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DICONO DI NOI



Polyphenolic Compounds and Antioxidant Activity of Cold-Pressed Seed Oil from Finola Cultivar of Cannabis Sativa L.

La ricerca dell'università di Messina sull'olio de La Canaperia Italiana, pubblicata su una rivista internazionale nel 2016.
La varietà Finola che spremiamo è molto ricca di polifenoli e molti altri antiossidanti.

PHYTOTHERAPY RESEARCH

Phytother. Res. (2016)

Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.5623

Copyright © 2016 John Wiley & Sons, Ltd.

Antonella Smeriglio,1,2 Enza M. Galati,1 Maria T. Monforte,1 Francesco Lanuzza,3 Valeria D’Angelo1 and Clara Circosta1*
1Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali, University of Messina, Messina, Italy
2Fondazione Prof. Antonio Imbesi: Borsa di Ricerca Scuola di Specializzazione in Farmacognosia, University of Messina, Messina, Italy
3Dipartimento di Economia, University of Messina, Messina, Italy

The aim of this study was to characterize the polyphenolic compounds and antioxidant activity of cold-pressed seed oil from Finola cultivar of industrial hemp (Cannabis sativa L.). Several methodologies have been employed to evaluate the in vitro antioxidant activity of Finola hempseed oil (FHSO) and both lipophilic (LF) and hydrophilic fractions (HF). The qualitative and quantitative composition of the phenolic fraction of FHSO was performed by HPLC analyses. From the results is evident that FHSO has high antioxidative activity, as mea-sured by DPPH radical (146.76 mmol of TE/100 g oil), inhibited β-carotene bleaching, quenched a chemically generated peroxyl radical in vitro and showed high ferrous ion chelating activity. Reactivity towards 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical cation and ferric-reducing antioxidant power values were 695.2 μmol of TE/100g oil and 3690.6 μmol of TE/100 g oil respectively. FHSO contains a significant amount of phenolic compounds of which 2780.4 mg of quercetin equivalent/100 g of total flavonoids. The whole oil showed higher antioxidant activity compared with LF and HF. Our findings indicate that the significant antioxidant properties shown from Finola seed oil might generally depend on the phenolic compounds, especially flavonoids, such as flavanones, flavonols, flavanols and isoflavones. Copyright © 2016 John Wiley & Sons, Ltd.

Keywords: Finola hempseed oil; Cannabis sativa L; antioxidant activity; flavonoids; polyphenols

INTRODUCTION

Industrial hemp (non-drug Cannabis sativa L. Family Cannabaceae) is a versatile plant that can be grown for its fibre, seed or oil. The Finola cultivar of industrial hemp is designed specifically as a, dioecious, oilseed variety, developed in Finland, known to contain less than 0.2% of 9-tetrahydrocannabinol in the whole plant. Finola seeds are an excellent source of oil for human foods. The typical seed weight for Finola is be-tween 12 and 15 g/1000 seeds, with smaller seed in the northern latitudes (>50°N) and larger seed in the south (<50°N). Finola, when sown between mid-May and early June near latitude 50°N, emits the seedlings 5–7 days after sowing and first true leaves, days 7–10. The beginning of flowering is 25 to 30 days and peak time of pollination at days 40–50. The 60–80% mature seeds are 90 to 100 days, and harvest time is 100 to 120 days after sowing. Hemp seed oil has been used as a food/medicine in China for at least 3000 years (de Padua et al., 1999; Callaway, 2004). It is considered to be one of the few seed oils that contains about 80% polyunsaturated fatty acids in a perfect 3:1 ratio of Omega-6 to Omega-3, which is suggested as optimal for human nutrition (Scorletti and Byrne, 2013). Hemp oil, in addition to its nutritional value, has demon-strated positive health benefits (Kaul et al., 2008; Prociuk et al., 2008). An unfortunate paradox of hempseed oil resides within its unsaturation, as this structural feature makes it both highly nutritious and chemically unstable (DeMan, 2000). Degradative pro-cesses in unsaturated oils were originally thought to begin with oxidation of the chemical double bond. Differences of stability between highly unsaturated oils can be observed and are at least partially attributable to the proportions of ancillary components within their seeds (Parker et al., 2003),which are co-expressed with the oil. These components include phenolic pigments that act as anti-oxidants, or specific anti-oxidants such as the tocopherols. Edible oils rich in natural antioxi-dants may play a role in reducing the risk of chronic diseases. The pathology of numerous chronic diseases involves oxidative damage to cellular components. Reactive oxygen species, capable of causing damage to DNA, lipids, and proteins, have been associated with carcinogenesis, coronary heart disease, and many other health problems related to advancing age (Marnett, 2000; Uchida, 2000). Minimizing oxidative damage may well be one of the most important approaches to the primary prevention of chronic diseases and ageing-associated health problems. Since antioxidants termi-nate direct reactive oxygen species attacks and radical-mediated oxidative reactions, appear to be of primary importance in the prevention of these diseases and health problems. Antioxidants have been detected in a

