Antioxidant and hepatoprotective effects of artichoke extracts and constituents in cultured rat hepatocytes

Pergamon

Toxicology

in Vitro 11 (1997) 669-672

Antioxidant and Hepatoprotective Effects of Artichoke Extracts and Constituents in Cultured Rat Hepatocytes R. GEBHARDT* Physiologisch-chemisches

Institut

and M. FAUSEL

der Universitst

Tiibingen,

D-72076

Tiibingen,

Germany

Abstract-Primary cultures of rat hepatocytes exposed to tert-butyl hydroperoxide (t-BHP) were used for characterizing the antioxidative and hepatoprotective potential of aqueous artichoke extracts (AK) and some selected constituents. Addition of t-BHP to the culture medium resulted in enhanced lipid peroxidation measured as production of malondialdehyde (MDA) and enhanced cytotoxicity detected by LDH leakage. Aqueous artichoke extracts added prior or simultaneously with t-BHP reduced both phenomena with EC& values of about 95 and I2 fig artichoke powder/ml, respectively. Furthermore, AE prevented the loss of intracellular GSH caused by t-BHP. Several polyphenolic and flavonoid constituents of AE were found to reduce MDA production. ECso values were 8. I, 12.5, 15.2, 28 pg/ml for caffeic acid, chlorogenic acid, cynarin and cynarosid, respectively. A similar ranking for AE and constituents was found when using the chemiluminescent xanthine oxidase assay for determination of the antioxidative potency, but EC% values were consistently lower. Concerning the hepatoprotective effect, that is prevention of LDH leakage, all constituents were almost equipotent and EC, values were lower than for MDA production. These results suggest (i) that lipid peroxidation is not the primary cause for cytotoxicity of t-BHP, (ii) that hepatocyte cultures exposed to t-BHP represent a suitable model system for screening plant extracts for antioxidant and hepatoprotective effects, and (iii) that AE have a marked antioxidative and hepatoprotective potential which can be ascribed, at least partially, to some ubiquitous and artichoke-specific polyphenolic and flavonoid compounds. 0 1997 Published by Elsevier Science Ltd

Abbreviations:AE = artichoke extracts; t-BHP = terr-butyl hydroperoxide; tration exerting half maximal response; LDH = lactate dehydrogenase; SD = standard deviation: W = Williams’ medium E.

INTRODUCTION By exposing

primary cultures of rat hepatocytes to various kinds of peroxides (e.g. t-BHP) and other prooxidants it is possible to mimic several aspects related to liver pathology characterized by increased lipid peroxidation and cytotoxicity due to oxidative stress (Gebhardt, 1995a; Sugihara et al., 1994; Yamamoto and Farber, 1992). Such exposed cultures provide a valuable in vitro system for investigating antioxidative and hepatoprotective properties of various chemicals (Dogterom et al., 1988; Glascott et al., 1992; Villa et al., 1992). Determination of lipid peroxidation through production of malondialdehyde (MDA), of the intracellular level of glutathione, and of lactate dehydrogenase (LDH) leakage represent simple endpoints that can help in assessing the relative effectiveness of different compounds to counteract the damaging conditions (Gebhardt, 1995b; Mertens et al., 1993; Morel et al., 1993). In the present study, we have used this model system to characterize the antioxidative and hepatoprotective potential of complex standardized

*Author for correspondence. 0887-2333/97/$17.00 + 0.00 0 SSDI 0887-2333(97)00078-7

1997 Published

EC, = effective concenMDA = malondialdehyde;

plant extracts from artichoke (Cynava scolymus L.) leaves and to identify some of the constituents responsible for these effects. In order to verify these results the xanthin oxidase chemiluminescence assay was applied as an additional in vitro method for measuring antioxidative properties.

MATERIALS AND METHODS

Isolation and cultivation

of rat hepatocytes

Male Sprague-Dawley rats (220-300 g) were used as hepatocyte donors. Rat hepatocytes were isolated according to Gebhardt et al. (1990). The isolated hepatocytes were suspended in William’s medium E (W) containing 10% newborn calf serum, 2 mM glutamine, penicillin (50 units/ml), streptomycin (50 pg/ml) and IO-’ M dexamethasone (Gebhardt et al., 1990). They were seeded in 1 ml culture medium at a density of 1.25 x 10’ cells/cm2 into six-well plates or 6 ml into petri dishes. From 2 hr onwards, serum-free medium was used. Exposure of hepatocyte cultures to t-BHP at 0.8 mM or the concentrations indicated for 40 min was started 2 hr after incubation in serum-free medium.

by Elsevier Science Ltd. All rights

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R. Gebhardt

670

and M. Fausel

Table I. Influence of t-BHP on MDA production and LDH leakage of cultured Concentration (mM)

