Author's Accepted Manuscript

Antidiabetic effects of flavonoids from So-

phora flavescens EtOAc extract in type 2
diabetic KK-ay mice
Xinzhou Yang, Jing Yang, Chan Xu, Mi Huang,
Qi Zhou, Jingnan Lv, Xinhua Ma, Changqiang
Ke, Yang Ye, Guangwen Shu, Ping Zhao

www.elsevier.com/locate/jep

PII: S0378-8741(15)00383-9
DOI: http://dx.doi.org/10.1016/j.jep.2015.05.043
Reference: JEP9545

To appear in: Journal of Ethnopharmacology

Received date: 30 January 2015

Revised date: 22 April 2015
Accepted date: 27 May 2015

Cite this article as: Xinzhou Yang, Jing Yang, Chan Xu, Mi Huang, Qi Zhou,
Jingnan Lv, Xinhua Ma, Changqiang Ke, Yang Ye, Guangwen Shu, Ping Zhao,
Antidiabetic effects of flavonoids from Sophora flavescens EtOAc extract in type
2 diabetic KK-ay mice, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.
jep.2015.05.043

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 Antidiabetic effects of flavonoids from Sophora flavescens EtOAc extract

in Type 2 diabetic KK-Ay mice

Xinzhou Yanga,b,1, Jing Yanga,1, Chan Xua, Mi Huanga, Qi Zhoua, Jingnan Lva, Xinhua Maa, Changqiang

Keb, Yang Yeb, Guangwen Shua *, Ping Zhaoa *

a
College of Pharmacy, South-Central University for Nationalities, 182 Min-Zu Road, Wuhan 430074, P.R.

China
b
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of

Sciences, 555 Zu-Chong-Zhi Road, Shanghai 201201, P.R.China

*
Corresponding authors at: College of Pharmacy, South-Central University for Nationalities, 182 Min-Zu

Road, Wuhan 430074, P.R. China. Tel.: +86 27 67841196; Fax: +86 27 67843220.
E-mail addresses: shuguangwen@whu. edu.cn (G. Shu); zping0124@163.com (P. Zhao).
1
These authors contributed to this article equally.
Abstract

Ethnopharmacological relevance: Bitter and cold Chinese medicines have been long used

for the treatment for diabetes mellitus (DM) for thousands of years in China. The roots of

Sophora flavescens Ait., one of bitter and cold Chinese medicines commonly used to remove

lung heat have been used to counteract DM and exerted good clinical effects for diabetic

patients in some folk hospitals in Fujian province, P.R. China. However, the corresponding

active principles and antidiabetic mechanism of this Traditional Chinese Medicine remain

unclear. Therefore, in this study, we aim at chemical profiling of the active principles,

validating the potential antidiabetic effects of the active EtOAc extract (SF-EtOAc) in vitro

and in vivo, and elucidating its probable antidiabetic mechanism as well as evaluating its

acute oral toxicity.

Materials and methods: An off-line semipreparative LC-NMR and LC-UV-ESIMS

protocol was developed to determine the chemical principles of the active EtOAc extract

rapidly and unambiguously. The effect of SF-EtOAc on the glucose transporter type 4

(GLUT4) translocation in L6 myotubes was examined. T2DM KKAy mice were induced by

high fat diet. SF-EtOAc was orally administration at the dose of 30, 60 and 120 mg/kg/d, for

21 days. Metformin was used as positive control. Body weight, plasma glucose, oral glucose

tolerance test, serum insulin and blood-lipid indexes were measured. Phosphorylation of the

AMP-activated protein kinase (AMPK) expression in liver was measured.

Results: We found that SF-EtOAc significantly improved oral glucose tolerance, increased

serum high density lipoprotein cholesterol (HDL-C) and reduced body weight, blood

glucose and other related blood-lipid indexes. Mechanistically, SF-EtOAc elevated

phosphorylation of AMP-activated protein kinase (AMPK) and stimulated membrane

translocation of GLUT4. Moreover, it was unveiled that oral median lethal dose (LD50) of

SF-EtOAc was more than 7500 mg/kg, suggesting that SF-EtOAc was practically non-toxic

for mice.

Conclusions: SF-EtOAc improves glucose tolerance, reduces hyperglycemia and resumes

insulin levels, at least in part, by activating GLUT4 translocation which may be modulated

by AMPK pathway. According to the results of the present study, SF-EtOAc possesses a

potent antidiabetic activity and could be used as a safe remedy for the treatment of diabetes.

Keywords: Antidiabetic; Sophora flavescens; KKAy mice; GLUT4; AMPK; off-line

HPLC-NMR

1. Introduction

Diabetes mellitus (DM) is a metabolic disorder syndrome of the endocrine system,

which is a chronic disease that occurs either when the pancreas does not produce enough

insulin or when the body cannot effectively use the insulin it produces (Yang et al., 2010).

