HORTSCIENCE 40(5):1204–1207. 2005. propandiol (Tris), 2,6-di-tert-butyl-4-methyl- phenol (BHT), DPPH, Folin-Ciocalteu reagent, catechin, chlorogenic acid, epicatechin, gallic Comparison of Antiproliferative acid, kaempferol, phloretin, quercetin, and quercitrin were purchased from Wako Pure and Antioxidant Properties among Chemicals Industry (Osaka, Japan). Phlorizdin dihydrate was from MP Biomedicals, Inc. Nineteen (Irvine, Calif.). Apple extracts. All apple cultivars were Yuko Yoshizawa1 maintained in the fi eld of the Department of Laboratory of Bio-organic Chemistry, Akita Prefectural University, Akita, Apple Research, National Institute of Fruit Tree 010-0195, Japan Science (Iwate, Japan). were harvested ripen (suitable for picking) on September to Kenji Sakurai November 2001 from the same growing block. Faculty of Biological Sciences, Akita Prefectural University, Akita, 010- The harvested dates for each are listed in Table 1. In total, 300 g of fresh fruit (two 0195, Japan to three fruit from each cultivar) were ho- Satoru Kawaii mogenized in 300 mL of ethanol. The ethanol extract was fi ltrated, concentrated in vacuo to Laboratory of Bio-organic Chemistry, Tokyo Denki University, Hatoyama, remove ethanol, and dissolved in distilled water Saitama, 350-0394, Japan to give 100 mL of apple extract, which was Masayoshi Asari used as stock solution (the concentration was 3 g fresh fruit equivalent/mL H2O). Kazuno Branch, Akita Fruit-tree Experiment Station, Kazuno, Akita, 018- Cells. HL-60 cells were obtained from 5201, Japan the Riken Gene Bank (Tsukuba, Japan), and were maintained in RPMI1640 medium Junichi Soejima supplemented with 10% fetal bovine serum National Institute of Fruit Tree Science, Tsukuba, Ibaraki, 305-8605 Japan (FBS). HL-60 cells in log phase (about 106 cells/mL) were diluted to 1.2 × 105 cells/mL Noboru Murofushi and preincubated for 18 h in 24-well plates Laboratory of Bio-organic Chemistry, Akita Prefectural University, Akita, (about 2 × 105 cells/mL). 010-0195, Japan Cell proliferation assay. The level of cell proliferation was measured by using alamar Additional index words. HL-60, antiproliferative activity, DPPH radical scavenging activity, Blue (Biosource International, Lewisville, Texas), an oxidation-reduction indicator. The level of proliferation was measured for HL-60 Abstract. Aqueous ethanol extracts prepared from 19 apple ( ×domestica Borkh.) cells grown in 96-well microtiter plates. Trip- cultivars were studied to explore their antiproliferative activity. Half of them showed strong licate plates were prepared. To each well 5 × inhibition on proliferation of human leukemic HL-60 cells, while the others were weak. 103 cells/100 µL of HL-60 cell suspension was Total polyphenols, 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, added, grown for 24 h, and then mixed with and total anthocyanins were measured and the results indicated that the antiproliferative 100 µL of medium containing serial dilution activity was more strongly correlated to the polyphenols and radical scavenging activity of samples to be assayed. Usually 200 µL of than to the anthocyanin content. Several polyphenols in ‘Jonathan’ were identifi ed and fi lter-sterilized apple extract (1/10 diluted from quantifi ed by high-performance liquid chromatography (HPLC) analysis. Among those the stock solution) was mixed with 600 µL compounds found during HPLC, catechin and epicatechin seemed partially responsible for of culture medium, and 4-fold serial dilution HL-60 antiproliferation. A careful examination on parentage of the apple cultivars tested was made in microtiter plates. Water-insoluble revealed that ‘Jonathan’ and its progeny showed high antiproliferation toward HL-60. standard samples were dissolved in DMSO This is the fi rst observation about the relationship between antiproliferative activity and (dimethylsulfoxide), and 8 µL of the solution parentage of apples, and the information would be useful to create new apple cultivars was added to 1 mL of the medium, then 4-fold that posses more anticancer potential. serial dilution were made in the microtiter plates, so that the fi nal DMSO concentration did not exceed 0.4% (v/v). After 3 d of incubation, Apples (Malus ×domestica Borkh.) are The health-promoting activities of apples can 20 µL of alamar Blue was aseptically added to recently indicated to have many health-promot- be infl uenced by their chemical components, each well, and incubated for 6 or 24 h. Cellular ing activities, especially anticancer, antiradical, which are the products of genetic interpretation; proliferation (% of untreated positive control) and antioxidant activities—most of these activ- thus, the effect of apple cultivars on biologi- was calculated with Eq. [1]: ities are believed to be due to their polyphenolic cal activities and mode of genetic inheritance ingredients (Boyer and Liu, 2004; Eberhardt of the activities were set as the object of our et al., 2000). On the other hand, many apple interest. To investigate these, we examined where A570 and A590 are the absorbance at 570 cultivars have historically been developed to antiproliferative and antiradical activities of 19 nm and 595 nm, respectively. achieve high production, better taste and fl a- apple cultivars, including the important ances- Total polyphenol analysis. The total phe- vor, longer shelf life, and reduction of labor. tors in apple breeding and the economically nolics were determined by Folin-Ciocalteu important progeny. Antiproliferative activity reagent primarily according to the method Received for publication 10 Nov. 2004. Accepted was studied using human leukemia HL-60 cells described in the literature (Prior et al., 1998; for publication 25 Mar. 2005. We thank Naomi and antiradical activity was examined by 1, Watanabe, Mikiko Otsu, Yuki Yokosawa, and Maiko Slinkard and Singleton, 1977), that was modi- Kikuchi for excellent technical assistance, Kiyotaka 1-diphenyl-2-picrylhydrazyl (DPPH) radical fi ed to use 96-well microtiterplate. To 20 µL of Kuroda, and Shu-hei Ikeda for discussion. This work scavenging system. 1/100 diluted sample from the stock solutions was supported in part by a grant-in-aid for scientifi c or 50, 40, 30, 20, and 10 mg·L–1 and a 0-blank research (14560027) to Y.Y. from the Ministry of Materials and Methods of standard series from gallic acid solutions in Education, Science, and Culture, Japan. 96-well microtiter plates were added 100 µL 1 Corresponding author; [email protected]. Reagents. 2-Amino-2-hydroxymethyl-1,3- of 1/100 diluted Folin-Ciocalteu stock reagent,

