Purification, structure, lipid lowering and liver protecting effects of polysaccharide from Lachnum YM281

Article abstract of DOI:10.1016/j.carbpol.2013.07.014

The extracellular polysaccharide (LEP) produced by Lachnum YM281 was obtained from the fermentation broth, and LEP-1b with molecular weight of 4.02 × 10⁴ Da was separated and sequentially purified through DEAE-cellulose 52 column chromatography

Full text of DOI:10.1016/j.carbpol.2013.07.014

Carbohydrate Polymers 98 (2013) 922–930
Contents lists available at SciVerse ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Purification, structure, lipid lowering and liver protecting effects of
polysaccharide from Lachnum YM281
∗
Tao Qiu, Xiaojing Ma, Ming Ye , Ruyue Yuan, Yanna Wu
Microbial Resources and Application Laboratory, College of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The extracellular polysaccharide (LEP) produced by Lachnum YM281 was obtained from the fermenta-
Received 7 March 2013
Received in revised form 3 July 2013
Accepted 7 July 2013
4
tion broth, and LEP-1b with molecular weight of 4.02 × 10 Da was separated and sequentially purified
through DEAE-cellulose 52 column chromatography and Sepharose CL-6B column chromatography.
1
13
GC–MS, IR and NMR ( H, C) spectroscopy analysis indicated that the repeat unit of LEP-1b was:
Available online 12 July 2013
β -
D
D
-Glcp
1
Keywords:
↓
3
Lachnum YM281
Extracellular polysaccharide
Structure
Lipid lowering
Liver protecting
β -
-Glcp
1
↓
6
→
3)-β -D
-Glcp-(1→3)-β-
D
-Glcp-(1→3)-β -
D
-Glcp-(1→3)-β -
D
-Glcp-(1→3)-β- -Glcp-(1→
D
The effects of LEP-1b on the serum lipids, liver lipids levels and aminotransferase activities of model
mice with hyperlipidemic fatty live were studied, and the results showed that LEP-1b had strong lipid
lowering and liver protecting effects on mice with hyperlipidemic fatty live.
©
2013 Elsevier Ltd. All rights reserved.
1
. Introduction
of polysaccharides with anti-hyperlipidemia activity could reduce
the risk of associated diseases (Thongngam & McClements, 2005).
High-fat food has a potential damage to liver, and when liver is dam-
aged, a great amount of fat will accumulate in liver cells, resulting
in fatty liver (Li et al., 2010; Wang, Cui, & Wei, 2010). Hence, in
the prevention or treatment of fatty liver and liver injury, the main
method is to lower the hepatic lipid and cholesterol levels.
Some Lachnum polysaccharides produced by saprophytic fungi
of Lachnum sp. was reported to have antioxidant and hyperglycemic
activities by our laboratory (Ye, Li, Yang, Zhu, & Lin, 2009; Ye et al.,
2011). We have also reported the backbone chains of the extracellu-
lar polysaccharides of Lachnum calyculiforme and Lachnum YM261
are (1 → 3)-␤-d-glucan, however, the structure of branch chains
and conformation of the lachnum polysaccharides remains unclear.
In the present study, specific structure of the Lachnum YM281 extra-
cellular polysaccharide were characterized by GC–MS, IR and NMR
spectroscopy analysis, and lipid lowering and liver protecting activ-
ities of LEP-1b were also demonstrated.
Fungal polysaccharides, including glucan, mannan and het-
eropolysaccharide, etc., are known to present a broad range of
biological activities, such as antioxidant, hypoglycemic, hypolipi-
demic, and liver protecting, etc., and have potential applications
in the fields of heath products and drugs (He & Zhang, 2004; Li,
Zhang, & Ma, 2010; Tseng, Yang, & Mau, 2008). In recent years, the
chemical structures, chain conformations and biological activities
relationships of fungal polysaccharides have drawn much atten-
tion. Suarez et al. (2006) reported that arabinogalactan with higher
molecular weight exhibited immunostimulatory activity, while one
with lower molecular weight did not. The bioactivities of fungal
polysaccharides are also associated with the helical structures. It
was reported that the immunoregulation and antitumor activi-
ties of some fungal polysaccharides, such as (1 → 3)-␤-d-glucan
form Pleurotus florida, Ganoderma lucidum or Amanita muscaria, are
related to the triple helical conformation (Bao, Wang, Dong, Fang,
&
Li, 2002; Rout, Mondal, Chakraborty, & Islam, 2008).
One key factor causing atherosclerosis, fatty liver and cardio-
cerebrovascular disease is the hyperlipidemia, hence, consumption
2. Materials and methods
2.1. Materials and reagents
∗ Corresponding author at: College of Biotechnology and Food Engineering, Hefei
University of Technology, Hefei 230009, China. Tel.: +86 0551 62919368;
fax: +86 0551 62919368.
Sepharose CL-6B and standard glucose were purchased form
Sigma Chemical Co. (St. Louis, MO, USA). DEAE-cellulose 52 was
purchased from Whatman Co. (Maidstone, Kent, UK). Standard
E-mail address: yeming123@sina.com (M. Ye).
0
144-8617/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carbpol.2013.07.014

T. Qiu et al. / Carbohydrate Polymers 98 (2013) 922–930
923
glucans with different molecular weight were obtained from
Shodex Co. (Tokyo, Japan). TC, TG, LDL-C, HDL-C, ALT, AST, MDA
and SOD kits were all purchased from Nanjing Jiancheng Bioengi-
neering Institute (Nanjing, China). Female Kunming mice (initial
weight at 20 ± 2 g) were purchased from Experimental Animal Cen-
ter of Anhui Medical University, Hefei, China (Certificate number:
Experimental Animal Standard No. 1 of Anhui Hospitals). The mice
(HPGPC) according to the methods reported by Ye et al. (2011). Glu-
cans with different molecular weight were used as standards for
molecular weight measurement. The molecular mass was calcu-
lated according to the calibration curve of standard dextrans. The
monosaccharide component of LEP-1b was obtained by acetylation
reaction followed by GC–MS analysis.
