Purification,structural elucidation,and in vitro antitumor effects of novel polysaccharides from Bangia fuscopurpurea

Jingna Wu,Xiaoting Chen,Kun Qiao,Yonghang Su,Zhiyu Liu,?aXiamen Key Laboratory of Marine Mediinal Natural Produts Resoures,Xiamen Medial College,361023,Xiamen,China

bFujian Universities and Colleges Engineering Research Center of Marine Biopharmaceutical Resources,Xiamen Medical College,361023,Xiamen,China

cFisheries Research Institute of Fujian,361013,Xiamen,China

Keywords:

Bangia fuscopurpurea

Polysaccharide

Structure,ovarian cancer

Antitumor effect

ABSTRACT

A water-soluble polysaccharide,designated BFP-3,was isolated from Bangia fuscopurpurea by hot water extraction,anion-exchange,and size-exclusion chromatography and tested to determine its antitumor activity.The structural characteristics of BFP-3 were investigated by chemical and spectroscopic methods,including partial acid hydrolysis,methylation analysis,one-and two-dimensional nuclear magnetic resonance,and gas chromatography-mass spectrometry.The results showed that BFP-3 was mainly comprised of rhamnose,arabinose,mannose,glucose,and galactose.Moreover,the weight-average molecular weight of BFP-3 was estimated to be approximately 333kDa.The backbone of BFP-3 was primarily composed of repeating 5-α-l-Araf-1→(4-α-d-Glcp-1)4→4,6-β-d-Manp-1units,and the side chains consisted of repeating β-d-Galp-1→(4-β-d-Galp-1)4→4,6-β-d-Galp-1→3,4-α-l-Rhap,β-l-Arap-1→(3-β-d-Galp-1)3,and β-l-Arap-1 units.Counting Kit-8 assays revealed that BFP-3 significantly inhibited the proliferation of A2780,COC1,SKOV3,HO-8910,and OVCAR3 ovarian cancer cells in vitro,indicating that BFP-3 could have potential applications in the treatment of ovarian cancer.

1.Introduction

Ovarian cancer is the leading cause of death from gynecological malignancy and the fifth most common cause of cancer-related death in developed countries[1].The ovaries are located in a difficult-to-image location,and there are currently no effective screening methods for the early detection of ovarian cancer[2].Approximately 70% of patients with ovarian cancer are diagnosed at a late stage,and the 5-year survival rate is about 40% for patients with all stages of ovarian cancer.Although therapeutic approaches for the treatment of cancer,such as chemotherapy and surgery,have rapidly developed in recent years,identification of successful treatments for ovarian cancer has been challenging owing to the high rate of advanced-stage diagnosis and acquisition of drug resistance in many cases[2,3].Cisplatin is a commonly used drug in the treatment of ovarian cancer,but often results in chemoresistance after repeated treatments,thereby limiting drug effectiveness[4].Accordingly,a search for safer and more effective compounds is needed.Various phytochemicals obtained from terrestrial sources have already been tested;however,only a few have been evaluated in clinical trials.Moreover,researchers have recently began searching for antitumor drugs from marine sources[5].

Among the many marine organisms, seaweeds are still regarded as undeveloped plant resources despite having been used as edible and medical sources for a long time[6].For decades,polysaccharides from marine algae,which have been reported to have broad-spectrum pharmacological effects and low toxicity,have been investigated as a promising source of anticancer drugs[7,8].Among marine algae,red and brown algae have been extensively studied as sources of polysaccharides,showing greater anticancer properties than green algae[9].Kang and colleagues[10,11]obtained a polysaccharide fromGracilariopsis lemaneiformiswith marked anticancer activity;this compound inhibited cell proliferation by inducing apoptosis and cell cycle arrest in a time-and concentration-dependent manner.Das Chagas Faustino Alves et al.[12]reported that a sulfated galactan fraction 1.0 from the red seaweedHypnea musciformis,composed mainly of polysaccharides and sulfate,possessed moderate antitumor activity in HeLa cells.Moreover,λ-carrageenan has been shown to have applications as a potent antitumor agent and an efficient adjuvant in cancer immunotherapy[13].

