J Korean Soc Food Sci Nutr 한국식품영양과학회지 43(3), 389~396(2014) http://dx.doi.org/10.3746/jkfn.2014.43.3.389 추출방법에따른쇠비름의항산화, 생리활성및 Acetylcholinesterase 저해활성 권유리 조성묵 황승필 권기만 김재원 윤광섭 대구가톨릭대학교식품공학전공 Antioxidant, Physiological Activities, and Acetylcholinesterase Inhibitory Activity of Portulaca oleracea Extracts with Different Extraction Methods Yu-Ri Kwon, Sung-Mook Cho, Seung-Pil Hwang, Gi-Man Kwon, Jae-Won Kim, and Kwang-Sup Youn Dept. of Food Science and Technology, Catholic University of Daegu, Gyeongbuk 712-702, Korea ABSTRACT The physiological properties of 70% ethanol extracts from Portulaca oleracea with different extraction methods (reflux extraction, RE; autoclave extraction, AE; low temperature high pressure extraction, LTPE) were investigated. The freeze-dried powder yields of RE, AE, and LTPE were 33.78%, 30.80%, and 11.05%, respectively. The color values of L and b were higher in LTPE, and the chroma values were higher in AE and LTPE compared to RE. The total polyphenolics and proanthocyanidin contents in LTPE were significantly higher than in other extracts. The amount of substances related to flavonoids contents was highest in RE (4.30 mg/g), followed by AE (4.06 mg/g), and LTPE (4.00 mg/g). DPPH radical scavenging ability with a concentration of 500 mg% (w/v) were in the following order; LTPE (88.87%)> RE (83.84%)> AE (80.67%). Further, the reducing power, ABTS radical scavenging ability, and nitrite scavenging activity was observed in the same tendency as seen with the DPPH radical scavenging ability. However, the ferrous ion chelating activity of RE (85.45%) and AE (83.88%) was significantly higher than that of LTPE (75.60%). α-glucosidase inhibitory activities of RE and LTPE with a concentration of 100 mg% were significantly higher than AE. Xanthine oxidase, and acetylcholinesterase inhibitory activities of LTPE were higher than the other extracts. These results suggest that the extracts from Portulaca oleracea have the potential to act as functional materials, and components of Portulaca oleracea could be effective in the prevention of Alzheimer's disease, and may be used to develop various functional food products. Key words: Portulaca oleracea, extraction method, antioxidant activity, acetylcholinesterase inhibitory activity 서 최근생활수준의향상으로식생활수준이개선되고있으며, 특히성인병예방에대한관심이높아짐에따라활성산소종 (reactive oxygen species, ROS) 을억제시킬수있는약용식물에대한관심이증대되고있다. 또한평균수명의증가로인한고령화현상이문제됨에따라치매와같은퇴행성질환에대한관심이증대되고있다. 이러한퇴행성질환은활성산소에기인된것으로알려지고있는데활성산소종은정상에너지대사과정에서는물론그외의여러기전에의해끊임없이생성되며동시에각종효소및비효소적항산화물질에의해제거되어정상생체에서는이들의생성과소거가균형을이루고있다. 그러나생체가과도한스트레스를받거나과음, 흡연, 피로또는각종질병상태에서는활성산소생성계와소거계사이의불균형에따른과잉의활성산소생성으로조직의손상에따른노화의촉진, 발암, 고혈압, 론 Received 8 November 2013; Accepted 4 December 2013 Corresponding author. E-mail: ksyoun@cu.ac.kr, Phone: +82-53-850-3209 동맥경화및당뇨등과같은각종질환을유발하는것으로보고되고있다 (1). 이러한질병의원인으로 superoxide anion radical(o 2), hydroxyl(oh), singlet oxygen( 1 O 2) 및 hydrogen peroxide(h 2O 2) 등과같은 free radical과의관련성이대두됨에따라식의약적인측면에서활성산소를감소시킬수있는천연식물자원을활용한소재개발이요구되고있는실정이다. 한편 Alzheimer's disease(ad) 의주증상인기억력장애는주로대뇌기저부의콜린성신경의손상에기인된것으로, 인체내전기적신호를전달하는신경전달물질인 acetylcholine(ach) 의향상이 AD에서인지기능개선에도움이된다고알려지면서 cholinergic agonist나 acetylcholinesterase(ache) 저해제를통한 ACh의농도증가에대한연구가진행되고있으나독성과부작용으로논란의여지로인하여천연소재로부터의새로운치매치료제개발을위한노력이경주되고있다 (2). 쇠비름 (Portulaca oleracea L.) 은쇠비름과 (Portulaca) 의한해살이식물로돼지풀, 도둑풀, 말비름이라고하며생약명으로는마치현 ( 馬齒莧 ), 오행초 ( 五行草고 ), 장명채 ( 長命采 ), 마치채 ( 馬齒採 ) 라고불린다. 우리나라각지의길가,
