Preparation Of Total Flavonoids From Eucommia Ulmoides Pollen And Its Anti-Aging Effect

Objective:To explore the preparation technology of total flavonoids of Eucommia ulmoides pollen,to establish the quality control method of total flavonoids of Eucommia ulmoides pollen,and to explore the anti-aging effect of total flavonoids of Eucommia ulmoides pollen.Methods:The suitable method for extracting total flavonoids from Eucommia ulmoides pollen was screened,and the crude flavonoid extract was purified by macroporous adsorption resin,and the sample concentration,sample volume,adsorption time,elution rate,elution solution and elution volume were investigated to optimize the purification process.The contents of total flavonoids in Eucommia ulmoides pollen before and after purification,rutin and isoquercetin after purification were detected,and 15 batches of total flavonoids in Eucommia ulmoides pollen were qualitatively identified by TLC and fingerprint to establish the quality standard of total flavonoids in Eucommia ulmoides pollen(draft).Effects of total flavonoids of Eucommia ulmoides pollen on aging mice: 60 male Kunming mice weighing 14～16g were randomly divided into 6 groups with 10 mice in each group: normal group,model group,vitamin C group(100mg/kg/d),high dose group(320mg/kg/d),medium dose group(160mg/kg/d)and low dose group(80mg/kg/d),except the normal group.The aging mice were induced by subcutaneous injection of D-galactose solution(D-galactose)into the neck and back.The mice were weighed once a week.Six weeks later,the animal behavior was tested by Morris water maze.After the behavior test was over,all the experimental animals were killed and samples were collected for examination.HE staining was used to observe the pathological changes of the brain and liver;The activity and content of superoxide dismutase(SOD),total antioxidant capacity(T-AOC)and MDA in serum,brain tissue and liver of experimental mice were detected by the kit.Immunohistochemical techniques(IHC)was used to detect nuclear factor erythroid 2‐related factor 2(Nrf2),Kelch‐like ECH‐associated protein 1(Keap1)and heme oxygenase-1(HO-1)in the hippocampus of mice;Western blotting(Western blot)was used to detect the expression of Nrf2 and Keap1 in mouse brain tissue;The gene expression levels of Nrf2,Keap1 and HO-1in mouse brain tissue were detected by real-time quantitative PCR(RT-qPCR).Results:(1)The best preparation technology of total flavonoids of Eucommia ulmoides pollen is as follows:Eucommia ulmoides pollen is extracted by ultrasonic extraction,the ratio of material to liquid is 1:20 with70% ethanol solution,soaking for 1 hour,ultrasonic extraction 3 times,each time 40 min,combining filtrate,the filtrate is concentrated and absorbed by HPD100 macroporous resin,the sample concentration is0.50mg/ml,the sample volume is 1BV,the adsorption time is 6 h,after the adsorption is completed,it is washed with distilled water until the eluent is colorless.The total flavonoids content of Eucommia ulmoides pollen was 50.4% after 50% ethanol solution was eluted at the elution rate of 1ml/min and the elution volume 3BV.After further purification by polyamide,the total flavonoids content reached 70.8%.(2)The TLC identification and Fingerprint identification methods of total flavonoids of Eucommia ulmoides pollen were established,and the quality standard draft of total flavonoids of Eucommia ulmoides pollen was established.(3)After the intervention of total flavonoids of Eucommia ulmoides pollen,the latency,the times of crossing the original platform,the swimming time and the percentage of distance in the platform quadrant of the mice in the behavior experiment were higher than those in the model group.among them,the latency,times of crossing the platform and swimming time in the platform quadrant of the middle dose group of total flavonoids of Eucommia pollen were significantly higher than those of the model group(P<0.05).The results of antioxidant indexes showed that after the intervention of total flavonoids of Eucommia ulmoides pollen,the activities of SOD and T-AOC in serum,brain and liver of mice were higher than those of the model group,and the contents of MDA in serum,brain and liver of mice were lower than those of the model group.The pathological state of brain and liver was improved compared with the model group.(4)The results of IHC,Western blot and RT-qPCR showed that the protein expression and gene expression levels of Nrf2 and HO-1 were higher than those in the model group,and there was significant difference between the middle dose group of total flavonoids of Eucommia pollen and the model group.Conclusion:(1)The total flavonoids of Eucommia ulmoides pollen were prepared by ultrasonic extraction,and the macroporous resin-polyamide method was used to enrich the total flavonoids with simple operation,stable process and high purity.The quality standard of total flavonoids in Eucommia ulmoides pollen was drawn up,which can effectively control the quality of total flavonoids in Eucommia ulmoides pollen.(2)The total flavonoids of Eucommia ulmoides pollen can improve the learning and memory ability of aging mice,increase the antioxidant level of mice,activate the Keap1-Nrf2 signaling pathway,regulate the level of oxidative stress in mice,and play an anti-aging role.