University of Nigeria Research Publications

MADUBUIKE, Kelechi G

Aut

hor

PG/M.Sc/03/34156

Title

The Anti-Inflammatory Activities of the Leaf Chloroform Extract of Plaisota Hirsute (K. SCHUM).

Facu

lty

Veterinary Medicine

Dep

artm

ent

Veterinary Physiology and Pharmacology

Dat

e

September, 2007

Sign

atur

e

AN EMPIRICAL ANALYSIS OF PURCHASING POWER PARITY: A CASE OF TWO ANGLOPHONE ECOWAS MEMBERS.

OGUANOBI CHIBUIKE R. (PG/M.SC./06/40895)

A RESEARCH WORK PRESENTED IN PARTIAL FULFILMENT

OF THE REQUIREMENTS FOR THE AWARD OF A DEGREE OF "r/ f

MASTER OF SCIENCE (M. SC.) IN THE DEPARTMENT OF

ECONOMICS, FACULTY OF THE SOCIAL SCIENCES,

UNIVERSITY OF NIGERIA, NSUKKA.

FEBRUARY, 2008.

THE ANTI-INFLAMMATORY ACTIVITIES OF THE LEAF CHLOROFORM EXTRACT OF Pdisutn Rirsritn (K. SCHUM)

MADURUIKE, KELECHI GIDEON

(PG/M. Sc/OYM 1 56)

DEPARTMENT OF VETERINARY PHYSlOLOGY AND PHARMACOLOGY

FACULTY OF VETERINARY MEDICINE UNIVERSITY OF NIGERIA

NSUKKA

SEPTEMBER, 2007

THE ANTT-TNFLAMMATORY ACTIVITIES OF THE LJEAF CHLOROFORM EXTRACT OF Pnlisotcl hirsufn (K. SCHUM)

MADUBUIKE, KELECHI GIDEON (PG/M.Sc/03/34156)

DEPARTMENT OF VETERINARY PHYSIOLOGY AND PHARMACOLOGY

FACULTY OF VETERTNARY MEDICINE UNIVERSITY OF NIGERIA

NSUKKA

SUPERVISOR: PROF. 1.U. ASUZU

SEPTEMBER, 2007

CERTIFICATION

I, MADUBUIKE. KELECHI GIDEON, a postgraduate student in the Department c

Veterinary Physiology and Pharmacology and with Registration Number PC/MSc/03/3415

has satisfactorily completed the requirements for course and research work for the de, uree c

Masters of Science (MSc) in Veterinary Pl~armacoiogy. The work embodied in this report i

original and has 11ot been submitted in part or 111I1 for any other diploma or degrec of this or an

other university.

P R O ~ I . U. ASUZU (Supervisor)

PROF P. A. ONYEYILI (External Esaminar)

DR S. C . UDEM (Head of Department)

PROF R. M. ANENE (Dean, Faculty of Veterinary Medicine)

DE,DICATlON

This work is a tribute to my dearly beloved wife. Chinenyc.

I'lie Anti-inflammatory Activities of thc Leaf Chluroform Extract of Palimfa hir.r~r(a (K

Schum).

A Dissertation submitted to the school of Postgraduate Studies of the University orNigeria in

partial fiilfilment for the award of the Degree of Master of Science.

(Veterinary Pharmacoiogy)

MADUBUIKE. KELECHI GlDEON

ACKNOWLEDGEMENTS

I an very grateful to Prof. I . U. Asuzu, my supervisor for his encouragement. advicl

support and invaluable input throughai~t this researcli work. His tolerance, kind criticisms an

corrections mere very helpfill in concluding this prqject.

My heartfelt appreciation goes to Dr. S. C. Udcrn of the Department of Veterinar

Physiology and Pharmacology and Dr. M. lgwebuike of h e Department of Veterinar

Anatomy, both of the University of Nigeria. Nsukka for technically putting me throi~gh soln

stages of this work. Dr. I. Ezeli of the IJepartment of Veterinary Parasitology and Entonlolog:

in above named institution was also very Iiefpful. I thank Mr. A. 0. Ozioko of Botan

Department, University of Nigeria, Nsukka for collecting and identifying the plant material.

sincerely acl<nowledge Dr A. 0. Anaga of the Department of Veterinary Physiology an

Pharmacology also of the same institution for supplying me with basic infor~nations about th

plant material as well as providing some laboratory animals for thc work.

I am very grateful to my parents, Mr Rr Mrs It. 0. Madubuike and my dear wifc

Chinenyc for their love, encouragement and perseverance thronghout the course of th

programme.

I equally appreciate Mr Parker Elljah Joshua of the Department of Biochemistr:

Ilniversity of Nigeria, for typing this manuscript and my friends and others whose names wet

not mentioned here, but in one way or the other contributed to the success of this rcsearc

work.

ABSTRACT

Ten plant sa~nples were screened for topical anti-inflarnniaory activity. Pali.so~c/ Iiirsu~u lea

(Family: Co~nnielinaceae) gave the highest activity. A gradicnt solvent extraction of the lea

was conducted using petroleum ether (40" - 60"), chloroform and methanol respectively. Th

different fractions were tested for anti-inflammatory activity, and the chloroform extrac

showed the highest effect. 3t was then sub~jectecl to fi~rtlier anti-inflarnmatury and analgesi

tests. using both topical and oral routes. All doses of'the extract (100, 200, 300 and 400 @kg

significantly (P<O.OOI) inhibited the crcllon oil-induced ear edema in mice. The extract (40

tng/kg and 800 nigitcg, pa.) sig~~ificantly (Pc0.05) inhibited the carrageenin-induced pa\

edema in rats, 3 hours following the administration of carrageenin. Oral administration of th

estract (200 mgtkg and 300 mgikg) for t h e consecutive days significantly [P<0.05) inhibite

the formation of granuloniatous tissues resulting from subcutaneous introduction of cotto

pellets in rats. All doses of the estracl (150. 300, 600 and 1,200 mg/kg, p.0.) significant1

(Pc0.05) decreased the acetic acid-induced writhing reflex in mice. The extract did nc

demonstrate any acutely toxic effect in mice within the dose range used; hence it was we

tolerated by the animals. In a l l the experiments, the effects of the exlract were dose-depender

and comparable to those of indornethacin, a standard anti-inflammatory drug used in th

experiments.

. . . . . . . . . Extract of P . hirsu~n . . . 2.6.7 Acetic acid-induced Writhing Test of the Chloroform

... ... ... Extract of P . hirszrla ... ...

... ... ... ... 2.6.8 Statistical Analyses 2.6.9 Preliminary Phytochemical Analyses of the Chloroform

. . . Extract of P. hirszt~n ...

... 2.6.9.1 Test for Carbohydrate

. . . 2.6.9.2 Test for Alkaloids

. . . 2.6.9.3 Test for Reducing Sugars

... 2.6.9.4 Test for Glycosides

... 2.6.9.5 Test for Saponins

. . . 2.6.9.6 Test for Tannins

. . . 2.6.9.7 Test for Flavonoids

. . . 2.6.9.8 Test for Resins

. . . 2.6.9.9 Test for Proteins

. . . 2.6.9.1 0 Test for Fats and Oil 2.6.9.1 1 Test for Steroids and Terpenoids 2.6.9.12 Test for Acidic Compounds

CHAPTER: RESULTS ... Extraction . . . ... ...

Extraction with petrolei~m ether ... Extraction with Chloroform . . . Extraction with Methanol ... Screening of Plants For Anti-Infla~nnialory Activity Effect of Different Estracts of Pirlisufrr hi/-sub on Csolon

. . . . . . ... Oil-induced Inflammation in Mice Dose-Response Effect of the Chloroform Extract of Prdisufn hirs~cta

... . . . ... on Croton Oil-induced Inflam~nation in Mice

... ... . . . ... . . . Acute Toxicity ... .. . Effect of the Chloroform Extract uf Pu~isisotu hiia~rkr on

. . . ... ... Carrageenin-induced Paw Edema in Rats Effect of the Chloroform Extract of P . I ' I ~ T S I I I L ~ on Cottoll

. . . . .. ... Pellet-induced Granuloma in Rats ... The Effect of P . hi~.sulu Chloroform Extract on Acetic Acid-induced

... . . . ... ... ... Writhing Reflex in Mice

... ... ... .. . Preliminary Phytochemical Analysis

. . . ... . . . CHAPTER FOUR: DISCUSSION AND CONCLUSION

viii

-. E tgul-e 1 . I

F ig~~re 1.2

Fig~r t : 3.1

Figure 3.2

Figure 3.3

Figure 3.4

Figure 3.5

Figure 3.6

LIST OF FIGURES PAGE

Palisotrr hirsutn in botanical garden (UNN) . . . ... . . . , . .