390 권유리 조성묵 황승필 권기만 김재원 윤광섭 채소밭, 빈터등지에서자생하며, 줄기의높이가 15~30 cm 내외로털이없고줄기의직경은 2~3 cm로가지가많이갈라져땅위로비스듬히퍼지면서자라는식물이다. 쇠비름은양념등으로버무려서먹기도하고약재로도활용되어왔으며과거선조들의민간요법에서는충독, 사독등의해독제로도사용되었고 (3), 아라비아반도에서는방부제, 항괴혈병제제, 진경제, 이뇨제, 구충제, 피부진정제로도사용되어왔다 (4). 또한오메가-3 지방산이나 norepinephrine, dopamine이많이들어있으며, 펙틴질을포함한수용성다당류나비타민류및아미노산, 유기산, 배당체, 알칼로이드성분등이다량함유되어있다 (5). 이와같은다양한유용성분들을함유하고있는쇠비름은강력한항균력을지닐뿐만아니라최근에는다양한유기용매별로추출및분획한쇠비름추출물에서항산화효과나항암, 항염증 (6), 담배의 nicotine 성분의제거효능 (7) 이보고되었으며, 또한비만및고지혈증의예방 (8), 항균작용을이용한장내세균억제 (9) 등의연구가보고되었다. 근래에는쇠비름이이질간균, 티푸스균, 대장균, 포도상균등에대한억제작용이현저하다고새롭게밝혀지고있으며더욱이급성장염의완치율은 90%, 만성장염완치율은 60% 인것으로알려져 천연항생제 라고평가된바있다 (10). 이와같이기능성소재활용에관한연구를통해쇠비름의우수성이밝혀져왔으나쇠비름의소비확대는크게진전되지않고있으며추출방법에따른항산화및생리활성에관한연구는전무한실정이다. 따라서본연구에서는쇠비름의이용가치를향상시키기위하여추출방법에따른항산화및생리활성을조사함으로써효과적인추출방법을제시하고, 산업화에적용할방법을모색및기능성소재로서의개발가능성을검토하고자하였다. 재료및방법재료본연구에서사용한쇠비름 (Portulaca oleracea) 잎은 2012년전북남원시에서수확한것을구입하였으며세척한후동결건조 (freeze dryer, FD SFDSM12, Samwon, Seoul, Korea) 한다음분쇄기 (IKA A11 basic, IKA Werke GmbH & Co. KG, Staufen, Germany) 를사용하여 40 mesh로분쇄한분말을 -50 C에보관하면서실험에사용하였다. 시료의추출및수율측정쇠비름의추출방법은분쇄시료 50 g에 10배의 70% 에탄올을가한후환류냉각추출 (reflux extraction, RE), 가압가열추출 (autoclave extraction, AE), 저온고압추출 (low temperature high pressure extraction, LTPE) 방법으로추출물을제조하였다. 환류냉각추출은분쇄시료와용매를넣은용기에냉각관을부착하여 80 o C의맨틀상에서 3시간씩 3 회반복추출하였다. 가압가열추출은 autoclave(df-100a, DOORI Scientific Co., Gyeonggi, Korea) 를이용하여 121 o C에서 15분동안추출하였으며저온고압추출은 rapid extractor(ft110, Benchtop rapid extractor, Armfield Ltd., Hampshire, UK) 를이용하여실온에서 1시간동안 8.0 bar 의압력하에서 2회반복추출하였다. 각각의추출물은 Whatman No. 1 여과지 (Toyo Ltd., Tokyo, Japan) 로여과한다음 rotary vacuum evaporator(rotary vacuum evaporator N-N series, EYELA, Tokyo, Japan) 로감압농축한후에동결건조 (freeze dryer, FD SFDSM12, Samwon) 하여분말시료를제조하였으며 -50 o C에보관하면서실험에사용하였다. 추출물각각의수율은추출액을동결건조시켜건물중량을구한다음추출액조제에사용한원료건물량에대한백분율로나타내었다. 색도측정추출분말의색도는표준백색판으로보정된 chromameter(cr-300 Minolta, Tokyo, Japan) 를이용하여측정하였다. Hunter scale에의한 L(lightness), a(rednessgreenness), b(yellowness-blueness), H o (hue angle) 및 chroma value를측정하였다. 폴리페놀, 플라보노이드및프로안토시아니딘함량측정폴리페놀함량은 Dewanto 등 (11) 의방법에따라추출물 100 μl에 2% sodium carbonate 2 ml와 50% Folin- Ciocalteu reagent 100 μl를가한후 720 nm에서흡광도를측정하였으며 gallic acid(sigma-aldrich Co., St. Louis, MO, USA) 의검량선에의하여함량을산출하였다. 플라보노이드함량은 Abdel-Hameed(12) 의방법에따라추출물 100 ml에 5% sodium nitrite 0.15 ml를가한후 25 C에서 6분간방치한다음 10% aluminium chloride 0.3 ml를가하여 25 C에서 5분간방치하였다. 다음 1 N NaOH 1 ml를가하고교반한다음 510 nm에서흡광도를측정하였으며 rutin hydrate(sigma-aldrich Co.) 의검량선에의하여함량을산출하였다. Vanillin-sulfuric acid법 (13) 에따라시료 200 μl에 1.2% vanillin 용액 500 μl와 20% sulfuric acid 500 μl를혼합하여 20분간방치한후 500 nm에서흡광도를측정하였으며 (+)-catechin(sigma- Aldrich Co.) 의검량선에의하여함량을산출하였다. DPPH radical 소거활성측정 Blois(14) 의방법에따라시료 0.2 ml에 0.4 mm DPPH (1,1-diphenyl-2-picryl-hydrazyl) 용액 0.8 ml를가하여 10분간방치한다음 525 nm에서흡광도를측정하였으며계산식, DPPH radical scavenging ability(%)=100- [(O.D of sample/ O.D of control) 100] 에의하여활성도를산출하였다.
