(i)

Title:- The Preliminary Phytochemical Investigation

of the Leaves of Croton Macrostachyus.

(Kuphorbiacae family)*

by MIDAMBOj B . KAGAI (MISS)

A project submitted in partial fulfilment of

the requirements for the award of the Bachelor

of Pharmacy degree of the University of Nairobi,

Kenya.

Supervisor DR. I. AL)L»AL - MENSAH

Department of Pharmacy,
Faculty of Medicine,
College of Health Sciences,
University of Nairobi.

June 1936.

o,NAIROBI Library
 (ii)

L>EU1 CATION

To the entire MIUAMBO family headed by Mr.

Erastus Midambo whose hard work and foresight has

seen many to success.

 (iii)

Acknowledgements i

My sincere gratitude is extended to iny

supervisor L)r . Addae-Mensah of the Pharmaceutical

chemistry section( department of Pharmacy whose

advice, guidance and continous encouragement

throughout this project was very much well taken

and appreciated. through his help and influence it

was possible to get the NMU and MS spectra run.

The assistance from the technical staff of the

department of Pharmacy, cannot be taken for granted

and more particularly Mr. ltichard Gibbson Mwalughu

whose help was highly appreciated.

t-ast, but not least, my appreciation goes to my

fellow working colleagues Miss Constance W andera and

•i1i s s Grace kar an j a who livened up the la bora t ory

atmosphere.
 (iv)

Table of Contents:

Tit le ...................... (i)

dedication.... ........ . . .(ii )

Acknowledgement s .......... (iii)

Abstract.................. .

Introduction......... .....2 .

Chapter O n e ................ 8 .

Chapter T w o ................ 1 5 .

Chapter Three..............1 9 .

Chapter Four...............2k,

Appendix...... 29.

References......... .30 .
 1

ABSTRACT

during the preliminary investigation of the

petroleum ether extract of the leaves of Croton

rnacrostachyus . two compounds were isolated:-

i) . Probably 3-cyclohexyl eicosane.

ii) . A compound which is probably a triterpenoid.

An account of the traditional medicinal uses

of croton species in Africa is given as well as the

research work done on croton species in the period

1978 and 1979.

Research work already done on Croton niacrostac

prior to 1985 is also recorded.

 2

INTRODUCTION:

Croton Macrostachyus is a tree 20 to ^lOft tall

with grey bark in the family Euphorbiacea with yellow

white flowers and slightly three lobed fruits half an

inch in diameter. The tree is wide spread in tropical

Africa growing in Savannah forest [ 1 ,2] Proton

macrostachyus is found in the forested areas of East

Africa and it is known as follows in the local dialects.

Musundzu (Kakamega) ; Mutundu (ka 111ba and Kikuyu) ;

Mutuntu (Meru) and Muwulugu (llehe) C3~) • The plant has

been used as antihelmintic for tapeworm and as a purgative.

The ash from burnt leaves is licked for coughs. The

juice from a fresh leaf is applied on fresh wounds to

hasten blood clotting. Juice from boiled roots is drunk

for malaria and venereal diseases. The bai'k peeled from

stems and roots, boiled in water, is used to bath newly

born babies as a remedy against skin rashes. The seed

and resins are howover considered poisonous [ 3] . The

C-
Chagga use the leava-juice with fresh green leaf of

Embelia Kjlimandscharica as an antihelmintic. The plant is

considered an abbysinian taenifuge. The bark which is

said to contain crotin is used in East Africa as a

 3

pur g a t i v e ( 2 ).

Other species in the genus croton have been shown

to contain active principles having medicinal or

toxic properties and have also been used in traditional

medicine in Africa.

The seed of Croton Elliottianus is a purgative

in man with O.lg to 0.2g. producing a mild effect and

o.^ig. a drastic effect. The fixed oil produced by the

seed also has the purgative effect but is less irritant

than the seed. The Masai use it as a purgative after

mixing the bark with curdled milk. Small doses in man

are diuretic and so is the oil. Both seed and oil are

mildly antihelmintic but have no cholagogue action with

the systemic action of haemolysis and haemorrhagic

spots in the tissue, the action of oil in these respects

being feebler than the seed. £ %J .

