A Natural Antioxidant Mixture from Spinach Does Not Have Estrogenic or Antiestrogenic Activity in Immature CD-1 Mice

The use of natural antioxidants and flavonoids in nutritional and pharmaceutical applications is increasing. Because some phytochemicals such as genistein, found in soy products, have estrogenic activity, we investigated the estrogenic potential of a natural antioxidant mixture (NAO) isolated from spinach leaves, using an in vivo uterotrophic bioassay and an in vitro transcriptional activation assay for the estrogen receptor (ER). Outbred female CD-1 mice (17 d old) were given subcutaneous injections of 17β-estradiol or genistein [500 and 500,000 μg/(kg · d), respectively] as positive controls or NAO [1000 to 1,000,000 μg/(kg · d)] for 3 d. Uterine wet weight/body weight ratios were determined. Both 17β-estradiol and genistein significantly increased uterine wet weight ratios compared with untreated controls, but NAO did not. Histological examination of the uterus showed that 17β-estradiol and genistein increased epithelial cell height, number and gland development, but NAO did not. Estrogenic activity of NAO was investigated in vitro using the ER transcriptional activation assay. BG1Luc4E2 cells expressing ER were stably transfected with a luciferase reporter gene responsive to estrogens. 17β-estradiol dose dependently increased luciferase activity; NAO had no effect. When NAO was tested for antiestrogenic activity, it did not lessen the effects of 17β-estradiol. These data suggest that NAO does not have estrogenic or antiestrogenic activity. Thus, an antioxidant mixture has been identified that does not have potentially adverse estrogenic activity.