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Hypoglycemic Activity of Eriobotrya japonica Seeds in Type 2 Diabetic Rats and Mice

Article (PDF Available) inBioscience Biotechnology and Biochemistry 72(3):686-93 · April 2008with275 Reads
DOI: 10.1271/bbb.70411} · Source: PubMed
Abstract
The hypoglycemic effects of Eriobotrya japonica seeds were investigated in type 2 diabetic Otsuka Long-Evans Tokushima fatty (OLETF) rats and KK-A(y) mice. The rats and mice were fed on a diet containing 10% powdered Eriobotrya japonica seeds with the coat intact for 4 months. Although the blood glucose concentration in the OLETF rats fed on the control diet without Eriobotrya japonica seeds was increased with time, the concentration in the OLETF rats fed on the diet with Eriobotrya japonica seeds was consistently low throughout the experimental period and was comparable to the level in Long-Evans Tokushima Otsuka (LETO) rats which are normal non-diabetic rats. Serum insulin was significantly lower in the OLETF rats fed on the Eriobotrya japonica seed diet than in those fed on the control diet at the termination of the experimental period. Eriobotrya japonica seeds suppressed the increment of blood glucose for 4 months and also effectively improved the glucose tolerance in the KK-A(y) mice, these actions being mainly exerted by the ethanol extract of the seeds. These results suggest that Eriobotrya japonica seeds had a hypoglycemic property and the effect is attributable to the components extracted by ethanol.
Hypoglycemic Activity of Eriobotrya japonica Seeds
in Type 2 Diabetic Rats and Mice
Kazunari TANAKA,
1;
y
Shoko NISHIZONO,
1
Nozomi MAKINO,
1
Shizuka TAMARU,
1
Osamu TERAI,
2
and Ikuo IKEDA
3
1
Graduate School of Human Health Science, Siebold University of Nagasaki,
1-1-1 Manabino, Nagayo-cho, Nishisonogi-gu n, Nagasaki 851-2195, Japan
2
Nagasaki Fruit Tree Experim ent Station, 1370 Onibashi-cho, Ohmura-shi, Nagasaki 856-0021, Japan
3
Graduate School of Agricultural Science, Tohoku University,
1-1 Amamiya-machi, Tsutsumidori, Aoba-ku, Sendai-shi, Miyagi 981-8555, Japan
Received June 28, 2007; Accepted November 30, 2007; Online Publication, March 7, 2008
[doi:10.1271/bbb.70411]
The hypoglycemic effects of Eriobotrya japonica seeds
were investigated in type 2 diabetic Otsuka Long-Evans
Tokushima fatty (OLETF) rats and KK-A
y
mice. The
rats and mice were fed on a diet containing 10%
powdered Eriobotrya japonica seeds with the coat intact
for 4 months. Although the blood glucose concentration
in the OLETF rats fed on the control diet without
Eriobotrya japonica seeds was increased with time, the
concentration in the OLETF rats fed on the diet with
Eriobotrya japonica seeds was consistently low through-
out the experimental period and was comparable to the
level in Long-Evans Tokushima Otsuka (LETO) rats
which are normal non-diabetic rats. Serum insulin was
significantly lower in the OLETF rats fed on the
Eriobotrya japonica seed diet than in those fed on the
control diet at the termination of the experimental
period. Eriobotrya japonica seeds suppressed the incre-
ment of blood glucose for 4 months and also effectively
improved the glucose tolerance in the KK-A
y
mice, these
actions being mainly exerted by the ethanol extract of
the seeds. These results suggest that Eriobotrya japonica
seeds had a hypoglycemic property and the effect is
attributable to the components extracted by ethanol.
Key words: Eriobotrya japonica seed; type 2 diabetes;
blood glucose; glucose tolerance; ethanol
extract
Eriobotrya japonica, known as loquat, is a fruit tree
belonging to the family Rosaceae. The leaves of
Eriobotrya japonica have been traditionally used as a
Kampo medicine named ‘pipaye’ in Chinese and
‘biwayo-to’ in Japanese, and they are used as antiussive,
anti-inflammatory, diuretic, expectorant, and analgesic
agents, and to treat women’s diseases and skin diseases.
