Identification of Phenolic Compounds in Petals of Nasturtium Flowers

Article pubs.acs.org/JAFC

Identification of Phenolic Compounds in Petals of Nasturtium Flowers (Tropaeolum majus) by High-Performance Liquid Chromatography Coupled to Mass Spectrometry and Determination of Oxygen Radical Absorbance Capacity (ORAC) G. Astrid Garzón,*,† David C. Manns,§ Ken Riedl,# Steven J. Schwartz,# and Olga Padilla-Zakour§ †

Departamento de Quı ́mica, Universidad Nacional de Colombia, AA 14490 Bogotá, Colombia Department of Food Science, New York State Agricultural Experiment Station, Cornell University, 630 West North Street, Geneva, New York 14456, United States # Department of Food Science, Parker Food Science Building, The Ohio State University, 2015 Fyffe Road, Columbus, Ohio 43210, United States §

S Supporting Information *

ABSTRACT: The contents and profile of polyphenols were analyzed in edible petals of nasturtium flowers (Tropaeolum majus) of three colors, and their oxygen radical absorbance capacities (ORAC) were compared. Three primary anthocyanins (ACNs) and 15 non-ACN phenolic compounds including hydroxycinammic acids (HCAs) and flavonoids (myricetin, quercetin, and kaempferol derivatives) were detected. Anthocyanin concentration was within 31.9 ± 21.7 and 114.5 ± 2.3 mg cyanidin-3glucoside (cy-3-glu)/100 g fresh weight (FW) in yellow and red petals, respectively. The concentration of HCAs varied between 33.3 ± 7.1 and 235.6 ± 8.1 mg chlorogenic acid equivalents/100 g FW for red and yellow flowers, respectively. Red flowers had the highest level of flavonoids (315.1 ± 2.4 mg myricetin equivalents/100 g FW) and the highest ORAC radical-scavenging activity. These results show the diversity and abundance of polyphenolic compounds in nasturtium flowers, which could be the basis for applications in functional foods, cosmetics, and pharmaceuticals. KEYWORDS: nasturtium flowers, Tropaeolum majus, phenolics, anthocyanins, ORAC

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INTRODUCTION

cottage cheese are consumed. Furthermore, leaves, stems, and flowers are used for the preparation of herbal vinegar.4 Nasturtium leaves are used in traditional medicine to treat diseases including cardiovascular disorders, urinary tract infections, asthma, and constipation.5−7 They are also used as an antibacterial, antiseptic, aperient, depurative, diuretic, expectorant, laxative, and stimulant.8 Studies evaluating the pharmacological effects of T. majus flowers showed that the extract possesses antithrombin activity.9 Recent studies10,11 reported that extracts from nasturtium leaves possess diuretic and antihypertensive activity in rats, whereas Goos et al. 12 demonstrated that a commercialized preparation of the leaves possesses antibacterial activity against urinary tract infections. In addition, the leaf extract has been shown to diminish the toxic blood and liver effects induced by diethyl maleate in rats.13 The therapeutic effects of the T. majus plant are believed to be related to the high levels of glucotropaeolin,14 carotenoids,1 vitamin C, and various phenolic compounds,10,15 primarily flavonoids.11 Most of the studies on the phytochemical composition of T. majus have been done on leaves. To our knowledge, data on

Edible flowers of ornamental plants have been used in human nutrition since ancient times not only because of their aesthetic appearance and seasoning effect but also because of their biologically active substances.1 Carotenoids, anthocyanins (ACNs), phenolic acids, and flavonoids are the most representative biologically active compounds found in petals of edible flowers.1 These compounds have an inhibitive effect on the free radicals and reactive oxygen species responsible for cell and tissue damage and the acceleration of pathological processes, such as cardiovascular diseases and cancer.2 Tropaeolum majus is an annual bushy, flowering plant (about 30 cm tall) from the order Brassicales, family Tropaeolaceae, native to the Andes from Bolivia to Colombia. Its leaves are nearly circular (3−15 cm diameter) with several veins radiating to the smoothly rounded or slightly lobed margin. With the common name nasturtium, the flowers are 2.5−6.0 cm in diameter, with five petals varying from yellow to orange to red. T. majus is widely cultivated as an ornamental and medicinal plant. Its leaves, flowers, and unripe green seeds are edible. Undeveloped flower buds and green seeds are used as a substitute for capers, and chopped unripe fruits are added to tartar sauces instead of horseradish. The garden nasturtium represents one of the most popular sources of edible flowers.3 It is added to soups, meats, pasta dishes, and paste spreads and also fried in pancake batter. In Canada, blossoms stuffed with © XXXX American Chemical Society

Received: July 16, 2014 Revised: November 26, 2014 Accepted: November 28, 2014

A

DOI: 10.1021/jf503366c J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry ACN composition in nasturtium flowers is very limited; only Garzón and Wrolstad16 have reported the main ACN composition of orange flowers. Furthermore, there is no information on their non-ACN phenolic composition. The aim of this study was to evaluate the potential use of nasturtium flowers as a nutraceutical and pharmaceutical ingredient by outlining the ACNs and non-ACN phenolics profile in the petals of three different varieties, namely, red, orange, and yellow. Their total phenolics content (TPC), antioxidant activity, and antimicrobial activities were also assessed.

