Discrimination of Alismatis Rhizoma based on chromatographic fingerprint,multiple components quantification,and pattern recognition analysis

A simple, sensible, and reliable HPLC–DAD method was first developed for fingerprint analysis of Alismatis Rhizoma, and then applied to analyze 85 samples from three main cultivated areas. In all, 40 common fingerprint peaks were designated, and six of which were definitely identified. Then, the combinatory analysis using similarity evaluation, principal component analysis, and orthogonal partial least square discriminant analysis revealed clear chemical consistency between samples from Fujian and Jiangxi provinces and substantial differences between those from Fujian/Jiangxi and Sichuan provinces. Furthermore, six components were dug out as potential chemical markers for distinguishing Alismatis Rhizoma from different areas, among which five were qualified for quantitative analysis. In conclusion, the combination of chemical fingerprint, multiple components quantification, and pattern recognition analysis was rather powerful and useful in discriminating Alismatis Rhizoma from different regions, which was a benefit for quality control.     

不同产地莪术挥发油的有效成分

用HP6890/5973气相色谱-质谱联用仪分析了四川、福建、浙江产的莪术挥发油的有效成分，结果表明不同产地莪术挥发油的成分差异较大，提示在选择莪术油为原粒制备制剂时要规定产地。     

Stability and structure studies on alisol a 24-acetate

Alisol A 24-acetate is one of the main active triterpenoid compounds isolated from Rhizoma Alismatis, which is a famous Traditional Chinese Medicine, and has been determined for the quality control of this crude drug. In this study, alisol A 24-acetate was found to be unstable in solvents and its stability in different solvents was investigated in detail. The results showed that alisol A 24-acetate and 23-acetate inter-transformed in solvents and the transformation rate was more rapid in protic solvents than in aprotic solvents. Moreover, both alisol A 24-acetate and 23-acetate were deacetylated to yield alisol A when kept in methanol for a long time. This is the first report on the structural transformation between alisol A 24-acetate, alisol A 23-acetate and alisol A. In addition, the single crystal X-ray structure of alisol A 24-acetate and the NMR data of alisol A 23-acetate were also reported for the first time.     

Changes in Triterpenes in Alismatis rhizoma after Processing Based on Targeted Metabolomics Using UHPLC-QTOF-MS/MS

Alismatis rhizoma (AR) has been used as an herbal medicine in China for over a thousand years. Crude AR, salt-processed AR (SAR), and bran-processed AR (BAR) are recorded in the Pharmacopoeia of the People′s Republic of China. However, the differences of chemical composition between crude AR and its processing products remain limited. In this study, triterpenes were identified from crude AR, SAR, and BAR by ultra-high performance liquid chromatography coupled with quadrupole time-of-flight-mass spectrometer (UHPLC-QTOF-MS/MS). Subsequently, the differences of triterpenes between the crude AR and processed ARs were compared via a targeted metabolomics approach. Finally, a total of 114 triterpenes were identified, of which 83, 100, and 103 triterpenes were found in crude AR, SAR, and BAR, respectively. After salt-processing, there were 17 triterpenes newly generated, 7 triterpenes with trends of increasing, and 37 triterpenes decreased. Meanwhile, 56 triterpenes including 21 newly generated and 35 with significant increases were observed in BAR. This study could be benefit to investigate the processing mechanism of AR, as well as support their clinical applications.     

Authentication of herbal medicines from multiple botanical origins with cross-validation mebabolomics,absolute quantification and support vector machine model,a case study of Rhizoma Alismatis

Accurate identification of the botanical origins of Rhizoma Alismatis (RA) is pivotal to its precise clinical usage. We herein present a strategy, by integrating untargeted metabolomics, data cross validation, absolute quantification, and vector machine model, for species discrimination and source recognition of Rhizoma Alismatis for the first time. An ultra-high-performance liquid chromatography/LTQ-Orbitrap mass spectrometry with precursor ions list-including data-dependent acquisition approach was developed for metabolite profiling. Holistic, continuous, and pattern recognition chemometrics could make it feasible to unveil forty-one markers, with “multi-duplicated and traceability samples comparison” cross validation to narrow down to twelve robust markers, some of which were further validated by absolute quantification. A support vector machine model was eventually developed to distinguish these two species and predict origins of commercially available varieties. This was the first report on systematic comparison and discrimination of two original species of RA. This integral strategy, in contrast to conventional approaches, renders more convincing data supporting for the discovery of multi-source chemical makers of traditional Chinese medicines (TCM).     

