| Objective:The content differences of total flavonoids,total phenols,anthocyanins and trace elements in Lycium ruthenicum Murr(LRM).from different places were compared,and the varieties with higher antioxidant activity were screened out;UPLC-MS-MS and multivariate statistical analysis methods were combined with the results of mouse behavior,antioxidant indexes and HE staining results of hippocampus tissue,using metabolomics research methods and techniques to explore the overall metabolite profile,metabolic profile changes,and biomarker populations of serum and hippocampal tissue after administration of alcohol extract of LRM.The mechanism of the LRM ethanol extraction of against Alzheimer’s Disease was investigated based on the metabolomics results.Method:1.The optimal extraction conditions of total flavonoids and phenols from LRM were proposed.The contents of total flavonoids,total phenols and anthocyanins were determined with colorimetric method.The trace elements in LRM were determined with ICP-MS.2.The 64 mice were randomly divided into 8 groups on average.One group was given normal saline by gavage as blank,and other group were given the extractions(equivalent of 0.5 g LRM/m L)of the different places LRM,then the effect on hypoxia tolerance under normal pressure was studied.The 72 mice were randomly divided into 9groups on average.The blank was same above,other 8 group were a model of myocardial oxygen consumption.One group of model was given normal saline by gavage as model,and other 7 group were given the extractions(equivalent of 0.5 g LRM/m L)of the different places LRM,then the effect on hypoxia tolerance was studied.The effects of the different concentration alcohol extracts of Chaidamu LRM on the hypoxia tolerance of normal pressure and model mice were studied in the same way.3.60 C57BL/6 mice were randomly divided into 6 groups.One group was given normal saline by gavage as blank,and AD group were subcutaneously injected Dgalactose(120 mg/Kg)and given the Al Cl3 solution(200 mg/Kg)by gavage.The blank and AD were feed 30 d according the above method.L、M、H group were given the LRM extraction(40、80、160 mg/Kg)and AA were given Vitamin C(100 mg/Kg)by gavage once a day for 30 d.4.After the last dose,the behavioral experiments of each group mice were performed,including new novel object recognition tral(NORT),morris water maze(MWM),passive avoidance tial(PAT),high plus maze(HPM).Finished the behavioral experiment,Blood was getted from the inner canthus and serum was separated.Brain was obtained by decollation,and then hippocampal and cerebral cortex tissues were separated on ice,while two whole brains of each group were reserved for HE staining.The content of SOD,MDA and ACHE were detected in the cerebral cortex homogenate.The serum and hippocampal tissues of mice were taken as samples for metabolomics study.Results:1.The total flavonoids content in the LRM of Chaidamu,Nuomuhong semi-wild A,Nomuhong semi-wild B,Nomuhong wild,Baron,Gansu and Ningxia were 60.40±0.49,40.20±0.15,54.80±0.14 5,4.86±0.18,48.31±0.28,44.56±0.23,41.69±0.30mg/g,respectively;total phenolic content were 33.23±0.09,32.76±0.13,30.59±0.13,30.41±0.45,24.47±0.18,28.54±0.12,30.16±0.11 mg/g,respectively;The anthocyanin content were16.30±0.28,19.13±0.08,19.05 ± 0.18,14.20± 0.13,11.62±0.48,12.24±0.05,15.42±0.16mg/g,respectively.The LRM contained a variety of essential trace elements,and the contents of heavy metals such as Pb,Cd and Cu were within the safe range prescribed by the Pharmacopoeia.2.Oxygen consumption experiments showed that the extracts from different habitats could have improving hypoxia tolerance in both normal pressure and increased myocardial oxygen consumption mice.The survival time of normal pressure mice was prolonged with different concentration of chaidamu extract,but there was no difference between groups.The survival time of the model group was significantly reduced in the hypoxia tolerance experiment of mice with increased myocardial oxygen consumption,indicating that the modeling was successful and the survival time of the mice was significantly prolonged in each drug administration group(P<0.01).3.Behavioral experiment:(1)The NORT results showed that the number of touching new objects by the AD group was significantly lower than that of the control group(P<0.01),and the behavior after administration extraction was improved.There was a significant difference between the drug-administered groups and the AD group(P<0.05).(2)HPM results: Compared with the control group,the AD group open time decreased(P<0.01).Compared with the AD group,the L and M groups were increased(P<0.01,P<0.05)and H,AA group were no significant difference from the AD group.(3)PAT results: In the adaptation phase of the first day,there was no significant difference in latency between the groups.Compared with the Control group,the AD group had a lower latency on the third day(P<0.05)and the AA group had an increased latency(P<0.05),but the other groups did not differ.