Isolation,purification,structural characteristic and antioxidative property of polysaccharides from A.cepa L.var.agrogatum Don

Hongcheng Liu,Hongxiu Fan,Jing Zhang,Shanshan Zhang,Wenting Zhao,Tingting Liu,Dawei Wang,*

a School of Food Science and Engineering,Jilin Agricultural University,Changchun,130118,China

b Scientific Research Base of Edible Mushroom Processing Technology Integration of Ministry of Agriculture and Rural Affairs,Changchun,130118,China

c Engineering Research Center of Grain Deep-processing and High-effeciency Utilization of Jilin Province,Changchun,130118,China

d Key Laboratory of Technological Innovations for Grain Deep-processing and High-effeciency Utilization of By-products of Jilin Province,Changchun,130118,China

Keywords:

ABSTRACT

1.Introduction

Allium cepa L.var.agrogatum Don(ALAP),which belongs to Liliaceae family,has been widely cultivated in Northeast China since the early 20th century.The yield of ALAP is 30000-45000 kg on average per hectare,and it has been exported to Russia,Japan,South Korea,and other countries.ALAP has a number of favourable characteristics,including resistance to storage,cold hardiness,and a short growth cycle[1,2].It has a spicy taste and can be eaten raw,in cooked dishes,and as a food additive.It is becoming more and more popular in China.The bulb is nearly spherical,the skin of the seed is reddish,and the leaves are dark-green and tubular,with a length of approximately 30 cm.The tillering ability of the bulb is very strong[3].Several spherical bulbs can be formed in a single plant,and each plant forms a larger bulb.Its irritating odour is stronger than that of ordinary onions,and this odour results mainly from organic sulphur compounds[4].The main organic sulphur compounds in ALAP are aliphatic hydrocarbon disulphide and trisulphide compounds.These organic sulphur compounds have been identified as thioethers(R-Sn-R(S-sulphides),thiosulphates(R-S(O2)-S-R),thiosulphinates(S-S(O)-R),and thiophenes[5].Recent pharmacological research have shown that its organic sulphur compounds exhibit antibacterial activity,diabetic alleviation,reduction of cholesterol and anti-cancer activity in addition to other beneficial functions[6-8].

Research on ALAP has mainly focused on the organic sulphur compounds and flavonoids.The polysaccharide content of ALAP is 13.16%(dry weight).To date,few reports are available on the polysaccharides from ALAP.Polysaccharides are important biological macromolecules composed of the same or different monosaccharides linked by glycosidic bonds and are an indispensable component of all living organisms.They have received increasing attention owing to their extensive functions[9].Liu et al.[10]obtained refined onion polysaccharides by deproteinising,bleaching,and DEAE-52 cellulose column purification,and the properties of polysaccharides and monosaccharide composition were measured.Ma et al.[11]extracted onion polysaccharides with four different solvents and examined the antioxidant effects of the four fractions.They found that onion polysaccharides extracted with dilute alkaline and chelating agent exhibited the highest scavenging activities for DPPH radicals,ABTS radical cations,hydroxyl radicals,superoxide anion radicals,as well as the antioxidant activities for reducing power and Fe2+chelating power.Li et al.[12]performed the cellulase-assisted extraction of garlic polysaccharides and showed that garlic polysaccharides can be used as antioxidants.Recent studies have reported that polysaccharides have a variety of antioxidative properties,and a number of different polysaccharide structures have been revealed.Therefore,we extracted,purified and characterise polysaccharide from ALAP and evaluated its antioxidant potential,to improve the practical applications and added value of this vegetable crop.

2.Materials and methods

2.1.Materials

ALAP bulbs were collected during August from Wulajie Town,Jilin City,China.Sephadex G-100 was purchased from Solarbio Biotechnologies(Beijing,China).Cellulose DEAE-52 was purchased from Whatman Ltd.(America).Monosaccharide standards(purity＞98%)were acquired from Dr.Ehrenstorfer GmbH(Germany).Phenol,sulphuric acid,sodium chloride,Tris-HCl,sodium hydroxide,salicylic acid,ascorbic acid(VC),ferrous sulphate,pyrogallol,absolute ethanol,hydrogen peroxide and 1,1-diphenyl-2-trinitrophenylhydrazine(DPPH)were of analytical grade.

