Preventive Effects of Peony Root Extracts on Oxidative Stress, Thrombosis and Atherosclerosis

(1)

서 론

, , ,

.

(reactive oxygen species; ROS) (reactive nitrogen species; RNS)

(oxid-

박순기², 이민자¹, 정현정^1,2, 이혜숙¹, 김혁^1,2, 나선택², 박선동^2,3, 박원환^1,2

1동국대학교 심혈관질환 기초의과학센터, ²동국대학교 한의과대학 진단학 교실, ³동국대학교 한의과대학 방제학 교실

Original Article

Preventive Effects of Peony Root Extracts on Oxidative Stress, Thrombosis and Atherosclerosis

Soon-Gi Park², Min-Ja Lee¹, Hyun-Jung Jung^1,2, Hye-Sook Lee¹, Hyuck Kim^1,2, Sun-Taek Na², Sun-Dong Park^2,3, Won-Hwan Park^1,2

1Cardiovascular Medical Research Center and ²Dept. of Diagnostics,

3Dept. of Prescriptionology, College of Oriental Medicine, Dongguk University

Objectives: There is currently increased interest in the identification of natural antioxidant compounds derived from various plants. Peony Root (PR) is used worldwide for the treatment of many types of cardiovascular disease including atherosclerosis and hypertension. It has been used in Korean traditional medicine for the treatment of glycosuria, hypertension and cancer. However, to date, no studies concerning the antioxidant properties of PR have been conducted. Therefore, this study was conducted to evaluate the in vitro scavenging activity, inhibitory effect of LDL oxidation of pro-oxidant reactive species and anti-thrombosis effect in response to treatment with PR using various screening methods including biological and non-biological oxidants.

Methods: In this study, the antioxidant activity of extract from PR was studied with in vitro methods by measuring the antioxidant activity by TEAC, measuring the scavenging effects on reactive oxygen species (ROS) [superoxide anion, hydroxyl radical] and on reactive nitrogen species (RNS) [nitric oxide and peroxynitrite] as well as measuring the inhibitory effect on Cu²⁺-induced human LDL oxidation and the inhibitory effect on collagen-induced platelet aggregation.

Results: The PR extracts were found to have a potent scavenging activity of oxidative stress [DPPH, superoxide anion, hydroxyl radical, nitric oxide and peroxynitrite, etc.] as well as an inhibitory effect on LDL oxidation and on platelet aggregation.

Conclusions: The PR extracts have anti-oxidative and anti-atherosclerotic effects in vitro system, which can be used for developing pharmaceutical drugs against oxidative stress and atherosclerosis.

Key Words : Peony Root (PR), anti-oxidative, anti-atherosclerotic, cardiovascular diseases

접수：2008년 10월 9일 수정：2009년 3월 9일 채택：2009년 3월 11일

교신저자：박원환(Won-Hwan Park)

경주시 석장동 707번지 동국대학교 심혈관 센터, 한의과 대학 진단학 교실

Tel：+82-54-770-2373, Fax：+82-54-770-2376, E-mail：[email protected]

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ative stress) (nitrosative stress) ,

, DNA

, , , ,

1,2)

. ,

1 .

,

, ,

LDL (low density lipoprotein;

) (chol-

esteryl ester) (fatty streak)

3)

. LDL

3-5% triacyl glyceride, 40-44%

, 20-24% phospholipid, 9-

10% 20-26%

. LDL in vivo

LDL

. LDL ,

,

4)

.

5)

.

LDL LDL

. LDL

6)

. ,

LDL .

LDL BHT, BHA

probucol

.

α- C

7)

.

. ,

.

,

.

, , .

streptokinase, urokinase

,

30

⁸⁾

.

,

. tPA (tissue-type plasminogen activator)

( )

(3)

. 2-4

9)

. ,

,

.

( , Peony Root, PR) Paeonia lactiflora PALL.

,

. paeoniflorin, paeonol, peaonin,

, , , tannin, , , ,

, peaoniflorin . ,

PR

, , , ,

, ,

. PR ,

10)

. PR

11)

, Cytochrome P450

12)

, resv-

eratrol tyrosinase lipoxygenase

13)

.

,

LDL in vitro

,

, .