Pdisotn l~ i rmtn plant . . . . . . . . . .. . . - - . . . . , ,

Percentage inhibition of oederna by cl~loroform extracts of different plants tested at 1000 pg/ear -. . . . . , . . ...

Percentage inhibitiun of oedenia by different extracts of Pnlisoicr lril-suw, using the mouse ear oedcma mod~i . . . . . . . . .

Percentage inhibition of oedema by varying doses of the chloroforni extract of P. i i i~~~zr tcr using the nlouse ear oedema model . . . . . .

Effect of the chloroform extract of P. hirsrrtn on carrageenin-induced

paw oedema in rats . . . , , , ... . . . ... ...

Percentage inhibition of cotton pellet-induced granuloma in rats by varying doses of chloroform extract of P. /uksu!ci . , . . . .

Antinociceptive effect of the chlosofortn extract of P. hir.w~n ...

LIST OF TABLES

"fable 3.1 Preliminary phytochemical analysis of thc

chloroform extract of P. / I L ~ . Y L ~ ~ L I

PAGE

... ... . . . ... 43

LIST OF ABBREVIATIONS

NSAID

WHO

N AD

MPO

HETAB

TMR

PBS

HzSOj

KOH

Non-steroidal anti-inflammatory drug

World Health Organisation

Nicotinamicle adenine dinucleoticte

Myeloperoxidase

Hexadecyltrimethylammonium bromide

Tetramethylbenzidine

Phosphate buffered saline

Tetraoxosulphate (VI) acid

Potassium hydroxide

CHAPTER ONE

1.1 INTRODUCTION AND LITERATURE REVIEW

Inflammation is common to almost all diseases that i~avolve microbiologic, chemical or

physicat injury to living tissue (Lees. 1998). It may bc defined as the complex

pathophysiologic response of vascuIarised tissue to injury (Thompson, 2005). The injury

may result from various stimuIi including thermal, chemical or physical damage;

ischemia; infectious agents; antigen-antibody interactions and other biological processes.

The inflammatory response is typically characterised by the five clinical signs: heat,

redness, swell i~~g, pain and loss of function (Ringer. 1997).

The inflammatory response which is stereotyped, homeostatic, and usually beneficial to

the organism (patient) involves the isolati~n and elimination of the injurious agent, repair

of tissue damage at the site of injury, and restoration of function. The need for anti-

inflammatory drugs arises when the inflatnmatory response becomes inappropriate,

aberrant, or sustained, and when it causes tissue destruction (Kvan, 1994).

Although anti-inflammatory drugs are uscd extensively to treat both acute and chronic

inflammatory conditions, none is curative. In general. they suppress rather than abolish

inflammatory reactions. thereby providing symptomatic relief [Lees, 1998), usualIy

accompanied with some undesiring side effects. There is therefore the need for new anti-

inflammatory drugs that will be safer and more potent than those currently available in

the market. Plant materials, mainly used by herbalists and traditional doctors to solve

most of the local medical problems in about eighty perccnt of the world's rural

population (Ayensu, 1983) no doubt offer a great ray of hope. In recognition of the high

esteem patients hold on traditional medicine, the World Health Organisation (WHO) has

called the attention of many countries to the ever increasing interest of the public in the

use of herbal medicines and encouraged countries to identify and exploit those aspects of

traditional medicine that provide safe and effective remedies (Akah et al., 1997).

Many drugs such as penicillin, vincristin, morphine, strychnine, ipecacuanha, quinine and

a host of other drugs have been produced from the plant. For instance, aspirin, which is

probably the world's most widely used drug could not have been developed without the

chemical blue print supplied by willow bark (Wachtel, 1983). In Nigeria, traditional

medicine occupies a unique position in health care delivery, especially among the rural

populace. However, the activities of the herbalists are surrounded with a lot of secrecy

and lack scientific procedure, hence the need to standardised the practice of traditional

medicine.

An evaluation of common plants used as anti-inflammatory agents in Nigerian traditional

medicine found that the water extract (infusion) of Palisota hirszrta significantly inhibited

the increase in rat paw circumference caused by 1% carrageenin, a standard phlogistic

agent (Akah et al., 1994). A preliminary phytochemica1 analysis of the extract revealed

the following chemical constituents: alkaloids, tannins, saponins, glycosides, flavonoids

and steroids. Further studies showed that the methanolic leaf extract of P. hirsuta

significantly protected the liver of carbon tetrachloride-poisoned rats. This effect was

suspected to be associated with the free radical

scavenging activity of the extract, an action pvsscsscd by several oriental plants. The

treated rats also demonstrated signs of centra! nervous system depression,

Based on these findings and the folkloric uses of P. hi,:w~u in the treatment of boil and

relieve of pain. the present study was designed to investigate the anti-inflammatory

properties of P. hirstrtu leaf in rats.

1.2 LITERATURE REVIEW

1.2.1 General Description of the Plant

Puliso/ci hirsufu belongs to the division of Slwrmirrophyta and class Angiospermue. Its

subclass is Dicoryledormceoe in the order of Curnmeiimdes of the family

Cor~zmelinneceae. It belongs to the genus Pdiso!tr and the specics hirsufu.

The plant P, hirsrt~n is known as 'thunb' in English, and 'Ikpere aturu' (Knee of the

sheep) in Tgboland. It is a robust herb, groning up to 3 in to 4 m in the tropical rain

forest. The stem is rigid with woody base. covered with soft brown hairs. The leaves arc

arranged in rosettes at the terminal of the stems. They are obviate to oblonceolate and

about 15 - 40 cnr and 4 - 1 1.5 cm broad. They are normally acute at the apex and narrow

at the base. tem~inating in flat densely hair petioles that are about 3 cin long. Its margin

and midribs have brown soft hairs. The under surface of the blade is dark green and

usually hairy. The fluorescence has loose spreading panicle of about 10-30 cm long with

many slender pink to whitish lateral branches of 1-2 cm long. The flowers are white to

purplish in colour and they open by 4:06 pm until dusk, The fruits arc glossy and black. It

is a weed of regrowth in farmlands in the rainforest zone (Okezie and Aguwa., 1987).

Figure. 1.1 : Palisota hirsuta ( in the Botanical garden, University of Nigeria, Nsukka )

4 ' w

Figure 1.2: Palisota himta plant ( stem and leaf )

1.2.2 Follclorie Uses of Pdisoto Irirslctn

Plant species of the family Commelh~ccrre are employed locally in treating various

aiIments by the natives living in places where the species are found. The famiIy is

medically endowed in nature, thus occupying an important position in traditional

medicine. The leaves of P. I.rir.sr~lrr have been reportedly used in the treatment of

toothaches in some parts of Igboland (Okezie and Aguwa. 1987). The leaves and stem of

the plant are taken as an infusion and recipe in the treatment of rheumatoid arthritis by

the natives of Ugwu in Bende Local Government Area of Abia State. The plant is also

used to relieve pain. treat boils, cough, gonorrhoea and also used as an antiseptic.

1.2.3 Natural Products and Anti-inflammatory Activity

Natural products of plant origin have been associated with anti-inflainn~atory activity.

The most important of these are flavonoids (Pifferi, 1972). Flavonoids are polyphenolic

compounds possessing 15 carbon atoms: two benzene rings joined by a linear three

carbon chain. They occur in fruits, vegetables, nuts, seeds, flowers, leaves and bark of

plants. Their high chemical reactivity is responsible for their affinity for membrane

phospholipids, and hormone receptors and for their ability to inhibit enzyme and

scavenge for free radicals (Pifferi, 1972).