number of plants and foods, including seed oils (Kalt et al., 1999; Iauk et al., 2014; Munro et al., 2015; Santana et al., 2015). Hemp seed oil shows excellent oxidative stability suggesting the possible presence of phenolic compounds that act as antioxidants in the cold-pressed seed oil (Abuzaytoun and Shahidi, 2006). The present study was conducted to evaluate the polyphenolic profile and antioxidant properties of cold-pressed Finola hempseed oil (FHSO) in order to determine its potential application in health promotion and disease prevention from oxidative damages.

MATERIALS AND METHODS

Chemicals. The 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), 2,2′-azobis (2-methylpropiona-midine) dihydrochloride (AAPH); fluorescein disodium salt; sodium phosphate dibasic (Na2HPO4); potassium phosphate monobasic (KH2PO4); random methylated β-cyclodextrin; Folin–Ciocalteu reagent; sodium carbon-ate; Iron(III) chloride hexahydrate; Iron(II) chloride tetrahydrate; sodium acetate; aluminium chloride; 2,4,6-Tris(2-pyridyl)-S-triazina; potassium peroxydisul fate; 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS+); β-carotene; ethylene-diaminetetraacetic acid (EDTA); 1,1-diphenyl-2-picry lhydrazyl radical (DPPH); Tween 40; linoleic acid, butylhydroxytoluene (BHT); and ferrozine were pur-chased from Sigma-Aldrich (Milan, Italy). Methanol and acetic acid were HPLC-grade and were purchased from Merck (Darmstadt, Germany). All polyphenolic reference compounds and tocopherols were purchased from Extrasynthese (Genay, France). All other chemicals used were analytical grade and obtained from either Sigma-Aldrich or Merck.

Plant seed oil and sample preparation. Oil of food grade quality, cold-pressed from seeds of Finola cultivar of indus-trial hemp (Cannabis sativa L.), was provided by Scotto & DAulerio (Sativa Molise, Italy). The FHSO sample prep-aration was carried out according to Huang et al. (2002) with some modifications. Briefly, 1 g of the cold-pressed seed oil was dissolved in 1 mL of acetone. An aliquot of sample solution was appropriately diluted in a random methylated β-cyclodextrin solution at 7% in acetone/water (50:50, v/v) to enhance the solubility of lipo-philic fraction (LF) in aqueous solution and used for the as-sessment of antioxidant activity. The in vitro antioxidant properties of both (LF and hydrophilic fraction (HF) were also determined. Separation of LF and HF was performed by the method of Minioti and Georgius (2010) with some modifications in the following way: 10 g of FHSO sample was diluted 1:1 (v:v) in n-hexane. Ten millilitres of metha-nol : water 8:2 (v:v) mixture was added for three times, and extraction procedure was carried out vortexing for 3 min. After centrifugation at 5000 rpm for 5 min at 4°C, the two fractions were separated. For determination of the phenolic profile by reversed-phase HPLC coupled with diode array and fluorescence detection (RP-HPLC-DAD-FLU), the phenolic fraction of Finola seed oil has been iso-lated by liquid–liquid extraction according to Montedoro et al. (1992) with some modifications. Briefly, 10 g of the

cold-pressed seed oil was added with 20 mL of methanol/water mixture (8:2 v/v) for five times. The frac-tions were collected and concentrated in vacuum under a gentle stream of nitrogen at < 35°C until syrup consistency. The extract was added with 10 mL of acetonitrile and washed thrice with 10 mL of hexane in order to remove the lipidic fraction. The sample was then brought to dry-ness with a gentle stream of nitrogen. Before injection, the phenolic extract was solubilized with 10 mL of metha-nol and filtered through a PTFE syringe filter 0.2 μm.

