초록 ▼

느타리버섯균사체배양물중의 숙취해소 및 혈중알콜농도감소 물질의 분리·동정 및 작용기작 구명을 위해 다음과 같은 실험을 하였으며, 혈중알콜감소효과가 큰 분획물로부터 nicotinamide adenine dinucleotide (NAD), genestein-O-β-D-glucan, diadzein-O-β-D-glucan, β-D-glucan이 분리ㆍ동정되엇다. 이들 분리된 화합물은 in vivo (mouse)에서 alcohol degydrogenase (ADH), acetaldehyde dehydrogenase (ALDH) 활성증진효과를

느타리버섯균사체배양물중의 숙취해소 및 혈중알콜농도감소 물질의 분리·동정 및 작용기작 구명을 위해 다음과 같은 실험을 하였으며, 혈중알콜감소효과가 큰 분획물로부터 nicotinamide adenine dinucleotide (NAD), genestein-O-β-D-glucan, diadzein-O-β-D-glucan, β-D-glucan이 분리ㆍ동정되엇다. 이들 분리된 화합물은 in vivo (mouse)에서 alcohol degydrogenase (ADH), acetaldehyde dehydrogenase (ALDH) 활성증진효과를 보였으며, in vitro 실험에서 genestein-O-β-D-glucan, diadzein-O-β-D-glucan, β-D-glucan첨가에 의한 ADH와 ALDH의 활성증진효과를 확인하였다.
1. 느타리버섯균사체액체배양
숙취해소효능 및 혈중알콜농도 감소효과를 최대화 할 수 있도록 하는 느타리버섯균사체 배양방법으로써 대두박, 황백당, 무기염 (MgSO₄, KH₂PO₄)을 기본배지로하여 소량배양에서 대량배양까지의 배양기술을 확립하였다.
2. 느타리버섯균사체추출균
사체배양물을 60℃에서 3시간 동안 자가분해시킨 다음, 100℃에서 다시 10시간동안 가열 추출한 다음, 배지중의 잔존 여과물을 규조토 여과법으로 제거하였다.
3. 느타리버섯균사체 추출물 (PO) 분획
가. 유기용매에 의한 분획
1) 유기용매별 분획
느타리버섯균사체추출물 (PO)을 hexane, chloroform, methanol (POM)로 분획하여, 이들 각 분획물의 혈중알콜감소효과를 in vivo (mouse)로 실험한 결과, POM 분획이 혈중알콜 감소효과가 가장 좋았다 (고형분 4.8 mg처리시 대조구에 비해 95.82%의 감소).
2) Methanol 분획물 (POM)을 다시 ethanol 농도별로 분획
Ethanol농도 40, 50, 60, 70, 80%의 용액에서 침전되는 물질을 분리하여, in vivo (mouse)로 실험한 결과, 80%에서 침전되는 분획물 (POM-80)이 혈중알콜감소 효과가 가장좋았다 (고형분 1.5 mg처리시 대조구에 비해 70.94%의 감소).
3) DEAE column에 의한 분획
POM-80을 DEAE-cellulose column으로 분리하였다. Elution된 각 분획물을 in vivo (mouse)로 실험한 결과 Tube 4와 5 분획물이 혈중알콜감소 효과가 가장좋았다 (대조구에 비해 Tube 4는 319.30%, Tube 5는 199.07%의 감소효과를 보임).
나. β-D-Glucan, glycoprotein, glycolipids
in vivo (mouse) 실험결과 β-D-Glucan은 74.7%의 혈중알콜농도 감소효과가 있었으며, glycoprotein은 47.2%, glycolipid 71.8%의 혈중알콜 감소효과가 있었다.
4. 혈중 알콜농도감소효과가 큰 분획물의 분리 및 동정
상기 분획물 중에서 혈중알콜농도 감소효과가 가장 크게 나타난 POM-80을 HPLC로 분리하여 얻은 RT 9분 peak (POM-80-9M)를 UV/VIS, IR로 확인한 결과 여기에는 β-D-glucan, β-D-glucan-O-genistein, β-D-glucan-O-daidzein, NAD가 함유되어 있었다. 이들 daidzein-O-β-D-glucan, genistin-O-β-D-glucan의 분자량은 18,000～20,000정도였다.
5. 혈중알콜농도감소 기작구명
가. Mouse 간 microsomal fraction의 alcohol dehydrogenase (ADH), acetaldehyde dehydrogenase (ALDH)활성도 증가POM-80처리구에서 121.4% 로 대조구 (commercial ADH 36 unit처리)보다 21.4%가 증가하였으며, ALDH의 경우 POM-80-9M 화합물을 23.4 μg처리하였을 때 23.4%가 증가하였다.
나. in vitro에서 POM-80의 첨가가 ADH activity (NADH생성량)에 미치는 영향:POM-80 1,182 μg을 처리한구에서의 ADH활성 (NADH생성량)은 352.52%로 약 3.5배가량 활성이 높아졌다.
다. NAD 첨가에 따른 ADH activity 증진효과: in vitro에서 NAD 1.4 ml까지 계속 ADH activity가 증가하였다.
라. Mouse liver microsome ADH와 ALDH측정
Gel electrophoresis로 POM-80 처리구와 POM-80-9M 처리구에서 ADH (40KDa), ALDH (35KDa) 생성량이 증가함을 확인하였다.