Flavonoids are subdivided into twelve structural groups namely: Flavones, Flavonones.

Flavonols, Flavononol, Isoflnvones, Anthucyanines, Anthocyanfdine, Leucoanthocyanin.

Chalcones, Dihydrochalcones, Aurones and Catechin. All flavonoids have a high

absorbance in the 250-270 nrn range where proteins and nucleic acids have absorptivity.

Flavones and flavonols absorb significantly in the range of 330-350 nm where

nicotinamide adcnine dinucleotide (NAD') and NADP' cofactors absorb strongly while

anthocyanine strongly absorb in the range of about 520-560 nm. It is probably in the area

of visual perception and light absorbance that strongest case can be made for the

unambiguous function of flavonoids (Harboul-ne, 1972).

Flavonoids exhibit a wide spectrum of pharmacological activities including antibacterial,

anti-inflammatory, anti-allergic, immunomodulatory. antiviral, antineoplastic, antiulcer

and vasoprotective effects among others (Das, 1989). Akaraz and Jilnenez (1 988)

reported that a number of flavonoids have exhibited anti-inflammatory activities. Most of

their pharmacological activities are attributed to their antioxidant property. However.

their therapeutic use is still limited because clinical efficacy is sometimes impaired by

persistent degradation, low absorption and failure to reach the target organs (Havsten,

1983)

1.2.4 Pathaphysiology of Inflammation

Inflammation occurs in three distinct phases - acute, subacute and chronic (or

proliferative) (Thompson, 3005). In the acute phase, chemical mediators released at the

site of injury cause vasodilation and increased capillary permeability. Protein-rich fluid

containing many components of plasma, including fibrinogen, kinins, con~plementary and

immunoglobulins that mediate the inflamn1.atory response exude from the capillaries into

the interstitial space (Lees, 1998). The subacute phase is characterised by movement of

phagocytic cells to the site of injury (Thompson, 2005). Leukocytes, platelets and RBC

adhere to the endothelial cell surfaces in response to adhesion ~nolecules released from

activated endothelial cells (Wallace, 2003). Neutrophils are the first cells to infiltrate the

site of injury. Rasophils and eosinopl~ils are more prevalent in allergic reactions or

parasitic infections (Lees, 1998). As the inflamn~atury process continues, macrophages

predominate, actively removing damaged cells or tissue . If the cause of injury is

eliminated, acute inflammation may be followed by a period of tissue repair. Blood clots

are removed by fibrinolysis and damaged tissues are regenerated or replaced with

fibroblast, collagen or endothelial cells. However, inflammation may become chronic,

leading to fiirther tissue destruction and fibrosis (Thompson, 2005).

1.2.5 Chemical Mediators of Inflammation

Biochemical mediators released during inflammation intensify and propagate the

inflammatory response. These mediators are soluble, diffusible molecules that can act

locally and systemically (Thompson. 2005). The cell damage associated with

inflammation induces cell membranes to cause leukocytes to release lysosomal enzymes;

arachidonic acid is then liberated from precursor compounds, and various eicosanoids are

synthesized (Wagner et al., 2004). The cyclooxygenase pathway of arachidonate

metabolism produces prostaglandins, which have a variety of effects on blood vessels,

nerve endings and on cells involved in inflammation. while its lipoxygenase pathway

yields leukotrienes, which have a powerf~d cl~emotactic effect and promote

bronchoconstriction and aIterations in vascular permeability (William ef al., 1993).

Kinin, neuropeptides and histamine are also released at the site uf tissue injury, as are

complement components, cytokines and other products of leukocytes and platelets

(Wagner et al., 2004; Wallace, 2005). Stimulation of the neutrophil membranes produces

oxygen-derived free radicals. Superoxide anion is formed by the reduction of molecular

oxygen. which may stimulate the psoduction of other reactive n~olecules such as

hydrogen peroxide and hydrosyl radicals, The interaction of these substances with

arachidonic acid results in the generation of chemotactic substances, thus perpetuating the

inflammatory process (Harris, 1990).

1.2.6 Anti-inflammatory Drugs

1.2.6.1 Nonsteroidal Anti-inflammatory Drugs (NSAIDs)

All NSAIDs except for acetaminophen, are antipyretic, analgesic and anti-inflammatory.

They are routinely used for the relief of pain and inflammation associated with

osteoarthritis in dogs and horses and for colic, navicular disease and faminitis in horses

(Thompson, 2005). There is also an increase in the use of NSAlDs for the relief of

perioperative pain in companion animals. Some of the NSAIDs includes: aspirin,

acetaminophen, phenylbutazone, ineclofenanlic acid ketoprofen, etodolac, vedaprofen,

meloxicam, deracoxib etc. They act primarily to reduce the biosynthesis of prostaglandin

by inhibiting the enzyme cyclooxygenase. This is also the basis of mast of their undesired

actions which include: gastrointestinal (GI) irritation, GI ulceration, and GI bleeding

(Ritter et al., 1995). Thonlpson (2005) reported bIood ciyscrasins in dogs, cats. and horses

following long term NSAID therapy. Acetaminophen administratio11 in cats is associated

with Meinz body anaemia, methaemoglobin, hepatic failure, and death. Administration of

phenylbutazone is also associated with bone marrow dyscrasias.

1.2.6.2 Corticosteroids

Corticosteroids are the inost commonly used anti-inflarnluatory drugs (Thompson, 2005).

They have been used in 60-70% of rheumatoid arthritis palients where they exhibit

prompt and dramatic effects (Hellman and Stone, 2U04). Some of the commonly used

corticosteroids include the short to medium-acting glucocorticoids e.g. cortisol, cortisone,

prednisone, prednisolone, methylprednisolone and meprednisonc. Intermediate-acting

glucocorticoids include: trianxinolone, paramethasone and fluprednisolonc, while long-

acting, glucocorticoids include betarnethasone and dexalnethasone (Thompson, 2U05).

While corticosteroids can be highly effective in suppressing or preventing inflammation,

their anti-inflammatory effects are inherently linked with suppression of thc immune

response, hyperglycaemia, muscle wasting and redistribrition of the body fat, espccially

when employed in long tenn therapy (Hellman and Stone, 2004 ; Thompson, 2005).

1.2.6.3 Dietary Manipulation of Inflammation

It has been demonstrated that dietary manipulation which substitutes unsaturated fatty

acids (such as eicosapentaenoic acids, found in marine fish) causes the alternative fatty

acids to be metabolised, changing the finaI prostaglandin and leukotriene products of thc

inflammatory process. The results of clinical studies suggest that therapy with dietary

eicosapentaenoic acid decreases both lnornillg stifiless and the number of tender joints in

patients with rheumatoid arthritis and erythn~a associated with psoriasis (Hellman and

Stone, 2004).

1.2.7 Methods of Inducing Inflammation (Inflammatory Models)

1.2.7.1 Mouse ear oedema test

Oedema caused by skin irritation was adapted by Tubaro et al. (1985) and Tragni ut ui.

(1985). The technique involves the application of croton oil (an irritant) on the inner

surface of the right ear of anaesthetised mice. This is achieved by dissolving 75 pg of

croton oil in 15 111 of acetone per ear. Six hours later, the mice are killed by cervical

dislocation and a plug (6 mm) is taken from both the treated and untreatcd ears. The

oedematous (inflammatory) response is qaantified as weight difference between the two

car plugs. The anti-inflammatory activity is evaluated as percent oedema reduction in the

animals treated with the substances under test with respect to control animals, treated

with the irritant alone.

1.2.7.2 Carrageenin paw oedema test

Winter et 01. (1962) described the induction of oeden~a in the hind paw of rats, using

carrageenin, a phlogistic agent. Adult Wister rats are used after a 12 hour fast. Animals

are deprived of water only during the experiment. Inflammation is induced by injecting

O.lml of a 1% (w/v) solution of carrageenin into the subplanter surface of the right hind

paw of the rats. Oedema formation is quantified as foot increase and measured by water

displacement using a plethysmometer, at 1 hour intervals for 8 hours after carrageenin

injection. The anti-i~~flammatory activity is calculated at each time of observation as

percent inhibition of oederna in the animals treated with the substances under test in

con~parison to control animals.

