Myricitrin, a flavonol rhamnoside of myricetin extracted from the Chinese bayberry (Myrica rubra Siebold) plant, has been used in Japan since 1992 as a flavour modifier in snack foods, dairy products, and beverages. It is affirmed as generally recognised as safe (GRAS) by the US Flavour and Extract Manufacturer Association (FEMA) and is considered safe by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) at current estimated dietary exposures. In anticipation of expanded marketing, 97% pure myricitrin was fed to male and female Sprague–Dawley rats at dietary concentrations of 0.5%, 1.5% and 5.0% in a 90-day toxicity study. There was increased food consumption and decreased body weight gain in males exposed to 5% myricitrin. Blood values were within laboratory reference ranges except for mean increases in basophils in low- and high-dose males and serum phosphorus in high-dose males. In the absence of abnormal clinical or histopathological changes, these changes are not considered adverse. Based on the 90-day rat toxicity study, the no observed adverse effect level (NOAEL) is 2926 mg kg–1 day–1 in males and 3197 mg kg–1 day–1 in females. Gavage administration of myricitrin resulted in blood levels of myricitrin within 1 h after single oral doses of 250, 500 or 1000 mg kg–1 body weight, indicating direct absorption of the glycosylated form of this flavonoid. Blood levels of myricetin, a metabolite of myricitrin, were not present in rats dosed orally with 1.6 mg kg–1 myricetin, but were present only at 12 or 24 h in one of five, in three of five, and in four of five rats dosed with 250, 500 and 1000 mg myricitrin kg–1 body weight, respectively, possibly a result of hepatic conversion of myricitrin to myricetin and enterohepatic recirculation of the resulting myricetin. The current studies further support prior safety assessments of myricitrin as a food flavouring.
flavour modifier, flavonol, myricetin, GRAS
Myricitrin is the rhamnose glycoside of myricetin, a naturally occurring flavonol isolated from the fruit, bark or leaves of the Chinese bayberry (Myrica rubra Siebold) and other medicinal plants. As with other polyphenols, it has a long history of use for its anti-oxidant, free-radical scavenging, anxiolytic, antinociceptive and anti-inflammatory properties and has been available from the Japanese market since 1992. The pharmacological properties are largely based on the inhibition of subtypes of protein kinase C, p38MAPK signalling, and inhibition of nitric oxidase synthetase, cyclooxygenase 2, TNF alpha, and myeloperoxidase activities (Schwanke et al. 2013). In Japan myricitrin is used as a flavour modifier in snack food, dairy products and beverages. Myricetin, the aglycone of myricitrin, structurally differs from quercetin by the presence of a hydroxyl group at the 5ʹ position on the B ring. The structures of myricitrin, myricetin and quercetin are present in Figure 1.
Figure 1. Structural formulas for myricitrin, myricetin and quercetin.
As a flavonoid,the bioavailability of dietary myricitrin is expected to occur following hydrolysis with release of the aglycone (myricetin) in the small intestine (Cook & Karmazyn 1996; Bravo 1998; Aherne & O’Brien 2002; Del Rio et al. 2010). Flavonoids and metabolites not absorbed in the small intestine are then acted on by colonic microflora with cleavage of conjugating moieties and ring fission of aglycones, absorption of resultant phenolic acids, phase II hepatic metabolism with enterohepatic circulation and urinary excretion (Blaut et al. 2003; Del Rio et al. 2010).
For myricitrin specifically, early studies in rats show that intestinal microorganisms breakdown myricitrin to myricetin, 3,4,5-trihydroxyphenylacetic acid and 3,5-dihydroxyphenylacetic acid (Smith & Griffiths 1970) with urinary excretion of ring-fission hydroxyphenylacetic products (Griffiths & Smith 1972). Once absorbed, the liver is the main site of myricetin metabolism while intestinal wall and kidney are secondary sites of metabolism (Ong & Khoo 1997). In a more recent study using intestinal loops in anaesthetised rats, myricitrin was absorbed by intestinal epithelial cells as a glycoside, not as the aglycone, with release into mesenteric blood as glucuronide or sulphate conjugates of myricitrin (Matsukawa et al. 2012). In simulated digestion experiments mimicking the human gastrointestinal system, myricitrin is stable at acidic pH 1.8 conditions and is degraded at alkaline pH 8.5, but without losing its inhibitory effect on induced low-density lipoprotein oxidation (Yokomizo & Moriwaki 2005). Myricitrin metabolites produced in vitro by human intestinal bacteria are indicative of dehydroxylation to quercetin-3-O-rhamnoside and deglycosylation to quercetin and myricetin (Du et al. 2014).
Myricitrin is affirmed as generally recognised as safe (GRAS) by the US Flavour and Extract Manufacturer Association (FEMA; Smith et al. 2009) and is considered to be of no safety concern based on current estimated dietary exposures by JECFA (2014). Highly purified commercially available myricitrin contains a small amount of myricetin, the aglycone of myricitrin. Myricetin and structurally related flavonoids such as quercetin have shown genotoxicity and DNA damage in several studies (Hardigree & Epler 1978; MacGregor & Jurd 1978; Hatcher & Bryan 1985; Sahu & Gray 1993; Hobbs et al. 2015). Previously conducted 3- and 12-month dietary studies of Chinese bayberry extract in rats were without toxicity (Yoshino et al. 2001). However, in those studies the test material was only 29.8% pure and contained 0.44% myricetin. Thus, the actual exposure to myricitrin may not have been sufficiently high enough to ensure safety. In anticipation of an expanded market as a flavouring agent, a 90-day rat toxicity study and a single-dose toxicokinetic (TK) study to define the safety of > 97% pure myricitrin are reported herein.
