!

a a a a a,b"

a "
b "
!
2016 1 13 "
2016 6 3 "
2016 7 1 "

"

(STZ)
"
α
HMG4CoA (3- -3 A)
(EAF)
50 100 200mg/kg EAF

"
EAF ； EAF

PCR EAF STZ

，
-1 -2 -1

1"
1.

） (IDF) ×
3.66 2012 ； 2030
5.22

Forsythia suspensa (Thunb.) Vahl Lian-Qiao

Shp2

2"
2.
2.1

130906

1:4 。
Whatman

5.3% 6.7% 7.2% −20°C

2.2
α
200μL （ Sigma4Aldrich
04400μg/μL 400μL 2U/mL,"Sigma4Aldrich
100mM pH"6.8 37°C 15 200μL
1% 100mM pH"6.8 37°C
20 1mL DNS,"Sigma4Aldrich,"
10 ± 540nm
(%)=(A0 A1)/A0 100 A0
± A1 ± IC50 （
％ "
HMG4CoA (3- -3 A) HMG4CoA
" Sigma4Aldrich 50μg
HMG4CoA 400μM 400μM
1mM 5mM 120mM 0.2mL 37°C
10 340nm ±
(%)=(B0 B1)/B0 100 B0
± B1 ± 5μM
Sigma4Aldrich ％ ＆

3"
 (EAF)
"
2.3" " ""

648 20425g
12 ± 12 "
2.4" " ""
(EAF)
480 ／ 5 n"="5
0 250 500 1000 2000mg/kg
(EAF) 2 0.5 4 8
12 24 48 72 1
EAF 2000mg/kg
200mg/kg EAF ／ "
2.5" " ""
180"mg/kg"STZ Sigma4Aldrich
STZ 300"mg/dL
／ "
2.6" " "
／ 50 20 n"="10 I
II
III " ％ 5mg/kg
Sigma4"Aldrich USA IV
50"mg/kg EAF;" V
100"mg/kg EAF;" VI 200"mg/kg EAF;"
VII 200"mg/kg EAF
28 "
2.7" " ""

4"
 " OneTouch"UltraVue
Johnson Johnson ， ""
2.8" " OGTT ""
EAF 4 EAF
16 OGTT 2g/kg
0 30 60 90 120 OneTouch"
UltraVue Johnson Johnson ， ""
2.9" " ""
4 EAF 、
TC TG HDL
TC TG
“ ACP
" ALP ALT AST
Beyotime "
GSH4Px SOD
CAT
“ ""
2.10" " RNA RT4PCR" "
Trizol RNA
Invitrogen USA NanoDrop®"ND41000 Thermo Pittsburgh PA
RNA primerScript"TM"RT 500ng"RNA cDNA Takara
" PCR SYBR； ExTaqΠ Takara IQ5 PCR
" BioRad Hercuies CA " PCR 41 INS41
42 INS42 41 PDX4"1 GCK
464 G6P PHC β4
Sangong"Biotechnology ：
1 " "
2.11" " "

5"
 ±SEM ” SPSS14.0
Journal of Ethnopharmacology 192 (2016) 256–263
” ANOVA 5 p<0.05 "

ide (Sigma- Table 1 1" qRT4PCR mRNA " "

ass of sulfo- Primers used for the analysis of mRNA expression by qRT-PCR. "
strated with
Genes Primer sequences (from 5′ to 3′) Product Accession
, mice were size (bp) number
"
" ( 5’ 3’)"
; Group VI "
/kg b.w. EAF β-actin Forward GATTACTGCTCTGGCTCCTAGC 147 NM_007393
Reverse GACTCATCGTACTCCTGCTTGC "
ere admini-
INS-1 Forward TCTTACCCCCTGATGCTGTGA 138 NC_000085 "
tration was Reverse TTCCCTGAGGTTCCTGTTGCT
"
INS-2 Forward TGAAGTGGAGGACCCACAAGT 108 NC_000073
Reverse ACAATGCCACGCTTCTGCT "
vel
PDX-1 Forward TCCAAAACCGTCGCATGAA 101 NC_000071 "
Reverse ACAATCTTGCTCCGGCTCTTC
red weekly. "
ed from tail G6P Forward TCTTGTTTGGTTTCGCGCTT 120 NC_000077
Reverse TGTCAAGGTGGACCCATTCTG
"
ble glucose
PHC Forward GGTGCCCTCTCCCCTTAAAAA 121 NM_011044 "
Medical Ltd.,
Reverse CCACCATATCCGCTTCCAAAG "
GCK Forward CATCCTGCTCAACTGGACCAA 121 NM_010292 "
Reverse CATTGCCACCACATCCATCTC
"

