Biological Macromolecules 

Purification, antitumor and anti-inflammation activities of an
alkali-soluble and carboxymethyl polysaccharide CMP33 from
Poria cocos

Abstract
A carboxymethyl polysaccharide CMP33 (15.23×104 Da) was isolated from edible
and pharmaceutical mushroom Poria cocos using alkaline extraction followed by
DEAE-52 and Saphadex-G200 + Saphadex-G150 column chromatographies. The
structure analysis showed that CMP33 was composed of glucosyl residues
containing a backbone chain of (1→3)-linked glucose residues and side chains of
(1→6) and (1→2)-linked glucose residues, and possessed triple-helix structure.
Bioassay results revealed that CMP33 displayed a dose-dependent inhibition on 5
cancer cells (HepG-2, MCF-7, SGC-7901, A549) in the range of 31.25-1000 μg/mL,
but low cytotoxicity on normal liver cells L-O2. Moreover, CMP33 stimulated NO
release and cytokine secretion (IL-1β, IL-6 and TNF-α), and also inhibited
LPS-stimulated overproduction of NO, IL-6, TNF-α and IL-1β, in RAW264.7 cells.
These results suggested that CMP33 possessed anticancer, anti-inflammation and
immune-stimulation activities, and potential for developing as a bioactive ingredient
in functional foods.
Key words: Poria cocos; carboxymethyl polysaccharide; antitumor;
anti-inflammation; immunostimulation
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1 Introduction
Poria cocos is a kind of edible and pharmaceutical mushroom that grows around the
roots of pine trees. It is widely used in the formulation of nutraceuticals, tea
supplements, cosmetics, and functional foods [1]. The main active ingredients in
Poria cocos include polysaccharide, triterpenes and steroids. Due to their various
biological functions, P. cocos polysaccharides have attracted great attention in past
decades, such as antioxidant, anti-inflammation, tumor growth and metastasis
inhibition, and immunomodulation [2].
Notably, the dominant product in the P. cocos sclerotium extract is the
water-insoluble (1 → 3)-beta-D-glucan with little bioactivities [3]. The water
solubility of polysaccharides plays a key role in their bioactivity. In order to alter the
solubility of polysaccharides, chemical modification has been reported to enhance
their aqueous solubility and improve bioactivity, including carboxymethylation,
sulfation, hydroxylation, formylmethylation, aminoethylation and phosphorylation
[4]. For example, Wang et al.[5] investigated the antitumor activities of five
beta-glucan derivatives from Poria cocos sclerotium. They found that the parent
polysaccharide PCS3-II had no anticancer activity, but their sulfated,
carboxymethylated, methylated, hydroxyethylated or hydroxypropylated derivatives
exhibited anticancer activity on S180 cells and gastric cancer cells (MKN-45 and
SGC-7901) in vitro and in vivo.
However, the antitumor and anti-inflammation activities of carboxymethylated Poria
cocos polysaccharide with triple-helix conformation were few investigated. In this
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study, we isolated a water-insoluble polysaccharide using alkaline extraction from
Poria cocos, subsequently, the carboxymethylation and purification were performed,
then, the structural characterization, antitumor and anti-inflammation activities of the
polysaccharide with triple-helix conformation were investigated.
2 Materials and methods
2.1 Materials and reagents
The dried Poria cocos was from a commercial market in Guangzhou, China. Five
cancer cells (human breast cancer cell MCF-7, human liver cancer cell HepG-2,
human lung cancer cell A549, human stomach cancer cell SGC-7901 and human
colon cancer cell HT-29), and human normal cell LO-2, were purchased from
Animal Experimental Center of Sun Yat-sen University, Guangzhou, China. Murine
macrophage RAW 264.7 cell line was provided by People’s Hospital of Guangdong
province, Guangzhou, China. DMEM and RPMI-1640 media were from Gibco
Company, USA. McCoy’s 5A medium was from Jiluo Biopharmaceutical Co. Ltd,
Hangzhou, China. DEAE-52, Saphadex-G150 and Saphadex-G200 were from Yueye
Biotechnol.Co.Ltd, Shanghai, China. Rhamnose, arabinose, fucose, mannose,
glucose, xylose, galactose and Lipopolysaccharide (LPS) was purchased from Sigma
Chemical Co.,USA. Mouse IL-1β, IL-6 and TNF-α Elisa kits were purchased from
Xinbosheng Biotechnol. Co. Ltd, Guangzhou, China. Other chemicals were of
analytical grade.
