Extraction Methods, Chemical Componsition And Biological Activities Of Propolis Essential Oil

Propolis is a resinous material collected by honeybees (Apis mellifera L.) from leaf buds and cracks in the bark of the plants and mixed with pollen, wax and bee enzymes. It possesses a broad spectrum of biological activities. The chemical composition of propolis is very complicated, containing great amounts of bioactive compounds such as flavonoids, phenolic acids and terpenoids. Essential oil is an important part of propolis which contains great amounts of bioactive ingredients, and may contribute a lot for propolis'pharmacological activities. However, the researchs on propolis essential oil were far from sufficient. The studies on chemical composition and biological activities of propolis essential oil can provide foundations for more comprehensive evaluation of the biological activities of propolis and better use of propolis.In this study, four procedures for the extraction of essential oils from propolis were compared, including steam distillation extraction, solvent extraction, ultrasonic assisted extraction and microwave assisted extraction. The Uniform design was used to optimize ultrasonic assisted extraction process of propolis essential oil and the orthogonal design was used to optimize microwave assisted extraction process of propolis essential oil. The chemical components of essential oil from Brazilian and Chinese propolis were separated and identified by gas chromatography-mass spectrometry (GC-MS). Antimicrobial activities of propolis essential oil were measured by disc diffusion method and minimum inhibitory concentration (MIC) determination. Antioxidant properties of propolis essential oil were measured by both cell-free and cell-based experimental models:2,2-diphenyl-l-picrylhydrazyl (DPPH) assay, oxygen radical absorbance capacity (ORAC) assay and the assay to inhibit the H2O2-induced lysis of rabbit red blood cells (RBC). The effects on oxidative stress and anxiety-like behavior of propolis essential oil were evaluated with restraint stress animal model. Results showed that:1. Among the four procedures chosen for extraction, ultrasonic assisted extraction and microwave assisted extraction were suitable for the extraction of propolis essential oil. With these two methods, essential oil was of a high yield and quality.2. The optimal extracting procedure for ultrasonic assisted extraction was as follows:at the extraction temperature of 59℃with 1:15 feed ratio and 62min of the extraction time, the essential oil yield reached 8.70% for Chinese propolis and 8.75% for Brazilian propolis.3. The optimum technological parameters for microwave assisted extraction were obtained as:35℃of microwave extraction temperature,15min of microwave extraction time,1:10 of the solid to liquid ratio and 2 times of extraction. Under such optimal conditions, it was found that the essential oil content was 11.21% in Brazilian propolis and 9.73% in Chinese propolis.4. Fifty-one components were identified from Brazilian propolis essential oil and the major components were terpenoids:Longifolene, a-eudesmol,β-eudesmol, P-Caryophyllene and Guaiol. Forty-seven components were identified from Chinese propolis essential oil and the major components were aromatic compound and terpenoids:Cinnamic acid,β-eudesmol, a-eudesmol, and y-eudesmol. Twenty-one compounds were found existing in both of Chinese and Brazilian propolis.5. The essential oil from both Brazilian and Chinese propolis showed good to moderate antimicrobial activity against G-positive bacteria and fungi, while showed slight activity on G-negative bacteria. The essential oil from both Brazilian and Chinese propolis showed antioxidant activities in vitro. The IC50 of DPPH free radical were 178.3μg/ml and 309.5μg/ml respectively. The relative ORAC values of them were 0.82μMTrolox eq/g and 0.88μMTrolox eq/g, while the IC50 of H2O2 induced RBC hemolysis were 0.871 mg/ml and 0.879 mg/ml respectively.6. Essential oil of propolis can significantly decrease MDA level and increase SOD activity in serum of restraint stress mice, ameliorate liver function by improving the oxidative stress status. It can also significantly increase SOD and CAT activity and the GSH level, and decrease MDA and NO level as well as XOD activity in liver tissue.7. Propolis essential oil can significantly increase the Open Arm Entry percentage and Open Arm Time percentage, and decrease the protect Head-dipping percentage and protect rearing percentage of restraint stress mice in elevated plus-maze test, which showed no significant effects on the number of activities in spontaneous locomotor activity test. Propolis essential oil also depressed the serum level of CORT and ACTH, decreased the content of MDA and improved SOD and GSH in brain tissues of restraint stress mice.Essential oil is an important component of propolis which contains great amounts of bioactive ingredients and possesses various biological activities. The present work provides additional data about the propolis essential oil obtained by microwave extration and reveals the interesting character of its chemical composition, antimicrobial activity and antioxidant acitvity in vitro, ameliorative effects on oxdative stress and anxiety. As propolis essential oil is a complex mixture of chemicals which may differ by plant sources, more investigations and further studies are necessary to elucidate its components and the exact site to achieve their potential anxiolytic effects.