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solution was recorded at 765 nm on a Shimadzu UV−visible spectrophotometer, model UV 160 U (Kyoto, Japan), using 1 cm disposable cells. All measurements were performed in triplicate. Acid Hydrolysis of Flavonols and Cleanup via Solid-Phase Extraction (SPE). Confirmative determination of non-ACN aglycones (kaempferol or luteolin) was achieved following a modification of the protocol described by Durst and Wrolstad19 for acid hydrolysis of ACNs. Approximately 1 mg of the pigment was mixed with 15 mL of 2 M HCl in a screw-cap test tube. The mix was flushed with nitrogen, capped, hydrolyzed for 45 min at 100 °C, and subsequently cooled in an ice bath. The hydrolyzed extract containing the polyphenolics was cleaned using a 20 mL capacity high-load C-18 cartridge (Alltech Association, Inc., Deerfield, IL, USA). After methanolic conditioning and sample loading, the column was washed with water to remove sugars, acids, and other water-soluble compounds. The methanolic eluent containing the aglycones was subsequently evaporated using the Buchi rotoevaporator at 40 °C. The extract was freeze-dried until HPLC- photodiode array (PDA) analysis. HPLC-PDA Analysis. All processed and freeze-dried extracts were reconstituted in 1 mL of 20% aqueous MeOH (v/v) and passed through a 0.2 μm nylon filter. For ACN, non-ACN phenolics, and aglycones, an Agilent 1260 Infinity series HPLC (Agilent Technologies, Santa Clara, CA, USA) was used. The equipment consisted of a G1322A inline degassing unit, a G1312B binary pump, a G1329B autosampler, a G1316A thermostated column compartment, a G4212B diode array detector fitted with a 10 mm path, and a 1 μL volume Max-Light cartridge flow cell. The instrument was controlled using an Agilent Chemstation software version B.04.02, service pack 1 with the spectral software module. Chromatographic separations were performed on a C18 Kinetex column (4.6 mm × 100 mm, 100 Å pore size, 2.6 μm particle size; Phenomenex, Torrance, CA, USA). The mobile phase composition for all analyses was (A) H2O/H3PO4 (99.5:0.5) and (B) ACN/H2O/H3PO4 (50:49.5:0.5). Chromatographic conditions for phenolic separations were as described previously.20 ACNs were monitored at 520 nm, whereas non-ACN phenolics were monitored at 320 and 360 nm for hydroxycinammic acids (HCAs) and flavonols, respectively. HPLC-Electrospray Ionization Quadrupole Time-of-Flight Mass Spectrometry. Initial reconstitution of the freeze-dried extracts followed similar protocols as described above. Separation of phenolic compounds was conducted under the following conditions: Detection was performed using a quadrupole/time-of-flight mass spectrometer (QTof Premier, Micromass Limited, Manchester, UK) equipped with an electrospray ionization (ESI) source operated in both positive and negative modes of polarity. ESI conditions included a capillary voltage of 3.2 kV for positive mode, 2.8 kV for negative, 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. During experiments employing collisionally induced dissociation (CID), argon was held at a pressure of 3.0 × 10−3 mbar. To screen for potential parent/daughter ion relationships, both positive and negative ESI mode quadrupole time-of-flight mass spectrometry (QTOF-MSe) runs were conducted. For QTOF-MSe experiments two functions were involved, one performed at low collision energy (LE) (5 eV) and the second at high collision energy (HE) to induce fragmentation (25 eV). Coincident retention times for a given pair of ions (one from LE, another from HE), where in HE the lower mass ion is enhanced and the higher mass ion is reduced, imply a parent/daughter MS/MS relationship. Highenergy spectra for each of the ions in Table 3 with reported fragment ions are provided in the Supporting Information. The accurate masses of the parent and daughter ions combined with the ultraviolet−visible (UV−vis) spectra, retention times, and a comparison of these properties with those of available authentic standards and with those reported in the literature16,21−23 lead to the tentative identifications of phenolics investigated in this study. Quantification of Anthocyanins and Non-anthocyanin Phenolics. In addition to calculation of monomeric ACN concentration by the pH differential method, ACN quantification was based on peak areas extracted from HPLC-PDA monitored at 520

