[CANCER RESEARCH 42. 1193-1198. April 1982]0008-5472/82/0042-OOOOS02.00

Isolation and Identification of Kahweol Palmitate and CafestolPalmitate äsActive Constituents of Green Coffee Beans ThatEnhance Glutathione S-Transf erase Activity in the Mouse1

Luke K. T. Lam,2 Velta L. Sparnins, and Lee W. Wattenberg

Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota 55455

ABSTRACT

Glutathione (GSH) S-transferase is a major detoxificationenzyme system that catalyzes the binding of a variety of elec-trophiles, including reactive forms of chemical carcinogens, toGSH. Green coffee beans fed in the diet induce increased GSHS-transferase activity in the mucosa of the small intestine and

in the liver of mice. A potent compound that induces increasedGSH S-transferase activity was isolated from green coffee

beans and identified as kahweol palmitate. The correspondingfree alcohol, kahweol, and its synthetic monoacetate are alsopotent inducers of the activity of GSH S-transferase. A similar

diterpene ester, cafestol palmitate, isolated from green coffeebeans was active but less so than was kahweol palmitate.Likewise, the corresponding alcohol, cafestol, and its monoacetate showed moderate potency as inducers of increased GSHS-transferase activity. Kahweol palmitate and cafestol palmitate were extracted from green coffee beans into petroleumether. The petroleum ether extract was fractionated by preparative normal-phase and reverse-phase liquid chromatographiessuccessively. Final purification with silver nitrate-impregnatedthin-layer chromatography yielded the pure palmitates of cafestol and kahweol. The structures were determined by examination of the spectroscopic data of the esters and their parentalcohols and by derivative comparison.

INTRODUCTION

GSH3 S-transferase has been studied extensively as a major

detoxification enzyme system that catalyzes the binding of awide variety of electrophiles to GSH (3,10). Since most reactiveultimate carcinogenic forms of chemical carcinogens are electrophiles, GSH S-transferase may play a significant role in

carcinogen detoxification. Enhancement of the activity of thissystem potentially could increase the capacity of the organismto withstand the neoplastic effects of chemical carcinogens.Experiments have been carried out to determine the correlationbetween increased GSH S-transferase activity in a target organ

of chemical carcinogenesis and its response to the carcinogen.For this purpose, the forestomach of the mouse was used.Members of several classes of inhibitors of BP-induced neoplasia of the mouse forestomach were studied for their effects onthe GSH S-transferase activity in that structure. Five of the

1 Supported by USPHS Contract NOI-CP-85613-70 and Grant CA-09599.

Presented in part at the 182nd American Chemical Society National Meeting,New York, N. Y., August 23 to 28, 1981 (14).

2 To whom requests for reprints should be addressed.3 The abbreviations used are: GSH, glutathione: BP, benzo(a)pyrene; PE,

petroleum ether; LC, liquid chromatography; TLC, thin-layer chromatography;NMR, nuclear magnetic resonance.

Received October 7, 1981 ; accepted January 4, 1982.

compounds increased the GSH S-transferase activity of theforestomach between 78 and 182%. They were p-methoxy-phenol, 2-fe/t-butyl-4-hydroxyanisole, coumarin, a-angelica-

lactone, and benzyl isothiocyanate (18). All 5 compoundsinhibited BP-induced neoplasia of the forestomach (13, 20,21). These data suggest that the capacity to enhance GSH S-

transferase activity might be used as a method of identifyingcompounds or natural products likely to inhibit BP or othercarcinogens detoxified in a similar manner (18).

