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International Journal of Biological Macromolecules 89 (2016) 707–716
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International Journal of Biological Macromolecules
journa l homepage: www.e lsev ier .com/ locate / i jb iomac
ntioxidant, antitumor and immunomodulatory activities ofater-soluble polysaccharides in Abrus cantoniensis
haowei Wu a, Xiong Fu a, Lijun You a, Arshad Mehmood Abbasi a,c, Hecheng Meng a,∗,ong Liu b,∗, Rana Muhammad Aadil a,d
School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, ChinaSchool of Applied Chemistry and Biological Technology, Shenzhen Polytechnic, Shenzhen 518055, ChinaDepartment of Environmental Sciences, COMSATS Institute of Information Technology, Abbottabad 22060, PakistanNational Institute of Food Science and Technology, University of Agriculture, Faisalabad 38000, Pakistan
r t i c l e i n f o
rticle history:eceived 20 October 2015eceived in revised form 18 February 2016ccepted 3 April 2016vailable online 4 April 2016
eywords:olysaccharides
a b s t r a c t
Abrus cantoniensis is a vegetative food in tropical areas of Asia and claimed as folk beverages and soupsconsumed for cleansing liver toxicants and preventing liver diseases. Polysaccharides (ACP-I and ACP-II)were extracted with hot water from A. cantoniensis, and isolated by DEAE cellulose chromatography. Thechemical properties as well as antioxidant, antitumor and immunomodulatory activities of ACP-I andACP-II were investigated. The results showed that the ACP-I (9.09 kDa) contained only glucose and ACP-II (38.45 kDa) consisted of rhamnose, arabinose, galactose and glucose. ACP-II exhibited higher oxygenradical absorbance capacity (ORAC) and hydroxyl radical prevention capacity (HRPC) than ACP-I with
brus cantoniensisntitumor
mmunomodulatory
ORAC values and HRPC values of 53.42 ± 3.32 �mol Trolox equiv./g DW and 34.84 ± 5.07 �mol Troloxequiv./g DW. Besides, in the wound healing assay, ACP-II exhibited potent migration inhibitory effectson MCF-7 cells. ACP-II could also significantly stimulate the proliferation of splenocytes and thymocytes,and enhanced NO production of peritoneal macrophages. These findings suggest that the polysaccharideACP-II in A. cantoniensis could be served as a novel potential functional food.
© 2016 Published by Elsevier B.V.
. Introduction
Genus Abrus is represented by 17 species which are distributedorldwide [1]. Only four species are cultivated in China, namely,
brus cantoniensis Hance, Abrus mollis Hance, Abrus pulchellus Wallnd Abrus precatorius Linn. The dominant variety of Abrus in Chinas A. cantoniensis and A. mollis, A. cantoniens, called “Jigucao” orChicken bone herb”, is well-known in Chinese folk as edible herbnd vegetative food consumed as the beverage and soup [2]. It isne of the main constituent of a popular folk drink, the red-cannedhinese herbal tea “Wong Lo Kat”. In southern China, particularly inuangdong and Guangxi areas people use this herbal tea as appe-
izer, especially after taking hotpot, barbecue, and fried food. Inummer, Cantonese would add this herb into soup together with
ork and chicken, or cook it alone in hot water as family drink. Theseeverage or soup have good curative effects for controlling inter-∗ Corresponding authors.E-mail addresses: [email protected] (H. Meng), [email protected]
D. Liu).
ttp://dx.doi.org/10.1016/j.ijbiomac.2016.04.005141-8130/© 2016 Published by Elsevier B.V.
nal heat, removing toxicity, dissipating blood stasis, and preventingjaundice as well as treating infectious hepatitis [3].
Previous studies have reported that extracts of Abrus speciesrevealed a wide range of pharmacological activities such asantioxidant properties [4], hepatoprotective effects [5], antitumoractivity [6], gastroprotective effect [7] and immunomodulatoryactivities [8]. It has been revealed that phytochemicals such asalkaloids, flavonoids, phenolic compounds, terpenoids, sapogenoland saponins, were components responsible for these activities[9]. Among these components, abrine (N-methyl-l-tryptophan),known as alkaloid, has been considered as the bioactive compoundfor hepartoprotective effects of A. cantoniensis [3]. Three flavonoidC-glycosides, including vicenin-2, isoschaftoside, and schaftosidewere reported to be the major bioactive compounds of A. mollisfor the antioxidant, anti-inflammatory, and the antiplatelet activi-ties as well as the hepatoprotective effect [10]. C-3 ketone and thelupine skeleton are important for the �-amylase inhibitory activityof A. precatorius [11].
