University of Nigeria Research Publications

MICHAEL, Izu A

Aut

hor

PG/M.Sc/80/697

Title

The Effect of Quercetin- 3- from the Leaves of Azadirachta Indica (Neem, Dogonyaro) on Rat

Liver.

Facu

lty

Biological Sciences

Dep

artm

ent

Biochemistry

Dat

e

November, 1984

Sign

atur

e

T I T L E - T H E KFFEXT O F QUERCFTIN-3-CLYCOSIDPS PHOI4 T H E LEAVES OF

AZADIRACHTA IIJDICA (NWI, DOCONYARO) ON wr LIVER.

P R E S U i T E D BY:

MR. N. I. AIUZODO ( P G / M s ~ / 8 0 / 6 9 7 )

I N PARTIAL FUWILhENT FOR T U AWARD OF' THE DEGREE O F

MASTER O F S C I E N C E S I N PEIARMACOLOGICAL BIOCIIElvlISTRY OF

T H E UNIVERSITY OF NIGERIA, NSULKA.

SUPERVISORS :

1) ~ k . 0. OBIDOA,

SGNIOR LECTURER,

DE2MTMiZNT OF B I O C B f i u S T R Y

2) DR. M. M. IWU,

SENIOR LECTURER,

DEPAXTMEZQ O F P W d C O G I J O S Y .

TilIS PROJBCT REPORT HAS BEEN APPROVED FOR THE AWARD O F

MASTER OF S C I E N C E DK;RU I N PHARMACOLOGICBL BIOCHEMISTHY

I N THE DEPARTl2ENT O F BI0CHEXISTR.Y O F !CEH U N I V E R S I T Y O F

N I G E R I A , N S U W .

Thie' work i a an original investigation by the candidate,

Mr. Michael I. Anazodo, and has not been published i n any

journal or work i n th i s Univereity or anywhere i n the world.

:-2 - 7, S U P E R V I S O R --- Heed, Department of Biochemietry External Examiner

-- Internal Examiner

CEIAPTm ON& LITERATURE REVIEW

Introduction .. Medicinal planter and k raditional medicine

Some remedies in traditional medici-?e

1.2.10. 8 .

1.3 The role of the l i ver in drug

(renobiotic) metabolism

1.4 Hepatic uiaroeomel eneyne systems that

catdlyae:' drug metaboli8m

1.5 Microsomal oxidative reaction8

1.6 Eneyme specificity . .

PAG4

i

ii

iii

T U L E OF1 C(iN!:',GiI ( ':r)$p~)

1.7.0 The mixed-function oxidase and its environment

1.7.1 Reaponse to chemical adveraity . . 1.8.0 The role of cytochrome F-450 i n drug metabolism

i

1.8.1 U.DPH - dependent oxidation . 1 .9 Enzyme induction . . . .

1 .'I 0.2 Morphology . . . .

1.10.4 Phemnacological uses . . . . 1.11 Purpose of study and rationale . .

CHAPTB TWO: IIA!l'EHIALS AND MElXODS

2.0 Materials . . . . 2.01 Plant materiala . . .

2.03 Animal feed . . .

2.05 Preparation of reagents/buf f era . . 2.06 Extraction of Quercetin-Wlycoaides .. 2.07 Phytochernical etudiee . . . 2.08 Qualitative t e s t s . . . .

TBULE OF CONTENT (CONTD. ) . -- P.4GE -. --

Alkeloids

F lavonoids

Anthraquinones

Fro t e i n s . . . 32

Identifi3nb'?.on and c h a r a c t e r i z a t i o n of t h e i s o l a t e s 32

T r ~ a t q c n t o f animals wi th t h e e x t r a c t 33

2+ I s o l a t i o n of microsonesby Ca p r e c i p i t a t i o n 34

Standard curve f o r t h e e s t ima t ion of p r o t e i n 37

Stnntiard curvc f o r t h e e s t ima t ion of

inorgnnic phosphate . . . . Standard curve f o r t h e e s t i n a t i o n of

paruinophcnol . . . . D ~ t e r m i n a t i m of phenobarbitone

s l e e p i n g t l u c . . .. Detern ina t io : of glucose-6-phos@atasc a c t i v i t y

De te rn ina t ion of NADPH - cytochrome C

( P-4.50) roductase a c t i v i t y

Determination of a n i l i n e hydroxylase a c t i v i t y

3.00 RESULTS . r

C H ~ L P I ' E ; ~ FOE: D I S C U S S I O N

4.00 D I S C U S S I O N .

REFETiEN C E S

PAGE

DEDICATION

DEDICATED TO MY BELOVED

MOTHER AND MY THREE

SISTERS CHINELO, NJIUEKA

AX11 OGECIIUKWJ .

-4 would l i k e t o express my sincere g ra t i tude t o a l l who made

t h i s project possible. My profound & r a t i t U d ~ goes to my

supervisors, Dr . 0. Obidoa, * . .* '

Biochemistry, University of ,..iA

L Senior Lacturer, Department

Senior Lecturer, Department of

Nigeria, Naukka and Dr . M. FA. I n ,

of Phasmacognosy, University of

Nigeria, Nsukka f o r t h e i r continued i n t e r e s t and expert academic

" advice.

I am a l so g r a t e fu l t o Dr . 0. F, C, Nwodo, Mr. Chibuaor Nnubia,

M r . F. C. Chilaka, M r . Awiwa Onyeneke, Nr . Oraegbuna, M r . Nwokedi,

w d dl members of the academic s t a f f of t h i s Department f o r their

contributions i n making t h i e piece of work a success,

Final ly , I must express my immense gra t i tude t o D r .

' Onyehodir i of the Faculty of Veterinary Medicine, University of

Nigeria, Nsukka f o r helping me i n the h i s to log ica l aspect , and t o

Mr. C. C. Abena of the Animal Breeding Section of the Faculty of

Veterinary Pledicine, f o r making avai lable the r a t s f o r the

~esea rch .

I$. I. Anazodo ( ~ r . )

iii

An aqueous e x L r a c t from t h e l e a v e s o f A c a d i r a c h t a i nc l i ca

( N e e r i i , Dogonyaro) c o n t c i ~ ~ l i n g two qui3rcct in-3=yl_ycusideswas

' ad1nirli;tered ( i p ) t o male a l b i n o r a t s i n t h e i n v c s t i y a t i o n : ;

Sephadex LH 20 column chromatoyraphy. F r e p a r z t i v e ti-dn la-ycr

chr~c jmatoyraphic t e c h ~ . l i q u e was used f o r t h e p u r i r ' i c a t i o n o f t h e

i s o l a t e s . F u r t h e r c h a r a c t e r i z a t i o n was by a ccjrnbj n a t i o n of

s p e z t r o s c o p i c a n d , c h e m i c a l methods , i n c l u d i n g U l t r a v i o l e t (UL');

I n f r a r e d (IR); P r o t o n Magne t i c Resonance (PMR); E l e c t r o n I o n i z a t i o n

Mass Spec t ra ( E I K S ) and d i r e c t ch roma tograp f i i c compar i son w i t h

r e f e r e n c e m a t e r i a l s .

These a n a l y s e s showed t h a t t h e e x t r a c t c o n , i s t e d e n k i ~ r l y

of qucrcet i n -3 - rhamnoc~ lucos ide ( K u t i n ) hnd qucrcctin--3-~'k1a1l!r~~>;,i.~1~

Q Q u r z : - c i t r i n ) . T h i s r e p o r t r e p r e s e n t s t h e f i r s t i s o l a t i o n cf

ilabbnoids ( 3 , 3 ' , d 8 , 5 , 7-pent~hydroxyflavon-3-rutinc~si~dtf ( i < u t i r \ )

t t 1 . i ~ r ncd ic i r i a l l y i m p o r t a n t s p e c i e s .

t h i s e x t r a c t , l i v e r t o body w e i g h t (4.10 - .e 0.38 f o r t r e a t e d r a t : ,

and 3.30 - + 0.61) and microsomal p r o t e i n t o l i v e r w e i g h t 7.44 - t 0.24 f o r t h e c o n t r o l r a t s ) r a t i o s were d~iferent between t r e a t e d

and u n t r e a t e d r a t s . Phenoba rb i tone - induced s l e e p i n g times were

s h o r t e r i n t r e a t e d a n i m a l s ( a t l e a s t l o % ) , NADPH cy toch rome C

(P -450) r e d u c t a s e a c t i v i t y

' decreased ( 1 .19x1 o - ~ + 0.40 and 4.76 x low3 2 0.63 umole/minute/mg

protein for test and control animals respectively) while aniline

hydroxylase activity increased in the treated animals (5.40 0.34

and 4.40 f 0.05 nmoles/hour/mg protein for treated and untreated

rats respectively). The extract also showed a concentration

dependent effect on aniline hydroxylase activity. Histological

studies on the liver showed hyperaria of the blood vessels as well

as increased cellularity suggesting increased activity of the liver.

These reeults suggest possible alterations in the

biochemistry of the rat liver cells following the administration of

the aqueous extract of A. (~eem). The chemical, bioche~aical

and pharmacological importance of these findings are dso

discussed.

CHAPTER ONE

LITERATURE REVIEW

1.00 INTRODUCTION

1.8 MEDICINAL YLIA"riLaJD TRADITIONAL MEDICINE.

More than ~ W O thirclSof t h e p lan t s p e c i e s s o far

i d e n t i f i e d a r e found i n t h e t r o p i c a l reg ions of t h e world. About

70% of t h i s populat ion a r e used i n t h e t h i r d world c o u n t r i e s i n

t h e p r a c t i c e of t r a d i t i o n a l medicine.

One of t h e major c r i t i c i s m s aga ins t t r a d i t i o n a l medicine

p r a c t i t i o n e r s ia t h a t t h e i r po t ions are n e i t h e r s tandard ized , n o r

are they dispensed t o the pnt ie i i t s i n speci f ied doses no r i n

s t r i c t l y r egu la t ed q u a n t i t i e s . The problem of s t anda rd iz ing a

crude drug prepara t ion , however, is n o t just t h a t of spec i fy ing

t h e anouni; o f t ho decoct ion t o bt. taken by tho pi1titm.t. I t i a

a l s o irnportan'l; that a l l s t a g e s of p repa ra t ions a r e si;andardizt.d.

These stages i n c l u d e c o l l e c t i o n of t h e plant, t h e e x t r a c t i o n

procedure und the f i n a l dosage form.

1 2.0 SOME REI'IEDIES IN TTIBDITIONA.L MEDICINE.

I n many African homes, t e e t h a r e cleaned i n t h e morning by chewing

the r o o t o r the alin,-&em ~ . . c o r t a i n plants u n t i l they acqu i r e

brush-like ends. The bm&t-U)oa&i~ then used t o brush the t e e t h

thrr- ;ugk~ly. I ~ ~ v e s t i g a t i o n s carr ied out on t h e an t imic rob ia l

a c t i v i t y of these chewing s t i c k s showed tha t they all possesa

antimicrobial a c t i v i t y ag i i n s t o r a l microb id flora, but t o t

varying degrees.

?.v , The antimicrobial a c t i v i t y of Zanthox~lilm zan thox~lo idsg (Odebiyi ,+" '* /-# .w '*

* .@% .,.- I and ~ofowora,l979) he8 been ehown t o be due t o befizoic acid

der iva t ives which are ac t ive st a of about 5 and a lkaloids

( canthin-6-one, berberine , chelerythrine, w d a four th unidentif ied

compound) which a r e ac t ive at a pH of 7.5 ( see s t ruc tures below).

I n the root of th is plant ex i s t s , therefore, compounds which w i l l

be ac t ive during heavy tooth decay (high @ - alkal ine) as well as

a f t e r a drink of, say, lime juice (low $I-acidic).

p o p u l a r trcalnic:nt f o r d i a r r h o e a ( D a l z i c l , 135C;. The

a n t l r n i c r o b i a l a c t i v i t y o f t h e water-: n t u r a t e d o i l has

b r e n shown t o be l ~ r o p o r t i o n a l t o t h e thyrnol c o n t e n t

( E l - s a i d c t a1 ?963).

" 1. 2. 3 !v:ONTi',?IOX T'CI.,;LNTCC;i

The plant FTontcr~~on to~cnt .o : ;n is w e l l known f o r i t : :

use i n parts of S o u t h America a s a tc:i drunk by women

f o r a b o u t 10 days during their menst r i in l cyc l e a s a

form of contrac~ptive, t o f a c i l i t a t e l a b o u r , s t i m u l a t e

m e n s t r u a t i o n , o r f o r t h e t e r m i n a t i o n o f e a r l y pregnancy.

