J. Sci. FoodAgric. 1984,35,6670

Biodegradation of Caffeine: Formation of Theophylline and Theobromine from Caffeine in Mature Coffea arabica Fruits"

Takeo Suzukib and George R. Waller"

Department of Biochemistry, Oklahoma Agricultural Experiment Station, Oklahoma State University, Stillwater, Oklahoma 74078, USA

(Manuscript received I 9 January 1983)

The level of theophylline in mature and ripened fruit is 20-50 times that in the immature green fruit of Coffea arabica L. Biodegradation of caffeine occurs in the mature, ripened coffee fruits through theophylline and theobromine as the first biodegradation products. It is now clear that theophylline is associated primarily with caffeine biodegradation, whereas theobromine is involved in both biosynthesis and biodegradation of caffeine.

1. Introduction

Senanayaka and Wijesekera' reported the pattern of formation of theobromine and caffeine (Figure 1) in cacao plants during the growth stages of the bean. Caffeine formation in coffee berries during fruit development,* as well as that in tea leaves during the growth of the seedling^,^.^ has been studied, but comparable data on the dimethylxanthines in coffee and tea plants are lacking. Much less is known about the metabolism of the methylxanthines and their roles in plants than theobromine and caffeine.

Both in-vivo and in-vitro studies with tea and coffee plants4-' have shown that theobromine is synthesised from N7-methylxanthine and transformed to caffeine. In contrast, theophylline can be synthesised from N'methylxanthine in ~itro,~,~,~ but there is no definitive evidence for the in-vivo process. In contrast with reports on the biosynthesis of the methylxanthines, Kalberer' reported N3- andor N7-methylxanthine, allantoin, allantoic acid, urea, and C 0 2 as biodegradation products of caffeine when radioactive (N-methyl-labelled as well as ring-labelled) caffeine was administered to old leaves of coffee plants. This paper indicates that the dimethylxanthines can also be synthesised from caffeine by demethylation.

This paper describes the pattern of formation of theobromine, theophylline (1,3-dimethylxan- thine), and caffeine in coffee berries during the growth stages of the bean and shows evidence that theophylline arises from caffeine breakdown in excised mature coffee berries. The level of theophylline in mature and ripened fruit is 20-50 times that in the mature green fruit. Biodegradation of caffeine occurs in the mature, ripened coffee fruits through theophylline and theobromine as the first biodegradation products. These results support and extend the findings of Kalberer' to include the role of dimethylxanthines.

2. Materials and methods

[8-'4C] Theophylline (47.5 Ciimol) was purchased from New England Nuclear, USA. The [8-I4C] caffeine was synthesised from [8-I4C] theophylline according to the methylation procedure of Hef t~nan ,~ and purified by repeated paper chromatography and thin layer layer chromatography

Journal Article No. J-4066 of the Agricultural Experiment Station, Oklahoma State 1 lniversity, Stillwater, Oklahoma. Present address: Department of Sericulture and Applied Biology, Kyoto University of Industrial Arts and Textile Fibers.

To whom correspondence should be addressed. Matsugasaki, Kyoto 606, Japan.

66

Biodegredation of caffeine 61

Trivial name Ri R3 R7

Xanthine H H H 1,3-Dimethylxanthine Theophylline CH3 CHI H 3,7-Dimethylxanthine Theobromine H CHI CH3 1,7-Dimethylxanthine Paraxanthine CHa H CHs 1,3,7-Trimethylxanthine Caffeins CHs CH3 CHs

R3

Figure 1. Some naturally occurring methylated xanthines.

(99.9% radiochemically pure; 3.8 Ci/mol). Coffeu arabica L. trees were obtained through the Plant Introduction Division of the US Department of Agriculture, and grown at 23-32°C in a greenhouse at Oklahoma State University. They have been producing coffee fruits since 1977.

2.1. Harvesting fruits Fruits were detached at various stages ranging from 10 days after petal fall to maturity (about 8 months), dried at 100°C for 1.5 h and then at 80°C for 6 h, and placed in a desiccator overnight.

2.2. Administration of [S-14C] caffeine and [S-I4C] theophylline Each excised coffee fruit was fed through the petiole with the appropriate radioactive chemical (500 nCi in 50 pl of water) for 12 h, and incubated at 23°C in distilled water for various periods, four coffee berries in each incubation.

