Studies On Quality Control Method And Pharmacokinetics Of Menispermi Rhizoma

Menispermi Rhizoma,also called BeiDouGen(Chinese name),is prepared from the dried rhizome of Menispermum dauricum DC.(family Menispermaceae).Menispermi Rhizoma is officially listed in the Chinese Pharmacopoeia since 1977 due to its definite effect of heat-clearing and detoxicating,wind-dispelling pain-relieving.It was used for the treatment of sore throat,enteritis,dysentery and rheumatoid arthralgia.In this study,the chemical basis,quality control method and pharmacokinetics of the active constituents of Menispermi Rhizoma were studied systematically using modern instrumental analytical techniques.1.Phytochemical studies on the Menispermi Rhizoma was performed by using modern separation and purification technology.By means of various chromatographic methods such as silica gel,Sephadex LH-20,ODS column chromatography,and macroporous adsorption resin column chromatography,20 compounds were isolated from the ethanol extract of Menispermi Rhizoma.On the basis of physico-chemical data and spectroscopic methods including 1H-NMR,13C-NMR,HMBC,HSQC,NOE and MS,their structures were identified,which included eight oxoisoaporphine alkaloids:oxoisoaporphine A(1),oxoisoaporphine B(2),menisporphine(3),6-O-demethylmenisporphine(4),bianfugedine(5),bianfugecine(6),dauriporphine(7),dauriporphinoline(8);two bisbenzyltetrahydroisoquinoline alkaloids:daurisoline(9),dauricicoline(10);two protoberberine alkaloids:dauricoside(11),stepholidine(12);two morphinane alkaloids:acutumine(13),acutumidine(14);two isoquinolone alkaloids:thalifoline(15),N-methylcorydaldine(16);one aporphine alkaloid:menisperine(17);two nitrophenanthrenes:aristolochic acid(18),aristoloterpenate I(20);one sesquiterpene lactone:aristolactone(19).Among them,compounds 1 and 2 are determined as new compounds.Compounds 18-20 are isolated from Menispermi Rhizoma for the first time.The 20 compounds were all the main ingredient of Menispermi Rhizoma,which laid the material foundation for the quality control and pharmacokinetic study of Menispermi Rhizoma.2.Using two universal detectors(UV and MS),methods were developed for simultaneous determination of multiple oxoisoaporphine alkaloids.(1)A novel and reliable method based on microwave-assisted extraction(MAE)followed by HPLC-UV was established for the simultaneous determination of six oxoisoaporphine alkaloids,including bianfugedine,menisporphine,6-O-demethylmenisporphine,bianfugecine,dauriporphine and dauriporphinoline.An orthogonal experiment method was employed in order to optimize the MAE conditions.The total contents of the six alkaloids were taken as the index points to evaluate the extraction efficiency under different factors and levels.The optimal MAE was performed at 60℃ for 11 min with ethanol-water(70:30,v/v)as the extracting solvent.Separations were carried out with a YMC C18 reversed-phase column(250 mm × 4.6 mm,i.d.,5 μm).The mobile phase consisted of 1%aqueous formic acid and acetonitrile containing 1%formic acid.A gradient elution program was used.All calibration curves showed good linearity within the test ranges.The precision was evaluated by intra-and inter-day tests,which revealed relative standard deviation(RSD)values less than 3.4%.The recoveries for the quantified compounds were between 98.0 and 101.8%.This method was applied to determine the amounts of the six compounds in Menispermi Rhizoma from five sources.