Changes in the phenolic composition of citrus fruits and leaves prepared by gamma irradiation of budsticks

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1 Life Siene Journl 212;9(3) Chnges in the phenoli omposition of itrus fruits nd leves prepred y gmm irrdition of udstiks Min Young Kim 1,2,*, Soon Je Im 1, Jong Hyun Kim 1, In-Jung Kim 1,2, Hyo Yeon Lee 1,2, Dong-Sun Lee 1,2, Youn Ji Lee 1, Ji Hee Byun 1, Ji Hye Kim 1, Ji Young Kim 1, Seo Rin Jeong 1, Jung Hyun Kim 3, Seo Hyun Moon 4 1. Fulty of Biotehnology, College of Applied Life Siene nd 2. Sutropil Hortiulture Reserh Institute, Jeju Ntionl University, Jeju , Repuli of Kore 3. Deprtment of Tourism nd Food Servie Cuisine, Cheju Tourism College, Jeju 695-9, Repuli of Kore 4. DNA Forensi Center, Ntionl Forensi Servie, Seoul , Repuli of Kore jeffmkim@jejunu..kr Astrt: Citrus mutnts were indued y the irrdition of itrus udstiks with 12 Gy of olt ( 6 CO) gmm irrdition. Three mutnt plnts demonstrting improved fruit qulity were seleted nd ompred with wild-type itrus plnt for evlution of the phenoli omposition, suh s totl phenolis, totl flvonoids, flvonoid distriution nd D-limonene. The results show tht irrdition indued hnges in totl phenoli nd flvonoid ontents of the fruit peel nd pulp, nd leves of itrus mutnts, s well s in D-limonene ontent. HPLC nlysis demonstrted tht hesperidine, nrirutin nd rutin were vrily distriuted in itrus mutnts. The otined results implite tht gmm irrdition my ontriute to vritions in phenoli omposition. [Min Young Kim, Soon Je Im, Jong Hyun Kim, In-Jung Kim, Hyo Yeon Lee, Dong-Sun Lee, Youn Ji Lee, Ji Hee Byun, Ji Hye Kim, Ji Young Kim, Seo Rin Jeong, Jung Hyun Kim, Seo Hyun Moon. Chnges in phenoli omposition of itrus fruits nd leves prepred y gmm irrdition of udstiks. Life Si J. 212;9(3): ] (ISSN: ) Keywords: itrus; muttion; gmm irrdition; phenoli ompounds 1. Introdution Gmm irrdition hs een used s n effetive plnt reeding method, whih n gretly indue high muttion numers nd modify physiologil hrteristis to rete new mutnts with improved properties (Nito et l., 25; Predieri, 21; Srto et l., 26). Free rdils produed y gmm irrdition n use hnges in struture, funtion nd metolism of plnt ells, whih resultntly produe higher mounts of ommerilly useful metolites nd develop eonomilly signifint vrieties (Kim et l., 24; Wi et l., 27). Mny useful mutnts were otined y gmm irrdition t vegettive propgted plnts (Ahloowli nd Mluszynski, 21; Predieri, 21). Citrus fruits rnk first in the world with respet to prodution mong the fruits (Terol et l., 21). The itrus rop is lso vlule ontriutor to the eonomy of Jeju islnd, Kore, with nnul yields of more thn.6 million tons. Citrus fruit prodution nd mrket demnd depends on severl ftors like life yle of fruit-ering trees, disese resistne, extended hrvesting period, mximum yield of higher fruit qulities (e.g., size, olor, texture nd nutritionl vlue of fruit) re importnt onsidertions. To meet the requirements of the fruit industry nd the onsumers, itrus muttion reeding pproh vi gmm ry irrdition to develop new itrus ultivrs hs stedily inresed (Bermejo et l., 211; Chudhuri, 22; Deng, 25; Ling et l., 28; Rz et l., 23; Wu, 1986). However, limited informtion exists on the potentil nd extent of enhning prodution of itrus seondry metolites using gmm irrdition, even though mny of the effets of gmm irrdition on seedlessness, pollen germintion, fruit qulity nd growth of itrus hve reeived onsiderle ttention (Bermejo et l., 211; Chudhuri, 22; Rz et l., 23; Wu, 1986). In the present study, the levels of iotive onstituents were thus nlyzed for nonirrdited nd irrdition-indued itrus in order to ompre their properties nd hnges. 