s performed 3. !
3. Results
EAF admin- 3.1" " α4 HMG4CoA "
ven to mice 3.1. α-amylase and HMG-CoA reductase activities α4 HMG4CoA
e measured
2 3 EAF
ation with a Enzyme activity analysis showed that extract fractionated by
n & Johnson EAF α4 IC50 117.36±6.2μg/mL ％ （
different organic solvents had different effects on the activities of
αp<0.05
-amylase and HMG-CoA reductase (Tables 250mg"EAF HMG4CoA
and 3). Among the
76.51±4.16 p<0.05
three fractions, EAF had the greatest inhibition on both enzymes.
The IC 50 of EAF for α-amylase was 117.36
EAF " 76.2 μg/mL, which was
hanized by even
3.2" " lower
" (p o0.05) than that of the positive control acarbose,
rdiac punc- showing it was a powerful inhibitor to the enzyme. At the same
； 4 STZ
high density time, 50 mg EAF inhibited 76.51 74.16% of the activity of HMG-CoA
II I p<0.05
of glycogen, reductase, which was significantly higher (p o0.05) than that of
al assay kits II
Chloroform and n-butanol EAF on these data, EAF
fractions. Based V VI
was
China). The selected for further in vivo studies.
phosphatase
ate amino-
eyotime In- 3.2. Body weight 6"

roxide dis- As expected, diabetic mice showed reduced body weight gain
(Table 4). The body weight of diabetic mice (group II) became
significantly lower (p o0.05) than that of normal mice (group I)
one week after the induction of diabetes by STZ. Oral adminis-
tration of EAF (group V and VI) significantly increased (p o0.05)
body weight in diabetic mice when compared
p<0.05 EAF to that of
。 the EAF
dia-
betic control mice (group
VII II),
vs suggesting
I " that EAF is anti-diabetic.
However, EAF had no effect on body weight in normal mice (Group
"
VII vs. Group I). Y. Zhang et al. / Journal of Ethno
2" α4 """""""" 3" HMG4CoA "
Table 2 " Table 3
Effects of F. suspensa fruit extract on α- Effects of F. suspensa fruit extract on HMG-
"
amylase activity. CoA reductase activity.
"
Fractions/standard IC50 (μg/mL) Fractions/standard Inhibition rate (%)
" " "
Chloroform " 347.917 17.19a " Chloroform 33.64 716.6c
Ethyl acetate " " " "7
117.36 " "6.2
" " c" " Ethyl acetate 76.517 4.16b
n-butanol " NI " n-butanol 13.477 0.84d
Control " NI Control 0
"
Acarbose（ b Simvastatin 93.577 6.48a
" 265.45 7 12.32

Note: IC50, concentration of extract to

" Note: Data are presented as mean 7 SEM.
a-d
inhibit 50% of α-amylase activity. NI, no " Values with different letters are
inhibition. significantly different. n¼ 3, p o 0.05.
Data are presented as mean 7 SEM. "
a-c
3.3. letters
Values with different Bloodare
glucose ±SEM"
significantly different. n ¼ 3, p o 0.05. a4d " n=3,"p<0.05"
As shown in Fig. 1, diabetic mice (groups II-VI) had significantly
IC50 50%α4 (p o0.05) higher
" blood glucose when compared to that of normal
NI "
mice (group I). Administration of EAF for one week significantly
decreased
±SEM" blood glucose in diabetic mice. The reduction in blood
glucose increased with the increase of EAF dosages. In normal
a4c n=3,"p<0.05"
mice, 200 mg/kg b.w. EAF did not further reduce blood glucose
" when compared to that of control.
3.3" " "
3.4. Oral glucose tolerance
1 I II4VI p<0.05
EAF ／ 2)
Oral glucose tolerance test (Fig. EAFshowed that, as expected,
diabetic mice had worse glucose tolerance
200mg/kg EAF when compared to
normal mice, which had much higher (p o0.01) blood glucose
"
after oral administration of 2 g/kg b.w. of glucose within 120 min.
3.4" " " Administration of EAF for 4 week obviously improved mouse
glucose
2 tolerance
； and the blood glucose decreased with the in-
” crease of EAF dosages.
2g/kg Excitingly, blood
120glucose became the same
as that of normal mice for diabetic mice administrated with
p<0.01 EAF"4
200 mg/kg b.w. of EAF. ／

3.5. Antihyperlipidemic effects 7"

Induction of diabetes by STZ significantly increased serum TG,

 EAF 200mg/kg EAF
"
3.5" " "
STZ TG TC TG
TC HDL p<0.05 5 4 EAF
p<0.05 TC TG TG TC HDL
TC TG HDL TG TC EAF 200mg/kg
EAF
STZ TG EAF STZ
TC "
5 STZ
p"<0.05 EAF
。
Zhang et al. / Journal of Ethnopharmacology 192 (2016) 256–263
Y. Zhang et al. / Journal of Ethnopharmacology 192 (2016) 256–263
p"<0.05 " 259
259

