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Magnoliae Cortex extract protects PC 12 cells from cytotoxicity induced by hydrogen peroxide or 6-hydroxydopamine through enzyme induction

Alzheimer’s and Parkinson’s disease are neurodegenerative disorders of unknown cause for which there is no cure or way of preventing or slowing its progression. Various genetic and environmental factors are thought to be involved in the onset of neurodegenerative diseases. Oxidative stress, such as the generation of reactive oxygen species and lipid peroxidation, is a major factor in initiating the disease process. However, oxidative stress in cells is known to be suppressed by drug-metabolizing enzymes, such as NAD(P)H:quinone oxidoreductase 1 (NQO1), and antioxidant enzymes, such as catalase. Here, we used PC12 cells, which are a recognized model for neuronal cell death and neurite extension, to investigate whether Magnoliae Cortex derived from the Magnoliaceae plant family can induce these enzyme activities. Our results reveal that Magnoliae Cortex extract induces the activity of both NQO1 and catalase. In addition, the cytotoxic effect of hydrogen peroxide and 6-hydroxydopamine was significantly suppressed by pretreatment of the cells with Magnoliae Cortex extract. Based on our findings, we conclude that induction of these enzyme activities by Magnoliae Cortex extract leads to an enhancement of its cytoprotective effect.


INTRODUCTION
Oxidative stress is thought to be involved in the development of a variety of neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis and Parkinson's disease (Andersen, 2004;Lin and Beal, 2006). However, because the pathogenic mechanism of neurodegenerative diseases is not fully understood, no therapy has been established to prevent progress of the disease itself. Oxidative stress refers to a state of disruption in the cellular balance between the amount of reactive oxygen species (ROS) and the endog-enous pool of antioxidants that results in an excessive accumulation of ROS (Sies and Cadenas, 1985). ROS are highly reactive and will readily modify proteins, lipids and nucleic acids, leading to extensive cellular damage.
Several defense mechanisms against oxidative stress are present within cells. Nonetheless, brain cells are particularly susceptible to oxidative stress because the adult human brain represents only 2% of total body weight, but accounts for 20% of oxygen consumed by the entire body. The fact that brain cells are vulnerable to oxidative stress supports the notion that ROS are involved in the development of neurodegenerative diseases. Vol. 6 No. 3 A number of factors are known to protect cells from the effects of oxidative stress. For example, dietary antioxidants, such as vitamin E, vitamin C, β-carotene and flavonoids (Aruoma, 1994), attenuate the levels of ROS. Drug-metabolizing enzymes, such as NAD(P)H:quinone oxidoreductase 1 (NQO 1) and glutathione S-transferase, and antioxidant enzymes, such as catalase, superoxide dismutase and glutathione peroxidase, are also known to suppress oxidative stress. NQO1 is thought to protect cells against oxidative stress caused by the products of dopamine metabolism, which include ROS, quinones and semiquinones (Zafar et al., 2006). Catalase reduces oxidative stress in cells by converting hydrogen peroxide (H 2 O 2 ) into oxygen and water. These observations suggest it may be possible to prevent neurodegenerative diseases caused by oxidative stress using compounds that can induce drug-metabolizing and antioxidant enzymes (Van Muiswinkel and Kuiperij, 2005).
Potent inducers of drug-metabolizing and antioxidant enzymes have been isolated from natural products (Morimitsu et al., 2002;Chen et al., 2004). Most studies have focused on antioxidants derived from fruits and vegetables because these represent a ready source of such compounds for daily intake. Based on these studies, many active constituents, such as sulforaphane (Zhang et al., 1992), curcumin (Balogun et al., 2003), isothiocyanates (Nakamura et al., 2000) and resveratrol (Chen et al., 2005), were isolated and identified from natural products.
In addition to foods, such as fruits and vegetables, another source of natural antioxidants are herbal medicines that contain many active constituents. Traditional herbal medicines are used as multi-component drugs, although the efficacy and mechanism of action of individual components are often unknown. Recently, the pharmacological action of traditional herbal medicines and elucidation of their effective constituents have been studied in many laboratories. The traditional herbal medicine Magnoliae Cortex is known to have efficacy in diseases of the central nervous system. In this study, we investigated the effects of Magnoliae Cortex extract on protection against oxidative stress in PC12 cells, which are frequently used as a model system for neuronal cell death.

Materials
Magnolia Cortex extract was kindly provided by Tsumura Co. Ltd. (Tokyo, Japan). Rat adrenal medulla PC12 cells were purchased from American Type Culture Collection (Manassas, VA, USA). 6-Hydroxydopamine (6-OHDA) was purchased from Sigma-Aldrich (St Louis, MO, USA). CellTiter-Glo ® Luminescent Cell Viability Assay kit was obtained from Promega Co. (Madison, WI, USA). Hydrogen peroxide was purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Cell culture Petri dishes (collagen type I coated ware) and 24-well cell culture multidisc (collagen type I coated ware) were from AGC Techno Glass Co., Ltd. (Shizuoka, Japan). Bio-Rad Protein Assay was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). All other reagents were of the highest grade commercially available.

