Antimicrobial and antioxidant activities of Flammulina velutipes polysaccharides and polysacchride

Carbohydrate Polymers 161(2017)26–32

Contents lists available at ScienceDirect

Carbohydrate

Polymers

j o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /c a r b p o

l

Antimicrobial and antioxidant activities of Flammulina velutipes polysacchrides and polysacchride-iron(III)complex

Ya-ru Dong,Shu-jie Cheng,Guo-hong Qi,Zhi-ping Yang,Shi-yu Yin,Gui-tang Chen ?

Department of Food Quality and Safety,China Pharmaceutical University,639Longmian Avenue,Nanjing 210009,China

a r t i c l e

i n f o

Article history:

Received 23November 2016Received in revised form 21December 2016

Accepted 28December 2016

Available online 29December 2016

Chemical compounds studied in this article:Hydroxyl (PubChem CID:961)DPPH,Free radical (PubChem CID:2735032)

Superoxide anion (PubChem CID:5359597)Malondialdehyde (PubChem CID:10964)2,2 -Azobis(3-ethylbenzothiazoline-6-sulfonic acid)(PubChem CID:73857778

Iron chloride (FeCl 3)(PubChem CID:24380)

Keywords:

Flammulina velutipes polysaccharides Polysacchride-iron (III)complex Antimicrobial activity Antioxidant activity

a b s t r a c t

FVP is polysacchrides obtained from Flammulina velutipes .A polysacchride named FVP2was isolated from FVP by DEAE cellulose-52chromatography and Sephadex G-100size-exclusion chromatography.FVP-Fe and FVP2-Fe were synthesized by neutralization of FeCl 3carbohydrate solution.The antibacterial and antifungal activities of FVP,FVP2,FVP-Fe,FVP2-Fe were investigated and their antioxidant effects on hydroxyl,2,2-diphenyl-1-picrylhydrazyl (DPPH),superoxide anion,2,2 -azobis-(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS)radicals,reducing power,inhibition of malondialdehyde (MDA)were assessed in vitro.The results suggested that FVP-Fe and FVP2-Fe signi?cantly suppressed the growth of bacteria Staphylococcus aureus ,Escherichia coli ,and Bacillus subtilis ,and have relatively strong antioxidant activity to scavenge superoxide anion radical.In addition,FVP exhibited strong antioxidant activity to eliminate hydroxyl,DPPH,ABTS radicals,had high reducing power and inhibited the MDA production of health mice liver homogenate induced by auto-oxidation and Fe 2+-H 2O 2system.

?2016Elsevier Ltd.All rights reserved.

1.Introduction

Flammulina velutipes as one of the most popular mushrooms,with high nutrition levels of vitamins,amino acids,polysacchrides and ?ber,has been widely cultivated and consumed all over the world,especially in China and Japan (Jing et al.,2014;Yang et al.,2012;Zhang,Xiao,Deng,He,&Sun,2012).The polysaccharides of F.velutipes (FVP)are the major active component in F.velutipes that have a lot of biological activities such as enhance immunity,improving memory,antioxidant activity,resisting decrepitude and suppressing tumor (Pang et al.,2007;Sheng et al.,2007;Shi et al.,2012;Wu,Duan,Liu,&Cen,2010;Zhang et al.,2013).Iron de?-ciency anemia (IDA)is one of the highest incidences of nutrient de?ciency diseases,and the basic method to prevent IDA is to take enough iron (Fretham,Carlson,&Georgieff,2011;Szeto,Harrison

?Corresponding author.

E-mail address:cpucgt@https://www.360docs.net/doc/1015134586.html, (G.-t.Chen).

&Innis,2012).At present,the popular iron supplements include two types,namely inorganic iron and organic iron.Ferrous sulfate is the representative of inorganic iron (Liu,Lin,Chang,Yang,&Chen,2004).Although it is an effective iron supplement,it has a lot of side effects,such as gastrointestinal irritation,oral odor and iron poi-soning,https://www.360docs.net/doc/1015134586.html,anic compounds represented by polysacchride-iron complex,with high bioavailability and little gastrointestinal irrita-tion,is a kind of effective iron supplement to prevent IDA (Wang,Chen,Wang,&Xing,2015;Shi et al.,2013;Zhang,Wang et al.,2012).The polysacchride-iron (III)complex of F.velutipes (FVP-Fe)was synthesized by neutralization of an FeCl3carbohydrate solution in order to achieve a new effective and bioactive iron supplement.Microorganisms such as bacteria and fungi usually cause food spoilage and poisoning,which decrease the shelf life of food and endanger the health of consumers (Sokmen et al.,2004).Some polysacchrides extracted from plants have strong antibacterial effect (Zhu,Sheng,Yan,Qiao,&Lv,2012),but to the best of our knowledge,there were no report available in the literature regard-ing the antibacterial and antifungal activities of FVP and FVP-Fe.

https://www.360docs.net/doc/1015134586.html,/10.1016/j.carbpol.2016.12.0690144-8617/?2016Elsevier Ltd.All rights reserved.

