Structural Elucidation and Immune-Enhancing Effects of Novel …downloads.hindawi.com/journals/bmri/2019/7528609.pdf · 2019-07-30 · ResearchArticle Structural Elucidation and Immune-Enhancing

Research ArticleStructural Elucidation and Immune-Enhancing Effects of NovelPolysaccharide from Grifola frondosa

Yu-Ri Seo1 Dinesh K Patel2 Woo-Chul Shin1 Wan-Sup Sim3

Ok-Hwan Lee 3 and Ki-Taek Lim 1

1Department of Biosystems Engineering Kangwon National University Chuncheon 24341 Republic of Korea2The Institute of Forest Science Kangwon National University Chuncheon 24341 Republic of Korea3Department of Food Science and Biotechnology Kangwon National University Chuncheon 24341 Republic of Korea

Correspondence should be addressed to Ki-Taek Lim ktlimkangwonackr

Received 9 October 2018 Revised 19 February 2019 Accepted 28 March 2019 Published 16 April 2019

Academic Editor Yun-Peng Chao

Copyright copy 2019 Yu-Ri Seo et alThis is an open access article distributed under theCreative CommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Beta-glucan (120573-glucan) is amacromolecule structurewhere glucose unit has bonded through 120573-glycosidic bond at 1 and 3 positionsIt is well known as a natural immunomodulator without exhibiting any side effects via enhancing immunity Mushroom contains alarge amount of 120573-glucan and it has anticancerous and antioxidant efficacy Structure and physical properties of 120573-glucan are highlyinfluenced by the types of mushroom In particularGrifola frondosa has 120573-1 3 and 120573-1 6 bonds in their structure It has been notedthat 120573-glucan content also depends upon the size of mushroom particles The exact content of 120573-glucan and their immunologicalactivity by a particle size of G frondosa have yet to be fully elucidated Herein 120573-glucan contents were analyzed according to theparticle size of leaf mushroom followed by cell activation and immunoactivity analysis The highest 120573-glucan content was observedat a particle size of 20-30 120583m (2765 plusmn 030 ww) All samples showed sim 103 cell activation compared to the control and greater cellactivity was observed at higher concentrationThe significant increase in cytokines secretion was observed in the presence of 20-30120583m particle size of G frondosa compared to the control This study suggested that 20-30 120583m size is the suitable size of G frondosathat can be used as a health supplement and food additive to act as an immune booster hypotensive agent and hypoglycemic agent

1 Introduction

Much research on immune enhancement from natural prod-ucts has been carried out Notably there is a growing interestin the efficacy of specific physiologically active substancesfrom natural products and many studies on natural sub-stances with anticancer antioxidant and immunologicalactivity effects have been reported [1] Mushrooms are oneof the most frequently used natural products for immuno-logical activity [2] Many polysaccharides extracted frommushrooms are effective as anticancer drugs and for immu-nity control [3] A lot of edible mushrooms have becomeattractive as ldquofunctional foodsrdquo and as source materialsfor immunomodulators antitumor agents antibiotics andantihypertensive drugs [4] G frondosa one of the outstand-ing medicinal mushrooms with reported anticancer effectscontains various free amino acids and vitamins B1 B2 C and

D G frondosa is widely used in Japan China and Korea as atraditional food additive due to its texture delicious taste andexcellent aroma [5] Unlike other mushrooms 120573-1 3 bondsare added to 120573-1 6 bonds in G frondosa [6] Furthermore120573-glucan extracted from G frondosa has been reported toenhance antitumor activity and bone marrow toxicity bystrengthening the immune system [7] The key ingredient inimproving the immune system by affecting cellular immunerecovery is known as 120573-glucan in edible mushrooms [8] It isa valuable dietary fibre and is a polysaccharide of D-glucosemonomers linked by 120573-glycosidic bonds [9] The capacity of120573-glucan to activate immune effector response varies with itsstructure and particle size It has also been reported that thesize of 120573-glucan particles controls the production of specificcytokines [10] As interest in 120573-glucan increased variousstudies into solvent extraction [11] pH condition [12] andair classification [13] were conducted to improve the content

HindawiBioMed Research InternationalVolume 2019 Article ID 7528609 7 pageshttpsdoiorg10115520197528609

2 BioMed Research International

of 120573-glucan In particular a mushroom extract preparedfrom superfine particles has been reported to be useful forimmunity enhancement because the activity of lymphocyteswas effectively activated in nonclinical experiments [14] Gfrondosa induces enhanced production of cytokines suchas interleukin-1 interleukin-6 and tumor necrosis factor 120572[15] Besides it has been reported that D-Fraction extractedfromG frondosa activates interleukin-12 production and alsofor the T helper-1 cytokine interferon-120574 and the T helper-2cytokines interleukin-4 and interleukin-10 thereby elicitingantitumor activity [16] Studies on the effect of variousparticle sizes of G frondosa on immunity have continuedbut few have investigated the evaporation effect of 120573-glucanin microparticulated G frondosa In this study the contentof 120573-glucan was analyzed according to the particle size ofG frondosa (10-20 20-30 and 30-40 120583m respectively) Theoptimized microsize should be examined and compared tothe control sample Pulverizing G frondosa using an airclassifying mill could be an effective way to concentrate 120573-glucan content

