Antioxidant Activity of Phenolics in Leaves of ... - ACS Publications

Antioxidant Activity of Phenolics in Leaves of Three Red Pepper (Capsicum annuum) Cultivars ... For a more comprehensive list of citations to this art...
0 downloads 0 Views 1MB Size
Download PDF
Article pubs.acs.org/JAFC

Antioxidant Activity of Phenolics in Leaves of Three Red Pepper (Capsicum annuum) Cultivars Woo-Ri Kim,† Eun Ok Kim,† Kyungsu Kang,† Sarangerel Oidovsambuu,† Sang Hoon Jung,† Byung Sup Kim,‡ Chu Won Nho,† and Byung-Hun Um*,† †

Functional Food Center, Korea Institute of Science and Technology (KIST) Gangneung Institute, Gangneung 210-340, Korea Department of Plant Science, Gangneung-Wonju National University, Gangneung 210-702, Korea

‡

ABSTRACT: The antioxidant properties and phenolic profiles were first investigated in this paper on the leaves of three red pepper cultivars, Blackcuban (BCPL), Hongjinju (HPL), and Yeokgang-hongjanggun (YHPL). Of the ethanol extract of the three cultivars, BCPL showed potent antioxidant activities against the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and the 2,2azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical. Nine antioxidative compounds from the red pepper leaves were isolated and identified as one polyamine phenolic conjugate, N-caffeoylputrescine (1); three chlorogenic acid derivatives, 5-Ocaffeoylquinic acid (2), 5-O-caffeoylquinic acid methyl ester (4), and 5-O-caffeoylquinic acid butyl ester (9); one anthocyanin, delphinidin-3-[4-trans-coumaroyl-L-rhamnosyl(1→6)glucopyranoside]-5-O-glucopyranoside (3); and four flavone glycosides, luteolin-7-O-apiofuranosyl(1→2)glucopyranoside (5), luteolin-7-O-glucopyranoside (6), apigenin 7-O-apiofuranosyl(1→2)glucopyranoside (7), apigenin-7-O-glucopyranoside (8). 1 and 3 had the greatest potential for radical-scavenging activity and HepG2 cells protecting effect against oxidative stress. BCPL exhibited the highest content of 1 and 3. Of the three cultivars BCPL may be considered a good source of antioxidants. KEYWORDS: Capsicum annuum, red pepper leaves, phenolic compounds, anthocyanin, antioxidant effects

■

INTRODUCTION Recently, researchers have actively studied new sources to provide safe natural antioxidant additives for foods and cosmetics. This is, in part, because some synthetic antioxidants commonly used to preserve food and cosmetic base materials, such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and tert-butylhydroquinone (TBHQ), are suspected to be toxic.1 Thus, there is a need to identify newer antioxidants that can scavenge several free radicals and prevent multiple diseases, including cancer, atherosclerosis, diabetes, inflammation, and aging.2,3 Natural antioxidants have become the target of many studies searching for sources of potentially safe, effective, and cheap antioxidants.4 Pepper (Capcicum annuum L.), belonging to the plant family Solanaceae, is the most highly consumed worldwide season-free vegetable. In Korea, it is the most important spice in food, as it is one of the most important ingredients in kimchi, which is a traditional fermented food.5 The red pepper fruit contains a high amount of provitamin A (β-carotene and β-cryptoxanthin).6 With respect to flavonoids, most studies have concentrated only on flavonoid aglycones (quercetin and luteolin) obtained after hydrolysis of red pepper fruits.7 Red pepper consumption and research have primarily focused on its fruits. Traditionally, however, red pepper leaves have also been eaten as cooked vegetables after blanching in hot water. Red pepper leaves can be a good source of food and cosmeceutical materials, including antioxidants. Recently, red pepper leaves have been reported to have antimutagenic, antioxidant, antimicrobial, and tyrosinase inhibitory activities.8 In addition, two flavonoids, apigenin and apigenin 7-Oapiofuranosyl(1→2)glucopyranoside, have been identified in pepper leaves.9 © 2013 American Chemical Society

The present research aimed to investigate phenolic compound profiles and antioxidant properties in the red pepper leaves of three cultivars, Blackcuban (BCPL), Hongjinju (HPL), and Yeokgang-hongjanggun (YHPL). The isolation and identification of phenolic compounds from the red pepper leaves of three cultivars, as well as the evaluation of antioxidant properties (through DPPH and ABTS radical-scavenging activity) and HepG2 cells protecting effects against tert-butyl hydroperoxide (t-BHP) induced oxidative stress are reported in this paper.

■

MATERIALS AND METHODS

Plant Materials. Leaves of three red pepper cultivars (BCPL, HPL, and YHPL) were obtained from the Department of Plant Science at Gangneung-Wonju National University in Gangneung, Gangwondo, Korea. Seeds of three red pepper cultivars were sowed and cultured in a glass house (20−30 °C) for 60 days. The cultured seedlings were transplanted on May 24, 2011, and cultivated in the fields under an experimental vinyl house (20−35 °C) of Gangneung-Wonju National University in Gangneung. After 90 days of cultivation, the full-grown leaves were collected. The fresh pepper leave samples were immediately stored at −20 °C until analysis. General Procedure. 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH), 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and potassium persulfate for ABTS, as well as Folin− Ciocalteu’s phenol reagent, trolox, quercetin, tannic acid, gallic acid, trifluoroacetic acid (TFA), and formic acid, were purchased from Sigma Chemicals (St. Louis, MO, USA). Analytical grade acetonitrile Received: Revised: Accepted: Published: 850

July 10, 2013 October 1, 2013 October 2, 2013 October 2, 2013 dx.doi.org/10.1021/jf403006c | J. Agric. Food Chem. 2014, 62, 850−859

Journal of Agricultural and Food Chemistry

Article

and distilled HPLC grade water were purchased from Fisher Scientific (Pittsburgh, PA, USA). Column chromatography was performed over silica gel (Merck, Darmstadt, Germany), Sephadex LH-20 (Pharmacia, Uppsala, Sweden), and Amberlite XAD-7 (Sigma Chemicals). Sodium carbonate anhydrous (Na2CO2) and aluminum chloride (AlCl3) were purchased from Shinyo Pure Chemical Co. Ltd. (Osaka, Japan). UV−vis absorption spectra were measured on a Synergy HT Multimicroplate reader (Bio-Tek instrument, Winooski, VT, USA). The preparative HPLC separations were performed using a JASCO PU-2086 Plus pump, an MD-2015 detector, a Rheodyne injector with a 2 mL sample loop, and an FC204 fraction collector (Gilson Scientific Ltd., Luton, Bedfordshire, UK). 1H and 13C nuclear magnetic resonance (NMR) spectra were acquired on a Varian Unity INOVA 500 spectrometer for the 1D and 2D NMR experiments in CD3OD. The chemical shifts are expressed in parts per million (ppm), and the coupling constants are in hertz (Hz). An Agilent LC-MS (Agilent Technologies, Waldbronn, Germany) equipped with UV detectors, quaternary pump, and quadrupole LC-MS 6120 was used with ChemStation as the operation system program. Radical-Scavenging Activity (Offline Spectrophotometric ABTS+ and DPPH+ Assay). ABTS radical-scavenging activity of the test extracts and compounds were determined using an ABTS+ decolorization assay according to the method of Argoti et al.10 with minor modifications. To the ABTS liquid substrate system was added 2.45 mM potassium persulfate in a stoichiometric ratio of 1:0.5 (v/v). The mixture was allowed to stand in the dark at room temperature for 8 h. Then, the nine purified compounds (100 μL) were allowed to react with 100 μL of the ABTS radical solution. The reaction mixture was incubated for 5 min at room temperature in dark conditions, and the absorbance was measured at 734 nm. The change in absorbance with respect to the control (containing ABTS+ solution only without the sample, expressed as 100% free radicals) was calculated as the IC50 value. The known antioxidants ascorbic acid and trolox were used as positive controls. The free radical-scavenging activity of the test extracts and compounds were determined by the DPPH assay.10 Briefly, the reaction mixture was contained in an ethanol solution of DPPH (100 μL) and purified phenolic compounds of three different concentrations (100 μL). The reaction mixture was incubated for 1 h at room temperature in dark conditions, and the absorbance was measured at 517 nm. IC50 values against three radical-scavenging activities were determined by regression analysis of the results obtained at three different concentrations of the sample. Ascorbic acid and trolox were used as positive controls. Measurement of Total Phenolics and Flavonoids Content. Total phenolics was determined using the reported method.11 A 20 μL sample aliquot of extract or gallic acid/tannic acid standard (50−500 μg/mL) was mixed with distilled water (1.58 mL) followed by 2 N Folin−Ciocalteau’s reagent (20 μL). After the mixture had been vortexed and incubated at room temperature for 5 min, 20% sodium carbonate solution (100 μL) was added. Samples were vortexed and kept at room temperature for 30 min. Absorbance of the blue solution was recorded at 730 nm. The total phenolic content was expressed as milligrams of gallic acid equivalent (mg GAE/g) and tannic acid equivalent (mg TAE/g) per gram of sample. Total flavonoid content was determined by the aluminum chloride colorimetric method using quercetin as a standard.12 Briefly, the extracts were dissolved in EtOH. Then, the sample solution (100 μL) was mixed with an aqueous solution of 2% aluminum chloride (100 μL). After 5 min of incubation at ambient temperature, the absorbance of the supernate was measured at 435 nm. The total flavonoid content was expressed as milligrams of quercetin equivalents per gram of sample. Screening of Antioxidant Activity with Online HPLC-ABTS System. To test the antioxidant activity of the extracts directly, the online HPLC-ABTS screening system was applied. This system consisted of an Agilent 1200 analysis HPLC system (Agilent Technologies, Santa Clara, CA, USA) fitted with an additional pump to supply the ABTS radical solution. For the ABTS radical reagent, a 2 mM ABTS stock solution containing 3.5 mM potassium persulfate was