(2)An HGE-UPLC-MS/MS method was established for the simultaneous determination of eight compounds,oxo iso aporphine A,oxoisoaporphine B,menisporphine,6-O-demethylmenisporphine,bianfugedine,bianfugecine,dauriporphine,and dauriporphinoline.HGE was optimized by response surface methodology(RSM)to obtain the maximum extraction efficiency of eight alkaloids.The optimal HGE was performed at room temperature for 9.0 min with ethanol-water(74:26,v/v)as the extracting solvent.Separations were carried out with a Waters ACQUITY UPLC? BEH C18 column(50 mm ×2.1 mm,i.d.,1.7 μm).The mobile phase consisted of acetonitrile and 0.2%aqueous formic acid.A gradient elution program was used.Detection was performed with a triple-quadrupole tandem mass spectrometer with an electrospray source(ESI)in positive ionization mode.The multiple reaction monitoring(MRM)analysis was conducted by monitoring the precursor ion to product ion transitions.All calibration curves showed good linearity within the test ranges.The precision was evaluated by intra-and inter-day tests,which revealed relative standard deviation(RSD)values less than 3.2%.The recoveries for the quantified compounds were between 96.4 and 103.8%.This method was applied to determine the amounts of the eight compounds in Menispermi Rhizoma from five sources.The results of the two methods were consistent,it showed that the contents of menisporphine and 6-O-demethylmenisporphine were higher than other alkaloids,bianfugecine were inexistence or too low to be detected in a few samples.In addition,the contents of alkaloids varied greatly from sample to sample.A multi-component-assay quality control method,using ultra-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry(UPLC-MS/MS),has been developed for the simultaneous quantification of 22 bioactive constituents in Menispermi Rhizoma.Separations were carried out with a Waters ACQUITY UPLC? BEH C18 column(50 mm ×2.1 mm,i.d.,1.7 μm).The mobile phase consisted of acetonitrile and 0.1%aqueous formic acid.A gradient elution program was used.Detection was performed with a triple-quadrupole tandem mass spectrometer with an electrospray source(ESI)in positive ionization mode.All calibration curves showed good linearity within the test ranges.The precision was evaluated by intra-and inter-day tests,which revealed relative standard deviation(RSD)values less than 5.0%.The recoveries for the quantified compounds were between 94.4 and 105.5%.The developed method was successfully applied for simultaneous quantification of 22 components in 10 batches of Menispermi Rhizoma obtained from different regions of northern China.Hierarchical cluster analysis was performed to differentiate and classify the samples based on the contents of the 22 characteristic constituents.The results showed that the contents of analytes in Menispermi Rhizoma from different sources were widely varied.In terms of class of constituents,bisbenzyltetrahydroisoquinoline alkaloids were the most abundant class of constituents in all the analyzed samples,whereas oxoisoaporphine alkaloids were relatively low(micro components).Aristolochic acid and aristolactone were inexistence or relatively low and even hardly detected in a few samples.It is well known that aristolochic acid has strong renal toxicity.It could be speculated that the existence of it may be associated with the toxcity of Menispermi Rhizoma.Thus,the present work will be beneficial for quality control of Menispermi Rhizoma and disclosing the secrets of potential efficacy and toxicity of Menispermi Rhizoma.3.A sensitive and selective liquid chromatography-tandem mass spectrometry method has been developed and validated for simultaneous quantitation of dauricine,daurisoline,N-desmethyldauricine,dauricicoline,dauriporphinoline,bianfugecine,dauricoside,stepholidine,acutumine and acutumidine in rat plasma.After addition of internal standard(verapamil),plasma samples were pretreated by a single-step protein precipitation with acetonitrile.Chromatographic separation was performed on a Waters BEH C18 column with gradient elution using a mobile phase composed of acetonitrile and water(containing 0.1%formic acid).The linear ranges for dauricine,daurisoline,N-desmethyldauricine,dauricicoline,dauriporphinoline,bianfugecine,dauricoside,stepholidine,acutumine and acutumidine were 1-500,2-1000,0.5-250,0.5-250,0.1-50,0.01-5,0.5-250,0.1-50,2-1000,5-2500 ng/mL,respectively.The method was linear for all analytes over the tested ranges with correlation coefficients larger than 0.9914.The lower