Effects of Samiunkyungtang on inflammation and fecal enzymes in ulcerative colitis animal model

서 론

(inflammatory bowel disease, IBD)

(ulcerative colitis, UC) (crohn’s disease, CD)

. 20-30

1)

2) .

四味軟堅湯

김소연, 류봉하, 박재우

Original Article

Effects of Samiunkyungtang on inflammation and fecal enzymes in ulcerative colitis animal model

So-yeon Kim, Bong-Ha Ryu, Jae-woo Park

3rd Dept. of Internal Medicine, College of Oriental Medicine, Kyung-Hee University

Objectives: This study was aimed to find the effect of Samiunkyungtang on inflammation and microflora in an ulcerative colitis animal model.

Methods: We established four groups of normal, control, test 1, and test 2 and assigned 6 rats toeach group. The normal group was not treated by any process and fed by normal saline. The control & test groups were provided with 4% dextran sodium sulfate (DSS) treatment for 7 days. Samiunkyungtang extract was orally administered to test groups (test 1=25mg/kg, test 2 100mg/kg) 3 days after DSS treatment for 10 days. After DSS treatment finished, we sacrificed the mice and measured colon length and enzyme activities such as myeloperoxidase (MPO), alkine phosphatase (ALP), β-glucuronidase, β-glucosidase, chondroitinase, and tryptophanase.

Results: The colon lengths of test 1 and 2 groups were longer than the control group (p<0.05). Histologically, the crypts and superficial epitheliums of test 1 and 2 groups were regenerated. Goblet cells from all test groups were retrieved. The inflammatory biochemical marker, MPO and ALP activities in all test groups were highly reduced (p<0.01) compared to the control group. The activities of fecal bacterial enzymes in test groups such as β -glucuronidase, β-glucosidase, chondroitinase, and tryptophanase were reduced compared to the control group (p<0.01).

Conclusions: As a result of this experiment, Samiunkyungtang is considered to have an inhibitory effect on inflammation and fecal enzyme activity in DSS-induced colitis animal model. Our results indicate that Samiunkyungtang may possess therapeutic effect on the development of DSS-induced colitis.

Key Words : Samiunkyungtang , ulcerative colitis, dextran sodium sulfate (DSS), fecal enzymes

2008 4 18 2008 5 7

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(Tel : +82-2-958-9107, Fax : +82-2-958-9136,

E-mail : [email protected])

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dextran sodium sulfate

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재료 및 방법

1.

재료 1)

17)

1 (Table 1). 500g

2.5L 2 ,

24.2%

. 2)

ICR 6 ( , , )

1 .

AIN-93G (Dyets Inc., Bethlehem, PA, USA) ,

.

Herbal name Drug name Scientific name Amount(g)

海 藻 Sagassum Sargassum enerve Agardh 5.0

海 帶 Laminaria Thallus Laminaria Japonica Aresch 5.0

牡 蠣 Ostreae Testa Ostrea gigas Thunberg 5.0

夏 枯 草 Prunellae Herba Thesium chinense Turczaninow 5.0

Table 1.

2.

방법 1) (1)

Fermin ¹⁹⁾

Dextran sodium sulfate ( DSS)

. ,

, 25mg/

kg ( S25), 100mg/kg

( S100) 4

6 .

,

4% DSS(MW 36,000-50,000, Sigma,

USA) 7

. DSS 3

DSS 10

25mg/kg, 100mg/kg 10 1

. (2)

7 4

. ,

. 2) (Histological examination)

4% paraformaldehyde

48 12

. alcohol (dehydration) alcohol:xylene

. paraffin paraplast

, 4μm paraffin section xylene (deparaffinization) hematoxylin-

eosin .

3) (1)

cold saline , .

4 , 7000 rpm 15

. (2) Myeloperoxidase( MPO)

Krawisz ²⁰⁾ 50mM phosphate

buffer(pH6.0) 1ml cold 0.5%

hexadecyltrimethyl -ammonium bromide ,

3 .

. 100 398 o-dianisidine(0.129mg/ml)

, 0.0005% H2O2

25 , 492nm time course

. bovine serum albumin standard Bradford method ²¹⁾

. MPO 1unit 25 1 H2O2 1mmole

.

(3) Alkaline phosphatase( ALP)

Bessy ²²⁾ cold saline

,

. 100 0.5mM

MgCl 2 , 5.5mM sodium p-nitrophenylpho- sphate 50mM glycine buffer(pH 10.5)

1ml 37 water bath 1

. 0.02N NaOH 1ml 405nm

. bovine serum albumin standard

Bradford method .

4) (1)

1g 20ml

500rpm 5

8000rpm 30

. 0.1M potassium

phosphate buffer(pH 7.0) 10 .

(2) β-Glucuronidase

100 0.1M potassium phosphate buffer(pH 7.0) 380 , 10mM p-nitroph-

enyl-β-glucuronide 20 37 60

. 0.5N NaOH 500

, 3000rpm 405nm

. (3) β-Glucosidase

50 0.1M phosphate buffer 350 , 20mM p-nitrophenyl-β-glucopyranosied 100

37 60 . 0.5N

NaOH 400 ,

3000rpm 405nm

.

