University of Nigeria...University of Nigeria Research Publications Author OKORO, Azubuike Gabriel...
Transcript of University of Nigeria...University of Nigeria Research Publications Author OKORO, Azubuike Gabriel...
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University of Nigeria
Research Publications
OKORO, Azubuike Gabriel
Aut
hor
PG/M.Sc/85/3227
Title
Extraction, Purification and Antimicrobial Activities of Quaternary Alkaloids of the Plant Sphenocentrum Jollyanum Pierre
Facu
lty
Biological Sciences
Dep
artm
ent
Microbiology
Dat
e August, 1989
Sign
atur
e
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~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
BY:
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
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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
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iii
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, ' '
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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
and
t h e progres:, o f t h e e n t i r e f ami ly . 8
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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
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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.
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v i i
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.
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viii.
TABLES OF CONTENTS
Certification Pages
iii
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
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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)
Pages
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
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X.
Pages
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
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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
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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
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
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
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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
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xiv.
Pages
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
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Pages
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 .
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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
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2.
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
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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 ,
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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 $%rnily Menispermaceae and on
p u r i f i c a t i o n g i v e s a ye l low - brown co lou r . Its
mo lecu l a r weight i s 338 ( s e e s t r u c t u r a l formulae on
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5.
I n t h i s e r a o f i n c r e a s i n g r e s i s t a n c e o f micro-
o rgan i sms t o t h e a v a i l a b l e a n t i b i o t i c s and when a l o t
o f a t t e n t i o n i s b e i n g f o c u ~ s e d on a l t e r n a t i v e medic ine ,
i t i s o n l y n e c e s s a r y t h a t ac t iv .e i n v e s t i g a t i o n be
c a r r i e d o u t i n t o h e r b a l medic ine w i t h a view t o
t a p p i n g what eve r u s e f u l m a t e r i a l s t h a t cou ld be ob t a ined .
Sphenocentrum jollyanurn i s a p l a n t t h a t h a s been
e x t e n s i v e l y employed i n f o l k l o r i c medic ine i n t h e
t r e a tmen t o f v a r i o u s k i n d s o f a i l m e n t s . . jol lyanurn
c a l l e d I1oJ i -enyir l i n Igbo (a language o f t h e South ~ a s t *
peop l e o f N i g e r i a ) i s r e p u t e d t o c u r e a number o f
m i c r o b i a l i n f e c t i o n s among i t s o t h e r numerous
p o t e n t i a l i t i e s .
T h i s r e x e a r c h w i l l t h e r e f o r e a t t e m p t t o e x t r a c t ,
i s o l a t e and i d e n t i f y t h e a c t i v e c o n s t i t u e n t ( s ) t h a t a r e
r e s p o n s i b l e f o r i t s a n t i m i c r o b i a l a c t i o n . viith t h e
c h e d s t r y o f t h e s e compounds known and t h e i r o t h e r
p r o p e r t i e s de te rmined , t h e p o s s i b i l i t y o f i n c o r p o r a t i n g
them i n t o c l i n i c a l u s e might become more r e a l i s t i c .
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LITEHATUlU REVIEW 6.
The ~ a r n i l u Menis~ermaceae:
The term uMenispermaceaett was coined from two Greek
words - mene (moon) and sperma (seed) because t h e
crescent. moon$ 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
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7.
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
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8.
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
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9.
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 .
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10,
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
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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
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12.
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
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13-
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
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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) .
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15.
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.
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16.
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
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17.
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
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LO*
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)
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H&o Q
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 .
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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
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21.
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 .
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PALMATI NE
COLUMBAMINE
I4
FIG. 1 - Chemical structures of Protoberberine Alkaloids.
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23
~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
raxe.
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
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24
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
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25.
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
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26.
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.
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27.
( 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
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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 .
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29
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).
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30
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
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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
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32
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
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33
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
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34
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 , $aph.giureus, a. c o l i , s a l m o n e l l a g a l l i n a r u m .
