Inhibition of α-Glucosidase by a Semi-Purified Ethyl Acetate Fraction from Submerged-Liquid Culture of Agaricus blazei Murill

정관주(Kwan Ju Jung),문연규(Yeon Gyu Moon),권정민(Jung Min Kwon),안채린(Chae Rin Ahn),김정옥(Jeong Ok Kim),하영래(Yeong Lae Ha) 한국생명과학회 2011 생명과학회지 Vol.21 No.11

β-Glucan 분획을 제거한 신령버섯 균사체 액체 배양액(β-glucan-free HEAB)으로부터 새로운 형태의 α-glucosidase 활성저해 소재를 개발하였다. β-Glucan-free HEAB를 용매분획하여 10 mg/ml 농도에서 α-glucosidase 저해활성을 측정한 결과 EA분획이 가장 강한 활성(61.6% 저해)을 나타내었고, 경구혈당강하제인 acarbose (5 mg/ml; 39.5% 저해) 보다 강하였다. EA분획을 더 분획하여 α-glucosidase 저해활성이 80.4%인 분획을 얻었다. 이 분획에는 α-glucosidase 저해 활성을 갖는 daidzin 및 genistin과 같은 isoflavone과 isoflavone의 sugar conjugate된 물질이 함유되어 있었다. 따라서 α-glucosidase 저해 활성을 나타내는 β-glucan-free HEAB나 EA분획물은 인체의 혈당을 조절할 수 있는 소재로 활용될 수 있을 것이다. Natural anti-diabetic semipurified ethyl acetate fraction was isolated from the submerged-liquid culture of Agaricus blaze Murill (AB) in a medium containing soybean flakes. Hot-water extract of AB (HEAB) was prepared by extraction at 121℃ for 60 min, followed by filtering through a filter presser filled with diatomate. The β-glucan-free HEAB, which was a supernatant fraction from HEAB by precipitation in an 80% ethanol solution, was fractionated into hexane, chloroform, ethyl acetate, and butanol fractions. The inhibition of the α-glucosidase activity by fractions was 59.0, 17.0, 61.6, and 37.9%, respectively, suggesting that ethyl acetate fraction was the most active. A subfraction having a strong α-glucosidase inhibitory activity (80.4%) was isolated from the ethyl acetate fraction. This subfraction contained isoflavones (genistin and daidzin) and their conjugates with sugars as potent inhibiters of α-glucosidase activity. These results suggest that the ethyl acetate fraction or HEAB containing isoflavones and their sugars conjugates could be useful sources for controlling blood sugar levels in humans.

Hydrogen peroxide를 처리한 rat 배아심근 H9c2 세포에서 CLA의 oxidative stress 완화 효과

박재홍(Jae Hong Park),문연규(Yeon Gyu Moon),권정민(Jung Min Kwon),조용운(Yong Un Cho),김정옥(Jeong Ok Kim),하영래(Yeong Lae Ha) 한국생명과학회 2012 생명과학회지 Vol.22 No.12

