[CANCER RESEARCH 42. 1193-1198. April 1982] 0008-5472/82/0042-OOOOS02.00 Isolation and Identification of Kahweol Palmitate and Cafestol Palmitate äsActive Constituents of Green Coffee Beans That Enhance 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 compounds increased the GSH S-transferase activity of the forestomach between 78 and 182%. They were p-methoxy- Glutathione (GSH) S-transferase is a major detoxification phenol, 2-fe/t-butyl-4-hydroxyanisole, coumarin, a-angelica- enzyme system that catalyzes the binding of a variety of elec- lactone, and benzyl isothiocyanate (18). All 5 compounds trophiles, including reactive forms of chemical carcinogens, to inhibited BP-induced neoplasia of the forestomach (13, 20, GSH. Green coffee beans fed in the diet induce increased GSH 21). These data suggest that the capacity to enhance GSH S- S-transferase activity in the mucosa of the small intestine and transferase activity might be used as a method of identifying in the liver of mice. A potent compound that induces increased compounds or natural products likely to inhibit BP or other GSH S-transferase activity was isolated from green coffee carcinogens detoxified in a similar manner (18). beans and identified as kahweol palmitate. The corresponding In efforts at identifying dietary constituents that might protect free alcohol, kahweol, and its synthetic monoacetate are also against chemical carcinogens, the effects of natural products potent inducers of the activity of GSH S-transferase. A similar on GSH S-transferase activity were studied. During this inves diterpene ester, cafestol palmitate, isolated from green coffee tigation, it was found that consumption of diets containing beans was active but less so than was kahweol palmitate. powdered green coffee beans resulted in a very marked en Likewise, the corresponding alcohol, cafestol, and its monoac hancement of GSH S-transferase activity in the liver and mu etate showed moderate potency as inducers of increased GSH cosa of the small bowel of the mouse. The magnitude of S-transferase activity. Kahweol palmitate and cafestol palmi induction was as high as that obtained with any test compound tate were extracted from green coffee beans into petroleum or natural material previously investigated. The coffee beans ether. The petroleum ether extract was fractionated by prepar used in the original study were from Guatemala. In subsequent ative normal-phase and reverse-phase liquid chromatographies work, coffee beans from Brazil, Colombia, El Salvador, Mexico, successively. Final purification with silver nitrate-impregnated and Peru were all found to have a comparable enhancing effect thin-layer chromatography yielded the pure palmitates of ca on GSH S-transferase activity (17). Roasted coffee and instant festol and kahweol. The structures were determined by exam coffee were found to have a weaker inducing activity than did ination of the spectroscopic data of the esters and their parent the green coffee beans studied, i.e., slightly less than 50% as alcohols and by derivative comparison. much. Decaffeinated instant coffee showed activity similar to that of instant coffee. Investigations were then begun to identify INTRODUCTION the constituents of green coffee beans having the capacity to enhance GSH S-transferase activity. In the present study, it GSH3 S-transferase has been studied extensively as a major was found that the coffee constituent kahweol palmitate is a detoxification enzyme system that catalyzes the binding of a highly potent inducer of increased GSH S-transferase activity. wide variety of electrophiles to GSH (3,10). Since most reactive The palmitate of a closely related diterpene, cafestol, was ultimate carcinogenic forms of chemical carcinogens are elec active as an inducer of GSH S-transferase but less so than was trophiles, GSH S-transferase may play a significant role in kahweol palmitate. carcinogen detoxification. Enhancement of the activity of this system potentially could increase the capacity of the organism MATERIALS AND METHODS to withstand the neoplastic effects of chemical carcinogens. Experiments have been carried out to determine the correlation Extraction of Green Coffee Beans. Powdered green coffee beans between increased GSH S-transferase activity in a target organ (Guatemala) were placed in a large modified Soxhlet extractor and were extracted with various solvents of increasing polarity (Chart 1). of chemical carcinogenesis and its response to the carcinogen. PE (b.p. 60-70°) was used first, which was followed by benzene, ethyl For this purpose, the forestomach of the mouse was used. Members of several classes of inhibitors of BP-induced neopla acetate, methanol, and water. Each solvent extraction was carried out over a period of 7 days. The solvent of each extraction was removed sia of the mouse forestomach were studied for their effects on under reduced pressure. The crude extracts were dried under reduced the GSH S-transferase activity in that structure. Five of the pressure until constant weights were obtained. The activities of the extracts were monitored by the GSH S-transferase assay. 1 Supported by USPHS Contract NOI-CP-85613-70 and Grant CA-09599. Fractionation of the PE Extract. The active PE extract was sepa Presented in part at the 182nd American Chemical Society National Meeting, rated into 7 fractions by preparative LC using a Waters Associates New York, N. Y., August 23 to 28, 1981 (14). prepSOO liquid Chromatograph equipped with prepPak-500/silica col 2 To whom requests for reprints should be addressed. 3 The abbreviations used are: GSH, glutathione: BP, benzo(a)pyrene; PE, umns. The elution solvent was PE;ether (2:1, v/v). The active fraction petroleum ether; LC, liquid chromatography; TLC, thin-layer chromatography; from preparative LC was further fractionated into 6 subfractions by NMR, nuclear magnetic resonance. reverse-phase preparative LC. Two prep PAK-500/C18 columns in Received October 7, 1981 ; accepted January 4, 1982. series were used with 98% methanol as the eluting solvent. Silver

