| Dark tea,as a unique microbial fermented tea in China,is manufactured in many provinces,including Yunnan,Hunan,Hubei,Sichuan and so on.The tea breeds of raw material,processing technology and technical requirements are differently in the different producing areas.For this reason,there are many categories of dark tea with different quality characteristics.In the processing of dark tea,the microbial fermentation process generates special chemical composition that different from other teas,which establishes the material basis of various health effects.As the domestic and foreign scholars’ in-depth study of health functions of drak tea,combining with the good experience of consumers,dark tea consumption has gradually become a fashion.At present,gout or hyperuricemia has become a commonly occurring metabolic disease.However,most of the drugs have adverse reactions.Therefore,safe and effective measures for gout or hyperuricemia prevention and treatment are urgently warranted.For a long time,most studies on dark tea are focusing on weight-loss,hypolipidemic,hypoglycemic,anti-oxidation and so on,yet there are few studies and reports about dark tea anti-hyperuricemia.As a result,most consumers know little about this aspect,and which affects the comprehensive understanding of beneficial effects of dark tea.For these reasons,we chose six different kinds of dark tea which were used to verify whether dark tea had the effect of lowering levels of uric acid.Simultaneously,we intended to explain the mechanism of dark tea lowering uric acid levels from animal biochemical levels,molecular levels and metabolic levels.The main contents and results of this study were as follows:1.Establishment of animal model of hyperuricemia and exploration of effective dose of dark teaA mice model of hyperuricemia was successfully established by intraperitoneal injection of potassium oxonate at 300mg/kg.This method is simple,reproducible and has high success rate,which will be used in the study of the effects of dark tea on lowering uric acid levels.A mice model of hyperuricemia was successfully induced by oral administration of potassium oxonate at 250mg/kg for 7 days.This model has good stability,which will be used in the research on the mechanism and metabolomics.A rat model of hyperuricemia was successfully established by oral administration of adenine at 150 mg/kg and potassium oxonate at 150mg/kg for 21 days.This model is more stable and has induced significant renal damage,which is suitable for the study of drugs improving renal dysfunction and metabolomics.To explore the effective dose of dark tea,intraperitoneal injection of potassium oxonate at 300mg/kg was used to establishe hyperuricemic mice.At last,0.5g/kg/d,1g/kg/d and 2g/kg/d were determined as low dose,medium dose and high dose in the further research,respectively.2.Analysis of main chemical constituents of dark tea and its effects on hyperuricemic miceFirstly,we analyzed the main chemical composition of the six kinds of dark tea water extract and detected the content of phenolic compounds especially.We found that there were significant differences of the main chemical composition between the six kinds of dark tea water extract.Tea polyphenol content was highest in Guangdong chenxiang cake-tea(40.66%),while it was lowest in Danxia jiamu pu-erh ripened tea(16.57%).Water extracts of Danxia jiamu pu-erh ripened tea(9.05%)and Guangdong chenxiang cake-tea(9.0%)had more content of caffeine,while Danxia jiamu qingzhuan-tea(3.93%)had the lowest content of caffeine.Six kinds of dark tea water extract contained a certain amount of flavonoids(3.08%-5.56%),soluble sugars(10.79%-17.63%)and theabrownins(8.23%-14.76%),while they all had less theaflavins(0.07%-0.17%),thearubigins(0.06%-1.73%)and free amino acids(1.24%-2.11%).Through the detection of catechins and other phenolic compounds,we found that they all had a variety of phenolic compounds.SPF male Kunming mice(210,20±2g)were randomly grouped as follows:normal control group,model control group,allopurinol group(20mg/kg/d),dark tea extract high dose group(2g/kg/d),dark tea extract medium dose