Phytochemicals screening, antioxidant capacity and chemometric characterization of four edible flowers from Brazil

Journal Pre-proofs Phytochemicals screening, antioxidant capacity and chemometric characterization of four edible flowers from Brazil Romy Gleyse Chagas Barros, Julianna Karla Santana Andrade, Ubatã Corrêa Pereira, Christean Santos de Oliveira, Yara Rafaella Ribeiro Santos Rezende, Tais Oliveira Matos Silva, Juliete Pedreira Nogueira, Nayjara Carvalho Gualberto, Hannah Caroline Santos Araujo, Narendra Narain PII: DOI: Reference:

S0963-9969(19)30785-9 https://doi.org/10.1016/j.foodres.2019.108899 FRIN 108899

To appear in:

Food Research International

Received Date: Revised Date: Accepted Date:

18 June 2019 24 November 2019 15 December 2019

Please cite this article as: Gleyse Chagas Barros, R., Karla Santana Andrade, J., Corrêa Pereira, U., Santos de Oliveira, C., Rafaella Ribeiro Santos Rezende, Y., Oliveira Matos Silva, T., Pedreira Nogueira, J., Carvalho Gualberto, N., Caroline Santos Araujo, H., Narain, N., Phytochemicals screening, antioxidant capacity and chemometric characterization of four edible flowers from Brazil, Food Research International (2019), doi: https://doi.org/10.1016/j.foodres.2019.108899

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ϭ

Phytochemicals

screening,

antioxidant

capacity

and

chemometric

Ϯ

characterization of four edible flowers from Brazil.

ϯ

Romy Gleyse Chagas Barros, Julianna Karla Santana Andrade, Ubatã Corrêa Pereira,

ϰ

Christean Santos de Oliveira, Yara Rafaella Ribeiro Santos Rezende, Tais Oliveira Matos

ϱ

Silva, Juliete Pedreira Nogueira, Nayjara Carvalho Gualberto, Hannah Caroline Santos

ϲ

Araujo, Narendra Narain*.

ϳ ϴ

Laboratory of Flavor & Chromatographic Analysis, PROCTA, Federal University of Sergipe, 49100-000 – São Cristóvão – SE, Brazil.

ϵ ϭϬ ϭϭ

* Corresponding author: Narendra Narain

ϭϮ

E-mail: [email protected]

ϭϯ ϭϰ

Phone: 55-79-3194 6514

ϭϱ ϭϲ ϭϳ ϭϴ ϭϵ ϮϬ Ϯϭ ϮϮ Ϯϯ Ϯϰ Ϯϱ



Ϯϲ

 1 

Ϯϳ

Abstract

Ϯϴ

Edible flowers are receiving renewed interest as potential sources of bioactive

Ϯϵ

compounds. The present study aimed to investigate the presence of bioactive compounds and

ϯϬ

antioxidant activity of some exotic flowers present in Brazil such as Amaranthus

ϯϭ

hypochondriacus, Tropaeolum majus (red), Tropaeolum majus (orange) and Spilanthes

ϯϮ

oleracea L. The content of total phenolic compounds, flavonoids, condensed, hydrolysable

ϯϯ

tannins and antioxidante capacity were determined. The identification and quantification of

ϯϰ

the phenolic compounds was performed through the UHPLC-QDa-MS system. The

ϯϱ

compounds p-coumaric acid and ferulic acid were identified and quantified for the first time

ϯϲ

in all flowers. Tropaeolum majus (red) presented the hightest amounts of total phenolic

ϯϳ

compounds and hydrolysable tannins. Also, it presented the highest antioxidant capacity for

ϯϴ

ORAC and FRAP assays. Thus, this study showed the diversity and abudance of natural

ϯϵ

antioxidants present in edible flowers, which could be explored for application in functional

ϰϬ

foods and pharmaceuticals.

ϰϭ

Keywords: Edible flowers; phenolic content; flavonoid content; antioxidant capacity; mass

ϰϮ

spectometry.

ϰϯ

ϰϰ

ϰϱ

ϰϲ

ϰϳ

ϰϴ 2 

1. Introduction

ϰϵ

ϱϬ

The horticulture plants are very diverse group and include numerous cultivars,

ϱϭ

genotypes and accessions. They have been an indispensable part of human life for ages, since

ϱϮ

their fruits, seeds, leaves, flowers and even roots and branches have been used to meet

ϱϯ

personal and social needs such as food, medicine, and decoration (Alibabic et al., 2018;

ϱϰ

Gunduz and Ozbay, 2018; Shah et al., 2018). Several horticulture plants have been studied in

ϱϱ

vivo and in vitro models as source of bioactive compounds and are associated with lower

ϱϲ

incidence of chronic diseases such as cardiovascular diseases (Mendonça et al., 2019; Kumar

ϱϳ

& Goel, 2019) and cancer (Anantharaju et al., 2016).

ϱϴ

Of late, the interest in horticulture plants such as edible flowers has grown because of

ϱϵ

their wide use in cooking preparations to enhance the sensory and nutritional qualities of food

ϲϬ

by adding color, flavor and visual appeal. They are used in sauces, jellies, syrups, liqueurs,

ϲϭ

vinegars, honey, oils, crystallized flowers, ice cubes, salads, teas, other beverages and desserts

ϲϮ

(Koike et al., 2015). Several studies related to edible flowers revealed their in vitro and in vivo

ϲϯ

potential health effects due to the presence of natural antioxidants such as phenolic acids and

ϲϰ

flavonoids with pharmaceutical effects including anti-inflammatory properties (Sergent et al.,

ϲϱ

2010; Chen et al., 2015; Ambriz-Pérez et al., 2016), prevention of degenerative diseases and

ϲϲ

lower risk of developing different types of cancer by inhibiting its initiation and progression

ϲϳ

by modulating genes involved in key regulatory processes (Ukiya et al., 2015; Lin et al.,

ϲϴ

2015).

ϲϵ

Consolidated in the international market, the edible flower market has grown in Brazil.

ϳϬ

The main producing centers are the city of Sao Paulo followed by Minas Gerais, which

ϳϭ

together amount to at least about 27 thousand sauces produced per year. The country moves

ϳϮ

about $ 8.2 million in flower sales in the national and international market. The main 3 

ϳϯ

cultivated species are Tropaeolum majus and Viola tricolor, that are intended mainly for the

ϳϰ

gastronomic market (Soares et al., 2015).

ϳϱ

The characterization of the phenolic and antioxidant profiles of some edible flowers

ϳϲ

has been reported earlier in the literature for marigold (Miguel et al., 2016), rose (Barros et

ϳϳ

al., 2012), dahlia, French rose, alexandria rose, centurea (Benvenuti et al., 2015),chamomile,

ϳϴ

hibiscus (Chen et al., 2015), viola tricolor (Koike et al., 2015), begonia, petunia (Benvenuti et

ϳϵ

al., 2015), pansy and dandelion (González-Barrio et al., 2018). However, there are only a few

ϴϬ

publications concerning the functional properties of other edible flowers consumed in Brazil

ϴϭ

such as purple amaranth (Amaranthus hypochondriacus), red capuzin (Tropaeolum majus),

ϴϮ

orange capuzin (Tropaeolum majus) and jambu (Spilanthes oleracea L.).

