Antioxidant and antigenotoxic activities of different parts of persimmon (Diospyros kaki cv. Fuyu) fruit

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1 Journl of Medicinl Plnts Reserch Vol. 4(2), pp , 18 Jnury, 2010 Avilble online t ISSN Acdemic Journls Full Length Reserch Pper Antioxidnt nd ntigenotoxic ctivities of different prts of persimmon (Diospyros kki cv. Fuyu) fruit In-Cheol Jng 1, Eun-Kyung Jo 1, Myung-Suk Be 1, Hyun-Jung Lee 2, Gyeong-Im Jeon 2, Eunju Prk 2, Hyun-Gyun Yuk 3, Gwng-Hwn Ahn 4 nd Seung-Cheol Lee 1 * 1 Deprtment of Food Science nd Biotechnology, Kyungnm University, Msn , Republic of Kore. 2 Deprtment of Food nd Nutrition, Kyungnm University, Msn , Republic of Kore. 3 Food Science nd Technology Progrmme, Deprtment of Chemistry, Ntionl University of Singpore, Science Drive, Singpore , Republic of Singpore. 4 Sweet Persimmon Reserch Institute, Gyungsngnmdo Agriculturl Reserch nd Extension Services, Gimhe , Republic of Kore. Accepted 23 December, 2009 This study ws conducted to determine the ntioxidnt ctivities nd the ntigenotoxic effects of cetone, ethnol, methnol nd wter extrcts from four different prts (clyx, seed, peel nd flesh) of persimmon fruits. Antioxidnt ctivities were evluted by DPPH (1,1-diphenyl-2-picryllhydrzyl) rdicl scvenging ctivity (RSA), ABTS (2,2-zino-bis (3-ethylbenzothizoline-6-sulfonic cid) dimmononium slt) RSA nd reducing power (RP). Seed nd clyx extrcts showed significntly (p < 0.05) higher ntioxidnt ctivities nd phenolic contents thn peel nd flesh extrcts. This study lso showed tht ethnol ws more effective on the extrction of ntioxidnt compounds from persimmon compred to other solvents. The ntigenotoxic effects of the persimmon extrcts on DNA dmge induced by H 2 O 2 in humn leukocytes ws evluted by Comet ssy. All persimmon extrcts inhibited DNA dmge induced by 200 µm of H 2 O 2. Especilly, clyx nd seed extrcts showed stronger inhibition ctivity thn peel nd flesh extrcts. Therefore, these results suggest tht persimmon extrcts my hve beneficil effects on oxidnt nd protective effect ginst oxidtive DNA dmge. Key words: Persimmon extrct, ntioxidnt ctivities, ntigenotoxic effect. INTRODUCTION Oxidtive stress, cused by the imblnce of rective oxygen species (ROS) nd ntioxidtive defense systems, is considered s mjor etiologicl nd/or pthogenic gent of most degenertive diseses such s cncer, Alzheimer s, dibetes nd ging (Dtt et l., 2000). The ntioxidnts re of interest in the tretment of severl cellulr degenertions, nd they inhibit or dely the oxidtion process by blocking the initition or propgtion of oxidizing chin rections (Beher et l., 2006). Antioxidnts interfere with the production nd the ctivtion of free rdicls (Mchlin nd Bendich, 1987), resulting in protecting the humn body from free rdicls tht my cuse some chronic diseses including cncer * Corresponding uthor. E-mil: sclee@kyungnm.c.kr. Tel: Fx: nd crdiovsculr diseses (Kinsell et l., 1993). They re lso used to preserve food by retrding rncidity, discolortion or deteriortion due to uto-oxidtion (Hung et l., 2003). Though synthetic ntioxidnts, such s butylted hydroxynisole, butylted hydroxyltoluene nd propyl gllte, hve been commonly dded to food products to retrd lipid oxidtion, the demnd for nturl ntioxidnts hs incresed becuse of questions nd negtive perception of consumers bout the long-time sfety of synthetic ntioxidnts (Yu et l., 2002). Regulr consumption of fruit nd vegetbles contining nturl ntioxidnts is correlted with the decresed risk of diseses such s cncer nd crdiovsculr diseses (Michels et l., 2000). Persimmon (Diospyros kki L.), which belongs to the Ebencee fmily, is cultivted in wide re, including Estern Asi, Spin nd Isrel. Persimmon fruit is known to hve mny bioctive compounds such s polyphenols,

