phenolic compounds in common spices

LWT - Food Science and Technology xxx (2013) 1e6

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Comparison of methods for evaluation of the antioxidant capacity and phenolic compounds in common spices ska b, Zuzana Ciesarová c, Kristina Kukurová c, Ma1gorzata Przygodzka a, Danuta Zielin ski a, * Henryk Zielin a Division of Food Science, Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences, Tuwima 10, P.O. Box 55, 10-748 Olsztyn 5, Poland b University of Warmia and Mazury in Olsztyn, Plac Lodzki 4, 10-957 Olsztyn, Poland c VÚP Food Research Institute, Priemyselná 4, P.O. Box 25, 824 75 Bratislava 26, Slovak Republic

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 June 2012 Received in revised form 19 September 2013 Accepted 23 September 2013

The present study investigates the antioxidant capacity (AC) of ethanol and ethanol/water (1:1, v/v) extracts of selected spices, using cyclic voltammetry (CV), spectrophotometric and photochemiluminescence (PCL) methods. The ABTS test was used for the measurement of the scavenging capacity of the extracts against 2,20 -azinobis-(3-ethylbenzothiazoline-6-sulphonate) radical cations whilst a photochemiluminescence technique was employed to measure the scavenging capacity against superoxide anion radicals (O 2 ). The cyclic voltammetry (CV) method provided the reducing capacity values of spices. The total phenolics (TPC) and total flavonoids (TF) were determined in both types of spice extracts. With the exception of vanilla, ethanol/water (1:1, v/v) was a better extraction solvent of TPC and TF in all spices when compared to ethanol alone. The order of AC values of spices determined by the applied methods was as follows: ABTS test > PCL assay > CV method. The obtained results were useful for classification of the tested spices into groups with high antioxidant capacity (clove, cinnamon and allspice), middle (star anise and nutmeg) and low AC (anise, ginger, vanilla, fennel, cardamom, white pepper and coriander). In conclusion, these findings may be useful for the selection of spices for further applications in different food formulations to support their function as antioxidants. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Spices Phenolic compounds Antioxidant capacity Photochemiluminescence Cyclic voltammetry

1. Introduction Spices are used worldwide as aromatic and pungent food ingredients. They originate from dried, edible, aromatic plants which are used in the food industry and households to add flavour and aroma showed in meals. Spices are prepared from different plant parts such as bark (cinnamon), flower buds (clove), roots (ginger), fruits (pimento), fully ripe berries (white pepper) (Suhaj, 2006) and can be added to food in form of whole or ground material or as extracts. In general, spices are used not only for flavouring foods but also due to their antiseptic and medicinal showed properties. Recently, the antimicrobial (Tajkarimi, Ibrahim, & Cliver, 2010), antiinflammatory (Mueller, Hobiger, & Jungbauer, 2010), antimutagenic and anti-carcinogenic properties of spices were

* Corresponding author. Tel.: þ48 89 5234682; fax: þ48 89 5240124. ski). E-mail address: [email protected] (H. Zielin

reported (Kaefer & Milner, 2008). Many of their health protective properties are linked to their antioxidative properties hence, spices are used in pharmacological products as well as in herbal medicine (Bythrow, 2005; Fitzgerald, Stratford, & Narbad, 2003; Gray & Flatt, 2007; Pawar, Pai, Nimbalkar, & Dixit, 2011; Peter, 2001; Stoilova, Krastanov, Stoyanova, Denev, & Gargova, 2007; Sulaiman & Ooi, 2012; Wright, Van-Buren, Kroner, & Koning, 2007). The antioxidant capacity of spices is poorly described and only limited information is available (Hinneburg, Damien Dorman, & Hiltunen, 2006; Pellegrini et al., 2006; Suhaj, 2006). The antioxidant and reducing capacity of spices is closely related to the presence of chemical constituents with antioxidant activity, mainly to the phenolic compounds (Parthasarathy, Chempakam, & Zachariah, 2008; Peter, 2001, 2004; Steinhaus & Schieberle, 2005) (Table 1). For example, the major phenolic compounds identified in star anise are kaempferol, quercetin and their glycosides (Wang, Hu, Huang, & Qiu, 2011) whilst in nutmeg the presence of lignans, eugenol, apinene, b-pinene, b-caryophyllene, p-cymene and carvacrol was reported (Parthasarathy et al., 2008; Peter, 2001). In anise nine

