감초폴리페놀 추출효율에 있어 열처리, 에탄올 농도, 추출시간 및 용매비율이 미치는 영향 탐색

채정일,류경선,서강석,김경훈,오영균,장선식,최창원,최낙진,Chae, Jung-Il,Ryu, Kyeong-Seon,Seo, Kang-Seok,Kim, Kyung-Hoon,Oh, Young-Kyoon,Jang, Sun-Sik,Choi, Chang-Weon,Choi, Nag-Jin Korean Association of Organic Agriculture 2012 韓國有機農業學會誌 Vol.20 No.3

Effects of pretreatment and extraction conditions on total polyphenol yield from licorice root were investigated using statistical method. For pretreatment, heat treatment at $121^{\circ}C$ for 10 min was applied. Licorice root content in solvent (10, 20, and 30%) ethanol concentration (20, 40, and 60%) and reaction time (1, 2, and 3 h) were used as variables for extraction conditions. Two experiments, with heat treated and no treated licorice, were prepared with same experimental design. Box behnken design was employed and produced a total of 15 trials. Total polyphenol yield from licorice root was not affected by heat treatment. Among variables, licorice content in solvent showed most significant effect regardless of other variables (p<0.05). Finally, optimum conditions for the extraction of total polyphenol from licorice root was detected as following: 10% of licorice in solvent, 52% ethanol as solvent, 2 h of reaction time and non-heat treatment and the extraction yield from optimized condition was 17.6 mg/g licorice root.

토양 균주 발효 추출물 Nargenicin 및 그 유도체의 항생제 대체 효과능 평가

조승식,홍준희,채정일,심정현,나종삼,유진철,Cho, Seung-Sik,Hong, Joon-Hee,Chae, Jung-Il,Shim, Jung-Hyun,Na, Chong-Sam,Yoo, Jin-Cheol 한국유기농업학회 2014 韓國有機農業學會誌 Vol.22 No.3

본 연구진은 신규 미생물인 Nocardia sp. CS682 균주에서 항균물질인 nargenicin을 확보하고, 그 유도체 5종을 확보하여 그람양성, 음성 및 다약제 내성균에 대한 항균 효능 및 LPS로 자극된 대식세포에서의 nitric oxide 생성 억제능을 확인하였다. Nargenicin 유도체들은 nargenicin 및 vancomycin에 비교하여 우수한 항균 활성을 보여주었으며, compound 4 및 5는 광범위한 항균 효능 외에 nitric oxide 생성 억제능을 보여 항균-항염효과를 가지는 dual effector로써 감염, 면역 질환에 응용 가능성을 시사하였다. Nargenicin 유도체들은 향후 염증반응에서의 면역 조절 기작에 대한 추가 연구가 필요할 것으로 사료된다. IIn vitro antimicrobial and anti-inflammatory activities of nargenicin and its derivatives were investigated. Nargenicin, an unusual macrolide antibiotic with potent anti-MRSA (methicilin-resistant Staphylococcus aureus) activity, was purified from the culture broth of Nocardia sp. CS682. And variety of novel nargenicin derivatives was synthesized from nargenicin. Two compounds (4 and 5) exhibit a broad spectrum of antimicrobial activities against infectious bacteria. The antimicrobial activity of derivatives against fifteen organisms was assessed using the minimum inhibitory concentration (MIC). The MIC values were in the ranges of $0.15{\sim}80{\mu}g/mL$ (w/v) for compound 1 and 2, $5{\sim}80{\mu}g/mL$ (w/v) for compound 3, $1.25{\sim}40{\mu}g/mL$ (w/v) for compound 4, and $1.25{\sim}80{\mu}g/mL$ (w/v) for compound 5, depending on the pathogens studied. In vitro, we investigated cytotoxicity and inhibition of nitric oxide (NO) production of synthesized compounds 1-5 in Raw 264.7 cells. LPS-induced nitric oxide releases were significantly blocked by compound 3, 4 and 5 in a dose-dependent manner. At high concentrations ($5{\mu}g/mL$) compound 5 inhibited the NO production by 95%. Compound 4 inhibited the release of NO in LPS-activated Raw 264.7 cells by 75% at the concentration of $10{\mu}g/mL$. Compound 3 inhibited the release of NO in LPS-activated Raw 264.7 cells by 65% at the concentration of $100{\mu}g/mL$. On the other hand, nargenicin, compound 1 and 2 did not inhibit NO production. These results demonstrated that compound 4 and 5 displayed antimicrobial activity and blocked LPS-induced pro-inflammatory mediators such as NO in macrophages, which might be responsible for its therapeutic application.

