基于高效液相色谱-高分辨质谱技术的柴胡皂苷c化学转化产物结构与途径分析

利用高效液相色谱|(HPLC)-Q Exactive-Orbitrap高分辨质谱(HRMS)技术分析12-磷钨酸化学转化柴胡皂苷c(SSc)的产物结构和转化途径。HPLC可以快速分离结构相近的转化产物。利用HRMS获得产物分子及其碎片离子的精确质量数,鉴定产物结构并分析转化途径。结果表明,在12-磷钨酸产生的酸性环境中,SSc主要通过C₁₈位的环醚开环和羟基消除反应生成具有异环双烯结构的柴胡皂苷h(SSh),并伴随着C₃位的去糖基化反应生成去鼠李糖-SSc和去鼠李糖-SSh。转化反应6 h后趋于稳定。HPLC-Q Exactive-Orbitrap技术能够快速准确地分析柴胡皂苷及其转化产物结构,并可以区分转化得到的异环双烯和同环双烯柴胡皂苷同分异构体。     

柴胡皂苷A对抑郁模型大鼠脑中单胺类神经递质及其代谢产物含量的影响

在酸性条件下, 利用高效液相色谱法研究了柴胡皂苷A对抑郁模型大鼠脑中单胺类神经递质及其代谢物含量的影响. 实验结果表明, 柴胡皂苷A可使抑郁型大鼠脑中的高香草酸、去甲肾上腺素、多巴胺及5-羟色胺的含量升高, 这一结果有利于对柴胡皂苷治疗抑郁症的物质基础及相关机理进行更深入了解.     

苦瓜子蛋白的分离纯化及真性质研究

从苦瓜种子的粗提物苦瓜子蛋白丙酮分级沉淀干粉中,用CM-Spehadex C-50和SephadexG-75分离得到了两个核糖体失活蛋白,α-苦瓜子蛋白(C-momorcharin,α-MMC和β-苦瓜子蛋白(β-momorcharin,β-MMC),其等电点分别为9.10(α-MMC),9.32(β-MMC).用ESI-MS和MALDI-TOF-MS测定它们的分子量,分别为28625(α-MMC),29076(β- MMC)(+Na,29099);,28795(α-MMC),29074.7(β-MMC).它们都是糖蛋白,其生物活性的分析测定表明,都属于RNA N-糖苷酶,本文重点对β-苦瓜子蛋白的分离纯化及其性质进行详细报道,并对其N-端部分的氨基酸顺序进行了测定.     

苦瓜子蛋白的分离纯化及其性质研究

从苦瓜种子的粗提物苦瓜子蛋白丙酮分级沉淀干粉中,用CM-SephadexC-50和SephadexG-75分离得到了两个核糖体失活蛋白,α-苦瓜子蛋白(α-momorcharin,α-MMC)和β-苦瓜子蛋白(β-momorcharin,β-MMC),其等电点分别为9.10(α-MMC),9.32(β-MMC)。用ESI-MS和MALDI-TOF-MS测定它们的分子量,分别为28625(α-MMC),29076(β-MMC)(+Na,29099);28795(α-MMC),29074.7(β-MMC)。它们都是糖蛋白。其生物活性的分析测定表明,都属于RNAN-糖苷酶。本文重点对β-苦瓜子蛋白的分离纯化及其性质进行详细报道,并对其N-端部分的氨基酸顺序进行了测定。     

猪肝中单胺氧化酶B的分离纯化

依据单胺氧化酶B(monoamine oxidase B, MAOB)的疏水特性,建立了一种从猪肝中分离纯化MAOB的新方法。用含有1% Triton X-100的膜蛋白裂解液制备粗酶,以饱和度为20%～50%的硫酸铵反抽提进行粗提,再利用自制的配基密度为75.7 μmol/mL的苯基疏水色谱及Sepharose Q High Performance离子交换色谱进一步分离纯化,得到纯化倍数为18.2、酶比活为135 U/mg的MAOB。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析显示为相对分子质量约60 000的单一蛋白质带。采用高效液相色谱-电喷雾串联质谱对该酶进行鉴定,证实为MAOB。本研究所用分离纯化方法可以有效纯化MAOB, 为MAOB的深入研究提供技术支撑。     

SPAP2基因的克隆、融合表达及分离纯化

本文构建的表达载体pGex-2T-SPAP2CT在大肠杆菌中表达出可融性蛋白, 其分子量为46 000, 经纯化后得到产率为10%、 纯度大于90%的GST-SPAP2CT蛋白.     