Croton gratissimua has been used as a remedy for

fevers by the Transvaal Sotho. The charred and

powdered bark is used to treat bleeding gums by brushing

them with the powder. They also use the leaf as one of

the ingredients for 'smoking' rheumatic patients. The

Zulu use the plant as cathartic and an eruptive irrit

ant.
 k

Zulu use the plant as Cathartic and an eruptive

irritant. The bark is applied for its irritant action o

on the chest wall, in any painful respiratory condition

and for intercostal neuralgia. It is also used for

dropsy, indigestion and pleurisy. The Zulu also use

the powdered bark ’ as one of the ingredients of a remedy

inserted into the uterus for disorders of that organ.

The bark is said to contain the toxalbumin crotin- The

Kgatla make an eye lotion for animals from a cold infusion

of the leaf and use the root as a charm medicine. The

leaf, stem and the fruit yield an aromatic oil (calamus

like)

The Transvaal and East African people have used the

bitter bark of Croton gubouga as a malarial remedy. The

seed is also used. The powdered bark is usually made

into a pill and is said to produce benefit, but opium

may be an ingredient. The same bark has been used

as a fish poison in ^azaland and bast Transvaal

The seed and bark cause intense burning sensation in

throat and mouth. Salivation, slight nausea and slight

purgation is also seen and these effects are thought

/5
 5

to be due to the presence of an acrid principle.

^he bark has a slight but not unpleasant odour and

the dust from it produces sneezing with a burning

sensation in the throat and on the tongue. The Luvale

administer an infusion of the root to thin babies to

make them fat [*]• Another croton species used for

influenza and malaria treatment is Croton merry- hartii ♦

In Cast Africa, Croton megalocarpus's pounded bark

is soaked in water overnight and the extract drunk as

a remedy for intestinal worms and for the treatment

of whooping cough. The plant species is said to contain

a toxalbumin.. The Masai administer a decoction of the

bark with blood as a tonic. Tests for antibiotic activity

are negative. Another plant species used as

a vermifuge and purgative by the Nyamwezi is Croton

Mubango. 3 •

Croton j'sendopulchellua's roots are used as a

decoction for relief of asthma by the Nyamwezi. The

leaves are boiled and applied to chest for colds. Twigs

and leaves are used with the twigs of Teclea nobilis

in making a vapour bath for the treatment of syphilitic

/6.
 6

sores, at the same time the powdered root of

crossopteryx febrifuga being applied locally. The root

and leaf are thought to contain the toxalbumin crotin.

An infusion of the leaves is given to cattle as a

remedy for anthrax. The leaves are also burnt in among

crops as an insecticide [ 4

The powdered bark of Croton Sylv aticus is a Swati

remedy for gall sickness in cattle. The bark yields a

tanning matter used in Gazaland as a fish poison. The

root is a remedy for pleurisy and indigestion and is said

to be avoided by birds as they are fatally poisonous to

them. A decocttiS of leaves is used as a wash for body

swelling caused by kwashiorkor or Tuberculosis. The roots

are also pounded to make poultices for swellings. A

decoction from the bark of the roots is taken orally

as a remedy for tuberculosis. An infusion of the leaves

is also taken as a purgative £ 2 ,3 !.

e
Croton Scheffl/ri
r
is used as remedy for miscarriages

while croton Zambesicus is used by the Masai with Villosa

as a strengthening medicine. [ 3 ]-

/7 •
 7

The leaves of Croton riichotiamus are dried and

burnt for inhalation b y ^ i n fumigation of a patient

with fever. It is an excellent remedy for chest ailments.

The leaves are chewed or dried and smoked as cigarette

by the $ukuma. It also acts as a remedy for stomach

diseases ;chopped roots are added to soup nuide from goat's

meat and taken as tonic [3].

The strongly scented roots of croton jatrophoides

are used for colds and stomachache (2 ).

From the outgoing remarks it is important to

screen plants that are used as traditional remedies by

the various groups of people to find out more about

the pharmacology and chemistry of the active principles.

The present project is part of an on going programme

of investigating the East African crotons which have

hitherto received very little phytochemical and /or

pharmacological attention.

/£.
 8

CHAPTER ONE

RESEARCH NORK DONE ON CROTON GENERA (EUPHORIA ACE AE FAMILY)

IN THE PERIOD 1978-1979.

Before this period, various reserchers had done

some phytochemical screening of plants native to N.E.

Brazil for the presence or absence of alkaloids,

steroids, triterpenoids, hydroxy anthraquinone derivatives

flavonoids, saponin compounds and antibiotics. Some

of the croton species that were subjected to this

screening included Croton rlmnniifolius (leaf), croton

compostris and croton refusa The species

worked on in the period under review are discussed below.

Croton sublvratus and Croton columnaris.

