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Change in the Content of Phenolic Compounds and Antioxidant. Activity during Manufacturing of Black Raspberry (Rubus coreanus. Miq.) Wine. Jeong-Yong ...
Food Sci. Biotechnol. 22(5): 1237-1244 (2013) DOI 10.1007/s10068-013-0207-5

RESEARCH ARTICLE

Change in the Content of Phenolic Compounds and Antioxidant Activity during Manufacturing of Black Raspberry (Rubus coreanus Miq.) Wine Jeong-Yong Cho, Jong Hoon Jeong, Jin Young Kim, Soo Ro Kim, Seong Ja Kim, Hyoung Jae Lee, Sang-Hyun Lee, Keun-Hyung Park, and Jae-Hak Moon

Received: 8 January 2013 / Revised: 18 March 2013 / Accepted: 20 March 2013 / Published Online: 31 October 2013 © KoSFoST and Springer 2013

Abstract The changes in total phenolics, total anthocyanins, gallic acid (GA), and 3,4-dihydroxybenzoic acid (DHBA) contents during black raspberry (BR, Rubus coreanus) wine manufacturing were investigated. Total phenolic content gradually increased during BR wine manufacturing. The total anthocyanin content slightly increased, but the changes were not significant. The GA and DHBA in BR wine were detected as main compounds and their content greatly increased during manufacturing. The DPPH radicalscavenging activity was also increased during BR wine manufacturing. In particular, the antioxidant activity in the blood plasma of rats administered with BR wine concentrate was significantly higher than those administered with BR fruit extracts in inhibition of copper ion-induced cholesteryl ester hydroperoxide formation. These results indicate that an increase in antioxidant activity of BR wine may be due to low molecular phenolic compounds including GA and DHBA produced during the manufacturing. Keywords: black raspberry wine, Rubus coreanus, gallic acid, 3,4-dihydroxybenzoic acid, antioxidant activity

Jeong-Yong Cho, Jong Hoon Jeong, Jin Young Kim, Soo Ro Kim, Seong Ja Kim, Hyoung Jae Lee, Keun-Hyung Park, Jae-Hak Moon () Department of Food Science and Technology, Functional Food Research Center, Chonnam National University, Gwangju 500-757, Korea Tel: +82 62 530 2141; Fax: +82 62 530 2149 E-mail: [email protected] Sang-Hyun Lee Korea Pear Research Organization, Chonnam National University, Gwangju 500-757, Korea

Introduction Black raspberry (BR, Rubus coreanus Miquel) belongs to the Rosaceae family that includes R. fruticosus, R. foliosus, and R. lambertianus (1). This fruits have been widely used in various processed foods such as beverages and wines. Recently, there are reports on various biological effects of BR including bone-protection (2), anticancer (3), antimicrobial (4), antioxidant (5), hepatitis B virus inhibition (6), and anti-inflammatory (7) activities. The biologically active compounds including anthocyanins (8), triterpenoids (911), phenolic acids and flavonoids (5,12), and tannins (13) have also been identified from BR fruits. BR wine is made by the fermentation of ripened BR fruits and has deep red color and sweet taste and is a popular Korean traditional alcoholic beverage (14). Certain biological activities of BR wine have already been reported including serum cholesterol reduction (15), antioxidant and anticancer (16), and anti-proliferative and anti-inflammatory (8) effects. However, studies on the biological effects and chemical constituents of BR wine are very limited when compared to those of BR fruits. In addition, it has recently been reported that BR wine displays high antioxidant activity comparable to commercial grape red wines (8). We contemplated that various metabolites may be produced during the fermentation of BR wine and contribute to biological effects. However, investigation on chemical constituents of BR wine has not yet been performed. Therefore, as a primary approach to explore the chemical constituents present in BR wine, we have previously isolated 10 low molecular phenolic and volatile compounds from the ethyl acetate (EtOAc) layer of BR wine (17,18). The isolated compounds were identified as 4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid (DHBA), 4-(2-hydroxyethyl)-

