Binding of Flavonoids from Sophora flavescens to the Rat Uterine Estrogen Receptor

• Abstract
• Material and Methods
• Acknowledgements
• References

Abstract

Prenylflavonoids and lavandulylflavonoids were isolated from the roots of Sophora flavescens Aiton (Fabaceae). The ability of 8-prenylkaempferol (1), kushenol X (2), norkurarinone (3), leachianone A (4), kushenol C (5), maackiain (6) and a root-extract of S. flavescens to displace 17β-estradiol (E2) from rat uterine estrogen receptor (ER) was determined. Relative binding affinities (RBA) of prenylated flavonoids were weak with RBA values between 0.004 and 0.072. A lavandulyl or prenyl group at the position 8 enhanced binding to rat uterine ER.

The oriental crude drug ”Ku-shen” (S. flavescens) has been used in Asian countries for centuries as an antipyretic, analgesic, anthelmintic and stomachic agent. Various constituents such as alkaloids [1], pterocarpans and flavonoids [2], [3], [4] have been described from S. flavescens. The flavonoids, which have been termed kushen flavonoids, consist of various prenyl- and lavandulylflavonoids (Fig. [1]). They inhibit enzymes such as phospholipase Cγ1 [5], diacylglycerol acyltransferase [6], testosterone 5α-reductase [3] and tyrosinase [4]. In addition, these flavonoids exhibit cytotoxic activities (apoptosis) in several human tumour cell lines [7], [8]. We recently observed that root extracts of S. flavescens possess potent estrogenic activity in different model systems. In this communication, we report the estrogen-binding properties of isolated kushen flavonoids.

The ethyl acetate extract from S. flavescens roots displaces 17β-estradiol (E2) from estrogen receptors (ER) (Fig. [2]) with a relative binding affinity (RBA) of 0.056 as compared to 17β-estradiol (RBA = 100). For comparison, we have also analysed extracts from liquorice (Glycyrrhiza glabra) and hop (Humulus lupulus) that have well-known phytoestrogenic properties [9]. An ethyl acetate extract from the roots of G. glabra and an ethanol extract from the cones of H. lupulus exhibit an RBA of 0.08 and 0.046, respectively for rat ER. The binding affinity of S. flavescens extract is in the same range, strongly indicating that S. flavescens has phytoestrogenic properties.

8-Prenylkaempferol (1), kushenol X (2), norkurarinone (3), leachianone A (4), kushenol C (5) and maackiain (6) which were isolated from the S. flavescens extract (Fig. [1]), also bind to the estrogen receptor (ER) indicating that they may constitute part of the active principle. Especially 1 and 2 exhibit substantial binding affinities (Table [1]) while 6 was inactive. These activities, however, are lower than that of phytoestrogen 8-prenylnaringenin (Table [1]) used as positive control. Flavonoids 1 to 5 are characterised by 8-lavandulyl/-prenyl and 5-hydroxy groups. They were found to displace E2 from rat ER. Compound 6 is a pterocarpan with a 3-hydroxy group, which displayed no affinity for ER.

The presence of a 3-hydroxy group or other substituents at position 6 in kushenols reduced the inhibitory activity towards tyrosinase [4]. In contrast, the C-3 hydroxy flavonoids tested (except for kushenol) showed increased binding affinities for the estrogen receptor.

Prenyl or lavandulyl groups at position 8 in flavonoids (e. g., 8-prenylnaringenin) promote estrogenic activity [10], [11] whereas a prenyl group at position 6 (e. g., 6-prenylnaringenin) result in almost complete loss of estrogenic activity [10], [12]. A bioassay-guided fractionation of a root-extract from S. flavescens showed that kurarinone (a 5-O-methyl-substituted norkurarinone, 3) exhibited estrogenic activity, albeit its activity was 10 000-fold weaker than that of E2 [11].

