From Vegetable Waste to New Agents for Potential Health

Sep 1, 2017 - ... MCF-7, MDA-MB-231, Caco-2, and PC3 human cancer cells), and 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiaz...
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From Vegetable Waste to New Agents for Potential Health Applications: Antioxidant Properties and Effects of Extracts, Fractions and Pinocembrin from Glycyrrhiza glabra L. Aerial Parts on Viability of Five Human Cancer Cell Lines Francesca Aiello,† Biagio Armentano,† Nicoletta Polerà,† Gabriele Carullo,† Monica Rosa Loizzo,† Marco Bonesi,† Maria Stella Cappello,‡ Loredana Capobianco,§ and Rosa Tundis*,† †

Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Via P. Bucci 87036, Rende, Cosenza, Italy CNR, Institute of Science of Food Production (ISPA), Prov. le Lecce-Monteroni, 73100 Lecce, Italy § Department of Biological and Environmental Sciences and Technologies, University of Salento, 73100 Lecce, Italy ‡

ABSTRACT: Glycyrrhiza glabra cultivation and harvesting produces substantial quantities of aerial parts as waste. With the aim to prospect an innovative valorization of these byproducts, the aerial parts were harvested in May and October and analyzed for their chemical profile, antioxidant properties, and effects on viability of five cancer cell lines. Pinocembrin was the main constituent. A significant protection of lipid peroxidation was observed with the May total extract (IC50 of 4.2 ± 0.4 μg/mL at 30 min of incubation). The effects on viability of HeLa, MCF-7, MDA-MB-231, Caco-2, and PC3 human cancer cells were investigated. All samples shown a remarkable activity with IC50 values below 25 μg/mL. Samples from plants harvested in May exhibited greater activity than those harvested in October. MCF-7 and HeLa were the most sensitive cells with IC50 in the range 2.73−3.01 and 3.28−5.53 μg/mL, respectively. G. glabra aerial parts represent a good source of valuable products. KEYWORDS: Glycyrrhiza glabra, vegetable waste, human cancer cells, effects on cellular viability, antioxidant properties

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INTRODUCTION Glycyrrhiza glabra L. (Fabaceae), also known as licorice, grows in Europe, the Middle East, and Western Asia. G. glabra is one of the most extensively used medicinal plant from the ancient medical history. The Egyptian, Chinese, Greek, Indian, and Roman civilizations employed their dried rhizome and root in traditional medicine as an expectorant and carminative drug. Traditionally, G. glabra roots have widely been used worldwide in food and pharmaceutical products.1 In Traditional Chinese Medicine (TCM), G. glabra is included in several formulations.2 Several studies have demonstrated the antiulcer,3 antiinflammatory,4 antimicrobial,5 antifungal,5 antioxidant,6 antipyretic,7 antiviral,8 and anxiolytic9 activities of G. glabra. A number of phytochemicals have been isolated from the roots of G. glabra.10 These include mainly triterpene saponins, flavonoids, coumarins, and other phenols. Flavonoids isolated from G. glabra can be divided into a variety of classes such as chalcones, flavanones, flavones, isoflavans, and isoflavones. These phytochemicals are related to a broad spectrum of biological effects and are indispensable in a variety of cosmetic, nutraceutical, and pharmaceutical applications.11−14 The aerial parts of G. glabra, considered an agrochemical waste, are still scarcely used. The few phytochemical studies on the G. glabra aerial parts demonstrated the presence of some phenolic compounds that are present either in small traces or not at all in the roots. In this field, G. glabra aerial parts have already shown attractive biological activities. In particular, Bassyouni et al.15 showed how the ethyl acetate extract from the leaves of G. glabra inhibited the bacterial growth of clinical methicillin© XXXX American Chemical Society

