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 and , 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-arabino- furanoside (), 3-O-α-L-arabinopyranoside (), 3-O-β-D-xylopyranoside (reynoutrin), 3-O- β-D-glucopyranoside (isoquercitrin), 3-O-β-D-galactopyranoside () followed by kaempferol 3-O-α- L-arabinofuranoside (juglanin), 3-O-β-D-xylopyranoside and 3-O-β-D-glucopyranoside (). More- over, two further minor were isolated from the leaves, characterized by hydrolysis experiments, UV and 1H NMR spectroscopy, and identified finally as 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

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

first chromatographed on polyamide (eluent: H2O- Material for the studies MeOH with MeOH gradient). Fractions eluted with Material for the current investigations were the 70-80% MeOH (1.75 g) were next rechroma- selected flavonoid fractions, containing monosides tographed on silica gel (eluent: EtOAc-MeOH 9:1

* E-mail address: [email protected], 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, Chromatographic procedure: The analyses were 0.10, 0.10 and 1.05 g, respectively). The EA-4 performed using two gradient elution systems: S- fraction, after crystallization from MeOH, gave I and S-II, differed in composition of the mobile compound III (700 mg). Compounds I, II, III were phase, which consist of solvent A (0.5% water structurally determined as avicularin, reynoutrin and solution of ortophosphoric acid) plus solvent B hyperoside, respectively. Details of structural (MeOH) for S-I, and solvent A plus solvent C (ACN) determination and also isolation and identification for S-II. The gradient profiles were as follows: of diglycosides IV ñ VII from n-BuOH extract were S-I: 0-15 min, 40-53% B; 15-20 min, 53-60% described previously (2). B; 20-25 min, 60-80% B; 25-26 min, 80-40% B; 26-

Moreover, the samples of crude Et2O and 26.5 min 40% B (post time), EtOAc extracts of P. serotina leaves and flowers S-II: 0-10 min, 18% C; 10-17 min, 18-22% C; (coll. in June 2002) constitute materials for hydro- 17-20 min, 22% C; 20-22 min, 22-35% C; 22-23 lysis experiments and cochromatographic analysis, min, 35-50% C; 23-25 min, 50-18% C, 25-25.5 min, and were also prepared previously (2). 18% C (post time). In both systems the flow rate was 1.0 mL/min High-Performance Liquid Chromatographic and detection was effected at 350 nm. For retention analysis parameters of analyzed flavonoid monosides see Instrumentation: RP-HPLC analysis was Table 1. The examples of chromatograms are shown carried out on a Hewlett-Packard 1100 Series in Figures 1 and 2. instrument equipped with a quaternary pump (HP 1311 A), a vacuum degasser (HP 1322 A), TLC, PC and CC studies and other methods a UV/VIS detector (HP 1314 A), a 20 µL sample UV spectra with usual shift reagents (accor- injector (Rheodyne 7725 i) and using Hypersil ODS ding to the standard procedure (6)) were made on (HP, 125 × 4 mm, 5 µm) as the analytical column. Unicam 500, 1H NMR on Bruker 500 MHz (in