These findings show that prenyl- and lavandulylflavonoids of S. flavescens may exert estrogenic activities by binding to ER. However, their binding affinities were weak if compared to estrogen; they were 1400 to 25 000-fold lower than that of E2.

Material and Methods

Dried and chopped roots of S. flavescens were extracted with 70 % (vol) ethanol, followed by liquid-liquid partitioning with ethyl acetate-water. The resulting ethyl acetate extract was further fractionated by high-speed countercurrent chromatography (HSCCC) and medium pressure liquid chromatography (MPLC) to obtain the flavonoid constituents. The structures of the flavonoids 1 - 6 were determined with UV, IR, mass and NMR spectroscopy in comparison to published data [2], [3], [13], [14], [15], [16], [17], [18]. ¹H- and ¹³C-NMR spectra were determined using a Bruker Avance 200 spectrometer (200 MHz for ¹H and 50.3 MHz for ¹³C) in DMSO-d ₆. The chemical shifts refer to TMS (¹H-NMR) or to DMSO-d ₆ (¹³C-NMR, δ = 39.5 ppm), respectively. Mass spectrometry data were obtained using a Micromass Trio2000 (Waters-Micromass MS-Technologies, Milford, MA, USA) with a single quadrupole type detector. IR spectra were recorded on a Bruker IFS 28 FT-IR spectrometer.

8-Prenylkaempferol (1): C₂₀H₁₈O₆ (M_R : 354); UV: λ_max = 273, 326, 374 nm; IR (KBr): ν_max = 3350, 1608, 1552, 1513, 1180 cm^-1; ¹H-NMR: δ = 1.64 (s, 3H), 1.75 (s, 3H), 3.43 (m, 2H), 5.18 (t, 1H, J = 6.5 Hz), 6.30 (s, 1H), 6.94 (d, 2H, J = 8.8 Hz), 8.05 (d, 2H, J = 8.8 Hz), 10.2 (br. s, 3H), 12.42 (s, 1H); ¹³C-NMR: δ = 17.8 (C-5′′), 21.2 (C-1′′), 25.4 (C-4′′), 97.8 (C-6), 103.0 (C-10), 105.6 (C-8), 115.5 (C-3′, C-5′), 122.0 (C-1′), 122.6 (C-2′′), 129.4 (C-6′, C-2′), 130.9 (C-3′′), 135.5 (C-3), 146.7 (C-2), 153.5 (C-9), 158.3 (C-4′), 159.2 (C-5), 161.1 (C-7), 176.1 (C-4).

Kushenol X (2):C₂₅H₂₈O₇ (M_R: 440); UV: λ_max = 340, 298 nm; IR (KBr): ν_max = 3385, 1638, 1450, 1460, 1277 cm^-1; ¹H-NMR: δ = 1.46 (s, 3H), 1.53 (s, 6H), 1.92 (br. s, 2H), 2.39 (m, 3H), 4.46 (s, 1H), 4.53 (s, 1H), 4.65 (dd, 1H, J = 11.2, 5.8 Hz), 4.88 (t, 1H, J = 6.3 Hz), 5.31 (d, 1H, J = 11.3 Hz), 5.68 (d, 1H, J = 5.9 Hz), 5.96 (s, 1H), 6.25 (dd, 1H, J = 8.4, 2.2 Hz), 6.34 (d, 1H, J = 2.3 Hz), 7.15 (d, 1H, J = 8.5 Hz), 9.36 (s, 1H), 9.49 (s, 1H), 10.67 (s, 1H), 11.89 (s, 1H); ¹³C-NMR: δ = 17.6 (C-7′′), 18.4 (C-10′′), 25.5 (C-6′′), 26.3 (C-1′′), 30.9 (C-3′′), 46.3 (C-2′′), 70.4 (C-3), 77.6 (C-2), 95.2 (C-6), 100.4 (C-10), 102.3 (C-3′), 106.1 (C-5′), 106.4 (C-8), 110.8 (C-9′′), 113.8 (C-1′), 123.4 (C-4′′), 129.4 (C-6′), 130.6 (C-5′′), 147.7 (C-8′′), 157.1 (C-2′), 158.5 (C-4′), 160.4 (C-5), 160.9 (C-9), 164.8 (C-7), 198.6 (C-4).

