Elephant Garlic Extracts Inhibit Adipogenesis in 3T3-L1 Adipocytes

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코끼리마늘의 3T3-L1 지방세포 분화억제 효과

이슬기¹, 한동엽¹, 김수린^1,2, 이원영¹, 남주옥^1,2*

1경북대학교식품공학부

2경북대학교농업과학기술연구소

Received: November 25, 2019 / Revised: April 23, 2020 / Accepted: April 27, 2020

서 론

우리나라식생활에있어필수불가결한식재료인마늘은전 통적으로향신료뿐만아니라항암^,항산화및향균등다양 한약리효능을갖는민간치료제로도사용되어왔다^[1].또한 마늘의주요유효성분인^allicin은혈압강화^,혈중지질저하 및혈당감소효과등다양한생리적효능을갖는다고알려 져있다^{[2, 3].}그러나^,생마늘의경우특유의냄새와향으로 인해생리활성효과를나타내기충분한양을지속적으로섭 취하기어려울뿐만아니라과량섭취시위벽을자극^,조직 손상을야기할수있어있다[4, 5]. 반면, 마늘을가열숙성 한흑마늘의경우^,마늘특유의매운맛과향은감소하고단 맛이증가될뿐만아니라제조과정중화학적변환이일어 나면서 S-allylcysteine와같은특정생리활성물질이증가한

다고보고된바있다^[6].이러한생리활성물질들의함량차

이로인해흑마늘은일반마늘에비해항산화^,동맥경화및 체내지질개선효과가우수한것으로알려져있다^[2].

코끼리마늘⁽Allium ampeloprasum L.)은 일반 마늘 (Allium sativum L.)에비해냄새가적고인편이약¹⁰배가

량큰부추속에속하는식물이다^[7].코끼리마늘은일반마늘

에비해조직감과단맛이뛰어나보다다양한종류의식품 소재로이용하기에적합할뿐만아니라최근에는항산화^,항 당뇨^,및향균효과와같은약리효과가보고되고있어이의 활용가치에대한방향성이제시되고있는상황이다^[8−^10].

또한^,이전의한연구는코끼리마늘내유효성분분석을통 해일반마늘에비해발효된코끼리흑마늘에서총피루브산 의함량이큰폭으로증가함을밝힌바있으며코끼리마늘

은항비만활성이있는 scopoletin을포함하고있다고알려

진바있다^{[4, 11].}그러나^,아직까지대부분의국내연구는 코끼리마늘의생육에관한연구로한정되어있으며코끼리 마늘및코끼리흑마늘과같은다양한조리형태의코끼리마 늘의생리활성효과에대한연구는현저히부족한실정이다^. 만성적인잉여에너지는지방세포의과도한증식및분화 를야기함으로써궁극적으로는세포내지질축적하여비만 Elephant Garlic Extracts Inhibit Adipogenesis in 3T3-L1 Adipocytes

Seul Gi Lee¹,Dongyup Hahn¹,Soo Rin Kim^1,2, Won Young Lee¹, and Ju-Ock Nam^1,2*

1Department of Food Science and Biotechnology, ²Institute of Agricultural Science and Technology, Kyungpook National University, Daegu 41566, Republic of Korea

Elephant garlic (Allium ampeloprasum L.) has been reported to have several pharmacological effects. How- ever, its anti-adipogenic effect and the possible molecular mechanisms have not yet been reported. In this study, we demonstrate that elephant garlic extracts suppress adipogenesis in 3T3-L1 adipocytes. Raw and steamed elephant garlic extracts (REG and SEG, respectively) suppressed the differentiation of adipocytes and cellular lipid accumulation. Of note, the anti-differentiation effect of REG treatment on 3T3-L1 cells resulted in cytotoxicity, whereas SEG-treated cells displayed no such cytotoxicity. Additionally, SEG treatment significantly reduced the adipogenesis-related gene expression of PPAR γ, C/EBPα, adiponectin, Ap2, and LPL. To our knowledge, these results are the first evidence of the anti-adipogenic effects of elephant garlic extracts on 3T3-L1 adipocytes.