Y.-r.Dong et al./Carbohydrate Polymers161(2017)26–3227

The most of radicals in human body are oxygen radicals with spe-ci?c biological functions,and the right amount of free radicals is necessary to the organisms.However,when excessive free radi-cals produced in human body,they attack normal cells,protein, nucleic acid,lipid,sugar and other biological macromoleculars in human body resulting in diseases,aging and death(Ami′c et al., 2017,2014).Nowadays,the development of novel antioxidants to eliminate free radicals effectively is the hot topic of research. More and more polysaccharides from plant and their correspond-ing synthetic products were researched,not only because of their good immunoregulatory activities but also the effective antioxi-dant activities(Gao,Wang,Wang,&Wang,2013;Liu,Sun,Rao,Su, &Yang,2013;Wang,Li,&Chen,2009;Zhang et al.,2013).How-ever,few investigations focused on the comparison between FVP and FVP-Fe on their antioxidant activity.

The objective of this study was to evaluate the antibacterial activity of the FVP,FVP2,FVP-Fe and FVP2-Fe against Staphylococcus aureus,Escherichia coli,Bacillus subtilis and their antifungal activity against yeast,Rhizopus,Aspergillus.The study also determined their antioxidant activity,including hydroxyl,2,2-Diphenyl-1-picryl-hydrazy(DPPH),2,2 -azobis-(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS),superoxide anion radicals scavenging activity,reduc-ing power,inhibiting MDA production of health mice liver homogenate induced by auto-oxidation and Fe2+-H2O2system. 2.Materials and methods

2.1.Materials and chemicals

Fresh F.velutipes mushrooms were purchased from a local supermarket(Nanjing,China).The DPPH and ABTS were purchased from Sigma Chemical Co.(St.Louis,MO,USA).All other reagents were purchased from Nanjing Chemical Reagent Co.(Nanjing, China).

2.2.Preparation,puri?cation of FVP

In the previous study,polysacchrides of F.velutipes were obtained after hot water extraction,deproteinization,and alcohol precipitation.The extraction yeild was6.25%and the content of polysacchrides of sample was70.58%.Two main fractions named FVP1,FVP2were isolated from the sample by DEAE cellulose-52 chromatography and Sephadex G-100size-exclusion chromatog-raphy.The yeilds of FVP1and FVP2were22.86%,17.45%and the content of polysacchrides of them were98.80%,96.35%,respec-tively.

2.3.Synthesis of FVP-iron(III)complex

F.velutipes polysacchride iron(III)complex(FVP-Fe)was synthe-sized by neutralization of an FeCl3carbohydrate solution.Brie?y, 0.3mL of2M FeCl3was added drop wise under continuous stirring to the aqueous solution containing100mg of FVP and75mg of sodium citrate.The pH values were adjusted to7.0by the addition of2M of HCl or2M of NaOH solution.The temperature(50?C)was controlled by the constant temperature oscillation incubator(HZC-F250,Taicang Experimental Equipment Factory,Taicang,China). After1.0h,the reaction mixture was then centrifuged at4000g for10min.The supernatant was concentrated and then dialyzed in distilled water to remove unbound ions.Finally,the dialyzate was concentrated,precipitated by anhydrous ethanol,and then was freeze-dried.2.4.Antimicrobial activity

2.4.1.Bacteria,fungi and activation culture

The antibacterial and antifungal activities of FVP,FVP2,FVP-Fe, FVP2-Fe were determined against the pathogenic microorgan-isms,which were three bacteria including Staphylococcus aureus, Escherichia coli,Bacillus subtilis and three fungi including yeast,Rhi-zopus,Aspergillus supplied by School of Life and Science,China Pharmaceutical University,China.Beef extract peptone medium stable for bacteria,was composed of3.0g of beef extract,10g of petone,5.0g of sodium chloride,15–20g of agar(the liquid medium without agar),which were dissolved in1.0L of distilled water,adjusted to pH7.2–7.4,and autoclaved at121?C for20min. Potato dextrose agar medium(PDA)was made up of300g of potato, 20g of dextrose,15–20g of agar(the liquid medium without agar), which were dissolved in distilled water and autoclaved at121?C for20min.Sterile loop was used to inoculate the strains stored in EP tubes at?80?C and melted in37?C water bath onto the speci?c medium of agar slant in tubes.Agar slant of bacteria was incubated at37?C in digital biochemical incubator(SPX-100B-Z,Shanghai Boxun Medical Biological Instrument Corp.,Shanghai,China)for 24h and the agar slant of fungi was incubated at28?C in digital bio-chemical incubator for48h.Then complete strains of bacteria were inoculated on the beef extract peptone liquid medium in tubes, which were in the constant temperature incubator shaker(37?C, 150rpm,4h).And the complete strains of fungi were inoculated on potato dextrose liquid medium in tubes,which were in the constant temperature incubator shaker(28?C,150rpm,12h).The bacteria and fungi solution was used for expanding culture to produce the suspension of bacteria and fungi.