Norkurarinone (3): C₂₅H₂₈O₆ (M_R: 424); UV: λ_max = 340, 294 nm; IR (KBr): ν_max = 3385, 1604, 1498, 1380, 1178 cm^-1; ¹H-NMR: δ = 1.45 (s, 3H), 1.54 (s, 3H), 1.58 (s, 3H), 1.93 (m, 2H), 2.45 (m, 3H), 2.62 (dd, 1H, J = 17.0, 2.9 Hz), 3.13 (dd, 1H, J = 17.2, 13.1 Hz), 4.50 (s, 1H), 4.57 (s, 1H), 4.91 (t, 1H, J = 6.6 Hz), 5.51 (dd, 1H, J = 13.1, 2.6 Hz), 5.94 (s, 1H), 6.27 (dd, 1H, J = 8.3, 2.2 Hz), 6.35 (d, 1H, J = 2.3 Hz), 7.23 (d, 1H, J = 8.4 Hz), 9.36 (s, 1H), 9.62 (s, 1H), 10.66 (s, 1H), 12.11 (s, 1H); ¹³C-NMR: δ = 17.6 (C-7′′), 18.5 (C-10′′), 25.5 (C-6′′), 26.5 (C-1′′), 30.7 (C-3′′), 41.4 (C-3), 46.3 (C-2′′), 73.8 (C-2), 95.1 (C-6), 101.6 (C-10), 102.3 (C-3′), 106.3 (C-5′), 106.4 (C-8), 110.8 (C-9′′), 115.8 (C-1′), 123.3 (C-4′′), 127.6 (C-6′), 130.7 (C-5′′), 147.8 (C-8′′), 155.5 (C-2′), 158.3 (C-4′), 160.8 (C-9), 161.1 (C-5), 164.7 (C-7), 197.2 (C-4).

Leachianone A (4): C₂₆H₃₀O₆ (M_R: 438); UV: λ_max = 340, 293 nm; IR (KBr): ν_max = 3320, 1639, 1598, 1286 cm^-1; ¹H-NMR: δ = 1.45 (s, 3H), 1.53 (s, 3H), 1.58 (s, 3H), 1.92 (m, 2H), 2.43 (m, 3H), 2.60 (dd, 1H, J = 17.2, 2.9 Hz), 3.17 (dd, 1H, J = 17.1, 13.4 Hz), 3.73 (s, 3H), 4.48 (d, 1H, 2.4 Hz), 4.57 (m, 1H), 4.89 (t, 1H, J = 6.8 Hz), 5.53 (dd, 1H, J = 13.2, 2.6 Hz), 5.94 (s, 1H), 6.42 (dd, 1H, J = 8.2, 2.2 Hz), 6.46 (d, 1H, J = 2.1 Hz), 7.32 (d, 1H, J = 8.1 Hz), 10.1 (br. s, 2H), 12.10 (s, 1H); ¹³C-NMR: δ = 17.5 (C-7′′), 18.5 (C-10′′), 25.5 (C-6′′), 26.5 (C-1′′), 30.7 (C-3′′), 41.2 (C-3), 46.3 (C-2′′), 55.3 (OMe), 73.4 (C-2), 95.2 (C-6), 98.9 (C-5′), 101.5 (C-10), 106.4 (C-8), 106.8 (C-3′), 110.8 (C-9′′), 117.0 (C-1′), 123.3 (C-4′′), 127.7 (C-2′), 130.6 (C-5′′), 147.8 (C-8′′), 157.5 (C-4′), 159.0 (C-6′), 160.6 (C-9), 161.1 (C-5), 165.0 (C-7), 197.0 (C-4).

