Ultrasonics Sonochemistry 9 (2002) 37-44

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Ultrasound-assisted extraction of water-soluble polysaccharides

from the roots of valerian (Valeriana officinalis L.)
Z. Hrom'adkov'a a, A. Ebringerov'a a,*
, P. Valachovi~c b

a
Institute of Chemistry, Slovak Academy of Sciences, Dubravska Cesta 9, 842 38 Bratislava, Slovak Republic
b
Drug Research Institute Modra, 900 01 Modra, Slovak Republic
Received 6 November 2000; received in revised form 12 February 2001

Abstract
The insoluble plant residues, obtained after preparation of medicinal tinctures from the roots of valerian ( Valeriana officinalis L.)
by classical and ultrasound-assisted extraction with aqueous ethanol in a pilot plant, were subsequently treated with hot water
to isolate the accessible polysaccharide cell wall components. At almost equal amounts of the hot-water extractable material, the
yields of the recovered polysaccharides were lower in the ultrasonical experiment. This is due to the fact that a part of
accessible poly- saccharides were already solubilised by the aqueous ethanol and recoverable from the medicinal tincture. Therefore,
the net yield of extracted polysaccharides was enhanced in the ultrasonical procedure. This fact as well as the sugar composition
and structural features of the isolated polysaccharides suggest that ultrasonication have attacked the integrity of cell walls,
released and degraded its most accessible polysaccharides (pectic polysaccharides and starch) and increased also the
extractibility of its less accessible components - xylan, mannan and glucan. The water-soluble polysaccharide fractions from
both the conventional and ultrasoni- cal experiments exhibit significant immunostimulatory activities in mitogenic and
comitogenic thymocyte tests. © 2002 Elsevier Science B.V. All rights reserved.
Keywords: Extraction; Ultrasound; Polysaccharides; Valerian; Valeriana officinalis; lmmunostimulatory activity