维生素对微量元素吸收利用的影响

维生素A：维生素A能增强硒抑制癌症的作用。缺乏对微量元素吸收利用维生素A可妨碍铁的吸收利用。 维生素B族：有学者发现给缺乏维生素B6饮食的鼠组织中锌含量降低,而缺乏维生素B1和维生素B2并不产生此现象,已证明维生素眈缺乏引起的锌吸收障碍,是由于PA（色氨酸的代谢产物）减少所致。     

新型药物辅料2-羟丙基-β-环糊精的色谱分离纯化

利用薄层色谱方法对碱浓度为1.5%、β-环糊精∶环氧丙烷(摩尔比)=1∶21的条件下生成的2-羟丙基-β-环糊精进行了定性分析,并通过对展开剂的选择和优化,得到了3种能有效分离不同取代度2-羟丙基-β-环糊精的展开剂体系,分别为正丙醇-水-浓氨水(6∶3∶1,V/V),异丙醇-水-浓氨水(6∶4∶0.5,V/V)和乙醇-水-浓氨水(6∶3∶0.8,V/V)。通过以乙醇体系为洗脱剂的硅胶柱色谱对其进行分离纯化,得到了两种不同取代的2-羟丙基-β-环糊精。经过ESI-MS谱图分析,确定分离后产品分别为单取代和双取代2-羟丙基-β-环糊精。     

莲子草假隔链格孢毒素的分离纯化与结构鉴定

从莲子草假隔链格孢(Nimbya alternantherae)中分离纯化了一种毒素物质, 并对其进行了结构鉴定, 为研究其致病机理及进一步的化学合成奠定了基础.     

GMA-EDMA-TiO2扩张床介质合成的改进及其在复合干扰素分离纯化中的应用

通过合成条件的改进,得到了一种性能更好的扩张床介质GMA-EDMA-Lipo-TiO2(GELT),其平均密度为1.25g/ml,孔容为0.564ml/g,平均孔径为15.6nm,比表面积为127.58m2/g,渗磨圆球率为95%。研究表明GELT在氢氧化钠溶液是稳定的。同时讨论了甲苯致孔剂对孔结构的影响及二氧化钛添加量对湿真密度的影响;将GELT与二乙胺偶联得到阴离子树脂(GELT-DEAE),在静态和动态条件下,GELT-DEAE吸附量分别88mgBSA/ml、65mgBSA/ml。将GELT-DEAE应用于扩张床直接从发酵液中分离复合干扰素,一步活性收率为75%,后经SPSepharoseFF离子交换层析和CellufastGel01凝胶层析进一步纯化。最后得到的成品其比活为6.82×108IU/mg,与商品干复津(Infergen)的比活相当,纯度为95%。     

新型水稻黄单胞菌Harpin蛋白的纯化及其特性研究

报道水稻黄单胞(Xanthomonas oryzae)的两个致病变种(pv.oryzae和 pv.oryzicola)所产生的Harpin蛋白. 结果表明， 表达菌株HRF1和HRF2分别携带两个致病变种编码Harpin的hrf1和hrf2基因. 在IPTG诱导下, 两个表达菌株的无细胞破碎液均具有激发烟草叶片过敏反应(HR)的活性. 采用(NH4)2SO4沉淀、 阴离子交换层析、 Native-PAGE微量制备等方法, 分别纯化出分子量为15 600和15 300, pI皆为4.5左右的单一条带; 这两个单一组分符合典型Harpin蛋白的特征: 可激发烟草HR， 诱导烟草抗TMV， 对蛋白酶K敏感、 对热稳定; 放线菌素D、 环己酰亚胺和氯化镧等真核生物代谢抑制剂可消除它们的生物活性; 琼脂双扩散试验(ODD)血清反应表明， 两个Harpin蛋白有交叉反应.     