2.2 Alkaline extraction and carboxymethylation of crude polysaccharide
Five hundred grams of the dried Poria cocos were powdered and dissolved in
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distilled water, and subjected to alkali extraction with 1 kilograms of 2% NaOH
(room temperature, 12 h). By stirring the alkaline solution, hydrogen peroxide (120 g,
35%) was slowly added to remove the pigment. Then, chloroacetic acid (180 g) was
introduced for substitution reaction (60 ℃, 8 h). The extracts were neutralized with
acetic acid and precipitated with 75% ethanol. The crude polysaccharide was
collected and lyophilized to obtain a crude carboxymethyl polysaccharide.
2.3 Purification of crude carboxymethyl polysaccharide
The crude carboxymethyl polysaccharide (180 mg) was dissolved 5 mL of pure
water. The solution was applied to DEAE-52 column (2.6 cm × 40 cm) and eluted
with 0.1 mol/L NaCl at a flow rate of 0.45 mL/min. Fractions were collected every
12 min/tube and assayed with the phenol-sulfuric acid method. The eluting peaks
were collected, concentrated, dialyzed and lyophilized to obtain carboxymethyl
polysaccharide. Subsequently, 80 mg of the carboxymethyl polysaccharide was
dissolved in 2 mL of pure water and loaded onto a combination column:
Saphadex-G200 + Saphadex-G150 (1.6 cm×50 cm). Then, elution was performed
with 0.1 mol/L NaCl at a flow rate of 0.3 mL/min. Fractions were collected every 10
min/tube, concentrated, dialyzed and lyophilized to obtain pure carboxymethyl
polysaccharide CMP33.
2.4 Purity analysis and molecular weight determination
Ten miligrams of CMP33 were dissolved in 100 mL of distilled water. Taking 1 ml
of the solution was diluted to 2 mL in a tube. One mL of phenol (5%) was added,
and 5 mL of sulfuric acid was rapidly added, reacting in a boiling water bath (30
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min). The tube without standard glucose was used as blank control. The absorbance
was recorded at 490 nm. The content of polysaccharide was calculated based on
standard glucose curve.
The molecular weight was determined using a Gel Permeation Chromatography
(GPC, Water Breeze, Waters, USA) coupled with refractive index detector (Waters
2414, Waters Inc. USA). CMP33 was dissolved in 0.02 mol/L KH2PO4 (pH 6) to a
concentration of 1.0 mg/mL and filtered through 0.45 μm of microporous filter
membrane before injection into the GPC system with a TSK-Gel G-5000 column
(7.8 mm×300 mm, Supelco, USA) linked with a TSK-Gel G-3000 column (7.8
mm×300 mm, Supelco, USA) at 35℃, eluting at a flow rate of 0.6 mL/min for 30
min. The calibration curve was prepared with standard dextrans with different
molecular weights (1800, 2500, 4600, 7100 and2,000,000 Da). The mass of CMP33
was calculated by comparing the retention time of the standards.
2.5 Spectral analysis
CMP33 (1 mg) was dissolved in distilled water to a concentration of 1.0 mg/mL, and
scanning was conducted in the range of 200-400 nm on Ultraviolet visible
spectrophotometer (UV2300, Tianmei Scientific Instrument Ltd, Shanghai, China).
CMP33 (2 mg) was ground with spectroscopic grade potassium bromide (KBr)
powder and pressed into 1 mm pellets. The FT-IR spectrum was recorded on a
Fourier transform infrared spectrophotometer (Nexus, Thermo Company, USA) in
the range of 4000-400 cm-1
2.6 Monosaccharide Composition Analysis
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CMP33 (10 mg) was hydrolyzed with 4 mL of trifluoroacetic acid (TFA, 2 mol/L)
for 8 h at 110℃ in a sealed task. The solution was cooled to room temperature,
methanol was added, concentrated and vaporized at 45℃ (repeating for 3-5 times)
to completely remove TFA.
Each of the seven monosaccharide standards and the hydrolyzed CMP33 were
reacted with 10 mg of hydroxylamine hydrochloride and 0.5 mL of pyridine at 90 °C
for 30 min. The mixture was cooled to room temperature and was acetylated with 0.5
mL of acetic anhydride at 90 °C for 30 min.
