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1 Journal of Life Science 2014 Vol. 24. No ~1324 ISSN (Print) ISSN (Online) DOI : Isolation of nticarcinogenic Isoflavone-conjugated Glycoproteins from a Submerged Liquid Culture of garicus blazei Mycelia by the utolysis Process So Young Kim 2, Young Suk Kim 3, Joung Soon Jang 4, oh Hyun Kim 1, bdur Md. Rakib 1, Gon Sup Kim 5, Jeong Ok Kim 3 and Yeong Lae Ha 1 * 1 Department of pplied Chemistry, Institute of griculture and Life Science, Gyeongsang National University, Jinju , Korea 2 Department of Food and Nutrition, International University of Korea, Jinju , Korea 3 HK iotech, Co. Ltd., Jinju , Korea 4 Department of Internal Medicine, Chung-ng University College of Medicine, Seoul , Korea 5 Laboratory of iochemistry, School of Veterinary Medicine, Gyeongsang National University, Jinju , Korea Received November 7, 2014 /Revised December 8, 2014 /ccepted December 9, 2014 Most beta-glucans obtained from various fruit bodies of mushrooms and mushroom mycelial cultures have high-molecular weight glycoproteins, conjugated with beta-glucans. We report that isoflavone-conjugated glycolproteins (designated as gluvone) were isolated and exhibited stronger anticarcinogenic activities. garicus blazei mycelia (M) was cultured in a liquid medium containing soybean flakes for 14 days. The liquid culture was autolyzed by incubating at 53 (ph 5.5) for 3 h. crude glycoprotein (CGP) fraction with a cytotoxic effect on a mouse ascite cancer cell line (S-180) and a human breast cancer cell line (MCF-7) was isolated from the autolyzed M cultures by 80% ethanol treatment. Gluvone was isolated from the CGP with Sephadex G-75 column chromatography. It exhibited a stronger anticancer effect than CGP against the S-180 cell-induced female ICR mouse ascites carcinogenesis. Gluvone with 9,400 daltons was identified as a glycoprotein conjugated with isoflavone. ccording to HPLC and GC analysis, in conjunction with 1 H-NMR spectral analysis, it contained 60% carbohydrates (glucose, fructose, and ribose), 31% protein, and 2% isoflavone (daidzein and genistein), which is a novel material. These results indicate that a strong anticarcinogenic gluvone was isolated from the autolyzed product of a submerged liquid culture of M, suggesting that autolysis could be a useful tool to produce antitumor agents. Key words : garicus blazei mycelia (M), autolysis, MCF-7 cancer cells, S-180 cancer cells, submergedliquid culture 서 버섯은독특한맛과향기를갖는기호성식품이면서항암 효과, 항콜레스테롤효과등의생리활성을갖기때문에민간 전통한약으로도이용되고있다 [9, 20, 34, 40]. 특히표고버섯 (Lentinus edodes) [24, 45, 46], 구름버섯 (Coriolus versicolor)[32], 갓버섯 (Lepiota procera) [17], 잎새버섯 (Grifola frondosa) [8, 33] 및잔나비걸상버섯 (Ganoderma applanatum) [42] 등은항암효 과가뛰어난버섯으로보고되었고, 이들로부터분리한항암 성다당체는주로당단백질 (glycoprotein: GP) 로보고되었다. 암을치료및예방할목적으로항암성버섯자실체로부터 GP 를분리할수있지만, 이는고비용, 장시간및비균질화상 *Corresponding author *Tel : , Fax : * ylha@gnu.ac.kr This is an Open-ccess article distributed under the terms of the Creative Commons ttribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 론 태이기때문에, 저비용, 고효율및균질화된 GP를항암성버섯균사체액체배양으로생산하고있다 [14, 21]. 액체배양에이용되는버섯균은신령버섯 (garicus blazei: ), 목질진흙버섯 (Phellinus linteus), 송이버섯 (Tricholoma matsutake), C. versicolor, L. edodes, 느타리버섯 (Pleurotus ostreatus) 균등이이용되고있지만, 버섯균사체 (M) 배양물의항암효과가가장우수하였다 [7, 24, 41, 48]. 버섯은국내에서신령버섯, 아가리쿠스버섯, 또는흰들버섯으로불리며, 일본국립암센터는동물실험에서고형암에대한억제율이 99.4%, 완전치유율이 90% 인것으로보고하였다 [48]. 이들의주활성성분은 β-(1,6)-glucosyl 가지를가진 β-(1,3)-glucan으로고형암뿐만아니라복수암, S상결장암, 난소암, 유방암, 폐암, 간암등에효과가있고 [10, 13, 15, 26, 28, 29], 버섯에함유된 GP는탄수화물이 50.2% 이고단백질이 43.3% 로이들탄수화물과단백질복합체가항암활성이있다고하였다 [3, 6, 16, 27, 31, 40]. Mizuno 등 [30] 과 ack 등 [2] 은버섯균사체로부터중요한항암성 β-d-glucan을분리하였고, 그활성정도는평균분자량, 구조및용해도등에따라차이가있다고보고하였다 [2, 31, 38].