2 Materials and Methods

21 Preparation of Materials The mushrooms (G frondosaRepublic of Korea) were treated by an air classifying mill(ACM-AL Daega Republic of Korea) that had a pulverizerand a separator Mushroom samples were pulverized afterpassing an impeller rotating at high speed in the air classifyingmilling machine and a classifier analysis software (Master-sizer S v310 Malvern UK) was used to classify particle sizeby centrifugal force and drag force Three fractions labeledas G1 G2 and G3 (10-20 20-30 and 30-40 120583m respectively)were obtained through air classification In the process thefeed mass was kept constant at 13 kg and the rotating speedwas set at 3600 g

22 120573-Glucan Analysis The quantitative analysis of 120573-glu-can was performed using a 120573-glucan kit (Megazyme IncChicago) [17] The content of 120573-glucan was calculated fromthe difference between total glucan (120573-glucan and 120572-glucan)and 120572-glucan To measure total glucan 20 mL of 12 Msulfuric acid was added to each of the three samples and leftfor 2 h Over this period 10 mL of water was added to eachsample and boiled in a water bath at 100∘C for 5 min Thesamples were then cooled at room temperature Each samplewas subsequently transferred to a 100 mL volumetric flaskand 6 mL of 10M potassium hydroxide (KOH) was addedThe samples adjust the pH 5 using 200 mM sodium acetatebufferThe samples were centrifuged at 1500 g for 10minTheobtained supernatants were transferred into a test tube and01 mL mixture solution (exo-1 3-120573-glucanase120573-glucosidasein 200 mM sodium acetate buffer) was added The sampleswere incubated at 40∘C for 60 min and 30 mL of GOPODReagent was added Absorbance was measured at 510 nmTo measure 120572-glucan 100 mg of each sample was added toa tube with 2 mL of 2 M KOH and stirred for 20 min in anice bath Then 8 mL of 12 M sodium acetate buffer (pH 38)and 02 mL of amyloglucosidaseinvertase were added The

samples were incubated at 40∘C for 30 min Next 01 mL ofthe sodiumacetate buffer and 30mLofGOPODreagentwereadded before incubation at 40∘C for 20 min Absorbance wasmeasured at 510 nm

23 Isolation and Structural Characterization of 120573-GlucanIsolation of 120573-glucan fromG frondosa was done as describedby Chakraborty et al [18] In brief washing of G frondosaparticles (20-30 120583m) was done with water followed by theethanol and dried it in an air oven at 45∘C for 48 h Afterthis extraction of 120573-glucan was performed by boiling thedried particles in 4 NaOH solution for 30 min and kept itovernight at 4∘C followed by centrifugation at 4000 rpm at8∘C for 1 h The supernatant was separated and precipitatedwith 110 (vv) ethanol and kept it for 48 h at 4∘C Theprecipitate was collected by centrifugation and repeatedwash with ethanol and acetone The obtained material wasextensively dialyzed with cellulose bag to remove the smallmolecules as well as alkali The precipitate was collectedby centrifugation and freeze-dried The structural propertiesof the extracted material were characterized by 1H-NMRmeasurement (400MHz JNM-ECZ400SL1) inMe2SO-D2O(61) and matrix-assisted laser depositionionization (time-of-flight) MALDI-TOF mass spectrometry (Bruker Autoflexspeed TOFTOF) as described earlier [19]

24 Cytotoxicity Test For the study of cytotoxicity cellviability experiment was performed with human mesenchy-mal stem cells (hMSCs) The hMSCs isolated from bone(Korean Cell Line Bank Republic of Korea) were culturedwith proliferative medium (90 DMEM 10 FBS and 1antibiotics) in a culture plate Cell viability was analyzed usingWST-1 assay (EZ-Cytox Cell Viability Assay Kit DaeillabService Co Ltd Republic of Korea) Before adding thesamples (G1 G2 and G3) the hMSCs were cultured in 96-well plates for 24 h The samples were added to each well atdifferent concentrations (10 50 and 100 120583gml) The viabilityof cultured hMSCs at 24 h after sample adding was assayedusing EZ-Cytox according to the described procedure Thecell medium with the untreated sample was set as a controlThe absorbance was measured at 450 nm (OD450) using thespectrophotometer

25 Antibody-Based Cytokine Array System Human Cytoki-ne Array C kit was purchased from Ray Biotech (AAH-CYT-1-2Norcross GA)The experiment was performed accordingto the manufacturerrsquos instructions The array membranescould detect 23 different growth factorscytokines at a timeTo investigate the immune effects of G1 G2 and G3 oncells 22 times 106 cellswell were seeded into 100 mm cultureplate Cell medium without samples served as the controlCells were grown with samples for 14 days The cells werethen starved to exclude the influence of serum cells for 24h Cell-free supernatants were obtained and analyzed bythe human cytokine arrays The membranes were treatedwith a blocking buffer and then treated with a sampleat 4∘C overnight The membranes were then washed withwash buffer and biotinylated antibody cocktail was added

BioMed Research International 3

0

5

10

01 1 10 100 1000

Volu

me D

ensit

y (

)

Size (m)

G1G2G3

Figure 1 Particle size analysis of the G frondosa powder by airclassifying mill

The membranes were incubated at 4∘C overnight and thenwashed followed by incubation with the HRP-Streptavidinat 4∘C overnight Finally the membranes were rewashed anddeveloped by incubation with detection buffer for 5 minChemiluminescence of the membranes was measured usingthe Chemidoc XRS system (BR170-8265 Bio-Rad USA)Relative protein expression was obtained by comparing thesignal intensities

