1

서 론

50% .

,

. Inonotus obliquus Fus-

coporia obliquus ,

‘Chaga’ , ‘

( )’ .

(Eumycota), (Basidiomycotina),

(Eubasidiomycetes), (Hymenomycet-

idae), (Aphyllophorales),

(Hymenochaetaceae), (Inonotus)

‘ ’ ‘ ’

.

,

문병혁, 이원철

동국대학교 한의과대학 내과학교실

Original Article

Studies on the Anti-cancer Activity of Chaga Mushroom Extract

Byung-Hyuk Moon, Won-Chul Lee

Dept. of Oriental Medicine The Graduate School, Dong-Guk University

Objectives: This study was to investigate the anti-oxidation and anti-cancer activity of Chaga mushroom extract.

Extraction condition optimization and beta-glucan analysis and anti-cancer activity tests were also done.

Methods: Optimum extraction conditions for Chaga mushroom extract were at a temperature of 90 and 2hrs with

10 times of water. Extraction yield and economics were best under these conditions.

Results: Anti-oxidation activity was the highest with the fraction of 100,000 MWCO and IC50 value was 13 / , and this value was comparable to that of vitamin E, alpha-tocopherol. Among the fractions from various organic

solvents, ethyl acetate fraction showed the highest anti-oxidation activity with IC50 value of 7 / . For anti-cancer

activity, chloroform fraction showed little anti-cancer activity and ethyl acetate fraction showed the best anti-cancer

activity with IC50 1.5 / for stomach cancer cells. Anti-cancer activities for different molecular weight fractions

were the best in the fraction of molecular weight less than 100,000Da, and IC50 values for stomach cancer cells and

liver cancer cells were 1.7 and 1.4 / , respectively.Conclusions: From these results, we can conclude that the extract of Chaga mushroom could be a good source for

functional food and natural anti-cancer medicine .

Key Words : Inonotus obliquus, anti-oxidation, anti-cancer, beta-glucan

Received：31 May 2007 Revised：26 March 2009 Accepted：8 June 2009

Correspondence to：문병혁(Byung-Hyuk Moon)

부산시 북구 구포2동 1087-1 한성빌딩 4층 구남한의원

Tel：+82-51-363-3232, Fax：+82-51-363-3288, E-mail：mbh9866@hanafos.com

30 4 (2009 7 )J Korean Oriental Med 2009;30(4):01-12

30 4 (2009 7 )

2

,

,

1).

16

,

(herb) .

, ( )

(herb)1).

, , , ,

, , , flavonoid, steroid2),

polyphenol2), terpenoid, inositol, inotodiol

3)

4), butulin

5), melanin

6).

β-glucan

,

.

19907-9)

.

.

10-14),

15,16),

1),

16,17),

10,18),

12),

19)

20).

.

. 10

.

,

.

실험재료 및 방법

1. 원재료의 준비

.

, ,

.

, ,

.

2. 추출물의 제조방법

3 .

, ,

,

.

, 600nm (Shimadzu co., UV-MINI

1,240).

3. 추출물의 분획 제조

Ultrafiltration(UF) Pall Maxisette

AT

,

(Fig. 1). 0.45

(458)

1 :

3

ultrafiltration

(Eyla co., NE) ,

( , PVTFD 20R)

.

Fig. 1

chloroform, ethylacetate, n-buthanol

2 , 2 ,

.

.

4. 항산화 활성의 측정

DPPH

. DPPH 1,1-diphenyl

2-picrylhydrazyl radical radical

. DPPH

(Sigma co.) .

.

Ethanol 0.1 mM DPPH(Sigma, D-9132)

2 ethanol 2 10

30 520nm

.

( ) .

EDA(electron donating ability) = (Cabs - Sabs)/Cabs 100

- Cabs: Negative contol( ethanol )

- Sabs: Sample

- Positive control: Vitamin E(Fluka, 95240)

.

5. Polyphenol의 분석

Polyphenol

.

1 / 0.2 ,

Water Extract(WE)

[Solvent Fraction]

Chloroform Phase(CP)

Top Phase

Ethylacetate Phase(EP)

Ethylacetate

n-Buthanol

Bottom Phase

n-Buthanol Phase(BP)

Water Phase(WP)

MWCO 100,000

MWCO 300,000

MWCO 300,000

UF1

UF2

UF3

[UF Fraction]

CHCI3

Fig. 1.

