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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


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

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 397

activity through increasing glucose metabolism and insulin secre- C and triglyceride (TG) were purchased from Changchun Huili
tion and promoting pancreatic cell proliferation [23]. Wang et al. Biotechnology Co., Ltd. (Changchun, China). The assay kits for
explored the hypoglycemic effect and the possible mechanism pyruvate kinase, hexokinase, alanine transaminase (ALT), aspartate
of a sulfated polysaccharide fucoidan extracted from Saccharina transaminase (AST), gamma-glutamyl transpeptidase (GGT), cata-
japonica, and the results suggested that fucoidan exhibited a lase (CAT) superoxide dismutase (SOD), glutathione (GSH) and the
considerable hypoglycemic effect, possibly by stimulating pan- enzyme-linked immunosorbent assay (ELISA) kit for insulin were
creatic release of insulin and/or by reducing insulin metabolism the products of Nanjing Biotechnology Co. Ltd. (NanJing, China).
[24]. One polysaccharide fraction (RLP-1) obtained by purifying Other commercially available reagents and solvents were of the
the crude polysaccharides extracted from a traditional Chinese highest grade unless otherwise indicated.
herb Rosae Laevigatae Fructus displayed hyperlipidemia possibly
through regulating PPAR-mediated lipid metabolism [25]. Another 2.2. Preparation of the polysaccharides from LJP
polysaccharide fraction, EPF2, isolated from the crude polysaccha-
rides extracted from Enteromorpha prolifera, was found to display As reported before [20,21], LJPs were extracted and puried with
high hypolipidemic activity and this activity might be attributed to some procedural adjustments. The ower buds stripped from L.
its antioxidant potential [26]. japonica was dried for 15 min in a domestic microwave oven at
Lonicera japonica Thunb. (Caprifoliaceae), known as Jin Yin 900 W until a constant weight (200 g), crushed into powder using a
Hua or Ren Dong in China, is recognized as edible and medicinal grinder (LX-04, Xian Jinzhen Machinery Co., Ltd. China), and soaked
food [27,28]. The edible buds and owers could be made into liquor in water (1:20, w/v) at for 180 min in an 80 C water bath. After
and tea in folk diet [29,30]. In addition, the plant could also be used three cycles, the incorporate extraction solution was ltrated and
as cosmetics and ornamental groundcover, and has been used for concentrated to 10% of the original volume with a rotary evap-
the treatment of various diseases, including arthritis, colds, enteri- orator under reduced pressure, and then it was precipitated via
tis, fever, infections, pains, sores, swelling, and diabetes mellitus for adding four times of volume of 95% (v/v) ethanol slowly by stirring
over 3000 years in Chinese medicine [3133]. In Chinese medicine, at 4 C for 24 h. The resultant polysaccharide sediment was further
the genuine producing areas of the plant are Fengqiu County, Xinmi rened by dissolution and precipitation three times. The rened
City of Henan province [27,28]. Modern pharmacological stud- pellets were completely dissolved in appropriate volume of water
ies have shown that L. japonica possesses wide pharmacological and intensively dialyzed for 4 days against water (cut-off Molec-
activity, such as anti-allergic, anti-inammatory, neuroprotective ular Weight 8000 Da). The retentate portion was deproteinized
effects [3436]. Besides, one pectin of the plant owers displayed by the freeze-thaw process (FD-1, Henan Yuhua Instrument Co.,
inhibitory effect on BxPC-3 and PANC-1 pancreatic cancer cells Ltd. China) for repeating 10 times and then followed by ltration.
