Borage, Calendula, Cosmos, Johnny Jump Up, and Pansy Flowers: Volatiles, Bioactive Compounds, and Sensory Perception

European Food Research and Technology https://doi.org/10.1007/s00217-018-3183-4

ORIGINAL PAPER

Borage, calendula, cosmos, Johnny Jump up, and pansy flowers: volatiles, bioactive compounds, and sensory perception

Luana Fernandes1,2,3 · Susana Casal2 · José A. Pereira1 · Ricardo Malheiro1 · Nuno Rodrigues1 · Jorge A. Saraiva3 · Elsa Ramalhosa1

Received: 27 June 2018 / Accepted: 28 October 2018 © Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract The aim of the present work was to study the main volatile and bioactive compounds (monomeric anthocyanins, hydrolysable tannins, total flavonoids, and total reducing capacity) of five edible flowers: borage (Borage officinalis), calendula (Calendula arvensis), cosmos (Cosmos bipinnatus), Johnny Jump up (Viola tricolor), and pansies (Viola × wittrockiana), together with their sensory attributes. The sensory analysis (10 panelists) indicated different floral, fruity, and herbal odors and taste. From a total of 117 volatile compounds (SPME–GC–MS), esters were most abundant in borage, sesquiterpenes in calendula, and terpenes in cosmos, Johnny Jump up, and pansies. Some bioactive and volatile compounds influence the sensory perception. For example, the highest content of total monomeric anthocyanins (cosmos and pansies) was associated with the highest scores of colors intensity, while the floral and green fragrances detected in borage may be due to the presence of ethyl octanoate and 1-hexanol. Therefore, the presence of some volatiles and bioactive compounds affects the sensory perception of the flowers.

Keywords Edible flowers · Volatile compounds · Sensory analysis · Bioactive compounds

Introduction and fragrances of flowers are analyzed through their volatile essential oils [1]. Currently, there are some studies that Edible flowers are becoming more popular in recent years have applied solid-phase microextraction (SPME) method to due to the interest of consumers and professional chefs. analyze volatile compounds in fresh flowers [2–5], having a Flowers not only look great, but also add color, aroma, and more clear perception of the real flower volatiles by avoid- flavor to drinks and dishes. It is known that fragrance/aroma ing the interferences of newly formed compounds induced and appearance are attributes that affect consumers’ pref- by extraction condition or enzymatic action. Edible flow- erences, being important quality factors. Usually, flavors ers have a complex flavor, without single compound that accounts for a distinctively flavor. The characteristic flavor of a flower is mainly due to the association of several vol- * Susana Casal atile constituents, which are mostly made up of terpenes, [email protected] esters, alcohols, carbonyls, and alkane compounds [2, 4, 6]. * Elsa Ramalhosa However, there are others nonvolatile chemical constituents, [email protected] such as phenolic compounds, sugars, and organic acids, that have a variable impact on the volatility of aroma compounds 1 Centro de Investigação de Montanha (CIMO)/School of Agriculture, Polytechnic Institute of Bragança, Campus de and taste and consequently on their sensory perception [7]. Stª Apolónia, 5300‑253 Bragança, Portugal Thus, the present work had two objectives. The first one 2 LAQV@REQUIMTE/Laboratory of Bromatology was to quantify the main bioactive compounds [monomeric and Hydrology, Faculty of Pharmacy, Porto University, Rua anthocyanins, flavonoids, total reducing capacity (TRC) and Jorge Viterbo Ferreira, 228, 4050‑313 Porto, Portugal hydrolysable tannins] and volatile compounds by headspace 3 Organic Chemistry, Natural Products and Agrifood solid-phase microextraction coupled to gas chromatogra- (QOPNA), Chemistry Department, University of Aveiro, phy–mass spectrometry (HS-SPME-GC–MS) present in five Campus Universitário de Santiago, 3810‑193 Aveiro, common fresh edible flowers (borage, pansies, Johnny Jump Portugal

