International Journal of Biological Macromolecules 102 (2017) 396404

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Hypoglycemic and hypolipidemic effects of a polysaccharide from

ower buds of Lonicera japonica in streptozotocin-induced diabetic
rats
Dongying Wang a, , Xiangmei Zhao b , Yulan Liu a
a
College of Food Science and Technology, Henan University of Technology, Zhengzhou 450001, China
b
Department of Emergency, Zhengzhou University Peoples Hospital, Zhengzhou 450003, China

a r t i c l e i n f o a b s t r a c t

Article history: Hyperglycemia and dyslipidemia are classic features for diabetes mellitus (DM). In this study, one fraction
Received 22 January 2017 of the crude polysaccharides extracted from Lonicera japonica ower buds (LJP) were investigated for its
Received in revised form 15 February 2017 hypolipidaemic and hypoglycaemic activities by means of streptozotocin (STZ)-induced diabetic rats.
Accepted 12 April 2017
Interestingly, after orally administrated with 800 mg/kg body weight (B.W.) LJP for 42 days, the food and
Available online 15 April 2017
water intake and the levels of sugar and insulin in blood for the diabetic rats were drastically decreased,
while the contents of liver and skeletal muscle glycogen and the concentrations of hepatic pyruvate
Keywords:
kinase and hexokinase were obviously increased (p < 0.01 or p < 0.05). The levels of total cholesterol
Hypoglycemic
Hypolipidemic (TC), triglyceride (TG), low-density lipoprotein-cholesterin (LDL-C) and very-low-density lipoprotein-
Polysaccharide cholesterin (VLDL-C) were signicantly descended, while high-density lipoprotein-cholesterin (HDL-C)
was signicantly ascended (p < 0.01 or p < 0.05). In addition, the oxidant stress in liver was restored as
well. The results suggested that LJP could be considered as an ingredient of functional foods for diabetes,
and this is the rst report about the hypoglycemic and hypolipidemic effects of the polysaccharides
extracted from Lonicera japonica.
2017 Elsevier B.V. All rights reserved.

1. Introduction dence of DM is becoming a serious threat to the health of people

Diabetes mellitus (DM) is a progressive and complex metabolic
disorder which is mainly characterized by hyperglycaemia and dys-
lipidaemia including hyperlipoidemia [1,2]. The underlying cause
of DM is the defective production or action of insulin. In diabetes,
the body either fails to properly respond to its own insulin or does
not make enough insulin, or both [36]. Due to the ready availability
of large quantities of calorie rich foods and the technology driven
reduction in routine daily exercise, the prevalence of diabetes has
drastically increased recently [7]. According to the International
Diabetes Federation (IDF), more than 382 million people suffering
from diabetes in 2014, and this number was estimated to increase
to 592 million by 2035 [8]. Currently, despite the presence of known
anti-diabetic medicines in the pharmaceutical market including
biguanides and sulfonylureas, DM and its related complications
has been a major medical problem, and the rapidly increasing inci-
Corresponding author.
E-mail addresses: dywang@haut.edu.cn (D. Wang), uyi zhao@163.com
(X. Zhao), liuyl7446@sina.com (Y. Liu).
all over the world [911]. Apart from contraindications and high
prices, frequently-used synthetic anti-diabetic drugs are associated
with adverse effects and even toxicity after long-term use [1214].
The potential adverse effects of these anti-diabetic drugs including
low blood sugar, stomach upset, weight gain, lactic acidosis, skin
rash or itching, tiredness or dizziness, metal taste, gas & bloating,
diarrhea, lactic acidosis, liver disease and anemia risk, swelling of
legs or ankles, etc. [1517]. These inadequacies have prompted a
continuous search for other alternative drugs and natural therapies
with low toxicity and high efciency [18,19].
As an important class of bioactive biomacromolecules obtained
from many natural plants, polysaccharides have been widely
studied in the biochemical and medical areas due to spe-
cic bioactivities, including antioxidantive, hepatoprotective,
immunostimulatory, antiviral and antitumor property [10,2022].
Furthermore, of special interest considering the increasing demand
for safe and efcient anti-diabetic agents from natural origins,
polysaccharides have often been reported to exert interesting reg-
ulatory functions in humans. For example, Zhu et al. reported
that a polysaccharide extracted from the fruit of Lycium barbarum
revealed remarkable hypoglycemic effects and insulin-sensitizing

