Antioxidant activity, total flavonoid, total phenolic and anthocyanin contents of Cynara scolymus L. leaves and Trigonella foenum-graecum L. seeds

Annals o he Academy o Romanian Scien is s Se ies on Science and Technology o In o ma ion ISSN 2069-1149 Volume 6, Numbe 1/2017 174 ANTIOXIDANT ACTIVITY, TOTAL FLAVONOID, TOTAL PHENOLIC AND ANTHOCYANIN CONTENTS OF CYNARA SCOLYMUS L. LEAVES AND TRIGONELLA FOENUM-GRAECUM L. SEEDS Szabolcs VÍGH 1 ,2, Zol án CZIÁKY 2 , László Tamás SINKA 2, Cip ian PRIBAC 3, Liana MOŞ 3, Viole a TURCUŞ 3, and End e MÁTHÉ 2, 3, 4 * Abs ac . In he p esen compa a i e s udy we a e analyzing he an ioxidan ac i i ies, o al la onoid, o al phenolic and o al an hocyanin con en s o a ichoke (Cyna a scolymus L.) and enug eek (T igonella oenum-g aceum L.) ex ac s. Aqueous and alcoholic ex ac s we e made om equal amoun s o a ichoke lea es and enug eek seeds. The ob ained esul s a e indica ing ha he aqueous a ichoke and enug eek ex ac s an ioxidan capaci ies a e simila , while he co esponding alcoholic ex ac s an ioxidan capaci ies a e g ea ly educed. The o al phenolic con en o alcoholic a ichoke and enug eek ex ac s was much g ea e han in case o he co esponding aqueous ex ac s. The an hocyanin ype o la onoids we e no de ec ed in any o ou ex ac s made o a ichoke lea es and enug eek seeds. In case o a ichoke lea o enug eek seed ex ac s, he o al la onoid and o al phenolic con en s, p esumably, ha e e y li le i any in ol emen in he an ioxidan capaci ies o a ichoke and enug eek, espec i ely. Keywo ds: a ichoke, enug eek, DPPH, la onoid, polyphenols, an hocyanin 1. In oduc ion A ichoke Teas and ex ac s made om he lea es o he a ichoke plan a e a adi ional onic o he li e and gallbladde [1, 2]. Bo h labo a o y and clinical s udies s ongly sugges a ichoke lea ex ac o be a good he apeu ic op ion in dyspep ic synd ome (i i able s omach, ne ous gas opa hy, la ulence, i i able colon, unc ional bilia y ac disease), [2]. In Eu ope he plan is widely used o ea 1 Resea che , Ins i u e o Ag icul u al Sciences, Uni e si y o Nyí egyháza, Sos ói s . 31/B, H- 4432, Nyí egyháza, Hunga y 2 Resea che , Ag icul u al and Molecula Resea ch Ins i u e, Uni e si y o Nyí egyháza, Sos ói s . 31/B, H-4432, Nyí egyháza, Hunga y 3 Resea che , “Vasile Goldiş” Wes e n Uni e si y o A ad, Facul y o Medicine, Li iu Reb eanu S .91-93, RO-310414, A ad, Romania 4 Resea che , Facul y o Ag icul u e and Food Sciences and En i onmen al Managemen , Uni e si y o Deb ecen, Böszö ményi s . 138, H-4032 Deb ecen, Hunga y * co esponding au ho end e.ma [email protected] 177 Szabolcs VÍGH, Zol án CZIÁKY a e ioscle osis and lowe choles e ol. Se e al s udies ha e hin ed a he abili y o a ichoke ex ac o lowe lipid le els. I wo ks by a ec ing choles e ol syn hesis in he li e a se e al poin s in he syn he ic pa hway and by inc easing he elimina ion o choles e ol [1]. The plan chemical cons i uen cyna in ac s as lipolysis-inhibi o in much he same manne as nico inic acid (niacin), wi hou he gas ic upse and lushing ha niacin supplemen s could cause. The diu e ic p ope ies o he a ichoke a e also use ul in kidney disease [2]. Fenug eek Fenug eek (T igonella oenum-g aecum) is a medicinal ood plan ha has he bene icial e ec o lowe ing blood suga . The bi e seeds called “me hi” in India, a e used as a condimen , and he lea es a e used in eas. Fenug eek con ains po en an ioxidan s ha ha e bene icial e ec s on he li e and panc eas, making i use ul in he ea men o diabe es, high choles e ol, and diges i e diso de s [3, 4]. The ha d, b own, ed and yellow seeds a e he pa used medicinally and in cooking. T adi ional uses included b onchial p oblems, ube culosis, gou , gene al body pain, swollen glands, skin p oblems and low libido. 