Hindawi Publishing Corporation Journal of Biomedicine and Biotechnology Volume 2012, rticle !

" #$%$#&, ' pages doi(10)11**+2012+#$%$#&

Research Article ,tructural Characteri-ation and ntio.idati/e cti/ity of 0ow12olecular13eights Beta11,%14lucan from the 5esidue of 6.tracted 4anoderma lucidum 7ruiting Bodies
Pai17eng 8ao,1 ,hwu1Huey 3ang,2 3ei19ing Hung,% 0iao,% Chun12ao 0in,&, * and 3en1Bin :ang%
1

:u1Han

Division of Cardiology, Wan Fang Hospital, 111 Hsing-Long Road, Section 3, aipei 11!, ai"an # Core Facility Center, $ce of Research and Develop%ent, aipei &edical 'niversity, #() W*-Hsing Street, aipei 11), ai"an 3 +eno%ics Research Center, Acade%ia Sinica, 1#, Acade%ia Road, Section #, aipei 11(, ai"an $rthopedics Research Center, aipei &edical 'niversity Hospital, aipei 11), ai"an ( School of &edicine, aipei &edical 'niversity, aipei 11), ai"an Correspondence should wbyang;gate)sinica)edu)tw 5ecei/ed 1 2011 be addressed to 3en1Bin :ang,

ugust 2011< 5e/ised 10 ,eptember 2011<

ccepted 1& ,eptember

cademic 6ditor( 2une=a-u !inuma Copyright > 2012 Pai17eng 8ao et al) 9his is an open access article distributed under the Creati/e Commons ttribution 0icense, which permits unrestricted use, distribution, and reproduction in any medium, pro/ided the original wor= is properly cited) 9he ma?or cell wall constituent of +anoder%a l*cid*% @+. l*cid*%A is /11,%1glucan) 9his study e.amined the polysaccharide from the residues of al=aline1e.tracted fruiting bodies using high1performance anion1e.change chromatography @HP 6CA, and it employed nuclear magnetic resonance @B25A and mass spectrometry @2,A to confirm the structures) 3e ha/e successfully isolated low1molecular1weight /11,%1glucan @024A, in high yields, from the waste residue of e.tracted fruiting bodies of +. l*cid*%) 9he %1@&,*1dimethylthia-ol121ylA12,*1diphenyl tetra-olium bromide @299A assay e/aluated the capability of 024 to suppress H2 C2 1 induced cell death in 5 32#&)$ cells, identifying that 024 protected cells from H2 C2 1induced damage) 024 treatment decreased H2 C2 1induced intracellular reacti/e o.ygen species @5C,A production) 024 also influenced sphingomyelinase @,2aseA acti/ity, stimulated by cell death to induce ceramide formation, and then increase cell 5C, production) 6stimation of the acti/ities of neutral and acid ,2ases in vitro showed that 024 suppressed the acti/ities of both neutral and acid ,2ases in a concentration1 dependent manner) 9hese results suggest that 024, a water1soluble /11,%1glucan recycled from e.tracted residue of +. l*cid*%, possesses antio.idant capability against H2 C2 1induced cell death by attenuating intracellular 5C, and inhibiting ,2ase acti/ity)

1) !ntroduction
Beta11,%1"1glucans constituents of fungi, algae, and higher plants) 9heseare glucans e.hibit di erent solubility in water1based solutions D1, 2E) +. l*cid*% is the most popular medicinal mushroom, with immunomodulating acti/ity, applied in the treatment of a /ariety of diseases, including

cancer, in numerous sian countries D%F*E) Pre/ious research has isolated se/eral ma?or substances with potent bioacti/1 ities, such as polysaccharides @in particular, /1 1,%1glucanA or protein1con?ugated /11,%1glucan D1, #F'E) Polysaccharides from +. l*cid*% are not linear in structure, comprising /1 @1 #A1"1glucosyl1lin=ed side

chains on a /11,%1"1glucan bac=bone) Bumerous studies ha/e described their functions DGF12E)

