Caffeine: Production by Plant (Coffea spp.)

2016

Caffeine and Man

During evolution Homo sapiens has selected from the plant kingdom’s vast diversity a few species containing caffeine and related purine alkaloids [PA] and has manufactured them into pleasant “stimulants”. This process occurred in different civilizations from East to West and resulted in six “self-prescribed” drugs which are coffee (Coffea arabica L. and Coffea canephora Pierre ex Froehner), tea (Camellia sinensis (L.) O. Kuntze), cocoa (Theobroma cacao L.), mate (Ilexparaguariensis St. Hil.), guarana (Paullinia cupana H.B.K.) and cola (Cola nitida Schott et Endl.). Since they are taken daily or at least very frequently, caffeine, the active principle, is a regular component of the human diet. For the major dietary caffeine sources Barone and Roberts () suggest caffeine content values as follows; 85, 60 and 3 mg of caffeine per 5-oz cup for ground roasted, instant, and decaffeinated coffee respectively; 40. and 30 mg per 5-oz cup for leaf or bag tea and instant tea respectively; 18 mg per 6-oz glass for colas; 4 mg per 5-oz cup for cocoa or hot chocolate; and 5 mg per 8-oz glass for chocolate milk. From product usage and consumption analyses, the same authors estimate that the mean daily intake is approximately 3 mg/kg for all adults in the general population.

Because of the never-ending debate on the risk of caffeine consumption to human health, great effort has been made to understand metabolism, phar-macokinetics, physiological and behavorial effects of purine alkaloids (). Additionally we recommend the study of Kihlman’s () book entitled Caffeine and Chromosomes dealing with the effects of purine alkaloids on the genome of microorganisms and eukaryotic cells.

One aspect of caffeine pharmacology, however, is important in the context of this review, namely the differential mode of action on different classes of organisms. In man, caffeine is to date considered as acting antagonistically to endogenous adenosine, which mediates blood vessel dilatation and inhibits platelet aggregation and hormone-induced lipolysis (). By contrast, in insects the effects of caffeine and related purine alkaloids are due primarily to inhibition of phosphodiesterase activity and to an increase in intracellular cyclic adenosine monophosphate (). Moreover, evidence has accumulated that PA may function as natural insecticides.

Botany of Coffee

According to Chevalier (1947) and Leroy (1967) the genus Coffea L. sensu strictu is divided into two sections, Eucoffea and Mascarocoffea (). The section Eucoffea consists of coffee species originating in Africa and includes the two cultivated species of economic importance Coffea arabica L. and Coffea canephora Pierre ex Froehner, whereas in the section Mascarocoffea () those coffee species are grouped which grow on the islands in the occidental part of the Indian Ocean. Altogether, about 100 coffee species have been described, two thirds of them are Mascarocoffeas reported to contain no caffeine (Bertrand 1901). Despite the phenotypic diversity within the genus Coffea, all coffee trees cytogenetically examined so far are, with the exception of the tetraploid autogamous species C arabica, diploid (2n = 22) and self-sterile.

Ethiopia is the place of origin of Coffea arabica, the Arabica coffee tree, where it still grows wild (). From here it was taken to southern Arabia, cultivated and its beans first used for the preparation of a beverage some 800 years ago. At the end of the 17th and at the beginning of the 18th century Coffea arabica was spread all over the world by the colonists. During this process of propagation and domestication the genetic variabiliy present in the “gene center” of its botanical origin was not exploited. Therefore the cultivated Arabicas originate from a very few genotypes only and are genetically extremely uniform. After the coffee rust (Hemileia vastatrix B. & Br) had destroyed the plantations (1876) in Java and Sumatra, Dutch agronomists in 1900 introduced to Java the species Coffea canephora Pierre ex Froehner which had been discovered in Congo.

This species, also termed Coffea robusta, is characterized by a high tolerance towards coffee rust and by its vigorous growth. Today about three-fourths of the world’s harvest is Arabica and the rest is Robusta. The ecophysiological requirements of these two cultivated species are quite different. Coffea robusta is adapted to the conditions in the tropical rain forest at low sea level (constantly high temperatures and high humidity) whereas Coffea arabica grows optimally in climate regions comparable to those in the mountains of its homeland. Moreover, Robusta beans have a higher caffeine content (1.2-4.0%) than the Arabica beans (0.6-1.9%) (), and the so-called organoleptic qualities of Arabica are rated superior to Robusta. In order to combine the high productivity and the rust resistance of Robusta with the organoleptic value and low caffeine content of Arabica, the novel hybrid Coffea arabusta was bred ().