prepared in water and diluted 8-fold in HPLC grade water. This solution was incubated overnight in darkness at room temperature for radical stabilization. Antioxidant capacity was measured according to the previous method.13 Briefly, 10 μL of three cultivars of pepper leaf extract solutions was injected into the online HPLC-ABTS system. The individual compounds were separated with a column (Kromasil 100-5 C18, 250 × 4.6 mm, 5 μm) in the solvent system of 0.1% TFA in acetonitrile (A) and 0.1% TFA in water (B) with a 1 mL/min flow rate. The gradient conditions were as follows: 0−10 min, 20% A; 10− 30 min, 20−50% A; and then returning to the initial conditions. The column was kept at 25 °C during the entire sequence. The ABTS radical solution was supplied with a flow rate of 0.5 mL/min. The chromatogram was recorded at 330 and 530 nm as a positive peak, and the visible detector was set at 734 nm to measure the decrease of ABTS radicals as a negative peak. The data were analyzed by ChemStation software (Agilent Technology). Extraction and Isolation of Anthocyanin and Phenolics from Red Pepper Leaves. The leaves (1 kg) of three different red pepper cultivars were extracted three times with EtOH containing 0.1% HCl in a dark environment during 24 h at room temperature. The acidic ethanol solvent was evaporated under reduced pressure to afford the BCPL (76.10 g), HPL (49.99 g); and YHPL (45.27 g) extracts. The BCPL extract was resuspended in water containing 0.1% HCl and was partitioned with n-hexane (n-Hex), methylene chloride (CH2Cl2), ethyl acetate (EtOAc), n-butanol (n-BuOH), and water (H2O). All fractions were then concentrated in vacuo (n-Hex fraction, 11.1 g; CH2Cl2 fraction, 3.06 g; EtOAc fraction, 3.56 g; n-BuOH fraction, 29.69 g; H2O fraction, 24.77 g). The EtOAc fraction was chromatographed by a preparative HPLC system (Waters prep-HPLC, Hydrosphere C18 column (250 × 20 mm, 5 μm)) eluting with an ACN/0.1% FA in H2O (10:0 to 100:0, v/v) gradient condition. Three fractions were obtained as follows: A (0.31 g), B (0.16 g), and C (0.27 g). The A and B fractions were purified by the preparative HPLC system (JASCO PU-2086 Plus pump, MD-2015 detector); compound 4 (1.62 mg) and compound 9 (5.80 mg) were obtained, respectively. The C fraction was chromatographed by the preparative HPLC system to yield three subfractions (EA-C-1−EA-C-3). Subfractions EA-C-1 (60 mg), EA-C-2 (27.5 mg), and EA-C-3 (86.95 mg) were further chromatographed by the same purification procedure on preparative HPLC and yielded a pure compound 7 (3.37 mg) and compound 8 (80 mg). The purple n-BuOH fraction (3 g) was chromatographed for anthocyanin by Amberlite XAD-7 column chromatography eluting with acidic MeOH containing 0.1% HCl/H2O (40:60 to 70:30) to yield two fractions (Bu-A and Bu-B). The Bu-B fraction (1.2 g) was chromatographed by the preparative HPLC system to yield two subfractions (Bu-B-1 and Bu-B-2). Subfraction Bu-B-1 (84.2 mg) was rechromatographed by Sephadex LH-20 column chromatography eluting with MeOH containing 0.1% TFA, and a pure compound 1 (21.76 mg) and compound 3 (32 mg) were separated. The extracts of HPL and YHPL were not isolated as the same chemical components from the HPLC chromatogram, and the LC-MS data of HPL and YHPL as BCPL are presented in Figure 2. The YHPL extract was resuspended to isolate the compounds except for the same chemical components of BCPL in water. It was partitioned three times with n-Hex, CH2Cl2, EtOAc, n-BuOH, and H2O. All fractions were then concentrated in vacuo (n-Hex fraction, 16.14 g; CH2Cl2 fraction, 5.76 g; EtOAc fraction, 1.7 g; n-BuOH fraction, 10.26 g; H2O fraction, 14.0 g). The EtOAc fraction was purified by the preparative HPLC system, and compound 2 (56.66 mg) and compound 5 (7.83 mg) were obtained. The H2O fraction (2 g) was fractionated using vacuum liquid chromatography with silica-C18. Elution was carried out successively with solvents of increasing nonpolar MeOH/H2O (5:95 to 100:0, v/v) to afford compound 6 (4.08 mg). Cell Culture and Cell Viability Assay. Human hepatoma HepG2 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). HepG2 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, UT, USA), 100 units/mL penicillin, and 100 μg/mL streptomycin. The cells were cultured in a 851

