| Purpose: Bergamot is the dried fruit of Citrus medica L.var.sarcodactylis Swingle.,also known as “bergamot”,“five-finger orange” and “miluo orange”.In China,it has been cultivated and used for medicine and food for a long time,which are distributed mainly in Sichuan,Guangdong,Zhejiang,Fujian and other places.The 2020 edition of the Chinese Pharmacopoeia only uses a single component of hesperidin as the quality control index for bergamot,which not only lacks specificity but also cannot fully evaluate the quality of bergamot.Research on the activity of bergamot also focuses on the effects of broad categories of ingredients,and seldom involves specific active ingredients.The relationship between its ingredients and efficacy is still unclear,which not only makes it difficult to control the quality test indicators of bergamot but also affects the quality evaluation of bergamot and clinical efficacy,these factors have hindered further development of bergamot.This article aims to establish the research method of bergamot Q-markers based on the new concept of “quality markers”.By determining its efficacy Q-markers and establishing related Q-markers HPLC containing test methods,it provides a scientific basis for further quality control of bergamot.Methods:(1)UPLC-Q-Orbitrap HRMS was used to carry out comprehensive composition identification of bergamot.Perform mass spectrometry analysis based on the compound’s fitted molecular formula,accurate molecular weight,and characteristic fragment ions to clarify the chemical identity.(2)Through the application of traditional Chinese medicine serum medicinal chemistry research methods and UPLC-Q-Orbitrap HRMS technology,combined with the identification results of bergamot to screen the blood prototype components of bergamot,the purpose of clarifying the effective material basis of bergamot can be achieved,which not only clarifies the medicinal substance basis of bergamot but also lays the theoretical foundation for the optimal quality index of bergamot.(3)The results of network pharmacological analysis showed that the 6 active ingredients of bergamot(isoscopoletin,7-hydroxycoumarin,hesperidin,diosmin,5,7-dimethoxycoumarin,bergapten)may act on the 6 core targets of STAT3,AKT1,TNF,PTGS2,ESR1,and VEGFA to regulate PI3K/Akt,MAPK,NF-κ B,TNF,VEGF signaling pathways and estrogen levels,thereby participating in cell the biological processes of factor response,regulation of apoptosis,regulation of immune response,regulation of inflammatory response and other biological processes play the role of promoting angiogenesis,reducing the body’s inflammatory response,inhibiting tumor necrosis factor,improving the immune system,and enhancing the body’s defense capabilities,thereby realizing “same treatment for different diseases” effect on four diseases : chronic gastritis,functional dyspepsia,peptic ulcer and depression.isoscopoletin,7-hydroxycoumarin,hesperidin,diosmin,5,7-dimethoxycoumarin and bergapten that play a key role in “same treatment for different diseases” was initially identified as bergamot Q-markers.(4)The method of combining HPLC chromatographic fingerprint qualitative and multi-index content determination is used to evaluate the measurability of the preliminarily determined bergamot Q-markers.As a result,the main Q-markers HPLC content determination method is established,which can be used for subsequent quality Provide basis for control research.Results:(1)This article identified 83 chemical components from the bergamot alcohol extract,covering all the known component types of bergamot,including flavonoids,coumarins,organic acids,terpenes,amino acids and other compounds.Among them,16 components were accurately identified by comparison with the reference substance.(2)Through a series of tests,bergamot serum medicinal chemistry research methods have been established,including the optimal dosage of bergamot samples(12.6 g/kg),blood collection methods(retroorbital venous plexus blood collection and abdominal aortic blood collection),and most The best blood collection point(1 h),the preparation of serum samples and the pretreatment method(methanol precipitation method),this laid the method foundation for the follow-up research.By using UPLC-Q-Orbitrap HRMS technology,26 blood prototype components of bergamot were screened through serum medicinal chemistry research,mainly including flavonoids and coumarins.(3)The results of network pharmacological analysis showed that the 6 active ingredients of bergamot(isoscopoletin,7-hydroxycoumarin,hesperidin,diosmin,5,7-dimethoxycoumarin,bergapten)may act on 6 core targets including STAT3,AKT1,TNF,PTGS2,ESR1,VEGFA,and regulate PI3K/Akt,MAPK,NF-κB,TNF,VEGF signaling pathways and estrogen levels,thereby participating biological processes such as the response to cytokines,the regulation of apoptosis,the regulation of immune response,and the regulation of inflammatory response play a role in promoting angiogenesis,reducing the body’s inflammatory response,inhibiting tumor necrosis factor,improving the immune system,and enhancing the body’s defense capabilities.To achieve “same treatment for different diseases” effect on four diseases :chronic gastritis,functional dyspepsia,peptic ulcer and depression.7-hydroxycoumarin,isoscopoletin,diosmin,hesperidin,5,7-dimethoxycoumarin,bergapten were preliminary framed bergamot Q-markers.(4)Established a preliminary framed bergamot Q-markers HPLC content measurement method.The methodological results show that isoscopoletin,7-hydroxycoumarin,hesperidin,diosmin,5,7-dimethoxycoumarin,bergapten have a good linear relationship with their respective peak area integral values within the range of 0.010～0.051 mg/m L,2.472～12.360 mg/m L,0.827～4.135 mg/m L,1.060～5.300 mg/m L,2.524～12.580mg/m L,0.635～3.176 mg/m L;the average sample recovery rates are 97.48%,98.63%,100.17%,98.25%,98.12%,99.17%;RSD values are 1.09%,1.22%,2.03%,1.70%,1.25%,1.18%,respectively.Obtained measurable active ingredients with a clear mechanism of action 7-hydroxycoumarin,isoscopoltin,diosmin,hesperidin,5,7-dimethoxycoumarin and bergapten as definite bergamot Q-markers.Conclusion: This article first conducts in vitro component identification and serum medicinal chemistry studies of bergamot to further explore the material basis of bergamot’s medicinal effects,then use the method of network pharmacology to study the internal mechanism of the blood components to play the role of “same treatment for different diseases”,not only clarified the efficacy of bergamot Q-markers but also established a related Q-markers HPLC test method.The Q-markers research method embodied in this article can not only establish a quality standard for Traditional Chinese Medicine with a clear mechanism related to the efficacy of bergamot,and provide a basis for the follow-up research and development of bergamot,but also apply to the research of other Traditional Chinese Medicines with unclear material basis and unclear mechanism of action,and provide the basis for the Q-markers research of similar Chinese Medicine. |
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