The Research On The Active Compositions Of Essential Oils Of Propolis

Propolis is a resinous product collected by honeybees (Apis mellifera) from leaf buds and cracks of various plant sources, mainly from poplar(Populus)genus and, to a lesser extent, beech, horsechestnut, birch and conifer trees, mixed by bees with beeswax to form a sealing material in their honeycombs, smooth out the internal walls, and protect the entrance against intruders. In general, it is composed of 50% resin and vegetable balsam, 30% wax, 10% essential and aromatic oils, 5% pollen, and 5% various other substances, including organic debris. Recently, Some of the considerable biological activities of propolis extracts were reported, such as antibacterial, anti-inflammatory, antifungal, antitoxin, antiprotozoan, antiviral, Imunostimulant, anticancer, antiproliferative, antioxidative, scavenging free radicals, antiradiation, cardioprotective effect, anaesthetic, and antilipemic, et al. 10% Essential oils of Propolis, which is one of the mostly effective active components of propolis, it had occasionally been reported to possess antimicrobial activity and antilipid peroxidative action. The thesis focused on the extraction, separation, identification of physical and chemical properties and chemical compositions, and, antimicrobial activity and antioxidant activity were investigated, which will provide experimental datas for developing and applying essential oils of propolis as a resource of cosmetic or medicine. The main results and conclusions are as follows:The extract methods of essential oils from propolis were studied with the traditional and modern technologies, and chemical compositions were assessed by GC/MS. The results of different methods, such as hydrodistillation extraction (H), petroleum ether solvent extraction (P), ultrasound-assisted extraction (U), microwave-assisted extraction (M), and supercritical CO₂ extraction (S) showed that the order of extract rates was as follow: M>U>P>S>H. The optimal conditions for essential oils by microwave-assisted extraction can be concluded as follows: 32 min at 30℃, the ratio of solvent to material is 11 by using petroleum ether as the solvent, and the yield of extract was 13.83%, at these conditions, compared with petroleum ether solvent extraction, microwave-assisted extraction needs only 2/45 of the time, and achieves 1.51 times yield for extraction of essential oils from propolis. The relative content of terpene and ester in essential oils of propolis extracted by hydrodistillation (HDE) is the highest, the relative content of terpene and ester reaches 80.1%, and the main constituents of HDE areα-Eudesmol(30.28%),γ-Eudesmol(10.99%), Isomaltol(9.25%), Eicosane(8.76%),α-Bisabolol(7.77%), Guaiol (6.15%), Z-14-Nonacosane (3.98%), Eudesma-3,7(11)-diene (3.45%), Nonacosane (3.21%), Longifolenaldehyde (1.90%), (-)-Calamenene (1.74%), Curcumene (1.68%), Cedrol (1.51%), 2H-2, 4a-Ethanonaphthalen-8(5H)-one, hexahydro-2, 5, 5-trimethyl-(1.51%), etc. Then, the relative content of terpene and ester in essential oils of propolis extracted by supercritical CO₂ extraction(SE) achieves 26.43%, the main active constituents of SE areα-Eudesmol(6.89%), Lanosta-8,24-dien-3.beta.-ol, acetate (5.31%),γ-Eudesmol(2.97%), Lupeol(2.55%), etc.The ethanol dissolution method and systematic solvent extraction method were adopted to separate the active compositions of essential oils from propolis residue. The results of dissolution method showed that the main active compositions such as terpene and ester were accumulated with a relative content from 1.18% to 9.57%; the results of systematic solvent extraction method showed that the method was effective, terpene and ester, 4 kinds of flavanoids, and Oleic Acid with a relative content of 38.71%, 45.26%, 51.02% in three separated samples.The methods from national standard and prc codex for identification of physical and chemical properties of essential oils from propolis were adopted, and 13 kinds of index were determinated.The free radical scavenging activity of essential oils of propolis were examined by comparing to those of known antioxidants such as BHT, VE, and ethanol extract(EEP), water extract(WEP), supercritical CO₂ extract(SEP) of Propolis, using the method of DPPH and chemiluminescence; Total flavonoid contents in extract of propolis were determined by the method of aluminum chloride colorimetry, Total polyphenol contents in extract of propolis were determined by the Folin-Ciocalteau colorimetry method; Antioxidation mechanism of propolis also had been discussed. The results showed that five different extract of essential oils from propolis all had certain antioxidant activity, but weaker than EEP, WEP, and SEP, Generally speaking. The research also obtained a discovery that SE showed strong potential antioxidant activity, the 50% inhibitory concentration of SE to DPPH radical, super oxygen-anion radical and hydroxyl radical were 50μg/mL, 0.75mg/mL and 1.3mg/mL. propolis extracts had relatively strong DPPH radical scavenging activity were correlated with the 4 parts of chemical compositions, such as terpene and ester in essential oils of propolis, total polyphenol and flavonoid contents two part main in EEP, and several water-soluble chemical compositions with low content in WEP; Inhibition activity of propolis extract on super oxygen-anion radical and hydroxyl radical were mainly correlated with total flavonoid contents.The antimicrobial activity of essential oils of propolis was determinated by paper-disc method and tube dilution method, and it mechanism also had been discussed. The results showed that the essential oil displayed a broad antimicrobial spectrum, the antimicrobial activity of SE and SEP both were stronger than EEP, and essential oils of propolis extracted by P, U, and SE exerted a much stronger antimicrobial effect against Aspergillus niger than EEP. Unexpected or expected, all the essential oils of propolis had effect on Escherichia coli, and the MIC of HDE and SE for Escherichia coli, Bacillus subtilis, Proteus vulgaris, and Staphylococcus aureus indicated were 0.0391-0.625mg/mL. Aiming at Escherichia coli and Staphylococcus aureus, the strong antimicrobial activity of SE could be due to three aspects, which were exteaction method, synergistic action of polyphenol especially flavonoid and terpene and ester, the last one, mode of action, maybe. Further researchs of mechanism are expected.