| Propolis is a viscous material prepared by bees collecting buds and secretions from plants and mixed with wax and enzyme. Propolis has been widely used in medicine, food and other industries due to its biological activity. The biological and pharmacological properties of propolis is attribute to its chemical composition which is complex and variable between source of plant, ecoclimatic, harvest time, extraction method. Analyze and identify the chemical composition of propolis with HPLC is the common method, which has defects as cost longer time and much elution solvent to analysis, lower accuracy and so on. Propolis utilization possess a strong odor, poor water solubility, no secondary use of the residue after extraction. These disadvantages result in limit of its application, poor storage stability,shelf-life extension. To establish a fast effective analytical method for propolis and improve the applied properties, 31 WEPs from different locations and 2 gums were analyzed by UPLC and isolated and identified the unknown compounds in EEPs and WEPs by UPLC-Q-TOF-MS. And we prepared EEP liposomes and EEP microcapsules by thin film evaporation-DHPM and emulsification gelation/complex coacervation method. Specific results are as follows:(1) The separations were conducted at a temperature of 35 °C on a Agilent ZORBAX Eclipse Plus C18 column(50 mm×2.1 mm, i.d.,1.8 μm), Gradient elution was consisting of 0.1% aqueous formic acid and methanol and elution time was 20 min. UV detection was at 256 and 280 nm. Using UPLC, We analyzed the chemical composition of 31 WEPs, 2 WEGs, 8 EEPs and 8 WEP-Rs. All information achieved by UPLC, had suffered a linear discriminant analysis(LDA).The results showed that the kinds and contents of detected chemical references varied dramatically in these WEPs. 3,4-dimethoxy cinnamic acid, cinnamic acid, naringenin, coffee acid, ferulic acid, pinocembrin, catechin, epicatechin and chrysin presence in more than 70% WEPs; WEP from HN had the most special chromatographic profile which significantly different from others. There are still caffeic acid, p-coumaric acid, ferulic acid, cinnamic acid, naringenin in WEP-R. Different raw propolis were well classified base on the geographical regionalization in China and color by LDA.(2)Based on the previous literature on propolis and relevant databases, 31 mixture of EEPs and WEPs from different location is subjected to UPLC-Q-TOF-MS analyze. 918 common substances were identified from 31 propolis, including caffeic acid, pinocembrin, chrysin which already reported in previous study. And 15 compounds were preliminary identified by precise molecular weight and MS/MS.(3)The thin film evaporation-dynamic high pressure microfluidization was used to prepare EEP liposomes with soybean lecithin and cholesterol as the membrane materials. The characterization of prepared EEP liposomes was examined by dynamic light scattering, transmission electron microscopy, DPPH radical scavenging activity, low temperature storage test The results showed that the average size was 68 ± 14 nm, encapsulation efficiency was 97.80±5.21%, the appearance is uniform spherical, Ke value was 2.53±0.05%. The liposomes obtained were incubated at 4 ℃ for 3 and 6 months and its encapsulation efficiency fell 16.74% and 28.77%, PDI fell 6.61% and 14.32%, AAI was 2.78 and 2.71, respectively. Contents change of 7 compounds in EEP liposome were 1.08% ~ 6.63% analyzed by HPLC after 180 d storage time, lower than 5.55% ~ 19.42% in EEP. This study highlights the protection of liposome for EEP.(4)EEP calcium alginate-chitosan microcapsules were prepared by emulsification/intemal gelation-complex coacervation, the characterization of microcanpsules were investigated by dynamic light scattering instrument, confocal laser scanning microscope, FITR, X-ray diffraction, a simulated gastrointestinal tract model and a temperature-accelerated test. The results show that the microcapsules observed by CLSM were spherical with a mean particle size of 265 ± 25 nm. The encapsulation efficiency and loading capacity values was 72.80±3.8% and 19.96±2.4%, respectively. FITR and X-ray diffraction showed that sodium alginate and chitosan form the microcapsules. The in vitro release of EEP microcapsules in 8h was 46.60±0.80%. Contents change of 7 compounds in EEP microcapsules was 14.01%-39.68% analyzed by HPLC after a temperature-accelerated test, lower than 37.01%~77.45% in EEP. The prepared microcapsules has a small particle and great stability with a fitting release rate to the enteric system. The active substances inside EEP microcapsules were well protected.In conclusion, a rapid analysis method of propolis by UPLC is established, the combination method of propolis extracted with alcohol and water was developed to best utilize; The mixture of EEP and WEP was quantified by UPLC-Q-TOF-MS which can achieve a comprehensive profiles of propolis and provide the theoretical data for further development and utilization. EEP liposomes and EEP microcapsules solves the problem of instability during storage. Propolis can be used as a new delivery system for drug control and sustained release; it can effectively encapsulates the active ingredients and sustained release. EEP microencapsulation can widely used in cosmetics, agriculture, food technology and textiles. This study result was: UPLC and UPLC-Q-TOF-MS can identify the chemical composition of propolis quickly, the prepared EEP liposomes and microcapsules has the application possibility in pharmaceutical, cosmetic industries. |
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