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


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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.
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Central European Journal of Biology
* E-mail:
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
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
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
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
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
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.
Table 2. Inuenceof incubationtime,temperature,andshaking on
Absorbance Relative
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
range 5−50μg 20−90mg
equation y=0.0199x+0.0118 y=0.0236x−0.0127
R20.991 0.9899
Table 3. Linearityofthephenol-sulfuricacidmethod.
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.
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
Figure 2. Polysaccharidecontent in % of glucose equivalents in
collected in summer and autumn period, respectively.
Each column represents the average for ve individual
Optimization and use of a spectrophotometric method
for determining polysaccharides in
Echinacea purpurea
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... After precipitation, washing of polysaccharides with 80% ethanol is recommended. [5][6][7] Wagner described a general procedure of polysaccharide isolation from different plants [8] and in our previous research we optimized it for quantification of Echinacea purpurea polysaccharides. [7] Another method to remove mono-, di-, and oligosaccharides from polysaccharides is dialysis with semipermeable membranes [9] or ultrafiltration, where the membrane separates molecules with higher molecular weight from molecules with lower molecular weight. ...
... [5][6][7] Wagner described a general procedure of polysaccharide isolation from different plants [8] and in our previous research we optimized it for quantification of Echinacea purpurea polysaccharides. [7] Another method to remove mono-, di-, and oligosaccharides from polysaccharides is dialysis with semipermeable membranes [9] or ultrafiltration, where the membrane separates molecules with higher molecular weight from molecules with lower molecular weight. ...
... Such ethanol dissolves mono-, di-and oligosaccharides but not polysaccharides. [7] The precipitate was then dried (40 C, p ¼ 0 mbar) and weighed. ...
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This article describes optimized isolation procedures that enable simple quantification of polysaccharides in herbal sirup. Isolation of polysaccharides present in the herbal sirup was made by both precipitation and ultrafiltration. Quantification of sirup polysaccharides was performed with gravimetric and spectrophotometric methods. The first step of the spectrophotometric method was separation of polysaccharides from mono- and oligosaccharides by precipitation or ultrafiltration followed by quantitative determination by the colorimetric phenol-sulfuric acid method performed in glass tubes or in microplates. The content of polysaccharides was expressed as sucrose or pectin equivalents according to the standard curves of these standards. Results of the optimized gravimetric procedure were moderately repeatable. We demonstrated the linearity and appropriate repeatability of the phenol-sulfuric acid method in microplates but not in glass tubes. Thus, the method with ultrafiltration followed by phenol-sulfuric acid method in microplates had good repeatability, accuracy, and linearity. This simple method was shown to be useful for determining polysaccharides in herbal sirup.
... Pharmacological species as antioxidants react with free radicals, which loss their unpaired electrons and become diamagnetic. The activity of diamagnetic molecules is lower than paramagnetic free radicals, the risk of modification of chemical structures in tissues decreases, and their functions are not destroyed (Jaroszyk, 2008;Bartosz, 2006). ...
... Herb is particularly valued because of an immune. E. purpureae also exhibits properties such as anti-inflammatory, antibacterial, antiviral, antifungal, antioxidant, diuretic, cholagogue, and antispasmodic, and stimulates the synthesis of collagen and elastin (Kočevar et al., 2012;Schapowal, 2013). ...
... It has the prophylactic effect and helps in the treatment of respiratory infections, flu, and tonsillitis. It is also recommended by recurrent infections of the urinary tract and inflammation of the ascending cholangitis (Kočevar et al. 2012;Moraes et al., 2011). ...
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The effect of UVA (315–400 nm) irradiation on Echinaceae purpureae interactions with free radicals was examined by the use of electron paramagnetic resonance (EPR) spectroscopy. The changes of antioxidant properties of E. purpureae with time of UV irradiation from 10 to 110 min (10 min steps) were determined. DPPH as the paramagnetic reference was used in this study. Changes of EPR signals of the reference after interactions with nonirradiated and UV-irradiated E. purpureae were detected. Interactions of the tested E. purpureae samples caused decrease of the EPR signal of DPPH as the result of its antioxidant properties. The decrease of the amplitude of EPR line of DPPH was lower for interactions with UV-irradiated E. purpureae. EPR examination confirmed antioxidant properties of E. purpureae. The weaker antioxidant properties of E. purpureae after UV irradiation were pointed out. E. purpureae should be storage in the dark. The tests bring to light usefulness of electron paramagnetic resonance with microwave frequency of 9.3 GHz (an X-band) in examination of storage conditions of pharmacological herbs.