Determination of polyphenolic   profile by RP-HPLC-DAD-FLU. The determination of polyphe-nol compounds was performed as described by Garrido et al. (2008) with some modifications. An Agilent high-performance liquid chromatography system (1100 se-ries) equipped with a UV–Vis photodiode-array detec-tor (DAD) (G1315) and a fluorescence detector (G1321) coupled with a control system (G1323) equipped with an LC pump (G1312) and an auto-injector (G1313) was used. Separation was performed

consisting of solvent A (water/acetic acid, 97:3, v/v), sol-vent B (methanol) was applied at a flow rate of 1.0 mL/ min as follows: 0–3 min 100% A, 3–9 min 97% A, 9– 24 min 88% A, 24–30 min 80% A, 30–33 min 80% A, 33–43 min 70% A, 43–63 min 50% A, 63–66 min 50% A, 66–76 min 40% A, 76–81 min 40% A, 81–110 min 100% B, 110–120 min 40% A, 120–125 min 60% A and equilibrated 15 min for a total run time of 140 min. Sam-ples were filtered before through a 0.22 μm ptfe filter, and the injection volume was 20 μL. The detection condi-tions were set at 270 nm for phenolic acids and flavanones, 330 and 370 nm for flavonols, isoflavones and flavones. The UV spectra of the different compounds were re-corded from 200 to 400 nm. The wavelengths used for fluorescence detection of flavan-3-ols were λex: 276 nm and λem: 316 nm. Data acquisition was performed using ChemStation A.10.01 software (Agilent, USA). The iden-tification of the phenolics was made according to UV-visible spectra, retention time, co-chromatography with commercially available standards. Quantification was car-ried out by external standard calibration curves (Table 1).

Validation of analytical method. The HPLC method has been validated according to International Council for Harmonisation of Technical Requirements for Pharma-ceuticals for Human Use Harmonised Tripartite Guide-line 2005 (International Conference on Harmonisation of Technical Requirements for Registration of Pharma-ceuticals for Human Use, 2005), in terms of selectivity, linearity, limit of detection and quantitation and preci-sion. A good analytical method should be able to mea-sure accurately the analyte in the presence of suspected interferences such as its own degradation products and any co-eluting compounds. The chromato-graphic separation of polyphenols do not show any overlap of each other (base-line separations); also, as highlighted into chromatogram, no interferences are found at the retention times of chemicals from matrix constituents. A test solution with different concentra-tions of pure reference standards was prepared and analysed by using the analytical parameters described earlier. The detection limit was calculated as the amount of chemicals that resulted in a peak three times higher with respect to the baseline noise. The linear calibration range, regression equation, quantification limit and pre-cision, express as relative standard deviation percent (RSD%), of compounds of interest were calculated and the results were listed in Table 2.

POLYPHENOLIC COMPOUNDS AND ANTIOXIDANT ACTIVITY OF FHSO

Table 1. Phenolic compounds identified in methanol extract of Finola Hemp Seed Oil (FHSO). Date are expressed as μg/100 g of FW and as means ± SD (n = 3) of three independent experiments

Compounds

Peak

Rt (min)

λ (nm)

Methanol extract

Phenolic acids

 

 

 

 

 

Hydroxybenzoic acids

 

 

 

 

 

 

 

 

 

 

 

Gallic acid

1

6.68

268

0.031

± 0.0021

Protocatechuic acid

2

13.20

258; 293

0.306

± 0.0120

4-Hydroxybenzoic acid

3

23.25

253

0.197

± 0.0174

Vanillic acid

5

31.19

262; 291

0.078

± 0.0028

Hydroxycinnamic acids

 

 

 

 

 

Chlorogenic acid

6

31.756

291; 319

0.103

± 0.041

trans-p-Cumaric acid

8

42.575

309

0.278

± 0.0124

Flavonoids

 

 

 

 

 

Flavanones

 

 

 

 

 

Eriodictyol-7-O-glucoside

9

47.785

284; 327

0.530

± 0.0310

Naringenin-7-O-glucoside

10

53.843

283; 332

1.445

± 0.065

Eriodictyol

15

60.774

287

0.929

± 0.0540

Naringenin

17

66.997

289

29.744

± 1.2350

Naringin

18

69.423

284; 330

3.156

± 0.2135

Flavonols

 

 

 

 

 

Quercetin-3-O-rutinoside

11

55.292

256; 357

3.908

± 0.1752

Quercetin-3-O-glucoside

12

55.596

254; 354

0.840

± 0.0421

Kaempferol-3-O-glucoside

13

60.444

266; 349

6.501

± 0.2256

Kaempferol-3-O-rutinoside

14

60.614

266; 350

3.434

± 0.1830

Isoflavones

 

 

 

 

 

Daidzein

16

63.697

248; 302

1.574

± 0.0885

Genistein

19

69.632

261; 332

1.528

± 0.0655

Flavones

 