재료 및 방법

1. 재료 1)

Organic solvents, 2,2 -Azinobis (3-ethylbenzothia- zoline-6-sulphonate) diammonium salt (ABTS), DPPH (α,α'-diphenyl-β-picryl-hydrazyl), phosphate buffered saline tablets (PBS) low-density lipoprotein (LDL), fibrinogen, thrombin Merck (Darmstadt, Germany) , sodium chloride (NaCl), potass- ium chloride (KCl), dihydrorhodamin 123 (DHR 123), thiobarbituric acid (TBA), trichloroacetic acid (TCA), copper sulfate (CuSO

4

), sodium borate, hydrochloride (HCl), 4,5-Diaminofluorescein (DAF-2), 3-morpholi- nosydnonimine hydrochloride (SIN-1), sodium dodecyl sulfate (SDS), collagen, eugenol sodium citrate Sigma Chemical Co. (St. Louis, MO, USA)

. 6-carboxy-2',7'-dichlorofluorescein diacetate (DCFH-DA) Molecular Probes (Eugene, OR), Trolox

Fluka BioChemika (Buchs, Swizerland) , peroxynitrite Cayman Chemical Co. (Ann Arbor, MI,

USA) .

1 .

2)

2006

( ) ( , )

.

100 g 3 L round flask

2 L 3

. 1 mm-pore-size filter (Whatman No. 2) . Buchi R rotary vaccum evaporator (Flawil, Switzerland)

6.94 g ( : 6.94%).

(4)

2. TEAC (Trolox Equivalent Antioxidant Capacity) assay 에 의한 항산화 효과의 측정

PR TEAC

. 10 μL PR (1 mg/mL)

trolox standard ABTS

⁺

1.0 mL

1 Amersham Bioscience UV/Visible spectr- ophotometer (Buckinghamshire, UK) 734

nm . 6 monitoring

1 mM trolox

trolox standard curve

TEAC .

3. DPPH 에 의한 유리기 소거 효과의 측정 PR

DPPH (α,α'-diphenyl-β-picryl-hydrazyl) 16 mg 100

mL 100 mL

2.5 mL

PR 0.5 mL 528 nm

Molecular Device microplate reader (Sunnyvale,

CA, USA) .

4. Superoxide anion 소거 효과의 측정 PR superoxide anion

PR , 30 mM EDTA (pH 7.4), 50 mM NaOH 30 mM hypoxanthine

1.42 mM nitro blue tetrazolium (NBT) 30 μL, 100 μL, 10 μL, 200 μL

. 3 0.5

U/mL xanthine oxidase 100 μL 50 mM phosphate buffer (pH 7.4)

3 mL . 20

560 nm microplate reader .

5. DCFDA assay 에 의한 hydroxyl radical 소거 효과의 측정

PR OH

2´,7´-dichlorodihydofluorescein diacetate (DCFDA)

. DCFDA

esterase

2´,7´-dichlorodihydrofluorescein (DCFH) , DCFH

2´,7´-dichlorofluorescein (DCF)

. 10 μL menadione 10

μL potassium phosphate buffer (pH 7.4) 130

μL OH , 125 μM DCFDA

esterase DCFDH 50 μL

20 .

excitation wavelength 485 nm emission wavelength 530 nm Molecular Device fluo- rescence microplate reader (Sunnyvale, CA, USA)

.

6. NO radical 소거 효과의 측정

PR NO radical DAF-2 assay

. PR

NO indicator 4,5-diaminofluorescein (DAF-2)

2 NO

490-495 nm green

triazolofluorescein . DAF-2

NO . 1 mg DAF-2

550 μL DMSO , 50 mM

phosphate buffer (pH 7.4) 1 : 400

. NO SIN-1 DAF-2 96 well plate , DAF-2 NO

10 excitation wave length 485 nm emission wave length 530 nm fluorescence

microplate reader .

7. Peroxynitrite 소거 효과의 측정

PR peroxynitrite

96-well microplate PR

90 mM NaCl, 5 mM KCl 100 μM dieth- ylenetriaminepenta acetic acid 10 μM DHR 123

sodium phosphate buffer (pH 7.4)

(5)

. 10 μM ONOO fluorescence microplate reader excitation 500 nm, emi-

ssion 536 nm . ONOO

peroxynitrite SIN-1

superoxide anion nitric oxide ONOO

.

8. RT-PCR 에 의한 항산화효소계 활성 측정

PR HepG2 24

RNA .

1X PBS , 1 mL total

RNA isolation reagent 5 . 0.2 mL chloroform

10 , 4 , 12,000 rpm 15 min

.

0.5 mL isopropanol

10 . 12,000 rpm 8

RNA pellet ,

DEPC 75% 1 mL 10,000

rpm 5 .

RNA 10 ,

50 μL DNase/RNase

UV/Visible spectrophotometer total RNA

.