However, most of these compounds are small molecules, andlittle information is available for the macromolecules from A. can-toniensis. Polysaccharides, as widely distributed macromolecules,have also attracted great attention owing to their antioxidant, anti-
708 S. Wu et al. / International Journal of Biological Macromolecules 89 (2016) 707–716
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ig. 1. Characterization of ACP-I and ACP-II. (A) Monosaccharide composition analonosaccharide; (b) and (e) for ACP-I; (c) and (f) for ACP-II. Peak 1 represents for
PGPC chromatogram of ACP-I and ACP-II and the standard curve of molecular wei
umor and immunomodulatory activities [12], anti-hyperglycemic13] and hepatoprotective effects [15] with low toxicity andnsignificant side effects [14]. The most attracting bioactivities
y HPLC-ELSD assay and HPAEC-PAD assay (B); (a) and (d) the mixture of standardose, 2 for arabinose, 3 for xylose, 4 for mannose, 5 for galactose, 6 for glucose. (C)) FT-IR spectra of ACP-I and ACP-II.
are the immunomodulatory and anti-cancer effects [16]. In addi-tion, numerous studies have revealed the positive correlationbetween bioactivities of the tested compounds in A. cantoniensis
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ig. 2. Antioxidant activities of ACP-I and ACP-II. (A) ORAC measurement on the flueasurement on the fluorescein consumption induced by hydroxyl radical genera
oncentrations and the net area under the fluorescence decay curve of treated grou
9]. For instance, the anti-proliferative activities and gastroprotec-ive effect of bioactive compound in A. cantoniensis were reportede due to the antioxidant activities [7,9]. Therefore, it is meaning-ul to explore the anti-tumor, immunomodulatory effects as wells antioxidant activities of polysaccharide in A. cantoniensis.
In the present investigation, two water-soluble polysaccharidesACP-I and ACP-II) were isolated and purified from A. cantoniensis.he chemical structure of ACP-I and ACP-II were characterized byigh performance gel-permeation chromatography (HPGPC), higherformance liquid chromatography-evaporative light scatteringetector (HPLC-ELSD), high performance anion-exchange chro-atography with pulsed amperometric detection (HPAEC-PAD)
nd Fourier-transform infrared (FT-IR) spectroscopy. Additionally,ntioxidant, antitumor and immunomodulatory activity as well asheir correlation were also investigated.
. Materials and methods
.1. Materials
A. cantoniensis were purchased from Foshan city, Guangdong
rovince, China, sliced and grinded into fine powder before extrac-ion. Male BALB/c mice of 8–10 weeks old were purchased fromhe Experimental Animal Center of Sun Yat-Sen University inuangzhou, China.ein consumption induced by peroxyl radicals (ROO•) generated by ABAP. (B) HRPC the mixture of H2O2 and Co (II). Insert: linear dependence relations between theus control group.
2.2. Chemicals
Monosaccharide standards of glucose (Glc), xylose (Xyl), ara-binose (Ara), galactose (Gal), mannose (Man) and rhamnose(Rha) were purchased from Sigma Chemical Co., St. Louis,Mo., USA. DEAE-52 cellulose was purchased from Waterman(Maidstone, Kent, UK). 6-Hydroxy-2,5,7,8-tetramethylchronman-2-carboxylic acid (Trolox), fluorescein disodium (FL), 2,2′-azobis (2-amidinopropane) dihydrochloride (ABAP), hydrocorti-sone, Lipopolysaccharide (LPS) and Concanavalin A (ConA) wereobtained from Sigma Chemical Company. WME medium, DMEMmedium, Hank’s balanced salt solution (HBSS), insulin, peni-cillin, streptomycin and gentamicin were purchased from GibcoBiotechnology Co. Hydrogen peroxide (30%), cobalt(II) chloridehexahydrate and other reagents were used of analytical grade.
2.3. Isolation and purification
300 g of dried powder in triplicate was extracted with 9000 mLof distilled water at 92 ◦C for 3 h. After centrifugation at 4500 rpmfor 15 min, supernatants were combined and concentrated to onetenth of the initial volume by rotary vacuum evaporator at 45 ◦C.
Then the extract was deproteinized by Sevag method for six times[17], followed by decoloration with the D354FD macroporous anionexchange resin (Zhengguang Resin Co., Ltd., Hangzhou, China) at50 ◦C for 30 min. Resulting solution was precipitated by the addition
710 S. Wu et al. / International Journal of Biologic
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ig. 3. Antiproliferative activity and cytotoxicity of ACP-I and ACP-II towards humanepatoma HepG2 cells (A) and human breast MCF-7 cells (B). The values are pre-ented as the mean ± SD of triplicates. *p < 0.05, **p < 0.01 versus control group.
nhydrous ethanol to a final concentration of 75% (v/v) and wasept overnight at 4 ◦C. A. cantoniensis crude polysaccharides (ACP)ere collected and freezed-dried after centrifugation at 4500 rpm
or 15 min.ACP (500 mg) was re-dissolved in deionized water and loaded
nto a DEAE-52 (OH−) cellulose column (2.6 × 100 cm; Whatman,SA) equilibrated with deionized water. ACP was fractionated andluted by deionized water and 0.2 M NaCl solution at the flowate of 0.6 mL/min, resulting in two separate fractions, ACP-I andCP-II, respectively. These polysaccharide fractions were furtheroncentrated, dialyzed (cut-off: 3.5 kDa) and lyophilized in a vac-um freeze drier. Sugar content of ACP-I and ACP-II was determinedy phenol-sulfuric acid method as explained before [18]. Possi-le endotoxin contamination was analyzed using a chromogenicAL/TAL endpoint assay kit as described before [19].
