anosylisorhamnetin, 3-O-glucopyranosylisorhamnetin, 7-O-galactopyranosylluteolin ... luteolin (III) [2, 6], quercitrin (IV) [2, 7], isoquercitrin. (V) [2, 6, 7], and .... were visualized on paper by spraying with o-toluidine ... Elution with a 19 : 1 chloro-.
Russian Journal of Bioorganic Chemistry, Vol. 26, No. 11, 2000, pp. 787-789. Translatedfrom Bioorganicheskaya Khimiya, VoL 26, No. 11, 2000, pp. 877-880. Original Russian Text Copyright 0 2000 by guldashev, Batirov.
A Novel Isoflavone Glycoside from Caragana alaica M. P. Yuldashev 1 and E. Kh. Batirov Yunusov Institute of the Chemistry of Plant Substances, Academy of Sciences of Republic of Uzbekistan, ul. Akademika Abdullaeva 77, Tashkent, 700170 Republic of Uzbekistan ReceivedApril 11,2000; in finalform,June 23, 2000 Abstract--3-O-Rhamnopyranosylisorhamnetin, 3-O-glucopyranosylisorhamnetin, 7-O-galactopyranosylluteolin, quercitrin, isoquercitrin,wistin, and a novel isoflavonoid,3'-hydroxy-6,4'-dimethoxy-7-O-13-D-glucopyranosylisoflavone, were isolated from the aerial parts of Caragana alaica. Of these, the previously described compounds were identifiedon the basis of their physicochemicaland spectral characteristics, whereas the spectral analysis and conversion to a known compound, cladrastin, allowed the structural elucidation of the novel isoflavoneglycoside. Key words: Caragana alaica, 3'-hydroxy-6,4'-dimethoxy-7-O-fl-D-glucopyranosylisoflavone, 3-O-rhamnopyranosylisorhamnetin, 3-O-glucopyranosylisorhamnetin, 7-O-galactopyranosylluteolin INTRODUCTION
RESULTS AND DISCUSSION The C. alaica aerial parts were gathered at the beginning of fructification period in the neighborhood of Irkeshtam settlement. Column chromatography on silica gel of the ethanol extract of the aerial parts of the plant gave the previously reported flavonoids--3-Orhamnopyranosylisorhamnetin (I) [2, 3], 3-O-glucopyranosylisorhamnetin (II) [3-5], 7-O-galactopyranosylluteolin (III) [2, 6], quercitrin (IV) [2, 7], isoquercitrin (V) [2, 6, 7], and wistin (VI) [2, 7, 8J--and also a new isoflavone glycoside (VII).
Alaic pea shrub (Glycyrrhiza glabra A. Pojark (Fabaceae family)) is a low shrub with highly ramified branches covered with yellowish-greenish rind and small single or, rarely, ternate barbs located at the leaf bases. Caragana alaica grows at the stony slopes of mountains, canyons, and river banks and benches [1]. This plant was not earlier subjected to phytochemical study. OR
"o O oH
(I) R = CH3 (IV) R = H
/
HO~,H2 O- ~
OH
~O.. (III)
OH
O
]
Ho/F-o I
-~'OH
OH
0
OH OH OR O ~ O H
/
HO~/~I
(II) R = CH3 (V) R=H
. %Ao OH
O I HO~IH2 I o~HO~
R2OI~,H2 O -
HC)~---]/
RZOb~ C
OH
,-,
,
O~R?~ ~ u . ~ l
OR2
H
3
~__ 0
~
R
(VI) RI= R2= H
(VII) R, = OH, R2 = H OCH3(IX) RI = OC(O)CH3, R2 = C(O)CH3
O
ITo whomcorrespondenceshouldbe addressed;phone:+7 (371) 137-6022 1068-1620/00/2611-0787$25.00© 2000MAIK"Nauka/ Interperiodica"
788
YULDASHEV, BATIROV
The UV spectrum of (VII) (~'max 257, 262, 287 sh, and 317 nm) is characteristic of isoflavone derivatives. The ~H NMR spectrum of (VII) contains the H2 proton signal as a singlet at 8.07 ppm, which is also characteristic of isoflavones [2]. The signals of two protons of ring A, H5 (8.29 ppm, s) and H8 (6.75 ppm, s); the signals of three protons of ring B, H5' (6.94 ppm, d, J = 8.5 Hz), H6' (7.23 ppm, dd, ij = 8.5 Hz and 2j = 2.5 Hz), and H2' (7.65 ppm, d, J = 2.5 Hz); and also the signals of the anomeric proton (5.70 ppm, d, J = 6.5 Hz), other protons of the carbohydrate moiety (4.05-4.50 ppm, m), and the protons of two methoxy groups (3.64 ppm, 3 H, s and 3.76 ppm, 3 H, s) were also observed. The acidic hydrolysis of glycoside (VII) gave aglycone (VIII) (7,3'-dihydroxy-6,4'-dimethoxyisoflavone, C17H1406, [M] + --- 314) [2, 8] and glucose. As the previously reported glycosides (II), (V), and (VI) isolated from this plant contained D-glucopyranose, we also referred the glucose obtained by the hydrolysis of (VII) to the D-series. Acetylation of glucoside (VII) with acetic anhydride in pyridine gave pentaacetyl derivative (IX) characterized with the intensive peaks of the fragmentary ions of a tetraacetylhexose residue with m[z of 331, 329, 271, and 169 along with the molecular ion with m/z = 686 [9]. Thus, compound (VII) contains one carbohydrate residue. The mass spectrum of aglycone (VIII) contains the peaks of ions (a + 1)÷ (m/z = 167) and b (mlz = 148), which result from the retrodienoic cleavage of the molecular ion with mlz = 314 (see Scheme 1). This indicates the presence of one methoxyl and one hydroxyl group in each aromatic ring (A and B). A comparative analysis of the UV spectra of compounds (VII) and (VIII) in the presence of sodium ace-