2 Journal of Life Science 2014, Vol. 24. No 본연구에서는지금까지 버섯자실체나 M 배양물로부터분리된항암성 GP와는상이한분자량이적고 isoflavone이결합된 GP, 즉, isoflavone-conjugated glycoprotein (Gluvone으로명명 ) 을분리하였다. M을액체배양하고, 이액체배양물이가지고있는효소를이용한자가분해로항암성이우수한 Gluvone을분리하였다. Gluvone의항암성은 mouse 복수암세포주인 S-180 세포, 인체유방암세포주인 MCF-7 세포및 S-180 세포로유도한 mouse 복수암에대해확인하였다. 재료및방법재료 버섯균주는농촌진흥청 (Suwon, Korea) 에서분양받아사용하였다. 대두박은태평양화학 (Seoul, Korea), 황백당은제일제당 (Seoul, Korea), KH 2PO 4, MgSO 4 7H 2O는 Shinyo사 (Osaka, Japan) 에서구입하였다. 7주령 female ICR mouse는 Life science사 (Daegu, Korea) 에서, S-180 세포와 MCF-7 세포는 Korea Cell Line ank (Seoul, Korea) 에서분양받아사용하였다. Fructose, galactose, glucose, maltose, ribose, sucrose, xylose, acetic acid, potato dextrose, daidzin, genistin, daidzein, genistein은 Sigma-ldrich사 (St. Louis. MO) 에서구입하였다. Diethyl ether, ethanol, hexane은 J. T. aker사 (Phillipsburg. NJ) 에서 HPLC grade를구입하였고, 그외사용된시약은 1급이상이었다. 버섯균사체배양 PD배지조제및배양 Potato dextrose ( g) 에황백당 30 g, acto-agar 20 g을첨가하고, 증류수를사용하여 1 l로보정하고살균 (121, 15 min) 한후, petri-dish에분주하여 PD 배지로사용하였다. 버섯균주를 PD배지에일정량접종하여항온기 (25 ) 에서배양하면서균사생장을측정하였다. 액체배양배지조제및배양기본배지 (1 l) 에황백당 20 g, 대두박분해물 ml, MgSO g, KH 2PO g을첨가하였다. 이액체배지를 500 ml 용량의삼각플라스크에 300 ml 분주하고살균 (121, 30 min) 하여액체배지로사용하였다. 액체배지에 PD 배지에서활성화한 M을접종하고진탕항온기 (120 rpm, 25 ) 에서 14일배양하였다. 배양물의자가분해자가분해조건상기에서배양한 M 액체배양물을반응온도 (53, 63 ), 반응 ph (4.5, 5.5, 6.5, 7.5), 반응시간 (1, 3, 5, 15, 24 hr) 에서진탕 (120 rpm) 하면서자가분해정도를측정하였다. 이들반응에서자가분해최적조건을확립하였다. 자가분해물의활성성분분획 M 액체배양물을자가분해 (53, ph 5.5, 120 rpm, 3 hr) 하였다. Fig. 1 에준하여자가분해한 M 배양물을열수추출 ( ) 하여여과한여액을진공농축 (10 배 ) 하였다. 이시료에 80% ethanol 용액이되도록 ethanol을첨가하고진탕 (120 rpm, 15 min) 한다음, 4 에서 24 hr 방치하였다. 침전물을분리하여활성성분분석시료 (crude glycoprotein: CGP) 로사용하였다. 활성성분분석 Fig. 1에서분리된 CGP를 Fig. 2에준하여 Sephadex G-75 column을이용하여분획하였다. CGP 시료 1 ml ( mg/ ml) 를 1% NaCl이충진된 Sephadex G-75 column (5 cm Mycelium Submerged Liquid Culture utolyze (53, ph 5.5, 120 rpm, 3 hr) Cook (hot water,, 2 hr) Filtrate Supernatant Filtrate Concentrate (1/10) dd EtOH 3 vol Centrifuge (10,000 rpm, 30 min) Precipitate dd EtOH 3 vol centrifuge (10,000 rpm, 30 min) Repeat (x 3) Crude glycoprotein (CGP) Cytotoxicity (MCF-7, S-180 cell) Fig. 1. flow chart for the isolation of CGP fraction from the submerged-liquid culture of M by autolysis. Crude glycoprotein (CGP) Fractionation - Sephadex column (G-75) chromatography - mobile phase: 1% NaCl 1 Tube #12 50 TLC, HPLC, GC, NMR Dialysis (membrane 6,000~8,000, 24 hr) C18 cartridge (clean-up:80% MeOH) Gluvone (Isoflavone-conjugated glycoprotein) ssay (MCF-7 cell, Mouse aseites Cancer) ctivity test Fig. 2. flow chart for the isolation of Gluvone from CGP.