Proteomics-based Identification of Components in the Adventitious Roots of Panax Ginseng C. A. Mayer related to Energy Metabolism and Antibiotic Effects

조진형,전영주,이라함,심정현,채정일,Cho, Jin-Hyoung,Jeon, Young-Joo,Lee, Ra-Ham,Shim, Jung-Hyun,Chae, Jung-Il Korean Association of Organic Agriculture 2014 韓國有機農業學會誌 Vol.22 No.1

Korean Panax ginseng C. A. Meyer (P. ginseng) is a well-known and one of the most important tonic herbs used in traditional Korean medicine. The pharmacological effects of P. ginseng have been reported by many researchers. Nevertheless, little is known between the mechanism of action and the active compounds. In this study, we performed a comprehensive proteomic analysis and protein categorization in order to understand the physiological characteristics of the major components in the adventitious roots of P. ginseng. Whole proteins extracted from the cultured adventitious roots of P. ginseng were separated by two-dimensional polyacrylamide gel electrophoresis (2-DE). Among the 1000 spots which were detected by silver staining, 113 spots were labeled and identified by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF MS). Our results showed that 40 proteins were identified among the 113 spots, with a hit ratio of 35.3%. A number of proteins identified on the 2-DE gels (30%; 16 spots) were involved in energy metabolism. These proteomic data will be helpful to better understand the physiological and pharmacological effects of P. ginseng.

구강편평세포암종 세포에서 감초 유래 Liquiritigenin의 항증식, 세포주기 정지 및 세포사멸 유도

곽아원(Ah-Won Kwak),윤구(Goo Yoon),채정일(Jung-Il Chae),심정현(Jung-Hyun Shim) 한국생명과학회 2019 생명과학회지 Vol.29 No.3

Liquiritigenin (LG)은 licorice 뿌리에서 분리된 chiral flavonoid이다. LG는 항산화, 항암 및 항염증 효과를 포함하여 다양한 생물학적 활성을 가지고 있다. 구강편평세포암종에서 LG의 항암 활성은 아직 밝혀지지 않았다. 본 연구에서는 구강편평상피암 세포(HN22)에서 LG의 항암 효능을 시험하였다. HN22 세포에 LG를 처리하여 MTT분석으로 세포 생존율을 평가하였으며, Annecin V/7-Aminactinomycin D 염색, 세포주기 및 Multi-caspase 활성을 Muse™ cell Analyzer로 분석하여 세포사멸 유도를 확인하였다. 분석결과, 구강편평상피암 HN22 세포에 LG를 처리시 G2/M 세포주기 정지를 일으켰으며, Western blotting 통하여 cyclin B1 및 CDC2 발현 감소와 p27 발현증가를 확인하였다. LG는 활성산소종의 생성을 유발하고, CCAAT/enhancer-binding protein homologous protein(CHOP) 및 78-kDa glucose regulated protein (GRP78)의 발현을 농도의존적으로 유도하였다. HN22 세포에 LG의 처리는 미토콘드리아 막전위의 손실(△Ψm)을 일으켰다. LG를 처리한 HN22 세포의 단백질 분석결과 apoptotic protease activating factor-1 (Apaf-1), cleaved Poly (ADP-Ribose) Polymerase (C-PARP) 및 Bax의 발현을 증가함을 확인하였다. 따라서 우리의 결과는 LG이 구강편평상피암 세포의 세포사멸을 유도하여 항암제 역할을 할 수 있는 천연 화합물임을 시사한다. Liquiritigenin (LG) is a chiral flavonoid isolated from the roots of licorice. It exhibits multiple biological activities including anti-oxidant, anti-cancer, and anti-inflammatory effects. In particular though, the anti-cancer activity of LG in oral squamous cell carcinoma has yet to be elucidated, and LG-induced apoptosis in oral squamous cell carcinoma remains poorly understood. In the present study, we tested the role of LG in inducing apoptosis in oral squamous cell carcinoma cells. LG treatment of HN22 cells resulted in a dose-dependent inhibition of cell viability as detected by a 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide assay. The induction of apoptosis in terms of Annexin V/7-Aminoactinomycin D staining, sub-G1 population, and multi-caspase activity were assessed with a Muse<SUP>TM</SUP> Cell Analyzer. Flow cytometric analysis revealed that LG treatment resulted in G2/M arrest in cell cycle progression and downregulation of cyclin B1 and CDC2 expression in a concentrationdependent manner. It also resulted in significant upregulation of p27. In addition, LG was seen to trigger the generation of reactive oxygen species and induce CCAAT/enhancer-binding protein homologous protein and 78-kDa glucose-regulated protein in concentration-dependent upregulation. The LG treatment of HN22 cells led to a loss of mitochondrial membrane potential (△Ψm); it also reduced the levels of anti-apoptotic protein and increased the expression of apoptotic protease activating factor-1, cleaved poly (ADP-ribose)polymerase and Bax. Overall, our results indicate that the pro-apoptotic effects of LG in HN22 cells depend on the activation of both intrinsic and extrinsic signaling pathways. Thus, our results suggest that LG constitutes a natural compound with a potential role as an anti-tumor agent in oral squamous cell carcinoma.

