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University of Nigeria

Research Publications

OKORO, Azubuike Gabriel

PG/M.Sc/85/3227

Extraction, Purification and Antimicrobial Activities of Quaternary Alkaloids of the Plant Sphenocentrum Jollyanum Pierre

Biological Sciences

Microbiology

e August, 1989

~XTRACTLON, P U R I P I CAT ION AND ANTlI\1I CKOBI AL

ACT I V l T I l i S OF QUAT GRNARY ALKALOIDS O F THE

PLANT SPHQTOCENTKUIVI JOLLY rLNUlvI PIERRIT

OKOLO, A Z U B I l G GABKIXL

(PG/M. SC/85/3227)

DEPARTVUNT OF MICROBIOLOGY

UNIVBRSITY O F N I G E R I A

NSUlClGi

AUGUST, 1989

S~13erv.i sor:

A 1):roject sub!ni t ied . t o -the

Department of i : ~ I i c r u b ~ . o l o ~ y , i n p a r t i a l fu1Pi.l. rnsrlt o f the req~xirerncnts for t h c n.v~arCt of

M r . Azubike G. Okolo, a post -graduate s t u d e n t i n t h e

Department o f Microbiology, s p e c i a l i s i n g i n I n d u s t r i a l

Microbiology h a s s a t i s f a c t o r i l y completed t h e requi rements

f o r cou r se and research . work f o r t h e degree o f

Master o f Sc ience (M.Sc ), i n I n d u s t r i a l Plicrobiology.

The work covered i n t h i s t h e s i s i s o r i g i n a l and h a s

no t been submi t ted i n p a r t o r f u l l f o r any diploma o r

d e u e c o f t h i s o r o t h e r U n i v e r s i t i e s .

DWARTMENT OF MICHOBIOLOGY UNIVEHSITY OF N.IGERIA '

NSUKKA, ' '

To t h e memory o f M r . Geof f rey 0. Okolo

M y f a t h e r

t h e s t e a d f a s t n e s s and perseverance o f

Mrs. F e l i c i a U. Okolo

My mother

t h e progres:, o f t h e e n t i r e f ami ly . 8

Poe t i c E x ~ r e s s i o n

J e v i l You Know

(. . . . .For Ant imicrobia l P l a n t s )

Whooping Cough and f e s t e r i n g s o r e s

Red eyes, sandy f a c e with oozing pus

Swollen g e n i t a l s ho t l i k e h e l l Aches andpai% i n ou r g u t s we l l E v i l scheme of i n v i s i b l e forms Crawling i n ou r system l i k e worms Gentamycin, p e n i c i l l i n even b a c i t r a c i n Products from your dear K i t and Kin; , You g i x e t h e scourge; you g i v e t h e c u r e Treachery s e a r s through o u r h e a r t s l i k e a saw I w i l l go down t h e road of my a n c e s t o r s To d ioco v e ~ . what made them g r e a t d o c t o r s I s h a l l hang t h e t r e a s u r e o f my f a t e On t h e wall o f he rbs , r o o t s and l e a v e s till d a t

Those g i v e r o f l i f e t o a l l who b rea the You have given u s food and room t o bread Your benevolence you l e t l o o s e on u s T i l l e t e r n i t y , we s h a l l p reach your cause.

Zubie 1l . th December, 1988

ABSTRACT

The r o o t s and s tems o f s ~ h e n o c e n t r u m jOllsanum were

s e p a r a t e l y sun-dr ied and ground i n t o f i n e powders. The

p r e sence o f t a n i n s , oleogums, f l a v o n o i d s , g l y c o s i d e s ,

v o l a t i l e o i l s and a l k a l o i d s was de t e rmines i n t h e p l a n t

m a t e r i a l . Three a l k a l o i d s were subsequen t l y e x t r a c t e d from

t h e p l a n t , i d e n t i f i e d as pa lma t ine , columbamine and

j a t r o r r h i z i n e and p u r i f i e d , u s i n g t h i n l a y e r

chromatography (TLC) and p r e p a r a t i v e t h i n l a y e r

chromatography r e s p e c t i v e l y . 8

The a l k a l o i d s produced v e r y s i g n i f i c a n t a n t i m i c r o b i a l

a c t i v i t y on F s c h e r i c h i a C o l i , ~ t a ~ h s l o c o c c u s a u r e u s ,

Pseudomonas a e r u a i n o s a K l e b s i e l l a pneumonia, p ro t e u s

v u l g a r i s , A a ~ i l l u s s u b t i l i s and u n d i d % d b i c a n s .

The minimum i n h i b i t o r y c o n c e n t r a t i o n s (M.1.C) o f t h e

a l k a l o i d s were small (0.016 - 0.8mg/rnl) on t h e t e s t

microorganisms. The a l k a l o i d s were obv ious ly

b a c t e r i o s t a t i c a t low concentration^ b u t b a c t e r i c i d a l a t

h i g h e r c o n c e n t r a t i o n s .

he t h r e e a l k a l o i d s had t h e i r o p t i m a l a n t i m i c r o b i a l

a c t i v i t y a t 400C b u t l o s t a c t i v i t y s u b s t a n t i a l l y a t h i g h

t e m p e r a t u r e s (00 - 1 0 0 ~ ~ ) . A l l t h r e e a l k a l o i d s l o s t abou t

50% o f t h e i r a c t i v i t y a t t h e ex t remes o f pH, t h e i r op t ima l

a c t i v i t y b e i n g w i t h i n t h e pH r ange o f 6 - 9.

T e t r a c y c l i n e and a m p i c i l l i n used as c o n t r o l s showed

o v e r a l l h i g h e r a n t i m i c r o b i a l a c t i v i t y o v e r t h e a l k a l o i d s

w i t h t h e e x c e p t i o n o f j a t r o r r h i z i n e . However t h e a n t i b i o t i c s

confornled t o t h e same p a t t e r n o f a c t i v i t y . A l l t h e a

a l k a l o i d s l o s t o v e r 60% o f t h e i r a n t i m i c r o b i a l a c t i v i t y

when s t o r e d open a t room t empe ra tu r e o v e r a p e r i o d o f

seven weeks, a s h a r p c o n t r a s t w i t h what happens w i t h t h e

a n t i b i o t i c s ( T e t r a c y c l i n e and a m p i c i l l i n ) . The e f f e c t

o f t h e a l k a l o i d s on t h e c e l l - w a l l morphology o f t h e

t e s t microorganisms shows t h a t t h e mechanism o f b

b i o l o g i c a l a c t i o n of t h e a l k a l o i d s i n v o l v e s t h e c e l l - w a l l

o f t h e t e s t microorganisms.

TABLES OF CONTENTS

Certification Pages

Dedication

Poetic Expression (Devil You Know)

Abstract

Table of Contents

Likt of Tables

List of Figures

List of plates

1. INTRODUCTION

2. LITERATURE REVIEW The family Menispermaceae

Distribution of the Family Menispermaccae

Phytochemistry

Alkaloids of the Family Menispermaceae

Non Alkaloidal Constituents of the Family Menispermaceae

Definition of Alkaloids

Nomenclature and Classification of Alkaloids

The Benzylisoquinolinc Alkaloids

The Aporphine Alkaloids

The Morphinandienone Alkaloids

The Pavine Alkaloids

The Bisbensylisoquinoline Alkaloids

The Protoberberine Alkaloids

Occurrence and Distribution of Alkaloids

Solubility of Alkaloids Role of Alkaloids in Plants Pharmacolo~ical Activities of the Alkaloids of

~~henoc&trum '011 anum ~nticholinesferase %ct E Adrenocorticotrophic Effect Anaphylactic Effect Other Antimicrobial Activity Other Constituents of Sphenocentrum j ollyanum Plants with Antimicrobial Activity

vi - vii viii - x xi - xii xiii - xiv

3, MATERIALS AND METHODS Microorganism

Authentic Samples of Alkaloids

Plant Materials Used Preliminary Tests

Test for Glycosides

Test for Saponins Test for Flavonoids

Test for Anthracene Derivatives Test for Cyanogenetic Glycosides

Test for Cardiac Glycosides

Test for Steroidal Aglycone

Test for Resinous Matter

Tests for Oleogum

Test for Tanins

Test for Alkaloids

EXTRACTION Separation

Preparation of Thin T,ayer Chromatography (TLC) plates Development

Observation

Visualization

Identification Isolation

Screening for Antimicrobial Activity

Cup-plate method Determination of Minimum Inhibitory Concentration (MIC) Determination of Minimum Cidal Concentra- tion (MCC)

47 41 47 - 42 43 43 43 44 45 45 46 47 47 48 48 49 50 51

51 52 - 54 55 55 55 56 57 58 59 - 60

61 Determination of the Effect of Temperature on the Antimicrobial Activity of the Alkaloids 62

Determination of the Effect of pH on the Antimicrobial Activity of the Alkaloids 63

Determination of t h e E f f e c t of

Open Storage on t h e Antimicrobial

A c t i v i t y of t h e Alkaloids

Determination of t h e E f f e c t of t h e

Alkaloids on t h e Cell-Morphology

of t h e t e s t Microorganisms

4. RESULTS

5 . DISCUSSION AND CONCLUSION

6. ACKNOWLEDGEMENT

7. REFERENCES

8. APPENDIX

LIST OF TABLES Page 1. Geographical D i s t r i b u t i o n o f Af r i can

Menispermaceae Spec ies (accord ing t o T,oupin, 1962)

2. Alka lo ids From Niger ian Menispermaceae Spec ies

3. Chemical C o n s t i t u e n t s P r e s e n t i n E x t r a c t s of Stem and Root of t h e P l a n t Sphenocentrum jollyanum

4. I d e n t i f i c a t i o n of P u r i f i e d Compounds i n F r a c t i o n s of Whole Root and Root Rark E x t r a c t s of Sphenocentrum jollyanurn on TLC p l a t e s

5. I d e n t i f i c a t i o n of P u r i f i e d Compounds i n F r a c t i o n s of Whole Stem and Stem Rark E x t r a c t s of Sphenocentrum ,jollyanurn on TLC p l a t e s

6. I n h i b i t i o n o f Microbia l Growth bv Sphenocentrum jollyanum (Root and j ark) Crude E x t r a c f s

7. Ant imicrobia l A c t i v i t y of P u r i f i e d Alka lo ids of Spenocentrum jollyanum p l a n t

8. Minimum I n h i b i t o r y Concent ra t ion (MIc) of the P u r i f i e d Alkaloids of Sphenocentrum j ollvanum p l a n t

9. Minimum C i d a l Concent ra t ion (Mcc) of t h e P u r i f i e d Alka lo ids of Sphenocentrum jollyanum p l a n t

10. The E f f e c t of Temperature on t h e Antimicro- b i a l A c t i v i t y of T e t r a c y c l i n e

11. The E f f e c t of Temperature on t h e Antimicro- b i a l A c t i v i t y of Ampic i l l i n

12. The E f f e c t of Temperature on t h e Antimicro- b i a l A c t i v i t y of Palmat ine

13. The E f f e c t of Temperature on t h e Ahtimicro- b i d A c t i v i t y of t h e Combined P u r i f i e d Alka lo ids

14. The E f f e c t of Temperature on t h e Anti- mic rob ia l A c t i v i t y of J a t r o r r h i z i n c

15. The E f f e c t of Temperature on t h e Anti- mic rob ia l A c t i v i t y o f Columbamine

16. The E f f e c t o f pH on t h e Ant imicrobia l A c t i v i t y of T e t r a c y c l i n e

The Ef fec t of pH on the Antimicrobial Ac t iv i ty of Ampicill in

The Ef fec t of pH on t h e Antimicrobial Ac t iv i ty of Palmatine

The Effect of pH on t h e Antimicrobial Ac t iv i ty of t h e Combined Pu r i f i ed Alkaloids

The Ef fec t of pH on t h e Antimicrobial Ac t iv i ty of J a t r o r r h i z i n e

The Ef fec t of pH on t h e Antimicrobial Ac t iv i ty of Colurnbamine

The E f f ec t o f Open microbial Ac t iv i t y

The E f f ec t of Open microbial Ac t iv i ty

The E f f ec t of Open microbial Ac t iv i ty pur i f i ed Alkaloids

The E f f ec t of Open microbial Ac t iv i ty

Storage on t h e Anti- of Tetracycl ine

Storage on t h e Anti- of Ampicillin

Storage on t he Anti- of Palmatine

Storage onl t h e Anti- of t he Combined

Storage on t h e Anti- of J a t r o r r h i z i n e

Storage on t h e Anti- of Columbamine

I n t e r p r e t a t i o n of t he Chromatogram of t h e Stem Ext rac t s

I n t e r p r e t a t i o n of t h e Chromatogram of t h e Root Ext rac t s

Methods of Preparat ion of pH Buffers 1 - 14

x i i i . L I S T OF FIGIJRES Pages

S t ruc tu ra l Formulae of Palmatine, Columbamine and Ja t ro r rh i z ine ( ~ l k a l o i d s of Sphenocentrum j ollyanum) 22

Flow-Chart of t he Extract ion Procedure 53 The Effect of Temperature on the Anti- microbial Act iv i ty of Palmatine 83

The Ef fec t of Temperature on the Anti- microbial Act iv i ty of Columbamine 84

The Ef fec t of Temperature on the Anti- microbial Act iv i ty of J a t ro r rh i z ine 85

The Ef fec t of T,mperature on t h e Anti- microbial Act iv i ty of t he Combined Alkaloids 86

The Ef fec t of pH on the Antimicrobial Act iv i ty of Palmatine 87

The Ef fec t of pH on the Antimicrobial Act iv i ty of Columbamine

The Ef fec t of pH on the Antimicrobial Act iv i ty of J a t ro r rh i z ine 89

The Ef fec t of pH on the Antimicrobial Act iv i ty of the Combined Alkaloids 90

The Ef fec t of Open SLurage on the Anti- microbial Act iv i ty of Palmatine 91

The Ef fec t of Open Storage on the Anti- microbial Act iv i ty of Columbamine 92

The Ef fec t of Open Storage on the Anti- microbial Act iv i ty of J a t ro r rh i z ine 93

14 The Ef fec t of Open Storage on the Anti- microbial Act iv l ty of t h e Combines Alkaloids 94

16. The Effect of Temperature on the Anti- microbial Ac t iv i t i e s of t h e Three Alkaloids and the Combined Alkaloid 95

16. The Ef fec t of pH on the Antimicrobial A c t i v i t i e s of t he Three Alkaloids and the Combined Alkaloid 96

17. The E f f e c t of Open Storage on t h e Anti- microbial A c t i v i t i e s of t h e Three Alkaloids and t h e Combined Alkaloid 97

18. The E f f e c t of t h e P u r i f i e d Alkaloids on t h e C e l l Morphology of - Em - c o l i 98

19. The E f f e c t of t h e P u r i f i e d Alkaloids on t h e C e l l Morphology of Staph. aureus 99

20. The Chromatogram of t h e Stem E x t r a c t s I55

21. The Chromatogram of t h e Root E x t r a c t s 156

P l a t e 1 : Zone of I n h i b i t i o n a t t h e Optimum tempera ture of a n t i m i c r o b i a l A c t i v i t y f o r Palmatine. 100

P l a t e 2:

Zone o f I n h i b i t i o n a t t h e Optimum tempera ture of Ant imicrobia l A c t i v i t y f o r Ampicil l in. 100

P l a t e 3: Zone o f l n h i b i t i o n ~ at t h e Optimum pH o f Ant imicrobial A c t i v i t y f o r Palmatine. 101

P l a t e 4: Zone of I n h i b i t i o n a t t h e Optimum pH o f Ant imicrobia l

A c t i v i t y f o r J a t r o r r h i z i n e . 8 101

P l a t e 5 : Zone o f I n h i b i t i o n . at t h e Optimum pH of Ant imicrobia l A c t i v i t y o f Columbamine.

P l a t e 6: Zone o f I n h i b i t i o n a f t e r t h e 4 t h Week o f Open S torage Of Palmatine. 102

P l a t e 7: Zone o f I n h i b i t i o n a f t e r t h e 4 t h Week o f Open S torage o f t h e Combined P u r i f i e d Alkaloids .

P l a t e 8: Zone o f I n h i b i t i o n a f t e r t h e 4 t h Week o f Open

S to rage o f J a t r o r r h i z i n e .

P l a t e 9 : Zone o f I n h i h i t i o n a f t e r t h e 4 t h Week o f Open

S to rage o f Te t r acyc l ine .

INTRODUCTION.

During t h e p a s t f i f t y y e a r s , o rgan ic chemis t s have

syn thes i zed a l a r g e number o f i n t e r e s t i n g chemical

subs t ances many o f which have proved u s e f u l i n t h e

t r ea tmen t o f d i s eaces . T h i s had been h a i l e d as t h e

t h e r a p e n t i c r evo lu t ion . Yet t h e a b i l i t y o f man t o

s y n t h e s i z e new chemical subs t ances i s very l i m i t e d

(sofowara, 1979)~. ~ u r i n g t h e p a s t f i f t y m i l l i o n y e a r s ,

however, o r l o n g e r - p l a n t s hav.e been a b l e t o s y n t h e s i z e

a v.ery much wider r ange of chemical subs t ances , many

of which have been and can be o f tremendous va lue i n t h e

t r ea tmen t and p reven t ion o f d i sease .

It i s imposs ib l e t o know how many p l a n t s p e c i e s t h a t

e x i s t through o u t t h e world today - a l though w e l l

over 250,000 s p e c i e s o f f lower ing p l a n t s ( t h e most

advanced group i n t h e evo lu t iona ry s c a l e ) has been

recognised. laus us & R7, , 1970)

Of a l l l i v i n g organisms, p l a n t s a r e unique i n as

much as t h e y need f o r t h e i r e x i s t e n c e very s imple

chemical subs t ances - carbon d iox ide ( ~ 0 ~ ) and oxygen

(02) from t h e a i r and mine ra l salts d i s s o l v e d i n water

from t h e i r s o i l o r aquieous environment. The presence

o f c h l o r o p h y l l enab le s them t o u t i l i z e d i r e c t l y energy

from t h e sun and t o conver t t h i s l i g h t energy i n t o

chemical energy by b ind ing i t i n t o chemical subs t ances

u n t i l r e q u i r e d f o r use. his p roces s known as

pho tosyn thes i s , c o n v e r t s hydrogen, C02 and water i n t o

carbohydra tes . Tbnn Sy , s e v i e s o f w e l l - de f ined

o x i d a t i v e phosphory la t ions and o t h e r p roces ses t h e s u g a r s

a r e conver ted t o o t h e r o rgan ic subs t ances found i n p l a n t s

and i n so doing i n c o r p o r a t e n i t r o g e n , su lphur e t c from

t h e i n o r g a n i c s a l t s t o g i v e p r o t e i n s and g l y c e r i d e s

t o g e t h e r wi th many phenol ic compounds, s t e r o i d s ,

t e r p e n e s , a l k a l o i d s e t c ( ~ y l e r & a. , 1981 )

These sub tances d i f f e r from s p e c i e s t o s p e c i e s s o t h a t

t h e r e i s a wide v a r i a t i o n o f chemical subs t ances

produced i n t h e p l a n t Kingdom. 8

It can e a s i l y be demonstrated t h a t t h e po lysaccha r ides ,

p r o t e i n s and g l y c e r i d e s a r e e s s e n t i a l r e s e r v e . Sources

of energy f o r t h e p l a n t , bu t i t i s l e s s easy t o a p p r e c i a t e

t h e r o l e o f t h e secondary p l a n t p roduc t s u s u a l l y p r e s e n t

i n small amounts only . Yet t h e y must p l a y a r o l e i n t h e

p l a n t matabol ic p rocesses , o r t h e y would accumulate i n

q u a n t i t y , s i n c e p l a n t s have no means o f removing u s e l e s s

waste products . (Tyle r &&, 1981). The f a c t i s

however t h a t t h e s e chemical subs t ances can and a r e

b e n e f i c i a l t o an imals i n c l u d i n g man. Indeed, t h e e n t i r e

animal Kingdom depend on p l a n t s f o r t h e i r source o f energy.

Some of t h e s e chemical subs t ances can be u t i l i z e d f o r

t h e t r ea tmen t o f d i s e a s e and as t h e r e a r e many

subs t ances which occur n a t u r a l l y and encompass a f a r

wide r r a n g e t h a n i s prov ided by t h e o r g a n i c chemis t , i t

i s obv ious t h a t t h e p l a n t Kingdom o f f e r s a b e t t e r

o p p o r t u n i t y o f p r o v i d i n g r e a l l y u s e f u l med i c ina l agen t s .

S ince t h e p l a n t Kingdome i s ex t remely v a s t , f o r

t h e i n v e s t i g a t i o n s o f m i c r o b i o l o g i s t s , p h a r c o l o g i s t s ,

pharmacognos i s t s e t c t o be e f f e c t i v e t h e r e would have

t o be c a r e f u l s c i e n t i f i c p l a n n i n g based upon o u r p r e s e n t

knowledge r e g a r d i n g t h e u s e o f p l a n t s f o r t h e t r e a t m e n t

o f d i s e a s e .

The concep t o f t h e u s e o f d r u g s t h a t i nvo lv , e s d i a g n o s i s 8

d a t a and f i g u r e s , epidemiology, p o p u l a t i o n c o n t r o l e t c i s

a modern one, b u t even i n t h e p a s t , t h e r e were many

d i f f e r e n t ways o f u s i n g p l a n t s . The p a s s i n g o f t i m e

h a s n o t o b l i t e r a t e d them and i n d e e d t h i s c o u l d h a r d l y

be t h e c a s e when, one c o n s i d e r s t h e d i f f e r e n t s-Lages o f

development i n o u r many c i v i l i z a t i o n s t h roughou t t h e

world today (Sofowara, 1979).

Sphenocentrum .iollyanum P i e r r e i s found i n c l o s e d

f o r e s t s i n I v o r y Coast , Ghana and N ige r i a . According t o I r v i n e (1961 ), i t i s an e r e c t sh rub w i t h g l a b r o u s

b r a n c h l e t s and v a r i a b l e l e a v e s on t h e same shoo t . Leaves

a r e o b r a t e e l l i p t i c and e n t i r e o r pa lma te ly lobed . The

apex i s acumina te as w e l l as t h e l o b e and t h e p e t a l e i s

s u l c a t e . F lowers o c c u r i n a x i l l a r y c l u s t e r s and are

y e l l o w i s h - whi t e w h i l e t h e f r u i t s a r e a v o i d - e l l i p s o i d ,

broad a d puberulous . The r o o t s a r e ye l low i n c o l o u r .

Remarkable powcrs iii, c la imed f o r t h i s p l a n t i n

h e a l i n g wounds which a r e f i r s t washed w i th a d e c o c t i o n

o f t h e l e a f y twigs and covered w i t h t h e powdered bark.

The p l a n t i s a l s o chewcd as an a p h r o d i s i a c (Iwu, 1986)

The r o o t s and f r u i t s o f $phenocentrum i o l l ~ a n u ~ a r e

used i n some p a r t s o f South Eas t N i g e r i a f o r b o i l s and

eye i n f e c t i o n s . The r o o t s when chewed have a s o u r t a s t e

and s t i m u l a n t p rope r ty . They c u r e l o s s o f a p p e t i t e and

impa r t a sweet t a s L e t o t h i n g s a f t e r w a r d s . The f r u i t s , a r e e d i b l e and when stewed w i th l i m e a r e used f o r

coughs ( I r v i n e , 1961).

O k a r t e r (1976) r e p o r t e d t h e p r e s e n c e o f p a l m a t i n e

and j a t r o r r h i z i n e , two p r o t o b c r b e r i n e a l k a l o i d s from

i n Sphenoc entrum . i o l l~anum. P d m a t i n e i s a q u a t e r n a r y

a l k a l o i d o f common, occu r r ence i n t h e Menispermacease.

It h a s a mo lecu l a r weight o f 352 and m e l t i n g p o i n t

o f 241'~. I t ' s UV spectrum i s t y p i c a l o f a p r o t o b e r b e r i n e

and a b s o r p t i o n at 436 nm i n n e u t r a l o r a c i d s o l u t i o n

a g r e e s w i t h t h e ye l l ow c o l o u r o f t h i s a l k a l o i d .

( c i l b e r t & &. , 1973).

J a t r o r r h i z i a e i s a l s o a q u a t e r n a r y a l k a l o i d . It i s o f

common o c c u r r e n c e i n t h e $%rnily Menispermaceae and on

p u r i f i c a t i o n g i v e s a ye l low - brown co lou r . Its

mo lecu l a r weight i s 338 ( s e e s t r u c t u r a l formulae on

I n t h i s e r a o f i n c r e a s i n g r e s i s t a n c e o f micro-

o rgan i sms t o t h e a v a i l a b l e a n t i b i o t i c s and when a l o t

o f a t t e n t i o n i s b e i n g f o c u ~ s e d on a l t e r n a t i v e medic ine ,

i t i s o n l y n e c e s s a r y t h a t ac t iv .e i n v e s t i g a t i o n be

c a r r i e d o u t i n t o h e r b a l medic ine w i t h a view t o

t a p p i n g what eve r u s e f u l m a t e r i a l s t h a t cou ld be ob t a ined .

Sphenocentrum jollyanurn i s a p l a n t t h a t h a s been

e x t e n s i v e l y employed i n f o l k l o r i c medic ine i n t h e

t r e a tmen t o f v a r i o u s k i n d s o f a i l m e n t s . . jol lyanurn

c a l l e d I1oJ i -enyir l i n Igbo (a language o f t h e South ~ a s t *

peop l e o f N i g e r i a ) i s r e p u t e d t o c u r e a number o f

m i c r o b i a l i n f e c t i o n s among i t s o t h e r numerous

p o t e n t i a l i t i e s .

T h i s r e x e a r c h w i l l t h e r e f o r e a t t e m p t t o e x t r a c t ,

i s o l a t e and i d e n t i f y t h e a c t i v e c o n s t i t u e n t ( s ) t h a t a r e

r e s p o n s i b l e f o r i t s a n t i m i c r o b i a l a c t i o n . viith t h e

c h e d s t r y o f t h e s e compounds known and t h e i r o t h e r

p r o p e r t i e s de te rmined , t h e p o s s i b i l i t y o f i n c o r p o r a t i n g

them i n t o c l i n i c a l u s e might become more r e a l i s t i c .

LITEHATUlU REVIEW 6.

The ~ a r n i l u Menis~ermaceae:

The term uMenispermaceaett was coined from two Greek

words - mene (moon) and sperma (seed) because t h e

crescent. moon$ shape of t h e seed of t h e p l a n t s i s a

c h a r a c t e r i s t i c f e a t u r e o f t h e family (Hutchinson & &Lo,

1974),

There i s probably no family i n t h e vegetable kingdom

so completely heteromorphous as t h e Menispermaceae,

Generally i t s s p e c i e s a r e climbing and occas iona l ly

e r e c t shrubs o r small t r e e s , They a r e dioecious, t h e i r

1eav.es a r e usua l ly simple, e n i i r e , a l t e r n a t e , o f t en p e l t a t e ,

p e t i o l a t e, e x i s t i p u l a t e , r a r e l y t r i f o l i a t e o r palmately

lobed o r nerwed, The in f lo rescence may be cymose,

pan icu la te , racemose, c a p i t u l a t e , f a s c i c u l a t e and t h e

f lowers a r e small, unisexual and r e g u l a r i n shape, The

f r u i t s a r e more o r l e s s pulpy drupes, while t h e seeds

are o f t e n c u r ~ e d and horseshoe-shaped and may o r may not

hav.e an endosperm. (Hutchinson & a. , 1974)

The family Menispermaceae i s medicinal ly endowed by

n a t u r e and t h e r e f o r e occupies an important p o s i t i o n as

source of medicinal agen t s , This goes t o add more

credence t o t h e f a c t t h a t orthodox medicine (modern

medicine) s t i l l depends t o some extent on t h e p l a n t

kingdom d e s p i t e t h e a v a i l a b i l i t y o f hundreds o f s y n t h e t i c

chemical substances,

The family Menispermaceae comprises 65 genera and

350 s p e c i e s ; m a j o r i t y o f which a r e found i n t h e t r o p i c a l

and s u b t r o p i c a l r e g i o n s o f t h e world, Some genera a r e

found i n t h e warm temperate r eg ions , In o t h e r words

t h e members o f t h e f a r n i 3 y a=e g e n e r a l l y d i s t r i b u t e d

throughout t h e t r o p i c s wi th a few s p e c i e s i n warm tempera te

r eg ions ; i n t r o p i c a l Af r i ca mainly o c c u r r i n g i n evergreen

f o r e s t s ( ~ r e a s e and Evans, $982).

