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Russian Journal of Plant Physiology, Vol. 48, No. 5, 2001, pp. 693700. Translated from Fiziologiya Rastenii, Vol.

48, No. 5, 2001, pp. 801808.


Original Russian Text Copyright 2001 by Solovchenko, Chivkunova, Merzlyak, Reshetnikova.

METHODS

A Spectrophotometric Analysis of Pigments in Apples


A. E. Solovchenko, O. B. Chivkunova, M. N. Merzlyak, and I. V. Reshetnikova
Department of Cell Physiology and Immunology, Faculty of Biology, Moscow State University,
Vorobevy gory, Moscow, 119899 Russia;
e-mail: mnm@6.Cellimm.bio.msu.ru
Received June 26, 2000

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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SOLOVCHENKO et al.

Comparison of the completeness of Chl and Car extraction from


the peel of ripe apples (the content of Chl is ca. 3 nmol/cm2)
with methanol, acetone, and with the Folch system (see Materials and Methods)
Pigment(s)
Chl a
Chl b
Car

The fraction of extracted pigments, %


methanol

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

* Mean value with standard deviation (data of three replicate


assays). The amount of pigments after threefold extraction by the
Folch method was taken equal to 100%.

to Folch et al. [20]. In the first case, extracts were


obtained by the rapid homogenization of disks with a
mortar and pestle in 10 ml of methyl alcohol or acetone.
The peel homogenate was centrifuged in a glass test
tube at 3000 g for 510 min. For extraction by the Folch
method, the disks were homogenized with a mortar and
pestle in 10 ml of chloroformmethanol (2 : 1, v/v)
mixture; the homogenate was passed through a paper
filter. Then, distilled water was added to the amount of
0.2 of the extract volume, and the diluted filtrate was
centrifuged in glass test tubes for 10 min at 3000 g to
complete the separation of ChF and WMF.
To saponify Chl and lipids, ChF was evaporated to
dryness in a rotary evaporator, and the dry residue was
dissolved in 4 ml of methanol. Then, 360 l of 1 M
aqueous solution of KOH was added, and the solution
was incubated in darkness for 12 h. Unsaponified lipids
(including Car) were extracted with three portions of nhexane, 2 ml each; the extracts were combined, completely evaporated, and the residue was dissolved in
chloroform. Pheophytins a and b were obtained by adding HCl to corresponding Chl solutions [13]. The position of peaks and the ratio between the individual peaks
in the absorption spectra of Chl and Pheo were close to
those described in the literature [13, 19]. For an analysis of Anth, HCl was added to methanol extracts or
WMF to a final concentration of 0.1% [10]; in some
experiments, Anth were extracted with methanol containing 0.1% HCl.
Flavonoids were separated by TLC on 5565 cellulose plates (Merck, Germany) in a n-butanol : acetic
acid : water (4 : 1 : 5, v/v/v, upper phase) system [10].
Car were separated by TLC on Silufol silica gel plates
(Kavalier, Czech Republic) in a hexane : chloroform :
benzene (10: 20: 1, v/v/v) system [21].
Absorption spectra were recorded with a Hitachi
150-20 spectrophotometer (Japan) with a spectral resolution of 2 nm. The spectra were digitized and processed in spreadsheets using an IBM-compatible PC.
To assess the absorption spectra of Car and lightabsorbing impurities, the absorption spectra of Chl a
and b were subtracted from the absorption spectra of

the extracts. The residual absorption spectra were


calculated from the equation
A ( ) = A 0 ( ) [ ( )C a + b ( )C b ],

(1)

where A() is the residual and A0() is the initial


absorption spectra, a() and b() are the spectra of the
absorption coefficients of Chl a and b, and Ca and Cb
are concentrations of Chl a and b, respectively [15,
22, 23].
The contents of Chl and Car were determined spectrophotometrically using the values of absorption coefficients in methanol, acetone [13], or chloroform [19];
for Anth, the value of the molar absorption coefficient
at 530 nm was taken equal to 30 mM1 cm1 [10].
a and b preparations were from Fluka (Switzerland),
rutin was from Chemapol (Czech Republic), and the
solvents (Chimed, Russia) were of a chemically pure
grade. Chloroform was purified by washing with an
equal volume of distilled water.
RESULTS
Acetone and methanol extracts (particularly after
their concentration) were often turbid, and preliminary
chloroform extraction of the cuticular lipids of the disks
eliminated this turbidity. In further experiments, we
compared the effects of CaCO3 and MgO on the extent
of Chl pheophytinization during extraction. With
CaCO3, spectral patterns characteristic of Pheo were
observed: clear peaks at 535 and 560 nm and between
590 and 620 nm [13, 15]. When the disks were ground
with MgO, the absorption spectra did not show any significant amount of Pheo (data not shown). Hence, in all
subsequent experiments, peel disks were treated with
chloroform before homogenization, and pigments were
extracted in the presence of MgO added to the amount
specified in the Materials and Methods.
The comparison of various methods for pigment
extraction showed that acetone and the Folch system
provided for a more complete extraction of Chl and Car
from apple peel than methanol extraction, which
extracted only 84 and 63% of Chl a and Car, respectively (table). Repeated extraction by the Folch method
resulted in almost complete extraction of the pigments.
In further experiments, we compared the spectral
properties of the extracts. The absorption bands of Chl a
(the main peak at 662666 nm) and Chl b (a shoulder
near 650 nm) were present in the red region of the
absorption spectrum (Figs. 13). In the blue region pronounced absorption bands near 430440 and 470
480 nm due to light absorption by Chl and Car [13, 15]
were present. The ChF spectra (Fig. 3, curve 1) also
contained a shoulder at 420 nm. The methanol and acetone extracts (Figs. 1, 2) strongly absorbed light in the
short-wavelength region at wavelengths below 425 nm.
In the red region, the absorption spectra of methanol
extracts and ChF (Figs. 1, 3, curves 1) are probably the
sum of Chl a and b absorption spectra and do not reveal

