The coffee species Coffea dewevrei and Coffea arabica have marked differences in caffeine metabolism and the control of the caffeine content during fruit ripening and leaf aging is still not clear. The aim of this work was a detailed investigation on the alkaloid degradation in young and aged leaves, and immature and mature fruits of these species. Young and aged leaves, and immature and mature fruits were fed with [2-C-14] caffeine. After an incubation period they were extracted for [2-C-14] caffeine and metabolites and analysed by reversed-phase liquid chromatography and radiocounting of collected fractions. In leaves and fruits of Coffea dewevrei there were higher degradation rates of caffeine. In both species, compared to young tissues, aged leaves and mature fruits presented lower capacity to degrade the alkaloid, what was shown by the low radioactivities detected in the metabolites formed in the degradation pathway. Radioactivity was detected in 7-methylxanthine, which is also a precursor in the caffeine biosynthesis. The data support the conclusion that the ratio between biosynthesis and biodegradation controls the variation of the caffeine content during fruit ripening and leaf aging in C. arabica and C, dewevrei.33121957196

Mazzafera, P

Vitoria, AP

Pesquisa Agropecuária Brasileira

Repositorio da Producao Cientifica e Intelectual da Unicamp

Pesq. agropec. bras., Brasília, v.33, n.12, p.1957-1961, dez. 1998
CAFFEINE DEGRADATION IN LEAVES 1957
CAFFEINE DEGRADATION IN LEAVES AND FRUITS
OF COFFEA ARABICA AND COFFEA DEWEVREI1
ANGELA PIERRE VITÓRIA2 and PAULO MAZZAFERA3
ABSTRACT - The coffee species Coffea dewevrei and Coffea arabica have marked differences in
caffeine metabolism and the control of the caffeine content during fruit ripening and leaf aging is still not
clear. The aim of this work was a detailed investigation on the alkaloid degradation in young and aged
leaves, and immature and mature fruits of these species. Young and aged leaves, and immature and
mature fruits were fed with [2-14C] caffeine. After an incubation period they were extracted for
[2-14C] caffeine and metabolites and analysed by reversed-phase liquid chromatography and radiocounting
of collected fractions. In leaves and fruits of Coffea dewevrei there were higher degradation rates of
caffeine. In both species, compared to young tissues, aged leaves and mature fruits presented lower
capacity to degrade the alkaloid, what was shown by the low radioactivities detected in the metabolites
formed in the degradation pathway. Radioactivity was detected in 7-methylxanthine, which is also a
precursor in the caffeine biosynthesis. The data support the conclusion that the ratio between biosyn-
thesis and biodegradation controls the variation of the caffeine content during fruit ripening and leaf
aging in C. arabica and C. dewevrei.
Index terms: caffeine metabolism, coffee, methylxanthines.
DEGRADAÇÃO DE CAFEÍNA EM FOLHAS E FRUTOS DE COFFEA ARABICA E COFFEA DEWEVREI
RESUMO - As espécies de café, Coffea dewevrei e Coffea arabica, apresentam diferenças marcantes
quanto ao metabolismo de cafeína, e ainda não é claro o controle do conteúdo desse alcalóide durante o
amadurecimento dos frutos e envelhecimento das folhas. O objetivo deste trabalho foi fazer uma
investigação detalhada da degradação de cafeína em folhas jovens e velhas, e em frutos imaturos e
maduros dessas espécies. Folhas jovens e velhas, e frutos imaturos e maduros de Coffea arabica e
Coffea dewevrei foram incubados com [2-14C] cafeína. Após a incubação procedeu-se a extração de
[2-14C] cafeína e seus metabólitos, que foram analisados por cromatografia líquida de fase reversa e
determinação da radioatividade de frações coletadas. Em folhas e frutos de C. dewevrei houve maior
degradação de cafeína. Em ambas espécies de café, em comparação com tecidos jovens, folhas velhas e
frutos maduros mostraram ter menor capacidade em degradar o alcalóide, sendo isto mostrado pelas
radioatividades detectadas nos metabólitos formados na via de degradação da cafeína. Foi detectada
radioatividade em 7-metilxantina, que também está presente na via biossintética de cafeína. Os dados
suportam a conclusão de que a razão entre biossíntese e biodegradação controla a variação do conteúdo
