大豆异黄酮β-葡萄糖苷酶产生菌的选育及产酶条件研究

以豆豉为材料筛选产大豆异黄酮β-葡萄糖苷酶的菌株,并用京尼平甙法检测相对酶活。以相对酶活最高的野生菌为出发菌(经镜检为杆菌),通过UV和LiCl诱变育种,利用单因素和正交实验进行产酶条件研究确定最佳条件为:黄豆粉1%、酵母膏1%、麸皮2%、KH2PO40.1%、NaCl0.5%、起始pH值7.5、装液量30ml、发酵时间48h、发酵温度37℃、转速70r/min。在该条件下,用京尼平甙法测得该菌株的相对酶活为0.826,水杨苷法测得绝对酶活为5.49μg/ml。

延年康口服液中四羟基二苯乙烯葡萄糖苷的含量测定

目的 :建立延年康口服液中 2 ,3,5 ,4’ 四羟基二苯乙烯 2 O β D 葡萄糖苷的含量测定方法。 方法 :采用反相高效液相色谱法 ,Nova PakC1 8(4μm ,1 5 0mm×3.9mm )色谱柱 ,流速 :0 .8mL·min-1 ,乙腈 水 (2 0∶80 )为流动相 ,检测波长为 32 0nm。结果 :2 ,3,5 ,4’ 四羟基二苯乙烯 2 O β D 葡萄糖苷的线性范围为0 .0 4 5 6～ 0 .2 736 μg(r =0 .9991 ) ,平均回收率为98.6 9%,RSD为 1 .91 %(n =6 )。结论 :方法简便、快速、准确 ,适合该口服液中 2 ,3,5 ,4’ 四羟基二苯乙烯 2 O β D 葡萄糖苷的含量测定。

HPLC测定番泻叶中5种主要化学成分的含量

目的：建立番泻叶中主要化学成分的含量测定方法。方法：采用HPLC，DiamonsilC18柱（4.6mm×250mm，5μm），流动相乙腈-1％冰醋酸梯度洗脱（10：90-15：85-18：82-20：80-25：75），流速1mL·min^-1，柱温40℃，检测波长270nm。结果：5种化学成分均达到基线分离，各成分都有较宽的线性范围和良好的线性关系。结论：本法快速、灵敏、准确，可靠，重复性好，可用于中药番泻叶药材的质量控制。

HPLC测定制首乌及首乌延寿片中二苯乙烯苷的含量

目的建立原药材制首乌及首乌延寿片中2,3,5,4'-四羟基二苯乙烯-2-O-β-D-葡萄糖苷(THPG)的含量测定方法.方法采用HPLC法,Diamonsil C18色谱柱 (4.6 mm×250 mm,5 μm);流动相为乙腈-水(24∶76),检测波长320 nm,柱温30℃,流速1 ml·min-1.结果回归方程为:Y=7.356×103X-2.126×104(r=0.9998),线性范围80～630 ng.平均回收率为98.32%,RSD＝0.57%.结论方法简便、准确、快速、灵敏、重复性好,可用于原药材制首乌及首乌延寿片制剂的质量控制.

废弃烟叶化学成分研究(一)

[目的]对废弃烟叶有效化学成分进行研究。[方法]以柱色谱、薄层色谱纯化,采用UV、IR、MS、NMR等波谱方法进行结构鉴定。[结果]从石油醚、乙醇混合浸提液中分离得到12个化合物,经波谱解析得到其中6个化合物的结构,即正二十四烷、对羟基正二十烷酸苯乙酯、β-谷甾醇、豆甾醇、东莨菪苷、豆甾醇-3-O-葡萄糖苷。[结论]这些化合物均首次从烟叶中分离得到。

