针灸治疗多囊卵巢综合征致不孕症临床疗效观察

目的就针灸治疗多囊卵巢综合征致不孕症的临床疗效进行观察与探究。方法选择广州市天河区冼村街社区卫生服务中心自2013年4月至2015年5月期间收治的多囊卵巢综合征致不孕症患者66例,依据患者来院先后顺序平均分为参照组和观察组,每组患者33例。参照组患者予以常规治疗,观察组患者采用针灸治疗,最后比对两组患者的临床治疗效果。结果经不同方法治疗后,观察组患者各项指标的改善程度好于参照组,妊娠率和排卵率也均高于参照组,组间统计后差异显著。结论在多囊卵巢综合致不孕症患者治疗中应用针灸,其临床效果显著,具有临床应用价值。     

小檗碱的药物代谢动力学

小檗碱是中药三棵针的主要有效成分之一，为了研究小檗碱的药物代谢动力学，本文采用胶束增敏荧光法对小檗碱在兔体内的血药浓度进行２４ｈ监测，求出毓药物代谢动力学参数，结果表明小檗碱在动物体内的Ｋａ为１．６３７ｈ＾－１，ｔ１／２（α） ０．３８６９ｈ，ｔ１／２（β）为６．７６ｈ，Ｋ２１为０．１５５３ｈ＾－１，Ｋ１２ｏ ０．０３８４ｈ＾－１，ＡＵＣ为２１１。９４μｇ．ｈ／ｍｌ。     

探究多囊卵巢综合征患者冻融胚胎移植子宫内膜准备方案的选择

目的探讨多囊卵巢综合征患者冻融胚胎移植子宫内膜准备的两种方案的效果。方法选择2014年3月到2016年3月间在深圳武警医院选择行冻融胚胎移植子宫内膜准备的700例多囊卵巢综合征患者作为研究对象,对所有患者的临床资料进行回顾性分析,根据患者子宫内膜准备方案分为人工组(n=350)和促排卵组(n=350),对比两组患者冻融胚胎移植效果。结果人工组患者胚胎复苏率、优质胚胎率、移植胚胎数、子宫内膜厚度、胚胎种植率、妊娠率与促排卵组相比均无差异,P0.05。结论人工子宫内膜与促排卵内膜效果相似,均可推广运用。     

微量量热法研究黄连中小檗碱类生物碱对金黄色葡萄球菌生长代谢的影响

采用微量量热法研究中药黄连中具有抑菌作用的3种小檗碱类生物碱成分对金黄色葡萄球菌生长代谢的影响．在不同给药条件下,以表达功率．时间曲线（热谱曲线）的特征参数生长速率常数k、半数抑菌浓度（IC50）、最大输出功率（Pmax）及达峰时间（tp）和总产热量（Qt）为指标,对3种小檗碱类生物碱成分抑制金黄色葡萄球菌生长代谢程度进行客观地量化评价．结果表明,k、Pmax和Qt均随3种小檗碱类生物碱成分浓度的增加而相应的减小;tp随3种小檗碱类生物碱成分浓度的增加而相应的延长;其IC50分别为小檗碱101．4μg／mL、巴马汀241．0μg／mL和药根碱792．3μg／mL,即抑菌活性大小顺序为小檗碱〉巴马汀〉药根碱．初步构效关系研究提示,位于苯环的C2、C3上的亚甲二氧基或甲氧基较羟基更有助于提高其抑菌活性．     

表面增强拉曼光谱研究小檗碱与DNA的相互作用

用表面增强拉曼光谱研究了小檗碱（BER）与小牛胸腺DNA（ct DNA)的相互作用,并对重要谱峰进行了归属。在Ag胶体系中,小檗碱分子的拉曼信号增强显著,表明小檗碱阳离子键合到银胶粒子的表面。加入ct NA之后,小檗碱分子的大部分SERS带的强度进一步增加,而仅有少数几个带的信号强度趋于消失,可能是小檗碱分子的异喹啉部分键合到DNA的小沟槽,小檗碱与DNA的相互作用模式主要是通过静电力及疏水相互作用,吸收光谱表明,Ag胶体系的存在并未改变小檗碱分子与DNA的相互作用模式。     