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Please cite this article in press as: Przygodzka, M., et al., Comparison of methods for evaluation of the antioxidant capacity and phenolic compounds in common spices, LWT - Food Science and Technology (2013), http://dx.doi.org/10.1016/j.lwt.2013.09.019

2

M. Przygodzka et al. / LWT - Food Science and Technology xxx (2013) 1e6

Table 1 Chemical constituents with antioxidant capacity in spices. Spices

Components

Nutmeg

Sabinene, a-pinene, b-pinene, terpinen-4-ol, g-terpinene, myristicin, campfene, myrcene, a-hellandrene, a-terpinene, limonene, 1,8-cineole, p-cymene, terpinolene, transsabinene hydrate, copaene, linalool, cis-sabinene hydrate, cis-p-menth-2-en-ol, cis-piperitol, safrole, methyl eugenol, eugenol, elemicin (Parthasarathy et al., 2008; Peter, 2001) trans-Anethole, fenchone, estragole, limonene, camphene, a-pinene, fenchyl alcohol, anisaldehyde, myristicin, dillapiole (Hossain et al., 2008; Lu et al., 2011; Parthasarathy et al., 2008) Chlorogenic acid isomers, trans-anethole, estragole, anise ketone, other components in minor concentrations: bcaryophyllene, anisaldehyde, anisic acid, linalool, limonene, a-pinene, acetaldehyde, p-cresol, creosol, hydroquinine, bfarnasene, g-himachalene ar-curcumene (Lv et al., 2012; Peter, 2001; Wojdy1o et al., 2007) Eugenol, eugenol acetate, b-caryophyllene, a-cububene, acopaene, isoeugenol, nerolidol, farnesol (Kaefer & Milner, 2008; Peter, 2001) Vanillin, vanillic acid, p-hydroxybenzaldehyde, phydroxybenzoic acid, linoleic acid, acetic acid, hexadecanoic acid, p-hydroxybenzyl alcohol, vanillyl alcohol, acetovanillone, anisic acid (Parthasarathy et al., 2008) 1,8-Cineole, a-terpinyl acetate, limonene, borneol, methyl eugenol, a-pinene, b-pinene, sabinene, myrcene, aphellandrene, g-terpinene, p-cymene, terpinolene, linalool, linalyl acetate, a-terpineol, a-terpinyl acetate, citronellol, nerd, geraniol, trans-nerolidol (Agbor et al., 2006; Peter, 2001) a-Pinene, linalool, b-damascenone, eugenol, skatole, mcresol, guaiacol, piperonal (Steinhaus & Schieberle, 2005) a-Zingiberene, geranial, geraniol, b-bisabolene, nerol, 1,8cineol, a-terpineol, borneol, b-phellandrene, linalool, methyl nonyl ketone, camphene (Parthasarathy et al., 2008) Kaempferol, quercetin and their glycosides (Wang et al., 2011), trans-anethole, a-pinene, camphene, b-pinene, linalool, safrole, anisaldehyde, acetoanisole, estragole, limonene, p-allylanisole, anisyl methyl ketone, 1,8-cineole, p-cymene, terpinen-4-ol, a-terpineol, d-3-carene, aphellandrene, b-phellandrene (Parthasarathy et al., 2008; Peter, 2004) Methyl eugenol, eugenol, myrcene, b-caryophyllene, apinene, b-pinene, (e)-b-ocimene, a-thujene, sabinene, d-3carene, a-phellandrene, limonene, b-phellandrene, pcymene, a-terpinene, b-terpinene, terpinolene, 1,8-cineole, linalool, terpinen-4-ol, p-cymen-8-ol, a-terpineol, p-menth2-en-1-ol, a-humulene, a-selinene, b-selinene, d-cadinene, b-elemene, allo-aromadendrene, germacrene, spathulenol, caryophyllene oxide, viridiflorol, humulene oxide, transcadinol, trans-muurolol, a-uurolol, a-cadinol, selin-11-en4-ol, caryophylla-2(12), 6(7)-dien-5-ol, chavicol, myristicin, elemicin (Kaefer & Milner, 2008; Peter, 2004) Linalool, a-pinene, g-terpinene, geranyl acetate, camphor, geraniol, b-pinene, camphene, myrcene, limonene, p-cymol, dipentene, a-terpinene, n-decylaldehyde, borneol, acetic acid esters (Peter, 2004) Cinnamaldehyde, eugenol, eugenol acetate, cinnamyl acetate, cinnamyl alcohol, linalool, methyl eugenol, benzaldehyde, cinnamaldehyde, benzyl benzoate, monoterpene, hydrocarbon, caryophyllene, safrole, pinene, phyllandrene, cymene, cineol (Peter, 2001)