In efforts at identifying dietary constituents that might protectagainst chemical carcinogens, the effects of natural productson GSH S-transferase activity were studied. During this inves

tigation, it was found that consumption of diets containingpowdered green coffee beans resulted in a very marked enhancement of GSH S-transferase activity in the liver and mu

cosa of the small bowel of the mouse. The magnitude ofinduction was as high as that obtained with any test compoundor natural material previously investigated. The coffee beansused in the original study were from Guatemala. In subsequentwork, coffee beans from Brazil, Colombia, El Salvador, Mexico,and Peru were all found to have a comparable enhancing effecton GSH S-transferase activity (17). Roasted coffee and instant

coffee were found to have a weaker inducing activity than didthe green coffee beans studied, i.e., slightly less than 50% asmuch. Decaffeinated instant coffee showed activity similar tothat of instant coffee. Investigations were then begun to identifythe constituents of green coffee beans having the capacity toenhance GSH S-transferase activity. In the present study, it

was found that the coffee constituent kahweol palmitate is ahighly potent inducer of increased GSH S-transferase activity.The palmitate of a closely related diterpene, cafestol, wasactive as an inducer of GSH S-transferase but less so than waskahweol palmitate.

MATERIALS AND METHODS

Extraction of Green Coffee Beans. Powdered green coffee beans(Guatemala) were placed in a large modified Soxhlet extractor andwere extracted with various solvents of increasing polarity (Chart 1).PE (b.p. 60-70°) was used first, which was followed by benzene, ethyl

acetate, methanol, and water. Each solvent extraction was carried outover a period of 7 days. The solvent of each extraction was removedunder reduced pressure. The crude extracts were dried under reducedpressure until constant weights were obtained. The activities of theextracts were monitored by the GSH S-transferase assay.

Fractionation of the PE Extract. The active PE extract was separated into 7 fractions by preparative LC using a Waters AssociatesprepSOO liquid Chromatograph equipped with prepPak-500/silica col

umns. The elution solvent was PE;ether (2:1, v/v). The active fractionfrom preparative LC was further fractionated into 6 subfractions byreverse-phase preparative LC. Two prep PAK-500/C18 columns in

series were used with 98% methanol as the eluting solvent. Silver

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L. K. T. Lam et al.

Green Coffee Beans

PE(8%) 0H EtOAc MeOH H2O

Prep LC-silica

sÀ23(20%)4567PrepLC-C,sB

C(38%)D7 l|TLC-Sllica Gel-AgNO 3

1Palmitic

Acid(1C)isaponificationCafestolPalmiticAcid(2c)1Kahweol

<2a>4

Chart 1. Extraction and isolation scheme for kanweol and cafestol palmitates0H, benzene; EtOAc, ethyl acetate. PrepPAK-500/silica columns were used forthe normal-phase preparatory (Prep)LC. The eluting solvent was PE:ether (2:1,v/v). PrepPAK-500/C18 columns were used for the reverse-phase preparatoryLC. The eluting solvent was 98% methanol (MeOH). The developing solvent forsilver nitrate-impregnated Silica Gel GF thin-layer plates was PEiether (2:1 ).

nitrate-impregnated TLC was used for the final purification of individual

active ingredients.Saponification of Compounds 1 and 2. A sample of Compound 1

(or Compound 2), isolated from silver nitrate-impregnated TLC plates,

was dissolved in 10% aqueous ethanolic potassium hydroxide at roomtemperature. The reaction mixture was warmed to 50-60° for 0.5 hr.

It was then poured into ice, and the aqueous solution was extracted 3times with ether. The organic layers were combined and dried overanhydrous magnesium sulfate. The ether was removed under reducedpressure. The neutral product was crystallized from PE:ether.

The aqueous layer after ether extraction was acidified with 6 Nhydrochloric acid and then extracted with 3 volumes of ether. Theether was dried over anhydrous magnesium sulfate. The solvent wasremoved under reduced pressure, and the acid thus obtained wasdried under reduced pressure overnight.

Spectroscopic Studies. IR spectra were recorded on a BeckmanAcculab 5 spectrophotometer. UV spectra were recorded on a Beckman Model 25 spectrophotometer. Proton NMR spectra were determined on a Briiker WM 250 spectrometer at 250.13 MHz with tetra-methylsilane as internal standard. Gas chromatography-mass spec-troscopy was determined on an LKB9000 spectrometer. High-resolution mass spectroscopy were recorded on a double-beam AEI-MS-30spectrometer. Melting points were determined on a Fisher-Johns Mel-

Temp apparatus and were uncorrected.Assay for in Vivo Enhancement of GSH S-Transferase Activity.