ϴϯ

Tropaeolum majus is widely cultivated as an ornamental and medicinal plant. This

ϴϰ

species belongs to the Tropaeolaceae family and presents edible parts like leaves, flowers,

ϴϱ

and unripe green seeds. Undeveloped flower buds and green seeds are used as a substitute for

ϴϲ

capers, and chopped unripe fruits are added to tartar sauces instead of horseradish. The

ϴϳ

capuzin garden represents one of the most popular sources of edible flowers and it is

ϴϴ

consumed in various countries such as Brazil, Canada, India and France (Panizza, 1997).

ϴϵ

Tropaeolum genus are used in traditional medicine to treat diseases including cardiovascular

ϵϬ

disorders, urinary tract infections, asthma, and constipation. (Lorenzi & Matos, 2002).

ϵϭ

Belonging to the Asteraceae family, Spilanthes oleracea L. is a native plant of the

ϵϮ

Amazon region. Its flowers are very peculiar and have large use in local cuisine, especially

ϵϯ

due to the intoxicating damping of the mucous membranes. In popular medicine, jambu is

ϵϰ

used for anesthetic purposes, especially to treat toothache, as it contains the presence of

ϵϱ

spilanthol (Shanley, 2005). Amaranthus hypochondriacus is an ancient plant native to South

ϵϲ

and Central America and belonging to the family Amaranthaceae. Extensively cultivated 4 

ϵϳ

during the five centuries of the Aztec civilization in Mexico is made up of small vegetables

ϵϴ

and broad leaves with an inflorescence. Traditionally, flowers are used to treat toothache and

ϵϵ

fever, and are also used to color some foods like corn and Chicha (Costa & Borges, 2005).

ϭϬϬ

Regarding previous data on phenolic compounds in these species, Bazylco et al.

ϭϬϭ

(2013) conducted a study with hydroethanolic extracts of Tropaeolum majus L. and identified

ϭϬϮ

that the co-dependent relation of the antioxidant and anti-inflammatory effects are based on

ϭϬϯ

the esters group compounds such as quinic and cinnamic acids. Garzón et al. (2009) reported

ϭϬϰ

the main anthocyanins and antioxidant activity of Tropaeolum majus L. flowers, and showed

ϭϬϱ

an excellent ability to eliminate free radicals due to the presence of phenolic compounds and

ϭϬϲ

ascorbic acid. Noori et al. (2015) determined the flavonoid content of Amaranthus genus,

ϭϬϳ

where they evaluated the possible effect of compounds such as isorhamnetin, kaempferol,

ϭϬϴ

quercetin and rutin. Navarro-González et al. (2014) made an attempt to identify the phenolic

ϭϬϵ

compounds of Spilanthes oleracea L. and detected some quercetin and cynarine derivatives.

ϭϭϬ

Taking into consideration these aspects of research and lacking data on these flowers,

ϭϭϭ

the present study was aimed to determine the presence of bioactive compounds and to identify

ϭϭϮ

and quantify the phenolic compounds, carotenoids and organic acids present in edible flowers

ϭϭϯ

from Brazil such as purple amaranth, red capuzin, orange capuzin and jambu through the

ϭϭϰ

UHPLC-QDa-MS system, as well as to evaluate their antioxidant capacity.

ϭϭϱ

2. Materials and Methods

ϭϭϲ

2.1.Chemicals

ϭϭϳ

Sodium carbonate, sodium hydroxide, acetone, hydrochloric acid, phosphoric acid,

ϭϭϴ

diethyl ether were obtained from Synth (São Paulo, Brazil) and sodium nitrite, monobasic

ϭϭϵ

sodium phosphate, aluminum chloride, tannic acid from Dinamica (São Paulo, Brazil).

ϭϮϬ

Ethanol, methanol, potassium iodate and ferric chloride were purchased from Neon (São 5 

ϭϮϭ

Paulo, Brazil). Folin-Ciocalteu phenol reagent, 2,2-difenil-1-picrilhidrazilo (DPPH), 2,4,6-

ϭϮϮ

tripyridyl-s-triazine

ϭϮϯ

(Trolox), 2,2ƍ-azobis(2-methylpropionamidine) dihydrochloride (AAPH), fluorescein, formic

ϭϮϰ

acid (HPLC grade), acetonitrile (HPLC grade), ethyl acetate (HPLC grade), methanol (HPLC

ϭϮϱ

grade) and the phenolic standards such as acacetin (C16H12O5), apigenin (C15H10O5),

ϭϮϲ

biochanin A (C16H12O5), caffeic acid (C9H8O4), chlorogenic acid (C16H18O9), chrysin

ϭϮϳ

(C15H10O4), daidzein (C15H10O4), ferulic acid (C10H10O4), gallic acid (C7H6O5), p-coumaric

ϭϮϴ

acid (C9H8O3), vanillic acid (C8H8O4), (+)catechin (C15H14O6), (−)epicatechin (C15H14O6),

ϭϮϵ

ethyl gallate (C9H10O5), kaempferol (C15H10O6), luteolin (C15H10O6), naringenin (C15H12O5),

ϭϯϬ

protocatechinic acid (C7H6O4), quercetin-3-glucoside (C21H20O12), rutin (C27H30O16), vanillin

ϭϯϭ

(C8H8O3), canthaxanthin (C40H52O2), ȕ-carotene (C40H56) and lycopene (C40H56) were

ϭϯϮ

supplied from Sigma Aldrich (St. Louis, MO, USA).

(TPTZ),

ϭϯϯ

2.2.Bioactive compounds

ϭϯϰ

2.2.1. Samples

6-hidroxy-2,5,7,8-tetramethylchroman-2-carboxylic

acid

ϭϯϱ

The samples of edible flowers were obtained from Ervas Finas Horticulture, São

ϭϯϲ

Paulo, Brazil. The flowers purple amaranth (Amaranthus hypochondriacus), red capuzin

ϭϯϳ

(Tropaeolum majus), orange capuzin (Tropaeolum majus) and jambu (Spilanthes oleracea L.)

ϭϯϴ

were cultivated in a greenhouse where these were fortified with natural fertilizers, non-toxic

ϭϯϵ

antifungal agents and natural enemies (Trichogramma) to fight purges and insects. The

ϭϰϬ

flowers were transported in plastic containers maintained at 5 °C to the Laboratory of Flavor

ϭϰϭ

and Chromatographic Analysis, Federal University of Sergipe, São Cristóvão, Brazil.

ϭϰϮ

Subsequently, the samples were freeze-dried under the following conditions: first, the flowers

ϭϰϯ

were frozen in freezer at íௗƒ& IRU ௗK WKHQ the samples were placed in a freeze dryer

ϭϰϰ

(Christ Alpha 1–2 LD Plus) and dried at íௗƒ& SUHVVXUH RI ௗPEDU YDFXXP RI 6 

ϭϰϱ

0.42ௗmbar for 48ௗh. After freeze-drying, the samples were triturated using a mortar and pestle

ϭϰϲ

and stored at 4 °C until analysis. 2.2.2. Extraction

ϭϰϳ

ϭϰϴ

The extraction for the analysis of total phenolic compounds and flavonoids was

ϭϰϵ

performed according to the method reported by Andrade et al. (2017) with some

ϭϱϬ

modifications. 0.5 g of each lyophilized flower were mixed with 20 mL of 12% ethanol. The

ϭϱϭ

mixture was transferred to ultrasound (Unique model USC-1400A) at a frequency of 40 KHz,

ϭϱϮ

and left at room temperature (25 ± 2 °C) for 60 min. All extracts were centrifuged (Eppendorf

ϭϱϯ

Centrifuge, 5810 R) at 24 °C at 12.000 rpm for 15 min and the supernatants collected.

ϭϱϰ

The method used for extraction of tannins was recommended by Rhazi et al. (2015).