Hydrogen peroxide (H2O2)를 처리한 rat cardiomyoblast H9c2 cell (rat 배아심근 세포)에서 conjugated linoleic acid (CLA)의 oxidative stress 경감 효과를 연구하였다. H9c2 세포는 DMEM/F-12 배지에서 배양(37℃, 5%CO2)하였다. CLA (10, 20, 30, 40, 50 μM)는 배양 6일간 H9c2 세포 증식을 거의 직선적으로 촉진하였다. 따라서 CLA (20 μM, 50 μM)를 H9c2 cell에 2일간 처리한 후 H2O2 (10 μM; 각각 1시간 및 2시간 처리)의 독성을 조사한 결과 CLA (20 μM, 50 μM)는 10 μM H2O2 1시간 처리에서 세포독성을 대조에 비해 유의성 있게 억제하였다(p<0.05). 또한 이 CLA는 apoptotic DNA 분포를 대조 세포에 비해 유의성 있게 감소 시켰다(p<0.05). 따라서 이 결과는 CLA가 H2O2에 의한 apoptosis를 억제함으로서 oxidative stress로부터 H9c2 세포를 보호한다고 사료되고, 이 CLA는 cardiac diseases를 보호할 수 있는 물질로 사용될 수 있을 것이다. 그러나 많은 연구가 수행되어야 할 것이다. Conjugated linoleic acid (CLA) exhibits several beneficial biological activities including anticarcinogenesis and body-fat reduction. Now, we report that CLA ameliorated the oxidative stress in rat cardiomyoblast cells, H9c2, treated with hydrogen peroxide (H2O2). Cells were cultured in DMEM/F-12 media at 37℃ with humidified atmosphere of 5% CO2. The cells, cultured for 48 hrs, were seeded at a density 3.5×103 cell/well in a 24 well-plate and incubated for 24 hr. Using these cells, two experiments were performed: the cytotoxicity test of CLA (10, 20, 30, 40, and 50 μMs), and the oxidative stress amelioration test of CLA (20 and 50 μMs) against cells treated with H2O2 (10 and 50 μMs) for 1 and 2 hrs. CLA enhanced the growth of H9c2 cells at any concentrations of CLA and at any incubation times (up to 6 days), indicating that CLA acts as a growth stimulant. No protective effect of CLA (20 and 50 μMs) was seen in cells treated 50 μM H2O2 for 1 and 2 hr, but these CLA concentrations ameliorated (p<0.05) the adverse effect of 10 μM H2O2 in cells treated for 1 hr. These CLA concentrations significantly (p<0.05) reduced the proportion of apoptotic cells, relative to control cells. These results suggest that CLA protected H9c2 cells from the oxidative stress of H2O2 through the suppression of cell apoptosis and could be a useful compound for the prevention of cardiac diseases caused by oxidative stress.

추출방법에 따른 한약재의 인체신경모세포 SK-N-SH 보호 효과

Jung Min Kwon(권정민),Yeon Gyu Moon(문연규),Young Suk Kim(김영숙),Ji Young Jung(정지영),Yeong Lae Ha(하영래),Jae Kyung Yang(양재경) 한국생명과학회 2011 생명과학회지 Vol.21 No.8

한약재의 추출방법에 따른 인체신경모세포 SK-N-SH에 대한 보호 효과를 연구하였다. 당귀, 건지황, 작약 및 천궁을 시료로 사용하였고, 열수추출(환류냉각, 5 시간), 증숙추출(100℃ 및 120℃, 90분 후 열수추출) 방법과 에탄 올추출(환류냉각, 5 시간)방법을 비교하였다. 추출물을 농도별로 SK-N-SH 세포에 2 시간 처리한 후 H₂O₂로 250 μM로 2 시간 산화스트레스를 유발한 다음 세포독성 및 apoptosis와 caspase-3의 발현 정도를 측정하였다. 모든 약재의 열수추출물이 다른 추출물보다 세포 증식을 촉진하였고, apoptosis를 억제하였다. 한약재 열수추출물 1 ㎍/㎕ 농도까지는 세포증식을 촉진하였지만, 그 이상의 농도에서는 오히려 감소시켰다. 열수추출물은 다른 추출물보다 총페놀성 화합물이 많이 함유되어 있었고, 항산화능이 높았다. 또한, 이와 같은 효과는 당귀의 열수추출물이 다른 약재 열수추출물보다 우수하였다. 본 연구결과는 약재의 열수추출법이 인체신경모세포인 SK-N-SH의 증식과 세포사멸 억제를 위해 가장 우수한 방법이었고, 당귀 열수추출물이 가장 우수한 효과를 지니고 있어 기억력 보호나 상실억제제로 활용할 수 있을 것이다. Extraction methods of medicinal plants were evaluated for growth enhancing effects of human neuroblastoma SK-N-SH cells. Hot-water extraction (reflux for 5 hr), hot-water extraction post treatment (100℃ or 120℃, 90 min) and ethanol extraction (reflux for 5 hr) methods were applied to Angelica gigas, Rhemania glutinosa, Paeonia lactiflora and Cnidium officinale samples to extract their constituents. Cells were treated for 2 hr with various concentrations of extracts (0, 0.5, 1, 2, 4 ㎍/㎕ media) prior to H₂O₂ (250 μM) treatment for 2 hr to provide oxidative stress. Cell viability, caspase-3 expression and apoptosis were measured for cells treated with sample extracts. Hot-water extract exhibited a stronger growth enhancing and apoptosis protecting ability than other extracts. These activities were shown at less than 1 ㎍/㎕ concentration, and not greater than 2 ㎍/㎕ concentration. Hot-water extract contained more polyphenolic compounds than other extracts coming along with stronger antioxidant activity. The efficacy of antioxidant activity was stronger in the hot-water extract of Angelica gigas than other hot-water extracts of medicinal plants. These results suggest that hot-water extraction is an appropriate method to extract materials for growth enhancing and apoptosis protection of SK-N-SH cells, and hot-water extracts of Angelica gigas might be useful materials for protection from aging brain cells.