Fennel

Anise

Clove

Vanilla

Cardamom

White pepper Ginger

Star anise

Allspice

Coriander

Cinnamon

chlorogenic acid isomers were recently identified (Lv et al., 2012; ski, & Czemerys, 2007). Allspice and clove buds Wojdy1o, Oszmian are both rich sources of eugenol (Kaefer & Milner, 2008; Peter, 2001, 2004). Fennel was shown to be rich in trans-anethole, fenchone, estragole, limonene, camphene, a-pinene, fenchyl alcohol, anisaldehyde, myristicin and dillapiole (Hossain, Brunton, Barry-Ryan, Martin-Diana, & Wilkinson, 2008; Lu, Yuan, Zeng, & Chen, 2011; Parthasarathy et al., 2008). White pepper contains lignans,

alkaloids, flavonoids, aromatic compounds and amides (Steinhaus & Schieberle, 2005) while limonene, caffeic acid, 1,8-cineole and its esters, a-terpinyl and linalyl acetates have been reported as the most abundant bioactive compounds in cardamom (Agbor, Vinson, Oben, & Ngogang, 2006; Peter, 2001). The wide spectrum of chemical constituents with antioxidant capacity in spices is shown in Table 1, which provides a comprehensive view on the diversity of the compounds. The relationship between antioxidant properties of spices and health has an increasing impact on food innovation due to the popularity of the concept of functional food (Balestra, Cocci, Pinnavaia, & Romani, 2011; Ciesarová, Suhaj, & Horvátková, 2008). For that reason there is the need to identify suitable methodologies for the characterization of their antioxidative potential. A great multiplicity of methods for the determination of antioxidant capacity has been reported such as the ABTS assay, DPPH radical scavenging activity, oxygen radical absorption capacity, ferric reducing antioxidant power and sensitive electrochemical and photochemiluminescene approaches (Katalinic, Milos, Kulisic, & Juki, 2006; Magalhaes, Segundo, Reis, & Lima, 2008; Prior, Wu, ska, Wiczkowski, & Pisku1a, 2008). & Schaich, 2005; Zielin Amongst them, rapid electrochemical methods for antioxidant assays have emerged in the past decade based on the fact that one of the modes of action of antioxidants is the ability to donate electrons (Chevion, Roberts, & Chevion, 2000). To the best of our knowledge, voltammetric, photochemiluminescent or spectrophotometric methods have never been applied to the same samples to study the antioxidant capacity of spices. In this study a collection of spices was selected due to their common use as aromatic and pungent food ingredients. The first aim of this work was to investigate the antioxidant capacity (AC) of ethanol or ethanol/water (1:1, v/v) extracts of selected spices, using cyclic voltammetry (CV), spectrophotometric and photochemiluminescence (PCL) methods. The second objective was to determine whether the content of total phenolics and total flavonoids correlate with antioxidant and reducing capacity of spices. 2. Material and methods 2.1. Chemicals 2,20 -Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 6-hydroxy-2,5,7,8-tetramethylchroman-2carboxylic acid (Trolox), (þ)-catechin, gallic acid, aluminium chloride hexahydrate (AlCl3 6H2O, 98%) and sodium nitrite (NaNO2, 97%) were obtained from Sigma (Sigma Chemical Co., St. Louis, MO, USA). PCL ACW (Antioxidant Capacity of Water-soluble substances) kit was supplied by Analytik Jena AG (Jena, Germany). Ethanol (96%), FolineCiocalteu reagent and sodium carbonate (Na2CO3) were purchased from POCh, Gliwice, Poland. Sodium hydroxide (NaOH) was obtained from Chempur (Poland). Water was purified using the Mili-Q-system (Milipore, Bedford, USA). 2.2. Materials Commercially available spices were provided by local firms from the Slovak and Czech Republic (Mäspoma spol. s r.o., Zvolen, Slovak Republic, Johann Kotányi Ltd., Praha, Czech Republic). The spices included anise (Pimpinella anisum L.), star anise (Illicium verum Hook f.), white pepper (Piper guineense L.), fennel (Foeniculum vulgare Mill.), cardamom (Elettaria cardamomum L.), clove (Syzygium aromaticum L.), coriander (Coriandrum sativum L.), nutmeg (Myristica fragrans Houtt.), allspice (Pimenta dioica L.), cinnamon (Cinnamomum zeylanicum), vanilla (Vanilla Mill.), ginger (Zingiber officinale Rosc.) and spice mix used for ginger cake and bread preparation. According to the producer’s declaration, spice mix