Usually,polysaccharides bioactivities are correlated with their chemical properties such as molecular sizes,monosaccharides constituent,and features of glycosidic linkages[14].A sulfated mannan(N6)and two xylomannans(N3 and N4)were isolated from red algaeNemalion helminthoides.These mannans had a linear backbone of(1→3)-linked α-D-mannopyranose units with sulfate groups at the 4-or 6-position,D-xylose side-chains were linked at 2-position in the xylomannans.The N3 and N4 exerted marked immunomodulatory activity,whereas this activity was not determined with N6[15].Fang and colleagues reported a novel water-soluble polysaccharide(LJP-31)fromLaminaria japonica,which exhibited significant immunomodulatory activity.The LJP-31 was mainly made up of arabinose,mannose,glucose and galactose in a molar ratio of 1.0:7.8:6.6:0.8,and the mannose was linked by(1→4)and(1→3,6)glycosidic bond,the glucose was linked by(1→4)glycosidic bond,and the other two monosaccharides were linked by(1→)glycosidic bond[16].

Bangiais a primitive red alga distributed throughout the world,with widespread marine populations present along northern and southern coastal areas[17].Bangia fuscopurpureaexists in the rocky intertidal zone,where it is subjected to wide fluctuations in external salinity due to ebb and flow tides,long periods of desiccation,and long exposure to high irradiance[18].Owing to its high nutrition and desirable flavor,B.fuscopurpureahas been cultured in Putian,Fujian province,China since the 1990s,making this location the only known area ofBangiacultivation in worldwide[19].

Accordingly,in this study,we evaluated a novel water-soluble polysaccharide fromB.fuscopurpurea,designated BFP-3,which had not been reported previously.Here,we describe the isolation,structural characterization,and evaluation of the bioactivities of BFP-3.

2.Materials and methods

2.1.Materials

B.fuscopurpureawas collected in September 2016 from Xiuyu District,Putian City(Fujian province,China).Human ovarian cancer cell lines(A2780,COC1,SKOV3,HO-8910,and OVCAR3)were provided by Meixuan Biological Science Co.,Ltd.(Shanghai,China).Diethylaminoethyl(DEAE)-Sepharose Fast Flow and Sephacryl S-100 High-Resolution were purchased from Science and Technology Development Co.Ltd.(Beijing,China).Dextrans,dimethyl sulfoxide and monosaccharide standards were from Sigma(St.Louis,MO,USA).Trifluoroacetic acid,acetic acid,acetic anhydride,sodium borohydride,chloroform,NaOH,methyl iodide,formic acid,methanol,trifluoroacetic acid,acetic anhydride and sodium sulfate were of analytical reagent grade and were from Sinopharm chemical reagent Co.,Ltd(Shanghai,China).

2.2.Extraction,isolation,and purification of polysaccharides

The plants were washed to remove epiphytes and mud and sand debris and were then shadow dried.The dried materials were cut into small pieces and extracted twice with distilled water(1:40,m/m)at 100?C for 4h each time.The combined aqueous extract was concentrated to 25% of the original volume by a rotary evaporator at 60?C and precipitated by the addition of ethanol at a final concentration of 85%(V/V)at 4?C overnight.After centrifuging,the separated precipitate was dissolved in an appropriate volume of ultrapure water and deproteinated by an enzymatic method(neutral protease,56?C,4h)until no specific absorbance at 260nm(nucleic acids)and 280nm(proteins)was detected.Subsequently,the crude polysaccharides were collected by centrifugation and lyophilization. The crude polysaccharides(2g)were dissolved in distilled water(10mL)and fractionated by a DEAESepharose Fast Flow(50mm×500mm)column using distilled water and 0.2mol/L NaCl as the eluent on a AKTA Purea25. The total polysaccharide content was determined spectrophotometrically at 490nm using the phenol-sulfuric acid method.Two fractions(BFP-1 and BFP-2)were collected,dialyzed(molecular weight cutoff[MWCO]:3500Da),and freeze-dried.BFP-2 was detected as the main antiproliferative fraction and was further purified using a Sephacryl S-100 High-Resolution(3.0cm×120cm)column to give one target polysaccharide,BFP-3,which was then lyophilized for antitumor evaluation and structural analysis.