There are several reports of the leaves of Eriobotrya
japonica having a hypoglycemic action.
1–3)
On the other
hand, most seeds of Eriobotrya japonica are discarded
and not used. Hamada et al.
4)
have shown that an ethanol
extract of Eriobotrya japonica seeds had an antioxidative
action and was effective in reducing oxidative stress
from the adriamycin-induced renal disorder in rats.
Nishioka et al.
5)
have reported that a 70% ethanol or 70%
methanol extract of Eriobotrya japonica seeds inhibited
the development of liver fibrosis in rats. However, there
is little information on the effect of Eriobotrya japonica
seeds on diabetes mellitus. It has recently been observed
that several kinds of seed and their alcoholic or aqueous
extracts exerted a hypoglycemic effect on rats, rabbits,
and mice.
6–10)
Ravi et al.
7)
have shown an antihypergly-
cemic action of the seed kernel of Eugenia jambolana,
called Black plum or Indian Black berry, and its effect
might have been due to the antioxidative effect of
flavonoids in the seeds. Ahmad et al.
8)
have reported that
flavonoids in the seeds of Cuminum nigrum, kala zeera
(black aumin), exhibited considerable hypoglycemic
activity in normal and alloxan-diabetic rabbit s. Thus,
there is a possibility that polyphenols incorpor ated in the
seeds played an important role in the hypoglycemic
effects. Eriobotrya japonica leaves and seeds contain
such active constituents as flavonoids, ellagic acid,
tannins, and amygdalin. Amygdalin is one of the major
components in the seeds of this plant and has been
believed to exert effective physiological functions in the
body. Although there is a report that amygdalin had a
preventive effect on alloxan-induced diabetic mice, type
1 diabetic model animals,
11)
the effect of amygdalin
on type 2 diabetes is obscure.
We investigated in the present study the effects of
Eriobotrya japonica seeds and their ethanol extract on
y
To whom correspondence should be addressed. Fax: +81-95-813-5216; E-mail: katanaka@sun.ac.jp
Abbreviations: OLETF, Otsuka Long-Evans Tokushima fatty; LETO, Long-Evans Tokushima Otsuka; SD, Sprague-Dawley
Biosci. Biotechnol. Biochem., 72 (3), 686–693, 2008
the development of diabetes in Otsuka Long-Evans
Tokushima fatty (OL ETF) rats and KK-A
y
mice, both
type 2 diabetic model animals.
Materials and Methods
Plant material. Fresh and mature Eriobotrya japonica
(Mogi-loquat) fruits were collected from a tree in
Nagasaki Fruit Tree Experiment Station, Nagasaki
Prefecture, Japan. Their seeds with coat were separated,
lyophilized for 3 weeks, and ground in powder process-
ing machinery (P-3 Power Mill, Dalton Co., Tokyo,
Japan) to obtain a coarse powder (60–80 mesh). The
chemical composition of the powdered Eriobotrya
japonica seed preparation was determined. The crude
protein and lipid contents, which were respectively
assayed by the Kjeldahl method and Soxhlet method
with diethyl ether, were 4.9% and 2.4%. The sugar
content determined by the phenol/sulfonic acid method
as 72.1%. The seed preparation also contained 2.1% ash
which was measured by the direct ignition method
(540
C overnight). The content of dietary fiber analyzed
by the Prosky metho d
12)
was 7.0%, most of which
was water-insoluble dietary fiber. To determine the
polyphenols, the seed sample was extracted by 80%
methanol, concentrated, lyophilized, and then injected
into an HPLC column (Cosmosil 5C18-A R-II, 4:6
250 mm). Analyses were conducted with CH
3
CN:
MeOH:CH
3
COOH:H
2
O (113/20/5/862, v/v/v/v) as
the mobile phase at a flow rate of 0.4 ml/min. The
preparation of powdered seeds contained 2.16, 0.68,
0.12, 0.09, and 0.04 mg/g of (+)-catechin, epicatechin,
epigallocatechin, epicatechin gallate, and chlorogenic
acid, respectively. The amount of amygdalin was
measured by HPLC in a TSKgel ODS-80Tm column
(4:6 250 mm). The seed sample homogenized with a
0.05
M citrate solution was applied to the column under
the following HPLC conditions: mobile phase , acetoni-
tryl:H
2
O:0.2 M phosphate buffer at pH, 4.0 (16/79/5,
v/v/v); flow rate, 0.8 ml/min. The amount of amygdalin
included in the seed preparation was 20.0 mg/g. A
hundred gram of freeze-dried powdered seeds was
extracted with 500 ml of 95% ethanol by shaking for
8 h at room temperature (<40
C), and then the super-
natant and residue were separately collected. This
procedure was conducted ten times. The ethanol fraction
was collected and evaporated to dryness at room
temperature in a rotary evaporator under reduced
pressure. The yields of the ethanol extract and residue
were approximately 15 g and 85 g/100 g (w/w in terms
of the dried starting material), respectively.
