Anti-obesity Effects of the Water Extract from Orostachys japonicus in High-fat Diet-Fed Mice

(1)

와송추출물의 비만유발 ICR 마우스에서의 항비만 효과

이경선¹, 이형선²*

1고려대학교일반대학원의생명융합과학과

2중원대학교의료보건대학임상병리학과

Received: July 13, 2017 / Revised: September 18, 2017 / Accepted: October 17, 2017

서 론

최근우리나라에있어서중요한건강문제로대두되고있 는비만은동물성식품과가공식품의섭취량의증가와육체

적인활동저하등에의해발생되고있다^{[1]. 2014}년국민건

강^·영양조사에따르면국민¹인당하루평균영양소지방섭

취량이^{47.9 g}으로국민건강영양조사를처음시작한¹⁹⁶⁹년

에조사된^{16.9 g}에비하여약^2.8배이상증가하였다^[2].이 러한식생활의변화는비만인구의증가를초래하여그와관 련된고혈압^,고지혈증^,동맥경화^,심근경색등과같은순환 기계질환의발생이증가되었다^.이들중특히고지혈증은 심혈관계질환중관상동맥질환의가장중요한원인인자로

알려져있다^[3].고지혈증을유발하는환경적요인중에는고

지방식이요인의영향이크다고알려져있으며^,특히동물

성지방섭취로인한에너지과잉으로비만증에걸릴확률이

매우높다고보고되고있다^[4].따라서^,식이요법의중요성이

인식되면서식이섬유소나올리고당을함유한저칼로리저지 방식품을섭취하려는요구가날로증가하고있으며^,독성이 없고안정성이확보된기능성식품에대한관심이높아져이 를이용한비만예방및치료에대한많은연구개발이진행 되고있다^[5].

와송⁽Orostachys japonicus)은암속^,옥송^,작엽하초^,바이 솔등으로불리는돌나무과의다년생초본식물로오래된기 와지붕에서잘자란다^[6].과거전통의약품으로이용되었으 며 fatty acid ester류, flavonoid류, triterpenoid류^sterol류

및 aromatic acid류등의다양한생리활성성분을포함하고

있는것으로보고되고있다^{[7, 8].}면역^,항산화^,항균^,항고 지혈증^,항당뇨^,항암^,항염증효과등다양한생리활성효과 를가지고있는것으로입증된바가있다^[9−^15].또한^{, Kim} 등에의해게재한와송의유기용매별추출물을활용하여랫 트비만모델에적용한논문이있다^.이경우는본연구자의 식이제조의자율급식과달리추출물을존대를활용하여위 Anti-obesity Effects of the Water Extract from Orostachys japonicus in High-fat Diet-Fed Mice

Kyungsun Lee¹ and Hyeong-Seon Lee²*

1Department of Integrated Biomedical and Life Science, Graduated School, Korea University, Seoul 02841, Republic of Korea

2Department of Biomedical Laboratory Science, Jungwon University, Goesan 28024 , Republic of Korea

We investigated the anti-obesity effects of the water extract from Orostachys japonicus in high-fat-fed mice.

For the analysis, 24 mice were divided into four groups: normal (N) group, high-fat diet (HFD) group, high- fat diet supplemented with 2.5% O. japonicus (HFD-OJL) group, and high-fat diet supplemented with 5% O.

japonicus (HFD-OJH) group. The body weight gain and food efficiency ratio increased with a high-fat diet, but significantly decreased in the HFD-OJ groups compared with those in the HFD group. Levels of serum triglyceride, total cholesterol, and LDL-cholesterol were significantly lower, but the HDL-cholesterol level was significantly higher in the HFD-OJH group. The adipocytes within the epididymal adipose tissue were found to have expanded in the HFD group. However, the adipocytes in the HFD-OJ groups were compara- ble to those of the N group. These results suggest that O. japonicus has potential as an anti-obesity agent by decreasing body weight and lowering the serum lipid levels in obese mice.

Keywords: Orostachys japonicus, high fat diet, adipocyte size, anti-obesity, mice

*Corresponding author

Tel: +82-43-830-8861, Fax: +82-43-830-8679 E-mail: [email protected]

(2)

내경구투여방식을이용하여활성을확인하였으며^,한가지 단편적인농도를지정하여실험하여활성을확인하였다^[16].

이러한다양한생리활성평가를근거로하여와송성분이비 만예방효과를가지고있을것으로기대할수있는소재이 다^.하지만와송에대한다양한연구에도불구하고아직까지 와송에대한비만관련연구는매우미비한실정이다^.따라서 본실험에서는고지방식이로비만이유도된^ICR마우스에 서와송의열수추출물이전반적인지질대사에미치는영향 을비교하여효능을평가하고^,항비만활성의기능성식품 개발을위한기초자료로활용하고자한다^.