1.2.7.3: Cotton pellet granuloma test

A teclinique for inducing subacute inflan~n~ation was adapted by Hideo and Masanao

(1975). It involves preparing cotton rolls into 5 mrn section. Four cotton pellets weighing

30 mg each are sterilized and impregnsted with 0.4 rnl of 5% aqueous solution of

ampicillin. Under ether anaesthesia, the pellets are introduced subcutaneousIy through a

skin incision in the back of the animals (Wister rats). Food and water are given to the

animals ucl libitzrrn for five consecutive days. 011 the fifth day, the animals are sacrificed

with chloroform, the granulomas (cotton pellets) removed. dried for 24 hours at 60°c,

and the dry weight determined. The difference between the initial and the final dry

weights are considered to be the weight of granulornatous tissue. This technique is based

on the fact that subacute inflammation results in formation of granulomatous tissue.

1.2.7.4 Detection of Myeloperoxidase (MPO) Activity by Microtitre Assay

A microtitre system based on kinetic assays of MPO was used (Andrews and Krinsky.

1982; Stark et al., 1992). This method utilizes two reagents: Reagent 1 comprises 100

mM sodium acetate, pH 6.0, 1% hexadecyltrimethylainrnoni~lm bromide (HETAB) and

20 n1M ethylenediaminetetracetate acid; Reagent 2 comprises 1.0 mM H20L: 1%

HETAB. 3.2 mM 3, 3, 5, 5-tetramethylbenzidine (TMB), A 100 pl volun~e of cell

suspension in phosphate-buffered saline (PRS) and 100 pl of reagent 1 are added to

microtitre wells. After 20 niinutes of incubation at room temperature to allow HETAB to

lyse the cells and to liberate MPO from the neutrophil granules, 100 p1 of reagent 2 is

added by n~ultichannel pipette. The optical densities of all wells OF the plate are recorded

by an automated microplate reader at G30 nm.

This method is based on the fact that the lysosome granules of mammalian

polymorphonuclear cells contain the enzyrrie'myeloperoxidase (Lam, 1997) and that

during inflnm~natory reactions, these cells infiltrate the site of injury (Lees, 1998).

CHAPTER TWO

MATERIALS AND METHODS

2.1 CHEMICALS

Thc following chemicals (analytical grade) were used.

Acetone (sigma, Spain), Methanol (Sigma-Aldrich, Germany), Petroleum ether 40-60'~

(Sigma-Aldrich, Germany), Chloroform (Sigma-Aldrich, Germany), Carrageenin (Sigma,

Spain). Croton oil (Sigma, Spain), Acetic acid (Sigma, Spain).

2.2 INSTRUMENTS AND GLASSWARES

2.2.1 Instruments

Analytical weighing balance (Metler H30 , Switzerland), Electric oven (Gallenltamp.

England), National Blender (Japan), Dissecting set, stainless steel wire cages, waterbath,

hammer, cooking knife, micropipette (Filmpipette@ Labsystems, Finland), Intubation

tubes.

2.2.2 Glasswares

Beakers, Burettes. Pipettes, Test-tubes, Conical Flasks. Funnels, Measuring Cylinders.

Wash bottles, Glassrods, Ceramic mortar and pestle

2.3 DRUGS

Indomethacin (Sigma, Spain)

Ketamine HCI (Rotexmedia, Germany).

2.4 ANIMALS

Rats:

Out bred Albino Wister rats of both sexes suppliecl by Mrs C. Eleanya. of the Department

of Pharmacology, University of Nigeria, Nsulcka were used for the experiments. The

weight of the rats varied between 75 and 230 g. They were kept in clean stainless steel

wire mesh cages. Standard commercial pelleted feed (Guinea Feed@, Nigeria) and clean

drinking water were given to them ad libi/vnz.

Mice:

Outbred Albino mice weighing between 16 and 40 g supplied by Dr. A. 0. Anaga of the

Faculty of Veterinary Medicine and Mrs 0. Eleanya of the Faculty of Pharmaceutical

Sciences, both of the University of Nigeria, Nsukka, were uscd for the experiments. They

were housed in clean plastic cages, supplied with clean drinking water and fed with

standard comn~ercial pelleted feed (Guinea Feed@ , Nigeria).

2.5 COLLECTION AND EXTRACTION OF PLANT MATERIALS

(a) Based on foIkloric uses and with the assistance of traditional healers, fresh

sa~nples of some Nigerian plants were collected in June, 2004. They were

identified by Mr. A. 0. Ozioko of the Botany Department, University of

Nigeria, Nsukka. The plant materials included: the leaves of C70stu.s qfir,

Ficus hmingii , Urenu lohulo, Rifchieu c.q~pnr.oides, Pa/isoia hirszrfct, und

Seluginellu nzysorus. Also collected were the roots of Ur-enn lohuta, Ritchiea

c~ryyuroides, and the bulb of C,'r.inzm jugus.

The samples were cut into small pieces with a cooking knife and dried under mild

sunlight. Subsequently, they were pulverised into coarse powder in the Depaflment of

Crop Science. University of Nigeria. Nsukka. Twenty gratns of each sample was

separateIy macerated in 200 ml of chloroform for 24 hours with intermittent shaking.

They were then filtered into clean conical flasks, allowed to evaporate to dryness and

preserved for preliminary examination (screening) for anti-inflammatory activity.

Large quantity of P. hii*szlta leaves was collected in Nsukka town, in May, 2005. by Mr.

A. 0. Ozioko of Botany Department, University of Nigeria, Nsukka. The fresh leaves

were dried under mild sunlight, then pulverized into coarse powder in the Department of

Crop Science, University of Nigeria, Nsultlta. Seven hundred grammes of the pulverized

sample were macerated in 3.5 litres of petl-ole~nl ether (40-60'~) for 24 hoi~rs with

intermittent shaking. Thereafter filtration was done with the help of filter papers and

funnel into a weighed beaker. The solvent was allowed to evaporate at room temptmture.

The marc was recovered, dried over night at room temperature and 500 g of it was

macerated in 3.0 litres of chloroforn~ and shaken at I hour intervals for 24 hours. The

resulting extract was obtained by filtering through filter paper into a weighed beaker and

allowed to evaporate to dryness at room temperature. Again the marc was recovered,

dried as before and extracted with methanol, using the same method. The filtrate this time

was dried in an electric oven at 4 0 ' ~ .

Determination of the Percentage Yield of the Extracts

The percentage yield of the extracts were calculated using the formula

GI % Yic~ld(wi'~~l)=(-) 100

17

Where

a is the weight of recovered extract

b is the weight of plant ~naterial used

2.6 WHOLE ANIMAL EXPERIMENTS

2.6.1 Screening of Plants for Anti-inflammatoty Activity

The extracts were tested for anti-inflammatory activity following the method of T'ubaro ef

LII. (1985). Cutaneous inflammation was induced by applying 75 pg of crcton oil

dissolved in 15 p1 of acetone per ear, on the inner surface of the right ear (surface:about 1

cm2) of anaesthetiscd mice.

'Thirty three (33) mice weighing between 21 and 27g and divided into eleven groups (A-

K) of t h e e mice each. Also. the extracts and in.domethacin were separately weighed into

test-tubes, after which the inflammation-inducing solution was prepared by dissolcing the

croton oil in acetone. The extracts and indomethacin were dissolved with the phlugistic

solution (croton oil in acetone), sealed, labelled and placed in ice to avoid evaporation.

The animals were anaesthetised with ketamine HCl (145 mg/kg, i.p.) and treated as

follows:

( I 000 pg) of the chloroform extract of Corns nfir IleafS, Fjctn Ihoningii (leaf), Ritchietr

~zrppuroides (leaf). R. cupparoi~ks (root), Uwna luhuta (leafi, U. lobcrta (root), pnlisotcr

hiisutu (leaf). Selugineh mysoros (leaf) and C3intm7 jrrgtts {bulb) respectively, clissolved

in 15 p1 of the phlogistic solution and appEied to the inner surface of the right ear. Group

K had indomethacin (0.1 mg/ear) dissdved in I5 pl of the phlagistic solution applied to

the inner surface of the right ear.

