(T1030-05-29) Radioprotective Effects of a Standardized Tocotrienol Rich Extract from Annato Seeds (Bixa orellana L.)

Purpose: For the past decades, the US Government Radiation and Nuclear Countermeasures Program has been seeking the development of radioprotectant and radiomitigator agents. Nevertheless, currently there is no drug that can provide muti-organ protection against lethal doses of radiation, that is affordable, does not show toxicity, can be stockpiled and can be easly and rapidly administered. In this research we have prepared an standarized a tocotrienol rich extract, from annatto seeds, Bixa orellana L. In this research we have tested the standardized extract in vitro and in vivo to asses its potential as a radioprotective agent.

Methods: Dried seeds of annatto were extracted with hexanes and and enriched tocotrienol extract, was prepared using column chromatography in silica gel 70-230 mesh, eluting with hexane-ethyl acetate (9:1) The extract was standardized to contain 60% of tocols. The antioxidant activity was evaluated in rat liver microsomes by measuring inhibition of lipid peroxidation using TBARS kits from Cayman Chemical (Ann Arbor, Michigan). For clonogenic cell survival assay, H9C2 cells were plated in 60-mm dishes with 5 ml of complete medium and allowed to attach and incubate at 37°C for 24h. Cells were then exposed to radiation. Cellular oxygen consumption rates (OCR) were measured at 37°C using an XF96 extracellular analyzer (Seahorse Bioscience). Survival studies were performed using CD2F1 male mice. The mice were feed vitamin E deficient diets for seven days before irradiation, to minimize the effect of dietary tocols on the radioprotection study. Twenty-four hours before total body radiation, mice received subcutaneous injection of 0.1 mL of vehicle or extract (adjusted to contain 300 mg/kg body weight). The vehicle was 5% Tween in PEG 400 solution. Injections were administrated subcutaneously near the femur with a 23-gauge needle. Mice received total body irradiation in two different single doses (8.5 Gy and 9.5 Gy) using a Mark I Model 68A cesium-137 animal irradiator (Shepherd & Associates, San Fernando California, CA).

Results: An efficient method was developed to enrich the tocotrienol content  from 0.2 % in the annatto seeds to 60 % of tocotrienols in the standardized extract. The extract contains δ-tocotrienol and γ-tocotrienol, in a 10:1 ratio. The extract inhibits lipid peroxidation of Wistar rat liver microsomes (Figure 1), it also protects H9C2 rat cardiomyocytes against H₂O₂ induced oxidative injury (Figure 2), used as a pre-treatment. For the radioprotection survival studies mice were monitored for 30 days with all observed deaths occurring between days 10 -19. Remarkably, the extract provided 100% survival at both the 8.5 Gy (LD50) and 9.5 Gy (LD100) doses of irradiation, (Figure 3 & 4). These results show that our tocotrienol-rich fraction of annatto displays profound radioprotective properties against lethal doses of radiation in vivo, when mice were pre-treated subcutaneously 24 hours prior to irradiation.

Conclusion: An annatto extract, containing higher concentration of the natural tocotrienols, was prepared by extraction, purification, and standardization. In the tests of its radioprotective efficacy, all mice treated with a single subcutaneous dose of annatto standardized extract survived a total body irradiation (TBI) at 8.5 and 9.5 Gy, which are otherwise lethal doses. This study suggest that annatto standardized extract has the potential to protect humans against radiation and may be promising option to develop into an economical and efficacious radioprotectant product.

Acknowledgments: The University of Arkansas has received patent protection for parts of this research. A potential royalty stream may occur consistent with the University of Arkansas policy. This research was supported in part by grants from the National Center for Research Resources award 1ULl RR029884, the IDeA Networks of Biomedical Research Excellence (INBRE), UAMS start-up funds, and NIH/NIAID grant AI67798. ENP was a recipient of a graduate scholarship from SENESCYT Ecuador.

Inhibition of TBHP induced lipid peroxidation in Wistar rat liver microsomes by DT3 and enriched extract. Data are means ± SEM of triplicate experiments



H9C2 heart cardiomyocytes were treated with 5 uM DT3 or 5 uM extract or vehicle control (DMSO) overnight. Cells were then exposed to 100 uM H2O2 for 4h in the presence or absence of tocotrienols. Cellular bioenergetic profiles were determined using XF96 Seahorse Extracellular Flux Analyzer



(A) Survival curve for CD2F1 irradiated mice at 8.5 Gy dose. (B) Survival curve for CD2F1 irradiated mice at 9.5 Gy dose.