Efficacy of different Cynara scolymus preparations on liver complaints

Journal of Ethnopharmacology 86 (2003) 203–211

Efficacy of different Cynara scolymus preparations on liver complaints E. Speroni a,∗ , R. Cervellati b , P. Govoni c , S. Guizzardi c , C. Renzulli a , M.C. Guerra a a

Department of Pharmacology, University of Bologna, Via Irnerio 48, Bologna 40126, Italy Department of Chemistry G. Ciamician, University of Bologna, Via Selmi 2, Bologna 40126, Italy Department of Experimental Medicine, Histology section, University of Parma, Via Volturno 39, Parma, Italy b

c

Received 15 April 2002; received in revised form 3 February 2003; accepted 12 February 2003

Abstract Cynara scolymus leaves extracts have long been used in folk medicine for their choleretic and hepatoprotective activities, that are often related to the cynarin content. These therapeutic properties are also attributed to mono- and di-caffeoylquinic acids and since commercial C. scolymus preparations can differ for their activities, we studied four extracts to evaluate, if present, a relationship between the hepatobiliary properties of the different preparations and their content in phenolics. The antioxidant activity of the commercial preparations examined was also considered in an in vitro system. The results showed that the extract with the highest content in phenolic derivatives (GAE) exerted the major effect on bile flow and liver protection. Also the results of the antioxidant capacity (BR) of the different preparations are in good agreement with the results obtained in vivo. On the contrary, administering rats with doses of chlorogenic acid, equivalent to those present in this extract, we did not observe any choleretic or protective action. An histopathological analysis of liver sections confirmed the biochemical results. Perhaps caffeoyl derivatives have a role in the therapeutic properties of C. scolymus extracts, as reported in literature for “in vitro” studies, but when administered alone, they are not so effective in exerting this action. © 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cynara scolymus; Phenolic content; Hepatic damage; Choleresis; Histopathologic studies; Briggs–Rauscher reaction

1. Introduction Cynara is a genus of seven annual taxa belonging to the family of Asteraceae, found mostly in all central and southern countries of Europe. Extracts from artichoke, Cynara scolymus L., have been used in folk medicine against liver complaints and the extracts or its constituents have been claimed to exert a beneficial action against hepato-biliary diseases (Gebhardt, 1997; Chinou and Harvala, 1997) and to improve liver regeneration after partial hepatectomy (Adzet et al., 1987; Preziosi, 1962). At least some of these effects are due to the antioxidative potential of artichoke extracts or their constituents that are mainly flavones, flavanones, flavonols, coumarins, phenolic acids and that can be present in different amounts and combinations (Skakun and Stepanov, 1976; Faure et al., 1990; Cao et al., 1997). The therapeutical activity of the crude extract is probably first of all due to the phenolic structure of these substances, that is responsible of the free radical mediated processes

∗ Corresponding

author. Tel.: +39-051-2091793; fax: +39-051-248862. E-mail address: [email protected] (E. Speroni).

inhibition (Fraga et al., 1987; Hertog and Hollmann, 1998). Free radical and lipid peroxidation are known to play an important role in a great number of pathological states (Brent and Rumack, 1993; Knight, 1995). For centuries, C. scolymus, has also been used for its strong choleretic activity causing substantial increase in the amount of bile excreted and an increase in the concentrations of biliary acids of the bile. These therapeutic activities could be related to mono- and di-caffeoylquinic acids content since extensive evidences reported choleretic and hepatoprotective activities of these compounds, but such effects have not been investigated yet (Gadgoli and Mishra, 1997; Gorzalczany et al., 2001). Commercial products containing artichoke total extracts, can differ for their therapeutic properties, as for the methodologies of preparation as for the different content in polyphenolic compounds. The purpose of this study was to evaluate the effect of four different artichoke commercial preparations on bile flow (BF) and compare them. Since artichoke total extract is frequently expressed in cynarin content, we determined in each extract tested for choleretic activity the amount of cynarin and also of total caffeoyl derivatives expressed as chlorogenic acid. Moreover, we tested the antioxidant

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properties with tests “in vitro”. The commercial extracts have been evaluated for their choleretic activity. Therefore, we investigated the “in vivo” hepatoprotective effects of C. scolymus leaves total extract using CCl4 intoxicated rats as experimental model.

