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OBJECTIVE: To carry out HPTLC and HPLC fingerprint analysis of 18 batches of Ganoderma samples using two kinds of reference substance of Ganoderma extract, G. lucidum Extract Reference Substance(CZERS) and G. sinense Extract Reference Substance(ZZERS). METHODS: HPTLC Fingerprint was used to analyze triterpene acids and sterols in Ganoderma with chloroform-acetonitrile-methanol-formic acid (13∶2∶0.5∶0.5, develop 3 times) and cyclohexane-ethyl acetate-methanol-formic acid (15∶5∶0.5∶0.5, develop 2 times) respectively. HPLC Fingerprint analysis was conducted using Kromasil 100-5 C18 column (4.6 mm×250 mm, 5 μm) kept at 25 ℃. Mobile phase A was acetonitrile and B was 0.02% phosphoric acid; gradient elution procedure was as follows: 0-40 min, 29%→33% A; 40-70 min, 33%→65%A; 70-105 min, 65%→100%A; 105-120 min, 100% A; flow rate was 1.0 mL•min-1. DAD detector was adopted with detection wavelength set at 244 nm. The injection volume was 10 μL. RESULTS: By using ERS and fingerprint analysis, G. lucidum, G. sessile and G. lucidum could be distinguished. The components of G. lucidum in different species and growth patterns were different. CONCLUSION: There are many varieties of G. lucidum, which can be divided into wild and artificial cultures, and the culture media of artificial culture are different, which leads to the difference of individual components of different G. lucidum. Fingerprint analysis based on ERS of specific varieties are more suitable for the overall quality control of G. lucidum.
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Objective:To establish a method for qualitative analysis of components in Perilla frutescens leaves and stalks by liquid chromatography-mass spectrometry (LC-MS)，so as to explore the substance basis of pharmacodynamics differences between P.frutescens leaves and stalks.Method:P. frutescens leaves and stalks were extracted by 80% methanol-water ultrasound. The samples were analyzed by UPLC-Q-Exactive-Orbitrap-MS comprehensively. Halo-C18 column (2.1 mm×100 mm，2.7 μm) was used for gradient elution with 0.05% formic acid aqueous-0.05% acetonitrile formate as mobile phase in positive and negative ion modes. The flow rate was 0.3 mL·min-1，the column temperature was 40 ℃，and the injection volume was 5 μL.Result:The chemical compound in P. frutescens was deduced and identified based on the retention time of chromatography，and the exact molecular weight，excimer ion peaks，fragment ions and reference materials in Xcalibur software. The chemical composition of P. frutescens was identified by Mass Frontier 7.0 software. Totally 4 amino acids，7 phenylpropanoids，10 flavonoids，12 triterpenoids，7 organic acids，4 fatty acids，10 unknown compounds and 54 compounds were identified. Among them，6 triterpene acids, including glochidone, were identified in P. frutescens for the first time. The structures of five characteristic compounds were analyzed. There were 45 constituents in P.frutescens leaves and 32 constituents in P. frutescens stalks. They had 23 common constituents.Conclusion:LC-MS can identify the components of P. frutescens rapidly and effectively. This study provides an important theoretical basis for the quality control of different parts of P. frutescens and the development and utilization of P. frutescens.
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OBJECTIVE： To establish the method for content determination of 6 kinds of triterpene acids such as haw acid， corosolic acid， betula acid， betulonic acid， oleanolic acid and ursolic acid in Tibetan medicine Rubus biflorus. METHODS： Pre-column devrivatization HPLC-FLD-APCI/MS method was adopted. 2-（7H-dibenzo[a，g]carbazol-7-yl） ethyl-4-methylbenzene- sulfonate was used as the pre-column derivatization reagent．Hypersil C18 column was used with the mobile phase consisted of 5％ acetonitrile water solution-acetonitrile （gradient elution） at the flow rate of 1.0 mL/min. The excitation wavelength of fluorescence was 300 nm and the emission wavelength was 395 nm. The column temperature was 35 ℃， and sample size was 10 μL. Under atmospheric-pressure chemical ionization （APCI） source in positive-ion mode， pressure was 60 psi， the drying gas flow rate was 9 L/min， the dry gas temperature was 350 ℃， the gasification temperature was 450 ℃， and the capillary voltage was 3 500 V. RESULTS： The linear range of haw acid，corosolic acid，betula acid，betulonic acid，oleanolic acid and ursolic acid were 0.025-6.4 μg/mL（r≥0.999 6）. The quantitative limits were 5.11， 4.78， 4.42， 4.22， 4.29， 4.51 ng/mL； and detection limits were 1.42， 1.27， 1.30， 1.28， 1.16， 1.22 ng/mL， respectively. RSD of precision test was less than 5％， stability and repeatability tests were all less than 2％（no betulonic acid detected）. The recovery rates were 97.90％-100.55％（RSD＝1.00％，n＝6）， 97.95％-102.95％（RSD＝1.74％，n＝6）， 96.00％-101.20％（RSD＝2.00％，n＝6）， 93.25％-104.20％（RSD＝4.25％，n＝6）， 92.20％-103.30％（RSD＝3.58％，n＝6）， 97.80％-103.50％（RSD＝2.03％，n＝6）， respectively. CONCLUSIONS： The method is accurate， reliable and exclusive， and can be used for simultaneous determination of 6 kinds of triterpene acids in Tibetan medicine R. biflorus.
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An ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS) method was developed to evaluate the chemical consistency of triterpene acids in ethanol extracts of Poria and acetic ether extracts thereof. First, high resolution mass spectrometry data were obtained with Full scan mode, by comparing with MS data from the reference compounds and literatures, a total of 23 components were unequivocally or tentatively identified in ethanol extracts and acetic ether extracts thereof. Then, a mimic multiple reaction monitoring (mMRM) mode was established using UPLC-QTOF-MS/MS to quantify the triterpene acids in ethanol extracts and acetic ether extracts thereof. Eleven components were absolutely quantified with reference compounds, while 12 components without reference compounds were relatively quantified with peak areas, the transfer and enrichment rate of triterpene acids during liquid-liquid extraction were calculated. It was found all of the 23 triterpene acids identified in Poria ethanol extracts could be transferred into acetic ether extracts with high transfer and enrichment rate. The present study provides not only scientific evidence for further extraction of triterpene acids in Poria by acetic ether, but also an approach for comprehensive evaluation of the chemical consistency of herbal medicine extracts before and after the liquid-liquid extraction.
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OBJECTIVE: To establish a method for determination of six triterpene acids in solid dispersion tablets of Eriobotryae Folium total triterpenoid acids by HPLC-ELSD.
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OBJECTIVE: To study the optimum extraction condition of triterpene acids in ldregs of leaves of Eriobotrya japonica (Thunb.) Lindl. METHODS: Single factor analysis and orthogonal experimental design were used to select the concentration of ethanol (A), solid-liquid ratio (B), extraction time (C), and extraction times (D) by L9(34) orthogonal test. RESULTS: The concentration of ethanol was the main process factor, and extraction times was the secondary factor. The optimum condition was A2B1C3D1, which included using 90% ethanol as the extraction solvent with 7 times the amount of φ, and 80°C heat back for 2 times, 1.5 h for each time. CONCLUSION: Ethanol has good extraction effect for triterpene acids in loquat residue. This study provides important information for the comprehensive utilization of leaves of Eriobotrya japonica (Thunb.) Lindl. resources by pharmaceutical manufacturers.