• BMB Reports
• /
• 제35권2호
• /
• pp.206-212
• /
• 2002

The NS2B-NS3(pro) polyprotein segment from the dengue virus serotype 2 strain 16681 was purified from overexpressing E. coli by metal chelate affinity chromatography and gel filtration. Enzymatic activity of the refolded NS2B-NS3(pro) protease complex was determined in vitro with dansyl-labeled peptide substrates, based upon native dengue virus type 2 cleavage sites. The 12mer substrate peptides and the cleavage products could be separated by reversed-phase HPLC, and were identified by UV and fluorescence detection. All of the peptide substrates (representing the DEN polyprotein junction sequences at the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 sites) were cleaved by the recombinant protease NS2B-NS3(pro). No cleavage was observed with an enzymatically inactive S135A mutant of the NS3 protein, or with a modified substrate peptide of the NS3/NS4A polyprotein site that contained a K2093A substitution. Enzymatic activity was dependent on the salt concentration. A 50% decrease of activity was observed in the presence of 0.1M sodium chloride. Our results show that the NS3 protease activity of the refolded NS2B-NS3(pro) protein can be assayed in vitro with high specificity by using cleavage-junction derived peptide substrates.

• 대한본초학회지
• /
• 제23권3호
• /
• pp.27-32
• /
• 2008

Objectives : In this paper, we describe that $^1H-NMR$ spectroscopy may be superior to the conventional HPLC for the quantitative analysis of hesperidin from Citrus unshiu. Methods : $^1H-NMR$ spectra (400 MHz) were recorded in $DMSO-d_6$ using a Varian UNITY Inova AS 400 FT NMR spectrometer. One hundred milligram of powdered Citrus unshiu was weighed out and mixed with 1 ml of $DMSO-d_6$ with sonication for 30 min (room temperature). The extracts were filtrated through a 0.45 ${\mu}m$ PVDF filter and 0.5 ml of filtrated extract used for quantitative $^1H-NMR$ measurement (added 1 mg of dimethyl terephthalate as internal standard). The quantity of hesperidin was calculated by the ratio of the intensity of the compound to the known amount of internal standard. For HPLC analysis, the half gram of plant material was extracted with 60 ml of MeOH for 2 hours. The extracts were made 100 ml volume and analyzed by a Waters HPLC system using a YMC ODS column. The total flow rate was 1.0 ml/min with a sample volume 10 ${\mu}l$ and UV detection at 280nm. Results : The contents of hesperidin in Citrus unshiu was determined $5.33{\pm}0.06$% in the quantitative $^1H-NMR$ method and $5.15{\pm}0.12%$ in HPLC method. Using the quantitative $^1H-NMR$ the contents of hesperidin can be determined in much shorter time than the conventional HPLC measurements. Conclusions : From those results, the advantages of quantitative $^1H-NMR$ analysis are that can be analyzed to identify and quantify, and no reference compounds required for calibration curve. Besides, it allows rapid and simple quantification for hesperidin with an analysis time for only 10 min without any pre-purification steps.

• Bulletin of the Korean Chemical Society
• /
• 제30권1호
• /
• pp.137-144
• /
• 2009

In this study, quantitative and pattern recognition analyses for the quality evaluation of Herba Epimedii using HPLC was developed. For quantitative analysis, five major bioactive constituents, hyperin, epimedin A, epimedin B, epimedin C, and icariin were determined. Analysis was carried out on Capcell pak $C_{18}$ column ($250{\time}4.6$ mm, 5 ${\mu}m$) with a mobile phase of mixture of acetonitrile and 0.1% formic acid, using UV detection at 270 nm. The linear behavior was observed over the investigated concentration range (2-50 ${\mu}g/mL;\;r_2\;>$ 0.99) for all analytes. The intraand inter-day precisions were lower than 4.3% (as a relative standard deviation, RSD) and accuracies between 95.1% and 104.4%. The HPLC analytical method for pattern recognition analysis was validated by repeated analysis of one reference sample. The RSD of intra- and inter-day variation of relative retention time (RRT) and relative peak area (RPA) of the 12 selected common peaks were below 0.8% and 4.7%, respectively. The developed methods were applied to analysis of twenty Herba Epimedii extract samples. Contents of hyperin, epimedin A, epimedin B, epimedin C, and icariin were calculated to be 0$\sim$0.79, 0.69$\sim$1.91, 0.93$\sim$9.58, 0.65$\sim$3.05, and 2.43$\sim$11.8 mg/g dried plant. Principal component analysis (PCA) showed that most samples were clustered together with the reference samples but several apart from the main cluster in the PC score plot, indicating differences in overall chemical composition between two clusters. The present study suggests that quantitative determination of marker compounds combined with pattern-recognition method can provide a comprehensive approach for the quality assessment of herbal medicines.