Urinary biomarker and treatment mechanism of Rhizoma Alismatis on hyperlipidemia

Rhizoma Alismatis (RA), a diuretic in Asia and Europe, was found to possess anti‐hyperlipidemic activity. Since the biomarkers and mechanisms of RA in the treatment of hyperlipidemia are inadequate, ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight synapt high‐definition mass spectrometry and multivariate data analysis were employed to investigate the urinary metabolomics of RA on hyperlipidemic rats induced by high‐fat diet. The metabolic profile of RA‐treated hyperlipidemic group located between control and diet‐induced hyperlipidemic groups. Nineteen metabolites with significant fluctuations were identified as potential biomarkers related to the hyperlipidemia and anti‐hyperlipidemia of RA using partial least‐squares‐discriminate analysis, heatmap analysis and correlation coefficient analysis. The fluctuations of these biomarkers represented disturbances in amino acid metabolism, purine metabolism, pyrimidine metabolism and energy metabolism. After RA treatment, these perturbed metabolites were restored to normal or nearly normal levels. RA can alleviate high‐fat diet‐induced dysfunctions in these metabolic pathways.     

Stimulation quantification of four natural lipase inhibitors from Alismatis Rhizoma by high-performance thin-layer chromatography method

The lipase inhibitory activities of four main components from the rhizomes of Alisma orientale (Sam.) Juz. were evaluated by an in situ high-performance thin-layer chromatography (HPTLC)‒bioautographic assay taking orlistat as control standard. The order of relative activity was alisol B 23-acetate > alisol B > alisol A > alisol C 23-acetate. With that, an accurate, efficient and sustainable HPTLC method was developed to simultaneously determine the four lipase inhibitors from the methanolic extracts of Alismatis Rhizoma (AR). The method was carried out on HPTLC glassed plates (20 × 10 cm) coated with silica gel 60 F₂₅₄ (0.2 mm thickness) using a mixture of cyclohexane and ethyl acetate (1:1, V/V) as the mobile phase. The R_F values found for alisol B 23-acetate, alisol C 23-acetate, alisol B and alisol A were 0.62, 0.42, 0.28 and 0.09, respectively. The method was validated for specificity, linear range, precision, stability, and recovery. The results determined by scanning densitometry showed no significant difference to the results obtained by HPLC. The developed method was verified to be trustworthy for the evaluation of quality markers in AR.

     

Systematic chemical profiling of a multicomponent Chinese herbal formula Huo Luo Xiao Ling Dan by ultra high performance liquid chromatography coupled with electrospray ionization quadrupoletime‐of‐flight mass spectrometry

Huo Luo Xiao Ling Dan, a Chinese herbal formula consisting of 11 different herbs, has been used in folk medicine for the treatment of arthritis and other chronic inflammatory diseases. However, the chemical compositions of Huo Luo Xiao Ling Dan are not completely characterized. In the present study, an ultra high performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight mass spectrometry method in positive and negative ion modes was employed to identify biochemical constitutes in Huo Luo Xiao Ling Dan. As a result, a total of 76 compounds including alkaloids, monoterpene glycosides, iridoids, phenolic acids, and tanshinones, coumarins, lactones, flavones, and their glycosides, triterpenes, and triterpene saponins were characterized by comparing the retention time and mass spectrometry data with reference standards within 5 ppm error or by reference to the reference literature. These results would provide the basis for a further in vivo study of Huo Luo Xiao Ling Dan and information for potential new drug candidates for treating arthritis and other chronic inflammatory diseases.     

Simultaneous determination of 19 constituents in Cimicifugae Rhizoma by HPLC–DAD and screening for antioxidants through DPPH free radical scavenging assay

Cimicifugae Rhizoma (sheng ma) is a well‐known traditional Chinese medicine, which has been demonstrated to possess anti‐inflammatory, antipyretic and analgesic activities. In the present study, a simple and efficient HPLC–DAD (high‐performance liquid chromatography coupled with diode array detection) method was developed and validated for simultaneous quantification of 19 chemical components (including 16 phenolic acids, one coumarin and two alkaloids) in Cimicifugae Rhizoma. The result indicated that this method could effectively evaluate the quality of Cimicifugae Rhizoma and provide a valuable reference for further study. Additionally, the antioxidant activity of Cimicifugae Rhizoma was evaluated by DPPH (1,1‐diphenyl‐2‐picrylhydrazyl) radical scavenging assay. The results showed that the content of phenolic acids and antioxidant activity exhibited significant correlation coefficients.     