Beta11,%1"1glucans in +. l*cid*% ha/e high molecular weights and low solubility in neutral water solutions) 9his creates the need to degrade glucan into smaller si-es for fur1 ther application) Polysaccharide e.traction from +. l*cid*% produces a large amount of residue as waste material) 9here1 fore, the de/elopment of a HgreenI method of production, which recycles used materials and low /olumes of waste, is of high importance) 9he present studyJs methods for isolating /11,%1"1glucan are ecient and low cost when compared with other e.traction methods, pro/iding high1 Kuality products from the residue of +. l*cid*%, which may be useful in commercial applications) C.idati/e stress is implicated in the production of supero.ide radicals in biological systems) 5C, released from

2 phagocytic cells initiates a wide range of to.ic o.idati/e reac1 & tions and e.cessi/e formation of 5C, causes cell apoptosis) dministration of antio.idants, such as medicinal herbs, can protect cells by attenuating in?ury caused by o.idati/e stress D1%E) lmost all cell types e.press sphingomyelinase @,2aseA) !ntracellular ceramide production increases the hydrolytic acti/ity of ,2ases D1&E) Ceramide is an intracellular lipid mediator that responds to /arious stimuli including stress) 9he inflammatory o.idant, H2 C2 , induces rapid increases in ceramide le/els because of hydrolysis of sphingomyelin @,2A in plasma membranes, which contributes to the induction of /arious types of cell death) 9his study demonstrates that /11,%1glucan acti/ity is capable of interfering with ,2ases, and identifies /11,%1glucan deri/ing from +. l*cid*% as a potential antio.idant source) 9his study e.panded the application of +. l*cid*%, reusing the waste materials from the e.tracted residue of fruiting bodies to obtain high1purity /11,%1glucan in high yields) 3e also e.plored the biofunctions of low1 molecular1 weight1+. l*cid*% /11,%1glucan< an ecient antio.ida1 ti/e agent that eliminates 5C, in H2 C2 1treated cells by interfering with ,2ases) 5esults indicated that low1molec1 ular1weight /11,%1glucan has potential future use as a biomedicine) 9he technology used during polysaccharide degradation might also ha/e potential application on other medicinal herbs to release /arious glycans for biological assay)

Journal of Biomedicine and Biotechnology

% C"&G0

2 1 0 1 11 21 9ube @2 m0A %1 &1

7igure 1( ,i-e e.clusion chromatography diagram of recycled /1 1,%1glucan from the waste residue of +. l*cid*%. ,amples were isolated using a ,ephade. 411* column @H24( 11F1%< 024( 1&F 20< C,4( 2%F2G tubesA, and the sugar content of each tube was determined using phenol1sulfuric acid analysis @C"&G0 nmA)

2) 2aterials and 2ethods

#.1. Recycling and 0solation of /-1,3-+l*cans fro% 12tracted Resid*e of +. l*cid*%. 9he dried fruiting bodies of +. l*cid*% were ground and e.tracted using dilute BaCH solution at *0 C for 12 h) 9he e.tract was centrifuged to pro/ide water1insoluble part @waste residue, '0 to 'GLA and water1soluble part @soluble heteroglycan e.tracts, 11 to 20LA D1*E) 9he waste residue was further degraded by 2 B HCl at *0 C for & h to pro/ide hydrolysate @&*LA and insoluble residue @**LA) 9his glucan1containing hydrolysate underwent purification by gel1filtration chromatography @,ephade. 411*A to obtain three fractions< named high1 molecular1weight1glucan @H24A in 1'L, low1molecular1 weight1glucan @024A in #0L and oligosaccharide of glucose @C,4A in 22L yield, respecti/ely) 7igure 1 displays the chromatographic diagram of recycled /11,%1glucan from the waste residue of +. l*cid*%) #.#. 0nstr*%ents in Str*ct*ral Deter%ination of /-1,3+l*can. High1performance anion1e.change chromatogra1 phy @HP 6CA was used for the composition analysis of /11,%1glucans) 4lucose released by the acidic hydrolysis of 024 @& B 97 11% C # hA and characteri-ed using HP 6C1P " with =nown monosaccharides as standard, a "ione. Bio0C system @"ione., ,unny/ale, C , O, A with a CarboPac P 10 analytical column @&.*