Vol.:(0123456789)1 3 European Food Research and Technology up, calendula, and cosmos), together with their organoleptic Gas chromatography–mass spectrometry (GC–MS) analysis appreciation. The second objective was to determine possible relationships between the volatile and phenolic com- A Shimadzu GC-2010 Plus gas chromatographer equipped pounds identified in the five flowers with the tasters’ sensory with a mass spectrometer Shimadzu GC/MS-QP2010 SE perception. detector was used for volatiles determination. A TRB-5MS (30 m × 0.25 mm × 0.25 µm) column (Teknokroma, Spain) was used. The injector was set at 220 °C and the manual Materials and methods injections were made in splitless mode, with helium (Praxair, Portugal) at a linear velocity of 30 cm/s and a total flow Samples of 24.4 mL/min as mobile phase. The oven temperatures were the following: 40 °C (1 min); 2°C/min until 220 °C (30 min). The ionization source was maintained at 250 °C Borage officinalis Calen- White borage ( ), yellow calendula ( with ionization energy of 70 eV and with an ionization cur- dula arvensis) Cosmos bipinnatus) , purple cosmos ( , pur- rent of 0.1 kV. All mass spectra were acquired by electron Viola tricolor ple Johnny Jump up ( ), and red pansies ionization in the m/z 35–500 range. The full-scan MS spectra Viola wittrockiana ( × ) flowers were obtained from a Portu- fragments were compared with those obtained from a data- guese store, located in the Northeast of Portugal, that sells base (NIST 11), and with those of commercial standards edible flowers. In Fig. 1, the five studied flowers are pre- acquired from diverse producers (see Table 1). For qualita- sented. Around 20 g of each flower was used to perform the tive purposes, the areas of the chromatographic peaks were analyses described below. determined integrating the re-constructed chromatogram from the full-scan chromatogram using for each compound Volatile compounds the ion base (m/z intensity 100%). For semi-quantification purposes, volatile amounts were calculated by the ratio of Headspace solid‑phase microextraction (HS‑SPME) fibers each individual base ion peak area to the area of the internal standard base ion peak area and converted to mass equiva- For the HS-SPME, a fiber coated with divinylbenzene/car- lents on the basis on the internal standard mass added. boxen/polydimethylsiloxane (DVB/CAR/PDMS; 50/30 µm) Bioactive compounds was selected, based on a preliminary assay conducted with other fiber (PDMS; 100 µm), both from Supelco (Bellefonte, Extraction USA). The selection of the fiber was based on the highest qualitative (number of volatiles extracted) and quantitative data (peak areas) obtained for a sample (data not shown). Fresh samples were extracted with water:acetone (6:4; v/v), at 37 °C, for 30 min under agitation (IKA, RCT Model B, Staufen, Germany) at a frequency of 1000 rpm, following the HS‑SPME methodology used by Li et al. [9]. The extracts were filtered, concentrated in the rotary evaporator (Stuart RE3022C, The HS-SPME was carried out according to the method- Staffordshire, United Kingdom), frozen at − 18 °C, and lyo- ology applied by Almeida et al. [8], with some modifica- philized (48 h, Coolsafe, Lynge, Denmark). The obtained tions. First, fresh petals of borage, calendula and cosmos extracts were redissolved in water:acetone (6:4; v/v) to a (0.20–0.30 g), whole pansy flowers and four flowers of concentration of 50 mg extract/mL and preserved under Johnny Jump up (0.70–1.00 g) (cut a few mm below the freezing until further analysis. Each extraction treatment calix, being the pedicels wrapped in aluminum foil to mini- was performed in triplicate. mize water loss) were placed in 50 ml vials (except pansies in 100 ml vials). Then, 4-methyl-2-pentanol was added Monomeric anthocyanins, total flavonoids, hydrolysable as internal standard (10 ppm in methanol), being the vials tannins, and total reducing capacity immediately sealed with a polypropylene cap with silicon septum. The volatiles were released at room temperature The total monomeric anthocyanins, total flavonoids and (± 25 °C) during 5 min. After that, the 30/50 µm DVB/CAR/ hydrolysable tannins contents, as well as the total reducing PDMS fiber was exposed during 30 min at room temperature capacity (TRC) of the edible flowers extracts were deter- for volatiles adsorption, and then inserted into the injection mined following the methodologies used by Fernandes port of the GC system for thermal desorption and recondi- et al. [10]. All measurements were performed in triplicate. tioning (10 min at 280 °C). For each sample of flower, the The results for monomeric anthocyanins were expressed in HS-SPME analysis was performed in quintuplicate. mg cyanidin-3-glucoside/g dried weight (mg Cy 3-glu/g

1 3 European Food Research and Technology

Fig. 1 Visual appearance of the Edible flowers five edible flowers

Borage (Borago officinalis L.)

Johnny Jump up (Viola tricolor)

Pansies (Viola ×wittrockiana)

Calendula (Calendula arvensis)

Cosmos (Cosmos bipinnatus)

DW), flavonoids in mg of quercetin equivalent/g dried Sensory analysis weight (mg QE/g DW), hydrolysable tannins in mg of tannic acid equivalent/g dried weight (mg TAE/g DW) For sensory analysis, ten tasters (six females and four males, and TRC in mg gallic acid equivalent/g dried weight (mg from 29 to 45 years) from the Polytechnic Institute of Bra- GAE/g DW). gança, Portugal (including teachers, students, and other staff) agreed to participate in the evaluation sessions. The panel is subject to periodic training and updates in sensory analysis,

1 3 European Food Research and Technology Pansy n.d 2.83 ± 2.89 n.d n.d 0.53 ± 0.31 0.53 ± 0.30 n.d 3.89 n.d n.d n.d 0.16 ± 0.06 n.d 4.09 ± 1.60 1.80 ± 0.85 1.57 ± 0.60 7.62 n.d 0.85 ± 0.38 0.54 ± 0.11 n.d 1.39 n.d n.d n.d 8.99 ± 4.43 n.d n.d n.d n.d Johnny Jump up Jump Johnny 14.1 ± 3.0 6.5 ± 1.7 n.d n.d n.d n.d 7.0 ± 2.2 27.6 n.d 0.76 ± 0.20 3.83 ± 1.49 n.d 18.8 ± 10.8 n.d n.d 0.19 ± 0.05 23.6 0.39 ± 0.24 0.39 ± 0.05 1.09 ± 0.18 0.20 ± 0.05 2.07 2.32 ± 1.37 n.d n.d n.d n.d n.d 1.03 ± 0.16 3.08 ± 0.94 Cosmos n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d 10.0 ± 2.97 n.d 10 n.d 1.81 ± 0.25 1.16 ± 0.41 n.d 2.97 n.d n.d n.d n.d n.d n.d 1.41 ± 0.13 n.d Calendula 32.8 ± 14.5 n.d n.d n.d n.d n.d n.d 32.8 7.94 ± 5.17 3.06 ± 0.84 n.d 0.42 ± 0.16 n.d n.d 1.98 ± 0.78 n.d 13.4 n.d 0.91 ± 0.26 n.d n.d 0.91 n.d n.d n.d n.d n.d n.d 9.43 ± 2.01 n.d Edible flowers* (µg/100 g of flower) Borage n.d 85.5 ± 22.9 67.8 ± 8.0 29.4 ± 5.4 n.d n.d n.d 183 n.d n.d n.d n.d n.d n.d 3.58 ± 0.60 n.d 3.58 n.d n.d n.d n.d n.d 477 ± 71 84.8 ± 9.9 n.d 24.9 ± 3.8 24.1 ± 4.8 101 ± 10 n.d d ID S/MS S/MS S/MS MS MS S/MS MS S/MS S/MS S/MS S/MS S/MS MS S/MS S/MS MS MS MS MS MS MS S/MS MS MS MS MS S/MS c 67 56 57 57 57 41 91 44 41 77 43 91 41 57 41 57 57 57 57 71 57 57 88 88 55 88 43 QI ( m/z ) b lit 859 870 979 110 801 859 960 804 854 854 858 901 948 997 1033 1066 1068 LRI 1203 1042 1054 1102 1068 1100 1200 1400 1500 1005 a 867 871 980 798 859 958 799 853 853 858 902 942 999 1032 1071 1073 1107 LRI 1204 1041 1058 1102 1073 1103 1201 1401 1499 1007 standard deviation) standard ± rose sugar der, melon der, Sensory description Moldy, earthy Moldy, Fruity, floral, herbal, sweet floral, Fruity, Herbal, spicy carrot Citrus, green, rose Green Floral, herbal, green,Floral, fatty Floral, spicy, honey, lilac, honey, spicy, Floral, Fruity, herbal, grassy Fruity, Green, apple-like Green, Fruity, woody almond, burnt woody Fruity, Floral, green, fatty, lemon green,Floral, fatty, peel orange Floral, hyacinth Floral, Fresh cut grassFresh - laven Citrus, fruity, floral, Fruity, floral, citrus, fatty citrus, floral, Fruity, --- Alkane, fusel Alkane Alkane Green Fruity Fruity Fruity Fruity Acidic Fruity Green Compound ( Z )-3-Hexen-1-ol 1-Hexanol 1-Octen-3-ol 2-Ethyl-1-hexanol ( E )-2-Octen-1-ol 1-Octanol 2-Phenylethanol Σ of alcohols Hexanal ( E )-2-Hexenal Benzaldehyde Decanal Phenylacetaldehyde ( E )-2-Octenal Nonanal Octanal Σ of aldehydes Undecane Dodecane Tetradecane Pentadecane Σ of aliphatic hydrocarbon Butyl 2-methylbutanoate Butyl Ethyl butanoate Methyl 2-methylbutanoate Methyl Ethyl isovalerate Ethyl Ethyl pentanoate Ethyl Ethyl 2-methylbutanoate Ethyl Ethyl hexanoate Ethyl Hexenyl acetate Hexenyl Volatile profile of five edible flowers, expressed in µg/100 g of flower (mean in µg/100 g of expressed flowers, edible of five profile Volatile 1 Table Chemical class Alcohols Aldehydes Aliphatic Hydrocarbons Esters

