Phenolic Compounds in Food and Their Effects on Health II

Chapter 16

Molecular Characterization of Quercetin and Quercetin Glycosides in Allium Vegetables Their Effects on Malignant 1

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Cell

Transformation 1

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Terrance Leighton , Charles Ginther , Larry Fluss , William K. Harter , Jose Cansado , and Vicente Notario Downloaded by UNIV OF MISSOURI COLUMBIA on May 7, 2013 | http://pubs.acs.org Publication Date: October 1, 1992 | doi: 10.1021/bk-1992-0507.ch016

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Department of Biochemistry and Molecular Biology, University of California, Berkeley, CA 94720 Department of Radiation Medicine, Georgetown University Medical Center, Washington, DC 20007-2144

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Flavonol levels in the edible portions of Allium vegetables (leeks, shallots, green onions, garlic and onions) range from 1 g / k g of vegetable. Shallots contained uniformly high concentration of total flavonols, >800 mg/kg i n each of five independent samples. However, onions varied widely i n the amounts of flavonoids they contained. White onions contained no detectable flavonols, but 20 cultivars of yellow and red onions contained between 60 mg/kg and >1000 mg/kg. Individual samples of leeks, garlic, and scallions contained undetectable levels of flavonols. The primary flavonols present i n shallots were quercetin 4'-glucoside and quercetin aglycone; flavonols identified in onions were three quercetin diglucosides, quercetin 4'-glucoside, querce tin aglycone, and, in some cases, isorhamnetin monoglycoside or kaempferol monoglycoside. The relative concentrations of the various onion flavonols varied significantly i n different onion cultivars. Treatment of vegetable flavonol extracts with human gut bacterial glucosidases (fecalase) efficiently deglycosylated the quercetin glucosides with concomitant release of free quercetin. Quercetin selectively inhibits the growth of transformed tumorigenic cells (ras/3T3 and H35) and prevents the neoplastic transformation of NIH/3T3 cells with the oncogene H-ras. Flavonols are widely distributed in edible plants, primarily i n the form of flavo nol glycosides (1). It has been estimated that humans consuming high fruit and vegetable content diets ingest up to 1 g of these compounds daily (2). Glycosidases are found in both saliva and in intestinal tract bacteria that catalyze the removal of the sugar moieties from a wide array of flavonol glycosides, yielding the cognate aglycones (5-5). Among the most biologically active and common dietary flavonols is quercetin (3,3',4',5,7-pentahydroxyflavone). Recent investigations of the potential effects of quercetin on human health began following the identification of quercetin as a mutagen in the Amestest (6). The glycosides of quercetin were also found to be mutatgenic i n the Ames-test, but only after activation by removal of the sugar moiety to release 0097-6156/92/0507-0220$06.00/0 © 1992 American Chemical Society

In Phenolic Compounds in Food and Their Effects on Health II; Huang, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

Downloaded by UNIV OF MISSOURI COLUMBIA on May 7, 2013 | http://pubs.acs.org Publication Date: October 1, 1992 | doi: 10.1021/bk-1992-0507.ch016