The trend in research at this time was a deliberate

search into anti ulcer, anticancer/co-carcinogenic

components in the plants. Mishima e_t al studied the

furanoditerpene, plaunol (I) and its acetate were prepared

by extraction of Croton sublvratus and croton columnaris

and additional chemical treatment. Plaunol (I) and its

acetate had antipeptic ulcer activity (data given in rats)

/9
 9

i d )

Pharmacological screening directed towards finding

antipeptic ulcer substances of plant orip_;in led to findings

that the acetone extract of crude drug of Croton

snblyratus by Eiichi Kitizawa trt al. Of) . ^ Two new

diterpene lactones named plaunol A (Hi) and plaunol(IV)

showed significant inhibitory activities against

reserpine induced ulcer in mouse and shay-ulcer in rat.

From anti-reserpine active fraction, 18 -hydroxy geranyl

geraniol (V ) was isolated and from anti-shay active

fraction the new diterpene lactones designated plaunol

A and plaunol B were isolated by silica gel column

chromatography. The structures of plaunol A and geranyl

geraniol were determined by X-ray analysis while that

of plaunol B was determined by chemical and spectral data.

/10
 10
Oil

lO l
)

The relationship between co-carcinogens of the

diterpene ester type froi.: this plant and oesophageal cancer

ill Curacao was then investigated by Weber et al C(Q .

Three highly irritant and tumour promoting croton factors

and the corresponding 3 cryptic croton factors

were isolated and characterised as new esters of

l 6 -hydroxy and *t-deoxy, l 6 -liydroxy phorbol. A

suggestion was put forward that tumour promoters of the

phorbol ester type, ingested through the wide spread and

frequent use of Croton flavens . may be causally related

to the high rate of oesophageal cancer in uracao.

 11

Croton tliasii*

L>e alvarega ejt a_l worked on the ground wood and

diasin (VI) was isolated and its structure determined

by 1 11 and 13 C NMR analysis and chemical means.

Hiasin (VI)

Croton californicus»

The furanoid diterpene (-) hardwicki&fj* acid and

the long chain 1-triacontanol ( C H _ ( C H w e r e

first reported in this species*’fruit Q Q •

Croton niveus.

Kojas E.T. and Hodrijruer H.L. isolated Nivenolide (VII)

a diterpene lactone from this plant and the structure

determined by chemical and spectral means D-p} •

v D
I _
 12

Croton draconoides •

Alkciloids of this croton species were studied by

Uettole et a_l and found to contain thaliporphine and

glaucine (VJL1I). Taspine was the only cilkaloid in the

latex Q.l3 .

glaucine (VIII).

Croton Spnderianus:

Within this same period (197&-79) 1 research on

essential and fatty oils of various crotons have also

been reported. The essential and fatty oils of Croton

sonderianus (stem and leaves) were studied by Craveiro

et a 1 (1 2 ) who reported the presence of oc-pinene, ^ - p i n e n e ,

myrcene, 2 -terpineol, caryophyllene, humulene, Cv-elemene,

^-elemene, calamenene and several unidentified

seisquiterpenes alcohols and hydrocarbons. The fatty acids

yielded hexadecanoic, cis- 9-octadenoic and 9 -methyl-

heptadecanoic acid.
......................... / 1 3 .
 13

C r o t o n cirgvrophylloitles (leaves)

The volatile constituents of this croton were

investigated and found to contain o<- pinene ,sabinene

1 ,}> cineole, ^-eleinene, fi-caryophyllene, 2 -humulene,

^-elcmene and an unidentified sesquiterpene alcohol and

a sesquiterpene hydrocarbon. For the first time fi-

olenene and sabinene were reported in this plant £ 15j•

C rot on_aff. Zehntnerjs

The same sort of work as above was also done here

on the anise-like flavour of this plant and showed

r
that estragote, the major constituent of essential oils

in this species occurs together with minor quantities

fn
of camphor, trans-anethole, isoboipeol, caryophylene,

i-elenene, safrole, methyl isoeugenol heptadecane and

eicosane. This was the first report of these compounds

in the genus. 15 predominant volatile constituents of the

oil wereidentified by Gas-chromatography-mass

spectroscopy
 The sort of work exemplified here, shows the high

potential in the study of the various constituents of

the croton species in question - croton macrostachvus -

which is one of the most widely used croton species in

Kenya. Some work has already been done but on judging

from amount of work done, there is still greater chance

of exploitation of the field.

 15

CHAPTER T W O .

