Evaluation of the Mutagenicity Potential of Trace-Rutinosidase Variety of Tartary Buckwheat ( Fagopyrum tataricum Gaertn . ) Using the Ames Test

To ensure the safety of “Manten-Kirari”, a non-bitter and trace-rutinosidase variety of Tartary buckwheat, we evaluated its mutagenic activity in a bacterial reverse mutagenicity assay, the Ames test. Salmonella typhimurium TA100, TA1535, TA98, TA153, and Escherichia coli WP2 uvrA were employed as test bacteria. The flour of “Manten-Kirari” was dissolved at 12 50,000 μg/mL in DMSO and investigated. The number of revertants did not differ compared to the negative control for all concentrations tested, whereas that in the positive control, the number of revertants was increased with or without metabolic activation for each bacterial strain tested. These results suggested that the flour of the Tartary buckwheat “Manten-Kirari” was not genotoxic.


Introduction
Rutin is a flavonoid and is widely distributed throughout the plant kingdom [1]- [4].Rutin is reported to have various effects such as strengthening the blood capillaries [5] [6], as an antioxidant [7]- [9] and to have alpha-glucosidase inhibitory activities [10].In addition, the clinical effects of rutin in a double-blind crossover study are investigated and reductions in serum myeloperoxidase and cholesterol levels [11], mucosal symptoms, headache, and tiredness are observed [12].For these reasons, attention has been focused on buckwheat as a rutin-rich material for food products [13] [14].
Buckwheat is the only known cereal to contain rutin in its seeds.Among cultivated buckwheat species, Tartary buckwheat (Fagopyrum tataricum Gaertn.)contains approximately 100-fold greater rutin in the seeds than common buckwheat (Fagopyrum esculentum Gaertn.).However, Tartary buckwheat seeds also contain extremely high rutinosidase activity [15]- [17] (Figure 1).This activity is sufficient to hydrolyze the rutin present in buckwheat flour (approximately 1% -2% [w/w]) within a few minutes of the addition of water [15]- [17].However, the flour of Tartary buckwheat, also known as "bitter buckwheat", is characterized by strong bitterness, thereby limiting its use in food products.
Recently, our research group develops a Tartary buckwheat variety named "Manten-Kirari" [18] [19].The variety is developed by crossing between trace-rutinosidase line and "Hokkai T8", which is the reading Tartary buckwheat variety in Japan."Manten-Kirari" flour exhibits rutinosidase activity about two or three orders of magnitude less than that of the common variety of Tartary buckwheat.Therefore, the majority of the rutin in "Manten-Kirari" is not hydrolyzed.As a result, the rutin concentration in foods containing "Manten-Kirari", such as noodle or pound cake, is much higher than doughs made with other varieties, in which almost all the rutin content is hydrolyzed [20].In addition, the flour and food products lack the characteristic bitterness of other varieties.Therefore, "Manten-Kirari" is a promising ingredient for rutin-rich food products.
Currently, very limited information regarding the safety of Tartary buckwheat or rutin is available.Wilson et al. [21] report that intravenous and intraperitoneal injections of 30 to 50 mg/kg in rats and guinea pigs, and intravenous injections of 100 to 200 mg/kg in rabbits, show no deleterious effects.Currently, rutin, such as found in "Manten-Kirari", is not widely consumed in large amounts.Therefore, to ensure the safety of "Manten-Kirari", it is necessary to evaluate its mutagenic activity.In this context, we investigate the mutagenic activity of "Manten-Kirari" flour in a bacterial reverse mutagenicity assay (Ames test).

Flour Preparation
Seeds of the Tartary buckwheat variety "Manten-Kirari" (trace rutinosidase variety) were milled using a test mill (Quadrumat® Junior, Brabender® GmbH & Co., Duisburg, Germany) at the flour milling percentage of 63%.The flour was stored at −20˚C until used for experiments.

Rutin Hydrolysis of "Manten-Kirari" Flour in DMSO
Tartary buckwheat flour (100 mg) and 1.0 mL of DMSO were suspended and incubated at 37˚C for 3 hours.Next, to extract rutin and quercetin from the DMSO-suspended flour, 7.2 mL of methanol and 1.8 mL 0.1% phosphoric acid were added to the mixture and stored at 37˚C for 16 hours.After extraction, the sample was centrifuged at 5000 g, 10 min at 20˚C, the resultant supernatant was analyzed using HPLC [17], rutin and quercetin concentrations were determined.The extracts were filtered through a 0.45 mm filter and applied to HPLC.HPLC was performed on an CAPCELL PAK C18 column (SHISEIDO, Japan) at a flow rate of 1.5 ml/min.Elution gradient program was 0 -20 min linear gradient from solvent A [methanol-water-phosphoric acid (30:69.7:0.3)] to solvent B [methanol].A chromatograph was monitored at 360 nm.

