基于GC-MS和UPLC-QTOF/MS技术的灵芝孢子粉化学成分分析

采用极性不同的6种溶剂（石油醚、乙酸乙酯、丙酮、乙醇、甲醇和水）、按索氏提取法逐级萃取破壁灵芝孢子粉,并同时运用气相色谱-质谱联用（GC/MS）和超高效液相串联四极杆飞行时间质谱（UPLC-QTOF/MS）技术对各萃取物进行化学成分分析与鉴定。结果表明：GC/MS共鉴定出101种化合物,其中酸类10种、酯类40种、醇类7种、酮类6种、酚类2种、烃类18种、甾类9种和杂原子化合物9种;UPLC-Q-TOF/MS共推断出40种化合物,其中倍半萜类1种、二萜类1种、三萜类9种、生物碱类4种、酰胺类7种、有机酸类9种以及其他化合物9种。两种测定方法间共有化合物仅1种,仅存在于5种有机溶剂（石油醚、乙酸乙酯、丙酮、乙醇和甲醇）萃取物之一的化合物共105种,2种或2种以上萃取物共有的化合物共31种,实验方法较好地实现了样品中化合物组分的充分分离,扩大了可检测化合物的范围。研究结果为灵芝孢子粉中化学成分的系统分析与鉴定、及灵芝孢子粉的化合物谱图库的完善提供了基础资料,为相关药理、药效分析及灵芝的药用模式真菌研究提供参考。     

Synthesis,characterization and in vitro anti-invasive activity screening of polyphenolic and heterocyclic compounds

Invasion is the hallmark of malignant tumors, and is responsible for the bad prognosis of the untreated cancer patients. The search for anti-invasive treatments led us to screen compounds of different classes for their effect in an assay for invasion. Thirty-nine new compounds synthesized in the present study along with 56 already reported compounds belonging mainly to the classes of lactones, pyrazoles, isoxazoles, coumarins, desoxybenzoins, aromatic ketones, chalcones, chromans, isoflavanones have been tested against organotypic confronting cultures of invasive human MCF-7/6 mammary carcinoma cells with embryonic chick heart fragments in vitro. Three of them (a pyrazole derivative, an isoxazolylcoumarin and a prenylated desoxybenzoin) inhibited invasion at concentrations as low as 1 microM; instead of occupying and replacing the heart tissue within 8 days, the MCF-7/6 cells grew around the heart fragments and left it intact, when treated with these compounds. At the anti-invasive concentration of 1 microM, the three compounds did not affect the growth of the MCF-7/6 cells, as shown in the sulforhodamine B assay. Aggregate formation on agar was not stimulated by any of the three anti-invasive compounds, making an effect on the E-cadherin/catenin complex improbable. This is an invasion suppressor that can be activated in MCF-7/6 cells by a number of other molecules. Our data indicate that some polyphenolic and heterocyclic compounds are anti-invasive without being cytotoxic for the cancer cells.     

佛波醇制备工艺优化及其衍生物的合成表征与细胞毒活性研究

目前佛波醇制备过程比较繁琐,本研究首先对制备工艺进行优化,使制备周期缩短至3天。然后以佛波醇、二十碳五烯酸、二十二碳六烯酸、花生酸为原料,设计合成了18个新化合物,运用1H NMR,13C NMR,HR-MS对化合物进行结构表征,并测试了这些化合物对人正常胚肺成纤维细胞(MRC-5)的毒性。结果显示13个化合物对正常细胞毒性较大(IC50<38.12μmol/L),5个化合物毒性较小(IC50> 100μmol/L)。佛波醇的12位羟基、13位羟基分别与长链饱和脂肪酸形成单酯时毒性较低,实验结果为佛波醇的结构修饰提供参考。     

High-throughput determination of identity, purity, and quantity of combinatorial library members using LC/MS/UV/ELSD

We have used a high-throughput LC/MS/UV/ELSD method to rapidly determine the absolute quantity and purity of 42 organic compounds from seven lead discovery libraries. A general calibration curve generated from a different set of 42 compounds with seven different scaffolds was used in this analysis. We have also studied 33 organic compounds with different molecular weight (MW) by LC/MS/UV/ELSD to investigate the effect of MW on ELSD response and the accuracy for purity and quantity measurement using UV(214) and ELSD. A general ELSD calibration curve from these compounds was also generated to quantify 42 library compounds. Purity measurement by ELSD underestimates the amounts of impurities due to a reduced ELSD response from smaller molecular weight impurities often produced in library synthesis. Absolute quantity determination by ELSD is more accurate (RSD 28%) than that by UV(214) (48%) using a calibration curve generated from the same set of compounds with diverse MWs. Error assessment for the measurement of absolute quantity of a class of commercial compounds and a class of representing reference compounds from seven diverse lead discovery libraries shows that structurally related compounds should be used to generate calibration curves to sustain smaller deviation.     