resistant Staphylococcus aureus. Three dihydrostilbenes isolated from the leaves of Sicilian G. glabra demonstrated to be good antioxidant compounds and ligands of cyclooxygenase-2.16 The aim of this work is to prospect a potential nutraceuticalpharmaceutical use of the aerial parts of G. glabra, now considered as agrochemical waste. The aspects regarding the use of wastes have gained increasing interest because these are high value products and their recovery may be attractive. The byproducts represent an interesting source of compounds with a variety of bioactivities. The transformation of byproducts into “products” with high benefit makes it possible for industries to reduce their treatment cost, even to make additional profits, and thus to improve their competitiveness. We have harvested the aerial parts of G. glabra in two periods such as May and October. The first period coincides with the plant flowering, while the second period coincides with the harvesting of roots and simultaneously the removing of the aerial parts as a waste product. The extracts were subjected to phytochemical screening and were tested in a series of in vitro experiments such as 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium (MTT) assay to study the effects on cellular viability (HeLa, MCF-7, MDA-MB-231, Caco-2, and PC3 human cancer cells), and 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3ethylbenzothiazoline-6-sulfonic acid (ABTS), ferric reducing Received: July 3, 2017 Revised: August 18, 2017 Accepted: August 22, 2017

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DOI: 10.1021/acs.jafc.7b03045 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Pinocembrin. 1H NMR (CDCl3, 300 MHz): δ (ppm) 12.13 (b. s. 2H), 7.5−7.3 (m, 5H), 6.1 (s, 2H), 5.42 (dd, 1H, J = 3.0, 13.0 Hz), 3.10 (dd, 1H, J = 13.0, 17.2, Hz), 2.82 (dd, 1H, J = 3.0, 14.0 Hz). 13C NMR: δ (ppm) 195.7, 164.9, 164.3, 164.1, 138.2, 128.88, 128.86, 128.0, 126.13, 126.11, 103.0, 95.9, 95.5, 79.1, 43.2. Natural pinocembrin has a steric structure of S-configuration and a specific rotation [α]D ⟨15⟩ of −45.3 (c, 0.9, acetone as solvent). The n-hexane-soluble fractions of G. glabra were analyzed and the relative peak areas for individual constituents averaged. Quantification was computed as the percentage contribution of each compound to the total amount presented. The relative percentage was calculated using a Shimadzu GC17A GC equipped with a flame ionization detector (FID) and controlled by a Borwin Software (Shimadzu, Milan, Italy), and a fused silica 30 m HP-5 capillary column (i.d. 0.25 mm, length 30 m, film thickness 0.25 μm).19 The temperature was initially of 50 °C, then raised to 280 °C at a rate of 13 °C/min, and finally held at that temperature for 10 min. The percentage composition of the samples was computed by the normalization method from the GC peak areas, calculated by means of 3 injections from each sample, without using correction factors using an external standard method. Constituents of the n-hexane fractions were analyzed by a Hewlett-Packard 6890 gas chromatograph interfaced with a Hewlett-Packard 5973 Mass Selective, using electron impact (EI) (Agilent Technologies, Milan, Italy) and equipped with an HP-5 MS capillary column (30 m length, 0.25 mm i.d., 0.25 μm film thickness). The ionization voltage is 70 eV. The same column was used. The gas chromatography conditions were the same as reported for GC analysis. Cell Cultures. Human colorectal adenocarcinoma (Caco-2) cells were obtained from prof. Tiziano Verri (University of Salento, Italy). The remaining cancer cell lines were purchased from the ATCC from the American Type Culture Collection (ATCC) (Manassas, VA). Human uterine cervix adenocarcinoma (HeLa) and human colorectal adenocarcinoma (Caco-2) cells were maintained in MEM medium containing 10% FBS (fetal bovine serum), 1% L-glutamine, 1% Eagle’s nonessential amino acids, and 1% penicillin-streptomycin. Human breast cancer ER+ cells (MCF-7) were sustained as previously described.20 MDA-MB-231 triple negative breast adenocarcinoma cell line were maintained in DMEM-F12 containing 10% FBS and 1% penicillin/streptomycin. PC3 androgen-independent human prostate cancer cell line were grown in DMEM-F12 supplemented with 1% penicillin/streptomycin and 10% FBS.21 MCF-10A human mammary epithelial cells were maintained in DMEM-F12 supplemented with 1% glutamine, 0.5 mg/mL hydrocortisone, 10% horse serum (HS), 1% penicillin/streptomycin, 20 ng/mL human Epidermal Growth Factor (hEGF), and 0.1 mg/mL cholera enterotoxin (complete medium). All the cell lines were cultured at 37 °C in 5% CO2 in a humidified atmosphere. All tested samples were dissolved in DMSO and diluted in appropriate medium supplemented with 2% DCC-FBS (dextrancoated charcoal-treated newborn calf serum serum) to obtain the working concentration. Cell Viability Assay. Cell viability was determined by using the MTT assay, as previously described.22 Briefly, MCF-7, HeLa, and MCF-10A cells (3 × 104 cells/well) were seeded in a 24-well plate and cultured in complete medium overnight. Before treatment, the culture medium was switched to DMEM F-12 or MEM or DMEM according to cell line, supplemented with 2% charcoal-stripped (CS) FBS. Then, the cells were untreated or treated with different concentrations of samples. DMSO was used as the vehicle control. After 72 h, the fresh MTT reagent (final concentration 0.33 mg/mL) was added and incubation was continued for 2 h. Each experiment was executed in triplicate and the optical density was measured at 570 nm in each well, including the control. After shaking the plates for 15 min, absorbance was measured at 570 nm in each well, including the controls. A minimum of three experiments containing five different concentrations of samples and doxorubicin in triplicate was combined for the calculation of IC50 values. The absorbance readings were used to determine the IC50 using GraphPad Prism 7 Software (GraphPad Inc., San Diego, CA).