Two New Cyclic Pentapeptides from the Marine‐Derived Fungus Aspergillus versicolor

Two new cyclic pentapeptides, named versicotides A ( 1 ) and B ( 2 ), were obtained from a marine‐derived fungus strain ZLN‐60, identified as Aspergillus versicolor. Their structures were established on the basis of chemical and spectroscopic evidence. Versicotides are new cyclic pentapeptides which contain an L ‐alanine residue, two anthranilic acid (=2‐aminobenzoic acid) residues, and two N‐methyl‐L ‐alanine residues. Antitumor activities were evaluated by the SRB and MTT methods.     

Purification and characterization of an exoinulinase from Aspergillus fumigatus

An extracellular exoinulinase was purified from the crude extract of Aspergillus fumigatus by ammonium sulfate precipitation, followed by successive chromatographies on DEAE-Sephacel, Sephacryl S-200, concanavalin A-linked amino-activated silica, and Sepharose 6B columns. The enzyme was purified 25-fold, and the specific activity of the purified enzyme was 171 IU/mg of protein. Gel filtration chromatography revealed a molecular weight of about 200 kDa, and native polyacrylamide gel electrophoresis (PAGE) showed an electrophoretic mobility corresponding to a molecular weight of about 176.5 kDa. Sodium dodecyl sulfate-PAGE analysis revealed three closely moving bands of about 66, 62.7, and 59.4 kDa, thus indicating the heterotrimeric nature of this enzyme. The purified enzyme appeared as a single band on isoelectric focusing, with a pI of about 8.8. The enzyme activity was maximum at pH 5.5 and was stable over a pH range of 4.0–9.5, and the optimum temperature for enzyme activity was 60°C. The purified enzyme retained 35.9 and 25.8% activities after 4 h at 50 and 55°C, respectively. The inulin hydrolysis activity was completely abolished with 1 mM Hg⁺⁺, whereas EDTA inhibited about 63% activity. As compared to sucrose, stachyose, and raffinose, the purified enzyme had lower K _m (0.25 mM) and higher V _max (333.3 IU/mg) values for inulin.     

Two New Secoanthraquinone Derivatives from the Marine‐Derived Endophytic Fungus Aspergillus wentii EN‐48

Two new secoanthraquinone derivatives, wentiquinones A ( 1 ) and B ( 2 ), together with the eight known compounds 3 – 10 , were isolated from the culture extracts of Aspergillus wentii EN‐48, an endophytic fungus derived from an unidentified marine brown algal species of the genus Sargassum. The structures of these compounds were elucidated on the basis of extensive spectroscopic analysis.     

Effect of Agave americana L. on the human,and Aspergillus oryzae S2 α-amylase inhibitions

Among phenolic compounds, Agave americana L. extract contained puerarin (38.4%) and p-coumaric acid (12.29%) (pCa). From the Lineweaver–Burk plots, pCa and puerarin demonstrated a competitive and a non competitive inhibitions towards human α-amylase activity, respectively. PCa exhibited a higher human inhibitory activity with an IC₅₀ of 98.8 μM which was about 2.3 times than acarbose. Puerarin (IC₅₀ = 3.87 μM) and pCa (IC₅₀ = 10.16 μM) also showed an excellent inhibition for Aspergillus oryzae S2 α-amylase activity. The inhibitions of the described biocatalysts compounds towards both amylases were significantly decreased when they were pre-incubated with starch. The binding modes of these compounds were evaluated in silico. The binding efficiency order of these molecules in terms of polar contact numbers for both enzymes was in agreement with the in vitro studies. These findings provided a rational reason to establish the isolated compounds capability as therapeutic target for hyperglycaemia modulation and antifungal therapy.     

Application of centrifugal partition chromatography coupled with evaporative light scattering detection for the isolation of saikosaponins‐a and ‐c from Bupleurum falcatum roots

Centrifugal partition chromatography (CPC) coupled with evaporative light scattering detection (ELSD) was applied to separate saikosaponins‐a and ‐c preparatively from Bupleurum falcatum roots. The two‐phase solvent system composed of ethyl acetate/n‐butanol/methanol/water (15:1:3:15 by volume) was used to yield saikosaponins‐a (36.1 mg) and ‐c (28.7 mg) from 370 mg of saponin‐rich extract. The purities of isolated compounds were 96.6 and 97.3% for saikosaponins‐a and ‐c, respectively. Structure identification of these compounds was accomplished by comparison of spectroscopic data of ESI‐MS, ¹H NMR, and ¹³C NMR with those of previously reported values.     

