Food Sci. Biotechnol. 21(4): 1081-1086 (2012) DOI 10.1007/s10068-012-0140-z
RESEARCH ARTICLE
Jaboticabin and Flavonoids from the Ripened Fruit of Black Rasberry (Rubus coreanum) Jeong-Yong Cho, In Yoon, Da-Hwa Jung, Sook Hee Hyun, Kye-Han Lee, Jae-Hak Moon, and Keun-Hyung Park
Received: 27 January 2012 / Revised: 15 March 2012 / Accepted: 16 March 2012 / Published Online: 31 August 2012 © KoSFoST and Springer 2012
Abstract The ethyl acetate (EtOAc) layer of ripened fruits of black raspberry (Rubus coreanum) had higher DPPH radical scavenging activity than was detected in other layers. Six phenolic compounds were purified and isolated from the EtOAc layer of ripened black raspberry fruits via octadecyl silane (ODS)-column chromatography and amide column-HPLC using a guided DPPH radical scavenging assay. These compounds were identified as 4hydroxybenzoic acid (1), 2-O-(3',4'-dihydroxybenzoyl)4,6-dihydroxyphenylmethylacetate (2, jaboticabin), phloridzin (3), kaempferol 3-O-β-D-glucopyranoside (4), quercetin 3O-β-D-glucuronic acid methyl ester (5), and quercetin (6), based on MS and NMR analysis. Three compounds (1, 5, and 6) had been identified previously in this plant, but other compounds (2-4) were newly isolated from this plant. Compound 2, 5, and 6 evidenced higher DPPH radical scavenging activity than α-tocopherol at the same concentration. Keywords: black raspberry fruit (Rubus coreanum), jaboticabin, flavonoid, DPPH radical scavenging activity
Introduction Korean black raspberry (bokbunja, Rubus coreanus Miquel.) belongs to the Roseceae family, which includes R. Jeong-Yong Cho, In Yoon, Da-Hwa Jung, Sook Hee Hyun, Jae-Hak Moon, Keun-Hyung Park () Department of Food Science & Technology, and Functional Food Research Center, Chonnam National University, Gwangju 500-757, Korea Tel: +82-62-530-2143; Fax: +82-62-530-2149 E-mail:
[email protected] Kye-Han Lee Division of Forest Resources and Landscape Architecture, College of Agriculture & Life Sciences, Chonnam National University, Gwangju 500-757, Korea
fruticosus, R. foliosus, and R. lambertianus (1). The fruits of the black raspberry have been employed in traditional folk remedies for liver protection as well as for the treatment of enuresis, asthma, and spermatorrhea in Korea (2). Additionally, the fruits have been employed in a variety of processed foods in Korea. For example, black raspberry wine, which is made via the fermentation of the ripened fruits, is a popular Korean alcoholic beverage (3). Many studies have noted that the fruit exerts biological effects, including anticancer (4), antimicrobial (5), antioxidant (6, 7), and hepatitis B virus inhibitory (8) activities. Moreover, triterpenoids (niga-ichiogoside F1, F2, suavissimoside R1, and coreanoside F1) (9,10), catechins, and tannins (procyanidin B-4 and sanguiin H-4) (11,12) have been identified in the black raspberry. Recently, niga-ichiogoside F1 and 23-hydroxytormentic acid, which have been reported to exert antinociceptive and anti-inflammatory effects, were also isolated from the unripened fruits of black raspberry (13). Moreover, the anti-rheumatoid arthritis effects of the compounds in Freund’s complete adjuvant reagent-induced rats have been previously reported (14). Recently, we purified and isolated the antioxidants from black raspberry fruits using a guided DPPH radical scavenging assay. Five phenolic antioxidative compounds were previously identified (6,7) in the course of our investigation of chemical constituents from the fruits of black raspberry. In the present study, 6 phenolic compounds, including jaboticabin, were isolated. This paper describes the isolation, structural elucidation, and DPPH radical scavenging activity of 6 phenolic compounds from the methanol extract of black raspberry fruits.
