- Email: [email protected]
Antioxidant properties of butein isolated from Dalbergia odorifera
Biochimica et Biophysica Acta 1392 (1998) 291^299
Antioxidant properties of butein isolated from Dalbergia odorifera Zhi-Jiao Cheng a , Sheng-Chu Kuo b , Shiuh-Chuan Chan b , Feng-Nien Ko a , Che-Ming Teng a; * a
Pharmacological Institute, College of Medicine, National Taiwan University, No. 1, Jen-Ai Rd., Sect. 1, Taipei, Taiwan b Graduate Institute of Pharmaceutical Chemistry, China Medical College, Taichung, Taiwan Received 10 March 1998; accepted 18 March 1998
Abstract The antioxidant properties of butein, isolated from Dalbergia odorifera T. Chen, were investigated in this study. Butein inhibited iron-induced lipid peroxidation in rat brain homogenate in a concentration-dependent manner with an IC50 , 3.3 þ 0.4 WM. It was as potent as K-tocopherol in reducing the stable free radical diphenyl-2-picrylhydrazyl (DPPH) with an IC0:200 , 9.2 þ 1.8 WM. It also inhibited the activity of xanthine oxidase with an IC50 , 5.9 þ 0.3 WM. Besides, butein scavenged the peroxyl radical derived from 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH) in aqueous phase, but not that from 2,2-azobis(2,4-dimethylvaleronitrile) (AMVN) in hexane. Furthermore, butein inhibited copper-catalyzed oxidation of human low-density lipoprotein (LDL), as measured by conjugated dienes and thiobarbituric acid-reactive substance (TBARS) formations, and electrophoretic mobility in a concentration-dependent manner. Spectral analysis revealed that butein was a chelator of ferrous and copper ions. It is proposed that butein serves as a powerful antioxidant against lipid and LDL peroxidation by its versatile free radical scavenging actions and metal ion chelation. z 1998 Elsevier Science B.V. Keywords: Antioxidant; Lipid peroxidation; Low density lipoprotein; Butein; Xanthine oxidase; (Dalbergia odorifera)
1. Introduction Butein (2P,4P,3,4-tetrahydroxychalcone), isolated from Dalbergia odorifera T. Chen [1], is a chalcone compound belonging to the £avonoid subclass. The structures of chalcones are similar to curcumin, a known antioxidant [2]. Chalcones exhibit the basic structure with two benzene rings linked through an ,L-unsaturated carbonyl group. They are a group of secondary plant metabolites occurring widely throughout the plant kingdom and are common components of the human diet. Although their importance to human health has remained long unclear,
* Corresponding author. Fax: +886 (2) 2322-1742.
some chalcones have been found to possess anti-in£ammatory, antibacterial, antifungal, and antitumor activities [3,4]. Some derivatives of hydroxychalcones exhibit antioxidative activity and inhibit the activities of xanthine oxidase [5]. It is believed that the biological e¡ects of chalcones mainly come from their antioxidant [6] and metal ion chelating [7] properties. Recent studies also indicate that chalcones inhibit cAMP-phosphodiesterase [8] and glutathione Stransferases [9]. Because many physiological and pharmacological functions have been reported for a variety of chalcones, it is interesting to understand the antioxidant properties of chalcones. Butein has been reported to be an inhibitor of xanthine oxidase, and exhibits inhibitory e¡ects on lipid peroxidation in rat liver mi-
0005-2760 / 98 / $19.00 ß 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 5 - 2 7 6 0 ( 9 8 ) 0 0 0 4 3 - 5
BBALIP 55263 5-6-98
292
Z.-J. Cheng et al. / Biochimica et Biophysica Acta 1392 (1998) 291^299
crosomes [5]. However, the antioxidant properties of butein have not been fully addressed. The aim of this study was to elucidate the antioxidant pro¢le of butein using various experimental models. Furthermore, its protective e¡ects against LDL lipid peroxidation were also evaluated. 2. Materials and methods 2.1. Materials Butein (Fig. 1) was isolated from Dalbergia odorifera T. Chen, as described previously [1], and dissolved in DMSO. Its structure was identi¢ed by UV, IR, MS and NMR. Butylated hydroxytoluene (BHT), K-tocopherol, desferrioxamine mesylate, 2thiobarbituric acid (TBA), tetramethoxypropane (TMP), B-phycoerythrin, 2-deoxyribose, 1,1-diphenyl-2-picrylhydrazyl (DDPH), xanthine oxidase (grade IV, from buttermilk), nitroblue tetrazolium (NBT), catalase, superoxide dismutase (SOD, type I, from bovine liver), D-mannitol, ascorbic acid and hydrogen peroxide (30% solution) were purchased from Sigma (St. Louis, MO). CuSO4 , 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH) and 2,2-azobis(2,4-dimethylvaleronitrile) (AMVN) were obtained from Wako Pure, Japan. cis-Parinaric acid was purchased from Molecular Probes, USA. 2.2. Lipid peroxidation assay of rat brain homogenate Rat brain homogenate was prepared from the brains of freshly killed Wistar rats. The lipid peroxidation in the presence of iron ions was measured by the thiobarbituric acid method, as described previously [10]. Tetramethoxypropane was used as a standard, and the results were expressed as nano-
moles of malondialdehyde (MDA) equivalents per milligram protein of rat brain homogenates. 2.3. DPPH scavenging activity An ethanolic solution of the stable nitrogen centered free radical DPPH (100 WM) was incubated with the test compounds, and the absorbance monitored spectrophotometrically at 517 nm. The concentration (IC0:200 ) of the test compounds that induced a decrease of 0.200 in absorbance during 30 min observation was taken as the free radical scavenging potency [11]. 2.4. Superoxide scavenging activity Superoxide anion was generated by xanthine/xanthine oxidase and measured by the NBT reduction method [12]. Test compounds were incubated in 50 mM KH2 PO4 /K2 HPO4 (pH 7.4) containing K2 H2 -EDTA (0.3 mM), NBT (0.6 mM), xanthine (0.1 mM) and xanthine oxidase (0.02 U/ml). Production of superoxide anion was monitored spectrophotometrically at 560 nm. Superoxide dismutase (100 U/ml) was used as a reference inhibitor. The e¡ect of test compounds on the activity of xanthine oxidase was determined by measuring uric acid formation at 295 nm using the previous condition [13]. A molar extinction coe¤cient, 11 000 M31 cm1 , for uric acid was used for calculation. 2.5. Scavenging of the hydroxyl radical generated by the ascorbate/iron ion/H2 O2 system The reaction mixture contained 2.8 mM 2-deoxyribose, 1.4 mM H2 O2 , 20 WM FeCl3 and 100 WM EDTA without or with test compounds in 10 mM KH2 PO4 -KOH bu¡er, pH 7.4. The reaction was triggered by the addition of 50 WM ascorbate and the mixture incubated at 37³C for 60 min. Solutions of FeCl3 , ascorbate and H2 O2 were made up in deaerated water immediately before use. The extent of deoxyribose degradation by hydroxyl radical was measured with the thiobarbituric acid method [14]. 2.6. Scavenging of hydrogen peroxide
Fig. 1. The chemical structure of butein.