ϭϱϱ

An aliquot of 0.5 g of each sample was mixed with 20 mL of 80% methanol and shaken

ϭϱϲ

(SOLAB, Brazil, SL 222) at 200 rpm, 25 °C, for 10 min. The mixture was collected, filtered

ϭϱϳ

and was maintaned in a rotary evaporator at 40 °C for evaporation of methanol. To separate

ϭϱϴ

the phenolic and non-phenolic phases, two drops of HCl (6N) were added in the remaining

ϭϱϵ

volume of water and extraction with diethyl ether was carried out (3 x 5 mL) with the aid of a

ϭϲϬ

separatory funnel. The tannins phase (aqueous phase) containing condensable and

ϭϲϭ

hydrolyzable tannins was adjusted to 10 mL of deionized water.

ϭϲϮ

For carotenoids extraction, 1 g of each sample was dissolved in 2 mL of acetone.

ϭϲϯ

The mixture was then transferred to ultrasound sonicator (Unique model USC-1400A) at a

ϭϲϰ

frequency of 40 KHz, 25 °C, for 30 min (Gomes et al., 2018). Organic acids extraction was

ϭϲϱ

performed according to the method proposed by Lee (1993) with modifications. The samples

ϭϲϲ

(0.5 g) were diluted in 10 mL of monobasic sodium phosphate (0.01 M) acidified with

ϭϲϳ

phosphoric acid and acetonitrile (99:1).

7 

The extracts were centrifuged for 15 min at 12.000 rpm, at a temperature of 22 °C

ϭϲϴ ϭϲϵ

and then filtered through a 0.45 ȝm filter. All extracts were stored in an amber bottle and kept in a freezer (-18 °C) until analysis.

ϭϳϬ

2.2.3. Determination of condensed tannins

ϭϳϭ

ϭϳϮ

The total content of condensed tannins was determined according to the methodology

ϭϳϯ

described by Rhazi et al. (2015) with modifications. A 0.5 mL aliquot of the aqueous extract

ϭϳϰ

was added to 3 mL of 4% vanillin-methanol solution and 1.5 mL of HCl. The solution was

ϭϳϱ

stored in a dark room for 15 min. The absorbance was read at 500 nm (Molecular Devices,

ϭϳϲ

Sunnyvale, CA, USA; SpectraMax M2) and the results were expressed in milligrams of

ϭϳϳ

quercetin (QE) equivalent per grams of sample. The total content of condensed tannins was

ϭϳϴ

determined through a catechin curve prepared from concentrations varying from 0 to 0.3

ϭϳϵ

mg/mL. 2.2.4.

ϭϴϬ

Determination of hydrolysable tannins

ϭϴϭ

The total content of hydrolysable tannins was determined by the potassium iodate test

ϭϴϮ

described by Rhazi et al. (2015). One milliliter of the aqueous extract was homogenized with

ϭϴϯ

5 mL of 2.5% aqueous solution of KlO3 previously heated for 7 min at 30 °C. The mixture

ϭϴϰ

was kept in a water bath at 30 °C for 2 minutes. Absorbance was measured at 550 nm in

ϭϴϱ

spectrophotometer (Molecular Devices, Sunnyvale, CA, USA; SpectraMax M2). A

ϭϴϲ

calibration curve was constructed using a tannic acid solution ranging from 0 to 7 mg/mL.

ϭϴϳ

The results were expressed as milligrams of tannic acid equivalent (TAE) per grams (mg

ϭϴϴ

TAE/g). 2.2.5. Identification and quantification of phenolic and flavonoid compounds by

ϭϴϵ

UHPLC-QDa-MS

ϭϵϬ

8 

ϭϵϭ

The obtained extracts were analyzed in an UPLC liquid chromatograph Acquity Class

ϭϵϮ

H (Waters) coupled with PDA detector and simple quadrupole type (QDa) mass spectrometer,

ϭϵϯ

equipped with electrospray ionization in negative mode for all the compounds and mode of

ϭϵϰ

acquisition Selected Ion Monitoring (SIM).

ϭϵϱ

Chromatographic separation was performed according to the methodology reported by

ϭϵϲ

Barros et al. (2017) with some modifications, on an Ascentis Phenyl (15 cm x 4.6 mm, 5 ȝm;

ϭϵϳ

Supelco analitical) column. The mobile phase consisted of: solution A (deionized water with

ϭϵϴ

0.1% formic acid) and solution B (acetonitrile with 0.1% formic acid) at a flow rate of 0.35

ϭϵϵ

mL/min, at a temperature of 40 °C and injection volume of 5 ȝL. The elution was performed

ϮϬϬ

in gradient mode, according to the following events: 0-15 min, 100% A; 15-25 min, 75% A;

ϮϬϭ

25-35 min, 60% A; 35-45 min, 50% A; 45-55, 30% A; 55-60 min, 0% A.

ϮϬϮ

Quantification was done from calibration curves constructed for each of the flavonoid

ϮϬϯ

and phenolic acid standards, and the respective concentrations were determined through the

ϮϬϰ

areas of the bands of the target compounds as a function of the calibration curve. The curve

ϮϬϱ

concentration range was varied from 0.02 to 1 mg/mL. The limits of detection (LOD) were

ϮϬϲ

calculated, taking into consideration a signal-to-noise (S/N) ratio of > 3. The analytes

ϮϬϳ

concentration that were above the detection limit, but below the limits of quantification

ϮϬϴ

(LOQ), were designated as traces (TR). The UHPLC-MS parameters, retention time and

ϮϬϵ

linearity limits for polyphenolic compounds are presented in Table 1. 2.2.6. Identification and quantification of carotenoids by UFLC-DAD

ϮϭϬ

Ϯϭϭ

The identification and quantification of carotenoids were performed according to the

ϮϭϮ

method proposed by Gomes (2018) with modifications. The obtained extracts were analyzed

Ϯϭϯ

in an Ultra-Fast Liquid Chromatograph (Shimadzu), equipped with two LC-20AD pumps,

Ϯϭϰ

SIL-20A auto-injector, CTO-20A column oven, CBM-20A system controller and diode array 9 

Ϯϭϱ

detector SPD-M20A. Chromatographic separation was performed through a Kinetex C18

Ϯϭϲ

(250 cm x 4.6 mm, 5 ȝm) Phenomenex column. The mobile phase consisted of

Ϯϭϳ

methanol/ethyl acetate/acetonitrile (50:40:10). The elution was performed in isocratic mode,

Ϯϭϴ

with a flow of 1 mL/min, temperature of 30 °C, time of 20 min and injection volume of 10

Ϯϭϵ

ȝL.

ϮϮϬ

The standard calibration curves were: A – canthaxanthin (y = 1E+06x – 5299.9, r2 =

ϮϮϭ

0.994), B - ȕ-carotene (y = 345263x + 1876.4, r2 = 0.9988) and C – lycopene (y = 62942x –

ϮϮϮ

1725.3, r2 = 0.9933). The concentrations of standard reagents were varied from 0.02 to 1.00

ϮϮϯ

mg/mL. 2.2.7. Identification and quantification of organic acids by HPLC-DAD

ϮϮϰ

ϮϮϱ

The identification and quantification of organic acids were determinated according

ϮϮϲ

to the methodology proposed by Lee (1993) with modifications. The extracts were analyzed

ϮϮϳ

in a High Performance Liquid Chromatograph (Shimadzu), equipped with a quaternary pump

ϮϮϴ

LC-20AT, SIL-20A auto-injector, CTO-20A column oven, CBM-20A system controller and

ϮϮϵ

diode array detector SPD-M20A. Chromatographic separation was performed through a VP-

ϮϯϬ

ODS C18 (250 cm x 4.6 mm, 5 ȝm) Shimadzu column. The mobile phase consisted of

Ϯϯϭ

monobasic sodium phosphate (0.01 M) acidified with phosphoric acid and acetonitrile (99:1).

