Study On Preparation Of Zingerone Self-microemulsion And Its Mechanism Of Anti-liver Cancer

Liver cancer is one of the most common malignant tumors in the world,which has been widely attention.For the treatment of liver cancer,it is often necessary to implement individualized and comprehensive treatment for liver tumor stages of discretionary.Zingerone,a cheap nontoxic compound widely available in many herbal medicines,can interfere with the energy metabolism of hepatocytes and inhibit the proliferation of hepatocellular carcinoma cells.However,due to its poor water solubility and low bioavailability in rats,the medical application and clinical effect of zingerone are greatly restricted.To address this problem,a self-microemulsifying drug de1ivery system（SMEDDS）was selected as the carrier of zingerone,which improved the solubility and oral bioavailability of zingerone by enhancing drug bypass transport and avoiding the first-pass effect of portal vein.With the superior performance,SMEDDS can greatly enhance the anti-tumor effect in vitro.The anti-liver cancer mechanism of zingerone and Z-SMEDDS was investigated by MTT assay,flow cytometry and western blot.The study and specific results are included as following parts:（1）Firstly,a HPLC method was established to detect the content of zingerone fin vitro and in vivo analysis,and methodological investigation was carried out,which proved that the method is accurate and reliable,linear range 0.5μg/mL to 250μg/mL.For different excipients,a single factor experiment combined with pseudo-ternary phase diagram was used to screen out suitable excipients for self-microemulsion and optimize the prescription.The best prescription was obtained as follows:zingerone 6.61%,ethyl oleate 10%,Tine 80 81%,PEG400 9%.（2）Z-SMEDDS were prepared by reversed-phase microemulsion method.The Z-SMEEDS prepared under the optimal prescription had good morphology and uniform dispersion.The average particle size of Z-SMEDDS was 17.29±0.07 nm,and the Zeta potential was-22.81±0.29 m V.The PDI was 0.17±0.01,and the encapsulation rate was 97.96%±0.02%,which showed good stability after one month of storage at room temperature.The in vitro release results showed that the cumulative release rate of Z-SMEDDS was significantly higher than that of free zingerone.The cumulative release rate of Z-SMEDDS in the four media was above 70%in vitro,which indicated that SMEDDS can remarkly improve the water solubility of zingerone.（3）Eight male SD rats were randomly divided into zingerone bulk drug group and Z-SMEDDS group,with 4 rats in each group.The dosage was 300 mg/kg by intragastric administration,and pharmacokinetic analysis was conducted in vivo.The pharmacokinetic data showed that the relative bioavailability of Z-SMEDDS was increased by 7.63 times compared with gingerone bulk drug group,t_1/2 was extended from 16.20 min to 212.4 min,MRT was extended from 22.2 min to 276 min,and the C_max of Z-SMEDDS was 46.99μg/mL.The C_max of Z-SMEDDS was increased to 99.98μg/mL,AUC_0-24h of bulk drug and preparation were 19.21μg/mL and146.58μg/mL,respectively.（4）In vitro anti-tumor experiment results showed that the IC50 value of gingerone was 134.1μg/mL after 48 hours of treatment,and 31.80μg/mL for Z-SMEDDS.The anti-tumor effect of Z-SMEDDS group was significantly improved compared with the active drug group,and significantly different from the positive control（5-FU）.Calcein-AM/PI double staining showed that the survival rate of cells treated with free zingerone,self-microemulsion and 5-FU decreased with the increase of drug concentration and action time.DAPI assay revealed that the zingerone treated cells showed apoptosis and nuclear shrinkage,and fragmentation appeared with the increase of concentration.The results of flow cytometry demonstrated that the apoptosis rate of free zingerone group was 20.37%,and that of Z-SMEDDS group was 73%,and the early apoptosis was more significant than that of API group.Western blot results showed that the expression of BAX was significantly up-regulated and the expression of Bcl-2 was down-regulated in Z-SMEDDS group compared with zingerone bulk drug group at the same drug concentration.These results indicate that Z-SMEDDS can improve the solubility of zingerone,thus increasing the uptake of zingerone by cells,and finally achieving the purpose of enhancing the anti-tumor effect.（5）The tumor spheres were cultured by the suspension drop method.The results showed that the tumor spheres were round and closely linked under the condition of 1000 cells/20μL,and the experiment was reproducible.The results of calcein-AM/PI staining showed that the survival rate of the tumor pellets treated with the same concentration of free zingerone,Z-SMEDDS and 5-FU decreased with the increase of action time.The results of flow cytometry showed that the apoptosis rate of zingerone free drug group was 30.6%,and that of Z-SMEDDS group was 73.9%.The Z-SMEDDS group accounted for a higher proportion of early apoptosis,which could restrain the growth of tumor cells more effectively.Western blot results showed that the expression of Bcl-2protein was significantly decreased,while the expression of BAX protein was significantly increased,which was basically consistent with the trend of two-dimensional experiment in vitro.