4.00
0.25
0.06
0.9770
48
Table 11: Result of acute toxicity (LD50) test (Methanol Extract)
Stage one. Dose (mg/kg) Mortality
10 0/3
100 0/3
1000 0/3
Stage two. Dose (mg/kg) Mortality
1000 0/1
1600 0/1
2900 0/1
5000 0/1
LD50 = > 5000 mg/kg
49
Table 12: Result of acute toxicity (LD50 ) test (Diethylether Extract)
Stage one. Dose (mg/kg) Mortality
10 0/3
100 0/3
1000 0/3
Stage two. Dose (mg/kg) Mortality
1000 0/1
1600 0/1
2900 0/1
5000 0/1
LD50 = > 5000 mg/kg
50
Table 13: Mean paw volume ± SEM (ml) Treatment Dose (mg/kg)
P.O.
Mean Paw volume ± SEM (ml)
0hr 1hr 2hr 3hr 4hr
10% Tween 80 0.40ml 1.27 ± 0.18 0.70 ± 0.00 0.73 ± 0.19 0.83 ± 0.07 0.90 ± 0.15
Methanol Extract 50 0.93 ± 0.04 0.60 ± 0.06* 0.70 ± 0.16 0.80 ± 0.02 0.88 ± 0.17
100 0.80 ± 0.00 0.70 ± 0.16 0.83 ± 0.16 0.97 ± 0.18 1.06 ± 0.54
Diethyl ether
Extract
50 0.87 ± 0.04 0.77 ± 0.12 0.63 ± 0.21* 0.72 ± 0.05 0.53 ± 0.19
100 0.90 ± 0.10 0.70 ± 0.10 0.60 ± 0.10 0.35 ± 0.44 0.31 ± 0.28
Acetyl Salicylic
Acid
100 1.03 ± 0.14 0.77 ± 0.40 0.58 ± 0.09 0.73 ± 0.11** 0.62 ± 0.18
Diclofenac 100 0.97 ± 0.20 0.63 ± 0.04 0.53 ± 0.04 0.40 ± 0.16 0.45 ± 0.20
*Significant relative to control reading at p < 0.05
**Significant relative to control reading at p < 0.01
(n=5)
SEM = Standard error of mean
51
Table 14: Percentage inhibition of acute inflammation of the rat paw Treatment Dose (mg/kg)
P.O.
Percentage inhibitions in edema
+1hr +2hr +3hr +4hr
10% tween 80 0.40ml _ _ _ _
Methanol Extract 50 14.3 4.11 3.91 2.83
100 0.0 -13.7 -16.2 -17.8
Diethyl ether
Extract
50 -10.0 0.0 13.8 40.7
100 0.0 17.8 52.4 65.6
ASA 100 -10.0 0.0 12.7 31.6
DCF 100 10 27.4 49.6 53.7
ASA = Aspirin (Acetyl Salicylic Acid)
DCF = Diclofenac
52
CHAPTER FOUR
DISCUSSION AND CONCLUSION
The calculated yield of aqueous methanol extract was 25.00%. This
showed that methanol is good extraction solvent for the material. This
research work is directed towards discovery of chemical entity responsible for
the observed antimicrobial and anti-inflammatory effect of the leaf extract but
for us to achieve this, the knowledge of good developing solvent for the crude
drug is indispensible. From the result of the trial TLC of the methanol extract,
different solvent systems were used. This included n-hexane/methanol (1:1),
n-hexane/methanol (2:1) and 5 drops of acetic acid in this case the resolution
was very poor. N-hexane/chloroform (1:2) and 5 drops of acetic acid gave fair
result, yielding 5 bands. The best solvent system was that of n-
hexane/chloroform (1:2) which yielded 10 bands as shown in Table 3. The
solvent system n-hexane:chloroform (1:2) was found to be a good
chromatographic eluent for the methanol extracts, most especially the major
antimicrobial components.