2. Ma e ials and me hods The a ichoke d ied lea es we e p oduced by he TTDR 2000 L d., Hunga y. 10 g o sample was ex ac ed o 5 min wi h 200 ml o boiling dis illed wa e (Aqueous A ichoke ex ac , AqA). I was allowed o cool o oom empe a u e (RT), il e ed and s o ed in e ige a o . Ano he 50 g o d ied a ichoke lea es we e ex ac ed wice wi h 500 ml aqueous 50% e hanol solu ion by s i ing o 4 h s on a magne ic s i e (Alcoholic A ichoke ex ac , AlA). This ex ac was il e ed, e apo a ed unde acuum and s o ed in e ige a o The enug eek seeds we e p oduced by TRIGONELLA MED. LTD., Mosonmagya ó á , Hunga y. Fenug eek seeds we e d ied a 40 °C and powde ed. 15 g o he powde was ex ac ed wi h 300 ml o boiling dis illed wa e o 5 min. The mix u e was allowed o cool a RT, il e ed and s o ed in e ige a o (Aqueous Fenug eek ex ac , AqF). O he 15 g o powde ed enug eek seeds we e e luxed on a magne ic s i e wi h 300 ml aqueous 80% e hanol o 3 h s (Alcoholic Fenug eek ex ac , AlF). The mix u e was il e ed and he il a e was concen a ed by o a y e apo a o and s o ed in e ige a o . 2.1. De e mina ion o an ioxidan capaci y by he DPPH me hod The DPPH (2,2- Diphenyl-1-pic ylhyd azyl, Sigma-Ald ich) me hod was used o de e mine he an ioxidan capaci y o a ichoke and enug eek ex ac s. The me hod is based on he sca enging o DPPH adical h ough he ac ion o an an ioxidan ha decolou izes he DPPH solu ion. The p ocedu e was desc ibed by ANTIOXIDANT ACTIVITY, TOTAL FLAVONOID, TOTAL PHENOLIC AND ANTHOCYANIN CONTENTS OF CYNARA SCOLYMUS L. LEAVES AND TRIGONELLA FOENUM-GRAECUM SEEDS 179 Molyneux (2004) [5]. 200 µl o ex ac we e added o 300 µl o me hanol and 2400 µl DPPH solu ion (22.6 µg/ml). The mix u e was le o s and a oom empe a u e o 30 minu es in he da k and il e ed (0.45 µm) be o e he abso bance was measu ed a 517 nm. (UV/VIS Spec ome e , Pe kin Elme Lambda 35). All he measu emen s we e pe o med in iplica e. The esul s we e exp essed as asco bic acid equi alen s by means o he dose- esponse calib a ion cu e. The sample concen a ion p o iding 50 % inhibi ion (IC50) was calcula ed om he g aph o inhibi ion pe cen age agains sample concen a ion. Each ex ac was measu ed in iplica e. 2.2. Quan i ica ion o o al la onoids The o al la onoid con en o he ex ac was de e mined by he aluminium chlo ide colo ime ic me hod ollowing he p ocedu e desc ibed by Dae-Ok Kim e al. (2003) [6]. We ha e made a eagen solu ion con aining 5 ml (10g/ml) o aluminium chlo ide, 5 ml o KOAc (po assium ace a e, 1M/l), 75 ml o me hanol and 140 ml o dis illed wa e (AS). 0.5 ml o he ex ac was mixed wi h 4.5 ml o AS, il e ed (0.45 µm) and he abso bance was de e mined a 415 nm in he spec opho ome e (Pe kin Elme Lambda 35). The concen a ions we e calcula ed based on he equa ion ob ained om he s anda d u in cu e (10, 40, 70, 100 µg/ml). Resul s we e exp essed as mg u in equi alen s. Each ex ac was measu ed in iplica e. 2.3. De e mina ion o o al phenolic con en To al phenolic con en was de e mined using he me hod de eloped by Single on and Rossi (1965) [7]. To al phenolic con en was es ima ed by he Folin-Ciocal eu colo ime ic me hod, using gallic acid as a s anda d phenolic compound. 