1."EAF
n HMG-
on HMG-
"
te (%)
rate (%)
c
±SEM " a4e
6c
6
d
4d
b
n=10
a
8a p<0.05"
n77SEM.
SEM.
ers are
tters are
"
05.
.05.
"
mg/dL " " " "
sII-VI)
II-VI)had
hadsignificantly
significantly Fig.
Fig.1.
1. Effects
Effects of
of EAF
EAF on
on blood
blood glucose
glucose in streptozotocin-induced diabetes
in streptozotocin-induced diabetes mice.
mice.
ared totothat
pared thatofofnormal
normal Data
Dataare
arepresented
presented asas mean
mean 7 7 SEM.
a–e
SEM. a–eValues with different
Values with different letters
letters at
ataasingle
singletime
time "
one
oneweek
weeksignificantly
significantly point
pointare
are significantly different. n ¼ 10, p o0.05.
significantly different. o0.05.
he reduction
reductionin inblood
The
F dosages.
AF dosages. In
blood
In normal
normal
"
rreduce
reduceblood
blood glucose
glucose
2. EAF"4
a4d

wedwedthat,
that,as
asexpected,
expected, n=10 p<0.05"
ecewhen
when compared
compared to to
o0.01) blood
po0.01) blood glucose
glucose "
glucose within120
ucose within 120min.
min.
ously improved mouse
usly improved mouse "
decreased with the
ecreased with the in-
in-
ucose becamethe
cose became thesame
same "
ece administrated
administrated with
with

"
mg/dL "
" "
Fig.2.
Fig. 2. Oral
Oralglucose
glucose tolerance
tolerance of
of mice
mice after
after oral
oral administration
administration of
of EAF
EAF for
for 44weeks.
weeks.
a–d
ylyincreased
increasedserum
serumTG,
TG, a–d Values with different letters at a single time point are significantly different.
Values with different letters at a single time point are significantly different.
decreased serum
serum HDL
HDL nn¼10,
¼10,ppoo0.05.
0.05.
decreased
malmice
mal mice(Table
(Table5).
5).Ad-
Ad- 8"
ed(po
(po0.05)
0.05)serum
serumTC,
TC, insulin is
insulin is decreased
decreased significantly
significantly in
in STZ-induced
STZ-induced diabetic
diabetic mice
mice as
as
d
reased HDL in diabetic
eased HDL in diabetic
compared to
compared to normal
normal control
control mice
mice (p(p o0.05).
o0.05). The
The EAF
EAF treatment
treatment
C, TG,HDL
HDLand
andhepatic
hepatic increased serum
increased serum insulin
insulin and
and hepatic
hepatic glycogen
glycogen but
but these
these levels
levels
, TG,
were still significantly lower than that of normal control mice
 n-butanol 13.477 0.84d
Control 0
Simvastatin 93.577 6.48a

Note: Data are presented as mean 7 SEM.

a-d
Values with different letters are
significantly different. n¼ 3, p o 0.05.

3.3. Blood glucose

3.6" "
As shown "
in Fig. 1, diabetic mice (groups II-VI) had significantly
Fig. 1. Effects of EAF on blood glucose in streptozotocin-induced diabetes mice.
(p o0.05) higher blood glucose when compared to that of normal Data are presented as mean 7 SEM. a–eValues with different letters at a single time
mice (group I). Administration of EAF for one week significantly point are significantly different. n ¼ 10, p o0.05.
STZ
decreased blood glucose in diabetic mice. The reduction in blood ACP ALP AST
glucose increased with the increase of EAF dosages. In normal
ALT
mice, 200 mg/kg b.w. EAF did not further reduce blood glucose p<0.05 6 EAF"4
when compared to that of control.

3.4. Oral glucose tolerance

ACP ALP AST ALT p<0.05 "
7 MDA
Oral glucose tolerance test (Fig. 2) showed that, as expected, STZ
diabetic mice had worse glucose tolerance when compared to
normal mice, which had much higher (p o0.01) blood glucose
after oral administration of 2 g/kg b.w. of glucose within 120 min.
p<0.05 EAF MDA p<0.05
Administration of EAF for 4 week obviously improved mouse
glucose tolerance and the blood glucose decreased with the in- GSH4Px SOD CAT p<0.05 "
crease of EAF dosages. Excitingly, blood glucose became the same
3.7" "
as that of normal mice for diabetic mice administrated with
200 mg/kg b.w. of EAF.
"
RT4PCR
3.5. Antihyperlipidemic effects EAF 3
Fig. 2. Oral glucose tolerance of mice after oral administration of EAF for 4 weeks.

