| Phytolaccae Radix(PA)is the dried root of Phytolacca acinosa Roxb.or Phytolacca americana L,which has the effect of expelling water and swelling,PA is used for edema,fecal and urine stoppage.It is also used for various ascites in clinical practice such as renal,cardiac,hepatic ascites and so on.Un-processing PA is toxic and can cause severe gastrointestinal irritation,such as abdominal pain and diarrhea.So vinegar-processing PA is more commonly used in clinical application for its reduced toxicity and side effects.PA is generally processed with vinegar to reduce toxicity,but its mechanism of detoxification is unknown.Previous research of our laboratory group found that PA has both diuretic effect and intestinal toxicity,its toxicity has reduced while diuretic effect retained,after processing with vinegar.Further research found that the n-butanol fraction is considered to be both the main pharmacodynamic and the toxic fraction of PA.The n-butanol fraction of PA is mainly composed of triterpenoid saponins.The composition of saponins in PA has changed after processing with vinegar,some components decreased,and some components did not change significantly,so it is speculated that the toxicity reduction after vinegar processing and the retention of efficacy may be related to the structural transformation of saponins,but the specific transformation mechanism and its correlation with toxic effects are still unclear.We establishes the toxicity and efficacy evaluation model in our research,taking the intestinal inflammatory toxicity and ascites-removing efficacy of PA as the research object to study the toxicity and pharmacodynamic changes of total extracts and total saponins after PA processing with vinegar,then separate and purify the saponins of PA to further study for the structural transformation regularity and changes in toxicity and efficacy after simulating vinegar-processing,the correlation between the change of composition structure and the change of toxicity and efficacy in the process of vinegar-processing,explore the transformation mechanism of toxicity to efficacy in the vinegar-processing of PA and identify the quality markers related to the effect of raw and vinegar-processing PA.The main research works are as follows:1.Study on the changes of efficacy and toxicity of total extracts of PA after processing with vinegarEstablished a mouse model of H22 cancerous ascites and taken the ascitic fluid weight,urine weight,fecal water content in mice with ascites,aquaporin 1-4(AQP1-4)expression of kidney,aquaporin 1、3 expression of colon as indexes to evaluate the medicinal effect of PA and PA after vinegar-processing for removing ascites.At the same time,the intestinal toxicity of PA was evaluated by the release levels of inflammatory factors TNF-α and IL-1β in the jejunum and ileum of normal mice after administration.The results of the efficacy experiment showed that compared to the model group,the total extracts of both PA and vinegar-processing PA can significantly increase the urine weight and fecal water content,and reduce ascitic fluid weight,which indicates that the ascites-removing effect of PA is related to its diuretic and laxative effects.And it also can significantly down-regulated the expression of renal aquaporins AQP1-4 and colonic aquaporins AQP1 and 3.Toxicity results show that,compared to normal group,the total extract of PA can significantly increase the release of inflammatory factors in the jejunum and ileum of normal mice,causing intestinal inflammation,after processing with vinegar,the inflammatory factors were significantly reduced.The above results indicated that the effect of removing ascites was retained while the intestinal toxicity was reduced in PA after processing with vinegar.2.Study on the difference of efficacy and toxicity of the total saponins in PA after processing with vinegarAfter the total extract of PA is extracted by the system solvent,the n-butanol fraction that can enrich saponins is further purified by macroporous resin column chromatography to obtain the fraction of the total saponins fraction in PA(TSPA).According to the efficacy and toxicity indicators screened out in the animal model,the differences in the efficacy of ascites-removing and intestinal inflammation toxicity of the total saponins of PA processing with vinegar were evaluated.The results of the efficacy experiment showed that compared with the model group,the total extract and the total saponin group of PA could significantly increase the urine weight and fecal water content of the ascites model mice,and reduce the ascites weight,can significantly inhibit the expression of AQP 1-4 in kidney and AQP1 and 3 in colon,and there was no significant difference in the efficacy of removing ascites between the total saponins group and the total extract group of PA.The results of the toxicity test showed that,compared with the normal group,the total saponins of PA can significantly increase