Materials and Methods General experimental procedures NMR spectra were
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obtained using an unitINOVA 500 spectrometer (Varian, Walnut Creek, CA, USA) with tetramethylsilane as the internal standard in CD3OD and DMSO-d6. All MS spectra were obtained via analysis using a LC electrospray ionization tandem mass spectrometer (LC-ESI-MS/MS, Synapt HDMS; Waters, Milford, MA, USA) equipped with an ultraperformance LC (Acquity UPLC; Waters) and Acquity BEH column (2.1×50 mm; Waters). Column chromatography was carried out using octadecyl silane (ODS) gel (170 mesh, YMC, Kyoto, Japan). HPLC analysis was conducted using Discovery® RP Amide C16 (25 cm×10 mm, 5 µm, Waters). DPPH and α-tocopherol were purchased from SigmaAldrich (St. Louis, MO, USA). Other chemicals were of reagent grade, and were obtained from commercial sources. Plant material The black raspberry (bokbunja, Rubus coreanus Miquel.) fruits were collected in June 2005 from Gochang county, 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 of Chonnam National University located on Bogildo. Extraction and isolation The fresh ripened fruits (1.08 kg) were extracted with methanol (MeOH, 11,000 mL) at room temperature for 24 h. The MeOH extracts were then filtered through No. 2 filter paper (Whatman International, Maidstone, UK) and concentrated in vacuo at 38oC. The MeOH extracts (93.42 g) were suspended in H2O (1,000 mL) and successively partitioned with chloroform (CHCl3, 1,000 mL, 3 times), ethyl acetate (EtOAc, 1,000 mL, 3 times), and water-saturated n-butanol (BuOH, 1,000 mL, 3 times). Each layer was evaporated in vacuo at 38oC. The EtOAc layer (1.49 g) was fractionated via chromatography on an ODS column (100 g, 2.5×94 cm) and subsequent elution of a mixture of H2O and MeOH (8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 8:2, v/v, each step 1,000 mL). The active 50% MeOH fraction (129.6 mg) was injected onto an amide column-HPLC apparatus (45% MeOH containing 0.1% trifluoroacetic acid, TFA) to obtain compounds 1 (tR 10.7 min, white powder, 0.3 mg), 2 (tR 26.2 min, white powder, 1.8 mg), 3 (tR 34.7 min, yellowish powder, 2.7 mg), 4 (tR 42.9 min, yellowish powder, 0.7 mg), 5 (tR 53.8 min, yellowish powder, 0.8 mg), and 6 (tR 57.6 min, yellowish powder, 1.6 mg). Assay of DPPH radical scavenging The free radicalscavenging activities of the isolated compounds and αtocopherol were assessed using the DPPH radical according to a modified version of the method described by Abe et al. (15). In brief, a methanol solution (200 µL) of the isolated compounds (20 µM) was added to DPPH
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radical ethanol solution (1,800 µL, final concentration, 100 µM). The solution was then mixed and permitted to stand for 30 min in darkness. The free radical scavenging activity of each compound was then quantified via the decolorization of DPPH at 517 nm. The DPPH radical scavenging activities of the isolated compounds were also determined as the percentage decrease in absorbance as shown by a blank test. An assay for the purification of antioxidative compounds was conducted by spraying DPPH reagent on a TLC (Silica gel 60 F254, 0.25 mm thickness, Merck, Darmstadt, Germany) in accordance with a modified version of the method described by Takao et al. (16). Each fraction purified by ODS column chromatography or amide column-HPLC was spotted onto the plate and developed with solvent mixture of MeOH/CHCl3 =1:1 (v/v). After spraying 200 mM DPPH ethanol solution onto the plate, the fractions visualized as decolorizations of the spot were regarded as reflective of antioxidative activity.