• KSBB Journal
• /
• 제21권2호
• /
• pp.129-132
• /
• 2006

Artemisia princes var. orientalis의 수용성 추출물이 식품, 화장품, 의약품 등의 항균제로 이용될 수 있는지에 대해 연구 하였다. 순수한 물을 이용한 추출물에서는 항균성을 찾기 힘들었고, 쑥의 줄기와 잎 부분에서 메탄올로 추출한 후 물로 추출한 물이 Staphylococcus aureus에 대해서 항균성을 크게 나타냈으며, 잎이 줄기 부분보다 더 큰 항균성을 나타냄을 확인했다. 항균성을 갖는 물질을 밝혀내기 위해 쑥의 주된 항균 성분이라 알려진 caffeic acid와 비교 실험을 하였다. UV 특성곡선에서 caffeic acid와 유사한 phenolic compound의 profiling은 확인할 수 있었으나 caffeic acid의 disc diffusion method를 이용한 실험에서는 항균능력을 나타내지 않았다. 물질규명을 위해 추출물을 세 부분으로 분획 하였고, 이 중에서 Staphylococcus aureus에 대해 항균성은 UK 2에서 크게 나타났다. 본 연구는 Artemisia princeps var. orientalis 의 메탄올 후 물 추출물이 항균제로써의 가능성을 알아봄으로써 식품 또는 화장품의 보존료나 항균제의 이용성이 있음을 충분히 확인하였다.

• 한국식품영양과학회지
• /
• 제45권11호
• /
• pp.1685-1690
• /
• 2016

시판 과일 주스와 발효식초의 총 페놀 함량, 플라보노이드함량, 유기산 함량, 그리고 xanthine oxidase(XO) 저해 활성을 비교하였다. 과일 주스와 식초에 함유된 총 페놀(mg gallic acid equivalent/100 mL)과 플라보노이드(mg catechin equivalent/100 mL) 함량은 각각 82.8~157.6 mg과 15.7~85.8 mg으로 측정되었다. 주스류에서는 사과 주스, 식초류에서는 흑초가 각각 가장 높은 총 페놀 함량과 플라보노이드를 함유하였다. 유기산의 함량 중 citric acid, malic acid, ascorbic acid는 주로 과일 주스에서 검출되었다. Acetic acid가 주성분인(688.3~4,413.7 mg/100 mL) 식초류 중에서 흑초는 acetic acid뿐 아니라 citric acid, malic acid, tartaric acid를 함유하여 유기산의 분포가 가장 다양하였다. XO 저해 활성은 과일 주스보다 식초가 평균 34.2% 더 높게 측정되었으며, 흑초는 64.9%로 가장 높은 저해 활성을 나타내었다. 특히 유기산의 종류 중에서 아세트산의 함량과 XO 저해 활성 간에 높은 정의 상관성이 존재하여($R^2=0.7192$) 식초류의 XO 저해 활성의 주요 지표물질로 나타났다.

• Journal of Pharmaceutical Investigation
• /
• 제34권6호
• /
• pp.453-457
• /
• 2004

Atenolol and related compounds found in raw materials and commercial products were analyzed by reversed-phase high-performance liquid chromatography. A mixed solution of phosphate buffer (3.4 g/l, pH 3.0), tetrahydrofurane and methanol (800:20:180, v/v/v) including sodium octanesulfonate (1 g/l) and tetrabutylammonium-hydrogensulfate (0.4 g/l) was used as mobile phase at the flow rate of 0.25 ml/min. Detection was carried out at UV 226 nm. Atenolol related compounds, such as bis ether, tertiary amine and blocker acid were identified by comparing the retention time of the standard. The within-day and between-day precisions of the separated compounds were less than 1.2% and 3.4%, respectively. The contents of related compounds of the tested samples were under the limit prescribed in the European Pharmacopoeia. The pattern of the related compounds showed that atenolol raw materials and products could be classified in three different groups, indicating that the materials originated from different source or treated in different way.

• Environmental Engineering Research
• /
• 제17권4호
• /
• pp.205-210
• /
• 2012

Hydroperoxyl radical/superoxide anion radical ($HO_2{\cdot}/O_2^-{\cdot}$, $pK_a$=4.8) as an intermediate is of considerable importance in oxidation processes. Hence, the method of detecting $HO_2{\cdot}/O_2^-{\cdot}$ with high sensitivity is necessary to be developed. To achieve this objective, this study newly employed terephthalate (TA) as a probe for the measurement of $HO_2{\cdot}/O_2^-{\cdot}$ in the kinetic method presented in our previous study. This method was based on the hydroxylation of TA to produce mainly hydroxyterephthalic acid or hydroxyterephthalate (OHTA), which was analyzed by fluorescence detection (${\lambda}_{ex}$=315nm, ${\lambda}_{ex}$=425nm). The life-time of $HO_2{\cdot}/O_2^-{\cdot}$ and its concentration formed from the photolysis technique of $H_2O_2$ were reported in this study. At range of pH 2-10, the life-time of $HO_2{\cdot}/O_2^-{\cdot}$ was 51-422 sec. In particular, an increase in the life-time with pH was observed. The sensitivities of the kinetic method by using TA were always higher with 1.7-2.5 times at pH 8.0 than those by using benzoic acid. From these results, this study can contribute to understanding the basic functions of $HO_2{\cdot}/O_2^-{\cdot}$ in oxidation processes.

