| Trapa bispinosa Roxb.has thousands of years of history as a folk medicinal fruit in my country,and its shell has medicinal value.Ganoderma lucidum is an edible and medicinal fungus with a very wide range of pharmacological activities.The microbial transformation of Ganoderma lucidum as a fermentation strain can promote the synergy of the active ingredients of the Trapa bispinosa Roxb.shell.In this project,the single factor experiment combined with the response surface experiment method,Optimization of the process of removing solid-state fermentation of Trapa bispinosa Roxb.shell with Ganoderma lucidum as strain;the pre-fermentation purified polysaccharide（P1）and the fermentation purified polysaccharide（P2）were prepared,and the characterization and comparative analysis were carried out;the alcohol extracts before and after fermentation（AE1,AE2）,compare the content of active substances such as polyphenols and flavonoids in AE1 and AE2,and analyze their components by UPLC-QTOF-MS/MS technology;take mouse macrophages（RAW 264.7）as the object of investigation,comparative analysis of fermentation Differences in the ability of pre-and post-polysaccharides to stimulate immune activity and the antioxidant activity of alcohol extracts.The main results are as follows:（1）Response surface optimization The optimal process of Ganoderma lucidum fermentation Trapa bispinosa Roxb.shell:Ganoderma sinense fermentation,fermentation time 2 d,temperature 14℃,humidity 75%.The yield of water extract obtained by the optimal process was 4.355±0.01613%,and the scavenging rate of DPPH free radical was 82.3592±0.05918%.（2）The total polysaccharide content of Trapa bispinosa Roxb.shell crude extract after fermentation of Ganoderma lucidum was 18.484±0.090%,which was significantly higher than that before fermentation（9.810±0.364%）（P<0.05）.The ratios were average,and the weight-average molecular mass of the main fraction of P2 was concentrated at 2.019×105±1.112 u.The average molecular masses of P1and P2 are 6.004×106u and 7.812×104u,respectively.P1 is composed of 7 kinds of monosaccharides:mannose,glucuronic acid,galacturonic acid,glucose,galactose,arabinose,and fucose.Compared with P1,P2 increases rhamnose and xylose.The main components of both are Glucose accounted for 86.817%（P1）and63.191%（P2）,respectively.Scanning electron microscope（SEM）showed that P1was uniformly granular,and P2 was crystalline.Fourier transform infrared（FT-IR）analysis showed that the“CH2”vibration intensity of the polysaccharide skeleton of P2 was stronger than that of P1,and the amide I peak shifted to the lower wavenumber direction,and theβ-sheet amide I peak of P1 changed to none.In the regular amide I peak,the vibration intensity of the hydroxyl group also increased.Both P1 and P2 have triple helix structure,and the content of P2 is higher.（3）Compared with AE1,the content of active substances such as polyphenols and flavonoids in AE2 has an upward trend.UPLC-MS/MS analysis showed that there were 58 substances in the AE1 part and 63 substances in the AE2 part.The main active ingredients of AE2 are:ellagic acid,1,3,6-Tri-O-galloyl-β-D-glucose,chalconaringenin,naringenin,gallocatechin,indole,ganoderol A,et al.Compared with the main components of AE1,substances such as gallocatechin,cynaroside and ganoderol A were added,and substances such as Myricetin,Maltol and6-Gingerol were reduced.After fermentation,the newly generated secondary metabolites are witch hamamelitannin,pinocembrin,betulinic acid,beta-boswellic acid,3-epioleanolic acid,chimonanthine,3’,4’-anhydrovinblastine and lamiide;kuwanon H,arjunolic acid and myrcene disappeared after fermentation.（4）Both P1 and P2 stimulation can significantly increase the secretion of pro-inflammatory factors such as NO,i NOS,TNF-α,IL-2,IL-10 in RAW264.7cells,and stimulate immune activity,and P2 is more effective.Both P1 and P2could significantly up-regulate the expression of p50,p52 and p65 genes in the NF-κB pathway,while down-regulating the expression of p100 and ikkα,and P2was significantly stronger than P1.After AE1 and AE2 treatment,the secretion of T-AOC and T-SOD in RAW264.7 increased significantly,and the secretion of MDA decreased significantly,and the effect of AE2 was more obvious than that of AE1.AE2 can significantly increase the m RNA expression of gpx2,gpx5,gclc,and gclm genes in the Nrf2/Keap1-ARE pathway,and can significantly down-regulate the expression of keap1;AE1 only up-regulates the expression of gpx2,gpx5,and gclc genes.The fermented water chestnut shell of Ganoderma lucidum can improve the ability of the polysaccharide part to stimulate immune activity,and its effect is related to the NF-κB pathway;it can also significantly improve the antioxidant capacity of the alcohol extract,and its effect is related to the Nrf2/Keap1-ARE pathway. |
