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Scavenging of reactive oxygen species and inhibition of arachidonic acid metabolism by silibinin in human cells
Life Sciences, Vol. 58, No. 18, pp. lS91-1600, 1996 copyright 0 1996 Elsevier science Inc. Printed ia the USA. All riehts raerwd
om-nas/%-su.oo t .oo
PII SOO24-3205(96)00134-S
SCAVENGING ARACHIDONIC
OF REACTIVE OXYGEN SPECIES AND INHIBITION OF ACID METABOLISM BY SILIBININ IN HUMAN CELLS
Carola Dehmlow, lnstitut fiir Physiologische
Niels Murawski,
and Herbert de Groot
Chemie, Universittitsklinikum, D-451 22 Essen, Germany
(Received
Hufelandstrasse
55,
in final form March 5, 19%)
Summarv The effects of the flavonoid silibinin, which is used for the treatment of liver diseases, on the formation of reactive oxygen species and eicosanoids by human platelets, white blood and endothelial cells were studied. Silibinin proved to be a strong scavenger of HOCI (K&o 7 p.M), but not of 02- (IC50>200 PM) produced by human granulocytes. The formation of leukotrienes via the 5-lipoxygenase pathway was strongly inhibited. In human granulocytes ICso-values of 15 PM and 14.5 PM silibinin were detected for LTB4 and LTC4/D4/E4/F4 formation, respectively. In contrast to this, three- to fourfold silibinin concentrations were necessary to halfmaximally inhibit the cyclooxygenase pathway. For PGE2 formation by human monocytes an I&-value of 45 PM silibinin was found. I&o-values of 69 p.M and 52 yM silibinin were determined for the inhibition of TXB2 formation by human thrombocytes and of 6-K-PGF1, formation by human omentum endothelial cells, respectively. Thus, the deleterious effects of HOCI that can lead to cell death, and those of leukotrienes that are especially important in inflammatory reactions, can be inhibited by silibinin in concentrations that are reached in vivo after the usual clinical dose. Silibinin is thought not only to display hepatoprotective properties but might also be cytoprotective in other organs and tissues. Key Words: cyclooxygenase
siiibinin,
reactive
oxygen
species,
free
radical
scavenger,
myeloperoxidase,
Slipoxygenase,
Flavonoids are natural compounds widely distributed in the plant kingdom to which high pharmacological potencies have been attributed (1). Silibinin is a member of this group that has found its way into therapeutical use. It is the most active of three isomers that can be isolated from the seeds of the milk thistle, Si/yburn Marianum (2). Silibinin displays hepatoprotective properties and can be used clinically for the treatment of toxic liver damage and chronic liver disease. The protective effects of silibinin have been studied in various models of experimental liver damage (3-8) and in human clinical studies (9). It is usually assumed that silibinin’s main mechanism of action is an inhibition of lipid peroxidation processes due to its free radical scavenging properties. In addition, a membrane stabilizing effect and an enhancement of protein *Corresponding author: H. de Groot, lnstitut fiir Physiologische Chemie, klinikum, Hufelandstrasse 55, D-45122 Essen, Germany, Tel.:49-201-723 49-201-723 5943
Universit&s4101, FAX:
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Effects of Silibmin on Human Cells
biosynthesis have been suggested properties (10).
as contributing
factors towards
Vol. 58, No. 18, 1996
its hepatoprotective
Both the scavenging of reactive oxygen species and the inhibition of arachidonic acid metabolism are important features of a hepatoprotective drug, as these mediators may significantly contribute to liver damage during inflammatory processes. The damaging potential of reactive oxygen species is often mediated via lipid peroxidation that leads to the breakdown of cellular membranes (11). Eicosanoids are mediators that play an especially in inflammatory reactions, important role in signal transduction, immunological disorders, and septic shock (12). So far the effect of silibinin on the formation of reactive oxygen species and eicosanoids has not been studied in human cells. Using rat Kupffer cells we were recently able to show a strong inhibition of LTB4 formation (I&c 15 pM), only a minor effect on PGE;! formation (IC50>200 PM), and an inhibition of 02- and NO formation by silibinin (IC5c around 80 PM) (13). Preliminary studies with phagocytic cells from human liver confirmed the pronounced inhibition of LTB4 formation. These results prompted us to study the effect of silibinin on the formation of reactive oxygen species and the various metabolites of the 5-lipoxygenase and cycloxygenase pathway of arachidonic acid metabolism by human platelets, white blood and endothelial cells. Materials
and
Methods
Materials: Silibinin was kindly provided by Madaus AG (Cologne, Germany). Histopaque-1 119, Histopaque-1077, phorbol-12-myristate-13-acetate (PMA), calcium ionophore A 23187, the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (FMLP), zymosan A, and lipopolysaccharides (LPS) were from Sigma (Steinheim, Germany). Luminol (5’-amino-2,3-dihydro-1,4_phthalazinedione), lucigenin (9,9’-bis-Nmethylacridinium nitrate), sodium azide, and superoxide dismutase were purchased from Boehringer (Mannheim, Germany). Leukotriene B4 (3H) radioimmunoassay kit was purchased from Du Pont-New England Nuclear (Dreieich, Germany). Leukotriene C4/D4/E4/F4 (3H) radioimmunoassay kit, Prostaglandin E2 (sH) radioimmunoassay kit, Thromboxane BP (3H) radioimmunoassay kit, and 6-Keto-Prostaglandin Fl, (sH) radioimmunoassay kit were from Paesel and Lorei (Frankfurt, Germany). Ethanol, dimethyl sulfoxide (DMSO), and all other chemicals were from Merck (Darmstadt, Germany). Isolation of mononuclear cells and granulocytes: EDTA-blood was collected from healthy donors. Mononuclear cells and granulocytes were isolated from whole blood using two-step density gradient centrifugation. Briefly, 3 ml Ficoll (density 1.077 g/ml) were layered onto 3 ml Ficoll (density 1.119 g/ml). 6 ml of whole blood were carefully layered onto the gradient and centrifuged at 22°C for 30 min at 700xg in a 15 ml conical polypropylene tube. Plasma was discharged, mononuclear cells were collected from the plasma/Histopaque-1077 interphase and granulocytes from a separate layer above the erythrocyte pellet. Remaining erythrocytes were removed by hypotonic lysis; after 30 set, isotonicity was restored with hypertonic saline. Mononuclear cells and granulocytes were washed by centrifugation and resuspended in phosphate buffered saline (PBS) at the desired concentration. Viability, measured by trypan blue exclusion, was always above 95%. Isolation of platelets: Citrate-blood was collected from healthy donors. Undiluted blood was layered onto the Ficoll gradient and centrifuged as described above. The uppermost layer that contains mononuclear cells and platelets was transferred into
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Effects of Silibinin on Human Cells
