Acta Poloniae Pharmaceutica Drug Research, Vol. 62 No. 6 pp.

435441, 2005 ISSN 0001-6837

Polish Pharmaceutical Society

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC

IDENTIFICATION OF FLAVONOID MONOGLYCOSIDES
FROM PRUNUS SEROTINA EHRH.
MONIKA OLSZEWSKA*

Department of Pharmacognosy, Faculty of Pharmacy, Medical University of d,

1 Muszyski St., 90-151 d, Poland

Abstract: Five minor flavonoid monosides, glycosides of quercetin and kaempferol, together with three
previously isolated compounds were identified cochromatographically in P. serotina Ehrh. leaves and flowers
(inflorescences) using RP-HPLC and TLC techniques and finally determined as quercetin 3-O--L-arabino-
furanoside (avicularin), 3-O--L-arabinopyranoside (guaijaverin), 3-O--D-xylopyranoside (reynoutrin), 3-O-
-D-glucopyranoside (isoquercitrin), 3-O--D-galactopyranoside (hyperoside) followed by kaempferol 3-O--
L-arabinofuranoside (juglanin), 3-O--D-xylopyranoside and 3-O--D-glucopyranoside (astragalin). More-
over, two further minor flavonols were isolated from the leaves, characterized by hydrolysis experiments, UV
and 1H NMR spectroscopy, and identified finally as isorhamnetin 3-O--arabinofuranoside and isorhamnetin
3-O--xylopyranoside, the rare natural products.

Keywords: Prunus serotina Ehrh., HPLC, flavonoids, leaves, flowers, isorhamnetin 3-O--arabinofuranoside,
isorhamnetin 3-O--xylopyranoside

Prunus serotina Ehrh. (American black
cherry), the largest of the native cherries repre-
senting the subgenus Padus of the family Rosaceae,
is a very interesting plant with potential use for
production of antioxidant extracts, which can be the
biologically active basis for cosmetics and
phytopharmaceuticals (1). In the previous paper (2)
the flavonoids have been recognized as the main
chemical components of P. serotina leaves and
flowers, which may be connected with the expected
antioxidant activity of these plant materials. From
the leaf flavonoid complex seven dominant
compounds (I VII) were isolated and structurally
determined as three quercetin monosides:
hyperoside, avicularin, reynoutrin, three quercetin
biosides: 3-O-rutinoside, 3-O-neohesperidoside and
3-O-(2-O--L-rhamnopyranosyl)--D-galactopy-
ranoside as well isorhamnetin 3-O-rutinoside (2).
The current paper presents the results of the
chromatographic and isolation studies of further
flavonoids, which occurs in P. serotina leaves and
flowers in minute amounts.
EXPERIMENTAL
Material for the studies
Material for the current investigations were the
selected flavonoid fractions, containing monosides
(the fractions E-1 E-4, separated from the Et2O
extract and the fractions EA-2 and EA-3, separated
from the EtOAc extract), which were obtained
chromatographically (CC) from the leaves of P.
serotina as follows: the powdered leaf sample
(600 g, collected in the Botanical Garden in d in
October 2001) was preextracted with petrol
followed by CHCl3 in Soxhlet apparatus and then
exhaustively extracted with boiling MeOH and 70%
MeOH. Combined methanol extracts were
evaporated, dissolved in water and partitioned
between Et2O, EtOAc and n-BuOH. The Et2O
extract (2.6 g) was submitted to CC on polyamide
(eluent: C6H6-MeOH with MeOH gradient) to yield
five fractions: E-1 E-5 (0.02, 0.08, 0.05, 0.20 and
0.64 g, respectively). Fraction E-5 was rechroma-
tographed under the same conditions and gave
compounds I (150 mg) and II (85 mg), finally
purified by crystallization from MeOH. The EtOAc
(8.0 g) and n-BuOH (7.0 g) extracts were separately
submitted to chromatographic gel filtration on
sephadex columns (using MeOH as eluent) to
separate flavonoid and proanthocyanidins fractions.
The EtOAc-flavonoid fraction (3.85 g) was then
first chromatographed on polyamide (eluent: H2O-
MeOH with MeOH gradient). Fractions eluted with
70-80% MeOH (1.75 g) were next rechroma-
tographed on silica gel (eluent: EtOAc-MeOH 9:1

