Characterization of Polyphenols from Lycium ... - ACS Publications

Article pubs.acs.org/JAFC

Characterization of Polyphenols from Lycium ruthenicum Fruit by UPLC-Q-TOF/MSE and Their Antioxidant Activity in Caco‑2 Cells Tao Wu,*,†,‡ Haiyang Lv,‡,§ Fengzhong Wang,*,§ and Yi Wang∥ †

Key Laboratory of Food Bio-technology, School of Food and Bioengineering, Xihua University, Chengdu, 610039, China Institute of Agro-products Processing Science and Technology, Chinese Academy of Agricultural Sciences, Beijing 100193, China ∥ Xi’an Manareco New Materials Co. Ltd., Xi’an, 710086, China §

ABSTRACT: The fruit of Lycium ruthenicum Murr. (LRF) has long been used in folk medicine. Nevertheless, detailed information related to its polyphenol compositions remains scarce. In this study, we confirmed that the total phenolic and anthocyanin contents of LRF fruit extracts (LRFEs) were 4906.5 ± 60.6 mg of gallic acid equivalents/100 g DW and 787.6 ± 34.1 mg of cyanindin-3-glucoside equivalents/100 g DW, respectively. A characterization of LRFEs was performed by ultrahigh performance liquid chromatography/quadrupole time-of-flight mass spectrometry using an MSE data acquisition. A total of 26 polyphenols were tentatively identified, of which 19 represent the first reports of these polyphenols in LRFEs. Furthermore, the cellular antioxidant array showed that LRFEs could protect Caco-2 cells against H2O2-induced oxidative damage based on microscopic fluorometric imaging. KEYWORDS: antioxidant activity, Lycium ruthenicum Murr., MSE, polyphenols

■

INTRODUCTION Polyphenols are naturally occurring compounds found largely in fruits, vegetables, and herbs. The antioxidant activities of polyphenols are explained by their redox properties, which causes them to act as hydrogen donors, reducing agents, and single oxygen quenchers. The food industry has become increasingly interested in polyphenols with antioxidant properties that may help to prevent several major human diseases such as arteriosclerosis, cardiovascular disease, and cancer. 1 Compared with some synthetic antioxidants, natural polyphenols are more acceptable to consumers.2 This situation has created a growing interest in the analysis and identification of natural antioxidants with the goal of finding new resources that could be incorporated into foods. The genus Lycium (Solanaceae) includes approximately 80 species. In particular, L. ruthenicum Murr., a wild thorny shrub, grows primarily in the typical salinized desert in northwestern China. Lycium spp. grow up to 1.5 m tall and produce 1−2 cm long, purple ellipsoid berries. Fruits of L. ruthenicum Murr. (LRF), also called “black goji berry” in China, have long been recorded in the Tibetan medical classic Jing Zhu Ben Cao as a component in traditional medicinal preparations. An evergrowing body of scientific research indicates that LRF possesses a variety of biological activities, and can be used in attenuating radiation injury, in combating fatigue, and in the treatment of heart disease, abnormal menstruation, and menopause.3−6 Currently, many LRF products are sold through the online health food market, and the fruits are praised for uses related to human well-being and anti-aging, although they are usually very expensive. Among the chemical constituents of LRF, polysaccharides have been well documented as the major bioactive compounds.5,7−10 However, little is known about the composition of polyphenols in LRF. The only two known reports on this topic show that 14 anthocyanins have been detected by electrospray ionization mass spectrometry,11 and © XXXX American Chemical Society

six anthocyanins were purified and identified by nuclear magnetic resonance spectrum in LRF.12 Over the past two decades, an increasing number of researchers have studied compounds in complex plant mixtures using high-performance liquid chromatography coupled to quadrupole time-of-flight (Q-TOF) mass spectrometry.13−15 The combination of these two techniques provides high separation capacity and high efficiency in determining fragmentation patterns, which facilitates the elucidation of the characteristic structure of target and/or nontarget compounds using accurate mass measurements. The fragmentation behaviors of polyphenols have been extensively investigated for the characterization of unknown compounds even without reference standards.16,17 Normally, it is essential to perform several MS experiments for obtaining structural information by initially conducting low collision energy (LE) experiments, followed by higher collision energy (HE) MS/MS experiments to obtain fragment ions. Therefore, the traditional MS/MS approach generally requires multiple steps for obtaining the fragmentation patterns of target compounds. Alternatively, the MSE data acquisition (where E represents every data) method provides two parallel alternating scans using either LE to obtain molecular ion information or HE to obtain full-scan accurate mass fragment, precursor ion, and neutral loss data.18 All of these data can be obtained from a single analytical run. Use of the MSE technique can greatly reduce investigation time. The aim of this study was to comprehensively identify and quantify the nature of LRF extracts (LRFEs). Ultraperformance liquid chromatography coupled with Q-TOF high-resolution Received: January 4, 2016 Revised: February 26, 2016 Accepted: March 1, 2016

