高效液相色谱柱前衍生化方法分析血浆中3-O甲基葡萄糖（英文）

目的：本文首次建立了用1-苯基-3-甲基-5-吡唑啉酮（PMP）作为柱前衍生化试剂对3-O-甲基-葡萄糖（3-OMG）进行衍生化的方法,并应用此方法对大鼠血浆中的3-OMG进行分析。方法：70℃水浴时,3-OMG在0.4 mol/L的NaOH条件下和PMP进行反应。衍生物采用反相高效液相色谱法进行分析,紫外检测波长为245 nm。结果：线性系数在0.991以上,最低定量限0.0078 mg/mL。回收率在98%～105%之间,且日间与日内变异不高于12%。本方法成功应用于糖尿病大鼠血浆中3-OMG的分析。结论：本方法简便、高效,适合对正常和糖尿病大鼠的血浆3-OMG进行分析。     

柱前衍生化HPLC分析蒲公英多糖的单糖组成

目的 合理优化衍生方法测定蒲公英多糖中单糖的组成及摩尔比。方法 采用1-苯基-3-甲基-5-吡唑啉酮(PMP)柱前衍生,用反相高效液相色谱法进行测定。色谱条件为：ZORBAX-C₁₈柱;流动相：0.1 mol·L^-1的磷酸盐缓冲液(pH 6.7)-乙腈(83∶17);流速：1 mL·min^-1;检测波长：245 nm;进样量：20 μL;柱温：室温。结果 蒲公英多糖由D-鼠李糖、葡萄糖、D-半乳糖、D-木糖和D-阿拉伯糖组成,以不同方法提取的各单糖含量的摩尔比为：水提取法,Rha∶Glc∶Gal∶ Ara=0.088∶0.500∶0.190∶0.340;超声提取法,Rha∶Glc∶Gal∶Ara=0.050∶0.350∶0.080∶0.320;酶提取法,Rha∶Glc∶ Gal∶Ara=0.270∶0.630∶0.330∶0.460;超声协提取同酶法,Rha∶Glc∶Gal∶Ara =0.078∶0.550∶0.130∶0.170。结论 改进后的衍生化方法和等度洗脱的色谱方法具有操作简单、快速、重复性好等特点,可用于蒲公英多糖中单糖组成和摩尔比的测定。     

虫草多糖中单糖组成的柱前衍生化HPLC分析

建立了柱前衍生化HPLC法测定虫草多糖中单糖的组成及摩尔比。采用C18柱,以0.1mol/L磷酸盐缓冲液(pH6.7)-乙腈(83∶17)为流动相,检测波长254nm。衍生化试剂为1-苯基-3-甲基-5-吡唑啉酮。所测3批样品中虫草多糖由甘露糖、葡萄糖、半乳糖3种单糖组成,单糖的摩尔比约为2∶2∶1。平均回收率分别为101.0%、98.6%和99.2%,RSD分别为1.9%、1.6%和1.2%。     

Assessment of the opiate properties of two constituents of a toxic illicit drug mixture

The intravenous use of an illicit synthetic drug preparation has caused permanent parkinsonism in a number of addicts. Chemical analysis has revealed the ingredients to be two related compounds 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenyl-4-propionoxypiperidine (MPPP). The opiate properties of these two compounds have been assessed using in vitro receptor binding techniques as well as behavioral tests indicative of opiate action, including analgesia, catatonia, respiratory depression and the loss of righting and corneal reflexes. All opiate activity was found to reside with MPPP, which proved to be a potent mu-type agonist. It is concluded that the opiate properties of MPPP alone explain repeated abuse of MPTP/MPPP mixtures by heroin addicts.     

