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EFFECT OF THE FLAVANOLIGNANS OF Silybum marianum L. ON LIPID PEROXIDATION IN RAT LIVER MICROSOMES AND FRESHLY ISOLATED HEPATOCYTES ENRICA BOSISIO, CINZIA BENELLI and ONDINA PIROLA Institute of Pharmacological Sciences, Faculty of Pharmacy, University of Milan, Italy Received in final form 17 September 1991

SUMMARY The effect of several flavanolignans (silicristin, silidianin, silybin and isosilybin) present in silymarin, the extract of Silybum marianum fruits, was tested on lipid peroxidation in rat liver microsomes and freshly isolated hepatocytes . In microsomes lipid peroxidation was generated by ADP/Fe" and NADPH . All flavanolignans inhibited peroxidation in a concentration dependent manner . In hepatocytes lipid peroxidation was induced by ADP/Fe'+ complex and cell damage was evaluated as LDH activity released in the medium . The inhibition of the peroxidative process by flavanolignans was also evident in this model, even if with a potency order different from that found in microsomes . In contrast, the effect on LDH release was significant only for silybin and isosilybin, the other compounds being inactive on this parameter . KEY WORDS :

flavanolignans, lipid peroxidation, microsomes, hepatocytes .

LDH, lactate dehydrogenase ; GSH, glutathione ; MDA, malondialdehyde ; TCA, trichloroacetic acid ; DMSO, dimethylsulphoxide . ABBREVIATIONS :

INTRODUCTION The hepatoprotective properties of the extract of Silybum marianum fruits in folk medicine have been known for many years and they are due to a flavonoidic component known as silymarin [1, 2] . Silymarin is constituted chiefly by silybin, silicristin and silidianin [1], silybin being quantitatively the most important . The crude extract of the seeds contains also the dehydro derivatives of the three compounds and isosilybin [3] . Several studies were devoted to investigating the effects of silymarin and its main constituent silybin with several models of experimental liver toxicity Correspondence to : Dr Enrica Bosisio . Institute of Pharmacological Sciences, Via Balzaretti, 9 . 20133 Milano . Italy . 1043-6618/92/020147-08/$03 .00/0

© 1992 The Italian Pharmacological Society

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induced by various agents (galactosamine, CC14, ethanol, phalloidin and GSH depleting agents) [4-10] . A comparative study on the activity of the constituents of silymarin was reported by Hikino et al . [11] in cultured rat hepatocytes, treated with galactosamine or CC14. Although animal and human studies showed the beneficial effects of silymarin and silybin in reducing the damage caused by various toxic agents [12], the mechanism of action of these drugs is not completely clarified. A membrane stabilizing activity was suggested for silymarin [13, 14], mediated by radical scavenger and/or antioxidant properties, evidenced both in vivo [9] and in vitro [15, 16] . More recently it was shown that silymarin increased the availability of glutathione [10, 17] . Up to now a complete study on the effect of single flavanolignans on the peroxidative processes caused by free radicals is lacking . In this study flavanolignans of Silybum marianum were assayed on lipid peroxidation induced by ADP/Fe" complex and NADPH in rat liver microsomes . The compounds tested were silymarin, silybin, silidianin, silicristin and isosilybin . As reference compounds, quercetin and (+)-catechin were used . Secondly, the drugs were tested in freshly isolated hepatocytes incubated in the presence of ADP/Fe 3+ . This experimental model has some advantages with respect to microsomes, since the system maintains membrane integrity, intracellular organization and the endogenous mechanisms protecting against radical peroxidation [18] .

MATERIAL AND METHODS Chemicals

Collagenase and bovine serum albumin (fraction V) were from Sigma Chemicals (St Louis, USA) . Hyaluronidase and kits for the determination of LDH were from Boehringer (Mannheim, Germany) . Silybin, silidianin, silicristin, isosilybin (mol . wt=482) and silymarin (that contains about 60% of flavanolignans) were a gift of Inverni della Beffa (Milan, Italy) . (+)-Catechin and quercetin were obtained from Fluka (Buchs, Switzerland) . All other chemicals and reagents were from Merck (Darmstadt, Germany) and were of analytical grade . Microsome isolation

Male Sprague-Dawley rats weighing 175-200 g were housed under normal light (7 a .m.-7 p.m.) and were allowed free access to food and water . Animals were sacrificed by decapitation after overnight fast . Liver microsomes were isolated essentially as described in [19] and stored at -80 °C, sealed under nitrogen atmosphere . All buffers were bubbled with nitrogen before use . Protein concentration in the microsomal suspension (3-4 mg/ml) was determined according to Bradford [20], using bovine serum albumin as standard . Lipid peroxidation in microsomes

The degree of lipid peroxidation was estimated by measuring MDA in the

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incubation media . The procedure was as follows . Microsomal suspension (0 .6-0 .8 mg of protein) was preincubated for 5 min at 37 °C and the reaction started by addition of the 200µM NADPH in 0 .1 M potassium phosphate, final volume of I ml [19] ; 0 .4 mm ADP and 16 pm FeSO 4 were added to the incubation mixture [21] . The reaction was stopped after 20 min by addition of I ml of 30% TCA and 0 .1 ml of 5 N HCl . MDA was determined by the thiobarbituric method [21], using malonaldehyde-tetramethylacetale as routine standard . Basal peroxidation was evaluated by omitting the radical inducing agent . Compounds to be tested were added to the incubation mixture before the preincubation in concentrations ranging between 0 .1 and 1000 pM with 10 pl of DMSO . The antilipoperoxidative effect was expressed as inhibition of MDA formation, setting the values obtained without inhibitor as 100% activation . Mean values of 3-6 replications of four increasing concentrations were used for the calculation of the dose-effect curve for each substance .

