Extraction And Purification Of Ursolic Acid,Preparation And Characterization Of Ursolic Acid Nanoparticles

Ilex-latifolia Thunb is used as food and drug in our country's folk,having a long historical standing.It comes from many provenances and contains a variety of chemicals,such as triterpenes and glycosides,polyphenols,flavonoids,amino acids,aroma,volatile oils and so on.Ursolic acid(UA)is a pentacyclic triterpenoid.Which exists in Ilex latifolia Thunb.Pharmacological studies have shown that UA has a wide range of pharmacological effects,for instance anti-hepatitis,anti-bacterial,anti-tumor,anti-ulcer,decreasing blood sugar effects and so on.Although the effects of UA is remarkable,there has not been prescribed preparations clinically yet.The main reason for this is poor water solubility and low oral bioavailability of UA.Therefore,in this paper,we extracted UA by ionic liquids(ILs)based ultrasonic-assisted and purifed it antisolvent recrystallization(ASR).Following using emulsion solvent evaporation(ESE)method to prepare UANs,and investigating its physical and chemical properties,exploring the improvement of oral bioavailability.ILs based ultrasonic-assisted extraction was used to extract UA from Ilex latifolia Thunb.At first,we investigated the effects of different extraction solvents on extraction ratio.And then,the single-factor method was carried out on these factors who influenced extraction ratio:ILs concentration,solid-liquid ratio,ultrasonic power,ultrasonic temperature,ultrasonic time.The optimal conditions of extraction UA is:ILs concentration 0.4 mol/L,solid-liquid ratio 1:40,ultrasonic power 500W,ultrasonic temperature 40 ?,ultrasonic time 30min;the mean extraction ratio is 2.41 ±0.35mg/g.Generating precipitation with acid and alkali,and then,the content of UA crude extract was 29.89 ± 2.23%.The content of UA could be increased by ASR.By means of the single-factor method,the effect of main factors on the content of UA was investigated.The main factors affecting the outcome of purification were drug concentration,volume ratio of antisolvent to solvent,recrystallization temperature,stirring time and stirring rate.The optimal conditions were determined as:drug concentration 50 mg/mL,volume ratio of solvent to antisolvent 1:6,recrystallization temperature 20 ?,stirring time 1 min and stirring rate 1500r/min;the content of UA reached 92.18± 1.87%.Ursolic acid particles(UANs)were prepared by ESE method,and followed by freeze-drying.The organic phase is trichloromethane containing 30%(v/v)ethanol,the aqueous phase is ultrapure water,poloxamer 188 is as surfactant and cryoprotectant.The optimal conditions for preparing nanoparticles were screened out using single-factor experiment.The particle size was used as the basis for optimization experiment.The following six main parameters having significant influences on particle size were picked out,included the concentration of poloxamer 188,volume ratio of organic to water phase,homogenate speed and homogenate time,as well as homogenization pressure and cycles,the optimal conditions of preparation UANs:0.5‰ of poloxamer 188,1:4 of volume ratio of organic to water phase,7000 rpm of homogenate speed for 2 min and a homogenization pressure of 500 bar for 6 times.Based on the optimal conditions,the mean particle size(MPS)was 157.5 ± 28.0 nm and zeta potential of 20.33±1.67 mV.And then,the morphology of UANs powder obtained was presented by scan electronic micro-scope(SEM).To estimate the UANs weather and how changes in surface chemical character and physical structure,using X-ray diffraction(XRD),thermal gravimetric analyzer(TG)and differential scanning calorimetry(DSC).SEM showed that UANs were nearly spherical with uniform particle size distribution.By the XRD,TG and DSC,we could acquaint UANs had the same chemical structure as the raw UA but had been amorphous state.The residual contents of trichloromethane and ethanol were separately less than the ICH limit for class III and class II solvents.The result of solubility test figured that the equilibrium solubility of UANs was 13.48 times in SGF,11.79 times in SIF and 23.99 times in deionized water than raw UA.Accordingly,the dissolution rate of UANs in SGF and SIF had an apparent enhancement.The oral bioavailability of UANs increased 2.68 times than raw UA.UANs improved antioxidant activity toward cells compared with raw UA,and EC50 of UANs reduced 37.5 times than raw UA.From results shown above,UANs possesses a value of application on enhancement oral bioavailability.