Optimization and use of a spectrophotometric method for determining polysaccharides in Echinacea purpurea

Abstract

Echinacea purpurea is one of the most widely used immunostimulant plants. Its main active compounds are polysaccharides, glycoproteins, caffeic acid derivatives, alkamides, and melanins. The article describes an optimized extraction procedure that enables spectrophotometric quantification of polysaccharides from Echinacea purpurea. The extraction procedure can be widely applied as it demonstrated to be useful for determining polysaccharide content in flowers and leaves, in summer and autumn plants, in plants with green and red stem, and in plants from two different plantations. A significantly higher content of polysaccharides in flowers in comparison to leaves, as well as in plants with green stems in comparison to plants with red stems was determined. Statistical differences were absent in plants collected in different seasons and growing at different plantations.

Full text

Central European Journal of Biology
* E-mail: samo.kreft@ffa.uni-lj.si
Research Article
1University of Ljubljana, Faculty of Pharmacy,
Department of Pharmaceutical Biology,
1000 Ljubljana, Slovenia
2The Institute for Hop Research and Brewing,
3310 Žalec, Slovenia
3Agriculture and Forestry Chamber of Slovenia,
1000 Ljubljana, Slovenija
Nina Kočevar Glavač1, Iztok Jože Košir2, Janko Rode3, Samo Kreft1,*
Optimization and use of a spectrophotometric
method for determining polysaccharides
in
Echinacea purpurea
1. Introduction
Echinacea purpurea is one of the most widely
used immunostimulant plants for treating u and
common cold. For medicinal treatment, root and
herb extract as well as pressed juice of fresh aerial
plant parts are used. The main active compounds
are polysaccharides, glycoproteins, caffeic acid
derivatives (cichoric acid), alkamides, and melanins
[1-3]. Protein-free polysaccharide preparations called
EPS (Echinacea polisacharide fraction), PSI (35 kDa
4-O-methylglucuronoarabinoxylan), PSII (50 kDa
acidic arabinorhamnogalactan), and arabinogalactan
have been studied [4-6]. These polysaccharides affect
phagocytosis, chemotaxis, and production of cytokines
in granulocytes and macrophages in vitro [4,7]. In animal
studies, it was shown that EPS enhances phagocytosis
[7] and arabinogalactan enhances resistance against
systemic infections with Listeria monocytogenes
and Candida albicans in mice [8,9]. Polysaccharides
puried from Echinacea purpurea tissue culture injected
intravenously into patients undergoing chemotherapy
for gastric cancer showed lower leucopoenia [10].
Distribution of alkamides, polyenes/ynes, and
phenolics within the same plant is well documented
[11-13]. Isolation, separation and analytical determination
of these compounds has also been demonstrated using
high-performance liquid chromatography [14-16], capillary
electrophoresis [17-19] and liquid chromatography
with mass spectrometry (LC-MS) [20]. In contrast,
only limited analytical data exist on polysaccharides
in plant tissue. General procedure of polysaccharide
isolation was described by Wagner [7] with only a
few compounds being characterized, such as puried
polysaccharides [7], methylglucuronoarabinoxylan, and
arabinorhamnogalactan [5].
Cent. Eur. J. Biol. • 7(1) • 2012 • 126-131
DOI: 10.2478/s11535-011-0091-z
126
Received 01 June 2011; Accepted 29 September 2011
Keywords: Echinacea purpurea • Extraction • Polysaccharides • Spectrophotometry
Abstract: Echinacea purpurea is one of the most widely used immunostimulant plants. Its main active compounds are polysaccharides,
glycoproteins, caffeic acid derivatives, alkamides, and melanins. The article describes an optimized extraction procedure
that enables spectrophotometric quantication of polysaccharides from Echinacea purpurea. The extraction procedure can
be widely applied as it demonstrated to be useful for determining polysaccharide content in owers and leaves, in summer and
autumn plants, in plants with green and red stem, and in plants from two different plantations. A signicantly higher content of
polysaccharides in owers in comparison to leaves, as well as in plants with green stems in comparison to plants with red stems
was determined. Statistical differences were absent in plants collected in different seasons and growing at different plantations.
© Versita Sp. z o.o.