The content of hydrogen peroxide was measured
BBALIP 55263 5-6-98
Z.-J. Cheng et al. / Biochimica et Biophysica Acta 1392 (1998) 291^299
indirectly using a catalase-based method, with a Clark-type electrode (YSI model 5331, Yellow Spring Instruments, OH, USA). The reaction mixture contained 0.5 or 1.0 mM H2 O2 and test compound in 50 mM potassium phosphate bu¡er with 0.2 mM NaCl, pH 7.4. After the mixture was incubated at 25³C for 40 min, catalase (5.8 U/ml) was added and O2 production was monitored polarographically for 0.8 min. After the experiments, the amount of H2 O2 remaining was calculated using a standard curve made for O2 production vs. H2 O2 concentration (0.1^2.0 mM). 2.7. Scavenging of peroxyl radicals in aqueous solution and in hexane The ability of the test compounds to scavenge hydrophilic or lipophilic peroxyl radicals was evaluated by the method described by Tsuchiya et al. [15]. A decrease in B-phycoerythrin £uorescence (Ex 540 nm, Em 575 nm) by AAPH-generated peroxyl radicals was monitored in 20 mM Tris-HCl (pH 7.4) using a £uorescence spectrophotometer (Hitachi, Model F4000). The stoichiometric factors of the test compounds with peroxyl radicals derived from AAPH (25 mM) were calculated according to the lag time during which £uorescence loss was protected. The rate of peroxyl radical formation from AAPH is 1.6U106 /s at 40³C [16]. Ascorbic acid was used as a positive control. The trapping activity of test compounds on lipophilic peroxyl radicals was assessed in a hydrophobic environment (hexane) at 40³C. cis-Parinaric acid, in ethanol, was added to a ¢nal concentration of 30 WM, and its £uorescence monitored (excitation 304 nm, emission 421 nm). AMVN (¢nal concentration 100 mM in chloroform solution) was then added and the quenching of the £uorescence monitored. The test compound was added 2 min after the initiation of AMVN-induced cis-parinaric acid oxidation. K-Tocopherol was used as a positive control. 2.8. Human LDL isolation and oxidation Human low-density lipoproteins (LDLs, d = 1.019V1.063 g/ml) were isolated from citrated plasma by adding 2.5 mM EDTA and using a sequential density gradient ultracentrifugation method
293
at 4³C [17]. LDLs were dialyzed against PBS containing 1 mM EDTA, sterilized by ¢ltration through a 0.22 Wm Millipore ¢lter, and stored at 4³C in sealed vials £ushed with nitrogen until use. Before peroxidation experiments, LDLs were extensively dialyzed with PBS to remove all traces of EDTA. The protective e¡ects of test compounds on the peroxyl radicalinduced oxidation of cis-parinaric acid incorporated into LDL were determined as previously described by Laranjinha et al. [18]. The reaction mixture contained 15 Wg/ml LDL protein and 0.75 WM cis-parinaric acid in PBS and the reaction was triggered by AAPH (10 mM) at 37³C. cis-Parinaric acid degradation was monitored by a £uorescence spectrophotometer (Hitachi, Model F-4000; excitation 324 nm, emission 413 nm). Ascorbic acid was taken as a positive control. Under the experiments of copper-catalyzed LDL oxidation, the reaction mixture contained the vehicle or test compound and LDL (adjusted to 100 Wg LDL protein/ml or 200 Wg LDL protein/ml only for electrophoretic mobility assay) in PBS. The oxidative reaction was carried out at 37³C and induced by adding a freshly prepared CuSO4 solution (¢nal concentration 5 WM). Conjugated diene formation was continuously monitored at 234 nm [19]. To assess the extent of lipid peroxidation, the formation of thiobarbituric acid-reactive substance (TBARS) was measured and expressed as MDA equivalents/mg LDL protein. The extent of aldehyde-modi¢ed lysine in oxidized LDL was monitored by determining the £uorescence intensity (excitation 350 nm, emission 420 nm) [20]. The change on surface charge of the LDL protein was evaluated by an increase in electrophoretic mobility, which was determined using the Lipo¢lm lipoprotein electrophoresis kit supplied by Sebia (France). 2.9. Iron and copper chelation study Iron and copper chelations were determined by adding 200 WM of FeSO4 or 100 WM CuSO4 to the solution containing 100 WM of test compound. After incubation at room temperature for 10 min, the absorption spectra (190V600 nm) were recorded. The chelation of iron (or copper) with test compound was evaluated by absorbance change and/or spectral shift after incubation [21].
BBALIP 55263 5-6-98
294
Z.-J. Cheng et al. / Biochimica et Biophysica Acta 1392 (1998) 291^299
2.10. Determination of protein contents The protein contents of the rat brain homogenates and LDLs were determined as described by Bradford [22] using a Bio-Rad protein assay kit. 20 Wl sample was diluted with 800 Wl deionized water and mixed with 200 Wl dye reagent concentrate and the absorbance was measured at 595 nm. Bovine serum albumin was used as a standard. 2.11. Statistical analysis All experimental data are shown as means þ SE and accompanied by the number of observations. Statistical analysis was performed using Student's t-test, and the signi¢cant di¡erence was set at P 6 0.05. The IC50 value was obtained by regression analysis. 3. Results 3.1. Inhibition of lipid peroxidation in rat brain homogenate
Fig. 2. The inhibitory e¡ects of butein on Fe2 -induced lipid peroxidation in rat brain homogenate. Rat brain homogenates were preincubated with DMSO (0.5%, basal and control) or various concentrations of butein at 37³C for 10 min, then Fe2 (200 WM) was added, except for basal, and incubated for another 30 min. Data are presented as means þ S.E. (n = 6). *P 6 0.05, **P 6 0.01, ***P 6 0.001, as compared with the control.
In unstimulated control experiments, the amount of TBARS formed in rat brain homogenate was 2.1 þ 0.4 nmol/mg protein. In control experiments stimulated by the addition of Fe2 (200 WM), the amount of TBARS was 12.7 þ 1.3 nmol/mg protein. Butein caused a concentration-dependent inhibition of iron-induced lipid peroxidation in rat brain homogenates with an IC50 , 3.3 þ 0.4 WM (Fig. 2). It produced a nearly complete inhibition of ironinduced peroxidation at a concentration of 10 WM. BHT also inhibited this iron-induced lipid peroxidation with an IC50 , 1.3 þ 0.1 WM. Control experiments indicated that butein did not a¡ect the measurement of TBARS because the absorbance at 532 nm was not a¡ected by adding butein to brain homogenate that was either intact or already oxidatively modi¢ed (data not shown).
Metal ions caused a spectral shift and absorbance change of butein (Fig. 3). The Fe2 -butein complex has peaks at 286 nm and 422 nm while the Cu2 butein complex has peaks at 286 nm and 454 nm.
3.2. Iron and copper chelation study
3.4. Superoxide anion scavenging action
Flavonoids have been known to form chelates with a number of metal ions [23]. Butein alone has peaks at 260 nm and 395 nm.
Superoxide anion generated by the xanthine/xanthine oxidase system was monitored by the reduction of NBT. The initial rate of NBT reduction
3.3. Interaction of butein with stable free radical, DPPH In the DPPH assay system, the free radical scavenging activity of antioxidants was expressed as IC0:200 . The scavenging activities of butein, K-tocopherol and BHT were all in a concentration-dependent manner with IC0:200 values of 9.2 þ 1.8, 11.9 þ 0.2 and 14.5 þ 2.5 WM (n = 6), respectively. Apparently, butein was as potent as K-tocopherol and BHT in scavenging DPPH radicals.
BBALIP 55263 5-6-98
Z.-J. Cheng et al. / Biochimica et Biophysica Acta 1392 (1998) 291^299
295
3.6. E¡ect of butein on deoxyribose degradation caused by hydroxyl radical Butein is poorly soluble in aqueous solution at pH 7.4. Therefore, the butein preparation was made in alkaline solution and then the pH adjusted to 7.4 immediately before use. Our results indicated that butein, at 100 WM, did not inhibit the hydroxyl radical-induced deoxyribose degradation. 3.7. E¡ects of butein on peroxyl radical in aqueous solution and in hexane Fig. 3. Absorption spectra of butein and metal ionbutein complex in PBS at pH 7.4. a, absorption spectrum of butein; b, absorption spectrum of Fe2 -butein complex; c, absorption spectrum of Cu2 -butein complex. Note that the absorption spectra in this ¢gure were obtained 10 min after incubation of butein (100 WM) with Fe2 (200 WM) or Cu2 (100 WM).
(0.218 þ 0.002 OD/min, n = 6), caused by xanthine oxidase (0.02 U/ml) generated superoxide anion, was almost completely inhibited by superoxide dismutase (100 U/ml; 0.007 þ 0.002 OD/min, n = 6). Butein (1V10 WM) did not cause direct reduction of NBT since the absorbance of NBT was not changed by butein (data not shown). However, it inhibited the NBT reduction in the xanthine/xanthine oxidase system with an IC50 , 7.1 þ 1.0 WM. Since compounds inhibiting xanthine oxidase also a¡ect NBT reduction, butein was also investigated for its direct e¡ect on xanthine oxidase by measuring the amount of uric acid formation. The initial rate of uric acid formation caused by xanthine oxidase (0.02 U/ml) was 0.228 þ 0.010 OD/min (n = 6). Butein also inhibited xanthine oxidase-induced uric acid formation in a concentration-dependent manner with an IC50 , 5.9 þ 0.3 WM.
Fluorescence of B-phycoerythrin (excitation 540 nm, emission 575 nm) rapidly declined by the attack of peroxyl radicals derived from AAPH (25 mM). This £uorescence loss was delayed by adding butein or ascorbic acid (Fig. 4). Based on the delay in £uorescence decay, we calculated the stoichiometry factor of peroxyl radical with butein or ascorbic acid by the following equation: Stoichiometry AAPHRtime=antioxidant where [AAPH] is the concentration of AAPH (M), [R] is the rate of peroxyl radical production by AAPH, [time] is the duration of consumption of anti-
3.5. The interaction with H2 O2 In our assay system, butein (100 WM) did not react with H2 O2 (0.5V1.0 mM) directly. It did not cause a signi¢cant loss of H2 O2 because the rate of O2 production, as measured in a catalase-based assay, was not changed after 40 min incubation with butein (data not shown).