The chromatograms were recorded on a HP 3396 B DMSO-d6, TMS as internal standard). reporting integrator, set at 7 mm/min of chart speed. Preparative column chromatography (CC) was Standards and solvents: The standards of performed on polyamide SC6 (Roth), silica gel 60 flavonol monoglycosides (NMR spectroscopy grade (MN) and sephadex LH-20 (Fluka); analytical TLC purity) were isolated earlier as follows: avicularin, on silica gel 60 precoated plates and polyamide guajiverin, reynoutrin, isoquercitrin, juglanin and aluminium sheets (Merck), PC on Whatman No. 1. kaempferol 3-O-β-D-xylopyranoside from Prunus For TLC and PC the following solvent systems in spinosa flowers (3, 4), hyperoside from Prunus volumetric ratios were employed (vol. ratios): serotina leaves (2) and astragalin from Scopolia S-1: EtOAc / HCOOH / H2O (18:1:1) lurida leaves (5). The standard solutions for analysis S-2: n-BuOH / AcOH / HCOOH / H2O (100: were prepared by dissolving 1-2 mg of each 27:1:5, organic phase) glycoside in 50 mL of MeOH. The solvents used S-3: n-BuOH / AcOH / H2O (4:1:5, organic such as MeOH, acetonitrile (ACN), water and phase) ortophosphoric acid (Merck) were of HPLC grade S-4: CHCl3 / AcOEt / MeOH (14:3:3) purity. S-5: EtOH 96% / NH4OH 25% / H2O (20:1:4). Sample preparation: The analyzed flavonoid Flavonoids were visualized by UV light 366 fractions (20-200 mg) were dissolved in 2-20 mL of nm, with NH3 vapors and by spraying with 1% AlCl3 MeOH, respectively, filtered through a syringe in MeOH. Sugars were detected by spraying with Whatman PTFE filter (13 mm, 2 µm) and next 2-10 aniline phthalate solution in n-BuOH and heating at µL of the solutions prepared in such a way were 105OC. The TLC (on silica) retention parameters of injected into the HPLC system. determined flavonoids are listed in Table 1. Peak identification: The identification of the separated peaks of flavonoids was first carried out Isolation of isorhamnetin 3-O-α-arabinofura- cochromatographically by direct comparison with noside (VIII) and isorhamnetin 3-O-β-xylopyra- the retention parameters of standard solutes. Next, noside (IX) the inner standard method was used (addition of The E-1 fraction was submitted to CC on standard solution to the analyzed fraction) and the polyamide (eluent: C6H6-MeOH with gradient of peaks were identified by the observed increase of MeOH in the range 20-30%) to yield compounds their intensity. The above-mentioned procedure was VIII (5 mg) and IX (3 mg), finally purified on done separately for each standard. sephadex (MeOH as eluent). High-performance liquid chromatographic identification... 437

Total acid hydrolysis 5′), 6.44 (1H, s, H-8), 6.18 (1H, s, H-6), 5.62 (1H, s, 1-2 mg of the glycosides VIII and IX and 20 H-1″), 4.15 (1H, m, H-2″), 3.86 (3H, s, OMe-3′), ″ mg of the crude leaf and flower Et2O and EtOAc 3.70 (1H, dd, J=5.1 and 4.3 Hz, H-3 ), 3.45 (1H, m, extracts were refluxed separately with 5% HCl for J=5.4 Hz, H-4″), 3.24 (2H, m, partially overlapped ″ 2 h. The hydrolysates were extracted with Et2O and with H2O signal, 2H-5 ). the obtained extracts were washed with water, evaporated to dryness and resolved in MeOH. Isorhamnetin 3-O-β-xylopyranoside (IX) Identification of the aglycones was done by coPC Amorphous yellow powder, m.p. 194-197OC; λMeOH (S-3) and coTLC (S-4, on polyamide) with TLC Rf 0.57 (S-1), 0.73 (S-2); UV max nm: 260, standards of quercetin (Rfs: 0.78 (S-3) and 0.06 (S- 268sh, 300sh, 354; NaOMe 271, 329, 412; AlCl3

4), isolated from Prunus spinosa (3)), isorhamnetin 267, 301, 360sh, 403; AlCl3-HCl 267, 300, 362, 401;