2. Mteril nd Methods 2.1. Gmm irrdition nd plnt mintenne The itrus ultivr, Citrus unshiu Mr. v. Miygw, whih is most widely ultivted in Jeju islnd, Kore, ws seleted s the originl plnt mterils for muttionl reeding experiments. One yer old shoots of itrus were exposed to gmm irrdition t the doses of 12 Gy from olt ( 6 CO) soure t the Institute for Nuler Siene nd Tehnology, Jeju Ntionl University nd were then grfted onto one yer old sour ornge (C. urntium L.) seedling. Afterwrds, young irrdited tree were mintined for out 1 yer t nursery nd then seleted mutnt plnts were moved to the min grden in Sutropil Hortiulture Reserh Institute t Jeju Ntionl University where the rest of the study ws rried out. Both itrus wild-type (WT) nd mutnt (Mut) plnts were mnged in sme wy with respet to fertiliztion, irrigtion nd disese ontrol lifesienej@gmil.om

2 Life Siene Journl 212;9(3) The growing onditions nd degree of ripeness t hrvest were ontrolled to eliminte the effets of environmentl onditions on the different WT nd Mut ultivrs Smple preprtion Fully mture itrus fruits with leves were hrvested on ripening in Novemer, 21. Three different Mut ultivrs were used in the present study. Aording to the morphologil hrteristis, inluding fruit shpe, size nd olor, nd ontents of sugr nd id, whih were determined in detil s desried previously (Oh nd Kim, 211), test sujets were divided into four groups: WT, itrus derived from non-irrdited shoots; Mut I, itrus mutnts with omprtively high sugr/id rtio; Mut II, itrus mutnts with red olor; Mut III, itrus mutnts with rough shpe. The fruit peel nd pulp, nd leves of WT nd Mut itrus were seprted, disseted, weighed, lyophilized nd then ground into fine powder using lender. Portions (25 g) of the powered smples were suessively extrted with 25 ml of methnol in the 25 ºC shking inutor for 24 h nd susequently purified y using.45 μm memrne filter (Wters, Milford, MA, US), whih were stored t 4 ºC until further use Anlysis of totl phenoli nd flvonoid ontents Contents of totl phenol nd flvonoid were determined y the modified method desried previously (Senevirthne et l., 21). For totl phenol quntittion, 3 μl of itrus WT nd Mut methnoli extrts were mixed thorough with 3 μl of 95% ethnol, 15 μl of distilled wter, 15 μl of Folin-Ciolteu regent nd 3 μl of sturted sodium ronte solution (5%). After 6 min stnding, the sorne ws red t 725 nm ginst lnk in Spetr MR miroplte reder (Dynex Tehnologies, In., Chntilly, VA, US). Content of phenols ws lulted on the sis of the lirtion urves of glli id, nd ws expressed s mg glli id per 1 g dry mtter. To determine totl flvonoids, eh extrt in methnol (15 μl) ws mixed with 4.5 μl of 5% NNO 2, 6 μl of distilled wter nd 4.5 μl of 1% AlCl 3. After inution for 6 min, 6 μl of NOH solution (4%) ws dded to the mixture nd mde up to finl volume of 15 μl with distilled wter. The sorne ws mesured 15 min lter t 51 nm. Content of flvonoids ws lulted on the sis of the lirtion urves of rutin nd ws expressed s mg rutin per 1 g dry mtter HPLC nlysis of flvonoids A 2 μl liquot of itrus WT nd Mut methnoli extrts ws injeted into Shim-pk VP-ODS (C 18 ) olumn (4.6 m 15 mm 5 μm; Shimdzu, Tokyo, Jpn) of the Wters high performne liquid hromtogrphy (HPLC) system equipped with 626 pump, 486 UV-VIS detetor nd utosmpler (Wters, Milford, MA). The moile phse for the HPLC system ws (A) wter/eti id (99.5/.5, v/v): (B) etonitrile/eti id (99.5/.5, v/v) t flow rte of 1 ml/min. The moile phse progrm onsisted of four periods: -1 min, 2% A; 1-16 min, 45% A; 16-2 min, 75% A; nd 2-22 min, 2% A. The olumn ws operted t 4 C, nd the eluent ws monitored with single-hnnel UV detetor t wvelength of 28 nm. The flvonoids were identified y ompring their retention times nd UV spetr with those of uthenti stndrds (ChromDex, Irvine, CA, US). The ontent of eh flvonoid ws lulted from the