Fig. 4. Fluorescence study of the e¡ects of butein and ascorbic acid on AAPH-generated peroxyl radicals. Fluorescence of Bphycoerythrin (5 nM) was monitored at 540 nm excitation and 575 nm emission in 20 mM Tris-HCl (pH 7.4) at 40³C. [AAPH] = 25 mM, butein (3 WM, a; 1 WM, b; 0.3 WM, c), ascorbic acid (3 WM, e) or DMSO (0.5%, d) was added at arrow. Inset: concentration dependence of the inhibition period of £uorescence quenching.
BBALIP 55263 5-6-98
296
Z.-J. Cheng et al. / Biochimica et Biophysica Acta 1392 (1998) 291^299
butein concentration (Fig. 5, inset). It seems that butein was more e¡ective than ascorbic acid in the prevention of AAPH-induced cis-parinaric acid oxidation in LDL. 3.9. E¡ects of butein on TBARS and conjugated diene formation and electrophoretic mobility of the LDL stimulated by Cu 2+
Fig. 5. cis-Parinaric acid (0.75 WM) oxidation in human LDL induced by AAPH (10 mM) in the absence or presence of butein. Butein (3 WM, a; 1 WM, b; 0.3 WM, c; 0.1 WM, d), DMSO (0.5%, e) or ascorbic acid (5 WM, f) was added to the reaction mixture at the time indicated by the arrow. Inset: concentration dependence of the inhibition period of £uorescence quenching.
oxidant (s), and [antioxidant] is the concentration of butein or ascorbic acid (M). The stoichiometry factors of butein and ascorbic acid were calculated to be 1.4 þ 0.1 and 0.7 þ 0.1, respectively. That is 1.4 molecules of AAPH-derived peroxyl radicals scavenged by each butein molecule during the delay time. In hexane, the £uorescence of cis-parinaric acid (excitation 304 nm, emission 421 nm) was rapidly declined by the attack of peroxyl radicals derived from AMVN (100 mM). The £uorescence loss could be delayed by K-tocopherol but not by butein.
Butein inhibited the Cu2 -induced TBARS formation of LDL with an IC50 , 6.3 þ 0.2 WM. In addition, butein dose-dependently prolonged the lag phase of conjugated diene formation as shown by the plot of butein concentration vs. lag time (Fig. 6, inset). During the oxidation of unsaturated fatty acid of LDL, reactive aldehydes are formed and bound to the lysine residue of apoB. The modi¢cation of apoB would increase not only the electrophoretic mobility but also the £uorescence (excitation 350 nm, emission 421 nm) intensity of LDL. The LDL incubated with copper ion showed increases in electrophoretic mobility (Fig. 7) and £uorescence intensity (from
3.8. E¡ect of butein on cis-parinaric acid oxidation in LDL As described previously by Laranjinha et al. [18], the £uorescence intensity of cis-parinaric acid was barely detected in the bu¡er solution without LDL. Adding LDL to the bu¡er resulted in a dramatic increase in £uorescence intensity. It may imply that cis-parinaric acid incorporated into the LDL particles. Under this condition, the peroxyl radicals derived from AAPH would quench the £uorescence. Addition of butein or ascorbic acid to the system inhibited the decline of the £uorescence (Fig. 5). According to the length of the delay period, we found that the inhibition periods are proportional to the
Fig. 6. Conjugated diene formation of LDL exposed to Cu2 in the absence or presence of butein. LDL protein (35 Wg/ml) was incubated with 5 WM Cu2 (control, b) for the indicated times in the absence or presence of butein (0.1 WM, F ; 0.2 WM, R; 0.5 WM, S; 1.0 WM, 8). Conjugated dienes were monitored at 15 min intervals at 234 nm and absolute values of absorbance are plotted. The data shown are from a single experiment of four independent experiments. Inset: concentration dependence of the inhibition period of diene formation.
BBALIP 55263 5-6-98
Z.-J. Cheng et al. / Biochimica et Biophysica Acta 1392 (1998) 291^299
Fig. 7. E¡ect of butein on electrophoretic mobility change of human LDL. LDL protein (200 Wg/ml) was incubated with CuSO4 (5 WM) in phosphate-bu¡ered saline at 37³C for 12 h in the absence or presence of various concentrations of butein. Then, the reaction was stopped by 50 WM BHT, and the samples were subjected to electrophoresis analysis. (1) standard; (2) native LDL; (3) ox-LDL. LDL oxidized in the presence of 1 WM butein (4); 3 WM butein (5); 10 WM butein (6); 30 WM butein (7); 100 WM butein (8).
3.1 þ 0.1 to 62.8 þ 3.4 AU). However, we found that butein would quench the £uorescence of oxidatively modi¢ed LDL, so we further investigated the e¡ects of butein on the apoB modi¢cation of LDL by electrophoretic mobility change. In the presence of butein, we found that 30 WM butein could obviously reduce the electrophoretic change of oxidatively modi¢ed LDL (Fig. 7). 4. Discussion Rat brain homogenates are usually used as a preparation to evaluate the antioxidant activities of compounds on lipid peroxidation [24,25]. It is known that transition metal ions are involved in both initiation and propagation of lipid peroxidation [26]. Rat brain homogenates exposed to ferrous ion exhibited lipid peroxidation in air by a mechanism which may involve site-bound iron-mediated decomposition of lipid hydroperoxides to yield alkoxyl or peroxyl radicals, leading to the chain reaction of lipid peroxidation [27]. In this system, we found that butein e¡ectively inhibited ferrous ion-induced lipid peroxidation. Its potency was comparable to BHT, a typical antioxidant in food. The antiperoxidative activity of butein may be attributed to metal ion chelation, free radical scavenging or membrane stabilization. Desferrioxamine, a potent iron chelator, could inhibit iron-induced lipid peroxidation to 50% at a concentration approximately half of the
297
iron added to rat brain homogenates [28]. Although butein also directly chelated ferrous ion, it needed 30-fold less than desferrioxamine to inhibit the lipid peroxidation to the same degree. Moreover, according to the DPPH assay, butein acted as a direct free radical scavenger with a potency (IC0:200 , 9.2 þ 1.8 WM) similar to K-tocopherol, a chain-breaking antioxidant. Hence, we believe that the metal ion chelating property of butein is not mainly component contributed to its anti-peroxidation activity in the brain homogenates. Generation of peroxyl radicals is a necessary step in the formation of TBARS during lipid peroxidation [29]. We assessed the peroxyl radical scavenging activity of butein by halting the AAPH- and AMVNinduced £uorescence decay of B-phycoerythrin and cis-parinaric acid in aqueous and lipophilic phases, respectively. Our results indicated that butein scavenged the hydrophilic but not the lipophilic peroxyl radicals. Consequently, the localization of antioxidants and the generation site of radicals should be taken into account as evaluating their antioxidant activity. In AAPH-initiated peroxidation of LDL, chain-initiating radicals are generated in aqueous phase and chain-propagating lipid peroxyl radicals are located within membranes [30]. Butein e¡ectively scavenged peroxyl radicals in LDL solution and PBS but not in hexane. We suggest that butein is an effective membrane antioxidant located in the interface between membranous and aqueous phase. However, butein could not exert its antioxidant activities in an extremely hydrophobic phase such as hexane. It is well known that superoxide anions could damage biomacromolecules directly or indirectly by forming hydrogen peroxide, hydroxyl radical, peroxynitrite or singlet oxygen during pathophysiological events such as ischemia-reperfusion injury [31]. In the xanthine/xanthine oxidase system, butein inhibited NBT reduction and uric acid formation in a concentration-dependent manner with IC50 , 7.1 þ 1.0 WM and 5.9 þ 0.3 WM, respectively. Clearly, butein is an e¡ective inhibitor of xanthine oxidase. It is usually accepted that the superoxide anion generated from xanthine oxidase in hypoxia leads to ischemia-reperfusion injury [32]. Therefore, butein would be expected to prevent injury induced by hypoxia. The oxidation of LDL has been suggested to be a key event in human atherosclerosis [33]. The detailed
BBALIP 55263 5-6-98
298
Z.-J. Cheng et al. / Biochimica et Biophysica Acta 1392 (1998) 291^299
mechanism of LDL oxidation in vivo is unknown and remains to be clari¢ed. In copper-catalyzed LDL oxidation, the appropriate mechanism also has not been clearly de¢ned. However, one suggestion is that copper initially catalyzed the breakdown of the lipid hydroperoxides in the surface phospholipids or in the core cholesteryl ester, subsequently, modi¢cation of phospholipids and cholesteryl esters were formed and propagated. Eventually, the lysine residues of apoB were modi¢ed by the highly reactive products of oxidized fatty acid (i.e., malondialdehyde and 4-hydroxynonenal). In such oxidized LDL, its surface charge tends to be negative and the characteristic £uorescent chromogen is exposed [34]. In our assay systems butein not only prevented AAPH-initiated cis-parinaric acid oxidation in LDL solution but also inhibited copper-catalyzed lipid peroxidation. Since butein could directly chelate copper, these results imply that butein is not only an e¡ective metal ion chelator but also a powerful chain-breaking antioxidant in the LDL systems. Thus butein would be a potential protector against the oxidative modi¢cation of LDL. In conclusion, this study veri¢es that butein is a natural antioxidant against lipid peroxidation in rat brain homogenates and human LDL. Its antioxidant activities are primarily attributed to its free radical scavenging actions and metal ion chelation. It will be interesting to further investigate the e¡ects of butein on various radical-mediated injury models in vitro and in vivo. Acknowledgements This work was supported by a research grant of the National Science Council of Taiwan (NSC 872314-B-002-159-M25). References [1] S.M. Yu, Z.J. Cheng, S.C. Kuo, Endothelium-dependent relaxation of rat aorta by butein, a novel cyclic AMP-speci¢c phosphodiesterase inhibitor, Eur. J. Pharmacol. 280 (1995) 69^77. [2] K. Elizabeth, M.N.A. Rao, Oxygen radical scavenging activity of curcumin, Int. J. Pharmaceut. 58 (1990) 237^ 240.
