Chapter 7
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Anticancer Activity and Flavonoid Content of Various Citrus Juices 1
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Satoru Kawaii , Yasuhiko Tomono , Kazunori Ogawa , Minoru Sugiura , Masamichi Yano , and Yuko Yoshizawa 2
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Laboratory of Bio-organic Chemistry, Tokyo Denki University, Hatoyama, Saitama 350-0394, Japan National Institute of Fruit Tree Science, Okitsu, Shimizu, Shizuoka 424-0204, Japan Laboratory of Bio-organic Chemistry, Akita Prefectural University, Akita 010-0195, Japan 2
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It is commonly accepted that cancer formation can be prevented by the consumption of certain foods; thus flavonoids in Citrus fruits and juices are among the most prominent anti-cancer agents. From a viewpoint of health-promotion by dietary habits, cancer preventative activity in the Citrus juices is more important than that found in their inedible parts. However, few studies have focused on the biological activities in Citrus juices, possibly because the measurable activities in juices tend to be hindered by the more abundant substances, such as sugars.In order to eliminate the masking, we prepared the readily extractable fractions of Citrus juices by adsorbing on porous polymer resin, and successive elution from the resin with ethanol and acetone. Among 34 Citrus juices examined, King (C. nobilis) exhibited potent differentiation-inducing activity toward HL-60 leukemia cells, and the active principles were identified as four polymethoxyflavones.
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© 2004 American Chemical Society In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
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77 Flavonoids are important secondary plant metabolites, and are mainly present in plant tissues in relatively high concentrations as sugar conjugates. Epidemiological studies have indicated that flavonoid consumption is associated with a reduced risk of cancer. Citrus is a rich source of flavonoids. Determination of Citrusfruitswith high concentrations of individual flavonoids is desirable in order to study their biological properties. In a recent literature review on Citrus flavonoids, a broad spectrum of biological activity including anticarcinogenic and antitumor activities was discussed {1,2). From a viewpoint of health-promotion by dietary habits, cancer preventative activity in the Citrus juices is more important than that found in their inedible parts. However, a few studies have focused on the biologically active substances in Citrus juices, possibly because the measurable activity of juice tended to be hindered by the more abundant substances, which had no activity in vitro, such as sugars. This study was intended to survey the manifestation of anticancer activity and the content of major flavonoids in the representative and/or economically important Citrus species according to Tanaka's classification. We precisely evaluated the influence of cultivar on flavonoid composition by principal component analysis. Experimental A readily extractable flavonoid fraction was prepared from various Citrus juices, involving adsorption on Diaion HP-20 (Mitsubishi Chemicals, Tokyo, Japan), a porous polymer resin, and successive elution from the resin with ethanol and acetone. This method led to substantially decrease in sugar content in that fraction. Various Citrus juices were subjected to screening for anticancer effect, which was assayed by the differentiating activity against human promyelocytic leukemia cells (HL-60), and to high-performance liquid chromatography (HPLC) analysis of 24 flavonoids. The influence of cultivar flavonoid composition was examined using principal component analysis.
Results and Discussion Screening for HL-60 differentiating activity of Citrus juices The readily extractable fraction with reduced sugar content was prepared from Citrus juice. The feasibility of the physiological screening on the readily
In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
Downloaded by PENNSYLVANIA STATE UNIV on July 18, 2012 | http://pubs.acs.org Publication Date: December 1, 2003 | doi: 10.1021/bk-2004-0871.ch007
78 extractable fraction in order to identify Citrus species and cultivars rich in anti cancer substances was therefore demonstrated. The readily extractable fractions from 34 Citrus juices were examined for their HL-60 differentiating activity by monitoring nitro blue tetrazolium (NBT) reducing cell. Total activity was obtained from a division of the amount of fraction yield by the minimum effective dose, i . e. 200 μg/ml. C nobilis (common name; King, total activity; 5860), C. bergamia (common name; Bergamot, total activity; 5120), and C. limettioides (common name; Sweet lime, total activity; 4545) exhibited strong activity, whereas C. depressa (common name; Shiikuwasha, total activity; 2293) and C. erythrosa (common name; Kobeni mikan, total activity; 1157) showed moderate activity (3). The HL-60 cells (4), established from an acute myeloid leukemia patient, are blocked at a certain step of the cellular maturation process and displayed a high proliferation ability. Terminal differentiation of HL-60 can be monitored by changes of morphological, biochemical, and immunological properties. Certain compounds, known to be efficacious cancer preventative agents, including interferon, retinoic acid, l a , 25-dihy(fcoxyvitamin D , are potent inducers of HL-60 cell differentiation, and appear to be clinically effective against myeloproliferative disorders and human colon, mammary and lung xenografts and melanoma (5). 3
Isolation of active compounds from King juice The readily extractable fraction prepared from 200 mL of King juice was subjected to purification of active principles under a guide of HL-60 differentiating activity. The fraction was partitioned between diethyl ether (Et 0) and water, and the bioactive Et 0 fraction was chromatographed on a silica gel column eluted with CHC1 . Active fraction was finally purified by HPLC to give four active compounds. These compounds were nobiletin (isolation yield; 12.7 mg), heptamethoxyflavone (30.4 mg), natsudaidain (12.0 mg), and tangeretin (11.8 mg). Nobiletin was identified by direct comparison of nuclear magnetic resonance (NMR) and mass spectral data with authentic compounds; heptamethoxyflavone, natsudaidain, and tangeretin were identified by comparison of H - and C-NMR spectral data with those reported in the literature {2,6). These four polymethoxyflavones as active principles have already been isolated and identified from die fruit peels of C. nobilis as HL-60 differentiation inducer (7). King juice was indicated to be rich in polymethoxyflavones. Test medium of the readily extractable fraction of King contained 1.8 μΜ of polymethoxyflavones (nobiletin; 0.41 μΜ, heptamethoxyflavone; 0.76 μΜ, natsudaidain; 0.33 μΜ, tangeretin 0.33 μΜ). Assuming that these 2
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In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
Downloaded by PENNSYLVANIA STATE UNIV on July 18, 2012 | http://pubs.acs.org Publication Date: December 1, 2003 | doi: 10.1021/bk-2004-0871.ch007
79 polymethoxyflavones had same potency of inducing activity of NBT reduction, the total concentration of polymethoxyflavones was very close to the minimum effective concentration examined for these flavonoids (2.5 μΜ). We, therefore, considered that these polymethoxyflavones mainly contributed the differentiation inducing activity of the readily extractable fraction of King juice. The 27 Citrus flavonoids listed in Figure 1 were examined for their HL-60 differentiation-inducing activity. The rank order of potency of the flavonoid was judged by the percentage of of HL-60 cell which reduced NBT at a concentration of 10 μΜ. The decreasing sequence of the differentiationinducing activity is given as follows; natsudaidain > luteolin > tangeretin > quercetin > apigenin > heptamethoxyflavone > nobiletin > acacetin > eriodictyol > taxifolin. The percentages of NBT reducing cells were 62.5 ± 4.0, 58.5 ± 4.0, 53.0 ± 4.1, 51.8 ± 1.9, 44.8 ± 2.9, 39.3 ± 2.7, 37.3 ± 2.9, 36.8 ± 1.1, 36.3 ± 1.9, and 34.0 ± 1.9, respectively (
