acute and subacute toxicity studies on rutin-rich tartary ... · rutin, a type of flavonol, is...
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175
J Nutr Sci Vitaminol, 61, 175–181, 2015
Rutin, a type of flavonol, is widely distributed in the plant kingdom (1–4). Some reports have shown that rutin strengthens fragile blood capillaries (5, 6), and has antioxidative (7, 8), antihypertensive (9) and a-glu-cosidase inhibitory activities (10). The clinical effects of rutin-rich food have also been reported; administra-tion leads to reductions in serum myeloperoxidase and cholesterol levels (11), mucosal symptoms, headache and tiredness (11). Among cereals, Tartary buckwheat is known to contain large amounts of rutin (approxi-mately 1–2% [w/w]) in its seeds (12). For this reason, buckwheat has gained attention as a rutin-rich material for food products (13, 14). However, Tartary buckwheat seeds contain markedly higher rutinosidase activity (Fig. 1) (15–17). Rutinosidase is a b-glycosidase that releases the disaccharide rutinose from rutin, and Tar-tary buckwheat seeds contain at least two rutinosidase isozymes (15–17). The rutinosidase activity in Tartary buckwheat seeds is sufficient to hydrolyze rutin in the flour within a few minutes after the addition of water.
Recently, we developed a new variety of Tartary buck-wheat designated ‘Manten-Kirari.’ The rutinosidase activity of ‘Manten-Kirari’ is about two or three orders of magnitude less than that of other Tartary buckwheat
varieties (18, 19). Therefore, most of the rutin in ‘Man-ten-Kirari’ will remain present without hydrolysis. As a result, rutin concentration in ‘Manten-Kirari’ dough is much higher when compared to dough from other vari-eties. Wilson et al. (20) reported that intravenous and intraperitoneal injections of 30 to 50 mg/kg in rats and guinea pigs, and intravenous injections in rabbits of 100 to 200 mg/kg, were innocuous. However, to date, foods containing large amounts of rutin, such as ‘Man-ten-Kirari,’ have not been eaten very widely. Therefore, to ensure the safety of ‘Manten-Kirari,’ it is necessary to evaluate its toxicity. In this paper, we assessed the acute and subacute toxicity of ‘Manten-Kirari’ dough.
MATERIALS AND METHODS
Preparation of flour and investigation of rutin concentra-tion. ‘Manten-Kirari’ flour and common buckwheat flour were obtained using a test mill machine with a flour milling percentage of 63%. Rutin in the flour was extracted three times using methanol containing 0.1% phosphoric acid at 80˚C for 8 h, followed by analysis using HPLC (17). In addition, rutinosidase isozymes in ‘Manten-Kirari’ flour were investigated using the in-gel detection method (21) to confirm the absence of con-taminating rutinosidase in the normal variety, which possesses large amounts of rutinosidase.
Acute and Subacute Toxicity Studies on Rutin-Rich Tartary Buckwheat Dough in Experimental Animals
Tatsuro Suzuki1, Toshikazu Morishita2, Takahiro Noda2 and Koji Ishiguro2
1 National Agriculture and Food Research Organization Hokkaido Agricultural Research Center, Hitsujigaoka, Toyohira, Sapporo, Hokkaido 062–8555, Japan
2 National Agriculture and Food Research Organization Hokkaido Agricultural Research Center, Memuro Upland Farming Research Station, Shinsei, Memuro, Kasai-gun, Hokkaido 082–0081, Japan
(Received August 4, 2014)
Summary In order to investigate the toxicity of rutin-rich dough from the Tartary buck-wheat variety ‘Manten-Kirari,’ acute and subacute toxicity studies (10,000 and 5,000 mg/kg flour, respectively) were performed using rats. In the acute toxicity study, no toxic symp-toms were observed and no rats died during the test. Body weight in the ‘Manten-Kirari’-treated group was not significantly different when compared with that of the control group. On pathologico-anatomic observation, no unusual symptoms were observed in the ‘Manten-Kirari’-treated group when compared with the control group. In the subacute toxicity study, no toxic symptoms were observed and no rats died during the test. Body weight and food intake in the ‘Manten-Kirari’-treated and common buckwheat groups were not significantly different when compared with the control group. However, some investigated properties, such as urine protein and serum albumin, were significantly different in the ‘Manten-Kirari’ and common buckwheat groups when compared with the control group. However, these changes were not caused by toxicity, but by transient changes. On pathologico-anatomic observation, some abnormalities were observed in the liver, kidneys, heart, lung, bronchi and pituitary gland in some rats. However, the incidental rates in the ’Manten-Kirari’ and common buckwheat groups did not differ when compared to controls. Therefore, these abnormalities may be caused by natural generation. Based on these results, we concluded that dough at a dose of 5,000 mg flour/kg is at a non effect level.Key Words acute toxicity, subacute toxicity, Tartary buckwheat, rutin, food