Materials and methods
Ninety-day repeated-dose toxicity study
A repeated-dose 90-day oral toxicity study was conducted at Integrated Laboratory Services (ILS), Inc. (Research Triangle Park, NC, USA) in male and female Sprague– Dawley rats following good laboratory practices, Organisation for Economic Co-operation and Development (OECD) Guideline #408, and Eika No. 29 (Japan Ministry of Health and Welfare). Greater than 97% pure myricitrin, containing 0.16% myricetin, was obtained from San-Ei Gen, F.F.I., Inc. (Osaka, Japan) and added to Purina Certified 5002 meal diet (Ralston Purina Company, St. Louis, MO, USA) at dose concentrations of 0.5%, 1.5% and 5.0%. Dose formulations were within acceptable criteria for both concentration and uniformity (Tables 1 and 2). Stability of myricitrin in the diet stored between 0 and 30° C was at least 56 days. Control diet was Purina Certified 5002 meal without added myricitrin. Ten males and 10 females were assigned to each dietary group based on equivalent body weight following a 7-day acclimatisation period. Dose levels were selected following a 14-day range-finding study without abnormal clinical observations, changes in body weights or selected tissue weights.
Toxicokinetic (TK) study
A TK study of myricitrin (> 97% pure as above) was conducted at ILS in accordance with the USFDA’s Good Laboratory Practice Regulations (21 CFR Part 58). This study was designed to satisfy the Testing Guideline No. 417: Toxicokinetics (OECD 2010), specifically timecourse studies; plasma/blood kinetics, as well as all applicable standard operating procedures of ILS. Twenty male Sprague–Dawley rats were allocated to one of four designated dose groups and administered a single oral dose of one of three dose levels of myricitrin (250, 500 and 1000 mg kg–1 body weight) or one dose level of myricetin (1.6 mg kg–1 body weight) in corn oil. The concentration and uniformity of myricitrin and myricetin in corn oil was within acceptable limits (Table 3). The top dose of myricitrin (1000 mg kg–1 body weight) was selected due to the low toxicity and the limit dose as stipulated in the test guideline (OECD 2010). Because myricetin, the aglycone of myricitrin, has some demonstrated genotoxicity, one group of rats was administered 1.6 mg kg–1 myricetin based on the amount of myricetin present in the highest dose of myricitrin. Blood was collected prior to and at 1, 3, 6, 12 and 24 h following dose administration. Plasma was analysed for myricitrin and myricetin concentrations by Applied Biosystems API-3000 LC/MS/MS (Applied Biosystems, Grand Island, NY, USA).
Both studies were conducted within the same Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC)-accredited specific pathogen-free facility. All procedures were in compliance with Animal Welfare Act Regulations (9CFR 1-4) and the Guide for the Care and Use of Laboratory Animals (ILAR 2011), and with approval of the ILS Institutional Animal Care and Use Committee.
Group mean and standard deviations (SDs) were calculated and reported. All data were analysed – final body weight, body weight gain, food consumption (g kg–1 day–1 ), absolute and relative (to body weight) tissue weights, neurotoxicological endpoints, urinalysis endpoints, and clinical pathology endpoints – using Statistical Analysis System version 9.2 (SAS Institute, Cary, NC, USA). First, studentised residual plots were used to detect possible outliers in the data. Homogeneity of variance was analysed using Levene’s test. If the data were heterogeneous, then appropriate transformations (log, square root, multiplicative inverse) were performed and the data reanalysed for homogeneity of variance. Data were then analysed using a one-way analysis of variance (ANOVA) and myricitrinexposed groups were compared with the appropriate control group using Dunnett’s test. Heterogeneous datasets were analysed using a Dunn’s test to compare exposed groups with the concurrent control group. Finally, dosedependent changes were evaluated using a linear regression model.
TK parameters were derived using non-compartmental methods employing a validated installation (validated on 20 September 2013) of Phoenix WinNonlin® version 6.3 (Pharsight Corporation, St. Louis, MO, USA). Area under the curve (AUC) values for myricitrin concentration of each animal were determined and the resulting values were compared among groups via appropriate t-tests.
Ninety-day repeated-dose study
All rats survived to the scheduled termination at 90–93 days without any clinical evidence of moribundity. There were no myricitrin-related clinical abnormalities during the study. Group mean initial and final body weights as well as body weight gain are presented in Table 4. While there were no statistically significant changes in final body weight, there was a 10.5% reduced body weight gain in males in the 5.0% dose group. There were no changes in feed consumption in females exposed to myricitrin compared with concurrent controls. There was a significant increase in food consumed by male rats exposed to 1.5% and 5.0% myricitrin compared with concurrent controls with a significant dose-dependent trend (Table 5).