dx.doi.org/10.1021/jf403006c | J. Agric. Food Chem. 2014, 62, 850−859

Journal of Agricultural and Food Chemistry

Article

humidified atmosphere of 5% CO2 at 37 °C. For chemical treatments, HepG2 cells were cultured in DMEM without FBS to reduce direct interaction between the phytochemicals and FBS. Protective activities of the isolated compounds from the three pepper cultivar leaves against t-BHP-induced cell death were measured using the cell viability assay as previous described.14 Briefly, HepG2 cells (1 × 104 cells/well) were plated in 96-well plates and incubated for 24 h. Cells were then preincubated with the isolated (10−20 μg/mL) compounds from three pepper cultivar leaves and epigallocatechin gallate (EGCG, 10 μM) in serum-free DMEM for 24 h. Cells were washed with Dulbecco’s phosphate-buffered saline (DPBS) and treated with t-BHP (250 μM) in serum-free DMEM for 3 h. Then, cell viability was measured using the EZ-Cytox Cell Viability Assay Kit (Daeil Lab Service, Seoul, Korea). Quantification of Phenolics by HPLC. The nine phenolic compounds isolated from red pepper leaves of the three different cultivars were quantified by an HPLC method. HPLC analysis was performed with an Agilent 1200 HPLC system consisting of a binary pump (G1312A), autosampler (G1367B), diode array detector (G1315D), and degasser (D1379B). The separation of each compound was carried out on a Kromasil 100-5 C18 column (250 × 4.6 mm, 5 μm) with 1 mL/min flow rate in a solvent system of acetonitrile containing 0.1% TFA (A) and water containing 0.1% TFA (B). The gradient condition used was as follows: 0−10 min, 20% A; 10−30 min, 20−50% A; and then returning to the initial conditions. The chromatogram was detected at 330 nm. Calibration curves were constructed with each compound in a range of 0.1−1000 μg/mL. A high linearity (r2 > 0.999) was obtained for each standard curve. Statistical Analysis. Data were expressed as the mean ± standard deviation (SD). Statistical analysis included one-way analysis of variance (ANOVA) followed by Tukey’s Honestly Significant Difference (HSD) test, and Pearson correlation test for correlation between variables were performed using IBM SPSS statistics 21. p values HPL > YHPL. On the other hand, the order of total flavonoid content was found to be YHPL > BCPL > HPL. Online HPLC−ABTS+ Assays as Activity Guide. To investigate the major compounds and to measure their ABTS radical-scavenging activity, the ethanol extracts of the three red pepper cultivar leaves were subjected to the online HPLC− ABTS+ screening system.15 This methodology allows rapid pinpointing of antioxidants in complex mixtures through postcolumn mixing of separated analytes with radical solutions. The resulting reduction signal was detected as a negative peak by an absorbance detector (at 734 nm for ABTS+ scavenging activity). After this online radical-scavenging screening of the extracts, it was easy to detect the active components present in each extract. Without isolation by the online HPLC−ABTS+ assay system, peaks 1, 2, and 4 would have been easily overlooked because of their small quantity (Figure 2). These compounds were negligible in HPLC chromatogram at 330 nm, but it was very active in the online HPLC-ABTS+ assay system. Hence, the online ABTS assay system was an efficient method for the purification of antioxidants based on activity-guided chromatographic separation and isolation from natural resources. As shown in Figure 2, the BCPL chemical components were almost similar to HPL, but YHPL was different from BCPL and HPL. In addition, the anthocyanin from the chromatogram at 530 nm existed only for BCPL. Therefore, this indicated that major and minor antioxidants (peaks 1−9) were isolated using preparative HPLC and open column chromatography. Isolation and Identification of Anthocyanin and Phenolic Compounds. To identify peaks 1, 3, 4, and 7−9 of the HPLC chromatogram of the BCPL extract and peaks 1, 2, 5, and 6 of the HPLC chromatogram of the YHPL extract, we partitioned with n-Hex, CH2Cl2, EtOAc, n-BuOH, and H2O. The EtOAc and n-BuOH fractions of BCPL and EtOAc and H2O fraction of YHPL were subjected to silica gel RP-C18 chromatography and reverse phase preparative-HPLC. The resulting compounds 1−9 were isolated from BCPL and YHPL (Figure 3). Compounds 1−9 were characterized and identified by UV and ESI-MS, as well as 1D and 2D NMR. The spectral data of compounds 1−9 agreed with earlier published data: compound 1,16 compound 2,15 compound 3,17 compound 4,18

■

RESULTS AND DISCUSSION Radical Scavenging Activity of the Extracts of Three Red Pepper Cultivar Leaves. The antioxidative activities of the ethanol extracts from the pepper leaves of three different cultivars (BCPL, HPL, and YHPL) were evaluated by DPPH and ABTS assay with ascorbic acid and trolox as standards. As shown in Table 1, the DPPH radical-scavenging activity (IC50) Table 1. Radical-Scavenging Activity of the Extracts of Three Pepper Cultivar Leavesa radical-scavenging activity IC50 (μg/mL) extract BCPL HPL YHPL ascorbic acid trolox

DPPH 49.2 62.2 54.3 1.3 1.6

± ± ± ± ±

6.1b 3.4a 4.5ab 0.2c 0.6c

ABTS+ 26.2 41.9 33.7 0.3 0.3

± ± ± ± ±

3.2b 3.1a 3.9ab 0.1c 0.0c

a Values represent the means ± SD from triplicate experiments. Different letters in the same column denote a significant difference according to Tukey’s test, p < 0.05.

of BCPL was 49.2 ± 6.1 μg/mL, lower than that of YHPL (54.3 ± 4.5 μg/mL) or HPL (62.2 ± 3.4 μg/mL). Similarly, the ABTS radical-scavenging activity (IC50) of BCPL was 26.2 ± 3.2 μg/mL, lower than that of YHPL (33.7 ± 3.9 μg/mL) or HPL (41.9 ± 3.1 μg/mL). Therefore, the order of antioxidant effects by radical-scavenging activity of the three red pepper leaves was BCPL > YHPL > HPL. Content of Total Phenolics and Total Flavonoids. Folin−Ciocalteu’s assay is a frequently used screening method 852

dx.doi.org/10.1021/jf403006c | J. Agric. Food Chem. 2014, 62, 850−859

Journal of Agricultural and Food Chemistry

Article

Figure 1. Total phenolic (A) and flavonoid contents (B) of three red pepper cultivar leaves. The total phenolic content was expressed as gallic acid equivalent (GAE) and tannic acid equivalent (TAE). The total flavonoid content was expressed as quercetin equivalents. BCPL, Blackcuban; HPL, Hongjinju; YHPL, Yeokgang-hongjanggun; FW, fresh weight. Each bar represent the mean ± SD from triplicate experiments. Different letters in the bar denote a significant difference according to Tukey’s test, p < 0.05.

Figure 2. Chromatogram and UV spectrum of the online HPLC-DAD-ABTS screening system. The ethanol extracts from red pepper leaves of three different cultivars, Blackcuban (BCPL), Hongjinju (HPL), and Yeokgang-hongjanggun (YHPL), were analyzed with an HPLC system, and the eluates from the HPLC column were reacted with ABTS radical reagent. The analytes were detected at 330 and 530 nm as a positive peak, and their ABTS radical-scavenging activity was recorded at 734 nm as a negative peak.

H-12); 13C NMR (125 MHz, CD3OD) δ 169.6 (C-9), 148.9 (C-4), 146.8 (C-5), 142.4 (C-7), 128.1 (C-1), 122.2 (C-2), 118.2 (C-8), 116.4 (C-3), 115.0 (C-6), 40.5 (C-14), 39.6 (C11), 27.6 (C-12), 25.9 (C-13). 5-O-Caffeoylquinic acid (2): pale yellow powder; ESI-MS m/z 355 [M + H]+; 1H NMR (500 MHz, CD3OD) δ 7.52 (1H, d, J = 15.9 Hz, H-7′), 7.04 (1H, d, J = 1.8 Hz, H-2′), 6.94 (1H, dd, J = 8.2 Hz, 1.7 Hz, H-6′), 6.78 (1H, d, J = 8.2 Hz, H-5′), 6.22 (1H, d, J = 15.9 Hz, H-8′), 5.28 (1H, dd, J = 11.7 Hz, 7.4

compound 5,19,20 compound 6,21 compound 7,22 compound 8,13 and compound 9.21 The structural characteristics of isolated phenolics 1−9 are detailed below. N-Caffeoylputrescine (1): apricot powder; ESI-MS m/z 251 [M + H]+; 1H NMR (500 MHz, CD3OD) δ 7.39 (1H, d, J = 15.6 Hz, H-7), 7.00 (1H, d, J = 1.9 Hz, H-6), 6.90 (1H, dd, J = 8.2 Hz, 1.9 Hz, H-2), 6.76 (1H, d, J = 8.1 Hz, H-3), 6.37 (1H, d, J = 15.6 Hz, H-8), 3.34 (2H, d, J = 6.4 Hz, H-11), 2.96 (1H, t, J = 7.2 Hz, 7.2 Hz, H-14), 1.70 (2H, m, H-13), 1.63 (2H, m, 853

dx.doi.org/10.1021/jf403006c | J. Agric. Food Chem. 2014, 62, 850−859

Journal of Agricultural and Food Chemistry

Article

Figure 3. Chemical structures of phenolic compounds (1−9) isolated from red pepper leaves. 1, N-Caffeoyl putrescine; 2, 5-O-caffeoylquinic acid; 3, delphinidin-3-[4-(trans-coumaroyl)-L-rhamnosyl(1→6)glucopyranoside]-5-O-glucopyranoside; 4, 5-O-caffeoylquinic acid methyl ester; 5, luteolin-7O-apiofuranosyl(1→2)glucopyranoside; 6, luteolin-7-O-glucopyranoside; 7, apigenin-7-O-apiofuranosyl(1→2)glucopyranoside; 8, apigenin-7-Oglucopyranoside; 9, 5-O-caffeoylquinic acid butyl ester.

