of phenolic compounds and antioxidant properties of Glycyrrhiza glabra L . rhizomes and roots

The present work aims to characterize and quantify the phenolic composition and to evaluate the antioxidant activity of Glycyrrhiza glabra L. (commonly known as licorice) rhizomes and roots. The antioxidant potential of its methanol/water extract could be related to flavones (mainly apigenin derivatives), flavanones (mainly liquiritin derivatives), a methylated isoflavone and a chalcone, identified in the extract. Lipid peroxidation inhibition was the most pronounced antioxidant effect (EC50 1⁄4 0.24 0.01 mg mL 1 and 22.74 2.42 mg mL 1 in TBARS and b-carotene/linoleate assays, respectively), followed by free radical scavenging activity (EC50 1⁄4 111.54 6.04 mg mL ) and, finally, reducing power (EC50 1⁄4 128.63 0.21 mg mL ). In this sense, licorice extract could be used as a source of antioxidants for the pharmaceutical, cosmetic and/or food industries.


Introduction
Environmental factors, such as pollution, smoking, certain drugs, poor diet, sedentary lifestyle and stress-inducing agents, are considered the main external aggressors for human bodies, increasing cell deterioration and, in the long term, contributing to aging and several diseases/disorders.Furthermore, the normal metabolism also produces high quantities of oxidant molecules, through different chemical reactions.][3] Plants are widely used to improve health and even to treat various diseases.6][17][18] Its antioxidant properties have also been reported, either in aqueous, 7,19,20 ethanol, [20][21][22] methanol 20,[23][24][25] or methanol/water 26,27 extracts.There are several studies that focused on the phenolic characterization of Glycyrrhiza sp. 23,24,26,27Nevertheless, information on the quantication of these compounds is scarce.
The aim of this work was to characterize and quantify the phenolic composition and evaluate the antioxidant properties in methanol/water extracts of Glycyrrhiza glabra L. (rhizomes and roots).

Samples
Dried rhizomes and roots of Glycyrrhiza glabra L. were supplied by Soria Natural (Garray -Soria, Spain).The samples were obtained in the autumn 2012 and certied as clean products, with monitored parameters for pesticides, herbicides, heavy metals and radioactivity.For each analysis, three different samples were used and the assays were performed in triplicate.

Extraction procedure
The extraction was performed by stirring the sample (1 g) with 30 mL of methanol/water (80 : 20, v/v) at 25 C and 150 rpm for 1 h, and ltered through Whatman no. 4 paper.The nal residue was then extracted with an additional 30 mL portion of the extraction solvents mixture.The combined extracts were evaporated at 35 C under reduced pressure (rotary evaporator Büchi R-210, Flawil, Switzerland) and then lyophilized (Free-Zone 4.5, Labconco, Kansas City, MO, USA).The lyophilized extracts were re-dissolved in methanol/water (80 : 20, v/v), performing a stock solution with a concentration of 20 mg mL À1 , from which several dilutions were prepared.

