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Structure prerequisite for antioxidant activity of silybin in different biochemical systems in vitro.

Abstract

Structural analogues (flavanone: 2-4 and flavone: 5 and 6, respectively) of silybin (1a) were synthesized and tested for inhibitory activity on [O.sub.2.sup.*-] release and PKC translocation in PMA-stimulated neutrophils as well as xanthine oxidase activity in order to identify the molecular structures responsible for the antioxidant property of silybin. Concerning the prevention of hem-mediated oxidative modification of LDL by silybin, the hydroxyl radical scavenging activity of its structural analogues was also determined. We demonstrated that the basic skeleton of 1a (4) is responsible for its inhibitory activity on [O.sub.2.sup.*-] release in PMA-stimulated neutrophils via inhibition of PKC translocation, since introduction of a double bound and hydroxyl groups at C-5 and C-7 position (5 and 6) did not result in further increase in inhibition of [O.sub.2.sup.*-] release. It has been shown that the presence of the phenolic hydroxyl group at C-5 and C-7 of 1a is essential for the inhibition of xanthine oxidase activity. Moreover, introduction of a double bond into the C-ring of 2 and 3, resulting in flavone derivatives (5 and 6), markedly enhanced the antioxidant effect in all the tested systems. Finally, silybin (1a) and its flavon derivatives (5 and 6) directly scavenged hydroxyl radicals as well. On the basis of these results it might be concluded that different moiety of silybin is responsible for inhibition of overproduction of [O.sub.2.sup.*-] in stimulated neutrophils, xanthine oxidase activity, and for prevention of hem-mediated oxidative modification of LDL.

[c] 2005 Elsevier GmbH. All rights reserved.

Keywords: Silybin; Oxidative stress; Antioxidants; Protein kinase C; Xanthine oxidase; Low density lipoprotein

Introduction

Recently, it has been demonstrated that several groups of phenolic compounds including flavanolignans, such as silybin, isosilybin, silydianin and silychristin display very potent antioxidant and anticancer activity (Zi et al., 1998; Afaq et al., 2002; Dhanalakshmi et al., 2002; Singh et al., 2002, 2003; Gallo et al., 2003; Agarwal et al., 2003), and prevents hepatocytes from ethanol induced damage (van Pelt et al., 2003). The pharmacological activities of flavonoids are assumed to derive mainly from the inhibition of certain enzymes involved in the formation of reactive oxygen species (ROS). In fact, many flavonoids have been reported to be potent inhibitors of ROS production enzymes such as xanthine oxidase (Cos et al., 1998; Sheu et al., 1998). Furthermore, flavonoids inhibit superoxide anion formation in stimulated neutrophils (Limasset et al., 1993; Lu et al., 2001; Selloum et al., 2001; Varga et al., 2001), and inhibit Cytochrome P-450 enzyme activity (Beckmann-Knopp et al., 2000) or scavenge superoxide anions (Furuno et al., 2002).

We have previously demonstrated that silybin (1a) inhibits the release of superoxide anion ([O.sub.2.sup.*-]) in phorbol-myristate-acetate (PMA)-stimulated neutrophils and modification of its lipophilicity significantly alters its action (Varga et al., 2001). It was also demonstrated that silybin (1a) inhibits protein kinase C (PKC) translocation and NADPH oxidase activity in PMA-stimulated neutrophils in a concentration dependent manner, and an increase in lipid solubility by methylation of the phenolic hydroxyl groups of 1a (1a [right arrow] 1b) improves its antioxidant ability presumably via enhanced penetration through the membrane lipid bilayer (Varga et al., 2004).

Flavonoids have been recently classified into several classes according to their ability to inhibit xanthine oxidase activity and scavenge superoxide anion formation (Cos et al., 1998; Furuno et al., 2002). In order to rank silybin (1a) in this classification and get further insights in the structure prerequisite of its antioxidant activity we set our sights on the study of 1a and its related compounds (2-6) regarding their effect on xanthine oxidase activity, free radical (e.g. superoxide anion and hydroxyl radicals) scavenging ability and prevention of hem-mediated modification of low density lipoprotein (LDL). Considering the fact that [O.sub.2.sup.*-] production involves the activation of PKC in a wide variety of cells including neutrophils (Gopalakrishna and Jaken, 2000), and silybin (1a) has been found to be its potent inhibitor (Varga et al., 2004), we determined the PKC activity in PMA stimulated neutrophils exposed to silybin's analogues (2-6).