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Green Coffee Beans GSH S-transferase activity. The inductive effects on the small bowel mucosa 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 PE(8%) 0H EtOAc MeOH H2O to previously published procedures (18). All steps were done at 0-4°. Prep LC-silica The tissues to be studied were homogenized in 0.1 M sodium phosphate sÀ267PrepLC-C,sB3(20%)45 buffer (pH 7.5). The homogenate was centrifuged at 100,000 x g for 1 hr. The supernatant was used for the assay of GSH S-transferase activity. The activity was determined spectrophotometrically at 30° C(38%)D7 l with 1-chloro-2,4-dinitrobenzene as substrate according to the proce |TLC-Sllica Gel-AgNO 3 dure of Habig ef al. (8). 1Palmitic RESULTS

Isolation Procedure Acid(1C)isaponificationCafestolPalmiticAcid <2a>4 (2c)1Kahweol The activity of the enzyme GSH S-transferase in the liver and mucosa of the small bowel of the mouse was enhanced by the Chart 1. Extraction and isolation scheme for kanweol and cafestol palmitates 0H, benzene; EtOAc, ethyl acetate. PrepPAK-500/silica columns were used for addition of 20% green coffee beans to the diet. The enhance the normal-phase preparatory (Prep)LC. The eluting solvent was PE:ether (2:1, ment of enzyme activity in the liver was approximately 5 times v/v). PrepPAK-500/C18 columns were used for the reverse-phase preparatory LC. The eluting solvent was 98% methanol (MeOH). The developing solvent for that of the control (Chart 28). A slightly smaller enhancement silver 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. 1 Saponification of Compounds 1 and 2. A sample of Compound 1 (or Compound 2), isolated from silver nitrate-impregnated TLC plates, X was dissolved in 10% aqueous ethanolic potassium hydroxide at room temperature. The reaction mixture was warmed to 50-60° for 0.5 hr. It was then poured into ice, and the aqueous solution was extracted 3 times with ether. The organic layers were combined and dried over anhydrous magnesium sulfate. The ether was removed under reduced pressure. The neutral product was crystallized from PE:ether. I 2 The aqueous layer after ether extraction was acidified with 6 N hydrochloric acid and then extracted with 3 volumes of ether. The ether was dried over anhydrous magnesium sulfate. The solvent was removed under reduced pressure, and the acid thus obtained was dried under reduced pressure overnight. GCB PE BZ EA ME WA RC RS CON Spectroscopic Studies. IR spectra were recorded on a Beckman Acculab 5 spectrophotometer. UV spectra were recorded on a Beck 3 18 man Model 25 spectrophotometer. Proton NMR spectra were deter B mined 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-resolu tion mass spectroscopy were recorded on a double-beam AEI-MS-30 spectrometer. Melting points were determined on a Fisher-Johns Mel- Temp apparatus and were uncorrected. I - J, Assay for in Vivo Enhancement of GSH S-Transferase Activity. 1 12 Female ICR/Ha mice from the Harlan-Sprague-Dawley Company (In dianapolis, Ind.) were used in all experiments. Mice were randomized 10 by weight at 7 weeks of age into the groups to be used in a particular protocol. In the initial stages of the fractionation, the extracts were taken 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 would contain the equivalent of 20% powdered green coffee beans. In each experiment, 20% crude powdered green coffee beans were included »L as a positive control as well as a control in which there were no additions. The diets were fed for 12 days at which time the mice were killed. The mucosa of the proximal half of the small bowel and the liver were taken for the determination of GSH S-transferase activity. In later 0 GCB PE B Z EA ME WA RC RS CON stages of the fractionation procedure in which smaller