... A negative control sample was prepared with distilled water. 12 ...
Introduction Nowadays several plant species, such as Echinacea angustifolia L., can be considered natural important sources for therapeutic applications. Echinacea angustifolia is one of the most known medicinal plants, it shows protective and preventive effects against many chronic diseases, thanks to immunostimulant properties, mostly due to its polysaccharides and antioxidants. However, the optimisation of green extraction techniques to respect the environment is, currently, a hard challenge for the recovery of secondary metabolites. Methodology Hydro‐enzymatic extraction has been performed for the first time, it was compared with other different extraction techniques, for their efficacy in bioactive compound recovery. Phytochemical characterisation has been carried out through high‐performance liquid chromatography diode array detector (HPLC‐DAD) analysis and the antioxidant activity has been also measured. Results The highest extractive yields and the strongest antioxidant activity was obtained by cellulase and xylanase enzyme extraction. The enzymatic extraction with pectinase enzyme led to a higher polysaccharide content in comparison with the literature. The hydro‐enzymatic extraction method and analytical conditions allowed the identification and quantification of two compounds, to the best of our knowledge, for the first time in E. angustifolia root extract. Conclusion The optimised extraction methods discussed in this work led to a higher polysaccharides content, in comparison to previous literature results. The enzymatic extraction seems to be the best extraction technique, in terms of antioxidant yield and efficacy in bioactive compound recovery.
... Total soluble polysaccharides were also determined spectrophotometrically at 730 nm with the phenol-sulfate method [185]. This kind of reaction was optimized and used also by Glavac et al. [186]. ...
Echinacea purpurea (L.) Moench, Echinacea angustifolia DC. var. angustifolia and Echinacea pallida (Nutt.) Nutt. are frequently used as medicinal plants and their preparations are among the most widely used herbal medicines. The extracts from these species have shown a highly complex chemical composition, including polar compounds (caffeic acid derivatives, CADs), non-polar compounds (alkylamides and acetylenic secondary metabolites; essential oil) and high molecular weight constituents (polysaccharides and glycoproteins). All these chemical classes of compounds have demonstrated to possess interesting biological activities. In the light of all the above, this paper is focused on the analytical techniques, including sample preparation tools and chromatographic procedures, for the chemical analysis of bioactive compounds in medicinally used Echinacea species. Since sample preparation is considered to be a crucial step in the development of analytical methods for the determination of constituents present in herbal preparations, the strength and weakness of different extraction techniques are discussed. As regards the analysis of compounds present in Echinacea plant material and derivatives, the application of different techniques, mainly HPLC, HPLC-ESI-MS, HPLC-ESI-MS/MS, HPCE, HPTLC and GC, is discussed in detail. The strength, weakness and applicability of the different separation tools are stated.
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In this study, a fusion model to combine electronic nose and electronic tongue was developed to detect submerged fermentation of Tremella aurantialba (T. aurantialba). Three chemical indicators of T. aurantialba fermentation were detected by traditional measuring methods as a comparison, including ergosterol content, reducing sugar content and polysaccharide content. Principal component analysis (PCA) and layer fusion in the feature were applied on electronic nose and electronic tongue data. Support vector machine regression (SVR) model was established for quantitative determination of the three chemical indicators. The SVR model was combined with PCA for testing of ergosterol content, reducing sugar content, and polysaccharide content. The correlation coefficient of the test set for ergosterol content, reducing sugar content, and polysaccharide content were 0.9946, 0.9945, and 0.9934, respectively. The root mean square error of prediction of the models for the three indicators were 2.7765 μg/mL, 29.1405 mg/mL, and 0.4858 mg/mL, respectively. The results proved that the combined system could be used to predict the ergosterol content, reducing sugar content, and polysaccharide content during submerged fermentation of T. aurantialba. In conclusion, detection of T. aurantialba fermentation based on electronic nose and electronic tongue technologies achieved satisfactory results and, therefore, offers broad application prospects in the liquid state industry. Practical applications The fermentation broth of T. aurantialba has a variety of pharmacological effects. The submerged fermentation of T. aurantialba is very complex. The existing detection technology needs a cumbersome process of sample preparation and costs a lot of time, which cannot meet the need of rapid detection. Electronic nose combined with electronic tongue could detect the odor and taste changes during the T. aurantialba fermentation quickly. The fusion techniques showed good performances in the quantitation of the main indicators of T. aurantialba. The method can be used to determine the state of fermentation instead of the traditional methods. It further improves the accuracy and scientificity of quantitative evaluation for T. aurantialba fermentation. Data fusion technique to combine electronic nose and electronic tongue could be applied in the liquid state fermentation industry in the future.
