| Paclitaxel is one of the active components of Taxus,which can inhibit the proliferation and growth of tumor cells.Taxol comounds can be isolated from the bark,leaves and fruits of Taxus.However,the growth cycle of Taxus mairei is long,and its very low content can not meet the market demand.Taxus is a first-class protected plant in China.Under the premise of protecting Taxus resources,the development of efficient and simple extraction technology is one of the key points in the utilization of Taxus resources.There are few reports on the technology of efficiently obtaining the effective components of paclitaxel at home and abroad.At present,there are many limiting factors in the clinical application of paclitaxel,such as poor water solubility,large side effects,low bioavailability and so on.Therefore,the development of a versatile carrier for accurate delivery of paclitaxel is expected to become a means of clinical treatment of liver cancer.In this study,the branches and leaves of Taxus cuspidata were extracted efficiently and greenly,and high purity paclitaxel was obtained by separation and purification.A novel targeted nanoparticle drug delivery system containing paclitaxel was developed for the treatment of liver cancer.The results of this study are as follows:1?A stable and reliable method for the determination of paclitaxel HPLC was established.the precision,repeatability,stability and RSD values of sample recovery were all less than 2%.The micellar solution of N-(3-hydrogenated abietic acid acyl-2-hydroxy)propyl-NMagneN-triethanolammonium chloride(HREOA)was used as extraction solvent Paclitaxel from branches and leaves of Taxus cuspidata was extracted by ultra-high pressure auxiliary surfactant HREOA method.The micellar solution of N-(3-hydrogenated rosinosy-2-hydroxy)propyl-N,N,N-triethylenehydroxyl ammonium chloride(HREOA)was used as extraction solvent Paclitaxel from branches and leaves of Taxus cuspidata was extracted by ultra-high pressure auxiliary surfactant HREOA method.Taking the extraction rate of paclitaxel as the index by single factor and response surface method,the optimum extraction conditions were obtained as follows:the mass fraction of surfactant was 1.40%,the ratio of liquid to solid was 35:1 mL/g,the extraction pressure was 94 MPa,and the extraction time was 6 min.Under the optimal extraction conditions,the extraction rate of paclitaxel was 87.164%.Compared with the traditional extraction methods,the ultra-high pressure extraction method needs the shortest extraction time,the highest extraction rate of the target products,relatively low power consumption and relatively low CO2 emissions.Ultra-high pressure extraction is an efficient,time-saving,environment-friendly and energy-saving method for the extraction of paclitaxel.2?The extraction mechanism of paclitaxel from branches and leaves of Taxus cuspidata with high pressure-assisted micellar solution was preliminarily explored.The structure of plant cells is different from that of all organelles.Under certain pressure,organelles with monolayer membranes are more likely to rupture than organelles with bilayer membranes.Controlling the pressure range is beneficial to improve the extraction efficiency of the target product.The fiber structure of the material was observed by scanning electron microscope(SEM).It was found that more and larger hollow holes appeared on the surface of the material blade when the extraction pressure was 100 MPa,and the change of the structure was not significant when the pressure continued to increase.The soaking treatment in the early stage of the material makes the cells expand and full,which can provide a favorable spatial structure for the diffusion of effective components.However,the effects of different extraction solvents on the extraction of paclitaxel under high pressure are also different Compared with the organic solvent ethanol,1.4%HREOA aqueous solution has a better effect of extracting paclitaxel,and natural environmental protection is conducive to the sustainable development of the environment.The contents of cellulose,hemicellulose and lignin in the materials before and after high pressure extraction tended to decrease compared with those before extraction.3?The crude extract of paclitaxel was extracted step by step with ethyl acetate,petroleum ether and chloroform and the purity and recovery of paclitaxel were 2.85%and 93.6%,respectively.Then,the purification process of crude paclitaxel was studied by alumina chromatography column,and the optimum conditions were obtained as follows:adsorption time 30 min,diameter-height ratio 1:8,elution speed 1.5 mL/min.Under these conditions,the purity and recovery of paclitaxel were 38.4%and 132.5%,respectively.Finally,the purification process of crude paclitaxel was studied by recrystallization,and the optimum conditions were obtained as follows:the concentration of paclitaxel was 40 mg/mL,the volume ratio of antisolvent to solvent was 15 min,the deposition temperature was 25? and the deposition time was 3 min.Under these conditions,the purity and recovery of paclitaxel were 84.5%and 83.1%,respectively.The paclitaxel product with purity ? 98%was obtained by secondary recrystallization.4?The optimum preparation conditions of PHBV-PTX-NPs,were prepared by emulsion solvent volatilization method.According to the experiments of single factor method and response surface method,the volume ratio of water phase to oil phase was 7:1,the concentration of PHBV was 30 mg/mL,the concentration of PVA was 1.6 mg/mL,homogenization time was 7.5 min,homogenization pressure was 80 MPa,homogenization times were 7 times.The PHBV-PTX-NPs were prepared by emulsion solvent evaporation method.The process was optimized by single factor and response surface method.The optimum conditions were as follows:volume ratio of water phase to oil phase 7:1,PHBV concentration 30 mg/mL,PVA concentration 1.6 mg/mL,homogenization time 7.5 min,homogenization pressure 80 MPa,homogenization times 7 times.The particle size of PHBV-PTX-NPs prepared under the optimal conditions is 62.3 nm.The coating was formed on the surface of nanoparticles by oxidative self-polymerization of dopamine,and the targeted ligand RGD peptide was grafted by Michael addition reaction.The RDG-PDA-PHBV-PTX-NPs,with targeting and pH response were obtained,which were spherical particles with a particle size of 124.6±7.7nm.The successful encapsulation of PDA on PHBV-PTX-NPs was identified by Fourier transform infrared spectroscopy.The results of X-ray diffraction,differential scanning calorimetry and thermo gravimetric analysis show that PTX exists in the nanoparticles in an amorphous state.The RDG-PDA-PHBV-PTX-NPs can stably exist in physiological saline solution and red blood cell suspension in vitro,and has the ability to release PTX sensitively to pH,which proves that RDG-PDA-PHBV-PTX-NPs can be injected intravenously.5?Through the high-connotation cell imaging analysis system and infrared imaging system,it is verified that RGD-PDA-PHBV-PTX-NPs has significant targeting to HepG2 tumors.The blank vector was proved to be non-toxic to LO2 of normal hepatocytes by MTT experiment The IC50 values of the RGD-PDA-PHBV-PTX-NPs on HepG2 and SMMC-7721 hepatoma cells were 0.78 ± 0.04 ?g/mL and 16.1 ± 0.97 ?g/mL,respectively.The tumor inhibition rate of tumor-bearing Hep G2 mice treated with RGD-PDA-PHBV-PTX-NPs through tail vein for 14 days was 86.56%,which proved that it had targeted function and excellent chemotherapeutic effect.During the treatment of the RGD-PDA-PHBV-PTX-NPs,there was no significant change in body weight and no serious organ damage.The RGD-PDA-PHBV-PTX-NPs based on targeting function and pH sensitive release is safe and low toxic,and it is expected to be a potential nano-delivery system for the treatment of HepG2 cell carcinoma. |
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