应用化学 外文翻译 药物化学

原 文题目： Concentration of avonoids and phenolic compounds in aqueous and ethanolicpropolis extracts through nanoltrationAbstract Propolis has a variable and complex chemical composition with high concentration of avonoids andphenolic compounds present in the extract. The extract varies with the solvent used in extraction. Ethanol extracts more phenolic acid and polar compounds than water. Before their use in industry, extracts must be concentrated but the use of high temperatures can degrade some compounds. Membrane pro-cesses is an option that allows concentration at low temperatures. Nanoltration was carried out withaqueous and ethanolic extracts and each extract results in two distinct fractions: permeate and retentate. The capacity of the membrane to retain the compounds was veried by spectrophotometric analysis: for aqueous solution, the membrane retained around 94% of the phenolic compounds and 99% of the avonoids, while for the ethanolic solution these values were 53% and 90%, respectively. Ferulic acid retentionindex was 1.00 and 0.88 to aqueous and ethanolic solutions, respectively. Thus, the nanoltration processshowed high efciency in the concentration of propolis extracts.1 IntroductionOver the last few decades, interest in functional foods has beengrowing fast, leading to the discovery of new functional components or processes that can improve food processing, as well as products that may help to retard aging or avoid diseases. In this context, bee products have gained the attention of consumers and researchers, due to their chemical compositions and functional properties. Propolis is one of the bee products with functional properties, but it cannot be consumed as a food because it is a resinous substance. It is prepared from the buds and exudates of certain trees and plants. These substances are transformed by the addition of wax and the enzyme glucosidase present in the bee saliva in order to form propolis (Bankova et al., 2000; Park et al., 1998). The product obtained is used by honeybees to protect the hives against invaders and contamination, to seal holes and to maintain the temperature. Some important characteristics have been reported for this substance, such as anti-microbial and antioxidant effects, anesthetic properties and others. Due to these characteristics, which can bring health benets, propolis is considered a functional ingredient and is used in food, beverages, cosmetics and medicine to improve health and prevent diseases (Burdock, 1998; IFIC, 2009). There are over 150 constituents in propolis, including polyphenols, terpenoids, steroids and amino acids. Flavonoids are one of the most important groups and can represent around 50% of the propolis contents,depending on the region where it is collected, since its characteristics is inuenced by plants and weather (Krell, 1996; Park et al., 1998). Kumazawa et al. (2004) tested the antioxidant activity of propolis from various geographic origins and showed different activities for each sample. Other studies indicated that the propolis from Europe and China contained many kinds of avonoids and phenolic acids, whereas the Brazilian samples had more terpenoids and prenylated derivatives of p-cumaric acid (Bankova et al., 2000). Finally, each combination of compounds in the propolis of acertain origin can represent specic characteristics in the nal product.The most common propolis extracting process uses ethanol as the solvent. However, this has some disadvantages such as the strong residual avor, adverse reactions and intolerance to alcohol of some people (Konishi et al., 2004). Researchers and industry areinterested in producing a new type of extract with the same compounds extracted by the ethanolic method, but without the disadvantages. Water has been tested as the solvent, but resulted in a product containing less extracted compounds (Park et al., 1998). Konishi et al. (2004) tested water with a combination of some tensoactive compounds to replace part of the alcohol used in propolis extraction and all the tests were efcient in extracting it, and the product showed good anti-microbial activity.Depending on the application, the solvent in propolis extracts must be reduced or eliminated. The processes that are used today, as lyophilization, vacuum distillation and evaporation, have some disadvantages like the use of high temperatures and high energy consumption. Lyophilization requires large amounts of energy, since the sample needs to be maintained at 20 C for at least 24 h, and energy is also required for the sublimation of the solvent used during preparation of the extract. Moreover, the method often requires a previous stage of concentration, maintaining the product at 70 C until part of the solvent is evaporated.Vacuum distillation requires great amounts of energy to generate the vacuum, and can lead to loss of compounds of low molecular weight, which can be removed together with the solvent evaporated in the system. Evaporation maintains the extract underheating at 70 C, until all the solvent is removed. This process, in addition to the high demand for energy, can degrade the avonoid and phenolic compounds in propolis, due to the temperature used. However, it is the process that gives greater ease of implementation in companies due to low cost on the equipment required compared with the previous cases. The use of membrane concentration processes has been growing due to certain advantages, such as: low temperatures, absence of phase transition and low energy consumption (Matta et al., 2004). This procedure is based on the principle of selective permeation of the solute molecules through semi-permeable, polymeric or inorganic membranes. The driving force for mass transfer across the membrane in most membrane processes, such as a microltration, ultraltration, nanoltration and reverse osmosis is mechanical pressure (Maroulis