• 제목/요약/키워드: 2-D LC MS/MS

검색결과 184건 처리시간 0.031초

금전초 추출물 및 분획물의 항산화 활성 및 세포 보호 효과 (Antioxidative and Cellular Protective Effects of Lysimachia christinae Hance Extract and Fractions)

  • 김아랑;정민철;정혜인;송동기;서영빈;전영희;박소현;신혁수;이상래;박수남
    • 공업화학
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    • 제29권2호
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    • pp.176-184
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    • 2018
  • 본 논문에서는 금전초로부터 50% 에탄올 추출물, 에틸아세테이트 분획 및 아글리콘 분획을 제조하였고 이들의 항산화 활성, 세포 보호 효과 및 성분 분석에 대한 연구를 수행하였다. 항산화능 평가에서 50% 에탄올 추출물, 에틸아세테이트 분획 및 아글리콘 분획의 라디칼 소거 활성($FSC_{50}$)은 각각 146.8, 22.2 및 $27.2{\mu}g/mL$이었고, 총 항산화능($OSC_{50}$)은 29.3, 2.9 및 $4.5{\mu}g/mL$이었다. 에틸아세테이트 분획의 자유라디칼 소거 활성 및 총 항산화능이 가장 크게 나타났다. 또한 $^1O_2$로 유도된 적혈구 광용혈에 대한 세포 보호 효과(${\tau}_{50}$)는 금전초 50% 에탄올 추출물, 에틸아세테이트 분획 및 아글리콘 분획 $5{\mu}g/mL$에서 각각 26.9, 57.5 및 103.9 min을 나타냈다. 특히 아글리콘 분획물의 ${\tau}_{50}$은 (+)-${\alpha}$-tocopherol (37.7 min)보다 훨씬 큰 세포 보호 효과를 나타내었다. UVB로 유도된 세포 손상에서 에틸아세테이트 분획물은 최대 90.1%까지 세포 생존율을 증가시켰다. 과산화수소로 유도된 세포 손상에서도 에틸아세테이트 분획물은 $5-25{\mu}g/mL$에서 농도 의존적으로 세포 보호 효과를 나타내었다. 금전초 에틸아세테이트 분획물의 성분 분석을 위해 TLC, HPLC, UV-vis spectrum, LC-MS로 분석한 결과, quercetin, kaempferol 및 그 배당체가 주성분들로 확인되었다. 결론적으로 금전초가 자외선에 노출된 피부를 보호하는 항산화제로서 작용할 수 있고, 기능성 화장품 원료로 응용 가능성이 있음을 시사한다.

유통 식육 중 성장호르몬제 초산메드록시프로게스테론의 분석 (Analysis of Medroxyprogesterone Acetate in Meats)

  • 이윤애;심지연;이륜경;김승환;오현숙;조태용
    • 한국식품위생안전성학회지
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    • 제25권3호
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    • pp.226-231
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    • 2010
  • 본 논문은 합성형 성장호르몬으로 천연 프로게스테론에 비해 20~30배 강하게 작용하는 초산메드록시프로게스테론에 대한 정량한계, 표준물질의 직선성, 회수율 등의 유효성 평가 및 내부표준물질 선정을 통한 분석방법 최적화를 식품공전의 시험법을 기초로 수행하고, 이를 바탕으로 국내 유통식육 내 초산메드록시프로게스테론의 잔류량을 조사하였다. 그 결과 초산메드록시프로게스테론의 검출한계는 1.5 ug/kg, 정량한계는 5.0 ug/kg 으로 나타났으며 표준물질의 좋은 직선성 ($r^2$= 0.9968)을 확인 하였다. 내부표준물질의 비교결과 식품에 오염될 우려가 없는 progesterone-D9으로 선정하였다. 이를 시험한 결과 7.69분의 머무름 시간을 가지며, 정량이온은 m/z 324-100, 정성이온은 m/z 324-113으로 설정하였다. 내부표준물질에 대한 정량의 회수율 실험결과 67.5~109.5%로 높아진 회수율을 나타낸 것을 알 수 있었다. 이와 함께 전국의 서울특별시 및 6개 광역시에서 수거한 국내산 쇠고기 46건, 수입산 쇠고기 43건, 국내산 돼지고기 60건, 수입산 돼지고기 12건과 국내산 닭고기 35건의 초산메드록시프로게스테론의 잔류량을 실태 조사한 결과 검출된 경우는 한건도 없었다. 따라서, 초산메드록시프로게스테론에 대한 유통 식육은 안전한 것으로 조사되었다.

