The Inhibitory Effects of Bimanbang-1(肥滿1號方) on the Obese-mouse Fed High-fat Diet

(1)

서 론

The Inhibitory Effects of Bimanbang-1( 1 ) on the

Obese-mouse Fed High-fat Diet

Shin Hong-jung, Yoon Il-ji

Dae-jeon University, College of Oriental Rehabilitation medicine

Objective: This study investigated the effects of Bimanbang-1 ( 1 : here in after referred to BMB-1) on the obese gene and obese inhibitory.

Methods: C57BL/6 mice were induced by high fat diet. Mice were divided into three groups (normal, high fat diet with control, high fat diet with BMB-1 extract) and fed for 15 weeks. Items of this experimental study are as follows:

body weight change, final body weight, the weight change of the adipocytes in body, the level change of LFT, NEFA and creatinine, the expression of β3AR and leptin gene in primary adipocytes, the production change of leptin in primary adipocytes, the expression of β3AR, leptin and TNF-α in adipocytes tissue.

Result:

1. All experimental groups showedthat the weight change decreased considerably and the high density group showedthat the final weight decreased considerably.

2. The high density group showed that the amount of the adipocyte in weight decreased considerably.

3. All experimental groups showedthat the amount of ALT decreased considerably, and AST decreased in the high density group. However, the amount of creatinine and glucose did not increase considerably.

4. All experimental groups showed that the amount of total cholesterol, LDL-cholesterol, triglyceride, and NEFA decreased, and HDL-cholesterol increased considerably.

5. The high density groups showedthat the amount of leptin decreased considerably.

6. All experimental groups showed that the revelation of β3AR in primary adipose cell and 3T3-L1 cell increased considerably, and that the revelation of leptin and TNF-α in primary adipose cellsand 3T3-L1 cells decreased considerably.

7. All experimental groups showed that the size of adipocyte in adipocytes tissue decreased.

8. All experimental groups showed that the adipose vacuoles in liver tissue decreased considerably.

Conclusion: The findings suggest that Bimanbang-1 causes weight loss and histological change, thus it may be effective to treat obesity.

Key Words : BMB-1 (Bimanbang-1), obese-mouse, obese gene, obese inhibitory, adipocyte, liver tissue.

2008 3 14 2008 5 9

: , 2 1136

(Tel:+82-42-470-9136, Fax:+82-42-470-9008, E-mail:[email protected])

肥滿號方高脂肪食餌肥滿誘導白鼠

신홍중, 윤일지

Original Article

(2)

1).

,

2), ,

, ,

3).

1 ,

,

.

재료 및 방법

1. 재료 1)

C57BL/6 (8 ,

, 30 , 20 24 )

( ) (Bio-

serv, U.S.A.)

22±2 1

. kg

(Scheme 1, 2).

2)

1 ( BMB-1)

, 1

(Scheme 3).

2. 방법 1)

BMB-1 2 1,300 2

rotary vacuum

evaporator .

freeze dryer 16.4

.

. 2)

( ),

( ), reductil ( ), BMB-1

( ) 15

Components Percentage(%)

Crude protein 22.1

Crude fat 8.0

Crude fiber 5.0

Calcium oxide 8.0

Calcium 0.6

Phosphorus 0.4

Scheme 1.

Components (%)

Casein, High protein 26.0

DL-Methionine 0.4

Sucrose 16.2

Corn Starch 16.0

Beef Tallow 30.0

Cellulose 5.0

Mineral Mix, AIN-76 4.5

Calcium Carbonate 0.4

Vitamin Mix, Teklad 1.3

Choline Citrate 0.2

Scheme 2.

(3)

. 8 28 g . ,

, reductil 10 /

, BMB-1 500 / , 250

/ 8

. 3) 7

0.1 g .

4)

(1) ,

Ether

3,000 rpm 15

0.1 g

(2) ALT, AST, glucose

AST, ALT, glucose JSCC UV method

.

한 약 명 생 약 명 용량(g)

곽 향 Pogostemonis herba 5

소 엽 Perillae folium 4

백 지 Dioscoreae tokoro rhozoma 2

대복피 Arecae pericarpium 2

복 령 Poria 4

후 박 Magnliae cortex 4

백 출 Atractylodis macrocephalae rhizoma 4

진 피 Fraxini cortex 4

반 하 Pinelliae rhizoma 4

길 경 Platycodi radix 2

자감초 Glycyrrhizae radix 2

계혈등 Spatholobi caulis 6

우 슬 Achyranthis bidentatae radix 8

천 궁 Candil rhizoma 8

원 지 Polygalae radix 2

창 포 Acorigraminei rhizoma 2

산조인 Ziziyphi soinosae semen 4

죽 여 Bambusae caulis taeniam 2

결명자 Cassiae semen 8

의이인 Coicis semen 4

갈 근 Puerariae radix 2

향부자 Cyperi rhizoma 2

계 지 Cinnamomi ramulus 4

Total 89

Scheme 3.