* E-mail address: molszewska@pharm.am.lodz.pl, Tel.: (+42) 677 91 69, Fax: (+42) 678 83 98

435
436 MONIKA OLSZEWSKA
v/v) to afford four fractions: EA-1 EA-4 (0.12,
0.10, 0.10 and 1.05 g, respectively). The EA-4
fraction, after crystallization from MeOH, gave
compound III (700 mg). Compounds I, II, III were
structurally determined as avicularin, reynoutrin and
hyperoside, respectively. Details of structural
determination and also isolation and identification
of diglycosides IV VII from n-BuOH extract were
described previously (2).
Moreover, the samples of crude Et2O and
EtOAc extracts of P. serotina leaves and flowers
(coll. in June 2002) constitute materials for hydro-
lysis experiments and cochromatographic analysis,
and were also prepared previously (2).
High-Performance Liquid Chromatographic
analysis
Instrumentation: RP-HPLC analysis was
carried out on a Hewlett-Packard 1100 Series
instrument equipped with a quaternary pump (HP
1311 A), a vacuum degasser (HP 1322 A),
a UV/VIS detector (HP 1314 A), a 20 L sample
injector (Rheodyne 7725 i) and using Hypersil ODS
(HP, 125 4 mm, 5 m) as the analytical column.
The chromatograms were recorded on a HP 3396 B
reporting integrator, set at 7 mm/min of chart speed.
Standards and solvents: The standards of
flavonol monoglycosides (NMR spectroscopy grade
purity) were isolated earlier as follows: avicularin,
guajiverin, reynoutrin, isoquercitrin, juglanin and
kaempferol 3-O--D-xylopyranoside from Prunus
spinosa flowers (3, 4), hyperoside from Prunus
serotina leaves (2) and astragalin from Scopolia
lurida leaves (5). The standard solutions for analysis
were prepared by dissolving 1-2 mg of each
glycoside in 50 mL of MeOH. The solvents used
such as MeOH, acetonitrile (ACN), water and
ortophosphoric acid (Merck) were of HPLC grade
purity.
Sample preparation: The analyzed flavonoid
fractions (20-200 mg) were dissolved in 2-20 mL of
MeOH, respectively, filtered through a syringe
Whatman PTFE filter (13 mm, 2 m) and next 2-10
L of the solutions prepared in such a way were
injected into the HPLC system.
Peak identification: The identification of the
separated peaks of flavonoids was first carried out
cochromatographically by direct comparison with
the retention parameters of standard solutes. Next,
the inner standard method was used (addition of
standard solution to the analyzed fraction) and the
peaks were identified by the observed increase of
their intensity. The above-mentioned procedure was
done separately for each standard.
Chromatographic procedure: The analyses were
performed using two gradient elution systems: S-
I and S-II, differed in composition of the mobile
phase, which consist of solvent A (0.5% water
solution of ortophosphoric acid) plus solvent B
(MeOH) for S-I, and solvent A plus solvent C (ACN)
for S-II. The gradient profiles were as follows:
S-I: 0-15 min, 40-53% B; 15-20 min, 53-60%
B; 20-25 min, 60-80% B; 25-26 min, 80-40% B; 26-
26.5 min 40% B (post time),
S-II: 0-10 min, 18% C; 10-17 min, 18-22% C;
17-20 min, 22% C; 20-22 min, 22-35% C; 22-23
min, 35-50% C; 23-25 min, 50-18% C, 25-25.5 min,
18% C (post time).
In both systems the flow rate was 1.0 mL/min
and detection was effected at 350 nm. For retention