::ahn e t al (14181) showed that t h e p l ~ n t c o n t a i n s t r a c e

amounts (somctirncs a s l i t t l e as 0.0075 percent. i n t h ~

I

is r e l a t e d t n 2,oapntonal !-ut is n o t s c t i v e . Roth com-

nunds are new and have n o t been found i n any o t h ~ r p l a n t .

L o a p t a n 0 2 Kon t an01

1 .2.4 HACENIA &3YSSINIC4 4

i I a ~ u n i y pb?rssinica is used as a t a e n i c i d e i n E th iop ia while

.m2 h&Q&&&& is used f o r expe l l i ng hbohornis , whipworme and legworr,

The aqueous e x t r ~ c t of both p l a n t s have been shown t o be a n t i -

he lmin t i c i n v i t r o ( ~ b e ~ a z and Tecle , 1980).

1.2.5 RAUWOLFfA Xw-mA: This and some other Rauwolfia spec lea a r e

ubiqui tous components of h e r b a l p repa ra t ions used throughout

Af r i ca t o t r e a t mental ly d i s t u r b e d p a t i e n t s . The r o o t i s g iven

i n t h s form of a decoct ion o r soaked i n l o c a l l y d i s t i l l e d gin.

The r o o t s of rrauwolfia spec i e s , e s p e c i a l l y E . -tori% and z. m m , are known t o con ta in t h e a l k a l o i d r eee rp ine which

possesses hypotensive and s e d a t i v e p r o p e r t i e s . Th i s roo t i s used

i n t t ~ b l e t fo rm i n modern medicine t o t r e a t s t a t e s of anx ie ty

and hypertcr\sion. Its use i n t r a d i t i o n a l medicine a l l over

Af r i ca f o r managing mental ly d i s tu rbed p a t i e n t s , t h e r e f o r e , c m

be j u s t i f i e d on a s c i e n t i f i c b a s i s , a l though t h e presence of

r e s e r p i n e i n t h e r o o t s o f . these. plants; .ma$ dt be known t o t h e

t rad i t i oad medical p r a c t i t i o n e r s .

1.2.6 D I C I T & I S ~ N Dropsy i s a cond i t i on i n which t h e

i n e f f i c i e n t working of t h e h e a r t l e a d s t o r e t e n t i o n of f l u i d and

hence t o gene ra l swe l l i ng of t h e body. Th i s was e f f e c t i v e l y

t r e o t e d i n &gland with a decoct ion c o n s i s t i n g of a mixture of

some 20 p l an t s . It was l a t e r discovered t h a t t h e only a c t i v e

ingredient was from the leaves of Foxglove - DinitaLlp Emuj-ea

( ~ e Strange, 1977). Since then t h i s p lant and the re la ted species

D. l anatq have been thoroughly investigakil culd t h a i r pharmscologically - ac t ive cons t i tuen t s a r e now known t o be s t e r o i d a l e lycosides having

cardiotonic ac t iv i ty . ' Digaxxn and d ig i tox in are both i ao la ted from

D ,. and bot'n compounds are o f f i c i a l drugs i n B r i t i s h and

. < other Pharmacopoeia.

DJ&Q&j&

Ygi d i g i toxose

digi toxose

1.2.7 CINCHONA SPECIES

The a c t i v e an t imala r i a l cons t i tuen t of Cinchona bark wae

i d e n t i f i e d as t h e a lka lo id quinine. - Quinine

Unfor tmntc ly this drug produces unders i rab le t o x i c s i d e e f f e c t s

such .:s f.rnp.:ir?d h e x i n g on prolonged use. ( ~ r e a s e and Evans,

1970). kJc:,;myts t o modify t h e structure of t h e drug showed t h a t . , .

* - 4 . ' ,&p,,..' $v.'.,". :< , , *.**, n o t n l l .:>?::'x of '32 molecule of qu in ine were necessary f o r ,?. .

* T +- "

@"*' -* , * a n t i r 1 C ~ r i o . l a c t i v i t y . Syn the t i c a n t i m a l a r i a l drugs were, t h e r e f o r e , *:$

devel ?p,?d. vLicln r e t c i n e d t h e a c t i v i t y of qu in ine with t h e t o x i c

ef f ef:trraducsd, for example chloroquine.

1 .2.6 ERYTHI~OXYWJ! COCA: . coca leaf . was showed by South limerican

in dim^ l c n g rgo t o prevent hunger and i n c r e a s e endurance wh i l s t

t h e ?a3ourr?rs vorked i n t h e f i e l d s . Yhc drug i s habi t -forning.

R e s e a x h on the leavos l e d t o t h e d iscovery of t h e a l k a l o i d

cocatne. Becmsc of t he

Cocaine.

t o x i c n a t u x ? coca leaf i s seldom used i n medicine but t h e

alka205? coo,iine i s employed a s a l o c a l a n a e s t h e t i c i n t h e form

of i 5s 'y~?roc?lJ.orid.e, benzoate o r borate.

t The use in traditional medicine of those various plant extracts

all over Africa and beyond for managing or %he treatme<t of verious

8 * v.,. C . ~ ailments, therefore, can be justified on a scientific basis, although

..,I**' . - I , .. ,*the active principles of the differant plants may not be known to the

-.- .." .&. - A +I..' .,.s L

.I..' . . traditional medical practitioners.

1.7 THE ROLE CF LIVER IN DRUG (XENOBIOTIC) MCTABOLa

The administration of several drugs and environmental chemicals

(foreign substances) can inhibit or potentiate the pharmacological

and toxicological action of other xenobiotics by altering the

activity of the drug metabolising enzyme systems in the liver

microsomes. The fate arid effects of xenobimtics in the liver Elre

modulated by several factors including diets, other xenobiotics,

age, sex, species and pathological state of the animal,

A consistent and prominent response of the liver cells to

drugs and chemicals is an increase in the smooth endoplAsnlic

reticulum (cameron .g$. &, 1973). On exposure to a large number of

chemicals, including steroids, polycyclic aromatic hydrocarbons,

anaesthetic agents, and insecticides, a proliferation of the smooth

endoplasmic reticulum is evident, and is acconpanied by an increased

activity of the drug metabolising enzymes, especially the microsomal

mixed function oxidases ( ~onneg, 1967; Schutlo-Herrnann, 1974).

R variety o f r~actionfl ( I ~ U & a, 1977). s w t h y & & o l m t h p t r n ~ m d

o t h e r polycycl ic hydrocarbons such as beneo(a) pyrene induce a

d i f f e r c n t bmoprotein, cytochrome P-448 ( LU & & 1973).

Itecent s t u d i e s have ind ica t ed t h e complexity of t h e induct ion

p a t t e r n of: ~n ic rosona l membrane p r o t e i n s t h a t are produced fo l lowing

- , the admin i s t r a t i on of such chemicals. From t h e experimental

obse rva t ions us ing SDS-PAGE t o ana lyse t h e polypept ides of t h e

l i v e r microsomes, i t became obvious t h a t a number of i nduc t ion

pa t t e r n s were produced by t rea tment with va r ious agents ( ~ a m e r o n

a, 1979). A miuimm of twelve d i f f e r c n t p a t t e r n s were ev iden t ,

and i t was suggested t h a t t h e hepcrtocnrcinogan, 2-ucctylaminofluorene

( 2-AA17),ns wel l as am dyes and n i t rosamine hepatocarcinogens

appear t o induce a h a o m p r o t e i n which d i f f e r s f r o n t h a t induceu by

pheno bcrbi tone and 3-methyl-cholanthrone ( Cameron & A, 1 976 ) . Vith r e spec t t o . x e n o b i o t i c t o x i c i t y , t h e l i v e r i s t h e major

organ f o r t h e body's defence mechanisms. The microsomal

metabol i s ing enzymes r m d e r s ~~Nxx p9lar t h e lipophilic foreign

and ,endogenous molecule in the liver. Tho i n c r e a s e i n

a c t i v i t y of t hese enzymes t h a t occurs a f t e r b i r t h is undoubtedly

an important defence mechanism. T h i s is evident from t h e f a c t t h a t

t h e d u r a t i o n and i n t e n s i t y of a c t i o n of many drugs are l a r g e l y

determined by the speed a t which they a r e b io t r ans fo rhed i n the body

by t h e eilqwes of t h e l i v e r microsomes.

1.4 HEPATIC liJ,CI,2i,t^j>lUL ENZYNE SY STUl TH1.T' CLTALYSES DRUG MBTABOLI 314 :

,,.. . . 2;: . .*.+. a:.:' ' The mic rosom~l drug metabol i s ing enzyme system of t h e l i v e r i s &* 2-.

h-+.rurp* i e,- '?

found i n t h e endoplasmic re t icu lum of the c e l l s . These enzymes r+w. -

?* d rkwL ., *. c a t a l y s e both t h e r educ t ive s p l i t t i n g of t h e azo-linkage and t h e

ox ida t ive Nademethylation of t h e amino azo dye. The r e a c t i o n

r e q u i r e s niootinami.de adenine d inuc lco t ide phonphate (NNIP+),

4. nico t i n m i d e adenine d i n u c l c o t i d e (NU ) , and melocular oxygen.

Heconbinction of t h e va r ious c e l l f r a c t i o n s of l i v e r homogenates

revealed. i;Ae requirement of both s o l u b l e and microsomal f r a c t i o n s .

Fu l l a c t i v i t y was obtained when t h e s o l u b l e f r a c t i o n was rep laced

- , by a NADPII gene ra t ing system, Glucoso-6-phosphate dehydrogenase o r

i s o c i t r i c w i d , IUF' and i s o c i t r a t e dehydroganasa o r by NADPH i t s e l f .

Magnesim i o n s were requi red f o r ful l . a c t i v i t y . The requirement of

both a redvciilg agent and molecular oxygen p l a c e s t h e r e a c t i o n wi th in

t h e e x t e r n a l mixed func t ion oxidase c l a s s i f i c a t i o n ason on, 1957, 1965)

which means t h a t t h e enzyme c a t a l y s e s t h e consumption of one molecule

of oxygen pe r molecu1.e of s u b s t r a t e w i t h one atom of oxygen appearing

i n t h e s ~ t b s - t m t e and the o t h e r ,etm, .. . . u.Wrgoin,~ equivalen-t redrlctions.

9

k wide v a r i e t y of o x i d a t i v e r e a c t i o n s a r e known t o occur i n

nicrosomes; deamination, 0-,N- and S d e a l k y l a t i o n , hydroxyla t ion

of a l k y l end a ~ j 1 hydrocarbons, e p o x i d a t i ~ n , formation of alkylol

d e r i v a t i v e s , 1;-hydroxylation, N- and S-oxidation and dehalogena t i on .

Azo and n i t r o r educ ta se a c t i v i t i e s are a l s o found i n h e p a t i c

Becaiisc. t h e r e cannot be i n advance a s p e c i f i c micronomal

system f o r every poss ib l e compound t h a t e n t e r s the body, and s i n c e

t h e numbor o f metabol ic pathways by which drugs can be biotransformed

a r e l i m i t e d , .the enzymes a r e non-specif ic and act on molecular

groups 'rfither than p a r t i c u l a r compounds. Sometiuiies t h e concurrent

admin i s t r a t i on of s e v e r a l drugs causes competi t ion f o r t h e s e

pathways. Sone conlpounds such as SKF 5258 ( a p r o t e i n s y n t h e s i s

i n h i b i t o r ) , w i l l i n h i b i t t h e s y n t h e s i s of microsomal enzymes and so

prolong t h e acticn of drugs like phenobarbitone, though t h i s

i n h i b i t i o n may be succeeded by s t imula t ion . S i m i l a r l y i f t h e l i v e r

i s d a ~ a g e d , o r i n imnature bab ie s where the enzymes a r e d e f i c i e n t ,

t h e metabolism of drugs such a s pethidiiie, morphine and

chloramphenicol can be g r e a t l y prolonged.

