Characterization Of The Chinese Propolis Chemical Composition And Screen And Evaluation Of The Antioxidant Components

Crude propolis,as a balmy and resinous substance,is produced by honeybees(Apis mellifera ligustica)from leaf buds and cracks in the bark of certain plants mixed with their saliva and beeswax.The chemical compositions of Chinese propolis often vary largely due to their raw material coming from resinous substance,usually depending on geographical region,floristics and collection time.This makes the biological activities,such as antioxidative and antibacterial properties,changeable.Therefore,it is necessary to comprehensively analyze the chemical composition,screen and evaluate the antioxidant components of Chinese propolis for their quality control to evaluate the quality of propolis.Firstly,it is the first time to apply Py-GC/MS to analyze the chemical composition of crude propolis.Furthermore,samples obtained from different regions of China were also compared basing on the chemical composition of crude propolis.Secondly,the orthogonal design was used to optimize ultrasound assisted extraction process of flavonoids in crude propolis to get the ethanol extract of propolis(EEP).The content of the total polyphenol,the total flavonoids,the total dihydrogen flavonoids and the content of 17 compounds in EEP were determined.The total antioxidant activity of EEP was also studied.Finally,the on-line HPLC-ABTS method was optimized to study the antioxidant activity of EEP,screen the antioxidant components in Chinese propolis,and try to distinguish the propolis from the gum of poplar tree.The main results of this study are as follows:1.Py-GC/MS method has been successfully applied for the analysis of chemical composition in Chinese crude propolis samples.The optimum analysis conditions were below: 550 ?C for pyrolysis temperature,120 meshes for sample size,sample amount 0.1 mg,split ration 50:1,the initial column temperature of 40 ?C,heated up to 300 ?C(remained 10 min)at the rate of 10 ?C /min.Furthermore,the difference of chemical composition for propolis from different geographical origins was compared.The difference of more categories chemical composition can be detected by Py-GC/MS.Especially,the more kinds and higher content of flavonoids were found in the propolis obtained from Shandong province.However,the higher content of triterpenoids was found in the propolis obtained from Zhejiang province.Several categories of chemical compounds in crude propolis were characterized in a single run without any cumbersome pretreatment,including flavonoids,terpenoids,acids,esters,hydrocarbons,phenols and alcohols,aldehydes and ketones,and others.It is a new method for the evaluation of proplis quality.2.The optimal extracting procedure for ultrasonic assisted extraction was as follows: the ultrasonic power of 59 KHz,the extraction temperature of 22±3 ?,feed ratio l:15,18 min of the extraction time,4 times for extraction times with the 65% ethanol aqueous solution.The contents of the total polyphenol,the total flavonoids,the total dihydrogen flavonoids and 17 compounds in EEP indicated that Chinese propolis was rich in polyphenol and flavonoids compared with the Brzail green propolis.The major flavonoids were pinobanksin-3-O-acetate,pinocembrin,chrysin,galangin and pinobanksin.The dominant phenolic acids and their esters were caffeic acid,p-coumaric acid,caffeic acid phenethyl ester and p-coumaric acid benzyl ester.The correlation analysis showed that the antioxidant activity of EECP was positively correlated significantly with the contents of the total polyphenol and the total flavonoids.The antioxidant activity of EECP was positively correlated significantly with the content of the caffeic acid,pinobanksin,kaemferol,pinobanksin-3-O-acetate,chrysin,caffeic acid phenethyl ester,galangin,caffeic acid cinnamyl ester,ferulic acid benzyl ester but not correlated significantly with the content of p-coumaric acid,and p-coumaric acid benzyl ester,and partly correlated with ferulic acid and pinocembrin.3.A convenient and valid on-line HPLC-ABTS method was established and optimized to screen the c antioxidant components and evaluate the antioxidant contribution of the single polyphenol to the antioxidant activity of Chinese propolis.The optimum conditions were 4.5 m for length of reaction coil and 0.2 mL/min for ABTS flow rate with the absorption of 0.750±0.025 in the 734 nm for ABTS·+ solution.The active fingerprints of Chinese propolis were established and 10 negative peaks with larger areas and good resolution were selected as the common active peaks.Six compounds of them were identified as caffeic acid,kaempferol,galangin,pinobanksin 3-acetate,caffeic acid phenethyl ester,and caffeic acid cinnamyl ester.Caffeic acid,kaempferol,galangin were the major contributors for all EECP.Pinobanksin 3-acetate,caffeic acid phenethyl ester,and caffeic acid cinnamyl ester were also the main contributors for EECP except propolis obtained from Heilongjiang,Jilin and Liaoning province.The correlation analysis between the total antioxidant activity by the on-line method and the single antioxidant activity by the on-line method for 10 common active compounds showed that caffeic acid,kaempferol,pinobanksin 3-acetate and caffeic acid phenethyl ester are positively correlated significantly with higher correlation coefficients with the antioxidant activity of EECP,and caffeic acid cinnamyl ester,galangin were positively correlated significantly with lower correlation coefficients with the antioxidant activity of EECP.The six compounds can be recommended as mark components.