DM may lead to various complications such as renal failure, cardiovascular disease,

blindness or other liver disease (He et al., 2011). Type 2 diabetes, resulting from the body’s

ineffective use of insulin, comprises 90% of people with diabetes around the world, and it is

largely the result of excess body weight and physical inactivity (Wild et al., 2004). Type 2

diabetes (T2DM) and its associated complications are major health and economic burdens

worldwide, and the burdens are expected to continue to increase.

Currently available therapies for T2DM include insulin and various oral antidiabetic

agents such as sulfonylureas, biguanides, α-glucosidase inhibitors, and glinides, which are
used as monotherapy or in combination to achieve better glycemic regulation (Levetan, 2007;

Jung et al., 2006). A number of anti-diabetic drugs are losing effectiveness against this

disease, showing drug resistance and serious side effects, such as heart failure or liver

disease, including biguanides and thiazolidinedione (Goodwin et al., 2008; Pirat et al., 2012).

Managing diabetes without any side effects is still a challenge. Therefore, the search for

more effective and safer natural hypoglycemic agents has continued to be an important area

of investigation. The traditional Chinese medicines (TCMs) have been used to counteract

DM (recognized as “Xiao Ke Zheng” in ancient China) for thousand years based on unique

theory system with few side effects, and it has been attracting more and more attention for its

dialectical therapy (Liu et al., 2013; Yang et al., 2014).

Sophora flavescens Ait. is a shrub distributed in East Asian and some European

countries, and has been traditionally used as herbal medicine and functional food ingredient

for thousands of years (Vina et al., 2012). As one of traditional Chinese medicines, the roots

of S. flavescens have been widely used as medicinal herbs in a variety of herbal formulations

to treat a range of diseases, such as various virus infections, cancer, cardiac arrhythmia, ulcer,

inflammatory disorder, fever, and skin diseases. Meanwhile, this traditional Chinese

medicine and its complex preparations have been long used for the treatment of diabetes and

exerted good clinical effects for diabetic patients in some folk hospitals in Fujian province,

P.R. China (Shi et al., 1999; Jung et al., 2008). However, this TCM’s antidiabetic mechanism

and the corresponding active principles remain unclear. In the present study, we aim at

chemical profiling of the active principles, validating the potential antidiabetic effects of
SF-EtOAc in vitro and in vivo, and elucidating its probable antidiabetic mechanism as well

as evaluating its acute oral toxicity.

2. Materials and Methods

2.1. Instruments and reagents

The NMR spectra were recorded in DMSO-d6 on an AVANCE III 500 MHz

spectrometer equipped with 1.4 mm heavy wall Micro NMR tubes (NORELL, Landisville,

USA). The active volume of the 1.4 mm heavy wall Micro NMR tube is about 150 µL.

Direct injection ESI MS and LC-PDA-ESIMS analyses were recorded on a Waters

ACQUITY SQD MS system (Waters, Milford, MA, USA) connected to a Waters 1525

HPLC with a 2998 Photodiode Array Detector (Waters, Milford, MA, USA) and a Waters

SunfireTM C18 column (5 µm, 4.6 × 150 mm) (Waters, Ireland). Semi-preparative HPLC was

carried out on a Waters 2535 HPLC fitted with a 2998 Photodiode Array Detector and a 2707

Autosampler (Waters). Separations were performed on a Waters SunfireTM C18 column (5

µm, 10 × 150 mm) (Waters, Ireland). All the solvents used for chromatography were of high

performance liquid chromatography (HPLC) grade and all the other chemicals were of

analytical-reagent grade. HPLC-grade methanol and acetonitrile were purchased from Merck

Chemical Company (Darmstadt, Germany). Sephadex LH-20 gel was obtained from GE

Health Care (Uppsala, Sweden). Silica gel GF254 precoated glass plates (1.00 mm, Yantai

Jiangyou Silica Development Co., Ltd, Yantai, China) were used for preparative TLC.

2.2. Plant material and sample preparation

 The roots of Sophora flavescens Ait were collected from Lingyuan City, Liaoning

province, P.R. China in Sept, 2012, and identified by Professor Dingrong Wan of College of

Pharmacy, South-Central University for Nationalities, Wuhan, P.R. China. A voucher

specimen (No. SF20120520) is deposited in College of Pharmacy, South-Central University

for Nationalities. Air-dried roots of S. flavescens (200 g) were ground and then extracted

sequentially by maceration at room temperature with n-hexane (3 × 1500 mL, 3 h each),

followed by ethyl acetate (3 × 1500 mL, 3 h each) and methanol (3 × 1500 mL, 3 h each).

The solvents were evaporated at reduced pressure to yield 3.7 g, 13.5 g, and 21.3 g of

n-hexane, ethyl acetate and methanol fractions, respectively.

