| Taxus cuspidata is mainly distributed in Japan,Korea,Russia and Northeast China.Its branches,leaves,bark and roots contain the natural anticancer drug paclitaxel,which is one of the main raw materials for paclitaxel extraction.The common extraction methods of paclitaxel from Taxus cuspidata have many disadvantages,such as long time,high energy consumption,low safety and large use of organic solvents,which bring great difficulties to the large-scale production of paclitaxel.In addition,paclitaxel,as a natural anticancer drug,has a good inhibitory effect on the breast cancer,ovarian cancer and lung cancer.At present,paclitaxel is mainly given by the injection in the clinical application,but there are many defects in the injection,such as easy to produce adverse reactions,not suitable for long-term administration and so on.Oral administration is more safe and suitable for long-term administration,but the poor water solubility and low oral bioavailability of paclitaxel seriously limit its application in the clinical treatment.In view of the above problems,this study selected the Taxus cuspidata with the strong regeneration ability as the extraction raw material,and established an extraction process with the high efficiency,high safety and little damage to the structure of paclitaxel.The solid-phase extraction and reversed-phase column chromatography methods with the simple operation and ideal separation effect were used to separate and purify paclitaxel in the extraction solution to obtain the paclitaxel with high-purity.In addition,a porous starch-based paclitaxel oral delivery system was developed,which significantly improved the oral bioavailability of paclitaxel.The specific research results are as follows:1.In this study,the ultra-high pressure technology was used as the extraction method,and the single factor and response surface methods were used to optimize the extraction conditions of paclitaxel from Taxus cuspidata by the ultra-high pressure technology:the ratio of material to liquid was 1:16.22(g/mL),the ethanol content in the solvent was 91.36%,the pressure holding time was 5.64 min,and the extraction pressure was 139.85 MPa.Under this condition,the actual extraction rate of paclitaxel was 98.97 μg/g.After purification,the purity of paclitaxel was increased from 0.0591%to 98.13%,and the paclitaxel with higher purity was obtained.2.In this study,PPS was prepared by the direct adsorption of paclitaxel on porous starch with acetone as the solvent,and according to the results of single factor optimization experiment,the optimal preparation conditions of PPS were determined:the adsorption time was 30 min,the concentration of paclitaxel solution was 50 mg/mL,the mass ratio of paclitaxel to porous starch was 1:4.In addition,on the basis of the optimal PPS preparation process and the anti-solvent recrystallization technology,the porous starch suspension with paclitaxel acetone solution was added to 10 times volume aqueous solution containing 0.5%HPMC to prepare PNPS.After testing,the drug loading and entrapment efficiency of PPS were 9.94%and 44.15%respectively,while that of PNPS were 14.13%and 53.77%respectively.The results of SEM and EDS analysis showed that paclitaxel in PPS was evenly adsorbed on the inner wall of porous starch,while paclitaxel in PNPS was loaded on the inner wall and surface of porous starch in the form of nanoparticles.FTIR results showed that the paclitaxel in PNPS and PPS can form hydrogen bonds with porous starch.The results of XRD and DSC showed that the paclitaxel in PPS and PNPS was loaded into porous starch as the amorphous state.The residual amount of acetone in PPS and PNPS was 0.015%and 0.024%,respectively,which were far less than the requirement limit of Chinese pharmacopoeia for class Ⅲ solvent and met the safety standard.3.In vitro evaluation showed that the saturated solubility of PPS in artificial gastric juice,artificial intestinal juice and deionized water were 3.94,3.57 and 26.02 times of paclitaxel,respectively.In addition,that of PNPS in artificial gastric juice,artificial intestinal juice and deionized water were 3.61,3.39 and 24.42 times of paclitaxel,respectively.The results of in vitro dissolution showed that the dissolution of paclitaxel was limited by the hydrogen bond between porous starch and paclitaxel in artificial gastric juice.The cumulative release of PPS was lower than that of raw paclitaxel at 0-2 h,and then the release of paclitaxel increased slowly under the influence of saturated solubility,while the release of PPS increased steadily and gradually exceeded that of raw paclitaxel.At 24 h,the cumulative release of raw paclitaxel and PPS was 20.18%and 60.11%respectively.Different from PPS,the binding force between paclitaxel and porous starch in PNPS was greatly reduced,and the cumulative release of PNPS in artificial gastric