Studies On The Chemical Constituents And Biological Activities Of Fructus Sophorae And Nelumbinis Plumula

The thesis focused on the chemical constituents and biological activities study of Fructus Sophorae and Nelumbinis Plumula.Identification of 131 constituents in Fructus Sophorae and 100 constituents in Nelumbinis Plumula were performed using liquid chromatography combined mass spectrometry.It was confirmed that the main chemical composition in Fructus Sophorae and Nelumbinis Plumula were flavonoids and alkaloids,respectively.The total flavonoids in Fructus Sophorae and the total alkaloids in Nelumbinis Plumula were prepared respectively.Furthermore,the anti-osteoporosis effect of flavonoids and the lipid regulating biological activity were screened respectively.Moreover,the research on flavonoids in Fructus Sophorae was systematically conducted,and 51 monomer compounds were isolated and identified.Sophorabioside,an isoflavonoid with high content,good solubility and strong anti-osteoporosis activity,was evaluated for its safety and main pharmacokinetic properties.The following innovative results were obtained.1.For the first time,based on UPLC-Q/TOF-MS technology combined with UNIFI new natural product platform,the chemical constituents were analyzed and identified.A total of 131 constituents were identified in Fructus Sophorae,including 81flavonoids?19 isoflavones,37 flavonols,19 flavonoids,3 dihydroisoflavones,2dihydroflavonols and 2 anthocyanins?,18 triterpenoids,7 glycosides,5 steroids,2carbohydrates,2 anthraquinones and 15 other types compounds.And the flavonoids were the main constituents.A total of 100 constituents were identified in Nelumbinis Plumula,including 35 alkaloids,34 flavonoids,4 sugars,5 glycosides,3 steroids,1nucleotide,18 other types compounds.And the alkaloids and flavonoids were the main constituents.2.For the first time,total flavonoids and total alkaloids with high content were extracted and prepared.The extraction process of flavonoids in Fructus Sophorae was optimized with the response surface method.The preparation conditions were as follows:70%ethanol,extraction temperature was 70°C,extraction time was 3.5 h,solid-to-liquid ratio was1:8?g/m L?.The extraction and macroporous adsorption resin were used to get flavonoids with content more than 70%.In the other hand,the extraction process of alkaloids in Nelumbinis Plumula was established.After extracted by n-hexane,the pH value was adjusted to 3 in order to remove acid-insoluble impurities,the pH value was then adjusted to 9-10,and the solution was extracted by dichloromethane repeatedly.After repeated recrystallization with ethanol-water,the alkaloids were obtained with the content more than 80%.3.The hypolipidemic effect of alkaloids in Nelumbinis Plumula were screened for the first time.The results indicated that the alkaloids could reduce triglyceride?TG?,cholesterol total?CT?and low-density lipoprotein cholesterol?LDL-cho?significantly,and could increase the level of HDL-cholesterol?HDL-cho?significantly.4.A total of 51 compounds were separated and identified from Fructus Sophorae.These compounds are:Genistein?1?,Genistin?2?,Sophoricoside?3?,Sophorabioside?4?,Genistein-4',7-diglucoside?5?,BiochaninA?6?,Sissotrin?7?,Oroboside?8?,Pratensein?9?,Prunetin?10?,Tectorigenin?11?,Tectoridin?12?,Formononetin?13?,Ononin?14?,Daidzein?15?,Daidzin?16?,Puerarin?17?,3'-Methylorobol?18?,Dehydroferreirin?19?,Cajanin?20?,Kaempferol?21?,Sophoraflavonoloside?22?,Nicotiflorin?23?,Kaempferol-3-O-?2?-O-?-D-glucosyl?-?-D-rutinoside?24?,Kaempferol-3-O-?-D-sophorosid-7-O-?-L-rhamnoside?25?,?-Rhamnoisorobin?26?,Populnin?27?,Astragalin?28?,Quercetin?29?,Quercetin-3-O-?-D-sophoroside?30?,Rutin?31?,Quercitrin?32?,Isoquercitrin?33?,Isorhamnetin?34?,Hyperoside?35?,Myricetin?36?,Myricitrin?37?,Choerospondin?38?,Naringenin?39?,Naringin?40?,Luteolin?41?,Genkwanin?42?,Apigenin?43?,Chrysin?44?,Baicalin?45?,Diosmetin?46?,Thevetiaflavone?47?,Gallic acid?48?,Caffeic acid?49?,Ethyl caffeate?50?,Daucosterin?51?.Among them,compounds 6,7,9,10,14,18-20,23,27,30,32,33,35-51 was separated from Fructus Sophorae for the first time,compounds 18,19,27,30,35,36,38,40,42,47,50 was separated from the genus sophora for the first time.5.The anti-osteoporosis effect and underlying mechanism of the flavonoids in Fructus Sophorae were discussed for the first time by using Molecular pharmacology and molecular docking technique.The influence of 12 componds including compound1,2,3,4,12,21,22,23,31,38,47 on the proliferation,differentiation and mineralization effects of osteoblasts MC3T3-E1 were evaluated by MTT assay combined with ALP and ARS staining.The results showed that seven compounds such as genistein,sophoricoside,sophorabioside,tectoridin,kaempferol,sophoraflavonoloside and rutin had the obvious anti-osteoporosis effect.In the other hand,in case of inhibition of estrogen receptors,the effect of proliferation and differentiation of osteoblasts of the above active compounds would be significantly reduced.In the molecular docking study,stable hydrogen bonds were observed between the effective compounds and the estrogen receptor??ER??.The good space and electrical complementarity provided the further validation for the underlying mechanism.In a word,sophorabioside and other compounds may act as anti-osteoporosis agents by binding to estrogen receptors to produce estrogen-like effects.6.The studies on the anti-osteoporosis effect of sophorabioside were carried on for the first time.The results showed that sophorabioside has a significant inhibitory effect on weight gain in ovariectomized model rats,and could elevated serum Ca,Mg,P content,increased BMD content,and bone load capacity of model rats.7.For the first time,the acute toxicity in mice and the main pharmacokinetics of sophorabioside were evaluated.The maximum tolerated dose in mice admininstrated with sophorabioside is 2.0 g/kg.The compound has linear kinetic characteristics in rats:C_max and AUC are linear with the dosage,the peak time(T_max)is about 6 h,the elimination half-life(T_1/2)is about 8 h,the elimination rate?CL?is approximately 8.7L/h/kg.It was suggested that it would take longer time to distribute and would eliminate more slowly.The absolute bioavailability is about 19%,plasma protein binding rate is 50.7%.The main metabolic pathway in vivo was deglycosylation resulting in forming monosaccharide glycosides or aglycones.Then I-phase and II-phase metabolism such as deglycosylation,dediglycosylation,methylation,hydrogenation,sulfation,phosphorylation,acetylation,and amino acid formation occurred.In summary,the systematic study on the chemical composition and biological activity of Fructus Sophorae and Nelumbinis Plumula were performed,which will provide scientific data and theoretical basis for clarifying the chemical composition and expanding medicinal application.