Results and Discussion Isolation and identification of the antioxidative compounds The MeOH extracts (93.42 g) of fresh black raspberry fruits (1.08 kg) were successively partitioned with CHCl3, EtOAc, and BuOH. The antioxidative activities of these layers were subsequently evaluated using a DPPH radical. The EtOAc layer evidenced higher antioxidative activity than other layers (data not shown). Therefore, the EtOAc layer (1.49 g) was fractionated via ODS column chromatography by elution with a mixture of H2O and MeOH. The fractions were subsequently developed via TLC and sprayed with a DPPH free radical ethanol solution. All of the fractions evidenced DPPH radical scavenging activity (data not shown). In particular, the 50% MeOH fraction exhibited stronger DPPH free radical scavenging activity than the other fractions. Therefore, the 50% MeOH fraction was purified via HPLC (RP amide C16 column, 45% MeOH containing 0.1% TFA) to obtain 6 compounds (1-6) (Fig. 1). The structure of the isolated compounds was then elucidated by NMR and MS spectral data as follows: Compound 1: White powder; 1H-NMR spectrum (500 MHz, DMSO-d6) 6.98 (2H, d, J=8.0 Hz, H-2, 6), 6.63 (2H, d, J=8.0 Hz, H-3, 5); ESIMS (negative) m/z 137 [M-H]−. Compound 2: White powder; 1H- and 13C-NMR data see Table 1; ESIMS (negative) m/z 333.06 [M-H]-, 301.03 [MCH3OH-H]−. Compound 3: Yellowish powder; 1H-NMR (500 MHz, CD3OD) spectrum δ 7.06 (2H, d, J=7.8 Hz, H-2, 6), 6.58 (2H, d, J=7.8 Hz, H-3, 5), 5.97 (1H, br. s, H-3'), 6.18 (1H, br. s, H-5'), 2.88 (2H, t, J=7.8 Hz, H-α), 3.45 (2H, m, H-
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Fig. 1. Isolation procedure of antioxidative compounds from the EtOAc layer of black raspberry fruits.
β), 5.03 (1H, d, J=7.0 Hz, H-1"), 3.91-3.39 (4H, m, H-2"H-5"), 3.91 (1H, d, J=11.5 Hz, H-6"a), 3.81 (1H, dd, J= 6.0, 13.0 Hz, H-6"b); 13C-NMR (125 MHz, CD3OD) spectrum δ 134.0 (C-1), 130.5 (C-2, 6), 116.2 (C-3, 5), 156.2 (C-4), 31.0 (C-α), 47.1 (C-β), 206.8 (C=O), 107.0 (C-1'), 162.5 (C-2'), 98.5 (C-3'), 167.7 (C-4'), 95.6 (C-5'), 166.0 (C-6'), 102.3 (C-1"), 74.9 (C-2"), 8.6 (C-3"), 71.3 (C-4"), 78.6 (C-5"), 62.6 (C-6"); ESIMS (positive) m/z 459 [M+Na]+. Compound 4: Yellowish powder; 1H-NMR (500 MHz, CD3OD) spectrum δ 6.85 (2H, d, J=8.0 Hz, H-2', 6'), 7.58 (2H, d, J=8.0 Hz, H-3', 5'), 6.22 (1H, br. s, H-6), 6.41 (1H, br. s, H-8), 5.21 (1H, d, J=7.5 Hz, H-1"), 3.44-3.81 (6H, m, H-2"-H-6"); ESIMS (positive) m/z 449 [M+H]+, 471 [M+Na]+. Compound 5: Yellowish powder; 1H-NMR (500 MHz, CD3OD) spectrum δ 7.58 (1H, br. d, J=9.0 Hz, H-6'), 7.63 (1H, br. s, H-2'), 6.85 (1H, d, J=9.0 Hz, H-5'), 6.21 (1H, br. s, H-6), 6.41 (1H, br. s, H-8), 5.23 (1H, d, J=7.5 Hz, H-1"), 3.75-3.45 (4H, m, H-2"-H-5"), 3.66 (3H, s, -OCH3); 13CNMR (125 MHz, CD3OD) spectrum δ 158.6 (C-2), 132.4 (C-3), 179.4 (C-4), 163.2 (C-5), 100.1 (C-6), 166.2 (C-7), 94.9 (C-8), 159.6 (C-9), 105.8 (C-10), 123.6 (C-1'), 116.0 (C-2'), 146.1 (C-3'), 150.0 (C-4'), 116.2 (C-5'), 123.0 (C6'), 104.9 (C-1"), 75.5 (C-2"), 77.3 (C-3"), 72.9 (C-4"), 75.5 (C-5"), 170.8 (C-6"), 53.0 (-OCH3); ESIMS (positive) m/z 493 [M+H]+, 515 [M+Na]+. Compound 6: Yellowish powder; 1H-NMR (500 MHz, CD3OD) spectrum δ 7.62 (1H, br. d, J=8.8 Hz, H-6), 7.70 (1H, br. s, H-2), 7.24 (1H, d, J=8.8 Hz, H-5), 6.20 (1H, br. s, H-6), 6.45 (1H, br. s, H-8); ESIMS (negative) m/z 301 [M-H]−.