1 3 European Food Research and Technology Pansy 85.2 ± 47.8 n.d n.d n.d n.d n.d n.d 2.57 ± 1.01 n.d n.d n.d n.d 1.07 ± 0.25 n.d n.d n.d n.d 97.8 n.d 13.5 ± 10.9 7.46 ± 2.77 8.79 ± 4.42 29.8 n.d n.d n.d n.d n.d n.d n.d Johnny Jump up Jump Johnny n.d n.d n.d 0.59 ± 0.13 24.0 ± 6.0 n.d n.d 7.63 ± 3.13 n.d 2.09 ± 0.29 n.d 4.22 ± 0.91 16.9 ± 2.53 n.d n.d n.d n.d 59.5 n.d n.d 0.91 ± 0.31 n.d 0.91 n.d n.d n.d n.d n.d n.d n.d Cosmos 7.91 ± 0.66 n.d n.d n.d n.d n.d n.d n.d n.d n.d 1.66 ± 0.19 0.55 ± 0.09 n.d n.d n.d 1.78 ± 0.61 n.d 13 n.d n.d 4.82 ± 1.49 n.d 4.82 n.d n.d n.d n.d n.d n.d n.d Calendula n.d n.d n.d n.d n.d n.d n.d n.d n.d 4.82 ± 2.45 1.07 ± 0.39 n.d n.d n.d n.d n.d n.d 15.3 n.d n.d n.d n.d n.d 2.36 ± 0.58 6.31 ± 1.61 1.77 ± 1.04 17.7 ± 3.06 1.19 ± 0.38 21.5 ± 6.5 9.73 ± 1.77 Edible flowers* (µg/100 g of flower) Borage n.d 19.3 ± 7.3 13.8 ± 4.3 34.1 ± 6.5 832 ± 132 3.41 ± 0.50 1.59 ± 0.36 n.d 9.37 ± 1.16 40.8 ± 7.3 n.d n.d n.d 49.6 ± 16.0 9.99 ± 2.94 n.d 7.93 ± 2.21 1734 26.9 ± 6.53 n.d n.d n.d 26.9 n.d n.d n.d n.d n.d n.d n.d d ID S/MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS S/MS MS S/MS S/MS MS MS MS MS MS MS MS c 43 88 67 55 43 88 88 88 43 43 57 43 43 81 81 105 115 105 QI ( m/z ) 120 149 103 120 131 121 161 105 161 161 b lit 983 890 986 1009 1090 1090 1093 1168 1186 1186 1191 LRI 1192 1197 1235 1236 1269 1296 1395 1455 1460 1097 1338 1348 1375 1376 1385 1391 a 986 887 988 1016 1091 1091 1098 1168 1187 1189 1189 LRI 1192 1197 1235 1240 1267 1296 1395 1452 1460 1089 1335 1348 1369 1374 1384 1381 1387 spicy lavender, melon lavender, sweet citrus, pepper, woody citrus, pepper, Sensory description Fruity, herbal, citrus, green,Fruity, Wintergreen, almond floral Fruity Fruity Fruity, chamomile, minty, minty, chamomile, Fruity, Green Fruity Minty, sweet Minty, Apple peel Fruity, floral, floral, anise, green, Fruity, Herbal Green Wintergreen, mint Wintergreen, Waxy Fruity, grape, waxy Fruity, Floral Honey, cinnamon Honey, Soap, gasoline Soap Blackcurrant, boiled fruit, Fruity, green. Baked, fatty green. Baked, Fruity, Woody Herbal, wax – Woody, spicy Woody, Herbal Woody pound 1 Compound Hexyl acetate Hexyl Methyl benzoate Methyl Diethyl malonate Diethyl Ethyl heptanoate Ethyl Ethyl benzoate Ethyl Hexenyl butanoate Hexenyl E )-octenoate Ethyl-(4 Methyl salicylate Methyl Hexyl butanoate Hexyl Ethyl octanoate Ethyl Thymol methyl ether methyl Thymol Hexyl 2-methylbutanoate Hexyl Ethyl salicylate Ethyl Ethyl nonanoate Ethyl Ethyl decanoate Ethyl Gerany acetone Gerany )-Ethyl cinnamate ( E )-Ethyl Σ of esters 3-Octanone 3-Heptanone 6-Methyl 5-hepten-2-one 6-Methyl 2-Nonanone Σ of ketones δ-Elemene α-Cubebene α-Ylangene α-Copaene Sesquiterpene-like com - Sesquiterpene-like β-Bourbonene β-Copaene (continued) 1 Table Chemical class Ketones Sesquiterpenes