1 7.0 SXED-FUNCTIOPJ OOXIDBSE SYSTEM AIvD ITS E W I R O N I J I ~ T

> More than 4 x 1 0 ~ foreign compounds/cliemicals exist in our

environment, Plany of these drugs and environmental pollutants also -.. -* called xanobiotics are highly toxic and s growing number of these

*' * /IF

&* r' chemicals are being shown to cause mutations, cancer and birth defects. -9.- < ' ur+rd_l_l

&" ,A< 1.7.1 HESI%NS% TO CHi3QLCL.L iiDVE3ISITY

Most dmgs are metabolized in the liver, and metabolizing enzymes

can occur iri the soluble, mitochondrial, or microsomal fractions,

The most coumon routes--of drug metabolisn involve oxidation, reduction,

hydrolysis, and conjugution. Very often a drug is subjected to

severel competing pethways simultaneously, and the extent of formation

of the various metabolites depends on the reletive rates of the

various~interactions. In addition, very common metabolic reactions

proceed sequentially, and oxidation, redu-ction or hydrolyeis reactions -

( also called phase L reactions) are followed by conjugation ( phase - 2 reactions) of these derivatives.

Many xenobiotics are so hydrophobic that they would remain in - -

the organisn indefinitely were it not for phase and phase drug -

metabolizing enzymes. During phase netobolism, polar groupa

(such ae -011) are introduced into the parent molecule, thereby

presenting the phase a conjugating enzymes with substrate.

Phenobnrbitonc 13

Phenobarbitone alcohol

The phase 11 enzymes use the polar groups as "handle' f o r at taching

other very water-soluble moieties such a s glucuronic acid, sulphate

rad ica l , or the amino acid,glycine among other conjugating agents.

Phase Reaction

0 COOH nicrosonal

1 u FOOH 0

Phenobarbitone Glucurcnic

alcohol acid

'*

I@L OH H (OH

Glucuronyl der ivat ive

U

The phase 1 products and especia l ly the phase conjugates a r e

su f f i c i en t l y polar and are e a s i l y excretable by the organism. Living

things often respond t o chemical advers i ty by induction of these

enzymes.

1.8,Q THE ROLE OF CYTOCHROME P-450 IN DRUG METABOLISM

Omura - e t .- a 1 (1964) r e p o r t e d t h e p r e s e n c e o f a c a r b o n

+ monoxide b i n d i n g p igment i n l i v e r microsomes which t h e y

named cy toch rome P-450 b e c a u s e o f t h e a b s o r p t i o n peak a t

450nm o f t h e r e d u c e d CO-complex o f t h e pigment. From t h e

p h o t o c h e m i c a l a c t i v a t i o n s p e c t r a o f a number of CO-inhib i ted

microsomal mixed- func t ion o x i d a s e s , o n e of t h e roles o f

cy toch rome P-450 h a s been d e m o n s t r a t e d t o b e t h e a c k i v a t i o n

o f oxygen f o r many microsomal o x i d a t i o n r e a c t i o n s . NADPH

r e d u c e s a component i n microsomes which reacts w i t h

m o l e c u l a r oxygen t o form an ' a c t i v e oxygen ' i n t e r m e d i a t e

which is t h e n t r a n s f e r r e d t o t h e d r u g ( G i l l e t t e , 1963).

Cytochrome P-450 i s t h e r e f o r e s a i d t o be a m u l t i s u b s t r a t e

h a e m o p r o t e i n which f u n c t i o n s i n t h e o x i d a t i v e me tabo l i sm

- . of a bro'ad s p e c t r u m o f o r g a n i c compounds ( E s t a b r o o k & 2, 1973).

'k: ZL < XOOH A

The schelne above i e , t h e hypo the t i c cyc l e of reduct ion and

oxida t ion r e a c t i o n s t h a t occur dur ing cytochrorne P-450 func t ions

I n most ox ida t ive r e a c t i o n s ca t a lysod by t h e l i v e r rnicroso~nes,

t h e s u b s t r a t e combines f i r s t with t h e oxidized form of a carbon

monoxide s e n s i t i v e haemoprotein P-450. The substrate-P-450 complex

i s then reduced by an e l e c t r o n t r a n s f e r r e d from NADPH by a f l a v o p r o t e i n ,

NADPH-cytochrome c r e d u c t a s e . T h i s i n t u r n r e a c t s w i t h m o l e c u l a r

oxygen t o form a s u b s t r a t e - c y h c h r o n r e P-450-0 Complcx, 2-

Al though t h e s e q u e n c e of events a f t e r t h i s s tage is rrot t

c o m p l e t e l y known, i t i s b e l i e v e d t h a t a second e l e c t r o n reduces

subs t ra te -P-450-0 -complex t o form an ' a c t i v e oxygen ' 2

i n t e r m e d i a t e t h a t decomposes t o t h e p r o d u c t and t h e o x i d i z e d

P-450. S i n c e l i v e r microsorrle c o n t a i n c o n s i d e r a b l y more P-450

t h a n FAD m o l e c u l e s ( s t o i c h i o m e t r y i s 1 0 cy toch rome P-450: 1

R e d y c t a s e or 1 0 0 : 1 0 Yang et&, 1 9 7 8 ) and i n t h e a b s e n c e of

a known m o b i l e c a r r i e r be tween t h e t w o enzymes, i t a p p e a r s t h a t

o n e r e d u c t a s e m o l e c u l e has t o i n t e r a c t w i t h many P-450 m o l e c u l e s

f o r e f f i c i e n t c a t a l y s i s (Nebert & al, 1981) . P h o s p h o l i p i d i s

an i n t e g r a l p a r t o f t h i s enzyme sys tem.

C o n t i n u e d a d m i n i s t r a t i o n of some d r u g s or e n v i r o n m e n t a l

c l ~ c m i c a l s s t i m u l a t e s t h e i n c r e a s e d s y n t h e s i s o f t h e rnicroso::~al *!'

i I n d u c t i o n o c c u r s a f t e r a few d a y s of a d m i n i s t r a t i o n

of t h e d r u g b u t c e a s e s w i t h i n s e v e r a l weeks a f t e r t h e d r ~ y

administration h a s been withdrawn. Such d r u g s w i l l t h e r e f o r e

e n h a n ~ e t h e i r own rtilie of m e t a b o l i s m , and owing t o t h e non-

s p e c i f i c i t y o f t h e microsomal enzymes a c t i v i t y , t h e rne iabol i sm

of o t h e r d r u g s and even no rma l c o n s t i t u e n t s

of t h e body may ba a f f ec t ed . An i nc reas ing nu ibe r of drugs known

t o induce e n z p e s i n t h i s form inc lude t h e b a r b i t u r a t e s , meprobamate,

c a f f e i n e and o thers .

r When an enzyue t h a t a c t s on a drug is induced, t h e drug is

metabolized more ra2 id ly . There is an acce lorh tod r a t e of disappear-,

ance of t h e drug, and t h e me tabo l i t e s a r e f o m c d more quickly.

The consequence on t h e organism depends on t h e r e l a t i v e a c t i v i t y

of t h e drug and its metabol i tbs . I f t h e me tabo l i t e s have l i t t l e

o r no e f f e c t , enzyme induc t ion speeds t h e tea l ina t ion of a c t i o n

of t h e drug ( c o n m y & a, 1960). But i f t h e n e t a b o l i t e s have

t h e same e f f e c t e.s t h e drug, o r when t h e o b s e ~ v e d e f f e c t s of t h e

d r w are those o f the metabol i tes , enzyme induct ion nay i n t e n s i f y

t h e s e .ePf e c t s by a c c e l e r a t i n g t h e product ion of t h e metabol i tes .

When t h e drug is no t apprec iab ly metabolizec! by t h e induced enzymes,

one would expect 110 change i n t h e du ra t ion o r i n t e n s i t y of ac t ion .

7 10 AZADIhACHTA I N D I C q

1 .1g, 1 TAXONOPiY ( I -~utchinsen & a, 1958)

Kingdon P l a n t q

Phyllum Angiospermae

Sub-phylluot Dicotyledonae

Family Neliaceae

Genus Azadirachta

Spec ies I n d i c a

1 2 MORPHOLOGY t

i n d i c q belongs t o t h e f ami ly of Meliaceae p l a n t s

t h a t grow wild i n t h e dry f o r e s t of Dacca, Burna and I n d i a , bu t is

n a t u r a l i z e d i l l I J ige r i a and o t h e r West African coun t r i e s . I t i s

planted both f o r ornamental and medicinal purposes and r e c e n t l y i n

Northern Nige r i a and o t h e r Sahel reg ions t o check encroachment by

t h e ~ a h u G a d e s e r t . Members of t h e Meliaceae f ami ly a r e t r e e s o r

shrubs , mostly with hard scented wood, v e r y r a r e l y subherbaceous.

They a r e mainly a t r o p i c a l f ami ly r e a d i l y recognized by t h e i r

pinmate l a n d d a r l eaves and t h e i r s tanena, which a r e u s u a l l y connato

forming a tube resembling a co ro l l a .

The pi.nnate leaves ; l e a f l e t s 5-8 pairs, Ovate-Lanceolate t o

Lanceolate and f a l c a t e , are very asymmetrical a t base, Long-ncuinnte ,

coa r se ly serrated on mergins and occas~ iona l ly lobed , with up t o

about 1 1 cm long and 3 cm broad, glaborous. Thc pan ic l e s are

many-flowered and auxiliary; p e d i c l e s about 1,5 nm long; s e p a l s

ovale-sub o r b i c u l a r , about 1 mm long; p e t a l s ob lanceola te , 5-6 mm

long; stamina1 colunn about 4 - 5 mm long, a n t h e r s wi th in t h e lobed

apex; f r u i t s e l l i p s o i d , 1 -seeded, g labrous , 1.2-20cm long.

W i r a c l a a d i c ~ is an evergreen tree &aterows up t o 80 f e e t

h igh , with abw-dant p a n i c l e s of whi te f lowers , and yellow f r u i t s ;

introduced Por shade and firewood, and known as "Neem". Th i s p l a n t

i s a na t ive of India , now widely d i s t r i b u t e d and o f t e n n a t ~ r ~ l l z e d 9

i n t rop ic31 and sub t rop ica l countr ies .

1.10.3 i'llYT0CHEi:i:IGTRY I d *

' . ?

#& +*

n .i ,&- Butterworth and Iqorgan (1971) i s o l a t e d an a c t i v e substance , I -

*T-- #- ..*" ., F azad i rach t in from t h e saedsof w a c h t a , u u . The Sten bark

e-*, . ,pL7%

.,nimbinin (C 1 0 ) nimbosterol and about 6% of tannin ( Ind. harm. 2.f 30 9

Codex Vol. 1 , 1953). It was shown t h a t t h e leaves e x t r a c t contain8

nimbolide (C 0 ) a melicacin with t h e fol lowing bas ic s t ruc tu re : 2?30 7

/- I I I

NIMBOLI DE

1,10,4 PHARMACCLOGICU USES .

Many workers have published accounts of c e r t a i n a s p e c t s of the

pharmacological app l i ca t ions of Azadl.rachta .. . j;CldiCtl, and the a c t i v e

p r i n c i p l e s i s o l a t e d from i t ( ~ u t t o r w o r t h and Morgan 1971 ) . Nimbidin,

a compound i s o l a t e d from t h e a i l seeds of A. m, h a s been screened

in comparison with two standard antinflammatory agents, phenylbutaaone

( a non-steroid) and prcdnisolone ( a steroid) against various

experimental models of inflammation and it was found to reduce greatly

acute paw oeclema in rats induced by phlogistic agents, carrageenin

and kaoiin. In acute phase of inflammation, nimbidin (40mg/kg) wtrs

found to possess significant activity comparable to phenylbutasone

( I 00mg/k) . '

The oil expressed from its seeds is also said to be a stimulant

alternative and effective in rheumatism and skin diseases. Recently,

it was reported that nimbidin possessed significant antigastric ulcer I 1

activity in experimental animals, where it was shown to prevent ul.~ration ;

by reducing gastric secretion. Okpanyi and Ezeukwu in 1981 found that

- the extr&t of A. Sndica has a pronounced antiinflammatory (rat paw

oedema) and a fairly good antipyretic effect ( pyrogen induced

hyporpyrexia in rabbits).

Pillai and Santhakumari in 1981 clearly demonstrated significant

antiarthritic and antiinflamatory properties of nimbidin, a compound

with multiple pharmacological actions. However, the oral acute

toxicity of A. indica extract indicates a very low LD in the range 50

of 13 g/kg in mice.

CHAPTER TWO

MATERIALS AND Mg_H,O_DP

Tb.c: f r c s h l eaves of dogonyaro (m were c o l l e c t e d <."

from t h e p l a n t s around t h e Un ive r s i t y of Niger ia , Nsukka Campus. . Y' i

' ++.p-, 4 . 0 2 ANIKALS .+ ;jA. , &?,.