ing to the methods described by Ha=omori et al) D1#E) 9he alditol acetates and partially methylated alditol acetates were separated by 4C12, using a HP1 capillary column @dimethyl silo.ane, %0 cm 200 mm from Hewlett Pac=ardA and a BPM1$0 column @,46 nalytical ,cience Pty 0td), Victoria %1%&, ustraliaA on PolarisN !on 9rap 4C12,+2, ,ystem @9hermo 7isher ,cientific, !nc), 3altham, 2 02&2*%, O, A) 9he o/en temperature was increased from %' C to 1*0 C at *0 C per min, then to 2%0 C at % C per min and to 2#0 C for * min, with carrier gas @HeA at a flow rate of 1 m0+min) B25 studies, 1 H+1% C B25, and other 1" and 2" e.periments were performed on a Bru=er 7ourier transform spectrometer @ V1#00A eKuipped with a * mm "C! dual cryoprobe) ,pectra were obtained at 2G' 8 with solutions of polysaccharide in "2 C) 9he HC" signal was achie/ed by a presaturation for 0)* s) B25 diusion ordered spectroscopy @"C,:A e.periments were carried out using the standard pulse seKuence on the same V1#00 BB spectrometer for determination of molecular weight) 9he eKuation for "C,: e.periments was as follows D1$E<
2 2 2

0 = 00 eDy g

3 @ 34 %A

0.&G m2 s1 D = '.2 10G &" . @1A

mm 2*0 mmA and a CarboPac P 10 guard column @& mm *0 mmA was used) 4as chromatography1mass spectrometer @4C12,A was used for analysis of saccharide composition and lin=ages accord1

Journal of Biomedicine and Biotechnology 2ass spectrometry was used for analyses of molecular weight and lin=ages) 9he molecular weights of the sodium adducts of oligosaccharides D2 P BaEP were determined using 2 0"!19C7 2, @ pplied Biosystems, 7oster City, C ) O, A) 9he glycan was mi.ed with 10 mg+m0 of 2, *1 dihydro.yben-oic acid @2,*1"HBA and 10 m2 BaCl in the ratio of * ( * ( %) !n 2, mode, the spectra were accumulated at an a/erage of 1,000 to 2,000 shuts at a sample concentration of 1 mg+m0) #.3. Cell C*lt*re. 9he mouse monocyte1macrophage cell line, 5 3 2#&)$, was cultured in "262 supplemented with 10L 7B,, 100 O+m0 penicillin, and 100 mg+m0 strep1 tomycin) Conditions were maintained in a humidified incubator @G*L air with *L CC2 A at %$ C)

#.-. Cell 5ia6ility Assay. 299 assay was conducted to assay cytoto.icity and cell /iability, as described pre/iously D1'E, dependent on the con/ersion of yellow tetra-olium salt to purple forma-an product) Cells @1 10* cells+m0A were grown on a G#1well plate) Cells were treated with 024 @0F 200 7g+m0A or 024 @0F1*0 7g+m0A combined with 200 72 H2 C2 for 2& h, after which an 299 solution @1 mg+m0 in PB,A was added, and cells were incubated for 2 h) fter the supernatant was discarded and "2,C was added to the well, the absorbance at *$0 nm was measured using a spectrophotometer @9hermo Varios=an 7lash, Vantaa, 7inlandA) #.(. Detection of 0ntracell*lar R$S For%ation. 0e/els of cellu1 lar o.idati/e stress were detected using the fluorescent probe, "C7" , as described pre/iously D1GE) fter being treated with 20 72 H2 C2 in the absence or presence of 024 @100 7g+m0A, 5 32#&)$ cells were treated with 20 72 "C7" for %0 min and then washed in PB,) "C7" is mainly trapped in the cytoplasm and is o.idi-ed to the highly fluorescent dichlorofluorescein @"C7A by intracellular 5C,) "C7 fluorescence intensities in cells were obser/ed using fluorescence microscopy @Clympus !M$1, 9o=yo, JapanA) #.!. S*ppression of 8e*tral and Acid S&ase Activities 6y L&+. Beutral ,2ase @n,2aseA acti/ity was estimated using an mple. 5ed ,2ase assay =it @!n/itrogenA, as described pre/i1 ously D20E) Briefly, the assay mi.ture contained the following components in a total /olume of 200 70) 9he en-yme source @0)0& O+m0 ,2ase, 1 O+m0 H5P, 0)2 O+m0 choline o.idase, ' O+m0 al=aline phosphatase, and *00 72 ,2 at pH $)&A in the absence or presence of 024 @0F200 7g+m0A was pre1 pared) 7or the n,2ase assay, assay mi.tures were incubated at %$ C for %0 min) ssay of acid ,2ase @a,2aseA acti/ity was performed at pH *)0) 9he reaction mi.ture contained 0)& O+m0 ,2ase, 024 @0F200 7g+m0A, and *00 72 ,2 in 100 70 of *0 m2 sodium acetate, pH *)0, and cell samples were incubated at %$ C for %0 min) 100 70 wor=ing solution @consisting of 100 72 mple. 5ed, 1 O+m0 H5P, 0)2 O+m0 choline o.idase, and ' O+m0 al=aline phosphatase in 100 m2 9ris1HCl, pH ')0A was added, and cells were incu1 bated at %$ C for %0 min) 7luorescence was measured using a microplate reader @9hermo Varios=an 7lashA with e.citation at *&* nm and emission at *G0 nm)

