Cocarcinogenic Principles from the Seed Oil of Croton tiglium
and from Other Euphorbiaceae
Erich Hecker
Biochemisches Institut am Deutschen Krebsforschungszentrum, Heidelberg, Germany
Introduction
The spurge family or Euphorbiacea includes some 280 genera and 8000 species which occur in tropical and in temperateregions all over the world. These succulent or nonsucculentplants range from herbs and shrubs to tree and cactus types.Many of them contain a milky juice which is more or lesstoxic, especially for cold-blooded animals, and can produce adermatitis similar to that from poison ivy. The fruits are usually three-celled capsules, each cell containing a single seed fromwhich in some species toxic, vesicating, and irritant seed oilsmay be obtained. The largest genera of the spurge family arethose of Croton, with about 700 species, and of spurge orEuphorbia, with about 1600 species.
Investigations Concerning the Active Principles from CrotonOU
Croton tiglium is a leafy shrub native to Southeast Asia.From the seeds of Croton tiglium 50—60%by weight of croton oil may be obtained by either extraction or expression.Croton oil is toxic to amphibia (9) and fish (41); used internally it is a drastic cathartic (9), and on skin it is an irritantand vesicant (12). Diluted with a suitable inactive vehicle, croton oil was used as a counterirritant. However, it acts sopowerfully that the oil was deemed unsafe for use either as acathartic or as a counterirritant. Several efforts toward theisolation of the toxic and vesicant principles never went beyond a methanol-soluble fraction called "croton resin," which
accounts for most of the toxic and vesicant activity of crotonoil (9, 12,41).
In 1941, in experiments on the skin of mice, Berenblumdetected what he first called the "cocarcinogenic" activity ofcroton oil and of "croton resin" (3). Some years later, after an
important modification of this trial by Mottram (34), Berenblum and Shubik (6—8)devised their well-known experimentof treating the skins of mice with one single subcarcinogenicdose of carcinogenic hydrocarbon followed by repeated applications of croton oil. From the results of such and similarother experiments, the "two-stage hypothesis" of skin
carcinogenesis was derived (4).In the years to follow, this type of experiment on mouse
skin became one of the most important although controversialapproaches in the analysis of the mechanism of carcinogenesis.[In the following, such experiments will be referred to as"Berenblum experiments" (21)]. The interpretation of the
first or "initiation" stage of Berenblum experiments as the
result of essentially irreversible biologic events was readily accepted. However, the interpretation of the second or "promotion" stage remained controversial for many years, espe
cially after a weak but definite tumorigenic activity of crotonoil was detected (see Refs. 10, 36). In case of irreversibletumorigenic events caused by croton oil, the oil would be justanother carcinogen. In case of reversible tumorigenic eventscaused by croton oil, a special type of cocarcinogenic activitywould have been demonstrated which could be called tumor-promoting activity (10). If the nature of the biologic activityof croton oil could be established definitely in the sense of atumor promoter rather than as a carcinogen, Berenblum experiments might provide useful models for investigations into thebiochemical mechanism of tumorigenesis(19, 21).
Since croton oil is a multicomponent mixture of lipids, theoil as such is not suited for investigations into the nature of itsbiologic activities and their relation to tumorigenesis (18,19).Necessarily, then, Berenblum (3) and many others tried topurify and isolate the cocarcinogenic or tumor-promoting principles from the oil. All of these attempts also got stuck with"croton resin," which represents an extraordinary combina
tion of nasty biologic and most unusual chemical properties.Some years ago we succeeded in the isolation (13, 18, 24,
28, 30) of the active principles from croton oil and its chemical as well as biologic characterization (2, 21, 22), employingbiologic assays for toxic, irritant, and tumor-promoting activities combined with smooth and efficient separation tools suchas liquid-liquid extraction methods. As an introduction toproblems currently under investigation in our laboratory at theGerman Cancer Research Center in Heidelberg, I shall summarize very briefly some results which have been publishedalready. Also it may be noted that the more general term"cocarcinogen" will be used rather than "tumor-promoter"
until it will be possible to definitely characterize the biologicactivity of the pure active principles in a satisfactory manner.
