Preparation And Evaluation Of Self-Microemulsifying Drug Delivery System For LianXiang Prescription

1.Research PurposeMicroemulsion drug delivery systems,hereinafter referred to as MEDDS has suchcharacteristics as strong dispersion of drug,low interfacial tension,which can promote the solubility of insoluble drugs,permeability absorption of drugs through membranes,thestability of drugs as well as the targeting of drugs.Apart from microemulsion,theself-microemulsifying drug delivery systems,hereinafter referred to as SMEDDS,is mainlycharacterized by its automatic formation of microemulsion under physiological conditions.The world witnesses only a few reports on the research of traditional Chinesemicroemulsion prescriptions,most of which are made from monomer active ingredientextracted from traditional Chinese medicine,some of which are made from the mixture oftwo kinds of essential oils.Up till now there is no such report as research onmicroemulsification of traditional Chinese medicine compound including two (or above)extracts with different polarity.At present,traditional Chinese medicine from which a largeamount of monomer active ingredient can be extracted is rarely-seen,while prescriptionswith effective parts or components take up a relatively huge proportion in traditionalChinese medicine.Therefore,it is more practical to proceed from effective parts orcomponents as traditional Chinese microemulsion prescriptions.LianXiang prescription consists of total-alkaloid of Coptis chinensis Franch.andessential oil of Eugenia caryophyllata Thunb.,whose function is to reduce pathologicalfire,cure inflammation,smooth Qi and relieve pain.It plays a key and effective role incuring chronic gastritis,enteritis,gastric duodenum ulcer and abdominal pain.Therefore,itis granted with patent for invention by the State.LianXian prescription consists of a fewnumber of extracts with specific and clear ingredients,which,as the research object,facilitates the analysis of experiment results and further research.In this prescription,total-alkaloid of Coptis chinensis Franch or berberine chloride are typical in that they havelow solubility in water or most of the oils,and poor absorption if taken orally.This paper aims to probe into the problems and solutions,such as promotingsolubility and absorbtion of insoluble drugs in water or most of oils,in respect of the preparation and evaluation of SMEDDS for the model traditional Chinese medicinecompound;this paper also attempts to explore the effective approaches to promote thestability and bioavailability of LianXiang prescription through research on the SMEDDS ofthe prescription;thus laying a foundation for further study on compatibility of traditionalChinese medicine compound and its function mechanism.2.Methods and Results2.1 Research on Quality Control of the Preparation Materials2.1.1 MethodsThe total-alkaloid of Coptis chinensis Franch.is prepared from the purchased Coptismedicine according to the original ways of preparation of LianXiang prescription.HPLC isused to determine the content of berberine chloride in the total-alkaloid of Coptis ChinensisFranch.The operating conditions:Kromasil C18 (250mm×4.6mm,5μm)column;UVD170U detector;mobile phase:acetonitrile:0.1mol/L NaH₂PO₄:0.025mol/L sodiumdodecyl sulphate (56:22:22);measuring wavelength:265nm.Purchased essential oil of Eugenia caryophyllata.GC is used to determine the contentof eugenol in the essential oil.The operating conditions:SE-54 quartz capillary column(15m×0.25mm×0.33μm);flame ionization detector(FID);column temperature:110℃;gasifying temperature:250℃;detector temperature:180℃;gas carrier:nitrogen;flow rate:10ml/min,determination by internal standardization.2.1.2 ResultsBerberine chloride appears a linearity in the range of 0.026μg—0.416μg,withcorrelated coefficient r=0.9996;the average recovery is 98.2%,RSD=2.04% (n=6).Thedetermination results for the three batch samples of the total-alkaloid are 75.7%,RSD=1.30%(n=5);76.9%,RSD 0.97%(n=3);74.1 %,RSD 1.55%,respectively.Eugenol appears a linearity in the range of 0.578mg/ml～4.624mg/ml,with thecoefficient r=0.9996;the average recovery is 98.0%,RSD=2.66% (n=6).Thedetermination results for the three batch samples of the essential oil are:78.7%,RSD=1.54% (n=5);75.6%,RSD=1.17% (n=3);79.3%,RSD 1.48%.2.2 