Research On Liver-specific Targeting Of Angelica Sinensis Polysaccharide ASP1in Vitro And In Vivo

Angelica polysaccharide is the main biologically active component of theAngelica sinensis (Oliv.) Diels, has significant activity in the anti-anemia, anti-tumor,liver damage, and immune function. In this study, the angelica polysaccharide, namedASP1, was obtained by water extraction and acid-base deproteinization and thenisolated and purified by ethanol precipitation, ultrafiltration and Sephadex G50. ASP1was labeled by FITC for the further study. Research on liver-specific targeting ofASP1was undertaken in vitro and in vivo.Part Ⅰ Extraction, isolation and purification of ASP1The raw angelica polysaccharide was obtained by water extraction and acid-basedeproteinization. After ethanol precipitation and ultrafiltration, the small pigment andoligosaccharide were removed. Then the ultrafiltration product was further purified bysize-exclusion chromatography on Sephadex G50to get the refined angelicapolysaccharide, named as ASP1. The purity of ASP1was identified by the assaying ofsaccharide, the measurement of molecular weight, KI-I2test and UV test. Thesaccharide content, measured by phenol-sulfuric acid method, was90.8%. Themolecular weight, measured by HPGPC, was8.780×104Da. The ASP1was free fromstarch, protein and nucleic acid shown in KI-I2test and UV. The results indicated thatASP1was high purity homogeneous polysacharides.Part Ⅱ Preparation,analysis and stability in samples of ASP1-FITCASP1was labeled by FITC. The yield was83%. The labeling efficiency wasevaluated by UV, FS and HPGPC. The UV test showed that the characteristicabsorption peak near494nm appeared in spectrogram of FITC-labeled ASP1but theunlabelled one. The degree of substitution by FITC was1.18%(w/w). TheFITC-labeled ASP1, test by FS, has strong fluorescence intensity and λex=494nm，λem=520nm. The molecular weight, measured by HPGPC, was8.780×104Da. Andit showed minute difference before and after the labeling. The stability of ASP1-FITCin0.01M PBS and palsma samples in vitro and vivo were evaluated by HPGPG-FLD.The results showed that both the molecular weight and fluorescence intensity of ASP1-FITC had no obvious change in20h in0.01M PBS and palsma samples under37℃，60rpm and dark. The molecular weight of ASP1-FITC did not change eitherafter admistration by tail vein for2h. We succeeded in labelling ASP1with FITC.The labelled ASP1had a high yield, appropriate substitution, high fluorescenceintensity and good stability. It was suitable for the research on liver-specificedtargeting of ASP1in vitro and in vivo.Part Ⅲ Research on liver-specific targeting of ASP1in vivo.1. Establishment of method to determinate the content of ASP1in biological samples.A sensitive, efficient and accurate method was established and validated for thedetermination of content of ASP1in biological samples on the base of fluorescence ofASP1. The linear calibration curve covered a concentration range of0.25μg/ml to200μg/ml with a lower limit of quantification of0.20μg/ml. The extraction recoveryof ASP1was determined at low, medium and high concentrations (0.5,2.5,25.0μg/ml), the recovery rate is91.98%~114.20%. The samples had a good stabilitywhen kept for3days under4℃and7days under-20℃away from light. Themethod has been successfully applied to the study of pharmacokinetics and tissuedistribution of ASP1in rat.2. Pharmacokinetics and tissue distribution of ASP1in ratDetermine the plasma concentration in rat after administration a dosage of12mg/kg ASP1through caudal vein. Then use DAS2.1.1to fit the compartmental modeland calculate the pharmacokinetic parameters. Results showed that twocompartmental model fitted the best, t1/2=15.931min， CL=0.001L/min/kg， MRT（0-）=23.536min. The sampling time of tissue distribution was determined at30min,60min and120min according to the concentration time curve andpharmacokinetic parameters. Tissue distribution results showed that ASP1concentration from high to low in turn for liver, plasma, intestine, heart, spleen,lung, stomach, kidney and brain at120min after administration. The plasmaconcentration declined to1/60of Cmaxand was far below the concentration in liver.ASP1concentration in liver was30times higher than that in other tissues. So wecame to the conclusion that ASP1has good liver-specific targeting. The study result of our group showed that particle size of ASP1ranged between0.6μm and1.2μm andcould passive target to liver and spleen. However, ASP1concentration in spleen hadbeen very low after administration, which showed that ASP1had bad passivetargeting to spleen. Many studies have proved that one drug has the similar passivetargeting to liver and spleen. So it implied that active targeting but passive targeting ispredominant.3. Liver tissue slice observationThe experimental group was administrated a dosage of120mg/kg ASP1. Thecontrol group was administrated equivalent NS. Both were put to death at120min.The liver tissues slices was prepared and observed under confocal laser scanningmicroscopy. The results showed that the fluorescence intensity of experimental groupwas obviously higher than that of control group. In experimental group, most offluorescence was observed in cytoplasm but rarely in intercellular substance, whichmeant that ASP1was endocytosed by hepatocyes. The result further confirmed thatASP1had strong active liver targeting effect.Part Ⅳ Research on liver-specific targeting of ASP1in vitro.Use the digestive method of collagenase perfusion to obtain rat hepatocytes andthen construct ASP1liver-specific targeting model in vitro. The cell viability ofobtained rat hepatocytes was above90%, among which the parenchymal hepatic cellshad a large proportion. Add50μg/mL,100μg/mL and200μg/mL ASP1into1×106rathepatocytes, respectively, as experimental group. And add the medium of samevolume as the control group. The results showed that fluorescence intensity ofexperimental group cells increased with the concentration of ASP1andsignificantly(contrasted with the control group,*p﹤0.05). Contrasted with the controlgroup, the targeting rate, cell number with fluorescence intensity increased accountedfor the proportion of the total cell number, also increased with the ASP1concentration.The targeting rate of50μg/mL,100μg/mL and200μg/mL ASP1were73.93%,94.83%and98.23%, respectively. Research on liver-specific targeting of ASP1in vitro alsoconfirmed that ASP1had strong active targeting effect on rat hepatocytes.