The Open Horticulture Journal, 2014, 7, 1-5 1

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1 Send Orders for Reprints to The Open Horticulture Journl, 214, 7, Open Access Incresed Antioxidnt Activity, Despite Reduced Rosmrinic Acid Accumultion in Methnolic Extrcts, of Spermint (Menth spict L.) Plntlets Regenerted In Vitro Dni Fdel 1, Spiridon Kintzios 2,*, Athnsios S. Economou 1, Georgi Moschopoulou 2 nd Helen Isis A. Constntinidou 3 1 Deprtment of Horticulture, School of Agriculture, Aristotle University, Thessloniki, Greece; 2 Lortory of Plnt Physiology, Fculty of Agriculturl Biotechnology, Agriculturl University of Athens, Athens, Greece; 3 Deprtment of Crop Science, School of Agriculture, Aristotle University, Thessloniki, Greece Astrct: One hundred spermint (Menth spict) plntlets were regenerted from picl shoot segments of ten fieldgrown donor plnts. Although the ccumultion of rosmrinic cid nd totl phenolics in vitro ws lmost hlf thn in vivo, regenernts demonstrted tenfold-higher hydrogen peroxide compred to the donor plnts. This finding my hve een ssocited with the incresed ctivity of hydroxyphenylpyruvte reductse, key enzyme of the phenolic iosynthetic pthwy nd the incresed production of yet unidentified phenolic compounds in vitro. This process of in vitro culture ssocited with reduction of rosmrinic cid nd totl phenolics nd with n increse of the ntioxidnt cpcity indicted the possile promotion of in vitro-specific iosynthetic pthwys. Keywords: Menth spict L., ntioxidnt ctivity, in vitro regenertion, phenolics, rosmrinic cid. INTRODUCTION Spermint (Menth spict L., Lmicee) is herceous perennil plnt, which is worldwide cultivted for its leves, contining essentil oils (giving the chrcteristic minty rom when crushed) nd phenolic compounds with ntioxidnt properties [1]. Among them, rosmrinic cid, - O-cffeoyl-3,4-dihydroxyphenyllctic cid, is the most prominent ntioxidnt phenolic constituent of the Lmicee, Borgincee nd Apicee fmilies [2, 3], with plethor of phrmceuticl properties. As hyrid, spermint is rrely propgted y seeds nd s consequence, is exclusively propgted y vegettive cuttings nd micropropgtion [4-6]. So fr, the ntioxidnt ctivities of spermint hve een strictly ssocited with its rosmrinic cid content [7], while no study so fr hs reported the ccumultion of rosmrinic cid, totl phenolics nd ntioxidnt properties of spermint plntlets derived from in vitro cultures. Therefore, the im of the present study ws to investigte the rosmrinic cid nd totl phenolic content of spermint (Menth spict L.) plnts regenerted in vitro, long with the ntioxidnt ctivity of their methnolic extrcts. MATERIALS AND METHODS Explnt Preprtion One hundred spermint plntlets were regenerted in vitro from picl shoot segments of ten donor plnts, following modifiction of previously descried protocol [8]. *Address correspondence to this uthor t the Lortory of Plnt Physiology, Fculty of Agriculturl Biotechnology, Agriculturl University of Athens, Athens, Greece; Tel: ; Fx: ; E-mil: skin@u.gr Plntlet Regenertion For shoot formtion, the nodl segments were plced verticlly for four weeks in Murshige nd Skoog (MS) medium contining 3 % (w/v) sucrose,.8% (w/v) gr,.1 % (w/v) myoinositol nd supplemented with 1 mg/l 1-nphtlene cetic (NAA) nd 9 mg/l 6-enzyldenine (BA). The ph ws djusted to 5.8. The in vitro