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Phyton (Austria)
Vol. 25
Fasc. 1
113-121
28. 2. 1985
Investigations on Carotenoids in Embryophyta
III Representatives of the Hepaticae
B . CZECZUGA *)
With 1 Figure
Received March 27, 1984
.
•
Summary
CZECZUGA B. 1985. Investigations on carotenoids in Embryophyta III.
Representatives of the Hepaticae. — Phyton (Austria) 25 (1): 113—121, with
1 figure. — English with German summary.
By means column and thin-layer chromatography the occurrence of
carotenoids and their content was determined in thalli of 10 species from
the Hepaticae. 19 carotenoids were found. Moreover quantitative and
qualitative differences were found in the content of carotenoids in thalli
of the investigated Hepaticae.
i
Zusammenfassung
' .
CZECZUGA B. 1985. Untersuchungen über Carotinoide in Embryophyta III.
Vertreter der Hepaticae. — Phyton (Austria) 25 (1): 113—121, mit 1 Abbildung. — Englisch mit deutscher Zusammenfassung.
An den Thalli von 10 Arten aus der Klasse der Hepaticae wurden
säulen- und dünnschichtchromatographisch Vorkommen und Menge der
Carotinoide bestimmt. Es wurden 19 Carotine gefunden. Weiters bestehen
quantitative und qualitative Unterschiede im Carotinoidgehalt der Thalli
der untersuchten Lebermoose.
Introduction
The past investigations on carotenoids in the phylum Bryophyta
were mainly concerned with mosses, whereas only few papers have
refered to these pigments regarding few species of Hepaticae. Thalli
of Marchantia polymorpha, however, were analyzed by several authors
*) Prof. Dr. B. CZECZUGA, Department of General Biology, Medical
Academy, PL-15-230 Bialystok, Poland.
Phyton, Vol. 25, Fasc. 1, 1985.
$
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114
and therefore only for this species of Hepaticae carotenoids has been
given a fuller account (SUIRE 1975, GOODWIN 1980).
In view of all these data I started the analysis of individual
carotenoids occurrence in thalli of available Hepaticae representatives
with expectation that the obtained results will enrich our knowledge
in this field.
'".
M a t e r i a l and Methods
Investigations were carried out on 10 Hepaticae spezies collected
in Bialystok region: Chiloscyphus polyanthus (L.) CORDA, Conocephalum conicum (L.) DTJM., Lophocolea heterophylla (SCHRAD.) DUM.,
Lunularia cruciata (L.) DUM., Marchantia polymorpha L., Metzgeria
pubescens (SCHRAD.) RADDI, Odontoschisma denudatum (MART.) DUM.,
Pellia fabbroniana RADDI, Riccia fluitans L. and Trichocolea tomentella (EHRH.) DUM.
The thalli were cleaned of all organic debris, macerated and
placed into dark glass bottles and covered with acetone thus replacing
the air above the fluid in the bottle by nitrogen. The samples were kept
in a refrigerator until removed for Chromatographie analysis of the
carotenoid content.
• The carotenoid pigments were extracted by means of 95% acetone
a dark room. Saponification was carried out by means of 10% KOH
in ethanol at a temperature of about 20° C for 24 hours in the dark
in a nitrogen atmosphere.
Columnar and thin-layer chromatography, described in detail in
our previous (CZECZUGA 1980 a) were used for the separation of the
various carotenoids. A glass column (Quickfit — England) approximately 1 cm 0 and 15—20 cm in length, filled with A12O3) was used in
column chromatography. The extract was passed through the column
after which the different fractions were eluted with the solvent. Silicagel was used for the thin-layer chromatography, with the appropriate
solvent systems, the Rf-values being determined for each spot. For
identification of carotenoids co-chromatography was applied using
authentic carotenoids (Hoffmann-La Roche & Co. Ltd., Basel, Switzerland and Sigma Chemical Company, USA).
The pigments were identified by the following methods: a) behaviour on column chromatography, b) absorption spectra of the pigments
in various solvents were recorded using a Beckman spectrophotometer
model 2400 DU, c) the partition characteristics of the carotenoid between
hexane and 95% methanol, d) comparison of Rf on thin-layer chromatography, e) the presence of allylic hydroxyl groups was determined
by the acid chloroform test, and f) the epoxide test. ;.
>
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115
Quantitative determinations of the concentrations of carotenoid
solutions were made from the quantitative absorption spectra. These
determinations were based on the extinction coefficient E [l°/o/cm] at
the wavelengths of maximal absorbance in petroleum ether or hexane.
Differences between any two means were tested using the Student's t-test.