Animals. Male Otsuka Long-Evans Tokushima fatty
(OLETF) rats, a model of spontaneous non-insulin
dependent diabetes mellitus, and male Long-Evans
Tokushima Otsuka (LETO) rats, the non-diabetic con-
trol model of OLETF rats, were kindly pres ented by
Otsuka Pharmaceutical Co., Tokushima, Japan. Male
KK-A
y
mice, a type 2 diabetic model, were purchased
from Clea Japan, Tokyo, Japan. Male Sprague-Dawley
(SD) rats were obtained from Clea Japan for the toxicity
study. The rats and mice were individually housed in
stainless-steel and plastic cages, respectively, under a
controlled atmosphere (temperatur e, 22 1
C; humid-
ity, 55 5%; light cycle, 0800-2000), fed with a
commercial pellet diet (CE-2, Clea Japan), and had free
access to water.
All animal studies were carried out under the guide-
lines for animal experiments at Siebold University of
Nagasaki (Nagasaki), and Law No. 105 and Notification
No. 6 of the government of Japan.
Toxicity experiment. To assess the toxic effects of
the powdered Eriobotrya japonica seed preparation, 6
female and 6 male SD rats of 5 weeks old were
orally administered with 8000 mg of Eriobotrya japon-
ica seeds/k g of body weight per day for a period of
4 weeks.
Effects of dietary Eriobotrya japonica seeds on
diabetic OLETF rats (experiment 1). Five-week-old
OLETF rats and LETO rats were fed on a commercial
diet (CE-2, Clea Japan) for 3 months. When the rats
were 4 months old, the glucose concentration in blood
obtained from the tail vein was measured with a
commercial kit (Glucose C-II Test, Wako Pure Chemi-
cal Industries, Osaka, Japan) after 6 h of fasting. The
OLETF rats were divided into two groups of equal body
weight and blood glucose value, and were then given the
experimental diet. The control diet was prepared
according to the formula recommended by the American
Institute of Nutrition
13)
(Table 1). The powdered Erio-
botrya japonica seeds were added at the level of 10% to
the control diet at the expense of sucrose. Since the
powdered seed preparation contained 72% sugar, 5%
protein, and 2% fat, the replacement of 10% sucrose by
the seed preparation is not considered to have changed
the total energy intake. The OLETF rats were fed on
either the control diet or the seed diet, and the LETO rats
were fed on the control diet. The value of glucose in
blood withdrawn from the tail vein was measured every
month after 6 h of fasting. The food and water intake
was recorded daily and every other day, respectively.
After feeding the experimental diets for 4 months, the
rats were anesthetized with sodium pentobarbital after
6 h of fasting, and the blood and liver were collected.
The concentrations of serum cholesterol, triglycerides,
phospholipids, and free fatty acids were enzymatically
assayed by using Cholesterol C-II Test, Triglyceride G-
Test, Phospholipid B-Test, and NEFA C-Test kits
(Wako Pure Chemical Industries), respectively. Serum
insulin was determined by a radioimmunoassay (Insulin
‘Eiken’ radioimmunoassay kit, Eiken Chemical Co.,
Tokyo, Japan), and liver lipids were extracted according
to the method of Folch et al.