http://dx.doi.org/10.1016/j.ijbiomac.2017.04.056
0141-8130/ 2017 Elsevier B.V. All rights reserved.
 D. Wang et al. / International Journal of Biological Macromolecules 102 (2017) 396404
activity through increasing glucose metabolism and insulin secre-
tion and promoting pancreatic cell proliferation [23]. Wang et al.
explored the hypoglycemic effect and the possible mechanism
of a sulfated polysaccharide fucoidan extracted from Saccharina
japonica, and the results suggested that fucoidan exhibited a
considerable hypoglycemic effect, possibly by stimulating pan-
creatic release of insulin and/or by reducing insulin metabolism
[24]. One polysaccharide fraction (RLP-1) obtained by purifying
the crude polysaccharides extracted from a traditional Chinese
herb Rosae Laevigatae Fructus displayed hyperlipidemia possibly
through regulating PPAR-mediated lipid metabolism [25]. Another
polysaccharide fraction, EPF2, isolated from the crude polysaccha-
rides extracted from Enteromorpha prolifera, was found to display
high hypolipidemic activity and this activity might be attributed to
its antioxidant potential [26].
Lonicera japonica Thunb. (Caprifoliaceae), known as Jin Yin
Hua or Ren Dong in China, is recognized as edible and medicinal
food [27,28]. The edible buds and owers could be made into liquor
and tea in folk diet [29,30]. In addition, the plant could also be used
as cosmetics and ornamental groundcover, and has been used for
the treatment of various diseases, including arthritis, colds, enteri-
tis, fever, infections, pains, sores, swelling, and diabetes mellitus for
over 3000 years in Chinese medicine [3133]. In Chinese medicine,
the genuine producing areas of the plant are Fengqiu County, Xinmi
City of Henan province [27,28]. Modern pharmacological stud-
ies have shown that L. japonica possesses wide pharmacological
activity, such as anti-allergic, anti-inammatory, neuroprotective
effects [3436]. Besides, one pectin of the plant owers displayed
inhibitory effect on BxPC-3 and PANC-1 pancreatic cancer cells
growth at the concentration of 1 mg/mL with inhibitory ratio
of 66.7% and 52.1%, respectively [37]. Another polysaccharide,
LJW0F2, puried from the plant owers was demonstrated to
inhibit A42 aggregation in a dose-dependent-manner, and atten-
uate the cytotoxicity induced by A42 aggregation in SH-SY5Y
neuroblastoma cells [38]. Quite interestingly, Pan et al. has demon-
strated that L. japonica could increase the content of sugar and
high-density lipoprotein-cholesterin (HDL-C) in blood, reduce the
level of serum total cholesterol (TC) and atherosclerosis index (AI)
in mice-induced by alloxan [39]. 70% ethanol extract of L. japonica
had anti-diabetic effects in type II diabetic rats via the peroxisome
proliferator-activated receptor gamma (PPAR-) regulatory action
[40]. However, until now, the hypoglycemic and hypolipidemic
effects of the polysaccharide extract from the plant havent been
detailly explored.
Accordingly, the present study was undertaken to investigate
the hypoglycemic and hypolipidemic effects of the polysaccharide
obtained from the ower buds of L. japonica (LJP) in streptozotocin
(STZ)-induced diabetic rats.
2. Materials and methods
2.1. Materials and chemicals
The ower buds of L. japonica Thunb. was obtained from Yuzhou
Chinese Medicine Market (Henan province, China), which was har-
vested from the countryside region of Fengqiu County (Henan
province, middle part of China), and identied by Prof. Fan Wen-
chang in Guangdong Food and Drug Vocational College according
to the identication standard of Pharmacopeia of China (2015 edi-
tion). Voucher specimens of the plant materials were deposited
at the College of Food Science and Technology, Henan Univer-
sity of Technology, Zhengzhou, China. -Glucosidase (EC 3.2.1.2,
100 U/mg) from Saccharomyces cerevisiae, -amylase (EC 3.2.1.1,
P3.70 U/mg) from Bacillus subtilis and STZ were purchased from
Sigma-Aldrich Co. (St. Louis, Mo, USA). Assay kits for TC, HDL-
397
C and triglyceride (TG) were purchased from Changchun Huili
Biotechnology Co., Ltd. (Changchun, China). The assay kits for
pyruvate kinase, hexokinase, alanine transaminase (ALT), aspartate
transaminase (AST), gamma-glutamyl transpeptidase (GGT), cata-
lase (CAT) superoxide dismutase (SOD), glutathione (GSH) and the
enzyme-linked immunosorbent assay (ELISA) kit for insulin were
the products of Nanjing Biotechnology Co. Ltd. (NanJing, China).
Other commercially available reagents and solvents were of the
highest grade unless otherwise indicated.
2.2. Preparation of the polysaccharides from LJP
As reported before [20,21], LJPs were extracted and puried with
some procedural adjustments. The ower buds stripped from L.
japonica was dried for 15 min in a domestic microwave oven at
900 W until a constant weight (200 g), crushed into powder using a
grinder (LX-04, Xian Jinzhen Machinery Co., Ltd. China), and soaked
in water (1:20, w/v) at for 180 min in an 80 C water bath. After
three cycles, the incorporate extraction solution was ltrated and
concentrated to 10% of the original volume with a rotary evap-
orator under reduced pressure, and then it was precipitated via
adding four times of volume of 95% (v/v) ethanol slowly by stirring
at 4 C for 24 h. The resultant polysaccharide sediment was further
rened by dissolution and precipitation three times. The rened
pellets were completely dissolved in appropriate volume of water