[FIGURE 1 OMITTED]

Materials and methods

Compounds

Silybin (1a) was purchased from Sigma (Sigma Co, St. Louis, MO, USA). Flavanone (2-4) and flavone (5 and 6) derivatives were prepared as described previously (Czompa et al., 2000). Structures of these compounds are presented in Fig. 1.

Flavonoid stock solutions and diluted working solutions were prepared in DMSO except for the case when hydroxyl radical scavenging activity was studied (see below). From these solutions, 5 [micro]l was added to the samples to achieve the required concentrations. The same volume of DMSO was added to the control in all the experiments.

PMNL separation

It was performed according to the method of Boyum (Boyum, 1968) and published procedures (Varga et al., 2001). A PMNL purity over 95% and cell viability greater than 95% were microscopically ascertained by Giemsa staining and trypan blue exclusion, respectively.

Assay of superoxide generation

[O.sub.2.sup.*-] was assessed spectrophotometrically by measuring the reduction of Cytochrome C (Type IV, Sigma Co., St. Louis, MO, USA) with the methods of Babior (Babior et al., 1970) in a micro-assay using 96-well microplate and an ELISA reader (Anthos Labtec, Wien, Austria) as was published in detail previously (Varga et al., 2001). The cells were preincubated with the tested molecules for 20 min at 37[degrees]C and were stimulated by PMA at a final concentration of [10.sup.-7] M. The absorbances were measured at 550 nm for Cytochrome C reduction and at 492 nm for baseline correction. Each experiment was performed in triplicates. The results are expressed as percentage of the controls.

PKC activity in the cytosol

It was determined in the PMA-activated neutrophils using a non-radioactive Elisa kit from Calbiochem (Calbiochem-Novabiochem Co. San Diego, CA, USA) according to the protocol of kit and as previously published (Varga et al., 2004). Cell free cytosol was prepared from 5 x [10.sup.6] cells/ml after preincubation with silybin (1a) and its analogues (2-6) at a concentration of 20 [micro]M for 20 min at 37[degrees]C in Hanks' Balanced Salt solution (HBSS, Sigma Co., St. Louis, MO, USA). Afterwards, neutrophils were stimulated with PMA (Sigma, St. Louis, MO, USA) at [10.sup.-7] M, for 3 min at 37[degrees]C. The reaction was stopped by 10 volume of ice-cold phosphate buffer. After centrifugation, cells were resuspended in the sample preparation buffer (part of the kit containing 50 mM Tris-HCl, 50 mM [beta]-mercaptoethanol, 10 mM EGTA, 5mM EDTA, 1 mM PMSF, 10 mM benzamidine, pH = 7.5) and were disrupted. The cell lysate was centrifuged (4[degrees]C, for 1h) and the supernatant was used immediately in the kit. PKC activity was determined in the presence of calcium, phosphatidylserine and ATP (final concentrations in reaction mixture: 25 mM Tris-HCl (pH = 7.0), 0.3 mM Mg[Cl.sub.2], 0.1 mM ATP, 2 mM Ca[Cl.sub.2], 50 [micro]g/ml phosphatidylserine, 0.5 mM EDTA, 1 mM EGTA, 5 mM [beta]-mercaptoethanol). All experiments were performed in duplicates in neutrophils of three different donors. Protein content of cytosol fraction was determined by the method of Lowry (Lowry et al., 1951).

Xanthine oxidase activity

It was determined by two independent methods.

(a) Spectrophotometrical method measuring uric acid formations (Sheu et al., 1998), using xanthine as substrate (at a final concentration of 60 [micro]M) and xanthine oxidase (0.044 U/ml) in the presence and the absence of the tested molecules (1a, 2-6) at concentration ranges of 1-100 [micro]M. Xanthine oxidase was preincubated with the tested compounds for 10 min at room temperature. Reaction was started by the addition of xanthine. Formation of uric acid was followed at 295 nm in a spectrophotometer for 3 min (Hewlett Packard type: 8453, USA). Concentration of the formed uric acid was determined by a calibration curve prepared from known concentrations of uric acid. All experiments were performed in triplicates. Results are expressed as mean [+ or -] SD of three independent experiments.