Gefitinib-민감성 또는 내성 비소세포폐암 세포에서 Licochalcone C에 의한 자가포식 유도

오하나(Ha-Na Oh),윤구(Goo Yoon),채정일(Jung-Il Chae),심정현(Jung-Hyun Shim) 한국생명과학회 2019 생명과학회지 Vol.29 No.12

감초(Glycyrrhiza inflata)의 뿌리에서 분리된 Licochalcone (LC)은 항염증 및 항종양과 같은 많은 약리학적 효과를 가지고 있다. 현재까지 LCC는 구강암과 방광암에서 연구되었지만 폐암에서의 연구는 밝혀진 바 없다. 또한, 암에서 LCC에 의해 유도된 autophagy에 대한 연구는 없었다. 본 연구는 gefitinib-민감성 또는 내성을 갖는 폐암세포에 대한 LCC의 효과 및 작용 메커니즘을 조사하기 위해 고안되었다. MTT 분석 데이터는 LCC가 비소세포폐암 세포주인 HCC827 (gefitinib-민감성) 및 HCC827GR (gefitinib-내성)에서 세포생존율을 유의하게 억제함을 보여주었다. 흥미롭게도, Annexin V/7-aminoactinomycin D 이중 염색 및 세포주기 분석에서 가장 높은 농도의 LCC 처리는 apoptosis를 유도하는 비율이 약 10%였다. LCC는 비소세포폐암 세포주에서 세포주기 G2/M 관련 단백질인 cyclin B1 및 cdc2의 발현을 감소시킴으로써 G2/M 정지를 야기하였다. LCC의 처리는 autophagy marker 단백질인 microtubule-associated protein 1 light chain 3 (LC3) 및 autophagy과정에 관여하는 단백질인 autophagy-related gene (Atg)5의 발현을 증가시킴으로써 autophagy를 유도하였다. 또한, LCC는 reactive oxygen species (ROS)의 생성을 증가시켰으며, ROS 억제제인 N-acetyl-L-cysteine (NAC)에 의해 세포생존율이 부분적으로 회복되었다. Western blotting 분석에서, NAC과 LCC의 동시처리에 의해 cdc2의 발현이 증가하고 LC3의 발현은 감소되었다. 이러한 결과는 LCC가 비소세포폐암에서 ROS-의존적 G2/M 정지 및 autophagy를 유도함으로써 항종양 효과에 기여할 수 있음을 나타낸다. 결론적으로, LCC 치료는 비소세포폐암에 대한 잠재적 치료제로서 유용할 수 있다. Licochalcone (LC), isolated from the roots of Glycyrrhiza inflata has multiple pharmacological effects including anti-inflammatory and anti-tumor activities. To date, Licochalcone C (LCC) has induced apoptosis and inhibited cell proliferation in oral and bladder cancer cells, but lung cancer has not yet been studied. In addition, no study reported LCC-induced autophagy in cancer until now. The present study was designed to investigate the effect of LCC on gefitinib-sensitive and -resistant lung cancer cells and elucidate the mechanism of its action. The 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay data showed that LCC significantly inhibited cell viability in non-small cell lung cancer (NSCLC) HCC827 (gefitinib-sensitive) and HCC827GR (gefitinib-resistant) cell lines. Interestingly, Annexin V/7-aminoactinomycin D double staining and cell cycle analysis showed an apoptosis rate within about 20% at the highest concentration of LCC. LCC induced G2/M arrest by reducing the expression of the cell cycle G2/M related proteins cyclin B1 and cdc2 in NSCLC cell lines. Treatment of LCC also induced autophagy by increasing the expression of the autophagy marker protein microtubule-associated protein 1 light chain 3 (LC3) and the protein autophagy-related gene 5 involved in the autophagy process. In addition, LCC increased the production of reactive oxygen species (ROS), and the cell viability was partially restored by treatment with the ROS inhibitor N-acetyl-L-cysteine. In western blotting analysis, the expression of cdc2 was increased and LC3 was decreased by the simultaneous treatment of NAC and LCC. These results indicate that LCC may contribute to anti-tumor effects by inducing ROS-dependent G2/M arrest and autophagy in NSCLC. In conclusion, LCC treatment may be useful as a potential therapeutic agent against NSCLC.

