Journal of Ethnopharmacology 96 (2005) 79–85

Antioxidant effect of Inonotus obliquus

Yong Cuia,1 , Dong-Seok Kima,b , Kyoung-Chan Parka,∗
a Department of Dermatology, Seoul National University,

Bundang Hospital, 300 Gumi-Dong, Bundang-Gu, Seongnam-Si, Kyoungki-Do 463-707, Republic of Korea
b Research Division for Human Life Sciences, Seoul National University, 28 Yongon-Dong, Chongno-Gu, Seoul 110-744, Republic of Korea

Received 10 December 2003; received in revised form 18 August 2004; accepted 19 August 2004
Available online 28 October 2004

Abstract

The mushroom Inonotus obliquus (Fr.) Pilát (Hymenochaetaceae), has been widely used as a folk medicine in Russia, Poland and most of
the Baltic countries. The purpose of this study was to elucidate the antioxidant capacities of Inonotus obliquus. Four extracts from the fungus
were evaluated for antioxidant activity against the 1,1-diphenyl-2-picrylhydrazyl (DPPH), superoxide, and peroxyl radicals. The polyphenolic
extract had a strong antioxidant activity, and the extract containing triterpenoids and steroids presented a relatively strong antioxidant effect.
The polysaccharide extract, however, was inactive. The protective effects of these four extracts were assessed against hydrogen peroxide-
induced oxidative stress using a human keratinocyte cell line, HaCaT. Our results show that the polyphenolic extract protected these cells
against hydrogen peroxide-induced oxidative stress, while the polysaccharide, triterpenoid and steroid extracts were ineffective. Additionally,
the remnant polyphenolic and low molecular weight polysaccharide extracts showed a weakly protective effect at a concentration of 50 ␮g/ml.
Our results indicate that Inonotus obliquus has the capacity to scavenge free radicals at concentrations higher than 5 ␮g/ml and that the
polyphenolic extract can protect cells against oxidative stress.
© 2004 Elsevier Ireland Ltd. All rights reserved.

Keywords: Inonotus obliquus; Antioxidant; Hydrogen peroxide

1. Introduction and most of the Baltic countries (Huang, 2002). In Western

Siberia, Inonotus obliquus has been used to alleviate worms,
The mushroom Inonotus obliquus (Fr.) Pilát (Hy- tuberculosis, liver or heart diseases, stomach ailments, and
menochaetaceae), is a black parasitic fungus that grows on also as an internal cleansing agent. However, its pharmaco-
living trunks of the mature birch, and is mainly found at logical actions have not been well documented, in spite of its
latitudes of 45◦ N–50◦ N. Traditionally, it has been used for increasing usage.
the treatment of gastrointestinal cancer, cardiovascular dis- It has long been recognized that many naturally occur-
ease and diabetes since the 16th century in Russia, Poland ring substances in plants have antioxidant activities. Of these
substances, the phenolics which are widely distributed, have
the ability to scavenge free radicals by single-electron trans-
Abbreviations: AAPH, 2,2 -azobis(2-amidinopropane)dihydrochloride;
DCF, 2,7-dichlorofluorescin-diacetate; DPBS, Dulbecco’s phosphate-
fer (Hirano et al., 2001). Recently, it was reported that
buffered saline; DPPH, 1,1-diphenyl-2-picrylhydrazyl; MTT, 3-(4,5- the melanin complex obtained from Inonotus obliquus con-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; NBT, nitroblue tains a strong antioxidant and exhibits genoprotective activity
tetrazolium chloride (Babitskaia et al., 2000). In addition, it has been shown that
∗ Corresponding author. Tel.: +82 31 787 7311; fax: +82 2 3675 1187.
it protects against the effects of gamma radiation (Rasina,
E-mail address: gcpark@snu.ac.kr (K.-C. Park).
1 Present address: Department of Research Supporting Center for Medical 2002) and increases catalase activity in HeLa S3 tumor cells
Science, Dong-A University College of Medicine, Busan 602-714, Republic (Rzymowska, 1998). Mushrooms usually contain a wide va-
of Korea. riety of free radical scavenging molecules, such as polysac-

