Development And Evaluation Of Daidai Flavones Self-microemulsifying Pellets

Object:The effective site of Daidai flavones was used as raw material,and developed into self-microemulsifying pellets(SME pellets).The absorption characteristics of Daidai flavones SME pellets were studied by rat isolated everted gut,and the oral bioavailability was evaluated in the rats.The quality analysis and evaluation methods were established to offer firm experimental and theoretical basis for further developing the antioxidantive and quality controled products of Daidai flavones SME pellets and fully using the daidai fruits which is the abundant medicine resources in Fujian.Methods:1.The effective site of Daidai flavones was separated and purified preparation procedure which had been optimized by our research group before.The Daidai flavones with the stabilized content of total flavones up to 70%,was used as raw material and developed into Daidai flavones SME pellets based on self-microemulsifying drug delivery systems(SMEDDS).The kinds and dose ranges of oil?surfactant and cosurfactant were determined through the equilibrium solubility experiment and ternary phase;The prescription of Daidai flavones SMEDDS was given by simple grid method and the preparation method was optimizated by single factor analysis;The Daidai flavones SME pellets were prepared with optimized solid ingredients via extrusion/spheronization technique,and the preparation method was optimized using the orthogonal experiment.2.The preparation was evaluated by appearance,size distribution,droplet size?stability and influencing factors after forming micro emulsion,etc.The formula compositions were identified by thin layer chromatography(TLC);the methods for the determination of total flavonoids by UV method and characteristic ingredients of naringin and neohespiridin by HPLC method were established,While a HPLC analysis model of feature maps was constructed for the comprehensive evaluation and control the inherent quality of Daidai flavones SME pellets.3.To establish dissolution determination methods for the Daidai flavones SME pellets,With the total flavonoids as evaluation indexes and naringin and neohespiridin as characteristic ingredients.To study the dissolution influence of Daidai flavones SME pellets in different media,the dissolution of Daidai flavones SME pellets in different media,namely,distilled water,hydrochloric acid solution(pH 1.0),acetic acid salt buffer(pH 4.5)and phosphate buffer(pH 6.8)were studied by UV and HPLC methods.The zero and first-order dynamic equation,Higuchi equation were applied to simulate the Daidai flavones-SMEDDS pellets release data.Meanwhile the similarily of release profile were evaluated by bootstrap f2 method.4.The isolated everted gut was used as model,naringin and neohespiridin which are representative compositions of Daidai flavones SME pellets,the concentrations of naringin and neohespiridin in rat duodenum?jejunum and ileum in the same administration dosage,and the jejunum absorption in different administration dosage were determined by high performance liquid chromatography with mass detector(HPLC-MS).Evaluation the absorption efficiency of the Daidai flavones SME pellets by comparing the jejunum absorption between the self-microemulsifying pellet and the effective part extractive of flavones from Daidai fruits.5.To develop a HPLC method for simultaneously determining the content of naringin and neohespiridin in rat plasma,and to evaluate the oral bioavailabilitym of Daidai flavones SME pellets by comparing the pharmacokinetics with the suspension of Daidai flavones effective parts.Results:1.The ratio of Daidai flavones SME pellets formula was:SMEDDS(Daidai flavones powder:Lauroglycol FCC:Tween 80:Transcutol HP=3:4:10:6,w/w)?microcrystalline cellulose(MCC)and lactose was 10:12:5(w/w).The preparation was as follows:according to the formula,Daidai flavones powder was dissolved in cosurfactant of transcutol HP,then surfactant of Tween 80 and oil of Lauroglycol FCC were added and mixed until forming uniform brownish black Daidai flavonoids self-microemulsifying concentrated solution.The concentrated solution were added into MCC and lactose,followed by addition of water until a mass suitable for extrusion was obtained(water:total mixture=0.3?0.4:1,w/w);Eventually,the wet mass was extruded at 24 HZ/min in an axial screen extruder equipped with a die of 1mmthickness with 1mm diameter circular holes.The extrudate was spheronized for 3min at 24 HZ/min.The produced pellets were then dried at 40? in a drying oven and the size range at 16-30 sieves as product.The preparation process of Daidai flavones SME pellets in this research has the advantages of simple and high yield.2.The size of Daidai flavones SME pellets in this research mainly distribute at 16-30 sieve and the yield up to 92.43%,the end product was uniform spherical pellets.The droplet sizes after forming micro emulsion were nearly the same in different dispersion mediums(hydrochloric acid solution(pH 1.0),acetic acid salt buffer(pH 4.5),phosphate buffer(pH 6.8)and distilled water),in different rotate speeds(50?75?100 r·min-1,distilled water as dispersion medium),also in different temperatures(25??37??50?,distilled water as dispersion medium).The droplet sizes of three lots of Daidai flavones SME pellets were between 78.8-85.8 nm and were no