1 3 European Food Research and Technology Pansy n.d n.d 0.20 ± 0.05 n.d n.d n.d n.d n.d n.d n.d 1.92 ± 1.20 n.d n.d n.d n.d n.d n.d 0.31 ± 0.09 n.d n.d n.d n.d n.d n.d n.d 2.43 n.d n.d Johnny Jump up Jump Johnny n.d n.d n.d n.d 4.23 ± 0.05 n.d n.d n.d n.d n.d 0.72 ± 0.19 n.d n.d n.d n.d n.d n.d 2.36 ± 0.50 n.d n.d 0.08 ± 0.07 n.d n.d 0.17 ± 0.05 n.d 7.56 n.d 0.49 ± 0.15 Cosmos n.d n.d n.d n.d n.d n.d n.d 0.40 ± 0.08 n.d n.d 2.30 ± 0.27 n.d n.d 1.06 ± 0.57 n.d n.d n.d n.d n.d n.d 1.88 ± 1.45 1.44 ± 1.55 n.d n.d n.d 7.08 13.7 ± 2.0 n.d Calendula 3.69 ± 0.92 3.11 ± 1.58 n.d 5.90 ± 3.37 118 ± 26 7.70 ± 2.84 0.66 ± 0.30 n.d 0.37 ± 0.36 3.11 ± 1.86 26.4 ± 13.9 2.13 ± 0.42 9.82 ± 2.03 22.6 ± 8.2 5.86 ± 2.56 2.73 ± 0.80 13.5 ± 1.8 n.d 0.55 ± 0.19 10.8 ± 2.4 31.5 ± 4.4 6.46 ± 1.20 1.15 ± 0.25 n.d 0.70 ± 0.42 338 7.72 ± 2.16 n.d Edible flowers* (µg/100 g of flower) Borage n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d d ID MS MS MS MS MS MS MS MS MS MS S/MS MS MS MS MS MS MS S/MS MS MS MS MS MS MS MS MS MS c 93 93 93 41 41 93 161 105 161 204 120 QI ( m/z ) 105 161 161 161 161 161 105 161 105 161 159 161 105 157 161 104 b lit 889 954 1391 1400 1409 1419 1434 1439 LRI 1441 1454 1479 1483 1485 1500 1505 1518 1523 1538 1542 1583 1640 a 890 949 1387 1392 1398 1405 1415 1425 1434 1440 1441 LRI 1443 1449 1470 1474 1477 1481 1486 1496 1507 1513 1518 1521 1534 1538 1576 1635 Sensory description Citrus, fruity Woody Woody, balsamic Woody, Floral, woody Floral, Woody, warm, tea warm, Woody, Woody, spicy Woody, Woody Woody, spicy, Woody, sweet Citrus, fruit Woody Woody, spicy Woody, Woody Woody, citrus, sweet Woody, Herbal, spicy Thyme, medicine, woody Thyme, Woody Woody Woody Herbal Balsamic, gasoline Camphor pound 2 pound 3 pound 4 pound 5 pound 6 pound 7 Compound β-Cubebene Sesquiterpene-like com - Sesquiterpene-like Longifolene α-Gurjunene α-Caryophyllene Sesquiterpene-like com - Sesquiterpene-like ( E )-α-bergamotene α-Guaiene Aromadendrene Sesquiterpene-like com - Sesquiterpene-like β-Caryophyllene Sesquiterpene-like com - Sesquiterpene-like Sesquiterpene-like com - Sesquiterpene-like γ-Muurolene Alloromadendrene Germacrene D α-Muurolene ( E,E )-α-Farnesene Sesquiterpene-like com - Sesquiterpene-like Calamenene δ-Cadinene α-Cadinene α-Calacorene Caryophyllene oxide Caryophyllene Epi-α-cadinol Σ sesquiterpenes Styrene Camphene (continued) 1 Table Chemical class Terpenes

1 3 European Food Research and Technology Pansy n.d 9.47 ± 4.34 3.69 ± 1.47 n.d 7.32 ± 2.65 n.d n.d 10.4 ± 4.3 188 ± 122 n.d 7.89 ± 4.14 n.d n.d n.d n.d n.d n.d n.d n.d n.d 1.24 ± 0.64 4.48 ± 1.24 1.47 ± 0.77 0.53 ± 0.19 2.44 ± 0.92 n.d 24.6 ± 16.0 Johnny Jump up Jump Johnny 1.92 ± 0.48 19.6 ± 3.9 11.4 ± 2.3 5.17 ± 0.82 106 ± 20 n.d 0.64 ± 0.19 3.86 ± 0.38 56.9 ± 6.2 n.d 10.1 ± 1.8 1.55 ± 0.21 n.d n.d n.d n.d n.d n.d n.d n.d 1.42 ± 0.20 n.d 1.48 ± 0.21 n.d n.d n.d 1.34 ± 0.89 Cosmos n.d n.d n.d 2.91 ± 2.87 3.10 ± 1.24 n.d n.d 36.0 ± 6.6 5.83 ± 0.61 n.d 7.70 ± 6.02 n.d n.d 6.17 ± 1.22 19.4 ± 12.0 0.91 ± 0.15 n.d 6.87 ± 4.20 n.d n.d n.d 1.14 ± 0.50 3.34 ± 2.22 n.d n.d n.d 1.16 ± 0.41 Calendula 788 ± 97 268 ± 33 n.d 98.6 ± 8.0 21.8 ± 8.3 66.6 ± 14.7 11.8 ± 1.2 n.d 40.7 ± 2.5 9.29 ± 2.25 n.d n.d 0.76 ± 0.30 n.d 111 ± 7 0.66 ± 0.19 37.5 ± 1.0 n.d 1.73 ± 1.68 3.65 ± 1.19 1.57 ± 0.29 n.d 1.23 ± 0.22 n.d n.d 4.56 ± 2.29 n.d Edible flowers* (µg/100 g of flower) Borage n.d n.d n.d n.d n.d n.d n.d 42.1 ± 6.6 9.11 ± 1.39 n.d n.d 4.46 ± 0.97 n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d d ID MS S/MS S/MS MS MS MS MS MS S/MS MS MS MS MS MS MS S/MS MS MS MS MS MS S/MS MS MS MS MS S/MS c 93 93 93 93 41 93 68 93 93 93 93 93 71 93 91 41 69 95 71 71 QI ( m/z ) 121 119 117 121 121 112 108 b lit 930 939 979 975 990 1002 1017 1024 LRI 1029 1050 1050 1059 1070 1068 1088 1096 1059 1110 1114 1114 1132 1139 1144 1152 1165 1171 1171 a 929 934 976 975 992 1001 1015 1023 LRI 1028 1051 1052 1059 1067 1060 1088 1098 1106 1109 1112 1117 1130 1141 1143 1151 1163 1170 1171 phor, citrus, pine phor, spicy, sweet spicy, oily, peach oily, herbaceous peely spicy Sensory description Woody, green, herbal Woody, Fruity, green, woody, cam - green, woody, Fruity, Woody Pepper, turpentine, woody Pepper, Balsamic, fruity, lemon, Balsamic, fruity, Flowery, citrus, sweet Flowery, Anise, floral, fruity, minty, minty, fruity, Anise, floral, Balsamic, citrus, fruity, Balsamic, citrus, fruity, lemon, spicy Citrus, fruity, minty, orange, orange, minty, Citrus, fruity, Sweet, tropical Sweet, fruits Flowery, sweet Flowery, Fruity, lime Fruity, Balsamic Woody, balsamic Woody, Citrus, terpenic, woody, Citrus, terpenic, woody, Floral, freesia Floral, Gasoline, turpentine Turpentine Thujonic Woody Herbal Camphor, medicine Camphor, Sweet, herbal Sweet, Minty Mentholic, minty sweet ---- Peppermint, mentholic Compound α-Thujene α-Pinene β-Pinene Sabinene β-Myrecene α-Phellandrene α-Terpinene ρ-Cymene Limonene ( E )-β-Ocimene β-Ocimene γ-Terpinene ( Z) -Sabinene hydrate (E) -Sabinene hydrate ρ-Cymenene Linalool δ-Terpinene 1,3,8- p -Menthatriene β-Thujone Perillene Alloocimene (-)-Champor Neo-Allo-ocimene L-Menthone Neomenthol 3-Thujen-2-one Menthol (continued) 1 Table Chemical class