MATERIALS AND METHODS

Reagents. For the high-performance liquid chromatography (HPLC) work, cyanidin-3-glucoside (cy-3-glu) and pelargonidin-3sophoroside (pg-3-soph) were purchased from Extrasynthese (Lyon, France), cyn-3-soph was from Polyphenols (Sandnes, Norway), and 5caffeoylquinic acid (5-CQA) was from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). The Folin−Ciocalteau reagent was purchased from MP Biomedicals, LLC (Illkirch, France). For the antioxidant studies, gallic acid, fluorescein sodium salt, and 2,2′-azobis(2methylpropionamidine) dihydrochloride (AAPH) were obtained from Sigma-Aldrich (St. Louis, MO, USA); Trolox (6-hydroxy2,5,7,8-tetramethylchroman-2-carboxylic acid) was obtained from Acros Organics (Morris Plains, NJ, USA); and both sodium phosphate monobasic and sodium phosphate dibasic were purchased from Fisher Scientific (Fair Lawn, NJ, USA). For the antimicrobial studies, trypticase soy agar was purchased from Becton, Dickinson and Co. (Sparks, MD, USA). Various HPLC grade organic solvents for extractions and subsequent analyses were purchased from Fisher Scientific (Pittsburgh, PA, USA). Plant Material. Petals of orange nasturtium flowers were collected from open fields around different locations in Bogotá, Colombia, in June of 2013, whereas petals of red and yellow flowers were collected from gardens in Geneva, NY, USA, during the summer of 2013. All petals were lyophilized and stored at −80 °C until analyzed. Polyphenolic Extraction. For the extraction and isolation of ACNs and other polyphenolics, the freeze-dried plant material was milled. The powder (0.5 g) was placed in a centrifuge tube and mixed with 1:1 (w/v) 70% aqueous acetone under a nitrogen atmosphere. The tube was capped, and ultrasound-assisted extraction at a fixed frequency of 50 kHz was performed for 10 min in a chilled water bath (B-2200R-1; Branson, Shelton, CT, USA). The sample was centrifuged for 20 min at 4 °C at 3000g using an Eppendorf 5810 R centrifuge (Hamburg, Germany) to pellet the solids. The acetone extract was then transferred to a separate vial, and five successive reextractions were performed on the pellet as described. The pooled extracts were partitioned with chloroform (1:2 acetone/chloroform, v/ v) under nitrogen atmosphere. After centrifugation, the aqueous portion containing the ACNs and non-ACN phenolics was recovered and separated from the residual acetone in a Buchi rotoevaporator set at 40 °C and resolubilized in water. Extracts were stored under nitrogen at −80 °C until needed. Determination of Monomeric Anthocyanin Content, and Total Phenolics. The extracted monomeric ACN pigment content was determined by the pH differential method.17 Absorbencies were read at 490 and 700 nm for orange flowers and at 510 and 700 nm for red flowers. For comparison purposes, pigment content was calculated as cy-3-glu equivalents using an extinction coefficient (ε) of 26900 L cm−1 mol−1 and a molar mass of 449.2 g mol−1. Measurements were performed in triplicate with standard deviations reported about the means. Total phenolics content was determined as gallic acid equivalents (GAE)/100 g FW.18 A 20 μL sample aliquot of extract or gallic acid standard (50−500 mg/L) was mixed with 1.58 mL of water followed by 100 μL of Folin−Ciocalteau’s reagent. After vortexing and incubating at room temperature for 8 min, 300 μL of a 20% (w/v) aqueous sodium carbonate solution was added. Samples were vortexed and held at room temperature for 2 h. The absorbance of the blue B


Article

Journal of Agricultural and Food Chemistry Table 1. Anthocyanin Composition of Nasturtium Flowers (Tropaeolum majus)a mg Cy-3-glu/100 g DW anthocyaninb dp-3-dihexosidec cy-3-sophc pg-3-sophc total ACNc total monomeric ACNd

yellow 95.1 24.8 125.7 245.5 NDe

± ± ± ±

80.7A 18.3A 86.2A 167.3A

mg Cy-3-glu/100 g FW

orange 35.9 10 439.6 485.4 873.5

± ± ± ± ±

red

17.2A 8.7A 231.5A 237.6AB 342.8A

591.6 76.0 212.7 880.3 1292.2

± ± ± ± ±

yellow 53.1B 6.2B 65.2A 18.3B 56.3A

12.4 3.2 16.3 31.9 ND

± ± ± ±

10.5a 2.4a 11.2a 21.7a

orange 4.3 1.2 52.7 58.2 108

± ± ± ± ±

2.1a 1.0a 27.8a 28.5a 37.2a

red 76.9 9.9 27.7 114.5 168.0

± ± ± ± ±

6.9b 0.8b 8.5a 2.3b 7.3a

Results are expressed as mean ± SD (n = 3). Values in rows with different letters are significantly different at the 95% confidence level. bIndividual compounds presented as percentage of total peak area monitored at 520 nm. dp, delphinidin; cy, cyanidin; pg, pelargonidin; soph, sophoroside. c Calculated by HPLC-PDA. dCalculated by pH differential. eND, nondetectable. a

nm and external calibration curves. For comparison purposes, cy-3-glu was used as a standard. Quantification of total and individual flavonoids was based on peak areas extracted from HPLC-PDA monitored at 360 nm using external calibration curves of quercetin, kaempferol, and myricentin, whereas quantification of quinic acid esters was based on the peak area at 320 nm by external calibration curves of 5-CQA. Levels of all flavonoid derivatives were calculated using the calibration curve of the respective free flavonoids. All samples were analyzed in triplicate, and for comparison purposes, values are expressed as mg/100 g DW and mg/100 g FW. Determination of Antioxidant Activity. The automated ORAC assay was conducted using 96-well microplates according to the method described by Prior et al.24 with modifications. An aliquot (25 μL) of a petal extract or a trolox standard was mixed with 150 μL of 4 nM fluorescein in a 75 mM NaH2PO4 buffer at pH 7.4. Following 30 min of incubation at 37 °C, 25 μL of AAPH was added to each well and the fluorescence was recorded 485/528 nm (emission/excitation) once a minute for 3 h using a Synergy HT fluorescence microplate reader (Biotek Instruments, Winooski, VT, USA). The calibration curves consisted of various concentrations of Trolox (0−100 μM) made up in a phosphate buffer. Results were calculated on the basis of differences in areas under the fluorescence decay curve between the blank, samples, and standards. Final ORAC values were expressed as μmol of Trolox equivalents (TE)/100g DW and μmol of TE/100 g FW. Antibacterial Activity. The antibacterial activity of the extracts against foodborne pathogenic strains including Salmonella enterica, serovar Hartford, Escherichia coli ATCC 43895, Listeria monocytogenes 104025, Staphylococcus aureus 9144, and Bacillus cereus F 4550 was determined using the spot-on-lawn assay.25 A 50 μL aliquot of an overnight culture of the respective strain was inoculated in 7 mL of liquid TSB agar media (0.7% agar) and poured onto plates containing a TSA agar base. After the agar in the plates solidified, 20 μL of each extract was spotted onto this lawn, and plates were incubated overnight at 37 °C for 24 h. After incubation, the inhibition zones were evaluated. Statistical Analyses. Quantitative data are presented as mean values with the respective standard deviation. Significant (P < 0.05) differences among means were identified using the least significant difference (LSD) multiple-range test after a multifactor analysis of variance (ANOVA). All analyses were performed with Statgraphics plus, version 2.1.

