| Fructus Aurantii is a kind of dried and immature fruit belonging to its Citrus aurantium L.and cultivated variety,which has the effect of regulating qi,removing stagnation and bloating,mainly treat chest hypochondriac Qi stagnation,swelling pain,food accumulation cannot digest and other symptoms.Because of the strong dryness of crude products,clinical often used after processing.Jiangxi is a genuine producing area of Fructus Aurantii,and the processing technology of Fructus Aurantii has local characteristics.Common Chinese herbal pieces of Aurantii Fructus except crude and stir-fried with bran contained in “Chinese Pharmacopoeia”2015 Edition,there still have stir-fried pieces with chaff contained in the 2008 edition of the Processing Specification of Jiangxi Traditional Chinese Medicine Pieces,“ZhangBang” stir-fried with honey bran and "Jianchang-Bang” featured pieces of stir-fried with chaff.Traditional Chinese Medicine Theory holds that Aurantii Fructus has the function of “reducing dryness and increasing efficiency” after bran and chaff processing,but there is no report on the processing mechanism of Aurantii Fructus pieces with Jiangxi characteristics.The elaboration of the processing mechanism of stir-fried Aurantii Fructus with bran in Pharmacopoeia is mostly at the level of chemical composition and pharmacological effect,but the research on the key link of the organic relationship between the two aspects is not deep enough,and lacks the necessary biological function verification to the pharmacological effect,it is difficult to fully and systematically explain.In order to solve these problems,we have carried out the following research in this thesis.1.ObjectiveThe influence of different processing methods on the changes of chemical constituents in Aurantii Fructus is studied by modern component analysis and detection technology,and screening the Chinese herbal pieces of Aurantii Fructus.Using pharmacological and molecular biology techniques,the material basis and mechanism of the effects of Aurantii Fructus on “dryness” and “relieving epigastric distention and flatulence” are clarified.On this basis,the relationship between ingredient changes and pharmacodynamics differences before and after processing of Aurantii Fructus is discussed in order to explain the processing mechanism of the dominant varieties of Aurantii Fructus.2.Method 2.1 Study on the Material Basis of Different Processed Products of Aurantii Fructus with Jiangxi CharacteristicsThe processed products of Aurantii Fructus with Jiangxi characteristics were used as the research object,the crude products of Aurantii Fructus and stir-fried Aurantii Fructus with bran in Pharmacopoeia are used as control.The contents of quality markers(Q-Marker)hesperidin,neohesperidin,naringin and synephrine in different processed products of Aurantii Fructus are determined by high performance liquid chromatography.UHPLC-Q-TOF/MS chromatography combined with Peakview software is used to identify the chemical constituents of alcohol extract and to explore the effects of different processing methods on the chemical constituents;Volatile oil and aromatic water are extracted by steam distillation,is and the volatile components and relative contents of volatile oil and aromatic water are detected by GC-MS.The difference in chemical constituents of volatile oil and aromatic water of Aurantii Fructus and the effects of different processing methods on volatile components are compared with principal component analysis.In addition,the content of Q-Marker components and the chromatographic peak area of chemical markers are used as indicators,the multi-index weighted scoring method is used to weight the different processed products,and the dominant pieces of Aurantii Fructus are screened through comprehensive analysis.2.2 Study on "Dryness" of Aurantii Fructus and the Mechanism of Reducing Dryness by Gran ProcessingTo establish pathological models of slow transit constipation(STC)and gastric mucosal injury in rats.Taking the body mass,constipation index,water consumption,urination volume,renal aquaporin 3(AQP3)content and the ratio of serum Cyclic Adenosine monophosphate(cAMP)to serum Cyclic Guanosine monophosphate(cGMP)as evaluation indexes,effect of crude and processed products of Aurantii Fructus with different doses on the water metabolism of healthy rats is observed.The gastric mucosal injury index,contents of serum tumor necrosis factor-?