K l e b s i e l l a ~ n e u m o n i a e and I;. a l b i c a n s . Aladesanmi d.,
(1986) c a r r i e d o u t phytochemical s t u d i e s on two p l a n t s
normal ly used i n f o l k l o r e medic ine i n N i g e r i a ; Marsdenia
l a t i f o l i a and P l e i a s c e r a s b a r t e r i and went f u r t h e r t o
show t h a t t h e a n t i b a c t e r i a l a c t i v i t y u s u a l l y a s c r i b e d t o
t h e p l a n t s i s due t o t h e p r e sence i n them o f a n t i b a c t e r i a l
a l k a l o i d s , f l a v o n o i d s , s apon in s , t a n i n s and s t e r o l s .
F lavonoids have a l s o been r e p o r t e d t o p o s s e s s
a n t i m i c r o b i a l a c t i v i t y . lCiski & &. , (1983) e s t a b l i s h e d
a n t i - b a c t e r i a l ac1; iv i ty f o r t h e f l av ,ono id c i r c i m a r i t i n
i s o l a t e d from t h e l e a v e s o f S a l v i a p a l e s t i n a (Lah i a t aceae ) .
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35. C i r s i m a r i t i n showed a high a c t i v i t y a g a i n s t s t a p h au reus ,
s t a p h epidernidis , &. c o l i , Kleb. pneutnonia, P ro t eus vu1r;aris
and &. aeruginosa , A similar a c t i v i t y was r e p o r t e d by
M i t s che r e t a le , (1 980) f o r t h e i s o f l a v o n o i d s h i s p a g l a b r i d i n
A, h i s p a g l a b r i d i n 13, g l a b r i d i n and g l a b r o l i s o l a t e d from
Glycyr rh iza c l a b r n var t y ~ i c a . Apar't from i ' lavonoids,
ano ther c l a s s o f c o n s b i L u c n t ~ reporbed Lo e x h i b i t a n t i -
mic rob ia l a c t i v i t y a r e t h e e s s e n t i a l o i l s .
Janssen & dm, ( 1 987) gave an extensiv4e l i b e r a t u r e review
o f e s s e n t i a l o i l s which posses s a n t i m i c r o b i a l a c t i v i t y and
i n a r e s e a r c h c a r r i e d ouL by Onaviunmi and Ogunlana (198$)
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
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36.
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
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37
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.
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38
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
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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
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40.
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).
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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
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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
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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 .
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( i )
( i i )
( i i i )
44.
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
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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
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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%)
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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
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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
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49.
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 ) .
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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.
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51
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 .
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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 .
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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
ash
lzS0L
( extract d i t h CH C13
evaporate to evaporate dryness
I
alkaloidal mixture stage
L B B (quaternary
alkaloids 1
dissolve in MeOH
dissolve in MeOH
stage 3A
I
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
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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
20 1
2. Benzene 20
Chloroform
Benzene
35
Methanol
~ t h y l a c e t a t e
45
Methanol
8
Chloroform
Chloro form
85
Methanol
4
Acetone
4
Chloroform
50
Methanol
10
Acet ic ~ c i d
1
Isopropanol
35
Acet ic a c i d
1
Ammonia
1
Net; hanol
1
14et hanol
9
Ace t ic a c i d
1
Diethylamine
1
Ammonia
Water
Water
Die thylamine
1
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55.
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 -
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56
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
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57.
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 .
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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
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59.
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.
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' 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
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61.
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
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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
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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
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64.
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 .
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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
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~ 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 - -
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6'7.
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.
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- 68.
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
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69.
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 ' ~
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'70.
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
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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,
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-
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
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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
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'14.
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
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75.
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 .
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'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
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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
1
2
3
Quatenary I3
3a
4
Mixture C
1 a
Un iden t i f i ed Compound
Un iden t i f i ed Compound
Palmat ine
Palmat ine
J a t r o r r h i z i n e
Un iden t i f i ed Compound
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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
1
L
Q u a t e r n a r y I3
3
4
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
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79.