Ames Test
Salmonella typhimurium TA100 [22], TA1535 [22], TA98 [23], TA1537 [24], and Escherichia coli WP2 uvrA were used for the Ames test.Phenotype confirmation, genotype and mutation type detected are shown in Table 1.A preliminary experiment was conducted to optimize the test solution.Tartary buckwheat flour did not completely dissolve in any of the following solutions tested: water, acetone, N,N-dimethylformamide, 1,4-dioxisane and 1,4-epoxybutane.Of these, DMSO resulted in the best dispersion condition; therefore, we employed DMSO as the test solution.Tartary buckwheat flour and DMSO were mixed (12 -50,000 μg flour/mL_DMSO) and subjected to sonication for approximately 2 minutes.The S9 microsomal fraction was used as a metabolic activation system.As a positive control, B[a]P and 2AA were used in the presence of S9, while AF-2, NaN 3 and ICR-191 were used in the absence of S9.The DMSO was used as a negative control.A standard preincubation assay [25] [26] was performed.In the plate incorporation method, two replicates were conducted per dose group.
In 2 mL of the overlay agar without S9, 0.1 mL of bacterial culture (2.3 -5.7 × 10 9 bacteria), 0.1 mL of sample solution and 0.5 mL of 100 mM sodium phosphate buffer (pH 7.4) were added and mixed.Bacterial concentration was calculated using optical density.In the S9 added overlay agar, 0.5 mL of S9 mix was substituted for 0.5 mL of the sodium phosphate buffer, and the remainder of the protocol was as for the culture medium without S9 mix.Next, the molten overlay agar was added to the minimum salts agar containing 0.6% (w/v) agar and 0.5% (w/v) NaCl.After allowing the medium to harden, the plates were incubated at 37˚C for 48 h and the number of revertants was recorded.These experiments were performed at BML, Inc. (BML General Laboratory, Kawagoe, Saitama, Japan) under contract from the New Drug Research Center, Inc. (Eniwa, Hokkaido, Japan).

Rutin Hydrolysis of "Manten-Kirari" Flour in DMSO
Although the rutinosidase activity in "Manten-Kirari" is two or three orders magnitude less than that of the common variety, some rutinosidase activity remains.Therefore, we investigated the hydrolysis of rutin by trace amounts of rutinosidase in "Manten-Kirari" flour in DMSO.Rutin concentration of DMSO-suspended flour were almost same compared with intact flour (Figure 2).In addition, aglycone of rutin quercetin, which is the product of rutinosidase activity, was not increased in DMSO-suspended flour (Figure 2).This indicates that the rutin in "Manten-Kirari" was not hydrolyzed in DMSO.Therefore, the extract contained rutin as the major polyphenolic flavonoid.

Ames Test
To date, there have been few reports dealing with the mutagenic activity of rutin or Tartary buckwheat.Therefore, prior to start detailed examination, we first investigated a range of Tartary buckwheat concentrations, 12 to 50,000 μg/mL, as shown in Table 2.The positive controls showed an increase in the number of revertants both with and without S9 compared to the negative control.Also, the S9 mix or sample solution was confirmed as sterile.In the tested samples, colony numbers in "Manten-Kirari" flour did not differ from the negative control at all concentrations with or without metabolic activation for all bacteria tested.Notably, we observed precipitation with Tartary buckwheat concentrations >3130 μg/mL.In response, we used the concentration range 200 -3130 μg/mL for a detailed examination, and the results are shown in Table 3.The Ames test showed no    increase in the number of revertants for each bacterial strain tested with or without S9.In this paper, all experiments were performed in duplicate; therefore, statistical analysis could not be applied.However, the results of "Dose optimization of Tartary buckwheat flour for bacterial mutagenic assessment with or without S9" (Table 2) and "Effect of various doses of Tartary buckwheat flour on bacterial mutagenicity with or without S9" (Table 3) were almost identical in the number of revertants for each sample.Therefore, although statistical analysis could not be conducted, the results show high reproducibility.From these results, it is suggested that the flour of the Tartary buckwheat variety does not exhibit genotoxicity.In addition, dough at a dose of 5000 mg flour/ kg is non effect level at acute and subacute test using experimental animals [27].From these results, "Manten-Kirari" flour should be safe.The concentration of rutin in "Manten-Kirari" flour is about 15 mg/g flour.Among several crops, Tartary buckwheat contains a notably high polyphenol concentration.Some papers have described the effect of polyphenols on mutagenic activity, assessed by the Ames test [28].Therefore, we hypothesized that rutin may affect the number of revertants in the Ames test.However, in this experiment, "Manten-Kirari" flour did not affect the number of revertants in the Ames test, suggesting that rutin is not mutagenic.
"Manten-Kirari" is a promising Tartary buckwheat variety for use in rutin-rich food products; therefore, the results of our mutagenesis analysis provide important information for optimizing its use in the food industry.

Figure 2 .
Figure 2. Rutin residual ratio in DMSO-suspended flour.There is no significant difference between flour and DMSO-suspended flour in rutin concent.

Table 1 .
DNA sequence specificity of microbial test strains.

Table 2 .
Consideration of dose level for Tartary buckwheat flour on microbials tested with or without S9.

Table 3 .
Effect of different dose of Tartary buckwheat flour on microbials tested with or without S9.