Preparation Of Apigenin Nanoparticles And Preliminary Study On Anti-inflammatory And Anti-Tumor Activity In Vitro

Apigenin(API)is known as parsley or celanthin,is a natural and widely distributed flavonoid,and its rich pharmacological activity has been concerned and recognized by domestic and foreign researchers.However,API is particularly poorly soluble,almost insoluble in water and low in organic solvents,resulting in low bioavailability.In this paper,API nanoparticles were prepared by anti-solvent precipitation method,the preparation conditions were optimized and the preparation results were characterized.The saturated solubility and dissolution of raw API,physical mixture of API and API nanoparticles were compared.To investigate the anti-inflammatory and anti-tumor activities of raw API and API nanoparticles on RAW264.7 macrophages in mice and HepG2 cells.The results indicated that:1.Based on single factor experiments and response surface methods,the process conditions of API nanoparticles were optimized.The optimum technological conditions were determined as follows:tween 80 as surfactants and concentration 5 mg/mL,API concentration 18.8 mg/mL,antisolvent/solvent volume ratio 10:1,the precipitation temperature 45℃,the dropping speed 1 mL/min,the stirring speed 1200 r/min,the stirring speed 5 min,the freeze-drying method was used and 2-Hydroxypropyl-β-cyclodextrin(HP-β-CD)(API:HP-β-CD,1:7,g/g)was selected as a cryoprotectant.API nanosuspension with a MPS of 180.5 nm was obtained under the optimum conditions,and the yield of API nanoparticles is 76.25%..2.The API nanoparticles were characterized using various analytical techniques such as SEM,FTIR,XRD,DSC and TG.The experimental data showed that the surface of API nanoparticles was attached with HP-β-CD,and the crystal structure becomes amorphous.What’s more,the particle size decreased,which led to a larger specific surface area and easier dehydration and decomposition.3.The results showed that the saturation solubility of raw API,physical mixture of API with HP-β-CD and API nanoparticles in artificial gastric juice were 0.91 μg/mL,8.51 μg/mL and 21.56 μg/mL,respectively,and in artificial intestinal juice were 0.85 μg/mL，17.51 μg/mL and 63.64 μg/mL,respectively.In artificial gastric juice and artificial intestinal juice,the dissolution rates of raw API,API nanoparticles and physical mixture of API with HP-β-CD were 13.46%,45.81%,82.24%;12.13%,38.43%,67.48%,respectively.Therefore,the saturated solubility and dissolution rates of API nanoparticles were obviously higher than the others.In artificial gastric juice,the saturation solubility and dissolution rates of API nanoparticles were 23.69 times and 6.11 times of raw API,respectively;in artificial intestinal juice,the saturation solubility and dissolution rates of API nanoparticles were 74.87 times and 5.56 times of raw API,respectively.4.In this paper,RAW264.7 murine macrophage and HepG2 hepatoma cells were selected for preliminary studies of anti-inflammatory and anti-tumor activity,respectively.In the concentration range of 7.82-125 μg/mL,the Griess and ELISA method was used to detect the secretion of NO and IL-10 after LPS-induced inflammation in the RAW264.7 cells by raw API and API nanoparticles,respectively.It was found that raw API and API nanoparticles inhibited the secretion of NO and IL-10,and the inhibitory ability of API nanoparticles was stronger compared with the raw API.The Annexin V-FITC/PI double staining method was used to detect the effect of raw API and API nanoparticles on the apoptosis rate of HepG2 cells.At the concentrations of 15.63 μg/mL and 62.5 μg/mL,the survival rate of API nanoparticles treated HepG2 cells was lower than that of raw API treated HepG2 cells,and the increase of apoptosis rate was proportional to the concentration of apigenin.It was verified that raw API and API nanoparticles had anti-inflammatory effects and anti-tumor effects,and the inhibitory effect of API nanoparticles was stronger compared with the raw API.In summary,this study used the anti-solvent precipitation method to successfully prepare API nanoparticles,which have greater solubility,higher dissolution rate,and better anti-inflammatory and anti-tumor activities than the raw API.