9he water1soluble component contained heteroglycan, glycoprotein and others, and earlier studies ha/e reported its immunomodulating function D%, #, 10E) 9he recycling of /11,%1glucan from the residue of +. l*cid*% was of interest due to the abundance of /11,%1glucan in the cell walls of fungi and wide application

%) 5esults and "iscussion

3.1. 0solation of /-1,3-+l*cans fro% 12tracted Resid*e of +. L*cid*%. 9his studyJs aim was to obtain 024 from the e.tracted residue of +. l*cid*% and to e.amine its antio.ida1 ti/e acti/ities) l=aline e.tracted the ground fruiting bodies of +. l*cid*% to release a water1soluble component @20LA and a water1insoluble residue @'0LA)

in medicinal food D1, 2, &E) Hydrolysis of the waste material, a water1insoluble residue, by strong acidic degradation @2 2 HCl, *0 C, & hA obtained 024 as low molecular weight glucans) 9his process pro/ided smaller si-ed beta1 glucans by narrowing down the high1molecular1weight1 polyglycans, with the hydroly-ed glucans becoming water soluble) 7ur1 ther purification of the hydrolysate using si-e1e.clusion chromatography @,ephade. 411*A pro/ided three fractions< named H24, 024, and C,4) 9he isolated process is shown in 7igure 1) 9he 024 is also obtained from the hydrolysate by ethanol precipitation @6tCH+H2 C = *+1A or by ultrafiltration @O7A with a molecular cuto *00 "a to remo/e low1molecular1weight1components for large1 scale isolation) ,tructural analyses and biological assays used the prepared 024 following collection and lyophili-ation) 3.#. Str*ct*ral Analysis of L&+. 9he isolated /11,%1glucan from the residue of e.tracted +. l*cid*% fruiting bodies was a white powder and relati/ely pure, despite containing compounds of /arious si-es due to chemical degradation producing a /ariety of molecular weights) 7or composition analysis, acidic hydrolysis @& B 97 , 110 C, & hA further degraded 024 and determined the monosaccharide con1 struct in HP 6C) Procedures identified the only glucose present in the 024 fraction, suggesting that 024 is a glucan from the cell wall of +. l*cid*%) 0in=ages analysis used 4C12, according to the methods described by Ha=omori et al) D1#E) 5esults showed that 024 contains a high ratio of 2,&,#1 trimethyl acetyl glucitol @1 % lin=ed glucoseA and trace amounts of 2,%,&,#1tetramethyl acetyl glucitol @terminal glucoseA and 2,&1dimethyl acetyl glucitol @1 # lin=ed glucoseA) B25 @7igure 2A, at 2G' 8, elucidated the structure of 024 is /11,%1glucan) 9he 1 H B25 spectrum of 024 protons H1FH# appeared at &)'2, %)*G, %)'2, %)**, %)*0, %.G$#a , and %.'0#b ppms, and its 1% C B25 spectrum appeared at 10%)0 @C11A, $%)2 @C12A, '*)2 @C1%A, #')& @C1&A, $#)% @C1 *A, and #0)G @C1#A ppms, respecti/ely) 9hese signals are in agreement with results from pre/ious analyses D11, 21E) s shown in 7igure 2, the tiny signals of /11,#1glucan appeared at 102)2 @C11A, $%)G @C12A, $#)* @C1%A, $0)& @C1&A, $#)' @C1*A, and #G)$ @C1#A ppms and the protons H1FH# at &)$0, %)&0, &)1*, &)0*, %)G0, &.#0#a , and %.$'#b ppms D22E) 3.3. &olec*lar Weights of L&+ as Deter%ined 'sing 8&R and &ALD0- $F &S. 5ecently, in/estigators de/eloped B25 "C,: as a useful tool for measurement of molecular weights, including those of polysaccharides D1$, 2%E) Osing the eKuation in the e.perimental section, the estimated molecular weight of 024 is %G$G "a) 9he signal at &)' ppm, log D = G.0, is H2 C with molecular weight = 1' @7igure %A) 9he signal of anomeric protons on 024 merged in the HC" at &)' ppm) 9he con/ergent dots in the same hori-ontal, at log D = 10.0, indicated that 024 has a narrow range in terms of si-e) 6.cepting the HC" signal, no dots appeared in the parallel line in the "C,:

