不同产地黄芪药材的UPLC/Q-TOF-MS指纹图谱研究

目的:采用超高效液相色谱串联四极杆飞行时间质谱仪(UPLC/Q-TOF-MS)建立黄芪药材的指纹图谱,初步鉴定其主要色谱峰,并结合主成分分析(PCA)模式识别方法评价不同产地药材质量。方法:用ACQUITY UPLC BEH C18色谱柱,以水-乙腈-异丙醇体系梯度洗脱,使用ESI离子源,正、负离子模式下采集数据。应用Markerlynx软件进行不同产地药材的PCA分析。结果:在18 min内建立了黄芪药材的UPLC/Q-TOF-MS指纹图谱,结合PCA分析,可以区分不同产地的药材,并找出包括毛蕊异黄酮,芒柄花素,黄芪皂苷Ⅰ、Ⅱ等8个差异最大的化合物。结论:UPLC/Q-TOF-MS方法所得谱图重现性和特征性较好,可以用于黄芪指纹图谱的快速鉴别。应用化学计量学方法可以较全面地反映不同产地药材学成分的差异,为其质量控制提供实验依据。     

Myocardial lipidomics profiling delineate the toxicity of traditional Chinese medicine Aconiti Lateralis radix praeparata

Ethnopharmacological relevance

The lateral root of Aconitum has been popularly used in traditional Chinese medicine (TMC) known as Fuzi which is beneficial for the treatment of various diseases, such as rheumatism, painful joints, syncope and bronchial asthma. However, it has a potential carditoxicity with a relatively narrow margin of safety.

Aim of the study

This paper was designed to explore the mechanisms of Fuzi's toxicity and find out potential tissue-specific biomarkers of toxic effects.

Material and methods

A myocardial lipidomics based on ultraperformance lipid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UHPLC/Q-TOF MS) was developed to compare three cardiac lipid extraction methods and investigate the changes of lipids in mice heart of three different dosage groups. In addition, we concurrently inspected the biochemical parameters in plasma, observed the histology of the heart and recorded the electrocardiogram (ECG).

Results

The cardiotoxicity of Fuzi was dose-dependent, and the high-dose group obviously manifested the heart damage in histology and a certain degree of arrhythmia. Significant changes of 14 lipid metabolites which primarily involved in phospholipid metabolism, sphingolipid metabolism, saturated fatty acid oxidation and unsaturated fatty acid peroxidation were identified and considered as the potential biomarkers of Fuzi toxicity.

Conclusion

The lipidomics approach is helpful to search potential tissue-specific biomarkers and understand the underlying mechanisms of Fuzi toxicity on the heart.     

利用UHPLC/Q-TOF MS技术进行知母中7个甾体皂苷的含量测定(英文)

本文利用UHPL C/Q-TOFMS技术建立了知母中7个主要甾体皂苷的含量测定方法,它们分别为知母皂苷N,知母皂苷E1,知母皂苷BⅡ,知母皂苷B,知母皂苷Ⅰ,知母皂苷A2和知母皂苷AⅢ。色谱柱为ACQUITY UPLC HSST3柱(2.1mm×100mm,1.8μm),流动相为乙腈–水(0.1%甲酸)梯度洗脱,分析时间为18分钟;LOQ和LOD分别为0.18–0.75ng/μL和0.05–0.22ng/μL;7个甾体皂苷分别在一定范围内呈现良好的线性关系,相关系数分别为0.9902–0.9979,且日内及日间精密度均低于5%,回收率为97.13%–101.98%。该方法快速、准确、重现性好,适用于知母药材及知母配方颗粒中7个甾体皂苷的含量测定。     

UPLC/Q-TOF-MS/MS分析蛇床子提取物大鼠体内外化学成分

目的采用超高效液相色谱与串联四级杆飞行时间质谱仪联用技术(U PLC/Q-TOF MS/MS)对蛇床子超临界提取物的体内外成分进行分析和鉴定。方法用ACQUITY UPLC BEHC18色谱柱,以0.1%甲酸水溶液(A)-乙腈(B)为流动相梯度洗脱,检测波长254 nm,使用ESI离子源,在正离子模式下采集数据;采用与标准品对比和质谱数据分析及检索文献的方法鉴定化学成分。结果检测出蛇床子超临界提取物中9个化合物,其中5个成分经过鉴定确认均为香豆素类,大鼠口服蛇床子提取物的血浆中检测出欧前胡素和蛇床子素两个原形成分。结论 UPLC/Q-TOF MS技术快速有效,具有较高的灵敏度和分辨率,适宜于鉴定中药复杂的体内外活性成分。     

蛇床子超临界提取物肝肾毒性及代谢组学的初步研究

目的 以常规毒理学指标研究蛇床子超临界提取物的毒性,比较蛇床子组、模型组和空白组的尿液代谢表型差异.方法 将SD大鼠随机分为蛇床子组、四氯化碳组和空白组,给药后收集尿液和血液,采用全自动生化仪测定血清中与肝脏、肾脏功能代谢有关的各项指标,用超高效液相色谱-四级杆飞行时间质谱(UPLC/Q-TOf MS)技术测定大鼠的尿样,利用主成分分析(PCA)对其数据进行处理.结果 与空白组比较,蛇床子组中碱性磷酸酶(ALP)活性明显下降,而谷丙转氨酶(GPT)、谷草转氨酶(GOT)、总胆红素(TBI)、血尿素氮(BUN)、血肌酐(CRE)都略有上升.与四氯化碳组比,蛇床子组中GPT、GOT、ALP都存在非常显著性差异.代谢组学分析组间表型差异明显.结论 蛇床子超临界提取物具有一定的毒性,在临床使用时应予注意.     

