Antioxidative Activities of Wen-pi-tang-Hab-Wu-ling-san (WHW$^{(R)}$) in vitro

서 론

,

1) . Superoxide, hydrogen peroxide, hydroxyl radical

, , , DNA

, , , ,

, , , , ,

, , ,

2,3) . Antioxidative Activities of Wen-pi-tang-Hab-Wu-ling-san (WHW ^® ) in vitro

Jin-Ki Jung, Yong-Ki Park

Department of Herbology, College of Oriental Medicine and Oriental Medicine Drug R&D Center, Dongguk University, Gyeongju 780-714, Republic of Korea.

Objectives: The objective of this study was to investigate the antioxidant effects of manufactured Wen-pi-tang-Hab- Wu-ling-san (WHW

) in vitro.

Methods: WHW

was prepared by the pilot manufacture of WHW water extract from a GMP system appointed company. Antioxidative activities were determined by in vitro tests as follows: the scavenging activities of oxygen free radicals including 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, superoxide anion, hydrogen peroxide and nitric oxide radicals, as well as ferrous ion chelating capacity and Trolox equivalent antioxidant capacity (TEAC).

Results: WHW

significantly scavenged oxygen free radicals such as DPPH (IC

=115.28 ± 0.25 / ), superoxide anion (IC

=8.56 ± 0.08 / ), hydrogen peroxide (IC

=240.36 ± 3.41 / ) and nitric oxide (IC

=162.28 ± 0.21 / ) radicals. WHW

also showed ferrous ion chelating activity (IC

=543.19 ± 4.85 / ) and Trolox equivalent effects (IC

=45.311 / ) in TEAC and ORAC assay, respectively.

Conclusion: This study demonstrates that WHW

has strong antioxidative properties through free radical scavenging activity. These data suggest that WHW

be used as an antioxidant agent.

Key Words : Traditional Korean medicine, Wen-pi-tang-Hab-Wu-ling-san, antioxidation, free radicals

加減溫脾湯合五 散 完製

정진기, 박용기

동국대학교 한의과대학 본초학교실동국대학교 한방신약개발센터

Original Article

Received：3 July 2009 Revised：31 August 2009 Accepted：31 August 2009

Correspondence to：박용기(Yong-Ki Park)

경북 경주시 석장동 707번지 동국대학교 한의과대학 본초학교실

Tel：+82-54-770-2661, Fax：+82-54-770-2281, E-mail：[email protected]

4,5) .

6) .

.

(Wen-pi-tang-Hab-Wu-ling- san; WHW)

.

7)

8-11)

,

9-12)

.

creatinine BUN

13) , TGF-β

14) ,

creatinine ¹⁵⁾

.

16) ,

17) , ¹⁸⁾ ,

19) .

TGF-β

20) ,

21,22)

, ²³⁾

.

WHW (WHW ^® )

in vitro ,

.

재료 및 방법

1. 材料 1)

WHW ^® 1

( ) ( , )

. 2)

1,1-diphenyl-2-picrylhydrazyl (DPPH), nitro blue tetrazolium salt (NBT), phenazine methosulfate (PMS), dimethyl sulphoxide (DMSO), thiobarbituric acid (TBA), trichloro acetic acid (TCA), sodium nitroprusside, sulphanilic acid, N-(1-Naph thyl) ethylene diaminedi hydrochloride, Ethylene dia -minetetra acetic acid (EDTA), 2,2’-azino-bis(3-ethyl ben zthio zi o zline-6-sulphonic acid (ABTS), FeSO 4

7H 2 O, deoxyribose, 2,2’-azobis(2-amidinopropane) dihydro chloride (AAPH), and trolox (6-hydroxy-2,5,7,8 -tetra methylchroman-2-carboxylic acid), vitamin C

Sigma (St. Louis, MO, USA) .

2. 方法 1) WHW ^®

WHW ^® 1 14

GMP ( ) (

, ) 50 3 Batch

Pilot . WHW ^®

24% , 4 3

. 2) DPPH radical assay

WHW ^® DPPH Gyamfi ²⁴⁾

. ,

2 absolute ethanol 0.2 mM

DPPH 1 37 30

. 517 nm

% .

3) Superoxide radical scavenging assay WHW ^® superoxide radical(O 2 -

)

Robak ²⁵⁾ . ,

1 20 μM PMS, 156 μM NADH, 25 μM nitro blue tetrazolium 0.1M phosphate buffer(pH

7.4) 1.9 2 .

560 nm .