14)
The cholesterol,
triglyceride, and phospholipid levels in the liver were
Hypoglycemic Activity of Loquat Seed 687
measured by the methods of Sperry and Webb,
15)
Fletcher,
16)
and Rouser et al.,
17)
respectively.
Effects of dietary Eriobotrya japonica seeds, the two
fractions obtained from ethanol extraction of the seeds,
and amygdalin on diabetic KK-A
y
mice (experiment 2).
After 4-week-old male KK-A
y
mice had been fed on a
commercial diet (CE-2, Clea Japan) for one week, they
were divided into five groups of equal body weight and
blood glucose level. The mice were then given one of
five experimental diets for 4 months. The composition of
the control diet was identical to that in experiment 1.
The powdered Eriobotrya japonica seeds were added at
the level of 10% to the control diet at the expense of
sucrose, this being served as the seed diet. Since the
powdered seeds were separated to a 15% extract and
85% residue by ethanol extraction, 1.5% of the extract
or 8.5% of the residue was added to the control diet to
serve as the ethanol extract diet or the ethanol residue
diet, respectively. Amygdalin constituted approximately
2% in the powdered seed, so 0.2% amygdalin was added
to the control diet. The valu e of blood glucose from the
tail vein was measured enzymatically every month after
6 h of fasting. Four months later, the mice were killed by
cardiac puncture under sodium pentobarbital anesthesia
after 6 h of fasting, and the liver was excised. Serum and
liver lipids were determined as already described above.
At a week before their sacrifice, the mice were orally
administered with 2 g of glucose/kg of body weight
after 6 h of fasting. Blood samples were collected from
the tail vein at 0, 30, 60, and 120 min after admin-
istration.
Statistical analysis. All data are expressed as the
mean SEM. Statistical analyses were performed by
one-way ANOVA followed by Tukey’s procedure.
Values are considered to be significantly different
when the p value was less than 0.05.
Results
In the toxic study, none of the female or male SD rats
tested orally with 8000 mg of Eriobotrya japonica
seeds/kg of body weight per day showed any unusual
weight gain or any visible symptoms of toxicity. At the
termination of the feeding period, no abnormality of the
internal organs was apparent. It is therefore considered
that the LD50 value for the oral infusion of Eriobotrya
japonica seeds would be more than 8000 mg/kg of body
weight.
Effects of dietary Eriobotrya japonica seeds on
diabetic OLETF rats (experiment 1)
Since the OLETF rats had been fed on a comme rcial
diet for a prelimi nary feeding period for 3 months and
gained more body weight than the LETO rats, the weight
of the OLETF rats at the initiation of feeding with the
experimental diets was higher (p < 0:01) than that of
the LETO rats (Table 2). The body weight gain of the
OLETF rats fed on the control diet or the seed diet was
more than that of the LETO rats fed on the control diet
for the feeding period, the difference being significant
(p < 0:05) between the LETO rats and OLETF rats
fed on the seed diet. The food intake and relative liver
weight were sign ificantly higher (p < 0:05) in the
OLETF rats than in the LETO rats, although they were
identical between the control and seed groups of OLETF
rats. Although the water intake by the OLETF rats fed
on the control diet increased with increasing time, that
by the OLETF rats fed on the seed diet did not increase,
as was the case with the LETO rats (Fig . 1A).
Blood glucose in the LETO rats fed on the control diet
maintained a low level throughout the feeding period
(Fig. 1B). Blood glucose in the OLETF rats fed on the
control diet consistently increased from the second
month after starting the experimental diet, the difference
from the value for the LETO rats being significant
from the second month to the termination of the
experimental period (p < 0:05 in the second month
and p < 0:01 in the third and 4th months) . Feeding
the seed diet effectively suppr essed the increase of
blood glucose concentration, the level being the same as
that in the LETO rats throughout the experimental
period and significantly lower (p < 0:05) than the
control group of OLETF rats from the second month
to the 4th month.
