*Correspondingauthor:E-mail:[email protected];Phone:
+886-2-2736-1661ext.6160;Fax:+886-2-2378-0134.
Introduction
Yam(Dioscorea species)isamemberofthemono- cotyledonousfamilyDioscoreaceaeandisastaplefoodin
WestAfrica,SoutheastAsia,andtheCaribbean(Akoruda,
1984).Thefreshtuberslicesarewidelyusedasfunctional
foodsinTaiwan,andthedriedslicesareusedastraditional
Chinesemedicines(Liuetal.,1995).Yamtubercontains
mucilages,mannan-proteinmacromolecules(Misakietal.,
1972;TsaiandTsai,1984).Recently,yamtubermucilage
wasreportedtoexhibitantioxidant(Houetal.,2002;Lin
etal.,2005),angiotensinconvertingenzymeinhibitory
activities(Leeetal.,2003)andhypoglycemicactivities
(Hikinoetal.,1986;BaileyandDay,1989).Furthermore,
Chineseyam(D. alatacv.TainongNo.2)feedingre- sultedinantioxidanteffectsinhyperhomocysteinemiarats
(Changetal.,2004).
Manyisolatedpolysaccharidesarereportedtohave
immunomodulatoryactivities(BrownandGordon,2003;
Feizi,2000),andmedicinalmushrooms(Wasser,2002)
have been intensively investigated for their beneficial ef- fectsasimmunomodulatoryandantitumoragents.Len- tinan (Len), the (1→3)-β-glucanisolatedfromLentinus edodes,hasbeendemonstratedtohaveananti-tumor
activityagainstSarcoma180in vivoandin vitro(Zhang
etal.,2005).Reishi(Ganoderma lucidum)polysaccha- rideswerereportedasimmunepotentiators(Changand
Lu,2004;ZhuandLin,2005;Hsu,etal.,2004).Thecold- waterextractsofdietarymushrooms,includingHypsizigus mamoreus,Agrocybe aegerita,andFlammulina velutipes,
wereshowedtohaveantiproliferativeactivityagainst
humanleukemicU937cells(Ouetal.,2005).Theim- munomodulatoryactivitybyanisolatedα-glucan-protein
complexfrommyceliumofTricholoma matsutakehasalso
beendocumented(Hoshietal.,2005).Severalfood-grade
microalgae,includingSpirulina platensis,Aphanizomenon flos-aquae,andChlorella pyrenoidosa,arealsoknownto
containpolysaccharides,potentimmunostimulatorsofhu- manmonocytesandmacrophages(Pughetal.,2001).In
thisstudy,orallyadministeredmucilagesfromthreedif- ferentTaiwaneseyamcultivars,includingDioscorea alata
L.cv.Tainong1(TN1),Dioscorea alataL.cv.Tainong2
Immunostimulatory activities of yam tuber mucilages
Huey-FangSHANG
1,Huey-ChuanCHENG
2,Hong-JenLIANG
3,Hao-YuLIU
4,Sin-YieLIU
5,and
Wen-ChiHOU
4,*
1 Department of Microbiology and Immunology, Taipei Medical University, Taipei, Taiwan
2 Mackay Memorial Hospital, Taipei 104 and Mackay Medicine, Nursing and Management College, Taipei 112, Taiwan
3 Department of Food Science, Yaunpei University of Science and Technology, Hsinchu 300, Taiwan
4 Graduate Institute of Pharmacognosy, Taipei Medical University, No. 250, Wu-Hsing Street, Taipei 110, Taiwan
5 Taiwan Agricultural Research Institute, Council of Agriculture, Executive Yuan, Wu-Feng, Taichung, Taiwan
(ReceivedMay9,2006;AcceptedAugust15,2006)
ABSTRACT. The purified mucilages from three Taiwanese yam cultivars, including Dioscorea alataL.cv.
Tainong1(TN1),D. alataL.cv.Tainong2(TN2),andD. alataL.var.purpurea(Roxb.)cv.Ming-Jen(MJ),
andthecommerciallentinan(Len)wereusedtoevaluatetheimmunostimulatoryeffectsonthemurineinnate
andadaptiveimmunity.BALB/cmiceweregroupedandadministratedorallywith0.5mlofTN1,TN2,MJ
dailyfor5weeks.Thepositiveandnegativecontrolswerefedwithlentinananddistilledwater,respectively.