1 3 European Food Research and Technology Pansy n.d 0.75 ± 0.25 n.d 0.77 ± 0.23 n.d n.d n.d 263 18.4 ± 12.1 n.d 18.4 Johnny Jump up Jump Johnny n.d n.d 0.78 ± 0.11 n.d n.d n.d n.d 223 n.d 0.29 ± 0.11 0.29 Cosmos n.d n.d n.d n.d n.d 0.59 ± 0.43 n.d 109 17.8 ± 2.7 n.d 17.8 Calendula 1.08 ± 0.24 n.d 2.85 ± 0.10 n.d 0.54 ± 0.07 12.0 ± 3.7 1.88 ± 0.56 1494 n.d n.d n.d Edible flowers* (µg/100 g of flower) Borage n.d n.d n.d n.d n.d n.d n.d 55.7 n.d n.d n.d d ID MS MS MS MS MS MS MS c 71 93 71 91 148 148 161 QI ( m/z ) 119 138 b lit 872 1177 1182 1088 1186 1195 1463 1534 LRI 1148 a 872 1174 1180 1083 1186 1194 1460 1528 LRI 1146 - musty, spicy, sweet, ter sweet, spicy, musty, penic, woody Sensory description Fruity, herbaceous, licorice, Fruity, Mentholic Fruity, herbal, pine Fruity, woody sweet, Menthol Licorice, anise ------Geranium ---- Compound Terpin-4-ol Isomenthol Terpinolene Neoisomenthol Estragole ( Z )-Cadina-1(6),4-diene ( E ) - Cadina-1,4-diene Σ of terpenes ρ-Xylene Veratrole Σ of others compounds (continued) Identification method (S—identified with standard; MS—identified by comparingby with (S—identified spectrum mass method Identification MS—identified standard; 11) NIST with database LRI Lit—linear retention index reported in the index [ 37 ] literature LRI Lit—linear retention

LRI—linear retention index obtained index LRI—linear retention

Quantification ions 1 Table Chemical class a b c d Other compounds . not detected not n.d . as internalstandard using 4-methyl-2-pentanol semi-quantification from are *Values