integrted pek re if the smple nd the orresponding stndrd GC-MS nlysis of D-limonene A Shimdzu QP21 Ultr gs hromtogrphi mss spetrometri (GC-MS) system, utosmpler nd rel time nlysis softwre were used for the nlysis. A Restek Rtx-5MS pillry olumn (3 m.25 mm i.d..25 μm) ws used for seprtion. The injetor temperture ws 25 C. The temperture progrm ws strted t 5 C for 2 minute nd heted t 1 C/min to 8 C, inresed y 2 C/min up to 14 C nd then y 4 C /min up to 28 C. Smple injetion ws done in split mode (split rtio 2:1). High purity helium ws used s rrier gs t flow rte of 1 ml/min. Mss spetrometry ws run in full sn mode (m/z 45-5) with 2 C MS soure temperture nd 2.5 minutes solvent ut time. D-limonene ws indentified y ompring the retention times nd mss spetr otined with those of uthenti stndrd (Sigm) Sttistil nlysis Results re presented s mens ± stndrd devition. Sttistil omprisons were mde y nlysis of vrine (ANOVA) proedure followed y Dunn s multiple rnge tests (SPSS 12.). P <.5 ws onsidered signifintly different. After multiple omprisons, the mens in the following tle nd figures were followed with different smll letter -d sed on their vlues nd sttistil differenes. 3. Results nd Disussion Dt in Figure 1 show tht the Gmm irrdition signifintly ffeted the totl phenoli nd flvonoid ontent of itrus when ompred with lifesienej@gmil.om

3 Life Siene Journl 212;9(3) WT smples. The peel nd pulp extrts of itrus Mut I nd II hd signifintly higher totl phenoli ontent thn the extrts of itrus WT, wheres lower totl phenoli ontent ws noted for those of Mut III (Figure 1A). The itrus Mut I peel hd the highest totl phenoli ontent (23786 mg/1 g) whih is 37% higher thn tht of WT peel (Figure 1A). Totl flvonoid ontent in the peel ( mg/1 g) nd pulp ( mg/1 g) of itrus WT, Mut I nd Mut II were lmost in the similr quntity (Figure 1B). However, the flvonoid ontents of itrus Mut III peel nd leves were signifintly higher thn those of WT, while flvonoid ontent of Mut III pulp ws signifintly lower (p <.5) (Figure 1B). In this study, itrus peel nd pulp extrts hd reltively higher totl phenoli nd flvonoid ontents s ompred with the lef extrts, exept tht of Mut III group (Figure 1). A B Totl phenolis (mg/1 g) Totl flvonoids (mg/1 g) Lef Peel Pulp d Lef Peel Pulp Fig. 1. Totl phenoli (A) nd flvonoid (B) ontents in the leves, peel nd pulp extrts of itrus derived from non-irrdited nd irrdited shoots. Dt presented re in men ± stndrd devition (n = 3) whih with different letters ove the rs re signifintly different t p <.5. Flvonoids re widely distriuted group of phenoli ompounds, whih hve wide rnge of iologil effets, suh s inhiition of key enzymes in mitohondril respirtion, protetion ginst oronry hert disese nd nti-inflmmtory, ntitumor, nd ntimiroil tivities (Wng et l., 28). Citrus fruit ontins high levels of the flvnones, s well s flvonol, whih re very rre in other plnts (Gttuso et l., 27). In the present study, four flvnones (nringin, neohesperidin, hesperidin nd nrirutin) nd one flvonol (rutin) were identified in lef extrts, while hesperidin, nrirutin nd rutin were the min flvonoids deteted in itrus peel nd pulp extrts (Tle 1). The mounts of hesperidin in peel ( mg/1 g), pulp ( mg/1 g) nd leves ( mg/1 g) of itrus in this work were the highest ompred to mounts of other flvonoids. The mounts of nringin, hesperidin nd rutin were signifintly lower ut nrirutin ws signifintly higher in lef extrts of ll Mut groups thn WT (p <.5). Neohesperidin ourred in the lef extrts of ll experimentl groups with similr distriution ptterns ( mg/1 g) (Tle 1). Here, otined results of peel nd pulp showed tht these two prts hd very similr flvonoids tendenies without remrkle differenes lthough peel ontined higher level of investigted flvnones thn pulp. The peel nd