Screening of Self-microemulsifying formulation for Essential oil of Clove2.2.1 MethodsThe solubility of total-alkaloid of Coptis chinensis Franch.in essential oil of clove andother auxiliary materials is tested.They are also subject to microemulsifying test for theinitial screening of oil phase,surfactant and so on.Surfactant and cosurfactant with certainoil phase will be screened through the study of ternary phase diagram and in accordancewith the size of self-microemulsifying areas.2.2.2 ResultsEssential oil of clove has been selected as individual oil phase for the reason that the solubility of the total-alkaloid in the essential oil is much higher than any other kind of oil.As Tween 80 and Cremophor RH60 are able to form microemulsion with high content ofoil with a less amount of surfactant,they have been chosen for further screening.There islittle difference between the solubility of the total-alkaloid of Coptis chinensis Franch.inethanol,1.2-propylene glycol,glycerol and PEG400,therefore they have been chosen forfurther screening of cosurfactant.The systems of essential oil of clove-Tween 80-ethanol,essential oil of clove-RH60-ethanol and essential oil of clove-RH60-propylene glycol leadto relatively large self-microemulsifying areas,as a result,they have been selected forfurther test.2.3 Test and Optimization of the Self-microemulsifying Formulation for LianXiangPrescription2.3.1 MethodsTotal-alkaloid of Coptis chinensis Franch.is added to the optional system ofmicroemulsion for essential oil of clove to test its influence on the self-microemulsifyingareas.The influence of pH on the self-microemulsifying areas is tested with pH6.8PBS or0.1mol/L HC1 as dilution media.Such samples as those failed to form microemulsion at thevicinity of self-microemulsifying areas at 37℃are tested at 25℃for purposes ofobserving the influence of temperature on the self-microemulsifying areas.Systems with relatively large self-microemulsifying areas have been chosen.Optimalformulae are selected on condition that the match proportion of LianXiang prescriptionremains the same;the larger the proportion of essential oil of clove,the smaller theproportion of surfactant;the border of the self-microemulsifying areas has been avoided.On the basis of the formulae selected through study of ternary phase diagram,with particledimension and polydispersity as evaluation index,the type and contents of surfactant andcosurfactant have been tested so as to select the optimal self-microemulsifying formula.2.3.2 ResultsAddition of total-alkaloid of Coptis chinensis Franch.have broadened,to some degree,the size of self-microemulsifying areas of three optional systems;the sizes ofself-microemulsifying areas of three optional systems have not shown any significantchanges with pH6.8 PBS or 0.1mol/L HCl as dilution media;samples that failed to formmicroemulsion at the vicinity of self-microemulsifying areas at 37℃have become clearand bright,demonstrating the broadening of the self-microemulsifying areas.The formulae selected by phase diagram analysis:total-alkaloid of Coptis chinensisFranch:essential oil of clove:RH60:ethanol=40:20:60:20(w/w);the total-alkaloid:the essential oil:RH60:propylene glycol=40:20:60:20 (w/w);the total-alkaloid:the essential oil:Tween 80:ethanol=40:20:60:20 (w/w).Result on the basis ofparticle dimension and polydispersity as evaluation index:the total-alkaloid:the essentialoil:RH60:propylene glycol=40:20:60:20 (w/w).The microemulsion formed in thisformula has smaller particle dimension and polydispersity index (17.4 d.nm,0.176). The particle dimension of microemulsion will be shorter with the increase of theamount of surfactant;the particle dimension is shorter when ethanol is applied ascosurfactant and RH60 as surfactant than when Tween 80 is applied.Both the particledimension and polydispersity are larger when RH60 is applied as surfactant and ethanol isapplied as cosurfactant than when propylene glycol is applied.Addition of total-alkaloid ofCoptis chinensis Franch.has narrowed the particle size of the microemulsions.2.4 Preparation of SMEDDS of LianXiang PrescriptionFirst mix the prescribed propylene glycol,RH60 and essential oil of clove,churn themup,then put prescribed total-alkaloid of Coptis chinensis Franch.and mix them up,finallystore