BioFactors 21 (2004) 109112 IOS Press

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Chaga mushroom extract inhibits oxidative DNA damage in human lymphocytes as assessed by comet assay
Yoo Kyoung Parka, , Hyang Burm Leeb,c, , Eun-Jae Jeona , Hack Sung Jungb and Myung-Hee Kanga,
a Department

of Medical Nutrition, Kyunghee University, 1 Hoekidong, Dongdaemoonku, Seoul 130-701, South Korea b Department of Food and Nutrition, Hannam University, 133 Ojeong-dong, Daedeok-gu, Daejeon 306-791, Korea c School of Biological Sciences, Seoul National University, Kwanak-gu, Seoul 151-742, Korea d Seson Biotech, Daejeon 305-710, Korea

Abstract. The Chaga mushroom (Inonotus obliquus) is claimed to have benecial properties for human health, such as anti-bacterial, anti-allergic, anti-inammatory and antioxidant activities. The antioxidant effects of the mushroom may be partly explained by protection of cell components against free radicals. We evaluated the effect of aqueous Chaga mushroom extracts for their potential for protecting against oxidative damage to DNA in human lymphocytes. Cells were pretreated with various concentrations (10, 50, 100 and 500 g/mL) of the extract for 1 h at 37 C. Cells were then treated with 100 M of H2 O2 for 5 min as an oxidative stress. Evaluation of oxidative damage was performed using single-cell gel electrophoresis for DNA fragmentation (Comet assay). Using image analysis, the degree of DNA damage was evaluated as the DNA tail moment. Cells pretreated with Chaga extract showed over 40% reduction in DNA fragmentation compared with the positive control (100 mol H2 O2 treatment). Thus, Chaga mushroom treatment affords cellular protection against endogenous DNA damage produced by H2 O2 . Keywords: Chaga extract, comet assay, human lymphocyte, Inonotus obliquus, oxidative DNA damage

1. Introduction Mushrooms have long been included in the human diet. There are many compounds in mushrooms including active polysaccharides, some of which are unique. Chaga mushroom, one of the many known medicinal mushrooms, has been used primarily in Russia, Eastern Europe and Japan to treat early stages of stomach and lung cancer, and it has no documented side effects. In these northern latitudes it is a fungal parasite of living trees, growing primarily on branch stubs on birches, but sometimes on elm, alder, or beech trees. Studies have shown anti-tumor effects of Chaga mushroom water extracts in vitro [4,5] as well as antimicrobial and antiviral activities [6,7]. A recent report suggested that a hot

Corresponding author. Tel.: +82 42 629 7491; E-mail: mhkang@hannam.ac.kr. These authors contributed equally to this work.

0951-6433/04/$17.00 2004 IOS Press and the authors. All rights reserved

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Y.K. Park et al. / Chaga mushroom extract inhibits oxidative DNA damage in human lymphocytes

water extract of Chaga mushrooms might suppress cellular proliferation in a time-dependent manner in human stomach cancer cell lines [3]. Ham et al., using the Ames test, also showed strong anti-mutagenic and anti-cytotoxic effects of ethanol extract, ethyl acetate fraction, water fraction and other sub-fractions from Chaga mushrooms [1,2]. It is widely believed that oxidative nuclear DNA damage over the human lifespan contributes signicantly to the age-related development of major cancers, such as those of the colon, prostate and breast. The presence of DNA damage has thus become one of the most sensitive biological markers for evaluating the oxidative stresses resulting from the imbalance between free radical generation and control through antioxidant systems. The aim of this work was to evaluate the effect of Chaga mushroom extracts on the potential for protection against oxidative damage to DNA produced by H 2 O2 treatment in human lymphocytes. 2. Methods 2.1. Sample preparation Chaga mushrooms were directly obtained from native birch plants grown at Baektu Mountain, North Korea. Two parts of the fruiting bodies including the sclerotium, a compact mass stored with reserve food material and the mycelium, a mass of interwoven laments that forms in the vegetative portion, were used as aqueous extracts. Samples of sclerotium and mycelium were prepared by heating in water (twice at 85C for 3 h), ltering (No.1 lter paper) followed by freeze-drying. 2.2. Cell preparation and treatment Lymphocyte isolation for the Comet assay was performed using Histopaque 1077-1 (Sigma Chemical Co., Korea), followed by a brief wash with phosphate buffered saline (PBS). Cells were pretreated with various concentrations of sclerotium or mycelium extracts (10, 50, 100, 500 g/mL) or with 100 mg/mL of the known antioxidants ascorbic acid (Vitamin C), Trolox and -tocopherol for 1 h at 4 C. Cells were harvested and then treated with 100 M of H 2 O2 for 5 min to induce oxidative stress. 2.3. Analysis Endogenous lymphocyte DNA damage was analyzed using the alkaline Comet assay (single-cell gel electrophoresis) with little modication. Images of 100 randomly selected cells (50 cells from each of two replicate slides) were analyzed. Measurements of DNA density were performed using image analysis (Kinetic Imaging, Komet 4.0, Liverpool, U.K.), determining tail moment (TM, calculated as the percentage of total cellular DNA in the tail and as tail length) and relative DNA damage scores were calculated as TM values normalized against positive controls treated with 100 M H 2 O2 or against negative untreated controls. 3. Results and discussion Figure 1 shows the inhibitory effect of Chaga mushroom extracts expressed as relative TM scores of DNA damage. Pretreatment with sclerotium extracts at 10, 50, 100 and 500 mg/mL reduced the