Female ICR/Ha mice from the Harlan-Sprague-Dawley Company (In

dianapolis, Ind.) were used in all experiments. Mice were randomizedby weight at 7 weeks of age into the groups to be used in a particularprotocol. In the initial stages of the fractionation, the extracts weretaken to dryness and added to a semipurified diet consisting of 27%vitamin-free casein, 59% starch, 10% corn oil, 4% salt mix (U.S.P.

XIV), and a complete mixture of vitamins (Teklad, Inc., Madison, Wis.).The amount of each extract added was such that the diets wouldcontain the equivalent of 20% powdered green coffee beans. In eachexperiment, 20% crude powdered green coffee beans were includedas a positive control as well as a control in which there were noadditions. The diets were fed for 12 days at which time the mice werekilled. The mucosa of the proximal half of the small bowel and the liverwere taken for the determination of GSH S-transferase activity. In later

stages of the fractionation procedure in which smaller amounts of testmaterial were available, the assay technique was modified. The testmaterials (2.5 mg), dissolved in cottonseed oil, were given by P.O.intubation, 0.2 ml/mouse. The animals were killed 28 hr later. Mucosaof the proximal half of the small bowel was taken for determinations of

GSH S-transferase activity. The inductive effects on the small bowelmucosa under these conditions are about one-third of the magnitude

as in the feeding procedure. Liver is much less responsive.GSH S-transferase activity in the cytosol was determined according

to previously published procedures (18). All steps were done at 0-4°.

The tissues to be studied were homogenized in 0.1 M sodium phosphatebuffer (pH 7.5). The homogenate was centrifuged at 100,000 x g for1 hr. The supernatant was used for the assay of GSH S-transferaseactivity. The activity was determined spectrophotometrically at 30°

with 1-chloro-2,4-dinitrobenzene as substrate according to the proce

dure of Habig ef al. (8).

RESULTS

Isolation Procedure

The activity of the enzyme GSH S-transferase in the liver and

mucosa of the small bowel of the mouse was enhanced by theaddition of 20% green coffee beans to the diet. The enhancement of enzyme activity in the liver was approximately 5 timesthat of the control (Chart 28). A slightly smaller enhancement

I 2

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GCB PE BZ EA ME WA RC RS CON

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„B

GCB PE B Z EA ME WA RC RS CON

Chart 2. Activity of green coffee beans (GCB) and their solvent extracts onthe enhancement effect of GSH S-transferase in the mucosa of the small bowel(A) and in the liver (B) of mice. BZ, benzene; EA, ethyl acetate; ME, methanol;WA, water; RC, reconstituted coffee beans; PS, residue of extraction; CON,control. Bars, S.D.

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Kahweol and Cafestol Palmitates in Green Coffee Beans

was found ¡nthe mucosa of the small bowel (Chart 2A). Theoverall magnitude of enzyme activity in the latter organ, however, was much lower than that in the former. PE extraction for7 days successfully removed most of the ingredients that hadinducing activity. Subsequent extractions by benzene showedactivity that affected only the mucosa of the small bowel buthad little effect in the liver. The other solvent extracts wereinactive. When the extracts were combined with the residue ofextraction, a slight increase of activity was observed in comparison with the original green coffee beans (Chart 2).