Six hours after induction of intlammation, the mice were sacrificed by cervical

dislocation and a plug (6 mm) was taken from both the treated and the untreated ears. The

ocdernatous response was quantified as weight difference between the two p l ~ gs. The

anti-i~iflammatory activity was evaluated as percent oedema reduction in the animals

treated with the substances under test with respect to control animals, treated with thc

irritant alone. This was calculalted according to the following forn~ula:

Where

x is the mean oedema of the treated group

y is the mean oedema of the control group

2.6.2 Test of Different Estracts of Pdisotn ltirsutn for Anti-inflammatory Activity

Five groups of Albino mice weighing betwecn 20 and 28 g, each group having fivz mice,

were used to detennine which of the extracts of P. himutu will exhibit the highest anti-

inflammatory activity, using the mouse ear oedema model as described by Tubaro c/ al.

(1985).

Group A: Served as control and received only the phlogistic solution. containing 75 rng

of croton oil dissolved in 15 pI of acetone, applied with the help of a micropipetfe, to the

inner surface of the right ear of mice anaesthetised with 145 mgkg of Ketamine HC1

intraperitoneally.

Group B: Petroleum ether extract of P. I?~J.SLI/U was mixed with 15 p1 of the

inflammation-inducing solution and applied as in group A.

Group C: Chlorofornl extract of P. hirsufn was dissolved in 15 p1 of the inflammation-

inducing solution and applied to the inncl- surface of the right car of anaesthetised mice

using a micropipette.

Group D: Methanol leaf extract of P IZ~~SZI /L I was dissolved in 15 111 of the pl-logistic

solution and applied to the inner surface of the right ear of anae~he~ised mice.

Group E: Served as positive control. One hundred (100) microgram per ear of

indornethacin was dissolved in IS ,u1 of the inflamnlation-inducii~g solution and applied

as in group D.

After six hours, the mice were sacrificed by cervical dislocation and 6 inn1 diameter plugs

were obtained from both the treated and untreated ears and weighed with a Metler

Analytical balance. The differencc between the right (treated) ear and the left (uidreated)

ear was quantified as the oedematous response. The anti-inflammatory activity was

evaluated as percent oederna reduction in the animals treated with the extracts and

indomethacin with respect to control animals.

2.6.3 Rose - Response Effect of the Chloroform Extract of P. Izirsrttrr

The method of Tubaro ef al. (1985) was adopted for this study. Thirty-six (26) mice

weighing between 21 and 30 g were used for the study. The mice were divided into six

groups of six mice per group. The negative control group received 75 pg of croton oil

dissolved in 15 pl of acetone (inflammation-inducing solution) on the inner surface of the

right ear. The test groups had varying doses: 100, 200, 300, and 400 pg/eas of the

chloroform extract of P. hirsz.rtm, dissolved in 15 p1 of the phlogistic solution, applied to

the same site.

Six hours later. the animals were sacrificed by cervical dislocation and 6 mm diameter

plugs were taken from both ears and weighed. 'The difference in weight, between the

treated and untreated ears was quantified as the sedematous response. The anti-

inflainmato~y activity was determined as ~)crcent oedema inhibition in the test groups

with respect to the negative control animals (untreated group).

2.6.4 Acute Toxicity Test of the Chloroform Leaf Extract of P. kirsiifn

Five groups of mice of both sexes weighing between 16 and 38 g, with each group having

five mice were used to determine the acute toxicity of the chloroform leaf extract of P

hii-s~{tu. Four groups (A-D) were treated orally with varying doses of the extract (250

nlglkg. 500 mglkg, 1000 mg/kg and 2000 mglkg). Group E served as control wi-h each

mouse receiving 0.5 ml of distilled water. The animals were allowed access to feed and

water r r d lihilum for 24 hours. They were observed for signs of acute toxicity and death.

2.6.5 Carrageenin-induced Paw Oedema Test of the Chloroform Extract of P.

11 irs z4tu

The experiment was conducted following the method of Winter et cd. (1962:t. Acute

inflammation was induced by injecting 0.05 ml of 0.6% carrageenin in 0.9% sodium

chloride (norinal saline) in the pIantar region of the right hind paw of the i~nimals.

Oedema formation was quantified as foot volume increase and measured by water

displacement using a graduated test-tube mounted on a stand.

Six groups of Albino Wister rats of both sexes weighing between 98 and 110 g. each

group having six rats, were used for the experiment. The study was set up as folloivs:

Group A served as the positive control and was given 10 n~glkg indomethacin by gastric

intubation, one hour before induction of inflamn~ation. Groups B, C, D and E were

treated with 100, 200, 400 and 800 mglkg respectively. of the chloroforn~ extract of P.

hirsuta by gastric intubation, one hour before induction of inflammation. Group F served

as negative control and received equivaleilt volume of water.

I11 a11 the animals, the volun~e of water displaced by the right hind paw of the rats before

the administration of the phlogistic agent was determined. After carrageenin hjection, the

volume of water displaced by the treated paw was measured at one hour intervals for six

hours. 'The anti-inflammatory activity was calculated at each time of observation as

percent inhibition of oederna in the animals treated with the test substances in comparison

with the control animals. This was calculated using the following formular:

u - X % Irz/~ihi/iorz = 100 (1 - ------)

h - y

Where

b is the mean paw volume of the control rats after carrageenin injection

y is the mean paw volume of the control rats before carrageenin injection

x is the mean paw volume of treated rats before carrageenin injection

a is the mean paw volume of treated rats after carrageenin injection

2.6.6 Cotton- pellet Granuloma Test of the Chloroform Extract of P. Itirsrrfn

The method described by Iiideo and Masanao (1975) was adopted for the experiment.

Thirty-six adult Albino Wister rats of both sexes, weighing between 180 and 220 g were

uscd for the study. They were weighed, marked and divided into six groups (A-I:), each

consisting of six rats. Cotton pellets, each weighing 30 ing were prepared by clolciing

sterile cotton wool into about 5 mm sections. They were tied in a nylon bag and sta-ilized

again in boiling water for one hour. 'The rats were anaesthetised in a glass hooc, using

chloroform and cotton wool. About 2 cm diameter section was shaved on either side of

the dorsal midline of each rat. The shaved areas were disinfected with phenol ( 1 ~ 0 ~ ' ~ ' )

and methplated spirit. Using surgical blade and forceps, single skin incisions were made

and the cotton pellets inserted subcutaneously.

Animals in groups A. B, C and D were treated with the chloroform extract of P. hirstrtu

(50 mglkg. 100 mglkg, 200 mglkg and 300 mglkg respectively) by gastric intubation,

once daily for five days. Group E received indomethacin (10 mgtkg) once daily for five

days, using the same route. Group F served as negative control and the rats were given

ecpivalent volume of distilled water daily for five days by gastric lavage.

On the fifth day, the animals were sacrificed by cervical dislocation, The cotton pellets

were removed and dried for 24 hours at 60 'c: in a hot air oven. The dry weights were

then determined. The differences between the initial and final dry weigh.:s were

considered to be the weight of granuloiuatous tissues produced. The anti-intlainmatory

activity was quantified as percent reduction in granuloinatous tissue formed in the treated

groups wit11 respect to control.

t, 'A:

2.6.7 Acetic acid-induced Writhing Test of the Chloroform Extract of P.

hirslrtcr

The test was performed as described by Knster el a!. (1959). Thirty-six oulbred Albino

mice weighing between 21 and 27 g were used for the experiment. The mice were

divided into six groups (A-F), each consisting of six mice. Groups A, B, C and [I werc

treated with the chloroform extract sf P. hir.s.u(c/ (150 mg/kg, 300 rngkg, 600 mgthg and

1200 mgkg respectively) by gastric intubation. Group E received indomethatin (10

mgfkg) orally. while animals in group F served as control and were give11 equivalent

volume of distilled water. One hour later, all the aniinals were injected intraperitmeally

with 10 mI/kg of 0.7% acetic acid in distilled water. Ten minutes later, the number of

writhes (abdominal constrictions) was recorded for fifieen minutes by visual observation

of the animals. The antinociceptive effect was evaluated as percent reduction in the

number of abdominal c~nstrictions in the treated groups with respect to control.