WORK PREVIOUSLY DONE ON CROTON MACROSTACHYUS .

In the course of a continuing search for tumour

inhibitors of plant origin. alcoholic extracts of the

fruits of Croton niacrostacliyus showed significant

inhibitory activity in lovis Lung carcinoma in mice

(LL) . Kupchan e_t aT in 1$)68 Cl 5^ worked on the isolation

and structural elucidation of crotepoxide (IX)

x—»/
a novel

tumour inhibitory cyclohexane diepoxide derivative from

this plant.

fractionation of the ethanol extract guided by assay

against LL revealed that an active principle, was

concentrated successively in methanol layer of a 10%

aqueous methanol/skellysolve B partition and in the 1 -

butanol layer of 1 -butanol/water partition. further

fractionation involving silicic acid chromatography

yielded the crotepoxide (C^gHgOg)

M.P. 15 0 - 1 5 1 °C.

= +7^ (Cl. 70 CHCl3 );

. MftOH
27^ (6=1050 and 2 8 ln^u (6 = 860)
Max

/16.
 16

cnci_
> 3.35, 5.71,5. 7 8 ,6 .2 4 ,6 .3 1 ,6 .8 $, 7 .2 9 ,7 .8 9
max
8.20,9.00,10.24 and 11.24 p.

I''-*-- signals (in CLCl^) at 2.28 (5H ,m ,aromatic) ;

4.27 (lH,d,Jxy = 9.5:t/. > CliOAC) ; 5 . 4 2 and 5 .7 5

(211, doublets J = 12.0HZ CH o0C0Ph) ; 6.32 (Iil,d,

JdC ~ “ »5 ‘1Z); 6.5 6 (IH,d ,d , = 2 .5 and

4*°H Z )» 6.90(IH,d,d, JAa = 4.0 and J Ay = 1 .5H Z )

/.uo (3*1 ,s , acetate) and 7»95 (3H ,S ,acetate).

Crotepoxide belongs to a small group of naturally

occuring highly oxygenated cyclohexane derivatives, other

members of which are terreic acid (X), Epoxydone (XI)

senepoxide (XJLi!) seneol (XIII) and Shikimic acid (XIV) .

However crotepoxide has the diepoxide functionality. This

function has been shown earlier to confer tumour inhibitory

activity on other classes of synthetic compounds.

Investigations are underway to determine the significance

of various structural features in relationship to the

turr.our inhibitory activity of crotepoxide.

00CCH.

\
^ CCV ° \ - x /
— O - c— c — C— c — c
C i*otepoxide (IXl f I I I
H h a H H
X Y A d C-
./17.
 17

o C 001 I

■OH
HO Shikimic acid
OH (XIV)

^eneol (XIII)

CH OH

Epoxydone (XI)

Odak e_t al 0 _(D then embarked on the total

synthesis of the dl crotepoxide (usually isolate^ from

fruits of croton macrostachvus and from the leaves

and stem of piper futadzura) by effecting it from

epoxide (Xl) in 9 steps.

 18

Another undertaking was the short terra in vitro

assay for promotor substances using human lytnpho-

blasto.id cells latently infected with lipstein-Darr

\ t*
viruslused to assay such substances) on e^phorbiaceae

family and more particularly in croton macrostachyus

and croton megalocaruus»

Most of the work reported in the period under

survey has been done in Brazil( America, Japan and

virtually none in East Africa where the plant occurs

in plenty. More so, in the research done some parts

of the plant had not been studied like the twigs and

leaves. Therefore bearing in mind the variation in

components (quantitative and qualitative) in a plant

with variation in geographic and morphological

differences the research project was and is a course

worth taking.

/19.
 19

CHAPTER THREE - THE PRESENT INVESTIGATION

RESULTS AND DISCUSSION.

The petroleum ether extract of the leaves of

Croton m.ncrostachvus was separated into seven main

fractions SK.-1 , BE--2, BK -3 ,BK~5, iik-6 , BK -7 on a

chromatographic column- silica gel; Benzene * chloroform

(1 :1 ) as eluting solvent and eluting further with more

solvents with increasing polarity. The characteristics

of the various fractions are shown in table 1 .

Table is

Colour of Melting Yield (g)

,r .. Crystals Point ‘°C
traction

Elh- 1 whit e crystals

70-71 O.D 67 O

BK -2 white crystals
74-76 0.2156
dK -3 white crystals
70-7*1 0.0623
i3K-*i cream crystals
68-72 0.0304

ill; -5 light green

76 -78 0.2628
i3K-6 cream crystals
75-77 0.0150
13K-7 Brown crystals
72-7** 0.0855

No further work was done on fractions tiK-l,i3K-3,

BK-4, BK-3, BK- 6 , 3K-7.

