Theobromine, and Theophylline

Caffeine (melting point 238°C, sublimes at 178°C density 1.23), theobromine, and theophylline are xanthine derivatives classified as central nervous stimulants, but differing markedly in their properties. They can be extracted from a number of natural sources. 

Caffeine has been isolated from waste tea and from the decaffeiniza-tion of coffee by extraction at 70°C, using rotating countercurrent drums and an organic solvent, frequently trichloroethylene. The solvent is drained off, and the beans steamed to remove residual solvent. The extraction solvent is evaporated, and the caffeine is hot-water-extracted from the wax, decolorized with carbon, and recrystallized. 

Caffeine is also manufactured synthetically by, for instance, the methy-lation of theobromine and also total synthesis by methylation and other reactions based upon urea. 

Caffeine is used by the pharmaceutical industry and also by the soft-drink industry for cola-style drinks. 

Calcium acetate is manufactured by the reaction of calcium carbonate or calcium hydroxide with acetic acid. 

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This experiment describes a quantitative analysis for caffeine, theobromine, and theophylline in tea, pain killers, and cocoa. Separations are accomplished by MEKC using a pH 8.25 borate-phosphate buffer with added SDS. A UV detector set to 214 nm is used to record the electropherogram. An internal standard of phenobarbital is included for quantitative work. 

Care must be exercised in the choice of acid employed in chloramine T — mineral acid reagent since the detection sensitivity and also the color of the fluorescences produced depend to a significant extent on the choice of acid. This is illustrated for the purine derivatives caffeine, theobromine and theophylline in Figure 1 and Table 1. 

If the fluorescence was excited at a wavelength of = 313 nm, then the detection limits were 200 ng (caffeine, theophylline) to 400 ng (theobromine) substance per chromatogram zone. At an excitation wavelength X = 365 nm the detection limits were appreciably lower at 100 ng substance per chromatogram zone for theobromine and theophylline. The sensitivity remained unchanged in the case of caffeine. 

Figure 33-8. Caffeine, a trimethyixanthine. The di-methylxanthines theobromine and theophylline are similar but lack the methyl group at N-1 and at N-7, respectively.

Caffeine was conceived for a wide range of readers interested in the effects on human health, nutrition, and physiological function of the methylxanthine beverages and foods—tea, coffee, mate, cola beverages, and cocoa and chocolate products. These products supply one or more of the dietary methylxanthines—caffeine, theobromine and theophylline— and are an integral part of the diet of many people in many countries. The interest in the health effects of both the methylxanthines in isolation and in the products containing them has grown rapidly in recent years. 

The methylxanthines of interest are caffeine (1,3,7-trimethylxanthine), theophylline (1,3-dimethylxanthine), and theobromine (3,7-dimethylxanthine) and they occur in coffee, tea, mate, cocoa products, and cola beverages. This chapter is an introduction to their chemistry, isolation, and biosynthesis. While the class of methylxanthines is large and comprised of more members than these three, this chapter will essentially be limited to caffeine, theobromine, and theophylline. 

The analysis of the methylxanthines (caffeine, theobromine, and theophylline) is important in the areas of nutrition and clinical chemistry. These three compounds compose the majority of the alkaloids present in coffee, tea, cocoa, cola nuts, and guarana. 

There are numerous methods in the literature for the determination of caffeine, theobromine, and theophylline in food matrices, including coffee, tea, and cocoa. Until recently, methods have emphasized the determination of the major methylxanthines in a commodity, for example, caffeine in coffee or theobromine in cocoa. Present methods range from being specific for one of the compounds in a single matrix to being an all-encompassing assay of major and minor methylxanthines in food products. 

HPLC allows a quantitative determination with relatively simple extractions. In many cases, extraction only involves a heating of the commodity with water, followed by filtration and injection onto an HPLC column. In the determination of caffeine, theobromine, and theophylline in cocoa, coffee, or tea, as well as in other foods, there is scarcely a month that passes without a new paper on this assay. Kreiser and Martin provide typical conditions for analysis.28 In their studies, samples were extracted in boiling water and filtered prior to injection onto the HPLC column. The HPLC conditions used a Bondapak reversed phase column and a mobile phase of water methanol acetic acid (74 25 1) with detection at 280 nm. This method is accurate, precise, and conserves time. It has also been adopted by the AOAC as an official method for the determination of theobromine and caffeine in cocoa beans and chocolate products.29... 

Franzke, C.L., Grunert, K.S., and Griehl, H., Estimation and contents of theobromine and theophylline in mate, cola and cocoa. Zeitschrift fuer Lebensmitteluntersuchung und Forschung, 139,85,1969... 

There is also confusion in the literature concerning theobromine and theophylline levels in tea beverage. The most reliable data indicate 0.16 to... 