Keywords: 3T3-L1 adipocytes, Allium ampeloprasum L, anti-adipogenic, elephant garlic, steamed elephant garlic

*Corresponding author

Tel: +82-53-950-7760, Fax: +82-53-950-7762 E-mail: [email protected]

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을 유발할 수 있다^[12].이러한 지방세포의 분화과정은 peroxisome proliferator-activated receptor γ^(PPARγ) CCATT/enhancer binding protein α^(C/EBPα⁾등의 전사

인자에의해주로조절된다^[13].

본연구에서는코끼리마늘을생마늘^,흑마늘^,흑마늘열수 추출세가지방식으로조리이후다양한용매를이용하여추 출한코끼리마늘추출물들이^3T3-L1지방전구세포의분화 및관련조절인자의발현에미치는영향을조사하였다^.

재료 및 방법

용매별 시료의 제조

본연구에서는경상북도군위군에서재배및수확된코끼 리마늘을군위기술센터로부터지원받아생코끼리마늘^(Raw Elephant Rarlic, REG), 흑코끼리마늘(Steam Elephant Garlic, SEG) 및흑코끼리마늘열수추출물(Steam Elephant Garlic Extract, SEGE)과같은세가지종류의코끼리마늘추 출물을제조하였다^.생코끼리마늘^(REG)의경우껍질을제거 하고세척^,건조및갈아균질화하였다^.흑코끼리마늘^(SEG) 및흑코끼리마늘열수추출물^(SEGE)의경우찜기를이용하 여^{2 L}의증류수와함께가열후¹²시간숙성하였다^.숙성된 흑코끼리마을은증류수없이혹은증류수와함께^1:1비율 로갈아균질화하였으며본연구에서는이를각각흑코끼 리마늘^,흑코끼리마늘열수추출물이라고명명하였다^.균질화 된 각 코끼리마늘은 70% hexane, 70% ethyl alchol, 및 butanol에^1:1비율로혼합후진탕배양기를이용하여³⁰시

간동안^{200 rpm}에서용해하였다^.용해된추출물은회전식

진공농축기를이용하여완전건고후^DMSO를이용하여일 정한농도로희석, membrane filter (0.22-um pore size)로 제균후항암및항비만효과분석을위한실험시료로사용 하였다

세포배양

3T3-L1 지방전구세포는한국세포주은행^(KCLB)으로부터

분양받은후, 10% Newborn Calf Serum, 1% antibiotics를

포함하는^DMEM배지에^{5% CO}2가 공급되는³⁷℃

incubator에서배양하였다^.

세포 생존율 측정

3T3-L1 지방전구세포를 96-well plates에 각^well당 4 × 10³으로분주하여^{5% CO2}가공급되는 incubator 에서 하룻동안배양하였다^.다음날, REG, SEG, SEGE를^{0, 2, 4,} 8 mg/ml의농도로⁴⁸시간동안처리하였으며^,실험대조군 에는동량의^DMSO를추가하였다^.처리시간종료후^,각배 지를제거하고^{0.4 mg/ml}의 MTT (3-(4,5-dimethylthiazol-

2-yl)-2,5-diphenyl tetrazolium bromide) 를각^well에추가 하여^incubator에서³시간 배양한뒤 iso-propyl alcohol을 이용하여생성된^formazan을용해시킨뒤, infinite reader 를이용하여^{595 nm}에서흡광도를측정했다^.측정값은³번 은반복하여평균값으로얻었다^.

지방세포 분화 유도

지방세포분화는이전의연구에서서술된바와동일하게 방법을이용하여진행하였다[13]. 간략히, 3T3-L1 지방전구 세포(preadipoycytes)를성숙한지방세포(mature adipocytes)로 분화유도하기위해서, 6-well plate에분주뒤^,세포가^100%

confluent가 되었을 때 MDI solution (0.5 mM IBMX, 0.25μM DEX, 167 nM insulin, and 100 µM indomethacin)

이포함된^{10% FBS}를포함하는^DMEM배지로교체해주

었다^.분화유도²틀뒤, 167 nM insulin의^insulin이포함된 배지로교체해주었다^.배양중인세포의배지는²일간격으 로새로운^insulin이포함된배지로교체해주었다^.코끼리마 늘의지방세포분화억제효과를관찰하기위하여^,분화기간 동안⁽총⁸일) REG, SEG 또는^SEGE를 2, 4 mg/ml의농도 로처리하였다^.