.4. Characterization of ACP-I and ACP-II
.4.1. Determination of molecular weightMolecular weight of purified polysaccharides was determined
y high performance gel-permeation chromatography (HPGPC)n an Agilent 1260HPLC system (Agilent, USA) equipped with aSK-GEL G4000PWxl column (7.8 × 300 mm; Tosoh, Tokyo) and aefractive index detector (RID) (Agilent, USA) using the method
xplained by Xu et al. [20]. A set of pullulan standards with molec-lar weights of 5.90, 9.60, 21.1, 47.1, 107, 200, 340, and 788 kDaShodex Standard P-82; Macherey-Nagel, Germany) were used foraking the calibration curve. Measurements were performed at
al Macromolecules 89 (2016) 707–716
35 ◦C and 0.02 M potasium phosphate buffer (pH 6.0) was used asmobile phase with a flow rate of 0.6 mL/min.
2.4.2. Monosaccharide composition analysis of ACP-I and ACP-IIThe monosaccharide compositions of ACP-I and ACP-II were
determined by HPLC-ELSD and HPAEC-PAD analysis as reportedearlier [21]. Briefly, ACP-I and ACP-II fractions were hydrolyzedwith 2 M trifluoroacetic acid at 110 ◦C for 4 h. Excessive acid wasremoved by co-evaporation with 200 �L methanol to dryness forthree times.
One part of the hydrolysate was analyzed by the Agilent HPLCinstrument with an Alltech 3300 evaporative light scattering detec-tor (ELSD) (Alltech Associates, USA). The hydrolysate was analyzedby Asahipak NH2P-50 4E column (4.6 × 250 mm; Shodex, Japan)coupled with a guard column of NH2P-50G column (4.6 × 50 mm;Shodex, Japan), which was eluted with a mixture of acetonitrile andwater in a ratio of 80:20 (v/v, %) at a flow rate of 0.8 mL/min. Resultswere compared and quantified using the following monosac-charide standards: l-rhamnose, l-arabiose, d-xylose, d-mannose,d-galactose and d-glucose.
Another part of the hydrolysate was measured by high perfor-mance anion exchange chromatography (HPAEC) analysis, whichwas carried out on an ICS-5000 system (Dionex, USA) coupledwith a pulsed amperometric detector (PAD) using a CarboPacPA1 column (4.0 × 250 mm, Dionex, USA) coupled with its guardcolumn (4.0 × 50 mm) under a gradient elution (mobile phase:H2O/100 mM NaOH) at 1 mL/min. Sugar identification was carriedby comparison with reference sugars (l-rhamnose, l-arabiose, d-xylose, d-galactose and d-glucose).
2.4.3. FT-IR spectral analysisIR spectra for polysaccharides were recorded with a Vector 33
FT-IR spectrophotometer (Bruker, Ettlingen, Germany) as men-tioned before [22]. The dried samples were ground and pressedinto KBr pellets for measurement in the wave number range of4000–400 cm−1.
2.5. Antioxidant activity assays
2.5.1. Determination of oxygen radical scavenging capacity(ORAC)
The antioxidant activity of ACP-I and ACP-II was estimated byoxygen radical absorbance capacity (ORAC) assay [23], using fluo-rescein (FL) as the flurescent probe. Briefly, 20 �L of blank, Troloxstandard solutions (6.25–50 �M) and purified polysaccharide solu-tions were transferred to triplicate wells in a black 96-well plate(Corning Scientific). After incubation at 37 ◦C for 10 min, 200 �Lof fluorescein (0.96 �M) was added to each well and incubated at37 ◦C for another 20 min. Fluorescein consumption was immedi-ately measured after adding 20 �L of ABAP (119.4 mM) to each wellat every 2.5 min for 50 cycle on a Fluoroskan Ascent FL plate reader(Thermo, USA) with the excitation at 485 nm and the emission at538 nm. Final values were calculated using the linear relationship(Y = a + bX) between Trolox concentration (X) (�M) and the areaunder the fluorescence versus time curve for samples minus thearea under the curve for the blank (Y). ORAC values were expressedas micro mole Trolox equivalent per gram of dried sample (�molTrolox equiv./g DW). Data were presented as mean ± SD of threereplicates.
2.5.2. Estimation of hydroxyl radical prevention capacity (HRPC)HRPC assay was performed as explained by Moore et al. [24].
Briefly, 20 �L of blank, Trolox standard, or polysaccharide dilutionswith 75 mM PBS (pH 7.4) were added to a black 96-well plate in trip-licate, followed by the addition of 200 �L fluorescein (0.96 �M) toeach well and incubated at 37 ◦C for 20 min. Then 10 �L of freshly
S. Wu et al. / International Journal of Biological Macromolecules 89 (2016) 707–716 711
Table 1Correlation coefficient matrix between antioxidant, antitumor and immunomodu-latory activities.
