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Flavonoids from Astragalus hamosus
To cite this Article: Krasteva, Ilina, Platikanov, Stefan, Nikolov, Stefan and Kaloga, Maki , 'Flavonoids from Astragalus hamosus', Natural Product Research, 21:5, 392 395 To link to this article: DOI: 10.1080/14786410701236871 URL: http://dx.doi.org/10.1080/14786410701236871
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Natural Product Research, Vol. 21, No. 5, 1 May 2007, 392–395
Flavonoids from Astragalus hamosus ILINA KRASTEVA*y, STEFAN PLATIKANOVz, STEFAN NIKOLOVy and MAKI KALOGAx yFaculty of Pharmacy, Department of Pharmacognosy, 2 Dunav St., 1000 Sofia, Bulgaria zInstitute of Botany, Bulgarian Academy of Sciences, 23 Akad. G. Bonchev St., 1113 Sofia, Bulgaria xInstitut fu¨r Pharmazie, Pharmazeutische Biologie, Ko¨nigin Luise Strasse 2þ4, D-14195, Berlin, Germany (Received 6 March 2006; in final form 5 May 2006) A new flavonol glycoside 7-O-methyl-kaempferol 40 - -D-galactopyranoside (rhamnocitrin 40 - -D-galactopyranoside) (1) was isolated from the aerial parts of Astragalus hamosus. The known flavonols hyperoside (2), isoquercitrin (3) and astragalin (4) were also identified. Structures of the compounds were elucidated by chemical and spectral methods. Keywords: Astragalus hamosus; Fabaceae; Flavonoids
1. Introduction Astragalus hamosus L. (Fabaceae) is used in herbal medicine as emollient, demulcent, aphrodisiac, diuretic, laxative, and good for inflammation, ulcers, and leucoderma. It is useful in treating irritation of the mucous membranes, nervous affections, and catarrh [1]. Previous phytochemical study afforded the isolation of rhamnocitrin-3-O-glucoside from the aerial parts of A. hamosus [2]. Rutin, astragalin, and isoquercitrin have been obtained in callus and suspension cultures of the plant by high-performance liquid chromatography (HPLC) method [3]. This article describes the isolation and structure elucidation of new flavonol glycoside 7-O-methyl-kaempferol, 40 - -D-galactopyranoside (rhamnocitrin 40 - D-galactopyranoside), and known flavonoids hyperoside, isoquercitrin, and astragalin from the introduced samples from A. hamosus.
*Corresponding author. Tel.: þ7359 2 9236552. Fax: þ359 2 9879874. Email:
[email protected] Natural Product Research ISSN 1478-6419 print/ISSN 1029-2349 online ß 2007 Taylor & Francis http://www.tandf.co.uk/journals DOI: 10.1080/14786410701236871
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2. Results and discussion The ethyl acetate extract of A. hamosus herbs was subjected to separation and purification on Sephadex LH-20 column chromatography and preparative thin layer chromatography (TLC), and led to the isolation of new flavonol glycoside (1) together with three known flavonoids hyperoside (quercetin-3- -D-galactopyranoside) (2), isoquercitrin (quercetin-3- -D-glucopyranoside) (3), and astragalin (kaempferol-3- D-glucopyranoside) (4). Structural assessment of 1 was effected by analysis of MS and 1H and 13C NMR spectroscopic data. Allocation of signals was facilitated by COSY, NOE, and HETCOR experiments. UV spectral data of compound 1 with diagnostic shift reagents suggested the likely presence of 7, 40 -disubstituted flavonol glycoside with free hydroxyl groups at 3 and 5 positions [4,5]. Acid hydrolysis of 1 gave rhamnocitrin and galactose. The negative ion FAB-MS spectrum showed the quasi molecular ion [M H] at m/z 461, corresponding to the molecular formula C22H22O11, and one important ion peak appeared at m/z 299 [(M H) 162] indicating the loss of the galactosyl unit. The positive ion FAB-MS of 1 exhibited peaks at m/z 463 [M þ H]þ and sodium adduct at m/z 485 [M þ Na]þ. The EIMS of 1 showed a fragment at m/z 300, generated from the [M]þ and significant peaks at m/z 167 and m/z 121 [RDA product]þ. These fragments were recorded in the HR-EIMS at m/z 300.06325 (Calcd 300.063390 for C16H12O6), m/z 167.03428 (Calcd 167.03444 for C8H7O4) and m/z 121.02881 (Calcd 121.02896 for C7H5O2). Fragment m/z 167 for C8H7O4 in MS spectrum indicated 5-hydroxy, 7-methoxy substitutions in ring A [6]. NMR experiments further confirmed the previous conclusions. The 13C NMR spectrum revealed 15 carbon signals of the flavone skeleton, six carbon resonances of the hexosyl unit and an additional signal at C 56.54 indicating a methylated flavonoid [7]. The 1H NMR spectrum showed two meta-coupled signals at 6.36 and 6.71 (d, J ¼ 1.80 Hz, each) demonstrating the presence of a 1,2,3,5-tetrasubstituted benzene ring. Moreover, two-proton doublets at 6.97 and 8.21 (J ¼ 9.00 Hz) corresponded to two sets of ortho aromatic protons. 4′
O
H 5″
1″
6″
O O
H3CO
HO
4″ 2″
7
OH
OH OH
3
OH
5
OH
O
1
From the NOE experiment the three-proton singlet at 3.94 correlated to both the H-6 signal at 6.36 and the proton H-8 at 6.71 indicating the linkage of the methoxy group to C-7 of the aglycone. The coupling constant of the anomeric proton ( 5.36) was 7.60 Hz, indicating that the galactopyranosyl unit had the R-configuration at the anomeric center. The attachment of the galactopyranosyl moiety to C-40 of the aglycone was evident from correlations between the anomeric proton H-100 and
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H-30 and H-50 in the NOE spectrum [8]. On the basis of all these data, compound 1 was identified as 7-O-methyl-kaempferol 40 - -D-galactopyranoside or rhamocitrin 40 - -D-galactopyranoside. The known compounds hyperoside (2), isoquercitrin (3), and astragalin (4) were identified by direct comparison with authentic samples, acid hydrolysis, and UV spectra using diagnostic shift reagents [4,5].