 

 

 

 

Apigenin

20

75.484

268; 330

1.234

± 0.0745

Flavanols

 

 

 

 

 

Catechin

4

25.277

279

5.284

± 0.2542

Epicatechin

7

35.944

279

10.157

± 0.6548

 

 

 

 

 

 

Total phenolic content. Total phenolic content was deter-mined according to the Folin–Ciocalteu method with some modifications (Trombetta et al., 2010). Briefly, 50 μL of FHSO (range 10–1.25 mg/mL), LF (range 100–12.5 mg/ml) and HF (range 1000–125 mg/mL) sample solution was added to Folin–Ciocalteu reagent (500 μL) followed by de-ionized water (450 μL). After 3 min, sodium carbonate (500 μL, 10% w/v) was added; samples were left in the dark at room temperature for 1 h vortexing every 10 min, and the absorbance was then measured at 785 nm using a UV-VIS Spectrophotometer (Shimadzu UV-1601). Results were expressed as milligrams of gallic acid equivalents/100 g of fresh weight (FW).

Determination of total flavonoids. Total flavonoids were de-termined according to Kumaran et al. (2007) with some modifications. Briefly, aliquots (0.2 mL) of FHSO (range 12.5–1.562 mg/mL), LF (range 25–3.125 mg/mL) and HF (range 500–62.5 mg/mL) samples were mixed with 0.2 mL

of AlCl3 (2 mg/mL) and 1.2 mL of sodium acetate (50 mg/ mL). Absorbance at 440 nm was recorded after 2.5 h. Flavo-noid content is expressed as milligrams of quercetin equivalents/100 g of FW.

Tocopherol profile. The n-hexane solution of the cold-pressed Finola seed oil was used to evaluate the α- β-γ- and δ-tocopherol concentrations in the oil. The determination was carried out by normal phase HPLC with spectrofluorimetric detection; HPLC separation of tocopherols was performed using a Chromspher 5 Si (Varian), 250 × 2 mm i.d. with 5 μm particle size, at room temperature. The mobile phase was composed of n-hexane: isopropanol (99.8:0.2, v/v), and the flow rate was 0.3 mL/min. The wavelengths for fluorescence de-tection were λex: 295 nm and λem: 330 nm. Qualitative analysis of tocopherols α, β, γ and δ was carried out com-paring the chromatographic behaviour and the UV exci-tation and emission spectra of the components with pure standard solutions. Quantification was carried out by external standard calibration curves.

  1. ABTS.+ radical cation scavenging activity. The ABTS assay was performed as described by Morabito et al. (2010) with some modifications. Briefly, the reaction mixture was prepared by mixing 4.3 mM potassium per-sulfate and 1.7 mM ABTS solution 1:5 (v/v) following by 12–16 h incubation in the dark at room temperature.

A. SMERIGLIO ET AL.

 

Table 2. Parameters for method validation

Polyphenols

 

 

Parameters

 

 

 

 

 

 

 

 

 

 

 

 

R.S.D1

R.S.D (%),

 

 

 

Calibration

Coefficient

(%),n = 6

n = 6

 

 

 

range (μg/L)

regression R2

within-day

between-day

LOD2 (μg/L)

LOQ3 (μg/L)

 

 

 

 

 

 

 