growth at the concentration of 1 mg/mL with inhibitory ratio Finally, the extracts were centrifuged at 3000 r/min for 10 min to
of 66.7% and 52.1%, respectively [37]. Another polysaccharide, remove insoluble material and the supernatant was lyophilized
LJW0F2, puried from the plant owers was demonstrated to in the freeze-dry apparatus to afford the rened polysaccharide,
inhibit A42 aggregation in a dose-dependent-manner, and atten- LJPs. After got LJPs, according to Chen et al.s method [1], the crude
uate the cytotoxicity induced by A42 aggregation in SH-SY5Y extract was directly dissolved in the distilled water and separated
neuroblastoma cells [38]. Quite interestingly, Pan et al. has demon- by a DEAE-52 cellulose anionexchange chromatography column
strated that L. japonica could increase the content of sugar and (300 mm 26 mm, GE Healthcare, UK). After loading with sample,
high-density lipoprotein-cholesterin (HDL-C) in blood, reduce the the column was eluted with distilled water, followed by stepwise
level of serum total cholesterol (TC) and atherosclerosis index (AI) elution with increased concentration of NaCl (0.2, 1.0 and 1.5 M,
in mice-induced by alloxan [39]. 70% ethanol extract of L. japonica respectively) at 4 mL/5 min/1 tube. The main eluted fraction (LJP)
had anti-diabetic effects in type II diabetic rats via the peroxisome was collected for subsequent experiments.
proliferator-activated receptor gamma (PPAR-) regulatory action
[40]. However, until now, the hypoglycemic and hypolipidemic 2.3. Analysis of chemical characterization of LJP
effects of the polysaccharide extract from the plant havent been
detailly explored. As reported previously, the total carbohydrate content of LJP
Accordingly, the present study was undertaken to investigate was measured by the phenol-H2 SO4 colorimetric assays using
the hypoglycemic and hypolipidemic effects of the polysaccharide glucose as the standard. The uronic acid content was deter-
obtained from the ower buds of L. japonica (LJP) in streptozotocin mined by the vitriol-carbazole colorimetric assays with glucuronic
(STZ)-induced diabetic rats. 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
2. Materials and methods using gas chromatography (GC) method on a HP5890 instru-
ment (Hewlett-Packard Component, USA) with a column HP-5
2.1. Materials and chemicals (30 m 0.32 mm 0.25 m) as described by Oosterveld et al. [41].

The ower buds of L. japonica Thunb. was obtained from Yuzhou 2.4. Assessment of enzymes linked to carbohydrate hydrolysis
Chinese Medicine Market (Henan province, China), which was har-
vested from the countryside region of Fengqiu County (Henan 2.4.1. -Amylase activity
province, middle part of China), and identied by Prof. Fan Wen- The -amylase inhibition was performed using the method
chang in Guangdong Food and Drug Vocational College according previously described [42]. First, 500 L of starch solution (0.5%,
to the identication standard of Pharmacopeia of China (2015 edi- 20 mM sodium phosphate buffer with 6.7 mM sodium chloride,
tion). Voucher specimens of the plant materials were deposited pH 6.9) was pre-mixed with 10 L test sample in the buffer
at the College of Food Science and Technology, Henan Univer- at different concentrations. The reaction was started by adding
sity of Technology, Zhengzhou, China. -Glucosidase (EC 3.2.1.2, 10 L of 1 unit -amylase in cold distilled water to the mix-
100 U/mg) from Saccharomyces cerevisiae, -amylase (EC 3.2.1.1, ture. Following incubation at 65 C for 5 min, the reaction was
P3.70 U/mg) from Bacillus subtilis and STZ were purchased from terminated by addition of 600 L of the DNS reagent (1% 3,5-
Sigma-Aldrich Co. (St. Louis, Mo, USA). Assay kits for TC, HDL- 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 42 days with the same treatment. Liquid and food intake were mon-
all the tests were performed in triplicate. The inhibitory activity itored daily, and body weight, levels of blood glucose (ACCU-CHEK
calculated by the following formula: -amylase inhibitory activ- Active, Roche Diagnostics GmbH, Mannheim, Germany) were mon-
ity (%) = (1 Asample /Acontrol ) 100. Here the Acontrol and Asample are itored weekly throughout the whole experimental period. At the
the absorbance values of the blank sample and the test samples end of the experiment (day 42), the rats were fasted for 14 h, and
checked after cooled down to ambient temperature, respectively. blood samples were collected from the ophthalmic vein, incubated
Acarbose was used as the positive control. at room temperature for 30 min, centrifuged and the serum samples
were stored at 70 C for further assay. Subsequently, the rats were
2.4.2. -Glucosidase activity euthanized. The liver and skeletal muscle were harvested, washed
The -glucosidase inhibitory activities of the LJP were conducted in ice-cold physiological saline to remove the blood, snap-frozen in
according to the reported method previously [42]. Concisely, 5 L liquid nitrogen and stored at 70 C for further assay.