발효, 세포배양, 생물공정 세포 크기 차이를 이용한 유세포 분석을 통한 인간배아줄기세포 유래 기능성 혈관세포의 확립

이지혜 ( Ji Hye Lee ),김주미 ( Ju Mi Kim ),정형민 ( Hyung Min Chung ),채정일 ( Jung Il Chae ) 한국미생물생명공학회 2011 한국미생물·생명공학회지 Vol.39 No.4

Human embryonic stem cells have been highlighted as a valuable cellular source in the regenerative medicine field, due to their pluripotency. However, there is the challenge of the establishment of specific functional cell type forms of undifferentiated human embryonic stem cells (hESC). To establish and purify functional cell types from hESCs, we differentiated undifferentiated hESCs into vascular lineage cells and sorted the specific cell population from the whole cell population, depending on their cell volume, and compared them with the non-sorted cell population. We observed that about 10% of the PECAM positive population existed in the VEGF induced differentiating human embryoid body (hEB), and differentiated hEBs were made into single cells for cell transplantation. After making single cells, we performed cell sorting using a fluorescence-activated cell sorter (FACs), according to their cell volume on the basis of FSC region gating, and compared their therapeutic capacity with the non-sorted cell population through cell transplantation into hindlimb ischemic disease model mice. 4 Weeks after cell transplantation, the recovery rate of blood perfusion reached 54% and 17% in the FSC regions of sorted cells- and non-sorted cells, respectively. This result suggests that derivation of a functional cell population from hESCs can be performed through cell sorting on the basis of cell volume after preliminary differentiation induction. This approach may then greatly contribute to overcoming the limitations of marker sorting.

Effect of Mature Miscanthus sacchariflorus var. No. 1 on In Vitro Rumen Fermentation Characteristics and Its Dry Matter Digestibility

Sang Buem Cho(조상범),David Tinotenda Mbiriri,Sung Jin Oh(오성진),A Reum Lee(이아름),Jin Ho Yang(양진호),Chae Hwa Ryu(유채화),Chang Min Park(박창민),Yun Ho Moon(문윤호),Jung-Il Chae(채정일),Nag-Jin Choi(최낙진) 한국초지조사료학회 2012 한국초지조사료학회지 Vol.32 No.2