Hepatocyte preparation

Hepatocytes were isolated by liver perfusion according to Cighetti et al . [22] . Cells were suspended in Krebs-Henseleit buffer, pH 7 .4, added with 2% albumin and 5 mm glucose, to give a final concentration of 3x106 cell/ml . Cell viability was determined by trypan blue exclusion test and LDH activity . Preparations with viability over 90% were used .

Lipid peroxidation in hepatocytes

Radical production was induced by ADP/Fe' + complex and cell damage was estimated by measuring LDH release into the incubation media ; lipid peroxidation was evaluated as MDA formation as described for microsomes . Cells (12x10 6/sample) were incubated for 60 min at 37 ° C in the presence of 4 mm ADP-160 ,um FeCl 3 , final volume 4 ml [23-25] . At the end of the incubation, aliquots of cell suspensions were treated with 30% TCA for MDA evaluation [20] ; incubation media were separated by centrifugation and aliquots were taken for LDH evaluation [26] . Compounds to be tested were added before the starting of the incubation at concentrations of 50-100-500 ,um in DMSO (1% of solvent per sample) . Basal peroxidation was measured by omitting the pro-oxidant agent and the inhibitory effect of the compounds was evaluated with respect to induced peroxidation, calculated as the difference between total and basal values . Results are the mean of three determinations .

Statistical anal N'Sis lC,,, values were derived from the dose-effect curves, calculated according to the regression analysis (least-square method) by plotting the log concentration against the % inhibition ; confidential limits were also calculated . In experiments with the hepatocytes . significance of the difference was estimated with the Dunnett test .

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RESULTS Effects offlavanolignans on lipid peroxidation in liver microsomes Basal peroxidation in microsomes incubated without the pro-oxidant agent corresponded to the formation of 7 .5±4 .0 nmol of MDA/mg protein, (mean±SD, n=12) . MDA synthesis was elevated 4 .4-fold over the basal values when ADP/Fe 21 was added. All the tested compounds inhibited MDA production in a concentration dependent manner . IC50 values are reported in Table I . Silicristin, silybin, isosilybin and silymarin showed an activity comparable with that of catechin ; silidianin was much less active . Quercetin was the most active flavonoid .

Effect offlavanolignans on lipid peroxidation and LDH release in hepatocytes Basal production of MDA was 1 .85±0 .94 nmol/10 6 cell (mean±sD, n=6) and increased to 7 .73±1 .51 nmol/10 6 cell in hepatocytes treated with ADP/Fe' + . All the compounds under study inhibited the formation of MDA in a concentration dependent manner (Table II) . Considering the effect obtained at the concentration of 100 ,um, the potency of inhibition decreased in this order : silymarin>silicristin=silidianin>isosilybin=silybin . Therefore, for silidianin, isosilybin and silybin the order of potency observed in this model does not follow the same pattern as the one found in microsomes . As regards LDH release, the enzyme activity measured in the medium of control hepatocytes was 0 .33±0 .18 units/10 6 cell (mean±SD, n=6) and rose to 0 .48±0 .20 units/106 cell in samples treated with ADP/Fe' + . The effects of the compounds on this parameter are reported in Tables III and IV . Silymarin, silicristin and silidianin did not affect the release of LDH induced by ADP/Fe' + , even at 500 ,um (Table III) . All the other substances significantly

IC 50 (pg/ml and

pm)

Table I of flavanolignans on peroxidation induced by ADP/Fe" and NADPH in rat liver microsomes

Compound

ADP/Fe 21 ,ug/ml

Catechin Quercetin Silymarin Silybin Silidianin Silicristin Isosilybin

12 .7 1.2 24 .2 16 .3 102 .0 11.8 15 .4

(11 .5-13 .9) (1 .1-1 .3) (23 .3-25 .2)* (13 .1-20 .3) (80 .3-129 .7) (10 .4-13 .4) (14 .2-16 .9)

PM 43 .6 (39 .7-47 .9) 3 .5 (3 .2-3 .8) 30 .1 (29 .0-31 .4)* 33 .8 (27 .1-42 .2) 211 .7 (166 .6-269 .0) 24 .4 (21 .5-27 .7) 32 .0 (29 .5-35 .1)

Confidence limits (P=0 .05) in parentheses . *Silymarin contains about the 60% of flavanolignans and micromolarity was calculated considering the amount of flavanolignans present in the extract and the mol . wt of 482 .

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The ouabain-resistant " 6Rb uptake (approximately 40% of the total "Rb uptake) did not differ between the groups . Note that serum from SHR showed a more pronounced stimulatory effect on the Na-K pump than that from WKY in the VSMC derived from WKY (open circles) (11 .5±1 .0 for WKY serum versus 13 .5± 2 .1 [x103 c .p .m ./10 5 cells] for SHR serum ; n=12, P

Effect of the flavanolignans of Silybum marianum L. on lipid peroxidation in rat liver microsomes and freshly isolated hepatocytes.

The effect of several flavanolignans (silicristin, silidianin, silybin and isosilybin) present in silymarin, the extract of Silybum marianum fruits, w...
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