A

DOI: 10.1021/acs.jafc.6b00035 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 1. Base peak chromatogram of the Lycium ruthenicum fruit extract obtained by an MSE data collection technique method (UPLC-Q-TOF/ MSE) in negative (A) and positive (B) mode. 10000g for 20 min. The supernatant was filtered through mediumspeed filter paper. The filtrate was concentrated in a RE-2000A rotary evaporator (Yarong, Shanghai, China) at 40 °C, 0.1 MP conditions, and then dried using the freeze-dryer to produce extracts of LRFEs. The extraction yield was 27.8%. Total Phenolic Content. The total phenolic content (TPC) was obtained according to the method of Wu et al.19 The results were expressed as gallic acid equivalents (mg) per 100 mg of dry weight extract (GAE mg/100 g DW). All standard dilutions and samples were conducted in triplicate. Total Anthocyanin Content. The pH shift method was used to estimate the total anthocyanin content (TAC) in the samples as previously described.20 The total anthocyanin content was calculated and expressed as cyanindin-3-glucoside (mg) per 100 g DW (mg CYG/100 g DW). All analyses were conducted in triplicate. UPLC-Q-TOF/MSE Analyses. The separation was performed on a Waters Acquity UPLC HSS T3 C18 column (2.1 × 100 mm, 1.8 μm) using a Waters Acquity UPLC system (Waters Corp., Milford, MA, USA), operating at a flow rate of 0.3 mL/min throughout the gradient. The column temperature was 35 °C, and the injection volume was 2 μL (1 mg/mL). The eluents were A, 0.1% of formic acid in water, and B, acetonitrile. Separation of polyphenols was conducted under the following conditions: 0−2 min, 2% B; 2−8 min, 2−10% B; 8−23 min, 10−14% B; 23−40 min, 14−70% B; 40−40.5 min, 70−100% B; 40.5− 42 min, 100% B.

mass spectrometry methods along with MSE data acquisition (UPLC-Q-TOF/MSE) was developed for the analysis of structures based on fragmentation patterns using the accurate mass measurements of the deprotonated molecules and product ions. In addition, the preliminary antioxidant activities of LRFEs in human intestinal epithelial Caco-2 cells were also evaluated.

■

MATERIALS AND METHODS

Chemicals and Reagents. HPLC grade solvents including methanol, formic acid, and acetonitrile were purchased from Thermo Fisher Scientific Inc. (Shanghai, China). All reagents were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA) unless otherwise specified. Plant Materials and Extraction Procedure. Fresh ripe fruits were collected in August 2014 from the Qaidam Basin, Qinghai Province, China, dried using an FD-1A-80 freeze-dryer (Boyikang, Beijing, China), and then kept at −20 °C. The dried fruit samples were ground and passed through a 60 mesh filter. The powdered fruit (10 g) was placed in a flask and mixed with 200 mL of 70% methanol by ultrasound-assisted extraction for 2 h at room temperature. The extract was collected and centrifuged (Heraeus Sepatech Labofuge 200, Thermo Fisher, Waltham, MA, USA) at B


Article


Table 1. Polyphenols Tentatively Identified in Lycium ruthenicum Fruit Extract Using the MSE Data Collection Method (UPLCQTOF/MSE) peaka

tRb

1

6.78

515.1430

2

7.85

529.3088

3

7.90

4

LE, m/z

molecular formula

HE, m/z, (%)

Negative Mode 515.1384(100), 353.0860(22), 191.0547(87) C25H24O12

structural assignment

error (ppm)

3,4-di-O-CQA

45.62

refc 23, 24 25

C28H42N4O6

N,N-bis-dhc-spermine

10.58

353.1024

529.3088(100), 407.2649(21), 365.2569(44), 243.2176(3) 353.0860(100), 191.0547(43)

C16H18O9

trans-neo-CQA

41.35

8.23

515.1384

515.1384(43), 353.0860(21), 191.0547(100)

C25H24O12

3,5-di-O-CQA

36.69

5

8.41

515.1430

515.1384(47), 353.0860(20), 191.0547(100)

C25H24O12

4,5-di-O-CQA

45.62

6

8.73

353.0860

353.1024(62), 191.0604(100)

C16H18O9

trans-CQA

−5.10

7

9.33

353.0860

353.1024(100), 191.0575(34)

C16H18O9

trans-crypto-CQA

−5.10

8

9.49

634.2982

634.2982(50), 472.2484(100), 308.1988(15) 632.2831(75), 470.2300(100), 291.1728(11) 771.2039(100), 609.1458(38), 301.0360(9), 300.0278(4) 472.2528(100), 350.2096(32), 470.2300(100), 334.1785(54), , 291.1728(18) 470.2300(100), 334.1785(45), 291.1728(5) 470.2300(100), 334.1785(37), 291.1728(13) 468.2116(100), 332.1600(59), 289.1565(10) 609.1458(100), 301.0494(27), 271.0247(12), 255.0278(4) 593.1488(100), 285.0278(43) 623.1656(100), 315.0530(45)

350.2096(9),

C31H45N3O11

N,N-bis-dhc-spermidine-Hex

0.16

334.1785(45),

C31H43N3O11

dhc-caffeoyl-spermidine-Hex

0.95

462.0812(10),

C33H40O21

quercetin-O-Rut-Hex

6.35

308.1988(65) 308.1988(4)

C25H35N3O6 C25H33N3O6

N,N-bis-dhc-spermidine dhc-caffeoyl spermidine isomer

6.56 0.64

25 29

308.1988(3),

C25H33N3O6

dhc-caffeoyl spermidine isomer

0.64

29

308.1988(2),

C25H33N3O6

dhc-caffeoyl spermidine isomer

19.56

29

306.1816(15),

C25H31N3O6

N,N-dhc-spermidine

5.13

23

300.0278(40),

C27H30O16

quercetin-O-Rut

0.49

33

kaempferol-O-Rut isorhamnetin-O-Rut

3.0 7.0

31 34

Pet-3-O-[6-O-(4-O-(4-O-cis-Glu-Cou)rha)-Glu]-5-O-[glu] Pet-3-O-[6-O-(4-O-(4-O-tra-Glu-Cou)rha)-Glu]-5-O-[glu] Del-3-O-[6-O-(4-O-(trans-cou)-rha)-βglu]-5-O-[glu] Pet- 3-O-[6-O-(4-O-(trans-p-caffeoyl)rha)-glu]-5-O-[glu] Pet-3-O-[6-O-(4-O-(4-O-Cou)-rha)Glu]-5-O-[glu] isomer Pet-3-O-[6-O-(4-O-(4-O-Cou)-rha)Glu]-5-O-[glu] isomer Mal-3-O-Rut-Cou-5-O-Glu