◦
were kept at 23 ± 2 C with a humidity of 55 ± 5% and a light-dark
2
2
.5. Structure elucidation of LEP-1b
cycle of 14 h:10 h. The high fat diet consisting of 78.6% standard lab-
oratory diet, 10% egg yolk, 10% pig fat, 1% cholesterol, 0.2% sodium
cholate and 0.2% methylthiouracil was prepared by our laboratory
.5.1. Periodate oxidation and Smith degradation
LEP-1b (25 mg) was dissolved in 25 mL of 15 mM NaIO solution
4
(Li et al., 2010; Yu et al., 2003). Common-used lipid lowering drug of
◦
and the mixture was kept in the dark at 4 C. Subsequently, 500 ␮L
of reactive mixture were taken out every 6 h, diluted 200 times with
distilled water, and read in a spectrophotometer at 223 nm until the
absorbance to a constant value. Ethylene glycol (1 mL) was added
to terminate the reaction. The periodic acid consumption was cal-
simvastatin was purchased form Merck & Co., Inc. (NJ, USA). Other
chemicals and reagents were of analytical grade and purchased
from Shanghai Zhenqi Chemical Reagent Co., Ltd. (Shanghai, China).
2.2. Microorganism and cultivation
culated based on the standard curve of NaIO . Reaction mixture
4
of 2 mL was titrated with 0.001 M NaOH solution to determine the
production of formic acid.
After periodic acid oxidation, the reaction mixture was added
with ethylene glycol (1 mL), dialyzed against distilled water for
The fruiting bodies of Lachnum YM281 were collected from
Huangshan Mountain, Anhui, China. Lachnum YM281 was isolated
by our laboratory and preserved in China Center for Type Culture
Collection (CCTCC) with the collection number of M 2011196.
Fermentation medium contained glucose 3.0%, yeast extract
4
8 h, reduced by 80 mg of NaBH4 addition in the dark for 24 h.
Then, the reaction mixture was neutralized with 0.1 M acetic acid
and dialyzed against distilled water again. The dialyzed product
was dried, hydrolyzed, acetylated and analyzed by GC–MS analysis
according to our previous method (Ye et al., 2011). GC–MS con-
ditions: silica capillary column: HP-5 (30 m × 0.25 mm × 0.25 ␮m);
0
.5%, KH PO 0.1%, MgSO ·7H O 0.1% and vitamin B 0.005%. The
2
4
4
2
1
activated strain was inoculated in a 250 mL Erlenmeyer flask con-
◦
taining 50 mL fermentation medium at 180 rpm, 25 C for 4 days.
Then the seed medium was transferred to a 5 L fermentor con-
taining 3.5 L fermentation medium and cultivated for 10 days at
◦
◦
temperature program: initially at 50–250 C at a rate of 10 C/min
◦
1
80 rpm, 25 C.
◦
and then held for 20 min; inlet temperature: 260 C; ion source:
EI, 70 eV; molecular weight: 35–650 amu s ; helium flow speed:
−
1
2
.3. Extraction and purification of LEP-1b
1
mL/min.
Three-fold volume of 95% ethanol was mixed with fermenta-
2
.5.2. Methylation analysis
The method descried by Needs and Selvendran (1993) with
tion broth after its suction filtration and concentration. Then the
◦
solution was precipitated at 4 C for 12 h before centrifuged for
minor modification was used for methylation reaction. Briefly,
amounts of 28.5 mg LEP-1b was dissolved in 0.15 mL water, and
then mixed with 4.5 mL of DMSO and 1 g of 3 A molecular sieve.
After blending and sealing, the container with the above mixture
was placed in a desiccator for 24 h to remove water. Then the mix-
ture was filtered into the reaction flask in dry environment. The
reaction solution was added with 75 mg of NaOH powder, aerated
1
0 min at 4000 rpm. After supernatant was removed, sediment was
dissolved with distilled water. Crude extracellular polysaccharides
were obtained after freeze-drying. Before purification, the crude
extracellular polysaccharides of Lachnum YM281 was decolored
and deproteinized by our previous method (Ye et al., 2011).
DEAE-cellulose 52 column chromatography and Sepharose
CL-6B column chromatography was applied to fractionate the
polysaccharides in order. Firstly, crude polysaccharides (100 mg)
was re-dissolved in 5 mL distilled water and applied to a DEAE-
cellulose 52 column (1.6 cm × 60 cm) equilibrated with distilled
water. Then, the polysaccharide was fractionated and eluted with
different concentrations of stepwise NaCl solutions (0, 0.1, 0.5 M)
at a flow rate of 0.5 mL/min. The main fraction (20 mg) was
obtained and then further fractionated by Sepharose CL-6B col-
umn (1.6 cm × 60 cm) chromatography with 0.9% NaCl solution at
a flow rate of 0.55 mL/min. The samples were partly collected
using an automated step-by-step fraction collector and detected
tube by tube by the phenol–sulfuric acid method using glucose
as the standard, and the elution curves were plotted. As results,
three fractions of polysaccharides (LEP-1, LEP-2 and LEP-3) were
obtained. LEP-1 was concentrated, dialyzed and further purified
through a column of Sepharose CL-6B to isolate LEP-1a and LEP-1b.
The resulted LEP-1b was the main component of the crude polysac-
charide of Lachnum YM281 and was then concentrated, dialyzed
and lyophilized for further study.
with N , and treated with ultrasound for 30 min at room temper-
2
ature. After that, the reaction solution was added with 0.75 mL
of CH I, aerated with N , and treated with ultrasound for 2 h at
3
2
room temperature. N2 was aerated to remove the remained CH3I.