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PBS. The nucleated ceils were sedimented by centrifugation at 22°C for 10 min at 1OOxg. Platelets were washed by centrifugation and resuspended in PBS at a concentration of 5x106 cells/ml (14). Culture of omentum endothelial cells: Human omentum endothelial cells were enzymatically harvested from samples of omental fat by a slightly modified method according to Kern et al. (15). Monolayer cultures were incubated in a humidified 5% Con-air atmosphere at 37°C with Ml99 medium supplemented with 35% MEM medium and 15% inactivated human serum. Cells were used for experiments between the second and fourth subpassage. Chemiluminescence measurements: Chemiluminescence was detected with the ARGUS-50 photon-counting imaging system, Hamamatsu Photonics (Herrsching, Germany). Measurements were performed with 0.5x1 0s granulocytes/0.5 ml Hank’s balanced salt solution (HBSS) at 37°C for 30 min. To enhance the chemiluminescence signal either luminol (50 PM), which reacts with different reactive oxygen species, or lucigenin (100 PM), which reacts specifically with 02-, were added. Then phorbol-12myristate-13-acetate (PMA, 1 yM) and silibinin (l-200 pM) were added. In all experiments silibinin was dissolved in ethanol. 5 pl silibinin solution were added to vehicle alone was added to controls. achieve the final concentration, Chemiluminescence was assessed as counts/2 cm?30 set (16). To determine the reactive oxygen species responsible for the luminoland lucigenin-enhanced chemiluminescence signal, different radical scavengers were used. To luminolenhanced measurements sodium azide (50 PM), ethanol (50 mM), and DMSO (50 mM) were added after PMA-stimulation. To lucigenin-enhanced measurements SOD (20 pg/ml) was added after PMA-stimulation. Incubation procedure for determination of arachidonic acid metabolites: For all experiments the different cell types were preincubated for three minutes at 37°C with l-25 PM silibinin to determine 5-lipoxygenase products and with l-l 00 pM silibinin to determine cyclooxygenase metabolites, respectively. Then arachidonic acid metabolism was stimulated. At the end of the incubation time with silibinin and stimulus the reaction was stopped by placing the tubes in an ice-bath. In incubations for LTB4 and TXBz measurements, 1 ml ice-cold buffer (0.9% NaCI, 0.1% gelatin, 0.1% sodium azide in 10 mM Tris/HCI buffer, pH 8.6) was also added to 1 ml cell suspension. To remove cells, samples were centrifuged at 4°C for 15 min at 1400xg. The supernatants were decanted and frozen immediately at -80°C. Eicosanoids present in the supernatants were measured within 48 h. LTB4 formation: Three different stimuli were used to activate 1x10s granulocytes/ml: A 23187 (20 PM), FMLP (20 PM), and opsonized zymosan A (10 mg/ml). After 5 min of incubation at 37°C with A 23187 or FMLP, and 10 min with opsonized zymosan A the reaction was stopped. To opsonize zymosan, it was boiled for 30 min in PBS. Then it was incubated for 30 min with pooled normal human serum at 37°C. It was washed twice with PBS and resuspended in PBS at a concentration of 100 mg/ml. LTCd/Dd/Ea/Fs formation: (20 pM) for 30 min.
5x106
granulocytes/ml
PGEa formation: 5x10s mononuclear cells/ml After 8 h of incubation the reaction was stopped. TXBz formation:
were
stimulated
were stimulated
with
with
A 23187
10 pg/ml
LPS.
1 pM A 23187 was used to stimulate 5x106 platelets/ml for 30 min.
Effects of Silibinin on Human Cells
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Vol. 58, No. 18, l!B6
with 6-K-PGF1, formation: Human omentum endothelial cells were stimulated 10 pM A 23187. After 60 min of incubation the supernatants of the monolayer cultures were transferred into tubes and treated as described above. Eicosanoid assays: Eicosanoids present in the samples were measured by radioimmunoassay with the use of commercial test kits according to the manufacturer’s instructions. Briefly, 100 p.1sample and 100 pl of sH eicosanoid were transferred into tubes, 100 ul antibody to the particular eicosanoid under investigation were added. The mixture was incubated for 2 h at room temperature or overnight at 4°C. Charcoal suspension was added and the samples were centrifuged at 2000xg for 15 min. The radioactivity in the supernatant was counted in a liquid scintillation counter. Statistics: Data are given as means f S.D.. Statistical analysis was performed using a one-way analysis of variance (ANOVA). A P value of less than 0.05 was considered significant. Results Measurement of reactive oxygen species Freshly isolated human granulocytes exposed to PMA (1 PM) showed a strong chemiluminescence signal in the presence of either luminol (50 uM) (Fig. 1A) or lucige-
120000-
PMA
*
100 pM silibinin
,,....*.*. ./I
PJA
80000
100000 -
100 pJ silibinin
”
80000 P a or‘ 60000-
luminol lucigenin
0 0
,,,,,““,““,““,““,“‘,, 5 10
15 time (min)
20
25
30
“9 ......
‘,S’..
60000 -
Od
-._.-
..._.
.j
30 time (min)
Fig. 1 Effect of silibinin on luminoland lucigenin-enhanced chemiluminescence of PMA-activated human granulocytes. Human granulocytes were incubated in HBSS at 37°C. (A) Luminol (50 PM), PMA (1 PM), and silibinin (100 PM) were added where indicated. Chemiluminescence was mainly due to the formation of HOCI as indicated by the fact that it was possible to almost completely inhibit it by sodium azide. (B) Lucigenin (100 PM), PMA (1 PM), and silibinin (100 FM) were added where indicated. Chemiluminescence was mainly due to the formation of 02- as indicated by the fact that it was almost completely inhibited by superoxide dismutase. The black line represents a measurement where silibinin was added, the dotted line a measurement where it was not added.
Vol. 58, No.
18, 1996
Effects of Silibinin on Human Cells
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nin (100 PM) (Fig. 1B) acting as the chemiluminescence enhancer, indicating the formation of significant amounts of reactive oxygen species. Luminol can react with different reactive oxygen species. In the case of PMA-activated human granulocytes the reaction with hypochlorite (HOCI) is most important (17). This is also suggested by the fact that adding sodium aride (50 pM) led to a decrease in the chemiluminescence signal of 90%. Sodium azide is a potent inhibitor of the enzyme myeloperoxidase, present in granulocytes, that catalyzes the reaction of Hz02 and Cl- to HOCI. On the other hand, addition of ethanol (50 mM) and dimethyl sulfoxide (50 mM), that are very effective scavengers of OH-radicals, had no effect on the chemiluminescence intensities (data not shown). Lucigenin, on the other hand, reacts only with 02‘, which was confirmed by the almost complete elimination of the chemiluminescence signal after addition of superoxide dismutase (data not shown). Luminol-enhanced chemiluminescence was strongly decreased by adding silibinin, indicating a significant inhibition of HOCI release. This inhibitory effect of silibinin was clearly concentration-dependent with an ICsc-value of 7 uM silibinin (Fig. 2A.). In contrast to this, only a small effect of silibinin on lucigenin-enhanced chemiluminescence, and therefore on 02- formation was detectable. Under these conditions the IQ-value was well above 200 yM silibinin (Fig. 28).
Ol~,,,,,,,,,,,,,,~,,,, 0
50
01,,,,,,,..,,,,,,,,,,, loo
150
silibinin (vh.4)
Effect of silibinin granulocytes.