de cafeína observada durante o amadurecimento dos frutos e envelhecimento das folhas em C. arabica
e C. dewevrei.
Termos para indexação: metabolismo da cafeína, café, metilxantinas.
INTRODUCTION
Kalberer (1965) fed Coffea arabica leaves with
four differently labelled caffeine and detected
3 or/and 7-methylxanthine, allantoin, allantoic acid
and urea as alkaloid metabolites. Later, Suzuki &
Waller (1984a, 1984b) showed in fruits of the same
species that caffeine was firstly converted to theo-
1 Accepted for publication on May 29, 1998.
Granted by Fundação de Amparo à Pesquisa do Estado de
São Paulo-FAPESP.
2 Bióloga, M.Sc., Dep. Fisiologia Vegetal, IB, Universi-
dade Estadual de Campinas, Caixa Postal 6109,
CEP 13083-970 Campinas, SP. Bolsista do CNPq.
3 Eng. Agr., Dep. Fisiologia Vegetal, IB, Universidade
Estadual de Campinas.  Bolsista do CNPq. E-mail:
pmazza@obelix.unicamp.br
Pesq. agropec. bras., Brasília, v.33, n.12, p.1957-1961, dez. 1998
A.P. VITÓRIA and P. MAZZAFERA1958
phylline (1,3-dimethylxanthine) and theobromine
(3,7-dimethylxanthine), being the first dimethylxan-
thine the main degradation pathway. Therefore, the
caffeine catabolic pathway was established as: caf-
feine ® theobromine/theophylline ® 3-/7-methyl-
xanthine ® xanthine ® uric acid ® allantoin ® al-
lantoic acid ® glyoxilic acid + urea ® CO2 + NH4.
Mazzafera et al. (1991) and Mazzafera (1993) in-
vestigated [8-3H] caffeine degradation in immature
fruits of several coffee species observing that in
C. dewevrei the alkaloid was rapidly metabolized when
compared to the other species. Enzymic studies car-
ried out by Mazzafera et al. (1994) suggested that the
ratio between biosynthesis and biodegradation might
determine caffeine content in the coffee tissues. This
might explain why caffeine content is high in imma-
ture fruits and young leaves of C. arabica and low
in immature fruits and leaves of C. dewevrei
(Mazzafera et al., 1991; Mazzafera & Magalhães,
1991).
Recently, Ashihara et al. (1996) showed that the
caffeine catabolic pathway in C. arabica leaves was
very similar to fruits (Suzuki & Waller, 1984a, 1984b)
and that ageing leaves degraded less caffeine than
young ones. In addition, they observed also that
7-methylxanthine could be a product from xanthine
methylation, this being more pronounced in aged
leaves. This monomethylxanthine had not been de-
tected by Mazzafera et al. (1991) and Mazzafera (1993)
in the same coffee species and in C. dewevrei.
C. dewevrei and C. arabica have marked differ-
ences in caffeine metabolism and control of the caf-
feine content during fruit ripening and leaf aging is
still not clear.
The aim of this work was a detailed investigation
on the alkaloid degradation in young and aged leaves,
and immature and mature fruits of these species.
MATERIAL AND METHODS
C. dewevrei leaves and fruits were collected from a tree
growing in a living collection at the Instituto Agronômico
de Campinas, SP, Brazil. Leaves and fruits of C. arabica
were collected from a tree growing at the experimental area
of our department. Immature fruits were considered those
with ivory and opaque endosperm, as described by Mendes
(1941). Mature fruits were considered those with hard-
ened endosperm. Young leaves were collected from the
first leaf pair, and old leaves from the fifth and sixth leaf
pairs.
For the radioisotopic feeding experiments,
[2-14C] caffeine was synthezised by methylation of
[2-14C] xanthine (specific activity 52.4 mCi.mmol-1, Sigma
Chemical Company, St Louis, USA) with dimethylsulfate,
according to Heftmann (1971).
The fruits were fed with [2-14C] caffeine using the pro-
cedure used by Mazzafera et al. (1991). The amount of
radioactivity given varied according to the absorption of
the solution by the fruits (Table 1). After 72 hours, of
incubation the fruits were extracted for caffeine and
metabolites.
Leaf disks were obtained from leaves with a cork-borer
(2 cm diameter) weighed and infiltrated under vacuum with
water containing the labelled caffeine and 0.1% Tween 20.
After infiltration, they were quickly blotted dry and
weighed again. By this way it was possible to estimate the
[2-14C] caffeine infiltrated. The disks were transferred to a