Table 1. Composition of the Experimental Diets
Control diet Eriobotrya japonica seed diet
g/kg of diet
Casein 200 200
Corn oil 100 100
Mineral mixture (AIN-76)35 35
Vitamin mixture (AIN-76)10 10
Cellulose powder 50 50
DL-Methionine 3 3
Choline bitartrate 2 2
Cornstarch 150 150
Powdered Eriobotrya japonica seeds 0 100
Sucrose 450 350
688 K. T
ANAKA et al.
The concentrations of serum cholesterol, triglycer-
ides, phospholipids, and free fatty acids at sacrifice were
significantly higher (p < 0:05) in the OLETF rat group
than in the LETO rat group. There were no differences
in these parameters between the cont rol diet and the seed
diet with the OLETF rats (Table 2). The serum insulin
level in the OLETF rats fed on the control diet was
appreciably higher (p < 0: 01 ) than in the LETO rats fed
on the control diet. The OLETF rats fed on the seed
diet showed a significantly lower level of serum insulin
(p < 0:05) than the OLETF rats fed on the control diet.
The liver cholesterol and triglyceride levels were the
same between OLETF rats fed on the control diet and
the seed diet, these values being significantly higher
(p < 0:05) than those for the LETO rats. The liver
phospholipid concentration in the LETO rats was
significantly higher (p < 0:05) than that of the two
groups of OLETF rats.
Table 2. Effects of Dietary Eriobotrya japonica Seeds on the Growth Parameters, Serum Lipids and Insulin, and Liver Lipids in LETO and OLETF
Rats (experiment 1)
Rat
LETO OLETF
Diet
Control Control Eriobotrya japonica seeds
Growth parameters
Initial body weight (g) 423 11
a
609 16
b
609 26
b
Final body weight (g) 538 17
a
780 25
b
838 26
b
Body weight gain (g) 116 16
a
171 40
ab
229 21
b
Food intake (g/day) 19:1 0:5
a
29:1 0:8
b
27:6 0:9
b
Relative liver weight 2:67 0:08
a
3:82 0:17
b
3:72 0:15
b
(g/100 g of body weight)
Serum lipids and insulin
Cholesterol (mmol/l) 4:23 0:16
a
7:54 0:31
b
7:82 0:37
b
Triglycerides (mmol/l) 0:97 0:12
a
3:04 0:23
b
3:19 0:23
b
Phospholipids (mmol/l) 2:48 0:17
a
4:80 0:11
b
5:13 0:19
b
Free fatty acids (mEq/l) 0:66 0:04
a
1:00 0:04
b
1:14 0:05
b
Insulin (mU/ml) 24:9 2:8
a
294 49
b
156 27
c
Liver lipids
Cholesterol (mmol/g) 7:79 0:60
a
11:3 1:9
b
12:9 1:1
b
Triglycerides (mmol/g) 16:2 1:7
a
134 21
b
157 18
b
Phospholipids (mmol/g) 38:7 1:1
a
34:4 0:7
b
34:9 0:8
b
Each value is the mean SEM of 6 rats.
abc
Different letters in the same column indicate statistical difference, p < 0:05.
A
0
40
80
120
160
200
0234
Months
g/2 days
LETO-control
OLETF-control
OLETF-seeds
B
a
a
a
b
b
b
b
b
b
0
5
10
15
20
25
0234
Months
mmol/l
LETO-control
OLETF-control
OLETF-seeds
11
Fig. 1. Effects of Dietary Eriobotrya japonica Seeds on the Water Intake (A) and Blood Glucose (B) Levels in LETO and OLETF Rats
(experiment 1).
Each value is the mean SEM of 6 rats.
ab
Different letters in the same time period indicate statistical difference, p < 0:05. LETO-control,
Long-Evans Tokushima Otsuka rats fed on the control diet; OLETF-control, Otsuka Long-Evans Tokushima fatty rats fed on the control diet;
OLETF-seeds, Otsuka Long-Evans Tokushima fatty rats fed on the Eriobotrya japonica seed diet.
Hypoglycemic Activity of Loquat Seed 689
Effects of dietary Eriobotrya japonica seeds, the two
fractions obtained from ethanol extraction of the seeds,
and amygdalin in diabetic KK-A
y
mice (experiment 2)
There were no differences in body weight gain, food
intake, water intake, and relative liver weight among the
5 groups (Table 3).