2 156 J. Med. Plnt. Res. crotenoids, dietry fiber nd minerls. For exmple, persimmon contins 70 mg of vitmin C, while pple contins only 4 mg in 100 g of its flesh prt (New Food Composition Tble Editing Committee, 2009). Persimmon hs been trditionlly used for mny medicinl purposes such s blood pressure-lowering effect, diuretic effect nd coughs, nd it is probbly involved in the reduction of degenertive humn diseses (Steinmetz nd Potter, 1996). Although there re some reports regrding ntioxidnt ctivity of fruits nd peels of persimmon (Fuki et l., 2009; Lee et l., 2008; Jung et l., 2005), little work hs been done on the comprtive reserch bout the functionl ctivity of different prts of persimmon fruit. Thus, the im of this study ws to evlute the ntioxidnt nd ntigenotoxic ctivities of four different prts (clyx, flesh, peel nd seed) of persimmon extrcted by four different solvents (cetone, ethnol, methnol nd wter). MATERIALS AND METHODS Mterils Persimmon (D. kki cv. Fuyu) fruits were provided from Sweet Persimmon Reserch Institute (Gimhe, Kore). L-Ascorbic cid (Vitmin C), 2,2-zino-bis (3 - ethylbenzothizoline-6-sulfonic cid) dimmononium slt (ABTS), butylted hydroxyltoluene (BHT), dimethyl sulfoxide (DMSO), trichlorocetic cid (TCA) nd 1,1- Diphenyl-2-picrylhydrzyl (DPPH) were purchsed from Sigm Chemicl Co. (St. Louis, MO, USA). Folin-Cioclteu regents were from Wko Pure Chemicl Industries, Ltd. (Osk, Jpn). Hydrogen peroxide, potssium chloride, potssium ferricynide nd potssium phosphte were lso purchsed from Sigm Chemicl Co. (St. Louis, MO). All the other orgnic solvents nd chemicls used in this study were of nlyticl grde. Preprtion of extrcts from persimmon fruits Fresh persimmon fruits were divided into four prts (flesh, peel, clyx nd seed), nd ech prts were freeze-dried with freeze dryer (Ilshin Lb., Seoul, Kore). The dried persimmon fruit prts were ground in mill nd pssed through 25-mesh sieve. Ech 5 g of dried prts (flesh, peel, clyx nd seed) ws extrcted with 100 ml of four different solvents (cetone, ethnol, methnol nd wter) overnight t room temperture nd filtered through Whtmn No. 1 filter pper. were then removed by evportion in vcuo, nd the dried extrcts were obtined. Ech dried extrct ws redissolved in DMSO with concentrtion of 50 mg/ml for the experiments, nd diluted with DMSO when needed. All smples were plced in glss bottle nd stored t 4 C until used. Extrction yield ws clculted by the following formul: Extrction yield (%) = [dried extrct weight/dried smple weight] 100 DPPH rdicl scvenging ctivity (RSA) The DPPH RSA of ech extrct ws determined ccording to the method of Jeong et l. (2004). After 0.1 ml liquot of extrct dissolved in DMSO ws mixed with 0.9 ml of mm DPPH in ethnol for 30 min, the opticl density (OD) of the smple ws mesured t 517 nm using spectrophotometer (Shimdzu UV- 1601, Tokyo, Jpn). RSA ws expressed s percentge inhibi- tion nd it ws clculted by the following formul: DPPH RSA (%) = [1 (smple OD/control OD)] 100 ABTS rdicl scvenging ctivity (RSA) The ABTS RSA ws evluted with the method of Muller (1985). Ech extrct (0.1 ml), potssium phosphte buffer (0.1 ml, 0.1 M, ph 5.0) nd hydrogen peroxide (20 µl, 10 mm) were mixed nd preincubted t 37 C for 5 min. After pre-incubtion, ABTS (30 µl, 1.25 mm, in 0.05 M phosphte-citrte buffer, ph 5.0) nd peroxidse (30 µl, 1 unit/ml) were dded to the mixture, nd then it ws incubted t 37 C for 10 min. The OD level ws obtined with multiplte reder (Sunrise RC/TS /TS Color-TC/TW/BC/6Filter, Tecn Austri GmbH, Grödig, Austri) t 405 nm, nd the ABTS RSA ws clculted by the following formul: ABTS RSA (%) = [1 (smple OD/control OD)] 100 Reducing power (RP) The RP of ech extrct ws determined ccording to the method of Oyizu (1986). The extrct (1.0 ml), sodium phosphte buffer (1.0 ml, 0.2 M, ph 6.6) nd potssium ferricynide (1.0 ml, 10 mg/ml) were mixed nd incubted t 50 C for 20 min. Trichlorocetic cid (1.0 ml, 100 mg/ml) ws dded to the mixture nd then the mixture ws centrifuged t 13,400 g for 5 min. The superntnt (1.0 ml) ws mixed with distilled wter (1.0 ml) nd ferric chloride (0.1 ml, 1.0 mg/ml), nd then its bsorbnce ws mesured t 700 nm. Totl phenolic contents (TPC) TPC in the ech extrct were determined ccording to the method of Gutfinger (198. Ech extrct (1.0 ml) t concentrtion of the 1,000 µg/ml ws mixed with 1.0 ml of 2% N 2CO 3 fter stnding 3 min nd 0.2 ml of 50% Folin-Cioclteu regent dded to the mixture fter stnding for 30 min. The mixture ws centrifuged t 13,400 g for 5 min. The bsorbnce ws mesured t 750 nm nd TPC were expressed s gllic cid equivlents. DNA dmge by lkline Comet ssy Leukocytes were isolted s frction of mononucler cells (contining lymphocytes nd monocytes) from nonymous buffy cot preserves by grdient centrifugtion with HISTOPAQUE (Sigm, St. Louis, MO, USA). Leukocytes were incubted with ech extrcts in DMSO (50 µg/ml) for 30 min t 37 C in drk incubtor. For oxidtive stimulus, they were then resuspended in PBS with 200 µm H 2O 2 for 5 min on ice. After ech tretment, smples were centrifuged t 250 g for 5 min nd wshed with PBS. One percent of DMSO without oxidtive stimulus ws treted for negtive control. The leukocytes were then mixed with 75 µl of 0.7% low melting grose, nd dded to the slides precoted with 0.5% grose. The slide ws then immersed in lysis solution (2.5 M NCl, 100 mm EDTA, 10 mm Tris nd 1% sodium lurylsrcosine; 1% Triton X- 100 nd 10% DMSO) for 1 hr t 4 C. The slides were then plced into n electrophoresis tnk contining 300 mm NOH nd 10 mm N 2EDTA (ph 13.0) for 40 min. For electrophoresis of the DNA, n electric current of 25 V/300 ± 3 ma ws pplied for 20 min t 4 C. The slides were wshed three times with neutrlizing buffer (0.4 M Tris, ph 7.5) for 5 min t 4 C, nd then treted with ethnol for nother 5 min before stining with 50 µl of ethidium bromide (20 µg/ml). Mesurements were crried out by n imge nlyzer

