Research On Anti-fatigue And Anti-depressive Pharmacological Effects And Mechanism Of Total Flavonoids Of Mulberry Leaves

In today's fast-paced lifestyle,physiological fatigue and pathological fatigue have an increasingly prominent influence on human work efficiency and quality of life,especially for pilots,astronauts,pilots and other special occupational groups,long-term sleep deprivation will easily lead to fatigue,affect physical and mental health,and even bring safety hazards.In addition,many diseases,including depression,schizophrenia,Parkinson's disease,Fibromyalgia,amyotrophic lateral sclerosis,autoimmune diseases,infectious diseases,metabolic diseases,cancer can be accompanied by severe fatigue,and fatigue has become a serious public health problem that harms human health.At present,physiological fatigue treatment mainly includes special nutritional supplement,sleep adjustment and kinesitherapy,while pathological fatigue?such as chronic fatigue syndrome?mainly includes moderate exercise and cognitive psychotherapy,supplemen ted by nutritional supplement and symptomatic drug therapy.Depression is the most i mportant type of affective mental disorders,with high prevalence and suicide rate char acteristics,causing serious economic burden to the society.From 2005 to 2015,the tot al number of patients with depression increased by 18.4%;In 2015,the World Health Organization listed depression as the single largest cause of disability globally.Traditio nal Chinese medicine has a very long history of understanding fatigue and depression,combining traditional Chinese medicine theory with modern pharmacology,studying a nd developing safe and effective anti-fatigue and anti-depression modern Chinese medi cine,which will plays an important role in drug development and worldwide disease treatment.ObjectiveTo screen safe and effective anti-fatigue active drugs,and to evaluate their anti-fatigue efficacy and preliminary mechanism in animal models of exercise fatigue and pathological fatigue.In addition,in view of the high comorbidities of depression and fatigue,we research the antidepressant effects of effective anti-fatigue drugs.MethodsExperiment research 1:Screening of anti-fatigue active drugs.Aceording to body weight,BALB/c mice were randomly divided into solvent control group,positive drug gro-up and test drug groups with different concentrations:Mulberry Leaves Extract?MLE?,Total Flavonoids in Mulberry Leaves?TFML?,Total Flavonoids in Bamboo Leaves?TFBL?,Tea Polyphenols?TP?,and Theanine?TA?.All groups were treated with cont-inuous intragastric administration for 7 days or 14 days respectively,and weight bearing swimming experiment was carried out to detect the exhaustion latency of mice in each group,C57BL/6J mice were selected and randomly divided into normal control group,solvent control group,positive drug group and each test drug administration group?TFML,TFBL,TA?.After treatment for 14 days of continuous intragastric admini?stration,a treadmill experiment was conducted to detect the movement distance of each group of mice;C57BL/6J mice were selected and randomly divided into normal control group,solvent control group,and Lipopolysaccharide?LPS?fatigue model group,positive drug group,TFML group,TFBL group according to body weight.Except the positive drug American ginseng was treated with intragastric administration for 14 days,the rest of the drugs were treated with intragastric administration 24 hours after int-raperitoneal injection of LPS.After 1 hour,a tread-mill experiment and a grip test were performed to detect the movement distance and muscle strength of each group of mice.Experiment research 2:Study on anti-fatigue effect and mechanism of total flavonoids in Mulberry leaves.BALB/c mice were randomly divided into solvent control group,positive drug group,and TFML group according to body weight.A continuous administration for 7 days or single-dose administration were treated respectively following with weight bearing swimming experiments 1 h later;C57BL/6J mice were randomly d-ivided into normal control group,solvent control group,positive drug group and TFML group according to body weight,and treadmill experiment was used 1 hour later after single administration;BALB/c mice were randomly divided according to body weight into solvent control group and reserpine with LPS model group,positive drug group,TFML group.After 14 days of continuous intragastric administration,behavioral paradigms of weight bearing swimming test,grip test,forced swimming test,squint score,Y-maze test,and Von Frey method for mechanical pain domain test were used.Testing the blood biochemical indexes and hepatic glycogen in mice to explore the possible mechanism of TFML on exercise fatigue.Testing the hypothalamic monoamine neurotransmitter in mice,observing the colon