2.4.2.Antibacterial and antifungal activity assay

Antibacterial and antifungal activities were evaluated by Oxford cup method(Pei et al.,2014;Su et al.,2014).Two milliliter of bacte-ria suspension’s diluted inoculum(106CFU/mL)added into100mL of sterile beef extract peptone liquid medium,and2.0mL of fungi suspension’s diluted inoculum(106CFU/mL)added into100mL of potato dextrose liquid medium,were shaken well in the constant temperature incubator shaker at150rpm for5min,then mixed with100mL of the appropriate agar medium with3.0g of agar. The mixture was poured into the petri dishes(90×15mm)quickly, and cooled in the super clean bench.Sterile oxford cups(6mm inside diameter,8mm outside diameter)?lled with10mg/mL of FVP,FVP2,FVP-Fe,FVP2-Fe were placed on the agar medium,four cups on an agar medium and one of them?lled with sterilized water as blank control.In addition,every microorganism had two parallel experiments,the bacterial agar mediums were incubated at37?C for24h and the fungal agar mediums were cultured at28?C for48h. The bacterial and fungal inhibitory activities were determined by measuring the diameter of inhibition zone after taking down oxford cups,the data was recorded as means±SD.

2.5.Antioxidant activity assay

The antioxidant activity was assessed by seven different tests, including hydroxyl radical,DPPH radical,ABTS radical and superox-ide anion radical scavenging activities,reducing power,inhibiting the MDA production of liver homogenate induced by auto-oxidation and Fe2+-H2O2system respectively.In addition,ascorbic acid was used as a positive control.

2.5.1.Hydroxyl radical scavenging activity

Spectrophotometric method was used to determine the hydroxyl radical scavenging activity,which was investigated according the methods of Xu et al.(2012)and Wang,He,and Liu (2008).In the reaction system,1.0mL of sample solution including

28Y.-r.Dong et al./Carbohydrate Polymers161(2017)26–32

FVP,FVP2,FVP-Fe,FVP2-Fe and ascorbic acid(0,0.1,0.2,0.3,0.4, 0.5,0.6,0.7,0.8,0.9,1.0mg/mL)was mixed with1.0mL of FeSO4 (6mM),1.0mL of H2O2(6mM),then incubated at37?C for10min. One milliliter of salicylic acid–ethanol(6mM)was added into the mixture,shook well and incubated at37?C for30min before the absorbance of the mixture was recorded at510nm by ultraviolet and visible spectrophotometer(TU-1901UV/VIS spectrophotome-ter,Beijing Purkinje General Instrument Co.,Ltd.,Beijing,China). Deionised water displaced sample was as blank and ascorbic acid as a positive control.The hydroxyl radical scavenging activity was calculated as follows:

Hydroxyl radical scavenging activity(%)=[1?(A1?A2)/A0]×100 Where A1was the absorbance of sample solution or ascorbic acid, A2was the absorbance of mixture,but the deionised water instead of H2O2,and A0was the absorbance of the blank,sample replaced by deionised water.

2.5.2.DPPH radical scavenging activity

The DPPH radical scavenging activity was determined by the method previously described by Chen,Ma,Liu,Liao,and Zhao (2012),with a minor modi?cation.Two mmol/L DPPH solution was prepared in anhydrous ethanol in advance.Sample of1.0mL(at the concentration of0,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1.0mg/mL respectively)was added to1.6mL of distilled water and0.4mL of DPPH solution(2mM).The mixture was shaken and incubated at30?C for10min away from the light in the water bath.Then deionised water was used as blank,the absorbance of the reaction system was determined at517nm,and the ascorbic acid was the positive control.The DPPH radical scavenging activity was assessed by the following equation:

DPPH radical scavenging activity(%)=[1?(A1?A2)/A0]×100 Where A1was the absorbance of sample or ascorbic acid with DPPH solution,A2was the absorbance of mixture,but the deionised water instead of DPPH solution,and A0was the absorbance of the blank, sample replaced by deionised water.

