Combined Hyphenated Chromatography Techniques With Chemometric Algorithms To Research “Citrus Reticulatae Pericarpium-fructus Aurantii”

Citrus reticulatae pericarpium and Fructus aurantii are used frequently in Traditional Chinese Medicines,its often combined as drug pair.At present,the researches on Citrus reticulatae pericarpium and Fructus aurantii mainly focused on the effective components and biological activities of single herbs.There are little studies on the compatibility mechanism,pharmacodynamic substance basis and the extraction process.In this paper,taking the pair of Citrus reticulatae pericarpium and Fructus aurantii as research objects,using chromatography-mass spectrometry combined with chemometrics method to analyze the effective components and extraction process optimization:（1）In order to compare the changes of components in Citrus reticulata Blanco and citrus reticulata’chachiensis’between 3-5 years,researched the changes of flavonoids content and the components in citrus reticulata’chachiensis’（Chapter 2-3）.Due to the technical bottlenecks and non-ideal operating conditions of modern analytical instruments,the retention time drift and co-elution conditions are unavoidable,which makes it very difficult to accurately identify and quantify the components.In this paper,we proposed a modified multi-channel peak calibration based on Haar CWT combined with trilinear decomposition,which aligning the retention time shifts without changing the peak shape of data,so as to ensure the accuracy of qualitative and quantitative results of subsequent second-order correction algorithm.Using alternating trilinear decomposition（ATLD）algorithm,multiple curve resolution alternating least square（MCR-ALS）algorithm and MCPA-ATLD,the data-sets of liquid chromatography-diode array detector（LC-DAD）and gas chromatography-mass spectrometry（GC-MS）,the slight retention time shift of interfering and non-interfering components were compared and analyzed.The results showed that the qualitative and quantitative analysis of both MCPA-ATLD and MCR-ALS obtains satisfactory results,but MCPA-ATLD analysis results without uncertainty of rotation and fewer constraints,simplified the process of data processing.In this study,the MCPA-ATLD was used to analyze the contents of flavonoids,aroma,sweet and bitter components of Citrus reticulata Blanco,and the citrus reticulata’chachiensis’between 3-5 years.The results suggested that the flavonoid glycosides showed a decreasing trend in dried citrus reticulata’chachiensis’,while polymethoxy flavonoids have no obvious change trend.The study on the changes of aroma content needs reliable data sources;the lib PLS software successfully revealed the key aroma components of the citrus reticulata’chachiensis’,including sabinene hydrate,carvacrol,α-terpineol and thymol.In conclusion,trilinear decomposition and the multi-channel peak alignment were used to qualitative/quantitative analysis,which improve the accuracy of second-order data greatly,provides a reference for quality control and theoretical research on medicinal properties of citrus reticulata’chachiensis’.（2）Clarify the components of"Citrus reticulatae pericarpium-Fructus aurantii"and compound preparation is the basic step for the quality control（Chapter 4）.In this study,the gas chromatography-mass spectrometry（GC-MS）and comprehensive-two-dimensional gas chromatography-time-of-flight mass spectrometry（GC×GC-TOF/MS）were used to separate and detect the volatile components of"Citrus reticulatae pericarpium-Fructus aurantii"in 8 batches of Chaihu shugan wan（CSW）.The results showed that the main volatile components of"Citrus reticulatae pericarpium-Fructus aurantii"in different batches of CSW were D-Limonene,linalol andα-terpinol.At the same time,a total of more than 400 compounds were detected by GC×GC-TOF/MS,while only more than 100 compounds were identified by GC-MS,indicating that GC×GC-TOF/MS has higher resolution and peak capacity.After comparing the components of big honey pill（BP-CSW）and water honey pill（WP-CSW）by intelligent clustering of modulation peaks and multi-component spectral correlation chromatography（ICMP-MSCC）,the difference of volatile components could be clearly observed,provides a basis for the quality control of CSW.（3）In order to promote the comprehensive application of"Citrus reticulatae pericarpium-Fructus aurantii",the supercritical CO₂ extraction（SCE）process of polymethoxy flavonoids in"Citrus reticulatae pericarpium-Fructus aurantii"was optimized（Chapter 5）.Firstly,the Box-Behnken Design（BBD）Design method of response surface optimization（RSM）was applied to investigate the effects of different factors（temperature,pressure and time）on the extraction of polymethoxy flavonoids from SCE.Secondly,using liquid chromatography-diode array detector-mass spectrometry instrument（LC-DAD-MS）to make qualitative analysis for SCE extract ingredients.Finally,constructing the model of nobiletin content,through investigation the model stability by analysis of variance,making analysis for single/multiple factors,obtained the optimal extraction conditions.As a result,we identified 11 compounds from the SCE extraction.Response surface analysis showed that the optimal extraction conditions were as follows:extraction pressure of 200 bar,extraction temperature of55℃,extraction time of 120 min,and the content of nobiletin in the extracts was 0.941mg/g.