(459)

30 4 (2009 7 )

4

2 0.2 Folin

ciocalteu’s phenol reagent

3 . 3 Na2CO3

0.4 ,

4 , 1

725nm . polyp-

henol tannic acid

. tannic acid 1

50% methanol 1 0, 50, 100, 150, 200,

300ppm 725nm

.

6. 당조성 분석

HPLC . HPLC

YMC-Pack Polyamine II

, acetonitrile : = 75

: 25 1 /min. , HPLC

Shimadzu LC-10A .

RI , 26

.

.

7. Beta-glucan의 분석

Beta-glucan ‘Mushroom and Yeast

Beta Glucan Kit’ .

.

1) Total glucan

100

. 1.5 HCl(37%)

tube vortex 30 water bath

45 β-glucan

15 vortex . 10

tube , vortex .

, 100

5 , 2

. , 2N KOH

10 . 100 volumetric flask ,

200mM sodium acetate buffer tube washing

volume .(1,500 g,

10min.) 0.1 glass tube

, 0.1 Bottle 1 [exo-1,3 β-glucanase

(100U/ ) β-glucosidase (20U/ ) ]

, vortex 40 1 . 3

GOPOD mixture(glucose reagent buffer + glucose

oxidase, peroxidase, 4-aminoantipyridine) 4

0 20 510nm

.

2) Alpha-glucan

100 2

KOH , . 20 ice water

bath . 1.2M Sodium acetate buffer(pH3.8)

8 bottle 2 (50%

glycerol amyloglucosidase 1630U/ invertase

500U/ solution) 0.2 . 4

0 water bath , 30 ,

. Alpha-glucan 10%

100 volumetric flask volume

100 .

Alpha-glucan 10% ,

. 10.3

. 0.1 tube , 3

GOPOD reagent 40 20

510nm .

,

reagent blank D-glucose standard solution

.

reagent blank:

0.2 acetate buffer + 3 GOPOD Mixture

D-glucose standard solution:

0.1 D-glucose standard (1 / ) +

0.1 acetate buffer + 3 GOPOD mixture

3)

total glucan (% w/w) = E F/W 90 α-glucan

= E F/W 90 (final volume 100 )

(460)

1 :

5

= E F/W 9.27 (final volume 10.3 )

β-glucan = total glucan - α-glucan

E = reaction absorbance - blank absorbance

F = a factor to convert absorbance to of

D-glucose

= 100( of D-glucose standard) / GOPOD

absorbance for 100 of D-glucose

standard)

W =

8. 항암활성의 분석

1)

(1) AGS ( )

: RPMI 1640 with L-glutamine(GIBCO

11875-093, 300 /L), 25mM

HEPES(GIBCO 15630-080), 25mM

sodium carbonate(GIBCO 25080-094), FBS

10%, 1% Penicillin/streptomycin

solution(GIBCO 15140-122)

Growth pattern: monolayer

1 2 3 1 × trypsin(SIGMA T4174)

PBS(KH2PO4 2.1g/

, NaCl 90g/ , Na2HPO4 7.26g/ ) washing

.

(2) HepG2 ( )

: DMEM(GIBCO 12561), 10% FBS,

1% Penicillin/streptomycin solution

Growth pattern: monolayer

1 2 3 1 × trypsin

PBS washing .

(3) HeLa ( )

: DMEM, 10% FBS, 1% Penicillin/streptomycin

solution

Growth pattern: monolayer

1 2 3 1 × trypsin

PBS washing .

(4) Calu-3 ( )

: MEM(GIBCO 12561),

10% FBS(GIBCO 16000-044),

1% MEM vitamin(SIGMA M6895),

10% FBS,

1% Penicilln/streptomycin solution

Growth pattern: monolayer

1 2 3 1 × trypsin

PBS washing .

2) - MTT assay

1 × trypsin solution cell

trypsin 96well

plate 1 × 105cells/ 180 1 .

Test 20 , 24

. 5 / MTT 20 , 4

. . DMSO 150

30 550nm

.

: PBS

PBS 10%

DMSO .

결과 및 고찰

1. 추출조건의 결정

,

.

.