세포 내 지질 염색(Oil Red O staining, ORO)

분화가완료된성숙한^3T3-L1지방세포의세포내^lipid droplet을확인하기위해^{, ORO}염색을수행하였다^.분화가 완료된^3T3-L1세포를 0.6% Glutaraldehyde로상온에서 1시간동안고정, 60% isopropyl alcohol로세척한후^0.4%

ORO soultion으로상온에서²⁰분간염색하였다^.염색된세 포를 촬영하고 100% isopropyl alcohol로 ORO solution을

용해하여^{570 nm}에서흡광도를측정하여정량화하였다^.

세포 내 mRNA 발현 수준 측정

세포 내^{total RNA}를 RNAiso Plus reagent (TaKaRa Bio, Japan)를이용하여분리한이후, complimentary DNA (cDNA) 합성은 Prime-Script RT reagent Kit (TaKaRa Bio)를이용하여제조사의메뉴얼에따라수행하였다^{. Real-} time PCR은 SYBR Green (TOYOBO, Japan)과 함께 Cycler iQ^TM Real-Time PCR Detection System (Bio-Rad Laboratories, USA) 기기를이용하여수행하였다^{. PCR}조 건은⁹⁴℃에서³⁰초(denaturation), 60℃에서³⁰초 (annealing), 72℃에서⁴⁵초(extension)로³⁵번으로설정하 였다^{. Primer}서열은^{Table 1}에도시된바와같다^.

세포 내 단백질 발현 수준 측정

세포를 lysis buffer (Biosesang, Korea)에용해한뒤⁴℃ 에서³⁰분간방치한후세포용액을 12,000 rpm에서¹⁵분

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간 원심 분리하였다^.동량의 단백질⁽³⁰μ^g)을^{10% SDS} polyacrylamide gel에의해 분리시켰다^{. gel}내의 단백질을 Nitrocellulose Membrane (Amersham Protran Premium 0.2μm NC, GE Healthcare Life Sciences, Germany)에 전달시킨뒤 5% skim milk로¹시간동안^blocking하였다^. 그후^{, 1}차항체를⁴℃에서 overnight 처리하였고²차항체 는 상온에서¹시간 반응시켰다^. 반응 후^{, enhanced} chemiluminescence reagent (GEHealthcare, UK)를^membrane

에 도포하고 FUSION Solo apparatus (Vilber Lourmat) 를이용하여검출하였다^.

통계처리

3회반복한실험결과는평균및표준편차를표시하였으

며^,각군간의통계적유의성에대한검증은^SPSS통계프

로그램을이용하여일원배치분산분석을실시^,유의성여부 를판정하였다^(*p < 0.05, **p < 0.01).

Fig. 1. Effects of REG, SEG, and SEGE on the lipid accumulation of 3T3-L1 adipocytes. (A) The effect of REG, SEG, and SEGE on lipid accumulation in 3T3-L1 adipocytes. Lipid droplets were stained by Oil Red O solution and photographed at 100× magnification. (lower rightinsets were photographed with a microscope at 400× magnification). The negative control (NC) are preadipocytes and the positive control (CON) are fully differentiated adipocytes. (B) The absorbance of stained cells measured at 495 nm.

Table 1. Sequences and accession numbers for primers used in RT-PCR.