[3] R.J. Anto, K. Sukumaran, G. Kutta, M.N.A. Rao, V. Subbaraju, R. Kuttan, Anticancer and antioxidant activity of synthetic chalcones and related compounds, Cancer Lett. 97 (1995) 33^37. [4] C. Chin, P. Nagaratnam, Cytotoxic e¡ect of butein on human colon adenocarcinoma cell proliferation, Cancer Lett. 82 (1994) 65^72. [5] S. Sogawa, Y. Nihro, H. Ueda, T. Miki, H. Matsumoto, T. Satoh, Protective e¡ects of hydroxychalcones on free radicalinduced cell damage, Biol. Pharmacol. Bull. 17 (1994) 251^ 256. [6] L. Larson, The antioxidants of higher plants, Phytochemistry 27 (1988) 969^978. [7] T.W. Wu, K.P. Fung, L.H. Zeng, J. Wu, A. Hempel, A.A. Grey, N. Camerman, Molecular properties and myocardial salvage e¡ects of morin hydrate, Biochem. Pharmacol. 49 (1995) 537^543. [8] U.R. Kuppusamy, N.P. Das, E¡ects of £avonoids on cyclic AMP phosphodiesterase and lipid mobilization in rat adipocytes, Biochem. Pharmacol. 44 (1992) 1307^1315. [9] K. Zhang, N.P. Das, Inhibitory e¡ects of plant polyphenols on rat liver glutathione S-transferases, Biochem. Pharmacol. 47 (1994) 2063^2068. [10] F.N. Ko, C.H. Liao, Y.H. Kuo, Y.L. Lin, Antioxidant properties of demethyldiisoeugenol, Biochim. Biophys. Acta 1258 (1995) 145^152. [11] A. Mellors, A.L. Tappel, The inhibition of mitochondrial peroxidation by ubiquinone and ubiquinol, J. Biol. Chem. 241 (1966) 4353^4356. [12] M. Nishikimi, N.A. Roa, K. Yagi, The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen, Biochem. Biophys. Res. Commun. 46 (1972) 849^854. [13] I. Fridovich, Quantitative aspects of the production of superoxide anion radical by milk xanthine oxidase, J. Biol. Chem. 245 (1970) 4053^4057. [14] B. Halliwell, J.M.C. Gutteridge, Formation of a thiobarbituric-acid-reactive substance from deoxyribose in the presence of iron salts, FEBS Lett. 128 (1981) 347^352. [15] M. Tsuchiya, G. Scita, H.J. Freisleben, V.E. Kagan, L. Packer, Antioxidant radical-scavenging activity of carotenoids and retinoids as compared to K-tocopherol, Methods Enzymol. 213 (1992) 460^472. [16] K. Teras, D. Niki, Damage to biological tissues induced by radical initiator 2,2-azobis(2-aminopropane) dihydrochloride and its inhibition by chain breaking antioxidant, Free Radical Biol. Med. 2 (1986) 193^201. [17] R.J. Havel, H.A. Eder, L.H. Bragdon, The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum, J. Clin. Invest. 34 (1955) 1345^ 1353. [18] J.A.N. Laranjinha, L.M. Almeida, V.M.C. Maderira, Lipid peroxidation and its inhibition in low density lipoproteins: quenching of cis-parinaric acid £uorescence, Arch. Biochem. Biophys. 297 (1992) 147^154. [19] H. Esterbauer, J. Gebicki, H. Puhl, G. Jurgens, The role of
BBALIP 55263 5-6-98
Z.-J. Cheng et al. / Biochimica et Biophysica Acta 1392 (1998) 291^299
[20]
[21]
[22]
[23]
[24]
[25]
[26]
lipid peroxidation and antioxidants in oxidative modi¢cation of LDL, Free Radical Biol. Med. 13 (1992) 341^390. U.P. Seinbrecher, Oxidation of human low density lipoprotein results in derivatization of lysine residues of apoprotein B by lipid peroxide decomposition products, J. Biol. Chem. 262 (1987) 3603^3608. A. Rahman, S.M.H. Shahabuddin, J.H. Parish, Complexes involving quercetin, DNA and Cu(II), Carcinogenesis 11 (1990) 2001^2003. M.M. Bradford, A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248^254. I.B. Afanas'ev, A.I. Dorozhko, A.V. Brodskii, V.A. Kostyuk, A.I. Potapovitch, Chelating and free radical scavenging mechanisms of inhibitory action of rutin and quercetin in lipid peroxidation, Biochem. Pharmacol. 38 (1989) 1763^ 1769. J.M. Braughler, R.L. Burton, R.L. Chase, J.F. Pregenzer, E.J. Jacobsen, F.J. VanDoornik, J.M. Tustin, D.E. Ayer, G.L. Bundy, Novel membrane localized ion chelators as inhibitors of iron-dependent lipid peroxidation, Biochem. Pharmacol. 37 (1988) 3853^3860. A.B. Kozlov, E.A. Ostrachovitch, I.B. Afanas'ev, Mechanism of inhibitory e¡ects of chelating drugs on lipid peroxidation in the rat brain homogenates, Biochem. Pharmacol. 47 (1994) 795^799. B. Halliwell, J.M.C. Gutteridge, Oxygen toxicity, oxygen radicals, transition metal and diseases, Biochem. J. 219 (1984) 1^5.
299
[27] J.M. Braughler, R.L. Chase, J.F. Pregenzer, Oxidation of ferrous iron during peroxidation of various lipid substrates, Biochim. Biophys. Acta 921 (1987) 457^464. [28] C.M. Teng, G. Hsiao, F.N. Ko, D.T. Lin, S.S. Lee, N-Allylsecoboldine as a novel antioxidant against peroxidation damage, Eur. J. Pharmacol. 303 (1996) 129^139. [29] J.M.C. Gutteridge, B. Halliwell, The measurement and mechanism of lipid peroxidation in biological systems, Trends Biochem. Sci. 15 (1990) 129^135. [30] Y. Yamamoto, E. Niki, J. Eguchi, Y. Kamiya, H. Shimasaki, Oxidation of biological membranes and its inhibition. Free radical chain oxidation of erythrocyte ghost membranes by oxygen, Biochim. Biophys. Acta 819 (1985) 29^36. [31] R. Radi, J.S. Beckman, K.M. Bush, B.A. Freeman, Peroxynitrite-induced membrane lipid peroxidation: the cytotoxic potential of superoxide and nitric oxide, Arch. Biochem. Biophys. 288 (1991) 481^487. [32] R.C. Kukreja, M.L. Hess, The oxygen free radical system: from equations through membrane-protein interactions to cardiovascular injury and protection, Cardiovasc. Res. 26 (1992) 641^655. [33] D. Steinberg, S. Parthasarathy, T.E. Carew, J.L. Khoo, J.L. Witztum, Modi¢cations of low-density lipoprotein that increase its atherogenicity, New Engl. J. Med. 320 (1989) 915^ 924. [34] W. Jessup, S.M. Rankin, C.V. De Whalley, J.R.S. Honlt, J. Scott, D.S. Leak, K-Tocopherol consumption during lowdensity lipoprotein oxidation, Biochem. J. 265 (1990) 399^ 405.
