Study On The Effect Of Expelling Water Retention With Drastic Purgative And The Toxicity Mechanism On Basis Of“YOU-GU-WU-YUN”Theory Of Diterpenoids In Kansui Radix Stir-baked With Vinegar

Kansui(short for the dried roots of T.N.Liou ex T.P.Wang),which was bitter,cold,toxic,and recorded in Shennong-Bencao,has multiple medical applications for edema,fullness,chest and abdomen ponding,retention of phlegm and fluid embolism.Kansui is widely used in clinical treatment of ascites due to cirrhosis,nephritis edema,infiltration of pleurisy and ascites due to advanced schistosomiasis in China for centuries.Kansui has serious irritant to the oral cavity,gastrointestinal tract,skin and eye mucous membrane,and inflammation,promoting tumor toxicity.Ancient Chinese medicine often used vinegar method to decrease the toxicity,easing its resistance,based on the guiding principle“YOU-GU-WU-YUN”theory.However,modem research of Euphorbia kansui paid more attention to toxicity and toxic mechanism of reduction in normal body,still could not scientifically reveal the connotation of toxic drug application based on“YOU-GU-WU-YUN”theory,severely restricted the clinical application.At the same time,how the effect of expelling water retention with drastic purgative change before and after stir-baking was also not clear meanwhile reducing the toxicity.The model doctors had no idea of its change of effect thus abandoned it fearing of its toxicity and fierce purgation,seriously restricted the clinical application of Euphorbia kansui.Therefore,the toxicity and effect on the animals under normal and pathological state of kansui before and after stir-baking with vinegar and the active ingredient groups of kansui were studied based on“YOU-GU-WU-YUN”theory,in order to further elucidate the substance basis of expelling water retention with drastic purgative of Kansui Radix stir-baked with vinegar and to provide a basis for the mechanism of“YOU-GU-WU-YUN”theory.The main content of this paper includes the following parts:Part one:Literature SearchThrough the review of the related literature at all times and in all over the world,processing technology,chemical constituents,pharmacological effects,toxicology,clinical application of Kansui Radix stir-baked with vinegar and ascites syndrome pathogenesis,drastic water-expelling traditional Chinese medicine were summarized,The progress of Kansui Radix stir-baked with vinegar and drastic water-expelling traditional Chinese medicine for the treatment of ascites was reviewed in this paper,providing a reference for the further development of traditional Chinese medicine.Part two:Investigation of the toxicity and effect on animals under normal and pathological state of Kansui Radix before and after stir-baking with vinegarThe experiment studied in the ways of liver,stomach and intestine pathological morphology,liver function,oxidative damage,urine volume,volume of ascites,urinary sodium,potassium chloride levels,urine pH value,RAAS system,and ADH to explore the differences of effect of expelling water retention with drastic purgative and toxicity of kansui before and after stir-baking with vinegar on the normal and cancerous ascites model rats.The results showed that,crude and stir-baked with vinegar of Kansui Radix and their extracts could induce tissue injury to a certain degree on normal rats,leading to high activity of AST,ALT and ALP,concentration decrease of GSH,SOD,concentration increase of MDA,LDH in serum.And they could significantly reduce the tissue damage caused by model,reduce the concentration of AST,ALT,ALP,MDA.LDH.and increase the concentration of GSH,SOD in serum of model rats.Crude and stir-baked with vinegar of Kansui Radix and their extracts could increase the amount of urine and decrease the ascites volume,the content of urine sodium,potassium and chloride levels,urine pH,PRA,Ang Ⅱ,ALD and ADH in serum of model rats significantly.On normal rats,stir-baking with vinegar could reduce the toxicity of Kansui Radix;the toxicity of crude and stir-baked with vinegar of Kansui Radix decreased on model rats,and they could even significantly alleviate the damage caused by model.Crude and stir-baked with vinegar of Kansui Radix could expel water retention with drastic purgative impactfully,and had good effect on the symptoms of model rats.Part three:Investigation of the toxicity and effect in animals under normal and pathological state of active ingredient groups of Kansui Radix stir-baked with vinegarWith decompression silica gel chromatography.active ingredient of Kansui Radix stir-baked with vinegar were separated as group A,B,C,D.With normal and cancerous ascites model rats as the research object,the liver,stomach and intestine pathological morphology.liver function,oxidative damage,inflammatory injury,apoptosis,urine volume,volume of ascites,urine sodium and potassium chloride level urine pH,PRA,Ang Ⅱ,ALD,ADH,AQP8 expression and immune inflammatory indexes were explored to study the effect of expelling water retention with drastic purgative and toxicity of each group of active ingredient.Results showed that group B and C could cause different degrees of tissue injury,lead to high activity of AST,ALT and ALP,concentration decrease of GSH,SOD,concentration increase of MDA,LDH in serum,and expression increase of NF-κB,ICAM-1,caspase 3.8.9.Bax.expression decrease of E-cadherin,Bcl-2 in liver,stomach and intestine.And it could significantly reduce the tissue damage caused by model,reduce the concentration of AST,ALT,ALP,MDA,LDH,increase the concentration of GSH,SOD in serum,and decrease NF-κB,ICAM-1,caspase 3,8,9,Bax,increase E-cadherin,Bcl-2 expression in liver,stomach and intestine.Group B and C could increase the amount of urine and decrease the ascites volume,urine sodium,potassium and chloride levels,urine pH,PRA,Ang Ⅱ,ALD and ADH content in serum,increase gastrointestinal AQP8 expression,and decrease content of ascites immune anti-inflammatory cytokines IL-2,IL-6,TNF-α,INF-γ、VEGF of model rats significantly.Compared with that on normal animoals,the toxicity of active ingredient group B and C decreased on model rats,and could even significantly alleviate the damage caused by model.Active ingredient group B and C could expel water retention with drastic purgative impactfully,and had good effect on the symptoms of model rats.The mechanism might be related to the regulation of RAAS and immune inflammation.Part four:Qualitative analysis of active ingredient groups of Kansui Radix stir-baked with vinegarQualitative analysis of active ingredient groups of Kansui Radix stir-baked with vinegar was done with UPLC-Q-TOF/MS.Ingenane type diterpenes were mainly found in group B,and Jatrophane type diterpenes were mainly found in group C.That two might be the main toxicity-effect components of Kansui Radix stir-baked with vinegar.Part five:Component separation of ethyl acetate part of Kansui Radix stir-baked with vinegarWith silica gel,preparative HPLC and TLC as main methods,fourteen compounds were isolated from ethyl acetate extract of Euphorbia kansui and twelve of them were elucidated.Five ingenane-type diterpenes were named as 3-O-(2’E,4’Z-decadienoyl)-20-O-acetylingenol(EK-01),3-O-(2’E,4’E-decadienoyl)-20-O-acetylingenol](EK-02),3-O-(2,3-dimethylbutanoyl)-13-O-dodecouldoyl-20-deoxylingenol(EK-03),3-O-benzoyl-20-deoxyingenol](EK-04),5-O-benzoyl-20-deoxyingenol(EK-05),three jatrophane-type diterpene was named as kansuinin A(EK-08),kansuinin B(EK-09),kansuinin C(EK-10),two triterpenes such as euphol(EK-06),epi-kansenone(EK-07),other types such as p-sitosterol(EK-11),isoscopletin(EK-12).