Bloodsamplesweredrawnfromtheretroorbitalsinusonday7and21,andthelymphocytesubpopulation,
phagocytosisofgranulocyteandmonocytewereanalyzedbyflowcytometry.Themiceweresacrificedon
day36,andthesplenocyteswereharvestedfordeterminationsofnaturalkiller(NK)cellcytotoxicityactiv- ity.ThestimulationindexonthephagocytosisofperitonealmacrophagesandtheRAW264.7celllinebyyam
mucilagewerealsodeterminedin vitro.Theresultsshowedthatallthreemucilages,especiallyMJyam,could
elevatethenumberofThelpercellsintheperipheralbloodandenhancethephagocyticactivityofgranulo- cyte,monocytesandmacrophagesboth ex vivoandin vitrotests.Increasedspleniccytotoxicactivityfollow- ing the administration of mucilages from MJ yam was observed. Furthermore, the production of specific anti- ovalbumin(OVA)antibodyandOVA-stimulatedspleniccellproliferationwerealsoenhancedbyallmucilage
groups.Itissuggestedthatthetubermucilagemayfunctionasanimmunomodulatorysubstance.
Keywords: Dioscorea;Immunostimulatory;Lentinan;mucilage;Nkcell;Phagocyticactivity;Yam.
(TN2),andD. alata L.var.purpurea (Roxb.)cv.Ming-Jen
(MJ),wereevaluatedfortheirimmunostimulatoryeffects
ontheinnateandadaptiveimmunityofBALB/cmice.
MATERIAL AND METHODS
Plant materialsLocalfreshtubersofTaiwaneseyam,includingD. ala- taL.cv.Tainong1(TN1),D. alataL.cv.Tainong2(TN2),
andD. alata L.var.purpurea (Roxb.)cv.Ming-Jen(MJ),
werekindlyprovidedbyDr.Liu,Sin-Yie(TaiwanAgricul- turalResearchInstitute,Wu-Feng,Taichung,Taiwan).The
TN1 tuber is a round or elliptic shape with white fleshes inthebrownpeel.TheTN2tuberisacylindraceousshape
with white fleshes in the brown peel. The MJ tuber is a cylindraceous shape with purple fleshes in the purplish-red peel.
Extraction and purification of yam tuber mucilage
Afterwashingandpeeling,theyamtuberswerecutinto
strips for mucilage extraction and purification as described elsewhere (Hou et al., 2002; Lee et al., 2003). Briefly, yam tuberwashomogenizedwithfourvolumes(W/V)of50
mMTris-HClbuffer(pH8.3)containing1%vitaminC.
Aftercentrifugationat14,000×gfor30min,thesuperna- tants were mixed with isopropanol to a final concentration of70%,andstirredquicklyat4ºCovernight.Theprecipi- tates were filtrated and dehydrated with 100% isopropanol, then,rinsedwithacetone.Afterdryingat40ºCinanoven,
thecrudemucilage(CM)wasgroundandcollectedfor
further purification by both SDS and heating procedures.
OnegramofCMpowderwasdissolvedin200mldistilled
waterandkeptina50ºCwaterbath.Fortymililitersof5%
SDSsolution(dissolvedin45%ethanol)wereaddedto
theCMsolution.Themixturewasstirredgentlyat50ºC
for30min,then,atroomtemperatureforanother2h.
Themucilagesolutionwasthenplacedinanicebathto
quicklyreducethetemperatureinordertoprecipitatethe
SDS-proteincomplex.Aftercentrifugationasabove,the
supernatantswereprecipitatedwithisopropanolanddried
ina40ºCovenasdescribedearlier.Themucilagewas
againground,dissolved,andthenheatedinboilingwater
for20min.Aftercentrifugation,thesupernatantswere
mixed with isopropanol to a final concentration of 70%.
The partially purified mucilage was filtrated, dehydrated, rinsedwithacetone,dried,andthencollectedforfurther
use.