추출방법에따른쇠비름의생리활성특성비교 391 ABTS radical 소거활성측정 Re 등 (15) 의방법에따라 7.4 mm ABTS[2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt] 와 2.6 mm potassium persulfate를혼합하여실온 암소에서 24시간동안방치하여 radical을형성시킨다음실험직전에 ABTS 용액을 732 nm에서흡광도가 0.700± 0.030이되도록 phosphate buffer saline(pbs, ph 7.4) 으로희석하여사용하였다. 희석된용액 950 μl에추출물 50 μl를가하여암소에서 10분간반응시킨후 732 nm에서흡광도를측정하였으며계산식, ABTS radical scavenging ability(%)=100-[(o.d of sample/ O.D of control) 100] 에의하여활성을산출하였다. 환원력측정 Saeedeh와 Asna(16) 의방법에따라시료 1 ml에 0.2 M phosphate buffer(ph 6.6) 2.5 ml와 1% potassium ferricyanide 용액 2.5 ml를가한후 50 o C에서 30분간반응시켰다. 다음 10% trichloroacetic acid(tca) 용액 2.5 ml를가한후 1,650 g에서 10분간원심분리하였으며, 상징액 2.5 ml에증류수 2.5 ml와 0.1% FeCl 3 용액 0.5 ml 를가한후 700 nm에서흡광도를측정하였다. 아질산염소거활성측정 Kato 등 (17) 의방법에따라 1 mm NaNO 2 용액 1 ml에시료추출액 1 ml를가하고 0.1 N HCl과 0.2 M citrate buffer(ph 2.5) 를가하여총부피를 10 ml로조정하였다. 다음에 37 o C에서 1시간반응시킨후 1 ml를취하여 2% 초산용액 3 ml와 30% 초산용액으로용해한 Griess reagent(1% sulfanilic acid : 1% naphthylamine=1:1) 0.4 ml 를순차적으로가한후실온에서 15분간방치, 520 nm에서흡광도를측정하였다. 대조구는 Griess reagent 대신증류수를사용하였으며계산식, nitrite scavenging activity (%)=100-[(O.D of sample/ O.D of control) 100] 에의하여산출하였다. 철이온 (Fe 2+ ) 에대한 chelating 효과측정 Yen 등 (18) 의방법에따라시료추출액 1 ml, 80% ethanol 0.8 ml, 2 mm FeCl 2 4H 2O[iron(II) chloride tetrahydrate] 용액 0.1 ml, 5 mm ferrozine[3-(2-pyridyl)- 5,6-diphenyl-1,2,4-triazine-4',4''-disulfonic acid] 용액 0.1 ml를첨가한다음혼합하여실온에서 10분간반응시킨후 562 nm에서흡광도를측정하였다. 대조구는대표적 chelating agent인 ethylenediaminetetraacetic acid (EDTA) 를사용하였으며계산식, ferrous ion chelating effect(%)=100-[(o.d of sample/ O.D of control) 100] 에의하여산출하였다. α-glucosidase 저해활성측정 Kim 등 (19) 의방법에따라시료 50 μl, 1 uint/ml α- glucosidase 0.05 ml와 200 mm potassium phosphate buffer(ph 7.0) 50 μl를잘혼합하여 37 o C에서 10분간전처리하였다. 다음에 3 mm ρnpg(ρ-nitrophenyl α-dglucopyranoside) 100 μl를첨가하여 37 o C에서 10분간반응시킨후 0.1 M Na 2CO 3 0.75 ml로반응을정지시켜 405 nm에서흡광도를측정하였으며, 저해활성은 α-glucosidase inhibitory activity(%)=[1-(c Abs-S Abs)/ (C Abs- B Abs)] 100: C Abs: 대조구흡광도, S Abs: 시료흡광도, B Abs: 시료무첨가구의흡광도 에의하여산출하였다. Xanthine oxidase 저해활성측정 Stirpe 등 (20) 의방법에따라추출물 0.1 ml와 0.1 M potassium phosphate buffer(ph 7.5) 0.6 ml에 2 mm xanthine 기질액 0.2 ml를첨가하고 xanthine oxidase (0.2 uint/ml) 0.1 ml를가하였다. 