Effects of carvacrol on defects of ischemia-reperfusion in the rat liver.

Abstract

Many plants found in nature have been used to treat various illnesses. One such plant is oregano (Kekik in Turkish). Health beneficial effects of carvacrol obtained from oregano oil have been shown scientifically. We have investigated the comparative effects of carvacrol in the liver of rats subjected to ischemia-reperfusion defect, with silymarin. To test the effects we formed four groups using male Wistar albino rats. Group I was control. The other three groups of animals were administered 60 min prior to surgical operation single doses of physiological serum, carvacrol and silymarin, respectively. Group II, III and IV animal were subjected to 45 min long liver ischemia and 60 min reperfusion. Blood and tissue samples were collected for biochemical and histological analysis following the test.

AST and ALT values obtained after biochemical analysis of the serums showed statistically significant difference in group II than the other three groups. A statistical evaluation of the serum AST levels among the groups II, III and IV showed that both groups III and IV which had no difference in between were significantly different in a positive way from group II (p < 0.001). As to the serum ALT levels, difference between group II and group III (p < 0.001) and group II and group IV (p < 0.01) was found significant. No statistical difference was observed in groups I, III and IV for GSH, MDA and CAT levels of the liver. A statistical evaluation of the GSH level in group III and group IV and was found to be significantly different from group II (p < 0.001) without any difference between them. A similar evaluation for MDA and CAT levels among the revealed no difference between group III and group IV, however, group II showed difference with group II and group IV (p < 0.05).

Histological findings were in harmony with the biochemical results. We conclude that carvacrol protects the liver against defects caused by ischemia and reperfusion, and carvacrol is not hepatotoxic at the applied dosage.

[C] 2007 Elsevier GmbH. All rights reserved.

Keywords: Liver; Ischemia/reperfusion; Carvacrol; Silymarin; Rat

Introduction

Any functional defect occurring in the liver which is one of the most important organs of the human body affects all the systems. Chemical substances, medicines, alcohol, liver tumors, viral liver diseases and ischemia occurring after surgical operations are likely causes of liver injury. Hepatic ischemia which is a frequent problem encountered in clinical conditions such as liver transplantations, liver deficiency and liver surgery results in functional and structural defects in hepato-cytes (Debonera et al., 2001; Okatani et al., 2003). Blockage of the veins to control bleeding during partial hepatectomy results in the undesired ischemia (Watanabe et al., 2001; Seo and Lee, 2002). Prolongation of the period of ischemia is an important risk factor for complications in liver transplantations (Kaibori et al., 2000). As in many surgical operations, ischemia occurring during hepatectomy (min. 60 min in Pringle's maneuver) causes serious tissue injury (Nakano et al., 1998; Zapletal et al., 2001). Reperfusion following ischemia results in the passage of toxic products to the systemic circulation. This worsens the liver damage (Ozturk et al., 2002). Furthermore, oxidants formed and circulated as a result of I/R in another organ bring about negative effects especially in the lungs and the liver (Tang et al., 1997; lijima et al., 1997; Serteser et al., 2002).

Medicinal treatment is necessary for a rapid recovery of liver injury due to various reasons. However, medicines used in the treatment of liver have too many side effects. Therefore, natural products offer hope for the development of new liver medicines. Most of such medicines are plant based. For instance, silymarin has been used for liver health. Positive effects of silymarin in liver regeneration is well documented. Silymarin is a plant derived flavonoid which is extracted from the fruits and seeds of the milk thistle (Silybum marianum L. Gaertn.) belongs to the family of Asteraceae. Silymarin is a flavonolignan and consists of a mixture of mainly three flavonoids, silybin (silibinin), silydianin and silychristin. Silibinin is the major component (70-80%) found in silymarin and is thought to be the most biological active compound. Pharmacological studies revealed that silymarin is non-toxic even at higher physiological doses, which suggests its safe use for the treatment of various diseases (Katiyar, 2005). Oregano water (kekik suyu), a hydrosol of oregano, is used as a folk medicine in Turkey for liver health. Oregano oil contains carvacrol as the main constituent. Several useful attributes of carvacrol have been scientifically proven in in vivo studies, however, there is no research into liver protective and liver healing effects of carvacrol. We, therefore, have decided to investigate in vivo effects of carvacrol in the livers of rats subjected to I/R, in comparison with silymarin.