2.2.Extraction of water-soluble polysaccharides from ALAP

The water-soluble polysaccharides were prepared by the ultrasonic microwave-assisted extraction method[10].Fresh ALAP bulbs were peeled off,roughly chopped and mashed by a domestic blender.The mashed bulbs of ALAP was homogenised with distilled water(in ratio of bulbs:distilled water=1:17,g/mL)and extracted by ultrasonic microwave-assisted extraction at 80°C for 16 min(microwave power 200 W,ultrasonic power 360 W).All water extracts were combined,filtrated and concentrated to 1/4 of the original volume by the rotary evaporator at 50°C,-0.09 MPa,and the proteins were removed from the concentrated extract by the Sevage[13]method.The polysaccharide solution was precipitated by adding 90%ethanol to its concentration of 72%(V/V)ethanol.The mixture was stored at 4°C in the refrigerator for overnight.After centrifugation,the collected precipitate was dissolved in distilled water,followed by lyophilization.Finally,protein-free polysaccharide of ALAP was obtained and the yield was 11.92%.

2.3.Isolation and purification of ALAP

Purification of the protein-free ALAP was performed by a previously reported procedure with the following modifications:200 mg of ALAP was re-dissolved in ultrapure water and then injected into a DEAE-52(2.6 cm×30 cm)column[14].The sample was fractionated by eluting with ultrapure water followed by a gradient of 0.1-0.5 mmol/L sodium chloride solution,and the eluting rate was set at 1.0 mL/min.An automatic collector was used to obtain the eluent for 4.0 min per tube and an aliquot of every four tubes was tested for the polysaccharide content by the phenol-H2SO4colorimetry method.The main polysaccharide fractions(ALAP-1 and ALAP-2)were collected,concentrated in a rotary evaporator,dialyzed in ultrapure water and finally freeze-dried(shown in Fig.1).ALAP-2 was subjected to further purification through a Sephadex G-100 gel permeation chromatography column.After ALAP-2 was loaded onto the column,the sample was eluted by ultrapure water with an eluting rate of 1.0 mL/min.The main fraction was combined,concentrated and freeze-dried to yield a pure polysaccharide named ALAP-21,which was used for further structural analysis and bioactivity evaluation.

2.4.Physicochemical characterizations of ALAP-21

Content of the total sugars of ALAP-21 was measured with the phenol-H2SO4colorimetric method[15].The ultraviolet spectrum of ALAP-21(1 mg/mL)was recorded by an Unico UV2100 spectrophotometer(USA)from 180 to 500 nm for detection of the impurity content.The nature of ALAP-21 was verified through the following methods:I2-KI reaction,FeCl3reaction and sulfuric acidcarbazole method[16,17].

2.5.Monosaccharide composition of ALAP-21

The monosaccharide component of ALAP-21 was determined by an Agilent 5975-6890 N gas chromatography-mass spectrometer(GC-MS,Agilent,USA)according to the reported method with some modifications[18].For hydrolysis,10.00 mg ALAP-21 was accurately weighed and added with 4 mol/L trifluoroacetic acid(2 mL).The tubes were then vacuum sealed,followed by hydrolysis at 110°C for 4 h.The hydrolysate was cooled at 37°C and then centrifuged to remove the remaining debris.The separated supernatant was evaporated at reduced pressure,with repeated addition of anhydrous ethanol to remove excess trifluoroacetic acid.For derivatization,6 mg of hydrolysed product of ALAP-21 was accurately weighed and added with 2 mL of dried pyridine and 0.2 mL salinization reagent(BSTFA:TMCS=99:1,V/V).After shaking,the hydrolysate was derivatized at 50°C for 30 min.After filtration through a 0.45 μm polytetrafluoroethylene membrane,the derivative was analyzed by GC-MS system.Derivatizations of monosaccharide standards with ribitol as the internal standard was performed as above.