D i s t r i b u t i o n o f t h e Family Menis~ermaceae

Geographical ly , t h e Menispermaceae family l e a n s

towards t h e t r o p i c s , They are as p r e v i o u s l y s t a t e d widely

d i s t r i b u t e d throughout t h e t r o p i c s w i t h a number of s p e d i e s

i n . t h e warm tempera te r eg ions , Even though Nige r i a b o a s t s

o f a d i v e r s e c l i m a t e , t h e Nigerian.. Menispormaceae a r e

n e v e r t h e l e s s seen t o be d i s t r i b u t e d so p r e d i c t a b l y , t h a t

t h e y giv.e c redence t o t h e i r t r o p i c a l l ean ing .

T h e i r h a b i t a t i n c l u d e c l o s e d f o r e s t - Sphenocentrum

jollyanurn and ~ i o s c o r e o ~ h ~ l l u m cumins i i ; Dry dec idous

f o r e s t - S t e ~ h a n i a d i n k l a ~ e i and Tr ic1 i & p b c o r d a t q ;

Semi-desert scrub; - Tinosporq bakig; and secondary f o r e s t

wi th . undergrowth of dec idous evergreen p h i ~ i o c a r ~ a ,

recemifera ,

Most of t h e Niger ian s p e c i e s a r e a l s o widespread i n

many Afr ican c o u n t r i e s p a r t i c u l a r l y i n most West Afr ican

c o u n t r i e s where t h e y occur mainly i n evergreen f o r e s t a r e a s ,

Ini Niger ia , m a j o r i t y a r e d i s t r i b u t e d i n t h e Southern p a r t s

wi th a few such as Tinosporq b a k i s f avour ing t h e drier

North, It i s impor t an t t o n o t e t h a t chasmanthera

d e ~ e n d e n s i s r e p o r t e d t o he commonly p l a n t e d i n n a t i v e

g a r d e n s as a med ic ina l p l a n t wh i l e on t h e o t h e r hand a

few l i k e Jat e o r h i z a Macrantha a r e c u l t i v a t e d ( s p e c i a l l y

i n Ghana). (Table 1 i n appendix) .

PHY'I'O C 1IMI S'L'HY :

The l i v i n g organism ( u s u a l l y p l a n t ) may be

c o n s i d e r e d a b i o s y n t h e t i c l a b o r a t o r y , n o t o n l y f o r

chemical compounds C ~ a r b o h y d r a t e s , p r o t e i n s and

f a t s ) t h a t a r e u t i l i z e d f o r food by man and an imal

bu t a l s o f o r a m u l t i t u d e o f compounds ( a l k a l o i d s , b

g l y c o s i d e s , v o l a t i l e o i l s e t c . ) t h a t e x e r t p h y s i o l o g i c

e f f e c t s . These chemical compounds g i v e p l a n t d r u g s

t h e i r t h e r a p e u t i c p r o p e r t i e s . Drugs a r e used as such

i n t h e i r c r u d e form o r may be e x t r a c t e d , t h e r e s u l t i n g

p r i n c i p l e s b e i n g employed as med ic ina l agen t s . It i s

obv ious t h a t any s t u d y o f t h e d r u g s o f p l a n t o r i g i n

must embrace a thorough c o n s i d e r a t i o n o f t h e s e chemica l

e n t i t i e s c lau us & &., 1970). The u s u a l te rm f o r t h e s e

e n t i t i e s i s c o n s L i t u e n t s ; b u t s i n c e t h e p l a n t o r animal

i s composed of many chemical compounds i t i s common

p r a c t i c e t o s i n g l e o u t t h o s e compounds t h a t a r e

r e s p o n s i b l e f o r t h e t h e r a p e u t i c e l f e c t s and c a l l them

a c t i v e c o n s t i t u e n t s . These a c t i v e c o n s t i t u e n t s a r e

d i f f e r e n t i a t e d from i n e r t c o n s t i t u e n t s which a l s o o c c u r

i n p l a n t drugs . C e l l u l o s e , s u b e r i n and c u t i n a r e u s u a l l y

r e g a r d e d as i n e r t m a t t e r i n p l a n t d rugs ; i n a d d i t i o n ,

s t a r c h . albumin. co lour in f i m a t t e r s ( ~ i m e n t s ) and o t h e r

s u b s t a n c e s may have no d e f i n i t e pharmaco log ica l a c t i v i t y and

a l s o a r e c o n s i d e r e d i n e r t c o n s t i t u e n t s .

Of ten t h e p r e s e n c e o f i n e r t s u b s t a n c e s may modify o r

p r e v e n t t h e a b s o r b a b l i t y o r po tency o f t h e a c t i v e

c o n s t i t u e n t s . To e l i m i n a t e t h e u n d e s i r a b l e e f f e c t s o f

i n e r t matter i n t h e c r u d e d r u g o r i t s p r e p a r a t i o n s , a c t i v e

p r i n c i p l e s a r e e x t r a c t e d , c r y s t a l l i z e d and p u r i f i e d f o r

t h e r a p e u t i c use. A t t i m e s t h e y a r e c h e m i c a l l y a n a l y s e d

and t h e i r e m p i r i c a l formulae c a l c u l a t e d , t h i s i s known as

phy tochemis t ry (sofoviara, 19'79). b

These c o n s t i t u e n t s have been r e f e r r e d t o as "secondary"

p l a n t s u b s t a n c e s , No c o m p l e t e l y s a t i s f a c t o r y e x p l a n a t i o n

h a s been advanced Tor t h e i r p r e s e n c e i n a g r e a t number o f

p l a n t f a m i l i e s ; n e v e r t h e l e s s , t h e i r o c c u m e n c e i n r e l a t e d

g e n e r a and t h e i r chemica l r e l a t i o n s h i p s i n d i c a t e t h a t t h e y

may be a f a c t o r i n t h e b a s i c metabol ism o f p l a n t s .

P h a r m a c o l o g i u a l l y a c t i v e c o n s t i t u e n t s a r e t h o s e t o

which t h e t h e r a p e u t i c a c t i v i t y o f t h e d rug i s a t t r i b u t e d .

They may be e i t h e r s i n g l e chemica l s u s t a n c e s o r m i x t u r e s

o f p r i n c i p l e s , t h e s e p a r a t i o n o f which i s n e i t h e r p r a c t i c a l

n o r a d v a n t a g e o u s ( ~ o f o w a r a , 1979). Even though

phytochernis t ry d e l v e s i n t o t h e c h e m i s t r y o f t h e s d a c t i v e

c o n s t i t u e n t s , t e s t s f o r t h e i r a n t i - m i c r o b i a l a c t i v i t i e s must

n o t n e c e s s a r i l y go i n t o such s p e c i f i c i t i e s i n i t i a l l y .

The s i n g l e chemical subs t ances u s u a l l y ob t a ined from p l a n t s

i n c l u d e s u g a r s , s t a r c h e s , p l a n t a c i d s , enzymes, g lycos ides ,

a l k a l o i d s , p r o t e i n s , hormones and v i t a m i n s whi le t h e

mix tu re s a r e f i x e d o i l s , f a t s , waxes, v o l a t i l e o i l s , r e s i n s ,

o l e o r e s i n s , gum r e s i n s and balsams, o lau us & a,, 1970).

~ l k a l o i d s o f t h e Family Menispermac eae:

Some members o f t h e Af r i can Menispermeceae fami ly have

been i n v e s t i g a t e d f o r t h e i r a c t i v e c o n s t i t u e n t s and among

them a r e p l a n t s p e c i e s which a r e indegenous t o N ige r i a ,

The p r e v i o u s i n v e s t i g a t i o n s o f t h e fami ly show d i f f e r e n t 8

t y p e s o f a l k a l o i d s and o t h e r chemical c o n s t i t u e n t s and i t

i s worth whi le t o e s t a b l i s h a comparat ive review of t h e

members w i t h p a r t i c u l a r emphasis on t h e Niger ian, s p e c i e s ,

Uowever i n f o r m a t i o n on t h e o t h e r chemical c o n s t i t u e n t s i s

ve ry v a s t and i s n o t as r e l e v a n t t o t h i s work as t h e

a l k a l o i d s , so emphasis i s p l a c e d on t h e a l k a l o i d s .

j ' i r s t l y , t h e a l k a l o i d s o f t h e Menispermaceae were

reviewed by Tornita (1952) and Thornber, (1970). S ince t hen ,

t h e nunber o f a l k a l o i d s occu r r ences r e p o r t e d have t r e b l e d

and t h e growth o f i n t e r e s t i n a l k a l o i d a l b i o s y n t h e s i s and

chemical taxonomy prompts a new survey cilif t h i s f i e l d , A

l o t i s known about t h c a l k a l o i d s o f t h e Menispermaceae, but

by f a r t h e g r e a t e s t c o n t r i b u t i o n , t o this knowledge came from

t h e group of Japanese workers l e d by T o n i t a (1966).

The number o f Afr ican s p e c i e s o f t h e Samily so f a r

i n v e s t i g a t e d i s about 24 and they have y i e l d e d about 98

d i f f e r e n t a l k a l o i d s t h e s - t ruc Lures o f which have been

1 1 . e s t a b l i s h e d ; and of t h e s e , 15 s p e c i e s have been r epo r t ed

found so far i n N ige r i a (Ohir i , 1983).

Many o f t h e Niger ian s p e c i e s of t h e family have been

thoroughly i n v e s t i g a t e d e s p e c i a l l y t hose t h a t belong t o t h e

t r i b e s ' f r i c l i s i e a e and Tinosporeae by workers e s p e c i a l l y

those from ~ h a n a and Niger ia .

Among t h e Niger ian s p e c i e s of the ~ r i h e Tinosporeae

i n v e s t i g a t e d was Chasmanthera dependens which t h r i v e s a t

t h e f o r e s t margin and savannahs of N ige r i a where t h e bark

i s used l o c a l l y i n t h e t rea tment of v e n e r a l d i s e a s e s and

as a g e n e r a l t on i c . Except f o r a r e p o r t by O l i v e r (1960) as

b t o t h e presence o f be rbe r ine i n . t h e r o o t s o f Chasmantihera work

dependens, very l i t t l e phytochemicalLwas done on t h i s

s p e c i e s u n t i l t h e i n v e s t i g a t i o n s c a r r i e d o u t by O h i r i g& &., (1982, 1903). In. t h e i r work no b e r b e r i n e was i s o l a t e d from

t h e r o o t 2 however t h e stern was exhaus t ive ly i n v e s t i g a t e d

and s e v e r a l a l k a l o i d s were i s o l a t e d and i d e n t i f i e d . They

inc luded f i v e qua te rnary a l k a l o i d s , n i n e t e r t i a r y non-

phenol ic a l k a l o i d s and f i v e t e r t i a r y a l k a l o i d s . T h i s woCk

y i e l d e d twenty one a l k a l o i d s i n a l l and e igh teen o f them were

i d e n t i f i e d .

y inospora s p e c i e s have a l s o been du ly i n v e s t i g a t e d

by t h e group of workers, B i s s e t and N.waiwu (1983). ~ h e s e

p l a n t p a r t s r evea l ed t h e presence o f some qua te rnary

a l k a l o i d s among o t h e r c o n s t i t u e n t s , l i k e d i t e r p e n o i d b i t t e r

p r i n c i p l e s , po lysaccha r ides and s t e r o i d a l compounds.

Tinospora b a k i s which i s $he on ly one o f t h e ~ i n o s p o r a

s p e c i e s so far found t o be indegenous t o Niger ia , i s

r e p o r t e d t o c o n t a i n two pro t o b e r i b e r i n e a l k a l o i d s namely

palmat ine (Beauguesne, 1938) and b e r b e r i n e (Thornber, 1970).

Ibwever t h e r i n o s p o r a s p e c i e s as a whole c o n t a i n mainly

t h e p r o t o b e r b e r i n e a l k a l o i d s e s p e c i a l l y b e r b e r i n e and

palmat ine which f r e q u e n t l y occu r bu t may be masked by t h e

aporphine, magmoflorine and by cho l ine , whi le b e r b e r i n e

and pa lmat ine a r e found i n t h e stems o f a nurnber o f s p e c i e s ,

pa lmat ine o c c u r s in t h e r o o t s o f more s p e c i e s than does ;

be rbe r ine (Brisset and Mwaiwu, 1983). b

The Niger ian s p e c i e s o f the t r i b e Tinosporeae,

g ioscoreophyl lum cumminsii, Ko lobo~e ta lum aur icu la tum and

;&hi~iocar .va r acemi fe ra have been i n v e s t i g a t e d t o some e x t e n t ,

So f a r t h r e e qua t e rna ry a l k a l o i d s , magnoflor ine ,

j a t r o r r h i z i n e and columbamine have been i s o l a t e d from Q,

cumminsii ( ~ d e s i n a & a,, 1978).

According t o Dv~urna-Badu & &, , (1 980) t h e t h r e e

a l k a l o i d s namely O-methylflav"inanthine, N-methylcoridine

and magnoflor ine have been i s o l a t e d and i d e n t i f i e d from t h e

d r i e d 1 e a v . e ~ and t w i g s o f Yclobopetalum auricu1ai;um. Also

O-methylf lavinanthine has a l s o been i s o l a t e d and i d e n t i f i e d

from @. r acemi fe ra ( ~ a c k i e dm, 1974). - The r e c e n t i n v e s t i g a t i o n s c a r r i e d o u t by O h i r i & &., (1983)

on t h e stem o f s y n c l i s i a scabr ida--Miers y i e l d e d a number

o f a l k a l o i d s - cyc lean ine , nor-cycleanine and c o c s o l i n e ;

a new a l k a l o i d was a l s o cyc l ean iae - N - oxide was a l s o

i s o l a t e d and c h a r a c t e r i s e d . P r i o r t o t h i s work,

Scheirimann & &., ( i 980 ) r e p o r t e d t h e i s o l a t i o n o f

cyc l ean ine from t h e r o o t s 01 t h i s p lank; t h i s was followed

by t h e r e p o r t o f Wambebe & &., (1982) on t h e pharmacology

of t h i s c o n s t i t u e n t .

F i n a l l y , Okar te r (1976) had been a b l e t o i s o l a t e and

i d e n t i f y two p ro tobe rbe r ine qua t e rna ry a l k a l o i d s - palmat ine and j a t r o r r h i z i n e - f r h t h e r o o t s , stem and l e a v e

of Sphenocentrum j o l l ~ a n u m p i e r r e (Okar te r , 1976).

~t t h i s p o i n t , i t w i l l be more i n keeping wi th t h e n a t u r e fi

o f t h i s work t o g i v e a t a b u l a t e d summary o f t h e a l k a l o i d s

o f t h e Niger ian spec i e s . a able 2 i n - appendix).

Nan-Alkaloidal C o n s t i t u e n t s of t h e Family Menis~ermaceae ;

N o n ~ n l k a l o i d a l subs t ances found among members o f the

Niger ian Menispermaaeae i n c l u d e t h e d i t e r p e n o i d h i t t e r

p r i n c i p l e s and o t h e r s such as sweet p r i n c i p l e s , s t e r o l ,

po lysaccha r ides , phenol ic g lucos ides , f a t t y a c i d s ; s t e r o i d a l

compounds and e s s e n t i a l o i l d . The d i t e r p e n o i d b i t t e r

p r i n c i p l e s a r e u s u a l l y C-20 compound^ and t h o s e i s o l a t e d s o

f a r from the family i n c l u d e columbin, i s o co lub in ,

chasmanthin, palmarin , j -a teor in , i s o i a t e o r i n , f i b s a u c i a ,

t i nospor ide and t i n o p h g l l i n e . Others l i k e t i n o sporan,

c o r d i f o l i d e and p i c r o r e t i n have a l s o beer1 i s o l a t e d bu t they

a r e y e t t o be e l u c i d a t e d . Most o f them have been r e p o r t e d

t o occur i n l o c a l l y a v a i l a b l e s p e c i e s o f t he family. For

i n s t a n c e columbin which i s so f a r t h e most abundant i n t h e

Family Menispermaceae and iLs epimer isocolumbin occur i n t he

dry seeds o f pioscoreoph?rllum curnmiasii (Ramstad & &., 1975). Th i s was t h e f i r s t r e p o r t o f isoco1umb;in~ i n p l a n t s

and p re l imina ry t e s t s i n d i c a t e t h a t i t i s a s s t r o n g l y b i t t e r

as columbin. ~ o l u r n b i n h a s been r e p o r t e d t o occu r i n t h e

r o o t s o f J a t e o r h i z q palrnats, (Bar ton and Dov; 1956);

Tinospora s p e c i e s ( B i s s e t and Nwaiwu, 1983) and p - i c l i s i q

subcorda ta (Barton and Dov, 1956) and from t h e s e e d s

o f Sphenocentrum j o l l ~ a n u m ( ~ i l b e r t & &., 1967).

chasmanthin, j a t e o r i n , i s o j a t e o r i n and isocolumbin were

r e p o r t e d i s o l a t e d from r o o t s o f J a t e o r h i z a palmata as minor

congeners o f Columbin (Barasubramanian< g& &. , 1962).

The ~ i n o s p o r a s p e c i e s a r e r e p o r t e d t o c o n t a i n a v a r i e t y o f

b i t t e r p r i n c i p l e s o f d i t e r p e n i o d o r i g i n . They i n c l u d e

columbin, p i c r o r e t i n , t i n o s p o r i d e , c a r d i f o l i d e e t c , I n

a d d i t i o n t o t h e s e d i t e r p e n o i d s , t h e presence o f f l avono ids

hav.e been r e p o r t e d i n p inospora malabr ica (Pralrash ai ld

Lornnn, 1982). The f l avono ids a r e t h e dibendoyl e than.e,

t i nospor inone and a l l y loxyf l avone .

Fukuda & &., (1983) i s o l a t e d and i d e n t i f i e d from

d r i e d l e a v e s o f F inospora t u b c r c u l a t a , N- t ransfe ru loy l

tyrgmine, N - c i s f e r u l o y l t y r m i n e and a new phenol ic

g lucos ide t i n o t u b e r i n e . Sonie ,S.t_ep_h_a-nia s p e c i e s c o n t a i n

o t h e r chemical subs t ances i n a d d i t i o n t o t h e i r a l k a l o i d s .

For i n sbance v . e r a t r i c a c i d and r u t i n were r e p o r t e d i n

s t e p h a n i a a b y s s i n i c a ( ~ ~ a a l & &. , 1966) .

Moreover p r e l i m i n a r y s t u d i e s o f ~ t e ~ h a n i a h e r n a n d i f o l i a

showed t h e p r e sence o f s t e r o i d s and f a t s , wh i l e a m ix tu r e o f

s t e r o l s hav.e been i s o l a t e d from Sphenocentrum j o l l ~ a n u m

( G i l b e r t & am, 1967)

o f m: These a r e known t o e x e r t marked p o s i o l o g i c a c t i v i t y

i n man and t o p o s s e s s many p r o p e r t i e s . However, a p r e c i s e

d e f i n i t i o n 01' t h e term "a lka lo id1I ( a l k a l i - l i k e ) i s somewhat

d i f f i c u l t because t h e r e i s no c l e a r c u t boundary between

a l k a l o i d s and n a t u r a l l y o c c u r r i n g complex m i n e s . ( ~ r e a s e e

and Evans, 1903).

The g roup i s a v e r y v ,a r i ed one bu t a l l c o n t a i n a b a s i c

n i t r o g e n . T h e i r n i t r o g e n atom may be p r imary , secondary ,

t e r t i a r y o r qua t e rna ry .

R q u a t e r n a r y a l k a l o i d i s one i n which t h e t h r e e hydrogen,

atoms hav.e been comple t e ly r e p l a c e d by o t h e r g roups ,

t e r t i a r y and ~ e c o n d a r y a l k a l o i d s have two and one hydrogen

a tomfs ) r e p l a c e d r e s p e c t i v e l y .

Consequent ly , i t i s o n l y t h e chemica l p r o p e r t i e s o f a b a s i c

n i t r o g e n t h a t u n i f y t h e many c l a s s e s of' a l l r a l o i d s ( ~ r e a s e

and Evans, 1983). Desp i t e t h e d i f f i c u l t y o f o b t a i n i n g a

p r e c i s e d e f i n i t i o n , a l k a l o i d s a r e g e n e r a l l y d e s c r i b e d as

a l a r g e g roup o f compounds found o c c u r r i n g i n p l a n t s ,

b i o s y n t h e t i c a l l y d e r i v e d from amino a c i d s and many a r e

pharmaco log iua l ly ac Live.

f lomenclnture and ~ l a s s i f i c a t i o n o f A lka lo id s :

Ey agreement , chemica l r u l e s d e s i g n a t e t h a t t h e names

o f a l l a l k a l o i d s shou ld end i n - i n e . A p r e f i x o r s u f f i x

i s added t o t h e name o f a p r i n c i p a l a l k a l o i d t o d e s i g n a t e

a n o t h e r a l k a l o i d from t h e same sou rce e.g. q u i n i n e ;

q u i n i n d i n e , hydroquinhne ('prier & &., 1976).

A l k a l o i d s a r e named from v a r i o u s s o u r c e s which include: .

Gener ic name o f p l a n t y i e l d i n g them e.g.

Hydra s t i ne from from l t y d r a s t i s c anadens i s ,

A t rop ine from B t ropa be l l adonna .

S p e c i f i c name o f p l a n t y i e l d i n g them e.g. 6

Cocaine from mythroxy lum Coca; Be l l adon ine

from At ropa bel ladonna.

Common name o f d rug p l a n t y i e l d i n g them e.g.

E r g o t m i n e from =got produced by c l a v i c e p s

purpurea .

P h y s i o l o g i c a l a c t i v i t y e.g. m e t i n e from

Cephae l i s ipecacuanha so named because i t c a u s e s

emes i s ; Morphine ( a n a r c o t i c ) from Papaver somniferum

i s named a f t e r , Morpheus, t h e ' Greek god o f dreams.

Name o f d i s c o v e r e r e. $. P e l l e t i e r i n e from Punica

granaturn was d i s c o v e r e d by P e l L e t i e r .

Apar t from the c l a s s i f i c a t i o n based on t h e

s u b s t i t u t i o n 1 o f t h e hydrogen s t oms i n t h e n i t r o g e n ,

atom o f t h e a l k a l o i d , a l k a l o i d s can f u r t h e r be

c l a s s i f i e d i n t o v a r i o u s s t r u c t u r a l g roups namely

Benzy l i soqu ino l ines , Aporphines, Morpk-liaandionnes,

pav ines , B i sbenzy l i soqu ino l ines and t h e p ro tobe rbe r ines .

The Benzy l i soau ino l ine A lka lo id s :

The benzy l i soqu ino l ine a l k a l o i d s p o s s e s s i s o q u i n o l i n e

s k e l e t o n based on 1 - b e n z y l i s o q ~ i n o l i n e

They a r e o f two groups based on t h e qua t e rna ry r i n g system

and t e r t i a r y base. Examples o f p l a n t s p e c i e s i n N ige r i a

c o n t a i n i n g a l k a l o i d s o f t h i s group i n c l u d e ~ r i c l i - s i a

f u n i f e r a which c o n t a i n s ob longine ( q u a t e r n a r y ) and Cocculus

pendulus which has as one of i t s c o n s t i t u e n t s c o c l a u r i n e

( t e r t i a r y ) .

The Aporphine Alka lo ids :

The aporphine- type a l k a l o i d s g e n e r a l l y c o n s t i t u t e t h e

l a r g e s t group of t he i s o q u i n o l i n e a l k a l o i d s . They a r e

abundant i n most p l a n t f a m i l i e s and s e v e r a l rev iews on t h e

aporph ines have appeared i n l i t e r a t u r e , t h e most complete

o f which i s by Manske (1954). Since t h e n , a r e l a t i v e l y

l a r g e number o f new aporphine a l k a l o i d s have been i s o l a t e d

and many o t h e r s syn thes i zed . A l l t h e aporphine a l k a l o i d s

a r e based on t h e 4H-dibenzoquinol ine s t r u c t u r e o r i t s

M-methylder iva t ive commonly known a s a p o r p h i n e s n u c l e u s .

The numbering system i s as i n d i c a t e d below s o as t o confo

w i t h t h e r i n g i n d e x ( P a t t e r s o n & &., 1960).

An example o f an a p o r p h i n e i s s t e p h a r i n e from s t e p h a n i a

Aporpkine s k e l e t o n

The Morphinandienone A l k a l o i d s ;

The morphinandienones a r e rare i n t h e Family

Menisperrnaceae. I n a d d i t i o n t o t h e i r a s s i g n e d morphine

c k e l e t o n t h e s e g roup o f a l k a l o i d s p o s s e s s one o r more

d i o n e s u b s t i t u t i o n s i n t h e s t r u c t u r e . P a l l i d i n e , a

member o f t h e g roup h a s r e c e n t l y been i s o l a t e d from

t h e s t ems o f Chasrnanthera dependens ( O h i r i & &., 1983)

P a l l i d i n e The Pavine Alka lo ids :

AS f a r as t h e Niger ian Menispermaceae a r e concerned,

t h e pavine group of alkaloidsafi!%:new on t h e scene. They a r i s e

from 3-hydroxybenzyl isoquinol ines . A p ro to type of t h i k

group of a l k a l o i d s c a l l e d Uisnorargemonine was r e c e n t l y

i s o l a t e d from t h e s tems o f ~ h a s m a n t h e r a dependens (Oh i r i

e t a1 1983). ,, -* ?

This was t h e f i r s t r e p o r t o f t h e i s o l a t i o n o f any member of

t h i s g r o u p of a l k a l o i d s from t h e Family. 1

The B i s b e n z ~ l i s o q u i n o l i n e ~ l k a l o i d s :

Thi s i s ano the r l a r g e group of i s o q u i n o l i n e s . However,

they a r e no t widespread among t h e members o f t h e ~ a m i l y

Menispermac eae .

20 . The B i sbenzy l i soqu ino l inos a r e b u i l t up from two benzyl iso-

q u i n o l i n e u n i t s l i n k e d by e t h e r b r idges ; and i n a d d i t i o n

t o t h i s e t h e r l i n k a g e , methylenoxy b r i d g i n g o r d i r e c t carbon - carbon bonding i s a l s o found between t h e benzy l i soqu ino l ine

u n i t s (Guha e t al., 1979). F u n i f e r i n e from t h e p l a n t

~ r i c l i s i a f u n i f e r a i s a t y p i c a l example o f a

a l k a l o i d . I 5 ' I .

~ h s bisb .enzyl i soquinol ine s k e l e t o n

The P ro tobe rbe r ine Alka lo ids :

This group o f i s o q u i n o l i n e s a r e widely d i s t r i b u t e d among

t h e Nigeria.n s p e c i e s o f t h e jlamily Menispermaceae. The

p r o t o b e r b e r i n e s have a s y ~ t em o f nomenclature t h a t has no t

been s t a n d a r d i s e d and i s o f t e n , confusing, p a r t l y because t h e r e

a r e i n f a c t ' two s e r i e d of a l k a l o i d s , one based upon t h e

qua,ternary r i n g system and t h e o t h e r upon t h e t e r t i a r y base.

A l l known p r o t o b e r b e r i n e s - con ta in at l e a s t f o u r oxygen

f u n c t i o n s (OH, OCH3, OCH20) which must be a t t a c h e d t o C2, c3 Cg and CI0 p o s i t i o n s of t h e s t r u c t u r a l ske le ton . ylhese

oxygen f u n c t i o n s determine which s p e c i f i c a l k a l o i d t h a t would

e v e n t u a l l y emerge.

A t y p i c a l example of t h e p r o t o b e r b e r i n e a l k a l o i d i s

b c r b e r i n e which h a s been is01 ~ ' : e d from ~ i n o s p o r a balr is among

numerous o t h e r members oi' Lhe ~ ' a rn i l y Menispermac eae.

a l k a l o i d s pa lma t ine , and j a t r o r r h i z i n e hav.e been i ~ o l a t e d

from Dhenocen t rum .jollyanurn as w e l l as from o t h e r p l a n t s

o f t h e j'arnily wh i l e columbamine which o c c u r s i n q u i t e a

number o f s p e c i e s i s h i g h l y thought t o o c c u r i n

~ p h e n o c e n t r u m .jollyanurn as wel l .

PALMATI NE

COLUMBAMINE

FIG. 1 - Chemical structures of Protoberberine Alkaloids.

~t t h i s p o i n t , i t i s p e r t i n e n t t o conclude from t h e

preceeding review of t he a l k a l o i d s , t h a t b i sbenzy l i soqu ino l ine

a l k a l o i d s a r e t h e most. abundant ly occurr i i lg a l k a l o i d s of' t h e

Niger ian Menispermaceae p l a n t s p e c i e s , fol lowed by t h e

aporphines and the protoDel-berines; t h e benzy l i soqu ino l ines ,

pav ines and ~norphinadicnones a r e on t h e oLher hand r a l i ~ e r

Occurrence and D i s t r i b u t i o n of' Alka lo ids :

Most a l k a l o i d s occur i n n a t u r e as w e l l de f ined

c y s t a l l i n e subs t ances which u n i t e with a c i d s t o form saAts.