The Hydrophilic Fraction from Croton Oil. From the methanol-soluble hydrophilic fraction of croton oil, which roughlyresembles what earlier investigators called "croton resin," an
unexpectedly high number of eleven molecularly uniformcompounds has been obtained. They are highly toxic in frogsand represent essentially all the irritant and cocarcinogenicactivities of the oil as assayed in mice. These compounds havebeen identified as until then unknown diesters of the sameparent alcohol phorbol, each with one short- and one long-chain fatty acid (see Chart 1). Also these eleven diesters may
R^ = long, R2 = short chain Fatty acid residue compound group A
Rj = short, R2 = long chain Fatty acid residue compound group B
Chart 1. Structure and stereochemistry of the biologically activephorbol-12,13-diesters from croton oil.
be divided into two groups, A1—A4 and Bj—B7 the individual
diesters within each group all showing practically identical Rfvalues (0.3 and 0.4 respectively) in thin-layer chromatography.
The structure of phorbol has been worked out (23, 31) asshown in Chart 1. Thus phorbol was until now an unknowntetracyclic diterpene exhibiting 8 centers of asymmetry at Catoms 4, 8, 9, 10, 11, 12, 13, and 14. The six oxygen functions reside in a tertiary a-ketol (acyloin) group at C-3 and C-4involving the five- and the seven-membered rings, in ana-glycol group at C-12 and C-13 involving the six- and thethree-membered rings and in a tertiary hydroxyl at C-9 interconnecting the seven- and the six-membered rings. Also, therelative configuration and the conformation of seven of theeight asymmetric centers, excepting C-10, was established(23). Finally, in the fall of last year (32), using a bromine-containing derivative of phorbol prepared in our laboratory,the structure shown in Chart 1 was confirmed and completedwith respect to relative configuration of H-10 by X-ray diffraction analysis in the laboratory of Prof. Hoppe in Munich.Furthermore, by this method the absolute configuration ofphorbol was determined as seen in Chart 1. On the lower rightside of Chart 1, the three-dimensional structure of phorbol isshown. A British group (37) also confirmed the structure andrelative stereochemistry of phorbol by X-ray diffraction analysis of a bromine derivative of phorbol different from the onewhich was used by us.
With the structure of phorbol unraveled, two problems remained regarding the biologically active phorbol diesters: (a)which two hydroxyls out of the five of phorbol are esterifiedin the diesters isolated from the oil; and more specifically (b)what kind of isomerism is involved in those two pairs of compounds A2 and B7 as well as A3 and B4 which consist ofindividuals with different RF values, but nevertheless exhibitidentical molecular formulae and the same acid components?
Since the analytic methods applied indicate that the twoester functions involve the a-glycol group of phorbol, studieshave been undertaken (2, 29) aimed at the partial synthesis ofphorbol-12,13-diesters in order to solve the remaining prob
lems. The essence of these investigations is summarized inChart 2. As recorded on the left side of Chart 2, partial synthesis starts from phorbol (I), which is easily obtained fromcroton oil (11). Esterification with either long-chain fatty acidchlorides or with acetic anhydride yields phorbol-12,13,20-triacylates ila or phorbol-12,13,20-triacetate lib respectively.In a subsequent step by base-catalyzed transesterification,phorbol-12-monoesters such as Ilia and Ilio are prepared.Acetylation of long-chain fatty acid 12-monoesters (Ilia)yields mixed functional triesters (IVa). In a similar reaction,phorbol-12-monoacetate Ilio is esterified in the 13 and 20position with long-chain fatty acid chlorides to give mixedfunctional triesters (IVè). Finally, in the triesters IVa and IVbthe ester group at C-20 may be removed selectively by acid-catalyzed transesterification. Depending on the starting material used, this step yields either phorbol-12,13-diesters of TypeVa with the acetyl residue in the 13 position and the long-chain acyl residue in the 12 position, or Type Vb phorbol-12,13-diesters with inverse positions of the acyl residues.Through these and similar routes (11), unlimited amounts ofphorbol-12,13-diesters are now available. In order to take careof the numerous requests from the scientific community, wehave persuaded a German chemical company Th. Schuchardt,München,to make available Compound A, commercially byour partial synthesis (11).
In order to study systematically their physical, chemical, andbiologic properties, we have prepared a series of each of theisomerie phorbol-12,13-diesters of Type Va and Vb (11). Intheir physical and chemical properties, characteristic differences exist depending clearly upon the type of 12,13-isomerunder consideration, as summarized in Table 1. For example,the diesters of Type Va all show the same RF value; however,it is smaller than the RF value of the diesters of Type Ve.There is also a characteristic difference between the two typesof positional isomers with respect to the melting points oftheir 4 -nitroazobenzene-carboxylic acid-{4)-esters and theirIR-spectra. As a rule, it was found that, in mass-spectrometricfragmentation, acyloxy residues attached to C-12 in phorbol-12,13-diesters leave the molecule as radicals, whereas acyloxyresidues attached to C-13 leave the molecule as a completeacid F (see Table 1).