BBALIP 55263 5-6-98
Antioxidant properties of butein isolated from Dalbergia odorifera Zhi-Jiao Cheng a , Sheng-Chu Kuo b , Shiuh-Chuan Chan b , Feng-Nien Ko a , Che-Ming Teng a; * a
Pharmacological Institute, College of Medicine, National Taiwan University, No. 1, Jen-Ai Rd., Sect. 1, Taipei, Taiwan b Graduate Institute of Pharmaceutical Chemistry, China Medical College, Taichung, Taiwan Received 10 March 1998; accepted 18 March 1998
Abstract The antioxidant properties of butein, isolated from Dalbergia odorifera T. Chen, were investigated in this study. Butein inhibited iron-induced lipid peroxidation in rat brain homogenate in a concentration-dependent manner with an IC50 , 3.3 þ 0.4 WM. It was as potent as K-tocopherol in reducing the stable free radical diphenyl-2-picrylhydrazyl (DPPH) with an IC0:200 , 9.2 þ 1.8 WM. It also inhibited the activity of xanthine oxidase with an IC50 , 5.9 þ 0.3 WM. Besides, butein scavenged the peroxyl radical derived from 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH) in aqueous phase, but not that from 2,2-azobis(2,4-dimethylvaleronitrile) (AMVN) in hexane. Furthermore, butein inhibited copper-catalyzed oxidation of human low-density lipoprotein (LDL), as measured by conjugated dienes and thiobarbituric acid-reactive substance (TBARS) formations, and electrophoretic mobility in a concentration-dependent manner. Spectral analysis revealed that butein was a chelator of ferrous and copper ions. It is proposed that butein serves as a powerful antioxidant against lipid and LDL peroxidation by its versatile free radical scavenging actions and metal ion chelation. z 1998 Elsevier Science B.V. Keywords: Antioxidant; Lipid peroxidation; Low density lipoprotein; Butein; Xanthine oxidase; (Dalbergia odorifera)
1. Introduction Butein (2P,4P,3,4-tetrahydroxychalcone), isolated from Dalbergia odorifera T. Chen [1], is a chalcone compound belonging to the £avonoid subclass. The structures of chalcones are similar to curcumin, a known antioxidant [2]. Chalcones exhibit the basic structure with two benzene rings linked through an ,L-unsaturated carbonyl group. They are a group of secondary plant metabolites occurring widely throughout the plant kingdom and are common components of the human diet. Although their importance to human health has remained long unclear,
* Corresponding author. Fax: +886 (2) 2322-1742.
some chalcones have been found to possess anti-in£ammatory, antibacterial, antifungal, and antitumor activities [3,4]. Some derivatives of hydroxychalcones exhibit antioxidative activity and inhibit the activities of xanthine oxidase [5]. It is believed that the biological e¡ects of chalcones mainly come from their antioxidant [6] and metal ion chelating [7] properties. Recent studies also indicate that chalcones inhibit cAMP-phosphodiesterase [8] and glutathione Stransferases [9]. Because many physiological and pharmacological functions have been reported for a variety of chalcones, it is interesting to understand the antioxidant properties of chalcones. Butein has been reported to be an inhibitor of xanthine oxidase, and exhibits inhibitory e¡ects on lipid peroxidation in rat liver mi-
0005-2760 / 98 / $19.00 ß 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 5 - 2 7 6 0 ( 9 8 ) 0 0 0 4 3 - 5
BBALIP 55263 5-6-98
292
Z.-J. Cheng et al. / Biochimica et Biophysica Acta 1392 (1998) 291^299
crosomes [5]. However, the antioxidant properties of butein have not been fully addressed. The aim of this study was to elucidate the antioxidant pro¢le of butein using various experimental models. Furthermore, its protective e¡ects against LDL lipid peroxidation were also evaluated. 2. Materials and methods 2.1. Materials Butein (Fig. 1) was isolated from Dalbergia odorifera T. Chen, as described previously [1], and dissolved in DMSO. Its structure was identi¢ed by UV, IR, MS and NMR. Butylated hydroxytoluene (BHT), K-tocopherol, desferrioxamine mesylate, 2thiobarbituric acid (TBA), tetramethoxypropane (TMP), B-phycoerythrin, 2-deoxyribose, 1,1-diphenyl-2-picrylhydrazyl (DDPH), xanthine oxidase (grade IV, from buttermilk), nitroblue tetrazolium (NBT), catalase, superoxide dismutase (SOD, type I, from bovine liver), D-mannitol, ascorbic acid and hydrogen peroxide (30% solution) were purchased from Sigma (St. Louis, MO). CuSO4 , 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH) and 2,2-azobis(2,4-dimethylvaleronitrile) (AMVN) were obtained from Wako Pure, Japan. cis-Parinaric acid was purchased from Molecular Probes, USA. 2.2. Lipid peroxidation assay of rat brain homogenate Rat brain homogenate was prepared from the brains of freshly killed Wistar rats. The lipid peroxidation in the presence of iron ions was measured by the thiobarbituric acid method, as described previously [10]. Tetramethoxypropane was used as a standard, and the results were expressed as nano-
moles of malondialdehyde (MDA) equivalents per milligram protein of rat brain homogenates. 2.3. DPPH scavenging activity An ethanolic solution of the stable nitrogen centered free radical DPPH (100 WM) was incubated with the test compounds, and the absorbance monitored spectrophotometrically at 517 nm. The concentration (IC0:200 ) of the test compounds that induced a decrease of 0.200 in absorbance during 30 min observation was taken as the free radical scavenging potency [11]. 2.4. Superoxide scavenging activity Superoxide anion was generated by xanthine/xanthine oxidase and measured by the NBT reduction method [12]. Test compounds were incubated in 50 mM KH2 PO4 /K2 HPO4 (pH 7.4) containing K2 H2 -EDTA (0.3 mM), NBT (0.6 mM), xanthine (0.1 mM) and xanthine oxidase (0.02 U/ml). Production of superoxide anion was monitored spectrophotometrically at 560 nm. Superoxide dismutase (100 U/ml) was used as a reference inhibitor. The e¡ect of test compounds on the activity of xanthine oxidase was determined by measuring uric acid formation at 295 nm using the previous condition [13]. A molar extinction coe¤cient, 11 000 M31 cm1 , for uric acid was used for calculation. 2.5. Scavenging of the hydroxyl radical generated by the ascorbate/iron ion/H2 O2 system The reaction mixture contained 2.8 mM 2-deoxyribose, 1.4 mM H2 O2 , 20 WM FeCl3 and 100 WM EDTA without or with test compounds in 10 mM KH2 PO4 -KOH bu¡er, pH 7.4. The reaction was triggered by the addition of 50 WM ascorbate and the mixture incubated at 37³C for 60 min. Solutions of FeCl3 , ascorbate and H2 O2 were made up in deaerated water immediately before use. The extent of deoxyribose degradation by hydroxyl radical was measured with the thiobarbituric acid method [14]. 2.6. Scavenging of hydrogen peroxide
Fig. 1. The chemical structure of butein.
