1

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

2

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

5

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

6

1.6.2 Non-steroidal Anti-inflammatory Agents- - - - -

10

1.6.3 Anti-inflammatory Herbs- - - - - - - -

11

1.7 The Aim and Objectives of the Study- - - - - -

13

CHAPTER TWO: EXPERIMENTAL

2.1 Materials and Methods- - - - - - - -

14

2.1.1 Apparatus and Instruments (Equipment)- - - - -

14

2.1.2 Reagents and Solvents- - - - - - - -

14

2.2 Plant Material- - - - - - - - - -

14

2.2.2 Micro-organisms- - - - - - - - -

15

2.3 Extraction- - - - - - - - - -

15

2.4 Thin Layer Chromatography of Aqueous Methanol Soluble Fraction -

15

2.4.1 Trial Analytical TLC- - - - - - - -

15

2.4.2 Thin Layer Chromatography of the Diethylether Extract- - -

16

2.5 Preparation of Stock Sample Solution in DMSO- - -

16

2.6 Antimicrobial Test on Aqueous Methanol and Diethyl ether Extracts-

16

7

2.7 Determination of Minimum Inhibitory Concentration (MIC)- -

17

2.8 Media- - - - - - - - - - -

17

2.8.1 Nutrient Agar- - - - - - - - -

17

2.9 Preparative TLC of Extracts - - - - - - -

17

2.10 Isolation of TLC Bands - - - - - - -

18

2.11 Antimicrobial Screening of the TLC Bands - - - - -

18

2.11.1 Microbiological Test on Ear Swab a Patient with Otitis Media- -

18

2.12 Phytochemical Test on All the Extracts- - - - -

19

2.13 UV Spectral Determination- - - - - - -

21

2.14 Acute Toxicity (LD50) Test- - - - - - -

21

2.14.1 Egg Albumin-induced Edema in Rats- - - - -

22

2.15 Statistical Analysis- - - - - - - - -

23

CHAPTER THREE: RESULTS

3.1 Extraction Yield- - - - - - - - -

24

3.2 Phytochemical Analysis- - - - - - - -

24

8

3.3 Chromatographic Separation- - - - - - -

24

3.3.1 Chromatographic Fractionation of the Methanol Extract- - -

24

3.4 Phytochemical Results of the TLC Fractions- - - - -

24

3.5 The Rf Values of TLC Bands - - - - - - -

24

3.6 Antimicrobial Activity- - - - - - - -

25

3.6.1 Anti-microbial Screening of the Extracts- - - - -

25

3.6.2 Anti-microbial Screening of the TLC Fractions- - - -

25

3.6.3 Minimum Inhibitory Concentration (MIC) of Standard Drug

and TLC Fractions- - - - - - - -

25

3.7 Result of Acute Toxicity Test- - - - - - -

24

3.8 Result of Anti-inflammatory Analysis- - - - - -

25

CHAPTER FOUR: DISCUSSION AND CONCLUSION- - -

40

References- - - - - - - - - - -

45

Appendices- - - - - - - - - - -

49

9

LIST OF TABLES

Table 1: Examples of Some Anti-inflammatory Herbs- - - -

12

Table 2: Classes of Phytocompounds present in the methanolic and

diethyl ether extracts and powdered leaves- - - - -

26

Table 3: Trial TLC of methanol extract- - - - - -

27

Table 4: Result of phytochemical analysis of the TLC bands- - -

28

Table 5: The Rf values of the TLC bands- - - - - -

29

Table 6: Culture and sensitivity analysis of ear swab of patient

with otitis media- - - - - - - - -

30

Table 7: Result of the antimicrobial screening of methanolic and

diethyl ether extracts- - - - - - - -

31

Table 8: Result of antimicrobial screening of TLC fractions- - -

32

Table 9: Average Minimum Inhibitory Concentration of TLC bands- -

33

Table 10: Values of X2, IZD, logarithms of concentration, MIC of TLC

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fractions with significant activities and standard drugs- - -

34

Table 11: Result of acute toxicity (LD50) test (methanol extract) - -

36

Table 12: Result of acute toxicity (LD50) test (diethyl ether extract)- -

37

Table 13: Mean paw volume ± SEM (ml)- - - - - -

38

Table 14: Percentage Inhibition of Acute Inflammation of the rat paw-

39

LIST OF FIGURES

Fig. 1: Chemical Structures of Some Glucocorticoids- - -

10

Fig. 2: Chemical Structures of Non-steroidal

Anti-inflammatory Drugs- - - - - -

11

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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

16

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─

19

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.

21

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)

22

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

23

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

24

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.

26

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

69

APPENDIX IX

70

APPENDIX X

71

APPENDIX XI

Band 10