26 Statistical Analysis Data are presented as mean plusmn stan-dard deviation All statistical analyses were carried out usingSPSS Statistics (IBM SPSS Statistics 23 IBM Inc USA)Statistical significance between control and treatment groupswas compared with a one-way analysis of variance (ANOVA)Statistical significance was considered lowastp lt 005

3 Results and Discussion

31 Analysis of 120573-Glucan Content in Different Particle SizesThe three different sizes of G frondosa studied in this workwere obtained by air classifying mill Figure 1 shows thegranulometric distribution of the three different particle sizesby Malvern software The accompanying 10 50 and 90values from the cumulative undersize curves are presented inTable 1 The d50 of samples (G1 G2 and G3) was 183 228and 327 120583m respectively Also high yields were maintainedat 97 in the pulverization120573-glucan contents of three different particle sizes in

microparticulated G frondosa are shown in Table 2 Theexperiment was repeated three times for each sample The 120573-glucan contents of G1 and G3 were 1880 plusmn 078 and 1767plusmn 033 respectively The highest average 120573-glucan contentwas found in a particle size of 20-30 120583m (G2 2765 plusmn 030)The 120573-glucan content of G frondosa was generally reportedto be 652 [20] Furthermore the 120573-glucan contents of Gfrondosa were measured as 25991 in a recent study [21]These results indicated that the microsized mushroom couldconcentrate the 120573-glucan contents Besides it was shown thatthe 120573-glucan content in a particle size of 20-30 120583mwas more

Table 1 Granulometric distribution of G frondosa samples

Sample Parameters on Particle Size Yield ()ltd10 (120583m) ltd50 (120583m) ltd90 (120583m)

G1 394 183 668 97G2 409 228 858 97G3 667 327 902 97

Table 2 120573-glucan contents via particle size of G frondosa

Sample 120573-glucan ( ww)(1) RSD ()(2)

G1 1880 plusmn 078 416G2 2765 plusmn 030 109G3 1767 plusmn 033 190(1)All values are expressed as mean plusmn SD in triplicate(2)Relative standard deviation

significant than the 120573-glucan content of various mushroomspulverized to a capacity of 100 120583m or less [22]

32 Structural Analysis of 120573-Glucan A 1H-NMR spectrumof 120573-glucan extracted fromG frondosa has been presented inFigure 2 It is well known that anomeric proton signal of the120573-glucan has occurred in the range of 4-6 ppm dependingupon the chemical moieties present in their structure [23]The appearance of the peaks at 5 and 45 ppm in the1H-NMR spectrum confirms the presence of the 120573-(1997888rarr3)and 120573-(1997888rarr6)-D-glucan linkage in the extracted materialrespectively

MALDI-TOF mass spectrum of 120573-glucan extracted fromG frondosa has been presented in Figure 3 MALDI-TOFmass analysis confirmed the presence of the 120573-(1997888rarr3) glucanstructure in the extract with the molecular mass gap of 162Da (one hexose unit) as observations have been reportedearlier [24 25] 25-Dihydroxybenzoic acid (25-DHB)matriximproved the polysaccharide signals intensity in theMALDI-TOF mass spectrometry However it was interesting to notethat in-between some peaks molecular gap was 648 DaThis was 4-fold higher than one hexose unit suggestingthe presence of oligomer structures in the polymer chainMass spectrum of 120573-glucan extracted from the G frondosaindicates that glucans are composed of a mixture of glucoseunits with molecular weights around 6678 sim 24068 Dawith the degree of polymerization (DP) = 4 sim 15 which issimilar to the earlier reported values of glucan extracted frommushrooms [26] On the basis of these experiments (NMRand Mass data) we concluded that extracted material hascomposed of hexose units with 120573-(1997888rarr3) and 120573-(1997888rarr6)-D-glucan linkage and presumable structure of glucans is giveninside the mass spectrum

33 Cytotoxicity Test The in vitro cell culture experimentswere performed with hMSCs (bone cell) to investigate theeffect of sample concentration and the three different particlesizes on cell proliferation responses The concentration ofsamples was set at 10 50 and 100 120583gml TheWST-1 assayingof hMSCs cells on an individual has been carried out after


O

OO

OO

OO

OHOH

OHOH

OH

OH

OH

1

1 0

2

2

12

12

3

3

3

3

4

4

5

5

5

56

6

46

46 2

22

-(1 rarr 6)-(1 rarr 3)

Glycosidic Linkage

Figure 2 A 1H-NMR spectrum of 120573-glucan extracted from G frondosa

OH

OHOHOHOH

OHOHOH

OHOH

OHOH OH

OHOH

（2（（2（（2

（2（（2（

（2（

（2（

OOOO

OOO

OOO

OOO

O O

1

1 1

2

2 2

3

33

4

44

5

5 5

6

66

m

n

Glycosidic Linkage

Hexose Unit

Oligomer Unit

Polysaccharides Chain

648

162

162

750 1000 1250 1500 1750 2000

mz

2250 2500 27500

1000

2000

3000

4000

Inte

ns [

au]