3 Jng et l. 157 (Kinetic Imging, Komet 4.0, U.K) nd fluorescence microscope (LEICA DMLB, Germny) to determine the percentge of fluorescence in the til (til intensity, TI; 50 cells from ech of two replicte slides). Sttisticl nlysis All mesurements were performed in triplicte, nd nlysis of vrince ws conducted by the Generl Liner Model using SAS softwre (1995), Student-Newmn-Keul s multiple rnge tests were used to compre the significnt differences of the men vlues mong tretments (p < 0.05). The dt for Comet ssy were the mens of three determintions nd ws nlyzed using the SPSS pckge for Windows (Ver. 12). The men vlues of the DNA dmge (til intensity) from ech tretment were compred using one-wy nlysis of vrince (ANOVA) followed by Duncn's multiple rnge test. A p-vlue less thn 0.05 were considered sttisticlly significnt. RESULTS AND DISCUSSION Extrction yields The extrction yields of four different prts of persimmon were presented in Tble 1. Reltively higher extrction yields were obtined from flesh nd peel prts thn those of clyx nd seed prts. Among solvents, methnol ws most effective solvent on the extrction, exhibiting the highest extrction yields t most prts, except for seed. The highest extrction yield ws found in the methnol extrct from the flesh prt with 91.02%, while the wter extrct of seed prt (2.02%) hd the lowest extrction yield. These results showed tht the extrction yield vried by solvents nd persimmon prts, indicting tht ech prt consists of different components. DPPH rdicl scvenging ctivity (RSA) The scvenging ctivity on DPPH free rdicl is common method to evlute the ntioxidtive ctivity of ntioxidnts. The DPPH RSA of extrcts from persimmon fruits ws evluted by compring with scorbic cid nd BHT, which re well-known nturl nd synthetic ntioxidnts, respectively. As shown in Tble 2, extrcts from seed nd clyx prts hd reltively strong DPPH RSA (low IC 50 vlue), exhibiting high ntioxidnt cpcity compred to extrcts from peel nd flesh prts. Ethnol extrct of seed nd cetone extrct of clyx showed the lowest IC 50 vlues with bout 48 µg/ml, which ws only 1.78 fold lower thn tht of vitmin C (IC 50 = µg/ml) nd 6.46 fold higher thn tht of BHT (IC 50 = µg/ml). Except for wter, most solvents were effective to extrct ntioxidnts from clyx nd seed. Extrct from peel prt showed higher DPPH RSA thn tht from flesh prt. Similr with our results, Choi et l. (2006) reported tht 70% ethnol extrct of persimmon peel showed significnt higher DPPH RSA thn tht of flesh. Ahn et l. (2002) demonstrted tht persimmon seed extrct showed higher DPPH RSA thn grpe seed extrct. The present results showed tht extrct from persimmon clyx by cetone hd the strongest DPPH RSA. This might be due to the difference on ( the mount nd the form of tnnin which is the min phenolic compound of persimmon fruit (Gu et l., 2008), (2) the mount of ntioxidnt crotenoid compounds (Veberic et l., 2010) nd (3) the mount of vitmin C (Hosotni et l., 2004). ABTS rdicl scvenging ctivity (RSA) Like the results of DPPH RSA, the scvenging effect of extrcts from four prts of persimmon fruit on the ABTS rdicls lso followed this order; clyx > seed > peel > flesh (Tble 3). The highest ABTS RSA ws detected in ethnol extrct of clyx (IC 50 = µg/ml), exhibiting similr IC 50 vlue compred with scorbic cid nd BHT (IC 50 = nd µg/ml, respectively). The ABTS method is suitble for monitoring of lipophilic ntioxidnts such s crotenoids nd lipophilic extrcts of nutritionl components (Nicolett et l., 1999). This fct cn explin why wter (most polr solvent) extrcts from clyx, seed nd peel prts exhibited the lower ABTS RSA compred with other solvent extrcts. In flesh prt, however, wter extrct showed the highest ABTS RSA, indicting tht ntioxidnt compounds differ by prts of persimmon fruits. Reducing power (RP) The