1. Introduction increased component extraction was achieved in a

Ultrasonical treatment is well established in the pro-
cessing of plant materials, particularly, for extracting
low molecular substances [1-4]. Few reports have docu-
mented also its viability in improving pectin techno-
logy [5] and extracting plant polysaccharides such as
the immunologically active xylan from corn cobs [6,7]
and gum-like heteroxylan from corn hulls [8] without
observation of substantial changes in the functional
properties of the mentioned biopolymers.
Herbal tinctures are often employed in medical treat-
ments. Their commercial production is time-consuming
and, therefore, not very effective. A comparative study
of the yield and composition of aqueous ethanol extracts
obtained from the herbs-sage and valerian by conven-
tional and ultrasound-assisted method have shown a
more efficient extraction using the later method [9]. An
*
Corresponding author. Tel.: +421-75941-0284; fax: +421-75941-
0222.
2.1. Materials
E-mail address: chemebri@savba.sk (A. Ebringerov'a).
1350-4177/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved.
Pll: S 1350 - 4177 ( 01 ) 00093 - 1
shorter time, particularly, in the case of sage. The
mechano- chemical effect of ultrasound is believed to
accelerate the extraction of organic compounds from
plant materials due to disruption of cell walls and
enhanced mass transfer of the cell contents [10]. This
was recently [11] docu- mented by the increased
extractibility of water-soluble polysaccharides from the
ethanol-insoluble plant residue of sage obtained from the
ultrasound-assisted process in comparison to that from
the classical extraction.
The aim of this study was to compare the yield,
com- position and properties of water-extractable
polysac- charides of the ethanol-insoluble plant residues
obtained by the classical and ultrasound-assisted
preparation of medicinal tinctures from the roots of
valerian.
2. Experimental
Air-dried roots of valerian (Valeriana officinalis L.)
were crushed to a particle size of 2-3 mm and used for
 38 Z. 2rom 'dkov ' et al. 3 4ltrasonics Sonochemistry 9 520026 37844
the preparation of medicinal tincture by classical and
optimised ultrasound-assisted extraction methods as
previously described [9]. Both classical and
ultrasonical experiments were performed in the pilot
plant of the drug factory Mediplant (Modra, Slovakia)
using iden- tical conditions to those used for industrial
purposes (cylindrical shaped glass extractor, bath size
of 56 l, height of 85 cm and diameter of 29 cm,
intervals of circulation, flow rate). The crushed roots
were loaded into the percolator and 60% aqueous
ethanol was added at the solid-liquid ratio of 1:6.7. A
ceramic perforated plate was laid on the top of the
plant material. The circulation of solvent was achieved
using a peristaltic pump operating for 8 h per day
(short mode [9]). The rest of the day, extraction
proceeded at static macera- tion conditions. The
extraction process was carried out for three days, then
the tincture was separated off. The ultrasound-assisted
extraction was performed in a same device and at the
same conditions used for the classical extraction.
Ultrasound was applied for 2 h at the be- ginning of
each extraction day. The remaining plant residues
from both experiments were dried on air and used as
the starting material in the presented study. Zymosan,
a particulate yeast �-glucan was purchased from
Likospol Ltd. (Bratislava, Slovakia).
The ultrasound disintegrator USD 600 (Ultragen
Nitra, Slovak Republic) operating at a frequency of
20 kHz with input power of 600 W and ultrasound in-
tensity of about 1 W/cm2. The immersion stainless steel
transducer was of the horn-type with the height of 79 cm
and diameter of 5 cm.
2.2. Methods
Moisture content was determined by drying at
105°C to constant mass. All yields and composition
analyses were calculated on moisture-free basis.
Methods used for qualitative (paper chromatography)
and quantitative (gas-liquid chromatography) analysis
of sugars in form of their corresponding alditol
trifluoroacetates have been described in previous
papers [12,13]. Protein was calculated from the
nitrogen content (%N x 6.25) as- sayed using an
elemental analyser model 240 (Perkin- Elmer).
FT-lR spectra were measured in KBr pellets (2 mg
sample/200 mg KBr) using a Nicolet-Magna 750
spec- trophotometer operating at 4 cm -1 resolution.
NMR spectra in D20 were recorded at 40°C on a FT-
NMR AVANCE DPX-300 Bruker spectrometer
(75.46 MHz) equipped with a selective excitation unit
and gradient enhanced spectroscopy kit (GRASP) for
generation of Z-gradients up to 50 G/cm in a 5 mm
inverse probe. The two-dimensional NMR experiments
were performed using Bruker software XWlN/NMR
version 1.3 on a Silicon Graphics lNDY Computer
system. The mole- cular properties were characterised
by HPGPC on GM
Biospher 1000 + 300 columns using a commercial in-
strument (Laboratorn'1 P~r'1stroje, Praha, Czech
Repub- lic) with Rl- and UV254-detectors. The columns
were calibrated with pullulan standards P10-P800
(Shodex Standard P-82, Macherey-Nagel, Germany).
The immunostimulatory activity of the polysaccha-
rides was determined by mitogenic and comitogenic in
vitro tests using rat thymocytes and phytohaemaggluti-
nin (PHA) as described in detail in a previous paper [8].
2.3. Large-scale hot water extraction of the plant residue
The hot water extraction of the plant residue from the
classical and ultrasonical experiments was performed
according to the scheme in Fig. 1 in the same device as
used for the medicinal tincture preparations in the pilot
plant of the Mediplant factory. The air-dry material (2.5
kg) was treated for 3 h with 30 l of hot distilled water
thermostated at 62 ± 5°C. Then, the hot water extract
(HWE) was let to flow out through a gauze-protected
tap located bellow the bottom of the extractor. The yield
of the extracted material was determined by lyophilisa-
tion of an aliquot volume of HWE. The polysaccharide
component of HWE was separated by dialysis in a cel-
lophane bag as the non-dialysable portion and recov-
ered by lyophilisation (fraction P1). From an other
aliquot of HWE, polysaccharides were precipitated with
ethanol (1:3, v/v), separated by decantation and dialysed
from distilled water. The non-dialysable portion was
separated into the soluble (ws-P2) and insoluble (wis-P2)
subfractions by centrifugation and lyophilised.
2.4. Isolation of polysaccharides from the medicinal
tincture
The tincture (1000 ml) was exhaustively dialysed
against distilled water and then the non-dialysable por-
tion was separated by centrifugation and lyophilised
yielding fraction TP.
The codes ""c"" and ""u"" were used in the text to sign
polysaccharide fractions obtained by the classical and
ultrasonical experiments, respectively.
3. Results and discussion
3.1. Effect of ultrasonication on the extractibility of
valerian root polysaccharides
The plant residues, remaining after the classical and
ultrasound-assisted extraction of valerian roots by
aqueous ethanol for three days, were treated with hot
water according to the scheme in Fig. 1 with the aim
extract polysaccharide components. The yield of the
HWE material was about the same in the classical and
 Z. 2rom 'dkov' et al. 3 4ltrasonics Sonochemistry 9 520026 37844
Fig. 1. lsolation scheme of polysaccharides from valerian roots with and without application of ultrasound during the preparation of ethanolic
medicinal tincture.
ultrasonical experiments representing 11.9% and 11.5%,
respectively, of the dry plant residue. From HWE,
polysaccharide fractions P1 and P2 were isolated by
dialysis and by ethanol precipitation with subsequent
dialysis, respectively. As illustrated in Fig. 2, the yields
of the polysaccharides separated by the two different
isolation techniques were lower in the ultrasonical ex-
periment. This is opposite to the results reported for the
plant residue of sage treated under similar extraction
conditions [11], where both the yields of HWE and ex-
tracted polysaccharides were higher in the ultrasonical
experiment.
Note that lesser effects of ultrasonication at the
same
experimental conditions have been already reported for
the medicinal tincture preparations from valerian roots
in comparison to those reported in the case of sage [9].
However, significant yield differences were found only
39
during the first day of extraction and insignificant ones
after three-days extraction used in the present study.
ln order to look for possible polysaccharide losses,
the medicinal tinctures (MT) obtained by both proce-
dures from valerian roots after three days of extraction
were subjected to dialysis which is able to separate
polymeric material from the tincture. As shown in Fig.
3, the tinctures from the ultrasonical and classical ex-
traction procedures differ in their dry mass content,
being 8.6 and 7.5 g/100 ml, respectively. Dialysis of MT
yielded the non-dialysable polymeric fraction (TP)
which again was higher in the case of the ultrasonical
experiment, i.e. 5.8% versus 4.8%, related to the dry
matter of the tinctures. This clearly indicates that some
polymeric material had been already dissolved in the
60% aqueous ethanol, particularly in the case of the
ultrasonic experiment. The analytical data in Table 1
40 Z. 2rom' dkov ' et al. 3 Ultrasonics Sonochemistry 9 520026
37844