2.5.

3.Superoxide anion radical scavenging activity

Superoxide anion radical scavenging activity was assessed by the method of Qiao et al.(2009)with little modi?cation.Before the determination,8mmol/L pyrogallic acid was prepared with 10mmol/L hydrochloric acid.Three milliliter of pH8.2Tris-HCl buffer solution(0.05M)and50?L of pyrogallic acid(8mM)were mixed with1.0mL of sample solution including FVP,FVP2,FVP-Fe,FVP2-Fe and ascorbic acid(0,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8, 0.9,1.0mg/mL).Shake the mixture and determine its absorbance at325nm immediately,using deionised water as blank.The absorbance of?rst minute and sixth minute were recorded.The formula of superoxide anion radical scavenging activity was as fol-lows:

Superoxide anion radical scavenging activity(%)=[1?(A1?A2)/A0]×100 Where A1was the oxidation rate of mixture with sample solu-tion and ascorbic acid,A0was the autoxidation rate of the pyrogallic acid.

2.5.4.ABTS radical scavenging activity

ABTS radical scavenging activity was evaluated according to method of Liang et al.(2012)with some modi?cation.ABTS could be oxidized by K2(SO4)2to generate glaucous cationic ABTS radicals. If the reaction system existed electron-antioxidant,it could react with ABTS and fade the glaucous ABTS,resulting in the decrease of the absorbance at734nm.0.3841g of ABTS and0.0662g of K2(SO4)2were dissolved in deionised water.Then the solution was transferred to100mL volumetric?ask,diluted with deionised water and mixed,which was incubated at room temperature for 12–16h in dark place away from light.The stock solution includ-ing7.0mmol/L ABTS and2.45mmol/L K2(SO4)2was diluted with 0.01mol/L pH7.4PBS to the right concentration(0.7absorbance at 734nm).In the reaction system,1.0mL of H2O and2.0mL of the ABTS solution were added into1.0mL of sample solution includ-ing FVP,FVP2,FVP-Fe,FVP2-Fe and ascorbic acid(0,0.1,0.2,0.3, 0.4,0.5,0.6,0.7,0.8,0.9,1.0mg/mL).Shaking well and place the solution at the room temperature away from light for6min,then determine the absorbance of the mixture at734nm.In addition, the deionised water was used as blank and ascorbic acid as positive control.The ABTS radical scavenging activity was calculated as the following equation:

ABTS radical scavenging activity(%)=[1?(A1?A2)/A0]×100 Where A1was the absorbance of sample solution or ascorbic acid with ABTS solution,A2was the absorbance of mixture,but the deionised water instead of ABTS solution,and A0was the absorbance of the blank,sample replaced by deionised water.

2.5.5.Determination of the reducing power

The determination of reducing power referenced the method of Wu et al.(2015)with some modi?cation.One milliliter of sam-ple solution including FVP,FVP2,FVP-Fe,FVP2-Fe(0,0.1,0.2,0.3, 0.4,0.5,0.6,0.7,0.8,0.9,1.0mg/mL)was mixed with2.0mL of PBS(0.2M,pH6.6)and2.0mL of potassium ferricyanide(1%,W/V), which was incubated at50?C in water bath for20min.Adding 2.0mL of trichloroacetic acid(TCA)into the solution to stop the reaction.After centrifugation(4000rpm,10min),3.0mL of super-natant was reacted with0.5mL of FeCl3(0.1%,W/V)for10min, then measured at700nm by ultraviolet spectrophotometer.50?L different concentration of ascorbic acid was used as positive con-trol and deionised water as blank.The reducing power could be assessed as follows:

Reducingpower=A1?A0

Where A1was the absorbance of sample solution or ascorbic acid, and A0was the absorbance of the blank.

2.5.6.Inhibiting the MDA production of health mice liver homogenate induced by auto-oxidation

One milliliter of mice liver homogenate(4%,W/V)was mixed with1.0mL of sample solution including FVP,FVP2,FVP-Fe,FVP2-Fe(0,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1.0mg/mL),which was incubated at37?C in water bath for1.5h.The mixture was added 1.0mL of TCA(10%,W/V)and1.0mL of thiobarbituric acid(TBA, 0.67%,W/V),mixed,boiled for15min then cooled with water.After centrifugation(4000rpm,5min),the supernantant was measured at532nm,deionised water as the blank.The inhibition ratio of MDA was valuated as follows:

T he inhibition ratio of MDA(%)=[1?(A1?A2)/A0]×100

The inhibition ratio of MDA(%)=[1?(A1?A2)/A0]×100

Where A1was the absorbance of the mixture with sample solution or ascorbic acid,A2was the absorbance of sample solution with deionised water,and A0was the absorbance of the blank,sample replaced by deionised water.

