Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 62 No. 6 pp. 435ñ441, 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 £Ûdü, 1 MuszyÒski St., 90-151 £Ûdü, 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-α-L-arabinofuranoside (avicularin), 3-O-α-L-arabinopyranoside (guaijaverin), 3-O-β-D-xylopyranoside (reynoutrin), 3-Oβ-D-glucopyranoside (isoquercitrin), 3-O-β-D-galactopyranoside (hyperoside) followed by kaempferol 3-O-αL-arabinofuranoside (juglanin), 3-O-β-D-xylopyranoside and 3-O-β-D-glucopyranoside (astragalin). Moreover, 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-α-arabinofuranoside and isorhamnetin 3-O-β-xylopyranoside, the rare natural products. Keywords: Prunus serotina Ehrh., HPLC, flavonoids, leaves, flowers, isorhamnetin 3-O-α-arabinofuranoside, isorhamnetin 3-O-β-xylopyranoside
(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 £Ûdü 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 rechromatographed 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: H2OMeOH with MeOH gradient). Fractions eluted with 70-80% MeOH (1.75 g) were next rechromatographed on silica gel (eluent: EtOAc-MeOH 9:1
Prunus serotina Ehrh. (American black cherry), the largest of the native cherries representing 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 ñ 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-α-L-rhamnopyranosyl)-β-D-galactopyranoside 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
* E-mail address:
[email protected], Tel.: (+42) 677 91 69, Fax: (+42) 678 83 98
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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 ñ 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 hydrolysis 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 µL sample injector (Rheodyne 7725 i) and using Hypersil ODS (HP, 125 × 4 mm, 5 µm) 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-β-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 µm) and next 2-10 µL 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: SI 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; 2626.5 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 (according 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. α-arabinofuraIsolation of isorhamnetin 3-O-α β-xylopyranoside (VIII) and isorhamnetin 3-O-β 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 (S4), 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 Larabinose (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 (S5)).
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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″). β-xylopyranoside (IX) Isorhamnetin 3-O-β Amorphous yellow powder, m.p. 194-197OC; MeOH TLC Rf 0.57 (S-1), 0.73 (S-2); UV λmax 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 δ, 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-5″a), 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-5″b). RESULTS AND DISCUSSION
α-arabinofuranoside (VIII) Isorhamnetin 3-O-α Amorphous yellow powder, m.p. 220-225OC; MeOH TLC Rf 0.72 (S-1), 0.80 (S-2); UV λmax 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 δ, 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-
Flavonols are of particular interest to phytochemists 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:
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.
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MONIKA OLSZEWSKA
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 elution 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
by fractionated extraction and/or preparative chromatographic separation), can help in overcoming 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
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High-performance liquid chromatographic identification...
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 concentrations), followed by five sugars: L-arabinose, D-
Figure 3. Structures of isolated flavonoids. VIII: isorhamnetin 3-O-α-arabinofuranoside, IX: isorhamnetin 3-O-β-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 3galactoside, which were eluted as one peak. The SII 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 galactosides), 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, respectively. 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]*
in fractionated P. serotina leaf extracts
S-I
E-1 E-2 E-3 E-4 EA-2 EA-3 standards 1
hyperoside
+
+
2
reynoutrin
+
+
3
guaijaverin
+
4
avicularin
5
astragalin
+
6
kaempferol 3-O-β-Dxylopyranoside
+
7
juglanin
+
8
isoquercitrin
9
isorhamnetin 3-O-βxylopyranoside
10
isorhamnetin 3-O-αarabinofuranoside
+
+
TLC retention factors Rf*
S-II
real real standards S-1 samples samples
S-2
10.86
10.85
15.56
15.56
0.25
0.52
11.84
11.82
-
-
0.46
0.70
12.36
12.40
-
-
0.43
0.69
12.86
12.87
-
-
0.65
0.80
13.91
13.84
21.40
21.36
0.34
0.62
15.62
15.56
-
-
0.61
0.77
16.26
16.20
-
-
0.74
0.84
10.86
10.85
16.30
16.35
0.30
0.57
+
-
16.62
-
-
0.57
0.73
+
-
17.15
-
-
0.72
0.80
+ +
+
+
* 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.