Inhibition rate (%)= 1

[ ]

× 100

[ ]

(461)

30 4 (2009 7 )

6

, ,

. Fig. 2

. 10

10 .

.

.

3

Fig. 3 .

85 .

85 ,

8

0 90 .

3

.

Fig. 1

,

.

Fig. 4 .

, n-buthanol

fraction .

100,000

.

Extraction Time(hrs)

Abso

rban

ce(60nm

)

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

00 2 4 6 8 10 12

2 5 7 10 15 20

Yield

(%

w/w)

35

30

25

20

15

10

5

0

Temperature(℃)

0 20 40 60 80 100 120

Fig. 2. Fig. 3.

(462)

Water Extract(WE)

Chloroform Phase(CP)

Ethylacetate Phase(EP)

n-Buthanol Phase(BP)

Water Phase(WP)

4.2%

17.3%

29.5%

49.5%

44.2%

35.8%

20.0%

UF1

UF2

UF3

Fig. 4.

1 :

7

,

.

.

15% 70%

,

.

2. 항산화 활성

DPPH

radical , free

radical

.

quercetin,

kaempferol, catechin flavonoids

, , , , , 21).

Table 1 .

50% radical IC50

, 100 3

0 2 .

radical 2

.

90

.

Table 2

. ethylacetate

,

Extraction Temperature(℃) IC50(㎍/㎖)30 12

50 13

70 16

80 17

90 19

100 25

* Soluble solids concentration was 2.1%(w/v) for each sample.

Table 1.

Fraction Name IC50(㎍/㎖)Inner part 18

Outer part 100

Water extract 19

Chloroform fraction >300

Ethylacetate fraction 7

n-Buthanol fraction 18

Water fraction 35

Ultrafiltration-1 13

Ultrafiltraion-2 47

Ultrafiltration-3 >300

* Solubles concentration was 2.1%(w/v) for each sample.

Table 2.

(463)

30 4 (2009 7 )

8

100,000Da

.

ethylacetate

,

.

.

ethylacetate

.

IC50( / ) 18 , 100

.

3. 성분함량의 비교

1) Polyphenol

Polyphenol tannin

, 21,22)

. flavonoids polyphenol

tannins ,

polyphenol23).

polyphenol Table 3

. polyphenol

, ethyl-

acetate fraction

. polyphenol

(hydrophobicity) flavo-

noid .

2

flavonoid ethylacetate

fraction24).

n-buthanol fraction , polyphenol

.

,

.

2)

Fraction NameYellow part(inner part)

Polyphenol(%, w/w)

Black part(outer part)

Polyphenol(%, w/w)

Water extract 12.4 15.5

Chloroform fraction 12.8 16.3

Ethylacetate fraction 36.2 31.6

n-Buthanol fraction 21.6 20.1

Water fraction 15.6 13.9

* Solubles concentration was 2.1%(w/v) for each sample.

Table 3.

Sugars Extract Acid hydrolyzed crude beta-glucan

Rhamnose 21.7 10.3

Fucose 26.8 2.1

Glucose 1.1 48.2

Arabinose 9.6 6.7

Xylose 3.5 11.3

Mannose 1.2 16.8

Galactose 36.1 4.6

Total(%) 100.0 100.0

Content of sugars in Ext.(%) 4.2 70.1

Table 4.

(464)

1 :

9

10% 9 ethanol

/ /

(Crude beta-glucan)

HPLC .

Table 4 .

,

.

galactose, fucose, rhamnose

, glucose, xylose, mannose,

rhamnose .

β-glucan

4.2%

,

70.1% .

30%

, .

3) Beta-glucan

Beta-glucan

β-glucan 1).

, Table 5

. β-glucan 1,3-1,6 1,3-1,4

,

. kit

1,3-1,6 .

β-glucan

(Table 5). β-glucan

40 50% ,

20%

.

β-glucan

4 12 ,

SOD(Superoxide dismutase) 3201).

4. 항암활성 비교

25,26).

Fraction Name Beta-glucan content(%, w/w)

Water extract 46.6

Chloroform fraction <1.0

Ethylacetate fractoin <1.0

n-Buthanol fraction 10.5

Water fraction 65.2

Ultrafiltration-1 10.5

Ultrafiltration-2 49.5

Ultrafiltration-3 33.0

Table 5.