parameters of analyzed flavonoid monosides see
Table 1. The examples of chromatograms are shown
in Figures 1 and 2.
TLC, PC and CC studies and other methods
UV spectra with usual shift reagents (accor-
ding to the standard procedure (6)) were made on
Unicam 500, 1H NMR on Bruker 500 MHz (in
DMSO-d6, TMS as internal standard).
Preparative column chromatography (CC) was
performed on polyamide SC6 (Roth), silica gel 60
(MN) and sephadex LH-20 (Fluka); analytical TLC
on silica gel 60 precoated plates and polyamide
aluminium sheets (Merck), PC on Whatman No. 1.
For TLC and PC the following solvent systems in
volumetric ratios were employed (vol. ratios):
S-1: EtOAc / HCOOH / H2O (18:1:1)
S-2: n-BuOH / AcOH / HCOOH / H2O (100:
27:1:5, organic phase)
S-3: n-BuOH / AcOH / H2O (4:1:5, organic
phase)
S-4: CHCl3 / AcOEt / MeOH (14:3:3)
S-5: EtOH 96% / NH4OH 25% / H2O (20:1:4).
Flavonoids were visualized by UV light 366
nm, with NH3 vapors and by spraying with 1% AlCl3
in MeOH. Sugars were detected by spraying with
aniline phthalate solution in n-BuOH and heating at
105OC. The TLC (on silica) retention parameters of
determined flavonoids are listed in Table 1.
Isolation of isorhamnetin 3-O- -arabinofura-
-xylopyra-
noside (VIII) and isorhamnetin 3-O-
noside (IX)
The E-1 fraction was submitted to CC on
polyamide (eluent: C6H6-MeOH with gradient of
MeOH in the range 20-30%) to yield compounds
VIII (5 mg) and IX (3 mg), finally purified on
sephadex (MeOH as eluent).
 High-performance liquid chromatographic identification...
Total acid hydrolysis
1-2 mg of the glycosides VIII and IX and 20
mg of the crude leaf and flower Et2O and EtOAc
extracts were refluxed separately with 5% HCl for
2 h. The hydrolysates were extracted with Et2O and
the obtained extracts were washed with water,
evaporated to dryness and resolved in MeOH.
Identification of the aglycones was done by coPC
(S-3) and coTLC (S-4, on polyamide) with
standards of quercetin (Rfs: 0.78 (S-3) and 0.06 (S-
4), isolated from Prunus spinosa (3)), isorhamnetin
(Rfs: 0.85 (S-3) and 0.29 (S-4), obtained from Pyrus
communis (7)) and kaempferol (Rfs: 0.88 (S-3) and
0.17 (S-4), (3)). The remaining aqueous solutions
were evaporated to dryness, resolved in MeOH and
the sugars were identified by coPC (S-3) and
coTLC (S-5) with authentic standards of L-
arabinose (Rfs: 0.17 (S-3), 0.44 (S-5)), D-glucose
(Rfs: 0.15 (S-3), 0.44 (S-5)), D-galactose (Rfs: 0.13
(S-3), 0.36 (S-5)), L-rhamnose (Rfs: 0.31 (S-3),
0.57 (S-5)) and D-xylose (Rfs: 0.19 (S-3), 0.54 (S-
5)).
Isorhamnetin 3-O- -arabinofuranoside (VIII)
Amorphous yellow powder, m.p. 220-225OC;
TLC Rf 0.72 (S-1), 0.80 (S-2); UV maxMeOH
nm: 255,
267sh, 304sh, 355; NaOMe 272, 328, 415; AlCl3
268, 300sh, 365sh, 404; AlCl3-HCl 267, 300sh, 360,
400; NaOAc 272, 320sh, 395; NaOAc-H3BO3 254,
267sh, 305sh, 358. 1H NMR , ppm: 12.61 (1H, s,
OH-5), 7.65 (1H, d, J=1.6 Hz, H-2), 7.59 (1H, dd,
J=1.6 and 8.5 Hz, H-6), 6.90 (1H, d, J=8.5 Hz, H-
Figure 1. HPLC chromatograms of flavonoid standard solutions. I: elution system S-I, II: elution system S-II. The peaks correspond to
numbering of compounds in Table 1.
437
5), 6.44 (1H, s, H-8), 6.18 (1H, s, H-6), 5.62 (1H, s,
H-1), 4.15 (1H, m, H-2), 3.86 (3H, s, OMe-3),
3.70 (1H, dd, J=5.1 and 4.3 Hz, H-3), 3.45 (1H, m,