Trial ORAC HRPC HCPR MCPR MCAMR SPI TPI NOSPM
ORAC 1.000HRPC 0.395 1.000HCPR −0.904** −0.245 1.000MCPR 0.697** 0.589** −0.324 1.0000MCAMR 0.216 0.241 −0.325 −0.086 1.000SPI 0.831** 0.214 −0.790** 0.325 0.556* 1.000TPI 0.769** −0.235 −0.618** 0.049 0.586* 0.670** 1.000NOSPM 0.848** 0.214 −0.838** 0.508** 0.716** 0.888** 0.593** 1.000
ORAC, oxygen radical absorbance capacity; HRPC, hydroxyl radical preventioncapacity; HCPR, HepG2 cell proliferation ratio; MCPR, MCF-7 cell proliferation ratio;MCAMR, MCF-7 cell anti-migration rate; SPI, splenocyte proliferation index; TPI,t*t
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Fig. 4. Effects of ACP-I and ACP-II on the migration of human breast MCF-7 cells bywound healing assay. MCF-7 cells were treated with different concentration levelsof ACP-I and ACP-II (0, 125, 250, 375 �g/mL). (A) Images of ACP-I or ACP-II treatedMCF-7 cells at 0, 8, 16, 24 and 32 h compared with control group. (B) Cell migration
hymocyte proliferation index; NOSPM, NO secretion from peritoneal macrophage;*, correlation is significant at the 0.01 level (2-tailed); *, correlation is significant athe 0.05 level (2-tailed).
repared hydrogen peroxide (1.1 M) was added into appropriateells using an automatic dispenser assembled on the fluorescenceicroplate reader. After shaking for 5 s, initial reading of fluores-
ence intensity was recorded and reaction was started by 10 �Lf cobalt chloride solution (Co = 9.2 mM) automatically dispensedith dispenser. Fluorescence intensity was recorded after everyinute for 40 min with the excitation at 485 nm and emission at
38 nm. Trolox solutions at the concentration of 0.25, 0.50, 0.75 and.0 mM were used as standards and 75 mM phosphate buffer (pH.4) was used as blank. The HRPC values were calculated using aegression equation between the Trolox concentration and the netrea between the fluorescence versus time curve of the treatmentnd the blank and expressed as �mol Trolox equiv./g DW. Dataere presented as mean ± SD of three replicates.
.6. Cell line cultures
Human liver cancer line HepG2 (ATCC HB-8065) and humanreast cancer line MCF-7 (ATCC HTB-22) were purchased from ATCCompany. HepG2 cells were cultured in growth medium (WMEupplemented with 5% fatal bovine serum FBS, 10 mM Hepes,
mM l-glutamine, 5 �g/mL insulin, 0.05 �g/mL hydrocortisone,0 units/mL penicillin, 50 �g/mL streptomycin, and 100 �g/mLentmycin). MCF-7 cells were cultured in DMEM supplementedith 10% (v/v) FBS and 50 units/mL of penicillin and 50 �g/mL of
treptomycin and 100 �g/mL gentmycin. HepG2 and MCF-7 cellsere maintained at 37 ◦C in a humidified atmosphere containing
% CO2.
.7. Antitumor assay
.7.1. Cytotoxicity and antiproliferative activityMethylene blue colorimetric method was performed to evalu-
te the cytotoxicity and antiproliferative effects of ACP-I and ACP-IIractions on HepG2 and MCF-7 cells as demonstrated earlier [25].he cells were cultured in 96-well plates at a density of 4 × 104
nd 2.5 × 104 cells/well for cytotoxicity and antiproliferative activ-ty assay respectively, and incubated for 6 h at 37 ◦C. Old medium
as replaced with 100 �L fresh medium in each well including con-rol (without polysaccharides) and with polysaccharides at variousoncentrations (31.2–375 �g/mL). Blank wells were treated with00 �L of fresh medium without cells. After incubation at 37 ◦Cor 24 h and 72 h to measure the cytotoxicity or antiproliferativectivity respectively, the medium was removed and the cells were
ashed with PBS. About 50 �L of methylene blue solution (HBSSontaining 0.6% methylene blue and 1.25% glutaraldehyde) wasdded in each well followed by incubation at 37 ◦C for another 1 h.he staining was removed by washing 96-well microplate in deion-
rate at 16, 24 and 32 h are represented as the means ± SD obtained from three inde-pendent experiments. Significance difference with control group was described as*p < 0.05,**p < 0.01.
ized water until the water was clear. Then 100 �L elution buffer(49% (v/v) PBS, 50% (v/v) ethanol, and 1% (v/v) acetic acid) wasadded and 96-well microplate was oscillated for 20 min at roomtemperature before taking reading on microplate reader at 570 nm.
Cytotoxicity was calculated by using formula as given below:
Ratio (%) = [(Abs1 − Abs2) Abs1] × 100
HepG2 cell proliferation ratio (HCPR) and MCF-7 cell prolifera-
tion ratio (MCPR) were calculated using the formula:Ratio (%) = Abs2
Abs1× 100
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here Abs1 is the absorbance of control, and Abs2 is the absorbancef treated cells. If cytotoxicity value of tested polysaccharidexceeded 10% compared with the control, the corresponding con-entration was considered to be cytotoxic.
.7.2. Wound healing assayCell migration assay was performed as described by Park et al.