Magnoliae Cortex extract
Magnoliae Cortex (100 mg) was added to 2 mL of methanol, and the mixture was vigorously stirred at room temperature for 10 min and then centrifuged at 1,000 × g for 10 min. The resulting supernatant was transferred to another container and 2 mL of methanol added to the residue. The same process was repeated one more time and the resultant supernatants were combined and used as Magnoliae Cortex extract.

Induction of enzyme activity
Dose-and time-dependent enzyme induction by Magnoliae Cortex extract were performed under the following conditions. PC12 cells were seeded at a density of 2 × 10 6 cells/dish in a 100 mm diameter cell culture Petri dish and cultured at 37°C under 5% CO 2 for 96 hr. After culturing, the medium was removed and the cells washed with 1 × PBS (-). Ten ml of serum-free medium containing Magnoliae Cortex extract (0-200 µg/mL) was then added. Methanol was added as a control to a final concentration of 0.2%. The cells were then cultured at 37°C under 5% CO 2 conditions for a further 24 hr. Alternatively, Magnoliae Cortex extract was added to a final concentration of 100 µg/mL. As a control, methanol was added to a final concentration of 0.1%. Cells were then cultured at 37°C under 5% CO 2 for 12, 24 or 48 hr.

Cytotoxicity
PC12 cells were seeded at a density of 6.6 × 10 4 cells/ well in a 24-well cell culture dish and cultured at 37°C and 5% CO 2 for 72 hr. After culturing, the medium was removed, and 400 μL of serum-free medium containing Magnoliae Cortex extract (0-300 µg/mL) added to each well. As a control, methanol was added to a final concentration of 0.2%. The cells were then cultured at 37°C under 5% CO 2 conditions for 24 or 48 hr.

Enzyme source
After culturing the cells for a specified period of time, the medium was removed and the cells were washed with 1 × PBS (-). Four mL of 1 × PBS (-) was added and cells were detached from the culture dish using a cell scraper. The resulting cell suspension was centrifuged at 500 × g for 3 min. The cell pellet was resuspended in 300 µL of 50 mM Tris-HCl buffer (pH 7.4) containing 2 mM EDTA and 125 mM sucrose and then subjected to ultrasonic treatment (10 sec). The resultant homogenate was centrifuged at 15,000 × g for 15 min, and the supernatant used as the enzyme source.

Protein concentration
Protein concentrations were determined using a Bio-Rad Protein Assay kit according to the manufacturer's instructions.

Cell viability
Cell viability was established using a CellTiter-Glo ® Luminescent Cell viability Assay kit according to the manufacturer's instructions and as previously described (Nishiyama et al., 2010).

Cytotoxicity induction
PC12 cells were seeded at a density of 6.6 × 10 4 cells/ well in a 24-well cell culture dish and cultured at 37°C and 5% CO 2 for 72 hr. After culturing, the medium was removed, and 300 µL of serum-free medium containing Magnoliae Cortex extract (0-200 µg/mL) was added to each well. After culturing for 24 hr, 100 μL of serumfree medium containing H 2 O 2 (250 µM or 500 µM) or containing 6-OHDA (100 µM or 200 µM) was added to each well. After 3 hr (H 2 O 2 ) or 24 hr (6-OHDA) incubation, cell viability was measured according to the methods described above.

Cytotoxicity
Cytotoxic effects of Magnoliae Cortex extract on PC12 cells were determined (Fig. 1). PC12 cells were incubated with Magnoliae Cortex extract at various concentrations for 24 and 48 hr. No significant cytotoxicity was observed up to 200 µg/mL of extract after 24 hr treatment. However, a decrease in cell viability was observed when cultured cells were exposed to extracts at a concentration of 300 µg/mL and the survival rate was 65.0 ± 16.0%. At 48 hr, no significant cytotoxicity was observed up to 100 µg/mL of extract. However, a marked decrease in cell viability was observed after 48 hr incubation with extract at ≥ 200 µg/mL, and the cells were almost all killed after treatment with extract at 300 µg/mL.

Enzyme induction
The concentration-dependent effects of Magnoliae Cortex extract on NQO 1 and catalase activities were determined (Figs. 2 and 3). NQO 1 and catalase activities were significantly increased after treatment with Magnoliae Cortex extract in a concentration-dependent manner. After treatment at the highest concentration of extract (200 µg/mL), the activities of NQO 1 and catalase were 1.71 ± 0.16-fold and 3.38 ± 0.17-fold greater than that of the control, respectively. In addition, the time-dependent effects of Magnoliae Cortex extract on NQO 1 and catalase activities were measured. Both NQO 1 and catalase activity increased until 24 hr after treatment with Magnoliae Cortex extract, and thereafter showed a slight decrease. The induction rate of NQO 1 and catalase activity after 24 hr treatment was 1.74 ± 0.09-fold and 2.16 ± 0.12-fold greater than that observed for the control, respectively.