2.3. LC-PDA-ESIMS method

Analysis was carried out on a Waters Sunfire C18 column (5 µm, 4.6 × 150 mm)

equipped with a Waters Sunfire C18 cartridge as a precolumn. Water (A) and acetonitrile (B)

containing 0.1% formic acid were used as mobile phases. The gradient program was used as

follows: 0-20 min, 10% to 100% B; 20-25 min, 100% B. The analysis was performed at the

flow rate of 1.0 mL/min. UV-Vis spectra were recorded with the range of 200-500 nm at a

spectral acquisition rate of 10 scans per second. The eluent was split at a 1 : 5 ratio before

the mass spectrometer. ESI-MS were recorded in both positive and negative ion modes. The

capillary voltage was 4000 V, the capillary exit voltage was 140.0 V, and the skimmer

voltage was 40 V. The nebulizer gas pressure was set to 40 psi, the dry gas flow to 10.0

L/min and the dry temperature to 320 °C. Mass range was set from 120-1500 m/z. Data

acquisition and processing were achieved with MassLynx™ 4.0 software (Waters, Milford,

USA).
2.4. Chemical characterization

0.4 g of the ethyl acetate fraction was dissolved in 2.0 mL of DMSO and the solution

was filtered. Separation of the ethyl acetate extract was carried out with the same solvent

system and similar optimized gradient elution to that of LC-PDA-ESIMS. The optimized

gradient program used as follows: 0-20 min, 10% to 50% B; 20-30 min, 50%-100% B; 30-36

min, 100% B. The flow rate was set at 5.0 mL/min and the injected volume of extract was

200 µL at a concentration of 200 mg/mL in DMSO. A total of 11 peak-based fractions were

collected manually. 10 injections were carried out, and corresponding fractions were

combined. Final purification were performed as follows: peaks 2, 4–7 and 9–11 were filtered

through a Sephadex LH-20 column (400 × 10 mm, MeOH containing 0.1% formic acid) to

yield pure compounds 3 (2.7 mg), 6 (3.4 mg), 7 (4.7 mg), 8 (4.2 mg), 9 (48 mg), 13 (3.1 mg),

14 (2.8 mg) and 15 (1.7 mg). Peak 1 was purified by preparative TLC

(CH2Cl2:MeOH:formic acid—100:15:0.5) to yield compounds 1 (2.1 mg) and 2 (2.3 mg).

Peak 3 was purified by preparative TLC (CH2Cl2:MeOH:formic acid—100:10:0.5) to give

compounds 4 (2.4 mg) and 5 (2.6 mg). Peak 8 was also purified by preparative TLC

(CH2Cl2:MeOH:formic acid—100:7:0.5) to give compounds 10 (3.8 mg), 11 (3.5 mg) and 12

(4.6 mg), respectively. The NMR spectra were recorded in DMSO-d6 on an AVANCE III 500

MHz spectrometer equipped with 1.4 mm heavy wall Micro NMR tubes. Typically, 1H

spectra were obtained at 128 scans. 1H-1H COSY spectra were obtained at the scan range of

4-32. The HSQC and HMBC spectra were obtained at the scan range of 16-96. Compounds

1-15 were dissolved in DMSO-d6 or MeOH-d4 with the amount range of 0.8-3.0 mg for

NMR tests.
2.5. Insulin-responsive aminopeptidase (IRAP) translocation assay

L6 rat skeletal muscle cells (L6 cells) were transected with pIRAP-mOrange cDNAs

(presented by Professor Tao Xu, Chinese Academy of Sciences) using Lipofectamine 2000

as per manufacturers protocol. L6 cells stably expressing IRAP-mOrange (L6

IRAP-mOrange) were cultured in α-MEM supplemented with 10% fetal bovine serum at 37

o
C in 5% CO2. Before starting the experiment, L6 IRAP-mOrange was seeded in 48-well

plates, and incubated until 100% confluence and then starved in serum-free-α-MEM for 2 h.

The cells were imaged with a laser-scanning confocal microscope LSM 510 (Carl Zeiss, Jena,

Germany) to monitor the dynamics of IRAP-mOrange translocation. Images were taken after

addition of 100 nM insulin or 10 µg/mL test samples, using 555 nm excitation laser every 5

minutes in 25 min.

2.6. Assay of glucose uptake

Glucose uptake assay was performed on L6 cells with a glucose uptake cell-based assay

kit (Cayman Chemical, USA). L6 cells were seeded with 5 × 104 cells/well in 100 µL

α-MEM medium in 96-well plate. After 12 h incubation, cells were treated with different

concentrations of SF-EtOAc (5 µg/mL, 10 µg/mL or 15 µg/mL) or vehicle control in 100 µL

glucose-free α-MEM medium containing 150 µg/mL 2-NBDG. Plates were incubated at

37 °C with 5% CO2 for 30 min. At the end of the treatment, plates were centrifuged for five

minutes at 400 x g at room temperature. The supernatant was aspirated, and then 200 µL of

cell-based assay buffer was added to each well. Plates were centrifuged for five minutes at

400 x g at room temperature. Then the supernatant was aspirated, and 100 µL of cell-based
assay buffer was added to each well. The 2-NBDG taken up by cells was detected with

fluorescent filters (excitation/emission = 485/535 nm).