(4)

(3) Creatinine

Creatinine enzymatic colorimetry method

.

(4) Cholesterol

Total cholesterol enzymatic colorimetry method

, LDL-Cholesterol direct enzymatic

assay .

(5)

Triglyceride free fatty acid enzymatic colorimetry method

. 5)

(1) in vitro

3T3-L1 2%

(fetal bovine serum, FBS) RPMI 1640 1 / collagenase IV

, 37 , shaking 30

. cell strainer

. ACK 37 5

, 0.04% trypan blue

. 3 24 well plate

(1×105cells) BMB-1

(100 / , 50 / ) 8

RNAzolB total RNA .

(2) in vivo

ELISA C57BL/6 high-fat diet 15

.

2% (fetal bovine serum, FBS) RPMI 1640 1 / collagenase

IV 37 , shaking

30 5

. cell strainer

.

ACK 37 5

0.04%

trypan blue .

10% FBS-DMEM 7

24 well plate 5×105 BMB-1(100 / , 50 / )

48 .

TNF-α leptin enzyme-

linked immuno-sorbent assay (ELISA Endogen,

U.S.A.) . well

100 , 1

2 washing

antibody Avidin-HRP conjugated 100 1

. TMB 100

30 50 stop

ELISA reader 450 nm .

leptin β3AR C57BL/6

. RNAzolB 700

, RNAzolB 1/10 CHCl3

(chloroform) , 15 vortex ,

15 . 4 13,000 rpm

15

iso-propanol 15

, 1 70% EtOH

washing .

(5)

total RNA diethyl pyrocarbonate(DEPC)

20 RT-PCR .

(reverse transcription) total

RNA 3 75 5 ,

2.5 10 mM dNTPs mix, 1 random sequence hexanucleotides(25 p / 25 ), RNA inhibitor 1 RNase inhibitor(20 U/ ), 1 100 mM DTT, 4.5 5×RT buffer(250 mM Tris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl2)

, 1 M-MLV RT(200 U/ ) DEPC

20 . 20

37 1

first-strand cDNA , 95

5 M-MLV RT

cDNA polymerase chain

reaction(PCR) .

cDNA 1 20

PCR (10 mM Tris- Cl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 7.5 mM 4NTPS, 20 p sense, antisense primers, 2 units Taq polymerase)

. PCR turbo thermocycler system predenaturation 3 min, denat- uration 94 , 1min ; annealing 55 , 1 ; extention

72 , 1min 30 . primer

annealing 1% agarose gel .

6) Ether

liver adipocyte

tissue .

10 % 48

12

.

60 % 100%

, xylene

. (microtome)

3 4

hematoxyline eosin (H&E)

.

3. 통계처리

SPSS 11.0 unpaired student's

t-test P<0.05, P<

0.01 P<0.001 .

결 과

1. 체중에 미치는 영향

15 29.2±0.8 g ,

, 15 40.2±2.2 g .

28.6±0.3 g ,

15 BMB-1 500 / 32.6±

0.9 g, 250 / 35.1±1.6 g

, (p<0.05,

p<0.001) (Fig. 1).

9.3±0.8 g , 20.3

±2.0 g, 7.2±0.6 g .

BMB-1 500 /

12.2±0.9 g, 250 / 15.2±

1.8 g , 500 /

(p<0.001, p<0.01) (Fig. 2).

(6)

2. 체중내 adipocyte 중량에 미치는 영향

adipocyte 2.1±0.3 g,

10.9±2.2 g, 0.8±0.1 g

, BMB-1 500 /

3.4±0.9 g, 250 / 5.9±1.6 g

500 / (p<0.01)

(Fig. 3).

3. ALT, AST 및 glucose 에 미치는 영향

ALT 27.4±3.8 IU/L,

65.1±5.0 IU/L

.

21.7±3.6 IU/L, BMB-1 500 / 42.0±5.9 IU/L, 250 / 33.7±2.1 IU/L

(p<0.01, p<0.001)

Body weight(g)

45

40

35

30

35

20

15

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Ilormal Control Reductll BMB-1 : 500mg/kg BMB-1 : 250mg/kg

Time (weeks)

Fig. 1.

25

20

15

10

5

0

Final incrase body weight(g)

WT Control Reductil 10mg/kg

BMB-1 500mg/kg

BMB-1 250mg/kg

High-Fat diet - + + + +

*

Fig. 2.