(Rfs: 0.85 (S-3) and 0.29 (S-4), obtained from Pyrus NaOAc 274, 323, 402; NaOAc-H3BO3 260, 268sh, 1 δ communis (7)) and kaempferol (Rfs: 0.88 (S-3) and 300sh, 354. H NMR , ppm: 12.53 (1H, s, OH-5), 0.17 (S-4), (3)). The remaining aqueous solutions 7.84 (1H, d, J=1.8 Hz, H-2′), 7.53 (1H, dd, J=1.8 were evaporated to dryness, resolved in MeOH and and 8.5 Hz, H-6′), 6.90 (1H, d, J=8.5 Hz, H-5′), 6.34 the sugars were identified by coPC (S-3) and (1H, s, H-8), 6.11 (1H, s, H-6), 5.35 (1H, d, J=7.2 coTLC (S-5) with authentic standards of L- Hz, H-1″), 3.82 (3H, s, OMe-3′), 3.66 (1H, dd, J=5.0 ″ (Rfs: 0.17 (S-3), 0.44 (S-5)), D-glucose and 11.5 Hz, H-5 a), 3.21-3.40 (2H, m, partially ″ ″ (Rfs: 0.15 (S-3), 0.44 (S-5)), D-galactose (Rfs: 0.13 overlapped with H2O signal, H-3 and H-4 ), 3.18 ″ (S-3), 0.36 (S-5)), L-rhamnose (Rfs: 0.31 (S-3), (1H, dd, J=8.4 and 8.4 Hz, H-2 ), 2.98 (1H, dd, ″ 0.57 (S-5)) and D-xylose (Rfs: 0.19 (S-3), 0.54 (S- J=10.9 and 10.4 Hz, H-5 b). 5)). RESULTS AND DISCUSSION Isorhamnetin 3-O-α-arabinofuranoside (VIII) Amorphous yellow powder, m.p. 220-225OC; Flavonols are of particular interest to phyto- λMeOH TLC Rf 0.72 (S-1), 0.80 (S-2); UV max nm: 255, chemists as they have been shown to possess a wide

267sh, 304sh, 355; NaOMe 272, 328, 415; AlCl3 bioactive potential and are regarded as one of the

268, 300sh, 365sh, 404; AlCl3-HCl 267, 300sh, 360, most numerous and widespread groups of natural

400; NaOAc 272, 320sh, 395; NaOAc-H3BO3 254, polyphenols found in plants (8). The variety of 267sh, 305sh, 358. 1H NMR δ, ppm: 12.61 (1H, s, flavonols that can occur in plant materials is large, OH-5), 7.65 (1H, d, J=1.6 Hz, H-2′), 7.59 (1H, dd, and their analysis creates different challenges. The J=1.6 and 8.5 Hz, H-6′), 6.90 (1H, d, J=8.5 Hz, H- 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. 438 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 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 by fractionated extraction and/or preparative with UV detection, as is usual, interferences with chromatographic separation), can help in over- other substances may exist, especially with other coming some of these obstacles, also permitting the phenolics, present in higher amounts and/or with identification of minor and trace components. The higher extinction coefficients, current paper presents the application of this method ¡ in samples that possess a complex flavonoids to identification of P. serotina minor flavonoids. composition, it is difficult to obtain satisfactory The chromatographic screening analysis of P. separations in a single run, particularly when plant serotina flavonoid fraction exhibited the presence of materials are rich in polyglycosylated compounds, large number of compounds, among which the which exhibit similar polarity and often differ only monosides appeared to be dominant. All the occured in the characteristic of interglycosidic linkages, monoglycosides showed UV absorption properties ¡ the accurate identification of substances in characteristic for 3-O-substituted flavonols. The main the chromatograms is impossible as no standards are monoglycosidic components (hyperoside, avicularin, available, that is a major problem when dealing with reynoutrin) as well as diglycosides were isolated polyglycosides, which usually requires their chromatographically and identified previously (2). previous isolation. For identification of further monosides The HPLC analysis of flavonoids, performed (occurred as minor or trace elements) the samples of in fractionated extracts of plant materials (obtained the crude leaf and flower Et2O and EtOAc extracts High-performance liquid chromatographic identification... 439 were first submitted to total acid hydrolysis. In the xylose, D-galactose, D-glucose and L-rhamnose hydrolysates three aglycones were detected, namely (only in traces). Next, the containing monosides and quercetin (as dominant component), isorhamnetin fractionated flavonoid fractions, which were and kaempferol (in significantly lower concen- obtained previously from Et2O and EtOAc P. trations), followed by five sugars: L-arabinose, D- 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-

