• 약학회지
• /
• 제51권4호
• /
• pp.259-263
• /
• 2007

We previously reported the isolation of a sialic acid-specific lectin eluted from the bark of Maackia fauriei using alkaline buffer on a fetuin-affinity column. Application of a borate-based elution buffer in the present study increased the specific activity of purified lectin from crude protein extract by 2.6-fold, whilst only slightly decreasing the recovery by 1.13%. The biological properties of the lectin eluted with borate buffer were the same as those of the lectin eluted with alkaline buffer such as in terms of the hemagglutination activity, hemagglutination inhibition activity, molecular mass, purity, and cytotoxicity to human breast cancer cells. A prepared biotin-labeled lectin conjugate was used to investigate the binding to various glycoproteins. Our results indicate that eluting with borate buffer is more efficient than using alkaline buffer to isolate the lectin adsorbed in a fetuin-affinity column.

• Journal of Microbiology and Biotechnology
• /
• 제29권7호
• /
• pp.1043-1052
• /
• 2019

Active lipase-producing bacterium Burkholderia gladioli Bps-1 was rapidly isolated using a modified trypan blue and tetracycline, ampicillin plate. The electro-phoretically pure enzyme was obtained by purification using ethanol precipitation, ion-exchange chromatography, and gel filtration chromatography. The molecular weight was 34.6 kDa and the specific activity was determined to be 443.9 U/mg. The purified lipase showed the highest activity after hydrolysis with $p-NPC_{16}$ at a pH of 8.5 and $50^{\circ}C$, and the $K_m$, $k_{cat}$, and $k_{cat}/K_m$ values were 1.05 mM, $292.95s^{-1}$ and $279s^{-1}mM^{-1}$, respectively. The lipase was highly stable at $7.5{\leq}pH{\leq}10.0$. $K^+$ and $Na^+$ exerted activation effects on the lipase which had favorable tolerance to short-chain alcohols with its residual enzyme activity being 110% after being maintained in 30% ethanol for 1 h. The results demonstrated that the lipase produced by the strain B. gladioli Bps-1 has high enzyme activity and is an alkaline lipase. The lipase has promising chemical properties for a range of applications in the food-processing and detergent industries, and has particularly high potential for use in the manufacture of biodiesel.

• Journal of Ginseng Research
• /
• 제43권3호
• /
• pp.377-384
• /
• 2019

Background: Inflammation is widespread in the clinical pathology and closely associated to the progress of many diseases. Triterpenoid saponins as a key group of active ingredients in Panax notoginseng (Burk.) F.H. Chen were demonstrated to show antiinflammatory effects. However, the chemical structures of saponins in the leaves and stems of Panax notoginseng (PNLS) are still not fully clear. Herein, the isolation, purification and further evaluation of the antiinflammatory activity of dammarane-type triterpenoid saponins from PNLS were conducted. Methods: Silica gel and reversed-phase C8 column chromatography were used. Furthermore, preparative HPLC was used as a final purification technique to obtain minor saponins with high purities. MS, NMR experiments, and chemical methods were used in the structural identifications. The antiinflammatory activities of the isolated saponins were assessed by measuring the nitric oxide production in RAW 264.7 cells stimulated by lipopolysaccharides. Real-time reverse transcription polymerase chain reaction was used to measure the gene expressions of inflammation-related gene. Results: Eight new minor dammarane-type triterpene oligoglycosides, namely notoginsenosides LK1-LK8 (1-8) were obtained from PNLS, along with seven known ones. Among the isolated saponins, gypenoside IX significantly suppressed the nitric oxide production and inflammatory cytokines including tumor necrosis $factor-{\alpha}$, interleukin 10, interferon-inducible protein 10 and $interleukin-1{\beta}$. Conclusion: The eight saponins may enrich and expand the chemical library of saponins in Panax genus. Moreover, it is reported for the first time that gypenoside IX showed moderate antiinflammatory activity.