Experimental animals
FiveweeksoldmaleBALB/cmicewerepurchased
fromNationalLaboratoryAnimalCenter(Taipei,Taiwan)
and divided randomly into five groups (n=8). Each group washousedindividuallyinwire-bottomedstainlesssteel
cagesinatemperature-andhumidity-controlledroom
(at22ºC)witha12-hlight/darkcycleandfreeaccessto
AIN-76feedsandwater.Allanimalexperimentalproce- duresfollowedthepublishedguidelines(COA,2004).For
assessmentofinnateimmunity,each0.5mlofTN1,TN2,
MJ(10mg/m)andcommercialLen(0.05mg/ml)weread- ministratedorallyonceadayfor5weeks.Distilledwater
wasusedforthecontrolgroup.Bloodsamplesweredrawn
fromtheretroorbitalsinusondays7and21,andthelym- phocytesubpopulationandbothphagocytosisofgranulo- cyte, and monocyte were analyzed by flow cytometry as describedbelow.
Lymphocyte subpopulation assay
Thelabeledprimaryantibodiesusedforlymphocyte
subpopulation,includingTcell,Bcell,Thelpercell,and
cytotoxicTcell,byflowcytometry(Viauetal.,2005)
wereshowedinTable1.Alllabeledmonoclonalantibod- ieswerepurchasedfromSerotecCompany(Oxford,UK).
Fiftymicrolitersofbloodisolatedfromtheretroorbital
sinusondays7and21wereputintheFalcontube(Falcon
2052). Labeled monoclonal antibodies (5 μl) were added andincubatedatroomtemperatureunderlightprotec- tionfor20min.Afterlysisandwashing,thelymphocyte
subpopulation was determined by the flow cytometry and analyzedbyCellQuestsoftware(BectonDickinsonFACS
CaliburTM,CA).
Phagocytosis of granulocyte and monocyte FITC-labeledEscherichia coli (MolecularProbes,
USA)powder(5mg)wassuspendedin0.5mlofHank’s
balancedsaltsolution(HBSS)andusedforphagocytosis
analysis by flow cytometry (Butcher et al., 2001; Gaforio etal.,2002).Onehundredmicrolitersofbloodfromthe
retroorbitalsinusondays7and21weremixedwith20
μl of FITC-labeled E. coli solutionat37ºCfor10min.
TheFalcontubewasimmersedinanicebathtostopthe
phagocytosis.Onehundredmicrolitersofoftrypanblue
(1.25mg/ml)wereaddedtoquenchtheresidualFITC- labeledE. coli. After lysis and washing, 5 μl of propidium iodide(PI,2mg/ml)wereaddedfor10min,andthe
phagocytosisofgranulocyteandmonocytewasdeter- mined by flow cytometry (Becton Dickinson FACS Cali- burTM,CA).
Table 1. The labeled primary antibody used for lymphocyte subpopulation assay by flow cytometry.
Lymphocytesubpopulation Surfacemarker Monoclonalantibodyused
Tcell CD3+ Phycoerythrin(PE)-anti-mouseCD3
Bcell CD19+ Fluoresceinisocyanate(FITC)-anti-mouseCD19
Thelpercell CD4+ FITC-anti-mouseCD4
Tcytoticcell CD8+ PE-anti-mouseCD8
Phagocytosis of peritoneal macrophage and the RAW264.7 cell line in vitro
Theperitonealmacrophageswereobtainedat3day
afterintraperitonealinjectioniorwith2mlofthioglycol- latebroth(BectonDickinson,CA)(ChoiandHwang,
2002;Choietal.,2004).RAW264.7cellline(Sosroseno
etal.,2003)wasculturedinDulbecco’smodifiedeagle
medium(DMEM,GibcoBRL,USA)containing5%fetal
bovineserum(FCS,GibcoBRL,USA),adjustedto2×106 cell/ml,andseededina96-wellplate(100µl/well).Ten
microlitersofTN1,TN2,MJ(1mg/ml),andcommercial
Len(0.1µg/ml)wereaddedtothe96wellsandcultured
ina5%CO2humidifiedincubatorat37ºCfor60min.
