藤黄灰链霉菌-H103发酵液中抗真菌活性成分的分离纯化

通过大孔树脂吸附等方法对藤黄灰链霉菌H103发酵液中的抗真菌活性成分进行了分离纯化,得到了纯度较高的活性物质的结晶,并且建立了通过大孔树脂吸附-结晶的分离纯化路线以及反相高压液相色谱-蒸发光散射(HPLC-ELSD)的检测方法,为进一步的理化性状研究打下了一个良好的基础。实验表明,最佳吸附树脂为X-5树脂,洗脱剂为50%乙醇,HPLC条件为:反相色谱柱Agilent 20RBA×310SB-C18(150mm×4.6mm i.d,5μm),以乙腈(A)-水(B)为流动相,梯度洗脱,0~4.0m in,V(A):V(B)=20∶80,4.0~9.5m in,V(A)∶V(B)=45∶55,此后V(A):V(B)=80∶20,流动相流速:0.8mL/m in,柱温:30℃,ELSD条件:漂移管温度115℃,载气流速(空气)2.3L/m in。     

虎眼万年青总皂苷的纯化及抗氧化活性研究

对虎眼万年青总皂苷的纯化工艺及抗氧化活性进行研究。以比吸附量和比洗脱量为指标,筛选大孔树脂型号,进一步优化工艺条件。结果表明,AB-8型大孔树脂对虎眼万年青总皂苷具有良好的吸附和洗脱效果,最佳纯化条件为药材量与树脂用量比不超过7∶1,用水、30%乙醇除杂,70%乙醇洗脱,在此条件下得到的虎眼万年青总皂苷纯度为55.18%。对纯化后虎眼万年青总皂苷的抗氧化活性进行研究,发现虎眼万年青总皂苷对羟自由基、超氧自由基阴离子和DPPH自由基的IC_(50)分别为2.43、2.28和0.05 mg/m L,具有良好的抗氧化活性。     

川栀子中京尼平苷的提取吸附工艺研究

研究了川栀子中有效成分京尼平苷的吸附提取工艺。通过正交实验设计,综合考察了提取时间、提取溶媒和料液比(栀子质量(g)与溶剂体积(m L)之比)等因素的影响,优化得到京尼平苷的最佳提取工艺为:水提2次,料液比1∶6,提取时间为1 h。选用HP-20、HPD-100、HPD-450、D-101、D-101A五种不去同型号的大孔吸附树脂,测定其对京尼平苷吸附性能,最终选定HPD–100型号的大孔吸附树脂作为最佳吸附材料。     

大孔吸附树脂分离纯化地黄中梓醇工艺的研究

利用大孔吸附树脂分离提取地黄中梓醇。以地黄粗提液中梓醇含量为指标,高效液相色谱(HPLC)为含量测定方法,考察九种不同极性大孔吸附树脂对梓醇的吸附和解吸附性能,筛选出最佳树脂D101进行分离实验。结果表明,D101大孔吸附树脂的静态吸附容量为69.2mg/g干树脂,其吸附等温线符合Langmuir和Freundlich吸附等温式。采用5%乙醇作为洗脱剂,洗脱液减压浓缩后进行硅胶柱层析分离,氯仿：甲醇（8：2）梯度洗脱得到梓醇单体,纯度达90%以上,梓醇得率为6%。     

大孔树脂吸附法分离海金沙总黄酮的研究

用大孔树脂吸附法分离海金沙中的总黄酮,考查了解吸剂浓度、吸附固液比(g/mL)、吸附温度和吸附时间对海金沙总黄酮提取率的影响。结果表明:用70%乙醇作解吸剂,在吸附固液比为1∶6(g/mL),50℃温度下吸附90 min,总黄酮提取率为1.81%。     

大孔吸附树脂法去除山银花总皂苷的工艺研究

本文采用大孔吸附树脂法对山银花总皂苷的去除工艺进行了研究。以山银花总皂苷含量为指标,比较了4种大孔树脂对山银花总皂苷的吸附和解吸附性能,并对优选出的树脂进行吸附工艺参数的优化。结果表明,HPD-100型大孔树脂具有较好的吸附性能,其最佳吸附工艺条件为:药液上样浓度0.65 g/m L,流速2 m L/min,药液p H值为4,吸附12 h。经蒸馏水和30%乙醇洗脱各5 BV后,收集两者的洗脱液,总皂苷去除率和总咖啡酰奎宁酸富集率均接近90%,确定了本实验所建立的方法能有效去除山银花总皂苷,富集总咖啡酰奎宁酸。     