Hz, H-3), 4.14 (1H, t, J = 3.6 Hz, 3.6 Hz, H-5), 3.74 (1H, dd, J = 7.0 Hz, 2.6 Hz, H-4), 2.19 (2H, m, H-2), 2.01 (2H, m, H-6); 13 C NMR (125 MHz, CD3OD) δ 175.4 (C-7), 168.3 (C-9′), 149.6 (C-4′), 147.2 (C-7′), 146.7 (C-3′), 127.5 (C-1′), 123.0 (C-6′), 116.5 (C-5′), 115.0 (C-2′), 114.9 (C-8′), 76.1 (C-1), 72.4 (C-4), 72.0 (C-3), 70.3 (C-5), 38.0 (C-6), 37.8 (C-2). Delphinidin 3-[4-trans-coumaroyl-L-rhamnosyl(1→6)glucopyranoside]-5-O-glucopyranoside (3): amorphous purple powder; ESI-MS m/z 920 [M + H]+; 1H NMR (500 MHz, CD3OD) δ 8.86 (1H, s, H-4), 7.76 (2H, s, H-2′, H-6′), 7.56 (1H, d, J = 15.9 Hz, H-7′′′′′, 7.40 (2H, d, J = 8.6 Hz, H-2′′′′′, H-6′′′′′), 6.98 (1H, s, H-8), 6.97 (1H, s, H-6), 6.80 (2H, d, J = 8.6 Hz, H-3′′′′′, H-5′′′′′), 6.24 (1H, s, H-8′′′′′), 5.52 (1H, d, J = 7.8 Hz, H-1‴), 5.19 (1H, d, J = 7.8 Hz, H-1″), 4.90 (1H, m, H-4′′′′), 4.70 (1H, s, H-1′′′′), 4.05 (1H, m, H-6‴), 3.94 (1H, m, H-6″), 3.87 (1H, m, H-3′′′′), 3.85 (1H, d, J = 3.3 Hz, H5‴), 3.81 (1H, d, J = 3.8 Hz, H-2‴), 3.80 (1H, m, H-6″), 3.79 (1H, m, H-2′′′′), 3.77 (1H, m, H-5′′′′), 3.71 (1H, s, H-6″), 3.70 (1H, m, H-2″), 3.63 (1H, m, H-5″), 3.60 (1H, m, H-3″), 3.59 (1H, m, H-4″), 3.48 (1H, m, H-4‴), 1.00 (3H, d, J = 6.3 Hz, H-6′′′′); 13C NMR (125 MHz, CD3OD) δ 169.7 (C9′′′′′), 169.2 (C-7), 164.4 (C-2), 157.0 (C-9), 156.6 (C-5), 147.7 (C-4′), 147.4 (C-4′′′′′), 146.2 (C-3), 145.8 (C-3′), 145.7 (C-5′), 133.3 (C-4), 131.5 (C-2′′′′′), 131.4 (C-6′′′′′), 127.26 (C-1′′′′′), 117.2 (C-3′′′′′, C-5′′′′′), 116.92 (C-7′′′′′), 114.9 (C-8′′′′′), 113.2 (C-3′, C-6′), 112.9 (C-10), 112.8 (C-1′), 105.7 (C-6), 102.9 (C-1″), 102.1 (C-1″), 101.9 (C-1′′′′), 97.2 (C-8), 78.38 (C-3‴), 77.8 (C-5″), 77.7 (C-3″), 77.4 (C-5‴),

75.2 (C-4′′′′), 74.6 (C-2″), 74.2 (C-2‴), 71.9 (C-2′′′′), 71.2 (C-4‴), 70.7 (C-4″), 70.3 (C-3′′′′), 67.6 (C-5′′′′), 66.9 (C6‴), 61.9 (C-6″), 18.15 (C-6′′′′). 5-O-Caffeoylquinic acid methyl ester (4): oale yellow powder; ESI-MS m/z 369 [M + H]+; 1H NMR (500 MHz, CD3OD) δ 7.52 (1H, d, J = 15.9 Hz, H-7′), 7.04 (1H, d, J = 2.1 Hz, H-5′), 6.95 (1H, dd, J = 8.2 Hz, 2.1 Hz, H-6′), 6.78 (1H, d, J = 8.2 Hz, H-2′), 6.22 (1H, d, J = 15.9 Hz, H-8′), 5.27 (1H, m, H-3), 4.15 (1H, m, H-5), 3.73 (1H, dd, J = 7.3 Hz, 3.1 Hz, H4), 3.69 (3H, s, H-9), 2.21 (2H, m, H-6), 2.16 (2H, m, H-2); 13 C NMR (125 MHz, CD3OD) δ 175.3 (C-7), 168.3 (C-9′), 149.7 (C-4′), 147.2 (C-7′), 146.9 (C-3′), 127.7 (C-1′), 123.0 (C-6′), 116.5 (C-2′), 115.0 (C-5′), 114.9 (C-8′), 75.5 (C-1), 72.8 (C-4), 72.4 (C-3), 70.0 (C-5), 52.7 (C-9), 38.0 (C-6), 37.6 (C-2). Luteolin-7-O-apiofuranosyl(1→2)glucopyranoside (5): brown powder; UV (MeOH) λmax 210, 253, 262sh, 348 nm; ESI-MS m/z 581 [M + H]+; 1H NMR (500 MHz, DMSO-d6) δ 7.45 (1H, dd, J = 8.4 Hz, 2.2 Hz, H-6′), 7.42 (1H, d, J = 2.2 Hz, H-2′), 6.89 (1H, d, J = 8.3 Hz, H-5′), 6.76 (1H, s, H-8), 6.75 (1H, s, H-3), 6.42 (1H, d, J = 2.2 Hz, H-6), 5.35 (1H, s, H-1‴), 5.19 (1H, d, J = 7.3 Hz, H-1″), 3.91 (1H, d, J = 9.3 Hz, H-4‴), 3.73 (1H, m, H-2‴), 3.70 (1H, d, J = 10.4 Hz, H-6″), 3.66 (1H, d, J = 9.3 Hz, H-4‴), 3.51 (1H, d, J = 9.2 Hz, H-2″), 3.48 (1H, s, H-5″), 3.47 (1H, m, H-6″), 3.31 (1H, m, H-4″), 3.29 (1H, m, H-5‴), 3.25 (1H, m, H-3″); 13C NMR (125 MHz, DMSO-d6) δ 181.9 (C-4), 169.8 (C-9), 164.5 (C-2), 162.1 (C-7), 156.9 (C-5), 150.1 (C-3′), 145.7 (C-4′), 121.2 (C-1′), 119.0 (C-6′), 854