Sontag, G., Krai, K., Bestimmung von Coffein, Theobromine and Theophyllin in Tee, Koffee and Getranken durch Hochdrucksflussigkeits chromatographie mit electrochemischem Detektor. Microchim. Act a, 11 39, 1980. 

Theobromine and theophylline concentrations are relatively low in coffee the values in green coffee beans are, respectively, 20 and 5 mg/kg.3... 

Theobromine and theophylline also occur in mate, although several investigators have failed to find these dimethylxanthines.8 Likely levels of these substances in the dried product are theobromine, 0.3% theophylline, 0.004%.5... 

The principal psychoactive constituent of guarana is caffeine (2.5-5%), although theobromine and theophylline are present in smaller amounts (Gruenwald et al. 1998). Depending on the preparation,... 

Caffeine pharmacokinetics are nonlinear. For example, when comparing a 500 mg dose to a 250 mg dose, the clearance is reduced and elimination half-life is prolonged with the higher dose (Kaplan et al. 1997). Thus, larger doses prolong the action of the drug. Active metabolites of caffeine are paraxanthine, and to a lesser degree, theobromine, and theophylline. Urinary metabolites are I-methylxanthine, l-methyluric acid, and an acetylated uracil derivative. 

After the nucleic acid purines adenine and guanine, the next most prominent purine in our everyday lives is probably caffeine. Caffeine, in the form of beverages such as tea, coffee, and cola, is one of the most widely consumed and socially accepted natural stimulants. Closely related structurally are theobromine and theophylline. Theobromine is a major constituent of cocoa, and related chocolate products. Caffeine is also used medicinally,... 

Walton et al. (2001a) examined data for compounds eliminated by the cytochrome P450 isoenzymes CYP1A2 in humans. Absorption, bioavailabihty, and route of excretion were generally similar between humans and the test species for each of the substances (caffeine, paraxanthine, theobromine, and theophylline). However, interspecies differences in the route of metabolism, and the enzymes involved in this process, were identified. The magnitude of difference in the internal dose, between species, showed that values for the mouse (10.6) and rat (5.4) exceeded the fourfold default factor for toxicokinetics, whereas the rabbit (2.6) and the dog (1.6) were below this value. 

A variety of xanthines including caffeine, theobromine, and theophylline have been found from food materials including.coffee, chocolate, and tea (419-420). Theophylline determination in sera has been much studied. The technique allows the determination of theophylline at serum levels of 1.5-20 mg/liter theophylline with sample sizes ranging from 50 to 10 /xl (42 -425). Hill (426) assayed theophylline using 50 /xl of serum and an analysis time of 8 min with good interbatch precision and accuracy. Alternative methods which allow the determination of as little as 0.1 mg/ml (427) or 20 ng theophylline in 10 ml serum have been described (428). 

Many alkaloids are generally known to have stimulating properties, such as caffeine, cathine, theobromine and theophylline. These alkaloids are considered in many different medical applications. 

Several purine derivatives are found in nature, e.g. xanthine, hypoxanthine and uric acid. The pharmacologically important (CNS-stimulant) xanthine alkaloids, e.g. caffeine, theobromine and theophylline, are found in tea leaves, coffee beans and coco. The actual biosynthesis of purines involves construction of a pyrimidine ring onto a pre-formed imidazole system. 

PURINES. [CAS 120-73-0]. Derivatives of the dicyclodiureide of malonic and oxalic acids. The dicyclodiureide is uric acid and the parent compound is purine so that uric acid is 2,6,8-trioxypurine or the keto form of 2,6,8-trihydroxypurine. Caffeine, theobromine, and theophylline me other important purine compounds. 

The purine alkaloids caffeine, theobromine, and theophylline (Figure 6.135) are all methyl derivatives of xanthine and they commonly co-occur in a particular plant. The major sources of these compounds are the beverage materials such as tea, coffee, cocoa, and cola, which owe their stimulant properties to these water-soluble alkaloids. They competitively inhibit phosphodiesterase, resulting in an increase in cyclic AMP and subsequent release of adrenaline. This leads to a stimulation of the CNS, a relaxation of bronchial smooth muscle, and induction of diuresis, as major effects. These effects vary in the three compounds. Caffeine is the best CNS stimulant, and has weak diuretic action. Theobromine has little stimulant action, but has more diuretic activity and also muscle relaxant properties. Theophylline also has low stimulant action and is an effective diuretic, but it relaxes smooth muscle better than caffeine or theobromine. 

Guarana contains tannins and has been used to treat diarrhea. It also contains a high concentration of caffeine, which, in conjunction with the alkaloids theobromine and theophylline, gives guarana diuretic and stimulant properties. Guarana is used to treat fatigue, mild depression, headache, and migraine. 

For theobromine and theophylline, the most basic nitrogen is the one with the non-bonded lone pair available in an sp2-hybridized orbital. 

Both theobromine and theophylline can form /V-glycosides with sugars at the same N position noted above as the most basic nitrogen. 

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