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,1
Life Science Faculty, Bar-Ilan University, Ramat-Gan, Israel;
Laboratory of Molecular Toxicology and
** Laboratory of Experimental Pathology; National Institute of Environmental Health Sciences, National Institutes of Health/DHHS; Research Triangle Park, NC 27709
1To whom correspondence should be addressed. E-mail: newbold1{at}niehs.nih.gov.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • phytochemicals • uterotrophic bioassay • antioxidants • flavonoids • environmental estrogens
In recent years, the naturally occurring polyphenolic antioxidants have received increased attention as cancer-preventing agents (1). Natural antioxidants such as epigallocatechin gallate (green tea polyphenol) are being tested for their efficacy in chemoprevention of skin and prostate cancers (2,3). Studies suggest that factors such as structural variation and number of polyphenolic groups may influence the effectiveness of these chemicals in impairing signaling pathways associated with tumor promotion (4). However, considerable evidence exists for the role of antioxidants in fruits and vegetables in the maintenance of health and in disease prevention (5).
Spinach is an important dietary vegetable often associated with beneficial health effects. Fresh spinach contains 
1 g/kg of total flavonoids and other phenolic constituents that act as antioxidants due to the free radical scavenging properties of their hydroxyl groups. (6).
Natural antioxidant mixture (NAO) is a water-soluble extract obtained from spinach leaves that has been shown to have antioxidative (7), antiproliferative (8) and anti-inflammatory properties (9) in biologic systems. Further, NAO has been reported to show protective effects in various animal models of disease such as doxoroubicin-induced cardiotoxicity (10), skin papilloma (11), prostate cancer (8) and LPS-induced septic shock (12,13). Based on NMR spectroscopy, the major active components of NAO have been identified as glucuronic acid derivatives of flavonoids, trans and cis isomers of p-coumaric acid and meso-tartarate derivatives of coumaric acid, and uridine (7). Each component showed antioxidant activity when the rate and extent of linoleic acid autooxidation was tested (7). Although each isolated fraction exhibited antioxidant activity, the combination of the fractions had a synergistic effect; NAO is a powerful antioxidant that is extremely effective in protecting against oxidation damage (7).
Many properties of NAO have been elucidated. It, as well as a glucurinated flavonoid recently isolated and purified from NAO and having the chemical structure of 6-(3,4-dihydroxy-phenyl)-9-hydroxy-7-methoxy-dioxolo [4,5-
] chromen-8-one 4'-ß-glucuronid (1,3), shows scavenging activity for reactive oxygen species, such as superoxide, OH anion radical and singlet oxygen (14). It is water-soluble, highly stable at high temperature and has a long shelf life (6). It is not genotoxic and lacks toxicity; the lethal dose at which 50% die (LD-50) of NAO in mice is 1500 mg/kg (6).
Because other phytochemicals with antioxidative properties such as genistein, found in soy products, show estrogenic activity (15) and bind preferentially to estrogen receptor beta (16), we tested the estrogenic potential of NAO in established in vivo and in vitro bioassays. Knowledge of the estrogenic activity of NAO is important because the treatment of developing organisms with estrogenic compounds has long-term deleterious effects (17,18).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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17ß estradiol and genistein (Sigma Chemicals Co., St. Louis, MO) were dissolved in saline and propylene glycol (1:1). NAO, a water-soluble antioxidant, is composed of a mixture of natural molecules extracted and purified from spinach leaves as described previously (7). Briefly, NAO was prepared as follows: spinach leaves were homogenized with an equal amount of water. The resulting supernatant was collected and purified by ultrafiltration using a 3000-pore-size membrane. The filtered fraction was collected, lyophilized, and stored at 4°C until diluted and further tested.
Standardization of NAO.
Recovery of NAO comprised 3 g from 1 kg of wet spinach leaves. Every NAO batch was characterized by three assays. Each batch was tested for antioxidant activity using thiobarbituric acid and xylenol orange assays (7) to monitor malondialdehyde and hydroperoxide levels. Each batch was analyzed by HPLC to determine the amount and concentration of active components such as cumaric acid derivatives and specific flavonoids. The actual concentration of active ingredients in each preparation of NAO stock solution was determined spectrophotometrically using a typical spectrum of NAO with a maximal absorption peak of cis- and trans-cumaric acid at 310 nm and two absorption peaks of flavonoids at 275 and 350 nm (7).