1 3 European Food Research and Technology especially for the evaluation of olive oils, cheeses, and table of three Echinacea species. Flamini et al. [12] also detected olives. To evaluate the sensory profile of the five flowers monoterpenes in high proportion, namely, 90.5 and 93.3% under study in the present work, the flowers were examined in whole flowers and petals of Citrus deliciosa, respectively. without any condiments, bread, crackers, etc. After a careful Within the 36 terpenes identified, limonene was detected in evaluation of the perceived flavors (minimum three flowers all studied flowers. However, the most abundant identified of each species/panelist), the tasters were asked to fill out a terpene was different for each edible flower. In more detail, questionnaire aimed at determining the performances of the for borage and cosmos, the main terpene was ρ-cymene; in edible flowers. Different organoleptic characteristics (color, calendula, it was α-thujene followed by α-pinene; in Johnny spiciness, sweetness, astringency, bitterness, taste, and odor) Jump up, it was β-myrecene followed by limonene; and in were included in the evaluation scheme and were expressed pansy, it was limonene. in a scale of 1–10. Esters were the second chemical class in terms of diversity in borage (16 compounds) and Johnny Jump up (nine Statistical analysis compounds) flowers. Between the esters identified, ethyl benzoate was the most abundant in both flowers (832 and The SPSS Statistic software, version 18.0 (SPSS Inc., Chi- 24.0 µg/100 g flower, respectively). It is reported that ethyl cago, USA), was used for the statistical treatment of the data. benzoate has a pleasant odor that could be described similar The normality and variance homogeneity were checked by to wintergreen or mint, and it is frequently used in pharmacy, Shapiro–Wilk and Levene tests, respectively. As the data fol- cosmetic, and food industry [13]. lowed a normal distribution, analyses of variance (ANOVA) Sesquiterpenes were the second chemical class most or ANOVA Welch were carried out to evaluate if there were abundant in calendula concerning the number of the com- significant differences (p < 0.05) between samples. ANOVA pounds quantified, being 28 compounds detected, but seven was applied when homogeneity of variances was observed, of those were just tentatively identified as sesquiterpene-like while ANOVA Welch was applied for the other cases. In compounds. α-Caryophyllene (118 µg/100 g flower) was the addition, significant post hoc analyses were performed major sesquiterpene, followed by δ-cadinene (31.5 µg/100 g (Tukey HSD test if variances in the different groups were flower), β-caryophyllene (26.4 µg/100 g flower), and identical or Games-Howell test if they were not). All analy- γ-muurolene (22.6 µg/100 g flower). In the other flowers, ses were performed in triplicate. β-caryophyllene was always detected, except in borage; and it was the most abundant in cosmos and pansy. On contrary, α-caryophyllene was the most abundant sesquiterpene Results and discussion in Johnny Jump up flowers. This particular compound is one of the 12 most common volatile compounds detected Volatile compounds in floral scents. Furthermore, some sesquiterpenes were only detected in one flowers’ species, such as: caryophyl- The volatile composition of the five edible flowers is lene oxide in Johnny Jump up, longifolene in pansies, and described in Table 1. In total, 117 volatile compounds were α-guaiene in cosmos. In calendula, several sesquiterpenes identified, belonging to different chemical classes. Calen- were detected solely in this flower, such as β-bourbonene dula flowers presented the highest number of identified and α-muurolene. compounds (62), followed by Johnny Jump up (42), pansy Some alcohols were also identified, such as 1-hexanol (34), and cosmos (29). Borage showed the lowest diver- and (Z)-3-hexen-1-ol, the most abundant. (Z)-3-Hexen-1-ol sity of compounds identified (24). The volatile compounds is produced in small amounts by the plants and it acts as an were distributed by seven chemical classes, namely, alcohols attractant to many predatory insects [14]. (7); aldehydes (8); aliphatic hydrocarbons (4); esters (25); Aldehydes were present in flowers in small amounts. ketones (4); sesquiterpenes (32); and terpenes (36). Nonanal was the most abundant in cosmos and borage, phe- In general terms, the five edible flowers analyzed showed nylacetaldehyde in Johnny Jump ups and (E)-2-octenal in differences in the volatile profiles considering qualitative and pansies. Nonanal is an attractant for some insects and repel- quantitative results. Terpenes were the major chemical class lent to others depending on its concentration [15–17]. in almost all edible flowers studied (values between 55.7 and Ketones and aliphatic hydrocarbons were the compounds 1494 µg/100 g flower). This was expected, because these with lower representativeness (number of compounds identi- compounds have important roles in plants, such as pollinator fied) in the edible flowers studied, although Johnny Jump up attraction, direct and indirect defense against insects, bacte- and pansies were the ones that presented the major number ria, fungi, and in intra/inter-plant signaling [11]. Mazza and of aliphatic hydrocarbons and ketones, respectively. Among Cottrell [6] also observed that terpenes were the most iden- ketones, 6-methyl-5-hepten-2-one was the only compound tified volatile compounds (82–91%) for flowers and stems identified in three flowers (cosmos, Johnny Jump ups, and

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Table 2 Total reducing Flowers Parameters capacity, monomeric anthocyanins, flavonoids, and TRC Hydrolysable tannins Flavonoids Total mono- hydrolysable tannins of five (mg GAE/g DW) (mg TAE/g DW) (mg QE/g DW) meric anthocya- edible flowers expressed in dry nins weight (mg Cy 3-glu/g DW)

Borage 5.6 ± 0.6b 74.7 ± 10.4b 40.4 ± 5.5b 0.15 ± 0.05a Calendula 1.1 ± 0.2a 3.7 ± 1.8a 0.35 ± 0.10a 0.07 ± 0.01a Cosmos 12.4 ± 1.1c 82.9 ± 7.4b 44.5 ± 3.3b 4.18 ± 0.09d Johnny Jump up 15.8 ± 0.8d 79.2 ± 14.8b 68.9 ± 8.5c 0.64 ± 0.17b Pansy 18.0 ± 1.0e 78.4 ± 6.7b 98.8 ± 3.5d 1.47 ± 0.06c

Values are expressed as: mean ± standard deviation Values with the same letter in the same column are not statistically different (p > 0.05)

