Protective effects of Pycnogenol® on carbon tetrachloride-induced hepatotoxicity in Sprague–Dawley rats

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Abstract

Oxidative damage is implicated in the pathogenesis of various liver injuries. In the present study the ability of Pycnogenol® (PYC) as an antioxidant to protect against CCl4-induced oxidative stress and hepatotoxicity in rats was investigated. Four experimental groups of six rats each were constructed: a vehicle control group received the respective vehicles (distilled water and corn oil) only; a CCl4 group received a 14-day repeated intraperitoneal (i.p.) dose of distilled water and then a single oral dose of CCl4 at 1.25 ml/kg; and the CCl4&PYC 10 and CCl4&PYC 20 groups received a 14-day repeated i.p. dose of PYC 10 and 20 mg/kg, respectively, and then a single oral dose of CCl4 at 1.25 ml/kg. Hepatotoxicity was assessed 24 h after the CCl4 treatment by measurement of serum aminotransferase (AST) and alanine aminotransferase (ALT) activities, hepatic malondialdehyde (MDA) and glutathione (GSH) concentrations, and catalase, superoxide dismutase (SOD), and glutathione-S-transferase (GST) activities. The results were confirmed histopathologically. The single oral dose of CCl4 produced significantly elevated levels of serum AST and ALT activities. Histopathological examinations showed extensive liver injuries, characterized by extensive hepatocellular degeneration/necrosis, fatty changes, inflammatory cell infiltration, congestion, and sinusoidal dilatation. In addition, an increased MDA concentration and decreased GSH, catalase, SOD, and GST were observed in the hepatic tissues. On the contrary, PYC treatment prior to the administration of CCl4 significantly prevented the CCl4-induced hepatotoxicity, including the elevation of serum AST and ALT activities and histopathological hepatic lesions, in a dose-dependent manner. Moreover, MDA and GSH levels and catalase, SOD, and GST activities in hepatic tissues were not affected by administration of CCl4, indicating that the pretreatment of PYC efficiently protects against CCl4-induced oxidative damage in rats. The results indicate that PYC has a protective effect against acute hepatotoxicity induced by the administration of CCl4 in rats, and that the hepatoprotective effects of PYC may be due to both the inhibition of lipid peroxidation and the increase of antioxidant activity.

Introduction

Liver disease is considered to be a serious health problem, as the liver is an important organ for the detoxification and deposition of endogenous and exogenous substances. Steroids, vaccines, and antiviral drugs, which have been employed as therapies for liver diseases, have potential adverse effects, especially when administered for long terms. Therefore, herbal products and traditional medicines with improved effectiveness and safety profiles are needed as a substitute for chemical therapeutics. It is reported that a number of herbal products have been shown to protect against liver injury, and many possess one or a combination of antioxidant, antifibrotic, immune modulatory, or antiviral activities (Seeff et al., 2001, Lee and Jeong, 2002, Shin et al., 2006). In recent years, there has been a substantial increase in the use of so-called complementary and alternative therapies that utilize herbal medicines by patients with liver disease (Strader et al., 2002, Fogden and Neuberger, 2003, Hanje et al., 2006).

Pycnogenol® (PYC), a standardized extract from the bark of the French maritime pine (Pinus maritima), is used extensively in dietary supplements, multi-vitamins, and health products because of its direct, strong antioxidant activity (Rohdewald, 2002). Major constituents of PYC are polyphenols, specifically monomeric and oligomeric units of catechin, epicatechin, and taxifolin (Rohdewald, 2002, Grimm et al., 2006). It is well documented that polyphenols comprise a wide area of natural substances of plant origin, and almost all of them exhibit a marked antioxidant activity (Bors and Michel, 2002, Cai et al., 2002).