spectrum indicating that protons in 024 are connected, and 024 is a homogeneous glucan with dierences in narrow range si-es)

H* H1 H#b H#a H% H& H2

Q12.* Q12 Q11.*

*)*

&)* @ppmA

&

%)*
Q11 Q10.*

1)#*1 %)0''

%)G2

Q10 QG.* QG
C#

@aA
C1 C% C* C2 C&

Q'.* Q'

G 10* 100 G* G0 '* '0 @ppmA @bA $* $0 #*

'

&

9he structure1acti/ity relationship @, 5A of 024 when e.erting antio.idati/e ef1 fects was of interest) Osing intracellular ceramide as a mar=er to determine the hydrolytic acti/ity of ,2ases e/aluated the

7igure 2( 9he 1 H B25 spectrum @aA and 1% C B25 spectrum @bA of 024) 9hese signals were in agreement with those of /11,%1 glucan from pre/iously results D11E)

2 0"!19C7 2, analysis confirmed the presence of /1 1,%1glucan in 024) 7igure & shows the molecular mass of 024 with gaps as 1#2 "a @one he.ose unitA between pea=s to pea=s) Ose of 2,*1"HB as a matri. substantially enhanced polysaccharide signals in the 2 0"! mass spectrometry D2&E) 9his study obtained positi/e ions of glucan with unam1 biguous signals, obser/ing 024 as sodiated ions @D4lcn P BaEP A where n is the number of glucose units @#1*A) 9he mass region of ion pea=s had a pea=1to1pea= mass dierence of 1#2)1 "a, consistent with the repeating unit of the /1 @1 %A1glucan) 7or e.ample, when determining the degree of polymeri-ation @"PA of 024 using 2 0"!1 9C7, the a/erage 024 mass appeared at "P = G @1&GG)1 "a, D4lcG P BaEP A as a base pea=) 9he molecular weights of 024 showed an a/erage of 1,*00 "a in 2 0"!19C7 2,, with slight dierences from those obtained using the "C,: e.periment) Based on these results, this study aimed to reco/er low molecular weight /11,%1glucan from waste materials of +. l*cid*% residue) 9hese recycled /11,%1glucans might ha/e potential commercial application as low1cost medicinal food) lthough some 1"1glucans are commercially a/ailable, they ha/e di erent / molecular weights, biological acti/ities, and structures) Pre/ious studies ha/e identified the biological acti/ities of /11,%1glucan containing +. l*cid*% D*F'E) How1 e/er, the antio.idati/e acti/ities of low molecular weight glu1 cans ha/e yet to be fully understood)

log D

@ppmA

7igure %( 9he 1" B25 "C,: e.periment of 024) Based on the eKuation, the calculated molecular weight of 024 is %G$G "a) 9he signal of anomeric protons merged in the HC" at &)' ppm and the signal, at log D = G.0, is H2 C with molecular weight = 1')