Fig. 2. Yields (related to the dry plant residue) of the HWE and
polysaccharide fractions, separated from the extract by dialysis (P1)
and by ethanol precipitation with subsequent dialysis (P2), for the
classical and ultrasound-assisted extraction procedure. P2 represents
the sum of subfractions ws-P2 and wis-P2. The inserted numbers in-
dicate the polysaccharide yields in percent related to the dry mass
content of HWE.
Fig. 3. Effect of ultrasonication on the (a) dry mass of the medicine
tincture (MT) and (b) yield of the polymeric fraction (TP), separated
from the tincture by dialysis, for the classical and ultrasound-
assisted extraction procedure. The inserted numbers indicate the
protein con- tent in %.

Fig. 2 further demonstrates that the yield of poly-

show that the polymeric fraction from the classical ex-
periment (TP/c) contains about equal amounts of pro-
tein and polysaccharides, whereas that from the
ultrasonical experiment (TP/u) was entirely composed of
polysaccharides, accompanied with only ~ 5% of pro-
tein. The obtained results suggest that due to the me-
chanochemical effect of ultrasonication on the plant cell
walls, a part of the polysaccharide components became
highly accessible and/or more depolymerised and thus
soluble in 60% aqueous ethanol. This fact can explain
the unexpected lower yield of polysaccharides released
by the subsequent hot water extraction of the ethanol-
insoluble plant residue in the case of the ultrasonic ex-
periment. Therefore, it is to conclude that the net yield
of extractable polysaccharides was enhanced by the
ultrasonical treatment.
saccharides from both experiments depends also on the
used isolation method. The ethanol precipitation with
subsequent dialysis yielded nearly a twice higher amount
(fraction P2) when compared to that obtained by direct
dialysis of the hot water extract (fraction P1). Very
probably, some low molecular mass substances were co-
precipitated with the polysaccharides by ethanol and
resist in the precipitate during the following dialysis
step.
Further evidence of cell wall disruption by ultraso-
nication was derived from the chemical composition of
the isolated polymeric fractions (Table 1) and their
structural features. Due to the higher proportion of
polysaccharides solubilised in the medicinal tincture
from the ultrasonical experiment, the protein content of
its polymeric fraction, TP/u, is much lower than in the

