| Licorice is a greatly nutritious and curative botanical medicine,which is widely used as food and medicine.So far,nearly 400 chemical components have been isolated from licorice,including about 300 flavonoids and more than 20 triterpenoids.Licorice flavonoids have pharmacological activities such as antioxidant,anti-inflammatory,anti-tumor and skin whitening,because of its poor water solubility and low bioavailability,the application of licorice flavonoids in food and pharmaceutical fields is greatly limited.At present,the research on licorice flavonoids mainly focuses on the pharmacological activity,extraction and purification,while the study on solubilization is less,and traditional alcohol extraction,water extraction and macroporous adsorption resin methods are used for the extraction and purification of licorice flavonoids,which generally have the disadvantages of high cost,time-consuming,and toxic residual reagents.In order to better develop and utilize licorice flavonoids,this study performed efficient green extraction of flavonoids from urals licorice roots,further separated and purified to obtain high-purity licorice flavonoids,prepared licorice flavonoid nanoparticles by anti-solvent recrystallization method and improved its bioavailability.The test results are as follows:1.The extraction of licorice flavonoids was studyed by ultrasonic microwave-assisted micelle extraction using lauryl sodium sulfate as extractant.The extraction process parameters were optimized by single factor and response surface methods.The extraction rate of licorice flavonoids was used as an indicator.The final optimal extraction process parameters were that the mass fraction of lauryl sodium sulfate was 2%,the liquid-material ratio was 21,the microwave power was 832 W,and the time was 10 min.Under this optimal condition,the extraction rate of licorice flavonoids reached 3.65%.Licorice flavonoids were repeatedly extracted three times with 80%of ethanol hot reflux method,and the extraction rate reached3.71%.2.Ethyl acetate was used to pre-treat the extract,the extraction was repeated three times,and the ethyl acetate phase was combined.The crude licorice flavonoids obtained after drying had a purity of 36.47%and a recovery of 92.80%.Crude licorice flavonoids were determined by HPLC.The contents of echinatin,isoliquiritigenin and glycyrrhizin in crude licorice flavonoids were 0.55%and 0.56%,respectively.Crude licorice flavonoids were purified by metal complexation method,and the single factor method was used to optimize the process parameters.The optimal process parameters were that licorice flavonoids concentration was 2 mg/mL,and the mass ratio of licorice flavonoids-calcium chloride was 1:0.3,the solution pH was 10.The purity of the crude licorice flavonoids obtained under this optimal condition was 63.56%,and the recovery was 77.27%.The contents of echinatin and isoliquiritigenin in crude licorice flavonoids were 0.81%and 1.46%,respectively.The anti-solvent recrystallization method was further used to purify the crude licorice flavonoids.The process parameters were optimized by single factor and response surface methodology.The optimal process parameters were finally obtained that the time was 1 min,the temperature was 27?,the antisolvent-solvent ratio was 12,and the concentration of licorice flavonoids was 82 mg/mL.Under this optimal condition,the purity of licorice flavonoids was 90.32%,and the recovery was 88.98%.The content of echinatin,isoliquiritigenin,and glycyrrhizin in licorice flavonoids were 1.12%and 2.42%,respectively.3.The anti-solvent recrystallization method was used to prepare licorice flavonoids nanoparticles.The influence of different factors on the particle size of licorice flavonoids nanosuspension was investigated.The process parameters were optimized by single factor experimental method.The content of poloxamer 188 was 0.3%,the deposition temperature was 40?,the stirring speed was 750 r/min,the dropping speed was 5 mL/min,the volume ratio of antisolvent-solvent was 12,the deposition time was 20 min,and the concentration of licorice flavonoids 50 mg/mL.Under these optimal conditions,the particle size of the licorice flavonoids nanosuspension was 95 nm,and the particle size of the licorice flavonoids nanoparticles powder after lyophilization was 108.2 nm.The morphological characterization of raw licorice flavonoids and licorice flavonoids nanoparticles was performed by scanning electron microscopy.Compared with the raw licorice flavonoids,the licorice flavonoids nanoparticles showed a uniform spherical morphology,and the particle size was much smaller than the raw licorice flavonoids' 41.8 ?m.According to XRD,TG,and DSC analysis,it can be concluded that licorice flavonoids nanoparticles did not form new crystals,and basically existed in an amorphous form.Licorice flavonoids nanoparticles were tested for solvent residues.The methanol content of licorice flavonoids nanoparticles was 22.94 ppm,and the residual methanol content was lower than the ICH limit of 3000 ppm for class ? solvent methanol.It meets ICH guidelines and can be used in pharmaceuticals.4.The saturated solubility of echinatin,licochalcone A and total flavonoids in raw licorice flavonoids and licorice flavonoids-HP-?