amounts of test material were available, the assay technique was modified. The test Chart 2. Activity of green coffee beans (GCB) and their solvent extracts on the enhancement effect of GSH S-transferase in the mucosa of the small bowel materials (2.5 mg), dissolved in cottonseed oil, were given by P.O. (A) and in the liver (B) of mice. BZ, benzene; EA, ethyl acetate; ME, methanol; intubation, 0.2 ml/mouse. The animals were killed 28 hr later. Mucosa WA, water; RC, reconstituted coffee beans; PS, residue of extraction; CON, of the proximal half of the small bowel was taken for determinations of control. Bars, S.D.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1982 American Association for Cancer Research. Kahweol and Cafestol Palmitates in Green Coffee Beans was found ¡nthe mucosa of the small bowel (Chart 2A). The overall magnitude of enzyme activity in the latter organ, how .8.6.4.2.0.6.4.2------Axfeih:•: ever, was much lower than that in the former. PE extraction for 7 days successfully removed most of the ingredients that had :^SÃŒ:|[è1jgTrr*-F*J inducing activity. Subsequent extractions by benzene showed activity that affected only the mucosa of the small bowel but Õ 1.4 - had little effect in the liver. The other solvent extracts were inactive. When the extracts were combined with the residue of 1| extraction, a slight increase of activity was observed in com o parison with the original green coffee beans (Chart 2). S k The PE extract contained a small amount of caffeine which 1? could be removed by dissolving the dried extract in a small ëç volume of PE and filtering. The caffeine-free PE extract was 2 E then fractionated by preparative LC to give 7 fractions. Fraction VB WE 3 contained materials that gave similar enhancement of activity to that of the PE extract. The weight of Fraction 3 was 20% O that of the PE extract. The other fractions were all inactive in the GSH S-transferase assay (Chart 3). All attempts to further fractionate Fraction 3 with normal-phase preparative LC re sulted in subfractions of mixtures of similar components. Anal ysis of Fraction 3 by reverse-phase high-performance liquid chromatography (Ci8-/iBondapak, eluted with methanol) indi cated 6 peaks (Subfractions A to F) that were well separated. AB RC CON Subfractions A to F were separated by preparative LC. All 6 Chart 4. Activities of the reverse-phase preparatory LC subtractions of Frac subfractions were active in enhancing GSH S-transferase ac tion 3 on the enhancement effect of GSH S-transferase in the mucosa of the tivity in the mucosa of the small bowel of the mouse (Chart 4). small bowel of mice. PC, reconstituted Fraction 3; CON, control. Bars, S.D. Subfraction C, which constituted approximately 38% of Frac-

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8i.co CChart CON CON4), PA C,fied 5. Activities of Subfraction saponi-7c)components (| toB1A1and the o 6420B_ S-transferaseproducts of Compound 1 (|a.i\isits on the enhancement effect of GSH acid;CON, in the mucosa of the small bowel of mice.i '/V authentic palmitic S.D.tioncontrol. Bars,

ßp| r andwas3, showed the highestactivity in theenzyme assay pifíiSJplj IÌ3|il purification.Tochosen for further Pi=l tiSii Ka! IÄ8K further separate Subfraction C intoits pure components, GCB PE 1 234567 CON TLC (Silica Gel G) plates impregnated with silver nitrate were Chart 3. Activities of the different fractions separated from the PE extract used (Chart 1). These silver nitrate-treated plates were able to (PE) on the enhancement effect of GSH S-transferase in the mucosa of the small bowel (A) and in the liver (B) of mice. GCB, green coffee beans; CON, control. separate Subfraction C into 4 components, 3 of which were Bars, S.D. active (Chart 5A). The activity of Compound 1 was similar to

