| Ginsenoside Rg3(3β,12β,20(R)-dihydroxydammar-24ene-O-[β-D-glucopyranosyl (1â†'2)-β-D-glucopyranoside]), a minor ginsenoside from thePanax ginseng, has been shown multiple pharmacological activities, includingdecrease toxicity, improve symptoms of Qi–deficiency, increase immune function,memory improvement, and inhibit proliferation, infiltration and metastasis of thetumor cells. However, Rg3has poor solubility and oral bioavailability, which limits itsclinical activity. The plasma concentration of Rg3after oral administration is low.Previous researches obtained some human pharmacokinetic parameters, includingCmaxof (166) ng/ml, Tmaxof (0.66) h an3.2mg/kg from the oral experiments. Itis widely accepted that low plasma concentration and short half-life of drugs hadmany reasons, firstly, the drug absorbed by gastrointestinal was low; secondly, thedrug is metabolized quickly by the enzymes and bacteria in the gastrointestinal tract;thirdly, the drug can be absorbed by the gastrointestinal mucosal, but is metabolizedby the enzymes in intestinal or liver, which is called first-pass effect. Therefore, thepurposes of this paper are to increase its bioavailability and to improve efficacy.In order to improve Rg3biodistribution in vivo, a safer and more effectiveformulation of Rg3is highly desired. Liposomes contain one or more amphiphilicbilayers and an internal aqueous space, and can be used as drug carriers. Liposomeshave been used to formulate a variety of drugs. Hydrophobic chemotherapeutic agents,such as paclitaxel and docetaxel, can incorporate into the liposomal bilayer, leading toimproved solubility, prolonged circulation time, altered biodistribution in vivo, andreduced side-effects.The following parts are included in this paper:1. The physicochemical properties of Rg3, including n-octanol/water partitioncoefficient, the solubilities in various solvent, stability were determined. The resultsindicate that: Rg3is insoluble in water, ether or chloroform and soluble in methanol, alcohol and dichloromethane. The concentration of the methanol solution at roomtemperature and accelerated under the experimental conditions decreased slightly, andrelatively stable nature.2. Preparation of Rg3-lipsome. Rg3-Liposomes (L-Rg3) were prepared bypolycarbonate membrane extrusion method. Based on a single-factor test, threefactors, namely ratio of lipid to drug, ratio ePC to cholesterol, and total lipidconcentration mg/ml, were selected to optimize the composition of L-Rg3. Theselected factors were subjected to analysis by the response surface methodology(RSM) with a three factor-three coded level Box–Behnken design (BBD). Therelationship between each factor and index was fitted using data processing softwareDesign-Expert trial version8.0.6. The set of optimum conditions, determined using theRSM optimization approach, were tested experimentally according to the modelequation. Three batches of liposomes were prepared according to the optimizedformulation. Then EE of each batch were determined. The mean experimental EE82.47%was close to the predicted results89.69%(lipid concentration56.82mg/ml;ratio of lipid to drug9.11, ratio lipid to cholesterol4.03). The characteristics of L-Rg3were investigated. The results indicated that the liposome were mainly unilamellarand stability of liposome were investigated. The results of stability indicated thatL-Rg3were stable at4or25for12months.3. The pharmacokinetics and tissue distribution of L-Rg3were investigated. Tostudy the pharmacokinetics behavior and tissue distribution of liposomal Rg3, a stable,rapid, and accurate LC-MS method for the determination of Rg3in biological samplewas developed. Thirty Wistar rats were divided into six groups of5animals each. Therats in each group were given Rg3or L-Rg3i.v. at a dose of0.25,0.5or1mg/kg. Theplasma concentration vs time of Rg3obtained after the injection. After injection ofL-Rg3, the Rg3concentration was still measurable after12h, while the drug injectedfree in solution was not detectable even after5h. The liposomes enhanced the Cmaxand AUC of Rg3by1.19-and1.52-fold, respectively (p<0.05). Rg3was extensively distributed in all tissues assayed. The liposome enhanced the concentration of Rg3inall the tested tissues. Compared to Rg3solution, L-Rg3significantly enhanced theliver and lung absorption of the drug (P<0.05).4. In pharmacodynamics, we studied the anti-tumor effect of L-Rg3and Rg3fortwo cancer cell lines (A549and HepG-2) in vitro by MTT assay. Proliferation ofA549and HepG-2cells were both inhibited in concentration-dependent manners.IC50of Rg3and L-Rg3for A549cell were respectively126.43μg/ml and84.68μg/ml;IC50of Rg3and L-Rg3for HepG-2cell were respectively99.74μg/ml and65.87μg/ml. No cytotoxicity of the unloaded liposome was observed in either cell-linestudied. In vivo anti-tumor experiment, A549bearing mice was used as animal model,inhibitive rate of tumor and life quality were used to evaluate the efficacy followingadministration at a dose of1mg/kg. Tumor volume, inhibitive and survival rates weredetermined. Treatment with Rg3and L-Rg3both showed decrease in tumor volumecompared with that of the controls during treatment period (control group,1335mm3,p <0.05). Inhibitive rate of tumor in the L-Rg3group (59.16%) was higher Rg3group(50.86%)(P <0.05). At21days after the treatments, the mice in the control, L-Rg3,and Rg3group were all alive. There were no significant abnormalities in psychosis,status of activity, reaction to stimulation, appetite or depilation of mice in the Rg3,control or L-Rg3group. The percent weight loss was used to evaluate the toxicity ofdifferent formulations. The percent weight loss was not above15%, which indicatedthat the toxicity of liposome was low. However, the mice relative to the experimentalgroup, the control group lost more weight. In addition, CD34immunohistochemicalanalysis was used for measuring microvessel density (MVD) of the tumor tissues.Compared with the control group, MVD value in the ginsenoside Rg3and L-Rg3group decreased significantly, especially in the liposome group (P <0.05). Inconclusion, the anti-tumor activities of L-Rg3in vivo and in vitro were superior toRg3.5. We also studied the pharmaceutical safety of L-Rg3to determine if this formulations was safe via iv administration. The results indicated that no hemolysisand no hypersensitivity were found in the formulations, in addition, the formulationwas tolerated for mice at maximal concentration and maximal injection volume,which showed the safety of this formulation. |
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