3 1318 생명과학회지 2014, Vol. 24. No cm) 에 loading 한후 10 ml씩 50개를분취하였다. 이때사용된 mobile phase는 1% NaCl이었다. 분리한활성성분인 Gluvone의총탄수화물함량은페놀-황산법 [44], 총단백질함량은 radford 법 [23] 으로측정하였다. Gluvone의성분분석구성당분석구성당은가수분해한다음 HPLC [43] 및 GC [47] 로분석하였다. Gluvone 시료 10 mg을가수분해 (10 ml 6N-HCl, 6 hr) 한다음 10 ml의 6N-NaOH로중화한후단당류를 Dionex HPLC [column, Rezex RCM-Monosaccharide column ( mm); column temp., 75 ; mobile phase, deionized water; detector, RI; flow rate, 0.6 ml/min] 로분석하였다 [43]. 또한 Gluvone 시료 10 mg을가수분해 (1 M H 2SO 4, 90, 8 hr) 한다음, CaCO 3 로중화하였다. 그여액을원심분리하고, 농축시킨다음건조하였다. 건조된시료를 acetyl화 (c 2O- Pyridine, 90, 30 min) 하여생성된 alditol acetate를 GC (Model 6890, gilent Co. Ltd., C) 로분석하였다 [47]. 칼럼은 HP-5 capillary column (25 m 0.32 mm, i.d.) 을사용하였고, helium을 carrier gas로사용하였다. 온도는 150~240 (2 /min) program하였고, 최종온도에서 10분간분석하였다. Isoflavone 조성 Isoflavone의조성은 HPLC로분석하였다 [35]. Gluvone 시료 10 mg을가수분해 (10 ml 6N-HCl, 6 hr) 한다음 10 ml의 6N-NaOH로중화한후 methanol로추출하여 HPLC로분석 [Column, C18 column ( cm); column temp., 25 ; mobile phase, 0.1% ammonium acetate와 methanol (2:8 8:2); detector, UV (254 nm); flow rate, 1 ml/min] 하였다. 표준물질은 daidzin, genistin, daidzein, genistein을이용하였다. 아미노산조성시료 10 mg을 6N-HCl로가수분해 (6N-HCl 1 ml, N 2 gas 충진, 110, 24 hr) 하였다. 이가수분해물을냉각하고, deionized water 3 ml을첨가하여 HCl을감압으로제거 (3회반복세척 ) 하고건조하였다. 이시료를 0.3 M lithium hydroxide에용해하고여과한다음, 5 ml로정용한뒤 0.22 μm membrane filter로여과하고, 여액을아미노산분석기 (Hisashia Hirano) 로분석 [Column, Ultrapac 11 cation exchange resin (6 mm 200 mm); buffer flow, 45 ml/hr; ninhydrin flow, 35 ml/hr; column temp., 50~80 ; buffer step, 4 step; reaction bath temp., 130 ; buffer ph range, ; injection volume, 40 μl] 하였다. 분자량측정 TSK column ( cm) 이장착된 Dionex HPLC [P-680 pump; column temp., 25 ; mobile phase, deionized water; detector, RI; flow rate, 1 ml/min] 로분석하였다. Standard 물질로 dextran (5,000, 12,000, 25,000), maltoheptaose, maltotriose) 을이용하여 log scale로계산하였다. 암세포배양및세포독성측정 MCF-7 및 S-180 세포는 10% FS와 penicillin-streptomycin이함유된 RPMI 1640 배지를사용하여항온기 (5% CO 2, 37 C) 에서배양하였다 [1]. 세포는 Nunc culture dishe (35 mm, i.d., Rochester, NY) 에서 80% confluency까지배양하고 0.25% trypsin-2 mm EDT 처리한후, 원심분리 (1,000 rpm, 10 min) 하여세포를회수하였다. 세포를 96-well culture plate (Nunc) 에 well 당 세포를 seeding하고배지를첨가하여 24 hr 배양하였다. 배양된세포에서 serum의효과를제거하기위해배지를제거하고 5 μg/ml transferrin, 5 ng/ml sodium selenite와 0.1 mg/ml S가함유된배지를첨가하여 24 hr 배양하였다. serum-starved 세포에시료 (50 μg/ml deionized water) 50 μl를첨가하여 ascorbic acid (50 ng/ml), α-tocopherol phosphate (20 ng/ml) 가함유된 RPMI 1640 배지에서 48 hr 배양하였다, 세포독성은 MTT assay로측정하였다 [1]. 시료가처리된세포에 MTT 용액 (5 mg/ml PS) 을처리하고 4 hr 반응시킨후, MTT 용액을제거하고 200 μl DMSO를첨가하여 MTT formazan crystal을용해하였다. 570 nm에서흡광도를 nthos 2020 microplate reader (Wals, ustria) 로측정하여시료의세포독성을측정하였다. Mouse 항복수암실험 Mouse 항복수암실험은 Lee 등 [22] 의방법에준하여실시하였다. Female ICR mouse (6-7주령) 를 cage당 10마리씩넣고 ( 이때, cage당 mouse 평균무게가같게무작위로분배 ), 온도 (22±1 ) 와습도 (55±5%) 가유지되는사육장에서 1주일사육한후실험에사용하였다. 물과사료 (Chow diet) 는자유롭게먹도록하였다. Female ICR mouse 복강에서계대배양된 S-180 세포 ( 세포 /ml PS) 을복강에 0.1 ml 주사하여복수암을유발하였다. 복수암유발후 2일마다시료 0.2 ml (10 mg 시료함유 ) 을 mouse의복강에주사하였다. S-180 세포복강투여후 42일동안생존한 mouse의수와생존일수를기록하였다. 이동물실험은경상대학교동물실험윤리강령 (GR M0004) 에따라수행하였다. 통계처리 Data는평균 (Mean)± 표준편차 (SD) 로나타내었고, data의통계처리는 SS program을이용하여 NOV (nalysis of variance) test와 Duncan's multiple range test를하였다.