and intensively dialyzed for 4 days against water (cut-off Molec-
ular Weight 8000 Da). The retentate portion was deproteinized
by the freeze-thaw process (FD-1, Henan Yuhua Instrument Co.,
Ltd. China) for repeating 10 times and then followed by ltration.
Finally, the extracts were centrifuged at 3000 r/min for 10 min to
remove insoluble material and the supernatant was lyophilized
in the freeze-dry apparatus to afford the rened polysaccharide,
LJPs. After got LJPs, according to Chen et al.s method [1], the crude
extract was directly dissolved in the distilled water and separated
by a DEAE-52 cellulose anionexchange chromatography column
(300 mm 26 mm, GE Healthcare, UK). After loading with sample,
the column was eluted with distilled water, followed by stepwise
elution with increased concentration of NaCl (0.2, 1.0 and 1.5 M,
respectively) at 4 mL/5 min/1 tube. The main eluted fraction (LJP)
was collected for subsequent experiments.
2.3. Analysis of chemical characterization of LJP
As reported previously, the total carbohydrate content of LJP
was measured by the phenol-H2 SO4 colorimetric assays using
glucose as the standard. The uronic acid content was deter-
mined by the vitriol-carbazole colorimetric assays with glucuronic
acid standard. The protein content was quantied by the Brad-
ford method with bovine serum albumin (BSA) standard [20,21].
Besides, the monosaccharide composition analysis was conducted
using gas chromatography (GC) method on a HP5890 instru-
ment (Hewlett-Packard Component, USA) with a column HP-5
(30 m 0.32 mm 0.25 m) as described by Oosterveld et al. [41].
2.4. Assessment of enzymes linked to carbohydrate hydrolysis
2.4.1. -Amylase activity
The -amylase inhibition was performed using the method
previously described [42]. First, 500 L of starch solution (0.5%,
20 mM sodium phosphate buffer with 6.7 mM sodium chloride,
pH 6.9) was pre-mixed with 10 L test sample in the buffer
at different concentrations. The reaction was started by adding
10 L of 1 unit -amylase in cold distilled water to the mix-
ture. Following incubation at 65 C for 5 min, the reaction was
terminated by addition of 600 L of the DNS reagent (1% 3,5-
dinitrosalicylic acid, 12% sodium potassium tartrate in 0.4 M NaOH).
398 D. Wang et al. / International Journal of Biological Macromolecules 102 (2017) 396404
The -amylase inhibitory activity was determined at 540 nm, and
all the tests were performed in triplicate. The inhibitory activity
calculated by the following formula: -amylase inhibitory activ-
ity (%) = (1 Asample /Acontrol ) 100. Here the Acontrol and Asample are
the absorbance values of the blank sample and the test samples
checked after cooled down to ambient temperature, respectively.
Acarbose was used as the positive control.
2.4.2. -Glucosidase activity
The -glucosidase inhibitory activities of the LJP were conducted
according to the reported method previously [42]. Concisely, 5 L
of -glucosidase solution (10 units/mL, 0.1 mol/L potassium phos-
phate buffer, pH 6.9) was pre-mixed with test sample at different
concentrations in 1 mL of potassium phosphate buffer (0.1 mol/L,
pH 6.9). Following incubation at 37.5 C for 20 min, 10 L p-nitro
phenyl glucopyranoside (pNPG, 10 mmol/L) as substrate was added
to the mixture to start the reaction. The reaction was carried
out at 37 C for 10 min and stopped by adding 4 mL of 0.1 mol/L
sodium carbonate solution. The amount of released product (p-
nitro phenol, PNP) was measured at 410 nm, and the inhibitory
activity was calculated using the following equation: -glucosidase
inhibitory activity (%) = (1 Asample /Acontrol ) 100. Here the Acontrol
and Asample are the absorbance values of the blank sample and of
the tested samples, respectively. Acarbose was used as the positive
control and all the tests were conducted in triplicate.
2.5. In vivo study
2.5.1. Animals management and induction of experimental
diabetes
Healthy male Sprague-Dawley rats with a body weight of weight
200 20 g, and rodent chow composed of 40% corn our, 26% wheat
our, 10% bran, 10% sh meal, 10% bean cake, 2% mineral, 1%
coarse, and 1% vitamin complex were obtained from Experimen-
tal Animal Center of Zhengzhou University (Zhengzhou, China). All
experimental procedures of animals were approved by the Ethics
Committee for Animal Experimentation of the University, and car-
ried out in accordance with the Regulations of Experimental Animal
Administration issued by the State Committee of Science and Tech-
nology of Peoples Republic of China. All the animals were housed
under the standard conditions with 12/12 h light-dark cycle at a
temperature of 22 2 C and a humidity of 60 5%. All of them
could get tap water and rodent chow ad libitum. After one week of
acclimatization, diabetes was induced by a single intraperitoneal
injection (ip.) of STZ immediately after it dissolved in cold citrate
buffer (0.1 M, pH = 4.0) in fasted rats at a dose of 60 mg/kg body
weight. After 72 h, rats with fasting blood glucose of 250 mg/dL
or more were used for the animal study. Non-diabetic controls
received a cold citrate buffer injection. Treatment with the LJP
started on the third day after STZ injection and continued until day
42.
2.5.2. Design of animal experiments
The rats were divided into 5 groups, with 10 rats on each group.
All the rats were allowed free access to tap water rodent chow as