Gallic acid

6.25–100

0.9991

0.111

3.273

1.080

3.497

Protocatechuic acid

6.25–100

0.9990

0.652

1.677

0.953

3.843

4-Hydroxybenzoic acid

6.25–100

0.9990

3.043

5.628

1.037

3.123

Vanillic acid

6.25–100

0.9993

0.487

2.502

1.220

2.732

Chlorogenic acid

6.25–100

0.9990

2.346

5.009

1.045

3.150

trans-p-Cumaric acid

6.25–100

0.9991

0.806

2.656

1.366

3.219

Eriodictyol-7-O-glucoside

6.25–100

0.9997

0.295

2.163

0.670

2.235

Naringenin-7-O-glucoside

6.25–100

0.9991

0.716

2.003

1.049

3.497

Eriodictyol

6.25–100

0.9994

2.085

5.671

1.953

4.843

Naringenin

6.25–100

0.9990

0.546

2.619

1.037

3.123

Naringin

6.25–100

0.9993

0.226

2.071

1.220

3.732

Quercetin-3-O-rutinoside

6.25–100

0.9990

0.235

1.762

0.450

1.250

Quercetin-3-O-glucoside

6.25–100

0.9991

0.392

2.206

0.366

1.219

Kaempferol-3-O-glucoside

6.25–100

0.9994

0.165

1.103

0.359

1.170

Kaempferol-3-O-rutinoside

6.25–100

0.9997

2.402

3.936

0.654

2.179

Daidzein

6.25–100

0.9991

0.111

1.440

0.949

2.947

Genistein

6.25–100

0.9995

0.058

1.149

1.053

3.743

Apigenin

6.25–100

0.9990

0.313

2.188

0.872

2.423

Catechin

6.25–100

0.9993

0.542

2.361

0.452

1.502

Epicatechin

6.25–100

0.9990

0.423

2.729

0.745

2.150

1Relative standard deviation.

2Limit of detection.

3Limit of quantification.

Before use, the ABTS+ solution was diluted with phos-phate buffer (pH 7.4) in order to obtain an absorbance of 0.7 ± 0.02 at 734 nm. Aliquots of FHSO (range 50– 6.25 mg/mL), LF (range 100–12.5 mg/mL) and HF (range 500–62.5 mg/mL) sample solution (50 μL) were added to 1 mL of ABTS+ solution and incubated in the dark at room temperature for 6 min; the absorbance was then re-corded at 734 nm using an UV-VIS Spectrophotometer (Shimadzu UV-1601). Trolox was used as reference com-pound (15.5–250 μM), and results were expressed as μmoles of trolox equivalents (TE)/100 g of FW.

DPPH radical scavenging activity. The DPPH free radical scavenging activity was performed according to Ramadan and Morsel (2006) for FHSO and LF samples and according to Rapisarda et al. (1999) for HF sample with some modifi-cations. Freshly prepared DPPH toluene (1.5 mL) or meth-anol solution (10 4M), respectively, was mixed with 37.5 μL of FHSO (range 1–0.125 mg/mL), LF (range 500–62.5 mg/ mL) and HF (range 1000–125 mg/mL) sample, and the mix-ture was vortexed for 10 s at room temperature. The de-crease in absorption at 517 nm, against a blank of pure toluene or methanol without DPPH, was measured after 20 min using an UV-VIS Spectrophotometer (Shimadzu UV-1601). The Trolox was used as reference compound and results were expressed as mmoles of TE/100 g of FW.

β-carotene bleaching method. The β-carotene bleaching as-say was performed according to Aidi Wannes et al. (2010) with some modifications. β-Carotene (0.4 mg) dissolved in 0.4 mL of chloroform and linoleic acid (40 μL) were mixed

with 400 μL of Tween-40. Chloroform was removed at 40° C under a vacuum. The resulting mixture was diluted with 50 mL of oxygenated water. Aliquots of this emulsion (5 mL) were mixed with 0.2 mL of FHSO (range 25– 3.125 mg/mL), LF (range 50–6.25 mg/mL) and HF (range 500–62.5 mg/mL) sample at several concentrations. An emulsion without β-carotene was used as control. The reac-tion mixture was initially recorded at the starting time (t = 0) at 470 nm and then incubated at 50°C for 120 min re-cording the absorbance every 20 min. The BHT was used as reference compound and results were expressed as mmoles of BHT/100 g of FW.

Chelating capacity on Fe2+. The Fe2+chelating capacity was evaluated as described by Zhao et al. (2006) with some mod-ifications. Briefly, 0.05 mL of FeCl24H2O solution (1.8 mM) was added to 0.1 mL of each FHSO (range 6.25–0.781 mg/ mL), LF (range 12.5–1.562 mg/mL) and HF (range 500– 62.5 mg/mL) sample solubilized in methanol/water mixture (2:1, v/v) and incubated at room temperature for 5 min. A mixture of 0.1 mL of ferrozine solution (4 mM) was added to the reaction mixture, and sample volume was adjusted to 3 mL with deionized water vortexing and incubating for 10 min at room temperature. The absorbance was then read at 562 nm using an UV-VIS Spectrophotometer (Shimadzu UV-1601). The EDTA was used as reference compound, and results were expressed as μmoles of EDTA equivalent/100 g of FW.