2.3.Homogeneity and molecular weight

The homogeneity and molecular weight of BFP-3 were determined by high-performance gel-permeation chromatography(HPGPC)on a Waters 515 HPLC system. A sample solution(2mg/mL,20μL)was injected for each run and eluted with 0.2mol/L NaCl solution at 40?C at a flow rate of 0.8mL/min.Data were analyzed using GPC software.The homogeneity of BFP-3 was determined based on the peak shape of the HPGPC chromatogram.Eight analytical standards of dextran(molecular weights 5,12,25,50,80,150,270,and 410kDa)were used for the calibration curve.The molecular weight was identified on the basis of the standard dextran.

2.4.Monosaccharide composition analysis

The monosaccharide composition was measured by gas chromatography-mass spectrometry(GC–MS)on a Shimadzu GCMS-QP2010.Brie fly,2mg BFP-3 was hydrolyzed with trifluoroacetic acid(TFA,2mol/L,1mL)at110?C for 90 min, and the excess acid was then removed by evaporating with methyl alcohol(MeOH)after hydrolysis.The hydrolysates were then reduced with NaBH4(60mg) for 8 h and acetylated with aceticanhydride(1mL)at100?C for 1h.The standard monosaccharide was derived based on the same method.The acetylated derivatives were dissolved in 3mL anhydrous chloroform(CHCl3)for GC–MS analysis.

The chromatographic conditions were as follows:HP-5 capillary column(30m×0.25mm×320μm),increasing temperature from 120?C to 250?C at 3?C/min and holding at 250?C for 5min.The temperatures of both the injector and detector were set at 250?C.Helium was used as a carrier gas.The operating MS conditions were as follows: electron impact ionization,70eV;scan range,m/z50–550 amu;source temperature,230?C.The instrument was tuned occasionally using the autotune procedure;EI mass spectra were measured in the total ion monitoring mode, and the peak area(TIC)data were used for quantitative determination.

2.5.Partial acid hydrolysis

Based on the fact that glycosyl linkages of the side chain were more labile to acid than those of the main chain[20],the main chain and the side chain of BFP-3 were determined by partial hydrolysis.BFP-3(50mg)was hydrolyzed with 0.2mol/L TFA(10mL)at 100?C for 1h,and the residue was then dialyzed(3500 Da cut-off membrane)with ultrapure water.The retained and permeated fractions were assigned as BFP-3?0.2I and BFP-3?0.2E,respectively.After the BFP-3?0.2I was further hydrolyzed with 0.6 mol/L TFA(100mL)at 100?C for 1h,the residue was dialyzed(MWCO:3500Da)with ultrapure water. The retained and permeated fractions were named BFP-3?0.6I and BFP-3?0.6E,respectively.To determine the compositions of monosaccharides in BFP-3?0.2I,BFP-3?0.2E,BFP-3?0.6I,and BFP-3?0.6E,the samples were subjected to GC–MS analysis as described in section 2.4.

2.6.Methylation analysis

Prior to methylation,BFP-3 was reduced to the corresponding neutral sugars[21].The methylation analysis of the reduced BFP-3 was carried out using the modified method as described previously[22].BFP-3(10mg)was dissolved in dimethyl sulfoxide(DMSO;10mL),reacted with sodium hydroxide(NaOH)powder(5mg)and iodomethane(1mL)for 2h.4.0mL distilled water was added to terminate the reaction.The reaction mixture was extracted with CHCl3,and the solvent was then removed by vacuum evaporation.The methylation procedure was repeated three times.

The permethylated polysaccharide was hydrolyzed by treatment with methanoic acid(HCOOH,88%,3mL)at 100?C for 3h,evaporated to dryness,and further hydrolyzed with 2mol/L TFA(4mL)at 100?C for 6h.The mixture was evaporated to dryness,reduced with NaBH4,and acetylated with acetic anhydride(AC2O),and the methylated alditol acetates were analyzed by GC–MS.