Composition of NAO.
Flavonoids and p-couaric acid derivatives were active components of the aqueous extract of spinach leaves (7). Based on 1H and 13C NMR spectroscopy, four of the seven hydrophobic fractions isolated from spinach extract were identified as glucuronic acid derivatives of flavonoids, three additional fractions as trans and cis isomers of p-coumaric acid and others as meso-tartarate derivatives of p-coumaric acid. The molecular weight of NAO, based on NMR analysis, was 500 to 1000. Active compounds that were identified by NMR can be separated into three categories of chemicals: flavonoid derivatives; coumaric acid derivatives; and hydrophilic components, one identified as uridine. The flavonoids (14) are similar in structure to genistein, which was previously reported to show estrogenic activity (15).
For dosing of mice, 500 mg of NAO was dissolved in 2.5 mL saline and added to 2.5 mL propylene glycol (saline:propylene glycol, 1:1) to make a stock solution (100 g/L); this concentration was the highest dose tested (1,000,000 µg/kg) and was serially diluted in saline:propylene glycol (1:1) for the lower doses (1000, 10,000 and 100,000 µg/kg). Higher concentrations of NAO could not be tested due to lack of solubility.
Immature uterotrophic bioassay.
Timed pregnant female CD-1 mice [Crl:CD-1(ICR)], obtained from the breeding colony at the National Institutes of Environmental Health Sciences (NIEHS; Research Triangle Park, NC), delivered their young on day 19 of gestation. All litters were standardized to 10 female pups per dam and housed in a temperature-controlled room (21 to 22°C) with a 12-h light and 12-h dark cycle. Mice consumed ad libitum fresh reverse osmosis/deionized water and NIH-31 feed (19). Animal procedures complied with NIEHS/NIH animal care guidelines. As previously described (20), pups were weaned on day 17, housed 5 per cage, and injected subcutaneously with varying doses (a minimum of 5 mice per dose) of NAO dissolved in saline:propylene glycol (1:1) on days 17, 18 and 19. Both 17ß-estradiol and genistein (500 and 500,000 µg/kg, respectively) were used as positive controls; the complete dose response of mice to 17ß-estradiol and genistein has been previously reported (15). Control mice were untreated. Uterine wet weight/body weight ratios were determined on day 20. We were careful not to lose uterine luminal fluid because fluid imbibition is an important estrogenic response (21). Uterine tissues were fixed in neutral buffered formalin and processed for histological examination.
Estrogen receptor transcriptional activation assay.
Estrogenic activity. The BG1Luc4E2 cell line was stably transfected with a luciferase reporter gene responsive to estrogen or chemicals that are estrogen agonists as previously described (15). The cell line was grown for 4 d in estrogen-free DMEM (GIBCO Chemical Co., Gaithersburg, MD) containing 5% fetal calf serum that was carbon/dextran filtered to remove endogenous estrogen activity. Just prior to dosing the BG1Luc4E2 cells, NAO dissolved in DMSO (<0.1%) was suspended in cell culture medium at doses ranging from 1.0 to 50,000 µg/L. Solutions of NAO were used to expose monolayers of cells grown in 96 well culture plates. In addition to the dilutions of NAO, a standard curve for 17ß-estradiol was generated (0.0001 to 50,000 µg/L). The plates were incubated in a humidified CO2 incubator to induce optimal expression of luciferase activity. Following incubation, media were removed and the cells were microscopically observed for viability. Luciferase activity induction was measured (20). Microscopic examination of the cells following exposure to the sample extracts did not indicate cellular toxicity.
Antiestrogenic activity. Various concentrations of NAO were added to a known amount of 17ß-estradiol (0.0125 µg/L) and results were compared with those for 17ß-estradiol alone.
Statistical analysis.