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 2.5.3. Measurement of the serum insulin levels
concentrations in 1 mL of potassium phosphate buffer (0.1 mol/L, The serum insulin was estimated by the competitive inhibition
pH 6.9). Following incubation at 37.5 C for 20 min, 10 L p-nitro method of ELISA according to the kit manufacturers instructions.
phenyl glucopyranoside (pNPG, 10 mmol/L) as substrate was added The level of insulin resistance and the function of islet cells
to the mixture to start the reaction. The reaction was carried were appraised by homeostasis model assessment (HOMA) in
out at 37 C for 10 min and stopped by adding 4 mL of 0.1 mol/L accordance with Matthews et al.s method [43]. Values of blood
sodium carbonate solution. The amount of released product (p- glucose and serum insulin were expressed in mmol/L and pmol/L,
nitro phenol, PNP) was measured at 410 nm, and the inhibitory respectively. Among them, HOMA-IR = (serum glucose serum
activity was calculated using the following equation: -glucosidase insulin)/22.5 [44].
inhibitory activity (%) = (1 Asample /Acontrol ) 100. Here the Acontrol
and Asample are the absorbance values of the blank sample and of 2.5.4. Measurement of liver and skeletal muscle glycogen levels
the tested samples, respectively. Acarbose was used as the positive Frozen liver and skeletal muscles were processed for glycogen
control and all the tests were conducted in triplicate. analysis immediately after its removal from the 70 C freezer.
Glycogen was extracted from the tissues as described [6,17]. The tis-
2.5. In vivo study sue was weighed, homogenized in 33% KOH, and boiled at 100 C for
30 min with occasional stirring by automatic homogenate machine.
2.5.1. Animals management and induction of experimental After cooling, 96% EtOH was added, and then, the samples were
diabetes heated to boiling and cooled in an ice bath to aid glycogen precipi-
Healthy male Sprague-Dawley rats with a body weight of weight tation. The homogenate was centrifuged at 4000 r/min for 15 min,
200 20 g, and rodent chow composed of 40% corn our, 26% wheat after the supernatant discarded, the resulting pellet was washed
our, 10% bran, 10% sh meal, 10% bean cake, 2% mineral, 1% and resolubilized in water. Glycogen content was assayed by treat-
coarse, and 1% vitamin complex were obtained from Experimen- ment with an iodine reagent, and the absorbance was measured
tal Animal Center of Zhengzhou University (Zhengzhou, China). All at 460 nm. The results were expressed as mg of glycogen per g of
experimental procedures of animals were approved by the Ethics tissue.
Committee for Animal Experimentation of the University, and car-
ried out in accordance with the Regulations of Experimental Animal
2.5.5. Measurement of liver pyruvate kinase and hexokinase
Administration issued by the State Committee of Science and Tech-
levels
nology of Peoples Republic of China. All the animals were housed
Levels of pyruvate kinase and hexokinase in liver supernatant
under the standard conditions with 12/12 h light-dark cycle at a
were measured with the commercial enzymatic kits following the
temperature of 22 2 C and a humidity of 60 5%. All of them
instructions on the kits.