1204 HORTSCIENCE VOL. 40(5) AUGUST 2005

AAugustBook.indbugustBook.indb 11204204 66/14/05/14/05 112:09:122:09:12 PPMM followed after 5 min by the addition of 80 tive and antiradical activities. Figure 1 sum- total polyphenols (R = –0.66) than to total

µL of 7.5% (w/v) Na2CO3 solution. After 1 marizes the antiproliferative activity of 19 apple anthocyanins (R = –0.33), whereas no sig- h at room temperature, a microplate reader cultivars based on their ED50 (50% effective nifi cant correlation was found between DPPH (Benchmark Plus, BioRad Laboratories) mea- dose) values. Eleven cultivars strongly inhib- antiradical activity and total polyphenols (R sured the absorbance at 765 nm. The results ited the cellular proliferation of HL-60 with = –0.07) and total anthocyanins (R = 0.01).

were expressed as milligrams of gallic acid ED50 values of <20.0 mg fresh fruit equivalent However, there was a signifi cant relationship equivalent per gram fresh fruit. per well of microtiter plate. Table 1 summarizes (R = –0.26) between antiproliferative activity Total anthocyanin analysis. The total an- the DPPH radical scavenging activity of apple and DPPH antiradical activity, as indicated in thocyanin was estimated by a pH differential cultivars, and indicates 14 cultivars showed Fig. 2c, suggesting the involvement of other un- method (Cheng and Breen, 1991). Absorbance potent radical scavenging activity, whereas identifi ed compounds on antiproliferative and was measured at 510 and 700 nm in the mixture ‘’, ‘Kitaro’, ‘Kotaro’, ‘ (bagging), antiradical activities. Fruit color had no effect of 1/10 diluted stock solutions and buffers of pH and ‘Sansa’ had only weak activity. on antiproliferative and antiradical activities.