activity power (FRAP), and β-carotene bleaching assays to investigate the antioxidant properties.

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MATERIALS AND METHODS

Chemicals, Reagents, and Instrumentation. Solvents of analytical grade, thin layer chromatography (TLC, silica gel), silica gel (0.040−0.063 and 0.063−0.200 mm) were obtained from VWR International s.r.l. (Milan, Italy). Organic solutions were dried over MgSO4 and evaporated on a rotary evaporator under reduced pressure. Melting points were obtained using a Gallenkamp melting point apparatus. 1H and 13C NMR spectra were recorded on a Bruker 300 MHz spectrometer with TMS as an internal standard: chemical shifts are expressed in δ values (ppm) and coupling constants (J) in hertz (Hz). Gas chromatography (GC) analyses were performed by using a Shimadzu GC17A GC equipped with a flame ionization detector (FID) and controlled by Borwin Software (Shimadzu, Milan, Italy). Gas chromatography−mass spectrometry (GC−MS) analyses were performed by using a Hewlett-Packard 6890 gas chromatograph interfaced with a Hewlett-Packard 5973 Mass Selective, using electron impact (EI) (Agilent Technologies, Milan, Italy). Tween 20, ascorbic acid, Folin-Ciocalteau reagent, sodium carbonate, butylated hydroxytoluene (BHT), quercetin, chlorogenic acid, propyl gallate, 2,2diphenyl-1-picrylhydrazyl (DPPH), tripyridyltriazine (TPTZ), 2,2′azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) solution, 6hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 3(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium (MTT), β-carotene, doxorubicin, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and dextran-coated charcoal-treated newborn calf serum serum (DCC-FBS), linoleic acid, cholera enterotoxin, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich S.p.a. (Milan, Italy). Plant Materials. The aerial parts of Glycyrrhiza glabra L. were collected in the Botanic Garden, University of Calabria, Rende (Cosenza province, Southern Italy) (39° 35′ 74″ N, 16° 22′ 94″ E) in May and October 2015 and were identified by Dr. N. G. Passalacqua, Natural History Museum of Calabria and Botanic Garden (CLU), University of Calabria. Extraction Procedure. The fresh aerial parts of G. glabra, collected in May (1430 g) and October (786 g), were exhaustively extracted by maceration with methanol (5 × 2 L) to give 140.78 and 92.69 g of the respective dried residue (yield of 9.84 and 11.93%, respectively). The total extract was suspended in methanol and partitioned with nhexane to yield an n-hexane-soluble fraction (yield of 1.12 and 1.03% for May and October, respectively) and a methanol-soluble fraction (yield of 6.79 and 10.43% for May and October, respectively). Isolation and Characterization. The G. glabra methanol-soluble fraction was investigated for its total phenols content as previously reported.17 The fraction was mixed with 0.2 mL of Folin-Ciocalteau reagent, 2 mL of water, and 1 mL of sodium carbonate (15%). After 2 h of incubation at room temperature, the absorbance was measured at 765 nm. The total phenols content was expressed as milligrams of chlorogenic acid equivalents/g of extract. The total flavonoids content of G. glabra methanol-soluble fraction was determined as previously described.17 Absorbance was read at 510 nm. The total flavonoid content was expressed as milligrams of quercetin equivalents/g of extract. Specific amounts of methanol-soluble fraction was subjected to silica gel (0.063−0.200 mm) flash column chromatography eluted with dichloromethane/methanol gradient system. Fractions exhibiting similar TLC profiles were grouped into 10 major fractions (F1−F10). Starting from 2.0 g of methanol-soluble fraction, 0.120 g of fraction F1 was obtained. This fraction was further purified by flash column chromatography (silica gel 0.040−0.063 nm) using a n-hexane/ethyl acetate gradient system affording a consistent amount of pinocembrin (5,7-dihydroxyflavanone) (70 mg), mp of 195.4 °C. The structure of pinocembrin was elucidated by comparison of its NMR spectral data with those previously reported.18 B