Enzymatic Synthesis and Characterization of Galactosyl Trehalose Trisaccharides

  • Kim, Bong-Gwan;Lee, Kyung-Ju;Han, Nam-Soo;Park, Kwan-Hwa;Lee, Soo-Bok
    • Food Science and Biotechnology
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    • 제16권1호
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    • pp.127-132
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    • 2007
  • [ ${\alpha},\;{\alpha}$ ]-Trehalose was efficiently modified by a transgalactosylation reaction of Escherichia coli ${\beta}-galactosidase$ using lactose as a donor to yield two galactosyl trehalose trisaccharides. The reaction products of trehalose by the enzyme were observed by thin layer chromatography (TLC) and high performance anion exchange chromatography (HPAEC) and were purified by BioGel P2 gel permeation chromatography and recycling preparative HPLC. Liquid chromatography-mass spectrometry (LC-MS) and ^{13}C$ nuclear magnetic resonance (NMR) analyses revealed that the structures of the main products were $6^2-{\beta}-D-galactosyl$ trehalose (1) and $4^2-{\beta}-D-galactosyl$ trehalose (2). A reaction of 30%(w/v) trehalose and 15%(w/v) lactose at pH 7.5 and $45^{\circ}C$ resulted in a total yield of approximately 27-30% based on the amount of trehalose used. The galactosyl trehalose products were not hydrolyzed by trehalose. In addition the mixture of transfer products (9:1 ratio of 1 to 2) showed higher thermal stability than glucose, lactose, and maltose, but less than trehalose, against heat treatment over $100^{\circ}C$ at pH 4 and 7. It also exhibited better thermal stability than sucrose at pH 4 alone.

Purification and Identification of a Natural Antioxidant Protein from Fertilized Eggs

  • Yang, Shaohua;Wang, Lulu;Wang, Ying;Ou, Xiaoqian;Shi, Zhaoyuan;Lu, Chongchong;Wang, Wei;Liu, Guoqing
    • 한국축산식품학회지
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    • 제37권5호
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    • pp.764-772
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    • 2017
  • Fertilized hen eggs are rich in a variety of bioactive ingredients. In this study, we aimed to obtain an antioxidant protein from fertilized eggs and the radical scavenging abilities on 1, 1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl radical (${\bullet}OH$), superoxide anion ($O^{2-}{\bullet}$) were used to evaluate the antioxidant activity of the purified protein. During 20 d of incubation, the radical scavenging ability of protein extracted from fertilized eggs exhibited significantly differences and the protein on day 16 showed higher antioxidant capacity. Based on this, the antioxidant protein of the samples on day 16 were isolated for the follow-up study. With a molecular weight 43.22 kDa, the antioxidant protein was purified by Diethylaminoethyl cellulose -52 (DEAE-52) column and Sephadex G-100. The LC-MS analysis showed that the purified protein molecular weight was 43.22 kDa, named D2-S. The sequence of amino acids was highly similar to ovalbumin and the coverage reached to 84%. The purified protein showed a radical scavenging rate of $52.34{\pm}3.27%$ on DPPH and $63.49{\pm}0.25%$ on ${\bullet}OH$, respectively. Furthermore, the C-terminal amino acid sequence was NAVLFFGRCVSP, which was consistent with the sequence of ovabumin. These results here indicated that purified protein may be a potential resource as a natural antioxidant.

Saccharomyces cerevisiae Hsp30 is Necessary for Homeostasis of a Set of Thermal Stress Response Functions

  • Thakur, Suresh;Chakrabarti, Amitabha
    • Journal of Microbiology and Biotechnology
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    • 제20권2호
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    • pp.403-409
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    • 2010
  • Saccharomyces cerevisiae Hsp30 is a plasma membrane heat shock protein that is induced by various environmental stress conditions. However, the functional role of Hsp30 during diverse environmental stressors is not presently known. To gain insight into its function during thermal stress, we have constructed and characterized a ${\Delta}hsp30$ strain during heat stress. $BY4741{\Delta}hsp30$ cells were found to be more sensitive compared with BY4741 cells, when exposed to a lethal heat stress at $50^{\circ}C$. When budding yeast is exposed to either heat shock or weak organic acid, it inhibits Pma1p activity. In this study, we measured the levels of Pma1p in mutant and Wt cells both during optimal temperature and heat shock temperature. We observed that $BY4741{\Delta}hsp30$ cells showed constitutive reduction of Pma1p. To gain further insights into the role of Hsp30 during heat stress, we compared the total protein profile by 2D gel electrophoresis followed by identification of differentially expressed spots by LC-MS. We observed that contrary to that expected from thermal-stress-induced changes in gene expression, the ${\Delta}hsp30$ mutant maintained elevated levels of Pdc1p, Trx1p, and Nbp35p and reduced levels of Atp2p and Sod1p during heat shock. In conclusion, Hsp30 is necessary during lethal heat stress, for the maintenance of Pma1p and a set of thermal stress response functions.