Isolation and identification of two isomeric forms of malonyl-coenzyme A in commercial malonyl-coenzyme A

Two isomers of malonyl-coenzyme A (malonyl-CoA) were detected in a commercial preparation of malonyl-CoA. These compounds were separated by preparative high-performance liquid chromatography (HPLC) and characterized by HPLC/ultraviolet (UV)/mass spectrometry. Both compounds had a UV absorbance maximum at 259-260 nm. Both compounds underwent negative electrospray ionization to produce a [M-H](-)quasi-molecular ion at m/z 852 and both compounds underwent collision-induced dissociation to produce a characteristic fragment at m/z 808, all consistent with the structure of malonyl-CoA. Nuclear magnetic resonance spectrometry showed that the two chromatographically distinguishable malonyl-CoAs are structural isomers: the major component is the naturally occurring malonyl-CoA and the contaminant is 3'-dephospho- 2'-phospho-coenzyme A.     

蝉虫草样品的分级萃取与化学成分分析

利用索氏萃取技术,依次采用石油醚、乙酸乙酯、丙酮、无水乙醇和甲醇等5种溶剂对蝉虫草纯粉进行分级萃取,运用傅里叶变换红外光谱分析法和气相色谱-质谱联用技术对5级萃取物进行分析鉴定。傅里叶变换红外光谱分析显示萃取物中含有与烯烃类、羧酸类、酯类、醇类和酮类等化合物相关的C-H、C=O、C-O和C=C等官能团。气相色谱-质谱联用技术共鉴定出有机小分子化合物34种,以酯类和脂肪酸类为主,多为碳链长度为15-20的长链脂肪酸及对应的酯,其中十八碳不饱和脂肪酸相对含量高达28.95%;分别存在于两种或以上萃取物中的有机化合物共有11种;仅存于石油醚萃取物中的化合物6种,乙酸乙酯萃取物中3种,丙酮萃取物中2种,无水乙醇萃取物中6种,甲醇萃取物中6种。在一定极性范围内,利用溶剂的极性梯度变化,可实现蝉虫草中活性物质的按极性梯度分离;采用分级萃取技术可有效分离蝉虫草中部分化学成分。鉴定结果充实了蝉虫草中化合物的种类资源,为蝉虫草中活性物质谱图库的完善、构效关系的建立及蝉虫草产品的利用开发提供支撑。     

Regulation of Substances in Allantoic and Amniotic Fluid of the Chicken Embryo

Traditionally, the avian allantois has been considered a respiratory organ and a dumping ground for metabolic wastes. We tested the hypothesis that the allantoic fluid is also a depot for free amino acids and related compounds. To gain further insight in the specific role of the allantoic fluid, we included plasma and the amniotic fluid in this study. The work was carried out in 13- and 14-day-old chicken embryos. Using an HPLC-fluorometric technique, 40 of the 41 amino acids and related compounds investigated were detected. The amniotic fluid contained 32 compounds, while plasma and allantoic fluid contained 38 and 39 compounds, respectively. The glucose concentration was determined with a hexokinase technique. It was highest in plasma and lowest in the amniotic fluid. We identified three barriers that hyper- and hyporegulate a number of compounds: (1) a blood/allantois barrier, (2) a blood/amnion barrier, and (3) an allantois/amnion barrier. Compared with plasma and allantoic fluid, the amniotic fluid is a mostly hyporegulated environment.     