200
0
50
100 silibinin (PM)
150
200
Fig. 2 on the formation of HOC1 and 02- by human
Experimental details were the same as in Figure 1. Maximum chemiluminescence intensities after PMA-activation and addition of silibinin were calculated as percentages of (A) HOCI formation and (B) 02formation in controls where silibinin was not added. Values are means + S.D. of 4-6 experiments; * kO.05 compared to controls without silibinin.
LTB4 formation Freshly isolated human granulocytes activated with the calcium ionophore A 23187 (20 uM) formed 4558 pg LTB&Os cells within 5 min of incubation. Longer incubation times did not result in higher LTB4 amounts, after 30 min of incubation the amount of
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Effects of Silibinin on Human Cells
Vol. 58, No.
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LTB4 detected remained in the same range. Treatment of the cells with silibinin led to a strong inhibition of LTB4 formation in a dose-dependent manner. The I&o-value found here was 15 uM silibinin (Fig. 3A). This inhibitory effect of silibinin on LTB4 formation was independent of the stimulus used. When LTB4 formation of granulocytes was stimulated with either FMLP (20 pM) or opsonized zymosan (10 mg/ml) similar ICsovalues of silibinin (~20 uM) were determined (data not shown).
LTCa/Dd/Ea/Fa
formation
The effect of silibinin on the LTCd/Db/Eb/Fh formation of human granulocytes was similar to that found for LTB4 formation. 30 min after stimulation with A 23187 (20 yM) 2940 pg LTC~/D~/E~/F&O~ granulocytes were detected. The LTC4/D4/E4/F4 formation was inhibited by silibinin in a dose-dependent fashion with an I&o-value of 14.5 uM (Fig. 38).
A
0
5
Effect of metabolites
10 15 silibinin CM)
20
25
0
Fig. 3 silibinin on the formation by human granulocytes.
5
10
15
20
25
silibinin (PM)
of
54ipoxygenase
(A) Human granulocytes were incubated in PBS for 5 min at 37°C. LTB4 formation was stimulated by A 23187 (20 PM). (B) Human granulocytes were incubated in PBS for 30 min at 37°C. LTC4/D4/E4/F4 formation was stimulated by A 23187 (20 PM). Leukotriene concentrations were calculated as percentages of leukotriene formation by controls in which no silibinin was added. Values are means f S.D. of 4-6 experiments; *PcO.O5 compared to controls without silibinin.
PGE2 formation Incubation of freshly isolated human monocytes with LPS (10 ug/ml) for’8 h led to the formation of 238 pg PGEz/los cells. PGE2 was already detectable after 1 h of incubation but increased with a prolonged incubation period. PGEs formation was also inhibited by silibinin in a dose-dependent manner but significantly higher silibinin concentrations were necessary relative to the effect on leukotriene formation. The I&ovalue determined was 45 pM silibinin (Fig. 4A).
a-
100
0
20
40
40
60
silibinin (PM)
silibinin (PM)
Fig. 4 Effect of silibinin on the formation of cyclooxygenase metabolites by human mononuclear cells, platelets, and endothelial cells.
C
00
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Effects of Silibinin on Human Cells
Vol. 58, No. 18, 1996
60
silibinin (PM)
60
100
(A) Human mononuclear cells were incubated in PBS for 8 h at 37°C. PGE2 formation was stimulated by LPS (10 ug/ml). (B) Human platelets were incubated in PBS for 30 min at 37°C. TXB2 formation was stimulated by A 23187 (1 PM). (C) Human omentum endothelial cells were incubated in PBS for 60 min at 37°C. 6-K-PGF1, formation was stimulated by A 23187 (10 CLM). Eicosanoid concentrations were calculated as percentages of eicosanoid formation by controls in which no silibinin was added. Values are means + S.D. of 4-6 experiments; * PcO.05 compared to controls without silibinin.
TXB2 formation Human thrombocytes release TXA2 upon stimulation. This unstable eicosanoid rapidly reacts to the stable TXB2 (12). TXB2 was determined in our study after incubating thrombocytes using A 23187 (1 PM) as the stimulant. TXB2 was already detectable after 5 min incubation time. After 30 min 1477 pg TXBz/lOs cells were determined. Again rather high silibinin concentrations were necessary to half-maximally inhibit TXB2 formation. Here an IC5c-value of 69 uM silibinin was found (Fig. 48).
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6-K-PGF1,
Effects of Silibinin on Human Cells
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formation
Prostacyclin, another product of the cyclooxygenase pathway mainly formed by endothelial cells, is also a very unstable compound. Within seconds it is converted to 6K-PGFt, that is detectable with the help of radioimmunoassays (12). Human omentum endothelial cells cultured in monolayers formed 1484 pg 6-K-PGFl,/lOs cells within 1 h after stimulation with A 23187 (10 PM). After 4 h of incubation time the 6-K-PGFt, formation further increased to 3881 pg 6-K-PGFl,/lOs. To determine the effect of silibinin on 6-K-PGFt, formation, an incubation time of 1 h was chosen. Silibinin inhibited 6-K-PGFt, formation in a dose-dependent manner with an ICso-value of 52 PM silibinin (Fig. 4C).
Discussion In this study human cells were used to investigate the effect of the hepatoprotective flavonoid silibinin on the liberation of reactive oxygen species and the formation of eicosanoids. In both cases inhibitory effects were detected that did not affect the whole class of metabolites but displayed a strong predominance: HOCI formation was preferentially inhibited as compared to 02- formation; leukotriene production was strongly inhibited again at low concentrations, while only minor effects on the cyclooxygenase pathway were found at these concentrations. The preferential inhibition of HOCI formation by silibinin is in accordance with the results of Mira et al. (18). In their experiments in non-enzymatic systems micromolar concentrations of silibinin dihemisuccinate rapidly reacted with OH radicals and with HOC1 but not with 02-. An I&-,-value of about 5 uM was estimated for the scavenging of HOCI in a luminol-enhanced chemiluminescence assay. We determined almost the same ICso-value (IC5o 7 FM) in a similar assay with activated human granulocytes (Fig. 2A). Scavenging effects of silymarin and silybinin in the umolar concentration range on superoxide and alkoxyl radicals were also shown by Pascual et al. in nonenzymatic in vitro systems (19). As the effect of silibinin on HOCI formation can be observed in cellular and in non-enzymatic systems, it is unlikely that silibinin interferes with the enzyme myeloperoxidase. The same holds true for a possible reaction of silibinin with the cellular membrane as explanation for the observed effects. More probable is a direct reaction of the flavonoid with HOCI. The much weaker effect of silibinin on 02‘ formation is therefore also thought to be due to a scavenging effect, not to a reaction with the 02- producing enzyme NADPH-oxidase. This direct scavenging effect has also been proposed by Comoglio et al. for ethanol-derived free radicals using a silybin-phospholipid complex (20). Inhibitory effects of silibinin on the cyclooxygenase and 5-lipoxygenase pathways were detected in our study but silibinin concentrations that led to halfmaximal inhibitions were quite different from each other. A strong inhibition of the 5lipoxygenase pathway occurred. Silibinin halfmaximally inhibited LTB4 production at a concentration of 15 PM, for LTC4/D4/E4/F4 production an ICao-value of 14.5 uM was found (Fig. 3A,B). Three- to fourfold silibinin concentrations were necessary to achieve the same inhibitory effect on the formation of metabolites of the cyclooxygenase pathway. Here ICso-values of 45 PM, 69 PM, and 52 uM silibinin were determined for the inhibition of PGE2, TXB2, and 6-K-PGFt, formation, respectively (Fig. 4A,B,C). These IC:so-values both for the inhibition of leukotriene and prostaglandin formation are similar to those we found using rat Kupffer cells to determine the effect of silibinin on LTB4 and PGE2 formation (13). All metabolites of the 5-lipoxygenase pathway under investigation were inhibited with almost the same ICr,o-values of silibinin. As no predominance for either product was found, it can be concluded that silibinin interferes directly with the 5-lipoxygenase.
