Edible Flowers: A Rich Source of Phytochemicals with Antioxidant and

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Edible Flowers: A Rich Source of Phytochemicals with Antioxidant and Hypoglycaemic Activity Monica Rosa Loizzo, Alessandro Pugliese, Marco Bonesi, Maria Concetta Tenuta, Francesco Menichini, Jianbo Xiao, and Rosa Tundis J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b03092 • Publication Date (Web): 13 Aug 2015 Downloaded from http://pubs.acs.org on August 18, 2015

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

Phytochemical characterization (HPLC, GC, GC-MS

Malva sylvestris

Eight edible flowers

Antioxidant in vitro properties

-Amylase and -glucosidase inhibitory activity

Sambucus nigra

Hedysarum coronarium

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Edible Flowers: A Rich Source of Phytochemicals with Antioxidant and

2

Hypoglycaemic Properties

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Monica Rosa Loizzoa, Alessandro Puglieseb, Marco Bonesia, Maria Concetta Tenutaa, Francesco

5

Menichinia, Jianbo Xiaoc, Rosa Tundisa,*

6 7

a

8

b

9

c

10

Department of Pharmacy, Health and Nutrition Sciences, University of Calabria, 87036 Rende (CS), Italy

Department of Food Science, University of Parma, Parco Area delle Scienze, 43124 Parma, Italy

Research Center of Bio-separation Engineering Technology, Anhui Institute of Applied Technology, Hefei, Anhui,

230031, China; Institut für Pharmazie und Lebensmittelchemie, Universität Würzburg, 97074, Würzburg, Germany.

11 12 13

Running title

14

Edible flowers: chemistry and bioactivity.

15 16

Corresponding author. Address: Department of Pharmacy, Health and Nutrition Sciences,

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University of Calabria, I-87036 Rende (CS), Italy. Phone: +39 984 493246; Fax: +39 984 493107;

18

E-mail: [email protected] (R. Tundis)

19 20 21 22 23 24 25 26


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ABSTRACT: Edible flowers are receiving renewed interest as rich sources of bioactive

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compounds. Ethanol extracts of eight edible flowers were phytochemically characterized and

29

investigated for their bioactivity. Rutin, quercetin, luteolin, kaempferol, and myricetin were selected

30

as standards and quantified by HPLC. The fatty acids profile was analysed by GC and GC-MS.

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Antioxidant properties were evaluated by using different in vitro tests. The hypoglycaemic effects

32

were investigated via the inhibition of -amylase and -glucosidase. S. nigra exhibited the highest

33

radical scavenging activity (IC50 of 1.4 g/mL), followed by H. coronarium (IC50 of 1.6 g/mL).

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Both species contained high quercetin and rutin content. S. nigra extract exerted the highest activity

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in preventing lipid oxidation. M. sylvestris extract inhibited both-amylase and -glucosidase with

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IC50 values of 7.8 and 11.3 g/mL, respectively. These findings support the consumption of edible

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flowers as functional foods and their use as sources of natural antioxidants by food industry.

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Keywords: edible flowers; flavonoids; fatty acids; antioxidant activity; carbohydrate hydrolysing

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enzymes.



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INTRODUCTION

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The use of edible flowers is becoming popular as evidenced by an increase in the number of

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articles regarding this topic.1 Flowers have traditionally been used in cooking in various cultures,

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such as European and Asian (Victorian English, east Indians etc.) to improve appearance (color,

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odor and flavour), and nutritive value of meals. Flowers are served as a salad, to prepare cakes and

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drinks or as a side dish.2,3

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Flowers are rich in a great variety of natural antioxidants including flavonoids, anthocyanin

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and many other phenolic compounds.4 Epidemiological data has shown that a diet rich in

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antioxidant could prevent chronic diseases such as diabetes type II, cancer, cardiovascular and

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neurodegenerative disorders. During metabolism reactive oxygen species (ROS) and other free

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radicals are generated. These radicals are normally inactivated by endogenous antioxidant system.

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However, in particular conditions such as consequence of lifestyle or pathological situations, these

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free radicals could be accumulated generating the oxidative stress.5

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Diabetes has emerged as a major threat to health worldwide. Type 2 diabetes or non-insulin-

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dependent or adult-onset results from the body’s ineffective use of insulin. Type 2 diabetes

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comprises 90% of people with diabetes around the world, and is largely the result of excess body

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weight.6 There is growing evidence that excess generation of ROS, largely due to hyperglycaemia

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as a consequence of glycation reaction, causes oxidative stress in a variety of tissues. In type 2

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diabetic patients, oxidative stress is closely associated with chronic inflammation that may play a

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role in the development of complications in diabetes.7 -Amylase breaks down large insoluble

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starch molecules into absorbable molecules. This enzyme is found in the pancreatic juice and saliva.

