| Adinandra nitida Merr.ex H.L.Li,commonly known as Shiyacha,is mainly distributed in Guangdong,Guangxi,Guizhou and other places with a wide distribution area.In the folk of South China,the young leaves of bright leaf Yangtong are often used to make tea.They have unique flavor,generate saliva and quench thirst.They also have the effects of detoxification,hemostasis,anti-inflammatory and lowering blood pressure.They have high health care and medicinal value.The reason why Adinandra nitida has a variety of effects is that it contains flavonoids,caffeine,tea polyphenols,terpenes and other bioactive substances,of which flavonoids are the main active substances of Adinandra nitida,with a content of more than20%.The results showed that Adinandra nitida was rich in camellianin A with apigenin as aglycone.Apigenin has a variety of biological activities such as anti-tumor,anti-oxidation and anti-inflammatory.However,apigenin has poor water solubility(0.03 mg/ml)and low bioavailability,which seriously limits its wide application in medicine,health products,food and other industries.At present,there are many studies on the pharmacological activity of apigenin,but there are few studies on the extraction,purification and improvement of water solubility of apigenin.Most of the extraction methods of apigenin are traditional organic solvent reflux extraction,and most of the purification methods are column chromatography,which generally has the disadvantages of high cost,long time-consuming,low quality,and difficult to expand production.Based on the above reasons,in this study,apigenin and its glycosides were extracted from the leaves of Adinandra nitida,and apigenin in the extract was separated and purified to obtain high-purity apigenin,and its water solubility was improved in order to improve its oral bioavailability.The results are as follows:1.Apigenin was extracted by surfactant micellar enzyme pretreatment and ultrasonic micro wave synergistic extraction method.Alkyl glycoside10 and the compound enzyme with cellulose and pectinase were chosen as surfactant and enzyme,respectively.Single factor method was utilized to obtain the optimum enzyme pretreatment condition,and the optimum condition was:the solid-liquid ratio of 1:30,the enzyme concentration of 3%,the enzymatic hydrolysis temperature of 50℃,p H of 5,the extraction time of 3 h,the ratio of cellulase to pectinase of 4:2.The leaves of Adinandra nitida were treated under the optimum condition for ultrasonic microwave synergistic extraction.The optimum ultrasonic microwave extraction condition,determined by single factor combined with response surface methodology,was:microwave power of 800 W,microwave time of 8 min and surfactant concentration of 2%.Under the above condition,the model predicted that the extraction rate of apigenin was 18.53mg/g and the total extraction rate of apigenin was 22.18 mg/g.2.Using the cloud point property of surfactant micelles to separated apigenin.Sodium chloride was added to the extract to settle the surfactant,then ethanol was used to precipitate the surfactant,and finally ethanol was evaporated to obtain the crude apigenin whose purity was 33.50±1.55%and yield was 90.47±2.37%.Next,antisolvent recrystallization method was utilized to purify crude apigenin,using N-methylpyrrolidone as solvent and water as antisolvent.The effects of stirring time,temperature,volume ratio of antisolvent to solvent and apigenin concentration on the purity and yield of apigenin were investigated by single factor and response surface experiments,The optimum preparation process of high-purity apigenin was:temperature of 62.5℃,apigenin concentration of 109.2 mg/ml,stirring time of 19.15 min,volume ratio of antisolvent to solvent of 17.5.Under the best preparation process,high-purity apigenin was prepared with the purity of 98.47%and the yield of 86.65%.According to the result of high performance liquid chromatography,infrared spectroscopy,liquid chromatography-mass spectrometry and differential scanning calorimetry,we concluded that the purified sample was apigenin.3.The oral delivery system of apigenin was established,and the casein in milk was sued as carrier material to encapsulate apigenin.The effects of mass ratio of apigenin to casein,temperature,stirring time and apigenin concentration on the encapsulation efficiency and drug loading of apigenin-casein nanoparticles were investigated by single factor method.The best preparation proces of apigenin-casein nanoparticless was:mass ratio of apigenin to casein of3:5,temperature of 35℃,stirring time of 10 min,apigenin concentration of 0.6 mg/ml.Under the best condition,apigenin-casein nanoparticles was prepared with the entrapment efficiency of 48.56±3.45%and the drug loading of 23.37±2.51%.The average particle size was238.7±2.1 nm,the PDI was 0.182±0.015 and the zeta potential was-7.48±0.24 MV,indicating that the average particle size of apigenin-nanoparticles was small,evenly dispersed and stable.The morphology,surface chemical structure and crystal form of apigenin-casein nanoparticles were analyzed by SEM,FTIR,XRD,DSC and ~1H-NMR.SEM results showed that the morphology of apigenin casein nanoparticles was similar to spherical particles,and the particle size was uniform.FTIR and ~1H-NMR results showed that apigenin was hydrogen bonded to casein.The results of XRD and DSC showed that apigenin in apigenin casein nanoparticles was basically amorphous.4.The saturated solubility of apigenin-casein nanoparticles in water,artificial intestinal juice and artificial gastric juice