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Effects of Silibinin on Human Cells
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The precursor LTA4 can no longer
be synthesized sufficiently and levels of all leukotrienes produced from LTA4 decrease. The same holds true for the cyclooxygenase pathway, all three products generated from prostaglandin HP by action of different enzymes were inhibited at comparable silibinin concentrations. In this case, silibinin probably acts upon the cyclooxygenase, the formation of PGH2 from arachidonic acid is disturbed. Inhibitory effects of various flavonoids on arachidonic acid metabolism have also been found by other authors. Laughton et al. (21) showed inhibitory effects of flavonoids on 5 lipoxygenase and/or cyclooxygenase using rat leukocytes and liver microsomes. In mice the oral administration of flavonoids inhibited the formation of paw oedema and the formation of PGEz and LTB4 (22). Yoshimoto et al. found various flavonoids to be relatively selective inhibitors of arachidonate 5lipoxygenase using enzyme preparations from rat cells (23). All these studies did not make use of silibinin, and they worked with species other than humans. Flavonoids from Silybum Marianum were employed by Fiebrich and Koch and Baumann et al., but again no human cells were used. Fiebrich and Koch found effects of silymarin on lipoxygenase from soybeans and on prostaglandin synthase from rats (24, 25). Baumann et al. worked in cell-free systems with enzyme preparations from rats (26). These authors determined inhibitory effects on both enzymes with a predominance for %ipoxygenase, but rather high concentrations (>O.l mM) were necessaty to achieve effects. Taken together, two properties of silibinin that may contribute to its therapeutic action became obvious in our study with human cells. Firstly, as regards reactive oxygen species silibinin acted mainly as a scavenger of HOCI liberated by neutrophils. The reaction with 02- occurred only to a much lesser extent. HOCI is a powerful antibacterial but also cytotoxic agent produced by neutrophils. Already at low concentrations it oxidizes sulfhydryl groups, disturbs various protein functions, and leads to cell death (27, 28). It therefore plays an important role in inflammatory reactions, and scavenging of this toxic agent by silibinin may be sufficient to explain its cytoprotective properties. Secondly, an inhibition of arachidonic acid metabolism with a strong predominance for the 5lipoxygenase pathway became obvious. It is known that leukotrienes contribute significantly to tissue injuries (29), whereas prostaglandins are considered to display cytoprotective effects (30). Our results therefore suggest that silibinin should not only be hepatoprotective but might also exhibit cytoprotective properties as regards injuries to other organs and tissues. Silibinin concentrations that were necessary to inhibit HOCI production and the 5-lipoxygenase pathway are in the range of plasma concentrations that are reached after the usual clinical dose of 240 mg silibinin (0.4-l .3 FM),
Acknowledaements This work was supported by the Biotech Program of the EU. We wish to thank Christina Weeger for her excellent
technical
assistance
with the LTB4 measurements.
References 1. 2. 3. 4. 5.
B. HAVSTEEN, Biochem. Pharmacol. 32 1141-1148 (1983). P. MORAZZONI and E. BOMBARDELLI, Fitoterapia 66 3-42 (1995). M. MOURELLE, P. MURIEL, L. FAVARI and T. FRANCO, Fundam. Clin. Pharmacol. 9 183-191 (1989). P. MURIEL and M. MOURELLE, J. Appl. Toxicol. n 275-279 (1990). A. VALENZUELA and R. GUERRA, FEBS Lett. m 291-294 (1985).
1600
6. 7.
8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.
Effects of Silibinin on Human Cells
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P. MURIEL, T. GARCIAPINA, V. PEREZ-ALVAREZ and M. MOURELLE, J. Appl. Toxicol. X 439-442 (1992). R. CAMPOS, A. GARRIDO, R. GUERRA and A. VALENZUELA, Planta Med. % 417-419 (1989). A. VALENZUELA, C. LAGOS, K. SCHMIDT and L.A. VIDELA, Biochem. Pharmacol. 34 2209-2212 (1985). P. I. LANG, K. NEKAM, G. DEAK, G. MUZES, R. GONZALES-CABELLO, GERGELY, G. CSOMOS and J. FEHER, Ital. J. Gastroenterol. a 283-287 (1990). E. LENG-PESCHLOW and A. STRENGE-HESSE, Z. Phytother. 12 162-l 74 (1991). H. DE GROOT, Hepato-Gastroenterol. &I 328-332 (1994). K. DECKER, Semin. Liv. Dis. 5 175-190 (1985). C. DEHMLOW, J. ERHARD and H. DE GROOT, Hepatology in press (1996). M. HOFFMAN, D.M. MONROE and H.R. ROBERTS, Am. J. Clin. Pathol. 98 531533 (1 992). P.A. KERN, A. KNEDLER and R.H. ECKEL, J. Clin. Invest. II 1822-1829 (1983). A.K. CAMPBELL, TIBS 11 104-108 (1986). J. ARNOLD, S. HAMMERSCHMIDT and K. ARNOLD, Biochim. Biophys. Acta 1097 145-151 (1991). L. MIRA, M. SILVA and CF. MANSO, Biochem. Pharmacol. &j 753-759 (1994). C. PASCUAL, R. GONZALES, J. ARMESTO and P. MURIEL, Drug Develop. Res. a 73-77 (1993). A. COMOGLIO, A. TOMASI, S. MALANDRIO, G. POLI and E. ALBANO, Biochem. Pharmacol. a 1313-1316 (1995). M.J. LAUGHTON, P.J. EVANS, M. MORONEY, J.R.S. HOULT and B. HALLIWELL, Biochem. Pharmacol. 42 1673-1681 (1991). M.L. FERRANDIZ and M.J. ALCARAZ, Agents Actions 2 283-288 (1991). T. YOSHIMOTO, M. FURUKAWA, S. YAMAMOTO, T. HORIE and S. WATANABEKOHNO, Biochem. Biophys. Res. Commun. 116 612-618 (1983). F. FIEBRICH and H. KOCH, Experientia % 1548-1550 (1979). F. FIEBRICH and H. KOCH, Experientia a 1550-1551 (1979). J. BAUMANN, F. VON BRUCHHAUSEN and G. WURM, Prostaglandins 24.627639 (1980). B. HALLIWELL, Am. J. Med. g 14S-22s (1991). C.G. COCHRANE, Am. J. Med. 9l_ 23S-30s (1991). F.A. KUEHL and R.W. EGAN, Science m 978-984 (1980). J. QUIROGA and J. PRIETO, Pharmac. Ther. % 67-92 (1993).