The result of the phytochemical tests with the powdered dried leaves of
Crateva religiosa showed the presence of the following compounds:
carbohydrate, alkaloids, tannins, flavonoids, resins, proteins, oils, steroids and
terpenoids. Result of the phytochemical tests on the aqueous methanol extract
indicates the presence of the following classes of compounds; carbohydrate,
alkaloid, tannins, flavonoids, proteins and oil in moderate concentration while
resins, and steroids occur in low concentrations. The diethylether extract
showed the presence of resins, proteins, oil in moderate concentrations while
alkaloids, flavonoids, steroids and terpenoids occurred in low concentrations.
Phytochemical screening of the TLC bands showed that Band 10 had
steroids, terpenoids, fats and oil in low concentration. Band 9 had steroid and
terpenoid in moderate concentration while alkaloid, resin, fats and oil were
present in low concentration. Bands 5-8 had alkaloid, steroid, terpenoid, fats
53
and oil in equal proportion. Bands 1-3 had alkaloid, steroid, terpenoid, fats
and oil in equal proportion. While flavonoid was abundant in band 1 it is
present in small concentration in band 2. Band 3 has no flavonoid.
The Rf values of the fractionated TLC bands are given in Table 5. The
colours in the day light (different levels of yellow) as well as the Rf values of
the bands can be used as identification index for the various compounds
collected most especially bands 7 and 10 that had anti-microbial activity.
The results of the antimicrobial screening showed that the inhibitory
zone diameter of chloramphenicol (25 mm), Gentamycin (18 mm) were more
pronounced compared to that of the methanol extract against B. subtilis while
the diethylether extract did not inhibit the organisms under study. Increased
inhibitory zone diameter was observed with the methanol extract against S.
aureus (18 mm) though the activity which is a function of the IZD is still
slightly lower than those of the Gentamycin (22 mm) and Chloramphenicol
(28 mm). The methanol extract as well as the diethyl ether extract had no
observed activity against K. pneumonia that was sensitive to gentamycin and
chloramphenicol with Inhibition Zone Diameter of 14 and 20 mm
respectively. It was observed that Escherichia coli was resistant to both the
leaf extracts and the standard drugs while P. aeruginosa was resistant to the
diethyl ether extract as well as the standard drugs but slightly sensitive to the
methanolic extract IZD (14 mm). Sensitivity of S. typhae with
chloramphenicol IZD (28 mm) was more obvious but the same organism was
resistant to the methanol and the diethyl ether extracts as well as the
gentamycin. Observation equally shows that Candida albicans was resistant to
the methanolic and diethyl ether extract as well as the standard drugs.
Sensitivity of Staph. aureus to the methanol extract obviously justified the use
of the plant leaf extract in the treatment of infected otitis media by the local
people of the Eastern Nigeria.
54
The MIC values of TLC fractions that did not show a strong response to
the test organisms were not determined. From table 8, the antimicrobial
property of band 10 is perhaps more peculiar than any other fractions. Its
activities for B. subtilis and Staph. aureus were more pronounced with higher
inhibitory zone diameters (15 mm and 30 mm) respectively and with lower
MIC value of 1.06 0.10 mg/ml and 0.70 0.17 mg/ml respectively showing
that very little concentration will be required to elicit antimicrobial activity.
The maximum susceptibility for Band 10 was indicated by S. aureus, 0.70
0.17 mg/ml. The least was for B. subtilis which is 1.47 0.10 mg/ml. For
band 7, B. subtilis and P. aeruginosa were equally susceptible to band 7
averaging 1.47 0.17 mg/ml. Maximum susceptibility indicated by the
organism shows that Band 10 had greater susceptibility than band 7. When the
activities of tested bands were compared to S. aureus, it followed that the
organism was most susceptible to bands 10 and 7 than to any band. Band 10
was twice more active than band 7. Furthermore, band 10 is more active
against the organism S. aureus when compared to Chloramphenicol and
Gentamycin. The Minimum Inhibitory Concentration for band 10 against
Staph. aureus being 0.70 0.17 when the positive control is 0.94 0.11 and
0.98 0.10 mg/ml respectively for Gentamycin and Chloramphenicol.