大孔吸附树脂对金银花叶茎藤总黄酮的吸附性能

从金银花叶茎藤中提取总黄酮并用D-101大孔吸附树脂进行纯化,研究了D-101大孔吸附树脂对总黄酮的吸附及解吸附特性。结果表明,D-101树脂对金银花叶茎藤总黄酮分离纯化的最佳工艺参数为:上样液黄酮浓度0.538 mg/mL,静置吸附时间80 min,料液比1∶5(g∶mL),pH 2,流速为2 mL/min,以60 mL 75%的乙醇溶液洗脱,黄酮解吸率为94.5%,纯化后黄酮纯度为84.5%,是粗提液黄酮含量(16.8%)的5倍。金银花叶茎藤总黄酮在D-101树脂上的吸附等温线符合Langmuir等温吸附方程。吸附热力学参数表明吸附过程为自发、放热过程,吸附动力学可用Pseudo-second-order模型较好地拟合,30℃时其表观吸附速率常数为1.034×10-2g/mg.min。     

大孔吸附树脂纯化无柄金丝桃茎部总黄酮工艺研究

通过静态吸附筛选纯化无柄金丝桃茎部总黄酮的最佳树脂,并利用静态吸附解吸动力学确定纯化无柄金丝桃茎部总黄酮的工艺参数。实验结果显示AB8大孔吸附树脂为纯化总黄酮的最佳树脂。最佳工艺参数为:上样液浓度为1.30 mg/m L,体积为60 m L,p H=4.0,流速为1.00 m L/min,树脂柱径高比为1∶10,70%乙醇溶液(p H=7.0)为洗脱剂。经AB8树脂纯化,无柄金丝桃茎部总黄酮的纯度由30.26%提高到了55.70%,AB8大孔吸附树脂纯化无柄金丝桃茎部的总黄酮效果明显,其工艺参数简单可行。     

从蚯蚓中联合提取抗氧化酶SOD、CAT的方法研究

本研究采用闪式提取技术,固液比为1:4(m/V)的2.5 mmol/L pH 7.0磷酸缓冲液,提取转速5500 rpm,提取时间2 min,从蚯蚓体内提取出SOD、CAT,并通过羧甲基纤维素CM-22离子交换层析实现SOD和CAT的联合提取分离,SOD、CAT的活性回收率分别达到88.23%和69.5%。在纯化工艺中经过丙酮沉淀和柱层析技术得到蚯蚓SOD纯品,比活达到9352 U/mg,产物在SDS-PAGE上为单一条带,其亚基分子量约为17 kD;通过柱层析纯化了蚯蚓CAT,比活达到22606 U/mg。     

大孔吸附树脂分离纯化党参中苍术内酯Ⅰ和苍术内酯Ⅱ的工艺研究

采用大孔吸附树脂分离纯化党参药材中的主要活性成分苍术内酯Ⅰ和苍术内酯Ⅱ。以吸附量、吸附率、解吸附量、解吸附率为指标,筛选出较好的DM130型大孔树脂,并对苍术内酯Ⅰ和苍术内酯Ⅱ分离纯化的工艺条件进行了研究。结果表明用DM130型大孔吸附树脂对党参中的苍术内酯Ⅰ和苍术内酯Ⅱ进行分离纯化的最佳工艺条件为:30℃,供试品上样液的浓度为0.125 g/m L,上样液体积为200 m L,上样液流速为6 m L/min,95%乙醇作洗脱剂,洗脱剂流速为4 m L/min。经过纯化后,苍术内酯Ⅰ和苍术内酯Ⅱ的含量分别增加了32.3倍和25.6倍,表明DM130型大孔吸附树脂对党参中的苍术内酯Ⅰ和苍术内酯Ⅱ进行分离纯化的方法可行、有效。     

A rapid method for extraction of zearalenone and zeraralenols in fermented corn

A rapid extraction method is described for isolation of zearalenone and α- and β-trans-zearalenols from laboratory fermented corn. Corn fermented withFusarium crookwellense at 25°C for 2 weeks was agitated for 5 minutes in acetone. The acetone extract was evaporated to dryness and the remaining residue was chromatographed on a silica gel column with hexane:ethyl acetate (8:2). The products eluted with the hexane:ethyl acetate mixture in 1–1/2 column volumes and were identified by regular phase and C-18 reverse-phase thin layer chromatography. The products were verified by gas chromatography-mass spectroscopy. Product recoveries were 62.5–70% for the zearalenols and 70% for zearalenone in the range 0.5–50 mg/Kg.     

