莲子心碳苷黄酮与黄酮异构体的色谱质谱研究

采用电喷雾质谱法(ESI-MS),对从莲子心分离得到的碳苷类黄酮化合物进行质谱碎裂规律研究。结果表明,负离子模式下,六碳糖碳苷黄酮主要发生糖环裂解,通过丢失特征性的碎片(90 u、120 u、150 u)与氧苷黄酮区分;单糖取代的六碳醛糖氧苷黄酮直接丢失单糖部分(162 u),六碳醛糖种类无法通过质谱区分,但由于它们在液相上的保留时间不同,可通过液相色谱-质谱(LC-MS)联用方法分离鉴定;二糖取代的氧苷黄酮主要碎片离子通过丢失糖部分(146 u、162 u、308 u)所得,二糖的种类及连接方式可通过质谱图上的碎片离子峰及其相对丰度辨别。莲子心中多种碳苷黄酮和氧苷黄酮质谱的不同裂解规律,不仅有助于莲子心黄酮化合物的快速鉴定,而且可以通过与液相色谱联用实现莲子心中同分异构体的快速区分。     

蓝莓提取物中花青素和黄酮类活性成分的UPLC-TOF MS联用分析

使用超高效液相色谱/飞行时间质谱,分别在TOF/MS全扫描和Q-TOF/MS碎片扫描两种模式下分析不同厂家生产的蓝莓提取物中的的花青素和黄酮类活性成分。首先依据高分辨精确质量在"待筛查化合物数据表"中查出可能的化合物,再结合二级质谱的子离子碎裂特征对化合物进行定性分析,结合参考文献,从蓝莓提取物中共鉴定出19个花青素类化合物、8个黄酮类化合物。同时,本文还对蓝莓提取物中的花青素苷和黄酮苷的质谱裂解规律进行了探讨。这一分析方法方便快捷、灵敏度高、准确性好,可将其推广至其它天然活性成分的无对照品筛查研究之中。     

高产黄酮苷银杏悬浮培养细胞系选育和继代培养稳定性研究

银杏叶中主要有效成分黄酮苷和萜内酯,具有多种药理作用,银杏叶提取物(EGB)及其加工品具有广阔的市场前景[1,2].为满足市场需要,Carrier等[3]在90年代初就开始细胞培养生产黄酮苷和萜内酯的研究.细胞大规模培养生产黄酮苷的关键技术之一是选育性状稳定、生产能力强的细胞系.     

贵州传统发酵豆制品中水解银杏黄酮苷的微生物β-葡萄糖苷酶筛选

[目的]微生物β-葡萄糖苷酶法水解银杏黄酮苷具有重要意义,不过目前这方面的研究极少。因此,本文目的是筛选到水解银杏黄酮苷的酶活高的微生物β-葡萄糖苷酶,并分析其底物选择性机制。[方法]以银杏叶提取物作为唯一碳源富集培养,从贵州传统发酵豆豉中筛选产对银杏黄酮苷水解酶活高的β-葡萄糖苷酶的菌株,并对该菌株进行鉴定。然后比较此β-葡萄糖苷酶对不同底物的选择性,同时测定此酶水解银杏黄酮苷反应的米氏常数Km及最大反应速率Vmax。最后,对不同的底物进行分子对接,分析其底物特异性机制。[结果]结果表明,筛选到的菌株GUXN01所产β-葡萄糖苷酶水解银杏黄酮苷的酶活最高,被鉴定为枯草芽孢杆菌。此β-葡糖糖苷酶对β构型的糖类以及苷类等具有广泛的底物特异性和不同的选择性,尤其对银杏黄酮苷具有很好的亲和性。分子对接研究表明枯草芽孢杆菌β-葡萄糖苷酶对银杏黄酮苷和其他糖苷类具有不同亲和性和选择性的原因主要是酶结构和底物分子结构的相互作用力的差异导致的。[结论]这些发现为GUXN01所产的β-葡萄糖苷酶应用于水解银杏黄酮苷类生产相应苷元奠定了良好的基础。     