The f i r s t s e t of male a l b i n o r a t s were obtained from t h e animal

house of t h e Facu l ty of Medicine, Un ive r s i t y of Niger ia , Teaching

Hosp i t a l , Enugu. The second s e t from t h e animal house of t h e

Patholog;; Dopartnent , Facu l ty of Ve te r ina ry Medicine, Un ive r s i t y !

of Nigeria, Nsukkn Campue. The animals, two t o t h r e e weeks 016 were i of the same s t r a i n and were housed dur ing the experimental per iod

i n wire n e t s b o l i c cages: um OFMGBBI* 2,?3 AEImAL.-.D. W B B A B V

k a t feed p e l l e t s were obtained from t h e animal house of t h e

F a c u l t y of Ve te r ina ry Nedicine, Un ive r s i t y of Nigeria .

The chemicals and biochemicals used in this study

were products of Sigma Chemicals, St. Louis, U.S,A., and

f r o q May and Baker Ltd., nagenham, England end i nc lude

Petroleum e t h e r

C?l'\orof o m

Weklxmol

ilPthanol

Dragondorff's Reagent

Wagner's Iieagent

NADP+

NADPIS

I s o c i t r i c a c i d

Ani l ina hydrochloride

Glucose-6-Phosphate Dehydrogenase ( BDH )

Fol in-c ioca l teau s o l u t i o n

Amoniw~ molybdate

l lscorbic a c i d

Bovine 5;erum albumin

Cytochroine C Type 111

I s o c i t r a t e Dehydrogenase

25

2-05 PliLPARffTICI; OF ~~L~LS.~I;TS/~U~*'F:<:?S

The v a r i o u s b u f f e r . s o l u t i o n s f o r t h e r e s p e c t i v e prepa-

r a t i v e and a n a l y t i c a l p r o c e s s e s a re as o u t l i n e d below:

SOLUTIONS FOR FiICROSOME PHEPARkTTGk'

10 rnM Tris-HC1 pH 7.4 c o n t a i n i n g 0.25M Sucrose

KETHOD

E x a c t l y 120g Tris-(hydroxymethy1)-anino methane

and 85.575g Sucrose were d i s s o l v e d i n a small volume

of d i s t i l l e d water i n a l i t r e v o l u m e t r i c f l a s k .

This s o l u t i o n was t i t r a t e d wi th 0.01 M hydro-

c h l o r i c a c i d t o a pH 7.4, monitored on a Pye-Unicani

3 model 291 pH meter , and t h e n made up t o Idm . 10 mM Tris-HC1 p H 7.4 c o n t a i n i n g 150 mM KC1.

Exac t ly 11.184g o f potass ium c h l o r i d e and 1.2Gg

Tris-(hydroxymethyl)-aminomethane were d i s s o l v e d i n a

. s m a l l amount of d i s t i l l e d water i n a l i t r e vo lumet r i c

f l a s k . T h i s w a s t i t r a t e d w i t h 0.01 K H C 1 t o pH 7.4,

3 and t h e n made up t o Idm . N O W A L SALINE

E x a c t l y 9.0 g: Sodium c h l o r i d e was d i s s o l v e d ir. a 5,

l i trs v o l u m e t r i c f l a s k end made up t o 1 dm3 wi th

d i s t i l l e d water.

This was t o a f i n a l concentrat ion o f 8.0 mK. The volume

o f the supernatant recovered after c e n t r i f u g a t i o n a t 12,000g

9 .

was recorded.

CALCULATIONS

F% ' + 1 molar conta ins 110.9 g CaCIZ i n 1 dmJ *e+ - *

&, \ &+ .,,a b- . 0.008 M conta ins 110.9 x 0.008 g dm-3

if t h e volume recovered = A cm 3

110.9 x 0.008 x A 7000

A volume o f supernatant conta ins

DETERMINATION OF CYTOCHPnME C REDUCTASE ACTIVITY

PREPARATION GF REAGENTS

1 mM Potassium Phosphate Buffer pH

CHZMICALS

K2 H PO,,

K H2 PO4

D i s t i l l e d water

KLTHOD

Exactly 0.1361 g KHz PO4 (acid) was d i s s o l v e d i n a minimum

volume o f d i s t i l l e d water.

Exactly 0.1842 g K2 X PO4 ( b a s e ) w a s d i s s o l v e d i n a l i t t l e

volume of d i s t i l l e d wu te r - T h i s base was t i t r a t e d w i t h t h e 3

9 acid to a pH 7-6 and then made up t o 1 dm - . , b. 400 uM Cytochrome C

+s*P '-Exactly 0.4954 g c y t o c h r o n e C was dissolved i n d ~ s t i l l e d water &-r*

.*4 ' &* >, &x,. and made up t o 100 cm3 i n a v o l u m e t r i c flaak.

c. 1.2 mM NADPH

E x a c t l y 0.1 g NADPH w a s d i s s o l v e d i n d i s t i l l e d water a n d then

made up t o 100 cm3 i n a v o l u m e t r i c flaak.

GLUCCSE-6-I'HOSFHATASE ACTIVITY

PREPARATION O F HEAGSNTS

a. 0.1 M Sodium A c e t a t e B u f f e r pH 6.5

Sodium ac+tat e

0.1 M a c e t i c a c i d

g l a s s - d i s t i l l e d w a t e r

W r H O D

Exactly 13.608 g Sodium a c e t a t e crystal^ was d j s s o l v e d i n n

smell amount o f g l a s s - d i s t i l l e d w a t e r i n a vo lun iu t r i c flnck.

This s o l u t i o n was t i t r a t e d w i t h 0.1 M acetic a c i d . t o n pH 6.5,

3 and t h e n made up t o 1 dm .

m o u n t of g l a s s - d i s t i l l s d wator and then nade up t o 1 O O m L in a

, _ J - .t

Xxtlctly 20 c.?, c.tocl- s o l u t i o n of TCA was nadc up t o 100 I I I ~ s o l u t i o n +,&.'r;l ;-"' ',

1, I.i7#- w i tri glass-di:; ;:ill-zd wcter.

Z x m t l y 1C: Z - .rr~c:t -.=-,ic ac id c r y s t a l s was disso lved i n 100 m l of

so lu t ion .

2ixactly 18 m l of concentr?.ted s u l f u r i c a c i d was added t o 90 n l

p l a s s - d i s t i l l e d wc3.tcr.

2 , 03'1;i1C!i'Iii:ii 'T.%OCEplJIiE OF QUElCGTIN-WLYCCSIDZS

~ x I J G 20C p. k r y rlci&t of the l e a v e s of &riiracbti.. i n d i c a (ddeonyem)

w ~ s grouiid ::rid thcn pucl:cr! i r ~ u soxhle t apparatus .

;:-boct 3.50 m',. pet.solcur: s t h s r was used t o remove l i p i d mater ia l s .

The resu l t i i ig 3.od res idue was d r i e d and repackod i n t h e soxh le t

appara tus .

::bout 1.750 Iitrc!; o f m t h n n o l was r c f l m c d c o n s t a n t l y through t h e

lwf ~nat~ri.::..). foz* z b o u t G hours , while the soxh lc t e x t r a c t o r was

0 rsair:taini.d a t a consixwt i-enpurnture of a'iout 50 C. %he f i n a l

1 ...

t

methanolic ex t rac t was concentrated t o a semi-solid material

. . t under reduced pressure at 40°c i n a ro ta ry evaporator and then

par t i t ioned i n to water and chloroform (volume by volume), ..d4'

150ml water: 150ml chloroform.

This was mixed thoroughly i n sepa'rating funnel f o r about 30

minutes and then allowed t o s t m d overnight.

The lower chloroforu layer was run out and 10Gml water

added t o it. This was mixed as before and was allowed to stand

overni&t, The combined aqueous ex t rac t ( 220ml) was concentrated

a t a temperature of between 80' w d 90' C using a ro ta ry

evaporator,

Phytochemical t e s t s were subsequently carried out on t h i s

aqueous ex t rac t a s described bel-ow.

2,08 QUALITATIVE TEST (Iwu, 1978)

A knowledge of the chemical substances present i n A plant

i s obviously essen t ia l f o r an adequate biochemical understand:ing

of tilt: pharmacolqical ac t ions u.P the plant.

30 There have been agme r e p o r t s on the phytochemistry of A.

indica . i Icvcrtheless i t was found necessary t o s t i l l c a r r y out

phytochemical t e s t s on the sample co l l ec ted f o r t h i s work.

Alkaloids a r e pharmacologically important p l an t cons t i tuents .

.?A' W

F;,,8+d.'" . They have been reported t o possess autonomic, c e n t r a l nervous, I#

i A ~ < ,.w cardiovascular , u t e r ine , d i u r e t i c , an t imicrobia l , an t ineop lae t i c P*" d

f.t.,- and a n t i i n f l a m a t o r y a c t i v i t i e s . The genera l methods f o r t h e i r

ex t rac t ion and i d e n t i f i c a t i o n a r e based on t h e presence i n all

a lka lo id of a t l e a s t one bas ic n i t rogen atom i n the nucleus,

Th i s can f o m soluble salts with d i l u t e ac ids , and can bo

p rec ip i t e tod f r o n s o l u t i o n s by c e r t a i n bases and heavy metals.

METHOD OF DETECTION

To 5 m l por t ion of t h e aqueous e x t r a c t of t h e d r y l eaves

of A. a l d i c a obtained by a lcohol ext rac t ion , was added two drops

of Dragcndorf f' s reagent/Wagner ' s reagent o r Mayer ' s reagent.

The n ix tuse remained c l ea r .

b) FLAVONOIDS

These compounds which occur both i n t h e f r e e s t a t e , and

combined as glycosidos, a r e t h e l a r g e o t group of n a t u r a l l y

~ c c u r i n g p lant phenola. The union o f n C6 - C u n i t with an d ,/ 3

aromatic r i n g l e s d k t o the s t r u c t u r a l u n i t below, known a s t h e

"Flavonoid" - s t r u c t u r e , because i ts occurance i n na tu re is

most comon i n t h e flavones.

Fhe Flcivonoid S t r u c t u r e

The nr!turnlly occiiring f lavonoid conpounds a r e , except f o r

f lavoric. i t s e l f , hydroxylated ( phenolic) substances. The

g r e a t e s t rpmber of f lcvonoid compounds possess two d i s t i n c t

n u c l e i ; one, des igna ted as the A r i ng , i s comonly 2-, 2,4- o r

2,4,6-hydr.oxylated-ic, i n t h e t y p i c a l "acetate-derived" pa t te rn .

The o the r , t h c A r i n g , is nos t cormnonly 4-, 3.40, o r 3,4,5-

hydroxylated - t h a t is , i n t h e p a t t e r n c h a r a c t e r i s t i c of t h e

phenylpropme d c r i v o t i v e s , a r i s e v i a t h c Shikirnic a c i d pethway

( ~ e i s m a n T. N., 1963).

Flavonoids have been r epor t ed t o be a s soc i a t ed with

a n t i h e l m i n tic, m t i l ~ a l a r i a l , an t imic rob ia l and a n t i n e o p l a s t i c

a c t i v i t i e s .

METBOD OF DETECTION - To 5 n l of t h e e x t r a c t was added about t h r e e o r more drops

of 5$ equc.ous F o r r i c ch lo r ide so lu t ion .

Tb1=2 ~ i x t u r c vTas observed t o kum blue black. This ind ica ted

the pr~senc:? of f lavonoid compounds i l l t h e ex t rac t .

c ) ANTHRAC 'IT YCx q,:

rr .c.s-k: To 5 n l sample so lu t ion of t h e e x t r a c t was added

a few d.rops of 5% potass iun hydroxide s o l u t i o n

i n 507: methanol.

The colour of t h e mixture turned yellow-red

immediately i n d i c a t i n g t h e presence of t h e

anthraquinone g lycos ides in . t h e ex t rac t .

d ) PROTEINS,:

Test:; P ro te in was determined by the method of Lowry

,c& .a1 ( 1951 ) . The o p t i c a l dens i ty of t h e so lu t ions

was ncasured a t 750 nn using Coleman Jr.

Spectrophotorneter ( f o r low p ro te in concentrat ions) .

There was no colour development i n d i c a t i n g t h e

absence of protein.

