| Propolis is a strongly adhesive resinous substance collected by honeybees from buds and exudates of certain plants and mixed with maxillary gland secretion and beeswax. It can be used by bees to protect the entrance against intruders such as plant diseases, insect pests, pathogenic microorganism and so on, or as a kind of special substance to seal holes in their honeycombs and sterilize inside environment. Propolis abounds with flavonoids compounds, so it is called "the treasury of flavonoids". The flavonoids have shown a variety of biological effects, including antibiotic, sterilizing, anti-inflammatory, acesodyne, reducing blood fat, accelerating organ regeneration, promoting immunity and so on. Because their proportion to the effective ingredients of propolis is higher and their content is more convenient for determining, the flavonoids are usually selected as a representative of the effective ingredients of propolis. So the content of flavonoids is the most important quality index of propolis products. High hydrostatic pressure (HHP), which means cold isostatic superhigh hydraulic pressure that ranges from 100 to 800MPa or more, is currently considered as an attractive innovative non-thermal process that can effectively inactivate microorganisms and preserve fresh-like food products. Exploiting the effects of HHP in biotechnology has received increased interest during the last decade. HHP has been successfully applied in the processes of pasteurization and sterilization in the food and pharmaceutical industries. There have many advantages in extracting the effective ingredients from crude herbs with the help of HHP: (1) More kinds of solvent can be used. We can select appropriate solvents, such as water, lipophililic and hydrophilic organic solvent etc, according to the property of extract. (2) HHP processing is operated at room temperature without any heating process such as leaching at high temperature, distillation and so on, so some heat-sensitive effective ingredients of herbs will not lose their biologic activity. (3) Extraction time is very short, and there needs only a few minutes in one extracting course with the help of HHP. (4) The operation parameters of high-pressure equipment are easy to adjust and control. There needs only one equipment to extract many kinds of effective ingredients from raw herbs with different solvents. (5) Economization of energy. There only have little energy transform and energy exchange with outside during the course of duration and decompression, and almost no energy is consumed. (6) HHP processing is operated in an airtight environment, so the solvent can't vaporize to the surroundings. Moreover, there have no wastewater and exhaust gas. So it accords with the request of the environmental protection. In order to extract at room temperature, shorten extraction time and increase the extraction yield of flavonoids, we applied HHP processing to extracting flavonoids from propolis. To explore the mechanism of enhancing mass transfer by HHP and grope the optimum extraction process of HHP processing, four key technical problems are studied in this paper. 1. Studied theoretically on the mechanism of enhancing mass transfer by HHP in detail. Several conclusions could be obtained as follows: (1) The high hydrostatic pressure can increase significantly the flux and velocity of solvent pervasion. Because of the ultra-high hydrostatic pressure we used, even though the pore diameter is very small, the pressure difference between inner and exterior of cell is so large that it would lead the solvent flux to become very large, i.e., the ultra-high hydrostatic pressure can significantly increase the velocity of solvent pervasion and decrease pervasion time. So HHP processing can greatly shorten the extraction time. The higher hydrostatic pressure is, the more solvent would penetrate into the inner of cell and the more effective ingredients would enter into the solvent outside through the cell wall. Thus the higher extraction yield can be obtained.(2) The high hydrostatic pressure can lead to violent eddy diffusion within the cell during the course of duration and decompression, which will increase the concentration difference between inner and exterior of cell and then accelerate the speed of mass transfer. When pressurized, the pressure of both ends of pore on the cell wall is not equal, which would result in the forced flow. The velocity of solvent flow is in direct proportion to the pressure difference, i.e., the larger the latter is, the larger the former is. Then the onflow appears in the pore. The larger the velocity of flow is, the more violent the onflow is. So a large amount of swirls appear in the solvent within the cell and disperse the effective ingredients to the solvent violently. The soluble compositions of cell dissolve into solvent rapidly and form dense solution, which result in enormous concentration difference with the solvents around of the outside, so the solute would spread into solution around rapidly in order to balance the concentration difference and make it disappear at last. In a word, the high hydrostatic pressure can lead to violent eddy diffusion, increase