Gene name Accession no. Forward primer Reverse primer

Adiponectin NM_009605.4 GATGGCACTCCTGGAGAGAA TCTCCAGGCTCTCCTTTCCT

Ap2 NM_024406 AACACCGAGATTTCCTTCAA TCACGCCTTTCATAACACAT

C/EBPα NM_001287523 CAAGAACAGCAACGAGTACCG TCACTGGTCAACTCCAGCAC

LPL NM_008509 CTGGTGGGAAATGATGTGG TGGACGTTGTCTAGGGGGTA

PPARγ AB644275 GGAAGACCACTCGCATTCCTT GTAATCAGCAACCATTGGGTCA

β-actin EF095208 AGGCTGTGCTGTCCCTGTAT ACCCAAGAAGGAAGGCTGGA

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결과 및 고찰

REG, SEG 및 SEGE가 3T3-L1 지방전구세포 분화에 미치는 영향

지방방울(Lipid droplet)은지방전구세포가성숙한지방세 포로분화하는분화과정(adipogenesis)의가장큰특징중하 나이며지방방울은주로중성지방인 triglyceride (TG)로구

성되어있다^{[14]. TG}는포도당과같은중요한에너지원이지

만과다섭취할경우지방세포에흡수되어저장되고이는비 만의원인으로작용한다^.따라서^,본연구에서는^{REG, SEG} 및^SEGE가^3T3-L1지방전구세포가분화하는동안세포내 축적되는지방방울의축적에영향을미치는지조사하였다^. 처음으로^,코끼리마늘을세가지다양한용매(hexane, 70%

ethyl alchol, 및^butanol)에서추출한각코끼리마늘추출물 들을모두동일한농도로^3T3-L1세포에처리하였다^.그결 과^,세가지추출물에서전반적으로^Butanol분획층에서가 장 우수한 지방세포분화 억제효과를 보였다^{(data not} shown). 이에따라^,하위연구에서는^Butanol분획층을단 독으로사용하였다^.

다음으로^{, Butanol}층에서추출한^{REG, SEG}및^SEGE를 세포가분화하는기간내내 2, 4 mg/ml로처리하고^ORO염 색을수행하였다^.실험결과, REG, SEG, SEGE는모두지 방세포의지질축적을농도의존적으로억제하는것을확인하 였다(Figs. 1A, B). 세포내^ORO가염색된지방구를정량분 석한결과^,대조군(control, CON)과비교하여^{4 mg/ml}농도 의^{REG, SEG}및^SEGE처리는각각 57.3%, 64.8%, 91.0%

지방세포분화를억제하였다(Fig. 1B). 그러나^{, ORO}가염색 되지않은미분화세포의모양을확인했을때^REG를처리

한세포의경우세포모양이변형되면서세포손상이야기된

것을확인할수있었다^.그러므로^{, REG}의지방방울축적억

제효과는지방세포분화과정의억제를통해서가아닌외부 자극에의한세포사멸을유도함으로써발생된현상이라고 추측된다^.반면^SEG는세포손상을야기하지않으면서지방

세포의 분화를 억제하는것으로 보여지며^{, SEGE}의경우

SEG에비해지방세포분화억제능이현저히낮은것으로평

가된다^.

상기의결과를바탕으로우리는다양한방식으로조리및 다양한용매에서추출한코끼리마늘추출물중부탄올분획 층의^REG와^SEG가가장우수한지방세포분화억제능을갖 는다는것을확인하였으므로^,하위연구에서는부탄올분획

층의^{REG, SEG}의효능에관해집중적으로연구하였다^.

REG, SEG 및 SEGE가 3T3-L1 지방전구세포의 생존에 미치 는 영향

각마늘추출물이지방전구세포의생존에미치는영향을 규명하기위해^,우리는^REG및^SEG를⁰−^{8 mg/ml}의농도 로^3T3-L1지방전구세포에⁴⁸시간동안처리하였다^.그결 과^{, 8 mg/ml}농도의^REG처리는통계적으로유의하게지방 세포의생존을저해하는것을확인하였다^{(Fig. 2).}반면에^, SEG 는모든농도의처리구간에서지방세포의생존에영향 을미치지않음을확인하였다^.이러한결과는^{, REG}가지방 세포의분화기간동안세포의생존에영향을미침으로써지 방세포의분화를감소시킬수있다는가설에대한직접적인 증거를제공하고있다^.또한지방세포의분화는약⁸일간지

속되기때문에분화기간동안^REG처리의중첩에의해이

러한세포독성효과는보다증폭되었을것이라고예상한다^. SEG가 지방생성과정에 관여하는 유전자의 mRNA 및 단백 질 발현에 미치는 영향

지방세포분화과정에서^REG및^SEG가^3T3-L1지방세 포분화관련인자들의발현에미치는영향을알아보기위해 지방형성과정에관여하는주요전사조절인자인^PPARγ^{, C/}

