Mutagenic, Acute, and Subchronic Toxicity Studies of the Hesperetin-7-Glucoside-β-Cyclodextrin Inclusion Complex

Abstract

Hesperetin glucosides such as hesperidin and hesperetin-7-glucoside are abundantly present in citrus fruits and have various pharmacological properties. However, the potential toxicity of hesperetin glucosides remains unclear. An initial assessment of the safety of hesperetin-7-glucoside-β-cyclodextrin inclusion complex (HPTGCD) as a functional food ingredient was undertaken to assess toxicity and mutagenic potential. A bacterial reverse mutation assay (Ames test) using Salmonella typhimurium (strains TA98, TA1535, TA100, and TA1537) and Escherichia coli (strain WP2 uvrA) with HPTGCD (up to 5000 µg/plate) in the absence and presence of metabolic activation was negative. In a single oral (gavage) toxicity study in male and female rats, HPTGCD at dose up to 2000 mg/kg did not produce mortality nor clinical signs of toxicity or change in body weight. In a subchronic oral (dietary admix) toxicity study in rats receiving 0, 1.5, 3, and 5% HPTGCD for 13 weeks, no adverse effects were noted and the no-observed-adverse-effect level (NOAEL) was 5% in the diet (equivalent to 3267.7 mg/kg/day for males and to 3652.4 mg/kg/day for females). These results provide initial evidence of the safety of HPTGCD.

Keywords: acute toxicity; functional food ingredient; hesperetin-7-glucoside; mutagenicity; subchronic dietary toxicity.

Figure 1.

Industrial production method of hesperetin-7-glucoside–β-cyclodextrin inclusion complex (HPTGCD). HPTGCD was produced using a patented method to treat hesperidin (Citrus aurantium extract [hesperidin (HSP) purity: 90.3%] with naringinase [160 units/g, Amano Enzyme, Inc., Nagoya, Japan] and β-cyclodextrin [Cyclo Chem Co., Ltd., Kobe, Japan]) with the following steps: (1) HSP (33 mM) and β-cyclodextrin (βCD, 50 mM) were mixed with water in a tank, (2) The temperature and pH of the solution were adjusted to 73°C and pH4.5 using NaOH and H₂SO_4., (3) Naringinase digestion (30 units/DSN g) was conducted for 24 h at 73°C, pH 4.5, (4) After confirming a > 96% conversion rate to HPTG*, the solution including the product was filtrated and turned into powder using a spray dryer machine after sterilization. *The conversion rate (%) was performed by nearly 98% using high-performance liquid chromatography (HPLC) analysis.

Figure 2.

Changes in mean body weight in male and female Sprague–Dawley (SD) rats administered a HPTGCD-containing diet in the 13-week subchronic toxicity study. Each point represents the mean value of the animal groups fed with the indicated concentration (0%, 1.5%, 3.0%, and 5.0%) of HPTGCD in the diet.

Figure 3.

(a) Food consumption per animal/day in male and female SD rats administered an HPTGCD-containing diet in the 13-week subchronic toxicity study. (b) HPTGCD intake based on body weight of SD rats in each study group during the investigation period of 13 weeks for the subchronic toxicity study. Each point represents the mean intake per animal group fed with the respective dose (0%, 1.5%, 3.0%, and 5.0%) of HPTGCD in the daily diet.