4) Nitric oxide radical assay

WHW ^® nitric oxide radical(NO-)

Marcocci ²⁶⁾ . ,

sodium nitropusside 10 mM sodium nitropr- usside 20 mM phosphate buffer(pH 7.4)

. 2 0.5 sodium nitropr-

usside 25 150

. 0.5 sulphanilic acid reagent 1

5 1 Griess Illosvoy

(0.1% naphthylethylenediamine dihydrochloride)

30 .

540 nm .

5) Hydrogen peroxide decomposition assay WHW ^® hydrogen peroxide(H 2 O 2 )

Marcocci ²⁶⁾ . ,

1 4 80 mM H 2 O 2 5

phosphate buffer . 1

2 dichromate/aceticacid 10

532 nm .

6) Ferrous ion chelating capacity assay

WHW ^® (ferrous ion chelating

capacity) Decker ²⁷⁾ .

5 0.1 2 mM FeCl2 0.2

5 mM ferrozine

methanol 10

. Fe2+/ferrozine 562 nm

. (ferrous ion chelating capacity; %)

.

藥材名 학명

黨參 Codonopsis pilosula (Fr.) N

.

丹蔘 Salvia miltiorrhiza B

.

半夏 Pinellia ternata (Thunb.) Breitenbach

黃連 Coptis japonica M

.

吳茱萸 Evodia rutaecarpa (Juss.) B

.

淫羊藿 Epimedium koreanum N

大黃 Rheum palmatum L.

紫蘇葉 Perilla frutescens var. acuta K

甘草 Glycyrrhiza uralensis F

. 茵蔯 Artemisia capillaris T

.

茯 Poria cocos W

白朮 Atractylodes japonica K

.

猪 Polyporus umbellatus F

桂枝 Cinnamomum cassia P

Table 1.

7) Trolox equivalent antioxidant capacity(TEAC) assay

WHW ^® TEAC ²⁸⁾

. , pH

2,2’- azinobis(3-ethylbenzothiazo-thiazoline-6-sulfonate)

(ABTS) H 2 O 2 ABTS+

,

ABTS+ .

TEAC 2.45 mM K2S2O8

7 mM ABTS

12-48 . ascorbic

acid , Trolox

. , 1980 ABTS ⁺

6

734 nm . TAC

nmol Trolox equivalent . 8) Oxygen radical absorbance capacity(ORAC)

assay

ORAC assay Cao ²⁸⁾

. fluorescein , peroxyl

radical 2,2’-azobis(2-amidinopropane) di

hydrochloride(AAPH) . 20

16.7 nM β-phycoerythrin 120

15 32 mM

AAPH 60 excitation 485 nm

emission 530 nm 2 60

. 0, 0.02, 0.2,

1.0 2.0 nmol Trolox ,

AUC (area under the curve)

. ORAC AUC

nmol (AUCnet=

AUCsample/standard_AUCblank).

9)

50% 3

(mean) ± (standard

deviation; SD) ,

GraphPad Prism 4.0(GraphPad Software, San Diego, CA, USA) one-way analysis

p 0.05

.

결 과

1. DPPH radicals 소거 효과

WHW ^® DPPH

70 60 50 40 30 20 10 0

100 150 200 250 500 750 1000 Vit-C WHW (㎍/ml)

IC

=115㎍/ml

D P P H ra d ic al sc av en g in g ( % )

Fig. 1.

radicals radicals

. WHW ^®

1000 / 68.84 ± 0.53% vitamin-C

10 / 69.41 ± 0.47%

(IC 50 =115.28 / , Fig. 1).

2. Superoxide radicals 소거 효과 superoxide radical(O 2

- )

ROS

proton perhydroxyl

radical(H2O ) dismutation H 2 O 2

. WHW ^® O 2

- radicals , WHW ^®

O 2

- radicals

(IC 50 =8.564 / ; Fig. 2). WHW ^® 1000 / O 2

- radicals 94.83 ± 10.99%

, vitamin-C 10 / 50.22 ± 24.57%

**

***

***

***

***

45 40 35

20

10

0 5

WHW (㎍/ml) IC

=162.28㎍/ml

10

N itr ic o xid e ra d ic al sc av en g in g (% )

25 75 100 Vit-C

30 25

15

25

50 150

Fig. 3.

* *

125

100

75

50

25

0 50

WHW (㎍/ml) IC

=115㎍/ml

100

S up er o xid e ra d ic al sc av en g in g (% )

250 500 750 Vit-C

Fig. 2.

O 2

- radicals .

3. Nitric oxide radicals 소거 효과 Nitric oxide(NO)

ROS

1,2) . WHW ^® NO radicals (Fig. 3), WHW ^®

NO radicals

(IC 50 =162.28 / ). WHW ^® 100 /

NO radicals 38.11 ± 2.33% ,

vitamin-C 10 / 19.36 ± 0.74%

.