Completely methylated polysaccharide was obtained by repeating
above procedure for three times. Water (2 mL) was added to ter-
minate the reaction. The reaction solution was neutralized with
1
M acetic acid, dialyzed 48 h against distilled water, and dried. IR
spectroscopy was applied to check whether the polysaccharide was
−
1
completely methylated (without absorption peak at 3400 cm ).
The methylated polysaccharide was hydrolyzed, derived and ana-
lyzed by GC–MS followed the method described in Section 2.5.1.
2.5.3. Partial acid hydrolysis
Partial acid hydrolysis of LEP-1b was proceeded according to
the method by Tong et al. (2009) with minor modification. LEP-
1b (80 mg) was hydrolyzed with 8 mL of 0.05 M trifluoroacetic acid
◦
(TFA) for 16 h at 95 C, and dialyzed for 48 h against distilled water
after TFA was removed. The products in and outside the dialysis
sack were collected respectively. Followed by concentrated and
dried, the products were fractionated by Sephadex G-100 column
2
.4. Determination of homogeneity, molecular weight and
monosaccharide component of LEP-1b
(
1.6 cm × 60 cm) chromatography eluted with distilled water at a
The homogeneity and molecular weight of LEP-1b were deter-
mined by high performance liquid gel permeation chromatography
flow rate of 0.2 mL/min (Ye et al., 2011). Finally, the obtained com-
ponents were completely methylated, hydrolyzed, acetylated, and

9
24
T. Qiu et al. / Carbohydrate Polymers 98 (2013) 922–930
analyzed by GC–MS using the same method described in Section
measured every day, body weights were weighed every week, and
excrements were collected during the last 3 days of experiment.
Mice of each groups were kept fasting for 12 h before the last
gavage. Blood were collected one hour after the last gavage by enu-
cleating eye balls, and serum was prepared by centrifugation at
2
.5.1.
2.5.4. UV, IR and NMR analysis
The purified LEP-1b was applied to ultraviolet (UV) spectrum,
◦
infrared spectrum (IR) and nuclear magnetic resonance (NMR)
analysis. UV spectrum was recorded by scanning the LEP-1b
solution (5 mg/mL) in a Shimadzu MPS-2000 spectrophotome-
ter (Shimadzu, Japan) with wavelength of 190–400 nm. To detect
functional groups of LEP-1b, IR spectrum were obtained with
Nexus-6700 Fourier-transform infrared spectrometer (Thermo
Nicolet, USA). LEP-1b was ground, mixed with KBr powder (1/100),
pressed into pellets and detected in the frequency range of
4000 rpm for 10 min and preserved at −20 C until analysis. Livers
◦
were quickly excised, weighed and preserved at −80 C until anal-
ysis. The liver index (g/100 g bw) was calculated as liver weight
(g)/body weight (100 g).
2.6.2. Measurement of lipids in serum and liver
The TC, TG, HDL-C and LDL-C levels in serum were determined
using relevant diagnostic kits, and atherogenic index (AI) was cal-
−
1
1
4
000–400 cm . Bruker AV-500 spectrometer (Bruker, Germany)
culated as (TC − HDL-C)/HDL-C (Queiroz-Monici, Costa, Silva, Reis,
13
was used for H NMR and C NMR analysis with the frequency of
&
Oliveira, 2005).
13
5
00.13 MHz for 1H NMR and 125.76 MHz for C NMR, the temper-
The lipid in the liver tissue was extracted according to a previ-
ature of 300 K and the delay time of 2 s. Chemical shifts were given
in ppm.
ously reported method with minor modifications (Ding et al., 2010).
The contents of liver TC and TG were determined with kits. After
vacuum freeze-drying, the liver tissue was ground for the determi-
nation of liver fat content with Soxhlet extraction.
2.5.5. Congo red reaction
Conformation structure of LEP-1b was analyzed by Congo red
reaction according to the method described by He, Shao, Men,
and Sun (2010) with minor modification. The mixture solutions of
the LEP-1b (0.5 mg/mL) in 0–0.5 M NaOH (increasing stepwise by
2
.6.3. Determination of ALT, AST and SOD activities and MDA
content
The serum alanine aminotransferase (ALT), aspartate amino-
0.05 M increments) containing 91 ␮M of Congo red were prepared
transferase (AST), liver superoxide dismutase (SOD) activities and
liver methane dicarboxylic aldehyde (MDA) content were deter-
mined using commercial kits followed the kits instructions.
and analyzed with a UV-VIS spectrophotometer (HP, USA) in the
range of 400–700 nm, and the maximum absorption wavelengths
were recorded as a function of NaOH concentration. Congo red in
NaOH served as the negative control.
2
.6.4. Measurement of TBA, TC and fat excretions in excrement
The excrement of mice was weighed after freeze-drying. Then
2
2
.6. Lipid lowering and liver protecting effects of LEP-1b
total bile acid (TBA) content in excrement was determined by the
method of Wilson, Nicolosi, Rogers, Sacchiero, and Goldberg (1998).
Method raised by Kritchevsky and Tepper (2005) was used to
extract and determine the fat content in excrement. The TC content
of excrement was determined using kit. TBA (TC or fat) excretion
.6.1. Animal and treatments
Ninety female Kunming mice weighting 20 ± 2 g were used in
the animal experiments. After a week of adaptive feed, the mice
were randomly divided into 6 groups with 15 animals in each
group: high-dose LEP-1b group (200 mg/kg), medium-dose LEP-
(
mg/mouse/3 days) = excrement mass × TBA (TC or fat) content.
1
b group (100 mg/kg), low-dose LEP-1b group (50 mg/kg), normal
2
.6.5. Morphological observation of liver tissue
Fresh liver tissue masses (about 2 cm × 2 cm × 0.2–0.3 cm) were
control group, model control group, and positive control group
(
7 mg/kg simvastatin).
embedded in optimal cutting temperature (OCT) tissue freezing
medium and then sliced with freezing microtome with the thick-
ness of 8 ␮m. The sections were fixed in 10% formalin solution
for 1 min for conventional HE staining, and then observed with an
optical microscope, and photos were taken.