ϮϯϮ

The elution was performed in isocratic mode, with a flow of 1 mL/min, temperature of 40 °C,

Ϯϯϯ

time of 30 min and injection volume of 5 ȝL.

Ϯϯϰ

The standard calibration curves were: A – quinic acid (y = 1e+006x - 70752, r2 =

Ϯϯϱ

0.9596), B – succinic acid (y = 3.71e+006 x – 7.59e+003, r2 = 0.9930) and C – tartaric acid (y

Ϯϯϲ

= 4e+006x + 151364, r2 = 0.9924). The concentrations of standard reagents were varied from

Ϯϯϳ

0.02 to 1.00 mg/mL.

10 

Ϯϯϴ

2.2.8. Antioxidant capacity

Ϯϯϵ

2.2.8.1. DPPH assay

ϮϰϬ

The method was developed according to Brand-Williams et al. (1995) modificated by

Ϯϰϭ

Thaipong et al. (2006). The stock solution was prepared by mixing 2.4 mg of DPPH radical

ϮϰϮ

with 100 mL of methanol. The solution absorbance was adjusted at 0.7 ± 0.02 in 515 nm

Ϯϰϯ

using an UV–Vis spectrophotometer (Molecular Devices, Sunnyvale, CA, USA; SpectraMax

Ϯϰϰ

M2). 3.9 mL of DPPH radical were placed in a test tube and 100 µL of the antioxidants

Ϯϰϱ

extract were added (methanol was used as blank). The decrease in absorbance at 515 nm was

Ϯϰϲ

measured at 30 min. The calibration curve was prepared using solutions of 0 to 60 ȝmol

Ϯϰϳ

Trolox. The antioxidant capacity was expressed as the µmol TE/g. 2.2.8.2. FRAP assay

Ϯϰϴ

Ϯϰϵ

The ferric reducing antioxidant power (FRAP) of the extracts was determinated

ϮϱϬ

following the method suggested by Benzie & Strain (1996) modificated by Thaipong et al.

Ϯϱϭ

(2006). Stock solutions of acetate buffer (300 mM, where 3.1 g of C2H3NaO·3H2O was mixed

ϮϱϮ

with 16 mL of C2H4O2, pH 3.6), TPTZ (10 mM, mixture with 40 mM HCl) and FeCl3·6H2O

Ϯϱϯ

(20 mM) were prepared. The fresh working solution was prepared by mixing 25 mL of the

Ϯϱϰ

acetate buffer, 2.5 mL of the TPTZ solution and 2.5 mL of FeCl3·6H2O and then incubated in

Ϯϱϱ

a water bath at 37 °C before use. Each extract (150 ȝL) was allowed to react with FRAP

Ϯϱϲ

solution (2850 ȝL) and the mixture was incubated in a dark room for 30 min. The absorbance

Ϯϱϳ

was measured in a spectrophotometer (Molecular Devices, Sunnyvale, CA, USA; SpectraMax

Ϯϱϴ

M2) at 593 nm. Two calibration curves were prepared using solutions of 20 to 800 ȝmol of

Ϯϱϵ

Trolox (FRAP TE) and FeSO4 (FRAP FS). The results were expressed in ȝmol TE/g and

ϮϲϬ

ȝmol FeSO4/g. 2.2.8.3. ORAC assay

Ϯϲϭ

11 

ϮϲϮ

The oxygen radical absorbance capacity (ORAC) was estimated according to the

Ϯϲϯ

methodology reported by Prior et al. (2003) modificated by Thaipong et al. (2006). Each

Ϯϲϰ

extract (0.75 mL) was vortexed with a fluorescein working solution (1.25 mL, composed by

Ϯϲϱ

25 ȝL of fluorescein and 50 mL of phosphate buffer). The solution was heated at 37 °C for 15

Ϯϲϲ

min. Finally, the AAPH solution (0.75 mL) was added and the absorbance was measured by

Ϯϲϳ

using a spectrophotometer (Molecular Devices, Sunnyvale, CA, USA; SpectraMax M2) under

Ϯϲϴ

fluorescence conditions: excitation at 485 nm and emission at 520 nm. The calibration curve

Ϯϲϵ

was prepared using solutions of 0 to 50 ȝmol Trolox. The results were expressed as ȝmol

ϮϳϬ

TE/g. 2.2.9. Statistical analysis

Ϯϳϭ

ϮϳϮ

The statistical analysis was performed by analysis of variance (ANOVA), using SAS

Ϯϳϯ

software (SAS Institute, Cary, NC) Version 9.1.3. Significant differences between the mean

Ϯϳϰ

values were determined by Tukey's test at 95% of confidence level (p ” 0.05). The data matrix

Ϯϳϱ

containing the results on the total bioactive compounds, polyphenols and antioxidant activity

Ϯϳϲ

analysis in each of the investigated samples was submitted to chemometric evaluation

Ϯϳϳ

using XLSTAT software (Addinsoft Inc., Paris, FR, 2016). The Principal Component

Ϯϳϴ

Analysis (PCA) was based on the Pearson's correlation (pௗ”ௗ0.05) of data set method and was

Ϯϳϵ

used to evaluate the differences between the samples. All samples were analyzed in triplicate.

ϮϴϬ

All datas used to evaluate the parametric tests presented normal distribution as well as

Ϯϴϭ

homogeneous variances.

ϮϴϮ

3. Results and Discussion

Ϯϴϯ

3.1. Condensed and hydrolysable tannins

Ϯϴϰ

Figure 1 presents the condensed and hydrolysable tannins extracted from edible

Ϯϴϱ

flowers. For all samples analyzed, the hydrolyzed tannins presented a higher content when 12 

Ϯϴϲ

compared to condensed tannins. The highest condensed and hydrolysable tannins content was

Ϯϴϳ

exhibited by red capuzin (22.01 ± 0.25 mg CA/g and 66.11 ± 0.50 mg TAE/g, respectively), while

Ϯϴϴ

the lowest hydrolysable tannin content was detected in the jambu sample (12.51 ± 0.58 mg

Ϯϴϵ

TAE/g). The purple amaranth and jambu did not contain condensed tannins and exhibited only

ϮϵϬ

hydrolysable tannins, being the levels presented by purple amaranth greater than tha jambu

Ϯϵϭ

(20.83 ± 0.56 against 12.51 ± 0.18 mg TAE/g).

ϮϵϮ

When compared to important sources of condensed and hydrolysable tannins it was

Ϯϵϯ

evaluated that red capuzin presented higher contents of condensed tannins than beans, red

Ϯϵϰ

wine, nuts and chocolate. All the samples exhibited values greater than commom sources of

Ϯϵϱ

hydrolysable tannins as persimmon seeds, green tea and grape seeds (De La Rosa et al.,

Ϯϵϲ

2010). 3.2. Identification and quantification of phenolic compounds

Ϯϵϳ

Ϯϵϴ

The concentrations of the phenolic compounds present in the extracts of the different

Ϯϵϵ

edible flowers are presented in Table 2. Eighteen compounds were identified in the samples

ϯϬϬ

through the UHPLC-QDa-MS system, but only nine were above the quantification limits.