SEPAliA'li.i.j~-eeeee.C_~idi +CJ'E)iiI~iiTION ilND IDEbJTIFICATION OF THE 2 * 09 ---- .., -- CONSTI1;'_UXI'JTS OF THE I SOLATE:

2 The coi:icentrated. c..queous e x t r a c t remaining a f t e r t h e b io log ica l

t e s t ( 3 3 ~ ) was chrorilatographed over s i l i c a g e l G. ( ~ e r c k )

preparcd -rlth. wnter i n the r a t i o of 1 gram g e l t o 2 m l of

dietilled water.

The p l a t e s werc a c t i v a t e d f o r 30 minutes at o temperature of

1 loOc.

v ~ l u u s a r e g iven as t

1)istance t r a v e l l e d bv s o l u b Distance t r a v e l l e d by t h e so lvent ,

P l a t e s were viewed under s h o r t wave UV-light and components of t h e

..#. H" e x t r a c t s were i d e n t i f i e d by t h e i r r e spec t ive f lourescence co lours . .+#&-

'*' ' ,,L$->*

b , The components v e r e f u r t h e r c h ~ r a c t e r i z e d by t h o i n t e r p r c t a -

t i o n of t h e i r u l t r a v i o l e t (w i th s h i f t reagents ) , I n f r a r e d (IR),

proton miqmetic resonance (60 NHz) s p o c t r a and d i r e c t chronato-

graphic comparison with r e fe rence compounds.

11HEAThi31JT OF ANIMALS WITH THE; EXTRACT

An optirnwn dose of 400 ng per kg body weight of t h e

aqueous e x t r a c t was determined by t h o admin i s t r a t i on of va r ious

doses o f . t h c e x t r e c t t o t h e specimen animals. Th i s was determined

by monitor ing the change i n t h e l i v e r t o body weight r a t i o of t h e

animal. Th i s p a r t i c u l a r concent ra t ion was t h e r e f o r e choosen when

an apprec i ab le o r n o t i c e a b l e i n c r e a s e in t h e l i v e r t o body weight

of t h e animal was observed.

METHOD

h group of s i x male a l b i n o r a t s were used i n each set of

experi~!!ents. Three of t h e s e were used as test animals, and t h e

o t h e r three , as the c o n t r o l experiments ( i . e , t r e a t e d animals and

un t r ea t ed animals r e spec t ive l ly ) .

The ani~nals were weighed each day before t h e adminis t ra t ion

of t h e p l a n t e x t r a c t , so as t o be a b l e t o ~!etermine t h o

appropr i a t e dosage t o be administered. T h i s w a s beccmse of t h e

f a c t t h a t the weight of t h e rats va r i ed w i t h t i . 1 1 1 ~ .

Thuo the use of t h e dosage c o n t r o l formular

mg of dw/ka; body wt. of t h e animal x weieht of Animal i n gram - -4 -- 1 000 cnncsn t ra t ion ( mg/ml)

s o l u t i u n

T h i s gave t h e a ; > p o p r i a t e volume of t h e e x t r a c t t o be administered

t o an animal each time t h e t rea tment tras done.

Care was taken t o make s u r e t h a t n o t more than between

O.5ml and 0,61111 by volume of t h e drug was given a t a t i n e to

each animal, so as t o avoid a case of hernodilution.

I s o l a t i o n of t h e microsomal f r a c t i o i l of t he hc,p;itlc cel. I.

g e n e r a l l y employsa procedure of d i f f e r e r l t i a l cen t r i f ughtion.

Th i s procedure however r e q u i r e s u l t r a c e n t r i f u , y t i o n which no t

a l l 1aboriltor.y c m a f fo rd . Acid p r e c i p i t a t i o n and Gel f i l t r a t i o n

r e s u l t s i n t h e i n a c t i v a t i o n of a number of microsomal enzymes such a s

cytochrome P-450, Glucose-6-phosphatase and otherS(~angen & &., 1973

REAG

Sodium c h l o r i d e 0. 956 ( normal s a l i n e )

O . O l d 1 Tris-HCL pH 7.4 con ta in ing 0,251'1 sucrose

Calcium c h l o r i d e

O.OlLJ\i Tris-!EL pH 7.4 con-l;ai:rling 150mM KCL.

7 .

~.',:.ts vi;re k i l l e d by c e r v i c a l d i s l o c a t i o n 24 hours

n f t o r tl;.: ::.cl~iinistration of t h e l n s t d.ose of t h e Quercetin-3-

Glycor-:.<?do ~ l i x t u r o . The l i v e r s were quick ly exc ised and

p e r f u s A ~ r i t h ice-cold normal s a l i n e .

r.7 I J ~ l i y o r s were ninced and homogcnised i n ice-cold

in i : 1 , The wshed rnicro~oliiel p e l l e t s were then

suspcndr!c' 5.r; 1 O:.nl\I T r i u - H C 1 $1 7.4 contc3,ining 15031 K C 1 aml

storocl \aiTci,::r ic+col:! cond i t i on :for use.

10 LIVER HOFIOGENfiTE

I N 0,25M SUCifOS~ - 10 ~ $ 4 T R I S J I C 1 pH 7.4

CENTRIFUGE at 600 g for 10 n i n .

I P ; (KIPITATE DISCMDED ( CELL DEBHI S , NUCLEI )

Ltit i s o l i d C & 1 t o 8 1 9 1 2

S:'~rl. 1 c o n c m t r : . t i o n , stir --.--

-.mi c c n t r i f ugc st 25,OOGg

PARTICLE FaEE SUPEfrNATiiNT

( COIJTAINS SOlviE IiI BOSOMES

OCCASIONALLY)

D. 2 : n l rolin-cioc:-,I:to::u reagent

i n IlSRGENT D 5 m7 d i s t i l l , > d wa-L,!r

F. S tr.nii:.s.rd P r o t e i n ( 200 ug Bovine Serum 7 2

i i t i 1 d i s t i l l e d water)

st: rtc??rd c..: ~ ,vc f o r the es t in ia t ion of p ro t c in was

prc p m c d us in,^ boviqe serwn albumin u s t he r e f e r e n c e p ro t e in

n i x ::(:3.1 f o r 10 ~i imtes a t roon teixperaturc. 0.5 ml of

rc r f ren t D m:: nddcd and r ~ p i d l y nixed. The o p t i c a l d e n s i t y

of thc.: s o l u t i o n s .wxz ncesured a t 750 nn us ing Colenan Jr.

Spectr.ophotome-t;i;r ( ;'or low p r o t e i n concont ra t ion) .

- 2.13 D;.T.:gI&T1'3.i L j ' INORGANIC PHOSXIXE

LEILG ENTS

1. i0.0 R Ascorkjc ac id i n 100 n l g l a s s d i s t i l l e d water

t 2. 2.5 (1: Amoniur~ Nolybdate i n 100 n l glass d i s t i l l e d water

3 g 11 n,?O. : add 18 r n l conc H2S04 to -90ml water.

10 nl P.I12S04

20 nl g l a s s d i s t i l l e d water

i iaGENT k , 10 r ~ l . 2.5 $ L~moniun molybdate

6 . ~ a r a f i l ~ ~ / t r a t e r b a t h

1"IhTiIOD

I1h;! I?: . ' ibC: of F i s k e and Subbarow ( 1925) employed i n

t F : i s d e t a m i n z t i o n depends on t h e f o r n a t i o n of

p~.osphono?.-;bda-t;e and t h e reduct ion of t h i s conplexi

n l , , ..,: 2. t r e m l ; high s e n s i t i v i t y of t h i s r e a c t i o n r e q u i r e s

tl.;c: 1wo of' 2ci.d-wished g l a s s wares, because g l a s s wares

w-s;:ni' ~ v i t b . p h o ~ ~ h a t e conta in ing soaps u s u a l l y have

sv r4'i ci m t 14:cidues t o t u r n t h e reagents black i n t h e absence

rLj& u- ' 4' JTS A S PLiBETi'i(ED

One n i l l i n o l n r s o l u t i o n of p a i n o p h a n o l i n e thanol was

prepared by d i s so lv ing 0.0055 g of t h e s o l u t e i n 50 r n l of

li s e r i n l d i l u t i o n of t h e above s tock s o l u t i o n was

preparzd t o o b t a i n a range of c m c c n t r o t i o n s . -4.0 nmoles/rnl) 0.1~

Tris-IiC1 $1 7.6 b u f f e r s o l u t i o n was used.

To 2 rid of each concent ra t ion was added 2 n l I M

sodiutn cmbonatct s o l u t i o n s nixed thoroiighly, and then

0.5 !:rl fo l in -c ioca l t eau d i l u t e d 2 nl t o 5 nl. d i s t i l l e d

water. The co lou r was allowed t o develop by s t and ing t h e

n i x t u r ~ s f o r 20 x i n u t e s , and t h e o p t i c a l d e n s i t y ( O D )

was red. 2.1 6 X nm us ing a Coleman Jr. Spectrophotoncter .

2-1 2 D ~ T K I ~ ~ J I I T ~ I O F ~ G!.' H ~ ~ O B l r I i 1 3 I T O N E SLEEPILTG TIMd

111 :;iiiac:ls, mzyne induct ion is g e n e r a l l y denonstreted

as a dt-crcasc i l l t h e s l eep ing time induced by t h e d e r i v a t i v e s

of t h e b a r b i t u r i c a c i d e g . thiopentone, phenobcrbitonc;,

hexoberbi tone e t c .

Phonobarbi tone (S tock So lu t ion )

Keter f o r i n j e c t i o n

syringes ( 1 n l ) and needles

cages

chrononeter

METHOD - Tho s tock s o l u t i o n o f p h c n o b a r b i t o n e was d i l u t e d t o t h e

d c s i r c d concent ra t ion before admin i s t r a t i on t o t h e an ina l s .

An optimum dose of 50 mg/kg body weight of t h e animal

was L~.YCC~. The animels were weighed each t ime i.e. each day,

before every admin i s t r a t i on of the r equ i r ed dose of t h e

b a r b i t u r a t e . Adrainis t rat ion was i n t r a p e r i t o n e a l . The s l eep ing

t i n e o f t h e a n i u a l was recorded when t h e animal8 regained t h e i r

r i s i n g r e f l e x e s , i e . when it t r i e s t o change i t s s l eep ing pos i t i on ,

The s t a r t i n g poin t was noted as soon as t h e b a r b i t u r a t e was

a d u i n i s te red .

2.1 6 DEl!O,i;IIiIl,TIOli OF GLUCOSE-&PHOSRI~LT~SE ACTIVITY

E C : 1.1.1.49

Glucose-6-Phosphatase enzyne, found mostly i n t h e microsones

is re spons ib l e f o r t h e hydro lys i s of t h e h igh energy phosphate

bond of glucose-6-phosphate, t h e s p e c i f i c subs t r a t e .

Hydrolysis

Glucose-6-phosphatase

Glucose-6-phosphate Glucose no ie ty

Ttic first evidence f o r t h i s enzyuc was obtained by F a n t l and

Rome in l\;4.5. Hers e t - a 1 - in 1.951 demons-1;rated t h a t Glucose-6-

Phosplmtase i s a &rosonal enzyne, and i s theref o re , used as t h e

narker enzyne f o r t h e microsonal f r a c t i o n .

O,? irl Sodium a c e t a t e buffer $1 6,5

20 $ Tr ich lo roace t i c ac id

g 1;; 11 20 2 0.

2,5 ;; knmoniun molybdate

10 % Ascorbic ac id

Glass d i s t i l l e d water.

PliOCEDURE

Glucose-6-Phosphatase a c t i v i t y i n t h e microsonal f r a c t i o n

was bsseyed accord-j.ng t o t h e method described by Ologunde

( B.Sc. Thesis , Dqartrnent of Biochemistry, Univers i ty of

Nigeria , Nsukica 1978) a s a modif icat ion of t h e F i ske and

Subbarow' s method.

The enzyme a c t i v i t y was assayed i n a r e a c t i o n mixture

(mediun) conta in ing 1 , 8 m l of 0.1 M s o d i m a c e t a t e buffer ,

pH 6.5 and 0.1 m l of 5 r;?M Glucose-6-phosphate. 0.1 m l

microsonal f r a c t i o n ( d i l u t e d t o 2 mg pro te in per m l i n acid

washed t e s t tubes) was then added t o g e t a 2 ml mixture t o t a l

volune. Af te r incubating t h e r eac t ion mixture f o r 15 minutes,

.t ~11 20$ Tr ich lo roace t i c ac id was added t o i t t o s t o p the

r eac t ion znd then kept a t ice-cold condit ion f o r a minimum 7

of 10 minutes before t h e p ro te in hase was cent r i fuged o f f ,

Phosphate conten-L vas determined i n t h e supernatant by t h e

method of Il'isko and Subbarrow a s described before.