Growth and Productivity of Coffee Tissue Cultures

Coffea arabica

It is interesting to observe that the starting point of the PA in vitro formation studies was the question whether the caffeine in the coffee bean is synthesized on the spot or whether it is imported from the neighboring pericarp or leaves (). Keller et al. () therefore put halves of unripe coffee beans (endosperm tissue) on a nutrient agar and followed caffeine production in these primary callus cultures (). The longer the seed halves were in culture, the greater became the caffeine fraction of the medium. The sum of caffeine present in tissue and medium increased faster than the dry weight, so that after 31 d a caffeine content above 2% (initially 1.25%) was found. The authors assumed that caffeine formation was stimulated by the efflux which results in a lowered intracellular concentration and as a result in diminished product inhibition. To test this hypothesis Frischknecht et al. () added caffeine to the culture medium and noticed product inhibition when callus tissue caffeine concentration exceeded the limit of 900 to 1000 µg/ml tissue, i.e., 5 mM. These studies were not performed with seed tissue, but with callus cultures derived from inter-node segments of Coffea arabica, which have a fairly constant production ranging between 1.0 and 1.6%. The high productivity of callus cultures was also observed by Waller et al. ().

In contrast, PA formation by suspension cultures of Coffea arabica varies from cell line to cell line in the wide range of 0.03% to 0.7% (dry wt), i.e., 5 to 130mg/l medium (). From primary suspension cultures cell lines of high or low productivity may easily be initiated by selection on the basis of the cell aggregate size, which is usually in direct proportion to the alkaloid productivity. The low content of 0.038% found by Buckland and Townsley () is consistent with the “apple sauce” like morphology they described. After a lag phase of 4 to 5 days dry weight increases rapidly to about 650 mg per culture or 16 g/l. It is remarkable that PA formation accelerates during the entire cultivation period, which means that most of the final amount (about 70mg/l) is synthesized after the exponential period of growth.

Main PA in suspension cultures of Coffea arabica is caffeine. About 25% to 50% of the total content is present as theobromine, a value considerably higher than in the plant (). Other PA such as theophylline, paraxanthine and methyluric acids were not detected and 7-methylxanthine, the precursor of theobromine (), only in small concentrations.

The efflux of caffeine into the growth medium described above for callus cultures is also observed in submerged cultures. However, because of nonlimited diffusion, theobromine and caffeine are dispersed equal to the ratio of volume tissue to volume nutrient medium. This free exchange across the membranes was found to occur in suspension cultures under all experimental conditions. In the coffee leaf 40% to 60% of caffeine is fixed as molecular complex with chlorogenic acid (.) and cannot be washed out from water-infiltrated leaf discs (). It appears that the chlorogenic acid concentrations found within cultured cells () are too low for efficacious complexation.

Other Coffee Species

As mentioned in the introduction, the occurrence of PA in remarkable concentrations is restricted to the section Eucoffea within the genus Coffea. In addition to Coffea arabica, we brought five other Eucoffea species into culture in order to test their biosynthetic potential (). Besides Coffea arabica only Coffea canephora and C. congensis formed PA in vitro under standard conditions. It is very interesting that all three species belong to the same subsection, Erythrocoffea. Optimation studies as regards productivity have not yet been made.

Coffee Tissue Culture – a “Standard of Excellence”

Although the world’s demand for caffeine for medicaments and soft drinks is enormous, it is quite clear that caffeine produced by tissue culture is not of economic value. Large quantities are supplied by the decaffeination process.

Moreover, chemical synthesis is inexpensive. What, then, is the purpose of studying PA production in vitro? We are convinced that coffee cell suspension cultures are an ideal system to investigate the basic mechanisms involved in the formation of a secondary plant substance. The key advantages are as follows: PA are readily produced; the production characteristics are reliable and reproducible; only two compounds (theobromine and caffeine) are formed, which facilitates analysis; due to equal distribution between cells and medium, PA concentrations are directly and rapidly determined by HPLC in an aliquot of the liquid medium.