Statistical analysis was performed by ANOVA and Dunnett’s test (Stat View SE+ Graphics; Abacus Concepts, Berkeley, CA). Differences were considered significant at P < 0.05 using Dunnett’s test.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Estradiol and genistein (500 and 500,000 µg/kg, respecively) significantly increased the uterine wet weight/body weight ratio compared with untreated controls. The complete dose response for these two compounds has been previously reported (15). However, none of the NAO doses affected the uterine wet weight (data not shown) or the ratio (Fig. 1). Further, none of the NAO dose groups differed histologically from the control group (data not shown), whereas estradiol and genistein both increased uterine cell height, cell number and gland formation (15).
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Estrogenic activity. Microscopic examination of the cells following 24 h exposure to NAO did not indicate cell toxicity (Fig. 2A). The NAO did not induce luciferase activity relative to untreated controls, whereas estradiol increased it 50-fold (Fig. 2A). This indicates that NAO does not show estrogenic activity in this assay.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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We have previously shown that NAO isolated from spinach leaves can prevent lipid peroxidation in both plant and animal systems (6,7). Investigators have demonstrated beneficial effects of NAO in animal models of cardiotoxicity (10), LPS-induced septic shock (9,12,13), skin papilloma (11) and prostate cancer (8). We plan to test its potential beneficial use in our animal model of estrogen-induced uterine adenocarcinoma (23). However, before initiating the study and to further characterize the properties of NAO, the current investigation was conducted to determine the estrogenic activity of NAO. The results of the present study indicate that NAO, at doses comparable to genistein (15), does not show estrogenic activity; the possibility that it may show other hormonal activity continues to be tested.
Current literature highlights continuing interest in the protective biological effects of some natural antioxidants, especially from edible plants. For example, genistein isolated from soy products has been reported to have antioxidant effects, and therefore has been proposed as a preventative therapy for some disease processes. Genistein has other properties including estrogenic activity (15). We have shown that exposure to genistein during development causes long-term problems including reproductive tract neoplasia (24). Because estrogens and other environmental endocrine disrupting chemicals may be involved in fertility problems, early puberty, and cancers of the prostate, breast, ovary and uterus, it was important to determine whether NAO shows estrogenic activity before continuing with future experiments.
Because oxidative stress has been reported to play a key role in inflammatory processes, aging and carcinogenesis (25), interest continues in the protective biological effects of some natural antioxidants that may be candidates for cancer prevention therapy and extension of the human life span. Several studies have been conducted to elucidate the therapeutic potential of spinach antioxidants (6). Dietary intake of spinach extracts has been reported to beneficially affect subjects with various cancers, such as those of the ovary (26), lung (27), prostate (28), breast (29,30) and colon (30). During screening for inhibitors of lipid peroxidation, NAO was identified as a specific inhibitor of the lipoxygenase enzyme (6,7); it was subsequently studied for its efficacy as an antioxidant and found to be superior to other antioxidants such as vitamin E and green tea (6–8). The mechanism of its pharmacological effects is not understood, but it may involve direct effects such as inhibition of cellular proliferation and interference with the cell cycle, or it may be indirectly related to the scavenging of free radicals released in various organs stressed by oxidants, such as the liver, the spleen, the thymus and the reproductive tract. Elucidation of these mechanisms remains an important area of future study.
In summary, NAO does not show estrogenic or antiestrogenic activity. If no other hormonal or endocrine disrupting activity can be identified, NAO may be added to a growing list of dietary antioxidants that may be useful treatments in protecting against the toxic effects of environmental mutagens and carcinogens.
Manuscript received 28 May 2003. Initial review completed 19 June 2003. Revision accepted 15 August 2003.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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transcriptional activation assay for estrogenicity (A) or antiestrogenicity (B) in BG1Luc4E2 uterine cells treated with NAO or estradiol. The cells expressing ER were stably transfected with a luciferase reporter gene responsive to estrogens. Duplicate samples were analyzed. (A) Estradiol increases luciferase activity but NAO does not, indicating that NAO lacks estrogenicity. (B) Estradiol alone (solid bar) increases luciferase activity, but various concentrations of NAO in combination with estradiol (solid line) do not alter the effects of estradiol; therefore, this assay does not show NAO to be antiestrogenic.