Reduction of Visceral and Body Fats in Mice by Supplementation of Conjugated Linoleic Acid with γ-Oryzanol

Jae Il Byeon(변재일),Tae Woo Ohr(오태우),Young Suk Kim(김영숙),Yeon Gyu Moon(문연규),Cherl Woo Park(박철우),Jeong Ok Kim(김정옥),Yeong Lae Ha(하영래) 한국생명과학회 2008 생명과학회지 Vol.18 No.9

Conjugated linoleic acid (CLA)와 γ-oryzanol (OZ) 혼합물의 mouse 체지방 및 복부지방 감소에 관한 연구를 하였다. Female ICR mice (10주 령)을 몸무게 차이가 없도록 Control (70 μl olive oil + 30 μl CLA), CLA-OZ 1 (70 μl olive oil + 30 μl CLA + 0.5 ㎎ OZ), CLA-OZ 2 (70 μl olive oil + 30 μl CLA + 1.0 ㎎ OZ), OZ (100 μl olive oil + 1.0 ㎎ OZ) 및 Olive oil (100 μl olive oil)로 구분하였다. 처리시료는 매일 4주간 경구투여 하였고 식이와 물은 자유롭게 먹게 하였다. 시료처리 4주 후에 mice의 몸무게를 달고 탈골법으로 sacrifice하여 전체몸무게, 복부지방 무게, 장기와 복부지방을 제거한 나머지 부분 무게 (empty carcass weight: ECW)를 측정하였다. CLA (control) 처리는 olive oil 처리에 비해 유의성 있게 몸무게, ECW, 복부지방 및 체지방을 감소시켜 CLA가 복부지방 및 체지방을 감소시킴을 알 수 있었다. CLA-OZ 1 처리는 OZ 처리에 비해서는 복부지방 및 체지방을 유의성 (p<0.05) 있게 감소시켰으나 CLA (control) 처리에 비해서는 유의성이 없었다. 또한 CLA-OZ 2 처리는 control 처리 및 OZ 처리에 비해 복부지방 및 체지방을 유의성 (p<0.05) 있게 감소시켰다. 이 결과는 CLA와 OZ를 혼합하여 처리한 mice에서 이들을 각각 단독으로 처리한 mice에서보다 복부지방 및 체지방이 많이 감소되어 OZ가 CLA의 지방감소 작용에 상승효과가 있음을 의미한다. The synergistic effect of conjugated linoleic acid (CLA) and γ-oryzanol (OZ) on the reduction of visceral and body fats was investigated in mice. Female ICR mice, 10 weeks of age, were acclimated for one week and then randomly divided into 5 treatment groups by body weights: Control (70 μl olive oil + 30 μ CLA), CLA-OZ 1 (70 μl olive oil + 30 μl CLA + OZ 0.5 ㎎), CLA-OZ 2 (70 μl olive oil + 30 μl CLA + OZ 1.0 ㎎), OZ (100 μl olive oil + OZ 1.0 ㎎), and Olive oil (100 μl olive oil). Samples were daily intubated, p.o., for 4 weeks. Food and water were ad libitum. Four weeks later, mice were sacrificed by neck dislocation, followed by measuring whole body weight, empty carcass weight (ECW), which is weight without organs and visceral fats, visceral fats, body fats and protein content. Mice treated with CLA (control) sample maintained significantly, p<0.05, lower whole body weight, ECW, visceral and body fats, relative to mice treated with olive oil sample, indicating that CLA reduces the visceral and body fats. The CLA-OZ 1 treatment significantly reduced, p<0.05, visceral and body fats as compared to OZ treatment, but not significantly different from control treatment. Meanwhile, CLA-OZ 2-treated mice maintained significantly, p<0.05, lower visceral and body fats than control and OZ-treated mice. Protein contents in mice were not affected by any other treatments. These results suggest that OZ enhanced the reduction of visceral and body fats in mice by CLA.