(b) Fluorimetric method using 2-amino-4-hydroxypteridine (AHP, Sigma St. Louis, MO, USA) as substrate at a final concentration of [10.sup.-5] M (Haining and Legan, 1967). Experimental conditions were the same as described above. Fluorescence of the hydrolysis product of AHP was measured at excitation wavelength of 345 nm and emission wavelength of 390 nm. Results are expressed as percentage of fluorescence in control, which contained the same volume of DMSO as samples. All experiments were performed in triplicates. In this system, compound 4 could not be measured due to its autofluorescence.

Hem-mediated oxidative modification of LDL

It was measured as described previously (Ujhelyi et al., 1998). Namely, LDL was separated from the plasma of healthy donors by gradient centrifugation (50.2 Ti rotor, Beckman Instruments, 300 000g, 4[degrees]C, 3 h). Test molecules (at concentrations of 20-100 [micro]M) were added to the LDL samples and preincubated for 15 min at room temperature. Measurement of the oxidative resistance of LDL was based on the kinetics of hem-catalyzed lipid peroxidation and delta T at [V.sub.max] values (in sec) were determined. All experiments were performed in triplicates. Results are expressed as mean [+ or -] SD of three independent measurements.

Hydroxyl radical scavenging activity

Hydroxyl radical was produced in the presence of 20 [micro]M Fe[Cl.sub.3], 1.4mM [H.sub.2][O.sub.2], 2.8 mM deoxyribose, 100 [micro]M EDTA, and 100 [micro]M ascorbate, in 10 mM phosphate buffer, pH = 7.4, as was published earlier (Sanz et al., 1994). For these experiments test compounds of 1a, 2-6 were dissolved in a small amount of 1 M aqueous NaOH diluted immediately by phosphate buffer to achieve the appropriate concentrations. Deoxyribose degradation by hydroxy radical was measured with the thiobarbituric acid method (Okhava et al., 1979). All experiments were performed in triplicates.

Determination of TBARS

Three hundred microliter of sample was added to 600 [micro]l of acidic thiobarbituric acid reagent. The samples were heated at 100[degrees]C for 15 min, and after cooling the TBARS was extracted by 3 ml of butanol (Okhava et al., 1979). The absorbance of extract was recorded at 532 nm and the results are expressed as percentage of control. All determinations were performed in triplicates.

Statistical analysis

Data were expressed as means [+ or -] SD. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by t-test, paired or unpaired for multiple comparisons. Differences were considered significant at p < 0.05.

Results

Effect of 2-6 on superoxide anion release by human neutrophils

Silybin (1a) and its structural analogues (2-6) inhibited the PMA stimulated [O.sub.2.sup.*-] release in human neutrophils as shown in Fig. 2a. Flavanone derivatives possessing hydroxyl groups at C-5 and C-7 position (2, 3) have been found to exhibit the same order of inhibitory activity on PMA stimulated [O.sub.2.sup.*-] release as was found for silybin (1a). It is noteworthy that removal of the hydroxyl group at C-5 and C-7 of 3 (3 [right arrow] 4) led to an increased inhibition of [O.sub.2.sup.*-] release in PMA stimulated neutrophils. The same effect was achieved by the introduction of a double bound in the ring C of flavanones (2, 3) resulting in flavone derivatives 5 and 6, respectively. It has to be noted that weak pro-oxidant effect was observed (data not shown) when compounds were used at low concentrations (less than 5 [micro]M). This might be due to the fact that cells in culture may behave differently from cells in vivo and that is the pro-oxidant effect of medium (Halliwell, 2003) which might overlap using antioxidants at high concentrations.

[FIGURE 2 OMITTED]

Since [O.sub.2.sup.*-] production in human neutrophils requires stimulation and translocation of PKC from the cytosol to the membrane fraction (Gopalakrishna and Jaken, 2000) and silybin (1a) inhibits this activation process (Varga et al., 2004) the effects of its structural analogues (2-6) on PKC activity were also examined. The results showed that inhibition of [O.sub.2.sup.*-] production in PMA-stimulated neutrophils paralleled with the inhibition of the PKC translocation from cytosol to membrane fraction e.g. with increased remaining PKC activity in the cytosol (Fig. 2b). These results prove that flavanone (2-4) and flavone (5 and 6) analogues of silybin (1a) attenuate [O.sub.2.sup.*-] production in PMA-stimulated neutrophils by a mechanism in which PKC is involved, and that in this respect basic skeleton of 1a (4) was the most potent.