재료 및 방법

와송추출물 준비

경남밀양와송농장에서재배한건와송을농장에서직접 구입하였다^.건조된와송은파쇄하여가루로만들고증류수 를이용하여침지및열수추출을³회반복실시하여상층 을 얻어냈다^.상층은 여과지(Whatman No. 1, Whatman International Ltd., Germany)를이용하여여과후회전식 감압농축기(Laborota 4000, Heidolph, Germany)를이용하 여농축후동결건조기를이용하여^,건조된형태의와송열 수추출물분말을획득하였다^{(Fig. 1).}

실험동물 사육 및 식이

실험동물은생후⁴주령의수컷^ICR종마우스를코아텍

(Korea)에서구입하여¹주일동안환경에적응시킨후⁶마

리씩⁴개의군으로나누었다(Table 1). 실험동물들은물과실 험식이를자유롭게섭취하도록하였고^,실험식이는^Research

Diets (New Brunswich, USA)의^AIN-95식이를변형하여 정상식이(10% fat kcal)와고지방식이(60% fat kcal)를구입 하여섭취하도록하였다^.식이조성은^{Table 2}와같다^{. 8}주 동안고지방식이를공급하여비만을유도하였으며^{, 9}주에서 14주까지와송열수추출물의효과를확인하기위하여총칼

로리^60%고지방식이에^2.5%와^5%의와송열수추출물분

말을첨가한식이를조제하여자유급식하였다. 실험환경은 온도²²±¹℃^,습도⁵⁰±^5%로유지하였고^,밤낮을¹²시간으 로조절하였다^.실험의전과정은오송첨단의료산업진흥재 Table 1. Experimental design.

Groups Diet composition

N¹⁾ Normal diet

HFD²⁾ High fat/High cholesterol diet

HFD-OJL³⁾ High fat/High cholesterol diet + 2.5% OJ HFD-OJH⁴⁾ High fat/High cholesterol diet + 5% OJ

1)AIN-93 modified diet with 4% fat (10% fat calories).

3)HFD + 2.5% of O. japonicus extract powder.

4)HFD + 5% of O. japonicus extract powder.

Fig. 1. Procedure of extraction from O. japonicus.

Table 2. Composition of experimental diets.

Ingredient

Normal

diet High fat diet

N¹⁾ HFD²⁾ HFD- OJL³⁾

HFD- OJH⁴⁾

Casein 200 200 200 200

L-cystine 3 3 3 3

Corn starch 315 - - -

Maltodextrin 35 125 125 125

Sucrose 350 68.8 68.8 68.8

Cellulose 50 50 50 50

Soybean oil 25 25 25 25

Lard 20 245 245 245

Mineral mix 10 10 10 10

Dicalcium phosphate 13 13 13 13

calcium carbonate 5.5 5.5 5.5 5.5

Potassium citrate 16.5 16.5 16.5 16.5

Vitamin mix 10 10 10 10

Choline bitarate 2 2 2 2

FD&C yellow dye #5 0.05 - - -

FD&C blue dye #1 - 0.05 0.05 0.05

OJ - - 19.34 38.68

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단실험동물센터의동물실험윤리위원회의승인을받아실시 되었다(KBIO-IACUC-2016-061).

체중 및 사료 효율 측정

실험기간중동물의상태관찰을위해주¹회체중을주 기적으로측정하였다^.식이는주²회동일한시간에공급하 였고전에남은사료의양과새로공급한사료의양을측정 하여그차이로섭취한식이량을계산하였다^.사료효율^(FER;

food efficiency ratio)은실험기간에증가한체중증가량을 같은기간동안에섭취한식이량으로나누어산출하였다^. 시료 채취 및 처리

실험종료전실험동물은최소¹²시간절식시킨후희생 하였으며^,복부를개복한후하대정맥에서혈액을채취하여 혈청을 분리하였다^. 채취한 혈청은 total cholesterol, triglyceride, low-density lipoprotein (LDL)-cholesterol, high-density lipoprotein (HDL)-cholesterol의농도를자동 화학분석기(200FR, TOSIBA, Japan)를이용하여정량측정 하였다^.혈액의 동맥경화 지수(Atherogenic Index, AI)는 Hablund의계산법인^{AI = [(}총콜레스테롤량 −^HDL콜레스

테롤량^)/HDL콜레스테롤량^)]식을이용하여계산하였다^.

심혈관위험지수(Cardiac Risk Factor, CRF)는^Rosenfeld의

계산법에따라^{CRF =}총콜레스테롤양^/HDL콜레스테롤양

의식을사용하였다^{[17, 18].}혈액채취후^PBS용액을조직 으로들어가는혈관에주입하여혈액을제거한후부고환을

적출하여생리식염수로헹구고^10%중성포르말린에고정후 조직 파라핀 블록을 제작하여 H&E (hematoxylin and

eosin) 염색을실시하였다^.염색된고환지방조직은광학현

미경¹⁰⁰배로관찰되였다^. 통계처리

본실험결과는 SPSS 21.0 프로그램(IBM-SPSS, USA)을이 용하여평균과표준편차로표시하였고^,각군간의통계적

유의성검증은t-test를이용하여분석하였다. 각그룹간의

유의성은p < 0.05와p < 0.01 수준에서검증하였다^.