2.7.Nuclear magnetic resonance(NMR)spectroscopy

The1H NMR spectra,13C NMR spectra,and two-dimensional spectra(heteronuclear single quantum coherence[HSQC]and nuclear Overhauser spectroscopy[NOESY])were recorded using a Bruker Avance 500 MHz spectrometer(Germany),which was operated at 500MHz.BFP-3 was exchanged and dissolved in D2O,then freeze-dried,and redissolved in D2O for NMR analysis[23,24].

2.8.Antitumor assays

Cell viability was evaluated with a Cell Counting Kit-8(CCK-8)from Dojindo Laboratories(Tokyo,Japan)according to the manufacturer’s instruction.Brie fly,cells were seeded in 96-well plates at a concentration of 1×104cells/well with complete culture medium.After culturing the cells for 12 h in an incubator,different amounts of compound were added in 96-well plates to final concentrations of 0.05,0.1,0.2,0.4,0.8,1.6,3.125,6.25,12.5,25,50,and 100 μg/mL;medium without added compound was used as a negative control.Cells were incubated at 37?C for 48h,10μLCCK-8 solution was added to each plate,and the plates were incubated for an additional 2 h. The absorbance(optical density[OD])of each well was measured with a microplate reader at 450 nm.According to the formula for cell viability,we obtained the relationship between the compound concentration and the proliferation inhibition rate and then calculated the half-maximal inhibitory concentration(IC50)values of the compound:

2.9.Statistical analysis

SPSS 17.0 statistical software(SPSS,Inc.,Chicago,IL,USA)was used for data analysis.Experimental data were expressed as the mean±standard deviation.

3.Results

3.1.Purification and screening of polysaccharide fractions with antiproliferative activity

The crude polysaccharide(BFP)was isolated from the hot water extract ofB.fuscopurpureaand then subjected to ethanol precipitation,deproteinization,and lyophilization.We then attempted to screen antitumor polysaccharides from BFP by bioactivity-directed purification procedures.The antitumor potency of fractions BFP-1 and BFP-2 from the DEAE-Sepharose Fast Flow column(Fig.1A)was compared by evaluating the inhibition ratios in A2780,COC1,SKOV3,HO-8910,and OVCAR3 cells.As shown in Fig.2,the BFP-2 fraction eluted with 0.5mol/L NaCl exhibited higher antitumor activity against A2780,COC1,SKOV3,HO-8910,and OVCAR3 cells,with IC50values of 23.69,40.51,36.45,38.42,and 30.91μg/mL,respectively.

According to thein vitrocell viabilities of A2780,COC1,SKOV3,HO-8910,and OVCAR3 cells treated with BFP-3 at different concentrations,BFP-3 exhibited potential antiproliferative activities in all human ovarian cancer cell lines at all concentrations.In addition,the IC50values were as low as 5.39,17.12,14.56,10.08,and 13.85μg/mL,respectively(Fig.2).Thus,we concluded that the antitumor activity of the polysaccharide was significantly enhanced after purification.This outcome was consistent with that reported by other authors[23].

Fig.1.Isolation and purification of crude polysaccharide from B.fuscopurpurea.(A)Crude polysaccharide was applied to a DEAE-Sepharose Fast Flow column;(B)BFP-2 was applied to a Sephacryl S-100 High-Resolution column.

Fig.2.Effect of polysaccharide fractions from the B.fuscopurpurea on the proliferation of(A)A2780,(B)COC1,(C)SKOV3,(D)HO-8910 and(E)OVCAR3 cells lines.Cell viability was measured by CCK-8 assay after treatment at various concentrations(0.05–100μg/mL)of BFP-1,BFP-2 and BFP-3.All the data are means±SD(n=3).The IC50(F)values were calculated by Bliss method.