The PE extract contained a small amount of caffeine whichcould be removed by dissolving the dried extract in a smallvolume of PE and filtering. The caffeine-free PE extract was

then fractionated by preparative LC to give 7 fractions. Fraction3 contained materials that gave similar enhancement of activityto that of the PE extract. The weight of Fraction 3 was 20%that of the PE extract. The other fractions were all inactive inthe GSH S-transferase assay (Chart 3). All attempts to furtherfractionate Fraction 3 with normal-phase preparative LC re

sulted in subfractions of mixtures of similar components. Analysis of Fraction 3 by reverse-phase high-performance liquidchromatography (Ci8-/iBondapak, eluted with methanol) indi

cated 6 peaks (Subfractions A to F) that were well separated.Subfractions A to F were separated by preparative LC. All 6subfractions were active in enhancing GSH S-transferase ac

tivity in the mucosa of the small bowel of the mouse (Chart 4).Subfraction C, which constituted approximately 38% of Frac-

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AB RC CONChart 4. Activities of the reverse-phase preparatory LC subtractions of Frac

tion 3 on the enhancement effect of GSH S-transferase in the mucosa of the

small bowel of mice. PC, reconstituted Fraction 3; CON, control. Bars, S.D.

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5. Activities of SubfractionC,fiedproducts of Compound 1 (|a.i\isits

components (| toB1A1

pi|;:;;;:;

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and thesaponi-7c)on the enhancement effect of GSHS-transferase

in the mucosa of the small bowel of mice.i '/V authentic palmiticacid;CON,

control. Bars,S.D.tion

3, showed the highestactivity in theenzyme assayandwaschosen for furtherpurification.Tofurther separate Subfraction C intoits pure components,

GCB PE 1 234567 CON

Chart 3. Activities of the different fractions separated from the PE extract(PE) on the enhancement effect of GSH S-transferase in the mucosa of the smallbowel (A) and in the liver (B) of mice. GCB, green coffee beans; CON, control.Bars, S.D.

TLC (Silica Gel G) plates impregnated with silver nitrate wereused (Chart 1). These silver nitrate-treated plates were able to

separate Subfraction C into 4 components, 3 of which wereactive (Chart 5A). The activity of Compound 1 was similar to

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L K. T. Lam et al.

that of the control level. Compound 2 showed the greatestpotency in enhancing the activity of GSH S-transferase. Administration of Compound 2 resulted in an increase of GSH S-transferase activity that was 4 times that of the control. Compounds 3 and 4 were able to induce the enzyme activity totwice the control level. Compounds 1 and 2, the componentsthat had the highest R( values, were isolated on preparativeTLC plates impregnated with silver nitrate. Compound 1, whichwas present in much higher quantity than were the other 3components, was easily purified and characterized as a purecompound. The behavior of this set of compounds duringfractionation and TLC separation indicated that they belongedto the same class of organic compounds with only minorstructural differences. The structural determination of any oneof these compounds will facilitate the identification of theremaining components. Since Compound 1 was isolated in itspure form in sufficient quantity, structural determination wasstarted with this compound. Spectroscopic evidence and derivative comparison indicated that Compound 1 was the palmi-

tate of cafestol (2). Component 2, which has a molecular weightthat is 2 mass units lower than that of Compound 1, wasidentified to be the palmitate of kahweol (1). The previouslyknown coffee constituents, cafestol and kahweol, could beisolated as the free alcohols from mild alkaline hydrolysis ofCompounds 1 and 2, respectively. While cafestol palmitateshowed very little activity as an enzyme inducer, cafestol andits monoacetate showed activity that was approximately 1.5times above control level (Chart 50) (see "Addendum"). Kah

weol and its monoacetate showed activity that was similar tothat of kahweol palmitate (Chart 6). Palmitic acid (Compound1c) was inactive as an inducer of the activity of GSH S-transferase (Chart 56).