DOI: 10.1021/acs.jafc.7b03045 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Briefly, values were log-transformed, normalized, and nonlinear regression analysis was used to generate a sigmoidal dose−response curve to calculate IC50 values for each cell line. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) Radical Scavenging Activity Assay. DPPH radical scavenging activities of G. glabra extracts, fractions, and pinocembrin were determined according to the method previously described.23 A mixture of DPPH methanol solution (1.0 × 10−4 M) and G. glabra samples (1−1000 μg/mL) was prepared. The absorbance was read at 517 nm (UV−vis Jenway 6003 spectrophotometer). The DPPH radicals scavenging activity was calculated as follows: [(A0 − A1/A0) × 100], where A0 is the absorbance of the control and A1 is the absorbance in the presence of the sample. The positive control was ascorbic acid. 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) Radical Scavenging Activity Assay. The ABTS solution was mixed with potassium persulfate and left in the dark for 12 h before use.23 The ABTS solution was diluted with methanol to an absorbance of 0.70 at 734 nm. The G. glabra samples (1−1000 μg/ mL) were added to the ABTS solution, and the absorbance was measured after 6 min. Ascorbic acid was the positive control. β-Carotene Bleaching Test. The β-carotene bleaching test was done following the procedure previously described.23 Concisely, βcarotene solution was added to linoleic acid and 100% Tween 20. The emulsion was mixed with 200 μL of G. glabra extracts, fractions, and pinocembrin (1−1000 μg/mL). The tubes were placed in a water bath at 45 °C. The absorbance was read at 470 nm against a blank at t = 0 and after 30 and 60 min of incubation. Propyl gallate was the positive control. Ferric Reducing Activity Power (FRAP) Assay. This assay is based on the redox reaction that involves TPTZ (2,4,6-tripyridyl-striazine)-Fe3+ complex. FRAP reagent was prepared as reported previously.23 The absorption was measured at 595 nm. All the samples were tested at the concentration of 2.5 mg/mL. BHT (butylated hydroxytoluene) was the positive control. Relative Antioxidant Capacity Index (RACI). RACI was used as an integrated approach to evaluate and compare the antioxidant capacity of samples. Herein, data obtained from ABTS, DPPH, FRAP, and β-carotene bleaching tests were used to calculate RACI value for G. glabra samples. Statistical Analysis. The concentration giving 50% inhibition (IC50) was calculated by nonlinear regression with the use of Prism GraphPad Prism, version 4.0 for Windows (GraphPad Software, San Diego, CA). The concentration−response curve was obtained by plotting the percentage inhibition vs concentration. One-way analysis of variance test (ANOVA) followed by a multicomparison Dunnett’s test were applied.