Table 1
Analytical data of polysaccharide fractions isolated from the plant residue of Valeriana roots obtained after classical and ultrasound-assisted ex-
traction with aqueous ethanol
Fraction Yield%a Neutral sugar composition (mole%) GalA%
Rha Ara Xyl Man Glc Gal m.r.b
Classical extraction
TP 44.7 21.2c 13.2 5.2 16.6 28.9 14.8 0.49
+d
P1 nd 5.8 29.9 6.5 9.1 38.2 10.5 2.67 ++
ws-P2 8.3 3.0 36.1 3.5 4.2 20.6 32.7 1.01 28e
wis-P2 1.3 6.5 24.9 7.1 8.5 27.0 26.1 0.66 +
Ultrasound-assisted extraction
TP 4.2 9.6c 29.1 8.5 9.0 34.5 9.2 2.68 ++
P1 nd 14.5c 11.8 8.2 21.2 31.8 12.6 0.64 ++
ws-P2 10.3 8.3 26.2 3.4 8.5 18.3 35.3 0.61 32e
wis-P2 1.1 8.5 17.4 12.2 15.8 23.9 22.4 0.43 +
a
Related to the ethanol-insoluble plant residue.
b
Mole ratio.
c
Contains minor amounts of fructose.
d
GalA, D -galacturonic acid estimated by paper chromatography.
e
GalA, D -galacturonic acid estimated by alkalimetric titration.
 Z. 2rom 'dkov ' et al. 3 Ultrasonics Sonochemistry 9 520026 41
37844
case of that from the classical experiment, TP/c. ln ac-
cord, the neutral sugar composition of TP/u is similar to
that of the HWE polysaccharides, P1/c, from the clas-
sical experiment. Also, an arabinose-rich component
appeared in the hot water extract only in the case of the
classical procedure (P1/c), whereas it was already solu-
bilised in the medicinal tincture from the ultrasonical
procedure (TP/u).
ln contrast, great similarities are in sugar composi-
tion of the ethanol-precipitated fractions (ws-P2) from
both experiments. Both contain mainly galactose, ara-
binose, and rhamnose comprising 60-70% of the neutral
sugars, besides of glucose, mannose and galacturonic
acid. Xylose and mannose, typical constituents of the
less accessible cell wall polysaccharide components of
herbal plants [14], are accumulated in both water-
insoluble subfractions (Table 1) and appear in higher
amounts in that from the ultrasonical experiment. This
is in accord with differences in the solubility of these
fractions. Whereas, ~90% of P2/c is water soluble, it
was only 60% in the case of P2/u due to the presence of
the insoluble xylan and mannan components. These
results are a further support of the mechanochemical Fig. 4. FT-lR spectra of the aqueous ethanol-soluble fractions (1) TP/
attack of ultrasound on cell walls. c and (2) TP/u, and HWE fractions (3) ws-P2/c and (4) ws-P2/u ob-
tained from the classical (c) and ultrasonical (u) experiments.

