Lipase-mediated lipid removal from propolis extract and its antiradical and antimicrobial activity Hyein Parka, Song Hwan Baeb, Yooheon Parka, Hyeon-Son Choia,*, Hyung Joo Suha,*

a

Department of Food and Nutrition, Korea University, Seoul 136-703, South Korea

b

Department of Food and Biotechnology, Hankyong National University, Anseong, Anseong

456-749, South Korea

*Corresponding authors. Tel.: +82 2 940 2764; Fax: +82 2 940 2859. E-mail address: [email protected] (H.J. Suh), [email protected] (H.S. Choi)

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jsfa.6874 This article is protected by copyright. All rights reserved

Abstract BACKGROUND: Propolis contains many antioxidants such as polyphenols and flavonoids. However, propolis–derived lipid components interrupt an efficient isolation of antioxidants from propolis extract. We examined the effectiveness of various lipase treatments for the removal of lipids from propolis extract, and evaluated the biological features of the extract.

RESULT: Lipase OF and Novozyme 435 treatments did not reduce fatty acid level in propolis extract. However, the Lipozyme TL IM-treated propolis extract showed a significant decrease of fatty acid level, suggesting the removal of lipids. Lipozyme RM IM also significantly decreased the fatty acid level of the extract, but the reduction of polyphenols and flavonoids, which are antioxidants, was accompanied. In Lipozyme TL IM treatment, an increase of active flavonoids, such as Artepillin C and kaempferide, was observed with a slight increase of ferric reducing/antioxidant power (FRAP) radical scavenging activity. In addition, antimicrobial activity toward skin health-related bacteria such as Staphylococcus epidermidis and Propionibacterium acnes was enhanced by Lipozyme TL IM treatment.

CONCLUSION: Lipozyme TL IM treatment effectively removes lipids from propolis extract and enhances antibacterial activity. Therefore, we suggest that Lipozyme TL IM is a useful lipase for lipid removal of propolis extract.

Keywords: propolis, lipid removal, Lipozyme TL IM, antimicrobial effect

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INTRODUCTION Propolis is a brownish, resin-like material produced by honeybees. It is a natural product that is collected from the exudates and buds of living plants to form a mixture of wax and bee enzymes. Raw propolis usually consists of 50% resin and vegetable balsam, 30% wax, 10% essential and aromatic oils, 5% pollen, and 5% organic debris.1 Propolis has been used for medicinal purposes, such as the treatment of wounds and tumors, throughout history. In ancient Egypt, it was used for mummification. Propolis still receives credit for its beneficial effects on various biological functions today. Many studies have shown that propolis has varied physiological effects including anticancer, antioxidant, antibacterial, anti-inflammatory, and antiviral activities.2-6 For this reason, propolis is widely used as a popular remedy in folk medicine. It is available, either in refined form or combined with other natural products, in cosmetics and health foods.7 The biologically active constituents of propolis are polyphenols, and aromatics, with flavonoids considered to be the most active. However, the application of propolis to food has been limited owing to its low water solubility, which is attributed to the lipid components, such as beeswax and hydrophobic resins, in raw propolis. Therefore, the efficient extraction and isolation of active compounds from propolis requires the removal of beeswax and resin. However, physical removal of propolis lipids can cause the loss or low recovery of active components from propolis. To enhance the yield of active compounds, use of strong hydrophobic solvent should be accompanied, but it also makes difficult to recover active compounds from the propolis extract. At present, the adoption of enzymes in food processing is increasing. In particular, chemical interesterification is a widely used technology in the food industry. The use of lipases as biocatalysts for the modification of triacylglycerols (TGs) has many potential benefits for the development of novel lipids that reflect the specificity of the lipases. Several

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studies have reported the production of TGs, which are valuable in nutrition, food, and pharmaceutical applications, via enzymatic processes.8, 9 In addition, Wang et al. 10 produced biodiesel from corn oil using immobilized Lipozyme TL IM. Lipase OF has been used to develop conjugated linoleic acid (CLA) isomers via selective esterification.11 A recent study obtained new fats containing fatty acids similar to those found in human milk from TGs using Lipozyme RM IM, a fungal lipase.12 However, the use of TGs and waxes in edible or mechanical oils has proven problematic.10, 12 Their use can cause turbidity in edible oils such as sunflower oil