0378-8741/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2004.08.037
80 Y. Cui et al. / Journal of Ethnopharmacology 96 (2005) 79–85

Fig. 1. Preparation of Inonotus obliquus extracts.

charides and polyphenols (Liu et al., 1997; Mau et al., 2002). 2-yl)-2,5-diphenyl tetrazolium bromide) were obtained from
However, little is known about the cytoprotective effects of Sigma Co. (St. Louis, MO, USA) and 2,7-dichlorofluorescin-
Inonotus obliquus against oxidative stress. diacetate (DCF) was purchased from Calbiochem (San
In this study, the antioxidant activity of the extracts ob- Diego, CA, USA), while analytical grade ethanol and
tained from Inonotus obliquus was evaluated and compared ethyl acetate were obtained from Merck (Darmstadt,
with that of l-ascorbic acid. Extracts of the soluble polysac- Germany).
charide and polyphenolic components of the mushroom were
prepared according to previously described methods (Mizuno
2.2. Cell line
et al., 2000; Thang et al., 2001). This study was under-
taken in order to evaluate the antioxidant activity of Inonotus
Human HaCaT keratinocytes (kindly provided by Dr. N.E.
obliquus, as assessed by its ability to scavenge free radicals
Fusenig, DKFZ, Heidelberg, Germany) were incubated in
and to protect human keratinocytes from oxidative stress.
DMEM supplemented with 10% FBS, 1 mM sodium pyru-
vate, 50 ␮g/ml streptomycin and 50 ␮g/ml penicillin at 37 ◦ C
in 5% CO2 .
2. Materials and methods

2.1. Materials 2.3. MTT assay

Dulbecco’s modified Eagle’s medium (DMEM), Ham’s Cell viability was determined by the MTT assay (Pagè
F-12, fetal bovine serum (FBS, Hyclone, Logan, UT, USA), et al., 1988). For the MTT assay, 20 ␮l of MTT solution
and Dulbecco’s phosphate-buffered saline (DPBS) were pur- (5 mg/ml) was added to each well of a 96-well plate, and incu-
chased from Gibco Ltd. (Grand Island, NY, USA). 1,1- bated for 4 h. The supernatant was removed, and the formazan
Diphenyl-2-picrylhydrazyl (DPPH), nitroblue tetrazolium crystals produced were dissolved in 200 ␮l of dimethyl-
chloride (NBT), hypoxanthine, xanthine oxidase, and 2,2 - sulfoxide, and quantified by measuring their optical den-
azobis (2-amidinopropane) dihydrochloride (AAPH), hydro- sity at 540 nm using an ELISA reader (TECAN, Salzburg,
gen peroxide, l-ascorbic acid, MTT (3-(4,5-dimethylthiazol- Austria).
 Y. Cui et al. / Journal of Ethnopharmacology 96 (2005) 79–85 81