2.3. Analytical procedure Coffee fruits were ground with 30 ml of 0.0125~ H2S04 with sea sand in a mortar, the mixture refluxed for 30 min, and the residue and sea sand removed by centrifuging at lo4 g for 20 min. The residue was re-extracted with 20 ml of 0 .0125~ H2S04 and washed with 20 ml of water; the washings were combined with the supernatant. The combined supernatant solutions were shaken with chloroform (4 times, 35 ml portions) and the extracts (which contained caffeine, theobromine, and theophylline) were analysed by thin layer chromatography on silica gel plates (Merck 60F-254) in chloroform: methanol (9: 1) and 1-butanol: acetic acid: water (25 : 4: 10). The acid-soluble fraction remaining after chloroform extraction was subjected to paper chromatography on Whatman 3MM paper in 1-butanol : acetic acid: water (4: 1 : 1) and ethanol: acetic acid: water (81 : 5 : 14), or to chromatography on silica gel plates, as before, to characterise the monomethylxanthines and other water-soluble materials. The incubating water was treated in the same way as the supernatant from the berries.

To purify and identify the products of metabolism in coffee fruits, they were chromatographed on paper or thin-layer plates as described. Caffeine and dimethylxanthines on the silica gel plates were located by ultraviolet quenching. For the water-soluble compounds, purines were detected by ultraviolet quenching or spraying with Ehrlich's reagent. The eluates were used for either further purification by chromatography or mass spectrometric analysis (MS). In MS, enough eluate was placed in a vial for use in a direct probe and analysed with a LKB-9000 GUMS instrument."," The known compounds were run by the same techniques.

Radioactivity measurements were made in a PRIAS liquid scintillation counter using Instagel from the Packard Instrument Co.

68 T. Suzuki and G . R. Waller

3. Results and discussion 3.1. Pattern of formation of the methylxanthines in coffee fruits Table 1 shows the pattern of formation of caffeine and related dimethylxanthines in coffee fruits during the growth stages of the bean. The concentration of theobromine was the highest in the 10 day and 1 month stages and remained smaller but constant throughout fruit development. Caffeine showed a similar pattern, with a sharp decrease after 10 days, and another decrease at 7-8 months. In contrast, theophylline was absent until the 5 month stage, when the fruit was yellow; beyond this stage it increased slightly. However, in contrast with these dimethylxanthines, paraxanthine (1,7-dimethylxanthine) was not found in C. arabica fruits throughout development, although it has been reported in young C. arabica seedlings" and callus cultures."

3.2. Biodegradation of caffeine by excised coffee fruits The biodegradations of [8-14C] caffeine and [8-14C] theophylline are shown in Table 2. The uptakes of the radioisotopes were 94% and 88% respectively in the 12 h incubation period. An additional 12 h was allowed for metabolism to continue, then the metabolism part of the experiment was terminated. The aqueous incubating water and the fruit (acid-soluble extract, water-soluble extract and plant residue) were then analysed. The recovered radioactivity, which was divided between the aqueous, acid-soluble, and plant-residue fraction, was considerably less than the fruit uptake, the difference presumably being the amount lost as 14C02. The [8-14C] caffeine gave theophylline, theobromine, N3- and N7-methylxanthines, allantoin, allantoic acid, and urea. The [8-14C] theophylline was shown to form N3-methylxanthine, allantoin, allantoic acid, urea, and an unknown compound, but no N'-methylxanthine. Additional experiments using [8-14C] caffeine and [8-'4C] theophylline with incubation up to 55 h gave similar results. There was little incorporation of theophylline into caffeine, probably indicating that the red fruit had little biosynthetic capacity. N'-Methylxanthine was not found in either of the experiments reported.

The change in theophylline levels (Table 1) indicates that fruit at the red-brown-black stage might be degrading caffeine rather than synthesising it. Table 2 clearly shows that biodegradation of

Table 1. Caffeine, theobromine and theophylline contents of individual coffee fruit at different stages of growth (1980)

Alkaloid levels (pg g-1 dry wt) Age Growth

(months) stage Caffeine Theobromine Theophyiline

0 . 3 Green 9200 146 - 1 Green 8000 145 - 4 Green 7900 55 -

5-6 Yellow-red 7500 59 28 7-8 Brown-black 6100 56 56

Table 2. Biodegradation of [8-14C]caffeine and [8-14C]theophylline in mature coffee fruit

Radioactivity recovered (nCi/fruit)

Fruit uptake of Distribution of radioactivity (nCi/fruit) Compound radioactivity Dried

administered (nCi/fruit) A B residue Cf Tb Tp MX 3-MXAA Urea Unknown

[8-14C]Caffeine 470 200 - 1 . 2 191 0 . 4 0 . 5 0 . 3 - 1 . 1 0 . 6 0 .0 - 220 208 0 . 6 0 . 7 0 . 4 - 5 . 4 0 . 9 0 .0

