NMR Spectroscopic Analysis Reveals Extensive Binding Interactions of Complex Xyloglucan Oligosaccharides with the Cellvibrio japonicus Glycoside Hydrolase Family 31 α‐Xylosidase

The study of the interaction of glycoside hydrolases with their substrates is fundamental to diverse applications in medicine, food and feed production, and biomass‐resource utilization. Recent molecular modeling of the α‐xylosidase CjXyl31A from the soil saprophyte Cellvibrio japonicus, together with protein crystallography and enzyme‐kinetic analysis, has suggested that an appended PA14 protein domain, unique among glycoside hydrolase family 31 members, may confer specificity for large oligosaccharide fragments of the ubiquitous plant polysaccharide xyloglucan (J. Larsbrink, A. Izumi, F. M. Ibatullin, A. Nakhai, H. J. Gilbert, G. J. Davies, H. Brumer, Biochem. J. 2011 , 436, 567–580). In the present study, a combination of NMR spectroscopic techniques, including saturation transfer difference (STD) and transfer NOE (TR‐NOE) spectroscopy, was used to reveal extensive interactions between CjXyl31A active‐site variants and xyloglucan hexa‐ and heptasaccharides. The data specifically indicate that the enzyme recognizes the entire cello‐tetraosyl backbone of the substrate and product in positive enzyme subsites and makes further significant interactions with internal pendant α‐(1→6)‐linked xylosyl units. As such, the present analysis provides an important rationalization of previous kinetic data on CjXyl31A and unique insight into the role of the PA14 domain, which was not otherwise obtainable by protein crystallography.     

Simultaneous Extraction and Determination of Characteristic Steroidal Saponins and Homoisoflavonoids in Zhejiang Ophiopogon japonicus

Zhejiang Ophiopogon japonicus (ZOJ) is a specific variety of Ophiopogon japonicus with characteristic steroidal saponins and homoisoflavonoids, which are also main pharmacodynamic constituents with clinical effects, including curing inflammation and cardiovascular diseases. However, few analysis methods were applied to simultaneously and quantitatively determine two kinds of its constituents, and hazardous organic solvents are mostly used for extraction. In this study, a new validated simultaneous extraction and determination method for four characteristic steroidal saponins and homoisoflavonoids in ZOJ was established by ionic liquid–ultrasonic extraction (IL-UAE) combined with HPLC-DAD-ELSD analysis, which can be used for the quality control of ZOJ. Chromatographic separation was performed with a DAD wavelength at 296 nm, and the ELSD parameters of the drift tube temperature (DTT), atomizer temperature (AT), and nitrogen gas pressure (NGP) were set at 20% heating power, 70 °C, and 25 psi, respectively. The optimal IL-UAE conditions were 1 mol/L [Bmim]CF₃SO₃ aqueous solution, a liquid–material ratio of 40 mL/g, and an ultrasonic time of 60 min. The proposed method is reliable, reproducible, and accurate, which were verified with real sample assays. Consequently, this work will be helpful for the quality control of ZOJ. It can also present a promising reference for the simultaneous extraction and determination of different kinds of constituents in other medicinal plants.     

花鲈同工酶的组织特异性研究

采用聚丙烯酰胺垂直平板电泳对花鲈的心、肝、眼球、视网膜、肌肉、肾、鳃、胸鳍等8个组织的10种同工酶(LDH、MDH、ME、α-GPDH、ADH、SDH、SOD、G—6—PDH、EST、GDH)进行了分析，结果认为：10种同工酶在各个组织中的表达频率，以肝脏最高，其次为心脏、肌肉、视网膜，较低的为眼球、锶、肾、鳍组织．其中LDH、MDH、及SOD三种同工酶都能在8个组织中得到表达，但表达的强度有所差异．基因座位表达程度，以肝脏、肌肉两组织为最全面．     

Spirostanol Saponins from the Fibrous Roots of Ophiopogon japonicus (Thunb.) Ker‐Gawl

Three new steroidal saponins, (25R)‐ruscogenin‐3‐yl α‐L ‐rhamnopyranosyl‐(1→2)‐[β‐D ‐xylopyranosyl‐(1→4)]‐β‐D ‐glucopyranoside ( 1 ), diosgenin‐3‐yl 2‐O‐acetyl‐α‐L ‐rhamnopyranosyl‐(1→2)‐[β‐D ‐xylopyranosyl‐(1→4)]‐β‐D ‐glucopyranoside ( 2 ), and pennogenin‐3‐yl 2‐O‐acetyl‐α‐L ‐rhamnopyranosyl‐(1→2)‐[β‐D ‐xylopyranosyl‐(1→4)]‐β‐D ‐glucopyranoside ( 3 ) were isolated from the fibrous roots of Ophiopogon japonicus (Thunb .) Ker‐Gawl . Their structures were determined by spectroscopic methods including IR, HR‐ESI‐MS, and 1D‐ and 2D‐NMR. All of these three steroidal saponins exhibited weak cytotoxicities against Hela and Hep2 cell lines.     