contained cinnamon, pepper, clove, anise, coriander, fennel and nutmeg. 2.3. Preparation of extracts from spices

3

temperature-controlled spectrophotometer with a CPS-Controller (Shimadzu, Tokyo, Japan). The antioxidant capacity was expressed as mmol Trolox/g dry matter (DM). 2.6.2. Superoxide radical anion (O2) scavenging capacity assay The photo-induced chemiluminescence (PCL) assay, carried out using the method proposed by Popov and Lewin (1999), was performed to measure the antioxidant activity of spice extracts against superoxide anion radicals (O2) generated from luminol, a photosensitizer, under exposure to UV light. The detailed protocol was ska et al. (2008). The antioxidant carecently described by Zielin pacity of spice extracts was determined using the ACW analytical kit for measuring the antioxidant capacity of hydrophilic compounds and a PhotochemÒ apparatus (Analytik Jena, Leipzig, Germany). Antioxidant capacity was expressed as mmol Trolox/g dry matter (DM).

Approximately 100 mg of dried and pulverized spice was extracted with 1 mL of ethanol or with a mixture of ethanol/water (1:1, v/v) by sonication (30 s). The mixture was vortexed for 30 s, then sonicated and centrifuged for 5 min (5000g, 4 C). This step was repeated five times (the residue was each time re-suspended in 1 mL of fresh solvent). The supernatants were combined and collected in a 5 mL volumetric flask. Three independent extractions were carried out on each spice sample. Finally, extracts were kept at 20 C prior to further determination of the total phenolics (TPC) flavonoids (TF), and antioxidant capacity by ABTS, PCL and CV assays. 2.4. Determination of total phenolic content (TPC) TPC of the crude spice extracts (20 mg/mL of solvent) was determined according to Shahidi and Naczk (1995). Briefly, 0.25 mL of the extract was mixed with 0.25 mL FolineCiocalteu reagent, previously diluted with distilled water (1:1, v/v), 0.5 mL saturated sodium carbonate (Na2CO3) and 4 mL of water. The mixture was incubated at room temperature for 25 min and centrifuged at 2000g for 10 min. Supernatant absorbance was measured at 725 nm using a spectrophotometer (UV-160 1PC, Shimadzu, Tokyo, Japan). TPC was standardized against gallic acid (GA) and expressed in terms of mg GA equivalents (GAE)/g dry matter (DM). The linearity range for this assay was determined as 0.02e0.3 mg GA/mL (R2 ¼ 0.99), giving an absorbance range of 0.10e1.90 AU. 2.5. Determination of total flavonoid content (TF) TF content was determined with a colourimetric method according to Jia, Tang, and Wu (1998). Briefly, 0.25 mL of ethanol or ethanol/water extracts of spices was diluted with 1.25 mL of distilled water. Then 75 mL of aqueous NaNO2 solution with the concentration of 5 g/100 mL was added, and the mixture was allowed to stay at room temperature. After 6 min, 150 mL of AlCl3 6H2O solution (10 g/100 mL) was added, and the mixture was allowed to stand for another 5 min. After that, 0.5 mL of 1 mol/L NaOH was added. The solution was thoroughly mixed, and the absorbance measured immediately against the prepared blank sample at 510 nm using a spectrophotometer (UV-160 1PC, Shimadzu, Tokyo, Japan). TF content was standardized against (þ)-catechin (CA) and the results were expressed as mg CA/g dry matter (DM). The linearity range for this assay was determined at 0.01e1.0 mg CA/mL (R2 ¼ 0.99).