Kushenol C (5): C₂₅H₂₆O₇ (M_R: 438): UV: λ_max = 361, 268 nm; IR (KBr): ν_max = 3378, 1650, 1611, 1241 cm^-1; ¹H-NMR: δ = 1.45 (s, 3H), 1.55 (s, 3H), 1.62 (s, 3H), 2.02 (m, 2H), 2.51 (m, 1H), 2.74 (d, 2H, J = 6.8 Hz), 4.48 (s, 1H), 4.57 (s, 1H), 4.96 (t, 1H, J = 5.9 Hz), 6.25 (s, 1H), 6.32 (m, 2H), 7.41 (d, 1H, J = 8.7 Hz), 9.67 (s, 1H), 10.50 (s, 1H), 12.55 (s, 1H); ¹³C-NMR: δ = 17.6 (C-7′′), 18.4 (C-10′′), 25.5 (C-6′′), 26.7 (C-1′′), 30.8 (C-3′′), 46.5 (C-2′′), 97.3 (C-6), 103.4 (C-10), 103.9 (C-3′), 104.8 (C-8), 106.4 (C-5′), 111.0 (C-9′′), 123.1 (C-4′′), 130.0 (C-6′), 130.8 (C-5′′), 147.5 (C-2), 148.6 (C-8′′), 154.4 (C-3), 158.1 (C-4′), 158.9 (C-9), 160.4 (C-5), 161.1 (C-7), 177.6 (C-4).

Maackiain (6): C₁₆H₁₂O₅ (M_R: 284); UV: λ_max = 311, 290 nm; IR (KBr): ν_max = 3571, 1620, 1500, 1473, 1148 cm^-1; ¹H-NMR: δ = 3.58 (m, 2H), 4.23 (m, 1H), 5.51 (m, 1H), 5.91 (s, 1H), 5.95 (s, 1H), 6.26 (d, 1H, J = 2.3 Hz), 6.47 (dd, 1H, J = 8.4, 2.3 Hz), 6.52 (s, 1H), 6.97 (s, 1H), 7.24 (d, 1H, J = 8.5 Hz), 9.63 (s, 1H); ¹³C-NMR: δ = 39.5 (C-6a), 65.7 (C-6), 77.9 (C-11a), 93.2 (C-10), 101.0 (O-CH₂-O), 102.8 (C-4), 105.3 (C-7), 109.6 (C-2), 111.3 (C-11b), 118.4 (C-6b), 132.0 (C-1), 141.0 (C-8), 147.4 (C-9), 153.7 (C-10a), 156.3 (C-4a), 158.7 (C-3).

[2,4,6,7 - ³ H]-Estradiol (89 Ci/mmol) ([³ H]-E2) was obtained from Amersham Bioscience Europe (Freiburg, Germany). 17β-Estradiol (E2) was obtained from Riedel-de Haën (Seelze, Germany). E2, flavonoids 1 - 6 and S. flavescens extract were dissolved in dimethyl sulfoxide (DMSO). Radioactive steroid solutions were prepared in TRIS-HCl buffer (0.01 M, pH 7.4 for ER).