Chromophoric determination of putrescine, spermidine and spermine with dabsyl chloride by high-performance liquid chromatography and thin-layer chromatography

A fast and sensitive method for the determination of putrescine, spermidine, spermine and ammonia by high-performance liquid chromatography (HPLC) with dabsyl chloride is described. These compounds are converted to their chromophoric dabsyl derivatives and are separated by a normal-phase chromatographic column (μPorasil, 10 μm) with 2% acetone in chloroform as isocratic mobile phase. The sensitivity of the method is 20 pmoles. The present method was shown to be a straightforward procedure for estimating polyamines in various rat tissues.The chromophoric derivatives of polyamines are also well separated by thin-layer chromatography (TLC) on silica gel, and the combination of the HPLC and TLC procedures provides a reliable method for qualitative and quantitative analysis of polyamines.     

Analysis of coenzyme Q(10) in human plasma by column-switching liquid chromatography

A new method of determining coenzyme Q10 in human plasma was developed based on column-switching high performance liquid chromatography (HPLC). CoQ10 was quantitatively extracted into 1-propanol with a fast one-step extraction procedure, after centrifugation, the supernatant was cleaned on an octadecyl-bonded silica column and then transferred to reversed-phase column by a column-switching valve. Determination of CoQ10 was performed on a reversed-phase analytical column with ultraviolet detection at 275 nm and the mobile phase containing 10% (v/v) isopropanol in methanol at a flow-rate of 1.5 ml/min. The sensitivity of this method allows the detection of 0.1 microg/ml CoQ10 in plasma (S/N=3). The linearity between the concentration and peak height is from 0.05 to 20 mg/l. The reproducibility (R.S.D.%) of the method is less than 2% (within day) and less than 3% (between day), the average recovery is 100.9 + 2.1%, it takes only 30 min to complete an analysis procedure, suitable for the determination of CoQ10 in human plasma especially for batch analysis in clinical laboratories. Finally, the method was applied to determine the plasma CoQ10 levels in healthy subjects, hyperthyroid and hypothyroid patients.     

长裙竹荪抑菌活性物质的分离纯化及其抑菌效果

长裙竹荪Dictyophora indusiata是珍贵的食药用真菌,具有很强的抑菌作用,在天然防腐剂开发方面具有广阔的应用前景。本研究以长裙竹荪的抑菌活性为指标,通过萃取、3次不同流动相的硅胶柱层析、1次反相柱层析和薄层层析法对竹荪提取物进行分离纯化,得到一个抗菌活性强的单体化合物。根据核磁共振波谱等数据分析,推断该化合物为间苯三酚。以巨大芽孢杆菌和肠炎沙门氏菌为供试菌,用平板打孔法及原位抑菌法测定该化合物的抑菌效果,结果表明：该化合物对这两种菌有很强的抑制作用,半抑制浓度分别为83.06μg/mL和51.58μg/mL。本研究首次从长裙竹荪中获得具有抗菌活性的单体化合物间苯三酚,为竹荪天然抗菌物质的开发提供理论依据。     

Purification, stabilization, and characterization of rat hepatic triglyceride lipase

