Inhibitory Effect And Mechanism Of Saponins From Rhizoma Anemarrhenae On Toxicity Of β-amyloid Protrin

OBJECTIVE:To study the possible mechanism of Anemarrhena asphodeloides against Alzheimer’s disease,find out the potential substances of anti-Alzheimer’s disease,enrich the comprehensive utilization and development of Anemarrhena asphodeloides,save resources and protect the environment.METHODS:Macroporous resin was used to optimize the enrichment process of total saponins in Anemarrhena asphodeloides roots.The effects of total saponins in Anemarrhena asphodeloides roots and saponins B-Ⅱ on nematode longevity,oxidative stress tolerance and heat stress tolerance,reactive oxygen species（ROS）andβ-amyloid protein aggregated in vitro and through the nematode in vivo,respectively,using wild-type Caenorhabditis elegans（N2）and AD transgenic nematode CL4176 as to explore the potential anti-Alzheimer’s disease activity and possible mechanism of TSFAA and TSBⅡ,β-amyloid imprinting analysis and regulation of genes related to Alzheimer’s disease were performed.RESULTS:（1）After optimizing the conditions,AB-8 macroporous resin was the best resin for the enrichment of total saponins from Anemarrhena asphodeloides.The optimum enrichment process was as follows:0.2 g·mL^-1 sample solution,1 mL·min^-1sample adsorption,0.2 mol·L^-1 Na OH solution for impurity removal,then water washing to neutral effluent,and finally 3 BV 70%ethanol elution.The total saponin elution rate and purity of Anemarrhena asphodeloides were 67.93%and 57.61%respectively.（2）By inducing paralysis of Aβgene expression induced by warming of AD transgenic nematode CL4176,it was found that TSFAA-0.2 concentration group had the best effect in reducing the paralysis rate of transgenic nematode significantly;compared with control group,all concentration groups of Anemarrhenoside B-Ⅱ had certain anti-paralysis effect on nematode,which had certain statistical significance.Among the TSBⅡ groups,TSBⅡ-2 concentration group had the best effect.（3）The longevity test of AD transgenic nematodes showed that the drug concentration groups had a certain effect on prolonging the longevity of nematodes,indicating that the drug had no toxic effect on nematodes,and there was a significant difference compared with the control group（P<0.05）.（4）Compared with the control group,the TSFAA and TSBⅡ groups had a slight effect on prolonging the death of Caenorhabditis elegans,which had statistical significance.The results showed that TSFAA and TSBⅡ groups could prolong the thermal damage of nematodes through heat stress;TSFAA and TSBⅡ groups had a certain effect on control in oxidative stress experiment,and had a certain degree of significant effect.Sex difference indicated that TSFAA and TSBⅡ could prolong the thermal damage of nematode through oxidative stress.（5）By inducing paralysis of Aβgene expression induced by warming of AD transgenic Caenorhabditis elegans CL4176,it was found that TSFAA and TSBⅡ could reduce ROS production in nematode compared with model group,and there were significant differences.（6）After incubation with Aβ_1-42 protein standard,it was found that the total saponins and B-Ⅱ of Anemarrhena asphodeloides could reduce the accumulation ofβ-amyloid protein to a certain extent,and there were significant differences（P<0.05）.Therefore,we can know that saponins from Rhizoma Anemarrhenae can inhibit the aggregation ofβ-amyloid protein in vitro.（7）All concentration groups of total saponins of Anemarrhena asphodeloides could reduce the accumulation and deposition ofβ-amyloid protein in Caenorhabditis elegans（P<0.05）,and all concentration groups of saponin B-Ⅱ of Anemarrhena asphodeloides could reduce the accumulation and deposition ofβ-amyloid protein in nematode（P<0.05）.Thus,we can know that saponins from the root of Anemarrhena asphodeloides can inhibit the accumulation and deposition ofβ-amyloid proteins in Caenorhabditis elegans.（8）Qrt-PCR assay showed that TSFAA-0.2 could significantly up-regulate the expression of sod-3,hsp-16.2,daf-16 and down-regulate the expression of skn-1 and amy-1 compared with model group;TSBⅡ-2 could significantly up-regulate the expression of sod-3,hsp-16.2,daf-16 and down-regulate the expression of skn-1 and amy-1 compared with model group.TSFAA and TSBⅡ can enhance the tolerance of oxidative stress and heat stress,reduce the aggregation and deposition of protein Aβ,and protect neurons and cells from toxicity.CONCLUSION:TSFAA and TSBⅡ groups could reduce the accumulation and deposition of Aβprotein in nematodes,and to some extent retard oxidative and thermal damage,and regulate the genes such as amy-1,hsp-6.2,daf-6,sod-3 and skn-1 to AD.