was studied.It was found that the saturated solubility of apigenin-casein nanoparticles in corresponding media was 826.05,108.20 and 422.96 times higher than that of apigenin original drug,respectively.The results of in vitro dissolution showed that in the artificial gastric juice,the cumulative release of apigenin-casein nanoparticles was only 64.33±1.28%at 720 min,which was 2.62 times that of apigenin powder,and almost completely dissolved at 360 min.In the artificial intestinal fluid,the cumulative release of apigenin-casein nanoparticles reached 97.43±0.36%at 720 min,which was 3.56 times that of apigenin(27.35±0.53%),and almost completely dissolved at 360 min.The results of in vitro simulated digestion experiment show that the small intestine and large intestine are the main digestive parts,because apigenin is stable in acidic environment,low solubility,unstable in alkaline environment,high solubility and easy to be decomposed by enzymes.The residual amount of apigenin after simulated digestion in the mouth,stomach,small intestine and large intestine is basically that apigenin-casein nanoparticles are higher than that of apigenin,indicating that the encapsulation of casein has a protective effect on apigenin.5.The hypoglycemic effect of apigenin-casein nanoparticles on diabetic mice was investigated.The results showed that apigenin-casein nanoparticles had no effect on body weight and fasting blood glucose to normal mice,but could promote the weight gain of diabetic mice.Moreover,the medium dose and high dose of apigenin-casein nanoparticles could significantly reduce the fasting blood glucose of diabetic mice and increase glucose tolerance of diabetic mice,suggesting that apigenin-casein nanoparticles has hypoglycemic effect.6.The pharmacokinetics of raw apigenin,physical mixing of apigenin and casein and apigenin-casein nanoparticles were investigated.Moreover the cytotoxicity of apigenin-casein nanoparticles to SD rats were investigated.The results of pharmacokinetic study showed that the peak time of the maximum plasma concentration of apigenin-casein nanoparticles group,apigenin casein physical mixing group and raw apigenin group were 0.5 h,4 h and 2 h respectively,and the maximum plasma concentration were 5.37,1.93 and 1.86μg/m L,respectively,the maximum plasma concentration and peak time of apigenin casein nanoparticles group were better than those of original drug group and apigenin casein physical mixing group.The maximum plasma concentration of apigenin in apigenin casein nanoparticles group was 2.88 times higher than that of original drug group and 2.78 times higher than that of physical mixing group,respectively.Das2.0 software was used to fit the data.Raw apigenin group conforms to the two compartment model with weight coefficient w=1,the physical mixing of apigenin casein group conforms to the one compartment model with weight coefficient w=1,and apigenin-casein nanoparticles group conforms to the two compartment model with weight coefficient w=1/cc.The AUC of apigenin-casein nanoparticles group was 3.0 times than that of apigenin original drug group and 2.39 times than that of physical mixing group,respectively.The results showed that the amorphous morphology and nano size of apigenin-casein nanoparticles were helpful to improve the water solubility,dissolution rate and bioavailability of apigenin.After intragastric administration of raw apigenin and apigenin-casein nanoparticles,the drug concentrations in tissues at 0.5,1,2,4,8 and 12 h were analyzed.From the experimental results,apigenin was distributed in a certain amount in the heart,liver,spleen,lung,kidney and brain of rats.Due to the absorption,metabolism and excretion of drugs in rats,the distribution proportion of drugs in various organs is not invariable.The heart and liver are always the main distribution organs of drugs,followed by the kidney,and the least is the spleen,lung and brain.In the hearts of rats,the content of apigenin of casein-nanoparticles group was 1.16 times that of raw apigenin group;In rat liver,the content of apigenin of apigenin-casein nanoparticles group was 1.12 times that of raw apigenin;In rat spleen,the content of apigenin of apigenin-casein nanoparticles group was 1.10 times that of apigenin group;In rat lung,the content of apigenin of apigenin-casein nanoparticles group was 1.33 times that of apigenin group;In rat kidney,the content of apigenin of apigenin-casein nanoparticles group was 1.07 times that of apigenin technical group;In rat brain,the content of apigenin of apigenin-casein nanoparticles group was 1.29 times that of apigenin technical group.The results showed that the concentration of apigenin casein nanoparticles in rat tissues was higher than that of apigenin powder group under the same dosage and time.The cytotoxicity experiment investigated the effect of apigenin-casein nanoparticles to rat organ coefficient and the cytotoxicity to the liverof rats after intragastric administration for 14days.The experimental results showed that the rats organ coefficients of heart,liver,spleen,lung and kidney in low,medium and high dose group has no obvious difference to those in the control group.Moreover,there were no obvious difference of the tissue structure and cell morphology of the liver between low,medium and high-dose group.Pathological changes induced by drugs were not found.Even the dose of 1000 mg/kg SD had no toxic effect on rats,indicating that apigenin-casein nanoparticles had good biosafety. |
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