不同栽培方法和生长时间的美花石斛中多糖含量的比较

目的 测定不同栽培方法、生长时间美花石斛中多糖的含量.方法 栽培美花石斛分为激素处理组、光照处理组和对照组,并采集不同生长时间段的石斛药材,采用蒽酮-硫酸比色法测定其多糖含量,测定波长625_(nm).结果 多糖的线性范围为5.0～17.5 μg·mL~(-1)(r=0.9997),平均加样回收率为100.77%,RSD=1.57%(n=6),各实验组多糖含量均呈递增趋势,增长幅度各有差异.结论 葸酮-硫酸比色法快速、准确、灵敏,可作为美花石斛药材质量的检测手段之一;处理措施对美花石斛的多糖累积产生了影响.     

Antidepressant-like effect of ursolic acid isolated from Rosmarinus officinalis L. in mice: Evidence for the involvement of the dopaminergic system

Ursolic acid, a constituent from Rosmarinus officinalis, is a triterpenoid compound which has been extensively known for its anticancer and antioxidant properties. In the present study, we investigated the antidepressant-like effect of ursolic acid isolated from this plant in two predictive tests of antidepressant property, the tail suspension test (TST) and the forced swimming test (FST) in mice. Furthermore, the involvement of dopaminergic system in its antidepressant-like effect was investigated in the TST. Ursolic acid reduced the immobility time in the TST (0.01 and 0.1 mg/kg, p.o.) and in the FST (10 mg/kg, p.o.), similar to fluoxetine (10 mg/kg, p.o.), imipramine (1 mg/kg, p.o.) and bupropion (10 mg/kg, p.o.). The effect of ursolic acid (0.1 mg/kg, p.o.) in the TST was prevented by the pretreatment of mice with SCH23390 (0.05 mg/kg, s.c., a dopamine D₁ receptor antagonist) and sulpiride (50 mg/kg, i.p., a dopamine D₂ receptor antagonist). The administration of a sub-effective dose of ursolic acid (0.001 mg/kg, p.o.) in combination with sub-effective doses of SKF38393 (0.1 mg/kg, s.c., a dopamine D₁ receptor agonist), apomorphine (0.5 μg/kg, i.p., a preferential dopamine D₂ receptor agonist) or bupropion (1 mg/kg, i.p., a dual dopamine/noradrenaline reuptake inhibitor) reduced the immobility time in the TST as compared with either drug alone. Ursolic acid and dopaminergic agents alone or in combination did not cause significant alterations in the locomotor and exploratory activities. These results indicate that the antidepressant-like effect of ursolic acid in the TST is likely mediated by an interaction with the dopaminergic system, through the activation of dopamine D₁ and D₂ receptors.     

Fucoidan protects against dopaminergic neuron death in vivo and in vitro

Parkinson's disease is a neurodegenerative disorder of uncertain pathogenesis characterized by a loss of dopaminergic neurons in substantia nigra pars compacta, and can be modeled by the neurotoxin 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP). Oxidative stress may contribute to MPTP- and Parkinson's disease-related neurodegeneration. Fucoidan is a sulfated polysaccharide extracted from brown seaweeds which possesses a wide variety of biological activities including potent antioxidative effects. Here we investigated the effect of fucoidan treatment on locomoter activities of animals, striatal dopamine and its metabolites and survival of nigral dopaminergic neurons in MPTP-induced animal model of Parkinsonism in C57/BL mice in vivo and on the neuronal damage induced by 1-methyl-4-phenylpyridinium (MPP⁺) in vitro, and to study the possible mechanisms. When administered prior to MPTP, fucoidan reduced behavioral deficits, increased striatal dopamine and its metabolites levels, reduced cell death, and led to a marked increase in tyrosine hydroxylase expression relative to mice treated with MPTP alone. Furthermore, we found that fucoidan inhibited MPTP-induced lipid peroxidation and reduction of antioxidant enzyme activity. In addition, pre-treatment with fucoidan significantly protected against MPP⁺-induced damage in MN9D cells. Taken together, these findings suggest that fucoidan has protective effect in MPTP-induced neurotoxicity in this model of Parkinson's disease via its antioxidative activity.     