The blood glucose level in the KK-A
y
mice fed on the
control diet gradually increased after the initiation of the
experimental diet (Fig. 2). The blood glucose level in
the mice fed on the seed diet maintained a low level
throughout the feeding period, the differences for the
control group being significant (p < 0:05) in the second
and 4th months. The ethanol extract of Eriobotrya
japonica seeds effectively suppressed the elevation of
blood glucose, the level being identical to that in the
mice fed on the seed diet throughout the experimental
Table 3. Effects of Dietary Eriobotrya japonica Seeds, the Two Fractions Obtained from Ethanol Extraction of the Seeds, and Amygdalin on the
Growth Parameters, and Serum and Liver Lipids in KK-A
y
Mice (experiment 2)
Group Control Seeds Extract Residue Amygdalin
Growth parameters
Initial body weight (g) 25:0 0:325:2 0:424:9 0:425:0 0:425:1 0:5
Final body weight (g) 46:7 1:147:4 1:048:6 1:247:2 1:247:5 0:7
Body weight gain (g) 21:7 1:122:2 0:823:7 1:022:2 0:922:4 0:9
Food intake (g/day) 4:08 0:19 4:13 0:17 4:23 0:17 4:18 0:18 4:41 0:23
Water intake (g/day) 21:4 1:318:5 1:218:9 0:820:6 1:019:1 1:3
Relative liver weight 4:64 0:11 5:04 0:35 4:88 0:25 4:59 0:20 4:62 0:27
(g/100 g of body weight)
Serum lipids (mmol/l)
Cholesterol 2:74 0:25 3:48 0:32 2:83 0:14 2:83 0:22 2:80 0:40
Triglycerides 1:49 0:09 2:06 0:42 2:74 0:11 2:74 0:09 2:74 0:32
Phospholipids 2:25 0:11 2:80 0:28 2:28 0:16 2:31 0:18 2:25 0:29
Liver lipids (
mmol/g)
Cholesterol 27:1 1:7
a
22:9 1:0
b
21:5 0:8
b
22:2 0:5
b
21:4 2:8
b
Triglycerides 62:9 11:6
ab
114 13
a
130 20
a
105 10
ab
46:0 11: 9
b
Phospholipids 35:6 1:637:7 1:438:7 1:335:6 1:334:6 4:3
Each value is the mean SEM of 6 mice.
ab
Different letters in the same column indicate statistical difference, p < 0:05.
Control, Seeds, Extract, Residue, and Amygdalin indicate the control group, the Eriobotrya japonica seed group, the seed ethanol extract group, the seed ethanol-
treated residue group, and the amygdalin group, respectively.
a
a
a
b
a
b
c
ab
ab
ab
bc
0
5
10
15
20
25
30
0234
Months
mmol/l
Control
Seeds
Extract
Residue
Amygdalin
1
Fig. 2. Effects of Dietary Eriobotrya japonica Seeds, Two Fractions Obtained from the Ethanol Extract of the Seeds, and Amygdalin on the Blood
Glucose Level in KK-A
y
Mice (experiment 2).
Each value is the mean SEM of 6 mice.
abc
Different letters in the same time period indicate statistical difference, p < 0:05. Control, Seeds,
Extract, Residue, and Amygdalin indicate the control group, the Eriobotrya japonica seed group, the seed ethanol extract group, the seed
ethanol-treated residue group, and the amygdalin group, respectively.
690 K. T
ANAKA et al.
period. On the other hand, the residue obtained from
ethanol extraction of the Eriobotrya japonica seeds
produced a significant elevation of the blood glucose
level (p < 0:05) in the 4th month as compared with the
ethanol extract of the seeds. Amygdalin did not induce
any increment of the blood glucose level until the third
month, the value in the second month being significantly
lower (p < 0:05) than that of the control group.
However, amy gdalin increased blood glucose in the
4th month to a level comparable to that of the control
group.