ϯϬϭ

It was verified that the same compounds (caffeic acid, chlorogenic acid, p-coumaric

ϯϬϮ

acid, epicatechin, ferulic acid, kaempferol and rutin) were identified in the orange and red

ϯϬϯ

capuzin flowers, but these flowers had different contents. The purple amaranth was the only

ϯϬϰ

flower that contained the catechin compound, while jambu was the only one that possessed

ϯϬϱ

naringenin. All flowers contained the compounds p-coumaric acid, ferulic acid and

ϯϬϲ

kaempferol. However, none of the edible flowers presented the compounds vanillic acid and

ϯϬϳ

vanillin.

13 

ϯϬϴ

Rutin and chlorogenic acid were the major compounds detected in the two capuzin

ϯϬϵ

variations. Similarly, the compounds acacetin, apigenin, biochanin A, chrysin, daidzein, ethyl

ϯϭϬ

gallate, gallic acid, naringenin and protocatechinic acid were detected in both samples, but

ϯϭϭ

only at trace levels. Gárzon et al. (2014) and Koike et al. (2015) identified polyphenols in red

ϯϭϮ

and orange capuzin samples and highlighted the presence of compounds such as chlorogenic

ϯϭϯ

acid, mirecetin, quercetin and kaempferol.

ϯϭϰ

Many studies in vitro and in vivo have shown that rutin has excellent properties to

ϯϭϱ

prevent neurodegenerative disorders, cardiovascular disease and skin cancer (Babazadeh et

ϯϭϲ

al., 2017; Frutos et al., 2019). However, the health benefits of rutin may be influenced by its

ϯϭϳ

quantity and bioavailability. Rutin concentration varies between plant species and specific

ϯϭϴ

parts of the plant, in addition to varying in relation to the plants geographic origin. Normally,

ϯϭϵ

the daily intake of rutin ranges from 1.5 to 70 mg/kg, depending on the nutritional habits of an

ϯϮϬ

individual (Frutos et al., 2019). Thus, it would be necessary to consume 12 g of petals of red

ϯϮϭ

capuzin or 70 g of orange capuzin to achieve maximum daily intake.

ϯϮϮ

Chlorogenic acid has been reported to possess antioxidant, anticancer, anti-

ϯϮϯ

inflammatory activity and acts as a cholesterol-lowering agent (Parveen et al., 2011), as well

ϯϮϰ

as a number of protective properties against degenerative diseases (Niggeweg et al., 2004 ).

ϯϮϱ

Coffee is considered to be the main source of chlorogenic acid in the human diet, and it is

ϯϮϲ

estimated that a 100 mL tea-cup provides 100 mg of total chlorogenic acids (Stefanello et al.,

ϯϮϳ

2019). Thus, 100 g of red or orange capuzin petals would provide an amount of chlorogenic

ϯϮϴ

acid comparable to a 100 mL coffee cup.

ϯϮϵ

The purple amaranth presented higher values of p-coumaric acid and rutin. The

ϯϯϬ

compounds acacetin, apigenin, biochanin A, chrysin, daidzein, ethyl gallate, gallic acid, and

ϯϯϭ

protocatechinic acid were detected but below the limit of quantification. Noori et al. (2015) 14 

ϯϯϮ

detected the presence of the compounds viz. isorhamnetin, kaempferol, quercetin and rutin in

ϯϯϯ

aerial parts of purple amaranth. The jambu contained principal compounds such as ferulic

ϯϯϰ

acid and p-coumaric acid, and the compounds acacetin, apigenin, biochanin A, daidzein,

ϯϯϱ

luteolin, protocatechinic acid and rutin at trace levels. Navarro-González et al. (2014)

ϯϯϲ

attempted to identify jambu compounds, and reported the presence of quercetin, rutin and

ϯϯϳ

cynarine.

ϯϯϴ

The p-coumaric acid exhibed in vivo evidences of its action as a neuroprotective,

ϯϯϵ

antiapoptotic (Guven et al., 2015) and anticancer agent (Kong et al., 2013). In addition, it acts

ϯϰϬ

in the prevention of cardiovascular diseases as evidenced by the in vivo study of Roy &

ϯϰϭ

Stanely (2013). The estimated daily intake of p-coumaric acid for humans is 15 mg/day

ϯϰϮ

(Luceri et al., 2007), and therefore 80 g of purple amaranth or 120 g of jambu are able to

ϯϰϯ

provide the daily requirement of this compound.

ϯϰϰ

Ferulic acid is an abundant antioxidant and may induce in vitro and in vivo prevention

ϯϰϱ

and treatment of various disorders linked to oxidative stress, such as Alzheimer's disease,

ϯϰϲ

diabetes, cancer, cardiovascular disease and atherosclerosis. It is estimated that the daily

ϯϰϳ

intake of ferulic acid is approximately 150-250 mg/day (Zhao & Moghadasian, 2008), and

ϯϰϴ

hence it is necessary to consume on average 100 g of jambu to reach the stipulated values.

ϯϰϵ

Xiong et al. (2014) analyzed common edible flowers from China and reported rutin

ϯϱϬ

concentrations in the flowers Prunus persica, Flos carthami and Lavandula pedunculata.

ϯϱϭ

These results are bellow to those found for the two variations of capuzin reported in this

ϯϱϮ

study. All flowers studied showed p-coumaric acid values higher than Lilium brownii var

ϯϱϯ

viridulum and Flos lonicerae. Finally, they showed higher values for chlorogenic acid when

ϯϱϰ

compared to Chrysanthemum morifolium and Flos rosae rugosa.

15 

ϯϱϱ

As regards to the profile of quantified phenolic compounds in edible flowers, it was

ϯϱϲ

observed that the orange and red capuzin presented four distinct classes of phenolic

ϯϱϳ

compounds (hydroxycinnamic acids, flavonols and flavan-3-ols), whereas purple amaranth

ϯϱϴ

presented compounds belonging to the classes of hydroxycinnamic acids, flavonols, flavan-3-

ϯϱϵ

ols and jambu to hydroxycinnamic acids, flavonols and flavanones. 3.3. Identifiation and quantification of carotenoids

ϯϲϬ

ϯϲϭ

In relation to the carotenoid contents, only ȕ-carotene was detected in red capuzin and

ϯϲϮ

orange capuzin. The other flowers did not show the presence of this compound. The flowers

ϯϲϯ

under study did not contain carotenoids such as lycopene and canthaxanthin.

ϯϲϰ

In addition to the known provitamin A activity of ȕ-carotene, its consumption has been

ϯϲϱ

attributed to the reduction of the risk of developing chronic diseases viz. certain types of

ϯϲϲ

cancer, cardiovascular diseases, macular degeneration and cataract (Rodriguez-Amaya, 2019).