For GC-MS analysis,1 μL of sample derivative was injected into the GC-MS system at 280°C in 30:1 shunt injection mode with the helium gas flowrate set at 1 mL/min.The sample derivatives were separated on a HP-5 capillary column and the initial temperature maintained at 80°C for 3 min.Then the column temperature was programmed from 80°C to 280°C at 10°C/min,holding for 8 min.The parameters of the mass spectrometer were set as follows:EI source,230°C ion source temperature,150°C quadrupole temperature,70 eV electron energy,1200 V electron multiplier voltage and m/z 20-550 amu mass scan range.

2.6.FT-IR analysis

The infrared spectrum of ALAP-21(KBr pellet)was determined with a Shimadzu IR restige-21 spectrophotometer(Japan)[19].The dried sample(2 mg)was grounded with 200 mg KBr dry powder(spectroscopic grade),and the mixture was pressed into a disk of 1.0 mm thickness.FT-IR spectroscopy analysis was performed in the wavenumber range of 4000-400 cm-1.

Fig.1.(A)DEAE-52 cellulose chromatographic profile for ALAP using deionized water and 0.1,0.3,0.5 mmol/L NaCl solution as eluents;(B)Sephadex G-100 chromatographic profile for ALAP-2 eluted with deionized water.

2.7.NMR analysis

The NMR analysis of ALAP-21 was performed following a previously described method[20].A Bruker Avance 600 spectrometer(600 MHz,Germany)was applied to record the NMR spectra of ALAP-21 with tetramethylsilane(TMS)as internal standard.The sample was dissolved in D2O in a NMR tube at 27°C.Chemical shifts are expressed in ppm.

2.8.Antioxidant activity of ALAP-21

2.8.1.DPPH radical scavenging assay

The activity of scavenging DPPH radicals of ALAP-21 was analyzed by the procedure described by Xie et al.[21].In brief,0.5 mL of the ALAP-21 aqueous solution at concentrations of 0.1,0.2,0.3,0.4 mg/mL and 0.5 mg/mL was mixed with 1.5 mL distilled water and 2.0 mL ethanolic solution of DPPH radical(0.2 mmol/mL).After shaking vigorously,the reaction solution was left to stand at room temperature for 30 min under light-shaded condition.The UV absorption for the reaction solution was determined at 517 nm with VC as a reference standard and 95% ethanol as a blank.The capability of ALAP-21 to scavenge DPPH radicals was expressed as[1-(A2-A1)/A0]×100%.

Where A2,A1and A0refers to the absorbances of the samples with DPPH solution,samples without DPPH solution and the blank,respectively.

2.8.2.Hydroxyl radical scavenging assay

The capability of ALAP-21 to scavenge hydroxyl radicals was analyzed by the procedure described in reference[22].First,1 mL of ALAP-21 aqueous solution with varying concentrations(0.2-1.0 mg/mL)were added with 1 mL of 9 mmol/L FeSO4solution,1 mL of 9 mmol/L salicylic acid-ethanol solution,1 mL H2O2solution(9 mmol/L).The reaction solution was allowed to stand at 37°C for 0.5 h,then was determined by UV spectrometry at 510 nm.VCof the same concentration was taken as a reference standard.The capability of ALAP-21 to scavenge hydroxyl radicals was expressed as[A0-(A2-A1)]/A0×100%.

Where A2stands for the absorbance value of reagent blank where salicylic acid was replaced by ultrapure water,A1stands for the value of the sample,and A0stands for the value of sample blank where the sample was replaced by ultrapure water.

2.8.3.Determination of superoxide anion scavenging activity

Determination of the scavenging ability to superoxide anion radicals was performed by the procedure described in a previous study[23].1 mL of ALAP-21 solution of different concentrations(0.2-1.0 mg/mL),3.9 mL ultrapure water and 4.5 mL Tris-hydrochloric acid buffer(pH 8.2,50 mmol/L)were mixed together and left to stand for 0.5 h in a 25°C water bath.Then 0.3 mL of 0.045 mol/L 1,2,3-phentriol was added to the mixture to start the reaction.Absorbance value at 325 nm was recorded at a time interval of 10 s over a period of 5 min,using Tris-hydrochloric acid buffer and ultrapure water instead of the polysaccharide sample as the blank control.The capability of ALAP-21 to scavenge superoxide anion radicals was expressed as(1-A1/A0)×100%.

Where A1and A0refers to the rates of absorption changes of the sample and blank control,respectively.