Five known compounds were identified as 4-hydroxybenzoic acid (1) (17), phloridzin (3) (18), kaempferol 3-O-β-Dglucopyranoside (4) (17), quercetin 3-O-β-D-glucuronic acid methyl ester (5) (19), and quercetin (6) (7) (Fig. 2). These compounds were identified via comparisons of the 1 H- and 13C-NMR spectral data as reported in the relevant literature based on the NMR and MS spectroscopic data. Identification of compound 2 Compound 2 was obtained as an amorphous white powder. The molecular ion peak at m/z 333.06 [M-H]− and fragment ion peak at m/z 301.03 [M-CH3OH-H]− were noted on ESI-MS (negative) spectrum, thereby indicating that the molecular weight of 2 is 334. The 13C-NMR (Table 1) and HSQC spectra revealed 16 carbon signals, including 2 carbonyl carbons at δ 174.5 (C8) and 166.4 (C-7'), 12 sp2 carbons at δ 158.7-101.2, a methoxyl group at δ 52.4 (-OCH3), and a methylene carbon at δ 30.0 (C-7). From the MS, 13C-NMR, and HSQC spectra, the molecular formula of 2 was suggested to be C16H14O6. The 1H-NMR spectrum (Table 1) showed the presence of tri-substituted aromatic ring proton signals [δ 7.51 (1H, br. s, H-2'), 6.87 (1H, d, J=7.5 Hz, H-5'), 7.52 (1H, br. d, J=7.5 Hz, H-6')] and tetra-substituted aromatic proton signals [d 6.26 (1H, d, J=2.5 Hz, H-3), 6.14 (1H, d, J=2.5 Hz, H-5)]. This data was consistent with a pentahydroxylated structure like the A and B rings of quercetin. In addition, the presence of a methoxyl proton signal at δ 3.55 (3H, s, -OCH3) and a methylene proton signal at δ 3.49 (2H, s, H-7) was observed. All of the protonated carbons were assigned on the basis of the results of the HSQC analysis. The important long-range correlations between protons and carbons were determined by the
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Fig. 2. Chemical structures of antioxidative compounds isolated from the black raspberry fruits and important HMBC correlations (arrow) of 2. Table 1. NMR spectroscopic data of jaboticabin (2) Position
δH (int., mult., J in Hz)
δC
1 2 3 4 5 6 7 8 -OCH3 1' 2' 3' 4' 5' 6' 7'
6.26 (1H, d, 2.5) 6.14 (1H, d, 2.5) 3.49 (2H, s) 3.55 (3H, s)
107.3 158.7 101.2 158.5 102.3 152.5 30.0 174.5 52.4 121.8 118.0 146.6 152.6 116.2 124.5 166.4
7.51 (1H, br. s) 6.87 (1H, d, 7.5) 7.52 (1H, br. d, 7.5) -
HMBC spectrum (Fig. 2). In particular, the presence of the cross-peaks from the proton signals of H-7 (δ 3.49, 2H, s) and the methoxyl group (δ 3.55, 3H, s) to the carbonyl carbon signal of C-8 (δ 174.5) and from the proton signal at δ 3.49 (H-7) to carbon signals at δ 107.3 (C-1), 158.3 (C-2), and 152.5 (C-6) indicated that the partial structure of 2 was methyl 2,4,5-trihydroxyphenylacetate. Moreover, the HMBC correlations of the tri-substituted aromatic ring proton signals at δ 7.51 (1H, br. s, H-2'), and 7.52 (1H, d, J=7.5 Hz, H-6') to a carbon signal at d 166.4 (C-7') revealed
that another partial structure of 2 was 3,4-dihydroxybenzoic acid. According to the results of the MS and NMR spectroscopic data, 2 was suggested to be an esterified structure of methyl 2,4,5-trihydroxyphenylacetate and 3,4dihydroxybenzoic acid, which is constituted of a number of depsides composed of 2 or more monocyclic aromatic units linked by an ester bond (20). When the 1D-NMR spectra of 2 were compared with those of depsides including methyl 2,4,6-trihydroxyphenylacetate and 3,4dihydroxybenzoic acid, 2 was consistent with the 2-O(3',4'-dihydroxybenzoyl)-4,6-dihydroxyphenylmethylacetate isolated from jaboticaba (Myrciaria cauliflora) (21), as recently reported in the relevant literature. Therefore, the structure of 2 was elucidated as 2-O-(3',4'-dihydroxybenzoyl)4,6-dihydroxyphenylmethylacetate (jaboticabin) (Fig. 