The Research Of Sustained-release Dropping Pills Of Compound Venenum Bufonis

Objective:Based on the antitumor effect of Venenum Bufonis and Ganoderma lucidum,the topic plans to prepare the compound Venenum Bufonis sustained-release dropping pills with synergistic antitumor function,and then completes the research of composition selectio、antitumor mechanism and molding technology,and then studys on quality standard and preliminary stability of preparation.Methods:1、Preformulation study:The HPLC analysis methods of Venenum Bufonis were established by taken cinobufagin and resibufogenin as indicators.The UV analysis methods of Ganoderma lucidum were established by taken ganoderma amylose and triterpenes as indicators.It laid the foundation for the preparation procedure,quality evaluation and preparation stability research.2、In vitro pharmacodynamics research:Determined by MTT screening the best proportion of Venenum Bufonis/Ganoderma lucidum in antitumor effect,and provide evidence for selecting the ratio of compound controlled-release formulations.Cell inhibition rate as the index,combining with the golden section method for cell pharmacodynamics experiment,respectively to Venenum Bufonis and Ganoderma lucidum ratios into following groups:1:0,0.618:0.382,0.528:0.472,0.472:0.528,0.382:0.618,1:0.Determined by MTT method to observe different groups,different dosage concentration on the effect of HepG2 cells growth,so as to screen out the the best proportion of Venenum Bufonis/Ganoderma lucidum antitumor effect.3、Antitumor mechanism:Through FITC Annexin Apoptosis Detection kit I detected the degree of apoptosis of human hepatocellular carcinoma HepG2 cells;Detect the cell cycle after Venenum Bufonis and Ganoderma lucidum on HepG2 by FCM;Study the effect of Venenum Bufonis and Ganoderma lucidum on HepG2 to bax mRNA、bcl-2 mRNA、Caspase-3 mRNA and p53 mRNA;Using enzyme-linked immunosorbent assay(ELISA),detect the change of protein kinase activity and second messenger concentration after Venenum Bufonis and Ganoderma lucidum effect,and detect thechanges of signal path.4、Preparation technology research:The appearance,roundness,pills weight difference,dissolution of dropping pills as the indexes,using single factor experiment and L9(34)orthogonal test optimization of preparation technology.The topic uses the comprehensive evaluation method to study the best preparation technology and fits the release curve to study the releasing mechanism.5、Quality standard:Prepare the compound Venenum Bufonis sustained-release dropping pills,and study the character、qualitative identification、content and release in vitro;Respectively study the three batches of samples of the appearance、the roundness and qualitative identification of Venenum Bufonis and Ganoderma lucidum;Determine the cinobufagin and bufogenins content of the pills by HPLC;Determine the contents of Polysaccharides and Triterpenes by UV.Through the dissolution test,the samples were studied about the 12 h accumulative release rates in vitro.6、Preliminary stability study:The influence factors for three batches of compound sustained-release dropping pills were inspected and accelerated stability were also observed.Results:Respectively established the analysis methods in vitro of Venenum Bufonis and Ganoderma lucidum.The results showed strong specificity,good linear relationship and high precision,the recovery rate of also conformed to the requirements of the determination.Compared with Venenum Bufonis and Ganoderma lucidum alone,the joint application has better antitumor effect and has much higher antitumor effect than the effect of Venenum Bufonis and Ganoderma lucidum alone.With the increasing of the ratio of Venenum Bufonis and Ganoderma lucidum,the results showed the inhibitory rate was increasing as well,and showed the best inhibitory rate in 0.472:0.528,so determine the best ratio of 0.472:0.528.From Antitmor mechanism study,the results showed that Venenum Bufonis and Ganoderma lucidum combined can’t act on the cell cycle.Venenum Bufonis、Ganoderma lucidum respective and associated treatment also can induce apoptosis of HepG2,and compared with control group,it had significant difference,compared with them alone had significant different in middle and high concentration and in a dose dependent manner;The q-PCR results showed they can raise the level of bax mRNA,Caspase-3 mRNA and p53 mRNA,but cut the level of bcl-2 mRNA.Through detection of signal path,the result shows that can improve the concentration of cAMP,activate the activity of PKA,at the same time reduce the concentration of cGMP,and inhibit activity of PKC.Ultimately determine the best preparation technology for sustained-release dropping pills of compound Venenum Bufonis:methyl silicone oil as condensate,stearic acid:PEG4000 1:5,drug:accessories 1:2,the melting temperature 80℃,the distance of dropping 8cm,the condensate temperature 50℃ to 5℃.After the optimized preparation,the roundness and in vitro release effect of compound sustained-release dropping pills were good,and appearance and content uniformity.The releasing mechanism met Higuchi andRitger-Peppas equation.From the study of quality standard,the samples of compound sustained-release dropping pills were brown solid preparation,uniform appearance and good roundness.TLC identification has the good specificity.The results showed the content of cinobufagin and resibufogenin respectively shall not be less than 14.10 mg/g、8.93 mg/g,and the metastasis rate respectively was 84.72%、76.65%;The results of polysaccharide and triterpenoid respectively shall not be less than 15.50 mg/g and 34.53 mg/g.The metastasis rate respectively was 72.49%、84.82%.The results of release showed that it released 30%before 2 h,released 50%in 6h and 80%in 12 h,which conformed tothe requirements of sustained-release preparation.Based on the investigation of the influence factors,the study showed that high humidity factor had a certain influence on the stability of preparation,and should be sealed storage at dry places.According to the accelerated stability test for the prepration,the testing indexes compared with "0",no obvious change of the contents,but the accumulative releaserate of preparation was slightly decreased.This was a certain relationship with the aging problem of dropping pills.Conclusion:This paper adopted the methods of MTT and golden section,and make sure the best antitumor ratio of Venenum Bufonis/Ganoderma lucidum was 0.472:0.528.The results confirmed that the Venenum Bufonis and Ganoderma lucidum joint application can improve the anti-tumor effect.It can active signal transduction pathway of cAMP-PKA and inhibite cGMP-PKC,and then promotes tumor cell apoptosis,achieves the antitumor effect.The preparation technology is simple、feasible.The completion of this project provides a reliable quality control method for preparation,provides an optimistic outlook for clinical treatment of liver cancer,and provides accurate,effective foundation to purpose to treat the livercancer.