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Establishment Of HPLC Fingerprint Of Euphorbia Lunulata Bge And The Intestinal Absorption,Percutaneous Absorption And Pharmacokinetics Study Of Its Compounds

Posted on:2018-11-27Degree:MasterType:Thesis
Country:ChinaCandidate:M F CenFull Text:PDF
GTID:2334330533967271Subject:Pharmacology
Abstract/Summary:PDF Full Text Request
ObjectiveEuphorbia lunulata Bge is a traditional Chinese medicine in China,in order to further develop and utilization of its medicinal resources,and taking comprehensive understanding of its clinical efficacy of material basis,Euphorbia lunulata Bge was as the research object,its quality control method and related compositions in rat intestinal absorption,percutaneous absorption and in vivo pharmacokinetics study were conducted in the present study.Methods(1)15 batches of Euphorbia lunulata Bge samples were collected from different regions in China.The separation was performed with an Agilent Zoxbax SB-C18 column(5 ?m particle size,4.6×250 mm i.d.).The UV detection was set at 254 nm.The mobile phase was consisted of methanol and 0.2% phosphoric acid in water with gradient elution,and the flow rate was 1.0 mL/min.A high performance liquid chromatography method was established to investigate fingerprint of Euphorbia lunulata Bge.This method was combined with herbal medicine fingerprint similarity evaluation system and two chemometrics methods-hierarchical clustering analysis and principal components analysis to classify and distinguish the attributions of Euphorbia lunulata Bge samples from various regions.(2)The separation was performed with an Agilent Zoxbax SB-C18 column(5 ?m particle size,4.6×250 mm i.d.).The UV detection was set at 254 nm.The mobile phase was consisted of methanol and 0.2% phosphoric acid in water with gradient elution,and the flow rate was 1.0 mL/min.A high performance liquid chromatography method was established to simultaneously quantitative analysis of gallic acid,esculetin,hyperoside,quercetin and kaempferol of 15 batches of Euphorbia lunulata Bge samples obtained various regions.(3)The rat everted gut sac was used to investigate the intestinal absorption properties of different concentrations of Euphorbia lunulata Bge aqueous extract.Gallic acid,esculetin and hyperoside were as the target ingredients,the samples were analyzed by the established HPLC method.The cumulative absorption(Q),absorption rate(Ka)and the apparent permeability coefficient(Papp)of gallic acid,esculetin and hyperoside in duodenum,jejunum,ileum and colon were calculated.(4)Using Franz diffusion cells,isolated mice skin as in vitro transdermal barrier,30% ethanol-saline solution as received solution,gallic acid,esculetin and hyperoside as indicated compounds,the samples were detected by the HPLC method.The transdermal absorption of Euphorbia lunulata Bge aqueous extract without penetration enchancers and the effect of different penetration enchancers(azone,bornel,propylene glycol)in different concentrations on the transdermal absorption of the aqueous extract of Euphorbia lunulate Bge were investigated.The cumulative penetration amount,percutaneous rates,enhanced rate and permeability coefficient were calculated.(5)The rats were intragastric administration of Euphorbia lunulate Bge aqueous extract at the single dose of 1.2 g/kg,2.4 g/kg and 4.8 g/kg,respectively.The HPLC method was used to analyze the concentration of gallic acid,esculetin and hyperoside in rat plasma,and their pharmacokientics parameters were calculated.Results(1)HPLC fingerprint of Euphorbia lunulata Bge aqueous extract was established.Using hyperoside as the internal standard peak,32 common peaks were obtained.Five characteristic peaks were identified with the standards,and they were gallic acid,esculetin,hyperoside,quercetin and kaempferol,respectively.The data of HPLC fingerprint from 15 batches of Euphorbia lunulata Bge samples from different regions were evaluated by the similarity,hierarchical clustering analysis and principal componetsanalysis.The results showed that the similarity were in the range of 0.886~0.994,the content of the constituents were different and 15 batches of Euphorbia lunulata Bge samples could be classified into two clusters.(2)The linear calibration curves were constructed within the range of 5~250 ?g/mL(r=0.9999),10~200 ?g/mL(r=0.9998),20~500 ?g/mL(r=0.9998),5~80 ?g/mL(r=0.9999)and 2~50 ?g/mL(r=0.9998)for gallic acid,esculetin,hyperoside,quercetin and kaempferol,respectively.The result indicated that the content of the Euphorbia lunulata Bge herbal medicine from various regions was significant.The content ranges of gallic acid,esculetin,hyperoside,quercetin and kaempferol of 15 batches of Euphorbia lunulata Bge herbal medicines were 0.16~0.97 mg/g,0.95~1.27 mg/g,1.56~2.04 mg/g,0.32~0.46 mg/g and 0.12~0.26 mg/g,respectively.The method was simple,rapid and accurate for the quantitive analysis.