다음 37 o C에서 15분간반응시킨후 1 N HCl 1 ml를가하여반응을종결시킨후, 반응액중에생성된 uric acid의양을 292 nm에서흡광도를측정하였으며계산식, xanthine oxidase inhibitory activity(%)=100-[(o.d of sample/ O.D of control) 100] 에의하여산출하였다. Acetylcholinesterase 저해활성측정 Ellman 등의방법 (21) 과 Sandahl과 Jenkins 방법 (22) 을변형하여측정하였다. AChE와 acetylthocholine iodine (ATC) 은 100 mm phosphate buffer(ph 8.0) 에녹여각각 0.25 U/mL와 75 mm을제조하였으며, 발색시약은 39.6 mg의 DTNB[5,5-dithiobis(2-nitrobenzoic acid)] 와 15 mg의 sodium bicarbonate를 100 mm phosphate buffer(ph 8.0) 10 ml에녹여제조하였다. 추출물 30 μl에 phosphate buffer(100 mm, ph 8.0) 2.8 ml, AChE(0.25 U/mL) 30 μl, DTNB 100 μl를넣고혼합한후, 37 o C에서 10분간 preincubation을한다음기질 ATC 30 μl를첨가하여 37 o C에서 3분간반응시킨후 412 nm에서흡광도를측정하였다. 대조구로서는추출물대신 100 mm phosphate buffer(ph 8.0) 30 μl를가하여측정하였으며, 저해활성은계산식, acetylcholinesterase inhibitory activity(%)=[1-(c Abs-S Abs)/ (C Abs-B Abs)] 100, C Abs: 대조구흡광도, S Abs: 시료흡광도, B Abs: 시료무첨가구의흡광도 에의하여산출하였다. Thin layer chromatography(tlc) 에의한정성분석 TLC에의한정성분석은추출물동결건조분말을 80% 메탄올에녹여 0.45 μm syringe filter(whatman, Rockland, MA, USA) 로여과후 TLC 분석을위한시료로사용하였으며, 각각의시료는 silica plate(aluminum sheet silica gel 60 F254, Merck, Darmstadt, Germany) 에점적한후 tol-
392 권유리 조성묵 황승필 권기만 김재원 윤광섭 uene/acetone/formic acid(6:6:1, v/v/v) 의혼합용매로전개하였다. 표준품 (chlorogenic acid, rutin, tannin, β-carotene; Sigma-Aldrich Co.) 으로정성분석하였으며, 성분확인은각성분들의 R f 값과자외선 (UV-254 nm, UV-366 nm) 을이용한띠의색상으로확인하였다. TLC에의한 AChE의저해활성효과는 Yang 등 (23) 의방법을참고하여측정하였으며, 효소액은 acetylcholinesterase(e.c. 3.1.1.7, Sigma product No. C3389, Sigma-Aldrich Co.) 1,000 Unit을 50 mm tris-hydrochloric acid buffer(ph 7.8) 150 ml에녹인다음효소의안정화를위해 150 mg bovine serum albumin을첨가하여 4 o C를유지하도록하였다. 각각의시료는 silica plate에점적한후 toluene/acetone/formic acid(6:6:1, v/v/v) 의혼합용매로전개하였으며건조하여용매를충분히날려준다음효소액을균일하게분사하여충분히말려주었다. 다음 1-naphthyl acetate(1.5 mg/ml) 를분사하고 37 o C에서 20분간반응시킨후 0.05% Fast Blue B Salt를분사하여발색시키고밴드를확인하였다. 통계처리모든실험은 3회반복으로행하여평균치와표준편차로나타내었고, 유의성검증은 version 12의 SPSS(12, SPSS Inc., Chicago, IL, USA) software를이용하여 Duncan's multiple range test를행하였다. 결과및고찰수율및색도추출방법을달리한쇠비름의수율및색도는 Table 1과같다. 수율은 RE(reflux extraction), AE(autoclave extraction) 및 LTPE(low temperature high pressure extraction) 가각각 33.78%, 30.80% 및 11.05% 로추출방법에따라유의적인차이를나타내었으며, RE>AE>LTPE 순으로높은수율을나타내었다. 환류냉각추출과가압가열추출에서높은수율을나타내는것은열처리공정에의하여불용성세포벽의수용화에의해수용성식이섬유가증가되는현상과더불어, 수용화과정중에식물조직의구조적인변화에따라불용성식물세포벽으로부터식이섬유성분의용해가용이해짐에따른결과로사료된다 (24). 한편 Kang 과 Lee(25) 와 Kim 등 (26) 의연구에서환류냉각추출및가압가열추출이저온고압추출에비해높은수율을나타내는결과와일치하는것으로보아본연구에서도높은온도와압력에의하여막투과성이증대되어보다많은성분이쉽게용출되어지는것으로판단된다. 색도에서는밝기를나타내는 L값과황색도를나타내는 b값의경우 LTPE에서가장높았으며, AE 및 LTPE에서색상을나타내는 H o 값및채도를나타내는 chroma value는증가하였다. 폴리페놀, 플라보노이드및프로안토시아니딘함량추출방법을달리한쇠비름의폴리페놀, 플라보노이드및프로안토시아니딘의함량을측정한결과는 Table 2와같다. RE, AE 및 LTPE의폴리페놀함량은 g당 18.62 mg, 17.11 mg 및 22.16 mg으로저온고압추출의경우환류냉각및가압가열추출에비해각각 19.01% 및 29.51% 높은함량을나타내었다. 