Y.K. Park et al. / Chaga mushroom extract inhibits oxidative DNA damage in human lymphocytes

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(a)
DNA damage (Relative score)
d 100 80 60 40 20 a 0 P N 10 50 100 500 Sclerotium (ug/ml) c b b bc

(b)
DNA damage (Relative score)
d 100 80 60 b 40 20 a 0 P N 10 50 100 500 Mycelium (ug/ml) b b

Fig. 1. Protection potential of Chaga mushroom extracts of (a) sclerotium and (b) mycelium on oxidative DNA damage to human lymphocytes induced by H2 O2 treatment. Different letters represents signicant differences at P < 0.05 by ANOVA. P, positive control (100 mol H2 O2 ); N, negative control (no H2 O2 ).

P N

d a b b c b b
0 20 40 60 80 100

100 ug/ml

1 2 3 4 5

DNA damage (Relative score)

Fig. 2. Comparison of the antioxidant activities of two Chaga mushroom extracts and antioxidant vitamins (all at 100 g/mL) on oxidative DNA damage to lymphocytes induced by H2O2 treatment. Different letters represents signicant differences at P < 0.05 by ANOVA. Key: P, positive control (100 mol H2 O2 ); N, negative control (no H2 O2 ); 1, sclerotium; 2, mycelium; 3, ascorbic acid (vitamin C); 4, Trolox; 5, -tocopherol.

degree of DNA damage by 44%, 67%, 76% and 62%, respectively, compared with the positive controls (Fig. 1(a)). Similarly, pretreatment with mycelium extracts at 10, 50, 100 and 500 mg/mL reduced the degree of DNA damage by 34%, 61%, 71% and 71%, respectively (Fig. 1(b)). Food products containing phytochemicals categorized as non-nutrient antioxidants have been reported to play equally important roles as other antioxidative vitamins. To compare the antioxidant activities of Chaga extracts with other chemicals known to have strong antioxidant activities, we repeated the experiment at doses of 100 mg/mL each of ascorbic acid, Trolox and -tocopherol. The protective effects against DNA damage are shown in Fig. 2. Compared with the damage level of the positive control, except for ascorbic acid all the other antioxidants showed over 50% reduction in the level of DNA damage. Our results thus demonstrate that Chaga mushroom extracts afford protection against cellular DNA damage produced by H 2 O2 in healthy human lymphocytes at levels similar to those

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provided by other known antioxidants. Although we did not aim to identify the main active ingredient (s) responsible for this protective action, others have reported similar anti-genotoxic effects of Chaga fractions and extracts soluble in ethanol [1,2]. Evidence from the literature, although obviously incomplete, is consistent with the view that increased oxidative DNA damage contributes to subsequent cancer development, and that diets rich in antioxidants might contribute to delay or prevent cancer development. Our observations here suggest that Chaga mushroom extracts might represent a valuable source of biologically active compounds with potential for protecting cellular DNA from oxidative damage in vitro. Understanding the chemical properties of the extracts as well as the mechanisms by which they might effect such protection are thus important issues anddeserve further study. Acknowledgement This work was supported by Korea Research Foundation Grant (KRF-2003-050-C00019). References
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