cultures were sucultured two times four week intervls nd were mintined in wlk-in growth room with temperture of 24 ± 2ºC nd light of 16 h dily from cool-white fluorescent lmps t photosynthetic photon flux density of 8 mol m -2 s -1. At the end of the 12-week culture period, regenerted shoots were seprted nd individully trnsferred for four weeks to growth regultor-free MS medium for rooting. Rooted plntlets were susequently trnsferred to pots contining soil nd pet (1:1). In vitro 16 weeks old regenerted plntlets were sujected to further nlysis. Determintion of Totl Phenolics nd Rosmrinic Acid Concentrtion The determintion of totl phenolics concentrtion in the regenerted plntlets ws performed s reported previously [8]. One hundred l of ech phenolic liquot ws ligned nd drop of pure rosmrinic cid for identifiction ws spotted t 1 cm from the low edge of TLC. The TLC ws run in system of C 4 H 1 O: CH 3 COOH: H 2 O (3:1:1). After UV identifiction, rosmrinic cid ws isolted, collected nd centrifuged t 13 rpm for 5 min. The superntnt ws collected nd the totl volume ws djusted to 5 μl. The concentrtion of rosmrinic cid ws expressed in terms of mg RA equivlent / g fw t 333 nm on Ultrviolet-Visile (UV-VIS) nd HPLC method ws pplied to methnolic extrcts hving / Benthm Open

2 RA concentrtion(mg/g f.w.) 2 The Open Horticulture Journl, 214, Volume 7 Fdel et l. the est 18 nd the worst 1 rosmrinic cid contents nd the correltion mong dt ws studied [9]. Hydrogen Peroxide nd Key Enzymes Activity The estimtion of hydrogen peroxide scvenging ility of phenolic extrcts ws done y replcement of titrtion method with simple titrtion using NS 2 O 3 [1]. The enzyme ctivity of L-phenyllnine mmoni lyse (PAL) nd Hydroxyphenylpyruvte reductse (HPPR) in the plnt phenolic iosynthetic pthwy ws estimted y mesuring respectively the production of trns-cinnmic cid nd hydroxyphenyllctic cid [11, 12]. PAL ctlyzes the oxidtive desmintion of phenyllnine, resulting into the formtion of trns-cinnmic cid, while HPPR is responsile for the reduction of 4-dihydroxyphenylpyruvte to 4- dihydroxyphenyllctte [13, 14]. Sttisticl Anlysis The tretments of ech experiment were setup in completely rndomized design. The men of two reding for ech smple ws registered. Sttisticl nlysis ws sed on nlysis of vrince (ANOVA). Significnt differences (p<.1) mong the mens were determined y Duncn s multiple rnge test. RESULTS AND DISCUSSIONS Shoot Prolifertion, Rooting nd Plntlet Production Ten shoots from ech nodl culture were otined. At the end of the 12 week culture period, regenerted shoots rooted t essentilly 99% were seprted nd individully trnsferred for four weeks to growth regultor-free MS medium. Rooted plntlets were susequently trnsferred to pots contining soil nd pet (1:1). In vitro regenertion nd genetic trnsformtion of Menth species were lso discussed [4]. Other reporters found tht BA proved est for shoot regenertion nd growth responses of some cultivrs of nodl explnt culture of Menth rvensis [8]. Totl Phenolic nd Rosmrinic Acid Concentrtion The verge totl phenolics nd RA concentrtion in the field-grown donor spermint plnts ws lmost doule thn A in regenernts (Fig. 1A, B). Considerle vrition in totl RA concentrtion ws oserved oth in the donor plnt nd the regenernts. Consequently, regenernts were clssified ccording to their RA content into