E-mail: [email protected]
Suzuki T et al.176
Animals. Crl:CD(SD)(SPF) rats (male and female) were purchased from Charles River Laboratories Japan, Inc. (Yokohama, Japan). Animals were maintained in a room at 2263˚C under a relative humidity of 50620% with a 12 h/12 h light-dark cycle with 13–17 air purges per day. Rats were housed in steel cages. Animal experiments in this study were performed at New Drug Research Center, Inc. (Eniwa, Japan). In addition, all experiments conformed to the relevant laws, and were approved by the Animal Experimental Committee (Nos. 130122A and 130117A).
Acute toxicity. Rats were acclimated for 7 d before the acute test. Healthy rats were randomly divided into two groups (‘Manten-Kirari’ flour and control) with 10 animals (5 males and 5 females in each group). Animals subjected to this test were aged 6 wk, and weighed 184–196 g (males) and 136–154 g (females). Animals were fasted for 15 h. To make dough, the ‘Manten-Kirari’ flour was mixed with 0.5% sodium carboxymethyl cellulose at 250 mg/mL, which was then administrated orally at a dose level of 10,000 mg flour/kg body weight using a gavage. As a control, 0.5% sodium carboxymethyl cellu-lose was administrated. Administration was divided into two doses at 2-h intervals in order to reduce the harm to animals. Clinical signs of the animals were closely observed for the first 4 h, and were observed more than once per day up to 14 d after administration. Animals were weighed just after administration, and at 1, 3, 7, 10 and 14 d after administration. At 14 d after admin-istration, surviving rats were subjected to euthanasia under isoflurane anesthesia with death from exsangui-nation, and were then subjected to pathologico-ana-tomic observation.
Subacute toxicity. Rats were acclimated for 13 d before the subacute test. Healthy rats were randomly divided into three groups (‘Manten-Kirari’ flour, common buckwheat flour and control) with 12 animals (6 males and 6 females in each group). Animals subjected to this test were aged 6 wk. To make dough, ‘Manten-Kirari’ flour was mixed with 0.5% sodium carboxymethyl cellu-lose at 250 mg/mL, and was then administrated orally at a dose of 5,000 mg flour/kg of body weight using a gavage. As a control, 0.5% sodium carboxymethyl cellu-lose was administrated. Administration was performed once per day. Clinical signs of animals were observed more than once per day for 28 d after the beginning of the test. Animals were weighed three times per week in the first week, and twice per week after that. In the final week, 3 h feces and urine were collected individually under fasted conditions. Collected samples were inves-tigated for pH, protein, glucose, occult blood, Ketone
bodies and urobilinogen using test paper (SIMENS Mul-tisticks; Siemens Healthcare Diagnostics Ltd., Malvern, PA). Bilirubin was also investigated using a microscope with Sternheimer-Malbin staining. Subsequently, 21 h urine was also collected individually. Collected samples were subjected to investigation for specific gravity, color and ion concentration (Na, K and Cl). On the final day of administration, after a 15-h fast, we took blood samples under isoflurane anesthesia. Collected blood was sub-jected to investigation for number of leukocytes, num-ber of red cells, hemoglobin content, hematocrit, blood platelet count, neutropenia rate, lymphocyte rate, mono-cyte rate, eosinophile rate, basophil rate and reticulocyte rate using a Sysmex XT-2000iV Automated Hematology Analyzer (Sysmex Corporation, Hyogo, Japan). Plasma was subjected to investigation for prothrombin time and activated partial thromboplastin time using a CA-530 (Sysmex Corporation). In addition, plasma was sub-jected to investigation of total protein concentration, albumin concentration, globulin concentration, glucose concentration, total cholesterol concentration, triglycer-ide concentration, total bilirubin, blood urea nitrogen, creatinine concentration, AST, ALT, g-GTP and ion con-centration (Ca P, Na, K and Cl) using a Hitachi Model 7070 Automated Analyzer (Hitachi, Ltd., Tokyo, Japan). After blood samples were obtained, animals were sub-jected to anesthesia before being sacrificed by exsangui-nation, and then heart, spleen, thymus, lung, salivary glands, liver, kidneys, brain, pituitary gland, adrenal gland, uterus, ovaries and testes were weighed and fixed in 10% formalin solution. In addition, heart, spleen, lung, bronchi, stomach, duodenum, liver, pancreas, kidneys, brain, pituitary gland, adrenal gland, uterus, ovaries and testes were embedded in paraffin, and were subjected to pathologico-anatomic observation using a microscope after staining with hematoxylin-eosin.