I n p l a n t s , they do no t only e x i s t a s sa l t bu t a l s o as N-

ox ides o r i n f r e e s t a t e (Trease and Evans, 1983). They a r e

most o f t e n colour ' less , however, a few coloured ones a r e

known.

The e l a b o r a t i o n of a l k a l o i d s i s n o t l o c a l i s e d i n

c e r t a i n s p e c i f i c o rgans bu t appea r s t o be a c h a r a c t e r i s t i c

o f a l l o rgans ( i n c l u d i n g t h e s eeds ) . ~ c c o r d i n g t o Manske

and Holnles (1 g>O), i n t h e f i r s t y e a r o f growth, a l k a l o i d s

seem t o be q u i t e evenly d i s t r i b u t e d amongst the v a r i o u s

organs bu t ~ i t h ~ i n c r e a s e d age, t h e r e appea r s t o be a

l o c a l i s a t i o n of' t h e s e bases i n a few organs.

From a v a i l a b l e d a t a , a l k a l o i d s appear t o have a

r e s t r i c t e d d i s t r i b u Lion i n t he p l a n t kingdom, Ty le r & a,, (1976) have found t h a t among t h e angiosperms, t h e

Leguminosae, Papaverac eae, Ranumclaceae, ll'ubiac eae ,

Menispermaceae, Solanaceae and Berbar idaceae a r e

o u t s t a n d i n g f o r p l a n t s y i e l d i n g a l k a l o i d s .

S o l u b i l i t y of' A l k a l o i d s :

A knowledge o f t h e s o l u b i l i t y of a l k a l o i d s and - t h e i r

s a l t s i s o f c o n ~ i d e r a b l e impor tance pha rmaceu t i c a l l y . Not

on ly a r e a l k a l o i d s s u b s t a n c e s o f t en admir i i s t e red i n s o l u t i o n

bu t a l s o t h e d i f f e r e n c e s i n s o l u b i l i t y between a l k a l o i d s and

t h e i r s a l t s p rov ide methods f o r t h e i s o l a t i o n of a l k a l o i d s

from t h e p l a n t and t h e i r s e p a r a t i o n from accompanying non-

a l k a l o i d a l subst;ances. While t h e s o l u b i l i t i e s o f d i f f e r e n t

a l k a l o i d s and sa l t s show c o n s i d e r a b l e v a r i a t i o n s ( a s might 6

be e x p e c t e d ) , i t i s t r u e t o cay t h a t t h e f r e e bases a r e

f r e q u e n t l y s p a r i n g l y s o l u b l e i n wa t e r b u t q u i t e s o l u b l e

i n o r g a n i c s o l v e n t s . There a r e o f c o u r s e e x c e p t i o n s t o t h e s e

g e n e r a l i z a t i o n s , some ba se s b e i n g r e a d i l y e x t r a c t e d w i th

wa te r ( c a f f e i n e ) whi le o t h e r s such as ~ o l c h i c i n ~ e a r e s o l u b l e

i n a c i d s , n e u t r a l o r a l k a l i n e wa t e r (Trease and Evans, 1983).

Role :

S e v e r a l a rguments have been made on t h e f u n c t i o n o f

a l k a l o i d s i n p l a n t s t h a t c o n t a i n them.

(i) Pt h a s been proposed t h a t a l k a l o i d s may be

n i t r o g e n o u s waste p r o d u c t s ( l i k e u r e a i n man).

However, some peop le have a rgued t h a t t h e l a r g e

s t r u c t u r a l f o r ~ n u l a e e l a b o r a t e d by most a l k a l o i d s

make t h i s a very l a b o u r i o u s way o f was te d i s p o s a l .

O t h e r s a r g u e t h a t t h e l a r g e s t r u c t u r e s cou ld be

l i k e n e d t o an e l i m i n a t i o n p r o c e s s l i k e c o u p l i n g

o f d r u g s w i t h o t h e r m o l e c u l e s t o e x c r e t e them.

A l k a l o i d s a r e t h u s s e e n i n t h i s l i g h t as end

p r o d u c t s o f d e t o x i f i c a t i o n r e a c t i o n s r e p r e s e n t i n g

a m e t a b o l i c l o c k i n g up o f compounds t h a t a r e

o t h e r w i s e ha rmfu l t o p l a n t s .

The second argument i s t h a t a l k a l o i d s may be

n i t r o g e n o u s r e s e r v e s c a p a b l e o f s u p p l y i n g n i t r o g e n

and o t h e r n e c e s s a r y e l e m e n t s t o t h e p l a n t . There

i s however, no ev idence t o p rove t h a t a l k a l o i d s a r e

u t i l i z e d by p l a n t s e x c e p t as a n i n t e r m e d i a t e i n

b u i l d i n g u p o t h e r e l a b o r a t e m o l e c u l e s and a r e more b

p r o b a b l e t o be end p r o d u c t s o f m e t a b o l i c r e a c t i o n s .

A l k a l o i d s are a l s o though t t o be a d e f e n s i v e mechanism

o f p r o t e c t i n g t h e p l a n t s a g a i n s t i n s e c t s and

h e r b i v o r e s . T h i s l o g i c i s however d i f f i c u l t t o

s u s t a i n s i n c e a l k a l o i d s form good b e v e r a g e s and most

a r e h a r m l e s s t o w i l d a n i m a l s d e s p i t e t h e i r b i t t e r

t a s t e and p h a r m a c o l o g i c a l a c t i v i t y .

They have a l s o been s e e n as r e g u l a t o r y growth f a c t o r s

o f p l a n t s as w e l l as s t i m u l a n t s f o r such p h y s i o l o g i c

a c t i v i t y as metabolism and r e p r o d u c t i o n .

The b a s i c i t y o f a l k a l o i d s may have a r o l e i n t h e

maintenance o f i o n i c b a l a n c e o f t h e p l a n t system.

O f a l l t h e s e arguments , none i s a l l embracing o f what

a l k a l o i d s do i n p l a n t s b u t t h e most a c c e p t e d t h e o r y i s t h a t

a l k a l o i d s a r e used as i n t e r m i a d i a t e s i n p l a n t metabol ism and

when. used i n man, t hey e x e r t e x t e n s i v e p h y s i o l o g i c a c t i v i t y

which i n c l u d e s a m n e s t o t h e r s , an t im ic r0 .b i a l a c t i v i t y ,

i n c r e a s e i n b lood p r e s s u r e , d e c r e a s e . i n blcP,dpressure,

myotic ef f cc t , r nyd r i a t i c e f f e c t , a n a l g e ~ i c and n a r c o t i c

a c t i o n , s t i m u l a t i o n o f c e n t r a l ne rvous sys tem, e t c .

One o f such a l k a l o i d - producing p l a n t s t h a t ha s been

and i s st i l l be ing u sed i n some c o u n t r i e s f o r c u r a t i v e

pu rpose s i s Sphenocentrum . i o l l ~ a n u m P i e r r e . It be longs

t o t h e t r i b e Pen i an theae and Family Menisperrnaceae.

Pharmaco1oe;ical A c t i v i t i e s o f t h e A l k a l o i d s :

I n t h e Menispermaceae i n g e n e r a $ , t r i b e s w i t h gene ra3 6

conka in ing p r o t o b e r b e r i n e s a r e commonly used as a n t i b i o t i c s

and a n t i p e r i o d i c s among o t h e r pu rpose s h ha mar and S a l g a r ,

1974).

Palmat ine ha s been found t o p o s s e s s a n a l g e s i c ,

a n t i a r r h y t h m i c , p o s i t i v e i n o t r o p i c , a d r e n o c o r t i c o t r o p i c ,

a n i i c h o l i n e s t e r a s e and b a c t e r i c i d a l p r o p e r t i e s .

( i ) b n t i c h o l i n e s t e r a s e J;:f f e c t r

Drugs t h a t i n h i b i t o r i n a c t i v a t e a c e t y l c h o l i n e s t e r a s e

are c a l l e d a n t i a c e t y l c h o l i n e s t e r a se s , Chin and Kunitomo

(1965) r e v e a l e d t h a t pa lma t ine h a s an a n t i c h o l i n e s t e r a s e

e f f e c t i n expe r imen t a l an imals . It c a u s e s a c e t y l c h o l i n e

( ~ c h ) t o accumulate a t t h e c h o l i n o c e p t i v e s i t e s and t h u s i s

p o t e n t i a l l y c a p a b l e o f p roduc ing e f f e c t s e q u i v a l e n t t o

c o n t i n o u s s t i m u l a t i o n o f c h o l i n e r g i c f i b r e s th roughout t h e

c e n t r a l and p e r i p h e r a l ne rvous system.

( i i ) Adrenocorticotrophic Effec t :

Comparative s tudy on t h e adrenocort icotrophic hormone

(ACTH) r e l ea s ing a c t i v i t i e s of palmatine and te t rahy-

chopalmatine w a s repor ted by Savona - e t -* a 1 9 (1966). I n r a t s , subcutaneous (SC) i n j e c t i o n of 25-50rng/kg palmatine

deple ted ascorb ic acid. Pretreatment with sodium pen tobarb i t a l

(4.0rng/kg) given in t rapen tonea l ly caused complete

i n h i b i t i o n of adrenal ascorbic acid. Another research

work c a r r i e d ou t by Chin and Kunitomo (1965) showed t h a t

palmatine i n h i b i t e d t he e f f e c t of a d r e n a l h e when t e s t e d

on t he perfus ion prepara t ions of Laewen - Trendelenburg's

toad leg . It a l s o has an e f f e c t i v e an t agon i s t i c a c t i on

on 5'-hychoxytryptamine (5-HT).

( i i i ) Anaphylactic Effec t : 6

Palmatine has been found t o cause anaphylaxix when

i n j e c t e d subcutaneously i n t o ra ts , . It was repor ted t h a t

subcutaneous i n j e c t i o n of palmatine caused oodema of t h e

paws i n rats. Pretreatment of r a t s with diphenhydramine

(75mg) o r Smg of chlorpromazine s i g n i f i c a n t l y i nh ib i t ed

t h e ocdema of t h e kind induced by so i n j e c t i o n of

palmatine.

( i v ) Other Antimicrobial Act iv i ty :

Palmatine i s genera l ly known t o possess a n t i -

microbial a c t i v i t y - in -v i t ro . Hangiwa and Harada (1962)

showed t h a t palmatine has b a c t e r i o s t a t i c a c t i on aga in s t

S-taphylococcus aureus, The ant i -fungal a c t i v i t y

exhibi ted by palmatine w a s a l s o repor ted by Horn and

Jatrorrhizine possesses antifertility, antifungal,

anti-bacterial and arbortefecient properties. It is also a

central nervous system depressant.

(i) Antifertility Effect:

The antifertility effect of Jatrorrhizine was reported

by Farnsworth - et -* a1 (1975). The compound has abortefacient and

uterine stimulant actions. It was also shown to elicit a

stimulant response when tested against uterine muscles either

in -vitro or in-vivo and to produce contraction in isolated - - -- guinea-pig ileum. b

(ii) Antimicrobial Effect:

Hangiwa and Harada (1962) reported the antibacterial

effect of jatrorrhizine and this was strengthened by the

report of its antifungal activity by Horn and Steffen (1968).

Other Constituents of Sphenocentrurn jollyanum:

Columbin, a bitter principle which had been found in

such plants like Jateorrhiza palmata has been isolated from

the seeds of Sphenocentrum jollyanum. It is known to cause

inflammation of the skin (Gilber - et -* a1 9 1967)

The petroleum ether extract of Sphenocentrum jollyanurn usually

yields a mixture of campesterol, Beta-sitosterol and

stigmosterol each of which can be used as a precusor in the

microbiological synthesis cf steroids.

Extended flavonoids and biflavanones have been extracted from

Sphenocentrurn jollyanum and were shown to possess aphrodisiac

effect. They exhibited a stirnulatory effect on the .

sexual organs of male volunteers and t h i s e f f e c t did not

appear t o be dependent on t h e emotional s t a t e of the pa t i en t

(IWU, 1986). P lan t s With Antimicrobial Activity:

The a r t and science of herbal medicine have been

prac t i sed within t he length and breath of Africa i n

p a r t i c u l a r and the world i n general and i s s t i l l being widely

pract ised even today, I n s p i t e of t h e severe setback suffered

by the p l an t medicine with the advent of t h e a n t i b i o t i c s ,

a number of p l an t s have survived t h e neglect and subsequent

abandonment t h a t saw most medicinal p lan t s out of the o f f i c i a l

records. The anti-microbial p lan t s t h a t survived t h i s

setback a r e qu i t e few but a r e s t i l l ac t ive ly used i n most

nat ions , even i n t he so-called developed nat ions , For ins tance

Pinus p a l u s t r i s (Long Leaf pine) which belongs t o t he family

Pinaceae and abundant i n the United Stakes of America i s

s t i l l being used i n some p a r t s of t h e world a s an an t i s ep t i c .

Turpentine, an e x t r a c t of t he ? l an t was used i n cure of t h e

treatment of gonorrhoea and ur inary disease (wren, 1970),

Juniperus oxycedrus (p r i ck ly juniper) of t h e family

Cupressaceae i s found mainly i n t he Mediterranean region.

Today, it i s applied t o old wounds and u lcers , I t ' s ex t r ac t ,

cade o i l i s uded i n t he treatment of p a r a s i t i c sk in diseases

l i k e eczema, p so r i a s i s and p r u r i t i c dermatoses. It i s a l s o

an i n ~ r e d i e n t i n a n t i s e p t i c soaps ( ~ r i e v e , 1967).

Ephreda major and E. S in ica both of t he family

Ephredoraceae and found around Spain, S i c i l y and Pakistan

i s s t i l l widely used t o Gure colds, coughs and eruptions

due t o in fec t ions . Liquidambar s t y r a c i f l u a (sweet gum)

grows widly i n t he United S t a t e s of America. I t belongs

t o t he family Hamamelidaceac. The leaves , r i c h i n tannin

a r e chewed t o curb diarrhoea and t o r e l i e v e sore-throat .

I n La t in America, the g u m i s popular a s a remedy f o r

gonorrhoea orto ton, 1974) . Acacia senegal (gum acacia) occurs wild on dunes i n ,

Senegal and Mauritania and throughout t r o p i c a l Africa.

This gum once a valued a r t i c l e of commerce among e a r l y

Egyptians is s t i l l u t i l i z e d l o c a l l y a s a remedy f o r

gonorrhoea, dysentry, sore n ipp les and nodular leprosy

(Wren, 1970).

Styrax benzoin ( ~ e n z o i n t r e e s ) belongs t o t h e family

Styracaceae and has a long h i s t o r y of medical use.

However, today when d i l u t e d i n water, t h e alcohol

so lu t ion of benzoin becomes milky and under t he name

" v i r g i n ' s milk" has been used t o hea l cracked nipples and

has a l s o been employed i n feminine hygiene. The a lcohol ic

ex t r ac t of benzoin i s incorporated i n t o benzoin t i n c t u r e

( f r i a r ' s balsam o r Tur l ing ton ls balsam). This t i n c t u r e

i s commonly employed a s an a n t i s e p t i c sol - e t -. a1 1960).

Other p l an t s t h a t a r e s t i l l widely employed i n t h e treatment

of i n f e c t i o n s i n c l u d e s t r o ~ h a n t u s m a t u s (smooth S t r o p h a n t u s )

o f t h e f ami ly Apocynaceae used i n N i g e r i a a g a i n s t ringworm,

Ca r i ca PaPaya (paw-paw) of t;he fami ly Car icaceae a l s o used

i n t h e t r e a t m e n t o f ringworm and eczema,

( ~ h y m e ) o f t h e f ami ly L a b i a t e employed as an a n t i s e p t i c and

fumiga t ing h e r b (Osol & &., 1960) e t c .

More r e c e n t l y , modern workers have devoted a l o t o f t ime

t o t h e a n t i m i c r o b i a l p r o p e r t i e s o f h i g h e r p l a n t ; t h i s i n t e r -

d i s c i p l i n a r y r e s e a r c h can be b road ly c a t e g o r i z e d i n t o two.

T h e f i r s t c a t e g o r y d e a l s w i t h t h e e x t r a c t i o n , i s o l a t i o n and

t h e c h a r a c t e r i z a t i o n o f a n t i - m i c r o b i a l a c t i v e c o n s t i t u e n t s

from p l a n , t s r e a d i l y r ecogn i sed i n h e r b a l medic ine as

p o s s e s s i n g a n t i - m i c r o b i a l a c t i v i t y . The second i n v o l v e s

random s c r e e n i n g o f both, corninon and r a r e p l a n t s f o r a p o s s i b l e

i d e n t i f i c a t i o n o f t h e ones p o s s e s s i n g a n t i - m i c r o b i a l a c t i v i t y .

such s c r e e n i n g r a r e l y goes i n t o t h e i n t r i c a c i e s o f i s o l a t i n g

and charac t e r i z i n g t h e s p e c i f i c a n t i m i c r o b i a l p r i n c i p l e s

i n i t i a l l y . Subsequent work however may g e t down t o such

s p e c i f i c s . Both approaches a r e impor t an t and i n f a c t a r e

complimentary.

m n e h c h i and Sorrnaghi (1980) sc reened 140 e x t r a c t s from

138 s p e c i e s of I r a n i a n p l a n t s aqd found l e s s t h a n 50% of them

a c t i v e a g a i n s t v a r i o u s micro-organisms.

S i m i l a r programmes (works) a r e be ing c a r r i e d o u t a l l

ove r t h e world eventhough obv ious ly w i t h l e s s number o f

p l a n t s p e c i e s . Obscure Nepalese p l a n t s were s t u d i e d f o r

f o r t h e i r o v e r a l l rneulc lua l v a l u e and t h e i r a n t i - m i ~ x a $ a

m i c r o b i a l ~ r o p e r L i e s i n p a r t i c u l a r (Manandhar, 1980)-

Holdsworth & &., (1 980) su rveyed and s c r e e n e d t r a d i t i o n a l

m e d i c i n a l p l a n t s o f Papua New Guinea and found some o f

them u s e f u l . I n U t t a r Bradesh d i s t r i c t o f I n d i a , Chandra

and Pandey (1983) c o l l e c t e d p l a n t s c l a imed t o have m e d i c i n a l

v a l u e s s e q u e l Lo s c r e e n i n g them. Compreher~sivc r e v i e w s o n

m e d i c i n a l p l a n t s i n g e n e r a l and a n t i - m i c r o b i a l p l a n t s i n

p a r t i c u l a r have been p u b l i s h e d ( Inyang, 1986; T y l e r , 1987)-

A number o f workers have gone beyond s c r e e n i n g and

p u b l i ~ h i n g r e v i e w s based on e x i s t i n g f a c t s and l i t e t a t u r e s ,

t h e y have c a r r i e d o u t e x t r a c t i o n s o n some p l a n t s p r o b a b l y

i n a b i d t o i s o l a t e and i d e n t i f y t h e a c t i v e p r i n c i p l e ( s )

r e s p o n s i b l e f o r a n t i - m i c r o b i a l a c t i v i t y . However some o f

t h e s e r e s e a r c h e r s s e l d o m > g e t t o t h e s p e c i f i c a c t i v e

c o n s t i t u e n t .

Ben jamin and Lamikara (1980) have e x t r a c t e d a n anti-

m i c r o b i a l i n g r e d i e n t from t h e p lank Mornordica char anti^ a

p l a n t u s e d i n N i g e r i a f o r Lhe t r e a t m e n t o f s k i n d i s e a s e s .

S t u d i e s c a r r i e d o u t on Sudanese m e d i c i n a l p l a n t Lupinus

t e r m i s hav,e shown t h a t a n a l c o h o l i c e x t r a c t o f t h e p l a n t i s

v e r y e f f e c t i a e a g a i n s t eczema. An i n c o r p o r a t i o n o f t h e

s t h e n o l i c e x t r a c t s o f t h e s e e d s i n t o an o in tment shows a n

e f f i c a c y a g a i n s t eczema comparable w i t h c o r t i c o i d

( ~ n t o u n & &. , 1987; ~ n t o u n and Taha, 1931 ); a hydro-

a l c o h o l i c e x t r a c t o f t h e p l a n t camomile showed broad

spectrum a c t i v i t y a g a i n s t b a c t e r i a , i s o l a t e d from t h e

vag ina and mouth. It i n h i b i t s . compleLely t h e growth of

~ h y l o c o c c u s au reus , $ t r cp tococcus mutans and

~ t r p t o c o c c u s s a l i v a r u s whi le e x e r t i n g a b a c t e r i c i d a l

e f f e c t on B a c i l l u s megathcrium, Lep tosp i r a

i c te rohaemorrhag ica and grichomonas v a g i n a l i s ( ~ i n c o , 1983)

E a r l i e r i n 1979, Boakye - Yiadom showed t h a t aqueous

e x t r a c t s o f t h e r o o t s o f p y p t o l e p i s Sanau ino len t s o f t h e

fami ly Asclepiadaceae have been t e s t e d a g a i n s t f o u r

u r i n o g e n i t a l p a t hogens; Me i sae r i a gonorrhoeae, & s c h e r i c k a

c o l i , Pseudomonas ae rug inosa and Candida a l b i c a n s and

found t o p o s s e s s s t r o n g an t i -mic rob ia l a c t i v i t y a g a i n s t

them excep t E. ae rug inosa

I n N ige r i a a n e x t r a c t o f f r u i t s and s e e d s o f C a r i c a papaya

which i s p r o t e i n - l i k e ~ x n - t ~ d a n ~ a n t i - b a c t e r i a l e f f e c t on

s t a p h au reus , B a c i t l u s ce r eus , a. c o l i , &. aerugigo-sa

and hig gel la f l e x n e r i . The e x t r a c t was proved t o be

b a c t e r i c i d a l (meruwa, 1982).

Many workers have done exhaus t i ve phytochemical

i n v e s t i g a t i o n s i n t o p l a n t s and subsequent ly proved t h e

a c t i v e c o n s t i t u e n t s of t h e p l a n t s t o be an t i -microh ia .

xy lop i c a c i d and two o t h e r d i t e r p e n e i s o l a t e s e x t r a c t e d from

t h e f r u i t s o f J y l o p i a a e t h i o p i a of t h e fami ly Annonaceae

have been shown t o have a n t i - m i c r o b i a l p r o p e r t i e s . They

were t e s t e d a g a i n s t s t a p h au reus , E. c o l i , & aerug inosa ,

B a c i l l u s s u b t i l i s and Candida a l b i c a n s and were a l l found

t o be a c t i u e (Boalrye - Yiadn?l el; &. , 1977)

A S e s q u i t e r p e n i c a c i d , c o s t i c . a c i d showed a n

a n t i - b a c t e r i a l e f fec - t on Be s u b t i l i s grown on a chemica l ly

d e f i n e d medium. his a c i d w a s i s o l a t e d by Rao and Alvarez

(1 981 ) from t h e a e r i a l shoo t o f mpa to r i u rn c a ~ i l l i f ' o l i u m .

hus sa in , el; al. , ( 1 979) i s o l a t e d e thy lga l1a l ; e and g a l l i c a c i d

from t h e a e r i a l p a r t s o f Minosa hamata (Mimosaceae) and

showed t h e s e t o have a n t i - m i c r o b i a l a c t i v i t y a g a i n s t

M~cobac t e r i um s m e m a t i ~ , s t a p h a r e u s , &. c o l i and s.

Rao & dm, (1982) proved t h a t t h e a n t i m i c r o b i a l a c t i v i t y 4

a s c r i b e d t o QaemonoroDs d raco (Palmaceae) i s due t o t h e

p r e sence o f Lhe two p r i n c i p l e s t hey i s o l a t e d , dracohodin

and d r aco rub in . These showed a c t i v i t y a g a i n s t M,

s m e ~ m a t i s , $aph.giureus, a. c o l i , s a l m o n e l l a g a l l i n a r u m .

K l e b s i e l l a ~ n e u m o n i a e and I;. a l b i c a n s . Aladesanmi d.,

(1986) c a r r i e d o u t phytochemical s t u d i e s on two p l a n t s

normal ly used i n f o l k l o r e medic ine i n N i g e r i a ; Marsdenia

l a t i f o l i a and P l e i a s c e r a s b a r t e r i and went f u r t h e r t o

show t h a t t h e a n t i b a c t e r i a l a c t i v i t y u s u a l l y a s c r i b e d t o

t h e p l a n t s i s due t o t h e p r e sence i n them o f a n t i b a c t e r i a l

a l k a l o i d s , f l a v o n o i d s , s apon in s , t a n i n s and s t e r o l s .

F lavonoids have a l s o been r e p o r t e d t o p o s s e s s

a n t i m i c r o b i a l a c t i v i t y . lCiski & &. , (1983) e s t a b l i s h e d

a n t i - b a c t e r i a l ac1; iv i ty f o r t h e f l av ,ono id c i r c i m a r i t i n

i s o l a t e d from t h e l e a v e s o f S a l v i a p a l e s t i n a (Lah i a t aceae ) .

35. C i r s i m a r i t i n showed a high a c t i v i t y a g a i n s t s t a p h au reus ,

s t a p h epidernidis , &. c o l i , Kleb. pneutnonia, P ro t eus vu1r;aris

and &. aeruginosa , A similar a c t i v i t y was r e p o r t e d by

M i t s che r e t a le , (1 980) f o r t h e i s o f l a v o n o i d s h i s p a g l a b r i d i n

A, h i s p a g l a b r i d i n 13, g l a b r i d i n and g l a b r o l i s o l a t e d from

Glycyr rh iza c l a b r n var t y ~ i c a . Apar't from i ' lavonoids,

ano ther c l a s s o f c o n s b i L u c n t ~ reporbed Lo e x h i b i t a n t i -

mic rob ia l a c t i v i t y a r e t h e e s s e n t i a l o i l s .

Janssen & dm, ( 1 987) gave an extensiv4e l i b e r a t u r e review

o f e s s e n t i a l o i l s which posses s a n t i m i c r o b i a l a c t i v i t y and

i n a r e s e a r c h c a r r i e d ouL by Onaviunmi and Ogunlana (198$)

i n N ige r i a , they r e p o r t e d t h e a n t i m i c r o b i a l a c t i v i t y o f

t h e e s s e n t i a l o i l s o f cymbonogo c i t r a t u s (Lemon g r a s s ) .

Dispersed i n dimethyl su lphoxide (DMSO) t h e o i l s showed

a c t i v i t y a g a i n s t both G r a m p o s i t i v e and GraILncgative

organisms, however they showing g r e a t e r a c t i v i t y a g a i n s t

t h e former.

From t h e number of a v a i l a b l e l i t e r a t u r e , i t can be

c o r r e c t l y i n f e r r e d t h a t a l k a l o i d s form t h e l a r g e s t c l a s s o f

a c t i v e i n g r e d i e n t s i s o l a t e d from p l a n t s t h a t have

a n t i m i c r o b i a l a c t i v i t i e s A lka lo ids have been, L -

i s o l a t e d from p l a n t s l i k e r h a l i c t r u m ruffosum (WU- an e t a l , ,

1976) and ~ r i c l i s i a d i c t y o p h y l l a ( s p i f f & a&, 1981)

and shown to posses s very s t r o n g a n t i m i c r o b i a l a c t i v i t y ,

A r e l a t i v e l y s imple ~ ~ t ~ - c ~ r b o l l n . e a l k a l o i d harmine from

Peganurn harmala ( ~ y g o p h y l l a c e ~ e ) have been s h e m t o posses s

very s t r o n g b r o a d - s p e c t r w ac t i v i t y a g a i n s t a number

of micro-organisms (~1-shamma & &i., 198 1 ) . some a l k a l o i d s can be regarded as t t a n t i b i o t i c s l l i n t h a t

they have been s u b j e c t e d t o &I vivo t e s t s , o r even employed

i n t h e t r ea tmen t o f r e a l d i s eases . 13erberine i s a yel low

a l k a l o i d which occu r s i n numerous p l a n t s . It was f i r s t

i s o l a t e d more than a cen tury ago from t h e p l a n t

Xanthoxylon cava. Its g r e a t e s t use h a s been a s a n amoebicide

i n t h e t rea tment o f amoebic dysen t ry caused by ~:nt;amoeba

h i s t o l y t i c a ( ~ u b b i a h and nmin, 1967).

Berber ine h a s a l s o been used i n exper imenta l c h o l e r a

i n f e c t i o n s ( ~ u t t a & a., 1972)and i n two human c h o l e r a 6

epidemics a l l due t o y i b r i o c h o l e r a e ( L a h i r i and Uut ta , 1967).