plastic box containing a sheet of foam wet with water at
the bottom. The box was kept opened until the excess of
water had been evaporated from the disks. Then the box
was closed and maintained under constant laboratory light
(300 mE.m-2.s-1). After 48 hours they were extracted for
caffeine and metabolites.
The same procedure was used for the extraction of
caffeine and metabolites from leaves and fruits. The mate-
rial was frozen with liquid nitrogen and finely ground in
mortar with pestle. The powder was transferred to cap
sealed tubes, 500 mg of MgO was added and the extraction
was done with 10 mL of 0.0125 N  aqueous  H2SO4 in
80oC  water-bath for one hour. The tubes were centrifuged
at 1,200 g and the supernatants reserved. Two following
extractions were made with 10 mL of 0.0125 N aqueous
H2SO4 and the supernatants combined. Then they were
fractionated with 3 x 20 mL of chloroform and the com-
bined chloroform fractions were dried under  reduced pres-
sure at 25oC. The compounds solubilized in 2 mL of dis-
tilled water were filtered in 0.22 mm membrane for subse-
quent analysis in HPLC (high performance liquid chroma-
tography). Most of the caffeine, theobromine and theo-
phylline was expected to be found in these fractions.
The remaining aqueous phase were dried at 80oC and
the residues solubilized in 2 mL of distilled water.
C18 Sep-Pak cartridges (Millipore - Waters Associates)
were used for cleaning up the samples. The first eluting
liquid and a 2 mL further 50% aqueous methanol washing
were combined, filtered in 0.22 mm membranes and used
for analysis in HPLC. In these fractions it was expected to
find monomethylxanthines, xanthine, uric acid, allantoin
and allantoic acid.
Pesq. agropec. bras., Brasília, v.33, n.12, p.1957-1961, dez. 1998
CAFFEINE DEGRADATION IN LEAVES 1959
In the samples from  the chloroform  phase, 10 mg of
caffeine, theobromine and theophylline as internal stan-
dards were added. The compounds were separated by re-
versed-phase liquid chromatography using a Shimadzu
HPLC system with a Spherisorb ODS2 column (5 mm,
4 mm x 250 mm). The solvent (12.5% methanol in 0.5%
aqueous acetic acid) was delivered by a LC-10AS pump at
a flow rate of 1 mL.min-1 and the compounds eluting from
the column were detected in SPD-10A UV-detector oper-
ating at 280 nm. The signals from the detector were inte-
grated in a C-R6A Chromatopac recorder. Caffeine, theo-
bromine and theophylline eluting from the column, moni-
tored by the UV-detector and the recorder were collected
in scintillation vials. After drying at 80oC, 5 mL of scintil-
lation fluid was added to the vials and the radioactivity
determined in a LS 6000TA scintillation counter (Beckman
Instruments).
In the samples from the aqueous phase, 10 mg of
allantoin, allantoic acid, uric acid, xanthine,
7-methylxanthine, 3-methylxanthine, theobromine, theo-
phylline, caffeine as internal standards were added. At
beginning of the chromatography, 0.5% aqueous acetic acid
was used as solvent. The solvent was delivered at a
0,5 mL.min-1 flow rate and the detector at 280 mm. After
20 minutes, enough time for the elution of the
monomethylxanthines, allantoin, allantoic acid, uric acid
and xanthine, the solvent was changed to 12.5% methanol
in 0.5% aqueous acetic acid and the flow was increased to
1 mL.min-1 in order to detect caffeine, theobromine and
theophylline. Allantoin, allantoic acid and uric acid elute
very close from the HPLC, therefore, they were collected
as only one fraction. The eluting compounds, monitored
by the UV-detector and the recorder, were collected in
scintillation vials and processed as above for the determi-
nation of radioactivity.
RESULTS AND DISCUSSION
Previous data in the literature have shown that
caffeine degradation in C. arabica fruits and leaves
proceeds slowly (Suzuki & Waller, 1984a, 1984b;
Mazzafera et al., 1991; Mazzafera et al., 1994;
Ashihara et al., 1996).
Suzuki & Waller (1984b) observed that caffeine
degradation in ripening fruits was slow compared to
immature fruits. The fruits were incubated with
[2-14C] caffeine and after the incubation period 93%
of the radioactivity was still present in caffeine. How-
ever, fruits incubated with [8-14C] theophylline
showed much less radioactivity in this
