| Background:Gout is a disease of recurrent acute arthritis caused by MSU crystallizing,depositing and then interacting with tissue microenvironment.It is reported that the prevalence of gout in China is 1%-3%,with a rising trend year by year.it is often associated with some diseases such as obesity,sugar,lipid metabolic disorder,hypertension,atherosclerosis and coronary heart disease,even serious disability.Acute episode treatment of gout should be given priority to resist gouty arthritis,but its intermittent period of treatment is given priority to lower uric acid.At present,the treatment of gout is given priority to with western medicine,such as Allopurinol,Benzbromarone,Colchicine,but some adverse reactions may occur with the treatment.To study the therapeutic effect of herbal compound on Gout would be of great academic and clinical value for its less adverse reactions and other characteristics.The Er-Qin-Ju-Ling decoction derived from the clinical experience of national physician master Yan Zhenghua treating of rheumatism combined with our lab’s experience to prevention and treatment of hyperuricemia for more than 20 years.However,its prescription composition has not yet been optimized,and its extraction technology,acute toxicity and effect resisting to gout have not been fully elucidated.Thus,by means of orthogonal design combined with pharmacology and analytical chemistry methods,this research will optimize the prescription,extraction technology of Er-Qin-Ju-Ling decoction,observe its acute toxicity,reveal its effects on hyperuricemia and acute gouty arthritis.Objectives:1)Observe the effects of Er-Qin-Ju-Ling decoction under the traditional water extraction based on orthogonal design on the level of serum UA,TNF-α in rats,and the joint swelling degree and take them as evaluation indexs respectively to optimize its prescription interventing with hyperuricemia and acute gouty arthritis.2)Determine extract content,total polysaccharides,total coumarin,total iridoid glycosides,esculin,gentiopicroside and cichoric acid of Er-Qin-Ju-Ling decoction and optimize the process for Er-Qin-Ju-Ling decoction by comprehensive score.3)Observe the acute toxicity of Er-Qin-Ju-Ling decoction and its effects on the levels of serum UA,ADA,XOD and discuss its mechanism from the approach uric acid formation;Observe its effects on the auricle swelling,twisting reaction induced by acetic acid and hot plate reaction in mices;Observe its effects on the joint swelling degree,the levels of TNF-α,IL-1β of tissue in gouty arthritis rats to discuss its mechanism.Research contents and methodsThis paper consists of two parts,literature review and experimental research.Literature review mainly gather the etiology and epidemiology progress of gouty arthritis and hyperuricemia,the treatment progress of hyperuricemia and gouty arthritis and the progress in chemical constituents and pharmacological effect for anti-gout of Gentianae Macrophyllae Radix,Fraxini Cortex,Cichorii Herba,Poria cocos of Er-Qin-Ju-Ling decoction in recent more than 20 years.The experimental research part by means of orthogonal design combined with pharmacology and analytical chemistry methods,optimize prescription and extraction technology of Er-Qin-Ju-Ling decoction,and then reveal the influence of Er-Qin-Ju-Ling decoction on acute gouty arthritis and hyperuricemia.Experimental study section is divided into three chapters:The first chapter:The research on prescription optimization of Er-Qin-Ju-Ling decoction for anti-gout based on orthogonal design.In the orthogonal design,take Gentianae Macrophyllae Radix,Fraxini Cortex,Cichorii Herba,Poria cocos of Er-Qin-Ju-Ling decoction as its four factors,set up three levels for its every factor;Observe the influence of traditional extracted-water Er-Qin-Ju-Ling decoction based on the orthogonal design on the levels of UA and TNF-α in hyperuricemia rats induced by 10%fructose water and the joint swelling degree in acute gouty arthritis rats induced by3%MSU water,and then,take them as evaluation indexs respectively to optimize the best prescription of Er-Qin-Ju-Ling decoction.The second chapter:The research on extraction technology optimization of Er-Qin-Ju-Ling decoction based on orthogonal design.In the orthogonal design,take ethanol concentration,extraction time,extraction times and solid-liquid ratio as its four factors,set up three levels for its every factor;determine extract content,total polysaccharides,total coumarin,total iridoid glycoside,esculin,gentiopicroside and cichoric acid of Er-Qin-Ju-Ling decoction based on the orthogonal design,and then optimize the extraction technology for Er-Qin-Ju-Ling decoction by comprehensive score.The third chapter:The research on acute toxicity and intervention effect of Er-Qin-Ju-Ling decoction on acute gouty arthritis and hyperuricemia.Observe the acute toxicity of Er-Qin-Ju-Ling decoction and its effects on the levels of serum UA,ADA,XOD to discuss its mechanism from the approach uric acid formation;Observe its effects on the auricle swelling,twisting reaction induced by acetic acid and hot plate reaction in mices;Observe its effects on the joint swelling degree in gouty