(3)The peaks were detected in the intestinal incubation solution,and 17 peaks from the Euphorbia lunulata Bge aqueous extract were confirmed through comparing to the chemical fingerprint.Three peaks were identified as gallic acid,esculetin and hyperoside,respectively,and their intestinal absorption properties were conducted.Gallic acid,esculetin and hyperoside of Euphorbia lunulata Bge aqueous extract in different concentrations were linear absorption in different intestinal segements,and the square of coefficient correlation exceed 0.90,which was consistent with first order rate process.The subsequence of accumulative absorption content(Q)of esculetin and hyperoside in four intestinal segements was duodenum > jejunum > ileum > colon.The subsequence of accumulative absorption content(Q)of gallic acid in four intestinal segement was jejunum > duodenum > ileum > colon.The Ka of gallic acid,esculetin and hyperoside increased along with the raised concentration of the Euphorbia lunulata Bge aqueous extract.The Papp values of these three ingredients in different concentrations of Euphorbia lunulata Bge aqueous extract samples were no significant difference(P>0.05).The Papp values of gallic acid of different concentrations of samples in four intestinal segements were in the range of 1×10-6~1×10-5(cm/s).The Papp values of hyperoside of different concentrations of samples in duodenum,jejunum and ileum were in the range of 1×10-6~1×10-5(cm/s),the esculetin of different concentrations of samples in duodenum and jejunum were in the range of 1×10-6~1×10-5(cm/s).(4)The result of transdermal absorption of Euphorbia lunulata Bge aqueous extract showed that the percutaneous rates of gallic acid,esculetin and hyperoside without adding penetration enchancers were 0.649 ?g/cm2/h,0.845 ?g/cm2/h and 2.283 ?g/cm2/h,respectively.The cumulative penetration amount and percutaneous rates of gallic acid,esculetin and hyperoside were enhanced when adding different penetration enchancers(azone,bornel and propylene glycol).The data of percutaneous rates indicated that the accumulative permeation amount of these constituents was increased linearly as the time increased,the square of coefficient correlation exceed 0.90,which was consistent with first order rate process.The transdermal absorption was increased as the concentration of the penetration enchancers increased.The subsequence of different penetration enchancers with the same concentration for gallic acid transdermal absorption was propylene glycol > azone > bornel,for esculetin and hyperoside transdermal absorption was azone > bornel > propylene glycol.The penetration effect of enchancers used in combination was stronger than one penetration enchancers used alone.(5)There were 19 peaks detected in the rat plasma,compared with the standards,three peaks were identified as gallic acid,esculetin and hyperoside,respectively.The pharmacokinetics properties of these constituents were conducted.Gallic acid,esculetin and hyperoside were achieved better separation in rat plasma without endogenous substances interfering.The concentrations of gallic acid,esculetin and hyperoside were in the range of 0.02~10 ?g/mL,0.05~10 ?g/mL and 0.15~180 ?g/mL,respectively.The peak area and plasma concentration were in good linearity with r>0.9995.The extracted recovery was over 90%,the inter-and intra-day precision of RSD was <10%,indicated that the method was sensitive and consistent with CFDA requirement of pharmacokientics study.The method can be used to quantify gallic acid,esculetin and hyperoside in rat plasma.The rats were intragastric administration of Euphorbia lunulate Bge water extract at the dose of 1.2 g/kg,2.4 g/kg and 4.8 g/kg,respectively.The Cmax of gallic acid were(4.284 ± 0.233)mg/L,(9.028 ± 0.243)mg/L,and(18.056 ± 0.487)mg/L,respectively.The Tmax of gallic acid were(1.500 ± 0.000)h,(1.500± 0.000)h and(1.500 ± 0.000)h,respectively.The AUC0-? of gallic acid were(11.639 ± 1.103)mg/L·h,(23.578 ± 1.634)mg/L·h and(47.537 ± 3.777)mg/L·h,respectively.The Cmax of esculetin were(4.005 ± 0.163)mg/L,(7.933 ± 0.346)mg/L and(15.201 ± 0.220)mg/L,respectively.The Tmax of esculetin were(0.250 ± 0.000)h,(0.250 ± 0.000)h and(0.250 ± 0.000)h,respectively.The AUC0-?of esculetin were(14.537 ± 1.949)mg/L·h,(28.679 ± 3.892)mg/L·h and(54.596 ± 2.549)mg/L·h,respectively.The Cmax of hyperoside were(3.081 ± 0.134)mg/L,(5.268 ± 0.673)mg/L and(10.083 ± 0.708)mg/L,respectively.The Tmax of hyperoside were(0.500 ± 0.000)h,(0.500 ± 0.000)h and(0.500 ± 0.000)h,respectively.The AUC0-?of hyperoside were(3.848 ± 0.977)mg/L·h,(5.587 ± 1.000)mg/L·h and(10.725 ± 0.825)mg/L·h,respectively.The statistical analysis showed that the pharmacokinetic characteristics of gallic acid,esculetin and hyperoside were linear in three dosages.ConclusionThe HPLC fingerprint of Euphorbia lunulata Bge aqueous extract combined with multi-compounds quantitative was preliminarily established for evaluation quantity of Euphorbia lunulata Bge.The constituents and their contents of Euphorbia lunulata Bge from various regions were significant.The difference produced may be related to the different plants growing environments,such as temperature,soil quality,sun-shine,precipitation and harvest time.Besides,the characteristic of intestinal absorption,transdermal absorption and in vivo pharmacokientics study of Euphorbia lunulata Bge aqueous extract were conducted.The results show that gallic acid,esculetin and hyperoside can enter to the blood,indicated that these active constituents may be related to pharmacological action,and the passive absorption of these constituents in the intestine.The study provided a theoretical basis for dosage forms design of Euphorbia lunulata Bge extract and provided the experimental reference for development its related new drugs.Moreover,the present study enriched the pharmacokinetic studies of the chemical constituents of herbal medicines and made a meaningful exploration for quality control and pharmacokinetics study of Euphorbia lunulata Bge.
Keywords/Search Tags:Euphorbia lunulata Bge, HPLC, fingerprint, intestinal absorption, transdermal absorption, pharmacokinetics
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