2.6.3. The cyclic voltammetry (CV) method A potentiostat/galvanostat (GAMRY, Warminster, PA, USA) was used for voltammetric experiments as it was recently reported in ska et al., 2008). Cyclic voltammetric experiments detail (Zielin were performed on ethanol/water (1:1, v/v) and ethanol spice extracts mixed with 0.2 mol/L sodium acetate-acetic buffer (pH 4.5) at a ratio of 1:1 (v/v) in ethanol or a mixture of ethanol/water (Cosio, Buratti, Mannino, & Benedetti, 2006). The measurements were performed at room temperature in the apparatus cell (volume 200 mL), to which respective extracts mixed with the buffer solution were introduced. 100 mL of each extract and 100 mL of buffer solution were used in the assay. The cyclic voltammograms were acquired in the range of 100 to þ1200 mV at a scanning rate of 100 mV s1 and at 2 mV intervals. The total charge was measured below the anodic wave curve of the voltammogram. Ethanol or ethanol/water (1:1, v/v) solutions of Trolox within the concentration range of 0.05e2.50 mmol/L were used and the results were expressed as mmol Trolox/g DM. The total charge under the anodic wave of the background signal (solvent þ supporting electrode) was subtracted from the total charge under the anodic wave obtained for each sample measured within the range of þ100 to þ1100 mV. Triplicate samples were run for each set. 2.7. Statistical analyses The results of the chemical analyses are illustrated as the means and the standard deviation of three independent measurements. Statistical analysis was performed using Student’s t-test with the significance level set at p < 0.05. The Statistica 7.1.30.0 software (Statsoft Inc., USA) for Windows was used. The correlation analysis between TPC and TF and antioxidant capacity of spices was performed by each applied method and the Pearson correlation coefficient was calculated.

2.6. Determination of the antioxidant capacity of spices 3. Results and discussion 2.6.1. Scavenging of 2,2-azinobis-(3-ethylbenzothiazoline-6 sulphonate) radical cation (ABTS þ) assay The method described by Re et al. (1999) was used to determine the antioxidant capacity of spice extracts. To perform the mea surements, the ABTS þ solution was diluted with a mixture of ethanol/water, to the absorbance level of 0.70 0.02 at 734 nm. For the spectrophotometric assay, 1.48 mL of the ABTS þ solution and 20 mL of the respective extract or the Trolox solution were mixed, and absorbance was measured directly after 6 min at 734 nm and at 30 C. The standard curve was plotted based on the length of the lag phase versus Trolox concentrations within the range of 0.1e 2.5 mmol/L of Trolox standard solutions in a mixture of ethanol/ water. Measurements were carried out using the UV-160 1PC

3.1. Total phenolics (TPC) and flavonoids content (TF) of spices The TPC content as determined by the FolineCiocalteu reagent is shown in Fig. 1. The studies showed that a mixture of ethanol/water (1:1, v/v) was a better extraction solvent of TPC compared to ethanol alone. About twofold (cinnamon, clove, spice mix and cardamom), fourfold (ginger, fennel and anise), eightfold (allspice and coriander) and twenty fold (star anise) increases in TPC were noted in this case. Our findings are in agreement with the results reported by Liu, Qiu, Ding, and Yao (2008) who showed also high TPC in clove among other spices. No change in TPC after extraction by both solvents was noted for nutmeg and white pepper while in


4


TPC (mg GAE/g DM)

200

150

100

50

se Ca rd am on te pe pp er Co ria nd er

el

ni

W hi

A

lla

Fe nn

ge r in G

Va ni

ix

eg ut m

N

ve

ic em

Cl o

Sp

St ar an ise Ci nn am on A lls pi ce

0

Fig. 1. Effect of solvent extraction on the total phenolics content (TPC) of spices. The light-grey columns indicate TPC contents after extraction performed with mixture of ethanol/water (1:1, v/v) (n ¼ 3). The dark columns indicate TF contents after extraction performed with ethanol (n ¼ 3).