To establish model mice with hyperlipidemic fatty liver, normal
control group was given standard laboratory diet and the other 5
groups were fed with high fat diet for 2 weeks. After diet inducing,
4
randomly, respectively. Blood samples were collected by removing
the eye balls, and centrifuged at 4000r/min for 10 min at 4 C. The
mice from the control group and each model group were selected
◦
◦
2.6.6. Statistical analysis
serum was obtained and preserved at −20 C. Livers were quickly
◦
All data were expressed by mean ± SD after statistical analysis.
Software of SPSS13.0 was used for t-test of differences between
groups. Differences were considered statistically significant when
p < 0.05 and extremely statistically significant when p < 0.01.
excised, weighed and preserved at −80 C until analysis. Then, total
cholesterol (TC), triglyceride (TG), low-density lipoprotein choles-
terol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) in
serum and liver of the selected mice were determined respectively.
The results showed that, when compared with control group, the
serum and liver TC, TG, LDL-C and fat contents were increased sig-
nificantly after 2 weeks of high fat feeding (p < 0.01). The liver was
soft and exhibited color of gray-yellow or pale. The liver cut sur-
face was greasy, showing obvious fatty liver symptoms. The above
results indicated that model mice with hyperlipidemic fatty liver
had been successfully established (Ding, Gao, Chao, & Xia, 2010).
From the 3rd week, mice of the three LEP-1b groups were given
with LEP-1b at the doses of 50,100 and 200 mg/kg respectively by
gavage. Mice of the normal control group and model control group
were given normal saline of the same volume, once a day for 4
weeks consecutively. For mice of positive control group, 7 mg/kg of
simvastatin was given by gavage. During the administration period,
normal control group was fed with standard laboratory diet and
the other 5 groups were fed with high fat diet. Food intakes were
3. Result and discussion
3.1. Isolation, purification and molecular weight of LEP-1b
Crude LEP was prepared from the fermentation broth of
Lachnum YM281 by concentration, alcohol precipitation, depro-
teinization, decoloration and dialysis. The production of LEP
reached 6.38 g/L in the fermentation medium. Then, the crude LEP
was firstly fractionated through an anion-exchange chromatog-
raphy of Cellulose DEAE-52 to obtain three independent elution
fractions of LEP-1, LEP-2 and LEP-3 (Fig. 1A). The neutral polysac-
charide (LEP-1), the main fraction of the crude LEP, was eluted by
distilled water. The fraction of LEP-1 was collected, concentrated
and fractionated by Sepharose CL-6B column chromatography. As

T. Qiu et al. / Carbohydrate Polymers 98 (2013) 922–930
925
GC–MS. Sole peak at the retention time of 19.296 min was observed
on its total ion chromatogram (TIC) and its mass spectral data was
consistent with that of acetyl derivative of glucose. The presence
of single sugar moiety suggested that the LEP-1b was a glucan only
composed of glucose.
The results of periodate oxidation showed that LEP-1b con-
sumed 0.29 mol of periodate and produced 0.14 mol of formic acid
per mole of glucosyl residues with a molar ratio of 2.07:1. This
suggested the existence of 1→ or 1 → 6 glycosidic linkages and
inexistence of 1 → 2, 1 → 4, 1,2 → 6, 1,4 → 6 glycosidic linkages,
which could consume periodate but produce no formic acid (He
et al., 2010). No erythritol was observed, and a large amount of
glucose and certain amount of glycerol were detected from GC–MS
data of derivatives from LEP-1b after periodate oxidation and Smith
degradation. Glucose was the main product in the Smith degrada-
tion of LEP-1b, which indicated that a great amount of glycosidic
linkages, such as 1 → 3, 1, 3 → 6, 1, 2 → 3 and 1, 2 → 4 existed in LEP-
1
b were not oxidated in periodate oxidation. Formation of glycerol
also revealed that 1→ or 1 → 6 glycosidic linkages were existed in
LEP-1b (Wang, Yin, Lv, Gao, & Wang, 2010).
The methylated product of LEP-1b was hydrolyzed, acetylated
and analyzed by GC–MS. As shown in Table 1, 2,3,4,6-Me -Glc,
4
2
,4,6-Me -Glc and 2, 4-Me -Glc were detected with molecular ratio
3 2
of 1.07:5.15:1.00 and retention time of 17.023 min, 18.665 min
and 19.493 min, respectively. The high proportion of 2,4,6-Me -Glc
3
implied that the backbone chain of LEP-1b was composed of 1,3-
linked Glc. The presence of 2,4-Me -Glc revealed the existence of
2
1
1
,3,6-linked Glc, indicating that there was a branch chain in LEP-
b, and the branched chain was linked at C6 of the backbone chain
of the glucan. 2,3,4,6-Me -Glc indicated the existence of the non-
4
reducing terminal glucose residues (Dong, Jia, & Fang, 2006).
The partial acid hydrolysis products of LEP-1b were dialyzed
and the products in and outside the dialysis sack were concen-
trated and dried, respectively. Both of components in the dialysis
sack (backbone chain, named as LEP-1b-1) and outside the dial-
ysis sack (branch chain, named as LEP-1b-2) were detected to be
homogeneous component by Sephadex G-100 column chromatog-
raphy. LEP-1b-1 and LEP-1b-2 were analyzed with the method of
methylation analysis. The results listed in Table 1 showed that LEP-
1
b-1 was composed of 1,3-linked Glc and 1,3,6-linked Glc with a
molecular ratio of 1:3.96, and LEP-1b-2 was composed of 1,3-linked
Glc and terminal Glc with a molecular ratio of 1:1.08. Combining
the results of methylation analysis, LEP-1b was speculated to con-
sist of glucose residues linked by 1 → 3 glycosidic linkage, and each
repeat unit was constituted by seven glucose residues. That is, in
each repeat unit of LEP-1b, there were one backbone chain (five glu-
cose residues linked by 1 → 3 linkage) and one branch chain (two
glucose residues linked by 1 → 3 linkage), and the branch chain was
linked at C6 of the backbone chain by 1 → 6 linkage.