Extensive scientific research on Echinacea species revealed that the bioactive components most likely responsible for the immunostimulatory activities of Echinacea were alkamides, caffeic acid derivatives, glycoproteins, and polysaccharides. Three species, namely Echinacea purpurea, E. angustifolia and E. pallida, are predominantly used in herbal supplement preparations. Also, different plant parts are shown to have different amounts of the bioactive components. In addition, the phytochemistry, quality control based analysis of bioactive constituents, as well as addressing the safety concerns related to Echinacea herbal supplement preparations are discussed.
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A rapid and quantitative method of quality assurance for marker phytochemicals in products containing material derived from Echinacea species has been developed. In order to assess the efficiency of extraction of phytochemicals from the roots and aerial parts of Echinacea purpurea and E. angustifolia, a study of solvent mixtures and extraction methods was carried out to determine the recovery of known compounds from plant materials. Ultrasonic extraction of dried samples with methanol:water (7:3) or ethanol:water (7:3) gave good yields of cichoric acid, echinacoside and the alkamides, undeca-2E,4Z-diene-8,10-diynoic acid isobutylamide and a mixture of dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamides (recoveries of 89%, 85%, 80% and 90%, respectively). The HPLC separation of the phenolic compounds cichoric acid, chlorogenic acid and echinacoside was also improved by careful attention to the pH of the mobile phase. A shortened HPLC column allowed turnaround times of 22 min for phenolic components and 15 min for alkamides with lower solvent use. Assessment of commercial raw materials from the North American market using the new method was useful for confirmation of species and showed a very large variation in concentration of markers in the products sold in this market. Copyright © 2000 John Wiley & Sons, Ltd.
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Interest in dietary fiber is a consequence of the belief that di- etary fiber contributes positively to the health/quality of life of the consumer. The physiological effects of dietary fiber are what make it of interest to the consumer, food nutritionists, and regu- lators. Because dietary fiber is a multicomponent mixture, it is essential that there is a clear definition and a methodology to allow measurement of the defined components. Over the past thousands of years, the human diet has changed from one based on a wide range of coarse plant materials with little animal products to one in which the plant components are limited to few plant products (which are highly milled and pro- cessed) and a larger portion of animal products. The need for coarse foods of plant origin to combat constipation can be traced back to Hippocrates in the 4th century bc (5), who commented on the laxative action of the outer layers of cereal grains.
The concentrations of methyl glycosides, oligosaccharides, glycopeptides, and glycoproteins can be accurately determined by using calibration curves composed of the appropriate monosaccharide(s) obtained with a modified version of the colorimetric phenol-sulfuric acid method. Calibration curves of mu g sugar vs. 490 nm for Man, Glc, or Gal are shown to provide reliable determinations (typically +/-3-4%) of corresponding methyl glycosides and linear and branched-chain oligosaccharides containing the corresponding reactive hexose residue. For complex oligosaccharides containing a known mixture of reactive hexose units, the appropriate mixture of monosaccharides are shown to provide equally accurate calibration curves for concentration determinations. In the case of the soybean agglutinin, which is a tetramer possessing one Man9 oligomannose-type chain per subunit, the protein concentration was determined from the Man calibration curve which agreed with that obtained from the molar extinction coefficient of the protein.
Purple coneflower (Echinacea purpurea) is an immunostimulating drug, containing multiple substances. The most important in activity are polysaccharides, caffeic acid derivatives (cichoric acid), alkamides and glycoproteins. It is not clear yet, which substances are responsible for activity. Cichoric acid is an appropriate marker of the quality of E. purpurea containing product, because it has immune stimulatory effects and it is susceptible to degradation. In this work, an improved capillary electrophoresis method for determining cichoric acid in dried press juice from purple coneflower was developed. The optimal conditions were: electrophoretic buffer-75mM borate, pH 8.8; injection 20mbar for 20s; separation at 20kV; detection at 350nm, temperature 35 degrees C.