26]. Briefly, MCF-7 human breast cancer cells were seeded into 24-ell plates, at 80–90% confluency, the cell monolayer was scratchedith a 200 �L micropipette and washed with PBS twice to remove
ell debris. The remaining cells were treated with fresh mediumontaining different concentrations of ACP-I or ACP-II. Woundedrea were observed and captured by microscope and cell migrationate was calculated by ImageJ software version 1.48v (http://rsb.nfo.nih.gov/ij/). MCF-7 cells anti-migration rate (MCAMR, %) wasresented as the percentage of cell covering area to total woundedrea, which was equal to the wounded area of corresponding 0 h.
.8. Immunomodulatory activities
.8.1. Animal cell isolation and cultureCells’ isolation and culturing was carried out as reported before
27]. Spleen and thymus cells were aseptically removed fromALB/c mice and gently homogenized with a syringe piston theninced through a 200 mesh cell strainer (BD Biosciences) to achieve
ingle cell suspension. After lysed the red blood cells with Tris-H4Cl lysis buffer, the cells were washed with PBS, adjusted to aensity of 5 × 106 cells/mL in DMEM containing 10% FBS (v/v) andept on ice for further use.
Peritoneal macrophages were obtained from the peritonealavity of mice as described earlier [28]. Mice was injected intra-eritoneally with 3% Brewer thioglycollate (BD Biosciences) threeays before scarification and sterile peritoneal lavage with 10 mLBS without calcium and magnesium. After centrifugation at 200gor 10 min the cells pellets were suspended in cold DMEM complete
edium and adjusted to a concentration to 2 × 106 cells/mL thenept on ice.
.8.2. Splenocyte and thymocyte proliferation testThe effects of polysaccharides fractions ACP-I and ACP-II on
he splenocyte and thymocyte proliferation were determinedsing the MTT assay [29]. Splenocyte or thymocyte suspension5 × 106 cells/mL) was seeded in to 96-well culture plate in thebsence and presence of polysaccharide fractions (31.2, 62.5, 125,50 and 375 �g/mL). Wells containing ConA (5 �g/mL) or LPS10 �g/mL) alone served as positive control and with medium alones blank. After incubation at 37 ◦C for 48 h, the culture was treatedith 20 �L MTT (5 mg/mL) and kept for 4 h, supernatant was dis-
arded and 100 �L DMSO was added in each well to dissolve theesulting formazan for 20 min. The extent of lymphocyte cell pro-iferation was measured at 570 nm using microplate reader. Theplenocyte proliferation index (SPI) and thymocyte proliferationndex (TPI) were calculated from the ratio of the optical densityOD) values of treated group to the control group.
.8.3. Determination of nitric oxide (NO) production100 �L of mouse peritoneal macrophages (2 × 106 cells/mL) was
eeded in 96-well plate and allowed to adhere for 2 h at 37 ◦C in 5%O2 humidified incubator. Non-adherent cells were removed byashing with medium and adherent macrophages were further
ultured with fresh medium alone or media containing vari-
us concentrations of polysaccharides (31.2, 62.5, 125, 250, and75 �g/mL) and LPS (10 �g/mL, as positive control) for 48 h. At thend of the culture period, nitrite content (NO) secreted by peri-oneal macrophage (NOSPM) was determined by a NO assay kital Macromolecules 89 (2016) 707–716
(Nanjing Jiancheng Bioengineering Institute, Nanjing) according tothe manufacturer’s instruction [30].
2.9. Statistical analysis
Data were expressed as means ± standard deviations (SD) (n = 3)of three replicates. One way ANOVA was used to determine the sta-tistical differences between the experimental groups and controlgroup and P values of 0.05 or less were considered to be statisticallysignificant. Correlation coefficients among the tested bioactivitiesof ACP-I and ACP-II under the same concentration were also calcu-lated. All data were reported as the mean ± SD for three replicates.
3. Results and discussion
3.1. Isolation and purification of polysaccharides
About 12.8% A. cantoniensis crude polysaccharide (ACP) wasobtained from hot water extract of A. cantoniensis. Two fractions ofwater soluble polysaccharides called ACP-I and ACP-II were isolatedby DEAE-52 cellulose column, which were eluted by the deionizedwater and 0.2 M NaCl solution respectively. ACP-I was appeared aswhite plate powder while ACP-II was obtained as primrose yellowloose powder.
3.2. Characterization of ACP-I and ACP-II
Total carbohydrate content of purified polysaccharides ACP-I and ACP-II fractions was 98.3% and 93.7%, respectively. Thesepolysaccharide solutions were found to be free from endotox-ins. ACP-I was considered as glucan, which could be inducedfrom the results of HPLC-ELSD and HPAEC-PAD analysis. Themonosaccharide composition of ACP-II was analyzed by HPLC-ELSDsystem as given in Fig. 1A, which indicated that ACP-II is a het-eropolysaccharide, and consisted of four monosaccharides suchas rhamnose, arabinose, galactose and glucose with a molar ratioof 1:3.41:3.62:10.91. Results obtained from HPAEC-PAD analysis(Fig. 1B) also verified the presence of four monosaccharides forACP-II with molar ratio of 1:3.46:4.56:10.22. Glucose appeared tobe the predominate monosaccharide, followed by galactose andarabinose.
