连翘鲜果粗酶对连翘酯苷A的酶解作用

探讨连翘鲜果粗酶对连翘酯苷A含量的影响,为连翘的炮制提供依据。采用高效液相色谱法(HPLC)分析不同浓度粗酶以及在不同孵育温度、孵育时间条件下,粗酶对连翘酯苷A酶解程度的影响,并利用水煮杀酶的方式测试了不同炮制时间对连翘果实中酶的灭活作用。结果表明,连翘果实中存在对连翘酯苷A有酶解作用的酶;用沸水煮的方法炮制连翘时,水煮数分钟即可达到杀酶保苷作用。此外,通过HPLC分析确认连翘酯苷A在粗酶液的作用下分解产物之一为咖啡酸。     

连翘红茶中连翘脂素的提取方法研究

以连翘红茶为原料,对其提取分离连翘脂素方法进行研究。以连翘脂素的质量浓度作为指标,采用超高压液相色谱进行分析检测,比较不同柱材料和重结晶条件对连翘脂素的分离纯化能力。最佳方法:利用75%乙醇回流提取,水洗两次,D101大孔吸附树脂70%洗脱7BV,重结晶四次,四道工序七步操作即可得到了纯度98%的连翘脂素,总收率49%。整个提取分离方法具有得到的连翘脂素纯度高,回收率高,不使用易燃有毒的有机溶剂,对设备的要求低,操作步骤少且简单,大孔吸附树脂可重复多次使用等特点。     

微波法提取连翘苷的工艺研究

以连翘叶为原料,连翘苷含量为检测指标,以提取溶剂、液料比、微波功率、提取时间为因素,采用四因素三水平L9(34)正交设计优选出连翘苷的最佳提取条件。结果表明,最佳提取条件为用液料比10∶1的75%乙醇微波(300 W)提取3次,每次120 S,得到连翘苷含量为0.52%。提取工艺合理可行,稳定可靠。     

炮制方法对连翘主要化学成分连翘酯苷的影响

探讨炮制方法对连翘中化学成分的影响,为连翘的合理炮制提供依据.采用HPLC法分析比较清蒸炮制时间对连翘化学成分含量影响.连翘炮制品与生品比较化学成分有明显变化,尤以连翘酯苷的变化最明显.炮制对连翘中化学成分有较大影响,以连翘酯苷为考察指标评价炮制方法,蒸10～15 min连翘质量最佳.     

连翘苷荧光传感器的构建和应用

以柠檬酸为碳源,三聚氰胺和甲醛为双掺杂剂合成碳量子点,并对合成的量子点进行透射电镜(TEM)、傅里叶变换红外光谱(FTIR)、X射线粉末衍射(XRD)、紫外吸收光谱(UV)和荧光光谱(RF)表征,结果表明该量子点粒径均一,发光稳定,适合用作荧光探针。优化了碳量子点含量、pH、反应时间以及温度等影响因素,结果显示检测波长λmax=425 nm时,连翘苷在0.008~0.030 mg/mL范围内有良好的线性关系,1/△f=0.00005(1/c)+0.0003(R2=0.9948),加标回收率在92.5%~106.3%之间,RSD<5%,且干扰物质、反应时间、温度等因素在一定范围内对实验结果的测定无影响。该方法可用于药剂中连翘苷含量的测定。     

毛细管电泳法分离测定芦丁、槲皮素和连翘苷

用毛细管电泳紫外检测法同时测定了芦丁、槲皮素和连翘苷，研究了各种条件的影响，得到了优化的实验条件，在20mmol/L的Na2B4O7(H3B03调节至pH8．40)-30mmol/L十二烷基硫酸钠-10％乙腈(1 9)的缓冲溶液中，分离电压为12kV时，芦丁、槲皮素和连翘苷在10min内得到了良好的分离，检测波长为254nm，芦丁、槲皮素和连翘苷分别在0．01-1．0mg/mL，0．01-1．0mg／mL和0．05-1．0mg/mL质量浓度范围内与电泳峰高呈现良好线性关系，检测下限分别为0．005mg/mL，0．005mg/mL和0．01mg／mL，应用于实际样品的测定。     

