ПРИКЛАДНАЯ БИОХИМИЯ И МИКРОБИОЛОГИЯ, 2007, том 43, № 4, с. 495-500
УДК 582.287
ANTIOXIDANT ACTIVITIES OF CULTURED Armillariella mellea © 2007 r. L. T. Ng*, S. J. Wu**, J. Y. Tsai***, M. N. Lai****
*Department of Biotechnology, Tajen University, Yanpu Shiang, Pingtung, Taiwan; e-mail: lthuang@mail.tajen.edu.tw
**Department of Nutritional Health, ***Graduate Institute of Biotechnology, Chia-Nan University of Pharmacy and Technology, Tainan, Taiwan ****Kang Jian Biotech Co., Ltd., Nantou Hsien, Taiwan Received February 07, 2006
This study aimed to evaluate the antioxidant activities of a cultured medicinal fungus - Armillariella mellea (Vahl. ex Fr.) Karst. (AM). Three antioxidant assay systems, namely cytochrome c, xanthine oxidase inhibition and FeCl2-ascorbic acid stimulated lipid peroxidation in rat tissue homogenate tests, were used. Total flavonoid and phenol contents of AM extracts were also analyzed. Results showed that both aqueous (AM-H2O) and etha-nolic (AM-EtOH) extracts of solid state cultured AM showed antioxidant activities in a concentration-dependent manner. At concentrations 1~100 |g/ml, the free radical scavenging activity was 73.7~92.1% for AM-H2O, and 60.0~90.8% for AM-EtOH. These extracts also showed an inhibitory effect on xanthine oxidase activity, but with a lesser potency (IC50 - 9.17 |g/ml for AM-H2O and 7.48 |g/ml for AM-EtOH). In general, AM-H2O showed a stronger anti-lipid peroxidation activity on different rat's tissues than AM-EtOH. However, both AM extracts displayed a weak inhibitory effect on lipid peroxidation in plasma. Interestingly, the anti-lipid peroxidation activity of AM-H2O (IC50 - 6.66 |g/ml) in brain homogenate was as good as a-tocopherol (IC50 -5.42 |g/ml). AM-H2O (80.0 mg/g) possessed a significant higher concentration of total flavonoids than AM-EtOH (30.0 mg/g), whereas no difference was noted in the total phenol content between these two extracts. These results conclude that AM extracts possess potent free radical scavenging and anti-lipid peroxidation activities, especially the AM-H2O in the brain homogenate.
Oxidative stress is regarded as a fundamental cause of various chronic and degenerative diseases, including ischemia-reperfusion injury, liver disease, inflammation and renal failure [1, 2], diabetes mellitus [3], cancer [4], heart disease [5], and neuronal degeneration such as Alzheimer's disease [6] and Parkinson's disease [7]. Compounds that can scavenge free radicals are shown to be effective in ameliorating the progress of these diseases. Reactive oxygen species (ROS) such
as O2 (superoxide anion), .OH (hydroxyl radical), H2O2 (hydrogen peroxide) and *O2 (singlet oxygen), are highly reactive and are formed in all cells as unwanted metabolic by-products of normal aerobic metabolism. They are known to damage various biological molecules such as proteins, lipids, and DNA [8]. Exposure to external sources such as cigarette smoke, pollutants, chemicals, and environmental toxins may also result in an increase in the number of free radicals in the body.
Cells are protected from ROS induced damage by the body's own enzymatic and non-enzymatic antioxidants [9]. The enzymatic antioxidants (i.e. superoxide dismutase, catalase and glutathione peroxidase) are able to convert excessive ROS into non-toxic compounds [10, 11], whereas the non-enzymatic antioxi-dants such as vitamins C and E, carotenoids, flavonoids and polyphenols, which mainly derived from fruits and vegetables, are believed to be responsible for free radical scavenging and inhibition of lipid peroxidation in
the body. It is well known that an imbalance between the amount of ROS and antioxidant enzymes can result in health problem. Therefore, research on using natural antioxidants to inhibit lipid peroxidation and to protect biomolecules from damage by the free radicals has received great attention recently.
Armillariella mellea (Vahl. Ex Fr.) Karst. (AM), a consumable fungus belonging to the family of Tricholo-mataceae, is a popular ingredient in traditional Chinese medicine preparations. Its growth has a strong symbiotic relationship with Gastrodia elata (also known as Tian Ma of family Orchidaceae), a slow growing and expensive medicinal herb in Asia. Recently, biologists in mainland China have carried out experiments to cultivate AM in view of replacing the supply of G. elata [12]. Preliminary studies also showed that AM possesses similar pharmacological properties as G. elata. Traditionally, it is used for treating geriatric patients with palsy, dizziness, headache, neurasthenia, insomnia, numbness in limbs, and infantile convulsion [13].
At present, scientific information on the chemical and biological properties of AM is limited, many of its therapeutic properties are based on traditional beliefs. In chemical studies, fruiting bodies of AM were reported to contain armillaramide [12], melleolides K, L and M [14] and fibrinolytic metalloprotease [15].
