Screening And Preparation Of Anti-Alzheimer’s Disease Active Ingredients From Phellinus Igniarius And Astragalus Membranaceus Flowers,Stems And Leaves

Phellinus igniarius（P.igniarius）and Astragalus membranaceus（A.membranaceus）flowers,stems and leaves are widely used herbal medicines,which have been proved to have neuromodulatory,anti-inflammatory and antioxidant functions.At present,domestic and foreign research is mainly focused on anti-tumour functions,with relatively little research content on their active ingredients for the treatment of neurological disorders.According to the literature,P.igniarius,A.membranaceus flowers,stems and leaves have been reported to be more effective in health care and in reducing nerve damage.Alzheimer’s disease（AD）is currently the most common chronic neurodegenerative disease.Some studies have shown that extracts of P.igniarius,A.membranaceus flowers,stems and leaves have anti-AD effects,but the complexity of the components and the limited research on the relevant targets have shackled further investigation.In this paper,taking the P.igniarius and A.membranaceus flowers,stems and leaves as the research objects,and the active components and targets acting on AD were clearly investigated,the active components are screened step by step under the overall framework,and their active components are extracted,separated and purified by using mass spectrometry,chromatography and response surface technology.The potential anti-AD active components are screened by using multi-target and multi-angle combination of network pharmacology,molecular docking and molecular dynamics simulation and other molecular levels.A combination method for rapidly screening potential enzyme inhibitors from crude extracts of medicinal plants has been established.The specific contents are as follows:First,using 5-lipoxygenase（5-LOX）and acetylcholinesterase（ACh E）as target proteins,20 potential enzyme inhibitors in P.igniarius and A.membranaceus flowers,stems and leaves extracts were rapidly screened and identified using affinity ultrafiltration liquid chromatography mass spectrometry（AUF-LC-MS）technique.The active ingredients in P.igniarius and A.membranaceus flowers,stems and leaves extracts were screened by network pharmacology,and 16 of them had dual-target inhibitory ability;the active ingredients interacting with AD target genes were screened from the pairs using network pharmacology;the optimal extraction process of the active ingredients in P.igniarius and A.membranaceus flowers,stems and leaves was optimized by response surface methodology;the results of the activity screening were used as a guide for the separation and purification of the active ingredients in the extracts of P.igniarius and A.membranaceus flowers,stems and leaves by a combination of Semi-preparative HPLC and high-speed countercurrent chromatography（HSCCC）.The results showed that a total of 15 active ingredients were isolated by Semi-preparative HPLC,namely phellibaumin E,osmundacetone,genistin,9,10-dimethoxy-pterocarpane-3-O-β-D-glucoside,calycosin-7-glucoside and hyperoside（isolated from A.membranaceus flowers）,rhamnocitrin,wogonin,ononin,isoquercitrin,calycosin-7-glucoside and hyperoside（isolated from A.membranaceus stemsandleaves）,3-hydroxy-9,10-dimethoxyptercarpan,7,2′-dihydroxy-3′,4′-dimethoxyisoflavan,and baicalein with purity of 98.86%,99.51%,98.85%,99.37%,98.63%,98.55%,98.55 98.31%,98.53%,96.72%,96.11%,95.29%,98.25%,97.91%,95.46%and 95.23%,respectively.A total of five active ingredients were isolated by HSCCC,namely protocatechuic aldehyde,isorhamnetin-3-O-rutinoside,calycosin（isolated from A.membranaceus flowers）,calycosin（isolated from A.membranaceus stems and leaves）and soyasaponin,with purity of 98.89%,98.22%,98.45%,95.18%and 95.57%,respectively.Secondly,small molecules with strong dual-target inhibition were selected and their inhibitory ability against 5-LOX and ACh E was analysed by enzymatic reaction kinetics and their mechanism of action was evaluated by Lineweaver-Burk double inverse plotting.The results showed that all three active ingredients exhibited reversible inhibition against 5-LOX and ACh E,specifically against 5-LOX,with osmundacetoneasacompetitiveinhibitor,9,10-dimethoxy-pterocarpane-3-O-β-D-glucosideand3-hydroxy-9,10-dimethoxyptercarpan as non-competitive inhibitors,with inhibition constants K_i of 6.74×10^-5、7.13×10^-5and 7.62×10^-5 mol/L;For the inhibition of ACh E,osmundacetone,9,10-dimethoxy-pterocarpane-3-O-β-D-glucoside and3-hydroxy-9,10-dimethoxyptercarpan showed competitive inhibitors,with inhibition constants K_i of 8.37×10^-5、6.36×10^-5 and 7.21×10^-5 mol/L,respectively.The mechanisms of action of the three active ingredients on AD were further explored at the molecular level to reveal the potential biological mechanisms.Finally,molecular docking and molecular dynamics simulation were used to explore the mechanism of action of three active ingredients on AD and reveal the potential biological mechanism.Molecular docking results show that osmundacetone,9,10-dimethoxy-pterocarpane-3-O-β-D-glucosideand3-hydroxy-9,10-dimethoxyptercarpan can spontaneously and tightly bind 5-LOX and ACh E,with the docking binding energies of-5.4,-6.3 and-5.3 kcal/mol for 5-LOX and-6.9,-7.8 and-8.5 kcal/mol for ACh E,respectively.Molecular dynamics simulation further proves that osmundacetone,9,10-dimethoxy-pterocarpane-3-O-β-D-glucoside and 3-hydroxy-9,10-dimethoxyptercarpan can closely dock with 5-LOX and ACh E,forming a stable inhibitor-enzyme complex system,and the three active components have a stronger ability to inhibit ACh E.To sum up,the innovation of this paper is to take AD as the representative,through AUF and enzymatic reaction kinetics combined with molecular docking and molecular dynamics simulation to double screen and verify the activity of the compound,and explore the potential mechanism of the active components in the medicinal plants P.igniarius and A.membranaceus flowers,stems and leaves to treat AD from the experimental and virtual double-layer;Through the complementary application of Semi-preparative HPLC and HSCCC,the active components are systematically separated and purified,so as to rapidly prepare high-purity monomer compounds.Effective anti-AD active components can be screened from the P.igniarius and A.membranaceus flowers,stems and leaves,providing a convenient,universal and efficient new method for developing anti-AD drugs from medicinal plants.