Statistical analysis. Statistical analyses were per-formed by the Dunnett test or Kruskal-Wallis H test. p,0.05 was considered to be significant on the Kruskal-Wallis H test, and p,0.05 and ,0.01 were considered to be significant on the Dunnett test.
RESULTS
Rutin concentration in flourRutin concentration of ‘Manten-Kirari’ flour and
common buckwheat flour was 1,570 mg/100 g flour and 18.2 mg/100 g flour, respectively. These rutin con-centrations are usual for each species. In addition, we confirmed that rutin in this dough was not hydrolyzed during administration.
Fig. 1. Rutinosidase activity.
Toxicity in Rutin-Rich Tartary Buckwheat Dough 177
Acute toxicityIn the acute toxicity study, no rats died. In addition,
no toxic symptoms were observed during the test. The body weight of the ‘Manten-Kirari’-treated group was not significantly different when compared with that of the control group (Fig. 2). On pathologico-anatomic observation, no unusual symptoms were observed in the ‘Manten-Kirari’-treated group when compared with the control group.Subacute toxicity
In the subacute toxicity study, no rats died. In addi-tion, no toxic symptoms were observed during the test. Body weight and food intake in the ‘Manten-Kirari’-treated and common buckwheat groups were not sig-nificantly different when compared with those of the control group (Fig. 3). Food intake in the ‘Manten-Kira-ri’-treated group and common buckwheat group were not significantly different when compared with that of the control group (data not shown). The number of rats with a positive urine protein test was significantly higher in male rats of the ‘Manten-Kirari’ group and female
Fig. 2. Changes in rat body weight in acute toxicity test. A: male, B: female, Bars indicate SD.
Fig. 3. Changes in rat body weight in subacute toxicity test. A: male, B: female, Bars indicate SD.
Tabl
e 1
. R
esu
lts
of u
rin
e ex
amin
atio
n.
Uri
ne
volu
me
(mL/
21
h)
Spec
ific
grav
ity
(g/m
L)N
a(m
Eq/2
1 h
)K
(mEq
/21
h)
Cl
(mEq
/21
h)
Col
orpH
Uri
ne
pr
otei
n1
Glu
cose
1K
eton
e
bodi
es1
Occ
ult
bl
ood1
Uro
bilin
ogen
(Eh
rlic
h u
nit
/dL)
Bili
rubi
n1
Mal
eC
ontr
ol g
rou
p1
2.3
62
.37
1.0
66
0.0
11
.94
60
.18
23
.60
60
.24
02
.07
60
.24
0ye
llow
8.0
60
.00
00
00
.10
‘Man
ten
-Kir
ari’-
trea
ted
grou
p1
3.7
62
.24
1.0
56
0.0
01
.90
60
.35
23
.85
60
.58
12
.10
60
.28
7ye
llow
8.0
60
.03
*0
00
0.1
0
Com
mon
bu
ckw
hea
t-tr
eate
d gr
oup
17
.46
6.4
61
.05
60
.01
1.9
56
0.2
46
3.7
16
0.4
12
2.0
36
0.2
32
yello
w8
.06
0.0
00
00
0.1
0
Fem
ale
Con
trol
gro
up
8.0
06
3.5
11
.07
60
.01
1.2
96
0.2
73
2.4
96
0.4
32
1.3
86
0.2
38
yello
w8
.06
0.0
00
00
0.1
0‘M
ante
n-K
irar
i’-tr
eate
d gr
oup
7.5
76
1.0
81
.06
60
.01
1.1
96
0.1
55
2.4
46
0.4
56
1.2
76
0.2
42
yello
w8
.06
0.0
00
00
0.1
0
Com
mon
bu
ckw
hea
t-tr
eate
d gr
oup
7.9
36
0.9
31
.06
60
.01
1.1
56
0.1
65
2.2
46
0.3
38
1.1
46
0.2
46
yello
w8
.06
0.0
1*
00
00
.10
1 n
um
ber
of p
osit
ive
indi
vidu
als,
* p,
0.0
5, m
ean
s6SD
.