These physical and chemical differences may be used as atool for rapid identification of even very small amounts ofisomerie phorbol-12,13-diesters. Thus, reinvestigation (30) ofthe eleven phorbol diesters isolated from croton oil revealedthat all those diesters which have been classified via their RFvalues into Compound Group A carry their long-chain fattyacid residue in the 12 position and their short-chain fatty acidresidue in the 13 position. All phorbol diesters classified asCompound Group B show inverse positions of their fatty acidresidues. Consequently the isomerism concerning the naturalCompounds A2/B7 as well as A3/B4 was recognized as positional isomerism (11) as indicated in Table 2: CompoundA2 is 12-O-decanoyl-phorbol-13-acetate, whereas CompoundB7 is the corresponding positional isomer. Similarly Compound A3 carries the dodecanoyl moiety in the 12 positionand the acetyl moiety in the 13 position. Again for CompoundB4, simply the position of these acid residues has to be exchanged (see also Chart 1).
Chart 2. Pathway s for partial synthesis of phorbol-12,13-esters.
Also the two fractions isolated from croton resin by vanDuuren and his group (43) and sent to Heidelberg have beenidentified by similar means (Table 3). We found that his Fractions C and A both give single spots in thin-layer chromatog-raphy showing different RF values. Consequently these fractions belong to the A and B groups respectively (Table 1). Bymass spectrometry, Fraction C was found to be essentiallypure and identical with our Compound Aj, that is 12-0-tetra-decanoyl-phorbol-acetate-13. Van Duuren's Fraction A, how
ever, by mass spectrometry was revealed to be a mixturemainly consisting of the four phorbol-12,13-diesters, Bj, B2,B4, B7, and small amounts of Compound Aj.
With the molecularly uniform and chemically characterizedactive principles from croton oil at hand, the basic problem ofwhether it is a "carcinogen" or a "tumor promoter" may be
decided. Furthermore, interesting questions as to the relationsbetween chemical structure and biologic activity come up. Yetan assay system providing for quantitative biologic evaluationswas lacking: the quantitation and comparison of carcinogenicand/or cocarcinogenic activities from Berenblum experimentsis highly unsatisfactory as long as data such as percentage oftumor-bearing mice or tumors/mouse are used withoutadequate statistical treatment.
Table1PropertiesRF
value"NABS-20-esterIR
spectrumVa4|-OH
9§-OH121-OCOR13§-OCOCH320*-OH0.3m.p.
<100°C1710cm"1Mb4|-OH
9§-OH12j-OCOCH3131-OCOR20*-OH0.4m.p.
>140°C1705
u. 1736cm-1
Mass spectrumM-CH3COOH
M -CHjCOO*
M-N/VV COOH
Consequently, by thoroughly investigating dose-response relationships in Berenblum experiments, we developed a quantitative assay procedure for cocarcinogenic activity. As a resultit was found that probit analysis will provide an adequatestatistical model (21, 22). Thus, if the percentage of tumor-bearing animals is plotted on probability paper against a linearscale of the weeks of treatment, linear regression lines will beobtained as shown in Chart 3. In this manner average latencyperiods (tso) may be estimated with corresponding confidencelimits as indicated in Chart 3 and with a significance level agenerally set to 0.05. Since manual evaluation of such probitanalyses (21, 22) is both most laborious and tedious, suchprobit analyses were recently programmed (26) for our center's IBM 360/30 computer, in collaboration with the Institute
for Documentation, Information and Statistics.
Table 2
CompoundisolatedA2
B7A3B4Identical
with syntheticphorbol-12,13-diesterRf«0.3
0.40.30.4R!
(Cl2)Decanoyl
AcetylDodecanoyl
AcetylR2(Cl3)Acetyl
DecanoylAcetyl
Dodecanoyl
Identification of the isomerie phorbol-12,13-diesters isolated from croton oil as positional isomers.
"CH 2C12/acetone = 3/1.
Table 3van Duuren's
compound
c
A
RF"
0.2
0.3
Identical with:
Compound A, (12-TPA-13)
Mixture of 5 phorbol-12,13-diesters:Aj,B j,82)64,67
Chemical and physical properties of isomerie phorbol-12, 13-diesters Identification of van Duuren's phorbolesters by mass spectrometry.Type Va and Vb.NABS, 4'-nitroazobenzene-carboxylic acid-(4). 12-TPA-13,12-O-tetradecanoyl-phorbol-acetate-13.