Figure 3 shows the concentration and net change in
blood glucose levels of the mice after the oral admin-
istration of glucose in the week before sacrifice. The
blood glucose level in the mice fed on the control diet
rose to a peak value 30 min after glucose loading and
then decreased until 120 min, when the level was
appreciably higher than that before the administration
of glucose. The seed diet markedly suppressed the
increment of blood glucose concentration at 30, 60, and
120 min as compared with the control diet, the value at
120 min being almost restored to that at 0 min. The
ethanol extract of Eriobotrya japonica seeds was also
effective in suppressing the increment of blood glucose,
while the ethanol-treated residue of the seeds and
amygdalin did not suppress the increment of glucose
level at 30 and 60 min.
There were no differences in the serum cholesterol,
triglyceride, and phospholipid concentrations among
the 5 groups (Table 3), although the concentration of
hepatic cholesterol was significantly lower in the 4
experimental groups than in the control group. The
concentration of hepatic triglyceride tended to be
higher in the 3 groups that were fed with the Eriobotrya
japonica seeds than that in the control group. There was
no difference in hepatic phospholipid concentration
among the groups.
Discussion
OLETF rats established as an animal model of
human type 2 diabetes exhibit obesity, hyperinsuline-
mia, hypertriglyceridemia, and hyperglycemia.
18)
In the
present study, the blood glucose and water intake levels
increased with the passage of time in the OLETF rats
fed on the control diet, and such symptoms of diabetes
as hyperphagia, obesity, hyperinsulinemia, and hyper-
glycemia appeared in the 4th month after commencing
feeding with the experimental diets. Eriobotrya japonica
seeds effectively suppressed the enhancement of blood
glucose concentration and water intake, these levels
being the same as those in the LETO rats throughout the
experimental period. The seeds were also confirmed to
suppress hyperglycemia and improve glucose tolerance
in the KK-A
y
mice, type 2 diabetic animals. It was thus
elucidated that Eriobotrya japonica seeds had potent
antihyperglycemic activity.
Type 2 diabetes is a syndrome charact erized by
defective insulin secretion and insulin resistance.
19)
The
pancreatic cells in type 2 diabetes are forced to secrete
more insulin to overcome a loss of insulin sensitivity,
inducing in hyperinsulinemia. Therefore, insulin resist-
ance is thought to exert hyperinsulinemia. Because
feeding Eriobotrya japonica seeds decreased the serum
insulin concentration in OLETF rats when compared to
feeding the diet without the seeds, they are thought to
have improved the insulin resistance, which may be one
of the possible mechanisms for suppressing the blood
glucose level and improving glucose tolerance. Another
possibility is that the Eriobotrya japonica seeds reduced
or delayed glucose absorption in the intestines. Since the
Eriobotrya japonica seeds did not inhibit -glucosidase
activity in the intestines of normal SD rats (data not
shown), they might have modulated glucose absorption
but not digestive enzyme activities. Fenugreek (Trig-
onella foenum graecum) seeds and a hot-water extract
from defatted sesame (Sesamum indicum L.) seeds have
been reported to decrease or retard the glucose absorp-
Control
Seed
Extract
Residue
Amygdalin
0
10
20
30
40
0
30 60 90 120
min
mmol/l
min
0
10
20
0
30 60 90 120
mmol/l
A
B
a
b
b
a
b
b
b
b
c
c
a
a
ab
bc
a
b
b
a
ab
b
bc
c
a
ab
bc
Fig. 3. Effects of Dietary Eriobotrya japonica Seeds, Two Fractions
Obtained from the Ethanol Extract of the Seeds, and Amygdalin on
the Concentration (A) and the Change (B) of Blood Glucose after an
Oral Administration of 2 g of Glucose/kg of Body Weight in KK-A
y
Mice (experiment 2).
Each value is the mean SEM of 6 mice.
abc
Different letters in
the same time period indicate statistical difference, p < 0:05.
Control, Seeds, Extract, Residue, and Amygdalin indicate the
control group, the Eriobotrya japonica seed group, the seed ethanol
extract group, the seed ethanol-treated residue group, and the
amygdalin group, respectively.
Hypoglycemic Activity of Loquat Seed 691
tion in diabetic rats.