Effects of 2-6 on xanthine oxidase activity

The flavanone derivatives 2 and 3 proved to be similarly effective in the inhibition of xanthine oxidase activity measured either by the formation of uric acid (Fig. 3a) or by the generation of fluorescence product from AHP (Fig. 3b) as was found for silybin (1a). In contrast to silybin (1a) and its analogues 2 and 3, the flavanone derivative 4 containing no hydroxyl group proved to be a very weak inhibitor of xanthine oxidase (Fig. 3a). Interestingly, flavone derivatives 5 and 6 almost totally blocked uric acid formation at a concentration less than 5 [micro]M in both systems. I[C.sub.50] values of the tested molecules are shown in Table 1, where the first row shows I[C.sub.50] measured by uric acid formation and second one shows I[C.sub.50] measured by AHP hydrolysis. Both methods gave similar results; flavanone derivatives 2 and 3 have I[C.sub.50] values in similar range as was found for silybin (1a), and flavone derivatives 5 and 6 had extremely low I[C.sub.50] values, while flavanone derivative 4 had I[C.sub.50] > 100 [micro]M.

[FIGURE 3 OMITTED]

Michaelis-Menten constants were also determined by Lineweaver-Burk plots in the xanthine/xanthine oxidase system. Results showed that not only silybin (1a) (Sheu et al., 1998) but its structural analogues (2-6) altered both [V.sub.max] and [K.sub.m] values suggesting a non-competitive-uncompetitive mechanism in the inhibition of xanthine oxidase activity (Table 2).

[FIGURE 4 OMITTED]

In order to determine whether these molecules (2-6) possess a direct superoxide scavenging activity [O.sub.2.sup.*-] formation induced by xanthine/xanthine oxidase system was measured. Reduction of Cytochrome C in the presence of silybin and its analogues (1a and 2-6) is shown in Fig. 4. Since, I[C.sub.50] values for Cytochrome C reduction were higher for all the tested molecules (Table 3) compared to I[C.sub.50] values for uric acid formation (Table 1), it can be concluded that none of them have direct superoxide scavenging activity.

Prevention of oxidative modification of LDL

In physiological circumstances, oxidative modification of LDL is one of the most important events in the initiation of atherosclerosis. Since natural antioxidants such as Vitamin E (Halliwell and Guttridge, 1999) or silybin (1a) (Locher et al., 1998; Skottova et al., 1999; Brown et al., 1998; Varga et al., 2004) prevent the oxidative modification of LDL, we investigated the effects of silybin analogues (2-6) on hem mediated oxidative modification of LDL. Results show that not only silybin (1a) but also its flavone analogues (5 and 6) prevented LDL from hem-mediated oxidative modification, while its flavanone analogues (2-4) were ineffective at concentration of 20 [micro]M (Fig. 5). It has to be noted that flavanone derivatives 2 and 3 also enhanced the oxidative resistance of LDL (from 3390 to 5900 s and 8500s, respectively) at high concentration (100 [micro]M) but compound 4 was ineffective at even this high concentration.

Hydroxyl radical scavenging activity

Since hydroxyl radical formation is involved in the oxidative modification of LDL (e.g. oxidative destruction of polyunsaturated fatty acids) direct hydroxyl radical scavenging properties of silybin (1a) and its analogues (2-6) were also studied. As shown in Fig. 6, silybin (1a) slightly and its flavone analogues (5 and 6) significantly inhibited hydroxyl radical formation. It is noteworthy that flavanone derivatives 2-4 were found to be equally ineffective (Fig. 6). These results suggest that prevention of LDL from hem-mediated oxidative modification might be connected to the hydroxyl radical scavenging activity of silybin (1a) and its flavone analogues 5 and 6. However, in the case of silybin (1a), which possesses only a weak direct hydroxyl radical scavenging activity, its lignane moiety as well as its hydroxyl group at C-3 might also play a role in the prevention of hem-mediated LDL oxidation.

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]

Discussion

Silymarin, a mixture of flavanolignans (silybin, isosilybin, silychristin, silydianin) isolated from the seeds of the violet-flowered Silybum marianum has high human acceptance being used clinically and consumed as a dietary supplement around the world for its strong antihepatotoxic efficacy (Flora et al., 1998; Wellington and Jarvis, 2001). Silymarin is well tolerated and largely free of any adverse effects; non-toxic in acute, subchronic and chronic tests even at large doses; and there is no known L[D.sub.50] for silymarin in animal studies (Flora et al., 1998; Wellington and Jarvis, 2001). A wide range of studies have shown strong cancer chemopreventive and anticancer efficacy of silymarin and sylibin in different animal tumor bioassay systems as well as in cell culture models of epithelial cancers such as skin (Ahmad et al., 1998; Singh et al., 2002), prostate (Dhanalakshmi et al., 2002), and breast (Bhatia et al., 1999) cancers.