-CD(1:0,1:1,1:2,1:3,1:4,1:5,1:6,1:7,1:8,1:9)nanoparticles lyophilized powders were tested by UV and HPLC method.Echinatin and licochalcone A in raw licorice flavonoids had a saturated solubility of 0.0198 mg/mL and 0.0021 mg/mL.The saturated solubility of echinatin in licorice flavonoids-HP-?-CD(1:0,1:1,1:2,1:3,1:4,1:5,1:6,1:7,1:8,1:9)nanoparticles were 0.14,0.46,0.63,0.70,0.79,0.73,0.76,0.77,0.72,0.69 mg/mL,the saturated solubility of licochalcone A in licorice flavonoids-HP-?-CD(1:0,1:1,1:2,1:3,1:4,1:5,1:6,1:7,1:8,1:9)nanoparticles were 0.82,1.61,1.70,1.73,1.78,1.77,1.77,1.80,1.75,1.76 mg/mL.Based on the experimental results,the optimal condition of lyophilization was 1:4 of licorice flavonoids-HP-?-CD.The saturated solubility of total flavonoids in raw licorice flavonoids was 8.03 mg/mL,the saturated solubility of total flavonoids in licorice flavonoids-HP-?-CD(1:0,1:1,1:2,1:3,1:4,1:5,1:6,1:7,1:8,1:9)nanoparticles were 30,170.23,181.21,183.23,200.25,197.34,196.78,198.26,186.23,187.23 mg/mL.The optimal conditions of the experimental results are that the ratio of licorice flavonoids-HP-?-CD was 1:4,which was consistent with the above experimental results.The saturated solubility of total flavonoids in licorice flavonoids nanoparticles lyophilized powder was 25 times that of the raw licorice flavonoids.The vitro dissolution rates in artificial intestinal juice and artificial gastric juice of echinatin,licochalcone A and total flavonoids in raw licorice flavonoids and licorice flavonoid nanoparticles were measured.The maximum dissolution rates in artificial intestinal juice of echinatin and licochalcone A in licorice flavonoids nanoparticles were 51.33%and 73.44%,which were 4.09 times and 69.66 times that of echinatin and Licochalcone A in the raw licorice flavonoids,at 720 min,the maximum dissolution rates in artificial gastric juice of echinatin and Licochalcone A in licorice flavonoids nanoparticles were 56.84%and 97.46%,respectively,which were 2.63 times and 8.92 times that of echinatin and Licochalcone A in the raw licorice flavonoids.At 720 min,the maximum dissolution rate in artificial intestinal juice of total flavonoids in licorice flavonoids nanoparticles was 75.82%,which was 9.63 times that of total flavonoids in raw licorice flavonoids,at 720 min,the maximum dissolution rate in artificial gastric juice of total flavonoids in licorice flavonoids nanoparticles was 60.65%,which was 11.66 times that of total flavonoids in raw licorice flavonoids.Therefore,the experimental results showed that the prepared licorice flavonoid nanoparticles exhibited higher dissolution rate,which greatly improved the water solubility of licorice flavonoids.5.The antioxidant activity of raw licorice flavonoids and licorice flavonoids nanoparticles in rats was measured.When the dose was 300 mg/kg and the stomach was administered for 28 days,the MDA content of raw licorice flavonoids and licorice flavonoids nanoparticles were 2.18 nmol/mL and 1.12 nmol/mL.When the dose was 300 mg/kg,and the stomach was administered for 28 days,the CAT activity of raw licorice flavonoids and licorice flavonoids nanoparticles were 6.09 U/mL and 6.96 U/mL.When the dose was 100 mg/kg,and the stomach was administered for 28 days,the GSH-PX activity of raw licorice flavonoids and licorice flavonoids nanoparticles were 8.36 U/mg and 9.39 U/mg.When the dose was 300 mg/kg,and the stomach was administered for 28 days,the T-SOD activities of raw licorice flavonoids and licorice flavonoids nanoparticles were 370.76 U/mL and 392.85 U/mL.The experimental results showed that licorice flavonoids nanoparticles had stronger antioxidant activity.6.The bioavailability of echinatin and Licochalcone A in raw licorice flavonoids and licorice flavonoid nanoparticles in rat was measured.At 60 min after gavage,the maximum plasma concentrations of echinatin and Licochalcone A in licorice flavonoids nanoparticles reached 4.98 ng/mL and 24.72 ng/mL,at 90 min after gavage,the maximum plasma concentration of echinatin and licochalcone A in licorice flavonoids nanoparticles reached 67.62 ng/mL and 242.94 ng/mL.The bioavailability of echinatin and licochalcone A in licorice flavonoids nanoparticles were 10.63 times and 6.54 times that of echinatin and licochalcone A in raw licorice flavonoids.7.The toxicity experiment of licorice flavonoids nanoparticles in rats was measured.During the experiment,all groups of rats did not show abnormal behavior.According to the histopathological observation of the liver,compared with the control group,no obvious drug-induced pathological changes were found in the tissue structure and cells.The experimental results showed that when the dose reached 800 mg/kg,licorice flavonoids nanoparticles were not toxic to SD rats and showed good biological safety. |
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