肾结石患者尿NAG、γ-GT和β_2-MG水平升高的意义及机制探讨

目的:探讨肾结石患者尿N-乙酰-β-葡萄糖苷酶(NAG)、γ-谷氨酰胺转移酶(γ-GT)和β2-微球蛋白(β2-MG)升高的意义及机制。方法:选择40例肾结石患者(志愿者)和30例健康无结石志愿者,用生化分光光度法测定尿NAG、γ-GT和Cr,以及血浆超氧化物岐化酶(SOD)、丙二醛(MDA)和一氧化氮(NO),用放免法测定尿β2-MG、血浆内皮素(ET-1)和血清肿瘤坏死因子(TNFα)。结果:结石组尿NAG、γ-GT和β2-MG显著高于健康对照组(P<0.05)。结石组血中ET-1、TNF-α和MDA值显著升高,而血SOD、NO值显著降低,两组之间有显著性差异(P<0.05)。结论:肾结石形成可能与肾小管功能损伤相关,氧自由基、局部血流动力学改变和炎症反应在在肾小管损伤中发挥了作用。

Chemical Constituents from Pueraria lobata

Pueraria lobata ( Wild. ) Ohwi ( Ye-Ge in Chinese ) is a perennial herb ofthe genus Pueraria, which belongs to the Leguminosae family. It is commonly employed to relievefever and dysentery, promote the production of body fluid, reduce stiffness and pain of the nape,and for the treatment of cardiovascular diseases, e. g. hypertension, myocardial infarction, andarrhythmia. Previous phytochemical studies on P. lobata reported a number of bioactive isoflavones,e. g. daidzein, daidzin, and puerarin. Further investigation of its root has led to isolation offourteen compounds and their structures were identified as daidzein, ononin, daidzin, 3'' -methoxypuerarin, puerarin, pueroside B, daidzein-8-C-apiosyl- (1-6)-glucoside, 3''-hydroxy-puerarin,puerarinxyloside, daidzein-7, 4'' -O-glucoside, puerarin-4''-O-glucoside, mirificin-4''-O-glucoside,sissotorin, and pueroside C. Compounds 11 and 13 were isolated from the root of P. lobata for thefirst time.

α-Glucosidase Inhibitors from Glycyrrhiza uralensis Fisch.

Aim To screen for α-glucosidase inhibitor from Glyeyrrhiza uralensis Fisch.. Methods Glycyrrhizic acid, glycyrrhetinic acid, flavonoids of glycyrrhiza, alkaloids of glycyrrhiza, and glycyrrhiza polysaccharides were isolated from the root of Glycyrrhiza uralensis Fisch. respectively. Three compounds were isolated from the flavonoids of glycyrrhiza as guided by the α-glucosidase inhibitory test in vitro. Moreover, the characteristics of inhibitory kinetics of glycyrol and glycyrrhetinic acid were investi- gated. Results The flavonoids of glycyrrhiza and glycyrrhetinic acid had the strongest α-glucosidase inhibitory activity. Glycyrol,β-sitosterol and liquifitin were isolated and identified. Glycyrol was a fast- binding, reversible, noncompetitive α-glucosidase inhibitor, showing IC50 at 0.26 μg·mL^-1 Glycyrrhetinic acid was a fast-binding, irreversible α-glucosidase inhibitor, showing IC50 at 102.4 μg·mL^-1. Conclusion Glycyrol is an effective α-glucosidase inhibitor.

Glycosides from Erigeron breviscapus

A new glycoside, gamma-pyranone-3-O-beta-D-[6'-(4'-hydroxy-3',5'-dimethyoxybenzoyl)]-glucopyranoside (erigeside D, 2), together with nine known compounds, viz., erigeside I (1), erigeside A (3), erigeside B (4), erigeside II (5), scopolin (6), icariside B-2 (7), blumenol C glucoside (8), (+)-syringaresinol O-beta-D-glucopyranoside (9) and scutellarein-7-O-beta-D-glucuronide methyl ester (10), was isolated from Erigeron breviscapus (Vant.) Hand.-Mazz. Their structures were established on the basis of spectral evidence. According to the beta-glucopyranosyl moiety in 7, the absolute configuration of 7 was determined by X-ray crystallographic analysis. Compounds 7, 8 and 9 were isolated for the first time from this genus, an compound 6 was first obtained from this plant.