ntioxidnt bility of certin compounds is ssocited with their RP (Jyprksh et l., 200, thus the RP my serve s significnt indictor of potentil ntioxidnt ctivity (Meir et l., 1995). RP ws determined by mesuring the reduction of the Fe 3+ form of Fe 3+ / ferricynide complexes to the ferrous (Fe 2+ ) form. The RP of extrcts ws evluted nd listed in Tble 4. Extrcts from clyx nd seed prts showed significntly (p < 0.05) higher RP thn peel nd flesh prts, regrdless of solvents. For exmple, ethnol extrct from seed hd the highest RP (IC 50 = µg/ml), nd methnol extrct from flesh showed the lowest RP (IC 50 = µg/ml). Overll trend ws very similr with other ntioxidnt ctivities described before. Totl phenolic contents (TPC) Tble 5 shows TPC of ech extrct t concentrtion of 1 mg/ml. Extrcts from the clyx nd the seed of persimmon fruits showed significntly (p < 0.05) higher TPC thn extrcts from peel nd flesh prts. The highest TPC ws found in cetone extrct from seeds with mg GAE/g, probbly due to high mount of ntioxidnt compounds including phenolic compound such s tnnin (Gorinstein et l., 1994). For solvents, cetone ws most effective solvent to extrct phenolics from clyx, seed nd peel, wheres wter extrct showed the highest

4 158 J. Med. Plnt. Res. Tble 1. Extrction yields from ech prt of persimmon by solvents. Clyx Seed Peel Flesh Methnol cw 7.87 dx bw w Ethnol cx 6.80 dy by x Acetone 9.68 bz 2.02 dz 3.48 cz z Wter by 9.12 dw x cy (Unit: %) Different letters within row (-d) nd column (w-z) re significntly different (p < 0.05). Tble 2. DPPH rdicl scvenging ctivity of extrcts from different prts of persimmon by solvents (Unit: IC 50 ). Positive controls Clyx Seed Peel Flesh Vit-C BHT Methnol ± 2.14 y ± 0.52 by ± cy ± dz Ethnol ± 1.93 bx ± 0.72 w ± cz ± dy Acetone ± 1.19 w ± 1.55 bx ± 7.77 cw ± dx ± ± 3.98 Wter ± z ± cz ± bx ± dw All mesurements were done in triplicte, nd ll vlues re mens ± stndrd devition. Different letters within row (-d) nd column (w-z) re significntly different (p < 0.05), n=3. IC 50 (µg/ml): Concentrtion for scvenging 50% of DPPH rdicls. Tble 3. ABTS rdicl scvenging ctivity of extrcts from different prts of persimmon by solvents (Unit:IC 50 ). Positive controls Clyx Seed Peel Flesh Vit-C BHT Methnol ± 0.37 x ± 0.96 bx ± cx ± dz Ethnol ± 0.38 w ± 0.56 by ± cy ± dx Acetone ± 0.86 y ± 2.09 by ± 3.56 cw ± dy ± ± 1.12 Wter ± 1.73 z ± bz ± dz ± cw All mesurements were done in triplicte, nd ll vlues re mens ± stndrd devition. Different letters within row (-d) nd column (w-z) re significntly different (p < 0.05), n=3. IC 50 (µg/ml): Concentrtion for scvenging 50% of ABTS rdicls. Tble 4. Reducing power of extrcts from different prts of persimmon by solvents (Unit: IC 50 ). Positive controls Clyx Seed Peel Flesh Vit-C BHT Methnol ± by ± 0.67 y ± 6.03 cy ± dz Ethnol ± 2.60 bx ± 0.37 x ± cz ± dz Acetone ± 3.17 by ± 0.47 y ± cx ± dy ± ± 0.22 Wter ± 6.15 z ± 9.62 bz ± cz ± dx All mesurements were done in triplicte, nd ll vlues re mens ± stndrd devition. Different letters within row (-d) nd column (x-z) re significntly different (p < 0.05), n=3. IC 50 (µg/ml): Concentrtion for incresing vlue in opticl density. TPC in flesh prt. The order of TPC of extrcts from four different prts of persimmons nerly coincided with tht of them, indicting tht there ws correltion between the TPC nd other ntioxidnt properties of extrcts.