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, and the wax in diesel fuel has often been found to form crystals below

certain temperatures that block fuel filters or lines.10 Winterization, a process to remove or reduce such a wax or TG content, has been used for the improvement of oil palatability and dewaxing of fuel oil, etc.9 It is usually achieved via filtration or centrifugation in solvent. Therefore, if winterization via enzymatic process were applicable to propolis containing large amounts of beeswax, bee propolis could be used much more widely in research and industry. In this study, we adopted an enzymatic winterization procedure to remove the lipid components of propolis extract by using lipases, which possess the ability, via esterification reactions, to reconstruct heavier lipid compounds. For this purpose, we used the following conventional lipases: Lipase OF, Lipozyme TL IM, Lipozyme RM IM, and Novozym 435. We examined the effect of lipase treatments for the removal of lipids from propolis extract, and evaluated the characteristics of the extract. MATERIALS AND METHODS Materials Raw propolis was collected in China. Ethanol was obtained from Daejung Chemical Co., Ltd (Chungbuk, Korea) and α-β-γ-cyclodextrin was from Bionic Trading Corporation (Ansan, South Korea). Lipase OF was from Meito Sangyo, Ltd (Nagoya, Japan), and Lipozyme RM

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IM, Lipozyme TL IM, and Novozym 435 were purchased from Novozymes (Bagsvaerd, Denmark). Standard flavonoids, including the compounds caffeic acid, p-coumaric acid, apigenin, galangin, chrysin, and catechin, were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA), kaempferide was purchased from INDOFINE Chemical Co., Inc. (Somerville, NJ, USA), and Artepillin C was obtained from Wako (Osaka, Japan). A GasPak EZ Anaerobe Pouch for the antibacterial assay was purchased from Becton, Dickinson and Company (Sparks, USA) and a sterilized 96-well cell culture plate was obtained from SPL Life Sciences Co., Ltd (Pocheon, Korea). Thioglycollate medium was from Difco, BD Biosciences (San Jose, CA, USA), and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), Folin–Ciocalteu phenol reagent, N-succinyl-(Ala)3-p-nitroanilide, and 2,4dinitrochlorobenzene (DNCB) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Basic extraction by water or ethanol with hexane pre-treatment Raw propolis, lyophilized after freezing at −80°C, was ground prior to hexane pretreatment and ethanol extraction. Samples were pre-treated with 100 mL of hexane (Daejung Chemical Co.; Chungbuk, Korea) by shaking at 140 rpm in horizontal water bath for 2 hr. Hexane fraction was discarded from centrifugation at 3000 rpm for 20 min, and samples were dried in the hood for overnight. Hexane pre-treated samples were extracted by incubation with 100 mL of absolute ethanol (Daejung Chemical Co.; Chungbuk, Korea) or water for every 5 g of crude propolis while shaking at 140 rpm for 2 h. The insoluble portion was then separated twice by centrifugation at 3000 rpm for 20 min. Enzyme treatment after ethanol extraction The enzymatic reactions were carried out at 50°C in a horizontal shaking water bath at 140 rpm for 2hr. Lipase OF, Lipozyme TL IM, Lipozyme RM IM, or Novozym 435 with an This article is protected by copyright. All rights reserved

enzyme mass concentration (w/v) of 5% of the substrate was added to propolis extract (40 mL). Enzyme treated propolis extracts were centrifuged at 3000 rpm for 20 min to remove formed insoluble parts. Each experiment was performed in triplicate and the results are expressed as mean values ± standard deviation. Gas chromatography (GC) analysis of total fatty acid contents The fatty acid methyl esters (FAMEs) were prepared by saponification and methylation

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and analyzed in a gas chromatograph (Varian 3900; USA) equipped with a Supelco-2560 column (length 100 m, internal diameter 0.25 mm, film thickness 0.2 μm), with helium carrier gas from 100–240°C at a rate of 3°C/min. The injection temperature and the flameionization detector temperature were set at 225°C and 270°C, respectively. Peak areas of the fatty acids were calculated as the percentage of total area, and the peaks were identified by comparing the retention times with those of a standard mixture of methyl esters. Determination of acid value The acid value was determined in accordance with the method described by Leibovitz and Ruckenstein.13 It was calculated by measuring the volume of 0.1 N potassium hydroxide solution required to turn the titration solution pink, and used in the following formula: Acid value (mg of KOH) = [(T − B) × 5.611 × F] / S

where T is the titration volume of 0.1 N KOH on the sample (mL), B is the titration volume of 0.1 N KOH on the blank (mL), S is the weight of the sample (g), and F is the factor of KOH solution.