Suzuki T et al.178
Tabl
e 2
. R
esu
lts
of b
lood
inve
stig
atio
n.
Red
cel
lsR
etic
ulo
cyte
ra
teH
emog
lobi
nH
emat
ocri
tM
ean
co
rpu
scu
lar
volu
me
Mea
n
corp
usc
ula
r h
emog
lobi
n
con
cen
trat
ion
Mea
n
corp
usc
ula
r vo
lum
eLe
uko
cyte
s
(31
04/m
L)(%
)(g
/dL)
(%)
Volu
me
(fL)
(pg)
(g/d
L)(3
10
2/m
L)
Mal
eC
ontr
ol g
rou
p8
19
61
73
.42
60
.29
15
.36
0.4
44
.76
1.3
54
.56
0.5
18
.76
0.2
34
.36
0.3
77
61
0‘M
ante
n-K
irar
i’-tr
eate
d gr
oup
80
26
22
3.7
16
0.3
01
5.0
60
.24
4.2
60
.85
5.1
61
.51
8.8
60
.53
4.0
60
.57
66
20
Com
mon
bu
ckw
hea
t-tr
eate
d gr
oup
79
36
31
3.7
66
0.3
71
5.2
60
.74
4.2
61
.95
5.8
60
.91
9.1
60
.33
4.3
60
.48
16
20
Fem
ale
Con
trol
gro
up
79
66
33
3.4
76
0.9
41
5.1
60
.64
3.6
61
.75
4.8
61
.71
9.0
60
.53
4.6
60
.36
46
22
‘Man
ten
-Kir
ari’-
trea
ted
grou
p7
92
63
33
.61
60
.94
14
.76
0.7
42
.26
2.0
53
.36
1.9
18
.66
0.6
34
.96
0.4
58
61
6C
omm
on b
uck
wh
eat-
trea
ted
grou
p8
18
62
52
.94
60
.36
15
.16
0.5
43
.36
1.1
52
.96
1.3
18
.56
0.4
35
.06
0.3
54
66
.1
Diff
eren
tial
wh
ite
bloo
d co
un
t (%
)P
late
lets
Pro
thro
mbi
n
tim
eT
hro
mbo
plas
tin
ti
me
Eosi
nop
hile
r
ate
Neu
trop
enia
Bas
oph
il
rate
Mon
ocyt
e r
ate
Lym
phoc
yte
rat
e(3
104
/mL)
(s)
(s)
Mal
eC
ontr
ol g
rou
p0
.83
60
.21
2.3
63
.10
.06
0.0
2.7
76
0.7
84
.16
3.7
12
76
7.7
13
.96
3.0
20
.56
2.7
‘Man
ten
-Kir
ari’-
trea
ted
grou
p1
.00
60
.51
3.4
63
.10
.06
0.0
2.9
46
0.6
82
.66
3.7
11
16
20
.41
5.4
61
.42
5.3
68
.8C
omm
on b
uck
wh
eat-
trea
ted
grou
p0
.85
60
.51
3.5
64
.00
.06
0.0
2.9
06
1.2
82
.76
5.0
12
46
7.5
14
.36
1.9
21
.86
1.8
Fem
ale
Con
trol
gro
up
1.6
36
0.8
12
.16
7.1
0.0
60
.02
.27
60
.98
4.0
67
.01
27
66
.79
.76
0.2
15
.86
0.5
‘Man
ten
-Kir
ari’-
trea
ted
grou
p1
.28
60
.5 9
.16
1.7
0.0
60
.02
.48
60
.48
7.2
62
.31
25
65
.49
.86
0.5
15
.26
0.9
Com
mon
bu
ckw
hea
t-tr
eate
d gr
oup
1.6
56
0.5
13
.16
6.5
0.0
60
.03
.30
61
.88
2.0
68
.41
30
61
1.2
9.5
60
.21
5.6
60
.8
* p,0
.05
, mea
ns
6SD
.
