Kurarinol, tyrosinase inhibitor isolated from the root of Sophora flavescens

Phytomedicine: International Journal of Phytotherapy & Phytopharmacology, August, 2008 by Y.B. Ryu, I.M. Westwood, N.S. Kang, H.Y. Kim, J.H. Kim, Y.H. Moon, K.H. Park

Abstract

It is well known that flavanones, sophoraflavanone G 1, kurarinone 2, and kurarinol 3, from the root of Sophora flavescens, have extremely strong tyrosinase inhibitory activity. This study delineates the principal pharmacological features of kurarinol 3 that lead to inhibition of the oxidation of L-tyrosine to melanin by mushroom tyrosinase (I[C.sub.50] of 100 nM). The inhibition kinetics analyses unveil that compounds 1 and 2 are noncompetitive inhibitors. However similar analysis shows kurarinol 3 to be a competitive inhibitor. Compounds 1 and 2 exhibited potent antibacterial activity with 10 [micro]g/disk against Gram-positive bacteria, whereas kurarinol 3 did not ostend any antibacterial activity. Interestingly, kurarinol 3 inhibits production of melanin in S. bikiniensis without affecting the growth of microorganism. It is thus distinctly different from the other tyrosinase inhibitors 1 and 2. In addition, kurarinol 3 manifests relatively low cytotoxic activity (E[C.sub.50] > 30 [micro]M) compared to 1 and 2. To account for these observations, we conducted molecular modeling studies. These suggested that the lavandulyl group within 3 is instrumental in the interaction with the enzyme. More specifically, the terminal hydroxy function within the lavandulyl group is most important for optimal binding.

[C] 2007 Elsevier GmbH. All rights reserved.

Keywords: Root of Sophora flavescens; Lavandulylated flavanone; Mushroom tyrosinase; Kurarinol; Molecular modeling

Introduction

Melanin helps to protect the skin from damaging ultraviolet radiation from the sun. However, high concentrations of melanin in the skin can result in hyperpigmentation such as freckles and moles. Tyrosinase is the rate-limiting enzyme in melanin production, which is fashioned in melanocytes which are located within the basal epidermis (Espin and Wichers, 2001). Therefore, tyrosinase inhibitors have attracted considerable interest in medicinal and cosmetic products, primarily in relation to the treatment of hyperpigmentation (Chen and Kubo, 2002). Tyrosinase (monophenol, polyphenol oxidase; EC 1.14.18.1) catalyzes the hydroxylation of L-tyrosine to 3,4-dihydroxyphenyalalanine (L-DOPA) and the subsequent oxidation of L-DOPA to dopaquinone (Fenoll et al., 2002). The 0-quinone product is then transformed into melanin in a series of nonenzymatic reactions (Schoot-Uiterkamp et al., 1976). The active site of tyrosinase consists of two copper atoms and it can also adopt one of three states: 'met' (Em), 'deoxy' (Ed), and 'oxy' (Eo) (Ros et al., 1996). Structural models have been proposed for the active site of each of these three forms of tyrosinase (Prota, 1998). All interaction between tyrosinase and its substrates are suggested to take place at the binuclear copper site. The two substrates for this enzyme, monophenol and 0-diphenol, have been found to react with different oxidation states of the same coupled binuclear copper sites (Rodriguez-Lopez et al., 2001; Sanchez-Ferrer et al., 1995). Sophora flavescens, known as the Chinese drug 'Kushen', has been used as a stamachic, antipyretic analgesic, and insecticide (Murakoshi et al, 1982). This species continues to be a rich source of new secondary metabolites with a broad spectrum of biological activities, including cytotoxic, antibacterial, and apoptosis modulator properties (Ko et al., 2000; Kuroyanagi et al., 1999). Previous studies of metabolites from the root of Sophora flavescens have already reported the isolation and structural determination of a series of lupin alkaloid and prenylated or lavandulylated flavanones (Murakoshi et al., 1984; Wu et al., 1985). Several papers including ours reported that lavandulylated flavonone 1-3 have a strong tyrosinase inhibitory activity (Ha et al, 2001; Kim et al., 2003; Son et al., 2003).