Determination of total phenolic compounds and flavonoids

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The total polyphenol (TP) content was determined using the Folin–Ciocalteu method14 adapted to a micro scale. Distilled water (0.79 mL), appropriately diluted propolis extract (0.01 mL), and Folin–Ciocalteu reagent (0.05 mL) were added to a 1.5-mL Eppendorf tube and mixed. After exactly 1 min, 0.15 mL of sodium carbonate (20 g/100 mL) was added, and the mixture was mixed and stand at room temperature in a dark place for 120 min. The absorbance at 750 nm was read, and the total polyphenol concentration was calculated from a calibration curve (r2 = 0.9990), using gallic acid as the standard (50–800 mg L-1). The total flavonoid (TF) content was determined using the p-dimethyl amino cinnamaldehyde (DMACA) method15 adapted to a micro scale. Appropriately diluted propolis extract (0.2 mL) and 0.1% DMACA (1 mL) in 1 N HCl-containing methanol were added to a 1.5-mL Eppendorf tube, mixed, and left at room temperature for 19 min. The absorbance at 640 nm was measured. The DMACA reagent was prepared immediately before use. Absorbance values were calibrated using catechin as the standard (4–50 μg mL-1). High-performance liquid chromatography (HPLC) analysis of active flavonoid Levels of 7 major flavonoids were analyzed by HPLC using an Agilent HPLC system (Agilent; Arlington, MA, USA) equipped with a quaternary solvent delivery system, an autosampler, and an ultraviolet (UV) detector16 with an EC 250/4.6 NUCLEOSIL 100-5 C18 (250 × 4.6 mm, 5 μm; Macherey-Nagel GmbH & Co., KG, Germany) column. UV absorption was measured at 290 nm and 340 nm. Gradient elution was employed using solvent A (5% acetic acid) and solvent B (100% methanol) at 30°C. The following flavonoids were used as standards: caffeic acid, p-coumaric acid, apigenin, chrysin, galangin, kaempferide, and Artepillin C. Antioxidant study

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In vitro antioxidant activity of the propolis extracts was evaluated using an improved ABTS scavenging assay as described by Re et al..17 Lipase-treated or untreated extract was centrifuged and the supernatant was used for the assay. Briefly, a purple ABTS radical cation solution (7.00 mM) was prepared by mixing ABTS with potassium persulfate (2.45 mM) in H2O overnight in the dark at room temperature. The ABTS radical cation solution was diluted with ethanol or phosphate-buffered saline (PBS) (pH 7.4) to an absorbance (414 nm) of 1.40– 1.50 for assay. The extracts (50 mL) were added to 1 mL of the diluted ABTS radical cation solution, and after 90 min, the remaining amount of ABTS was determined at 414 nm using a spectrophotometer. The ferric reducing/antioxidant power (FRAP) assay was performed as described by Maksimović et al..18 Appropriately diluted extract (0.1 mL) was transferred into test tubes, and 3.0 mL of freshly prepared FRAP reagent (25 mL 300 mM acetate buffer, pH 3.6 + 2.5 mL 10 mM 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ) in 40 mM HCl + 2.5 mL 20 mM FeCl3·6H2O) was added. The absorbance at 593 nm was recorded after 5 min incubation at room temperature. A blank contained 0.1 mL of solvent instead of extract. Relative activities were calculated from the calibration curve of Iron

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sulfate heptahydrate standard solutions

(0.4–2 mM) under the same experimental conditions. All measurements were conducted in triplicate. Antimicrobial study of microbes harmful to the skin Propionibacterium acnes strain ATCC 6919 was grown under anaerobic conditions in an anaerobic gas-generating pouch (GasPak EZ; Becton, Dickinson and Company; Sparks, USA) in a CO2 incubator at 37°C. The cultures were incubated for 48–72 h on thioglycollate medium broth (Difco; BD Biosciences; San Jose, CA, USA). Staphylococcus epidermidis ATCC 12228 was cultured in the dark at 37°C. The cultures were incubated for 18–24 h on

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Mueller-Hinton broth (Difco; BD Biosciences; San Jose, CA, USA). Antimicrobial assays were carried out on a sterilized 96-well cell culture plate (SPL Life sciences Co. Ltd.; Pocheon, Korea). Prior to analysis, 230 µL of the medium was placed in the 96-well cell culture plate, and 10 µL of the microbes from the culture and 10 µL of the propolis ethanolic extract were added. The difference of absorbance was monitored for 48 h at 600 nm. Statistical analysis Data are expressed as means ± standard deviations (SD). Statistical analyses were performed using SPSS v12.0 (SPSS, Inc.; Chicago, IL, USA). Comparisons of results of all groups were performed using one-way analysis of variance (ANOVA) and Tukey’s multiple range tests, and significance was assessed at P < 0.05. RESULTS Basic extraction by water or ethanol Extraction of propolis was performed using water or 70% ethanol with or without hexane pre-treatment. The chemical composition and antioxidant activity of propolis extracts are shown in Table 1. Our data showed that the solid content was higher in the 70% ethanol extract than in the water extract, and hexane pre-treatment led to greater solid content in both water and 70% ethanol extracts than without pre-treatment. This result was also observed with respect to the levels of polyphenol and flavonoid, which were reflected in the solid content. In particular, the solid content and flavonoid levels in the ethanol extraction with hexane-pretreatment (HEE) group showed a 4-fold increase (12.34 ± 0.09 mg/mL) and a 9fold increase (457.77 ± 10.78 mg mL-1), respectively, compared with the levels (2.9 ± 0.14 mg mL-1 and 51.4 ± 0.95 mg mL-1) in the water extraction

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group. This result showed that

HEE is a more efficient method of propolis extraction compared with other methods.