high, medium nd low RA ccumultors (Tle 1). Single fctor ANOVA nlysis ws done (Tle 2) nd the differences etween mens were tested ccording to Duncn s multiple rnge test (Tle 3). A high positive correltion ws oserved etween totl phenolic nd RA concentrtion in high nd medium RA ccumultors (Tle 4). Although the process of in vitro culture ws ssocited with reduction of RA nd totl phenolics, the ntioxidnt cpcity ws incresed, t lest s fr s the scvenging ctivity of hydrogen peroxide is concerned, thus indicting the possile promotion of in vitrospecific iosynthetic pthwys. On the contrry, the correltion in low RA ccumultors ws considerly lower. The correltion etween HPLC nd TLC results ws highly significnt (r 2 =.96). As fr s in vitro vrition of RA ccumultion is concerned, there re only few previous reports in the literture. For exmple, n oservtion of medium-dependent vrition of RA ccumultion in Ocimum mericnum in vitro shoot cultures ws ssocited with induced genetic vrition s reveled y RAPD (Rndom Amplifiction of Polymorphic DNA) nlysis [9]. Similr studies were conducted nd RA reched 178 g/g dry weight in Ocimum silicum [8]. Other reporters hve lso reported 1.8% RA equivlent from Slvi officinlis using ethnol wter mixtures [15]. Hydrogen Peroxide nd Key Enzymes Activity The H 2 O 2 -scvenging ctivity of the in vitro regenernts ws ten times higher thn tht of the donor plnts (Fig. 2). It is importnt to mention tht ssying the inhiition of hydrogen peroxide-induced oxidtion hs een frequently used for evluting the ntioxidnt properties of mint species, s reviewed previously [16]. It is possile tht other ntioxidnt ssys, such s the mesurement of ferrous iron cheltion nd the 1, 1-diphenyl-2-picryl-hydrzyl free rdicl scvenging ctivity provide different results regrding the ntioxidnt efficcy of spermint extrcts, s lredy reported previously [17]. Nevertheless, it hs een lso demonstrted tht there exists wide degree of vrition etween different phenolic compounds in their effectiveness s ntioxidnts B Totl Phenolic concentrtion (mg RAE/g f.w) Regenernt Regenernt Fig. (1). Accumultion of totl phenolics (A) nd rosmrinic cid (B) in spermint donor plnts nd regenernts. Verticl rs correspond to stndrd errors. Columns mrked with different letters indicte sttisticlly different vlues (p<.1).

3 Incresed Antioxidnt Activity, Despite Reduced Rosmrinic Acid Accumultion The Open Horticulture Journl, 214, Volume 7 3 Tle 1. Phenolic nd rosmrinic cid (RA) ccumultion in spermint donors nd plntlets (mg/g fw ± stndrd error). Numers followed y different letters indicte sttisticlly different vlues (p<.1). Ech Numer is n Averge of the numer of plntlets. RA ccumultors Numer of plntlets Phenolics Rosmrinic Acid ±.44 d.72 ±.41 d Regenernt* ±.17 e.39 ±.3 e High ± ±.23 Medium ± ±.14 Low 2.56 ±.7 c.5 ±.4 c *High, medium nd low RA ccumultors together. Tle 2. Single fctor ANOVA tle of rosmrinic cid ccumultor extrcts (donor nd regenernts). Source of Vrition SS df MS F P-vlue F crit Between groups E Within groups Totl Tle 3. Duncn s multiple rnge test tle. Totl phenolic ccumultion in donor plnts nd regenernts (expressed s equivlent rosmrinic cid concentrtion (mg/g fw ± stndrd error). Phenolics Regenernt High Medium Low Men ± SE 5.5 ± ± ± ±.7 LSR SSR [18, 19]. Although some reserchers hve reported positive correltion etween free