小檗碱与人血清白蛋白结合的电喷雾离子阱质谱研究

以小檗碱与人血清白蛋白间的非共价结合作用为例,提出了一种应用电喷雾离子阱质谱(ESI-ITMS)研究蛋白-配体非共价结合稳定常数与结合计量比等特性的新方法。根据小檗碱与人血清白蛋白间的ESI-ITMS滴定实验结果,小檗碱与人血清白蛋白形成复合物的最大表观化学结合计量比为4.4∶1;根据ESI-ITMS滴定实验结果的Scatchard方程分析,小檗碱-人血清白蛋白复合物的表观稳定常数K值为1.29×105L/mol,结合位点数n为5.1;根据作用温度对复合物热力学常数△H、△S的影响,确定小檗碱与人血清白蛋白间的作用力主要为疏水作用力。实验结果与应用荧光法得到的结果基本一致。     

Cu(Ⅱ),Ni(Ⅱ)存在条件下盐酸小檗碱与牛血清白蛋白相互作用的研究

使用荧光光谱和紫外光谱研究Cu（Ⅱ）,Ni（Ⅱ）存在的条件下对盐酸小檗碱（berberine chloride dihydrate,BC）与牛血清白蛋白（BSA）相互作用的影响。得到的猝灭常数Ksv表明,金属离子Cu（Ⅱ）或Ni（Ⅱ）的存在不影响其猝灭机理。基于Fcirster非辐射能量转移机理,获得了盐酸小檗碱与牛血清白蛋白的212位色氨酸残基问的距离：讨论了金属离子与BSA的结合构型对BC与BSA相互作用的影响。     

生物样品中小檗碱的电化学分析新方法

由于小檗碱氧化电位较高(>1.1 V vs. Ag/AgCl),进行生物样品中小檗碱的选择性电化学分析具有挑战性。基于溴百里酚蓝(BTB)和小檗碱的分子间非共价作用,通过单壁碳纳米管(SWNTs)将BTB修饰在电极表面并通过电化学聚合产生poly-BTB,利用聚BTB(poly-BTB)电极过程可逆以及氧化还原电位低的特点,以poly-BTB同时作为小檗碱的识别元件和电化学探针建立了小檗碱的电化学分析新方法。循环伏安和电化学阻抗谱结果表明,poly-BTB和小檗碱的分子间作用导致小檗碱结合在poly-BTB/SWNTs修饰电极表面,从而引起修饰电极的峰电流下降。在最优化的实验条件下,poly-BTB/SWNTs修饰电极的电流下降率和小檗碱的浓度在0.05～1μmol/L和1～100μmol/L范围内呈良好线性关系,检测限为0.022μmol/L。动物实验结果表明,该方法对生物样品具有很好的选择性,可用于血浆和肝脏匀浆中小檗碱含量的测量,为小檗碱相关的生理病理研究提供了一种简单有效的方法。     

小檗碱与牛血清白蛋白相互作用的核磁共振研究

采用核磁共振波谱技术(NMR)研究小檗碱与牛血清白蛋白(BSA)的相互结合作用.比较不同浓度小檗碱在一定浓度的BSA溶液中的化学位移、弛豫时间和扩散系数的变化情况.研究表明,小檗碱环上芳香质子与BSA的结合作用较强,其它烷基质子与BSA结合作用较弱;BSA的结合使得小檗碱各个质子的1HT1弛豫时间明显减少,质子在溶液中...     

基于超高效液相色谱-四极杆飞行时间质谱联用技术的血瘀模型大鼠血浆代谢组学分析

采用盐酸肾上腺素加冰水浴建立急性血瘀大鼠模型，使用超高效液相色谱-四极杆飞行时间质谱（UPLC-Q-TOF/MS）检测空白对照组与血瘀模型组中血浆代谢物，用主成分分析（PCA）、有监督偏最小二乘法判别分析（PLS-DA）及正交偏最小二乘法判别分析（OPLS-DA）对代谢组学数据进行多维统计分析，筛选潜在生物标志物。与对照组相比，在血瘀模型组大鼠血浆中检测出46个差异代谢物，血瘀模型组中乙酰胆碱、N6，N6，N6-三甲基-L-赖氨酸、胞嘧啶、乙酰肉碱等21个代谢物显著上调，吲哚丙酸、LysoPC（14：0）等25个代谢物显著下调，可能与脂质代谢、半乳糖代谢、亚油酸代谢、不饱和脂肪酸生物合成、糖酵解、花生四烯酸代谢等通路有关。代谢产物可作为血瘀证研究中的重要标记物，该研究结果有助于揭示血瘀证的发病机制，可为临床血瘀疾病的诊断及选用药物治疗提供思路，为后续治疗手段提供参考依据。     

