Preparation Of Mixture Propolis For Livestock And Poultry

Propolis is a gelatineous mixture of various amounts of resins collected by the honeybee from branches, leaf buds and barks of plants, saliva and other secretions of the bees, beeswax and pollen. Propolis has various physiological effects such as anti-bacterial, anti-viruses, anti-fungi, inhibition of carcinoma tumour cells, anti-oxidant, anti-ulcer, anti-fatigue, anti-anaphylaxis, immunostimulating, hepatoprotection, tissue regeneration. Propolis has been widely used in medicine clinic and health care products. In veterinary clinic, studies and application of propolis have been a little. The extraction and purification technology of active constitutent of propolis was mainly studied for preparing MIXTURE PROPOLIS for livestock and poultry. The studies of pharmacy of MIXTURE PROPOLIS were as follows.1Study on the preparation technology of MIXTURE PROPOLISThe extraction technology of active constitutent of propolis was mainly studied by alcohol extracting and water precipitation. The standard of quality control was that the extraction rate of chrysin, galangin and total flavonoids were all no less than80%. We selected the optimal condition of alcohol extracting and water precipitation by orthogoral test. The extraction technology of active constitutent of propolis were as follows. Take propolis powder100g which has been pulverizated through a20screen mesh sieve, use80%alcohol metered volume to500mL, extract for4days under40℃, and mix more than2times each day. Then stand (0～4℃) for24hours, filter for supernatant; again stand (0～4℃) for24hours, filter for supernatant. Adjust pH of supernatant to8.0～8.5with ammonia water, then add purification water with sodium sulfite (3g) and edathamil disodium (0.3g), mix, end at50%alcohol, stand (0～4℃) for24hours, filter for supernatant. The supernatant was then evaporated to a thick paste, continue to emplay.The preparation technology of MIXTURE PROPOLIS:Use900mL solvent (alcohol: glycerin:stabilizer=7.8:2:0.2) to dissolve the thick paste, adjust pH to7.5～8.5with ammonia water, filter, add solvent above to1000mL, bulk and seal at last. The standard of quality control of10%(10g propolis/100mL) MIXTURE PROPOLIS was that the content of chrysin was no less than1.6mg/mL, the content of galangin was no less than0.8mg/mL and the content of total flavonoids was no less than12mg/mL. The solvent was that78%alcohol,20%glycerin and2%stabilizer.Three samples were produced in the semi-works production of MIXTURE PROPOLIS in GMP workshop. The preparation technology of this preparation was feasible. The quality of product was stable with more than95%yield.2Study on the method of quality control of MIXTURE PROPOLISThe quality of propolis was evaluated by the content of chrysin and galangin in the Pharmacopoeia of People’s Republic of China (2010, subdivision). The quality of propolis was evaluated by the content of total flavonoids in national standards of People’s Republic of China of propolis. The method of quality control of MIXTURE PROPOLIS was studied considering the two countries quality control standards of propolis.The quality control of chrysin and galangin in MIXTURE PROPOLIS was studied by TLC and HPLC. Silica gel G plate was used in TLC. The developing solvent was the mixture of trichloromethane, methanol and butanone (9.4:0.3:0.3). The color developing reagent was aluminum trichloride test solution. The results were observed under ultraviolet lamp (365nm). The chromatogram of samples have the same homologous fluorescence spots as the chromatogram of reference substance. The spots were sharp and separated from each other. The method of TLC has good repetitiveness and specificity. The conditions of HPLC were as follows. The separation was performed on ZORBAX-SB-C18HPLC column with mobile phase consisted of0.15%orthophosphoric acid-Methanol (36:64) at flow rate of1.0mL·min-1under25℃. The detection wavelength was268nm. The injection volume was20uL. The linear range of chrysin was1～24μg·mL-1whose linear equation was Ar=129998C-19260(R2=0.9997) with an average recovery of101.49%(RSD=0.92%, n=6). The linear range of galangin was1～24μg·mL-1whose linear equation was Ar=73356C-21955(R2=0.9995) with an average recovery of100.76%(RSD=1.62%, n=6). The method is simple, rapid, accurate, reliable and reproducible for the content determination of chrysin and galangin in propolis and MIXTURE PROPOLIS.The quality control of total flavonoids in MIXTURE PROPOLIS was studied by UV. The detection wavelength was360nm. The linear range of rutin was5～25μg·mL-1whose linear equation was A=0.0319C-0.0191(R2=0.9995) with an average recovery of100.31% (RSD=0.58%, n=6). The method is simple, accurate, reliable and suitable for the content determination of total flavonoids in propolis and MIXTURE PROPOLIS.3Study on the stability of MIXTURE PROPOLISThe stablitity of MIXTURE PROPOLIS was studied according to the requirement of Veterinary Pharmacopoeia of People’s Republic of China (2010, subdivision). The results of highlight experiment:at5days and10days, the characters of this preparation have not changed; pH decrersed by0.11at5days, pH decrersed by0.12at10days compared with the pH at0days; the content of chrysin and galangin respectively decrersed by1.14%and5.80%at5days and increased by6.46%and1.74%at10days; the contet (RSD<2%)of total flavonoids were all stable. In preliminary accelerated stability test (40℃), results showed that the characters of this preparation including quality characters, pH, identification and contet were all stable during the inspection of three months. In preliminary long-term stability (25℃), results showed that the characters of this preparation including quality characters, pH, identification and contet were all stable during the inspection of three months.