were collected from different locations across Bogotá, Colombia. Conversely, the yellow and red samples were collected in localized small gardens in the city of Geneva, NY, USA. This variability is expected as it has been reported that plant phenolics vary in response to environmental factors such as light intensity and nutrient availability.27 The TPC in red flowers was 908.7 ± 284.6 mg GAE/100 g FW, equivalent to the TPC in orange flowers (687.7 ± 161.3 mg GAE/100 g FW) and significantly higher than that one in yellow flowers (538.4 ± 6.5 mg GAE/100 g FW) (p < 0.01). The lower TPC content of yellow flowers is driven by the low content of ACNs. The three samples showed TPC values comparable to tropical fruits with known elevated phenolic concentrations. Vasco et al.28 tested 17 fruits from Ecuador, and Rufino et al.29 studied 18 tropical fruits from Brazil. According to their classification, fruits with a TPC > 500 mg GAE/100 g FW are considered high. According to Vasco’s classification, at 1010 mg GAE/100 g FW, banana passion fruit (Passiflora mollissima L.) displayed the highest TPC. Likewise, Rufino et al. found that camu-camu (Myrciaria dubia) and acerola (Malpighiae marginata) had 1176 and 1063 mg GAE/100 g FW, respectively. Therefore, these fruits were placed at the top of the analyzed fruits as excellent sources of polyphenols. Anthocyanin and Non-anthocyanin Polyphenolic Composition. Petals from red, orange, and yellow flowers shared similar ACN profiles (Figure 1). Delphinidin-3-dihexoside (dp-3-dihesoxide), cy-3-soph, and pg-3-soph were detected in each sample. The results from the HPLC-ESI-QTOF-MSe analysis of ACNs are shown in Table 2. Delphinidin-3-dihexoside with parent ions at m/z 627.158, [M + H]+, and m/z 625.146, [M − H]−, resulted in HE delphinidin aglycone ion signals at m/z 303.051, [M + H − X]+, and m/z 301.033, [M − H − X]−, arising from a 324 amu loss, suggesting a dihexoside moiety. Accordingly, this compound was labeled delphinidin-3-dihexoside (dp-3-dihexoside). Cyanidin-3-soph was characterized by molecular ions at m/z 609.15, [M − H]−, and m/z 611.162, [M + H]+, with the resultant HE daughter ions at m/z 285.037 and 287.058, corresponding to the cyanidin aglycone. Identification of pg-3-soph was determined via molecular ions at m/z 595.168, [M + H]+, and m/z 593.157, [M − H]−, and aglycone ion signals at m/z 271.063, [M + H − X]+, and m/z 269.042, [M − H − X]−. These last two ACNs were also confirmed on the basis of congruence of retention time and UV−vis properties with authentic cy-3-soph and pg-3-soph standards. These findings confirm previous investigations on the ACN composition of orange nasturtium flowers (Garzón and

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RESULTS AND DISCUSSION Anthocyanin and Total Phenolics Content. The means of total ACN as determined by the pH differential method were 108.8 ± 37.2 and 168.0 ± 7.3 mg cy-3-glu/100 g FW in orange and red flowers, respectively (Table 1). These averages are comparable to the range of 70.3−201.0 mg cy-3-glu/100 g FW and the mean of 137 mg cy-3-glu/100 g FW for 21 blackberry samples taken from different origins as reported by Chiang and Wrolstad.26 The high SD for the average value in the orange flowers may be due to the varied geographic sampling site as the samples C


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Journal of Agricultural and Food Chemistry

Figure 1. HPLC-PDA profile of anthocyanins in nasturtium flowers (Tropaeolum majus) detected at 512 nm: (A) yellow flowers; (B) orange flowers; (C) red flowers. Peaks: 1, delphinidin-3-dihexoside; 2, cyanidin-3-sophoroside; 3, pelargonidin-3-sophoroside. Figure 2. HPLC-PDA profile of hydroxycinnamic acids in nasturtium flowers (Tropaeolum majus) detected at 320 nm: (A) yellow flowers; (B) orange flowers; (C) red flowers. Peaks: 1, 3-caffeoylquinic acid (neochlorogenic acid); 2, cis-3-p-coumaroylquinic acid; 3, trans-3-pcoumaroylquinic acid; 4, 5-caffeoylquinic acid (chlorogenic acid); 5, cis-5-p-coumaroylquinic acid; 7, trans-5-p-coumaroylquinic acid.

Wrolstad16) and provide the first report of the ACN profile of red and yellow flowers. The HPLC-PDA profile of non-ACN phenolics in flowers of T. majus is shown in Figure 2, whereas the HPLC-ESI-QTOFMSe results from the analysis of these compounds are shown in Table 3. Non-anthocyanin phenolics included HCAs and flavonols. Six HCAs belonging to chlorogenic acid family (esters of trans-cinnamic acids and quinic acid) were found. The first and fourth eluting compounds with λmax 323 and 322 nm, respectively, and a difference in tR of 1.56 min yielded molecular ions [M − H]− at m/z 353.087 and [M − H + Na]+ and at m/z 375.069 with an HE fragment at m/z 191.053 identical to the 5-CQA standard. The fragment at m/z 191.053 corresponds to the loss of hydrogen from quinic acid typical of chlorogenic acids and their derivatives in tandem mass spectrum extracted ion chromatogram (EIC).22 Because the order of elution between neochlorogenic acid (3-CQA) and chlorogenic acid (5-CQA) has been established in reversephase chromatography,21 peaks 1 and 4 were identified as 3CQA and 5-CQA, respectively.