1.0 and 4.5, using A = (A510 – A700)pH1.0 – (A510 It is very natural for us to speculate that the According to the antiproliferative activity, – A700)pH4.5 and molar extinction coeffi cient of antiproliferative and DPPH radical scavenging the apple cultivars were classifi ed as those with cyanidin-3-glucoside of 29,600. The results activities due to the polyphenolic components, strong (EC50 < 3 mg), medium (EC50 was 3 to were expressed as micrograms of cyanidin- since much literature reported the anticancer 5 mg), and weak activity (EC50 > 5 mg). The 3-glucoside equivalent/g fresh fruit. and antiradical effect of polyphenolic com- results summarized in Table 2 demonstrated DPPH radical scavenging activity. The pounds. Thus, total polyphenols and total that ‘Jonathan’ and the most of its progeny scavenging activity of apple extracts against anthocyanins were measured by the methods were classifi ed as the cultivars with strong DPPH radical was measured according to the described in Materials and Methods, and the activity. method described previously (Yamaguchi et relationships between antiproliferative activity A close examination of parentage relation- al., 1998). Each 0.05 mL of 1/100 diluted stock and contents of polyphenols and anthocyanins ships of ‘Jonathan’ revealed a characteristic solutions of apple was added to 1.95 mL of are summarized in Fig. 2a and b, respectively. feature. As indicated in Table 2, ‘Jonathan’ is 100 mM Tris-HCl buffer (pH 7.4) and 3.0 mL The results showed that the antiproliferative the parent of ‘Akane’, ‘Tsugaru’, ‘’, of 100 µM DPPH in ethanol, and the mixture effect was more negatively correlated to the ‘Himekami’, and ‘Hokuto’ and is the grand- was kept at 25ºC under dark for 20 min. The absorbance at 517 nm was measured. Deionized water was used as blank experiment, and BHT (5 µg·mL–1) was used as positive control. The scavenging activity of DPPH radical (%) was calculated with Eq. [2]:

Each assay contains extract derived from 1.5 mg fruit. Identifi cation and quantifi cation of poly- phenols. Identifi cation of polyphenols was performed by HPLC according to the method modifi ed from literature (Lattanzio et al., 2001). A HPLC (L-7100; Hitachi) equipped with a UV detector, an autosampler, and an integra- tor was used. The analysis was performed on a Cadenza CD-C18 (150 mm × 4.6 mm i.d.) (Imtakt Corporation, Kyoto, Japan) with the solvent fl ow 1.0 mL·min–1. Mobile phase A was 5.0% acetic acid and B was methanol. The binary linear gradient method was used as follows: 1) linear increase in B from 5% to 15% from 0 to 20 min, 2) linear increase in B Fig. 1. ED values of 19 apple cultivars on HL-60 antiproliferation. from 15% to 40% from 20 to 30 min, and 3) 50 linear increase in B from 40% to 99% from Table 1. 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity and harvest time of apple 30 to 50 min. After washing the column with cultivars. 99% methanol in 5.0% acetic acid, the program Apple cultivar DPPH radical scavenging activity (%) Harvest turned to initial condition and the system was 1 Akane 37.0 Late September re-equilibrated for 10 min. The peaks were 2 52.0 Early November compared with those of the apple polyphenols 3 52.4 Mid-October described in the literature (Lattanzio et al., 4 Himekami 51.7 Late September 2001; Lee et al., 2003; Tsao et al., 2003; van 5 Hokuto 49.2 late October der Sluis et al. 2001), and the amounts were 6 52.0 Mid-November calculated from standard curves obtained from 7 Jona gold 51.4 Mid-October authentic samples. The antiproliferative activi- 8 Jonathan 51.2 Mid-October 9 Kitaro 21.2 Mid-October ties of these polyphenols were also examined 10 Kotaro 33.1 Late October according to the method described above. 11 Mutsu (bagged) 24.1 Late October 12 Mutsu (without bagging) 48.8 Late October Results and Discussion 13 Orin 46.0 Late October 14 Sansa 39.6 Early September Our group had been interested in anticancer 15 Santaro 52.4 Late September activity of various fruit earlier (Yoshizawa et 16 Sekai-ichi 48.2 Mid-October al., 2000, 2004). In this paper, the important 17 Senshu 52.0 Early October 18 Star king Delicious 43.2 Mid-October ancestors in apple breeding program and their 19 Tsugaru 45.4 Mid-September progeny were examined for their antiprolifera-