Hepatic triglyceride lipase (H-TGL) was purified to near homogeneity from heparin-containing rat liver perfusates with the following column chromatography steps: heparin-Sepharose affinity chromatography, anion-exchange chromatography on DEAE-Sephacel, and gel filtration on Ultrogel AcA 34. A final specific activity of 45,000 μmol fatty acid/mg/h was obtained with an overall 31% recovery of catalytic activity. The heparin-Sepharose step resulted in a 20-fold purification, while the DEAE and gel filtration steps led to further purification with complete recovery of activity. An extensive survey of various detergents as potential stabilizers of H-TGL activity led to the selection of Triton N-101 for use in the column buffers of the DEAE and gel filtration steps. Relative to initial H-TGL activity upon dilution in buffer without detergent, recoveries between 90 and 100% were consistently obtained with Triton N-101-containing buffers following a 24-h incubation at 20°C. In contrast after a 24-h incubation at 20°C those control samples lacking detergent were at least 95% inactivated. The highly purified H-TGL exhibited a single major band by sodium dodecyl sulfate-electrophoresis. The use of DEAE chromatography and stabilization of H-TGL with Triton N-101 are the improvements in purification that resulted in an 8-fold enhancement in specific activity relative to the highest previous report of purification from rat liver perfusates.     

大孔吸附树脂对栀子中环烯醚萜苷类成分的富集行为

采用大孔吸附树脂对栀子中环烯醚萜苷类成分的富集行为进行研究,结果表明,D101大孔吸附树脂柱层析适合分离纯化栀子中环烯醚萜苷类成分,采用80%乙醇洗脱,紫外-可见分光光度法进行定量测定。使用该方法富集后,80%乙醇洗脱液干燥后总固体物中环烯醚萜苷类成分含量可达到70%以上。     

Evaluation of different primary recovery methods for E. coli-derived recombinant human growth hormone and compatibility with further down-stream purification

Due to advances in fermentation technology, it is now possible to obtain fermentation broth with over 30% solids. The high solid content makes the clarification step difficult, especially at large scale. The primary protein recovery step is challenging due to the heterogeneous solution of soluble and insoluble material. In this study, we compare different primary recovery routes and the compatibility with the initial capture chromatography step. The primary recovery routes studied are standard clarification by centrifugation and extraction in aqueous two-phase systems. The compatibility of the feed streams from the different primary recovery steps with the first chromatography step is addressed. An anion-exchange column was used as the first capture column in the purification process. The aqueous two-phase system was composed of a random copolymer of ethylene oxide and propylene oxide (EOPO) in combination with a waxy starch. The target protein in this study was human growth hormone (hGH) produced in recombinant Escherichia coli. The purity of hGH in the top phase after aqueous two-phase extraction was found to be significantly higher than in clarified homogenate supernatant and increased as the EOPO polymer concentration in the aqueous two-phase system increased. Stability of the supernatant and EOPO top phases and hGH were determined by turbidity measurements and LC-MS assay. All of the feed-streams from the primary recovery steps were compatible with the anion-exchange chromatography step; however, the capacity of the resin was strongly dependent on the purity of the load. Different process aspects, e.g., resin capacity, viscosity, purification, and yield of hGH and scalability are compared.     

葛根异黄酮的快速分离检测方法研究

以SDS正丁醇-正庚烷-水组成的微乳体系作为展开剂,通过聚酰胺薄层层析,探讨不同类型微乳液对葛根异黄酮分离检测效果的影响。结果表明,选择含水量为70％的微乳液作为展开剂,分离效果明显,一次检测出12个黄酮化合物;与以三氯甲烷-甲醇-水(7:2．5:0．25)为展开剂的硅胶薄层相比,微乳薄层色谱对葛根异黄酮的检测效果和灵敏度显著提高,为实验研究中常规检测提供了简便快捷的方法。     

紫茎泽兰9-羰基-10,11-去氢泽兰酮分布积累动态

9-羰基-10,11-去氢泽兰酮为紫茎泽兰（Eupatorium adenophorum）的主要致肝脏毒性成分及杀虫的生物活性成分。从紫茎泽兰叶片中分离提纯得到9-羰基-10,11-去氢泽兰酮（Euptox A）标准品,建立了高效液相色谱法测定紫茎泽兰中Euptox A含量的分析方法。采用C18反相色谱柱,柱温30°C,以甲醇-水（60：40,v/v）为流动相、流速为0.8 mL.min–1、检测波长为255 nm进行测定。Euptox A在紫茎泽兰中的添加回收率为97.3%–103.7%,检测限为0.4μg.g–1。利用建立的方法测定Euptox A在紫茎泽兰体内分布与积累的动态变化规律。结果表明,Euptox A主要分布在紫茎泽兰的叶片中,且在营养生长期积累量高,生殖生长期积累量低。该方法快速、简捷,可用于紫茎泽兰原料及其产品中Euptox A成分的测定。