이는고압하에서막투과성이증가함에따라폴리페놀화합물의용출이용이해짐에따른결과로사료된다. 한편열처리의경우결합형페놀화합물을유리형으로전환시켜용출을쉽게하거나고분자페놀화합물을저분자로분해해시료에서추출되는페놀성물질의함량을증가 (27) 시키는것으로보고된바있으나열처리에따른유용성분과항산화활성은식물체의종류나결합구조, 소재에따른 Table 1. Yields, and Hunter's color value of Portulaca oleracea extracts with different extraction methods Samples 1) Yields (%, DB 2) ) 33.78±1.21 3)a4) 30.80±0.89 b 11.05±.0.57 c Hunter's color value L (lightness) a (redness) b (yellowness) H o (hue angle) Chroma value RE AE LTPE 49.98±0.29 c 55.28±0.81 b 58.29±0.83 a 0.73±0.01 a 0.08±0.09 c 0.39±0.08 b 9.36±0.35 c 12.42±0.23 b 16.52±1.79 a 85.63±0.06 b 89.63±0.42 a 89.13±0.71 a 9.35±0.35 c 12.42±0.23 b 16.52±1.79 a 1) SE, stirrer extraction; RE, reflux extraction; AE, autoclave extraction; LTHP, low temperature high pressure extraction. 2) DB: dry basis. 3) Values are means±standard deviation of triplicate determinations. 4) Different superscripts within a column (a-c) indicate significant differences (P<0.05). Table 2. Total polyphenol, flavonoid and proanthocyanidin contents of Portulaca oleracea extracts with different extraction methods Measurement RE 1) AE 2) LTPE 3) Polyphenols (mg GAE 4) /g) Flavonoids (mg RHE 5) /g) Proanthocyanidins (mg CHE 6) /g) 18.62±0.32 7)b8) 4.30±0.02 a 3.38±0.07 b 17.11±0.30 c 4.06±0.04 b 3.35±0.10 b 1-3) See Table 1. 4-6) GAE, gallic acid equivalents; RHE, rutin hydrate equivalents; CHE, catechin hydrate equivalents. 7) Values are means±standard deviation of triplicate determinations. 8) Different superscripts within a row (a-c) indicate significant differences (P<0.05). 22.16±0.26 a 4.00±0.07 b 3.70±0.12 a
추출방법에따른쇠비름의생리활성특성비교 393 성분의변화모두에영향을미치는것으로알려지고있다 (28). Woo 등 (29) 은톱풀 (Achillea alpina) 과울릉미역취 (Solidago virgaurea) 어린잎을가압열처리 (121 o C, 15 min) 하였을때폴리페놀함량은감소된다고하였으며본연구에서도가압가열추출의경우고온에의한성분의손실이있는것으로나타났다. 플라보노이드함량은 g당 4.30 mg, 4.06 mg 및 4.00 mg으로가압가열추출및저온고압추출의경우유사하였고환류냉각추출에서는유의적으로높은함량을나타내었다. 프로안토시아니딘함량에서는 RE, AE 및 LTPE가 g당각각 3.38 mg, 3.35 mg 및 3.70 mg으로저온고압추출이환류냉각및가압가열추출에비해각각 9.47% 및 10.45% 높은함량을나타내었다. 이상의결과추출방법에따른페놀화합물함량의유의적인차이를나타내었으며저온고압추출시페놀화합물등의유용성분의추출함량증대에좋은영향을미칠수있을것으로판단된다. DPPH radical 소거활성, ABTS 라디칼소거활성및환원력추출방법을달리한쇠비름의전자공여능, 환원력및 ABTS 라디칼소거활성을측정한결과는 Table 3과같다. 500 mg% 에서의 RE, AE 및 LTPE의전자공여활성은각각 83.84%, 80.67% 및 88.87% 로저온고압추출이환류냉각및가압가열추출에비해유의적으로높은활성을나타내었다. 한편양성대조군으로합성산화방지제인 BHT(butylated hydroxytoluene, 95.38%) 보다는전반적으로낮은활성을나타내었으나쇠비름추출물의농도가증가함에따라그활성역시증가 (data not shown) 하기때문에천연기능성소재로써의활용도는높을것으로사료된다. 