The Quality Evaluation Of Fuzi Decoction And Pharmacodynamic Study About Treat Chronic Heart Failure

Objective:The purpose of this study was to optimize the extractive technique of alkaloid in Fu Zi decoction via orthogonal experiment.Establish the fingerprint of Fu Zi decoction and determine the content analysis of Fu Zi decoction which basis of the results of orthogonal experiment for quality assessment.The animal models of CHF was established by constriction of abdominal aorta and identify the physical index of CHF rat model after intragastric administration to point out the pharmacodynamic action of Fu Zi decoction.Using UPLC/MS to investigate the chemical compounds of Fu Zi decoction which is a foundation about the effect of Fu Zi decoction against CHF were put forward for next research.Methods: 1.HPLC-UV method for the simultaneous determination of benzoylmesaconine,benzoylaconine and benzoylhypacoitine content.The column chromatography for Waters XTerra MS C18(150 mm × 4.6 mm,5 ?m),flow phase A for acetonitrile and B is 0.1 mol·L-1 ammonium acetate solution(p H = 6.97),gradient elution(0 ~ 15 min,5% ~ 34% A;15 ~ 30 min,34% ~ 37% A;30 ~ 32 min,37% ~ 65% A).Detection wavelength was 235 nm.The flow rate was 1 m L·min-1;column temperature was 30?,injection volume was 10 ?L.Using the orthogonal experiment to optimize the extractive technique of Fu Zi decoction and establish the fingerprint of it.Column chromatography for BDS HYPERSIL C18(250 mm × 4.6 mm,5 ?m)flow phase A is acetonitrile and B is 0.04 mol·L-1 ammonium acetate solution(p H = 10),gradient elution(0 ~ 15 min,10% ~ 27% A;15 ~ 50 min,27% ~ 60% A;50 ~ 60 min,60% A).Detection wavelength was 240 nm.The flow rate was 1 m L·min-1;column temperature was 30?,injection volume was 10 ?L.2.122 SD rats were adaptive feed one week and exclude rats of non-normal,randomly divided remained rat into blank group,sham operation group and model group.The animal model of CHF was established by constriction of abdominal aorta.After being treated for 8 weeks,observing rats generally condition,analysis the data of ultrasonic examination and EGG,exclude rats of non-normal.82 SD rats were remained and randomly divided into sham operation group,low,medium and high of Fu Zi extraction by ethanol group,model group,Captopril group,high of Fu Zi decoction group and blank group.After being treated for 4 weeks by relat medicine,observing rats generally condition,analysis the data of ultrasonic examination,ECG and detected BNP,NTpro BNP,IL-6,TNF-?,ANG-II,ALD,CK,ET-1,LDH,PRA,CRP by ELISA.3.The UPLC/MS method was used to detect the chemical component in Fu Zi decoction.Column chromatography for ACQUITY UPLC BEH C18(100 mm × 2.1 mm,1.7 ?m),flow phase A for acetonitrile and B is 0.1% formic acid-water,gradient elution(0 ~ 5 min,10% ~ 20% A;5 ~ 30 min,20% ~ 80% A;30 ~ 33 min,80% A).The flow rate was 0.3 m L·min-1;column temperature was 30?,injection volume was 10 ?L.The optimized parameters of mass spectrometry were illustrated as below: HESI,spray voltage: +3.5 k V;sheath gas pressure: 40 arb;Aux gas pressure: 5 arb;capillary temp: 320 ?;scan range,m/z 100 –1500 in negative ion mode and positive ion mode.Results: 1.HPLC method for the simultaneous determination of benzoylmesaconine,benzoylaconine and benzoylhypacoitine content.The logistic regression analysis of benzoylmesaconine y=6609.2x-8.6951,the logistic regression analysis of benzoylaconine y=10481x-21.324,the logistic regression analysis of benzoylhypacoitine y=5988.4x+ 8.0685,all correlation coefficient are higher than 0.9990.The optimum technology conditions of extraction alkaloid from Fu Zi decoction in the view of economy were as follows: volume fraction of ethanol of 70%,extraction time of 2 hours,extraction 2 times and ratio of solid to liquid is 1:30.Compare the similarity of fingerprint of Fu Zi decoction by Traditional Chinese medicine(TCM)chromatographic fingerprint similarity evaluation software.The similarities between 10 batches Fuzi decoction and comparison products are higher than 0.91.2.By observing rats generally condition,analysis the data of ultrasonic examination and ECG.The rats were excluded because of the heart failure in the context of normal and the remained 82 rats can be used in pharmacodynamics study.The model group rats were markedly pathological changed.The results of the physical index which were compared of blank group,model group,sham operation group,low,medium and high of Fu Zi extraction by ethanol group,Fu Zi decoction group and Captopril group were indicate that Fu Zi decoction group,low,medium and high of Fu Zi extraction by ethanol group and Captopril group have significantly effect of rats.The Captopril group and high of Fu Zi extraction by ethanol group have an obviously improve in pathogenesis condition of rats.The medium of Fu Zi extraction by ethanol group also can improve the pathogenesis condition of rats.The low of Fu Zi extraction by ethanol group and Fu Zi decoction group have a little effect of the pathogenesis condition of rats.3.So 42 compounds can be inferred basis the UPLC-MS/MS analysis of Fu Zi decoction combined with the characteristic information of characteristic fragment,the pyrolysis law of chemical bond and compare the related literatures or reference substance data.Conclusion: This study established the simultaneous determination of benzoylmesaconine,benzoylaconine and benzoylhypacoitine content in Fu Zi decotion by HPLC method.The 10 different batchs of herb which from different drugstore is determine by this way and the total content of benzoyl-mesaconine,benzoylaconine and benzoylhypacoitine are higher then 0.010%.The diester alkaloid can not find in both of 10 batchs.This is an evidence of the 10 batchs herb can used in clinical safely and effective.The extraction technique of Fu Zi decoction via orthogonal experiment and establish the fingerprint of Fu Zi decoction.The results indicate that different batchs Fu Zi decoction have similiarty in chemical compounds and contents.The result of experment could provide a basis of active constituent in Fu Zi decoction for the quality evaluation of Fuzi decoction.The study was established model by constriction of abdominal aorta and researched the pathogenesis of CHF to study the mechanism of Fu Zi decotion in therapy of CHF.The study of pharmacodynamics in Fu Zi decoction pointed out that the Fu Zi decotion have an advantage affect of CHF which cause by constriction of abdominal aorta and have disadvantage in exacerbation of CHF.It is also give a viable guidance of clinical medication about CHF.UPLC/Q Exactive Focus were used to separate and identify the chemical compounds of Fu Zi decotion.42 chemical compositions in Fu Zi decotion were inferred,it is also indicate the advandage of MS in confirm or exclude potential molecular formulas which is a very fast and effective way to study of the basis pharmacodynamics substances in Fu Zi decoction.