Isolation Of Chemical Constituents From Pittosporum Glabratum And Evaluation Of Anti-neuroinflammatory Activity Of Sesquiterpene Glycosides From The Genus Pittosporum

The secondary metabolites of plants are an important source of natural drugs,as well as leading compounds for various clinical drugs,which can be applied to the treatment of various diseases in the human body.With the rapid increase of the global population and the intensification of population aging,Alzheimer’s disease（AD）,as a neurodegenerative disease（ND）in the elderly,has received widespread attention in the medical community.However,due to its complex pathogenesis and diverse pathological manifestations,the exact pathogenesis is still unclear.Currently,research has considered that neuroinflammation is an important factor leading to AD.The plants of the genus Pittosporum,belonging to the family Pittosporaceae,are widely distributed in many parts of southern China.Their characteristic components are polyesterified sesquiterpenoid glycosides,which have excellent anti-neuroinflammatory activities.In this paper,the following research results have been obtained by studying the chemical constituents and the anti-neuroinflammatory activity of sesquiterpenes from the same genus of Pittosporum:（1）The ethyl acetate extraction segment of the aerial part of Pittosporum glabratum Lindl.Var.neriifolium Rehd.et Wils.was separated and purified by various separation and purification methods,including forward and reverse phase chromatography,high-performance liquid chromatography,dextran gel chromatography,and combined with nuclear magnetic resonance（NMR）,high resolution mass spectrometry（HRESIMS）,circular dichroism（CD）,various chromatographic analysis techniques such as infrared spectroscopy（IR）and ultraviolet spectroscopy（UV）have been used to identify the structure of monomer compounds,and a total of 12 compounds have been obtained,which are:4aromadendrane-type sesquiterpenoid glycosides[including a new compound（1,named Pitglabroside A）and 3 known structural analogues（2-4）],3 lignans（5-7）,1phenylpropanoid（8）,2 benzaldehyde derivatives（9-10）1 diterpene（11）and 1 triterpene（12）.（2）The neuroinflammatory cell model is BV-2 microglia induced by LPS,and the NO release of cells treated with active compounds is detected by NO detection kit.The anti-neuroinflammatory activity of 4 sesquiterpenoid glycosides（1-4）from P.glabratum Lindl.var.niifolium Rehd.et Wils.was evaluated.The results showed that the 4 compounds had no significant toxicity to BV-2 cells and had good NO release inhibitory activity,with IC₅₀values of 6.06,4.68,18.75,and 17.66μM,respectively.The structure-activity relationship analysis showed that the anti-neuroinflammatory activity of aromadendrane-type sesquiterpene glycoside was significantly stronger than that of substituted angeloyloxy when it was substituted for senecioyloxy.（3）An evaluation of the anti-neuroinflammatory activity of 20 polyesterified sesquiterpenes discovered by the research team from other plants of the genus Pittosporum in the early stage,especially eudesmane-type sesquiterpenoid glycosides,showed that 14compounds had good anti-neuroinflammatory activity(8.05μM≤IC₅₀≤41.04μM)of which 7 compounds have an IC₅₀value of 20μM,lower than the positive control drug Indomethacin(IC₅₀=20.6μM).The analysis of the structure-activity relationship showed that the change of the outer ring double bond to the inner ring double bond at the C-1position of eudesmane-type sesquiterpene glycosides was conducive to the expression of anti-neuroinflammatory activity,while the oxidation ring opening of the inner ring double bond is not conducive to the expression of this activity;the methoxy group substituted at C-4position of eudesmane-type sesquiterpene glycosides isαWhen configured,it can enhance its anti-neuroinflammatory activity,andβWhen configured,it enhances its cytotoxicity;the anti-neuroinflammatory activity of chain sesquiterpene glycosides may be greater than that of most cyclic sesquiterpene glycosides.（4）In order to explore the mechanism of the anti-neuroinflammatory activity of eudesmane-type sesquiterpenoid glycosides in the secondary metabolites of Pittosporum,compound 13(IC₅₀=8.05μM)and other similar compounds 14-16,the effects of 4compounds on the expression of inducible nitric oxide synthase（i NOS）and cyclooxygenase-2（COX-2）were studied using Western blot.The results showed that all 4compounds inhibited the expression of i NOS and COX-2 in BV-2 in a dose dependent manner,compounds 13 and 14 significantly inhibited the expression of i NOS,while compounds 15 and 16 had similar inhibitory effects on the expression of i NOS and COX-2,both of which were lower than those of 13 and 14.Through structure activity relationship analysis,it was found that changes in the configuration of eudesmane-type sesquiterpenoid glycosides C-7 and C-10 significantly affect the expression of i NOS.In addition,the presence of C-5 peroxides significantly reduces the inhibition of i NOS expression.The results of protein molecular simulation docking experiments showed that the active compound interacted with Q263 in the i NOS center through intermolecular hydrogen bonds,further verifying the anti-neuroinflammatory activity of the active compound.