vanilla extracts a sevenfold higher content of TPC was noted in ethanol extracts indicating that active compounds present in vanilla have lower polarity and therefore they are more suitable for extraction by ethanol (Jager, Perfetti, & Diachenko, 2007). The ethanol/water extracts of star anise, cinnamon, allspice and clove showed the highest TPC values within the range of 140.8e 170.4 mg GAE/g DM while the lowest TPC values (2.2e10.8 mg GAE/ g DM) were obtained for extracts of nutmeg, ginger, vanilla, fennel, anise, cardamom, white pepper and coriander (Fig. 1). The TPC of spice mix (90.5 mg GAE/g DM) reflected the composition of the mix, containing cinnamon, pepper, clove, anise, coriander, fennel and nutmeg. The ethanol extract of cinnamon, clove and vanilla showed the highest TPC values (54.3e72.4 mg GAE/g DM). Our findings indicate the importance of solvent selection for TPC extraction. Previously this relation was reported by Hinneburg et al. (2006) who showed twice higher TPC in hydrodistilled extracts of cardamom, ginger, anise and fennel in comparison to the ethanol/ water extracts. For industrial purposes ethanol or aqueous ethanol would be probably better than the most commonly employed methanol (Suhaj, 2006). The total flavonoids content of spices is illustrated in Fig. 2. The mixture of ethanol/water (1:1, v/v) was a better extraction solvent of TF when compared to ethanol. The highest content of TF was noted in ethanol/water extracts of cinnamon and spice mix (96.8 and 64.2 mg catechin/g DM, respectively) while the lowest ones (1.1e2.5 mg catechin/g DM) were observed for white pepper, cardamom, coriander and fennel. In this study about twofold (anise) and threefold (star anise and allspice) higher TF content was noted after extraction with ethanol/water as compared to

TF (mg catechin/g DM)

100

75

50

25

se Ca rd am W on hi te pe pp er Co r ia nd er

ne l

ill a

An i

Fe n

Va n

ge r Gi n

m eg

Nu t

m ix

Cl ov e

Sp ice

St ar a

ni se Ci nn am on A lls pi ce

0

Fig. 2. Effect of solvent extraction on the total flavonoids (TF) content of spices. The light-grey columns indicate TF contents after extraction performed with mixture of ethanol/water (1:1, v/v) (n ¼ 3). The dark columns indicate TF contents after extraction performed with ethanol (n ¼ 3).

extraction by ethanol alone, while no significant differences in TF contents after extraction by either solvents from white pepper and fennel were observed. Exceptions were noted for clove, vanilla, ginger and cardamom. In this case, the use of ethanol extraction is recommended. Previously, ethanol was used by Takácsová, Kristianova, and Vinh (1999) for the extraction and isolation of antioxidant chemicals from ginger, nutmeg and coriander. TF highly contributed to the pool of phenolic compounds determined with the FolineCiocalteu (FC) reagent. The TF contribution to TPC as above 50% was noted for spice mix (71%), coriander (100%), nutmeg (94%), cardamom (67%), cinnamon (60%), ginger (60%) and vanilla (57%). The percentage of the contribution as well as a wide range of TPC values provided in this study reflects the diversity of the chemical constituents in spices as well as their ability to reduce the FC reagent (Prior et al., 2005). In this study TPC of spices were positively correlated with TF content (r ¼ 0.64, p ¼ 0.078) when ethanol/water was used for extraction. 3.2. The antioxidant capacity (AC) of spices provided by ABTS, PCL and CV assays The antioxidant capacity of spices determined against ABTS þ radical is shown in Table 2. The ethanol/water (1:1, v/v) and ethanol extracts were employed to determine the antioxidant capacity of spices, however the use of ethanol/water (1:1, v/v) extracts resulted in higher values of AC with the exception of nutmeg, ginger and vanilla. The ethanol/water extracts from clove, spice mix and cinnamon showed the highest ABTS scavenging capacity within the range of 1120e2071 mmol Trolox/g DM while AC of allspice, star anise and nutmeg ranged from 290 to 718 mmol Trolox/g DM. The ginger, vanilla, fennel, anise, cardamom, white pepper and coriander formed the group of spices characterized by the lowest AC values (14e74 mmol Trolox/g DM). These results were in agreement with those reported by Shan, Cai, Sun, and Corke (2005) and Hossain et al. (2008), despite of the fact that they used 80% methanol extract of spices. In our study, a positive correlation between TPC and TF content versus AC provided by the ABTS assay (r ¼ 0.67 at p ¼ 0.027 and r ¼ 0.65 at p ¼ 0.017, respectively when ethanol/water extracts were investigated) was noted. The correlation between the total phenols and antioxidative capacity has been previously reported by Zheng and Wang (2001). The rank of AC values of spices provided by the ABTS assay after extraction with ethanol/water (1:1, v/v) was as follows: clove z spices mix > cinnamon > allspice > star anise > nutmeg >> fennel z anise z cardamom z vanilla z ginger z white pepper > coriander. This similar rank was noted for AC values after extraction with ethanol. The antioxidant capacity of spices determined against super oxide radical anion (O2) is shown in Table 2. The AC of ethanol/ water (1:1, v/v) and ethanol extracts from spices represents their ability to scavenge O 2 radicals generated from luminol, a photosensitizer, when exposed to UV light (Popov & Lewin, 1999). The AC of star anise, cinnamon, nutmeg, cardamom and white pepper was 5e8 times lower when compared to those observed using ABTS after extraction with ethanol/water. In contrast, the AC of fennel, anise and coriander was comparable with the AC values obtained by the ABTS assay. Higher AC values after extraction with ethanol were noted for cinnamon, vanilla, ginger, nutmeg, white pepper and cardamom, thus supporting our findings related to the content of TPC and TF (Figs. 1 and 2, respectively). In this study, only a positive correlation was found after extraction with ethanol (r ¼ 0.76 at p ¼ 0.108 and r ¼ 0.66 at p ¼ 0.071). The rank of AC values of the spices provided by the PCL assay after extraction with ethanol/water (1:1, v/v) was as follows: clove > allspice > spices mix > cinnamon > star anise z fennel z anise z ginger > nutmeg > coriander > vanilla z white pepper z cardamom. The rank of AC