Rapid Characterization of the major chemical constituents from Polygoni Multiflori Caulis by liquid chromatography tandem mass spectrometry and comparative analysis with Polygoni Multiflori Radix

Polygoni Multiflori Caulis is a traditional Chinese medicine that has been used for a long time to treat sleep disorders. However, the multiple chemical composition analysis has not been reported. In this study, a simple, rapid and effective ultra‐high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry method was established to characterize the components of Polygoni Multiflori Caulis. In addition, a chemical comparative analysis was performed with Polygoni Multiflori Radix, another traditional Chinese medicine from the same plant, through multivariate statistical analysis and semi‐quantitative analysis to screen the difference in chemical ingredients between these two herbal medicines from same medicinal plant. A total of 33 peaks were detected within 25 min, and 28 of them were identified or tentatively characterized by comparing the retention time and mass spectrometry data. Based on the results, 12 characteristic components were screened out by multivariate statistical analysis, and their content change trends were compared by semi‐quantitative analysis.     

Analysis of Chuanxiong Rhizoma substrate on production of ligustrazine in endophytic Bacillus subtilis by ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry

Ligustrazine was the active ingredient of the traditional Chinese medicine Chuanxiong Rhizoma. However, the content of ligustrazine is very low. We proposed a hypothesis that ligustrazine was produced by the mutual effects between endophytic Bacillus subtilis and the Ligusticum chuanxiong Hort. This study aimed to explore whether the endophytic B. subtilis LB5 could make use of Chuanxiong Rhizoma fermentation matrix to produce ligustrazine and clarify the mechanisms of action preliminarily. Ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry analysis showed the content of ligustrazine in Chuanxiong Rhizoma was below the detection limit (0.1 ng/mL), while B. subtilis LB5 produced ligustrazine at the yield of 1.0268 mg/mL in the Chuanxiong Rhizoma‐ammonium sulfate fermentation medium. In the fermented matrix, the reducing sugar had a significant reduction from 12.034 to 2.424 mg/mL, and rough protein content increased from 2.239 to 4.361 mg/mL. Acetoin, the biosynthetic precursor of ligustrazine, was generated in the Chuanxiong Rhizoma‐Ammonium sulfate (151.2 mg/mL) fermentation medium. This result showed that the endophytic bacteria B. subtilis LB5 metabolized Chuanxiong Rhizoma via secreted protein to consume the sugar in Chuanxiong Rhizoma to produce a considerable amount of ligustrazine. Collectively, our preliminary research suggested that ligustrazine was the interaction product of endophyte, but not the secondary metabolite of Chuanxiong Rhizoma itself.     

Screening epidermal growth factor receptor antagonists from Radix et Rhizoma Asari by two‐dimensional liquid chromatography

Radix et Rhizoma Asari is a traditional Chinese medicine, and has many pharmacological effects, such as calming, analgesia, anti‐inflammation, antiarrhythmic, antihypertensive, antivirus, etc. But few studies have screened the active compounds from extracts of Radix et Rhizoma Asari for tumor therapy. In this study, a two‐dimensional liquid chromatography system was built to screen active compounds acting on epidermal growth factor receptor (EGFR) from Radix et Rhizoma Asari. The screening result showed that asarinin from Radix et Rhizoma Asari was the targeted component that could act on EGFR specificity. The competitive binding assay and molecular docking assay results showed asarinin binding with EGFR in similar manner as with gefitinib, which was used as a positive control drug. Then the antitumor effect of asarinin was studied through cell growth assay in vitro. The results showed that gefitinib and asarinin could inhibit highly expressed EGFR cell growth in a dose‐dependent manner in the range of dose from 0.10 to 102.4 μM. This two‐dimensional liquid chromatography system will be a useful method in drug discovery from natural medicinal herbs for searching potential antitumor candidates.     

Rapid discovery of absorbed constituents and metabolites in rat plasma after the oral administration of Zi Shen Wan using high‐throughput UHPLC–MS with a multivariate analysis approach

Zi Shen Wan is a typical formula consisting of three herbs, Phellodendri Amurensis Cortex, Rhizoma Anemarrhenae, and Cortex Cinnamomi, and has been widely used for treating prostatitis and infection diseases. However, it lacks in‐depth research of the constituents of Zi Shen Wan in vivo and in vitro. In this work, ultra high performance liquid chromatography coupled with quadrupole‐time‐of‐flight mass spectrometry and MassLynx software was established to characterize the chemical compositions of Zi Shen Wan in vivo and in vitro. In total, 92 peaks were characterized in vitro and 33 peaks were characterized in vivo based on mass spectrometry and tandem mass spectrometry data. Among the 33 compounds characterized in rat plasma, 22 prototype components absorbed in rat serum and 11 metabolites were identified in vivo. This work was fully reports the chemical constituents of traditional Chinese formula of Zi Shen Wan, it demonstrated that ultra high performance liquid chromatography combined with quadrupole time‐of‐flight mass spectrometry coupled to MassLynx software and multivariate data processing approach could be successfully applied for rapid screening and comprehensive analysis of chemical constituents in vitro and prototype components or metabolites in vivo of traditional Chinese medicine.     