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On the other hand, -glucosidase in the mucosal brush border of the small intestine catalyses

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the end step of digestion of starch and disaccharides that are abundant in the human diet.8 In order

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to reduce the levels of postprandial hyperglycaemia -amylase and -glucosidase inhibitors could

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be used. Several inhibitors of the carbohydrate hydrolysing enzyme have been isolated from plants

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to serve as an alternative drug with increased potency and lesser adverse effects than existing ACS Paragon Plus Environment

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synthetic drugs such as acarbose.9 Diabetes seems to influence the cholesterol absorption efficiency

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and synthesis with the respective non diabetic state. For this reason diabetic patients are often

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affected by high levels of cholesterol in the blood.10

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The objectives of this study are as follows: a) to investigate the phytochemicals content

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(phenols, flavonoids, carotenoids, anthocyanins, fatty acids) of different edible flowers (Anchusa

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azurea, Capparis spinosa, Cichorium intybus, Hedysarum coronarium, Malva sylvestris, Robinia

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pseudoacacia, Rosmarinus officinalis and Sambucus nigra) largely consumed in Italy; b) to

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evaluate the content of flavonoids namely rutin, quercetin, luteolin, kaempferol, and myricetin as

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standards; c) to study their potential antioxidant activities by using different in vitro assays namely

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DPPH, ABTS, FRAP and -carotene bleaching tests; d) to investigate the hypoglycaemic activity

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through the inhibition of carbohydrate -hydrolysing enzymes -amylase and -glucosidase.

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MATERIALS AND METHODS

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Chemicals and Reagents. Potato starch, sodium phosphate, sodium chloride, -amylase from

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porcine pancreas (EC 3.2.1.1), -glucosidase from Saccharomyces cerevisiae (EC 3.2.1.20),

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maltose, sodium acetate, potassium hydrogen carbonate, sodium potassium tartrate, 3,5-

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dinitrosalicylic acid, o-Dianisidine Color Reagent (DIAN), PGO Enzymes Solution (PGO), acetic

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acid, perchloric acid, basic bismuth nitrate, potassium iodide, phosphoric acid, sodium hydroxyde,

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HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), sodium phosphate monobasic,

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chlorogenic acid, quercetin, kaempferol, luteolin, rutin, myricetin, anhydrous sodium sulfate,

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sodium phosphate buffer, 2,2-diphenyl-1-picrylhydrazyl (DPPH), Folin-Ciocalteau reagent, Tween

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20,

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ethylbenzothiazoline-6-sulfonic acid (ABTS) solution, 6-hydroxy-2,5,7,8-tetramethylchroman-2-

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carboxylic acid (Trolox), potassium persulphate, -carotene, Tween 20, linoleic acid, FeCl3, FeSO4,

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butylated dimethyl sulfoxide (DMSO), hydroxytoluene (BHT), ascorbic acid, propyl gallate,

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cyanidin-3-glucoside, methaphosphoric acid, 2,6-dichloroindophenol, cholesterol, and 5-

tripyridyltriazine

(TPTZ),

sodium

potassium

tartrate


tetrahydrate,

2,2'-azino-bis(3-


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cholestane were purchased from Sigma-Aldrich S.p.a. (Milan, Italy). Acarbose from Actinoplanes

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sp. was obtained from Serva (Heidelberg, Germany). Solvents of analytical grade were obtained

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from VWR International s.r.l. (Milan, Italy).

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Plant Material and Extraction Procedure. Flowers of Sambucus nigra L. Hedysarum

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coronarium L., Cichorium intybus L., Malva sylvestris L., Anchusa azurea Mill., Capparis spinosa

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L., Robinia pseudoacacia L., and Rosmarinus officinalis L. were collected in Cosenza province

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(Calabria, Italy) during spring 2013. Flowers were cleaned by using distilled water; the petals are

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separated and kept at room temperature to drain. The petals are then exhaustively extracted by

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absolute ethanol at room temperature (48 h) for 3 times. The ethanol solutions are pooled together

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and evaporated to dryness using a rotary evaporator under reduced pressure to obtain the total

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extract.

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Total Phenolic Content. The total phenolic content was determined by the Folin-Ciocalteau

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method.11 Extract was mixed with 0.2 mL Folin-Ciocalteau reagent, 2 mL of water and 1 mL of

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15% Na2CO3. After 2 h incubation at 25°C the absorbance was measured at 765 nm using a UV-Vis

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Jenway 6003 spectrophotometer. The total phenolic content was expressed as mg of chlorogenic

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acid equivalents per g of dry extract.

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Total Flavonoid Content. The flavonoids content was determined as previously described.12

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Extract was added to 4 mL of distilled water. At zero time, 0.3 mL of 5% (w/v) sodium nitrite

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was added to the flask. After 5 min, 0.6 mL of 10% (w/v) AlCl3 was added, and then at 6 min 2 mL

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of 1 M NaOH were also added to the mixture, followed by the addition of 2.1 mL distilled water.

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Absorbance was read at 510 nm. The levels of total flavonoid content were expressed as mg of

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quercetin equivalents per g of dry extract.

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Total Antocyanin Content. Total monomeric anthocyanin content was determined using the

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pH-differential method13 with slight modifications. Briefly, 0.5 mL of the extract (1.5 mg/mL in

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water) was mixed with a) 3.5 mL of potassium chloride buffer (0.025 M, pH 1); b) 3.5 mL of

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sodium acetate buffer (0.025 M, pH 4.5). After 15 min, the raw absorbance of each solution was

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measured at 510 and 700 nm. The absorbance difference was calculated as follow: A= [(A510A700)

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pH 1.0  (A510A700) pH 4.5]. The anthocyanin content was calculated using the molar absorptivity

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() and molecular weights (MW) of cyanidin-3-glucoside (= 26,900; MW= 449.2). The

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concentration of monomeric anthocyanin was: (Absorbance × MW × Dilution Factor × 1000)/( ×

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1). Results are expressed as mg of cyanidin 3-glucoside equivalents/g of extract.