4 Journal of Life Science 2014, Vol. 24. No 결과 M 액체배양 PD배지에서계대배양중인 버섯균을지름 8 mm 크기로잘라배양할 PD 배지의중앙에올려놓고 24 항온기에서 14일간배양 ( 균사가약 90 mm 정도자람 ) 하였다. 이것을 M 액체배양원으로사용하였다. M을 14일간액체배양하면서 3, 7, 10, 14일에배양한배양액 ml를원심분리 (4, 5,000 rpm, 30 min) 하여균사체를회수하여무게를측정하였다. M의건물은배양기간에따라 4.1 g (3일). 5.8 g (7일), 13.1 g (10일), 20.0 g (14일) 으로증가하였다. 따라서 M을 14일간액체배양하여자가분해시료로사용하였다. M 액체배양물자가분해 M을 14일간액체배양한후, M 액체배양물을삼각플라스크 ( ml) 에넣고 53 와 63 의항온기에서 ph 4.5, 5.5, 6.5, 7.5의조건으로 1, 3, 5, 15, 24 hr 반응시킨다음자가분해효소활성을점도의변화로측정하였다 (Fig. 3). M 액체배양물은 53 의 ph 5.5에서 3 hr 반응에서점도가 18,750 ml/sec에서 1,500 ml/sec으로최대로감소 (12.5배) 하였다. 또한 M의다른반응조건 (53 와 63, ph와반응시간 ) 에서도 M 액체배양물의점도가감소되었지만, 그정도는 53 의 ph 5.5에서 3 hr 반응보다낮았다. 이결과로 M 액체배양물은자가분해되었음을알수있었고, 자가분해최적조건은 53 에서는 ph 5.5에서 3 hr 반응이었다. M 액체배양물을자가분해최적온도와 ph 조건 (53, ph 5.5) 에서 0, 1, 3, 5, 15, 24 hr 자가분해로분리한 CGP의 S-180과 MCF-7 세포에대한세포독성을비교하였다 (Fig. 4). 자가분해하지않은대조 CGP에비해 3 hr 자가분해하여얻은 CGP는 S-180 세포에대해 28%, MCF-7 세포에대해 42% 의세포독성을나타내었다. 그러나 3 hr 이하또는그이상반응시켜얻은 CGP의세포독성효과는증가하지않았다. 따라서세포독성을높이기위한 M 액체배양물의최적자 Table 1. Effect of CGP on the S-180 cell-induced mouse ascites carcinogenesis Treatment 1) Control CGP Gluvone Mean survival day 2) 19.2±0.5 c 22.3±1.0 b 26.9±0.9 a Survival rate Survival (%) 3) mouse 4) /10 1/10 3/10 1) Each treatment was consisted of 10 mice. Mice were given 10 mg sample/mouse/0.2 ml PS. Control mice were given S-180 cells and PS (0.2 ml). CGP represents a crude glycoprotein fraction obtained from the liquid culture of M by autolysis (53, ph 5.5, 120 rpm, 3 hr). Gluvone means isoflavone-conjugated glycoprotein purified from CGP by Sephadex G-75 column chromatography. 2) verage survival days of mouse until 42 days after treatment. Mean ± SD. Means with different small superscript letters represent significantly different at p<0.05 by Duncan s multiple range test. 3) Survival rate =[mean survival days of treatment mice/mean survival days of control mice]. 4) Numbers of mouse survived until 42 days after treatment. 기분해조건은 53, ph 5.5, 3 hr 이었다. Gluvone 의함암성 M 액체배양물의최적자가분해조건으로얻은 CGP 를 Sephadex G-75 column chromatography를이용하여분획하였다 (10 ml/tube) (Fig. 4). 각분획의 UV 흡광도를 280 nm 에서측정하였고, 280 nm에서흡광도를갖는 3개의분획 [1 (tube 5), 2 (tube 12) 및 3 (tube 19)] 을얻었다 (Fig. 4). CGP와 CGP로부터얻은분획 1, 2, 3의 MCF-7 세포에대한세포독성은 PS만을처리한대조군에비해분획 2가유의성있게 MCF-7 세포의생육을억제하였다 (Fig. 4C). 따라서이분획 2를 Gluvone으로명명하였다 (Fig. 2). S-180 세포로유도된 mouse의복수암에대해 Gluvone은강한항암효과를나타내었다 (Table 1). PS만을투여한대조처리군 mouse의평균수명은 19.2일이었고, 자가분해로얻은 Incubation time (hr) Incubation time (hr) Fig. 3. Effects of autolysis reaction conditions on the viscosity of the submerged-liquid culture of M at 53 ( panel) and 63 ( panel). Viscosity of M liquid culture autolyzed at ph 5.5 and 55 for 3 hr was significantly decreased, p<0.01, from that of other autolyzed conditions by Duncan s multiple range test. Detailed measurement condition was described in Materials and Methods.