• Journal of Electrical Engineering and Technology
• /
• 제9권5호
• /
• pp.1704-1711
• /
• 2014

Sandy and dusty weather condition often occurs in the high altitude areas of China, which may greatly influence the corona generated radio interference (RI) characteristics of the bundle conductors of 750 kV AC power transmission lines. Corona generated RI of the conductors of the 750 kV AC power transmission lines used in practice is measured by EMI receiver with a coupling circuit and a coupling capacitor connected between the high voltage side and the earth side in fine and sandy and dusty condition. The measuring frequency is 0.5 MHz, and the quasi-peak detection is used. RI excitation function is calculated based on the corona RI current measured by the EMI receiver. Corona generated RI characteristics were analyzed from sand concentration and sand particle size. The test result shows that the corona generated RI excitation function is influenced by the sandy and dusty condition. Corona discharge of the conductors is more serious in sandy and dusty condition with an ultraviolet (UV) detector. Corona generated RI excitation function increases with the increase of sand concentration and also increases with the increase of particle size.

• Natural Product Sciences
• /
• 제13권4호
• /
• pp.378-383
• /
• 2007

Phenathrene derivatives, such as batatasins, are well-known constituents in Dioscorea Rhizoma. Although phenanthrenes have been reported as representative compounds in this plant, standard markers for quality control have been focused on the polar constituents (saponins and purine derivatives). Herein, simple, rapid and reliable HPLC method was developed to determine 3,5-dimethoxyphenanthrene-2,7-diol (DMP) and batatasin-I (BA-I) as non-polar standard maker compounds of Dioscorea Rhizoma. DMP and BA-I were analyzed under optimized HPLC conditions [column: Columbus $5{\mu}$ C18 100A ($30{\times}4.6mm$ i.d., $5{\mu}m$; mobile phase: $H_2O$ with 0.025% $CH_3COOH$ (v/v) for solvent A and $CH_3CN$ with 0.025% $CH_3COOH$ (v/v) for solvent B, gradient elution; flow rate: 2 mL/min; detection: 260 nm), and each experiment was finished within 13 min. Good linearity was achieved in the range from 0.5 to $10.0{\mu}g/mL$ for each compound, and intra- and inter-day precision were in the acceptable levels. The recovery test were performed with three different Dioscorea Rhizoma samples (D. opposita, D. batatas and D. japonica), and showed its accuracy values in the range of 97.2 - 102.8% for three different concentrations of DMP and BA-I. The content levels of DMP and BA-I were ranged under 0.0020%. These results demonstrated that amounts of DMP and BA-I are easily determined with conventional HPLC-UV-DAD method although the content levels were lower than those of saponins and allantoin in Dioscorea Rhizoma. This HPLC method could be used for quality control of various Dioscorea preparations.

• 한국식품영양학회지
• /
• 제32권1호
• /
• pp.61-68
• /
• 2019

In this study, the contents of water-soluble vitamins $B_1$ (thiamin), $B_2$ (riboflavin), $B_3$ (niacin), and $B_{12}$ (cyanocobalamin) in namuls (wild greens), such as salads and side dishes, consumed in Korea were quantified by high-performance liquid chromatography (HPLC) with UV and fluorescence detection. All samples were prepared with recipes used in Korea. All analyses were performed under the quality control of vitamin $B_1$, $B_2$, $B_3$, and $B_{12}$. The vitamin $B_1$, $B_2$, and $B_3$ contents in namuls consumed in Korea were analyzed. The highest content of vitamin $B_1$ was $3.018{\pm}0.016mg/100g$ in Putgochudoenjang-muchim. The highest contents of vitamin $B_2$, $B_3$, and $B_{12}$ were $0.279{\pm}0.003mg/100g$ in Gul-muchim, $12.241{\pm}0.040mg/100g$ in Chamchi-salad, and $8.133{\pm}0.371{\mu}g/100g$ in Pijogae-muchim, respectively. These results showed that animal-based ingredients in salads provided for good intake of vitamin $B_{12}$. These results can be used as basic data for food composition tables and improvement of the national health of Koreans.