the release of inflammatory factors in the jejunum of normal mice,causing intestinal inflammation.After processing with vinegar,the levels of inflammatory factors were significantly reduced.The above results show that the total saponins of PA are both the pharmacodynamic fraction for dispelling ascites and the toxic fraction of intestinal inflammation,and the pharmacodynamic effect is still retained while the toxic effect is reduced after processing with vinegar.3.Analysis,Separation and Purification of chemical components of total saponins in PAIn order to further study the mechanism of detoxification and effectiveness of PA processing with vinegar,the components of total saponins in PA were analyzed,separated and purified.LSMS/MS was used to analyze the components of the total saponins in PA,and a total of 20 chemical components were speculated and identified,including 18 saponins.Four main triterpenoid saponins were isolated and identified from TSPA by various techniques such as forward silica gel column chromatography,medium pressure C18 reverse silica gel column chromatography,LH-20 gel column chromatography and high pressure preparative liquid phase.They are Esculentoside A,Esculentoside B,Esculentoside C and Esculentoside H,which content of TSPA was 83.3%analyzed by HPLC.4.Structural transformation and the correlation between toxicity and efficacy of saponins in PA after simulated processing with acetic acidThe isolated triterpenoid saponin compound in PA was simulated with acetic acid,and LCMS/MS was used to analyze the structural transformation and regularity of each component after simulated processing with vinegar.PA from different origins were collected,and the content changes of four saponins before and after processing with vinegar were determined by HPLC to verify the law of simulated processing.And on the basis of the research on the structural transformation of the above-mentioned saponins in PA after processing with vinegar,the correlation between the structural transformation and the change of toxicity and efficacy was investigated.Pharmacodynamic evaluation is used Western blot to determine the protein expression levels of AQPs in Human kidney-2 cells(HK-2)and Murine inner medullary collecting duct cells(mIMCD3).Toxicity evaluation is used ELISA kit to determine the release levels of TNF-α and IL-1β from macrophage cells(RAW264.7).The experimental results show that EsA and B are relatively stable and there is no obvious structural transformation after simulated processing with vinegar.C-28 ester bond of EsH was hydrolysised under heating with acetic acid and EsC undergoes hydrolysis of the glycosidic bond at the C-3 position.The results of content determination showed that the content of EsH,EsB and EsC decreased,while the content of EsA remained basically or slightly increased after processing with vinegar.The results of pharmacodynamic studies showed that the pro-inflammatory effects of EsH and EsC were reduced after simulated processing with vinegar,and the diuretic effects were enhanced or retained,which was consistent with the overall animal experimental results.It further indicated that the conversion of saponins may be the material basis of reducing toxicity and retaining efficacy in PA after processing with vinegar.Summary1.Starting from the intestinal inflammatory toxicity and ascites-removing efficacy of PA,and evaluating the toxicity and efficacy of the overall animal,the study showed that the intestinal toxicity was significantly reduced after processing with vinegar,while the ascites-removing efficacy was still retained,and the total saponins of PA are both the main pharmacodynamic and toxic fraction of PA.2.A variety of saponins were speculated and identified in the total saponins of PA by LC-MS/MS technology,and four triterpenoid saponins were obtained by further separation and purification.The isolated triterpenoid saponin compound in PA was simulated with acetic acid to analyze the structural transformation and regularity of each component after simulated processing with vinegar.The experimental results show that EsA and B are relatively stable and there is no obvious structural transformation after simulated processing with vinegar.C-28 ester bond of EsH was hydrolysised under heating with acetic acid and EsC undergoes hydrolysis of the glycosidic bond at the C-3 position.3.The differences in toxicity and efficacy of two compounds,EsH and EsC,which undergo structural transformation after simulated processing with vinegar,were evaluated by HK-2 cells,mIMCD4 cells and RAW264.7cells respectively.The results showed that the pharmacodynamic effects of EsH and EsC were enhaned or retained after the simulated processing,and the toxic effects were reduced.It indicated that the transformation of saponins may be the material basis of reducing toxicity and retaining efficacy in PA after processing with vinegar. |
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