Afterremovingthesupernatants,20µlofFITC-labeledE.
coli wasaddedfor2h.Twohundredmicrolitersoftrypan
blue(1.25mg/ml)wereaddedineachwellsoneminfor
quenching.Thestimulationindex(%)forphagocytosisof
peritonealmacrophageandRAW264.7cellsbyyammuci- lages was determined by fluorescent analyzer.
Splenocyte-mediated cytotoxicity assay
Treated mice were sacrificed at day 36 and splenocytes wereharvestedfordeterminationsofNKcellactivity
(Choi et al., 2004; Zhu and Lin, 2005). Briefly, isolated splenocyteswerewashedwithPBSfor3timesandadjust- edto4×106cell/mlwithRPMI-1640medium(GibcoBRL,
USA)aseffectorcells.TheYAC-1cells,astargetcells,
werewashedwithHBSSandadjustedto2×106cell/ml.
Target cells at 200 μl were mixed with 2 μl of DiOC18(3
mM)(MolecularProbes,USA)at37ºCfor20min.Target
cellswereresuspendedin200µlofRPMI-1640medium
(GibcoBRL,USA)forfurtheruses.Theeffectorcellswere
mixedwithtargetcells(40:1,20:1,10:1,and5:1)inse- rialdilutionsandwereco-culturedin5%CO2 humidified incubatorat37ºCfor2h.Thesupernatantswereremoved,
andthesamevolumeofPIsolution(0.2mg/ml)wasadd- ed.Thesplenocyte-mediatedcytotoxicitywasdetermined
byflowcytometry(BectonDickinsonFACSCaliburTM,
CA).
Effects of yam mucilage on the production of the specific anti-Ova antibody (adaptive immunity assessments)
Miceweredividedin5groups,eachconsistingof8
animalsandadministeredorallywith0.5mlofTN1,TN2,
MJ(10mg/ml)andcommercialLen(0.05mg/ml)once
adayfor5weeks.Theovalbumin(OVA,2mg/mlwas
usedonday0,and6mg/mlwasusedonday21)waspre- mixedwithanequalvolumeofaluminumadjuvant(2.5
mg/ml)formiceimmunizations.Eachmousewasinjected
intraperitoneallyonday0andday21with0.2mlofOVA/
aluminumadjuvant.Bloodsamplesweredrawnfromthe
retroorbitalsinusbeforeandondays7,32,and43after
immunization.Theproductionofspecificanti-OVAan- tibody was analyzed by ELISA method (Mlčková et al., 2001;Okamotoetal.,2003).The96-wellplatewascoated
withOVA(1to5µg/ml)andthenblockedwithbovine
serumalbumin(10mg/ml).Peroxidase-conjugatedgoat
anti-mouseIgG(dilutedin1:104)or-conjugatedgoatanti- mouseIgM(dilutedin1:104)wasaddedandincubayedat
roomtemperaturefor2h.Theamountsofanti-OVA-IgG
andanti-OVA-IgMweremeasuredbyaddinghydrogen
peroxideandTMBfor15minandthenrecordingat450
nmbyELISAreader(TECANSunrisemicroplatereader,
Männedorf,Switzerland).
Effects of yam mucilage on the splenocyte proliferations after specific Ag stimulation (adaptive immunity assessments)
Twenty-fourdaysafterthesecondbooster,themice
weresacrificed,andthesplenocyteswereharvestedfor
proliferationdeterminationsbyMTTassay.Briefly,iso- latedsplenocyteswerewashedwithPBSfor3times,
adjustedto1×106cell/mlwithRPMI-1640medium
(GibcoBRL,USA),andseededina96-wellplate(100
µl/well).FiftymicrolitersofOVA（1 μg/ml）wereadded
toeachtestwellandthenculturedina5%CO2humidi- fied incubator at 37ºC for 48 h. Five microliters of MTT (5mg/ml)werethenaddedunderlightprotectionfor4
hrs and 100 μl of 10% SDS in 0.01 N HCl were added for 18hrs.Absorbanceat595nmwasdeterminedbyELISA
reader(TECANSunrisemicroplatereader,Männedorf,
Switzerland).Thestimulationindex(%)forproliferation
ofsplenocytesbyOVAwascalculatedfollowingtheequa- tion:(A595withyammucilagetreatment)÷(A595without
yammucilagetreatment)×100%.
