Study On Anti-aging Effects Of Radix Astragali And Radix Astragali Preparata Based On Network Pharmacology And Liquid Chromatography Coupled With Mass Spectrometrytechnology

With the rapid development of population aging,aging is becoming a global public health problem.The development of potential clinical therapies and drugs to delay aging and treat aging-related diseases has become a hot research direction.Astragalus as a traditional Chinese medicine and medicinal and food homologous plant has a wide range of physiological and pharmacological effects.Both the root of Radix Astragali(RA)and the Radix Astragali preparata(RAP)have anti-aging pharmacological effects,but the material action basis and mechanism are not clear,and the difference in the anti-aging effect of the RA and RAP has not yet been clarified.Based on this,this subject research is proposed,and it is planned to explore the material basis and possible mechanisms of the anti-aging effects of RA and RAP based onliquid chromatography coupled with mass spectrometry(LC-MS),network pharmacology and molecular biology.In this study,the network pharmacology and molecular docking were first used to predict and verify the anti-aging active ingredients,action targets and pathways of RA.According to the value of degree,the main active ingredient of RA anti-aging may be quercetin,gardenial,isorhamnetin,3,9-di-O-methyl nissan pea sandalwood and kaempferol,etc.These active ingredients play an anti-aging role mainly by acting on targets such as Aktl,MAPK1,MAPK8,SIRT1,mTOR and CASP3,and regulating signal pathways such as PI3K/Akt,MAPK,AMPK,and Toll-like receptor.Then,the multi reaction monitor-ultraperformance liquid chromatography coupled with tandem mass spectrometry(MRM-UPLC-MS/MS)technology was used to analyze the in vitro substance composition of the water extracts of RA and RAP,17 compounds were detected from them,and the relative content of each compound was calculated according to the standard curve of astragaloside ?.The results showed that the contents of kaempferol,quercetin,astragaloside?,calycosin-7-O-?-D-glucoside,palmitic acid,calycosin-7-O-?-D-gluc oside-6"-O-acetate,formononetin-7-O-?-D-glucoside-6"-O-acetate,formononetin and astragaloside I were higher than those of RA.the contents of calycosin-7-O-?-D-glucoside-6"-O-malonate,linolenic acid,ononin,formononetin-7-O-?-D-gluc os ide-6"-O-malonate,c alyc osin,((6aR,11aR)-3-Hydroxy-9,10-d imethoxypterocarpan),astragaloside ? and soyaponin I were all reduced.It means that the contents of components of RA will change accordingly after being fried by honey.Ultraperformance liquid chromatography coupled with quadrupole time of flight mass spectrometry(UPLC-Q-TOF/MS)was used to analyze the serum samples of rats at different time points after taking water extracts of RA and RAP for three consecutive days.The results of total ion chromatogram revealed that there were some differences in the peak shapes and peak intensities between the serum metabolic profiles of RA and RAP.The sPLS-DA chart showed that the serum metabolites of RA and RAP at different time points have better aggregation within the group and greater separation between the groups,further explaining the differences between their serum metabolites and the generation of potential metabolic biomarkers.The distance from the point in the Loading chart to the central axis represents the contribution of the corresponding feature value.The larger the distance,the greater the contribution,and the points scattered around the periphery may become metabolic biomarkers.Based on the HMDB database,39 potential biomarkers were screened from blood samples of RA,and 10 blood-injecting components were identified,of which the quercetin original drug was in blood.The metabolic pathways are mainly glycerophospholipid metabolism and arachidonic acid metabolism.From the blood samples of RAP,40 kinds of potential biomarkers were screened,and 17 blood-injecting components were identified,of which the quercetin original drug was in blood.The metabolic pathways are mainly glycero phospholipid metabolism and primary bile acid biosynthesis.This indicated that the metabolic difference between RA and RAP after blood injection may be caused by arachidonic acid metabolism and primary bile acid biosynthesis.Finally,a mouse aging model was established by subcutaneous injection of D-galactose for 7 weeks,and RA,RAP and quercetin were given at the same time to study its protective effect on aging mice.The mice were divided into 5 groups:blank group(Con group),model group(Model group),RA protection group(RA group),RAP protection group(RAP group)and quercetin protection group(Quercetin group).Among them,the quercetin group was only used to verify that quercetin had a protective effect,and was not compared with the RA group and RAP group.The specific research was as follows:(1)Validation of D-galactose-induced aging modelAfter 7 weeks of modeling,the Y-maze experiment was used to evaluate the spatial memory ability of the mice through the autonomous alternation rate.The results showed that compared with the Con group,the autonomic alternation rate of the mice in the Model group was significantly reduced(p<0.01),which proved that the spatial learning and memory ability of the mice was reduced,and that the aging model was successfully established.The RA,RAP,and Quercetin protection groups had an autonomous alternation rate between the Con and Model groups,and the RAP group was higher than the RA group,which proves that extracts of RA and RAP and quercetin have anti-aging effects,and the anti-aging effect of RAP is stronger than RA.