could get tap water and rodent chow ad libitum. After one week of
acclimatization, diabetes was induced by a single intraperitoneal
2.5.6. Determination of serum lipid levels
injection (ip.) of STZ immediately after it dissolved in cold citrate
The serum TC, and TG and HDL-C concentrations were tested
buffer (0.1 M, pH = 4.0) in fasted rats at a dose of 60 mg/kg body
by commercial enzymatic kits following the instructions on the
weight. After 72 h, rats with fasting blood glucose of 250 mg/dL
kits. According to Friedewalds equation, low-density lipoprotein-
or more were used for the animal study. Non-diabetic controls
cholesterol (LDL-C) levels were calculated as TC-TG/5-HDL-C,
received a cold citrate buffer injection. Treatment with the LJP
whereas very-low-density lipoprotein-cholesterol (VLDL-C) levels
started on the third day after STZ injection and continued until day
were calculated as TG/5 [45]. The atherogenic index (AI) was cal-
42.
culated as (TC-HDL-C)/HDL-C [17].
2.5.2. Design of animal experiments
The rats were divided into 5 groups, with 10 rats on each group. 2.5.7. Determination of oxidative stress parameters in the serum
All the rats were allowed free access to tap water rodent chow as and liver
well. The distribution of the groups was as follows: Group 1: rats The levels of ALT, AST and GGT in serum, and the contents of
were administered intragastrically (ig.) with physiological saline CAT, SOD and GSH-Px in liver supernatant were tested following
(vehicle control) during the experimental period (normal control the instructions on the kits.
group, NC). Group 2: STZ-induced diabetic rats were administered
ig. with physiological saline (model control group, STZ). Group 3: 2.6. Statistical analysis
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 All the experiments were performed in triplicate. Unless other-
diabetic rats were administered ig. with 400 mg/kg B.W. LJP (STZ wise indicated, the results were expressed as means SD (standard
and middle dose of LJP, STZ + MLJP). Group 5: STZ-induced diabetic deviation). Mean values among treatment groups were compared
rats were administered ig. with 800 mg/kg B.W. LJP (STZ and high by the ANOVA test. p-Values of <0.01 and <0.05 were considered as
dose of LJP, STZ + HLJP). All groups were maintained for successive statistically signicant.
D. Wang et al. / International Journal of Biological Macromolecules 102 (2017) 396404 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,
Fig. 1. DEAE-52 column chromatography of crude polysaccharides of Lonicera the body weight of all the rats administrated with LJP had been
japonica (LJPs). (A) Eluted with 0.2 M NaCl; (B) eluted with 1.0 M NaCl; (C) eluted decreasing. Compared with the normal control group, all diabetic
with 1.5 M NaCl. 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
3. Results and discussion liquid intake were apparently higher in STZ group compared to
the non-diabetic controls (p < 0.01). However, the high dose of
3.1. Physicalchemical properties of LJP the polysaccharides tested in this experiment (STZ + HLJP group)
demonstrated a apparent decrease in the food and liquid intake
The crude polysaccharides (LJPs) was extracted from L. japonica compared to the STZ group (p < 0.01), getting closer to the intake
by means of a multistep purication procedure including hot-water levels of non-diabeticcontrol groups. The present result suggests
extraction and repeated ethanol precipitation. The extraction yield that the polysaccharide can effectively inhibit the STZ-induced food
of LJPs could reach about 6.9% (w/w) of the dried ower buds as well as water intake.
