[논문]동충하초균으로 발효한 인삼잎에서 분리한 중성다당의 면역활성 및 구조적 특성

차하영; 박혜령; 신광순

doi:10.9721/kjfst.2021.53.5.570

동충하초균으로 발효한 인삼잎에서 분리한 중성다당의 면역활성 및 구조적 특성
Immunostimulatory activity and structural characteristics of neutral polysaccharides isolated from ginseng leaves fermented by Cordyceps sinensis 원문보기

한국식품과학회지 = Korean journal of food science and technology, v.53 no.5, 2021년, pp.570 - 577

차하영 (경기대학교 식품생물공학과) , 박혜령 (경기대학교 식품생물공학과) , 신광순 (경기대학교 식품생물공학과)

초록
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동충하초균으로 발효한 인삼잎의 면역활성과 구조를 규명하기 위하여 조다당(GLF)를 분리하고 구성당과 당쇄 결합양식을 확인한 결과 C. sinensis 유래의 glucan이 주를 이루며 소량의 인삼잎 유래 pectic substances가 혼재되어 있을 것이라 추정하였다. 이온 교환 수지를 이용해 GLF로부터 중성다당체(GLF1)를 분리하였으며, 이를 lyticase, β-glucosidase 및 α-glucoamylase 효소를 처리한 결과, GLF1은 α-glucoamylase에 의해 가수분해되는 것을 확인함으로써 주로 α-glucan을 함유하고 있음을 추정할 수 있었다. 대식세포 분비능을 측정한 결과, 동충하초균으로 인삼잎을 발효하여 얻은 조다당 GLF가 단순 열수추출 조다당인 GLW보다 더 우수한 활성(Data not shown)과 수율을 보여주었으며, GLF와 GLF1 모두 농도 의존적으로 면역활성이 증가하는 경향과 동일 농도에서 유사한 높은 활성을 나타냈다. 한편 GLF1의 전체구조의 특성을 확인하기 위하여 isoamylase 및 α-amylase 효소를 처리한 결과, isoamylase 처리 획분인 PHI가 72.8%의 glucose로 구성되며 iodine-starch 반응이 증가하는 결과를 보였다. 반면, PHI의 αamylase 처리 후 분리한 PHIA1, PHIA2 및 PHIA3 획분에서는 iodine-starch 반응이 나타나지 않았으며, 이들의 대식세포 분비능을 확인한 결과 세 가지 획분 모두 어떠한 활성도 나타내지 않음을 확인하였다. 이상의 결과를 종합하면 동충하초 발효 인삼잎유래 중성다당은 α-(1→4)-glucan을 주쇄로 존재하며 C(O)-6 위치에서 측쇄가 연결되어 존재하는 α-glucan의 구조를 이루고 있으며, 이들의 면역활성은 α-glucan 전체 구조에서 기인하는 것임을 최종 확인하였다.

Abstract ▼ AI-Helper

This study elucidated the biological activities and structural characteristics of polysaccharides isolated from ginseng leaves fermented using Cordyceps sinensis (GLF). GLF comprised at least 18 glycosyl linkages, including 4-linked glucose residues (24.0%). To characterize the neutral polysaccharides in GLF, it was further fractionated by anion exchange chromatography, and the unabsorbed fraction (GLF1) was isolated. Peritoneal macrophages stimulated with GLF1 produced various cytokines in a dose-dependent manner. The properties and activities of the four subfractions (PHI, PHIA1-PHIA3) obtained after sequential enzymatic digestion were examined. PHI and PHIA3 primarily comprised glucose, whereas PHI exhibited an iodine-color reaction. Furthermore, the PHIA1-3 fractions indicated that cytokine production was completely inhibited. These results suggest at the immune activities of GLF1 may be due to the α-(1→4)-glucan branched at the C(O)6 position, which was produced by C. sinensis.

주제어

표/그림 (9)

그림 Fig. 1. Isolation and purification of immunostimulating polysaccharides from ginseng leaf fermented by Cordyceps sinensis.
그림 Fig. 2. Effect of GLF and GLF1 on IL-6, IL-12 and TNF-α production by murine peritoneal macrophage. Macrophages (2.0×10⁵ cells/ well) were treated at flat-bottomed 96-well microplate with GLF and GLF1 at a series of concentration (0.06-1,000 μg/mL) for 24 h. Not treated sample was used as the NC and LPS (1 μg/mL) as the PC. Means with different superscript letters (a-j) indicate significant differences at p<0.05 by Duncan's multiple range test.
표 Table 1. Chemical properties of GLF isolated from ginseng leaf
표 Table 2. Glycosidic linkage analysis of GLF isolated from fermented ginseng leaf
그림 Fig. 3. Reducing sugar measurement for confirmation of enzyme digestibility for GLF1 by lyticase, β-glucosidase and α- glucoamylase of GLF1. Means with different superscript letters (a-e) indicate significant differences at p<0.05 by Duncan's multiple range test.
$Fig. 4. Iodine-starch reaction of GLF1 and its subfractions derived by sequential enzymatic degradation.$ 그림 Fig. 4. Iodine-starch reaction of GLF1 and its subfractions derived by sequential enzymatic degradation.
$Table 3. Chemical properties of GLF1 and its subfractions derived by sequential enzymatic degradation$ 표 Table 3. Chemical properties of GLF1 and its subfractions derived by sequential enzymatic degradation
그림 Fig. 5. A possible structure of GLF1 purified from ginseng leaf fermented by Cordyceps sinensis.
$Fig. 6. Effect of GLF1 and its subfractions derived by sequential enzymatic degradation on IL-6, IL-12 and TNF-α production by murine peritoneal macrophage. Macrophages (1.4×10<sup>5</sup> cells/well) were treated at flat-bottomed 96-well microplate with sample at different concentration (25, 100 μg/mL) for 24 h. Not treated sample was used as the NC and LPS (1 μg/mL) as the PC. Means with different superscript letters (a-h) indicate significant differences at p<0.05 by Duncan's multiple range test.$ 그림 Fig. 6. Effect of GLF1 and its subfractions derived by sequential enzymatic degradation on IL-6, IL-12 and TNF-α production by murine peritoneal macrophage. Macrophages (1.4×10⁵ cells/well) were treated at flat-bottomed 96-well microplate with sample at different concentration (25, 100 μg/mL) for 24 h. Not treated sample was used as the NC and LPS (1 μg/mL) as the PC. Means with different superscript letters (a-h) indicate significant differences at p<0.05 by Duncan's multiple range test.

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