The analysis was performed using an Agilent 6890N GC with a FID injector
(Agilent Technologies, Palo Alto, CA, USA) equipped with a DB1701 capillary
column (30 m × 0.32 mm× 0.25 μm). Initial oven temperature was 180 °C for 2 min,
increased to 220 °C at 2 °C/min, held for 1 min, increased by 7 °C/min to a final
temperature of 250 °C. The derivatives were filtered through 0.45 μm of
microporous filter membrane before injection into GC system with an 1 μL of
injection volume.
2.7 Periodate oxidation and Smith degradation
The periodate oxidation analysis was performed as described before [6]. Specifically,
CMP33 (20 mg) was dissolved in 15 mmol/L of sodium periodate (NaIO4). The
mixture was kept at room temperature in dark. The absorbance was recorded at 223
nm every 6 h with a spectrophotometer. After equilibrium, ethylene glycol (1.5 mL)
was added to end the reaction. The amount of NaIO4 consumption was calculated
according to the decrease in absorbance at 223 nm. Formic acid production was
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determined by titration with 0.01 M NaOH.
The solution treated with ethylene glycol was dialyzed against tap water and then
distilled water, each for 24 h. After concentration, the mixture was reduced by
sodium borohydride (NaBH4) overnight. The pH value was adjusted to 6-7 and the
excess NaBH4 was neutralized with 50% acetic acid. The solution was dialyzed
against tap water and then distilled water, each for 24 h. After dialysis, the retentate
was concentrated and freeze-dried, hydrolyzed with TFA (2 mol/L) at 110°C for 8 h.
After acetylated, GC analysis was performed as described in Monosaccharide
Composition Analysis. Using the same protocol, glucose, glycerol and erythritol
were derivatized and used as control.
2.8 Nuclear magnetic resonance (NMR) Spectroscopy
CMP33 (15 mg) was dissolved in o.5 mL of D2O. The spectra of 1H-NMR,
13C-NMR, HSQC (heteronuclear single quantum coherence), HMBC (heteronuclear
multiple- bond correlation), H-H COSY (hydrogen-hydrogen correlation
spectroscopy) were recorded on a Bruker 600 MHz NMR spectrometer. The
software MestReNova-11.0.2 was used for data processing.
2.9 Triple-helix structure assay
Two milligrammes of CMP33 were added to distilled water (2 mL) and 100 μmol/L
of Congo Red dye (2 mL). Then, 4 mol/L of NaOH was slowly introduced to a final
concentration of 0.5 mol/L. Under different concentrations of NaOH, the maximal
absorbance within 400-600 nm was recorded on Ultraviolet visible
spectrophotometer (UV2300, Tianmei Scientific Instrument Ltd, Shanghai, China).

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The Congo Red solution without CMP33 was used as control.
2.10Antitumor activity in vitro
CMP33 was dissolved in DMEM or McCoy’s 5A media to a final concentration
1000 μg/mL, and filtered through 0.22 μm of microporous filter membrane for
experiment. Cancer cells (HepG-2, MCF-7, SGC-7901, A549) were cultured on
DMEM medium (containing 10% fetal bovine serum and 1% penicillin+
streptomycin), and HT-29 cells were cultured on McCoy’s 5A medium (containing
10% fetal bovine serum and 1% penicillin+ streptomycin). Logarithmic growth
phase cells (100μL) were seeded on 96-well microtiter plate, cultured for 48 h in a
37 °C humidified atmosphere with 5% CO2. Different concentrations (31.25-1000
μg/mL) of CMP33 and positive drug 5-flurouracil (5-FU) were added to each well
(200 μL), no drug was used as negative control, 5 replicates for each well, and
continued to be cultured for 48h. After PBS washing, 20 µL of MTT
(3-(4,5-dimethylthiazol-2-yl) 22,5-diphenyl- tetrazolium bromide) solutions (5
mg/mL) and media (180 μL) were added to each well, and cultured for additional 4 h
at 37 °C. Removing media containing MTT, DMSO (150 μL) was added, shaking for
15 min, the optical density (OD) was recorded at 490 nm with a microplate reader
(Model 550, Bio-Rad, USA). The percentage of inhibition was calculated by the
formula: Inhibition (%)=(1- OD (experimental group) / OD(control group)×100%.
2.11 Anti-inflammation activity in vitro
CMP33 was dissolved in RPMI-1640 media to a final concentration 1000 μg/mL,
and filtered through 0.22 μm of microporous filter membrane for experiment.