2. Materials and methods 2.1. Plant material We used four different C. scolymus total extract preparations commercially available. 2.2. Chemicals Hematoxylin (C.I. No. 75290) and eosin yellowish (C.I. No. 45380) were purchased from Chroma-Gesellschaft. Toluidine blue (C.I. No. 52040), biebrich scarlet (C.I. No. 26905), acid fuchsin (C.I. No. 42685), orange G (C.I. No. 16230) and light green SF yellowish (C.I. No. 42095) were purchased from Sigma Chemical Co. (St. Louis, MO). All other chemicals were of analytical grade and were purchased from Sigma. 2.3. Determination of cynarin, chlorogenic acid and other derivatives expressed as chlorogenic acid The crude extracts were analyzed in a Shimadzu HPLC equipped with a photodiode array detector (model SPD-M10 Avp). A Phenomenex Luna C18 (2) (150 mm × 4.6 mm) 3-␮m analytical column was used and the mobile phases were: phosphoric acid 0.01 M (solvent A) and acetonitrile:methanol = 2:1 (solvent B). Analyses were performed in the following gradient: solvent B from 10 to 20% over 15 min and then from 20 to 50% over the next 15 min. Detection at 330 nm. 2.4. Determination of total phenolic content expressed as gallic acid equivalent (GAE) The total phenol content of the four extracts was measured spectrophotometrically with Folin-Ciocalteu reagent using gallic acid as a calibration standard in the range 10–50 mg/l (Singelton and Rossi, 1965). 2.5. Determination of antioxidant activity based on BR oscillating reaction The BR oscillating system was discovered in 1973 (Briggs and Rauscher, 1973) and consists of the iodination and oxidation of an organic substrate (in general, malonic acid or its derivatives) by acidic iodate in the presence of hydrogen peroxide with Mn2+ ion as catalyst. Oscillatory behavior in the BR system can be easily followed potentiometrically using a

bright platinum electrode coupled with a suitable reference electrode. When an antioxidant scavenger of free radicals is added to an active oscillating BR mixture, there is an immediate quenching of oscillations, an inhibition time that linearly depends on the concentration of the antioxidant added, and subsequent regeneration of oscillations (Cervellati et al., 2000, 2001). Oscillations in the BR mixtures were followed potentiometrically by recording the potential of the mixture using a coupled bright platinum electrode (Hamilton, model P/N 238 945)–reference electrode (double-junction Ag/AgCl, Ingold, model 373-90-WTE-ISE-S7). Electrodes were connected to a pH multimeter (WTW, model pH 540 GLP) controlled by an IBM-compatible PC. The accuracy of the multimeter was ±1 mV. The suitable data acquisition Multi Achat II (WTW) was used. The multimeter was equipped with a temperature sensor with an accuracy of ±0.1 ◦ C. 2.6. Test animals Male Sprague–Dowley rats (150–180 g) were purchased from Harlan Italy (Correzzana, MI). They were housed in standard conditions (22 ± 1 ◦ C, humidity: 60 ± 55, 12:12 h light/dark cycle), and fed with standard diet and tap water ad libitum for 1 week prior to treatment. Procedures and animal comfort were controlled by the University Veterinary Service. 2.7. Bile flow Rats were starved for 18 h before the experiment with free access to water. Each extract was suspended in 0.5% carboxymethyl cellulose (CMC) in distilled water prior to oral administration. Eight groups of six rats each, were treated by gastric gavage with each of the four plant extracts at a dose of 1 and 2 g/kg. Two groups of six rats each received chlorogenic acid at a dose of 40 and 80 mg/kg, corresponding to the content of the caffeoyl derivative in 1 and 2 g/kg of the third extract (C), that presented the major content in chlorogenic acid. Moreover, two groups of six rats each received cynarin at a dose of 0.005 and 0.01 mg/kg, corresponding to the content in 1 and 2 g/kg of the first extract (A), that presented the major content in cynarin. Control animals received a similar volume of CMC solution (0.2 ml/100 g). Twenty minutes after the oral treatment with the extracts, animals were anesthetized with urethane (1.2 g/kg i.p). The abdomen was opened with a midline incision and the common bile duct exposed and cannulated just before the hepatic hilus in order to avoid contamination with pancreatic juice. Rectal temperature was monitored and maintained at 37 ± 0.5 ◦ C throughout the experiment using a warming lamp. Bile was collected by gravity in pretared vials at 20-min intervals for 120 min. BF was determined by weight assuming that the specific gravity of rat bile is 1.0.