Subsequently,the fraction BFP-2 was further separated using a Sephacryl-100 column and assigned as BFP-3(Fig.1B).The GPC spectrum(Fig.3)of BFP-3 showed a single and symmetrical peak,indicating that it was a homogeneous polysaccharide.Moreover,the weight-average molecular weight(MW)was approximately 333kDa.GC–MS analysis revealed that BFP-3 was a heteropolysaccharide,composed mainly of rhamnose(Rha),arabinose(Ara),mannose(Man),glucose(Glc),and galactose(Gal).

3.2.Structure of BFP-3

After partial acid hydrolysis of BFP-3,the monosaccharide components of the intercept and permeate products after the dialysis analyzed by using the GC–MS method, and the GC–MS profiles were presented in Fig.4.According to the results of partial acid hydrolysis(Table1),in which the monosaccharide compositions of BFP-3?0.2I and BFP-3?0.2E were compared,the molar ratios of Rha,Ara,and Gal in BFP-3?0.2E were higher than those in BFP-3?0.2I,whereas the molar ratios of Man and Glu were lower than that in BFP-3?0.2I.These results suggested that Rha,Ara,and Gal residues were more sensitive than Man and Glu residues to this mild acid,probable due to their linkages and location in the side chain.

Fig.3.High-performance gel-permeation chromatography(HPGPC)chromatogram of purified polysaccharide BFP-3.

Table 1Composition analysis of partial acid hydrolysis product.

BFP-3?0.2I was further hydrolyzed with 0.6mol/L TFA to obtain BFP-3?0.6I and BFP-3?0.6E.The monosaccharide composition analysis showed that the molar ratios of Rha and Gal in BFP-3?0.6I were lower than those in BFP-3?0.6E,whereas the molar ratios of Ara,Man,and Glu were higher than those in BFP-3?0.6E.In BFP-3?0.6I,Rha was completely hydrolyzed,whereas the molar ratio of Gal was only 0.09.Thus,we concluded that Rha and Gal were likely to be located in the side chain.The molar ratio of Ara in BFP-3?0.6I was higher than that in BFP-3?0.6E,indicating that Ara may be located in the backbone.

Methylation analysis, the popular method to determine theglycosyl linkages of polysaccharides, has been used to illustratethe structure of polysaccharide for a long time. After reduction, BFP-3 was subjected to linkage analysis by methylation,hydrolysis, reduction and acetylation, which were then determined by the GC–MS method. The results of methylation ofBFP-3 (Table 2) revealed that BFP-3 contained nine types oflinkages, including 1-linked-Arap, 1,5- linked-Araf, 1-linked-Galp,1,3,4-linked-Rha, 1,4-linked-Galp, 1,4-linked-Glcp, 1,3-linked-Galp, 1,4,6-linked-Manp, and 1,4,6-linked-Galp residues at a molarratio of 2:1:2:1:4:4:3:1:1; the Gal, Glc, and Man existed in thepyranose form, and the Ara existed in the pyranose and furanoseforms [25]. According to the monosaccharide composition, the BFP-3 polysaccharide was assumed to be a pectin [26], with a backbonecomprised of Rha and Gal, and branches comprised of Ara. However,the results obtained from the partial acid hydrolysis analysis predicted that the backbone of BPF-3 may have consisted of Man, Glu,and Ara. This information, along with the methylation analysis data,allowed us to suggest that 1,4,6-linked-Manp, 1,4-linked-Glcp, and1,5-linked-Araf residues were present in the backbone.

NMR was chosen to provide more exact structural information,including α-or β-anomeric configurations,linkage patterns,and sequences of the sugar residues[27].Allocation of signals and identification of sugar units were accomplished by combinations of two dimensional spectra and comparison with the chemical shifts with reported data[28].The1H and13C NMR spectra of BFP-3(Fig.5)were crowded in a narrow region of 3.2–5.5ppm(1H)and 60–110ppm(13C NMR),typical of polysaccharides,and the main anomeric proton signals at δ 5.11 and 5.25ppm and anomeric carbon signals at δ104.56,103.95,103.24,102.24,and 99.34ppm revealed that BPF-3 may contain five types of monosac-charide residues[29].The main signals at δ 83.39,81.78,80.93,79.85,and 76.42ppm were in a region of 75–85ppm,suggesting that the C-2,C-3,and C-4 of the polysaccharide may have been substituted,thereby causing a downfield shift.The signals at δ 68.67 and 62.33ppm,located in a high-field region,were likely to be the signal peaks of C-6(5).

