Study On The Effect And Mechanism Of Phytolaccae Radix After Vinegar Processing In Toxicity Reducing And Efficacy Retaining

Phytolacca radix,used as a traditional chinese diuresis medicine,is derived from the root of Phytolacca acinosa Roxb.or Phytolacca americana L.It is poisonous and has pharmacodynamic effects of diuresis,defecation and detumescence.Improper use can lead to toxic symptoms of nausea and vomiting,abdominal pain and diarrhea,dyspnea,arrhythmia,and serious cases can cause hematemesis,hematochezia,coma,cardiac and respiratory paralysis to death.Therefore,the clinical application must be processed to reduce toxicity.At present,it is mainly used to treat kidney edema,cirrhosis ascites and other diseases.The current processing method of phytolaccae radix collected in Chinese Pharmacopoeia is vinegar processing and is stipulated that the content of esculentoside A(EsA)should not be less than 0.20%.Previous studies found that the main toxic part of phytolaccae radix is n-butanol,and vinegar processing can reduce the toxicity of n-butanol.The main toxic components of phytolaccae radix are EsB and EsC.The vinegar-processing can significantly change the composition of saponins and reduce the content of toxic saponins.Studies have suggested that Esculentoside A(EsA)is the main active ingredient of phytolacca,which has diuretic and anti-inflammatory effects.It shows that many saponins in phytolacca are related to their toxicity andpharmacodynamics.However,the toxicity and pharmacodynamics of the saponins may vary due to their different structures.The diuretic effect of phytolaccae radix after vinegar processing is different in the existing literature study,and the mechanism of its detoxification and diuretic effect preservation after vinegar processing is still unclear,which needs further study.In this paper,the raw products and vinegar processing products of phytolacca were taken as research objects,and the saponins in the active parts of phytolacca were separated and purified.The changes of pharmacodynamics and toxicity of phytolaccae radix before and after vinegar processing and different saponins were evaluated at the level of whole animal and cell molecule.The correlation between the changes of composition before and after vinegar processing and the diuretic and toxic effects was analyzed.The mechanism of reducing toxicity and preserving diutetic effect of phytolaccae radix after vinegar processing was preliminarily clarified.The main experimental contents and results are summarized as follows:1.Study on the diuretic effect and the inflammatory toxicity of phytolaccae radix water extract before and after vinegar processingTo establish a model of renal diuretic efficacy and intestinal inflammatory toxicity in saline-loaded rats,the rats were given normal saline for water load before the administration.The diuretic effect of phytolacca on rats was investigated by measuring urine volume,urine electrolyte content,contents of plasma ADH(antidiuretic hormone),ALD(aldosterone)and ANP(atrial natriuretic peptide)and protein expression levels of renal aquaporins(AQPs)and Sodium transporters.At the same time,the intestinal water content and the mRNA expression of inflammatory factors TNF-alpha and IL-lbeta in intestinal segments of saline-loaded rats were used as indicators to evaluate the intestinal inflammatory toxicity of phytolaccaThe results showed that both before and after vinegar processing of phytolacca could significantly increase the urine volume and electrolyte content of saline-loaded model rats,reduce the content of ADH content and inhibit the expression of AQP1,AQP2,AQP3,NHE3 and ATP protein;the raw product can significantly inhibit the expression of AQP4 and NCC protein,the vinegar processing product can significantly reduce the content of ALD and increase the content of ANP.Compared with the raw product,vinegar processing product significantly increased urine volume,urinary potassium,urinary sodium and ANP content,and significantly inhibited the protein expressions of AQP3,NHE3 and ATP.The results of intestinal toxicity study showed that compared with the model group,the water content of jej unum and ileum and the mRNA expression levels of TNF-alpha and IL-lbeta in raw product group were significantly increased,but there was no significant difference in the water content of intestinal segments in vinegar processing product group.Compared with the raw product group,the expression levels of TNF-alpha and IL-1beta in jejunum and ileum of vinegar processing product group were significantly lower.The above experimental results showed that phytolaccae radix had strong diuretic effect before and after vinegar processing.Raw products could cause intestinal inflammation.After processing with vinegar,intestinal inflammation was alleviated,and diuretic effect could be preserved to achieve the purpose of detoxification and effect retaining2.Study on screening of diuretic and inflammatory active parts and separation and purification of components from phytolaccaCombining with the above experimental results of overall animal pharmacodynamics,the pharmacodynamics evaluation model of mouse renal medullary collecting duct 3 epithelial cells(mIMCD3)in vitro culture was established with AQP2 and AQP3 as diuretic pharmacodynamics evaluation indicators,and the toxicity evaluation model of mouse macrophages(RAW264.7)in vitro culture was established with the release levels of macrophage inflammatory factors TNF－alpha and IL-lbeta as inflammatory toxicity evaluation indicators to screen the effective and toxic parts of phytolacca.Ethyl acetate and n-butanol were used to extract the water extract of phytolacca.Ethyl acetate fraction,n-butanol fraction and water fraction of phytolacca were given to mIMCD3 and RAW264.7 cells respectively.The protein expressions of AQP2,AQP3 in mIMCD3 cells and the