The methanol-soluble fractions have been investigated for their total phenols and flavonoids content. Samples of May showed a higher total phenols (66.7 ± 2.3 vs 49.3 ± 1.9 mg equivalents of chlorogenic acid/g of extract) and flavonoids (38.1 ± 1.7 vs 19.9 ± 1.0 mg equivalents of quercetin/g of extract) content compared to samples of October. Successively, these fractions were subjected to chromatographic fractionation. Pinocembrin was isolated from the first fraction (F1) and identified as the main constituent. A yield of 3.0% was obtained from the aerial parts of G. glabra collected in May. This value is about 2 times greater than the aerial parts of October (1.7%). The dihydroxyflavanone pinocembrin is widely found in nature. In fact, it was previously isolated from different plant species, including Alpinia mutica, Centaurea eryngioides, Cistus incanus, Cleome droserifolia, Dalea elegans, Eriodictyon californicum, Helichrysum gymnocomum, Lippia graveolens, L. origanoides, Litchi chinensis, Lychnophora markgravii, Oxytropis falcate, Sparattosperma leucanthum, Syzygium samarangense, and Turnera dif f usa.24 In addition to pinocembrin, the presence of genistein, isoquercitrin, prunetin, 6-prenylnaringenin, licoflavanone, wighteone, and lupiwighteone in the aerial parts of G. glabra was demonstrated.25 The n-hexane-soluble fractions of G. glabra leaves collected in May and October were analyzed by GC and GC−MS. Data are reported in Table 1 where compounds are listed in order of elution on a HP-5 MS column. A total of 19 compounds have been identified as main constituents. Fatty acids, particularly methyl linolenate (36.5%), methyl palmitate (15.9%), and methyl stearate (12.1%), and phytol (12.4%), characterized the Table 1. Major Identified Components (%) of the n-HexaneSoluble Fractions of G. glabra Aerial Parts Collected in May and Octobera percentage (%) compound α-pinene β-pinene L-phellandrene p-cimene α-terpinene trans-caryophyllene (Z)-β-farnesene farnesol neophytadiene 2-pentadecanone 6,10,14trimethyl methyl palmitate methyl linoleate manoyl oxide methyl linolenate methyl stearate phytol docosanoic acid methyl ester stigmasterol β-sitosterol

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RESULTS AND DISCUSSION Phytochemicals Profile. G. glabra is one of the most important medicinal plants and has been used in traditional medicine for at least 4000 years. The roots of G. glabra, which contain triterpenes and flavonoids as the most representative classes of constituents, were the most used and investigated plant portions.10 In contrast, the aerial parts are considered as waste and are less studied. Herein, the aerial parts of G. glabra were collected in Calabria (Southern Italy) in two harvesting periods such as May and October. The aerial parts were extracted by maceration with methanol to yield a total extract. This extract was suspended in methanol and partitioned with n-hexane to yield a n-hexanesoluble fraction and a methanol-soluble fraction. The greatest extraction yield was obtained for samples of October with values of 11.8 and 10.4% for the total extract and the methanolsoluble fraction, respectively. Similar values were reported for the n-hexane-soluble fractions (1.1 and 1.0% for May and October, respectively).