Enzymatic Synthesis of Puerarin Glucosides Using Leuconostoc Dextransucrase

  • Ko, Jin-A;Ryu, Young Bae;Park, Tae-Soon;Jeong, Hyung Jae;Kim, Jang-Hoon;Park, Su-Jin;Kim, Joong-Su;Kim, Doman;Kim, Young-Min;Lee, Woo Song
    • Journal of Microbiology and Biotechnology
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    • 제22권9호
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    • pp.1224-1229
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    • 2012
  • Puerarin (P), an isoflavone derived from kudzu roots, has strong biological activities, but its bioavailability is often limited by its low water solubility. To increase its solubility, P was glucosylated by three dextransucrases from Leuconostoc or Streptococcus species. Leuconostoc lactis EG001 dextransucrase exhibited the highest productivity of puerarin glucosides (P-Gs) among the three tested enzymes, and it primarily produced two P-Gs with a 53% yield. Their structures were identified as ${\alpha}$-$_D$-glucosyl-($1{\rightarrow}6$)-P (P-G) by using LC-MS or $^1H$- or $^{13}C$-NMR spectroscopies and ${\alpha}$-$_D$-isomaltosyl-($1{\rightarrow}6$)-P (P-IG2) by using specific enzymatic hydrolysis, and their solubilities were 15- and 202-fold higher than that of P, respectively. P-G and P-IG2 are easily applicable in the food and pharmaceutical industries as alternative functional materials.

물푸레나무(Fraxinus rhynchophylla) 수피의 추출성분 (The Chemical Constituents of the Stem Barks of Fraxinus rhynchophylla)

  • 양은주;이동근;이종원;김예실;임선하;송경식
    • Applied Biological Chemistry
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    • 제50권4호
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    • pp.348-351
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    • 2007
  • 물푸레나무(F. rhynchophylla)의 수피를 95% EtOH로 추출하여 얻어진 추출물을 $CH_2Cl_2$, n-BuOH 및 $H_2O$ 순으로 분획하였다. 이 중 $CH_2Cl_2$, 가용성 분획에 대하여 silica gel column chromatography를 실시하여 1종의 sterol 화합물(1)을 분리하였다. 한편 n-BuOH 가용성 분획에 대해서도 silica gel, RP-18 및 Sephadex LH-20 등의 충진제를 사용하여 column chromatography를 행하여 3종의 phenylpropanoid 화합물(2-4)을 얻었다. 각 화합물의 구조는 $^1H-NMR$와 LC-MS data를 해석하고 문헌과 비교하여 daucosterol(1), caffeic acid(2), 6,8-dihydroxy-7-methoxycoumarin(3) 및 coniferaldehyde glucoside(4)로 동정하였다. 이들 화합물은 진피에서 처음으로 분리되었다.

QuEChERS법을 활용한 농산물 중 제초제 Tolpyralate의 최적 분석법 선발 및 검증 (Application and Validation of an Optimal Analytical Method using QuEChERS for the determination of Tolpyralate in Agricultural Products)

  • 이한솔;박지수;이수정;신혜선;김지영;윤상순;정용현;오재호
    • 한국환경농학회지
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    • 제39권3호
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    • pp.246-252
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    • 2020
  • BACKGROUND: Pesticides are broadly used to control weeds and pests, and the residues remaining in crops are managed in accordance with the MRLs (maximum residue limits). Therefore, an analytical method is required to quantify the residues, and we conducted a series of analyses to select and validate the quick and simple analytical method for tolpyralate in five agricultural products using QuEChERS (quick, easy, cheap, effective, rugged and safe) method and LC-MS/MS (liquid chromatography-tandem mass spectrometry). METHODS AND RESULTS: The agricultural samples were extracted with acetonitrile followed by addition of anhydrous magnesium sulfate, sodium chloride, disodium hydrogencitrate sesquihydrate and trisodium citrate dihydrate. After shaking and centrifugation, purification was performed with d-SPE (dispersive-solid phase extraction) sorbents. To validate the optimized method, its selectivity, linearity, LOD (limit of detection), LOQ (limit of quantitation), accuracy, repeatability, and reproducibility from the inter-laboratory analyses were considered. LOQ of the analytical method was 0.01 mg/kg at five agricultural products and the linearity of matrix-matched calibration were good at seven concentration levels, from 0.0025 to 0.25 mg/L (R2≥0.9980). Mean recoveries at three spiking levels (n=5) were in the range of 85.2~112.4% with associated relative standard deviation values less than 6.2%, and the coefficient of variation between the two laboratories was also below 13%. All optimized results were validated according to the criteria ranges requested in the Codex Alimentarius Commission (CAC) and Ministry of Food and Drug Safety (MFDS) guidelines. CONCLUSION: In conclusion, we suggest that the selected and validated method could serve as a basic data for detecting tolpyralate residue in imported and domestic agricultural products.