Labdane Diterpenes and Flavonoids from Leonurus japonicus

Chemical investigation of the aerial part of Leonurus japonicus led to the isolation and characterization of a new labdane‐type diterpene, named 3α‐acetoxy‐15‐O‐methylleopersin C ( 3 ), and a new acylated rutin derivative, named 2′′′‐syringylrutin ( 9 ), along with seven known compounds, including a labdane‐type diterpene and six flavonoids. The structures of the new compounds were established by spectroscopic methods.     

ent‐Kaurane Diterpenoids from Isodon japonicus

Two new 6,7‐seco‐ent‐kaurane diterpenoids, isojaponins A ( 1 ) and B ( 2 ), together with 18 known ent‐kaurane diterpenoids were isolated from the aerial parts of Isodon japonicus. The structures of the two new compounds were elucidated by extensive 1D‐ and 2D‐NMR spectroscopic methods in combination with MS experiments.     

毛细管电泳-电化学检测益母草及其冲剂中的黄酮类化合物

用毛细管电泳-电化学检测(CE-ED)法同时测定了益母草及三种益母草冲剂中的根皮苷、橙皮素、芦丁、山奈酚、洋芹素和槲皮素六种黄酮类化合物的含量。考察了实验参数对分离、检测的影响,得到了最佳试验条件。采用300μm碳圆盘电极为工作电极,检测电位为 0.95 V(vs.SCE),在80 mmol/L的Na2B4O7-H3BO3(pH 8.7)的运行缓冲液中,上述各组分在20 min内能完全分离。根皮苷、橙皮素、芦丁、山奈酚、洋芹素、槲皮素在5.00×10-7~1.00×10-4g/mL范围内均与其相应的峰电流呈线性关系,其检出限(S/N=3)分别为4.68×10-7、2.03×10-7、4.68×10-7、2.68×10-7、4.81×10-7、3.61×10-7g/mL。该法应用于实际样品的分析,结果令人满意。     

一维SEMDY和旋转坐标NOE差谱NMR新技术用于天然化合物中寡糖的结构研究

报告从日本续断根部的乙醇提取物中分得二个新的五糖三萜皂甙,应用一维ＳＥＭＤＹ和旋转坐标ＮＯＥ差谱等ＮＭＲ新技术互相配合的方法对它们的结构进行了研究,确定为：３－Ｏ－α－Ｌ－吡喃鼠李糖（１→３）－β－Ｄ－吡喃葡萄糖（１→３）－α－Ｌ－吡喃李鼠糖（１→２）－α－Ｌ－吡喃阿拉伯糖－常春藤甙元－２８－Ｏ－β－吡喃葡萄糖酯甙（１）,和３－Ｏ－「β－Ｄ－吡喃葡萄糖（１→４）」「α－Ｌ－吡喃鼠李糖（１→３）」－     

一维SEMDY和旋转坐标NOE差谱NMR新技术用于天然化合物中寡糖的结构研究

报告从日本续断根部的乙醇提取物中分得二个新的五糖三萜皂甙,应用一维SEMDY和旋转坐标NOE差谱等NMR新技术互相配合的方法对它们的结构进行了研究,确定为：3-O-α-L-吡喃鼠李糖（1→3）-β-D-吡喃葡萄糖（1→3）-α-L-吡喃鼠李糖（1→2）-α-L-吡喃阿拉伯糖-常春藤甙元-28-O-β-吡喃葡萄糖酯甙（1）,和3-O-[β-D-吡喃葡萄糖（1→4）] [α-L-吡喃鼠李糖（1→3）]-β-D-吡喃葡萄糖（1→3）-α-L-吡喃鼠李糖（1→2）-α-L-吡喃阿拉伯糖-齐墩果酸（2）·结果表明,一维SEMDY和旋转坐标NOE差谱技术互相配合的方法测定寡糖链结构十分有效,高度重叠的糖基1H-NMR信号可按一定规律分离,容易鉴别,糖基之间的连接顺序和连接位置可以准确测定,不需要对化合物进行化学降解或衍生化。     

花鲈工厂化育苗技术的研究

研究提高花鲈工厂化育苗成活率的技术，结果表明：（１）采用自选单胞藻营养强化轮虫和卤虫成体，按卤虫总量２．５．３．０％浓鱼油胶丸营养强化卤虫无节幼体，投喂花鲈仔稚幼鱼，能满足仔稚幼鱼生长发育的营养需求：（２）泼洒１ｐｐｍ呋喃西林或氯霉素能有效地治疗和预防仔稚幼鱼细菌性疾病的发生；（３）稚鱼阶段的适宜放养密度为１５００尾／ｍ３左右；（４）花鲈仔鱼经９０天培育，平均全长可达３８．５５ｍｍ，育成率达到５８．２１％，单位水体出苗量达到１５９９尾／ｍ３。