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The Tumor-Enhancing Principles of Croton Tiglium L.': (Cocarcinogenic) Materials Labeled A and C From The
The Tumor-Enhancing Principles of Croton Tiglium L.': (Cocarcinogenic) Materials Labeled A and C From The
Institute of Environmental
The extraction and isolation of 2 active cocarcinogenic agents from tiglium L. and their long-term biologic testing are described. Both tent cocarcinogens at very low dosages. The biologic activity of formed derivatives of the active materials is also reported. Phorbol
a semisynthetic compound derived from croton resin, showed notable
ity.
are discussed.
In our earlier reports, the fractionation, isolation, and preliminary biologic assay of 2 active tumor-enhancing
a week. Promoter solutions were applied onto the clipped dorsal skin with a No. 5 squirrel hairbrush, with
100 mg of solution delivered per application. Mice were individually weighed before painting was started and at regular intervals thereafter. They were observed regu larly, and their tumors were charted on appearance throughout the duration of the experiment. Papillomas
were counted only if they persisted for 4 weeks or more.
Papillomas
that
Persisted
for 4 weeks
or more and
and
then
regressed
were
counted
in the
were
cumulative
excised
number
confirmed
of
tumors
OF SEED
OF
CROTON
TIGLIUM
L.
Shelled croton seed (Croton tigliuin L., crushed in a Waring Blendor and extracted
with methyl
was distilled crystalline
The solvent
atmosphere oil by filtra at
Female Swiss mice (l\'iillerton Research Farms, l'sIiller ton, N. Y.) were vaccinated against ectromelia and put
chips in stainless steel cages, 10 to a cage, and were fed Purina laboratory chow and water ad libitum. The ani mal rooms were air-conditioned and were maintained at
7276F.
PROCED1JRE
SEED
EXTRACT
Solvent fractionation.The dark-brown extract was par titioned between hexane and methyl alcohol-water (9:1).
All methyl alcohol-water extracts were combined, and all
The backs of the mice were clipped free of hair 2 days before the initial treatment and then as needed for the duration of the experiment. DMBA,2 freshly recrystal
solution
1 This
(0.1 ml).
work was
This primary
supported by Grants
treatment
CA-05946,
was followed
CA-06989, and
hexane extracts were combined. No further work was done on the hexane extract. The methyl alcohol-water layer was filtered to remove an amorphous solid. The filtrate was evaporated to dryness in vacuum, and the residue was extracted with ether. The ether-soluble
fraction was dried, filtered, and taken to dryness. The
viscous
constituted Florisil
brown
resin
was labeled
croton resin
was
(4) and
chroma
thracene; A and C are fractions A and C from croton resin pre pared as described in this work; CR, croton resin; WSE, whole croton seed extract.
was subjected
1872
Cancer Research
yielded
distribution
1965
distribution in a 200-tube (20 mi/tube) machine, with 500 transfers. The solvent for the lower phase was methyl alcohol-water-acetic acid (94.5 : 5.0 :0.5); the upper phase was hexane. The progress of the distribution was fol lowed by thin-layer chromatography on silica gel plates with ether used as solvent ; spots were visualized by spray ing with 50 % sulfuric acid and brief heating with an infra red lamp. Tubes 016still contained complex mixtures;
Tubes
on a
was purified
same solvent
by countercurrent
system and machine
as that used for separation of the active materials. After 600 transfers, 4 gm of the crude acetate from the ether eluate yielded 1.2 gm of material (in Tubes 60100), which readily crystallized from ether to give 0.5 gm of an
acetate, gram This m.p. 198201C ; reported (9) m.p. (9 : 1) solvent, by m.p., mixed 198200C.
This product
chromato
RF 0.50; rn.p., and
1755 contained
thin-layer
a mixture RF 0.44
that
; these
gave 0.25,
only
2 spots
were
chromatogram
2 materials
[a]@ in methyl
142, reported
labeled
A and
C with
and
respectively.
The contents
of Tubes
1755were combined
and taken
infrared absorption spectrum with the acetate of Thomas and Marxer (9), a sample of which was provided by Dr.
A. Marxer.
lar formula
agree well
data
formula.
gm of C (recovery : 65 %).
Analysis.Calcuiated
active (A: [a]i@' + 40.3; C: [a]i' + 61.2), colorless amorphous materials. Both A and C show in their infrared spectra free hydroxyl and ester carbonyl absorp tion.