Toxicity in Rutin-Rich Tartary Buckwheat Dough 179Ta
ble
3.
Res
ult
s of
blo
od c
hem
ical
inve
stig
atio
n.
AST
(GO
T)
ALT
(GP
T)
ALP
g-G
TP
Tota
l pro
tein
Alb
um
inG
lobu
linA
/G r
atio
Glu
cose
(IU
/L)
(IU
/L)
(IU
/L)
(IU
/L)
(g/d
L)(g
/dL)
(g/d
L)(m
g/dL
)
Mal
eC
ontr
ol g
rou
p7
3.0
61
5.6
26
.76
1.8
66
34
63
6.5
0.3
36
0.5
25
.90
60
.18
2.6
36
0.1
63
.27
60
.23
0.8
16
0.1
01
55
62
2.9
‘Man
ten
-Kir
ari’-
trea
ted
grou
p7
1.8
66
.82
7.7
64
.63
59
36
11
1.5
0.0
06
0.0
05
.82
60
.15
2.5
26
0.0
83
.30
60
.18
0.7
76
0.0
51
47
61
2.1
Com
mon
bu
ckw
hea
t-tr
eate
d gr
oup
75
.86
9.0
30
.56
6.0
97
40
68
0.6
0.1
76
0.4
15
.93
60
.12
2.6
06
0.1
13
.33
60
.16
0.7
86
0.0
71
46
61
0.7
Fem
ale
Con
trol
gro
up
72
.26
7.2
20
.36
1.9
74
14
63
4.1
0.5
06
0.5
56
.07
60
.29
2.7
56
0.1
43
.32
60
.17
0.8
36
0.0
31
34
61
4.3
‘Man
ten
-Kir
ari’-
trea
ted
grou
p7
1.5
68
.22
0.0
62
.00
43
46
12
4.8
0.8
36
0.4
15
.95
60
.30
2.7
86
0.0
83
.17
60
.30
0.8
96
0.1
01
36
61
8.6
Com
mon
bu
ckw
hea
t-tr
eate
d gr
oup
62
.26
9.9
18
.86
1.9
43
79
64
4.0
0.8
36
0.4
16
.27
60
.18
2.9
36
0.0
8*
3.3
36
0.1
20
.88
60
.03
12
36
13
.0
Tri
glyc
erid
eTo
tal
chol
este
rol
Blo
od u
rea
n
itro
gen
Cre
atin
ine
Tota
l bi
liru
bin
Na
KC
lP
Ca
(mg/
dL)
(mg/
dL)
(mg/
dL)
(mg/
dL)
(mg/
dL)
(mEq
/L)
(mEq
/L)
(mEq
/L)
(mg/
dL)
(mg/
dL)
Mal
eC
ontr
ol g
rou
p4
2.0
67
.75
5.8
61
5.5
13
.26
1.4
20
.21
56
0.0
21
0.0
60
60
.00
91
47
61
.22
4.6
46
0.3
81
08
61
.17
8.1
36
0.6
91
0.3
60
.33
‘Man
ten
-Kir
ari’-
trea
ted
grou
p5
9.7
63
1.4
56
.86
10
.41
2.3
61
.31
0.2
28
60
.02
90
.06
06
0.0
13
14
66
0.8
34
.57
60
.20
10
76
0.8
88
.25
60
.47
10
.16
0.3
3C
omm
on b
uck
wh
eat-
trea
ted
grou
p6
2.3
62
2.0
56
.56
9.3
12
.26
1.6
50
.24
06
0.0
28
0.0
58
60
.00
81
46
60
.89
4.6
56
0.2
31
08
60
.81
7.8
56
0.4
91
0.3
60
.24
Fem
ale
Con
trol
gro
up
14
.26
2.9
59
.56
11
.71
3.5
62
.03
0.2
68
60
.03
50
.05
06
0.0
13
14
56
1.0
64
.76
60
.38
10
86
1.2
18
.67
60
.39
10
.06
0.1
7‘M
ante
n-K
irar
i’-tr
eate
d gr
oup
19
.26
5.6
57
.86
8.6
13
.96
2.2
20
.26
86
0.0
34
0.0
60
60
.01
41
44
60
.98
4.7
96
0.1
91
07
61
.32
9.3
36
0.5
3 9
.96
0.3
3C
omm
on b
uck
wh
eat-
trea
ted
grou
p1
7.5
67
.26
5.0
61
1.7
14
.66
1.1
20
.26
26
0.0
33
0.0
62
60
.01
01
45
61
.30
4.6
16
0.3
11
07
61
.13
8.7
26
0.6
61
0.2
60
.32
* p,0
.05
, mea
ns6
SD.