2.7. Animals

The 8-week-old male KK-Ay mice, weighing 28–32 g, and the 8-week-old male C57/BJ

mice, weighing 26–28 g, were from Beijing HFK Bioscience Co., Ltd. All animals were

maintained in laminar flow cabinets under specific pathogen-free (SPF) conditions in

facilities approved for Accreditation of Laboratory Animal Care and in accordance with

International Guidelines for Care and Use of Laboratory Animals and approved by the

Animal Ethical Committee of the Institute of Health and Epidemic Prevention (Wuhan, P.R.

China). The cages, bedding, food, and water were autoclaved. The two groups of mice were

housed separately and maintained on a 12-h light/dark cycle, temperature 23 ± 3 oC,

humidity 55 ± 15%. The KK-Ay mice (n = 60) were given a high-fat diet consisting of 40%

(wt/wt) fat (Medicience Co., Ltd., Yangzhou, China), orally, for 4 consecutive weeks (an

average weight of 43 g) to establish a type 2-like diabetic mice model while the C57/BJ mice

(n = 12) was given standard laboratory diet (Beijing HFK Bioscience Co., Ltd). Four weeks

later, the fasted blood glucose levels of the mice were tested. Fifty mice whose fasted blood

glucose levels ≥ 11.1 mmol/L were classified as T2DM. Male KK-Ay mice of 12 weeks old

were randomly divided into 5 groups: saline treatment (vehicle group), SF-EtOAc treatment

(30, 60, 120 mg/kg/day) and metformin treatment (200 mg/kg/day), while saline was

administrated orally to C57/BJ mice as a normal control. Each group was

consisted of 10 mice. T2DM mice were fed with high-fat diet in later study.

2.8. Determination of biochemical indexes

 During the experimental period, the body weights of the mice were measured daily.

Blood glucose levels were measured in tail blood using a glucometer (ONETOUCH Ultra,

Lifecan, USA). After 3 weeks of treatments, the mice were anesthetized and blood samples

were collected to determine biochemical parameters. After weighing, part of livers and

muscles were frozen in liquid nitrogen and stored at -80 oC for biochemical assays, and the

rest were fixed in 10% neutral buffered formalin, embedded by paraffin, then stained in

hematoxylin and eosin. The stained tissues were observed through an optical microscope and

were photographed. Blood samples from orbital venous plexus were collected into tubes.

Serums were prepared by centrifugating the blood at 1000 x g for 15 min at 4 oC. Serum

total cholesterol (TC), serum triglyceride (TG), serum high density lipoprotein cholesterol

(HDL-C), serum low density lipoprotein cholesterol (LDL-C) and free fat acid were

determined with automatic biochemical analyzer (Beckman Coulter, USA). The levels of

serum insulin were determined using radioimmunoassay with Radiation immune diagnostic

kits (Jiuding Medical Biological Engineering Co., Ltd, P.R. China). Partial livers and skeletal

muscle were collected and homogenized (10%, w/v) in cold normal saline. The tissue

homogenate was then mixed with a solution of chloroform/methanol (2:1, v/v), according to

a ratio of 1:1 (v/v). The prepared sample was centrifuged at 1200 x g for 10 minutes, and the

obtained substratum was used for measurement of total cholesterol, triglyceride and free

fatty acid according to the colorimetric methods (Lian et al., 2011).

2.9. Oral glucose tolerance test (OGTT)

After an overnight fast, 2 g/kg of glucose was administered orally to all tested mice

before OGTT at the 18th day of the treatment. The results of the OGTT were compared to
those of mice treated with either SF-EtOAc treatment group (30, 60, 120 mg/kg/day) or a

vehicle control group (saline). To assess the fasting blood glucose levels, blood samples were

collected from each subject at 0, 30, 60, and 120 min after administration of glucose. The

C57/BJ mice served as normal controls for comparison to the untreated diabetic mice.

2.10. Western blotting analysis

Skeletal muscle and liver were ground and lysed in RIPA buffer [50mM Tris-HCl (pH8),

150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1% SDS], supplemented with

complete protease inhibitor cocktail (Roche) and phosphatase inhibitor (Phosstop, Roche),

and then centrifuged at 8000 rmp, 4 oC for 12 min. Supernatant protein concentration was

determined with BCA assay kit (Abgent, USA). Protein samples denatured in SDS sample

buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, and 0.0025% bromophenol blue)

were subjected to SDS-PAGE and blotted onto pure nitrocellulose membranes (Pall, USA).

Blotted membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1%

Tween 20 for 1 h and then incubated with primary antibodies against AMPK (Cell Signaling

Technology), phosphoAMPKα (Thr172) (Cell Signaling Technology) for 16 h at 4 °C. After

three washes in Tris-buffered saline containing 0.1% Tween 20, the membranes were

incubated with anti-mouse or anti-rabbit IgG antibodies for 2 h. Immunoreactive signals

were detected and quantified with the Gel Image system (Aplegen, USA).