RP-HPLC Determination of Pinoresinol Diglucopyranoside in Qing′e Pill Extract

Aim To develop and determine pinoresinol diglucopyranoside in Qing'e Pill, atraditional Chinese compound preparation containing Eucommia ulmoides Oliv. as the principal drug,by a reverse-phase high-performance liquid chromatographic method (RP-HPLC) . Methods The extract ofQing'e Pill was refluxed with 75% ethanol, purified on an AB-8 macroporous adsorption resin columnand then injected into HPLC system. The HPLC assay was performed on an ODS analytical column with amixture of methanol-acetonitrile-water (24:3:78, V/V/V) as the mobile phase at a flow-rate of 1.0mL·min^(-1), and a UV detector set at 227 nm. Results Good linearity between peak area andconcentration was found in the range of 5.5 - 170 μg·mL^(-1) for pinoresinol diglucopyranoside ( r> 0.9998) . The average recovery was 99.3%. The intra-day assay RSD and the inter-day assay RSDwere 1.3% and 2.8%, respectively (n = 5). The content of pinoresinol diglucopyranoside in Qing'ePill was determined to be 0.446 +- 0.012 mg·g^(-1) (n = 10). Conclusion The RP-HPLC method wasproved to be sensitive, specific, accurate and precise for the determination of pinoresinoldiglucopyranoside in Qing' e Pill.

A MONOCLONAL ANTIBODY RECOGNIZING NON DERIVATIVE 13 HYDROXY GIBBERELLINS AND THEIR GLUCOSIDES *

The production and characterization of a monoclonal antibody (MAb AB10) against GA 3 glucoside as well as GA 3 is described. MAb AB10 was derived from an immunogen in which human serum albumin (HSA) was linked to GA 3 at carbon 3. This antibody showed high affinity for GA 3 glucoside as well as for 13 hydroxy gibberellins (GA 1, GA 3, GA 5, etc). The affinity of MAb AB10 for 13 hydroxy GAs was significantly reduced by methylation of the 7 oic acid but not by glycosylation of 3 hydroxyl group. Based on this antibody, both of competitive enzyme linked immunosorbent assays (ELISAs) for GA 3 glucoside and for GA 3 were developed. These two ELISAs displayed linear detection ranges from 0 2 pmol to 20 pmol. Using these assays, the fluctuation of GA 3 like and GA 3 glucoside like substances in the leaves of Rumex japonicus was investigated. The results indicated that the glycosylation of free GAs was connected with leaf senescence and that the function of 6 benzyl amino purine in retarding the leaf senescence was probably related to delaying the process of glycosylation of free GAs.

Inactivation Kinetics of β-N-Acetyl-D-Glucosaminidase from Prawn (Penaeus vannamei) by Formaldehyde

β-N-Acetyl-D-glucosaminidase （NAGase, EC.3.2.1.52） is chitinolytic enzymes and disintegrate dimmer and trimer a composition of oligomers of N-acetyl-β-D-glucosamine （NAG） into monomer. Prawn （P. vannamei） NAGase is involved in digestion and molting processes. Some pollutants in seawater affect the enzyme activity causing loss of the biological function of the enzyme, which affects the exuviating shell and threatens the survival of the animal. The effect of formaldehyde on prawn （P. vannamei） β-N-acetyl-D-glucosaminidase activity for the hydrolysis of pNP-NAG has been studied. The results show that formaldehyde, at appropriate concentrations, can lead to reversible inactivation of the enzyme, and the IC50 is estimated to be 1.05mol· L^-1. The inactivation mechanism obtained from Lineweaver-Burk plots shows that the inactivation of the enzyme by formaldehyde belongs to the competitive type. The inactivation kinetics of the enzyme by formaldehyde has been studied using the progress-of-substrate-reaction method described by Tsou, and the rate constants have been determined. The results show that k＋0 is much larger than k-0, indicating the free enzyme molecule is fragile in the formaldehyde solution.

Chemical Constituents of Rheum tanguticum Maxim.ex Balf.

Aim To study the chemical constituents of the root and rhizome of Rheum tanguticum Maxim. ex Balf. Methods Chemical constituents were isolated and purified by many chromatographic methods, and their structures were elucidated by MS, NMR, and others. Results Twenty compounds were isolated and their structures were identified as β-sitosterol, chrysophanol, aloe-emodin, physcion, rhein, emodin, etc. Conclusion Among these compounds, 4-(4′-hydroxyphenyl)-2-butanone, 4-(4′-hydroxyphenyl)-2-butanone-4′-O-β-D-(2″-...