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2.8. CCl4 administration The animals were divided into 14 groups consisting of six rats each. The control group was given 0.5% CMC suspension by gastric gavage (per os). The carbon tetrachloride group (positive control) received 0.5% CMC and 50% CCl4 in liquid paraffin (2.5 ml/kg body weight, per os) 60 min after the administration of the vehicle. The test groups were treated with 1 or 2 g/kg per os of the suspension of the four C. scolymus total extracts in 0.5% CMC. Two groups of six rats each received chlorogenic acid at a dose of 40 and 80 mg/kg, corresponding to the content of the caffeoyl derivative in 1 and 2 g/kg of the third extract (C), that presented the major content in chlorogenic acid. Two groups of six rats each received cynarin at a dose of 0.005 and 0.01 mg/kg, corresponding to the content in 1 and 2 g/kg of the first extract (A), that presented the major content in cynarin. Sixty minutes after the administration of the extracts or chlorogenic acid, the animals received CCl4 , 50% in liquid paraffin (2.5 ml/kg body weight, per os). Twenty-four hours after the toxin administration, blood samples were withdrawn by cardiac puncture and then the rats were sacrificed by an overdose of urethane. Blood samples collected in heparinized tubes were centrifuged at 3000 × g for 10 min to obtain plasma. Plasma samples were used to test aspartate transferase (AST) and alanine transaminase (ALT) activities. The liver of each rat was promptly removed and used to determine the tissue levels of lipid peroxide and for histopatological studies. 2.9. Aspartate transferase and alanine transaminase in plasma The activities of AST and ALT were determined according to the method of Wilkinson et al. (1972) using an assay kit. 2.10. Lipid peroxidation in liver tissue Liver peroxidation in liver samples was measured according to the method of Okawa et al. (1979) and modified by Jamall and Smith (1985); Rao and Mishra (1998). Rats were sacrificed by an overdose of urethane and the liver was immediately excised and chilled in ice-cold 0.9% NaCl and then perfused via portal vein with ice-cold 0.9% NaCl. After washing with 0.9% NaCl, 1 g of wet tissue was exactly weighted and homogenized in 9 ml of 0.25 M sucrose using a teflon homogenizer to obtain a 10% suspension. The cytosolic fraction was obtained by a two-step centrifugation at 2000 × g for 30 min at 4 ◦ C. A volume of the homogenate (0.20 ml) was transferred to a vial and mixed with 0.2 ml of a 8.1% (w/v) sodium dodecyl sulphate solution, 1.5 ml of a 20% acetic acid solution (pH 3.5) and 1.5 ml of a 0.8% (w/v) solution of TBA and the final volume was adjusted to 4.0 ml with distilled water. Each vial was tightly capped and heated in a boiling water bath for 60 min. The vials were then cooled under running water. Four milliliter of N-buthanol was added to each vial and centrifuged at