J=5.4 Hz, H-4), 3.24 (2H, m, partially overlapped
with H2O signal, 2H-5).
Isorhamnetin 3-O- -xylopyranoside (IX)
Amorphous yellow powder, m.p. 194-197OC;
TLC Rf 0.57 (S-1), 0.73 (S-2); UV max MeOH
nm: 260,
268sh, 300sh, 354; NaOMe 271, 329, 412; AlCl3
267, 301, 360sh, 403; AlCl3-HCl 267, 300, 362, 401;
NaOAc 274, 323, 402; NaOAc-H3BO3 260, 268sh,
300sh, 354. 1H NMR , ppm: 12.53 (1H, s, OH-5),
7.84 (1H, d, J=1.8 Hz, H-2), 7.53 (1H, dd, J=1.8
and 8.5 Hz, H-6), 6.90 (1H, d, J=8.5 Hz, H-5), 6.34
(1H, s, H-8), 6.11 (1H, s, H-6), 5.35 (1H, d, J=7.2
Hz, H-1), 3.82 (3H, s, OMe-3), 3.66 (1H, dd, J=5.0
and 11.5 Hz, H-5a), 3.21-3.40 (2H, m, partially
overlapped with H2O signal, H-3 and H-4), 3.18
(1H, dd, J=8.4 and 8.4 Hz, H-2), 2.98 (1H, dd,
J=10.9 and 10.4 Hz, H-5b).
RESULTS AND DISCUSSION
Flavonols are of particular interest to phyto-
chemists as they have been shown to possess a wide
bioactive potential and are regarded as one of the
most numerous and widespread groups of natural
polyphenols found in plants (8). The variety of
flavonols that can occur in plant materials is large,
and their analysis creates different challenges. The
most important issues are:
438
Figure 2. HPLC analysis of real samples of fractionated flavonoid extracts from P. serotina leaves: I: fr. E-1, II: fr. E-2, III: fr. E-3 (in elu-
tion system S-I), IV: fr EA-2 (in elution system S-II). For peak identification see Table 1.
when the analysis is carried out by HPLC
with UV detection, as is usual, interferences with
other substances may exist, especially with other
phenolics, present in higher amounts and/or with
higher extinction coefficients,
in samples that possess a complex flavonoids
composition, it is difficult to obtain satisfactory
separations in a single run, particularly when plant
materials are rich in polyglycosylated compounds,
which exhibit similar polarity and often differ only
in the characteristic of interglycosidic linkages,
the accurate identification of substances in
the chromatograms is impossible as no standards are
available, that is a major problem when dealing with
polyglycosides, which usually requires their
previous isolation.
The HPLC analysis of flavonoids, performed
in fractionated extracts of plant materials (obtained
MONIKA OLSZEWSKA
by fractionated extraction and/or preparative
chromatographic separation), can help in over-
coming some of these obstacles, also permitting the
identification of minor and trace components. The
current paper presents the application of this method
to identification of P. serotina minor flavonoids.
The chromatographic screening analysis of P.
serotina flavonoid fraction exhibited the presence of
large number of compounds, among which the
monosides appeared to be dominant. All the occured
monoglycosides showed UV absorption properties
characteristic for 3-O-substituted flavonols. The main
monoglycosidic components (hyperoside, avicularin,
reynoutrin) as well as diglycosides were isolated
chromatographically and identified previously (2).
For identification of further monosides
(occurred as minor or trace elements) the samples of
the crude leaf and flower Et2O and EtOAc extracts
 High-performance liquid chromatographic identification... 439