2.5.7.MDA production of health mice liver homogenate induced

by Fe2+-H2O2system

One milliliter of sample solution including FVP,FVP2,FVP-Fe, FVP2-Fe(0,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1.0mg/mL)was mixed with1.0mL of mice liver homogenate(4%,W/V),0.1mL of

Y.-r.Dong et al./Carbohydrate Polymers161(2017)26–3229 Table1

Antimicrobial activity of FVP,FVP2,FVP-Fe and FVP2-Fe.

Group Staphylococcus aureus Escherichia coli Bacillus subtilis Yeast Rhizopus Aspergillus

FVP7.99±0.01c8.00±0.01c7.99±0.01b8.00±0.017.98±0.028.00±0.01 FVP27.98±0.04c8.00±0.01c8.00±0.01b7.98±0.007.99±0.018.00±0.00 FVP-Fe15.45±0.22a11.87±0.26b13.91±1.13a8.00±0.017.94±0.107.98±0.04 FVP2-Fe13.07±0.45b12.29±0.04a12.45±0.99a8.00±0.047.99±0.018.00±0.01

Note:Data are the diameter of inhibition zone(mm)shown as means±SD(n=6).Values in the same row not sharing a common superscript letter are signi?cantly different at P<0.05.

FeSO4(6mM)and50?L of H2O2(60mM),which was incubated at

37?C for1.5h.The mixture was added1.0mL of TCA(10%,W/V)and

1.0mL of TBA(0.67%,W/V),mixed,boiled for15min then cooled

with water.After centrifugation(4000rpm,5min),the supernan-

tant was measured at532nm,deionised water as the blank.The

inhibition ratio of MDA was valuated as follows:

TheinhibitionratioofMDA(%)=[1?(A1?A2)/A0]×100

Where A1was the absorbance of the mixture with sample solution

or ascorbic acid,A2was the absorbance of sample solution with

deionised water,and A0was the absorbance of the blank,sample

replaced by deionised water.

3.Results and discussion

3.1.Effect on the antibacterial and antifungal activity

The rule about d,the diameter of inhibition zone was made to analyze the inhibitory activities of samples.When 6.5≤d<8mm,it indicated the sample had no inhibitory activ-ity,when8.0≤d<10.0mm,it indicated the sample had relatively strong inhibitory activity and when d≥10.0mm,meaning the sam-ple with high inhibitory effect.The antibacterial activity against Staphylococcus aureus,Escherichia coli,Bacillus subtilis was showed in Table1.The inhibition zones of FVP and FVP2against three bacteria were in the range of7.98±0.02to8.00±0.01,indicating that the two samples had no inhibitory effect on Staphylococcus aureus,Escherichia coli.While FVP-Fe exhibited high antibacterial effect on Staphylococcus aureus,Escherichia coli,and Bacillus subtilis (15.45±0.22,11.87±0.26,13.91±1.13,respectively).And FVP2-Fe was also effective against the three bacteria at the inhibition zones of13.07±0.45,12.29±0.04and12.45±0.99,respectively. In addition,Staphylococcus aureus was most susceptible to FVP-Fe with the biggest inhibition zone.

The results of antifungal activity against yeast,Rhizopus and Aspergillus were also described in Table1.It could be observed that FVP,FVP2,FVP-Fe and FVP2-Fe did not show inhibitory activ-ity against the three fungi,the inhibition zones in the range of 7.94±0.10to8.00±0.04.

In general,FVP and FVP2exhibited low inhibitory effect on bac-teria and fungi,while FVP-Fe and FVP2-Fe also had no antifungal activity against yeast,Rhizopus and Aspergillus.However,FVP-Fe and FVP2-Fe showed strong antibacterial activity against Staphylo-coccus aureus,Escherichia coli and Bacillus subtilis.

