Based On Chemical Constituents And Pharmacological Effects To Explore The Mechanism Of Wuling Prescription To Be Suitable For Powder Rather Than Decoction

Wulingsan Prescription was founded by Zhang Zhongjing, and first set out in the Treatise on Febrile Diseases. Original prescription records Polyporus eighteen zhu, peeled, Alisma one hundred twenty-six zhu, Atractylodes eighteen zhu, Poria eighteen zhu, Guizhi half two, peeled, the flavors, pound as powder, drinking hot water, day three. Drink warm water, sweating more. The modern clinical practice, although the prescription are also known as Wuling powder form, but they are all practical applications Wuling decoction form, and so far has not identified any party can prove that Wuling powder form and Wuling decoction form considerable clinical efficacy reported in the literature. Large number of clinical practice has proved that classical powder form has its clinical efficacy which decoction form is incomparable, and the application of powder form dosage is far less than its decoction.Have reported a randomized controlled clinical study of 41 patients with Wuling powder form and decoction form to treat Syndrome. To increase urine output and weight loss as an indicator, the results show the efficacy of Wuling powder form group was significantly better than the Wuling decoction form group. Thus, significantly stronger Wuling powder form of small doses is significantly stronger than Wuling decoction form with a large dose.With further research,the author found that a group of non-volatile low polar molecules in Wuling decoction form is missing up to 450 times than its powder form. Due to the research, we propose a hypothesis that Wulingsan was used by decoction form will result in a low polar component missing.We believe that even taking into account the mutual solubilization between the chemical composition of traditional Chinese medicine or solubilization phenomenon, water can not extracted low polar component completely, resulting in low polar chemical composition of herbs has not been extracted or have not been fully extracted. When the efficacy of the active ingredient in these low-polar components, the prescription should be in powder form rather than in decoction form. On terms powder form, non-volatile and volatile of low polar component will be lose in decocton form. For some other modern formulations, it has been noted that the loss of the volatile components and use of modern technology to preserve its volatile components, such as the use of cyclodextrin inclusion technology to prevent the loss of volatile oil and so on. If the use of modern research techniques confirmed why Wuling should be used by powder form but not soup form, and then reveal its scientific signification, it will prove that contemporary formulation of reform has not only lost some valuable experience of Chinese medicine, but also resulting in a huge waste of resources. Therefore, this experiment mainly for the major differences about low polar constituents between powder form and decoction form of Wuling prescription and its pharmacodynamic studies.Material foundation research is the first part in the research. In this experiment Wuling prescription extracted with 95% ethanol and then extracted with ethyl acetate and petroleum ether. Two compounds have ergosterol coumarin isolated from normal phase silica gel column and ODS column.We have found and assigned eight different chemical compositions between powder form and decoction form of wuling prescription, they are coumarin, cinnamic aldehyde, atractylenolide III, atractylenolide II, Chrysophanol, atractylenolide I,23-acetyl alisol B and ergosterol. By HPLC study, We found that coumarin in powder extract content decoction of 8.44 times as compared with powder, and decoction of coumarin loss up 88.16%; the content of powder extract 23-acetyl alisol B is 55.74 times decoction extract, and decoction 23-acetyl alisol B loss of about 98.21%; decoction extract atractylenolide II and atractylenolide III were 98.8% and 89.02% loss, while cinnamic aldehyde, chrysophanol, ergosterol and atractylenolide I are completely missing, detected nothing by HPLC. Although wuling capsule have taken mesures to reserved volatile components and added a variety of complex process, but low polar component in wuling capsule is missingseriously. Low-volatile non-polar chemical constituents are almost completely absent,and the loss of cinnamic aldehyde is still nearly 60% as much atractylenolide loss II also more than 40%, although Wuling capsule preparation process using cyclodextrin inclusion technology to reserve volatile components in Guizhi.In this experiment, Pharmacodynamic study is the second part. Three kinds ingredient of coumarin,23-acetyl alisol B and ergosterol which is missing in wuling decoction form.Through they affect AQPi and AQP4 mRNA and protein expression in HK-2 cell to assess the efficacy of the three ingredient. In MTT, select a different dose of drug, by the inhibition rate of less than 10% of nominal. High School low concentration of ergosterol selected are 25μM,12.5μM,6.25μM; coumarin