1.28

0.65

12, 35 12, 35 12

2.84

12

6.32

12

0.43

12

3.2

11

0.43

12

0.43

12

9

10.1

632.2831

10

10.55

771.2039

11 12

10.64 11.33

472.2528 470.2300

13

12.25

470.2300

14

12.98

470.2389

16

15.6

468.2161

19

21.53

609.1458

24 25

23.14 23.47

593.1488 623.1656

15

14.04

17

17.85

18

18.9

1095.3173, 933.2653 1095.3176 933.2655 919.2509

18.9

949.2635

20

21.6

933.2718

21

22.76

933.2655

22

22.81

947.2791

23

22.95

933.2655

26

23.59

933.2655, 771.2127

C27H30O15 C28H32O16 Positive Mode 1095.3173(93), 933.2655(21), 479.1201(17), C49H59O28 317.0681(100) 1095.3176(8), 933.2655(59), 479.1201(11), C49H59O28 317.0681(100) 919.2509(58), 757.1933(18), 465.1056(10), C42H47O23 303.0494(100) 949.2635(20), 479.1201(5), 317.0645(17) C43H49O24 933.2718(100), 771.2127(42), 317.0718(62) 933.2655(100), 771.2127(12), 317.0681(43) 933.2655(100), 771.2127(14), , 317.0645(57) 933.2655(100), 771.2127(14), , 317.0645(57) 933.2655(100), 771.2184(12), 317.0681(41)

479.1201(23),

C43H49O23

479.1201(7),

C43H49O23

479.1201(9)

C44H51O23

479.1201(9)

C43H49O23

479.1201(6),

C43H49O23

Pet-3-O-[6-O-(4-O-(4-O- Cou)-rha)Glu]-5-O-[glu] isomer Pet-3-O-[6-O-(4-O-(4-O- Cou)-rha)Glu]-5-O-[glu] isomer

1.28

23, 25 23, 24 23, 24 23, 25 23, 25

a Peak numbering refers to peaks in Figure 1. btR = retention time. cNumbers refer to numbering in reference, blank based on the fragmentation pattern. Abbreviations: LE, low collision energy; HE, higher collision energy; CQA, caffeoylquinic acid; dhc, dihydrocaffeoyl; Hex, hexose; Pet, petunidin; Glu, β-D-glucopyranoside; Cou, p-coumaroyl; Rha, α-L-rhamnopyranosyl; Del, delphinidin; Mal, malvidin; Rut, rutinoside.

Mass spectrometric data were acquired on an Xevo G2 Q-TOF mass spectrometer (Waters MS Technologies, Manchester, U.K.) using both positive and negative ion electrospray ionization. The scan range was from 100 to 1200 m/z. Electrospray ionization (ESI) conditions included a capillary voltage of 3.2 kV for positive mode, and 2.8 kV for negative mode; a cone voltage of 35 V, ion guide at 1 V, source temperature of 100 °C, and nitrogen desolvation gas temperature of 400 °C flowing at 600 L/h. For MSE experiments two functions were involved, one performed at LE (15 eV) and the

second at ramp HE (20−60 eV) to induce fragmentation. Structural characterization of the polyphenols was based on fragmentation pattern and earlier published literature. Cell Culture. The Caco-2 human intestinal cell line was purchased from the cell bank of Chinese Academy of Sciences. The cells were cultured in Dulbecco’s modified Eagle’s medium/F12 medium (DMEM/F12, Gibco, Grand Island, NY, USA), supplemented with 20% heat-inactivated fetal bovine serum (FBS, Sijiqing, Hangzhou, China), 50 units/mL penicillin−streptomycin (Gibco) in 50 cm2 C


Article


Figure 2. Typical MSE spectra of four polyamine derivatives tentatively identified in Lycium ruthenicum fruit at ramped collision energy (20−60 eV): (A) negative mode; (B) positive mode. (1) N,N-bBis(dihydrocaffeoyl) spermine; (2) N,N-bis(dihydrocaffeoyl) spermidine; (3) caffeoyl(dihydrocaffeoyl) spermidine; (4) N,N-dicaffeoylspermidine. D


Article

Journal of Agricultural and Food Chemistry plastic flasks (JET Bio-Filtration Products; Guangzhou, China). Cells were grown at 37 °C with 5% CO2 in a incubator (Thermo Forma, Waltham, MA). Cells were subcultured after reaching approximately 80% confluence and used between passages 18 and 24 in the experiments. Cellular Antioxidant Activity. The cellular antioxidant activity was determined using the procedures described by Mine et al.21 Following the treatment, 1 × 105 cells/mL were incubated in DMEM/ F12 medium with 5% FBS as treatment medium for 2 h at 37 °C with 0.03 μg/mL, 0.15 μg/mL, and 0.31 μg/mL of LRFEs, and then the cells were washed by Hanks balanced salt solution followed by the addition of 100 μM H2O2 with treatment medium for 6 h. The cells were washed with 100 μL of phosphate buffered saline and then resuspended in the buffered saline and incubated for 60 min at 37 °C with 10 μM DCFH-DA (dichlorodihydrofluorescein diacetate) added. The microplate was detected in a fluorescence microplate reader (Synergy H4 Hybrid Reader, Bio-Tek Instruments Inc., Winooski, VT, USA) at 37 °C. Emission at 538 nm was measured with excitation at 485 nm every 5 min for 1 h. Each plate also included triplicate positive control, negative control, and sample background wells: positive control wells contained cells treated with DCFH-DA and H2O2, negative control wells contained cells treated with DCFH-DA without H2O2, and sample background wells contained cells treated with sample and DCFH-DA without H2O2. The 2′,7′-dichlorodihydrofluorescein fluorescence was detected by flow cytometry with a FACSCalibur flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA), and the mean of the fluorescence intensity was measured as the geometric mean using the manufacturer’s analysis software. Cell Visualization Assessment. Cell staining was visualized using an ImageXpress Micro XL (Molecular Devices, LLC, Sunnyvale, CA, USA). Emission wavelength at 538 nm was measured with excitation wavelength at 485 nm. Statistical Analysis. All of the measured values were expressed as the mean value of three replicates with according standard deviation. Significant differences between means were carried out using an analysis of variance (ANOVA) followed by Tukey’s multiple comparison test via the SAS 9.1 program (SAS Institute, Inc., 2004).