碳苷黄酮及其药理活性研究进展

随着天然产物化学的不断发展,越来越多具有生物学功能和药理活性的植物碳苷黄酮得到了分离、纯化和鉴定。本文综述了近些年来从水果、蔬菜和药物植物中分离鉴定的碳苷黄酮及其清除自由基、抗氧化、抗辐射等广谱药理活性,以期为碳苷黄酮的研究和开发提供信息和指导。     

用胶原纤维分离含不同糖基数的黄酮苷类化合物北大核心CSCD

以戊二醛交联的胶原纤维做固定相,通过柱层析法研究其对含不同糖基数的黄酮苷类化合物（槲皮苷、芦丁、刺槐素）的分离特性。结果表明,在高浓度乙醇溶液中,胶原纤维对黄酮苷类化合物的吸附以氢键作用为主;在低浓度乙醇溶液中,吸附以疏水键作用为主。采用90%~70%~50%的乙醇水溶液进行分步洗脱,槲皮苷、芦丁和刺槐素能够得到分离,它们的纯度分别为99.39%、95.32%和94.23%,回收率分别为89.25%、80.36%和80.49%。戊二醛交联的胶原纤维分离材料具有良好的重复使用性能,在5次重复分离实验中,其分离性能无明显降低。     

用胶原纤维分离含不同糖基数的黄酮苷类化合物

以戊二醛交联的胶原纤维做固定相,通过柱层析法研究其对含不同糖基数的黄酮苷类化合物(槲皮苷、芦丁、刺槐素)的分离特性。结果表明,在高浓度乙醇溶液中,胶原纤维对黄酮苷类化合物的吸附以氢键作用为主;在低浓度乙醇溶液中,吸附以疏水键作用为主。采用90%~70%~50%的乙醇水溶液进行分步洗脱,槲皮苷、芦丁和刺槐素能够得到分离,它们的纯度分别为99.39%、95.32%和94.23%,回收率分别为89.25%、80.36%和80.49%。戊二醛交联的胶原纤维分离材料具有良好的重复使用性能,在5次重复分离实验中,其分离性能无明显降低。     

红背山麻杆化学成分的研究

采用柱色谱技术从红背山麻杆叶子的60％乙醇提取物中分离得到4个黄酮苷和2个其他类化合物.根据理化性质及波谱方法分别鉴定为:芹菜素-6-C-D-葡萄糖苷(1)、芹菜素-7-O-芸香糖苷(2)、芹菜素-7-O-β-(2″-O-α-鼠李糖基)葡萄糖醛酸苷(3)、木犀草素-7-O-α-L-鼠李糖(1→6)-β-D-葡萄糖苷(4)、没食子酸乙酯(5)、β-胡萝卜苷(6).以上化合物均为首次从该植物中分离得到,其中化合物1～4为首次从山麻杆属中分离得到的黄酮苷.     

茉莉酸和双氧水对斜生褐孔菌一株黑色素缺失突变体产生酚类化合物的影响

黑色素合成是桦褐孔菌Phaeoporus obliquus次生代谢过程中的一个重要事件。黑色素的过多合成势必影响其他酚类的积累。本文以茉莉酸和双氧水为调控因子,开展了该突变体深层发酵过程酚类化合物积累研究。正常培养条件下,每升发酵液积累酚类化合物最高可达317.45mg/L,其中包括苯甲酸衍生物(45.3mg/L),黄酮苷元(74.4mg/L),黄酮苷(160mg/L)以及硬毛素类化合物(18.5mg/L)。加入茉莉酸后,总酚最大值增加到620.45mg/L,其中苯甲酸衍生物增加到230.26mg/L,黄酮苷元增加到166.86mg/L,而黄酮苷和硬毛素类化合物却分别降低到98.5mg/L和7.1mg,双氧水的加入没有提高总酚的最大积累(315.69mg/L)。因此,与野生型相比,黑色素缺失使总酚的积累有所增加,随茉莉L酸的加入总酚积累最大值增加近一倍。虽然双氧水的加入提高了苯丙氨酸裂解酶活性,但总酚的积累却没有增加,这可能是由于一部分酚类化合物在抗双氧水氧化胁迫过程中被消耗。     