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1000 × g for 10 min. The fluorescence of the supernatant fraction was measured: excitation wavelength, 530 nm and emission wavelength, 590 nm. Due to peroxidative effect of CCl4 on tissue, livers of CCl4 -treated rats were used as positive control. 1,1,3,3-Tetraethoxypropan was used as standard for calibration of the curve. 2.11. Histopathological studies Large specimens of the liver, promptly removed after autopsy, were fixed in 10% formalin for 24 h and embedded in paraffin. Serial sections were stained with hematoxylin–eosin to show the common morphology of the tissues, especially the cells, and others sections with Masson’s trichrome method to value fibrous tissue formed in response to toxic insult to the liver. Small specimens were fixed in 1% paraformaldehyde plus 1.5% glutaraldehyde in sodium cacodylate buffer, pH 7.4 at 4 ◦ C, dehydrated in graded acetone and embedded in epoxy resin. Semi-thin sections were stained with toluidine blue. Ultra-thin sections were obtained with a diamond knife on a LKB ultramicrotome, collected on Cu–Ni–Au grids, stained with uranyl acetate and lead citrate and observed on a Philips EM 400T electron microscope. 2.12. Statistical analysis The data are expressed as mean ±S.D. and were analyzed by Dunnett’s test. Probability levels of less than 0.01 were considered significant. 3. Results 3.1. Caffeoyl derivatives present in the extracts We determined the total caffeoyl-derivatives content, cynarin and chlorogenic acid content, in four commercially available extracts of C. scolymus leaves. The results are summarized in Table 1. 3.2. Determination of total phenolic content expressed as GAE Fig. 1a shows the graphs Abs (765 nm) versus suitable dilutions for the four extracts. It can be see that the order of the phenolic content is: extract 3 > extract 4 > extract 1 > extract 2. Using the gallic acid calibration curve it was possible to calculate the mean GAEs (mg/l), in the explored dilution range. The values are: 1843 ± 42 extract 3, 1670 ± 43 extract 4, 1273 ± 21 extract 1, 402 ± 2 extract 2. 3.3. Determination of antioxidant activity based on BR oscillating reaction Fig. 1b shows the graphs tinhib /s versus the same dilutions for the four extracts as in Fig. 1a. Since inhibition time is

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Table 1 Cynarin, chlorogenic acid and remaining derivatives content of the four extracts tested Extract

1 2 3 4 a

Cynarin

Caffeoyl derivativesa

Chlorogenic acid

(mg/g)

(%)

(mg/g)

(%)

(mg/g)

(%)

0.005 7 × 10−5 8 × 10−5 5 × 10−4

0.5 0.007 0.008 0.05

2.5 0.079 40 4.45

0.25 0.008 4.00 0.45

14.7 0.367 77.1 15.3

1.47 0.037 7.71 1.53

The values were expressed as the sum of the areas corresponding to the remaining caffeoyl-derivatives content and expressed as chlorogenic acid.

strictly related to the antioxidant capacity, it can be seen that the order of the antioxidant power is: extract 3 > extract 4 > extract 1 > extract 2. These results demonstrate the very good correlation between the total phenolic content and the antioxidant capacity.

C. scolymus leaves, respectively, of extracts 1 and 3, that were the richest in the two compounds, administered at 1 and 2 g/kg. 3.5. Effects of C. scolymus leaves extracts on CCl4 -induced damage

3.4. Bile flow The effects of orally administered crude extracts of C. scolymus leaves, cynarin and chlorogenic acid, on BF are reported in Figs. 2 and 3. Control rats presented a slight regular decrease in BF level during the whole experiment. Extract 1, at 1 and 2 g/kg, seemed to have no effects on BF, that remained at control level during 2 h. On the contrary, extract 2, induced a marked stimulation of BF, within the first 20 min that decreased in the following 20 min until the end of the experiment. A significant but not strictly dose-dependent effect, was obtained with the extract 3. The increase was obtained already since 20 min after administration and persisted during 120 min. Sample 4 produced a little, but not significant, increase on BF at 2 g/kg, and then decrease to control values. At 1 g/kg, no effect was observed. Cynarin (0.005 and 0.01 mg/kg) and chlorogenic acid (40 and 80 mg/kg) showed no choleretic effects (Fig. 3a and b) at the doses corresponding to the content in

The results of antihepatotoxic activity of C. scolymus leaves extracts compared with chlorogenic acid are summarized in Table 2. In the CCl4 -treated control rats, serum AST and ALT were increased, respectively, to 2653 ± 148 and 2472 ± 148. In normal group, the values of AST and ALT were 81 ± 18 and 44 ± 8. The groups treated with 1 and 2 g/kg of extract 3, decreased significantly the high levels of AST and ALT, towards normalization when compared with the CCl4 control group (P < 0.05). In contrast, in the rats administered with extracts 1, 2 and 4 and with chlorogenic acid or cynarin, there was no significant recovery of serum AST and ALT. The effects of the four extracts and chlorogenic acid on hepatic lipid peroxidation, using CCl4 produced injury in rats, are reported in Table 3. MDA was increased up to 50% in CCl4 -treated group when compared with the normal group.