한편 ABTS 라디칼소거활성은추출방법별로 LTPE(91.37%)> RE(86.70%)> AE(83.19%) 순으로저온고압추출에서높은활성을보였으나농도대비양성대조군에비해현저히낮은 활성을나타내었다. 환원력은시료에존재하는 reductones 가제공하는수소원자가활성산소사슬에분해함으로써항산화활성을나타내는것으로항산화활성과직접연관된것으로알려졌으며 (30), 전자공여능과유사한경향을나타내었는데 LTPE(1.60) 가 RE(1.39) 및 AE(1.33) 에비해유의적으로높게수치를나타내었다. Osawa(31) 는식물로부터추출된 phenol 부류의화합물은항산화능을포함한다양한생리적효능을나타내고이효능은주로산화 환원력에의한효과로보고한바있으며, 페놀함량이높을수록항산화능이증가한다고보고하였는데이는본연구와비슷한결과를나타내었다. 따라서 LTPE의환원력이높은결과는쇠비름추출물에함유된페놀화합물에의한것이라고사료된다. 아질산염소거활성및철이온 (Fe 2+ ) 에대한 chelating 효과측정추출방법을달리한쇠비름의아질산염소거능및체내에서세포의지질및단백질의산화를촉진하는 Fe 2+ 의 chelating 효과를조사한결과는 Table 4와같다. RE, AE 및 LTPE의아질산염소거활성은각각 35.67%, 33.48% 및 40.33% 로저온고압추출, 환류냉각추출, 가압가열추출순으로높은활성을나타내었으며, 모든추출군이대조군 BHT (32.12%) 보다높은활성을나타내어향후기능성소재로서의개발가능성이있을것으로여겨진다. Takashi 등 (32) 의보고에따르면폴리페놀과플라보노이드화합물은종류에따라차이는있으나 phenol계유도체들이 nitroso화합물의생성을억제한다고하여본연구에서추출방법에따른소거활성의차이는페놀화합물의함량차이에의한결과라판단되며, 저온고압추출의경우위장내의낮은 ph 조건에서 nitrosamine 형성을보다효과적으로억제할수있을것으로여겨진다. 한편 ferrous ion chelating 효과에서는 RE Table 3. DPPH and ABTS radical scavenging ability, reducing power of Portulaca oleracea extracts with different extraction methods Measurement RE 1) AE 2) LTPE 3) Positive control, BHT DPPH radical scavenging ability (%) ABTS radical scavenging ability (%) Reducing power (OD 700) 83.84±0.11 4)b5) 86.70±0.74 b 1.39±0.06 b 80.67±0.87 c 83.19±1.13 c 1.33±0.03 b 88.87±1.01 a 91.37±0.99 a 1.60±0.05 a 95.38±0.69 98.15±0.25 2.13±0.12 1-3) See Table 1. The concentrations of positive control (BHT; butylated hydroxytoluene) solutions were measured at 50 mg%, and various extracts sample were measured at 500 mg%. 4) Values are means±standard deviation of triplicate determinations. 5) Different superscripts within a row (a-c) indicate significant differences (P<0.05). Table 4. Nitrite scavenging activity (NAS), and ferrous ion chelating activity (FICA) of Portulaca oleracea extracts with different extraction methods Measurement 1) RE 1) AE 2) 3) Positive control LTPE BHT EDTA NSA (%) FICA (%) 35.67±0.72 4)b5) 33.48±1.36 b 40.33±0.71 a 32.12±0.89 85.45±0.27 a 83.88±0.40 b 75.60±0.42 c - - 98.74±0.21 1-3) See Table 1. The concentrations of positive control (BHT: butylated hydroxytoluene, 50 mg%; EDTA: ethylenediaminetetraacetic acid, 100 mg%), and various extract samples were measured at 100 mg%. 4) Values are means±standard deviation of triplicate determinations. 5) Different superscripts within a row (a-c) indicate significant differences (P<0.05).