the mixture hermetically or fill them into capsules.2.5 Methods for Content Determination of SMEDDS2.5.1 MethodsHPLC is applied to determine the contents of berberine chloride in SMEDDS;GC isapplied to determine the contents of eugenol in SMEDDS.The operating conditions are thesame as the quality control of the materials.2.5.2 ResultsBerberine chloride appears a linearity in the range of 0.026μg—0.416μg,withcorrelated coefficient r=0.9996;the average recovery is 97.7%,RSD=1.91%(n=6).The results of berberine chloride content for the three batch samples are 21.4%,RSD=1.82% (n=3);21.2%,RSD2.25%;22.6%,RSD2.33%.Eugenol appears a linearity in the range of 0.578mg/ml～4.624mg/ml,with thecoefficient r=0.9996;the average recovery is 98.6%,RSD=2.77% n=6).The results ofeugenol content for the three batch samples are:10.2%,RSD=2A5% (n=3);10.6%,RSD 2.43%;10.9%,RSD 2.37%.2.6 Investigation of Stability of the SMEDDS and Its Microemulsion2.6.1 MethodsVessels with lids containing the SMEDDS are put into refrigerator at the temperatureof 4℃.The outlook will be observed and the contents of berberine and eugenol will bedetermined.SMEDDS are put into the vessels with lid and placed in normal temperature withoutsunshine.They are subject to sampling and determination regularly.SMEDDS are added with water at 37℃,0.1mol/L HCl,pH6.8 PBS and 0.9%NaClrespectively.They are churned up,subject to sampling regularly and determined theabsorbency at 425nm.2.6.2 ResultsThere is no medicine crystallized from the SMEDDS after seven days at lowtemperature;the contents of berberine and eugenol show no significant changes.There is no medicine crystallized from the SMEDDS after six months at normal temperature;the contents of berberine and eugenol shows no significant changes.The absorbency of the SMEDDS in water,0.1mol/LHCl,pH6.8 PBS and 0.9%NaClrespectively shows no significant changes within six hours,demonstrating the stability ofmicroemulsion under designated conditions.2.7 Measurement of Self-microemulsifying Rate2.7.1 MethodsTake a proper amount of the SMEDDS,added with dilution media at 37℃,such asdistilled water,0.1mol/L HCl or pH6.8 PBS.Mix them round and sample them regularly soas to determine the contents of berberine chloride.2.7.2 ResultsThere is no significant difference between the self-microemulsifying rate of theSMEDDS in water,0.1mol/L HC1 or pH6.8 PBS;the SMEDDS is able to selfmicroemulsify completely within 10 minutes in designated media.2.8 Investigation of Relations of Self-microemulsifying Degree and the Volume ofDilution Media2.8.1 MethodsTake a proper amount of the SMEDDS,added with distilled water at 37℃.Churnthem up,put them still and absorb carefully and specifically the upper part of liquid so as todetermine the content of berberine chloride.Later a certain amount of distilled water will beadded for sampling and testing.2.8.2 ResultsThe SMEDDS is able to microemulsify completely at 37℃when the proportion ofwater to the SMEDDS stands at 40:1 or above.At 37℃,the saturation contents of thetotal-alkaloid in LianXiang microemulsion is 6.21mg/ml,when the proportion of water tothe SMEDDS stands at 20:1;5.84 mg/ml when the proportion stands at 30:1;5.38mg/ml when the proportion stands at 40:12.9 Investigation on Pharmacokinetics of the SMEDDS with Small Mice2.9.1 MethodsKM small mice are divided at random into the experiment group of LianXiangSMEDDS and the control group of LianXiang emulsion preparation.The single dosage ofPO small mice of the SMEDDS and the emulsion preparation is 400 mg/kg and 280 mg/kgrespectively,with the same amount of berberine.After PO administration,the eyeballs ofthe mice are to be extracted for blood.Plasma are separated and stored in refrigerator.Put the mixture of methanol and acetonitrile into a certain amount of plasma sample,churn them up in a swirl manner,leave them centrifugalized,separate the upper liquid anddry it on heated water.A certain amount of methanol are to be added to the leftovers,whichare swirled to dissolve,centrifugal.The upper liquid is to be extracted for sampling andtesting. With the help of HPLC,the changes of berberine concentration in plasma,with thepassage of time,of small mice in each group are to be tested after po.administration withthe preparations.The operating conditions:Kromasil C18 (250mm×4.6mm,5μm)column,Mobile phase:Acetonitrile:0.1mol/L NaH₂PO₄:0.025mol/L SDS (54:23:23);Measuring wavelength:265nm.AUC_0→tare obtained by trapezoid approach;C_max and t_max are educed according toexperiment data;k and t_1/2 are obtained by the slope of the tail-end straight line of logC-tcurve;the relative bioavailability F of the SMEDDS to emulsion preparation is calculatedWith F=(AUC_0→24h)_SMEDDS/(AUC_0→24h)_enulsion×100ï¼…2.9.2 ResultsBerberine chloride appears a linearity in the range of 10.4μg/L—156μg/L,withcorrelated coefficient r=0.9991;the precision (stipulated with RSD,n=5)of three sampleswith low,medium and high concentration is 12.9%;7.0%;9.6% respectively.Theprecision tested the next day is 10.4%;8.3%;10.2% respectively.The average recovery((%,(?)±s,n=5)of samples with low,medium and high concentration is 89.6±12.0;94.7±6.6;91.8±8.9respectively.The AUC_0→24h of LianXiang SMEDDS and the LianXiang emulsion preparation is 688.3±123.7(μg·h·L^-1)and 371.4±68.4 (μg·h·L^-1)respectively (t test,P=0.0001).C_maxis 107.1±24.8 (μg·L^-1)and 70.6±23.0(μg·L^-1)respectively (t test,P=0.0122).t_max is 1 h and 3 h respectively (Wilcoxon rank sum test,P=0.0022).t_1/2 is 13.5 h and 7.22 hrespectively.The relative bioavailability F(F=(AUC_0→24h)_SMEDDS/(AUC_0→24h)_enulsion×100ï¼…)of theSMEDDS to the emulsion preparation is 193.1±63.2.3.Conclusions3.1 HPLC to test the content of berberine chloride and GC to test the content of eugenol canbe the main way of quality control of raw materials (total-alkaloid of Coptis chinensis Franch.and essential oil of clove)and the preparation samples.3.2 The microemulsion formed in the optimal LianXiang formula SMEDDS (total-alkaloidof Coptis chinensis:essential oil of clove:RH60:propylene glycol)has smaller particledimension and polydispersity index,whose self-microemulsifying areas are not sensitive tothe change of media pH but highly susceptible to temperature;the types,content,andproportion of surfactant and cosurfactant exerts a great impact on particle dimension andpolydispersity;Addition of the total-alkaloid broadens the size of clove essential oilself-microemulsifying areas,shortens the particle dimension,facilitating the formation ofmicroemulsion.Such phenomenon as drops of microemulsion become smaller due todrug-load has been seen.It can be deduced that berberine shows a certain degree of hydrophile and lipophile nature or surface active in this system,which partly plays the roleof surfactant,the majority of which scatters on the interface between oil and water in themicroemulsion system.3.3 Preparation of LianXiang SMEDDS is mild and simple,which is easy to be put intoindustrialization.3.4 Essential oil of clove and berberine show good stability in SMEDDS;the stability ofmicroemulsion preparation is not susceptible to pH and the ionic strength of the media.3.5 The test approach of the relations of self-microemulsifying degree and dilution mediavolume established in this paper can be one of the ways to evaluate the quality of theSMEDDS.The saturation contents in LianXiang microemulsion of berberine are muchgreater than that in water at each designated proportions of H₂O to the SMEDDS.All themedicines in SMEDDS are easy to microemulsify in stomach and intestine.3.6 The absorption rate and degree of LianXiang SMEDDS are greater than that ofLianXiang emulsion preparation,that is to say,the absorption rate and degree of thepreparation are on the increase with the shortening of particle dimension.The t_1/2 of SMEDDS is longer than that of emulsion preparation,which can lengthen the circulationtime of medicine within the body.In conclusion,total-alkaloid of Coptis chinensis Franch.or berberine chloride showpoorly solubility in water or a majority of oils,with poor absorption if taken orally.However,they show relatively high solubility in the microemulsion system of cloveessential oil+ RH60+ propylene glycol.The essential oil of clove demonstrates goodstability in the SMEDDS.The in vivo absorption rate and degree of LianXiang SMEDDSby the small mice has been greatly promoted than that of LianXiang emulsion preparation.In short,microemulsifying is an effective way to promote the solubility ang bioavailabilityof the drugs in Chinese medicine prescription with low solubility in water or most of theoils.