5 Jng et l. 159 Tble 5. Totl phenolic contents of extrcts from different prts of persimmon by solvents Unit: mg GAE /g). Clyx Seed Peel Flesh Methnol ± 1.17 by ± 0.84 y 6.10 ± 0.13 cz 4.54 ± 0.13 dy Ethnol ± 0.91 by ± 0.37 x 6.92 ± 0.36 cy 4.27 ± 0.13 dz Acetone ± 1.06 bx ± 0.19 w ± 0.29 cw 5.29 ± 0.21 dx Wter ± 0.56 z ± 0.32 bz 9.35 ± 0.11 cx 8.60 ± 0.24 dw All mesurements were done in triplicte, nd ll vlues re mens ± stndrd devition. Different letters within row (-d) nd column (w-z) re significntly different (p < 0.05), n=3. GAE: gllic cid equivlents 50 d Methnol Ethnol Acetone Wter Til intensity (%) b b c b c c b cd bc bc 0 NC PC Clyx Se ed Pe e l Fle sh Figure 1. The effect of extrcts (50 µg/ml) from persimmon clyx, seed, peel nd flesh by methnol, ethnol, cetone nd wter on 200 µm H 2O 2 induced DNA dmge in humn leukocyte. Vlues re men with stndrd devition of triplicte experiments. NC, 1% DMSO treted negtive control; PC, 200 µm H 2O 2 treted positive control. Vlues not shring the sme letter re significntly different from one nother (p < 0.05) by Duncn s multiple rnge test. Protective effect of persimmon on oxidtive DNA dmge in humn leukocytes The genotoxic effects of H 2 O 2 nd the protective bility of ech extrct were ssessed in norml humn leukocytes by the comet ssy (Figure. At concentrtion of 50 µg/ml, the solvents (cetone, ethnol nd methnol) extrcts of clyx nd seed, nd methnol extrct of flesh exhibited the gretest protective effect on H 2 O 2 induced DNA dmge, showing insignificnt difference (p > 0.05) between extrcts nd DMSO treted negtive control. The extrcts from peel nd flesh hd reltively low protective effect compred to the extrcts from clyx nd seed. Active oxygen species or free rdicls, such s singlet oxygen ( 1 O 2 ), super oxide nion rdicl (O 2 - ), hydrogen peroxide (H 2 O 2 ), nd hydroxyl rdicl (OH ) re considered to cuse oxidtive dmge to DNA, which bring bout vriety of diseses, s well s ging (Sohl nd Weindruch, 1996). Persimmon contins mny bioctive compounds, such s polyphenols, flvonoids, nd crotenoids (New Food Composition Tble Editing Committee, 2009). Crotenoids, the most potent biologicl quenchers of singlet O 2, ct s chin-breking ntioxidnts (Liebler, 1993), nd flvonids inhibit the enzymes responsible for - O 2 production (Hnski et l., 1994). -Crotene nd flvonoids hve been reported to prevent DNA dmge ginst H 2 O 2 in humn lymphocytes ssessed by the Comet ssy (Astley et l., 2004; Noroozi et l., 1998). Likewise, numerous studies hve demonstrted tht polyphenolic compounds reduce DNA dmge in humn lymphocytes when ssessed with the Comet ssy (Kpiszewsk et l., 2005). However, though flesh extrcts showed the lowest TPC in every solvent (Tble 5), they exhibited higher ntigenotoxic ctivity thn clyx nd seed extrcts (Figure. Therefore, it my be stted tht ntigenotoxic effect of persimmon extrcts my be due to presence of some other constituents like -crotene (Tkhshi et l., 2006), tnnin (Lee et l., 2007) nd scorbic cid (New Food Composition Tble Editing Committee, 2009) insted of only polyphenolic compounds.

6 160 J. Med. Plnt. Res. Conclusion This study ws the first ttempt to show the difference in the functionl ctivities of persimmon clyx, seed, peel nd flesh. The results showed tht extrcts from clyx nd seed hd higher ntioxidnt ctivities nd ntigenotoxic ctivity compred to extrcts from peel nd flesh, regrdless of solvents. In ddition, this study reveled tht ethnol ws better solvent to extrct ntioxidnt compounds from persimmon compred with other solvents. Therefore, this study suggests the potentil use of persimmon extrcts by ethnol s good ntigenotoxic compounds nd nturl ntioxidnt by modulting oxidnt metbolism by stimultion of ntioxidnt ctivity. ACKNOWLEDGEMENT This study ws supported by Sweet Persimmon Export Reserch Orgniztion of Technology Development Progrm for Agriculturl nd Forestry, Ministry of Agriculture, Forestry nd Fisheries, Republic of Kore. REFERENCES Ahn HS, Jeon TI, Lee JY, Hwng SG, Lim YH, Prk DK (2002). 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