5 1320 생명과학회지 2014, Vol. 24. No S-180 MCF nm C Growth rate (%) Relative Cell Number (% of control) Incubation time time (hrs) (hr) Control NO autolysis #5 #12 #19 Sample D bs isoflavone glucan Fraction Number glucan protein Fig. 4. Cytotoxic and structural characteristics of CGP and Gluvone. panel, cytotoxicity of CGP against S-180 and MCF-7 cells; panel, fractionation of CGP by Sephadex G-75 column chromatography; C panel, cytotoxicity of Gluvone (tube 12) on MCF-7 cells; and D panel, 1 H-NMR spectrophotometric analysis of Gluvone (500 MHz/DMSO). ars with different capital letters represent significantly different at p<0.05 by Duncan s multiple range test (C panel). Detailed measurement and analysis conditions were described in Materials and Methods. CGP 처리군은 22.3 일, CGP 로부터분리한 Gluvone 처리군은 26.9일이었다. 또한최종 42일간사육한결과 10마리중대조군은 0마리, CGP 처리군은 1마리, Gluvone 처리군은 3마리가생존하여 Gluvone 처리군의수명연장효과가높게나타났다. CGP 처리군의수명연장효과는 PS만을투여한대조군보다는강하였지만, Gluvone 처리군보다는약하였다. 이결과로자가분해에의해항암성이증가하였지만 Gluvone의항암성이더강하였음을알수있었다. Gluvone의화학적구성 Gluvone의화학적구성을조사하기위하여먼저일반성분을분석하였다 (Table 2). Gluvone 시료의고형분함량은 82.6% 였고고형분중총탄수화물이 60.6%, 총단백질이 31.0%, 총 isoflavone이 2.0% 였다 (Table 2). 1 H-NMR 분석에서 ppm의 glucan-protein complex Table 2. Chemical composition of Gluvone 1) Ingredient Total solid (%) Carbohydrate Protein Isoflavone Others 60.6±2.3 2)a 31.0±1.5 b 2.0±0.1 d 6.4±0.8 c 1) Gluvone means isoflavone-conjugated glycoprotein purified from CGP by Sephadex G-75 column chromatography. 2) Mean ± SD (n=3). Means with different small superscript letters represent significantly different at p<0.01 by Duncan s multiple range test. signal, ppm에서 glucan signal, ppm에서 isoflavone signal을확인하였다 (Fig. 4D). 이결과로 Gluvone은 isoflavone이 conjugated된 GP임을예측할수있었다 [36, 37]. 총탄수화물이 60% 함유된 Gluvone의구성당을 GC (Fig. 5) 및 HPLC (Fig. 5) 로분석하였고, 그구성비는 glucose가 51%, fructose가 47%, ribose가 2% 였다 (Table 3). Gluvone의 glucose 이외의 fructose는배지에첨가한황백당으로부터유래한것으로생각되었다. 그리고소량의 ribose는 M이생육하면서생성하는효소들에의해합성된것인지는정확히알수없었다. Table 3. Composition of monosaccharides and isoflavones in Gluvone 1) Component Composition (%) 3) Monosaccharide 2) Isoflavone 2) Glucose Fructose Ribose Daidzin Genistin Daidzein Genistein 50.9±2.1 3)a 47.1±3.8 b 2.0±0.2 c 18.9±0.9 C 24.4± ± ±0.5 C 1) Gluvone means isoflavone-conjugated glycoprotein purified from CGP by Sephadex G-75 column chromatography. 2) Monosaccharides and isoflavones were isolated from Gluvone by HPLC 3) Mean ± SD (n=3). Means with different small superscript letters or same capital letters represent significantly different at p<0.01 by Duncan s multiple range test.