A c t i v i t y u n i t s were expressed h mole phosphate/l 5

rnin/ng prote in .

2.17 TIE D ~ ~ i i ' N I W f ~ f I O l l OF NADPH CYTOCIIROME C REDUCTASE ACTIVITY

INTilODUCTIO3

The NLDPH-cytochrome c ( P-450) reductase ( M.', 1,6.2.4)

i s a f l avopro to in enzyme and an i n t e g r a l conponent of t h e

nicrosomal. rionoozygenese system of which cytochrome P-4W

i s t h e turr i i iml oxidase. I t t r a n s f e r s e l e c t r o n s from NADPH

v i a i t s two f l avopro te in (FAD and FMN) p r o s t h e t i c groups t o

the haen iron of cytochrome P-450 where they a r e used t o

produce an a c t i v e oxygen spec ies involved i n t h e hydroxylation

of xenobiot ics and endogenous s u b s t r a t e s l i k e s t e r o i d s , f a t t y

a c i d s (onura -g- & 1965, 1966). The node of e l e c t r o n

t r a n s f e r has a l s o been s tud ied by Y w ( 1977).

In r a t l i v e r microsomes, the n m b e r of cytochrome P-450

nolecules can 5c 29-30 t i n e s more than t h a t of t h e reductase

showed by t h e s t u d i e s on the progesterone hydroxylat ing

and lU1ieow systern of R J i z o ~ u t h a t the

induct ion of cytochrome P-450 i s due t o t h e de novo syn thes i s

of t h e enzyme, where a s t h e inc rease of a c t i v i t y of t h e

reductase can be explained by t h e a c t i v a t i o n of t h e

p reex i s t ing cnzym.

REAGEMTS

1 r,~Jl'i Po tass iuo phosphate buffer pH 7.6

4.00 m o l a r cytochrone C Type I11

1 .2 lllM NADPH.

PROC EDUJU

Assay method was as descr ibed by S t r o b e l and Digma ( 1978).

The r e a c t i o n n i x t u r e contained 2.3 m l of 1 mM P o t a s s i m

phosphate buf fe r $i 7.6, and 0.3 n l of 400 m o l a r oxidized

cytochrone C , 0.3 n l of 1.2 mM NADRI and t h e r eac t ion was

s t a r t e d by t h e add i t ion of 0.1 m l nicrosori~al suspension t o g ive

a f i n a l vol.w.~o of 3.0 m l . A t t h i s v o l m e , t h i s amounted t o a

t o J x l p ro te in concentrat ion of 2.0 - 2.5 ~ i g p ro te in per ml.

The i i ~ c r e s ~ c i n absorbance a t 550 UI ( h i c a m Sp 500

0 spectror?hotonctcr) was followed a t a t c t q e r a t u r e of 27 C

and the i n i t i a l v e l o c i t i e s were recorded, s p e c i f i c a c t i v i t y

was calculated i n ~mole/ninute/mg p ro te in us ing an ex t inc t ion

c o e f f i c i e n t of 21 cmm2 d-' f o r ferrocytochrone C.

E. C . 1e99.1.1

CHEMI CkLS

NW>P"-

GI-ucose-6-Phosphate

0.1 M potassium phosphate buf fe r $ 7.4

h i l ? . n e hydrochloride

Sodium hydroxide

Phenol.

., ;o sml ceq;ireci ths irlclusion of an NADPH genera t ing system i n the

r e a c t ion medium ( ~chenkmann & C i n t i , 1 972).

of 0.1 M potassium phosphate buf fe r $ 7.4, 100 u l of 50 dl &$I2

i n 1.15$ K C 1 , 110 u l of cofac tor so lu t ion (10 mM NBDP+, 100 mM

glucose-Lphaephate, and 1.0 ZIJ, of C,lucose-6-phosphate de'nydrogetiese

i n 1 .15$ KC^), and 100 u l of 50mM a n i l i n e hydrochloride i n 1 .15$ K C 1

aa t h e subs t r a t e .

The reaction w f i s i n i t i a t e d by adding t h e s u b s t r a t e l a s t and then

incubated at 3 7 ' ~ i n a water bath. The r e a c t i o n w a s terminated a f t e r

70 minutes by adding 400 u l of 20% t r i c h l o r o a c e t i c acid. This m i x t u r e

was cent r i fuged t o remove prote in haze. To 700 u l a l i q u o t was added

700 u l of i molar Na2C03 solution, 700 u l of 0.5 1 NaOH i n & Phenol.

The o p t i c a l density of t h i s mixture was measured a t 630 run a f t e r

30 minutes. Enzyme a c t i v i t y wss expreseed as m o l e s paminophenol

formed h o u r 1 mg protein-1. Blanok as says contained 100 u l of 1 .15$

K C 1 ine tead of the a n i l i n e s u b s t r a t e Solution.

1tE:;UL'l'S

Y H Y T O C ; l A ~ ~ ~ , & STUUILS

Ph-J.tvc:llc. ! i c a l s t u d i e s on t h e n e t ' x n o l i c e x t r a c t showed fke

presence of Lhc f l evonoid conpounds by its c o l o u r r e a c t i o n (b lue -

b l a c k ) w i t h 5,; equeous F e r r i c chlor ic lc s o l u t i o n . No o b s e r v a b l e

c o l o u r ch~ni;c was n o t i c e d w i t h e i t h e r D r a p n d o r f f ' s , k y e r ' s

o r ' da~ncr ' a rr?ap;ent, i n d i c a t i v e of t h e absence of t h e a l k a l o i d s

i n t h e o>:trnct. F r o t c i n was a l s o a b s e n t as was r.ieasured by t h e

riwthod !;f Lowry & & ( 1 951 j . IIowovol-, t h e e x t r a c t pave a

yellow-red c o l o u r r e z c t i o n w i t h the e d d i t i o n of 5$ p o t e s s i w : ~

hydroxidi . s o l ~ i t i o n i n 509; methanol i n d i c a t i n l : t h e p resence of

Table I

Coi:.!;ounds I n d i c a t i o n

- indicc..tc.s presence of conpound

- i n d i c o t e n ebsence of ccr.lpound.

Table I shvws t h a t t h e aqueous extract cantained withor dkaloids

nor p ro t e ins , but a hiph concent ra t ion of the flnvonoids as wel l

a s s l ~ a l l ar;iount CJ the anthraquinone g lycos ides .

Fu r the r i d e n t i f i c a t i o n of t ho c o n s t i t u e n t s of t h e i s o l a t e . .

- .. . , . I : .3 , , - was c a r r i e d Qut by t h i n l a y e r chro~:iatographic techniques

. x f i a $ s * i d + R .-@' ps5L2& - ,.@@" on S i l i c a ..el C. (iicrk) 1m t h i ckness , wi th r e f e rence claterirrls.

THd SOL,VENT SYSTiGiS COIISIST OF:

S vs t e ~ : d

e thy l a ce t a t e : 5

xethyl e thy l ketone : 3

formic ac id : 1

d i s t i l l e d water : 1

Svsten E --- Benzene : 45

methanol : 8

ace t i c ac id : 4

Svstem C

1 -bu tmol

a ce t i c ac id

d i s t i l l c d water

Svstcn D

water

Svsten E

phenol : 3

d i s t i l l e d water : 1

N.B Sys tem C, D and E were run on paper. -

: 4

: 1 t o p layer,

: 5

Charac t e r i za t ion of theso cumpounds were done by physical methods

( s p e c t r a analyses)

t -: Yellow coloured powder.

UV ( , nm) : Methanol

Methanol + Sodium hydroxide 206,270,410

Kethanol + Aluminium c.hloride 275 3 0 5 ~ h , 430

Methanol + Aluminium c h l o r i d e + H C 1 270,300,403

3.90 - 3.15 ( 10 protons, rhamnoglycosyl),

0.80 (unresolved peak, 3, CH,<- rl~clmnosyl). 2

TLC: Compound A gave i d e n t i c a l Rf va lues w i th an autnen t i c sample

of Rutin ( quercetin-3-rhamnoglucoeide) i n f i v e so lven t systems.

Hvdrolvsis of _-wound A:

The chloroform l a y e r of t h e hydro lys i s product of

compound A, when p u r i f i e d on a s h o r t column of s i l i c a g e l

y i e lded a yellow compound which c r y s t a l l i z e d i n ethanol .

52

The melting poin t of t h e anhydrous form of t h i s compound

i s 184 - 186', while t h a t of the hydrated form i s 293 - 295'.

The product of hydorhysis was found i d e n t i c a l with querce t in

by comparison of t h e i r chromatographic mobi l i ty and colour

reac t ions .

C O ~ D O U ~ B: Yellow coloured amorphous powder.

Methanol + Sodium hydroxide 273,325,410

Methanol + Aluminium ch lo r ide 276,303sh9330ah,440

Methanol + Sodium a c e t a t e 275 324 , 380

5.10 ( d i s t o r t e d 2, IH, 3-1 rhamnoayl),

4.20-3.25.

(unresolved mu1 t i p le t sugar p ro tom)

Mass Spectra:

The E lec t ron I o n i z a t i o n Wass Spec t r a of t he compound

gave no fragment peaks but decomposed i n t h e i n l e t

chamber. The maas s p e c t r a was t h e r e f o r e t h a t of t h e

aglycone obtained. ( s e e compound A ) , a d me t hy l a t ed

wit'n methyl su lpha te and methyl iod ide .

: M/Z ( r e l a t i v e i n t e n s i t y percent)

CO-TLC :

The parent compound gave i d e n t i c a l Rf values i n f i v e

so lven t eystems and the same colour r e a c t i o n with An

a u t h e n t i c sample of quercetin-3-rhamnoside ( rquerci t r i n )

RESULTS OF TAE BIOCHEkICAL ASSAY: t

An optimal s u b l e t h a l dose of 400mg/kg body weight of the i s o l a t e

i n no more than 0.5ml of t h e aqueous e x t r a c t , was determined by

p r i o r adminis t ra t ion of va r ious doses of t h e drug t o white w i s t a r

; ;i alb ino r a t s , and by the subsequent monitoring of the change i n

l i v e r t o body weight r a t i o s .

'The' f i e u r e a 11, I11 and I V a r e standard curves f o r t h e

es t imat ion of pro te in , inorganic phosphate and paminophenol

respect ive ly .

a b l e I : Tabl;t of v a l u e s of t h e e f f e c t of e x t r l c t o n -

C o n t r o l A n i m a l s 1

T!ie av;:raeii. we i .gh t ( j a i i ~ (g r ; lm/dsy ) a i f f :.!-l-ii f;r : ii;-.

qr*c;c;J.; t . r t?atcd ( 2 . 5 + U. 1.0 g /day ) as com!.>,iriii: !,:-i Li7 th-. r ln!- r l , : t::5 - ( r . 0 - + O,GO c j / d d y ~ o

AL-though starvation can d e c r e a s e micrc)sorn,~i r i i i : : t : ~ l ! - f ~ ~ - ~ c . t i ~ r i

o::id;;;e a c t i v i t i t . ~ for t h e v s r i o u s s u b s t r a t ~ s i n t i - ) , : l i v ( - < r ~ j of

rnal;? rats ( P r o i a .et -- a1 19811, it is u n l i k e l y ti-!::t the ~.>Jc i i - j i ! l .

I.o:.s chs::ri/cd ~ A S a result of t h e 24 h o u r s s t a r v n t j . o n (35 ,Or l .+:?.05 -- f c r kr -.; l tsd animals and 1 2 , 5+2.10 fo r t h e un t r c ! r ; t -x i ) fa1 lnv i t t ( j -

t h e l a s t admin i s t r a t i on of t h e e x t r a c t had a major metabol ic

i n f luence on this experiment, because all animals l o s t weipht

i n t h c se:lc f a sh ion .

Iiowevc:~:, t h e r e s u l t a n t d i f f e r e n c e i n t h e weight l o s s i n t h e

t r e a t e d : r r jup 2.9 compared wi th t h e un t r ea t ed may be due t o a

certnii: e f f e c t of t h e drug on t h e o v e r a l l p a t t e r n of i t s netabolism.