2.9.Statistical analysis

The presented data were shown in the mean values of three replicates±standard deviation(SD).Statistical analysis of all the results were done using one-way ANOVA,and significant differences were evaluated based on Duncan’s multiple comparison test.Differences with P＜0.05 were regarded as statistically significant.

3.Results and discussion

3.1.Preparation and physiochemical characteristics of ALAP

After ultrasonic microwave-assisted water extraction,ethanolic precipitation and deproteinization,the crude polysaccharide ALAP was separated from ALAP and further purified by ion-exchange column DEAE-52.As shown in Fig.1(A),two polysaccharide fractions ALAP-1 and ALAP-2 were collected.ALAP-2 took the majority of ALAP(89.66%).As ALAP-1 represented the negligible quantity,only ALAP-2 were applied to Sephadex G-100 column to obtain high-purity polysaccharide for structural characterization and antioxidant activity determination.Fig.1(B)showed only one peak(ALAP-21)in the water elution,indicating that ALAP-21 was a homogeneous polysaccharide.

The content of the total sugars in ALAP-21 was 97.2% based on the phenol-H2SO4colorimetric method.ALAP-21 had negative responses to the I2-KI reaction and FeCl3reaction,indicating the absence of starch and polyphenols.No absorption was detected at 260 nm or 280 nm in the ultraviolet absorption spectrum,demonstrating no protein or nucleic acid in ALAP-21.The sulfuric acid-carbazole colorimetric assay showed that ALAP-21 contained uronic acid,which indicated that ALAP-21 can be classified as an acidic polysaccharide.Previous literature reported that[24,25]acidic polysaccharides usually exhibited stronger antioxidant potential than neutral polysaccharides,and therefore ALAP-21 might have antioxidant potential.

Fig.2.Formation of a trimethylsilyl derivative of monosaccharide standard by silylation.

Fig.3.Mass spectrum of a BSTFA-derivatized monosaccharide with the corresponding structure.

3.2.Monosaccharide composition analysis

Modern research shows that the biological activities of polysaccharides are different with the different monosaccharide compositions.Polysaccharides containing a higher amount of uronic acids are known to possess better biological activities,because the polysaccharide properties can be altered by uronic acids.Therefore,monosaccharide composition analysis is necessary in the study of the structure of polysaccharides.Pre-column derivatization by silylation is the common method for GC-MS analysis of monosaccharide.During the silylation reaction,the hydroxyl groups are converted into their corresponding trimethylsilyl(TMS)ethers via an SN2substitution reaction,increasing the volatility and stability of the monosaccharides.Examples of derivatization reaction and mass spectrogram of the monosaccharide derivatized with BSTFA were shown in Figs.2 and 3.The mass spectrogram showed that derivatized products were stable and fragments at m/z=204,217 and 191 were characteristic for the analyzed compounds.Chromatograms of mixed standard monosaccharides and ALAP-21 sample were shown in Fig.4(A)and(B).The monosaccharide composition was calculated based on retention time and standard monosaccharides.The sensitivity was expressed by determining the limits of detection(LOD)and quantification(LOQ).The LOD and LOQ were defined as signal to noise(S/N)=3 and 10 respectively[26,27],and were calculated after injection of standard samples with the lowest concentration that the GC-MS instrument could detect with acceptable response.Table 1 showed that LOD and LOQ ranged from 0.1 μg to 0.4 μg and from 0.4 μg to 1.2 μg for monosaccharide derivatives,respectively.The sensitivity was similar to the previously developed method[28].

Result of monosaccharide composition analysis showed that ALAP-21 was composed of glucose,galacturonic acid,mannose,galactose,arabinose,rhamnose,xylose,fructose and glucuronic acid with the molar ratio of 26.282:27.546:11.400:4.781:2.467:2.445:3.622:1.106:1.753,suggesting that ALAP-21 was an acidic heteropolysaccharide.As the contents of glucose and galacturonic acid were the highest in ALAP-21,the main backbone of ALAP-21 might be composed of glucose and galacturonic acid.The monosaccharide composition of ALAP-21 was different to that of other Allium species reported in previous literature.Golovchenko et al.isolated a pectic polysaccharide(AC-3)from Allium.cepa L.,and monosaccharide analysis revealed AC-3 was composed of galacturonic acid(GalA),arabinose(Ara),rhamnose(Rha),galactose(Gal),xylose(Xyl)and glucose(Glc)with the molar ratio of 71.0:1.8:1.7:17.2:0.8:0.4.A fructan consisted of Glc and Fru with a relative molar ratio of 1:8 has been identified from Welsh onion(Allium fistulosum L.)[29,30].According to the results,ALAP contained a different monosaccharide composition.