6 Journal of Life Science 2014, Vol. 24. No Fig. 5. nalysis of monosaccharides from Gluvone by GC ( panel) and HPLC ( panel). Detailed analysis condition was mentioned in Materials and Methods. 또한 Gluvone에단백질이 31% 함유되어있었는데, 이단백질의아미노산을산가수분해하여분석하였다, 그결과 sx (21.9%), Glx (12.5%), Lys (7.8%), Phe (7.8%), Gly (7.2%), Thr (5.7%), la (5.7%), His (4.9%), Ser (4.9%), Leu (4.7%), rg (3.9%) Val (3.8%), Pro (3.6%), Cys (0.9%), Met (1.0%), Ile (2.8%), Tyr (0.9%) 으로나타났다. Gluvone 구성성분인 isoflavone을 HPLC로분석하였다 (Fig. 6). Glucose 배당체인표준품 daidzin과 genistin의 retention time은각각 9.5와 12.6 min이었고, glucose가없는비배당체표준품 daidzein, genistein의 retention time은각각 18.2, 22.2 min이었다. Gluvone 분해물로부터의 retention time 9.5, 12.6 min는각각 daidzin과 genistin으로확인되었고, retention time 18.0, 22.0 min은각각 daidzein과 genistein 으로확인하였다. 또한이들 daidzin, genistin, daidzein, genistein의구성비는각각 18.9, 24.4, 37.4, 19.3% 로나타났다 (Table 3). Gluvone의분자량을 dextran 표준품과 maltoheptaose, maltotriose 등을이용하여측정하였다 (Fig. 6). Dextran 25,000은 retention time이 8.5 min, dextran 12,000은 8.8 min, dextran 5,000은 9.2 min으로나타났으며, maltoheptaose의 retention time은 9.8 min, maltotriose는 9.9 min으로나타났다. Gluvone의 retention time은 9.0 min으로표준품들의 retention time과비교하여 Gluvone의분자량이약 9,400 Da으로측정되었다. 고찰본연구에서는 M을대두박이함유된배지에서배양한액체배양물을자가분해 (53, ph 5.5, 3 hr, 120 rpm) 하여분자량이 9,400 Da 정도의 Gluvone을세계최초로얻었다 (18). 이 Gluvone은자가분해전보다 MCF-7 및 S-180 세포에대한세포독성이강하였고, S-180 세포로유발한 mouse 복수암에대한항암성이크게증가하였다. Gluvone의항암성은 isoflavone과자가효소분해의해 Gluvone의분자량이작아졌기때문인일것으로추정되었다. Mizuno 등 [30] 은버섯균사체로부터항암성 β-d-glucan을분리하였고, 그활성정도는고분자물질의평균분자량, 구조및용해도등에따라차이가있다고보고하였다. 또한 Liu 등 [25] 은 Saccharomyces cerevisiae 반응물을자가분해 (55, ph 5.5, 120 rpm, 24 hr) 하여항암성이큰 β-d-glucan 생산하였고, 이는가수분해효소의작용으로세포내생물고분자물질이저분자물질로변화된다고보고하였다. 이과정에관여하는효소는 phospholipase, proteinase와 ribonuclease라하였다. 또한 Cho와 Choi [5] 는먹물버섯의자가분해과정에서버 Fig. 6. Determination of molecular weight ( panel) and analysis of isoflavones of Gluvone by HPLC ( panel). Detailed measurement and analysis conditions were mentioned in Materials and Methods.

