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METHODS
AbstractMethods of pigment extraction using traditional polar organic solvents (acetone or methanol) were
compared to those employing a chloroformmethanol mixture. We found that, for spectrophotometric pigment
analysis in the apple peel, the cuticular lipids must be preliminarily extracted from the samples with chloroform
and MgO must be added during homogenization to prevent pigment degradation. The traditional extraction did
not result in the complete extraction of intact pigments, and such extracts contained a considerable amount of
light-absorbing impurities. The application of chloroformmethanol extraction allowed us to markedly reduce
the content of such impurities and to increase the accuracy and sensitivity of the measurement of the content of
chlorophylls and carotenoids. In addition, this extraction method proved useful for the analysis of phenolic substances (anthocyanins and flavonoids) in the water-methanol fraction of the extracts.
Key words: anthocyanins - carotenoids - chlorophylls - spectrophotometry - Malus domestica
INTRODUCTION
The amount and ratio of chlorophylls, carotenoids,
and anthocyanins in leaves and fruits of plants are
important and are sensitive indices of their physiological state and its changes during growth, development,
and when affected by various stresses [17]. Plant tissues also possess a high content of flavonoids, anthocyanins, and other phenolic substances. The study of the
quantitative aspects of the changes in the content of
these substances, which perform various important
roles in plants, is also of interest [712].
Pigments in plant tissues are generally quantified by
spectrophotometric methods [1, 2, 1315], which are
subject to interference from light-absorbing impurities
present in the extracts [15, 16]. Since apple peel has a
low content of Chl and Car [1], other light-absorbing
substances present in the conventional ethanol or acetone extracts [13, 14] can markedly contribute to the
absorption of peel extracts. In addition, apples contain
considerable amounts of cuticular lipids [17] and
organic acids [18], which can influence the extractability and spectral properties of pigments, as well as chlorophyll pheophytinization. Under certain conditions
(senescence, stress) some products of Chl degradation
appear in plant tissues, and these products can also arise
during extraction [3, 4, 15]. Thus, it is essential to avoid
pigment degradation during extraction, to remove lightabsorbing impurities, and to increase the sensitivity of
Chl and Car analysis. On the other hand, to analyze
Abbreviations: Anthanthocyanins; Carcarotenoids; ChF
chloroform fraction of the Folch extract; Chlchlorophyll;
Pheopheophytins; WMFwater-methanol fraction of the
Folch extract.
phenolic substances, such as flavonoids and anthocyanins, one must first eliminate the contribution of Chl
and Car to the total absorption.
To approach the problems associated with Chl and
Car analysis, Wellburn [19] suggested to extract pigments from plant tissues with a chloroformmethanol
mixture. Following this strategy, we used an extraction
system, proposed by Folch et al. [20], which is widely
used in lipid analysis and allows the easy separation of
the lipid- and water-soluble components of extracts
between the chloroform (ChF) and water-methanol
(WMF) fractions. We investigated the possibility of
using ChF for measuring the Chl and Car contents in
apples. We also demonstrated that the WMF, free of Chl
and Car, could be used for measuring the content of
Anth, flavonoids, and other phenolic substances in
fruits.
MATERIALS AND METHODS
Apples (Malus domestica Borkh., cv. Zhigulevskoe)
were collected in 19992000. The peel was cut from the
fruit surface and carefully freed from the pulp; ten disks
of 11 mm in diameter were cut out with a cork borer,
twice washed with distilled water for 1 min, and dried
between sheets of filter paper. To remove cuticular lipids, the disks were washed for 1 min with two 10-ml
portions of chloroform. To prevent chlorophyll pheophytinization, CaCO3 or MgO were added before disk
homogenization [13]: 200 mg for immature fruits, collected early in August, and 100 mg for ripe fruits, collected at later dates. Pigments were extracted using two
methods: (1) the conventional method with polar solvents (acetone or methanol) [1, 13, 19] or (2) according
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acetone
ChF
83.8 6.4*
93.2 4.0
62.8 11.1
93.8 2.4
92.1 1.7
94.2 2.0
95.2 1.3
96.0 1.4
96.0 1.5
(1)
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695
2.4
1
2.0
~
~
1
0.4
Absorbance
0.3
0.2
0.1
3
4
2
0
350
400
450
500
550
Wavelength, nm
600
650
700
Fig. 1. (1) Typical absorption spectrum of a methanol extract from the apple peel and (2) its residual spectrum obtained by subtracting the absorption spectrum of (3) Chl a and (4) Chl b from the original spectrum (curve 1).
