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.
Rutin is a flavonoid and is widely distributed throughout the plant kingdom [
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 [
Recently, our research group develops a Tartary buckwheat variety named “Manten-Kirari” [
Currently, very limited information regarding the safety of Tartary buckwheat or rutin is available. Wilson et al. [
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.
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 [
Salmonella typhimurium TA100 [
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, NaN3 and ICR-191 were used in the absence of S9. The DMSO was used as a negative control. A standard preincubation assay [
In 2 mL of the overlay agar without S9, 0.1 mL of bacterial culture (2.3 - 5.7 × 109 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).
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 (
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
Allele | Strains | DNA target | Revertion event | Reference |
---|---|---|---|---|
hisG46 | TA100 | -G-G-G- | Base-pair substitution | [ |
hisG46 | TA1535 | -G-G-G- | Base-pair substitution | [ |
trpE95 | wp2 uraA | A:T | Base-pair substitution | |
hisD3052 | TA98 | -C-G-C-G-C-G-C-G- | Frameshift | [ |
HisC3076 | TA1537 | -C-C-C-C-C-C-(+1 cytosine at run of C’s) | Frameshift | [ |
concentration (μg/ml) | TA100 | TA1535 | wp2 uraA | TA98 | TA1537 | |||||
---|---|---|---|---|---|---|---|---|---|---|
−S9 | +S9 | −S9 | +S9 | −S9 | +S9 | −S9 | +S9 | −S9 | +S9 | |
Negative controles 0 | 147 | 163 | 14 | 10 | 33 | 35 | 19 | 30 | 17 | 23 |
12 | 170 | 149 | 10 | 13 | 33 | 31 | 18 | 29 | 19 | 21 |
49 | 159 | 147 | 13 | 10 | 33 | 34 | 21 | 31 | 18 | 24 |
200 | 146 | 162 | 15 | 10 | 29 | 30 | 16 | 33 | 16 | 23 |
780 | 156 | 160 | 14 | 11 | 32 | 30 | 18 | 29 | 16 | 24 |
3130 | 161 | 164 | 11 | 10 | 32 | 31 | 16 | 27 | 16 | 19 |
12,500 | 159 | 175 | 11 | 11 | 32 | 31 | 19 | 29 | 15 | 19 |
50,000 | 148 | 169 | 10 | 11 | 32 | 28 | 16 | 28 | 14 | 19 |
Positive controls | 605 | 949 | 130 | 332 | 143 | 315 | 604 | 225 | 1820 | 95 |
Substance | AF-2 | B[a]P | NaN3 | 2AA | AF-2 | 2AA | AF-2 | B[a]P | ICR-191 | B[a]P |
Data are means of two independent experiments.
Concentration (μg/ml) | TA100 | TA1535 | wp2 uraA | TA98 | TA1537 | |||||
---|---|---|---|---|---|---|---|---|---|---|
−S9 | +S9 | −S9 | +S9 | −S9 | +S9 | −S9 | +S9 | −S9 | +S9 | |
Negative controles 0 | 152 | 162 | 15 | 13 | 33 | 40 | 19 | 29 | 16 | 19 |
200 | 153 | 168 | 14 | 17 | 31 | 42 | 21 | 24 | 14 | 23 |
390 | 166 | 156 | 12 | 14 | 30 | 32 | 18 | 30 | 16 | 25 |
780 | 162 | 169 | 13 | 14 | 34 | 32 | 17 | 22 | 16 | 19 |
1560 | 166 | 157 | 15 | 16 | 36 | 31 | 20 | 30 | 15 | 21 |
3130 | 169 | 163 | 16 | 15 | 27 | 35 | 23 | 32 | 15 | 19 |
Positive controls | 523 | 947 | 422 | 335 | 158 | 334 | 574 | 226 | 1710 | 85 |
Substance | AF-2 | B[a]P | NaN3 | 2AA | AF-2 | 2AA | AF-2 | B[a]P | ICR-191 | B[a]P |
Data are means of two independent experiments.
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” (
“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.
Tartary buckwheat flour of “Manten-Kirari” would not have mutagenesis.
We thank Dr. Mukasa for his useful advice for planning of experiment. We thank to Mr. S. Nakamura, and Mr. K. Abe and Mr. T. Fukaya for their assistance in the field. We also thank Ms. K. Fujii, Ms. M. Hayashida, and Ms. T. Ando for technical assistance. This work was partly supported by a grant from the Research Project on Development of Agricultural Products and Foods with Health-promoting benefits (NARO), Japan.
Tatsuro Suzuki,Toshikazu Morishita,Shigenobu Takigawa,Takahiro Noda,Koji Ishiguro, (2016) Evaluation of the Mutagenicity Potential of Trace-Rutinosidase Variety of Tartary Buckwheat (Fagopyrum tataricum Gaertn.) Using the Ames Test. Journal of Agricultural Chemistry and Environment,05,100-105. doi: 10.4236/jacen.2016.52011