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Moreover, the antioxidative effect (based on ABTS and FRAP) was enhanced in the hexane pre-treatment and 70% ethanol extract groups (Table 1), in which polyphenol and flavonoid levels were relatively increased compared with the other groups. The half maximal inhibitory concentration (IC50) value (0.36 ± 0.0 g) of HEE on ABTS was reduced by 2.7-fold compared with that of WE (1.08 ± 0.01 g), indicating that radical scavenging activity of HEE was much higher than that of WE. In addition, the FRAP value in the HEE group was 10-fold higher than that in the WE or water extraction with hexane-pretreatment (HWE) groups. These results showed that 70% ethanol extraction after hexane pre-treatment led to the highest antioxidative effect among the tested groups. The antioxidative effect was correlated to the chemical content, especially the polyphenol and flavonoid levels, observed in the tested groups. Effect of lipase treatment on solid content Since the HEE group showed the highest solid contents including polyphenols from propolis, we used HEE when investigating the effect of lipases on lipid removal and the characteristics of propolis extract. HEE extract was centrifuged before and after each lipase treatment to determine the effect of lipases on propolis chemical contents. The aqueous phase of the lipase-treated extract was dried by nitrogen purging to analyze dry weights, a process which has been referred to as relative removal of lipid in the propolis extract.19, 21 Treatment with Lipase OF and Lipozyme TL IM increased the dry weight of the propolis extract supernatant, whereas dry weights of Lipozyme RM IM- and Novozym 435-treated propolis extracts were slightly reduced (Fig. 1A). In particular, the dry weight of Lipase OF-treated propolis extract (39.24 mg mL-1) was increased by 1.8-fold compared with the control group (21.19 mg mL-1) (Fig. 1A). Our results showed that Lipase OF and Lipozyme TL IM promoted the release of solvent-soluble materials from propolis extract, suggesting that these

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lipases can hydrolyze the lipid materials in propolis extract, which can then be precipitated by centrifugation. By contrast, Lipozyme RM IM and Novozym 435 promoted the formation of lipids, leading to the decrease of dry weight in the supernatant. Effect of lipase treatment on acid value The amount of free fatty acid present in the propolis extract was determined using a modified Association of Official Analytical Chemists procedure,22 which uses ester bond breaking to represent lipase activity. Lipase OF-treated propolis extract had the highest acid value (53.52 mg of KOH) of all the groups (Fig. 1B). This result indicates that the lipid hydrolyzing activity of Lipase OF was stronger than that of the other lipases, correlating to the variation in dry weight. By contrast, treatment with Lipozyme RM IM or Novozym 435 decreased the acid value of propolis extract compared with the control, suggesting that these enzymes catalyze the esterification rather than hydrolysis of lipids in propolis, causing remobilization of the lipid. Treatment with Lipozyme TL IM resulted in an acid value that was similar to that of the control. Effect of lipase treatment on total fatty acid and palmitic acid contents We determined the effect of the lipases on the level of fatty acids in the lipase-treated propolis extract. Figure 2 shows the levels of palmitic acid and total fatty acid in the lipasetreated propolis extract. Palmitic acid, a major fatty acid in propolis wax, was used as the index of propolis lipid content based on previous reports.23 Lipase OF and Novozym 435 treatment slightly increased the palmitic acid level in the extract (Fig. 2B). The increase of palmitic acid with Lipase OF treatment correlated with the increase of solid content and acid value, showing that the enzyme hydrolyzed the lipids in the propolis extract to release fatty acids (palmitic acid). However, the total fatty acid contents of the lipase-treated extracts were not altered, and Novozym 435-treated extract showed a slight increase in total fatty acid and This article is protected by copyright. All rights reserved