Based on the distribution of molecule weight as determinedby HPGPC analysis on an Agilent system, ACP-I and ACP-II frac-tions were found to be homogeneous polysaccharides as shownin Fig. 1C. The average molecular weight of ACP-I and ACP-II was9.09 kDa and 38.45 kDa, respectively. ACP-I and ACP-II showed noabsorbance peaks at 280 and 260 nm by UV scanning and thisrevealed the absence of protein and nucleic acid in polysaccha-rides (data not shown). Results of FT-IR spectra are mentioned inFig. 1D, which indicated distinctive absorptions of polysaccharideswith a broad stretching intense characteristic peak at 3400 cm−1
for the hydroxyl group and a weak band at 2934 cm−1 for C Hstretching and bending. The bands in the region of 1024, 1078 and1149 cm−1 for ACP-I, 1028, 1080 and 1151 cm−1 for ACP-II wereobserved easily. The absorptions ranged from 1000 to 1200 cm−1
attributed the stretching vibrations of C O C and C O H, this sug-gested the presence of C O bonds in polysaccharides. Particularly,the absorption at 1024 cm−1 for ACP-I and 1028 cm−1 for ACP-IIshowed the presence of pyranose ring. Moreover, the character-istic absorption bands at 856 cm−1 and 897 cm−1 indicated thatACP-II contained both �- and �-type glycosidic linkages in its struc-ture and ACP-I contained only �-glycosidic linkages at 858 cm−1.
In addition, ACP-I showed strong absorption at 935 cm−1, whichis typical for d-glucose in the pyranose form. This is in consistentwith the result of monosaccharide composition of ACP-I by bothHPLC-ELSD assay and HPAEC-PAD analysis. However, there was no
S. Wu et al. / International Journal of Biologic
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Fig. 5. Immunomodulatory activities of ACP-I and ACP-II. (A) and (B) Effects ofACP-I and ACP-II on the lymphocyte proliferation, lymphocytes (5 × 105 cells/well)were stimulated with medium alone (control) or polysaccharides as well as LPS(10 �g/mL) or ConA (5 �g/mL) alone served as positive group for 48 h. Cell pro-liferation was measured by MTT assay. (C) Effects of ACP-I and ACP-II on NOproduction from mouse peritoneal macrophages. Macrophages (2 × 105 cells/well)were incubated with fresh medium alone (control) or polysaccharides as wella*
g
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s LPS (10 �g/mL) for 48 h. The values are presented as the mean ± SD (n = 3).p < 0.05, **p < 0.01 versus control group; #p < 0.05, ##p < 0.01 versus LPS positiveroup; �p < 0.05, ��p < 0.01 versus ConA group.
bsorption near 1750 cm−1 corresponding to a carboxylic group,ndicating the absence of uronic acids existed in polysaccharides32].
.3. Antioxidant activities of ACP-I and ACP-II
Crude polysaccharides obtained from A. cantoniensis have been
eported as excellent scavengers of free radicals [33]. However,ery few studies have been conducted on isolated polysaccharideractions. In our previous work, we have ever tried to estimate thentioxidant capacity of polysaccharides using a more biological rel-al Macromolecules 89 (2016) 707–716 713
evant and quantifiable method, cellular antioxidant activity (CAA)assay. However, this quantification, based on the comparison ofmedian effective dose (EC50) of quercetin standard and sampleswas failed, since there was no increase in the CAA unit with theincreased rate of polysaccharide content up to 20 unit. This mightbe due to high molecular weight of polysaccharides or their com-plex behaviour in the solution, that make it difficult to pass throughthe cell membrane for uptake or metabolism within 1 h [31]. Weestimated the antioxidant properties of ACP-I and ACP-II by ORACand HRPC assays in kinetic way. The kinetic protective behaviour byACP-I and ACP-II against fluorescent decay induced by ABAP pre-sented in Fig. 2A, revealed that the fluorescent decay curves forACP-I and ACP-II were above that of blank. Furthermore, both ACP-I and ACP-II showed excellent linear relationship between the netarea and concentrations (Fig. 2A) with the correlation coefficientsof R2 = 0.994 and R2 = 0.982, respectively.
Among the physiologically relevant ROS, hydroxyl radical isknown as the most reactive chemical species, which extremelyreacts with almost every type of biomolecules. Hence, it is imper-ative to evaluate the hydroxyl radical scavenging or preventioncapacity of foods, phytochemicals, and dietary supplements [24]. Inthe HRPC assay, hydroxyl radical served as free radical and gener-ated by a Co(II)-mediated Fenton-like reaction [34]. Kinetic studiesindicated that the inhibitory effects of polysaccharides on OH• rad-ical showed a complex behaviour as demonstrated in Fig. 2B. Thefluorescent decay curves of ACP-I were very close or even belowto that of the blank suggesting the generation of reactive species.Similar behaviour has been reported previously for some polysac-charides, such as water soluble polysaccharides extracted fromIsaria farinosa B05 were found to be lack of scavenging activitytowards hydroxyl radicals [35]. It was reported that chitosan sul-fate enhanced hydroxyl radical generation and showed negativescavenging values for hydroxyl radicals using spin trapping spec-troscopy directly [36].