Structural Identification

Compound 1. Compound 1 has been identified as the palmitate of cafestol (Chart 7). Mass spectrometry (70 eV) yielded:m/e 554 (M +, 71.2). The precise mass for CseHseCXiwas

Calculated: 554.4335Found: 554.4353

The loss of 18 mass units (-H2O) from 554 (m/e 536) clearly

indicated the presence of a hydroxy group. No other significantmass fragments were observed between m/e 554 and m/e298. The IR spectrum showed the presence of OH stretchingfrequencies at 3600 and 3450 cm"'. Intense C—Hstretchingfrequencies around 2900 cm"1 indicated the presence of a

large number of saturated carbon moieties. The carbonylstretching frequency at 1740 cm"1 together with the fragmen

tation pattern of the mass spectrum were indicative of an esterfunction. The 250-MHz proton NMR spectrum confirmed thepresence of large numbers of protons on saturated carbons. Atriplet centered at 2.36 ppm was the result of the méthylèneprotons adjacent to the carbonyl function of the ester (Compound 1). A singlet at 4.27 (2H) appeared to be the resonancepeak due to the 2 protons at C-17. Two doublets at 7.24 and6.20 ppm (J < 0.01 Hz), respectively, are the resonances fromthe protons at C-19 and C-18 on the furan ring.

Saponification of Compound 1 yielded an alcohol (Compound 1a) and an acid (Compound 1c). The acid was identified

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Chart 6. Activities of Compound 2 (2) and its derivatives and the derivativesof Compound 1 (|) on the enhancement effect of GSH S-transferase in themucosa of the small bowel of mice. 7a, cafestol; lb, cafestol monoacetate; 2a,kahweol; 20, kahweol monoacetate; CON, control. Bars, S.D.

CHzOR

1 R = C(CH2)UCH3

1a (Cafestol) R = HO

1b R=CCH3

92 R=C(CH2)14CH3

2a (Kahweol) R = HO

2b R=CCH3

Chart 7. Structure of cafestol palmitate (1) and kahweol palmitate (2); theirparent compounds, cafestol (ia) and kahweol (2g), and their monoacetates(lb, 20).

to be palmitic acid (M, 256) by mass spectroscopy and bymelting point.

Compound 1a. The molecular formula of the alcohol, Compound 1a, was determined by high-resolution mass spectros

copy to be C2oH28O3.

Calculated: 316.2038Found: 316.2027

Gas chromatography-mass spectroscopy of its trimethylsilane

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: K-' i'r -T

derivatives revealed mono- and ditrimethylsilane derivatives

having molecular ions at m/e 388 and m/e 460, respectively.The proton NMR spectrum at 250 MHz showed the absence ofaliphatic protons of the palmitic acid moiety. The resonancesat 7.24 and 6.20 ppm were unchanged. The singlet at 4.27,however, has changed into an AB quartet with J = 13.5 Hz.

This quartet is the result of hydrogen bonding of the hydroxygroups on C-1 6 and C-1 7 which makes the protons on C-1 7

magnetically nonequivalent. These spectral data and the IRspectrum of Compound 1a are consistent with the structure ofcafestol (4, 6, 15).

The monoacetate of Compound 1a, which was synthesizedby the treatment with acetyl chloride in pyridine, had an IRspectrum identical to that of cafestol monoacetate, Compound1b, reported by Djerassi ef a/. (5).

Compound 2. Compound 2 was found to be very similar instructure to Compound 1 and was identified as the palmitate ofkahweol (Chart 7). The molecular weight was determined to be552 by mass spectroscopy. The precise mass for CaeHgeC^was

Calculated: 552.4178Found: 552.4203

The IR spectrum of Compound 2 was almost identical to that ofCompound 1 except for a small peak at 3050 cm"1 which is

the stretching frequency of C—H bond on a carbon-carbon

double bond. The proton spectrum had 2 sets of doublets at7.26 and 6.25, respectively. These resonances are due to the2 vinyllic protons of the double bond at C-1 and C-2 of Com

pound 2. Saponification of Compound 2 yielded palmitic acidand an alcohol Compound 2a.

Compound 2a. Compound 2a has been identified as kahweol. Mass spectrometry yielded m/e 314 (M+ 10.4). The

molecular formula of Compound 2a was determined by high-

resolution mass spectroscopy to be

Calculated: 314.1882Found: 314.1872

Compound 2a is easily oxidized in the presence of air. Itsmonoacetate, Compound 2b, melts at 133.5-136°, which is

similar to the melting point of the monoacetate of kahweolreported by Kaufman and Sen Gupta (11,1 2).