PDX41 INS41 INS42 GCK mRNA p"

a–d
Induction of diabetes by STZ significantly increased serum TG, Values with different letters at a single time point are significantly different.
TC, and hepatic lipid (TG and TC) while decreased serum HDL n ¼10, p o 0.05.

(p o0.05) when compared to those of normal mice (Table 5). Ad-

<0.05 100mg/kg"EAF
ministration of the EAF for 4 weeks reduced (po 0.05) serum TC, V PDX41 INS41 INS42
insulin is decreased significantly in STZ-induced diabetic mice as
TG, and hepatic lipid (TG and TC) and increased HDL in diabetic compared to normal control mice (p o0.05). The EAF treatment

mRNA
mice. In addition, the changes in serum TC, TG, HDL and hepatic I
increased serum insulin and hepatic glycogen but these levels
were still significantly lower than that of normal control mice
EAF
lipid (TG and TC) were EAF dosage-dependent. Administration of
200 mg/kg b.w. of EAF to non-diabetic mice did not alter the lipid (p o0.05).
GCK
profile when compared to that of normal control mice. While mRNA "
glibenclamide normalized the STZ-induced increase in serum and 3.6. Biochemical parameters
"
hepatic TG. But the EAF normalized the STZ-induced increase of
hepatic TC. STZ-induced diabetic mice exhibited a significant increase
As shown in Table 5, the level of hepatic glycogen and serum (p o0.05) in serum creatinine concentration and the activities of
" 4"EAF "
Table 4
Effects of EAF on mouse body weight.

Group Pre-treated Post-treated "

" " 1 week
" 2 week
" 3 week
" 4 week
"
I 23.54 7 0.21 25.44 70.33a 27.52 7 0.40a 28.737 0.35a 29.377 0.45a
II 23.377 0.25 23.85 7 0.42d 24.377 0.59f 25.83 7 0.46e 26.54 7 0.58d
III 23.42 7 0.27 24.837 0.25b 26.78 7 0.22b 27.84 70.30b 28.3370.40b
IV 23.44 70.26 23.99 7 0.16d 24.987 0.23e 26.34 7 0.31d 26.79 7 0.38d
V 23.517 0.23 24.327 0.25c 25.78 7 0.38d 26.98 7 0.39c 27.357 0.45c
VI 23.46 7 0.32 24.637 0.19b 26.12 70.26c 27.52 70.44b 28.03 7 0.26b
VII 23.53 7 0.23 25.57 70.24a 27.34 7 0.52a 28.95 7 0.58a 29.36 7 0.47a

Note: Data are presented as mean 7 SEM.

a-f
Values with different letters within a column are significantly different. n ¼ 10, p o 0.05.

" ±SEM a4f n=10,"p<0.05"

"
260 Y. Zhang et al. / Journal of Ethnopharmacology 192 (2016) 256–263
" 5"STZ EAF "
Table 5
" EAF altered serum and hepatic lipid profiles, serum insulin and hepatic glycogen in streptozotocin-induced diabetes mice.
" Parameters (unites) Group I Group II Group III Group IV Group V Group VI Group VII
" TG" Serum TG (mmol/L) 1.25 7 0.14e 2.15 70.15a 1.29 7 0.17e 1.89 7 0.20b 1.54 70.18c 1.38 7 0.16d 1.20 7 0.14e
TC" Serum TC (mmol/L) 4.577 0.16e 9.34 70.43a 5.41 70.21d 7.83 7 0.46b 6.49 70.41c 5.82 7 0.22d 4.34 7 0.27e

" HDL"
Serum HDL (mmol/L)
Hepatic TG (mg/g protein)
3.78 7 0.18a
86.127 7.16de
1.98 7 0.17d
152.69 7 15.63a
3.54 70.20b
98.62 77.25d
2.727 0.11c
139.84 714.59b
3.15 70.14bc
121.65 7 11.36bc
3.37 70.13b
108.477 9.53c
3.94 7 0.15a
83.87 7 6.85e
TG" Hepatic TC (mg/g protein) 36.647 2.16d 93.45 76.48a 45.577 3.18c 80.497 4.26b 63.58 7 3.38c 43.487 2.84cd 30.89 7 2.14e

" TC" Serum Insulin (ng/mL)

Hepatic Glycogen (mg/g)
0.577 0.07a
4.02 7 0.26ab
0.26 70.04e
2.14 70.11e
0.497 0.08b
3.687 0.21b
0.317 0.06d
2.39 7 0.14d
0.37 70.07c
2.98 7 0.16c
0.43 7 0.09b
3.37 70.28bc
0.54 7 0.05a
4.43 7 0.22a
"
" "
a–e
Note: Data are expressed as mean 7 SEM. Values with different letters within a row are significantly different. n ¼5, po 0.05.