• 한국응용약물학회:학술대회논문집
• /
• 한국응용약물학회 1994년도 춘계학술대회 and 제3회 신약개발 연구발표회
• /
• pp.186-186
• /
• 1994

Many agonists have been known to activate the hydrolysis of membrane phospholipids through the bindings with corresponding receptors on the various cells. Diacylglycerol and inositol 1,4,5-trisphosphate(IP3) generated by the action of phosphoinositide-specific phospholipase C (PI-PLC) are well known second messengers for the activation of protein kinase C and the mobilization of Ca2+ in many cells. Three types of PI-PLC isozyme (${\alpha}$,${\gamma}$, and $\delta$) and several subtrpes for each type have been identified from mammalian sources by purification of enzymes and cloning of their cDNAs. Each type PI-PLC isozyme is coupled to different receptors and mediators, for example, ${\beta}$-types are coupled to the seven-transmembrane-receptors via Gq family of G-proteins and ${\beta}$-types directly to the receptor tyrosine kinases. Specific modulators for the signaling pathway through each type of PI-PLC should be very useful as potential potential candidates for lend substances in developing novel drugs. To establish the sensitive and convenient screening systems for searching modulators on PI-PLC mediated signaling, two kinds of approaches have been tried. (1) Establishment of in vitro assay condition for each type of PI-PLC isozyme: Overexpression by using vaccinia virus and purification of each isozyme was carried out for the preparation of large amounts of enaymes. Optimum and sensitive assay condition for the measurements of PI-ELC activities were established. (2) Development of the cell lines in which each type of PI-PLC is permanently overexpressed: A fibroblast cell line (3T3${\gamma}$1-7) in which PI-PLC-${\gamma}$1 was overexpressed by using pZip-neo expression vector was developed and used for the measurement of PDGF-induced IP3 formation. The responses for IP3 formed in 3T3${\gamma}$1-7 cells by the treatment of PDGF is 8 times more sensitive than those in control cells. 3T3${\gamma}$l-7 cell is useful for the screening of the inhibitors on the PDGF-induced cellular responses from large number of samples in a small volume(50 ${\mu}$l) and short time(5-15 min). Using these systems, we screened hundreds of herb-extracts for the inhibition of PDGF-induced IP3 formation and selected several extracts that showed the inhibition as the candidates for isolation and characterization of active substances. The determination of the acting point of selected extracts or fractions in the PDGF signaling pathway has been analyzing.

• Journal of Microbiology and Biotechnology
• /
• 제23권2호
• /
• pp.225-236
• /
• 2013

Among several bacteria examined, an antibacterial-producing Lactobacillus strain with probiotic characteristics was selected and identified based on 16S rRNA gene sequencing. Subsequent purification and mode of action of the antibacterial compounds on target cells including E. coli were investigated. Maximum production of the antibacterial compound was recorded at 18 h incubation at $30^{\circ}C$. Interestingly, antibacterial activity remained unchanged after heating at $121^{\circ}C$ for 45 min, 24 h storage in temperature range of $70^{\circ}C$ to room temperature, and 15 min exposure to UV light, and it was stable in the pH of range 2-10. The active compounds were inactivated by proteolytic enzymes, indicating their proteinaceous nature, and, therefore, referred to as bacteriocin-like inhibitory substances. Isolation and partial purification of the effective agent was done by performing ammonium sulfate precipitation and gel filtration chromatography. The molecular mass of the GFC-purified active compound (~3 kDa) was determined by Tris-Tricine SDS-PAGE. To predict the mechanisms of action, transmission electron microscopy (TEM) analysis of ultrathin sections of E. coli before and after antibacterial treatment was carried out. TEM analysis of antibacterial compounds-treated E. coli demonstrated that the completely altered bacteria appear much darker compared with the less altered bacteria, suggesting a change in the cytoplasmic composition. There were also some membrane-bound convoluted structures visible within the completely altered bacteria, which could be attributed to the response of the E. coli to the treatment with the antibacterial compound. According to the in vivo experiments oral administration of L. plantarum HKN01 resulted in recovery of infected BALB/c mice with Salmonella enterica ser. Typhimurium.