Chart 3. Average latency periods tso and relative tumor-promotingactivities of croton oil and Compound A.J. 14/14 NMRI mice, standard,i:0.1 MMdimethylbenz(a)anthracene (DMBA), TDSO = 2 p X tso, significance level a = 0.05.
Thus the regression lines as recorded in Chart 3 have beenobtained by a computerized procedure so as to fit in an optimal manner the weekly counts of tumor-bearing mice in ourstandard set up. Dimethylbenz(a)anthracene (DMBA) was usedas initiator (i = 0.1 /UM) followed by twice weekly applications of
either 500 Mgof croton oil (right side, Chart 3) as well as 10(middle, Chart 3) or l /ig (left side, Chart 3) of Compound A,.From the regression lines the average latency period tso maybe read in weeks. For example, with 500/ug of croton oil, tsowas found to be 10.7 weeks. The confidence limits have beencalculated to range from 10.2 to 11.3 weeks, as indicated inChart 3. If the single dose of cocarcinogen is expressed by p,the corresponding average total dose (tumor dose 50, TD50) issimply calculated by TDSO = 2 X pX tso. For example, in the
case of croton oil, TDSO is 10.7 mg. Depending on the singledoses used, for Compound At two TD50 values of 156 and 26jug respectively are being obtained. In order to compare theactivities of two compounds, their TD50 values may be compared provided the corresponding regression lines are parallelwithin a significance level set to a = 0.05. This is the case forthe regression lines in Chart 3 as checked by computer. Thus,at a single dose level of 10 pg of Compound Aj, the latter is69 times more active than croton oil. At a dose level of 1 /ag ofA!, this phorbol-12,13-diester is 420 times as active as crotonoil. The appearance of two TDSO values for Compound A,(Chart 3) indicates that the single dose of p = 10 jug is too
high.Within such data for both carcinogenic as well as cocarcino-
genic activities, a quantitative Berenblum experiment (21,22)may be performed as shown in Chart 4. The first group of 28mice was treated with an initial dose of 0.1 UM DMBA and,one week later, twice weekly with the same dose of DMBA.Tumors developed with an average latency period tso of 12 ±3 weeks corresponding to an average total TDSO of 2.4 ±0.6MM DMBA. A linear dose response relationship is obtained(Chart 4, Regression Line 1). The second group received the
initial dose of DMBA alone followed by twice weekly treatments with solvent. Essentially no tumors appeared within 60weeks. The third group received no initial DMBA followed bytwice weekly applications of 0.0016 MMof Compound A,.Again no tumors appeared within 60 weeks. However, a highnumber of tumors developed when the treatments used singlyin Experiments 2 and 3 were combined to furnish Experiment4. As may be seen from Regression Line 4 as compared toRegression Line 1 and from the corresponding average latencyperiods tso (Chart 4), Experiment 4 may be compared withExperiment 1 using DMBA alone. Thus in Experiment 4, tso isnot significantly different from the latency period in Experiment 1. The corresponding tumor doses show that DMBAproduces the same tumor yield if, on a molar base, about 60times as much DMBA as compared to Compound A j is used forthe sequential applications.
This set of experiments indicates a qualitative difference (21,22) between the biologic events caused by DMBA and Compound Aj respectively. Those induced by the typical carcinogen DMBA are essentially irreversible, as shown by thepersistence of the information "potential tumor cell" coined
by one single dose of DMBA such as 0.1 MMfor months, i.e.,many cell generations (21). However, the events induced by asingle dose of 0.0016 MM °fCompound Aj are practicallyreversible since the sequential application of such doses doesnot cause development of carcinomas and not even benigntumors. Thus, in the dosage used, Compound A, is not acarcinogen but a cocarcinogen to be classified as tumor pro-motor. In addition to the evidence presented, this statement isalso supported by histologie analysis of the tumors producedin both experiments 1 and 4. In the case of Experiment 1,approximately 80% of the tumors were found to be epithelialcarcinomas, whereas in the case of Experiment 4 more than90% of all tumors produced have been diagnosed as benignpapillomas.