Measured values of oxygen radical antioxidant capacity forACP-II was 53.42 ± 3.32 �mol Trolox equiv./g DW at 0.625 mg/mL,and the HRPC value was 34.84 ± 5.07 �mol Trolox equiv./g DW at1.25 mg/mL. ORAC value for ACP-I was 13.42 ± 3.32 �mol Troloxequiv./g DW at 2.5 mg/mL but hydroxyl radical antioxidant capac-ity assay for ACP-I demonstrated negative values even at a highconcentration of 10 mg/mL. Present analysis showed that ACP-II exhibited relatively strong oxygen radical antioxidant capacitycompared ACP-I and reported levels for polysaccharides purifiedfrom the leaves and stem of Rabdosia serra, Triticale bran and Lami-naria japonica at 10–40, 33.86, and 123.14 �mol Trolox equiv./g DWat 2.5 mg/mL [37]. It is pertinent to mention here that the antiox-idant activities of A. cantoniensis polysaccharides are not as highas small molecules such as polyphenols and flavonoids; however,considering the fact that these food polysaccharides are water sol-uble and can be consumed in larger quantities extensively as thebeverage and soup and make them beneficial for human health.
3.4. Cell proliferation assay
Antiproliferative activities and cytotoxicity of ACP-I and ACP-IItowards HepG2 and MCF-7 cells were evaluated in vitro and resultsare presented in Fig. 3. It was noted that both fractions showed nocytotoxicity towards HepG2 cells and MCF-7 cells at the concentra-tion lower than 375 �g/mL. Similarly, ACP-I and ACP-II exhibitedinsignificant antiproliferative effects against HepG2 cells and MCF-
7 cells at the concentrations of 31.25–375 �g/mL. However, ACP-IIshowed 75% proliferation rate towards HepG2 cells at the concen-trations 375 �g/mL compared with the control (100%) as given inFig. 3A.
7 ologic
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14 S. Wu et al. / International Journal of Bi
.5. Inhibitory effect of ACP-I and ACP-II on migration of MCF-7ells
Majority of the cancer cells move across from one site to anothern the host and grow to form secondary tumor [38]. The patientsf breast cancer some time suffer from tumor recurrence becauseven after complete resection of their localized cancer, subsequentacroscopic metastases develop again and cause metastatic dis-
ase, which leads to cancer-related death [39]. Wound healingssay was adopted to investigate the inhibitory migration effectsf polysaccharides ACP-I and ACP-II. Cells moving into the scarredegion reduced the wounded area [40], which was considered ashe total area between two edges of the scar at 0 h time point.ccording to this principle, the cell covering area between twodges of the scar was compared to detect the inhibitory effectsf the polysaccharide fractions on MCF-7 cell migration as shown
n Fig. 4A. In control group the scratch area was occupied by theigrating cancer cells without polysaccharide treatment, whereas
ewer cells moved into the surface area of the scar in the polysac-harides treated group. The quantitative results indicated thatCP-I and ACP-II had significant effects on MCF-7 cells migration
n dose and time-dependent ways (Fig. 4B). Compared with theigration rate of 82.30% of control for 32 h culture, migration rate
f ACP-I treated MCF-7 cells was 58.36%, 52.47% and 42.27% athe concentrations of 125, 250 and 375 �g/mL, respectively, andhat of ACP-II was 51.45%, 49.13% and 49.06%. Numerous stud-es have been reported that natural plant polysaccharides fromarious origins exhibit anti-metastatic effects such as Scutellariaarbata [41], Panax ginseng [42], Colocasia esculenta [16], Rosa rox-urghii Tratt [43]. It is well known that tumor metastasis occursn many steps including vessel formation, cell attachment, adhe-ion, migration, invasion and cell proliferation [44]. Thus, the wayolysaccharides involve in these events is an extremely complexechanism. Lee et al. [45] believed that polysaccharides contain-
ng arabinogalactan residues inhibit metastasis. Herein, ACP-1,ithout containing arabinogalcatan residues, displayed compara-
le anti-migration activity with ACP-II, indicating the possibilityf other factors except for arabinogalactan residues affecting thenti-migration activity of polysaccharides. Some polysaccharidesuch as Rosa roxburghii Tratt, P. ginseng and Sepiella maindroni inkolysaccharides [46] not only possess antimetastatic properties,ut also obviously inhibit metastatic tumor growth. Obviously,hese polysaccharides could suppress tumor growth as well as
etastasis. In the present study, as illustrated in Fig. 3B, no antipro-iferative activity and cytotoxicity on MCF-7 cells were observedor ACP-I and ACP-II within concentration range. Furthermore, noignificant correlation was observed between the anti-migrationate and the proliferation ratio of MCF-7 cells in Table 1. Theseesults indicated that the polysaccharide fractions dose not inhibithe growth of cancer cells directly but can cause significant inhibi-ion of migration in MCF-7 cells. Similar results were also observedn polysaccharides from Inonotus obliquus [44] and brown seaweedscophyllum nodosum [47]. Although the mechanism of their anti-etastatic action remains unclear, we believed that A. cantoniensis
olysaccharides can be considered a useful candidate in inhibitionf metastasis.
