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United States Department of Agriculture

Agricultural Research Service

Research Project: PHYTOESTROGENIC EFFECTS OF FUNGALLY INDUCED ISOFLAVONOIDS IN LEGUMES
2010 Annual Report


1a.Objectives (from AD-416)
1. Induction, purification, and characterization of isoflavonoids in soybean seed, root, plant and cell culture incubated with A. sojae and A. sojae cell wall extracts. Induced isoflavonoids will be produced in soybean using several different biotic elicitors developed from A. sojae. Elicitor experiments will initially focus on soybean seeds, roots and cell cultures. Once an effective elicitor system is developed in the laboratory, elicitor experiments to induce isoflavonoids will be conducted on soybean seeds within the pod of a developing plant. 2. Determine hormonal and antioxidant activities of individual and combinations of induced isoflavonoids with in vitro bioassays. Determine hormonal and antioxidant activities of induced isoflavones purified under Objective 1 using in vitro bioassays. Three established assays for the determination of estrogenic and antiestrogenic activity will be utilized. Additionally, the antioxidant activities of induced isoflavones will be determined using three assays. 3. Determine hormonal and anticancer effects of individual and combinations of induced isoflavonoids with in vivo animal model systems. Determine estrogenic, antiestrogenic and anticancer activities of induced isoflavones (discovered in Objective.
1)in two in vivo mouse models. Induced isoflavones that display in vitro activity in Objective 2 will be lead candidates for in vivo testing. The glyceollins represent potential beneficial antiestrogens that need to be tested in animal models to confirm activity observed in vitro. Two different mouse model systems will be utilized to confirm estrogenic, antiestrogenic and anticancer activity (breast and ovarian cancer). 4. Develop methods for the organic syntheses of the glyceollins I, II and III. Also, precursors and synthetic analogues of the glyceollins will be produced and characterized for beneficial hormonal activities. Computer modeling methods will be developed to assist in the development of structure-activity relationships that point toward the most active forms of the molecule. Accomplishment of objectives 2-4 will require seeking out additional scientific cooperators with the particular expertise needed, either within ARS or in other organizations.


1b.Approach (from AD-416)
Induced isoflavonoids will be produced in soybean using several different biotic elicitors developed from A. sojae. Elicitor experiments will initially focus on soybean seeds, roots and cell cultures. Once an effective elicitor system is developed in the laboratory, elicitor experiments to induce isoflavonoids will be conducted on soybean seeds within the pod of a developing plant. Isolated induced isoflavones will be tested for hormonal and antioxidant activities using several in vitro bioassays. Three established assays for the determination of estrogenic and antiestrogenic activity will be utilized. Additionally, the antioxidant activities of induced isoflavones will be determined using three assays. Induced isoflavones that display in vitro activity will be lead candidates for in vivo testing. The glyceollins represent potential beneficial antiestrogens that need to be tested in animal models to confirm activity observed in vitro. Two different mouse model systems will be utilized to confirm estrogenic, antiestrogenic and anticancer activity (breast and ovarian cancer). Also, organic syntheses methods for the glyceollins I, II and III will be developed. Precursors and synthetic analogues of the glyceollins will be produced and characterized for beneficial hormonal activities. Computer modeling methods will be developed to assist in the development of structure-activity relationships that point toward the most active forms of the molecule.


3.Progress Report
This is the final report for the project 6435-53000-001-00D terminated in November 2009.

