Profiling analysis of amino acids from hyperlipidaemic rats treated with Gynostemma pentaphyllum and atorvastatin

Context Gynostemma pentaphyllum (Thunb.) Makino has been used in traditional medicine for the treatment of hyperlipidaemic with a long history.

Objective: The objective of this study was to evaluate the influence of Gynostemma pentaphyllum (GP) and atorvastatin on amino acids from the plasma and liver tissue of hyperlipidaemic rats.

Materials and methods The rats were fed a high-fat diet continuously for 11 weeks for the construction of hyperlipidaemic model. The hyperlipidaemic rats were treated with Gynostemma pentaphyllum (120?mg/kg) and atorvastatin (1.8?mg/kg) for 4 weeks, and the rats were intragastric administration one time every day. Chromatographic separation was performed on a Shim-pack XR-ODSIII C18 analytical column (75?mm?×?2.0?mm i.d., 1.6?μm, Shmadazu Corp., Tokyo, Japan). The biomarkers of amino acids were identified by principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA).

Results After feeding with a high-fat diet, the TC and LDL-C values of the hyperlipidaemic mode rats increased dramatically (p?<?0.01). The established method allowed a target analysis of 12 kinds of amino acids. PCA studies showed that the plasma amino acids had not returned to normal after GP treatment, but those had recovered slightly after atorvastatin treatment. GP has almost no impact on the metabolism of amino acids, while atorvastatin can modify the metabolism of amino acids via self-regulatory mechanisms.

Discussion and conclusion UPLC/DAD combined with SCX-SPE can be successfully used for profiling analysis of amino acids. By the comparison of biomarkers following treatment with GP and atorvastatin, the influence of the two drugs on biomarkers is revealed.     

Metabolic profiling reveals therapeutic biomarkers of processed Aconitum Carmichaeli Debx in treating hydrocortisone induced Kidney-Yang deficiency syndrome rats

Ethnopharmacological relevance

Kidney-Yang Deficiency syndrome (KYDS) is a diagnostic pattern in traditional Chinese medicine (TCM) and clinical data showed that the unbalance in adrenal cortical hormone is the key issue in KYDS patients. The processed Ranunculaceae Aconitum carmichaeli debx (Bai-Fu-Pian in Chinese, BFP) is one of the most commonly used Chinese herbs for treating KYDS. The present study was conducted to explore the therapeutic biomarkers of the BFP in treating hydrocortisone administration induced KYDS rats.

Materials and methods

Thirty male Sprague-Dawley rats were randomly divided into five groups with six in each group. KYDS in rats was induced by i.p. injection of hydrocortisone at the dose of 10 mg/kg per day for 15 days as described previously. The rats with KYDS were administered orally, starting from the day of hydrocortisone administration stopped, with BFP extract at the dose of 0.32 g/kg, 0.64 g/kg and 1.28 g/kg per day respectively for 15 days. The blood samples were collected for the liquid chromatography quadruple time-of-flight mass spectrometry (LC-Q-TOF-MS) test, as well as radioimmunoassay to determine the concentrations of cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP) and adrenocorticotrophic hormone (ACTH). The metabolic responses to BFP administration were investigated by using the principal components analysis (PCA) and orthogonal partial least squares analysis (OPLS). Bioinformatics analyses were performed by using the Ingenuity Pathway Analysis (IPA). Variance analysis and linear regression analysis were used in this study.

Results

The signs and concentrations of cAMP, cGMP and ACTH in the model rats were similar to those previously described about KYDS rats and BFP treatment can reverse the changes. Seventeen significantly changed metabolites among different groups were identified. Thirteen metabolites were identified in the KYDS rats comparing to healthy rats with nine up-regulated and four down-regulated. After BFP treatment at three dosages, five up-regulated metabolites including phosphate, betaine, (4-hydroxyphenyl) acetaldehyde, 5-hydroxyindol-3-acetic acid and 5'-phosphoribosyl-N-formylglycinamide were dose-dependently reversed. The network analysis with IPA showed that four canonical pathways including superpathway of methionine degradation, purine nucleotides De Novo biosynthesis II, tyrosine synthesis and serotonin receptor signaling involved the therapeutic mechanism of BFP in treating the KYDS rats.