ϯϲϳ

The red capuzin and orange capuzin presented high concentrations of ȕ-carotene, especially

ϯϲϴ

when compared to the flowers Viola wittrockiana and Antirrhinum majus (González-Barrio et

ϯϲϵ

al., 2018) and other foods rich in ȕ-carotene such as carrot and pumpkin (Vargas-Murga et al.,

ϯϳϬ

2016). According to the NHMRC (2006), the estimated daily intake of ȕ-carotene should be

ϯϳϭ

6-18 mg/day, so approximately 1 g/day of the orange or red capuzin variation would be

ϯϳϮ

enough to supply the established daily value. 3.4.Identifiation and quantification of organic acids

ϯϳϯ

ϯϳϰ

The results obtained in relation to the profile of organic acids showed that purple

ϯϳϱ

amaranth is the flower with the larger diversity of organic acids and presented the levels for

ϯϳϲ

quinic acid that did not exhibited statistically significant diferences (p ” 0.05) with red

ϯϳϳ

capuzin. The lowest quinic acid value was exhibited by jambu. The succinic acid did not 16 

ϯϳϴ

expressed statistically significant diferences (p ” 0.05) between flowers. Only purple

ϯϳϵ

amaranth exhibed the tartaric acid compound (Table 3). 3.5.Antioxidant capacity

ϯϴϬ

ϯϴϭ

Antioxidant capacity of the extracts was conducted using the oxygen radical

ϯϴϮ

absorbance capacity (ORAC), as well as FRAP and DPPH assays. Capuzin (red) exhibited the

ϯϴϯ

greatest oxygen radical absorbance capacity (ORAC assay) and it was followed by capuzin

ϯϴϰ

(orange) and purple amaranth; while jambu had the lowest ORAC value (Table 4). The

ϯϴϱ

highest reducing capacity (FRAP) was exhibited by capuzin (red) extract, followed by purple

ϯϴϲ

amaranth. Capuzin (orange) and jambu extracts presented the lowest FRAP values that did not

ϯϴϳ

presented statistical significant diferences (p ” 0.05). The extract of purple amaranth exhibited

ϯϴϴ

the greatest free radical scavenging (DPPH assay), followed by orange capuzin and jambu,

ϯϴϵ

that presented a similar trend. Red capuzin demonsted the lowest DPPH level.

ϯϵϬ

The difference between the antioxidant capacity behaviors of the four flowers studied

ϯϵϭ

could be related to difference in the mechanisms accessed by these techniques. In FRAP

ϯϵϮ

methodology antioxidant capacity is based on the antioxidant ability to reduce the ion Fe3+

ϯϵϯ

(iron III) to Fe2+ (iron II) (Benzie & Strain, 1999; Gülçin et al., 2014). In DPPH method the

ϯϵϰ

antioxidant capacity is based on the antioxidant capacity of transferring H atoms to radicals

ϯϵϱ

(Schaich et al., 2015). ORAC procedure measures specifically the ability of compounds to

ϯϵϲ

scavenge oxygen free radicals, through the antioxidant capacity to quenching peroxyl radicals

ϯϵϳ

via hydrogen atom transfer or radical addition (Prior et al., 2003). Thus, these methodologies

ϯϵϴ

involve different mechanisms of action of the antioxidant agents against free radicals,

ϯϵϵ

indicating its ability to delay or inhibit the oxidation of a substrate through the reaction with

ϰϬϬ

iron (FRAP), donation of hydrogen (DPPH) or addition of hydrogen or radicals (ORAC).

17 

ϰϬϭ

When compared to the antioxidant capacity of other species of flowers, it can be

ϰϬϮ

observed that purple amaranth, orange capuzin, red capuzin and jambu presented lower values

ϰϬϯ

by the FRAP methodology when compared the results obtained for Calendula officinalis,

ϰϬϰ

Hibiscus sabdariffa L., Siraitia grosvenorii and Redartfulplum tea (Chen et al., 2015).

ϰϬϱ

In relation to the antioxidant capacity evaluated by the ORAC methodology, it was

ϰϬϲ

found that the variations of red and orange capuzin presented higher values than those

ϰϬϳ

presented by Garzón et al. (2015) for the same flowers. Navarro-González et al. (2014)

ϰϬϴ

described the antioxidant capacity of the jambu by the ORAC assay as being 10.77 ȝmol

ϰϬϵ

TE/g, or in other words, below the values detected in this study. However for the purple

ϰϭϬ

amaranth, there is no reported data on its antioxidant activity determined by ORAC method.

ϰϭϭ

As for DPPH assay, it was evaluated that the capuzin flowers (red and orange varietes)

ϰϭϮ

presented a free radical scavenging lower than those found for the same species by Garzón et

ϰϭϯ

al. (2009). All the flowers demonstrated lower levels than other flowers of common

ϰϭϰ

consumption as Calendula officinalis L., Chamomilia and Hibiscus sabdariffa L. (Chen et al.,

ϰϭϱ

2015).

ϰϭϲ

The evaluation of the data in relation to the analysis of total polyphenols, bioactive

ϰϭϳ

compounds and antioxidant capacity, indicated that there is a correlation between their level

ϰϭϴ

of occurrence and the profile of the samples. Table 5 presents the data of the Pearson

ϰϭϵ

correlation coefficient between total polyphenols, bioactive compounds and the antioxidant

ϰϮϬ

activity of the edible flowers extracts. Three bioactive compounds (caffeic acid, rutin and

ϰϮϭ

kaempferol) presented positive correlation (p ” 0.05) with the antioxidant activity, being the

ϰϮϮ

highest for rutin (r = 0.9291, 0.8828, 0.8728 for ORAC, FRAP TE, FRAP FS assays,

ϰϮϯ

respectively). These results suggest that rutin is one of the main contributors with positive

18 

ϰϮϰ

correlation with antioxidant activity in flowers, since an increase in the concentration of this

ϰϮϱ

compound increase the antioxidant activity. 3.6. Chemometric evaluation of the samples

ϰϮϲ

ϰϮϳ

The results of the Principal Component Analysis (PCA) generated for the four

ϰϮϴ

different species of edible flowers are presented in Figure 2. These results indicate their

ϰϮϵ

variability in respect to the bioactive compounds and antioxidant activity. The graphs of

ϰϯϬ

scores and loadings obtained (Figure 2A and B) showed that the main components F1 and F2

ϰϯϭ

were able to explain 90.9% of the total variation of the data, of which the component F1

ϰϯϮ

explained 63.16% and the component F2, 27.74%.

ϰϯϯ

In Figure 2A, the scores plot shows the distribution of the four different species of

ϰϯϰ

edible flowers studied in the two main components. While in Figure 2B, the loadings plot

ϰϯϱ

shows the distribution of the variables studied in the two main components. The total

ϰϯϲ

polyphenols (PC and FC), antioxidant activities (FRAP FS, FRAP TE, ORAC and DPPH) and

ϰϯϳ

some bioactive compounds (p-coumaric acid, catechin, epicatechin, kaempferol, caffeic acid,

ϰϯϴ

rutin, ȕ-carotene and chlorogenic acid) are grouped in F1 positive. These variables are present

ϰϯϵ

in higher values or restricted in the samples of purple amaranth, red and/or orange capuzin

ϰϰϬ

when compared to the jambu. On the other hand, the polyphenols, ferulic acid and naringenin,

ϰϰϭ

are allocated in F1 negative, since these were the compounds found in higher value or only in

ϰϰϮ

jambu. It can be inferred that the parameters FRAP FS, FRAP TE, ORAC, p-coumaric acid,

ϰϰϯ

epicatechin, kaempferol, caffeic acid, rutin, ȕ-carotene and chlorogenic acid characterize the

ϰϰϰ

samples of red and orange capuzin, the parameters catechin and DPPH characterize the purple

ϰϰϱ

amaranth, while naringenin and ferulic acid characterize the jambu sample.