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phenol, pyrocatechol, ethyl gallate, ethyl succinate, vanillic acid, ethyl 3,4-dihydroxybenzoate, furan-2-ol, and 4-(4hydroxyphenyl)butan-2(S)-ol. We expected that a few compounds of the identified phenolic constituents may be used as specific molecular marker compounds for quality control and evaluation of physiological activity in the manufacturing process of BR wine. However, there is no available study on behavior of phenolic compounds during BR wine manufacturing. In addition, investigation for correlations between phenolic compounds and biological activities during BR wine manufacturing has not yet been performed. In this study, we report change in the contents of total phenolics and total anthocyanins during the manufacturing of BR wine. In addition, phenolic compounds were analyzed by GC-MS and HPLC to specify (a) molecular marker compound(s) as an indicator for estimation of quality and physiological activity of BR wine. Moreover, antioxidant activities of BR wine and its fruits were also evaluated by DPPH radical-scavenging assay and copper ion-induced lipid peroxidation in rat plasma after oral administration of BR wine.

Materials and Methods Materials and chemicals The fruits of BR were collected in June 2009 from Naju, Korea and identified by Prof. Kye-Han Lee, Laboratory of Forest Ecology, College of Agriculture and Life Science, Chonnam National University. A voucher sample has been deposited in the warm-temperate forest arboretum located on Bogil Island, Chonnam National University. Folin-Ciocalteu’s phenol reagent was purchased from Nacalai Tesque Inc. (Kyoto, Japan). DPPH and cyanidin were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). The GA and DHBA were obtained from Acros Organics (Fair Lawn, NJ, USA) and Wako Pure Chemical Industries (Osaka, Japan), respectively. Other chemicals were of reagent grade and obtained from commercial sources. Preparation of BR wine The manufacture of BR wine was carried out according to the mass-production method of Yeonsudang Manufacturing Company in Gwangju, Korea. Briefly, the frozen BR fruits were thawed at room temperature. The ground BR fruits (20 kg, final volume 14 L) were filled in 20 L round glass jars and adjusted to 24oBx by supplements of high fructose corn syrup. After the treatment with carbon dioxide (final concentration, 100 ppm) for 6 h, the BR fruit solution was inoculated with dried yeast, and then fermented for 15 days at 20oC in an incubator (DS-31F; Dasol Scietific Co., Ltd., Hwaseong,

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Korea). After the treatment with carbon dioxide (final concentration, 100 ppm), the fermented BR fruit was macerated for 45 days at 15oC and filtered by the sieve (25 mesh) to remove seed. The filtrate was centrifuged at 1,000×g for 30 min to remove the pulp. The BR wine juice in steel was aged for 45 days at 15oC. The samples during the BR wine manufacturing were collected at 12 stages as follows: S1, BR fresh fruits; S2, the 7th day of fermentation; S3, the 15th day of fermentation; S4, the 10th day of maceration; S5, the 20th day of maceration; S6, the 30th day of maceration; S7, the 45th day of maceration; S8, BR wine juice after filtration; S9, BR wine juice after centrifugation; S10, the 15th day of aging; S11, the 30th day of aging; and S12, the 45th day of aging. Pretreatment of BR wine and fruits for chemical analysis and determination of antioxidant activity BR fruits (100 g) were homogenized (BM-2 Nissei biomixer; Nihonseiki Kaiseiki Ltd., Tokyo, Japan) with EtOH (50 mL) and filtered through a filter paper (No. 2; Whatman International, Maidstone, UK). The residue was extracted with 100% ethanol (EtOH, 50 mL) and then filtered (No. 2; Whatman). The combined filtrate was made up to 100 mL with EtOH. The samples obtained during BR wine manufacturing were respectively centrifuged at 1,000×g for 30 min. Each supernatant was filtered through a filter paper (No. 2; Whatman). Subsequently, fresh BR fruit extracts and samples collected in different steps of fermentation and aging during BR wine manufacturing were immediately stored at 70oC until analysis. Determination of total phenolic content Total phenolic contents in BR wine samples and fruits were measured by Folin-Ciocalteu’s method (19). Briefly, each water-diluted sample solution (0.5 mL, equivalent to 0.72 g of fresh fruit weight) was mixed with Folin-Ciocaltue’s phenol reagent (0.5 mL) and saturated Na2CO3 solution (0.5 mL). The mixture was incubated at room temperature for 60 min in the dark and then detected at 700 nm using a spectrophotometer (V-550; Jasco, Kyoto, Japan). Total phenolic contents of samples were quantified on the basis of the calibration curve of GA as a standard compound. Total phenolic content of BR wine was determined through triplicate experiments. Determination of total anthocyanin content Total anthocyanin content was measured according to colorimetric method (20). Briefly, each sample solution (0.5 mL, equivalent to 0.72 g of fresh fruit weight) was diluted with 20 mL of 1 N HCl solution. After 10 min, the absorbance of each sample solution was measured at 520 nm in a 10 mm cuvette using UV/VIS spectrophotometer (V-550;