Ferric reducing antioxidant power assay. The ferric reducing antioxidant power (FRAP) assay was performed according to Benzie and Strain (1996) with some modifications. The fresh working FRAP reagent was pre-pared daily by mixing acetate buffer (300 mM, pH 3.6), 2,4,6-Tris(2-pyridyl)-S-triazina solution (10 mM in 40 mM HCl) and FeCl36H2O solution (20 mM) was prepared at the ratio 10:1:1 (v/v/v), respectively. The reagent was warmed to 37 °C, and the initial absorbance was read. After addition of 50 μL of FHSO (range 10–1.25 mg/mL), LF (range 25–3.125 mg/mL) and HF (1000–125 mg/mL) sam-ple solution to the FRAP reagent, the absorbance was measured after 4 min at 20°C at wavelength of 593 nm by an UV-VIS Spectrophotometer (Shimadzu UV-1601). The antioxidant capacity of sample was determined by a calibration curve, using Trolox as reference compound (0.1–1 mM). Results were expressed in μmoles of TE/100 g of FW.

Oxygen radical absorbance capacity assay. Antioxidant activity of sample against 2,2′-azobis(2-amidinopropane)-dihydrochloride (AAPH) peroxyl radical was chemically examined by the oxygen radical absorbance capacity (ORAC) method according to Dávalos et al. (2004) with some modification. Briefly, 20 μL of FHSO (range 2– 0.25 mg/mL), LF (range 8–1 mg/mL) and HF (range 20– 2.5 mg/mL) sample diluted in 75–mM phosphate buffer solution pH 7.4 was mixed with 120 μL of fluorescein fresh daily solution (117 nM). After a pre-incubation time of 15 min at 37°C, 60 μL of freshly prepared AAPH solution (40 mM) was rapidly added. The fluorescence was re-corded every 30 s for 150 min (λex: 485; λem: 520) using a Fluorescence Plate Reader (FLUOStar Omega, BMG LABTECH), and the decrease in fluorescence was moni-tored. A blank using phosphate buffer instead of sample and calibration solutions of Trolox (12.5–50 μm) were also included in each assay. The ORAC value was calculated using the area under the fluorescence decay curves and was expressed as μmoles of TE/100 g of FW.

STATISTICAL ANALYSIS

All analyses were performed in triplicate and repeated three times. The data were recorded as mean ± standard deviation (SD) and analysed by one-way ANOVA using a SigmaPlot 12.0 software. P-values < 0.05 were considered statistically significant.

RESULTS AND DISCUSSION

There are increasing evidences that as antioxidants, poly-phenols may protect cell constituents against oxidative damage and, therefore, limit the risk of various degenera-tive diseases associated with oxidative stress (Pandey et al., 2009). Thus, antioxidant capacity is widely used as a parameter to characterize foods or medicinal plants and their bioactive components. In this study, the qualitative and quantitative composition of the phenolic fraction of FHSO was performed by reverse phase high-performance liquid chromatography (RP-HPLC), and the antioxidant activities of FHSO and of both LF and HF have been evaluated in a series of in vitro tests: DPPH and AB

assays, β-carotene bleaching method, ORAC assay, reduc-ing power and metal chelating activities.

Determination of polyphenolic profile by

RP-HPLC-DAD-FLU

The phenolic fraction of FHSO has been isolated by liquid–liquid extraction using methanol/water (80:20 v/v). The methanol extract is been analysed by reversed-phase HPLC using gradient elution with methanol–aqueous acetic acid. Detection of individual polyphenols that belong to one constituent class is been based on measurement of UV-Vis absorption at 270, 330 or 370 nm. Because some phenolic com-pounds show several absorption maxima, the use of si-multaneous multiple UV (photodiode array) is recommended for identification. Figure 1 and Table 1 show the compounds of the specific class of polyphe-nols identified and quantified in the methanol extract of FHSO. The flavonoids are contained in greater quantities compared with phenolic acids. Flavonoids most represented in the phenolic fraction of Finola seed oil are flavanones such as naringenin, flavanols such as epicatechin, flavonols and isoflavones such as kaempferol-3-O-glucoside and daidzein, respectively. Phenolic compounds may occur as free aglycones or glycoside derivatives. Thus, the methanol extracts be-fore acidic hydrolysis and after acidic hydrolysis were analysed. The presence of daidzein and genistein is verified in FHSO methanol extract before hydrolysis (Fig. 1) and after hydrolysis (Fig. 2). The analysis re-sult suggests that isoflavones daidzein and genistein are contained in cold-pressed Finola seed oil mainly as glycoside derivatives. HPLC analysis showed that phenolic fraction of FHSO contains significant amounts of flavonoids, especially as free aglycones, that have different phenolic structures and may di-rectly react with and quench stable DPPH and cation ABTS.+ radicals, absorb oxygen radicals generated by AAPH (ORAC), form chelating complex with transition metals and reduce ferric ions, providing hu-man health benefits.