The a n t i - b a c t e r i a l e f f e c t of b e r b c r i n e on B a c i l l u s a n t h r a c i s

aud 1. c h o l e r a e have been r e p o r t e d more r e c e n t l y (Amin

e t a1 1969, Modak g.L al., 1970a). - ' They a l s o a t t empted t o e x p l a i n i t s mode o f a c t i o n .

Another a l k a l o i d t h a t h a s been employed i n a c t u a l

t r ea tmex t and ha6 been i n v e s t i g a t e d a lmost as much as

be rbe r ine i s emetine. m e t i n e was f i r s t i s o l a t e d i n t h e

e a r l y p a r t o f t h e Igt;h, cen tury . It i s t h e a c t i v e

p r i n c i p l e o f t h e popula r medic ina l p l a n t of B o l i v i a and B r a z i l

Cephael is ipecacuanha from t h e family Hubiaceae. It i s

used as a n amoebiocide i n t h e t r ea tmen t of amoebic

dysen t ry by E. h i s t o l y t i c a but no t f o r t h e t r ea tmen t of

b a c i l l a r y dysen t ry ( i s h e r 1975). I n I n d i a , erne t i n e h a s

been s u c c e s s f u l l y employed a g a i n s t h r i l ha rz i a s i s , c h o l e r a

and v i r a l h e p a t i t i s (wren, 1970).

TWO l e s s e r known) a l k a l o i d s t h a t hawe come i n t o u se t o some

degree o f e x t e n t a r e i rehadiamine (Iliamine pregn-5-ene - 3 Beta, 20 ac - diamine) f i r s t i s o l a t e d from t h e West

A f r i c i a n rubber p l a n t j'untumina e l a s t i c a and Malouetine

( 3 - / 3 , 20 CK - b i s - ( t r i m e t h y l ammonium) - 5 CDC - pregnane a l s o i s o l a t e d from a West Afr ican p l a n t Malouetina

beauae r t i ana , hey have been used e f f e c t i v e l y i n

i n f e c t i o n s due t o s t a p h au reus , E. c o l i and some fung i

(Mahler and Green, 1970; S i l v e r &., 1970).

It would appear t h a t t h e bu lk of work on. a n t i m i c r b b i a l

p r i n c i p l e s o f p l a n t o r i g i n i s dons us ing b a c t e r i a

as t h e t e s t organisms, here a r e p l a n t s whose a c t i v e

c o n s t i - t u e n t s a r e known t o e x e r t s p e c i f i c a n t i f unga l o r

a n t i v i r a l a c t i v i t i e s . Some o f t h e s e p r i n c i p l e s a r e termed

an t i -mic rob ia l i n a very broad sense i n t h a t t h e i r a c t i v i t i e s

a r e a c t u a l l y an t i -protozoan. For i n s t a n c e , t h e e x t r , c t

gram t h e s e e d s of' P impine l la d i v e r s i f o l i a h a s been proved

t o e x e r t an a n t i funga l a c t i v i t y (Pandey & a&, 1983).

Bolbrer ia v e r t i c i l l a t a has been s u c c e s s f u l l y i n v e s t i g a t e d

as an an t i eczema t i c p r i n c i p l e (Benjamin, 1979). Lamikara

e t &,, (1981) showed t h a t Niger ian h e r b a l s o f t soaps - showed s t r o n g a n t i funga l a c t i v i t y . q u i t e r e c e n t l y Mi l l a rd

e t al., (198'?j! showed t h a t a p reny la t ed f lavanone from t h e -- stem bark of B r y t h r i n a be r t e roana e x e r t e d an a c t i v i t y on t h e

fungus C l a d o s ~ o r i u m cucumerinum.

A n a t u r a l l y o c c u r r i n g po lyene a l k a l o i d , ~ h i a r u b r i n e A h a s

been demons t ra ted t o have a n t i v i r a l a c t i v i t y (Hudson &. , 1986) Lycor inc , a n a l k a l o i d e x t r a c t e d by Ieven (1982)

from C l i v i a r n i n i a t a ( ~ m a r y l l i d a c e a e ) was found t o be

r e s p o n s i b l e f o r t h e pronounced a n t i v i r a l a c t i v i t y a g a i n s t t h e

p o l i o m y e l i t i s v i r u s . The e x t r a c t came from t h e r o o t s and

l e a v e s o f t h e p l a n t . Bedows and H a t f i e l d (1982) a l s o

demons t ra ted t h e a n t i v i r a l a c t i v i t y o f p o d o p h y l l o t o x i n

a n aqueous e x t r a c t o f l ?odo~hyl lum pet taturn.

P o d o a h y l l o t o x i n i n h i b i t e d t h e r e p l i c a t i o n o f m e a s l e s v i r u s

and Herpes s implex v i r u s t y p e I. A phytochemica l b

i n v e s t i g a t i o n o f Mlaia r o x b u r g h i a n a va r . Beddomei

( M e l i a c e a e ) r e s u l t c d I.il I h c i s o l a t i o n o f t h e a c t i v e

p r i n c i p l e and i t s c h r a c t e r i z a t i o n as (+ ) o d o r i n o l . It

e x h i b i t e d s t r o n g a n t i v i r a l a c t i v i t y a g a i n s t ~ a n i k h e t

d i s e a s e v i r u s (HDU) i n c h i c k embryos ( J o s h i &., 1987).

N a t u r a l l y - o c c u r r i n g p h o t o s e n s i t i z e r s have been known even

t o e x e r t a n a n t i - b a c t e r i o p h a g e a c t i v i t y (Hudson & &., 1987).

Some o f t h e a n t i - p r o t o z o a n a c t i v i t y e x h i b i t e d by

n a t u r a l l y o c c u r r i n g a c t i v e p r i n c i p l e s hav,e been documented.

Bhutani & aJ., (1987) r e p o r t e d a comparison i n t h e

potency o l phenant h r o l i n d o l i z i d i n e a l k a l o i d s and emet ine

as ant i -amoebic d rugs . They found two o f them e f f e c t i v e .

Chan & d., (1986) were more s p e c i f i c e x t r a c t s o f t h e

p l a n t Eurycoma l o n ~ i f o l i a were t e s t e d and found a c t i v e

a g a i n s t m u l t i - d r u g r e s i s t a n t T h a i l a n d s t r a i n (K-I) of

Plasmodium f a l c i ~ a r u m 9 v i t r o . Also known t o d i s p l a y i n -

v i t r o a n t i - m a l a r i a l a c t i v i t y i s a n e x t r a c t o f t h e p l a n t

Brucea j avan icq . Again, t h e a c t i v i t y was a g a i n s t a m u l t i -

d rug r e s i s t a n t s t r a i n of Y. f a l c i p a r u m (Pavanand e t a l . ,

1986).

There a r e r e p o r t s o f a c t i v e p r i n c i p l e s which a r e n o t

t h e cornrmnly o c c u r r i n g t y p e s b e i n g r e s p o n s i b l e f o r a n t i - a c t i v e

m i c r o b i a l a c t i v i t y . s i n c e t h e s e L p r i n c i p l e s a r e much

r a r e r t h a n t h e a l k a l o i d s , f l a v o n o i d s , e s s e n t i a l o i l s 4

e t c . , t h e i r i d e n t i f i c a t i o n i s a l o t more complex and

d i f f i c u l t . I n 1986, har rib & &., c a r r y i n g o u t an

i n v e s t i g a t i o n found that; e x t r a c t s o f t h e p l a n t

s o l e n o s t emma a r ~ : e l showed r e a s o n a b l e broad-spectrum

a n t i - b a c t e r i a l a c t i v i t y . The f o u r compourld~ i s o l a t e d from

t h e p l a n t which were r e s p o n s i b l e f o r t h e a c t i v i t y have n o t

been i d e n t i f i e d , I s h i g u r o & a., (1986) i s o l a t e d two

cotnpounds s imply r e f e r r e d t o as a n t i b i o t i c compounds, and

proved t h e i r a n t i m i c r o b i a l a c t i v i t y . he compounds were

S a r o t h r a l e n A and B from Lhe p l a n t , Kypericum japonicum.

From t h i s p l a n t , I s h i g u r o & &. , (1 987) i s o l a t e d t h r e e

o t h e r a n t i b i o t i c compounds, S a r o a s p i d i n A, B and C which

e x e r t e d a pronounced a n t i m i c r o b i a l a c t i v i t y ,

A unique a c t i v i t y e x h i b i t e d by O r i e n t a l f o l k m e d i c i n a l

p l a n t Z i z v ~ h i - frutus i s t h e i n h i b i t i o n % o f i n s o l u b l e g l u c a n

fo rmat ion by Carc inogen ic bac te r ium ~ t r e ~ t o c o c c u s mutans.

'I'his i s i n d e e d a major s t r i d e i n the p r e v e n t i o n o f d e n t a l

c a r i e s (Kohda & &., 1986).

F i n a l l y , pe rhaps one o f t h e most e x h a u s t i v e work i n

t h e f i e l d o f n a t u r a l l y - o c c u r r i n g an t i -m ic rbob i a l

a g e n t s was l h e one r e p o r t e d by Ahmad eC; &. , (1906). They

i s o l a t e d an a l k a l o i d , j u l i f l o r i n e from t h e p l a n t Proso p i s

~ L ~ f l o r ' ~ and t e s t e d i t f o r a n t i m i c r o b i a l a c t i v i t y

a g a i n s t s i x t e e n s p e c i e s o f b a c t e r i a , six o f which were

Gram-negative, t h e remaining t e n were f i ram-posi t ive , *

J u l i f l o r i n e e x h i b i t c d a broad spect rum a c t i v i t y . T h i s

group o f workers went f u r t h e r and f avou rab ly compared t h e

e f f i c a c y o f j u l i f l o r i n e as an a n t i - m i c r o b i a a g e n t s w i t h

t h o s e o f a m p i c i l l i n and t e t r a c y c l i n e , bo th o f them broad

spectrum a n t i b i o t i c s of proven e f f i c a c y ,

Recent ly i t i s becoming an a t t r a c t i v e a r e a o f r e s e a r c h

t o c u l t i v a t e t h e s e med i c ina l p l a n t s and va ry ing t h e

c ropp ing c o n d i t i o n s w i t h a view t o improving t h e q u a n t i t y

and qua l iEy o f t h e i r a c t i v e c o n s t i t u e n t s . The u l t i m a t e

purpose o f t h i s r e s e a r c h i s t o make u s e f u l med i c ina l

p l a n t s r e a d i l y a v a i l a b l e and t h e i r c o l l e c t i o n b o t h

f o r l a b o r a t o r y r e s e a r c h and f o r i n d u s t r i a l - s c a l e p r o c e s s i n c

a l o t e a s i e r (Luanra tana and G r i f f i n , 1980; Luanra tana

and riffi in, 1981 and Kapoor., 1982).

MII'I'~L'HIHLL ANL, lih1l'k1Ul)S

E s c h e r i c h i a c o l i

~ t a p h y l o c o c c u s a u r e u s

Pseudomona a e r u g i n o s n

B a c i l l u s s u b t i l i s

K l e b s i e l l a pneumonLae

P r o t e u s v u 1 ~ l ; a r i s

~ a n d i d a a l b i c a n s

These were c o l l e c t e d from t h e C u l t u r e C o l l e c t i o n b

Unit of Lhe Department o f Ivlicrobiology, U n i v e r s i t y o f

N ige r i a , Nsukka. They were s t o r e d on n u t r i e n t a g a r s l a n t s

( excep t f o r G. a l b i c a n s which was s t o r e d on Sabouraud agar

0 s l a n t ) a b 4 C and s~hs~qu-: : ' i ;y ~ u b c u l t u r e d t o

ma in t a in v i a b i l i t y .

Authen t ic samples o f a l k a l o i d s :

Palmat i n e

J a t r o r r h i z i n e

Columbamine

Te t r ahyd ropa lma t ine

These were o b t a i n e d from t h e Department o f

Pharmacognosy , F a c u l t y o f Pharmaceu t ica l Sc i ences ,

U n i v e r s i t y o f N i g e r i a Nsukka.

S l a n t M a t e r i a l s Used:

The p l a n t $phenocentrurn jollyanurn P i e r r e was

c o l l e c t e d from U n i v e r s i t y oi' N ige r i a , Nsuklra farm and from

f o r e s t s w i t h i n Nsulrka i n October , 19% arid i d e n t i f i e d by

42 . h i r e A.O. Ozioko, Department of Botany, U n i v e r s i t y of

N i g e r i a , Nsukka. A voucher specimen h a s been d e p o s i t e d

at t h e Medic ina l P l a n t s ! Research Labo ra to ry , ~ e p a r t m e n l o f

Pharmacognosy, F a c u l t y of Pharmaceu t ica l s c i e n c e s ,

Un ive r s i t y o f N i g e r i a , Nsukka.

The sterns and r o o t s were s e p a r a t e d and a quar l l i ty o f t h e i r

respective b a r k s s c r a p e d o f f . They were a l l subsequen t l y

but s e p a r a t e l y sun-dr ied and ground i l l t o f i n e powders,

These f i n e powders were code-named as fo l l ows :

l<M, f o r r o o t bark powder

M A , f o r whole r o o t powder

U U , f o r stem bark powder

SSA, f o r whole s tem powder

P a i r n i n a r y y e s t 6 :

Cherra;Bcal i les ts :

A l l t h e p r e l i m i n a r y t e s t s were c a r i i e d o u t a c c o r d i n g t o

t h e methuds d e s c r i b e d by T r e a s e and Evans (1982).

P y e s t fer: GI-:

One gram each o f t h e powdered whole r o o t (MA), r o o t bark

(REB), whole stem (SSA) and s tem b a r k (SEB) was shaken i n

a t e s t t u b e w i t h abou t lOml o f h o t w a t e r and f i l t e r e d .

To t h e f i l t r a t e was added lml ~ e h l i n g l s s o l u t i o n

( l i ' eh l ingls s o l u t i o n I and 11 i n e q u a l p a r t s ) . i nhe

mixtuxe was cooled . The r e s u l t i n g reddish-brown

p r e c i p i t a t e was f i l t e r e d o f f . To t h e f i l t r a t e 11111

o f 37; H2S04 was added and a g a i n b o i l e d under r e f l u x f o r

3 0 minu tes . i lhe m i x t u r e was c o o l e d and f i l t e r e d . To t h e

f i l t r a t e , sodium hydrox ide s o l u t i o n was added u n t i l t h e

f i l t r a t e was j u s t a l k a l i n e t o l i t m u s paper . ( r e d l i t m u s

paper t u r n e d b l u e ) .

Two n?l o f i ?eh l ing1s s o l u t i o n was added and t h e m i x t u r e

b o i l e d . A b r i c k r e d p r e c i p i t a t e was formed i n t h e

b o i l i n g m i x t u r e i n d i c a t i n g a p o s i t i v e r e s u l t .

T e s t f o r s a p o n i n s :

One gram each o f t h e powdered r o o t and stem (RSA, kEB,

SSA and SEB) o f t h e p l a n t was b o i l e d w i t h lOml o f w a t e r

and f i l t e r e d . About 5 m l of t h e f i l t r a t e was d i l u t e d t o

20ml and t h i s d i l u t e d f i l t r a t e used f o r t h e f o l l o w i n g

t e s t s .

( i i )

( i i i )

T e n i d o f t h e d i l u t e d f i l t r a t e was chaken

v i g o r o u s l y i n a t e s t - t u b e . P e r s i s t e n c e oi' foam f o r

a b o u t 30 m i n u t e s i n d i c a t e s a p o s i t i v e r e s u l t .

Two d r o p s o f o l i v e o i l were added t o l O m l o f

d i l u t e d f i l t r a t e i n a t e s t - t u b e and shaken.

L m u l s i f i c a t i o n o f o i l i n d i c a t e s a p o s i t i v e r e s u l t .

F i v e ~ d s o f 107; r e d blood c e l l s ( W C ) i n normal

s a l i n e was p l a c e d i n two t e s t - t u b e s . FYveidlof

no~mal . s a l i n e was added t o one o f t h e t e s t t u b e s b and 5ml each o f t h e ground p l a n t m a t e r i a l s was added

t o t h e o t h e r t e s t - t u b e . Both t u b e s were shaken

g e n t l y and s t o r e d . O c c u r e ~ c e o f haemolys i s o f t h e

fi&C i n t h e p r e s e n c e o f t h e p l a n t m a t e r i a l s i n d i c a t e d t h e

p r e s e n c e o f capon ins .

T e s t f o r F l a v o n o i d s :

About O.5gram each o f powdered p l a n t m a t e r i a l s (MA,

HEB, SSA and SEB) was b o i l e d w i t h l5ml of e t h y l a c e t a t e and

f i l t e r e d . The f i l t r a t e was used f o r t h e f o l l o w i n g t e s t s :

( i ) ~wo; .mlof f i l t r a t e was added t o l r n l o f d i l u t e ( lo$)

ammonia. A y e l l o w c o l o u r a t i o n o f t h e ammonia l a y e r rvhich

d i m i n i s h e s i n i n t e n s i t y on a d d i t i o n o f 25% H2 SO i n d i c a t e s 4 a p 0 s i t i v . e r e s u l t .

( i i ) 0 n e . d - o f f i l t r a t e was added t o lml o f 1 % aluminium

c h l o r i d e s o l u t i o n .

I f t h e aqueous l a y e r t u r n s ye l low, t h e n f l a v o n o i d s a r e

p r e s e n t ,

( i i i ) ~hreetrnd- of f i l t r a t e was evapora ted t o d rynes s and

t h e r e s i d u e d i s s o l v e d i n l m l o f methanol and few d rops of

f e r r i c c h l o r i d e soluLion. E;'oxsmation ol' a d a r k g reen

c o l o u r a t i o n con f i rms the p resence o f f l avono ids ,

Tes t f o r Anthracene De r iva t i ve6

Born t rager s Tes t

About 0.2gra.m each o f powdered p l a n t maLer ia l s (HEB, SEB,

M A and SSA) was b o i l e d wi th 6 m l of 2076 H S O f o r 3 minutes 2 4

and f i l t e r e d , 'L'he f i l t r a L c was added 4ml o f e t h e r i n a

s e p a r a t i n g f 'unnel and -to t h e e t h e r l a y e r was added l m l b

amrr~onia s o l u t i o n . A rose - red c o l o r a t i o n i n t h e ammonia

l a y e r i n d i c a t e s t h e przs--~Gt: o f an th r acene de r iva t i v . e s .

Modi f l e d Born t r a g e r ' s 'I 'est

About 0,Zgram each o f powdered plank m a t e r i a l s each was

pu t i n a t e s t - t u b e , yo t h i s t e s t t u b e was added 10m1,

d i s t i l l e d wa te r , 4 d rops o f 5% sodium ca rbona t e and 20ml.

of' f e r r i c c h l o r i d e , The t e s t - t u b e was b o i l e d f o r about

14 minutes , The mix tu re was f i l t e r e d arid coo led , bout

25ml o f e t h e r was added t o t h e f i l t r a t e i n a s e p a r a t i n g

funnel . he e t h e r e x t r a c t was washed w i th l>ml o f wa te r

and l a k e r shaken w i t h 2ml o f 15% ammonia,

Absence of rose-red c o l o r a t i o n i n t h e ammonia l a y e r

conf i rms t h e absence o f t h e a n t h r a c e n e d e r i v a t i v e s i n t h e

p l a n t ,

Tes t f o r cyanogene t ic Glycodides :

TWO gram of powdered p l a n t m a t e r i a l s each was p laced i n a

small f l a s k and covered w i th enough water . A s u i t a b l y

impregnated f i l t e r e d paper ( impregnated w i th sodium

p i c r a t e s o l u t i o n ) was suspended i n t h e f l a s k and t h e f l a s k

covered. The f l a s k was hea t ed and p l aced i n a c o o l p l a c e

f o r about 2 hours .

The orange-red c o l o r a t i o n o f t h e sodium p i c r a t e

paper s t r i p i n d i c a t e s t h e p r e sence cyanogene t i c g l y c o s i d e s .

Tes t f o r c a r d i a c g l y c o s i d e s :

K e l l e r - K i l l i a n i T e s t :

One gram each o f powdered p l a n t m a t e r i a l s each was b o i l e d

wi th l O m l o f 70% a l c o h o l f o r 2 minutes . 5 m l o f t h e

f i l t r a t e was added t o 1 0 m l o f wa te r and 0.5rnl o f l e a d

a c e t a t e s o l u t i o n . The mix tu re was shaken and f i l t e r e d .

The f i l t r a t e was e x t r a c t e d w i t h chloroform. The chloroform

e x t r a c t w a s d iv ided i n t o 3 p a r t s .

I n t h e f i r s t p a r t , t h e chloroform l a y e r was evapora ted t o

dr.yness and r e s i d u e d i s s o l v e d i n 3 m l o f glacria.1 a c e t i c a c i d

c o n t a i n i n g 2 d rops of 57; f e r r i c c h l o r i d e . T h i s was

c a r e f u l l y tran:;ferred t o 2ml o f c o n c e n t r a t e d s u l p h u r i c

a c i d i n a t e s t - t u b e . A reddish-brown r i n g o c c u r r i n g a t t h e

i n t e r p h a s e o f t h e s u l p h u r i c a c i d and g l a c i a l a c e t i c

i n d i c a t e s t h e p r e sence o f c a r d i a c , g lycos ide s .

Legal s Keac t i o n :

'The chloroform e x t r a c t o b t a i n e d from t h e above r e a c t i o n

was evapora ted t o d rynes s and t h e r e s i d u e d i s s o l v e d i n 2

d rops of p y r i d i n e . A drop of sodium n i t r o p r u s s i d e (2%)

47 was added t o g e t h e r w i th a d rop of' 2076 sodium hydroxide.

Presence of deep-red c o l o r a t i o n shows t h a t c a r d i a c

g l y c o s i d e s a r e p r e s e n t i n t h e p l a n t m a t e r i a l s .

Kedde Tes t - One,rnlof t h e chloroi'crm e x t r a c t o b t a i n e d bci'ore t h e

K e l l e r - K i l l i a n i t e s t was evapora ted t o d rynes s and

d i s s o l v e d i n methanol . Oneml o f 2% s o l u t i o n oP 3 , 5 - d i n i t r o b e n z o a c i d i n methanol and 1 m l o f 5.7% of .sodium

hydroxide s o l u t i o n were added.

The appearance o f an immediate v i o l e t c o l o u r which f a d e s

th rough reddish-brown t o brownish ye l low i s i n d i c a t i v e

of t h e p r e sence of' c a r d i a c g lycos ide s .

T e s t f o r S t e r o i d a l Aducone :

One gram each of ' powdered p l a n t m a t e r i a l s was p laced

i n a c o n i c a l f l a s k t o g e t h e r w i th 20ml. o f 60% \v/V e t h a n o l

and 10ml. of 1376 W/V l e a d a c e t a t e s o l u t i o n . The mix tu re was

b o i l e d f o r 2 minu tes , coo led and f i l t e r e d i n t o a s e p a r a t i n ~

f l a s k and thon e x t r a c t e d t w i c e wi th l5ml. chloroform. Mueml

o f t h e ch loroform e x t r a c t was p l aced i n a c o n i c a l flask and

evapora ted t o d rynes s i n a wate r bath . Twonil o f 3, 5

d i n i t r o b e n z o i c a c i d s o l u t i o n i n e t h a n o l was added and lml.

o f 1.0 N N a O H

A wine r e d c o l o r a t i o n i n d i c a t e s t h e p r e sence of' s t e r o i d a l

aglycone.

y e s t s f o r Hesinous Mat te r :

( i ) About 0.1 gram each of (REB, sEB, HSA and SSA) was

e a c h added 1 0 m l . o f l i g h t pe t ro leum e t h e r and g e n t l y

b o i l e d o v e r a w a t e r b a t h , f i l t e r e d and e q u a l volume o f

1576 copper a c e t a t e s o l u t i o n added. I f t h e p l a t

m a t e r i a l s c o n t a i n r e s i n s , t h e n t h e p e t r o l e u m e t h e r l a y e r

becomes g r e e n co loured .

( i i ) bout 0 - 5 gram each o f (HEB, SEB, HSA and SSA) was

added t o 5ml o f a c e t i c a c i d a n h y d r i d e i n a t e s t tube .

The t u b e was warmed and f i l t e r e d . One d r o p o f 80%

s u l a h u r i c a c i d was added. The f o r m a t i o n o f \ p u r p l e

c o l o r a t i o n shows t h a t t h e r e a r e r e s i n s i n t h e p l a n t

m a t e r i a l s , b

T e s t f o r Oleogum:

bout 0 , lgram each 01 t h e powdered p l a n t m a t e r i a l s

each w a s mixed w i t h a b o u t 0.5 gram sand and t r a n s f e r r e d

t o a t e s t - t u b e c o n t a i n i n g 3 m l e t h e r . The m i x t u r e was

wel l -shaken, f i l bered i n t o an. e v a p o r a t i n g d i s h and a l lowed

t o e v a p o r a t e t o t h i n , f i l m , Few d r o p s o f bromine w a t e r

were added t o t h e f i l m .

The a p p e a r a n c e o f a v i o l e t c o l o r a t i o n i n d i c a t e s t h e

p r e s e n c e o f oleogum.

mst Lor ~ a n n i n s ;

~ i v e gram. each o f t h e powdered p l a n t m a t e r i a l s was

added t o 40ml w a t e r b o i l e d and f i l t e r e d . The f i l t r a t e

w a s used f o r t h e f o l l o w i n g t e s t s :

( i ) Two d r o p s o f h'eC1 were added t o 5 j m l o f f i l t z a t e . 3 The f o r m a t i o n o f a b l u e p r e c i p i t a t e i s a p o s i t i v e

t e s t f o r t a n n i n s ,

( i i ) One n ~ l o f po tass ium dichromate was added t o 5ml of

f i l t r a t e . I f a brown p r e c i p i t a t e i s l'orned, t h e n

t h e t e s t i s p o s i t i v e f o r t ann ins .

( i i i ) F i v e : r d o f f i l t r a t e was added t o I m l o f l e a d sub

a c e t a t e s o l u t i o n ,

'rhe fo rmat ion o f a d i r t y whi te p r e c i p i t a t e shows

t h a t t h e p l a n t m a t e r i a l s c o n t a i n t ann ins .

x e s t f o r . a l k a , l o i d s :

Two gram each o f powdered p l a n t m a t e r i a l s was p l aced + i n a c o n i c a l Ilask. Twentyml o f 5% s u l y h u r i c a c i d i n

e t h a n o l (50%) was added. he mix tu re was b i l d f o r

2 minu t e s and f i l t e r e d , The f i l t r a t e was p l aced i n a

separaLing f l a s k and made a l k a l i n e w i th d i l u t e a~iilnonia

(8%-v/v). The s o l u t i o n was e x t r a c t e d w i th equa l volumes

0 f C H C l 3. The chloroform s o l u t i o n was e x t r a c t ed with

two 5 m l q u a n t i l i e s o f 576 s u l p h u r i c a c i d . The f o l l o w i n g

t e s t s were c a r r i e d o u t wi th t h e f i n a l a c i d e x t r a c t .

(i) To 2ml o f a c i d e x t r a c t w a s added 0.5rnl o f Meyer's

r e agen t (po t a s s ium mercur ic i o d i d e s o l u t i o n ) . Formation

of a cream p r e c i p i t a t e s t h e p r e sence o f a l k a l o i d s i n t he

p l a n t m a t e r i a l s .

( i i ) To 2 m l of a c i d e x t r a c t was added O . 5 m l o f

Dragendorff s r e a g e n t (Bismuth potass ium i o d i d e

s o l u t i o n ) .

50 . I f orange-yel low p r e e i p i t a t e forms, t h e p l a n t

m a t e r i a l s c o n t a i n a l k a l o i d s .

( i i i ) To 2ml o f a c i d e x t r a c t was added O . 5 m l o f Wagner's

r e a g e n t ( i o d i n e i n po tass ium i o d i d e s o l u t i o n ) .

A rcddish-brown p r e c i p i t a t e indiccl t ies t h o p r e s e n c e

o f a l k a l o i d s i n t h e p l a n t m a t e r i a l s .

( i v ) yo 2ml o f a c i d e x t r a c t was added 0.5ml o f p i c r i c

a c i d s o l u t i o n and t h e m i x t u r e a l l o w e d t o s t a n d .

The f o r m a t i o n o f a yel lovi p r e c i p i t a t e i n d i c a t e s t h e

p r e s e n c e o f a l k a l o i d s i n t h e p l a n t m a t e r i a l s .