GAS CHROMATOGRAPHIC DETERMINATION OF FATTY ACIDS OBTAINED BY HYDROLYSIS OF A AND C
i\I.W., 490. Found: C, 63.74; H, 6.79; M.W. (by os mometer), 467 ; CCH3, 15.13 % ; saponification num ber, 100. Ultraviolet absorption in methyl alcohol:
Xmax 238 mj@ (max 8150) ; reported (9) Amax 233 Ifl@i (fmax
8000), Amax 324 m@ (@max53). The infrared absorption spectrum shows a sharp hydroxyl band at 3430 cm1,
The materials
line hydrolysis, tatively
mixtures
to alka
respectively,
at 1640 cm'.
and carbon-carbon
double
bond absorption
were performed by integration of the areas under the curves of the known and unknown mixtures. On hy
drolysis, A yielded tiglic (6.0 % of total acids), caprylic
(6) and
The
was
separated, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under nitrogen
steam bath ; 188 mg of a red residue were obtained.
material was purified by chromatography on thick silica gel plates with ether-hexane (6 :4) used as solvent. Two major bands appeared with RF 0.44 (AD-i) and RF 0.40
(AD-2).
extracted with ethyl alcohol. This product contained inorganic material, which was removed by chromatography
on acid-washed Florisil and elution with methyl alcohol. The phorbol was crystallized from ethyl alcohol, m.p.
239241C, reported m.p. 238240C (3), [a]@ + 118
rately and extracted with ether. The ether extracts were taken to dryness to give 80 mg of AD-i and 68 mg of AD-2. These materials were crystallized from ether hexane to give orange needles; AD-i, 65 mg, m.p. 120
121C; AD-2, 52 mg, m.p. 151i52C.
in dioxane. Analysis.Calculated
Crystalline ester from C.A total of 230 mg of C was converted to the 4-carboxy-4'-nitroazobenzene derivative
as described above for A. The product was also purified
M.W., 364.
mometer),
by thin-layer
chromatography
only,
373 ; CCH,
was crystallized
from ether-hexane
absorption
to give red needles, i65 mg, m.p. i23124C. his T material was labeled CD. The analytical data on the 3 crystalline esters derived from A and C are given in
Table 1.
Phorbol acetate from the 4-carboxy-4'-nitroazobenzene
m.p. 151
deriva
tive, m.p. i23124C, prepared from C, were hydrolyzed with barium hydroxide in absolute methyl alcohol by
refluxing for i hr. The methyl alcohol was removed
Enhancement
by Croton
tiglium
L.
1873
TABLE 1
ANALYTIcAL DATA OF 4-CARBOXY-4'-NITROAZOBENZENE DERIVATIVES OF A AND C
No.AD-iAD-2CDm.p.12021C15152C12324CMolecular Compound
formulaC@,H61N,O11C47H@,N,O12C@,H63N,O11m
.w.Calculated856842870FoundOsmometer875855883Rast857836AnalysisCalculatedC67.3567.0467.64H7.187.067.30N4
a All 3 materials
showed
an inflection
on the short
wavelength end absorption at 235 m@. under nitrogen; 10 ml of dry pyridine and 30 ml of acetic acid-acetic anhydride (1 : 1) were added, and the solutions Multiple
motion
tumors
were obtained
with croton
in the initiation-pro
resin, A, and C (at the
experiments
were refluxed for 4 hr. The solvents were removed in vacuum. The residues were extracted with ether, and
the ether extracts were chromatographed on acid-washed Florisil. The ether eluates were taken to dryness, and aliquots were chromatographed on silica gel plates with ether-hexane (9 : 1) used as solvent. Phorbol acetate, m.p. 198201C, used as a marker. was The derivatives
levels) ; the range was 38papillomas/ animal. Very few of these tumors re
of benign tumors were obtained in
with
croton
by
oil 6 months
which time 1
after
animal
the begin
in the
experiment,
group
regressed.
already
The
bore
a papilloma,
papillomas
which
subsequently
after the
2 other
appeared
accidental DMBA,
determine
croton
layer chromatography. The product was an amorphous solid but gave only 1 spot on a chromatogram; it was used as such for biologic testing.
RESULTS AND DISCUSSION
2 to
tumor
would
response and rate of 1st tumor appearance. From the results given in Table 2 it appears that the higher dosage does not significantly enhance tumor response.
The results obtained in initiation-promotion experi ments with DMBA used as initiator are summarized in Table 2. With croton resin and fractions A and C at the
higher dosage level, the 1st tumors appeared
tate, phorbol acetate, and the hydrogenated polyphenolic ester described earlier (10) were tested for biologic ac These materials were applied at a dosage of 5 at 385i tivity.
days from the date of initiation. The high activity of the croton resin and of A and C is clearly indicated in
these results. Even at the very low dosages of 0.5
@@g/application times weekly and were preceded by a 3 phorbol acetate and the hydrogenated polyphenolic ester
showed DMBA no activity. However, of the 20 animals given followed by phorbol myristate acetate, 11 bore
single treatment with 150 @g DMBA 14 days before of After 30 weeks of testing, @g the beginning of promotion.