From the hemicellulosic material of Echinaceae purpurea a homogeneous 4-O-methyl-glucuronoarabinoxylan was isolated by ethanol fractionation, ion-exchange chromatography and gel filtration. Sugar and methylation analysis revealed that the polysaccharide contained a (1 → 4)-linked β-d-xylan backbone with branching points at C-2 and C-3. Further characterization of the structure was performed by periodate oxidation and Smith degradation, methanolysis, uronic acid determination, basic degradation and 13C NMR spectroscopy. The 4-O-methyl-glucuronoarabinoxylan showed immuno-stimulating activity in several in vitro immunological test systems.
Samples of Echinacea purpurea. (L.) Moench were taken from 25 plantations at two harvesting times (July and October). Five shoots from each plantation were measured and weighed. The contents of cichoric and caftaric acid were determined in flowers, leaves, and stems of samples harvested in July. All morphological parameters decreased with increasing age of the plantation, but age had no influence on the cichoric and caftaric acid contents. The average weight of leaves and stems in 6-year-old plantations was more than sixfold lower than those from 1-year-old plantations. In flowers, the reduction was fourfold. Cichoric and caftaric acid contents in leaves differed significantly between the regions, but the region had no influence on the morphological parameters. Irrigated plantations yielded more than 50% higher weights of leaves and stems and 25% higher weights of flowers. Irrigation had no influence on cichoric and caftaric acid contents.
From the medium of Echinacea purpurea cell cultures three homogeneous polysaccharides, two neutral fucogalactoxyloglucans with mean Mr of 10 000 and 25 000 and an acidic arabinogalactan with a mean Mr of 75 000, have been isolated by DEAE-Sepharose CL-6B, DEAE-Trisacryl M and Sephacryl S400 column chromatography. Their structures were elucidated mainly by methylation analysis, partial acidic and enzymatic hydrolysis and 13C NMR spectroscopy. The fucogalactoxyloglucan of mean Mr25 000 enhances phagocytosis in vitro and in vivo. The arabinogalactan specifically stimulates macrophages to excrete the tumour necrosis factor (TNF).
A liquid chromatography-particle beam/mass spectrometry (LC-PB/MS) method with electron impact (EI) and glow discharge (GD) ionization sources is presented for the determination of caffeic acid derivatives in echinacea tinctures. In this work, two commercially available echinacea ethanolic extracts were used as the test samples for the separation, identification, and quantification of the caffeic acid derivatives (caffeic acid, chlorogenic acid, cichoric acid, and caftaric acid), which are suggested to have beneficial medicinal properties. Detailed evaluations of the two primary controlling parameters for EI (electron energy and source block temperature) and GD (discharge current and pressure) sources were performed to determine optimal instrument operation conditions. The mass spectra obtained from both ion sources provide clear and simple molecular fragmentation patterns for each of the target analytes. The absolute detection limits for the caffeic acid derivatives were determined to be at subnanogram levels for both the EI and GD sources. The separation of the caffeic acid derivatives in echinacea was accomplished by reversed-phase chromatography using a C(18) column and a gradient elution system of water containing 0.1% trifluoroacetic acid and methanol, with an analysis time of less than 40 min. A standard addition method was employed for the quantification of each of the caffeic acid derivatives in the tincture.
From the high molecular weight fraction of an aqueous extract from roots of Echinacea purpurea L. Moench, arabinogalactan-proteins (AGPs), a class of proteoglycans proposed to be involved in cell differentiation and plant growth, were purified and characterized with regard to amino acid composition and structure of the polysaccharide moiety. The protein content of the AGP was 5.0 % (w/w) with the dominating amino acids Glx, Hyp, Asx, Ser, Thr and Ala. The highly branched polysaccharide moiety shows a linkage composition typical of AGPs with 1,3-, 1,6- and 1,3,6-linked galactopyranosyl residues and arabinofuranosyl residues predominantly as terminal and 1,5-linked residues. Terminal units of glucuronopyranose acid were also detected. Furthermore, a new method for the localization of AGPs in plant tissue has been developed. The synthetic (beta- D-Glc)(3) Yariv phenylgycoside (betaGlcY) is known to specifically bind to AGPs. For immunolocalization, polyclonal betaGlcY-antibodies have been generated and were used to label Yariv-treated thin sections of roots from E. purpurea. After addition of the FITC-conjugated secondary antibody, the sections were analyzed by confocal laser scanning microscopy. AGPs are detected mainly in the central cylinder in the area of the xylem. Cell walls of vessels and tracheids are strongly labelled, especially at the inner area of the wall. Furthermore, there is intense labelling of the pit canals.