3. Experimental section 3.1. General experimental procedures Melting point is uncorrected. UV spectra were recorded on a ‘‘WPA-LIGHTWAVE’’ spectrometer with diagnostic shift reagents. 1H NMR (400 MHz) and 13C NMR (100.6 MHz) spectra were recorded on Bruker DPX-400 and Bruker AMX-400 instruments using TMS as internal standard. EIMS, HR-EIMS, and FAB-MS spectra were carried out Varian MAT CH7A, Finnigan MAT 711 and Finnigan MAT CH5DF, respectively. Thin-layer chromatographic study was carried out on silica gel plates (Kieselgel G, F254, type 60, Merck, Germany), using the solvent systems, eluted with EtOAc/HCOOH/H2O (100 : 10 : 40) for glycosides, CHCl3/MeOH (9 : 1) for aglycones and Me2CO/CHCl3/MeOH/H2O (75 : 10 : 10 : 5) for sugars. The spots were visualized by spraying with NTS/PEG reagent (for flavonoids) and aniline/hydrogen phthalate reagent, followed heating at 110 C (for sugars). Column chromatography (CC) was carried out with Sephadex LH-20 (Pharmacia, Sweden). Preparative TLC was carried out on silica gel plates (Kieselgel 60, 0.5 mm thick, Merck, Germany), eluted with S1 (EtOAc/MeCOEt/HCOOH/H2O, 5 : 3 : 1 : 1) and S2 (EtOAc/AcOH/H2O, 10 : 2 : 3).
3.2. Plant material Aerial parts from introduced seeds samples of A. hamosus were collected in October 2001 at the Experimental field, Institute of Botany, BAS Sofia. The plant was identified by Dr D. Pavlova from the Department of Botany, Faculty of Biology, Sofia University, where voucher specimen has been deposited (SO 102680).
3.3. Extraction and isolation Air-dried powdered aerial parts of the plant (500 g) were defatted with n-hexane and extracted with MeOH/H2O (9 : 1) and (1 : 1). The extracts were filtrated, concentrated under reduced pressure, and successively partitioned with CHCl3, EtOAc, and n-BuOH. The ethyl acetate extract (4.9 g) was chromatographed on a Sephadex LH-20 column (using methanol as solvent) to give three main fractions A–C. Pure compound 1 (30 mg) was obtained from fraction B by crystallization. Fraction A was purified by chromatography on Sephadex LH-20 column, followed by preparative TLC with solvent system S1 to afford 4 (10 mg). By chromatography on Sephadex LH20 followed by preparative TLC with solvent system S2 9 mg of 2 and 8 mg of 3 were obtained from the fraction C.
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3.3.1. Rhamnocitrin 40 -b-D-galactopyranoside (1). Pale yellow powder; m.p. 205–206 C; UV (MeOH) max: 270, 301 sh, 352; (þNaOMe) 270, 352; (þNaOAc) 270, 360; (þAlCl3) 275, 356, 398; (þAlCl3 þ HCl) 275, 356, 398; FAB-MS (negative): m/z 461 [M H], m/z 299 [(M H) 162]; FAB-MS (positive): m/z 485 [M þ Na]þ, m/z 463 [M þ H]þ; HR-EIMS: m/z 300.06325 (Calcd 300.063390 for C16H12O6), m/z 167.03428 (Calcd 167.03444 for C8H7O4) and m/z 121.02881 (Calcd 121.02896 for C7H5O2); 1H NMR (400 MHz, acetone-d6): 12.44 (1H, s, OH-5), 9.11 (1H, br s, OH-3), 8.21 (2H, d, J ¼ 9.00 Hz, H-20 , H-60 ), 6.97 (2H, d, J ¼ 9.00 Hz, H-30 , H-50 ), 6.71 (1H, d, J ¼ 1.80 Hz, H-8), 6.36 (1H, d, J ¼ 1.80 Hz, H-6), 5.36 (1H, d, J ¼ 7.60 Hz, H-100 ), 3.94 (3H, s, OCH3-7), 3.92 (H-600 b), 3.89 (br s, H-400 ), 3.79 (1H, dd, J ¼ 7.60, 8.30 Hz, H-200 ), 3.73 (H-600 a), 3.63 (H-300 ), 3.54 (H-500 ); 13C NMR (100 MHz, acetoned6): 158.42 (C-2), 135.70 (C-3), 179.41 (C-4), 162.67 (C-5), 98.83 (C-6), 166.89 (C-7), 93.05 (C-8), 157.94 (C-9), 106.60 (C-10), 122.63 (C-10 ), 132.31 (C-20 ), 115.89 (C-30 ), 161.08 (C-40 ), 115.89 (C-50 ), 132.31 (C-60 ), 56.54 (C-7-OCH3), 105.26 (C-100 ), 72.84 (C-200 ), 74.86 (C-300 ), 69.46 (C-400 ), 76.85 (C-500 ), 62.01 (C-600 ).
3.4. Acid hydrolysis A methanolic solution of glycosides (3 mg) were refluxed with 2 N HCl (5 mL) for 1 h. The aglycones and sugars were identified by TLC with authentic samples.
Acknowledgements The authors are grateful to Mrs U. Ostwald, Institute of Organic Chemistry, FU-Berlin, for providing the MS spectra.
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