May 0.3 tr tr tr tr 0.4 0.4 0.2 2.3 tr

± 0.01

± ± ± ±

0.02 0.03 0.01 0.1

October

identification method

± ± ± ± ± ± ± ± ± ±

1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2 1,2 1,2 1,2 1,2

0.5 0.3 0.5 1.1 0.5 0.3 0.8 1.2 2.5 2.3

0.01 0.02 0.02 0.03 0.02 0.01 0.05 0.4 0.1 0.1

15.9 ± 1.2 3.2 ± 0.6 − 36.5 ± 3.7 12.1 ± 0.8 12.4 ± 1.0 0.8 ± 0.01

4.5 ± 0.3 1.2 ± 0.2 12.5 ± 0.9 11.3 ± 0.8 8.7 ± 0.9 16.5 ± 1.3 1.7 ± 0.2

1,2 1,2 1,2 1,2 1,2 1,2 1,2

tr tr

8.1 ± 1.0 12.9 ± 1.1

1,2 1,2,3

Data are expressed as mean ± standard deviation (S.D.) (n = 3). 1, comparison of retention times; 2, comparison of mass spectra with MS libraries, 3, comparison with authentic compounds. Compositional values 400 μM), pinocembrin treatment did not induce any effects on nontumorigenic MCF-10A breast epithelial cells (Table 3). In order to evaluate the contribution of the flavanone to the activity of the fraction, we have tested also fraction F1 without pinocembrin. IC50 values of 3.4, 11.9, 7.4, 12.7, and 8.2 μg/mL were found against HeLa, MCF-7, PC3, MDA-MB-231, and Caco-2 cell lines, respectively. Interestingly, obtained results confirm the importance of the synergistic action of the compounds. In fact, in all tested cell lines the IC50 values are better than those reported for pinocembrin alone but not than those reported for fraction F1. The anticancer activity of pinocembrin is well documented in the literature. This compound has been reported to inhibit growth of several human cancer cell lines and to induce apoptosis.3 G

DOI: 10.1021/acs.jafc.7b03045 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 4. Antioxidant Activity of G. glabra Total Extracts and Fractionsa β-carotene bleaching test (IC50 μg/mL) DPPH (IC50 μg/mL)

ABTS (IC50 μg/mL)

FRAPb (μM Fe(II)/g)

30 min

May total extract methanol fraction n-hexane fraction

38.3 ± 1.5 26.4 ± 1.7 66.2 ± 2.6

9.3 ± 0.5 6.2 ± 0.3 35.8 ± 2.7

14.1 ± 1.2 26.8 ± 1.9 2.3 ± 0.9

4.2 ± 0.4 6.2 ± 0.9 10.7 ± 0.8

4.5 ± 0.7 7.7 ± 0.8 14.1 ± 0.4

October total extract methanol fraction n-hexane fraction

32.3 ± 1.8 63.4 ± 2.2 147.9 ± 3.7

5.4 ± 0.9 5.4 ± 0.6 39.0 ± 5.6

29.7 ± 1.4 21.3 ± 1.1 7.7 ± 0.9

6.4 ± 0.6 4.9 ± 0.3 5.9 ± 0.7

9.5 ± 0.7 7.6 ± 0.5 5.1 ± 0.3

1.8 ± 0.06

5.0 ± 0.8 1.0 ± 0.04

0.09 ± 0.004

Positive Control BHT ascorbic acid propyl gallate

60 min

RACI value 0.12 −0.83 −0.39

0.14 0.02 1.90

63.1 ± 4.3

Data are expressed as means ± SD (n = 3). bAt 2.5 mg/mL. DPPH: One-way ANOVA ***p < 0.0001 (F = 304, R 2= 0.92), Dunnett’s test *p < 0.01. ABTS: One-way ANOVA ***p < 0.0001 (F = 7297, R 2= 0.99), Dunnett’s test *p < 0.01, °p > 0.05. β-Carotene-bleaching test: (30 min) Oneway ANOVA ***p < 0.0001 (F = 577.9, R 2= 0.99), Dunnett’s test *p < 0.01; (60 min). One-way ANOVA ***p < 0.0001 (F = 703, R 2= 0.99), Dunnett’s test *p < 0.01. FRAP: One-way ANOVA ***p < 0.0001 (F = 4412, R 2= 0.99), Dunnett’s test *p < 0.01. a

The most abundant component of G. glabra aerial parts, pinocembrin, was screened for radical scavenging potential, lipid peroxidation protection, and ferric reducing ability. As reported in Table 5, this compound showed DPPH radicals

