Comparison of Antiproliferative and Antioxidant Properties Among
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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 Apple Cultivars (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). Apples 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 cultivar 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, Jonathan Texas), an oxidation-reduction indicator. The level of proliferation was measured for HL-60 Abstract. Aqueous ethanol extracts prepared from 19 apple (Malus ×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 ‘Akane’, ‘Kitaro’, ‘Kotaro’, ‘Mutsu (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’, ‘Jonagold’, 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.