MDA production (pmol,mg protein)

II 0.5 0.x I .o I.5 2.0

251 837 1098 II59 I562 1929

& I IT i_ 177 * 247 i_ 206 k 225 -i_ 387

76k I68 k 372 i 534 * 878 i II43 +

*Percentage of total hepatocellular LDH activity. Walues represent means f SD from duplicate determmatmns Table 2 Comparison determined

of EC,,, values ofaruchoke with the t-BHPjhepatocyte

hepatocytes

LDH leakage (mU:ml)

o/b”

IO 42 104 166 241 32s

6 I3 29 42 69 91

m three to eight expenments.

extract,. polyphenolic compounds and tlavonea model or the xanthme oxldase assay EC,,, values (jcg/ml)

MDA production

Materul Artichoke extract I Artichoke extract 2 Caffelc acid Chlorogenic acid Cynarm Cynaroside

“Determined from dose-response computer-aided analysis.

Preparation

qf the artichoke

LDH leakage

curves

with

extract.s

The standardized powder (Hepar SL” forte, Sertiirner Arzneimittel GmbH, Giitersloh. Germany) prepared from native extracts of artichoke (Qnara scolwnus L.) leaves (4.5:1) was used for preparation of aqueous extracts by dispersing 100 mg artichoke powder in 8 ml W, shaking for 20 min at room temperature, centrifuging at 6000 g and filtering through sterile filters with 0.2 pm pore size. All dilutions of the stock extract were made with W and supplemented to the normal culture medium. Concentrations are expressed as pg artichoke powder dry weight per ml. Determination

of’ qytoto.xicity

utzd other

Xanthine

II 13 70 8.6 IO 3 9.2

93 98 XI 12.5 IS.2 28

ctnal~~ticul

at

oxidase assay 69 81 0.6 I.5 2.4 4.7

least

IO different

concentrations

by

Aqueous artichoke extracts (AK) prepared from a powdered extract of artichoke leaves, reduced both MDA production and LDH leakage dose dependently when added to the culture medium during the exposure to t-BHP (Fig. 1). Addition of AE prior to t-BHP produced almost the same protective effect (not shown). Interestingly, the reduction of LDH leakage always showed lower EC,, values than the reduction of MDA production (Table 2) indicating that lipid peroxidation reflected by the liberation of MDA was not the single cause for the cytotoxic response. AE also prevented the loss of intracellular GSH caused by t-BHP (Table 3). Using the xanthine oxidase assay as a cell-free biochemical technique for the screening of antioxidants, slightly

procedures

Cytotoxicity of the compounds tested was determined by means of the LDH leakage assay as described (Gebhardt, 1994). Production of MDA was measured by the TBA assay as modified by Mertens et al. (1993). The cellular content of total GSH was determined according to Brehe and Burch (1976). The xanthine oxidase chemiluminescence assay was performed according to Videla (1983) with slight modifications.

600 500

RESULTS 0.001

0.01

Concentration

0.1

e

100

f

300

2

200

f

100

= 5 =

0 0

Exposure of rat hepatocyte cultures to different concentrations of t-BHP for 40 min resulted in stimulated lipid peroxidation measured as production of MDA in a concentration-dependent manner (Table 1). Simultaneously, an increasing LDH leakage could be observed indicating the cytotoxic response of the hepatocytes to t-BHP (Table 1). For the screening of antioxidants the concentration of 0.8 mM t-BHP was chosen which caused a well pronounced but still submaximal cytotoxic response.

r

r

1

(mglml)

Fig. 1. Influence of aqueous artichoke extracts on MDA production (0- - -0) and LDH leakage ([I- - - []) induced by t-BHP in cultured rat hepatocytes. Hepatocytes were cultured for a total of 4 hr and then exposed to t-BHP (0.8 mM) for 40 min with or without AE added in various concentrations to the culture medium. The short lines represent the basal values of MDA production (left: solid) and LDH leakage (right: dashed). Values represent means + SD of triplicate determinations of a representative experiment.

Antioxidant effects of artichoke extracts Table

3.