5

Table 2 Antioxidant capacity of spices as determined by three different methods. Against ABTS þ radicals (mmol Trolox/g DM)

Spices

After ethanol/water extraction (1:1, v/v) Star anise Cinnamon Allspice Clove Spices mix Nutmeg Ginger Vanilla Fennel Anise Cardamom White pepper Coriander

500.4 1119.9 718.8 2071.1 1991.3 289.8 39.4 46.3 73.7 61.6 46.1 43.1 14.1

14.7c,B 199.2e,A 10.8d,B 75.5f,B 61.9f,B 14.1b,A 0.8a,A 3.6a,A 2.8a,B 0.8a,B 2.1a,B 0.1a,A 2.9a,B

After ethanol extraction 66.8 1057.1 280.2 926.8 124.2 358.5 52.8 102.3 26.8 12.7 23.7 40.4 5.6

1.3abc,A 19.3g,A 1.5d,A 64.2f,A 0.5c,A 19.2e,B 0.2abc,B 14.7bc,B 0.4ab,A 0.8a,A 0.4ab,A 2.1ab,A 0.5a,A

Photochemiluminescence assay (PCL) (mmol Trolox/g DM)

Cyclic voltammetric method (CV) (mmol Trolox/g DM)

After ethanol/water extraction (1:1, v/v)

After ethanol/water extraction (1:1, v/v)

67.7 177.4 269.3 886.1 195.2 37.6 53.35 14.1 64.6 64.5 5.4 9.0 22.6

10.2e,B 5.2f,A 22.9h,B 28.9i,B 2.1g,B 1.9c,A 4.26d,B 0.8ab,A 0.1de,B 0.2de,B 0.2a,A 1.0a,A 0.7b,B


0.7c,A 17.1h,B 7.7f,A 3.9i,A 0.2bc,A 5.6d,B 6.0e,A 0.5g,B 0.7c,A 0.2bc,A 0.4ab,B 0.1c,B 0.3a,A

139.6 39.8 40.7 128.9 14.7 85.2 103.3 159.3 36.9 44.3 67.2 64.9 10.6

7.9h,B 1.4 bc,A 0.3bc,B 0.9g,B 0.4a,B 0.9e,B 7.5f,B 1.2i,B 0.9b,B 1.3c,b 0.8d,B 0.8d,B 1.1a,A