Materials and methods

Plant extract

The plant substance tested in this study, carvacrol (2-methyl-5-(l-methyl ethyl) phenol), was isolated from steam distillated essential oil of Origanum onites L. collected from West Anatolia (Fig. 1). For the isolation, fractional distillation was performed using a lab-size glass fractional distillation unit containing column packed with S/S Knit Mesh packing material (2.8 cm x 1.35 m). Reflux ratio was adjusted at 10/1 to 20/1 and the medium pressure was 8-10 mm Hg. Carvacrol-rich fractions were bulked to obtain carvacrol with 99% purity (GCMS).

[FIGURE 1 OMITTED]

Silymarin used in our experiment containing 70-80% silibinin was purchased from Sigma (Cat no S0292).

Animals

Male Wistar albino rats, weighing between 230 [+ or -] 30 g, were used after 2 weeks of adaptation. They were housed in polycarbonate cages in an air-conditioned room (12L/12D, 22 [+ or -] 2 [degrees]C, 50 [+ or -] 5% humidity). Water and food supplied ad libitum.

The experimental design and procedures were approved by the Institutional Ethical Committee for Animal Care and Use at the Eskisehir Osmangazi University, Eskisehir, Turkey.

Experimental protocols

The rats were randomly divided into four groups, each containing 8 animals.

Group I normal (sham operation). Each animal received 0.5 ml physiological serum 60 min prior to surgical operation and was subjected only to laparotomy in order to make hepatic veins and bile duct clearly visible. The animals were killed after 105 min.

Group II (I/R-untreated). The animals were subjected to I/R and received 0.5 ml saline solution intraperitonally.

Group III (I/R and carvacrol treated). The animals were subjected to I/R and received carvacrol (73 mg/kg) (MSDS; NIOSH).

Group IV (I/R and silymarin treated). The animals were subjected to I/R and received silymarin (100 mg/kg) (Magliulo et al., 1973; Oliveira et al., 2001).

Group II animals received saline solution intraperitonally 60 min prior to I/R; Group III and IV animals received carvacrol and silymarin, respectively, followed by 50 mg/kg thiopental anaesthesia (Tang et al., 1997; Crocenzi et al., 2000, 2003) and laparotomy to make hepatic veins and bile duct visible by clearing the tissues around them. By the help of sterile microvascular bulldog clamps liquid passage was blocked through hepatic veins and bile duct for 45min to bring about ischemia and 60 min for reperfusion. Blood samples were taken intracardiacally in order to measure AST and ALT levels.

Following the experiment, parts of liver were kept at -80[degrees]C in order to measure the levels of MDA, GSH and CAT (Watanabe et al., 2001; Nakano et al., 1998; Shah et al., 1999). The remaining parts was fixed in 10% neutral formalin for histological investigations. Four micrometer thick sections in paraffin taken from each liver sample were stained with standard H&E. Samples were investigated by light microscopy by using Spot Advanced Software (V. 3.2.4; Diagnostic Instruments, Sterling Heights, USA). Sections were digitally photographed using a Spot insight color 3.2.0 diagnostic camera.

Statistical analysis

The results were expressed as the mean [+ or -] SE of eight animals per group. One way analysis of variance (ANOVA) and TUKEY test were used for the analysis and comparison of data within and between groups (SPSS 12.0 for windows). Differences were considered significant p < 0.05.

Results

Biochemistry

Results of AST, ALT, GSH, MDA ve CAT levels in the blood and liver tissues of Group I, II, III and IV animals are summarized in Table 1 and Figs. 2A, B, C and D.