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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-α-L-arabinofuranoside, 3-Oα-L-arabinopyranoside, 3-O-β-D-xylopyranoside, 3-O-β-D-glucopyranoside and 3-O-β-D-galactopyranoside (avicularin, guaijaverin, reynoutrin, isoquercitrin and hyperoside, respectively), as well kaempferol 3-O-α-L-arabinofuranoside, 3-O-β-D-glucopyranoside (juglanin and astragalin, respectively) and kaempferol 3-O-β-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-β-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 îquercetin 3-O-glucoside not identical with isoquercitrinî, 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 3position 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 δ 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 singlet at δ 5.62 ppm, which indicated the α-configuration of arabinosyl residue and suggested its occurrence in the furanose form (8, 11). The abovementioned fact was evidenced by the location of the H-2″-signal at δ 4.15 ppm (multiplet) and by unseparated signal of 5″ methylene group, recorded as a multiplet at δ 3.24 ppm (11). Moreover, all the sugar region pattern was in agreement with those
recorded and published for quercetin and kaempferol 3-O-α-L-arabinofuranosides (3, 8, 9). Consequently, VIII was identified as isorhamnetin 3-O-α-arabinofuranoside. In the 1H NMR spectrum of IX the anomeric proton of xylosyl residue was recorded at δ 5.35 ppm as a doublet with diaxial coupling constant J1,2 = 7.2 Hz, assignable to the β-pyranose form of sugar. This suggestion was proved by the strongly anisochronous 5″ methylene responses (observed as two double doublets with ∆δ 0.68 ppm), characteristic 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-β-D-xylopyranoside, reynoutrin (2) and another polyphenolic xylopyranosides (12-14) . Finally, IX was determined as isorhamnetin 3-O-βxylopyranoside. 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 α-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-α-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-α-xylopyranoside from Vaccinium macrocarpon (9). Finally, the current study was the first time isolation of isorhamnetin 3-O-α-arabinofuranoside and 3-O-β-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. 502-13847 (198) of the Medical University of £Ûdü. REFERENCES 1. Golz-Berner K., Zastrow L.: PGT Int. Appl. NO 2001026617A1, Patent, CA 134, 300647 g (2001). 2. Olszewska M.: Acta Pol. Pharm. 62, 127 (2005). 3. Olszewska M., Wolbiú M.: Acta Pol. Pharm. 58, 367 (2001). 4. Olszewska M., Wolbiú M.: Acta Pol. Pharm. 59, 133 (2002). 5. Nowak S., Wolbiú M.: Acta Pol. Pharm. 59, 275 (2002).
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6. Mabry T.J., Markham K.R., Thomas M.B.: The Systematic Identification of Flavonoids, Springer, Berlin-Heidelberg-New York 1970. 7. RychliÒska I., Gudej J.: Acta Pol. Pharm. 59, 53 (2002). 8. Markham K.R., Geiger H.: in The Flavonoids. Advances in research since 1986, Harborne J.B. (ed.), Chapman and Hall, Cambridge 1994. 9. Vvedenskaya I.O., Rosen R.T., Guido J.E., Russell D.J., Mills K.A., Vorsa N.: J. Agric. Food Chem. 52, 188 (2004). 10. Power F.B., Moore C.W.: J. Chem. Soc. 97, 1099 (1910). 11. Zapesochnaya G.G.: Khim. Prir. Soed. 15, 21 (1979). 12. Wen-Sheng Y., Hong L., Xin-Min Ch., Lei Y.: Phytochemistry 31, 4385 (1992).
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13. H¸bner G., Wray V., Nahrstedt A.: Planta Medica 65, 636 (1999). 14. Nakanishi T., Iida N., Inatomi Y., Murata H., Inada A., Murata J., Lang F.A., Iinuma M., Tanaka T.: Phytochemistry 65, 207 (2004). 15. Geiger H., De Groot-Pfleiderer W.: Phytochemistry 18, 1709 (1979). 16. Niemann G.J.: Z. Naturforsch. 35 C, 514 (1980). 17. Budzianowski J., Skrzypczak L., Weso≥owska M.: Sci. Pharm. 58,15 (1990). 18. Ma Y.-L., Lei Z.-H., Kong Q., Huang Q.-D., Feng Y., Tai B.-S., Yahara S., Nohara T.: Stud. Plant Sci. 6, 302 (1999), CA 131, 16413w (1999). 19. Eskalieva B.K., Burasheva G. Sh.: Chem. Nat. Comp. (Khim. Prir. Soed.) 38, 102 (2002). Received: 20.08.2005