Statistical analysis
Meansoftriplicatesweremeasured.Student’st-testwas
usedforcomparisonbetweentwotreatments.Adifference
betweenthecontrolandeachtreatmentwasconsidered
statistically significant when P<0.05(*)orP<0.01(**).
RESULTS AND DISCUSSION
Yamtubercontainsmucilagesthatwerereportedtobe
amannan-proteinmacromolecule(Misakietal.,1972;
TsaiandTai,1984).Ourpreviousreportrevealedthatthe
total recovery of purified yam tuber mucilages was about 48%thatofthecrudemucilages(Houetal.,2002).Inthis
report,mucilagesfromthreecultivarsofnativeTaiwanese
yamwerereportedtohaveimmunomodulatoryactivities
ininnateimmunityandadaptiveimmunity.
Lymphocyte subpopulation assay
TheresultsshowedthatthenumberofTcellsinTN2
orMJyammucilages-treatedanimalsisincreasedascom- pared with the control group in the first week (Figure 1A) andMJyammucilages-treatedonesinthethirdweek(Fig- ure1B)(P<0.01).Thiscellincrementwasduetoelevated
numberofThelpercellsandTcytotoxiccells(Figures1C
and1D)(P<0.05).TheincreasedThelpercellswerealso
foundafterglutaminesupplementsinratswithgut-derived
sepsis(Yehetal.,2004).Sinclair(1998)foundthatthe
the third, but not the first week as compared with the con- trol(P<0.01).Inthethirdweek(Figure2B),thephago- cyticactivitiesofgranulocyteandmonocytepopulations
significantly increased in the Len (P<0.01),TN1(P<0.05),
TN2(P<0.05),andMJ(P<0.01)groups.Ourpresentdata
revealthattheoraladministrationofnativeTaiwanese
yammucilagescanelevatethephagocyticcellpopulations
ofBALB/cmiceex vivo.Inmammals,phagocytosisisa
veryimportantdefenseagainstpathogeninvasionsand
apoptoticcellscavenging,whichisperformedbyphago- cyteslikemacrophages,dendriticcells,andgranulocytes
(Stuartetal.,2005).Monocytesandotherleukocytesare
recruited to the inflammatory site and differentiate in ad- vance to inflammatory macrophages (Van den Berg et al., 2001).
isolatedpolysaccharidesfromAstragalus membranaceus
(Huang-gi,Chineseherb)elevatedtheTcellandTcyto- toxiccellnumbers.TheincreasedThelpercellsubpopula- tionsinTh1orTh2subsets(MosmannandSad,1996)and
thesecretedcytokineswillbefurtherinvestigated.
Phagocytosis of granulocyte and monocyte isolated from blood of BALB/c mice
In the first week (Figure 2A), the phagocytic activity of thegranulocytepopulationshowednoapparentdifferenc- esfromthecontrolgroup.However,phagocyticactivity
bymonocytepopulationsignificantlyincreasedinTN1,
TN2,andtheMJgroupsascomparedwiththatinthecon- trol(P<0.05forTN1AndTN2andP<0.01forMJ).The
phagocyticactivityinLen-treatedanimalswaselevatedin
Figure 1.Theeffectsofyammucilagesonthelymphocytesubpopulations.Thebloodfromtheretroorbitalsinusonday7andday21
was analyzed for both T and B cells (A and B) and T cell subsets (C and D) using flow cytometry. Means of triplicates were measured.
A difference between the control and each treatment was considered statistically significant when P<0.05(*)orP<0.01(**).Barrep- resentsstandarddeviation.
Figure 2.Theeffectsofyammucilagesonthephagocytosisofgranulocyteandmonocyteonday7(A)andday21(B).Meansoftrip- licates were measured. A difference between the control and each treatment was considered statistically significant when P<0.05(*)or
P<0.01(**).Barrepresentsstandarddeviation.