2.6.8 Statistical Analyses

The Pharmacological data obtained were analysed using the Analysis of Variance-

ANOVA.

2.6.9 Preliminary Phytochemical Analyses of the Chloroform Extract of P.

lz irsrrtu

The test was carried out according to the procedures outlined by Harbourne (1973) .

Trease and Evans (1989).

2.6.9.1 Test for Carbohydrate

To 0.1 g of the extract 2ml of distilled water was added, boiled and filtered. To the

filtrate, few drops of Molisch's reagent were added. Concentrated sulphuric acid was then

gently poured down the side of the test-tube to form a lower layer. The column was

observed for the presence of a purple interfacial ring.

2.6.9.2 Test for Alkaloids

To 20 ml of 3% sulphuric acid in 5% ethanol, 2 g of the extract was added, then heated

on boiling water bath for 10 minutes, cooled and filtered. Few drops of Meyer's reagent,

Dragendoff s reagent, Wagner's reagent and picric acid solution (1%) were separately

added to 2ml of the filtrate.

The remaining filtrate was placed in 100 n ~ l separating funnel and made alkaline with

dilute ammonia solution. The aqueous alkaline solution was separated and extracted with

5 ml portions of dilute sulphuric acid. The extract was tested separately with few drops of

Mayer's. Wagner's, Dragendoff s reagents and picric acid solution. The mixtures were

observed for colour change and the presence of precipitate.

2.6.9.3 Test for Reducing Sugar

To 5 1111 of a mixture of equal parts of Fehling's solution I and 11, 5 ml of aqueous extract

was added, then heated on a water bath for Sminutes. The mixture was observed for the

presence of brick red precipitate.

2.6.9.4 Test for Glycosides

To 0.1 g of the extract in a test-tube 51111 of 3% Sulphuric acid was added and boiled for

15 minutes on a water bath, then cooled and neutralized with 20% potassium hydroxide

solution. 10 ml of a mixture of equal parts of Fehling's solution I and I1 was added and

boiled for 5 minutes. The mixture was obscn~ed for a more dense brick red precipi~ate.

2.6.9.5 Test for Saponins

To 0.25 g of the extract 20 1111 of distilled water was added and boiled on a hot water bath

for 2 minutes. The mixture was filtered t~hi le hot and allowed to cool. The filtrate was

used for the following tests.

1 . To 5 nil of the filtratc 15 1~11 of distilled water was addcd and shaken

vigorously. The mixture was observed for the presence of stable froth (foam)

2. To 5 ml of the filtrate was added 5 ml of Fehling's solution (equal parts of I

and 11) and the content were heated on a water bath. The mixture was

observed for reddish precipitate. Further heating with sulphuric acid was done

and observation was made for the appearance of brick red precipitate.

2.6.9.6 Test for Tannins

To l g of the extract 20 rnl of water was added, boiled, filtered and used for the following

tests:

1 . To 3 nil of the filtrate, few drops of ferric chloride were added. The mixtl~re was

observed for a greenish black precipitate.

2. To a little of the filtrate was added lead acetate solution. The mixture was

observed for a reddish colour.

2.6.9.7 Test for Flavonoids

To 0.2 g of the extract 10 ml of ethylacetale was added and heated on a water bath for 3

minutes. The mixture was cooled and filtered. To 4 ml of the filtrate 1 rnl of dilute

ammonia solution was added and shaken. The layers were allowed to separate and the

ammoniacal layer was observed for appearance of yellow colour.

2.6.9.8 Test for Resins

1. To 0.2 g of the extract 15 ml of 96% ethanol was added and filtered. The

alcoholic extract was then poured into 20 ml of distilled water in a beaker and

observed for the presence of precipitale.

2. To 0.2 g of the extract 15 ml of chloroforin was added and filtered. The extract

was concentrated to dryness. The residue was re-dissolved i n 3 rnl of acetone

and another 3 ml of concentrated hydrochloric acid was added. The mixture

was heated in a water bath for 30 minutes and observed for a pink colour

which changcs to magenta red.

2.6.9.9 Test for Proteins

To 0.5 g of the extract 20 1111 of distilled water was added and filtered. The filtrate was

used for the following tests:

1. To a little portion of the filtrate in n test-tube, two drops of Million's reagent were

added. The mixture was observed for thc presence of white precipitate.

2. A crystal of copper sulphate was added to 2 nd of the filtrate, followed by Z drops

of potassiunl hydroxide solution. Thc mixture was observed for a purple or pink

colour.

3. To a little portion of the filtrate was added a few drops of picric acid and observed

for a yellow precipitate.

2.6.9.10 Test for Fats and Oil

The extract (0.1 g) was pressed between a filtere paper and the paper was observccl. A

control was also prepared by placing two (2) drops of olive oil on a filter paper.

Translucency of the filter paper indicated the presence of fats and oil.

2.6.9.1 1 Test for Steroids and Terpenoids

A quantity of ethanol was added to 1 g of the extract and refluxed for a few minutes and

filtered. The filtrate was concentrated to 2.5 ml on a boiling water bath. Hot Distilled

water (5 ml) was added to the concentrated solution. The mixture was allowed to stand

for 1 hour and the waxy matter was filtered off. The filtrate was extracted with 2.5 ml of

chloroform using a separating funnel. To 0.5 ml of the chloroform extract in a test tube

was careft~lly added 1 ml of concentrated sulphuric acid to form a lower layer. A reddish

brown interface showed the presence of steroids.

Another 0.5 ml of the chloroform extract was evaporated to dryness on a water bath and

heated with 3 nd of concentrated sulphuric acid for 10 minutes on a \vater bath. A grey

colour indicated the presence of terpenoids.

2.6.9.12 Test for Acidic Compounds

A small quantity of the extract was placed in clear dry test-tube and sufficient water

added. This was warmed in a hot water bath, and then cooled. A piece of water-wetted

litmus paper was dipped into the filtrate and the paper observed for a change of colour

from blue to red.

CHAPTER THREE

RESULTS

3.1 EXTRACTION

3.1.1 Extraction with petroleum ether

Weight of pulverized sample used = 700 g

Weight of extract recovered = 2.64 g

3.1.2 Extraction with Chloroform

Weight of pulverized sample used = 500 g

Weight of extract recovered = 8 g

3.1.3 Extraction with Methanol

Weight of pulverized sample used = 500 g

Weight of extracted recovered = 13 1 g

131 100 % Yield (1.01 w ) = -- x -

500 1

The petroleum ether extract of Pulisotu hir.rri/u was oily and brownish in colour

The chloroform extract was greenish and sticky while its methanolie extract was dark

green in colour with the greenish pigment of the leaves adhering to the wall of the flask

while the remaining extract was decanted.

3.2 SCREENING OF PLANTS FOR ANTI-INFLAMMATORY ACTIVITY

The chloroform extract of each of the plants screened significantly (P<0.01) inhibited the

mouse ear edema induced by croton oil (Fig.3.1). Whereas Crinum jcgus, Urenu lohlr/a,

Selnginellu mysorus, Costus i!fer and Rftckicu cr~pparoides (root) reduced the edematous

response by about 32-52%, Riichica cclpparoides (leaf) and Ficus thoningii exhibited

83.78% and 95.05% inhibition of edema. The extract of Pulisotu hirsttta provoked the

highest anti-inflammatory activity (96.40% edema inhibition).

Group treatments

Figure 3.1 : Percentage inhibition of oedema by chloroform extracts of different plants tested at 1000 pglear.

3.3 EFFECT OF DIFFERENT EXTRACTS OF Pnlisotcr ltirsritn ON CROTON

OIL-INDUCED INFLAMMATION IN MICE

When compared to the control, 300 mg/ear of the different extracts of P, hirsutci

significantly (P<O.O I ) inhibited the edematous response to croton oil topically applied on

the ear of mice. While the petroleum ether extract and the mcthanolic extract exhibited

edema inhibition of 14,4% and 25.6% respectively, the chloroform extract provoked a

53.8% edema inhibition. Indornethacin, a standard anti-inflammatory drug tested at 100

&ear provoked 32.29%1 inhibition (Fig. 3.2).