limits of quantification(LLOQ)were 1 ng/mL for dauricine,2 ng/mL for daurisoline,0.5 ng/mL for N-desmethyldauricine,0.5 ng/mL for dauricicoline,0.1 ng/mL for dauriporphinoline,0.01 ng/mL for bianfugecine,0.5 ng/mL for dauricoside,0.1 ng/mL for stepholidine,2 ng/mL for acutumine,5 ng/mL for acutumidine,respectively.The extraction recoveries of the analytes and verapamil were above 77.4%.The specificity,matrix effect,precision,accuracy and stability of this method can meet the requirements of biological sample analysis.The validated method was successfully applied to the pharmacokinetics study of the ten alkaloids in rats after oral administration of Menispermi Rhizoma extract.The Cmax of dauricine was 187.5±73.2,170.2±72.2 μg/L,AUC0-∞was 6462.2±1880.2 μg·h/L,t1/2 was 20.53±3.76 h,Tmax was 0.32±0.25,9.40±2.41 h;The Cmax of daurisoline was 209.7±114.8,333.2±165.3 μg/L,AUC0-∞ was 9893.7±3726.0 μg·h/L,t1/2 was 16.03 ±2.59 h,Tmax was 0.52± 0.32,10.20±2.49 h;The Cmax of N-desmethyldauricine was 27.3 ± 10.0,40.5 ± 7.8 μg/L,AUC0-∞ was 1650.9±177.3 μg·h/L,t1/2 was 21.87 ± 4.05 h,Tmax was 0.43 ± 0.30,12.00 ± 0.00 h;The Cmax of dauricicoline was 14.4±4.2,28.1±5.8 μg/L,AUC0-∞ was 895.2±171.7 μg·h/L,t1/2 was 16.60±2.55 h,Tmax was 0.43 ±0.30,12.00 ± 0.00 h;The Cmax of dauriporphinoline was 16.3±7.6μg/L,AUC0∞ was 33.2 ±15.7 μg·h/L,t1/2 was 7.70±4.61 h,Tmax was 0.42 ± 0.19 h;The Cmax of bianfugecine was 2.1 ±0.8 μg/L,AUC0-∞ was 19.7 ±12.4 μg·h/L,t1/2 was 8.28±2.25 h,Tmax was 0.43±0.30 h;The Cmax of dauricoside was 91.5±51.5 μg/L,AUC0-∞ was 284.0±113.9μg·h/L,t1/2 was 8.02 ± 1.29 h,Tmax was 0.30±0.07 h;The Cmax of stepholidine was 3.3 ±1.1,2.6 ±0.8 μg/L,AUC0-∞ was 90.3 ± 22.4μg·h/L,t1/2 was 15.55 ±4.05 h,Tmax was 0.42 ±0.19,14.40 ± 5.37 h;The Cmax of acutumine was 375.0 ±186.7,117.1 ± 53.4 μg/L,AUC0-∞ was 4232.2± 2772.4 μg·h/L,t1/2 was 11.21±5.99 h,Tmax was 0.26±0.10,10.00±2.74 h;The Cmax of acutumidine was 1628 ± 534,1343 ± 469 μg/L,AUC0-∞ was 29129 ±10028 μg·h/L,t1/2 was 5.59 ± 2.19 h,Tmax was 0.55±0.31,11.00 ± 2.24 h.The results indicated that the pharmacokinetic characteristics of the 10 alkaloids showed significant difference owing to the discrepancy of their structures.And,the pharmacokinetics of one analyte in rats after oral administration of extract was different from that after oral administration of the neat compound.The results might be helpful for further clarification of the relationship between the bioactive constituents and the mechanisms of Menispermi Rhizoma in efficacy and toxicity.4.A UPLC-MS/MS method has been developed and validated for simultaneous quantitation of dauricine,dauricicoline,dauricoside,acutumine and acutumidine in rat urine.After addition of internal standard(verapamil),urine samples were pretreated with acetonitrile.Chromatographic separation was performed on a Waters BEH C18 column with gradient elution using a mobile phase composed of acetonitrile and water(containing 0.1%formic acid).The linear ranges for dauricine,dauricicoline,dauricoside,acutumine and acutumidine were 5-2500,2.4-1200,2-1000,6-3000,10-5000 ng/mL,respectively.The method was validated in urine samples,which showed good linearity over a wide concentration ranges(r≥0.9902).The lower limits of quantification(LLOQ)were 5 ng/mL for dauricine,2.4 ng/mL for dauricicoline,2 ng/mL for dauricoside,6 ng/mL for acutumine,10 ng/mL for acutumidine,respectively.The extraction recoveries of the analytes and verapamil were above 76.4%.The specificity,matrix effect,precision,accuracy and stability of this method can meet the requirements of biological sample analysis.The validated method was successfully applied to the urine excretion study of the five alkaloids in rats after oral administration of Menispermi Rhizoma extract.The experimental results showed that the mean cumulative excretion in urine within 144 h was 335118±63965 ng for dauricine,54564±13915 ng for dauricicoline,8860±2019 ng for dauricoside,16371±5155 ng for acutumine,83130±24784 ng for acutumidine.