N.K. Glavač
et al.
The aim of our work was to optimize a method for
isolation and quantication of polysaccharides from
Echinacea purpurea, and to determine content in
owers and leaves, in summer and autumn plants, in
plants with green and red stem, and in plants from two
different plantations.
2. Experimental Procedures
2.1 Plant material
Aerial plant parts of Echinacea purpurea Moench were
collected during owering time (at the beginning of July
and October). First group of 20 samples was obtained
from The Institute for Hop Research and Brewing
(Žalec, Slovenia): 5 plants with red stem and 5 plants
with green stem collected in summer and autumn
harvest, respectively. Second group of samples was
obtained from two different plantations and collected in
summer and autumn period, respectively. Flowers and
leaves were collected separately (5 samples of each of
two organs from each of two plantations and each of
two harvests = 40 samples). Plant material was dried at
room temperature, cut into 1cm particles, and powdered
in the mortar.
2.2 Polysaccharide isolation
Various isolation protocols were tested. The basic method
of polysaccharide isolation was adopted from literature
[7] and optimized. All extraction steps were performed
by sonication procedure as the studies have shown that
ultrasound-assisted extraction indicates more efcient
extraction in shorter time and at lower temperatures
owing to disruption of cell walls and enhanced mass
transfer of cell contents such as polysaccharides [21].
Inuence of solvent volume, time of extraction, and
ethanol precipitation were then studied. The optimal
isolation method was: 50 mg of powdered herbal drug
was extracted with 10 mL of petroleum ether for 10 min
at room temperature in an ultrasonic bath and centrifuged
at 3400xg 10 min. After removal of supernatant, the plant
material in the sediment was extracted with methanol
(10 mL for 10 min at room temperature). Petroleum ether
and methanol extracts contained colored substances
(chlorophyll, carotene and other pigments). Removal of
these compounds was important, to prevent interaction
with the colorimetric assay for polysaccharides.
Supernatant was discarded and the pellet extracted
with 10 mL of 0.5 M sodium hydroxide rst for 10 min
in an ultrasonic bath and then at room temperature for
24 h. For comparison, some samples were analyzed
by using a method that contained a precipitation step.
Precipitation was carried out by adding 8 mL of ethanol to
2 mL of supernatant incubating for 1 h at −20°C, and then
centrifuging for 10 min at 3400xg. The precipitate was
dissolved in 2 mL of distilled water, sonicated for 5 min,
and centrifuged for 10 min at 3400xg. The supernatant
was analyzed spectrophotometrically. Precipitation
with 80% ethanol selects for polysaccharides but the
oligosaccharides, disaccharides and monosaccharides
remain soluble [22].
2.3 Determination of polysaccharide
concentration
Various protocols for color reaction were tested as
described in the results section. The optimal method
was: 200 μL of polysaccharide solution (containing
5−50 μg of polysaccharides) were diluted with 300 μL of
distilled water and 500 μL of 5% (w/w) phenol solution
was added. Then, 2.5 mL of 96.5% sulfuric acid was
added rapidly and the mixture was shaken. After 60 min
incubation at 70°C, absorbance was measured at
490 nm (spectrtophotometer: Perkin Elmer Lambda
Bio+). A negative control sample was prepared with
distilled water.
2.4 Thin layer chromatography (TLC) analysis
of sugars in polysaccharide hydrolysate
A sample of 10 mL of polysaccharide extract was
dried on a rotary evaporator. The sample was treated
with 2 mL of 2 M triuoroacetic acid and heated under
reux for 1 h. Following incubation, 10 mL of water was
added, the mixture was dried on a rotary evaporator and
this procedure was repeated twice. The residue was
dissolved in 0.5 mL of 80% ethanol and 1 μL was applied
on a Silicagel 60 TLC plate (Merck). The plate was
developed with acetonitrile:water (85:15) and derivatized
by spraying with 0.5% thymol and 5% sulphuric acid in
ethanol and heating for 5 minutes at 120°C.
2.5 Statistical evaluation
ANOVA, t-test, and linear regression were calculated
using Microsoft Ofce Excel 2003.
3. Results and Discussion
3.1 Optimization of polysaccharide extraction
When performing the polysaccharide isolation process
with 10 mL of solvents (petroleum ether, methanol and
sodium hydroxide solution) per 50 mg of herbal drug
in comparison to 1 mL per 50 mg of herbal drug, the
amount of extracted polysaccharides was 9% higher.
The increased volumes of petroleum ether and methanol
resulted in more effective removal of pigments, while the
increased volume of sodium hydroxide and sonication
127