2.4. Mushroom and extraction procedure 10 ␮l of extract (diluted to final concentrations of 50, 10,
and 5 ␮g/ml), 170 ␮l activated DCF solution and 20 ␮l of
Powdered Inonotus obliquus (Fr.) Pilát (100 g) was ex- 600 mM AAPH (adjusted to a final concentration of 60 mM).
tracted with 80% ethanol at room temperature overnight, and The reaction was initiated by adding the AAPH solution.
then freeze-dried (Fa, 3.53 g). The residual fraction was dis- After 10 min, the absorbance was read at 490 nm using an
solved in distilled water in a boiling water bath for 4 h. The ELISA reader (TECAN, Salzburg, Austria). The inhibition
aqueous phase was evaporated and reduced to half its volume rate was determined versus l-ascorbic acid.
and then mixed with 95% ethanol (1:4, v/v), and the precipi-
tated fraction was freeze-dried (Fb, 8.5 g). The aqueous phase 2.8. Protective effects of Inonotus obliquus extract after
was then evaporated to remove the ethanol and mixed with treatment with hydrogen peroxide
ethyl acetate (2:1, v/v). The upper and lower aqueous phases
were then evaporated and lyophilized (Fc, 1.53 g; Fd, 3.9 g, Human HaCaT cells were seeded in 96-well plates at a
respectively) (Fig. 1). The freeze-dried extracts were recon- density of 6000 cells/well. The cells were grown to near
stituted in 80% ethanol (Fa, 100 mg/ml) and DPBS (Fb, Fc, 70–80% confluence, and then synchronized by incubation
Fd fraction, each 20 mg/ml). These stock solutions were ster- in DMEM containing 0.5% FBS for 8 h. Inonotus obliquus
ilized by filtration through a 0.45 ␮m pore membrane (Sarto- extracts were added at concentrations of 10 and 50 ␮g/ml,
rius AG, Goettingen, Germany) and kept in the dark at 4 ◦ C. respectively. After 16 h, the cells were washed and 1 mM of
hydrogen peroxide was added. The cell viability was mea-
2.5. Scavenging effect on DPPH radicals sured by means of the MTT assay. To compare the data, the
cytotoxic effects of Inonotus obliquus extracts were also eval-
Various concentrations of the stock solutions (diluted to uated. l-Ascorbic acid and catalase were used as positive
final concentrations of 50, 10, and 5 ␮g/ml) were mixed with controls.
0.25 mM DPPH in ethanol, to produce a final DPPH concen-
tration of 0.1 mM. The mixture was vigorously shaken and
2.9. Statistical analysis
left to stand for 10 min in the dark, and its absorbance was
measured at 517 nm. l-Ascorbic acid was used as the control
The significance of the differences between the results
(McCune and Johns, 2002).
was assessed using the Student’s t-test, and significance was
accepted for p-values <0.01.
2.6. NBT/XO (superoxide scavenging) assay

The scavenging potential of the mushroom extract

3. Results
for superoxide radicals was analyzed using a hypoxan-
thine/xanthine oxidase generating system coupled with NBT
3.1. Scavenging of DPPH radicals by Inonotus obliquus
reduction, as previously described (Kirby and Schmidt,
1997), with a slight modification involving the use of 96-well
Four extracts were prepared from Inonotus obliquus as de-
plates. Briefly, the reaction mixture contained 134 ␮l buffer
scribed in the Section 2 (Fig. 1). The different extracts showed
(50 mM KH2 PO4 /KOH, pH 7.4), 2 ␮l of 100 mM Na2 EDTA,
variable DPPH radical-scavenging activities. The Fa and Fc
20 ␮l of 3 mM hypoxanthine, 2 ␮l of 10 mM NBT, and 10 ␮l
extracts exerted free radical scavenging effects in a dose-
of extract. The microplates were read 2.5 min after adding
dependent manner. In particular, the Fc (polyphenolic) extract
32 ␮l of xanthine oxidase (1 unit per 10 ml buffer) at 540 nm
showed strong antioxidant activity at concentrations higher
using an ELISA reader (TECAN, Salzburg, Austria). The su-
than 5 ␮g/ml, but its effect was less than that of l-ascorbic
peroxide scavenging activity was expressed as the percentage
acid (Table 1). The Fa extract, which contained triterpenoids
inhibition compared to the blank (buffer instead of extract).
and steroids, also had a relatively strong antioxidant effect.
l-Ascorbic acid was used as a positive control.
The Fb extract, containing soluble polysaccharides, and the
Fd extract, containing remnant polyphenolic compounds and
2.7. DCF/AAPH assay
low molecular weight polysaccharides, were almost inactive
(Table 1).
An azo initiator, AAPH, was used to produce peroxyl rad-
icals, and the scavenging activity of the mushroom extracts
was monitored via the spectrophotometric analysis of 2,7- 3.2. NBT/XO (superoxide scavenging) assay
dichlorofluorescin-diacetate (Valkonen and Kuusi, 1997).
The activation of DCF was achieved by mixing DCF (3.41 ␮l The superoxide radical scavenging activity of the extracts
of 50 ␮g/ml solution) and NaOH (1.75 ml of 0.01N solu- was investigated (Table 1). All of the extracts showed dose-
tion) and allowing the mixture to stand for 20 min before dependent superoxide radical scavenging activity. In particu-
adding 18.25 ml of sodium phosphate buffer (25 mM, pH lar, the Fc extract had the highest scavenging activity, which
7.2) (Cathcart et al., 1983). The reaction mixture contained was higher than that of l-ascorbic acid.
82 Y. Cui et al. / Journal of Ethnopharmacology 96 (2005) 79–85