Oxidative stress is defined in general as excess formation and/or insufficient removal of highly reactive molecules such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) (Johansen et al., 2005, Valko et al., 2007). ROS include free radicals such as superoxide anion (radical dotO2), hydroxyl (radical dotOH), peroxyl (radical dotRO2), hydroperoxyl (radical dotHRO2) as well as nonradical species such as hydrogen peroxide (H2O2) and hydrochlorous acid (HOCl). RNS include free radicals like nitric oxide (radical dotNO) and nitrogen dioxide (radical dotNO2), as well as nonradicals such as peroxynitrite (ONOO), nitrous oxide (HNO2) and alkyl peroxynitrates (RONOO). The effects of antioxidants on oxidative stress are mainly measured through certain observable biomarkers, including the enzymatic activities of catalase, SOD, GSH-Px, and GSH-reductase, as well as thiobarbituric acid reactants (TBARS) levels, an indirect measurement of free-radical production (Maritim et al., 2003, Johansen et al., 2005, Valko et al., 2007).

Previous studies showed that PYC is a very potent antioxidant to scavenge reactive oxygen and nitrogen species such as superoxide anion radical, hydroxyl radical, lipid peroxyl radical (LOOradical dot), peroxynitrite radical, and singlet oxygen (1O2). It has also been shown to bind to proteins, thereby affecting both structural and functional characteristics of key enzymes and other proteins involved in metabolism; to participate in the cellular antioxidant network, especially in prolonging the lifetime of ascorbyl radical, possibly through a one-electron reduction of dehydroascorbic acid; and to protect endogenous vitamin E and glutathione (Packer et al., 1999, Maritim et al., 2003). It has been claimed that PYC has diverse beneficial effects on a wide range of medical conditions, including inflammation, diabetes, asthma, hypertension, attention deficiency hyperactivity disorder, cancer, immune disease, and others (Rohdewald, 2002). Despite the favorable pharmacological properties of PYC, the protective capacity of PYC against hepatotoxicity has not previously been explored.

PYC may have a protective effect on the deteriorated hepatic function that results from free radicals in the toxic chemical-induced hepatotoxicity. To test this hypothesis, the present investigation examined the ability of PYC to protect against carbon tetrachloride (CCl4)-induced oxidative stress and hepatotoxicity. We selected CCl4 as a model hepatotoxicant because this chemical is a potent hepatotoxin and a single administration can rapidly lead to both oxidative stress via the excessive production of free radicals and acute liver injuries such as centrilobular necrosis and steatosis in rats (Recknagel et al., 1989, Janakat and Al-Merie, 2002, Weber et al., 2003). Because CCl4 is a common contaminant found in both ambient outdoor and indoor air, the human health risk is very high. It is therefore important to investigate the antioxidant activity of PYC against CCl4-induced hepatic oxidative damages.

Section snippets

Animal husbandry and maintenance

Thirty male Sprague--Dawley rats aged 8 weeks were obtained from a specific pathogen-free colony at Bio Genomics Inc. (Seoul, Republic of Korea) and used after 1 week of quarantine and acclimatization. The animals were housed in a room maintained at a temperature of 23 ± 3 °C and a relative humidity of 50 ± 10% with artificial lighting from 08:00 to 20:00 and 13 to 18 air changes per hour. The animals were housed two per cage in stainless steel wire mesh cages and were allowed sterilized tap water

Effects of PYC on CCl4-induced hepatotoxicity

The single oral dose of CCl4 caused severe hepatotoxicity in rats, as evidenced by the significant elevation of serum AST and ALT activities and the increased incidence of histopathological hepatic injury after the administration of CCl4. In contrast, no treatment-related effects on the absolute and relative weights of the liver and necropsy findings were observed in the CCl4-treated rats (data not shown). The protective effects of pretreatment with PYC on the CCl4-induced elevation of serum

Discussion

It has been reported that PYC shows a number of beneficial effects against various types of degenerative diseases in humans, largely because the major ingredients of PYC, polyphenols, have very potent antioxidant activity (Packer et al., 1999, Rohdewald, 2002). Therefore, we considered that PYC is useful in the prevention of various liver injuries induced by oxidative stress. In the present study, the capability of PYC to protect against CCl4-induced hepatotoxicity and oxidative stress was

Acknowledgements

This work was supported by the Regional Research Centers Program (Bio-housing Research Institute), granted by the Korean Ministry of Education&Human Resources Development.

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