T h i s i s 1lo.i\rcvc~, s u b j e c t t o f u r t h e r invest iga. t ion.

I n t h e tzble I V below i s presented t h e e f f e c t of Quercetin-3-

y l y c o s i d ~ ? ~ (iil. 'c.:c. r e l a t i v e l i v e r w e i ~ l i t s and t h e microson~al

prcjtein cni~-t;o;>-i; :,rhich were d i f f e r e n t ia t h e two groups.

Table I V s The t c h l e of va lues f o r t h e e f f e c t of e x t r a c t on

relative l i v e r weights and r e l a t i v e m i c r o s o ~ ~ a l

1:ro-tein content

Value Trea ted animals Untreated animals

--- . - . -- , - - -- - ----

Hepatic Microsma1

( r;y pro t e in per 0.47 + 0.60 7.44 + 0.24

l i v e r v e i , ~ J L )

$&: Groups of 3 r a t s were t r e a t e d with the e x t r a c t f o r 4 days

and another 3 un t r ea t ed as t h e c o n t r o l experiments were

r e p l i c a t e d a t least 7 t imes and d u p l i c a t e de te rmina t ions were

made in a l l ca ses , and r e s u l t 8 expressed as nean 2 atandard

d e r i v a t i o n (sD), It was observed t h a t bo-L;h f a c t o r s were h ighe r

i n rats t r e a t e d v i t h t h e e x t r a c t compared wi th t h e un t r ea t ed rats.

WF'ECT CF :".id &??J&T OIi THE PHi3NOBiiIiBITOXJ--INDUCED SLEEPING TIME

The man v a l u s o f t h e phenobarbitone-induced sleeping t ime

decreased i n t h e t r e a t e d r a t s conpared t o t h a t of t h e un t r ea t ed -

animals i n t e b l e 1.

Table V

Phenobarbitone-induced s l eep ing time - - - - ..a

Time i n m i n u t e s Day Experimental C o n t r o l

EFFhY:ll OF I ~ _ U E ; { C ~ I I L ~ - G L Y C -.- -- - - OSIDES ON NADAI_C~OCRhOkE P-450

LEDUCY-Sd M D --- i d l I L I l ~ I 3 - - -- HYDIiOXYLASE A C T I V i ' i ' I E s

Pican vr 1 v . t ~ f o r t h e hllDPH cytochroue C ( P-450) reductaso

a c t i v i t y i l l t h e unt rea ted animals was found t o be hlgher , and t h e

a n i l i n e hydroxylase a c t i v i t y i n t h e l i v e r nicrosomes was however,

found t o be h ighe r i n t h e l i v e r s of rats t r e a t e d wi th t h e e x t r a c t

, ; n i l i n e hydrc;xyla:;e -1

( , l ~no l r . s I \ mg. p r o t e i n )

. , c - t i v i ity were n o t found to he d i f f ecerit in rat : ; izr s L i k . . ( \ . i L ' I

C o n t r o l cu~it:):.. .L:;

,-.-

C._l_l_

V ~ l u c --

i-:y;;:r Tollnd to ci;ncen t r a t e within th:: micro:;ornal. f rdct i .0~ i-!f

Experimental a n i m a l s

I 1 : T h e tctble of v a l u e s fo r the? c ~ n c e r l t r ~ t i ~ ~ c of

Glrlcose-6- p'nospha t a se activity in t h e nicrsscxla.1

and t i~r: s u p e r n a t a n t f r a c t i o n s r e s p e c t i v r ~ l y .

V a l u e E x p e r i m e n t a l Animal --- -

fraction) r ,.,,, t ; - ,.-.' J. A. ,.\. ---*.. . --. - ..--.--- -.-.

T h e c fEr .c t of tnc qut . rce t in -3-g lycos ic les rnixI:urcs f rom

A. i n d i c-;-i lc:.:,lv :s on ttic c - t c t i v i t y of a n i l i n 2 h y d r o x y l . a s e -

'The t a b l e

h y d r o x y l a s e a c t i v i t y .

L x t r c i c t A n i l i n e h y d r o x y l a s e l..ivl L. c j / q G ~ : j y -1

( m q / t n 1) A c t i v i t y ( umolc h mg p r o t c in) i1ci:jht

! n ; : j r ~ i . f i c * t i o n e u c h r o m a t i n s were s c e n I.n thr. cel l .; .in(; i,.h L.;

was i n d i c a t i v e of inc~-cased n u c l e a r a c t i v i t y of 1 . *, i TI ,-, ,- cr$lls. These f e c l t u r e s arc, i n c o n t r a s t t o kk~osc s f L l l~ - . c o n t r o l

whe re t h e r e were h e t e r o c h r o m a t i n s c o v e r i n q rw; i r ly th,: cJntiri?

n u c l e u s . I n t h e s e cells o n l y n u c l e u s was v i s i b l e , 'this i s

corn;:~onl y inc l ica t i v e of r e d u c e d h e p a t i c ac ti.v.i. L y , ..;c?c?

pno t o g r a p h s below.

The t e s t e x t r a c t from A z a d i r a c m i n d i c q l eaves was

chror~ato;:rai?hed over s i l i c a g e l and e l u t e d wi th va r ious r a t i o s

t of c: thylacetate - methanol mixtures. h o t 1 a prel iminary Tw

screen , only two major compounds were de tec ted . These compounds

werc found t o be f l avono ids from t h e i r co lour r e a c t i o n s with

, >> 1.

,dwr).@' "Y 5F F . ~ U E O U S f r r i c c h l o r i d e s o l u t i o n (blue-black) and ammonia y&G,. ,d * vapcu- ( pl l ow t u r n m e t o brown on hea t ing ) . Although

anthraquinoues were found i n t h e crude drug, t hey were n o t

de t cc t ed i l l the chromatogram.

Thc two compounds k and B were p u r i f i e d by TLC us ing t h e

maxim. a-i; 206, 256 and 356nn. The 356nm absorp t ivn band

su{:;;y?st the presence o f an 6(, B u n ~ t a t u r a t e d Ketone

* ( n - ?LT t r a n s i t i o n occuring n o r n a l l y a t 310 - 350m)

i n d i c a t i v e of t he c e n t r a l nucleus of querce t in . The ban&

a t 736, 25611~1 sug+st benzene (200, 255nn) and phenol ic

@ ( 21 1, 270ni:i) ZI - t r a n s i t i o n s . Both moie t ies a r e

present i r l t h e que rce t in ( r i n g s X and z) .

= rhamnose = Q u e r c i t r i n

R = r u t i n o s e = r u t i n .

These k:nl?s s h i f t e d on add i t i on of a l k a l i n e reagei i ts (sodium

hydrcxifii>,, i i lurniniw c h l o r i d e and sodium a c e t e t e ) t o s h o r t e r

wavelc!n,.-t2.s. The rc%ct io i l of t h e s e s h i f t r eagen t s conf i r r n t h e

f 1avo:lnirl n n t u r ~ of

?he broad band

v i b r a t i o n s poss ib ly

t h i s conpound

-1 a t 3400 cm i s c h a r a c t e r i s t i c f o r 0 - EI i n d i c a t i v e of t h e phenolic 0 - H, whi le t h a t

a t 1??0 &;T' shows ke ton ic 6 0 v i b r n t i o n s f o r the r i n g Y 6 0

end 1 .:',8 on-' band i n d i c a t e s C - 11 arormt ic s t r e t c h i n g v ib ra t ions .

Th i s ::auld he f o r both r i n g s and Z. S u b s t i t u t i o n s i n t h e

i l r ~ m i ; ~ i c r i n e s a r e i nd ica t ed by t h e bands i n the rcgion

- 1 " 0 - ""G CE which i n d i c z t e s d i - OT t r i s u b s t i t u t i o n s .

""1.2 n r n spec tril~l c o n f i r m t h e arornatic n u c l e i wi th t h e

peaks ~t 7.40, 6.30 an<. 6.38. These indicct tes t h e pro tons on

r i n g s X ;mtl Z. Tho p?c?lcs a t 4.25, 3.90 - 3.15 sad 0.80 i n d i c a t e

OH, r>g! ~no{,~lycosyl n r d s a t u r a t e d ( i n rhamnosyl) - protons 3

r e spsc ' l v ~ l y .

As ~ ' i ~ n t l ~ ~ r : e d e : i r l i e r (page 51 ) t h e aglyconc p a r t of compound A

rave i q t l m t i cc ; Sf va lue v i t h que rce t in when both compounds were

chron:.! +c f~ :?yhe(? t 9;:c ther. Compound A was subsequent ly i d e n t i f i e d

3s ri,14:ir? by 7- d i r ~ ~ c i . a - TLC with a u t h e n t i c saml les of que rce t in

g lycos ides . S imi l e r ly , conpound B wns i d e n t i f i e d as quercetin-3-

rhamos ide ( q u e r c i t r i n ) froci i t s colour r c a c t i o n s and - TLC.

Sur.:i,crily, f r o n t h e a n a l y s i s of and comparison of t h e s p e c t r a

E l t r a v i o l e t (w) ; I n f r c r e d ( IR) ; Proton Magnetic gesonance

7 ( PMB) : 2nd Elec t ron I o n i z a t i o n Mas. Spec t r a ( E ~ N s ~ with those

published f o r t h e c o ~ . p o u ~ d s , and by d i r e c t &l- TLC with t h e

r e f e rence ma te r i a l s , t h e i d e n t i t y of t h e two cotlpounds were

. es t ab l i shed . Q u e r c i t r i n (1: r rhamnoside) , hespe r id in and l iut in >++ 1 *&+nw**

ry 1 - k3 9 **: 1 >. a r e a l l a c t i v e d i u r e t i c substances. Rutin i s va luab le i n t h e

I )

t rea tment of c a p i l l a r y f r a g i l i t y sin, G . M. 1959).

k dose of 400mg/kg body weight of t h i s e x t r a c t was chosen

as t h e o p t i ~ r t ~ s u b l e t h a l dose f o r t h i s study. Th i s was a r r i v e d

a t by the> admin i s t r a t i on of va r ious doses o f t h e e x t r a c t and,

t h e biolu{ricnl pmametcr of l i v e r t b body weight r a t i o was

monitored. The l i v e r weight t o body weight r a t i o f o r t h e t r e a t e d

r a t s was ,.',.I0 - + 0.38 and 3.30 2 0.81 f o r t h e c o n t r o l rats and t h e

rnicro~~i:ic?l p ro t e in increased apprec iab ly (8.47 & 0,60 f o r t h e

t r c a t e d r?ts and 7.44 + 0.24 f o r t h e c o n t r o l rats) ,

S i g n i f i c e n t changes were c l e a r l y demonstra,ted i n the a c t i v i t i e s

of some wzymes of t h e !,!icrosomal f r a c t i o n s fo l lowing t h e admini-

s t r a t i o n cr" t h i s p l an t e x t r a c t . The decrease of phenobnrbitone-induced

sleepin? t i n e froti, 405 A 2-15 ( f o r un t r ea t ed r a t s ) t o

360 + 5.06 minutes ( f o r t h e t r e a t e d r a t s ) suggested t h a t t h e r e

was probably 3.n i n c r e a s e i n t h e a c t i v i t j e s of t h e enzymes r e spons ib l e

w r h i tone-induced s l e e p i n g tirric a s w e l l a s t h d t o f the l i v c r : L o

hody w e i g h t at: r a t i o and t h e m i c r o s o m a l p r o t e i n c o r ~ t e n t may be

d i t h e r by d i r e c t s y n t h e s i s uf t h e s e enzyme c o m p o n e n t s or t h e

a t i r n u l a t i o n of t h e enzyme a c t i v e sites (increased m e t a b o l i s m o f

p h e n o b a r b i t o n e ) b r o u g h t a b o u t by biochemi .ca1 chanc j r !~ i n d u c e d by

t l ~ e p r e s m c c crf t h e s u l : ; t . r a t e . However, s i n c e a y r a d u a ' l d e c r e a s e

i n the s l e e p i n g t i m e o f t h e r a t s was o b s e r v e d w i t i r subsequen t

a:.triii nis t r a t i o n o f t h e e x t r a c t , i t was i n f e r r e d ti\ '> t t h e i n c r e a s e

i n a c t i 6 i . t ~ was d u e t o t h e s y n k h e s i s o f t h e n e e d e d p r o t e i n t o cope

:!.I. t h the c h e m i c a l a d v e r s i t y w i t h i n i t s s y s t e m ,

i n c , t there was a decrease i n t l ~ e a c t i v i t y of NAC:F'H-cytochrorne C

- 1 ( i ' -450) r ~ d u c t n s e (1.19 x - 0.40 umole niinol iarj prote i i l f o r

c n p i t r i o m t a l r a t s , and 4.76 x 10'-~+ 0.63 urnole rnin-' rn(j p r ~ t c i n - 1 - ccnkrol a n i m a l s 1: T h i s e v i d e n c e s u p p o r t e d thc: s u q q e s t i o n t h a t t11c

experimental r a t s , and 4.40 + 0.05 f o r t h e c o n t r o l r a t s ) showed - f u r t h e r t h a t t h e r e d u c t a s e d i d n o t c o n t r o l t h e rate of k h c rnic:roscm,~l

r w n o o x y g e n a s e a c t i v i t y . However, b o t h enzymes a re an i n t e g r a l

carnponent o f t h e microsoma1 monooxygenase system of w h i c h c y t o c t ~ r o m e

F-450 i s t h e t e r m i n a l o x i d a s e .