Table 1
Scavenging effects of Inonotus obliquus extracts for DPPH, superoxide and peroxyl radicals
Radical and extract Fa Fb Fc Fd l-Ascorbic acid
concentration (␮g/ml)
DPPH
5 12.4 ± 2.6 4.5 ± 0.9 30.6 ± 1.8 4.9 ± 0.9 75.2 ± 1.0
10 22.2 ± 3.3 3.8 ± 2.9 60.1 ± 4.2 5.3 ± 2.6 88.1 ± 0.3
50 58.7 ± 6.4 11.7 ± 0.9 81.8 ± 1.0 11.0 ± 0.5 90.8 ± 0.2
Superoxide radical
5 19.9 ± 11.7 9.8 ± 9.9 55.1 ± 1.7 33.5 ± 6.8 26.3 ± 4.9
10 28.8 ± 12.9 38.1 ± 12.3 80.9 ± 2.9 55.1 ± 4.3 44.9 ± 8.8
50 61.9 ± 3.3 73.3 ± 4.3 91.5 ± 1.4 75.0 ± 3.5 78.8 ± 1.0
Peroxyl radical
5 10.1 ± 7.8 5.4 ± 9.3 42.9 ± 7.5 14.8 ± 9.4 89.9 ± 2.3
10 20.2 ± 4.7 3.5 ± 10.9 63.1 ± 2.6 23.3 ± 11.1 94.6 ± 1.9
50 55.5 ± 2.6 34.7 ± 7.2 77.0 ± 4.3 66.3 ± 12.4 95.6 ± 1.3
The scavenging effects were expressed as the percentage inhibition (mean ± S.D., n = 4) compared to the blank (buffer instead of extract). l-Ascorbic acid
was used as a positive control. Fa: triterpenoids and steroids; Fb: polysaccharides; Fc: polyphenolic extract; Fd: remnant polyphenolic compounds and low
molecular weight polysaccharides.

3.3. DCF/AAPH assay 0.5 mM, while treatment with 1 mM hydrogen peroxide re-
sulted in significant cell death (Fig. 3). Based on these data,
The peroxyl radical scavenging effects were also exam- the cells were treated with 1 mM hydrogen peroxide and the
ined. All of the extracts showed dose-dependent activity. In extracts were added in order to assess their protective effects
particular, the Fc extract presented the strongest effect, al- with regard to cell survival.
though its scavenging effect was lower than that of l-ascorbic
acid (Table 1). 3.5. Protective effects of Inonotus obliquus extract

3.4. Cytotoxic effects of Inonotus obliquus and hydrogen Hydrogen peroxide (1 mM) decreased the cell viability to
peroxide 45.6% of that of the control. Catalase was strongly protective
against hydrogen peroxide-induced cell death, but l-ascorbic
The Fa and Fc extracts of Inonotus obliquus were cytotoxic acid showed only a negligible effect. The protective effects
at concentrations higher than 100 ␮g/ml. However, the Fb and of Inonotus obliquus were also examined. The Fa and Fb ex-
Fd extracts were only cytotoxic at concentrations exceeding tracts were not protective against hydrogen peroxide-induced
400 ␮g/ml (Fig. 2). Thus, we used concentrations lower than oxidative stress. However, the Fc extract was strongly effec-
100 ␮g/ml for this experiment. To assess the protective effects tive at a concentration of 50 ␮g/ml, and this effect was com-
of the extracts against hydrogen peroxide-induced cell death, parable to that of catalase at a concentration of 0.4 mg/ml.
hydrogen peroxide was added to cultured cells. The MTT as- The Fd extract was slightly protective at a concentration of
say showed that hydrogen peroxide was not cytotoxic below 50 ␮g/ml (Fig. 4).