7 1322 생명과학회지 2014, Vol. 24. No. 12 섯의세포질및세포벽이분해된다고보고하였으며, 분해과정에관련된효소로는 chitin 분해효소인 chitinase와밀접한관계가있다고보고하였다. 본연구에서도 M 액체배양물의자가분해로배양물의점도가 18,750 ml/sec에서 1,500 ml/sec(12.5배감소 ) 으로감소하였고, 이배양물로부터분리한 Gluvone은강한항암효과 (S-180과 MCF-7 세포및 S-180 세포로유발한 mouse 복수암 ) 를나타내었다. 버섯으로부터유래한 GP는 β-(1,3)-glucopyranose polymer에 β-(1,6)-glucopyranose의가지를갖고있는 β-d-glucan이단백질에 conjugation되어있어, 분자량이클수록항암효과가크다고알려져있다 [12, 23, 46]. 그러나본연구에서분리한 Gluvone은분자량이 9,400 Da로서분자량이수십만에달하는 GP보다작지만항암성이우수한것은 Liu 등 [25] 과 Cho와 Choi [5] 가보고한바와같이배양물에함유된여러효소에의해 GP 분자량이감소되어항암성이증가한다는보고와일치하였다. 또한 Gluvone의항암성이우수한것은 GP에 isoflavone이 conjugation되어있어 isoflavone의시너지효과일것으로추정되지만앞으로더연구가수행되어야할것이다. 항암성을갖는 GP의항암효과는손상된면역기능을회복시키거나촉진시키기때문인것으로알려져있다 [40, 48]. 이와같은활성을갖는 GP는 Lentinan ( 표고버섯자실체 ), PS-K (Krestin) [ 구름버섯 (Coriolus versicolor) 균사체 ], Schizophyllan [ 치마버섯 (Schizophyllum commune) 균사체 ], Licovek (C. versicolor의균사배양물 ), Kp 및 Calvaine ( 상황버섯균사체배양물 ) 등이보고되었다 [4, 11, 19, 22]. 그러나본연구에서분리한 Gluvone은이들 GP와는구조나특성이다르다. Gluvone에는 isoflavone이 conjugation되어있다는것이큰장점이다. 현재 isoflavone이 glucose에 conjugation되어있는지, 단백질에 conjugation 되어있는지는확실하지않아더연구가되어야할것이다. 결론적으로, 본연구에서는 M 액체배양물을자가분해 (53, ph 5.5, 120 rpm, 3 hr) 하여얻은 Gluvone은 MCF-7 및 S-180 암세포에대한강한세포독성을나타내었고, S-180 세포로유발한 mouse 복수암을강하게억제하였다. 이 Gluvone은분자량이약 9,400 Da의중분자로서 GP에 isoflavone 이 conjugation 되어있다. 따라서이 Gluvone은인체암예방및치료물질로활용할수있을것이다. 감사의글이연구는한국연구재단연구과제 (2013R ) 와중소기업청연구과제 (C ) 에의해수행되었음에감사드립니다. References 1. bdur, R. M., Kim, Y. S., Jang, W. J., Jang, J. S., Kang, S. J. and Ha, Y. L Preventive effect of t,t-conjugated linoleic acid on 12-O-tetradecanoyl-phorbol-13-acetate-induced inhibition of gap junctional intercellular communication in human mammary epithelial MCF-10 cells. J gic Food Chem 59, aek, S. J., Kim, Y. S., Chun, U. H., Lee, E. S. and Lee, J. W Properties of the high and low molecule of the proteoglycan extracted from Ganoderma lucidum IY009. Korean J Mycol 29, Chang, H. L., Chao, G. R., Chen, C. C. and Mau, J. L Non-volatile taste components of garicus blazei, ntrodia camphorata and Cordyceps militaris mycelia. Food Chem 74, Chihara, G., Hamuro, J., Maeda, Y., rai, Y. and Fukuoka, F Fractionation of the polysaccharides with marked antitumor activity, especially lentinan from Lentinus edodes (erk) Sing (an edible mushroom). Cancer Res 30, Choi, H. T., and Cho, C. W Ultrastructural studies on the autolysis of Coprinellus congregatus. Korean J Microbiol 41, Choi, J. M. and Koo, S. J Effects of β-glucan from garicus blazei Murill on blood glucose and lipid composition in db/db mice. Korean J Food Sci Technol 32, Gao, C., Zhong, L., Jiang, L., Geng, C., Yao, X. and Cao, J Phellinus linteus mushroom protects against tacrin-induced mitochondrial impairment and oxidatve stress in HepG2 cells. Phytomedicine 20, Cui, F. J., Tao, W. Y., Xu, Z. H., Guo, W. J., Xu, H. Y., o, Z. H., Jin, J. and Wei, Y. Q Structural analysis of anti-tumor heteropolysaccharide GFPS1b from the mycelia of Grifola frondosa GF9801. ioresour Technol 98, Dilani, D. D. S., Sylvie, R., Francoise, F., li, H.. and Kevin, D. H Medicinal mushrooms in supportive cancer therapies: an approach to anti-cancer effects and putative mechanisms of action. Fungal Divers 55, Dong, Q., Yao, J., Yang, X. T. and Fang, J. N Structural characterization of water-soluble β-d-glucan from fruiting bodies of garicus blazei Murr. Carbohydr Res 337, Han, M. W., Chung, K. S., Kim, S. S., Kim, H. S., Kim, K. Y. and Kim, K. H Effect of Kp, an antitumor protein-polysaccharide from mycelial culture of Phellinus linteus on the humoral immune response of tumor-bearing ICR mice to sheep red blood cells. rch Pharm Res 16, Irina, S., nissa, S., Sami, S., Thierry, G., Philippe, L., Catherine, M. G., Sarah, D., Pavel, S. D., lexander, O. C., lexander, S. S. and natolii, I. U Chemical structure and biological activity of a highly branched (1 3,1 6)-β- D-glucan from Isochrysis galbana. Carbohydr Polym 111,

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9 1324 생명과학회지 2014, Vol. 24. No Sugiyama, K., Saeki, S. and Ishguro, Y Hypercholesterolemic activity of ningyotake (Polyporus confleuens) mushroom in rats. J Jpn Soc Nutr Food Sci 45, Sung, J. M., Moon, H. W. and Park, D. S Growth condition of liquid culture by Pleurotus ostreatus. Korean J Mycol 27, Taichih, U., Yoshio, I. and Takashi, M Isolation and characterization of two kinds of heterogalactan from the fruit bodies of Ganoderma applanatum by empolying a column of concanvalin a sepharos-4. Carbohydr Res 92, Takuma, S. and Nobuo, T Further study of the structure of lentinen, antitumor polysaccharides from Lentinus edodes. Carbohydr Res 47, Tatiana, I. I., nna, V. S. and Tatiana, N. Z ntioxidant activity of fucose-containing sulfated polysaccharides obtained from Fucus evanescens by different extraction methods. J ppl Phycol DOI 10.7/s Unursaikhan, S., Lina, Z., Xiaojuan, X., Xufeng, Z., and Fanbo, Z Effects of molecular structure on antitumor activities of (1 3)-β-D-glucans from different Lentinus edodes. Carbohydr Polymers 63, Wasser, S. P Medicinal mushrooms as a source of antitumor and immunomodulating polysaccharides. ppl Microbiol iotechnol 60, Yun, J. S., Jeon, H. S., Kim, I. S., Lee, H. J., Lee, H. J., Hyun, J. Y. and Kim, J Simultaneous determination of fructose, glucose, and sucrose in honey and commercial drinks by GC and GC/MS. Korean J Food Sci Technol 45, Zhang, M., Cui, S. W., Cheung, P. C. K. and Wang, Q ntitumor polysaccharides from mushrooms: a review on their isolation process, structural characteristics and antitumor activity. Trends Food Sci Technol 18, 초록 : 신령버섯균사체액체배양물의자가분해에의한항암성 isoflavone-conjugated glycoprotein 분리김소영 2 김영숙 3 장정순 4 김보현 1 라키브압두르 1 김곤섭 5 김정옥 3 하영래 1 * ( 1 경상대학교대학원응용생명과학부, 2 한국국제대학교식품과학과, 3 HK바이오텍, 4 중앙대학교내과학교실, 5 경상대학교수의과대학생화학교실 ) 신령버섯균사체 (garicus blazei mycelia: M) 를대두박이함유된액체배지에배양하고, 이것을자가분해 (53, ph 5.5, 120 rpm, 3 hr) 하여항암성이강한 isoflavone-conjugated glycoprotein (Gluvone 이라명명 ) 을분리하였다. Gluvone 은지금까지알려진당단백질과는달리분자량이작고 (9,400 Da), isoflavone이결합되어있다는점이다르다, Gluvone 은 60% 탄수화물 (glucose, fructose, ribose), 31% 단백질및 2% isoflavone (daidzein, genistein) 으로구성되어있었다. 이 Gluvone은 S-180 복수암세포, MCF-7 인체유선암세포에대한독성이강하였고, S-180 세포로유발한 mouse 복수암을강하게억제하였다.