cells almost have no significant toxicity, when given mix dose of 100uM, cells inhibition rate of coumarin is only 10.33%. Because of its non-cytotoxic, so they chose high school low concentration are 100μM,50μM,25μM; high school and low 23-acetyl alisol B concentrations are 6.25 μM,1.5625μM,0.390625μM; the concentration of positive control drug of hydrochlorothiazide is 12.5μM, and the cell inhibition rate was 7.69%. The experiment also set up LDH leakage rate indicators to detect the damage in HK-2 cells. With the increasing concentration of the compound, the extent of damage to cells gradually increased. The maximum dose of ergosterol, coumarin and 23-acetyl alisol B are 25uM, 100uM,6.25uM, LDH leakage rate of the maximum dose are 60.81%, 44.37%,76.68%, hydrochlorothiazide and the normal group are 61.02% and 33.15%. Including positive control drug, the cell injury severity descending order are 23-acetyl alisol B, hydrochlorothiazide, ergosterol and coumarin. Cell experiments of three compounds show that AQP1 and AQP4 expression in HK-2 cells high-dose of ergosterol treatment group was significantly lower than normal control group and even much lower than hydrochlorothiazide-treated group, while protein expression of AQP1 in HK-2 cells is similar to hydrochlorothiazide treatment groups; Protein expression of AQPi and AQP4 in HK-2 cells which is treated by coumarin high low are successively increased, and is higher than hydrochlorothiazide treated group and lower than the normal group, wherein the high-dose treatment group is similar to hydrochlorothiazide group on AQPi expression; Protein expression of AQP1 and AQP4 in HK-2 cells which is treated by high-dose of 23-acetyl alisol B are significantly reduced and lower than hydrochlorothiazide-treated group, low-dose group were higher than hydrochloro-thiazide group and lower than the normal group. These results indicate that ergosterol, coumarin,23-acetyl alisol B in certain doses are able to significantly to reduce the expression of AQPi and AQP4 mRNA and protein in HK-2 cells.This part of the experiment has been studied three chemical composition of the diuretic effects on wistar rats, and the three chemical composition are ergosterol, coumarin and 23-acetyl alisol B which losed in wuling pescription in decoction form.Specific steps are as follows:Animals Filters:Male rats 180-200g Wistar, advance placed in metabolic cages for one days to adapt to the environment. To prohibit drinking in rats for 18h before the experiment, then feed deionized water 25ml/kg, and immediately placed in metabolic cages to collect 2h urine. When the rat’s urine output exceed 40%, we believe that is the qualified one. The screening of qualified animals were divided into five groups, with eight rats in each group. Each treatment group as follows:normal control group,2.5ml 1% saline was administered; ergosterol group (35mg/kg), fed with the same amount of saline containing the appropriate concentration of ergosterol; coumarin group (50mg/kg), fed with the same amount of saline containing the appropriate concentration of coumarin:23-acetyl alisol group B (50mg/kg), fed with the same amount of saline containing the appropriate concentration of 23-acetyl alisol B; hydrochlorothiazide group (1 Omg/kg), containing the appropriate concentration of hydrochlorothiazide equivalent amount of saline. The rats administered orally once daily, and administered in a volume of 25ml/kg, with intragastric administration of 8 days. On first day and seventh day, the rats were placed in metabolic cages quickly to collect urine within 6h after given drugs, and record the volume of urine. One hour after given drugs on eighth day, Rats were injected sodium pentobarbital of 50mg/kg to anesthetize, and to take the kidney with suitable save. Detection index:urine and the expression of AQP1, AQP2, AQP4 mRNA and protein.The results showed that urine output of ergosterol group were significantly increased on first day and seventh day. Compared with negative control group, the groups of coumarin and 23-acetyl alisol B were not significantly different. Ergosterol play an important role on diuretic effect within 6 hours. Expression of AQP1, AQP2 and AQP4 mRNA and protein in rat kidney which treated by ergosterol was significantly reduced. For coumarin and 23-acetyl alisol B, urine of Wistar rats had no significant lower compared with negative control group, but the expression of AQP4 mRNA and protein in kidney of Wistar rat were significantly reduced, indicating that the coumarin and 23-acetyl alisol B had some impact on lose cloth water in rats body.Through the above study, we found that,compared with Wulingsan powder form, Wulingsan decoction form lost a lot of low polarity chemical composition, and these chemical compositions have diuretic activity and these active ingredients have a certain impact on the expression of aquaporins.It provides a new idea and research directionon on basic research effective substance of wuling prescription in powder form rather than in decoction form,also provides reference for clinical drug dosage form selection.