E x t r a c t i o n :

E x t r a c t i o n , S e p a r a t i o n and I s o l a t i o n were c a r r i e d

o u t a c c o r d i n g t o methods d e s c r i b e d by O h i r i (1983) ,

The p l a n t m a t e r i a l s , Ikg each o f SSA and FiSA and 5O0g

e a c h o f k U A and sEB were e x h a u s t i v e l y e x t r a c t e d w i t h

methanol a t room t e m p e r a t u r e by Marce ra t ion , f o r f i v e

days. The s o l v e n t was rernoved from t h e f i l t r a te under

r educed p r e s s u r e , Brovmish y e l l o w v i s c o u s masses were

ob ta ined . These were d i s s o l v e d i n 3% Hcl, warmed and

f i l t e r e d . The r e s i d u e s wcre k e p t . The aqueous e x t r a c t s

were made s l i g h t l y a l k a l f - n e w i t h 1076 sodiurn c a r b o n a t e

s o l u t i o n . The l i b e r a t e d b a s e s were e x t r a c t e d w i t h

ch lo ro fo rm, washed w i t h w a t e r and d r i e d o v e r anhydrous

sodium s u l p h a t e , The orange-yel low f i l t r a t e s were

e v a p o r a t e d t o d r y n e s s l e a v i n g dark brownish-yel low

a l k a l o i d a l m i x t u r e , A.

The aqueous l a y e r s were e x t r a c t e d wi th .n-butanol.

The n-butanol e x t r a c t s were washed w i t h wate r and d r i e d

ove r anhydrous sodium su lpha t e . When t h e s o l v e n t s were

removed, brownish-yellow a l k a l o i d a l mix ture B, were

ob ta ined . The aqueous e x t r a c t s were a160 evapora ted

l e a v i n g mix tu re C. The t h r e e m i x t u r e s A, B and c from

each o f t h e p l a n t m a t e r i a l s . (RED, SM3, RSA and SSA)

were s e p a r a t e l y d i s s o l v e d i n methanol and used f o r t h i n

l a y e r chromatography (TLC).

, ? e ~ a r a t i o n :

P r e p a r a t i o n o f Thin Layer Chromatography (TLC) p l b t e s

~ h r o m a t o g r a p h i c p l a t e s o f s i z e 10 x 2Ocm were coa t ed 4- wi th S i l i c a g e l RF 254 366 (Merck) i n t h e fo l l owing

manner. Twenty gram o f s i l i c a g e l (Merck) was mixed

wi th 40ml o f water . A s l u r r y o f t h e mix tu re was made and

spread by mechanical dev ice o n t o t h e prewashed and oven

d r i e d 'I'LC p l a t e s . The t h i c k n e s s of t he s i l i c a g e l c o a t

was 0.25cm. The p l a t e s were a l lowed t o d ry i n t h e open

air . Afterward, they were ac t i u a t e d i n t h o oven a t 105'~

f o r one hour. The s i l i c a g e l adsorben t s e rved as t h e

s t a t i o n a r y phase. Using a p e n c i l , a mark w a s made about

2.5cm froin t h e bottom edge of ' t h e 'I'LC p l a t e . his was

t h e s t a r t i n g p o i n t . Another lnark was made a f t e r a

s u i t a b l e d i s t a l l c e from t h e s t a r t i n g p o i n t ( 1 % ~ ) t o

i n d i c a t e t h e d i s t a n c e t h e s o l v e n i would be a l l o w e d t o

t rav .e l . Th i s was t h e s o l v e n t f r o n t .

52 . C a p i l l a r y t u b e s o f s u i t a b l e d i a m e t e r s were washed

and d r i ed . A d i f f e r e n t c a p i l l a r y t u b e was used f o r each

mix tu r e t o p r even t con t amina t i on of t h e e x t r a c t s . A

c a p i l l a r y t u b e was dipped i n t o t h e a p p r o p r i a t e e x t r a c t

( A , B y C ) and a d rop was s p o t t e d on t h e s t a r t i n g p o i n t on

t h e TLC p l a t e . The s p o t was a l lowed t o d ry and t h e

p rocedure was r e p e a t e d u n t i l a s u f f i c i e n t amount o f

e x t r a c t has beer1 p l a c e d on. t h e adsorben t .

The p l a t e so p r epa red was p l aced i n a t a n k p r e v i o u s l y b

e q u i l i b r a t e d wi th t h e a p p r o p r i a t e s o l v e n t system. T h i s

was ach i eved by pou r ing t h e s o l v e n t sys tem i n t o t h e

t ank , c o v e r i n g t h e t a n k ( a i r - t i g h t ) and a l l o w i n g i t

t o s t a n d at room t empe ra tu r e f o r abou t t h i r t y minutes .

The s o l v e n t sys tem which i s t h e mobi le p h a ~ e was a l lowed

t o move up t h e p l a t e t o t h e r e q u i r e d d i s t a n c e , thab

i I>cm from t h e s t a r t i n g p o i n t . The s o l v e n t sys tems

used were p r o p e r l y s e l e c t e d because t h e y , i n con j u n c t i o n

w i th o t h e r o p e r a t i n g c o n d i t i o n s such as t h e n a t u r e

o f development, t h e chamber a tmosphere and t h e

t empe ra tu r e , c o n t r o l s e p a r a t i o n .

material

I extract w i t h

f i l t r a t e

$ to dryness

T wi th lo'/. Na2C03

warm w i th I

3% HCI filter I

stage 3 B M 1 \ extract with

HCI extract 1 residue

n- butanol, dry with

! I bbsify to p H 9 '

stoge iz

( extract d i t h CH C13

evaporate to evaporate dryness

alkaloidal mixture stage

L B B (quaternary

alkaloids 1

dissolve in MeOH

stage 3A

dissolve in MeOH

CH C13

ext roc t 1

s t c j e LA

FIG. 2 - Flow chart of the extraction - procedure.

ulkaloidal mixt ure

A ( tert iary

alkaloids 1

The v a r i o u s s o l v e n t sys tems t r i e d f o r e f f e c t i v e

s e p a r a t i o n of t h e c o n s t i t u e n t s i nc lude :

1 . D ie thy l e t h e r Chloroform

2. Benzene 20

Chloroform

Benzene

Methanol

~ t h y l a c e t a t e

Methanol

Chloroform

Chloro form

Methanol

Acetone

Chloroform

Methanol

Acet ic ~ c i d

Isopropanol

Acet ic a c i d

Ammonia

Net; hanol

14et hanol

Ace t ic a c i d

Diethylamine

Ammonia

Die thylamine

Observat ion :

The f i r s t five s o l v e n t sys tems gave poor r e s o l u t i o n s .

he compounds were e i t h e r n o t r e s o l v e d o r were

incomple te ly reso lved . I n o t h e r i n s t a n c e s , t a i l i n g

was prcdomi~lant whi le some s p o t s were d i s t o r t e d . his

was probably due t o t h e us? o f exce:;r;i.v(:lg yo l - a r

compounds o r unequal d i s t r i b u t i o n o f p o l a r i t y between

t h e s o l v e n t s . ~ h e s e f i r s t f i v e s o l v e n t s were

subsequent ly d i scarded . he remaining s i x s o l v e n t s

gave b e t t e r r e s o l u t i o n s . c l e a r l y de f ined , regular-shaped

s p o t s were ob ta ined . Each s p o t could be i d e n t i f i e d s i n g l y '

under U V l i g h t . However, e t h y l a c e t a t e , I sopropanol ,

~rnlnonia (9, 7, 3 ) gave t h e s h a r p e s t r e s o l u t i o n and f o r

t h i s r ea son , subsequent TLC r u n s were made i n t h i s s o l v e n t

system.

V i s u a l i z a t i o n :

The p l a t e was removed from t h e t a n k and d r i e d i n t h e

air . Each s p o t was i d e n t i f i e d under U V l i g h t o r by

sp ray ing w i t h a s u i t a b l e r eagen t . I n t h i s c a s e , t h e

spray r eagen t used was Dragendor f f ' s r eagen t . The c o l o u r s

o f t h e s e p a r a t e d components under UV were noted. The

reddish-brown c o l o u r s o b t a i n e d confirmed t h e presence

o f a l k a l o i d s as co~nponents o f t h e stem and r o o t .

I d e n t i f i c a t i o n :

&f v a l u e s f o r t h e s e p a r a t e d components were

c a l c u l a t e d . A Hrf va lue i s parameter used i n d i f f e r e n t i a -

t i n g v a r i o u s s p o t s . It may be d e f i n e d as t h e p e r c e n t a g e o f

t h e d i s t a n c e t r a v e l l e d by t h e s p o t d i v i d e d by t h e

d i s t a n c e t r a v e l l e d by Lhe s o l v e n t f r o n t . Measurement was

made t o t h e c e n t r e o f t h e s p o t f o r round s p o t s w h i l e f o r

e l onga t ed s p o t s , measurement was t o t h e f r o n t o f t h e spo t .

Under s t a n d a r d c o n d i t i o n s , Ikf v a l u e s i s a c o n s t a n t f o r a

p a r t i c u l a r compound. However, i n p r a c t i c e , v a r i a t i o n s

o f H r f v a l u e s o c c u r s o i t i s d e s i r a b l e t o r u n r e f e r e n c e

compounds a l o n g s i d e unlmown mix tures .

A d d i t i o n a l l y , t h e s e p a r a t e d components were compared , by TLC w i t h a u t h e n t i c samples o f pa lma t ine , j a t r o r r h i a i n e ,

columbamine and ~ e t r a h y d r o p a l m a t i n e .

11SOLAl'lOLi :

An i s o l a t i o n p rocedure was c a r r i e d o u t f o r t h e

q u a t e r n a r y f r a c t i o n (mix tu re B ) u s i n g p r e p a r a t i v e t h i n

l a y e r chromatography (prep. TLC).

he procedure f o r p r e p a r a t i v e TLC i s as d e s c r i b e d f o r

Simple TLC on page . >.lo., e x c e p t t h a t i n s t e a d o f s p o t t i n g

t h e sample t o be i s o l a t e d ; i t i s s l r e a k e d .

The s t r e a k i n g was r e p e a t e d s e v e r a l t i m e s u n t i l a

s u f f i c i e n t amount o f ~ n i x t u r e was p l aced on t h e adsorben t .

The p l a t e s were t h e n developed as p r e v i o u s l y d e s c r i b e d

( a s t e r s e p a r a t i o n ) i n e t hy l ace t a l i e , i s o p r o p m o l , anlmonia

(9 , '7, 3) s o l v e n t system. The p l a t e s were removed from

t h e t a n k a f t e r development, a i r - d r i e d f o r abou t 1 minute

and t h e d i s c r e t e bands o f m i g r a t i o n observed. The bands

were t h e n s e p a r a t e l y s c r a p e d o f f a l o n g w i t h t h e silica

g e l adso rben t u s i n g a s p a t u l a . Each hand was sc r aped

i n - t o a d i f f e r e n t c o n i c a l f l a s k . A number o f t h i s

i s o l a t i o n procedure was c a r r i e d o u t t o i n c r e a s e t h e

q u a n t i t y o f Lhe i s o l a ' t e d compound. To t h e s i l i c a g e l

w i th t h e i s o l a t o d compound i n a c o n i c a l f l a s k , methanol

was added. The i s o l a t e d compound d i s s o l v e d i n t h e

me-L hanol l e a v i n g t h e s i l i c a g e l und i s so lved , t h e compound

was then f i l t e r e d o u t usin.g Whatman 112. 1 f i l t e r paper .

&fore methanol vias added t o t h e c o n i c a l f l a ~ k t;o w a ~ h o u t

any remain ing compound, and a g a i n t h e compound was f i l t e r e d b

o u t . ~ l l t h e f i l t r a t e s o f t h e same compound were t hen

pooled t o g e t h e r and r e - f i l t e r e d s ev . e r a l t i m e s u n t i l c l e a r

ye l low Co brownijsh yel low s o l u t i o n were ob t a ined .

The f i l . t r a t e were t hen s p o t t e d a long w i t h a u t h e n t i c samples

on TLC p l a t e s and developed. T h e i r Hrf v a l u e s were

c a l c u l a t e d and t h e r e s u l t s recorded. 'Three a l k a l o i d s were

i s o l a t e d and code-named ,yn I, I1 and I11 f o r pa lma t ine ,

columbanine a n d , j a t r o r r i h i z i n e r e s p e c t i v e l y . They were

subsequen t ly evapora ted and s - to red at about 4'~.

~ c r e e n i n f ; or An t imic rob i a l AC t i v i t x :

( 1 ) Each o f t h e f o u r powdered p l a n t s was ex . t r ac t ed i n t h e

c o l d wi th wa.ter alld methanol. The f i l t r a t e s ( e x t r a c t s )

were s c r eened f o r t h e i r anLi -microb ia l a c t i v i t y u s i n g t h e

cup-p la te method. Methanol s e r v i n g a s c o n t r o l was a l s o

t e s t e d f o r a p o s s i b l e a n l i - m i c r o b i a l a c t i v i t y .

58 c u p - P l a t e Method:

~ h c c u p - p l a t e method d e s c r i b e d by ( ~ u g n a n i and

Ezenwanne 1985) was used t o d e t e r m i n e t h e z o n e s o f

i n h i b i t i o n exhi b:i Led by Liie t e ~ t micro-organisms. E'reshly

p r e p a r e d n u t r i e n t a g a r (NA) p l a t e s were f l o o d e d w i t h

an 18 h o u r n u t r i e n t b r o t h c u l t u r e o f t h e t e s t o r g a n i s m s

and d r a i n e d o f f comple te ly . A w e l l i s made i n t h e

middle of' t h e p l a t e by means o f a n 8 m m a g a r b o r e r . Eacli

o f t h e e x t r a c t s was i n t r o d u c e d i n t o t h e w e l l by means o f

a P a s t e u r p i p e t t e . The n u t r i e n t a g a r p l a t e s were sub- b

s e q u e n l l y i n c u b a t e d a t 3 7 ' ~ f o r 24 h o u r s and t h e d i a m e t e r

o f t h e v a r i o u s zones o f i n h i b i t i o n ( a r e a s a round t h e

w e l l s n o t overgrown by t h e t e s t micro-organisms) were

measured w i t h a r u l e r and r e c o r d e d .

( i i ) The second l e v e l o f ~ c r e e n i n g s were c a r r i e d o u t

a t each s t a g e i n t h e e x t r a c t i o n p r o c e d u r e t o e n s u r e t h a t

t h e p l a n t m a t e r i a l s s t i l l r e t a i n e d t h e i r a n t i - m i c r o b i a l

a c t i v i t y . The method used was a l s o c u p p l a t e as p r e v i o u s l y

d e s c r i b e d .

( i i i ) The i s o l a t e d p u r e a l k a l o i d s were t e s t e d f o r t h e i r

a n t i - m i c r o b i a l a c t i v i t y and t o d e t e r m i n e t h e spec t rum o f

t h e i r a c t i v i t y . E ' O r t h i s t e s t , s even ~ n i c r o - o r g a n i s m s i n

18 hour n u t r i e n t b r o t h c u l t u r e were t o f l o o d n u t r i e n t a g a r

p l a t e s a f t e r which s e l l s were made i n t h e n u t r i e n t a g a r i n

t h e p l a t e s . One hundred rng o f p a l m a t i n e was d i s s o l v e d i n

> m l s o f me thano l and t h e r e s u l t a n t s o l u t i o n was i n t r o d u c e d

i n t o a l l t h e w e l l s f o r bach o f t h e t e s t - o r g a n i s g s u s i n g

t h e cup -p l a t e t e chn ique and i n c u b a t i n g at 3 7 ' ~ f o r 24

hours , t h e d i ame te r o f t h e zones o f i n h i b i t i o n were

measured and recorded .

The same c o n c e n t r a t i o n of ' columbamine was a l ~ o used

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

E'or j a t r o r r h i z i n e 2.>mg of t h e e x t r a c t i n l m l of' methanol

was i n t r o d u c e d i n t o t h e v a r i o u s p l a t e s f looded viilh t h e

t e s t micro-orgaliisms. The p l a t e s were t h e n i ncuba t ed a t

3 7 ' ~ f o r 24 hou r s and the d i a m e t e r s o f Lhe i r zones o f

i n h i b i t i o n were measured.

A l l t h e t h r e e samples o f t h e a l k a l o i d s (Pa lmat ine , 4

Colombamine and J a t r o r r h i z i n e ) were mixed a c c o r d i n g t o t h e

c o n c e n t r a t i o n s s t a t e d above and cup -p l a t e method was used t o

de te rmine i t s zones o f i n h i b i t i o n on t h e v a r i o u s a g a r p l a t e s

of t h e t e s t -mic ro-organ i sms were measured.

Known a n t i - b i o t i c s w i t h broad spect rum a c t i v i t y , P e n i c i l l i n

and AlopicilPin were used a t a c o n c e n t r a t i o n o f 2.5rng/ml

(wa t e r ) a s comparisc,ls. l l l e i r zones o P i n h i b i t i o n were

measured u s i n g t h e cup -p l a t e method.

De te rmina t ion oi' Dlinimurn I n h i b i t o r y Concen t r a t i on ( P I I C ) :

The Flinimurn I n h i b i t o r y Concen t rq t i on ( M I C ) o f a l l t h e

a l k a l o i d s on t h e t e s t micro-organisms were de te rmined

u s i n g t h e method d e s c r i b e d by Cruickshank e t al . , (1975) .

Y1he minimum i n l l i b i t o r y c o n c e n t r a t i o n i s t h e l owes t

c o n c e n t r a t i o n o r t h e h i g h e s t d i l u t i o n of' an a n t i - m i c r o b i a l

a g e n t o r d i s i n f e c t a n t which w i l l i n h i b i t t h e growth of micro-

organisms.

' I l k was de te rmined w i t h t h e i s o l a t e d p u r e

s a ~ n p l e s (Pa lmat ine , Colurnbamine and j a t r o r r h i z i n e ) a s

w e l l as w i t h t h e m i x t u r e o f t h e samples. The i - i l C s of ' t h e

knowq a n t i b i o t i c s ( A m p i c i l l i n and ~ e t r a c y c l i n e ) were a l s o

de te rmined and used as comparisons. One hundred mg o f

p a l m a t i n e was d i s s o l v e d i n > m l o f methauol t o o b t a i n a

c o n c e n t r a t i o n o f 20mg/ml.

One hundred mg of Columbamine was d i s s o l v e d i n ljml 01

rne.thanol Lo o b t a i n a c o n c e n t r a t i o n o f ZOmg/ml.

Ten rng o f J a t r o r r h i z i n e was d i s s o l v e d i n 5ml o f methanol

t o o b t a i n a c o n c e n t r a t i o n o f 2mg/ml. b

'I'he a n t i b i o t i c s ( a m p i c i l l i n and t e t r a c y c l i n e ) were each

used a t a c o n c e n t r a t i o n of 2mg/ml o b t a i n e d by d i s s o l v i n g

10mg o f e a c h i n ljml o f d i s t i l l e d water .

Four m l o f n u t r i e n t b r o t h were p l a c e d i n each o f f i v e

t e s t t u b e s used f o r t h e t e s t s on each a l k a l o i d and 9ml

o f n u t r i e n t b r o t h were p l a c e d i n each o f f iv . e t e s t - t u b e s

and used f o r t h e t e s t s on each o f t h e a n t i b i o t i c s . One m l

o f each of' t h e a l k a l o i d s was i n t r o d u c e d i n t o t h e f i r s t s e t

of t e s t - t u b e s and t h o r o u g h l y mixed. One m l o f t h e m i x t u r e

from each o f t h e a l k a l o i d s i n n u t r i e n t b r o t h was

withdrawn and i n t r o d u c e d i n t o t h e second s e t o f t e s t - t u b e s ,

ab.d s o on u n t i l t h e l a s t s e t o f t e s t - ' t u b e s from where 1 n i l

was wilhdrarm and discarded.

Yhis wa:; .i-t:.;lcc-:Led for l, l ln a l ~ L i b i o t i c s u s i n g t h e t e s t - t u b e s

c o n t a i n i n g 9ml each of n u . t r i e n t b r o t h . About 0 . 2 ~ 1 o f

each o f t h e t e s t micro-organism was p l a c e d i n a l l t h e f i v e

t e s t - t u b e s l a b e l l e d s p e c i f i c a l l y f o r i t and i n c u b a t e d at

3 7 ' ~ f o r 24 hours . ~ f t e r i n c u b a t i o n , t h e t u b e s s e r e

examined f o r growth as c h a r a c t e r i z e d by t u r b i d i t y o f t h e

n u t r i e n t b r o t h . he las t t e s t - t u b e c o n t a i n i n g c l e a r n u t r i e n t

b r o t h ( w i t h o u t t u r b i d i t y ) c o n t a i n s t h e minimum i n h i b i t o r y

c o n c e n t r a t i o n (WIC) o f t h e a l k a l o i d o r a n t i b i o t i c as t h e

c a s e may be. I n o t h e r words, t h e t e s t - t u b e w i t h t h e c l e a r

n u t r i e n t b r o t h t h a t immcdiat e l y p r e c e e d s t h e t u r b i d

n u t r i e n t b r o t h con t , a ins t h e I ' l lC of t h e compound b e i n g

t e s t e d .

Ue te rmina t ion of prinimum C i d a l c o n c e n t r a t i o n (MCCL:

The Ninimum C i d a l C o n c e n t r a t i o n ( M C C ) o f a l l

a l k a l o i d s on t h e t e s t micro-organisms were de te rmined

u s i n g t h e method d e s c r i b e d by Cruickshank & a., ( 19'75)

A l l t h e a l k a l o i d s and a n t i b i o t i c s were s e r i a l l y d i l u t e d a s

d e s c r i b e d under M I C d e t e r m i n a t i o n . The t e s t micro-

organisms were added and the m i x t u r e i n c u b a t e d f o r 24

hours . From each of' t h e t e s t - t u b e w i t h o u t t u r b i d

n u t r i e r i t b r o t h 2mls o f t h e n u t r i e n t b r o t h were withdrawn

and s e p a r a t e l y i n t r o d u c e d n? t o f r c s h l y p r e p a r e d i l u t r i e n t

a g a r p l a t e s . The n u t r i e n t b r o t h was v i g o r o u s l y shaken p r i o r

t o t h i s wi thdrawal . The f l o o d e d n u t r i e n t a g a r 2 l a t e s

were t h e n d r a i n e d o f f e x c e s s n u t r i e n t b r o t h and

i n c u b a t e d a t 370C f o r 48 hours . The minimum c i d a l

c o n c e n t r a t i o n s (MCC) were c o n t a i n e d i n t h e t e s t t u b e s

w i t h t h e h i g h e s t d i l u t i o n s t h a t gave no growth on t h e

n u t r i e n t a g a r p l a t e s a f t e r i n c u b a t i o n f o r 40 h o u r s a t

p e t e r m i n a t i o n of ' llle E f f e c t o f Temperature on t h e Anti- p i c r a b i a l ~ c t i v i t y of' t h e A l k a l o i d s :

The e f f e c t s of ' t e m p e r a t u r e varia .Lion on ti le a n t i -

m i c r o b i a l a c t i v i . t i e s o f t h e a l k a l o i d s were de-i;er*mined

acccjlvuinll; t o t h e methods d e s c r i b e d by Eneru~va (1 982). 0 The i n i t i a l tempera . ture of ' t h e a l k a l o i d s was 29 C. That

was t h e t e m p e r a t u r e f o r t h e a n t i b i o t i c s used f o r

comparison. Each of t h e a l k a l o i d , was d i s s o l v e d i n

methanol t o a c h i e v e a c o n c e n t r a t i o n o f 2.0mg/ml f o r

J a t r o r r h i z i n e ZOmg/ml f o r columbamine grid Pa lmat ine .

The c o n c e n t r a t i o n o f t h e a n t i b i o t i c s was 2mg/ml. b

Each o f them was inbroduced i n t o a . t e s t - t u b e and immersed

i n a t r o u g h o f i c e - s a l t m i x t u r e u n t i l a t e m p e r a t u r e of'

(loC was o b t a i n e d .

Each o f them was t h a n withdrawn and i t s d i a m e t e r o f zone

of i n h i b i t i o n measured on &. c o l i t h e most s u s c e p t i b l e

t e s t organism u s i n g t h e c u p - p l a t e method d e s c r i b e d above.

The i c e - s a l t u i ix ture was l e f t t o thaw f o r a w h i l e u n t i l

a t e m p e r a t u r e o f 1 0 ' ~ was a t t n i n e d and t h e measurement o f

t h e d i a m e t e r o f zone o f i n h i b i t i o n was a g a i n done. T h i s

was a l s o r e p e a t e d a t ZOOC. The t u b e s were then tnkcn from

t h e i c e - t r o u g h and at 30°c, t h e d ia rue te r s o f t h e i r zones

of i n h i b i t i o n were measured. The t u b e s were s u b s e q u e n t l y

t r a n s i ' e r r e d t o a . tempera ture- regula ' ted wa te r -ba th and

t h e d iamebers o f t h e i r zones o f i n h i b i . b i o n were measured

a t i n t e l - v a l s o f 1 0 ' ~ - ( 4 0 ° ~ , 50°c, 7o0c, 80°c, 90°c and

l()o°C). fljhe d i a m e t e r s of t h e i r v a r i o u s Zones of

i n h i b i t i o n were r e c o r d e d .

De te r ln ina t ion o f the Il:ffcct o f p H on t h e A n t i m i c r o b i s l A c t i v i t y o f t h e A l k a l o i d s :

pH b u f f e r s 1 - 14 were p r e p a r e d followinl; . t h e t a b l e

s e t o u t i n t h e append ix ( t a b l e 30) . Each o f t h e

a l k a l o i d s was d i n s o l v c d i n methanol and s u b s e q u e n t l y i n

each 01' Lhc pH b u f ' f e r s (1 - 14) t o a c h i e v e t h e c o n c e n t r a -

t i o n s of' ~Omg/ml f o r Pa lmat ine and Columbaniine and 2.Omg/ml

f o r J a t r o r r h i z i n e . A corrccn- t ra t ion of ' 2.0mg/1nl was a l s o

o b t a i n e u for3 each of t h e two a n t i b i o l i c s . The d i a m e t e r

o f t;he zone o f i n h i b i t i o n ol' each o f t h e a l k a l o i d and b

a n l i b i o t i c undel, Lhe v a r i o u s pH buL'fers (1 - 14) was

measur-ed u s i n g t h e c u p - p l a t e nlethod. 'the i n i L i a 1 pH

v a l u e s o f tile a l k a l o i d s and a n t i b i g t i c s b e f o r e t h e

e x p c ~ :L~.IL'I~ L x-L:II:.;o(I L r o m s l i g h t l y a l k a l i n e t o n e u t r a l as

measured w i t h t;he (we Unican, Model 290) pkl meter . ?'he

t e s t was c a r r i e d o u t on 4. c o l i t h e most s u s c e p t i b l e t e s t

organism.

J)eterminat ion o f Lhe FXf'ect o f O ~ e n S t o r a g e on t h e

A n t i m i c r o b i a l A c t i v i t y o f i h e ~ l k a l o i d s :

A p o r t i o n o f each o f - the a l k a l o i d s and t h e

a n t i b i o t i c s were p l a c e d i n open p e t r i - d i s h e s and. k e p t on

1ab .o ra to ry s h e l v e s . samples o f each of t h e a l k a l o i d s

a i i t i b i o t i c s were withdrawn a t i n t e r v a l s one week and t h e

d i a m e t e r s o f t h e i r r e s p e c Live zones o f i n h i b i t i o r i on t h e

most s u s c e p t i b l e micro-organism g. c o l i were measured

u s i n g t h e c u p - p l a t e t e c h n i q u e . These t e s t s were c a r r i e d

f o r a t o . t a l p e r i o d o f seven weeks. The c o n c e n t r a t i o n s o f

t h e a l k a l o i d s and a n t i b i o t i c s were t h e same as t h e o n e s

used i n p r e v i o u s e x p e r i m e n t s .

D e t e r m i n a t i o n o f t h c Eff'ect of' Lhe A l k a l o i d s on t h e C e l l -

IL'Ior~~h~10,q'y of t h e t e s t Plicro-orrranisms :

Each o f t h e t e s t micro-organisms was i n o c u l a t e d

i n t o n u t r i e n t b r o t h o r Sabouraud b r o t h i n t e s t t u b c s

and c i d a l c o n c e n t r a t i o n s ol ' t h e a l k a l o i d s (as d e t e r m i n e d

from t h e MCC) each was i n t r o d u c e d i n t o t h e v a r i o u s n u t r i e n t

b r o t h t e s t - t u b e s c o n t a i n i n g t h e m i c r o - o r ~ a n i s m s . The

m i x t u r e was w e l l shaken and i n c u b a t e d a t 37 '~ . Samples o f

t h e a l k a l o i d o f a n t i b i o t i c - t r e a t e d m i c r o b i a l c u l t u r e s b

were withdrawn at 12 h o u r l y i n t e r v a l s a n d g ram-s ta ined .