竹叶中黄酮类化合物丰度的文献调研

(淡)竹叶具有很高的药用和食用价值,黄酮类化合物作为标志性的功能组分,其含量高低决定了竹叶的开发利用价值。本文综述了自1994~2013年间国内外公开发表的58篇文献,对涉及竹亚科18个属、91种竹叶的黄酮类化合物含量水平进行了分析研究。结果表明,干竹叶的总黄酮含量分布在4.60~25.43 mg/g之间,其中四种特征性碳苷黄酮(荭草苷、异荭草苷、异牡荆苷和牡荆苷)的总量为1.23~2.49 mg/g;倭竹属(Shibataea Makino ex Nakai)的总黄酮含量最高,思簩竹属(Schizostachyum Nees)最低;刚竹属(Phyllostachys Sieb.et Zucc.)叶片的总黄酮含量平均为16.34 mg/g,四种碳苷黄酮的含量合计为1.38 mg/g,其中异荭草苷含量最高(0.55 mg/g)。纵观整个研究现状,对刚竹属毛竹[P.heterocycla var.pubescens(Nazel)Ohwi]叶中黄酮类化合物的研究文献最多,涉及面最广。本研究结果以期为竹叶黄酮的开发利用提供一些帮助和指导。     

HPLC analysis of benzo[a]pyrene-albumin adducts in benzo[a]pyrene exposed rats. Detection of cis-tetrols arising from hydrolysis of adducts of anti- and syn-BPDE-III with proteins

Quantitation of protein-benzo[a]pyrene adducts represent a more sensitive analysis method than quantitation of benzo[a]pyrene-DNA adducts. By accurate analysis of benzo[a]pyrene-protein adducts several different molecular adduct forms can be studied. Male Wistar rats were injected i.p. with benzo[a]pyrene, and serum albumin was isolated and subjected to acid hydrolysis at 90 degrees C for 3 h. The hydrolysate was analyzed by HPLC with fluorescence detection. The HPLC profiles obtained after albumin hydrolysis from benzo[a]pyrene exposed animals were compared to similar HPLC profiles from in vitro adducted bovine serum albumin (BSA) and direct hydrolysis of both r-10,t-9-dihydrodiol-c-7,8-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (syn-BPDE-III) and r-10,t-9-t-dihydrodiol-t-7,8-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE-III). After acid hydrolysis of albumin from benzo[a]pyrene exposed rats, 6 fluorescent peaks were separated. Four of the peaks were isomers of benzo[a]pyrene-tetrahydrotetrols, (+/-)-benzo[a]pyrene-r-7,t-8,9,10-tetrahydrotetrol, (+/-)-benzo[a]pyrene-r-7,t-8,9,c-10-tetrahydrotetrol, (+/-)-benzo[a]pyrene-r-7,t-8,c-9,t-10-tetrahydrotetrol and (+/-)-benzo[a]pyrene-r-7,t-8,c-9,10-tetrahydrotetrol. In addition we found two fluorescent peaks, named X1 and X2 with retention times similar to the benzo[a]pyrene-tetrols. The unknown fluorescent peaks reacted similar to the four known tetrols in both dose response experiments and time course experiments. Fluorescent material with retention times equal to X1 and X2 were found after acid hydrolysis of syn-BPDE-III and anti-BPDE-III in acid and in hydrolysates from BSA treated in vitro with syn-BPDE-III and anti-BPDE-III. The ratio X1/X2 was relatively constant indicating epimerization equilibrium between these to species. Synchronous fluorescence analysis of fractions containing X1 or X2 from both in vivo and in vitro experiments showed fluorescence spectra characteristic of benzo[a]pyrene tetrols using a wavelength difference of 34 nm.     