Cell lines Cancer site Water extractChloroform

fraction

Ethylacetate

fraction

n-Buthanol

fraction

Water

fraction

AGS Stomach 4.8 215 1.5 21 6.5

HepG2 Liver 9.3 320 2.3 15 8.4

HeLa Cervical 6.5 299 4.4 31 9.9

Calu-3 Lung 3.1 189 2.9 8.4 5.9

Table 6.

(465)

30 4 (2009 7 )

10

. 1

.

(Cytotoxic effect)

MTT assay 50%

(IC50) .

Table 6

. chloroform fraction

. Ethylacetate fraction

ethylacetate fraction

.

, ethylacetate fraction27). n-Buthanol

fraction water fraction

, chloroform

fraction .

, water fraction

. fraction

(hydrophobicity)

1

.

, chloroform fraction

, ,

water fraction n-buthanol fraction

chloroform fraction

, ethylacetate fraction

fraction

.

.

Table 7

UF-1, UF-2 UF-3

, 100,000Da

UF-1

. (AGS) - 1.7mg/ , HepG2(

) - 1.4mg/ , HeLa( ) - 3.7mg/ ,

Calu-3( ) - 2.8mg/ ,

,

ethylacetate fraction

.

100,000

Da

. , 100,000 300,000Da

UF-2 300,000

UF-3 UF-1

, .

, tannin, phenol

,

.

Cell lines Cancer site Ultrafiltration-1 Ultrafiltration-2 Ultrafiltration-3

AGS Stomach 1.7 2.8 65

HepG2 Liver 1.4 3.5 88

HeLa Cervical 3.7 4.1 71

Calu-3 Lung 2.8 3.2 103

Table 7.

(466)

1 :

11

결 론

,

, ,

.

1.

10 90 2 2

.

2.

100,000Da

IC50 13 / .

3. ethylacetate

fraction IC50 7 /

.

4. polyphenol

25% .

5.

galactose, fucose, rhamnose

, ethanol

70% .

6. β-glucan

300,000Da 60%

.

7. chloroform

fraction

, ethylacetate fraction , IC50

1.5mg/ .

8.

100,000 Da

, , IC50

1.7mg/ , 1.4mg/ .

.

,

.

참고문헌

1. , , , ,

.

2003:10-35.

2. Shivrina AN. Chemical characteristics of compo-

unds extracted from Inonotus obliquus. Chem.

Abstr. 1967;66:17271-9.

3. Kier L. Triterpenes of Poria obliqua. J. Pharm.

Sci.. 1961;50:471-4.

4. Ludwiezak RS, Wrzeciono U. Forschungen uber

die cgemischen bestandteil de Inonotus obliquus.

IV. Ergosterol Rocz. Chem. 1975;34:1701-5.

5. Kahlos K, Hiltunen R. Identification of some

lanostane type triterpenes from Inonotus obliquus.

Acta. Pharm. Fenn. 1983;92:220-4.

6. Kukulyanskaya TA, Kurcheno NK, Kurcheno

VP, XBabitskaya VG. Physicochemical Properties

of melanins Produced by the Sterile Form of

Inonotus obliquus(“Chagi”) in Natural and

Cultivated Fungus. Applied Biochemistry and

Microbiology. 2002;38(1):58-61.

7. Chang HY.. Mycelial characteristics for the

artificial cultivation of Inonotus obliqua(Pers)

Pilot. Korean J. Plant Res. 2002;15(1):135-43.

8. Kahlos K, Toikka R, Hiltunen R. Effect of Some

Glucosamine Derivatives on the Production of

Fungal Volatiles of Inonotus obliquus in votro.

Planta Med. 1992;58, Suppl. Issue 1:A610.

9. Kahlos K, Tikka VH. Antifungal activity of

cysteine, its effect on C-21 oxygenated lanosterol

derivatives and other lipids in Inonotus obliquus,

in vitro. Appl. Microbiol. Biotechnol. 1994;42:

385-90.

10. Ham SS, Oh SW, Kim YK, Shin KS, Chang HY,

Chung GH. Antimutagenic and Cytotoxic Effects

of Ethanol Extract from the Inonotus obliquus.

J. Korean Soc. Food Sci. Nutr. 2003;32(7):1088

-94.