Table 2 Effects of four different extract of Cynara scolymus commercially available, cynarin and chlorogenic acid on serum AST and ALT in CCl4 intoxicated ratsa Parameter

AST (U/l)

Control (0.5% CMC) CCl4 per os Extract 1 (1 g/kg) Extract 1 (2 g/kg) Extract 2 (1 g/kg) Extract 2 (2 g/kg) Extract 3 (1 g/kg Extract 3 (2 g/kg) Extract 4 (1 g/kg) Extract 4 (2 g/kg) Chlorogenic acid (40 mg/kg) Chlorogenic acid (80 mg/kg) Cynarin (0.005 mg/kg) Cynarin (0.001 mg/kg)

81.93 2653.05 2354.89 2340 2101 1997.45 1510.50 1327.96 2389.32 2156.58 2212.60 1983.78 2300.45 2007.78

± ± ± ± ± ± ± ± ± ± ± ± ± ±

18.13 148.06 89.92 67.45 56.34 78.56 23.22 85.46 57.32 34.90 67.34 79.05 76.89 87.06

Protectionb (%)

ALT (U/l)

−43.06%∗ −49.94%∗

44.30 2471.69 2134.89 2156.56 2234.78 2190.70 1109.35 1039.56 2341.23 2002.43 1956.32 1879.54 1890.78 1956.90

± ± ± ± ± ± ± ± ± ± ± ± ± ±

Protectionb (%) 8.06 148.38 109.78 78.56 31.16 102.78 60.54 102.58 78.54 84.31 73.21 75.09 67.09 78.90

Data are expressed as mean ± S.D. (n = 6). Calculated by considering the difference in the mean levels between control and CCl4 -treated groups as 100% protection. ∗ P < 0.01, compared with the CCl control by Dunnett’s test. 4 a

b

−55.12∗ −57.94∗

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Fig. 1. (a) Total phenol content of the four Cynara scolymus extracts evaluated as gallic acid equivalents (GAE)/L. (b) Straight lines of tinhib vs. concentration of the four extracts tested.

Pretreatment with 1 and 2 g/kg of extract 3 reduced CCl4 -induced MDA production by 50%, when compared with CCl4 control group (P < 00.5). This decrease was not strictly dose dependent. In contrast, pretreatment with the other three extracts or chlorogenic acid did not reduce significantly the production of MDA.

3.6. Histological examination Specimens from control animals showed normal microarchitecture of the liver, divided into exagonal lobules oriented around the terminal tributaries of the hepatic vein (central veins). The hepatic parenchyma was organized into cribriform, anastomosing sheets of hepatocytes, seen in mi-

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Fig. 2. (a) Effect of four Cynara scolymus total extracts (1 g/kg p.o.) and CMC solution on bile flow in the rat. Results are expressed as mean ± S.D. (n = 6). (∗ ) P < 0.05 vs. control, Dunnett’s test. (b) Effect of four C. scolymus total extracts (2 g/kg p.o.) and CMC solution on bile flow in the rat. Results are expressed as mean ± S.D. (n = 6). (∗ ) P < 0.05 vs. control, Dunnett’s test.