1 . 2 2 . 9 1 . 1 7 . 2 3 . 1 120 6 .1 0 . 0

[8-14C]Theophylline 440 30 - 0 . 8 0 . 7 0 .0 1 1 - - 220 0 . 8 0 . 2 0 .0 82 -

Abbreviations: A = Aqueous incubating water, B = acid-soluble extract, Cf = caffeine, Tb = theobromine, Tp = theophylline, MX= N3-niethylxanthine and N7-methylxanthine, 3-MX= N3-methylxanthine, AA = allantoin and allantoic acid.

T 9 A

0

t 0 A

t

70 T. Suzuki and G . R. Waller

caffeine occurs in the mature, ripened coffee fruits through theophylline and theobromine as the first biodegradation products. Table 2 also shows that theophylline is degraded in mature fruits and that the products of theophylline metabolism are identical to those of caffeine metabolism with the exception of caffeine, 7-methylxanthine, and an unknown. These results and the work of Kalberer' indicate that caffeine can be degraded in coffee fruits through the steps outlined in Figure 2.

The results (Table 2 ) indicate that the biodegradation of caffeine to dimethylxanthines was slow and may be the rate-limiting step. The rapid degradation of xanthine to urea via uric acid, allantoin and allantoic acid in tea leaves has been r e p ~ r t e d , ' ~ and the authors have made similar observations in the coffee

Acknowledgements

T. Suzuki thanks the Kyoto Kogei-Sen-i University in Japan for granting a leave of absence, which was spent at Oklahoma State University. This work was supported by National Science Foundation Grant PCM-78-23160. The authors also thank Howard Van Woert and Bryan Smith for technical assistance and Otis C . Dermer for critical reading of this manuscript.

References 1. Senanayake, U. M.; Wijesekera, R. Theobromine and caffeine content of the cocoa bean during its growth. 1. Scr. Food

Agric. 1971, 22, 262-263. 2. Keller, H.; Wanner, H.; Baumann, T. W. Kaffeinesynthese in Fruchten und Gewebekulturen von Coffeu urubicu. Plunru

3 . Konishi, S.; Ozasa, M.; Takahashi, E. Metabolic conversion of N-methyl carbon of a-glutamylmethylamide to caffeinc in tea plants. Plunt Cell Physiol. 1972, 13, 365-375.

4. Suzuki, T.; Takahashi, E. Biosynthesis of caffeine by tea leaf extract. Biochem. J. 1975, 146, 87-96. 5. Suzuki, T.; Takahashi, E. Biosynthesis of purine nucleotidcs and methylated purines in higher plants. Drug Mefub. Rev.

1977, 6, 213-242. 6. Roberts, M. F.; Waller, G. R. N-Methyltransferases and 7-rnethyl-~-nucleoside hydrolase activity in Coffeu urubicu and

the biosynthesis of caffeine. Phyfochemisrry 1979, 18, 451-455. 7. Waller, G. R.; Suzuki, T.; Roberts, M. F. Cell-free metabolism of caffeine in Coffeu urubicu. Proc. 9th Internut. Colloq. Sci.

Tech. Coffee 1981,627-635. 8. Lalberer, P. Breakdown of caffeine in lcaves of Coffeu urubicu L. Nature 1965, 205, 597-598. 9. Heftman, E. Synthesis of ~affeine-2-'~C. 1. Labelled Compds. 1971, 7 , 463-465.

10. Hignite, C. In BiochemiculApplicution of Mass Spectrometry (Waller, G . R., Ed.) Wiley-Interscience. New York, 1972, p. 434.

11. Chou, C. H.; Waller, G. R. Isolation and identification by mass spectrometry of phytotoxins in Coffeu urubicu. Bot. Bull. Acud. Sin. (Tuipei) 1981, 21, 25-34.

12. Suzuki, T.; Waller, G. R. Metabolism of caffeine in sterile callus tissue cultures of Coffeu urubicu. Proc. 5th Int. Plunt Tissue Cell Culture 1982, 385-386.

13. Suzuki, T.; Takahashi, E. Metabolism of xanthine and hypoxanthine in the tea plant (Theu sinensis L.). Biochem. 1. 1975, 146, 79-85.

14. Suzuki, T.; Waller, G. R. Biodegradation of caffeine by excised fruits of Coffeu urabicu L. Fed. Proc. 1981,40, 1645.

1972, 108, 339-350.