*'--. . t . of xanobiotics and endogenous substrates such as steroids, f a t t y

.q ,.! .,.,.;* ;. acids, environmental wlutants a i d the quercetin-3-glycosi/ies al<:ct,.

' p n JT However, since microeomes contail1 enzymes which oxidize h4wIil ard

u t i l i z e molecular oxygen in t'ne absence of drugs, the t~laljrsi:; i s

grerltly complicated. Whether or not a drug st iuulates or depresso~

hIWF1-1 oxidation would seem to depend upon whether or not i t s t i ~ 11 t x i .

or depl-esses cytochronce P-450 reductase a c t i v i t y ; t h i a in t u r : ~ wo . l . 1

o r hid no effect on the reductaso.

Despite the ncclained antimalarial property of nzadi.racb:e 6 . i 2 i . r ' 2 t

by patrons uf herbal medicine, tiie extracts showed ~ : o aril;i&.!r, . l i t ) . c

i n fec ted a lb ino r a t s ÿ el la 1 9 7 6 ) ~ However, Ekanern ( 1978) reported 9

t h a t the a n t i n a l a r i a l property of A. &&&Q t o be due t o a reduction

i n t h e p a r a s i t e count i n chloroquine - s e n s i t i v e s t r a i n s of 2. p::*...4 ' -

. me' - i n fec ted mice t r e a t e d with a decoction of t h e leaves.

,A- ', -. d@" I.

:.* Since the an t ima la r i a l p r o p e r t i e s of Azadirachta e x t r a c t mey o r m y

n o t be due t o d i r e c t a n t i - p a r a s i t i c a c t i v i t y , i t was considered

. necessary t d st-.~dy t h e parasi te-host c e l l biochemistry during

treatment wi th Neem f o r a poss ib le explanat ion of the m t i n a l a r i a l

property. The s ign i f i cance of t h e oxidat ion reduction, o r "redox"

s t a t u s of r ed blood c e l l s on p a r a s i t e and h o s t c e l l biochemistry

during ma la r i a l i n f e c t i o n has been inves t iga ted by Etkin and o the r s

( ~ t k i n , 1975; Etkin and Eaton, 1 9 7 5 ) ~ They observed t h a t increased

- l e v e l s o'f red c e l l oxida t ion a t t endan t upon p h X f l 0 d i ~ l i n f e c t i o n

i:, a cona i s t en t f e a t u r e of malaria . It has been suggested that " .

while plasmodium p a r a e i t e s themselves a?pear t o be respons ib le f o r

genera t ing o x i d a l t s , excessive oxidat ion may, i n t h e long run, be

de t r imenta l t o continued and success fu l ma la r i a l i n f e c t i o n ( ~ t k i n and

Eaton, 1975). An appropr ia te redox balance nus st t i lerefore be maintained

i n o rde r t o ensure red c e l l i n t e r g r i t y and pmper metabolic

funct ioning which i s a l s o e s s e n t i a l f o r t h e development of the

plasmodium pa ras i t e . In e f f e c t : , under induced physiological

condit ion where excess oxidat ion occurs and cannot be compensated,

a v a r i e t y of danaging e f f e c t s w i l l ensue and w i l l r e s u l t i n t h e

d e s t r u c t i o n of both the red blood c e l l s (haenolys is ) and t h e

malaria pa ras i t e s .

For ma1,zr ia~infec t ion t h e r e l evan t redox r e a c t i o n would be:

a) Conversion of haenoglobin t o an oxidized fo rn , nethaenoglobin.

b) Conversior~ of the ( reduced) enzyme cof a c t o r s n icot inanide

adenine dinuc1eot;ide (NADH) and n ico t inan ide adenine

d inuc leo t idc phosphate (NADPH) t o t h e i r oxidized counterpar ts ,

NAD+ and: NADP-'

c ) Conversion of t h e compound g lu ta th ione (m) t o i ts

oxidized counterpar t (GS - SG).

The ?resent s tudy demonstrates t h a t t h e aqueous e x t r a c t

from t h e l eaves of kzadi rachta i n d i c a adn in i s t e red in t ra-

perMkmeally induces s e l e c t i v e l y s i g n i f i c a n t changes i n t h e

biochemistry of t h e l i v e r i n the s h o r t period of treatment. The

suppression of NADPH cytochrorne C ( P-450) reductase a c t i v i t y by

75$ is b i o l o g i c a l l y and s t a t i s t i c a l l y s i g n i f i c a n t , s i n c e i t is

genera l ly considered t o be the ra te- l imi t ing component of t h e

microsonal nonooxygenase system ( ~ i k a n s at d, 1981 1. The

probable induct ion of cytochrome P-450 haenoproteins i s a l s o

suggested by t h e decrease i n phenobarbitone sleepimg time and t h e

e l eva t ion of ra i - l ine hydroxylase a c t i v i t y i n t r e a t e d r a t s . This

e f f e c t when considered independently n igh t n o t in f luence t h e

o v e r a l l phamcco-kinet ics and t o x i c i t y of both endogenous and

exogenous substances involved i n malar ia i n f e c t i o n , s ince i t has

been shown t h e t the reductase alono does not control the r a t e of

s u b s t r a t e oxidetion ( ~ a t s u b a r a a, 1976). However, when t h i s

r e s u l t i s conbined with the evidence presented e a r l i e r (Etkin 19'8:l;

Iwu, 1983), i t would appear t h a t t h e mode of ac t ion of t h e

e x t r a c t of A e a d i r a c h t ~ A n d i c ~ i s by redox perturbation i n the

form of t h c i n r o s i t i o n of s u b s t a n t i a l oxidant s t r e s s during

malar ia l infec t ion.

I t has been suerested t h a t the increased oxidation during

m a l a r i d i n f c c t i o n might explain the biochemical bas i s f o r t h e

protec t ion agains t fu ln inan t malaria observed i n people with

inher i t ed de f ic ienc ies of glucose-6-l~hoephate dehydrogenase

(G-6-PD). This enzyme i s responsible f o r ca ta lys ing the f i r s t

of a series of r eac t ions t h a t generate t h e reducing compounds

NLDH, Mk.lIi3! and GSH (Etkin , 1981 ) . It has a l s o been shown t h a t

t h e sane pheiionenon is responsible f o r tho non-susceptibi l i ty

t o malaria a t t a c k of animals with v i t ~ m i n E deficiency aton on

& ah, 1976). Supplementation of vitamin E i n such aninala

resu l t ed i n a l o s s of r es i s t ance t o plasmodia1 infect ion. The

generat ion of oxidants may a l s o explain the mode of ac t ion of

some syn the t i c an t imala r i a l drugs t h a t act on the endoery-

th rocv t i c Torn of the disease.

The toxicological implicat ion of t h i s f inding has not been

deternined but it i s obvious t h a t per turbat ion i n t h e redox

s t a t u s may or may n o t l e a d t o enhanced metabolism o f xenobictics

sometime w i t h ha rmfu l e f f e c t . I t i s t h e r e f ' o r e i m p e r a t i v e t h a t

t t h e e f f e c t i v e d o s e and t o x i c i t y s f t h e s e v e r y p o p u l a r a n t i -

m a l a r i a l a g e n t s s h o u l d b e d e t e r m i n e d t o m i n i m i s e t h e a t t e n d a n t

L-isk of s u c h u n c o n t r o l l e d m e d i c a t i o n . The i n d i s c r e r n i n a t e u s e

of h e r b a l r e m e d i e s p o s e s s e r i o u s h e a l t h h a z a r d s f o r o b v i o u s

r e a s o n s . However, i n t h e a b s e n c e o f a c o u r s e and effect

a s s o c i a t i o n w i t h any s p e c i f i c i a t r o g e n i c d i s e a s e or p a t h o l o g i c a l

p r o c e s s i t r e m a i n s a c o n v e n i e n t and c h e a p e r a l t e r n a t i v e i n

d e v e l o p i n g c o u n t r i e s .

The r e s u l t s p r e s e n t e d 3.n t h i s work emphas i ze t h a t :

a ) t h e aqueous e x t r a c t of i n d i c a c o n t a i n s a t l eas t

2 ~ h a r ~ ~ ~ a c o l o g i c a l l y a c t i v e a g e n t s querce t in-3- rhnmnos ide

( y u e r c i t r i n ) and quercetin-3-rhamnoglu~oside ( R u t i n ) ,

b) t h e e x t r a c t o f - A. i n d i c a w a s found t o i n c r e a s e t h e

microsornal p r o t e i n c o n t e n t ; t o have r educed t h e phcno-

a n i l i n e h y d r o x y l a s e a c t i v i t $ r , a s w e l l as a r c d u c t i ~ n

of t h e NADPH-cytochrome c (P-450) r c d u c t a s e a c t i v i . t y ,

which may p o s s i b l y p l a y a role i n t h e b i o c h e m i c a l

mechanism o f i ts a n t i m a l a r i a l a c t i o n .

I t i s hoped t h a t t h e t o x i c i t y t e s t i n g s of t h e s e a g e n t s

w i l l l e a d t o t h e deve lopmen t o f d r u g s f o r t h e i r employment

i n t h e t r e a t m e n t o f m a l a r i a by more c o n v e n t i o n a l rnctl~ods.

STATISTICAL Ai\!liLYSIS (c leaver e t a l , 1974) --

Where ji equals t h e mean values 4 n i s t h e number of values

and S, t h e standard deviat ion.

SIGNIFICANCE TESTING: STUDEBTS - TEST --.

The'pseudonyn ' s tudent ' concealed the i d e n t i t y of W.S.

Gosse t , who worked f o r an Edwardian brewery. H i s employers

forbade him t o publish h i s s t a t i s t i c a l work under h i 8 own name

i n or:isr t o conceal from t h e i r competitors t h e advanced

nature o f t h e i r product t e s t ing .

DEFINITION

'STATISTIC ----- This i s fiefined a s t h e r a t i o of the d i f fe rence of t h e

means o f two sanples t o the standard devia t ion of the means.

There a r e two inpor tant p r a c t i c a l s i t u a t i o n s i n which the

knowledge o f s i g n i f i c a n t t e s t i n g w i l l be useful .

a. t o !mow how observations f ron d i f f e r e n t samples

should be t r ea ted .

b. t o e s t a b l i s h whether a change i n experiliiental

condit ions has changed the resu l t .

Student t - t c s t of s ign i f i cance was used f o r a l e v e l of

a i&f icancs qf 5% ( P = 0.05) or 5% confidence level .

The s i g n i f i c a c c d i f fe rence between two nonpaired va r iab les

was calculated from the f orrnular ( ~ n e d c c o r & A, 1967).

n 4 n2 -2 1

wherc nl = np = 3, the number of animals s a c r i f i c e d i n

each experklcnta l group f o r one s e t of experiments,x and 1

X2 are t h e d i X e r e n t means with standard devia t ions S and 1

S calculatcc! frcm t h e n and n nmber of var iables . 2 1 2

Thc ' t ' clistribuztion t a b l e gave a s i g n i f i c a n t

d i f fe rence ol" i2.776 f o r 5% l e v e l of s ign i f i cance ( p = 0.05)

agains t (n, -; n2 - 2 ) degrees of frecdon. Thus means

d i f f e r i n g by B.776 o r nore a r e s i g n i f i c a n t l y d i f fe ren t .

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a 4

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