Micronization Of Magnolia Bark Extract By RESS As Well As Dissolution And Pharmacokinetics Evaluation

1.ObjectsRapid expansion of supercritical solutions(RESS) is a new method for preparing microparticles which has developed rapidly in recent years.The micron- and even nano-particles obtained by this method which could greatly enhance the drugs solubility and bioavailability.There are a lot of research about RESS,but no study is reported on preparing Chinese medicine microparticles by this technology.The purpose of this study is to discuss the feasibility and superiority of RESS method in the field of Traditional Chinese Medicine.Magnolia Bark extract(MBE) obtained by supercritical carbon dioxide(scCO₂) extraction technology and the MBE particles prepared by conventional mechanical milling were taken as the experiment material.2.Methods2.1.Preparation of micronized MBE particlesThirty grams of MBE particles were filled in a 300 mL stainless steel vial placed in the extraction unit.Carbon dioxide was brought to the desired operating pressure and temperature conditions through controlling water bath temperature and the pump flow rate.The supercritical carbon dioxide was then allowed to pass through the extraction unit to dissolve MBE particles(balance 30min) and to form a supercritical solution. In the expansion chamber,rapid phase change of sprayed supercritical solution across the nozzle into the atmospheric conditions induced the high supersaturation of the solute and resulted in the formation of microparticles.Nanoparticles were obtained by expanding supercritical solution to aqueous conditions of 50mL distilled water. 2.2.Optimization of RESS processAn orthogonal L₉(3)³ test design was used to investigate the optimal preparation condition of microparticles with the particle size and the total-phenol amount as indexes.Extraction temperature varied from 30 to 50℃,pressure from 200 to 300 bar, and nozzle size from 50 to 200μm.The influence of extraction temperature,pressure and nozzle size on particle characteristics was studied.The experiments were conducted in triplicate under each processing condition.Nine tests were carried out at varied extraction temperature(30,40,50℃),extraction pressure(200,250,300bar) and nozzle size(50,100,200μm).The range of each factor level was based on our previous results.2.3.Characterization of nanoparticles2.4.1 Particle morphologyThe nanoparticles suspend in water.The particle morphology analysis was carried out using atomic force microscopy(AFM,SHIMADZU SPM-9500J3,Japan).2.4.2 Particle Size DistributionThe nanosuspension was analyzed in DSL on ZETASIZER 3000HS(Malvern, Germany;Cell Type:Capillary cell,Detector Angle=90.0 Wavelength=633.0nm)2.4.Characterization of microparticles2.4.1 Particle morphologyThe microparticles were evaluated morphologically by SEM(S-3000N,Japan). Pictures were taken at 20 kV and magnifications of 1000×and 2000×.2.4.2.Particle Size DistributionParticle size of microparticle samples was determined by image analysis of photomicrographs(SPM,Japan).240 particles were selected and analyzed by statistical software SSPS 13.0.2.4.3.Differential Scanning CalorimetryDSC(NETZSCH DSC204,Germany) was used for thermal analysis.The operating conditions in the closed-aluminum pan system were as follows:sample weight,4.0 mg;heating rate,10℃/min;from 30 to 250℃,nitrogen gas flow,20 ml/min.2.4.4.Dissolution studiesDissolution studies were performed following the Ch.P.X C rotation-basket method in 900mL of methanol-water(15:85) at 37℃and 100 rpm(Xu et al.,2006). Accurately weighted samples containing the equivalent of 100 mg of total-phenol(the amount of Magnolol and Honokiol) were spread in the dissolution medium.Then, 5mL dissolution medium were pipeted at certain time(5,10,20,30,45,60,90min) and passed through a 0.45μm meberane filter.The withdrawn dissolution medium was replaced with equal quantities of fresh dissolution medium to maintain a constant volume.The concentrations of total-phenol in the withdrawn samples were determined by HPLC.2.4.5.Pharmacokinetics evaluationTwo groups of SD rats are oral administration with microparticles and MBE particles respectively and then determine the contents of total-phenol in plasma using HPLC at different time.The results are compared and analyzed by statistical software SPSS 13.0.3.Results3.1.Preparation of mieropartieles and nanopartielesAt the beginning of our work,co-precipitates in power form couldn't be obtained in one step by RESS method.The microparticles was viscosity and sticky.This might be caused by MBE particles which contained some butyraceous or sticky ingredients. These ingredients were dissolved in the scCO₂ and precipitated combined with drug particles in the expansion process.We tried to obtain microparticles in powder form. Firstly,we added certain amount of theβ-CYD to MBE(β-CYD:MBE particles 2:1 w/w) and took the mixture as the experiment stuff.Theβ-CYD was not soluble in scCO₂ fluid that it would not precipitate combined with EBM in the expansion chamber.Secondly,the MBE materials were also refined in recycling by scCO₂ extraction technology until the total-phenol amount of magnolol and honokiol were up to 80.4%before being mixed withβ-CYD.Nanoparticles were obtained by expanding supercritical solution to aqueous conditions of 50mL distilled water.3.2.Optimization of preparation parameters of microparticlesExtraction pressure,extraction temperature and nozzle size are generally considered to be the most important factors that affect the particle size and yield(%) of total-phenol amount.In the present study,all selected factors were examined using an orthogonal L₉(3)³ test design.The total evaluation index was used to analysis by statistical method.The analysis results of orthogonal test for microparticles, performed by statistical software SPSS 13.0.The smallest mean size of microparticles sample was 4.7±2.1μm and maximum yield(%) of total-phenol amount was 90.3±1.0%.In view of orthogonal analysis,we adopted statistical software to calculate the values of K,k and