Conclusions

Five therapeutic biomarkers (phosphate, betaine, (4-hydroxyphenyl) acetaldehyde, 5-hydroxyindol-3-acetic acid and 5'-phosphoribosyl-N-formylglycinamide) and two corresponding canonical pathways (amino acid metabolism and purine nucleotide metabolism) were identified to be involved in the therapeutic mechanism of BFP treating the KYDS.     

红外光谱结合化学计量学鉴别美洲大蠊及其他虫类药材研究

目的基于红外光谱法结合化学计量学建立美洲大蠊及其他虫类药材(全蝎、水蛭)粉末的鉴别方法,为美洲大蠊的鉴别提供一定的依据。方法运用傅里叶变换红外光谱仪采集3种虫药药粉的红外光谱数据,对已得光谱数据二阶求导后运用纵坐标归一化法、标准化法等预处理方法对光谱进行优化,采用化学计量学,包括系统聚类分析(hierarchicalcluster analysis,HCA)、主成分分析(principal component analysis,PCA)以及偏最小二乘判别分析(partial least squares discriminant analysis,PLS-DA)进一步对光谱数据进行分析。结果美洲大蠊与全蝎、水蛭的红外指纹图谱有差异。美洲大蠊在1 711、1 410、712 cm-1处吸收峰明显,全蝎在1 753、1 400、1 168、717 cm-1处吸收峰明显,水蛭在1 558、1 457、1 400、669 cm-1处吸收峰明显,且水蛭在1 753～1 711cm-1处明显无吸收峰。3种虫类药材在1 800～1 700 cm-1处峰形差异明显较大。二阶导数图谱中,主要峰的位置相同,但共有峰的强度不同。运用HCA分析方法发现3种虫类药材能够被快速区分,运用PCA与PLS-DA分析方法发现3种虫类药材分布在不同区域。结论本研究能够简便、快速的鉴别美洲大蠊与全蝎、水蛭等虫类药材,为美洲大蠊的质量控制及评价提供参考。     

基于指纹图谱通便灵胶囊的化学计量学分析

目的 通过HPLC建立通便灵胶囊指纹图谱，并借助化学计量学分析，对其进行系统、科学的质量评价。方法 采用JADE-PAK ODS柱（250 mm×4.6 mm，5 μm），以流动相为甲醇（A）-0.1%甲酸溶液（B），进行梯度洗脱，流速1.0 mL·min^–1，柱温30℃，检测波长340 nm，建立通便灵胶囊的指纹图谱，通过ChemPattern软件确立共有峰，利用UPLC-Q/TOF-MS对共有峰进行成分确认，并进行化学计量学分析。结果 通过建立的通便灵胶囊指纹图谱确定14个共有峰，主成分分析得分图显示4个厂家的样品能够明显区分开，偏最小二乘判别分析载荷图显示化学成分松果菊苷、山柰酚-3-O-β-D-槐糖苷和异鼠李素-3-O-龙胆二糖苷对4个厂家样品的区分有较大贡献。结论 不同厂家样品存在较大差异，有可能会造成药效的不一致，此方法可为通便灵胶囊的质量控制提供参考依据。     