and Saravacos, 2003). Nanoltration is a unit operation that permits many applications, such as solvent recovery from ltered oil, exchange of solvents in the chemical industry (Geens et al., 2006), concentration and purication of ethanolic extracts of xantophylls, which is important in both the pharmaceutical and food industries (Tsui and Cheryan, 2007) and inwine concentration (Banvolgyi et al., 2006), as well as in juice concentration (Vincze et al., 2006) in the food industry.The objective of this study was to investigate the membrane concentration of propolis extracts using water and ethanol as the solvents, exclusively, verifying the quality of the concentrated products in terms of the retention of avonoids and phenolic compounds during processing. The process was evaluated according to the permeate ux, inuence of temperature and pressure and concentration factor. The results obtained for each solution were compared to verify the viability of developing a new propolis product, based on water as the solvent.2. Materials and methods2.1. PropolisRaw propolis was obtained from Apis mellifera beehives in the State of So Paulo, Brazil, and was acquired in a single batch, in order to minimize the variability associated with the vegetation used for its production and the weather conditions. It was stored under refrigeration (4 C) until use in the preparation of extracts.The propolis produced in this region is characterized as group 12 (Brazil has 12 different groups of propolis, with distinct characteristics) and presents a great amount of soluble substances, antimicrobial activity against Staphylococcus aureus and Streptococcusmutans and greater anti-inammatory activity than samples from other parts of the country, which can be associated with the higher concentrations of avonoids and phenolic compounds found in this group (Park et al., 2002).The ethanolic propolis solution was prepared from crude propolis previously comminuted in a bench blender with a 500 W motor, homogenized, weighed on a semi-analytical balance and mixed with 80% ethanol. The mixture was kept at room temperature for 7 days and manually stirred once a day. After this period, the sample was centrifuged (Beckman Allegra 25-R, Beckman Coulter, German) at 8800 g for 20 min. The supernatant was ltered and refrigerated for 3 h at 4 C and then ltered again for wax removal. Finally, the esulting extract was stored at room temperature in the dark.Preparation of the aqueous solutions followed the same procedures, using deionized water. Each solution was prepared in a proportion of 20% propolis and 80% solvent. Both extracts were evaluated with respect to their avonoid and phenolic compounds contents, to be compared with the concentrated products.2.2. Determination of total avonoidsThe total avonoid content of the propolis solutions was determined by the aluminum complexation method (Marcucci et al., 1998). In this procedure, the extracted solutions were diluted in the proportion of 1:10 (0.5 mL) and mixed with 0.1 mL of 10% aluminum nitrate, 0.1 mL of 1 mol/L potassium acetate and 4.3 mL of 80% ethyl alcohol. The samples were kept at room temperature for 40 min and the absorbance read at 415 nm. Quercetin was used as the standard to produce the calibration curve. The mean of three readings was used and the total avonoid content expressed in mg of quercetin equivalents (mg/g).2.3. Determination of the phenolic compoundsThe polyphenols in the propolis solutions were determined by the FolinCiocalteau colorimetric method (Kumazawa et al., 2004). According to this procedure, the extracted solution was previously diluted in the proportion of 1:10 (0.5 mL) and then mixed with 0.5 mL of the FolinCiocalteau reagent and 0.5 mL of 10% Na2CO3. The absorbance was read at 760 nm after 1 h of incubation at room temperature. Gallic acid was used as the standard to produce the calibration curve. The mean of three readings was used and the total phenolic content expressed in mg of gallic acid equivalents (mg/g).2.4. HPLC determinationThe compounds present in the initial extract, permeate and concentrated products, were determined by HPLC as described by Parket al. (1998). Three hundred microliters of each solution were injected into a liquid chromatograph (Shimadzu, Tokyo, Japan) connected to a diode-array detector at 260 nm. The mobile phase was water/acetic acid (19:1, v/v) (solvent A) and methanol (solvent B), with a constant ow rate of 1 mL/min. The gradient started at30% solvent B, passing to 60% at 45 min, 75% at 85 min, 90% at 95 min and back to 30% at 105 min. The column was maintained at a constant temperature of 30 C and the chromatograms processed using the computer software Chromatography Workstation(Shimatzu Corporation, Tokyo, Japan). The initial and concentrated samples were diluted in 1.5 mL of distilled water and the permeate sample was injected without dilution. The following authentic standards of phenolic acids and avonoids (Extrasynthese, Genay, France) were examined: q-cumaric acid, ferulic acid, cinnamicacid, gallic acid, quercetin, kaempferol, kaempferide, apigenin, isorhamnetin, rhamnetin, sakuranetin, isosakuranetin, hesperidin, hesperetin, pinocembrin, chrysin, acacetin, galangin, myricetin, tectochrysin and artepillin C, as they correspond to the most usual compounds present in propolis.2.5. Membrane concentrationIn this study, the propolis extracts were concentrated using a tangential ltration system on a pilot scale, with a nanoltration membrane as seen in the schematic diagram shown in Fig. 1. The experiments were performed on pilot equipment that permits the batch circulation mode, which means that both permeate and concentrate could be carried back to the feed tank. The permeate was totally removed just in a single experiment, where it was necessaryto obtain the concentrated product of the process. The nanoltra tion module is equipped with a