Application of pathways activity profiling to urine metabolomics for screening Qi‐tonifying biomarkers and metabolic pathways of honey‐processed Astragalus

Honey‐processed Astragalus, a widely used Qi‐tonifying and immunomodulating herb in traditional Chinese medicine, has strengthened the tonic effects and achieved fewer side effects compared with astragali radix in clinical application. Here, we focus on Qi‐tonifying biomarkers and pathways of honey‐processed Astragalus using urine metabolomics that provide the basis for building the linkage between metabolites in rat urine and its symptoms. The spleen Qi deficiency model group, normal group, astragali radix group, and honey‐processed Astragalus group were implemented to evaluate Qi‐tonifying effects. Twelve potential biomarkers were screened by multivariate statistical analysis by using ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry. Furthermore, pathways activity profiling showed unique pathways that are primarily involved in tryptophan metabolism, tricarboxylic acids cycle, and methionine metabolism. The results demonstrated that metabolomics coupled with pathway activity profiling were promising tools. It might serve as a novel methodological clue to systematically dissect the underlying Qi‐tonifying mechanism of honey‐processed Astragalus.     

泽泻三萜类成分的提取工艺优化及活性评价

建立泽泻中三萜类成分的最佳提取工艺,并考察其对老年性耳聋小鼠血清生理学指标的影响.通过正交试验优化泽泻三萜类成分的提取工艺,并用紫外分光光度法(UV)测定含量.采用D-半乳糖建立小鼠老年性耳聋模型,灌胃给予泽泻三萜类成分提取物后,测定小鼠血清丙二醛(MDA)含量和超氧化物歧化酶(SOD)活力.结果表明:泽泻三萜类成分的...     

基于气相色谱-质谱联用的代谢组学用于黄连治疗II型糖尿病的机理探索

将代谢组学的方法用于研究黄连治疗II型糖尿病的机理。II型糖尿病造模采用对大鼠灌胃脂肪乳并腹腔注射40 mg/kg链脲佐菌素的方法,大鼠分为正常组、模型组、黄连给药治疗组(10 g/kg)、二甲双胍给药治疗组(0.08 g/kg)。大鼠给药30天后,采集血样用于生化指标的检测,采集24 h尿样用于代谢组学的分析。与模型组相比,糖尿病大鼠给药黄连30天后,空腹血糖值(FBG)显著降低了59.26%,总胆固醇(TC)降低了58.66%,甘油三酯(TG)降低了42.18%。采用气相色谱-质谱联用技术(GC-MS)对大鼠尿样中的内源性物质进行了相对含量测定,主成分分析结果表明,正常组与模型组显著分离,黄连组处于正常组与模型组之间,更接近于正常组。发现12个代谢物与糖尿病有关,包括4-甲基苯酚、苯甲酸、氨基丙二酸等。给药黄连后,其中的7个代谢物发生显著性回调,与氧化应激状态相关的氨基丙二酸和L-抗坏血酸出现向正常组显著性调节的趋势。这些结果表明,黄连不仅具有降糖和降血脂的作用,而且具有抗氧化作用,在一定程度上可能会抑制糖尿病合并症的发生和发展。     

Systematic Qualitative and Quantitative Analyses of Wenxin Granule via Ultra-High Performance Liquid Chromatography Coupled with Ion Mobility Quadrupole Time-of-Flight Mass Spectrometry and Triple Quadrupole–Linear Ion Trap Mass Spectrometry

Wenxin granule (WXG) is a popular traditional Chinese medicine (TCM) preparation for the treatment of arrhythmia disease. Potent analytical technologies are needed to elucidate its chemical composition and assess the quality differences among multibatch samples. In this work, both a multicomponent characterization and quantitative assay of WXG were conducted using two liquid chromatography–mass spectrometry (LC-MS) approaches. An ultra-high performance liquid chromatography–ion mobility quadrupole time-of-flight mass spectrometry (UHPLC/IM-QTOF-MS) approach combined with intelligent peak annotation workflows was developed to characterize the multicomponents of WXG. A hybrid scan approach enabling alternative data-independent and data-dependent acquisitions was established. We characterized 205 components, including 92 ginsenosides, 53 steroidal saponins, 14 alkaloids, and 46 others. Moreover, an optimized scheduled multiple reaction monitoring (sMRM) method was elaborated, targeting 24 compounds of WXG via ultra-high performance liquid chromatography–triple quadrupole linear ion trap mass spectrometry (UHPLC/QTrap-MS), which was validated based on its selectivity, precision, stability, repeatability, linearity, sensitivity, recovery, and matrix effect. By applying this method to 27 batches of WXG samples, the content variations of multiple markers from Notoginseng Radix et Rhizoma (21) and Codonopsis Radix (3) were depicted. Conclusively, we achieved the comprehensive multicomponent characterization and holistic quality assessment of WXG by targeting the non-volatile components.     