ϰϰϲ

19 

4. Conclusion

ϰϰϳ

ϰϰϴ

This study analyzed the comprehensive profile of phenolic acids, flavones, flavonols,

ϰϰϵ

flavan-3-ols, isoflavones, organic acids and carotenoids of four edible flowers. Phenolic acids

ϰϱϬ

such as p-coumaric acid and ferulic acid were widely detected in flower samples under

ϰϱϭ

investigation for the first time. Kaempferol, rutin, catechin and epicatechin were observed to

ϰϱϮ

be the predominant flavonoids in edible flowers. Carotenoids were detected only in the

ϰϱϯ

samples of capuzin. There were more phenolic and carotenoid contents in red capuzin. The

ϰϱϰ

purple amaranth presented the hightest contents of organic acids. Chemometric

ϰϱϱ

characterization and its correlation have been performed for the first time to evaluate

ϰϱϲ

similarities between identified phenolic compounds and antioxidant capacity of edible flower

ϰϱϳ

samples. Thus, these flowers should serve as potential rich resources of natural antioxidants

ϰϱϴ

for use as functional food ingredients or pharmaceuticals for control of diseases caused by

ϰϱϵ

oxidative stress.

ϰϲϬ

Acknowledgements

ϰϲϭ

All authors gratefully acknowledge the financial support received from CNPq, Brazil,

ϰϲϮ

vide research project ‘Instituto Nacional de Ciência e Tecnologia de Frutos Tropicais’

ϰϲϯ

(Project 465335/2014-4) in developing this work. Authors (Romy Barros, Julianna Andrade,

ϰϲϰ

Ubatã Pereira, Yara Rezende, Taís Silva, Christean Oliveira, Hannah Araujo e Nayjara

ϰϲϱ

Carvalho) acknowledge and thank FAPITEC and CAPES (Ministry of Education, Brazil) for

ϰϲϲ

their fellowships.

ϰϲϳ

ϰϲϴ

ϰϲϵ 20 

ϰϳϬ

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ϲϳϰ

ϲϳϱ

ϲϳϲ

ϲϳϳ

ϲϳϴ

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ϲϴϬ

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ϲϴϱ

Figure captions

ϲϴϲ

Figure 1. Total tannins content in edible flowers extracts.

ϲϴϳ

Figure 2. Chemometric analysis: (A) PCA score plot and (B) PCA loading plot generated

ϲϴϴ

with the data set of the total bioactive compounds, polyphenols and antioxidant activity.

31 

70

Purple amaranth Capuzin (orange) Capuzin (red) Jambu

a

65 60 55 50 45

mg/g

40 35 30 25

a

c

20

b

15

d

10 5 0

b Condensed

Hydrolysable

Tannins 

Figure 1. Total tannins content in edible flowers extracts.

3.5 Purple amaranth 2.5

F2 (27.74 %)

1.5 0.5 Orange capuzin

-0.5

Red capuzin

-1.5 Jambu

-2.5 -3.5 -5

-4

-3

-2

-1

0

1

2

3

4

5

F1 (63.16 %)

(A) 2.5 2 1.5

p-coumaric acid (+)-Catechin

F2 (27.74 %)

1 0.5

(-)-Epicatechin FRAP FS FRAP TE PC FC ORAC Kaempferol ȕ-carotene

DPPH

0 Ferulic acid

-0.5

Naringenin

-1

Rutin -1.5

Caffeic acid

Chlorogenic acid

-2 -3.5

-3

-2.5

-2

-1.5

-1

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

F1 (63.16 %)

(B) Figure 2. Chemometric analysis: (A) PCA score plot and (B) PCA loading plot generated with the data set of the bioactive compounds, total polyphenols and antioxidant activity.

Table 1. UHPLC-QDa-MS parameters, retention time and lineary limits for phenolic compounds identified in edible flowers. Molecular formula

Compounds

Hydroxybenzoic acids Ethyl gallate

Molecular mass (g/mol)

Precursor ion

Retention time (min)

Collision energy (eV)

MS [M-H]-

Calibration equation*

Correlation coeficient (r2)

LOQ (mg/mL)

  C9H10O5



198.17



198

197



15



23.83

y = 2.20e+007 x + 3.15e+005  0.993179

 0.002

Gallic acid Protocatechinic acid

C7H6O5 C7H6O4

170.12 154.12

170 154

169 153

15 15

12.65 16.96

y = 1.42e+007 x + 4.28e+004 y = 9.31e+006 x + 6.87e+004

0.993477 0.995705

0.005 0.002

Vanillic acid Vanillin

C8H8O4 C8H8O3

168.14 152.15

168 152

167 151

15 15

21.77 25.44

y = 4.61e+005 x - 1.12e+003 y = 1.47e+006 x - 2.61e+003

0.992141 0.994991

0.002 0.002

21.31

y = 1.83e+007 x + 6.87e+004  0.992968

 0.002

Hydroxycinnamic acids Caffeic acid

  C9H8O4



180.16



180

179



15



Chlorogenic acid p-Coumaric acid

C16H18O9 C9H8O3

354.31 164.04

354 164

353 163

15 15

18.85 24.93

y = 2.29e+007 x - 3.14e+004 y = 8.79e+006 x - 1.47e+004

0.992627 0.992967

0.002 0.002

Ferulic acid

C10H10O4

194.18

194

193

15

25.92

y = 3.26e+006 x - 8.66e+003

0.99348

0.002

Flavonols Kaempferol Rutin

 C15H10O6

35.74 22.10

y = 5.14e+007 x - 4.66e+004  0.994287

 0.002

0.99066

0.002

43.39

y = 1.16e+007 x + 1.76e+004  0.994552

 0.002

 

C27H30O16

286.23 610.52



284.26



286 610

285 609



284

283



15 15



15



y = 9.36e+006 x + 8.62e+004



Flavones Acacetin

 C16H12O5

Apigenin Chrysin

C15H10O5 C15H10O4

270.05 254.22

270 254

269 253

15 15

35.30 43.07

y = 2.93e+007 x + 3.60e+005 y = 3.01e+007 x + 2.42e+005

0.993176 0.9902

0.002 0.002

Luteolin

C15H10O6

286.24

286

285

15

31.65

y = 2.51e+007 x + 5.19e+005

0.99066

0.002

19.76 20.98

y = 2.80e+007 x - 2.71e+004  0.992431

 0.002

Flavan-3-ols (+)-Catechin (−)-Epicatechin



  C15H14O6



C15H14O6

290.26 290.26



290 290

289 289



15 15



y = 2.38e+007 x - 2.10e+004

0.994851

0.002



Flavanones

















Naringenin

C15H12O5

Isoflavones Biochanin A

 C16H12O5

272.26

272

271

15

10.92

y = 1.37e+005 x + 1.13e+003

0.993856

0.005

44.70

y = 3.24e+007 x + 2.35e+005  0.993176

 0.023

30.81

y = 2.79e+007 x + 2.89e+005

 

284.26



284

283

Daidzein C15H10O4 254.23 254 253  y is the value of the peak area. x is the concentration of the standard compound. LOQ – Limit of quantification.



