Study On The Pharmacodynamic Material Basis Of The Seville Orange Flower In The Treatment Of Alzheimer's Disease

Objective:The pharmacological effects of ethanol-eluting parts of fresh seville o range flower and those eluted by AB-8 macroporous resin on SDAD were stud ied by measuring Morris water maze and physiological and biochemical indexe s in Sleep Disorder Alzheimer's Disease?SDAD?model rats.At the same time,the chemical constituents of seville orange flower were analyzed and identified by ultra-high performance liquid chromatography-quadrupole-time of flight mass spectrometry?UPLC-Q-TOF-MS?.The effective fractions of 70%ethanol were separated,purified and identified by various column chromatography methods a nd spectroscopy techniques,which provided the basis for the study of the phar macodynamic substances of seville orange flower for the treatment of Alzheime r's disease.Methods:1.Low-temperature vacuum freeze-drying method and ethanol impregnation method were used to obtain fresh seville orange flower buds and ethanol impregnate.The latter concentrated extract was eluted by AB-8 macroporous resin to obtain 30%ethanol elution part and 70%ethanol elution part.Sleep disorder induced by sleep deprivation and one-time intraventricular injection of A?amyloid protein were used to make model rats of Sleep Disorder Alzheimer's Disease?SDAD?.The pharmacological activities of different medication sites were studied through the determination of Morris water maze and physiological and biochemical indicators.2.Ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry?UPLC-Q-TOF-MS?was used to establish a XIC database of known chemical constituents in the seville orange flower,combined with the XIC Manager function of Peakview software.According to the determination of relative molecular weight and the theoretical accuracy of relative molecular weight,the molecular formulas of the compounds corresponding to each chromatographic peak were determined,which combined with the first and second order mass spectra of the related compounds,as well as isotope distribution and the law of compound splitting.Compared with the reference material and literature,the chemical constituents of 95%ethanol refluxing extracted fresh seville orange flower samples were identified.3.Silica gel column chromatography,polyamide resin,ODS-C₁₈8 column chromat ography and high performance liquid chromatography were used to separate,pu rify and enrich the effective elution fraction of 70%ethanol water from seville orange flower.The above monomer compounds were analyzed and identified by UV,IR,NMR and MS.Result:1.Compared with SDAD model group,seville orange flower can improve the learning and memory ability of the model rats,improve the mental state of the rats,increase the speed of body weight,increase the level of superoxide dismutase in serum,decrease the contents of acetylcholinesterase,malondialdehyde,glutamic acid,alanine aminotransferase,glutamic oxalase,NO,Ca²⁺and show good resistance.Oxidative damage and neuroprotective effect.2.By using the function of Formula finder in Peakview software,combining the first and second mass spectra of related compounds,isotope distribution and decomposition rules of compounds,and comparing with reference materials and literature,31 compounds with flavonoids,triterpenoids,coumarins and organic acids were identified and inferred.3.The effective elution fraction of 70%ethanol from seville orange flower wa s separated,purified and identified by column chromatography and NMR spectr oscopy.Twenty-three monomer compounds were identified,twelve of them wer e dihydroflavonoids:theaflavanoside??1?,2?s?-theaflavanoside??2?,theaflavano side??3?,Hesperidin?4?,neohesperidin?5?,Naringenin-7-O-?-D-gentiobioside?6?;2?s?-3?,4?-dimethoxy-5,7-dihydroxyflavanone-7-O-neohesperidoside?7?,poncirin?8?,6,8-dimethyl-3'-methoxy-5'-hydroxy-naringenin-7-O-?