Studies On The Intestinal Toxicity And Attenuated Mechanism After Being Processed Of Phytolaccae Radix

Thesis of Master Degree was supported by National scientific research project of traditional Chinese Medicine, "Research on the key technology of the integration of habitat processing and further processing of 30 Chinese Herbal Pieces" (NO.201507002)Radix phytolaccae (Chinese name is shanglu) is derived from the root of phytolacca acinosa Roxb. Or P.americana L. It is widely used as diuretic in traditional Chinese clinics.Because of its violently toxic potency,Radix phytolaccae has been long considered as toxic medicine.The patients have the pathological changes of digestive system, cardiovascular system, respiratory system and nervous system. Symptoms of them are nausea, vomiting, abdominal pain and diarrhea accompanied by dizziness, headache, elevated body temperature, heartbeat, shortness of breath, serious slurred speech, restless convulsions, coma and even death.Because of its toxicity,the medicinal materials must be processed before clinical use, and represented by "vinegar processed product".Processed with vinegar can reduce toxicity and the adverse reaction of the medicine.Diarrhea and intestinal mucosal irritation is the most widely toxic side effects of Phytolacca Radix.Previos study found that Esculentosides induced rabbit conjunctival edema and release of proinflammatory mediators from macrophages.To research the intestinal toxicity of Phytolacca Radix and after being processed with vinegar,the level of intestinal edema,water content of intestine and stool,the AQPs expression of colon and HT-29 cells were detected to isolate the toxic compounds from different polar fractions.The main research contents and work are as follows:1 The establishment of toxicity evaluation model of phytolaccaWe investigated the level of intestinal edema,water content of intestine and stool by the mouse intestinal model to compare the toxicity of different polar fractions of Phytolacca Radix.Diarrhea is the most commonly toxic side effects of Phytolacca Radix.It is well accepted that AQPs are responsible for osmotically driven transmembrane water movements,while the presence of an active water transport.The level of AQPs expression,combined with diarrhea in mice was used to research the toxic compounds of Phytolacca Radix.2 Comparative study on toxicity of different polarity fractions of Phytolacca alcohol extractsWe extract the 70% ethanol extracts of Phytolacca Radix by petroleum ether, dichloromethane,n-butanol to obtain the different polar fractions of Phytolacca.The model of mouse intestinal irritation and mouse diarrhea were used to evluate the toxicity of different polar fractions.The level of intestinal edema,the water content of duodenum and jejunum,the water content in stool and the level of AQP1,AQP3,AQP4 mRNA were detected.The results display that n-butanol fraction can increase level of intestinal edema,the water content of duodenum and jejunum,the water content in stool and the level ofAQP4 mRNA,decrease the expression of AQP1 and AQP3 mRNA.We summarize that the most of toxic components of Phytolacca transfer to the n-butanol extracts.3 Comparative study on toxicity of n-butanol fraction in Phytolacca before and after being processed by vinegarTo compare the intestinal toxicity of n-butanol fraction in Phytolacca Radix before and after being processed with vinegar,the level of intestinal edema,diarrhea degree and AQPs expression of colon were detected.The result show that level of intestinal edema,the water content of duodenum and jejunum,the water content in stool,the level of AQP4 mRNA and protein were decreased and the expression of AQP1,AQP3 mRNA and protein were increased compared with the n-butanol fraction before being processed. So the toxicity of n-butanol saturated with water fraction in Phytolacca Radix decreased obviously after being processed with vinegar.4 Comparative study on toxicity of the toxic enrichment fraction of PhytolaccaWe took comparative study on the toxicity of before and after purification of the composition of n-butanol based on the detection of water content of intestine and stool,the level of AQPs expression.The result shows that the water content in stool, the degree of diarrhea and the expression of AQP4 mRNA were increased and the expression of AQP1,AQP3 mRNA were decreased in mice administrated with ether supernatant of n-butanol fraction.The small polar fractions have most toxicity after purification and are the toxic enrichment faction of Phylocca.We speculated that the toxic compounds of Phytolacca were trasfered into ether supernatant.5 The separation analysis and identification of the toxic enrichment fraction of PhytolaccaWe took segregation on Silica gel column and C18 gel colum,purification on Sephadex LH-20 gel column and identified by HR-ESI/MS and 13C-NMR.We got six kinds of compounds, such as β-sitosterol、Esculentoside A、Esculentoside B、Esculentoside C、EsculentosideD、Esculentoside F.6 Comparative study on the toxicity of the compounds of the toxic enrichment fraction of PhytolaccaWe took comparative study on the toxicity of the compounds of Esculentoside A、 Esculentoside B、Esculentoside C、Esculentoside D、Esculentoside F、n-butanol fractions and ether site based on AQP3 expression of HT-29 cells.The results show that Esculentoside C、 Esculentoside B-. n-butanol fractions and ether site can decrease the expression of AQP3 mRNA in HT-29 cells. We speculated Esculentoside C and Esculentoside B is the toxic compounds of Phytolacca.7 Comparison of saponins in crude and processed n-butanol fraction.We took comparative study on the charges of saponins in crude and n-butanol fraction by HPLC-MS/MS,combined with spectral data of isolated five saponins and software of PeakviewTM.The result show that no new saponins were generated in n-butanol fraction after being processed by vinegar,saponins peak area decreased except for EsA and the composition of saponins charged significantly. We speculated that the decrease of toxicity in n-butanol fraction associated with the reducation of saponins content and charges of saponins composition.8 Study on the content determination of Esculentoside C and Esculentoside B in the toxic enrichment site of Phylocca,crude and processed phytolacca.EsC and EsB are the major toxic components of Phytolacca. We compared the contents of EsC and EsB in crude and processed phytolacca and the toxic site. The results show that the contents of EsC increased from 2.05%(n-Butanol) to 2.59%(ether site) by segregation,and reduced from 0.12% to 0.05% after being processed with vinegar, the contents of EsB increased from 2.28%(n-Butanol) to 16.46%(ether site) by segregation,and reduced from 0.19% to 0.10%.We speculated that the processing method with vinegar is reasonable and scientific.