Cyclothiazide and AMPA receptor desensitization: analyses from studies of AMPA-induced release of [3H]-noradrenaline from hippocampal slices

1. Responses in brain produced by the activation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) subtype of ionotropic receptor for L-glutamate are often rapidly desensitizing. AMPA-induced desensitization and its characteristics, and the potentiating effect of cyclothiazide were investigated in vitro by analysing AMPA-induced release of [³H]-noradrenaline from prisms of rat hippocampus.
2. AMPA (1–1000 μM) stimulated the release of [³H]-noradrenaline in a concentration-dependent manner that was both calcium-dependent and tetrodotoxin-sensitive, and attenuated by the AMPA-selective antagonists, NBQX (1 and 10 μM), LY 293558 (1 and 10 μM) and GYKI 52466 (10 and 30 μM).
3. By use of an experimental procedure with consecutive applications of AMPA (100 μM, 28 min apart), the second response was reduced, indicative of receptor desensitization, and was reversed by cyclothiazide in a concentration-dependent manner (1–300 μM). The concentration-response curve for AMPA-induced release of [³H]-noradrenaline was shifted leftwards, but the reversal by cyclothiazide of the desensitized response was partial and failed to reach the maximal response of the first stimulus.
4. Observations made with various schedules of cyclothiazide application indicated that the initial AMPA-evoked response was already partially desensitized (150% potentiation by 100 μM cyclothiazide) and that the desensitization was not likely to be due to a time-dependent diminution and was long-lasting (second application of cyclothiazide was ineffective).
5. Co-application of a number of drugs with actions on second messenger systems, in association with the second AMPA stimulus, revealed significant potentiation of the AMPA-induced release of [³H]-noradrenaline: forskolin (10 μM, +78%), Rp-cAMPS (100 μM, +65%), Ro 31-8220 (10 μM, + 163%) and thapsigargin (100 μM, +161%).
6. The AMPA receptor-mediated response regulating the release of [³H]-noradrenaline from rat hippocampal slices was desensitized and cyclothiazide acted to reverse partially the desensitization in a concentration-dependent manner. Since the time-course of desensitization was longer lasting than that noted in previous electrophysiological studies, multiple events may be involved in the down-regulation of AMPA receptor activity including receptor phosphorylation and depletion of intracellular Ca²⁺ stores.

     

Discrimination of polysaccharides from traditional Chinese medicines using saccharide mapping—Enzymatic digestion followed by chromatographic analysis

Polysaccharides isolated from traditional Chinese medicines (TCMs) exhibit multiple pharmacological activities. However, quality control of polysaccharides is a challenge because of their complicate structure and macro-molecular mass. In this study, saccharide mapping based on specific enzymatic digestion of polysaccharides and chromatographic analysis was proposed to discriminate the polysaccharides from different TCMs. Endo-carbohydrase such as glucanase, arabinanase, xylanase, galactanase, cellulase, amylase and pectinase were used for enzymatic digestion of polysaccharides from 9 TCMs namely Panax ginseng, P. notoginseng, P. quinquefolium, Cordyceps sinensis, C. militaris, Ganoderma lucidum, G. sinense, Astragalus membranaceus and Angelica sinensis. By using high performance size exclusion chromatography (HPSEC) as well as derivatization with 1-Phenyl-3-methyl-5-pyrazolone (PMP) and HPLC analysis, the enzymatic hydrolysis properties of polysaccharides and their saccharide mapping were determined. The polysaccharides from 9 TCMs were firstly successfully distinguished based on their characteristic saccharide maps, which is helpful to improve the quality control of polysaccharides.     

2-(5-甲基-2-苯基-4-(口恶)唑)-乙醇的合成

以丙酰乙酸乙酯为起始原料,先溴化得到4-溴丙酰乙酸乙酯,再在甲苯中与苯甲酰胺回流环合得到2-(5-甲基-2-苯基-4-(口恶)唑)-乙酸乙酯,最后经氢化铝锂还原,得到目标杂环中间体2-(5-甲基-2-苯基-4-(口恶)唑)-乙醇.该合成方法反应步骤少,原料便宜,目标物的总收率为67.3%.     