morphology in mice and detecting the changes of intestinal flora to explore the possible mechanism of TFML on pathological fatigueExperiment research 3:Study on antidepressant effect and mechanism of total flavonoids in Mulberry leaves.BALB/c mice were randomly divided into solvent control group,positive drug group and TFML group according to body weight.A continuous administration for 7 days or single-dose administration were treated respectively following with forced swimming experiment and mouse tail suspense on experiment 1 h later.Pharmacological models?yohimbine toxicity enhancement test,5-hydroxy-L-tryptophan?5-HTP?induced mouse head shaking behave-or test,reserpine depletion test?were used for possible mechanism of TFML anti-depression effect.Results1.After screening research,it was found that TFML?TFBL and TP have anti-fatigue activity:?1?The results of weight bearing swimming experiments in mice showed that TFML?200 mg/kg?administered continuously for 14 d can significantly prolon-g the exhaustion latency of mice;TP?200 mg/kg?continuous admiistration for 7 d significantly increased the exercise endurance of normal mice.?2?The results of the mouse treadmill experiment showed that TFML?100 mg/kg,20m g/kg,400 mg/kg?and TFBL?50 mg/kg,100 mg/kg?were administered in a single dose and continuously.TFML and TFBL can significantly increase the experimental movin-g distance of mice running platform?3?On the fatigue model caused by LPS,single administration of TFML?200 mg/kg?and TFBL?100 mg/kg?can significantly increase the running distance of the running platform of mice.The results of the grip test experiment show that TFML?200 mg/k g?can significantly improve the decease of muscle strength in mice caused by LPS.2.TFML has anti-fatigue effect in sports fatigue:?1?The weight-bearing swimming results of mice showed that a single administration of TFML?200 mg/kg?has no significant effect on the exhaustion latency of mice.Continuous administration of 7 d can significantly increase the exhaustion latency of mice.?2?The results of the mouse treadmill experiment showed that a single administration of TFML?100 mg/kg,200 mg/kg,400 mg/kg?can significantly increase the moving distance of mice.3.LPS and reserpine can be used to duplicate the mouse fatigue model:?1?After a single intraperitoneal injection of LPS?0.4,0.8,1.2 mg/kg?after 8 h,12 h,and 24 h,there is a tendency to reduce the latent period of weight bearing swimming exhaustion in mice.Among them,LPS 0.4 mg/kg injected after 24 h can significantly reduce the exercise tolerance of mice.?2?Continuous intraperitoneal injection of reserpine?0.2 mg/kg,0.4 mg/kg,0.8 mg/kg?for 14 days can significantly reduce the exhaustion latency of weight-bearing swimmi-ng mice.4.TFML can improve some symptoms of LPS-induced mouse fatigue model:a single intraperitoneal injection of LPS can significantly shorten the exhaustion incubation period of the weight-bearing swimming experiment in mice,reduce the muscle strength in the grip test,increase the mice the immobility time of the forced swimming experiment,increase the squint score of the mouse24 h later.Continuous administration of TFML?200 mg/kg,400 mg/kg?for 14 days can significantly improve the exercise endurance of mice against decreasing induced by LPS,reverse the decreas-e of mouse muscle strength caused by LPS,shorten the lack of forced swimming caused by LPS Movement time.And it had a tendency to improve the squinting behavior of mice.5.TFML can improve some symptoms of mouse fatigue model caused by reserpine:After 14 days of continuous intraperitoneal injection of reserpine,the reserpine model significantly reduced the exhaustion latency of mice in the weight-bearing swimming experiment.In the grip test,the reserpine model significantly reduced the mice Muscle strength.In the Y maze experiment,there is a tendency to damage the learni-ng and memory of mice,but there is no significant difference.In the forced swimming experiment,the cumulative immobility time of the mice is significantly increased.In the mechanical pain threshold test experiment,it significantly increased the sensitivety of mice to mechanical pain.TFML?200 mg/kg?with 14 days of continuous administration can significantly improve the drop in exercise tolerance caused by reserpine.TFML?200 mg/kg,400 mg/kg?can significantly reverse the decreasing of muscle strength of mice caused by reserpine,ameliorate depression-like behavior caused by reserpine in mice forced swimming test.TFML?200 mg/kg?can significantly reduce Reserpine induced pain sensitivity in mice.6.TFML can improve the accumulation of metabolites in the serum of mice caused by exercise:Compared with the normal control group,the content of NH3 and LAC in the serum of the mice increased significantly after the exhaustion of the treadmill