Application Of High Performance Liquid Chromatography-Mass Spectrometry In Analysis Of Polyphenols From Anoectochilus Roxburghii (Wall.) Lindl And Artichoke (Cynara Scolymus L.)

Polyphenolic compounds, including flavonoids and phenolic acids, are species of compounds that have a variety of biological activities. The biological activities of polyphenolic compounds in natural products are closely related to their molecular structure, composition and content. Identification and quantification of polyphenolic compounds in natural products can provide the theoretical basis for researching pharmacological mechanism of action. In this dissertation, the extraction methods of the total polyphenols from Anoectochilus roxburghii (wall.) Lindl and Artichoke (Cynara scolymus L.) were studied, and the high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis methods were developed for identification, quantification and comparison of polyphenolic compounds. The main results were as follows:(1) The effects of extraction solvent, concentration and extraction techniques (ultrasound and reflux) on extraction yields of flavonids from Anoectochilus roxburghii (ARL) were investigated respectively. The experiment result shows that the optimal extraction conditions were ultrasonic assisted extraction with 80% ethanol aqueous solutions. The chromatographic conditions such as the composition of mobile phase and column temperature on the separation were researched, and samples analysis were performed on a ZORBAX Eclipse XDB-C18 column by gradient elution with acetonitrile and water (containing 0.03% formic acid) as mobile phase at a flow rate of 0.5 mL/min.10 compounds were identified or tentatively characterized by online ESI/MSn and the comparison with reference compounds and literature data. The limits of detection (LODs) of five known flavonoids were in the range of 0.129-0.223 μg/mL. All calibration curves showed good linear regression (R2> 0.9995) within the testing concentration ranges. The recoveries were from 98% to 106% with RSD values less than 3.49%. By comparing the flavonoids in Yunnan wild ARL, Taiwan tissue culture ARL and Fujian tissue culture ARL, the results showed that Fujian tissue culture ARL contain lots more flavonoids than others and have potential as alternative Yunnan wild ARL. It was demonstrated that the established method was suitable for quality control of Anoectochilus roxburghii, owing to the advantages of enhanced selectivity and high sensitivity.(2) The effects of extraction solvent and concentration on extraction yields of polyphenols from Artichoke were determined individually. The optimal experiment conditions were ultrasound assisted with 80% methanol aqueous solutions. The HPLC conditions for the separation of polyphenolic compounds in Artichoke from Yunnan was researched, and the separation and analysis were carried out on a ZORBAX Eclipse XDB-C18 column by gradient elution with acetonitrile and water (containing 0.08% formic acid) as mobile phase at a flow rate of 0.5 mL/min.19 compounds were identified or tentatively characterized by online multistage mass spectrometry and the comparison with reference compounds and literature data. The calibration curves of chlorogenic acid, cynaroside and silibinin showed good linear regression (R≥20.9998) within the testing concentration ranges, and the limits of detection (LODs) were 0.153 p.g/mL,0.336μg/mL and 0.313μg/mL, respectively. The recoveries were from 98% to 102% with RSD values less than 1.60%, and the content in Artichokes sample was 29.79 mg/g,10.25 mg/g and 58.72 mg/g, respectively.