Preparation Of Trichosanthes Sustained-release Pellets And Its Antithrombotic Effect

Objective:To determine the best formulation and preparation process,and then to prepare the qualified Trichosanthes sustained-release pellets,and establish its quality standard.To study the pharmacokinetic characteristics of Trichosanthes sustained-release pellet s in rats.To explore the antithrombotic effect and mechanism of Trichosanthes sustained-release pellets.Methods:1)The optimal formulation and preparation technology of drug loaded layer of Trichosanthes pellets were determined by single factor investigation and orthogonal test with taking the binder,solvent,drug loading and the speed of the peristaltic pump of the fluidized bed as the research object.The optimal formulation and preparation process of sustained-release layer of Trichosanthes pellets were determined by single factor experiments with taking ethyl cellulose（EC）,polyethylene glycol4000（PEG 4000）,talcum powder,coating weight gain and curing time as research objects.The suitable blank pellets were selected and coated with Glatt Mini fluidized bed to prepare Trichosanthe sustained-release pellets.2)The appearance,particle size and water content of the Trichosanthes sustained-release pellets were investigated.The che mical fingerprints of different batches of Trichosanthes sustained-release pellets were established by high performance liquid chromatography（HPLC）,and the similarity was calculated by the similarity evaluation system of chromatographic fingerprints of traditional Chinese medicine.According to pharmacopoeia of the people’s Republic of China[2015 edition,Vol.4,general rule 0931,method 2（paddle method）],the content of 3,29-dibenzoyl karounitriol（3,29-DK）in 1,2,4,6,8,10,12 and 24 h was determined,the concentration range was determined by establishing standard curve,and the precision,repeatability and stability were investigated for met hodological verification.The release rate of 3,29-DK in the Trichosanthes sustained-release pellets was used as the index to evaluate the release effect in vitro.3)Healthy male SD rats were divided into two groups:Trichosanthes sustained-release pellets group and Trichosanthes extract s group.Blood was collected from the orbit after single gavage.The concentrations of 3,29-DK in sustained-release pellets and extracts of Trichosanthes were determined by LC-MS/MS after the treatment of plasma samples,the standard curve of3,29-DK was established,and the specificity,minimum detection limit,precision and accuracy,recovery rate,stability,matrix effect and other methodological studies were carried out.The contents of 3,29-DK in the plasma of rats in different time points of the two groups were determined,and the datas were processed by Das 2.0 software to calculate the pharmacokinetic parameters.4)Healthy male SD rats were randomly divided into 7 groups,Trichosanthes extr acts group（0.79 g/kg/d）,high,medium and low dose groups of Trichosanthes sustained-release pellets（2.52 g/kg/d,1.26 g/kg/d,0.252 g/kg/d）,Compound Danshen Dropping Pill s group（85 mg/kg/d）,model group and normal group（given the same volume of normal saline）,each group with 6 rats,and were administered continuously for 7 days.On the 8th day,the high,middle and low dose groups of Trichosanthes sustained-release pellets were administered 7.5 h before operation,and the other groups were admin istered 1h before operation.The artery-vein bypass thrombus model was established in rats,the time of the model was 15minutes.Then thrombus was taken out and blood was taken from abdominal aorta.The dry weight of thrombus was measured,the contents of thromboxane B ₂（TXB₂）and 6-keto-prostaglandin F_1a(6-Keto-PGF_1a)in plasma were measured.The antithrombotic effect of sustained-release pellets was evaluated and its mechanism of action was analyzed.Results:1)The best formulation and preparation process of drug loaded layer of Trichosanthes pellets were 5%binder,60%ethanol,35%drug loading,adjusting the peristalsis speed of the peristaltic pump of the fluidized bed to 0.3 r/min can form the drug loaded layer of Trichosanthes pellets.The best formulation and preparation process of sustained-release layer of Trichosanthes pellets were 5%EC,1.5%PEG 4000,1.25%talcum powder,dissolved with anhydrous ethanol,10%weight gain by coating and6 h curing time.The Trichosanthes sustained-release pellets were prepared by loading the drug layer and the sustained-release layer on the blank core.2)The color of Trichosanthes sustained-release pellets was uniform without moisture absorption,deliquescence and caking,which met the quality requirements;the particle size of Trichosanthes sustained-release pellets was 0.663 mm,and the water content was 6.7%,which also met the requirements;the chromatographic fingerprint similarity of 10 batches of Trichosanthes sustained-release pellets was（0.989±0.009）（n=10）;the validation of methodology met the requirements,3,29-DK content was 0.14mg/g（n=3）.3)The linear relationship of 3,29-DK in Trichosanthes sustained-release pellets was good in the concentration range of 0.5～512 ng/m L,and the methodological validation was in accordance with the requirements.AUC_（0-t）was（172.12±8.12）μg/L*h,AUC_（0-）∞was（190.06±14.54）μg/L*h,MRT_（0-t）was（15.51±0.40）h,MRT_（0-）∞was（20.74±1.32）h,t_1/2 was（12.21±2.37）h and T_max was（7.67±0.82）h.Compared with Trichosanthes extract s group,there was statistical significance（P<0.05）,the results showed that the blood concentration of Trichosanthes sustained-release pell ets in healthy SD rats was relatively stable,the absorption was slow,and the pellets had sustained-release effect.4)From the results of the study on the thrombus model of artery-vein bypass in rats,we can see that:compared with the model group,each administration group has inhibition effect on thrombus;compared with the Trichosanthes extracts group,the medium and h igh dose groups of Trichosanthes sustained-release pellets could inhibit the thrombus formation better and had statistical significance（P<0.05）.Compared with the normal group,the TXB ₂ level in the model group increased significantly（P<0.01）,the 6-keto-PGF1аlevel decreased significantly（P<0.01）,and the TXB ₂/6-keto-PGF1аvalue had significantly difference（P<0.01）.Compared with the model group,the TXB ₂ level of the Compound Danshen Dropping Pill s group was significantly decreased（P<0.01）,the 6-keto-PGF1аlevel was significantly increased（P<0.01）,and the TXB₂/6-keto-PGF1аvalue was decreased（P<0.05）;the TXB ₂ level of high,medium and low dose groups of Trichosanthes sustained-release pellets could all be decrease the level of TXB ₂（P<0.05 or P<0.01）,and increase the level of 6-keto-PGF1а（P<0.01）,so that the TXB ₂/6-keto-PGF1аvalue tend to the normal level and had statistical significance（P<0.01）.Conclusion:The formulation of Trichosanthes sustained-release pellets is reasonable,the preparation process is simple and easy to operate with good repeatability,the in vitro release rate meets the requirements of Chinese Pharmacopoeia（2015 Edition）,and the quality standard of Trichosanthes sustained-release pellets is established.Pellets have sustained-release effect and antithrombotic effect,and antithrombotic effect has a certain dose-response relationship,the mechanism of antithrombotic effect may be related to the inhibition of platelet aggregation and the improvement of endothelial cell function.