본 연구는 반추동물의 조사료 자원으로서 거대억새를 개발하기 위한 목적으로 수행되었다. 우리나라에서 새롭게 개발된 품종인 거대억새 1호를 완숙기 이후에 채취하여 in vitro 반추위 발효를 이용해 반추위내 pH, 암모니아태 질소, 가스발생량, 휘발성 지방산 생성량 및 건물소화율을 조사하였으며, 볏짚과 비교하여 평가하였다. 거대억새는 볏짚에 비하여 유의적으로 높은 반추위내 pH를 나타내었다 (p<0.01). 암모니아태 질소의 경우 배양 12시간 이후에는 두 처리구간의 유의적인 차이를 나타내지 않았다 (p>0.05). 배양 6시간 이후 부터는 거대억새의 가스발생량이 볏짚에 비하여 유의적으로 낮게 나타났다 (p<0.05). 휘발성 지방산 생성량에 있어 acetate, propionate, butyrate, valerate 및 총생상량에서 볏짚이 거대억새보다 높게 나타났다. 그러나 iso-butyrate와 iso-valerate에서는 두 조사료원별 차이는 발견되지 않았다. 건물소화율에 있어 배양 12~24시간 사이의 거대억새 소화율이 볏짚에 비하여 유의적으로 나타났다. 결론적으로 거대억새의 이용성은 볏짚의 약 80% 수준인 것으로 나타났다. This study was conducted to develop Miscanthus as a new roughage resource for ruminant animals. Miscanthus sacchariflorus var. No 1, a newly developed germtype in Korea, was harvested at late maturity stage and its effect on rumen pH, ammonia nitrogen, gas production, volatile fatty acid (VFA) production and digestibility were evaluated using in vitro rumen fermentation. The effects of Miscanthus were compared with rice straw. Miscanthus showed significantly higher pH compared to rice straw (p<0.01). As for ammonia nitrogen, there was no significant difference after 12 h of incubation (p>0.05). Gas production in Miscanthus was significantly lower than that of rice straw in overall incubation time (p<0.05) after 6 h of incubation. In VFA production, acetate, propionate, butyrate, valerate and total VFA production in Miscanthus were lower than those in rice straw. However, production of iso-butyrate and isovalerate were not different in between two forage materials. Dry matter digestibility of Miscanthus was significantly lower than rice straw (p<0.05) during 12~24 h of incubation. As a result, the availability of Miscanthus as roughage source showed approximately 80% that of rice straw.

인간 재조합 인터루긴-32 면역조절작용에 대한 유세포 분석

이광수,김영관,채정일,심정현,김은미,강형식,김수현,윤도영,명평근 충남대학교 생물공학연구소 2006 생물공학연구지 Vol.12 No.-

Xenotransplantation of porcine organs has the potential to overcome the severe shortage of human tissues and organ available for human transplantation. however, it remains various hurdles for clinical xenotransplantation. In pig and mouse xenotransplantation, porcine xenograft evoke a strong cellular rejection response in immunocompetent host and grafts are destroyed within a week. This cellular immune response could involved both T cells and NK cells. A number of groups have shown that human NK cells can recognize and damage porcine endothelial cells. In addition, human T cells can respond to porcine endothelial cells through both direct and indirect mechanisms. Cellular rejection of porcine tissues requires T cells, particularly CD4^(+) cells. A new cytokine recombinant human interleukin-32α,β(IL-32α,β) has a role innate and acquired immune system. In order to investigate the role of recombinant mouse IL-18 and recombinant human IL-32α,β in xenograft rejection, we transplanted the PK(15) cells to C57BL/6 mice with or without intraperitoneal injection of recombinant mouse IL-18 or recombinant human IL-32 α,β. It was analyzed the population of NK cell, T cell and B cell in the C57BL/6 mice transplanted with PK(15) cells and recombinant mouse IL-18 or recombinant human IL-32α,β by flow cytometry analysis. As a result, lymph node and thymus of PK15/IL18, PK15/IL32α and PK15/IL32β injected group were increased to T cell activation population than normal injected groups. CD8^(+) T cells were decreased in lymph node of PK15/IL18, PK15/IL32α and PK15/IL32β injected groups. CD4^(+) T cells were increased in lymph node cell of PK15/IL32α and PK15/IL32β injected group and also, B cell population were increased in lymph node cell and spleen of PK15/IL18, PK15/IL32α and PK15/IL32β injected group. Therefore, we suggest that recombinant mouse IL-18 and recombinant human IL-32α,β suppress xenograft rejection in cellular xenotransplantation.

인간 재조합 인터루킨-32의 면역조절작용에 대한 유세포 분석

이광수,김영관,채정일,심정현,김은미,강형식,김수현,윤도영,명평근 충남대학교 형질전환복제돼지연구센터 2007 논문집 Vol. No.10