juice was significantly higher than that of raw paclitaxel,and the cumulative release of PNPS in 24 hours was 67.12%.In the artificial intestinal fluid,the porous starch was hydrolyzed by the α-amylase,which reduced the inhibition of paclitaxel release in PPS.The cumulative release of PPS and PNPS was significantly higher than that of raw paclitaxel at 0-24 h.At 24 h,the cumulative release of raw paclitaxel,PPS and PNPS was 34.17%,70.22%and 72.44%.The results of in vitro digestion simulation experiments showed that the degradation rates of raw paclitaxel,PPS and PNPS were 1.91%,1.18%and 1.35%after the oral simulation digestion.The degradation rates were 23.54%,15.65%and 17.16%after the gastric simulated digestion.The degradation rates were 31.11%,24.49%,and 26.37%after the intestinal mimic digestion.The degradation rates were 42.13%,38.97%and 38.24%,after the simulated colon digestion,respectively.According to the above results,porous starch can protect paclitaxel in the gastric environment,and the strength of the protective effect is affected by the binding force between paclitaxel and porous starch.4.Pharmacokinetic studies show that compared with that of raw paclitaxel,the absorption of PPS and PNPS in rats was significantly improved.The AUC values of PPS and PNPS were 2.94 and 5.42 times higher than that of raw paclitaxel,respectively.The results of tissue distribution showed that the highest contents in the rat heart of the PPS and PNPS groups were 1.20 and 1.58 times that of the raw paclitaxel group,4.14 and 3.84 times of the raw paclitaxel group in the liver,4.01 and 4.56 times of the raw paclitaxel group in the spleen,1.54 and 1.89 times of the raw paclitaxel group in the lung,2.98 and 3.15 times of the raw paclitaxel group in the brain,and 1.44 and 1.89 times of the paclitaxel in the kidney.The results showed that paclitaxel can be quickly dispersed to various organs after administration.Paclitaxel was mainly distributed in liver and heart before 4 h,and in kidney after 4 h.5.This study investigated the inhibitory effect of raw paclitaxel,PPS and PNPS on LLC cell line proliferation and solid tumor growth.The results of MTT experiments showed that the in vitro IC50 values of raw paclitaxel,PPS and PNPS on LLC cell lines were 17703.41 μM,95.10 μM and 85.68 μM,respectively.The results of solid tumor inhibition experiments showed that the administration of raw paclitaxel,PPS and PNPS had no significant effect on the body weight of C57BL/6 tumor-bearing mice.After the end of the treatment,the tumor volume of the tumor-bearing mice in each group gradually increased.The order of the tumor volume size increase range from day 1 to day 21 after the end of treatment was as follows:control group>PTX-L group>PTX-H group>PPS-L group>PNPS-L group>PPS-H group>PNPS-H group.On the 7th day after treatment,the tumor weight and volume inhibition rate were as follows:3.09%and 28.43%in PTX-L group,8.25%and 61.90%in PTX-H group,5.15%and 65.25%in PPS-L group,29.90%and 78.36%in PPS-H group,24.74%and 69.67%in PNPS-L group,34.02%and 84.81%in PNPS-H group.On the 14th day after treatment,the tumor weight and volume inhibition rate were as follows:14.70%and 18.69%in PTX-L group,16.18%and 39.00%in PTX-H group,20.59%and 43.94%in PPS-L group,51.71%and 72.99%in PPS-H group,33.09%and 46.49%in PNPS-L group,58.09%and 80.87%in PNPS-H group.On the 21th day after treatment,the tumor weight and volume inhibition rate of each group were as follows:4.75%and 4.69%in PTX-L group,10.11%and 26.06%in PTX-H group,10.87%and 31.01%in PPS-L group,21.90%and 53.27%in PPS-H group,20.06%and 41.59%in PNPS-L group,22.36%and 54.82%in PNPS-H group.Compared with the survival curves of C57BL/6 tumor bearing mice in each group,the order of survival time from long to short was PNPS-L group(42 days),PPS-L group(37 days),PNPS-H group(36 days),PPS-H group(35 days),PTX-H group(34 days),control group(30 days)and PTX-L group(29 days).The results of HE staining showed that PPS and PNPS could inhibit the necrosis and liquefaction of tumor tissue,and the inhibition effect of high dose group was better than that of low dose group.In addition,according to the results of HE staining sections of heart,spleen,lung and kidney,when the dosage was 5 mg/kg,the raw paclitaxel,PPS and PNPS had no obvious damage to the liver of C57BL/6 tumor bearing mice.When the dosage was 15 mg/kg,the raw paclitaxel had no obvious damage to the liver of C57BL/6 tumor bearing mice,but the liver of C57BL/6 tumor bearing mice in PPS and PNPS groups had slight inflammation.Based on the above results,it can be inferred that PPS and PNPS can enhance the inhibition of solid tumor in C57BL/6 tumor bearing mice inoculated with LLC cell line compared with the raw paclitaxel,and the effect is better when the dosage is 5 mg/kg. |
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