Fig. 2. Cytotoxic effects of Inonotus obliquus extracts on HaCaT cells. Cells Fig. 3. MTT reduction assay of H2 O2 cytotoxicity. HaCaT cells were treated
were treated with the extracts for 24 h. Cell viability was measured by the with H2 O2 at different concentrations for 6 h. Data are averages ± S.D. (bars)
MTT assay. Data are averages ± S.D. (bars) of triplicate determinations. of triplicate determinations.
 Y. Cui et al. / Journal of Ethnopharmacology 96 (2005) 79–85 83

Fig. 4. Protective effects of Inonotus obliquus extracts against H2 O2 (1 mM) induced oxidative stress as determined by the MTT assay. (* p < 0.01, ** p < 0.001
vs. H2 O2 alone).

4. Discussion tively high DPPH radical scavenging activity. These results

suggest that the triterpenoids and steroids in the Fa extract
In the present study, we found that Inonotus obliquus ex- may account for the free radical scavenging effect of Inono-
erts an antioxidant activity and protects cells against oxidative tus obliquus (Kim et al., 1999). Mau et al. (2002) reported
stress. These findings provide a pharmacological explana- that the methanolic extracts obtained from several medicinal
tion for some of its uses in folk medicine. Inonotus obliquus mushrooms, such as Ganoderma lucidum and Ganoderma
has long been used to improve overall health and prevent tsugae, showed high DPPH free radical scavenging activ-
various diseases, such as cancer, cardiovascular disease and ity. However, this scavenging activity (67.6–74.4%) was only
diabetes. In the literature, it has been reported that the an- obtained at a relatively high concentration (0.64 mg/ml). In
tioxidant activity of plants is responsible for their therapeu- contrast, the scavenging activities of the Fa (58.7%) and Fc
tic effect against cancer, cardiovascular disease and diabetes (81.8%) extracts of Inonotus obliquus were effective at a
(Anderson et al., 2004; Stanner et al., 2004). Thus, the action lower concentration (50 ␮g/ml) in our study. On the other
of Inonotus obliquus seems to be at least partially associated hand, the Fb polysaccharide extract and the Fd extract, con-
with its antioxidant effect. taining remnant polyphenolic compounds and low molecular
Traditionally, Inonotus obliquus has been taken in the form weight polysaccharides, were almost inactive.
of a hot water extract prepared from a small piece of the mush- The superoxide radical scavenging activity of the Inonotus
room (1–2 g) or one tablespoon of crushed mushroom. This obliquus extracts was also generally quite high. This was es-
produces an aqueous extract, which is taken at a dose of three pecially the case for the Fc extract, whose activity was higher
cups per day. In this study, we showed that the polyphenolic than that of l-ascorbic acid. It is well known that polyphenolic
extract had the strongest antioxidant activity among the four compounds are able to efficiently scavenge superoxide radi-
kinds of extracts, as well as exhibiting a strong protective cals (Valentao et al., 2002). These phenolic compounds may
activity against hydrogen peroxide-induced cell damage at a react with the superoxide radical via a one-electron transfer
concentration of 10–50 ␮g/ml. Our study showed that 1.5 g mechanism or by a hydrogen abstraction mechanism to form
of the Fc extract can be obtained from 100 g of total weight the corresponding semiquinone (Wang et al., 1996). Further-
of the mushroom and that the Fc extract was not cytotoxic more, polyphenolic crude extracts are known to have a cer-
at these doses. These data showed that only a small amount tain inhibitory activity towards xanthine oxidase (Costantino
of the polyphenolic components could be obtained by the et al., 1992).
traditional methods of extract preparation. Among the four extracts, the polysaccharide extract, Fb,
The different extracts of Inonotus obliquus showed vari- also showed superoxide radical scavenging activities. It has
able radical scavenging activities. The Fc extract, which con- been reported that the polysaccharide extracts of Ganoderma
tains polyphenolic components, showed strong anti-oxidant lucidum and Grifola umbellata possess superoxide radical
activity, while the Fa extract, which contained triterpenoids scavenging activity (Liu et al., 1997). The superoxide rad-
and steroids including lanosterol, inotodiol, trametenolic acid ical scavenging activity of polysaccharide extracts appears
and ergosterol peroxide (Kahlos et al., 1989), also had a rel- to depend on the amount of peptides present in the form
atively strong antioxidant effect. Triterpenoids and steroids of polysaccharide–peptide complexes. For example, lentinan
have been previously isolated from Inonotus obliquus (He and schizophyllan, which contain only trace amounts of pep-
et al., 2001), but their activities have not previously been tides in the polysaccharide samples, have almost no scav-
reported. In our experiment, the Fa extract showed a rela- enging effect, whereas polysaccharide krestin and polysac-
84 Y. Cui et al. / Journal of Ethnopharmacology 96 (2005) 79–85