Activity of Band 10 was slightly better than Band 7 with respect to B. subtilis
but much lower than those of Gentamycin and Chloramphenicol. In summary,
therefore, this research on Crateva religiosa reveals a great potential for its
use in various bacterial infection involving Staph. aureus especially in cases
of otitis media.
The diethyl ether extract showed progressive dose dependent inhibition
of egg albumin induced inflammation. This suggests that anti–inflammatory
constituent of this plant resides in this extract. The diethyl ether extract 50
mg/kg showed more activity than aspirin at the same dose after 3–4 hours.
55
While at 100mg/kg the same extract was quite outstanding in its anti-
inflammatory effect over aspirin and diclofenac after 3–4 hrs.
The wavelengths of maximum absorption, max of the TLC band are
given in appendix XI (spectra I-X). The range of max is between 200 nm to
665 nm. This is mainly in the visible region (>400 nm). We have few
absorption in the UV region (200-400 nm). Specific compounds can however,
not be assigned to the bands. Information which will be derivable from the
UV/VIS spectroscopy at this level might be that these compounds are highly
unsaturated. Further isolation and purification using chromatographic,
chemical and other spectroscopic methods will then be necessary to
unequivocally identify the compounds. The deductions made from the spectra
of the different fractions showed that the compounds present were UV active.
Exhaustive extraction of the leaf powder of Crateva religiosa yielded
two extracts, these are the aqueous methanol extract and the diethyl ether
extract. Acute toxicity test using the aqueous methanol and diethyl ether
extracts in mice established an oral LD50 > 5,000mg/kg which suggests that
the leaf extract is relatively safe (Lorke, 1983). Flavonoids, and some group of
terpenoids which are known to possess anti–inflammatory effects (Gabor,
1972; Barik et al, 1992) were present in these extracts.
It has been suggested that the probable mechanism of action of
flavonoid compounds is due to their inhibitory action on arachidonic acid
metabolism, cycloxygenase and 5–lipogenase pathway (Hajare et al, 2000,
Sharma et al, 1996). Though the suppression of inflammation I hr. post
injection of phlogistic agent suggests that the agent is likely to posses an
antihistamine effect whereas subsequent suppression after then suggests an
inhibition of arachidonic acid pathway (Willoghby and Flower, 1993). Like
the phlogistic agent induced edema, egg albumin – induced edema is probably
mediated by histamine, 5HT, kinins, polymorphonuclear leukocytes,
56
postranoids nitric oxide, neuropeptides and cytokines (Raychaudhuri et al,
1991)
Phytochemical analysis showed that the diethylether extract contained
mainly alkaloids, flavonoids, resins, protein, oil, steroids and terpenoids
(Table 2). Alkaloids and flavoniods have been shown to possess anti-
inflammatory activity in the adjuvant-induced arthritis model (Gabor 1972).
The diethyl ether extract showed dose dependent anti-inflammatory activity,
which was found to be statistically significant at higher concentration in acute
egg-albumin induced rat paw oedema model. These results were significant
when analysed statistically.
CONCLUSION
The aqueous methanol extract of the dried leaves of Crateva religiosa
demonstrated anti-microbial activities against Staph. aureus and Bacilus
subtilis. The diethylether extract of the leaves of Crateva religiosa also
showed a marked anti-inflammatory activity in rats. This may explain its
popular use in traditional medicine in the treatment of otities media.
However, further work needs to be done to elucidate and characterize
the anti-microbial and anti-inflammatory principle and the mechanism and site
of action. Further research is also necessary to evaluate its spectrum of
activity.
57
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APPENDIX I
62
APPENDIX II
63
APPENDIX III
64
APPENDIX IV
65
APPENDIX V
66
APPENDIX VI
67
APPENDIX VII
68
APPENDIX VIII
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APPENDIX IX
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APPENDIX X
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APPENDIX XI
Band 10