• 한국식품영양학회지
• /
• 제27권3호
• /
• pp.532-537
• /
• 2014

황백(Cortex Phellodendri: CP)은 황벽나무(Phellodendron amurense)의 건조된 수피로부터 얻어진다. 이 수피는 한국의 전통 한약제로서 설사, 황달, 무릎과 발의 통증, 요도관 및 피부 감염증에 폭넓게 사용되어 왔다. 이들 기능성 성분의 분리 및 정제는 박층 크로마토크래피, 컬럼 액체 크로마토크래피 및 HPLC와 같은 여러 분석법들이 동양의 약초연구에 이용되어 왔다. 본 연구는 CP로부터 berberine을 분리하기 위해 향류분배 크로마토크래피법(CPC)으로 효과적으로 수행하였다. 두 용매의 CPC 최적조성은 n-butanol: acetic acid: water(4:1:5 v/v/v)이었다. 이동상의 유속은 1,000 rpm 회전력에서 상승법으로 분당 3 mL 속도로 전개시켰다. CPC에서 분리된 분획분은 prep-HPLC로 정제하였다. $^1H$-NMR 스펙트럼은 4.10과 4.20 ppm에서 $3H(-OCH_3)$, 6.10 ppm에서 2H의 ($-OCH_2O-$) proton signal의 공명이 관찰되었다. 2개의 방향족 proton은 이중결합 패턴을 보였다. H-11과 H-12 doublet은 각각 7.98과 8.11에서 나타났다. $^{13}C$-NMR 스펙트럼에서는 C2와 C3의 methylenedioxy group($-OCH_2O-$), C9과 C10에 methoxy group($-OCH_3$)이 4개의 치환된 형태로 보였다. 분리 정제된 berberine의 화학구조는 $^1H$, $^{13}C$-NMR, ESI-MS 데이터 분석으로 확인하였다.

• 생명과학회지
• /
• 제11권6호
• /
• pp.561-567
• /
• 2001

능이버섯[Sarcodon aspratus(Berk.)S.Ito]의 fibrin 분해 활성물질을 분리정제하기 위하여(N $H_4$)$_2$S $O_4$침전법, DE52 anion exchange column chromatography, Sephacryl-S2000 gel filtration chromatography 및 Mono S cation FPLC를 행하였으며 정제된 효소의 특성을 측정하였다. 혈전용해 요소의 활성물질은 DE52 anion exchange colum chroma-tography에 NaCI의 농도가 0.2M 정도에서 용출되었으며 계속된 Sephacryl-S200 gel fitration chromatography 및 Mono S cation EPLC를 실시한 결과 단일 Peak를 얻었고 혈전용해효소의 특이활성은 55.2 U/mg protein으로 조효소액으로 비하여 11.3 배 증가하였으며 수율은 49.5%이었다. 또한 Mono S cation EPLC에서 얻은 활성획분을 12% SDS-PAGE로 전기영동한 결과 단일 band를 얻었으며 gel filtration의 결과와 비교함으로서 정제된 능이의 혈전용해 효소의 분자량은 29.300 Da인 것으로 확인되었다. 능이로 부터 정제한 혈전용해효소는 pH가 높아질수록 효소활성이 증가하였으며 pH 10.5의 알카리성에서도 안정하였으며 6$0^{\circ}C$이상의 온도에서는 효소활성이 급격히 실활하기 시작하였지만 8$0^{\circ}C$에서 25%의 상대활성을 보였다. 또한 본 효소는 C $u^{2+}$이온 $Co^{3+}$ 이온 등 중금속에 의하여 68%및 38%활성이 저해되었으며 $Ca^{2+}$이온 또는 $Mg^{2+}$의 초딤색 인 EDTA및 serine protease inhibitor이 PMSF에 의하여 활성이 저해되었었다. 이러한 결과들은 능이의 혈전용해효소가 Ca.sup 2+/또는M $g^{2+}$에 의하여 활성이 증가하는 serine protease임을 암시해 주고 있다.

• 생명과학회지
• /
• 제25권8호
• /
• pp.870-879
• /
• 2015

109종의 뽕잎 종류 중 국상 품종 뽕잎으로부터 α-amylase와 α-glucosidase 저해작용을 가지는 페놀성화합물을 분리하여 저해활성을 검토하였다. 국상 뽕잎의 물추출물에서 α-amylase와 α-glucosidase 저해 활성이 각각 93.8%와 48.7%를 나타내었다. 국상뽕잎 추출물의 페놀화합물의 함량은 물 추출물에서 9.7±0.2 mg/g, 60% ethanol 추출물에서 14.3±0.2 mg/g으로 나타났으며, 국상뽕잎 물 추출물 200 μg/ml의 농도에서 α-amylase와 α-glucosidase 저해활성이 각각 100%와 82.6%로 나타났다. 국상뽕잎으로부터 α-amylase와 α-glucosidase 저해활성을 가지는 천연물을 분리하기 위하여 국상뽕잎 물추출물을 Sephadex LH-20과 MCI-gel을 이용하여 normal phase type인 EtOH→H₂O와 reverse phase type인 H₂O→MeOH로 유기용매의 농도를 증가시키며 gradient로 용출하여 정제하였다. α-Amylase와 α-glucosidase 저해활성을 가지는 정제물은 FAB-MS, NMR과 IR spectrum을 구조 분석한 결과 quercetin으로 동정되었다.