dx.doi.org/10.1021/jf403006c | J. Agric. Food Chem. 2014, 62, 850−859

Journal of Agricultural and Food Chemistry

Article

2, 4, and 8 have been isolated from Ligularia fischeri var. spiciformis Nakai, Lonicera confuse, B. parviflora, Euphorbia ebracteolata, and leaves of Lonicera japonica, Erigeron breviscapus, and Sarcandra glabra.13,18 Compound 3 has been isolated from eggplant.17 In addition, compound 5 has been isolated from celery seeds, celery husk, Campanula patula, Centaurea cyanus, hot pepper fruits, Astragalus eremophilys, and Astragalus vogelii,24,25 and compound 7 has been isolated from Crotalaria podocarpa, Conocaryum calleryanum, and Artabotrys hexapetalus and leaves of Capsicum annuum.9,22,26 Finally, compounds 6 and 9 have been isolated from the leaves of Codonopsis nervosa and Elsholtzia rugulosa, flowers of Ligustrum lucidum, and leaves of Callicarpa nudiflora.21,27−30 Antioxidant Activities of Anthocyanin and Phenolic Compounds Isolated from Three Red Pepper Cultivar Leaves. The antioxidant activities of the isolated compounds 1−9 were assayed using the different assay systems: (1) scavenging activity of free radicals based on chemical trapping using DPPH and ABTS assay methods; (2) protective activity of compounds against t-BHP-induced HepG2 cell death. The free radical chain reaction is widely accepted as a common mechanism of lipid peroxidation. Radical scavengers may directly quench and react with peroxide radicals to terminate peroxidation chain reactions. Assays based upon the use of DPPH and ABTS+ are the most popular spectrophotometric methods for the determination of the antioxidant capacity of foods, beverages, and vegetable extracts.31 In the DPPH assay, the stable DPPH in alcoholic solution reduces to the yellow diphenylpicrylhydrazine due to the formation of the nonradical from DPPH by accepting an electron or hydrogen radical in the presence of an antioxidant. The DPPH radicalscavenging activities of nine phenolic compounds isolated from red pepper leaves are shown in Table 2. Most of the phenolic compounds significantly scavenged the DPPH radical in a dosedependent manner. Among the nine phenolics, compounds 3 (IC50 = 3.5 ± 0.4 μM) and 1 (IC50 = 4.8 ± 0.0 μM) showed the highest DPPH free radical-scavenging activity, of which

115.8 (C-5′), 113.2 (C-2′), 108.4 (C-1‴), 105.3 (C-10), 102.9 (C-3), 99.1 (C-6), 97.7 (C-1″), 94.4 (C-8), 79.4 (C-2‴), 76.8 (C-5″), 76.2 (C-2‴), 75.9 (C-2″), 73.8 (C-5‴), 72.8 (C-3″), 69.6 (C-4″), 64.1 (C-4‴), 60.4 (C-6″). Luteolin-7-O-glucopyranoside (6): brown powder; ESI-MS m/z 449 [M + H]+; 1H NMR (500 MHz, CD3OD) δ 7.42 (1H, s, H-2′), 7.41 (1H, d, J = 4.5 Hz, H-6′), 6.90 (1H, d, J = 8.3 Hz, H-5′), 6.79 (1H, s, H-8), 6.60 (1H, s, H-3), 6.49 (1H, s, H-6), 6.49 (1H, s, H-7), 5.07 (1H, d, J = 6.7 Hz, H-1″), 3.94 (1H, d, J = 11.8 Hz, H-6″), 3.73 (1H, m, H-6″), 3.55 (1H, m, H-5″), 3.51 (1H, m, H-3″), 3.50 (1H, m, H-2″), 3.42 (1H, m, H-4″); 13 C NMR (125 MHz, CD3OD) δ 184.1 (C-4), 167.0 (C-2), 164.9 (C-7), 162.9 (C-5), 159.0 (C-9), 151.3 (C-3′), 147.2 (C4′), 123.4 (C-1′), 120.5 (C-2′), 116.8 (C-5′), 114.2 (C-6′), 107.2 (C-10), 104.1 (C-3), 101.6 (C-1″), 101.1 (C-6), 96.0 (C8), 78.4 (C-5″), 77.9 (C-3″), 74.7 (C-2″), 71.3 (C-4″), 62.4 (C-6″). Apigenin 7-O-apiofuranosyl(1→2)glucopyranoside (7): yellow powder; UV (MeOH) λmax 210, 268, 340 nm; ESI-MS m/z 565 [M + H]+; 1H NMR (500 MHz, CD3OD) δ 7.89 (2H, d, J = 8.7 Hz, H-2′, H-6′), 6.93 (2H, d, J = 9.0 Hz, H-3′, H-5′), 6.81 (1H, dd, J = 2.0 Hz, 15.0 Hz, H-8), 6.66 (1H, d, J = 1.2 Hz, H-3), 6.48 (1H, d, J = 16.5 Hz, 2.0 Hz, H-6), 5.46 (1H, d, J = 1.6 Hz, H-1‴), 5.15 (1H, d, J = 7.3 Hz, H-1″), 4.04 (1H, d, J = 9.7 Hz, H-4‴), 3.95 (1H, t, J = 1.5 Hz, 1.5 Hz, H-2‴), 3.93 (1H, m, H-6″), 3.82 (1H, d, J = 9.6 Hz, H-4‴), 3.71 (1H, m, H6″), 3.68 (1H, m, H-2″), 3.66 (1H, m, H-3″), 3.54 (1H, m, H5″), 3.54 (2H, m, H-5‴), 3.41 (1H, t, J = 9.2 Hz, 9.2 Hz, H-4″); 13 C NMR (125 MHz, CD3OD) δ 184.1 (C-4), 168.1 (C-9), 166.9 (C-2), 164.0 (C-7), 163.0 (C-4′), 159.0 (C-5), 129.6 (C2′, C-6′), 123.2 (C-1′), 117.0 (C-3′, C-5′), 110.9 (C-1‴), 107.2 (C-10), 104.2 (C-3), 101.1 (C-6), 100.1 (C-1″), 95.9 (C-8), 80.7 (C-3‴), 78.6 (C-2″), 78.4 (C-3‴), 78.3 (C-5″), 78.1 (C2‴), 75.4 (C-4‴), 71.2 (C-4″), 65.8 (C-5‴), 62.5 (C-6″). Apigenin-7-O-Glucopyranoside (8): yellow powder; ESI-MS m/z 433 [M + H]+; 1H NMR (500 MHz, CD3OD) δ 7.89 (2H, d, J = 8.3 Hz, H-2′, H-6′), 6.93 (2H, d, J = 8.3 Hz, H-3′, H-5′), 6.82 (1H, s, H-8), 6.66 (1H, s, H-3), 6.49 (1H, s, H-6), 5.08 (1H, d, J = 6.1 Hz, H-1″), 3.94 (1H, d, J = 11.7 Hz, H-6″), 3.72 (1H, m, H-6″), 3.55 (1H, m, H-5″), 3.51 (1H, m, H-3″), 3.50 (1H, m, H-2″), 3.41 (1H, m, H-4″); 13C NMR (125 MHz, CD3OD) δ 184.1 (C-4), 166.9 (C-2), 165.0 (C-7), 163.1 (C5), 163.0 (C-4′), 159.1 (C-9), 129.8 (C-2′, C-6′), 123.1 (C-1′), 117.0 (C-3′, C-5′), 107.1 (C-10), 104.1 (C-3), 101.7 (C-1″), 101.1 (C-6), 96.0 (C-8), 78.2 (C-5″), 77.8 (C-3″), 74.6 (C-2″), 71.3 (C-4″), 62.4 (C-6″). 5-O-Caffeoylquinic acid butyl ester (9): pale yellow powder; ESI-MS m/z 411 [M + H]+; 1H NMR (500 MHz, CD3OD) δ 7.52 (1H, d, J = 15.9 Hz, H-7′), 7.04 (1H, d, J = 2.0 Hz, H-5′), 6.95 (1H, dd, J = 8.2 Hz, 2.0 Hz, H-6′), 6.78 (1H, d, J = 8.2 Hz, H-2′), 6.21 (1H, d, J = 15.9 Hz, H-8′), 5.26 (1H, m, H-3), 4.14 (1H, m, H-5), 3.75 (1H, dd, J = 7.0 Hz, 2.7 Hz, H-4), 2.22 (2H, d, J = 5.4, H-2), 2.17 (2H, m, H-9), 1.99 (2H, m, H-6), 1.61 (2H, m, H-11), 1.34 (2H, m, H-12), 0.89 (3H, t, J = 7.4 Hz, 7.4 Hz, H-13); 13C NMR (125 MHz, CD3OD) δ 175.0 (C-7), 168.1 (C-9′), 149.7 (C-4′), 147.3 (C-7′), 146.9 (C-3′), 127.6 (C-1′), 123.0 (C-6′), 116.5 (C-2′), 115.0 (C-5′), 114.9 (C-8′), 75.5 (C-1), 72.4 (C-4), 72.1 (C-3), 70.1 (C-5), 38.0 (C-2), 37.9 (C-6), 37.6 (C-9), 31.6 (C-10), 20.0 (C-11), 14.1 (C-12). All of these compounds (1−9) were identified for the first time in this paper as constituents of pepper leaves, except for compound 7.9 Compound 1 has been found in the leaves of Nicotiana tabacum and yellow bell pepper,16,23 and compounds

