原文题目：Concentration of flavonoids and phenolic compounds in aqueous and ethanolicpropolis extracts through nanofiltrationAbstractPropolis has a variable and complex chemical composition with high concentration of flavonoids 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. Nanofiltration 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 verified by spectrophotometric analysis: for aqueous solution, the membrane retained around 94% of the phenolic compounds and 99% of the flavonoids, 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 nanofiltration processshowed high efficiency 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 thissubstance, such as anti-microbial and antioxidant effects, anesthetic properties and others. Due to these characteristics, which can bring health benefits, 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 characterist ics is influenced 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 flavonoids 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 specific characteristics in the final product.The most common propolis extracting process uses ethanol as the solvent. However, this has some disadvantages such as the strong residual flavor, adverse reactions and intolerance to alcohol of some people (Konishi et al., 2004). Researchers and industry are interested 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 efficient 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 À20 °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 flavonoid 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 microfiltration, ultrafiltration, nanofiltration and reverse osmosis is mechanical pressure (Maroulis and Saravacos, 2003). Nanofiltration is a unit operation that permits many applications, such as solvent recovery from filtered oil, exchange of solvents in the che mical industry (Geens et al., 2006), concentration and purification of ethanolic extracts of xantophylls, which is important in both the pharmaceutical and food industries (Tsui and Cheryan, 2007) and in wine 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 flavonoids and phenolic compounds during processing. The process was evaluated according to the permeate flux, influence 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 São 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 underrefrigeration (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 Streptococcus mutans and greater anti-inflammatory activity than samples from other parts of the country, which can be associated with the higher concentrations of flavonoids 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 filtered and refrigerated for 3 h at 4 °C and then filtered 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 flavonoid and phenolic compounds contents, to be compared with the concentrated products.2.2. Determination of total flavonoidsThe total flav onoid 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 flavonoid 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 Folin–Ciocalteau 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 Folin–Ciocalteau reagent and 0.5 mL of 10% Na2CO3. The absorbance was readat 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 flow 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 flavonoids (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 filtration system on a pilot scale, with a nanofiltration 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 nanofiltra tion module is equipped with a NF90 membrane (Osmonics, Minnetonka, USA) which is composed of polyamide and polysulphone, with 0.6 m2 of filtration area and 98% rejection of MgSO4 ina test performed by manufacturing with a spiral module at 20 °C and 6.0 bar. Approximately5.0 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 permeatewas 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 nanofiltration unit where 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 (20–45 °C) and pressures (2.0–5.0 bar), in order to evaluate the influence of these parameters on the permeate flux and the concentrated product quality. In these experiments, both the permeate and retentate were maintained under re-circulation in closed systems. The permeate flux was calculated acco rding 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 filtration process was measured according to the quantity of flavonoids and phenolic compounds present in permeate, evaluated as described in Sections 2.2–2.4, and the efficiency was measured according to the flux 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 experimentalconditions. 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 flux of the solution under study with the permeate flux when water is used as feed solution, under different pressures. Usually a variation in system pressure will cause a change directly proportional to the perme ate flux. The fouling influence was measured by comparison of the permeate flux of the aqueous propolis extract with the flux of distilled water only, increasing the pressure from 1.0 to 5.0 bar.3. Results and discussionThe 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 flux 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 flux began to decrease, after around 15 min of processing. The rate of decrease was higher for the alcoholic extract than for theaqueous extract, evidencing a greater rate of fouling with the alcohol solution. After 20 min of processing, the permeate flux tended to stabilize, that is, concentration polarization already occurred and fouling did not increase with time. The permeate flux in the stable region was about 12.0 L/h m2and 25.0 L/h m2for alcoholic and aqueous solutions, respectively. The difference between the permeate flux 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 difficult to achieve, and this reduces the flux rate. Some of these compounds form a kind of wax which can cause more fouling in membrane.Tsui and Cheryan (2007) used nanofiltration to puri fy alcoholic corn extracts in the production of xanthophylls, and obtained a permeate flux of around 10.0 L/h m2when working at 27 bar and 50 °C. Hossain (2003) studied the membrane concentration of anthocyanins from blackcurrant pomace extracts using ultr afiltration, obtaining a maximum permeate flux of 17.3 L/h m2 at 1.4 bar and 18 °C. Using nanofiltration a permeate flux 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 flux 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 nanofiltration processes, but was sufficient to carry out this concentration procedure.Fig. 3 shows the difference between the curve of the permeate flux for the aqueous propolis extract and the curve of the permeate flux for distilled water to measure the degree of fouling in the process with the aqueous propolis solution.The difference between the permeate fluxes 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 flux wasaffected by a pressure variation, showing t hat the flux changed linearly with the pressure in the region studied. In this pressure range of operation, the concentrated products did not present significant variation among the experiments.By increasing the temperature from 20 to 45 °C and maintaining the pressure at 6.0 bar it was possible to determine the relation ship between the temperature and the permeability of the mem brane. Permeate flux increased proportionally, by around 8% per degree