A metabolomic study of the urine of rats with Alzheimer's disease and the efficacy of Ding‐Zhi‐Xiao‐Wan on the afflicted rats

As a well‐known traditional Chinese medicine formula, Ding‐Zhi‐Xiao‐Wan has long been used for the routine treatment of Alzheimer's disease. However, the mechanism of Ding‐Zhi‐Xiao‐Wan in treating Alzheimer's disease is unclear. Therefore, a nontargeted metabolomics method based on ultrahigh performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry has been established to explore the metabolic variations in the urine of Alzheimer's disease rats and investigate the therapeutic mechanism of Ding‐Zhi‐Xiao‐Wan on Alzheimer's disease. To develop a better rat model of Alzheimer's disease, amyloid β25‐35 was injected into the bilateral hippocampus of Sprague–Dawley rats. Multivariate analysis approaches were applied to differentiate the urine components between the four groups. Thereafter, a targeted metabolomics method was used to verify the identified endogenous metabolites and determine the mechanism of action of Ding‐Zhi‐Xiao‐Wan. Altogether, 26 potential biomarkers were found, of which 15 biomarkers (10 of which are potential biomarkers found in nontargeted metabolomics) were identified. The results show that Ding‐Zhi‐Xiao‐Wan mainly affects the pathways of taurine and hypotaurine metabolism, tryptophan metabolism, and phenylalanine metabolism. Ding‐Zhi‐Xiao‐Wan might play a role in the treatment of Alzheimer's disease by mediating antioxidative stress, regulation of energy metabolism, improvement of intestinal microbes, and protection of nerve cells.     

Rapid characterization of the chemical constituents and rat metabolites of the Wen‐Jing decoction by ultra high performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

The Wen‐Jing decoction, a traditional Chinese medicine formula, has been used as a blood‐activating and stasis‐eliminating drug to treat gynaecological syndromes, such as dysmenorrhea, amenorrhea, and menstrual disorders. However, its pharmacodynamic material basis and mechanism of action have not been thoroughly elucidated to date. The goal of this study was to characterize and identify multiple constituents and metabolites in Wen‐Jing decoction. An ultra high performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry method was established and validated in the present study for the first time. A total of 101 compounds, including 11 monoterpene glycosides, 19 flavonoids, 49 triterpene saponins, 5 phthalides, 3 phytoecdysones, and 14 others, were unambiguously or tentatively characterized by comparing their retention times and MS data with reference standards or with data reported in the literature. After oral administration of Wen‐Jing decoction, 27 compounds, including nine prototype compounds and 18 metabolites were detected in rat plasma. Thus, the ultra high performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry method was found to be efficient for in‐depth structural elucidation of chemical compounds in complex matrices of herbal medicines, which will provide useful chemical information for quality control and mechanism‐of‐action research.     

Urinary metabonomics study of Wu-tou-tang and its co-decoction with Pinelliae Rhizoma in adjuvant-induced arthritis rats

Wu-tou-tang was applied to treat rheumatoid arthritis,rheumatic arthritis,and joint pain for thousands of years.Aconiti Radix Cocta is the primary component of Wu-tou-tang,and Aconiti Radix and Pinelliae Rhizoma is one of the famous combination taboos in traditional Chinese medicine.A urinary metabonomics method based on the ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS) had been established to quantify the changes of the endogenous metabolites in the urine of adjuvant-induced arthritis rats treated by Wutou-tang and Wu-tou-tang and Pinelliae Rhizoma(WP) co-decoctions.There was a clear separation between Wu-tou-tang and WP co-decoction treated groups in the PCA model.16 potential biomarkers had been identified which correlated with how Pinelliae Rhizoma influenced the therapeutic effect made by Wu-tou-tang.Results showed that it could reduce the therapeutic effects or make some side effects when Pinelliae Rhizoma is added in Wu-tou-tang.This is the first time to use metabonomics to investigate the reason of combination taboo.