om-nas/%-su.oo t .oo
PII SOO24-3205(96)00134-S
SCAVENGING ARACHIDONIC
OF REACTIVE OXYGEN SPECIES AND INHIBITION OF ACID METABOLISM BY SILIBININ IN HUMAN CELLS
Carola Dehmlow, lnstitut fiir Physiologische
Niels Murawski,
and Herbert de Groot
Chemie, Universittitsklinikum, D-451 22 Essen, Germany
(Received
Hufelandstrasse
55,
in final form March 5, 19%)
Summarv The effects of the flavonoid silibinin, which is used for the treatment of liver diseases, on the formation of reactive oxygen species and eicosanoids by human platelets, white blood and endothelial cells were studied. Silibinin proved to be a strong scavenger of HOCI (K&o 7 p.M), but not of 02- (IC50>200 PM) produced by human granulocytes. The formation of leukotrienes via the 5-lipoxygenase pathway was strongly inhibited. In human granulocytes ICso-values of 15 PM and 14.5 PM silibinin were detected for LTB4 and LTC4/D4/E4/F4 formation, respectively. In contrast to this, three- to fourfold silibinin concentrations were necessary to halfmaximally inhibit the cyclooxygenase pathway. For PGE2 formation by human monocytes an I&-value of 45 PM silibinin was found. I&o-values of 69 p.M and 52 yM silibinin were determined for the inhibition of TXB2 formation by human thrombocytes and of 6-K-PGF1, formation by human omentum endothelial cells, respectively. Thus, the deleterious effects of HOCI that can lead to cell death, and those of leukotrienes that are especially important in inflammatory reactions, can be inhibited by silibinin in concentrations that are reached in vivo after the usual clinical dose. Silibinin is thought not only to display hepatoprotective properties but might also be cytoprotective in other organs and tissues. Key Words: cyclooxygenase
siiibinin,
reactive
oxygen
species,
free
radical
scavenger,
myeloperoxidase,
Slipoxygenase,
Flavonoids are natural compounds widely distributed in the plant kingdom to which high pharmacological potencies have been attributed (1). Silibinin is a member of this group that has found its way into therapeutical use. It is the most active of three isomers that can be isolated from the seeds of the milk thistle, Si/yburn Marianum (2). Silibinin displays hepatoprotective properties and can be used clinically for the treatment of toxic liver damage and chronic liver disease. The protective effects of silibinin have been studied in various models of experimental liver damage (3-8) and in human clinical studies (9). It is usually assumed that silibinin’s main mechanism of action is an inhibition of lipid peroxidation processes due to its free radical scavenging properties. In addition, a membrane stabilizing effect and an enhancement of protein *Corresponding author: H. de Groot, lnstitut fiir Physiologische Chemie, klinikum, Hufelandstrasse 55, D-45122 Essen, Germany, Tel.:49-201-723 49-201-723 5943
Universit&s4101, FAX:
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Effects of Silibmin on Human Cells
biosynthesis have been suggested properties (10).
as contributing
factors towards
Vol. 58, No. 18, 1996
its hepatoprotective
Both the scavenging of reactive oxygen species and the inhibition of arachidonic acid metabolism are important features of a hepatoprotective drug, as these mediators may significantly contribute to liver damage during inflammatory processes. The damaging potential of reactive oxygen species is often mediated via lipid peroxidation that leads to the breakdown of cellular membranes (11). Eicosanoids are mediators that play an especially in inflammatory reactions, important role in signal transduction, immunological disorders, and septic shock (12). So far the effect of silibinin on the formation of reactive oxygen species and eicosanoids has not been studied in human cells. Using rat Kupffer cells we were recently able to show a strong inhibition of LTB4 formation (I&c 15 pM), only a minor effect on PGE;! formation (IC50>200 PM), and an inhibition of 02- and NO formation by silibinin (IC5c around 80 PM) (13). Preliminary studies with phagocytic cells from human liver confirmed the pronounced inhibition of LTB4 formation. These results prompted us to study the effect of silibinin on the formation of reactive oxygen species and the various metabolites of the 5-lipoxygenase and cycloxygenase pathway of arachidonic acid metabolism by human platelets, white blood and endothelial cells. Materials
and
Methods
Materials: Silibinin was kindly provided by Madaus AG (Cologne, Germany). Histopaque-1 119, Histopaque-1077, phorbol-12-myristate-13-acetate (PMA), calcium ionophore A 23187, the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (FMLP), zymosan A, and lipopolysaccharides (LPS) were from Sigma (Steinheim, Germany). Luminol (5’-amino-2,3-dihydro-1,4_phthalazinedione), lucigenin (9,9’-bis-Nmethylacridinium nitrate), sodium azide, and superoxide dismutase were purchased from Boehringer (Mannheim, Germany). Leukotriene B4 (3H) radioimmunoassay kit was purchased from Du Pont-New England Nuclear (Dreieich, Germany). Leukotriene C4/D4/E4/F4 (3H) radioimmunoassay kit, Prostaglandin E2 (sH) radioimmunoassay kit, Thromboxane BP (3H) radioimmunoassay kit, and 6-Keto-Prostaglandin Fl, (sH) radioimmunoassay kit were from Paesel and Lorei (Frankfurt, Germany). Ethanol, dimethyl sulfoxide (DMSO), and all other chemicals were from Merck (Darmstadt, Germany). Isolation of mononuclear cells and granulocytes: EDTA-blood was collected from healthy donors. Mononuclear cells and granulocytes were isolated from whole blood using two-step density gradient centrifugation. Briefly, 3 ml Ficoll (density 1.077 g/ml) were layered onto 3 ml Ficoll (density 1.119 g/ml). 6 ml of whole blood were carefully layered onto the gradient and centrifuged at 22°C for 30 min at 700xg in a 15 ml conical polypropylene tube. Plasma was discharged, mononuclear cells were collected from the plasma/Histopaque-1077 interphase and granulocytes from a separate layer above the erythrocyte pellet. Remaining erythrocytes were removed by hypotonic lysis; after 30 set, isotonicity was restored with hypertonic saline. Mononuclear cells and granulocytes were washed by centrifugation and resuspended in phosphate buffered saline (PBS) at the desired concentration. Viability, measured by trypan blue exclusion, was always above 95%. Isolation of platelets: Citrate-blood was collected from healthy donors. Undiluted blood was layered onto the Ficoll gradient and centrifuged as described above. The uppermost layer that contains mononuclear cells and platelets was transferred into
