Study On Optimization Of Extraction Conditions And Stability Of Total Flavonoids From Propolis

Part I: Optimization of extraction conditions of total flavonoids from propolisObjective: To study the effect of different conditions on the extraction rate of total flavonoids from propolis,and to obtain the optimal combination of extraction time,ratio of liquid to material,ultrasonic power,and solvent pH at the time of the highest extraction rate.Methods: The organic solvent extraction and ultrasonic extraction were used to extract the propolis gum.Rutin was used as the reference substance.The absorbance of the total flavonoids of propolis obtained in various conditions were determined by UV spectrophotometer at a wavelength of 415 nm.The concentration and extraction rate of the total flavonoids of propolis was calculated by the standard curve.Taking the total flavonoids extraction rate of propolis as evaluation index,the effect of four factors-extraction time,liquid to material ratio,ultrasonic power and solvent p H on the extraction rate of total flavonoids in propolis was obtained by single factor test,and orthogonal test was carried out on the basis of single factor test,the optimum process of extracting total flavonoids from propolis was selected according to the results of the experiment.Results: In the different levels of various experimental factors designed,the best extraction process was extraction time 40 min,material-liquid ratio 30:1,ultrasonic power 70 kHz,pH value of the solvent 6.0,The primary and secondary effects of various factors were solvent p H>ultrasonic power>extraction time>liquid-to-liquid ratio.Conclusion: The p H value of the extraction solvent has a significant effect on the extraction rate of total flavonoids from propolis.The extraction rate is the highest when the p H value is6.0.This experiment provides a reference for the extraction and utilization of total flavonoids from propolis.Part II: Study on the stability of total flavonoids from PropolisObjective: To study the effects of temperature,stored light conditions,pH value,oxidant（H₂O₂ solution）and reducing agent（Na₂SO₃）,food additives,and metal ions on the stability of total flavonoids from propolis.Method:1.The total flavonoids from propolis were incubated at 4°C,20°C,30°C,40°C,50°C,60°C,70°C and 80°C for 1 h,respectively,then the changes of absorbance values at each temperature were measured.2.The total flavonoids from propolis were respectively placed in cold and dark places,indoors and outdoors illumination,and the absorbance values were measured and recorded at five time points: 1 d,7 d,15 d,30 d and 60 d.3.An aqueous solution of pH 2-12 was prepared with hydrochloric acid or sodium hydroxide and added to the total flavonoids from propolis.The change of absorbance after 6 h was determined.4.The total flavonoid solution from propolis containing different hydrogen peroxide and sodium sulfite were prepared within the scope of food safety,and the absorbance of the solution was determined after 6 h.5.The total flavonoid solution from propolis containing citric acid,malic acid,ascorbic acid,sucrose and sodium benzoate was prepared within the scope of food safety,and the absorbance of the solution was determined after 6 h,12 h and 24 h.6.The total flavonoid solution from propolis containing different concentrations of KCl,NaCl,CaCl₂,MgCl₂,A1C1₃,CuCl₂,FeCl₂ and FeCl₃ was prepared,and the absorbance of the solution was determined after 6 h.Result:1.The absorbance value of the total flavonoid from propolis below 4-40°C was not changed significantly,and the absorbance value was decreased obviously at 40°C.2.The absorbance value of the total flavonoid from propolis was increased slightly with the prolonged storage time in the dark,and decreased significantly more than 7 days under indoor and outdoor strong light.3.The absorbance value of the total flavonoid from propolis was not changed significantly within 5-8 pH value,and decreased significantly when the p H value was in the range of less than 5 and greater than 8.4.Under the effect of different concentrations of hydrogen peroxide,the absorbance value of the total flavonoid from propolis was not changed significantly,while sodium sulfite reduced significantly the absorbance value.5.In addition to sucrose,other food additives can change the absorbance value or change the color and clarity of the total flavonoids from propolis.6.The color,clarity,and absorbance of the total flavonoid solution from propolis containing KCl,Na ,and MgCl₂ showed no significant changes.The solution containing CaCl₂,A1C1₃,CuCl₂,FeCl₂ and FeCl₃ caused the color of the total flavonoids from propolis deepened,and the clarity decreased or even became turbid,the absorbance inability measured.Conclusion: The stability of the total flavonoid from propolis is better below 40°C,and the effect of light on the stability is greater,the stability has a close relationship with the pH value.The effect of oxidant on the stability is not significant,but the effect of reducing agent is very obvious,and it is possible that Sodium sulfite interfere with the combination of Al³⁺and flavonoids,resulting in a deviation in the determination of absorbance.The food additives had different effects on the stability except for sucrose.The effects of K⁺,Na⁺ and Mg²⁺ on the stability were less,the stability was greatly influenced by Ca²⁺,A1³⁺,Cu²⁺,Fe²⁺,Fe³⁺.