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)
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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
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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
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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
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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 - --
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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 - - - . .
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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
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-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 ' -- --
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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
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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 . -
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- 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 . - --
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-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 . - ---
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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, - -
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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 ,
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Time ( in weeks) - Ampicillin, - Tetracycline, - Jatrorrhizine.
FIG. 13 - The effect of open storage on the
antimicrobial activity of Jatrorrhizine on 5. col i .
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- 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 . - -
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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 . - -
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-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
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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 . - -
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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.
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Time (hou Morphology Descript ion
Normal
gram - positive
SwoIlen
gram- var iable
Rough
appearance
gram -variable
Rough
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.
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I
- - , .-- -- .'. - - . : - ..+ d - - D - -
Plate 1: Zone of inhibition at the Optimum temperature L*. of Antimicrobial Activity for Palmatine.
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' . 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
3
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4 t h Week of Open. Alkaloids .
the 4th . . . . . .. S%m,
Week
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Pla t e 9 : Zone of Inhibitiorrhafter t h e 4 th . Week of Open Storage ~f Tetracycline.
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DISCUSALON HNU CONCLUSION
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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
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1106
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
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107.
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 -
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108.
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)
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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) .
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- -- -
110.
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 .
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~ 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
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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
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113.
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
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114.
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.
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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
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116.
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.
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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 .
1 A . , 1 U l J . . , H 1 L l J i J . . ( 1978) :
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
B e r b e r i n e s u l p h a t e : A n t i m i c r o b i a l a c t i v i t y ,
b i o a s s a y and mode ol' a c t i o n .
Can. J. 14icrobiol . fi 1067 1 1070
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ANTOUN, M.D., TAHA, O.M.A., f1981):
Studies on Sudanese Medicinal plants 11:
Evaluation of an extract of Lupinus termis seeds in Chronic eczema Journal of N,tural Products &(2) 179 - 183
ANTOUN, M.D., ELKHAWAD, A.O., TAHA, O.M.A., (1977) : Studies on Sudanese Medicinal Plants I: The effect of an extract of Lupinus termis seeds in Chronic eczema
Journal of N3tural Products 40(4) 337 - 339
AYIM, J.S., DWUMATBADU, D., FIAGBE, N.Y., ATEYA, A.M., SLATKIN, D.J., KWAPP, J.E., SPIFF, P.L., (1977)
Constituents of West African Medicinal Plants XXI: b
Tiliafunlmine, a new iminobisbenzyliso-quinoline alkaloid from Tiliacora funifera Lloydia 40 561 - 566
AYNECHI , Y. , SORMAGHI , M .H. , ( 1980) : Screening of Iranian plants for antimicrobial activity Acta Pharm. Suec, 17 32: - 346 - -
BEAUGUESNE L . , ( 1 983) : Some medicinal Menispermaceae of the species Tinospora and Cocculus
Bull. S,i. Pharmacol & 7 - 11
Thro' Chem. Abstr. 32: 3089
BEDOWS , E . , HATFIELD, G.M. , ( 1982) : An investigation of the antiviral activity of Podophyllum pellatum Journal of Natural Products 45(1) 725 - 729
>
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119. BENJAMIN, T.V., (1979):
Inves t iga t ion of Borreria v e r t i c u l l a t a an a n t i eczematic p l an t of Nigeria.