3.2. Effect of ultrasonication on the structural and
biological active properties of isolated valerian polysac-
charides
Certain absorption bands in the 1200-1000 cm-1 of
the FT-lR spectrum of biopolymers [15] can be used
for an approximate identification of polysaccharides in
plant materials when combined with chemical analysis
data. As seen in Fig. 4, the absorption bands of
proteins at 1660 cm-1 (amide l) and 1550 cm-1 (amide
ll) are more pronounced in the FT-lR spectrum of
TP/c than in that of TP/u, what is in accord with the
high protein content of the former fraction (Table 1).
The spectrum of TP/u contains, in addition, absorption
bands at 3002, 2950, 2920, 2850, 1740, and 1710 cm-
1
, indicative of lipids [16]. The FT-lR spectra of the
ws-P2 subfraction from both experiments show a
similar pattern in accord with their similar sugar
composition. They contain ab- sorption bands typical
of pectin (polygalacturonan) at 1730 cm-1 v(C00H),
1145 cm-1 v(C-0-C), 1100, 1048
and 1019 cm-1 attributed to v(C-0H), v(C-C) and
ring
vibrations. The dominating band at 1078 cm -1 is indi-
cative of arabino-3,6-galactan type ll, present in pectic
polysaccharides [14]. However, the other characteristic
bands at 1034, 1043 and 1156 cm -1 attributed to man-
nan, xylan, and starch are overlapped by the vibrations
of the prevailing pectic polysaccharides.
The 13C-NMR spectra of both ws-P2 subfractions
had similar spectral patterns and that from the ultra-
sound experiment is shown in Fig. 5. Using the
hetero-
correlation NMR technique - HSTC, the several signals
in the anomeric region and some of other signals could
be tentatively identified (Table 2) by comparison with
spectral data reported for various plant polysaccha-
rides [17-24]. A group of 13C-NMR signals are seen at
b 110.1, 108.3-108.0, 104.6-104.0, 101.4, 101.0, and
100.3-99.9. The first two were assigned to the anomeric
carbons of terminal o-L -arabinofuranose residues con-
nected with galactan and arabinan, respectively. The
further signals are assignable to C-1 of terminal and
3,6-linked �-D -galactopyranose, et-L -rhamnopyranose,
4,6-linked et-D-glucopyranose, and 4-linked et-D -galac-
turonic acid units, respectively. The results suggest the
presence of 3,5-arabinan and arabino-3,6-galactan (type
ll) connected with pectin molecules, and starch-like et-
glucan as the main polysaccharide components. The
pectin is partially methylesterfied (degree of esterifica-
tion ~33%) and 0-acetylated (0Me:0Ac ~ 3.6:1) as
estimated from the signal areas of the corresponding
carbon atom resonances (Table 2).
The molecular properties of the aqueous ethanol- and
water-soluble fractions are shown in Table 3. 0nly
minor differences were found in the molar mass distri-
bution of the corresponding ws-P2 fractions from the
classical and ultrasonical experiments. However, a low
molar mass was estimated in the case of the TP fraction
isolated from the medicinal tincture of the ultrasonical
experiment. The results confirm that ultrasonication
have not only attacked the integrity of the cell walls
rendering their components more accessible to extrac-
tion, but have also caused their depolymerisation into
fragments of lower molar mass soluble in hot water and
even aqueous ethanol.
42 Z. 2rom 'dkov ' et al. 3 Ultrasonics Sonochemistry 9 520026
37844

Fig. 5. 13C-NMR spectrum (in D20) of ws-P2/u and inserted is the anomeric region of its HSQC-NMR spectrum. A, et-L-arabinofuranose; GaA,
et-
D-galactopuranosyl uronic acid; Ga, �-D-galactopyranose; G, et-D -glucopyranose; R, et-L -rhamnopyranose.

Table 2 date have not been investigated. Using the mitogenic

13
C/1H-NMR (in D20) cross-peaks from the HSQC spectrum of ws-
P2 of the ultrasonical experiment
Chemical shifts b (ppm) Assignments
13 1
C H
174.8 C-6 et-GalpA
171.7 C-6 methylesterified et-GalpA
110.1 5.23 C-1 terminal et-Araf--- Gal,
108.3 5.08 C-1 terminal et-Araf--- Ara,
5-linked et-Araf
3,5-linked et-Araf
108.0 5.10 C-1
104.6 4.63 C-1 terminal �-Galp
104.0 4.50 C-1 3,6-linked �-Galp
101.5 4.91 C-1 et-Rhap
101.0 4.98 C-1 4,6-linked et-Glcp
110.3-99.9 5.12-5.0 C-1 4-linked et-GalpA
84.8 4.12 C-4 terminal et-Araf
83.6 4.28 C-3 3,5-linked et-Araf
83.0 4.24 C-4 3,5-linked et-Araf
82.2 4.12 C-2 terminal et-Araf
82.0 3.86 C-3 3,6-linked �-Galp
79.8 4.45 C-4 4-linked et-GalpA
77.7 3.94 C-3 terminal et-Araf
72.7 4.73 C-5 4-linked et-GalpA
71.6 5.14 C-5 methylesterified et-GalpA
53.9 3.80 CH3 methyl ester group
21.5 2.08 CH3 acetyl group
17.9 1.28 C-6 et-Rhap