It has been previously reported that flavanoids (Cos et al., 1998) and sylimarin (Sheu et al., 1998) possess a very potent inhibition on xanthine oxidase activity. It was also demonstrated that silybin (1a) and its derivative (1b) of increased lipid solubility inhibit [O.sub.2.sup.*-] release in PMA-stimulated human neutrophils (Varga et al., 2001) via inhibition of PKC translocation and NADPH oxidase activity (Varga et al., 2004). Structure of polyphenols, flavonolignans and other antioxidants represents usually an aromatic ring and several hydroxyl groups (Halliwell and Guttridge, 1999). However, the underlying mechanism of action remains to be elucidated.

In the present study, in order to identify the structure responsible for the antioxidant effect of silybin (1a) we examined the antioxidant activity of its related compounds such as flavanone (2-4) and flavone derivatives (5 and 6). It was found that flavanone derivatives 2 and 3, except 4, have similar inhibitory effects on both [O.sub.2.sup.*-] production induced by PMA in human neutrophils and xanthine oxidase activity compared to silybin (1a) itself. These results suggest that neither the C-3 hydroxyl group nor the hydroxymethyl and 3-methoxy-4-hydroxyphenyl groups of silybin (1a) play a determinant role in these processes. Furthermore, flavanone derivative 4 proved to be a weak inhibitor of xanthine oxidase, no scavenger of hydroxyl radical and ineffective in the protection of hem-mediated LDL oxidation. Therefore it could be concluded that the hydroxyl groups at C-5 and C-7 position of the flavanon skeleton are essential for the antioxidant activity of 1a concerning xanthine oxidase. In contrast, flavanone 4 was found to be a very potent inhibitor of [O.sub.2.sup.*-] production and PKC translocation in PMA-stimulated neutrophils. On these bases, it is presumable that the C-5 and C-7 hydroxyl groups in the ring A of flavanones do not play any role in the inhibition of PKC activity and hence in the reduction of [O.sub.2.sup.*-] production. Several investigators demonstrated that structure of flavonoids play an important role in the inhibition of signal transduction enzymes such as PKC (Gopalakrishna and Jaken, 2000; Williams et al., 2004). It appears that the number of hydroxyl groups on the B ring and the degree of saturation of the C ring are important determinants of this particular bioactivity (Williams et al., 2004). Our results reinforced the importance of C2-C3 double bond in the inhibition of PKC activity in PMA stimulated neutrophils.

It is noteworthy that the introduction of a double bond at C2-C3 in the ring C of 2 or 3, resulting in the corresponding flavone derivatives (5 and 6, respectively), improved antioxidant activity of the molecules in the tested system. On the basis of these results it is presumable that the introduction of a double bond in the ring C of silybin (1a [right arrow] 7) would improve not only its xanthine oxidase inhibitory activity, but also the prevention of LDL from hem-mediated oxidative modification. Since, ROS production is induced by hem in the presence of hydrogen peroxide and all effective molecules (1a, 5, 6) scavenged hydroxyl radicals, it is presumable that their activity towards the prevention of LDL oxidation might be strongly connected to their hydroxyl radical scavenging activity. Our results considering the inhibition of the hem-protein mediated oxidative degradation of LDL are consistent with the finding that luteolin, with similar structure to flavone derivatives of 5 and 6, proved to be a very potent inhibitor of hem-protein mediated degradation of LDL though quercetin with 3-OH group on the C ring, possessed the highest activity (Brown et al., 1998). The importance of the C2-C3 double bound and 3-OH group in the C ring considering the hydrogen-donating capacity of flavanone and flavone derivatives was previously discussed (Rice-Evans et al., 1996). On that system quercetin was more potent than luteolin and rutin suggesting that the 4-keto group functions in conjugation with the C2-C3 double bound in hydrogene-donating.

Moreover, the results presented here suggest that none of the tested molecules, including silybin (1a), have superoxide anion scavenging capability; therefore the decreased [O.sub.2.sup.*-] production in PMA-stimulated neutrophils might be due to the inhibition of PKC translocation from the cytosol to membrane fraction.