DISCUSSION

In the present investigation, a potent inducer of increasedGSH S-transferase activity has been isolated from green coffee

beans. This compound is kahweol palmitate. A related compound, cafestol palmitate, was also active as an inducer ofGSH S-transferase activity but less so than kahweol palmitate.

The active ingredients from green coffee beans were almosttotally extracted into PE when the extraction was carried outfor 7 days or more. The PE extract was able to induce increasedGSH S-transferase activity in both the mucosa of the small

intestine and the liver of the mouse. On an equivalent weightbasis, the activity of the PE extract was approximately 40%higher than that of the green coffee beans. This increase inactivity was probably due to the greater availability of the

Kahweol and Cafestol Palmitates in Green Coffee Beans

inducing compounds resulting from their extraction. Benzene,the second solvent used in the extraction scheme, was able toextract substances that had inducing activity for the mucosa ofthe small bowel but were inactive in the liver of the mouse.

The first fractionation of the PE extract by normal-phase

preparative LC was able to eliminate 80% of the inactivesubstances. Attempts to manipulate the solvent composition tofurther separate the components in the active Fraction 3 werenot successful. Reverse-phase high-performance liquid chro-

matography, however, was able to resolve this fraction into 6distinct peaks. Peak C, which constitutes approximately 38%of Fraction 3, was isolated in the preparative scale using prepPAK-500/Ci8 columns. Preliminary UV spectral data on

Subfraction C indicated the presence of conjugated doublebonds in the molecules. Since silver nitrate-impregnated TLChad been used successfully to separate olefins, it was anticipated that the technique might be applied to further separatecomponents in Subfraction C (19). At least 4 well-separatedcomponents were found by this method which led to the finalidentification of the active ingredients.

The diterpenes, cafestol and kahweol, were isolated fromthe unsaponifiable portion of the PE extract of green coffeebeans in 1938 and 1932, respectively (1, 16). The structure ofcafestol was elucidated in the reports of Djerassi ef a/. (4, 6)and others (15). In the present investigation, Compound 1aobtained by the saponification of Compound 1 was identical tocafestol by spectroscopic and derivative comparison. Thestructure of kahweol was elucidated by Kaufmann and SenGupta (11,12). Compound 2a obtained by the saponificationof Compound 2 was shown to have the same structure as thatof kahweol. The exact location of the double bond was questioned recently by Gershbein and Baburao (7). Our NMR datafavor the double bond at C-1 and C-2 which is consistent with

the structure proposed by Kaufmann and Sen Gupta. Althoughthe existence of these 2 diterpenes has been known since1932, their palmitate esters have not been well characterized.Previous work has not established any biological effects ofcafestol or kahweol (2, 7, 9, 22).

The concentration of kahweol palmitate in the active Fraction3 is estimated at 10 to 15%. The remaining active constituentsin Subfractions A, B, and D to F are most probably due to fattyacid esters of kahweol. Preliminary TLC data indicated thepresence of cafestol and kahweol in all the active fractions inthe isolation scheme.

Several compounds that are potent inducers of increasedGSH S-transferase activity inhibit carcinogen-induced neoplasia (18). 3-ferf-Butyl-4-hydroxyanisole is among the most po

tent of these compounds and inhibits a wide variety of chemicalcarcinogens. When compared under the same experimentalconditions, i.e., a single p.o. administration 24 hr prior tosacrifice, kahweol palmitate is more than 3 times as potent inenhancing GSH S-transferase activity of the mucosa of thesmall intestine as 3-ferf-butyl-4-hydroxyanisole. It remains to

be determined whether kahweol palmitate and related diterpene esters will have carcinogen-inhibitory capacities. The

constituents of green coffee are complex mixtures of a verylarge number of chemicals. The composite biological effects ofcoffee on occurrence of neoplasia may not be the result of oneor 2 discrete components alone. Thus, it is not possible at thepresent time to assume a dominant biological significance of

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L. K. T. Lam et al.

the diterpene esters in the complex mixture of coffee constituents.