2). DPPH radical scavenging activity of the isolated compounds The antioxidative activities of the isolated compounds (final concentration, 20 µM) were evaluated via the DPPH radical scavenging method. Quercetin (6) evidenced significantly higher DPPH radical scavenging activity than α-tocopherol and other isolated compounds (Table 2). Flavonoids (5, 6) containing the catechol moiety in the B ring exhibited more profound radical scavenging activity than 3 and 4, which contained a monohydroxy phenyl group. It was reported previously that the catechol structure in the B ring of flavonoids is an important factor for the radical scavenging effect when compared to that of the monohydroxy phenyl group (22,23). Therefore, it is likely that the attenuation of the radical scavenging activity
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Table 2. DPPH radical scavenging activities of compounds isolated from the ripened black raspberry fruits Compounds1)
DPPH radical scavenging activity (%)
4-Hydroxybenzoic acid (1) Jaboticabin (2) Phloridzin (3) Kaempferol 3-O-β-D-glucopyranoside (4) Quercetin 3-O-β-D-glucuronic acid methyl ester (5) Quercetin (6) α-Tocopherol
9.82) 35.6 1.2 1.0 32.9 55.6 29.2
1)
α-Tocopherol was used as a positive control; final concentration of compounds: DPPH, 100 µM; isolated compounds, 20 µM; control compound (α-tocopherol), 20 µM 2) Data are representative of 2 experiments.
of 3 and 4 is caused by a lack of the hydroxyl group in the C-3' position. Additionally, quercetin (6), which contains a free hydroxyl group at the C-3 position, evidenced significantly higher DPPH radical-scavenging activity than quercetin 3-O-β-D-glucuronic acid methyl ester (5), which is etherified at C-3. These results indicate that the free hydroxyl group of the C-3 in flavonol is an important factor involved in free radical scavenging, as reported previously (22,23). Compound 2 and 5 containing the catechol moiety of the benzene ring evidenced similar DPPH radical scavenging activities (Table 2). Furthermore, 2 and 5 exhibited more profound DPPH radical scavenging activity than α-tocopherol, thereby indicating that these compounds may prove useful in the prevention of human diseases attributed to free radical damage. In this study, 6 antioxidative compounds were isolated and identified from the ripened black raspberry fruits. Among them, compound 5 was identified from the leaves of this plant (24). Compound 1 and 6 were previously isolated from the fruits of this plant (6,7). We believe that this study is the first to identify the other compounds (2-4) from the ripened black raspberry fruits. Recently, compound 2 (jaboticabin) was identified almost at the same time from the fruits of jaboticaba (21), juice of cranberry (Vaccinium macrocarpon) (25), and the leaves of kiwifruit (Axtinidia chinensis) (26). They reported that jaboticabin had DPPH radical scavenging activity, inhibitory effects against cigarette smoke-induced inflammation in human small airway epithelial cell, and cytotoxic activity against HT29 colon cancer cells (21). It has been reported previously that quercetin and its glycosides exert a variety of biological effects, including antioxidant (27), anti-inflammatory (28), and anticancer (29) effects. We demonstrated that jaboticabin (2) and quercetin derivatives (5 and 6) isolated from the black raspberry fruits exhibited higher DPPH radical scavenging activity than α-tocopherol at the same concentration. Therefore, the antioxidative activity of black raspberry fruits may be attributed to various phenolic compounds including jaboticabin and flavonoids.
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