No absorption peak was observed between 260 and 280 nm in
the UV spectrogram of LEP-1b. This suggested that there was no
protein or nucleic acid in the purified LEP-1b (Sun & Liu, 2009). The
FT-IR spectrogram of LEP-1b was shown in Fig. 2, the absorption
Fig. 1. Chromatography of LEP on Cellulose DEAE-52 Column (A), LEP-1 on
Sepharose CL-6B column (B) and high performance liquid gel permeation chro-
matogram of LEP-1b (C).
−
1
peak at 3394.10 cm represented the O H stretching vibration.
a result, two fractions of LEP-1a and LEP-1b were obtained, respec-
tively (Fig. 1B). Main fraction of LEP-1b was collected, dialyzed
and dried. Single symmetrical elution peak of LEP-1b by HPGPC
showed that LEP-1b was a homogeneous component and no other
polysaccharide was present. According to the calibration curve of
the elution times of standards, the molecular weight of LEP-1b was
−1
The peak at 2937.06 cm was induced by C H stretching vibra-
−1
tion. The intense absorption band at 1654 cm corresponds to the
bending mode of the absorbed water in LEP-1b (Shi, Xiao, Deng,
−1
Xu, & Sun, 2011). The band at 1421 cm was attributed to bending
vibration of C H or O H (Cao, Yuan, Sun, & Sun, 2011). And the
−1
absorption peaks between 1250 and 950 cm indicated that the
4
estimated to be 4.02 × 10 Da (Fig. 1C).
sugar rings were pyranose rings (Sun, Liu, Yang, & Kennedy, 2010;
Zou, Zhang, Yao, Niu, & Gao, 2010).
3.2. Structure elucidation of LEP-1b
1H NMR spectrum of LEP-1b was shown in Fig. 3. The signal
peaks between 3.57 ppm and 4.14 ppm suggested the existence of
sugar rings. The chemical shift of H1 proton (less than 4.95 ppm)
indicated that sugar rings of LEP-1b were pyranose rings and
After hydrolysis of LEP-1b with TFA and acetylation, the
monosaccharide composition of the LEP-1b was analyzed by

9
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T. Qiu et al. / Carbohydrate Polymers 98 (2013) 922–930
Table 1
GC–MS data for methylation analysis of LEP-1b, LEP-1b-1 and LEP-1b-2.
Group
Methylated sugar
2,3,4,6-Me4-Glc
MS data of main fragments, m/z
Linkage type
Molar ratio
LEP-1b
43, 45, 71, 87, 101, 117, 129, 145, 161, 205
43, 45, 87, 101, 117, 129, 139, 161
43, 71, 87, 101, 117, 129, 139, 161, 189
Terminal Glc
1,3-Linked Glc
1,3,6-Linked Glc
1.07
5.15
1.00
2
2
,4,6-Me3-Glc
,4-Me2-Glc
LEP-1b-1
LEP-1b-2
2,4-Me2-Glc
,4,6-Me3-Glc
43, 71, 87, 101, 117, 129, 139, 161, 189
43, 45, 87, 101, 117, 129, 139, 161
1,3,6-Linked Glc
1,3-Linked Glc
1.00
3.96
2
2,4,6-Me3-Glc
,3,4,6-Me4-Glc
43, 45, 87, 101, 117, 129, 139, 161
43, 45, 71, 87, 101, 117, 129, 145, 161, 205
1,3-Linked Glc
Terminal Glc
1.00
1.08
2
(Wang, Cui, et al., 2010; Wang, Yin, Lv, Gao, & Wang, 2010). The
chemical shifts of C , C , C , C5 and C were ␦71.07, ␦78.65, ␦67.21,
2
3
4
6
␦
73.15 and ␦63.68, respectively. The chemical shift of C , higher
3
than 78 ppm, indicated that C3 of glucose residues in LEP-1b were
substituted C . The chemical shift of C and C at ␦78.65 and ␦63.68
3
3
6
suggested the existence of ␤-glucopyranose residues. In addition,
the ␦67.21 signal revealed the existence of →6)-␤-D-Glcp-(1→, and
the strong signal peak at ␦63.68 implied that C6 of most glucose
residues in LEP-1b were un-substituted C6 (He et al., 2010). These
results of NMR spectra were consistent with that of IR analysis.
All the above results deduced that the backbone chain of LEP-
1
b was consisted of ␤-1,3-D-Pyran glucose residues, every five
glucose residues constituted a repeat unit, and each repeat unit
contained a branch chain (two D-Pyran glucose residues linked
by ␤-1,3-linkage), which was linked at C6 of the backbone chain
by ␤-1,6-linkage. The proposed structure of repeat unit of LEP-1b
was shown in Fig. 5. The glycosidic linkages of LEP-1b are almost
the same as some active ␤-d-glucan linkages reported previously,
whereas the branch chains and repeat units are different.