Figure 3 MALDI-TOF mass spectrum of G frondosa glucans using 2 5-DHB as a matrix The gap with 162 Da between peaks represents ahexose unit in the extract Fragmented ions appeared at DP = 4-15 with 6678 8300 11660 18145 19766 and 24060 Da respectively

incubating bone cells for 2 days (24 h after addition) ina proliferative medium at 37∘C The results of the WST-1cytotoxicity test for different particle sizes are shown in (Fig-ure 4) From this figure each of the microparticulated mush-room samples induced a concentration-dependent increasein cell proliferation G frondosa samples caused a significantincrease (lowastp lt 005) in cell viability at 50 and 100 120583gml Thecell viability at 10 120583gml of G1 was almost the same (p = 0611)but was higher at the other conditions (50 and 100 120583gml) Inthis regard the proliferation of cells was most accelerated at100 120583gml of G3 (increase 126 compared with the controlgroup) These results have confirmed noncytotoxicity for thethree different concentrations of G1 G2 and G3

34 Protein Expression of Antibody-Based Cytokine ArrayThe cytokine expression profile using a human cytokineproteinmembrane array is shown in Figure 5Themembranewas exposed only to the culture supernatant of hMSCs Byusing the antibody array (Figure 5(a)) the expression of 23different antibodies in cell-free supernatants with or withoutG frondosa treatment The quantitation of cytokine wasdetermined by calculating the intensity of the spot Sevencytokines were detected as spots on the membranes (IL-6IL-8 GRO abg GRO alpha MCP-2 CCL5 and MIP-1)(Figure 5(b))Themean for the signal densities for each of themembranes was calculated (Figure 5(c)) Interleukin 6 (IL-6)was upregulated about 126-177 in hMSCs with G frondosa


0

1

2

3

4

Cel

l via

bilit

y

ControlG1

G2G3

10 ＡＧl 100 ＡＧl50 ＡＧl

lowastlowastlowast

lowastlowast lowast lowast lowast lowast

G frondosa

Figure 4WST-1 assay of hMSCs (bone cell) cultured for different particle sizes ofG frondosa Values aremeans plusmn SD for three concentrationsper sample lowastp lt 005 compared with the control group

POS POS

IL-1 alpha

TGF beta 1

IL-13

IL-2

IL-15

TNF alpha

NEG NEG GCSF

IL-3 IL-5 IL-6 IL-7 IL-8 IL-10

IFN-gamma

TNF beta

MCP-1 MCP-2 MCP-3(CCL2) (CCL8) (CCL7)

BLANK BLANK BLANK BLANK

(CXCL8)

(CXCL9)

(CXCL1)

MIG

GM-CSF GROabg

GRO alpha

CCL5

POS

(a)

Control G1

G2 G3

(b)

MIP-1 delt

a IL6 IL8

GRO abg

GRO alpha

CCL5

MCP-20

2000

4000

6000

8000

10000

12000

ControlG1

G2G3

lowast

lowast lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast lowast

lowastlowast

lowast

lowast

lowast

lowast

(c)

Figure 5 Representative profile of the release of cytokines from hMSCs activated by microparticulated G frondosa (a) Template showingthe location of antibodies for protein spotted onto the human cytokine antibody array kit (b) Representative expression of various antibodiesin the cell-free supernatants with or without G frondosa treated (c) Relative protein levels detectedwith the cytokine array Values are meansplusmn SD for three concentrations per sample lowastp lt 005 compared with the control group

treatment Although interleukin-8 (IL-8) was decreased bythe addition of G1 it was increased by about 108 by additionof G2 and G3 Furthermore the addition of G2 was inducedthat the three cytokines (chemokine ligand 1 (CXCL1) CC-chemokine ligand 5 (CCL5) and GRO abg) were increased

compared to control When G2 and G3 were added to thecells they induced monocyte chemotactic protein-2 (MCP-2) production These results showed the immunomodulationeffect on various cytokine levels expressed in the hMSCswith G frondosa treatment The characteristic features of


the cytokines are their functional pleiotropy and redundancywhich include interferons interleukins stimulating factorsand growth factors and their regulating activities such asproliferation and differentiation are highly depended uponthe nature of the cells involved [27] These cytokines mayexhibit the proinflammatory or anti-inflammatory activitiesor both depending upon the target microenvironments[28] IL-1120573 TNF120572 IL-6 IL-15 IL-17 and IL-18 are themost crucial group of cytokines that exhibit inflammatoryactivities whereas IL-4 IL-10 and IL-13 are known asanti-inflammatory cytokines It was noted that the role ofinflammatory and anti-inflammatory cytokines in variousinflammation concerning their signaling pathways is stillambiguous [29ndash31]

4 Conclusions

G frondosa has various physiological activities 120573-glucan anessential component of G frondosa affects immune activity120573-glucan content in G frondosa depends on particle sizeraw materials and extraction conditions In this study theparticle size of G frondosa was microsized to concentrate on120573-glucan content (10-20 20-30 and 30-40120583m) Furthermorethe immunostimulatory activity of G frondosa was verifiedthrough cell activation ability and cytokine expression 120573-glucan contents of the microsized G frondosa were higherthan generally known 120573-glucan contents for other particlesizes It was confirmed that the microsized G frondosaeffectively concentrated the contents of 120573-glucan There wasalso a significant difference in cell proliferation in the treatedcells compared to the control Higher cytokine productionwas observed in the treated cells than in control In particularthe addition of G2 tended to increase all seven cytokines (IL-6 IL-8 GRO abg GRO alpha MCP-2 CCL5 and MIP-1)expressed The addition of G frondosa of particle sizes at 20-30 30-40 120583m induced MCP-2 production but the additionof particle sizes of 10-20 120583m did not lead to expression Theexpression of cytokines showed substantially higher cytokineproduction in the treated cells than in control As a result themicrosized G frondosa promoted cytokine production and isconsidered to be an excellent medicinal mushroom in termsof enhancing immunity It is suggested that the increasedexpression of various cytokines could be useful in the controlof immunity through anticancer and antitumor effects