using the method. After separated by a DEAE-52 column, three
polysaccharide fractions were obtained, and the second main frac- 3.4. Effects of LJP on serum levels of sugar, insulin, and HOMA-IR
tion (B, LJP) was selected for subsequent experiments (Fig. 1). The
total carbohydrate content and total uronic acid content of LJP The ower buds of L. japonica has long been frequently involved
were approximately 76.1% (w/w) and 14.7% (w/w), respectively. in the treatment of DM in Chinese medicine [27]. In this study,
Moreover, the protein content of LJP was 4.6% (w/w), indicating compared with the normal control group, STZ-induced diabetic rats
that the LJP was a protein bounded polysaccharide extract and the exhibited a obvious increase of glucose over the complete period
oating protein of it was almost removed after freeze-thaw pro- during the insulin tolerance test (p < 0.01, Table 2). Meanwhile, the
cess. Monosaccharide composition analysis exhibited that LJP was levels of plasma insulin and HOMA-IR were obviously increased in
composed of d-galactose, d-glucose, d-mannose, l-rhamnose and diabetic control rats (p < 0.01). The results revealed that the oral
d-xylose in the molar ratio of 0.46:0.32:0.25:3.71:0.27. In Fig. 1, LJP administration of LJP (800 mg/kg) for 6 weeks to diabetic rats not
was exhibited as a single symmetrical narrow peak, indicating that only signicantly decreased the levels of fasting serum glucose
the polysaccharide was homogeneous. (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
3.2. Effects of LJP on enzymes linked to carbohydrate hydrolysis (p < 0.01). As a result, the polysaccharide could elevate fasting blood
glucose and improve insulin resistance.
One therapeutic approach for treating diabetes is to effectively In the past few years, the plant extracts were found to display
suppress the postprandial blood sugar content by inhibiting the hpyerglycemic effects in alloxan- and sucrose-induced diabetic
activity of -amylase and -glucosidase so as to retard the absorp- mice [39,52]. Furthermore, as reported by Han et al., the 70%
tion of glucose in the digestive tract [18,4648]. During human ethanol extract of L. japonica attenuated the hyperglycemia, alle-
digestive process, dydrolysis of dietary starch is the main source viated the insulin resistance, and restored the -cell damage of
for blood glucose, and the contribution of -amylase is a prerequi- pancreas in the type II diabetic rats. Moreover, the anti-diabetic
site for the initiation of this process. Secreted from the pancreas effects were strongly involved in the regulation of PPAR- activa-
and salivary glands, -amylase can digest starch by breaking it tion as a potential mechanism [40]. Tzeng et al. demonstrated that
into much smaller oligosaccharides. And then, located in the brush- ethanol extract of L. japonica down-regulated the protein expres-
border surface membrane of intestinal cells, -glucosidase is able sion of p38 mitogen-activated protein kinase in the kidney of
to degrade the oligosaccharides into monosaccharides which are diabetic rats [53]. Compared with the these reports, we go further
absorbed by the intestinal epithelia that leads to the increase of to verify the the anti-diabetic effect of the polysaccharide extracted
the blood glucose level [16,49,50]. Therefore, inhibitors of these from the plant, so that the natural compound (s) responsible for the
enzymes delay or prolong carbohydrate digestion time, causing hypoglycemic activity was rstly expounded. Intriguingly, take the
a reduction in the rate of glucose absorption and consequently administration route (decoction of water) in clinic and the solubil-
blunting the postprandial plasma glucose rise [42,51]. Currently, ity of the polysaccharide, LJP appears to be more reliable to exert
inhibition of -amylase and -glucosidase is considered as one of the anti-diabetic effect of the herb medicine.
the most effective approaches for the treatment of diabetes mel-
litus [16,51]. In the study, the polysaccharide exhibited inhibitory 3.5. Effects of LJP on liver and skeletal muscle glycogen levels
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- For mammals, as the primary intracellular form of storable glu-
ues of acarbose in the -amylase inhibition assay and -glucosidase 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 401

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 0.44 0.13 and 1.02 0.28 mmol/L of NC group rats to 3.0 0.1,
kinase and hexokinase with 37.6 5.8, 5.0 0.8 U/g protein, respec- 3.4 0.2, 1.60 0.13 and 2.08 0.38 mmol/L of STZ group (p < 0.05),
tively (p < 0.05). Administration of LJP to diabetic rats resulted in while HDL-C was markedly reduced from 0.79 0.12 mmol/L to
the restoration of the pyruvate kinase content (107.7 6.9 U/g pro- 0.71 0.13 mmol/L (p < 0.05). However, the levels of plasma TG, TC,
tein, p < 0.01), along with a obvious recovery of the activity of the LDL-C and VLDL-C were signicantly reduced to 1.6 0.2, 1.7 0.2,
hexokinase (8.8 0.7 U/g protein, p < 0.05). 0.47 0.07 and 1.03 0.32 mmol/L in diabetic rat administrated
Pyruvate kinase and hexokinase are key enzymes in glucose with 800 mg/kg LJP (STZ + HLJP group, p < 0.05), while HDL-C was
metabolism. The former is an important enzyme in maintaining signicantly elevated to 0.79 0.07 mmol/L (p < 0.05). That means
glucose homeostasis and is considered as a marker in regulat- after treated with LJP for 42 days, levels of TC, TG, LDL-C and VLDL-
ing glucose hepatic release/uptake according to glycaemia level, C in diabetic SD rats were decreased while HDL-C levels were
while the latter is a rate-limiting enzyme for aerobic oxidation increased, indicating that LJP had benecial effect on dyslipidemia.