well. The distribution of the groups was as follows: Group 1: rats
were administered intragastrically (ig.) with physiological saline
(vehicle control) during the experimental period (normal control
group, NC). Group 2: STZ-induced diabetic rats were administered
ig. with physiological saline (model control group, STZ). Group 3:
STZ-induced diabetic rats were administered ig. with 200 mg/kg
B.W. LJP (STZ and low dose of LJP, STZ + LLJP). Group 4: STZ-induced
diabetic rats were administered ig. with 400 mg/kg B.W. LJP (STZ
and middle dose of LJP, STZ + MLJP). Group 5: STZ-induced diabetic
rats were administered ig. with 800 mg/kg B.W. LJP (STZ and high
dose of LJP, STZ + HLJP). All groups were maintained for successive
42 days with the same treatment. Liquid and food intake were mon-
itored daily, and body weight, levels of blood glucose (ACCU-CHEK
Active, Roche Diagnostics GmbH, Mannheim, Germany) were mon-
itored weekly throughout the whole experimental period. At the
end of the experiment (day 42), the rats were fasted for 14 h, and
blood samples were collected from the ophthalmic vein, incubated
at room temperature for 30 min, centrifuged and the serum samples
were stored at 70 C for further assay. Subsequently, the rats were
euthanized. The liver and skeletal muscle were harvested, washed
in ice-cold physiological saline to remove the blood, snap-frozen in
liquid nitrogen and stored at 70 C for further assay.
2.5.3. Measurement of the serum insulin levels
The serum insulin was estimated by the competitive inhibition
method of ELISA according to the kit manufacturers instructions.
The level of insulin resistance and the function of islet cells
were appraised by homeostasis model assessment (HOMA) in
accordance with Matthews et al.s method [43]. Values of blood
glucose and serum insulin were expressed in mmol/L and pmol/L,
respectively. Among them, HOMA-IR = (serum glucose serum
insulin)/22.5 [44].
2.5.4. Measurement of liver and skeletal muscle glycogen levels
Frozen liver and skeletal muscles were processed for glycogen
analysis immediately after its removal from the 70 C freezer.
Glycogen was extracted from the tissues as described [6,17]. The tis-
sue was weighed, homogenized in 33% KOH, and boiled at 100 C for
30 min with occasional stirring by automatic homogenate machine.
After cooling, 96% EtOH was added, and then, the samples were
heated to boiling and cooled in an ice bath to aid glycogen precipi-
tation. The homogenate was centrifuged at 4000 r/min for 15 min,
after the supernatant discarded, the resulting pellet was washed
and resolubilized in water. Glycogen content was assayed by treat-
ment with an iodine reagent, and the absorbance was measured
at 460 nm. The results were expressed as mg of glycogen per g of
tissue.
2.5.5. Measurement of liver pyruvate kinase and hexokinase
levels
Levels of pyruvate kinase and hexokinase in liver supernatant
were measured with the commercial enzymatic kits following the
instructions on the kits.
2.5.6. Determination of serum lipid levels
The serum TC, and TG and HDL-C concentrations were tested
by commercial enzymatic kits following the instructions on the
kits. According to Friedewalds equation, low-density lipoprotein-
cholesterol (LDL-C) levels were calculated as TC-TG/5-HDL-C,
whereas very-low-density lipoprotein-cholesterol (VLDL-C) levels
were calculated as TG/5 [45]. The atherogenic index (AI) was cal-
culated as (TC-HDL-C)/HDL-C [17].
2.5.7. Determination of oxidative stress parameters in the serum
and liver
The levels of ALT, AST and GGT in serum, and the contents of
CAT, SOD and GSH-Px in liver supernatant were tested following
the instructions on the kits.
2.6. Statistical analysis
All the experiments were performed in triplicate. Unless other-
wise indicated, the results were expressed as means SD (standard
deviation). Mean values among treatment groups were compared
by the ANOVA test. p-Values of <0.01 and <0.05 were considered as
statistically signicant.
 D. Wang et al. / International Journal of Biological Macromolecules 102 (2017) 396404
Fig. 1. DEAE-52 column chromatography of crude polysaccharides of Lonicera
japonica (LJPs). (A) Eluted with 0.2 M NaCl; (B) eluted with 1.0 M NaCl; (C) eluted
with 1.5 M NaCl.
3. Results and discussion
3.1. Physicalchemical properties of LJP
The crude polysaccharides (LJPs) was extracted from L. japonica
by means of a multistep purication procedure including hot-water
extraction and repeated ethanol precipitation. The extraction yield
of LJPs could reach about 6.9% (w/w) of the dried ower buds
using the method. After separated by a DEAE-52 column, three
polysaccharide fractions were obtained, and the second main frac-
tion (B, LJP) was selected for subsequent experiments (Fig. 1). The
total carbohydrate content and total uronic acid content of LJP
were approximately 76.1% (w/w) and 14.7% (w/w), respectively.
Moreover, the protein content of LJP was 4.6% (w/w), indicating
that the LJP was a protein bounded polysaccharide extract and the
oating protein of it was almost removed after freeze-thaw pro-
cess. Monosaccharide composition analysis exhibited that LJP was
composed of d-galactose, d-glucose, d-mannose, l-rhamnose and
d-xylose in the molar ratio of 0.46:0.32:0.25:3.71:0.27. In Fig. 1, LJP
was exhibited as a single symmetrical narrow peak, indicating that
the polysaccharide was homogeneous.