These advantages make coffee cell suspension cultures extremely suitable for designing bioreactors and for optimizing parameters of cell immobilization (). It is well known that most tissue culture systems produce plant biochemicals only in small and insufficient quantities. Selection of favorably producing cell lines is laborious and unsatisfactory because of the limited stability of the production characteristics. In the long run it will be necessary to find new ways for improving in vitro productivity of pharmaceutical-ly important substances. One possibility we propose is to introduce the stress situation, to which plants in the natural habitat are exposed, into tissue culture, because stress factors may have a highly modulating effect on secondary metabolism. In this respect coffee tissue culture may also act as a “standard of excellence” by means of which stress factors can be screened for their effectivness. Thus it was reported lately by Frischknecht and Baumann () that production of caffeine was stimulated by stressors such as high light intensity and – depending on the culture type – high NaCl concentration. It is very surprising that a “low-producing/small aggregate type” culture could be triggered by high light intensity to produce 100 times the PA amount of the control and yielded more than 450 mg/1.

The Caffeine-Free Coffee Plant

A review on coffee would be incomplete without touching on the problem of the caffeine-free coffee. One major concern of biotechnology will be the improvement of the so-called cup quality, a term referring to the organoleptic characteristics appreciated when coffee is drunk. Unfortunately it appears very difficult to find a correlation between the chemical composition of a coffee bean or of the final coffee beverage and the desired organoleptic impressions. “The quality can therefore be defined only in negative terms, as absence of defects” (). One exception is the level of caffeine in a beverage, which may be regarded as a component of cup quality, since an increasing number of consumers (21%, USA 1984) prefer coffee low in caffeine or caffeine-free. Despite the existence of dozens of coffee species devoid of caffeine, coffee is today industrially decaffeinated by the use of organic solvents, supercritical carbon dioxide, or aqueous liquids. One of the reasons why the naturally caffeine-free Coffea species from Madagascar cannot be used for the preparation of a beverage is that they accumulate an extremely bitter-tasting principle in the beans. The chemical nature was identified as the diterpene glycoside named mascaroside ().

Genetic manipulation using techniques of tissue culture and molecular biology will allow us in the next future to “create” a caffeine-free coffee plant. However, in doing this one has to bear in mind that PA or diterpene glycosides are of vital importance for the coffee plant as chemical defence agents (). Based upon ecological considerations a “new” coffee plant should – in place of caffeine – contain another PA in order to be protected against infection and predation. From our studies with several species belonging to different sections of Coffea () as well as with various other PA-containing members of the plant kingdom, we can conclude that in all PA-producing species the genes for the following biosynthesis chain are present:

Heteroxanthine ( = 7-methylxanthine) -» theobromine -» caffeine -» theacrine -» methylliberine -» liberine.

Therefore appropiate selection or manipulation should result in a plant pharmacologically less active but toxic enough to survive in the natural habitat or in a plantation without extreme phytosanitary protection.

Prerequisites for successful genetic manipulation would be (1) mass regeneration of coffee plantlets by embryoid formation or from protoplasts, (2) the development of a selection system, (3) a better understanding of PA biosynthesis and (4) a more profound knowledge concerning toxicology and teratology of PA other than caffeine and theobromine. With respect to mass propagation of embryoids, the progress in coffee tissue culture made in various laboratories of the world is encouraging; studies on coffee plant regeneration from protoplasts should be given high priority. A selection system for caffeine-free mutants could be a bioassay taking advantage of the special traits of caffeine against fungi (fungitoxicity) or against bacteria (inhibition of the UV dark repair mechanism). As regards PA biosynthesis, the methyltransferase catalysing the reaction theobromine -» caffeine and the corresponding expression mechanisms should preferably be studied. A new hypothesis regarding the key reaction leading from primary metabolism to PA synthesis will be published soon (). The pharmacology as well as the teratology of the methylxanthines theobromine and caffeine has already been studied (), and both compounds have been found harmless. Therefore a caffeine-free coffee plant containing theobromine in place of caffeine would not produce any hazards for human health. In contrast, methyluric acids which might be thought to replace caffeine – the beans of the wild cocoa Theobroma grandiflorum for instance are chemically protected by theacrine () – have not yet been characterized for their pharmacological and toxicological effects on human beings. Kihlman () has written a comprehensive article as regards theacrine actions on plants, Chinese hamster cells, and E. coli. Nevertheless the methyluric acids must still be regarded as valuable candidates which may replace caffeine yielding a caffeine-free coffee protected against attack by herbivores and pathogens.

 

Selections from the book: “Medicinal and Aromatic Plants I”, 1988.