dimethylxanthine compared with the [2-14C] caffeine
incubations, indicating that the conversion of caf-
feine to theophylline is the limiting step in caffeine
degradation. Incubations with [2-14C] xanthine
showed a very high metabolism to uric acid, allantoin
and allantoic acid. In [2-14C] caffeine incubations ra-
dioactivity was detected in both theophylline and
theobromine, however less in the first dimethyl-
xanthine, suggesting that the main route for caffeine
degradation was via theophylline and subsequently
to 3-methylxanthine.
The results in the Table 1 show that fruits of
C. dewevrei metabolized caffeine more extensively
than C. arabica fruits. Similar to the data of Suzuki &
Waller (1984b), for both species radioactivity was
also detected in theobromine and theophylline, with
higher levels in the first dimethylxanthine, indicating
theophylline as the main route for caffeine degrada-
TABLE 1. Radioactivity distribution among [2-14C] caffeine metabolites in mature and immature coffee fruits
incubated for 72 hours1.
Coffee
species
Radioactivity
applied
(cpm)
Caf
(%)
Thb
(%)
Thf
(%)
7mx
(%)
3mx
(%)
Xan
(%)
Ua + All + Ala
(%)
&DUDELFD
   Immature
   Mature
1,071,000
1,250,000
91.4
92.9
4.8
6.0
2.2
1.1
nd
nd
nd
nd
1.6
nd
nd
nd
&GHZHYUHL
   Immature
   Mature
843,000
899,000
2.9
3.9
73.5
71.3
1.6
3.6
1.3
1.0
11.6
7.7
1.4
3.8
7.7
8.8
1 Applied radioactivity refers to [2-14C] caffeine; radioactivities associated with the compounds are expressed as % of recovered radioactivity;
Caf: caffeine; Thb: theobromine; Thf: theophylline; Xan: xanthine; 7mx: 7-methylxanthine; 3mx: 3-methylxanthine; Ua: uric acid; All: allantoin;
Ala: allantoic acid; nd: not detected.
Pesq. agropec. bras., Brasília, v.33, n.12, p.1957-1961, dez. 1998
A.P. VITÓRIA and P. MAZZAFERA1960
tion. After 72 hours of incubation, immature fruits of
C. arabica accumulated less radioactivity in theo-
bromine and more in theophylline than mature fruits,
suggesting a more intense caffeine degradation al-
though the differences of radioactivity in this alka-
loid were not pronounced. Radioactivity was de-
tected in xanthine only in immature fruits, also de-
noting a higher caffeine degradation.
Similarly to Mazzafera et al. (1991) and Mazzafera
(1993), a significant accumulation of radioactivity in
theobromine was observed in fruits of C. dewevrei
(Table 1). These results are not sufficient to affirm
that caffeine degradation via theobromine proceeds
with higher intensity than in C. arabica. This might
be only a consequence of a more accelerate caffeine
degradation, and a limited degradation of theobro-
mine to 3-methylxanthine.
Much less radioactivity was present in caffeine
in immature and mature fruits of C. dewevrei
(Table 1). Except for theophylline, in mature fruits
less radioactivity accumulated in the other
methylxanthines. This, in addition to the higher ra-
dioactivity in caffeine, suggests lower degradation
rate of the alkaloid in mature fruits of C. dewevrei.
The data of this work  with C. arabica leaves
(Table 2) are in agreement with those of Ashihara
et al. (1996). These authors observed that caffeine
degradation was similar in mature (third leaf pair) and
aged leaves (near the base of the shoot), but lower
than in young leaves (first leaf pair). Here it was
observed that, compared to C. arabica, caffeine was
more metabolized in C. dewevrei leaves, particularly
in young ones, with expressive accumulation of
radioactivity in theobromine. However, this accumu-
lation was proportionally lower than in fruits.
In C. arabica fruits and leaves, consecutive
methylation of 7-methylxanthine and theobromine
leads to caffeine biosynthesis (Suzuki & Waller,
1984b; Ashihara et al., 1996). However, from studies
with fruits (Suzuki & Waller, 1984a, 1984b) it was
not clear whether during caffeine degradation,
7-methylxanthine was also a product of theobromine
demethylation. Using tritiated caffeine at position 8,
which is lost after xanthine conversion to uric acid
Mazzafera (1993) concluded that 7-methylxanthine
was not formed from theobromine in mature leaves
and immature fruits of C. dewevrei. On the other hand,
Ashihara et al. (1996) reported that in aged leaves
of C. arabica 7-methylxanthine was newly