Analysis of phenolic compounds
Phenolic compounds were determined by HPLC (Hewlett-Packard 1100, Agilent Technologies, Santa Clara, USA) as previously described by the authors. 28Double online detection was carried out in the diode array detector (DAD) using 280 nm and 370 nm as preferred wavelengths and in a mass spectrometer (MS) connected to the HPLC system via the DAD cell outlet.Peaks were tentatively identied based on their UV-vis and mass spectra and comparison with data reported in the literature.Quantication was performed from the areas of the peaks recorded at 280 and 370 nm using calibration curves (1-100 mg mL À1 ) obtained with phenolic standards of the same group.The results were expressed in mg per g of extract.
2.5.Evaluation of antioxidant activity 2.5.1.DPPH radical-scavenging activity (RSA) assay.The capacity to scavenge the "stable" free radical DPPH, monitored according to the method of Hatano et al., with some modications, 29 was performed by using an ELX800 Microplate Reader (Bio-Tek Instruments, Inc; Winooski, USA).The reaction mixture in each one of the 96-wells consisted of one of the different concentration solutions (30 mL) and methanolic solution (270 mL) containing DPPH radicals (6 Â 10 À5 mol L À1 ).The mixture was le to stand for 30 min in the dark.The reduction of the DPPH radical was determined by measuring the absorption at 515 nm.The radical scavenging activity (RSA) was calculated as a percentage of DPPH discolouration using the equation: RSA (%) ¼ [(A DPPH À A S )/A DPPH ] Â 100, where A S is the absorbance of the solution when the sample extract has been added at a particular level, and A DPPH is the absorbance of the DPPH solution.The extract concentration providing 50% of antioxidant activity (EC 50 ) was calculated from the graph of DPPH scavenging activity against extract concentrations.Trolox was used as positive control.
2.5.2.Reducing power (RP) assay.RP was determined according to the method of Oyaizu, with some modications. 30he present methodology is based on the capacity to convert Fe 3+ into Fe 2+ , measuring the absorbance at 690 nm, by using the microplate Reader mentioned above.At the different concentration solutions (0.5 mL) were added sodium phosphate buffer (200 mmol L À1 , pH 6.6, 0.5 mL) and potassium ferricyanide (1% w/v, 0.5 mL).The mixture was incubated at 50 C for 20 min, and then, trichloroacetic acid (10% w/v, 0.5 mL) was added.In a 48-wells, the obtained mixture (0.8 mL), and also deionised water (0.8 mL) and ferric chloride (0.1% w/v, 0.16 mL) was joined, and the absorbance was measured at 690 nm.The extract concentration providing 0.5 of absorbance (EC 50 ) was calculated from the graph of absorbance at 690 nm against extract concentrations.Trolox was used as positive control.The tubes were shaken and incubated at 50 C in a water bath.As soon as the emulsion was added to each tube, the zero time absorbance was measured at 470 nm in a spectrophotometer (AnalytikJena, Jena, Germany).b-Carotene bleaching inhibition was calculated using the following equation: (Abs aer 2 h of assay/initial Abs) Â 100. 31The extract concentration providing 50% of antioxidant activity (EC 50 ) was calculated from the graph of b-carotene bleaching inhibition against extract concentrations.Trolox was used as positive control (Fig. 2).
2.5.4.Lipid peroxidation inhibition (LPI) through thiobarbituric acid reactive substances (TBARS) assay.LPI was evaluated by the decreasing in thiobarbituric acid reactive substances (TBARS).Porcine brains were obtained from official slaughtering animals, dissected, and homogenized with a Polytron in ice-cold Tris-HCl buffer (20 mM, pH 7.4) to produce a 1 : 2 (w/v) brain tissue homogenate, which was centrifuged at 3000g for 10 min.An aliquot (0.1 mL) of the supernatant was incubated with the different solution concentrations (0.2 mL) in the presence of FeSO 4 (10 mM; 0.1 mL) and ascorbic acid (0.1 mM; 0.1 mL) at 37 C for 1 h.The reaction was stopped by the addition of trichloroacetic acid (28% w/v, 0.5 mL), followed by thiobarbituric acid (TBA, 2%, w/v, 0.38 mL), and the mixture was then heated at 80 C for 20 min.Aer centrifugation at 3000g for 10 min to remove the precipitated protein, the colour intensity of the malondialdehyde (MDA)-TBA complex in the supernatant was measured by its absorbance at 532 nm.The inhibition ratio (%) was calculated using the following formula: inhibition ratio (%) ¼ [(A À B)/A] Â 100%, where A and B were the absorbance of the control and the compound solution, respectively. 32The extract concentration providing 50% of antioxidant activity (EC 50 ) was calculated from the graph of TBARS formation inhibition against extract concentrations.Trolox was used as positive control.