charopeptide, which are obtained from mushrooms, such as and characterization of polyphenol type-a polymers from cinnamon
Ganoderma lucidum and Grifola umbellate which have lower with insulin-like biological activity. Journal of Agricultural and Food
polysaccharide/peptide ratios, exhibited the strongest scav- Chemistry 52, 65–70.
Babitskaia, V.G., Shcherba, V.V., Ikonnikova, N.V., 2000. Melanin com-
enging effects (Liu et al., 1997). However, the mechanism plex of the fungus Inonotus obliquus. Prikladnaya Biokhimiya i
underlying the free radical scavenging activity exerted by Mikrobiologiya 36, 439–444.
polysaccharides is still not fully understood. Cathcart, R., Schwiers, E., Ames, B.N., 1983. Detection of picomole
The protective effect exerted by the extracts on hydro- levels of hydroperoxides using a fluorescent dichlorofluorescein assay.
gen peroxide-induced cell death was assessed. The results Analytical Biochemistry 134, 111–116.
Costantino, L., Albasini, A., Rastelli, G., Benvenuti, S., 1992. Activ-
showed that the polyphenolic extract showed strong antiox- ity of polyphenolic crude extracts as scavengers of superoxide rad-
idant activity and was the most strongly protective against icals and inhibitors of xanthine oxidase. Planta Medica 58, 342–
hydrogen peroxide-induced cell damage. The effect of the 344.
polyphenolic extract at a concentration of 50 ␮g/ml was com- He, J., Feng, X.Z., Lu, Y., Zhao, B., 2001. Three new triterpenoids from
parable to that of catalase at a concentration of 0.4 mg/ml. In Fuscoporia obliqua. Journal of Asian Natural Products Research 3,
55–61.
contrast, l-ascorbic acid showed negligible effects. The Fa Hirano, R., Sasamoto, W., Matsumoto, A., Itakura, H., Igarashi, O.,
and Fb extracts did not show any protective effect against Kondo, K., 2001. Antioxidant ability of various flavonoids against
hydrogen peroxide-induced oxidative stress, although they DPPH radicals and LDL oxidation. Journal of Nutritional Science
showed some free radical scavenging ability. These results and Vitaminology (Tokyo) 47, 357–362.
suggest that hydrogen peroxide-induced cell death is related Huang, N.L., 2002. Inonotus obliquus. Edible Fungi of China 21, 7–8.
Kahlos, K., Kangas, L., Hiltunen, R., 1989. Ergosterol peroxide, an
not only to free radicals, but also to unresolved signaling active compound from Inonotus radiatus. Planta Medica 55, 389–
pathways. 390.
Our results and many previous reports show that the Kim, S.W., Park, S.S., Min, T.J., Yu, K.H., 1999. Antioxidant activity
polyphenolic extracts of various plant and fungal species, in- of ergosterol peroxide (5,8-epidioxy-5␣,8␣-ergosta-6,22E-dien-3␤-ol)
cluding Inonotus obliquus, have strong antioxidant activity. In Armillariella mellea. Bulletin of Korean Chemical Society 20,
819–823.
However, the prooxidant and cytotoxic effects of these pheno- Kirby, A.J., Schmidt, R.J., 1997. The antioxidant activity of Chinese herbs
lic components have been also reported in the literature (Liu for eczema and of placebo herbs. Journal of Ethnopharmacology 56,
et al., 1997). High concentrations of phenolic compounds 103–108.
may inhibit cell proliferation, and simultaneous exposure to Liu, F., Ooi, V.E., Chang, S.T., 1997. Free radical scavenging activi-
hydrogen peroxide and phenolics has been shown to lead to ties of mushroom polysaccharide extracts. Life Sciences 60, 763–
771.
the amplification of proliferation inhibition (Liu et al., 1997). Mau, J.L., Lin, H.C., Chen, C.C., 2002. Antioxidant properties of several
