ISOFRIAXIDIN, X CYTOTOXIC COUhIXRIN FROhI

- 1 I I C M S D R A ELA T A (EUPHORBIACEAE)
ROBERTP. BORRIS,GEOFFREI-1.CORDELL
and

XORMA?;

R.

FIRNSVORTH

D e p a r t m e n t of Piiermacognos? nnd Pharmacology, College of Pharmacy,

l l i i r e r s t t y of Illznois a t the J l e d i c a l Center, Chacago, I L 60612, L'.S.A.
elate (Didr.) Muell,-AIrg. ( E u p h ~ r b i a c e a e ) ~
collected in Peru during -1prll of 1976,ywas
air dried and milled to a coarse powder.

I n a continuing search for plants

having anticancer activity, the Peruvian plant Micrandru data (Euphorbiaceae) IT-as examined. The chloroform-soluble fraction of the methanol
extract of the roots showed actirity
against the P-385 lymphocytic leukemia in mice,' and a previouq paper
hay identified thoye compounds responsible for most of this activity
(2) . 2
G.8-Dimethoxy-i-hydroxycoumarin (isofrasidin) (1), u-hich displays
marginal cytotoxicity, ha< now been
isolated from the tn-igs of M . elata.

EXTI~.~CTIOS
.ISD ISITI-XL

FR.\CTIOS.LTIOS

.-

The plant material was exhaustively estracied 11-ith petroleum ether ibp 30-60").
.liter removal of t h e petroleum ether, t h e
marc m-as exhaustivel>- extracted with
methanol, and the methanolextract n-as concentrated in mzciio a t 40'. Three kg of the
dark brom-n, semi-solid residue (3.2 kg total)
was partitioned between distilled n a t e r (38
liters) and chloroform (95 liters): t h e
chloroform-soluble fraction v-as dried
( S a n S 0 4 )and concentrated in i'ocilo a t 40" t o
yield 338 g of a dark-green, solid residue.

cH30Q
HO

EXPERIMEKTAL3
PLASTir%rERIiL.-The plant material,
comprising 31.5 kg of the tm-igs of J l i c r a n d r a

OCH,

'The extracts. fractions and compounds

n-ere tested under the auspices of the Drug
Research and Development Branch of the
r a t i o n a l Cancer Institute (1). An isolate
is considered active if it s h o w an EDja<4
pg/ml in the KB or P-388 cell culture 1'11
rsifro, and a T ' C 2 1 3 0 5 in T i y o .

SEP.\R.ITIOS OF THE CHLOROI'ORJI FR.\CTIOS.

-Three hundred and thirty g of the chloroform-soluble fraction x i s redissolved in

chloroform, 350 g of Mh--silica gel-P 'UT-254j
was added, the solvent removed in m c i i o ,
and the adsorbed extract chromatographed
on a column of lIS-silica gel-P UT-254
packed in benzene (3.5 kg, 12x90 e m ) . Elution was initiated m-ith benzene, follon-ed by
benzene-chloroform mist ures, chloroform,
chloroform-methanol mixtures of increasing
polarity, and methsnol. .I total of 56 fractions (4liters eachj were collected.

*Since t h e acceptance of this manuscript

the plan: material previously identified as
Czoiziiia s p r u c e m a Baill., has been reidentified as M i c r a n d r a e l n f a iDidr.1 Muell.-Arg.
311elting points were determined using a
Kofler hot plate and are uncorrected. The
u v spectra n-ere obtained n-ith a Beckman
model DB-G spectrophotometer. The I R
spectra m-ere determined Kith a Beckman
model ir-18-4 spectrophotometer n-ith pol>-styrene calibration a t l G O l em-': absorption
bands are recorded in n-ave numbers (cn1-l).
pmr spectra n-ere recorded in CDCli n-ith
a T-arian T-GO.l instrument! operating a t
GO MHz Kith a Sicolet >\Iode1 TT-7 Fourier
Transform attachment. Tetramethylsilane
m-as used as an internal standard and cheniical shifts are reported in 8 ippml. 1Iass
spectra were obtained n-ith a T-arian X I T 1123 double-focusing spectrometer operating a t i 0 eT- and 1-10".