group(1g/kg/d),dark tea extract low dose group(0.5g/kg/d).To study the effect of dark tea on lowering serum levels of uric acid,allopurinol,high dose dark tea extract,medium dose dark tea extract and low dose dark tea extract were intragastrically administered to hyperuricemic mice for 7 days.At the last day,intraperitoneal injection of potassium oxonate at 300mg/kg was used to induce hyperuricemic mice.In results,six kinds of dark tea all could reduce the serum uric acid levels in hyperuricemic mice in different degrees,and Guangdong chenxiang cake-tea showed the best effects.In addition,six kinds of dark tea all could inhibit the activity of liver XOD in mice with hyperuricemia.Simultaneously,five kinds of dark tea could inhibit the activity of liver ADA.Inhibiting the activities of liver XOD and ADA,thereby reducing the production of uric acid in the body,may be one of the reasons that dark tea could lower the serum uric acid levels.3.Study on the mechanism of dark tea lowering uric acid levelsTo investigate the mechanism of dark tea lowering uric acid levels,Pu-erh ripened tea was researched from two hands that reducing uric acid production and promoting uric acid excretion.SPF male Kunming mice(60,20±2g)were randomly grouped as follows: normal control group,model control group,allopurinol group(20mg/kg/d),Pu-erh ripened tea extract high dose group(2g/kg/d),Pu-erh ripened tea extract medium dose group(1g/kg/d),Pu-erh ripened tea extract low dose group(0.5g/kg/d).Hyperuricemia was induced in the mice by oral administration of potassium oxonate for 7 days.Serum levels of uric acid,liver xanthine oxidase(XOD)and adenosine deaminase(ADA)activities were detected.At the same time,expression of renal m URAT1,m GLUT9,m OAT1 and m OCT1 protein and m RNA were measured by western blotting and q RT-PCR,respectively.Pu-erh ripened tea,at all doses,significantly lowered serum uric acid levels.At the high and medium doses,it also markedly inhibited liver XOD and ADA activities.Furthermore,m URAT1 and m GLUT9 protein and m RNA expression levels were significantly down-regulated by Pu-erh ripened tea treatment.These results suggested that Pu-erh ripened tea can resist to hyperuricemia,possibly by inhibiting,concurrently,uric acid production and renal urate reabsorption.4.Metabolic effects of dark tea on hyperuricemic mice studied by serum metabolomics based on GC-MSTo investigate the metabolic effects of dark tea on hyperuricemic mice,Pu-erh ripened tea was tested in hyperuricemic mice using serum metabolomics based on GC-MS.SPF male Kunming mice(60)were randomly grouped as follows: Normal control group,model control group,allopurinol group(20 mg/kg/d),Pu-erh ripened tea extract high dose(2g/kg/d)group,Pu-erh ripened tea extract medium dose(1g/kg/d)group,Pu-erh ripened tea extract low dose(0.5g/kg/d)group.A mouse hyperuricemia model was developed by oral administration of potassium oxonate for7 d.Serum metabolomics,based on GC-MS,was used to generate metabolic profiles from normal control,hyperuricemic and allopurinol-treated hyperuricemic mice,as well as hyperuricemic mice given Pu-erh ripened tea at three doses.Through screening for differential metabolites between the normal control group and the model control group,twelve potential biomarkers associated with hyperuricemia were identified,which were primarily involved in pyrimidine,amino acid,purine,fatty acid and energy metabolism.Differential metabolites were analyzed and selected between allopurinol group,Pu-erh ripened tea at three doses-treated groups and the model control group,respectively.We found that Pu-erh ripened tea and allopurinol differed in their metabolic effects in the hyperuricemic mice.Levels of glutamic acid,hydroxyacetone,4-hydroxybutyrate,indolelactate,L-allothreonine,nicotinoylglycine,isoleucine,L-cysteine and glycocyamine,all involved in amino acid metabolism,were significantly changed in hyperuricemic mice treated Pu-erh ripened tea.Thus,modulating amino acid metabolism might be the primary mechanism of anti-hyperuricemia by Pu-erh ripened tea. |
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