It has to be mentioned, however, that without initiation andby subsequent applications of single doses of Compound Ajlarger than those used in Experiment 3, after a long averagelatency period Compound Aj will produce tumors (21, 22,26). However, this is not to be evaluated as an expression oftrue carcinogenic activity, that is to say, cumulative action ofirreversible biologic events. Rather, and according to the reversibility of the biologic events demonstrated above, it is tobe understood as the result of cumulative action of reversiblebiologic events. In other words, doses of Compound A, greaterthan 0.0016 MMmay be too high in order to allow the effectof one single dose decline to zero before the next single dose isapplied, as it is the case in Experiment 3 (Chart 4). Accordingly Compound A! as found in independent experiments (E.Hecker, unpublished) did not show any initiating effect indoses comparable with initiating doses of DMBA. Also, tumorsproduced by long-term applications of tumorigenic doses ofCompound A, have been diagnosed histologically to be almostexclusively papillomas rather than carcinomas (26). Similardata, as shown in Chart 4 for Compound Aj, have been obtained for all the phorbol diesters isolated (21, 22, 26; E.Hecker, unpublished).
With regard to the two-stage technic, in 1964 Berenblum (5)made the statement: "We still have very far to go before we
) Standard deviation. *•)0.1ml , **+) 29-41 werk 2-3/22
Chart 4. Quantitative Berenblum experiment with initially 0.1 /IM dimethylbenz(a)anthracene (DMBA) and twice weekly 0.0016/LiMA.«StrainNMRI, 14/14 mice/group; solvent: acetone p.a.
reach the degree of precision which the biochemist enjoys." I
think more quantitative data on carcinogenic as well as tumor-promoting activities in terms of TD50 values will contribute agreat deal to this goal. Such data have already proved to beindispensable as a quantitative background for investigationsinto the biochemical mechanism of skin carcinogenesis as wellas into the relations between chemical structure and biologicactivity going on in our laboratory. For example, the parentalcohol phorbol does not show any activity either in mice asirritant or tumor promoter or in frogs as a toxic agent (21,24, 25), whereas phorbol-12,13,10-triacetate shows a slight butdefinite irritant and tumor-promoting activity. However, quantitatively it does not approach the activity of croton oil,whereas Compound Aj proved to be much more active thancroton oil (Chart 3). Furthermore, the tumor-promoting activity of some synthetic 12-0-acyl-phorbol-l 3-acetates as afunction of chain length of their long-chain fatty acid residuesin the 12 position has been investigated. For some of theseesters, TD50 values and confidence limits are given in Chart 5.AllTDso are significantly different and the corresponding regression lines parallel as checked by computer. Dependingupon the number of C atoms in the O-acyl-residue, a minimumvalue of TD50 is clearly reached with fourteen C atoms. Therefore Compound A, is the most active tumor-promotingphorbol-12,13-diester isolated from croton oil known so far.
Aiming at better quantitative data for the irritating activities,the ear redness assay in mice as used during the isolation procedure was reinvestigated: reading and differentiating the threedegrees of redness which define the Irritation Unit or IU (20)is difficult. Also statistical evaluation with respect to standarddeviation and significance level of the IU is not possible.Therefore, the assay was rearranged and extended in order todetermine an irritation dose 50 (ID50) (25), still using thedetermination of the IU as a pilot experiment. In the mainexperiment starting with the irritation unit, a series of 5 dilutions is prepared in geometric degression. Each of the 5 resulting concentrations is applied to one ear of each of six mice(sex ratio 1:1). After 24 hours the redness of the ears treatedis recorded differentiating only "redness" or "no redness."
The cumulative percentage of mice with red ears plotted on
0.9
0.8
0.7
0.6
0.5
l10 12 K 16 18
chain length 12-0-acyl( number ot C-atoms ) ""*
Charts. TDSQ and chain length of 12-0-acyl-groups in some 12-0-acyl-phorbol-13-acetates, synthetic. 14/14 NMRI mice, standard, i:0.1dimethylbenz(a)anthracene (DMBA), p:0.02, significance level û= 0.05.
probability paper against the logarithm of the correspondingdoses reveals linear regression lines. The ID50 may thus bedetermined, including the standard deviation a for a significance level a set to 0.05. Thus, for example, the followingID50 have been obtained: 0.5 /^g/ear for croton oil, 0.01Mg/ear for Compound Aj and 2.2 /^g/ear for phorbol-12,13,20-triacetate. By comparison, Compound Aj is about50 times as irritating as croton oil, and phorbol triacetate issomewhat less active than croton oil. The sequence of theseID50 values corresponds with the sequence of TDSO values forthese compounds as mentioned above. Also it may be notedhere that, contrary to some earlier statements in the literature(14, 40) the irritant and the tumor-promoting activities cannotbe divorced throughout the entire purification procedure ofcroton oil.