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Total Carotenoid Content. The total carotenoid content was measured at 450 nm.11 Extracts

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were added to 0.5 mL of 5% NaCl, vortexed for 30 s and centrifuged for 10 min at 4500 rpm. The

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supernatant (100 L) was diluted with 0.9 mL of n-hexane and measured at 460 nm. -Carotene

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was used as a standard. The total carotenoids contents were determined in triplicate and expressed

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as -carotene equivalents in mg per g of dry extract.

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HPLC analysis. Rutin, quercetin, luteolin, kaempferol, and myricetin were quantified by HPLC.

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Analyses were carried out in an HPLC system HP 1100 equipped with a pump, UV-vis detector

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(280 nm), column oven, injector and a C18 RP column (Phenomenex Luna 5 μm C18, 250 × 4.60

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mm). Water/formic acid (0.1%) (A) and methanol (B) were used as mobile phase as follows: 3 min

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100% A; 10 min 80% A; 60 min 100% B; 70 min 100% A). A flow rate of 1 mL/min was applied.

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The content was calculated from the integrated peak area of the extract and the corresponding

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standard.

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Fatty Acids Profile. Fatty acids were determined by Gas Chromatography-Mass Spectrometry

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(GC-MS) using a Hewlett-Packard 6890 gas chromatograph equipped with an HP-5 column and

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interfaced with a Hewlett Packard 5973 Mass Selective. Ionization of the sample components was

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performed in electron impact mode (EI, 70 eV). The carrier gas was helium and the oven

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temperature program was as follows: the initial temperature was 50 °C (for 5 min), then a

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13 °C/min ramp to 250 °C and held for 10 min. Fatty acids were identified by comparison of their

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mass spectra with those of standards and those stored in Wiley 138 and NIST 98 libraries. Results

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were expressed in relative percentage of each fatty acid.

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DPPH Radical Scavenging Activity Assay. DPPH radical scavenging activity was determined

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according to the technique previously described.14 A mixture of DPPH methanol solution (1.0 × 10-

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4

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The bleaching of DPPH was determined by measuring the absorbance at 517 nm (UV-Vis Jenway

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6003 spectrophotometer). The DPPH radicals scavenging activity was calculated as follows:

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[(A0A1/A0) × 100], where A0 is the absorbance of the control and A1 is the absorbance in the

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presence of the sample. Ascorbic acid was used as positive control.

M) and flower extracts (1-1000 g/mL in methanol) was prepared and kept in the dark for 30 min.

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ABTS Radical Scavenging Activity Assay. ABTS radical cation (ABTS+) solution was mixed

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with potassium persulphate and left in the dark for 12 h before use.14 The ABTS+ solution was

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diluted with methanol to an absorbance of 0.70 ± 0.05 at 734 nm. After addition of extract (1-1000

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g/mL in methanol) to the ABTS+ solution, absorbance was measured after 6 min. Ascorbic acid

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was used as positive control.

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-Carotene Bleaching Test. The -carotene bleaching test was done following the procedure

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previously describe.14 Briefly, -carotene solution was added to linoleic acid and 100% Tween 20.

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The emulsion was mixed with 200 L of samples (1-1000 g/mL in methanol). The tubes were ACS Paragon Plus Environment

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placed at 45 °C in a water bath. The absorbance was measured at 470 nm against a blank at t= 0, 30

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and 60 min. Propyl gallate was used as positive control.

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Ferric Reducing Activity Power (FRAP) Assay. The FRAP test is based on the redox reaction

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that involves TPTZ (2,4,6-tripyridyl-s-triazine)-Fe3+ complex.15 FRAP reagent was prepared by

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mixing 10 mM TPTZ solution with 40 mM HCl, 20 mM FeCl3 and 0.3 M acetate buffer. The

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absorption was measured at 595 nm. The FRAP value represents the ratio between the slope of the

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linear plot for reducing Fe3+-TPTZ reagent by extracts compared to the slope of the plot for FeSO4.

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Extracts were dissolved in methanol and tested at 2.5 mg/mL. BHT was used as positive control.

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Carbohydrate-hydrolysing Enzymes Inhibitory Activity. The -amylase and -glucosidase

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inhibition assays were performed using the method previously described.16 Acarbose was used as

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positive control. In -amylase inhibition test a starch solution (0.5% w/v) was obtained by stirring

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potato starch in 20 mM sodium phosphate buffer with 6.7 mM sodium chloride. The enzyme

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solution was prepared by mixing 25.3 mg of -amylase from porcine pancreas (EC 3.2.1.1) (10

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units/mg) in 100 mL of cold distilled water. A mixture of enzyme solution and samples (1-1000

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g/mL in DMSO) was prepared and added to starch solution at 25 °C for 5 min. The generation of

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maltose was quantified using 3,5-dinitrosalicylic acid at 540 nm.