10,20)
However, at present, the precise
mechanism(s) and site(s) of hypoglycemic activities are
still to be determined, and further investigation is
necessary.
Since the ethanol extract fra ction of the seeds induced
suppression of hyperglycemia and improved glucose
tolerance, the components which contributed to the
hypoglycemic effects are considered to have been in
the ethanol extract fraction. Amygdalin was one of the
major components in the ethanol extract of Eriobotrya
japonica seeds. Amygdalin has traditionally been be-
lieved in Japan to be a useful component for human
health and to have an antidiabetic effect. In the present
study, amygdalin was less effective in lowering blood
glucose and also did not improve the glucose tolerance
in KK-A
y
mice (Figs. 2 and 3). Although there is a
possibility that amygdalin retarded the incidence of
diabetes, it does not appear to have had effective
hypoglycemic activity. Therefore, amygdalin in the
Eriobotrya japonica seeds should be excluded as a
candidate of the effective components for improving
diabetes.
Eriobotrya japonica seeds also contain several kinds
of flavonoids, e.g., (+)-catechi n, epicatechin, epigallo-
catechin, epicatechingallate, and chlorogenic acid. The
development of diabetes has been indicated to be in part
due to tissue damage by increased oxidative stress.
21,22)
Since flavonoids have an antioxidative effect, those
contained in Eriobotrya japonica seeds might have
exerted a protective role in the development of diabetes
by reducing oxidative stress. Ahmad et al.
8)
have shown
that the total flavonoids extracted from Cuminum nigrum
seeds produced a significant and consistent hypoglyce-
mic effect in normal and diabetic rabbit s.
Several reports
23,24)
have indicated that the dietary
fiber contained in seeds lowers blood glucose and
improves glucose tolerance. It is known that water-
soluble dietary fibers such as pectin and guar gum
decrease or delay glucose absorption by increasing
the viscosity of the intestinal contents, resulting in a
reduction in the blood glucose concentration. The
hypoglycemic effects of black plum seeds and fenugreek
seeds have been suggested to be due to water-soluble
gummy fiber and pectin, respectively, but not water-
insoluble dietary fiber.
20,25,26)
The content of dietary
fiber in the Eriobotrya japonica seeds used in this
experiment was 7.0 g/100 g dry weight, most of which
was water-insoluble dietary fiber. Therefore, the dietary
fiber in Eriobotr ya japonica seeds is not thought to have
been the major contributor for reducing blood glucose
and improving glucose tolerance.
Elevated serum triglyceride and/or free fatty acid
levels are known to induce insulin resistance in the liver
and peripheral tissues.
27,28)
In the current experiment,
the serum and liver triglyceride and serum free fatty
acid conce ntrations were higher in the OLETF rats fed
on the control diet than in the LETO rats fed on the
control diet. Eriobotrya japonica seeds did not decrease
these lipid levels in the OLETF rats. The seeds slightly
reduced the hepatic cholesterol concentration in the
KK-A
y
mice, but it did not alter the serum and hepatic
triglyceride levels. These observations suggest that
the improvement of glucose metabolism by Eriobotrya
japonica seeds was not induced through any alteration of
lipid metabolism.
Our preliminary toxicity studies have revealed no
visible signs and symptoms of toxicity of Eriobotrya
japonica seeds in normal SD rats. However, since
Eriobotrya japonica seeds contain amygdalin, which is
degraded to cyanide,
29)
it cannot be discounted that a
large intake of Eriobotrya japonica seeds could cause
cyanide poisoning. Therefore, when Eriobotrya japon-
ica seeds are used as a functional food additive,
amygdalin might need to be removed from the seeds.
In conclusion, this study found that Eriobotrya
japonica seeds induced a hypoglycemic effect through
improving the insulin resistance, and that this action was
exerted by its ethanol extract, showing promise in the
development of a novel functi onal food for suppressing
hyperglycemia. Further studies are in progress to isolate
the active components in Eriobotrya japonica seeds and
to elucidate the mechanism of action for its hypoglyce-
mic effe ct.
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Hypoglycemic Activity of Loquat Seed 693
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