Uteri were excised from freshly killed rats (Sprague-Dawley). Tissues were immediately homogenised in ice-cold homogenisation buffer [10 mM, pH 7.4 TRIS-HCl, 1 mM EDTA, 10 % glycerol and 0.5 % protease inhibitor cocktail (P 2714 Sigma)]. The homogenate was centrifuged for 60 min at 100,000 × g at 4 °C. The supernatant was used for binding studies. From the Scatchard analysis, the maximal number of binding sites (5 × 10 ^{- 5} μg/mL) and dissociation constant (1 × 10 ^{- 5} μg/ml) were determined. Rat uterine ER (in the rat uterus mainly ERα is expressed) was mixed with [³ H]-E2 [7.4 × 10 ^{- 4} μg/mL], buffer, E2, flavonoids or extract of S. flavescens. Determinations were performed in triplicate. After 4 h incubation at 4 °C, binding was terminated by addition of dextran-coated charcoal (DCC, dextran T70 0.625 %, active charcoal 1.25 %) under constant stirring for 10 min at 0 °C. Then, unbound [³ H]-E2 was removed with DCC by centrifugation for 10 min at 1,500 × g. The supernatant was mixed with scintillation cocktail (100 μL/4 mL UltraGold), and radioactivity was measured in a liquid scintillation counter [LKB Wallac 1209 (Rackbeta)]. Effective concentration 50 (EC₅₀) was determined by competitive analysis. The relative binding affinity (RBA) was calculated as ratio of the EC₅₀ of E2 to the EC₅₀ of the flavonoid or plant extract.

Acknowledgements

A PhD fellowship from the Deutscher Akademischer Austausch Dienst e. V. (DAAD) for P.I.H. (A/00/10 938) is gratefully acknowledged.

References

• 1 Chen X, Yi C, Yang X, Wang X. Liquid chromatography of active principles in Sophora flavescens root.  J Chromatogr B. 2004;  812 149-63
  CrossrefPubMedSearch in Google Scholar
• 2 Ryu S, Lee H, Kim Y, Kim S. Determination of isoprenyl and lavandulyl positions of flavonoids from Sophora flavescens by NMR experiment.  Arch Pharmacol Res. 1997;  20 491-5
  PubMedSearch in Google Scholar
• 3 Kuroyanagi M, Arakawa T, Hirayama Y, Hayashi T. Antibacterial and antiandrogen flavonoids from Sophora flavescens .  J Nat Prod. 1999;  62 1595-9
  CrossrefPubMedSearch in Google Scholar
• 4 Son J, Park J, Kim J, Kim Y, Chung S, Lee S. Prenylated flavonoids from the roots of Sophora flavescens with tyrosinase inhibitory activity.  Planta Med. 2003;  69 559-61
  Thieme ConnectPubMedSearch in Google Scholar
• 5 Lee H, Ko H, Ryu S, Oh W, Kim B, Ahn S. et al . Inhibition of phospholipase C gamma 1 by the prenylated flavonoids from Sophora flavescens .  Planta Med. 1997;  63 266-8
  PubMedSearch in Google Scholar
• 6 Chung M, Rho M, Ko J, Ryu S, Jeune K, Kim K. et al . In vitro inhibition of diacylglycerol acyltransferase by prenylflavonoids from Sophora flavescens .  Planta Med. 2004;  70 258-60