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SOLOVCHENKO et al.
2.0
2
1.8
1.6~
~
0.6
Absorbance
0.4
3
0.2
4
0
350
400
450
500
550
Wavelength, nm
600
650
700
Fig. 2. (1) Typical absorption spectrum of an acetone extract from the apple peel and (2) its residual spectrum obtained by subtracting the absorption spectrum of (3) Chl a and (4) Chl b from the original spectrum.
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697
1.2
1.0
0.8
0.6
Absorbance
2
0.4
5
0.2
1
4
0
350
400
450
500
550
Wavelength, nm
600
650
700
Fig. 3. (1) Typical absorption spectrum of the chloroform fraction of a Folch extract from the apple peel, (2) its residual spectrum
obtained by subtracting the absorption spectrum of (3) Chl a and (4) Chl b from the original spectrum, and (5) absorption spectrum
of the unsaponified fraction in chloroform.
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SOLOVCHENKO et al.
1.0
(a)
0.8
1
3
0.6
Absorbance
0.4
2
0.2
0
250
300
350
400
450
2.4
4
(b)
~
~
0.6
0.5
0.4
0.3
0.2
4
0.1
3
0
400
450
500
550
Wavelength, nm
600
650
700
Fig. 4. Typical absorption spectra of the water-methanol fraction of Folch extracts from the apple peel.
(a) Spectra of peel extracts taken from (1) the sun and (2) shade sides of an apple and (3) spectrum of the methanol solution of pure
rutin. Spectra 1 and 2 were recorded with eightfold diluted WMF. (b) Absorption spectra of (1, 2) the methanol extract and (3, 4)
the water-methanol fraction of a Folch extract. Spectra 2 and 4 were recorded after the addition of HCl. Spectra of undiluted extracts
are shown.
The Folch system provided for the highest sensitivity of the analysis and the complete extraction of Chl
and Car. To measure the content of these pigments at
levels of about 0.40.5 nmol/cm2 fruit surface, no more
than 10 cm2 of fruit surface is required, whereas conventional analysis requires four to seven times more [1,
2]. The sensitivity of analysis in ChF is increased when
more concentrated solutions and microcuvettes are
used for pigment spectrophotometry.
The study of phenolic substances in fruit peel is of
significant interest. These compounds can serve as a
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light filter to absorb UV-radiation and act as antioxidants [712]. In acetone and methanol extracts, their
absorption spectrum overlaps with Chl and Car bands
(Figs. 1, 2), which makes their spectral analysis difficult. The low absorption of WMF in the range of 500
750 nm (Fig. 4b, curve 3) indicates the absence of Chl
and Car in these fractions, which allows for a more
accurate characterization of the substances that absorb
in the near UV region (Fig. 4a). The content of phenolic
substances (including flavonoids) in the peel taken
from the sun side was higher than in the peel from the
shade side of the fruits (cf. curves 1 and 2 in Fig. 4a),
probably due to fruit acclimation to high insolation
[7, 12].
Conventionally, the Anth content of plant tissues is
measured by photometry of methanol or water-methanol extracts with HCl added [10, 25]. In this case, Pheo
and other products of Chl degradation may significantly
contribute to the absorption of extracts at 530 nm
(Fig. 4). Correcting for the presence of Pheo [25] can
reduce such error. However, taking into account the
probable formation of other products of Chl degradation [3, 4, 25], such correction is not sufficient. We
found that, in the presence of HCl, Anth had similar
spectral properties in methanol extracts and in the
WMF (Fig. 4, curves 2, 4). However, WMF allows for
a more correct measurement of Anth, because the WMF
does not contain Chl (Pheo) and Car. It should be taken
in account that, at a high Anth content (above
3 nmol/cm2), repeated extraction with acid methanol is
needed for its complete extraction.
Thus, the strong points of the method suggested
include the higher sensitivity and selectivity of Chl and
Car analysis, as compared to the conventional analysis,
and the opportunity for the differential measurement of
Anth and other phenolic substances in the same extract.
It seems likely that, with the use of the above-mentioned modifications and with the appropriate precautions, Folchs extraction method could be successfully
used for measuring the pigment content in other plant
tissues.
ACKNOWLEDGMENTS
This work was supported by the Russian Foundation
for Basic Research, project no. 97-04-48471.
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