synthesized from xanthine, but they did not give
evidence that this monomethylxanthine was a
product of theobromine degradation. Therefore,
7-methylxanthine detected here might be from
xanthine and not from theobromine.
The role of 7-methylxanthine in caffeine degrada-
tion is still unclear. However, its re-utilisation
in caffeine biosynthesis might be possible, as
suggested in a recent work of Ashihara et al. (1997)
with tea, where it was observed that xanthine was
salvaged for caffeine biosynthesis, by formation of
3-methylxanthine.
TABLE 2. Radioactivity distribution among [2-14C] caffeine metabolites in young and aged coffee leaves
incubated for 48 hours1.
Coffee
species
Radioactivity
applied
(cpm)
Caf
(%)
Thb
(%)
Thf
(%)
7mx
(%)
3mx
(%)
Xan
(%)
Ua + All + Ala
(%)
&DUDELFD
     Young
     Aged
446,000
726,000
76.6
84.1
13.3
11.7
10.1
2.1
nd
2.1
nd
nd
nd
nd
nd
nd
&GHZHYUHL
     Young
     Aged
412,000
1,501,000
32.1
64.1
50.4
24.5
4.1
6.9
3.0
nd
nd
1.6
5.4
1.2
5.0
1.7
1 Applied radioactivity refers to [2-14C] caffeine; radioactivities associated with the compounds are expressed as % of recovered radioactivity;
Caf: caffeine; Thb: theobromine; Thf: theophylline; Xan: xanthine; 7mx: 7-methylxanthine; 3mx: 3-methylxanthine; Ua: uric acid; All: allantoin;
Ala: allantoic acid; nd: not detected.
Pesq. agropec. bras., Brasília, v.33, n.12, p.1957-1961, dez. 1998
CAFFEINE DEGRADATION IN LEAVES 1961
CONCLUSIONS
1. Coffea dewevrei leaves and fruits show higher
caffeine degradation than Coffea arabica ones.
2. In both species, aged leaves and mature fruits
show lower capacity to degrade caffeine.
3. 7-methylxanthine, which is also involved in the
caffeine biosynthesis, is a product of the alkaloid
degradation in aged leaves of Coffea arabica and
young leaves of Coffea dewevrei.
4. The biosynthesis and biodegradation ratio
controls the caffeine content variation during fruit
ripening and leaf aging in Coffea arabica and Coffea
dewevrei.
REFERENCES
ASHIHARA, H.; GILLIES, F.M.; CROZIER, A. Catabo-
lism of caffeine and related purine alkaloids in leaves
of tea (Camellia sinensis L.). Plant and Cell Physi-
ology, v.38, n.4, p.413-419, 1997.
ASHIHARA, H.; MONTEIRO, A.M.; MORITZ, T.;
GILLIES, F.M.; CROZIER, A. Catabolism of caf-
feine and related purine alkaloids in leaves of Coffea
arabica L. Planta, v.198, n.3, p.334-339, 1996.
HEFTMANN, E. Synthesis of caffeine-2-14C. Journal
of Labelled Compounds and Radiophar-
maceuticals, v.7, n.4, p.463-465, 1971.
KALBERER, P. Breakdown of caffeine in the leaves of
Coffea arabica L. Nature, v.205, n.4971, p.597-598,
1965.
MAZZAFERA, P. 7-methylxanthine is not involved in
caffeine catabolism in Coffea dewevrei. Journal of
Agricultural and Food Chemistry, v.41, n.10,
p.1541-1543, 1993.
MAZZAFERA, P.; CROZIER, A.; MAGALHÃES, A.C.
Caffeine metabolism in Coffea arabica and other
species of coffee. Phytochemistry, v.30, n.12,
p.3913-3916, 1991.
MAZZAFERA, P.; CROZIER, A.; SANDBERG, G. Stud-
ies on the metabolic control of caffeine turnover in
developing endosperms and leaves of Coffea arabica
and Coffea dewevrei. Journal of Agricultural and
Food Chemistry, v.42, n.7, p.1423-1428, 1994.
MAZZAFERA, P.; MAGALHÃES, A.C.N. Cafeína em
folhas e sementes de Coffea e Paracoffea. Revista
Brasileira de Botânica, v.14, n.2, p.157-160, 1991.
MENDES, A.J.T. Cytological observations in Coffea. VI.
Embryo and endosperm development in Coffea
arabica L. American Journal of Botany, v.28, n.9,
p.784-789, 1941.
SUZUKI, T.; WALLER, G.R. Biodegradation of caffeine:
formation of theophylline and theobromine from
caffeine in mature Coffea arabica fruits. Journal of
the Science of Food and Agriclture, v.35, n.1,
p.66-70, 1984a.
SUZUKI, T.; WALLER, G.R. Biosynthesis and biodegra-
dation of caffeine, theobromine, and theophylline in
Coffea arabica L. fruits. Journal of Agricultural
and Food Chemistry, v.32, n.4, p.845-848, 1984b.


Caffeine degradation in leaves and fruits of Coffea arabica and Coffea dewevrei

http://repositorio.unicamp.br/jspui/bitstream/REPOSIP/78280/1/WOS000078333800002.pdf

Caffeine degradation in leaves and fruits of Coffea arabica and Coffea dewevrei

Abstract

Similar works

Full text

Available Versions

Repositorio da Producao Cientifica e Intelectual da Unicamp