In this study, our aim was to examine the antioxidant activities of different AM extracts prepared from solid state cultured AM mycelia.
MATERIALS AND METHODS
Chemicals. L-(+)-Ascorbic acid, thiobarbituric acid (TBA), xanthine, xanthine oxidase and cytochrome c were purchased from Sigma Chemical Co. (USA). a-Tocopherol (used as a standard), dimethylsulphoxide (DMSO) and ferrous chloride were obtained from Wa-ko Pure Chemical Industries (Osaka, Japan). All other chemicals used were of analytical grade.
Test animals. Male Sprague-Dawley rats weighing 150 g (about 6 week of age) were used. They were purchased from the Animal Center of the National Laboratory of Animal Breeding and Research Center (Taiwan) and were housed in a controlled environment with temperature maintained at 22 ± 3°C and humidity at 55 ± 5% under a 12 : 12 h light/dark cycle. Animals were fed a standard laboratory diet and tap water ad libitum until use. They were treated in accordance with the guidelines of the National and Institute's Animal Care Committee.
Armillariella mellea culture and growth conditions. The cultured AM (CCRC36393) mycelia, which were grown in solid culture, were obtained from Kang Jian Biotech Co. (Taiwan). The solid fermentation medium comprised 200 g of pulverized maize, which was placed in the 1 l polypropylene culture flask. After adding 400 ml of water, the culture material was sterilized for 1 h at 121°C. Following cooling to room temperature, the medium was inoculated with 5 g of stock culture over the surface. Fermentation was carried out at 25°C for 2 months. The AM mycelia was collected and then lyophilized. The dried AM mycelia was ground to powdered-form and kept in an air-tight plastic bag until use.
Preparation of extracts. 100 g of AM powder was extracted with 1 l of boiling water for 1 h. The extract was filtered with filter paper (Advantec No. 1, Japan) while the residue was re-extracted under the same conditions twice. The filtrates were combined and then concentrated and lyophilized. For the ethanol extract, it was prepared by soaking 100 g of AM powder with 1 liter of ethanol (95%) at room temperature for 6 days. After filtering the extract with filter paper (Advantec No. 1, Japan), the filtrate collected was concentrated and lyophilized. The dried powdered-extract was stored at 4°C until use. The average yield obtained for aqueous AM extract (AM-H2O) and ethanol AM extract (AM-EtOH) was about 24% and 20%, respectively.
Preparation of liver and brain homogenates. On the test day, rats weighing about 200 g, were sacrificed to obtain the liver and brain, 2 g of liver and brain tissues were immediately removed and sliced into pieces. The tissue samples were homogenized with 10 ml of
150 mM KCl-Tris-HCl buffer pH 7.2, and then centri-fuged at 500 g for 10 min to give a supernatant of liver or brain homogenate. The supernatant was collected and the protein content of the supernatant was determined by the method of Lowry et al. [16].
Plasma sample preparation. Blood was obtained from the posterior vena cava of the animals with a hep-arinized tube and then centrifuged at 1000 g at 4°C for 15 min. The plasma sample was collected and stored at -80°C until analysis.
Free radical scavenging activity assay. Free radical scavenging activity was assayed spectrophotometri-cally by the reduction of cytochrome c method as described by McCord and Fridovich [17]. 10 mg of samples were dissolved in 1 ml of distilled water or DMSO and then diluted with 50 mM phosphate buffer (pH 7.8) to various concentrations (1 to 100 pg/ml). Then 0.07 units/ml of xanthine oxidase, 100 pM of xanthine and 50 pM of cytochrome c were added to these samples, which were mixed and incubated for 3 min at room temperature, followed by spectrophotometry determination at 550 nm.
Xanthine oxidase inhibition test. Xanthine oxidase inhibition activity was estimated by the formation of uric acid from xanthine-xanthine oxidase system [18]. 10 mg of samples were dissolved in 1 ml distilled water or DMSO, and then diluted with 50 mM KH2PO4 buffer (pH - 7.8) to various concentrations (1 to 100 pg/ml). After 100 pM of xanthine and 20 pl of xanthine oxidase (0.4 units) were added, samples were mixed and then incubated for 3 min at room temperature. Xanthine oxidase inhibition activity was determined by spectrophotometric measurement of uric acid production at 295 nm.
Anti-lipid peroxidation assay. The effect of AM extracts on rat liver and brain homogenates, and plasma induced with FeCl2-ascorbic acid for lipid peroxidation was determined by malondialdehyde-thiobarbituric acid (MDA-TBA) adduct according to Yoshiyuki et al. [19] and Wong et al. [20]. The reaction mixture containing 0.5 ml of liver or brain homogenate or plasma, 0.1 ml of Tris-HCl buffer (pH 7.2), 0.05 ml of 0.1 mM ascorbic acid, 0.05 ml of 4 mM FeCl2, and 0.05 ml of various concentrations of AM (1 to 500 pg/ml) or a-to-copherol was mixed in a capped tube and then incubated for 1 h at 37°C. After incubati
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