Structural analysis of choline phospholipids by fast atom bombardment mass spectrometry and tandem mass spectrometry

The structures of intact choline phospholipids were determined by positive and negative ion mode fast atom bombardment mass spectrometry, tandem mass spectrometry, and B2/E and B/E constant linked scan mass spectrometry. The molecular weight of the choline lipid could be clearly determined by the appearance of [M + H]+ or [M + Na]+ in the positive ion mode and triplet ions, e.g., [M - 15]-, [M - 60]-, and [M - 86]-, in the negative ion mode. The structures of the triplet ions were assigned to [M - CH3]-, [M - HN(CH3)3]-, and [M - CH2 = CHN(CH3)3]-, respectively, by the MS/MS of each triplet ion, and the origin of the triplet ions was found as the matrix-ion adduct to the target molecule by using the B2/E linked scan technique. The polar group could be identified by the existence of ions indicating glycerophosphocholine and its cleavage products and by the presence of the triplet ions in the negative ion mode. Positional determination of the distribution of constituent fatty acyl groups was carried out by comparing the intensity of deacylated ions from positions 1 and 2 in the positive ion mode and of the ions produced by MS/MS of the triplet ions. From the mass number of the [RCOO]- ion which appeared in the negative ion mode, the molecular weight and degree of unsaturation of the fatty acyl group were determined. The position of double bond(s) in the acyl group was determined from the MS/MS of the [RCOO]- ion.     

The analysis of multiple O-phosphoseryl-containing peptides by fast atom bombardment mass spectrometry

Summary Positive and negative ion FAB mass spectrometry were found to be useful for the structural analysis of phosphorylated peptides containing multiple O-phosphoseryl residues. The positive ion FAB mass spectra obtained for Ac-Ser(P)-Ser(P)-NHMe and Ac-Ser(P)-Ser(P)-Ser(P)-NHMe showed that -eliminative loss of H₃PO₄ from the Ser(P)-residue was a major event in the fragmentation of the two phosphopeptides and that successive losses of H₃PO₄ from the [M+H]⁺ ion occurred when the Ser(P)-cluster was located at the N-terminus. In contrast, the FAB mass spectrum of Ac-Glu-Ser(P)-Leu-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-NHMe showed only a single loss of H₃PO₄ from the [M+H]⁺ ion, with further losses of H₃PO₄ from internal Ser(P)-residues only occurring when fragmentation of the parent phosphopeptide generated daughter fragments that contained (part of) an N-terminal Ser(P)-residue. Negative ion FAB mass spectrometry also proved useful for the structural analysis of the three Ser(P)-peptides and showed high-intensity [M-H]^- ions along with minor [M-H-80]^- fragment ions.Abbreviations Ac acetyl - Ala dehydroalanyl - FAB-MS fast atom bombardment mass spectrometry - LSIMS liquid secondary ion mass spectrometry - NHMe N-methylamide - Ser(P) O-phosphoseryl - Thr(P) O-phosphothreonyl     

Differential solubilization of nuclear glucocorticoid receptors by DNAase I and DNAase II

This study analyzes the sensitivity of nuclear bound glucocorticoid receptors to solubilization from nuclei by DNAase I and DNAase II. Thymocytes were incubated with 10(-8) M [3H]dexamethasone, [3H]cortisol or [3H]triamcinolone acetonide, without or with 10(-6) M unlabelled dexamethasone, for 30 min at 37 degrees C and nuclei from these cells were digested with either DNAase I and DNAase II. DNAase I for 2 h at 3 degrees C leads to solubilization of 60% of the nuclear DNA and release of 10--20% triamcinolone acetonide-receptor, 30--40% dexamethasone-receptor and 85--90% cortisol-receptor. DNAase II at the same enzymatic concentration solubilizes only 10--20% of the nuclear DNA, but releases 40--50% triamcinolone-receptor, 60--70% dexamethasone-receptor and 100% cortisol-receptor. Release of nuclear bound dexamethasone-receptor by DNAase I parallels the solubilization of DNA, reaching maximum values by 2 h at 3 degrees C, whereas maximal release by DNAase II is obtained within 45 min when DNA solubilization is not complete. When nuclei initially extracted with DNAase I are re-extracted with DNAase II, greater than 65% of the DNAase I residual dexamethasone-receptors are solubilized, whereas DNAase I is ineffective in solubilizing DNAase II residual dexamethasone-receptors. DNAase I solubilizes only 30% of the 0.4 M KCl residual dexamethasone-receptor whereas DNAase II digests over 90% of this fraction. DNAase I extracts of nuclear dexamethasone-receptor chromatograph on G-100 Sephadex as a single radioactive peak just after the void volume, whereas DNAase II extracts of nuclear dexamethasone-receptor chromatograph as two peaks of radioactivity, one which is similar to the DNAase I solubilized receptor and a second broad peak of macromolecular bound radioactivity which is smaller in size.     