Assessment of the quality and structural integrity of a complex glycoprotein mixture following extraction from the formulated biopharmaceutical drug product

Biological drugs represent an important and rapidly growing class of therapeutics useful in the treatment of a variety of disorders ranging from cancer to inflammation to infectious diseases. Unlike single chemical entities, the recombinant production of these drugs in living cells confers considerable structural and chemical heterogeneity to the biologically derived protein product that constitutes the active pharmaceutical ingredient (API). In mammalian based expression systems, much of this diversity is conferred through heterogeneous protein glycosylation. These post-translational modifications can have significant effects on the structure, biological function, and pharmacological properties of the API. In addition, the bulk proteins that comprise the API are further formulated through the use of multiple excipients designed to ensure product stability, solubility, and lot-to-lot consistency. Unfortunately, these matrices can interfere with commonly available analytical methods used in the thorough chemical characterization of the biological drug product. At the same time, a demonstration of the suitable extraction of the bulk drug substance in a manner and form that does not destabilize the active ingredient or introduce any structural bias with direct reference to the original drug product is both critical and necessary. Here, we use recombinant human follicle stimulating hormone (follitropin alpha for injection) from a pharmaceutical source as an example to illustrate a suitable purification strategy to effectively extract the bulk drug substance from the formulated drug product with high purity and yield. We assess the suitability of this extraction method in preserving the structural integrity and overall quality of the drug substance relative to the formulated drug product, placing a particular emphasis on glycosylation as a key product attribute. In so doing, we demonstrate that it is possible to effectively extract the active pharmaceutical ingredient from a formulated biological drug product in a manner that is consequently sufficient for its use in comparability studies.     

Diarylheptanoids from Alpinia officinarum

A new diarylheptanoid, along with five known diarylheptanoids, was isolated from the rhizomes of Alpinia officinarum (Zingiberaceae). The structure of the new compound was determined to be trans,trans-1(3′-methoxy-4′-hydroxyphenyl)-7-phenyl-5-ol-4,6-dien-3-heptanone on the basis of spectral and chemical evidence.     

Exclusion of exogenous 5-methyl-2'-deoxycytidine from DNA in human leukemic cells. A study with [2(-14)C]- and [methyl-14C]5-methyl-2'-deoxycytidine

Modification of DNA-cytosine by a 5-methyl group is thought to be an important mechanism which regulates the expression of eukaryotic genes. This modification takes place after semiconservative replication. There is very little evidence, if any, that 5MeCyt could be naturally incorporated into mammalian DNA in semiconservative replication. We have clarified the possibility of incorporating 5MedCyd pharmacologically into human leukemic cells in vitro. To this end, we developed a novel small-scale synthesis method for 14C-labeled 5MedCyd starting from commercially available [14C]dThd derivatives. Particular attention was focused upon possible incorporation of radioactive 5MedCyd derivatives into the acid-soluble cellular fraction as well as into nucleic acids and protein in human cells. The results showed that [2(-14)C]- and [methyl-14C]5MedCyd were incorporated into human leukemic cells to a similar extent. The radioactivity originating from these compounds was incorporated mainly into the acid-soluble pool and nucleic acids. The exact nature of the intracellular radioactive molecules in RNA is not known, but the radioactive label in DNA hydrolyzate co-chromatographed exclusively with thymine. Hence, 5MedCyd is deaminated to thymidine before incorporating into DNA. This deamination had taken place already (partially) in the culture medium. Human leukemic cells do effectively protect their DNA from incorporation of exogenous 5MedCyd.     