palmitic acid levels (Fig. 2A and B). Treatment with Lipozyme RM IM or Lipozyme TL IM decreased the levels of total fatty acid and palmitic acid in the extract. In particular, the palmitic acid and total fatty acid levels of Lipozyme RM IM-treated extract were reduced by 20% and 15%, respectively, compared with the control. This result indicates that Lipozyme RM IM and Lipozyme TL IM esterify the fatty acids (palmitic acid). The fatty acid level in the Lipozyme RM IM-treated extract correlated with the dry weight and acid value, which were lower than in the control. However, the fatty acid level in the Lipozyme TL IM-treated extract decreased, although the extract had a higher dry weight compared with the control. This result suggests that the level of components other than fatty acids increased. Effect of lipase treatment on total polyphenol and flavonoid contents Polyphenols are known to be the active components in various biological activities of propolis 7. Therefore, we determined the effects of lipase treatment on the levels of total polyphenols and flavonoids in the propolis ethanol extract. Figure 4 shows the total polyphenol and flavonoids in the lipase-treated propolis extract. Lipase OF- and Lipozyme TL IM-treated propolis extracts showed no significant difference in levels of total polyphenols compared with the control (P < 0.05). However, treatment with Lipozyme RM IM or Novozym 435 decreased the level of total polyphenols in the propolis extract (Fig. 3B). This result indicates that Lipozyme RM IM removes polyphenols by esterification of fatty acids in the extract (Fig. 3B). Novozym 435 selectively decreased total polyphenols and flavonoids, which decreased the dry weight. Total flavonoids, unlike total polyphenols, were increased in Lipase OF and Lipozyme TL IM-treated extracts (Fig. 3A). In particular, the increase in total flavonoids in the Lipase OF group correlated with the increase of dry weight and acid value, suggesting that total flavonoids contribute to the increase in dry weight with Lipase OF. Lipozyme TL IM-treated extract showed a slight but insignificant increase in total

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flavonoids. These changes in the flavonoid content of the lipase-treated groups could affect the biological activities of the extracts. Accordingly, we examined the compositions of active flavonoids in the lipase-treated extracts using HPLC analysis (Table 2). Effect of lipase on active flavonoids Table 2 shows the active flavonoids in lipase-treated propolis. The total active flavonoid content was highest (1460.01 mg mL-1) in the Lipozyme TL IM-treated extract and was lowest in the Novozym 435-treated extract (1153.16 mg mL-1). Total flavonoids in the control, Lipase OF, and Lipozyme RM IM extracts were 1442.93 μg/mL, 1434.28 μg mL-1, and 1277.73 μg mL-1, respectively. Artepillin C and kaempferide were at their highest levels in the Lipozyme TL IM group and their lowest levels in the Novozym 435 group, compared with the other groups. Caffeic acid and p-coumaric acid levels were greatly reduced in the Lipase OF-treated extract. Our data show that lipase treatment can alter the flavonoid composition, and Lipozyme TL IM treatment leads to desirable changes in the active flavonoid content of the extract. Effect of lipase treatment on antioxidant effects The data reported above revealed changes in the flavonoid composition after lipase treatment. Accordingly, we determined antioxidant activity in lipase-treated extracts. Antioxidant activities of the sample groups based on ABTS and FRAP assays are shown in Fig. 3(C and D). Lipase OF and Lipozyme TL IM treatments did not significantly change the IC50 value according to the ABTS assay, compared with the control (Fig. 3C). This means there was no difference in the radical scavenging activity after treatment with either enzyme. By contrast, treatment with Lipozyme RM IM or Novozym 435 increased the IC50 values (0.40 mg mL-1 and 0.28 mg mL-1, respectively) according to the ABTS assay (Fig. 3C). This shows that Lipozyme RM IM and Novozym 435 treatments lead to a reduction in radical This article is protected by copyright. All rights reserved

scavenging activity, which correlates with the flavonoid-reducing effect of these enzymes. FRAP activity was increased by Lipase OF (0.56 mmol g-1) and Lipozyme TL IM (0.52 mmol g-1) treatments (Fig. 3D), although there was no significant difference between the control and Lipozyme TL IM values. Lipozyme RM IM and Novozym 435 treatments slightly decreased FRAP activity in the propolis extract (Fig. 3D). These results show that Lipase OF and Lipozyme TL IM treatments can enhance radicalscavenging activity in propolis, which may be explained by the increased amount of antioxidant compounds, especially flavonoid compounds. Effect of lipase treatment on antimicrobial effects One of the best-known biological characteristics of propolis is its antimicrobial activity. We determined the antimicrobial activity of lipase-treated extracts on skin health-related bacteria, such as Staphylococcus epidermidis and Propionibacterium acnes (Fig. 4). The Lipozyme TL IM-treated extract showed a significantly lower minimum inhibitory concentration (MIC) value against S. epidermidis, a resident skin microbe, compared with the control (Fig. 4A). This result shows that Lipozyme TL IM treatment enhances the inhibitory effect against S. epidermidis. By contrast, Novozym 435 treatment resulted in an increase in the MIC against S. epidermidis (Fig. 4A), revealing that Novozym 435 decreases antibacterial activity in propolis extract. In P. acnes-associated skin inflammations, such as acne or corneal ulcers, Lipozyme RM IM and Lipozyme TL IM treatments significantly decreased the MIC values by 1.2- and 1.6-fold, respectively, but the MIC values of Lipase OF- and Novozym 435-treated extracts were similar to that of the control (Fig. 4B). This result shows that Lipozyme RM IM and Lipozyme TL IM treatments increase the inhibitory effect against P. acnes. Lipozyme TL IM showed an inhibitory effect against the two tested bacteria.

