Antimicrobial Effect on the Periodontal Pathogens and Anti-inflammatory Effect of Eriobotryae Folium

서 론

, ,

Antimicrobial Effect on the Periodontal Pathogens and Anti-inflammatory Effect of Eriobotryae Folium

Mi Young Jeong ¹ , Young Hong Kim ² , Na Kyung Lee ² , Jin Yong Lee ³ , Yeek Herr ⁴ , Je Hyun Lee ⁵ , Sabina Lim ^1,2

1

WHO Collaborating Center for Traditional Medicine, East-West Medical Research Institute,

2

KyungHee University, Department of Meridian and Acupuncture, College of Eastern Medicine,

3

Kyung Hee University, Department of Oral Microbiology, College of Dentistry,

4

Kyung Hee University, Department of Periodontology, College of Dentistry,

5

Kyung Hee University, Department of Korean Medicine Dongguk University, Korea.

Objective: The purpose of this study was to evaluate on the antimicrobial effect on the periodontal pathogens and anti-inflammatory effect of Eriobotryae folium.

Eriobotryae folium are constituent herbs of Gagamgamroum, which has been used for a long time in oriental medicine as a herbal medicine for treating halitosis and toothache.

Method: Eriobotryae folium was prepared by extracting medicinal herb with water. We investigated antimicrobial activity by the minimum inhibitory concentration (MIC) test. We also investigated inhibition of IL-1β-induced collagenase (mmp-1), stromelysin-1 (mmp-3), interleukin-6 gene expression in human gingival fibroblasts using RT- PCR analysis.

Result: The antimicrobial effects of Eriobotryae folium was evaluated with MIC against periodontopathogens;

Porphyromonas gingivalis 2561, W50, A7A1-28, 9-14K-1, Prevotella intermedia 28, and Actinobacillus actinomycetemcomitans Y4. MICs of Eriobotryae folium were 1.25 / , 2.5 / , 0.625 / , 1.25 / , 10 / and 10 / . The anti-inflammatory effect of Eriobotryae folium was evaluated with influence of herbs on the IL-1β -induced expression of mmp-1, mmp-3, and interleukin-6. IL-1β increased mmp-1, mmp-3, and interleukin-6 mRNA levels. Eriobotryae folium significantly inhibited IL-1β-induced mmp-1, mmp-3, and interleukin-6 gene expressions in a dose-dependent manner.

Conclusion: These results suggested that Eriobotryae folium might reduce the excessive proteolytic capacity of the gingival fibroblast during inflammation and could be developed as a new drug for periodontitis.

Key Words : Periodontitis, Eriobotryae folium , collagenase (mmp-1), stromelysin-1 (mmp-3), interleukin-6

2008 4 1 2008 5 12

: , 1 ,

WHO

(Tel:+82-2-961-0324, Fax:+82-2-961-7831, E-mail:[email protected])

2005 ( )

.(KRF-2005-005-J00702)

枇杷葉

정미영

, 김영홍

, 이나경

, 이진용

, 허익

, 이제현

, 임사비나

Original Article

, , , , , 7

,

1,2)

. 30

Porphyromonas gingivalis, Prevotella intermedia, Actinobacillus actinomycetemcimitans

3,4)

.

, magnolol honokiol

, cytokine ,

, , ,

,

, Curcuma xanthorriza oil

, ,

5-11)

.

,

, (Eriobotryae Folium) (Rosaceae)

(Eriobotrya japonica Lindley) amygdalin, ursolic acid, oleanolic acid, malic acid, citric acid, tannin

,

, ,

12,13)

.

ursolic acid

, Staphylococus aureus

,

14-17)

.

cytokine, prostaglandin, collagenase

Matrix metalloproteinase(MMP) MMP-1,-3,-8,-9

MMP IL-1

tumor necrosis factor cytokine

18-22)

.

Porphyromonas gingivalis, Prevotella intermedia, Actinobacillus actinomyce- temcomitans

, (Human Gingival Fibroblast) IL-1β MMP-1, MMP-3, IL-6

MMP-1 MMP-3 IL-6

.

재료 및 방법

1.

비파엽추출물 제조

(Eriobotrya Japonica, )

. 92g

1000ml

2 .

DMSO (Dimethylsulfoxide) 20 mg/ml

.

2.

치주염유발균의 배양

Porphyromonas gingivalis 2561, W50, A7A1- 28, 9-14K-1 Prevotella intermedia

80% N , 10% H 10% CO2 37

(Shelcon Manufacturing Inc., Cornelius, OR, USA) hemin (5 μg/ml) vitamin K1 (0.2 μg/ml) harf-strength

BHI (HK-BHI) 48

, Actinobacillus actinomycetemcomitans 10% CO (Vision Co., Seoul, Korea) thioglycollate (Difco LAB. Co., Detroit,

MI, USA) .