Liver histology

In the liver sections of Group II widespread hyperemia, PMNL infiltration and vacuolization in hepatocytes were observed (Figs. 3 and 4). In liver sections of Group III and IV histological organization appeared to be protected but in the carvacrol group hyperemia was observed to continue partially (Fig. 5). In the carvacrol and silymarin groups vacuolization in hepatocytes and PMNL infiltration were not encountered (Figs. 5 and 6).

Discussion

In studies whereby the negative effects of ischemia on liver functions were demonstrated, it was indicated that amounts of enzymes like AST, ALT and ALP in serum had increased following ischemia (El-Ashmawy et al., 2005). In mice subjected to 45 min liver ischemia and reperfusion amount of ALT in serum was reported to increase significantly (Kawachi et al., 2000). The results of this study has some similarity with our results. Furthermore, in groups III and IV which had been subjected to I/R 60 min after carvacrol and silymarin injections, respectively, serum AST and ALT levels showed parallelism with the control group and were found to be lower than those of group II. This suggested a possible protective effect of carvacrol and silymarin in functional defects of hepatocytes.

Table 1. AST and ALT serum levels for group I, II, III and IV and mean
[+ or -] standard error (SE) figures for GSH, MDA and CAT in liver
tissue

Groups        AST (U/L)                 ALT (U/L)

I       101.25 [+ or -] 5.19       47.00 [+ or -] 3.83
II      154.63 [+ or -] 13.18     152.63 [+ or -] 23.19
III      91.00 [+ or -] 7.79 ***   79.75 [+ or -] 11.31 ***
IV      101.38 [+ or -] 6.45 ***   93.00 [+ or -]  7.31 **

P       II-III: p<0.001 ***       II-III: p<0.001 ***
        II-IV:  p<0.001 ***        II-IV: p<0.001 **

Groups      GSH (nmol/mg)            MDA (nmol/mg)

I       11.48 [+ or -] 1.16      4.92 [+ or -] 0.55
II       4.66 [+ or -] 0.58      6.80 [+ or -] 0.36
III     10.85 [+ or -] 0.85 ***  5.16 [+ or -] 0.34 *
IV      11.81 [+ or -] 0.78 ***  4.90 [+ or -] 0.35 *

P       II-III: p<0.001 ***      II-III: p<0.05 *
        II-IV:  p<0.001 ***       II-IV: p<0.05 *

Groups         CAT (U/mg)

I        124.38 [+ or -] 13.91
II        67.36 [+ or -] 6.71
III      117.63 [+ or -] 10.31 *
IV       126.25 [+ or -] 12.38 *

P        II-III: p<0.05 *
         II-IV:  p<0.05 *

Serum AST and ALT levels and GSH, MDA and CAT levels in liver tissue of
Group II (I/R -untreated) are statistically different from Group III
(carvacrol treatment) and Group IV (silymarin treatment).
There is no statistically difference between Group III and Group IV.
Each value was the mean [+ or -] SE of eight animals per group.
Differences at P values of <0.05 considered significant.

