| Flammulina velutipes is one of the most popular mushrooms in China, which contains plentiful nutrients and active substance and can be used for pharmaceutical and edible functions. A few reports have shown that F. velutipes was benefit for children growth and intellectual development. Although many researches have proved its improvement of learning and memory functions, the functional ingredients and mechanism haven’t been elucidated.In this study, response surface methodology, based on Box-Behnken design was used to optimize the ultrasonic extraction conditions of F. velutipes polysaccharides (FVP), and chromatographic and spectroscopy technologies such as high performance gel filtration chromatography (HPGFC), gas chromatography (GC), Fourier transform infrared spectrum (FT-IR), UV scanning spectrum (UV), and nuclear magnetic resonance (NMR), as well as chemical analysis methods were employed to characterized FVP structure. Hidden platform test and probe test of Morris water maze were applied to evaluate the improvement of scopolamine-induced memory impairments of FVP. Effects of FVP on the biochemical and neurological changes in hippocampus and cerebral cortex of rats were investigated, SOD and GSH-Px activities, content of some neurotransmitters such as acetylcholine (ACh),5-hydroxytryptamine (5-HT), dopamine (DA), and norepinephrine (NE), choline acetyltransferase (ChAT) and acetylchohnesterase (AChE) activities, phosphorylation of Calcium/calmodulin dependent protein kinase (CaMKⅡ) and extracellular regulated protein kinases (ERK), and expression of gap junction protein connexin36are were analyzed to clarify the mechanism. The following work was performed and the results are summarized:(1) Four solvent extracts of F. velutipes were prepared for AChE inhibitory activities assay. Results showed that the AChE inhibitory activity of water extract was significantly better than the other solvents extracts (ethanol, petroleum ether, and ethyl acetate), with19.75%inhibition rate. FVP as the main component in water extract was further shown to have20.0%AChE inhibition rate (2.0mg/mL). Box Behnken design was used to optimize the ultrasonic extraction conditions of FVP and four independent variables (ratio of water to raw material, ultrasonic power, ultrasonic time, and ultrasonic temperature) were taken into consideration. A quadratic model was constructed between ultrasonic conditions and yield of FVP, Y=-11.94+0.18·X1+0.03·X2+0.30·X3+0.25X4+6.88×10-5·X1X2+1.0×10-3·X1X3+1.2×10-3·X1X4-2.59×10-4·X2X3-1.11×10-4·X2X4-1.33×10-3·X3X4-5.99×10-3·X12-1.86×10-5·X22-2.87×10-3X32-2.06×10-3·X42。A modified optimization condition was ratio of water to material of25ml/g, ultrasonic power of620W, ultrasonic time of20min, and ultrasonic temperature of45℃, and a yield of FVP of8.33%was obtained under this condition, which is62.7%higher than that obtained with hot water extraction.(2) FVP was further purified by DEAE cellulose-52chromatography and Sephadex G-100size-exclusion chromatography to afford FVP-1and FVP-2, with total sugar content of98.65%and96.24%, respectively. Molecular weight of FVP-1and FVP-2was28kDa and268kDa, respectively. Uronic acid content was1.56%and3.42%, respectively. Sulfate content was0.09%and0.14%, respectively. FVP-1was composed of glucose, galactose, mannose and fucose in an approximate molar ratio81.3:12.1:3.6:3.0and FVP-2was composed of glucose, mannose, galactose, xylose and fucose in an approximate molar ratio57.9:12.0:15.1:9.5:5.5. Both FVP-1and FVP-2were triple helical structures. Ultraviolet spectrum indicated that FVP-1and FVP-2were without protein and nucleic acid. Characteristic absorptions of polysaccharides were observed in infrared spectra of FVP-1and FVP-2, which indicated the possible presence of pyranose ring in FVP-1and furanose ring in FVP-2, respectively, and glycosyl residues of FVP-1and FVP-2were mainly β-type glycosidic linkages.(3) Morris water maze was employed to evaluate effect of FVP on scopolamine-induced learning and memory impairments. Results showed that, compared with control,5mg/kg.bw ip scopolamine can injure the learning and memory of rats, presenting an increase in the escape latency and total swimming distance in hidden platform test, and decreased the numbers of crossing the platform and distance of moving around platform of rats in probe test.200mg/kg.bw and400mg/kg.bw FVP significantly decreased the escape latency and total swimming distance of rats in hidden platform test, and increased the numbes of crossing the platform and distance of moving around platform of rats in probe test. All these results indicated that FVP could improve scopolamine-induced learning and memory impairments, which afford scientific proof for learning and memory function of F.velutipe.(4) To clarify the learning and memory improvement mechanism of FVP, effects of FVP on the biochemical and neurological changes in hippocampus and cerebral cortex of rats were investigated. Results showed that5mg/kg.bw ip scopolamine decreased SOD and GSH-Px activities, as well as ACh,5-HT, DA, and NE contents in hippocampus and cerebral cortex. Decrease of ACh content owed to decrease of ChAT activity and increase of AChE activity. Expressions of Connexin36, p-ERK and p-CaMK Ⅱ in hippocampus and cerebral cortex were also decreased by scopolamine. Compared with scopolamine injured group rats, FVP reversed the decrease of SOD and GSH-Px activities induced by scopolamine, and increased ACh,5-HT, DA, and NE contents, especially effect of highest concentration FVP on ACh, DA, and NE contents in hippocampus. FVP increased ChAT activity and decreased AChE activity to raise ACh content. Moreover, FVP increased the expressions of Connexin36, p-ERK and p-CaMK Ⅱ in hippocampus and cerebral cortex. FVP achieves its learning and memory improving function through modulation of all the above mechanism. |
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