3.

항균활성 측정

Porphyromonas gingivalis, P. intermedia, A.

actinomycetemcomitans

(minimum inhibitory concentration, MIC) . (1/25) 96 well plate 0.1 ml

0.1 ml P. gingivalis, P. intermedia

48 ,

A. actinomycetemcomitans 10% CO2 3

7 24 . 570 nm

,

MIC .

well

agar . 48 37

MIC .

4.

치은섬유아세포 배양

3

. 40% (fetal

bovine serum<FBS>, Gibco Co., Grand Island,

NY, USA) 20% (penicillin G, streptomycin, amphotericin B , Gibco co.,Grand Island, NY, USA) α-minimum essential medium (α-MEM, L-glutamine , Gibco Co., Grand

Island, NY, USA) 3 .

60mm Petri dish (Corning Co., Midlasnd, MI, USA)

No. 15 scalpel

1mm² .

dish

pipette dish 2ml

37 , 5% CO ,

100% (Shel-Lab, USA) .

10% FBS 1% α

MEM

3 . Petri dish (Corning

Co., Midland, MD, USA) dish 2 ml

3 bench Pasteur pipette

dish

1,200

rpm 10 .

HBSS 2

60mm

petri dish .

5.

치은섬유아세포 활성도 측정

96 well tissue culture plates (Corning-Costar, U.S.A.) 12,500 cell/well

. 24~36 10% FBS

DMEM

. 2 MTS

assay . well

2 Cell Titer 96 Aqueous Cell Proliferation Assay kit(Promega, U.S.A.)

3-(4,5-dimethyl-thiazole-

w-yl)-2,5-diphenyl tetrazolium bromide 20

µl 4 .

ELISA plate reader(Microplate manager, BioRad,

U.S.A) 490nm

. 3

O.D.

.

6. RT-PCR

100mm dish(Corning-Costar, U.S.A.)

10% FBS DMEM

.

3 .

10% FBS DMEM

12-14 .

MMP-1, MMP-3, IL-6

5 .

, IL-1β

IL-1β , (100,

200, 500 μg/ml) IL-1β

12 . Trizol-reagent

(Promega, U.S.A.) RNA

. RNA reverse transcription-polym- erase chain reaction (RT-PCR) cDNA

, cDNA . primer

. MMP-1;'5-CAC AGC TTT CCT CCA CTG CTG CTG C-3' (forward), '5-GGC ATG GTC CAC ATC TGC TCT TGG C-3' (reverse): 396bp, MMP-3;'5-GAA AGT CTG GGA AGA GGT GAC TCC AC-3' (forward), '5-CAG TGT TGG CTG AGT GAA AGA GAC CC-3' (reverse): 284bp, IL-6;'5-ATG AAC TCC TTC TCC ACA AGC GC-3' (forward), '5-GAA GAG CCC TCA GGC TGG ACT G-3' (reverse): 628bp, GAPDH; '5-GTC TTC ACC ACC ATG GAG

AAG-3' (froward), '5-GTT GTC ATG GAT GAC CTT GGC-3' (reverse); 210bp. PCR

ethidium bromide 1% agarose .

7.

통계

± (Mean ±

standard error) , oneway - ANOVA Newman-Keuls

. p < 0.05 .

결 과

1.

비파엽 추출물의 치주염 원인균에 대한 최소

억제농도

Porphyro- monas gingivalis 2561, W 50, A7A1-28, 9-14K-1

Prevotella intermedia28,

Actinobacillus actinomycetemcomitans Y4 (minimum inhibitory

concentration, MIC) 1.25 /

, 2.5 / , 0.625 / , 1.25 / , 10 / ,

10 / (Table 1).

2.

비파엽 추출물의 세포활성도 효과

MTS assay

. 500 /

.

500 /

(Fig. 1).

3. IL-1

β의 시간 및 농도에 따른

과

의 발현 평가

MMP-1 MMP-3

IL-1β 100mm dish

IL-1β 24

0.1% FBS DMEM

24 IL-1β

0.01, 0.10, 1.00, 2.00 / IL-1β 12, 24, 36, 48

RT-PCR

MMP-1 MMP-3 ,

MMP-1 MMP-3

IL-1β 0.1 /

12 (Fig. 2).

4.