1874
Cancer Research
TABLE 2
TUMOR PROMOTION WITH CROTON SEED EXTRACT, CROTON RESIN, AND FRACTIONS A AND C T REATMENMEDIAN i
Primary
(j@g/100 (FRoM PRIMARY No. OF TREATMENT) TO 1STCUMULATIVE MICE WITHDAYS
TO
(pg/100
mg
acetone)DMBA acetone)Secondary
(150)CR (25)276491101711LifetimeDMBA (300)CR (25)254d38123252328DMBA (300)A (0.5)>43522241981435DMBA (300)A (5.0)365512731919435DMBA (300)C (0.5)>43510432291435DMBA (5.0)22551189209435DMBA (300)C (25)>3807319853380DMBA (150)WSE (150)None37000380DMBA (300)None42326630435DMBA
(150)Acetone30400380NoneAcetone>43500435NoneA
(0.5)>43500435NoneA (5.0)>43522410435NoneC
(0.5)>43500435NoneC
(5.0)4079130435NoneCR (25)43942610LifetimeNoneWSE (25)>37200372
a There
b Single
were
treatment.
20 Swiss
weekly
Millerton
beginning
female
14 days
mice/group
after primary
except
where
noted.
except for A and C at 0.5 @@g/appli
C Three
times
treatment
cation, fractions
d Thirty
which
were
treated
in this
twice work;
group.
weekly. WSE
were seed
as follows:
CR
is croton in this
resin; work.
A and
C are
A and
animals
C of this
extract
described
papillomas.
The
1st tumors
appeared
at 87 days
from
ported
by m.p.,
in the present
mixed m.p.,
work.
and
Our triacetate
infrared absorption
is identical
spectrum
the date of initiation. No tumors were observed in the control group, which received only phorbol myristate acetate. Materials A and C each constitute approximately 1% of the whole croton seed extract. The tumor response obtained with A and C at the lower dosage level, 1 j@g/ week, was only slightly lower than that obtained with the whole seed extract at a dosage level of 75 j.ig/week (see Table 2). It appears that A and C account for most of the tumor-promoting activity of the whole seed extract. However, it is likely that fractions B and D are also biologically active. These materials have not yet been
obtained free from A and C and therefore have not yet
with that prepared by Thomas and Marxer (9). Gas chromatographic analysis of A and C gave 4 acids in each case. Saponification numbers, however, suggest only 2 or 3 ester functions in A and C. It follows that both A and C contain small amounts of closely allied ma terials. In addition, A yielded two 4-carboxy-4'-nitro azobenzene derivatives. These 2 derivatives and that obtained from C differ in their molecular formulae by methylenes only. Substracting the derivative moiety, 3
molecular formulae for the original materials differ only materials are arrived
at:
that
C36H56O5. These
in the chain
suggest
length
ester,
m.p.
96C, described
earlier
A 4-carboxy-4'-nitroazobenzene
derivative,
C51H@.66.
N3O12, m.p. 8687C, been described earlier (6) ; this has molecular formula was later revised to C49H@N3O11. This compound is different in its m.p. from the 3 esters of the same type prepared in the present work. From the limited amount of information available in the brief
communications appears, however, of Hecker and co-workers (68), it that the active materials described are
polyhydroxy
to this effect
pound,
same
C26H28O6, isolated
compound, although
made. Hecker (6) prepared an acetate of the poly alcohol, to which the molecular formula C26H3409 was assigned; an m.p. for this compound was not given. This acetate is different in its optical rotation from that re
in the present
ACKNOWLEDGMENTS The croton seed used in this work was kindly supplied by the
Department of Agriculture of Ceylon. The countercurrent dis
by Croton
I. Croton
tiglium
Ibid.,
L.
17: 144452, 1934.
1875
in
K. V. The Poisons
Chief of Toxicology, Office of the Chief Medical Examiner of the City of New York. Samples of phorbol acetates were kindly sup plied by Dr. A. Marxer, of Ciba Limited, Basle, Switzerland.
This work was carried out with the assistance of L. Langseth and
6. Hecker, E. Uber das Toxische, Entzundliche und Cocarcino gene Prinzip des Crotonols. Angew. Chem., 74: 722, 1962.
7. Hecker, E., Bresch, H., and Szczepanski, Ch. v. Cocarcinogen
E. Arroyo.
REFERENCES 1. Bekersky, I. Spray Technique for the Preparation of Thin Layer Chromatography Plates. Anal. Chem., 55: 20162, 1963. 2. Cherbuliez, E., Ehninger, E., and Bernhard, K. Recherches sur la Graine de Croton. II. Le Principe Vesicant. Helv. Chim.
Acta, 15: 65870, 1932. 3. Flaschentrager, B., and Falkenhausen, F. V. The Poisons in Croton Oil. II. The Constitution of Krotophorbolone. Liebigs
Ann. Chem., 514: 28260, 1934. 4. Flaschentrager, B., and Wigner, G. The Poisons in Croton Oil. V. The Isolation of Croton Resin, Thin Oil and Phorbol from