Study On Purification Of Flavonoids From Tartary Buckwheat And Water Soluble Powder Of Rutin

In this paper,60.56%of crude flavonoids were obtained from the seeds of tartary buckwheat by ethanol extraction.In order to increase the content of total flavonoids,the total flavonoids were further purified by anti-solvent recrystallization method in this experiment,and the characterization,solubility,in vitro release profile,intestinal absorption and cell antioxidant activity of purified flavonoids were investigated.The experiment and the results are as follows:the Design Expert 8.0 software was used to optimize according to the Box-Behnken design and the following optimal conditions were obtained:stirring time 13.9 min,total flavonoid concentration 70.0 mg/mL,recrystallization temperature 38.0℃,water(The volume ratio of anti-solvent)to methanol(solvent)was 10:1.Under these conditions,the yield and purity of total flavonoids were 94.35%and 99.81%,respectively.The content of rutin was 86.37%,the content of kaempferol-3-O-rutinoside was 3.61%,and the content of quercetin was 5.21.%.The purified flavonoids were characterized by particle size and scanning electron microscopy(SEM).The results showed that the anti-solvent method not only increased the purity of the total flavonoids,but also reduced the size of the flavonoids(250-430 nm).Due to the decrease of particle size,the solubility of total flavonoids,in vitro release rate and intestinal absorption were all improved,but the results of cytotoxicity test showed that the purified buckwheat flavonoids did not have toxicity to cells due to improved absorption.In terms of antioxidants,the cellular antioxidation results showed that the antioxidant capacity of buckwheat flavonoids increased after purification due to increased absorbance and increased biocompatibility.In this paper,the above purified flavonoids were used as raw materials,rutin was recrystallized in ethanol in a new crystal form.Its water solubility and bioavailability were improved.In the experiment,rutin was completely dissolved in ethanol at 60℃,and when it was placed at 25℃,a new crystal form was precipitated due to supersaturation,rutin-ethanol solvate.First,in order to determine the change in chemical structure,rutin-ethanol solvate were assayed for rutin and ethanol by high performance liquid chromatography and gas chromatography.The experimental data showed thatthe hydrogen bonds were formed at the molar ratio of the two substances was 1:1.In the FTIR test,the characteristic peak of rutin changed,further confirming the presence of ethanol in the resulting crystals.To identify changes in the crystal structure of the rutin ethanolate,the rutin-ethanol solvate was characterized by microscopy,SEM,XRD,and DSC.The results showed that rutin ethanolate is a new kind of crystal that is different from rutin original drug and has different structural characteristics.The solubility test results show that due to the change of crystal structure,rutin ethanolate has better moisture absorption,so the dissolution rate is faster than rutin.In rat experiments,the bioavailability of rutin ethanolate is 1.67 times that of rutin,which means that the solvation of rutin improves the efficiency of oral absorption.In addition,the stability of the crystal structure of rutin ethanolate was investigated.The results showed that after the absorption of rutin ethanolate,it is easy to combine with water again to form hydrate,ie,rutin trihydrate,but the crystal structure will not be changed if it is stored under dry and sealed conditions.This structure is a metastable crystal different from rutin trihydrate.In this paper,hydroxypropyl-β-cyclodextrin(HP-β-CD)was used as the inclusion material and anti-solvent method was used to prepare rutin-hydroxypropyl-β-cyclodextrin inclusion complex.In vivo bioavailability and hypoglycemic effects were tested and the results are as follows:First,understand the dissolution equilibrium between rutin and hydroxypropyl-β-cyclodextrin and determine the molarity of rutin and hydroxypropyl-β-cyclodextrin.At a ratio of 1:1,rutin can be completely enclosed.Secondly,using anti-solvent recrystallization method combined with inclusion technology,using N,N-dimethylformamide(DMF)as solvent,acetone as anti-solvent.The effects of rutin concentration,inclusion time,inclusion temperature,recrystallization temperature,ratio of solvent and anti-solvent on the drug loading of rutin-hydroxypropyl-β-cyclodextrin inclusion complex,and to reach the optimal conditions:rutin Therutin concentration of 120 mg/mL,inclusion time is 60 min,inclusion temperature is 25 ℃,recrystallization temperature is 0 ℃,the solvent and anti-solvent ratio is 1:13.The drug loading of the inclusion complex was 28.93%±0.12%and the entrapment efficiency was 90.02%±1.3%.The inclusion complexes were characterized by SEM,FTIR,XRD,1H NMR and DSC.The results showed that rutin formed ainclusion complex with hydroxypropyl-β-cyclodextrin and existed in an amorphous form.In addition,solvent residue,solubility,in vitro dissolution,bioavailability and in vivo hypoglycemic tests were performed.The experimental results showed that the residual amounts of DMF and acetone in the rutin hydroxy propyl-β-cyclodextrin inclusion compound were 0.021%and 0.025%,respectively,which were in line with the human drug registration.Technical requirements The minimum standards required by the International Council for Coordination(ICH);the solubility and in vitro dissolution rate of rutin hydroxypropyl-β-cyclodextrin inclusion complexes are significantly higher than that of rutin in water-soluble test results.Oral bioavailability of rutin hydroxypropyl-beta-cyclodextrin inclusion compound is 2.24 times higher than that of rutin.After rutin has been included in the inclusion,the hypoglycemic activity is increased based on the increase of bioavailability.Increased blood glucose levels can be reduced to 8.6 mmol/L.