Widespread vacuolization observed during histological examination of the liver sections of Group II was interesting. Araya et al. (2003) reported the formation of vacuoles in hepatocytes and sinusoidal congestion in post-ischemic liver. The fact that vacuolization observed in group II was significantly decreased in group IV in which sinusoidal congestion had also disappeared supports the earlier studies which proved the hepato-protective effects of silymarin. The decrease of vacuolization in hepatocytes in Groups III and IV is an important finding to prove that carvacrol protects liver cells against I/R damage. Luper (1998) and Flora et al. (1998) reported that silymarin increased DNA and protein syntheses in hepatocytes. Svoboda and Hampson (1999) showed that carvacrol had no mutagenic effect. In another study, the absence of genotoxic effect of carvacrol was reported (Ipek et al., 2005). Furthermore, Flora et al. (1998) reported the protective effect of silymarin against genomic defects. In the light of such data, we are of the opinion that carvacrol protects hepatocytes similar to silymarin without any undue effect on their genetic make up. Liver GHS levels were reported to decrease in animals subjected to 60 min liver ischemia and 120 min reperfusion (Nakano et al., 1998). This study shows parallelism with our results. The fact that similar results were obtained in Group I and especially in Groups III and IV, and that GSH levels in these groups were higher than that of Group II clearly indicates the healing effect of carvacrol like silymarin in I/R damaged liver through its antioxidant activity. 30 min hepatic ischemia applied to rats were reported to decrease liver GSH level and increased the MDA level (Koken and Inal, 1999). Sener et al. (2003) reported the decrease of GSH level and the increase of MDA level in rats subjected to 45 min liver ischemia and 60 min reperfusion. In our study, MDA level was found to be similar in Group II, Group IV and the control group, while it was higher in Group II in liver I/R applied animals. In another study where rats were subjected to 60, 90, 120 and 180 min hepatic ischemia and 30 min reperfusion CAT activity was found to decrease similar to our findings (Gonzalez-Flecha et al., 1993). If evaluated according to liver GSH, MDA and CAT levels carvacrol showed important protective effect against I/R damage of the liver in a similar manner with silymarin.

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]

Shah et al. (1999) reported that oxidative damage caused mutagenic lesions in DNA. Ipek et al. (2003) showed antitumor and antimutagenic activities of carvacrol in an in vitro study. Therefore, the use of carvacrol against I/R injury can be important for the protection of genetic make up of the cells. In our study, carvacrol and silymarin showed similar activity in tolerating and healing the oxidative damage in I/R damaged livers of rats. Carvacrol has also been shown to have antimicrobial activity (Svoboda and Hampson, 1999; Bagamboula et al., 2004; Lanciotti et al., 2004). This feature can be significant for the use of carvacrol in liver surgery. We are of the opinion that carvacrol can be used in in vivo experiments related to liver ischemia. Our results have clearly shown that carvacrol protects the liver against I/R injury and is not hepatotoxic in the dose applied.

Acknowledgements

The study was supported by Eskisehir Osmangazi University Scientific Research Projects Committee (Project number: 200419025).

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Mediha Canbek (a), *, Mustafa Uyanoglu (a), *, Gokhan Bayramoglu (a), Hakan Senturk (a), Nilufer Erkasap (b), Tulay Koken (c), Sema Uslu (d), Canan Demirustu (e), Erinc Aral (f), K.Husnu Can Bascr (g)

(a) Department of Biology, Faculty of Science, Eskisehir Osmangazi University, 26480 Eskisehir, Turkey

(b) Department of Physiology, Faculty of Medicine, Eskisehir Osmangazi University, 26480 Eskisehir, Turkey

(c) Department of Biochemistry, Faculty of Medicine, Kocatepe University, 03200 Afyon, Turkey

(d) Department of Biochemistry, Faculty of Medicine, Eskisehir Osmangazi University, 26480 Eskisehir, Turkey

(e) Department of Biostatistics, Faculty of Medicine, Eskisehir Osmangazi University, 26480 Eskisehir, Turkey

(f) Department of Histology, Faculty of Medicine, Eskisehir Osmangazi University, 26480 Eskisehir, Turkey

(g) Department of Pharmacognosy, Faculty of Pharmacy, Anadolu University, 26470 Eskisehir, Turkey

Abbreviations: I/R, ischemia/reperfusion; H&E, haematoxylin and eosin; DNA, deoxyribonucleic acid; MDA, malondialdehyde; GSH, reduced glutation; CAT, catalase; PMNL, polymorphonuclear leukocytes; AST, aspartate transaminase; ALT, alanine transaminase; ALP, alkaline phosphatase; GCMS, gas chromatography and mass spectrometer analyses.

* Corresponding authors. Tel.: + 90222239 37 509/2849; fax: + 90 222 239 35 78.

E-mail addresses: [e-mail] (M. Canbek), [e-mail] (M. Uyanoglu).

0944-7113/$- see front matter (C) 2007 Elsevier GmbH. All rights reserved. doi: 10.1016/j.phymed.2007.11.022