(7)

(Table 1).

AST 113.3±16.6 IU/L,

200.8±6.7 IU/L

70.7±1.7 IU/L, BMB-1 500 / 112.1±9.5 IU/L, 250 /

172.5±17.4 IU/L 500 /

(p<0.001) (Table 1).

Glucose 106.1±5.3 / ,

161.1±7.8 / ,

151.7±9.3 / , BMB-1 500 / 149.5±7.8 / , 250 /

162.9±4.1 / (Table 1).

4. Creatinine 에 미치는 영향

Creatinine 0.27±0.04 / ,

0.16±0.01 / ,

14

Adipocytes tissue/body weight(g)

WT Control Reductil 10mg/kg

BMB-1 500mg/kg

BMB-1 250mg/kg

High-Fat diet - + + + +

* 12

10 8 6 4 2 0

*

Fig. 3.

Group Normal¹⁾

Fat diet Intake Control²⁾ ReducHigh til³⁾

10㎎/㎏ BMB-1⁴⁾

500㎎/㎏ BMB-1⁵⁾

250㎎/㎏

ALT(IU/L) 27.4 ± 3.8 65.1 ± 5.0 21.7 ± 3.6^† 42.0 ± 5.9^* 33.7 ± 2.1^† AST(IU/L) 113.3 ± 16.6 200.8 ± 6.7 70.7 ± 1.7^† 112.1 ± 9.5^† 172.5 ± 17.4 Glucose (㎎/㎗) 106.1 ± 5.3 161.1 ± 7.8 151.7 ± 9.3 149.5 ± 7.8 162.9 ± 4.1 1) Normal diet group

2) Only high-fat diet group

3) High-fat diet and Reductil 10 ㎎/㎏

4) High-fat diet and BMB-1 500 ㎎/㎏

5) High-fat diet and BMB-1 250 ㎎/㎏

Statistically significant value compared with control group data by t-test(*p<0.01, †p<0.001).

Table 1.

(8)

0.33±0.03 / , BMB-1 500 / 0.13±0.01 / , 250 / 0.15±0.01 /

(p<0.05 p<0.001) (Table 2).

5. Total cholesterol, LDL, HDL-Cholesterol, Triglyceride 및 NEFA 에 미치는 영향

Total cholesterol 93.8±4.4 /

, 161.2±3.0 /

. 134.0±3.8 / ,

BMB-1 500 / 127.8±3.3

/ , 250 / 131.6±7.1

/ (p<0.001,

p<0.01) (Table 3).

LDL-Cholesterol 19.2±1.4 /

, 47.4±2.6 / , 22.3±

0.3 / , BMB-1 500 /

28.3±1.7 / , 250 / 30.4±2.8 /

(p<0.001) (Table 3).

HDL-Cholesterol 85.3±3.6

/ , 68.8±4.6 / , 80.0±

2.5 / , BMB-1 500 / 120.2

±2.2 / , 250 / 143.0±3.2

/ ,

(p<0.001) (Table 3).

Triglyceride 119.2±15.9 /

, 106.9±3.2 / ,

35.3±2.0 / , BMB-1 500 / 51.5±5.1 / , 250 /

53.4±0.7 / ,

(p<0.001) (Table 3).

Group Normal

High Fat diet Intake Control Reductil

10㎎/㎏ BMB-1

500㎎/㎏ BMB-1

250㎎/㎏

Creatinine(㎎/㎗) 0.27 ± 0.04 0.16 ± 0.01 0.33 ± 0.03^† 0.13 ± 0.01^* 0.15 ± 0.01 Statistically significant value compared with control group data by t-test(*p<0.05 †p<0.001).

Table 2.

Group Normal

High Fat diet Intake

Control Reductil 10㎎/㎏ BMB-1 500㎎/㎏ BMB-1 250㎎/㎏

T.Chol.(㎎/㎗) 93.8 ± 4.4 161.2 ± 3.0 134.0 ± 3.8^‡ 127.8 ± 3.3^† 131.6 ± 7.1^† LDL-Chol.(㎎/㎗) 19.2 ± 1.4 47.4 ± 2.6 22.3 ± 0.3^‡ 28.3 ± 1.7^‡ 30.4 ± 2.8^‡ HDL-Chol. (㎎/㎗) 85.3 ± 3.6 68.8 ± 4.6 80.0 ± 2.5^† 120.2 ± 2.2^† 143.0 ± 3.2^‡ Triglyceride (㎎/㎗) 119.2 ± 15.9 106.9 ± 3.2 35.3 ± 2.0^‡ 51.5 ± 5.1^‡ 53.4 ± 0.7^‡

NEFA(uEq/L) 2349.0 ± 45.7 3039.0 ± 37.3 2489.0 ± 98.6^‡ 2649.0 ± 67.7^‡ 2883.7 ± 45.1^* Statistically significant value compared with control group data by t-test (*p<0.05, †p<0.01, ‡p<0.001).