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PBS. The nucleated ceils were sedimented by centrifugation at 22°C for 10 min at 1OOxg. Platelets were washed by centrifugation and resuspended in PBS at a concentration of 5x106 cells/ml (14). Culture of omentum endothelial cells: Human omentum endothelial cells were enzymatically harvested from samples of omental fat by a slightly modified method according to Kern et al. (15). Monolayer cultures were incubated in a humidified 5% Con-air atmosphere at 37°C with Ml99 medium supplemented with 35% MEM medium and 15% inactivated human serum. Cells were used for experiments between the second and fourth subpassage. Chemiluminescence measurements: Chemiluminescence was detected with the ARGUS-50 photon-counting imaging system, Hamamatsu Photonics (Herrsching, Germany). Measurements were performed with 0.5x1 0s granulocytes/0.5 ml Hank’s balanced salt solution (HBSS) at 37°C for 30 min. To enhance the chemiluminescence signal either luminol (50 PM), which reacts with different reactive oxygen species, or lucigenin (100 PM), which reacts specifically with 02-, were added. Then phorbol-12myristate-13-acetate (PMA, 1 yM) and silibinin (l-200 pM) were added. In all experiments silibinin was dissolved in ethanol. 5 pl silibinin solution were added to vehicle alone was added to controls. achieve the final concentration, Chemiluminescence was assessed as counts/2 cm?30 set (16). To determine the reactive oxygen species responsible for the luminoland lucigenin-enhanced chemiluminescence signal, different radical scavengers were used. To luminolenhanced measurements sodium azide (50 PM), ethanol (50 mM), and DMSO (50 mM) were added after PMA-stimulation. To lucigenin-enhanced measurements SOD (20 pg/ml) was added after PMA-stimulation. Incubation procedure for determination of arachidonic acid metabolites: For all experiments the different cell types were preincubated for three minutes at 37°C with l-25 PM silibinin to determine 5-lipoxygenase products and with l-l 00 pM silibinin to determine cyclooxygenase metabolites, respectively. Then arachidonic acid metabolism was stimulated. At the end of the incubation time with silibinin and stimulus the reaction was stopped by placing the tubes in an ice-bath. In incubations for LTB4 and TXBz measurements, 1 ml ice-cold buffer (0.9% NaCI, 0.1% gelatin, 0.1% sodium azide in 10 mM Tris/HCI buffer, pH 8.6) was also added to 1 ml cell suspension. To remove cells, samples were centrifuged at 4°C for 15 min at 1400xg. The supernatants were decanted and frozen immediately at -80°C. Eicosanoids present in the supernatants were measured within 48 h. LTB4 formation: Three different stimuli were used to activate 1x10s granulocytes/ml: A 23187 (20 PM), FMLP (20 PM), and opsonized zymosan A (10 mg/ml). After 5 min of incubation at 37°C with A 23187 or FMLP, and 10 min with opsonized zymosan A the reaction was stopped. To opsonize zymosan, it was boiled for 30 min in PBS. Then it was incubated for 30 min with pooled normal human serum at 37°C. It was washed twice with PBS and resuspended in PBS at a concentration of 100 mg/ml. LTCd/Dd/Ea/Fs formation: (20 pM) for 30 min.
5x106
granulocytes/ml
PGEa formation: 5x10s mononuclear cells/ml After 8 h of incubation the reaction was stopped. TXBz formation:
were
stimulated
were stimulated
with
with
A 23187
10 pg/ml
LPS.
1 pM A 23187 was used to stimulate 5x106 platelets/ml for 30 min.
Effects of Silibinin on Human Cells
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with 6-K-PGF1, formation: Human omentum endothelial cells were stimulated 10 pM A 23187. After 60 min of incubation the supernatants of the monolayer cultures were transferred into tubes and treated as described above. Eicosanoid assays: Eicosanoids present in the samples were measured by radioimmunoassay with the use of commercial test kits according to the manufacturer’s instructions. Briefly, 100 p.1sample and 100 pl of sH eicosanoid were transferred into tubes, 100 ul antibody to the particular eicosanoid under investigation were added. The mixture was incubated for 2 h at room temperature or overnight at 4°C. Charcoal suspension was added and the samples were centrifuged at 2000xg for 15 min. The radioactivity in the supernatant was counted in a liquid scintillation counter. Statistics: Data are given as means f S.D.. Statistical analysis was performed using a one-way analysis of variance (ANOVA). A P value of less than 0.05 was considered significant. Results Measurement of reactive oxygen species Freshly isolated human granulocytes exposed to PMA (1 PM) showed a strong chemiluminescence signal in the presence of either luminol (50 uM) (Fig. 1A) or lucige-
120000-
PMA
*
100 pM silibinin
,,....*.*. ./I
PJA
80000
100000 -
100 pJ silibinin
”
80000 P a or‘ 60000-
luminol lucigenin
0 0
,,,,,““,““,““,““,“‘,, 5 10
15 time (min)
20
25
30
“9 ......
‘,S’..
60000 -
Od
-._.-
..._.
.j
30 time (min)
Fig. 1 Effect of silibinin on luminoland lucigenin-enhanced chemiluminescence of PMA-activated human granulocytes. Human granulocytes were incubated in HBSS at 37°C. (A) Luminol (50 PM), PMA (1 PM), and silibinin (100 PM) were added where indicated. Chemiluminescence was mainly due to the formation of HOCI as indicated by the fact that it was possible to almost completely inhibit it by sodium azide. (B) Lucigenin (100 PM), PMA (1 PM), and silibinin (100 FM) were added where indicated. Chemiluminescence was mainly due to the formation of 02- as indicated by the fact that it was almost completely inhibited by superoxide dismutase. The black line represents a measurement where silibinin was added, the dotted line a measurement where it was not added.
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Effects of Silibinin on Human Cells
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nin (100 PM) (Fig. 1B) acting as the chemiluminescence enhancer, indicating the formation of significant amounts of reactive oxygen species. Luminol can react with different reactive oxygen species. In the case of PMA-activated human granulocytes the reaction with hypochlorite (HOCI) is most important (17). This is also suggested by the fact that adding sodium aride (50 pM) led to a decrease in the chemiluminescence signal of 90%. Sodium azide is a potent inhibitor of the enzyme myeloperoxidase, present in granulocytes, that catalyzes the reaction of Hz02 and Cl- to HOCI. On the other hand, addition of ethanol (50 mM) and dimethyl sulfoxide (50 mM), that are very effective scavengers of OH-radicals, had no effect on the chemiluminescence intensities (data not shown). Lucigenin, on the other hand, reacts only with 02‘, which was confirmed by the almost complete elimination of the chemiluminescence signal after addition of superoxide dismutase (data not shown). Luminol-enhanced chemiluminescence was strongly decreased by adding silibinin, indicating a significant inhibition of HOCI release. This inhibitory effect of silibinin was clearly concentration-dependent with an ICsc-value of 7 uM silibinin (Fig. 2A.). In contrast to this, only a small effect of silibinin on lucigenin-enhanced chemiluminescence, and therefore on 02- formation was detectable. Under these conditions the IQ-value was well above 200 yM silibinin (Fig. 28).
Ol~,,,,,,,,,,,,,,~,,,, 0
50
01,,,,,,,..,,,,,,,,,,, loo
150
silibinin (vh.4)
Effect of silibinin granulocytes.