.6. Immunomodulatory activities
Lymphocyte proliferation is an important indicator of immuneesponse [48]. Spleen or thymus cells were isolated from mice andtimulated by ACP-I and ACP-II with different concentrations of
1.2, 62.5, 125, 250, and 375 �g/mL. Treatment with the T cell mito-en ConA and B cell mitogen LPS alone were also investigated asositive group. The former was designed to evaluate T lymphocytectivity while the later one was for the assessment of B lymphocyteal Macromolecules 89 (2016) 707–716
activity. The polysaccharides fractions ACP-I and ACP-II showedsignificant facilitation to splenocyte proliferation compared to con-trol (p < 0.01) as mentioned in Fig. 5A. The facilitation induced byACP-II had a prominent-related increment at the concentrationranged 31.2–375 �g/mL with the proliferation index of 1.23, 1.33,1.36, 1.44 and 1.45 compared to the proliferation index of controlgroup (SPI: 1.00) and that of ACP-I. Likewise, in thymocyte prolif-eration test (Fig. 5B) ACP-I and ACP-II showed greater proliferationactivity compared to the control (TPI: 1.00, p < 0.01). Moreover,ACP-II demonstrated significant proliferation effect on splenocyteand thymocyte at the concentration of 250 �g/mL, which was evenhigher than that treated with LPS or ConA alone, suggesting a facil-itation on LPS-induced or ConA-induced lymphocyte proliferationby ACP-II.
Since stimulation of mouse macrophages with LPS leads toan inflammatory cascade through Toll-like receptor 4 (TLR4) andresulted in a series of products including TNF-�, NO and otherintermediates for eliminating the remaining microorganisms in theinflammatory loci [49]. Among those products, nitric oxide (NO)plays important role in the natural defence system and a varietyof physiological activity [50]. In the present study change in theNO production in macrophages culture with ACP-I and ACP-II wasinvestigated. Results indicated that both ACP-I and ACP-II induced asignificant increase of NO production in a dose-dependent manner(Fig. 5C), compared to the treatment with medium alone (NOSPM:2.45, p < 0.01). The NO production stimulated by ACP-II at 125, 250and 375 �g/mL was 7.24, 7.89 and 8.37 �M, respectively, indicat-ing stronger stimulating effect and was even higher than that of LPScontrol (NO production: 6.51 �g/mL).
3.7. Correlation analysis
Pearson’s correlation coefficients between the antioxidant, anti-tumor, and immunomodulatory activities in vitro were computedand results are presented in Table 1. The immunomodulatoryactivities including SPI, TPI and NOSPM showed relatively highpositive correlation with ORAC values (r = 0.831, 0.769 and 0.848,respectively), and a mild positive correlation with the MCF-7 cellsanti-migration rate (MCAMR, r = 0.556, 0.586 and 0.716, respec-tively). Although these results were carried out in vitro, it providesa clue that the antioxidant activity and cancer cells anti-migrationeffect of polysaccharides from A. cantoniensis might be relatedto their immonumodulatory effects or were mediated throughactivating the immune system of the host. Studies regardingpolysaccharides exerting antitumor activity or other bioactivitythrough their immunomodulatory effect have been perceived bymany researchers. Zheng et al. [51] reported that the enhancementof antioxidants and immune response was responsible for the anti-cancer effect of Purslane polysaccharides (PPs) in gastric cancer.Plant C. esculenta polysaccharide Taro-4-I exerted the anti-cancerand anti-metastatic activity of through immunostimulation [16].These results confirmed that further studies in vivo are necessaryto carried out for revealing their relationship.
4. Conclusion
Two water soluble polysaccharide fractions (ACP-I and ACP-II) were isolated and purified from A. cantoniensis by anionexchange chromatography. Compared with ACP-I, ACP-II possessedconsiderable antioxidant activities in ORAC and HPRC assays.Besides, ACP-II demonstrated significant anti-migration effects
on MCF-7 cells. Fraction ACP-II also exhibited immunomodula-tory activities deduced from their stimulating the proliferationof splenocytes or thymocytes and enhancing NO production ofperitoneal macrophages. Moreover, ORAC values and MCF-7 cells
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nti-migration activity of the polysaccharides showed high cor-elation with their immunomodulatory activities. This study firstemonstrated the antioxidant, antitumor and immunomodulatoryctivities of the polysaccharides from A. cantoniensis and their cor-elation of these activities. Further studies about elucidating thenderlying mechanism for the correlation are in progress.
cknowledgements
The authors are grateful for the financial support provided by theuangzhou Science and Technology Program (2013J4500036), theuangdong Science and Technology Program (2014B090904063
2012B050500003), the Leading Talents Program in Guangdongrovince (Ruihai Liu), the Fundamental Research Funds for the Cen-ral Universities (No. 2013ZM0049), and National Natural Scienceoundation of China (No. 31101222).
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