Effect of traditional Chinese medicine berberine on type 2 diabetes based on comprehensive metabonomics

A comprehensive metabonomic method, in combination with fingerprint analysis and target analysis, was performed to reveal potential mechanisms of berberine action in the treatment of patients with type 2 diabetes and dyslipidemia. Serum samples of 60 patients before and after treatment with either berberine or placebo were collected. Ultra-performance liquid chromatography and quadrupole time-of-flight tandem mass spectrometry (UPLC Q-TOF MS) coupled with pattern recognition analysis were used to identify changes in global serum metabolites. Compared with placebo, patients before and after berberine treatment could be separated into distinct clusters as displayed by the orthogonal signal correction filtered partial least-squares discriminant analysis (OSC-PLS-DA) score plot, which indicated changes in circulating metabolites after berberine treatment. Among them, free fatty acids changed markedly. These were further quantified by UPLC combined with single quadrupole mass spectrometry (UPLC SQ MS). There was a highly significant decrease in the concentrations of 13 fatty acids following berberine administration. 10 fatty acids also differed statistically from placebo. These results suggest that berberine might play a pivotal role in the treatment of type 2 diabetes through down-regulating the high level of free fatty acids and that comprehensive metabonomic measurements are potentially very useful for studying the mechanisms of action of traditional Chinese medicines.     

Development of chemical isotope labeling liquid chromatography mass spectrometry for silkworm hemolymph metabolomics

Silkworm (Bombyx mori) is a very useful target insect for evaluation of endocrine disruptor chemicals (EDCs) due to mature breeding techniques, complete endocrine system and broad basic knowledge on developmental biology. Comparative metabolomics of silkworms with and without EDC exposure offers another dimension of studying EDCs. In this work, we report a workflow on metabolomic profiling of silkworm hemolymph based on high-performance chemical isotope labeling (CIL) liquid chromatography mass spectrometry (LC-MS) and demonstrate its application in studying the metabolic changes associated with the pesticide dichlorodiphenyltrichloroethane (DDT) exposure in silkworm. Hemolymph samples were taken from mature silkworms after growing on diet that contained DDT at four different concentrations (1, 0.1, 0.01, 0.001 ppm) as well as on diet without DDT as controls. They were subjected to differential ¹²C-/¹³C-dansyl labeling of the amine/phenol submetabolome, LC-UV quantification of the total amount of labeled metabolites for sample normalization, and LC-MS detection and relative quantification of individual metabolites in comparative samples. The total concentration of labeled metabolites did not show any significant change between four DDT-treatment groups and one control group. Multivariate statistical analysis of the metabolome data set showed that there was a distinct metabolomic separation between the five groups. Out of the 2044 detected peak pairs, 338 and 1471 metabolites have been putatively identified against the HMDB database and the EML library, respectively. 65 metabolites were identified by the dansyl library searching based on the accurate mass and retention time. Among the 65 identified metabolites, 33 positive metabolites had changes of greater than 1.20-fold or less than 0.83-fold in one or more groups with p-value of smaller than 0.05. Several useful biomarkers including serine, methionine, tryptophan, asymmetric dimethylarginine, N-Methyl-D-aspartic and tyrosine were identified. The changes of these biomarkers were likely due to the disruption of the endocrine system of silkworm by DDT. This work illustrates that the method of CIL LC-MS is useful to generate quantitative submetabolome profiles from a small volume of silkworm hemolymph with much higher coverage than conventional LC-MS methods, thereby facilitating the discovery of potential metabolite biomarkers related to EDC or other chemical exposure.     

The Effect of Blood Contained in the Samples on the Metabolomic Profile of Mouse Brain Tissue: A Study by NMR Spectroscopy

(1) Recently, metabolic profiling of the tissue in the native state or extracts of its metabolites has become increasingly important in the field of metabolomics. An important factor, in this case, is the presence of blood in a tissue sample, which can potentially lead to a change in the concentration of tissue metabolites and, as a result, distortion of experimental data and their interpretation. (2) In this paper, the metabolomic profiling based on NMR spectroscopy was performed to determine the effect of blood contained in the studied samples of brain tissue on their metabolomic profile. We used 13 male laboratory CD-1^® IGS mice for this study. The animals were divided into two groups. The first group of animals (n = 7) was subjected to the perfusion procedure, and the second group of animals (n = 6) was not perfused. The brain tissues of the animals were homogenized, and the metabolite fraction was extracted with a water/methanol/chloroform solution. Samples were studied by high-frequency ¹H-NMR spectroscopy with subsequent statistical data analysis. The group comparison was performed with the use of the Student’s test. We identified 36 metabolites in the brain tissue with the use of NMR spectroscopy. (3) For the major set of studied metabolites, no significant differences were found in the brain tissue metabolite concentrations in the native state and after the blood removal procedure. (4) Thus, it was shown that the presence of blood does not have a significant effect on the metabolomic profile of the brain in animals without pathologies.     