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In -glucosidase inhibition test a maltose solution (4% w/v) was prepared by dissolving 12 g of

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maltose in 300 mL buffer of 50 mM sodium acetate. The enzyme solution was prepared by mixing

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1 mg of -glucosidase from Saccharomyces cerevisiae (EC 3.2.1.20) (10 units/mg) in 10 mL of ice-

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cold distilled water. Samples were dissolved in DMSO to give a final concentration ranging from 1

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to 1000 g/mL. Both control and samples were mixed to a maltose solution for 5 min at 37 °C.

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Then, -glucosidase solution was added and left for incubation at 37 °C for 30 min. Perchloric acid

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solution was used to stop reaction. The generation of glucose was quantified by the reduction of



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DIAN. The absorbance was measured at 500 nm.

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Statistical Analysis. The concentration giving 50% inhibition (IC50) was calculated by nonlinear

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regression with the use of Prism GraphPad Prism version 4.0 for Windows (GraphPad Software,

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San Diego, CA, USA). The dose-response curve was obtained by plotting the percentage inhibition

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versus concentration. Differences within and between groups were evaluated by one-way analysis

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of variance test (ANOVA) followed by a multicomparison Dunnett’s test compared with the

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positive controls.

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RESULTS AND DISCUSSION

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Phytochemicals profile. Eight edible flowers collected in Calabria (Italy) were investigated. The

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extraction yield, common names and colour, food and traditional medicinal use are presented in

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Table 1. Phytochemicals are a large group of plant-derived compounds hypothesized to be

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responsible for much of the disease protection conferred from diets. Among them phenolic

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compounds are attracting the attention of scientists because of their antioxidant, anti-inflammatory,

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anti-mutagenic, and anti-carcinogenic properties and their capacity to modulate some key cellular

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enzyme functions.17-19 The total phenolic content determined in this work was expressed in terms of

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mg chlorogenic acid/g dry extract. The results showed that among eight analysed edible flowers S.

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nigra exhibited the highest content with 228.5 mg chlorogenic acid/g dry extract. Significative

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values were also recorded for R. officinalis and C. intybus with 48.8 and 43.2 mg chlorogenic acid/g

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dry extract. A lower total flavonoid content than the total phenolic content was found amongst

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varieties of edible flowers, this condition is due to the presence of non-flavonoid phenolic

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compounds in plants.20 Correlation analysis evidenced a positive correlation between the total

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phenolic content and the total flavonoid content in all investigated species (r2= 0.771).

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HPLC was used to quantify some flavonoids of edible flowers extracts, namely rutin, myricetin,

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luteolin, quercetin and kaempferol. Except for A. azurea and M. sylvestris, rutin was the flavonoid

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most representative in R. pseudoacacia (28.4 mg/g), H. coronarium (28.2 mg/g dry), S. nigra (23.7 ACS Paragon Plus Environment

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mg/g) and C. intybus (20.3 mg/g) (Table 2). Qurcetin was identified only in C. spinosa, H.

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coronarium and S. nigra in which its value (23.6 mg/g) is comparable to rutin. Luteolin was

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revealed only in M. sylvestris flowers with value of 1.5 mg/g. All flower extracts are characterized

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by the presence of kaempferol (0.5-4.2 mg/g) and myricetin (0.6-3.5 mg/g dry extract) with the

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exception of C. intybus and H. coronarium, respectively.

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Recently, the flavonoids profile of ten common edible flowers from China was evaluated.21

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Agreed with our results the rutin and the quercetin are the main compounds. Anthocyanins have

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been categorized as the largest group of water-soluble pigments present in flowers.22 These natural

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pigments are a great interest in the food industry, due to their attractive colours and beneficial

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health effects, including anti-inflammatory, anti-artherogenic, anticancer, antidiabetic, and

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antioxidant activities.23 Humans consume a considerable amount of anthocyanin from plant-based

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food sources in daily life. C. spinosa showed the highest total anthocyanins content of 5.4 mg/g,

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followed by A. azurea (3.7 mg/g) and R. pseudoacacia (3.6 mg/g) (Table 2). M. sylvestris is

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characterized by the lower anthocyanins content (0.3 mg of cyaniding-3-glucoside equivalents/g).

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However, it is important to consider that anthocyanins often undergo degradative reactions during

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processing and storage.13

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Epidemiological study shows that the consumption of a carotenoids rich diet has been

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correlated with a lower risk for several diseases. Their beneficial effects are thought to be due to

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their role as antioxidants. The carotenoids that have been most studied in this regard are -carotene,

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lycopene, lutein, and zeaxanthin.24 C. spinosa and A. azurea showed the highest total carotenoids

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content with values of 3.4 and 3.0 mg/g, followed by R. pseudoacacia (2.8 mg/g).

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The results for fatty acid composition, total saturated fatty acids (SFA), monounsaturated fatty

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acids (MUFA), and polyunsaturated fatty acids (PUFA) of edible flower extracts are reported in

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Table 3. All samples are characterized by a content of fatty acids in the order SFA > PUFA >

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MUFA. The main SFA was palmitic acid (C16:0) (7.1-25.7% for R. officinalis and C. spinosa,

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respectively). ACS Paragon Plus Environment


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Linoleic acid and -linolenic acid are the two PUFAs identified in all flowers extracts. The

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extract of A. azurea is the major source of PUFAs with a total percentage of 13.3% compared to the

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other extracts (1.2-3.9%).