EBPα와 아디포카인(adipokine)인 adiponectin, ap2 (fatty acid-bindingproteins) 및 LPL (lipoprotein lipase)의

mRNA 발현수준을 비교분석하였다^.대조군과비교하여

SEG의처리는^PPARγ^{, C/EBP}α, Adiponectin, Ap2 및^LPL 의발현을모두농도의존적으로억제하였다^{(Fig. 3).}한편^, REG는 처리에 의한 다양한 지방세포 분화관련인자들의 mRNA 발현억제율은동일농도의^SEG에비해낮거나^ap2 의경우억제하지못함을확인하였다^.

또한 이러한^SEG및^REG에 의한^PPARγ와^C/EBPα

mRNA 발현량감소는단백질수준에서도동일하게나타났으

며이의발현억제율또한^REG에비해^SEG에서현저히높 Fig. 2. Effects of REG and SEG on the viability of 3T3-L1

preadipocytes. 3T3-L1 preadipocytes were treated with REG and SEG at various concentrations for 48 h. The viability of 3T3- L1 was determined by MTT assay. Values are mean ± S.D. of three experiments, with triplicate of each experiment.

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게나타남을확인하였다(Figs. 4A, B).

요약하자면^,상기의연구결과는흑코끼리마늘이^3T3-L1 지방세포의분화를억제함으로써생체외항비만효과를갖 는다는것을보여준다^.이는체중조절기능을갖는건강기능 식품원료로써혹은더나아가의약품의원료로써의코끼리 마늘의활용가능성을간접적으로제시한다^.또한^,흑코끼리 마늘과동일하게생코끼리마늘은지방세포분화억제효과를 가졌지만이는세포독성효과에기인한것으로판단된다^.이 러한독성효과는체중조절목적으로생코끼리마늘을섭취했 을때^,생체내조직손상및정상세포파괴등을부작용을동 반할수있다고추측할수있다^.그러나^,본연구결과를바 탕으로코끼리마늘을산업적으로활발히이용하기위해서는 생체내동물실험을통한안전성및효능입증과같은후속 연구가요구된다^.또한본연구에서는각마늘의조리방식

에따른생체외항비만효능수준의변화에관해서는명확 히제시하고있으나^,이러한차이점을야기하는추출물내 유효성분에관한연구결과는제시하고있지않다는점에서 연구의한계점을나타내고있다^.

요 약

부추속에속하는코끼리마늘⁽Allium ampeloprasum L.) 에대한몇가지약리효과는보고된바있으나^,이의항비만 및작용기전에대한보고전무한실정이다^.이에따라^,본연 구에서는코끼리마늘추출물이^3T3-L1지방세포의분화에 미치는영향을조사하여이의지방세포분화억제효능을규 명하였다^. 본 연구에서는 생코끼리마늘(Raw Elephant Rarlic, REG) 및 흑코끼리마늘(Steam Elephant Garlic, Fig. 3. Effect of REG and SEG on the mRNA expression of adipogenesis-related genes in 3T3-L1 adipocytes. 3T3-L1 adipocytes were treated with SEG or REG during the period of differentiation. After 8 days of differentiation, total RNA was isolated and real time reverse-transcription–polymerase chain-reaction (RT-PCR) confirmed the expression of adipogenic-related genes.

Fig. 4. Effects of REG and SEG on protein expression of adipogenic-related genes in 3T3-L1 adipocytes. (A) Cells treated with SEG or REG for the period of differentiation were lysed and total protein was extracted. The protein expression levels of PPARγ and C/EBPα were analyzed by western blot. An equal amount of loading was confirmed using β-actin. (B) Quantification of the protein expression.