■

fingerprint for the identification and authentication of fruits or fruit-derived products. Although no authentic standards were available for the mass confirmation, based on the known structure and neutral loss pattern of previous documents, a total of 26 polyphenols were tentatively identified (Table 1). Proposed identities of these compounds were based on their retention times, exact mass, and MSE fragmentation patterns in negative or positive modes. The identified polyphenols belonged to four groups: hydroxycinnamic acids, hydroxycinnamic acid amides (HAAs), flavonoid glycosides, and anthocyanins. Hydroxycinnamic Acids. Peaks 1, 4, and 5 exhibited the same pseudomolecular ion [M − H]− at m/z 515 in negative ionization. The fragmentation pattern at m/z 353 corresponds to the loss of a caffeic moiety [M − caffeoyl − H]−, and m/z 191 corresponds to the deprotonated quinic acid [M − 2 caffeoyl − H]−. According to the MS data,23 both were identified as dicaffeoylquinic acid isomers. The retention times of dicaffeoylquinic acids on a C18 reversed phase column increased in the following order: 3,4-di-O-caffeoylquinic acid < 3,5-di-O-caffeoylquinic acid < 4,5-di-O-caffeoylquinic acid.24 Therefore, peaks 1, 4, and 5 were provisionally identified as these three acids, respectively. Peaks 3, 6, and 7 displayed deprotonated molecules at m/z 353. A fragment ion [M − 162 − H]− at m/z 191 resulted from the loss of a caffeic acid unit. Therefore, they were tentatively identified as 3-caffeoylquinic acid isomers.23 The order of elution of these compounds has been established in reverse phase chromatography: trans-neo-chlorogenic acid < transchlorogenic acid < trans-crypto-chlorogenic acid.25 Therefore, peaks 3, 6, and 7 were tentatively identified as these three acids, respectively. Chlorogenic acid and its derivatives have received significant attention because of their potential roles in the regulation of glucose absorption and as part of the management of obesity and gluconeogenesis.26 The contributions of chlorogenic acid to glucose transport in skeletal muscle via the activation of AMPK have been revealed. 27 Potential uses of these compounds are suggested in functional foods, pharmaceuticals, and cosmetics. HAAs. HAAs are N-acylated biogenic amines conjugated with hydroxycinnamic acids via amide bonds. Notablely, four unique groups of HAAs were found in LRFEs including N,N-bisdihydrocaffeoyl spermine, N,N-bis-dihydrocaffeoyl-spermidine, dihydrocaffeoyl-caffeoyl-spermidine, and N,N-dihydrocaffeoylspermidine derivatives. Their typical spectra are presented in negative (Figure 2A) and positive (Figure 2B) mode by the HE MSE data collection. Peak 2 had deprotonated molecule at m/z 529 and was tentatively identified as N,N-bis(dihydrocaffeoyl) spermine. Deprotonated molecule produced fragment ions at m/z 407, m/z 365, and m/z 243 in a negative mode, matching those from the published spectrum in potato (Solanaceae family).25 Peak 11 with the precursor ion at m/z 472 produced two fragment ions at m/z 350 and 308 that are characteristic of N,N-bis(dihydrocaffeoyl) spermidine.25 Peak 8 with deprotonated ion [M − H]− at m/z 634 shared the same fragment pattern with peak 11 after a 162 amu loss, suggesting a hexose moiety. Hence, it was tentatively identified as a N,Nbis(dihydrocaffeoyl) spermidine hexoside. Peaks 12, 13, and 14 had the same parent ion at m/z 470 and were tentatively identified as caffeoyl (dihydrocaffeoyl) spermidine isomers. The fragment at m/z 308 is derived

RESULTS AND DISCUSSION

TPC and TAC. The data showed a high content of polyphenols (4906.5 ± 60.6 mg GAE/100 g DW) in LRFEs. Blueberries are known as one of the primary sources of polyphenols in the American diet. The TPC in LRFEs was significantly higher than that of blueberries (1622−3457 mg GAE/100 g DW).22 The TAC as determined by the pH differential method was 787.6 ± 34.1 mg CYG/100 g DW in LRFES, which was approximately 2−5 times higher than that of blueberries (140−318 mg CYG/100 g DW).22 Therefore, the high TPC and TAC indicated that the LRF is an excellent source of polyphenols. Although the main causes of this high content have not been investigated yet, it has been speculated that genomic difference and unique geographical conditions such as cold, drought, and strong UV-B could influence the TAC of LRF.11 Identification of Polyphenols by UPLC-Q-TOF/MSE Analysis. Mass spectral analysis and experiments were performed in the MSE mode for the purpose of obtaining structural information rapidly. The MSE technique takes advantage of two parallel alternating scans to significantly reduce analytical run time. Figure 1 shows the base peak chromatogram of LRFEs in negative (A) and positive (B) ESI mode under optimal conditions. All of these data were successfully obtained in a 30 min run. Chromatographic profiling could provide a relatively complete picture of metabolomic analysis, which is usually called analytical E