11. Cha JY, Jeon BS, Moon JC, Yoo JH, Cho YS.

(467)

30 4 (2009 7 )

12

Cytotoxic Effect of Inonotus obliquus Compo-

sition in HCT-15 Human Colon Cancer Cells and

AGS Gasteric Cancer Cells. J. Korean Soc. Food

Sci. Nutr. 2004;33(4):633-40.

12. Ra JH, Bae JH, Park HG, Kang KS. Studies on

a New Alimentotherapy for Diabetic Patients. J.

Korean Soc. Food Sci. Nutr. 2003;32(4):614-20.

13. Loviagina EV, Shivrina AN. On steroid compound

of the Chaga fungus. Biobimija. 1962;27:794-

800.

14. Kahlos K, Kangas L, Hitunen R, Antitumor

activity of some compounds and fractions frim

n-hexane extract of Inonotus obliquus. Acta.

Pharm. Fenn. 1987;96:33-40.

15. Ham SS, Oh SH, Kim YG, Shin KS, Chang HY,

Chung GH. Antioxidant and Genetoxic Activity

of Ethanol Extract from the Inonotus obliquus.

J. Korean Soc. Food Sci. Nutr. 2003;32(7):1071-5.

16. Song HS, Lee YJ, Kim SK, Moon WK, Kin DW,

Kim YS et al.. Downregulatory Effect of AGI-

1120(α-Glucosidase Inhibitor) and Chaga Mush-

room (Inonotus obliquus) on Cellular Active NF-

κB Activation and Their Anti-oxidant Activity.

Korean J. Pharmacogn. 2004;35(1):92-7.

17. Lee SJ, Seo DB. Chaga Mushroom has high

anti-oxidation capacity and redical removing

activity grown in the altitude 45 North. Food

Journal. 2004 Apr.;120-3.

18. Saitoh A, Sato C, Niiyama K. Anti mutagenic

Effects of Chaga. . 1996

Aug;15.

19. Ichimura T, Watanave O, Maruyama S. Inhibition

of HIV 1 protease by water soluble lignin

substance from an edible mushroom, Fuscoporia

obliqua. Biosci Biotechnol. Biochem. 1998;62:

575-7.

20. Chung GH, Composition containing Chaga Mus-

hroom extracts as effective component.. Korea

Pat. Publ.. 2003-0065964.

21. Lee KW, Kundu KJ, Kim SO, Chun KS, Lee HJ,

Surh YJ. Cocoa polyphenols inhibit phorbol

ester-induced superoxide anion formation in

cultured HL-60 cells and expression of cyclooxy-

genase-2 and activation of NF-kappaB and

MAPKs in mouse skin in vivo. Journal of

Nutrition. 2006;136(5):1150-5.

22. Joubert E, Winterton P, Britz TJ, Gelderblom

WCA. Antioxidant and pro-oxidant activities of

aqueous extracts and crude polyphenolic fractions

of rooibos(Aspalathus linearis). Journal of Agric-

ultural and Food Chemistry. 2005;53(26):10260-7.

23. Zongfu H, Wenli Y, Yaping Z. Studies on

scavenging of reactive oxygen species and

anti-lipis peroxidation by chlorogenic acid. Food

Science, China. 2006;27(2):128-30.

24. Banwart, WL, Porter PM, Granate TC, Hassett

JJ. HPLC Separation and wavelength area ratios

of more than 50 phenolic acids and flavonoids.

J. Chem. Ecol. 1985;11:383-95.

25. Hwang YJ, Noh GW, Kim SH. Effect of

Inonotus obliquus Extracts on Caspase-3 Activity

in Human Gastro-Intestinal Cancer Cell Lines.

The Korean Nutrition Society. 2003;36(1):18-23.

26. Cha JY, Chun BS, Park JW, Moon JC, Cho YS.

Effect of Fermented Compositions Containing

Inonotus obliquus with Houttuynia cordata on

Growth of Human AGS Gastric and HCT-15

Colon Cancer Cells. J. Korean Soc. Appl. Biol.

Chem. 2004;47(2):202-7.

27. Choi YH, Kim MJ, Lee HS, Hu S, Kwak SS.

Antioxidants in Leaves of Rosa rugosa. Korean

J. Pharmacogn., 1997;28(4):179-184.

(468)