pansies) but at low levels. This compound is formed from Sensory analysis the degradation of carotenoids and reported to contribute to off-flavors18 [ ]. Dodecane was the aliphatic hydrocarbon Figure 2 and Table 3 show the sensory profiles of the five present in all studied flowers, except in borage. studied edible flowers. The radar plots, for odors and tastes Regarding other compounds, veratrole was detected in detected by the panelists, are shown in Fig. 2. The odors Johnny Jump up and ρ-xylene in cosmos and pansies. detected in the flowers were divided into six classes, namely, floral (e.g., carnation, lilies, marigold, orange blossom, orchid, pollen, rose, and violet), fruity (e.g., banana skin, Total reducing capacity, hydrolysable tannins, total fig, mandarin, peach, and plum), herbal (e.g., green grass/ flavonoids, and monomeric anthocyanins leaves), marine (sea air), spicy (vanilla), and wood (cedar, acacia). For the taste, five classes were used, namely, flo- Table 2 shows the total reducing capacity (TRC), hydrolys- ral (lavanda, lilies, petals rose, and pollen), fruity (apple, able tannins, total flavonoids, and monomeric anthocyanins cherries, chestnut, grape seed, banana skin, walnut, and contents determined in the five studied edible flowers. Sig- cabbage), herbal (green grass/leaves and mint, parsley), nificant differences among them (p < 0.05) were observed. sweet (honey), and wood (camphor, cedar). It was observed Pansies showed the highest values of TRC (18.0 mg GAE/g that each flower had its individual sensory characteristics DW) and flavonoids (98.8 mg QE/g DW), while cosmos pre- (Fig. 1). Regarding odor, the panelists detected more floral sented the highest contents of hydrolysable tannins (82.9 mg fragrances in Johnny Jump up (fragrances of carnation, lil- TAE/g DW) and monomeric anthocyanins (4.18 mg Cy ies, pollen, rose, and violet), cosmos (fragrances of acacia, 3-glu/g DW). Nevertheless, Johnny Jump ups, pansies, and carnation, pollen, and violet), and pansies (fragrances of borage also presented high values of hydrolysable tannins, lilies, orchid, rose, and violet), while for calendula, a wood not being significantly different from cosmos. On contrary, odor was detected (fragrance of cedar). In borage, several calendula always presented the lowest values of all stud- fragrances were felt such as rose and violet (floral), green ied bioactive compounds. Intermediary values of TRC and leaves (herbal), and sea air (marine). Concerning taste, pan- monomeric anthocyanins were detected in Johnny Jump ups sies, and Johnny Jump up flowers showed a fruity flavor. In and of hydrolysable tannins and flavonoids in borage. When more detail, pansies tasted more like chestnut and walnut, expressing our results in fresh weight, cosmos, Johnny Jump while Johnny Jump up like cherries and walnut. Cosmos ups, and pansies presented the highest TRC. Pansies also presented a high lavanda taste, followed by parsley, camphor had the highest content of flavonoids, while cosmos showed green grass/leaves and mint tastes, a complex mixture of the highest contents of hydrolysable tannins and monomeric floral, herbal, and wood flavors. Regarding calendula, a high anthocyanins, followed by pansies. In our work, the values mixture of flavors was also detected by the panel, such as were equal to 11.6, 1.39, and 0.91 mg Cy 3-glu/100 g FW parsley, cabbage, grape seed, rose petals, cedar, cherries, and for pansies, borage, and calendula, respectively. Similar banana skin. On the other hand, borage showed the smallest results were reported by Benvenuti et al. [19]. These authors range of flavors, being the cabbage, and chestnut flavors the detected the following values for red pansies (12.4 mg Cy most intense. 3-glu/100 g FW), borage (1.43 mg Cy 3-glu/100 g FW), and Furthermore, each flower was classified according to calendula (0.47 mg Cy 3-glu/100 g FW). three sensory attributes including visual appearance, odor,

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Fig. 2 Odors and tastes detected by the panel of tasters for each edible flower species

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Table 3 Sensory evaluation of Sensory attributes Edible flowers the five edible flowers Borage Calendula Cosmos Johnny Jump up Pansies

Visual appearance Colors intensity 6.4 ± 1.1a 6.8 ± 0.5a 8.3 ± 0.7b 7.6 ± 1.1a,b 8.5 ± 0.8b (5.1–7.8) (6.3–7.3) (7.2–9.4) (5.0-8.3) (7.4–9.7) Physical integrity of 9.4 ± 0.6a 9.6 ± 0.4a 9.3 ± 0.3a 9.5 ± 0.8a 9.5 ± 0.6a the plant (8.5–10) (8.9–10.0) (9.1–10) (7.7–10) (8.3–10) Odor Odor sensation 8.0 ± 0.9a 7.8 ± 0.5a 8.4 ± 0.6a 8.6 ± 0.8a 7.6 ± 2.8a (6.5–9.2) (6.6–8.5) (7.5–9.2) (7.2–9.9) (1.5–9.7) Odor intensity 6.1 ± 0.9a 8.4 ± 0.7b 7.2 ± 1.4a,b 9.0 ± 0.6b 5.3 ± 2.7a (4.9–7.8) (7.7–9.8) (4.7-9.0) (8.1–9.9) (2.2–8.6) Taste Mouthfeel 5.8 ± 1.1a 6.2 ± 0.7a,b 7.5 ± 1.0b.c 6.8 ± 1.6a,b.c 8.4 ± 0.6c (4.0-7.2) (5.4-7.0) (6.3–9.2) (4.9–9.9) (7.4–9.3) Persistence 4.2 ± 1.1a 6.6 ± 0.8b,c 7.8 ± 1.0c 5.6 ± 2.0a,b 7.5 ± 1.6b,c (2.9–6.4) (5.3–7.6) (6.1–9.2) (1.5–7.6) (4.6–9.1) Bitterness 1.6 ± 0.4b 6.4 ± 1.1c 6.3 ± 1.0c 1.5 ± 1.2a,b 0.3 ± 0.4a (0.9–2.1) (4.4–7.8) (4.6–7.6) (0.1–3.7) (0.0–1.0) Astringency 1.4 ± 0.7a 4.4 ± 1.8b 4.2 ± 1.4b 1.6 ± 1.3a 0.3 ± 0.4a (0.7–2.4) (2.0-7.3) (1.8–5.9) (0.1–4.1) (0.0–1.0) Spiciness 0.3 ± 0.5a 2.6 ± 0.9b,c 3.6 ± 0.9c 0.3 ± 0.5a 1.7 ± 0.7b (0.0-1.5) (1.2–4.2) (2.7–5.3) (0.0-1.2) (1.0-2.3) Sweetness 3.5 ± 1.0b 0.5 ± 0.4a 0.7 ± 0.8a 2.6 ± 1.1b 6.4 ± 1.0c (2.1–5.1) (0.0-0.9) (0.0-2.6) (0.4–4.5) (5.2–7.8)