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Antioxidant activity. Considering that plant foods contain many different classes and types of

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antioxidants, the use of different methods that differ in their chemistry and in the way end points

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was recommended.25 For this reason in this study we applied different procedures, namely DPPH,

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ABTS, FRAP, and-carotene bleaching test. DPPH• is a stable free radical widely used as a method

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for studying the free radical-scavenging activity of natural antioxidants.26 The colour changes from

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purple to yellow and its absorbance decreases. Results of the DPPH• radical scavenging activity are

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given in Table 4. Both S. nigra and H. coronarium exhibited the highest radical scavenging activity

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with IC50 value of 1.4 g/mL followed by A. azurea and R. officinalis with IC50 values of 3.7 and

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4.7 g/mL, respectively (Figure 1). All these values are lower than those reported for positive

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control ascorbic acid (IC50 value of 5.0 g/mL). Interesting results were also obtained with C.

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intybus flowers (IC50 value of 7.4 g/mL). The scavenging capacities of the various edible flowers

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extracts for the ABTS+• radical were measured and compared. S. nigra and C. intybus showed the

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highest ABTS+• radical scavenging activity with IC50 values of 11.4 and 16.2 g/mL, respectively.

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Significant scavenging activity was also found with R. officinalis (IC50 value of 20.8 g/mL).

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In -carotene bleaching test the presence of extracts with antioxidant activity can hinder the

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extent of -carotene bleaching by neutralizing the linoleate free radical and other free radicals

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formed in the model.27 S. nigra, A. azurea and R. officinalis exerted the highest activity with IC50

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values of 2.2, 3.5 and 3.5 g/mL at 30 min of incubation and values of 6.1, 7.1 and 4.7 g/mL at 60

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min of incubation, respectively (Figure 2). A promising inhibition of lipid peroxidation was also

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found in M. officinalis that showed IC50 values of 8.5 and 10.5 g/mL at 30 and 60 minutes of

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incubation, respectively. The correlation analysis revealed that rutin was positively correlated with


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-carotene bleaching test (r2= 0.0952 at 30 min incubation and r2 = 0.1406 at 60 min of incubation).

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The FRAP assay measures the reducing potential of phytonutrients. Generally, the reducing

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properties are associated with the presence of compounds, which exert their action by breaking the

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free radical chain through donating a hydrogen atom.28 The ferric reducing ability of edible flower

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extracts expressed as FRAP values M Fe(II)/g are shown in Table 4. S. nigra flower extract had

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the highest FRAP value of 83.8 M Fe(II)/g followed by R. officinalis with FRAP value of 59.9 M

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Fe(II)/g. These values are better than those found with the positive control BHT (63.2 M Fe(II)/g).

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Correlation analysis evidenced a positive correlation between the total phenolic content FRAP test

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in all investigated species (r2= 0.442). In particular, both quercetin and rutin are positively

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correlated with FRAP assay.

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Based on the increase in the consumption of flowers, researchers have intensified their studies to

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assess the phytochemical profile and bioactivity with particular reference to antioxidant

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properties.29 The flowers of Antigonon leptopus, Bougainvillea glabra, Cosmos sulphurous, and

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Tagetes erecta largely used in Thailand for preparation of tea and salads were recently

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investigated.30 T. erecta extract showed a promising cellular antioxidant activity and total reducing

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capacity. However, the extract of A. leptopus demonstrated a more potent radical scavenging

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ability. In a study that included 19 Chinese edible flowers used as tea Paeonia lactiflora, P.

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suffruticosa, and Rosa rugosa exhibited the stronger antioxidant activity probably due their high

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polyphenolic contents.31

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A commercial S. nigra flowers extract was previously investigated for its potential antioxidant

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capacity.32 This extract exerted significantly DPPH• and OH• radical scavenging activity in a more

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effective manner than rutin. A significant inhibition of conjugated dienes formation and lipid

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peroxidation was also observed. C. intybus and S. nigra flowers from Czech Republic were

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previously investigated for their total phenolics and radical scavenging activity.33 The whole plant

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of A. azurea, M. sylvestris and C. intybus exerted radical scavenging effects in DPPH test, inhibition

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of hydrogen peroxide at concentration of 0.2 g/mL, and Fe2+-chelating activity greater than 70%.34 ACS Paragon Plus Environment


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More recently, M. sylvestris collected in Portugal was analysed for their total phytochemical

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content and antioxidant activity.35 Flowers extract showed an EC50 of 0.55, 0.17, and 0.11 mg/mL

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in DPPH, FRAP and -carotene bleaching test, respectively. The methanol extract of A. azurea and

301

C. intybus showed a radical scavenging activity of 0.02 and 1.11 mg/mL, respectively by using

302

DPPH assay.36 Both extracts exhibited also ferric reducing ability (EC50 of 0.01 and 0.57 mg/mL

303

for A. azurea and C. intybus, respectively), and protection of lipid peroxidation (EC50 of 0.02 and

304

0.45 mg/mL for A. azurea and C. intybus, respectively).

305

Quantified flavonoids are able to exert antioxidant activities in different in vitro assays (Table

306

6).37-48 In DPPH radical scavenging activity test quercetin and rutin exhibited IC50 values of 7.3 and

307

10.3 M, respectively.37A different trend in potency was observed in ABTS assay with IC50 values

308

of 19.62 and 2.83 M for quercetin and rutin, respectively.38,39 Luteolin that was found only in M.