The Hydrophobic Fraction of Croton Oil. With quantitativemeasures at hand such as TDSO and ID50, we began to followup an interesting observation which we made some years ago.At the very beginning of our fractionation procedure (27, 30),croton oil is split into two fractions (Chart 6): (a) A hydrophilic neutral fraction, from which the eleven phorbol-12,13-diesters have been isolated. In Chart 6 this fraction accounts for4.9% by weight of the croton oil and shows considerable irritantactivity (IDSO 0.03 jug/ear) compared to croton oil (ID50 0.19/Lig/ear). (b) A hydrophobic fraction, in Chart 6 accounting for95% by weight of croton oil. This fraction shows an ID50more than 100 times higher than the IDSO of the hydrophilicfraction and about 20 times higher than the ID50 of crotonoil. It can therefore be considered as showing practically noactivity. Most interestingly, however, this fraction containswith 0.6% an even higher relative amount of phorbol than theactive hydrophilic fraction.
Knowing about the low irritating as well as tumor-promotingactivity of phorbol-12,13,20-triacetate as mentioned above, wesuspected that phorbol might possibly be present in the hydro-phobic fraction of croton oil as 12,13,20-triesters. In this case,
Cocarcinogenic Principles of Croton Oil
as experienced in our synthetic work (11), an acid-catalyzedtransesterification would selectively cleave the esterbond inthe 20 position (Chart 2) and produce biologically active phor-bol-12,13-diesters. Indeed, by acid-catalyzed transesterificationof the hydrophobic fraction, an "activated hydrophorbic fraction" was obtained (Chart 6) with an IDSO of approximately
0.1 of that of the starting material. As shown in Chart 6, thisactivated hydrophobic fraction was subjected to a fractionation procedure similar to that developed for the hydrophilicfraction of croton oil (13, 27, 30). Thus two highly activeCompound Groups A' and B' have been obtained (Chart 6) the
latter still being under investigation in our laboratory. AfterCraig distribution (Chart 7), Compound Group A' was shown
to consist of the same highly irritant phorbol-12,13-diestersAj, A2, A3, and A4 as well as the inactive monopalmitin (30)which have been found already in Compound Group A fromthe hydrophilic fraction of croton oil. In addition, however, anas yet unknown phorbol diester A'S with an ID50 of 0.0525
jug/ear was isolated (Chart 7). By the methods described abovefor characterization of phorbol esters, Compound A'S was
found to contain tiglic- and «-butyric acid with the longer
CROTON OIL (100%)
HYDROPHIUC NEUTRAL FRACTION I 4.9 '/. )
IDjo 0 03 jjg/ear. PHORBOL 05V.
HYDROPHOBIC FRACTION 195%)
ID50 i 3 ug /ear, PHORBOL 0 6 V.
TRANSESTERIFICATION
PHORBOLESTERS ArA4 and B^ - By ACTIVATED HYDROPHOBIC FRACTION 193%)
Chart 7. Craig distribution of Compound Group A (ID50:0.045 /Jg/ear). System: CC14:CH3OH:H2O (2:1.0:0.15), V = 12/10, z = 1020. Singlewithdrawal procedure, n = 1600, T = 20 C. Fractions combined: battery 4, phases withdrawn 9.
chain tiglyl residue in the 12 position and the shorter chain«-butyryl residue in the 13 position (Chart 1). Investigationsaiming at the determination of the TD50 of this new phorbol-12,13-diester are currently going on.
Investigations Concerning the Active Principles fromEuphorbia Species
In experimental carcinogenesis the choice of tumor promoters has always been very limited. In fact, besides somesynthetic compounds of relatively weak potency, only thephorbol-12,13-diesters from croton oil show an interestinglyhigh tumor-promoting activity. However, despite their multiplicity, the chemical nature of these tumor promoters is relatively uniform. It may be interesting, therefore, to ask thequestion as to how many other kinds of tumor-promotingprinciples nature has to offer. Thus we became interested inthe biologically active principles of the genus of spurge orEuphorbia closely related to the genus of Croton. From thespecies of Euphorbia presently under investigation inHeidelberg, I would like to report some of our results obtainedwith Euphorbia lathyris and Euphorbia ingens.
Seed Oil from Euphorbia lathyris. Euphorbia lathyris is anherb growing in Southern Russia, the Mediterranean region ofEurope, and in Northern America. The three-celled capsuleswhich contain the seeds are similar in shape but smaller thanthose of Croton tiglium.