Total phenolic content and determination of total flavonoids

Table 3 shows the total phenolic content and total flavo-noids in FHSO and in both LF and HF expressed as mil-ligrams of gallic acid equivalents or as milligrams of quercetin equivalents per 100 g of FW, respectively. The content of total phenolic compounds in FHSO was signif-icantly higher (267.5 ± 8.84) than that from LF (67.6

± 3.10) and HF (13.2 ± 0.21). Total flavonoids have major concentration in the FHSO (2780.4 ± 133.06) than the LF (1027.3 ± 78.01) and HF (157 ± 1.10). The HF results to be the less rich in total polyphenols and flavonoids. This is likely because of the higher content in FHSO of lipophilic polyphenols, mainly flavonoids, as free aglycones.

Tocopherol Profile

The cold-pressed Finola seed oil showed a total tocoph-erol contents corresponding to 114.04 mg/kg of oil. Thelowest in β-tocopherol (0.64 mg/kg) and the highest in γ-tocopherol (91.57 mg/kg) while α- and δ-tocopherol were 19.74 and 2.09 mg/kg, respectively. γ-Tocopherol is the major form of vitamin E in many plant seeds, and recent studies indicate that γ-tocopherol may be im-portant to human health (Jiang et al., 2001).

(2016)

POLYPHENOLIC COMPOUNDS AND ANTIOXIDANT ACTIVITY OF FHSO

Table 3. Antioxidant properties, total content of phenolic compounds and flavonoids of Finola hempseeds oil (FHSO), lipophilic (LF) and hydrophilic fractions (HF)

 

 

DPPH

TEAC

FRAP

Chelating activity

Total Phenols

Total Flavonoids

 

 

 

 

 

 

 

 

 

 

mmoles TE1/

μmoles TE1/

μmoles TE1/

μmoles EDTA/

mg GAE3/

mg QRC4/

Samples

 

100 g FW2

100 g FW2

100 g FW2

100 g FW2

100 g FW2

100 g FW2

 

 

 

 

 

 

 

 

 

 

 

 

 

 

FHSO

146.76

± 2.840

695.2

± 45.83

3690.6

± 15.25

158.2

± 2.11

267.5

± 8.84

2780.4

± 133.06

LF

0.125

± 0.0037

326.1

± 33.68

2092.6

± 64.41

42.4

± 2.05

67.6

± 3.10

1027.3

± 78.01

HF

0.038

± 0.0032

94.6

± 3.9

74.2

± 3.47

5.6

± 0.14

13.2

± 0.21

157

± 1.10

1TE,Trolox equivalents.

2FW, fresh weight.

3GAE, gallic acid equivalents.

4QRC, quercetin equivalents. Data are expressed as means ± SD (n = 3) of three independent experiments.

DPPH and ABTS.+ scavenging activity

Antioxidant properties, especially radical scavenging ac-tivities, are very important because of the deleterious role of free radicals in biological systems. These are highly reactive species, capable in the nucleus, and in the membranes of cells of damaging biologically rele-vant molecules such as DNA, proteins, carbohydrates and lipids (Yang Young and Woodside, 2001). In this study, the radical scavenging activity of FHSO, LF and HF was determined using DPPH and ABTS methods. DPPH and ABTS assays have been widely used to de-termine the free radical-scavenging activity of various plants and pure compounds. As presented in Fig. 3A and B, both the DPPH and ABTS inhibition percentage values were dose-dependent in the range of the tested concentrations for FHSO, LF and HF. Both the DPPH and ABTS radical inhibition decreases in the order FHSO >LF >HF. DPPH and ABTS values, expressed as mmoles or μmoles of TE/100 g of FW, are listed in Table 3. FHSO showed higher activities than the LF and HF fractions, and these differences were statistically significant (p <0.05). These data suggest that Finola cold-pressed seed oil may have the potential to protect biologically important components, such as membrane lipids, DNA and proteins, from radical-mediated oxida-tive damage.

Reducing power assay

The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity. The presence of reductants such as antioxidant sub-stances in the samples causes the reduction of Fe3+ to the ferrous form. For the measurements of the reductive ability, the Fe3+– Fe2+ transformation was investigated in the presence of FHSO, LF and HF using the method of Benzie and Strain (1996) with some modifications. Figure 4 depicts the reducing power of FHSO, LF and HF. Like the antioxidant activity, the reducing power of FHSO and LF increased with increasing concentra-tion. At the different concentrations, FHSO showed higher activity than LF and HF, and these differences were statistically significant (p <0.05). The reducing power values of all samples are listed in Table 3 and ex-hibited the following order: FHSO >LF > HF. The ac-tivity of FHSO to reduce ferric ions is an important property, because transition metals play an important role in catalyzing LDL oxidation in vitro and in vivo.

