Takatoshi Koda

A thirteen-week oral toxicity study of annatto extract (norbixin), a natural food color extracted from the seed coat of annatto (Bixa orellana L.), in Sprague-Dawley rats.

A subchronic oral toxicity study of annatto extract (norbixin), a natural food color, was conducted. Groups of 10 male and 10 female Sprague-Dawley rats were fed annatto extract at dietary levels of 0, 0.1, 0.3 and 0.9% for 13 weeks. There were no treatment-related adverse effects on body weight, food and water consumption, ophthalmology and hematology data. Blood biochemical analysis revealed changes in rats of both sexes confined to the 0.9% and 0.3% groups, including increased alkaline phosphatase, phospholipid, total protein, albumin and albumin/globulin ratio. Marked elevation in absolute and relative liver weights was also found in both sexes of the 0.9% and 0.3% groups, but not the 0.1% group. Hepatocyte hypertrophy was evident and an additional electron microscopic examination demonstrated this to be linked to abundant mitochondria after exposure to a dietary level of 0.9% annatto extract for 2 weeks. Thus, the No-Observed-Adverse-Effect-Level (NOAEL) was judged to be a dietary level of 0.1% (69 mg/kg body weight/day for males, 76 mg/kg body weight/day for females) of annatto extract (norbixin) under the present experimental conditions.

Absence of liver tumor promoting effects of annatto extract (norbixin), a natural carotenoid food color, in a medium-term liver carcinogenesis bioassay using male F344 rats

Modifying potential of annatto extract (norbixin) on liver carcinogenesis was investigated in male F344/DuCrj rats initially treated with N-nitrosodiethylamine (DEN). Two weeks after a single dose of DEN (200 mg/kg, intraperitoneally), rats were given annatto extract at dietary levels of 0, 0.03, 0.1 and 0.3%, or phenobarbital sodium at 0.05% as a positive control for 6 weeks. All animals were subjected to partial hepatectomy at week 3, and were killed at week 8. There were no deaths related to annatto extract ingestion, and the treatment had no effects on body weights, or food and water consumption. Statistically significant increases of absolute and relative liver weights were apparent in the 0.1 and 0.3% groups. However, annatto extract did not significantly increase the quantitative values for glutathione S-transferase placental form positive liver cell foci observed after DEN initiation, in clear contrast to the positive control case. The results thus demonstrate that annatto extract at a dietary level of 0.3% (200 mg/kg/day) lacks modifying potential for liver carcinogenesis in our medium-term bioassay system.

Analytical and Preparative Separation of Kaoliang and Lac Colors by pH‐Zone‐Refining CCC

Abstract The natural coloring agents Kaoliang and Lac colors were separated using pH‐zone‐refining countercurrent chromatography (pH‐z‐CCC) on an analytical or preparative scale. The main components of Kaoliang color have been reported to be apigeninidin and luteolinidin, however, the structures of other compounds present in the color have not been elucidated as it is difficult to separate the compounds using conventional chromatographic techniques. Therefore, the separation of this color was done by pH‐z‐CCC that isolated three compounds. LC/MS and 1H‐NMR analyses were used to determine the structures established as a halogenated phenyl compound, p‐coumaric acid and methylated apigenin. Lac color mainly consists of four anthraquinone derivatives, named laccaic acid A, B, C, and E. These four laccaic acids were separated on a large scale by pH‐z‐CCC. Purified Lac color (1 g) was submitted to pH‐z‐CCC with tridodecylamine as a ligand, and produced 790 mg, 41 mg, 109 mg, and 6 mg of laccaic acid A, B, C, and D, respectively, with a minimum purity of 96%.