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Therefore, our data suggest that Lipozyme TL IM treatment could increase the antimicrobial activity of propolis extract.

DISCUSSION Propolis has received a great deal of attention owing to its medicinal properties, such as its antimicrobial activity, which is resulted from the active polyphenols. However, its use has been problematic because of its insolubility and high wax content, which can disturb efficient extraction of active compounds. In particular, physical separation of lipids from propolis is simple but has some limitations due to the loss or low recovery of active polyphenols. In this study, we attempted to remove the lipids from propolis extract using lipases combined with hexane treatment, and examined the effects of lipase treatment on the extract characteristics. Before lipase treatment, we examined the effect of hexane treatment, and found that it improved extraction efficiency and the levels of chemical constituents. Hexane pre-extraction is recognized to facilitate the removal of wax and resin while retaining active components, and subsequent ethanol extraction can be effectively used to obtain active constituents of propolis, which is mostly composed of lipid-soluble materials. Lipases are known to facilitate both lipid hydrolysis (esterase activity) and esterification (lipid remobilization).24 The Lipozyme TL IM-treated extract showed a decrease in total fatty acid and palmitic acid levels compared with the control (Fig. 2), unlike the results for dry weight, which showed a significant increase (Fig. 1A). This result indicates that Lipozyme TL IM increases lipid esterification, which decreases fatty acid content and increases some other components, including flavonoids (Figs. 2 and 3). Zhang et al.20 reported esterification of ascorbyl acetate by immobilized Lipozyme TL IM, which is well known as an enzyme for synthesizing biodiesel via esterification.25 In addition, Lipozyme TL IM treatment improved

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the antibacterial activity toward S. epidermidis and P. acnes, which affect skin health (Fig. 4), and increased radical scavenging activity (Fig. 3D). This result indicates that Lipozyme TL IM treatment favorably affects the propolis extract by reducing fatty acids content but increasing flavonoid levels and biological functions such as radical scavenging and antibacterial activity. By contrast, Novozym 435 treatment increased the fatty acid level in the extract but decreased the solid content, acid value, total polyphenol, and total flavonoid levels. Therefore, this enzyme is considered to be unsuitable for propolis extraction. The slight increase in the radical scavenging activity of Lipozyme TL IM treatment (Fig. 3D) could have been resulted, in part, from the increased levels of flavonoids (Table 2). Torres et al.29 reported that the antioxidant effect of resveratrol was improved by lipasecatalyzed acylation. However, our ABTS and FRAP assays revealed that the lipase treatments did not show any clearly enhanced antioxidative effects in ABTS assays. Our antioxidative analyses may not be sufficient to determine the full effect of lipase treatment. We suggest that other assays including cell line–based assays be used to further investigate the antioxidative effect of lipase treatments on propolis extract. Moreover, higher levels of total flavonoids do not always seem to guarantee higher radical scavenging activity. Kumazawa et al. 26 reported that levels of specific flavonoids determined radical scavenging activity. They showed that 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity depends on the levels of caffeic acid and ferulic acid, regardless of total flavonoid levels. Artepillin C and kaempferide, which were increased by Lipozyme TL IM treatment (Table 2), are known to be active biological compounds. Shimizu et al. reported that Artepillin C reduced oxidative stress in vivo and had an anti-inflammatory effect.27 Kaempferide has antimicrobial effects on skin infection-related microbes such as Staphylococcus aureus.28 The levels and compositions of polyphenols and flavonoids in propolis vary with the place of

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origin, the species of bee, and peripheral environmental factors, among others. A recent study showed large variation in polyphenol and flavonoid levels, depending on region.26 Our data show that Lipozyme TL IM treatment induces the release of active flavonoids from propolis, which can be responsible for its antimicrobial effects (Fig. 4). The antibacterial effects of propolis are usually observed in Gram-positive bacteria, such as the bacteria we tested.30 Glinnik and Gapanovich30 reported that propolis was effective against S. aureus and S. epidermidis. Moreover, Shub et al.31 reported that antibiotic-resistant strains were inhibited by propolis. Aga et al.32 isolated derivatives of hydroxycinnamic acid and benzopyran as antimicrobial compounds from Brazilian propolis. However, the antimicrobial mechanistic mode of action of propolis is not similar to the action of antibiotics, and has not been properly investigated. Takaisi-Kikuni et al.33 reported that propolis inhibited bacterial growth by suppressing cell division. They also found that propolis destroyed the cytoplasmic membrane and the cell wall of bacteria, thus causing bacteriolysis and inhibition of protein synthesis. Therefore, the antibacterial mechanism is considered to be complex. In conclusion, we investigated lipase treatments and their effective use in propolis extraction. Our data show that Lipozyme TL IM lowers the level of fatty acids, which can interfere with the extraction of active components, and increases the level of flavonoids. Moreover, Lipozyme TL IM-treated extract has improved radical scavenging activity and antibacterial effects. Therefore, we suggest that Lipozyme TL IM is a useful lipase for extraction of propolis active components . REFERENCES 1.