croscopic sections as cords of cells. Between the cords of hepatocytes, vascular sinusoids could be observed (Fig. 4A). In samples from liver of CCl4 -treatred rats presence of steatosis was evident. Fatty change appeared as clear vacuoles within hepatocytes, some of them as small vacuoles in the cytoplasm around the nucleus, while there were also many ballooned hepatocytes with cleared spaces that displaced the nucleus to the periphery of the cell. Some areas showed contiguous cells rupture and necrotic tissue. It was evident also a migration of leukocytes and some small areas of fibrosis (Fig. 4B). In electron microscopy images of the hepatocytes, there was delation of the endoplasmic reticu-

lum with detachment and disaggregation of polysomes, cell swelling with big clear vacuoles and mitochondrial injury (Fig. 5A) (Majno and Joris, 1996; Robbins, 1999). Similar aspects were present in specimens from rats treated with chlorogenic acid, both at 40 (Fig. 6A) and 80 mg/kg (Fig. 6B) and with cynarin (data not reported). In rats treated with 1 g/kg of C. scolymus, these abnormal accumulatios of triglycerides was less evident and only few “balloon-like” cells were present (Fig. 6C). The hepatic parenchyma of rats treated with 2 g/kg of C. scolymus showed a normal anatomy (Fig. 6D). Electron microscopy showed no damage to smooth and rough

E. Speroni et al. / Journal of Ethnopharmacology 86 (2003) 203–211 Table 3 Effects of four different extracts of Cynara scolymus commercially available, cynarin and chlorogenic acid on lipid peroxidation in CCl4 intoxicated ratsa Materials

Liver homogenate MDA level (nmol/g) wet liver (mean ± S.D.)

Control (0.5% CMC) CCl4 per os Extract 1 (1 g/kg) Extract 1 (2 g/kg) Extract 2 (1 g/kg) Extract 2 (2 g/kg) Extract 3 (1 g/kg) Extract 3 (2 g/kg) Extract 4 (1 g/kg) Extract 4 (2 g/kg) Chlorogenic acid (40 mg/kg) Chlorogenic acid (80 mg/kg) Cynarin (0.005 mg/kg) Cynarin (0.01 mg/kg)

55.86 109.1 98.67 88.56 76.46 73.23 56.13 53.99 78.89 75.45 89.31 85.39 84.39 87.67

± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.03 0.05 0.04 0.04 0.03 0.06 0.06 0.05 0.07 0.06 0.06 0.02 0.07 0.06

Protectionb (%)

48.55%∗ 50.50%∗

Rats were administered once with 1or 2 g/kg of C. scolymus leaves extract, 40 and 80 mg/kg of chlorogenic acid (corresponding to the content in chlorogenic acid present in 1 or 2 g/kg of the lyophilizate 3, with the higher content in this caffeoyl derivative), cynarin at a dose of 0.005 and 0.01 mg/kg, corresponding to the content in 1 and 2 g/kg of the first extract (A), that presented the major content in cynarin and then CCl4 p.o. a The data are expressed as mean ± S.D. (n = 6). b Calculated by considering the difference in the mean levels between control and CCl4 -treated groups as 100% protection. ∗ P < 0.01, compared with the CCl control by Dunnett’s test. 4

Fig. 3. (a) Effect of chlorogenic acid (40 mg/kg), cynarin (0.005 mg/kg) and CMC solution on bile flow in the rat. Results are expressed as mean ± S.D. (n = 6). P < 0.05 vs. control, Dunnett’s test. (b) Effect of chlorogenic acid (80 mg/kg), cynarin (0.01 mg/kg) and CMC solution on bile flow in the rat. Results are expressed as mean ± S.D. (n = 6). P < 0.05 vs. control, Dunnett’s test.

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endoplasmic reticulum and mitochondria were unaltered (Fig. 5B).