Results
14 carotenoids were identified in thalli of the Hepaticae investigated (Table 1 and Fig. 1), of which the presence of lycopene (Marchantia
polymorpha, Odontoschisma denudatum, Pellia fabbroniana), a-cryptoTable 1
List of the carotenoids from the investigated Hepaticae species
Carotenoid
Structure
Seraisystematic name
(see Fig. 1)
lycopene
lycoxanthin
a - carotene
ß - carotene
a - cryptoxanthin
ß - cryptoxanthin
lutein
lutein epoxide
A-X-A
A- X—B
C-X-D
D-X-C
C-X-F
D-X-F
E-X-F
E- X— K
1|>, ij) - carotene
ij>,ty- caroten - 16 - ol
ß, e - carotene
ß, ß - carotene
ß, e - caroten - 3' - ol
ß, ß - caroten - 3 - ol
ß, E - carotene - 3,3'-diol
5,6 - epoxy - 5,6 - dihydro - ß, e - carotene —
3,3' - diol
ß - carotene epoxide D - X - I
5,6 - epoxy - 5,6 - dihydro - ß, ß - carotene
zeaxanthin
F-X-F
ß, ß - carotene - 3,3' - diol
antheroxanthin
F - X - K 5,6 - epoxy - 5,6 - dihydro - ß, ß - carotene 3,3' - diol
a - doradexanthin
E - X - H 3,3' - dihydroxy - ß, e - carotene - 4 - one
F - X - H 3,3' - dihydroxy - ß, ß - carotene - 4 - one
adonixanthin
K - X i - L 5,6' - epoxy - 6,7 - didehydro - 5,6,5',6' neoxanthin
tetrahydro - ß, ß - carotene - 3,5,3' - triol
K-X-K
5,6,5',6' - diepoxy - 5,6,5',6' - tetrahydro
violaxanthin
- ß, ß - carotene - 3,3' - diol
F - X : - M 5,8 - epoxy - 5,8 - dihydro - ß, ß - carotene mutatoxanthin
3,3' - diol
M - X 2 - M 5,8,5',8' - diepoxy - 5,8,5',8' - tetrahydro
auroxanthin
- ß,ß - carotene - 3,3' - diol
N - Y - N 4',5' - didehydro - 4,5' - retro - ß, ß rhodoxanthin
carotene - 3,3' - dione
apo -12' K - X3
5,6 - eposy - 3 - hydroxy - 5,6 - dihydro - 12'
violaxanthal
- apo - ß - carotene - 12' — al
8*
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116
xanthin (Trichocolea tomentella), auroxanthin (Marchantia polymorpha,
Odontoschisma denudatum), mutatoxanthin (Marchantia polymorpha,
Odontoschisma denudatum, Pellia fabbronina), a-doradexanthin (Trichocolea tomentella, Metzgeria pubescens) and rhodoxanthin (Conocephalum conicum, Lophocolea heterophylla collected in March) is
worthy of note. The carotenoids found in thalli of all investigated He-
Y -
Fig. 1. Constituent parts of the carotene molecules from the investigated
Hepaticae (see Tab. 1).
paticae species were: ß-carotene, ß-cryptoxanthin, lutein (epoxy or
free) and zeaxanthin (Table 2). The Total carotenoid contents oscillated between 8,0 (Pellia fabbroniana) and 62,6 mg/g dry weight
(Lophocolea heterophylla).
Table 3 shows the results of the carotenoid content analysis in
thalli of Marchantia polymorpha collected in various months. The
carotenoids occurring in thalli through the whole period of the investigations are ß-cryptoxanthin, lutein epoxide and zeaxanthin. A relatively big amount of lutein epoxide is worthy of note. Regarding the
total carotenoid content, however, small amounts of carotenoids in
spring and autumn, and the accumulation of carotenoids in summer
4.7
6.7
42.0
5.1
45.7
+
52.3
+
10.0
6.9
44.2
19.6
2.3
2.2
6.4
23.4
9.5
3.1
Total amount
(mg/g dry weight)
C. conicum
10.2
Ch. polyanthus
lycopene
a - carotene
ß - carotene
ß - carotene epoxide
a.\- cryptoxanthin
ß - cryptoxanthin
lutein
lutein expoxide
zeaxanthin
antheroxanthin
violaxanthin
auroxanthin
rhodoxahthin
mutatoxanthin
a - doradexanthin
apo — 12' — violaxanthal
Carotenoid
42.0
15.0
3.6
56.5
10.8
2.6
6.6
4.9
L. cruciata
62.6
21.9
4.0
11.7
33.3
18.5
0.8
9.8
18.9
5.0
5.8
20.6
8.6
9.8
20.8
7.9
5.7
9.2
6.6
11.5
4.9
11.3
63.7
5.9
1.5
12.5
13.4
3.3
2.8
44.4
22.4
9.1 .