(2)Measurement of physiological indicatorsDuring the experiment,the weight of the five groups of mice increased,but the rate of increase was different.Compared with the Con group,the weight of the model group increased,but the rate was much lower than that of the Con group,indicating that as the aging time prolonged,the body weight gradually decreased.RA,RAP,and Quercetin-protected mice gained weight between the Con and Model groups,suggesting that protection of extracts of RA and RAP and quercetin can slow the trend of weight loss.The brain coefficient and liver coefficient of the mice were calculated,and it was found that compared with the Con group,the brain coefficient and liver coefficient of the mice in the Model group were significantly reduced(p<0.01).Compared with the Model group,the brain coefficient and liver coefficient of the RA,RAP,and Quercetin protection groups increased to varying degrees,and the increase in the RAP group was slightly higher than that in the RA group.This shows that D-galactose can cause brain tissue atrophy in mice,reduce metabolic capacity,and accelerate aging.After being protected by extracts of RA and RAP extract and quercetin,aging can be delayed and the effect of RAP was stronger than RA.(3)Determination of biochemical indicatorsThe mouse liver SOD,MDA and GSH indexes were measured respectively.As a result,compared with the Con group,SOD activity and GSH content in the Model group were significantly reduced(p<0.01,p<0.001),and MDA content was increasedsignificantly(p<0.01);The SOD activity and GSH content of the RA,RAP,and Quercetin protection groups increased significantly,and the MDA content decreased significantly comparing with the Model group.This shows that D-galactose can promote the accumulation of free radicals in mice,reduce the activity of antioxidant enzymes,and accelerate aging.The extracts of RA and RAP and quercetin can improve the free radical scavenging ability of aging mice and delay aging.(4)Morphological observationH&E staining was used to stain the brains of different groups of mice to observe histopathological changes.The results showed that the nerve cells of the Con group were tightly arranged,with large nuclei and clear nucleoli.Compared with Con group,some nerve cells in the brain of the Model group showed obvious vacuole changes,mild edema and mild inflammatory infiltration.In contrast,the pathological abnormalities of the RA,RAP,and Quercetin treatment groups were alleviated,and the effect of RAP was the most obvious,which was almost normal.(5)Metabolomics analysisUPLC-Q-TOF/MS was used to analyze mouse serum samples.The results showed that the Con group,Model group,RA,RAP,and Quercetin protection groups had good aggregation within the groups,large separation between groups,and accompanied by the generation of metabolic markers.The pathway results showed that the first five metabolic pathways related to the anti-aging effects of RA are glycerophospholipid metabolism,tryptophan metabolism,linoleic acid metabolism,?-linolenic acid metabolism and sphingolipid metabolism.The first five metabolic pathways related to the anti-aging effects of RAP are glycerophospholipid metabolism,arachidonic acid metabolism,sphingolipid metabolism,linoleic acid metabolism,and ?-linolenic acid metabolism.It can be seen that there are differences in the metabolic pathways in which RA and RAP play an anti-aging effect.(6)Western blot analysisFrom the results of network pharmacological prediction,the PI3K/Akt pathway was selected as the research object from the main anti-aging pathway of RA,and anti-PI3K antibody and anti-Akt1 antibody were used to perform western blot on different groups of liver tissues.The results showed that,the expression of PI3K protein in the Model group was significantly reduced(p<0.01)and the expression of Aktl protein was significantly reduced(p<0.05)comparing with the Con group;compared with the Model group,the PI3K and Aktl protein expression increased(p<0.05),and the increase in RAP group was slightly larger than that in RA group.This indicates that the PI3K/Akt1 pathway of the Model group mice is inhibited,extracts of RA and RAP and quercetin can activate PI3K,promote Aktl expression,inhibit cell apoptosis,and delay aging.(7)RT-PCR analysisAccording to the results of RT-PCR,the gene expression of PI3K in the Model group was significantly down-regulated(p<0.05)comparing with the Con group.Gene expression of PI3K was significantly up-regulated in RA,RAP and Quercetin groups(p<0.05)comparing with the Model group.Nevertheless,there was no apparent change in Akt1 gene expression both the Model group and the treatment group.This shows that D-galactose can down-regulate the PI3K gene expression,and the administration of RA,RAP and quercetin can increase the PI3K gene expression,but D-galactose,RA,RAP and quercetin had no effect on the expression of Aktl gene.This topic studied the anti-aging mechanism of RA and RAP by network pharmacology and liquid chromatography-mass spectrometry.The results show that RA/RAP can clear ROS in aging mice,increase SOD activity and GSH content in liver,reduce MDA content,thereby inhibiting oxidative stress and play an anti-aging effect.In addition,RA and RAP can promote the expression of PI3K gene,resulting in increased expression of PI3K protein,and induce phosphorylation of Aktl protein,inhibit oxidative stress,and exert anti-aging effects.The difference is that there are differences in the energy metabolism pathways of RA/RAP,RA inhibits TDO activity in the blood,thereby reducing Trp consumption,promoting the increase of Trp content,and delaying aging.RAP inhibits the activity of cPLA2 in the blood,reduces the release of AA,causes its content to decrease,and plays an anti-aging effect.