3.2. Effects of LJP on enzymes linked to carbohydrate hydrolysis
One therapeutic approach for treating diabetes is to effectively
suppress the postprandial blood sugar content by inhibiting the
activity of -amylase and -glucosidase so as to retard the absorp-
tion of glucose in the digestive tract [18,4648]. During human
digestive process, dydrolysis of dietary starch is the main source
for blood glucose, and the contribution of -amylase is a prerequi-
site for the initiation of this process. Secreted from the pancreas
and salivary glands, -amylase can digest starch by breaking it
into much smaller oligosaccharides. And then, located in the brush-
border surface membrane of intestinal cells, -glucosidase is able
to degrade the oligosaccharides into monosaccharides which are
absorbed by the intestinal epithelia that leads to the increase of
the blood glucose level [16,49,50]. Therefore, inhibitors of these
enzymes delay or prolong carbohydrate digestion time, causing
a reduction in the rate of glucose absorption and consequently
blunting the postprandial plasma glucose rise [42,51]. Currently,
inhibition of -amylase and -glucosidase is considered as one of
the most effective approaches for the treatment of diabetes mel-
litus [16,51]. In the study, the polysaccharide exhibited inhibitory
activity against both -amylase and -glucosidase with IC50 values
of 61.2 3.1 and 45.6 1.9 g/mL, respectively, while the IC50 val-
ues of acarbose in the -amylase inhibition assay and -glucosidase
399
inhibition assay were 59.4 3.3 and 39.7 2.6 g/mL, respectively.
In 2013, Zhang et al. reported that the methanol extract from the
ower buds of L. japonica showed a potent -glucosidase inhibitory
activity with maltose as a substrate in enzyme assay, where the
extract was found to be rich in polyphenols and the main pheno-
lic compounds, 3,5-dicaffeoylquinic acid, was found to be the main
maltase inhibitor in the plant [33]. Compared with polyphenols, the
polysaccharide exerted inhibitory activity against both -amylase
and -glucosidase. Therefore, the polysaccharide seems to more
interesting and expressive in the anti-diabetic study of the plant.
3.3. Effects of LJP on body weight and food and water intake
Table 1 summarised general physiological parameters: initial
and nal body weight as well as the food and water consump-
tion of all the groups of rats. With the experiment carried out,
the body weight of all the rats administrated with LJP had been
decreasing. Compared with the normal control group, all diabetic
groups presented a signicant loss of body weight at the end of
the treatment, whose weight gain was 88.9% (p < 0.01). Food and
liquid intake were apparently higher in STZ group compared to
the non-diabetic controls (p < 0.01). However, the high dose of
the polysaccharides tested in this experiment (STZ + HLJP group)
demonstrated a apparent decrease in the food and liquid intake
compared to the STZ group (p < 0.01), getting closer to the intake
levels of non-diabeticcontrol groups. The present result suggests
that the polysaccharide can effectively inhibit the STZ-induced food
as well as water intake.
3.4. Effects of LJP on serum levels of sugar, insulin, and HOMA-IR
The ower buds of L. japonica has long been frequently involved
in the treatment of DM in Chinese medicine [27]. In this study,
compared with the normal control group, STZ-induced diabetic rats
exhibited a obvious increase of glucose over the complete period
during the insulin tolerance test (p < 0.01, Table 2). Meanwhile, the
levels of plasma insulin and HOMA-IR were obviously increased in
diabetic control rats (p < 0.01). The results revealed that the oral
administration of LJP (800 mg/kg) for 6 weeks to diabetic rats not
only signicantly decreased the levels of fasting serum glucose
(p < 0.01), but also signicantly restored the changes in the level
of insulin resistance to near normal at the end of the study period
(p < 0.01). As a result, the polysaccharide could elevate fasting blood
glucose and improve insulin resistance.
In the past few years, the plant extracts were found to display
hpyerglycemic effects in alloxan- and sucrose-induced diabetic
mice [39,52]. Furthermore, as reported by Han et al., the 70%
ethanol extract of L. japonica attenuated the hyperglycemia, alle-
viated the insulin resistance, and restored the -cell damage of
pancreas in the type II diabetic rats. Moreover, the anti-diabetic
effects were strongly involved in the regulation of PPAR- activa-
tion as a potential mechanism [40]. Tzeng et al. demonstrated that
ethanol extract of L. japonica down-regulated the protein expres-
sion of p38 mitogen-activated protein kinase in the kidney of
diabetic rats [53]. Compared with the these reports, we go further
to verify the the anti-diabetic effect of the polysaccharide extracted
from the plant, so that the natural compound (s) responsible for the
hypoglycemic activity was rstly expounded. Intriguingly, take the
administration route (decoction of water) in clinic and the solubil-
ity of the polysaccharide, LJP appears to be more reliable to exert
the anti-diabetic effect of the herb medicine.
3.5. Effects of LJP on liver and skeletal muscle glycogen levels
For mammals, as the primary intracellular form of storable glu-
cose, glycogen is mainly stored in skeletal muscles and the liver for
400 D. Wang et al. / International Journal of Biological Macromolecules 102 (2017) 396404