  Thieme ConnectPubMedSearch in Google Scholar
• 7 Ko W, Kang T, Kim N, Lee S, Kim Y, Ko G. et al . Lavandulylflavonoids: a new class of in vitro apoptogenic agents from Sophora flavescens .  Toxicol In Vitro. 2000;  14 429-33
  CrossrefPubMedSearch in Google Scholar
• 8 Kim Y, Min B, Bae K. A cytotoxic constituent from Sophora flavescens .  Arch Pharm Res. 1997;  20 342-5
  PubMedSearch in Google Scholar
• 9 Liu J, Burdette J, Xu H, Gu C, van Breemen R, Bhat K. et al . Evaluation of estrogenic activity of plant extracts for the potential treatment of menopausal symptoms.  J Agric Food Chem. 2001;  49 2472-9
  CrossrefPubMedSearch in Google Scholar
• 10 Milligan S, Kalita J, Pocock V, Van De Kauter V, Stevens J, Deinzer M. et al . The endocrine activities of 8-prenylnaringenin and related hop (Humulus lupulus L.) flavonoids.  J Clin Endocrinol Metab. 2000;  85 4912-5
  CrossrefPubMedSearch in Google Scholar
• 11 Naeyer A, Berghe W, Pocock V, Milligan S, Haegeman G, Keukeleire D. Estrogenic and anticarcinogenic properties of kurarinone, a lavandulyl flavanone from the roots of Sophora flavescens .  J Nat Prod. 2004;  67 1829-32
  CrossrefPubMedSearch in Google Scholar
• 12 Milligan S, Kalita J, Heyerick A, Rong H, De Cooman L, De Keukeleire D. Identification of a potent phytoestrogen in hops (Humulus lupulus L.) and beer.  J Clin Endocrinol Metab. 1999;  84 2249-52
  CrossrefPubMedSearch in Google Scholar
• 13 Komatsu M, Tomimori T, Hatayama K, Mikuriya N. Studies on the constituents of Sophora species. IV. Constituents of Sophora angustifolia Sieb. et Zucc.  Yakugaku Zasshi. 1970;  90 463-8
  PubMedSearch in Google Scholar
• 14 Woo E, Kwak J, Kim H, Park H. A new prenylated flavonol from the roots of Sophora flavescens .  J Nat Prod. 1998;  61 1552-4
  CrossrefPubMedSearch in Google Scholar
• 15 Iinuma M, Tanaka T, Mizuno M, Shirataki Y, Yokoe I, Komatsu M, Lang F. Two flavanones in Sophora leachiano and some related structures.  Phytochemistry. 1990;  29 2667-9
  CrossrefPubMedSearch in Google Scholar
• 16 Wu L, Miyase T, Akira U, Kuroyanagi M, Noro T, Fukushima S. Studies on the constituents of Sophora flavescens AITON.  Chem Pharm Bull. 1985;  33 3231-6
  PubMedSearch in Google Scholar
• 17 Kinoshita T, Ichinose K, Takahashi C, Ho F, Wu J. Chemical studies on Sophora tomentosa: the isolation of a new class of isoflavonoid.  Chem Pharm Bull. 1990;  38 2756-9
  CrossrefPubMedSearch in Google Scholar
• 18 Hatayama K, Komatsu M. Studies on the constituents of Sophora species. V. Constituents of the root of Sophora angustifolia Sieb. et ZUCC.  Chem Pharm Bull. 1971;  19 2126-31
  PubMedSearch in Google Scholar

