| Cancer has become the most common disease which threats human health. Liver cancer has become one of the three factors in cancer death, and the death rates are still increasing.After unremitting efforts from several generations of medical and pharmic workers, chemotherapy can be used for controlling cancer and has received certain curative efficacy, but the side effects of chemotherapeutic drugs limit their application. Therefore, to discover and develop Traditional Chinese medicines that have therapeutic selectivity or that can preferentially kill cancer cells with few side effects to normal cells is an important tendency for cancer therapy. Many researches suggest that Traditional Chinese medicines may be used alone or in combination to prevent the occurrence and metastasis of cancer, or even to treat cancer. But Traditional Chinese medicine have complicated composition, widely effect and poor selectivity to specific tumor. Emodin has widely sources,exists in rhubarb genera,fagopyrum esculentum, buckthorn and senna. Emodin has widely pharmacological effects including antibacterial, anti-inflammatory, anti-tumor and protecting liver and kidney. Oleanolic acid is pentacyclic triterpene acid naturally and has obvious inhibitory effect on liver cancer cells. The study is to make reference to the theory of the research of targeted drug delivery led by traditional Chinese medicine,which combined modern new pharmacy technology with Traditional Chinese medicine guide theory applys in targeting drug delivery system, and used oleanolic acid as the surface material modifying emodin liposome targeting preparation,to make liposome more specific and selective,thus improves the curative effect.The first part:Objective:A high performance liquid chromatography(HPLC) method has been established for the determination of encapsulation efficiency of oleanolic acid-emodin liposome.Methods:Separation was achieved using an Agilent Zorbax C18 column(250 mm×4.6 mm,5 μm).The mobile phase consisted of 0.1% phosphoric acid solution with methanol (20:80, v/v). The UV detection was conducted at 210 nm for all compounds. Results:There has a good linear relation of emodin in the range of 2.50～160.0 0μg/mL(r=0.9996) and the average recoveries were 94.34%～96.60% with the RSD below 4.87%.The linear range of oleanolic acid was in the range of 2.50-160.00 μg/mL(r=0.9995) and the average recoveries were 91.23%～95.15%with the RSD below 4.31%.Conclusion:The established HPLC method is simple,fast,good specificity and thus is suitable for the determination of encapsulation efficiency of oleanolic acid-emodin liposome.The second part:Objective:To prepare oleanolic acid-emodin liposome (OA-EM-LP).Method:To prepare liposome by film hydration method and separate liposome and free drugs by low temperature ultracentrifugation. Central composite design-response surface method were used to optimize the formulations,and composited the overall desirability by the encapsulation efficiency of EM and OA as indicators to optimized the best formulation. Result:The optimum conditions of formulation were phosphatidylcholine/OA-EM(1:1) 26.32,phosphatidylcholine/cholesterol 16.68 and hydration medium pH6.37.The encapsulation efficiency of OA and EM were 89.13% and 86.16%,respectively.Conclusion:The OA-EM-LP has good encapsulation efficiency after being optimized with central composite design-response surface method.The third part:Objective:A high-performance liquid chromatography (HPLC) method was developed and validated for determination of emodin in mouse plasma and tissue (heart,liver,spleen,lung and kidney) after tail intravenous injection.Targeted evaluation indexes and studied the liver targeting of emodin enhanced by oleanolic acid by combined Traditional Chinese medicine guide theory with modern new pharmacy technology.Methods:Separation was achieved using a Thermo Syncronis C18 column(250 mm×4.6 mm,5 μm).The mobile phase consisted of 0.1%phosphoric acid solution with methanol (15:85, v/v) at a flow rate of 1.0 mL/min. The UV detection was conducted at 254 nm for all compounds. The mice were injected liposome by Tail intravenous with EM dosage 5 mg/kg.Detect the concentration of EM in plasma and tissues(heart,liver,spleen,lung and kidney).Calculate the pharmacokinetic parameters. Study the targeted evaluation of two kinds of liposomes (emodin liposome and oleanolic acid-emodin liposome)with the targeted evaluation indexes te,re,TEc.Results:The separation of emodin and impurity in biological samples was good.The linear range of EM was in the range of 0.02～8.00 μg/mL(r=0.9988) in plasma,0.05～1.60 μg/mL(r=0.9990) in heart,0.05～12.80 ug/mL(r=0.9999) in liver,0.05-3.20 μg/mL(r=0.9991) in spleen,0.05～ 6.40 μg/mL(r=0.9998) in lung and 0.05 ～ 6.40 μg/mL(r=0.9996) in kidney. The within-day RSD and between-day RSD were less than 9.87% and 9.95%,respectively.The stability RSD was below 9.76% and the average recoveries were 70.02%～76.95%, with the RSD below 7.69%.t1/2 and clearance of emodin liposome group were 8.96 h,974.8 mL/h·kg,respectively.t1/2 and clearance of oleanolic acid-emodin liposome group were 9.37 h,854.14 mL/h·kg,respectively.te-OA-EM and TEcOA-EM of oleanolic acid-emodin liposome group were both higher than te-EM and TEcEM of emodin liposome group, and the re of liver was more than one.Conclusion:The established HPLC method is Simple, good specificity and high sensitivity and thus is suitable to the determination of emodin in mouse plasma and tissue (heart,liver,spleen,lung and kidney). Oleanolic acid-emodin liposome has the ability of liver targeting,the liver targeting of emodin enhanced by oleanolic acid. |
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