Peaks 2, 3, 5, and 7 produced parent ions at m/z 337.092, [M−H]−, which fragmented in HE daughter ions at m/z 163.037 ([p-coumaric acid-H]−) and m/z 191.053 ([quinic acid-H]−). On the basis of the strong signal at m/z 163.037, peaks 2 and 3 were tentatively labeled as cis-3-p-coumaroyl quinic acid (CoQA) and trans-3-p-CoQA, respectively.21 In contrast, based on the prominent signal at m/z 191.053 and the later elution of compounds 5 and 7,21 they were labeled as cis-5p-CoQA and trans-5-p-CoQA.21 Figure 3 is the HPLC-PDA profile of flavonols in nasturtium flowers. Myricetin conjugates (peaks 6, 8, and 10) with tR = 7.16, 7.75, and 7.96 min, respectively, exhibited UV−vis absorption maxima at 354−356 nm. Compound 6 provided molecular ions at m/z 641.138, [M − H]−, and m/z 665.135, [M + H + Na+]+. The HE fragment [M − H − X]− at m/z 317.03 was produced after cleavage of a dihexoside (324 amu)

Table 2. HPLC-ESI-QTOF-MSe Analysis of Anthocyanins Present in Nasturtium Flowers (Tropaeolum majus) peak

tR (min)

λmax (nm)

LE MSe[M − H]−m/z

HE MSe[M − H]−m/z

LE MSe[M + H]+m/z

HE MSe[M + H]+m/z

tentative peak assignment

1 2 3

5.75 7.37 9.14

524 515 503

625.146 609.15 593.157

300.026, 301.033 284.031, 285.037 268.036, 269.042

627.158 611.162 595.168

303.051 287.058 271.063

delphinidin-3-dihexoside cyanidin-3-sophoroside pelargonidin-3-sophoroside

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Table 3. HPLC-ESI-QTOF-MSe Analysis of Non-anthocyanin Phenolics Present in Nasturtium Flowers (Tropaeolum majus) peak

tR (min)

λmax (nm)

LE MSe[M − H]− m/z

HE MSe[M − H]− m/z

1

4.0

323

353.087, 375.069

191.053, 179.030

ND

ND

2 3 4 5 6 7 8 9 10 11 12 13 14 15

4.73 4.93 5.56 5.57 7.16 7.44 7.75 7.77 7.96 8.41 9.05 9.44 9.71 10.41

315 308 322 320 354 320 356 356 356 345 344 350 344 350

337.092, 337.092, 353.087, 337.092, 641.138 337.092, 683.15 625.145 479.083 609.151 651.157 505.099 447.092 489.105

163.037, 191.053 163.037,191.053 191.053, 179.030 163.037, 191.053 316.015, 317.03 163.037, 191.053 316.015, 317.03 300.026, 301.032 316.015, 317.03 284.032, 285.039 284.032, 285.039 300.026, 301.033 284.032, 285.039 284.032, 285.039

ND ND ND ND 665.135 ND ND 649.141 503 633.145, 611.165 ND ND 471.091 ND

ND ND ND ND 319.047 ND 319.047 303.053 319.047 287.058 303.059 303.053 287.057 287.057

359.073 359.073 375.069 359.073 359.073

LE MSe[M + H]+ m/z

moiety from the corresponding myricetin aglycon. As such, this peak was tentatively identified as myricetin dihesoxide. Peak 8, with a loss of 366 amu from a molecular ion at m/z 683.15, [M − H]−, agrees with the loss of an acetyl-dihexoside moiety. Peak 10 yielded a molecular ion at m/z 479.083 in the negative ESI mode and a HE fragment at m/z 317.03. The loss of 162 amu from the aglycon revealed the cleavage of a hexose moiety. Quercetin derivatives (peaks 9 and 13) showed UV−vis absorption maxima at 356 and 350 nm, respectively. Peak 9 with tR = 7.77 and parent ions at m/z 625.144, [M − H]−, and m/z 649.14, [M + H − X + Na+]+, had a typical HE fragment at m/z 301.033, [M − H − X]−, and was identified as a quercetin dihexoside on the basis of the loss of a dihexoside moiety (324 amu). Peak 13 with tR = 9.44 had a molecular ion at m/z 505.099 in the negative ESI mode and an HE fragment 301.033. The loss of 204 amu equates to the loss of an acetyl hexoside moiety. For compounds 11, 12, 14, and 15, a λmax between 344 and 350 nm was observed. The molar mass of kaempferol and luteolin is the same; therefore, they will yield the same parent ion mass fragmentation pattern in MS. Furthermore, whereas these two compounds, under certain conditions and columns, have been known to commonly coelute,30 they did not under the conditions employed for this study. Therefore, acid hydrolysis is important when distinguishing between these two compounds. As such, this work unequivocally identified each of these compounds as kaempferol derivatives. From a bioactive prospective, it is essential to differentiate between these two flavonoids because their differential bioactivity may be attributed to their ability to react with reactive oxygen species and their interaction with membrane lipid bilayers. Tsuchiya31 indicated that in the structure−membrane interactivity, 3-hydroxylation of the C ring and 5,7-dihydroxylation of the A ring provide flavonoids with the greatest ability to interact with membranes and protect them. Therefore, kaempferol would have a better protective effect on the lipid bilayer than luteolin. The HE fragmentation of peak 11 led to the kaempferol aglycone at m/z 285.039 in a negative mode and m/z 287.05 in the positive mode. The loss of 32 amu is assumed to be from the cleavage of a dihexoside. A sodium adduct at m/z 633.145 was seen in the positive ESI mode. Peak 12 with tR = 9.05 had [M − H]− at m/z 651.157 with a fragment at m/z 285.039