(TNF-?),interleukin-6(IL-6)and interleukin-8(IL-8)levels are used as evaluation indexes to observe the effects of each drug group on gastric mucosal injury in healthy rats.Real-time fluorescence quantitative polymerase chain reaction amplifier(RT-PCR)is used to detect the expression of c-kit and SCF mRNA in gastric antrum and colon tissues of rats in each group.2.3 Study on relieving epigastric distention and flatulence of Aurantii Fructus and Synergistic Mechanism of Gran ProcessingEstablish a pathological model of functional dyspepsia(FD)in rats.Taking gastric remnant rate,intestinal propulsive rate and contents of serum motilin(MTL),vasoactive intestinal peptide(VIP),calcitonin gene-related peptide(CGRP)as evaluation indexes,effect of crude and processed products of Aurantii Fructus with different doses on gastrointestinal function of functional dyspepsia rats is observed.Western blot and Immunohistochemistry(IHC)are used to detect the levels of gastrin(GAS)and somatostatin(SS)protein expression and the distribution of positive cells in hypothalamus and antrum respectively.2.4 Study on Processing Mechanism of Stir-fried Aurantii Fructus with Honey Bran Based on Material Base-Target-Signaling PathwaySTC and FD are selected as the disease models of "dryness" and "relieving epigastric distention and flatulence" effects of Aurantii Fructus.The C-kit/SCF signaling pathway-related SCF,c-kit,PLC,RyR,and IP3 R proteins are selected as STC disease target proteins,and GAS and SS are selected as target proteins for FD diseases.Using network pharmacology correlation analysis methods,through active molecular screening,target prediction,molecular docking,network analysis and pathway analysis,the chemical constituents of crude and processed products of Aurantii Fructus are molecularly docked with related target proteins.Establish a “material basis-target-signal pathway” network to explore the material basis and processing mechanism of “reducing dryness and increasing efficiency ”of stir-fried Aurantii Fructus with honey bran.3.Result 3.1 Study on the material basis of different processed products of Aurantii Fructus with Jiangxi characteristicsAurantii Fructus can increase the content of hesperidin and decrease the content of naringin and synephrine in different degrees after processing.However,the content of neohesperidin did not show a homogeneous trend after processing.Stir-fried with honey bran could increase the content of neohesperidin in Aurantii Fructus,while stir-fried with bran,honey bran and bran could reduce the content of neohesperidin in varying degrees.The contents of hesperidin,neohesperidin,naringin and synephrine were taken as the indexes for comprehensive weighting score.It was found that stir-fried Aurantii Fructus with honey bran had the highest comprehensive score in processed products of Aurantii Fructus.In the ethanol extract of different processed products of Aurantii Fructus,a total of 55 chemical constituents were identified.By the PLS-DA analysis found that there were 14 chemical markers with significant differences between raw and processed products of Aurantii Fructus.The peak area of chemical markers was used as an index for comprehensive weighted scoring;it was found that stir-fried Aurantii Fructus with honey bran had the highest comprehensive score.To detect the volatile components of Aurantii Fructus and found that D-limonene and gamma-Terpene were the main volatile components in Aurantii Fructus,and stir-fried Aurantii Fructus with honey bran had the highest content of these components.There is a significant difference between volatile oils and aromatic fragrances.DLimonene,gamma-Terpene,Linalool,(1S)-(-)-?-Pinene,(1S)-(-)-?-Pinene are chemical markers of volatile oil and fragrance components;The volatile components were affected by different processing methods.The gamma-Terpene,2-Furanmethanol,5-ethenyltetrahydro-.alpha.,.alpha.,5-trimethyl-,cis-andBenzene,1-methyl-4-(1-methylethyl)-were the main components before and after processing.3.2 Study on the "dryness" of Aurantii Fructus and the mechanism of reducing dryness by stir-fried with bran.In the aspect of water metabolism,the same dosage group compared with the blank group,the order of difference was raw Aurantii Fructus > stir-fried Aurantii Fructus with bran>stir-fried Aurantii Fructus with honey bran,and high dose group > low dose group;In the aspect of gastric mucosal injury,there was no significant difference between different doses of raw Aurantii Fructus,stir-fried Aurantii Fructus with bran,stir-fried Aurantii Fructus with honey bran and blank group.when contrasted with blank controlled group,the expression of c-kit and SCF mRNA in antrum and colon of raw Aurantii Fructus was significantly decreased(P<0.01);Compared with the model group,there was no significant difference in all the indicators in the high-dose group of raw Aurantii Fructus;group of stir-fried Aurantii Fructus with bran and group of stir-fried Aurantii Fructus with honey bran C-kit and SCF mRNA