31P and 13C NMR studies of oxygen transfer during catalysis by 3-deoxy-D-manno-octulosonate cytidylyltransferase from Escherichia coli

[18O]3-Deoxy-D-manno-octulosonate (KDO), labeled at the anomeric oxygen, was prepared by exchange with [18O]H2O and used to follow the route of oxygen transfer during cytidine 5'-monophosphate-3-deoxy-D-manno-octulosonate (CMP-KDO) formation catalyzed by 3-deoxy-D-manno-octulosonate cytidylyl-transferase (CMP-KDO synthetase). The 31P-NMR signal of the phosphoryl group of CMP-KDO (-5.85 ppm), which appeared as a single resonance when CMP-KDO formation took place with unenriched KDO, appeared as two peaks when CMP-KDO formation took place in the presence of a mixture of [16O]-and [18O]KDO. These results demonstrate the retention of 18O during CMP-KDO formation. Confirmation that the labeled oxygen in CMP-KDO was retained in the "bridge" position between CMP and KDO came from 13C-NMR studies of CMP-KDO formed in the presence of 90% [2-13C, 18O] KDO. The prominent C-2 KDO resonance in CMP-KDO, which is normally a doublet at 101.4 ppm (Kohlbrenner, W.E., and Fesik, S.W. (1985) J. Biol. Chem. 260, 14695-14700), appeared as four peaks when a mixture of [2-13C,16O]- and [2-13C, 18O]KDO was used, confirming the direct bonding of 18O to the C-2 of KDO in CMP-KDO. These results are consistent with a nucleophilic displacement mechanism for CMP-KDO formation.     

High pressure liquid chromatography of cyclic nucleotide phosphodiesterase from purified human T-lymphocytes

The cyclic nucleotide phosphodiesterase (EC 3.1.4.17) in extracts of purified human peripheral blood T-lymphocytes was examined by ion exchange high pressure liquid chromatography. Four peaks of activity were isolated. The first peak of activity selectively hydrolyzed cyclic GMP. The following 3 peaks of activity (Ia, IIa and IIIa) were selective for cyclic AMP. The selective low Km cyclic AMP-phosphodiesterase inhibitor, Ro 20-1724 (d,1-1,4-[3-butoxy-4-methoxybenzyl]-2-imidazolidinone), did not inhibit the activity in Ia whereas it did inhibit the activity in IIa and IIIa (IC50 = 17 microM). The authors conclude that ion exchange high pressure liquid chromatography described in this communication is a useful method for the isolation of different forms of cyclic nucleotide phosphodiesterase activity from human T-lymphocytes.     

Biochemical analyses of secretory and excretory products of adult Dipetalonema viteae in culture

Radioisotopically labeled glucose and pyruvate were employed to elucidate biochemical mechanisms utilized by the filariid Dipetalonema viteae during cultivation. Adults isolated from amicrofilaremic hamsters were incubated at 37 C in a mixture of NCTC135:IMDM (NI), with either D-[14C-(U)]glucose or [1-14C]pyruvate, under a gas phase of 5% CO2/N2 for 3 days. Labeled organic acids were separated and quantified by ion exchange chromatography. High performance liquid chromatography (HPLC) was used for separation and quantification of the 23 free amino acids in the NI medium. Ion exchange chromatography revealed that lactate was the major glycolytic end product, accounting for 90-97% of the original carbon utilized. Small amounts of radioactivity were recovered in succinate and variably in acetate fractions. HPLC analysis demonstrated that some amino acids increased, some decreased, and some remained at the initial concentration. Alanine exhibited the greatest change, consistently increasing from 2 to 4 times the original concentration. Analyses of purified amino acid peaks revealed radioactivity only in the alanine peak, accounting for 2-4% of the original carbon utilized.     