14.2i,A 1.5e,B 0.4c,A 3.0h,A 0.8a,A 0.2g,A 2.1f,A 2.7j,A 0.4bc,A 1.0d,A 0.9bc,A 0.8e,A 0.2ab,A

Data expressed as means standard deviations of three independent extractions (n ¼ 3). Means in each column followed by different lower case letter indicate significant differences (p 0.05). Means in the same row and for each assay followed by a different capital letter indicate significant differences due to the extraction (p 0.05).

values of the spices provided by the PCL method was very similar to that obtained by the ABTS assay and a highly positive correlation was observed for both the ethanol/water and ethanol extracts (r ¼ 0.78 at p ¼ 0.076 and r ¼ 0.89 at p ¼ 0.395, respectively). The similarity of both ranks provided by ABTS and PCL assays enabled the discrimination of the tested spices into those with high antioxidant capacity (clove, cinnamon and allspice), middle (star anise and nutmeg) and low AC (anise, ginger, vanilla, fennel, cardamom, white pepper and coriander). In this study, the antioxidant capacity of spices was measured by cyclic voltammetric method (CV) which is very useful for the rapid screening of antioxidant capacity of food sample of different origin. The representative cyclic voltammograms of the ethanol/water extracts from clove, allspice, ginger and vanilla (20 mg/mL) were recorded from 100 to þ1200 mV at a scanning rate of 100 mV s1 (Fig. 3). These samples represented spices with high and average AC values as measured by ABTS and PCL assays. The area under the curve (AUC) was taken to reflect the total antioxidant capacity of the spices (Chevion

et al., 2000; Ragubeer, Beukes, & Limson, 2010). The higher AUC indicated a higher antioxidant capacity of the investigated extracts. The results are compiled in Table 2. The AC of spices was about twofold higher after extraction with ethanol/water as compared to extraction by ethanol alone (allspice, clove, spices mix, ginger, fennel and cardamom). For the rest of the spices, their AC was only slightly higher after extraction with ethanol/water with the exception of cinnamon. The AC of spices evaluated by the CV assay was lower when compared to those values provided by the ABTS and PCL assays. Moreover, no correlation was noted between AC values provided by CV and ABTS or PCL assays. The rank of AC values of spices provided by the CV method after extraction with ethanol/water (1:1, v/v) was as follows: vanilla > star anise > clove > ginger > nutmeg > cardamom > white pepper > anise z allspice z cinnamon z fennel > spices mix > coriander. The high AC values determined by CV for vanilla, may indicate the presence of reducing species rather than radical scavengers. A previous study reported the highest antioxidant activity for vanilla in the peroxidase-based assay while cinnamon exhibited a higher percentage of inhibition of oxidation than anise, ginger, nutmeg and vanilla (Murcia et al., 2004).

35

4. Conclusion 30

The studies showed that aqueous ethanol/water (1:1, v/v) was a better extraction solvent of TPC and TF when compared to ethanol with the exception of vanilla. The ethanol/water extractable TPC and TF were correlated with the AC of spices provided by both ABTS and PCL assays while no correlation was noted with the AC as determined by the CV method. The order of AC values of spices determined by applied methods was as follows: ABTS test > PCL assay > CV method. Based on the obtained results, the spices used in this study can be divided into groups with high antioxidant capacity (clove, cinnamon and allspice), middle (star anise and nutmeg) and low AC (anise, ginger, vanilla, fennel, cardamom, white pepper and coriander). In conclusion, these findings may be useful for the selection of spices for further application in different food formulations to support their function as antioxidants.

vanilla 25 clove

Ia/ µA

20 15

ginger

10

allspice

5 0 -5 -0.1

0.1

0.3

0.5 0.7 E/ V vs. Ag/AgCl

0.9

1.1

1.3

Fig. 3. Cyclic voltammograms of the ethanol/water extracts from clove, allspice, ginger and vanilla. Measurements were performed on ethanol/water extracts (20 mg/mL) mixed with 0.2 mol/L sodium acetate-acetic buffer (pH 4.5) at a ratio of 1:1 (v/v) in the mixture of ethanol/water at a ratio of 1:1 (v/v); scan rate 100 mV s1.

Acknowledgements This work was supported by grant No. 2012/07/N/NZ9/02250 from the National Science Centre and by the Slovak Research and Development Agency under the contract Nos. LPP 0310-09 and SK-


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