200
0
50
100 silibinin (PM)
150
200
Fig. 2 on the formation of HOC1 and 02- by human
Experimental details were the same as in Figure 1. Maximum chemiluminescence intensities after PMA-activation and addition of silibinin were calculated as percentages of (A) HOCI formation and (B) 02formation in controls where silibinin was not added. Values are means + S.D. of 4-6 experiments; * kO.05 compared to controls without silibinin.
LTB4 formation Freshly isolated human granulocytes activated with the calcium ionophore A 23187 (20 uM) formed 4558 pg LTB&Os cells within 5 min of incubation. Longer incubation times did not result in higher LTB4 amounts, after 30 min of incubation the amount of
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Effects of Silibinin on Human Cells
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LTB4 detected remained in the same range. Treatment of the cells with silibinin led to a strong inhibition of LTB4 formation in a dose-dependent manner. The I&o-value found here was 15 uM silibinin (Fig. 3A). This inhibitory effect of silibinin on LTB4 formation was independent of the stimulus used. When LTB4 formation of granulocytes was stimulated with either FMLP (20 pM) or opsonized zymosan (10 mg/ml) similar ICsovalues of silibinin (~20 uM) were determined (data not shown).
LTCa/Dd/Ea/Fa
formation
The effect of silibinin on the LTCd/Db/Eb/Fh formation of human granulocytes was similar to that found for LTB4 formation. 30 min after stimulation with A 23187 (20 yM) 2940 pg LTC~/D~/E~/F&O~ granulocytes were detected. The LTC4/D4/E4/F4 formation was inhibited by silibinin in a dose-dependent fashion with an I&o-value of 14.5 uM (Fig. 38).
A
0
5
Effect of metabolites
10 15 silibinin CM)
20
25
0
Fig. 3 silibinin on the formation by human granulocytes.
5
10
15
20
25
silibinin (PM)
of
54ipoxygenase
(A) Human granulocytes were incubated in PBS for 5 min at 37°C. LTB4 formation was stimulated by A 23187 (20 PM). (B) Human granulocytes were incubated in PBS for 30 min at 37°C. LTC4/D4/E4/F4 formation was stimulated by A 23187 (20 PM). Leukotriene concentrations were calculated as percentages of leukotriene formation by controls in which no silibinin was added. Values are means f S.D. of 4-6 experiments; *PcO.O5 compared to controls without silibinin.
PGE2 formation Incubation of freshly isolated human monocytes with LPS (10 ug/ml) for’8 h led to the formation of 238 pg PGEz/los cells. PGE2 was already detectable after 1 h of incubation but increased with a prolonged incubation period. PGEs formation was also inhibited by silibinin in a dose-dependent manner but significantly higher silibinin concentrations were necessary relative to the effect on leukotriene formation. The I&ovalue determined was 45 pM silibinin (Fig. 4A).
a-
100
0
20
40
40
60
silibinin (PM)
silibinin (PM)
Fig. 4 Effect of silibinin on the formation of cyclooxygenase metabolites by human mononuclear cells, platelets, and endothelial cells.
C
00
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60
silibinin (PM)
60
100
(A) Human mononuclear cells were incubated in PBS for 8 h at 37°C. PGE2 formation was stimulated by LPS (10 ug/ml). (B) Human platelets were incubated in PBS for 30 min at 37°C. TXB2 formation was stimulated by A 23187 (1 PM). (C) Human omentum endothelial cells were incubated in PBS for 60 min at 37°C. 6-K-PGF1, formation was stimulated by A 23187 (10 CLM). Eicosanoid concentrations were calculated as percentages of eicosanoid formation by controls in which no silibinin was added. Values are means + S.D. of 4-6 experiments; * PcO.05 compared to controls without silibinin.
TXB2 formation Human thrombocytes release TXA2 upon stimulation. This unstable eicosanoid rapidly reacts to the stable TXB2 (12). TXB2 was determined in our study after incubating thrombocytes using A 23187 (1 PM) as the stimulant. TXB2 was already detectable after 5 min incubation time. After 30 min 1477 pg TXBz/lOs cells were determined. Again rather high silibinin concentrations were necessary to half-maximally inhibit TXB2 formation. Here an IC5c-value of 69 uM silibinin was found (Fig. 48).
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6-K-PGF1,
Effects of Silibinin on Human Cells
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formation
Prostacyclin, another product of the cyclooxygenase pathway mainly formed by endothelial cells, is also a very unstable compound. Within seconds it is converted to 6K-PGFt, that is detectable with the help of radioimmunoassays (12). Human omentum endothelial cells cultured in monolayers formed 1484 pg 6-K-PGFl,/lOs cells within 1 h after stimulation with A 23187 (10 PM). After 4 h of incubation time the 6-K-PGFt, formation further increased to 3881 pg 6-K-PGFl,/lOs. To determine the effect of silibinin on 6-K-PGFt, formation, an incubation time of 1 h was chosen. Silibinin inhibited 6-K-PGFt, formation in a dose-dependent manner with an ICso-value of 52 PM silibinin (Fig. 4C).
Discussion In this study human cells were used to investigate the effect of the hepatoprotective flavonoid silibinin on the liberation of reactive oxygen species and the formation of eicosanoids. In both cases inhibitory effects were detected that did not affect the whole class of metabolites but displayed a strong predominance: HOCI formation was preferentially inhibited as compared to 02- formation; leukotriene production was strongly inhibited again at low concentrations, while only minor effects on the cyclooxygenase pathway were found at these concentrations. The preferential inhibition of HOCI formation by silibinin is in accordance with the results of Mira et al. (18). In their experiments in non-enzymatic systems micromolar concentrations of silibinin dihemisuccinate rapidly reacted with OH radicals and with HOC1 but not with 02-. An I&-,-value of about 5 uM was estimated for the scavenging of HOCI in a luminol-enhanced chemiluminescence assay. We determined almost the same ICso-value (IC5o 7 FM) in a similar assay with activated human granulocytes (Fig. 2A). Scavenging effects of silymarin and silybinin in the umolar concentration range on superoxide and alkoxyl radicals were also shown by Pascual et al. in nonenzymatic in vitro systems (19). As the effect of silibinin on HOCI formation can be observed in cellular and in non-enzymatic systems, it is unlikely that silibinin interferes with the enzyme myeloperoxidase. The same holds true for a possible reaction of silibinin with the cellular membrane as explanation for the observed effects. More probable is a direct reaction of the flavonoid with HOCI. The much weaker effect of silibinin on 02‘ formation is therefore also thought to be due to a scavenging effect, not to a reaction with the 02- producing enzyme NADPH-oxidase. This direct scavenging effect has also been proposed by Comoglio et al. for ethanol-derived free radicals using a silybin-phospholipid complex (20). Inhibitory effects of silibinin on the cyclooxygenase and 5-lipoxygenase pathways were detected in our study but silibinin concentrations that led to halfmaximal inhibitions were quite different from each other. A strong inhibition of the 5lipoxygenase pathway occurred. Silibinin halfmaximally inhibited LTB4 production at a concentration of 15 PM, for LTC4/D4/E4/F4 production an ICao-value of 14.5 uM was found (Fig. 3A,B). Three- to fourfold silibinin concentrations were necessary to achieve the same inhibitory effect on the formation of metabolites of the cyclooxygenase pathway. Here ICso-values of 45 PM, 69 PM, and 52 uM silibinin were determined for the inhibition of PGE2, TXB2, and 6-K-PGFt, formation, respectively (Fig. 4A,B,C). These IC:so-values both for the inhibition of leukotriene and prostaglandin formation are similar to those we found using rat Kupffer cells to determine the effect of silibinin on LTB4 and PGE2 formation (13). All metabolites of the 5-lipoxygenase pathway under investigation were inhibited with almost the same ICr,o-values of silibinin. As no predominance for either product was found, it can be concluded that silibinin interferes directly with the 5-lipoxygenase.