Brief Introduction of Research Progresses in Control and Biocontrol of Clubroot Disease in China

  • He, Yueqiu;Wu, Yixin;He, Pengfei;Li, Xinyu
    • 한국균학회소식:학술대회논문집
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    • 한국균학회 2015년도 춘계학술대회 및 임시총회
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    • pp.45-46
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    • 2015
  • Clubroot disease of crucifers has occurred since 1957. It has spread to the whole China, especially in the southwest and nourtheast where it causes 30-80% loss in some fields. The disease has being expanded in the recent years as seeds are imported and the floating seedling system practices. For its effective control, the Ministry of Agriculture of China set up a program in 2010 and a research team led by Dr. Yueqiu HE, Yunnan Agricultural University. The team includes 20 main reseachers of 11 universities and 5 institutions. After 5 years, the team has made a lot of progresses in disease occurrence regulation, resources collection, resistance identification and breeding, biological agent exploration, formulation, chemicals evaluation, and control strategy. About 1200 collections of local and commercial crucifers were identified in the field and by artificiall inoculation in the laboratories, 10 resistant cultivars were breeded including 7 Chinese cabbages and 3 cabbages. More than 800 antagostic strains were isolated including bacteria, stretomyces and fungi. Around 100 chemicals were evaluated in the field and greenhouse based on its control effect, among them, 6 showed high control effect, especially fluazinam and cyazofamid could control about 80% the disease. However, fluzinam has negative effect on soil microbes. Clubroot disease could not be controlled by bioagents and chemicals once when the pathogen Plasmodiophora brassicae infected its hosts and set up the parasitic relationship. We found the earlier the pathogent infected its host, the severer the disease was. Therefore, early control was the most effective. For Chinese cabbage, all controlling measures should be taken in the early 30 days because the new infection could not cause severe symptom after 30 days of seeding. For example, a biocontrol agent, Bacillus subtilis Strain XF-1 could control the disease 70%-85% averagely when it mixed with seedling substrate and was drenching 3 times after transplanting, i.e. immediately, 7 days, 14 days. XF-1 has been deeply researched in control mechanisms, its genome, and development and application of biocontrol formulate. It could produce antagonistic protein, enzyme, antibiotics and IAA, which promoted rhizogenesis and growth. Its The genome was sequenced by Illumina/Solexa Genome Analyzer to assembled into 20 scaffolds then the gaps between scaffolds were filled by long fragment PCR amplification to obtain complet genmone with 4,061,186 bp in size. The whole genome was found to have 43.8% GC, 108 tandem repeats with an average of 2.65 copies and 84 transposons. The CDSs were predicted as 3,853 in which 112 CDSs were predicted to secondary metabolite biosynthesis, transport and catabolism. Among those, five NRPS/PKS giant gene clusters being responsible for the biosynthesis of polyketide (pksABCDEFHJLMNRS in size 72.9 kb), surfactin(srfABCD, 26.148 kb, bacilysin(bacABCDE 5.903 kb), bacillibactin(dhbABCEF, 11.774 kb) and fengycin(ppsABCDE, 37.799 kb) have high homolgous to fuction confirmed biosynthesis gene in other strain. Moreover, there are many of key regulatory genes for secondary metabolites from XF-1, such as comABPQKX Z, degQ, sfp, yczE, degU, ycxABCD and ywfG. were also predicted. Therefore, XF-1 has potential of biosynthesis for secondary metabolites surfactin, fengycin, bacillibactin, bacilysin and Bacillaene. Thirty two compounds were detected from cell extracts of XF-1 by MALDI-TOF-MS, including one Macrolactin (m/z 441.06), two fusaricidin (m/z 850.493 and 968.515), one circulocin (m/z 852.509), nine surfactin (m/z 1044.656~1102.652), five iturin (m/z 1096.631~1150.57) and forty fengycin (m/z 1449.79~1543.805). The top three compositions types (contening 56.67% of total extract) are surfactin, iturin and fengycin, in which the most abundant is the surfactin type composition 30.37% of total extract and in second place is the fengycin with 23.28% content with rich diversity of chemical structure, and the smallest one is the iturin with 3.02% content. Moreover, the same main compositions were detected in Bacillus sp.355 which is also a good effects biocontol bacterial for controlling the clubroot of crucifer. Wherefore those compounds surfactin, iturin and fengycin maybe the main active compositions of XF-1 against P. brassicae. Twenty one fengycin type compounds were evaluate by LC-ESI-MS/MS with antifungal activities, including fengycin A $C_{16{\sim}C19}$, fengycin B $C_{14{\sim}C17}$, fengycin C $C_{15{\sim}C18}$, fengycin D $C_{15{\sim}C18}$ and fengycin S $C_{15{\sim}C18}$. Furthermore, one novel compound was identified as Dehydroxyfengycin $C_{17}$ according its MS, 1D and 2D NMR spectral data, which molecular weight is 1488.8480 Da and formula $C_{75}H_{116}N_{12}O_{19}$. The fengycin type compounds (FTCPs $250{\mu}g/mL$) were used to treat the resting spores of P. brassicae ($10^7/mL$) by detecting leakage of the cytoplasm components and cell destruction. After 12 h treatment, the absorbencies at 260 nm (A260) and at 280 nm (A280) increased gradually to approaching the maximum of absorbance, accompanying the collapse of P. brassicae resting spores, and nearly no complete cells were observed at 24 h treatment. The results suggested that the cells could be lyzed by the FTCPs of XF-1, and the diversity of FTCPs was mainly attributed to a mechanism of clubroot disease biocontrol. In the five selected medium MOLP, PSA, LB, Landy and LD, the most suitable for growth of strain medium is MOLP, and the least for strains longevity is the Landy sucrose medium. However, the lipopeptide highest yield is in Landy sucrose medium. The lipopeptides in five medium were analyzed with HPLC, and the results showed that lipopeptides component were same, while their contents from B. subtilis XF-1 fermented in five medium were different. We found that it is the lipopeptides content but ingredients of XF-1 could be impacted by medium and lacking of nutrition seems promoting lipopeptides secretion from XF-1. The volatile components with inhibition fungal Cylindrocarpon spp. activity which were collect in sealed vesel were detected with metheds of HS-SPME-GC-MS in eight biocontrol Bacillus species and four positive mutant strains of XF-1 mutagenized with chemical mutagens, respectively. They have same main volatile components including pyrazine, aldehydes, oxazolidinone and sulfide which are composed of 91.62% in XF-1, in which, the most abundant is the pyrazine type composition with 47.03%, and in second place is the aldehydes with 23.84%, and the third place is oxazolidinone with 15.68%, and the smallest ones is the sulfide with 5.07%.