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that of the control level. Compound 2 showed the greatest potency in enhancing the activity of GSH S-transferase. Ad ministration of Compound 2 resulted in an increase of GSH S- transferase activity that was 4 times that of the control. Com 4.0-£5oa¡3.0|"5*>1 pounds 3 and 4 were able to induce the enzyme activity to twice the control level. Compounds 1 and 2, the components that had the highest R( values, were isolated on preparative TLC plates impregnated with silver nitrate. Compound 1, which was present in much higher quantity than were the other 3 components, was easily purified and characterized as a pure compound. The behavior of this set of compounds during fractionation and TLC separation indicated that they belonged to the same class of organic compounds with only minor structural differences. The structural determination of any one of these compounds will facilitate the identification of the remaining components. Since Compound 1 was isolated in its 2.0in pure form in sufficient quantity, structural determination was started with this compound. Spectroscopic evidence and de coZ"wcoV rivative comparison indicated that Compound 1 was the palmi- tate of cafestol (2). Component 2, which has a molecular weight that is 2 mass units lower than that of Compound 1, was LOcoX8---mpia•m*TVATffijIMI•'.•'-•'.• identified to be the palmitate of kahweol (1). The previously known coffee constituents, cafestol and kahweol, could be isolated as the free alcohols from mild alkaline hydrolysis of Compounds 1 and 2, respectively. While cafestol palmitate showed very little activity as an enzyme inducer, cafestol and its monoacetate showed activity that was approximately 1.5 times above control level (Chart 50) (see "Addendum"). Kah 2a CON la 1b 2a 2b CON weol and its monoacetate showed activity that was similar to Chart 6. Activities of Compound 2 (2) and its derivatives and the derivatives that of kahweol palmitate (Chart 6). Palmitic acid (Compound of Compound 1 (|) on the enhancement effect of GSH S-transferase in the 1c) was inactive as an inducer of the activity of GSH S-trans mucosa of the small bowel of mice. 7a, cafestol; lb, cafestol monoacetate; 2a, kahweol; 20, kahweol monoacetate; CON, control. Bars, S.D. ferase (Chart 56).

Structural Identification CHzOR

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

Calculated: 554.4335 Found: 554.4353 9 The loss of 18 mass units (-H2O) from 554 (m/e 536) clearly 1 R = C(CH2)UCH3 2 R=C(CH2)14CH3 indicated the presence of a hydroxy group. No other significant 1a (Cafestol) R = H 2a (Kahweol) R = H mass fragments were observed between m/e 554 and m/e O O 298. The IR spectrum showed the presence of OH stretching 1b R=CCH3 2b R=CCH3 frequencies at 3600 and 3450 cm"'. Intense C—Hstretching frequencies around 2900 cm"1 indicated the presence of a Chart 7. Structure of cafestol palmitate (1) and kahweol palmitate (2); their parent compounds, cafestol (ia) and kahweol (2g), and their monoacetates large number of saturated carbon moieties. The carbonyl (lb, 20). stretching frequency at 1740 cm"1 together with the fragmen tation pattern of the mass spectrum were indicative of an ester function. The 250-MHz proton NMR spectrum confirmed the to be palmitic acid (M, 256) by mass spectroscopy and by presence of large numbers of protons on saturated carbons. A melting point. triplet centered at 2.36 ppm was the result of the méthylène Compound 1a. The molecular formula of the alcohol, Com protons adjacent to the carbonyl function of the ester (Com pound 1a, was determined by high-resolution mass spectros pound 1). A singlet at 4.27 (2H) appeared to be the resonance copy to be C2oH28O3. peak due to the 2 protons at C-17. Two doublets at 7.24 and 6.20 ppm (J < 0.01 Hz), respectively, are the resonances from the protons at C-19 and C-18 on the furan ring. Calculated: 316.2038 Found: 316.2027 Saponification of Compound 1 yielded an alcohol (Com pound 1a) and an acid (Compound 1c). The acid was identified Gas chromatography-mass spectroscopy of its trimethylsilane

