| AIMSOur aims were (1) to synthesize galactosides(GnDE); (2) to prepare Oleanolic acid solid lipid nanoparticles (OA-SLN) and the nanoparticles modified by GnDE (OA-GnSLN); (3) to establish HPLC analytical method for the determination of OA in biological samples; (4) to observe the distribution of OA, OA-SLN and OA-GnSLN in serum and viscera homogenates of the mice; (5) to study the differences of OA, OA-SLN and OA-GnSLN for liver-targeting in order to develope a new preparation of OA . The liver injury model in mice by injecting intraperitoneally CCl4 and detected the therapeutic effects of OA-G10SLN on injuried liver was detected, which may be the basis of clinical usage of oleanolic acid.METHODS1 Synthesis and identification of galactosides(GDE).The reaction of PnSE [C18H37(OCH2CH2)nOH,n=2,10] with tetraacetylgalactosyl bromide respectively gave corresponding galactosides, and the acetyl groups were removed to afford GnDE (n=2,10). The chemical structures of GnDE were identified by HNMR spectroscopy.2 OA-SLN and OA-GnSLN were prepared by thin layer ultrasonic technique. The constituents of formulation were dissolved in 15mL of chloroform in a round-bottomed flask 100 mL. After removal of the solvent by rotary evaporation in vacuo, a lipid film was obtained on the flask wall and subsequently re-suspended in 10 mL of 60 mg·mL-1 mannitol solution. The suspension was shattered by ultrasonics for 40min. Finally, the products were filtrated by 0.45μm filter membrane and put in cillin-bottles. Drug loading (DL) and entrapment efficiency (EE) were determined by Sephadex G-50 columnchromatography and RP-HPLC. The formulation was optimized by orthogonal design test.3 To establish HPLC analytical method for the determination of OA in biological samples, 0.1 mL of serum or viscera homogenates was entracted with methyl tert-butyl ether(MTBE1.0mL×2) The orgaric phase were combined and blowed to dryness. The residue was dissolued in 100μL methanol and 20μL of the solution was injected into the HPLC instrucment for analysis. HPLC analysis was performed using a Kromasil column . with methanol/H2O(90:10) the mobile phase and a flow rate of 1mL/min,and determinated at 210nm.4 To study the distribution of OA and OA-GnSLN, 120 mice were randomly into 24 groups, with 5 mice in each group. Mice in six group injected with OA-G2SLN, six groups with OA-G10SLN ,six groups with OA-SLN, the remaining six groups with OA-sol. The sample was injected into the caudal vein of each mice at a dosage of 20mg·kg-1. At the predetermined time, blood samples were collected from the ocular vein after removing eyeball. Serum was separated by centrifugation. The mice were then killed and each tested organ was removed, accurately weighed and homogenized. The concentration of OA serum and homogenized tissue samples were used to detect the OA by HPLC. The targeting efficiencies of the three studied samples was obtained.5 The CCl4-induced acute liver injury model in mice was established and OA-G10SLN was injected into the caudal veiv of the mice. at three dosages: 2.5mg·kg-1·d-1,12.5mg·kg-1·d-1 and 25mg·kg-1·d-1 for 7 days. The mice were killed and their sera were obtained to detect the concebtration of ALT and AST. Their liver were stained with HE to evaluate the effects of OA-G10SLN on acute liver injury.RESULTS1 The structures of GnDE were confirmed by HNMR spectroscopy .2 Both OA-SLN and OA-GnSLN were ivory colloid solution. Under TEM, spherical particles with an average diameter of 100.3±57.3nm for OA-SLN, 105.0±45.7nm for OA-G2SLN, and 97.3±40.2nm for OA-G10SLN were observed respectively. The DL of OA-SLN, OA-G2SLN and OA-G10SLN were 8.17%,8.06% and 8.01% respectively. EE of OA-SLN,OA-G2SLN and OA-G10SLN were 98.29% ,93.46% and 94.66% respectively. There was no significant difference among DL and EE of these three types of SLN.3 All the curves were linear over a range from 12.5 to 200.0μg·mL-1. The minimal detectable drug concentration was 1.0μg·mL-1(S/N≥3). The average extraction recovery rate of OA were all more than 95%. The methodological recovery rates of OA ranged from 91% to 109%. The within-day and between-day precisions were less than 10%. The assay is economic, simple, rapid and specific in detecting the concentration of OA in blood and the tasted tissues.4 Targeting efficiency for OA-SLN, OA-G2SLN and OA-G10SLN were 0.67,1.25,4.89 and 5.42 respectively. It was proved that the liver targeting effects of OA-SLN and OA-GnSLN were better than the effect of OA-sol.5 Compared with the model group and OA solution group ,ALT,AST in OA-G10SLN group at all three dosages decreased more significantly.The result confirmed that OA-G10SLN has more favourable therapeutic efficacy in curing hepatic injury of mice caused by carbon tetrachloride.ConclusionsGnDE were synthesized and OA-GnSLN for the first time in this study, on the basis of which OA-SLN and OA-GnSLN were prepared. The distribution of OA, OA-SLN and OA-GnSLN in serum and viscera homogenates of the mice indicated that OA-SLN and OA-GnSLN study possed good liver-targeting property, especially the OA-G10SLN. The pharmacodynamic suggested OA-G10SLN had obvious therapeutic effects on the acute liver injury. |
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