Effects of α-glucosidase Inhibitors on the Function of Mulberry Leaf Extract as an Additive in Feedstuff

The mulberry juice contains high concentrations of α-glucosidase inhibitors that affect glycometabolism and cause diarrhea in animals, thereby affecting the de-velopment and application of mulberry （Morus alba L.） as feedstuff resources. ln this study, the effects of mulberry leaf extract with and without removal of mulberry juice on starch metabolism were analyzed and compared. The results showed that mul-berry leaf extract with removal of mulberry juice exhibited significantly lower inhibi-tion rate on starch metabolism compared with mulberry leaf extract without removal of mulberry juice. ln animal feeding trials, piglet feedstuff was added with 10% mul-berry leaf powder; compared with mulberry leaf powder without removal of mulberry juice, experimental piglets fed with mulberry leaf powder with removal of mulberry juice exhibited significantly improved weight gain and significantyl reduced diarrhea rate.

Expression Activity of CLCuV Bidirectional Promoter in Agrobacterium tumefaciens

Cotton leaf curl virus (CLCuV) belongs to the subgroup III of geminiviruses with single strand DNA genome. Study demonstrated that the bidirectional promoter of CLCuV had activity in Agrobacterium tumefaciens (Smith et Townsend) Conn. This is the first report for the activity of the bidirectional promoter of geminivirus in A. tumefaciens. Results showed that the activity of the complementary sense promoter was stronger than that of virion sense promoter, and was detected 2-fold higher than that of CaMV 35S promoter in A. tumefaciens. Moreover, the promoter 5' deletion analysis indicated that the mean GUS activity driven by a 287 nucleotides complementary sense promoter fragment (from-287 to the translation initiation site) is 4 times higher than that driven by the whole complementary sense promoter in A. tumefaciens. This result suggested that there might exist negative regulatory elements in this deleted fragment. The function of other cis-elements included in CLCuV complementary sense promoter was also discussed in this paper.

Use of Glucosyl 3,5-Dinitrobenzoate in Synthesis of Glucosides and Related Oligosaccharides

To investigate a new glycosylation method. Methods In the presence of TMSOTfas catalyst, 1-O-(3, 5-dinitrobenzoyl)-2, 3, 4, 6-tetra-O-benzyl-α-D-glucopyranose 1 reacted with aseries of carboxylic acid, phenols, alcohols and saccharides respectively to give the correspondingglycosylation products. The compounds were determined by ~1H NMR and ^(13)C NMR spectra. ResultsThe α-glu-co-pyranosides and related oligosaccharides were prepared in high yields. Conclusion The3, 5-dinitro-benzoyl group was found to be a good leaving group at the anomeric position andO-glucopyranosides and oligosaccharides were stereoselectively synthesized in good yield.

Ultrasonic Extraction of Polysaccharides from Laminaria japonica and Their Antioxidative and Glycosidase Inhibitory Activities

In the present study, ultrasonic extraction technique （UET） is used to improve the yield of polysaccharides from Lami- naria japonica （LJPs）. And their antioxidative as well as glycosidase inhibitory activities are investigated. Box-Behnken design （BBD） combined with response surface methodology （RSM） is applied to optimize ultrasonic extraction for polysaccharides. The optimized conditions are obtained as extraction time at 54 min, ultrasonic power at 1050 W, extraction temperature at 80℃ and ratio of material to solvent at 1：50 （g mL-1）. Under these optimal ultrasonic extraction conditions, an actual experimental yield （5.75% ＋ 0.3%） is close to the predicted result （5.67%） with no significant difference （P〉0.05）. Vitro antioxidative and glycosidase inhibitory activities tests indicate that the crude polysaccharides （LJP） and two major ethanol precipitated fractions （LJP1 and LJP2） are in a concentration-dependent manner. LJP2 （30%-60% ethanol precipitated polysaccharides） possesses the strongest α-glucosidase in- hibitory activity and moderate scavenging activity against hydroxyl radicals （66.09% ±2.19%, 3.0 mg mL-l）. Also, the inhibitory activity against a-glucosidase （59.08% ± 3.79%, 5.0 mg mL-1） is close to that of acarbose （63.99% ± 3.27%, 5.0 mg mL-l）. LJP 1 （30% ethanol precipitated polysaccharides） exhibits the strongest scavenging activity against hydroxyl radicals （99.80%q-0.00%, 3.0mg mL-1） and moderate a-glucosidase inhibitory activity （47.76%± 1.92%, 5.0 mgmL-1）. LJP shows the most remarkable DPPH scav- enging activity （66.20%±0.11%, 5.0mgmL-1） but weakest a-glucosidase inhibitory activity （37.77%±1.30%, 5.0mgmL-1）. How- ever, all these LJPs exert weak inhibitory effects against a-amylase. These results show that UET is an effective method for extract- ing bioactive polysaccharides from seaweed materials. LJP 1 and LJP2 can be developed as a potential ingredient in hypoglycemic agents or functional food for the management of diabetes. This study provides scientific evidence and advances in the preparation technology and a hypoglycemic activities evaluation method for seaweed polysaccharides, especially glycosidase inhibition in com- bination with an antioxidative activity evaluation method.