levels of TNF-alpha and IL-lbeta in RAW264.7 cells were detected.The results showed that the n-butanol fraction of phytolacca had the strongest inhibitory effect on the protein expressions of AQP2 and AQP3 in mIMCD3 cells.Compared with other extracts,the n-butanol fraction significantly increased the levels of TNF-alpha and IL-1beta released by macrophages,suggesting that the n-butanol fraction of phytolacca was not only the main part of diuretic effect but also the main part of inflammatory toxicityTaking n-butanol part of phytolacca as the research object,the compounds of n-butanol fraction were separated by many types of separation methods such as silica gel column chromatography,C18 column chromatography and Sephadex LH-20 gel column chromatography Results showed that there are three main monomer compounds isolated and purified from the n-butanol fraction of phytolacca,and their structures were identified by NMR as EsculentosideA(EsA)?EsculentosideB(EsB)and EsculentosideH(EsH).3.Study on diuretic efficacy and inflammatory toxicity of EsculentosidesApply the in vitro pharmacodynamic evaluation model of the mouse kidney inner medullary collecting duct 3 epithelial cells(mIMCD3)and inflammatory toxicity evaluation model of mouse macrophages(RAW264.7)mentioned above,set the protein expression of AQP2 and AQP3 in mIMCD3 cells and the levels of TNF-alpha and IL-lbeta in RAW264.7 cells as indicators to compare the diuretic effects and inflammatory toxicities of EsA,EsB and EsHThe results showed that EsA could significantly inhibit the expression of AQP2 protein,while EsB and EsH could significantly increase the expression of AQP2 protein,all of which could significantly inhibit the expression of AQP3 protein.AQP2 distributes in the parietal membrane and intracellular vesicles of renal collecting duct cells.AQP3 is located outside the basement membrane,providing an excretion pathway for water reabsorbed by AQP2.AQP2 is the dominant protein in water reabsorption,indicating that EsA is the main component of diuretic efficacy of phytolacca;EsA could significantly inhibit the release of TNF-alpha and IL-1beta from RAW264.7 cells,EsB and EsH could significantly promote the release of TNF-alpha,and EsH is more toxic than EsB,indicating that EsA was nontoxic and EsH was the main component of inflammation toxicity of phytolacca.According to the experimental results of pharmacodynamics and toxicity,EsA is the main diuretic active ingredient and EsH is the main inflammatory toxic ingredient of phytolacca.4.Study on the effects of Esculentosides components processed by vinegarEsA and EsH,the main diuretic active and the main inflammatory toxic ingredients of phytolacca,were used as the research obj ects to prepare simulated vinegar processing.The changes of components of EsA and EsH after the preparation of simulated vinegar processing were qualitatively analyzed by HPLC-ELSD.The results showed that EsA and EsH did not show any new components when heated with water;EsA partially decomposed into new compounds during simulated vinegar processing;EsH changed its chemical structure during simulated vinegar processing,and could be converted into new compounds similar to EsA during simulated vinegar processing.By comparing the structures of EsA and EsH,it was found that the ester bond at EsH C-28 was more than that of EsA to connect one molecule of glucose.Combining the results of simulating the changes of vinegar processing,it was inferred that EsH could hydrolyze ester bond under acidic conditions of vinegar processing,remove one molecule of glucose,and convert to EsA.The contents of EsH and EsA in phytolacca were 0.27%and 0.60%,and the contents of EsH in phytolacca after vinegar processing decreased 45.88%significantly while the EsA remained unchanged,decreasing 2.51%Combining with the above experimental results on the changes of saponins,pharmacodynamics and toxicity before and after vinegar processing,the preliminary mechanism of the toxicity reduction of phytolacca by vinegar processing is revealed as follows:during the vinegar processing,the saponins EsH,which mainly causes inflammation toxicity,can hydrolyze ester bonds under acidic conditions to produce EsA with strong diuretic activity,so as to achieve the processing purpose of reducing toxicity and retaining efficacySummary:the effect and mechanism of vinegar-process on reducing intestinal inflammation toxicity and retaining diuretic efficacy of phytolacca were clarified through animal experiments,the main diuretic and inflammatory toxic part of phytolacca was n-butanol part,which are screened out by cell model,and three triterpenoid saponins,EsA,EsB and EsH,were isolated and identified from the part of n-butanol by means of column chromatography;And EsA can significantly inhibit the expression of AQP2 and AQP3 in renal cells,EsH can significantly promote macrophages to release inflammatory factor TNF-alpha.Therefore,EsA is the main diuretic active ingredient of phytolacca,EsH is the main inflammatory toxic ingredient.By comparing the structure and preparation of simulated vinegar processing,it was found that vinegar processing could hydrolyze the ester bond of EsH and then transform it into EsA.The content of EsH in phytolacca decreased significantly and EsA remained unchanged after vinegar processing.The results showed that vinegar processing could transform the main inflammatory toxic ingredients into the main diuretic active ingredients of phytolacca,so as to achieve the processing purpose of reducing toxicity and retaining efficacy.This paper demonstrated the rationality and scientificity of detoxification and diuretic effect retaining of phytolacca processed by vinegar,and preliminarily clarified the processing mechanism of phytolacca processed by vinegar,providing a basis for the production,quality control and clinical application of stalks.