비파엽에 의한 치은섬유아세포

발현에 미치는 영향

MMP -1, MMP-3 mRNA

, IL-1β , 100, 200 500 / MIC ( ㎎/㎖)

Eriobotryae Folium

P. gingivalis 2561 1.250

P. gingivalis W 50 2.500

P. gingivalis A7A1-28 0.625

P. gingivalis 9-14K-1 1.250

P. intermedia 28 10.000

A. actinomycetemcomitans Y4 10.000

MIC : minimum inhibitory concentration P. gingivalis : Porphyromonas gingivalis P. intermedia : Prevotella intermedia

A. actinomycetemcomitans : Actinobacillus actinomycetemcomitans Table 1.

2

1

0

〮

〮

control 50ug 100ug 200ug 500ug

500ug 200ug 100ug 50ug control

Fig. 1.

IL-1β

5 , MMP-1

5.5%, 11.2%, 22.8%

(p<0.05), MMP-3

15.4%, 45.1% (p<0.001), 84.4% (p<0.001) (Fig. 3).

MMP1

MMP3

GAPDH

396bp

284bp

210dp

Eriobotryae Folium(㎍)

- 0

+ 0

+ 100

+ 200

+ 500

A

* *

* 1.5

1.0

0.5

0.0

Optical density(% of control)

MMP-1

IL-1β (0.1㎍/ml) Eriobotryae Folium(㎍)

- +

0 0 100 200 500

+ + +

* 0.75

0.50

0.25

0.00

Optical density (% of control)

MMP-3

- +

0 0 100 200 500

+ + +

**

**

B C

Fig. 3. β

β

β

MMP1

MMP3

GAPDH

0 12 24 36 48

396bp

284bp

210dp

Time (h) A

MMP1

MMP3

GAPDH

396bp

284bp

210dp

0 0.01 0.1 1 2

Concentration (㎍/ml) B Fig. 2.

β

5.

비파엽에 의한 치은섬유아세포

발현에 미치는 영향

IL-6 cytokine mRNA

, IL-1β , 100, 200 500 /

IL-1β

5 , IL-6

8.1%, 57.2% (p<0.001), 91.6% (p<0.001) (Fig. 4).

고 찰

,

, , ,

23)

.

“ ( )

” ,

. , , , ,

, , 7

,

“( , , . ,

, , , .)”

, .

. “

(

)”

.

Staphylococus aureus

15)

.

1970 (Magnolia cortex)

,

IL-6

GAPDH

628bp

210dp

Eriobotryae Folium(㎍)

- 0

+ 0

+ 100

+ 200

+ 500

A

Optical density (% of control)

0.5

- +

0 0 100 200 500

+ + +

**

**

0.4 0.3 0.2 0.1 0.0

B

Fig. 4. β

β

β

β

. collagenase

7)

.

Anthony Van Leeuwenhoek(1632-1723) . Jordan Keyes(1964)

, Löe Theilade (1965, 1966)

.

Socransky(1970) ,

1976 Loesche, Slots Newman

25)

.

.

tetracycline, metronidazole clindamycin

.

26)

.

Porphyromonas gingivalis 2561, W 50, A7A1-28, 9-14K-1, Prevotella intermedia28, Actinobacillus actinomyc-

etemcomitans Y4 (MIC)

( ) Porph-

yromonas gingivalis MIC 0.625 / ~

2.5 / Prevotella

intermedia Actinobacillus actinomycetemcomitans

10 / .

.

cytokine, prostaglandin,

collagenase ,

cytokine

cytokine prostaglandin collagenase

. Cytokine

cytokine

. Cytokine interleukin-1

, T-cell, B-cell, ,

collagenase-1 (MMP-1) stromelysin(MMP-3)

27-29)

. Type

,

. MMP-1

MMP-8

30)

. MMP-3 Type

proMMP-8 -9

31)

. Cytokine interleukin

-6(IL-6)

32)

.

cytokine IL-1β

MMP-1, MMP-3 , IL-1β

MMP-1 MMP-3

IL-1β MMP-1 MMP-3

,

MMP-1 MMP-3

.

. IL-1β

IL-6

IL-6 .

.

MMP-1, MMP-3 IL-6

MMP-1 MMP-3 IL-6

.

결 론

MMP-1 MMP-3 cytokine IL-6

.

, Porphyromonas

gingivalis MIC 0.625 / ~2.5 /

.

, IL-1β

MMP-1 MMP-3

MMP-1 MMP-3

.

, IL-1

β IL-6

IL-6 .

Porphyromonas gin-

givalis MMP-1 MMP-3

IL-6 cytokine .

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