394 권유리 조성묵 황승필 권기만 김재원 윤광섭 (85.45%)> AE(83.88%)> LTPE(75.60%) 의순으로환류냉각추출및가압가열추출에서높은활성을나타낸반면항산화활성의경향과는상반되는결과를보였는데, 이는금속이온을제거할수있는물질과 radical을제거할수있는물질의차이에따른결과라사료된다. 대표적인금속이온 agent인 EDTA(98.74%) 의수준에는미치지는못하였으나높은활성을나타내어소재로서의이용가능성이예견되었으며, 특히쇠비름추출물의경우단일성분이아닌추출물임을고려해볼때추후분리및구조동정을통하여체내에생성된 ferrous ion을효과적으로제거시킬수있는천연물로활용할수있을것으로사료된다. α-glucosidase, xanthine oxidase 및 acetylcholinesterase 저해활성측정추출방법을달리한쇠비름추출물들의 α-glucosidase, xanthine oxidase(xo) 및 acetylcholinesterase(ache) 저해활성을측정한결과는 Fig. 1과같다. 일반적으로 α- glucosidase는소장상피세포의미세융모막 (brush border membrane) 에존재하는효소로서, 소장에서음식물중의전분을포도당과같은당으로분해하여흡수시킨다. α-glucosidase 저해제는이당류의분해효소를가역적으로억제하여탄수화물의흡수를지연시키는역할을하며, 소장전체에포도당이흡수되어식후혈당상승을완만하게한다 (33). 5 mg/ml 농도에서 α-glucosidase 저해활성은 RE, AE 및 LTPE가각각 48.90%, 18.23% 및 46.04% 로환류냉각및저온고압추출에서가장높은반면가압가열추출에서는낮은활성을나타내었다. 한편양성대조군인 acarbose (78.57%) 에비해낮은저해활성을보였으나환류냉각및저온고압추출의경우혈당의급격한증가를예방할수있는기능성소재로서의개발가능성이높을것으로판단된다. XO는 xanthine 또는 hypoxanthine으로부터 uric acid를형성하여혈장내에과량존재하게되면골절에축적되어심한통증을동반하는통풍과신장에침착되어신장질환을유발하는효소로알려져있다. 따라서 XO 저해활성은 free radical의생성을억제하므로항산화, 항노화및항암등의효과를기대할수있는생물학적중요한효소라고할수있 다. 5 mg/ml 농도에서 XO 저해활성은 RE, AE 및 LTPE가각각 71.81%, 77.87% 및 84.52% 로저온고압추출에서가장높은활성을나타내었으며저온고압의경우대조구 (86.45%) 에근접하는저해활성을나타내었다. 체내수많은신경전달물질중에서대표적인 acetylcholine(ach) 은시냅스 (synapse) 와시냅스사이의신경전달에관계하는중요한신경전달물질로신경세포안에존재하던 choline과 acetyl CoA가 acetyltransferase(chat) 라는효소에의해생성된다. ACh은신경말단에서분비되어수용체에결합함으로써신경세포의신호를전달한후 AChE에의해 acetate와 choline으로분해가되고, 분해된 choline은 carrier에의해다시신경계로부분흡수된다. 신경세포가손상된치매환자의경우생성되는 ACh의양은적고 AChE의작용은계속되어신경전달에이상이생기게되면서결국치매환자는학습능력및기억력감퇴와인지력저하등의같은병리현상이발생한다 (34). 쇠비름추출물의 AChE 저해활성은 5 mg/ml 농도에서 RE, AE 및 LTPE가각각 22.19%, 24.79% 및 31.03% 로저온고압추출에서가장높았고다음으로가압가열추출, 환류냉각추출순의저해활성을나타내었으나저해제인 tacrine(87.17%) 에비해서는전반적으로낮은활성을보였다. 반면쇠비름추출물의경우단일성분이아닌추출물인점으로볼때치매예방및개선제로서의가능성도기대된다. TLC에의한정성분석및 AChE 저해활성확인추출방법을달리한쇠비름추출물각각을 TLC에 10 μl 씩 spotting하여표준물질을자외선으로비교한결과 (I, II) 및 AChE 저해활성을확인한결과 (III) 는 Fig. 2와같다. RE, AE 및 LTPE 각각의추출물에서수종의미세한 spot이확인되었으며각각의표준품 chlorogenic acid(r f 0.07), rutin(r f 0.04), tannin(r f 0.02), β-carotene(r f 0.88) 과비교하여동일한위치에서 band가확인되었다. 한편 Fast Blue B Salt를분사하여보라색을발색시킨 plate(iii) 의경우 AChE 억제효능을나타내는 white band(r f 0.47) 를확인하였으며, UV-365 nm에서관찰하였을때동일한위치에서 blue zone(35) 을나타내이물질로인해 AChE 저해활성이 α-glucosidase inhibitory activity (%). 100 80 60 40 20 0 78.57 a (I) 48.90 b 46.04 b 18.23 c AC RE AE LTPE XO inhibitory activity (%). 120 100 80 60 40 20 0 (II) 86.45 a 84.52 a 77.87 ab 71.81 b BHT RE AE LTPE AChE inhibitory activity (%). 120 100 80 60 40 20 0 (III) 87.17 a 31.03 b 22.19 c 24.79 c TA RE AE LTPE Fig. 1. α-glucosidase, xanthine oxidase (XO), and acetylcholinesterase (AChE) inhibitory activity of Portulaca oleracea extracts with different extraction methods (100 mg%, dry basis). Values are means±standard deviation of triplicate determinations. Bars/mean values with different letters are significant differences (P<0.05). The concentrations of positive control (AC, acarbose; BHT, butylated hydroxytoluene; TA, tacrine) solutions were measured at 100 mg%.