Table 6
±SEM a4e
Effects of EAF on serum biochemical parameters in streptozotocin-induced diabetes mice. n=5,"p<0.05"
Parameters (unites) Group I Group II Group III Group IV Group V Group VI Group VII

d a c b bc c
Creatinine (mg/dL) 0.23 7 0.04 0.45 70.04 0.26 7 0.03 0.36 7 0.05 0.3370.04 0.28 7 0.05 0.25 70.04cd
ACP (K.A.) 4.89 7 0.22e 9.747 0.31a 5.36 7 0.24d 7.54 7 0.32b 6.28 7 0.37c 5.79 7 0.26cd 4.977 0.26e
ALP (K.A.) 12.37 70.34g 25.497 0.74a 14.277 0.78e 18.43 71.04b 16.39 70.95c 15.017 0.95d 13.337 0.97f
AST (U/mL) 73.717 2.69d 98.34 73.74a 78.69 7 3.08c 89.62 7 3.92b 85.36 7 2.87bc 80.317 3.17c 74.137 3.26d
ALT (U/mL) 53.477 2.13e 93.58 73.38a 60.89 7 2.74d 81.69 7 3.61b 71.58 73.25c 64.397 3.15d 9"
56.447 2.47e

Note: Data are expressed as mean 7 SEM. a–g

Values with different letters within a row are significantly different. n ¼ 5, p o0.05.
 Table 5
ALT
EAF (U/mL)
altered 53.477
serum and hepatic 2.13e serum93.58
lipid profiles, 73.38
insulin
a
and hepatic60.89 7 2.74
glycogen
d
81.69 7 3.61b diabetes
in streptozotocin-induced 71.58 73.25c
mice. 64.397 3.15d 56.447 2.47e

Note: Data are(unites)

Parameters expressed as meanGroup
7 SEM.
I
a–g
ValuesGroup
with different
II lettersGroup
within
IIIa row are Group
significantly
IV different. n ¼ 5,V p o0.05.
Group Group VI Group VII

e a e b c d
Serum TG (mmol/L) 1.25 7 0.14 2.15 70.15 1.29 7 0.17 1.89 7 0.20 1.54 70.18 1.38 7 0.16 1.20 7 0.14e
Serum TC (mmol/L) 4.577 0.16e 9.34 70.43a 5.41 70.21d 7.83 7 0.46b 6.49 70.41c 5.82 7 0.22d 4.34 7 0.27e
ACP,
SerumALP,
HDLAST and ALT when
(mmol/L) 3.78 7compared
0.18a to
1.98normal
7 0.17d control mice
3.54 70.20b 4. Discussion
2.727 0.11c 3.15 70.14bc 3.37 70.13b 3.94 7 0.15a
(Table
Hepatic6).
TG Administration
(mg/g protein) of EAF 7.16
86.127 for 4 weeks
de
152.69led to a significant
7 15.63 a
98.62 77.25d 139.84 714.59 b
121.65 7 11.36 bc
108.477 9.53 c
83.87 7 6.85e
d a c b c cd e
Hepatic TCin
decrease (mg/g protein)
serum 36.647
creatinine 2.16
content, 93.45
ACP, 76.48
ALP, 45.577
AST and ALT ac-3.18 Forsythia (Thunb.)
80.497 4.26suspense63.58 7 3.38 Vahl is43.487
one of the traditional
2.84 Chi-
30.89 7 2.14
Serum Insulin (ng/mL) 0.577 0.07a 0.26 70.04e 0.497 0.08b 0.06d 0.37 70.07c 0.43 7 0.09b 0.54 7 0.05a
tivities o0.05) nese0.317
medicines commonly used herbs and is mainly used for
Hepatic (p
Glycogen in diabetic
(mg/g) mice.
4.02 7 0.26ab 2.14 70.11e 3.687 0.21b 2.39 7 0.14d 2.98 7 0.16c 3.37 70.28bc 4.43 7 0.22a
" 6"STZ
As shown in Table 7, MDA significantlyEAF increased, but the ac- clearing and
Values with different letters within a row the
" detoxifying
pharmacology
(Atmakuri and Dathi, 2010). According to
research,
n ¼5, poit is also antioxidant, as well as anti-
tivities of antioxidant enzymes significantly decreased (p o0.05)
a–e
Note: Data are expressed as mean 7 SEM. are significantly different. 0.05.
bacterial, antiviral, anti-inflammatory, liver protective, antiemetic,
in liver and pancreas of STZ-induced diabetic mice when com-
Table 6
pared to those of control mice. EAF administration reduced MDA
"
choleretic, diuretic and so on (Rouf et al., 2001). Previously, it was
Effects of EAF on serum biochemical parameters in streptozotocin-induced diabetes mice. reported that Forsythia suspense (Thunb.) Vahl could be used to
level (p o0.05) and increased the activities of GSH-Px, SOD, and
"
CAT (p o0.05)
Parameters in liver and
(unites) Grouppancreas
I of diabetic
Group II mice. Group III
treat diabetes, but Group
Group IV
the underlying
V
mechanism
Group VI
is unclear
Group VII
(Hong
et al., 2015; Nawash et al., 2013). For this reason, the antidiabetic
"
Creatinine (mg/dL) 0.23 7 0.04 d
0.45 70.04 a
0.26 7 0.03 c
and
b
antihyperlipidemic
0.36 7 0.05 bc
activities of F.0.28
0.3370.04 c
0.25 70.04cdon
7 0.05 were evaluated
suspensa
ACP (K.A.) 4.89 7 0.22e 9.747 0.31a 5.36 7 0.24d 7.54 7 0.32b 6.28 7 0.37c 5.79 7 0.26cd 4.977 0.26e
3.7. Gene expression in 12.37 pancreas STZ-induced diabetic mice in this study with the purpose to un-
ALP (K.A.) 70.34gand liver 25.497 0.74a 14.277 0.78e 18.43 71.04b 16.39 70.95c 15.017 0.95d 13.337 0.97f
AST (U/mL) 73.717 2.69 d
98.34 73.74 a
78.69 7 3.08 c derstand the underlying
89.62 7 3.92 b
85.36 7rational.
2.87 bc
80.317 3.17 c
74.137 3.26d
ALT (U/mL) analysis revealed
RT-PCR 53.477that2.13e EAF altered
93.58 73.38 a
60.89 7 2.74
gene expression in
d
HMG-CoA
81.69 7 3.61b reductase (HMGCR)
71.58 73.25 c
is the rate-limiting
64.397 3.15d enzyme
56.447 that
2.47 e