Fig.4.GC–MS profiles of partial acid hydrolysis of(A)BFP-3-0.2I,(B)BFP-3-0.2E,(C)BFP-3-0.6I,(D)BFP-3-0.6E,and Methylation analysis of(E)BFP.

Table 2Methylation analysis data for BFP-3.

Fig.5.NMR spectra of the purified polysaccharide BFP-3.(A)1HNMR spectrum of BFP-3;(B)1CNMR spectrum of BFP-3.Samples were dissolved in D2O and mainly examined at 30?C.Spectra were obtained using with a Bruker Avance 500MHz spectrometer,1H NMR and1C NMR was determined at 125Hz and 500Hz,respectively.

The sugar residues were assigned by applying1H/13C HSQC and1H/1H NOESY(Fig.6).The assignments of the protons and carbons(by HSQC measurements)of the residues are summarized in Table 3.The results obtained from the monosaccharide composition and methylation results indicated higher proportions of 1,4-linked-Glcp,1,4-linked-Galp,and 1,3-linked-Galp from the HSQC.According to HSQC spectra and document data[30],the anomeric proton and carbon signals at δ5.24/102.3,4.43/104.2,and 4.52/104.3ppm corresponded to the 1,4-linked-Glcp,1,4-linked-Galp,and 1,3-linked-Galp residues,respectively.The anomeric proton and carbon signal at δ 5.11/99.3ppm were likely assigned to Man.

From the1H-1H NOESY spectrum of BFP-3,the anomeric proton signal(δ 4.32ppm)of β-d-Galp-1 was correlated to the H-4(δ 3.70ppm)of the 4-β-d-Galp-1,which indicated the sequences βd-Galp-1→4-β-d-Galp-1.The anomeric proton signal(δ 4.43ppm)of→4-β-d-Galp-1 was correlated with H-4(δ 3.70ppm)within the same linkage,which suggested the sequences of →4-β-d-Galp-1→4-β-d-Galp-1→.The anomeric proton signal(δ 4.43ppm)of →4-β-d-Galp-1→ was correlated with the H-4(δ 3.71ppm)of→4,6-β-d-Galp-1→,which indicated the sequences →4-β-d-Galp-1→4,6-β-d-Galp-1→.The anomeric proton(δ 4.34ppm)of→4,6-β-d-Galp-1→ was corrected to the H-4(δ 4.18ppm)of→4)-α-l-Rhap-(1→,which indicated the sequences →4,6-β-d-Galp-1 →3,4)-α-l-Rhap-(1→.The anomeric proton(δ 4.86ppm)of →3,4)-α-l-Rhap-(1→ was corrected to the peak of H-6b(δ 3.72ppm)of→4,6-β-d-Manp-1→,which indicated the sequence→3,4)-α-l-Rhap-(1→4,6-β-d-Manp-1→.Similarly,the anomeric proton(δ 4.85ppm)of β-l-Arap-1→ was corrected to the H-4(δ 3.75ppm)of→3-β-d-Galp-1→,which indicated the sequence β-l-Arap-1→3-β-d-Galp-1→.The anomeric proton(δ 4.85ppm)of →3-β-d-Galp-1→ was corrected to the H-3(δ 3.75ppm)within the same linkage,which indicated the sequences→3-β-d-Galp-1→3-β-d-Galp-1→.The anomeric proton(δ 4.85ppm)of →3-β-d-Galp-1→ was corrected to the H-6b(δ 3.69ppm)of →4,6-β-d-Galp-1→,which indicated the sequence →3-β-d-Galp-1→4,6-β-d-Galp-1→.Additionally,the anomeric proton(δ 5.24ppm)of →4-α-d-Glcp-1→ was corrected to the H-4(δ 4.52ppm)within the same linkage,which indicated the sequences→4-α-d-Glcp-1→4-α-d-Glcp-1→.The peak was also related to the H-4(δ 4.15ppm)of→4,6-β-d-Manp-1→,which indicated the sequences 4-α-d-Glcp-1→4,6-β-d-Manp-1→.Furthermore,combined with the results of partial acid hydrolysis,we deduced that the →5)-α-l-Araf-(1→ was likely in the backbone,which could demonstrated the sequences→5-α-l-Araf-1→4-α-d-Glcp-1→4,6-β-d-Manp-1→.

