The Research On Extraction And Application Of Flavonoids In Propolis

The thesis focused on the extraction, determination, separation of flavonoid inpropolis, and its application. The toxicity and function of products were studied. Themain results were as follows:Ultrasonic method was used in extraction of propolis flavonoids, the optimumconditions were obtained from the experiments: alcohol concentration 95%,temperature 35℃, proportion of propolis and solvent 1:10, time 20 minutes. Supercritical CO₂ was adopted to extract the active ingredients of propolis by measuringextraction rate, The best processing conditions of super critical CO₂ for theingredients propolis extraction at a pilot scale were: pressure 30Mpa; temperature55℃; time 4 hours; CO₂ flow of 50 kg/h; the extraction rate up to 37.50%. The besttechnology of flavonoids in propolis by ethanol extracting lay in that, ethanolconcentration of 95%, ratio of propolis and solvent 1:8, time 24 hours, temperature25℃, using 160-200 mu phosphate-activated carbon at 60℃Stir 1 h adsorptting thelead, filtering wax after frozen 8 hours under -18℃, lead content of propolisextraction under 0.4 ppm, wax content being 0.2% or less in this wayThe total flavanoids of ethanol extration and SCF-CO₂ extraction from propoliswere determined respectively by HPLC and three kinds spectrophotometry, andconcluded that the content of total flavanoids of ethanol extration changed from 5341mg/100g to 8080mg/100g in five samples and that of SCF-CO₂ extraction changedfrom 1990 mg/100g to 2316 mg/100g in three samples by spectrophotometry under415 nm, this measuring result was the minimum in four methods, not satisfyingrequirements. The result of spectrophotometry under 360 nm higher than the HPLCmethod was more reasonable. The result of spectrophotometry under 510 nm onethanol extraction was maximum in four methods and that was 41388mg/100g-45998 mg/100g, but the flavonoids content of supercritical CO₂ extractionwas from 6752 to 7667 mg/100g. Taking into account the competitive of health foodfinalized 510 nm methed as flavonoids content determination methed of Propolisproducts. The organic compounds of the ethanol extraction and supercritical CO₂ extraction from propolis were identified and quantified by GC/MS, the results shewthat there were 9 kinds of flavanoids in the ethanol extraction and its contentamounted to 39.73%; 6 kinds of flavanoids in supercritical CO₂ extraction and itscontent amounted to 25.32%. They had the same four kinds of flavanoids.Separatting strong polarity flavonoids from propolis by low concentration ofalcohol re-dissolved ethanol extraction, experiments proved that the yield ofseparation was 22.5%, and the trial more than 20%. This method could enrichflavonoids, providing raw materials for the future development of the high-flavonoidpropolis health food. Making ethanol extraction of propolis by frozen, crystallization,filtration, dissolution, recrystallization, the higher flavonoids can be attained. Theflavonoids were qualitatived and quantitatived by IR and HPLC. It proved thatChrysin's content was relatively high. Propolis Soft Capsule internal contents ofPropolis Liquid of Company were often found yellow sand-like material, the adoptionof the separation and identification methods, we could identify the yellow sand-likematerial composition.The experiment proved that the best formulation of ginseng Propolis Liquid wasmade of 7.5% propolis extracts of ethanol, 4% of the ginseng extraction, 30%PEG400 and 58.5 percent 75% of the consumption of alcohol. The best formula ofPropolis Soft Capsule of high content flavonoids was made of propolis ethanolextract 30 percent, PEG40050.5% glycerol 14.5%, purified water five percent。Thebest formula of oil-soluble propolis soft capsules was made of ethanol extract 20%,soybean oil 61%, beeswax 6.5 percent, lecithin 2.5%, starch 10 percent. Product threebatches of samples under optimum conditions and observe stability of samles forthree months at 38℃and relative humidity 75% of the environment and itscomposition and effectiveness of health science and other indicators were consistentwith stability requirements.Acute toxicological test LD₅₀ of ginseng propolis liquid was more than 20 ml/kgBW, so Ginseng Propolis Liquid was a non-toxic level. Three genetic toxicity testswere the Ames test, mice bone marrow PCE cell mouse micronucleus test and spermabnormality test, and three results were negative. 30 day feeding trial results were innormal range in the choice of test projects. In function experiments of Ginseng Propolis Liquid, the result of mice cellularimmune function experimental spleen lymphocyte transformation test was positive;result of delayed type hypersensitivity test was positive. The result of experimentalmice cellular immune function experimental antibody producing cells was positive;result of mice half hemolytic value was positive. Mice monocytes-macrophagefunction test results were positive results; test result of mouse macrophagephagocytosis chicken erythrocytes was positive. The result of natural killer cellactivity was positive. Judging from the above results, samples had enhanced immunefunction.