Article


Figure 3. Proposed fragmentation scheme of N,N-bis(dihydrocaffeoyl) spermidine in positive mode.

rutinoside-hexose based on the loss of a rutinoside moiety (308 amu) and hexose moiety (162 amu), respectively. Peak 24 had an [M − H]− ion at m/z 593 and yielded a fragment ion at m/z 285 [M − rutinose − H]−. The m/z 285 ion suggests that kaempferol or luteolin (isomer of kaempferol) is an aglycon. Kaempferol derivatives have been reported to be present in Lycium species. Thus, peak 24 was provisionally identified as kaempferol-3-O-rutinoside.31 Peak 25 had an [M − H]− ion at m/z 623 and was proposed as isorhamnetin-3-O-rutinoside. The ion at m/z 315 suggests that isorhamnetin is an aglycon based on the loss of a rutinoside unit (308 amu) [M − 308 − H]−.34 Anthocyanins. A total of nine anthocyanins were found in this study. Peak 15 shared the same fragment pattern with peak 17. Both of them produced parent ions [M]+ at m/z 1095, which fragmented into daughter ions at m/z 933 [M − hexose]+ and m/z 479 [M − (p-coumaroyl) − rutinoside]+, and yielded an aglycon pentunidin at m/z 317. Furthermore, peak 17 exhibited higher polarity than peak 20. Previous researchers have concluded that the cis-p-coumaroyl derivatives were eluted earlier than its trans configuration on a reversed phase column.35 Compared with a recent mass spectral analysis,12 peaks 15 and 17 were tentatively identified as petunidin 3-O-[6O-(4-O-(4-O-cis-(β- D -glucopyranoside)-p-coumaroyl)-α- L rhamnopyranosyl)-β-D-glucopyranoside]-5-O-[β-D-glucopyranoside] and petunidin 3-O-[6-O-(4-O-(4-O-trans-(β-D-glucopyranoside)-p-coumaroyl)-α-L-rhamnopyranosyl)-β-D-glucopyranoside]-5-O-[β-D-glucopyranoside]. Accordingly, peaks 20, 21, 23, and 26 produced the same parent ions at m/z 933 [M]+, which fragmented into daughter ions at m/z 771 [M − hexose]+, yielding a pentunidin aglycon at m/z 317. When the mass spectrum was compared to reports from the literature,12 they were tentatively identified as four petunidin 3-O-[6-O-(4-O-pcoumaroyl)-α-L-rhamnopyranosyl)β-D-glucopyranoside]-5-O-[β-D-glucopyranoside] isomers. To the best of our knowledge, except for the two cis−trans pcoumaroyl, other two isomers were found from LRF for the first time. Peak 18 corresponded to two different coeluted compounds. Compound 1 had a fragmentation pattern at m/z 919 [M]+, 757 [M − hexose]+, 465 [M − hexose − hexose]+, and yielded a delphinidin aglycon at m/z 303. In comparison of the mass spectrum,12 it was designated as delphinidin 3-O-[6-O-(4-O-

from the loss of a dihydrocaffeoyl molecule; these compounds have been reported previously in Solanum melongena.28 Peak 9 with deprotonated ion [M − H]− at m/z 632 shared the same fragmentation pattern with peaks 12, 13, and 14 after a 162 amu loss, and thus it was tentatively identified as (dihydrocaffeoyl) caffeoyl spermidine hexoside. Peak 16 yielded parent ion [M − H]− at m/z 468, and fragmented at m/z 332, 306, and 289; based on the rules in the literature mentioned,23 it was identified as N,N-dicaffeoylspermidine. HAAs have been previously identified by MS using negative mode.29 In contrast to negative mode (Figure 2A), positive ion mode offers more reliable identification due to predictable fragmentation (Figure 2B). All amides break along the amide bond producing acylium ion and protonated amine. In brief, only a typical proposed fragmentation scheme for N,Nbis(dihydrocaffeoyl) spermidine is shown in Figure 3. The ion at m/z 472 represents protonated N-dihydrocaffeoyl spermidine, while the most characteristic fragment was related to the base peak at m/z 163 and represents the caffeoyl ion. After the cleavage of the C−N bond from the ion neutral complex, the ion at m/z 310 was obtained and subsequently loses ammonia to produce the fragment ion at m/z 293. HAAs are involved in the defense of plants against pathogens, and most are present in the Solanaceae.30 Caffeoyl putrescine and dicaffeoyl putrescine were recently discovered in Lycium intricatum Boiss.31 A total of eight HAAs that were previously undescribed from Lycium species were detected in LRFEs. With these unique HAAs, we may be able to distinguish L. ruthenicum from other Lycium species. In addition, it has been reported that polyamines have anti-inflammatory, αglucosidase inhibitory, cytotoxic, and antitubercular activities,32 which suggested that LRF has great potential as a new source for natural health products. Flavonoid Glycosides. Peak 19 had an [M − H]− ion at m/z 609 and yielded an ion at m/z 301 [M − 308 − H]−. The m/z 271 and 255 ions in the MSE analysis showed that a quercetin aglycon, and not ellagic acid aglycon33 was present. So it was identified as quercetin-3-O-rutinoside. Peak 10 with parent ions [M − H]− at m/z 771 had a typical fragment at m/z 609 [M − 162 − H]− and 301 [M − 162 − 308 − H]−, and these were identified as quercetin-3-OF