mouse pancreas and liver (Fig. 3). The mRNA expression of pan- catalyzes the conversion of HMG-CoA to CoA and mevalonic acid
Note: Data are expressed as mean 7 SEM. a–gValues with different letters within a row are significantly different. n ¼ 5, p o0.05.
creatic PDX-1, INS-1, INS-2 and hepatic GCK significantly decreased (MVA) in the de novo synthesis of cholesterol (Goldstein and
(p o0.05) in diabetic mice when compared to that in normal mice. Brown, 1990; Osaki et al., 2015). In vitro assay shows that EAF in- """
hibited HMG–CoA reductase activity. Lipid abnormality is another
Administration
ACP, ALP, AST and of 100
ALT mg/kg
when EAF (grouptoV)normal
compared restored the mRNA
control mice 4. Discussion of diabetes mellitus, manifested mainly by high
expression of pancreatic PDX-1, ±SEM
INS-1 and INS-2
(Table 6). Administration of EAF for 4 weeks led to a significant
a4g
in diabetic mice
characteristic n=5,"p<0.05"
serum TG, TC, hepatic lipid (TG and TC) and low HDL (Jain et al.,
to the same
decrease level creatinine
in serum as in normal non-diabetic
content, ACP, ALP,mice AST (group
and ALTI).ac- In Forsythia suspense (Thunb.) Vahl is one of the traditional Chi-
2010; Yu et al., 2009; Xue et al., 2009), which were observed in our
" addition,
tivities EAF significantly
(p o0.05) in diabeticincreased
mice. the mRNA expression of he- nese medicines commonly used herbs and is mainly used for
STZ-induced diabetic mice. Administration of the EAF for 4 weeks
patic
As GCK
shownin diabetic
in Table mice.
7, MDA significantly increased, but the ac- clearing and detoxifying (Atmakuri and Dathi, 2010). According to
reduced serum TG, TC, hepatic lipid (TG and TC) and increased
" 7"STZ EAF
tivities of antioxidant enzymes significantly decreased (p o0.05) "
the pharmacology research, it is also antioxidant, as well as anti-
bacterial, antiviral, anti-inflammatory, liver protective, antiemetic,
in liver and pancreas of STZ-induced diabetic mice when com-
Table 7 to those of control mice. EAF administration reduced MDA
pared choleretic, diuretic and so on (Rouf et al., 2001). Previously, it was
Effect of EAF on oxidative stress markers of liver and pancreas in streptozotocin-induced diabetes mice.
reported that Forsythia suspense (Thunb.) Vahl could be used to
level (p o0.05) and increased the activities of GSH-Px, SOD, and
treat diabetes, but the underlying mechanism is unclear (Hong
CAT (p o0.05)
Parameters in liver and
(unites) pancreas
Group I of diabetic
Group IImice. Group III Group IV Group V Group VI Group VII
"Liver
et al., 2015; Nawash et al., 2013). For this reason, the antidiabetic
and antihyperlipidemic activities of F. suspensa were evaluated on
"GSH-Px(U/mg protein)
3.7.
955.54 7 53.12b
Gene expression in pancreas
536.34 729.45e
and liver 9.487 0.34e
527.167 49.24e STZ-induced
646.75 734.51 d
768.94
diabetic mice7 42.76 c
in this study901.56 751.05 bc
1087.54 7to
with the purpose 54.54
un-
a