A Penicillium sp. F33 metabolite and its synthetic derivatives inhibit acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine acetyltransferase (a key enzyme in platelet-activating factor biosynthesis) and carrageenan-induced paw edema in mice

Acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine (lyso-PAF) acetyltransferase is a key enzyme in the biosynthesis of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) in inflammatory cells. Substances which inhibit this enzyme are of therapeutic interest. In this study, we screened for new inhibitors of lyso-PAF acetyltransferase with anti-inflammatory effects. In a metabolite from Penicillium sp. F33, we isolated an acetyltransferase inhibitor identified as dihydrofumigatin (2-methoxy-1,3,4-trihydroxy-5-methylbenzene) from high resolution mass spectrometer and NMR data. Dihydrofumigatin had strong acetyltransferase inhibitory activity, but was not stable in aqueous solution. Thus, we chemically synthesized its oxidized form fumigatin (3-hydroxy-2-methoxy-5-methyl-1,4-benzoquinone) and derivatives thereof, and evaluated their inhibitory effects. Strong inhibitory activity was observed for saturated fatty acid esters of fumigatin; the order of inhibition was 3-decanoyloxy-2-methoxy-5-methyl-1,4-benzoquinone (termed FUD-7, IC₅₀ = 3 μM) > 2-methoxy-5-methyl-3-tetradecanoyloxy-1,4-benzoquinone (termed FUD-8, IC₅₀ = 20 μM) > 3-hexanoyloxy-2-methoxy-5-methyl-1,4-benzoquinone (IC₅₀ = 139 μM). Interestingly, these compounds also significantly suppressed the gene expression of lyso-PAF acetyltransferase/LPCAT2 in mouse bone marrow-derived macrophages stimulated by lipopolysaccharide (LPS). We further evaluated the effect of these substances on anti-inflammatory activity in vivo using the carrageenan-induced mouse paw edema test. FUD-7 and FUD-8 at 2.5 mg/kg showed significant, 47.9–51.7%, inhibition stronger than that of prednisolone at 10 mg/kg (41.9%). These results suggest that FUD-7 and FUD-8 are potent inhibitors with anti-inflammatory activity.     

1-甲基-1,2,3,4-四氢异喹啉的简易合成

目的制备1-甲基-1,2,3,4-四氢异喹啉。方法以苯乙胺为原料,经酰化反应得乙酰苯乙胺,在多聚磷酸的作用下环合得1-甲基-3,4-二氢异喹啉,经硼氢化钠还原得1-甲基-1,2,3,4-四氢异喹啉。结果反应总收率80%,比文献收率提高了10%,产物结构由1H-NMR光谱确证。结论经酰化、环合、还原三步反应制备1-甲基-1,2,3,4-四氢异喹啉的方法简单,原料便宜,处理容易,副产物较少。     

Increased release of [3H]acetylcholine in vitro from the mouse hippocampus by a convulsant barbiturate

The effects of the convulsant barbiturate, 5-(2-cyclohexylidene-ethyl)-5-ethyl barbituric acid (CHEB), on the spontaneous release of [³H]acetylcholine (ACh) from mouse hippocampal slices in an in vitro superfusion system have been evaluated. The pattern of the release of [³H]ACh by a single treatment with CHEB or an elevated potassium concentration was similar, with the peak release occurring in the same fraction. A maximally effective concentration of CHEB (500 μM) caused a 177% stimulation of spontaneous release of [³H]ACh, while 50 mM KCl increased the release by 2100% above the baseline. The stimulation of the spontaneous release of [³H]ACh by CHEB was concentration-dependent with an EC₅₀ of 180 μM. The pattern of release of [³H]ACh induced by multiple treatment with CHEB and elevated potassium appeared to differ, suggesting that different pools of [³H]ACh in the cholinergic neurons might be affected by these treatments. The action of CHEB on the sponaneous release of [³H]ACh was unique among some other convulsant drugs that were studied. Another convulsant barbiturate, S(+)-1-methyl-5-phenyl-5-propyl barbituric acid [S(+)-MPPB], pentylenetetrazol and a convulsant benzodiazepine 1,3-dihydro-5-methyl-2H-1,4-benzodiazepine-2-one (Ro-5-3663) did not affect the spontaneous release of [³H]ACh. The relationship between the stimulation of release of ACh and the convulsant action of the barbiturates is discussed.     