HE MSe[M + H]+ m/z

tentative peak assignment 3-caffeoylquinic acid (neochlorogenic acid) cis-3-p-coumaroylquinic acid trans-3-p-coumaroylquinic acid 5-caffeoylquinic acid (chlorogenic acid) cis-5-p-coumaroylquinic acid myricetin dihexoside trans-5-p-coumaroylquinic acid myricetin acetyl dihexoside quercetin dihexoside myricetin hexoside kaempferol dihexoside kaempferol acetyl dihexoside quercetin acetyl hexoside kaempferol hexoside kaempferol acetyl hexoside

Figure 3. HPLC-PDA profile of flavonoids in nasturtium flowers (Tropaeolum majus) detected at 360 nm: (A) yellow flowers; (B) orange flowers; (C) red flowers. Peaks: 6, myricetin dihexoside; 8, myricetin acetyl dihexoside; 9, quercetin dihexoside; 10, myricetin hexoside; 11, kaempferol dihexoside; 12, kaempferol acetyl dihexoside; 13, quercetin acetyl hexoside; 14, kaempferol hexoside; 15, kaempferol acetyl hexoside.

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Journal of Agricultural and Food Chemistry Table 4. Content of Non-anthocyanin Phenolics in Nasturtium Flowers (Tropaeolum majus)a content (mg/100 g DW) phenolic compound hydroxycinnamic acids neochlorogenic acid cis-3-p-CoQA cis-5-p-CoQA chlorogenic acid trans-5-p-CoQA p-coumaroylquinic isomer 4 total flavonols myricetin dihexoside myricetin acetyl dihexoside myricetin hexoside total

quercetin dihexoside quercetin acetyl hexoside total

kaempferol kaempferol kaempferol kaempferol total

dihexoside acetyl dihexoside hexoside acetyl hexoside

yellow 283.2 405.3 639.2 246.6 141.3 96.9 1718.8

± ± ± ± ± ± ±

14.5A 51.6A 56A 36.2A 11.0A 6.6A 110.1A

0.8 2.3 3.1 6.0

± ± ± ±

0.1A 0.7A 1.6A 2.5A

8.2 ± 2.0A 34.6 ± 16.2A 42.8 ± 18.2A

36.9 31.8 15.8 143.1 227.6

± ± ± ± ±

10.9A 6.7A 8.2A 50.6A 58.5A

orange

content (mg/100 g FW) red

yellow

181.6 60.1 105.8 233.0 49.0 46.8 550.6

± ± ± ± ± ± ±

12.7B 41.3B 76B 41.6AB 15.1B 8.7B 74.9B

Chlorogenic Acid Equivalents 36.4 ± 6.2C 36.8 ± 1.a B 9.9 ± 6.3 52.7 ± 6.7a 35.4 ± 6.4B 83.1 ± 7.3a 138 ± 38.3B 32.1 ± 4.7a B 36.8 ± 10.0 18.4 ± 1.4a b ND 12.6 ± 0.9a 256.5 ± 54.4C 235.6 ± 8.1a

0.64 4.7 4.4 9.7

± ± ± ±

0.13A 3.0A 1.7A 4.7A

Myricetin Equivalents 2264.8 ± 9.3B ND 74.7 ± 8.7B 0.3 ± 0.1a 84.5 ± 0.3B 0.4 ± 0.2a B 2424.0 ± 18.3 0.8 ± 0.3a Quercentin Equivalents 117.4 ± 13.1C 1.1 ± 0.3a A 6.2 ± 2.3 4.5 ± 2.1a 123.6 ± 15.6B 5.6 ± 2.4a

50.1 ± 9.6B 30.4 ± 24.9A 80.5 ± 27.5AB

1198.9 121.2 38.4 33.5 1392.0

± ± ± ± ±

Kaempferol Equivalents 267.7 ± 77.7A 4.8 30.7 ± 0.71A 4.1 5.3 ± 1.3A 2.1 10.8 ± 2.0B 18.6 314.4 ± 81.7A 29.6

479.8B 17.7B 17.7B 23.4B 480.3B

± ± ± ± ±

1.4a 0.1a 1.1a 6.6a 7.6a

orange ± ± ± ± ± ± ±

1.5b 5.0b 9.1a 5.0a 1.8b 1.1b 13.9b

21.8 7.2 12.7 28.0 5.9 5.6 81.2

ND 0.6 ± 0.4a 0.5 ± 0.2a 1.2 ± 0.6a

6.0 ± 1.2b 3.7 ± 3.0a 9.7 ± 3.3ab

143.9 14.5 4.6 4.0 167.0

± ± ± ± ±

57.6b 2.1b 2.1b 2.8b 57.6b

red 4.7 1.3 4.6 17.9 4.8 ND 33.3

± ± ± ± ±

294.4 9.7 11.0 315.1

± ± ± ±

0.8c 0.8b 0.8b 5.0b 1.3b

± 7.1c

1.2 1.1b 0.0b 2.4b

15.3 ± 1.7c 0.8 ± 0.3a 16.1 ± 2.0b

34.8 4.0 0.7 1.4 40.9

± ± ± ± ±

10.1a 0.1a 0.2ab 0.3b 10.6a

Results are expressed as the mean ± SD (n = 3). Values in rows with different letters are significantly different at the 95% confidence level. bND, nondetectable.