However, in our study, a significant protective effect was ob- medicinal mushrooms. Journal of Agricultural and Food Chemistry
served upon pretreatment with the polyphenolic extract of 50, 6072–6077.
Inonotus obliquus. This discrepancy may arise from several McCune, L.M., Johns, T., 2002. Antioxidant activity in medicinal plants
factors, such as the extract concentration, experimental de- associated with the symptoms of diabetes mellitus used by the In-
digenous Peoples of the North American boreal forest. Journal of
sign, and the relationship between polyphenolic compounds Ethnopharmacology 82, 197–205.
and hydrogen peroxide. Mizuno, M., Shiomi, Y., Minato, K., Kawakami, S., Ashida, H., Tsuchida,
In conclusion, the polyphenolic extract shows the H., 2000. Fucogalactan isolated from Sarcodon aspratus elicits re-
strongest antioxidant activity among the four kinds of ex- lease of tumor necrosis factor-alpha and nitric oxide from murine
tracts obtained from Inonotus obliquus, and can protect cells macrophages. Immunopharmacology 46, 113–121.
Pagè, M., Bejaoui, N., Cinq-Mars, B., Lemieux, P., 1988. Optimization
against oxidative stress. It has been reported that the fungal of the tetrazolium-based colorimetric assay for the measurement of
melanin complex of Inonotus obliquus has strong antioxi- cell number and cytotoxicity. International Journal of Immunophar-
dant activity. These findings suggest that the Fc extract may macology 10, 785–793.
contain a strongly antioxidant melanin complex or related Rasina, L.N., 2002. Effect of cryosubstance Chagi on deposition or
polyphenolics (Babitskaia et al., 2000). isolation Of 90Sr and on the effect of prolonged external expo-
sure to gamma-radiation. Radiatsionnaia Biologiaa, Radioecologiia 42,
399–403.
Rzymowska, J., 1998. The effect of aqueous extracts from Inonotus
Acknowledgements obliquus on the mitotic index and enzyme activities. Bollettino Chim-
ico Farmaceutico 137, 13–15.
Stanner, S.A., Hughes, J., Kelly, C.N., Buttriss, J., 2004. A review of
This study was supported by a grant (01-PJ2-PG6- the epidemiological evidence for the ‘antioxidant hypothesis’. Public
01NA01-0002) from the 2001 Good Health R&D Project, Health Nutrition 7, 407–422.
Ministry of Health & Welfare, R.O.K. Thang, P.T., Patrick, S., Teik, L.S., Yung, C.S., 2001. Anti-oxidant effects
of the extracts from the leaves of Chromolaena odorata on human
dermal fibroblasts and epidermal keratinocytes against hydrogen per-
oxide and hypoxanthine-xanthine oxidase induced damage. Burns 27,
References 319–327.
Valentao, P., Fernandes, E., Carvalho, F., Andrade, P.B., Seabra, R.M.,
Anderson, R.A., Broadhurst, C.L., Polansky, M.M., Schmidt, W.F., Khan, De Lourdes Bastos, M., 2002. Antioxidant activity of Hypericum an-
A., Flanagan, V.P., Schoene, N.W., Graves, D.J., 2004. Isolation drosaemum infusion: scavenging activity against superoxide radical,
 Y. Cui et al. / Journal of Ethnopharmacology 96 (2005) 79–85 85

hydroxyl radical and hypochlorous acid. Biology and Pharmaceutical Wang, P., Kang, J., Zheng, R., Yang, Z., Lu, J., Gao, J., Jia, Z., 1996.
Bulletin 25, 1320–1323. Scavenging effects of phenylpropanoid glycosides from pedicularis on
Valkonen, M., Kuusi, T., 1997. Spectrophotometric assay for total per- superoxide anion and hydroxyl radical by the spin trapping method.
oxyl radical-trapping antioxidant potential in human serum. Journal Biochemical Pharmacology 51, 687–691.
of Lipid Research 38, 823–833.