SOD (U/mg protein) 17.677 0.59a 10.317 0.23e 12.36 7 0.43d 14.577 0.13c 15.96 70.51b 16.26 70.27b
CAT (U/mg protein) 186.45 715.46 ab
92.34 79.24 e
101.25 7 8.46 e derstand the
115.42 7 6.24
underlying
d rational.
139.43 7 7.16 c
153.94 78.92 b
197.36 716.93 a

RT-PCR
MDA analysis
(nmol/mg revealed
protein) 1.46 7that
0.04eEAF altered3.127 gene
0.12a expression in a
3.177 0.13 HMG-CoA
2.89 7 0.15b reductase (HMGCR)
2.48 70.25 c is2.12
the70.23
rate-limiting
d enzyme
1.17 7 0.31f that
mouse pancreas and liver (Fig. 3). The mRNA expression of pan- catalyzes the conversion of HMG-CoA to CoA and mevalonic acid
Pancreas
"
creatic PDX-1,protein)
GSH-Px(U/mg INS-1, INS-2 and 731.54
746.45 b
hepatic GCK significantly
372.54 369.25 7 29.14f (MVA)
745.35f decreased 425.45in the ede novo
7 23.69 synthesis
564.87 728.45 d of cholesterol
672.82 7 34.92c (Goldstein
797.24735.48 and
a

SOD (U/mg protein) 13.05 7 0.48a

(p o0.05) in diabetic mice when compared
7.32 7 0.25e 7.87 70.41e
to that in normal mice. de Brown,
8.1571990;
0.27d Osaki12.63et al., 2015).
70.36 b
In vitro
11.83 7assay
0.32bc shows that
9.98 EAF
7 0.37 c
in-
CAT (U/mg protein) 165.487 12.41a 84.55 78.24e 89.65 7 5.48 98.36 7
hibited 6.72d
HMG–CoA
c
119.54 7 9.14activity.
reductase 138.46
Lipid77.43 b
abnormality 156.497 10.94ab
is another
Administration of 100 mg/kg
MDA (nmol/mg protein) EAFd (group3.017
1.43 7 0.03 V) restored
0.14a the2.78
mRNA
7 0.18ab 2.95 7 0.17a 2.54 7 0.19b 2.12 70.35c 1.26 7 0.37e
characteristic of diabetes mellitus, manifested mainly by high
expression of pancreatic PDX-1, INS-1 and INS-2 in diabetic mice
Note: Data are expressed as mean 7 SEM. a–fValues with different letters within a rowserum TG, TC, different.
are significantly hepaticnlipid
¼ 5, p o (TG and TC) and low HDL (Jain et al.,
0.05.
to the same level as in normal non-diabetic mice (group I). In
2010; Yu et al., 2009; Xue et al., 2009), which were observed in our
addition, EAF significantly increased the mRNA expression of he-
patic GCK in diabetic mice. ±SEM a4f n=5,"p<0.05"
STZ-induced diabetic mice. Administration of the EAF for 4 weeks
reduced serum TG, TC, hepatic lipid (TG and TC) and increased

"
Table 7

4. !
Effect of EAF on oxidative stress markers of liver and pancreas in streptozotocin-induced diabetes mice.

Parameters (unites) Group I Group II Group III Group IV Group V Group VI Group VII

Liver
GSH-Px(U/mg protein) 955.54 7 53.12b 536.34 729.45e 527.167 49.24e 646.75 734.51d 768.94 7 42.76c 901.56 751.05bc 1087.54 7 54.54a
SOD (U/mg protein) 17.677 0.59a 9.487 0.34e 10.317 0.23e 12.36 7 0.43d 14.577 0.13c 15.96 70.51b 16.26 70.27b
CAT (U/mg protein) 186.45 715.46ab 92.34 79.24e 101.25 7 8.46e 115.42 7 6.24d 139.43 7 7.16c 153.94 78.92b 197.36 716.93a
MDA (nmol/mg protein) 1.46 7 0.04e 3.127 0.12a 3.177 0.13a 2.89 7 0.15b 2.48 70.25c 2.12 70.23d 1.17 7 0.31f