Mean ± standard error (minimum–maximum) Values with the same letter in the same line are not statistically different (p > 0.05) and taste (Table 3). Regarding visual appearance, two Association between sensory attributes descriptors were evaluated, color intensity, and physical and bioactive compounds integrity of the plant. Cosmos and pansies were the flowers with the highest scores of colors intensity, while all Sensory attributes of flowers are dependent on the content flowers showed good physical integrity (> 9). According of minor components like phenolic and volatile compounds. to the panel, all flowers revealed a pleasant odor sensa- Furthermore, each single component can contribute to dif- tion (> 7.5), with no significant differences between them. ferent sensory perceptions. Bioactive compounds, such as On contrary, concerning odor intensity, Johnny Jump up, phenolics, are plant metabolites and contribute to important calendula, and cosmos had the highest scores, while bor- organoleptic properties (color, bitterness, and astringency) age and pansies had the lowest. Concerning taste, all flow- [20]. Concerning flowers’ color, anthocyanins play an ers had a delightful mouthfeel (> 5), although pansies were important role. By observing Tables 2 and 3, the flowers that distinguished from the others with the highest value (8.4). presented the highest contents of total monomeric antho- Cosmos, pansies, and calendula had the most persistent cyanins, namely, cosmos and pansies (4.18 and 1.47 mg Cy flavor. In more detail, calendula and cosmos originated a 3-glu/g DW, respectively), were those that had the highest more bitter, astringent and spicy taste than borage, Johnny scores of colors intensity (8.3 and 8.5, respectively). Jump up, and pansies. In contrast, pansies had the sweet- Phenolic compounds are responsible for the bitterness est taste, followed by borage and Johnny Jump up. These and astringency of plants, being tannins more likely to be results were like those referred by Benvenuti et al. [19], astringent and flavonoids more bitter [21]. In this order, it who reported that calendula showed higher values of spic- was expected that pansies were the most bitter flowers fol- iness and bitterness than borage and pansy, as well as, lowed by Johnny Jump ups, because they had the highest borage and pansy were sweeter than calendula. Thus, our values of TRC and flavonoids. However, the panel reported results showed that the five studied flowers have a high low scores of bitterness for both flowers. These results can sensory biodiversity. This will allow their valorization, be due to the sweetness caused by the sugars present in the because these flowers can make the dishes more attractive nectar of the flowers (not analyzed), which may be an effi- and confer a peculiar taste and odor. cient masking agent of astringency and bitterness caused

1 3 European Food Research and Technology by the phenolic compounds [22]. In fact, both flowers were of the principal component of fragrant rose flowers [35, 36]; described by the panelists to have a sweet taste (6.4 for pan- however, this compound was only detected in Johnny Jump sies and 2.6 for Johnny Jump ups). up. The panel also mentioned some mint flavors when they On contrary, for cosmos, the panelists did not detect tasted both flowers, probably due to presence of menthol, large sweet notes (0.7), being bitterness (6.3) and astrin- isomenthol, neomenthol, neoisomenthol, and L-menthone gency (4.2) the most detected flavors. These results are in compounds. accordance with those obtained for some analyzed bioactive Therefore, the volatiles produced from flowers vary sig- compounds. In fact, cosmos presented the highest values of nificantly among species, contributing to the diverse range hydrolysable tannins (82.95 mg TAE/g DW) and intermedi- in fragrances and aromas found in the plant kingdom. ary values of flavonoids (44.55 mg QE/g DW). Calendula showed the lowest value of TRC (Table 2), but the panelists detected in this flower a great range of tastes and odors, Conclusions probably because the intensities of fruity and floral aromas/flavors seem to increase when the level of polyphenols The flowers analyzed showed statistical differences in their decrease [23]. sensory attributes (colors intensity, odor intensity, mouthfeel, persistence, bitterness, astringency, and sweetness) and Association between sensory attributes and volatile in the variety of volatiles detected, presenting calendula the compounds highest number of identified compounds (62), followed by Johnny Jump up (42), pansy (34), cosmos (29), and borage The description of the odor of each isolated volatile com- (24). Terpenes were the major chemical class in terms of pound was obtained from the literature [24–29] and is pre- diversity in almost all edible flowers studied. Regarding bio- sented in Table 1. In this section, it was analyzed the possible active compounds, the highest values of TRC and flavonoids relationship between volatile compounds obtained by GC–MS were determined in pansies, and hydrolysable tannins and and the sensory attributes, assessed by the panelists. First, it is monomeric anthocyanins in cosmos, having calendula the known that the volatile compounds present at higher concen- lowest. Some relationships were found between color, bit- tration are not necessarily the major contributors of odor [28]. terness and astringency with the presence of some bioactive In borage flowers, a high number of esters associated to fruity compounds. For example, more anthocyanins gave higher fragrances were detected [13, 26, 30]. In fact, the panelists intensities of color (pansies and cosmos), more bitterness detected some green and floral notes, and a fruity taste (chest- to a higher content of flavonoids (cosmos), and more astrin- nut, cabbage, and cherries). Those floral and green fragrances gency to higher levels of tannins (cosmos); however, regard- may be due to the presence of some volatile compounds, as ing taste, it was more difficult to take precise conclusions, ethyl octanoate and 1-hexanol as reported by Śliwińska et al. because some compounds can mask the presence of others. [30]. Calendula showed woody (cedrus), floral (marigold and Additional relationships were found between the presence of orange blossom), and fruity (banana, mandarin and peach) fra- some volatile compounds and the sensory perception, such grances that were reported by panel (Fig. 2). The woody odor as the high number of sesquiterpenes detected in calendula of calendula was probably due to the high levels of sesquiter- can be associated with the woody notes detected by the panel. penes detected, most of them described as contributing to a wood odor (Table 1). Furthermore, some sesquiterpenes (e.g., Acknowledgements The authors acknowledge the Portuguese Foun- calamenene, β-caryophyllene, α-copaene) give the sensory dation for Science and Technology (FCT, Portugal) for the financial perception of spicy, so probably, it was because of this that support provided by the research grant [SFRH/BD/95853/2013] and FCT/MEC for the financial support to QOPNA research Unit the panel detected a spicy taste for this flower (2.6, the second [FCT UID/QUI/00062/2013], through national funds and when highest). Mandarin and orange blossoms fragrances may be applicable co-financed by the FEDER, within the PT2020 Partner- due to the presence of α-pinene. According to the panelist ship Agreement, and to REQUIMTE through the Project [PEst/UID/ group, cosmos showed floral and fruity odors (Fig. 2). The QUI/50006/2013]. The authors are also grateful to FCT (Portugal) and FEDER under Programme PT2020 for financial support to CIMO fruity odor was probably due to the high levels of ρ-cymene (UID/AGR/00690/2013). (41.7 µg/100 g). Regarding Johnny Jump up and pansies, the panel detected floral fragrances for both flowers. β-Ocimene Compliance with ethical standards and 1-hexanol are important floral scents in different flowers [31–34] and their odor was described as floral [30], while Conflict of interest The authors declare that they have no conflict of β-myrecene is described as sweet and fruity. Therefore, these interest. components might play an important role in the sweet, floral Compliance with ethics requirements This article does not contain aroma of these two samples. Furthermore, the panel detected any studies with human or animal subjects. in both flowers notes of rose, wherein 2-phenylethanol is one

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