Table 31H and13C NMR spectra assignments for BFP-3(ppm).

Fig.6.Two dimensional spectra of the purified polysaccharide BFP-3.(A),(B)and(C)HSQC spectrum of BFP-3;(B)NOESY spectrum of BFP-3.Samples were dissolved in D2O and recorded on a Bruker Avance 500MHz spectrometer using standard pulse sequences with 5mm tubes at 30?C.

Based on the above results,the predicted primary structure of the repeat unit in BFP-3 was confirmed as shown in Fig.7.

4.Discussion and conclusions

Fig.7.The repeat unit structure of BFP-3.(A)The side chain structure of BFP-3;(B)The backbone structure of BFP-3.

In this study,a neutral polysaccharide,BFP-3,was fractionated and purified fromB.fuscopurpureaby DEAE-Sepharose Fast Flow and Sephacryl S-100 Chromatography.Characterization analysis of BFP-3 was carried out using GC–MS,partial acid hydrolysis,methylation,and one-/two-dimensional NMR.Its antitumor activity was evaluatedin vitro.Results revealed that BFP-3 was composed of five monosaccharides(Rha,Ara,Man,Glc,and Gal),with a weight-average Mw of 333kDa.Structure analysis showed that the backbone of BFP-3 was composed of repeating units of 5-α-l-Araf-1→(4-α-d-Glcp-1)4→4,6-β-d-Manp-1,and the side chains consisted of repeating units of β-d-Galp-1→(4-β-d-Galp-1)4→4,6-β-d-Galp-1→3,4-α-l-Rhap,β-l-Arap-1→(3-β-d-Galp-1)3,and β-l-Arap-1.Moreover,BFP-3 showed strong inhibitory activity against A2780,COC1,SKOV3,HO-8910,and OVCAR3 ovarian cancer cells in proliferation assaysin vitro.Our findings suggested that BFP-3 could be used as a therapeutic agent in the treatment of ovarian cancer.In previous reports,several studies have reported different structural polysac-charides fromB.fuscopurpurea.He et al.[31]applied boiling water extraction to obtain the crude polysaccharide,removed proteins by method of Sevage and precipitated by ethanol,and further purified by DEAE-Cellulose 32 and Sephadex G-200 column chromatography.The polysaccharide PY3 was mainly consisted of Gal and Xyl in a molar ratio of 11:1.It was a kind of polysaccharide with 6-sulfate-galactose and β-D-galactopyranose as repeating units[30].The extraction and elution methods were similar with our studies,but the extracted polysaccharides possessed different structures.In addition,Jiang and colleagues isolated a sulfated polysaccharide from ethanol extraction ofB.fuscopurpurea,mainly composed of Gal together with a small amount of uronic acid,Man,and Glu,with a molecular weight of 133.18kDa, which could inhibit α-amylase and α-glucosidase in a concentration-dependent manner[32].Actually,the extraction and isolation process not only affected the structure of polysaccharide,but also influence the activity of polysaccharide.Therefore,these manipulations used to extract and isolate might result in the structural differences among different studies.

This study provided insight into the chemical structure andin vitroanticancer activity of BFP-3,which could be beneficial for its future development for high-value applications.However,the biological activities of polysaccharides are known to depend not only on their structure and molecular size but also on their conformation[33].Therefore,additional conformational analyses ofB.fuscopurpureapolysaccharides are needed in the future.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was funded by the Science and Technology Project of Xiamen Medical College(K2016-36).