of glucose [12,23]. As reported, the decreased level of insulin Furthermore, the atherogenic index, markedly increased from
could give rise to the impairment in the activity of them in dia- 1.02 0.23 in normal control rats to 3.30 0.76 in STZ-induced dia-
betic mice [10,12,23,55]. Besides, previous studies on regulation betic rats (p < 0.05). After treated with 800 mg/kg LJP (STZ + HLJP
of L-type pyruvate kinase gene expression by dietary fructose in group), it signicantly descended to normal state with 1.06 0.22
normal and diabetic rats conrmed that some polysaccharides (p < 0.05), revealing that the polysaccharide may potently decrease
could slightly up-regulate pyruvate kinase gene transcription and the risk of atherosclerosis, coronary heart disease and other diabetic
markedly up-regulate pyruvate kinase gene transcription accom- complications. As displayed before, in Pan et al.s investigation, the
panied with insulin treatment [23]. In agreement with them, we water extract of L. japonica was found to increase of the level of
observed apparent decreases in hepatic pyruvate kinase and hex- HDL-C, reduce the level of TC in serum and AI in alloxan-induced
okinase activities in untreated rats, which was probably due to the diabetic mice [39]. However, in Wang et al.s study, the extract I
insensitivity of cells to insulin. Administration of LJP rats led to sig- of L. japonica was found to reduce the levels of TG in serum and
nicant increase in the activities of pyruvate kinase and hexokinase. liver, while it produced no effect upon the levels of TC, LDL-C and
The increased activities of pyruvate kinase and hexokinase may HDL-C in hyperlipidaemia mice and rats, and the extract II couldnt
contribute to the restoration of serum glucose and insulin levels in affect the levels of blood lipid and fat [52]. The results here dis-
LJP treated rats. 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
3.7. Effects of LJP on serum lipid prole
reduction in blood glucose levels (Table 2), improvement of plasma
lipid levels and atherogenic indices (Table 3) in LJP-supplemented
In diabetic condition, as we know, abnormal lipid metabolism,
STZ-induced diabetic rats may demonstrate antihyperglycemic and
always leading to accumulation of plasma TC, TG, LDL-C and VLDL-
hypolipidemic activity of LJP.
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 3.8. Effects of LJP on oxidative stress parameters in the serum and
its incidence of atherosclerosis [14,15,46,57,58]. Conversely, HDL-C liver
plays a key role in cholesterol transport from the periphery to the
liver reduces the risk of cardiovascular disease [17,59]. Recently, plenty of researches have demonstrated that dia-
In the study, as exhibited in Table 3, the results displayed betes mellitus is typically associated with increased generation
that the levels of plasma lipid prole including TC, TG, LDL- of free radicals or impaired antioxidant defence mechanism and
C and VLDC-C were markedly elevated from 1.6 0.2, 1.6 0.1, 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- the study demonstrated the antioxidant activity of the polysaccha-
sion of diabetes and its complications [3,12,60,61]. Furthermore, ride extracted from the plant in vivo for the rst time. The oral
the imbalance of oxidative stress in liver has been found to con- administration of the polysacharides for 42 days caused obviously
tribute to the glucose metabolism and to be associated with decreases of ALT, AST and GGT activity in serum and decreases of
the onset and development of DM [62,63]. Thus, the regulation CAT, SOD and GSH levels in liver. Therefore, in accordance with
of damaging ROS levels in DM by antioxidants and free rad- others, the antioxidative stress might be one of the underlying
ical scavengers represents an attractive benet during the its mechanisms for LJP to alleviate the high blood glucose status in
treatment [64]. In this context, the reducing levels of antioxi- diabetic rats [3,12,6063].
