Studies On Multi-component Anti-lung Cancer Microemulsion Of "Ling-Yi Fang"

"Ling-Yi Fang" is a clinical anti-lung cancer prescription,which composed of Ganoderma lucidum and Coix lacryma-jobi.The prescription is good at bettering-qi and nourishing-yin,eliminating dampness through diuresis,eliminating tumor and resolving hard lump.In this thesis,under the guidance of the theory of traditional Chinese medicine and modern medicine theory,effective components of "Ling-Yi Fang" are extracted and purified using the technical of supercritical CO2 extraction(SFE-CO2)and liquid-liquid extraction.The solubility of Ganoderma lucidum triterpenes and Coix seed oil are poor and oral absorption of Ganoderma lucidum polysaccharide and coixan are poor too.Microemulsion has many advantages such as improving the solubility of water-insoluble drugs and lipophilic drugs,promoting the absorption of macromolecular drugs in vivo and improving the bioavailability of the drug,etc.Coix seed oil also has a nature as the oil phase.Therefore,in order to solve the above problems,the present study used Coix seed oil as oil phase of microcmulsion loading Ganoderma lucidum triterpenes,with Ganoderma lucidum polysaccharides and coixan adding in water phase to prepare "Ling-Yi Fang"multi-component microemulsion with high drug loading.Its physical and chemical properties was charactered,and drug loading and stability was evaluated.Anti-tumor activity of"Ling-Yi Fang" multi-component microemulsion was studied through the cell and animal model;and its anti-tumor mechanism was discussed by tumor tissue sections and TUNEL apoptosis.The results of this study show that,"Ling-Yi Fang" multi-component microemulsion has many features such as small average particle size,evenly distribution,high drug loading,stable quality,improving anti-tumor activity of the effective components in vitro and in vivo and so on.Firstly,the chemical composition of Ganoderma lucidum and Coix lacryma-jobi,anti-tumor progress and traditional Chinese medicine microemulsion research progress are reviewed.The experimental part of the study mainly includes the following aspects:1.Species,origin choice and the extraction and purification of the effective components and characterization of each single herb in "Ling-Yi Fang"First,using HPLC,UV spectrophotometric method for the determination of the content of the effective components,and combining anti-tumor activity experimental in vitro by early team,varieties and origin of the experiment with herbs were preferred,which Jiangsudongtai’s Ganoderma lucidum and Hebei’s Coix lacryma-jobi are selected,finally.Ganoderma lucidum triterpenes,Coix lacryma-jobi triglycerides,Ganoderma lucidum polysaccharides and coixan are final effective component groups by extracting and purifying effective components using modern extraction and purification technology.(1)Ganoderma lucidum triterpenes and Coix lacryma-jobi triglycerides are extracted using supercritical CO2 extraction technology.Extraction rate and purity of Ganoderma lucidum triterpenes and Coix lacryma-jobi triglycerides are 3.75%,6.62%,61.50%,74.35%,respectively.(2)The polysaccharide in Ganoderma lucidum and Coix lacryma-jobi are extracted and purified using water extraction and alcohol precipitation,Sevag removing protein method.Extraction rate and purity of Ganoderma lucidum polysaccharides and coixan are 6.56%,8.40%,46.40%,64.42%,respectively.2.Preparation,characterization and quality assessment of "Ling-Yi Fang"multi-component microemulsionIn this study,Ganoderma lucidum triterpenes is loaded using the anti-tumor effective component-Coix seed oil,both as a drug and oil phase of microemulsion.Preparation process of "Ling-Yi Fang" multi-component microemulsion is preferred by single factor method combined with the pseudo-ternary phase diagrams.The average particle size,zeta potential and other parameters of the microemulsion are charactered,and the quality of the microemulsion is controlled by drug loading and stability study.(1)The preferable microemulsion prescriptions using average particle size as a main indicator are as follows:①Ganoderma lucidum triterpenes:Coix seed oil:1944CS:RH40:PEG400:water=75 mg:132.5 mg:267.5 mg:400 mg:of 200 mg:2600 mg(Microemulsion 1,ME-1).②Ganoderma lucidum triterpenes:Coix seed oil:RH40:PEG400:water=75 mg:of 400 mg:400 mg:200 mg:2600 mg(Microemulsion 2,ME-2).