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SEG) 추출물이지방세포의분화및지질축적을억제한다는 것을보여주었다^.주목할만한점은^{, REG}의경우지방세포

에독성을나타낸반면^{, SEG}는독성을나타내지않았다는

점이다^.추가적으로^{, SEG}는^PPARγ^{, C/EBP}α, Adiponectin,

Ap2 및^LPL와같은지방세포분화관련유전자의발현을통

계적으로유의하게억제하였다^.본연구는두종류의코끼리

마늘이^3T3-L1지방세포의분화를억제함을보고한최초의

연구이다^.

Conflict of Interests

The authors have no financial conflicts of interest to declare.

References

1. Kwon OC, Woo KS, Kim TM, Kim DJ, 2006. Physicochemical characteristics of garlic (Allium sativum L.) on the high tem- perature and pressure treatment. Korean J. Food Sci. Technol.

38: 331-336.

2. Kang MJ, Lee SJ, Shin JH, Kang SK, Kim JG, Sung NJ. 2008. Effect of garlic with different processing on lipid metabolism in 1%

cholesterol fed rats. J. Korean Soc. Food Sci. Nutr. 37: 162-169.

3. Elkayam A, Mirelman D, Peleg E, Wilchek M, Miron T, Rabinkov A, et al. 2003. The effects of allicin on weight in fructose- induced hyperinsulinemic, hyperlipidemic, hypertensive rats.

Am. J. Hypertens. 16: 1053-1056.

4. Kim D, Kim KH, Yook HS. 2015. Analysis of active components of giant black garlic. J. Korean Soc. Food Sci. Nutr. 44: 1672-1681.

5. Byun PH, Kim WJ, Yoon SK. 2001. Changes of functional prop- erties of garlic extracts during storage. Korean J. Food Sci. Tech- nol. 33: 301-306.

6. Kang JR, Lee SJ, Hwang CR, Kim IS, Sung NJ. 2013. Effect of black garlic and Gaeddongssuk (Artemisia annua L.) extracts on the lipid profile and hepatic antioxidant enzyme activities of exercised rats. J. Korean Soc. Food Sci. Nutrition 42: 869-876.

7. Youn CK, Kim KH, Ahn KS, Jaeng JH, Park YU, Kwon YH, et al.

2015. Effect of planting density on yield and growth character- istics of elephant garlic. Korean J. Plant Resour. 28: 541-545.

8. Lu X, Ross CF, Powers JR, Aston DE, Rasco BA. 2011. Determina- tion of total phenolic content and antioxidant activity of garlic (Allium sativum) and elephant garlic (Allium ampeloprasum) by attenuated total reflectance-fourier transformed infrared spec- troscopy. J. Agric. Food Chem. 59: 5215-5221.

9. Rahimi-Madiseh M, Heidarian E, Kheiri S, Rafieian-Kopaei M.

2017. Effect of hydroalcoholic Allium ampeloprasum extract on oxidative stress, diabetes mellitus and dyslipidemia in alloxan- induced diabetic rats. Biomed. pharmacother. 86: 363-367.

10. Hughes BG, Lawson LD. 1991. Antimicrobial effects of Allium sativum L. (garlic), Allium ampeloprasum L. (elephant garlic), and Allium cepa L. (onion), garlic compounds and commercial garlic supplement products. Phytother. Res. 5: 154-158.

11. Ham JR, Lee H-I, Choi R-Y, Sim M-O, Choi M-S, Kwon E-Y, et al.

2016. Anti-obesity and anti-hepatosteatosis effects of dietary scopoletin in high-fat diet fed mice. J. Funct. Foods. 25: 433- 446.

12. Lee SG, Kim JS, Min K, Kwon TK, Nam JO. 2018. Hispidulin inhibits adipogenesis in 3T3-L1 adipocytes through PPARγ pathway. Chem. Biol. Interact. 293: 89-93.

13. Lee SG, Kwon TK, Nam J-O. 2017. Silibinin inhibits adipogene- sis and induces apoptosis in 3T3-L1 adipocytes. Microbiol. Bio- technol. Lett. 45: 27-34.

14. Lee JA, Ahn E-K, Hong SS, Oh JS. 2012. Anti-obesity effect of ethyl acetate extracts from Agrimonia pilosa Ledeb. in 3T3-L1 preadipocytes (62.3). J. Immunol. 188: 62.63-62.63.