dant enzymes including ALT, AST and GGT in serum, and the Taken altogether, our data herein suggest that the polysac-
improving levels of CAT, SOD and GSH in liver could increase charide fraction produced by L. japonica has a clear potential to
the response of antioxidant defense systems to oxidative stress improve diabetes conditions and potentially prevent its multiple
(Table 4). Compared with the NC group (41.9 5.5, 79.7 9.6 complications through its ability to: (1) decrease hyperglycaemia,
and 36.1 4.9 IU/L, respectively), the levels of ALT (175.2 10.8 (2) decrease hyperlipidaemia, (3) increase antioxidant capacity in
IU/L), AST (377.7 17.7 IU/L) and GGT (137.5 12.4 IU/L) signif- serum and liver. Currently, the specic mechanism to illuminate
icantly increased (p < 0.01) in the STZ group, while in SZT + HLJP these possible health benecial properties at both functional and
group, these levels were signicantly decreased (p < 0.01) with molecular levels is under investigation.
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, 4. Conclusion
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 In smmary, according to our present ndings, our study demon-
protein) and GSH (4.5 0.5 mg/g protein) dramatically declined strated that LJP, the main fraction of polysaccharides extracted from
(p < 0.01) in the STZ group, while in SZT + HLJP group, they were the ower buds of L. japonica, displayed high inhibitory activies
dramatically ascended with 84.5 8.2, 300.9 23.0 U/mg pro- against -amylase and -glucosidase in vitro. While in in vivo
tein (p < 0.01) and 9.8 0.7 mg/g protein (p < 0.05), respectively, assays, LJP revealed a potent hypoglycemic effect on STZ-induced
compared to the STZ rats. These results displayed that the admin- diabetic rats for its ability to reduce glucose levels and relieve
istration of the polysaccharide dose-dependently and signicantly insulin resistance in blood, as well as ameliorate lipid metabolism
restored the dramatic changes of these levels of antioxidants in and oxidative stress. Further studies in animal models and human
diabetic rats. volunteers need to be done to substantiate these ndings. The
Hsu et al. have investigated the antioxidant activities of the results suggest that LJP could be considered as a potential candidate
L. japonica ower bud extracts prepared by water, ethanol and for developing an ingredient of functional foods in the treatment
supercritical uid extraction (SFE) techniques. Compared with the of diabetes and its complications.
extracts obtained by ethanol and SFE, the water extract was found
to possess much higher activities, with higher contents of polyphe- Conict of interest
nols and avonoids and higher amount of chlorogenic acid and
luteolin-7-O-glucoside [65]. During the evauation for the antioxi- The authors declare no conict of interest.
dant activities of ethanol extracts of owers, leaves and branches of
L. japonica, all the extracts have strong quenched DPPH free radical,
Acknowledgements
while ower extract revealed the most potential DPPH radical-
scavenging capacity [66]. Also, the strong antioxidant activity of the
This study was supported by the National Key Specialty
extracts were attributed to the high phenolic contents. Interesingly,
Construction of Clinical Project of China (081619232833) and
D. Wang et al. / International Journal of Biological Macromolecules 102 (2017) 396404 403

Key Project in Science and Technology of Henan Province [25] C.H. Yu, X.Y. Dai, Q. Chen, J.N. Zhang, L.L. Deng, Y.H. Liu, H.Y. Ying,
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