• 대한수의학회지
• /
• 제24권2호
• /
• pp.149-162
• /
• 1984

The research was conducted(1) to confirm the agent(s) responsible for the antimicrobial activity contained in the fermented tomato juice with L. acidophilus(2) to extract and purify the antimicrobial agent(s)(3) to find the biological, physical and chemical properties of the agent(s). The following results were obtained and summarized as followings; 1. The agent responsible for the inhibitory activity was confirmed by both well assay method using fermented tomato juice with L. acidophilus and turbidimetric technique using the cell-free filtrate or neutralized filtrate of tomato acidohilus culture and found exerted antimicrobial agent other than lactic acid. 2. The procedures of purification : The isolation and purification of antimicrobial agent from the lyophilized acidophilus tomato culture were carried out by (1) methanol extraction (2) acetone extraction, (3) Sephadex G-50 gel filtration (4) paper chromatography and (5) thin layer chromatography. 3. The biological, physical and chemical properties of antimicrobial agent: The biological, physical, chemical properties of the purified antimicrobial agent were: (1) The antimicrobial activity was strong against test organisms; Bacillus subtilis(ATCC 6633), Escheichia coli(ATCC 25922), Staphylococcus aureus(ATCC 167), Pseudomonas fluorescens(KFCC 32394), Proteus vulgaris and Shigella dysenteriae. (2) The pH value of the agent was 2.0 and the inhibitory activity was lost when it was neutralized at 7.0 of pH and the agent was heat stable at $121^{\circ}C$ for 60 minutes. (3) The ultraviolet light absorption spectra of methanol-acetone extract and TLC fraction exhibited a maximum absorption at 260nm and 224nm respectively. (4) The most purified agent from TLC plate increased about 130-fold in activity. (5) The agent isolated from TLC plate was free from $H_2O_2$ or lactic acid. 4. Bioautographic assy: By means of bioautography of the agent on silica gel of TLC plate a strong inhibitory activity against B. subtilis was demonstrated. 5. Mass spectrometry: The agent obtained from TLC plate was analyzed by mass spectrometry which show the parent peak at m/e 264 suggesting the molecular weight of the compound and molecular group such as [$C_2H{^+}_4$], [CO], [CH=NH], [$C_3{H^}4_7$], [$\begin{array}{rcl}O\\{\parallel}\\CH_3-C\\\end{array}$], [$C_6-H{^+}_{11}$], [$C_5H{^+}_{11}$], [$\begin{array}{rcl}O\\{\parallel}\\C_5H_7-C^+\\\end{array}$] were suggested.

• 생명과학회지
• /
• 제23권2호
• /
• pp.259-266
• /
• 2013

혈전용해효소를 생산하는 균주 분리를 위해서 우선 한국, 일본 등지에서 모은 21개의 청국장 시료를 준비하였고 총 268개의 균주를 분리하였다. 이 중에서 1% skim milk가 포함된 nutrient agar 배지에서 protease를 생산하는 bacteria를 분리하였고, 이 결과로 22개의 균주가 분리되었다. 균주들은 apiweb을 통해 생화학적 특성에 근거하여 동정하였다. 또한 세균동정을 위해 16S rRNA 염기서열 분석을 수행하였다. 분리된 대부분의 균주는 Bacillus subtilis와 Bacillus amyloliquefaciens였다. 혈전용해효소의 활성은 fibrin plate 방법에 의해 측정되었고 A2-14, A2-20, C1-05, C1-09, F2-01로 명명된 다섯 균주가 선택되었다. 이중에서 A2-20 균주는 강한 혈전용해 활성을 보였고 동정결과 Bacillus amyloliquefaciens에 가까웠다. A2-20 균주에 의해 생산되는 혈전용해효소는 균 상등액을 이용한 gel filtration과 ion exchange chromatography를 거쳐 부분정제 되었다. 부분 정제된 효소의 최적 pH는 7.0이었고, 최적 온도는 $35^{\circ}C$였다. 정제된 단백질의 분석은 SDS-PAGE와 zymography로 이루어졌다. 이와 더불어 혈전용해효소의 유전자적 분석도 수행되었으며 A2-20 균주가 생산하는 혈전용해효소의 부분적인 염기서열과 유전적 상동성을 보이는 서열을 규명하였다.