NF90 membrane (Osmonics, Minnetonka, USA) which is composed of polyamide and polysulphone, with 0.6 m2 of ltration area and 98% rejection of MgSO4 in a test performed by manufacturing with a spiral module at 20 C and 6.0 bar. Approximately 5.0 L of each solution permeated through the membrane over 30 min, this being the time necessary to complete the concentration in an open system, which means that the permeate was removed from the process. In the trials the permeate was removed and the retentate re-circulated until a concentration factor of around four. The concentration factor is calculated according to Eq. (1):Fig. 1. Schematic diagram of the nanoltration unitwhere Vf is the total volume used in the feed, Vc is the volume collected in the concentrate fraction and Fc is the concentration factor. Other experiments were carried out at different temperatures (2045 C) and pressures (2.05.0 bar), in order to evaluate the inuence of these parameters on the permeate ux and the concentrated product quality. In these experiments, both the permeate and retentate were maintained under re-circulation in closed systems. The permeate ux was calculated according to the following equation: J=Vp/t*Ap (2)where Vp is the permeate volume collected during the time intervalt and Ap is the membrane surface area of permeation. The quality of the ltration process was measured according to the quantity of avonoids and phenolic compounds present in permeate, evaluated as described in Sections 2.22.4, and the efciency was measured according to the ux permeate rate and retention index. This index measures the relation between the amounts of the compound of interest in permeate and in the concentrated solution, which demonstrates the ability of the membrane to retain this compound under the experimental conditions. The index is calculated according to Eq. (3), in which R is the retention index, Cp is the concentration of the compound of interest in the permeate, and Cr is the concentration of the same compound in the retentate:R=1-Cp/Cr (3)It is important to know the rate of fouling that occurs in the membrane process, and one way of measuring this is to compare the permeate ux of the solution under study with the permeate ux when water is used as feed solution, under different pressures. Usually a variation in system pressure will cause a change directly proportional to the permeate ux. The fouling inuence was measured by comparison of the permeate ux of the aqueous propolis extract with the ux of distilled water only, increasing the pressurefrom 1.0 to 5.0 bar.3. Results and discussion The membrane process was carried out with the aqueous and ethanolic solutions in a closed system, in which the retentate and permeate streams being conducted back and mixed in a feed tank isolated from the environment, to evaluate the variation in permeate ux with time. The temperature was maintained at 20 C and the pressure at 5.0 bar. The results are shown in Fig. 2.After stabilization of the process, the permeate ux began to decrease, after around 15 min of processing. The rate of decrease was higher for the alcoholic extract than for the aqueous extract, evidencing a greater rate of fouling with the alcohol solution. After 20 min of processing, the permeate ux tended to stabilize, that is, concentration polarization already occurred and fouling did not increase with time. The permeate ux in the stable region was about 12.0 L/h m2 and 25.0 L/h m2 for alcoholic and aqueous solutions, respectively. The difference between the permeate ux of these solutions can be explained by their different compositions: the alcohol extract contains more compounds of low molecular weight, thus its concentration is more difcult to achieve, and this reduces the ux rate. Some of these compounds form a kind of wax which can cause more fouling in membrane.Tsui and Cheryan (2007) used nanoltration to purify alcoholic corn extracts in the production of xanthophylls, and obtained a permeate ux of around 10.0 L/h m2 when working at 27 bar and 50 C. Hossain (2003) studied the membrane concentration of anthocyanins from blackcurrant pomace extracts using ultraltration, obtaining a maximum permeate ux of 17.3 L/h m2 at 1.4 bar and 18 C. Using nanoltration a permeate ux of 20 L/h m2 was obtained at 20 bar and 50 C in the concentration of red wine (Banvolgyi et al., 2006). The red wine concentration process is important since it can be considered a similar process to the concentration of alcoholic propolis, considering that they have similar compounds in solution and use alcohol as the solvent. The similarities between the processes allow a comparison between results. Low pressures (around 6 bar) were used in the propolis concentration process, when compared to other processes cited in the literature, but even so the values obtained for the permeate ux were similar to those obtained in the other processes. Therefore this process can be assumed to be viable, mainly because of the reduced energy requirements necessary to generate the lower pressure.The pressure adopted was not characteristic of nanoltration processes, but was sufcient to carry out this concentration procedure.Fig. 3 shows the difference between the curve of the permeate ux for the aqueous propolis extract and the curve of the permeate ux for distilled water to measure the degree of fouling in the process with the aqueous propolis solution.The difference between the permeate uxes of water and the propolis solution shows the amount of fouling in the process, under the same conditions of temperature and pressure. This parameter increased, reaching 32% at 5.0 bar. The procedure also provided information on how the ux was affected by a pressure variation, showing that the ux changed linearly with the pressure in the region studied. In this pressure range of operation, the concentrated products did not present signicant variati