负载型纳米TiO2／Al2O3复合载体负载SO4^2-催化剂的制备与催化性能研究

分别采用沉淀法,水解法和溶胶凝胶法制备了负载型纳米TiO2/Al2O3复合载体,同时在复合载体表面负载SO42-制成SO42-/TiO2/Al2O3固体酸催化剂,并将此催化剂用于α-蒎烯异构化反应中.用XRD、FTIR,TPD等手段对催化剂的晶相结构、比表面积、孔径分布、表面酸性等进行了表征.结果表明,三种方法所制备的催化剂均为纳米级且拥有着丰富而规则的孔结构,水解法制备的SO42-/TiO2/Al2O3催化剂中TiO2的平均粒径(10.0 nm)较小,比表面积(172.88 m2/g)较大,平均孔径为3.926 nm,表面酸中心数和酸强度均高于沉淀法和溶胶凝胶法制备的催化剂,在α-蒎烯催化异化反应中的具有较高的活性,α-蒎烯转化率为82.76%.     

甘西鼠尾草中三个新的萜类化合物

采用反复硅胶、葡聚糖凝胶、反相硅胶及MCI凝胶柱色谱等多种色谱方法分离纯化了甘西鼠尾草的化学成分,根据理化性质和波谱数据对化合物的结构进行了鉴定.由甘西鼠尾草的根和根茎的50%乙醇提取物中得到了2个新的二萜化合物,命名为甘西鼠尾三醇A(1)和甘西鼠尾三醇B(2),同时得到了一个新的单萜苷化合物,命名为甘西鼠尾甲苷(3)...     

Chemical investigation of metabolites produced by an endophytic Aspergillus sp. isolated from Limonia acidissima

Endophytic fungi are considered as a good source to produce important secondary metabolites with interesting bioactivities. In a continuation of our studies towards the search for environmentally friendly bioactive compounds from Sri Lankan flora, we investigated the secondary metabolites produced by the endophytic fungi Aspergillus sp. isolated from the seeds of the popular edible fruit Limonia acidissima L. of the family Rutaceae. The pure culture of the Aspergillus sp. was grown on potato dextrose broth media. After 4 weeks fermentation, fungal media were extracted with organic solvents. Chromatographic separation of the fungal extracts over silica gel, Sephadex LH-20 and RP-HPLC furnished flavasperone (1), rubrofusarin B (2), aurasperone A (3), fonsecinone D (4) and aurasperone B (5). Compounds 1–4 showed moderate activities in brine shrimp toxicity assay. This is the first report of the ¹³C NMR data of compounds 4 and 5.     

Regioselective Dichlorination of a Non‐Activated Aliphatic Carbon Atom and Phenolic Bismethylation by a Multifunctional Fungal Flavoenzyme

The regioselective functionalization of non‐activated carbon atoms such as aliphatic halogenation is a major synthetic challenge. A novel multifunctional enzyme catalyzing the geminal dichlorination of a methyl group was discovered in Aspergillus oryzae (Koji mold), an important fungus that is widely used for Asian food fermentation. A biosynthetic pathway encoded on two different chromosomes yields mono‐ and dichlorinated polyketides (diaporthin derivatives), including the cytotoxic dichlorodiaporthin as the main product. Bioinformatic analyses and functional genetics revealed an unprecedented hybrid enzyme (AoiQ) with two functional domains, one for halogenation and one for O‐methylation. AoiQ was successfully reconstituted in vivo and in vitro, unequivocally showing that this FADH₂‐dependent enzyme is uniquely capable of the stepwise gem‐dichlorination of a non‐activated carbon atom on a freestanding substrate. Genome mining indicated that related hybrid enzymes are encoded in cryptic gene clusters in numerous ecologically relevant fungi.