| BackgroundTaxus chinensis（T.brevifalia Nutt.）is one of the rare natural anti-tumor plants recognized in the world.Taxus chinensis seeds belong to the parts of taxus chinensis which are rich in resources and not fully utilized.They have the advantages of renewability,easy to obtain and long preservation time and so on.However,the research and application of its anti-tumor active ingredients are few at home and abroad.In addition,previous studies of our research group have shown that taxus chinensis seeds have certain anti-tumor activity,so it is necessary to carry out systematic studies on their anti-tumor activity.In addition,lung cancer is associated with a high incidence,low survival rate,the risk of brain metastases,and the limitations of conventional treatment.Therefore,it is necessary to find a new way to treat lung cancer.PurposesIn this study,the parts of taxus chinensis seeds with good anti-tumor effect were screened out and prepared into liposome inhalation.Meanwhile,the inhibitory effect of liposome inhalation on lung cancer was investigated,providing theoretical basis for the research and application of taxus chinensis seeds and liposome inhalation,and broadening ideas for the treatment of lung cancer.MethodsThe oil content of taxus chinensis seeds was calculated by degreasing the seeds with petroleum ether.The extraction process of defatted taxus chinensis seed was optimized by single factor experiment with extraction rate of extractum as index and extraction solvent,extraction time,solid-liquid ratio and extraction temperature as factors.Subsequently,AB-8 macroporous adsorption resin and silica gel（300-400 mesh）was used to isolate and purifiy taxus chinensis seeds extracts and at the same time,A549 cells were used to select the best antitumor sites by MTT method.The active site（B2）was analyzed by TLC,HPLC and LC-MS.HPLC in vitro analysis method of B2 was established,the equilibrium solubility of B2 in ultrapure water,p H7.4 PBS solution,ultrapure water containing 2%Tween80 and p H7.4 PBS solution containing 2%Tween80for 72 h was determined,and the oil/water distribution coefficient in ultrapure water was determined.The formulation of B2 liposome was optimized by single factor;the selected liposomes were evaluated in vitro.The in vitro dissolution was investigated by slurry method;the inhibitory effect of A549 cells was investigated by MTT assay;zebrafish was used to investigate safety of B2 and B2 liposome.The quality of liposome inhalation was evaluated;emodin was used as internal standard and ethyl acetate extraction was used as tissue sample treatment method to establish in vivo HPLC analysis method for the study of tissue distribution in mice.The tissue distribution of intraperitoneal administration of B2 liposome,intraperitoneal administration of B2liposome and atomization administration of B2 liposome in mice was investigated.A549 tumor bearing nude mouse model was constructed by subcutaneous inoculation.The growth,tumor volume,inhibitory rate,spleen index and pathological morphology of tumor tissue in nude mice were used as observation indexes to compare the therapeutic effects of abdominal injection of B2and B2 liposome and atomized administration of B2 liposome,respectively.ResultsThe oil content of taxus chinensis seeds was 44.11±0.65%,the extraction conditions were 60%ethanol,40 min extraction time,1:50 g/m L solid-liquid ratio and 60℃extraction temperature.The extraction rate of extractum was 40.87±1.24%.The optimal antitumor active site was selected as B2,the proportion of paclitaxel in B2 was 61.46±0.72%,and the proportion of cephalomannine was 5.07±0.32%.In addition,B2 also contained other components with anti-tumor effects,such as esculin,berberine,betaine and so on.HPLC conditions were established for in vitro analysis of B2 conforming to the methodological requirements,and a good linear standard curve of B2 was established in vitro with paclitaxel and trichinine as reference,respectively.The equilibrium solubility results showed that B2 was insoluble in water and 2%Tween80 increased the solubility of B2.The oil/water partition coefficient value of B2 is greater than 2.6,which is a highly hydrophobic substance,providing a prerequisite for the subsequent preparation of B2 into liposome to improve its water solubility.Firstly,the preparation method of B2 liposomes was determined as ethanol injection method.Then,the optimal prescription was selected by single factor experiment:the mass ratio of phospholipid to drug was 2:1,the mass ratio of lecithin to cholesterol was 2:1,and the volume of hydration medium was 30 m L.The particle size,PDI and