Chelating activity

Transition metals may act as catalysts that promote the generation of the first few radicals, which initiate the



Figure 3. Effect of different concentrations of Finola Hemp Seed Oil (FHSO), Lipophilic (LF) and Hydrophilic (HF) fractions in free radical scav-enging assays: (A) DPPH; (B) ABTS.

A. SMERIGLIO ET AL.

 

Figure 4. Correlation between antioxidant activity and several concentrations of Finola hempseed oil (FHSO), lypophilic (LF) and hydrophilic fractions (HF). The antioxidant activity was measured by either (A) ABTS, (B) DPPH, (C) FRAP and (D) EDTA assays.

oxidative chain reactions (Nawar, 1996). Chelating agents may reduce the availability of transition metals and inhibit the radical-mediated oxidative chain reac-tions in biological or food systems, and consequently im-prove human health, and food quality, stability and safety. The present study detected significant dose-dependent chelating activity against Fe2+ for all samples tested (Fig. 3). FHSO showed higher chelating capacity than the LF and HF fractions (Table 3), and these differ-ences were statistically significant (p <0.05). These re-sults suggest the potential of Finola cold-pressed seed

oil to prevent oxidative damage from free radical medi-ated oxidation Oxygen radical absorbance capacity

ORAC measures the capacity of antioxidants to protect a fluorescing protein from peroxyl radical attacks and is a widely used assay for estimating antioxidant activity (Huang et al., 2002). FHSO, compared with LF and HF, has shown the highest capacity to protect fluores-cein from peroxyl radical attaks. Figure 5 shows that



Figure 5. Fluorescence decay curves of fluorescein induced by AAPH in presence of reference compound (Trolox), Finola hempseed oil (FHSO), lypophilic (LF) and hydrophilic fractions (HF). Data are expressed as mean (n = 3) of three independent experiments.

POLYPHENOLIC COMPOUNDS AND ANTIOXIDANT ACTIVITY OF FHSO


Figure 6. Beta carotene bleaching curves of reference compound (BHT), Finola hempseed oil (FHSO), lypophilic (LF) and hydrophilic fractions (HF). Results are expressed as mean (n = 3) of three independent experiments.

inhibition of the fluorescence decay of fluorescein in pres-ence of reference compound (Trolox), FHSO, LF and HF decreases in the following order: Trolox >FHSO > LF >HF. These results suggest that Finola cold-pressed seed oil may serve as excellent dietary source of natural antioxidants for protection of important protein molecules from radical-mediated damage.

 

β-Carotene bleaching assay

In the β-carotene bleaching assay, the oxidation of linoleic acid produces free radicals because of the removing of hy-drogen atom from diallylic methylene groups of linoleic acid. The highly unsaturated β-carotene then will be oxi-dized by the generated free radical. Degradation of the or-ange coloured chromophore of β-carotene could be monitored spectrophotometrically. However, the pres-ence of antioxidant constituents could prevent the bleaching of β-carotene because of their ability to neutral-ize the free radicals. Figure 6 shows the inhibitory activity of reference compound (BHT), FHSO and derived frac-tions on β-carotene bleaching. LF fraction showed the best inhibitory performance while HF exhibited the low-est. The inhibitory activity on β-carotene bleaching showed the following order: BHT >LF >FHSO > HF.

CONCLUSION

The characterization of the bioactive components in functional foods and medicinal plants is required to provide scientific evidence for improved their utiliza-tion for achieve human health benefits. The results ob-tained in this study showed that FHSO contains significant amounts of polyphenols, especially flavonoids such as flavanones, flavanols, flavonols and isoflavones. Flavonoids are effective antioxidants be-cause of their free radical scavenging properties and because they are chelators of metal ions (Kandaswami and Middleton, 1994); thus, they may protect tissues against free oxygen radicals and lipid peroxidation. In this study FHSO clearly showed to have high antioxi-dant activity against various antioxidant systems in vitro. The significant antioxidant properties exhib-ited from Finola seed oil may depend on the presence of vitamin E and phenolic compounds, especially flavo-noids that are contained in greater quantities compared with other phenolic compounds. Moreover, Finola cold-pressed seed oil can be used as source of natural antioxidants and as a possible food supplement or in pharmaceutical applications.

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