2.11. Acute toxicity study

KM mice (male and female) weighing between 18 and 22 g were approved by the Hubei

Provincial Center for Disease Control and Prevention [certification No. SCXK (E)

2008-0005]. The animals were housed at 22 ± 2 oC, 45-75% relative humidity, where 12 h
dark-light cycles were maintained with free access to food and water. Based on findings

from sighting study, 7500 mg/kg/day dose was selected for the main study. In the main study,

male mice (n = 10) and female mice (n = 10) were orally administered a single dose of

SF-EtOAc concentrate (7500 mg/kg/day) dissolved in saline. The animals were observed for

14 days for clinical signs or mortality. On day 14 at completion all the animals were

euthanized followed and gross pathological examinations were undertaken.

2.12. Statistical analysis

Data were expressed as mean ± S.E.M. and analyzed by one-way ANOVA followed by

Tukey’s post hoc test using Graphpad prime 5.0 software. A probability (P) value of less than

0.05 was considered statistically significant.

3. Results

3.1. Chemical characterization

The LC-PDA-ESIMS experiment was performed based on the standard operation

procedure shown in 2.3 (Fig. 1A). Considering that some HPLC peaks showed partial

overlap, separation conditions were optimized for full resolution of the critical HPLC peaks.

The similar optimized conditions were also used in large scale preparative isolation of target

compounds. A typical semi-preparative HPLC chromatogram obtained with an injection of

40 mg of extract is shown in Fig. 1B. A total of 11 peaks were collected and submitted to

further purification with Sephadex LH-20 column and preparative TLC to afford 15

compounds for which 1D and 2D NMR spectra were recorded with 1.4 mm heavy wall

Micro NMR tubes. The purity was tested by analytical HPLC under same conditions as
shown in Fig. 1A. The major peak was determined as (−)-kurarinone (9) (Kim et al., 2006),

the main principle of lavandulyl flavonoid in S. flavescens. Most of the remaining

compounds were all structurally related 8-lavandulyl flavonoids (Fig. 2) which were

identified as kushenol H (1) (Son et al., 2003), kushenol K (2) (Shin et al., 2002), kurarinol

(5) (Kim et al., 2006), kushenol N (8) (Son et al., 2003), neokurarinol (10) (Kyogoku et al.,

1973), kushenol C (11) (Hillerns & Wink., 2005), sophoraflavanone G (12) (Kim et al.,

2006), leachianone A (13) (Kyogoku et al., 1973), kuraridine (14) (Kim et al., 2006), and

kushenol A (15) (Kim et al., 2006) by comparison of their MS, UV, and NMR spectra with

published reference data. Other residual compounds were characterized as calycosin (3) (Du

et al., 2006), (2S)-7,2',4'-trihydroxy-5-methoxy-8-dimethylallyl flavanone (4) (Zhang et al.,

2013), (2R)-3α,7,4'-trihydroxy-5-methoxy-8-dimethylallyl flavanone (6) (Quang et al., 2012)

and isoxanthohumol (7) (Kim et al., 2006), respectively.

Fig. 1 (A) The LC–PDA–ESIMS analysis of SF-EtOAc is shown at 320 nm with peak labeling

corresponding to compounds 1–15. (B) The optimized semi-preparative HPLC separation of the
SF-EtOAc (40 mg in 200 µL DMSO) is shown at 320 nm with peaks 1–11 collected for microprobe NMR

and further purified if required.

Fig. 2 Structures of flavonoids from SF-EtOAc.

3.2. Effects of SF-EtOAc on IRAP translocation quantity and glucose uptake in cells

As we know, insulin can cause a time-dependent increase in GLUT4-EGFP

fluorescence in the evanescent field (Bai et al., 2007). This is consistent with the time course

for insulin-stimulated IRAP translocation to the PM in L6 cell. In order to acquire the

potential activity of SF-EtOAc on glucose metabolism, we examined its effect on the

translocation of the IRAP to the cell plasma membrane (PM) (Fig. 3).The addition of

SF-EtOAc had obvious effect on the translocation of IRAP (Fig. 3A). In our experiments, we

assume that SF-EtOAc induced transportation of IRAP may be through AMPK pathway. In

order to prove this, we used the Compound C (an inhibitor of AMPK, Selleckchem, USA)

incubate L6 cells for 30 minutes, and then added SF-EtOAc. Result showed that SF-EtOAc

could not increase the translocation of IRAP with the prior adding of Compound C (Fig. 3B),

compared with SF-EtOAc-direct stimulation. The dose-response curves (Fig. 3C) showed
that SF-EtOAc exhibited this biological activity at concentrations as low as 0.1 ng/mL and

reached maximal effect at between 1µg/mL and 10 µg/mL. In the absence of SF-EtOAc (5

µg/mL, 10 µg/mL, 15 µg/mL), insulin (10 nM) stimulated a dramatic increase in glucose

uptake over basal levels (Fig. 4). The data indicated that SF-EtOAc could stimulate glucose

uptake significantly at the dose of 10 µg/mL and 15 µg/mL.

Fig. 3 SF-EtOAc stimulated IRAP trafficking in L6 cell. (A) L6 cells were infected with pIRAP-mOrange

in order to detect externalized IRAP by confocal microscopy. Confocal images in L6 cells incubated in the

absence (basal) or presence of SF-EtOAc for 25 minutes. (B) Data represent the fold increase in

fluorescence induced by insulin and SF-EtOAc between 0 and 25 minutes. (C) The dose-response curve

was constructed in L6 cells for SF-EtOAc at concentrations from 0.1 ng/mL to 10 mg/mL. Values are the

mean ± SEM of three independent experiments.