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Jasco). The calibration curves were constructed by using external standards of cyanidin (1-50 ppm). The content of total anthocyanin in BR fruits and samples collected during BR wine manufacturing was determined through triplicate experiments.

method. External standards of GA and DHBA were added to aliquots of BR wine at three different concentrations (n=3, six replicates). The contents of GA and DHBA in BR fruits and its wine samples collected during BR wine manufacturing was determined through triplicate experiments.

Qualitative analysis of phenolic compounds by GC-MS The extract of BR fruits and wine (equivalent to 230 mg of fresh fruit weight) was suspended in a mixture of H2O/ MeOH=90:10 [v/v, pH 2.65, by trifluoroacetic acid (TFA)]. The suspension mixture was subjected to solidphase extraction using octadecylsilane (ODS) cartridge (MEGA BE-C18, 1 g, 6 mL; Varian, California, LA, USA) and then eluted by using a solvent mixture of H2O/MeOH =90:10, 70:30, 50:50, and 0:100 (v/v, pH 2.65, each 10 mL). Each separated fraction was concentrated in vacuum. A portion of each fraction was trimethylsilylated by a mixture of pyridine/N,O-bis(trimethylsilyl)acetamide/ trimethylchlorosilane (10:5:1, v/v/v). The GC/MS analysis was performed on a GC-MS (QP2010; Shimadzu, Kyoto, Japan) equipped with VB-1 capillary column (0.25 mm× 60 m, 0.25-µm film thickness; Valco instruments Co., Inc., Houston, TX, USA). The oven temperature was held at 60oC and programmed to 240oC at 4oC/min. Helium was used as a carrier gas, the flow rate and the split ratio were 18 cm/sec and 1:20, respectively. Mass spectra were obtained at 70 eV (EI) with an ion source temperature of 200oC. The identification of the components was accomplished by comparison of GC retention times and mass spectra with compounds isolated from BR wine in previous studies (17,18).

DPPH radical-scavenging assay Free radical-scavenging activities of samples were evaluated according to the method described by Yamaguchi et al. (21). Briefly, each sample (50 mL, equivalent to 46 mg fresh fruits) was mixed with 100 mM Tris-HCl buffer (pH 7.4, 250 mL). The mixture was added to DPPH radical ethanol solution (250 mL; final concentration, 250 mM). The solution was mixed and allowed to stand for 20 min in the dark. The reaction solution was analyzed by HPLC (SPD-M20D; Shimadzu) operating at 517 nm. The remaining DPPH radicals were separated on an ODS-80Ts column (4.6 mm i.d.×250 mm, 5 mm; Tosoh). Elution was achieved by using a solvent mixture of MeCN/H2O=60:40 (v/v) as the mobile phase and the flow rate was 1.0 mL/min. The DPPH radicalscavenging activity of samples was determined as the percentage decrease in peak area of DPPH radical on HPLC chromatogram when compared to a blank test.