experiment.TFML?200 mg/kg?significantly reduced the increasing of NH3 in the serum of the mice after exercise7.TFML can protect the mouse colon from damage caused by LPS:Colonic HE staining results of mice showed that:LPS can cause colonic villus rupture,arrangement disorder,and thinner intestinal wall.TFML?200 mg/kg,400 mg/k g?can reverse all the changes listed below.8.The effect of TFML on the intestinal flora of mice with LPS fatigue modelAt the phylum level,Bacteroidetes and Firmicutes are the two species with the highest proportion in each group of samples.At the genus level,the results of species difference analysis showed that the content of g_norank_fMuribaculaceae and g_unclassified_fLachnospiraceae in the LPS group was significantly lower than that of the solvent control group.g_Lactobacillus,Prevotellaceae_UCG The content of 001 was significantly higher than that of the solvent control group.The AG group significantly increased the content of Muribaculaceae and Lachnospiraceae than the LPS group.Compared with LPS group,TFML group significantly increased the content of Muribaculaceae.The AG and TFML groups significantly reduced the content of g_Lactobacillus compared with the LPS group.At the species level,the results of species difference analysis showed that compared to the solvent control group,s_uncultured bacterium_gnorank_f Muribaculaceae,Alloprevotella,s_unclassified f Lachnosp-iraceae,s_uncultur ed_organism_gnorank_fMuribaculaceae in the LPS model group significantly reduced and s unclassified g Lactobacillus,s_uncultured_Bacteroidales bacterium_gnorank_fMuribaculaceae,s_unclassified g_Prevotellaceae_UCG-001 significantly increased.Compa-red with the fatigue model group caused by LPS,s_u-nclassified_g Lactobacillus in A-G group and TFML group was significantly reduced.in AG group,s_uncultured_orga-nism_gnorank_f Muriba-culaceae,s_unclassified_fLachnospiraceae increased significa-ntly and s unclassified_gPrevotellaceae_UCG-001 decrea-sed significantly.s_uncultured_bacterium_gnorank_f Muribaculaceae,s_gut_metagenome_gAlloprevotella in TFML group increased significantly.9.The effect of TFML on the intestinal microflora of reserpine fatigue model miceAt the phylum level,Firmicutes and Bacteroidetes are still the top two fungi.At the genus level,compared with the solvent control group,there was no significant difference among the bacteria in the reserpine fatigue model group.Compared with the fatigue model group caused by reserpine,g_Lachnospira-ceae NK4A136_group?Tricho-sporidae?,g_Candidatus Saccharimonas in AG group significantly reduced,g_Prevotell-aceae UCG-001?Prov-obacteria?,g_norank_fMuribacifidobacterium,_{Bifidobacterium}in AG group increased significantly,g_Candidatus_Sacchari-monas and g_Lact-obacillu s?lactic acid bacteria?increased significantly in TFML group.At the species level,compared with the solvent control group,s_uncultured bacterium_gAlistipes?unculture d Azusa bacteria?,s_uncultured_organism_gnorank f Muribaculaceae content in the res erpine fatigue model group mice significantly reduced;AG group and TFML group did not significantly reverse this trend10,TFML has antidepressant activity:In the behavioral despair model,compared with the solvent control group,TFML?100 mg/kg?administered for 7 days can significantly shorten the immobility time of tail dwelling and forced swimming in mice.TFML?400 mg/kg?single administration can significantly shorten the immobility time of tail suspension and forced swimming in mice.The results of spontaneous activity experiments showed that TFML treated continuously for 7 days or single administration in the range of the received dose had no excitatory or inhibitory effect on the central nervous system of mice.In the mo use 5-HTP induced head shaking experiment,a single administration of TFML 100 mg/kg can significantly increase the number of mouse head shaking.In yohimbin toxicity enhancement experiment,a single administration of TFML?100 mg/kg,200 mg/kg,400 mg/kg?had no significant effect on the mortality of mice caused by yohimbine.In the reserpine antagonism experiment,Compared with the model group,a single admi-nistration of TFML 400 mg/kg can antagonize the drooping of the eyelid caused by reserpine?2.5 mg/kg,ip?,but has no significant effect on the decrease of anal temperature and exercise.Conclusion1.TFML has anti-fatigue activity and significantly improves the performance in behaveoral fatigue paradigm of sports fatigue and chronic fatigue syndrome:The mechanism of sports fatigue may be related to the reduction of accumulation of metabolites and its effects on LPS/reserpine pathological fatigue may be related to the regulation of intestinal flora.2.TFML has antidepressant pharmacological activity,and its mechanism may be related to enhancing the function of the serotoninergic system.