Prof. Dr Michael Wink

Institut für Pharmazie und Molekulare Biotechnologie

Universität Heidelberg

Im Neuenheimer Feld 364

69120 Heidelberg

Germany

Phone: +49-6221-544-880

Fax: +049-6221-544-884

Email: wink@uni-hd.de

References

• 1 Chen X, Yi C, Yang X, Wang X. Liquid chromatography of active principles in Sophora flavescens root.  J Chromatogr B. 2004;  812 149-63
  CrossrefPubMedSearch in Google Scholar
• 2 Ryu S, Lee H, Kim Y, Kim S. Determination of isoprenyl and lavandulyl positions of flavonoids from Sophora flavescens by NMR experiment.  Arch Pharmacol Res. 1997;  20 491-5
  PubMedSearch in Google Scholar
• 3 Kuroyanagi M, Arakawa T, Hirayama Y, Hayashi T. Antibacterial and antiandrogen flavonoids from Sophora flavescens .  J Nat Prod. 1999;  62 1595-9
  CrossrefPubMedSearch in Google Scholar
• 4 Son J, Park J, Kim J, Kim Y, Chung S, Lee S. Prenylated flavonoids from the roots of Sophora flavescens with tyrosinase inhibitory activity.  Planta Med. 2003;  69 559-61
  Thieme ConnectPubMedSearch in Google Scholar
• 5 Lee H, Ko H, Ryu S, Oh W, Kim B, Ahn S. et al . Inhibition of phospholipase C gamma 1 by the prenylated flavonoids from Sophora flavescens .  Planta Med. 1997;  63 266-8
  PubMedSearch in Google Scholar
• 6 Chung M, Rho M, Ko J, Ryu S, Jeune K, Kim K. et al . In vitro inhibition of diacylglycerol acyltransferase by prenylflavonoids from Sophora flavescens .  Planta Med. 2004;  70 258-60
  Thieme ConnectPubMedSearch in Google Scholar
• 7 Ko W, Kang T, Kim N, Lee S, Kim Y, Ko G. et al . Lavandulylflavonoids: a new class of in vitro apoptogenic agents from Sophora flavescens .  Toxicol In Vitro. 2000;  14 429-33
  CrossrefPubMedSearch in Google Scholar
• 8 Kim Y, Min B, Bae K. A cytotoxic constituent from Sophora flavescens .  Arch Pharm Res. 1997;  20 342-5
  PubMedSearch in Google Scholar
• 9 Liu J, Burdette J, Xu H, Gu C, van Breemen R, Bhat K. et al . Evaluation of estrogenic activity of plant extracts for the potential treatment of menopausal symptoms.  J Agric Food Chem. 2001;  49 2472-9
  CrossrefPubMedSearch in Google Scholar
• 10 Milligan S, Kalita J, Pocock V, Van De Kauter V, Stevens J, Deinzer M. et al . The endocrine activities of 8-prenylnaringenin and related hop (Humulus lupulus L.) flavonoids.  J Clin Endocrinol Metab. 2000;  85 4912-5
  CrossrefPubMedSearch in Google Scholar
• 11 Naeyer A, Berghe W, Pocock V, Milligan S, Haegeman G, Keukeleire D. Estrogenic and anticarcinogenic properties of kurarinone, a lavandulyl flavanone from the roots of Sophora flavescens .  J Nat Prod. 2004;  67 1829-32
  CrossrefPubMedSearch in Google Scholar
• 12 Milligan S, Kalita J, Heyerick A, Rong H, De Cooman L, De Keukeleire D. Identification of a potent phytoestrogen in hops (Humulus lupulus L.) and beer.  J Clin Endocrinol Metab. 1999;  84 2249-52
  CrossrefPubMedSearch in Google Scholar
• 13 Komatsu M, Tomimori T, Hatayama K, Mikuriya N. Studies on the constituents of Sophora species. IV. Constituents of Sophora angustifolia Sieb. et Zucc.  Yakugaku Zasshi. 1970;  90 463-8
  PubMedSearch in Google Scholar
• 14 Woo E, Kwak J, Kim H, Park H. A new prenylated flavonol from the roots of Sophora flavescens .  J Nat Prod. 1998;  61 1552-4
  CrossrefPubMedSearch in Google Scholar
• 15 Iinuma M, Tanaka T, Mizuno M, Shirataki Y, Yokoe I, Komatsu M, Lang F. Two flavanones in Sophora leachiano and some related structures.  Phytochemistry. 1990;  29 2667-9
  CrossrefPubMedSearch in Google Scholar
• 16 Wu L, Miyase T, Akira U, Kuroyanagi M, Noro T, Fukushima S. Studies on the constituents of Sophora flavescens AITON.  Chem Pharm Bull. 1985;  33 3231-6
  PubMedSearch in Google Scholar
• 17 Kinoshita T, Ichinose K, Takahashi C, Ho F, Wu J. Chemical studies on Sophora tomentosa: the isolation of a new class of isoflavonoid.  Chem Pharm Bull. 1990;  38 2756-9
  CrossrefPubMedSearch in Google Scholar
• 18 Hatayama K, Komatsu M. Studies on the constituents of Sophora species. V. Constituents of the root of Sophora angustifolia Sieb. et ZUCC.  Chem Pharm Bull. 1971;  19 2126-31
  PubMedSearch in Google Scholar

Prof. Dr Michael Wink

Institut für Pharmazie und Molekulare Biotechnologie

Universität Heidelberg

Im Neuenheimer Feld 364

69120 Heidelberg

Germany

Phone: +49-6221-544-880

Fax: +049-6221-544-884

Email: wink@uni-hd.de