Table 2. Radical-Scavenging Activity of the Phenolic Compounds Isolated from Red Pepper Leavesa radical-scavenging activity IC50 (μM) no.

compound

DPPH+

ABTS+

1 2 3

N-caffeoylputrescine 5-O-caffeoylquinic acid delphinidin-3-[4-(transcoumaroyl)-L-rhamnosyl-(1→6) glucopyranoside]-5-Oglucopyranoside 5-O-caffeoylquinic acid methyl ester luteolin-7-O-apiofuranosyl-(1→ 2)-glucopyranoside luteolin-7-O-glucopyranoside apigenin-7-O-apiofuranosyl(1→2) glucopyranoside apigenin-7-O-glucopyranoside 5-O-caffeoylquinic acid butyl ester ascorbic acid trolox

4.8 ± 0.0f 37.0 ± 3.4d 3.5 ± 0.4f

9.2 ± 0.8f 68.1 ± 1.7bc 4.4 ± 0.3f

20.4 ± 1.1e

38.0 ± 0.8e

18.8 ± 1.5e

76.2 ± 8.6b

39.5 ± 4.7cd >200a

61.4 ± 8.7cd 133.9 ± 3.0a

4 5 6 7 8 9

153.7 51.7 7.4 6.4

± ± ± ±

13.2b 0.7c 1.1f 2.4f

63.9 55.6 1.7 1.2

± ± ± ±

4.4cd 0.5d 0.6g 0.0g

a Values represent means ± SD from triplicate experiments. Different alphabet letters in the same column denote a significant difference according to Tukey’s test, p < 0.05.

855

dx.doi.org/10.1021/jf403006c | J. Agric. Food Chem. 2014, 62, 850−859

Journal of Agricultural and Food Chemistry

Article

Figure 4. Protective effects of the compounds from pepper leaves against HepG2 cell death induced by tert-butyl hydroperoxide (t-BHP). Each bar represents the mean ± SD from triplicate experiments. Different letters over the bars denote a significant difference according to Tukey’s test, p < 0.05. 1, N-caffeoylputrescine; 2, 5-O-caffeoylquinic acid; 3, delphinidin-3-[4-(trans-coumaroyl)-L-rhamnosyl(1→6)glucopyranoside]-5-Oglucopyranoside; 4, 5-O-caffeoylquinic acid methyl ester; 5, luteolin-7-O-apiofuranosyl(1→2)glucopyranoside; 6, luteolin-7-O-glucopyranoside; 7, apigenin-7-O-apiofuranosyl(1→2)glucopyranoside; 8, apigenin-7-O-glucopyranoside; 9, 5-O-caffeoylquinic acid butyl ester.

activities were stronger than that of ascorbic acid (IC50 = 7.4 ± 1.1 μM) and trolox (IC50 = 6.4 ± 2.4 μM), but was not statistically significant. The scavenging activities of the remaining compounds were in the following order: 5 > 4 > 2 ≥ 6 ≥ 9 > 8 > 7. The scavenging effects on the ABTS radical also showed marked differences according to the compounds (Table 2). Among them, the highest inhibition effects on the ABTS radical cation were found in compounds 3 (IC50 = 4.4 ± 0.3 μM) and 1 (IC50 = 9.2 ± 0.8 μM), similar to the results obtained from the DPPH assay. Meanwhile, the antioxidant effects for the ABTS radical scavenging of two phenolic compounds were lower than those of ascorbic acid (IC50 =1.7 ± 0.6 μM) and trolox (IC50 = 1.2 ± 0.0 μM), a well-known antioxidant. The scavenging activities of the remaining compounds were in the following order: 4 > 9 ≥ 6 ≥ 8 ≥ 2 ≥ 5 > 7. In previous research, the radical-scavenging activities by DPPH assay of compounds 2,10 6,11 and 832 have been similarly reported with results of compounds isolated from pepper leaves as 54.8 ± 1.4, 37.9 ± 1.6, and 44.7 μM, respectively. The in vitro ability to protect HepG2 cells from oxidative damage was evaluated for the isolated nine compounds (Figure 4). The protective effects of the compounds against oxidative stress were evaluated; t-BHP treatment for 3 h significantly decreased cell viability (3.8 ± 2.4%) compared to the control (100 ± 4.0%). Pretreatments using the compounds (10 and 20 μg/mL) increased cell viability in a dose-dependent manner. Particularly, compounds 1, 3, and 4 enhanced cell viability up to 98.3 ± 9.2, 66.4 ± 2.9, and 50.8 ± 6.1% at a concentration of 20 μg/mL, respectively. Table 3 summarizes the correlation between DPPH and ABTS radical-scavenging activity and HepG2 cells protective effect. A positive correlation (p < 0.05)

was observed between DPPH and ABTS radical-scavenging activity, whereas a negative correlation (p < 0.01) was observed between DPPH and ABTS radical-scavenging activity and the protective effect of the compounds in the cell culture system. These results suggest that the cell protective effect was exerted through a radical-scavenging mechanism. Most of the phenolic compounds expected from compounds 7 and 8 isolated from red pepper leaves had potent protective effects against t-BHPinduced HepG2 cell death, as well as scavenging activity on DPPH and ABTS radicals. Therefore, 1 and 3 of the phenolic compounds included in red pepper leaves are potent free radical scavengers and may be considered an excellent source of antioxidants. Phenolic Contents in Three Red Pepper Cultivar Leaves. The content of nine phenolic compounds in three red pepper cultivar leaves was determined by HPLC analysis. As shown in Table 4, red pepper leaves of different cultivars contain different ratios and types of polyphenols. The sums of identified phenolic compounds 1−9 of BCPL, HPL, and YHPL are 6.23 ± 0.68, 4.16 ± 0.15, and 2.76 ± 0.13 mg/g, respectively (Table 4), which correspond to the total phenolic contents of the three extracts (Figure 1). The most abundant phenolic compound found in BCPL was compound 7 (3.53 ± 0.28 mg/g fresh weight), representing approximately 57% of the total phenolics composition, followed by 3 (0.86 ± 0.01 mg/g) > 1 (0.77 ± 0.05 mg/g) > 4 (0.34 ± 0.16 mg/g) > 6 (0.04 ± 0.01 mg/g) > 5 (0.02 ± 0.00 mg/g), and 9 (0.02 ± 0.01 mg/g). Furthermore, the main phenolic compounds in HPL were 7 (3.28 ± 0.09 mg/g), 2 (0.36 ± 0.02 mg/g), and 8 (0.26 ± 0.01 mg/g), representing approximately 79, 8.7, and 6.3% of the total phenolic composition, respectively. In contrast to BCPL and HPL, the major phenolic compounds of YHPL were 5 (1.83 ± 0.09 mg/g) and 6 (0.37 ± 0.01 mg/g), representing 66 and 13.3% of the total phenolics, respectively, followed by 2 (0.26 ± 0.01 mg/g) and 1 (0.26 ± 0.02 mg/g) > 7 (0.06 ± 0.00 mg/g). Therefore, flavonoid 7 was the main component in BCPL and HPL, whereas flavonoids 5 and 6 were the predominant constituents in YHPL. Anthocyanin 3 was found only in BCPL. Compound 3 was identified for the first time from pepper leaves. Anthocyanins have been categorized as the most important group of water-soluble natural pigment in crops

Table 3. Correlation of Bioactivities of Each Compounda correlation coefficient (r) DPPH DPPH ABTS cell protective effect a

