Hedyotis Diffusa Antitumor Flavonoids And Polysaccharides Purification And Liposome Complexes

ObjectiveTo make an in-depth development of Oldenlandia diffusa, which is commonly used in clinical as an anti-tumor medicine:separating and purifying its active site(polysaccharides and flavonoids), making a transformation of flavonoids in vitro, preparing the liposome of Aglycon and polysaccharide which is the best carrier for anti-tumor drugs with the advantages of good activity against its target and low toxic side effects, and evaluating the new preparation by indexs of Pharmacodynatics and stability.Methods1. Use appropriate methods of extraction and purification to obtain high purity active Polysassharides fraction, and make a preliminary study of its primary structure and physical and chemical property.2. Diffusa flavonoid glycosides is purified by macroporous, and then be transformed into aglycon by acid hydrolysis in vitro.3. Check resin sesidue of flavonoid aglycone by headspace-GC.4. HPLC-MS method is used to preliminarily analyze the basic nucleus and relative proportion of flavonoid aglycone.5. Comparing of encapsulation results of different Iiposomes technologies and preparing of liposome of flavonoid aglycone. 6. Proliposome with good stability is prepared by freeze drying technology (LCOD).7. To study the physical and chemical properties and preliminary stability of diffusa flavonoid aglycone on liposome complex.8. Anti-tumor experiment in vitro and in vivo are used respectively to study the pharmacodynamics of liposome complex.Results1. Diffusa polysaccharide was made by water extraction and multi-level alcohol precipitation,and from which impurities such as proteins and pigments were removed by tannic acid precipitation and activated carbon adsorption,and then high purity polysaccharide(PODⅡ) were obtained by means of ultrafiltration,DEAE-32 ion-exchange chromatography and Superdex 30 gelchromatography and so on.2. Diffusa flavonoids effective parts extracted by ethanol were purified by modified calcium hydroxide-sulfuric acid method, AB-8 macroporous resin technology,and then were transformed into flavonoid aglycone with the advantages of small molecular, low polarity, good fat-soluble and membrane permeability by mild acid hydrolysis technology.3. According to analysis of diffusa flavonoid aglycone by headspace-GC,we found that pretreatment of resinused (patented technology) could effectively remove the resin residue.No related residue was detected in the test.4. According to analysis of diffusa flavonoid aglycone by HPLC-MS, we also found that its basic nucleus was quercetin and kaempferol,with the content ratio of 3.2:1. 5. By taking encapsulation efficiency as main evaluation index, we mainly compared the film dispersion method and the reverse evaporation method,finding that the improved reverse evaporation method could produce flavonoid aglycone liposomes with good stability and encapsulation efficiency.6. With PODⅡfreeze-drying excipient, as well as effective components, we obtained flavonoid aglycone liposomes with good stability。7. Particle size distribution,morphology,pH,Zeta potential and other physical and chemical properties of liposome complexes were accurately determined;factors that affect the stability of liposomes were investigated, such as light, temperature;liposome complexes stability at room temperature were investigated by three months.8. Method in vivo was used to test that the liposome complexes inhibited the growth of solid tumors and prolonged the life of ascites tumor on S180 and H22, and the effect on tumor growth of human lung cancer A549;Method in vitro was used to investigated the growth inhibition effect to tumor CellsH460, Hela, Bel-7402 and MCF-7.The effect of LCOD on macrophage phagocytosis, lymphocyte proliferation reaction, and spleen NK cell activity in mice were investigated respectively, in order to study LCOD's regulation of immune function in mice.ConclusionOldenlandia diffusa aglycone liposome complex made with innovative ideas shows excellent antitumor activity,and has good stability.Polysaccharides and flavonoids ingredients play a role of supporting just and dispelling evil effect via compatible application according with the Traditional Chinese medicine characteristics of "multicomponent,multiple target point,mehrkanal",and plays a whole effect. This research has great academic value and good development and application prospect,and is of great significance to Oldenlandia diffusa's rational application,comprehensive utilization and industrialization development.