Stereoselective urinary excretion of S-(-)- and R-(+)-propranolol glucuronide following oral administration of RS-propranolol in Chinese Han subjects

AIM: To study the stereoselectivity of phase Ⅱ glucuronidation metabolism of side-chain propranolol in Chinese Han population. METHODS: Sixteen adult Chinese Han volunteers with an average age of 20 years were given a single oral dose of 20 mg racemic propranolol. Human urine at indicated time after administration was collected and S-(-)-propranolol glucuronide and R-(+)-propranolol glucuronide were determined simultaneously by using RP-HPLC. RESULTS: The mean values of k were 0.19±0.04 h-1 and 0.28±0.06 h-1, of t1/2 3.56±0.73 h and 2.45±0.50 h, of Tmax 2.21±0.45 and 1.75±0.33 h, and of Xu0-24 5.65±0.98 and 2.95±0.62 μmoL for S-(-)- and R-(+)-propranolol glucuronide, respectively. The cumulative excretion percentages in urine of closes were 14.7±2.46% and 7.68±1.60% for S-(-)-and R-(+)-propranolol glucuronide, respectively. The results showed the elimination rate constant k of S-(-)-propranolol glucuronide was less than that of R-(+)-propranolol glucuronide; and the elimination half-life (t1/2), Tmax and the cumulative excretion amount (Xu0-24) of R-(+)-propranolol glucuronide were significantly less than that of S-(-)-propranolol glucuronide. CONCLUSION: The propranolol glucuronidation of the side-chain undergoes stereoselective excretion in Chinese Han population after an oral administration of racemic propranolol.

Simultaneous separation and determination of four main isoflavonoids in Astragali Radix by an isocratic LC/ESI-MS method

A simple, reliable and rapid isocratic liquid chromatography(LC)-mass spectrometric detection(MS) coupled with electrospray ionization(ESI) method for simultaneous separation and determination of calycosin-7-O-β-D-glucoside, ononin, calycosin and formonometin in Astragali Radix was developed. After the samples were extracted with ethanol, the optimum separation conditions for these analytes were achieved using water and acetonitrile(70:30, v/v) containing 0.2%(v/v) acetic acid as a mobile phase and a 2.0 mm×150 mm Hypersil-Keystone C18 column. Selective ion monitoring(SIM) mode and [M+H]+ ions at m/z 447, 431, 285 and 269 were used for quantitative analysis of four main active components above mentioned. The calibration curves were linear in the range of 0.4-175.0 μg/mL for calycosin-7-O-β-D-glucoside, 0.2-146.0 μg/m L for ononin, 0.4-210.0 μg/mL for calycosin and 0.5-217.0 μg/mL for formonetion, respectively. The limits of quantification(LOQ) and detection(LOD) were 0.4 μg/mL and 0.08 μg/m L for calycosin-7-O-β-D-glucoside, 0.2 μg/mL and 0.06 μg/m L for ononin, 0.4 μg/mL and 0.1 μg/mL for calycosin, 0.5 μg/m L and 0.1 μg/m L formonetion, respectively. The standard recoveries were in the range of 96.5%-104.7%. The developed method has successfully been used for the determination of four main flavonoids in Astragali Radix from various sources and can be used for identification, differentiation and quality evaluation of Astragali Radix.