| Objective To clarify the mechanism of action of Wuling prescripton in Powder form rather than in decoction form,then to provide a scientific reference for clinical dosage form selection.Methods ①High performance liquid chromatography for the study of Jiangxi Alisma.Sichuan Alisma,Fujian Guizhi,Guangxi Guizhi,Guangdong Guizhi,Zhejiang Atractylodes,Hunan Baizhu.Anhui Baizhu.Yunnan Pork,Shaanxi Pork,Henan Pork and Anhui Fu A total of 14 medicinal materials from Fujian.Yunnan,and Hunan were analyzed,and the contents of 8 indicators in different origins were analyzed;②The components in plasma of decoction and powders of Wuling Prescription were analyzed by using Strip tool of MS data analysis technology and QI software technology.Then the missing components of Wuling Decoction were detected;③The rapid screening tool for Caco-2 cell was applied to study the absorption,metabolism and transport information of the five components,including Alisol B 23-acetate,cinnamaldehyde.ergosterol poria coccic acid and atractylenolide I,through the membrane of the small intestine;④The inhibitory effect of missing components in Wuling Decoction on cell edema by oxygen and sugar deprivation model of primary astrocytes through rats was tested.MTT was used to detect the damage effects of 6 medical combinations and oxygen glucose deprivation time on astrocytes themselves.The leakage rate of lactate dehydrogenase(LDH)was used to detect the cytotoxic effects of each group on oxygen glucose deprivation.Western Blot was used to detect the expression of AQP4 protein in astrocytes;⑤Nerve function score method was used to detect the missing compositions CAE on MCAO rats cerebral ischemia area and the improvement of neurological function.Water content in brain tissue determined by dry-wet weight method was used to estimate the degree of brain edema.Western Blot method was used to detect the protein expression of AQP4,Nrf2 and HO-1 in rats brain tissues,and to test the missing composition effect on the regulation of water channel proteins and the signaling pathways of Nrf-2/HO-1.Luminex technology was used to detect the expression of 1L-β,1L-6 and TNF-a factors in blood samples,then to detect the effect on inflammatory response.Results ①The results showed that the contents of Alisol B 23-acetate in alisma have great difference,the highest content in Sichuan.It was found that the content of cinnamaldehyde and coumarin in Guangdong was highest.The area that highest content of atractylodes was Zhe Jiang.The content of ergosterol was highest in Yunnan.Therefore,in the experiments.Sichuan alisma.Guangdong cassia twig,Zhe Jiang macrocephala,Yunnan poria and grifola were selected as the original medicinal materials;②The collected data by UPLC-MS were analyzed with QI software,and specify compound library component identification technology,47 kinds of completely missing ingredient and 6 kinds of partial missing component were selected.And 6 same chemical components of decoction and powders were researched by the distribution of content of blood absorption with time.Compared with powders,decoction has serious chemical component loss;③ The linear relationship,precision,method recovery and stability were all within the allowable error range after methodological investigation.In the absorption and transport experiment,according to the judgment criteria of absorption difficulty,poria cocos acid which the Papp value at the order of 10-7 cm/s was difficult to absorb,that the order of 10-5 cm/s of cinnamaldehyde.and Alisol B 23-acetate between the two order.The relationship between dose-effect and time-effect of absorption and transport was that the content of cinnamaldehyde in the tested compound was detected with the extension of time.The transport rate and speed of AP→BL and BL→AP showed a gradually increasing trend;④According to the cell inhibition rate and cell activity state,the best reoxygenation time for subsequent experiments was 6 h,and according to the factors of cell inhibition and solubility of the medicine itself,the concentrations of cinnamaldehyde,ergosterol and Alisol B 23-acetate in this experiment were 10 μg/mL,the concentrations of poria cocos acid and atractylenolide III were 0.01 μg/mL,and the concentrations of mixed medicines were 0.1 μg/mL.The leakage rate of LDH increased with the increase of medicine concentration.Compared with the model group,the cytotoxic effect on astrocytes could be reduced to different degrees in the medicine administration group.The leakage rate of LDH could be significantly reduced in the Alisol B 23-acetate,atractylenolide Ⅲ,poria coccus acid and the mixed group except the cumarin and ergosterol,with statistically significant differences(P<0.01).A certain amount of AQP4 protein is expressed in normal astrocytes.Compared with model group,cinnamicaldehyde,Alisol B 23-acetate,ergosterol.poria cocos acid,atractylenolide Ⅲ and mixed medicines can significantly reduce the amount of AQP4 protein expression of astrocytes,with statistically significant difference(P<0.01);⑤Compared with the MCAO model group,the CAE-L group.CAE-M group and CAE-H group could significantly improve the behavioral abnormalities and neurological dysfunction of the MCAO rats,with statistical significance difference(P<0.01).Compared with the Sham group,the water content of ischemic side(IH)brain tissue in MCAO model group increased significantly,with statistically significant difference(P<0.05).Compared with the MCAO model group,both CAE-H and CAE-M administration groups reduced the water content in the IH side brain tissue significantly(P<0.01/P<0.05).Compared with MCAO model group,CAE low.middle and high dose group can significantly reduce the expression of AQP4 protein,with statistically significant difference(P<0.01).Protein expression of AQP4 in CAE-L,CAE-M and CAE-H groups had no significant difference.Compared with Sham group,the expression of 1L-β,1L-6 and TNF-a factors in the MCAO model group was significantly increased,and the difference was statistically significant(P<0.01).Compared with the MCAO model group,the inflammatory factors 1L-β,1L-6 and TNF-a in the blood of the rats in the CAE-L,CAE-M and CAE-H groups were reduced to different degrees by gavage.Compared with Sham group,the relative expression levels of Nrf2 and HO-1 proteins in rats brain tissues of MCAO model group were increased and the expression levels of HO-1 protein were significantly increased,while the expression levels of Nrf2 protein were not significantly increased.Compared with the MCAO model group,the relative expressions of Nrf2 and HO-1 were significantly up-regulated in the CAE high,medium and low dose groups.Conclusions From the medicinal material base,loss of pharmacodynamic active ingredients inevitably leads to loss of overall efficacy.The missing components of Wuling Decoction have obvious efficacy,and the loss of these components will inevitably lead to the decline of clinical efficacy.The hypothesis that "The mechanism of Wuling Prescriptions which matched powders rather than decoction was the limited ability of water to extract the low-polarity components,which resulted in a large number loss of low-polarity molecular compounds in Wuling Decoction." get preliminary validation. |
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