The Enema Fuyankang Quality Control Of Its Pharmacodynamic, Pharmacokinetic Studies

Recurrent oral ulcer (ROU),refered as recurrent aphthous ulcer (RAU),is a limitations and isolated oral mucous membraneous ulcer which posses the feature that recurrent attacks,difficulty on healing,continuous attacks,lasting long time from years to decades.A lot ofstudy and research on etiopathogenisis of ROU had been reported,but there are notconclusive etiological factor about ROU.It has been presumed that origin of ROU wereinduced by composite factors such as genetic gene,immunization,infection,microelement,mental state and so on,and immunization is one of the most important factors in thepathogenesis of ROU.Splenogastric Hygropyrexia Syndrome is the common syndrome in the clinicaldiagnosis of ROU and we have explored and studied the nature of SplenogastricHygropyrexia syndrome.Professor LAO Shaoxian from the Guangzhou traditional Chinesemedicine university has researched the type of syndrome in clinical diagnosis of ROUthoroughly and summarized that Splenogastric Hygropyrexia Syndrome is the commonsyndrome by many years clinical experience.So combination XHS and HPXLT are appliedfor clinical treatment of ROU.In this paper,to explore the role of Splenogastric Hygropyrexia Syndrome in clinicalROU,Splenogastric Hygropyrexia Syndrome in ROU were analyzed,the herbal medicinewere applied,and therapeutic efficacy of QingReHuaShiFa were evaluated for therapeuticmechanism research and foundational theory exploration of ROU.Literature ResearchROU are called as 'recurrent aphtha'.By retrospective analysis of ancient pertinentliterature about recurrent aphtha,there are four period:establishing,sprouting,forming andconsummating in the theory of Splenogastric Hygropyrexia Syndrome.And the nature of by removing blood stasis,resolving hard mass,cleaning heat and detoxifying andanti-inflammatory and analgesic.Clinical application indicated that Fuyankang tablettreating on CPID administration by enema took effect faster,shorter treatment course and lower relapse rate than oral administration.Our previous work has shown that the content ofthe main effective component in the Fuyankang tablet such as berberine,salvianolic acid Band paeoniflorin were too low,their theoretical transfer rates were less than 10%.And,forthe Fuyankang enema which is our research subject was developed by several advancedextraction and concentration technology,the transfer rates are higher than 70%.In order tobetter evaluate and control the preparation,the method of quality control was investigatedby TLC,HPLC-DAD combined with the hplc Fingerprint Chromatography.In order to illustratethe significance and basie on the determining the route of administration,thepharmarcodynamics for treating on CPID and pharmarcokinetics of difference route ofadministration of the preparation were investigated.Research methods and contents1 Study on the quality controlIn the study,a qualitative identification method by TLC (Thin Layer Chromatography)was established for seven Chinese herbal including Radix Paeoniae Rubra,Radix SalviaeMiltiorrhizae,Phellodendron bark,Rhizoma Corydalis,Sophora flavescens Ait,rhizomacurcuma and Cyperus Rotundus L.The result showed that the established method wassimple and eclusive,the spot was clear with good repeatability.A RP-HPLC/DAD method was established for the simultaneous determination ofberberine and salvianolic acid B in Fuyankang enema,the chromatograhic separation wasperformed on a Kromasil C₁₈ column (250mm×4.6mm,5μmn)with a linear gradient elutionof acetonitrile-0.04 M potassium dihydrogen phosphate solution containing 0.1%phosphoric acid.The flow rate was 1.0ml/min and column temperature was set 30℃.Thedetection wavelength was 230 nm.The linear range was 0.0924～2.3100μg (r=0.9999)forberbeine,0.1000～2.5000μg (r=0.9999)for salvianolic acid B.The average recoveries ofhigh,middle and low concentration of berberine were 100.40%,99.51% and 99.15%respectively with RSD of 0.59%,0.67% and 1.02% (n=3)respectively.The averagerecoveries from salvianolic acid B were 101.52%,99.32% and 98.68% respectivelywith RSD being 0.60%,0.92% and 1.00%(n=3),respectively.A method was established for the determination of paeoniflorin in Fuyankang enema,the chromatograhic separation was performed on a Kromasil C₁₈ column (250mm×4.6mm,5μm)with an isocratic elution of acetonitrile-0.04 M potassium dihydrogen phosphatesolution (15:85,v/v)containing 