결과 및 고찰

체중증가, 식이섭취량 및 식이효율

실험동물은⁸주간고지방식이를공급하여비만을유도하 였으며^,그후⁶주간와송열수추출물^2.5%와^5%을포함하 는식이를투여하였다^.본연구자의와송추출물의식이첨가 농도는류등에의한논문을참고하였다^.류등에의한연구 결과와송의열수추출물을 250 mg/kg/day의용량으로¹²주 간반복투여하여그에대한장기독성을확인하였다^.그결과

다양한생화학적성상에서독성을보이지않았다^[19].이러

한선행연구를바탕으로와송추출물의실험농도를설정하 였다^.각그룹간체중증가량^,식이섭취량^,및식이효율등을 확인하였다(Table 3). 비만유도기간⁸주후각그룹간의체 중증가를확인한결과정상군^(N)에비하여고지방식이^(HFD)

Table 3. Body weight, food intake, and food efficiency ratio of the mice.

N¹⁾ HFD²⁾ HFD-OJL³⁾ HFD-OJH⁴⁾

Weight (g)

0 week 23.33± 1.330 23.25± 0.61 22.75± 0.52 23.83± 0.82

8^th weeks (start supplement) 44.83± 2.210 60.25± 3.17^d 60.00± 1.52^d 58.33± 1.40^d 14^th weeks (end supplement) 47.58± 2.620 70.83± 5.74^d 61.67± 1.69^bd 58.74± 1.04^bd Weight gain (g)⁵⁾ 2.75± 2.580 10.58± 6.92^c 01.67± 2.14^a 00.42± 1.39^bc Intake

Food intake (g/day) 4.42± 0.130 04.48± 0.17 04.25± 0.34 04.27± 0.22

FER⁶⁾ 0.015± 0.027 0.056± 0.043^c 0.008± 0.037^a 0.004± 0.038^a

5)Weight gain (g) = 8^th weeks body weight – 14^th weeks body weight.

6)Food efficiency ratio = [weight gain (g/day)] / [food intake (g/day)].

Values are mean± SD of six mice per group.

ap < 0.05 compared with the HDF group.

bp < 0.01 compared with the HDF group.

cp < 0.05 compared with the N group.

dp < 0.01 compared with the N group.

(4)

를섭취한나머지³그룹에서모두유의하게증가됨을확인 하였다^.이러한결과는⁸주간의고지방식이로인하여비만 이유도되었음을확인할수있었다^.따라서장기간의고열량 의고지방식이섭취는생쥐의체중을과도하게증가시켰으 며^,이같은결과는선행연구자의비만유도결과와거의일 치하였다^[5].비만유도후⁶주간²개의군에^2.5%와^5%의와 송열수추출물을첨가하여식이를공급하고체중증가양상 을관찰하였다^.그결과지속적인고지방식이^(HFD)만을섭

취한그룹보다와송추출물식이(HFD-OJL, HFD-OJH) 섭취 군에서체중증가량이유의적으로감소되었음을확인할수 있었다^.또한^,본연구에서군간의식이섭취량은^HFD군^{> N} 군> HFD-OJH 군> HFD-OJL 군의순으로나타났으나군 간의유의적인차이가없었음에도식이효율비에서와송추출 물식이군에서유의적인감소를확인할수있었다^.고지방 식이는칼로리밀도가높아체중을증가시켜식이효율이높 은결과를보여준다^.그러나와송추출물식이군에서의식이

Fig. 2. Total cholesterol (A), Triglyceride (B), LDL-cholesterol (C), HDL-cholesterol (D), atherogenic index (E), and cardiac risk factor (F) contents of serum in mice fed HFD-OJ for 6 weeks.

Values are mean± SD of six mice per group.

Atherogenic index (AI) = [(Total cholesterol-HDL-cholesterol)/HDL-cholesterol].

Cardiac risk factor (CRF) = Total cholesterol/HDL-cholesterol.

*p < 0.05 compared with the HDF group.

**p < 0.01 compared with the HDF group.

#p < 0.05 compared with the N group.

##p < 0.01 compared with the N group.

(5)

효율의유의적감소는와송추출물에의해일부영양소의소 화흡수율과이용률이저하되었기때문인것으로판단되었다^. 즉^,고지방식이를섭취하더라도와송추출물을같이섭취하 게되면일부영양소가소화흡수되지않고배설된다는의 미로해석할수있다^.이는와송추출물식이의급여가체중 감소를유도하여체중증가억제제로서의개발가능성이있 음을시사하고있었다^.