antio.idati/e acti/ity of isolated andby also indicated if 024s e.erts protecti/e eects024, on cells attenuating in?ury under o.idati/e stress) !nflammatory o.idants cause this in?ury by inducing rapid increases in ceramide le/els due to the hydrolysis of ,2 in plasma membranes) 9he study findings showed that 024 is capable of protecting cells against H2 C2 1induced apoptosis) 024 inhibited the acti/ity of ,2ases to decrease the le/els of intracellular ceramides, which may pro/ide a useful indicator of the antio.idati/e acti/ity of /1@1 %A1"1glucan) 3.-. 9rotective 1ects of L&+ against H2 $2 -0nd*ced &acrophage Death. Beta1@1 %A1"1glucan is the one of the ma?or components of polysaccharide from 4) lucidum, and has anti1inflammatory properties D*E) 9his study e.amined the antio.idant eects of 024) 299 assay e/aluated the cytoto.icity of 024 to 5 32#&)$ cells @7igure *@aAA, indi1 cating that 024 is not meaningfully cytoto.ic to 5 32#&)$ macrophages when 024 was added at 0F 200 7g+m0 for 2& h) Procedures also e/aluated the protecti/e ability of 024 against H2 C2 1induced in?ury) fter 2& h H2 C2 @20 72A treatment, 299 assay re/ealed &0L cell /iability, which increased to o/er '0L after cotreatment of cells with 024 @100 7g+m0< 7igure *@bAA) 5esults showed that 024 e.erts dose1dependent protecti/e eects against H2 C2 1induced cell death in 5 32#&)$ cells) 9he antio.idati/e acti/ities of the other acidic hydroly-ed fractions, H24 and 024, showed similar potency, but C,4 @wich contains monosaccharideA demonstrated reduced antio.idati/e acti/ity) 9his finding suggests that the molecular weights of beta11,%1glucan plays important roles in determining its biological acti/ity)

#00
CH2 CH C CH /

*00

CH

CH

CH2 CH CH CH2 CH CH2 CH C CH2 CH2 CH / C C C C C C C C / HC C / / HC CH CH CH CH CH HC CH n CH %

CH CH

CH2 CH C C /

CH

!ntensity @a)u)A

&00 1#2 %00

200

100

0 1000 1200 1&00 1#00 1'00 %4: 2000 2200 2&00 2#00

7igure &( 2 0"!19C7 with 2,*1"HB as matri., determined the molecular mass of 024) 9he gap with 1#2 "a between pea=s represents a he.ose unit in 024) ,odiated ions @D4lcn P BaEP A appeared at "P = # to 1* with 101%)1, 11$*)1, 1%%$)1, 1&GG)1, 1##1)2, 1'2%)2, 1G'*)2, 21&$)2, 2%0G)2, and 2&$1)% "a, respecti/ely)

1#0 1&0 120 Viability @LA '0 #0 &0 20 0 0 2*

024

120 100 Viability @LA '0 #0 &0 20

100

*0 100 @7g+m0A @aA

1*0

200

0 H2 C2 @20 72A 024 @7g+m0A

Q Q

P 0 @bA

P 2*

P *0

P 100

P 1*0

7igure *( Protecti/e abilities of 024 against H2 C2 1induced cytoto.icity in 5 32#&)$ cells) @aA 299 assay e/aluated the cytoto.icity of 024 to 5 32#&)$ cells) Cells were treated with 024 @at 0, 2*, *0, 100, 1*0, and 200 7g+m0A for 2& h) 5esults are presented as means ," for n = * replicates) @bA 9he protecti/e ability of 024 from H2 C2 1induced cytoto.icity in 5 32#&)$ cells) Cells were cotreated with H2 C2 @20 72A and 024 @0, 2*, *0, 100, and 1*0 7g+m0A for 2& h) 5esults are presented as the means ," for n = * replicates)

3.(. L&+ Red*ced H2 $2 -0nd*ced 0ntracell*lar R$S Levels. H2 C2 medication induces intracellular o.idati/e stress by increasing 5C, production) 9his study estimated intracel1 lular 5C, in e.ogenous H2 C2 1in?ured @20 72A macrophage cells) 7luorescence microscopy with "C7" , a redo.1sen1

siti/e fluorescent dye, /isuali-ed 5C, production) !n cells treated with H2 C2 alone @7igure #@bAA, the o.idi-ed "C7 fluorescent intensity was significantly greater than in untreated control 5 32#&)$ cells @7igure #@aAA) Cotreat1 ment with 024 @100 7g+m0A pro/ided mar=edly reduced

024 @100 7g+m0A H2 C2 @20 72A

Q Q

P P

@aA

@bA

@cA

7igure #( 024 reduced H2 C2 1induced intracellular 5C, le/els) 5 32#&)$ cells were treated with H2 C2 @20 72A and 024 @100 7g+m0A for %0 min, and intracellular 5C, le/els were measured using "C7" fluorescent staining and fluorescence microscopy) 0eft panels, untreated control< middle panels, H2 C2 added alone< right panels, co1treatment with H2 C2 and 024) !mages in the upper panels were captured using bright1field microscopy @original magnification 200.A, and those in the lower panels were captured using fluorescence microscopy)