0.1% phosphoric acid.The flow rate was 1.Oml/min andcolumn temperature was set 30℃.The detection wavelength was 230 nm.The linear range was 0.4992～6.24μg (r=0.9998)for paeoniflorin.The average recoveries of high,middleand low concentration of paeoniflroin were 99.94%,98.97% and 98.23%,respectively withRSD of 0.15%,0.28% and 0.38% (n=3)respectively.The methods established for determination of multiple index were applied to thestability test and quality control of Berberin,salvianolic acid B and paeoniflorin inFuyankang enema.The results indicated that Fuyankang enema was stable and can becontrolled,which will give scientific security for the stability and safty in clinicalmedication2 Study of the HPLC fingerprintThe RP-HPLC/DAD method of fingerprint of major chemicals in Fuyankang enemawas established.The chromatography condition was determined by choosing the bestcondition from different mobile phase,elution gradients,detect wave-length,chromatographic column,etc.The chromatograhic separation was performed on a KromasilC₁₈ column (250mm×4.6mm,5μm)with a linear gradient elution of acetonitrile-0.04Mpotassium dihydrogen phosphate solution containing 0.1% phosphoric acid.The flow ratewas 1.0ml/min and column temperature was set 30℃.The detection wavelength was 230nm.Under the condition,total of shared 13 peaks were determined as characters of the map.The relationship of the shared peaks between Fuyankang enema and medicinal herbsconfirmed the absorptive peaks specific for Radix Paeoniae Rubra,Radix SalviaeMiltiorrhizae,Phellodendron bark,Rhizoma Corydalis and Smilax glabra by comparativestudy of the formulation,medicinal materials and reference substance。The results of themethodological studies indicated that this HPLC fingerprint is of high precision,strongrepeatability and good stability,conforming to the establishment principle of TCMfingerprin,and the fingerprint can fully be used for controlling the final quality ofFuyankang enem.3 A preliminary investigation on the pharmacodynamicA preliminary investigation on the pharmacodynamic of the formulation by the relatedpharmcodynamics experiment according to its principal treated symptom,such as chronicpelvic inflammatory was developed.Experiments such as egg-albumin inducing rat pedalswelling,cotton-pellet inducing rat granuloma and dimethybenzene-inducing mouse earedema were carried out to evaluate the anti-inflammation effect of Fuyankang enema,theanalgesic effect was tested by frequency body torsion caused by acetic acid in mice,theinfluence of resveratrol on the function of celiac macrophage in rats was measured withcarbon particle clearance test.Promoting blood circulation and removing blood stasis of Fuyankang enema was evaluated by acute hypostasis model of ratsThe results indicate that the high,middle dose could significantly suppressxylene-induced mouse ear edema;High,middle,low dose of Fuyankang enema couldsignificantly suppress the rat granuloma induced by cotton-pellet and egg-albumin inducingrat pedal swelling;High dose of it could reduce torsion times in mice.Fuyankang enemacould improve clearance index and phagocytic index,but it is not obvious.The three doseof the formulation could improve the blood Theological property of rats of acute hypostasismodel.And,the above pharmacological action of Fuyangkang enema was superiority to theFuyankang tablet.The result show that Fuyankang enema have significantlyanti-inflammtory,promoting blood circulation and removing blood stasis,obviousanalgesic effect and certain immunopotentiation.4 Comparative pharmacokinetic study after rectal administration and administration by gavageA sensitive HPLC method for the determination of berine in rat plasma was developedand validated.The plasma samples of berberine was precipitatied with acetonitrile and thendetermined by HPLC-DAD.Separations were achieved on a Kromasil C₁₈ column(4.6mm×250mm,5μrn).The mobile phase was acetonitrile-0.04 M potassium dihydrogenphosphate solution containing 0.1% phosphoric acid (v:v=32:68)with a flow of 1ml/minand DAD detected at 265nm at 35℃.There was no endogenous interference from plasma or in all procedure in the HPLCchromatogram.A good linear relationship was obtained in the concentration range of0.01788～3.576μg/mL (r=0.9996).The intra-day and inter-day RSD were in the range of1.28～2.36% and 2.26～5.31%.The recovery of extract method ranged from 90.10～100.35%.The method was simple and good reproducibility,and