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Figure legends Figure 1. Dry weight (A) and acid value (B) of supernatant obtained after lipase treatment of propolis extract. Values are means ± standard deviations (SDs). Means with different superscript letters are significantly different at P < 0.05 based on Tukey’s multiple range tests. Control; propolis 70% ethanol extract.

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Figure 2. Total fatty acid (A) and palmitic acid (B) contents of supernatant obtained after lipase treatment of propolis extract. Values are means ± standard deviation (SDs). Means with different superscript letters are significantly different at P < 0.05 based on Tukey’s multiple range tests. Control; propolis 70% ethanol extract. Figure 3. Total flavonoid (A), total polyphenol (B), radical scavenging activities on ABTS radical (C) and FRAP (D) of supernatant obtained after lipase treatment of propolis extract. Values are means ± standard deviations (SDs). Means with different superscript letters are significantly different at P < 0.05 based on Tukey’s multiple range tests. Control; propolis 70% ethanol extract. For ABTS and FRAP assays, reactions were carried out on a shaker at 50°C and 140 revolutions per minute (rpm) for 12 h with 40 mL substrate, propolis ethanolic extract, and 2 g enzyme powder. Figure 4. Antimicrobial activity of supernatant obtained after lipase treatment of propolis extract against Staphylococcus epidermidis (A) and Propionibacterium acnes (B). Values are means ± standard deviations (SDs). Means with different superscript letters are significantly different at P < 0.05 based on Tukey’s multiple range tests. A; antimicrobial activity against S. epidermidis, B; antimicrobial activity against P. acnes.

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Table 1. Chemical composition of basic propolis extracted using water or ethanol Water extraction

70% ethanolic extraction

Analysis WE

HWE

EE

HEE

Solid content (mg/mL)

2.9 ± 0.14d

4.40 ± 0.03c

9.52 ± 0.05b

12.34 ± 0.09a

Polyphenol (mg/g)

19.69 ± 0.12d

26.68 ± 0.06c

50.59 ± 0.21b

51.36 ± 0.31a

Flavonoid (μg/g)

51.40 ± 0.95d

105.18 ± 0.95c

382.02 ± 11.41b

457.77 ± 10.78a

ABTS (IC50, mg/mL)

1.08 ± 0.01a

0.78 ± 0.01b

0.44 ± 0.01c

0.36 ± 0.00d

FRAP (mmol/g)

0.04 ± 0.00d

0.05 ± 0.00c

0.36 ± 0.00b

0.59 ± 0.01a

Values are means ± standard deviations (SDs). Means with different superscript letters are significantly different at P < 0.05 based on Tukey’s multiple range tests. WE; water extraction of propolis, HWE; water extraction of propolis after pre-extraction by hexane, EE; 70% ethanol extraction of propolis, HEE; 70% ethanol extraction of propolis after pre-extraction by hexane.

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Table 2. Active flavonoids of supernatant obtained after lipase treatment of propolis extract by HPLC assay Active flavonoid (μg/mL)

Control

Lipase OF 100.42 ±

Enzyme treatment Lipozyme Lipozyme RM IM TL IM 110.96 ± 36.80 ± 0.03d 0.07b

Novozym 435 111.35 ±

Caffeic acid

113.61 ± 1.37a

p-Coumaric acid

29.36 ± 0.00c

30.81 ± 0.01a

9.95 ± 0.00e

29.47 ± 0.02b

27.68 ± 0.04d

Apigenin

39.28 ± 0.00b

38.26 ± 0.17c

46.13 ± 0.11a

39.33 ± 0.27b

36.47 ± 0.12d

553.30 ±

525.88 ±

551.34 ±

456.62 ±

1.75a

0.62b

0.15a

0.33c

301.01 ±

291.11 ±

312.65 ±

249.30 ±

0.16c

0.33d

0.16a

0.09e

410.48 ±

367.87 ±

416.27 ±

271.73 ±

0.90ab

0.15c

6.59a

6.73d

1434.28±1.06

1277.73±0.05

1460.01±6.25

1153.16±7.25

b

c

a

d

Chrysin+

553.78 ± 0.00a

Galangin Kaempferide

310.92 ± 0.00b

Artepillin C

395.98 ± 0.00b

Total flavonoid

1442.93±1.37ab

0.21c

0.12ab

Values are means ± standard deviations (SDs). Means with different superscript letters are significantly different at P < 0.05 based on Tukey’s multiple range tests. An EC 250/4.6 NUCLEOSIL 100-5 C18 (250 × 4.6 mm, 5 μm) column was used. UV absorption was measured at 290 nm and 340 nm. Gradient elution was employed using solvent A (5% acetic acid) and solvent B (100% methanol) at 30°C.