the Zeta potential of B2 liposome were115.77±3.35 nm,0.240±0.012 and-29.79±0.95 m V respectively.The Dendahl phenomenon was obvious.The transmission electron microscope showed that the bilayer structure of B2 liposomes was obvious,and the encapsulation rate of B2 was 88.09±1.83%with Paclitaxel as the reference,and 82.80±0.77%with cephalomannine as the reference,which were relatively high.The XRD results further verified that almost all B2 was encapsulated in the liposomes.The drug loading of B2 was 22.02±0.46%with paclitaxel as reference.The drug loading of B2 was 20.70±0.19%with trichinine as reference,indicating that the drug loading of B2 liposome was high.In addition,the stability test also proved that B2 liposome was relatively stable at 4℃and 25℃for 10 days,and the storage time could be extended by preparing it into lyophilized powder.The results of in vitro dissolution experiment also showed that compared with B2,the dissolution amount and dissolution rate of B2 liposomes were significantly increased.The IC50 values of paclitaxel,B2 and B2liposome on A549 cells were 115.12±16.70 ng/m L,67.22±8.49 ng/m L and 31.14±1.91 ng/m L,respectively.The results showed that the inhibitory effect of B2 liposome on A549 cells was stronger than that of B2 and paclitaxel.Results the study of toxicity of zebrafish showed that B2and B2 liposome were relatively safe compared with paclitaxel,and the safety of B2 could be further improved after the encapsulation of B2 in liposome.The appearance,particle size,Zeta potential and PDI of B2 liposome inhalation did not change significantly,and the encapsulation rate decreased.The atomizing recoveries of B2 with Paclitaxel and Cephalomannine as the references were 85.41±1.05%and 81.78±0.30%,respectively.An in vivo HPLC method was established for the study of tissue distribution in mice,and a good linear standard curve of each tissue was established in vivo.The content of B2 in tissues at different time points was studied by intraperitoneal injection of B2 and B2 liposome and atomized administration of B2 liposome.The results showed that compared with intraperitoneal injection of B2,the concentration of B2 in tissues on intraperitoneal injection of B2 liposome was higher at different time points,and B2 was mainly distributed in liver,spleen and kidney.This indicates that liposome can improve the distribution of B2 in various tissues.Compared with the intraperitoneal injection group of B2 liposome,the content of B2 in the lung and brain of mice in the B2 liposome atomization group was significantly increased,which indicated that the atomization administration could increase the concentration of B2 in brain while the accumulation of B2 in the lung.The study showed that nude mice treated with intraperitoneal injection of B2 and B2 liposome experienced slight weight loss（5%）and good growth,while nude mice treated with atomized B2liposome experienced slight weight gain and better growth.The tumor inhibitory rates of B2intraperitoneal injection group,B2 liposome intraperitoneal injection group and B2 liposome atomization group were 51.33±4.82%,66.08±4.68%and 83.14±10.40%,respectively,indicating that B2 liposome could significantly improve the inhibitory effect of B2 on tumor in vivo under the same intraperitoneal injection.At the same time,the atomized B2 liposome had a more significant inhibitory effect on the tumor inoculated in nude mice.By comparing the spleen index of nude mice,it was found that the spleen index of nude mice in the B2 liposome atomization group was closer to that in the normal group,indicating that the effect of atomizing B2 liposome on spleen was minimal.By observing the HE stained sections of tumor tissue,it was found that the B2 liposome atomization group,B2 liposome abdominal injection group and B2 abdominal injection group could promote the necrosis of tumor cells.At the same time,the same intraperitoneal injection of B2 liposome can improve the inhibitory effect of B2 on lung cancer cells.It is worth noting that the administration of B2 liposomes through atomization can promote the necrosis of tumor cells.ConclusionB2 was better than paclitaxel in terms of its inhibitory effect on A549 cells and safety on Zebrafish.Liposome can improve the solubility of B2 in water,the inhibition effect on A549 cells,the safety and the tissue distribution of B2 in mice.Liposome inhalation can make B2 accumulate in the lung,increase the concentration of B2 in the brain,and has obvious inhibitory effect on tumor in vivo,which is a relatively novel approach for the treatment of lung cancer. |
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