Studies on the Anti-cancer Activity of Chaga Mushroom Extract

(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：[email protected]

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(herb) .

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²⁾

, polyphenol

²⁾

, terpenoid, inositol, inotodiol

³⁾

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, butulin

⁵⁾

, melanin

⁶⁾

. β-glucan

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16,17)

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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

(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) = (C

abs

- S

abs

)/C

abs

100 - C

abs

: Negative contol( ethanol )

- S

abs

: Sample

- Positive control: Vitamin E(Fluka, 95240) .

5. Polyphenol 의 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

UF1

UF2

UF3 [UF Fraction]

CHCI

3

Fig. 1.

(4)

2 0.2 Folin ciocalteu’s phenol reagent

3 . 3 Na

2

CO

3

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-glucan 의 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 )

(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(KH

2

PO

4

2.1g/

, NaCl 90g/ , Na

2

HPO

4

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 × 10

⁵

cells/ 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

[ ]

(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)

A b so rb an ce (6 0n m )

0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

0 2 4 6 8 10 12

2 5 7 10 15 20

Y ie ld (%

̢

w/

w ) 35 30 25 20 15 10 5 0

Temperature(℃)

0 20 40 60 80 100 120

Fig. 2. Fig. 3.

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.

(7)

,

.

15% 70%

, .

2. 항산화 활성

DPPH

radical , free

radical

. quercetin, kaempferol, catechin flavonoids

, , , , ,

21)

.

Table 1 .

50% radical IC

50

, 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.

(8)

100,000Da .

ethylacetate ,

.

ethylacetate .

IC

50

( / ) 18 , 100

.

3. 성분함량의 비교

1) Polyphenol Polyphenol tannin

,

21,22)

. flavonoids polyphenol

tannins ,

polyphenol

²³⁾

.

polyphenol Table 3

. polyphenol

, ethyl-

acetate fraction

. polyphenol

(hydrophobicity) flavo-

noid .

2 flavonoid ethylacetate

fraction

²⁴⁾

.

n-buthanol fraction , polyphenol

. ,

.

2)

Fraction Name Yellow 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.

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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) 320

1)

.

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

Table 5.

Cell lines Cancer site Water extract Chloroform 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.

(10)

. 1

. (Cytotoxic effect)

MTT assay 50%

(IC

50

) .

Table 6

. chloroform fraction

. Ethylacetate fraction

ethylacetate fraction .

, ethylacetate fraction

27)

. 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.

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결 론

,

, ,

. 1.

10 90 2 2

. 2.

100,000Da

IC

50

13 / .

3. ethylacetate

fraction IC

50

7 /

.

4. polyphenol

25% .

5. galactose, fucose, rhamnose , ethanol

70% .

6. β-glucan

300,000Da 60%

.

7. chloroform

fraction

, ethylacetate fraction , IC

50

1.5mg/ .

8. 100,000 Da

, , IC

50

1.7mg/ , 1.4mg/ .

.

,

.

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