phytochemicals might exert a synergistic effect, which could justify the interesting antiproliferative activity. Antioxidant Activity of G. glabra. Several studies proved that diseases such as cancer are mediated by oxidative stress. Therefore, antioxidant compounds may play a key role in preventing or slowing the progression of these diseases. In this study, different procedures, namely, ABTS, FRAP, DPPH, and β-carotene bleaching tests were applied to the investigation of the antioxidants effects of G. glabra total extracts, n-hexanesoluble fraction, and methanol-soluble fraction. Data for both harvesting periods (May and October) are reported in Table 4. In the DPPH test, comparable results were obtained for the total extract (38.3 and 32.3 μg/mL for May and October, respectively). The methanol-soluble fraction of May showed the highest radical scavenging effects (IC50 of 26.4 μg/mL). Better data were obtained in the ABTS test. In this assay, of particular interest is the activity of total extract and the methanol-soluble fraction of October IC50 value of 5.4 μg/mL). This value is comparable with that of ascorbic acid (IC50 of 5.0 μg/mL) used as a positive control. In both DPPH and ABTS tests, the n-hexane-soluble fractions of May and October were the less active with IC50 values of 66.2 and 147.9 μg/mL in the DPPH assay and 35.8 and 39.0 μg/mL in the ABTS assay. The analysis of the results obtained through the β-carotene bleaching test revealed the May total extract was the most active with an IC50 value of 4.2 and 4.5 μg/mL after 30 and 60 min of incubation, respectively. A significant protection of lipid peroxidation was observed also with October methanol fraction (IC50 of 4.9 μg/mL at 30 min of incubation). Through the FRAP test, the total extract of G. glabra of October possessed the highest antioxidant activity with a value of 29.7 μM Fe (II)/g, followed by the methanol fraction of May (IC50 value of 26.8 μM Fe(II)/g. Pinocembrin was inactive in the FRAP test. According to Sun and Tanumihardjo,42 we used a RACI as an approach to compare antioxidant results from all employed tests. On the basis of RACI results, the n-hexanesoluble fraction of October showed the highest value followed by the total extract of October and May (Table 4).

Table 5. Anti-Radicals and Antioxidant Properties of Pinocembrina β-carotene bleaching test (IC50 μg/ mL) DPPH (IC50 μg/mL)

30 min

60 min

pinocembrin

9.4 ± 0.6***

28.8 ± 2.6***

42.1 ± 3.7***

Positive Control ascorbic acid propyl gallate

1.7 ± 0.06 1.0 ± 0.04

0.09 ± 0.004

compound

Data are expressed as means ± SD (n = 3). DPPH: One-way ANOVA ***p < 0.0001 (F = 211.8, R 2= 0.99), Dunnett’s test ***p < 0.0001. β-Carotene-bleaching test: (30 min) One-way ANOVA ***p < 0.0001 (F = 8562, R 2= 0.99), Dunnett’s test ***p < 0.0001; (60 min). One-way ANOVA ***p < 0.0001 (F = 12223, R 2 = 0.99), Dunnett’s test ***p < 0.0001. a

scavenging activity with an IC50 value of 9.4 μg/mL. IC50 values of 28.8 and 42.1 μg/mL were reported after 30 and 60 min of incubation in the β-carotene bleaching test. The antioxidant activity of G. glabra is well documented. Cheel et al.43 investigated the DPPH radical scavenging ability and lipid peroxidation properties of G. glabra from Czech Republic infusion and two main abundant constituents liquiritin and glycyrrhizin. The infusion exerted a weak effect in a concentration-dependent manner, (10−100 μg/mL), a further increase concentration did not cause an increase DPPHscavenging activity. Both, compounds showed negligible effects. In the β-carotene bleaching method, licorice infusion at a concentration of 200 μg/mL retained about 83% of the initial β-carotene after 1 h and 64% after 2 h. Tohma and Gulçin44 compared the antioxidant potential of Turkish G. glabra aerial parts aqueous extract (AE-AP) and H