Combination of HPLC with organic and inorganic mass spectrometry to study the metabolic response of the clam Scrobicularia plana to arsenic exposure

Arsenic is a toxic element extensively studied in the marine environment due to differential toxicological effects of inorganic and organic species. In the present work, the bivalve Scrobicularia plana was exposed to As^V (10 and 100 μg/L) for 14 days to evaluate the metabolic perturbations caused by this element. Arsenic speciation and metabolomic analysis were performed in the digestive gland of the bivalve using two complementary analytical platforms based on inorganic and organic mass spectrometry. It has been observed the greater presence of the innocuous specie arsenobetaine produced in this organism as defense mechanism against arsenic toxicity, although significant concentrations of methylated and inorganic arsenic were also present, depending on the level of arsenic in aqueous media. Complementarily, a metabolomic study based on mass spectrometry and statistical discriminant analysis allows a good classification of samples associated to low and high As(V) exposure in relation to controls. About 15 metabolites suffer significant changes of expression by the presence of As(V): amino acids, nucleotides, energy‐related metabolites, free fatty acids, phospholipids and triacylglycerides, which can be related to membrane structural and functional damage. In addition, perturbation of the methylation cycle, associated with the increase of homocysteine and methionine was observed, which enhance the methylation of toxic inorganic arsenic to less toxic dimethylarsenic.     

Comprehensive Profiling by Non-targeted Stable Isotope Tracing Capillary Electrophoresis-Mass Spectrometry: A New Tool Complementing Metabolomic Analyses of Polar Metabolites

Mass spectrometry (MS) driven metabolomics is a frequently used tool in various areas of life sciences; however, the analysis of polar metabolites is less commonly included. In general, metabolomic analyses lead to the detection of the total amount of all covered metabolites. This is currently a major limitation with respect to metabolites showing high turnover rates, but no changes in their concentration. Such metabolites and pathways could be crucial metabolic nodes (e.g., potential drug targets in cancer metabolism). A stable-isotope tracing capillary electrophoresis–mass spectrometry (CE-MS) metabolomic approach was developed to cover both polar metabolites and isotopologues in a non-targeted way. An in-house developed software enables high throughput processing of complex multidimensional data. The practicability is demonstrated analyzing [U-¹³C]-glucose exposed prostate cancer and non-cancer cells. This CE-MS-driven analytical strategy complements polar metabolite profiles through isotopologue labeling patterns, thereby improving not only the metabolomic coverage, but also the understanding of metabolism.     

HPLC determination of berberine in plasma of patients with ischemic heart failure

Summary Berberine absorption by patients with ischemic heart failure (IHF) after oral administration and the relationship between clinical effect and plasma berberine concentration are studied. Plasma samples were pretreated by chloroform extraction. Berberine was determined on a μBondapak column with acetonitrile-phosphoric acid mobile phase and UV detection. The limit for berberine in plasma was 8 ng mL⁻¹ for an injection volume of 50 μL. Average berberine recovery was 96.5%. Results showed that improvements in symptoms were more significant for patients with plasma berberine concentration>0.1 mg L⁻¹ than for those with<0.1 mg L⁻¹. Plasma berberine monitoring may be helpful in the treatment of patients with IHF.     

Metabolomic investigation of gastric cancer tissue using gas chromatography/mass spectrometry

Gastric cancer screening or diagnosis is mainly based on endoscopy and biopsy. The aim of this study was to identify the difference of metabolomic profile between normal and malignant gastric tissue, and to further explore tumor biomarkers. Chemical derivatization together with gas chromatography/mass spectrometry (GC/MS) was utilized to obtain the metabolomic information of the malignant and non-malignant tissues of gastric mucosae in 18 gastric cancer patients. Acquired metabolomic data was analyzed using the Wilcoxon rank sum test to find the tissue metabolic biomarkers for gastric cancer. A diagnostic model for gastric cancer was constructed using principal component analysis (PCA), and was assessed with receiver-operating characteristic (ROC) curves. Results showed that 18 metabolites were detected differently between the malignant tissues and the adjacent non-malignant tissues of gastric mucosa. Five metabolites were also detected differently between the non-invasive tumors and the invasive tumors. The diagnostic model could discriminate tumors from normal mucosae with an area under the curve (AUC) value of 0.9629, and another diagnostic model constructed for clinical staging was assessed with an AUC value of 0.969. We conclude that the metabolomic profile of malignant gastric tissue was different from normal, and that the selected tissue metabolites could probably be applied for clinical diagnosis or staging for gastric cancer.     