309

sylvestris extract showed a promising protection of lipid peroxidation in -carotene bleaching test.

310 311

In vitro Hypoglycaemic Effects of Edible Flowers. The lowering of post-prandial

312

hyperglycaemia through the inhibition of key-enzymes linked to type 2 diabetes mellitus is a critical

313

therapeutic strategy used to control type 2 diabetes. In this study, the ability of edible flower

314

extracts to inhibit -amylase and -glucosidase enzymes are presented in Table 5.

315

All samples inhibited -amylase and -glucosidase in a concentration-dependent manner. Only

316

S. nigra was inactive against -amylase. M. sylvestris extract exhibited an interesting activity with

317

an IC50 value of 7.8 g/mL since the bioactivity is 6.4-fold lower than the commercial drug

318

acarbose (IC50 value of 50.0 g/mL). The same extract exhibited the highest -glucosidase with

319

IC50 value of 11.3 g/mL which was 3.1-fold lower than those reported for acarbose (IC50 value of

320

35.5 g/mL) (Figure 3). Plants from the Malva genus have been previously investigated for their

321

potential anti-diabetic effects. M. verticillata seed extract increased glucose uptake via the

322

activation of AMP-activated protein kinase (AMPK) in vivo.49 More recently, M. parviflora leaves


Page 15 of 28


323

n-hexane extract efficiently inhibited insulin resistance, lipid abnormalities and oxidative stress that

324

represent multiple targets involved in diabetes pathogenesis.50

325

A 1.7-fold higher bioactivity compared to acarbose was recorded with C. intybus flower extract

326

against -amylase (IC50 value of 29.3 g/mL). The whole plant of C. intybus demonstrated

327

hypoglycaemic activity in acute and chronic studies (125 mg/kg daily for 14 days to diabetic rats

328

attenuates serum glucose by 20%, triglycerides by 91% and total cholesterol by 16%).51 Authors

329

demonstrated that extract did not change insulin levels in serum, which ruled out the possibility that

330

C. intybus induced insulin secretion from pancreatic β-cells. However, C. intybus extract markedly

331

reduced the hepatic glucose-6-phosphatase activity causing a decrease in hepatic glucose

332

production, which in turn results in lower concentration of blood glucose in treated diabetic rats.

333

S. nigra extract showed a selective -glucosidase inhibitory activity with an IC50 value of 11.9

334

g/mL. The in vivo effects of S. nigra polyphenolic rich extract were previously investigated in

335

streptozocin-induced diabetes.52 Glycosylated hemoglobin and lipid peroxides values are reduced

336

by S. nigra extract. A promising IC50 value of 34.5 g/mL was also recorded for R. officinalis

337

extract.

338

Recently, the ability of different R. officinalis whole plant extract to inhibit -amylase was

339

investigated.53 IC50 values of 28.36, 34.11 and 30.39 g/mL for essential oil, ethyl acetate and

340

methanol extracts, respectively, were found. R. officinalis leaves significantly decreased the blood

341

glucose level at a dose of 10 g/day.54 Generally, the hypoglycaemic activity of our samples could be

342

ascribed to their total phenolic and flavonoid content.

343

Rutin, the main abundant flavonoid in our flower extracts, was positively correlated with both -

344

amylase (r2= 0.1201) and -glucosidase (r2= 0.1245). Flavonoids have been reported as potential

345

anti-diabetic agents by acting on various molecular targets and by regulating different signalling

346

pathways. Flavonoids showed to enhance insulin secretion, to regulate glucose metabolism in

347

hepatocytes, to reduce insulin resistance, to increase glucose uptake in skeletal muscle and adipose



348

Page 16 of 28

tissue, and to inhibit carbohydrate-hydrolysing enzymes.9,55,56

349

The effect of quercetin and its glycosylated conjugate rutin on -amylase and -glucosidase was

350

previously investigated (Table 6).57,58 Results revealed that both compounds inhibited the enzymes

351

in a dose-dependent manner. Rutin showed higher inhibition of α-amylase than quercetin (EC50

352

value of 0.043 vs 0.061 M). A similar trend was observed also against α-glucosidase (EC50 value

353

of 0.037 vs 0.038 M). Combinations of rutin and quercetin showed a higher carbohydrate-

354

hydrolysing enzymes inhibition than the flavonoids alone suggesting a synergistic effect. The most

355

promising combination resulted 75 % rutin and 25 % quercetin.

356 357

Luteolin, kaempferol and myricetin have greater inhibitory potency on the -glucosidase enzyme with IC50 values of 21, 12 and 5 M, respectively.58

358 359

Conclusions. The renewed interest in the use of flowers in cooking and to improve appearance

360

and nutritive value of meals has prompted the interest of researchers.59 In this context, the findings

361

of this study showed that eight commonly used edible flowers are a rich source of bioactive

362

compounds. S. nigra flower extract contains the highest amount of phenolic content and exerts

363

interesting antioxidant effects. M. sylvestris was demonstrated a very promising hypoglycaemic

364

activity in comparison to the commercial drug acarbose. Results support consumption of edible

365

flowers in diet as functional foods and encourage research to further in vivo study these matrixes to

366

develop more applications such as dietary supplements, functional ingredients, additives to prevent

367

the food oxidation, etc.