连翘脂素去甲基衍生物的合成

以连翘苷为原料,依次经水解反应、氯化铝/吡啶催化的去甲基化反应和差向异构化反应,合成了两个连翘脂素去甲基衍生物（1和2）,其结构经¹H NMR, ¹³C NMR, 2D NMR(COSY, NOESY, HSQC, HMBC), IR和MS(ESI)确证。经结构解析对比,1和2与连翘苷的大鼠体内次级代谢产物一致。     

连翘抗菌—谱效关系研究

建立连翘指纹图谱与抗菌活性之间的谱效关系,探索连翘抑菌主要活性成分。用高效液相色谱(HPLC)法研究不同产地连翘提取物的指纹图谱;以抑制金黄色葡萄球菌的活性比较不同产地连翘的生理活性差异;采用主成分、双变量相关、多元线性回归分析连翘的谱效关系。在连翘指纹图谱中,连翘酯苷A、咖啡酸、连翘苷和连翘脂素与连翘抑制金黄色葡萄球菌活性之间存在密切的关联性。连翘酯苷A、咖啡酸、连翘苷和连翘脂素可能为连翘抑菌活性的有效成分群。     

连翘叶清除亚硝酸盐和阻断亚硝胺形成及抗氧化活性研究

本文为了丰富连翘叶的保健功能,探究其生物活性的化学物质基础。利用D101型大孔吸附树脂对连翘叶水提取物进行了分离,制备出4个富集不同化学成分的组分;测定了各组分对亚硝酸盐的清除、亚硝胺形成的阻断能力和抗氧化活性,同时测定各组分的总多酚含量,并比较分析多酚含量与各生物活性之间的相关性。实验结果表明,40%乙醇洗脱部分(Fr2)总多酚含量高(45.40 g/100 g),具有较强的清除亚硝酸盐(IC50值为38.57μg·m L~(-1))、阻断亚硝胺形成(IC_(50)值为1.06 mg·m L~(-1))和清除DPPH?的能力(IC_(50)值为3.72μg·m L~(-1))。此外,Fr2对Fe~(3+)也具有较强的还原能力(以Trolox当量抗氧化能力计,2.46 mmol·g~(-1))。通过多酚含量与各生物活性之间相关性分析可知,多酚类物质为连翘叶清除亚硝酸盐、阻断亚硝胺形成能力和抗氧化活性的主要化学物质基础。同时研究结果表明,连翘酯苷A为连翘叶阻断亚硝胺形成的主要活性成分之一。     

连翘不同部位总酚含量测定及抗氧化活性比较研究

以山西产连翘为实验原料,采用单因素试验考察法优化了超声辅助提取连翘中总酚的最佳条件,即70%甲醇超声提取2次,10 min/次,料液比1∶20,此方法提取方便快捷,效率较高,为连翘总酚提取工艺提供一定参考价值。另外,在此条件下采用Folin-Ciocalteu法分别测定了老翘、青翘、连翘叶及连翘花中总酚含量,结果显示连翘叶中含量最高,花及果实次之;采用1,1-二苯基-2-苦肼基(DPPH·)清除法综合考察其抗氧化活性,结果与总酚含量顺序一致,二者之间呈明显正相关性,对于连翘资源的综合开发利用以及连翘中天然抗氧化剂的研究提供了重要的科学依据和指导作用。     

Interaction between phillygenin and human serum albumin based on spectroscopic and molecular docking

In this paper, the interaction of human serum albumin (HSA) with phillygenin was investigated by fluorescence, circular dichroism (CD), UV-vis spectroscopic and molecular docking methods under physiological conditions. The Stern-Volmer analysis indicated that the fluorescence quenching of HSA by phillygenin resulted from static mechanism, and the binding constants were 1.71×10(5), 1.61×10(5) and 1.47×10(4) at 300, 305 and 310K, respectively. The results of UV-vis spectra show that the secondary structure of the protein has been changed in the presence of phillygenin. The CD spectra showed that HSA conformation was altered by phillygenin with a major reduction of α-helix and an increase in β-sheet and random coil structures, indicating a partial protein unfolding. The distance between donor (HSA) and acceptor (phillygenin) was calculated to be 3.52nm and the results of synchronous fluorescence spectra showed that binding of phillygenin to HSA can induce conformational changes in HSA. Molecular docking experiments found that phillygenin binds with HSA at IIIA domain of hydrophobic pocket with hydrogen bond interactions. The ionic bonds were formed with the O (4), O (5) and O (6) of phillygenin with nitrogen of ASN109, ARG186 and LEU115, respectively. The hydrogen bonds are formed between O (2) of phillygenin and SER419. In the presence of copper (II), iron (III) and alcohol, the apparent association constant K(A) and the number of binding sites of phillygenin on HSA were both decreased in the range of 88.84-91.97% and 16.09-18.85%, respectively. In view of the evidence presented, it is expected to enrich our knowledge of the interaction dynamics of phillygenin to the important plasma protein HSA, and it is also expected to provide important information of designs of new inspired drugs.     