Phagocytosis of peritoneal macrophage and the RAW264.7 cell line
TheperitonealmacrophagesandtheRAW264.7cells
werefrequentlyusedforphagocyticanalysisin vitro(Choi
etal.,2004;Sosrosenoetal.,2003).Alltheyammuci- lagesenhancedphagocytosisbymurineperitonealmacro- phagesascomparedwiththecontrol(P<0.01forTN1and
P<0.05forTN2andMJ,Figure3).Similarly,alltheyam
mucilagesaugmentedphagocytosisbyRAW264.7cells
ascomparedwiththecontrol(P<0.01,Figure3).Choiet
al.(2004)reportedontheyammucilageswithstimulated
activityofphagocytosis.Ourpresentresultshowedthat
thestimulationindexofperitonealmacrophagetreated
withTN1,TN2,MJ,andcommercialLenwere2.62,1.34,
1.24,and3.48-folds,respectively(Figure3A),andthe
stimulationindexofRAW264.7celllinetreatedwithTN1,
TN2,MJ,andcommercialLenwere2.73,3.28,3.17,and
6.7-folds,respectively(Figure3B).Highmannuronicacid- containingalginatewasreportedtostimulatethemurine
peritonealmacrophagephagocytosis(Son etal.,2001).
FromtheresultsofFigures2and3,themucilagesfrom
threeTaiwaneseyamsmayexhibitastimulatoryeffect
onphagocyticactivitybygranulocyteandmonocyte(ex vivo),onperitonealmacrophages,andontheRAW264.7
cells(in vitro).
Figure 3.Theeffectsofyammucilagesonthephagocyticactivityofperitonealmacrophage(A)andtheRAW264.7cellline(B).
Means of triplicates were measured. A difference between the control and each treatment was considered statistically significant when P<0.05(*)orP<0.01(**).Barrepresentsstandarddeviation.
Figure 4.Theeffectsofyammucilagesonthesplenocyte-me- diatedcytotoxicity.Meansoftriplicatesweremeasured.Adif- ferencebetweenthecontrolandeachtreatmentwasconsidered
statisticallysignificantwhenP<0.05(*)orP<0.01(**).Bar
representsstandarddeviation.
Figure 5. Effects of yam mucilages on the production of the serum specific anti-ovalbumin antibody of IgM (A) or IgG (B). Means of triplicates were measured. A difference between the control and each treatment was considered statistically significant when P<0.05(*)
orP<0.01(**).Barrepresentsstandarddeviation.
Splenocyte-mediated cytotoxicity assay
Theresultsofthepresentstudyshowedthatoraladmin- istrationofmucilagespromotedNKcellactivity.TheMJ
mucilagesshowedsignificantdifferences(40:1,P<0.01;
20:1,10:1,5:1,P<0.05)fromthecontrolgroup.Several
reportshaveshownthatpolysaccharidescanactivateNK
cellactivity(Wasser,2002;Choietal.,2004;Vetvickaand
Yvin,2004;Hoshietal.,2005;ZhuandLin,2005).NK
cellsarelargegranularlymphocytesthatlyseavarietyof
transformed and infected cells and are sufficiently devel- opedtocontrolinfectionortumor(DiefenbachandRaulet,
2001).ThepotentialuseofMJmucilagesforanti-cancer
treatmentwillbefurtherinvestigated.
Effects of yam mucilage on the production of the specific anti-Ova antibody
Forevaluatingtheeffectsofmucilagesontheadap- tiveimmunity,theproductionofspecificIgM(Figure
5A)orIgG(Figure5B)againstOVAwasdetermined.
Itwasfoundthatoneweekafterimmunizationtheoral
administrationsofmucilagecouldelevatetheamounts
of specific IgM (Figure 5A) and specific IgG (Figure 5B) againstOVAcomparedtothecontrolgroup(P<0.01,for
TN1andTN2).AfterthesecondboosterofOVA(day21),
the specific IgM production (day 32 and day 43) remained constant, and no significant differences among groups ap- peared.However,theIgGproductionapparentlyincreased
at day 32, and the Len and TN1 groups significantly out- perfomedthecontrolgroup(P<0.01).Itwasfoundthat
thespecificantibodiesrapidlyresponsesagainstforeign
proteins(orantigens)inthepresenceofyammucilages.