can be used to pharmacokineticstudy for berberine.The pharmacokinetic study of berberine after rectal administration and administrationby gavage with single dose 4.73mg/kg (content of berberine).was investigated by usingabove HPLC method respectively.In preliminary test,the berberine in 8 Wistar ratsplasma could be detected after oral administration of Fuyankang enema,but the berberinecannot be detected in SD rats after oral administration and enema.So,we guess that theabsorption of berberine may has the Species Differences,the pharmacokinetic of theberberine in the preparation remains to further investigationA sensitive HPLC method for the determination of paeoniflorin in rabbit plasma wasdeveloped and validated after liquid-liquid extraction.The plasma samples of paeoniflorinwas extracted by ethyl acetate.and then determined by HPLC-DAD.Separations were achieved on a Kromasil C₁₈ column (4.6mm×250mm,5μm).The mobile phase wasacetonitrile-water (v:v=16:84)with a flow of 1ml/min and DAD detected at 230nm at 30℃.There was no endogenous interference from plasma or in all procedure in the HPLCchromatogram.A good linear relationship was obtained in the concentration range of0.195～24.96μg/mL(r=0.9999).The intra-day and inter-day RSD were in the range of1.06～1.96% and 1.82～4.87%,respectively.The recovery of extract method ranged from88.86～99.83%.The method was simple and good reproducibility,and can be used topharmacokinetic study for paeoniflorin.The pharmacokinetic study of paeoniflorin after rectal administration andadministration by gavage with single dose 5.91mg/kg (content of paeoniflorin).wasinvestigated by using above HPLC method respectively.Administration by gavage,the paeoniflorin wasn't detected in the rabbit plasma byHPLC method established,even when the dosage reached to 17.73mg/kg,paeoniflorincould be detected in rabbit plasma of a few time point,but the content was too low resultedin no quantitative analysis.So,the pharmacokinetic parameter of administration by gavagewasn't obtained.After rectal administration,the curve of paeoniflorin plasma concentration-timecould be described by a one-compartment model correlation pharmacokinetic parameterdescribed as follows:Ke:0.94381/h,Ka:3.19091/h,t_1/2(ka):0.2172h,t_1/2(ke):0.7344h,T(peak):0.5421h,C(max):5.2397μg/mL,AUC:9.2602(μg/mL)*h,CL/F(s):0.6382mg/kg/h/(μg/mL),V/F(c):0.6762(mg/kg)/(μg/mL).The result indicate the absorption of paeoniflorin after rectal administrationFuyankang enema is much more superiority to administration by gavage.ConclusionIn the paper,the qualitative identification method by TLC,multiple-markersquantitative identification method by RP—HPLC/DAD and analysis method of fingerprintconformed to correlation standards,and can be used to systemically quality control on theFuyankang enema.The investigation about the Fuyankang enema compared with the tabletrelated to it's pharmcodynamics experiment according to its principal treatedsymptom,such as chronic pelvic inflammatory was developed,for example:anti-inflammatory,analgesic effect,immune function and hemorheology.The results showthat Fuyankang enema have a significantly anti-inflammtory,promoting blood circulationand removing blood stasis,obvious analgesic effect and certain immunopotentiation betterthan those in Fuyankang tablet.Rectal administration of the preparation,the absorption of paeoniflorin is fast,T(peak)is short,C(max)is high.According to related documents,weguess that the absorption of paeoniflorin after rectal administration is better than that bygavage may be the rectal administration avoid the gastrointestinal tract and hepatic firstpass effect,efflux via p-gp and hydrolytic degradation in the intestinal brush border part orall.The result of the study provides the scientific basis for the determining the route ofadministration of the preparation,and we proposes that the change of route ofadministration may be improve the bioavailability of the drags which are similar topaeoniflorin and one of effective means of improving the curative effect.In conclusion,the means of quality control by fingerprint combined with qualityidentification,quantitative identification for multiple markers associate withpharmcodynamics and pharmacokinetic was developed to evalue the Fuyankang enemacomprehensively and scientifically in vivo and vitro,which has built sound foundation onthe developing the effective preparation for treatment on the CPID,and has importantvalue.