Substantial results were realized over the life of the project. The soybean compounds, called glyceollins, that are induced in activated soy have shown unique antiestrogenic activity that has correlated to inhibition of hormonally induced cancers. Initial research showed the glyceollins inhibited the growth of breast and ovarian cancer cells in lab and animal studies. Further research showed that the glyceollins also inhibited the growth of prostate cancer cells. These findings suggest that soy protein enriched in glyceollins may have distinct estrogen-modulating properties compared to standard soy protein. To test this idea, a postmenopausal primate model was used to evaluate the short-term effects of glyceollin-enriched soy protein and standard soy protein isolate in combination with estrogen. Compared to baseline, estradiol (a female hormone) treatment induced significantly greater breast proliferation in the control and soy protein group, but not in the glyceollin-enriched soy protein group. This change was accompanied by a reduction in estradiol-induced uterine area and markers of estrogen receptor alpha activity markers in the glyceollin-enriched soy protein group compared to the control group. These preliminary findings suggest that glyceollins may enhance the anti-estrogenic properties of standard soy protein isolate in reproductive tissues. Other glyceollin-enriched soy foods being tested are soy sprouts and roasted soybeans. Further research has led to unique methods to produce a soy isoflavone extract containing the glyceollins from seeds grown under conditions of stress. A second induced soy compound called glycinol has also been isolated from stress-induced soy. Glycinol has demonstrated potent estrogenic activity in several in vitro assays.

Further investigations using lab assays and animal testing have confirmed the active stress-induced soy compound to be glyceollin I (from a mixture of glyceollins I, II, and III). Also, the glyceollins have demonstrated inhibitory activity in animal testing against several breast cancer cell lines, including two cancer cell lines that do not react to the cancer agent tamoxifen, and also a lung cancer cell line. In contrast to tamoxifen, the glyceollins had no harmful effects and partially blocked the effects of female estrogen. These findings identify glyceollins as antiestrogenic agents that may be useful in the prevention or treatment of breast and ovarian carcinoma without harmful effects. In order to further study the glyceollins activity in animal models, we have successfully developed a synthetic scheme to produce glyceollin I and II. A synthetic route is desirable since the glyceollins (a mixture of glyceollin I, II, and III) have been difficult to purify as individual components. A large scale synthesis of glyceollin I beginning from nearly 250 grams of starting material has been completed.


4.Accomplishments
1. Glyceollins Identified as Antiestrogens With Anticancer Activity. New effective treatments for breast cancer utilize antiestrogens. Agricultural Research Service researchers at New Orleans, Louisiana in collaboration with scientists at Tulane University have identified the compounds called glyceollins I, II, and III in soybean plants grown under conditions of stress as therapeutic antiestrogens that inhibit the growth of estrogen-dependent cancers. Glyceollins are not strong plant estrogens which differ from the strong estrogens genistein and daidzein normally found in soy, which suggests that soy foods enriched in glyceollins may have distinct estrogen-modulating properties compared to standard soy foods. Initial animal studies demonstrated that the glyceollins inhibited breast and ovarian cancer cell proliferation. These findings suggest that glyceollins in diets or as a pharmaceutical may enhance the antiestrogenic properties of standard soy foods in reproductive tissues and prevent cancers.

2. Glyceollin I Is Active Component of Glyceollin Mixture. The glyceollins, a group of novel phytoalexins isolated from activated soy, consist of a mixture of 3 compounds. We have shown that the glyceollin mixture is effective as a potential antiestrogenic, therapeutic agent that prevents estrogenstimulated tumorigenesis and displays a differential pattern of gene expression from tamoxifen, the current drug used in therapy. We identified glyceollin I as the active component of the combined glyceollin mixture. Further comparison of the effects of glyceollin I to the antiestrogens, -hydroxytamoxifen and ICI 182,780 (fulvestrant), in MCF-7 breast cancer cells and BG-1 ovarian cancer cells on estradiol-stimulated expression of progesterone receptor and stromal derived factor established a novel inhibition of ER-mediated gene expression and cell proliferation/survival. Glyceollin I may represent an important component of a phytoalexin-enriched food (activated) diet in terms of chemoprevention as well as a novel therapeutic agent for hormone dependent tumors.

3. Glyceollin-Enriched Soy Foods Produced. The production of soy foods containing the glyceollins is needed. Preliminary results indicate that several different elicitors can be used to induce glyceollins to high concentrations in different soy foods. A glyceollin-enriched soy protein isolate was first produced in our laboratory. Other glyceollin-enriched soy foods being tested are soy sprouts and roasted soybeans and a soy isoflavone extract containing the glyceollins from seeds grown under conditions of stress. These foods offer potential health benefits, including anticancer activities and could therefore be included in diets.


Last Modified: 10/25/2014
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