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Kahweol and Cafestol Palmitates in Green Coffee Beans

derivatives revealed mono- and ditrimethylsilane derivatives inducing compounds resulting from their extraction. Benzene, having molecular ions at m/e 388 and m/e 460, respectively. the second solvent used in the extraction scheme, was able to The proton NMR spectrum at 250 MHz showed the absence of extract substances that had inducing activity for the mucosa of aliphatic protons of the palmitic acid moiety. The resonances the small bowel but were inactive in the liver of the mouse. at 7.24 and 6.20 ppm were unchanged. The singlet at 4.27, The first fractionation of the PE extract by normal-phase however, has changed into an AB quartet with J = 13.5 Hz. preparative LC was able to eliminate 80% of the inactive This quartet is the result of hydrogen bonding of the hydroxy substances. Attempts to manipulate the solvent composition to groups on C-1 6 and C-1 7 which makes the protons on C-1 7 further separate the components in the active Fraction 3 were magnetically nonequivalent. These spectral data and the IR not successful. Reverse-phase high-performance liquid chro- spectrum of Compound 1a are consistent with the structure of matography, however, was able to resolve this fraction into 6 cafestol (4, 6, 15). distinct peaks. Peak C, which constitutes approximately 38% The monoacetate of Compound 1a, which was synthesized of Fraction 3, was isolated in the preparative scale using prep by the treatment with acetyl chloride in pyridine, had an IR PAK-500/Ci8 columns. Preliminary UV spectral data on spectrum identical to that of cafestol monoacetate, Compound Subfraction C indicated the presence of conjugated double 1b, reported by Djerassi ef a/. (5). bonds in the molecules. Since silver nitrate-impregnated TLC Compound 2. Compound 2 was found to be very similar in had been used successfully to separate olefins, it was antici structure to Compound 1 and was identified as the palmitate of pated that the technique might be applied to further separate kahweol (Chart 7). The molecular weight was determined to be components in Subfraction C (19). At least 4 well-separated 552 by mass spectroscopy. The precise mass for CaeHgeC^ components were found by this method which led to the final was identification of the active ingredients. The diterpenes, cafestol and kahweol, were isolated from Calculated: 552.4178 Found: 552.4203 the unsaponifiable portion of the PE extract of green coffee beans in 1938 and 1932, respectively (1, 16). The structure of The IR spectrum of Compound 2 was almost identical to that of cafestol was elucidated in the reports of Djerassi ef a/. (4, 6) Compound 1 except for a small peak at 3050 cm"1 which is and others (15). In the present investigation, Compound 1a the stretching frequency of C—H bond on a carbon-carbon obtained by the saponification of Compound 1 was identical to double bond. The proton spectrum had 2 sets of doublets at cafestol by spectroscopic and derivative comparison. The 7.26 and 6.25, respectively. These resonances are due to the structure of kahweol was elucidated by Kaufmann and Sen 2 vinyllic protons of the double bond at C-1 and C-2 of Com Gupta (11,12). Compound 2a obtained by the saponification pound 2. Saponification of Compound 2 yielded palmitic acid of Compound 2 was shown to have the same structure as that and an alcohol Compound 2a. of kahweol. The exact location of the double bond was ques Compound 2a. Compound 2a has been identified as kah tioned recently by Gershbein and Baburao (7). Our NMR data weol. Mass spectrometry yielded m/e 314 (M+ 10.4). The favor the double bond at C-1 and C-2 which is consistent with molecular formula of Compound 2a was determined by high- the structure proposed by Kaufmann and Sen Gupta. Although resolution mass spectroscopy to be the existence of these 2 diterpenes has been known since 1932, their palmitate esters have not been well characterized. Previous work has not established any biological effects of Calculated: 314.1882 cafestol or kahweol (2, 7, 9, 22). Found: 314.1872 The concentration of kahweol palmitate in the active Fraction 3 is estimated at 10 to 15%. The remaining active constituents Compound 2a is easily oxidized in the presence of air. Its monoacetate, Compound 2b, melts at 133.5-136°, which is in Subfractions A, B, and D to F are most probably due to fatty similar to the melting point of the monoacetate of kahweol acid esters of kahweol. Preliminary TLC data indicated the reported by Kaufman and Sen Gupta (11,1 2). presence of cafestol and kahweol in all the active fractions in the isolation scheme. Several compounds that are potent inducers of increased DISCUSSION GSH S-transferase activity inhibit carcinogen-induced neopla sia (18). 3-ferf-Butyl-4-hydroxyanisole is among the most po In the present investigation, a potent inducer of increased tent of these compounds and inhibits a wide variety of chemical GSH S-transferase activity has been isolated from green coffee carcinogens. When compared under the same experimental beans. This compound is kahweol palmitate. A related com conditions, i.e., a single p.o. administration 24 hr prior to pound, cafestol palmitate, was also active as an inducer of sacrifice, kahweol palmitate is more than 3 times as potent in GSH S-transferase activity but less so than kahweol palmitate. enhancing GSH S-transferase activity of the mucosa of the The active ingredients from green coffee beans were almost small intestine as 3-ferf-butyl-4-hydroxyanisole. It remains to totally extracted into PE when the extraction was carried out be determined whether kahweol palmitate and related diter for 7 days or more. The PE extract was able to induce increased pene esters will have carcinogen-inhibitory capacities. The GSH S-transferase activity in both the mucosa of the small constituents of green coffee are complex mixtures of a very intestine and the liver of the mouse. On an equivalent weight large number of chemicals. The composite biological effects of basis, the activity of the PE extract was approximately 40% coffee on occurrence of neoplasia may not be the result of one higher than that of the green coffee beans. This increase in or 2 discrete components alone. Thus, it is not possible at the activity was probably due to the greater availability of the present time to assume a dominant biological significance of

