Journal of the Korean Society of Food Science and Nutrition (한국식품영양과학회지)

The Korean Society of Food Science and Nutrition (한국식품영양과학회)
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Pharmacological Activity of Chaga Mushroom on Extraction Conditions and Immunostimulating Polysaccharide

추출조건에 따른 차가버섯 생리활성 및 면역활성 다당

  • Received : 2012.06.19
  • Accepted : 2012.07.04
  • Published : 2012.10.31
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Abstract

To investigate the pharmacological activity of chaga mushroom (Inonotus obliquus) on extraction conditions, chaga was extracted using water (reflux at $50^{\circ}C$, decoction over $90^{\circ}C$, pressure at $121^{\circ}C$) or ethanol (reflux at 50, 70, or $90^{\circ}C$). When water extract was further fractionated into crude polysaccharide (IO-CP), yields of IO-CP (4.8~16.8%) were higher than those of ethanolic extracts (IO-E, 1.9~2.7%) at increased temperature. For antioxidant activity, crude polysaccharide (IO-CP-121) obtained by pressurized extraction showed the highest polyphenolic and flavonoid contents (35.10 mg TAE/g and 18.48 mg QE/g, respectively) as well as DPPH and ABTS free radical scavenging activities (26.08 and 27.99 mg AEAC/100 mg, respectively). Meanwhile, IO-CP-D (decoction) and IO-CP-50 (reflux) had more potent mitogenic effects (2.10- and 1.95-fold of saline control at 100 ${\mu}g/mL$) as well as intestinal immune system modulating activities (6.30- and 5.74-fold) compared to IO-CP-121, whereas ethanolic extracts showed no activity. Although no IO-CP showed cytotoxicity against RAW 264.7 cells at 0.1 mg/mL, IO-CP-121 significantly inhibited TNF-${\alpha}$ and NO production as pro-inflammatory factors in LPS-stimulated RAW 264.7 cells (29.2 and 63.5%, respectively). Ethanolic extracts also showed no cytotoxicity at 0.1 mg/mL, whereas inhibition of TNF-${\alpha}$ and NO production was significantly low compared to that of IO-CP-121. In addition, active IO-CP-D was further fractionated into an unadsorbed (IO-CP-I) and seven adsorbed fractions (IO-CP-II~VIII) by DEAE-Sepharose CL-6B column chromatography in order to isolate immunostimulating polysaccharide. IO-CP-II showed the most potent mitogenic effect and macrophage stimulating activity (4.51- and 1.64-fold, respectively). IO-CP-II mainly contained neutral sugars (61.86%) in addition to a small amount of uronic acid (2.96%), and component sugar analysis showed that IO-CP-II consisted mainly of Glc, Gal, and Man (molar ratio of 1.00:0.55:0.31). Therefore, extraction conditions affect the physiological activity of chaga, and immunostimulating polysaccharide fractionated from chaga by decoction is composed mainly of neutral sugars.

추출조건에 따른 생리활성 영향을 검토하기 위하여 차가버섯(Inonotus obliquus)을 물($50^{\circ}C$ reflux, $90^{\circ}C$ 이상 decoction, $12^{\circ}C$ 가압추출)과 에탄올(50, 70과 $90^{\circ}C$ reflux)로 추출한 후 물 추출물을 조다당획분(IO-CP)으로 분획하였을 때 IO-CP(4.8~16.8%)의 수율은 에탄올추출물(IO-E, 1.9~2.7%)보다 높았으며 추출온도 증가에 따라 그 차이는 더 컸다. 가압추출로 조제된 조다당획분(IO-CP-121)은 가장 높은 총 폴리페놀 및 플라보노이드 함량(35.10 mg TAE/g와 18.48 mg QE/g)과 DPPH와 ABTS를 이용한 자유 라디칼소거능(26.08과 27.99 mg AEAC/100 mg)을 나타내었다. IO-CP-D(decoction)와 IO-CP-50(reflux)은 IO-CP-121보다 유의적으로 높은 마이토젠(saline 대조군의 2.10과 1.95배, 100 ${\mu}g/mL$)과 장관면역 활성(6.30과 5.74배)을 보였으나 에탄올추출물 활성은 확인되지 않았다. 또한, 모든 IO-CP는 0.1 mg/mL 농도에서 RAW 264.7 세포주에 대하여 독성을 나타내지 않았으나, IO-CP-121이 LPS-자극 RAW 264.7 세포주에 대하여 가장 유의적으로 염증성 인자인 TNF-${\alpha}$와 nitric oxide(NO) 생성을 억제하였다(29.2와 63.5%). 에탄올추출물도 0.1 mg/mL 농도에서 독성을 보이지 않았으나 TNF-${\alpha}$와 NO의 생성 억제능은 IO-CP-121보다 현저히 낮음을 알수 있었다. 한편, 활성 다당획분을 분리하기 위하여 면역활성 획분인 IO-CP-D를 DEAE-Sepharose CL-6B column에서 비흡착 획분(IO-CP-I)과 7개의 흡착 획분(IO-CP-II~VIII)으로 분획한 결과, IO-CP-II가 가장 우수한 마이토젠과 마크로파지 활성(4.51과 1.64배)을 나타내었다. 이와 같이 분리된 면역활성 획분인 IO-CP-II는 주로 중성당(61.86%)과 함께 소량의 산성당(2.96%)을 포함하고 있는 다당류임이 밝혀졌으며, 주요 구성당으로서 Glc, Gal와 Man(molar ratio of 1.00:0.55:0.31)를 포함하고 있음을 확인할 수 있었다. 이러한 결과로부터 추출조건은 차가버섯 생리활성에 중요하게 작용하고 있으며, decoction 추출방법으로 제조하여 분획한 면역활성 획분으로부터 차가버섯 면역활성은 적어도 중성 다당류가 관여하고 있음을 알 수 있었다.

Keywords

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