obvious differences comparing the determination results after 4 h.In the TLC spots,the sample of Daidai flavones SME pellets showed the same color fluorescent spots in the same location comparing the reference substance of naringin and neohespiridin and without interference of negative sample;The UV Method for determination of total flavones was established,and the content of three lots of samples were 40.84 mg·g-1,40.49 mg·g-1,40.28 mg·g-1 respectively.The HPLC method for simultaneous determination of naringin and neohespiridin was established,and the naringin content of three lots of samples were 11.68 mg·g-1,11.31 mg·g-1,11.28 mg·g-1,respectively;the neohespiridin content of three lots of samples were 16.82 mg·g-1,16.45 mg·g-1,16.44 mg·g-1,respectively.The specific chromatogram of Daidai flavones SME pellets was established and the similarity were all greater than 0.900.3.The Daidai flavones SME pellets have the same dissolution behavior in four kinds of dissolution mediums,and drug can quick release by drugs release achieving up 85%within 15 min.the drug release of The Daidai flavones SME pellets were most following the first-order release kinetics.The f2 between naringin and neohesperidin in hydrochloric acid solution(pH 1.0),acetic acid salt buffer(pH 4.5),phosphate buffer(pH 6.8)and distilled water were 94.32,95.88,91.89 and 92.48,respectively,and it demonstrated that naringin and neohesperidin in Daidai flavones SME pellets can synchronous released.4.In rat duodenum,jejunum and ileum,the absorption of naringin and neohespiridin declined in turn,but it is no significant difference between duodenum and jejunum.(P>0.05).The natural logarithm of accumulative concentration of naringin and neohespiridin were both significantly correlated with sampling time in 3.6,7.2,12.0 mg·mL-1 doses,and the square of coefficient correlation exceed 0.9,which consistent with first order rate process.The Ka of naringin and neohespiridin increases along with the raised dosage of the Daidai flavones SME pellets(P<0.05),indicated they are the passive absorption.In the same administration dosage,the accumulative absorption amount of naringin and neohespiridin at 90 min in the Daidai flavones SME pellets was 1.3-fold that in the extractive of flavones of Daidai.Compared with the extractive of Daidai flavones,the intestinal absorption of the Daidai flavones SME pellets was significantly improved.5.The naringin pharmacokintic parameters of the Daidai flavones SME pellets and the suspension were followed as:AUC(0-24)were(17.710±6.588)and(9.139±1.982)mg·h·L-1,Cmax(4.816±1.329)and(0.917±0.204)mg·L-1,tmax(0.611±0.086)and(0.917±0.204)h,t1/2(13.078±6.382)and(11.678±6.919)h,respectively.And the neohesperidin pharmacokintic parameters were followed as:AUC(0-24)were(18.094±4.892)and(10.961±2.276)mg·h·L-1,Cmax(5.657±1.391)and(2.096±0.512)mg·L-1,tmax(0.583±0.091)and(0.806±0.222)h,t1/2(14.606±7.562)and(10.985±6.963)h,respectively.The naringin and neohesperidin relative bioavailability of the Daidai flavones SME pellets were 193.78%and 165.08%,respectively.The bioavailability of Daidai flavones were increased by making them into the self-microemulsifying pellets,and the flavones biopharmaceutical character were improved.Conclusion:According to the solubility problems of the Daidai flavones effective part,to enhance effective compositions dissolve speed and improve oral bioavailability of Daidai flavones,the Daidai flavones SME pellets with lowering the cholesterol efficacy were designed and developed based on SMEDDS.The prescription was determined by Daidai flavones solubility test,the construction of ternary phase and simple grid design,etc.The optimized preparation technology having the advantages of simple,stable and strong reproducibility was choosed by orthogonal experimental design.The Daidai flavones SME pellets are uniform spherical in shape and have good self-microemulsifying efficiency by characteristic evaluation of preparation appearance,size distribution and self-microemulsifying efficiency.To control the quality of samples,The TLC method for the identification of Daidai flavones SME pellets,a UV method for the determination of total flavones and HPLC method for simultaneously determination of naringin and neohesperidin in Daidai flavones SME pellets were established.The HPLC features mapping mode of Daidai flavones SME pellets was set up for the first time.and provides some beneficial method for the multi-index overall quality control system of Daidai flavones SME pellets preparating by TCM effective parts.This study used the total flavones,naringin and neohesperidin as the indexes to investigate the dissolution properties of Daidai flavones SME pellets in 4 different PH,and evaluate the dissolution rate and preliminary discusse their quickly release mechanism.The bioavailability of Daidai flavones were increased by making them into the self-microemulsifying pellets,and the flavones biopharmaceutical character were improved by The drug determination.in rat organisms.By the result of this research,the scientific theoretical and experimental basis were offered for fully developing and using the daidai fruits adaptation to local conditions which is the abundant mechanism resources in Fujian,and the new experimental basis was offered for further developing the safe and effective anti-oxidation products of daidai fruits.