rdicl-scvenging ctivity nd totl phenolic compounds in numer of plnt species [2, 21], it is possile tht the individul phenolic constituents my exhiit different ntioxidnt properties. This hypothesis ws further supported y the oserved ctivities of PAL nd HPPR in the plnt phenolic iosynthetic pthwy. Spermint regenernts demonstrted higher ctivity of oth enzymes compred to the donor plnts, indicting the possiility of n ltered regultion of the phenolic iosynthetic pthwy in vitro (Fig. 3). Although the ctivity of HPPR is closely relted with rosmrinic cid ccumultion, its overexpression my led to the inhiition of rosmrinic cid iosynthesis [13]. This mechnism could provide n explntion for the reduction of the concentrtion of rosmrinic cid in the in vitro regenernts, while the oserved chnges in the phenolic iosynthetic pthwy could e ssocited with the incresed formtion of constituents with H 2 O 2 scvenging properties. In ddition, leves of non-cclimtized regenernts (such s those investigted in the present study) my possess lessthn-complete photosynthetic pprtus; consequently, they my hve less-thn-optiml oxidtive pentose phosphte pthwy, which in turn will hve ffected secondry metolite synthesis in negtive wy [22]. Tle 4. Determintion coefficient (r 2 ) etween totl phenolic nd rosmrinic cid (RA) concentrtion in rosmrinic cid ccumultors (p<.1). Totl Phenolic & RA Concentrtion R 2 vlue Totl Phenolic & High RA ccumultor.91 Totl Phenolic & Medium RA ccumultor.94 Totl Phenolic & Low RA ccumultor.38 CONCLUSION In conclusion, the results of the present study suggest tht in vitro culture my ffect phenolic metolic pthwys in Menth spict. Further reserch in our group is imed towrds the identifiction of minor phenolic constituents formed in vitro s well s the moleculr dissection of the respective iosynthetic pthwys.

4 nm hydroxyphenyl lctic cid eq m mm trns cinnmic cid eq. 4 The Open Horticulture Journl, 214, Volume 7 Fdel et l Fig. (2). Hydrogen peroxide-scvenging ctivity (expressed s equivlent scoric cid concentrtion) of spermint donor plnts nd regenernts. Verticl rs correspond to stndrd errors. Columns mrked with different letters indicte sttisticlly different vlues (p<.1). A B Fig. (3). L-phenyllnine mmoni lyse (PAL) ctivity (A) (expressed s trns-cinnmic cid concentrtion) nd hydroxyphenylpyruvte reductse (HPPR) ctivity (B) (expressed s hydroxyphenyllctic cid concentrtion) of spermint donor plnts (lck columns) nd regenernts (lck columns). Verticl rs correspond to stndrd errors. Columns mrked with different letters indicte sttisticlly different vlues (p<.1). CONFLICT OF INTEREST The uthor(s) confirm tht this rticle content hs no conflicts of interest..62 Regenernt.146 Regenernt ACKNOWLEDGEMENTS Dni Fdel cknowledges the support of the Greek Ntionl Scholrship Foundtion. ABBREVIATIONS BA = 6-enzyldenine HPPR = Hydroxyphenylpyruvte reductse (EC ) MS = Murshige nd Skoog (1962) medium NAA = 1-nphthlenecetic cid PAL = L-phenyllnine mmoni lyse (EC ) RA = Rosmrinic cid TLC = Thin Lyer Chromtogrphy REFERENCES [1] Kntt SR, Chnder R, Shrm A. Antioxidnt potentil of mint (Menth spict L.) in rdition-processed lm met. Food Chem 27; 1: [2] Petersen M, Simmonds MSJ. Rosmrinic cid. Phytochemistry 23; 62: [3] Petersen M, Adullh Y, Benner J, et l. Evolution of rosmrinic cid iosynthesis. Phytochemistry 29; 7: [4] Shsny