혈중 지질농도 측정

고지방식이와와송추출물분말의첨가수준을달리하여⁶

주간급여한흰쥐의혈청내지질농도분석결과는^{Fig. 2}

에나타내었다^.피하의내장지방의증가는혈중중성지방농 도에영향을미치므로고지방식이에의한지방축척은중성 지방의함량이높아진다고알려져있다^[20].혈장내중성지

방(Triglyceride)은고지방식이에의해가장큰영향을받는

데고지방식이섭취그룹에비해^5%와송추출물식이군에서

중성지방농도가유의하게낮았으며^{, 2.5%}와송추출물식이

군은약간의감소를보이기는했으나유의성을확인할수는 없었다^.

혈중총콜레스테롤의함량의증가는관상동맥질환과지

방대사의이상징후로이용되고있다^.또한^LDL콜레스테

롤은주로동맥혈관벽에붙어동맥경화를일으켜심장질 환의원인이되는인자로혈중농도가증가할수록질환의위

험성이커짐을의미한다^.반면^HDL콜레스테롤은혈중동

맥경화를일으키는^LDL콜레스테롤을혈관으로부터간으로

이동시켜제거하는역할을하는좋은콜레스테롤의일종으

로알려져있다^[21].혈장내총콜레스테롤과^LDL콜레스

테롤의농도가고지방식이를섭취한군에서정상군에비해 유의성있게증가하여고지방혈증상태가되었을것으로추 정된다^.고지방식이만을섭취한군에비해와송추출물식이 군에서총콜레스테롤과^LDL콜레스테롤이농도의존적으 로감소하며^,특히^5%와송추출물식이그룹은정상군과거

의유사한수치를보인다^{. HDL}콜레스테롤의경우고지방

식이군의경우유의적으로감소하였으나^2.5%와송추출물 식이군에서정상군과유사하고^5%와송추출물식이군에서 는정상군과고지방식이군에비해유의적으로의미있는증 가를보인다^.

혈중높은농도의콜레스테롤수치가유지되면동맥경화 및심혈관계질환의발생률이증가하는데, 그에따른지표가 되는것이동맥경화지수(atherogenic index, AI)와심혈관위 험지수(cardiac risk factor, CRF)이다^.혈청내^AI와^CRF는 고지방식이군에비해와송추출물식이군에서농도의존적으 로유의적인감소효과를보였다^.

부고환 주위 지방세포의 형태학적 관찰

적출한부고환주위지방조직을고정하여 hematoxylin &

Fig. 3. Histological change of epididymal adipocyte in mice. Hematoxylin and eosin stained section of epididymal adipose tissue (×100). (A) N group, (B) HFD group, (C) HFD-OJL group, (D) HFD-OJH group.

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eosin 염색을하여크기를현미경으로관찰한결과는^{Fig. 3} 과같다^.부고환지방세포의면적은고지방식이군이정상군 에비하여상당히크게증가한것을확인할수있었다^{. 5%}

와송추출물식이군에서는거의정상군과유사한경향을확 인할수있었다^.이결과는와송추출물을첨가한식이군의 혈청내지질성분수준의감소효과와일치한다^.따라서와송 추출물은지방축척을억제시키는것으로사료된다^.

요 약

본연구에서는와송로부터추출한열수추출물의항비만 효과를알아보기위해⁴주령의수컷쥐를¹주일간적응시킨 후^{, 8}주간정상식이군을제외한모든군에고지방식이를제 공하여비만을유도하였다^.그후정상식이군^,고지방식이군^,

2.5% 와송추출물식이군^{, 5%}와송추출물식이군으로나누

어⁶주간식이투여를실시하였다^.체중증가량은고지방식 이군에서유의적으로증가하였으며^,와송추출물첨가식이 군에서유의적으로감소하였다^.이러한결과는식이섭취량 에서군간의유의적차이가없었음에도불구하고체중증가 량과식이효율비가와송추출물첨가식이군에서탁월한감 소효과를보였다^.고지방식이에의한혈청중총콜레스테

롤^,중성지방^{, LDL-}콜레스테롤의증가는와송추출물의섭취

로인해낮아졌으며^{, HDL-}콜레스테롤의현저한증가를가 져왔다^.부고환지방조직에서관찰한지방세포의크기또한 와송추출물섭취군에서정상군과매우유사한형태를보여 준다^.위의실험결과를종합할때^,고지방식이와함께공 급한와송추출물은체중감소및체지방축척의억제와더불 어혈중지방수준의개선으로다양한비만유래질병에대 한긍정적영향을미치는것으로판단된다^.

Acknowledgments

This work was supported by the Jungwon University Research Grant (Research number 2016-054).

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