Susceptibility and resistance of ruminal bacteria to antimicrobial feed additives

Susceptibility and resistance of ruminal bacterial species to avoparcin, narasin, salinomycin, thiopeptin, tylosin, virginiamycin, and two new ionophore antibiotics, RO22-6924/004 and RO21-6447/009, were determined. Generally, antimicrobial compounds were inhibitory to gram-positive bacteria and those bacteria that have gram-positive-like cell wall structure. MICs ranged from 0.09 to 24.0 micrograms/ml. Gram-negative bacteria were resistant at the highest concentration tested (48.0 micrograms/ml). On the basis of their fermentation products, ruminal bacteria that produce lactic acid, butyric acid, formic acid, or hydrogen were susceptible and bacteria that produce succinic acid or ferment lactic acid were resistant to the antimicrobial compounds. Selenomonas ruminantium was the only major lactic acid-producing bacteria resistant to all the antimicrobial compounds tested. Avoparcin and tylosin appeared to be less inhibitory (MIC greater than 6.0 micrograms/ml) than the other compounds to the two major lactic acid-producing bacteria, Streptococcus bovis and Lactobacillus sp. Ionophore compounds seemed to be more inhibitory (MIC, 0.09 to 1.50 micrograms/ml) than nonionophore compounds (MIC, 0.75 to 12.0 micrograms/ml) to the major butyric acid-producing bacteria. Treponema bryantii, an anaerobic rumen spirochete, was less sensitive to virginiamycin than to the other antimicrobial compounds. Ionophore compounds were generally bacteriostatic, and nonionophore compounds were bactericidal. The specific growth rate of Bacteroides ruminicola was reduced by all the antimicrobial compounds except avoparcin. The antibacterial spectra of the feed additives were remarkably similar, and it appears that MICs may not be good indicators of the potency of the compounds in altering ruminal fermentation characteristics.     

Synthesis and biochemical evaluation of novel and potent inhibitors of the enzyme oestrone sulphatase (ES)

In an effort to investigate the structural requirements for the inhibition of the enzyme oestrone sulphatase (ES), we have previously undertaken extensive structure-activity relationship studies. Using the data from molecular modelling and structure–activity relationship determination studies, we have designed a number of compounds based upon 4-sulphamated phenyl ketones. Here, we report the results of our study into a series of these compounds as potential inhibitors of ES. The results of the study show that these compounds are potent inhibitors the possessing greater inhibitory activity than 4-methylcoumarin-7-O-sulphamate derivative (COUMATE) (a potent non-steroidal inhibitor), but are weaker than oestrone-3-sulphamate (EMATE) and the recently reported 667- and 669-COUMATE, however, they provide good lead compounds in the search for potent inhibitors of ES. Furthermore, the compounds are observed to be irreversible inhibitors. From the consideration of the structure-activity relationship of these novel compounds, we have attempted to rationalise the significance of the log P factor in the inhibition of ES and suggest that a log P requirement of approximately 3.5 aids the inhibition through the rapid expulsion of the carbon backbone from the active site. We also propose that the same factor is responsible for the hydrolysis of oestrone sulphate reaction, appearing to be an irreversible process.     

Analysis of Relative Concentration of Ethanol and Other Odorous Compounds (OCs) Emitted from the Working Surface at a Landfill in China

Estimating odor emissions from landfill sites is a complicated task because of the various chemical and biological species that exist in landfill gases. In this study, the relative concentration of ethanol and other odorous compounds emitted from the working surface at a landfill in China was analyzed. Gas sampling was conducted at the landfill on a number of selected days from March 2012 to March 2014, which represented different periods throughout the two years. A total of 41, 59, 66, 54, 63, 54, 41, and 42 species of odorous compounds were identified and quantified in eight sampling activities, respectively; a number of 86 species of odorous compounds were identified and quantified all together in the study. The measured odorous compounds were classified into six different categories (Oxygenated compounds, Halogenated compounds, Terpenes, Sulfur compounds, Aromatics, and Hydrocarbons). The total average concentrations of the oxygenated compounds, sulfur compounds, aromatics, halogenated compounds, hydrocarbons, and terpenes were 2.450 mg/m³, 0.246 mg/m³, 0.203 mg/m³, 0.319 mg/m³, 0.530 mg/m³, and 0.217 mg/m³, respectively. The relative concentrations of 59 odorous compounds with respect to the concentration of ethyl alcohol (1000 ppm) were determined. The dominant contaminants that cause odor pollution around the landfill are ethyl sulfide, methyl mercaptan, acetaldehyde, and hydrogen sulfide; dimethyl disulfide and dimethyl sulfide also contribute to the pollution to a certain degree.     

Susceptibility and resistance of ruminal bacteria to antimicrobial feed additives.

