Preparation Of Oleanolic Acid Microparticles And Its Preventive Effect On Liver Injury Induced By CCl 4

Objective:First,establish and optimize the premix membrane emulsification method to prepare Oleanolic acid microparticles with small and uniform particle sizes.Then provide technical reference for more indissolvable ingredients from traditional Chinese medicines to prepare microparticles,and enlage the application range of stainless steel membrane.Besides,improve the dissolution rate of Oleanolic acid by minimizing the particle size and increasing the specific surface area,and promote its gastronintestinal absoption or change its administration route to improve its protection effect on liver.Method:Based on the preparation experience of Silybin PLGA Microspheres and Silybin Microparticles,prepare Oleanolic acid Microparticles using stainless steel premix membrane emulsification method.And investigate the factors affecting the particles formation by taking average particles size and polydispersity index as main indexes.Besides,evaluate the qualities of Oleanolic acid Microparticles from following aspects:redispersiblity,crystalline state,drug contention uniformity and residual impurities.Then,compare the Oleanolic acid Microparticles release behavior in vitro with Oleanolic acid using direct release method.Last,compare liver protection effects against acute liver injury by carbon tetrachloride between Oleanolic acid Microparticles and Oleanolic acid with different dosages and different administration methods,including intragastric administration and tail intravenous injection.The liver protection effects were evaluated by serum alanine transaminase,serum aspartate transaminase,and liver homogenate malondialdehyde,tumor necrosis factor a and interleukin I beta.Results:Under the condition of the oil phase concentration 0.3%,the volume ratio of oil phase and aqueous phase 1:4,the transmembrane pressure 0.6MPa,the optimum conditions are as follows:aqueous phase concentration 3%,the volum ratio of the emulsion after filtration and the curing liquid 1:5,take the physiological saline solution containing 2.4%Polyvinyl alchol as curing liquid,curing temperature 70℃,centrifuge 30 minutes at speed of 6500 r·min-1 and washing with pure water for 4 times and 10minutes each time,adding 3%mannitol as cryoprotectant..Oleanolic acid Microparticles were spherical under election microscopy,the average particles size was(352.4±6.23)nm,polydispersity index was(0.131±0.035).The Oleanolic acid Microparticles have good dispersibility and are wetted by water easily.And the results of XRD showed that the characterstic peaks of Oleanolic acid Microparticles and Oleanolic acid are different,indicating that crystal form of Oleanolic acid may has changed.The drug contents in three batches Oleanolic acid Microparticles prepared by optimum methods were(14.62±0.22)%,(16.93±0.42)%,(15.78±0.99)%respectively,and the average drug contents of these three batches was(15.78±1.16)%,the average content of residual polyvinyl alcohol was(1.21±0.12)%.The accumulative releasing rate of Oleanolic acid Microparticles and Oleanolic acid was(74.56±5.40)%and(55.57±3.20)%respectively in 6 hours in pH6.8 phosphate buffer solution containing 0.2%lauryl sodium sulfate.The accumulative releasing rate of Oleanolic acid Microparticles and Oleanolic acid was(88.70±2.03)%and(36.86±5.74)%respectively in 2 hours in pH6.8 phosphate buffer solution containing 0.05%lauryl sodium sulfate,while the physical mixture powders was(11.49±0.14)%.In condition of pH7.4,the accumulative releasing rate was respectively(71.07±4.04)%、(26.45±2.74)%and(8.55±1.36)%.The contents of malondialdehyde,tumor necrosis factor a,interleukin I beta in mice liver of each group are as follows:blank group were(1.32±0.32)nmol/mgprot,(13.74±3.51)ng/mgprot,(3.46± 1.03)ng/mgprot,model group were(3.58±0.90)nmol/mgprot,(19.39±3.21)ng/mgprot,(4.72±0.79)ng/mgprot,Oleanolic acid intragastric administration at 12.5mg/kg group were(2.22±0.72)nmol/mgprot,(14.58±6.58)ng/mgprot,(3.19±1.64)ng/mgprot,Oleanolic acid intragastric administration at 5mg/kg group were(3.33±1.27)nmol/mgprot,(21.00±4.61)ng/mgprot,(4.63±1.09)ng/mgprot,Oleanolic acid Micropaticles intragastric administration at 1.25mg/kg group were(3.53±1.28)nmol/mgprot,(20.66±4.68)ng/mgprot,(4.98±1.14)ng/mgprot,Oleanolic acid Micropaticles intragastric administration at 2.5mg/kg group were(2.54±0.33)nmol/mgprot,(18.02±4.21)ng/mgprot,(4.37±0.99)ng/mgprot,Oleanolic acid Micropaticles tail intravenous administration at 0.65mg/kg group were(2.52±0.39)nmol/mgprot,(14.91±2.38)ng/mgprot,(3.31±0.58)ng/mgprot,Oleanolic acid Micropaticles tail intravenous administration at 1.25mg/kg group were(2.50±0.29)nmol/mgprot,(15.53±4.25)ng/mgprot,(3.30±0.84)ng/mgprot respectively.Conclusion:The Oleanolic acid Microparticles parepared by stainless premix membrane emulsification method have a small amount of residual impurities,and its prepare method could be repeated well.Compared with Oleanolic acid,the Microparticles dissolve faster.The intragastric administration dosage of Oleanolic acid Microparticles is 1/2 of Oleanolic acid,and intravenous is 1/13 when playing the equal protection effects.These indicate that turning Oleanolic acid into Microparticles can reduce the dosage and change the way of administration,therefore enhance the protection effects on liver.