16. LEIGHTON ET AL.

Quercetin & Its Glycosides in Allium Vegetables

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free quercetin (7). These results were of general interest because compounds that are mutagens in the Ames-test are frequently also carcinogens. We recently suggested that the mutagenic effect of quercetin could be specific to the nature or the Ames-test and other in vitro test systems (8). In the presence of molecular oxygen and redox metals, such as iron and copper, quercetin can autooxidize producing hydrogen peroxide and superoxide (9) which can react by non-enzymatic Haber-Weiss reactions to form hydroxyl radicals, a highly reactive and mutagenic species. We have proposed that in vitro mutagenicity tests, like the Ames-test, provide conditions and components required for generation of hydroxyl radicals, but that such conditions and components usually do not exist under normal physiological conditions. Thus it is not surprising that although quercetin is mutagenic in several in vitro test systems, it does not appear to be carcinogenic. It is not reproducibly carcinogenic i n rats, mice or hamsters (10-14). Although some reports suggest that quercetin induces cancer in rats (15-16), these observations have not been confirmed. It does not possess cancer initiating (17) or promoting activity in two-stage skin carcinogenicity assays (13,18). It does not act as a tumor promot er in rat urinary bladder (19) or liver carcinogenesis assays (20), nor does it induce unscheduled D N A repair in the Williams' hepatocyte system (21). Quercetin also does not exhibit teratogenic activity in rats or mice at 400 mg/kg (1 mmole/kg) (22). To the contrary, quercetin has been reported to exhibit several types of anticarcinogenic activities: Inactivation of carcinogens by quercetin. Quercetin significantly reduces the carcinogenic activity of several cooked food mutagens including bay-region diol epoxides of benzo[a]pyrene, and heterocyclic amines (23-25). These carcinogens require activation by cytochrome P-450 dependent mixed function oxidases; quercetin inhibits these oxidases in vitro (26). Quercetin also inhibits the bind ing of polycyclic aromatic hydrocarbons to D N A in vitro (27), and in epidermal and lung tissue of S E N C A R rats (28-29). Of particular importance in defining the in vivo correlation between dietary quercetin inputs and inhibition of carcin ogenesis is the report that quercetin inhibits the induction of mammary cancer by 7,12-dimethylbenz(a)anthracene and N-nitrosomethylurea in rats fed querce tin containing diets (30). Tumor promoting compounds which are also inhibited by quercetin in clude 12-0-tetradecanoylphorbol-13-acetate (TPA) a phorbol ester (31-33), and aflatoxin B l (34). Quercetin also inhibits the cytotoxic effects of T-2 mycotoxins (35). Inhibition of cancer associated enzyme activities. Quercetin directly inhibits enzymatic activities associated with several types of tumor cells in vitro and in vivo including the calcium and phospholipid-dependent protein kinase (protein kinase C ) (36-40); TPA-induced lipoxygenase and ornithine decarboxylase in mouse epidermal tissue (41); the high aerobic glycolytic enzyme levels of tumor cells (42); the activity of the oncogene pp60 v-src, and other tyrosine kinases (43); cyclo-oxygenases and 15-lipoxygenase (44); cyclic G M P phosphodiesterase (45); and cytochrome P-450/P-448 monooxygenases (26). Quercetin also inhib its cigarette smoke inhalation toxicity (46). Synergistic enhancement of known anti-proliferative agents. Quercetin synergistically enhances the antiproliferative activity of the anticancer agents cisdiamminedichloroplatinum(II) (cis-DDP) (cultured Walker rat sarcinoma cells and Ehrlich cells), nitrogen mustard (39), and busulphan (47) in human tumor cell culture systems. A t 100 μ M concentrations of quercetin, the antiprolifera-