4The plant material n-as supplied through

the auspices of the Drug Research and Development Program of the S a t i o n a l Cancer
Institute by the Economic Botany Lahoratory: .Igricultural Research Service, B-IRCEsst, U . S . D . I . , Beltsville, AIaryland. -4
herbarium specimen documenting this collection is deposited in the Herbarium of t h e
Sati?nal -1rhoretum, -%gricultural Research
Service, United States Department of
Agriculture, Kashingt on, D .C.
51Iacherey-Sagel, Duren, R e s t Germany.
G41

642

JOURNAL
O F SATUR.4L

1SOL.iTION OF 1sOFR.ciIDIS.-Fraction 32
from the column, which n-as eluted with
ch1oroform:methanol (99:1), was concentrated in V ~ C Z at
~ G 40" t o give 2.5 g of residue.
Tlc of the fraction (silica
chloroform:
methanol, 9i:3) indicated the presence of
two major components (Rf=0.23, 0.18).
Fraction 32 (0.75 g) from the column n-as
rechromatographed on 30 g of hlX-silica
gel-P/UT-254, eluted successively with chloroform-methanol (19:l) (16 fractions, 25 ml
each) and methanol (3 fractions). Tlc
(silica gel, benzene-acetone, 3 : l ) indicated
the major component t o be primarily in
fraction 6.
Preparative chromatography of fraction 6
on precoated silica gel 60-F254 plates6
(20x20 em, 2 mm thick), developed tm-ice
with benzene-acetone (3:1), followed by
processing of an intense blue fluorescent
band a t Rf=O..il, afforded a yellow amorphous solid. Crystallization from benzene
afforded amber styloids of isofraxidin (1)
mp 146-148" (412 mg, 0.000013~,'i;ir, Y mar;
(KBr) 3340, 1i00, 1570 and 1290 ern-'; uv,
X max (MeOH) 342, 224 and 212 nm; X max
( M e O H f S a O H ) 400,234 and 214 nm: nmr GO
31Hz (CDC1,) 6 3.94 (3H, S, 6-OCHs), 4.09
(3H, S, 8-OCH,), 6.26 ( l H , d , J = 9 . 3 H z ,
3-H). 7.58 i l H . d . J = 9 . 3 Hz. 4-H). 6.65

conipfete--agreement with those published

for isofraxidin (3).
BIOLOGICAL
ACTIx-ITr.-Isofraxidin IXSC324637) was inactive in the KB test system
(EDjn>lOO pglrnl), but was active in the
P-388 lymphoc>-tic leukemia (in 2 i f 7 0 ) test
system (EDso=1.7 pg/ml).

DISCUSSIOS
Isofrasidin was first isolated by
Spath and Jerzmano\vska-Sienlciev-iczomi (4) from the bark of the ash
tree, F Y Q X ~ I Wexcelsior
S
L. (Oleaceae) ,
and has subsequently been obtained
from other species of Frarni?zzis ( 5 - i ) ,
Artemisia (8-14) and Achillea (15)
(Compositae), Eleutlrerococczbs (16)
(Araliaceae), Cldoraizthzis (3) (Chloranthaceae) , Erica (17) (Ericaceae),
and Parastreplzia (18) (Astereae). The
compound has been partially and totally synthesized (19, 20).
Preparations containing isofraxidin
have been shown t o possess choleretic
hlerck, Darmstadt, West Germany.

[VOL.

43,

NO.

activity in rats. In terms of this

activity, isofraxidin is several times
more potent than equal amounts of
dehydrocholic acid (21).
To our knonledge, this is the first
report of a simple coumarin derivative
possessing activity in the P-388 lymphocytic leukemia cell culture system.
Neither of the cytotoxic diterpenes
obtained from the roots of M. elata ( 2 )
could be detected by means of thinlayer chromatography of the chloroform extracts of the twigs.
ACKSON'LEDGMESTS
The authors would like t o thank the Economic Botany Laboratory, .4gricultural Research Service, BARC-East, U.S.D.A.,
Beltsville, Maryland, funded by the Yational Cancer Institute, for the provision and
identification of the plant material. The
plant material was supplied under contract
CLI-67090 with the Division of Cancer
Treatment, Sational Cancer Institute, Department of Health, Education and Welfare,
Bethesda, hlaryland. The authors Fish t o
thank l l r . C. T. Che of the University of
Illinois at the Medical Center for obtaining
the pmr spectral d a t a .
Recezted 22 Febritary 1980.

1.

2.
3.

4.
5.
6.
7.
8.

9.
10.

11.
12.

6E.

PRODUCTS

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R.L. Geran, N . H.

SEP-OCT

19801

BORRIS ET AI..

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(19T8 1 .

ISOFRASIDIS

643

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