The content of hydrogen peroxide was measured
BBALIP 55263 5-6-98
Z.-J. Cheng et al. / Biochimica et Biophysica Acta 1392 (1998) 291^299
indirectly using a catalase-based method, with a Clark-type electrode (YSI model 5331, Yellow Spring Instruments, OH, USA). The reaction mixture contained 0.5 or 1.0 mM H2 O2 and test compound in 50 mM potassium phosphate bu¡er with 0.2 mM NaCl, pH 7.4. After the mixture was incubated at 25³C for 40 min, catalase (5.8 U/ml) was added and O2 production was monitored polarographically for 0.8 min. After the experiments, the amount of H2 O2 remaining was calculated using a standard curve made for O2 production vs. H2 O2 concentration (0.1^2.0 mM). 2.7. Scavenging of peroxyl radicals in aqueous solution and in hexane The ability of the test compounds to scavenge hydrophilic or lipophilic peroxyl radicals was evaluated by the method described by Tsuchiya et al. [15]. A decrease in B-phycoerythrin £uorescence (Ex 540 nm, Em 575 nm) by AAPH-generated peroxyl radicals was monitored in 20 mM Tris-HCl (pH 7.4) using a £uorescence spectrophotometer (Hitachi, Model F4000). The stoichiometric factors of the test compounds with peroxyl radicals derived from AAPH (25 mM) were calculated according to the lag time during which £uorescence loss was protected. The rate of peroxyl radical formation from AAPH is 1.6U106 /s at 40³C [16]. Ascorbic acid was used as a positive control. The trapping activity of test compounds on lipophilic peroxyl radicals was assessed in a hydrophobic environment (hexane) at 40³C. cis-Parinaric acid, in ethanol, was added to a ¢nal concentration of 30 WM, and its £uorescence monitored (excitation 304 nm, emission 421 nm). AMVN (¢nal concentration 100 mM in chloroform solution) was then added and the quenching of the £uorescence monitored. The test compound was added 2 min after the initiation of AMVN-induced cis-parinaric acid oxidation. K-Tocopherol was used as a positive control. 2.8. Human LDL isolation and oxidation Human low-density lipoproteins (LDLs, d = 1.019V1.063 g/ml) were isolated from citrated plasma by adding 2.5 mM EDTA and using a sequential density gradient ultracentrifugation method
293
at 4³C [17]. LDLs were dialyzed against PBS containing 1 mM EDTA, sterilized by ¢ltration through a 0.22 Wm Millipore ¢lter, and stored at 4³C in sealed vials £ushed with nitrogen until use. Before peroxidation experiments, LDLs were extensively dialyzed with PBS to remove all traces of EDTA. The protective e¡ects of test compounds on the peroxyl radicalinduced oxidation of cis-parinaric acid incorporated into LDL were determined as previously described by Laranjinha et al. [18]. The reaction mixture contained 15 Wg/ml LDL protein and 0.75 WM cis-parinaric acid in PBS and the reaction was triggered by AAPH (10 mM) at 37³C. cis-Parinaric acid degradation was monitored by a £uorescence spectrophotometer (Hitachi, Model F-4000; excitation 324 nm, emission 413 nm). Ascorbic acid was taken as a positive control. Under the experiments of copper-catalyzed LDL oxidation, the reaction mixture contained the vehicle or test compound and LDL (adjusted to 100 Wg LDL protein/ml or 200 Wg LDL protein/ml only for electrophoretic mobility assay) in PBS. The oxidative reaction was carried out at 37³C and induced by adding a freshly prepared CuSO4 solution (¢nal concentration 5 WM). Conjugated diene formation was continuously monitored at 234 nm [19]. To assess the extent of lipid peroxidation, the formation of thiobarbituric acid-reactive substance (TBARS) was measured and expressed as MDA equivalents/mg LDL protein. The extent of aldehyde-modi¢ed lysine in oxidized LDL was monitored by determining the £uorescence intensity (excitation 350 nm, emission 420 nm) [20]. The change on surface charge of the LDL protein was evaluated by an increase in electrophoretic mobility, which was determined using the Lipo¢lm lipoprotein electrophoresis kit supplied by Sebia (France). 2.9. Iron and copper chelation study Iron and copper chelations were determined by adding 200 WM of FeSO4 or 100 WM CuSO4 to the solution containing 100 WM of test compound. After incubation at room temperature for 10 min, the absorption spectra (190V600 nm) were recorded. The chelation of iron (or copper) with test compound was evaluated by absorbance change and/or spectral shift after incubation [21].
BBALIP 55263 5-6-98
294
Z.-J. Cheng et al. / Biochimica et Biophysica Acta 1392 (1998) 291^299
2.10. Determination of protein contents The protein contents of the rat brain homogenates and LDLs were determined as described by Bradford [22] using a Bio-Rad protein assay kit. 20 Wl sample was diluted with 800 Wl deionized water and mixed with 200 Wl dye reagent concentrate and the absorbance was measured at 595 nm. Bovine serum albumin was used as a standard. 2.11. Statistical analysis All experimental data are shown as means þ SE and accompanied by the number of observations. Statistical analysis was performed using Student's t-test, and the signi¢cant di¡erence was set at P 6 0.05. The IC50 value was obtained by regression analysis. 3. Results 3.1. Inhibition of lipid peroxidation in rat brain homogenate
Fig. 2. The inhibitory e¡ects of butein on Fe2 -induced lipid peroxidation in rat brain homogenate. Rat brain homogenates were preincubated with DMSO (0.5%, basal and control) or various concentrations of butein at 37³C for 10 min, then Fe2 (200 WM) was added, except for basal, and incubated for another 30 min. Data are presented as means þ S.E. (n = 6). *P 6 0.05, **P 6 0.01, ***P 6 0.001, as compared with the control.
In unstimulated control experiments, the amount of TBARS formed in rat brain homogenate was 2.1 þ 0.4 nmol/mg protein. In control experiments stimulated by the addition of Fe2 (200 WM), the amount of TBARS was 12.7 þ 1.3 nmol/mg protein. Butein caused a concentration-dependent inhibition of iron-induced lipid peroxidation in rat brain homogenates with an IC50 , 3.3 þ 0.4 WM (Fig. 2). It produced a nearly complete inhibition of ironinduced peroxidation at a concentration of 10 WM. BHT also inhibited this iron-induced lipid peroxidation with an IC50 , 1.3 þ 0.1 WM. Control experiments indicated that butein did not a¡ect the measurement of TBARS because the absorbance at 532 nm was not a¡ected by adding butein to brain homogenate that was either intact or already oxidatively modi¢ed (data not shown).
Metal ions caused a spectral shift and absorbance change of butein (Fig. 3). The Fe2 -butein complex has peaks at 286 nm and 422 nm while the Cu2 butein complex has peaks at 286 nm and 454 nm.
3.2. Iron and copper chelation study
3.4. Superoxide anion scavenging action
Flavonoids have been known to form chelates with a number of metal ions [23]. Butein alone has peaks at 260 nm and 395 nm.
Superoxide anion generated by the xanthine/xanthine oxidase system was monitored by the reduction of NBT. The initial rate of NBT reduction
3.3. Interaction of butein with stable free radical, DPPH In the DPPH assay system, the free radical scavenging activity of antioxidants was expressed as IC0:200 . The scavenging activities of butein, K-tocopherol and BHT were all in a concentration-dependent manner with IC0:200 values of 9.2 þ 1.8, 11.9 þ 0.2 and 14.5 þ 2.5 WM (n = 6), respectively. Apparently, butein was as potent as K-tocopherol and BHT in scavenging DPPH radicals.
BBALIP 55263 5-6-98
Z.-J. Cheng et al. / Biochimica et Biophysica Acta 1392 (1998) 291^299
295
3.6. E¡ect of butein on deoxyribose degradation caused by hydroxyl radical Butein is poorly soluble in aqueous solution at pH 7.4. Therefore, the butein preparation was made in alkaline solution and then the pH adjusted to 7.4 immediately before use. Our results indicated that butein, at 100 WM, did not inhibit the hydroxyl radical-induced deoxyribose degradation. 3.7. E¡ects of butein on peroxyl radical in aqueous solution and in hexane Fig. 3. Absorption spectra of butein and metal ionbutein complex in PBS at pH 7.4. a, absorption spectrum of butein; b, absorption spectrum of Fe2 -butein complex; c, absorption spectrum of Cu2 -butein complex. Note that the absorption spectra in this ¢gure were obtained 10 min after incubation of butein (100 WM) with Fe2 (200 WM) or Cu2 (100 WM).
(0.218 þ 0.002 OD/min, n = 6), caused by xanthine oxidase (0.02 U/ml) generated superoxide anion, was almost completely inhibited by superoxide dismutase (100 U/ml; 0.007 þ 0.002 OD/min, n = 6). Butein (1V10 WM) did not cause direct reduction of NBT since the absorbance of NBT was not changed by butein (data not shown). However, it inhibited the NBT reduction in the xanthine/xanthine oxidase system with an IC50 , 7.1 þ 1.0 WM. Since compounds inhibiting xanthine oxidase also a¡ect NBT reduction, butein was also investigated for its direct e¡ect on xanthine oxidase by measuring the amount of uric acid formation. The initial rate of uric acid formation caused by xanthine oxidase (0.02 U/ml) was 0.228 þ 0.010 OD/min (n = 6). Butein also inhibited xanthine oxidase-induced uric acid formation in a concentration-dependent manner with an IC50 , 5.9 þ 0.3 WM.
Fluorescence of B-phycoerythrin (excitation 540 nm, emission 575 nm) rapidly declined by the attack of peroxyl radicals derived from AAPH (25 mM). This £uorescence loss was delayed by adding butein or ascorbic acid (Fig. 4). Based on the delay in £uorescence decay, we calculated the stoichiometry factor of peroxyl radical with butein or ascorbic acid by the following equation: Stoichiometry AAPHRtime=antioxidant where [AAPH] is the concentration of AAPH (M), [R] is the rate of peroxyl radical production by AAPH, [time] is the duration of consumption of anti-
3.5. The interaction with H2 O2 In our assay system, butein (100 WM) did not react with H2 O2 (0.5V1.0 mM) directly. It did not cause a signi¢cant loss of H2 O2 because the rate of O2 production, as measured in a catalase-based assay, was not changed after 40 min incubation with butein (data not shown).