Optimization and use of a spectrophotometric method
for determining polysaccharides in
Echinacea purpurea
resulted in more efcient extraction of polysaccharides
from the plant matrix. Efcacy of extractions performed
for 1, 2, 3, 4, and 24 hours with 0.5 and 2 M sodium
hydroxide solution is shown in Table 1. Extraction
efciency reaches the maximum after 4 hours, but
24 hours were selected for routine experiments to
assure complete extraction in all cases and more
practical organization of work.
After extraction of polysaccharides, precipitation
with 80% ethanol was performed in two ways, 1 hour
at −20°C and 24 hours at 4°C. There were only slight
differences in quantity of precipitated polysaccharides
as determined by the spectrophotometric analysis (data
not shown).
We performed additional tests to see the composition
of sugars in the polysaccharide. Composition of
precipitated polysaccharides as well as polysaccharides
that remain soluble in 80% ethanol was evaluated by
hydrolysis and thin-layer chromatography using xylose,
galactose, and glucose as standards. In both samples,
all of the three sugars and one additional unidentied
sugar were detected, and among them xylose was
prevailing (semiquantitatively determed as >50%).
The amount of glucose was low (semiquantitatively
determed as <10%). To further elucidate, if the glucose
was a part of heteroglycane or if it originated from the
starch contamination, we incubated the polysaccharide
solution with alpha-amylase. After precipitation, the
amount of polysaccharides determined in colorimetric
assay was equal with and without amylase. This
demonstrated that the isolated polysaccharide did not
contain signicant amount of starch.
3.2 Optimization of determination of
polysaccharide concentration
Polysaccharide concentration was determined by the
phenol-sulfuric method [23], which was modied and
optimized to improve repeatability. We found out that
poor repeatability of the method originates (besides in
incomplete reaction) also in heterogeneous solution,
which is formed after addition of sulfuric acid. In
the solution, two phases can be seen, which cause
diffuse scattering of a light beam during absorbance
measurement, moreover, additional problem is formation
of bubbles as a consequence of heating of the solution
caused by sulfuric acid. Good repeatability (relative
standard deviation, RSD=2.1%) of spectrophotometric
analysis was reached by prolonged incubation at higher
temperature (Table 2). The repeatability of overall
method (extraction and spectrophotometric analysis)
was RSD=3.8%.
Linearity of optimized phenol-sulfuric method
was good (Pearson correlation R2=0.991 for glucose
standard and R2=0.9899, and for Echinacea purpurea
polysaccharide extract) in a tested range (see Table 3).
3.3 Quantication of Polysaccharides in
Echinacea purpurea
Plants
Total content of polysaccharides was signicantly
higher in green stem plants (2-way ANOVA: P=0.04227;
t-test P=0.029) (Figure 1). It is obvious, that the same
factor, that increases the content of red pigment in the
stem, decreases the content of polysaccharides. This
might be due to genetic or environmental (light) factor.
On the other hand, both statistical tests showed no
signicant difference in polysaccharide content between
plants collected in summer or autumn (2-way ANOVA:
P=0.516772). Total content of polysaccharides was
signicantly higher in owers as compared to leaves
Extraction time 0.5 M NaOH 2 M NaOH
1 h 1.09 1.57
2 h 1.24 1.69
3 h 1.21 1.70
4 h 1.42 1.78
24 h 1.42 1.76
Table 1. Inuence of solvent molarity and extraction time on
efcacy of polysaccharide extraction determined
spectrophotometrically by measuring absorbance at
490 nm. All experiments were repeated three times.
Efcacyofextractionisexpressedasabsorbancyunits.
Table 2. Inuenceof incubationtime,temperature,andshaking on
repeatabilityofthephenol-sulfuricmethod.Allexperiments
wererepeatedthreetimes.
Incubation
Absorbance Relative
standard
deviation
(RSD)[%]
123
30min,roomtemperature 0.869 0.976 0.970 6.4
60min,roomtemperature 1.077 0.947 0.953 7.4
30min,50°C,shaking 1.044 0.982 0.954 4.6
60min,50°C,shaking 0.903 0.970 0.939 3.6
30min,70°C,shaking 0.889 0.847 0.862 2.5
60min,70°C,shaking 0.895 0.867 0.860 2.1
Glucose Echinacea
Concentration
range 5−50μg 20−90mg
Regression
equation y=0.0199x+0.0118 y=0.0236x−0.0127
R20.991 0.9899
Table 3. Linearityofthephenol-sulfuricacidmethod.
128