Fig. 4 Effects of SF-EtOAc on stimulation of glucose uptake in L6 cells. Results are means ± SEM of

three independent experiments. ***P≤ 0.001, **P≤ 0.01, compared to normal control.

3.3. Effects of SF-EtOAc on body weight, tissue weight and blood glucose levels
 KK-Ay mice was produced by the transfer of the yellow obese gene (Ay allele) into

glucose-intolerant black KK mice, an inbred mouse strain established from Japanese native

mice (Okazaki et al., 2002). After feeding with a regular high-fat diet, the KK-Ay mice

spontaneously become obese, hyperinsulinemic, and hyperglycemic. Therefore, KK-Ay mice

were widely used as an experimental model for type 2 diabetes mellitus. In the present

research, KK-Ay mice were used as T2DM animal model to evaluate antidiabetic effects of

SF-EtOAc in vivo. Throughout the experiment, the body weights of all the experimental

mice were monitored daily. Fig. 5A showed the observations of body weight of experimental

mice during the experiment. As shown in the Fig. 5A, the body weights of all the tested

groups (metformin and SF-EtOAc treated) were reduced up in 2 weeks. The body weights of

the vehicle control group were continuously increased, and at the end of the experimental

period, there was a little weight gain, compared to the initial weight. At the same time,

considerably lower body weights were observed in the SF-EtOAc treated groups during the

tested period, while fat and liver mass indicated a clear reduction (Fig. 5B). In addition, the

effect of SF-EtOAc on blood glucose level was investigated in diabetic mice. Significantly

lower blood glucose levels were observed in the SF-EtOAc treated groups, compared to that

of the control group (Fig. 5C).

Fig. 5 Effects of SF-EtOAc on body weight (A), tissue weight (B) and fasted blood glucose levels (C) afer

treatment. +++P ≤ 0.001, compared to normal control; ***P≤ 0.001, **P≤ 0.01, *P≤ 0.05, compared to T2DM

mice treated with vehicle.

3.4. Effects of SF-EtOAc on serum insulin and OGTT

Compared to the normal control group, serum insulin levels in the diabetic groups

were increased markedly. After 3 weeks of treatment, serum insulin in the SF-EtOAc treated

groups was decreased (Fig. 6A). The impaired glucose tolerance can be improved by

metformin and SF-EtOAc after treatments for 3 weeks. After glucose loading in OGTT, the

peak blood glucose levels were significantly increased at 30 min. Mice treated with

metformin and SF-EtOAc showed a remarkable improvement in overall glucose response

after glucose loading (Fig. 6B).

Fig. 6 Effects of SF-EtOAc on serum insulin and OGTT. +++P ≤ 0.001, compared to normal control; ***P≤

0.001, **P≤ 0.01, *P≤ 0.05, compared to T2DM mice treated with vehicle.

3.5. Effects of SF-EtOAc on TG, TC, FFA, HDL-C and LDL-C levels in the serum

The results for serum lipids are shown in Fig 7. The serum TG, TC, FFA, and LDL-C in

the groups treated with SF-EtOAc were significantly lower than those in the vehicle group (P

< 0.05), while the serum HDL-C level was significantly higher.

Fig. 7 Effects of SF-EtOAc on Total choleterol (A), Triglyceride (B) and Free Fatty Acid (C), High

density lipoprotein cholesterol (D) and Low density lipoprotein cholesterol (E) levels in the serum. +++P ≤
*** **
0.001, compared to normal control; P≤ 0.001, P≤ 0.01, *P≤ 0.05, compared to T2DM mice treated

with vehicle.
3.6. Effects of SF-EtOAc on tissue lipid content

The results for liver and skeletal muscle lipids are shown in Fig. 8. The TC, TG and

FFA contents in the groups treated with SF-EtOAc were significantly lower than those in the

vehicle group, after treatment with 30, 60, or 120 mg/kg of SF-EtOAc for 3 weeks.

Fig. 8 Tissue lipid content after treatment for 3 week. Total choleterol (A), Triglyceride(B) and Free Fatty

Acid (C) levels in mice liver tissue; Total choleterol (D), Triglyceride (E) and Free Fatty Acid (F) levels in

mice skeletal muscle tissue. +++P ≤ 0.001, ++P ≤ 0.01, +P ≤ 0.05, compared to normal control; **P ≤ 0.01, *P

≤ 0.05 compared to T2DM mice treated with vehicle.

3.7. Mouse liver and pancreas histological changes

In the diabetic mice groups, the hepatic steatosis appeared, empty lipid vacuoles were

presented in liver (Fig. 9). After oral administration of SF-EtOAc for 3 weeks, the degree of

hepatic steatosis was reversed, especially in the 120 mg/kg SF-EtOAc group. To determine

whether SF-EtOAc was capable of protecting pancreatic β-cells, we performed histological

examinations. The results showed the abnormity caused by diabetes was alleviated by

SF-EtOAc treatment via a dose-dependent manner (Fig. 10).