tate shows a 8-nm bathochromic shift of the short-wave band. This implies the presence of a free 7-OH group in the molecule of aglycone (VIII) [ 11 ]. To conclusively confirm the position of the glycoside residue in compound (VIII), this compound was methylated with dimethylsulfate in the presence of potassium carbonate. The acidic hydrolysis of the resulting methylation product yielded compound (X), which was identical to cladrastin (7-hydroxy-6,3',4'-trimethoxyisoflavone) in the physicochemical properties [ 13]. Thus, in glycoside (VII) the carbohydrate residue is attached to the C7-hydroxyl group and one of the methoxyl groups is located at C6 of ring A. For biogenetic reasons (the presence of a methoxyl group in the C4' position of wistin), the second methoxyl group of glycoside (VII) is likely to be located at C4'. This is corroborated by a comparable analysis of the ~H NMR spectra of glycoside (VII) and pratensein (5,7,3'-trihydroxy-4'-methoxyisoflavone) [12] registered in pyridine-ds. The similarity of the chemical shifts of the ring B protons of compound (VII) (7.65 ppm, H2'; 6.94 ppm, H5'; and 7.23 ppm, H6') and pratensein (7.63 ppm, H2'; 6.93 ppm, H5', and 7.20 ppm, H6') indicates an identical location of the hydroxyl groups in ring B of both flavonoids. The coupling constant value of the anomeric proton signal in the 1H NMR spectrum of glycoside (VII) (6.5Hz) points to the 4Crconformation of the monosaccharide unit and, hence, the 13-configuration of the glycoside center of D-glucose [ 14]. Thus, glycoside (VII) is a novel compound, 3'hydroxy-6,4'-dimethoxy-7-O-13-D-glucopyranosylisoflavone. 3-O-Rhamnopyranosylisorhamnetin (I), 3-0glucopyranosylisorhamnetin (II), 7-O-galactopyranosylluteolin (HI), quercitrin (IV), isoquercitrin (V), and wistin (VI) have for the first time been isolated from C. alaica.
EXPERIMENTAL OR IR spectra were registered on a Perkin-Elmer SysH O ~ O tem 2000 FT-IR spectrophotometer in KBr pellets. UV (VIII) R = H spectra were recorded in ethanol on a Perkin-Elmer OCH3 (X) R = CI-I3 Lambda 16 spectrophotometer. Mass spectra were CH30- ~ xl¢ taken on a MKh- 1310 instrument at the electron impact 0 ionization (50 eV, El-MS). IH NMR spectra were regb~ istered on a BS-567 (Tesla) spectrometer in pyridine-d5 at a working frequency of 100 MHz. Melting points were taken on a Boetius instrument. HO ~ . ~ O H + OH Column chromatography was performed on KSK 100/160 Bm silica gel. TLC was carried out on Silufol HC =C ~ ~ - O C H 3 UV-254 plates. Paper chromatography was performed on CH30- ~ / "C~o Filtrak no. 11 paper, n-Butanol-pyridine-water 6 : 4 : 3 was used as a developing system for TLC and paper b, mlz 148 (a + 1)÷, mlz 167 chromatography. The spots of flavonoids were visualized by treating the plates with ammonia gas. Sugars were visualized on paper by spraying with o-toluidine Scheme1. salicylate followed by heating to 90-100°C. ii
t/
RUSSIAN JOURNAL OF BIOORGANICCHEMISTRY
Vol. 26
No. 11
2000
A NOVEL ISOFLAVONE GLYCOSIDE FROM CARAGANA ALAICA