(4) Chondroitinase

100 Chondroitin sulfate A (0.1mg

/ml) 300 100

37 5 ,

3000rpm 500

. 0.4 N NaOH 100 0.4M potassium

borate 100 5

67mM p-dimethylaminobenzaldehyde 3ml

37 20

585nm .

(5) Tryptophanase

0.1ml complete reaction mixture (0.1M bicine(pH8.0), 4% pyridoxal 5-phosphate, 20% bovine serum albumin) 0.2ml, 0.02M tryptophan 0.2ml

37 30 . color reagent

(p-dimethylaminobenzaldehyde 14.7g, 95% ethanol 948ml, C-H 2 SO 4 52ml) 2ml

2000rpm 10

550nm .

3.

통계

±

student t-test

. p<0.05 #,

p<0.01 ##, p<0.05 *,

p<0.01 **, .

결 과

1.

육안적 형태변화 소견

1)

(A) Normal (B) Control (C) S25 (D) S100

Fig.1.

. ,

(Fig 1).

DSS 7

, 8.17±0.16cm

6.95±0.08cm

(p<0.01).

S25 7.22±0.23cm, S100 7.43

±0.20cm , p<0.05

. S25 22.1%, S100

39.3%

.(Fig. 2).

2)

hematoxylin-eosin

, ,

(neutral mucin) (goblet cell)

(acid mucin) .

* * C olo n le ng th ( cm )

10 9 8 7 6 5 4 3 2 1 0

N C S25 S100

Fig. 2.

(A) Normal (B) Control (C) S25 (D) S100

Fig. 3.

, .

,

. S25

S100 .

2.

효소의 활성

1) Myeloperoxidase(MPO) Alkaline phosphatase (ALP)

neutrophil

MPO 7

. 5.73

.

S25 3.25 , S100

2.58 ,

S25 43.3%(p<0.01),

S100 55%(p<0.01) (Fig. 4(A)).

ALP 1.64

. MPO S25 1.14 , S100 0.92

S25 30.5%(p<0.01), S100 43.8%(p<0.01)

(Fig. 4(B)).

2)

7

β -glucuronidase 2.17 , β-glucosidase 1.80 , chondroitinase 1.83 , tryptophanase 1.33

4

(p<0.01).

β-glucuronidase S25 20.4%, S100 27.7%

. β-glucosidase S25 11.6%

S100

**

Ac tiv ity ( A bs )

##

0.9

0.6

0.3

0.0

N C S25 S100

**

**

##

A cti vit y (A bs )

0.4

0.0

N C S25 S100

0.2

(A) (B)

Fig. 4.

32.9%(p<0.05) . Chondroi- tinase S25 35.9%(p<0.01), S100

28.3%(p<0.05) . Tryptophanase

S25 9.6%

S100 29.4%(p<0.05) .

고 찰

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2) .

31.3%

,

20.4±28.9 ²³⁾ .

,

.

, , , , ,

A cti vit y (A bs )

#

1.2

N C S25 S100

0.9

0.6

0.3

0.0

Ac tiv ity ( A bs )

##

1.6

N C S25 S100

1.2

0.8

0.4

0.0

*

(A) (B)

A cti vit y (A bs )

##

0.2

N C S25 S100

0.0 A cti vit y (A bs ) *

##

0.08

N C S25 S100

0.06

0.04

0.02

0.00

** *

0.1

(C) (D)

Fig. 5. β β

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24) .

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My- cobacterium paratuberculosis avium

,

psychrotropic bacteria

“cold chain hypothesis”

25) .

, (dysbiosis)

26) .

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,

, probiotics, prebiotics

27) .

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28) .

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10) , ¹¹⁾ , ¹²⁾ , ¹³⁾ ,

14) , ¹⁵⁾

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37)

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Dextran Sodium Sulfate(DSS)

. , , 2.5-10%

DSS

, ,

DSS ,

38) . DSS

38) . DSS

11) ,

38) . 4% DSS 7

.

Myeloperoxidase(MPO) DSS

39) . MPO

Alkine Phosphatase(ALP)

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MPO ALP

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β-glucuronidase, β-gluc- osidase, Tryptophanase, Chondroitinase

. β-glucuronidase

,

40) . β-gluc-

osidase cycasin

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rutin, sennoside, paeoniflorin, saponin

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Tryptophanase trypt-

ophan indole,

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4

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0.5-1%

2) .

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DNA , cytokine

48) .

β-glucuronidase tryptophanase

49) ,

. β-glucuronidase tryptophanase

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cytokine

.

aminosalicyclic acid(5-ASA, mesalamine, mesalazine), glucocorticosteroid(GCS),

16) . probiotics

50) .

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결 론

, 4% DSS

DSS 25

mg/kg, 100 mg/kg 2 ,

. ,

1.

, . 2.

,

, .

3. MPO ALP

S25 43.3%, S100 55%

. ALP S25 30.5%, S100

43.8% .

4. β-glucuroni-

dase

. β-glucosidase S100 32.9%

. Chondroitinase

S25 35.9%, S100 28.3%

. Tryptophanase S100 29.4%

.

DSS

.

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