Anti-tumor and Immunostimulatory Functions of Two Feruloyl Oligosaccharides Produced from Wheat Bran and Fermented by Aureobasidium pullulans

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1 Anti-tumor n Immunostimultory Functions of Two Feruloyl Oligoscchries Prouce from Whet Brn n Fermente y Aureosiium pullulns Xiohong Yu,,, * Runqing Yng, Zhenxin Gu, Shojun Li, c n Hongshun Yng,e Feruloyl oligoscchrie 1 (FO1) n feruloyl oligoscchrie 2 (FO2) were prouce from whet rn fermente y Aureosiium pullulns (A. pullulns) through one- n two-stge temperture n ph controlling processes, respectively. Here the nti-tumor n immunostimultory functions of FO1 n FO2 were further exmine. Both FO1 n FO2 inhiite the growth of cncer cells ut were non-toxic to norml cells in vitro. In S180 tumor-ering mice, oth FO1 n FO2 significntly inhiite the growth of trnsplnte tumors n promote thymus, spleen inexes, interferon-γ, n interleukin-3 prouction. They lso increse peripherl leukocyte count n one-mrrow cellulrity. The iologicl ctivity of FOs prepre y ifferent processes ws further etermine. Interestingly, FO2 possesse more potent nti-tumor n immunostimultory effect thn FO1 in ose-epenent mnner. At ose of 250 mg/kg, the tumor inhiition rtes for FO1 n FO2 were 22.42% n 44.85%, respectively. These ntitumor properties of FOs my e meite y their eneficil effects on immunity responses. These t suggest tht FOs from whet rn my e use s ntitumor gents in the future. Keywors: Whet rn; A. pullulns; Feruloyl oligoscchrie; Anti-tumor function; Immunostimultory effect Contct informtion: : College of Chemistry n Biologicl Engineering, Yn Cheng Institute of Technology, Yncheng , Chin; : College of Foo Science n Technology, Nnjing Agriculturl University, Nnjing , Chin; c: College of Biologicl Engineering, Henn University of Technology, Zhengzhou, , Chin; : Foo Science n Technology Progrmme, c/o Deprtment of Chemistry, Ntionl University of Singpore, Singpore , Repulic of Singpore; e: Ntionl University of Singpore (Suzhou) Reserch Institute, 377 Lin Qun Street, Suzhou Inustril Prk, Suzhou, Jingsu , P.R. Chin; *Corresponing uthor: yxh1127@163.com INTRODUCTION Feruloyl oligoscchries (FOs) hve wiespre presence in grmineous plnts. FOs re forme y the croxyl esterifiction of ferulic ci (FA) n sugr hyroxyls (Yun 2006). They re wter-solule n het-resistnt ue to their moleculr structures, i.e., ferulic cyl n hyrophilic oligoscchries (Yun 2006; Xie 2010). Previous reserch hs focuse on the preprtion methos n the ntioxint ctivity of FOs (Yun et l. 2006; Ou et l. 2007; Wng et l. 2010; 2011). For exmple, Xie (2010) n Oht et l. (1994) reporte tht FOs exhiite etter ntioxint ctivity thn ferulic ci n ietry fiers. Yun et l. (2006) foun tht FOs h strong ility to remove Fe 2+, H2O2, n hyroxyl-free ricls. In ition, reserch inicte FOs lso inhiit the growth of Stphylococcus ureus (Yun 2006), inuce prolifertion of Bifiocterium (Yun et l. 2005), inhiit non-enzymtic glycosyltion of protein (Yun Yu et l. (2014). Feruloyl oligoscchrie, BioResources 9(4),