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RAW264.7 cells were cultured on RPMI-1640 medium (supplemented with 8% fetal
bovine serum). Logarithmic growth phase cells (100μL, 1×105
/mL) were seeded on
96-well microtiter plate, cultured for 24 h in a 37 °C humidified atmosphere with 5%
CO2. Removing the culture medium, the test samples were added according to 4
groups: control group, medium (100 μL); LPS (lipopolysaccharide) group, medium
(100 μL) containing LPS (1 μg/mL); CMP33 group, medium (100 μL) containing
31.25-1000 μg/mL of CMP33; LPS+CMP33 group, medium (100 μL) containing
LPS (1 μg/mL) and 31.25-1000 μg/mL of CMP33, repeating for 5 times for each
group. After additional 24 h of culture, supernatant was collected.
2.12 Assays for cell viability, nitric oxide and cytokine production
Cell viability was measured by above-mentioned MTT method. The nitric oxide (NO)
concentration was assayed by Griess method.7 Specifically, the conditioned culture
medium (100 μL) was mixed with Griess reagent (100 μL) in a 96-well microtiter
plate. After 10 min of reaction, the OD value of the mixture was detected at 540 nm
by a microplate reader (Model 550, Bio-Rad, USA). Based on the reaction results
between NaNO2 (0~200 μmol/L) and Griess reagent, the standard curve of NO2- vs.
OD was plotted, the released NO and inhibition on NO by CMP33 were determined.
Levels of TNF-α, IL-1β, and IL-6 in the supernatant were quantified by an ELISA kit
(Xinbosheng Biotech Co., Ltd., Guangzhou, China) according to the manufacturer’s
instructions.
2.13 Statistical analysis
All experiments were repeated thrice at least. The data were processed as the mean ±

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standard deviation. One-way analysis of variance (ANOVA) was done by SPSS19.0
software. Student’s t tests were performed to determine any significant differences, p
values <0.01 and 0.05 were considered to be very significant and significant.
3 Results
3.1 Structural characterization of carboxymethyl polysaccharide CMP33
The crude carboxymethyl polysaccharide was purified using DEAE-52 column and
Saphadex-G200 + Saphadex-G150. After eluted by 0.1 mol/L of NaCl (Figure 1A-B),
a pure carboxymethyl polysaccharide CMP33 was obtained. The HPGPC
chromatogram (Figure 2A) showed a single and symmetrical peak (Figure 2A) and
indicated CMP33 were homogeneous. Its molecular weight was estimated to be
15.23×104 Da. The polysaccharide content of CMP33 was determined as 99% from a
glucose standard curve. The UV spectrum indicated that no absorbance was detected
at 260 or 280 nm and CMP33 was free of protein and nucleic acid.
After hydrolysis and acetylation, GC-MS was used to analyze the monosaccharide
composition of CMP33. As shown in Figure 2B, only one peak appeared and was
identified as glucose according to the retention time of authenticated standards.
The FT-IR spectra (Figure 2C) of CMP33 indicated the presence of characteristic
peaks in the range of 4000~1650 cm-1
for a polysaccharide. Among them, the
absorbance band at 3600~3200 cm-1
corresponded to the stretching vibration of O−H
bonds. The bands at 3000~2800 cm-1 were related to the stretching vibration of C−H
in sugar rings. The peaks at approximately 1630 cm-1
and 1420 cm-1 were
characteristic of carboxymethylation that were associated with the stretching
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vibration of asymmetric and symmetric carboxyl groups, respectively [8]. The
absorption peak at 890 cm-1 was characteristic of β-pyranose [9], CMP33 is
β-glucan.
In order to determine the type of glucosidic bonds of polysaccharide, periodate
oxidation and Smith degradation were used. Based on the established standard curve
of periodate oxidation, one mol of CMP33 consumed 0.26 mol of periodate, and
produced 0.048 mol of formic acid. Thus, CMP33 was calculated to contain 4.83%
of (1→6)-linked glucose, 78.62% of (1→3)-linked glucose, and 16.55% of (1→
2,4)-linked glucose residues. Then, Smith degradation was performed on the
periodate oxidized product. GC-MS analysis (Figure 2D) indicated the presence of
glucose and glycerol but absence of erythritol in the reaction mixture. Taken together,
CMP33 was primarily composed of (1→3)-linked glucose residues, and (1→
2,6)-linked glucose residues were also present in small amounts.