Article


Figure 4. Cellular antioxidant activities of LRFPs in Caco-2 cells. Cells were treated with 0.3 and 0.03 μg/mL of LRFPs for 1 h each, and then 10 μM DCFH-DA for 1 h. Bars with different letters have mean values that are significantly different (P < 0.05). Abbreviations: POS, positive control; NEG, negative control; LRFEs, extracts from Lycium ruthenicum Murr. fruit.

previously demonstrated to regulate obesity and enhanced insulin sensitivity associated with adipocytokine secretion and PPARγ activation in adipocytes.36 The food industries have shown interest in anthocyanin, not only because of its known properties that are beneficial to human health but also because the attractive anthocyanin colors are useful in preparing processed foods. Cellular Antioxidant Activity in Caco-2 Cells. Generally, the antioxidant effects of polyphenols can be evaluated by several in vitro chemical assays. However, the bioactivities and mechanisms are likely to be different in live cells. Compared to chemical assays, its characteristics are related to the uptake, metabolism, and location of antioxidants within cells. Caco-2

(trans-p-coumaroyl)-α-L-rhamnopyranosyl)-β-D-glucopyranoside]-5-O-[β-D-glucopyranoside]. Compound 2 had a fragmentation pattern at m/z 949 [M]+ and 479 [M − rutinose − hexose]+, and yielded a pentunidin aglycon at m/z 317. When compared with the mass spectrum, it was identified as petunidin 3-O-[6-O-(4-O-(trans-p-caffeoyl)-α-L-rhamnopyranosyl)-β-D-glucopyranoside]-5-O-[β-D-glucopyranoside].12 Peak 22 had parent ions at m/z 947 [M]+, which fragmented into daughter ions at m/z 785 [M − hexose]+, and yielded a malvidin aglycon at m/z 331, and was tentatively identified as malvidin-3-O-rutinoside (p-coumaroyl)-5-O-glucoside.11 Anthocyanins are water-soluble glycosides that are responsible for the dark purple color of LRF. Anthocyanins have been G


Article


(2) Embuscado, M. E. Spices and herbs: Natural sources of antioxidants − a mini review. J. Funct. Foods 2015, 18 (Part B), 811−819. (3) Liu, Z.; Shu, Q.; Wang, L.; Yu, M.; Hu, Y.; Zhang, H.; Tao, Y.; Shao, Y. Genetic diversity of the endangered and medically important Lycium ruthenicum Murr. revealed by sequence-related amplified polymorphism (SRAP) markers. Biochem. Syst. Ecol. 2012, 45, 86−97. (4) Ni, W.; Gao, T.; Wang, H.; Du, Y.; Li, J.; Li, C.; Wei, L.; Bi, H. Anti-fatigue activity of polysaccharides from the fruits of four Tibetan plateau indigenous medicinal plants. J. Ethnopharmacol. 2013, 150, 529−535. (5) Peng, Q.; Liu, H.; Shi, S.; Li, M. Lycium ruthenicum polysaccharide attenuates inflammation through inhibiting TLR4/ NF-κB signaling pathway. Int. J. Biol. Macromol. 2014, 67, 330−335. (6) Duan, Y.; Chen, F.; Yao, X.; Zhu, J.; Wang, C.; Zhang, J.; Li, X. Protective Effect of Lycium ruthenicum Murr. Against Radiation Injury in Mice. Int. J. Environ. Res. Public Health 2015, 12, 8332. (7) Peng, Q.; Lv, X.; Xu, Q.; Li, Y.; Huang, L.; Du, Y. Isolation and structural characterization of the polysaccharide LRGP1 from Lycium ruthenicum. Carbohydr. Polym. 2012, 90, 95−101. (8) Liu, Z.; Dang, J.; Wang, Q.; Yu, M.; Jiang, L.; Mei, L.; Shao, Y.; Tao, Y. Optimization of polysaccharides from Lycium ruthenicum fruit using RSM and its anti-oxidant activity. Int. J. Biol. Macromol. 2013, 61, 127−134. (9) Lv, X.; Wang, C.; Cheng, Y.; Huang, L.; Wang, Z. Isolation and structural characterization of a polysaccharide LRP4-A from Lycium ruthenicum Murr. Carbohydr. Res. 2013, 365, 20−25. (10) Peng, Q.; Xu, Q.; Yin, H.; Huang, L.; Du, Y. Characterization of an immunologically active pectin from the fruits of Lycium ruthenicum. Int. J. Biol. Macromol. 2014, 64, 69−75. (11) Zheng, J.; Ding, C.; Wang, L.; Li, G.; Shi, J.; Li, H.; Wang, H.; Suo, Y. Anthocyanins composition and antioxidant activity of wild Lycium ruthenicum Murr. from Qinghai-Tibet Plateau. Food Chem. 2011, 126, 859−865. (12) Jin, H.; Liu, Y.; Guo, Z.; Yang, F.; Wang, J.; Li, X.; Peng, X.; Liang, X. High-Performance Liquid Chromatography Separation of cis−trans Anthocyanin Isomers from Wild Lycium ruthenicum Murr. Employing a Mixed-Mode Reversed-Phase/Strong Anion-Exchange Stationary Phase. J. Agric. Food Chem. 2015, 63, 500−508. (13) Qiu, J.; Chen, L.; Zhu, Q.; Wang, D.; Wang, W.; Sun, X.; Liu, X.; Du, F. Screening natural antioxidants in peanut shell using DPPH− HPLC−DAD−TOF/MS methods. Food Chem. 2012, 135, 2366− 2371. (14) López-Cobo, A.; Gómez-Caravaca, A. M.; Cerretani, L.; SeguraCarretero, A.; Fernández-Gutiérrez, A. Distribution of phenolic compounds and other polar compounds in the tuber of Solanum tuberosum L. by HPLC-DAD-q-TOF and study of their antioxidant activity. J. Food Compos. Anal. 2014, 36, 1−11. (15) Talhaoui, N.; Gómez-Caravaca, A. M.; León, L.; De la Rosa, R.; Segura-Carretero, A.; Fernández-Gutiérrez, A. Determination of phenolic compounds of ‘Sikitita’ olive leaves by HPLC-DAD-TOFMS. Comparison with its parents ‘Arbequina’ and ‘Picual’ olive leaves. LWT-Food Sci. Technol. 2014, 58, 28−34. (16) Gonzales, G. B.; Raes, K.; Vanhoutte, H.; Coelus, S.; Smagghe, G.; Van Camp, J. Liquid chromatography−mass spectrometry coupled with multivariate analysis for the characterization and discrimination of extractable and nonextractable polyphenols and glucosinolates from red cabbage and Brussels sprout waste streams. J. Chromatogr. A 2015, 1402, 60−70. (17) Fabre, N.; Rustan, I.; de Hoffmann, E.; Quetin-Leclercq, J. Determination of flavone, flavonol, and flavanone aglycones by negative ion liquid chromatography electrospray ion trap mass spectrometry. J. Am. Soc. Mass Spectrom. 2001, 12, 707−715. (18) Plumb, R. S.; Johnson, K. A.; Rainville, P.; Smith, B. W.; Wilson, I. D.; Castro-Perez, J. M.; Nicholson, J. K. UPLC/MSE; a new approach for generating molecular fragment information for biomarker structure elucidation. Rapid Commun. Mass Spectrom. 2006, 20, 1989− 1994.