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Anti-adipogenic Pregnane Steroid from a Hydractinia-associated Fungus, Cladosporium sphaerospermum SW67

  • Lee, Seoung Rak;Kang, Heesun;Yoo, Min Jeong;Yu, Jae Sik;Lee, Seulah;Yi, Sang Ah;Beemelmanns, Christine;Lee, Jaecheol;Kim, Ki Hyun
    • Natural Product Sciences
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    • 제26권3호
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    • pp.230-235
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    • 2020
  • A pregnane steroid, 3α-hydroxy-pregn-7-ene-6,20-dione (1), was isolated from a Hydractinia-associated Cladosporium sphaerospermum SW67 by repetitive column chromatographic separation and high-performance liquid chromatography (HPLC) purification. The planar structure of 1 was elucidated from the analysis of the spectroscopic data (1D and 2D NMR spectra) and LC-MS data. The absolute configuration of 1 was determined by interpretation of ROESY spectrum of 1, together with the comparison of reported spectroscopic values in previous studies. To the best of our knowledge, this is the first report of the identification of the pregnane scaffold from C. sphaerospermum, a natural source. Compound 1 was evaluated for its effects on lipid metabolism and adipogenesis during adipocyte maturation and showed that compound 1 substantially inhibited lipid accumulation compared to the control. Consistently, the expression of the adipocyte marker gene (Adipsin) was reduced upon incubation with 1. Further, we evaluated the effects of 1 on lipid metabolism by measuring the transcription of lipolytic and lipogenic genes. The expression of the lipolytic gene ATGL was significantly elevated upon exposure to 1 during adipogenesis, whereas the expression of lipogenic genes FASN and SREBP1 was significantly reduced upon treatment with 1. Thus, our findings provide experimental evidence that the steroid derived from Hydractinia-associated C. sphaerospermum SW67 is a potential therapeutic agent for obesity.