Congo red analysis (Figure 2E) displayed that the maximum absorbance of
CMP33+Congo red mixture was obviously larger than that of Congo red alone in the
range of NaOH concentration 0-0.5 mol/L, indicating the formation of CMP33 and
Congo red complex and occurrence of red shift. This suggested that CMP33
possessed triple-helix structure. Moreover, 1D- (
1H- and 13C-NMR) and 2D-NMR
(HSQC and HMBC) analysis (Figure 3) confirmed that CMP33 contained
carboxymethyl and its backbone consisted of (1→3)-linked glucan (high intensity
signal at 4.77 ppm).
3.2 Antiproliferative activity of CMP33

Based on MTT assay, the antiproliferative activity of CMP33 was displayed in Table
1. The results showed that CMP33 exhibited a dose-dependent inhibition on 5 cancer
cells in the range of 31.25-1000 μg/mL, top 2 cells with good inhibition were
SGC-7901 and HT-29, with IC50 values of 79.6 and 113.3 μg/mL, respectively. At
1000 μg/mL, the inhibitory percentages of CMP33 on HT-29 and SGC-7901 were 84%
and 96%, respectively, which were close to the inhibitory percentages of 5-FU on
HT-29 and SGC-7901, 95% and 100.9%, respectively. But 5-FU exhibited
remarkable cytotoxicity on normal liver cells L-O2 with 73.6% of inhibition,
compared with CMP33 with 11.3% of inhibition only.
3.3 Anti-inflammation activity of CMP33
MTT method was used to measure the effects of CMP33 on proliferation of
macrophage cells RAW264.7. Figure 4A-B showed that LPS can significantly
stimulate growth of RAW264.7 compared with control (p<0.01). In the case of no
LPS induction, CMP33 had no cytotoxicity on RAW264.7 (proliferation>100%) at
concentration≤500 μg/mL, and a little inhibition (7.3%) on RAW264.7
(proliferation>90%) at 1000 μg/mL. In the presence of LPS induction, CMP33 had
no significant inhibition on LPS-stimulated growth of RAW264.7 (p>0.05) at <125
μg/mL, but displayed significant inhibition on LPS-stimulated growth of RAW264.7
at ≥125 μg/mL. Taken together, CMP33 had no obvious cytotoxicity on RAW264.7
at the concentration of 31.25-1000 μg/mL.
Figure4C indicated that NO production was increased significantly by LPS treatment
compared with control (p<0.01). CMP33 significantly reduced the LPS-stimulated

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overproduction of NO at concentration 31.25-1000 μg/mL compared with LPS group
(p<0.01), up to 52.3% inhibition on LPS-stimulated NO production at 1000 μg/mL.
In the absence of LPS stimulation (Figure 4D), CMP33 significantly induced NO
production in a dose-dependent manner at concentration of 62.5-1000 μg/mL
(p<0.05), the NO production in CMP33 treatment group was more than 5-fold NO
production in control group.
Then, the effects of CMP33 on the secretion of inflammatory cytokines like IL-6,
TNF-α and IL-1β were evaluated in LPS-activated RAW 264.7 cells (Table 2). The
results demonstrated that LPS significantly stimulated production of IL-6, TNF-α
and IL-1β compared with control (p<0.01). However, CMP33 significantly
decreased the LPS-stimulated overproduction of TNF-α and IL-1β when the
concentration was not less than 31.25 μg/mL (p<0.01), and also significantly reduced
the overproduction of IL-6 when the concentration was not less than 62.5 μg/mL
(p<0.01). The maximum inhibition percentages of CMP33 on IL-6, TNF-α and IL-1β
production were 48.0%, 79.7% and 51.8%, respectively. On the other hand, in the
absence of LPS stimulation, compared with control, CMP33 significantly enhanced
the production of TNF-α and IL-1β when the concentration was not less than 62.5
μg/mL (p<0.05), and significantly increased the production of IL-6 after
concentration of 31.25 μg/mL (p<0.01). The productions of IL-6, TNF-α and IL-1β
in CMP33 group were 12.3, 2.4 and 6.6 folds those in control group, respectively.
4 Discussion
Studies have evidenced that Poria cocos polysaccharides play a vital role in human
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health, including antioxidant, anticancer, anti-inflammatory, immunomodulation,
antivirals [1]. Many different polysaccharides have been isolated from Poria cocos.