200 µl o ex ac s we e added o 3000 µl o dis illed wa e , 500 µl o Folin-Ciocal eu eagen and 2000 µl o sodium ca bona e solu ion (15g/100ml), and he mix u e was allowed o s and o 20 min a RT. 4300 µl o dis illed wa e was added and he abso bance was measu ed a 765 nm a e 1 hou incuba ion pe iod wi h he spec opho ome e (Pe kin Elme Lambda 35). The o al la onoid con en s we e calcula ed om a calib a ion cu e wi h 50, 100, 300, 500, 750 µg/ml poin s and he esul s we e exp essed as mg gallic acid equi alen pe g d y weigh . Each ex ac was measu ed in iplica e. 2.4. De e mina ion o o al an hocyanins The o al an hocyanin (TA) con en was de e mined using an ea lie desc ibed me hod (Lee e al., 2005) [8]. I de ec s he o al monome ic an hocyanin 177 Szabolcs VÍGH, Zol án CZIÁKY concen a ion by he pH di e en ial me hod. The me hod is a apid and simple spec opho ome ic applica ion based on he an hocyanin s uc u al ans o ma ion ha occu s upon a change in pH (colo ed a pH 1.0 and colo less a pH 4.5). The s anda d solu ion was p epa ed by weighing 82.2 mg o cyanidin-3-glucoside chlo ide, dissol ing in dis illed wa e and dilu ing o a inal olume o 1 L in a olume ic lask. 0.3 ml o ex ac was added o 2 ml o bu e (pH=1 o pH=4.5) and 1.7 ml o dis illed wa e . Samples we e il e ed (0.45 µm), and abso bance eco ded using a Pe kin Elme Lambda 35 spec opho ome e a wa eleng hs o 520 and 700 nm, o solu ions a pH 1.0 and pH 4.5, espec i ely. Resul s we e exp essed as cyanidin-3-glucoside (% w/w) equi alen s. To al an hocyanin con en s we e calcula ed as ollows: l DFMWA TA Lmg            3 )/( 10 (1) whe e A = (A520nm – A700nm)pH 1.0 – (A520nm – A700nm)pH 4.5; MW (molecula weigh ) = 449.2 g/mol o cyanidin-3-glucoside; DF = dilu ion ac o ; l = pa h leng h in cm; ε = 26 900 mola ex inc ion coe icien , in L* mol–1 * cm–1 , o cyd- 3-glu; and 103 = ac o o con e sion om g o mg. Each ex ac was measu ed in iplica e. ANTIOXIDANT ACTIVITY, TOTAL FLAVONOID, TOTAL PHENOLIC AND ANTHOCYANIN CONTENTS OF CYNARA SCOLYMUS L. LEAVES AND TRIGONELLA FOENUM-GRAECUM SEEDS 179 3. Resul s and discussions The a ichoke and enug eek a e conside ed o ha e impo an heal h p omo ing e ec s, and many o such claims we e ela ed o hei an ioxidan p ope ies ha seemed o be co ela ed wi h he o al la onoid, o al phenolic and an hocyanin con en . The e o e we p oposed o analyze he a ichoke and enug eek an ioxidan ac i i y, o al la onoid, o al phenolic and an hocyanin con en s. All he expe imen s we e ca ied ou as we desc ibed in Ma e ials and me hods, and he ob ained esul s a e p esen ed in Table 1 and Figu e 1. Table 1) Means and s anda d e o o an ioxidan capaci y, la onoid-, phenolic- and an hocyanin con en o a ichoke and enug eek ex ac s (n=3) Spec opho ome ic me hods Aqueous a ichoke (AqA) Alcoholic a ichoke (AlA) Aqueous enug eek (AqF) Alcoholic enug eek (AlF) A e age an ioxidan capaci y (µl/ml) 23.43±0.25 1.80±0.16 29.83±0.41 5.91±0.18 A e age o al la onoid con en (mg/ml) 5.830±0.31 16.087±0.39 2.827±0.22 32.267±0.61 A e age o al phenolic con en (mg/g) 5.99±0.36 12.31±0.34 1.01±0.15 1.48±0.12 A e age o al an hocyanin con en (mg/l) 0 0 0 0 177 Szabolcs VÍGH, Zol án CZIÁKY Fig. 1. : (AC) an ioxidan capaci y, (TF) o al la onoid, (TP) o al phenolic, (AqA) aqueous a ichoke ex ac , (AlA) alcoholic a ichoke ex ac , (AqF) aqueous enug eek ex ac , (AlF) alcoholic enug eek ex ac F om he ob ained da a we can conclude ha he aqueous a ichoke and enug eek ex ac s an ioxidan capaci ies we e ela i ely high, while he alcoholic a ichoke and enug eek ex ac s an ioxidan capaci ies we e ma kedly