I n t . J. Crude Drug. Rcs. 17(4) 135 - 136
BHUTAN1 , K .KO, SHARMA, G.L., ALI , M., ( 1987) : Plant-based antiamoe'oic drugs P a r t I: Anti-
amoebic a c t i v i t y of phcnanthrol indolizidine
a lka lo ids
Planta Medica 53(6) 532 - 536
BOAKYE-YIADOM, K. (1979):
Antimicrobial p roper t i es of s ome West Afric
Medicinal P l an t s 11: Antimicrobial a c t i v i t y of aqueous e x t r a c t s of Cryptolepis Sanquinolenta
b I n t . J. Crude Drug Res. 17(2) 78 - 80
BOAKYE-YIADOM, K . , FIAGBE, N.Y., AYIM, J.S.K., (1977):
Antimicrobial p roper t i es of some West African
Medicinal p l an t s I V : Entimicrobial a c t i v i t y of
xylopic ac id and other cons t i tuen t s of t h e f r u i t s
of Xylopia ae th iopia (~nnonaceae)
Lloydia 40(6) 543 - 545
CHAN, E.L., O'NEILL, M . J . , PHILLIPSON, J .D. , WARHURST, D.C.,
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PlantaMedica 2 105 - 107 - CHANDRA, K . , PANDEY, H.C., (1983):
Col lec t ion of p l an t s around Agora-Dodital i n
Uttarkashi D i s t r i c t of U t t a r Pradesh
with medicinal values and Folklore claims
I n t . J. Crude Drug Res. 21 (1) 21 - 28
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CLAUS, E.P.,.TYLER, V.E., BRADY, L.R. (1970):
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DWIA-BADU, D. , ATEYA, A.M., SLATKIN, D.J .9 KNAPP, J.E.,
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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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Const i tuents of West African medicinal p l a n t s X X V I I :
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DtVUMA-RADU, R e , OKARTER, T., TACKIE, A.N., LOPEZ, J.,
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Fun i fc r ine N-oxide, 2 q e w a l k a l o i d from T i l i a c o r a
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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Comparative ant igonorrhoea a c t i v i t i e s of s i x
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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
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
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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
Miers
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,
Gabon
Angola
Kenya
*Gabon
Uganda
Z a i r e
Cameroun, Z a i r e
Z a i r e
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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
Gabon
Nige r i a
Cameroun, Gabon
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
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,
air e
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
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PLANTS
Tribe 2: PENATHEAE
Penianthus l o n g i f o l i u s
Miers
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
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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,
Zaire
Ghana, Kenya, Tanzania,
Mozambique
Ivory Coast, Ghana,Nigeria
Liber ia , Nigeria, Central
African Republic, Zaire
S i e r r a Leone, Liber ia ,
Ghana
Liber ia , Ivory Coast, ,Togo,
Gabon
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
Central African Republic,
Sudan, Zaire, Rwanda,Uganda,
Kenya, Tanzania, Zimbabwe,
Angola
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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
Diels
C. friesonum Diels - C, hirta klotzsch - C. insignis Alston - C, macrosepala Dfels -
C, nigrescens Df els - C. owariensis Beauv, ex -
nc.
84. C. pareira I.
85. C. rigidifolia (~n~l.)
Diels
86. 2. tenuipes Engl. 87. s. tonilosa F.geyex
Ha~ve y
88. C. tnincata Engl. -
COUNTRIES
Namibia, South Africa
Tanzania, Mozambique
Kenya, Tanzania
Zaire
Tanzania, Zimbabwe, Mozam-
bique
Ethiopia
Kenya
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
Zaire
Zimbabwe, South Africa
Tanzania
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Table 1 Contd.
PLANTS
Cocculus balfouri schweinf
C. hirsutus (L) Diels -
C. pendulus Diels -
Limaciopsis loangensis
Engl.
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
Central African Republic,
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
Zaire
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141.
Tablc 2: Alkaloids From Nigerian Menispermaceae
Species
PLANT SPECIES - --
I, Chasmanthera depen-
dens -
2, Cissam e l o s mucro- xr-- 7
CONSTITUENTS
J a t r o r r h i z i n e
Columbamine
Magnoflorine
Pseudo colum- bamine
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
Stems
I t
I1
I1
11
I1
11
11
11
I1
I1
I1
I 1
I1
11
11
I1
I1
Roots
Stem
REFERENCES
Fer re i ra 1965 Vecchict t i e t a1 - * '
(1981)
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Table 2 contd . 142.