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Disclosure

Yu-Ri Seo and Dinesh K Patel are equivalent authors

Conflicts of Interest

The authors state no conflicts of interest

Acknowledgments

This research was supported by Cooperative ResearchProgram for Fostering Local Specialized Industries (noR0006141) Ministry of Trade Industry and Energy andCooperative Research Program for Agriculture Scienceand Technology Development (no PJ012854012 017) RuralDevelopment Administration The authors also acknowledgethe Ministry of Education (no 2018R1A6A1A03025582)Republic of Korea for providing the financial support inthe Basic Science Research Program through the NationalResearch Foundation of Korea (NRF) This research wasalso supported by the BK 21 Plus through the NationalResearch Foundation of korea (KRF) funded by the Ministryof Education (no C1010546-010-07) Republic of Korea

References

[1] H Wagner ldquoSearch for plant derived natural products withimmunostimulatory activity (recent advances)rdquo Pure andApplied Chemistry vol 62 no 7 pp 1217ndash1222 1990

[2] S Patel and A Goyal ldquoRecent developments in mushrooms asanti-cancer therapeutics a reviewrdquo 3 Biotech vol 2 no 1 pp1ndash15 2012

[3] F S Reis A Martins M H Vasconcelos et al ldquoFunctionalfoods based on extracts or compounds derived from mush-roomsrdquo Trends in Food Science and Technology vol 66 pp 48ndash62 2017

[4] K-I Minato A Ohara and M Mizuno ldquoA proinflammatoryeffect of the 120573-Glucan from Pleurotuscornucopiae mushroomon macrophage actionrdquo Mediators of Inflammation vol 2017Article ID 8402405 9 pages 2017

[5] A Klaus M Kozarski J Vunduk et al ldquoBiological potentialof extracts of the wild edible basidiomycete mushroom grifolafrondosardquo Food Research International vol 67 pp 272ndash2832015

[6] N Ohno K Sato Y Adachi I Suzuk T Yadoma and SOikawa ldquoCharacterization of the antitumor glucan obtainedfrom liquid-cultured grifola frondosardquoChemical amp Pharmaceu-tical Bulletin vol 34 no 4 pp 1709ndash1715 1986

[7] Y Masuda M Inoue A Miyata S Mizuno and H NanbaldquoMaitake 120573-glucan enhances therapeutic effect and reducesmyelosupression and nephrotoxicity of cisplatin in micerdquo Inter-national Immunopharmacology vol 9 no 5 pp 620ndash626 2009

[8] P A Ayeka ldquoPotential of mushroom compounds asimmunomodulators in cancer immunotherapy a reviewrdquoEvidence-Based Complementary and Alternative Medicine vol2018 Article ID 7271509 9 pages 2018

[9] B Du Z Bian and B Xu ldquoSkin health promotion effects ofnatural Beta-Glucan derived from cereals andmicroorganismsa reviewrdquo Phytotherapy Research vol 28 no 2 pp 159ndash1662014

[10] M J Elder S J Webster R Chee D L Williams J S HillGaston and J C Goodall ldquo120573-glucan size controls dectin-1-mediated immune responses in human dendritic cells byregulating IL-1120573 productionrdquo Frontiers in Immunology vol 8p 791 2017

[11] L Saulnier S Gevaudan and J-F Thibault ldquoExtraction andpartial characterisation of 120573-glucan from the endosperms oftwo barley cultivarsrdquo Journal of Cereal Science vol 19 no 2 pp171ndash178 1994


[12] F Temelli ldquoExtraction and functional properties of barley 120573-glucan as affected by temperature and pHrdquo Journal of FoodScience vol 62 no 6 pp 1194ndash1201 1997

[13] Y Wu A Stringfellow and G Inglett ldquoProtein and 120573-glucanenriched fractions from high-protein high 120573-glucan barleys bysieving and air classificationrdquo Cereal Chemistry vol 71 no 3pp 220ndash223 1994

[14] J Shen H Ren C Tomiyama-Miyaji et al ldquoPotentiation ofintestinal immunity by micellary mushroom extractsrdquo Journalof Biomedical Research vol 28 no 2 pp 71ndash77 2007

[15] Y Adachi M Okazaki N Ohno and T Yadomae ldquoEnhance-ment of cytokine production by macrophages stimulated with(l997888rarr3)-120573-D-glucan grifolan (GRN) isolated from grifola fron-dosardquo Biological and Pharmaceutical Bulletin vol 17 no 12 pp1554ndash1560 1994

[16] N Kodama Y Murata and H Nanba ldquoAdministration ofa polysaccharide from Grifola frondosa stimulates immunefunction of normal micerdquo Journal of Medicinal Food vol 7 no2 pp 141ndash145 2004

[17] B VMcclear andMGlennie-Holmes ldquoEnzymic quantificationOF (1997888rarr3) (1997888rarr4)-120573-D-glucan in barley and maltrdquo Journal ofthe Institute of Brewing vol 91 no 5 pp 285ndash295 1985