Susceptibility and resistance of ruminal bacterial species to avoparcin, narasin, salinomycin, thiopeptin, tylosin, virginiamycin, and two new ionophore antibiotics, RO22-6924/004 and RO21-6447/009, were determined. Generally, antimicrobial compounds were inhibitory to gram-positive bacteria and those bacteria that have gram-positive-like cell wall structure. MICs ranged from 0.09 to 24.0 micrograms/ml. Gram-negative bacteria were resistant at the highest concentration tested (48.0 micrograms/ml). On the basis of their fermentation products, ruminal bacteria that produce lactic acid, butyric acid, formic acid, or hydrogen were susceptible and bacteria that produce succinic acid or ferment lactic acid were resistant to the antimicrobial compounds. Selenomonas ruminantium was the only major lactic acid-producing bacteria resistant to all the antimicrobial compounds tested. Avoparcin and tylosin appeared to be less inhibitory (MIC greater than 6.0 micrograms/ml) than the other compounds to the two major lactic acid-producing bacteria, Streptococcus bovis and Lactobacillus sp. Ionophore compounds seemed to be more inhibitory (MIC, 0.09 to 1.50 micrograms/ml) than nonionophore compounds (MIC, 0.75 to 12.0 micrograms/ml) to the major butyric acid-producing bacteria. Treponema bryantii, an anaerobic rumen spirochete, was less sensitive to virginiamycin than to the other antimicrobial compounds. Ionophore compounds were generally bacteriostatic, and nonionophore compounds were bactericidal. The specific growth rate of Bacteroides ruminicola was reduced by all the antimicrobial compounds except avoparcin. The antibacterial spectra of the feed additives were remarkably similar, and it appears that MICs may not be good indicators of the potency of the compounds in altering ruminal fermentation characteristics.     

The metabolism of [3-H]oleanolic acid and its monoglycosides in cytoplasm and vacuole of protoplasts isolated from Calendula officinalis leaves

Isolated protoplasts from C. officinalis leaves were supplied with [3-³H]oleanolic acid, its 3-O-monoglucoside and 3-O-monoglucuronide. Transformations of these compounds into two series of oleanolic acid glycosides, i.e. glucosides (derivatives of 3-O-monoglucoside) and glucuronides (derivatives of 3-O-monoglucuronide) in the extravacuolar space and the vacuole were investigated. In the cytoplasm free oleanolic acid is glycosylated to both monoglycosides and to higher glycosides. Monoglycosides are partly hydrolysed to free oleanolic acid and partly glycosylated to higher derivatives. The vacuole contains the same radioactive compounds as the extravacuolar space. However, it seems most likely that these compounds are transported there from the sites of their synthesis in the cytoplasm.     

Simultaneous determination of thioridazine and its S-oxidized and N-demethylated metabolites using high-performance liquid chromatography on radially compressed silica

A method for simultaneously quantifying thioridazine, northioridazine, thioridazine-2-sulfoxide, thioridazine-2-sulfone and thioridazine-5-oxide in serum and plasma is described. Following solvent extraction these compounds were separated by high-performance liquid chromatography on radially compressed silica gel and detected by UV absorbance at 254 nm. Chromatography time is less than 7 min. The relative retention of these compounds as a function of the methanol and methylamine content of the mobile phase is discussed. Practical limits of detection, based upon an assayed plasma or serum volume of 1 ml, were 20 ng/ml for thioridazine-5-oxide and 10 ng/ml for the other compounds. The coefficient of variation for all compounds was less than 13%. The method is compared with more conventional high-performance liquid chromatographic and gas chromatographic methodology.     

Synthesis and antidepressant-like activity evaluation of sulphonamides and sulphonyl-hydrazones

In this study a series of sulphonamides and sulphonyl hydrazones of maleimide, naphthalimide and phthalimide derivatives was synthesized. The antidepressant effect of these compounds was evaluated by the forced-swimming test in mice. The behavioural parameter observed in this test is a reduction in the immobility time, which is indicative of antidepressant activity. All compounds, except 8, 11 and 24, were active as antidepressants. The most active compound was the sulphonyl-hydrazone 10 which showed an activity of around 72.02% at 60 mg/kg, it thus being more active than imipramine (10mg/kg, ip), a commercial antidepressant. Other important results were obtained for the benzylnaphthalimide derivatives, the sulphonamides 21 and 22 showing activity of 64% at 10mg/kg, also being more active than imipramine. These results indicate that the sulphonamides and sulphonyl-hydrazone cyclic imide derivatives are potential compounds for use in the designing of new candidates for the treatment of depression.     