R.The factors influenced the mean size and yield(%) of total-phenol amounts were listed in a decreasing order as follows:C＞A＞B,C＞A＞B respectively,according to the R value.So the smallest mean size of microparticles was obtained when extraction pressure,extraction pressure and nozzle size were A₂B₂C₂ and the maximum yield of total-phenol amount was obtained when extraction pressure,extraction pressure and nozzle size were A₂B₃C₃.According to the result of analysis of variance table(data not shown),we found a change in the factor levels for each factor(A,B or C) had no statistically significant effect on particle size(P＞0.05),but a change in factor A or C had significant effect on yield of total-phenol amount(P＜0.05).No statistically significant change in particle size may associate with agglomeration of particles occurred in expansion zone as the particles had ample time to collide with each other and coagulate to form bigger particles.In view of both mean size and yield,we set the optimum technology as follows:A₂B₂C₃ (250bar,100μm,50℃).Through confirmatory test,we got the microparticles with mean size of 4.7μm and yield of total-phenol amount of 91.2%.The analysis results of orthogonal test for nanoparticles,performed by statistical software SPSS 13.0.The smallest mean size of nanoparticles sample was 201.0±2.4nm and maximum yield(g·L^-1) of total-phenol amount was 0.169±0.018g·L^-1.In view of orthogonal analysis,we adopted statistical software to calculate the values of K,k and R.The factors influenced the mean size and yield(%) of total-phenol amounts were listed in a decreasing order as follows:C＞A＞B,C＞A＞B respectively,according to the R value.So the smallest mean size of microparticles was obtained when extraction pressure,extraction pressure and nozzle size were A₁B₁C₁ and the maximum yield of total-phenol amount was obtained when extraction pressure,extraction pressure and nozzle size were A₂B₂C₃.According to the result of analysis of variance table(data not shown),we found a change in the factor levels for factor A,B had statistically significant effect on particle size (P＞0.05),but a change in factor A,C had significant effect on yield of total-phenol amount(P＜0.05).In view of both mean size and yield,we set the optimum technology as follows:A₂B₃C₃(250bar,200μm,50℃).Through confirmatory test,we got the microparticles with mean size of 303.0nm and yield of total-phenol amount of 0.172 g·L^-1.3.3.Characterization of microparticles3.3.1.Particle morphology and size distributionThe MBE particles were irregular particles and gathered together.The microparticles obtained under the optimum preparation conditions were hoar, irregular schistic and the average size was 4.7μm,which was approximately 11 times smaller than the size of MBE particles(55.3μm).The particle size distribution of the micronized MBE was from 0.2-24.1μm,which was narrower than that of the MBE particles,8.3-102.4μm.The SEM data showed all microparticles had similar morphologies and particle sizes.3.3.2.Result of Differential Scanning Calorimetry analysisThe obtained particles were analyzed by DSC.The melting point of unprocessed MBE was 65.0℃whereas RESS processed MBE had a melting point of 66.2℃.As seen in Fig.8,the peak of micronized particles was higher and narrower than that of material particles.This can be attributed to the reduction of particle size and purity elevation after RESS-SC processing.The sizes of microparticles and material particles were 4.7μm,55.3μm respectively.The together amount of magnolol and honokiol in microparticles and MBE particles were 91.2%(w/w),80.4%(w/w) respectively.There was no peak which corresponds toβ-CYD melting point and proves absence ofβ-CYD in final product.The MBE peak disappeared in the curve e, but quite a few magnolol and honokiol were detected by HPLC.These results demonstrated that magnolol and honokiol were entraped inβ-CYD and the viscosity and sticky substaces in EMB were also entraped inβ-CYD,which connected with the amount of magnolol and honokiol elevation in microparticles.3.3.3.Dissolution behaviorThe dissolution study was conducted with the together amount of magnolol and honokiol as index for a time period of 90min.The results suggested that drug dissolution was significantly enhanced by the RESS procedures.At 90min,the amount dissolved of raw MBE was 6.37μg·mL^-1,which was significantly lower than that of micronized MBE(14.77μg·mL^-1),according to the results of ANOVA (F=73.59,P=0.001).Overall,the present study showed that micronized MBE had higher dissolution rate than raw MBE,which was possibly due to particle size reduction of MBE.Furthermore,together amount of magnolol and honokiol elevation may act to enhance the dissolution rate,the amount of magnolol and honokiol in microparticles and MBE particles were 91.2%,80.4%respectively.Probably,it was not only the size of the particles that contributed to the dissolution,it was also the difference in weight,i.e.the MBE particles contained something else,which was removed(or rather not extracted) in the RESS process.That something could also contribute to a slower dissolution rate in the case of MBE particles.3.3.4.Pharmacokinetics evaluationTwo groups of SD rats are oral administration with microparticles and MBE particles respectively and then determine the contents of total-phenol in plasma using HPLC at different time.The results are compared and analyzed bystatistical software SPSS 13.0,which show the ACU value and C_max value of microparticles group is statistically significant higher than MBE particles group.4.ConclusionRESS technique was successfully applicable for fine particle production of scCO2 extract of traditional Chinese medicine with Magnolia Bark as the model herb. It could significantly micronize the particle size of MBE particles in one step process. Micronized MBE had a significantly faster dissolution rate than MBE particles.RESS technology provides an alternative,single step,uniform and convenient method to increase the traditional Chinese medicine dissolution rate and solubility.