Location: Food and Feed Safety Research2012 Annual Report
1a. Objectives (from AD-416):
Identify stress factors and modify biochemical feed stocks that can influence the production of legume phytochemicals. Test individual combinations of phytochemicals in human model systems. Characterize procedures that can enhance the levels of natural products having estrogenic, anticancer, or potential cancer preventative activities. Perform computer modeling of potential phytoalexin structures for PPAR agonist activity.
1b. Approach (from AD-416):
Soybean cultivars will initially be grown under well-controlled test tube conditions. After various growth periods, roots will be harvested and their natural products extracted. Selected compounds will be purified and identified using HPLC and various instrumental methods applicable to organic structural characterization such as MS, NMR, IR, and UV. These experiments will then be repeated in the presence of various stress factors such as cyst root nematodes, and in the presence of modified biochemical feed stocks such as aryl-substituted phenylalanine and cinnamic acid derivatives, in order to determine useful elicitation methods and to produce altered phytochemical materials, respectively. Interesting components will again be purified and identified, after which the pharmacologic properties for all materials will be assessed as either individual or combinations of phytochemicals using in vitro models for estrogenic, anticancer, and potential cancer preventative activities. The most promising natural products will be produced in larger quantities by scaling-up the appropriate controlled-growth conditions and by conducting chemical synthesis, as necessary, to support further pharmacological assessments in an expeditious manner. The latter will involve the use of in vivo models indicative of potential estrogenic activity, anticancer therapy, or cancer prevention in humans. Useful growing conditions connected to the most pharmacologically promising compounds or mixtures of components, will be extended to greenhouse settings and eventually to field environments.
3. Progress Report:
The single most significant accomplishment for the University of Toledo (UT) during FY 2006 involved the completed synthesis of glyceollin I (a compound produced in soybean with potentially important effects in human health) which was accomplished at the 10 mg level. Our overall strategy to synthesize the glyceollins at this point took advantage of ten similar chemical steps for each of the family members. The overall route has a yield of nearly 4% (percent conversion from starting materials) while also avoiding the wasteful use of solvents that was regarded as being undesirable in earlier synthetic routes. A large scale synthesis of glyceollin I beginning from nearly 250 grams of starting material was completed in FY 2009. An ongoing analysis of the effect of root cyst nematodes (a natural and common glyceollin-eliciting soy stress factor) on soybean plants growing in production farm fields has been initiated, and significant levels of glyceollins have been detected in these plants. The highest levels of glyceollin have been found in soy root tissues, where the plant comes in contact with nematodes, but detectable levels have also been found many other plant tissues, including leaves, stems, and seed pod casings. During FY 2010, both the Agricultural Research Service (ARS) and UT have demonstrated beneficial effects in, in vitro (in the lab outside of the biological surroundings) studies that suggest that the glyceollins may be additionally useful for the potential treatment of prostate cancer. A one-gram supply of synthesized glyceollin I was forwarded to the ARS for efficacy and side-effect toxicity studies in animals. A second, one-gram supply was prepared for use in in vitro stability studies and in vivo (in the animal system) pharmacokinetic (PK) studies (measuring body's reaction to drugs, including absorption and metabolism). A number of glyceollin evaluation efforts initiated earlier in the program were brought to completion in FY 2011. Stability studies indicated glyceollins are structurally stable in plasma, urine, simulated intestinal fluid, simulated gastric fluid (SGF), and aqueous buffers at a pH of 4 or above, but some breakdown was observed at or below pH 2. Two rat oral dosing studies were completed in FY 2011. A oral dosing study was performed at UT with an intravenous delivery of 2 mg/kg glyceollin I, followed by oral delivery of 20 mg/kg glyceollin I in the same animals after a 48 hr washout period.