-D-glucoside?9?,2?s?-3?-methoxy-5,7,4?-trihydroxy flavanone-7-O-neohesperidoside?10?,Narirutin?11?,nar ingin?12?;Three flavonols-3-O-glycosides:Kaempferol-3-O-?-D-rutinoside?13?,Is orhamnetin-3-O-?-D-rutinoside?14?,4'-methoxy-isoquercitrin?15?;Two other flavo noids:Isosinensetin?16?,6,7,8,3?4?-pentamethoxyflavone?17?;One phenylpropanoi d compound:Methyl ferulate?18?;One Triterpenoid:Limonin?19?;One Sesquit erpene Compound:Corchoionol C?20?;Three other compounds:1-phenyl-1,2-et hanediol?21?,5,7-dihydroxychromone?22?,3-hydroxymethyl furanoyl rhamnoside?23?.Conclusion:1.seville orange flower can significantly improve the mental state of animals,the speed of weight gain tends to be normal,the level of T-SOD in serum increases,the content of AchE and MDA decreases,the content of glutamic acid,alanine aminotransferase,glutamic oxaloacetate aminotransferase,NO and Ca²⁺decreases.It shows that seville orange flower can effectively scavenge oxygen free radicals,improve the body's antioxidant and neuroprotective ability,and improve the ability of the body.Learning and memory function of SDAD model rats.Experiments have proved that seville orange flower can effectively scavenge oxygen free radicals,improve memory impairment and sleep disorders,play a good role in the prevention and treatment of AD,and provide a theoretical basis for seville orange flower in clinical treatment of Alzheimer's disease.2.Ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry?UPLC-Q-TOF-MS?,as a new analytical technology,has high resolution,high sensitivity,qualitative ability and strong structural characterization ability.It can quickly and accurately detect the chemical constituents in seville orange flower,and evaluate the quality of medicinal materials more effectively and truly.3.The effective elution sites of 70%ethanol water were mainly flavonoids in the form of glycosides and some aglycones.Twenty-three monomer compounds were isolated,of which eight compounds were first found in Citrus genus:Nar ingenin-7-O-?-D-gentiobioside?6?,6,8-dimethyl-3'-methoxy-5'-hydroxy-naringenin-7-O-?-D-glucoside?9?,2?s?-3?-methoxy-5,7,4?-trihydroxy flavanone-7-O-neohesperi doside?10?,4'-methoxy-isoquercitrin?15?,Methyl ferulate?18?,Corchoionol C?20?,1-phenyl-1,2-ethanediol?21?,3-hydroxymethyl furanoyl rhamnoside?23?;13 comp ounds were isolated for the first time in this plant:theaflavanoside II?1?,2?s?-theavanoside IV?2?,theaflavanoside III?3?,2?s?-3?,4?-dimethoxy-5,7-dihydroxy flavanone-7-O-neohesperidoside?7?,6,8-dimethyl-3'-methoxy-5'-hydroxy-5'-hydroxy.-Naringenin-7-O-betbeta-D-pyranranosioside?9?,2?s?-3?-methoxy-5,7,4?-trihydrox y flavanone-7-O-neohesperidoside?10?,Kaempferol-3-O-?-D-rutinoside?13?,Isorha mnetin-3-O-?-D-rutinoside?14?,4'-methoxy-isoquercitrin?15?,ferulic methyl ester?18?,Methyl ferulate?18?,Corchoionol C?20?,1-phenyl-1,2-ethanediol?21?,3-hy droxymethyl furanoyl rhamnoside?23?;18 were isolated for the first time from generations for the first time:theaflavanoside II?1?,2?s?-theavanoside IV?2?,theaflavanoside III?3?,Naringenin-7-O-?-D-gentiobioside?6?;2?s?-3?,4?-dimetho xy-5,7-dihydroxyflavanone-7-O-neohesperidoside?7?,poncirin?8?,6,8-dimethyl-3'-m ethoxy-5'-hydroxy-naringenin-7-O-?-D-glucoside?9?,2?s?-3?-methoxy-5,7,4?-trihydr oxy flavanone-7-O-neohesperidoside?10?,Narirutin?11?,Kaempferol-3-O-?-D-ruti noside?13?,Isorhamnetin-3-O-?-D-rutinoside?14?,4'-methoxy-isoquercitrin?15?,Me thyl ferulate?18?,Limonin?19?,Corchoionol C?20?,1-phenyl-1,2-ethanediol?21?,5,7-dihydroxychromone?22?,3-hydroxymethyl furanoyl rhamnoside?23?.The abo ve studiespreliminarily clarify the material basis of pharmacodynamics of seville orange flower in the treatment of Alzheimer's disease,and provide a solid the oretical basis and scientific significance for further development of medicinal re sources of seville orange flower.