ABTS

cell protective effect

0.774*

−0.772** −0.832**

Correlation is significant at (*) p < 0.05 level or (**) p < 0.01 level. 856

dx.doi.org/10.1021/jf403006c | J. Agric. Food Chem. 2014, 62, 850−859

Article

such as fruits, vegetables, and flowers.33−35 These natural pigments are beneficial to the food industry due to their good properties as food colorants and beneficial human health effects, such as antioxidant, anti-inflammatory, anticancer, antidiabetic, and antiartherogenic properties.11,33 Compound 2 is well-known as a treatment material for its antihistamine, antiviral, antioxidative activity, and DNA-protective activity.13,36,37 Compound 6 is reported on for its antiproliferative, anti-LDL oxidative, and cytotoxic activity, as well as its gelatinase and collagenase inhibition, antifeedant activity, and anti-HBV activity.38−43 Compound 7 is well-known as a treatment material for its anticomplement, anti-inflammatory, liver protection, and antibacterial activities.44−46 Compound 8 is well-known as a treatment material as an antifungal, cytotoxic, antibacterial, and anti-HIV agent.47−49 Finally, compound 9 is well-known as a treatment material due to its antibacterial nature.50 Thus, they represent an effective antioxidant source for commercial food and pharmaceutical uses due to their content, disease treatment, and strong radicalscavenging activities. Individual and total phenolic compounds showed significant differences in cultivars, and BCPL was observed to have the highest phenolic content for compounds 1 and 3, as well as the highest antioxidant capacity (DPPH, IC50 = 49.2 ± 6.1 μg/mL; and ABTS, IC50 = 26.2 ± 3.2 μg/mL). On the basis of the above findings, it is suggested that strong antioxidant activities of BCPL of red pepper leaves were related with high contents of compounds 1 and 3. These results indicate that compounds 1 and 3 may be important factors for the quality of red pepper leaves. Future studies are required to evaluate the various beneficial health effects of phenolic compounds isolated, as well as for red pepper leaves in vitro and in vivo.

a Values represent the mean ± SD from triplicate experiments. Different letters in the same column denote a significant difference according to Tukey’s test, p < 0.05. FW, fresh weight; total, sum of nine compound contents; nd, not detected. 1, N-caffeoylputrescine; 2, 5-O-caffeoylquinic acid; 3, delphinidin-3-[4-(trans-coumaroyl)-L-rhamnosyl(1→6)glucopyranoside]-5-O-glucopyranoside; 4, 5-Ocaffeoylquinic acid methyl ester; 5, luteolin-7-O-apiofuranosyl(1→2)glucopyranoside; 6, luteolin-7-O-glucopyranoside; 7, apigenin-7-O-apiofuranosyl(1→2)glucopyranoside; 8, apigenin-7-Oglucopyranoside; 9, 5-O-caffeoylquinic acid butyl ester. bValues represent the sum of the phenolic components 1−9.

6.23 ± 0.68a 4.16 ± 0.15b 2.78 ± 0.13c 0.02 ± 0.01 nd nd 0.65 ± 0.16 0.26 ± 0.01 nd 3.53 ± 0.28a 3.28 ± 0.09a 0.06 ± 0.00b 0.04 ± 0.01b 0.03 ± 0.00b 0.37 ± 0.01a 0.02 ± 0.00b 0.10 ± 0.01b 1.83 ± 0.09a 0.77 ± 0.05a 0.12 ± 0.02c 0.26 ± 0.02b BCPL HPL YHPL

nd 0.36 ± 0.02 0.26 ± 0.01

0.86 ± 0.01 nd nd

0.34 ± 0.16 0.01 ± 0.00 nd

6 5 4 3 2 1 cultivar

content (mg/g FW)

Table 4. Content of Phenolic Compounds in the Leaves of Three Red Pepper Cultivarsa

7

8

9

sumb

Journal of Agricultural and Food Chemistry

■

AUTHOR INFORMATION

Corresponding Author

*(B.-H.U.) Phone: +82-33-650-3601. Fax.: +82-33-650-3629. E-mail: [email protected]. Funding

This work was supported by an intramural grant (2Z03850) from the Korea Institute of Science and Technology, Gangneung Institute. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We appreciate the assistance of Dr. H. L. Eum and Prof. S. J. Hong in the department of Plant Science, Gangneung-Wonju National University in Gangneung, Gangwondo, Korea, in obtaining the plant material.

■

REFERENCES

(1) Gharavi, N.; Haggarty, S.; El-Kadi, A. O. S. Chemoprotective and carcinogenic effects of tert-butylhydroquinone and its metabolites. Curr. Drug Metab. 2007, 8, 1−7. (2) Crozier, A.; Jaganath, I. B.; Clifford, M. N. Dietary phenolics: chemistry, bioavailability and effects on health. Nat. Prod. Rep. 2009, 26, 1001−1043. (3) Skrovankova, S.; Misurcova, L.; Machu, L. Antioxidant activity and protecting health effects of common medicinal plants. Adv. Food Nutr. Res. 2012, 67, 75−139. (4) Dimitrios, B. Sources of natural phenolic antioxidants. Trends Food Sci. Technol. 2006, 17, 505−512. 857

dx.doi.org/10.1021/jf403006c | J. Agric. Food Chem. 2014, 62, 850−859

Journal of Agricultural and Food Chemistry

Article

(5) Ku, K. M.; Kang, Y. H. Quinone reductase inductive activity of Capsicum annuum leaves and isolation of the active compounds. J. Korean Soc. Appl. Biol. Chem. 2010, 53, 709−715. (6) Minguez-Mosquera, M. I.; Hornero-Mendez, D. Separation and quantification of the carotenoid pigments in red peppers (Capsicum annuum L.), paprika, and oleoresin by reversed-phase HPLC. J. Agric. Food Chem. 1993, 41, 1616−1620. (7) Lee, Y.; Howard, L. R.; Villalon, B. Flavonoids and antioxidant activity of fresh pepper (Capsicum annuum) cultivars. J. Food Sci. 1995, 60, 473−476. (8) Ku, K. M.; Kim, H. S.; Kim, B. S.; Kang, Y. H. Antioxidant activities and antioxidant constituents of pepper leaves from various cultivars and correlation between antioxidant activities and antioxidant constituents. J. Appl. Biol. Chem. 2009, 52, 70−76. (9) Choi, J. G.; Hur, J. M.; Cho, H. W.; Park, J. C. Phenolic compounds from Capsicum annuum leaves showing radical scavenging effect. Saengyak Hakhoechi 2007, 38, 258−262. (10) Argoti, J. C.; Linares-Palomino, P. J.; Salido, S.; Ramirez, B.; Insuasty, B.; Altarejos, J. On-line activity screening for radical scavengers from Baccharis chilco. Chem. Biodiversity 2013, 10, 189− 197. (11) Ben, H. A.; Trigui, M.; Culioli, G.; Blache, Y.; Jaoua, S. Antioxidant constituents from Lawsonia inermis leaves: isolation, structure elucidation and antioxidative capacity. Food Chem. 2011, 125, 193−200. (12) Quettier-Deleu, C.; Gressier, B.; Vasseur, J. Phenolic compounds and antioxidant activities of buckwheat (Fagopyrum esculentum Moench) hulls and flour. J. Ethnopharmacol. 2000, 72, 35−42. (13) Wang, J.; Wang, N.; Yao, X.; Kitanaka, S. Caffeoyl quinic acid derivatives from Bidens parvif lora and their antihistamine release activities. Zhongcaoyao 2006, 37, 966−970. (14) Kang, K.; Jho, E. H.; Lee, H. J. Youngia denticulata protects against oxidative damage induced by tert-butylhydroperoxide in HepG2 cells. J. Med. Food 2011, 14, 1198−1207. (15) Shang, Y. F.; Kim, S. M.; Song, D. G.; Pan, C. H.; Lee, W. J.; Um, B. H. Isolation and identification of antioxidant compounds from Ligularia f ischeri. J. Food Sci. 2010, 75, C530−C535. (16) Mizusaki, S.; Tanabe, Y.; Noguchi, M. New aromatic amide, caffeoylputrescine from callus tissue culture of Nicotiana tabacum. Agric. Biol. Chem. 1970, 34, 972−973. (17) Ichiyanagi, T.; Kashiwada, Y.; Shida, Y.; Ikeshiro, Y.; Kaneyuki, T.; Konishi, T. Nasunin from eggplant consists of cis-trans isomers of delphinidin 3-[4-(p-coumaroyl)-L-rhamnosyl (1→6)glucopyranoside]5-glucopyranoside. J. Agric. Food Chem. 2005, 53, 9472−9477. (18) Zhang, W.; Thi, B. T. H.; Chen, W.; Kong, D.; Li, H. Studies on structure and activity of phenolic compounds from Erigeron breviscapus. Zhongguo Yaoxue Zazhi. (Beijing, China) 2002, 37, 579− 582. (19) Harborne, J. B. Comparative Biochemistry of the Flavonoids; Academic: New York, 1967; 383 pp. (20) Materska, M.; Piacente, S.; Stochmal, A.; Pizza, C.; Oleszek, W.; Perucka, I. Isolation and structure elucidation of flavonoid and phenolic acid glycosides from pericarp of hot pepper fruit Capsicum annuum L. Phytochemistry (Elsevier) 2003, 63, 893−898. (21) Lee, J. Y.; Moon, S. O.; Kwon, Y. J.; Rhee, S. J.; Park, H. R.; Choi, S. W. Identification and quantification of anthocyanins and flavonoids in mulberry (Morus sp.) cultivars. Food Sci. Biotechnol. 2004, 13, 176−184. (22) Wanjala, C. C. W.; Majinda, R. R. T. Flavonoid glycosides from Crotalaria podocarpa. Phytochemistry 1999, 51, 705−707. (23) Park, S.; Jeong, W. Y.; Lee, J. H. Determination of polyphenol levels variation in Capsicum annuum L. cv. Chelsea (yellow bell pepper) infected by anthracnose (Colletotrichum gloeosporioides) using liquid chromatography-tandem mass spectrometry. Food Chem. 2012, 130, 981−985. (24) Perrone, A.; Masullo, M.; Plaza, A.; Hamed, A.; Piacente, S. Flavone and flavonol glycosides from Astragalus eremophilus and Astragalus vogelii. Nat. Prod. Commun. 2009, 4, 77−82.