Alteration of yeast activity by gamma radiation

Yeast is an important component in microbe based industrial technologies. Due to the techno-economic reasons, the fermentation technique has acquired renewed interest. The effect of -radiation on the fermentation reaction has been investigated. The studies show that exposure of the fermentation mixture to -radiation at 5 kGy enhance alcohol production, whereas irradiation at higher doses, viz., 10 kGy and 25 kGy caused a considerable reduction in the alcohol yield. Therefore, low dose irradiation of fermentation mixtures can be applied for increasing the alcohol production by about 25%.     

Effect of High Pressure on Paracoccus denitrificans Growth and Polyhydroxyalkanoates Production from Glycerol

The performance of fermentation under non-conventional conditions, such as high pressure (HP), is a strategy currently tested for different fermentation processes. In the present work, the purpose was to apply HP (10–50 MPa) to fermentation by Paracoccus denitrificans, a microorganism able to produce polyhydroxyalkanoates (PHA) from glycerol. In general, cell growth and glycerol consumption were both reduced by HP application, more extensively at higher pressure levels, such as 35 or 50 MPa. PHA production and composition was highly dependent on the pressure applied. HP was found to decrease polymer titers, but increase the PHA content in cell dry mass (%), indicating higher ability to accumulate these polymers in the cells. In addition, some levels of HP affected PHA monomeric composition, with the polymer produced at 10 and 35 MPa showing considerable differences relative to the ones obtained at atmospheric pressure. Therefore, it is possible to foresee that the changes in polymer composition may also affect its physical and mechanical properties. Overall, the results of this study demonstrated that HP technology (at specific levels) can be applied to P. denitrificans fermentations without compromising the ability to produce PHA, with potentially interesting effects on polymer composition.

     

Ethanol from lignocellulosic wastes with utilization of recombinant bacteria

This article presents the advanced technology that has been developed by BioEnergy International of Gainesville, Florida, utilizing novel recombinant strains of bacteria developed by Lonnie Ingram of the University of Florida. The first commercial applications of these unique fermenting organisms convert 5-carbon sugars, as well as 6-carbon sugars, and oligomers of cellulose (e.g., cellobiose and cellotriose) directly to ethanol. The proposed systems that will be utilized for conversion of agricultural wastes, mixed waste papers, and pulp and paper mill waste in forthcoming commercial installations are now under design. This involves the extensive experience of Raphael Katzen Associates International, Inc. in acid hydrolysis, enzyme production, enzymatic hydrolysis, large-scale fermentation engineering, and distillation/dehydration. Specific examples of this advanced technology will be presented in different applications, namely:

Preparative isolation and purification of phillyrin from the medicinal plant Forsythia suspensa by high-speed counter-current chromatography

Forsythia suspensa (Thunb.) Vahl. has been used widely in traditional medicines to treat gonorrhea, erysipelas, inflammation, pyrexia and ulcer. It has also shown antioxidant activity, as well as antibacterial, antiviral, choleretic and antiemetic effects. A high-speed counter-current chromatography (HSCCC) method was developed for the preparative separation and purification of the bioactive molecule phillyrin from F. suspensa (Thunb.) Vahl. The crude phillyrin was obtained by extraction with 50% ethanol from the dried fruits of F. suspensa (Thunb.) Vahl. under sonication. Preparative HSCCC with a two-phase solvent system composed of n-hexane-ethyl acetate-ethanol-water (1:9:1:9, v/v/v/v) was successfully performed, and the components purified and collected were analyzed by high-performance liquid chromatography. The method yielded 5.6 mg phillyrin at 98.6% purity from 500 mg of the crude extract (1.2% phillyrin) with the recovery of 92% in a one-step separation.     

