Safety and Toxicological Evaluation of a Novel Citrus Sudachi Extract Powder

Abstract

Background: Citrus sudachi, an evergreen tree found primarily in the prefecture of Tokushima, Japan, is a widely used popular citrus fruit used in cooking and also consumed as a juice. Citrus sudachi peels are rich in flavonoids including sudachitin (5,7,4’-trihydroxy-6,8,3’-trimethoxyflavone), and exhibit potent antioxidant, antimicrobial and anti-diabetic properties, while several limonoids and their glucosides are found in its seeds. We examined the broad spectrum safety of a novel light yellow to golden yellow Citrus sudachi Extract Powder (organic, nutritive) from the dried fruit rind (25:1 herbs to extract ratio) containing no less than 1% sudachitin in various toxicology models in GLP-approved laboratories.

Methods: The acute oral toxicity study was conducted in female Sprague-Dawley rats by up and down procedure. The single dose acute dermal LD₅₀ of Citrus sudachi Extract Powder was assessed in both male and female rats. The primary skin irritation toxicity of Citrus sudachi Extract Powder was assessed in female New Zealand Albino rabbits to determine the potential for Citrus sudachi Extract Powder to produce irritation after a single topical application, while primary eye irritation index of Citrus sudachi Extract Powder was conducted in female New Zealand Albino rabbits. Ames’ bacterial reverse mutation assay was conducted to determine the ability of Citrus sudachi Extract Powder to induce reverse mutation at selected histidine loci in five tester strains of Salmonella typhimurium viz. TA1535, TA97a, TA98, TA100, and TA102 in the presence and absence of a metabolic activation system (S9) at the doses of 5000, 1500, 500, 150 and 50 mg/plate. Mutagenic potential of Citrus sudachi Extract Powder was also evaluated in vitro mammalian cell gene mutation test using the thymidine kinase gene of L5178 Tk+/- 3.7.2C mouse lymphoma cell line.

Results: The acute oral LD₅₀ of Citrus sudachi Extract Powder was found to be greater than 5000 mg/kg body weight. The single dose acute dermal LD₅₀ of Citrus sudachi Extract Powder was found to be greater than 2000 mg/kg body weight in both male and female rats. In primary skin irritation test, Citrus sudachi Extract Powder was found to be slightly irritating to the skin. The primary dermal irritation index (PDII) calculated for Citrus sudachi Extract Powder was found to be 0.8. In primary eye irritation test, the maximum mean total score (MMTS) of Citrus sudachi Extract Powder was found to be 2.7 and thus, Citrus sudachi Extract Powder was classified as minimally irritating to the eye. In both Ames’ bacterial reverse mutation assay and in vitro mammalian cell gene mutation test, no mutagenicity was observed.

Conclusion: Overall, these toxicological evaluations demonstrate the broad spectrum safety of Citrus sudachi extract powder.

Keywords: Citrus sudachi Extract Powder; acute oral toxicity; acute dermal toxicity; primary dermal irritation; primary eye irritation; Ames’ bacterial reverse mutation assay; in vitro mammalian cell gene mutation assay