Table 1
Effects of LJP on body weight and food and water intake of rats.a

Body Weight (g) Food Intake Liquid Intake

Initial Final (g/rat/day) (mL/rat/day)

NC 200.4 7.1 378.5 15.4 22.3 4.3 37.2 3.8

STZ 205.5 7.1 159.7 18.8b 34.7 2.4b 223.4 11.9b
STZ + LLJP 203.5 9.1 177.7 14.9b 31.8 2.7 255.8 9.9
STZ + MLJP 206.4 9.7 193.6 10.1b 31.4 3.3 195.1 5.8
STZ + HLJP 209.2 7.3 157.9 20.1b 22.3 2.3c 43.2 9.7c
a
Values are expressed as means SD (n = 10).
b
As compared to normal group: p < 0.01.
c
As compared with STZ-induced diabetic group: p < 0.01.

Table 2
Effects of LJP on fasting serum glucose and insulin concentrations, and insulin sensitivity index of STZ-induced rats at the end of week 6.a

Blood Sugar Insulin HOMA-IR

0 day 7th day 14th day 21th day 28th day 35th day 42th day 42th day 42th day

NC 5.6 0.1 5.6 0.1 5.7 0.1 5.6 0.1 5.9 0.2 5.9 0.3 6.0 0.2 24.4 0.8 6.5 0.7
STZ 18.9 0.2b 19.2 0.2b 19.3 0.3b 19.3 0.2b 19.3 0.2b 19.8 0.3b 19.9 0.2b 36.8 1.3b 32.5 2.9b
STZ + LLJP 18.8 0.2 19.0 0.1 19.0 0.1 19.3 0.1 18.4 0.2 18.2 0.2 17.8 0.1 32.5 0.9 25.7 1.6
STZ + MLJP 18.9 0.1 19.2 0.2 19.1 0.2 19.0 0.2 16.6 0.1 14.0 0.1d 13.2 0.2c 29.6 0.7d 17.4 2.5d
STZ + HLJP 19.2 0.1 18.5 0.2 17.1 0.3 16.0 0.3d 15.2 0.3d 13.1 0.2c 11.4 0.1c 26.1 0.7c 13.2 1.6c
a
Values are expressed as means SD (n = 10). Values of glucose and insulin are expressed in mmol/L and mU/L, respectively.
b
As compared to normal group: p < 0.01.
c
As compared with STZ-induced diabetic group: p < 0.01.
d
As compared with STZ-induced diabetic group: p < 0.05.

Fig. 2. Effects of LJP on hepatic and skeletal muscle glycogen after administration of STZ in rats. After the simplex treatment of STZ (60 mg/kg, ip), animals were given LJP
(200, 400, or 800 mg/kg, ig) once daily for 42 successive days, and then hepatic (A) and skeletal muscle glycogen (B) were determined. Values are expressed as means SD
for 10 rats in each group. Compared to normal group, # p < 0.05. Compared to the STZ-induced diabetic group, * p < 0.05, ** p < 0.01.