20/
 20

CHAKACTKUlSATIUN OF BK-2.

1'raction UK-2 was recrystallised from methanol

to give the white crystals with m.p. 7 ^“ 7 6 °C

(uncorrected). An attempt to deduce the structure of

BK-2 was done from 'H N M K , mass spectroscojiy (hS) and

lil(kDH) . The IW spectrum (fig. l) showed significant

peaks at I,l45-l/l85 cm 1 corresponding to C-H bending

alkane - 296 O- 285 O Cli ^ corresponding to C-ii

stretching alkane. It is clear from the IH spectrum

that there is no oxygen functionality such as OH

or C =0 in the compound.

TheNMR spectrum (fig 2) showed only one large

single peak at S=l.25 (singlet). In view of difficulties

with the instrument, no intergration could be done on

this peak. The only deduction from the spectrum is

that there are many methylene protons (-CH^-). No

other signals could be seen from the above data, it

can be concluded tentatively that the compound is a

hydrocarbon.

/ 21.
 21

The mass spectrum (fig. 3 ) supported this possibility,

It gave a molecular ion (N+ ) peak at 36^1 another peak

at 33(» due probably to loss of -CH -CH - and then

a typical hydrocarbon fragmentation pattern between

M/e = 25 8 and M/e=^ 5 * from the above tentative

conclusion, the M peak value and consultation of

literature [xe] the following possible compounds were

considered; 7 -cyclohexyl eicosane C 26H 52; 5'

cyclohexyl eicosane (C Jl v , . ,
2 b 52 /. 9-cyclohexyl eicosane

(Cg^H ); 2 cyclohexyl eicosane; ^i-cyclohexyl eicosane

(C o 6H 52); 1 *k-dimethyl - 3 -n-0 ctadenyl cyclohexane and

3 cyclohe^yleicosane ( C ^ H ^ ) ( ^ 1 ). Un the basis of

the fragmentation it is suggested that structure is

the 5 -cyclo hexyl derivative.

H
I ,
CH CH -C-(CH ) ,CH
3 2 j 2 16 3

3-cyclohexyl eicosane (kVl)

ihe following fragmentation pattern is proposed!*

M +M / e*364 M +- 28 (-CH -CHo ) ♦ C H o(CIli (CH )l6 CH

2 -v (2 2 2 5

* d M / e-5 3 8

- LJ M / e=8 ^

cii-CH 2h C h o =CH (CH ) CH M/e=25:

y <■

Loss of CH ‘ i ,
i' C i l = C H l C H } i 5(L U 5
Units
 22

EXAMINATION OF THE MOTHER LIqUOUS;

13K-2.1 - Kecrystallised from acetone to give cream

crystals (lOmg) and the K _ values in several solvent

systems determined (fig.4 and table 2 ). No standards

were available for comparison. This could have

been desirable in structure characterisation of UK-2.1.

UK-3-6.1

This is the combination of the BK-J.l, BK'i.l,

UK- 5 . 1 and UK6.1 mother liquors as from TLC they seemed

to be composed of similar components. After the

recrystalisation from methanol, it gave cream white

crystals with to.P 1 1 0 - 1 1 2 °C (uncorrected). The compound

gave a deep purple colour witli anisaldehyde on TLC,

suggesting a possible triterpenoid. Characterisation

of UK 5-6.1 was to be attempted. HNMK,MS data on this

compound is still being awaited to enable us to suggest

a possible structure.

/23
 23

Table 2 .

BK 3 - 6 .I (variuos spots obtained

1 , 2 , 3, & 4 )
Solvent System 1* 2* 3* V ________

(0 .60 ) 0.07 0 .19

O
0.49

O
l}ure chloroform!

Chloroform benzene (3:l) o.ok 0.07 0 .12 0.39

Chloroform Benzene (i:l) 0.22 0.29 0 .^ 5 0.82

0.56
CO
-

O
0

•
0
Tohtene: U:l)
•

Chloroform Ether (Ul) - 0.35 0.47 0.67

Toluene (i:l) _ 0.75 0.53 0.48

/2k.
 2k

CHAl'TLK FOUR - EXPERIMENTAL;

'H NMK was done on a Perkin-elmer instrument

at 60inHz in CL)Cl_ with Ti'»S as the internal reference.