120 100 !ntensity @LA

n,2ase acti/ity

120 100 !ntensity @LA '0 #0 &0 20

a,2ase acti/ity

'0 #0 &0 20 0 0 2* *0 @aA 100 1*0 200

2*

*0 @bA

100

1*0

200

024 @7g+m0A

024 @7g+m0A

7igure $( mple. red assay e/aluated 024Js ,2ase inhibitory acti/ity) 9he relati/e acti/ities of n,2ase @aA and a,2ase @bA were detected after treatment with 0, 2*, *0, 100, and 200 7g+m0 of 024 for %0 min) 9he relati/e acti/ity is presented as the means ," for n = % replicates)

"C7 fluorescence compared with fluorescence after addition of H2 C2 only @7igure #@cAA) !ncreasing 024 decreased 5C, production induced by addition of H2 C2 to 5 32#&)$ cells) 3.!. L&+ 0nhi6ited S&ase Activity. !n pre/ious studies, fac1 tors of inflammation, including 9B71;, 5C,, 0P,, amyloid1 /, !011/, and 0"0, stimulated ,2ase acti/ation acti/ity, which then produced ceramide and initiated ceramide signal pathways to transfer death signaling D2*F 2$E) 9his study e/aluated the eects of 024 on bacterial ,2ase acti/ity in vitro) !ncreasing 024 @0F200 7g+m0A reduced the

acti/ity of n,2ase, with an !C of appro.imately 120 7g+m0 *0 /alue @7igure $@aAA) 024 also had inhibitory eects on a,2ase acti/ity @7igure $@bAA) 9hese data suggest that 024 has

the potential to act as a ,2ase inhibitor) !n Cur pre/ious research identified that a polysaccharide from Cordyceps %ycelia possessed antio.idati/e ability and associated with ,2ase inhibitory eects, thus reducing le/els of ceramides produced from hydrolysis of sphingomyelin D20E) lthough findings did not fully elucidate the mechanism by which 024 e.erts antio.idati/e eects, 024Js potential as a ,2ase inhibitor may be of importance for its future applications)

&) Conclusion
9his study successfully isolated 024, a low molecular weight /11,%1glucan fraction, from the e.tracted residue of +. l*cid*% in high yields) chemical method degraded

the high1molecular1weight insoluble cell wall glucans into smaller si-ed compounds) 9his study also in/estigated the antio.idant acti/ity of isolated 024 /11,%1glucan to indicate the potential commercial /alue of +. l*cid*% glucans) 024 can protect cells by attenuating in?ury under o.idati/e stress caused by inflammatory o.idants) 9he study finding show that 024, a water1soluble /11,%1glucan with low molecular weight, recycled from e.tracted residue of +. l*cid*%, has acti/ity against H2 C2 1induced apoptosis) 024, therefore, is capable of e.erting antio.idant eects by inhibiting ,2ases) 9he recycling of /11,%1glucan from the remains of fruiting bodies could potentially pro/ide a food supplement source of antio.idants for maintenance of body health) 9his is the first study to report the antio.idati/e eects of low molecular weight /11,%1glucan from +. l*cid*%) 9he methods used ha/e potential application in the isolation of antio.idati/e polysaccharides from other medicinal herbs)

stress induced by "1galactose in mouse brain,I Life Sciences, /ol) '', no) 1*, pp) $1%F$1', 2011) DGE 3)19) Hung, ,)1H) 3ang, C)1H) Chen, and 3)1B) :ang, H,truc1 ture determination of /1glucans from +anoder%a l*cid*% with matri.1assisted laser desorption+ioni-ation @2 0"!A mass spectrometry,I &olec*les, /ol) 1%, no) ', pp) 1*%'F1**0, 200')

c=nowledgments
9his paper was supported by a 4rant1in1 id for scientific research from 4enomics 5esearch Center, cademia ,inica, 9aiwan 5)C)C), and a grant from 3an 7ang Hospital, 9aipei, 9aiwan 5)C)C) @10092O137H1011%A) P) 7) 8ao and ,) H) wang are contributed eKually to this wor=)

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