土壤微孔对有机物吸附/解吸的影响及其表征

土壤吸附是影响环境中有机化合物迁移、降解及生物有效性的重要过程,而微孔的存在是影响有机化合物慢吸附过程的重要因素之一,土壤孔隙结构(pore structure)及土壤微孔的研究对于理解发生在土壤中的吸附/解吸过程十分必要.综述了近年来土壤微孔对有机化合物吸附解吸行为影响的研究态势,包括土壤的孔隙结构及微孔的存在形式、微孔吸附有机化合物的吸附动力学和可能机理、土壤中微孔表征的技术方法、孔隙大小分布的计算以及对未来的研究展望,以期对土壤有机污染生物修复的深入研究提供理论依据.     

Structure based virtual screening, 3D-QSAR,molecular dynamics and ADMET studies for selection of natural inhibitors against structural and non-structural targets of Chikungunya

The transmission of mosquito-borne Chikungunya virus (CHIKV) has large epidemics worldwide. Till date, there are neither anti-viral drugs nor vaccines available for the treatment of Chikungunya. Accumulated evidences suggest that some natural compounds i.e., Epigallocatechin gallate, Harringtonine, Apigenin, Chrysin, Silybin, etc. have the capability to inhibit CHIKV replication in vitro. Natural compounds are known to possess less or no side effects. Therefore, natural compound in its purified or crude extracts form could be the preeminent and safe mode of therapies for Chikungunya. Wet lab screening and identification of natural compounds against Chikungunya targets is a time consuming and expensive exercise. In the present study, we used in silico techniques like receptor-ligand docking, Molecular dynamic (MD), Three Dimensional Quantitative Structure Activity Relation (3D-QSAR) and ADME properties to screen out potential compounds. Aim of the study is to identify potential lead/s from natural sources using in silico techniques that can be developed as a drug like molecule against Chikungunya infection and replication. Three softwares were used for molecular docking studies. Potential ligands selected by docking studies were subsequently subjected 3D-QSAR studies to predict biological activity. Based on docking scores and pIC₅₀ value, potential anti-Chikungunya compounds were identified. Best docked receptor-ligands were also subjected to MD for more accurate estimation. Lipinski’s rule and ADME studies of the identified compounds were also studied to assess their drug likeness properties. Results of in silico findings, led to identification of few best fit compounds of natural origin against targets of Chikungunya virus which may lead to discovery of new drugs for Chikungunya.

Communicated by Ramaswamy H. Sarma     

Antimicrobial activity of essential oils and structurally related synthetic food additives towards Clostridium perfringens

Aims:  To assess the potential of essential oils and structurally related synthetic food additives in inhibiting the growth of Clostridium perfringens for the control of necrotic enteritis in chickens.
Methods and Results:  The antimicrobial activity of essential oils/compounds was measured by determining the inhibition of bacterial growth. Thirty-three of 66 oils/compounds exhibited ≥80% inhibition. Seven with the highest potency were further studied. The oils/compounds had MIC₉₅ values between 167 and 425  μ g ml⁻¹. Most of them were tolerant to low pH (2·0) and exhibited minor or no inhibition of Lactobacillus isolates from the chicken intestine. When mixed with chicken ileal digesta, the oils/compounds retained their efficacy against C . perfringens , but had little effect on the total number of lactobacilli and anaerobic bacteria in the digesta.
Conclusions:  Some essential oils/compounds demonstrated good potential in controlling C . perfringens .
Significance and Impact of the Study:  This study has identified candidates of essential oils/compounds for in vivo studies for the control of necrotic enteritis in chickens.     

Extraction methods and chemical standardization of botanicals and herbal preparations

Botanicals and herbal preparations are medicinal preparations, containing a single or two or more medicinal plants. The focus of this review paper is on the analytical methodologies, which included the combination of sample preparation tools and chromatographic techniques for the chemical standardization of marker compounds or active ingredients in botanicals and herbal preparations. The common problems and key challenges in the chemical standardization of botanicals and herbal preparations were discussed. As sample preparation is the most important step in the development of analytical methods for the analysis of constituents present in botanicals and herbal preparations, the strength and weakness of different extraction techniques are discussed. For the analysis of compounds present in the plant extracts, the applications of common chromatographic techniques, such as HPLC, CE, HRGC/MS, HPLC/MS and HPLC/MS/MS are discussed. The strength, weakness and applicability of various separation tools are stated. Procedures for the identification of marker or active compounds in plant extracts, using HPLC/MS, were proposed. Finally, the effects of batch-to-batch variation of the medicinal plants are investigated and discussed.