They were s u b s e q u e n t l y examined under t h e mic roscope

wiLh a view t o d e t e c t i n g changes i n t h e morphology o f t h e

c e l l s . The f i n d i n g s r e c o r d e d .

Table 3 s h o w t h e chemical constituent^ found i n t h e

powddred p l a n t S ~ h e n o c e n t r u m .iollyanurn (whole roo t e x t r a c t ;

KSIL; r o o t ba rk e x t r a c t , REB; whole s t em e x t r a c t , SSA; and

stem bark e x t r a c t , SEB). A1 k a l o i d s , g l y c o s i d e s , s t e r o i d a l

ag lycones , s apon in s , f l a v o n o i d s , t a n i n s and r e s i n s a r c

p r e s e n t i n t h e p l a n t m a t e r i a l s whi le c a r d i a c g l y c o s i d e s ,

cyanogene t i c g l y c o s i d e s , a n t h r a c e n e d e r i v a t i v e s and

oleogums a r e ab sen t .

The chemica l c o n s t i t u e n t s p r e s e n t i n t h e f r a c t i o n s of

t h e r o o t e x t r a c t (whole r o o t and b a r k ) of' t h e p l a n t

m a t e r i a l were i d e n t i f i e d on 'I'LC p l a t e s . F igu re 21 ( i n '

append ix) shows t h a t t e r t i a r y A f r a c t i o n con t a ined

pa lma t ine and two u n i d e n t i f i e d compounds. Qua t e rna ry B

f r a c t i o n c o n t a i n e d pa lma t ine and j a t r o r r h i z i n e w h i l e t h e

mix tu re C f r a c t i o n c o n t a i n e d a compound t h a t cou ld no t be

i d e n t i f i e d able 4 ) A s shown i n f i g u r e 20 ( i n append ix ) ,

i n t h e whole s tem and stern bark e x t r a c t s , 'Te r t i a ry A

f r a c t i o n c o n t a i n e d two u n i d e n t i f i e d compounds and s o

does mix t u r e C f r a c t i o n . Pa lma t ine , j a t r o r r h i z i n e and

columbamine were i d e n t i i ' ied i n t h e Qua t e rna ry B f r a c t i o n

s able 5 ) .

The r o o t ba rk e x t r a c t (NEB) of t h e p l a n t e x h i b i t e d

an t i -m ic ro b i a l a c t i v i t y measured a s t h e d i a m e t e r s o f

zones c f i n h i b i t i o n . A v a l u e !range o f 25mm on &L

c o l i t o 12mm on c. a l b i c a n s was ob t a ined . Elor t h e whole - r o o t e x t r a c t o f t h e p l a n t , t h e d i a m e t e r s of zones of

~ r o w t h i n h i b i t i o n were 20mm on g. s u b t i l i s , lgmm on E. -

c o l i , 1 8 m m on b o t h &. a e r u g i n o s a and s t a p h aureus . lcor

K pneumoniae and G. a l b i c a n s , t h e d i a m e t e r v a l u e was -0

12mm. Stem b a r k e x t r a c t (SUE) had . . a n t i m i c r o b i a l

a c t i v i t y shown by t h e d i a m e t e r s o f zones o f i n h i b i t i o n

i n t h e o r d e r of' - i8.Tmm on 3. c o l i and 2. s u b t i l i s ; l8mm

on s t a p h a u r e u s and 12mm on 5 pneumonia and G. a l b i c a n s .

Diameters o f zones o f i n h i b i t i o n measured f o r whole s tem

e x t r a c t , SSA were 1 8 . 5 m m and 1 8 m m on &. c o l i and g.

s u b t i l i s r e s p e c t i v e l y . On s t a p h a n r e u s , & a e r u a i n o s a

and a. v u l ~ a r ' i s , Lilt-: v2111t! :v;i:; 17uu and on &. pneumoniae #

i t was I 3 m m .

N,o zrone o f i n h i b i t i o n was obse rved on c. a l b i c a n s p l a t e .

Methanol, t h e d i s s o l v i n g s o l v e n t used th roughou t t h e

t e s t s a l s o gave no zone o f i n h i b i t i o n able 6 ) . Table

7 shows t h e a n t i m i c r o b i a l a c t i v i t y o f t h e p u r i f i e d

a l k a l o i d s , p a l m a t i n e , columbamine, j a t r o r r h i z i n e and t h a t

o f t h e combined p u r i f i e d a l k a l o i d s . A c o n c e n t r a t i o n o f

25mg/rnl o f p a l m a t i n e gave d i a m e t e r measurements o f

i n h i b i t i o n i n t h e o r d e r o f 1 3 m m on E. a e r u ~ i n o s a ,

1 5 . 5 m m on &. v u l . ~ a r i s , 20mm on s t a p h aureus . 24mm on G.

a l b i c a n s and 25mm on &. pneumoniae. Diameter

measurements o f 26mm and 3Omm were o b t a i n e d on E. c o l i

and B. s u b t i l i s r e c p e c t i a e l y . Columbamine a t t h e same

c o n c e n t r a t i o n gave d i a m e t e r s o f zones o f i n h i b i t i o n ol' 29mm

on B. o u b t i l i s , 27.5rnm on ~LaLel? a u r e u s , 26mm on b o t h a. c o l i and K. pneumoniae. Diameter measurements of' l5mm - -

and 14mm were r e c o r d e d on &. a e r u g i n o s a and &. v u l g a r i s

r e s p e c t i v e l y . ~t a c o n c e n t r a t i o n o f 2.5mg/ml,

j a t r o r r h i z i n e showed d i a m e t e r s o f zoneg o f i n h i b i t i o n o f

4 5 m m on 3. c o l i and 40.5mm on El, s u b t i l i s . On &. a e r u g i n o s a , t h e d i a m e t e r o f t h e zone was 3 4 m m and on

v u l g a r i s i t was 25mrn. Diameter measurements r e c o r d e d

u s i n g t h e combined gxmf p u r i f i e d a l k a l o i d s were 24mm on

bo th L. c o l i and g. s u b t i l i s , 1 8 m m on s t a p h a u r e u s , 16mm

on bo th g. pneumoniae and G. a l b i c a n s and 14mm on &,

j terur;inosa.

Tho Minimum I n h i b i t o r y C o n c e n t r a t i o n o f p a l m a t i n e i n

mg/ml was 0.16 on E. c o l i and 0.8 on t h e c u l t u r ~ o f o t h e r

t e s t micro-organisms; 3. s u b t i l i s , &. g c r u ~ i n o s q , S t a p h

a u r e u s , K, pneurnoniae, a. v u l f f a r i s and 5;. a l b i c a n s . The

lvl lC o f columbarnine on S t a p h a u r e u s , & v u l g a r i s and B. s u b t i l i s was 0,8rng/ml w h i l e on G, a l b i c a n s , _K. pneumoniae

and on &. c o l i , i t was O,16mg/rnl. J a t r o r r h i z i n g e x h i b i t e d

on M I C o f 0.16mg/ml on &. c o l i and Ij, pneumoniae. On

o t h c r micro-organisms; 2. s u b t i l i s , &. h e r u ~ i n o s a ,

s t a p h a u r e u s , a. vulp ;a r l c and G. a l b i c a n s , t h e M I C was

0 .8mg/ml. 'Ithe combined p u r i i ' i e d a l k a l o i d s had a n Ibl lC

o f 4mg/ml on &. a e r u f f i n o s a and O.l6mg/ml on B. s u b t i l i s .

On E. c o l i , s t a p h a u r e u s , a. v u l g a r i s , &. pneumonia

and G. a l b i c a n s , t h e M I C was 0.8mg/ml. ~ e t r ~ a c y c l i n e and

a m p i c i l l i n were used as c o n t r o l s . ~ e t r a c y c l i n e had an

M I C o f 0.0002m&/ml on E. c o l i and g. s u b t i l i s . &b&l

a u r e u s and K. pneurnoniae r e t u r n e d on M I C o f 0.002mg/1nl.

he M 1 C f o r &. v u l f ~ : a r i s and a. a c r u ~ ; i n o ~ a was Oe02mg/

m l wh i l e t h e M I C f o r G. a l b i c a n s remained 0.2mg/mlm por

a m p i c i l l i n a M I C v a l u e o f 0.002mg/ml was r e c o r d e d on Em

c o l i Be s u b t i l i s and s t a p h a u r e u s . A v a l u e o f Om02mg/ - 9 - m l was r e c o r d e d on a. v u l a a r i s , &, pneumoniae a n d a. a e r u ~ ; i n o s q w h i l e a v a l u e o f 0.2mg/rnl was r e c o r d e d on

a l b i c a n s a able 8 ) .

I n able 9 , t h e Minimum ~ i d a l C o n c e n t r a t i o n , MCC o f

p a l m a t i n e , columbamine, and t h e combined p u r i f i e d

a l k a l o i d s on a l l t h e t e s t micro-organisms (E. c o l i ,

Re s u b t i l i s , he aerup- inosa , s t a p h a u r e u s , _K. neunionlae - E-7- and g. v u l m r i s and I;. a l b i c a g s ) was 4.0mg/mlm The MCC

o f j a t r o r r h i z i n e on a l l t h e s e micro-organism was Oe4rng/tnl.

T e t r a c y c l i n e and a m p i c i l l i n were used a s c o n t r o l s . The

MCC o f a m p i c i l l i n on c u l t u r e s o f E. v u l r : a r i s , I&

pmumoniae and Ps. a e r u g i n o s a was 0.2mg/ml w h i l e t h o s e of

a. c o l i , s t a p h a u r e u s and & s u b t i l i s was O.O2mg/ml,

b h a t o f C. a l b i c a n s was 2.Omg/ml. T e t r a c y c l i n e had i t s

MCC on c o l i and g. s u b t i l i s a s Oe002pg/ml, on Ern

pneumoniae and s t a p h a u r e u c as 0.02mg/ml and on &.

a e r u ~ i n o s a , v u l g a r i s and C. a l b i c a n s as 0.2mg/mle

The e f f e c t o f t e m p e r a t u r e on t h e a n t i m i c r o b i a l

a c t i v i t y o f p a l m a t i n e was measured a s d i a m e t e r o f zones o f

i n h i b i t i o n . A t a c o n c e n t r a t i o n o f 2Omg/ml on a c u l t u r e

o f &. c o l i d i a m e t e r mcasurernent o f l9mm, 18mm and 1 7 . 5 m m

were r e c o r d e d a t 80°c, 9 0 ' ~ and 1 0 0 ' ~ r e s p e c t i v e l y . A t

lo°C, ~ o O C , 5 0 ' ~ and 6o0c, Lhe meosuremeilt rclnained abou t

2Omm whi le a t 4 0 ' ~ i t was 21.>Oc ( m a t e 1 ) . Fig. l+

shows t h a t t h e d i a m e t e r s o f zones o f i n h i b i t i o n

e x h i b i t e d by t h e combined p u r i f i e d a l k a l o i d s were 23mm a t

40°C, 21mm a t ~ O ' C , and 2Omm a t 20Oc. A d i a m e t e r o f

1 8 . 5 m m was r e c o r d e d at 1 0 ' ~ and 70°c. ~t 8o°C, gooC and

I O O ~ C , d i a m e t e r o f 17mn1, l > m m and l2mm werc recorded .

A t a c o n c e n l r a t i o n o f 2mg/ml j a t r o r r h i z i n e e x h i b i t e d

a n a n t i m i c r o b i a l a c t i v i t y measured by t h e d i a m e t e r

o f zone o f i n h i b i t i o n i n t h c o r d e r o f 1 8 m m at 8o°C, 16mm

a t 9 0 ' ~ and 12mm at 1 0 0 ~ ~ . ~t loOc, a v a l u e of'i?2mm * was r e c o r d e d , a t 20°c i t was 24mm and a t 1+ooC i t was

32mm ( l ' ig , 5). Columbaminc at 20mg/ml e x h i b i t e d d i a m e t e r

measurements o f t h e o r d e r o f 1 5 m m , 1 4 m m and 12mm a t tio°C,

~ O O C and 10oOc, r e s p e c t i v e l y . ~t 5o0c, i t was 2lmm, 0

a t 60 C i t was 18mm and a t 70°c, 16mm. Uiameter o f

22mm and 25mm were r e c o r d e d a t ~ O O C and 4 0 ' ~ r e s p e c t i v e l y

(Fig. 6 ) .

T e t r a c y c l i n e and a m p i c i l l i n were used as c o n t r o l s .

A t a c o n c e n t r a t i o n o f ?mrr/ : l l , t e t r a c y c l i n e gavc d i a m e t e r

of zones o l i n h i b i t i o n of Z5mm a t loOc, 28mm a t 2 0 ' ~ and a t

40°C t h e mcasurernents were r e c o r d e d a s 29mm and 3 l m m

r e s p e c t i v e l y . .34mn1 was r e c o r d e d a t 50°c. A t ~ O O C and

1 0 0 ~ ~ o n l y 22mrn and 201nm r e s p e c t i v e l y were recorded .

For a m p i c i l l i n a t t h e same c o n c e n t r a t i o n , d i a m e t e r

measurements a t ~ O O C and 1 0 0 ~ ~ were a l s o 28mm and 2Omm

r e s p e c t i v e l y . A t 50°c, i t was 28mm w h i l e at loOc, 2 0 ' ~

and 310'~ Lhe measuren~en ts were 25mm, 27rnn1 and 30nm. 32mm

was r eco rded a t 4 0 ' ~ ( P l a t e 2 ) .

The e f f e c t o f pH on t h e a n t i m i c r o b i a l a c t i v i t y o f

pa lma t ine was measured as d i a m e t e r s o f zones o f

i n h i b i t i o n . A t a concenLra t ion o f 20mg/ml on a cu lLu rc

o f &. c o l i t he d i ame te r measurements were 17mtn , 18rnrn and

20mm a t Lhc pH 1 , 2 and 3 r e s p e c t i v e l y . ~t pH 5 and 6 ,

i t was 24mm ; (p laLe 3) wh i l e aL 7 and 8 i t was 1 6 m m .

~t 9 Llie d i ame te r mcasuremcnt w a s l l+mm and a t 1 1 , i t was

9mrn. ~t 12, 13 and 14, t h e r e was no zone o f i n h i b i t i o n

( i 7 lor t h e combined p u r i f i e d a l k a l o i d s t h e , d i a m e t e r s o f zones o f i n h i b i t i o n ranged from 1 4 m m t o

17 .5mm from pH 1 t o 6. A t 7 and 0, 18.5mn1 was r eco rded

as t h e d i ame te r measurement. ~t 1 1 , t h e measurement was

l5mm wh i l e a t 12 and 13, i t was 12mm. ~t t h e pH o f 14 ,

t h e d i a m e t e r measurement was 10mm a s shown i n f i g . 8.

J a t r o r r h i z i n e a t a c o n c e n t r a t i o n o f 2mg/ml as shown i n

f i g . 9 gave d i ame te r measurements t h a t ranged from 21mm

t o 20mm from p1-I I Lo . A t pII 8 and 9, t h e measurement

was 30mm ( p l a t e 4 ) . From pH 110 t o 14, t h e d i ame te r size^

f e l l from 28mm t o 20mm. For columbarnine a t 20mg/ml

shown i n Fig. 10, pH1 , and 2 gave d i ame te r measurements

of I B m m , whi le at pH v a l u e s 3 t o 6 , t h e d i ame te r s were

18.?mrn, l9mm, 19.5mm and 20mm r e s p e c t i v e l y .

~t t h e pH o f '2, t h e d i ame te r measurement was 22.>mm

( p l a t e 5 ) whi le from 1 1 t o 14, on ly a d i ame te r o f 9mm

w a s recorded. ~ e t r a c y c l i n e and a m p i c i l l i n a t a

c o n c e n t r a t i o n o f 2mg/ml were used as c o n t r o l s i n t h e s e

t e s t s . able 16 shows t h a t a t pH 1 , 2 and 3, t h e d i a m e t e r s

o f t h e zones o f i n h i h i l i o n measured were 1 8 m m , 22mm and

23mm r e s p e c t i v e l y . A t p K 5 and 6, i t was 30mm and a t 7

and 8, i t was j2nim. Diameter measurement o f 20mm was

r e c o r d e d a t pH w h i l e 1 6 m m was r e c o r d e d a t 13 and ?I+.

i i l l i 11 a L l i l , ; :l:ti: c o n c e n t r a t i o n gave a d i a m e t e r v a l u e

o f 9mm a t 13 arid 14. A t t h e pH o f 12, 1 1 and 10, a

d i a m e t c r s i z e was 22mm. pH v a l u e s 9 and 8 gave a d i a m e t e r

of 3Omm w h i l e a t pIi 7, t h c d i a m e t e r was 3 > m m . At 1 , 2

and 3, t h e d i a m e t e r s were 23mm, 24mm and 25mm r e s p e c t i v + y .

?'he s t o r a g e e r f ' e c t on t h e a n t i m i c r o b i a l a c t i v i t y of

p a l m a t i n e i s measured as t h e d i a m e t e r s o f zones o i

i n h i b i t i o n . P a l m a t i n e a t a c o n c e n t r a t i o n o f 2>rng/ml on a

c u l t u r e o f c o l i gave a v a l u e o f 3Omm a t t h e b e g i n n i n g

o f t h e t e s t . ~t t h e end o f t h e f i r s t week, i t came t o

28mm w h i l e at t h e end o f t h e second , t h i r d , f o u r t h and

f i f t h weeks t h e v a l u e s f e l l s t e a d i l y from 25mm t h r o u g h

21mmand 18mmto 1 T m r n r e s p e c t i v e l y ( p l a t e 6 ) . A t t h e

end of weeks 6 and 7 , t h e d i a m e t e r e i z e s f e l l t o 1 4 m m and

1 3 m m r e s p e c t i v e l y ( f i g . 1 1 ) . The combined ' p u r i f l c d

a l k a l o i d s gave zones o f i n h i b i t i o n whose d i a m e t e r s measured

23.9mrn and, 23mm a f t e r weeks 1 and 2. At t h e end o f t h e

t h i r d , f o u r t h , f i f t h anu s i x t h weeks, t h e d i a m e t e r v a l u e s

were 20mm, 1 9 m m , 18.>rnm and l 8 m m r e s p e c t i v e l y ( p l a t e 7) .

~t t h e o n s e t , a d i a m e t e r o f 24mm was r e c o r d e d b u t t h i s

f e l l t o 17mm a f t e r week 7 as shown i n f i g . 12,

A t a c o n c e n t r a t i o n o f 2.>nlg/ml, j a t r o r r h i z i n e ( f i g . 13)

gave a d i a m e t e r s i z e o f 49m1n a t t h e o n s e t o i Lhe e x p e r i m e n t ,

43mrn a t t h e end o f week 2 ; 40mm and 5 4 l n m a f t e r t h e t h i r d

and f o u r t h weeks r e s p e c t i v e l y ( p l a t e 8 ) . A f t e r t h e

f i f t h and s i x t h weeks, v a l u e s o f 23mm and 20mrli

r c s p c c t i v e l y wcre r e c o r d e d . columbamine used a t a

c o n c e n t r a t i o n o f 2>rnt~;/ml gave d i a m e t e r v a l u e s o f 26mm

b e f o r e t h e f i r s t week. A f t e r week 1 , 2 and 3, t h e v a l u e s

were ,?Lt.fjrnm, 22mm and lgrnm r e s p e c t i v e l y . A t khe end o f

Lhe i 'our th week t h e d i a m e t e r was 16mm and a f t e r t h e f i f t h

week, i t was 14mm. fit t h e cnd o f weeks 6 and ') t h e v a l u e s

f e l l t o 12mm and 1 0 m m r e s p e c t i v e l y ( f i g . 14) . b

' I ' e t r a c y c l i n e and a m p i c i l l i n a t a c o n c e n t r a l i o n o f 2111g/

m l u s e d as c o n t r o l s . ~ e t r a c y c l i n e e x h i b i t e d a

d i a m e t e r o f zone o f i n h i b i t i o n o f 4>1nin a t t h e o n s e t o f khe

Les t , t h i s f e l l t o 4 3 m m a f t e r t h e f i r s t week. From

weeks 2 t o 4, t h e rr~easurement was 42mm ( p l a t e 9 ) .

A l t e r week >, i t was 41m1ti and a t t h e end o f Lhe s i x t h arid

s e v e n t h week i t l e l l t o 40rnm. A m p i c i l l i n showed a

diarnoler measurement of 42mm at t h e o n s e t o f t h e t e s t .

A f t e r week I, a v a l u e o f 4O.5mm was r e c o r a e d . his f e l l

t o 40mm from t h e end o f ' week 2 t i l l t h e end 01 week 4.

A f t e r weeks 6 and 7, i t Y e l l a g a i n t o 39mm.

k i g u r e 15 s h o e s t h a t a t t e m p e r a t u r e o f loOc, t h e

combined a l k a l o i d had a d i a m e t e r o f zone o f i n h i b i t i o n o f

18.5mn1 w h i l e columbamine, p a l m a t i n e and j a t r o r r h i z i n e had

diameter s i z e s o f 19mm, 2Omm and 22mm r c s ~ e c t i v e l y * A t

73 . 3 0 ' ~ t h e d i a m e t e r ~ l e z s ~ , a ~ n t s were 21mm f o r Lhe combined

a l k a l o i d , 22mm f o r columbanlinc, 20mm f o r palma t i n e and

26.3 f o r j a t r o r r h i z i n e . ~t t e m p e r a t u r e o f 40°c a l l t h e

a l k a l o i d s had Lhe i r h i g h c s t a c t i v i t i e s , j a t r o l - r h i z l n e

(3Prnm ) , columbamine (25mm), p a l m a t i n e (21 . > m ) and t h e

combined a l k a l o i d (Z3mm). These f e l l t o v a l u e s o f

l 8 m m f 01' j a t r o r r h i z i n e , 1 31nm f o r columbamine, lgrnn~ f o r

p a l m a t i n e and l r / m m f o r t h e combined a l l c a l o i d a t t h e

te rnpera turc 01 WOc.

A t 1 0 0 ~ ~ , t h e d i a m e t e r 01 zone o f i n h i b i t i o n o f

j a t r o r r h i z i n e , columbamine and t h e combined a l k a l o i d , had f a l l e n t o 12mm w h i l e that o f p a l m a t i n e Was 17 .>mm.

A t pH 1 , j a t r o r r l i i z i n e had a d l a r l ~ e t e r o f zone o f

i n h i b i t i o n of' 2 1 rnm, columbamine had 1 tlmm, pa l lua t ine

had 1 7 m m and t h e combined a l k a l o i d had 14mm. PalrnaLine

had i t s h i ~ h e s t a c t i v l t y o f 24mm a t t h e p11 v a l u e s 01 5

and 6. A t t h e s e pfi v a l u e s , j a t r o r r h i z i n e had 28n1n1

columbamine, 19.5mw and 20mm and t h e combined a l l i d o l d

17 .5mm. For .colurnb;unine i t s h i g h e s t d l a ~ n e t e r

measurement o f 2~.>m1n was a t t h e pH o f 7 , aL whlch

p a l m a t m e had a ciia~ne Lor ~ i z e 01 I 6mm, j a t r o i a . r S h i ~ i n e ,

29.3mr11 and Lhe co~nbined a l k a l o i d s , I 8 . I . 'I'he h i g h e s t

diauleLer s i z e o f 30~111 tneasul-ed Lor j a l r o r r h i z i n e vias a t

Lhe pi1 v a l u e s of U m u 9 a t which p a l m a l i n e had d i a m e t e r

s i z e s 01 lbmm and 14mm, columbamlne had 19.5mm and 1 l rum

and t h e combined alkaloids had 18.5mm and l'[.>mm. ~t

p~ 14 ja t l .or . rh iz ine had a d i a m e t e r s i z e o f 2Omm

t h e r e s t o f t h e a l k a l o i ~ s had d i a m e t e r s i z e s i ihlch were

n o t r e t a r d e d as s i g n i f i c a r ~ t ( f i g . I b ) .

F i g u r e 17 shows t h a t b e f o r e t h e a l k a l o i d s were s t o r e d

open, j a t r o r r h i z i n e had a d i a m e t e r rueasurement o f 4 5 m m ,

p a l m a t i n e jOrnm, columbamine, 26mm and t h e combined

a l k a l o i d s 24mm. A t t h e second week o f s t o r a g e t h e

dianie t er v a l u e s were LtOrn~n f o r j a t r o r r h i z i n e , 21 mni f o r

p a l m a t i n e , and l(3mrn l o r bo th columbamine and t h e

con~bined a l l r a l o i d s . ~t t h e end o f t h e f o u r t h week o f

s t o r a g e t h e d i a m e t e r measurement f o r j a t r o r r h i z i n e had

f a l l e n t o %7mm, p a l m a t i n e had v a l u e o f 1 8 m m , t h e * combined a l l r a l o i d s had a v a l u e o f 1 Cjmm and columbamine

had 1 6 m m . ~ t the end oi' the s e v e n t h week t h e d i a m e t e r

s i z e s f e l l f i n a l l y t o j a t r o r r h i z i n e ( 1 7. >mln), combined

a l k a l o i d s ( lr?rnm), p a l m a t i n e ( l3nim) and columbamine (lomu).

The a c t i o n 01 t h e p u r i i ' i e d a l k a l o i d s on Lhe c e l l

morphology o f micro-organisms s u c h as &, c o l i , Pr.

v u l g a r i s , &. a c r u a i n o s a and &. pneumoniae showed t h a t a t

t h e o n s e t o f growth, t h e c e l l morphology a p p e a r e d normal

under m i c r o s c o p i c examinat ion . A f t e r 12 h o u r s o f growth,

t h e c e l l s became swol len and Lhen t u r n e d t o drum-s t ick

s h a p e s a f L e r 24 hours . A f t e r 36 h o u r s t h e d rum-s t i ck

c e l l s broke up becoming c o c c o i d i n s h a p e , f i n a l l y

becoming r o u g h l y shaped and clumping t o g e t h e r a f t e r 48

hours . ( F i g u r e 18 ).

For B. s u b t i l i s i n f i ~ u r e V and $tap11 a u r e u s i n

f i g u r e 19, t h e ac.t;ion o f - these p u r i f i e d a l l r a l o i d s on t h e i r

c e l l morphology showerq +.l . , .xi t i le c e l l s appeared normal a t

t h e o n s e t o f Lhe t e s t . They become s w o l l e n a f t e r 12

hours. n t t h e end o f 24 h o u r s , t h e i r appearance bccalne

rough, t h i s roughness became more pronounced a f t e r 36

hours . rilhey s h r i v e l l e d up a f t e r 48 hours . I n f i g u r e

V I I , C. a l b i c a n s showed a normal c e l l morphology a t t h e

o n s e t o f ' growt;h b u t appeared s w o l l e n a f t e r 12 h o u r s of'

conl;act w i t h t h e a l k a l o i d s . n f t e r 24 hours , t h e c e l l s

became more s w o l l e n and r c f r a c t i l e , f i n a l l y becoming B

rough ly shaped a f t e r 36 hours . hey s h r i v e l l e d up b u t

d i d n o t clump t o g e t h e r a f t e r 48 hours .

Note: Each o f t h e t e s t s r e q u i r i n g t h e measurement 01 t h e - d i a m e t e r s o f zones 01 i n h i b i t i o n was r e p l i c a t e d f o u r ( 4 )

t imes . Tlie r e s u l t s r e c o r d e d above a r e Incan v a l u e s o f

each s e t o f r e p l i c a t i o n s .

'I'ablc 3: chemica l C o n s t i t u e n t s P r e s e n t i n E x t r a c t s

Tes ted For O b s e r v a t i o n

c a r d i a c g l y c o s i d e s

~ y a n o g e n e t i c g l y c o s i d e s

S t e r o i d a l ag lycone

~ n t l l r a c ene d e r i v a t i v e s

A l k a l o i d s

ani ins

I ies ins

T a b l e 4: I d e n t i f i c a t i o n o f P u r i f i e d conpounds i n

F r a c t i o n s o f Whole Root and Root Bark

E x t r a c t s S~henocen t rum j o l l ~ a n u m

on 'llX P l a t e s

I'rac-Lions ikoovered P u r i f i e d compounds

T e r t i a r y A

Quatenary I3

Mixture C

Un iden t i f i ed Compound

Palmat ine

Un iden t i f i ed Compound

T a b l e 5:

I d e n t i f ' i c a t i o n of P u r i f i e d Compounds i n

F r a c t i o n s o f Whole Stem and Stem Bark

E x L r a c t s o f S ~ h e n o c e n t r u m .jollyanurn

on 'l'LC P l a t e s

F r a c t i o n s Recovered P u r i f i e d Compounds

T e r t i a r y A

Q u a t e r n a r y I3

U n i d e n t i f i e d compound

Palrnat i n e

J a t r o r r h i z i n g

Columbariiline

U n i d e n t i f i e d Compound

Table 6 :

I n h i b i t i o n o f Mic rob; a:. Growth by sphenocen trum

Jollyanurn (Root and Stem) Crude e x t r a c t s

Diameter of' Zone o f grovith i n h i b i t i o n (mm)

T e s t Micro-organisms - lUij

2. c o l i 25

R s u t t i l i s - 24

Ps. a e r u g i n o s a - 24

s t a p h . a u r e u s 18.5

Pr. v u l g a r i s - 18

&. p e u m o n i a e 13

C. a l b i c a n s 12

- means no zone o f growth i n h i b i t i o n .