Analysis of renal and hippocampal type I and type II receptors by fast protein liquid chromatography

Type I and Type II adrenal steroid receptors from rat renal and hippocampal cytosols were studied by the technique of Fast Protein Liquid Chromatography. Type I receptors were labelled with [3H]aldosterone plus excess RU26988, and Type II receptors with [3H]dexamethasone. On a Mono Q anion exchange column the molybdate-stabilized renal and hippocampal Type I receptors both eluted as single symmetrical peaks at 0.27 M NaCl, with a recovery of approximately 90% and 60-fold purification (renal) and 10-15-fold (hippocampal). Molybdate-stabilized Type II binding sites from both hippocampal and renal cytosols co-eluted with the Type I sites. On Superose gel filtration renal Type I receptor-steroid complexes consistently eluted two fractions later than hippocampal Type I complexes, suggesting that the renal complexes are smaller; Type II receptor-steroid complexes from both cytosols co-eluted, consistently one fraction behind hippocampal Type I sites. Sequential gel filtration and anion exchange chromatography achieved a 1000-fold purification of renal Type I binding sites, with an overall recovery of 10%.     

Localization and partial composition of the oligosaccharide units on the propeptide extensions of type I procollagen

Type I procollagen secreted by matrix-free chick embryo tendon cells was labeled with L-[3,3'-3H] cystine and purified by DEAE-cellulose chromatography. After bacterial collagenase digestion, the NH2- and COOH-terminal propeptides were partially characterized by ion exchange chromatography and gel filtration. Similar experiments were then conducted after labeling with either D-[6-3H] glucosamine, D-[2-3H] mannose, or D-[U-14C] glucose. On the basis of these studies and subsequent carbohydrate analysis, it was concluded that the COOH-terminal peptide contained greater than 90% of the radioactive carbohydrate which consisted predominantly of glucosamine and mannose with traces of galactosamine and galactose. Only radioactive glucosamine could be detected in the NH2-terminal propeptide. Under conditions which inhibit hydroxylation of lysine and glycosylation of hydroxylysine, unhydroxylated procollagen (protocollagen) could still be labeled with [3H] glucosamine and [3H] mannose. This suggested that glycosylation of the propeptides is at least initiated at the level of the rough endoplasmic reticulum.     

Charge movement by the Na/K pump in Xenopus oocytes

Pre-steady-state transient currents (1986. Nakao, M., and D. C. Gadsby. Nature [Lond.]. 323:628-630) mediated by the Na/K pump were measured under conditions for Na/Na exchange (K-free solution) in voltage- clamped Xenopus oocytes. Signal-averaged (eight times) current records obtained in response to voltage clamp steps over the range -160 to +60 mV after the addition of 100 microM dihydroouabain (DHO) or removal of external Na (control) were subtracted from test records obtained before the solution change. A slow component of DHO- or Na-sensitive difference current was consistently observed and its properties were analyzed. The quantity of charge moved was well described as a Boltzmann function of membrane potential with an apparent valence of 1.0. The relaxation rate of the current was fit by the sum of an exponentially voltage-dependent reverse rate coefficient plus a voltage- independent forward rate constant. The quantity of charge moved at the on and off of each voltage pulse was approximately equal except at extreme negative values of membrane potential where the on charge tended to be less than the off. The midpoint voltage of the charge distribution function (Vq) was shifted by -24.8 +/- 1.7 mV by changing the external [Na] in the test condition from 90 to 45 mM and by +14.7 +/- 1.7 mV by changing the test [Na] from 90 to 120 mM. A pseudo three- state model of charge translocation is discussed in which Na+ is bound and occluded at the internal face of the enzyme and is released into an external-facing high field access channel (ion well). The model predicts a shift of the charge distribution function to more hyperpolarized potentials as extracellular [Na] is lowered; however, several features of the data are not predicted by the model.