Preparation And Pharmacodynamics Study Of Myricitrin Proliposomes

Myricitrin is a kind of natural flavonoids,which widely exists in the bark,fruit and leaves of Myrica rubra Sieb.et Zucc.Myricitrin has a variety of pharmacological effects,including antioxidant,anti-inflammatory,hypoglycemic,hepatoprotective,neuroprotective and antinociceptive effects.However,myricitrin has poor water solubility and low oral bioavailability,which limits its clinical application.In order to improve the solubility and bioavailability of myricitrin,as well as its anti-gouty arthritis and anti-uric acid activities,the proliposome was proposed as a drug delivery system.This paper mainly elaborated from the following five chapters:Chapter 1 ReviewsIn this chapter,the physical and chemical properties of myricitrin,pharmacological activity and preparation of myricitrin were summarized;the research progress of proliposome was introduced,including its advantages,preparation methods and application in pharmaceutics;finally,gout was summarized,and its pathogenic factors and therapeutic drugs were introduced.The content of this chapter could lay a theoretical foundation for the research of this paper.Chapter 2 Pre prescription study of myricitrinIn this chapter,a HPLC method for the determination of myricitrin in vitro was established,and the results of intra-day and inter-day precision,recovery and stability all met the requirements of methodology.The equilibrium solubility of myricitrin in four release media（p H 1.2 HCl solution,p H 6.8 PBS buffer solution,p H 4.5 ABS buffer solution and p H 7.0 double distilled water）was determined.The results showed that myricitrin was a insoluble drug,the maximum solubility was not more than 33μg/m L,and the minimum solubility was only 21.63±1.04μg/m L.This part could provide a basis for further study on the in vitro release behavior of free myricitrin and myricitrin proliposomes（MPs）.Chapter 3 Preparation and in vitro evaluation of MPsIn this chapter,MPs were prepared by film dispersion-freeze drying method.Firstly,the effects of the mass ratio of phospholipid to cholesterol,the mass ratio of phospholipid to sodium cholate,the mass ratio of phospholipid to IPM,the amount of myricitrin,the type and amount of cryoprotectant on the particle size（PS）of MPs were investigated by single factor experiment.Then,according to the results of single factor experiment,orthogonal design experiment was carried out to further optimize the prescription that was determined as shown below:90 mg phospholipid,9 mg cholesterol,45 mg sodium cholate,60 mg IPM,15 mg myricitrin.The quality of MPs prepared by the optimal formulation was evaluated,including the determination of PS and Zeta potential by particle size analyzer,the determination of entrapment efficiency（EE%）and drug loading（DL%）by HPLC,and the in vitro release behavior by dialysis bag method.The results showed that the average PS of MPs was 32.62±0.26 nm,the polydispersity index（PDI）was 0.179±0.017,and the Zeta potential was 38.56±0.17m V;the MPs was spherical and well dispersed by transmission electron microscopy;the EE%and DL%were high with 93.44±2.65%and 4.17±0.25%,respectively.The results of in vitro release showed that the cumulative release rate of MPs was higher than that of myricitrin in various release media.Chapter 4 Pharmacokinetics of MPs in vivoIn this chapter,a HPLC method for the determination of myricitrin in vivo was established,with the precision,recovery and stability meeting the requirements of methodology.Male SD rats were intragastrically administered with free myricitrin and MPs at a dose of 300 mg/kg.Blood samples were collected from the orbit of rats at selected time points.After pretreatment,samples were injected for analysis to determine the plasma concentration of myricitrin.Calculations of the relevant pharmacokinetic parameters of MPs by BAPP2.3 pharmacokinetics software were carried out.The results showed that the plasma concentration of MPs was higher than that of free myricitrin at different time points.The plasma concentration of free myricitrin reached the peak at 8 h,C_max was 580.99±21.44 ng/m L,and the C_max of MPS（894.36±33.32 ng/m L）reached the highest at 10 h,increased by about 153.94%.Although there was no significant difference in T_1/2,the relative oral bioavailability of MPs（about 171.81%）was significantly higher than that of free myricitrin,indicating that MPs could increase its oral bioavailability.Chapter 5 Study of anti-gouty arthritis and anti-uric acidactivity of MPsIn this chapter,a HPLC method for the determination of uric acid（UA）in vivo was established and the methodology was investigated.The rats were divided into five groups:normal control group（normal saline）,model group（normal saline）,positive control group（allopurinol）,free myricitrin group（60 mg/kg myricitrin）and MPs group（60 mg/kg myricitrin）.One hour after modeling,the corresponding drugs were given by gavage.3 h after modeling,blood samples were taken to determine the concentration of UA by HPLC.The results showed that the precision,recovery and stability of the established UA analysis method met the requirements of methodology;free myricitrin and MPs could significantly reduce the blood UA level of hyperuricemia rats（P<0.01）,and MPs had better effect on reducing UA than free myricitrin;H&E staining results showed that MPs had better liver protection effect on hyperuricemia rats than free myricitrin.In addition,the rat model of gouty arthritis was successfully established by injecting monosodium urate solution into the joint of the right hind foot,and then the joint swelling degree and serum inflammatory factor levels were measured.The results showed that both free myricitrin and MPs had the activity of reducing the degree of joint swelling,reducing the levels of inflammatory factors IL-1β,IL-6 and TNF-α,and inhibiting the inflammatory reaction.And the effect of MPs was better than that of free myricitrin.Therefore,myricitrin could be used as a potential drug for reducing UA and anti-gouty arthritis,and its preparation as a proliposome could increase the efficacy.