3.2.Effect on the hydroxyl radical scavenging activity

Hydroxyl radical as the active substance of reactive oxygen,has strong oxidizing ability and can damage various macromolecules, such as lipid,carbohydrates,amino acid and nucleic acid,causing disorganization and apoptosis,which is the most harmful to human health.Therefore,scavenging hydroxyl radical is one of the posi-tive measures to prevent diseases.The experiment adopted Fenton method to establish the reaction model,where Fe2+can react with H2O2to produce much short-lived but high activity of hydroxyl rad-

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Fig.1.Hydroxyl radical scavenging activity of FVP,FVP2,FVP-Fe,FVP2-Fe and Vc. Data are shown with means with standard deviation.

icals.At this point,if excess salicylic acid joined the reaction system, hydroxyl radical attacked the benzene ring of salicylic acid to pro-duce colored2,3-dihydroxybenzoic acid,which displayed a strong absorbance peak at510nm.When the sample had the capability to eliminate the hydroxyl radicals,it could compete with salicylic acid to react with hydroxyl radicals,resulting in the decrease of the2,3-dihydroxybenzoic acid and the absorbance at510nm.The hydroxyl radical scavenging activities of FVP,FVP2,FVP-Fe,and FVP2-Fe were shown in Fig.1.In the test,the concentration of sample ranged from0.1mg/mL to1.0mg/mL,the scavenging activity of sample on hydroxyl radicals increased with the increasing concentration. In general,FVP’s capability of inactivating the hydroxyl radical was higher than the FVP-Fe’s.In addition,compared with other samples, the FVP had the strongest to scavenge hydroxyl radicals and FVP2 was close to the FVP.The hydroxyl radical scavenging activities of FVP-Fe and FVP2-Fe were relatively weak.When the concentra-tion was1.0mg/mL,the scavenging activities of FVP,FVP2,FVP-Fe, and FVP2-Fe were41.54%,40.65%,19.79%,21.32%respectively,and the scavenging activity of ascorbic acid as positive control was 99.68%.The capability of eliminating hydroxyl radical in the sam-ple was found in order FVP>FVP2>FVP2-Fe>FVP-Fe.The protein in polysacchrride could chelate Fe2+to reduce the generation of hydroxyl radicals,and the FVP had more protein than the other samples.In addition,the sample could provide protons to hydroxyl radicals and achieved the scavenging radical result.So the FVP and FVP2had the certain ability to inactivated hydroxyl radicals at the high concentration.

3.3.Effect on the DPPH radical scavenging activity

DPPH could produce more stable nitrogen-centered radicals in organic solvent,which were purple and had a strong absorbance peak at517nm.The content of DPPH radicals and the absorbance value had a linear relationship within a certain concentration range. In the reaction system,the hygrogen donating ability was thought to do with the capability of scavenging DPPH activity.If the sample had strong antioxidant ability,the absorbance decreased at517nm, the DPPH radicals scavenging activity was higher.The scaveng-

30Y.-r.Dong et al./Carbohydrate Polymers161(2017)

26–32

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Fig.2.DPPH radical scavenging activity of FVP,FVP2,FVP-Fe,FVP2-Fe and Vc.Data

are shown with means with standard deviation.

ing activities of FVP,FVP2,FVP-Fe,FVP2-Fe and ascorbic acid were

determined at various concentrations on the above principle and

the results were shown in Fig.2.Results showed that the scaveng-

ing effect of FVP and FVP2increased quickly with increasing the

concentration,FVP-Fe,FVP2-Fe were in a relatively slow rate,and

the positive control of ascorbic acid always had a high scaveng-

ing activity.The scavenging activity of FVP2was higher than that

of FVP in the concentration ranged from0.1mg/mL to0.9mg/mL.

However,the scavenging effect of FVP was slightly above FVP2 s

at1.0mg/mL,while DPPH radical scavenging activities of FVP,

FVP2,FVP-Fe,FVP2-Fe were44.32%,43.78%,20.00%,25.14%,respec-

tively.At the high concentration,the capability of eliminating DPPH

radicals descending order was FVP>FVP2>FVP2-Fe>FVP-Fe.The

results indicated DPPH radical scavenging activity of FVP was supe-

rior to that of FVP-Fe and FVP possibly contained some substances

reacting with DPPH radicals by donating hydrogens.

3.4.Effect on the superoxide anion radical(O2?)scavenging

activity

Oxygen molecules could get an electron to become superoxide

anion radical because of electron transfer on the nuclear mem-

brane,cytokines P450of endoplasmic reticulum,cytokines,which

could generate H2O2by disproportionated reaction with super-

oxide dismutase(SOD).The superoxide anion radical triggered

liposome oxidation by generating more active hydroxyl ions or

singlet oxygen.The superoxide anion radical damaged DNA and

inactivated creatine kinase,glutathione peroxidase,catalase,caus-

ing diseases and accelerating aging.It was necessary for human

body to eliminate the excess superoxide anion radicals.The exper-

iment adopted pyrogallol autoxidation method,where pyrogallic

acid could produce superoxide anion radicals in weak base solu-

tion and generated colored intermediates with a strong absorbance

peak at325nm in a speci?c oxidation rate.The antioxidant can

inactivate superoxide anion radicals,decrease the rate of pyrogallol

autoxidation,and reduce the production of colored intermediates,

so it can decrease the reaction mixture’s absorbance at325nm.The

scavenging effect of superoxide anion radicals of FVP,FVP2,FVP-Fe,

FVP2-Fe was presented in Fig.3.From the graph it was evident that

FVP-Fe had stronger capability to eliminate superoxide anion rad-

icals than FVP,and the FVP-Fe had the highest scavenging activity.