368 369

Sources of funding. The study was not specifically funded.

370 371

Conflict of interest statement. The authors declare that there are no conflicts of interest.

372 373

Acknowledgment. We thank Dr. N.G. Passalacqua of the Botany Department at the University ACS Paragon Plus Environment

Page 17 of 28


374

of Calabria (Italy) for samples identification. A voucher specimen for each specie has been retained

375

at the Herbarium of the University of Calabria (CLU).

376 377 378 379 380 381 382 383

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520 521 522 523 524


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525


Table 1. The usage of edible flowers and their extraction yield (%). Common Italian name Buglossa azzurra

Scientific name

Family

Anchusa azurea

Boraginaceae

Capparis spinosa

Capparidaceae

Cappero

Cichorium intybus

Asteraceae

Cicoria comune

Edible use

Traditional medicinal use

Violetlight blue Whiteviolet

Salad, soup, boil, fries Salad and preserved in vinegar and salt

Light blue

Salad, soup, boil, potage

Sulla comune Malva selvatica

Purple

Salad and fries with eggs, soup, boil

Antitussive, depurative, diaphoretic and diuretic Diuretic, antiseptic and protective of capillary vessels Laxative, diuretic, hypoglycaemic, depurative, disinfectant of urinary tract, and hepatoprotective Hypocholesterolemic and laxative

Violetwhite

Decoction

Emollient, and laxative

Fabaceae

Robinia

White

Liquor and jam, pancakes, honey,

Lamiaceae

Rosmarino

Purpleblue

Aromatic, on roast meat or potatoes

Caprifoliacee

Sambuco nero

White

Salad

Hedysarum coronarium

Fabaceae

Malva sylvestris

Malvacee

Robinia pseudoacacia

Rosmarinus officinalis

Sambucus nigra

Color

526 527 528 529 530


Antispasmodic, antiviral, cholagogue, diuretic, emetic, emollient, febrifuge, laxative, purgative and tonic. Antirheumatic, carminative and anti-eczema Hypolipidemic, antibacterial, antiviral, immunostimulatory, cardioprotective.

Extraction yield (%) 4.4 9.4

6.2

4.8 8.2

9.4

10.5

6.1


531

Table 2. Phytochemicals content of edible flowers extracts. Sample

A. azurea C. spinosa C. intybus H. coronarium M. sylvestris R. pseudoacacia R. officinalis S. nigra

532 533

Page 24 of 28

Phenols TCa

TCb

33.6 ± 0.9 33.6 ± 0.7 43.2 ± 0.2 48.4 ± 1.2 30.4 ± 0.8 75.2 ± 0.8 38.8 ± 1.3 228.5 ± 3.2

17.3 ± 0.2 19.4 ± 0.1 32.0 ± 0.2 38.6 ± 0.2 12.7 ± 0.1 45.3 ± 0.3 20.3 ± 0.1 64.2 ± 0.6

Rut ND 7.9 ± 0.8 20.3 ± 0.9 28.2 ± 0.8 ND 28.4 ± 0.9 3.7 ± 0.2 23.7 ± 0.6

Flavonoids HPLCc Que Lut ND ND 5.8 ± 0.2 ND ND ND 8.0 ± 0.6 ND ND 1.5 ± 0.1 ND ND ND ND 23.6 ± 0.7 ND

Kae 0.9 ± 0.03 0.5 ± 0.02 ND 0.5 ± 0.1 0.8 ± 0.05 2.4 ± 0.2 4.2 ± 0.3 1.0 ± 0.04

Myr 1.1 ± 0.02 0.6 ± 0.1 3.5 ± 0.6 ND 0.8 ± 0.02 2.1 ± 0.3 3.0 ± 0.4 2.2 ± 0.4

Anthocyanins TCd

Carotenoids TCd

3.7 ± 0.7 5.4 ± 1.9 1.2 ± 0.5 0.7 ± 0.1 0.3 ± 0.1 3.6 ± 1.0 1.1 ± 0.6 0.9 ± 0.2

3.0 ± 0.2 3.4 ± 0.9 2.5 ± 0.2 2.7 ± 0.4 1.3 ± 0.1 2.8 ± 0.6 1.5 ± 0.6 1.7 ± 0.3

Data represent means ± SD (standard deviation) (n= 3). Kae: kaempferol; Myr: myricetin; Lut: luteolin; Que: quercetin; Rut: rutin. TC: total content. amg of chlorogenic acid equivalents /g of extract; bmg of quercetin equivalents/g of extract; cmg/g of extract; dmg/g of extract. ND: not detected.