Table 3.

(9)

NEFA(Non-esterified fatty acid)

2349.0±45.7 uEq/L, 3039.0±37.3

uEq/L , 2489.0±98.6

uEq/L, BMB-1 500 / 2649.0±

67.7 uEq/L, 250 / 2883.7±

45.1 uEq/L

(p<0.001, p<0.05) (Table 3).

6. Leptin 에 미치는 영향

Leptin 5.1±1.6 / ,

26.7±3.2 / ,

11.2±2.8 / , BMB-1 500 /

14.7±4.0 / , 250 / 19.6

±5.3 / BMB-1 500 /

(p<0.05) (Table 4).

7. Primary adipose cell에서의 유전자 발현에 미치는 영향

1) β3AR

β3AR 1±0.131,

5.44±0.152, BMB-1 50, 100 / 1.99±0.335, 4.06±0.499

(p<0.001, p<0.05) . 2) Leptin

Leptin 1±0.042,

0.362 ± 0.068, BMB-1 50, 100 / 0.658±0.085, 0.421±0.107

, (p<0.01, p<0.05)

.

8. 3T3-L1 cell에서의 유전자 발현에 미치는 영향 1) β3AR

β3AR 1.00±0.026,

5.53±0.227, BMB-1 50, 100 / 1.38±0.057, 3.52±0.145

(p<0.001) .

2) Leptin

Leptin 1.00±0.038,

0.125±0.078, BMB-1 50, 100 /

0.563±0.098, 0.328±0.107 , (p<0.001, p<0.01) .

3) TNF-α

TNF-α 1 ± 0.044,

0.554±0.067, BMB-1 50, 100 / 0.797 ± 0.087, 0.656 ± 0.121

, (p<0.05)

.

9. 조직학적 변화

1) Adipocyte

adipose

adipocytes

1.9 3.5 .

Group Normal

High-Fat diet Intake

Control Reductil 10㎎/㎏ BMB-1 500㎎/㎏ BMB-1 250㎎/㎏

Leptin(㎍/㎖) 5.1 ± 1.6 26.7 ± 3.2 11.2 ± 2.8^† 14.7 ± 4.0^* 19.6 ± 5.3 Statistically significant value compared with control group data by T test(*p<0.05, †p<0.01).

Table 4.

(10)

BMB-1 C, D adipocytes

.

(Fig. 4).

2) Liver

.

,

.

, BMB-1

, ,

, BMB-1 500 / (Fig. 5).

고 찰

, ,

,

4).

A B C D

Fig. 5.

A B C D

Fig. 4.

(11)

.

(BMI 25)

1995, 1998, 2001, 2005 20.5, 26.3, 29.6, 31.7%

5,6).

1990

7). 1984 2002

, ,

,

8).

,

Hubber ⁹⁾ , , ,

, , Van

Itallie ¹⁰⁾ , ,

.

, , , ,

, , 4

11).

. FDA

reductil sibutramine norad-

renaline serotonin

. , ,

. Xenical Orlistat

lipase

,

, , ,

2,12)

.

,

.

13) “

”

, ¹⁴⁾

“ ”

, ¹⁵⁾ “

” , ¹⁶⁾ “

” , ¹⁷⁾

“ ”

.

,

.

,

2). 1990 , 1999

, , ,

,

, , , , , ,

, ³⁾.

(12)

1

, ,

,

, ,

, ( )

18).

, ,

, .

1

15 29.2±0.8 g ,

15 40.2±2.2 g .

28.6 ±0.3 g ,

BMB-1 4

, 8

15 500 / 32.6±

0.9 g, 250 / 35.1±1.6 g

(Fig. 1).

15

9.3±0.8 g , 20.3±

2.0 g, 7.2±0.6 g .

BMB-1 500 /

12.2±0.9 g, 250 / 15.2±

1.8 g ,

1

(Fig. 2).

Adipocyte

19,20)

. adipocyte

2.1±0.3 g,

10.9±2.2 g, 0.8±0.1 g

, BMB-1 500 /

3.4±0.9 g, 250 / 5.9±1.6 g

1 reductil

,

(Fig. 3).