For example, from fresh sclerotium of Poria cocos, Wang et al.[10] extracted six
polysaccharides PCS1, PCS2, PCS3-I, PCS4-I, PCS3-II, and PCS4-II with the
molecular weight 9.1-21.1×104 Da. Lu et al.[11] identified a water-soluble
1,6-branched (1,3)-a-D-galactan from P. cocos, which is mainly composed of
galactose. Huang et al.[12] purified a heteropolysaccharide from P. cocos with the
molecular weight 1.69 × 104 Da, which was composed of fructose, mannose, glucose
and galactose with mole ratio 1:2.36:5.49:2.34. However, little polysaccharide
derived from P. cocos was reported to have triple-helix structure, which is essential
for explaining the immunomodulatory activity of (1,3)-b-D-glucan [13-15]. In this
study, a carboxymethyl polysaccharide CMP33 with triple-helix structure
(Mw=15.23×104 Da) was obtained from the dried sclerotium of Poria cocos by alkali
extraction followed by purifications using DEAE-52 and Saphadex-G200 +
Saphadex-G150 chromatographies.
Polysaccharides are known to have anticancer activities. The polysaccharide with
moderate range of molecular mass from 2.0×104 to 40.0×104
are beneficial to
enhance the antitumor activities [16]. From cultured mycelia of Phellinus igniarius,
Li et al.[17] extracted water-soluble intracellular polysaccharides IPSW-1, IPSW-2,
IPSW-3, and IPSW-4 with the average molecular weights of 15.1-34.1×104 Da,
which were found to inhibit the growth of SW480 and HepG2 cells. From the
fruiting bodies of Coriolus Versicolor, Awadasseid et al.[18] reported a novel glucan

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CVG, which has a linear alpha-glucan chain composed of only (1 →
6)-alpha-D-Glcp. Their results indicated that CVG has antitumor activity towards
Sarcoma-180 cells by its immunomodulation activity. In this study, the identified
polysaccharide CMP33 exhibited anti-proliferative activity on 5 cancer cells, such as,
the inhibitory percentages of CMP33 on HT-29 and SGC-7901 were 84% and 96%,
respectively, at 1000 μg/mL, similar to 5-FU (95% and 100.9%, respectively). But
the cytotoxicity of 5-FU (73.6%) on normal liver cells L-O2 was 7-fold cytotoxicity
of CMP33 (11.3%) on L-O2.
Chronic inflammation is associated with several chronic diseases including obesity,
diabetes, atherosclerosis, neurodegenerative diseases, and cancers [19]. NO is
implicated in inflammatory responses in response to tissue injury [20]. The
activation of macrophages results in the production of a large amount of NO and
various cytokines, including TNF-α and IL-6, which induce the proliferation of other
immune cells, including B-cells and T-cells [21]. From the tetraploid Gynostemma
pentaphyllum, Niu et al.[22] isolated a novel polysaccharide (GPP-S), which
exhibited inhibitory activities on IL-1β and IL-6 gene expressions in RAW 264.7
mouse macrophage cells. From the common edible plant Brassica rapa, Chen et
al.[23] purified two polysaccharides BRNP-1 and BRNP-2, which were proved to
stimulate the proliferation, NO release, and cytokine secretion (IL-6 and TNF-α) of
RAW264.7 macrophages. Zha et al.[24] extracted a water-soluble polysaccharides
LMP-1 from Lepidium meyenii, they found that LMP-1 up-regulated the expression
of Toll-like receptor 4 (TLR4), Toll-like receptor 2 (TLR2) and IL-1β. In the present
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study, we found that the purified polysaccharide CMP33 possessed
immune-stimulating activity by inducing overproduction of NO, IL-6, TNF-α and
IL-1β in RAW264.7 cells, and also exhibited anti-inflammation activity by inhibiting
LPS-stimulated overproduction of NO, IL-6, TNF-α and IL-1β in RAW264.7 cells.
Declarations of interest
The authors report no declarations of interest.
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Legends
Figure 1 Elution curve by DEAE-52 cellulose column chromatography (A) and
Sephadex-G200 + Sephadex-G150 column chromatography (B).
Figure 2 Characterization of CMP33. (A) HPGPC, (B) GC, (C) infrared spectrum,
(D) Smith degradation and (E) Congo Red diagrams.
Figure 3 NMR diagrams of CMP33, (A) 1H, (B) 13C, (C) HSQC, (D) H-H COSY, (E)
HMBC.
Figure 4 (A) Effects G150 of CMP33 on proliferation of RAW264.7 cells without LPS
stimulation compared with LPS), (C) Effects of CMP33 on NO production in RAW264.7 cells
with LPS stimulation (*p＜0.05，**p＜0.01, compared with LPS group), (D) Effects