Study On The Screening Of The Chemical Constituents Of Ilex Latifolia And Ilex Kudincha And Their Nanomaterials

Both Ilex Latifolia and Ilex kudincha are evergreen trees of the Holly family,they mainly contain pentacyclic triterpene saponins and flavonoids.The current researches on the chemical components of these two medicinal materials mainly aim at their water extracts and ethanol extracts,the activity of monomer compounds has not been reported much,especially by gene chip detection and high-throughputscreening.Ten compounds were selected from the 40 monomer compounds isolated and identified in the laboratory in the early stage,Using MCF-7 cells as model,andhigh-throughput gene chips were used to detect the transcriptome data of drug action and analyze the differential genes and their regulatory relationships.In this paper,kaempferol-3-O-?-D-glucoside?has regulation effect on breast cancer?andLatifoloside A?has regulation effect on Alzheimer's disease?are the objectes of analysis.To clarify the key genes and pathways of its effects on diseases,andpreliminary verification of the active effects of the two components through in vitro experiments;preparation of the two active components;study of the two chemistry The method of preparing nanoparticles of ingredients,using the drug packaging rate as an indicator,and using scanning electron microscopy and other methods tocharacterize the nanoparticles,laid the experimental foundation for the application of the chemical components of wintergreen and Kudingcha wintergreen in drug research.The main research contents and results are summarized as follows:1.Research on the activity screening of 10 monomer compounds in Ilex Latifolia and Ilex kudinchaTen saponins and flavonoids were selected for activity screening,which were kaempferol-3-O-?-D-glucoside?kaempferol-3-0-?-D-rutinoside?Kudinoside N?Luteolin and Quercetin in Ilex kudincha;Latifoloside A?Latifoloside I andLatifoloside D in Ilex Latifolia;chlorogenic acid and isochlorogenic acid in two plants.Screening found that Latifoloside A?200 g/mL?has regulatory effect on Alzheimer's disease,and Kaempferol-3-O-?-D-glucoside?100 g/mL?has regulatory effect onbreast cancer.Latifoside A can achieve the role of FHL2 in maintaining the balance and differentiation of adult brain glial cells by down-regulating the expression ofinterleukin 1?IL-1?and up-regulating the expression of the second member of theFHL protein subfamily?FHL2?,thereby inhibiting the glial differentiation and inhibite the Early failure of the NSC library;Latifoside A can also downregulate the colonystimulating factor 1 receptor?CSF1?to inhibit the excessive activation of Microglia in the pathology of Alzheimer's disease,thereby inhibiting the formation anddevelopment of?-amyloid plaques.In the TNF signaling pathway,Kaempferol-3-O-?-D-glucoside can up-regulate NFKBIA gene to degrade the NF-KB gene and inhibit the occurrence anddevelopment of breast cancer.Kaempferol-3-O-?-D-glucoside can also promoteapoptosis,autophagy and mitochondrial autophagy by up-regulating the expression of BNIP3L and BNIP3 proteins.2.Effect of Latifoloside A on A?_1-42-42 aging human neuroblastoma cell SH-SY5Y and Kaempferol-3-O-?-D-glucoside on human breast cancer MCF-7 cellsTo investigate the protective effect of Latifoloside A on SH-SY5Y cells aged by A?_1-42,the experiment was divided into four groups:blank control group,Latifoloside A control group,A?_1-42-42 group,Latifoloside A protection group.The MTT resultshowed that the cell survival rate of the Latifoloside A control group was notsignificantly different from that of the blank control group without the addition of Latifoloside A,indicating that Latifoloside A was not toxic to cells.Compared with the blank control group,the cell survival rate of the A?_1-42-42 group decreasedsignificantly.In the protection group of Latifoloside A[Latifoloside A?5,10,20,40?mol/L?,A?_1-42],the cell survival rate was the highest?74.838±4.272?%when the concentration of Latifoloside A was 20?mol/L.MTS method was used to investigate the inhibitory effect of Kaempferol-3-O-?-D-glucoside on the proliferation of MCF-7 cells.After 48 hours of action,it could inhibit the proliferation of breast cancer MCF-7 cells in a dose-dependent manner.Under the effect of 6.2,12.5,25,50,and 100?mol/L concentration,the inhibition rate gradually increases as the drug concentration increased,The highest inhibition rate of Kaempferol-3-O-?-D-glucoside?100?mol/L?was 89.64%±2.432%.3.Separation,preparation,purification and confirmation of chemical components of Ilex Latifolia and Ilex kudinchaLatifoloside A and Kaempferol-3-O-?-D-glucoside were prepared by extracting the n-butanol extraction part of the dried leaves of Ilex Latifoloside and the n-butanol extraction part of the dry leaves of Ilex Kudingcha,respectively.,Confirmed byspectrum and high performance liquid chromatography as Latifoloside A?purity isgreater than 98%?;obtained compound K-1?401mg?,confirmed by spectrum and high performance liquid chromatography as Kaempferol-3-O-?-D-glucoside?Purity isgreater than 98%?.Provide materials for exploring the biological activity of chemical components and nanoparticle research.4.Preparation and characterization of nanoparticles using PLGA and?-PGA-PAE carrier materialsPLGA material and amphoteric polymer material?-PGA-PAE were used to wrap Latifoloside A and Kaempferol-3-O-?-D-glucoside respectively,scanning electronmicroscopy was used to observe the nanoparticle size,and high performance liquid chromatography was used to investigate the nanoparticle package rate.The diameters of Kaempferol-3-O-?-D-glucoside and Latifoloside A PLGA are 88±3.9 nm and89±4.3nm respectively;PLGA material with Latifoloside A and Kaempferol-3-O-?-D-glucoside's parcel rates were 65.7%and 66.2%.The diameters of Latifoloside A and Kaempferol-3-O-?-D-glucoside?-PGA-PAE nanoparticles are 99±3.4 nm and98±2.9 nm,respectively;?-PGA-PAE with Latifoloside A and Kaempferol-3-O-?-D-glucoside's parcel rates were 67.3%and 68.2%,respectively.After replacing thecentrifugation method with dialysis,the encapsulation rate of Latifoloside A andKaempferol-3-O-?-D-glucoside nanoparticles had increased,which were 71.3%and71.8%,respectively.