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Table 3. Summary in functional properties of propolis extract treated by various lipases

Analytical properties

Control

LipaseOF

Lipozyme RM IM

Lipozyme TL IM

Novozyme 435

21.19 ± 0.3c

39.24 ±0.1a

20.75 ±0.35c

27.91± 0.1b

19.24 ± 0.29c

35.62±0.4b

52.5±0.9a

27.1±1.0c

36.19±0.7b

29.5±0.6c

2.4±0.03b

2.41±0.04b

1.85±0.005d

2.2±0.01c

2.5±0.01a

1.51±0.005b

1.61±0.03a

1.16±0.005a

1.30±0.005c

1.59±0.03a

0.18±0.005b

0.25±0.006a

0.125±0.001c

0.207±0.002b

0.15±0.001d

108±5.1a

105±2.1ab

91.1±2.0b

106±1.8ab

88.3±4.5b

0.19±0.005c

0.21±0.002bc

0.46±0.005a

0.2±0.01c

0.28±0.05b

0.46±0.02bc

0.55±0.03a

0.42±0.015c

0.51±0.005ab

0.41±0.001c

14.0±1b

14.1±0.5b

14.7±1.2b

12.5±1.2c

28±1.0a

3.8±0.1a

3.7±0.5ab

2.7±0.15bc

2.2±0.4c

3.4±0.2abc

Dry weight (mg/mL) Acid value (mg of KOH) Total Fatty

F.A

acids

(g/100g) Palmitic acid

(g/100g) Total Flavonoids (mg/g) Total Polyphenols (mg/g) ABTS IC50 (mg) Iron sulfate heptahydrate

MIC (mg)

(mmol/g) S. epidermiditis P. acnes

Values are means ± standard deviations (SDs). Means with different superscript letters are significantly different at P < 0.05 based on Tukey's multiple range tests, MIC; minimum inhibitory concentration,. F.A; fatty acid

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60

80

B

Dry weight (mg/mL)

45 a

b

30 c

d

e

15

0

Acid value (mg of KOH)

A 60

a

40

b

b c

c

20

5 F ol M IM 43 LI n tr eO RM Co ym Enzyme as eT z e p i m o L y v ym oz No oz Lip Lip

0

5 F ol M IM 43 LI n tr eO RM Co ym as eT z e p i m o L y v ym oz No oz Lip Lip

Figure 1.

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4

2.0

B

3 b

a

b c

2

d

1

0

ol n tr Co

5 F M IM 43 LI eO RM ym as eT z e p i m L y vo ym oz No oz Lip Lip

Palmitic acid (g/100 g of raw material)

Total fatty acid (g/100 g of raw material)

A

a

a

b 1.5

c d

1.0

0.5

0.0

5 F ol M IM 43 ntr LI eO RM Co ym as eT z e p i m L y vo ym oz No oz Lip Lip

Figure 2.

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0.4

150

Total flavonoid (mg/g of raw material)

0.3

a b b

0.2

c

d 0.1

0.0

Total polyphenol (mg/g of raw material)

B

A

120

ab

ab b

b

90

60

0

5 F ol M IM 43 ntr LI M eO Co ym as eT eR oz Lip ym v ym z o z o N o Lip Lip

n Co

tro

l Li p

5 F IM IM 43 M eO TL ym eR me oz m y v y z o No oz Li p Li p

as

0.8

0.5

D

C a 0.4

b 0.3

bc

c

c

0.2

0.1

0.0

n Co

l tro L

5 F M IM 43 LI eO M eT zym eR m o m y v y oz No oz Lip Li p

s i pa

FRAP(mmol/g of raw material)

IC50 (ABTS, mg of raw material/mL)

a

a

0.6

ab

bc c

c

0.4

0.2

0.0 n Co

Figure 3.

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tro

l Li p

5 F M IM 43 LI eO M eT eR zym m o m y v y oz No oz Li p Li p

as

40

6.0

B

30

a

20 b

b

b c

10

0

5 F ol M IM 43 n tr LI eO M Co as eT zym eR m o Lip m y v y oz No oz Lip Lip

MIC (P.acnes, mg of raw material/mL)

MIC (S.epidermidis, mg of raw material/mL)

A

4.5

ab a abc bc

3.0

c

1.5

0.0

5 F ol M IM 43 ntr LI eO M Co as ym eT z eR m o Lip m y v y oz No oz Lip Lip

Figure 4.

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