educed as compa ed o hei co esponding aqueous coun e pa s. The aqueous ex ac compa ed o he alcoholic ex ac showed an app oxima ely 16 imes g ea e an ioxidan capaci y in case o a ichoke. In case o enug eek he aqueous ex ac showed a 5 ime g ea e an ioxidan capaci y han he alcoholic ex ac . The o al la onoid con en o a ichoke and enug eek alcoholic ex ac s exceeded subs an ially he la onoid con en seen in bo h plan s de i ed aqueous ex ac s. Acco dingly, he a ichoke speci ic alcoholic ex ac o al la onoid con en appea s 3 imes g ea e as compa ed o he aqueous ex ac . The enug eek o al la onoid con en o he alcoholic ex ac s looks almos 12 imes g ea e han he aqueous ex ac ela ed alues. Mo eo e , i he wo plan s speci ic alcoholic ex ac s a e compa ed, i looks ob ious ha he enug eek alues a e wo imes g ea e han he a ichoke alues o o al la onoid con en sugges ing ha he enug eek seeds con ain wice as much la onoid han he a ichoke. Su p isingly, when he aqueous ex ac s o a ichoke and enug eek a e compa ed, he a ichoke ex ac speci ic o al la onoid con en was app oxima ely double o ha seen in case o enug eek ex ac . The o al phenolic con en o a ichoke alcoholic ex ac exceeded mo e han 2 imes he alues obse ed o he a ichoke aqueous ex ac sugges ing ha he alcoholic a ichoke ex ac con ains wice as many phenolic compounds like he aqueous a ichoke ex ac . Howe e , he aqueous and alcoholic enug eek ex ac s seemed o con ain simila amoun o phenolic compounds. Su p isingly we we e unable o de ec an hocyanin in any o ou a ichoke and enug eek ex ac s. Conclusions (1).The aqueous a ichoke and enug eek ex ac s an ioxidan capaci ies a e almos he same, and he co esponding alcoholic ex ac s a e showing law alues sugges ing ha he alcoholic ex ac s an ioxidan capaci ies a e g ea ly educed. (2). The o al phenolic con en o alcoholic a ichoke and enug eek ex ac s was much g ea e han in case o he co esponding aqueous ex ac s, (3). I seems ha an hocyanin ype o la onoids a e no p esen in a ichoke lea es and enug eek seeds. ANTIOXIDANT ACTIVITY, TOTAL FLAVONOID, TOTAL PHENOLIC AND ANTHOCYANIN CONTENTS OF CYNARA SCOLYMUS L. LEAVES AND TRIGONELLA FOENUM-GRAECUM SEEDS 179 (4). In case o a ichoke lea o enug eek seed ex ac s, he o al la onoid and o al phenolic con en s, p esumably, ha e e y li le i any in ol emen in he an ioxidan capaci ies o a ichoke and enug eek, espec i ely. (5). Bo h a ichoke lea es and enug eek seeds should ha e an ioxidan s o he han hose ha we e assessed. I emains o ge iden i ied he an ioxidan p ope ies ensu ing compounds in he case o alcoholic ex ac s. Acknowledgmen We hank o he all membe s o he Ag icul u al and Molecula Resea ch Ins i u e om he Uni e si y o Nyí egyháza o hei suppo and meaning ul discussions. The esea ch expenses we e co e ed by he “In i o s udy o some plan ex ac s o na u al o igin wi h emphasis on hei an i- umo e ec s.” HURO/0801 Hunga ian-Romanian C oss Bo de Coope a ion 2007-2013 g an . R E F E R E N C E S [1] K. K a , Phy omedicine 4, 369 (1997). [2] R. Mabey, The New Age He balis (Simon & Schus e , New Yo k, 1988). [3] J. A. Duke, The G een Pha macy (Rodale P ess Emmaus, Pennsyl ania, 1997). [4] J. Ba nes and L. A. Ande son, He bal Medicines (Pha maceu ical P ess, London, 2007). [5] P. Molyneux, Songklanaka in J. Sci. and Technol. 26, 211 (2004). [6] D. O. Kim e al., J. Ag ic. Food Chem. 51, 6509 (2003). [7] V.L. Single on and J.A. Rossi, Ame . J. Enol Vi icul . 16, 144 (1965). [8] J. Lee e al., J. AOAC In . 88, 1269 (2005).