UPLC测定经典名方金水六君煎中11种成分

目的建立同时测定经典名方金水六君煎(JLD)中腺苷、鸟苷、5-羟甲基糠醛、阿魏酸、甘草苷、毛蕊花糖苷、芸香柚皮苷、橙皮苷、甘草酸铵、6-姜辣素、藁本内酯11种主要成分的UPLC方法。方法采用UPLC法,色谱柱为Acquity UPLC HSS T3 C18柱(100 mm×2.1 mm,1.8m),0.1%磷酸水溶液-乙腈为流动相,梯度洗脱;体积流量为0.3 m L/min;柱温25℃;进样量1.0μL;检测波长:腺苷、鸟苷、5-羟甲基糠醛、甘草酸铵为248 nm,阿魏酸为330 nm,甘草苷、毛蕊花糖苷、芸香柚皮苷、橙皮苷、6-姜辣素、藁本内酯为281 nm。采用SIMCA 14.1软件进行偏最小二乘法-判别分析(PLS-DA),以寻找样品间的质量差异成分,同时采用Hotelling’s T2和DMod X 2种统计量,为不同批次样品的质量设定控制范围。结果被测定的11种成分色谱峰均有良好的分离度,方法精密度、重复性的RSD均小于3%;在室温条件下24 h内稳定;各成分均有较宽的线性范围和良好的线性关系(r≥0.9992);平均加样回收率96.62%～103.15%,RSD均小于2%。将15批不同产地的样品分为3类,通过PLS-DA分析含量测定结果,发现芸香柚皮苷、甘草酸铵、藁本内酯和阿魏酸是影响不同批次JLD物质基准质量贡献较大的4种成分。结论建立的方法操作简便,结果准确、可靠,可用于JLD及其制剂的质量控制与评价。     

柱前衍生RP-HPLC测定菊米中17种氨基酸的含量

目的:探讨菊米中基本氨基酸组成和不同加工方式、不同采收期对氨基酸含量的影响,为菊米的质量和加工利用提供科学依据。方法:采用柱前衍生处理样品,建立HPLC方法,分析菊米中氨基酸组成及含量,评价不同加工处理和不同采收期的菊米质量差异。结果:菊米中含有17种必需和非必需氨基酸,机械加工样品氨基酸含量高于手工加工。通过t检验,除脯氨酸和胱氨酸外,其余15种氨基酸在两种加工方法处理菊米样品中均存在显著差异。通过氨基酸含量的偏最小二乘法-判别分析(PLS-DA),加工方法对天冬氨酸、谷氨酸、丝氨酸、缬氨酸和赖氨酸的影响最大(VIP1),两种加工方法区分明显,可以判断加工方法的优劣。不同采收时期的菊米样品氨基酸含量差异不明显(P0.05),说明采收时期对氨基酸成分影响较小。结论:利用柱前衍生-HPLC法分析菊米营养成分氨基酸可作为评价菊米质量评价的方法之一,采用机械加工可保证菊米的质量。     

Metabonomic study on the anti-osteoporosis effect of Rhizoma Drynariae and its action mechanism using ultra-performance liquid chromatography-tandem mass spectrometry

Ethnopharmacological relevance

Rhizoma Drynariae (RD) is an effectively traditional Chinese medicine which is usually used in treating osteoporosis, bone fracture, streptomycin ototoxicity and hyperlipemia. Up to now, studies on pharmacological mechanism of RD mostly focus on cell and gene level, little is known about its metabonomics study. The aim of this study is to establish the rats plasma metabonomic profiles of control, model and treatment group, then to investigate the anti-osteoporosis effect of RD and its action mechanism.

Method

A total of 21 Wistar rats was divided into three groups: control group, model group and treatment group. The model and treatment rats were injected prednisolone for 12 weeks, at the same time the treatment rats were orally administered RD extract at a therapeutic dose (10 g/kg, expressed as the weight of raw material) once daily throughout the experimental period, control group and model group were orally gavaged approximately volume normal saline solution. After 12 weeks, all plasma samples of three groups were collected and their metabolic profiling changes were analyzed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The resulting dataset was analyzed by principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). The identification of all potential biomarkers was performed using reference standard by comparing their mass spectra, MS/MS fragmentation and retention time. Furthermore, clinical biochemistry and biomechanics study were also carried out to ensure the success of the osteoporosis model and to investigate the anti-osteoporosis effect of RD.