Fig. 2. The IR spectra of LEP-1b.
saccharide residues were linked by ␤-glycosidic linkage (Li, Fan, &
Ding, 2011). C NMR spectrum (Fig. 4) showed that the anomeric
1
3
carbon signal of ␦102.82 was generated by C1 of →3)-␤-D-Glcp-
3.3. Conformational characterization of LEP-1b
(
1→ and indicated the sugar rings conformation was Pyran type
It has been reported that (1 → 3)-␤-d-glucans with a helical
conformation can form complexes with Congo red in dilute alka-
line solution as indicated by a shift in the maximum absorption
wavelength (ꢀmax) of Congo red (Dong et al., 2006). A strong
alkaline environment can destroy ordered helical structure into
a disordered structure by breaking the intra- and inter-molecular
hydrogen bonds. The greater the effect in the ꢀmax shift, the higher
the helical structure content is (Rout et al., 2008). In this way, a
shift in the maximum visible absorption of Congo red induced by
the presence of a polysaccharide was used for conformational stud-
ies. As shown in Fig. 6, the ꢀmax of the mixture of Congo red and
LEP-1b presented a shift of 15 nm (from 496 nm to 511 nm) in 0.1 M
NaOH solution and the ꢀmax decreased with the increase of NaOH
concentration. The curve was flattened at 503 nm and remained the
same when the NaOH concentration was higher than 0.35 M. This
result suggested that the LEP-1b had triple helix conformation and
the ordered triple helix structure lost at concentrations of 0.35 M
NaOH and higher.
Fig. 3. The 1H NMR spectra of polysaccharide LEP-1b at 27 C.
◦
Fig. 4. The 13C NMR spectra of polysaccharide LEP-1b at 27 C.
◦

T. Qiu et al. / Carbohydrate Polymers 98 (2013) 922–930
927
Fig. 5. The proposed structure of LEP-1b.
3
.4. Lipid lowering and liver protecting effects determination of
hyperlipidemia, and the abnormality of serum lipid also is the major
risk factor of atherosclerosis (Li et al., 2010).
LEP-1b
As shown in Table 3, the TC, TG and LDL-C contents in serum
and AI of the model control mice were all significantly higher
than those of the normal control mice (p < 0.01). There was only
a slight decrease of HDL-C in mice fed the high fat diet, but this
was insignificant when compared with that in mice fed the normal
diet (p > 0.05). This phenomenon was in agreement with stud-
ies of Hu et al. (2010) and the possible reason was still unclear.
After 4 weeks of lavage administration, the high-dose as well as
medium-dose LEP-1b and the positive control drug of simvastatin
all significantly lowered the TC, TG and LDL-C contents in serum
and AI of the model mice, and significantly increased the serum
HDL-C content (p < 0.01 or p < 0.05), exhibiting a dose–effect rela-
tionship. Compared with the model control mice, the TC, TG and
LDL-C contents and AI of the high-dose LEP-1b mice declined by
3
.4.1. Food intake and growth of mice
Compared with the normal control, mice in the model control
showed a significant increase in the body weight gain and feed-
ing efficiency (p < 0.05, p < 0.01, Table 2). Four weeks after being
given administrations, the body weight gain and feeding efficiency
of mice in the high-dose LEP-1b group, medium-dose LEP-1b group
and positive control group were significantly lower than those in
the model control group (p < 0.01 or p < 0.05), and there was no sig-
nificant difference in the body weight gain and feeding efficiency
between the high-dose LEP-1b mice and the normal control mice.
3
.4.2. Lipid content in serum, liver and excrement
Blood–lipid reduction, hepatic lipid reduction and repair of
5
8.39%, 19.65%, 76.22% and 78.51%, respectively, and the HDL-C
damaged liver tissues play important roles in the prevention,
alleviation and cure of hyperlipidemia, atherosclerosis and fatty
liver, and are of great significance in reduction of the cardio-
cerebrovascular disease incidence. Hence, to develop natural,
low-toxic and high-efficient lipid-reducing and liver-protecting
medicines is the international research focus at present. The TC,
TG, LDL-C and HDL-C contents in serum are important indicators
for analysis of lipid metabolism. TC and LDL-C contents being too
high or HDL-C content being too low are the major symptoms of
content increased by 19.53%, whereas there was no significant dif-
ference in AI between the high-dose LEP-1b mice and the normal
control mice. These results indicated that LEP-1b exhibited excel-
lent blood–lipid level and atherogenic index lowering effects on
model mice with hyperlipidemic fatty liver.
TC, TG and fat contents in liver are important indicators for
analyzing of fatty liver. Cholesterol in the body is converted to
bile acid mainly via liver and then excreted with feces. Bile acids,
which were secreted into the intestine, are conducive to emulsifi-
cation and absorption of lipids. Therefore, bile acid, cholesterol and
fat contents in excrement are also important indicators for lipid
metabolism situation analysis (Hu et al., 2010).
In this study, the TC, TG and fat contents in liver of the model con-
trol mice were significantly higher than those of the normal control
mice (Table 3). After 4 weeks of lavage administration, the TC, TG
and fat contents in liver of the high-dose as well as medium-dose
LEP-1b mice and the positive control mice were all significantly
lower than those of the model control mice (p < 0.01). The TC, TG
and fat contents in liver of the high-dose LEP-1b mice decreased
by 44.12%, 33.40% and 57.53% compared to the model control mice.
And there was no significant difference in the liver TC, TG and fat
contents among the high-dose, medium dose and low-dose LEP-
1
b groups and the normal control group. Additionally, the TBA, TC
and fat excretions of the model control mice were all significantly
higher than those of the normal control mice (Table 3). Four weeks
after being given with administration, the TBA, TC and fat excre-
tion of the high-dose LEP-1b mice and the positive control mice
were both significantly higher than those of the model control mice
(p < 0.01 or p < 0.05). The TBA and TC excretions of the high-dose
Fig. 6. Changes in the absorption maximum of the Congo red-LEP-1b complex at
various concentrations of sodium hydroxide solution.

9
28
T. Qiu et al. / Carbohydrate Polymers 98 (2013) 922–930
Table 2
Food intake and growth of mice in various groups.