the concentration difference badly, and thus make mass transfer more rapidly. This explanation has not been reported in the literature. (3) High hydrostatic pressure can also make some changes in cell form and cause the cells of some fragile materials to rupture. Under the high hydrostatic pressure, cell volume would shrink and cell form would change from irregular to regular, which would cause the cells of some fragile materials to rupture, so the cell serum lost out more completely. The process of mass transfer of the effective composition was enhanced greatly and the higher extraction yield can be obtained. (4) When decompressed rapidly, the violent cavitation will cause cells to inflate sharply. If the inflation exceeds the cell deformation limit, cell would rupture, and cell serum would lose out. The solvent would dissolve the effective ingredients fast and completely. Thus the extraction yield of the effective ingredients is very higher. It is the first time to put forward that decompression velocity (decompression time) is also an important factor of influencing extraction yield in this paper. This viewpoint is perfection and supplement to the theory of highhydrostatic pressure extraction method. 2. To understand the mechanism of enhancing mass transfer by HHP expressly and demonstrate the feasibility of applying HHP processing to extracting flavonoids from propolis, we had studied the effect of high hydrostatic pressure on flavonoids components of propolis during extracting by high performance liquid chromatography. (1) The comparison between HHP processing and extraction at room temperature (ERT) was studied in this paper. We found that the flavonoids components by HHP processing are the same as those obtained by ERT and the extraction yield of flavonoids components does not decrease at 600MPa, which indicated HHP does not destroy the structure or molecule of the flavonoids components. The theoretic basis of applying HHP processing to extracting effective ingredients from propolis was received. (2) Through carrying out a large number of experiments, we found the extraction yield of flavonoids components increased significantly with the increase of pressure and only 1min was necessary in a complete extraction course, which indicated that high hydrostatic pressure can increase significantly the velocity of mass transfer and then shorten greatly the extraction time. This has proved from experiment it is feasible to apply HHP processing to extracting flavonoids components from propolis and an experimental basis for the research of processing technology of extracting effective components from propolis with the help of high hydrostatic pressure was offered. 3. The high hydrostatic pressure processing technology of extracting flavonoids from propolis was studied. (1) By many probing experiments, we had confirmed five factors influencing the extraction yield of flavonoids of propolis and performed single-factor experiments to study them in detail. Then we adopted the uniform design method to optimize test scheme and the optimum regression equation was received through experiments and data analysis. The equation was:232y =?0. 8+0.13x2 +0.03x3+0.000074x1x3+0.0015x2x3?0.0012x2?0.0025x y : The extraction yield of flavonoids(%, w/w); x1 : Pressure leve(lMPa);x 2: Ethanol concentration(%);x 3: Solid/liquid ratio(g/ml)。From the optimum regression equation, we could obtain the optimum process parameters, which were 500MPa pressure, 75% ethanol concentration, 1:35 solid/liquid ratio and 1min extraction time. Then the preferable process parameters could also be obtained. When pressure was from 450 to 500MPa, ethanol concentration was from 65% to 75% and solid/liquid ratio was from 1:28 to1:35(g/ml), the extraction yield of flavonoids was relatively higher. (2) Compared with the conventional extraction methods, we found the extraction yield of flavonoids of propolis with the help of HHP was more than that of extraction at room temperature by 8% and than that of heat reflux extraction by 17%. The extraction time of HHP was very short, only one minute. It proved that it was very efficient for us to extracting effective components from propolis with HHP processing and no other extraction method can qualify for it. (3) Compared with heat reflux extraction, HHP processing is operated at room temperature and the content of beeswax in the extracted solution is relatively lower. So it not only can reduce greatly the consumption of chemicals of separation and purification, shorten the time of equipment taken by the procedures of separation and purification and so on, but also can avoid some heat-sensitive components losing their biologic activity because of heating. 4. Studied contrastively on the antioxidant activity of the ethanol extracts of propolis (EEP) of different extraction methods. (1) The contents of flavonoids in the EEP by HHP processing and extraction at room temperature were relatively higher. The content of the EEP by extraction at room temperature was appreciably higher than that obtained by HHP treatment, and that by heat reflux extraction was the least.
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