추출방법에따른쇠비름의생리활성특성비교 395 A B C D RE AE LTPE A B C D RE AE LTPE A B C D RE AE LTPE Fig. 2. Screening of 70% ethanol extracts of Portulaca oleracea with different extraction methods using TLC assay method. The concentrations of 10 mg/ml samples were loaded on the TLC plate, and developed in toluene/acetone/formic acid (6:6:1, v/v/v). A, chlorogenic acid; B, rutin; C, tannin; D, β-carotene. Ⅰ: UV-366 nm (without chemical treatment), Ⅱ: UV-254 nm (without chemical treatment), Ⅲ: white spots on the purple background represent the AChE inhibition when 1-naphthyl acetate (1.5 mg/ml) and Fast Blue B Salt (50 mg/100 ml) were sprayed, followed by 1,000 Unit/mL of AChE spray. Abbreviations: see Table 1. 나타나는것을확인하였다. TLC 방법에의한 AChE의저해활성은 naphthyl acetate가 AChE에의해 α-naphthol로전환되고, α-naphthol은 2개의 active site form을가지고있는 Fast Blue B Salt와반응하여 azo dye가생성되어보라색이나타나게되는원리로 AChE 저해를할수있는성분이존재할시 α-naphthol의생산은멈추게되고 azo dye의생산이억제됨에따라활성성분은 white band를띠게된다 (35). 한편 TLC 법에서 AChE 저해활성은알칼로이드성분의존재로나타나는것으로알려지고있는데현재 AChE 억제제로잘알려져있는 galantamine의경우 50% 이상이알칼로이드계열화합물로이루어져있으며또한대부분의천연물 ACheE 저해활성에관한성분추적에서도알칼로이드성분의함유에따라저해활성을나타내는것으로보고되고있다 (36). 쇠비름의경우 5가지종류의알칼로이드성분인 oleracein A, B, C, D 및 E를함유하는것으로알려진바있으며 (5), 본연구에서쇠비름추출물에서 ACheE 저해활성을나타내는것은알칼로이드성분에따른결과로추정된다. 한편쇠비름의경우퇴행성질환의하나인치매의예방가능성을보여주었으나이에대한생리활성성분추적과보다구체적인 AChE 저해메카니즘연구와효능검증이수행되어야할것으로판단된다. 요약쇠비름의활용및생리활성을증가시킬수있는적정추출방법을알아보고자환류냉각, 가압가열및저온고압추출법을이용하여추출한쇠비름 70% 에탄올추출물의생리활성을비교하였다. 추출수율은환류냉각추출, 가압가열추출, 저온고압추출순으로높은수율을나타내었다. 색도에서는저온고압추출에서명도와황색도가높았고, 채도의경우가압가열및저온고압추출에서높았다. 폴리페놀및프로안토시아니딘함량은저온고압추출이환류냉각및가압가열추출에비해높은함량을나타내었으며, 플라보노이드함량은가압 가열추출에서높은함량을나타내었다. 항산화활성은저온고압추출이환류냉각및가압가열추출에비해유의적으로높은활성을나타낸반면철이온에대한제거효과의경우환류냉각추출및가압가열추출에서높은활성을나타내었다. α-glucosidase 및 XO 저해활성은저온고압추출에서가장높은활성을나타내었다. 한편 AChE 저해활성에서는저온고압추출, 가압가열추출, 환류냉각추출순으로높은활성을나타내었고, TLC bioassay를통하여살펴본결과특정 compound들에의해 AChE 저해활성이나타났으며퇴행성질환의하나인치매의예방가능성을확인하였다. 이러한결과를종합해볼때저온고압추출물이소재활용가치가높을것으로사료되며천연항산화제및기능성증진을위한소재로이용가능할것으로판단된다. 감사의글 본논문은 2013년도대구가톨릭대학교학부교육선진화선도대학지원사업에의한것으로감사드립니다. REFERENCES 1. Danrong Z, Yuqiong C, Dejiang N. 2009. Effect of water quality on the nutritional components and antioxidant activity of green tea extracts. Food Chem 113: 110-114. 2. Hung TM, Thung PT, Nhan NT, Mai NTT, Quan TL, Choi JS, Woo MH, Min BS, Bae KH. 2011. Cholinesterase inhibitory activities of alkaloids from Corydalis tuber. Nat Prod Sci 17: 108-112. 3. Yook CS. 1989. Coloured medicinal plants of Korea. Academic Press, Seoul, Korea. p 164. 4. Habtemariam S, Harvey AL, Waterman PG. 1993. The muscle relaxant properties of Portulaca oleracea are associated with high concentrations of potassium ions. J Ethnopharmacol 40: 195-200. 5. Xiang L, Xing D, Wang W, Wang R, Ding Y, Du L. 2005. Alkaloids from Portulaca oleracea L. Phytochemistry 66: 2595-2601.
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