RIB = Root E x t r a c t (Bark)

RSA = Root E x t r a c t hole)

SEB = Stem E x t r a c t (Bark)

SSA = Stem E x t r a c t (Whole)

Table 7: A n t i m i c r o b i a l A c t i v i t y o f t h e P u r i f i e d A lka lo id s

o f $nhenocentrum :iollyanum p l a n t

Diameters o f zones of ' Grow-th I n h i b i t i o n (Mm)

T e s t P u r i f i e d Al l ra lo ids

i 1 Comb. Columb. Palmat. s?iLLmU. 7iI:lraIloids

25mg/ml 25mg/ml 2.5'mg/ml

I . E. c o l i 24 26 30 45'

2. B. s u b t i l i s 24 25 26 40.5

Table 8:

Ninimum I n h i b i - t i o n C o n c e n t r a t i o n ( M I C ) of ' t h e

P u r i f i e d A l k a l o i d s c .f Sphenocent run;

j o l l ~ a n u m plan- t

l1lh~y iqlCltOOhL;AiJ I S P I S M l l i I t l U ~ 4 LNllIBI1l 'OIIY C O I J L ~ , ~ J ' ~ ' ~ I ~ ~ ~ ' I O ~ . J ( m c c / m l 1

o m . u r n Palmat . J a t r o r r J,letraa - &P.. ; Alka. C Y C ~ .

I . a. c o l i 0.8 0.16 0.16 0.016 0.0002 0.002

2. K. pneumoniae 0.8 0.16 0.16 0.016 0.002 0.02

T e t r a c y c l . - TETRACYCLINE

Amp - AMPICILLIN

Table 9 :

P1ini.mum ~ i d a l c o n c e n t r a t i o n (MCC) o f t h e p u r i f i e d

~ l k a l o i d s o f $plienocentrum jollyanurn p l a n t

T e s t Microbrp;anisms

&* c o l i

B. s u b t i l i s - s t a p h a u r e u s

K. pneumoniae

Ps. a e r u ~ i n o s a - Pr. v u l y x r i s - C. a l b i c a n s -

Minimum C i d a l c o n c e n t r a t i o n ( ~ Q / m l )

Comb. Golu~rih. Palmat J a t r o r b s TeLracycl Rmp. Alka*

4.0 4.0 4.0 0.4 0 . 002 0.02

4.0 4.0 4.0 0 4 0.002 0.02

4.0 4.0 4.0 9.4 0.02 0.02

4.0 4.0 4.0 0 4 0.02 0.2

4.0 4.0 4.0 0.4 0.2 0.2 + 4.0 1t.0 4.0 0.4 0.2 0.2

4.0 4.0 4.0 0.4 0.2 2.0 I

1 . - 1 - - -1- - L -- 1-

2 0 4 0 60 8 0 100 120 Temperature ( ' C ) - Ampicillin, o-oietracycl ine, O--O Palmat ine .

-1 . FIG. 3 - The effect of temperature on the antimicrobial

act iv i ty of palmat ine or, E, c n l i - --

L L I I I

20 40 60 80 100 1% ---- Temperature ( ' C

-Ampici l l in, e--.. Tetracycline, o---o Comb. alkaloids.

FIG. 4 - The effect of temperature on the antimicrobial activity of the combined alkaloids on E . col i - - - . .

L ----- I I L I

0 2 0 4 0 60 80 100 120 Temperature ( O C )

*--+ Ampicil l in, t--. Tetracycline, o--o Jatrorrhizine

FIG. 5 - The effect of temperature on the antimicrobial activity of Jatrorrhizine on g- -- co 1 i

-L. L -I-L-

2 0 4 0 60 80 100 120 Temperature ( " 1

x-x Ampici l l in. t-. Tetracyclin, o---o Columbamine .

FIG. 6 - The effect of temperature on the antimicrobial activity of columhamine on E. col i ' -- --

N- Ampicillin, o-----e Tetracycline, c+---o Palmatine.

FIG. 7 - The e f f e c t of p1-l on the antimicrobial

ac t i v i t y o f Palmatine on - E. co -- f i

1 M Ampic i l l in , t---r Tetracycline, o----o Comb. Alkaloids.

FIG. 8 - The effec; o f p H on the ant imicrobial

act iv i ty o f the Combined Alkaloids on E . -

- Ampicil l in, - Tetracycline, - Ja t rorr hizine .

FIG. 9 - The effect o f pH on the antimicrobial

ac t iv i ty o f Jatrorrhizine r m E, coJ i . - --

-Ampic i l l in , -Tetracycline, o---o Columbamine.

FIG. 10 - The effect of p H on the antimicrobial

act iv i ty of Columbamine. on E. coi i . - ---

Time ( in weeks 1 ---- - Y Ampic illin, . .. - Tetracycline, o-----o Palmatine .

FIG. 1 1 - The effect o f open storage on the

antimicrobial activity o f Palmatine on E. coli, - -

I 1 1 I I I I

1 2 3 4 5 6 7 Time ( in weeks 1 - Ampicillin, - Tetracycline, - Combined Alkaloids.

FIG. 1 2 - T h e effect of open storage on the antimicrobial activity of Comb Alkaloids on - E . col - i ,

Time ( in weeks) - Ampicillin, - Tetracycline, - Jatrorrhizine.

FIG. 13 - The effect of open storage on the

antimicrobial activity of Jatrorrhizine on 5. col i .

- L 0 1 2 3 4 5 6 7

Time ( in weeks) - Ampicillin, Tetracycline, o-----o Columbamine.

FIG.. 14 -The e f f e c t o f open storage on the

antimicrobial activity o f Columbamine on E. COI i . - -

Temperature ( C ) - Jat rorrhizine, o--o Columbamine,- Palmat ine, -Comb. Alka.

FIG. 15 - The effect of temperature on the antimicrobial activities of the three alkaloids 8 the combined alkaloid on E , c o l i . - -

-Jot rorrhizine, -CoIumbamine, -Palmat ine, -Combined alkaloid.

FIG. 16 - The effect o f P H on the antimicrobial activities o f

the three alkaloids and the combined alkaloid

Time ( i n w e e k s ) - J a t rorrhizine, - Columbamine, - Palmat ine, *--*Combined Alkaloid.

FIG. 1 7 - T h e e f f ec t of open s t o r a g e on the antimicrobial a c t i v i t i e s of the three alkaloids and t he

combined alkaloid on E. col i . - -

Time ( hours )

FIG. 18- E f f e c t o f

Morphology

Descr ip t ion

Nor ma1

gram-negat i v e

Swollen

gram-variable

gram- variable

Coccoid

shaped

gram-varia ble

Rough shaped

a n d clumping

together

g r a m -variable

the pur i f ied alkaloids on the cel l morphology of E. Qo.

Time (hou Morphology Descript ion

Normal

gram - positive

SwoIlen

gram- var iable

appearance

gram -variable

appearance

gram-var iable

Shrivel led

and densely

clumped

together

FIG. 19 - E f f e c t of the pur i f ied alkaloids on the cell

morphology of Staph. aureus.

- - , .-- -- .'. - - . : - ..+ d - - D - -

Plate 1: Zone of inhibition at the Optimum temperature L*. of Antimicrobial Activity for Palmatine.

' . Plate 5: Zone oS Inh ib i t ion , at the An.tPmicrobia1 A c t i v i t y

he 4 t h Week o f

4 t h Week of Open. Alkaloids .

the 4th . . . . . .. S%m,

Pla t e 9 : Zone of Inhibitiorrhafter t h e 4 th . Week of Open Storage ~f Tetracycline.

DISCUSALON HNU CONCLUSION

DISCUG;jIOI\J ANI, C0NCLUS;;ION: - - -

Sphenocentrum jollyanum P i e r r e c o n t a i n s a g r e a t number

of chemical compounds t h a t a r e p h y s i o l o g i c a l l y a c t i v e

c o n s t i t u e n t s which account f o r t h e many u s e s o f t h e p l a n t

i n h e r b a l medic ina l p repa ra t ions .

~ l l t h e e x t r a c t s of t h e p l a n t were p u r i f i e d and

i d e n t i f i e d us ing s imple TLC and p r e p a r a t i v e TLC

r e s p e c t i v e l y . Using t h e s e methods, qua t e rna ry B

f r a c t i o n of t h e r o o t e x t r a c t was found t o c o n t a i n two

c o n s t i t u e n t s which correspond i n co lou r and Hrf v a l u e s

wi th palmat ine (ye l low compound) and j a t r o r r h i z i n e 6

(yellow-brown compound) while t h e same f r a c t i o n i n t h e

stem y i e l d e d columbamine (brown compound) i n a d d i t i o n t o

palmat ine and j a t r o r r h i z i n e . I n both r o o t and stem

e x t r a c t s mixture C was found t o g ive .;one a l k a l o i d a l

s p o t (orange-red wi th Dragendor 1 s reagen t ) which however

could n o t be i d e n t i f i e d .

T e r t i a r y A f r a c t i o n conta ined t h r e e c o n s t i t u e n t s , two

of which could no t b.e i d e n t i f i e d , t h e t h i r d was however

i d e n t i f i e d as palmat ine ( ' root e x t r a c t s ) . Since palmat ine

being a qua te rnary a l k a l o i d i s not expected t o be i n t h e

t e r t i a r y A f r a c t i o n , i t must have migra ted i n t o t h i s

f r a s t i o n dur ing t h e e x t r a c t i o n process . In t h e stem

e x t r a c t s , t h e two c o n s t i t u e n t s bf t h e t e r t i a r y A, f r a c t i o n

could n o t be i d e n t i f i e d . On t h e whole, two a l k a l o i d s

(palmat ine and j a t r o r r h i z i n e ) were i s o l a t e d from t h e

r o o t and stem e x t r a c t s whi le an a l k a l o i d (columbamine)

was i s o l a t e d from t h e stem e x t r a c t s only. The stem and

r o o t a r e t h e r e f o r e recommended f o r t h e e x t r a c t i o n o f

pa lmat ine and j a t r o r r h i z i n e whi le t h e e x t r a c t i o n of

columbamine should be r e s t r i c t e d t o t h e stem.

Palmat ine and j a t r o r r h i z i n e have p r e v i o u s l y been i s o l a t e d

and i d e n t i f i e d by Oka r t e r (1976) from t h e l e a v e s , stem and

r o o t s o f t h e same p l a n t . It i s p o s s i b l e t h a t t h e

i n a b i l i t y o f Oka r t e r t o i s o l a t e columbamine from t h e

p l a n t he used cou ld stem from t h e d i f f e r e n c e s i n t h e

geog raph ica l a r e a from where t h e p l a n t s were c o l l e c t e d .

Another p o s s i b l e r ea son f o r t h i s i s t h a t t h i s work made

use of t he two a v a i l a b l e s p e c i e s o f t h e p l a n t ( t h e one

whose l e a v e s a r e e n t i r e and t h e o t h e r with edges o f t h e

l e a v e s s e r r a t e d ) . Okar te r (1 976) however d i d n o t s p e c i f y

which s p e c i e s he used. I t i s t h e r e f o r e i n f e r r e d t h a t he used

a s p e c i e s t h a t does no t e l a b o r a t e columbamine. P r i o r t o

p u r i f i c a t i o n , t h e c rude e x t r a c t s of t h e p l a n t (stem and

r o o t ) showed a b i l i t y t o s t o p t h e growth, o f t h e t e s t I

microorganisms. T h i s confirmed t h e f a c t t h a t c e r t a i n I

c o n s t i t u e n t s o f t h e p l a n t a r e o f med ic ina l value .

However, the a n t i m i c r o b i a l a c t i v i t y e x h i b i t e d by t h e c rude

e x t r a c t s could no t be a t t r i b u t e d e i t h e r wholly o r p a r t i a l l y >

t o any o f t h e a c t i v e c o n s t i t u e n t s p r e s e n t i n t h e p l a n t .

The r e l a t i v e s i z e s o f t h e d i ame te r s o f t h e zones o f

i n h i b i t i o n showed t h a t t h e a n t i m i c r o b i a l a c t i v e c o n s t i t u e n t s

a r e . . no t e v e n l y d i s t r i b u t e d i n t h e p l a n t . The r o o t

e x - t r a c t s o b v i o u s l y gave l a r b e r d i a m e t e r s o f zones o f

i n h i b i t i o n on most c l Lh:, l e s t micro-organisms t h a n t h e

s tem e x t r a c t s . VJhereas; t h e e x t r a c t oi' Llic ba rk 01 Lhe r o o t s

showed s u p e r i o r a n t i m i c r o b i a l a c t i v i t y t o t h e e x t r a c t o f

t h e whole r o o t , such a d i f f e r t ~ i c e was n o t pronounced i n

t h e c a s e o f the s tem e x t r a c t s . From t h e s e r e s u l t s , i t i s

r e a s o n a b l e t o a s s e r t t h a t more o f t h c a n t i m i c r o b i a l

a c t i v e s u b s t a n c e s a r e l o c a t e d i n t h e b a r k o f t h e r o o t o f

t h e p l a n t . T h i s r e s u l t a g r e e s w i t h t h a t of' Ebana (1988).

The p u r i f i e d a l k a l o i d s showed g r e a t e r a n t i m i c r o b i a l b

a c t i v i t y a s measured by d i a m e t e r o f zones of i n h i b i t i o n

o v e r t h e c rude e x t r a c t s . T h i s coy ld be a t t r i b u t e d Lo t h e

p r e s e n c e i n t h e c r u d e e x t r a c t s o f o Lher c o n s t i Luent ( s )

which a r e a n t a g o n i s t i c t o t h e a u t i . ~ ~ l i . c r ~ o l ~ i::l L: I-haloids.

P a l m a t i n e a t a c o n c e n t r a t i o n o f 25rng/ml showed a

c o n s i d e r a b l y h i g h a n t i m i c r o b i a l a c t i v i t y , i t was a c t i v e

on a l l t h e t e s t m i c r o - o r ~ ; a n i s m s , showing Lhe h i ~ h c s t

a c t i v i t y on x. col-i., g. ~ u b t i l i s and G. a l b i c a n s . Its

l o v ~ e s t a c t i v i t y was on &. aeruf:i.noca, b u t t h e a c t i v i t y

was h i g h enough t o be t a k e n as s i g n i f i c a n t . At t h e same

c o n c e n t r a L i o n , c o l u m b m i n e chovied an a p p r e c i a b l y h i g h

a c t i v i t y a g a i n s t micro-organisms. ~ 1 1 Lhc t e s t micro-

o rgan i sms were s u s c e p t i b l e t o i t ' even t h o u ~ h t h e

s u ~ c e p t i b i l i t y v a r i e d widely. The G r a m p o s i t i v e b a c t e r i a ,

U. s u b t i l i s and S taph a u r e u s appeared t o be more s e n s i t i v e -

t o Llie a l k a l o i d . The I . e r r ~ i l s c e p t i b l e rnicro-organisms were

t h e Gram n e g a t i v e &. vu1p;ar is and &. a e r u p i n o s a .

However &. c o l i and K. pneumoniae and I;. a l b i c a n s (a

y e a s t ) showed a hit:$ degree o f s u s c e p t i b i l i t y t o

columbarniiie. J a t r o r r h i z i n e showed t h e h ighes - t a n t i m i c r o b i a l

a c t i v i t y a t t h e l e a s t c o n c e n t r a t i o n o f 2.5111g/mle ~ h c

most s e n s i t i v e micro-organisms t o t h e a c t i o n of'

j a t r o r r h i z i n e 'were &. c o l i and g. s u b t i l i s w h i l e - the

l e a s t s e n s i t i v e was z. vu1p;ar'is which was s t i l l even

q u i t e s u s c e p t i b l e t o i t s a-!;tack, The r e s t o f t h e t e s t

rnicro-organisms showed a g r e a t d e a l oi' s e n s i t i v i t y t o #

j a t r o r r h i z i n e . P a l m a t i n e (23rng/ml), columbanline (2Tmg/ml)

and. j a t r o r r h i z i n e (2,5mg/ml) were mixed i n e q u a l volums and

t h e r e s u l t a n t cornbina t ion had a ve ry h i g h a c t i v i t y

a g a i n s t E. c o l i arid B. s u b t i l i s , The a n t i m i c r o b i a l

a c t i v i t y vJas n o t as h i g h on t h e r e s t o f t h e t e s t micro-

organisms. The combina t ion was b a r e l y a c t i v e a g a i n s t

Pr. vu1r;ari.s and &. ~ e r u g i n o s n , From Lhese r e s u l t s , i t - a p p e a r s t h a t a l l t h e a l k a l o i d s and t h e i r m i x t u r e a r e

broad-spectrum i n a c t i v i t y showing t h e a b i l i t y t o i n h i b i t

and /o r k i l l bo th G r a m negai ; ive and G r a m p o s i t i v e b a c t e r i a

as w e l l as I;, a l b i c a n s . 'yhis i s i n agreement w i t h t h e

yjork of ' flhmed el; &, (1386) on t h e a l k a l o i d , j u l i f l o r i n ;

Although t h e P l l C s 01 p a l m a t i n e and colunibamine on Lhe

t e s t micro-organisms mere q u i t e g r e a t e r t h a n t h a t 01

a m p i c i l l i n and t e t r a c y c l i n e s u g g e s t i n g l o w e r a c t i v i l ; ~ , t h e

r e s u l t s show t i n t m a l l c o n c e n t r a t i o n s (0.16 - 0.8mg/ml)

o f t h e s e a l k a l o i d s i n h i b i t e d t h e g rowth o f p a m - p o s i t i v e

and Gram-negat ive b a c t e r i a e q u a l l y w e l l . T h e i r M I C s

compared v e r y P'avourably w i t h t h a t r e p o r t a d f o r t h e l e a f

o i J u ~ ~ e n i a u n i f l o r a by Adebajo & &., (1988).

The M 1 C o f j a t r o r r h i z i n e o n t h e t e s t microortj ;anisms on

t h e o t h e r hand was q u i t e low (0.08mg/ml - O.lGmg/ml)

and a l m o s t comparab le i n some c a s e s w i t h t h a t o f

t e t r a c y c l i n e and a m p i c i l l i n (0.02mg/rnl on IJs.

ae rup ; inosa ) . However when t h e t h r e e a l k a l o i d s were

mixed i n e q u a l volumes, , t he v a r i o u s M I C v a l u e s were

i d e n L i c a l w i t h t h o s e o f p a l m a t i n e a n d co lumban ine b u t B

not; as h i g h as t h a t f o r j a t r o r ' r h i z i n e . The a n t i m i c r o b i a l

p o t e n c y of' j a t r o r r h i z i n e was h i g h e r t h a n t h o s e o f

p a l m a t i n e and columbaminc and i s o b v i o u s l y

d i m i n i s h e d by t h e p r e s e n c e o f t h e two l a l t e r a l k a l o i d s

when i n a m i x t u r e .

The Minimum C i d a l C o n c e n t r a t i o n (MCC) o f p a l m a t i n e and

columbarnine was t h e same w i t h t h a t r e c o r d e d f o r t h e

combined a l k a l o i d s . Only t h e MCC o f j a t r o r r h i z i n e on

a l l t h e t e s t mic ro -o rgan i sms compared f a v o u r a b l y w i t h t h a t

o f a m p i c i l l i n (0.2rng/rnl on &. pneumoniae, &. a e r u x i n o s a

and a. v u l m l - i s ) and t e t r a c y c l i n e (0.2ni~/ml on - C , a l b i c a n s ) . - 'The r e s u l t s o f t h e N I C and t h e MCC t e s t s showed t h a t t h e

a l k a l o i d s a r e m i c r o b i o ~ t a l i c at l 3 w c o n c e n t r a t i o n s b u t

n i i c r o b i o c i d a l a t h i g h e r c o n c e n t r a t i o n . T h i s f i n d i n g

a g r e e s e n t i r e l y w i t h t h e r e p o r t o f work done on ~ a r i c q

papaya by Dneruwa ( 1982) .

- -- -

A l l t h e t e s t s on t h e a n t i m i c r o b i a l a c t i o n o f Lhe

a l k a l o i d s ( ~ a b l e s 6 , 7 8 and 9 ) shoed Lhat A. c o l i and B.

s u b t i l i s were t h e most s e n s i t i v e micro-ox.,r,misrns t o t h e

a c t i o n 01' t h e a l k a l o i d s .

Based on i t s s e n s i t i v i t y , 4. c o l i , a bac te r ium most

f r e q u e n t l y used i n m i c r o b i o l o g i c a l e x p e r i m e n t s was

chosen as t h e t e s t rnicroorganism f o r t h e r e s t o f t h e

t e s t s e x c e p t t h o s e on t h e ce l l -morphology o f t h e

microorganisms (Boalrye-Yiadom & &. , 19'77; Hussain & &., 1979, and $.,Yneruwa, 1982).

The a n t i m i c r o b i a l a c t i v i t y o f p a l m a t i n e remained 6

s t a b l e o v e r a wide r a n b e 01 t e m p e r a t u r e , showing o n l y a

s l i g h t i n c r e a s e a t 4CL0c. ~t ex t remely h i g h t e m p e r a t u r e s

t h e r c was a s l i g h t d e c r e a s e i n a c t i v i t y and t h e zones

o f i n h i b i t i o n formed a t Lhese h i g h t e m p e r a t u r e s were

r a p i d l y overgrown i n 24 hours . Columbamine and j a t r o r r h i z i n e

c o u l d n o t t e m p e r a t u r e i n c r e a s e a s much as

pa lmat ine . Illhe a n t i m i c r o b i a l a c t i v i t y i n t h e s e two

c a s e s were l o w e r A a t t h e exLremes o f t e m p e r a t u r e s b u t

0 was o p t i m a l a t Lhe te i r ipera tures o f 40 C. The micro-

organisms d i d n o t t e n d t o regrow a t t h e zones o f

i n h i b i t i o n obLaincd v ~ i t h j a t r o r r h i z i n e a t very h i g h

t e m p e r a t u r e s as was t h e c a s e w i t h p a l m a t i n e and

columbamine. he combinat ion oi' t h e t h r e e a l k a l o i d s

showed a f a i r l y good t o l e r a n c e of t e rnpera tu re i n c r e a s e ,

i t s pe& o f a c t i v i t y be ing at 40'~. T h i s conforms t o

t h e p a t t e r n s of a c t i v i t y of t h e i n d i v i d u a l a l k a l o i d s .

~ h c a n t i m i c r o b i a l a c t i v i t y o f t h e t h r e e a l k a l o i d s

( ~ i g s . '7, 8, 9 and 10) were s i g n i f i c a n t l y a f f e c t e d by

pH v a r i a t i o n s .

A l l t h e t h r e c a l k a l o i d s (Palma t i n e , Columba~nine and

J a t r o . r r h i z i n e ) e x h i b i t e d t h e came pa.t t e r n of a c t i v i t y a.L

v a r y i n g pII, 'I'hcr-c was remnrkable low a c t i v i t y at t h e

ex t remes o f pR w i t h the a l k a l i n e e f i e c t on Lhe a c t i v i t y

b e i n g more profound, 'Jhe r e s u l t s show LhaL palrnaLine

had opt i rna l a c t i v i t y a t pIl and 6 , j a t r o r r h i z i n e a t 'I aiid

8 and :olumbanline at 6 and 7.

IIowever when x i x e d i n e q u a l volumes t h e combined b

a l k a l o i d s showed a rcmarknble s t a b i l i t y o v e r ; v a r y i n g

pII. T h i s m i x t u r e j u s t had a small peak a t pH v a l u e s

of '/ and 8. I t i s i i i t e r e s t i n g t o o b s e r v e t h a t even a t

t h e cxLrernes of pH t h a t j a t r o r r h i z i n e s t i l l a

c o m p a r a t i v e l y h i g h e r ac t i v i j - y , i n f ' a c t enough t o cornpare

v e r y f a v o u r a b l y vrith a m p i c i l l i n and t e t r a c y c l i n e under

similar c o n d i t i o n s . These r e s u l t s s u g g e s t that . t h e

b e s t e x t r a c t i o n p rocedure f o r t h e i s o l a L i o n of' a l k a l o i d s

f o r an t i m i c r o b i a l work s h o u l d n o t employ the ex t remes

o f pH. The e f f e c t o f pH on t h e a n t i m i c r o b i a l a c L i v i t y o f

t h e s e a l k a l o i d s d i f i e r e p a r t l y w i t h Lhat r e c o r d e d by

m e r u w a (1982) i n h i s work on p r i c a papaya f r u i t

e x t r a c t s ,

P a l m a t i n e , columbaniinc and j a t r o r r h i z i n e s h a r e a

cornuion c h a r a c t e r i s L i c w i t h r e g a r d t o t h e s t a b i l i t y of

t h e i r a n i i m i c r o b i a i activities when s t o r e d i n t h e open,

'l1heir. a n t i m i c r o b i a l a c t i v i - t i e s decreased p r o g r e s s i v e l y

w i th in a s h o r t p e r i o d o f t ime. I n t h i s r e g a r d t h e

mixtu1.e of tihe a l k a l o i d s confomed. e n - t i r e l y t o p a t t e r n .

I n t e rms o f open s t o r a ~ e , t h e a l k a l o i d s do no t co~npare

favourab ly wi th t he a n l i b i o t i c s , t e t r a c y c l i n e and

a m p i c i l l i n bo th o f which r.el;ain a lmost 907; of t h e i r

: a n t i m i c r o b i a l a c t i v i t i e s a f t e r 7 weeks of s l o r a g e . In

t h e same t ime p e r i o d , t h e a l k a l o i d s had a l l l o s t about

60y; of t h e i r r e s p e c t i v e a n t i m i c r o b i a l a c t i v i t i e s .

Due .to . t h i s l o s s of a c t i v i t y i t a p p e a r s r e a s o n a b l e t o 4

c a r r y ou t t h e e x t r a c t i o n p r o c e s s w i t h i n t h e minimal

p o s s i b l e t ime , each e x t r a c t i n t h e p r o c e s s be ing s t o r e d

covered a t about 40C.

After. i n c u b a t i o n i n a medium c o n t a i n i n g each of Lhe

a l k a l o i d s , t h e r e was d i s t o r t i o n of' t h e c e l l morphology o f

a l l the t e s t micro-organisms, t h e Gram-netat ive and G r a m -

p o s i t i v e a n e s became Gram-variable. The (jram-negative

bac t e r i a became swol len , ext remely e longa t ed and f i n a l l y

bncanie amorphous and clumped t o g e t h e r . The Gram-posi Live

ones a l s o swe l l ed up and t h e r e was a p r o g r e s s i v e

roughening of' t h e i r shapes u n t i l they a l l clumped

toge the r . G. a l b i c a n s behaved j u s t l i k e t h e @'am-

p o s i t i v e b a c t e r i a i n t h i s r ega rd . These f i n d i n g s

sugges t t h a t t h e s i t e of ' a c t i o n o f t h e s e a l k a l o i d s v;L::-,

(not, l A- j at t h e c e l l wall. The a l k a l o i d s appea r t o

e x e r t a n t i m i c r o b i a l a c t i v i t y by i n h i b i t i n € ; t h e growth o f

and by k i l l i n g t h e s e n s i t i v e micro-organisms, However,

i t . h a s been e s t a b l i s h e d t h a t t h e ma jo r mechanism o f

b i o l o g i c a l a c t i o n o f p r o t o b e r b e r i n e a l k a l o i d s such as

p a l m a t i n e , colu.mbairrir~e arid j a t r o r r h i z i n e i s t h e i r bind in^

t o t h e DNA o f t h e t a r g e t c e l l by in terca1a;Lion o f t h e i r

q u a t e r n a r y c a t i o n ( U l r i c h o r a & &., 1983). To r e s o l v e

t h i s a p p a r e n t c o n t r o v e r s y , lnore t h a n one t a r g e t o r g a n e l l e

have been p roposed a s s i t e s o f a c t i o n f o r some a 1 k a l o i d . s

(Mahler and Green, lgr/O; s i l v e r & &., 1970). A n t i m i c r o b i a l

a c t i o n by an a l k a l o i d can be i n i L i a t e d a t t h e c e l l - 6

menibrane arid c e l l - w a l l s w i t h t h e subsequen t l o s s of' t h e

normal membrane p e r m e a b i l i t y f u n c t i o n . Smal l m o l e c u l e s

( i o n s , amino a c i d s , s u g a r s ) would f i r s t l e a k o u t from

t h e c e l l s . Next, t h e l o s s o f small m o l e c u l e s would

g r e a t l y d e c r e a s e c e l l u l a r metabol i sm i n c l u d i n g ene rgy

metabo l i sm, a c t i v e t r a n s p o r t and n u c l e i c a c i d and p roLe in

s y n l h e s i s . T h i s would i n i t s e l f be l e t h a l s i n c e ene rgy

methabolisrn and s y n t h e t i c would be r e q u i r e d f o r r e c o v e r y .

w i t h membrane b a r r i e r d e s t r o y e d and t h e i o n i c s t r e n g t h

o f Lhe c e l l r educed , the a l k a l o i d s have bcen e s t a b l i s h e d

t o c o n v e r t DNA t o Complex 1 i n - v i t r o . It i~ t h i s f i r s t

complex t h a t l c e d s t o profound a l t e r a t i o n i n UIJA c o n f o r -

mat ion r e s u l t i n g i n a n unwinding o f c o v a l e n t l y c l o s e d

c i r c u l a r Dldk, similar i n geometry t o t n a t brought abouL

by mutagen ic i n t e r c a l a t i n g d y e s s u c h as e t h i d i u m

bromide (Warr ing , 19'70; Warring and C h i s h o l i n , 1972;

Mahler and P e r l a n , 1972).

