ISSN 10681620, Russian Journal of Bioorganic Chemistry, 2011, Vol. 37, No. 7, pp. 862–865. © Pleiades Publishing, Ltd., 2011. Original Russian Text © A.A. Drenin, E.Kh. Botirov, Yu.P. Turov, 2010, published in Khimija Rastitel’nogo Syr’ja, 2010, No. 2, pp. 53–56.
LOWMOLECULARWEIGHT COMPOUNDS
A New Isoflavone Glycoside from Trifolium pratense L. A. A. Drenin1, E. Kh. Botirov, and Yu. P. Turov Surgut State University, ul. Energetikov 22, Surgut, 628412 Russia Received March 5, 2009
Abstract—The isoflavonoid composition of red clover (Trifolium pratense L.) has been studied. The following compounds have been detected: cyclopolyol (+)pinitol, not found in clover before; known isoflavones for mononetin, prunetin, genistein, and prunetin4'OβDglucopyranoside; isoflavone monogalactosides for mononetin7OβDgalactopyranoside, inermin3OβDgalactopyranoside, and genistein7OβD galactopyranoside; and a novel compound, prunetin4'αDglucopyranoside. The structures of these com pounds have been proven by UV, IR, 1H NMR, 13C NMR, mass, and circular dichroism spectra. (+)Pinitol is known to possess biological activity. Keywords: red clover (Trifolium pratense L.), isoflavonoids, prunetin4'αDglucopyranoside, (+)pinitol DOI: 10.1134/S1068162011070041 1
INTRODUCTION
Red clover (Trifolium pratense L.) occurs in mead ows, forest margins, and forest openings almost every where in Russia [1]. It is commonly used in folk medi cine. It has expectorative, softening, diuretic, sudorific, antiinflammatory, and antiseptic effects. Tincture of its flowering tops and leaves is taken for anemia, catarrhal diseases, cough, and coldrelated and rheumatic pains. In dermatology, clover herb is recommended for per os administration in the form of tincture for treating aller gic diseases, vasculites, and vitiligo and for external use in the form of lotions and heating pads for treating furunculosis and eczema [2, 3]. Dietary supplements with a wide range of thera peutic effects have been developed on the basis of red clover extracts [3]. In particular, Ateroklefit (Evalar, Russia), Kardiin (Nutrifarm, Russia), and their US equivalent Red Clover Plus (NutriCare) are used for prevention and adjunctive therapy of cardiovascular diseases. Dietary supplement Klever is used as an immunomodulating, antioxidant, antianemic, woundhealing, expectorative, antiallergic, bacteri cidal, vasodilatory, spasmolytic, diuretic, sudorific, and sedative medicine. Numerous studies of clover have revealed isofla vonoids biochanin A, formononetin, daidzein, prune tin, genistein, pratensein, pseudobaptigenin, caly cosin; glycosides of ononin, sissotrin, trifolin, isotrifo lin; volatile and fatty oils; vitamin C; carotene; etc. [1–3]. We investigated the composition of isoflavonoids from red clover growing in the Khanty–Mansi Auton omous Okrug, Russia. No studies of phenolic com 1 Corresponding author: email:
[email protected].