Influence of artichoke extracts on hepatocellular GSH in control and t-BHP-exposed cultures Cellular GSH content (nmol/mg protein)

Artichoke extracts (mgiml) 0 0.001 0.01 0.1 1.0

Control” 36 f 35 f 38 + 34 + 32 k

2 2

I 3 2

t-BHP 22 * 1’ 28 f 2c.d 31 f2+ 34 5 2d 33 * Id

“Data are means k SD from two to four determinations derived from three different experiments. hDetermined 2 hr after serum-free cultivation. ‘Different from respective control; P < 0.01. dDifferent from the corresponding value in the absence of artichoke extracts; P < 0.01.

lower ECwl values were determined for the AE than with the MDA assay (Table 2). Since AE contain several ubiquitous as well as artichoke-specific polyphenolic compounds and flavonoids that could in principle be responsible for the antioxidant properties, we compared the efficacy of some of them using the same experimental system. The EC, values for the reduction of the MDA production and for inhibiting the chemiluminescent xanthine oxidase assay increased in the order caffeic acid, chlorogenic acid, cynarin, and cynaroside (luteolin-7-O-glucoside) with a similar relative proportion, although the values for the xanthine oxidase assay were lower again (Table 3). The EC, values for the cytoprotective effects, however, were within a narrow range for all compounds tested.

DISCUSSION

Primary cultures of rat hepatocytes have been used in many studies on oxidative challenge by various compounds as well as on the effects of antioxidants (Gebhardt, 1995a; Glascott et al., 1992; Mertens et al., 1993; Morel et al., 1993; Ponsoda et al., 1995; Yamamoto and Farber, 1992). Among other advantages, cultured hepatocytes show elevated levels of GSH even after short cultivation periods compared with isolated hepatocytes which have not yet fully recovered from the loss of this important protective molecule during the isolation procedure (Kera et al., 1988). Since there is only a minor loss in cytochrome P-450 activities during the early period of cultivation the cultured cells are also competent to metabolize xenobiotics similarly to hepatocytes in viva (Coecke et al., 1993; Gebhardt et al., 1990; Ponsoda et al., 1995; Waxman et al., 1990). This is of particular importance when assessing the influence of complex mixtures such as extracts from artichoke leaves which might contain a wide variety of known and unknown constituents that undergo biotransformation. As shown herein, t-BHP-induced MDA production reflecting lipid peroxidation was considerably reduced and the corresponding loss of intracellular GSH was prevented by AE. Likewise, LDH

671

leakage due to hepatocyte necrosis was reduced, but interestingly with a much lower EC, value. This difference in EC, values indicates that lipid peroxidation induced by t-BHP is not directly responsible for the cytotoxic effect of this agent. The same conclusion could be derived from the biochemical xanthine oxidase assay, which led to a relative antioxidative potency of the extracts and the constituents tested similar to that determined with the MDA assay indicating a similar mode of action with respect to the inhibition of lipid peroxidation. In contrast, with respect to the inhibition of the necrotic response the EC, value were quite similar. The interpretation that lipid peroxidation is not the direct cause of cytotoxicity in the case of t-BHP contrasts with findings by De Groot et al. (1988) obtained with the carbon tetrachloride model, but is in line with observation by Dogterom et al. (1988) in a different model using t-BHP and other cytotoxic agents. Akcording to our results, a variety of artichokespecific compounds, like cynarin (actually formed during processing) and luteolin-7-O-glucoside as well as several more abundant polyphenolic compounds such as caffeic acid and chlorogenic acid, which are all contained in the AE, may contribute to the antioxidative and hepatoprotective potential of these extracts. In fact, since the content of flavonoids and polyphenolic compounds in the AE ranges within 0.2 to 0.8%, and 1.5 to 2.5%, respectively, these numbers match well with the results of the xanthin oxidase assay, while they appear a little low when compared with the results of the t-BHP assay. This difference may reflect the influence of additional phenomena in the more complex cellular assay such as uptake, metabolism and mode of interaction. In contrast to our findings with the t-BHP assay, Adzet et al. (1987), using the carbon tetrachloride model system, found that only cynarin and, to a lesser extent, caffeic acid could act hepatoprotective. Thus, the results of the present study further suggest that primary hepatocyte cultures provide a suitable in oitro system for screening complex plant extracts for possible antioxidative and hepatoprotective constituents. The pronounced antioxidative potential of the standardized AE, Hepar SL” forte, may be of considerable interest with respect to the therapeutic application of this phytopharmacon, because it may reduce the production of oxidized LDL (R. Gebhardt, unpublished observation) that plays a unique role in arteriosclerosis. Since artichoke extracts were also found to inhibit hepatocellular cholesterol biosynthesis in vitro (Gebhardt, 1995a) and to lower serum levels of cholesterol in patients (Fintelmann, 1996; Wegener, 1995), they might actually help to reduce the atherogenic risk in a dual manner. Thorough studies to further explore these aspects are currently designed.

672

R. Gebhardt

Ackno~ledgemenrs-This work was supported in part by Sertiirner Arzneimittel GmbH, Giitersloh, Germany, and by the Deutsche Forschungsgemeinschaft. REFERENCES

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