| Hippophae rhamnoides L.,a plant of the genus Hippophae of the Elaeagnus family,contains rich nutrients and functional components.It has the functions of invigorating spleen and eliminating food,relieving cough and expectoration,activating blood circulation and dispersing blood stasis.It has been reported in the literature that the functional components in Hippophae rhamnoides include polysaccharides,flavones,vitamins,lipids,triterpenoids and sterols and so on.However,traditional detection technology can only detect Hippophae rhamnoides according to the reference substance of efficacy ingredient,but can not identify the effective ingredient without reference substance.Because of the high price of reference substances and the limited variety of laboratories,the current research on the effective ingredients in Hippophae rhamnoides not comprehensive enoughIn this study,a rapid,accurate and high throughput screening technique was developed by making full use of modern chromatographic and high resolution mass spectrometry techniques.The effective components of Hippophae rhamnoides fruits,leaves and stems were comprehensively identified.A non-target analysis method for pentacyclic triterpenoids and structural analogues was developed and successfully applied to the screening and identification of unknown pentacyclic triterpenoids in Hippophae rhamnoides samples.This method provides basic data for the development and utilization of Hippophae rhamnoides resources in the future,and also lays a foundation for the quality control of Hippophae rhamnoides related products and the basic research on the substance of medicinal effects.The full text is divided into five chapters,which are summarized as follows:Chapter 1 The origin and pharmacological action of Hippophae rhamnoides were introduced.Triterpenoids,as one of the main active ingredients,were defined and classified.The progress in the detection technology of the important pentacyclic triterpenoids in the triterpenoids family was summarized.Chapter 2 In order to explore the overall difference of the functional components of the fruit stem and leaf of Hippophae rhamnoides,ultra-high performance liquid chromatography coupled to quadrupole Orbitrap high resolution mass spectrometry(UPLC-Q-Exactive)combined with database of Orbitrap traditional Chinese medicine library(OTCML)used in this study.The chromatographic column is Hypersil GOLD aQ column(2.1 mm×100 mm,1.9 ?m).Meanwhile 0.1%formic acid acetonitrile(A)-0.1%formic acid water(B)gradient elution as the mobile phase,flowing at rate 0.3 mL/min.High resolution mass spectrometry with electrospray ionization(ESI)set positive and negative scanning mode at the same time.Based on the retention time,molecular ion,secondary fragment ion spectrum information and literature data of more than 1200 kinds of traditional Chinese medicine compounds contained in OTCML database,then the structure was identified.As results,102 active components were identified,including 18 glycosides,21 flavonoids,18 terpenoids,13 phenylpropanoids,6 alkaloids,14 volatile components and 12 polyphenols.Seventeen of these compounds were first found in Hippophae rhamnoides.There were obvious differences in the distribution of different parts.Chapter 3 A method for simultaneous determination of 25 pentacyclic triterpenoids in Hippophae rhamnoides by ultra-high performance liquid chromatography coupled to quadrupole Orbitrap high resolution mass spectrometry(UPLC-Q-Exactive)was established.The Hippophae rhamnoides samples were extracted by optimization and separated by Acquity UPLC Hss T3 column(2.1×50 mm,1.8 ?m).The mobile phase composition,buffer pH value and mass spectrometry parameters were optimized and discussed.The results showed that the linear relationships of 25 pentacyclic triterpenoids were good in their respective linear ranges.The correlation coefficients were more than 0.9912,the detection limit was 0.3-1.5 ng/ml,and the quantitative limit was 1.0-5.0 ng/ml.The recovery was 80.04-104.08%and the relative standard deviation was 3.01-9.35%(n=6).The method has been successfully applied to the simultaneous determination and quantitative analysis of pentacyclic triterpenoids in Hippophae rhamnoides samples from different habitats.Chapter 4 A rapid and simple method was developed to accurately screen pentacyclic triterpenoids and structural analogues,including lupane,oleanane and ursane type.Multiple neutral loss markers scanning combined with substructure recognition(MNLSR)by ultra-high performance liquid chromatography coupled to electrospray ionization quadrupole Orbitrap high resolution mass spectrometry(UHPLC/ESI Q-Orbitrap)was developed.Hippophae rhamnoides was used as an example.This approach includes three systematic workflows:(?)targeted identification of 25 reference standards to deduce mechanism of fragmentation in both positive and negative modes.(?)statistical analysis for 8 neutral loss markers and 21 structural fragment markers of pentacyclic triterpenes by fragmentation mechanism information,(?)untargeted identification using multiple neutral loss markers scanning to discover new compounds and then confirming structural characterization by structural fragment markers.Using this approach,15 new compounds were identified and confirmed from Hippophae thamnoides samples.This study extended the application of mass spectrometry to satisfy the requirements of high-throughput identification of new resources medicine and food for pentacyclic triterpenes.Chapter 5 The problems urgently needed to be solved and the future research directions of non-target screening were prospected. |
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