(25) Lin, L. Z.; Lu, S.; Harnly, J. M. Detection and quantification of glycosylated flavonoid malonates in celery, chinese celery, and celery seed by LC-DAD-ESI/MS. J. Agric. Food Chem. 2007, 55, 1321−1326. (26) Li, T. M.; Li, W. K.; Yu, J. G. Flavonoids from Artabotrys hexapetalus. Phytochemistry 1997, 45, 831−833. (27) Long, F.; Deng, L.; Chen, Y. Study on the chemical constituents in the flowers of Ligustrum lucidum. Huaxi Yaoxue Zazhi 2011, 26, 97− 100. (28) Liu, B.; Deng, A. J.; Yu, J. Q.; Liu, A. L.; Du, G. H.; Qin, H. L. Chemical constituents of the whole plant of Elsholtzia rugulosa. J. Asian Nat. Prod. Res. 2012, 14, 89−96. (29) Aga, E. b.; Li, H. j.; Chen, J.; Li, P. Chemical constituents from the aerial parts of Codonopsis nervosa. Zhongguo Tianran Yaowu 2012, 10, 366−369. (30) Gao, F.; Wang, H.; Ye, W.; Zhao, S. Chemical constituents from the leaves of Callicarpa nudiflora. Zhongguo Yaoke Daxue Xuebao 2010, 41, 120−123. (31) Ak, T.; Gulcin, I. Antioxidant and radical scavenging properties of curcumin. Chem.−Biol. Interact. 2008, 174, 27−37. (32) Nazemiyeh, H.; Bahadori, F.; Delazar, A. Antioxidant phenolic compounds from the leaves of Erica arborea (Ericaceae). Nat. Prod. Res. 2008, 22, 1385−1392. (33) Wang, H.; Cao, G.; Prior, R. L. Oxygen radical absorbing capacity of anthocyanins. J. Agric. Food Chem. 1997, 45, 304−309. (34) Clifford, M. N. Anthocyanins − nature, occurrence and dietary burden. J. Sci. Food Agric. 2000, 80, 1063−1072. (35) Cooper-Driver, G. A. Contributions of Jeffrey Harborne and coworkers to the study of anthocyanins. Phytochemistry 2001, 56, 229− 236. (36) Xu, J. G.; Hu, Q. P.; Liu, Y. Antioxidant and DNA-protective activities of chlorogenic acid isomers. J. Agric. Food Chem. 2012, 60, 11625−11630. (37) Zhang, Y.; Shi, S. Method for extracting and purifying highpurity antiviral active caffeoyl quinic acid components from Chinese medicine Cynara. Central South Univ., People’s Rep. China 2010, 10. (38) Garritano, S.; Pinto, B.; Giachi, I.; Pistelli, L.; Reali, D. Assessment of estrogenic activity of flavonoids from Mediterranean plants using an in vitro short-term test. Phytomedicine 2005, 12, 143− 147. (39) Gupta, P.; Yadav, D. K.; Siripurapu, K. B.; Palit, G.; Maurya, R. Constituents of Ocimum sanctum with antistress activity. J. Nat. Prod. 2007, 70, 1410−1416. (40) Harinantenaina, L.; Matsunami, K.; Otsuka, H. Chemical and biologically active constituents of Pteris multif ida. J. Nat. Med. 2008, 62, 452−455. (41) Mohammed, M. M. D.; Chen, M.; Zhai, L.; Ali, I. N. The cytotoxic activity of Linum grandif lorum leaves. Eur. J Chem. 2010, 1, 110−114. (42) Tian, Y.; Sun, L. M.; Liu, X. Q.; Li, B.; Wang, Q.; Dong, J. X. Anti-HBV active flavone glucosides from Euphorbia humif usa Willd. Fitoterapia 2010, 81, 799−802. (43) Chen, Y. Y.; Pan, Q. D.; Li, D. P. New pregnane glycosides from Brucea javanica and their antifeedant activity. Chem. Biodiversity 2011, 8, 460−466. (44) Babenko, N. A.; Shakhova, E. G. Effects of Chamomilla recutita flavonoids on age-related liver sphingolipid turnover in rats. Exp. Gerontol. 2006, 41, 32−39. (45) Cottiglia, F.; Loy, G.; Garau, D. Antimicrobial evaluation of coumarins and flavonoids from the stems of Daphne gnidium L. Phytomedicine 2001, 8, 302−305. (46) Pieroni, A.; Pachaly, P. An ethnopharmacological study on common privet (Ligustrum vulgare) and phillyrea (Phillyrea latifolia). Fitoterapia 2000, 71, 89−94. (47) Tang, R.; Chen, K.; Cosentino, M.; Lee, K. H. Anti-AIDS agents. 12. Apigenin-7-O-β-D-glucopyranoside, an anti-HIV principle from Kummerowia striata. Bioorg. Med. Chem. Lett. 1994, 4, 455−458. (48) Guelcemal, D.; Alankus-Caliskan, O.; Karaalp, C.; Oers, A. U.; Ballar, P.; Bedir, E. Phenolic glycosides with antiproteasomal activity 858

dx.doi.org/10.1021/jf403006c | J. Agric. Food Chem. 2014, 62, 850−859

Journal of Agricultural and Food Chemistry

Article

from Centaurea urvillei DC. subsp. urvillei. Carbohydr. Res. 2010, 345, 2529−2533. (49) Chen, W. Q.; Song, Z. J.; Xu, H. H. A new antifungal and cytotoxic C-methylated flavone glycoside from Picea neoveitchii. Bioorg. Med. Chem. Lett. 2012, 22, 5819−5822. (50) Xu, D. d.; Jiang, X. Z.; Gao, Y.; Zheng, W.; He, M.; Gao, Q. P. Chemical constituents of effective fraction of honeysuckle on inhibition of Escherichia coli biofilms. Zhongguo Shiyan Fangjixue Zazhi 2012, 18, 122−125.

859

dx.doi.org/10.1021/jf403006c | J. Agric. Food Chem. 2014, 62, 850−859