CONCLUSION:

Yhat i s known i n a c t u a l f a c t i s t h a t some 01 t h e

c l a i m s by n a t i v e h e r b a l p r a c t i t i o n e r s a r c t r u e .

~ n L i m i c r o b i a l p r i n c i p l e s can be i s o l a t e d from m e d i c i n a l

p l a n t s and proved t o be at; e l f e c t i v e as t h e a n t i b i o t i c s

i n use. Chemotherapy which i s a common denominator f o r

t h e modern med ic ine and pharmacy a c t u a l l y s t a r t e d from

p l a n t medic ine . It t h e r e f o r e f o l l o w s t h a t i n t h i s p e r i o d

o f f r u s t r a t i o n when new and d r a n a t i c a l l y f a t a l d i s e r l s e s a r e

emerging, eniphasis s h o u l d a g a i n be l a i d i n t h e f o r e s t s

and juril;les. A greater ' . number o f a n t i b i o t i c s a r e

g e t t i n g i n c r e a s i n g l y i n a c t i v e t o t h e e v e r m u t a t i n g and

r e s i s t a n t m i c r o b r g a r i i m s s o a c o m p l e t e l y new f i e l d o f

a t i m i c r o b i a l s e a r c h s h o u l d be encouraged. From t h i s

p a r t i c u l a r work, t h r e e a l k a l o i d a l compounds have been

i d e n t i f i e d as posseks inf ; : a r i t i r n i c r o b i a l a c t i v i - L y , t h e i r

potency under p h y s i c a l and chemica l c o n d i t i o n s have been

de termined s o t h e i r i r i c o r p o r a t i o n i n t o c l i n i c a l u s e

shou ld r1o.L pose any s e r i o u s problem.

I am profound ly i n d e b t e d t o my S u p e r v i s o r , i r . A.C.

Ilmcruwa n o t j u s t f o r hic: gu idance th roughou t t h i s work,

b u t f o r n u l u r i n g my s e n s e o f independence t o a h i g h

l e v e l 01 m a t u r i l y .

My u n a l l o y e d g r a t i t u d e t o e s t o limy b ro the r1 I Ijr. John

Nwaiwu ( f o r m e r l y of ' t h e Uept. o f Pharmacognosy, U.li.ld. )

whose i d e a t h i s work was i n t h e f i r s t p l a c e anu f o r t h e

innumerab le s c i e n t i i ' i c ma t e r i a l s and l i t e r a t u r e s

he made a v a i l a b l e t o me. +

I owe a l o t t o L)rs Bato Okolo and C h a r l i e Okeke f o r

e v e r y t h i n g f r i e n d s s l a n d f o r .

I a m irniucnsely g r a t e l u l t o M r . E r i c Onyeogu who

b e a u t i f i e d t h i s work by t h e touch o f e x c e l l e n c e he

brought i n by way o f t h c d iagrams, and . t o Uro. A

( ~ r . E.C. Onyeneke) f o r i n t r o d u c i n g ille t o p r a c t i c a l

chromalography.

How can I f o r g e t 'the u n l ' a i l i n g s u p p o r t from my f a m i l y ;

Mama, who s t a r t e d i t a l l , illy b r o t h e r s Chi j i o k e and Chulrwuma

and ~ o s t e s p e c i a l l y Oluchukau who cor i l inucd t o s t a y

c l o s e , f i l i a l l y lny s i s t e r s c h i n y e l u (and h e r f a m i l y ) ,

Ogo, I l j ido and o u r Ndid i ; t i ~ c l r t a u n t i n g s and

u n f l i n c h i n ~ b e l i e f i n me made i t ~ j o ~ s i b l c .

I want t o thank my s p e c i a l s i s t e r O b i i (and h c r f a m i l y )

f o r Lhc l r moral and m a t e r i a l s u p p o r t th roughou t t h e

d u r a t i o n of t h i s work.

Yhey made i t l i g h t e r and s t o o d by me and Llicy a r e

f r i e n d s ; Ccebce, J u n i o r , Chuddie, I p h i e , Ben j i e , A c h i l e ,

c h i - c h i , Aus t in , Nkechi, Bosah e t c .

A l o t o f p o s i L i v e i u s p i r a t i o n calne fruin my c o l l e a g u e s

and s i s t e r s , Ebe le , Barbara and P r i s c i l l a .

I aJ:;c., g i ~ l i ~ l ~ ~ : . l e d g e t h e a s s i s t a n c e o f t h e t e c h n i c a l

s t a f f o f t h e Depts. of lv l ic robiology, B iochemis t ry

and Pharmacoposy a t U . N . N . and t h o s e a t t h c UNTH

who came t o my r e s c u e .

l ' i n a l l y , I reinember w i t h i n e s t i m a b l e b l i s s t h e b

l o v e o f t h e s e l i t t l e c h i l d r e n , chid inma, Derek,

I f e a n y i , chiamaka and Chioma.

V o l a t i l e o i l s a c t i v e a ~ n i n s t t h e r e s i s t a n t

gram neka Live o rgan i sms

J 'ourt h O A U / ~ ' ~ ~ < C ~ y i n ~ o s i u r n on A f r i c a n

Medici.nal P l a n t s and w a d i t i o n a l Ned-j.c:inc

flbu ja - K i g e r i a .

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The i s o l a t i o n of' q u a t e r n a r y a l k a l o i d s f'rom t h e

l e a v e s 01 ~ i o s c o r e o u h y l l u m cumminsi i ( ~ t a p f . )

Y l a n t a Diedica 217 - 221.

1 A , 1 1 A A , V U A (1986) :

Sn Libac L e r i a l a c L i v i t y o f j u l i f l o r i n e i s o l a t cd,

f'rom P r o s o p i s . i u l i f l o r q

P l a n t a Medica & 285 - 288.

P r e l i m i n a r y b i o l o g i c a l and phy t;ochemical

i n v e s t i g a t i o n 01 two N i g e r i a n Medic ina l p l a n t s

l n t . cT. Crudc Drw; Res. 2 0 ( 2 1 61 - 70.

~LL-LHA~+~~~~A, 11. , U~?AKLL, S. , 14'LY N14 , b. L. , ~ l l l ' X l l ~ l ~ , L. A. , Plilik, Y.li., l i A U , t i . ~ . H . , ~ 1 ~ 1 1 ' ~ ~ 1 ~ , A., SKAk'Lli, J.L.,

vui;OCfLU, 'l., NU, 1 , (1981 ) :

A n t i m i c r o b i a l a g e n t s i'rom higher . p l a n t s :

A n t i m i c r o b i a l a g e n t s from Peganum harmnla ~ e e d s .

" J o u r n a l o f N a t u r a l I J roduc t s 44 (6) 745 - 747

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of Xylopia ae th iopia (~nnonaceae)

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Phyochemistry - I 9 1564 - 1568

DWUMA-AADU, D. , AYIM, T.S.K., WITHERS, SeF., AGYEMANG, N.0.v

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f u n i f e r a ( ~ e n i s ~ e r r n a c e a e )

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DWUPIA-BADU, D., A Y I M , J.S.K., TACKZE, . A.N., KNAPP, J.E.9

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Addit ional a l k a l o i d s of T r i c l i s i a pa tens and

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medicinal p l a n t s

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A n t i b a c t e r i a l substance from Carica papaya f r u i t

e x t r a c t .

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134- Table 1

Geographical ~ i s t r i b u t i o n o f Af r i c an Menisperlnaceae

s p e c i e s ( a c c o r d i n g t o Troupin, 1962)

PLANTS

1 ' 1 1 I TRICLISEEAL

1 , p n i s o c v c l a b l e ~ h a r o s e ~ a l a

U i e l s

2 A. cyamosa Troupin

3. 8. J o l l y a n & ( p i e r r e ) D i e l s

4. Be i rmae t i a c a b i n d e n s i s

l ' roupin

. LWinetrum ao i cu l a tum

t r o u p i n

6. J. C a ~ i t u l i f l o r u m ( ~ i e l s )

Troupin

7. g. Cordii 'ol ium Magenot de

Plioge

E. guneatuin Keay

&, dc lagoense D i e l s

&, exe l l i anum ~ r o u p i n

&. fe r rug ineum Keay

EI;. glabrum u i e l s ex

Trcupin

& scai idens Plani;enot e t

Ptiege

undulatum I-liern

&. J i l l o s u m ( E x e l l )

Troupin

COUNTRIES

Zimbav~e ,PIozambiyue, Z a i r e

Z a i r e

b Tanzan ia

Cameroun

Ivory Coast

Ghana f

Mozambique, South A f r i c a

Rwanda, Uganda

S i e r r a Leone, L i b e r i a

Carneroun

L i b e r i a , Ghana, I vo ry Coast

Z a i r e , Angola

Angola, Z a i r e

Table 1 contd .

16. S y n c l i s i a s c a b r i d a Pl iers

1 '7. Syrrhonenia f a s i c u l a t u m

18. A. h e x a s t amiiieum keay

19. s. w e l w i t c h i i (Hie rn )

U i e l s

21. 2. d i e l s i a n a Hutch e t

llal z

- 22. . d i n k l a g e i Engl.

23. 2. e a l a e n s i s Troupin

24, 2, f u n i f e r a (! t iers) O l i v

25. x. g a b o n e n s i s Troupin

26, 2. g o s s i o e i l e n E x e l l

27. 2. k e n y e n s i s Troupin

28. 2. k l a i n e a n a ( P i e r r e )

D i e l s

29. 2 , l a t i f o l i a ~ r o u p i n

3 0 2. l a u x n t i i De Wild

3 1 - T. ----A l e h m b a c h i i -- Kngl.

32. z. l e o n a r d i i ~ r o u p i n

N i g e r i a , ~ a r n e r o u n , Gabon, C e n t r a l A f r i c a n Repub l ic , Z a i r e , Angola

Cameroun, Z a i r e , Gabon

Ghana, S i e r r a Leone

c e n t r a l A f r i c a n Repub l ic ,

Angola

Angola, c e n t r a l A f r i c a p

Republ ic

\ S i e r r a Leone, L i b e r i a , Ghana, I v o r y Coast

L i b e r i a , I v o r y Coast

Z a i r e

Ghana, N i ~ e r i a , C e n t r a l

A f r i c a n Hepubl ic , Angola,

Z a i r e , liwanda, Kenya,

E t h i o p i a , Tanzania ,

Zimbabwe, Mozambique,

Angola

*Gabon

Uganda

Z a i r e

Cameroun, Z a i r e

Z a i r e

Table 1 contd.

x. l e o n e n s i s D i e l s

2. l o u s i i Troupin

ma.crophylla ( p i e r r e )

D ie l s

T. n i g e r i c a Troupin - 2. odora t a U1gl.

2. o v a l i s D i e l s

r r i c l i s i a a n g u s t i f o l a

D ie l s

T. c o r i a c e a O l i v . - T. d i c t y o p h y l l a D i e l s -

2. l a n c e o l a t a Troupin

T. l o u s i i ~ r o u p i n

2. rnacrophylla Oliv.

2. p a t e n s Ol iv .

47. 2. s a c l e u x i i ( P i e r r e ) D i e l s

48. x. subcorda tq Ol iv .

Guinea, S i e r r a Leon,

Ivory Coast

S i e r r a Leone, L i b e r i a ,

Ghana, Za i re

Nige r i a

Cameroun, Gabon

Angola

E q u a t o r i a l Guinea

Nige r i a , Cameroun, Cen t r a l

Afr ican Republ ic , Gabon

Za i r e

Cameroun, S i e r r a Leone,

quat to rial Guinea

Senegal , Guinea, S i e r r a

Leone, Ivory Coast ,

C e n t r a l Afr ican Hepublic,

Tanzania, 5 ' .Moz,ambique

cameroun, c e n t r a l Afr ican

~ e p u b l i c , ~ a b o n , ~ n @ a

PLANTS

Tribe 2: PENATHEAE

Penianthus l o n g i f o l i u s

P. zenker i ( ~ n g l . ) Die l s 9

Sphenocentrum jollyanurn

Tribe 3 TINOSPOHEAE --

Chasmmthera dependens

Hochst

C. w e l n i r t s c h i i Troupin 9

Dialytheca &osswei ler i

Exe l l

Dioscoreophyllum cumminsii

Die l s

D. f iosswei ler i Exe l l - D. v e t k e n s i i Troupin

COUNTRIES

Nigeria , Camcroun,

Cent ra l Afr ican Republic,

Gabon, Angola, Za i re

S i e r r a Leone, L i b e r i a ,

Ivory Coast, Ghana,

Niger ia , Cameroun

Ivory Coast, Nigeria ,

Ghana, Benin

+ S i e r r a Leone, Zai re , Ghana,

Togo, Chad, Sudan, Somalia,

Niger ia , Uganda

Cameroun, Gabon, Cent ra l

Afr ican Republic, Angola,

Za i re

Angola

Guinea, S i e r r a Leone,

L i b e r i a , Congo, Nigeria ,

Togo, Sudan, Cameroun

Zai re , Angola

S i e r r a Leone, L i b e r i a , b

I v o r y Coast, Niger ia ,

Zimbawe

138. yable 1 Contd.

PLANTS I COUNTRIES

58. Jateorhiza macrantha

(Hook f .) Exell

59. - J. palmata (Cam) Miers

60. Kolobopetalum auriculatum

6 . &. cheva l ie r i Troupin

62. K. leonense Hutch c t Dalz - 63. K. Ovatum s tapf

64, Leptoterantha mayumbensis

Exel l

65. Platyt inospora buchholzii

Die ls

66. Rhigiocarya p e l t a t a Miege

67. R. racemifera Miers -

68, Sarcolophium suberosum

, Troupin

69, Syntriandrim p r c u s i i Engl,

70, Tinospora bakis Miers

71. T. c a f f r a (Miers)Troupin

Gabon, Congo, Nigeria,

Ghana, Kenya, Tanzania,

Mozambique

Ivory Coast, Ghana,Nigeria

Liber ia , Nigeria, Central

African Republic, Zaire

S i e r r a Leone, Liber ia ,

Liber ia , Ivory Coast, ,Togo,

Central African Republic,

Gabon, Angola

Camcroun

Ivory Coast

S i e r r a Leone, Liber ia ,

Ivory Coast, Nigeria, Benin

Cameroun, Gabon

Nigeria, Central African

Republic, Gabon, Zaire

Mauritania, Senegal, Sudan,

Chad, Nigeria, Somalie,

Kenya, Tanzania

Sudan, Zaire, Rwanda,Uganda,

Kenya, Tanzania, Zimbabwe,

Angola

able 1 Contd,

PLANTS

72. - T, fragosa verdoorn &

Troupin T. mossanbicensis Engl. 73. -

74, - T. oblongifolia (Engl.) Troupin

75. - T. penninervifolia Troupin 76, - T. tenera Miers

Tribe 4 MENISPERMEAE

Cissampelos ellenbeckii

C. friesonum Diels - C, hirta klotzsch - C. insignis Alston - C, macrosepala Dfels -

C, nigrescens Df els - C. owariensis Beauv, ex -

84. C. pareira I.

85. C. rigidifolia (~n~l.)

86. 2. tenuipes Engl. 87. s. tonilosa F.geyex

Ha~ve y

88. C. tnincata Engl. -

COUNTRIES

Namibia, South Africa

Tanzania, Mozambique

Kenya, Tanzania

Tanzania, Zimbabwe, Mozam-

Ethiopia

Mozambique

Tanzania

Cameroun, Nigeria, Central

African Republic, Zaire,

Senegql, Chad, Togo, Benin, Ethiopia, Sudan, Ghana

Tanzania .

Sierra Leone, Nigeria,

Ghana, Togo, Equatorial Guinea, G,bon, Congo,

Rwanda, Angola, Zaire

Zimbabwe, Mozambique

Central African Republic, Zaire, Sudan

Zimbabwe, South Africa

Tanzania

Table 1 Contd.

PLANTS

Cocculus balfouri schweinf

C. hirsutus (L) Diels -

C. pendulus Diels -

Limaciopsis loangensis

Stephania abyssinica Walp.

S. cyanantha Welwex Hiern -

S. dinklagei (~ngl.) Diels -

S. laetificata (~iers) -

S. mildbraedii Diels -

COUNTRIES

Namibia, Angola

Sudan, Kenya, Tanzania,

Mozambique, Zimbabwe

Guinea, Mauritania, Algeria,

Nigeria, Somalie, Cape

Verdc, Central African

Republic, Nigeria, Congo, Zaire

Gabon, Congo, Zaire t

Ethiopia, Guinea, Zaire,

Equatorial Guinea, Sudan,

Angola, Somalie, Kenya

Equatorial Guinea, Zaire,

Rwanda, Angola, Kenya,

Tanzania

Guinea, Sierra Leone,

Liberia, Nigeria, Ghana,

Togo, Cameroun, Gabon

Nigeria, Cameroun, Central

,African Republic, Gabon,

Zaire, Angola, Equatorial

Guinea

Tablc 2: Alkaloids From Nigerian Menispermaceae

Species

PLANT SPECIES - --

I, Chasmanthera depen-

dens -

2, Cissam e l o s mucro- xr-- 7

CONSTITUENTS

Columbamine

Magnoflorine

Pseudo columbamine

Palmatine

Tetrahydro- palmatine

Li r iodcnine

Lyscicarnine

Oxoglaucine

Glaucine

Ananaine

Nornuci f e r i n e

Norglaucine

0,O-dimethyl- corytuber ine

Govanine

Coreximine

Risnorargemo- ndne

P a l l i d i n c .>

Berberine

Isochondode- na r ine

PLANT PART

REFERENCES

Fer re i ra 1965 Vecchict t i e t a1 - * '

(1981)

Table 2 contd . 142.

REFERENCES

Vecch ie t t i e t a l , , (1981)

PLANT PART

Roots tt

PLANT SPECIES

3. Cocculus gendulus

4. Dioscoreophyllurn Cumminsii

5. Kolobopetalum aurlculatum

6. Penianthus zenker i

7. Rhigiocarya - racemifera

Ikram - e t * # a 1 (1981)

Gupta - e t * ' a 1 (1970)

J o s h i - c t a 1 * (1974)

CONSTITUENTS

Oxyacanthine

Palrnatine

S i n a c t i n e

Coclaurinc

Menisarine

Cocsuline

Penduline

Cocsoline

Columbamine

Magnoflorine

N-Methylc ory- d i n e

0-methylfla- v inanth ine

Magnoflorine

Palmatine

Li r iodeninc ,

Magnoflorine

Menisperine

0-methylfla- v inanth ine

Dwurna-Badu e t a 1 (1980) - -* '

Duah - e t - * ' a 1 (1981)

Tackie e t a l . , ( 1974: I t

PLANT SPECIES

9. Stephania d i n k l a p i

10. .Synclisia scabr ida

Sync l i s ia scabrida

11 . Ti l iacora fun i f e r a

PLANT CONSTITUENTS I PART

Palmatine Roots

Palmatine

Corydine I

R,S,L tt

Dicentr ine

Isocorydine

Roemerine I

Roots 11

Stephalgine 1 "

Norcorydine

dinklageine

Stepharine I

Stem 11

Steporphine I

N-mc"lylglau- t i n e I N-methylc ory- dine

Cycleanine I R & S

, Cycleanine-N- 1 oxide

Nor-cycleanine

Cocsuline

Cocsoline

I Nort i l iacor ine-

1 Funiferine

1 Ti l iacor ine

REFERENCE

Roots 11

Tackie e t g. , ( 79747-

Okarter ( 1976) 11

Ohir i (1983) 11

Tack~tg~,-t., Ohir i (1983)

Tackie e t a 1 ' (1974)-*'

Ohiri e t a1.,(1983)

Table 2 c o n t d .

PLANT SPECIES I CONSTITUENTS Nortiliaco- rine-A

12. Tinospora bakis

13. Triclisia dictyo- phNlla

14. Triclisia Subcor- data -

Funif erine-N- oxide

I I so-tetrandrine

Tiliafunimine

N,Nt-dimethyl- funiferine

1 Oblongine

I Berberine Phacnithinc N,Nt-dimethy- lphaenthine

I 0-methylmos- chatoline

Cocsulinc

I sotetrandrinc Stebisimine

Isogilletine- N-oxide

Obamegine

Tridictyo- phyclinc

ILANT 'ART

,caves

R , S&L tt

R & S Stems

Leaves

R , S&L

REFERENCE

Iwurna-Badu et al. -- ( 1977)

Tackie et al., ( I 980) t

Beauguesne, I 938

Thornber , 1970

Ohiri (1983)

Owusu et g., (19m)

Ohiri (1983)

Spiff et g., (1981T

Tackie et g., ( 19737-

T a b l e 2 contd .

- - . .

PLANT SPECIES CONSTITUENTS

Fangchinoline

Cocsuline

Tetrandrine

Magnoflorine

Palmatine

KEY - R = Roots; S

PLANT PART

Unspe c i f i e d

REFERENCES

Ohi r i , (1983)

'ackie - e t -* a 1 9 (1973)

= Stem; L = Leaves

1&6$ APPEMYIX

Table 10: The E f f e c t of T,mperature on t h e Anti-

Microbia l A c t i v i t y of T e t r a c y c l i n e

( 2mg/ml) Temperature oC Diameter o f zone of i n h i b i t i o n (mml

40 3 1 50 34 60 32

70 30 80 28.5

90 22 t

100 20

The t e s t microorganism used he re was - E. - c o l i

Table 11: The E f f e c t o f Temperature on t h e Anti-

Microbia l A c t i v i t y of Ampic i l l in

( 2mdml) Temperature oC Diameter of zone of i n h i b i t i o n (mml

The t e s t micro-organism used he re was - E. - c o l i

Table 12: The Effect of T,mperature on the

Antimicrobial Activity of Palmatine

( 20mg/ml) Temperature oC Diameter of zone of inhibition (mm)

The test micro-organism used fhere was E. coli - - Table 13: The Effect of Temperature on the Anti-

Microbial Activity of the Combined

Purified Alkaloids

Temperature oC Diameter of zone of inhibition (mm) 10 18.5 20 19

The test micro-organism used here was I E. - coli

Table 14: The Effect of Temperature on the Anti- Microbial Activity of Jatrorrhizine

( 2mg/ml)

Temperature oC Diameter of zone of inhibition (mm)

The test micro-organism used here was Eo coli -- Table 15: The Effect of Temperature on the Anti-

microbial Activity of Columbamine

Temperature oC

Diameter of zone of inhibition (mm)

The test micro-organism used here was - E. - coli

Table 16: 149*

The Effect of pH on the Antimicrobial

Activity of Tetracycline (2mg/ml)

Table 17: The Effect of pH on the Antimicrobial

Activity of Ampicillin (2mg/ml)

Diameter of zone of inhibition (mm) 23

i: 25 26.5

5 27 + 6 29

9 30 10 22 11 22 12 22 13 14 ; The test micro-organism used here was - E. - coli

Table 18: 150.

The Effect of pH on the Antimicrobial

Activity of Palmatine (20mg/ml)

Diameter of zone of inhibition (mm) 1 '/

The test micro-organigram here was - E. - coli

Table 19: The Effect of pH on the Antimicrobial Activity of the Combined Purified - - -

Alkaloids

14 15 16.5

17.5 17.5

18.5 18.5

10.0 t

Table 20: The E f f e c t of pH on t h e Antimicrobial

A c t i v i t y of J a t r o r r h i z i n e (2mg/ml)

Diameter of zone of i n h i b i t i o n (mm) 21

30 30 28

co The t e s t micro-organ gram here w a s g. li

Table 21: The E f f e c t of pH on t h e Antimicrobial A c t i v i t y of Columbamine (20mg/ml)

9 Diameter o f zone of i n h i b i t i o n (mm) It3

-- - -

The t e s t micro-organism herebwas E. - c o l i

152. Table 22: The Effect of Open Storage on the

Antimicrobial Activity of Tetra-

cycline ( 2. 5mg/ml) Time (Weeks) Diameter of zone if inhibition

0 45.0 I 43.0 2 42.0

3 42.0

4 42.0 5 41 .O 6 40.0 7 40,O

The test micro-organism here was - E. - coli

Table 23: The Effect of Onen Storage on the Antimicrobial Activity of Ampicillin

( 2.5mg/ml) Diameter of zone of inhibition (mm)

T a U e 24: he ~ f f e c t of Open s t o r a g e on t h e g n t i r n i c r o b i a l ~ c t i v i t y o f Pa lmat ine

C25mgbl)

p i ame te r o f zone of i n h i b i t i o n (mm)

1 a 15 1 4. 13

The t e s t micro-organism he re was E;. c o l i

able 25: The ~ f f e c t of Open s t o r a g e on t h e p t i m i c r o b i a l A c t i v i t y of t h e

Combined P u r i f i e d A l k a l o i d s yime (weeks) Diameter of zone o f i n h i b i t i o n (mm)

0 24 00

1 23 50

2 23.00

3 20.00

4 1g.00

5 1.8.50

6 18.00

7 17oOO -

he t e s t micro-organism h e r e was 8. c o l i

154. Table 26: he E f f e c t of Open s t o r a g e on t h e

~ n t i r n i c r o b i a l ~ c t i v i t ~ of J a t r o -

r r h i z i n e (2.5mg/ml)

Time (Weeks 2 Biameter of zone o f i n h i b i t i o n (mm)

0 45 1 43 2 40

3 34 4 27

5 23 6 20

7 17.5

4 The t e s t micro-organism used he re was a. c o l i

?'able 27: The E f f e c t of Open s to rage on t h e

~ n t i m i c r o b i a l A c t i v i t y of columbamine

(25mg/ml) rime (weeks 1 Diameter of zone o f i n h i b i t i o n (mm)

0 26,O

1 24.5

2 22.0

3 19.0

4 16,o 5 14.0 6 12.0

7 10.0

FIG. 20 - Chromatogram of stem extracts.

APPEND1 X -- - . .

Tet. A Quat. B Mixt.C Pal. Jat. Col. THP

FIG. 21 - Chromatogram of root extracts.

Table 28.

I n t e r p r e t a t i o n ol' the Chromatogram of t h e stein E x t r a c t s ,

E thy l Aceta te

45 Isopropanol Ammonia

35 15 CONS'I'I'I'UKN'I' NU113NH H r f Values

T e r t i a r y 8:

Yellow

Q.uat e rna ry B:

Yellow

Mixture c: Brownish

0 n1 p our12 :

Palmat ine (Yellow) 6

J a t r o r r h i z i n e (Yellow-BrownL) 7

C~lumbamin~e (Brown) 8

Tetrahydropalmat ine 9 38.0

Table 29.

I n t e r p r e t a t i o n o f t h e Chromatogram of t h e Root E x t r a c t s .

E thy l Ace t a t e

45 I sopropanol Ammonia

T e r t i a r y g :

Brown.

Yellow

Yellow 3 53.0

0.uat e rna ry 13:

Yellow

Yellow Brown 4 45.0

Mix-ture G:

Brownish Green

Compounds:

Pa lmat ine (Yellow)

J a t r o r r h i z i n e (Yellow-Brown ) 7

Columbamine (Brown) 8

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