cells represent an epithelial cell line derived from human intestinal tissue that is widely used as a cell model to study ROS-induced damage.37 In this study, to get further evidence of the inhibition of ROS in cells by LRFEs, we conducted a microscopic image assay by a fluorometric method using DCFH-DA probe, with the goal of visualizing the amount of ROS in the cells (Figure 4). In untreated cells (negative control), few red fluorescent spots were observed, which proved that normal cells possess lower amounts of ROS. In contrast, cells exposed to H2O2 (positive control) showed a more intense red fluorescent spot than the untreated cells (Figure 4A), which is a well-established cytological hallmark indicative of the amount of ROS in cells. This further confirms that significantly increased ROS inhibition was done by LRFPs with three different contents (0.03 μg/mL, 0.15 μg/mL, and 0.31 μg/mL) (P < 0.05) in a concentration-dependent manner (Figure 4B). It was reported earlier that the purple carrot or potato extract had much higher cellular antioxidant activity at 5−100 μg/mL.38 These results suggest that the LRFEs played a role in protecting the cells from extraneous stimuli and had stronger ability to scavenge free radicals in a cellular environment than that of common vegetables. In summary, results of this study showed that the LRF provide a good source of polyphenols. Furthermore, UPLC-QTOF/MSE was demonstrated to be a powerful analytical technique for the rapid separation and detection of polyphenols in LRF. Consequently, a total of 26 polyphenols were tentatively identified in LRF, based on accurate mass determination of the deprotonated/protonated ions which were obtained from the MSE fragmentation pattern. The phytochemical components showed that, in addition to commonly found hydroxycinnamic acid and flavonoid glycosides, LRF contained unique anthocyanin derivatives and hydroxycinnamic acid amides. Moreover, the Caco-2 cell based antioxidant activity and LRFEs was also evaluated, and the results showed that LRFEs could protect Caco-2 cells against H2O2-induced oxidative damage by microscopic fluorometric imaging. The overall results could explain partly the past and current usage of LRF as a medicine or functional food, as well as support the possible uses of LRFs as potential sources in the nutraceutical, pharmaceutical, or cosmetic industries.

■

AUTHOR INFORMATION

Corresponding Authors

*(T.W) Tel/fax: +86-28-87720200. E-mail: [email protected]. *(F.W.) Tel/fax: +86-10-62810295. E-mail: wangfengzhong@ sina.com. Author Contributions ‡

T.W and H.L. contributed equally to this research.

Funding

The talent recruitment program/project of Xihua University (2016) and innovation projects of science and technology of the Chinese Academy of Agricultural Sciences (2015) supported this study. Notes

The authors declare no competing financial interest.

■

REFERENCES

(1) Lewandowska, U.; Szewczyk, K.; Hrabec, E.; Janecka, A.; Gorlach, S. Overview of Metabolism and Bioavailability Enhancement of Polyphenols. J. Agric. Food Chem. 2013, 61, 12183−12199. H