N.K. Glavač
et al.
(2-way ANOVA: P=0.00002; t-test P=0.001) (Figure 2).
This implies that it is favorable to include high proportion
of owers in the herbal material for producing medicinal
products. There was a slightly higher concentration
of polysaccharides in autumn owers and leaves in
comparison to plant parts collected in summer, but
the difference was not statistically signicant (2-way
ANOVA: P=0.103; t-test P=0.110). There were also
no differences found between plants from different
plantations. Regarding polysaccharide content
determined without or with precipitation with ethanol,
it has to be emphasized that values measured without
precipitation were approximately 2-fold higher. This can
be attributed to the loss of smaller polysaccharides and
oligosaccharides due to inefcient precipitation or to
higher stringency in selectivity of the precipitation step.
These two variations of analytical method will enable
that the products which will be tested in future for their
(in vitro or in vivo) activity, will be characterized on the
content of the total polysaccharides and the precipitable
polysaccharides to determine which fraction of the
polysaccharides is more relevant for efcacy.
Using the modied and optimized method for
extraction and quantication of total polysaccharides
in Echinacea purpurea, we demonstrated that there
is a signicantly higher content of polysaccharides in
owers in comparison to leaves, and higher content
in plants with green stems in comparison to plants
with red stems. On the other hand no statistical
differences were found between plants collected
in different seasons or different plantations. In our
previous research, we similarly found no difference in
content of caffeic acid derivatives in plants harvested
in summer or in autumn [13]. A limitation to the
isolation and quantication method that we describe
is that it does not show the qualitative composition of
polysaccharides. However, since absorbance response
varies for different sugars, the high heterogeneity of
monosaccharide composition within a polysaccharide
makes metabolomic approaches for quantication
difcult and highly complex.
Here we demonstrated a simple and reliable method
that offers useful and important improvements of
polysaccharide determination in plant material suitable
for routine work.
Acknowledgements
The authors are grateful to Ms. Nina Kroič and Ms.
Katja Delić for their skilful laboratory assistance.
Figure 1. Polysaccharide content in % of glucose equivalents
between plants with red and green stems collected in
summer and autumn period, respectively, determined
withoutorwith precipitationwith ethanol.Each column
represents the average of analyses of ve individual
plants.Errorbarsrepresentstandarderrors.
Figure 2. Polysaccharidecontent in % of glucose equivalents in
owerandleafplantpartsfromtwodifferentplantations
collected in summer and autumn period, respectively.
Each column represents the average for ve individual
plants.Errorbarsrepresentstandarderrors.
129

Optimization and use of a spectrophotometric method
for determining polysaccharides in
Echinacea purpurea
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