4. Discussion Artichoke has been used for centuries in folk medicine for its choleretic activity and more recently the C. scolymus extract has been proposed for its antioxidant properties against liver complaints. Commercially available extracts do not always show the same therapeutic activity, depending on their relative content in active principles. Antioxidant properties of caffeoylquinic acids contained in C. scolymus leaves extracts, are supposed to be almost in part responsible for the therapeutical activity (Gebhardt, 1997; Fraga et al., 1987), even if usually its therapeutic activities are ascribed to the cynarin content. The aim of this work was to compare the pharmacological activities of four extracts, commercially available, of C. scolymus and to observe, if present, a relationship between their effects and the content in caffeoyl derivatives. It’s very well known that a lot of active principles, often structurally related, are present in plant extracts and they can exert their activity synergistically. With this aim, in order to explain the pharmacological activity in relation with the antioxidant power, the total phenolic content was evaluated. The four extracts tested, showed a different phenol content. For extract 3, which had the most abundant content in chlorogenic and caffeoyl derivatives, the highest value of GAEs was recorded. There is a good dependency of the antioxidative activity, (evaluated with BR inhibition times), on the amount of phenols present in the extracts, which confirms the significance of the phenolic constituents as free radicals scavengers. According to the results obtained, with the four C. scolymus extracts studied, we had dissimilar patterns for choleretic activity. In particular, extracts 1 and 4 did not show any choleretic activity at 1 and 2 g/kg, while, extract 2 reached only a maximun during the first 20 min, and then decreased rapidly. Only extract 3 was found to induce an increase of BF, gradual and sustained. Cynarin and chlorogenic acid, administered as pure compounds, did not show choleretic activity at the doses tested and, neither a reduction in serum ALT and AST, nor a decrease in MDA content in liver homogenate was observed, although, previous in vitro investigations had shown that as cynarin as chlorogenic acid, a caffeoyl derivative present in C. scolymus, protected hepatocytes against hepatic damage induced by CCl4 in primary cultured hepatocytes (Gebhardt, 1997; Zafar and Ali, 1998). Hepatotoxic compounds such as CCl4 are known to cause remarkable increase in serum transaminases and induce liver injury through lipid peroxidation by free radical derivatives of the compound (Farber et al., 1971; Recknagel et al., 1976; Gravela et al., 1979; Wolf et al., 1980; Yagi, 1987; Azri et al., 1992; Brent and Rumack, 1993; Fehèr and Prònai, 1993).

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Fig. 4. (A) Normal microarchitecture of the liver from control rats: the hepatic parenchyma is organized into sheets oriented around central vein. Hematoxylin–eosin (magnification: 114×). (B) Liver of CCl4 -treated rats: fatty change is evident within hepatocytes, with many ballooned cell, necrotic tissue and emigration of leukocytes. Masson’s trichrome method (magnification: 114×).

Fig. 5. (A) Electron microscopy image of rat liver cell after CCl4 intoxication, with swelling of endoplasmic reticulum, big clear vacuoles and mitochondrial injury (magnification: 4000×). (B) Rat treated with 2000 mg/kg of Cynara scolymus: electron microscopy image of liver cell shows no damage to smooth end rough endoplasmic reticulum and mitochondria unaltered (magnification: 10,000×).

Fig. 6. (A, B) Liver of rats treated with chlorogenic acid (1 and 2 g/kg): the toxic effect of CCL4 is evident. Masson’s trichrome method (magnification: 114×). (C) Rats treated with 1 g/kg of Cynara scolymus: only few “balloon-like” cells are present. Masson’s trichrome method, 114×. (D) The hepatic parenchyma of rats treated with 2 g/kg of C. scolymus shows a normal anatomy and no presence of steatosis. Hematoxylin–eosin (magnification: 114×).

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Extracts 1, 2 and 4 did not protect liver injury induced by CCl4 , towards normalization, so at least from the point of view of the parameters analyzed in this study, they were ineffective in hepatic disorders. On the other hand, extract 3 was found to be effective in MDA levels in liver as well as in serum enzymes levels. The findings on choleretic activity and antioxidant efficacy, of different C. scolymus extracts, show that the different composition of the extract in active principles may cause some different effects. Only one of the extracts showed a significant activity, which seems not to be related to a high content in caffeoyl derivatives, as demonstrated administering rats with chlorogenic acid. The toxic effect of CCl4 is an example of free-radical disease, due to its conversion by P-450 enzyme system to the highly reactive toxic free radical CCl3 , which attacks the membranes of smooth and rough endoplasmic reticulum. Histological and ultrastructural analysis are in good agreement with the phenol content and antioxidant power of the four extracts. Hepatocytes of rats treated with C. scolymus, with the highest phenol content, had no fatty change, probably due to their capacity to synthesize apoprotein to complex with triglycerides and thereby facilitate lipoprotein secretion.

Acknowledgements This research was supported by the Ministry of Instruction University and Research (MIUR 60%) grant.

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