[18] I Chakraborty S Mondal D Rout and S S Islam ldquoAwater-insoluble (1997888rarr3)-120573-d-glucan from the alkaline extractof an edible mushroom Termitomyces eurhizusrdquo CarbohydrateResearch vol 341 no 18 pp 2990ndash2993 2006

[19] R Tada THaradaNNagi-Miura et al ldquoNMRcharacterizationof the structure of a 120573-(1997888rarr3)-d-glucan isolate from culturedfruit bodies of Sparassis crispardquoCarbohydrate Research vol 342no 17 pp 2611ndash2618 2007

[20] P Rodrıguez-Seoane M J Gonzalez-Munoz E Falque andH Domınguez ldquoPressurized hot water extraction of 120573-glucansfrom Cantharellus tubaeformisrdquo Electrophoresis 2018

[21] M Sari A Prange J I Lelley and R Hambitzer ldquoScreeningof beta-glucan contents in commercially cultivated and wildgrowing mushroomsrdquo Food Chemistry vol 216 pp 45ndash51 2017

[22] O Ozcan and F Ertan ldquoBeta-glucan content antioxidant andantimicrobial activities of some ediblemushroom speciesrdquo FoodScience and Technology vol 6 no 2 pp 47ndash55 2018

[23] H-G Park Y Y Shim S-O Choi and W-M Park ldquoNewmethod development for nanoparticle extraction of water-soluble 120573-(1997888rarr3)-D-glucan from edible mushrooms sparassiscrispa and Phellinus linteusrdquo Journal of Agricultural and FoodChemistry vol 57 no 6 pp 2147ndash2154 2009

[24] T-W D Chan and K Y Tang ldquoAnalysis of a bioactive 120573-(1 997888rarr 3) polysaccharide (Curdlan) using matrix-assisted laserdesorptionionization time-of-flightmass spectrometryrdquoRapidCommunications in Mass Spectrometry vol 17 no 9 pp 887ndash896 2003

[25] J Wang G Jiang T Vasanthan and P Sporns ldquoMALDI-MS characterization of maltooligopolysaccharides fromdebranched starch amylopectin of corn and barleyrdquo Starch -Starke vol 51 no 7 pp 243ndash248 1999

[26] W-T Hung S-H Wang C-H Chen and W-B Yang ldquoStruc-ture determination of 120573-glucans from Ganoderma lucidumwith matrix-assisted laser desorptionionization (MALDI)mass spectrometryrdquo Molecules vol 13 no 8 pp 1538ndash15502008

[27] J Scheller A Chalaris D Schmidt-Arras and S Rose-JohnldquoThe pro- and anti-inflammatory properties of the cytokineinterleukin-6rdquo Biochimica et Biophysica Acta (BBA) - MolecularCell Research vol 1813 no 5 pp 878ndash888 2011

[28] D Su Z Lu M Shen X Li and L Sun ldquoRoles of pro- and anti-inflammatory cytokines in the pathogenesis of SLErdquo Journal ofBiomedicine and Biotechnology vol 2012 Article ID 347141 15pages 2012

[29] P Wojdasiewicz Ł A Poniatowski and D Szukiewicz ldquoTherole of inflammatory and anti-inflammatory cytokines in thepathogenesis of osteoarthritisrdquo Mediators of Inflammation vol2014 Article ID 561459 19 pages 2014

[30] D G Kim S W Seo B K Cho et al ldquoReview of currentapproaches for implementing metabolic reconstructionrdquo Jour-nal of Biosystems Engineering vol 43 pp 45ndash58 2018

[31] Y R Seo J W Kim H Seonwoo et al ldquoCellulose-basednanocrystals sources and applications via agricultural byprod-uctsrdquo Journal of Biosystems Engineering vol 43 pp 59ndash71 2018

Stem Cells International

Hindawiwwwhindawicom Volume 2018


MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom


of 120573-glucan In particular a mushroom extract preparedfrom superfine particles has been reported to be useful forimmunity enhancement because the activity of lymphocyteswas effectively activated in nonclinical experiments [14] Gfrondosa induces enhanced production of cytokines suchas interleukin-1 interleukin-6 and tumor necrosis factor 120572[15] Besides it has been reported that D-Fraction extractedfromG frondosa activates interleukin-12 production and alsofor the T helper-1 cytokine interferon-120574 and the T helper-2cytokines interleukin-4 and interleukin-10 thereby elicitingantitumor activity [16] Studies on the effect of variousparticle sizes of G frondosa on immunity have continuedbut few have investigated the evaporation effect of 120573-glucanin microparticulated G frondosa In this study the contentof 120573-glucan was analyzed according to the particle size ofG frondosa (10-20 20-30 and 30-40 120583m respectively) Theoptimized microsize should be examined and compared tothe control sample Pulverizing G frondosa using an airclassifying mill could be an effective way to concentrate 120573-glucan content

2 Materials and Methods

21 Preparation of Materials The mushrooms (G frondosaRepublic of Korea) were treated by an air classifying mill(ACM-AL Daega Republic of Korea) that had a pulverizerand a separator Mushroom samples were pulverized afterpassing an impeller rotating at high speed in the air classifyingmilling machine and a classifier analysis software (Master-sizer S v310 Malvern UK) was used to classify particle sizeby centrifugal force and drag force Three fractions labeledas G1 G2 and G3 (10-20 20-30 and 30-40 120583m respectively)were obtained through air classification In the process thefeed mass was kept constant at 13 kg and the rotating speedwas set at 3600 g