Quantitative analysis of GA and DHBA by HPLC The GA and DHBA contents in BR fruits and its wine samples collected during BR wine manufacturing were quantitatively analyzed by HPLC analysis. Each sample (equivalent to 7 g of fresh ripened fruits) concentrated in vacuum was suspended in distilled water (pH 2.8 by 1 N HCl, 10 mL) and then partitioned with a solvent mixture of EtOAc/EtOH (4:1, v/v, 10 mL). The upper layers were concentrated and the concentrates were respectively dissolved in 1.0 mL of MeOH and filtered through a Millipore membrane (0.45 µm; Berrica, MA, USA). The filtrates were subjected to the HPLC (SPD-M20D; Shimadzu, Kyoto, Japan) with photodiode array detector (PDA) operating at wavelength ranging from 190 to 800 nm. The GA and DHBA were separated on an ODS-80Ts column (4.6 mm i.d.×250 mm, 5 µm; Tosoh, Tyoko, Japan). Elution was achieved by using 8% MeOH (pH 2.8, by TFA) as the mobile phase and the flow rate was 1.0 mL/min. The calibration curves (n=6 point) were constructed using external standards of GA and DHBA (0.1-20 µg). Accuracy and reproducibility were evaluated using the standard spike

Determination of inhibitory effect of BR wine and fruit extracts against copper ion-induced oxidation of rat blood plasmas obtained after their oral administration The antioxidative activities of BR wine and fruits were evaluated by measuring their inhibitory effects against cholesteryl ester hydroperoxide (CE-OOH) formation in copper ion-induced oxidation of diluted rat blood plasma (22). Sprague-Dawley rats (male, 6-week age, 180-200 g; Samtako Bio Korea, Osan, Korea) were kept at 23oC under a 12 h dark/light cycle and fasted for 12-15 h prior to oral administration of sample. Rats were orally administrated 16% EtOH solutions (1.5 mL) prepared from BR wine and fruit extracts (equivalent to 4.3 g of fresh fruits). After 1 h, rats were anesthesized with diethyl ether, the abdomen wall was opened, and blood was collected from the abdominal aorta into heparinized tubes. Rat plasma was isolated by centrifugation (1,500×g) at 4oC for 20 min. Blood plasma (125 µL) of each group (n=5) was combined and the mixture (600 µL) was diluted 5-folds by PBS solution. The plasma mixture was oxidized by the addition of 240 µL of CuSO4 PBS solution (final concentration, 100 µM). The reaction mixture was incubated at 37oC for 7 h with continuous shaking. The concentration of CE-OOH was determined according to the method described by Arai et al. (23). Briefly, aliquots (100 µL) were withdrawn from the incubation solutions and mixed with 3 mL of MeOH containing 2.5 mM of 2,6-di-tert-butyl-4-methylphenol (BHT). The mixture was sonicated (Power Sonic 4200; Hwashin, Ulsan, Korea) for 1 min, and then neutral lipids

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were extracted with 3 mL of n-hexane by vortexing vigorously for 1 min. The upper layer (n-hexane) was collected and extraction of the lower layer with 3 mL of nhexane was repeated. The combined n-hexane phases were evaporated in a rotary evaporator at room temperature. The remaining lipids were dissolved in 100 µL of MeOH/ CHCl3 (95:5, v/v), and aliquots were subjected to CEOOH analysis by RP-HPLC using a TSK-gel Octyl-80Ts column (Tosoh). The effluent was monitored by UV detection at 235 nm (SPD-10A; Shimadzu). The mixture solution of MeOH/H2O (97:3, v/v) served as a mobile phase and the flow rate was constant at 1.0 mL/min. The concentration of CE-OOH was calculated from a standard curve of cholesteryl linoleate hydroperoxide. Detailed procedures for preparation of the CE-OOH standard have been published elsewhere (22). Statistical analysis All data were expressed as mean± standard deviation (SD) using the Statistical package for social sciences (SPSS, Armonk, NY, USA) 17.0 package programs. Statistical differences for the contents of total phenolics, total anthocyanin, GA, and DHBA and DPPH radical-scavenging activity during BR wine manufacturing were analyzed by Duncan’s multiple comparison test (p