were first submitted to total acid hydrolysis. In the
hydrolysates three aglycones were detected, namely
quercetin (as dominant component), isorhamnetin
and kaempferol (in significantly lower concen-
trations), followed by five sugars: L-arabinose, D-
Figure 3. Structures of isolated flavonoids. VIII: isorhamnetin
3-O--arabinofuranoside, IX: isorhamnetin 3-O--xylopyranoside.
xylose, D-galactose, D-glucose and L-rhamnose
(only in traces). Next, the containing monosides and
fractionated flavonoid fractions, which were
obtained previously from Et2O and EtOAc P.
serotina leaf extracts (2), were submitted to TLC
cochromatographic analysis with numerous
standards, corresponding with the hydrolysis results.
Eight standards detected as chromatographically
consistent with components of the analyzed
fractions were submitted to HPLC analysis.
To separate the flavonoid standards, and next
the samples of fractions from P. serotina, two
gradient elution systems were elaborated. The S-I
system, employing gradient of methanol, enabled
separation of nine flavonoid monosides, but without
separation of quercetin 3-glucoside and quercetin 3-
galactoside, which were eluted as one peak. The S-
II system, employing gradient of acetonitrile,
permits separation and identification of these two
quercetin hexosides. It must be pointed out that by
applying two elution procedures for the analysis of
prechromatographed fractions, in which flavonoid
pentosides (arabinosides and xylosides) were
separated from hexosides (glucosides and galacto-
sides), the total time of analysis was significantly
shortened. In purposed elution procedures the time
of single run was only 26.0 and 25.0 min, respec-
tively. Conversely, for example the time of single
run in HPLC analysis advocated in literature for

