| Purpose:The processing technology of fermented Arisaematis Rhizoma with bovine bile was optimized.On this basis,the changes of bile before and after processing Arisaematis Rhizoma were systematically evaluated from three aspects:medicinal property,dryness and efficacy.Methods:1 A HPLC method for the determination of glycocholic acid in bovine bile and a TLC method for the identification of cholic acid and glycocholic acid were established.The quality evaluation system of bovine bile was established by detecting the p H,total density,solids content and glycocholic acid content in 50 batches of fresh bovine bile.At the same time,the preservation conditions and freeze-thaw conditions were investigated.2 The content determination method of schaftoside and isoschaftoside in Arisaema cum Bile was established.The conversion rate of schaftoside,isoschaftoside and glycocholic acid was taken as the investigation index,and the fermentation days,fermentation temperature,fermentation humidity and bile dosage were taken as the investigation objects to optimize the processing technology of Arisaema cum Bile.3 A rat‘cold-heat’model induced by ice water stimulation and levothyroxine sodium was established.To investigate the effect of bovine bile on the‘cold-heat’model rats before and after processing（1）Food intake,water intake,anal temperature,body weight and ear vein congestion;（2）Colon pathological changes;（3）The related factors of nervous system and endocrine system in serum and energy metabolism related factors such as pyruvate and ATPase activity in rat liver;（4）The expression of TRPV1and TRPM8 in hippocampus,hypothalamus and dorsal root ganglion.4.To investigate the effect of bovine bile on normal rats before and after processing Arisaematis Rhizoma:（1）water intake,urine output,stool water content;（2）Pathological changes of colon,kidney and submandibular gland;（3）The expression of aquaporin in submandibular gland,kidney and colon was detected by immunohistochemistry.（4）whole blood,plasma viscosity and serum c AMP/c GMP and ATPase content;（5）Structural changes of‘toxic needle crystal’and its effect on rabbit eye irritation.5 The rat model of febrile convulsion was established to investigate the duration of（1）convulsion,convulsion rate,convulsion latency and convulsion level before and after the fermentation of bovine bile.（2）Pathological changes of hippocampus;（3）The expression of GABAAR and GRIA1（Glu A1）protein in hippocampus was detected by immunohistochemistry.（4）16 Sr RNA high-throughput sequencing was used to observe the changes of intestinal flora.（5）The expression of TNF-α,IL-1β,IL-6 and HMGB1in the brain.Result:1 The density of bovine bile was about 0.9762 g/m L,and the total solid content was about 0.5383 g/m L.The quality of bovine bile was stable within 6 months at-20℃～-80℃,and the dissolution method was room temperature or 37℃water bath.2 The optimal parameters for the fermentation of Arisaematis Rhizoma with bovine bile were 35°C;humidity 75%;the ratio of cow bile to Arisaematis Rhizoma was 300%,and the fermentation time was 7 days.3 Arisaema Rhizoma can increase the content of NE,T4,DA in serum and the content of pyruvate（PA）,Na+-K+/ATPase,Ca2+-Mg2+/ATPase in liver of rats with cold syndrome,decrease the content of 5-HT,improve the colon injury of cold syndrome,and down-regulate the expression of TRPM8 in hippocampus,hypothalamus and dorsal root ganglion.The effect of Arisaema cum bile on the corresponding indexes is opposite to that of Arisaematis Rhizoma,which can improve the colon injury of rats with heat syndrome and down-regulate the expression of TRPV1 in hippocampus,hypothalamus and dorsal root ganglion.4 The maximum tolerance dose of crude Arisaematis Rhizoma in mice was 108g/kg,and the maximum tolerance dose of Arisaematis cum bile in mice was 75.6 g/kg.The irritation and dryness of Arisaema cum bile were significantly decreased after bile processing（P<0.05）.5 The convulsion time was shortened and the latency was prolonged in the high-dose group（P<0.05）.The expression of GABAAR protein was significantly increased（P<0.05）,the expression of GRIA1 protein was significantly decreased（P<0.05）,and the contents of TNF-α,IL-1β,IL-6 and HMGB1 in brain and serum were decreased（P<0.05）.16 Sr RNA sequencing analysis showed that the intestinal flora of FS model rats changed significantly,and the richness and diversity of intestinal flora were significantly regulated after the intervention of Arisaematis Rhizoma（P<0.05）.The results of species composition analysis showed that Arisaema cum bile could significantly increase the richness of probiotics such as Lactobacillus and reduce the richness of pathogenic bacteria such as Bacteroidales.Conclusion:Bovine bile can be stored at-20°C～-80°C for 6 months.The processing technology of Arisaematis cum bile is reasonable and reliable,which can provide parameters for its industrial production.After the fermentation of bovine bile,the medicinal properties changed from hot to cold;toxicity,irritation and dryness effects were weakened;the anti-febrile convulsion effect of Arisaematis cum bile is better than that of Arisaematis Rhizoma,which may be related to the intervention of intestinal flora-mediated metabolites of GABA and other neurotransmitters that inhibit convulsion and the expression of inflammatory factors in the brain. |
