| Objectives:Pulsatilla saponin D(PSD)is an active ingredient with strong anti-tumor activity,which was found in the early research of the national "Research of anti cancer drug and prepatation on Pulsatilla total saponins" of "State Project for Essential Drug Research and Development"(No.2011ZX11102-001-19).The anti-tumor research of Pulsatilla chinensis extraction and its active ingredient attracted more and more researchers' attention recently.However,the present medicinal properties study of PSD only limited to the research of pharmacological activity,a comprehensive,systematic and detailed research of it could not be found either at home or abroad.Neither the related preparation research nor the commercially available products been studied.The preparation process,bioactivity,quality control,formulation studies and pharmacokinetic processes both in vivo and vitro were comprehensively and systematic studied herein on the basis of analyzing the prior data and references in order to provide experimental support and foundation for the further development of PSD.Methods:According to the traditional Chinese medicine of saponin in UV absorption characteristics,using DAD and ELSD two kinds of detector,PSD method for determination of HPLC;single factor and orthogonal test method were used the optimization of PSD preparation and purification process parameters;in vivo evaluation of PSD antitumor activity by MTT method,The antitumor mechanism were studied from inducing tumor cell apoptosis and autophagy aspects;using HPLC determinated the PSD of oil-water partition coefficient,the equilibrium solubility,albumin binding important physicochemical parameters rate;the single pass perfusion method and everted gut sac method were used to evaluate the PSD absorption in rat intestine,dynamic behavior used LC/MS/MS method;using the preparation method improved PSD bioavailability and reduced the hemolysis.Results:A HPLC system equipped with DAD and ELSD detectors was employed to establish the content determination method for PSD.A simple and accurate method was established and used for determining 10 batches of herbs gathered from different areas.Results showed that material originated from Jilin province possessed the highest content(0.26%).The best preparation conditions were determined to be as follows:70%ethanol extracted three times added with 8 volumes of solvent with each time 1 hour,alkali hydrolysis(pH11-12,90-100 ? for 5-7 hours)and D101 macroporous adsorption resin for collecting the 70%ethanol fraction,PSD sample purity>90%.MTT method was used to determine the growth inhibitory activities of PSD to 12 kinds of tumor cells such as A549,and found its great tumor inhibited activity.Ideal antitumor effects were found against human colorectal adenomas HT-29 cancer cells,with IC50 value 3.76?g/mL.The antitumor activity was further verified with H22 tumor bearing mice cells model in vivo,and proved to be dose-dependent.The antitumor mechanism were studied from inducing tumor cell apoptosis and autophagy aspects and found that PSD functioned through enhancing Caspase 3 and LC3-? protein expression.X-ray diffraction showed it was multiple crystal powder.The study found the solubility differed greatly in different salt buffer solutions and solvents.The solubility increased with pH values and reached to the maximum concentration 255.89 mg/mL in methanol,while only with slight solubility as to 2.38 mg/mL in water.The results showed that PSD had strong lipophicity and the oil/water partition coefficients parameter in water is 0.7,which indicated its good permeability.Equilibrium dialysis method was used to study the binding rate of PSD combined with different genera of plasma proteins and found high combined rate,especially those combined with human plasma albumin was nearly to 70%.Macroscopic observation and ultraviolet spectrophotometric methods were used together to evaluate the hemolytic property of PSD and found the highest concentration with no hemolysis was 2.5 mg/mL.The results showed that the best absorption part was colon in the form of passive diffusion with good correlation.The LC-MS/MS analysis method for analyzing PSD in plasma samples with notoginseng saponins R1 as internal standard were established herein.Non-compartmental model was employed for the statistical analysis of the PSD pharmacokinetic parameters changes of rats in vivo.Bimodal of curves were observed at intragastrical administrated with high(200 mg/kg),medium(100 mg/kg)and low(50 mg/kg)dose.The Tmax of PSD was 30 min,showed its quick absorption ability in rats intestinal,while the T1/2 was about 20 hours showed its slow metabolism in rats.Linear correlation could be found among the given dose,AUC and the peak concentration(r2 was 0.9999 and 0.9952,respectively).No significant difference was found of the half-life data at the high,medium and low doses,which showed the way of the PSD dynamics behavior in rats was in line with the linear dynamics process.Fast distribution could be detected by intravenous injection at the dose of 3 mg/kg and could reach to the peak blood concentrations within 1 min,while with fast eliminated speed too.The absolute bioavailability of PSD was less than 0.1%,which showed its low bioavailability.Solid dispersion,cyclodextrin and albumin nanoparticles were studied in the paper.Firstly,we estimated three solid dispersions carrier materials(PVPk30,F68 and PEG6000)and the influence to the solubility of PSD with different proportion.The results showed that the maximum solubility(7.06 mg/mL)could be obtained with mixed ratio 1:6 with PEG6000.The dissolution behaviors and the intestinal absorption and pharmacokinetic changes were studied both before and after madding into the solid dispersions and found that the dissolution rate increased significantly,the Papp absorption parameters and Ka values increased nearly three times,and the relative bioavailability increased by 224%.Secondly,the best technological parameters were determined to be HP-?-CD:PSD 10:1,inclusion temperature 45?,closure time 6 h.The PSD dissolution behavior and the pharmacokinetic changes were studied before and after the formation,which showed that the relative bioavailability increased by 264%.Finally,The dosage of BSA,the dose and time of the crosslinking agent and the dosage of ethanol were estimated,and the best condition was determined to be:the ratio of PSD-HP-?-CD and BSA was 5:1,the consumption of ethanol was 15 mL,the dosage of crosslink agent,0.25%glutaraldehyde is 75?L,and the crosslink time is 12 hours.The total coating rate of the made PSD-HP-?-CD-NP was 90%and the yielded rate was 90%.Nanoparticles were determined by laser granulometer and the mean particle size range was 180.8 nm,while the PDI value was 0.226.The highest non-hemolysis concentration and pharmacokinetic research of nanoparticles were compared and the results showed that the concentration increased from 2.5 to 8?g/mL.Conclusions:Through the above research,the PSD refining purification process is simple,environmental protection,stability,in vitro and in vivo efficacy show that PSD has strong anti-tumor activity,preparation special parameters shows that PSD belongs to the low solubility,high permeability,low bioavailability and hemolytic strong medicine,solid dispersion and cyclodextrin complexes improve PSD bioavailability by 2 times,albumin anoparticles decreases PSD hemolytic by more than 3 times,the above research conclusion PSD druggability greater risks. |
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