Fig. 4. Fluorescence study of the e¡ects of butein and ascorbic acid on AAPH-generated peroxyl radicals. Fluorescence of Bphycoerythrin (5 nM) was monitored at 540 nm excitation and 575 nm emission in 20 mM Tris-HCl (pH 7.4) at 40³C. [AAPH] = 25 mM, butein (3 WM, a; 1 WM, b; 0.3 WM, c), ascorbic acid (3 WM, e) or DMSO (0.5%, d) was added at arrow. Inset: concentration dependence of the inhibition period of £uorescence quenching.
BBALIP 55263 5-6-98
296
Z.-J. Cheng et al. / Biochimica et Biophysica Acta 1392 (1998) 291^299
butein concentration (Fig. 5, inset). It seems that butein was more e¡ective than ascorbic acid in the prevention of AAPH-induced cis-parinaric acid oxidation in LDL. 3.9. E¡ects of butein on TBARS and conjugated diene formation and electrophoretic mobility of the LDL stimulated by Cu 2+
Fig. 5. cis-Parinaric acid (0.75 WM) oxidation in human LDL induced by AAPH (10 mM) in the absence or presence of butein. Butein (3 WM, a; 1 WM, b; 0.3 WM, c; 0.1 WM, d), DMSO (0.5%, e) or ascorbic acid (5 WM, f) was added to the reaction mixture at the time indicated by the arrow. Inset: concentration dependence of the inhibition period of £uorescence quenching.
oxidant (s), and [antioxidant] is the concentration of butein or ascorbic acid (M). The stoichiometry factors of butein and ascorbic acid were calculated to be 1.4 þ 0.1 and 0.7 þ 0.1, respectively. That is 1.4 molecules of AAPH-derived peroxyl radicals scavenged by each butein molecule during the delay time. In hexane, the £uorescence of cis-parinaric acid (excitation 304 nm, emission 421 nm) was rapidly declined by the attack of peroxyl radicals derived from AMVN (100 mM). The £uorescence loss could be delayed by K-tocopherol but not by butein.
Butein inhibited the Cu2 -induced TBARS formation of LDL with an IC50 , 6.3 þ 0.2 WM. In addition, butein dose-dependently prolonged the lag phase of conjugated diene formation as shown by the plot of butein concentration vs. lag time (Fig. 6, inset). During the oxidation of unsaturated fatty acid of LDL, reactive aldehydes are formed and bound to the lysine residue of apoB. The modi¢cation of apoB would increase not only the electrophoretic mobility but also the £uorescence (excitation 350 nm, emission 421 nm) intensity of LDL. The LDL incubated with copper ion showed increases in electrophoretic mobility (Fig. 7) and £uorescence intensity (from
3.8. E¡ect of butein on cis-parinaric acid oxidation in LDL As described previously by Laranjinha et al. [18], the £uorescence intensity of cis-parinaric acid was barely detected in the bu¡er solution without LDL. Adding LDL to the bu¡er resulted in a dramatic increase in £uorescence intensity. It may imply that cis-parinaric acid incorporated into the LDL particles. Under this condition, the peroxyl radicals derived from AAPH would quench the £uorescence. Addition of butein or ascorbic acid to the system inhibited the decline of the £uorescence (Fig. 5). According to the length of the delay period, we found that the inhibition periods are proportional to the
Fig. 6. Conjugated diene formation of LDL exposed to Cu2 in the absence or presence of butein. LDL protein (35 Wg/ml) was incubated with 5 WM Cu2 (control, b) for the indicated times in the absence or presence of butein (0.1 WM, F ; 0.2 WM, R; 0.5 WM, S; 1.0 WM, 8). Conjugated dienes were monitored at 15 min intervals at 234 nm and absolute values of absorbance are plotted. The data shown are from a single experiment of four independent experiments. Inset: concentration dependence of the inhibition period of diene formation.
BBALIP 55263 5-6-98
Z.-J. Cheng et al. / Biochimica et Biophysica Acta 1392 (1998) 291^299
Fig. 7. E¡ect of butein on electrophoretic mobility change of human LDL. LDL protein (200 Wg/ml) was incubated with CuSO4 (5 WM) in phosphate-bu¡ered saline at 37³C for 12 h in the absence or presence of various concentrations of butein. Then, the reaction was stopped by 50 WM BHT, and the samples were subjected to electrophoresis analysis. (1) standard; (2) native LDL; (3) ox-LDL. LDL oxidized in the presence of 1 WM butein (4); 3 WM butein (5); 10 WM butein (6); 30 WM butein (7); 100 WM butein (8).
3.1 þ 0.1 to 62.8 þ 3.4 AU). However, we found that butein would quench the £uorescence of oxidatively modi¢ed LDL, so we further investigated the e¡ects of butein on the apoB modi¢cation of LDL by electrophoretic mobility change. In the presence of butein, we found that 30 WM butein could obviously reduce the electrophoretic change of oxidatively modi¢ed LDL (Fig. 7). 4. Discussion Rat brain homogenates are usually used as a preparation to evaluate the antioxidant activities of compounds on lipid peroxidation [24,25]. It is known that transition metal ions are involved in both initiation and propagation of lipid peroxidation [26]. Rat brain homogenates exposed to ferrous ion exhibited lipid peroxidation in air by a mechanism which may involve site-bound iron-mediated decomposition of lipid hydroperoxides to yield alkoxyl or peroxyl radicals, leading to the chain reaction of lipid peroxidation [27]. In this system, we found that butein e¡ectively inhibited ferrous ion-induced lipid peroxidation. Its potency was comparable to BHT, a typical antioxidant in food. The antiperoxidative activity of butein may be attributed to metal ion chelation, free radical scavenging or membrane stabilization. Desferrioxamine, a potent iron chelator, could inhibit iron-induced lipid peroxidation to 50% at a concentration approximately half of the
297
iron added to rat brain homogenates [28]. Although butein also directly chelated ferrous ion, it needed 30-fold less than desferrioxamine to inhibit the lipid peroxidation to the same degree. Moreover, according to the DPPH assay, butein acted as a direct free radical scavenger with a potency (IC0:200 , 9.2 þ 1.8 WM) similar to K-tocopherol, a chain-breaking antioxidant. Hence, we believe that the metal ion chelating property of butein is not mainly component contributed to its anti-peroxidation activity in the brain homogenates. Generation of peroxyl radicals is a necessary step in the formation of TBARS during lipid peroxidation [29]. We assessed the peroxyl radical scavenging activity of butein by halting the AAPH- and AMVNinduced £uorescence decay of B-phycoerythrin and cis-parinaric acid in aqueous and lipophilic phases, respectively. Our results indicated that butein scavenged the hydrophilic but not the lipophilic peroxyl radicals. Consequently, the localization of antioxidants and the generation site of radicals should be taken into account as evaluating their antioxidant activity. In AAPH-initiated peroxidation of LDL, chain-initiating radicals are generated in aqueous phase and chain-propagating lipid peroxyl radicals are located within membranes [30]. Butein e¡ectively scavenged peroxyl radicals in LDL solution and PBS but not in hexane. We suggest that butein is an effective membrane antioxidant located in the interface between membranous and aqueous phase. However, butein could not exert its antioxidant activities in an extremely hydrophobic phase such as hexane. It is well known that superoxide anions could damage biomacromolecules directly or indirectly by forming hydrogen peroxide, hydroxyl radical, peroxynitrite or singlet oxygen during pathophysiological events such as ischemia-reperfusion injury [31]. In the xanthine/xanthine oxidase system, butein inhibited NBT reduction and uric acid formation in a concentration-dependent manner with IC50 , 7.1 þ 1.0 WM and 5.9 þ 0.3 WM, respectively. Clearly, butein is an e¡ective inhibitor of xanthine oxidase. It is usually accepted that the superoxide anion generated from xanthine oxidase in hypoxia leads to ischemia-reperfusion injury [32]. Therefore, butein would be expected to prevent injury induced by hypoxia. The oxidation of LDL has been suggested to be a key event in human atherosclerosis [33]. The detailed
BBALIP 55263 5-6-98
298
Z.-J. Cheng et al. / Biochimica et Biophysica Acta 1392 (1998) 291^299
mechanism of LDL oxidation in vivo is unknown and remains to be clari¢ed. In copper-catalyzed LDL oxidation, the appropriate mechanism also has not been clearly de¢ned. However, one suggestion is that copper initially catalyzed the breakdown of the lipid hydroperoxides in the surface phospholipids or in the core cholesteryl ester, subsequently, modi¢cation of phospholipids and cholesteryl esters were formed and propagated. Eventually, the lysine residues of apoB were modi¢ed by the highly reactive products of oxidized fatty acid (i.e., malondialdehyde and 4-hydroxynonenal). In such oxidized LDL, its surface charge tends to be negative and the characteristic £uorescent chromogen is exposed [34]. In our assay systems butein not only prevented AAPH-initiated cis-parinaric acid oxidation in LDL solution but also inhibited copper-catalyzed lipid peroxidation. Since butein could directly chelate copper, these results imply that butein is not only an e¡ective metal ion chelator but also a powerful chain-breaking antioxidant in the LDL systems. Thus butein would be a potential protector against the oxidative modi¢cation of LDL. In conclusion, this study veri¢es that butein is a natural antioxidant against lipid peroxidation in rat brain homogenates and human LDL. Its antioxidant activities are primarily attributed to its free radical scavenging actions and metal ion chelation. It will be interesting to further investigate the e¡ects of butein on various radical-mediated injury models in vitro and in vivo. Acknowledgements This work was supported by a research grant of the National Science Council of Taiwan (NSC 872314-B-002-159-M25). References [1] S.M. Yu, Z.J. Cheng, S.C. Kuo, Endothelium-dependent relaxation of rat aorta by butein, a novel cyclic AMP-speci¢c phosphodiesterase inhibitor, Eur. J. Pharmacol. 280 (1995) 69^77. [2] K. Elizabeth, M.N.A. Rao, Oxygen radical scavenging activity of curcumin, Int. J. Pharmaceut. 58 (1990) 237^ 240.