a

2.6 times higher than values for total anthocyanins determined by HPLC. The authors attributed these results to the presence of ACN polymers that influence color as well as copigmentation reactions that involve the formation of weak complexes between anthocyanin glycosides and other noncolored components such as phenolic acids, flavonoids, and flavonol derivatives. This results in a hyperchromic response that may result in an overestimation of total anthocyanins in spectrophotometric assays. Orange flowers showed an abundance of pg-3-soph (52.7 ± 27.8 mg cy-3-glu equiv/100 g FW) and low concentrations of dp-3-dihesoxide and cy-3-soph. Conversely, red flowers presented high values of dp-3-dihesoxide (76.9.6 ± 6.9 mg cy-3-glu equiv/100 g FW) and pg-3-soph (27.7 ± 8.5 mg cy-3glu equiv/100 g FW), whereas yellow flowers showed small concentration of each ACN with similar concentrations of pg-3soph and dp-3-dihesoxide. The concentrations of phenolic compounds in the extracts from petals with different colors as determined by peak area in the HPLC-PDA chromatogram are shown in Table 4. The highest level of flavonols was found in red flowers, with myricetin derivatives being the dominant flavonoids (315.1 ± 2.4 mg myricetin equiv/100 g FW or 2424.0 ± 18.3 mg myricetin equiv/100 g DW) followed by kaempferol derivatives (40.9 ± 10.6 mg kaempferol equiv/100 g FW or 314.4 ± 81.7 mg kaempferol equiv/100 g DW) and quercetin derivatives (16.1 ± 2.0 mg quercetin equiv/100 g FW or 123.6 ± 15.6 mg quercetin equiv/100 g DW). The quercetin content in red flowers is comparable with the amount present in food and medicinal plants with a very high concentration of this

(acetyl-dihexoside moiety, 366 amu) and was identified as kaempferol acetyl dihexoside. Peak 14 had ions at m/z 447.092, [M − H]−, and m/z 471.091, [M + H + Na]+, which is consistent with kaempferol hexoside. Compound 15 was tentatively assigned as kaempferol acetyl hexoside on the basis of its molecular ion ([M − H]−) at m/z 489.105 as it showed a mass difference of 42 amu in relation to its nonacylated analogue kaempferol hexoside (peak 14). The non-ACN phenolic composition of petals from nasturtium flowers is comparable with the composition of T. majus seeds. In 2013, Bazylko et al.23 reported the presence of 3-p-CoQA, 4-CoQA, 5-CoQA, 3-CQA, 3-CQA, and 5-CQA in 60 aqueous ethanol extracts. With regard to the flavonoid composition, the authors found quercetin and kaempferol derivatives, but not myricetin conjugates. Seeds also contained a methoxyquercetin derivative, isoquercetin, and astragalin. Quantification of Anthocyanins and Non-anthocyanin Polyphenolics by HPLC. Despite the similarity in constituent ACNs among petals with different colors, there were variations in total ACN concentration and amount of each pigment. A significant difference (p < 0.05) in the amount of ACNs as influenced by color was found. The highest amount of pigment was found in red flowers (114.5 ± 2.3 mg cy-3-glu/100 g FW), followed by orange flowers (58.2 ± 28.5 mg cy-3-glu/100 g FW) and yellow flowers (31.9 ± 21.7 mg cy-3-glu/100 g FW). Whereas the value of ACN content as determined by HPLC was lower than the value determined by the pH differential method, similar results have been observed by other authors. Pacheco-Palencia et al.32 found that the concentration of açai (Euterpe oleracea) ACNs quantified by spectrophotometry was F