Petroleum ether extract

Chloroform extract

(3 00 pglear)

Methanol extract

Group treatments

lndomethacin

Figure 3.2: Percentage inhibition of oedema by different extracts of Pnlisorn hirsrriu,

using the mouse ear oederna model

3.4 DOSE-RESPONSE EFFECT OF THE CHLOROFORM EXTRACT OF

Pdisotn kirsrrtn ON CROTON OIL-INDUCED INFLAMMATION IN MICE

A11 doses of the extract (100, 200, 300 and 400 rng/kg) and indomethacin (100 mg/kg),

when compared with the control, significantly (P<0.001) reduced the mouse ear edema

induced by croton oil. The extract exerted its anti-inflammatory effect in a close-related

manner. The effects of the extract at 200, 300 and 400 rnglkg were greater than that of

indomethacin tested at 100 &kg (Fig. 3.3).

Group treatments

Indometliacin ( I00 &car)

Figure 3.3: Percentage inhibition of oedema by varying doses of the chloroform extract of P, hirmta using the mouse ear oedema model

3.5 ACUTE TOXICITY

No death was recorded after twenty-four hours of administration of the various doses of

the chloroform extract of P. hirsulcr. All the mice showed signs of dullness and

inappetance. At 2000 mglkg (highest dose) of the extract, the animals initially exhibited

signs of abdominal discomfort (retching and constriction of the abdominal muscle), but

became very dull and clustered together for about eight hours following oral

administration of the extract. The animaIs generally passed loose (pasty) stool.

3.6 EFFECT OF THE CHLOROFORM EXTRACT OF Pnlisoln Itirslr~fn ON

CARRAGEENIN-INDUCED PAW EDEMA IN RATS

The chloroform extract of P. ?zirsu/cr showed remarkable anti-inflammatory activity

against acute inflammation by suppressing the paw edema in a dose-related manner. In

the animals treated with extract of P. hir.wIu, and in those treated with indomethacin (10

mglkg), the reduction in edema was significant (Pc0.05) 120 minutes after carrageenin

administration. When compared with the control, 100 mg/kg and 200 mglkg of the

extract did not show any significant edema inhibition throughout the duration of the

experiment.

The maximum inhibition (55%) was achieved with 800 mgkg of the extract within four

hours of the induction of inflammation. In the control rats, there was a progressive

increase in paw edema after injection of' carrageenin. This reached maximal intensity

three hours following the injection of the phlogistic agent (Figure 3.4). The inhibitory

effect of the extract at 800 mg/kg against the cutaneous edematous response to

carrageenin is comparable to that of indomethacin (1 00 mglkg).

Time (hours) -

i -t- lndomethacin (1 0 mglkg) e 100 mglkg extract -+ 200 mglkg Extract

1 + 400 mglkg Extract . -- - - -

+ 800 mglkg Extract - -

+ Control (normal saline)

Figure 3.4: Effect of the chloroform extract of P. hirs~ltu on carrageenin-induced paw

oedema in rats

3.7 EFFECT OF THE CHLOROFORM EXTRACT OF P. liirsntn ON

COTTON PELLET-INDUCED GRANULOMA IN RATS

The oral administration of the chloroform extract of P. hii-sulu inhibited the development

of granulomatous tissue induced by dry cotton pellets inserted subcutaneously in rats.

Doses of 200 mg/kg and 300 mg/kg of the extract provol<ed inhibition of 19.0% and

25.4% respectively, which were significant ~vhen compared with the control. The lower

doses, 50 m g k g and 100 mg/kg of the extract exhibited 4.65% and 7.74% inhibitions

respectively. However, they were not significant when compared with the control.

Iildomethacin (10 mg/kg) produced a 40.3% inhibition of the granurnatous tissue. This

effect was significantly (Pc0.05) higher than that of the extract at the doses tested (Figure

3.5).

50 mglkg 100 mglkg 200 mglkg 300 mglkg indomethacin (1 0 mglkg)

Group treatments

Figure 3.5: Percentage inhibition of cotton pellet-induced granuloma in rats by varying doses of chloroforn~ extract of P hir..wlu.

3.8 THE EFFECT OF P. lrirsrttn CHLOROFORM EXTRACT ON ACETIC

ACID-INDUCED WRITHING REFLEX IN MICE

Varying doses of the chloroform extract of P. hirstrta (150, 300, 600 and 1200 mg/kg)

significantly (P<0.05) inhibited acetic acid-induced abdominal constriction in mice in a

dose-dependent manner. The lowest dose of the extract 1-50 mg/kg produced 12.57%

inhibition while the highest dose (1200 mgfkg) produced 38.98% inhibition of pain

induced by acetic acid in mice. 'The effect of indomethacin, administered at 10 mg/kg was

greater (40.26%) than that of the highest dose of the extract (38.78%) (Figure 3.61.

150 mglkg 300 mglkg 600 mglkg

Group treatments

Figure 3.6: Antinociceptive effect of the chloroform extract of P. I ~ ~ I - S L I I L I

3.9 PRELIMINARY PHYTOCHEMICAL ANALYSIS

A prelilnary phytochemical analysis of the ch lo ro fo r~~~ extract of P. hirsuru revealed that

it contains the following: carbohydrates, aIkaloids, glycosides, resins and flavonoids.

Whereas carbohydrates and alkaloids were present in small concentrations, glycosides

and flavonoids were present in moderately high concentrations. Resins were present in

high concentration (Table 3.1).

TabIe 3.1 : Preliminary phytochemical anaIysis of the chloroform extract of P. hirsufa

TEST

Test for Carbohydrate

Extract + Molisch's Reagent, then

Conc. H2SO4

Test for Alkaloids

Extract + Mayer's Reagent

Extract + Mayer's Reagent

Extract + Picric Acid

Extract + Dragendoff s Reagent

Test for Reducing Sugar

Extract + FehIing's Solution

I and IE

Test for Glycosides

Extract -I- 3% H2S04 + 20% KOH.

Then Fehling's Solutions I and I1

Test for Saponins

Extract + Boiled Water, then

shaken vigorously.

Extract + Fehling's Solutions I and

I1

Test for Tanins

Extract + Few Drops of Ferric

chloride.

Extract + Lead acetate Solution.

OBSERVATION

Purple interfacial ring

Milky Precipitate

Reddish Brown Precipitate

Yellow Precipitate

Brick Red Precipitate

No Precipitate

No Colour Change

Brick Red Precipitate

No Stable Froth (Foam)

No Precipitate

No Colour Change

No precipitate

No Colour Change

No Precipitate

INFERENCE

Test for Flavonoids

Extract + Dilute Ammoniacal

Solution -

Test For Resins

Extract + 96% Ethanol

Extract + Acetone -L Conc HCI + Heat in a Water Bath

Test for Proteins

Extract + Million Reagent

Extract -I- Copper sulphate crystal + KOH.

Extract + Picric Acid

Test for Acidity

Extract -k Hot Water + Litmus

Paper (Blue)

Yellow Colour in the

Ammoniacal Layer

Presence of Precipitate

Pink Colour which changed

to magenta red

No Precipitate

No Colour Change

No Precipitate

No Colour Change of the

litm~ls paper

+++ = Relative Abundance of compound

++ = Moderate Abundance of compound

+ = Relative Low Presence of compound

ND = Not Detected

CHAPTER FOUR

DISCUSSION AND CONCLUSION

The chloroform extract of Palisoru hiwtrf~t did not produce death at doses used in the

study. No death was recorded even at a high dose of 2000 mg/kg, which is an indication

that the extract was well tolerated by the mice. It showed that the extract was safe at the

dose range used in this study. However, the mice showed signs of dullness which

indicates that the extract depresses the central nervous system. This may also explain the

inappetance observed during the study. The loose stool (faeces) passed by the animals

following oral administration of the extract indicates that the extract could cause irritation

of the gastrointestinal tract, which is one of the side effects of non-steroidal anti-

inflammatory drugs (Wagner, 2004).