4. Hydrogen peroxide 소거 효과 Superoxide radical(O2 -) H 2 O 2

* **

* *

* 70

60

30 20

0 10

WHW (㎍/ml) IC

=543㎍/ml

F e

ch ela tin g a ct iv ity ( % )

50 40

10

50 100 200 500 1000 Vit-C

Fig. 5.

***

*** ***

*** ***

70 60

30 20

0 75

WHW (㎍/ml) IC

=243㎍/ml

H

O

sc av en g in g ( % )

50 40

10

100 150 250 500 Vit-C

Fig. 4.

Haber-Weiss Fenton

OH .

WHW ^® superoxide radical

, WHW ^® 250 / 500 /

OH 50%

(IC 50 =240.36 / , Fig. 4).

5. 금속이온 제거 효과

NO heme (Fe) ferrous

(autooxidation)

. WHW ^®

(Fig. 5), WHW ^® , 0.25 / 0.5 / 57.41 ± 1.91% 60.72± 0.266%

6

3

0 10

WHW (㎍/ml) WHW 1000 mg = 496.837 mmol

O xy g en ra d ic al ab so rb an ce c ap ac ity (n m o l Tr o lo x eq uiv ale n t)

5

2

25 75 150 500 Vit-C

4

1

Fig. 7.

100

50

0 20

WHW (㎍/ml) IC

=45.311㎍/ml

Trolox value = 25.551

A B TS ra d ic al in hib itio n A ct iv ity ( % )

75

25

50 100 200 500 1000 Vit-C

Fig. 6.

(IC 50 =543.19 / ).

6. Trolox equivalent antioxidant capacity(TEAC) assay

WHW (total antioxidant capacity; TAC) ABTS radical inhibition activity

TEAC assay . WHW ^®

TAC 20, 50, 100, 200, 500 1000 /

32.24 ± 7.82%, 53.84 ± 6.05%, 92.42 ± 0.16%, 94.36 ± 0.83%, 95.60 ± 1.11% 96.73 ± 0.12%

(IC 50 =45.311 / ; Fig. 6). WHW ^®

100 / vitamin C TAC

.

7. Oxygen radical absorbance capacity(ORAC) assay

TEAC ORAC assay AUC

free radical

.

WHW ^® ORAC , WHW ^® 25, 75,

150 500 / 4.77 ± 0.053, 4.97 ± 0.154, 5.07 ± 0.148 5.10 ± 0.019 nmol ORAC

(Fig. 7), WHW ^® ORAC

(Y=4.715+ 0.1052X) , WHW ^®

1000 / 496.83 nmol .

고 찰

free radical ,

, , , DNA

, ,

, , ,

1-5) .

6) . BHT, BHA

α-tocopherol, vitamin C, carotenoids, flavonoids,

SOD

. ,

2) .

. (WHW)

.

.

TGF-β1

19) ,

20,21)

,

22) ,

23) .

WHW

WHW ^® in vitro

. Free radical

superoxide radical(O2·), hydroxyl radical(OH·), hydrogen peroxide(H 2 O 2 ), nitric oxide radical(NO·), singlet oxigen( ¹ O 2 )

3,4) . free radical

, (SOD)

(GSH, vitamin C, vitamin E, , ,

, ) .

free radical

, SOD GSH

superoxide anion radical

25,30)

.

,

5,6) . WHW ^® DPPH, super- oxide, nitric oxide free radicals

. WHW ^®

hydrogen peroxide

. 1980

vitamin E

Trolox total radical trapping

antioxidant parameter(TRAP) ³¹⁾ .

AUT

ORAC TEAC . ,

ROO OH

phycoerythrin (PE) PE

Trolox TAC Trolox equivalent

. WHW ^®

ORAC TEAC assay , WHW ^®

.

WHW ^® free radicals

, WHW ^®

.

WHW ^{® 32)} , ³³⁾ , ³⁴⁾ ,

35) ,

36) .

37) gentamicin sulfate

38)

.

39) .

WHW ,

WHW

WHW ^® ,

.

결 론

WHW (WHW ^® )

in vitro .

1. WHW ^® DPPH radicals (IC 50 =115.28 / ).

2. WHW ^® nitric oxide radicals (IC 50 =162.28 / ).

3. WHW ^® superoxide radicals (IC 50 =8.56 / ).

4. WHW ^® ferrous ion chelating activity (IC 50 =543.19 / ).

5. WHW ^® 1000 /

496.83 nmol .

감사의 글

(B070044) .

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