Extraction and separation of flavonoids. The airdried disintegrated aerial parts of C. alaica (1.2 kg) gathered at the beginning of fructification period (July, 1992) in the neighborhood of Irkeshtam settlement (Alaic valley, Republic of Kyrgyzstan) were extracted with ethanol (7 x 4 l) at room temperature. The combined ethanol extract was concentrated in vacuum to 0.6 1 and diluted with water to 1.2 I. The aqueous-ethanol extract was subsequently shaken with petroleum ether (5 x 500 ml), chloroform (5 x 500 ml), ethyl acetate (9 x 500 ml), and butanol (8 x 500 ml). The extracts were evaporated to give the following fractions: 12.0 g from petroleum ether, 10.0 g from chloroform, 17.0 g from ethyl acetate, and 23.0 g from butanol. The ethyl acetate extract (17.0 g) was chromatographed on a Silica gel (340 g) column using a step gradiem (0 to 20%) of methanol in chloroform; 250-ml fractions were collected. Elution with a 19 : 1 chloroform-methanol system gave 0.48 g of 3-O-rhanmopyranosylisorhamnetin (I), 0.52 g of 3-O-glucopyranosylisorhamnetin (ID, and 0.32 g of 7-O-galactopyranosylluteolin (Ill). Elution with a 9 : 1 chloroform-methanol system afforded 0.34 g of quercitrin (IV) and 0.65 g of isoquercitrin (V). Elution with a 4 : 1 chloroformmethanol system yielded 0.38 g of wistin (VI) and 0.45 g of isoflavone glycoside (VII). 3'-Hydroxy-6,4'-dimethoxy-7-O-13-glucopyranosylisoflavone (VII), mp 178-180°C; UV, ~max, nm: 257, 262, 287 sh., and 317; for 1H NMR, see the Results and Discussion section. Acidic hydrolysis of glycoside (VII). A solution of glycoside (VII) (20 mg) in 5% aqueous methanol solution of HCI (20 ml) was refluxed for 4 h. The precipitated aglycone was filtered off and crystallized from methanol to yield 7 mg of 7,3'-dihydroxy-6,4'dimethoxyisoflavone (VIII), mp 233-235°C; El-MS, m]z, (I, %): 314 (100) [M]+, 299 (24) [M-CH3] +, 271 (16), 243 (11), 228 (4), 205 (10), 167 (11), 151 (5.5), 148 (5), 133 (12), 105 (23), 91, 71, and 69. Paper chromatography of the hydrolyzate showed the presence of glucose. Pentaacetate (IX). A solution of glycoside (VII) (18 mg) in a mixture of pyridine (1 ml) and acetic anhydride (3 ml) was kept for 3 h at room temperature. The standard treatment yielded 12 mg of pentaacetate (IX); EI-MS, m/z, 686 [M] +, 331,329, 271, and 169. Cladrastin from glycoside (VII). Dimethyl sulfate (0.4 ml) and anhydrous K2CO 3 (50 mg) were added to a solution of glycoside (VII) (40 mg) in anhydrous acetone (5 ml), and the reaction mixture was refluxed for
789
6 h. The precipitate was filtered off, the filtrate was evaporated, and the residue was dissolved in ethanol (5 ml). Concentrated HCI (0.5 ml) was added, the reaction mixture was heated in a water bath for 3 h, and the solvent was evaporated. The residue was diluted with water (15 ml) and extracted with chloroform, the extract was dried and evaporated, and the residue was crystallized from methanol to give 14 mg of cladrastin (X), mp 205-206°C. UV, ~max, nm: 263 and 318; +CH3COONa: 270 and 330. El-MS, mlz (I, %): 328 (100) [M] +, 313 (19), 310 (7), 283 (6), 167 (13), and 148 (8) (cf[13]). REFERENCES 1. Flora SSSR (Flora of the USSR), Moscow: Izd. AN SSSR, 1945, vol. 11, p. 349. 2. Mabry, T.I., Markham, K.R., and Thomas, M.B., The Systematic Identification of Flavonoids, Berlin: SpringerVerlag, 1970. 3. Iriste, V.A. and Blinova, K.F., Khim, Prirod. Soed., 1973, no. 3, p. 436. 4. Luk'yanchikov, M.S., Khim. Prirod. Soed., 1992, no. 1, pp. 139-140. 5. Yuldashev, M.P., Batirov, E.Kh., and Malikov, V.M., Khim. Prirod. Soed., 1987, no. 3, pp. 452-453. 6. Abdullaev, Sh.V., Sattikulov, A., Batirov, E.Kh., Kurbatov, Yu.V., and Malikov, V.M., Khim. Prirod. Soed., 1983, no. 1, p. 104. 7. Klyshev, L.K., Bandyukova, V.A., and Alyukina, L.S., Flavonoidy rastenii (Plant Flavonoids), Alma-Ata: Nauka, 1978. 8. Wong,E., lsoflavonoids. The Flavonoids, Harborne, J.B., Ed., London: Chapman and Hall, 1975, p. 743. 9. Kochetkov, N.K. and Chizhov, O.S., Mass-spektrometriya uglevodov (Mass Spectrometry of Carbohydrates), Moscow: Mir, 1975, p. 409. 10. Biochemical Applications of Mass Spectrometry, New York, 1980, vol. 1, p. 1859. 11. Markham, K.R., Techniques of Flavonoid Identification, London: Academic, 1982, p. 113. 12. Yusupova, S.S., Batirov, E.Kh., Kiyamitdinova, E, and Malikov, V.M., Khim. Prirod. Soed., 1986, no. 5, pp. 639-640. 13. Shamma, M. and Stiver, L.D., Tetrahedron, 1969, vol. 25, p. 3887. 14. Altona, C. and Haasnoot, C.A.G., Org. Magn. Reson., 1980, vol. 13, p. 417.
RUSSIAN JOURNAL OF BIOORGANICCHEMISTRY Vol. 26
No. 11
2000