Tabl
e 4
. R
elat
ive
wei
ght o
f in
tern
al o
rgan
s.
Bra
inP
itu
itar
y gl
and
Saliv
ary
glan
dsT
hym
us
Hea
rtLu
ngs
Live
r(%
)(%
)(%
)(%
)(%
)(%
)(%
)
Mal
eC
ontr
ol g
rou
p0
.50
66
0.0
22
.57
60
.24
0.1
65
60
.02
0.1
54
60
.03
80
.34
56
0.0
16
0.3
37
60
.02
12
.82
76
0.1
26
‘Man
ten
-Kir
ari’-
trea
ted
grou
p0
.50
26
0.0
52
.48
60
.59
0.1
76
60
.02
0.1
70
60
.02
80
.36
06
0.0
26
0.3
49
60
.02
92
.86
66
0.1
64
Com
mon
bu
ckw
hea
t-tr
eate
d gr
oup
0.4
78
60
.02
2.5
56
0.2
20
.17
06
0.0
10
.16
66
0.0
19
0.3
34
60
.02
60
.32
86
0.0
16
2.8
45
60
.19
6
Fem
ale
Con
trol
gro
up
0.8
43
60
.07
5.7
96
0.8
60
.19
86
0.0
20
.23
56
0.0
41
0.3
88
60
.02
10
.46
76
0.0
22
2.9
31
60
.11
1‘M
ante
n-K
irar
i’-tr
eate
d gr
oup
0.7
99
60
.05
5.7
46
0.8
20
.18
96
0.0
20
.19
26
0.0
17
0.3
71
60
.03
00
.44
96
0.0
10
2.8
67
60
.19
5C
omm
on b
uck
wh
eat-
trea
ted
grou
p0
.83
86
0.0
85
.23
61
.03
0.1
95
60
.02
0.1
99
60
.03
30
.36
76
0.0
26
0.4
66
60
.01
52
.87
76
0.1
96
Sple
enK
idn
eys
Adr
enal
gla
nds
Test
esO
vari
esU
teru
s(%
)(%
)(%
)(%
)(%
)(%
)
Mal
eC
ontr
ol g
rou
p0
.18
76
0.0
30
0.7
68
60
.03
01
5.9
63
.64
0.8
04
60
.05
‘Man
ten
-Kir
ari’-
trea
ted
grou
p0
.18
46
0.0
24
0.7
51
60
.03
11
7.6
61
.93
0.8
26
60
.08
Com
mon
bu
ckw
hea
t-tr
eate
d gr
oup
0.1
73
60
.02
70
.71
46
0.0
39
14
.86
2.2
5*
0.7
89
60
.06
Fem
ale
Con
trol
gro
up
0.2
08
60
.01
10
.85
06
0.0
61
34
.06
3.8
33
7.6
66
.07
0.2
23
60
.06
0‘M
ante
n-K
irar
i’-tr
eate
d gr
oup
0.2
11
60
.02
80
.83
26
0.0
68
29
.96
4.3
04
0.2
65
.35
0.2
10
60
.03
4C
omm
on b
uck
wh
eat-
trea
ted
grou
p0
.21
36
0.0
22
0.8
21
60
.06
62
8.8
63
.49
42
.96
1.9
20
.21
96
0.0
72
* p,0
.05
, mea
ns6
SD.