ADDENDUM

During the isolation procedure, the activity of cafestol palmi-tate as an inducer of GSH S-transferase activity was deter

mined using a single p.o. administration of 2.5 mg/mouse.Under these conditions, cafestol palmitate showed only marginal activity (Chart 5). A subsequent experiment using 4administrations of 2.5 mg cafestol palmitate (once a day for 4days with mice killed 24 hr after the last administration) resultedin a GSH S-transferase activity of the small bowel mucosa 41 %greater than that of the control. Further experiments werecarried out using cafestol and kahweol palmitates obtainedfrom the esterification of the alcohols with palmitoyl chloride.The semisynthetic esters used in these experiments were identical to the naturally occurring compounds by TLC, mass spec-

troscopy, and NMR. With the use of 4 daily administrations anda dose level of 5.0 mg/mouse, the GSH S-transferase activity

of the small bowel mucosa of mice receiving cafestol palmitatewas 3.9 times that of the controls, and the corresponding GSHS-transferase activity of mice receiving kahweol palmitate was

5.6 times that of the controls. These data indicate that cafestolpalmitate enhances GSH S-transferase activity but is less po

tent than kahweol palmitate.

ACKNOWLEDGMENTS

We thank Gerald Bratt for the NMR and Thomas Krick for the mass spectradeterminations. The technical assistance of Chester Yee is gratefully acknowledged.

REFERENCES

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2. Chakravorty. P. N.. Wesner, M. M., and Levin, R. H. Cafesterol II. J. Am.Chem. Soc., 65. 929-932, 1943.

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8. Habig, W. H., Pabst, M. J., and Jakoby, W. B. Glutathione S-transferases.J. Biol. Chem., 249: 7130-7139, 1974.

9. Hauptmann, H., Franca, J., and Bruck-Lacerda, L. Cafesterol. III. Thesupposed estrogenic activity of Cafesterol. J. Am. Chem. Soc., 65: 993-994, 1943.

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11. Kaufmann, H. P.. and Sen Gupta, A. K. Terpene als Bestandteile desUnverseifbaren von Fetten, II. FürKonstitution des Kahweols, I. Chem. Ber.,96: 2489-2498, 1963.

12. Kaufmann, H. P., and Sen Gupta, A. K. Terpene als Bestandteile desUnverseifbaren von Fetten, IV. FürKonstitution des Kahweols, II. Chem.Ber., 97. 2652-2660, 1964.

13. Lam, L. K. T., Pai, R. P.. and Wattenberg, L. W. Synthesis and chemicalcarcinogen inhibitory activity of 2-fert-butyl-4-hydroxyanisole. J. Med.Chem.. 22:569-571, 1979.

14. Lam, L. K. T., Sparnins, V. L., and Wattenberg, L. W. The isolation andidentification of coffee constituents that induce the activity of glutathione S-transferase in mice, MEDI Abstr. 85. Books of Abstracts, 182nd AmericanChemical Society National Meeting, New York, N. Y , August 23 to 28, 1981Am. Chem. Soc., Washington, D.C., 1981.

15. Scott. A. I., Sim, G. A., Ferguson G., Young, D. W., and McCapra, F.Stereochemistry of the diterpenoids: absolute configuration of cafestol. J.Am. Chem. Soc., 84: 3197-3199, 1962.

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1198 CANCER RESEARCH VOL. 42

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1982;42:1193-1198. Cancer Res   Luke K. T. Lam, Velta L. Sparnins and Lee W. Wattenberg 

-Transferase Activity in the MouseSEnhance Glutathione Palmitate as Active Constituents of Green Coffee Beans That Isolation and Identification of Kahweol Palmitate and Cafestol

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