Figure 3. Structures of isolated flavonoids. VIII: isorhamnetin tively. Conversely, for example the time of single 3-O-α-arabinofuranoside, IX: isorhamnetin 3-O-β-xylopyranoside. 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-β-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-β- + - 16.62 - - 0.57 0.73 xylopyranoside 10 isorhamnetin 3-O-α- + - 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, recorded and published for quercetin and containing several unseparated flavonol pentosides kaempferol 3-O-α-L-arabinofuranosides (3, 8, 9). and hexosides, amounted to 50 min (9). Consequently, VIII was identified as isorhamnetin In P. serotina HPLC investigations ten peaks of 3-O-α-arabinofuranoside. flavonol monosides were found on the chromatograms. In the 1H NMR spectrum of IX the anomeric Eight of them were identified with authentic standards proton of xylosyl residue was recorded at δ 5.35 α as follows: quercetin 3-O- -L-arabinofuranoside, 3-O- ppm as a doublet with diaxial coupling constant J1,2 α-L-arabinopyranoside, 3-O-β-D-xylopyranoside, = 7.2 Hz, assignable to the β-pyranose form of 3-O-β-D-glucopyranoside and 3-O-β-D-galactopyra- sugar. This suggestion was proved by the strongly noside (avicularin, guaijaverin, reynoutrin, isoque- anisochronous 5″ methylene responses (observed as rcitrin and hyperoside, respectively), as well kaem- two double doublets with ∆δ 0.68 ppm), charac- pferol 3-O-α-L-arabinofuranoside, 3-O-β-D-glucopy- teristic for a pentose in the pyranose form (8, 9, 11). ranoside (juglanin and astragalin, respectively) and Furthermore, all the sugar region pattern was in kaempferol 3-O-β-D-xylopyranoside. The presence of agreement with those recorded and published for the detected compounds was then determined quercetin 3-O-β-D-xylopyranoside, reynoutrin (2) chromatographically by the comparative TLC analysis and another polyphenolic xylopyranosides (12-14) . in the fractionated methanolic extract of P. serotina Finally, IX was determined as isorhamnetin 3-O-β- flowers (inflorescences). Five of the mentioned xylopyranoside. compounds (guaijaverin, isoquercitrin, juglanin, Isorhamnetin 3-O-monopentosides are very astragalin and kaempferol 3-O-β-D-xylopyranoside) rare compounds, hitherto detected only in several were found in the analyzed taxon for the first time. The genera, i.e. in the genus Vaccinium (9), Taxodium obtained results of isolation (2) and now presented (15), Larix (16), Solidago (17), Erysimum (18) and cochromatographic studies suggest that a flavonol Alhagi (19). The arabinosides were identified as the hexoside, isolated by Power et al. (10) as the main α-L-arabinopyranosides (15, 18, 19) or without flavonoid from of P. serotina leaves, and identified as determination of configuration and ring form of îquercetin 3-O-glucoside not identical with isoquer- sugar residue (16, 17). The only one hitherto known citrinî, was in fact probably a mixture of isoquercitrin isorhamnetin 3-O-α-L-arabinofuranoside was and hyperoside. isolated from Taxodium distichum (15). Similarly the Eight identified flavonols were accompanied xylosides were previously isolated mostly without by two other flavonoids (occured in E-1 fraction) full structural determination as 3-O-xylosides (8), not corresponding with standards. So, the fraction with the exception of isorhamnetin 3-O-α-xylo- was then chromatographed on polyamide and next pyranoside from Vaccinium macrocarpon (9). on sephadex LH-20, and flavonols were isolated as Finally, the current study was the first time compounds VIII and IX. isolation of isorhamnetin 3-O-α-arabinofuranoside Upon acid hydrolysis the compounds released and 3-O-β-xylopyranoside from the genus Prunus, as isorhamnetin and different sugars identified as well the first presentation of its NMR spectral data. arabinose and xylose, respectively. The UV spectra analysis indicated the site of glycosylation at the 3- Acknowledgments position of the aglycone in both cases (6). Because of small amounts of compounds only the 1H NMR The study is a part of the project No. 502-13- spectra were recorded. For isorhamnetin moieties 847 (198) of the Medical University of £Ûdü. the spectra showed the expected proton signals in aromatic regions and the methoxyl singlets at δ 3.86 REFERENCES and 3.82 ppm, respectively for VIII and IX (8). In the spectrum of compound VIII the 1. Golz-Berner K., Zastrow L.: PGT Int. Appl. NO anomeric proton resonance was observed as a sin- 2001026617A1, Patent, CA 134, 300647 g (2001). glet at δ 5.62 ppm, which indicated the α-configu- 2. Olszewska M.: Acta Pol. Pharm. 62, 127 ration of arabinosyl residue and suggested its (2005). occurrence in the furanose form (8, 11). The above- 3. Olszewska M., Wolbiú M.: Acta Pol. Pharm. 58, mentioned fact was evidenced by the location of the 367 (2001). H-2″-signal at δ 4.15 ppm (multiplet) and by 4. Olszewska M., Wolbiú M.: Acta Pol. Pharm. 59, unseparated signal of 5″ methylene group, recorded 133 (2002). as a multiplet at δ 3.24 ppm (11). Moreover, all the 5. Nowak S., Wolbiú M.: Acta Pol. Pharm. 59, 275 sugar region pattern was in agreement with those (2002). High-performance liquid chromatographic identification... 441