ALT, AST

19). ALT

27.4±3.8 IU/L, 65.1±5.0 IU/L

. 21.7

±3.6 IU/L, BMB-1 500 / 42.0±5.9 IU/L, 250 / 33.7±2.1 IU/L

(Table 1). ALT

transaminase , , ,

AST ,

AST 1/3

. ALT ,

21).

AST 113.3±16.6 IU/L,

200.8±6.7 IU/L

70.7±1.7 IU/L, BMB-1 500 / 112.1±9.5 IU/L, 250 / 172.5±17.4 IU/L

(Table 1).

Transaminase AST , , ,

. ,

AST ,

21). 1

.

(13)

22). glucose

106.1±5.3 / , 161.1±7.8 /

, 151.7±9.3 / , BMB-1

500 / 149.5±7.8 / , 250

/ 162.9±4.1 /

BMB-1 500 /

(Table

1). ,

1 glucose

.

, 1 g 9 kcal

. ,

2 . cholesterol

, VLDL-cholesterol triglyceride

. LDL-cholesterol

. total cholesterol

total cholesterol

23,24)

. Trig-

lyceride glycerin 3 ester

triglyceride

. triglyceride

triglyceride

3).

total cholesterol

93.8±4.4 / , 161.2±

3.0 /

. 134.0±

3.8 / , BMB-1 500 /

127.8±3.3 / , 250 / 131.6±7.1 /

(Table 3). LDL-

Cholesterol 19.2±1.4 / ,

47.4±2.6 / , 22.3±0.3

/ , BMB-1 500 /

28.3±1.7 / , 250 / 30.4±2.8 /

(Table 3).

HDL-Cholesterol 85.3±3.6 /

, 68.8±4.6 / , 80.0±

2.5 / , BMB-1 500 / 120.2

±2.2 / , 250 / 143.0±3.2

/ ,

(Table 3). Triglyceride

119.2±15.9 / , 106.9±

3.2 / , 35.3±2.0

/ , BMB-1 500 / 51.5±5.1

/ , 250 / 53.4±0.7 /

,

(Table 3). NEFA(non-esterified fatty acid)

25). NEFA

2349.0±45.7 uEq/L, 3039.0±37.3 uEq/L

, 2489.0±98.6 uEq/L,

BMB-1 500 / 2649.0±67.7 uEq/

L, 250 / 2883.7±45.1 uEq/L

(Table 3).

1

. Creatinine

. creatinine

(14)

,

26).

creatinine 0.27±0.04 / ,

0.16±0.01 / ,

0.33±0.03 / , BMB-1 500 / 0.13±0.01 / , 250 / 0.15±0.01 /

(Table 2).

reductil

, 1 creatinine

. Leptin

(thermogenesis) , (arcuate) (ventromedial nuclei)

leptin neuropeptide Y

,

22,27)

.

leptin

28), leptin

29).

leptin 5.1±1.6

ng/ , 26.7±3.2 ng/ ,

11.2±2.8 ng/ , BMB-1 500 / 14.7±4.0 ng/ , 250 /

19.6±5.3 ng/

(Table 4).

β3AR ,

,

leptin

29).

Primary adipose cell β3AR

1±0.131, 5.44±

0.152, BMB-1 50, 100 / 1.99±0.335, 4.06±0.499

. Leptin

1±0.042, 0.362±0.068,

BMB-1 50, 100 / 0.658

±0.085, 0.421±0.107 , .

3T3-L1 cell β3AR

1±0.026, 5.53±0.227, BMB-1 50,

100 / 1.38±0.057, 3.52±

0.145

. Leptin 1±0.038,

0.125±0.078, BMB-1 50, 100 /

0.563±0.098, 0.328±0.1.78 , .

TNF-α 1±0.044,

0.554±0.067, BMB-1 50, 100 /

0.797±0.087, 0.656±0.121 , .

adiopose lipid

droplets , plasma membrane

, signet-ring

white fat . ,

lipid vacuole(clear space) , unilocular polygonal

. adipose

adipocytes , BMB-1

adipocytes

(15)

.

(Fig.

4).

1

, 1

.

,

. , BMB-1

,

, .

(Fig. 5).

1

,

.

1

, 1

.

결 론

1 ,

, ,

,

. 1.

, BMB-1 500 /

.

2. adipocyte BMB-1 500 /

. 3. ALT

, AST BMB-1 500 /

. glucose creatinine

.

4. total cholesterol, LDL-cholesterol, triglyceride NEFA

, HDL-cholesterol .

5. leptin BMB-1 500 /

.

6. primary adipose cell 3T3-L1 cell β 3AR

, leptin TNF-α

. 7. adipocyte

. 8.

.

(16)

1

,

.

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