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The precursor LTA4 can no longer
be synthesized sufficiently and levels of all leukotrienes produced from LTA4 decrease. The same holds true for the cyclooxygenase pathway, all three products generated from prostaglandin HP by action of different enzymes were inhibited at comparable silibinin concentrations. In this case, silibinin probably acts upon the cyclooxygenase, the formation of PGH2 from arachidonic acid is disturbed. Inhibitory effects of various flavonoids on arachidonic acid metabolism have also been found by other authors. Laughton et al. (21) showed inhibitory effects of flavonoids on 5 lipoxygenase and/or cyclooxygenase using rat leukocytes and liver microsomes. In mice the oral administration of flavonoids inhibited the formation of paw oedema and the formation of PGEz and LTB4 (22). Yoshimoto et al. found various flavonoids to be relatively selective inhibitors of arachidonate 5lipoxygenase using enzyme preparations from rat cells (23). All these studies did not make use of silibinin, and they worked with species other than humans. Flavonoids from Silybum Marianum were employed by Fiebrich and Koch and Baumann et al., but again no human cells were used. Fiebrich and Koch found effects of silymarin on lipoxygenase from soybeans and on prostaglandin synthase from rats (24, 25). Baumann et al. worked in cell-free systems with enzyme preparations from rats (26). These authors determined inhibitory effects on both enzymes with a predominance for %ipoxygenase, but rather high concentrations (>O.l mM) were necessaty to achieve effects. Taken together, two properties of silibinin that may contribute to its therapeutic action became obvious in our study with human cells. Firstly, as regards reactive oxygen species silibinin acted mainly as a scavenger of HOCI liberated by neutrophils. The reaction with 02- occurred only to a much lesser extent. HOCI is a powerful antibacterial but also cytotoxic agent produced by neutrophils. Already at low concentrations it oxidizes sulfhydryl groups, disturbs various protein functions, and leads to cell death (27, 28). It therefore plays an important role in inflammatory reactions, and scavenging of this toxic agent by silibinin may be sufficient to explain its cytoprotective properties. Secondly, an inhibition of arachidonic acid metabolism with a strong predominance for the 5lipoxygenase pathway became obvious. It is known that leukotrienes contribute significantly to tissue injuries (29), whereas prostaglandins are considered to display cytoprotective effects (30). Our results therefore suggest that silibinin should not only be hepatoprotective but might also exhibit cytoprotective properties as regards injuries to other organs and tissues. Silibinin concentrations that were necessary to inhibit HOCI production and the 5-lipoxygenase pathway are in the range of plasma concentrations that are reached after the usual clinical dose of 240 mg silibinin (0.4-l .3 FM),
Acknowledaements This work was supported by the Biotech Program of the EU. We wish to thank Christina Weeger for her excellent
technical
assistance
with the LTB4 measurements.
References 1. 2. 3. 4. 5.
B. HAVSTEEN, Biochem. Pharmacol. 32 1141-1148 (1983). P. MORAZZONI and E. BOMBARDELLI, Fitoterapia 66 3-42 (1995). M. MOURELLE, P. MURIEL, L. FAVARI and T. FRANCO, Fundam. Clin. Pharmacol. 9 183-191 (1989). P. MURIEL and M. MOURELLE, J. Appl. Toxicol. n 275-279 (1990). A. VALENZUELA and R. GUERRA, FEBS Lett. m 291-294 (1985).
1600
6. 7.
8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.
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P. MURIEL, T. GARCIAPINA, V. PEREZ-ALVAREZ and M. MOURELLE, J. Appl. Toxicol. X 439-442 (1992). R. CAMPOS, A. GARRIDO, R. GUERRA and A. VALENZUELA, Planta Med. % 417-419 (1989). A. VALENZUELA, C. LAGOS, K. SCHMIDT and L.A. VIDELA, Biochem. Pharmacol. 34 2209-2212 (1985). P. I. LANG, K. NEKAM, G. DEAK, G. MUZES, R. GONZALES-CABELLO, GERGELY, G. CSOMOS and J. FEHER, Ital. J. Gastroenterol. a 283-287 (1990). E. LENG-PESCHLOW and A. STRENGE-HESSE, Z. Phytother. 12 162-l 74 (1991). H. DE GROOT, Hepato-Gastroenterol. &I 328-332 (1994). K. DECKER, Semin. Liv. Dis. 5 175-190 (1985). C. DEHMLOW, J. ERHARD and H. DE GROOT, Hepatology in press (1996). M. HOFFMAN, D.M. MONROE and H.R. ROBERTS, Am. J. Clin. Pathol. 98 531533 (1 992). P.A. KERN, A. KNEDLER and R.H. ECKEL, J. Clin. Invest. II 1822-1829 (1983). A.K. CAMPBELL, TIBS 11 104-108 (1986). J. ARNOLD, S. HAMMERSCHMIDT and K. ARNOLD, Biochim. Biophys. Acta 1097 145-151 (1991). L. MIRA, M. SILVA and CF. MANSO, Biochem. Pharmacol. &j 753-759 (1994). C. PASCUAL, R. GONZALES, J. ARMESTO and P. MURIEL, Drug Develop. Res. a 73-77 (1993). A. COMOGLIO, A. TOMASI, S. MALANDRIO, G. POLI and E. ALBANO, Biochem. Pharmacol. a 1313-1316 (1995). M.J. LAUGHTON, P.J. EVANS, M. MORONEY, J.R.S. HOULT and B. HALLIWELL, Biochem. Pharmacol. 42 1673-1681 (1991). M.L. FERRANDIZ and M.J. ALCARAZ, Agents Actions 2 283-288 (1991). T. YOSHIMOTO, M. FURUKAWA, S. YAMAMOTO, T. HORIE and S. WATANABEKOHNO, Biochem. Biophys. Res. Commun. 116 612-618 (1983). F. FIEBRICH and H. KOCH, Experientia % 1548-1550 (1979). F. FIEBRICH and H. KOCH, Experientia a 1550-1551 (1979). J. BAUMANN, F. VON BRUCHHAUSEN and G. WURM, Prostaglandins 24.627639 (1980). B. HALLIWELL, Am. J. Med. g 14S-22s (1991). C.G. COCHRANE, Am. J. Med. 9l_ 23S-30s (1991). F.A. KUEHL and R.W. EGAN, Science m 978-984 (1980). J. QUIROGA and J. PRIETO, Pharmac. Ther. % 67-92 (1993).