Table 1 Calculated detection(LOD)and quantification(LOQ)limits of the GC-MS detection.

3.3.Infrared spectroscopy

Fig.4.GC-MS chromatograms of standard monosaccharides(A)and component monosaccharides of ALAP-21(B).

3.4.NMR spectroscopy

NMR analysis was performed to get more detailed structure informations about ALAP-21,such as α/β anomeric configuration,the patterns and sequences of glycosidic linkages and positions of acetyl groups.Previous literatures reported that[35]the1H signals at 4.4-5.5 ppm in the1H-NMR spectrum were usually assigned as the anomeric protons of the sample,where the1H signals at 5.0-5.8 ppm indicated the anomeric protons of α-configuration,and the1H signals at 4.4-5.0 ppm were assigned to the β anomeric configuration.The signals of protons deriving from-CH3(C-6)of deoxy-sugar were generally in the region of 0.8-1.4 ppm,and the signals for-CH3protons of O-acetyl groups(-OCOCH3)were in the region of 1.8-2.2 ppm.The proton signals of methyl group binding to the carboxyl groups(-COOCH3)of uronic acid were often in the region of 3.0-3.8 ppm.In the spectrum of13C-NMR,the anomeric carbon signals generally appeared in the region of 95-110 ppm,where the13C signals at 97-101 ppm indicated the anomeric carbons of α-configuration,and signals in the region of 103-107 ppm were attributed to the β anomeric configuration.The13C signals in the regions of 22-23.5,53-56 and 61-68 ppm indicated the presence of-CH3of acetyl group,carbon of carbomethoxy group and carbon of O-methyl group,respectively[36].

In the1H and13C-NMR spectra of ALAP-21,the1H signals at 5.40,5.23,4.77,4.21 ppm and13C signals at 103.53,103.07,103.01,102.91,102.86 ppm indicated the presence of both α and β configurations in the ALAP-21.Confirmation of all the signals of individual monosaccharide residues using only1H-NMR spectroscopy(Fig.6(A))is limited because of relatively narrow signal distributions,overlapping proton shifts and coupling and splitting of peaks.Generally,the number of signals at the anomeric carbon region(95-110 ppm)indicates the types of monosaccharide residues in the polysaccharide.As shown in Fig.6(B),there were five signals at the anomeric carbon(C-1)region,which implied that ALAP-21 contained five kinds of monosaccharide residues,and they were labeled asA(δ 102.86 ppm),B(δ 102.91 ppm),C(δ 103.01 ppm),D(δ 103.07 ppm)andE(δ 103.53 ppm)(Table 2).As the anomeric13C signals of sugars in furanose form should appear in the region of 107-110 ppm[37],the C-1 signals shown in the13C-NMR spectrum of ALAP-21 indicated that all monosaccharides could be in the pyranose form.

Table 2 The 1H and 13C signals for the glycosidic linkes of ALAP-21 in the NMR spectra.

Fig.5.FI-IR spectra for ALAP-21.