Protective effects
PC12 cells either treated or untreated with Magnoliae Cortex extract were incubated with H 2 O 2 , which is a ROS, and with 6-OHDA, which is a neurotoxic substance. The cell survival rate was then measured (Fig. 4). Cell viability of the untreated PC12 cells decreased significant- pretreated with 200 µg/mL of Magnoliae Cortex extract and then exposed to 100 µM or 200 µM 6-OHDA gave cell viabilities that were 1.3-and 1.9-fold greater, respectively, than the untreated group.

DISCUSSION
Oxidative stress is thought to be involved in the onset of neurodegenerative diseases. Here, we focused on drug-metabolizing and antioxidant enzymes as a potential factor to reduce oxidative stress. Specifically, PC12 cells were treated with Magnoliae Cortex extract, which is known to have efficacy in diseases of the central nervous system, at non-cytotoxic concentrations. We investigated the induction effect of Magnoliae Cortex extract on NQO 1 and catalase activity in PC12 cells and also exam-  ined the cytoprotective effect of the extracts against H 2 O 2 and 6-OHDA induced toxicity. Hydrogen peroxide is a ROS, which is converted to the highly reactive hydroxyl radical via the Fenton reaction. The hydroxyl radical causes damage to cell function by modifying functional molecules such as proteins, lipids or DNA. Pretreatment of the cells with Magnoliae Cortex extract significantly inhibited the cytotoxic effect of H 2 O 2 in a concentration-dependent manner as compared with untreated PC12 cells. However, no such inhibition in the cytotoxic effect of H 2 O 2 was observed upon simultaneous treatment of the cells with Magnoliae Cortex extract and H 2 O 2 (results not shown). These observations suggest the drug-metabolizing and antioxidant enzymes induced by pretreatment of the cells with Magnoliae Cortex extract plays an important role in protecting against oxidative stress.
6-OHDA is a neurotoxin commonly used to selectively destroy dopaminergic neurons and noradrenergic neurons and is widely used to generate experimental models of Parkinson's disease. The molecular mechanism of neurotoxicity mediated by 6-OHDA in PC12 cells is thought to be due to H 2 O 2 and p-quinone generated during the metabolism of this compound (Saito et al., 2007). Pretreatment of the cells with Magnoliae Cortex extract significantly suppressed the cytotoxic effect of 6-OHDA as compared with the untreated PC12 cells. Pretreatment of PC12 cells with Magnoliae Cortex extract induced NQO 1 and catalase activity, resulting in enhanced detoxification of H 2 O 2 and p-quinone. These results suggest that the induction of drug-metabolizing and antioxidant enzymes by the Magnoliae Cortex extract leads to an enhanced cell protective effect.
Magnolia can elicit various biological effects such as a muscle relaxant (Watanabe et al., 1983), inducing sedation (Yang et al., 2007), lowering anxiety (Kuribara et al., 1998) and in the treatment of gastric ulcers . The efficacy of herbal medicines is exerted by synergistic effects of many active constituents found in herbal extracts. It is possible that Magnoliae Cortex induced enhancement of NQO1 and catalase activity may also be involved in the biological effects mentioned above. For example, gastric ulcers are associated with cytotoxicity caused by increased ROS and of gastric juice secretion caused by vasoconstriction due to catecholamines and subsequent increased blood flow due to vasorelaxation (Das et al., 1997). It is thought that the anti-gastric ulcer effect of Magnolia is based on ß-eudesmol, which inhibits gastric acid secretion by blocking the H 2 receptor (Nogami et al., 1986;Kimura et al., 1991). As demonstrated in this study, enhancement in the detoxification of ROS by induction of drug-metabolizing and antioxidant enzymes may also contribute to the anti-stress ulcer action of Magnoliae Cortex extract.
Studies to identify active constituents of traditional herbal medicines are being conducted in many laboratories. It is essential to identify the constituents of an extract that has enzyme-inducing ability in order to clarify the nature and mechanism of action of herbal medicines. Studies on the isolation and identification of active constituents involved in enzyme induction by Magnoliae Cortex extract are currently underway in our laboratory.

Conflict of interest----
The authors declare that there is no conflict of interest. or 24 hr (6-OHDA). After incubation, cell viabilities were determined using a CellTiter-Glo ® Luminescent Cell Viability Assay kit. *, significantly different from each control at P < 0.05 (Student's t-test).