expressions in the gastric sinus and colon were significantly increased(P<0.05,P<0.01).3.3 Study on the “relieving epigastric distention and flatulence” of Aurantii Fructus and the mechanism of synergism by stir-fried with bran.In the aspect of promoting gastrointestinal motility,the same dose group was compared with the model group,the order of difference was stir-fried Aurantii Fructus with honey bran> stir-fried Aurantii Fructus with bran > raw Aurantii Fructus,and low dose group> high dose group.Both Western blot and IHC results showed that GAS expression in hypothalamus and gastric antrum of the model group was significantly lower than that of the blank group(P<0.01),the expression of SS was significantly higher than that of blank group(P<0.01);The expression of GAS and SS in hypothalamus and gastric antrum of each dosage group was down-regulated to some extent compared with model group.Compared with the model group,there was no significant difference in the expression of GAS and SS in hypothalamus between the domperidone group and the model group(P>0.05),the expression of GAS and SS in gastric antrum was significantly up-regulated and down-regulated(P<0.01).In the processed products of Aurantii Fructus,the effect of stir-fried Aurantii Fructus with honey bran on GAS and SS in hypothalamus and antrum of FD rats was the best,and the effect of low dose group was better than that of high dose group.3.4 Study on processing mechanism of stir-fried Aurantii Fructus with honey bran based on the association of “Physical basis-target-signaling pathway”Molecular docking results show that naringenin,Scutellarein-4',7-dimethyl ether,5-Hydroxy-6,7,8,4'-tetraMethoxyflavone,Bicyclo[3.1.0]hex-2-ene,2-methyl-5-(1-methylethyl)-,3-Dodecyne,D-Limonene,Benzene1-methyl-3-(1-methylethyl)-and(-)-Linalool in Fructus Aurantii were dry constituents.In addition,eriodictyol-7-glucoside,naringenin,limonin,nomilinicacid,nobiletin,nomilinic,synephrine,5-Hydroxy-6,7,8,4'-tetraMethoxyflavone,neoponcirin,Caffeic acid,1R-.alpha.-Pinene,3-Dodecyne,Limonene,Benzene1-methyl-3-(1-methylethyl)-,alpha.-Cadinol and(-)-Linalool are active ingredients of Fructus Aurantii.Compared with raw Fructus Aurantii,56.25% of the active ingredients in stir-fried Aurantii Fructus with honey bran increased,while 75% of the dry ingredients decreased.4.ConclusionAfter processing,the chemical composition and content of Fructus Aurantii have changed,and the change is closely related to the processing method,the material basis of stir-fried Aurantii Fructus with honey bran as the characteristics of "Zhangbang" is the best.Therefore,stir-fried Aurantii Fructus with honey bran is selected as the best processed product for subsequent research.Pharmacodynamics studies have found that the dryness of Aurantii Fructus is mainly manifested in the damage of body fluid,and the intensity is related to the dose.No obvious direct stimulation is found in gastric mucosa of healthy rats.Both stir-fried Aurantii Fructus with bran and stir-fried Aurantii Fructus with honey bran can effectively alleviate the dryness effect of crude,and stir-fried Aurantii Fructus with honey bran is better than stir-fried Aurantii Fructus with bran.Both crude Aurantii Fructus and stir-fried with bran products can promote the recovery of gastrointestinal function in FD rats,and the best effect to promote gastric motility is stir-fried with honey bran.Analyzing the processing mechanism of "reducing drying and increasing efficiency" of stir-fried Aurantii Fructus with honey bran.It is believed that eight kinds of dry ingredients such as naringin can act on SCF?c-kit?PLC?RyR?IP3R proteins in normal rats gastrointestinal tract and destroy SCF/c-kit signaling pathway.Then affect the number and function of interstitial cells of the gastrointestinal tract Cajal,resulting in dry signs such as dry mouth,oliguria,constipation.However,stir-fried Aurantii Fructus with honey bran can reduce the content of dry ingredients of 3/4,which can reduce the influence of c-kit and SCF protein expression to a certain extent,and achieve the purpose of alleviating dryness.Then,more than half of the active ingredients increased after stir-fried with honey bran,which may be the material basis of stir-fried Aurantii Fructus with honey bran to enhance the effect of " relieving epigastric distention and flatulence".16 kinds of medicinal ingredients such as eriodictyol-7-glucoside can affect the expression of GAS protein and SS protein.The increase of active ingredients can enhance the effect of drugs on regulating the disordered secretion of GAS and SS in peripheral gastrointestinal hormones of FD rats and enhance the effect of drugs. |
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