AK, Khnuj SPS, Dhwn S, Ydv U, Shrm S, Kumr S. High regenertive nture of Menth rvensis internodes. J Biosci 1998; 23: [5] Li X, Niu X, Bressn RA, Weller SC, Hsegw PM. Efficient plnt regenertion of ntive spermint (Menth spict L.). In vitro Cell Div Biol Plnt 1999; 35: [6] Bht S, Mheshwri P, Kumr S, Kumr A. Menth species: In vitro regenertion nd genetic trnsformtion. Mol Biol Tody 22; 3: [7] Fletcher RS, Slimmon T, McAuley CY, Kott LS. Het stress reduces the ccumultion of rosmrinic cid nd the totl ntioxidnt cpcity in spermint (Menth spict L.). J Sci Food Agric 25; 85: [8] Kintzios S, Mkri O, Pngiotopoulos Em, Scpeti M. In vitro rosmrinic cid ccumultion in sweet sil (Ocimum silicum L.). Biotech Lett 23; 25: [9] Rdy MR, Nzif NM. Rosmrinic cid content nd RAPD nlysis of in vitro regenerted sil (Ocimum mericnum) plnts. Fitoterpi 25; 76: [1] Zheng W, Wng SY. Antioxidnt ctivity nd phenolic compounds in selected hers. J Agric Food Chem 21; 49: [11] Petersen M, Alfermnn AW. Two new enzymes of rosmrinic cid iosynthesis from cell cultures of Coleus lumei: hydroxyphenylpyruvte reductse nd rosmrinic cid synthse. Z. Nturforsch 1988; 43c: [12] Reignult Ph, Cogn A, Muchemled J, Lounes-Hdj Shroui A, Durnd R, Sncholle M. Trehlose induces resistnce to powdery mildew in whet. New Phytol 21; 149: [13] Petersen M, Alfermnn AW. Two new enzymes of rosmrinic cid iosynthesis from cell cultures of Coleus lumei: hydroxyphenylpyruvte reductse nd rosmrinic cid synthse, Z. Nturforsch C 1988; 43: [14] Petersen M, Husler E, Meinhrd J, Krwtzki B, Gertlowski C. The iosynthetic of rosmrinic cid in suspension cultures of Coleus lumei Benth. Plnt Cell Tiss Org Cult 1994; 38: [15] Durling NE, Ctchpole OJ, Grey JB, et l. Extrction of phenolics nd essentil oil from dried sge (Slvi officinlis) using ethnolwter mixtures. Food Chem 27; 11: [16] Murci MA, Ege I, Romojro F, Prrs P, Jiménez AM, Tome MM. Antioxidnt evlution in dessert spices compred with common food dditives. Influence of irrdition procedure. J Agric Food Chem 24; 52: [17] Dormn HM, Kosr K, Khlos Y, Hiltunen HR. Antioxidnt properties nd composition of queous extrcts from Menth species, hyrids, vrieties, nd cultivrs. J Agric Food Chem 23; 51:

5 Incresed Antioxidnt Activity, Despite Reduced Rosmrinic Acid Accumultion The Open Horticulture Journl, 214, Volume 7 5 [18] Rords K, Prenzler P, Tucker G, Swtsitng P, Glover W. Phenolic compounds nd their role in oxidtive processes in fruits. Food Chem 1999; 66: [19] Kur C, Kpoor HC. Antioxidnt ctivity nd totl phenolic content of some Asin vegetles. Int J Food Sci Tech 22; 37: [2] Zheng W, Wng SY. Antioxidnt ctivity nd phenolic compounds in selected hers. J Agric Food Chem 21; 49: [21] Wngensteem H, Smuelsen A, Mlterud BKE. Antioxidnt ctivity in extrcts from corinder. Food Chem 24; 88: [22] Lttnzio V, Crdinli A, Rut C, et l. Reltionship of secondry metolism to growth in oregno (Orignum vulgre L.) shoot cultures under nutritionl stress. Environ Exp Bot 29; 65: Received: Septemer 18, 213 Revised: Decemer 11, 213 Accepted: Jnury 28, 214 Fdel et l.; Licensee Benthm Open. This is n open ccess rticle licensed under the terms of the Cretive Commons Attriution Non-Commercil License ( licenses/y-nc/3./) which permits unrestricted, non-commercil use, distriution nd reproduction in ny medium, provided the work is properly cited.