Since the seeds of Euphorbia lathyris may be produced inlarge amounts while growing the plant on relatively unfertilesoil, its use as a fuel has been reported; the seeds and the seedoil have been used also as a cathartic of milder action thancroton oil (33). On skin the oil causes a mild burning (44). Thebreeding of Euphorbia lathyris has been investigated in Russiain the late thirties (15, 39), and the seed oil was recommendedas a raw material for the production of olein and soap (33,39).
The chemistry and toxicology of the oil regarding its biologically active constituents has been investigated only fragmen-tarily. Dublyanskaya (15) reported the isolation of a fractionwhich separates from the seed oil as crystals, m.p. 199, 3-199,7°C("Euphorbia steroid") and which she believed to be iden
tical with the toxic principles from the oil (16). Later on byfurther fractionation she differentiated "Euphorbia steroid"
and a colorless resin, within the methanol-soluble fraction ofthe oil, the latter mentioned as the chief toxic constituent ofthe original oil (17). Jaretzki and Koehler (33) tried to purifythe fraction "Euphorbia steriod" and demonstrated its phar
macologie inertness. Also they stated certain similarities between the toxicity in frogs of the methanol-soluble fractionsfrom Euphorbia lathyris oil and croton oil.
The fractionation procedure for Euphorbia lathyris oil developed in our laboratory since 1965 and followed by determinations of ID50 as well as TDSO is shown in Chart 8. The oilextracted from the seeds shows an IDSO of 13.5 ^g/ear andaTD50 of 142 mg/mouse: the oil is considerably less active thancroton oil (Charts 3, 6). After extraction of the oil withmethanol, a hydrophobic fraction is obtained with an IDSO ofmore than 100 ßg/earand without tumor-promoting activity.Both activities reside in the hydrophilic fraction constituting10.3% by weight of the oil. By treatment of this fraction withsodium carbonate, an inactive acid fraction was obtained aswell as a neutral fraction with about ten-fold the irritant activity of the oil, and about double the activity regarding tumorpromotion. Multistage Craig distribution of the neutral fraction (Chart 9) shows in Fractions 300-800 three crystallinecompounds Lj, L2, and L3 together with the withdrawnphases amounting to 5.7% of the oil (Chart 8), and with noirritating activity (i.e., IU > 100 Mg/ear). However, Fractions112-304 and 772-1020 show irritant activity (Chart 9), theID50 of these fractions being 0.3-0.6 jug/ear and 0.36 ¿Ãg/earrespectively (Chart 8). From the active Fractions 772—1020,an active compound Ls has been obtained by thick-layer chro-matography showing an ID50 of 0.065 pig/ear. During theseparation procedure, L5 may be partially converted into abiologically inactive Compound L4 with identical molecularformula.
In Table 4 some results of the chemical characterization ofthe biologically inactive Compounds LI—1,3 and of thebiologically active Compound LS have been summarized.According to its m.p., Compound LI is identical with thefraction "Euphorbia steroid." Whereas diverse empirical
formulae have been proposed by earlier investigators (15, 17,33,42), we definitely established the empirical formulaC32H4008 by mass spectrometry. Furthermore, we foundthat this compound carries three ester functions, two with
Chart 8. Preparation and fractionation of Euphorbia lathy ris oil.
acetic and one with phenylacetic acid. The parent alcohol is aditerpene C2oH3oOs [Ourisson and collaborators at theUniversity of Strasbourg (France) recently dealt with thechemical characterization of the fraction "Euphorbia steroid"
and have confirmed some of our results (G. Ourisson, privatecommunication)]. Similarly the other crystalline compoundsL2 and L$ are polyesters of diterpenic parent alcoholsdifferent from that of Compound LI , as shown in more detailin Table 4. The biologically active Compound LS does notcrystallize and was found to be a hexadecanoic acid monoesterof a fourth diterpenic parent alcohol C2oH2gOs (Table 4).
Further investigations of these compounds as well as theactive principles of Fractions 772-1020 of the Craig distribution are under way.
Latex from Euphorbia ingens. Some of the species of spurgeoccurring in tropical regions grow up to huge cactus-like trees,for example Euphorbia ingens. During the second World War,latex which may be obtained by precipitation of the milky
juice of Euphorbia ingens was investigated as a possible sourceof rubber by Warren and coworkers (1, 35) in South Africa.Juice as well as latex of Euphorbia ingens is highly toxic, andthey both produce irritation and blistering of the skin in amanner similar to that of croton oil. In 1961 Roe and Pierce(38) investigated Euphorbia ingens latex in Berenblum-typeexperiments and found a potent tumor-promoting activity.