Characterization of the phenolic compounds
The phenolic prole of Glycyrrhiza glabra, obtained aer methanol/water extraction, and recorded at 280 and 370 nm is shown in Fig. 1; compound characteristics and tentative identities are presented in Table 1.Eleven compounds were detected corresponding to the groups of avones, avanones and chalcones, as well as a possible isoavone.
Compound 1 presented a pseudomolecular ion [M À H] À at m/z 593, releasing MS 2 fragment ions at m/z 443 (loss of 120 u), 383 (apigenin + 113 u) and 353 (apigenin + 83 u), whereas no relevant fragments derived from the loss of complete hexosyl (À162 u) or pentosyl residues (À132 u) were detected.This fragmentation behaviour is characteristic of di-C-glycosylated avones. 33The compound was tentatively identied as apigenin-6,8-di-C-glucoside (vicenin-2) owing to its previous description in Traditional Chinese Medicine Formulae containing Glycyrrhiza roots and rhizomes. 34,35ompound 2 presented a pseudomolecular ion [M À H] À at m/z 563.A compound with the same mass was reported in licorice (dried roots and rhizomes of Glycyrrhiza species) by Xu et al. (2013) and identied as the di-C-glycosylavone isoschaoside (i.e., 6-C-arabinopyranosyl-8-C-glucopyranosylapigenin).However, the MS 2 fragmentation pattern of the compound observed in our samples would not match such a structure, but it points to the pentosyl residue is O-attached to a C-glycosylating hexose.This assumption is supported by the characteristic fragment detected at m/z 413 ([M À 150] À ), which according to 33 would be typical from that type of substitution.Further, the fragment ion at m/z 443 ([M À 120] À ) supported the presence of a C-attached hexose, whilst the absence of an ion [(M À H) À 90] À suggested a 6-C attachment. 33The pentose should not be attached on positions 6 00 , 4 00 or 3 00 of the hexose, otherwise the fragment [(M À H) À 120] À would not be produced.As for the rest of fragment ions, the one at m/z 323 [(M À H) À 150 À 90] À would result from the partial loss of the C-attached hexose from the ion at m/z 413, whereas those at m/z 311 [aglycone + 41] À and 293 [aglycone + 41 À 18] À are associated to mono-C-glycosyl derivatives O-glycosylated on 2 00 . 36All in all, peak 2 was tentatively assigned as apigenin 2 00 -O-pentosyl-6-C-hexoside.
As previously indicated, compound 10 should correspond to a derivative of the chalcone isoliquiritigenin bearing pentosyl + hexosyl residues.43]45 As for the liquiritigenin derivatives, it was not possible to conclude about the precise location of the glycosyl groups, so that compound 10 was assigned as (neo) licuroside.
Compound 8 presented a pseudomolecular ion [M À H] À at m/z 565 releasing a fragment ion at m/z 271 (À294 u, loss of a pentosyl and hexosyl moieties), and a UV spectrum coherent with a avanone.These characteristics match the structure of naringenin-7-O-apiosyl-glucoside reported in radix Glycyrrhizae by Wang et al. (2014), so that this identity was tentatively assumed for the compound.
Compound 9 ([M À H] À at m/z 561) was tentatively identied as glycyroside (i.e., formononetin-7-O-apiosylglucoside) owing to the previous identication of that isoavone in radix Glycyrrhizae by Wang et al. (2014).The presence of formononetin derivatives in Glycyrrhiza species has also been reported by various authors. 35,41,42,45,46inally, it was not possible to identify compound 11 with a pseudomolecular ion [M À H] À at m/z 591 that released two fragments at m/z 297 (À294, loss of a pentosyl and hexosyl moieties) and 282 (further loss of À15 u of a methyl residue), although its UV spectra with a maximum at 372 nm pointed to a chalcone aglycone.
(2011) presented quantitative results, although they cannot be compared with ours results since they are expressed differently (mg g À1 of dry plant), thus these authors revealed liquiritin apioside as the main avonoid present in their sample, which is in agreement with the sample studied herein.In our case, the results were expressed in mg g À1 of extract in order to relate the amounts of phenolic compounds found in the extract to the antioxidant activity.Therefore this study will add new data related to the quantication of these compounds, which are scarce in literature.