③Ganoderma lucidum triterpenes:Coix seed oil:RH40:PEG400:water:Ganoderma lucidum polysaccharides:coixan=75 mg:400 mg:400 mg:200 mg:2600 mg:131.2 mg:507.5 mg(Microemulsion ME-3).(2)Three kinds of prepared microemulsion are charactered.The average particle size of ME-1,ME-2 and ME-3 is 41.13 nm,47.59 nm and 69.92 nm,respectively;PDI is 0.199,0.156 and 0.060,respectively;Zeta potential is-13.74 mV,-16.98 mV and-11.30 mV,respectively.Three kinds of microemulsion with round appearance and uniform distribution are observed by transmission electron microscopy.(3)The drug loading and stability is important indicators of quality evaluation of microemulsion.Drug loading measured by this study of three kinds of prepared microemulsion are 4.18%,9.87%and 17.71%,respectively.Through dilution,pH,high-speed centrifugation,left for a long period of time and other conditions,the stability of three kinds of microemulsion is investigated,and the stability of the microemulsion is fine.3.The anti-lung cancer activity of "Ling-Yi Fang",multi-component microemulsion in vivo and in vitro(1)Growth inhibition in vitro on human lung cancer A549 cell of "Ling Yi Fang"multi-component microemulsionThe anti-tumor activity of different proportion of effective components in "Ling-Yi Fang" and prepared "Ling-Yi Fang" multi-component microemulsion is studied by using MTT assay.The experimental results show that the IC50 on A549 cell of the suspension 1,the suspension 2,the ME-1 and ME-2 are 1.42,1.55,0.77 and 0.62 mg crude drug-mL"1.The results showed that the prepared microemulsion significantly enhances the anti-tumor activity in vitro of Ganoderma lucidum triterpenes and Coix seed oil,and the IC50 on A549 cell of ME-2 is significantly less than the ME-1,which indicating that the anti-tumor activity in vitro of ME-2 is strong.So anti-tumor activity in vivo of ME-2 is further studied.(2)Pharmacodynamic evaluation of anti-tumor in vivoIn order to the anti-tumor activity of microemulsion containing polysaccharides,it is added to ME-2.pharmacodynamic evaluation of the suspension of effective component of"Ling Yi Fang" and the corresponding "Ling Yi Fang" multi-component microemulsion is researched with tumor inhibition rate and immune regulation index as evaluation indexes using solid tumor model in mice bearing Lewis animal lung cancer.Experimental results show that,in the dose of 30 g of crude drug-kg-1·d-1,the inhibition rate of ME-2,a suspension 2,the ME-3 and which ME-3 corresponding to the suspension are 67.41%,44.63%,57.25%and 45.89%,respectively;and it has significant anti-tumor effect(P<0.05)compared with the saline group.Compared with the suspension group,the microemulsion group has significant anti-tumor effect(P<0.05).Compared with cyclophosphamide and saline group,liver index,thymus index,spleen index and content of TNF-α,IL-6 in plasma of“Ling-Yi Fang"administration group showed significant increase(P<0.05),which indicates that Ganoderma lucidum triterpenes and Coix seed oil and related combination can not only synergistic kill tumor cells,but also synergistically enhance immune activity,so as to play the role of anti-tumor.It fully reflects the advantage of multi-component,muti-channel and anti-tumor of the compound.4 Anti-lung cancer mechanism of "Ling-Yi Fang" multi-component microemulsionIn this study,anti-cancer mechanism of "Ling-Yi Fang" multi-component microemulsion is preliminarily discussed by tumor tissue sections and TUNEL apoptosis.The results show that "Ling-Yi Fang" multi-component microemulsion has a mechanism of promoting tumor necrosis and tumor cell apoptosis in a dose-dependent manner.Besides,the research of microemulsion as the solvent system to extract the active ingredient Ganoderma lucidum(1)The fat-soluble and water-soluble components in Ganoderma lucidum are simultaneously extracted with microemulsion as a solvent.This study provides an experimental basis for the feasibility of extracting the multi-components in Ganoderma lucidum with microemulsion.(2)According to the dissolution characteristics of triterpenes and polysaccharides in Ganoderma lucidum,the optimal blank microemulsion was used as a solvent to sonicate the Ganoderma lucidum powder,and one-step method to prepare Ganoderma lucidum multicomponent microemulsion is explored.