of temperature, as shown in Fig. 4. This result may be attributed to the effect of temperature on the viscosity of the solution. Also, the composition of the concentrated products obtained at different temperatures showed no significant difference among them, thus, a higher flux can be obtained by increasing the temperature.The initial solutions, permeates and concentrates obtained by nanofiltration in open system were all subjected to a spectrophotometric analysis as described in Sections 2.2 and 2.3. The results for the aqueous solution indicated that this permeate only contained small amounts of phenolic compounds and flavonoids, while the permeate from the ethanolic solution showed greater amounts, mostly of low molar weight phenolic compounds. Considering the losses in the compounds of interest in the resulting permeate, as compared to the initial solution, it can be seen that the aqueous solution of propolis retained almost 99% of the flavonoids and 84% of the phenolic compounds. However, for the ethanolicsolution, these values were 90% and 53% for the fla vonoids and phenolic compounds, respectively,as shown in Table 1. The lower retention obtained can be explained by the occurrence of plasticization in the case of ethanolic solution. This phenomenon can cause a membrane swelling or dilation, which in turn can increase the membrane diffusivity and solubility, causing loss of compounds in process.The results for the determination of flavonoids and phenolic compounds carried out by spectrophotometric methods were verified by HPLC analysis, as described in Secti on 2.4. The substances were identified by a comparison of their retention times and ultraviolet spectra with those of standards in the literature. Chromato grams were obtained from the initial aqueous extract, and from the concentrated and permeated products, which are represented in Fig. 5a–c, where the numbers 1–3 indicate the peaks identified in the HPLC analysis.Table 2 shows the results of the quantitative analysis for all samples from the aqueous propolis solution. Comparing these data, it can be seen that there were no losses of ferulic acid to the permeate and only 20% of the caffeic acid present in the initial solution was lost to the permeate, this compound thus being the most abundant in the concentrated extract, of the compounds identified. All the aqueous solutions showed peaks located in the region thatrepresents a retention time of up to 20 min. This occurred since the water, being a polar material, only extracts polar compounds. The last peak identified in the permeated solution, probably does not represent an isolated compound but interference in the system, since this compound was not present in the other chromatograms. Ferulic acid was not identified in the permeated solution, indicating that no losses to the permeate occurred. The other peaks in the chromatograms represented compounds that could not be identified according to the standards in the literature. The permeated solution showed a small amount of compounds in low con centration, allowing to verify the efficacy of the membrane concentration process.Park et al. (1998) analyzed an aqueous propolis solution prepared in the laboratory, using HPLC, and obtained similar results to those presented in Fig. 5a, reporting peaks with low retention times that represent polar substances, and identifying the compounds quercetin and pinocembrin. In their experiment, the proportions of water and alcohol in the solvents for propolis extraction were varied. Initial solutions contained 0–90% of alcohol, through which it was demonstrated that increasing the proportion of alcohol in the solution also increased the amount ofextracted flavonoids and phenolic compounds in the propolis.The chromatograms obtained for the ethanolic solutions are presented in Fig. 5d–f. It was possible to identify peaks in the ethanolic solution throughout the process. Compounds with a retention time greater than 20 min are apolar and were extracted by ethanol, this being an advantage of the use of this solvent as compared to water, which does not extract apolars. On analyzing the chromatogram it can be seen that a considerable amount of cumaric acid was lost to the permeate (peak number 2 in Fig. 5f). This acid belongs to the phenolic acid class and has a low molar weight, which could explain the low retention capacity of the membrane for this compound. The results of the spectrophotometric analysis shown in Table 1 indicate a loss of phenolic compounds to the permeate, accounting for the cumaric acid and other compounds not being identified by the HPLC method. Using the data given in Tables 2 and 3 the retention index could be calculated from Eq. (2). These results can be observed in Table 4. The retention indexes verified that the membrane process retained the compounds of interest better when using aqueous solutions, since this resulted in smaller losses of the compounds studied to the permeate, as compared to such losses when working with the alcoholic solution. Cumaric acid was an exception, since only 56% of this compound was not lost to permeate. Despite the loss of compounds, the values obtained represent a high retention index and verify that the nanofiltration process is appropriate for propolis extracts.During their study on the concentration of red wine by nanofiltration, Banvolgyi et al. (2006) obtained a retention index of 88% for total acids, 50% for free sulfuric acids and 93% for the total extracts. Tsui and Cheryan (2007), working with the purification of xantophylls by nanofiltration obtained a retention index of 90% for total solids, 88% for proteins and 98% for xanthophylls, the major compound of their study.In the present study, the values obtained for the retention indexes were very similar tothose cited in the literature for similar processes..4 ConclusionsThe results showed that nanofiltration can be considered as a good alternative for concentrating propolis extracts, since the membrane retained most of the flavonoids and phenolic compounds, which are of major importance to propolis quality. Particularly in the case of the aqueous extract, it could be considered that there was no loss of compounds to the permeate solution, since almost 100% of the major compounds were retained. In the experiments with alcoholic propolis, the losses were considerable but this is a consequence of the higher amount of compounds extracted by alcohol. However, the method can be used for alcoholic solution since almost 90% of the flavonoids were retained. Application of this technology could increase the use of propolis in many industrial applications, it being feasible to use the aqueous extract in new research projects and in the development of new products with functional properties. Furthermore, this process allows removal of the solvent from the extract, reducing the disadvantages associated with using alcoholic extractions. It should be noted that compared with other concentration methods, in the membrane process the product is not submitted to high temperatures and there is no change in the physical state of the solvent, which means that the functional properties of the compounds of interest are preserved and the process as a whole is energy saving.译文题目：黄酮类化合物和酚类化合物在水中和乙醇蜂胶中通过纳滤膜的提取率关键词:蜂胶膜浓度黄酮类化合物酚类化合物纳滤摘要：蜂胶具有可变的、复杂的化学成分，且蜂胶中黄酮类化合物的浓度较高，酚类化合物存在于蜂胶萃取物。