pounds in this red clover population had been carried out before. MATERIALS AND METHODS Aboveground organs of red clover plants were sampled in Surgut raion near Saigatina Village in July, 2006. Belowground organs were sampled at the same site in late September. Weights of airdried pulverized belowground (0.5 kg) and aboveground (1.0 kg) parts of clover were extracted with five portions of 85% ethyl alcohol at room temperature. The extracts were condensed by evaporation in vacuo and treated consecutively with hexane, chloroform, ethyl acetate, and nbutanol. The fractions were resolved by chromatography in columns with silica gel. Ethanol–chloroform gradient was used as the mobile phase. Flavonoid I was isolated from the chloroform frac tion of belowground parts. The yield was 88.3 mg. Compounds II (250 mg) and III (500 mg) were iso lated from the ethyl acetate fraction. Compound IV (70 mg) was isolated from the chloroform fraction of the aboveground sample. The ethyl acetate fraction contained isoflavone V (45 mg) and isoflavonoid gly cosides VI (350 mg), VII (25 mg), and VIII (35 mg). Compound IX (600 mg) was isolated from the butanol fraction of the aboveground sample. Thinlayer chromatography (TLC) was carried out on Sorbifil PTLCPAUF plates with fixed silica gel. The following mobile phases were used for TLC: (1) chloroform–ethyl acetate 9 : 1, (2) chloroform– ethanol 9 : 1, (3) chloroform–ethyl acetate–ethanol 6 : 1 : 3, (4) ethyl acetate–ethanol–water 6 : 3 : 1, (5) nbutanol–ethanol–water 5 : 3 : 2. Flavonoid spots were examined under UV illumination in a
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UFS254/365 chromatogram illuminator at 254 and 365 nm or developed with 3% ethanol solution of van illin with concentrated hydrochloric acid at a 4 : 1 ratio. Monosaccharides were visualized by sprinkling chromatograms with sulfuric acid followed by heating at 100–110°C for 20 min. Melting temperatures of individual compounds were determined by the capillary method in sulfuric acid. Ultraviolet spectra were recorded with an SF2000 spectrometer. Infrared spectra were recorded with a PerkinElmer Spectrum 100 Fourier transform infra red spectrometer with an ITRMiracle sampling accessory by the ATR method at the resolution 4.00 cm–1 with apodization = strong. Circular dichro ism spectra were recorded in ethanol with a Jasco J20 spectropolarimeter. Mass spectra of acylated derivatives of flavonoids and their glycosides were recorded with a PerkinElmer Clarus 500 ms GC/mass spectrometer. Conditions: ionization energy 70 eV, sweep range 40–400 Da, sweep rate 3 scans/s. Chromatograph: GLC, SE54 column (L = 30 m, d = 0.25 cm), helium as the carrier gas. Heat control: isothermal heating at 80°C then heating to 270°C at the rate 10°C/min. Evaporator temperature 270°C. Gas flow rate 0.5 ml/min. 1H NMR and 13C NMR spectra were recorded in deuterated dimethyl sulfoxide (DMSOd6) and deu teropyridine (Pydd5) with a Bruker AVACE AV300 NMR spectrometer. Working frequencies 300 MHz for 1H NMR and 75 MHz for 13C NMR. Trimethylsilyl ethers of monosaccharides obtained by glycoside hydrolysis were identified in a Kristall gas–liquid chromatograph with an SE54 column (L = 15 m, d = 0.32 cm), helium as the carrier gas, and flame ionization detection (T = 280°C), Heat control: isothermal heating at 70°C, then heating to 200°C at the rate 4°/min. Evaporator temperature 250°C. Gas flow rate 3 ml/min. RESULTS AND DISCUSSION Four known isoflavonoids were isolated from the aboveground and belowground organs of red clover: formononetin (I), prunetin (IV), genistein (V), and prunetin4'OβDglucopyranoside (VI). Three new isoflavonoid galactosides were identified: formonone tin7OβDgalactopyranoside (II), inermin3O βDgalactopyranoside (III), and genistein7Oβ Dgalactopyranoside (VIII). The determination of their structures is described in [4, 5]. The UV spectrum of compound VII with the for mula C22H22O10 and melting temperature 259–260°C is characteristic of isoflavone derivatives. It has peaks ethanol at λ max 263, 292, and 335 nm. Spectra of samples supplemented with sodium acetate did not show any bathochromic shift of band II. This observation points RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