Groups
Normal control
Model control
Positive control
LEP-1b (200 mg/kg)
LEP-1b (100 g/kg)
LEP-1b (50 mg/kg)
Initial body wt (g/m)
Final body wt (g/m)
Weight gain (g/m/d)
Food intake (g/m/d)
Feeding efficiency (%)
20.53 ± 1.04
30.95 ± 0.82
0.25 ± 0.03
3.32 ± 0.31
7.50 ± 0.60
20.20 ± 1.13
33.53 ± 1.02
0.32 ± 0.03
2.94 ± 0.28
9.86 ± 0.78
20.33 ± 1.09
20.36 ± 1.20
29.66 ± 0.58
0.23 ± 0.03
3.05 ± 0.32
7.38 ± 0.74
20.17 ± 1.33
20.49 ± 1.30
*
*
*
***
*,****
****
***
*
30.22 ± 1.67
30.35 ± 2.34
32.53 ± 0.71
***
***
0.24 ± 0.06
0.24 ± 0.04
0.26 ± 0.05
3.06 ± 0.40
8.38 ± 1.11
3.10 ± 0.37
2.94 ± 0.29
*
*
***
****
***
7.81 ± 1.20
8.22 ± 0.98
Notes: Results are mean ± SD, n ≥ 10. m, mouse.
*
p < 0.05 vs. normal control group.
*
*
p < 0.01 vs. normal control group.
p < 0.05 vs. model control group.
p < 0.01 vs. model control group.
*
**
*
***
LEP-1b group were 14.91% and 26.91% lower than those of the pos-
itive control group, respectively. However, the fat excretion of the
high-dose LEP-1b group was 0.75% higher than that of the posi-
tive control group. There was no significant difference in the TBA,
TC and fat excretion between the low-dose LEP-1b mice and the
model control mice.
3.4.3. Liver protecting activity of LEP-1b
High fat food intake for a long time will cause triglyceride and
cholesterol to synthesize faster than its transporting rate and accu-
mulate in the liver cells, resulting in fatty liver. Fatty liver formation
is related to the oxidative stress and lipid peroxidation in liver. Lipid
peroxidation in liver interferes with liver lipid metabolism, causing
lipid deposition, which again increases lipid peroxidation, damag-
ing the liver cells, and thus leading to the significant increase of
the serum ALT and AST activities and the liver MDA content and
the significant reduction of the liver SOD activity (Luo et al., 2009;
Percario et al., 2008).
In this study, it was found that the ALT and AST activities in
serum of the model control mice were both significantly higher
than those of the normal control mice (Table 3). After 4 weeks of
lavage administration, the ALT and AST activities in mice serum of
the LEP-1b groups and the positive control group were significantly
lower than those of the model control group (p < 0.01), showing a
good dose–effect relationship. The ALT and AST activities in mice
serum of the high-dose LEP-1b group were 50.36% and 52.22% lower
than those of the positive control group, respectively, and with-
out significantly difference from those of the normal control group.
Compared with the normal control group, the liver SOD activity of
the model control mice had a significant decline, and the liver MDA
content and liver index both showed a significant rise (Table 3).
After 4 weeks of lavage administration, compared with the model
These results described above indicated that LEP-1b can
effectively reduce the hepatic lipid level and enhance the
excrement-lipid excretions of the model mice with hyperlipidemic
fatty liver. The reason may be that LEP-1b has the activity of inhib-
iting the synthesis of HMG-CoA reductase from cholesterol, thus
reducing the endogenous synthesis of cholesterol. Another reason
may be that LEP-1b has the capacity of inhibiting the entero-
hepatic cycle of bile acids and enhancing the metabolism and
decomposition of cholesterols through the way of up-regulating
the expressions of ileum FXR and hepatic CYP7A1 genes and down-
regulating the expressions of I-BABP and hepatic FXR. The third
reason may be that the transcriptional up-regulation of PPAR␣,
PPAR␥, Lpl and Lpic leads to the decrease of the fat synthesis rate
and the increase of the fat hydrolysis rate, thus accelerating the
metabolism and decomposition of triglyceride (García-Mediavilla,
Villares, Culebras, Bayón, & González-Gallego, 2003; Makishima
et al., 1999; Takahashi et al., 2002). However, the specific mech-
anism of lipid lowering and liver protecting of LEP-1b need further
investigation.
Table 3
Effect of LEP-1b in different doses on the serum, liver lipid, TBA, TC and fat excretions in excrement and repair effect of LEP-1b on the damaged liver of the model mice.