The chemical nature of the active principles of Euphorbiaingens latex is unknown. Only biologically inactive triterpenessuch as euphol and euphorbol have been isolated from thelatex (1,35).
In 1965 we began developing a fractionation procedureaimed at the isolation of the active principles of Euphorbiaingens latex (Chart 10). In a first stage the latex is extractedexhaustively with acetone leaving an insoluble and biologicallyinactive residue. The acetone-soluble fraction shows an ID50of 0.74 jLtg/ear (Chart 10). By O'Keeffe distribution a
hydrophobic fraction may be separated with little irritant
Chart 9. Craig distribution of the neutral fraction from Euphorbia lathyris oil. System: Petroleum ether:methanol:H2O (15:10:0.5), V = 10/10,z = 1010. Single withdrawal procedure, n = 2800, T = 20°C.Fractions combined: battery 4, phases withdrawn 9.
EUPHORBIA INGES LATEX
ACETONE INSOLUBLE RESIDUE
ACETONE EXTRACTION OOP'/.)
1050 07C>jg/*ar
O'KEEFFE-DISTRIBUTIONHYDROPHOBIC FRACTION 176V.)
1050 B 9 *jg/ear
HYDROPHILIC FRACTION (19.4V.)
1050 Ollpg/ear
CHROMATOGRAPHY
FRACTION K 7- BV.) FRACTION 1ÕÕI5-6*/.) FRACTION H l~«'/.l
activity, whereas the ID$Q in the hydrophilic fraction rises to0.11 M8/ear- By chromatography of this hydrophilic fractionon silica gel, three subfractions I-III may be obtained, ofwhich I and II contain only little irritant activity. Fraction IIIshows about the same ID$Q as the preceding hydrophilicfraction. It may be noticed from Chart 10 that the purificationof Fraction III by dry weight is about four-fold, whereas theIDso values of Fraction III and the hydrophilic fractionindicate practically no increase. Thus, during chromatographysome of the irritant activity may have been destroyed. As yet,by Craig distribution of Fraction III (Chart 10), a molecularlyuniform Compound Ij with an ID$Q of 0.9 /ng/ear has beenobtained beside another zone of biologic activity.
In Table 5 the chemical data and the IDso of the newirritant and tumor-promoting compounds as yet isolated from
Euphorbia lathyris oil and Euphorbia ingens latex are compared with Compound Aj from croton oU. Although isolatedfrom two quite different Euphorbia species, Compound Ij andCompound LS are identical, as may be seen from theirmolecluar formulae, Rp values, acid residues, IDso values, andthe molecular formulae of their parent alcohols (Table 5).However, they differ definitely from the biologically inactiveconversion product L<t, which may be obtained also fromCompound I \ under conditions similar to that described abovefor Compound L$. Whether the difference between Compounds I j /Lg and L4 is solely restricted to the location of thehexadecanoic acid ester group or whether the parent alcoholitself is involved in this conversion remains to be seen. It isestablished with certainty, however, that the two compoundsl! and Ls are chemically different from Compound Al as wellas all other phorbol-12,13-diesters from croton oil with respectto both ester groups and parent alcohols. The common featureof all these tumor promoters is the diterpene nature of theirpolyfunctional parent alcohol esterified with one or two fattyacids. Therefore, they constitute a defined group of cocarcino-gens or perhaps tumor promoters of plant origin. With theirqualitative and quantitative similarity of biologic activities, aswell as the obvious multiplicity of individual compounds ofsimilar chemical structure, they perhaps confront us with asituation similar to that during the Thirties, when thecarcinogenic hydrocarbons were detected as a defined group ofcarcinogens. Our new group of cocarcinogens and tumorpromoters is and will be of outstanding interest for both adeeper understanding of the mechanism of tumorigenesis andcarcinogenesis in animal experiments at both the biologic andthe molecular level, and for investigations into the environment of human beings regarding tumorigenesis and carcinogenesis.
ACKNOWLEDGMENTS
I would like to end with the expression of my appreciation to mycoworkers who joined me in this field with both lasting enthusiasm andutmost ability. They deserve the major credit for the work which I hadthe honor of describing in this lecture.
Chemical characterization of the diterpeneesters from Euphorbia lathyris oil."Ether/petroleumether = 3/1.''Ethylacetate/acetone = 2/1.cln the literature, "Euphorbia steroid."
Chemical and biologic characterization of the new irritant and tumor-promoting compounds from Euphorbia lathyrisoil and Euphorbia ingens latex. Conversion product L^ comparison with Compound Aj from croton oil.
"ethyl ether/petroleum ether = 3/1.
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