future use. Therefore, most of glucose disposal occurs in the liver storage in the liver and skeletal muscle. On the basis of these nd-
and skeletal muscle, and glucose homeostasis is mainly regulated ings, we hypothesized that LJP may modulate the critical enzymes
by the liver and skeletal muscle [6,17]. As showed in Fig. 2, hep- of glycolysis and glycogen synthesis in the liver and skeletal mus-
atic and muscle glycogen contents of all the diabetic rats (9.4 1.5 cle, and this could be the reection of insulin-sensitizing effect in
and 1.2 0.1 mg/g, respectively) were markedly lower than those the hepatic and skeletal muscle tissues [55,56].
of normal controlled rats (p < 0.05; 15.2 1.6 and 2.0 0.2 mg/g,
respectively). However, the groups treated with the 800 mg/kg
3.6. Effects of LJP on liver pyruvate kinase and hexokinase levels
polysaccharide, LJP, showed much higher glycogen concentra-
tions than model controlled group, with 15.3 2.0 (p < 0.01) and
As reported, insulin resistance is closely associated with
2.0 0.2 mg/kg (p < 0.05), respectively. As reported, the glycogen
decreased glucose utilization and down-regulation of hepatic
levels in various tissues are a direct reection of insulin sensitivity,
glycolytic enzymes expression, such as pyruvate kinase and hex-
as insulin promotes intracellular glycogen deposition. The insulin-
okinase. Thus, an increase in glucose utilization by increasing the
stimulated glycogen content is markedly reduced in animals with
activity of pyruvate kinase and hexokinase could alleviate insulin
insulin resistance, in proportion to the degree of insulin deciency
resistance [12]. As displayed in Fig. 3, compared with that of the nor-
[17,54]. Interestingly, LJP exhibited benecial effects on glycogen
mal control rats (124.5 7.5, 11.4 1.3 U/g protein, respectively),
 D. Wang et al. / International Journal of Biological Macromolecules 102 (2017) 396404
Fig. 3. Effects of LJP on liver pyruvate kinase and hexokinase after administration of STZ in rats. After the simplex treatment of STZ (60 mg/kg, ip), animals were given LJP
(200, 400, or 800 mg/kg, ig) once daily for 42 successive days, and then liver pyruvate kinase (A) and hexokinase (B) were determined. Values are expressed as means SD
for 10 rats in each group. Compared to normal group, # p < 0.05. Compared to the STZ-induced diabetic group, * p < 0.05, ** p < 0.01.
the model control rats apparently decreased the activity of pyruvate
kinase and hexokinase with 37.6 5.8, 5.0 0.8 U/g protein, respec-
tively (p < 0.05). Administration of LJP to diabetic rats resulted in
the restoration of the pyruvate kinase content (107.7 6.9 U/g pro-
tein, p < 0.01), along with a obvious recovery of the activity of the
hexokinase (8.8 0.7 U/g protein, p < 0.05).
Pyruvate kinase and hexokinase are key enzymes in glucose
metabolism. The former is an important enzyme in maintaining
glucose homeostasis and is considered as a marker in regulat-
ing glucose hepatic release/uptake according to glycaemia level,
while the latter is a rate-limiting enzyme for aerobic oxidation
of glucose [12,23]. As reported, the decreased level of insulin
could give rise to the impairment in the activity of them in dia-
betic mice [10,12,23,55]. Besides, previous studies on regulation
of L-type pyruvate kinase gene expression by dietary fructose in
normal and diabetic rats conrmed that some polysaccharides
could slightly up-regulate pyruvate kinase gene transcription and
markedly up-regulate pyruvate kinase gene transcription accom-
panied with insulin treatment [23]. In agreement with them, we
observed apparent decreases in hepatic pyruvate kinase and hex-
okinase activities in untreated rats, which was probably due to the
insensitivity of cells to insulin. Administration of LJP rats led to sig-
nicant increase in the activities of pyruvate kinase and hexokinase.
The increased activities of pyruvate kinase and hexokinase may
contribute to the restoration of serum glucose and insulin levels in
LJP treated rats.
3.7. Effects of LJP on serum lipid prole
In diabetic condition, as we know, abnormal lipid metabolism,
always leading to accumulation of plasma TC, TG, LDL-C and VLDL-
C as well as decreased HDL-C. Elevated levels of TC, TG, LDL-C,
and VLDL-C are considered as major risk factors for cardiovascu-
lar disease, such as coronary heart disease (hyperlipidaemia) and
its incidence of atherosclerosis [14,15,46,57,58]. Conversely, HDL-C
plays a key role in cholesterol transport from the periphery to the
liver reduces the risk of cardiovascular disease [17,59].
In the study, as exhibited in Table 3, the results displayed
that the levels of plasma lipid prole including TC, TG, LDL-
C and VLDC-C were markedly elevated from 1.6 0.2, 1.6 0.1,
401
0.44 0.13 and 1.02 0.28 mmol/L of NC group rats to 3.0 0.1,
3.4 0.2, 1.60 0.13 and 2.08 0.38 mmol/L of STZ group (p < 0.05),
while HDL-C was markedly reduced from 0.79 0.12 mmol/L to
0.71 0.13 mmol/L (p < 0.05). However, the levels of plasma TG, TC,
LDL-C and VLDL-C were signicantly reduced to 1.6 0.2, 1.7 0.2,
0.47 0.07 and 1.03 0.32 mmol/L in diabetic rat administrated
with 800 mg/kg LJP (STZ + HLJP group, p < 0.05), while HDL-C was
signicantly elevated to 0.79 0.07 mmol/L (p < 0.05). That means
after treated with LJP for 42 days, levels of TC, TG, LDL-C and VLDL-
C in diabetic SD rats were decreased while HDL-C levels were
increased, indicating that LJP had benecial effect on dyslipidemia.
Furthermore, the atherogenic index, markedly increased from
1.02 0.23 in normal control rats to 3.30 0.76 in STZ-induced dia-
betic rats (p < 0.05). After treated with 800 mg/kg LJP (STZ + HLJP
group), it signicantly descended to normal state with 1.06 0.22
(p < 0.05), revealing that the polysaccharide may potently decrease
the risk of atherosclerosis, coronary heart disease and other diabetic
complications. As displayed before, in Pan et al.s investigation, the
water extract of L. japonica was found to increase of the level of
HDL-C, reduce the level of TC in serum and AI in alloxan-induced
diabetic mice [39]. However, in Wang et al.s study, the extract I
of L. japonica was found to reduce the levels of TG in serum and
liver, while it produced no effect upon the levels of TC, LDL-C and
HDL-C in hyperlipidaemia mice and rats, and the extract II couldnt
affect the levels of blood lipid and fat [52]. The results here dis-
played that the polysaccharide not only decended the levels of TC,
TG, LDL-C and VLDL-C, and AI, but also ascended the level of HDL-
C in blood of STZ-induced diabetic rats. Therefore, the observed
reduction in blood glucose levels (Table 2), improvement of plasma
lipid levels and atherogenic indices (Table 3) in LJP-supplemented
STZ-induced diabetic rats may demonstrate antihyperglycemic and
hypolipidemic activity of LJP.
3.8. Effects of LJP on oxidative stress parameters in the serum and
liver
Recently, plenty of researches have demonstrated that dia-
betes mellitus is typically associated with increased generation
of free radicals or impaired antioxidant defence mechanism and
pathological consequences of diabetes. Increased oxidative stress
402 D. Wang et al. / International Journal of Biological Macromolecules 102 (2017) 396404

Table 3
Effects of LJP on serum levels of TC, TG, HDL-C, LDL-C and VLDL-C for lipid prole, and AI of rats.a

TC (mmol/L) TG (mmol/L) HDL-C (mmol/L) LDL-C (mmol/L) VLDL-C (mmol/L) AI

NC 1.6 0.2 1.6 0.1 0.79 0.12 0.44 0.13 1.02 0.28 1.02 0.23
STZ 3.0 0.1b 3.4 0.2b 0.71 0.13b 1.60 0.13b 2.08 0.38b 3.30 0.76b
STZ + LLJP 2.7 0.3 3.2 0.1 0.72 0.09 1.38 0.26 1.94 0.34 2.84 0.45
STZ + MLJP 2.0 0.3 2.5 0.2 0.74 0.08 0.80 0.26 1.52 0.35 1.78 0.50
STZ + HLJP 1.6 0.2c 1.7 0.2c 0.79 0.07c 0.47 0.07c 1.03 0.32c 1.06 0.22c
a
Values are expressed as means SD (n = 10).
b
As compared to normal group: p < 0.05.
c
As compared with STZ-induced diabetic group: p < 0.05.