Metabolic profile of phillyrin in rats obtained by UPLC‐Q‐TOF‐MS

Forsythia suspensa Vahl (Oleaceae) is an important original plant in traditional Chinese medicine. The air‐dried fruits of Forsythia suspensa have long been used to relieve respiratory symptoms. Phillyrin is one of the main chemical constituent of Forsythia suspensa. A clear understanding of the metabolism of phillyrin is very important in rational clinical use and pharmacological research. In this study, the metabolism of phillyrin in rat was investigated for the first time using an ultra‐high‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry (UPLC‐Q‐TOF‐MS) method. Bile, urine and feces were collected from rats after single‐dose (10 mg/kg) orally administered phillyrin. Liquid–liquid extraction and ultrasonic extraction were used to prepare samples. UPLC‐Q‐TOF‐MS analysis of the phillyrin samples showed that phillyrin was converted to a major metabolite, M26, which underwent deglucosidation, further dehydration and desaturation. A total of 34 metabolites were detected including 30 phase I and four phase II metabolites. The conjugation types and structure skeletons of the metabolites were preliminarily determined. Moreover, 28 new metabolites were reported for the first time. The main biotransformation route of phillyrin was identified as hydrolysis, oxidation and sulfation. These findings enhance our understanding of the metabolism and the real active structures of phillyrin. Copyright © 2015 John Wiley & Sons, Ltd.     

Study on glucose oxidase fermentation coupled with membrane dialysis

The repression of glucose oxidase (GOD) biosynthesis by catabolites during fermentation can be alleviated through membrane dialysis fermentation (MDF). The results show that the volumetric enzyme productivity of MDF was two times higher than that of the control (fermentation without dialysis), and its total enzyme activity was increased by 30–50%. The operation conditions of MDF, such as pore size of the membrane, initiating time for membrane dialysis, and volume of dialysate used, were optimized. The content of amino acids and organic acids in the fermentation broth and the dialysate in the reservoir were assessed by amino acid analyzer and ionic chromatography, respectively. The relationship among the contents of pyruvic acid, gluconic acid, and enzyme activity during fermentation was analyzed quantitatively. Furthermore, the effect of membrane dialysis technology applied to the low-yield strain was found to be more effective than that applied to the high-yield strain.     

Quantitative Determination of Phillyrin in Human Plasma Using HPLC with Fluorescence Detection

A simple, rapid, and selective high-performance liquid chromatography method for determination of phillyrin in human plasma was developed. After extracting from the plasma samples with ethyl acetate, the analyte was chromatographed on a C18 column with methanol–water (50:50, v/v, pH 2.86) as mobile phase. The fluorescence excitation and emission wavelengths were 277 and 315 nm, respectively. The linear range of the standard curve of phillyrin was 0.0313–8.0 μg mL^?1 (r > 0.999). The limit of detection was 6.31 ng mL^?1. The average recovery of phillyrin was 101.02% from plasma. The intra- and inter-day variabilities of phillyrin were <10.00%.     

左旋葡聚糖的制备与在生物技术领域的应用

左旋葡聚糖(LGA)作为一种极具潜力的新糖源,既可以加酸水解为葡萄糖后间接用于微生物发酵,也可以被真菌或细菌甚至构造的基因工程菌直接代谢,目前已经在发酵上展现出良好的应用前景。此外,LGA还可作为碳源,生产生物乙醇、丁二酸等。目前制备LGA普遍采用生物质热解法,该方法存在能耗高、产率低、产物难以提取等缺点,应努力发展在溶剂体系中分解纤维素制备LGA的方法以提高产率。今后的研究应致力于开发微生物代谢LGA的新过程、新方法、新菌种,为LGA的高效利用提供坚实基础。     

The use of tandem-mass spectrometry for pilot identification

Summary The direct analysis of mixtures by tandem-mass spectrometry assisted by off-line liquid chromatographymass spectrometry is already established as a rapid specific and extremely sensitive method that can be applied to samples of a variety of types for example, to analyses of main compounds and by-products in reaction mixtures, to products in medicated solutions or to fermentation products in more or less complex matrices. The techniques are demonstrated as pilot methods for obtaining structural information for identification or structure elucidation.