Promega (Madison, WI, USA) man- ufacture DNase

,

. 2 μL RNA DNase

DNA 37 30

. 75 DNase

.

RNA oligo dT primer 70

5 4 5

. RNA inhibitor (1 unit/μL)

15 μL oligo dT

5 μL RNA 25 5

anneal , 42 60 min extension

70 15 heat-inacti-

vate . cDNA polymerase

chain reaction (PCR) . PCR primers (Table 1).

9. Relative Electrophoretic Mobility (REM) Assay

Human LDL REM agarose gel electrophoresis PBS (pH 7.4) LDL (120 μ

g/mL) PR , 10 μM CuSO

4

37 12 ,

Genes Sequences Primers

Cu Zn SOD 5'-AGGGCATCATCAATTTCGAG-3' sense

5'-ACATTGCCCAAGTCTCCAAC-3' antisense

GPx 5'-GTACGGAGCCCTCACCATT-3' sense

5'-AGCCCAGAATGACCAGACC-3' antisense

GR 5'-TTACTGCAGTTCCCGGTAGG-3' sense

5'-CAGCAGCTATTGCAACTGGA-3' antisense

Catalase 5'-CCCTCTCATCCCAGTTGGT-3' sense

5'-TAGTTGGCCACTCGAGCAC-3' antisense

GAPDH 5'-GGAAGGACTCATGACCACAG-3' sense

5'-TTGGCAGGTTTTTCTAGACG-3' antisense

Table 1.

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3 μg LDL 0.7% agarose gel loading TAE buffer (40 mM Tris, 40 mM acetic acid, and

1mM EDTA) 85 V 1

. Gel Coomassie brilliant blue

R-250 , inhibition ratio (%)

.

10. Human LDL 산화 억제효과의 측정

PR human LDL

human LDL 0.5 mL (120 μg/mL) 10 mM PBS buffer (pH 7.4) 1.5 mL, 0.1 mM CuSO

4

40 μL 40 μL 37

30 . 20% TCA 1.5 mL,

0.05 M NaOH 0.67% TBA 1.5 mL

. 90 , 45 water bath

2,000 ×g 5 .

532 nm

IC

50

.

11. Fibrinolytic clear zone 의 측정

PR human fibrin

plate . 100 mm petri dish

thrombin (50 unit/mL) 100 μL ,

0.1 M phosphate (pH 7.5) buffer 0.5%

(w/w) 5 mL

.

30 .

PR 0.2 g

37 20 lysis zone

.

12. 혈소판 응집 억제활성의 측정

PR

Chlono Log aggregometer (Havertown, PA, USA) impedance . , Sprague Dawley (SD) male rat 3.8% sodium citrate

Becton Dicknson vaccutainer (Mountain

View, CA, USA) , 1,000 rpm 10

. (platelet rich plasma,

PRP) 2,300 rpm 10

(platelet rich pellet) washing buffer

(pH 6.5) , 2,300 rpm 10

. (platelet poor plasma, PPP) , suspending buffer (pH 7.4)

2,300 rpm 10 (washed

platelet) .

4 5×10

⁸

cells/mL . Washing buffer 138 mM NaCl, 2.7 mM KCl, 12 mM NaH

2

CO

3

, 0.36 mM NaH

2

PO

4

, 5.5 mM glucose 1 mM EDTA

, suspending buffer 138 mM NaCl, 2.7 mM KCl, 12 mM NaH

2

CO

3

, 0.36 mM NaH

2

PO

4

, 5.5 mM glucose, 0.49 mM MgCl

2

0.25% gelatin

. Cuvette PRP 400 μL, agonist (collagen) 50 μL, sample 10 50 μL ,

. collagen

aggregation (%) (A) , PR aggregation (%)

(B) inhibition (%)

.

A: control aggregation (%), B: sample aggregation (%)

13. 통계처리

SPSS version 14.0 for Windows (Chicago,

(7)

IL, USA) program

one-way

ANOVA p<0.05

Tukey test .

결과 및 고찰

1. TEAC assay 에 의한 항산화 효과

PR TEAC

Table 2 . PR TEAC

0.051±0.001 . TEAC

2,2 -azinobis (3-ethylbenzo-thiazoline 6-sulfonate)

. E analogue trolox

ABTS

⁺

TEAC

ABTS 650, 734, 820 nm

ABTS

⁺

potassium

persulfate ABTS

¹⁴⁾

.

