| BackgroundAs the sixth most common cancer in the world and the third leading cause of cancer death,liver cancer is a serious threat to human life and health,and China is the country with the heaviest burden of liver cancer in the world,so the prevention and treatment of liver cancer is particularly important and urgent.Chemotherapy is one of the main treatment tools for liver cancer.At present,most traditional chemotherapeutic drugs in clinical practice have limitations such as poor efficacy and high toxic side effects.The seeds of Taxus wallichiana var.mairei,an important tissue part of the Taxus Linn.,have the advantage of being abundant and renewable.The seeds of Taxus wallichiana var.mairei have been reported to contain substances with good antitumour effects,but there are few studies on their extraction and isolation and their formulation.Liposomes are widely used as well-established drug delivery systems for nano-formulations and are used in delivery studies of active compounds in natural plants,especially anti-tumour compounds.ObjectivesTo extract,isolate and purify compounds with inhibitory effects on hepatocellular carcinoma cells（Hep G2）from the seeds of Taxus wallichiana var.maire,and prepare them into a liposome delivery system to improve their solubility and in vivo distribution to enhance their anti-tumour efficacy and reduce their toxicity.This work will provide ideas for the development of clinical drugs and dosage forms for the treatment of hepatocellular carcinoma,and provide a new direction for the exploitation of the seeds of Taxus wallichiana var.mairei.MethodsThe crushed seeds were extracted by ultrasound-assisted ethanol extraction,and the obtained alcoholic extract was separated by silica gel column chromatography to obtain different eluted sites and screened for MTT antitumor activity;the site with better antitumor activity was purified by recrystallization to obtain a single compound,which was evaluated for MTT antitumor activity;and the compound was structurally identified using NMR spectroscopy and MS（identified as paclitaxelnine）.A single-factor experiment was conducted to optimize the ultrasound-assisted ethanol extraction process using the content of taxinine as an index,and the content of taxinine in the seeds of Taxus wallichiana var.mairei was calculated.Development of HPLC method for the in vitro analysis of taxinine and its methodological investigation;the equilibrium solubility and oil-water partition coefficient of taxinine in different p H media were determined.The taxinine-liposomes were prepared by the thin film dispersion method and the optimal prescriptions were optimized by orthogonal tests using the encapsulation rate as an index.The in vitro evaluation of appearance,particle size,polydispersity（PDI）,potential,encapsulation rate,drug loading,transmission electron microscopy,X-ray diffraction,stability and dissolution were also carried out;Inhibition of Hep G2 cells by taxinine and its preparations by MTT and in vivo safety evaluation by zebrafish larvae toxicity assay.To establish an in vivo HPLC method for the distribution of taxinine in mice;to investigate the in vivo distribution of taxinine in mice after intraperitoneal administration of taxinine-liposomes and free taxinine.ResultsAfter extraction and isolation,MTT activity screening,crystallization and purification,MTT activity verification and structure identification,the active compound with certain inhibitory effect on Hep G2 cell growth was finally obtained and identified as taxinine.The optimized extraction conditions were as follows:extraction solvent was 80%ethanol,extraction time was 4 h,extraction temperature was 70℃,material-to-liquid ratio was 20:150（g:m L）in one extraction.The content of taxinine in the seeds of Taxus wallichiana var.mairei was 0.114±0.002%.The established in vitro HPLC method for the determination of taxinine had good specificity and linearity,and the precision,recovery,repeatability and stability were in accordance with the methodological requirements.The solubility of in p H 1.2 hydrochloric acid solution（0.58±0.01μg/m L）,p H 6.8 phosphate buffer（0.82±0.02μg/m L）,p H 7.4 phosphate buffer（1.09±0.02μg/m L）and double-distilled water（0.38±0.01μg/m L）was very small.The results indicated that the water solubility of taxinine was poor and it was an virtually insoluble or insoluble drug.The oil-water partition coefficient Log P of taxinine in different media was greater than 1,indicating that its lipid solubility was better and its water solubility was worse,which provided a theoretical and experimental basis for the preparation of taxinine liposome nano-delivery system.The optimal prescription of taxinine-liposomes optimized by orthogonal tests was:3 mg taxinine,24 mg lecithin,7.5 mg cholesterol,9 mg TPGS,10 m L of double-distilled water was hydrated to obtain a milky white clarified solution.The particle size,PDI,zeta potential,encapsulation rate and drug loading capacity of were 186.76±0.08 nm,0.226±0.012,-44.34±0.77 m V,93.75±1.29%and 6.44±0.09%,respectively;Transmission electron microscopy results showed that the taxinine-liposomes were homogeneously distributed in a spherical or nearly spherical shape with a distinct bilayer structure.The X-ray diffraction results also showed that taxinine was well encapsulated in the liposomes.The taxinine-liposomes were stable at 25°C and4°C for 30 days.The in vitro dissolution test results showed that the cumulative dissolution of taxinine in liposomes was up to 85.77±2.43%,while that of free taxinine was only up to 17.21±2.08%.The dissolution rate and cumulative dissolution of taxinine-liposomes in all four media were significantly higher than those of free taxinine.The in vitro antitumor results of taxinine showed that the IC50 of taxinine-liposomes against Hep G2 cell was 8.85±0.75μg/m L and that of free taxinine was 20.06±1.43μg/m L under 72 h experimental conditions,and taxinine-liposomes significantly improved the growth inhibitory effect of free taxinine on Hep G2 cells.In zebrafish toxicity assays,taxinine-liposomes exhibited higher safe doses than the free taxinine and 5-FU.An in vivo HPLC method was developed for the determination of taxinine in various tissues of mice in accordance with the methodological requirements.The results showed that the drug concentrations in the tissues of mice in the taxinine-liposome group were increased to different degrees at 0.25 h,0.5 h,1.5 h and 3 h after intraperitoneal administration compared with those in the free taxinine group,which greatly improved the absorption and distribution of taxinine in the tissues（especially the liver）of mice.ConclusionTaxinine as an active compound in the seeds of Taxus wallichiana var.mairei with a significant inhibitory effect on Hep G2 cells,by preparing it into taxinine-liposomes can significantly improve the solubility and in vitro dissolution of hydrophobic taxinine,which can ultimately improve the inhibitory effect of taxinine on Hep G2 cells,safety and tissue distribution of taxinine in mouse liver.The taxinine-liposomes may provide new ideas for the treatment of hepatocellular carcinoma in clinical practice. |
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