As a lot of herbs were reported, during the last de-

cade, to contain various biologically active polysaccha-
rides [24-26], it was of interest to test for such activity
the ws-polysaccharide fractions of valerian, which up to
 Z. 2rom 'dkov ' et al. 3 Ultrasonics Sonochemistry 9 520026 41
and comitogenic in vitro thymocyte
37844 tests, the
immuno- stimulatory activity of the ws-P2
subfractions from both classical and ultrasonical
experiments was compared to that of the commercial
immunomodulator Zymosan, a baker"s yeast �-
glucan. Both fractions showed almost the same
response in these tests. As illustrated for ws-P2/ u in
Fig. 6, the direct mitogenic activity was comparable to
that of Zymosan. But the comitogenic activity was
substantially higher in comparison to Zymosan and
was similar to that previously reported for the water-
soluble polysaccharide fractions isolated from sage
with and without application of ultrasound [27].

. onclusions

The application of ultrasound during the preparation

of medicine tincture from valerian roots had not only a
positive effect on the extraction yield [9] but affected
also the extractibility and yield of HWE
polysaccharides. A considerable amount of easy-
extractable pectic poly- saccharides and starch was
released already during the ultrasound-assisted aqueous
ethanol extraction of the roots. Consequently, the yield
of the HWE polysac- charides was lower in the case of
the ultrasonical ex- periment.
This fact as well as the sugar composition and
structural features of the isolated polysaccharides sug-
gest that ultrasonication have attacked the integrity of
cell walls, released and degraded its most accessible
polysaccharides (pectic polysaccharides) and increased
also the extractibility of its less accessible
compo-
44 Z. 2rom 'dkov ' et al. 3 Ultrasonics Sonochemistry 9 520026
37844
Table 3
Molecular properties of polysaccharide fractions isolated from the Valeriana roots
Fraction Molar mass distributiona Peak lll
Peak l Peak ll Mw
b b
Mw Area % Mw Area %
Classical extraction
P1 >500,000 3 8,000 97
ws-P2 38,000 17 20,500 45 ~5,000
Ultrasound-assisted extraction
P1
~5,000 100
ws-P2 34,000 19 21,500 55 ~5,000
a
Measured on GM Biospher columns calibrated with pullulan standards P5-P800.
b
Calculated from the peak areas of the Rl-detected chromatogram.

Acknowledgements
This work was supported by the Slovak grant agency
VEGA (Project no. 2/7138) and the Slovak Academy of
Science (C0ST action). The authors thank Dr. J.
Alfo. ldi and Dr. E. Machov 'a, both from the lnstitute
of Chemistry of SAS, for the NMR and HPGPC mea-
surements.

Fig. 6. lmmunostimulatory activity of the ws-P2/u fraction, deter-
mined by the (a) mitogenic and (b) comitogenic thymocyte tests, in
comparison to that of the control - Zymosan.
nents - xylan, mannan and glucan. The water-soluble
polysaccharide fractions from both the conventional
and ultrasonical experiments exhibit significant immu-
nostimulatory activities in mitogenic and comitogenic
thymocyte tests.
However, the sonication effect was less pronounced in
the case of valerian when compared to that of similarly
treated sage, where the ethanol [9] and hot water ex-
tracts as well as isolated polysaccharide fractions [11]
were obtained in considerably higher yields. This may be
due to the different plant tissues used for extraction, i.e.
the aerial parts in the case of sage which are rather soft
in contrast to the roots of valerian, what probably af-
fects their different susceptibility to the ultrasonical
treatment. Therefore, the mechanochemical effect which
is responsible for the disruption of cell walls, enhance-
ment of mass transfer, and increased the extractibility of
cell wall polysaccharides might be less in the case of the
roots. Ultrasonication causes depolymerisation of the
solubilised polysaccharides yielding aqueous ethanol-
soluble fragments. lt is noteworthy that the water-solu-
ble polysaccharides from valerian roots represent a
novel potential polysaccharide-based immunomodula-
tor and the ultrasonical treatment has no adverse effect
on the biological response.
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