Suzuki T et al.180
Fig. 4. Slight or trace abnormalities on pathologico-anatomic observation. A–D: liver; E–G: kidneys; H: heart; I, J: lung and bronchi; K: pituitary gland; A, C, E, F and J: Common buckwheat-treated group; B, D, G, H, I and K: ‘Manten-Kirari’-treated group. Bar indicates 100 mm.
rats of the common buckwheat group, when compared with the control group (Table 1). The number of rats in which serum albumin was significantly higher in female rats of the common buckwheat group when compared with the control group (Tables 2 and 3). In the common buckwheat group, relative adrenal gland weight was sig-nificantly lower when compared with that of the control group (Table 4). There were no significant differences in the absolute organ weight in the ‘Manten-Kirari’ group and common buckwheat group when compared with the control group (data not shown). There were no sig-nificant differences in other parameters in the ‘Manten-
Kirari’ group and common buckwheat group when compared with the control group (Tables 1–4). Histo-pathologic examination revealed the following minimal or mild findings: minimal cell aggregation in the liver (Fig. 4A), minimal localized cellular infiltration of Glis-son’s capsule in the liver (Fig. 4B), minimal peripheral lobular vacuolation in the liver (lipid droplets) (Fig. 4C), minimal fibrosis near the membrane in the liver (Fig. 4D), minimal localized basophilic changes/atrophy in the renal tubules (Fig. 4E), minimal localized intersti-tial cellular infiltration in the kidneys (Fig. 4F), minimal hyaline cast in the kidneys (Fig. 4G), minimal localized myocardial degeneration/cell aggregation in the heart (Fig. 4H), minimal localized foam cell aggregation in the lungs and bronchi (Fig. 4I), mild localized mineraliza-tion in arterial walls in the lungs and bronchi (Fig. 4J), and minimal cyst in the anterior lobe of the pituitary (Fig. 4K). Figures 4A, C, E, F and J show the common buckwheat treated group, and 4B, D, G, H, I and K show the ‘Manten-Kirari’-treated group. However, we found no important or critical abnormalities on pathologico-anatomic observation.
DISCUSSION
In the subacute toxicity test, the number of rats that were positive for urine protein was significantly higher. However, on pathologico-anatomic observation, there were no important or critical abnormalities in the glom-erulus or renal tubules of the kidneys. This was there-fore regarded as a transient change, and not sample tox-icity. Although a significant increase in serum albumin was observed, the variation was minimal in comparison with physiological variability (Table 3). The incidence of some abnormalities on pathologico-anatomic obser-vation did not differ between the sample and control groups. These findings are considered to be attribut-able to natural occurrences. Based on these results, we concluded that a dose of 5,000 mg flour/kg is at a non effect level.
Common buckwheat has been utilized as a food since the Jomon period, and it is recognized to be safe. In the acute and subacute tests, there were no critical differ-ences between ‘Manten-Kirari’ flour and common buck-wheat flour. In this study, the dose was 10,000 mg/kg for the acute toxicity test and 5,000 mg/kg for the sub-acute toxicity test; in general, this is the maximum limit of this test. In addition, there were no critical differences between the common buckwheat group and the ‘Man-ten-Kirari’ group. Therefore, ‘Manten-Kirari’ flour may be safe at the same level as common buckwheat flour.
AcknowledgmentsWe thank Dr. Y. Mukasa, for his useful advice. We
thank Mr. S. Nakamura, Mr. K. Abe, and Mr. T. Fukaya for preparation of common and Tartary buckwheat. We also thank Ms. K. Fujii, Ms. M. Hayashida, and Ms. T. Ando for technical assistance.
Toxicity in Rutin-Rich Tartary Buckwheat Dough 181
REFERENCES
1) Sando C, Lloyd J. 1924. The isolation and identification of rutin from the flowers of elder (Sambucus canadensis L.). J Biol Chem 58: 737–745.
2) Couch J, Naghski J, Krewson C. 1946. Buckwheat as a source of rutin. Science 103: 197–198.
3) Haley T, Bassin M. 1951. The isolation, purification and derivatives of plant pigments related to rutin. J Am Phar-maceut Assoc Sci Ed 40: 111–112.