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Table 1. 1H and 13C NMR spectrometry of prunetin4'O αDglucopyranoside δC (ppm)
C atom Aglycone 2 3 4 5 6 7 8 9 10 1' 2'/6' 3'/5' 4' OCH3 OH (C–5) Glucose 1" 2" 3" 4" 5" 6"
153.6 121.4 180.3 157.7 98.6 164.9 92.6 157.5 105.3 122.5 130.1 116.1 162.1 56.2
100.3 73.1 76.5 69.6 77.0 60.2
δH (J, Hz) 8.46 s
6.41 bs 6.66 bs
7.50 d (8.1) 7.09 d (8.1) 3.86 s 12.92 s 5.36 d (4.5) 4.58–5.18 4.58–5.18 4.58–5.18 4.58–5.18 4.58–5.18
to the absence of a free phenolic hydroxyl at C7 [6]. A 10nm bathochromic shift of band II was observed in spectra recorded with aluminum chloride, which points to a phenolic hydroxyl at C5 [6, 7]. The infrared spectrum of VII has bands corre sponding to vibrations of hydroxy groups (3317 cm–1), the keto group of γpyrone (1645 cm–1), aromatic C–C bonds (1600 cm–1), and C–O bonds of glycosides (1015 cm–1). An aglycone and a monosaccharide were obtained by acid hydrolysis. The aglycone was identified as pru netin by comparison with a known reference sample by TLC, GLC of acetylated derivatives, and the mix ing test. The monosaccharide was identified as Dglu cose by GLC of TMS ethers with the presence of a ref erence sample. Acetylation of the isoflavone with acetic anhydride in pyridine yielded its pentaacetyled derivative VIIa. Its mass spectrum contains peaks of ions with m/z val ues 368 (M+, 2%), 284 (100), 255 (10), 166 (50), 138 (40), 118 (30), and others. The peaks of ions with m/z 166 and 118, formed by retrodiene cleavage, are characteristic of isoflavone derivatives possessing a hydroxyl in ring B and hydroxy and methoxy groups in ring A. The 13C NMR spectrum of glycoside VII in DMSOd6 shows bands of carbon atoms of aglycone and the sugar moiety (Table 1). The signal of the ano Vol. 37
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Table 2. 1H and 13C NMR spectrometry of compound IX C atom
δC (ppm)
1 2 3 4 5 6 OCH3
70.9 72.4 70.1 72.6 71.9 83.8 59.7
δH (J, Hz) 4.47 d (6.6) 4.52 d (4.8) 4.72 d (2.1) 4.63 d (2.1) 4.35 d (5.7) 3.00 t (9.3, 9.3) 3.62 s
comparison to that of cyclohexane is related to the presence of a methoxy group at the ipso carbon atom, and the shift of signals from other five protons of the ring, to five alcohol hydroxyl groups [10]. Compound IX was identified by comprehensive analysis of the 1H NMR spectrum, chemical shifts of protonsignals, and spin–spin coupling constants as (1R,2S,3s,4S,5S,6r)6methoxycyclohexane1,2,3,4,5 pentaol (pinitol). RO
OCH3 OR
RO
OR
H3
OR H4
H5 H1 OR OCH3
1H
meric proton of glucose in the NMR spectrum forms a doublet at 5.36 ppm with J = 4.5 Hz. This value of the spin–spin coupling constant of the ano meric proton and the paramagnetic shift of the sig nals of sugar protons by 1.5 ppm in comparison to the β structure argue for the αpyranose structure of glu cose [8, 9]. The chemical shifts of signals from other sugar protons are within 4.58–5.18 ppm. Thus, compound VII is prunetin4'OαDglu copyranoside. CH2OR O OR
2 2' 1'
3'
OR
OR O
4'
B
3
O C 4
6'
O
8
OR OR H2 H6
IX: R = H IXa: R = COCH3
Large amounts of pinitol are often found in legumes, but it is the first detection of this compound in red clover. Pinitol has been registered in United States as an antihyperglycemic and antidiabetic medicine [11]. In July, when the samples were taken, the pinitol content in the aboveground organs of red clover was 0.9%.