Table 1. Occurence and retention parameters of P. serotina flavonoid monosides.

Nr Compound Occurence HPLC retention times tR [min]* TLC retention

in fractionated P. serotina leaf extracts S-I S-II factors Rf*
real real
E-1 E-2 E-3 E-4 EA-2 EA-3 standards standards S-1 S-2
samples samples
1 hyperoside + + + + 10.86 10.85 15.56 15.56 0.25 0.52
2 reynoutrin + + 11.84 11.82 - - 0.46 0.70
3 guaijaverin + 12.36 12.40 - - 0.43 0.69
4 avicularin + 12.86 12.87 - - 0.65 0.80
5 astragalin + + + 13.91 13.84 21.40 21.36 0.34 0.62
6 kaempferol 3-O--D- + 15.62 15.56 - - 0.61 0.77
xylopyranoside
7 juglanin + 16.26 16.20 - - 0.74 0.84
8 isoquercitrin + 10.86 10.85 16.30 16.35 0.30 0.57
9 isorhamnetin 3-O-- + - 16.62 - - 0.57 0.73
xylopyranoside
10 isorhamnetin 3-O-- + - 17.15 - - 0.72 0.80
arabinofuranoside

* The mean parameters calculated for three-times analyses with relative standard deviations RSD = 0.5 1.0% for HPLC
and RSD = 2.5 4.5% for TLC.
440 MONIKA OLSZEWSKA
flavonoid complex from Vaccinium macrocarpon,
containing several unseparated flavonol pentosides
and hexosides, amounted to 50 min (9).
In P. serotina HPLC investigations ten peaks of
flavonol monosides were found on the chromatograms.
Eight of them were identified with authentic standards
as follows: quercetin 3-O--L-arabinofuranoside, 3-O-
-L-arabinopyranoside, 3-O--D-xylopyranoside,
3-O--D-glucopyranoside and 3-O--D-galactopyra-
noside (avicularin, guaijaverin, reynoutrin, isoque-
rcitrin and hyperoside, respectively), as well kaem-
pferol 3-O--L-arabinofuranoside, 3-O--D-glucopy-
ranoside (juglanin and astragalin, respectively) and
kaempferol 3-O--D-xylopyranoside. The presence of
the detected compounds was then determined
chromatographically by the comparative TLC analysis
in the fractionated methanolic extract of P. serotina
flowers (inflorescences). Five of the mentioned
compounds (guaijaverin, isoquercitrin, juglanin,
astragalin and kaempferol 3-O--D-xylopyranoside)
were found in the analyzed taxon for the first time. The
obtained results of isolation (2) and now presented
cochromatographic studies suggest that a flavonol
hexoside, isolated by Power et al. (10) as the main
flavonoid from of P. serotina leaves, and identified as
quercetin 3-O-glucoside not identical with isoquer-
citrin, was in fact probably a mixture of isoquercitrin
and hyperoside.
Eight identified flavonols were accompanied
by two other flavonoids (occured in E-1 fraction)
not corresponding with standards. So, the fraction
was then chromatographed on polyamide and next
on sephadex LH-20, and flavonols were isolated as
compounds VIII and IX.
Upon acid hydrolysis the compounds released
isorhamnetin and different sugars identified as
arabinose and xylose, respectively. The UV spectra
analysis indicated the site of glycosylation at the 3-
position of the aglycone in both cases (6). Because
of small amounts of compounds only the 1H NMR
spectra were recorded. For isorhamnetin moieties
the spectra showed the expected proton signals in
aromatic regions and the methoxyl singlets at 3.86
and 3.82 ppm, respectively for VIII and IX (8).
In the spectrum of compound VIII the
anomeric proton resonance was observed as a sin-
glet at 5.62 ppm, which indicated the -configu-
ration of arabinosyl residue and suggested its
occurrence in the furanose form (8, 11). The above-
mentioned fact was evidenced by the location of the
H-2-signal at 4.15 ppm (multiplet) and by
unseparated signal of 5 methylene group, recorded
as a multiplet at 3.24 ppm (11). Moreover, all the
sugar region pattern was in agreement with those
recorded and published for quercetin and
kaempferol 3-O--L-arabinofuranosides (3, 8, 9).
Consequently, VIII was identified as isorhamnetin
3-O--arabinofuranoside.
In the 1H NMR spectrum of IX the anomeric
proton of xylosyl residue was recorded at 5.35
ppm as a doublet with diaxial coupling constant J1,2
= 7.2 Hz, assignable to the -pyranose form of
sugar. This suggestion was proved by the strongly
anisochronous 5 methylene responses (observed as
two double doublets with 0.68 ppm), charac-
teristic for a pentose in the pyranose form (8, 9, 11).
Furthermore, all the sugar region pattern was in
agreement with those recorded and published for
quercetin 3-O--D-xylopyranoside, reynoutrin (2)
and another polyphenolic xylopyranosides (12-14) .
Finally, IX was determined as isorhamnetin 3-O--
xylopyranoside.
Isorhamnetin 3-O-monopentosides are very
rare compounds, hitherto detected only in several
genera, i.e. in the genus Vaccinium (9), Taxodium
(15), Larix (16), Solidago (17), Erysimum (18) and
Alhagi (19). The arabinosides were identified as the
-L-arabinopyranosides (15, 18, 19) or without
determination of configuration and ring form of
sugar residue (16, 17). The only one hitherto known
isorhamnetin 3-O--L-arabinofuranoside was
isolated from Taxodium distichum (15). Similarly the
xylosides were previously isolated mostly without
full structural determination as 3-O-xylosides (8),
with the exception of isorhamnetin 3-O--xylo-
pyranoside from Vaccinium macrocarpon (9).
Finally, the current study was the first time
isolation of isorhamnetin 3-O--arabinofuranoside
and 3-O--xylopyranoside from the genus Prunus, as
well the first presentation of its NMR spectral data.
Acknowledgments
The study is a part of the project No. 502-13-
847 (198) of the Medical University of d.
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Received: 20.08.2005