Fig. 9 Effects of SF-EtOAc on morphological features of mice livers. Optic microscopy: HE (200 x). (A)

Normal control. (B) KK-Ay mice treated with vehicle. (C) KK-Ay mice treated with metformin (200

mg/kg). (D) KK-Ay mice treated with SF-EtOAc (30 mg/kg). (E) KK-Ay mice treated with SF-EtOAc (60

mg/kg). (F) KK-Ay mice treated with SF-EtOAc (120 mg/kg).

Fig. 10 Effects of SF-EtOAc on morphological features of mice pancreas. Optic microscopy: HE (200 x).

(A) Normal control. (B) KK-Ay mice treated with vehicle. (C) KK-Ay mice treated with metformin (200

mg/kg). (D) KK-Ay mice treated with SF-EtOAc (30 mg/kg). (E) KK-Ay mice treated with SF-EtOAc (60

mg/kg). (F) KK-Ay mice treated with SF-EtOAc (120 mg/kg).

3.8. Effects on AMPK protein expression in hepatic tissue and skeletal muscle

AMPK is activated under a variety of conditions that signify cellular stress, usually in

response to a change in the intracellular ATP-to-AMP ratio (Lee et al., 2006). Active AMPK

orchestrates a variety of metabolic processes, most of which lead to reduced energy storage

and increased energy production (Hardie et al., 2011). To determine whether the effect of

SF-EtOAc in animals could activate AMPK, we examined the phosphorylation of AMPK in

liver. The results shown in Fig. 11 indicate that, when compared with the normal control

group, the expression of hepatic p-AMPK protein in the diabetic mice groups decreased.

After 3 weeks of SF-EtOAc administration, the expression of p-AMPK proteins increased

(Fig. 11).

Fig. 11 Effect of SF-EtOAc on AMPK phosphorylation in liver. +++P ≤ 0.001, compared to normal

control; ***P≤ 0.001, **P≤ 0.01, *P≤ 0.05, compared to T2DM mice treated with vehicle.

3.9. Acute toxicity study

KM mice were derived in 1944 from a pair of Swiss mice that had been introduced

from Hoffline Institution of Hindustan into Kunming of China (Shang et al., 2009).

Kunming mice are the most commonly used outbred mouse line in China. This type of mice

shows strong disease resistance and adaptability, high breeding coefficient and survival rate

(Shang et al., 2009). So, KM mice have been widely utilized in pharmacological,

toxicological, medicinal and biological research and testing. In the acute toxicity study, oral

LD50 of the SF-EtOAc concentrate in KM mice was found to be greater than 7500 mg/kg/day.

In addition, the 14-day observation period during the acute oral toxicity study on SF-EtOAc

did not produce mortality, adverse clinical features, excessive body weight lose or gain and
food intake, toxicologically relevant changes in hematology and clinical biochemistry as

well as any other toxic signs (data not shown). Necropsy at the end of study did not reveal

any pathological abnormalities. The results of acute oral toxicity study suggest that

SF-EtOAc concentrate is unlikely to be toxic at the tested dose of 7500 mg/kg/day for KM

mice.

4. Discussion

Many Chinese bitter and cold herbs have revealed good antidiabetic effects in vitro and

in vivo, as well as in clinical practices, such as Coptis chinensis (Turner et al., 2008),

Momordica charantia (Tan et al., 2008), Swertia macrosperma (Wang et al., 2013) and

Gardenia jasminoides (Zhang et al., 2006), etc. The roots of S. flavescens, one of bitter and

cold Chinese medicines commonly used to remove heat have been long used for the

treatment of diabetes and exerted good clinical effects for diabetic patients in some folk

hospitals in Fujian province, P.R. China (Shi et al., 1999; Jung et al., 2008). However, this

TCM’s antidiabetic mechanism and the corresponding active principles remain unclear.

Therefore, there is a need to validate the antidiabetic effects, elucidate the mechanism and

determine the active principles of S. flavescens.

In the present study, a cell-based GLUT4 translocation assay using stable L6 cells

expressing pIRAP-mOrange cDNAs was established to preliminarily discover plants

extracts, fractions and their isolated compounds with potential antidiabetic effects by means

of evaluating their effects on the translocation of GLUT4 to PM. During the screening,

SF-EtOAc exhibited a strong effect of stimulation on GLUT4 translocation by 2.2 fold in L6

cells, as insulin displayed 2.3 fold. Based on the finding in vitro, we predicted that

SF-EtOAc may have the antidiabetic potency in vivo. The in vivo data clearly showed the

antidiabetic activity of SF-EtOAc, including the amelioration of hyperglycemia and

hyperinsulinemia. We observed SF-EtOAc significantly alleviated hyperglycemia and

hyperinsulinemia in KK-Ay mice. OGTT further indicated that hyperglycemia and

hyperinsulinemia were considerably ameliorated by SF-EtOAc. T2DM patients are often

prone to suffer from cardiovascular diseases as a result of dyslipidemia (Charbonnel et al.,