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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).

any impurities that absorb light in the range of 660


700 nm. Usually, the long-wavelength region of
absorption spectra of acetone extracts cannot be represented as the sum of only Chl a and b absorption spectra
(Fig. 2, curves 1, 2).
ChF absorbed in the near UV region (Fig. 3, curve 1)
to a considerably lesser extent than the acetone and
methanol extracts. The residual spectra of ChF (Fig. 3,
curve 2), calculated from equation (1), displayed small
deviation from the base line in the red region. Three
peaks at 420, 450 and 480 nm, which correspond to the
I, II, and III absorption bands of Car [24], are clearly
seen in these spectra. Similar spectra of methanol and
acetone extracts exhibited only II and III bands, and
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band I was masked by strong absorption by impurities


(Figs. 1, 2, curves 2). As seen from Fig. 3, the residual spectrum of ChF (curve 2) is very similar to the
absorption spectrum of its unsaponified fraction (curve 5).
The shape of the spectrum; the positions of the absorption peaks at 426, 449, and 478 nm; and the relationship
between these peaks corresponded to neoxanthin, violaxanthin, and their esters, isolated with TLC (data not
shown), which are major Car in ripe apples [5].
We also analyzed the spectra of the WMF in the peel
taken from the sun and shade sides of the fruits (Fig. 4a).
As seen from Figs. 1 and 2, in the acetone and methanol
extracts, the spectrophotometry of the substances that
absorb in the near UV region is difficult because their
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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.

absorption spectra overlap with those of Chl and Car.


The WMF spectra (Fig. 4b) displayed no absorption
bands of Chl and Car; they comprised absorption bands
near 280 and 360 nm, characteristic of phenolic compounds and flavonoids, respectively [9]. It seems likely
that rutin markedly contributes to the absorption at
360 nm of the WMF in the peel taken from the sun side
of fruits (this spectrum is also shown in Fig. 3). TLC
data and previous reports have confirmed the presence
of considerable amounts of this flavonoid in the apple
peel [11].
We found that WMF can be used to analyze Anth.
The addition of HCl to this fraction or to the methanol
extract from the peel (Fig. 4b, curves 2, 4) resulted in
the appearance of a broad absorption band of Anth with
a peak near 530 nm [8, 10]. In the red spectral region of

the methanol extract (Fig. 4b, curve 2), an absorption


band of Chl degradation products was found at 650 nm;
this band was absent from the WMF spectrum (Fig. 4b,
curve 4). The absorption band of Anth, which was
observed in the visible region of the absorption spectra
of the methanol extract (Fig. 4b, curve 2), was broader,
stronger, and less symmetric, apparently due to a contribution of Chl (Pheo) and Car degradation products to
the short-wavelength absorption maximum. When the
Anth content was up to 33.5 nmol/cm2, the Folch
method provided for its sufficiently complete extraction
from the samples. This fact is evidenced by similar
optical density values of the absorption maximum in
this case and when acid methanol was used (Fig. 4b,
curves 2, 4). It should be noted that at higher Anth content in the fruits, an additional step of dry residue

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A SPECTROPHOTOMETRIC ANALYSIS OF PIGMENTS

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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.

extraction with methanol, containing 0.1% of HCl, was


necessary for complete Anth extraction.
DISCUSSION
Our data show that cuticular lipids should be
removed to obtain the extracts suitable for the spectrophotometric analysis of pigments. Apparently, the turbidity of acetone and methanol extracts is due to the
low solubility of nonpolar cuticular lipids in these solvents. In addition, to prevent Chl degradation, MgO
should be added in a larger amount in the case of immature fruits, which contain high quantity of organic
acids.
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The spectral analysis of methanol and acetone


extracts showed that they contain impurities that markedly contribute to light absorption in the wavelength
range used for the spectrophotometric determination of
Chl and Car (Figs. 1, 2). Such impurities inevitably
introduce errors. The ChF of fruit peel contained far
less light-absorbing impurities (Fig. 3) and, therefore,
provided for a higher accuracy of the spectrophotometric analysis of pigments.
In addition, the use of ChF has an obvious advantage
in Car analysis, because, in chloroform, the III absorption maximum of the total Car (480 nm) is shifted to the
long-wavelength region (Fig. 3, [19]), as compared to
its position in acetone and methanol solutions (470 nm,
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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.

[13]). This fact and a much lower contribution of Chl b


to the absorption at 480 nm in chloroform [19] provide
for a more correct estimation of the Car content. Our
data also show that the spectral characteristics of the
total Car in the apple extracts can by obtained by analyzing the residual spectra of ChF obtained by subtracting Chl absorption. It is worth noting that the
absorption spectra of the total carotenoids were qualitatively and quantitatively similar to the spectra of unsaponified lipids (Fig. 3). This allows us to carry out the
analysis of Car in ChF without additional saponification and chromatography steps [15, 22].

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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A SPECTROPHOTOMETRIC ANALYSIS OF PIGMENTS

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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