Index
Normal control
Model control
Positive control
LEP-1b (200 mg/kg)
LEP-1b (100 mg/kg)
LEP-1b (50 mg/kg)
*
*
*
*
****
*,****
**,****
**
TC
TG
HDL-C
LDL-C
AI
2.61 ± 0.24
0.99 ± 0.08
1.32 ± 0.07
0.84 ± 0.21
0.98 ± 0.14
7.45 ± 0.68
1.73 ± 0.14
1.28 ± 0.12
5.93 ± 0.66
4.84 ± 0.42
2.91 ± 0.11
1.24 ± 0.13
1.61 ± 0.06
0.93 ± 0.15
0.81 ± 0.04
3.10 ± 0.16
3.77 ± 0.34
6.62 ± 0.57
*,****
*,****
**,***
**,***
**
1.39 ± 0.13
1.45 ± 0.10
1.54 ± 0.17
*
*,***
**,***
Serum lipids (mmol/l)
1.53 ± 0.09
1.50 ± 0.06
1.41 ± 0.09
*
*
*
*
****
****
**,****
**,****
**
1.41 ± 0.05
2.61 ± 0.26
5.09 ± 0.58
****
**,****
**,***
1.04 ± 0.23
1.51 ± 0.17
3.71 ± 0.58
**
**
**
**,****
**,****
****
**,****
**,****
**,***
TC
TG
Fat
9.50 ± 0.46
4.88 ± 0.12
9.95 ± 0.32
40.19 ± 4.11
15.72 ± 1.00
26.61 ± 0.76
13.27 ± 1.16
8.79 ± 0.60
10.54 ± 1.05
22.46 ± 1.38
29.03 ± 2.47
33.85 ± 2.38
Liver lipids (␮mol/g,
**,****
**,****
**
10.47 ± 0.59
11.47 ± 0.84
14.47 ± 0.76
␮
mol/g, %)
*,****
**,****
**,****
11.30 ± 0.86
15.88 ± 0.65
18.19 ± 0.67
*
*
*
**,****
**,****
**,****
**
Excrement
mg/mouse/3 days)
TBA
TC
Fat
33.67 ± 1.48
1.04 ± 0.12
38.23 ± 1.81
9.33 ± 0.91
79.34 ± 4.18
67.51 ± 3.90
71.27 ± 4.63
39.87 ± 1.96
*
**,****
**,****
**,****
**
(
29.73 ± 2.4
21.73 ± 1.87
17.37 ± 1.70
10.36 ± 1.53
*
*
**,***
**,***
**
**
114.96 ± 11.72
279.22 ± 21.86
331.58 ± 24.61
334.08 ± 20.65
295.24 ± 33.48
277.75 ± 20.83
*
*
**,****
****
****
*,****
ALT
AST
45.35 ± 1.85
40.69 ± 1.10
123.64 ± 6.15
93.33 ± 6.03
46.33 ± 3.83
49.08 ± 5.34
63.08 ± 6.01
Serum (U/L)
*
*
**,****
****
*,****
**,****
130.75 ± 8.32
87.03 ± 3.82
41.58 ± 2.36
43.58 ± 1.83
59.75 ± 4.15
**
**
**
**,****
**,****
****
*,****
*,****
*,****
Liver (U/mg pro,
nmol/mg pro,
g/100 g bw)
SOD
233.29 ± 10.27
1.17 ± 0.08
115.88 ± 6.17
2.10 ± 0.09
6.88 ± 0.15
171.93 ± 9.22
1.56 ± 0.09
4.72 ± 0.14
248.14 ± 4.91
210.08 ± 11.99
175.41 ± 9.19
****
**,****
**,***
MDA
Liver
index
1.21 ± 0.08
1.62 ± 0.07
1.87 ± 0.10
****
****
*,****
4.83 ± 0.20
4.88 ± 0.26
4.93 ± 0.20
5.36 ± 0.35
Notes: Results are mean ± SD, n ≥ 10.
*
p < 0.05 vs. normal control group.
*
*
p < 0.01 vs. normal control group.
p < 0.05 vs. model control group.
p < 0.01 vs. model control group.
*
**
*
***

T. Qiu et al. / Carbohydrate Polymers 98 (2013) 922–930
929
Fig. 7. Optical micrographs of mice liver tissues slice (magnification 40×). (a) Normal control group; (b) model control group; (c) positive control group; (d) LEP-1b group
at 50 mg/kg; (e) LEP-1b group at 100 mg/kg; (f) LEP-1b group at 200 mg/kg. The black arrow indicates fat vacuoles.
control group, the high-dose, medium-dose as well as low-dose
LEP-1b and the positive control drug of simvastatin all significantly
increased the liver SOD activity and decreased the liver MDA con-
tent and liver index (p < 0.01 and p < 0.05). As shown in Table 3, the
liver SOD activity of the high-dose LEP-1b group was significantly
higher than that of the normal control group (p < 0.05), the liver
MDA content and liver index of the high-dose LEP-1b mice was
liver SOD activity. Additionally, optical micrographs of mice liver
tissues slices suggested significant liver steatosis alleviating effects
and liver tissue morphology restoring abilities of LEP-1b on liver
tissue of the model mice, which fully demonstrates that LEP-1b
can alleviate the peroxidation damage of liver tissues and promote
the recovery of damaged liver tissues.
2
2.44% lower and 4.26% higher than those of the positive control
4
. Conclusion
mice, respectively, and had no significant difference with those of
the normal control group.
LEP-1b from Lachnum YM281 was isolated and purified. The
The differences in mice liver tissues slices from normal control
group, model control group, positive control group and different
LEP-1b groups were also compared by optical microscope after
staining. As shown in Fig. 7, the liver cells of the normal mice
are regularly arranged and have normal morphology, showing no
symptoms of fat degeneration, whereas a great number of large
fat vacuoles and invasive necrosis of cells are observed in the mice
liver tissues of the model control group, showing obvious diffuse
hepatic steatosis and inflammatory change. Liver tissue morphol-
ogy of mice in the low-dose, medium-dose as well as high-dose
LEP-1b groups and the positive control group were significantly
recovered. The liver tissue morphology and arrangement of the
high-dose LEP-1b mice showed no significantly difference from
those of the normal mice, whereas there were still a small amount of
fat vacuoles in mice of the low-dose LEP-1b group and the positive
control group.
molecular weight, structure and conformation of LEP-1b were
studied and deduced. LEP-1b can also significantly decrease the
serum lipids (TC, TG and LDL-C) and liver lipids (TC, TG and fat)
levels of the model mice, reduce the serum ALT and AST activi-
ties and liver MDA content, and improve the liver SOD activity.
These indicate that LEP-1b has a great blood–lipid reduction and
hepatic lipid reduction effect and can promote the recovery of
damaged liver tissues, and can be used in the development of
lipid-reducing and liver-protecting drugs or health products. Fur-
ther works on structure–activity relationship of LEP-1b are in
progress.
Acknowledgments
This study was financially supported by the National Nat-
ural Science Foundation of China (No. 31070021) and the
Fundamental Research Funds for the Central Universities
(2012HGZY0020).
These results above indicated that LEP-1b effectively inhibited
the increase of the serum ALT and AST activities, liver MDA content,
and liver index of the model mice, and effectively improved the

9
30
T. Qiu et al. / Carbohydrate Polymers 98 (2013) 922–930
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