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PHENOLIC COMPOUNDS IN FOOD AND THEIR EFFECTS ON HEALTH II


tive activity of cis-DDP and nitrogen mustard were enhanced 5-10 fold. Since toxicity of these antiproliferative agents is high at therapeutic concentrations, their synergism with quercetin may be of clinical importance. Other quercetin mediated effects. Quercetin is reported to inhibit allergic and inflammation responses of the immune system (48-52), to inhibit the growth of many bacteria and fungi (53-54), and to be a vasoprotective and antithrombotic agent (55). Given these effects on human health, there is increasing interest in foods and beverages that contain quercetin. Allium vegetables contain high levels of quercetin and quercetin glycosides (1,2). We report here that these vegetables provide a useful model system to investigate the genetic and geographical fac tors which determine the quantitative levels and individual species of plant flavonols. Aglycone flavonols have greater pharmacological activity than glycosylated species (3-5). However, flavonol glycosides are the most abundant species in edible plants (1-5). For this reason we have investigated the in vitro molecular processing of onion flavonol glycosides by human gut bacterial enzyme systems. The role of quercetin or other compounds, in dietary anticarcinogenesis is uncertain unless a specific molecular target interaction can be established which is of relevance to the cancer-generating processes. We report here the devel opment of a D N A transfection system which allows the introduction of a single activated human oncogene (H-ras) into a "normal" cell line (NIH/3T3) leading to the malignant transfromation of these cells. We show that quercetin is capa ble of inhibiting this malignant transformation model when present i n the cell culture medium at μ M concentrations. Materials and methods Flavonol and vegetable sources. Cyanidin-chloride, fisitin, myrecetin, and quercetrin were generously provided by Dr. McGregor of the U.S.D.A. (Albany, C A ) . Other flavonol standards were obtained from commercial sources. Apignin, catechin and malvidin-3,5-diglucoside (Sigma), quercetin-3,5-rutinoside (Pfaltz and Bauer) and quercetin (Kodak and Sigma). Twenty cultivars of onions grown in different regions of the United States and Mexico were provid ed by R i o Colorado Seeds L t d (Table I). Other Allium vegetables were pur chased locally. Flavonoid Extraction. 250-1000 g of raw vegetable with inedible portions removed (the removed portion amounted to 10-15% of weight), were chopped, mixed with 1 ml butanol containing 0.75% glacial acetic acid/g of tissue, and blended for 2 min, incubated overnight at 4 C , and filtered through Whatman #1 filter paper. The filtrate was taken to dryness under vacuum at 65°C. The dried material was resuspended in 2.5-10 ml D M S O , and stored in foil covered screw-capped tubes. Cooked (boiled or fried) onions were extracted using an identical method. Whole onion skins (10 g) were extracted with 250 ml butanol containing 0.75% acetic acid. The butanol fraction was dried under vacuum at 65°C, and the residue was resuspended in 2.5-10 ml D M S O . e

Column chromatography on Sephadex LH-20. Flavonol D M S O extracts (1 ml) were mixed with 1 ml methanol containing 7.5% glacial acetic acid and applied to a Sephadex L H - 2 0 column (2.5 χ 65 cm). Fractions were eluted with a methanol/7.5% glacial acetic acid mobile phase at 1 ml/min, and 200 fractions of 3.5 m l were collected. The absorbance of the fractions at 365 nm was


LEIGHTON ET AL.

Quercetin & Its Glycosides in Allium Vegetables

Table I. Estimated Flavonoid Content of Onions and Other Allium Vegetables Vegetable Variety (Origin)

Flavonoid Content ( 365 /g) A

u n i t s

Yellow onion


Utah Hard Yellow Globe (Bakersfield, CA) Yellow segregant from white dehydrator (Bakersfield, CA) Rio Ringo (Mexico) onion #1 onion #2 onion #3 RCS 1506 (Rio Grande Valley, TX) Rio Ringo (Imperial Valley, CA) RCS1542 (Rio Grande Valley, TX) Rio Ringo (Rio Grande Valley, TX) RCS 1502-1 (Mexico) RCS 1507 (Mexico) Rio Enrique (Mexico) onion #1 onion #2 onion #3 RCS 1506 (Mexico) Rio Honda (Imperial Valley, CA) Yellow Sweet Spanish (Bakersfield, CA) Texas Early Gruno 502 (El Centro, CA) Yellow Bermuda # 1 (El Centro, CA) Yellow Bermuda #2 Rio Bravo (Rio Grande Valley, TX) RCS 1502-1 (Rio Grande Valley, TX) Rio Enrique (Rio Grande Valley, TX) RCS 1542 (Mexico) Ringer Select (Mexico) onion #1 onion #2 onion #3 Sweet Vidalia (Vidalia, G A) RCS 1553 (Rio Grande Valley, TX) Rio Estrella (Imperial Valley, CA) Deming (New Mexico) Red onion California Early Red (Bakersfield) Southport Red (Hollister) White onion White #1 White #2 White boiling Shallot Shallot #1 Shallot #2 Garlic Leeks Scallions (green onions)

34.4 15.3 13.5 11.5 12.6 12.3 12.2 11.8 10.6 10.3 9.6 9.2 9.3 8.7 9.3 9.1 8.7 8.5 8.2 8.1 7.7 7.6 6.7 6.2 7.5 5.8 6.2 6.1 5.8 3.5 2.1 22.9 9.6

Phenolic Compounds in Food and Their Effects on Health II

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