Abstract ▼

The compounds possesing the activities of sobering and the reduction of blood alcohol concentration in the extracts of mushroom (Pluorotus ostreatus) mycelia were identified and their mechanistic activities mere investigated. Nicotinamide adenine dinucleotide (NAD), genestein-O-β-D-glucan, diadzein-

The compounds possesing the activities of sobering and the reduction of blood alcohol concentration in the extracts of mushroom (Pluorotus ostreatus) mycelia were identified and their mechanistic activities mere investigated. Nicotinamide adenine dinucleotide (NAD), genestein-O-β-D-glucan, diadzein-O-β-D-glucan and β-D-glucan were identified from tile fraction showing the effects of sobering and the reduction of blood alcohol concentration. This fraction was prepared from the methanol extraction of mushroom mycelia (POM) and then precipitation with 80% ethanol (POM-80). Further separation was carried out by TLC, DEAE column chromategraphy, and HPLC (Bio Sep). The identification of these compounds were carried out using IR, UV/VIS, TLC and HPLC. The approximate molecular weight was determined with Toyopearl HW-65F column (HPLC). The isolated compounds enhanced the activities of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH), in vivo mouse experiment and in vitro.
1. Culture of mushroom mycelia (Pluorotus ostreatus)
Mushroom cycelia (Pluorotus ostreatus) was cultured to produce the culture medium containing the compounds possessing the sobering effects and the reduction of blood alcohol concentration.
2. Extraction of mushroom mycelia (Pluorotus ostreatus)
Mushroom mycelia (Pluorotus ostreatus) was autolyzed at the reaction condition of 60℃, 1hr, and then heated (100℃) for 10hrs. The extracts were filtered with diatomacious, designated (PO).
3. Fractionation of PO
A. Fractionations
1) Fractionation into organic solvents
PO was fractionated with hexane, chloroform, and methanol fraction (POM).The effcts on the reduction of blood alcohol concentration by treatment with POM was the best showing that the solid 4.8 mg treatment gave 95.8% reduction compared to the control (in vivo mouse experiment).
2) Precipitates of POM with ethanol (40, 50, 60, 70, 80%)
The treatment of precipitates of POM with ethanol (80%, solid 1.5 mg)(POM-80) showed 70.9% reduction of blood alcohol concentration, in vivo mouse experiment.
3) DEAE column chromatography of POM-80
Tube 4 and 5 showed the activity fer the reduction of blood alcohol concentration, 319.3% and 199.1%, respectively.
B. β-D-glucan, glycoprotein, glycolipids
β-D-glucan fraction reduced the blood alcohol concentration to 74.7%, in vivo (mouse) experiment. And glycoprotein and glycolipids reduced the blood alcohol concentration to 47.2% and 71.8%, respectively.
4. Isolation and identification of the compounds showing the activity for the reduction of blood alcohol concentration
The compounds related to the reduction of blood alcohol concentration were tentatively identified as β-D-glucan, genistein-O-β-D-glucan, daidzein-O-β-D-glucan and NAD. The identification was performed with UV/Vis. TLC, HPLC and IR. The molecular weights of genistein-O-β-D-glucan or daidzein-β-D-glucan was determined with Toyopearl HW65S column and was distributed to the range of 18,000～20,000 Da
5. Investigation of mechanism
A. The enhancement of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) in mouse microsomal fraction
The activity of ADH was increased to 121.4% by treatment of POM-80, so that the activity was increased by 21.39% compared to the treatemnt of commercial ADH 36 Unit. The activity of ALDH was increased by 23.4% by treatment of HPLC RT 9.717 min peak (POM-80-9M) (23.4μg).
B. The effects on the ADH activity (in vitro) by the treatment of POM-80.The activity of ADH was increased to the 352.5% by treatment of POM-80 (1,182μg).
C. The ADH activity was increased by addition of NAD up to 1.4 ml (in vitro experiment).
D. Gel electrophoresis was showed that the amount of ADH (40KDa), ALDH (35KDa) was increased by the addition of POM-80 (1,182 μg).

목차 Contents

• 표지...1
• 제출문...2
• 목차...3
• I. 연구개발결과 요약문...5
• 연구개발사업 최종보고서 요약문...5
• Project Summery...7
• II. 총괄연구개발과제 연구결과...9
• 1. 총괄연구개발과제의 최종 연구개발 목표...9
• 2. 총괄연구개발과제의 최종 연구개발 내용 및 결과...13
• 가. 느타리버섯균사체의 액체배양...13
• 나. 느타리버섯균사체 추출출 조제...13
• 다. 추출물의 일반성분...13
• 1) 건조물 함량...13
• 2) 무기물 함량...13
• 3) 총단백질과 구성아미노산의 함량...15
• 4) 지방산함량...16
• 5) 당함량...17
• 라. 추출물의 고분자화합물...18
• 1) 다당체(Polysaccharide)...18
• 2) 당단백질 (Glycoproteins)...22
• 3) 당지질 (Glycolipid)...23
• 4) 단백질과 펩타이드 (Protein and Peptide)...24
• 마. 추출물의 분획...25
• 1) 분획체계도...25
• 2) 유기용매에 의한 분획...25
• 바. Methanol 분획 (POM)의 세부분획...27
• 사. 혈중알코올농도 감소 효과...30
• 1) 임상실험...30
• 2) 동물실험...32
• 아. 유효성분의 분리 및 동정...39
• 1) TLC에 의한 분리...39
• 2) UV 흡광도분석...40
• 3) HPLC분석...44
• 4) IR분석...48
• 5) Isoflavone과 Isoflavone-aglycon의 정량...56
• 6) NAD?...58
• 7) POM-80의 분자량 측정...62
• 차. 혈중알콜농도감소 기작...63
• 1) NAD?첨가에 따른 ADH activity 증진효과...63
• 2) 동물실험...64
• 3) In vitro 실험...69
• 4) ADH 및 ALDH의 유전자발현...74
• 3. 총괄연구개발과제의 연구결과 고찰 및 결론...77
• 4. 총괄연구개발과제의 연구성과 및 목표달성도...79
• 5. 총괄연구개발과제의 활용계획...83
• 6. 참고문헌...84
• 7. 첨부서류...85