DOI: 10.1021/acs.jafc.7b03045 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry ethanol extract (EE-AP) by different in vitro assays. Roots aqueous (AE-RP) and ethanol (EE-RP) extracts were also investigated. Generally, extracts exhibited a comparable activity in all tests and in a concentration-dependent manner. Among data both extract showed the highest activity in ABTS test with percentage of inhibition of 80.3 and 98.7% at the maximum concentration tested for AE-AP and EE-AP, respectively. With the application of extract at a concentration of 30 μg/mL on lipid peroxidation of linoleic acid emulsion, the effect was found to be 87.9 and 83.6% for AE-AP and EE-AP, respectively. In the study of ferric reducing potential, the following order was registered: trolox > α-tocopherol > EE-AP > EE-RP> AE-AP > AE-RP. The antioxidant properties of Sybaris G. glabra Et2O, AcOEt, MeOH, and BuOH of the inner part and cortex root extracts were investigated.45 The values of radical scavenging activity of cortex extracts are in the range 90−98% (Et2O extract), 86− 90% (AcOEt extract), 67−92% (MeOH extract), and 83−97% (BuOH extract). Inner extract radical scavenging potential varied as follows: from 75 to 86% for the Et2O extract, from 5 to 15% for the AcOEt extract, from 28 to 81% for the MeOH extract, and from 77 to 89% for the BuOH extract. The SC50 value (50% free scavenging concentration value) was calculated for cortex extracts and was found in the range 96.97−334 μg/ mL for Et2O and MeOH extracts, respectively. All these values are higher than those of found with our sample from the same origins but obtained with the aerial parts. More recently, Dong et al.18 compared the antioxidant activity of G. glabra roots and leaves extract. Analysis of results evidenced that the total flavonoid content in the leaves extract was higher than that in the roots extract. This high content is reflected in the antioxidant activity since the ORAC value of the leaf extract was nearly 2 times higher than that of the root extract. The main abundant compound in leaf extract was pinocembrin that showed good oxygen radical antioxidant capacity with ORAC value of 13 904.28 μmol trolox equiv/g while liquiritin, the main compounds in the root extract, was less active (1121.01 μmol trolox equiv/g). The flavonoids of G. glabra have achieved a significant interest for their structural diversity and their important biological properties. Isoliquiritigenin, licoricidin, glabridin, and licorisoflavan A showed anti-inflammatory, antioxidant, and antitumor properties. Herein, we have focused our attention to pinocembrin, isolated as the most abundant compound from the methanolsoluble fraction. Pinocembrin has previously been reported to have an extensive spectrum of biological activities including antibacterial, antifungal, anti-inflammatory, antioxidant, antiviral, and vasorelaxant activities.46−48 The stronger hydrogen atom donation of pinocembrin was due to 2 OH groups on ring A. In the meantime, due to the extra 5-OH on ring A of the flavanone, the p−π conjugation was formed and the density of the electron cloud was improved on ring A, the ring A and the 7-OH on ring A were both activated.18 In summary, the aerial parts of G. glabra, until now considered vegetable waste, may be a potential source of phytochemicals with antiproliferative and antioxidant properties, confirming the role of plant-derived compounds as a source of anticancer drugs. Herein, extracts and fractions of G. glabra aerial parts showed a promising antiproliferative activity mainly against MCF-7 and MDA-MB-231 cell lines compared to the positive control doxorubicin. In particular, the n-hexanesoluble fraction taken from plants harvested in May exhibited

better antiproliferative activity in all cell lines tested. Remarkably, unlike doxorubicin, G. glabra extracts and fractions exhibited no antiproliferative effects on nontumorigenic MCF10A breast epithelial cells, demonstrating the specific inhibitory effect on cancer cells.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +39 984 493246. Fax: +39 984 493107. E-mail: rosa. [email protected]. ORCID

Francesca Aiello: 0000-0001-6846-5582 Rosa Tundis: 0000-0002-3713-4403 Funding

The study was not specifically funded. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. N. G. Passalacqua of the Botany Department at the University of Calabria (Italy) for sample collection and identification.

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ABBREVIATIONS USED ABTS, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); DPPH, 2,2-diphenyl-1-picrylhydrazyl; DMSO, dimethyl sulfoxide; FBS, fetal bovine serum; FRAP, ferric reducing activity power; IC50, concentration giving 50% inhibition; MTT, 3-(4,5dimethylthiazol-2-y1)-2,5-diphenyltetrazolium; RACI, relative antioxidant capacity index

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REFERENCES

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DOI: 10.1021/acs.jafc.7b03045 J. Agric. Food Chem. XXXX, XXX, XXX−XXX