534 535 536 537 538 539 540 541 542


Page 25 of 28

543


Table 3. Fatty acids composition of edible flowers extracts. Fatty acid C9:0 C14:0 C15:0 C16:0 C17:0 C18:0 C18:1 n-9 C18:2 n-6 C18:3 n-3 C19:0 C20:0 C22:0 C23:0 C24:0

544 545 546 547

Pelargonic acid Myristic acid Pentadecanoic acid Palmitic acid Heptadecanoic acid Stearic acid Oleic acid Linoleic acid -Linolenic acid Nonadecanoic acid Arachidic acid Behenic acid Tricosanoic acid Lignoceric acid ∑SFA ∑MUFA ∑PUFA

AA 11.6 ± 1.0 0.6 ± 1.0 14.3 ± 1.0 5.5 ± 1.0 5.8 ± 1.0 6.0 ± 1.0 7.3 ± 1.0 tr 1.7 ± 1.0 1.8 ± 1.0 35.5 5.8 13.3

CA 1.9 ± 1.0 1.6 ± 1.0 25.7 ± 1.0 tr 3.3 ± 1.0 0.4 ± 1.0 0.7 ± 1.0 0.5 ± 1.0 2.6 ± 1.0 5.9 ± 1.0 tr 41.0 0.4 1.2

CI tr 0.7 ± 1.0 1.8 ± 1.0 18.5 ± 1.0 0.4 ± 1.0 1.7 ± 1.0 1.0 ± 1.0 1.9 ± 1.0 0.6 ± 1.0 0.4 ± 1.0 0.8 ± 1.0 1.5 ± 1.0 0.3 ± 1.0 26.1 1.0 2.5

Abundance % HE MA 4.0 ± 1.0 6.4 ± 1.0 0.8 ± 1.0 1.0 ± 1.0 1.5 ± 1.0 7.7 ± 1.0 22.4 ± 1.0 4.4 ± 1.0 3.6 ± 1.0 0.4 ± 1.0 1.9 ± 1.0 1.3 ± 1.0 0.8 ± 1.0 0.7 ± 1.0 3.1 ± 1.0 tr 1.2 ± 1.0 1.2 ± 1.0 2.1 ± 1.0 1.0 ± 1.0 0.2 ± 1.0 22.8 34.5 0.4 1.9 2.0 3.9

RB 2.4 ± 1.0 9.1 ± 1.0 0.7 ± 1.0 2.3 ± 1.0 0.7 ± 1.0 2.7 ± 1.0 0.6 ± 1.0 0.8 ± 1.0 0.5 ± 1.0 0.3 ± 1.0 15.8 0.7 3.3

RS 3.7 ± 1.0 0.4 ± 1.0 7.1 ± 1.0 0.3 ± 1.0 2.2 ± 1.0 1.1 ± 1.0 1.5 ± 1.0 1.9 ± 1.0 0.5 ± 1.0 0.4 ± 1.0 14.6 1.1 3.4

SN 1.7 ± 1.0 1.2 ± 1.0 17.7 ± 1.0 1.5 ± 1.0 0.8 ± 1.0 1.3 ± 1.0 0.2 ± 1.0 1.8 ± 1.0 22.6 1.5 2.1

Data represent means ± SD (standard deviation) (n= 3); AA: Anchusa azurea; CA: Capparis spinosa; CI: Cichorium intybus; HE: Hedysarum coronarium; MA: Malva sylvestris; RB: Robinia pseudoacacia; RS: Rosmarinus officinalis; SN: Sambucus nigra. SFA: Saturated Fatty Acids; MUFA: Monounsaturated Fatty Acids; PUFA: Polyunsaturated fatty acids. tr: < 0.1%. -: not detected.

548 549 550 551 552 553



554

Table 4. Antioxidant activity of edible flowers extracts. Edible flowers

Anchusa azurea Capparis spinosa Cichorium intybus Hedysarum coronarium Malva sylvestris Robinia pseudoacacia Rosmarinus officinalis Sambucus nigra Positive control BHT Ascorbic acid Propyl gallate

555 556 557 558 559 560

Page 26 of 28

-carotene bleaching IC50 (g/mL)

DPPH IC50 (g/mL)

ABTS IC50 (g/mL)

FRAP test

3.7 ± 0.4 41.7 ± 1.1*** 7.4 ± 0.7 1.6 ± 0.8 124.6 ± 2.3*** 174.8 ± 4.8*** 4.7 ± 0.2 1.4 ± 0.3

30.2 ± 1.4*** 139.3 ± 3.9*** 16.2 ± 1.4*** 28.5 ± 2.2*** 108.9 ± 5.7*** 197.5 ± 4.2*** 20.8 ± 1.7*** 11.4 ± 1.2***

30 min 3.5 ± 0.8 36.8 ± 2.4*** 16.2 ± 1.2*** 17.3 ±1.5*** 8.5 ± 2.6*** 10.7 ± 1.2*** 3.5 ± 1.7 2.2 ± 0.6

60 min 7.1 ± 0.8*** 63.9 ± 2.9*** 21.2 ± 1.3*** 18.7 ± 1.6*** 10.5 ± 1.2*** 32.9 ± 2.5*** 4.7 ± 0.7 6.1 ± 0.8

26.1 ± 3.8*** 20.8 ± 4.1*** 52.4 ± 2.1*** 19.1 ± 1.9*** 6.3 ± 1.1*** NA 59.9 ± 4.9 83.8 ± 4.8***

5.0 ± 0.8 -

1.7 ± 0.3 -

1.0 ± 0.04

1.0 ± 0.05

63.2 ± 4.3 -

M Fe(II)/g

Data are expressed as means ± S.D. (n= 3). NA: not active. DPPH Radical Scavenging Activity Assay: One-way ANOVA ***p

Edible Flowers: A Rich Source of Phytochemicals with Antioxidant and

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