胰腺癌患者血清代谢组学研究

目的检测胰腺癌患者与健康者血清的代谢组学,筛选能够鉴别胰腺癌与健康者的差异代谢物,寻找潜在的胰腺癌早期诊断标志物。方法采用病例对照成组分析方法,以病理诊断为胰腺导管腺癌患者作为病例组,健康人群为对照组。应用高效液相色谱单级四级杆飞行时间串联质谱联用仪检测胰腺癌及健康者的血清代谢组学。通过单维及多维统计方法分析两组间代谢组学的差异,根据分子质量、代谢产物数据库和质谱／质谱（MS／MS）信息筛选差异代谢物。通过差异代谢物的受试者工作特征曲线下面积（AUC）评价它们鉴别胰腺癌及健康者的价值。结果共纳入胰腺导管腺癌患者及健康者各54例,年龄及性别相匹配。最终筛选出12种差异代谢物,其中胰腺癌患者血清中羟基花生四烯酸（HETE）、苯丙氨酸、乙酰肉碱、亚油酸、棕榈酰肉碱、亚油醇肉碱、尿嘧啶脱氧核苷酸（dUMP）和甘氨鹅脱氧胆酸8种代谢产物水平显著高于健康者;溶血磷脂酰胆碱[LysoPC（18：0）]、LysoPC（P-16：0）、溶血磷脂酸[LPA（182／0：0）]、LysoPC（14：0）4种代谢产物水平显著低于健康者（P值均〈0．05）。HETE、LysoPC（18：0）、苯丙氨酸、LPA（18：2／0：0）、dUMP的AUC分别为0．98、0．96、0．92、0．91,均大于CA19-9的AUC（0．90）。结论胰腺导管腺癌患者及健康者的血清代谢组共筛选出12种差异代谢物,其中HETE、LysoPC（18：0）、苯丙氨酸、LPA（18：2／0：0）、dUMP有可能是潜在的诊断标志物。     

Quadrupole-Time-of-Flight Mass Spectrometric Identification of Hemoglobin Subunits α, β, γ and δ in Unknown Peaks of High Performance Liquid Chromatography of Hemoglobin in β-Thalassemias

Abstract

This is the first report of quadrupole time-of-flight (Q-TOF) mass spectrometric identification of the hemoglobin (Hb) subunits, α, β, δ and γ peptides, derived from enzymatic-digestion of proteins in the early unknown peaks of the cation exchange chromatography of Hb. The objectives were to identify the unknown high performance liquid chromatography (HPLC) peaks in healthy subjects and in patients with β-thalassemia (β-thal). The results demonstrate the existence of pools of free globin chains in red blood cells (RBCs). The α-, β-, δ- and γ-globin peptides were identified in the unknown HPLC peaks. The quantification and role of the free globin pool in patients with β-thal requires further investigation. Identification of all types of Hb subunits in the retention time (RT) before 1?min. suggests that altered Hbs is the nature of these fast-eluting peaks. Relevancy of thalassemias to the protein-aggregation disorders will require review of the role of free globin in the pathology of the disease.     

Bioactive components on immuno-enhancement effects in the traditional Chinese medicine Shenqi Fuzheng Injection based on relevance analysis between chemical HPLC fingerprints and in vivo biological effects

Ethnopharmacological relevance

Shenqi Fuzheng Injection (SFI) is an injectable traditional Chinese herbal formula comprised of two Chinese herbs, Radix codonopsis and Radix astragali, which were commonly used to improve immune functions against chronic diseases in an integrative and holistic way in China and other East Asian countries for thousands of years.

Materials and methods

This present study was designed to explore the bioactive components on immuno-enhancement effects in SFI using the relevance analysis between chemical fingerprints and biological effects in vivo. According to a four-factor, nine-level uniform design, SFI samples were prepared with different proportions of the four portions separated from SFI via high speed counter current chromatography (HSCCC). SFI samples were assessed with high performance liquid chromatography (HPLC) for 23 identified components. For the immunosuppressed murine experiments, biological effects in vivo were evaluated on spleen index (E1), peripheral white blood cell counts (E2), bone marrow cell counts (E3), splenic lymphocyte proliferation (E4), splenic natural killer cell activity (E5), peritoneal macrophage phagocytosis (E6) and the amount of interleukin-2 (E7). Based on the hypothesis that biological effects in vivo varied with differences in components, multivariate relevance analysis, including gray relational analysis (GRA), multi-linear regression analysis (MLRA) and principal component analysis (PCA), were performed to evaluate the contribution of each identified component.

Results

The results indicated that the bioactive components of SFI on immuno-enhancement activities were calycosin-7-O-β-d-glucopyranoside (P9), isomucronulatol-7,2′-di-O-glucoside (P11), biochanin-7-glucoside (P12), 9,10-dimethoxypterocarpan-3-O-xylosylglucoside (P15) and astragaloside IV (P20), which might have positive effects on spleen index (E1), splenic lymphocyte proliferation (E4), splenic natural killer cell activity (E5), peritoneal macrophage phagocytosis (E6) and the amount of interleukin-2 (E7), while 5-hydroxymethyl-furaldehyde (P5) and lobetyolin (P13) might have negative effects on E1, E4, E5, E6 and E7. Finally, the bioactive HPLC fingerprint of SFI based on its bioactive components on immuno-enhancement effects was established for quality control of SFI.

Conclusions

In summary, this study provided a perspective to explore the bioactive components in a traditional Chinese herbal formula with a series of HPLC and animal experiments, which would be helpful to improve quality control and inspire further clinical studies of traditional Chinese medicines.