(4)MWM results: There was a significant difference in the escape latency between the AD group and the control group on the fourth and fifth days(P<0.05).Compared with the AD group,the L group escape latency was significantly reduced in the five-day(P<0.05),and the escape latency of the M group was significantly less on the third to fifth days(P<0.05),but there was no significant difference in the H group.The space exploration experiment results indicated that the number of shuttles in the model group was significantly different from that of the control(P<0.05),which was lower than that of the model group.The number of shuttles in the dose group was significantly increased(P<0.05).There were no significant difference between the M group,the H group compared with the AD group.The number of shuttles increased in the AA group compared with the AD group(P<0.05).4.Antioxidant index:(1)SOD activities: The content in the AD group was significantly decreased(P<0.01).Compared with the AD group,the L,M and H groups and the AA group increased(P<0.01,P<0.05).(2)MDA activities: Compared with the control group,the content of the AD group was increased(P<0.01),and the L,M and H and the AA group were lower than the model group(P<0.01).(3)ACh E activities: The AD group was higher than the control group(P<0.01),and the L,M,and H and the AA group were lower than the AD group(P<0.01).5.HE results: The results of HE staining showed that the brain tissue of the control group was basically normal,the cells were neatly and compactly distributed,the cytoplasm was round and full,the staining was purple-red,and the nuclei were blue-stained.Compared with the control group,the brain tissue structure of the AD group was loose,void-like or cavity-forming,in which a plurality of neuron cells only had their contours,the nucleus condenses,dissolves,disappears,and the number of cells was significantly reduced.Compared with the model group,the pathological morphology of the brain tissue of each drug group was improved to different extents.The number of infarcted neurons was lower than that of the model group,the membrane was intact,and the nucleus was clearly visible,it indicates that the degree of necrosis is reduced and the pathological changes of nerve cells are improved.6.Metabolomics results: Metabolomics results: 17 differential metabolites in serum were identified.The pathways involved in these metabolites including taurine and hypotaurine metabolism;phenylalanine metabolism;proline,leucine and Biosynthesis of isoleucine;degradation of proline,leucine and isoleucine;metabolism of niacin and nicotinamide;biosynthesis of pantothenic acid and Co A;metabolism of histidine;metabolism of anthraquinone;metabolism of arachidonic acid;Primary bile acid biosynth-esis and aminoacyl-t RNA biosynthesis.In addition to the 17 metabolites identified in the serum,four substances were identified in the tissue,namely cis-aconitic acid,hypoxanthine and glutamine,and the metabolic pathway involved was citric acid.Circulation(TCA cycle),sputum metabolism,and alanine,aspartate and glutamate meta-bolism.Conclusions:1.In the research,the contents of total flavonoids,total phenols and anthocyanins in the extracts of 7 species of LRM from Qinghai(Chaidamu,Nuomuhong and Balong),Gan Su Zhang Ye and Ningxia Zhongning were investigated.The results showed that the content of the components in the LRM extract represented the regional differences,and the content of total flavonoids,total phenols and anthocyanins in the extract of Qinghai LRM is higher.In addition,the LRM contained a variety of essential trace elements,and the contents of heavy metals such as Pb,Cd and Cu were within the safe range prescribed by the Ch P(2015).2.The extract of LRM from different habitats could significantly improve the ability of resist hypoxia in mice,and the effect of the extract of Chaidamu was obvious.Therefore,the anti-hypoxia tolerance of mice with different concentrations of Chaidamu LRM extract was studied.The results showed that each concentration increased the survival time of normal-pressure mice,but there was no difference between the groups;different concentrations of Chaidamu LRM extract significantly prolonged the survival time of the model mice with increased myocardial oxygen consumption.3.The antioxidant indexes results showed that the SOD activity of the model group decreased meanwhile MDA content and ACh E activity increased,and the SOD,MDA and ACh E levels tended to be normal group levels of after administration.The four behavioral experiments and the results of HE staining showed that the extract of Chaidamu LRM had a significant effect on the learning and memory ability of mice,and reduced the anxiety level of AD mice.4.Twenty-four metabolites were detected in serum metabolomics and hippocampal tissue metabolomics experiments in mice.The same metabolites in serum and hippocampal tissue samples included L-tryptophan,phenylalanine,and docosahexaene acid,arachidonic acid,hypoxanthine,taurocholic acid and adenine.The metabolic pathways involved were predicted,in which the same metabolic pathways were found in serum and hippocampal tissue samples were taurine and hypotaurine metabolism,purine metabolism,arachidonic acid metabolism,primary bile acid biosynthesis,and unsaturated fatty acids biosynthesis and tryptophan metabolism.The above-mentioned common metabolic pathways were closely related to the antioxidant effect,that is,the alcohol extract of LRM exerted its anti-alzheimer’s effect mainly through the antioxidant mechanism and combined with other metabolic pathways. |
PDF Full Text Request