Maria

¹⁵⁾

28 TEAC ,

TEAC 0.406, 0.192,

0.187, 0.163 TEAC

0.001 . Ginko biloba flavonol Camellia sinensis catechin TEAC Pietta

16)

TEAC

0.07 1.25 PR

.

2. DPPH 에 의한 유리기 소거 효과 ,

,

.

. ,

. DPPH (1,1-diphenyl-2-picrylhydrazyl) radical

17)

. DPPH

hydroxyl radical

. DPPH PR

Table 3 .

PR IC

50

450.19±

13.28 AA BHT

.

Sample Antioxidant effect

PR^a (1 mg/mL)

Positive control Trolox (1 mg/mL)

TEAC (mM Trolox equivalent) 0.051±0.001^b 1

aPR, water extract of Peony Root.

bEach value represents the mean ± SE of triplicate measurements.

Table 2.

Sample

Antioxidant effect PR^a Positive control

AA BHT

DPPH radical scavenging activities (IC50=μg/mL) 450.19±13.28^b 162.80±9.64 191.15±10.12

aPR, water extract of Peony Root.; AA, ascorbic acid.

Table 3.

(8)

,

18)

.

3. Superoxide anion 소거 효과 PR superoxide anion

NBT . , xanthine

oxidase hypoxanthine superoxide anion NBT

diformazan 560 nm

. , superoxide anion , NBT

diformazan .

SOD-

3,19)

. PR

superoxide anion Table

4 IC

50

0.45±0.01 mg/mL

AA (IC

50

=1.22 ±0.02 mg/mL)

superoxide anion .

4. Hydroxyl radical 소거 효과

.

, ,

ROS ROS

1,2)

.

cyclooxygenase prosta-

glandins lipoxygenase

leukotrienes hydroxyl radical

.

20)

. hydroxyl radical

, Haber-Weiss Fenton reduction

Fenton reduction hydroxyl radical

21)

. Hydroxyl radical ,

, DNA

thiamin guanosine

. , hydroxyl

radical

. Dye

Sample

AA BHT

Superoxide anion scavenging activities (IC50=mg/mL) 0.45±0.01^b 1.22±0.02 NA^c

cNA is not active.

Table 4.

Sample

AA BHT

Hydroxyl radical scavenging activities (IC50=μg/mL) 39.96±0.97^b 59.79±2.96 NA^c

cNA is not active.

Table 5.

(9)

ester DCFDA DCFDA assay microsome

ferritin ,

22)

. PR hydroxyl radical IC

50

39.96±0.97 μ

g/mL AA

(IC

50

=59.79±2.96 μg/mL)

(Table 5), PR hydroxyl

radical microsome

.

5. NO radical 소거 효과

NO (Nitric oxide; ) ,

L-arginine nitric oxide synthase

(NOS) citruline .

NO intercelluar messenger

,

.

superoxide anion

peroxynitrite (ONOO ) . NO

, ,

, , , , ARDS (

), AIDS , , ,

23)

. NOS const- itutive NOS (cNOS)

inducible NOS (iNOS)

. cNOS neuronal NOS

(nNOS) endothelial NOS (eNOS) .

nNOS eNOS Ca

²⁺

calmodulin NO .

nNOS

NO . eNOS

NO . iNOS Ca

²⁺

calmodulin

,

lipopolysaccharide (LPS), interferon-g (IFN-g), Alzheimer’s amyloid peptide

iNOS . iNOS ,

NO NO

24)

. PR

nitrosamine NO radical

Table 6 . PR

(IC

50

= 4.65±0.03 μg/mL) NO

radical ,

AA (IC

50

=59.79±2.96 μg/mL) PR . Lee

²⁵⁾

diethylether

68.34% 68.23% NO .

6. Peroxynitrite 소거 효과

Peroxynitrite superoxide anion NO

L-tyrosine tyrosine

nitration

Sample

AA BHT

Nitric oxide radical scavenging activities (IC50=μg/mL) 4.65±0.03^b 59.79±2.96 NA^c

bEach value represents the IC50, mean ± SE of triplicate measurements.

cNA is not active.

Table 6.

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. Peroxynitrite superoxide anion NO

peroxynitrite

.

, peroxynitrite , ,

,

, in vivo . in

vitro in vivo

, , DNA

, , ,

,

²⁶⁾

. In vitro peroxyn- itrite D(-)penicillamine ebselen

, , , ,

ascorbic acid, α -tocopherol, flavonoids, polyhydroxyphenol

27)

. peroxynitrite PR

RNS

. PR peroxy-

nitrite , PR (IC

50

=

5.55±0.36 μg/mL) peroxynitrite

AA (IC

50

=2.54±0.04 μg/mL) , PR peroxynitrite

(Table 7).