The scavenging activity of the four samples on superoxide anion

followed the order:FVP-Fe,FVP2-Fe,FVP,FVP2and were55.26%,

47.57%,15.04%and8.27%at the concentration of1.0mg/mL,respec-

tively.FVP-Fe might react with pyrogallol or donate hydrogen to

superoxide anion radicals to scavenge the radicals.The result

indi-

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i

n

g

a

c

t

i

v

i

t

y

(

%

)

Conce ntration(mg/mL)

Fig.3.Superoxide anion radical scavenging activity of FVP,FVP2,FVP-Fe,FVP2-Fe

and Vc.Data are shown with means with standard deviation.

20

40

60

80

100

00.20.40.60.81

S

c

a

v

e

n

g

i

n

g

a

c

t

i

v

i

t

y

(

%

)

Conce ntration(mg/mL)

FVP

FVP-Fe

FVP2

FVP2-Fe

Vc

Fig.4.ABTS radical scavenging activity of FVP,FVP2,FVP-Fe,FVP2-Fe and Vc.Data

are shown with means with standard deviation.

cated the FVP-Fe and FVP2-Fe relatively strong ability to inactivate

superoxide anion radicals.

3.5.Effect on the ABTS radical scavenging activity

ABTS could generate the stable glaucous cation radicals by

reacting with K2(SO4)2,which had a peak absorbance at734nm.

The reaction system shallowed and the absorbance decreased at

734nm when the added antioxidants inactivate ABTS radicals.Fig.4

showed the scavenging activity of FVP,FVP2,FVP-Fe,FVP2-Fe and

the positive control ascorbic acid.As shown in the?gure,ascor-

bic acid always maintained the high scavenging effect,and the

scavenging activities of samples increased with the increasing con-

centration.It was obvious that the FVP had signi?cantly higher

capability to eliminate ABTS radicals than the other three samples.

At the concentration1.0mg/mL,the FVP showed79.37%scaveng-

ing effect close to ascorbic acid,and the scavenging activities of

FVP2,FVP-Fe,FVP2-Fe were44.27%,36.39%,31.32%,respectively.

The results indicated that the FVP had strong capability to inacti-

vate ABTS radicals by providing hydrogens or electrons and FVP2

was second to the FVP.

3.6.Effect on the reducing power

Sample with strong reducing power could provide electrons to

achieve the effect of scavenging radicals,generating more stable

production.So the reducing power is connected with the antioxida-

tion,and the determination of reducing power was usually used to

evaluate the antioxidant ability.In the experiment,the antioxidants

donated electrons,resulting in the reduction of the potassium fer-

ricyanide to potassium ferrocyanide,which could react with ferric

Y.-r.Dong et al./Carbohydrate Polymers 161(2017)26–32

31

0.0

0.10.20.30.40.50.60.70.80.90 0.2

0.4 0.6 0.8

1

R e d u c i n g p o w e r (A )

Conce ntration(mg/mL)

FVP FVP-Fe FVP2FVP2- Fe Vc

Fig.5.Reducing power of FVP,FVP2,FVP-Fe,FVP2-Fe and Vc.Data are shown with

means with standard deviation.

ion to produce prussian blue,having a peak absorbance at 700nm.If the sample had stronger reducing power,more prussian blue was generated,higher absorbance was evaluated at 700nm and the rad-ical scavenging activity was higher.Fig.5depicted the reductive capability of FVP,FVP2,FVP-Fe,FVP2-Fe compared with ascorbic acid as positive control.As Fig.5showed,the reducing power of four samples and ascorbic acid increased gradually with increasing concentration,and ascorbic acid maintained a high growth rate.In addition,the reducing power of FVP was much higher than the other three samples.At 1.0mg/mL,the reducing power of FVP,FVP2,FVP-Fe,FVP2-Fe were 0.319,0.134,0.136,0.084,which were in the order:FVP >FVP2and FVP-Fe >FVP2-Fe.