22 120573-Glucan Analysis The quantitative analysis of 120573-glu-can was performed using a 120573-glucan kit (Megazyme IncChicago) [17] The content of 120573-glucan was calculated fromthe difference between total glucan (120573-glucan and 120572-glucan)and 120572-glucan To measure total glucan 20 mL of 12 Msulfuric acid was added to each of the three samples and leftfor 2 h Over this period 10 mL of water was added to eachsample and boiled in a water bath at 100∘C for 5 min Thesamples were then cooled at room temperature Each samplewas subsequently transferred to a 100 mL volumetric flaskand 6 mL of 10M potassium hydroxide (KOH) was addedThe samples adjust the pH 5 using 200 mM sodium acetatebufferThe samples were centrifuged at 1500 g for 10minTheobtained supernatants were transferred into a test tube and01 mL mixture solution (exo-1 3-120573-glucanase120573-glucosidasein 200 mM sodium acetate buffer) was added The sampleswere incubated at 40∘C for 60 min and 30 mL of GOPODReagent was added Absorbance was measured at 510 nmTo measure 120572-glucan 100 mg of each sample was added toa tube with 2 mL of 2 M KOH and stirred for 20 min in anice bath Then 8 mL of 12 M sodium acetate buffer (pH 38)and 02 mL of amyloglucosidaseinvertase were added The

samples were incubated at 40∘C for 30 min Next 01 mL ofthe sodiumacetate buffer and 30mLofGOPODreagentwereadded before incubation at 40∘C for 20 min Absorbance wasmeasured at 510 nm

23 Isolation and Structural Characterization of 120573-GlucanIsolation of 120573-glucan fromG frondosa was done as describedby Chakraborty et al [18] In brief washing of G frondosaparticles (20-30 120583m) was done with water followed by theethanol and dried it in an air oven at 45∘C for 48 h Afterthis extraction of 120573-glucan was performed by boiling thedried particles in 4 NaOH solution for 30 min and kept itovernight at 4∘C followed by centrifugation at 4000 rpm at8∘C for 1 h The supernatant was separated and precipitatedwith 110 (vv) ethanol and kept it for 48 h at 4∘C Theprecipitate was collected by centrifugation and repeatedwash with ethanol and acetone The obtained material wasextensively dialyzed with cellulose bag to remove the smallmolecules as well as alkali The precipitate was collectedby centrifugation and freeze-dried The structural propertiesof the extracted material were characterized by 1H-NMRmeasurement (400MHz JNM-ECZ400SL1) inMe2SO-D2O(61) and matrix-assisted laser depositionionization (time-of-flight) MALDI-TOF mass spectrometry (Bruker Autoflexspeed TOFTOF) as described earlier [19]

24 Cytotoxicity Test For the study of cytotoxicity cellviability experiment was performed with human mesenchy-mal stem cells (hMSCs) The hMSCs isolated from bone(Korean Cell Line Bank Republic of Korea) were culturedwith proliferative medium (90 DMEM 10 FBS and 1antibiotics) in a culture plate Cell viability was analyzed usingWST-1 assay (EZ-Cytox Cell Viability Assay Kit DaeillabService Co Ltd Republic of Korea) Before adding thesamples (G1 G2 and G3) the hMSCs were cultured in 96-well plates for 24 h The samples were added to each well atdifferent concentrations (10 50 and 100 120583gml) The viabilityof cultured hMSCs at 24 h after sample adding was assayedusing EZ-Cytox according to the described procedure Thecell medium with the untreated sample was set as a controlThe absorbance was measured at 450 nm (OD450) using thespectrophotometer

25 Antibody-Based Cytokine Array System Human Cytoki-ne Array C kit was purchased from Ray Biotech (AAH-CYT-1-2Norcross GA)The experiment was performed accordingto the manufacturerrsquos instructions The array membranescould detect 23 different growth factorscytokines at a timeTo investigate the immune effects of G1 G2 and G3 oncells 22 times 106 cellswell were seeded into 100 mm cultureplate Cell medium without samples served as the controlCells were grown with samples for 14 days The cells werethen starved to exclude the influence of serum cells for 24h Cell-free supernatants were obtained and analyzed bythe human cytokine arrays The membranes were treatedwith a blocking buffer and then treated with a sampleat 4∘C overnight The membranes were then washed withwash buffer and biotinylated antibody cocktail was added


0

5

10

01 1 10 100 1000

Volu

me D

ensit

y (

)

Size (m)

G1G2G3









G1 394 183 668 97G2 409 228 858 97G3 667 327 902 97









O

OO

OO

OO

OHOH

OHOH

OH

OH

OH

1

1 0

2

2

12

12

3

3

3

3

4

4

5

5

5

56

6

46

46 2

22

-(1 rarr 6)-(1 rarr 3)

Glycosidic Linkage


OH

OHOHOHOH

OHOHOH

OHOH

OHOH OH

OHOH

（2（（2（（2

（2（（2（

（2（

（2（

OOOO

OOO

OOO

OOO

O O

1

1 1

2

2 2

3

33

4

44

5

5 5

6

66

m

n

Glycosidic Linkage

Hexose Unit

Oligomer Unit


648

162

162

750 1000 1250 1500 1750 2000

mz

2250 2500 27500

1000

2000

3000

4000

Inte

ns [

au]