The highfield1H resonance at δ 1.17 ppm was attributed to the-CH3(C-6)of rhamnose,which revealed that ALAP-21 contained rhamnose.The proton signal for the-CH3of carboxyl group of GalA appeared at δ 3.82 ppm,and the proton signal for CH3-O group appeared at δ 3.54 ppm.Two proton signals showed up at δ 2.35 and δ 2.55 ppm were assigned to the-CH3of acetylated hydroxyl group of GalUA.In addition,the1H signals at δ 3.08-4.21 ppm were assigned as H-2 to H-6.Multiple chemical shifts showed up in the regions of δ 69.08-76.65 ppm and δ 60.29-63.76 ppm in the13CNMR spectrum of ALAP-21(Fig.6(B)),which corresponded to the C-2 to C-4 and C-6 of sugar rings without substituent.The carbon signal for the-CH3(C-6)of rhamnose appeared at δ 16.78 ppm,which indicated that ALAP-21 contained rhamnopyranosyl unit.The carbon signal observed at δ 23.69 ppm was attributed to the-CH3of O-acetyl group,which usually located on the C-2 or C-3 of GalUA residues,and the carbon signal for the C-6(carbonyl carbon)of O-acetyl group was at δ 178.40 ppm[38],which indicated that the carbonyl groups of GalUA in ALAP-21 were in the form of their methyl esters.The C-6 signal for the methyl-esterified carboxyl groups of GalUA was at δ 169.83 ppm,in addition to the-CH3carbon of the methyl-esterified carboxyl groups appeared at δC54.17 ppm[32].Moreover,the13C chemical shift for the O-CH3group was at δ 61.35 ppm.Table 1 showed all the1H and13C signals,which were assigned based on the literature and monosaccharide analysis.

3.5.Antioxidant activities of ALAP-21

3.5.1.Scavenging activity of DPPH radicals of ALAP-21

The DPPH radical scavenging model has been widely used for evaluation of antioxidant activities of various natural compounds due to its sensitivity to low concentrations and short reaction period.The DPPH scavenging capability is measured by the reduction in the ultraviolet absorption of DPPH,which is caused by antioxidants.As the DPPH radical accepts an electron or a free radical species,which is provided by antioxidant,the DPPH radical will lose this absorption feature,leading to a visually conspicuous discoloration from purple to yellow.It is well known that the reactional mechanism between DPPH free radical and antioxidant is connected with the structure of the antioxidant.The number of DPPH free radical molecules reduced by antioxidant in reaction period is proved equal to the number of the hydroxyl groups of the antioxidant[39].

It was observed that ALAP-21 possessed a noticeable DPPH free radical scavenging capability,and there was a dose-dependent relationship between the sample concentrations and the antioxidant activity(Fig.7(A)).Specifically,the DPPH radical scavenging effects of ALAP-21 increased from 23.32%-82.02%with sample concentrations increased within the range of 0.1-0.5 mg/mL,although the DPPH scavenging abilities of ALAP-21 were inferior to that of VC at the same concentrations.

3.5.2.Scavenging activity of hydroxyl radicals of ALAP-21

Hydroxyl radical is the most active free radical that can cause oxidative damage to biomolecules in living organs such as proteins,carbohydrates,lipids and DNA,resulting in disorganization and cell apoptosis[11,40].Thus,the hydroxyl radical scavenging ability is one of the most widely used indicators for assessment of antioxidant activity.As shown in Fig.7(B),the hydroxyl radical scavenging capability of ALAP-21 increased in a dose-dependent manner within the tested concentration range,while VC exhibited a relatively stronger capability to scavenge hydroxyl radicals.At the concentration of 1.0 mg/mL,the scavenging effects of ALAP-21 and VC on hydroxyl radical were 53.33% and 97.01%,respectively.

Fig.6.1H(A)and 13C(B)NMR spectra of ALAP-21.

Fig.7.In vitro antioxidant activity of ALAP-21;(A)Scavenging activity of ALAP-21 on DPPH radical;(B)Hydroxyl radical scavenging activity of ALAP-21;(C)Scavenging activity of ALAP-21 on superoxide anion radicals.

3.5.3.Superoxide anion radical scavenging activity

Superoxide anion radical is considered to be one of the most important free radicals in living organisms,and is also the precursor of other reactive oxygen species,which can cause harmful hydroxyl radical and lipid peroxidation[41].It can be formed through various biological and photochemical reactions.In addition to causing direct damage to biomolecules in living cells,the superoxide anion free radical can also decompose to generate hydroxyl radicals and singlet oxygens,which play a key role in aggravation and initiation of many diseases such as local oxidative stress,cellular damage,lipid peroxidation,inflammation as well as cancer[42].

As shown in Fig.7(C),ALAP-21 displayed a noticeable scavenging capability for superoxide anion radical in a dosedependant manner as its concentration increased from 0.2 mg/mL to 1.0 mg/mL,while VC showed a stronger superoxide anion radical scavenging effect at the tested concentrations.The maximum scavenging activities of ALAP-21 and VC reached 50.28%and 98.03%at 1.0 mg/mL,respectively,indicating that ALAP-21 has a moderate scavenging effect on superoxide anion radical.