Table 4
Effects of LJP on serum levels of ALT, AST and GGT, and hepatic levels of CAT, SOD and GSH for oxidative stress parameters of rats.a

Serum Liver

ALT (IU/L) AST(IU/L) GGT(IU/L) CAT (U/mg protein) SOD (U/mg protein) GSH (mg/g protein)

NC 41.9 5.5 79.7 9.6 36.1 4.9 83.1 7.5 302.9 16.7 9.8 0.4
STZ 175.2 10.8b 377.7 17.7b 137.5 12.4b 29.5 8.0b 169.0 16.1b 4.5 0.5b
STZ + LLJP 168.0 16.6 298.8 28.6 136.1 18.8 32.1 8.5 179.3 8.8 4.6 0.5
STZ + MLJP 92.1 6.8d 159.6 27.2d 82.2 11.3d 55.2 9.7 218.4 22.6d 7.2 0.7
STZ + HLJP 48.7 7.7c 81.9 15.1c 38.3 4.6c 84.5 8.2c 300.9 23.0c 9.8 0.7d
a
Values are expressed as means SD (n = 10).
b
As compared to normal group: p < 0.01.
c
As compared with STZ-induced diabetic group: p < 0.01.
d
As compared with STZ-induced diabetic group: p < 0.05.

is a widely accepted participant in the development and progres-
sion of diabetes and its complications [3,12,60,61]. Furthermore,
the imbalance of oxidative stress in liver has been found to con-
tribute to the glucose metabolism and to be associated with
the onset and development of DM [62,63]. Thus, the regulation
of damaging ROS levels in DM by antioxidants and free rad-
ical scavengers represents an attractive benet during the its
treatment [64]. In this context, the reducing levels of antioxi-
dant enzymes including ALT, AST and GGT in serum, and the
improving levels of CAT, SOD and GSH in liver could increase
the response of antioxidant defense systems to oxidative stress
(Table 4). Compared with the NC group (41.9 5.5, 79.7 9.6
and 36.1 4.9 IU/L, respectively), the levels of ALT (175.2 10.8
IU/L), AST (377.7 17.7 IU/L) and GGT (137.5 12.4 IU/L) signif-
icantly increased (p < 0.01) in the STZ group, while in SZT + HLJP
group, these levels were signicantly decreased (p < 0.01) with
48.7 7.7, 81.9 15.1, 38.3 4.6 IU/L, respectively, compared to
the STZ rats. Conversely, compared with the NC group (83.1 7.5,
302.9 16.7 U/mg protein and 9.8 0.4 mg/g protein, respectively),
the levels of CAT (29.5 8.0 U/mg protein), SOD (169.0 16.1 U/mg
protein) and GSH (4.5 0.5 mg/g protein) dramatically declined
(p < 0.01) in the STZ group, while in SZT + HLJP group, they were
dramatically ascended with 84.5 8.2, 300.9 23.0 U/mg pro-
tein (p < 0.01) and 9.8 0.7 mg/g protein (p < 0.05), respectively,
compared to the STZ rats. These results displayed that the admin-
istration of the polysaccharide dose-dependently and signicantly
restored the dramatic changes of these levels of antioxidants in
diabetic rats.
Hsu et al. have investigated the antioxidant activities of the
L. japonica ower bud extracts prepared by water, ethanol and
supercritical uid extraction (SFE) techniques. Compared with the
extracts obtained by ethanol and SFE, the water extract was found
to possess much higher activities, with higher contents of polyphe-
nols and avonoids and higher amount of chlorogenic acid and
luteolin-7-O-glucoside [65]. During the evauation for the antioxi-
dant activities of ethanol extracts of owers, leaves and branches of
L. japonica, all the extracts have strong quenched DPPH free radical,
while ower extract revealed the most potential DPPH radical-
scavenging capacity [66]. Also, the strong antioxidant activity of the
extracts were attributed to the high phenolic contents. Interesingly,
the study demonstrated the antioxidant activity of the polysaccha-
ride extracted from the plant in vivo for the rst time. The oral
administration of the polysacharides for 42 days caused obviously
decreases of ALT, AST and GGT activity in serum and decreases of
CAT, SOD and GSH levels in liver. Therefore, in accordance with
others, the antioxidative stress might be one of the underlying
mechanisms for LJP to alleviate the high blood glucose status in
diabetic rats [3,12,6063].
Taken altogether, our data herein suggest that the polysac-
charide fraction produced by L. japonica has a clear potential to
improve diabetes conditions and potentially prevent its multiple
complications through its ability to: (1) decrease hyperglycaemia,
(2) decrease hyperlipidaemia, (3) increase antioxidant capacity in
serum and liver. Currently, the specic mechanism to illuminate
these possible health benecial properties at both functional and
molecular levels is under investigation.
4. Conclusion
In smmary, according to our present ndings, our study demon-
strated that LJP, the main fraction of polysaccharides extracted from
the ower buds of L. japonica, displayed high inhibitory activies
against -amylase and -glucosidase in vitro. While in in vivo
assays, LJP revealed a potent hypoglycemic effect on STZ-induced
diabetic rats for its ability to reduce glucose levels and relieve
insulin resistance in blood, as well as ameliorate lipid metabolism
and oxidative stress. Further studies in animal models and human
volunteers need to be done to substantiate these ndings. The
results suggest that LJP could be considered as a potential candidate
for developing an ingredient of functional foods in the treatment
of diabetes and its complications.
Conict of interest
The authors declare no conict of interest.
Acknowledgements
This study was supported by the National Key Specialty
Construction of Clinical Project of China (081619232833) and
 D. Wang et al. / International Journal of Biological Macromolecules 102 (2017) 396404
Key Project in Science and Technology of Henan Province
(142102310417), as well as the Doctor Research Fund of Henan
University of Technology (2016BS035).
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