A combination of metabolomics and metallomics studies of urine and serum from hypercholesterolaemic rats after berberine injection

Berberine, long used as a remedy in China and India for intestinal infections, has been discovered in recent years in western countries and is now being used to treat ailments ranging from urinary tract infections to diabetes and obesity. In order to study the effect of berberine more deeply, a combined metabolomic and metallomic approach was developed in this study using the hypercholesterolaemic rat model, which involved the use of proton nuclear magnetic resonance for the analysis of rat urine to achieve metabolic fingerprinting and inductively coupled plasma mass spectrometry for the analysis of rat blood serum to achieve metallomic fingerprinting. The results obtained indicated that major metabolic processes like Krebs cycle, cholesterol metabolism and osmoregulation in hypercholesterolaemic rats are perturbed upon berberine injection. In addition, the changes of some elements, such as V, Mn, Na and K, revealed in the metallomic study may contribute to the search of new biomarkers for hypercholesterolaemic disease. We concluded that both the metabolomic and metallomic profiles of berberine-treated hypercholesterolaemic rats were different from those of the control group and that the selected metabolites and elements could probably be applied as potential biomarkers for the understanding of the effect of berberine on biochemical process in the animal model. Such a multi-analytical approach will potentially provide an information-rich platform for the elucidation of effects of xenobiotics and drug efficacy studies.     

A Comparative Study of Pentafluorophenyl and Octadecylsilane Columns in High‐throughput Profiling of Biological Fluids

In high‐throughput metabolomic profiling, chromatographic separation is crucial because a well‐performed chromatographic separation may reduce signal suppression from complex biological matrices and improve the discoverability of low‐abundance metabolites. We compared the performance of pentafluorophenyl (PFP )‐ and octadecylsilane (ODS )‐based columns in profiling biological fluids. Peak resolutions and consistencies were acquired using several reversed‐phase columns and were evaluated. Total and extracted ion chromatograms demonstrated that the PFP column achieved better analyte separations than the ODS column. Low relative standard deviations on peak areas and retention times (<10.2 and <0.9%, respectively) acquired using the PFP column evidenced the high reproducibility and consistency of this column. In our study, a PFP column was used for profiling metabolomes extracted from urine and serum samples. Metabolomic study revealed a metabolome difference in normal and overweight participants. In total, 26 lipid species were significantly perturbed and further identified. Choline‐containing lipids were the most abundant perturbed lipidome in overweight participants, followed by sphingolipids and various phospholipids. We recommend the use of PFP columns in high‐throughput metabolomic analysis to promote the development of basic biological and clinical research in the future.     

Metabolic alterations in triptolide‐induced acute hepatotoxicity

Triptolide, a major active constitute of Tripterygium wilfordii Hook. F, is prescribed for the treatment of autoimmune diseases in China. One of its most severe adverse effects observed in the clinical use is hepatotoxicity, but the mechanism is still unknown. Therefore, the present study applied an LC/MS‐based metabolomic analysis to characterize the metabolomic changes in serum and liver induced by triptolide in mice. Mice were administered triptolide by gavage to establish the acute liver injury model, and serum biochemical and liver histological analyses were applied to assess the degree of toxicity. Multivariate data analyses were performed to investigate the metabolic alterations. Potential metabolites were identified using variable importance in the projection values and Student's t‐test. A total of 30 metabolites were observed that were significantly changed by triptolide treatment and the abundance of 29 metabolites was correlated with the severity of toxicity. Pathway analysis indicated that the mechanism of triptolide‐induced hepatotoxicity was related to alterations in multiple metabolic pathways, including glutathione metabolism, tricarboxylic acid cycle, purine metabolism, glycerophospholipid metabolism, taurine and hypotaurine metabolism, pantothenate and CoA biosynthesis, pyrimidine metabolism and amino acid metabolism. The current study provides new mechanistic insights into the metabolic alterations that lead to triptolide‐induced hepatotoxicity.