桑叶多糖对糖尿病小鼠肾病的影响

目的: 研究桑叶多糖对链脲佐菌素所致的糖尿病小鼠肾病的作用及其机制。方法: 建立糖尿病小鼠模型,随机分为:模型组,二甲双胍0.032 g·kg^-1组,桑叶多糖0.25,0.5,1 g·kg^-1组。连续灌胃给药35 d,末次给药后,检查小鼠空腹血糖;于给药第28天,将各组小鼠给药后置于代谢笼中收集24 h尿液,检查尿量,检测24 h尿微量白蛋白含量;HE染色法观察肾脏病理学变化。通过电子显微镜观察肾组织中细胞结构的变化。测定血清中肌酐(Cr)、尿素氮(BUN)和总胆固醇(TC)的含量。蛋白免疫印迹法检测肾脏组织中转录因子(NF-κB)和转化生长因子-β1(TGF-β1)的表达。结果: 与模型对照组比较,桑叶多糖1 g·kg^-1组中糖尿病小鼠的血糖明显降低,并显著降低尿微量白蛋白含量、尿量及血清中Cr、BUN、TC(P<0.05)。桑叶多糖1 g·kg^-1组的小鼠肾组织的病变得到缓解。下调肾脏组织中NF-κB及TGF-β1的蛋白水平(P<0.05)。结论: 桑叶多糖对链脲佐菌素所致糖尿病小鼠肾脏损害有一定的保护作用,机制可能与其通过抑制肾脏组织中NF-κB及TGF-β1的蛋白表达有关。     

Studies on the regulatory mechanism of the tyrosine hydroxylase system in adrenal slices by using high-performance liquid chromatography with electrochemical detection

A new method was developed to study the tyrosine hydroxylase (TH) system in rat adrenal slices by high-performance liquid chromatography (HPLC) with electrochemical detection (ED). TH activity was measured by determining DOPA, formed in adrenal slices containing all of the components of the TH system in the presence of an inhibitor of aromatic l-amino acid decarboxylase (NSD-1055), using a new and highly sensitive HPLC-ED method. The properties of the TH system in the slices were also examined. High K⁺ (52 mM) stimulated the formation of DOPA in the slices; this stimulation was not observed in Ca²⁺-free medium. Furthermore, the addition of ethyleneglycol bis (β-aminoethylether)-N,N′-tetraacetic acid (EGTA) to a Ca²⁺-free medium not only abolished the stimulation by high K⁺ but also significantly inhibited the conversion of l-tyrosine to DOPA. This suggests that intracellular Ca²⁺ may regulate TH activity in the adrenal medulla. The enzyme in homogenates of normal adrenal slices had two different K_m values for the tetrahydropterin cofactor. Alteration of enzyme kinetics using homogenates of adrenal slices that had been treated in various ways suggests that changes in the proportions of the two forms of TH produced by intracellular Ca²⁺ may regulate the activity of this enzyme in the adrenal medulla.     

4-苯基-5-乙氧羰基-6-甲基-3,4-二氢嘧啶-2(1H)-酮的绿色合成研究

目的探索操作简便,环境友好的4－苯基－5－乙氧羰基－6－甲基－3,4－二氢嘧啶－2（1H）－酮的合成方法。方法 以苯甲醛、乙酰乙酸乙酯和尿素作为起始原料,在无溶剂和微波加热条件下,选择1－丁基－3－甲基咪唑氯盐和1－丁基－3－甲基咪唑－L－乳酸盐两种不同的离子液体分别催化Biginelli反应制备4－苯基－5－乙氧羰基－6－甲基－3,4－二氢嘧啶－2（1H）－酮,并比较两种离子液体的催化效果。结果两种离子液体在微波、无溶剂条件下均可催化Biginelli反应制得目标化合物,其中1－丁基－3－甲基咪唑－L－乳酸盐作为催化剂目标化合物收率较高。结论以离子液体－丁基－3－甲基眯唑－L－乳酸盐作为催化剂,经微波、无溶剂Biginelli反应制备4－苯基－5－乙氧羰基－6-甲基－3,4－二氢嘧啶－2（1H）－酮,是一种易于操作的绿色合成方法。