Evaluation of antioxidant activity
Fig. 1 shows the results of the antioxidant potential of the licorice extract using different assays: DPPH radical scavenging activity (RSA), reducing power (RP), b-carotene bleaching inhibition (CBI) and lipid peroxidation inhibition (LPI) in brain cell homogenates.The most pronounced effect was observed for LPI assay (EC 50 ¼ 0.24 AE 0.01 mg mL À1 ), followed by CBI (EC 50 ¼ 22.74 AE 2.42 mg mL À1 ).RSA and RP presented higher EC 50 values (meaning lower antioxidant activity), respectively, 111.54 AE 6.04 mg mL À1 and 128.63 AE 0.21 mg mL À1 .
The LPI capacity, accessed by using the TBARS assay, measures the malondialdehyde (MDA) formed as the split product of an endoperoxide of unsaturated fatty acids resulting from oxidation of a lipid substrate.The MDA is reacted with thiobarbituric acid (TBA) to form a pink pigment (TBARS) that is measured spectrophotometrically at 532 nm. 32A + TBA / MDA À TBA 2 MDA + TBA + A / MDA + TBA 2 This procedure involves two distinct steps: the substrate is oxidized with the addition of a transition metal ion such as copper or iron or a free radical source such as 2,2 0 -azobis (2-amidinopropane) dihydrochloride, and then the extent of oxidation is determined by addition of TBA and spectrophotometric measurement of the product (MDA-TBA 2 ).Oxidation is inhibited by the addition of an antioxidant and, therefore, a reduction in the absorbance is observed.In the present experiment, the studied methanol/water extract exerted strong inhibitory effects of lipid oxidation (e.g., exponential inhibition of TBARS formation, being these effects achieved at extremely low concentrations), which is in agreement with the results obtained by Jiang et al. 47 that reported the efficacy of licorice ethanolic extract to prevent lipid oxidation and protect sensory attributes of ground pork.
Concerning to the CBI assay, and taking into account the basis of the method, b-carotene undergoes a rapid discoloration in the absence of an antioxidant since the free linoleic acid radical attacks the b-carotene molecule, which loses the double bonds and, consequently, loses its characteristic orange colour.Antioxidants can donate hydrogen atoms to quench radicals and prevent decolourization of carotenoids, 48  The decolourization of b-carotene can be monitored by spectrophotometry at 470 nm. 49Regarding the obtained results for the CBI activity of the studied methanol/water extract, a more pronounced effect (EC 50 ¼ 23 mg mL À1 ) was observed than the one reported by Ercisli et al. (2008) for ethanolic extracts of licorice roots collected in Turkey (EC 58 ¼ 75 mg mL À1 ).The results reported by these authors ranged between 28.3% (25 mg mL À1 ) and 88.7% (800 mg mL À1 ).
2,2-Diphenyl-1-picrylhydrazyl radical (DPPH), a stable organic nitrogen radical which presents a deep purple colour, allows the determination and quantication of the reducing capacity of antioxidants toward DPPH.Representing the DPPH radical by Xc and the donor molecule by AH (being mainly phenolic compounds, they are proton donators), the primary reaction is: In the present reaction, XH is the reduced form and Ac is the free radical produced in this rst step.This latter radical will then undergo further reactions, which control the overall stoichiometry, that is, the number of molecules of DPPH reduced (decolorized) by one molecule of the reductant. 50When a solution of DPPHc is mixed with a substance that can donate a hydrogen atom, the reduced form of the radical is generated accompanied by loss of colour.Upon reduction, the colour of DPPHc solution fades and this colour change is conveniently monitored measuring the absorbance decrease at 515-528 nm. 51Thus, by using the present assay, the free radicals scavenger effect of licorice was accessed.The RSA obtained for the studied methanol/water extract (EC 50 ¼ 112 mg mL À1 ) was similar to some of the values reported by Cheel et al. (2012) for similar extracts prepared from samples harvested at different times (February -EC 70 ¼ 100 mg mL À1 , May -EC 60 ¼ 30 mg mL À1 , August -EC 50 ¼ 50 mg mL À1 , November -EC 50 ¼ 30 mg mL À1 ), and by Cheel et al. (2010) for extracts obtained by infusion (EC 49 ¼ 100 mg mL À1 ).However, it was lower than the RSA described by Tohma & Gulçin (2010) for aqueous (EC 52 ¼ 62 mg mL À1 ) and ethanol (EC 54 ¼ 50 mg mL À1 ) extracts obtained from roots of Turkish licorice samples.
RP assay, widely used due to its specicity to access the electron-donating potential of antioxidants, and consequent reduction of yellow ferric form to blue ferrous form.The resultant blue colour is linearly correlated with the total reducing potential of electron-donating antioxidants, being measured spectrophotometrically at 700 nm. 55The RP value obtained in the present study (EC 50 ¼ 129 mg mL À1 ) was similar to the one described by Tohma & Gulçin (2010) for aqueous (EC 45 ¼ 62 mg mL À1 ) and ethanolic (EC 76 ¼ 50 mg mL À1 ) extracts.
Numerous reports have conrmed the association between phenolic compounds and bioactive properties.Regarding G. glabra, avonoids saponins, coumarins, and stilbenoids have been related with its bioactive properties.Until now, licochalcone A, B, C, D and echinatin, some isoavones and derivatives, such as glabridin, an isoavan, hispaglabridin A, hispaglabridin B and 4 0 -O-methylglabridin, but also some chalcones, namely isoprenylchalcone derivative and isoliquiritigenin, were described as possessing potent antioxidant effects, not only inhibiting lipid peroxidation but also acting as radical scavengers and oxidative process preventers. 18,27,56,57Regarding our study, it is feasible to attribute the antioxidant potential observed for the tested extract to the most abundant phenolic compounds identied, namely liquiritigenin apiosylglucosides.Nevertheless, it is important to highlight that plant extracts are usually much more effective than isolated compounds, as it was proved by Cheel et al. (2010) for the case of licorice aqueous extract.The authors veried that, despite in some assays licorice extract evidenced a weak antioxidant activity, the major components identied (liquiritin and glycyrrhizin) presented negligible or even no effects.
Overall, licorice extract could be used as a source of antioxidants for pharmaceutical, cosmetic and/or food industries.Regarding its antioxidants contribution in daily diet, further studies are necessary in order to elucidate the mechanisms of in vivo antioxidant action, bioavailability and involved metabolic pathways.

2. 5
.3.b-Carotene bleaching inhibition (CBI) assay.CBI was evaluated though the b-carotene/linoleate assay.A solution of b-carotene was prepared by dissolving b-carotene (2 mg) in chloroform (10 mL).Two millilitres of this solution were pipetted into a round-bottom ask.Aer the chloroform was removed at 40 C under vacuum, linoleic acid (40 mg), Tween 80 emulsier (400 mg), and distilled water (100 mL) were added to the ask with vigorous shaking.Aliquots (4.8 mL) of the obtained emulsion were transferred into different test tubes containing different concentrations of the samples (0.2 mL).

Table 1
Retention time (R t ), wavelengths of maximum absorption in the visible region (l max ), mass spectral data, identification and quantification of phenolic compounds in Glycyrrhiza glabra methanol/water extract a a n.q.not quantied.