OCH3
9
A 10
OR
OR
7 6
5
OR
5'
VII: R = H VIIa: R = COCH3
Prunetin4'OαDglucopyranoside is a novel compound that has heretofore not been described in the literature. Compound IX C7H14O6 ([α]D (ethanol) + 65.63°) shows no light absorption in the UV and visible ranges. Its IR spectrum has vibration bands of hydroxy groups (3250–3600 cm–1), aliphatic C–C bonds (2300– 2950 cm–1), and C–O bonds (1175 cm–1). Acetylation of compound IX with acetyl chloride yielded its pentaacetylated derivative IXa. Its mass spectrum contains peaks with m/z 404 (M+, 3%), 345 (2), 285 (2), 243 (8), 182 (35), 150 (50), 109 (15), and 87 (45). This pattern corresponds to the structure of methoxyinositol. The 13C NMR spectrum in DMSOd6 has signals from six carbon atoms of the cyclohexane ring linked to an oxygencontaining group (70.1–83.8 ppm) and a signal from the carbon of the methoxy group with a chemical shift of 59.7 ppm. The 1H NMR spectrum shows signals from five oxygen atoms of the >CH–OR groups of the cyclo hexane ring at 4.34–4.73 ppm and signals of methoxy group protons at 3.62 ppm. The signal of the C6 pro ton is a triplet in the stronger field range at 3.00 ppm (Table 2) The paramagnetic shift of the H6 signal in
CONCLUSIONS (1) Red clover roots contain two isoflavonoid monogalactosides: formononetin7OβDgalacto pyranoside and inermin3OβDgalactopyranoside. They also contain formononetin (7hydroxy4'meth oxyisoflavone). (2) A new glycoside, prunetin4'OαDglucopy ranoside, was found in the aboveground organs of red clover. These organs also contain genistein7OβD galactopyranoside, formononetin, prunetin, genistein, and prunetin4'OβDglucopyranoside. (3) For the first time, the cyclopolyol (+) pinitol was isolated from the aboveground organs of red clo ver. Thus, red clover is a new promising source of this biologically active compound. (4) The structures of the compounds were estab lished from data on the products of their reactions (acetylation and hydrolysis) and by spectroscopic methods: UV, IR, 1H and 13C NMR, mass spectrome try, and circular dichroism spectrometry. REFERENCES 1. Rastitel’nye resursy SSSR: Tsvetkovye rasteniya, ikh khimicheskii sostav, ispol’zovanie. Semeistva Hydrangeaceae–Haloragaceae (Plant Resources of the USSR: Flowering Plants, Their Chemical Composition and Use. Families Hydrangeaceae–Haloragaceae), Sokolov, V.P., Ed., Leningrad, 1987, pp. 137–142. 2. K’osev, P.A., Polnyi spravochnik lekarstvennykh rastenii (Complete Handbook of Medicinal Plants), Moscow, 2005.
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A NEW ISOFLAVONE GLYCOSIDE FROM Trifolium pratense L. 3. Lekarstvennoe rastitel’noe syr’e. Farmakognoziya (Medicinal Plant Materials. Pharmacognosy), Yakov lev, G.P. and Blinova, K.F., Eds., St. Petersburg, 2004, p. 728. 4. Drenin, A.A., Botirov, E.Kh., and Petrulyak, E.V., Khim. Prir. Soedin., 2008, vol. 44, no. 1, pp. 21–23. 5. Drenin, A.A., Botirov, E.Kh., and Petrulyak, E.V., Khim. Prir. Soedin., 2008, vol. 44, no. 2, pp. 141–143. 6. Markham, K.R., Techniques of Flavonoid Identification, London, 1982, p. 113. 7. Litvinenko, V.I. and Maksyutina, N.P., Khim. Prir. Soe din., 1965, vol. 4, no. 3, pp. 420–425.
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8. Mabry, T.J., Markham, K.R., and Thomas, M.B., The Systematic Identification of Flavonoids, New York, 1970. 9. Stepanenko, B.N., Khimiya i biokhimiya uglevodov (monosakharidy) (Chemistry and Biochemistry of Car bohydrates (Monosaccharides)), Moscow, 1977. 10. Pliev, T.N., Molekulyarnaya spektroskopiya (Molecular Spectroscopy), in 5 vols., Vladikavkaz, 2004, vol. 3, pp. 573–600. 11. Misra, L.N. and Siddiqi, S.A., Curr. Sci., 2008, vol. 87, no. 11, p. 1507.
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