Article

Journal of Agricultural and Food Chemistry (19) Wu, T.; Yan, J.; Liu, R.; Marcone, M. F.; Aisa, H. A.; Tsao, R. Optimization of microwave-assisted extraction of phenolics from potato and its downstream waste using orthogonal array design. Food Chem. 2012, 133, 1292−1298. (20) Chaovanalikit, A.; Wrolstad, R. E. Total Anthocyanins and Total Phenolics of Fresh and Processed Cherries and Their Antioxidant Properties. J. Food Sci. 2004, 69, FCT67−FCT72. (21) Donno, D.; Beccaro, G. L.; Mellano, M. G.; Cerutti, A. K.; Bounous, G. Goji berry fruit (Lycium spp.): antioxidant compound fingerprint and bioactivity evaluation. J. Funct. Foods 2015, 18 (Part B), 1070−1085. (22) Pertuzatti, P. B.; Barcia, M. T.; Rodrigues, D.; da Cruz, P. N.; Hermosín-Gutiérrez, I.; Smith, R.; Godoy, H. T. Antioxidant activity of hydrophilic and lipophilic extracts of Brazilian blueberries. Food Chem. 2014, 164, 81−88. (23) García-Salas, P.; Gómez-Caravaca, A. M.; Morales-Soto, A.; Segura-Carretero, A.; Fernández-Gutiérrez, A. Identification and quantification of phenolic compounds in diverse cultivars of eggplant grown in different seasons by high-performance liquid chromatography coupled to diode array detector and electrospray-quadrupoletime of flight-mass spectrometry. Food Res. Int. 2014, 57, 114−122. (24) Oszmiański, J.; Wojdyl̷o, A.; Gorzelany, J.; Kapusta, I. Identification and characterization of low molecular weight polyphenols in berry leaf extracts by HPLC-DAD and LC-ESI/MS. J. Agric. Food Chem. 2011, 59, 12830−12835. (25) Narváez-Cuenca, C.-E.; Vincken, J.-P.; Zheng, C.; Gruppen, H. Diversity of (dihydro) hydroxycinnamic acid conjugates in Colombian potato tubers. Food Chem. 2013, 139, 1087−1097. (26) Cho, A.-S.; Jeon, S.-M.; Kim, M.-J.; Yeo, J.; Seo, K.-I.; Choi, M.S.; Lee, M.-K. Chlorogenic acid exhibits anti-obesity property and improves lipid metabolism in high-fat diet-induced-obese mice. Food Chem. Toxicol. 2010, 48, 937−943. (27) Ong, K. W.; Hsu, A.; Tan, B. Chlorogenic acid stimulates glucose transport in skeletal muscle via AMPK activation: a contributor to the beneficial effects of coffee on diabetes. PLoS One 2012, 7, e32718. (28) Wu, S.-B.; Meyer, R. S.; Whitaker, B. D.; Litt, A.; Kennelly, E. J. A new liquid chromatography−mass spectrometry-based strategy to integrate chemistry, morphology, and evolution of eggplant (Solanum) species. J. Chromatogr. A 2013, 1314, 154−172. (29) Shakya, R.; Navarre, D. A. Rapid screening of ascorbic acid, glycoalkaloids, and phenolics in potato using high-performance liquid chromatography. J. Agric. Food Chem. 2006, 54, 5253−5260. (30) Muroi, A.; Ishihara, A.; Tanaka, C.; Ishizuka, A.; Takabayashi, J.; Miyoshi, H.; Nishioka, T. Accumulation of hydroxycinnamic acid amides induced by pathogen infection and identification of agmatine coumaroyltransferase in Arabidopsis thaliana. Planta 2009, 230, 517− 527. (31) Abdennacer, B.; Karim, M.; Yassine, M. r.; Nesrine, R.; Mouna, D.; Mohamed, B. Determination of phytochemicals and antioxidant activity of methanol extracts obtained from the fruit and leaves of Tunisian Lycium intricatum Boiss. Food Chem. 2015, 174, 577−584. (32) Sun, J.; Song, Y.-L.; Zhang, J.; Huang, Z.; Huo, H.-X.; Zheng, J.; Zhang, Q.; Zhao, Y.-F.; Li, J.; Tu, P.-F. Characterization and Quantitative Analysis of Phenylpropanoid Amides in Eggplant (Solanum melongena L.) by High Performance Liquid Chromatography Coupled with Diode Array Detection and Hybrid Ion Trap Time-of-Flight Mass Spectrometry. J. Agric. Food Chem. 2015, 63, 3426−3436. (33) Hvattum, E. Determination of phenolic compounds in rose hip (Rosa canina) using liquid chromatography coupled to electrospray ionisation tandem mass spectrometry and diode-array detection. Rapid Commun. Mass Spectrom. 2002, 16, 655−662. (34) Galati, E. M.; Mondello, M. R.; Giuffrida, D.; Dugo, G.; Miceli, N.; Pergolizzi, S.; Taviano, M. F. Chemical characterization and biological effects of Sicilian Opuntia ficus indica (L.) Mill. fruit juice: antioxidant and antiulcerogenic activity. J. Agric. Food Chem. 2003, 51, 4903−4908.

(35) Downey, M. O.; Rochfort, S. Simultaneous separation by reversed-phase high-performance liquid chromatography and mass spectral identification of anthocyanins and flavonols in Shiraz grape skin. J. Chromatogr. A 2008, 1201, 43−47. (36) Song, H.; Wu, T.; Xu, D.; Chu, Q.; Lin, D.; Zheng, X. Dietary sweet cherry anthocyanins attenuates diet-induced hepatic steatosis by improving hepatic lipid metabolism in mice. Nutrition 2016, DOI: 10.1016/j.nut.2016.01.007. (37) Prosperini, A.; Juan-García, A.; Font, G.; Ruiz, M. Reactive oxygen species involvement in apoptosis and mitochondrial damage in Caco-2 cells induced by enniatins A, A 1, B and B 1. Toxicol. Lett. 2013, 222, 36−44. (38) Shi, Y.; Kovacs-Nolan, J.; Jiang, B.; Tsao, R.; Mine, Y. Antioxidant activity of enzymatic hydrolysates from eggshell membrane proteins and its protective capacity in human intestinal epithelial Caco-2 cells. J. Funct. Foods 2014, 10, 35−45.

I


Characterization of Polyphenols from Lycium ... - ACS Publications

Recommend Documents