Itmightbepossibletousetheyammucilagesasimmune
adjuvantsforimmunomodulations.
Effects of yam mucilage on the splenocyte proliferations
FollowingimmunizationwithOVA,miceweresacri- ficed on day 45, and the splenocytes were harvested for proliferationdeterminations.AsseeninFigure6,outof
yammucilagesinvestigated,TN1andMJsignificantly
increasedcellproliferationascomparedwiththecontrol
(P<0.05).Itmeantthattheyammucilagesmightbeacted
asmitogens.Theincreasedlymphocytesubpopulation
neededinvestigationsfurther.
Inconclusion,theeffectsontheinnateimmunity(Fig- ures1to4)andadaptiveimmunity(Figures5and6)of
oraladministrationsofthreemucilagesfromTaiwanese
yamsonBALB/cmiceseemsignificant.Itissuggested
thatthetubermucilagemightfunctionasanimmunostim- ulatorysubstance.Theproductionofcytokinesandanti- cancertreatmentswillbeinvestigatedinthefuture.
Acknowledgements.TheauthorswanttothanktheNa- tionalScienceCouncil(NSC94-2313-B-038-001,NSC
94-2313-B-038-004), Republic of China for financial sup- ports.
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山藥黏多醣免疫調節活性的研究
商惠芳
1鄭惠川
2梁弘仁
3劉皓宇
4劉新裕
5侯文琪
41臺北醫學大學醫學系微免學科
2馬偕醫院及馬偕醫護管理專科學校
3元培科技大學
4臺北醫學大學生藥學研究所
5農委會農業試驗所
從三種山藥「台農一號（TN1）、台農二號（TN2）及名間長紅（MJ）」抽取的黏多醣（10
mg/ml），以及香菇多醣（Len，0.05 mg/ml）進行餵食 BALB/c 小鼠五週，觀察對於非特異性免疫活 性與特異性免疫活性之免疫調節活性評估。非特異性免疫活性包含淋巴細胞亞群的分佈、顆粒性白 血球和單核球細胞的吞噬作用、自然殺手細胞活性的分析。同時亦測試山藥黏質多醣對老鼠腹腔巨 噬細胞與巨噬細胞株（RAW264.7）體外吞噬作用之影響。而特異性免疫活性之評估則以 OVA 抗原
（ovalbumin）免疫老鼠後，利用ELISA分析專一性抗體產生濃度的高低；並以MTT定量法測試老鼠 脾臟細胞受OVA刺激後的分裂能力。本研究結果顯示，TN2及MJ組老鼠周邊血液中Tcell（CD3＋） 總數顯著增加（p＜0.01），而其中增加的又以輔助T細胞（Thelpercell,CD4＋）（p＜0.05）為最顯 著。而BALB/c小白鼠周邊血液中單核球細胞的吞噬能力在第一週時MJ組（p＜0.05）、TN1和TN2
組（p＜0.01）有顯著性增加，而顆粒性白血球的吞噬能力則皆無明顯變化。但至第三週時，四種樣品 皆顯著促進顆粒性白血球之吞噬能力；而單核球細胞之吞噬能力除TN2 組外其餘三種樣品皆呈顯著增 加。而四種樣品在試管中亦均能促進老鼠腹腔巨噬細胞與巨噬細胞株（RAW264.7）之吞噬能力。自然 殺手細胞毒殺能力的測定結果發現：Effectorcell和Targetcell比例從5：1至40：1時，MJ組自然殺手 細胞毒殺能力均比對照組有顯著增加（p＜0.01）。以OVA抗原免疫BALB/c小白鼠後，結果發現香菇 多醣和三種山藥黏質多醣皆可促進專一性抗OVA抗體IgG和IgM之生成，而其中以TN1和香菇多醣 效果較顯著。比較OVA免疫後老鼠的脾臟細胞在OVA抗原刺激後之分裂能力，亦發現香菇多醣和三種 山藥黏質多醣組老鼠脾臟細胞增生的能力均較對照組強。
關鍵詞：免疫促進；香菇多醣；山藥黏多醣；自然殺手細胞；吞噬活性；山藥。