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1982 American Association for Cancer Research. L. K. T. Lam et al. the diterpene esters in the complex mixture of coffee constit and probable absolute configuration of cafestol. J. Am. Chem. Soc., 80. uents. 247-248, 1958. 5. Djerassi, C., Wilfred, E., Visco, L., and Lemin, A. J. Terpenoids. II. Experi ments in the cafestol series. J. Org. Chem., 18: 1449-1460, 1953. ADDENDUM 6. Finnegan, R. A., and Djerassi, C. Terpenoids. XLV. Further studies on the structure and absolute configuration of cafestol. J. Am. Chem. Soc., 82. 4342-4344, 1960. During the isolation procedure, the activity of cafestol palmi- 7. Gershbein, L. L., and Baburao, K. Effects of feeding coffee and its lipids on tate as an inducer of GSH S-transferase activity was deter regenerating and intact liver. Res. Commun. Chem. Pathol. Pharmacol., 28: 457-472, 1980. mined using a single p.o. administration of 2.5 mg/mouse. 8. Habig, W. H., Pabst, M. J., and Jakoby, W. B. Glutathione S-transferases. Under these conditions, cafestol palmitate showed only mar J. Biol. Chem., 249: 7130-7139, 1974. ginal activity (Chart 5). A subsequent experiment using 4 9. Hauptmann, H., Franca, J., and Bruck-Lacerda, L. Cafesterol. III. The supposed estrogenic activity of Cafesterol. J. Am. Chem. Soc., 65: 993- administrations of 2.5 mg cafestol palmitate (once a day for 4 994, 1943. days with mice killed 24 hr after the last administration) resulted 10. Jakoby, W. B. The glutathione S-transferases: a group of multi-functional in a GSH S-transferase activity of the small bowel mucosa 41 % detoxification proteins. Adv. Enzymol., 46: 383-414, 1978. 11. Kaufmann, H. P.. and Sen Gupta, A. K. Terpene als Bestandteile des greater than that of the control. Further experiments were Unverseifbaren von Fetten, II. FürKonstitution des Kahweols, I. Chem. Ber., carried out using cafestol and kahweol palmitates obtained 96: 2489-2498, 1963. from the esterification of the alcohols with palmitoyl chloride. 12. Kaufmann, H. P., and Sen Gupta, A. K. Terpene als Bestandteile des Unverseifbaren von Fetten, IV. FürKonstitution des Kahweols, II. Chem. The semisynthetic esters used in these experiments were iden Ber., 97. 2652-2660, 1964. tical to the naturally occurring compounds by TLC, mass spec- 13. Lam, L. K. T., Pai, R. P.. and Wattenberg, L. W. Synthesis and chemical carcinogen inhibitory activity of 2-fert-butyl-4-hydroxyanisole. J. Med. troscopy, and NMR. With the use of 4 daily administrations and Chem.. 22:569-571, 1979. a dose level of 5.0 mg/mouse, the GSH S-transferase activity 14. Lam, L. K. T., Sparnins, V. L., and Wattenberg, L. W. The isolation and of the small bowel mucosa of mice receiving cafestol palmitate identification of coffee constituents that induce the activity of glutathione S- transferase in mice, MEDI Abstr. 