Pancreas
GSH-Px(U/mg protein) 746.45 731.54b 372.54 745.35f 369.25 7 29.14f 425.45 7 23.69e 564.87 728.45d 672.82 7 34.92c 797.24735.48a
SOD (U/mg protein) 13.05 7 0.48a 7.32 7 0.25e 7.87 70.41e 8.157 0.27d 12.63 70.36b 11.83 7 0.32bc 9.98 7 0.37c
STZ CAT (U/mg protein)
MDA (nmol/mg protein)
165.487 12.41a
1.43 7 0.03d
84.55 78.24e
3.017 0.14a
89.65 7 5.48de
2.78 7 0.18ab
98.36 7 6.72d
2.95 7 0.17a
119.54 7 9.14c
2.54 7 0.19b
138.46 77.43b
2.12 70.35c
156.497 10.94ab
1.26 7 0.37e

"
Note: Data are expressed as mean 7 SEM. a–f
Values with different letters within a row are significantly different. n ¼ 5, p o 0.05.

HMG4CoA HMGCR HMG4CoA CoA

MVA ， EAF HMG4CoA
TG TC TG
TC HDL STZ EAF"4
TG TC TG TC HDL EAF
：
EAF "

10"
 Y. Zhang et al. / Journal of Ethnopharmacology 192 (2016) 256–263 261

"
"
"
mRNA

"
"
"
"

"
"
"
"
"
"
"
"
"
"
"
"
"
"
Fig. 3. Gene expression in pancreas and liver after oral administration of EAF for 4 weeks. The mRNA expression of PDX-1 (A), INS-1 (B) and INS-2 (C) in pancreas and G6P

"
(D), GCK (E) and PHC (F) in liver was determined by qRT-PCR. Data are expressed as mean 7 SEM. β-actin was used as an internal control. Group I (control, non-diabetic
mice); Group II (diabetic mice); Group III (diabetic mice with 5 mg/kg b.w. glibenclamide); Group IV (diabetic mice with 50 mg/kg b.w. EAF); Group V (diabetic mice with
100 mg/kg b.w. EAF); Group VI (diabetic mice with 200 mg/kg b.w. EAF); Group VII (non-diabetic mice with 200 mg/kg b.w. EAF). a–dValues with different letters are

"
significantly different. n ¼ 5, p o 0.05.

" 3" EAF"4

HDL. It is not known whether EAF had a direct effect on lipids or qRT4PCR
(Bhandari et al., 2008). In this study, in vitro enzyme activity assay PDX41
the present hypolipidemia is achieved due to controlled hy- show that F. suspense (Thunb.) Vahl had a strong inhibition on α-

A INS41 B INS42 C
perglycemia. From this study, we can conclusively state that active
fraction of EAF has beneficial effects on diabetic hyperlipidemia.
G6P D GCK E PHC F
amylase, which is used for bioactive fraction screening in this
study and the strong α-amylase inhibiting activity suggests that
α-Amylase is an enzyme catalyzes the hydrolysis of starch to EAF might be used as a dietary supplement to delay the digestion
mRNA
maltose, maltotriose, variousα-(1–6) and α-(1 ! 4) oligoglucans SEM β4 I
of polysaccharide to monosaccharides, leading to lower glucose
and related α-glucans, which are then catalyzed by α-glucosidase absorption and postprandial blood glucose (Ademiluyi and Oboh,
to yield glucose (Janeček et al., 2014; Sudha et al., 2011). Some
;" II
studies have shown that α-amylase is an antidiabetic target
;"2013).
III 5mg/kg
Glibenclamide is listed in National Institute for Health and Care
(Sudha et al., 2011; Lordan et al., 2013). Inhibition of its activity is Excellence (NICE) guidance (NICE, 2008) and Committee on Prac-
;" IV 50mg/kg
anti-hyperglycemia by delaying the absorption of glucose EAF ;" V
tice Bulletins Obstetrics (CPBO, 2013) as an antidiabetic agent. 100mg/kg"
EAF ;" VI 200mg/kg EAF ;" VII
200mg/kg EAF a4d ” " n=5
p<0.05"

11"
 α4 α4(146) α4(144)
α4 。 α4 α4

： α4
α4 EAF
"
NICE NICE 2008
CPBO 2013 β4

EAF
EAF
"

β STZ DNA 、
β STZ ；
STZ
PDX41 INS41 INS42 PDX41
β β PDX41
INS41 INS42
PDX41
β
PDX41 EAF PDX41
INS41 INS42 mRNA EAF
EAF
"
Wolff 1993 Zhao"et"al. 2011
El4Missiry
SOD GSH4Px CAT SOD
GSH4Px CAT
MDA EAF

12"
 MDA EAF
"

STZ
ACP ALP ALT AST ： STZ
ACP ALP ALT AST
/ EAF
ACP ALP ALT AST EAF STZ
GCK"mRNA EAF GCK
，
GCK
： GCK PHC mRNA EAF
EAF
"
5. !
"

STZ
"
!
!
： Z111021006 。
31571862 "
!
!
"

13"