HORTSCIENCE VOL. 40(5) AUGUST 2005 1205

6650-Breed.indd50-Breed.indd 11205205 66/21/05/21/05 55:04:53:04:53 PPMM parent of ‘Santaro’, ‘Kitaro’, ‘Kotaro’, and to catechin and epicatechin, but should be an In conclusion, the genetic characteristics of ‘Sansa’. ‘Jonathan’ itself and its next generation integrated effect of not only the ones found ‘Jonathan’ had an intimate relationship toward of hybrids were classifi ed as the strong group on HPLC but unidentifi ed compounds, such the antiproliferative property, and most of ‘Jona- according to their antiproliferative activities. as procyanidins. Kaempferol, phloretin, and than’ relatives (eight of nine cultivars) tested in Other progeny of ‘Jonathan’ also had medium quercitrin were reported as common apple this study showed strong or medium activity. and strong activities, with the exception of polyphenol compounds (Boyer and Liu, 2004), This is the fi rst observation of antiproliferative ‘Kotaro’. A difference of antiproliferative ac- and some of them showed activity to HL-60; activity related to the parentage of apples, and the tivity was found between ‘Kotaro’ and ‘Kitaro’, however, these were not detected from ‘Jona- information will be useful to create new apple although they have same parent, i.e., ‘Fuji’ and than’. The composition of procyanidins and cultivars with anticancer potential. ‘Hatsuaki’. The inconsistency can be explained other polyphenols in ‘Jonathan’ are currently as results of the highly heterozygous nature of being investigated. Literature Cited the cultivated apples. It is well known that there Boyer, J. and R.H. Liu. 2004. is a considerably wide range of expression of (a) Apple phytochemicals most characteristics in apple seedlings, and and their health benefi ts. the difference in fruit color, i.e., ‘Kitaro’ is 15 Nutr. J. 12:5–20. yellow and ‘Kotaro’ is red, also seemed to be Cheng, G.W. and P.J. Breen. the result of heterozygous nature. 1991. Activity of phenyl- ‘Golden Delicious’ was developed from a alanine ammonia-lyase chance seedling, perhaps from ‘’, uit) 10 (PAL) and concentration

and a relationship between ‘Golden Delicious’ h fr R = -0.66 of anthocyanins and phenolics in develop- and ‘Jonathan’ has not been clear. ‘Golden De- ing strawberry fruit. J. licious’ is the parent of ‘Tsugaru’, ‘Jonagold’, Amer. Soc. Hort. Sci.