0

1

2

3

4

Cel

l via

bilit

y

ControlG1

G2G3


lowastlowastlowast


G frondosa


POS POS

IL-1 alpha

TGF beta 1

IL-13

IL-2

IL-15

TNF alpha

NEG NEG GCSF


IFN-gamma

TNF beta



(CXCL8)

(CXCL9)

(CXCL1)

MIG

GM-CSF GROabg

GRO alpha

CCL5

POS

(a)

Control G1

G2 G3

(b)

MIP-1 delt

a IL6 IL8

GRO abg

GRO alpha

CCL5

MCP-20

2000

4000

6000

8000

10000

12000

ControlG1

G2G3

lowast

lowast lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast lowast

lowastlowast

lowast

lowast

lowast

lowast

(c)






4 Conclusions


Data Availability


Disclosure




Acknowledgments


References





































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















0

5

10

01 1 10 100 1000

Volu

me D

ensit

y (

)

Size (m)

G1G2G3









G1 394 183 668 97G2 409 228 858 97G3 667 327 902 97









O

OO

OO

OO

OHOH

OHOH

OH

OH

OH

1

1 0

2

2

12

12

3

3

3

3

4

4

5

5

5

56

6

46

46 2

22

-(1 rarr 6)-(1 rarr 3)

Glycosidic Linkage


OH

OHOHOHOH

OHOHOH

OHOH

OHOH OH

OHOH

（2（（2（（2

（2（（2（

（2（

（2（

OOOO

OOO

OOO

OOO

O O

1

1 1

2

2 2

3

33

4

44

5

5 5

6

66

m

n

Glycosidic Linkage

Hexose Unit

Oligomer Unit


648

162

162

750 1000 1250 1500 1750 2000

mz

2250 2500 27500

1000

2000

3000

4000

Inte

ns [

au]





0

1

2

3

4

Cel

l via

bilit

y

ControlG1

G2G3


lowastlowastlowast


G frondosa


POS POS

IL-1 alpha

TGF beta 1

IL-13

IL-2

IL-15

TNF alpha

NEG NEG GCSF


IFN-gamma

TNF beta



(CXCL8)

(CXCL9)

(CXCL1)

MIG

GM-CSF GROabg

GRO alpha

CCL5

POS

(a)

Control G1

G2 G3

(b)

MIP-1 delt

a IL6 IL8

GRO abg

GRO alpha

CCL5

MCP-20

2000

4000

6000

8000

10000

12000

ControlG1

G2G3

lowast

lowast lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast lowast

lowastlowast

lowast

lowast

lowast

lowast

(c)






4 Conclusions


Data Availability


Disclosure




Acknowledgments


References





































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















O

OO

OO

OO

OHOH

OHOH

OH

OH

OH

1

1 0

2

2

12

12

3

3

3

3

4

4

5

5

5

56

6

46

46 2

22

-(1 rarr 6)-(1 rarr 3)

Glycosidic Linkage


OH

OHOHOHOH

OHOHOH

OHOH

OHOH OH

OHOH

（2（（2（（2

（2（（2（

（2（

（2（

OOOO

OOO

OOO

OOO

O O

1

1 1

2

2 2

3

33

4

44

5

5 5

6

66

m

n

Glycosidic Linkage

Hexose Unit

Oligomer Unit


648

162

162

750 1000 1250 1500 1750 2000

mz

2250 2500 27500

1000

2000

3000

4000

Inte

ns [

au]





0

1

2

3

4

Cel

l via

bilit

y

ControlG1

G2G3


lowastlowastlowast


G frondosa


POS POS

IL-1 alpha

TGF beta 1

IL-13

IL-2

IL-15

TNF alpha

NEG NEG GCSF


IFN-gamma

TNF beta



(CXCL8)

(CXCL9)

(CXCL1)

MIG

GM-CSF GROabg

GRO alpha

CCL5

POS

(a)

Control G1

G2 G3

(b)

MIP-1 delt

a IL6 IL8

GRO abg

GRO alpha

CCL5

MCP-20

2000

4000

6000

8000

10000

12000

ControlG1

G2G3

lowast

lowast lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast lowast

lowastlowast

lowast

lowast

lowast

lowast

(c)






4 Conclusions


Data Availability


Disclosure




Acknowledgments


References





































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















0

1

2

3

4

Cel

l via

bilit

y

ControlG1

G2G3


lowastlowastlowast


G frondosa


POS POS

IL-1 alpha

TGF beta 1

IL-13

IL-2

IL-15

TNF alpha

NEG NEG GCSF


IFN-gamma

TNF beta



(CXCL8)

(CXCL9)

(CXCL1)

MIG

GM-CSF GROabg

GRO alpha

CCL5

POS

(a)

Control G1

G2 G3

(b)

MIP-1 delt

a IL6 IL8

GRO abg

GRO alpha

CCL5

MCP-20

2000

4000

6000

8000

10000

12000

ControlG1

G2G3

lowast

lowast lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowast lowast

lowastlowast

lowast

lowast

lowast

lowast

(c)






4 Conclusions


Data Availability


Disclosure




Acknowledgments


References





































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















4 Conclusions


Data Availability


Disclosure




Acknowledgments


References





































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of












































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















Documents

Structural Elucidation and Immune-Enhancing Effects of Novel …downloads.hindawi.com/journals/bmri/2019/7528609.pdf · 2019-07-30 · ResearchArticle Structural Elucidation and Immune-Enhancing