According to the classification of Cos et al. (1998), silybin (1a), its flavanone (2, 3) and flavone (5, 6) derivatives belong to family of B type flavonoids which inhibit xanthine oxidase activity but not scavenge [O.sub.2.sup.*-] radical formation. Moreover, the flavanone derivative of 4 represents family of F type flavanoids such as naringenin (Cos et al., 1998), inhibiting only PKC activity without affecting xanthine oxidase activity and scavenging [O.sub.2.sup.*-] radical.

In summary, structural modification of silybin (1a), resulting in flavanone derivatives 2-3, did not influence its inhibition capability concerning [O.sub.2.sup.*-] production in PMA-stimulated neutrophils and xanthine oxidase activity. However, the derivatives 5 and 6 containing identical flavone moiety proved to be more efficient in inhibiting [O.sub.2.sup.*-] production in PMA-stimulated neutrophils and xanthine oxidase activity. Furthermore, they possessed hydroxyl radical scavenging activity, and enhanced the oxidative resistance of LDL suggesting that the double bound at C2-C3 position in the ring C has crucial role in the antioxidant activity of silybin (1a). Finally, hydroxyl groups at C-5 and C-7 position in the ring A (compound of 4) are not necessary for the inhibition of [O.sub.2.sup.*-] production in PMA-stimulated neutrophils but are essential for inhibition of xanthine oxidase. These results suggest that the dehydrosilybin (7), a metabolite of 1a, might be important for the antioxidant activity of silybin (1a) under in vivo circumstances, and that a specific structural feature and substitution pattern of silybin might be responsible for inhibition of overproduction of [O.sub.2.sup.*-] in stimulated neutrophils, xanthine oxidase activity, and for prevention of oxidative modification of LDL.

Acknowledgement

Research work was sponsored by National Scientific Research Foundation of Hungarian Academy of Sciences (Grant numbers: OTKA T 29090, T 42550). The authors thank Ms. Gyongyi Sallai for her technical assistance.

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Zs. Varga (a,*), I. Seres (a), E. Nagy (a), L. Ujhelyi (a), G. Balla (b), J. Balla (a), S. Antus (c)

(a) First Department of Medicine, Division of Nephrology, Hungary

(b) Department of Neonatology, Health and Medical Science Center, Hungary

(c) Department of Organic Chemistry, University of Dehrecen. H-4012. Debrecen, Nagyerdei krt. 98. Pob.19. Hungary

Received 28 April 2004; accepted 10 June 2004

Abbreviations: PKC, protein kinase C; NADPH, nicotinamide adenine dinucleotide phosphate; PMA, phorbol myristate acetate; LDL, low density lipoprotein; Ca, calcium; PS, phosphatidylserine; DAG, diacyglycerol; HBSS, Hanks' balanced salt solution; PMSF, phenylmethylsulphonyl fluoride; EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene glycol-bis(2-aminoethylether)-N, N, N', N'-tetraacetic acid; AHP, 2-amino-4-hydroxypteridine

*Corresponding author. Tel.: 36 52 411 717/5833; fax: 36 52 414951.

E-mail address: vargazs@ibel.dote.hu (Zs. Varga).
Table 1. Concentrations of compounds required for 50% inhibition of
xanthine oxidase activity

 I[C.sub.50] measured I[C.sub.50] measured
 by xanthine ([micro]M) by AHP ([micro]M)

1a 32.2 35
2 25.1 30
3 9.6 12
4 >100 n.d.
5 <0.05 <2
6 <0.05 <2

First row shows the results in xanthine/xanthine oxidase and second row
shows the results in AHP/xanthine oxidase system.
n.d. not determined.

Table 2. Apparent inhibition constants and [V.sub.max] values for the
tested molecules concerning xanthin oxidase

 [V.sub.max] [K.sub.m] ([micro]M)

Control 1.464 9.31
1a 1.231 60.4
2 0-553 19.54
3 1.287 103.4
4 1.051 20.63
5 0.593 31.42
6 0.832 37.42

Table 3. Concentrations of tested compounds required for 50% inhibition
of superoxide anion production induced by xanthine oxidase in the
presence of cytochrome C

 I[C.sub.50] ([micro]M)

1a 58.3
2 71.7
3 75.3
4 80.8
5 7.2
6 7.3
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Author:Varga, Zs.; Seres, I.; Nagy, E.; Ujhelyi, L.; Balla, G.; Balla, J.; Antus, S.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Clinical report
Geographic Code:4EXHU
Date:Jan 1, 2006
Words:5283
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