arthritis rats,the levels of TNF-α,IL-1β in the tissue of gouty arthritis rats to discuss its mechanism.Results:1)The results on prescription optimization of Er-Qin-Ju-Ling decoction.The influence of four factors on the serum UA levels in rats is B>C>A>D,and among them,the influence of factor B,factor C,factor A have significant difference(P<0.01 or P<0.05);The influence of four factors on the serum TNF-α levels in rats is C>D>A>B,and among them,the influence of factor C has significant difference(P<0.01);The influence of four factors on joint swelling degree in rats is D>C>A>B,and among them,the influence of factor C and factor D have significant difference(P<0.01).Comprehensive analysis shows that A2B3C2D3(namely,chicory 9g,gentiana macrophylla 6g,cortex fraxini 6g,poria cocos 15g)is the optimum prescription.2)The results on extraction technology optimization of Er-Qin-Ju-Ling decoction.The influence of four factors on comprehensive scores of all ingredients is B>C>A>D,and among them,the influence of factor A,factor B,factor C and factor D have significant difference(P<0.01).Comprehensive analysis shows that B3C3A2D3(namely,namely,thanol concentration 50%,extraction time 90 min,extraction times 3 and solid-liquid ratio 1:13)is the optimum extraction technology.3)Acute toxicity and influence of Er-Qin-Ju-Ling decoction on the levels of serum UA,XOD and ADA in hyperuricemia rats.LD50 of Er-Qin-Ju-Ling decoction is 114.5176g·kg-1,equivalently human’s 191 times.Compared with normal group,model group serum UA levels and XOD activity levels were significantly elevated on the 14th and 21th day(P<0.05),model group serum ADA activity levels were significantly increased on the 21th day(P<0.05);compared with model group,benzbromarone group serum UA levels were significantly decreased on the 21th day(P<0.05);Er-Qin-Ju-Ling decoction high-dosegroup serum UA levels significantly decreased on the 14th,21th day(P<0.05,P<0.01),Er-Qin-Ju-Ling decoction high-dose group and low-dose group serum XOD level were significantly decreased on the 21th day(P<0.05),Er-Qin-Ju-Ling decoction high-dose group and middel-dose group serum ADA level significantly decreased on the 14th,21th day(P<0.05 or P<0.01).4)Influence of Er-Qin-Ju-Ling decoction on swelling auricle,twisting reaction and hot plate reaction in mice and swelling joint in ratsDexamethasone group auricle swelling degree in mice compared with control group were significantly lower(P<0.01)and Er-Qin-Ju-Ling decoction high and middle dose group significantly lower(P<0.01,P<0.05);15 min after gavage,injecting acetic acid in mice intraperitoneally,indomethacin group twisting frequency was significantly reduced(P<0.01),and Er-Qin-Ju-Ling decoction high-dose group twisting frequency significantly lower than the control group(P<0.01);30 min after gavage,Er-Qin-Ju-Ling decoction high,middle and low dose group pain threshold were significantly elevated(P<0.05);4h,8h,16h,24h,48h after modelling,model group rats joint swelling degree increased significantly compared with control group(P<0.05 or P<0.01),24h after modelling,joint swelling degree of colchicine group decreased significantly compared with model group(P<0.01),16h,24h after modelling,joint swelling degree of Tong-Feng-Ding group were significantly decreased compared with model group(P<0.05),24h after modelling,Er-Qin-Ju-Ling decoction high-dose group joint swelling degree decreased significantly compared with model group(P<0.01);model group rats ankle organization IL-1β levels increased significantly compared with the normal group(P<0.05),colchicine group IL-1β level decreased significantly compared with model group(P<0.01),and Er-Qin-Ju-Ling decoction high-dose group,IL-1β level decreased significantly compared with model group(P<0.05).Conclusions:Er-Qin-Ju-Ling decoction(chicory 9g,cortex fraxini 6g,gentiana macrophylla 6g,poria cocos 15g)extracted by the extraction technology(extraction time:90 min,extraction times:three times,ethanol concentration:50%,material liquid ratio:1:13),belongs to the non-toxic level.It can inhibit the activity of serum XOD,ADA levels to reduce the serum UA level,and inhibit swelling auricle induced by xylene,twisting reaction induced by acetic acid,increase the pain threshold value,and also can inhibit joint organization IL-1β levels rise to improve gouty arthritis rats ankle swelling,so as to achieve the purpose of the treatment of gout,and two Qin Juling soup smaller effect on renal function.Innovation points1.Optimize the prescription and extraction technology of Er-Qin-Ju-Ling decoction for the first time,obtain the optimum extraction technology of the optimum prescription to Er-Qin-Ju-Ling decoction,and reveal its safety and pharmacological effects for anti-inflammatory,analgesic and anti-gouty arthritis.2.Explore the effect mechanism of Er-Qin-Ju-Ling decoction on gouty arthritis and for hyperuricemia the first time,and find that the uric acid-lowing effect of Er-Qin-Ju-Ling decoction be related to its inhibition to XOD and ADA activity levels and that its effect for anti-gouty arthritis be related to its inhibition to IL-1β levels. |
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