7. RT-PCR 에 의한 항산화효소계 활성

ROS RNS

Sample

AA BHT

Peroxynitrite scavenging activities (IC50=μg/mL) 5.55±0.36^b 2.54±0.04 572.41±28.90

Table 7.

Human β-actin

Cu Zn SOD

GPx

GR

Catalase

1 2 3 4 5 6 7 8 - - - + + + - + + - + + H2O2

PR

Fig. 1.

μ μ

(11)

, ,

, , , ,

2)

. ROS RNS , , DNA,

1)

.

(SOD, GPx, GR, catalase, thiol enzyme) (glutathione, AA, tocopherol, phenolic compound, phenylpropanoids, carotenoids, flavonoids)

28)

. ROS RNS

, redox signal transduction

29)

. , ,

30)

. HepG2 H

2

O

2

PR

mRNA

. , Fig. 1 C

u Zn-SOD, GR catalase mRNA PR

, GPx .

PR

.

8. Relative Electrophoretic Mobility (REM) Assay

LDL

, phospholipids, free cholesterol, triglyceride B-100 (apo B-100)

. Apo B-100 500 kDa

single peptide chain . Mon-

omeric protein

. LDL

LDL apo B-100

, ROS RNS apo B-100 14 kDa 500 kDa apo B-100 . In vitro

1 2 3 4

A AA A

Relative electrophoreticmobility(protection %) 120 100

80 60

40 20

0

Native LDL Ox-LDL W-1 W-10

BBBB Fig. 2.

μ μ

(12)

conjugated diene thiobarbituric acid- reactive substance (TBARS)

.

Fenton Haber-Weiss

Cu

²⁺

ROS

, apo B-100

31)

. REM apo B-100

, REM LDL

. Fig. 2 Cu

²⁺

LDL REM PR

. Native LDL REM 1

, Cu

²⁺

REM 2.67

, PR 1, 10 μ

g/mL 2.44, 1.33 . , 10

μg/mL 80.0% LDL

AA (85.7%)

. PR

LDL PR

radical chain .

9. Human LDL 산화 억제효과 LDL

LDL ,

4,6)

.

LDL , ,

LDL LDL

5)

. LDL

,

LDL . ,

LDL ,

, (fibrous plaque), (complicated lesion)

5)

.

CuSO

4

-LDL PR

Table 8

PR IC

50

96.18±5.34 μg/mL

AA (IC

50

=21.09±0.62 μg/mL) BHT (IC

50

=26.31±0.22 μg/mL) ,

. Katsube

³²⁾

flavonoid glycoside LDL

lag phage

. PR

LDL

.

10. Fibrinolytic clear zone 의 측정

.

Sample

AA BHT

Inhibitory effect on Cu²⁺- induced LDL oxidation (IC50=μg/mL) 96.18±5.34^b 21.09±0.62 26.31±0.22

Table 8.

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urokinase, sterptokinase tPA

8,9)

. -

.

,

.

33)

,

³⁴⁾

,

³⁵⁾

,

³⁶⁾

, ,

.

PR fibrin

plate fibrin clot

(Fig. 3). , 20

fibrin . 15

20 1,324 mg

. , PR

.

11. 혈소판 응집 억제활성의 측정

,

100

80

60

40

20

0

Aggregation (%)

1 2 3 4 5 6

Fig. 4.

μ μ μ μ μ μ μ

μ μ μ μ μ μ μ μ μ

μ μ μ μ μ μ μ μ μ μ

Fig. 3.

(14)

.

,

37)

.

,

phospholipase

A

2

.

cyclooxygenase thromboxane A

2

(TXA

2

) TXA

2

. , TXA

2

38)

. PR

,

5 19.04% ,

,

94.58%

. PR 1, 2, 5 μg/mL

, 90.17%, 77.54%, 42.95%

. eugenol

, 25.68% 75%

PR eugenol

.

결 론

PR in vitro

TEAC , DPPH

, ROS (superoxide anion, hydroxyl

radical) RNS (NO radical, peroxynitrite) ,

, RNS

. , PR (C

u Zn-SOD, GPx, GR catalase) mRNA

RT-PCR , PR

. , human LDL PR

AA BHT ,

PR LDL

. PR

, 20

, PR

,

. , eugenol

PR eugenol

. PR

.

감사의 글

/

(grant #: R13-2005-01001-0)

.

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