4) Fabjan N, Rode J, Kosir I. 2003. Tartary buckwheat (Fagopyrum tataricum Gaertn.) as a source of dietary rutin and quercitrin. J Agric Food Chem 51: 6452–6455.
5) Griffith JQ, Couch JF, Lindauer A. 1944. Effect of rutin on increased capillary fragility in man. Proc Soc Exp Biol Med 55: 228–229.
6) Shanno R. 1946. Rutin: A new drug for the treat-ment of increased capillary fragility. Am J Med Sci 211: 539–543.
7) Jiang P, Burczynski F, Campbell C. 2007. Rutin and fla-vonoid contents in three buckwheat species Fagopyrum esculentum, F-tataricum, and F-homotropicum and their protective effects against lipid peroxidation. Food Res Int 40: 356–364.
8) Awatsuhara R, Harada K, Maeda T. 2010. Antioxidative activity of the buckwheat polyphenol rutin in combina-tion with ovalbumin. Molecular Med Rep 3: 121–125.
9) Matsubara Y, Kumamoto M, Iizuka Y, Murakami K, Okamoto H, Miyake H, Yokoi K. 1985. Structure and hypotensive effect of flavonoid glycosides in Citrus unshiu peelings. Agric Biol Chem 49: 909–914.
10) Li YQ, Zhou FC, Gao F. 2009. Comparative evaluation of quercetin, isoquercetin and rutin as inhibitors of alpha-glucosidase. J Agric Food Chem 57: 11463–11468.
11) Wieslander G, Fabjan N, Vogrincic M, Kreft I, Janson C, Spetz-Nyström U, Vombergar B, Tagesson C, Leanderson P, Norbäck D. 2011. Eating buckwheat cookies is associ-ated with the reduction in serum levels of myeloperoxi-
dase and cholesterol: A double blind crossover study in day-care center staffs. Tohoku J Exp Med 225: 123–130.
12) Kitabayashi H, Ujihara A, Hirose T, Minami M. 1995. On the genotypic differences for rutin content in tartary buckwheat, Fagopyrum tataricum Gaertn. Breeding Sci 45: 189–194.
13) Kreft I, Fabjan N, Yasumoto K. 2006. Rutin content in buckwheat (Fagopyrum esculentum Moench) food materi-als and products. Food Chem 98: 508–512.
14) Ikeda K, Ikeda S, Kreft I, Rufa L. 2012. Utilization of Tar-tary buckwheat. Fagopyrum 29: 27–30.
15) Yasuda T, Masaki K, Kashiwagi T. 1992. An enzyme degrading rutin in Tartary buckwheat seeds. J Jpn Soc Food Sci Technol 39: 994–1000.
16) Yasuda T, Nakagawa H. 1994. Purification and charac-terization of rutin-degrading enzymes in Tartary buck-wheat seeds. Phytochemistry 37: 133–136.
17) Suzuki T, Honda Y, Funatsuki W, Nakatsuka K. 2002. Purification and characterization of flavonol 3-glucosi-dase, and its activity during ripening in Tartary buck-wheat seeds. Plant Sci 163: 417–423.
18) Suzuki T, Morishita T, Mukasa Y, Takigawa S, Yokota S, Ishiguro K, Noda T. 2014. Discovery and genetic analy-sis of non-bitter Tartary buckwheat (Fagopyrum tatari-cum Gaertn.) with trace rutinosidase activity. Breeding Sci 64: 339–343.
19) Suzuki T, Morishita T, Mukasa Y, Takigawa S, Yokota S, Ishiguro K, Noda T. 2014. Breeding of ‘Manten-Kirari,’ a non-bitter and trace-rutinosidase variety of Tartary buckwheat (Fagopyrum tataricum Gaertn.) Breeding Sci 64: 344–350.
20) Wilson RH, Moratarotti T, Doxtader E. 1947. Toxicity studies on rutin. Exp Biol Med 64: 324–327.
21) Suzuki T, Honda Y, Funatsuki E, Nakatsuka K. 2004. In-gel detection and study of the role of flavonol 3-glucosi-dase in the bitter taste generation in tartary buckwheat. J Sci Food Agric 84: 1691–1694.