Out of the ten plants screened for anti-inflammatory activity, I! hirsu~a gave the highest

effect of 96.4% inhibition of oedema induced by croton oil, using the mouse ear oedema

model. This led to its being selected for fi~rther investigation. Following gradient

extraction of P. hirsuru leaf, the different fractions (Petroleum ether, chloroform, and

methanol) were tested for anti-inflammatory activity. The chloroform extract showed the

highest effect, provoking 53.8% oedema inhibition, against methanol and petroleum ether

extracts which exhibited 32.3% and 14.6% inhibitions respectively. This may indicate

that the anti-inflammatory activity of the plant is more in the chloroform than methanol

and petroleum ether extracts. The extraction of the anti-inflammatory principles by

chloroform suggests they should be of lipophilic nature. This characteristics enabled them

to cross the skin barrier and to exert their antiphlogistic effect (Asuzu et al., 1999).

In almost all the models of inflainrnation and analgesia used for testing the anti-

inflammatory activity of P. hirszrta extract, it was observed that the extract exerts its

effects in a dose-dependent manner. When applied topically, 200, 300, and 400 mglkg of

the extract compared favourably with indornethacin (1 00 mglkg), a standard nonsteroidal

anti-inflammatory drug. This justifies the folkloric use of the plant in the treatment of

boils.

The chloroform extract of P. I?irsutn significantly inhibited the carrageenin-induced paw

oedema in rats after oral administration. 'This effect was also dose-dependent and peaked

four hours after injection of carrageenin. 'The potency exhibited by the extract following

oral adn~inistration shows that the active components are stable a.nd not destroyed during

the "first pass effect" in the liver. The extract also significantly reduced pain induced by

systemic administration of acetic acid in mice, implying that it may have an anti-

inflammatory property similar to that of indomethacin. This explains why the natives

who drink infksions of P. hirsuta extract get relief from pain and tenderness of the joints

associated with rheumatoid arthritis and other related diseases.

The analgesic effect of the chloroform extract of P. hirs~itn even at the highest dose (1200

mgkg) was lower than that of indomethacin (10 mgikg). This shows that the extract

possesses a weak analgesic property.

The chloroform extract of P. hii-sula when administered orally at the doses of 200 and

300 mg/kg for five consecutive days significantly inhibited cotton pellet-irduced

granuloma in rats indicating that the extract also may have activity against the

proliferative stage of inflammation (Percz, 1996).

The results obtained so far with the chloroform extract of P. h i ~ m r u pol-tray the extract as

a potential effective anti-inflammatory agent. However, in all the experiments carried out

in this study, relatively higher doses of the extract were needed to achieve an effect

comparable to that of indomethacin. This would likely be due to the crude nature of the

extract used for the experiments.

Although the mechanisms by which P. hiuszrta chloroform extract exerts its anti-

inflammatory and analgesic properties are not known, inhibition of the cyclooxygenase

pathway of arachidonic acid metabolism, which is the general mechanism of action of

NSAIDs (Ritter, 1995) could be suggested. Also, scavenging of free radicals could be

hypothesized, since the extract contains flavonoids which have been shown to be strong

scavengers of free radicals (Robak and Gryglewski, 1988).

In conclusion, the obtained results indicate that the chloroform extract of Pnlisota hirstrtu

leaf possesses significant anti-inflammatory and analgesic properties following topical

and oral administration. This confirms the validity of its local use for medicinal purposes.

However, hither studies have to be done to identify the active component (s) of the plant

and to determine their mechanism(s) of action.

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APPENDICES

Table 1: Effects of the chloroform extract of different plants on mouse ear croton oil-

Weight of treated ear ( X I O - ~ g)

induced inflammation , - - 1 Weight of I Difference in (Dose of extract = 1000 wlear; dose of croton = 75 pgkar)

7-

Plant Extract Mean difference in weight of treated and untreated ear 5

SEN ( x 10.' g) 2.22 h 0.1 1

untreated ear (X g)

untreated ear ( ~ 1 0 " g)

A Control

weight of treated and

B CosCzts qf ir (leaf) C Ficus thoningii (leaf) D Ritchie~r

coppuroides (root) F Urenu Iohuta (leaf) F Ui-em lobata (root) Ci

mysorus (leaf) - J Crinzrnz jagus (bulb) K Indomethacin (100 p g / 4 1 *Values significantly different from the control: p<0.05

Table 2: Effects of different extracts of P. hirsuta on mouse ear croton oil-induced inflanmation (Dose of extract = 300 pg/ear; dose of csoton .= 75 pgkar)

Extracts

A Control

B Petroleum Ether

(7

Chlrvform

D Methanol

E Indomethacin ( 1 00 pglear)

Weight of treated ear ( X I 0-2 g)

Weight of untreated ear ( X 10.; g)

I

"Values significantly different from the control: p<O

Difference in weight of treated and untreated ear

gf

Mean difference in weight of treated and untreated ear f SEM (x I o-' g) 2.23 k 0.027

Table 3: Effects of varying doses of the chloroform extract of P. hirszrtu on mouse ear croton oil-induced inflammation.

A Control

Weight of treated ear ( x g)

4.92 5.03 4.87 5.60 5.61 5.75 4.60 4.67 5.09 5.05 4.95 4.85 3.80 4.27 3.96 4. I7 4.10 4.0 1 4.35 3.76 4.20 4.3 1 3.98 3.92 3.9 1 4.1 1 4.12 4.06 4.18 4.06 4.67 4.5 1 4.96 4.82 4.76 4.55

Weight of mtreated ear :X 10-3 g)

Difference in weight of treated and untreated ear ( X 10-j g)

* ~ a l u e s significantly

-- Mean difference in weight of treated and untreated ear * SEM ( x 1 0-3 g) 3.26 5 0.012

1 2.34 I

different from the control: p - 4

Table 4: EFfect of the chloroform extract of P. hirscrra on carrageenin-induced hind Paw i~lflammation in the rat.

1 l(0.02) I

i,(O.O5) * Values significantly different from the control: p < 0.05

Table 5: Effect of the chlorofoim extract of P. hir.s~r/ci on carrageenin-induced hind Paw inflalnlnation in the rat.

Group Treatment Extract 100 mglkg Extract 200 rngikg Extract 400 rnglkg Estract 800 mglkg Indomethacin 10 mglkg

Percentage Inhibition 4 hours 10

2 7

3 0

5 5

4 8

5 hours I 6 hours 3 hours 10

2 0

2 0

3 6

4 3

1 hour -

-

-

29

29

17 2 hours 5

11

20

3 2

30

17

16

26

45

4 5

I5

3 0

4 8

4 8

1____-

Table 6: Anti-nociceptive effects of P. hirsuta using the acetic acid-induced writhing reflex in mice.

Weight of animals (g)

Indomethacin (50 mglkg)

Volume of acetic acid (ml)

7-

-

-

-

I --

29.9 1 0.30 / 16 _ i

V a l u e s significantly different from the control: p<0.05

Number of abdominal constriction

Mean no. of abdominal constriction k SEM 26.50 * 1.09

56

Table 7: Effects of the chloroform extract of P. hirsrria on cotton pellet-induced granuloma in rats

Groups

4 Zontrol

3 50 111g/kg

-" ., 100 mglkg

1 200 mglkg

-?

!00 mg/kg

F Indomethacin (1 0 m g w

Weight of dried cotton I Weight of granulon~atous pellet (x 1 o - ~ g) tissues ( x 1 O'L g: Right side Left side

1 Left side

4.7 3.8 5.3 3 .O 4.4 3.9 3.6 3.0 2.7 4.6 5.8 5.2 4.6 4.4 3 -6 3.8 3.2 4.7 2.1 3.8 5.5 3.7 3.4 4.8 -- 3.8 2.3 4.6 3.4 2.6 5.5 __-- 4.5 2.8 2.2 2.1 2.5 2.8 3.4

( 2.9 1.9 2.6 1 1.4

*Values significantly different from the control: p<0.05

of granulomatous ( tissues :t SEh4 (x 1 o - ~ g) 4.3 * 0 . 2 4 ~ -