Based on the results of antioxidant activities of ALAP-21 determined in vitro,ALAP-21 had a strong DPPH radical scavenging ability and moderate scavenging abilities on hydroxyl radical and superoxide anion radical.Cheng et al.[43]studied the antioxidant effect of a purified polysaccharide isolated from A.sativum and found that the polysaccharide displayed the highest hydroxyl radical and superoxide anion radical scavenging abilities(45%and 58%)at the concentration of 6.3 mg/mL.Ma et al.[11]extracted four polysaccharide fractions from red onion(A.cepa L.)with four different solvents(hot buffer solution,chelating agent,dilute and concentrated alkali solution)and they found that the hot water extracted polysaccharide exhibited the highest superoxide anion radical scavenging ability(76.27%),while the chelating agent extract exhibited the highest scavenging abilities for hydroxyl radicals(65.12%)and DPPH radicals(93.68%)[11].The mechanism underlying free-radical scavenging by the polysaccharides of Allium species is still not fully deciphered,but many studies have demonstrated that the antioxidant activities of polysaccharides are dependent on the structure of the polysaccharides,including conformation,molecular weight,molecular configuration,branching degree,monosaccharide constituent and carboxyl groups[44,45].Previous studies reported that the appearance of uronyl/acetyl groups and polymerization degree/nature may improve the antioxidant activities of the water-soluble polysaccharides of rice and ragi[46].Patel et al.[47]found that the acid polysaccharide fraction isolated from psyllium husk and seed could scavenge more DPPH free radicals than the natural polysaccharide fraction,which indicated that the radical scavenging activities might be related to the carboxyl groups,possibly because the electron or hydrogen atom donating ability of carboxyl group was stronger than that of hydroxyl group.In addition,a positive correlation between uronic acid content and antioxidant effect was also found in brown alga[48],tea[49]and edible mushroom polysaccharides[24].Previous studies also reported that glucans of high molecular weight may exhibit higher radical scavenging abilities than those of low molecular weight[50].Moreover,glucan with β-(1→3)-linked backbone also played an important role for the potential antioxidant activities of polysaccharides[51].Thus,it can be assumed that there were some functional groups in the structure of ALAP-21,which could improve its antioxidant activity.

4.Conclusion

A new acid polysaccharide ALAP-21 was extracted from ALAP by ultrasonic microwave-assisted water extraction,followed by purification with DEAE-52 anion exchange and Sephadex G-100 columns,and its structural elucidation was conducted by chemical analysis,FT-IR and NMR spectroscopies.Structural analysis revealed that ALAP-21 was mainly composed of glucose,galacturonic acid,mannose,galactose,arabinose,rhamnose,xylose,fructose and glucuronic acid with a relative molar ratio of 26.282:27.546:11.400:4.781:2.467:2.445:3.622:1.106:1.753.Five kinds of glycosidic linkages,(1→4)-α-D-Glcp,(1→4)-α-DGalAp6Me3OA,(1→4)-β-D-Galp6OMe,(1→2)-α-L-Rhap,(1→4)-β-D-Manp were detected in the structure of ALAP-21.Based on the above data,ALAP-21 is an acetylated polysaccharide with a backbone mainly consisted of(1→4)-α-D-Glcp and(1→4)-α-D-GalAp6Me3OA residues,in which the methylated galacturonic acid was substituted by an acetyl group.In addition,the in vitro antioxidant activity experiments showed that ALAP-21 exhibited noticeable scavenging abilities toward DPPH,hydroxyl and superoxide anion free radicals.Thus,we may rationally assume that ALAP-21 might have important applications in functional food,cosmetics and therapeutic drug.However,further studies are necessary to give more insight in the sequence of glycosidic linkage in ALAP-21 and elucidate the relationship between the polysaccharide structure and its antioxidant activity,and such studies are in progress in our research group.

Acknowledgement

The study was financially supported by the National Science and Technology Support Program of China(Grant No.2013BAD16B08).We would like to thank Editage(www.editage.cn)for English language editing.