Results

Obvious separation trend between control and model group was found in PCA score plot, the anti-osteoporosis effect of RD can be indicated in PLS-DA score plot among these three groups. Six potential metabolite biomarkers, Lysophosphatidylcholines (C16:0 LPC, C18:0 LPC, C18:1 LPC and C18:2 LPC), tryptophane and phenylalanine, which were proved to be related with osteoporosis, were identified in the rats plasma. Compared with control group, level of all biomarkers increased significantly in model group, while that was much closer to normal in treatment group.

Conclusion

The anti-osteoporosis effect of RD has been reliably confirmed by the metabonomics method. The osteoporosis might be prevented by RD via intervening antioxidant-oxidation balance, tryptophane metabolism and phenylalanine metabolism in vivo in rats.     

基于智能视觉技术的白及饮片真伪快速辨识方法研究

目的 利用电子眼和色差仪分别对白及Bletillastriata及其伪品进行辨识,旨在建立白及真伪的快速辨识方法。方法 首先基于《中国药典》、地方标准和HPLC指纹图谱对多采集来源的134批白及及其伪品样品进行综合鉴别,以确定标杆辨识信息（Y）。然后基于电子眼和色差仪分别获取上述样品的智能视觉信息（X）。最后利用Matlab建立Y=F(X)模型,即分别建立白及真伪二分类（白及、非白及）和四分类（白及、天麻Gastrodiaelata、玉竹Polygonatumodoratum、黄花白及Bletilla ochracea）的主成分分析-判别分析（principal component analysis-discriminant analysis,PCA-DA）、偏最小二乘-判别分析（partial least squares-discriminant analysis,PLS-DA）、最小二乘-支持向量机（least squares-support vector machine,LS-SVM）和K最近邻（K nearest neighbor,KNN）辨识模型并验证。结果 经留一法交互验证...     

Multivariate analysis of NMR-based metabolomic data

Nuclear magnetic resonance (NMR) spectroscopy allows for simultaneous detection of a wide range of metabolites and lipids. As metabolites act together in complex metabolic networks, they are often highly correlated, and optimal biological insight is achieved when using methods that take the correlation into account. For this reason, latent-variable-based methods, such as principal component analysis and partial least-squares discriminant analysis, are widely used in metabolomic studies. However, with increasing availability of larger population cohorts, and a shift from analysis of spectral data to using quantified metabolite levels, both more traditional statistical approaches and alternative machine learning methods have become more widely used. This review aims at providing an overview of the current state-of-the-art multivariate methods for the analysis of NMR-based metabolomic data as well as alternative methods, highlighting their strengths and limitations.     

基于谱效相关法探究酒炙丹参增强抗凝血活性的物质基础

目的通过谱效相关法探究酒炙丹参增强抗凝血活性的物质基础。方法采用HPLC法建立丹参生品与酒炙品指纹图谱,并进行相似度评价。测定不同批丹参生品与酒炙品的体外抗凝血效价,包括活化部分凝血活酶时间(APTT)、凝血酶原时间(PT)、凝血酶时间(TT)和纤维蛋白原(FIB)含量。以丹参生品与酒炙品的HPLC图谱的峰面积及其抗凝血效价为基础,采用偏最小二乘判别分析(PLS-DA)法对HPLC图谱和抗凝血效价进行相关性分析。结果10批生丹参饮片HPLC指纹图谱相似度为0.991~0.999,10批酒丹参饮片与对照图谱的相似度为0.982~0.999。丹参酒炙后抗凝血活性显著增强。PLS-DA法分析得出,指纹图谱中7(丹酚酸B)、6(紫草酸)、5(迷迭香酸)、15(VIP均大于1)号峰是导致丹参酒炙后抗凝血活性增强的主要成分。结论该方法能较精准地找到丹参酒炙增强抗凝血活性的主要贡献成分。