[3] R.J. Anto, K. Sukumaran, G. Kutta, M.N.A. Rao, V. Subbaraju, R. Kuttan, Anticancer and antioxidant activity of synthetic chalcones and related compounds, Cancer Lett. 97 (1995) 33^37. [4] C. Chin, P. Nagaratnam, Cytotoxic e¡ect of butein on human colon adenocarcinoma cell proliferation, Cancer Lett. 82 (1994) 65^72. [5] S. Sogawa, Y. Nihro, H. Ueda, T. Miki, H. Matsumoto, T. Satoh, Protective e¡ects of hydroxychalcones on free radicalinduced cell damage, Biol. Pharmacol. Bull. 17 (1994) 251^ 256. [6] L. Larson, The antioxidants of higher plants, Phytochemistry 27 (1988) 969^978. [7] T.W. Wu, K.P. Fung, L.H. Zeng, J. Wu, A. Hempel, A.A. Grey, N. Camerman, Molecular properties and myocardial salvage e¡ects of morin hydrate, Biochem. Pharmacol. 49 (1995) 537^543. [8] U.R. Kuppusamy, N.P. Das, E¡ects of £avonoids on cyclic AMP phosphodiesterase and lipid mobilization in rat adipocytes, Biochem. Pharmacol. 44 (1992) 1307^1315. [9] K. Zhang, N.P. Das, Inhibitory e¡ects of plant polyphenols on rat liver glutathione S-transferases, Biochem. Pharmacol. 47 (1994) 2063^2068. [10] F.N. Ko, C.H. Liao, Y.H. Kuo, Y.L. Lin, Antioxidant properties of demethyldiisoeugenol, Biochim. Biophys. Acta 1258 (1995) 145^152. [11] A. Mellors, A.L. Tappel, The inhibition of mitochondrial peroxidation by ubiquinone and ubiquinol, J. Biol. Chem. 241 (1966) 4353^4356. [12] M. Nishikimi, N.A. Roa, K. Yagi, The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen, Biochem. Biophys. Res. Commun. 46 (1972) 849^854. [13] I. Fridovich, Quantitative aspects of the production of superoxide anion radical by milk xanthine oxidase, J. Biol. Chem. 245 (1970) 4053^4057. [14] B. Halliwell, J.M.C. Gutteridge, Formation of a thiobarbituric-acid-reactive substance from deoxyribose in the presence of iron salts, FEBS Lett. 128 (1981) 347^352. [15] M. Tsuchiya, G. Scita, H.J. Freisleben, V.E. Kagan, L. Packer, Antioxidant radical-scavenging activity of carotenoids and retinoids as compared to K-tocopherol, Methods Enzymol. 213 (1992) 460^472. [16] K. Teras, D. Niki, Damage to biological tissues induced by radical initiator 2,2-azobis(2-aminopropane) dihydrochloride and its inhibition by chain breaking antioxidant, Free Radical Biol. Med. 2 (1986) 193^201. [17] R.J. Havel, H.A. Eder, L.H. Bragdon, The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum, J. Clin. Invest. 34 (1955) 1345^ 1353. [18] J.A.N. Laranjinha, L.M. Almeida, V.M.C. Maderira, Lipid peroxidation and its inhibition in low density lipoproteins: quenching of cis-parinaric acid £uorescence, Arch. Biochem. Biophys. 297 (1992) 147^154. [19] H. Esterbauer, J. Gebicki, H. Puhl, G. Jurgens, The role of
BBALIP 55263 5-6-98
Z.-J. Cheng et al. / Biochimica et Biophysica Acta 1392 (1998) 291^299
[20]
[21]
[22]
[23]
[24]
[25]
[26]
lipid peroxidation and antioxidants in oxidative modi¢cation of LDL, Free Radical Biol. Med. 13 (1992) 341^390. U.P. Seinbrecher, Oxidation of human low density lipoprotein results in derivatization of lysine residues of apoprotein B by lipid peroxide decomposition products, J. Biol. Chem. 262 (1987) 3603^3608. A. Rahman, S.M.H. Shahabuddin, J.H. Parish, Complexes involving quercetin, DNA and Cu(II), Carcinogenesis 11 (1990) 2001^2003. M.M. Bradford, A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248^254. I.B. Afanas'ev, A.I. Dorozhko, A.V. Brodskii, V.A. Kostyuk, A.I. Potapovitch, Chelating and free radical scavenging mechanisms of inhibitory action of rutin and quercetin in lipid peroxidation, Biochem. Pharmacol. 38 (1989) 1763^ 1769. J.M. Braughler, R.L. Burton, R.L. Chase, J.F. Pregenzer, E.J. Jacobsen, F.J. VanDoornik, J.M. Tustin, D.E. Ayer, G.L. Bundy, Novel membrane localized ion chelators as inhibitors of iron-dependent lipid peroxidation, Biochem. Pharmacol. 37 (1988) 3853^3860. A.B. Kozlov, E.A. Ostrachovitch, I.B. Afanas'ev, Mechanism of inhibitory e¡ects of chelating drugs on lipid peroxidation in the rat brain homogenates, Biochem. Pharmacol. 47 (1994) 795^799. B. Halliwell, J.M.C. Gutteridge, Oxygen toxicity, oxygen radicals, transition metal and diseases, Biochem. J. 219 (1984) 1^5.
299
[27] J.M. Braughler, R.L. Chase, J.F. Pregenzer, Oxidation of ferrous iron during peroxidation of various lipid substrates, Biochim. Biophys. Acta 921 (1987) 457^464. [28] C.M. Teng, G. Hsiao, F.N. Ko, D.T. Lin, S.S. Lee, N-Allylsecoboldine as a novel antioxidant against peroxidation damage, Eur. J. Pharmacol. 303 (1996) 129^139. [29] J.M.C. Gutteridge, B. Halliwell, The measurement and mechanism of lipid peroxidation in biological systems, Trends Biochem. Sci. 15 (1990) 129^135. [30] Y. Yamamoto, E. Niki, J. Eguchi, Y. Kamiya, H. Shimasaki, Oxidation of biological membranes and its inhibition. Free radical chain oxidation of erythrocyte ghost membranes by oxygen, Biochim. Biophys. Acta 819 (1985) 29^36. [31] R. Radi, J.S. Beckman, K.M. Bush, B.A. Freeman, Peroxynitrite-induced membrane lipid peroxidation: the cytotoxic potential of superoxide and nitric oxide, Arch. Biochem. Biophys. 288 (1991) 481^487. [32] R.C. Kukreja, M.L. Hess, The oxygen free radical system: from equations through membrane-protein interactions to cardiovascular injury and protection, Cardiovasc. Res. 26 (1992) 641^655. [33] D. Steinberg, S. Parthasarathy, T.E. Carew, J.L. Khoo, J.L. Witztum, Modi¢cations of low-density lipoprotein that increase its atherogenicity, New Engl. J. Med. 320 (1989) 915^ 924. [34] W. Jessup, S.M. Rankin, C.V. De Whalley, J.R.S. Honlt, J. Scott, D.S. Leak, K-Tocopherol consumption during lowdensity lipoprotein oxidation, Biochem. J. 265 (1990) 399^ 405.
BBALIP 55263 5-6-98