We found that the most active compound 3 expounds a different mode of biological activity compared with other lavandulylated flavanones 1 and 2. Here, we report the kinetic behavior of 1-3, as well as their bacterial growth inhibition properties, their relative abilities to inhibit melanin production in S. bikiniensis, and also molecular modeling strudies to explain our results.

Materials and methods

General

Specific rotation values were measured on a Perkin-Elmer polarimeter. Proton and carbon nuclear magnetic resonance spectra were measured downfield relative to tetramethylsilan in either CD[Cl.sub.3], [CD.sub.3]OD, or acetone [d.sub.6] as indicated. 2D-NMR data were obtained on a Varian Inova-500 spectrometer and Bruker DRX-500. EI-MS data were collected on Jeol JMS-700 spectrometer. CD spectra were recorded on a JASCO J-715 spectropolarimeter. Elemental analysis was performed with LECO CHNS-932 elemental analyzer. Absorption measurements were recorded using a ADAP-2020.

Plant material

Sophora flavescens was collected in Hamyang (Korea) and identified by Prof. Myong Gi Chung. A voucher specimen (Park, K.H. 112) of this raw material is deposited at Herbarium of Gyeongsang National University (GNUC).

Extraction and isolation

Dried roots of Sophora flavescens (1 kg) were repeatedly extracted with methanol at room temperature. The methanolic extract of the root of S. flavescens showed high inhibitory activity. After filtration, the filtrates were concentrated to give a dark brown residue, which was partitioned between water and chloroform. The organic layer was concentrated to give a brown residue (27 g). The activity-guided purification of the fraction using repeated column chromatography on Si[O.sub.2] yielded 320 mg of compound 1, 410 mg of compound 2, and 270 mg of compound 3 (Fig. 1).

[FIGURE 1 OMITTED]

Compound 1: amorphous white powder, mp 178-180[degrees]C, [alpha]20D-71.4[degrees] (c 2.0, C[H.sub.3]OH); IR (KBr): 3369, 1612c[m.sup.-1]; (1)H NMR (500 MHz, C[D.sub.3]OD) [delta] 1.48 (3H, s, H-6"), 1.56 (3H, s, H-10"), 1.63 (3H, s, H-7"), 2.0 (2H, m, H-3"), 2.47 (1H, m, H-2"), 2.73 (1H, dd, J = 17.1, 2.8 Hz, H-3a), 2.85 (2H, m, H-1"), 2.97 (1H, dd, J = 17.1, 13.2 Hz, H-3b), 4.55 (2H, s, H-9"), 4.90 (1H, m, H-4"), 5.56 (1H, dd, J = 13.2, 2.7 Hz, H-2), 5.92 (1H, s, H-6), 6.35 (1H, m, H-5'), 6.37 (1H, d, J = 2.3 Hz, H-3'), 7.30 (1H, d, J = 8.2Hz, H-6') (13)C NMR (125 MHz, C[D.sub.3]OD) [delta] 76.3 (C-2, d), 43.7 (C-3, t), 199.4 (C-4, s), 103.7 (C-4a, s), 163.6 (C-5, s), 96.7 (C-6, d), 166.9 (C-7, s), 109.1 (C-8, s), 163.0 (C-8a, s), 118.8 (C-l', s), 157.1 (C-2', s), 108.1 (C-3', d), 159.9 (C-4', s), 103.8 (C-5', d), 129.1 (C-6', d), 28.4 (C-l", t), 48.7 (C-2", d), 32.8 (C-3", t), 125.2 (C-4", d), 132.5 (C-5", s), 18.3 (C-6", q), 26.3 (C-7", q), 150.1 (C-8", s), 111.6 (C-9", t), 19.6 (C-10", q) (Ryu et al., 1996, 1997).