85. Books of Abstracts, 182nd American was 3.9 times that of the controls, and the corresponding GSH Chemical Society National Meeting, New York, N. Y , August 23 to 28, 1981 S-transferase activity of mice receiving kahweol palmitate was Am. Chem. Soc., Washington, D.C., 1981. 5.6 times that of the controls. These data indicate that cafestol 15. Scott. A. I., Sim, G. A., Ferguson G., Young, D. W., and McCapra, F. Stereochemistry of the diterpenoids: absolute configuration of cafestol. J. palmitate enhances GSH S-transferase activity but is less po Am. Chem. Soc., 84: 3197-3199, 1962. tent than kahweol palmitate. 16. Slotta, K. H., and Neisser. K. Zur Chemie des Kaffees. III. Mitteil.: Die Gewinnung von Cafesterol und anderen Verbindungen aus dem Universeif barendes Kaffee-Öls. Ber., 77: 1991-1994, 1938. ACKNOWLEDGMENTS 17. Sparnins, V. L., Lam, L. K. T., and Wattenberg. L. W. Effects of coffee on glutathione S-transferase (GST) activity and 7.12-dimethylbenz(a)- We thank Gerald Bratt for the NMR and Thomas Krick for the mass spectra anthracene (DMBAHnduced neoplasia. Proc. Am. Assoc. Cancer Res.. 22: determinations. The technical assistance of Chester Yee is gratefully acknowl 114, 1981. edged. 18. Sparnins, V. L., and Wattenberg, L. W. Enhancement of glutathione S- transferase activity of the mouse forestomach by inhibitors of benzo- (a)pyrene-induced neoplasia of the forestomach. J. Nati. Cancer Inst., 66. REFERENCES 769-771, 1981. 19. Stahl, E. Thin-Layer Chromatography, Ed. 2. Berlin: Springer Verlag, 1969. 1. Bengis. R. O.. and Anderson. R. J. The chemistry of the coffee bean. I. 20. Wattenberg, L. W. Inhibition of carcinogenic effects of polycyclic hydrocar Concerning the unsaponifiable matter of the coffee-bean oil. Extraction and bons by benzyl isothiocyanate and related compounds. J. Nati. Cancer Inst., properties of kahweol. J. Biol. Chem., 47: 99-113, 1932. 58:395-398, 1977. 2. Chakravorty. P. N.. Wesner, M. M., and Levin, R. H. Cafesterol II. J. Am. 21. Wattenberg. L. W., Lam, L. K. T., and Fladmoe, A. Inhibition of chemical Chem. Soc., 65. 929-932, 1943. carcinogen-induced neoplasia by coumarins and a-angelicalactone. Cancer 3. Chasseaud, L. F. The role of glutathione and glutathione S-transferases in Res., 39: 1651-1654, 1979. the metabolism of chemical carcinogens and other electrophilic agents. Adv. 22. Wettstein, A., Fritzsche, H., Hunziker, F.. and Miescher, K. XXXII. Ãœber Cancer Res., 29. 175-274, 1979. Steroid. Zur Konstitution des Cafesterols. Helv. Chim. Acta, 24: 332E- 4. Djerassi, C.. Cais, M., and Mitscher, L. A. Terpenoids. XXXIII. The structure 358E, 1941.

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

Cancer Res 1982;42:1193-1198.

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