Article

Journal of Agricultural and Food Chemistry compound. White onion (Allium cepa) contains 113.0 ± 6.0 mg quercetin equiv/100 g DW, Ginkgo biloba leaf has 136.0 ± 6.0 mg quercetin equiv/100 g DW, and Calendula officinalis flower has 112.0 ± 6.0 mg quercetin equiv/100 g DW.30 On the other hand, the primary source of quercetin in the American diet (onions, tea, and apples) is either much lower or comparable to that of nasturtium flowers of any color.31,33 Quercetin has been shown to be an excellent antioxidant with anti-inflammatory, antiproliferative, and gene expression changing capacities in vitro.34 In addition, antioxidative and anti-inflammatory effects have also been demonstrated in vivo, especially in people with hypertension and sarcoidosis.34 This high antioxidant activity is attributed to its o-dihydroxy structure on the B ring and a −OH group at position 3, which results in the ability of quercetin to transfer a hydrogen atom to radicals.35 Kaempferol derivatives were the main flavonoids in orange flowers (167.0 ± 57.6 mg kaempferol equiv/100 g FW or 1,392.0 ± 480.3 mg kaempferol equiv/100 g DW). The second most abundant group of compounds in these petals were members of the chlorogenic acid family (81.2 ± 13.9 mg chlorogenic acid equiv/100 g FW or 550.6 ± 74.9 mg chlorogenic acid equiv/100 g DW), followed by small amounts of quercetin derivatives (9.7 ± 3.3 mg quercetin equiv/100 g FW or 80.5 ± 27.5 mg quercetin equiv/100 g DW) and myricetin conjugates (1.2 ± 0.6 mg myricetin equiv/100 g FW or 9.7 ± 4.7 mg myricetin equiv/100 g DW). Among 20 plant materials, Rosa damascene flower showed the highest kaempferol content (1480.0 ± 35.0 mg kaempferol equiv/100 g DW),30 comparable to the kaempferol content in orange flowers. The high flavonoid content of this flower seems to be the primary reason for its protective effects on the cardiovascular system.36 Although kaempferol has only one OH group, accounting for a lower antioxidant activity than quercetin, epidemiologically, in vitro and in vivo studies show a positive association between the consumption of kaempferol and their glycosides, and subsequent pharmacological activities, including anti-inflammatory, antimicrobial, anticancer, cardioprotective, neuroprotective, antidiabetic, antiallergic, and antiasthmatic activities.37 Several studies have explained that the presence of a double bond at C2−C3 in conjugation with an oxo group at C4 and the presence of hydroxyl groups or an acylated structure at C3, C5, and C4′ are the primary structural impetus for the antioxidant activity of kaempferol.37 It has been estimated that the human dietary average intake of this polyphenol is approximately 10 mg/day.38 Therefore, 10 g of fresh orange flowers would supply this intake. Myricetin has been reported as a bioactive flavonoid during in vivo trials. Administration of aqueous and ethanolic extracts of myricetin glycosides from Myrtus communis L. showed significant antihyperglycemic, anti-inflammatory, and antinociceptive effects in albino mice as compared with control groups and reference drugs.39 Similarly, the methanolic extract of Cotinus coggygria showed medicinal potential as an antigenotoxic and antihepatotoxic natural agent in albino Wistar rats. Because myricetin is the main flavonoid of this plant, the authors hypothesized that this compound could be responsible for the observed bioactivity and free radical scavenging activity.40 In contrast, the most representative polyphenolic compounds present in yellow flowers were hydroxycinammates. The total concentration of isomers of CQA and p-CoQA in this sample (235.6 ± 8.1 mg chlorogenic acid equiv/100 g FW) was

significantly higher (p < 0.05) than the concentration in red and orange flowers. Conversely, as compared to yellow flowers, the amount of myricetin and quercetin conjugates was significantly higher (p < 0.05) in red petals, whereas the concentration of kaempferol conjugates was significantly higher (p < 0.05) in orange petals. It has been reported that chlorogenic acid has antioxidant, anticarcinogenic, anti-inflamatory, and cholesterol-lowering activities41 as well as protective properties against degenerative age-related diseases in animals.42 Coffee is considered the main source of chlorogenic acids in the human diet; it is estimated that a 200 mL cup delivers 200 mg of total chlorogenic acids.43 Thus, 100 g of fresh yellow petals would provide an amount of chlorogenic acid comparable to a 200 mL cup of coffee. Generally, total phenolics, including ACNs and non-ACN polyphenols, were much lower in yellow flowers as compared with orange and red flowers. Mato et al.44 stated that in edible flowers, an increased content of ACNs is correlated with high levels of total flavonoids. Terahara and Yamaguchi45 explained that there is a common pathway of synthesis of flavonoids and ACNs; pale flowers lack enzymes such as flavanone 3hydroxylase, dihydroflavonol 4-reductase,44 and/or anthocyanidin synthase.46 Antioxidant Activity. The ORAC radical-scavenging activity of the red flowers (18719 ± 696 μmol TE/100 g FW) was significantly higher (p < 0.05) than those of the orange flowers (11790 ± 1570 μmol TE/100 g FW) and yellow flowers (7111 ± 3545 μmol TE/100 g FW). ORAC values reflect the capacity of the cumulative extracted antioxidants present to donate a proton to the AAPH radical. When components with an important antioxidant capacity are present in high concentrations, the ORAC value is driven by these high-performing compounds. Furthermore, synergistic effects among components with high antioxidant properties have been found.47 Red flowers exhibited the highest TPC and ACN concentrations as well as the highest antioxidant activity. This can be explained by the high concentration and specific chemical structure of phenolic compounds present in red flower extracts. It has been shown that cyanidin, delphinidin, and quercetin derivatives have a high radical-scavenging activity and an antioxidant capacity comparable to the activities of the well-known antioxidants α-tocopherol and Trolox.48 The extracts from T. majus petals did not exhibit any antimicrobial activity against any of the tested microorganisms. In a previous study,23 the authors observed no effect of nasturtium seed extracts on S. aureus ATCC 9341 or E. coli ATCC 25922. The authors hypothesized that the low content of benzyl isothiocyante (a compound with antimicrobial activity and breakdown product of glucotropaeloin) in the extract was responsible for the lack of antimicrobial activity. In conclusion, this comparative study revealed the profile of phenolic compounds in T. majus petals of different colors. Their antioxidant activity was also evaluated, and the results showed that they appear to be good sources of natural antioxidants that can be used as food additives or dietary antioxidants once their bioavailability and mechanism of action have been evaluated.

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ASSOCIATED CONTENT

S Supporting Information *

QTOF-ESI-MSe for fragment ions in HE for non-anthocyanin phenolics present in nasturtium flowers (Tropaeolum majus); labels in each figure correspond to peak numbers as reported in G


Article


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Table 3. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(G.A.G.) Phone: 571 316 5000, ext. 14457. Fax: 571 3165220. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Tom Gibson (Cornell University) for technical assistance.

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Identification of Phenolic Compounds in Petals of Nasturtium Flowers

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