+
6.2
4.0
4.0
3.8
8.1
4.3
3.5
21.4
19.3
8.8
9.8
30.9
2.0
L. hete- M. poly- M. pu- O. denu- P. fabrophylla morpha bescens datum broniana
Table 2
Carotenoid pigments in some species of Hepaticae
(% of total pigments, + = trace)
12.3
16.9
39.1
11.6
5.1
7.4
10.8
9.1
itans
13.7
17.5
9.2
26.7
9.8
8.7
11.6
16.6
tella
R.flu- P. tomen-
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Table 3
Carotenoid pigments in Marchantia polymorpha
(% of total pigments, + = trace)
Carotenoid
lycopene
lycoxanthin
a-carotene
ß-carotene
ß - cryptoxanthin
lutein
lutein epoxide
zeaxanthin
antheroxanthin
adonixanthin
neoxanthin
violaxanthin
mutatoxanthin
11
20
in
rv
9
V
.
17
VI
Month
17
VII
21
IX
14
X
18
XI
7.9
9.3
1.8
7.4
59.8
29.0
2.0
3.7
+
8.0
83.7
+
17.4
39.9
5.7
9.2
16.4
20.8
20.6
8.6
7.3
8.2
14.7
8.5
+
7.7
6.7
48.8 48.8
6.0 8.3
20.3
Total content
(mg/g dry weight)
2.0
XZ
4? '
1.1
4.3
1.8
4.0
16.4
15.3
58.8
5.5
18.3
74.1
4.6
;
6-6
7.0
H-2
10.5
25.0
20.7
2.0
10.8
6.1
17.8
9.3
7.3
can be observed. The chlorophyll a/b ratio [Table 4) was the highest
in spring (1.41), and the lowest in October (1.00).
, :
Table 4
Chlorophyll a/b ratio in Marchantia polymorpha
(n = 5, SD values rounded up to 2 decimal places)
1
9
17
21
14
1,41
1,32
1,09
1,05
1,00
April
May
August
September
October
±
±
±
±
±
0,01
0,02
0,01
0,01
0,01
Discussion
Investigations on carotenoids in mosses in general and in several
species of Hepaticae (CZECZUGA 1980 a, CZECZUGA & al. 1982) carried
out some time ago, revealad a carotenoid diversity. All the carotenoids
mentioned in this paper, except a-cryptoxanthin, a-doradexanthin and
rhodoxanthin were already found in other moss species (STRAIN 1958,
TAYLOR & al.
1972,
MUES & al.
1973,
SUIRE 1975,
SCHMIDT-STOHN 1977).
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Hi
a-cryptoxanthin, known earlier as physoxanthin (BODEA & al. 1978),
was found in algae (LIAAEN-JENSEN 1977, CZECZUGA 1979), lichen
(CZECZUGA 1983), and in higher plants (CZECZUGA 1978). The second of
the carotenoids a-doradexanthin belongs to the carotenoid group
common in animals, and can be often found in Crustacea (CZECZUGA
1980 b) and in fishes (CZECZUGA 1981).
Rhodoxanthin is characteristic of some species of club-mosses,
horsetails and ferns (UEDA & MOMOSE 1968), and in some species of
Potamogeton in the first place (NEAMTU & ILLYES 1978). It can be often
met in numeraus species of conifers (IDA 1981).
The thalli of Marchantia polymorpha in various months revealed
a relatively big amount of lutein epoxide being accumulated in thalli
in the latter of summer, in autum, and after winter period. The content
of this carotenoid in that period oscillated between 48,8% (August,
September) and 83,7% (April). This may be connected with thalli decay
of given Hepaticae. Lutein epoxide is very often the predominant carotenoid in higher plants, especially during autumn. The highest total
carotenoids content in thalli of Marchantia polymorpha was observed
during summer. Investigations carried out on Chlorophyll a/b relation
showed falling tendencies of the ratio in the summer-autumn period.
RAO & al. (1979) investigating this ratio in three other species of
Hepaticae reported its range to be from 1,1 to 1.2 and in thalli of
Marchantia polymorpha was observed above 2.0 (ARC- 1982). According
to MARTIN (1980), it oscillated between 1.4 and 2.1 in 11 species of mosses, instead of ranging, like in higher plants, from 2.0 to 4.0 (CHANG &
TROUGHTON 1972, ALBERTE & al. 1976, IDA 1981). The low Chlorophyll a/b ratio in mosses has been explained by MARTIN (1980) and
MARTIN & CHURCHILL (1982) as being the result of moss occurrence
in poor light. The assumption that the ratio gradually decreases from
spring to autumn could be also confirmed by our investigations. In
the place the specimens were taken, thalli of Marchantia polymorpha
were most lit up when trees were still leafless, that is in April and
at the beginning of May. It was at the beginning of April that the
chlorphyll a/b ratio was the highest — 1.41. This is also evident from
the data by YOKOHAMA and MISONOU (1980) and PEREZ-BERMUDEZ & al.
(1981) regarding marine benthos green algae. Corresponding to the
increasing depth of the sea (lowering light) the chlorophyll a/b ratio
diminishes turning towards 1.0. The chlorophyll a/b ration of algae
sampled in shallows (brighter light) fluctuates from 1.25 to 2.56.
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