2004). Insulin resistance (IR) is an important component leading to the development of type

2 diabetes (Ahonen et al., 2012). This event leads to elevated circulating FFA and lipid

accumulation in livers (Kovacs et al., 2005; Subramanian, 2012). On the side, skeletal

muscle is another important insulin-responsive tissue (Bansal et al., 2012). Diabetes mellitus

is usually associated with abnormal levels of serum lipids (Shokeen et al., 2008). In our

study, a significant rise of TG, TC and FFA levels was observed in serums, livers, and

skeletal muscles of T2DM mice, while level of HDL-C in serum was down regulated in

T2DM animals. These exceptions could be relived by SF-EtOAc treatment with a

dose-dependent manner. Histopathological examinations of mouse livers supplied the

evidence that SF-EtOAc dose-dependently and efficiently rescued liver steatosis associated

with T2DM. Moreover, the hypolipidermic activity of SF-EtOAc was similar to that of the

standard drug metformin.

AMP-activated protein kinase (AMPK) is a major regulator of glucose and fatty acid

catabolism (Fogarty et al., 2010), and plays a major role in the control of metabolic disorders

such as diabetes, obesity, and cancer (Carling, 2004; Kim et al., 2007), and it has emerged as
a therapeutic target for metabolic disorders (Zhang et al., 2009). The activation of AMPK is

tightly regulated and is dependent on both its phosphorylation by upstream kinases and its

dephosphorylation by phosphatases (Hardie, 2011). Activation of AMPK increases fatty acid

oxidation, inhibits lipid synthesis, and can improve insulin action (Ye et al., 2005). AMPK

activity is thought to be impaired in diabetes as AMPK stimulation in animal models has

been shown to improve glycemia. Increasing glucose uptake into the cells and suppressing

endogenous glucose production and lipolysis suggest AMPK activators as a new class of

antidiabetic agents (Moussa et al., 2012). Furthermore, AMPK pathway is a major regulatory

pathway of GLUT4 translocation which is an essential step for inducible glucose uptake into

muscle and fat. In this study, we observed the AMPK pathway was activated by SF-EtOAc,

suggesting SF-EtOAc with therapeutic potential for diabetes by targeting AMPK.

For the rapid and accurate structure elucidation of natural products, on-line LC-NMR

plays an important role and allows for providing the precise and complementary structure

information. But the high costs for LC-NMR machines and deuterated solvents restricted its

applications in discovery of bioactive natural products and drug discovery (Wolfender, et al.,

2005). Our approach will provide a rapid and economical protocol for investigations of

bioactive principles from the traditional Chinese medicines as well as Chinese ethnic

medicines based on the combination of off-line LC-NMR and LC-UV-ESIMS.

Toxicological screening is very important for the development of new drugs and for the

extension of the therapeutic potential of existing molecules. Acute toxicity testing is carried

out to determine the effect of a single dose on a particular animal species. Developmental

toxicity and neurotoxicity of two matrine-type alkaloids isolated from S. flavescens, matrine
and sophocarpine, in zebrafish have been reported recently (Lu et al., 2014), implying a

possible toxic effect of S. flavescens on animals. Considering that fact that the polarity of

alkaloids is relatively higher and alkaloids are insoluble in ethyl acetate, we used ethyl

acetate as the solvent to extract the herb S. flavescens to avoid possible toxicity. As expected,

it was unveiled that few alkaloids existed in SF-EtOAc. The 14-day observation period

during the acute oral toxicity study and body weight measurements did not indicate any toxic

effects. The oral acute toxicity study on SF-EtOAc indicated that its single dose oral

LD50 for KM mice was proposed to be greater than 7500 mg/kg/day for KM mice. Thus,

according to the common classification of the relative toxicity of chemicals, SF-EtOAc was

classified as relatively harmless or non-toxic substance (Gosselin et al., 1984).

5. Conclusion

According to the results of the present study, the ethyl acetate extract of the traditional

Chinese medicine, S. flavescens, possesses a potent antidiabetic activity and could be used as

a safe remedy for the treatment of diabetes. SF-EtOAc improves glucose tolerance, reduces

hyperglycemia and resume insulin levels, at least in part, by activating GLUT4 translocation,

possibly due to several lavandulyl flavonoid compounds (kurarinone, sophoraflavanone G,

kushenol H, kushenol K, kurarinol, etc., and their derivatives) present within the extract.

Moreover, the GLUT4 translocation may be modulated by AMPK pathway. All these

findings provide scientific evidence that SF-EtOAc has potential to be safe and effective

agent in the therapy of diabetes mellitus.

Acknowledgements

The work was financially supported by National Natural Science Foundation of China

grants (No. 81102798 and No. 31070744), the State Key Laboratory of Drug Research

Foundation (SIMM1403KF-07), Wuhan Youth Chenguang Program of Science and

Technology (No. 2013070104010028), and the Fundamental Research Funds for the Central

Universities (CZY14013).

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