REFERENCES
Vecch ie t t i e t a l , , (1981)
PLANT PART
Roots
tt
tt
tt
Roots
R,S,L
R & S
R,S&
Leave
tt
tt
L & S
11
tt
R & S
tt
tt
Roots tt
tt
tt
PLANT SPECIES
3. Cocculus gendulus
4. Dioscoreophyllurn Cumminsii
5. Kolobopetalum aurlculatum
6. Penianthus zenker i
7. Rhigiocarya - race- mifera
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
J a t r o r r h i z i n e
Magnoflorine
Magnoflorine
N-Methylc ory- d i n e
0-methylfla- v inanth ine
J a t r o r r h i z i n e
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)
I t
Tackie e t a l . , ( 1974: I t
I t
I t
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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
J a t r o r r h i z i n e
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
tt
, Cycleanine-N- 1 oxide
Nor-cyclea- nine
Cocsuline
Cocsoline
11
tt
tt
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)-*'
11
Ohiri e t a1.,(1983)
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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
L
Obamegine
Tridictyo- phyclinc
ILANT 'ART
Loots
tt
,caves
It
tt
Roots
tI
Roots
It
R , S&L tt
tt
It
R & S Stems
Leaves
It
R , S&L
11
Root
REFERENCE
Iwurna-Badu et al. -- ( 1977)
Tackie et al., ( I 980) t
tt
Beauguesne, I 938
Thornber , 1970
Ohiri (1983)
Owusu et g., (19m)
Ohiri (1983)
Spiff et g., (1981T
Tackie et g., ( 19737-
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T a b l e 2 contd .
- - . .
PLANT SPECIES CONSTITUENTS
Fangchinoline
Cocsuline
Tetrandrine
Magnoflorine
Palmatine
KEY - R = Roots; S
PLANT PART
Root
I t
II
Unspe c i f i e d
REFERENCES
Ohi r i , (1983)
'ackie - e t -* a 1 9 (1973)
= Stem; L = Leaves
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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
10 25
20 28
30 29
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
10 25
The t e s t micro-organism used he re was - E. - c o l i
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Table 12: The Effect of T,mperature on the
Antimicrobial Activity of Palmatine
( 20mg/ml) Temperature oC Diameter of zone of inhibition (mm)
10 20
20 20
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
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Table 14: The Effect of Temperature on the Anti- Microbial Activity of Jatrorrhizine
( 2mg/ml)
Temperature oC Diameter of zone of inhibition (mm)
10 22
The test micro-organism used here was Eo coli -- Table 15: The Effect of Temperature on the Anti-
microbial Activity of Columbamine
Temperature oC
10
20
30
40
50
60
70
80
90
100
Diameter of zone of inhibition (mm)
19
The test micro-organism used here was - E. - coli
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Table 16: 149*
The Effect of pH on the Antimicrobial
Activity of Tetracycline (2mg/ml)
Diameter of zone of inhibition (mm)
The test micro-organism used here was - E. - coli
Table 17: The Effect of pH on the Antimicrobial
Activity of Ampicillin (2mg/ml)
Diameter of zone of inhibition (mm) 23
2 24
i: 25 26.5
5 27 + 6 29
7 8
35 30
9 30 10 22 11 22 12 22 13 14 ; The test micro-organism used here was - E. - coli
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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
Diameter of zone of inhibition (mm)
14 15 16.5
17.0
17.5 17.5
18.5 18.5
17.5
17.0
15.0
12.0
12.0
10.0 t
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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
22
26
27
28
28
29.5
30 30 28
26
24
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
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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)
42,O
40.0
The test micro-organism used here was - E. - coli
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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)
30 28
25 21
1 a 15 1 4. 13
The t e s t micro-organism he re was E;. c o l i
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
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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
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FIG. 20 - Chromatogram of stem extracts.
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APPEND1 X -- - . .
Tet. A Quat. B Mixt.C Pal. Jat. Col. THP
FIG. 21 - Chromatogram of root extracts.
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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:
Brown
Yellow
Q.uat e rna ry B:
Yellow
Yellow
Brown
Mixture c: Brownish
0 n1 p our12 :
Green
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
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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
35 15
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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