6. Mabry T.J., Markham K.R., Thomas M.B.: The 13. H¸bner G., Wray V., Nahrstedt A.: Planta Systematic Identification of Flavonoids, Sprin- Medica 65, 636 (1999). ger, Berlin-Heidelberg-New York 1970. 14. Nakanishi T., Iida N., Inatomi Y., Murata H., 7. RychliÒska I., Gudej J.: Acta Pol. Pharm. 59, 53 Inada A., Murata J., Lang F.A., Iinuma M., (2002). Tanaka T.: Phytochemistry 65, 207 (2004). 8. Markham K.R., Geiger H.: in The Flavonoids. 15. Geiger H., De Groot-Pfleiderer W.: Phytoche- Advances in research since 1986, Harborne J.B. mistry 18, 1709 (1979). (ed.), Chapman and Hall, Cambridge 1994. 16. Niemann G.J.: Z. Naturforsch. 35 C, 514 9. Vvedenskaya I.O., Rosen R.T., Guido J.E., (1980). Russell D.J., Mills K.A., Vorsa N.: J. Agric. 17. Budzianowski J., Skrzypczak L., Weso≥owska Food Chem. 52, 188 (2004). M.: Sci. Pharm. 58,15 (1990). 10. Power F.B., Moore C.W.: J. Chem. Soc. 97, 18. Ma Y.-L., Lei Z.-H., Kong Q., Huang Q.-D., 1099 (1910). Feng Y., Tai B.-S., Yahara S., Nohara T.: Stud. 11. Zapesochnaya G.G.: Khim. Prir. Soed. 15, 21 Plant Sci. 6, 302 (1999), CA 131, 16413w (1979). (1999). 12. Wen-Sheng Y., Hong L., Xin-Min Ch., Lei Y.: 19. Eskalieva B.K., Burasheva G. Sh.: Chem. Nat. Phytochemistry 31, 4385 (1992). Comp. (Khim. Prir. Soed.) 38, 102 (2002).

Received: 20.08.2005