15 15



0.993801

0.002

Table 2. Quantification of carotenoid and phenolic compounds in extracts of edible flowers according to chemical classes. Flowers (g/g)

Compounds Hydroxybenzoic acids Ethyl gallate Gallic acid Protocatechinic acid Vanillic acid Vanillin Total Hydroxybenzoic acids Hydroxycinnamic acids Caffeic acid

Purple amaranth 

TR TR TR ND ND 0.00 (0.00%)



ND

Capuzin (orange) 

TR TR TR ND ND 0.00 (0.00%)

 119.67b ± 0.62



TR TR TR ND ND 0.00 (0.00%)

 342.39a ± 1.42



ND ND TR ND ND 0.00 (0.00%)



ND

ND

1134.10 ± 4.48

848.51 ± 3.88

24.13c ± 0.01

p-Coumaric acid

223.54a ± 0.47

161.19b ± 5.85

159.75b ± 0.87

129.09c ± 0.42

Ferulic acid

99.38b ± 2.13

72.58b ± 0.75

59.85b ± 0.92

1226.20a ± 34.06

322.92 (61.13%)

1487.54 (14.38%)

1410.50 (8.77%)

1379.42 (96.66%)

36.39c ± 0.34

54.42b ± 0.92

76.14a ± 0.42

33.97d ± 0.26

147.63c ± 3.47

1125.41b ± 9.40

5995.85a ± 9.45

TR

184.02 (31.66%)

1179.83 (11.40%)

6071.99 (37.77%)

33.97 (2.38%)

TR TR TR ND 0.00 (0.00%)

TR TR TR ND 0.00 (0.00%)

TR TR TR ND 0.00 (0.00%)

TR TR ND TR 0.00 (0.00%)

Rutin Total Flavonols Flavones Acacetin Apigenin Chrysin Luteolin Total Flavones

b

Jambu

Chlorogenic acid

Total Hydroxycinnamic acids Flavonols Kaempferol

a

Capuzin (red)

Flavan-3-ols (+)-Catechin (−)-Epicatechin

38.89 ± 0.41 35.42b ± 0.34

ND 35.70b ± 0.69

ND 37.04a ± 0.19

ND ND

Total Flavan-3-ols Flavanones Naringenin Total Flavanones Isoflavones Biochanin A Daidzein Total Isoflavones Carotenoids Cantaxantin -carotene

74.31 (7.21%)

35.70 (0.34%)

37.04 (0.23%)

0.00 (0.00%)

ND 0.00 (0.00%)

TR 0.00 (0.00%)

TR 0.00 (0.00%)

13.68 ± 0.23 13.68 (0.96%)

TR TR 0.00 (0.00%)

TR TR 0.00 (0.00%)

TR TR 0.00 (0.00%)

TR TR 0.00 (0.00%)

ND ND

ND 7643.21b ± 311.16

ND 8554.60a ± 331.09

ND ND

Lycopene Total Carotenoids

ND 0.00 (0.00%)

ND 7643.21 (73.88%)

ND 8554.60 (53.23%)

ND 0.00 (0.00%)

ND – not detected; TR – traces. Results expressed as mean ± standard deviation (n = 3). Mean values of edible flowers extracts followed by different superscript lowercase letters were significantly different (p 0.05).

Table 3. Quantification of organic acids in extracts of edible flowers. Organic acids (mg/g) Quinic acid Succinic acid Tartaric acid

Purple amaranth

Capuzin (orange)

24.04a ± 0.31 0.11a ± 0.00 36.37 ± 0.01

ND 0.44a ± 0.01 ND

Capuzin (red) 20.21a ± 0.21 0.52a ± 0.04 ND

Jambu 8.45b ± 0.62 0.37a ± 0.01 ND

ND – not detected. Results expressed as mean ± standard deviation (n = 3). Mean values of edible flowers extracts followed by different superscript lowercase letters were significantly different (p 0.05).

Table 4. Antioxidant capacity of the extracts of edible flowers. Flowers Samples Purple amatanth Capuzin (orange) Capuzin (red) Jambu

ORAC (mol TE/g) 271.51c ± 1.52 505.07b ± 0.24 744.47a ± 22.81 192.43d ± 6.88

FRAP mol TE/g 6.78b ± 0.01 4.48c ± 0.01 10.97a ± 0.01 4.37c ± 0.01

mol FeSO4/g 87.67b ± 0.04 72.89c ± 0.09 114.79a ± 0.08 67.60c ± 0.07

DPPH (mol TE/g) 58.74a ± 2.26 52.87b ± 1.04 49.38c ± 1.41 53.61b ± 0.35

Results expressed as mean ± standard deviation (n = 3). TE – Trolox equivalent. Means in each column followed by different superscript lowercase letters were significantly different (p0.05) among the edible flowers.



Table 5. Pearson’s correlation matrix for bioactive compounds, total polyphenols and antioxidant capacity of edible flowers extracts. Variables Caffeic acid Chlorogenic acid

Caffeic acid

Chlorogenic acid

pcoumaric acid

-0.2292

1

Ferulic acid

-0.5559

-0.6386

Kaempferol

0.9904

0.7086

-0.1610

Rutin

0.9834 0.4770

0.4638

-0.1535

-0.5623

0.9252

(+)-Catechin (-)-Epicatechin

Rutin

(+)Catechin

(-)Epicatechin

Naringenin

carotene

PC

FC

ORAC

FRAP TE

FRAP FS

DPPH

1

0.5473

0.6386

Naringenin

0.5104 0.4770

-0.5377

-0.6595

-carotene

0.8675

0.9246

-0.2286

PC

0.9143

0.6789

0.1095

FC

0.9520

0.4338

0.0030

ORAC

0.9749

0.7377

-0.0924

FRAP TE

0.8056

0.1160

0.1835

FRAP FS

0.8016 0.4005

0.1448

0.2419

-0.2513

0.6657

DPPH

Kaempferol

1 0.6160 0.1987 0.5001

p-coumaric acid

Ferulic acid

1 0.5775 0.4495 0.3074 0.9992 0.9993 0.5973 0.7595 0.5479 0.6458 0.4852 0.5503 0.2792

Values in bold presented correlation significant at p0.05.

1 0.9525 -0.4721

1 0.3932

1 0.3097

1

-0.5547

0.4629 0.4280

-0.3333

-0.9990

1

0.9226

0.7645

-0.5749

0.5962

-0.5749

1

0.9412

0.8774

-0.2288

0.7665

-0.7430

0.8673

1

0.9283

0.9743

-0.2534

0.5615

-0.5307

0.7335

0.8897

1

0.9938

0.9291

-0.4203

0.6532

-0.6248

0.9318

0.9608

0.9174

1

0.7498

0.8828

0.0160

0.5060

-0.4754

0.4566

0.7695

0.9106

0.7462

1

0.7537

0.8728 0.3811

0.0591

0.5708

-0.5413

0.4726

0.6744

0.2728

-0.3009

-0.3406

0.7915 0.1700

0.9057 0.2308

0.7568 0.3213

0.9959 0.1379

0.5848

-0.3696

1 0.0946

1

Highlights

• • • • •

p-coumaric acid and ferulic acid were identified and quantified for the first time in all flowers; Hydroxycinnamic acids and flavonols were the most rrepresented epresented classes of phenolic compounds; Capuzin species presented hightest content of ȕ-carotene; Purple amaranth was the flower with the larger dive diversity in organic acids; Red capuzin was the most promising flower in polyphenols and antioxidant capacity.

Conflict of Interest

•

There is no conflict of interest among all authors in publishing this research paper.

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Romy Gleyse Chagas Barros: Principal investigator involved with experimental research and manuscript write-up Julianna Karla Santana Andrade: Deatermination of antioxidant activity in samples Ubatã Corrêa Pereira: Determination of carotenoids compounds in diferente samples Christean Santos de Oliveira: UHPLC-QDa-MS parameters & determination of phenolic compounds Yara Rafaella Ribeiro Santos Rezende: Quantification of organic acids in diferente samples Tais Oliveira Matos Silva: Determination of carotenoids compounds and write-up of the paper Juliete Pedreira Nogueira: Chemometric analysis & write-up of statistical evaluations Nayjara Carvalho Gualberto: Identification of phenolic compounds Hannah Caroline Santos Araujo: Quantification of tannins Narendra Narain: Principal Supervisor and evaluation of results; correction of the manuscript