‘Mutsu’ (bagged and no bagging), ‘Orin’, and polyphenols (mg/g fres 5 116:865–869. ‘Sekai-ichi’ and is the grandparent of ‘Santaro’, Eberhardt, M.V., C.Y. Lee, ‘Kitaro’, ‘Kotaro’, ‘Sansa’, and ‘Senshu’. and R.H. Liu. 2000. ‘Golden Delicious’ also had strong activity, Antioxidant activity of although most of its progeny only possessed 0 fresh apples. Nature weak activity. In contrast to ‘Jonathan’, the 0 10 20 30 40 50 60 70 80 90 100 405:903–904. antiproliferative activity of ‘Golden Delicious’ Fukuda, H. 1994. Apple, p. ED 23–27. In: K. Konishi, seemed not to be inherited to its progeny. 50 (mg equivalent of fresh fruit/well) S. Iwahori, H. Kitagawa, Similarly, ‘Indo’ is the parent of ‘Mutsu’ and T. Yakuwa (eds.). and ‘Orin’ and is the grandparent of ‘Senshu’. Horticulture in Japan. Although the parentage of ‘Indo’ has not been Asakura Publ. Co., Ltd., known, so we cannot discuss its low activity, (b) Tokyo, Japan. all of its progeny also showed weak activ- 100 Lattanzio, V., D.D. Venere, V. ity. ‘Delicious’, which is also an important Linsalata, P. Bertolini, A. breeding ancestor, is the parent of ‘Fuji’ and Ippolito, and M. Salerno. ‘Sekai-ichi’ and the origin of ‘Starking Deli- 2001. Low temperature metabolism of apple phe- cious’, although it is not examined in this report. nolics and quiescence of ‘Starking Delicious’, which is a bud mutation

esh fruit) Phlyctaena vagabunda. of ‘Delicious’, showed strong antiproliferative R J. Agr. Food Chem. 49 activity, whereas other ‘Delicious’ progenies = -0.33 5817–5821. g/g fr µ

had weak antiproliferative activity. ( Lee, K.W., Y.J. Kim, D.O. ‘Hokuto’ had been reported to be a result anthocyanins Kim, H.J. Lee, and of the cross ‘Fuji’ × ‘Mutsu’ (Yamada et al., C.Y. Lee. 2003. 1987); however, recent fi ndings suggested that phenolics in apple and 0 ‘Mutsu’ could not be the pollen parent based on their contribution to the total antioxidant capac- the S-glycoprotein profi les (Sassa et al., 1994) 0 10 20 30 40 50 60 70 80 90 100 ity. J. Agr. Food Chem. as well as the self-incompatible genotyping ED 51:6516–6520. by allele-specifi c PCR (Sakurai et al., 1997). 50 Prior, R.L., G. Cao, A. Mar- Furthermore, the comparison of S-glycoprotein (mg equivalent of fresh fruit/well) tin, E. Sofi c, J. McEwen, profi les of ‘Hokuto’ with its parents suggested C. O’Brien, N. Lisch- that ‘Jonathan’ was the true pollen parent of ner, M. Ehlenfeldt, W. ‘Hokuto’ (Sassa et al., 1994). Its strong antip- (c) Kalt, G. Krewer, and C.M. Mainland. 1998. roliferative activity may be another evidence to 65 support the parentage of ‘Hokuto’. Antioxidant capacity as infl uenced by total phe- Bagging is an operation to induce fruit color- 55 nolics and anthocyanin ation for green skin in ‘Mutsu’ (Fukuda, 1994), content, maturity, and and the effect is not really relevant to the breed- variety of Vaccinium spe- (%)

R ing aspects. However, from our results, bagging 45 = -0.26 cies. J. Agr. Food Chem. ‘Mutsu’ decreased antiproliferation of HL-60 46:2686–2693. and DPPH radical scavenging activity. 35 Sakurai, K., S.K. Brown,

cal scavenging and N.F. Weeden. 1997. To specify polyphenols involving antipro- activity

liferative activity in ‘Jonathan’, its extract was di Determining the self-in-

analyzed by HPLC and the results are shown ra 25 compatibility alleles of Japanese apple cultivars. in Table 3. The comparison with standard HortScience 32:1258– compounds revealed that catechin, chlorogenic 15 1259. acid, epicatechin, phlorizdin, and quercetin 0 25 50 75 100 were in ‘Jonathan’, and their EC values for Fig. 2. Relationship between 50 ED HL-60 were as shown. The antiproliferative 50 total polyphenols and antip- activity of ‘Jonathan’ seemed partially due (mg equivalent of fresh fruit/well) roliferative activity.