3.7.Effect on the malondialdehyde (MDA)production of health

mice liver homogenate induced by auto-oxidation

Liver homogenate could generate free radicals by auto-oxidation under suitable condition,which attacked polyunsatu-rated fatty acids on the bio?lm and caused lipid peroxidation.MDA as the end product of lipid peroxidation,enhance the penetrabil-ity of cell membrane,changed the structure and function of cell membrane and caused damage to human body through the cross-linking reaction with protein or nuclear acid.The contents of MDA in organic body could not only evaluate the extent of lipid per-oxidation and the oxidation ability,but also re?ect the extent of cells damage.The mice liver homogenate would produce MDA at the temperature 37?C by auto-oxidation,existing in the form of enol,reaction with barbituric acid to generate the red adducts by the action of heat,which had a peak absorbance at 535nm.When the antioxidant was added into the reaction system,it inhibited free radicals and MDA,resulting in the decrease of red adducts and the lower absorbance at 535nm.The abilities of four samples and ascorbic acid to inhibit the MDA production of healthy mice live homogenate induced by auto-oxidation were expressed in Fig.6.The FVP and ascorbic acid showed obvious growth rate of inhibit-ing MDA with increasing tested concentration,while the other three samples’inhibition ratio increased slowly.At 1.0mg/mL,the inhibition ratios of FVP,FVP2,FVP-Fe and FVP2-Fe were 45.23%,10.42%,11.64%,7.03%,respectively.Above all,FVP had relatively strong capability to inhibit MDA in health mice liver homogenate by auto-oxidation.

3.8.Effect on the MDA production of health mice liver homogenate induced by Fe 2+-H 2O 2system

The homogenate of mice liver with Fe 2+

and H 2O 2could effec-tively induce the increase expression of free radicals,causing the increase of end products,such as MDA.The abilities of four sam-ples and ascorbic acid to inhibit the MDA production of healthy

-100

1020304050607080900.0

0.2

0.4

0.6

0.8

1.0

I n h i b i t i o n r a t i o (%)

Conce ntration(mg/mL)

FVP FVP-Fe F2F2- Fe Vc

Fig.6.Inhibition ratio of FVP,FVP2,FVP-Fe,FVP2-Fe and Vc to eliminate the MDA production of health mice liver homogenate induced by auto-oxidation.Data are shown with means with standard deviation.

01020304050607080901000.0

0.2

0.4 0.6 0.8 1.0

I n h i b i t i o n r a t i o (%)

Conce ntration(mg/mL)

FVP FVP-Fe FVP2FVP2- Fe Vc

Fig.7.Inhibition ratio of FVP,FVP2,FVP-Fe,FVP2-Fe and Vc to eliminate the MDA production of health mice liver homogenate induced by Fe 2+-H 2O 2system.Data are shown with means with standard deviation.

mice live homogenate induced by Fe 2+and H 2O 2system were expressed in Fig.7.The inhibition ratios of four samples and ascorbic acid increased gradually with increasing tested concen-tration,which were all higher than the MDA inhibition ratios of samples induced by auto-oxidation except for FVP2-Fe.At the con-centration of 1.0mg/mL,the inhibition ratio of FVP was 84.16%,close to the inhibition ratio of ascorbic acid (87.75%).In addition,the inhibition ratios of FVP2,FVP-Fe and FVP2-Fe were 25.20%,42.04%,3.73%,respectively.The inhibition ratios of samples were found in the descending order at he high tested concentration:FVP >FVP-Fe >FVP2>FVP2-Fe.In the experiments,FVP showed the high inhibition ratio of MDA and indicated that it could scavenge free radicals effectively.Moreover,the FVP-Fe had fairly strong ability to inhibite the MDA and also could eliminate free radicals ef?ciently.

4.Conclusion

In this study,FVP and FVP2exhibited low inhibitory effect on bacteria and fungi,while FVP-Fe and FVP2-Fe had no anti-fungal activity against yeast ,Rhizopus and Aspergillus .However,FVP-Fe and FVP2-Fe signi?cantly suppressed the growth of bac-teria Staphylococcus aureus ,Escherichia coli ,Bacillus subtilis and had relatively strong antioxidant activity to scavenge superoxide anion radical.FVP exhibited strong antioxidant activity to eliminate hydroxyl,DPPH,ABTS radicals,had high reducing power and inhib-ited the MDA production of health mice liver homogenate induced by auto-oxidation and Fe 2+-H 2O 2system.The results suggested that FVP was a signi?cantly strong and natural antioxidant.In addi-

32Y.-r.Dong et al./Carbohydrate Polymers161(2017)26–32

tion,FVP-Fe and FVP2-Fe might be used as effective antibacterial agents.

Acknowledgements

This work was supported by the National Found for Fostering Talents of Basic Science(No.J1310032)and National Natural Science Foundation of China(No.31101255).

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