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United States Department of Agriculture

Agricultural Research Service

Research Project: PHYTOESTROGENIC EFFECTS OF FUNGALLY INDUCED ISOFLAVONOIDS IN LEGUMES

Location: Food and Feed Safety Research

2008 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 Aspergillus 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 will be conducted on soybean seeds within the pod of a developing plant. 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. 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). 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 ARS research project is also the inhouse project of the following cooperative agreements involved in like research: McLachlan/Tulane University/6435-42530-001-01G (expired); Erhardt/Toledo University/6435-42530-001-02S; McLachlan/Tulane University/6435-42530-001-03G (new).

This past year, several new advancements were made in the area of stress-induced phytoestrogens in soybean. Further investigations using lab assays and animal testing have confirmed the 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 anti-cancer drug, 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 recently been initiated. Fortuitously, our novel synthetic step may also be useful for the production of related natural products that have already been advanced into clinical studies by other investigators. Taking advantage of this finding, we are now including these types of utilities in our chemical patent applications. At this point, we have firmly established a strong patent base around the intellectual property associated with the specific chemical production of the glyceollins, as well as providing further coverage for the various therapeutic applications that we have previously identified. Progress by cooperators was monitored through routine teleconferencing, meetings, and scientific presentations of information relating to the project at professional society meetings and conferences.

NP 107, Component: 6, Problem Statement: 4.1.3.


4.Accomplishments
1. Soybean and other plants produce higher levels of certain compounds during periods of stress or insect attack.

Our research has identified the compounds 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. Compared with the plant compounds genistein and daidzein, normally found in soy, purified glyceollins have displayed greater inhibition of estrogen’s effects on proliferation and estrogen receptor signaling in breast cancer cells. Glyceollins also are not strong plant estrogens which differ from the strong estrogens genistein and daidzein normally found in soy. These findings suggest that soy protein enriched in glyceollins may have distinct estrogen-modulating properties compared to standard soy protein. Initial animal studies demonstrated that the glyceollins inhibited breast and ovarian cancer cell proliferation. These findings suggest that glyceollins may enhance the antiestrogenic properties of standard soy protein isolate in reproductive tissues.

Results of this experiment address National Program 107, Component 6: Health Promoting Properties of Plant and Animal Foods, Problem Statement 4.1.3: Develop more nutritious plant and animal products for human consumption.

2. This past year, further progress was made confirming the potential therapeutic value of the stress-induced soy plant compounds glyceollins using an in vivo (in laboratory test animals) model.

Lab assays and animal testing have confirmed the active glyceollin compound to be glyceollin I. Also, the glyceollins have demonstrated inhibitory activity in animal testing against several breast cancer cell lines, including two cell lines that do not react to the anti-cancer drug, tamoxifen, and also a lung cancer cell line. In contrast to tamoxifen, the glyceollins had no harmful effects and partially blocked the effects of estrogen. These findings identify glyceollins as estrogen blocking agents that may be useful in the prevention or treatment of several estrogen-dependent carcinomas. Also, the glyceollins have shown inhibitory activity against prostate cancer cell growth in lab assays. In contrast to other soy plant compounds, this effect of glyceollins on prostate cancer appeared to be mediated through a pathway activated by estrogen. Hence, the glyceollins exerted multiple effects on prostate cancer cells that may be considered cancer preventive and the mechanisms of action appeared to be different from other soy-derived phytochemicals. The complete synthesis of glyceollins I has been completed on a small scale, and a larger scale process is nearing completion to produce large quantities necessary for further animal studies.

Results of this experiment address National Program 107, Component 6: Health Promoting Properties of Plant and Animal Foods, Problem Statement 4.1.3: Develop more nutritious plant and animal products for human consumption.

3. Further research has led to unique methods to produce a soy isoflavone extract containing the glyceollins from seeds grown under conditions of stress.

Several ongoing studies are examining the potential health benefits, including anticancer activities, of this soy extract. 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. Glycinol is a precursor to the glyceollins and is being used as the initial structure in computer modeling programs in the design of novel antiestrogens.

Results of this experiment address National Program 107, Component 6: Health Promoting Properties of Plant and Animal Foods, Problem Statement 4.1.3: Develop more nutritious plant and animal products for human consumption.


5.Significant Activities that Support Special Target Populations
None.


6.Technology Transfer

Number of Invention Disclosures Submitted1
Number of Non-Peer Reviewed Presentations and Proceedings5

Review Publications
Boue, S.M., Shih, F.F., Shih, B.Y., Daigle, K.W., Carter Wientjes, C.H., Cleveland, T.E. 2008. Effect of Biotic Elicitors on Enrichment of Antioxidant Properties and Induced Isoflavones in Soybean. Journal of Food Science. 73:H43-H49.

Last Modified: 9/29/2014
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