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AAugustBook.indbugustBook.indb 11206206 66/14/05/14/05 112:09:162:09:16 PPMM Table 2. Relationship between parentage and HL-60 antiproliferation. No Fruit color Name HL-60z Seed parent Pollen parent 8 Red Jonathan S Unknown 3 Yellow Golden Delicious S Unknown 6 Yellow Indo W Unknown 1 Red Akane S Jonathan Worcester 19 Red Tsugaru S Golden Delicious Jonathan 7 Red Jonagold M Golden Delicious Jonathan 4 Red Himekami S Fuji ( × Delicious) Jonathan 5 Red Hokuto S Fuji (Ralls Janet × Delicious) Jonathan 15 Red Santaro M Hatsuaki (Jonathan × Golden Delicious) Starking Delicious 9 Yellow Kitaro M Fuji (Ralls Janet × Delicious) Hatsuaki (Jonathan × Golden Delicious) 10 Red Kotaro W Fuji (Ralls Janet × Delicious) Hatsuaki (Jonathan × Golden Delicious) 14 Red Sansa S (Kidd’s Orange Red × Golden Delicious) Akane (Jonathan × ) 11 Red Mutsu (bagged) W Golden Delicious Indo 12 Yellow Mutsu (without bagging) W Golden Delicious Indo 13 Yellow Orin W Golden Delicious Indo 16 Red Sekai-ichi W Delicious Golden Delicious 2 Red Fuji W Ralls Janet Delicious 17 Red Senshu W Toko (Golden Delicious × Indo) Fuji (Ralls Janet × Delicious) 18 Red Starking Delicious S bud mutation of Delicious z S = strong activity (EC50 < 3 mg), M = medium activity (EC50 was 3 to 5 mg), W = weak activity (EC50 > 5 mg).

Sassa, H., N. Mase, H. Hirano, and H. Ikehashi. 1994. Table 3. The amounts and the antiproliferative effects to HL-60 of polyphenols in ‘Jonathan’. Identifi cation of self-incompatibility-related gly- Compound Amount in ‘Jonathan’ (µg·g–1 fresh fruit) EC (µM) coprotein in styles of apple (Malus xdomestica). 50 Theor. Appl. Genet. 89:201–205. Catechin 64 42.8 Slinkard, K. and V.L. Singleton. 1977. Total phenol Chlorogenic acid 202 80.2 analysis: automation and comparison of manual Epicatechin 787 15.5 z methods. Amer. J. Enol. Viticult. 28:49–55. Kaempferol ND 72.1 van der Sluis, A.A., M. Dekker, A. de Jager, and Phloretin ND 400 W.M.F. Jongen. 2001. Activity and concentration Phlorizdin 10 <500 of polyphenolic antioxidants in apple: effect of Quercetin Trace 9.2 cultivar, harvest year, and storage conditions. J. Quercitrin ND <500 Agric. Food Chem. 49:3606–3613. zND = not detected. Tsao, R., R. Yang, J.C. Young, and H. Zhu. 2003. Polyphenolic profi les in eight apple cultivars using high-performance liquid chromatography Yamaguchi, T., H. Takamura, T. Matoba, and J. Differentiation-inducing effects of small fruit (HPLC). J. Agr. Food Chem. 51:6347–6353. Terao. 1998. HPLC method for evaluation of juices on HL-60 leukemic cells. J. Agr. Food Yamada, M., C. Suzuki, M. Ishiyama, H. Kita- the free radical scavenging activity of foods by Chem. 48:3177–3182. yama, and T. Sato. 1987. New apple cultivars, using 1,1-diphenyl-2-picrylhydrazyl. Biosci. Yoshizawa, Y., K. Sakurai, S. Kawaii, J. Soejima ‘Natsumidori’ and ‘Hokuto’ (in Japanese with Biotechnol. Biochem. 62:1201–1204. and N. Murofushi. 2004. Antiproliferative and English summary). Bul. Aomori Apple Expt. Yoshizawa, Y., S. Kawaii, M. Urashima, T. Fukase, antioxidant properties of crabapple juices. Food Sta. 24:1–14. T. Sato, N. Murofushi, and H. Nishimura. 2000. Sci. Technol. Res. 10:41–44.

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