Analytical TLC was carried out with silica gel 60

0 rj/j 3 S the adsorbent. The plates were prepared by

the conventional methods. Column chromatography

was on s ilica gel 60(0 .0 6 3 - 0 .200mtn=70-230 mesh

ASTM)

IK spectrum was 1’ecorded on a Perkin-Llmer IK

spectrophotometer 727^* Information on the instrument

used for MS was not available. Melting point was

determined using Gallenkamp m.p. apparatus as well

as Koffler hot-stage m.p. apparatus.

Anisald-;hyde spray reagent (1 % v/v anisaldehyde,

156 v/v concentrated sulphuric acid in glacial acetic

acid) was used for visualisation of TLC spots. The

reagent was used when freshly prepared.

/25
 25

Extractions

The dried leaves of Croton Macrostachyus were

ground to powder in a mill. The plant material was

collected from Karatina about 200kni. North of Nairobi

about 2,OOOin above sea level in July/August 19 8 5 .

60 Og of this powder was extracted in a soxhlet

extractor with petroleum ether (boiling point range

60 - 80°C) for /l8 hours. The extract was concentrated

(in a rotary evajiorator to 150 ml and put in a

refrigerator for three days after which a green brown

solid deposited. This was then filtered, the solid

washed with methanol of the colouring material St

methanol soluble components to give a cream white solid

(*t.2 g). To find out the most suitable solvent system

for the seperation of the various compoents present

in the solid on column chromatography, the solid was

examined by thin layer chromatography(TLC) on microscope

slides using various solvent systems and silica gel

GF 25*» as adsorbent and anisaldehyde reagent for detection

The following solvent systems were found most

suitable and were then used in the column chromatography

 26

in the given order of increasing polarity.

1. Benzene: Chloroform (5:l)

2. Benzene: Chloroform (i:i)

3« Chloroform

4. Chloroform.: methanol (1 :1 )

5. Methanol

6. Ethyl Acetate.

Separation (column chromatography).

A column 45cm long was used with silica gel OF

254 (0.063_020Omni 720-250mesh ASTM) .

r
The c^Tude extract was dissolved in minimum amount

of chloroform and added to the top of the column

to form a uniform layer of the solution. The eluting

solvents were added in the given order and the column

left to drip at the rate of 20 to 30 drops per minute

and 30ml fractions collected. TLC was done to monitor

the separation of the various components from the

crude extract using silica gel and chloroform: benzene

(i:i) as eluting solvent.

/ 27»
 27

Fractions that seemed to contain similar

components were combined into 7 major fractions

UK- 1 , dK- 2 , dk- 3 , BK-4, dh- 5 , 13K-6, and dh - 7 . The

fraction were evaporated to dryness on the rotary

evaporator and recrystallised from methanol.

EXAMINATION UK T i l h S E P A R A T E D SPLITS

Hydrocarbon BK - 2 (Probably 3-cvclohexvl eicosane)

m .p .- 7 ^~ 76 °C lit (KJ3it) showed peaks at Umax

296 O- 285O cm"'*' (C-H stretch); l/l35-l/i^k5 cm (Oil bending)

■*T1 NMU (CDCl^ TMS as internal reference) ^ 1 .2 5 (S)*MS

M at m/z 56 ^. Other prominent peaks are indicated

in the discussion.

Data from the above spectroscopic and physical

methods was analysed and used in deducing the compound

Hk- 2 . ^>o further work was done on the other

f ractions•

EXA.M1Na T 1UN OF ThE N.OTliLU LlkUOHSi

The mother liquors were spotted on TLC and

developed in CHCl^: benzene (l:l) and the spots

compared.
 28

RR 2.1 The mother liquor was evaporated to dryness

on the rotary evaporator and then recrystallised

Id
in Acetone to give whitish crystals (lOmg)^ -Whose

TLC in various solvents was performed and the values

calculated.

dh3-6.1 This is the combination of mother liquors

dh 3 to iik6 , evaporated and recrystallised in methanol

to give cream ’•hite crystals (0 .028'ig) m.p 1 1 0 - 1 1 2 °C

(uncorrected). The TLC of the compound was in the

solvent systems shown in table 2 and similarly the

values calculated and tabulated in table 2 . Samples

for N>ilt and NS analysis were supplied, but at the

time of submitting this report, results of 'H NMU and

NS were still not available. No work was done on

d h - 1 and Uk - 7 mother liquors.

29
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P.LFLRLNCLS:

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