pulp of ll Mut groups ( nd mg/1 g, respetively) ontined signifintly higher onentrtion of nrirutin thn those of WT (6.8 nd 92.4 mg/1 g, respetively); In the peel nd pulp of Mut III, the flvnone glyoside pttern onsisted predominntly of hesperidin nd nrirutin, nd their ontents were signifintly higher thn WT (p <.5). Hesperidin hs een reported to redue plsm/hepti holesterol (Montforte et l, 1995; Mnh et l, 23) nd to suppress the oxidtive stress in vitro nd vivo (Miyke et l, 1998). Furthermore, it hs een shown to protet nimls ginst hemilly indued severl ners (Tnk et l, 1997; Berkrd et l, 1998). Nrirutin hs een demonstrted to hve n nti-llergi properties (Kuo et l, 24) nd therpeuti effet on ronhil sthm (Funguhi et l, 27). Citrus oils re mixtures of voltile omponents of terpeni hydroron nd oxygented ompounds (Cno nd Bermejo, 211). D-limonene is the priniple omponent of itrus oil, rnging from 88% to 95%, whih is often used s n dditive in food produts nd frgrnes, nturl pestiide nd inset repellent (Cno nd Bermejo, 211). It hs lso een studied for its nti-rinogeni properties (Crowell nd Gould, 1994) lifesienej@gmil.om

4 Life Siene Journl 212;9(3) Tle 1. Flvonoid ontent (mg/1 g dry weight) in the leves, peel nd pulp extrts of itrus derived from non-irrdited nd irrdited shoots Group Flvnone Flvonol glyside Nringin Neohesperidin Hesperidin Nrirutin Nringenin Hesperetin Rutin Leves WT 3.9± ± ± ±.65 nd * nd 254.1±2.7 Mut I 2.6± ± ± ± 1.16 nd nd 186.6±1.76 Mut II 21.7± ± ± ±.91 nd nd 134.1±2.72 Mut III 17.4± ± ±1.29 d 23.1 ±.3 nd nd 186.4±.99 Peel WT nd nd 463.1± ±1.51 nd nd 65.7±.47 Mut I nd nd 573.7± ±.61 nd nd 72.1±.93 Mut II nd nd 34.3± ±1.23 nd nd 64.±.87 Mut III nd nd 546.6±3.38 d 78.5±.61 d nd nd 66.1±.63 Pulp WT nd nd 178.8± ±.59 nd nd 65.7 ±.47 Mut I nd nd 145.1± ±.16 nd nd 72.1 ±.93 Mut II nd nd 15.4± ±.6 nd nd 64. ±.87 Mut III nd nd 276.3±2.7 d ± 1.53 nd nd 66.1 ±.63 * Not deteted; -d Vlues with different supersripts in olumn re signifintly different (p <.5) D-Limonene (mg/1 g) Fig. 2. D-limonene ontent in the leves (A) nd peel (B) extrts of itrus derived from non-irrdited nd irrdited shoots. Dt presented re in men ± stndrd devition (n = 3) whih with different letters ove the rs re signifintly different t p <.5. The gs hromtogrphi profiles nd D- limonene ontent re presented in Figure 2 in vlues of milligrms per 1 grm of peel (dried weight). Mut I (14679 mg/1 g) nd II (23844 mg/1 g) displyed signifintly higher D-limonene vlues thn WT (11222 mg/1 g) (p <.5). However, Mut III (1118 mg/1 g) showed similr ontent with WT with no signifint differenes (Figure 2). In this urrent study, we exmined the influene of gmm irrdition on the phenoli onstituents of itrus. Overll, gmm irrdition uses iohemil hnges in the fruits nd leves, nd seem to e le to influene the synthesis of phenoli ompounds, whih hve een reognized s importnt due to their iotive role nd heth enefits lthough it would e neessry to rry out more nlyses, nd with itrus mutnts otined from rdition reeding progrms, to eluidte onlusive vlues. Corresponding Author: Dr. Min Young Kim Fulty of Biotehnology College of Applied Life Sienes Jeju Ntionl University Jeju , Repuli of Kore E-mil: jeffmkim@jejunu..kr Aknowledgement This reserh ws supported y Agriulture Tehnology Development Progrm ( ), Ministry for Food, Agriulture, Forestry nd Fisheries, Repuli of Kore. Referenes 1. Ahloowli BS, Mluszynski M. Indued muttions-a new prdigm in plnt reeding. Euphyti 21; 118: Berkrd B, Koyunu H, Soyir G, Bykut F. Inhiitory effet of hesperidin on tumour initition nd promotion in mouse skin. Res Exp Med 1998; 198: Bermejo A, Prdo J, Cno A. Influene of gmm irrdition on seedless itrus prodution: lifesienej@gmil.om

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