2 2006), n stimulte immunity (Yun 2006; Akhtr et l. 2012). Xie (2010) showe tht FOs with polymeriztion egree of 3 to 6 coul significntly limit the growth of humn colon cncer cell HCT-116 in vitro. However, to te, there hs een no literture report tht focuse on the in vivo nti-tumor properties of FOs. The iologicl functions of FOs re closely relte to their purity n structure (Zhi et l. 2000; Yun 2006; Xie 2010). Whet rn (WB) is n importnt source of FOs (Yun et l. 2005). Previous reserch y the uthors showe tht component purity or the composition of feruloyl rinoxylns (FAXs), egree of polymeriztion or the numer of xylose, n structure of FOs cn e moulte y A. pullulns s fermenttion processes (Yu et l. 2013, 2014). However, the mnner in which the component purity n egree of polymeriztion of FOs ffect their ntitumor function in vivo is still unknown. In previous work y the uthors, two components (FO1 n FO2) were prouce from whet rn fermente y A. pullulns vi one- n two-stge ph n temperture controlling processes, respectively (Yu et l. 2013, 2014). In the current stuy, the WB FOs nti-tumor n immunostimultory functions were investigte in vivo n in vitro. Moreover, the effects of purity n egree of polymeriztion of FOs on the nti-tumor n immunostimultory functions of the two FOs were etermine. Becuse FOs cn e prouce esily n unntly from A. pullulns-fermente WB, it is fesile to use FOs from WB s potentil nti-tumor component in functionl foos. Therefore, this stuy provies oth theoreticl sis n prcticl metho to evelop FOs s potentil functionl component of foo. EXPERIMENTAL Mterils Rw mterils n regents Commercil fresh WB ws supplie y Qin Flour Co. (Yncheng, Jingsu, Chin) n store t 4 C until use. Fluorourcil injection (5-FU; tch numer: ; specifictions: 10 ml, 0.25 g) ws from Hengrui Meicine Co. (Linyungng, Jingsu, Chin). Mei (RPMI-1640, IMDM, n F-12) were otine from GIBCO/BRL Life Technologies (Grn Isln, NY, USA). Fetl ovine serum (FBS), 3- (4,5)-imethylthihizo (-z-y1)-3,5-i- phenytetrzoliumromie (MTT), ethylene imine tetrcetic ci (EDTA), imethyl sulfoxie (DMSO), hemtoxylin (H), n eosin (E) were purchse from Sigm Chemicl Co. (St. Louis, MO, USA). Cncer cell lines Humn lung enocrcinom cells A549, humn gstric cncer cells BGC-823, n humn heptom cells HepG2 were purchse from the Cell Bnk n Tumor Cell Bnk of the Chinese Acemy of Sciences (Shnghi, Chin). Srcom S180 cells were provie y Jingsu Provincil Institute of Cncer Reserch (Nnjing, Jingsu, Chin). Humn norml liver cells L-02 n humn norml stomch cells GES were purchse from the Institute of Biochemistry n Cell Biology, Shnghi Institute for Biologicl Sciences, Chinese Acemy of Sciences (Shnghi, Chin). Yu et l. (2014). Feruloyl oligoscchrie, BioResources 9(4),

3 Animls Five-week-ol femle ICR mice, gre clen with weight of 18 to 22 g, were purchse from the Comprtive Meicine Centre of Yngzhou University (Yngzhou, Jingsu, Chin; Certificte No. SCXK ). They were house for three ys uner pthogen-free conitions t 18 to 24 C n reltive humiity of 70% on 12 h light/12 h rk cycle. All experiments were crrie out ccoring to the governmentl legisltion of Chin for the use n cre of lortory nimls n were pprove y the Bioethics Committee of the Institute of Meicinl Plnt Development, Chinese Acemy of Meicl Sciences (Beijing, Chin). Methos Preprtion of FO1 n FO2 Drie WB ws crushe into flour, psse through 40-mesh sieve to remove strch, n issolve in istille wter. The ph of the mixture ws juste to 5.5 with the ition of 2% (v/v) sulfuric ci to solution contining 60 g/l WB, 10 g/l xyln n 1 g/l peptone. The mixture ws incute t 50 C for 2 h. The resulting WB solution ws use s the fermenttion meium for A. pullulns 2012 (Yu et l. 2014). The FO1 n FO2 were prepre y one- n two-stge ph n temperture processes to control the WB fermenttion y A. pullulns 2012, respectively (Yu et l. 2013, 2014). A yiel of 904 nmol of FO1 per liter of fermenttion roth ws otine uner optiml conitions of initil ph 6.0, inocultion quntity 4.50%, n fermenttion temperture 29 o C. FO2 yiel vi the two-stge fermenttions reche 1123 nmol/l fter fermenttion for 96 h y controlling the initil ph t 4.0 n temperture t 33 C, n t 36 h of fermenttion chnging the ph to 6.0 n temperture to 29 C. This process ws 12 h shorter thn one-stge fermenttion for proucing FO1. The structures of FO1 n FO2 re shown in Fig. 1. The FO1 structure ws reporte in Yu et l. (2014). FO2 ws chrcterize y gs chromtogrphy (GC), infrre spectroscopy (IR), n electrospry ioniztion mss spectrometry (ESI-MS) (Yu et l. 2014). It ws emonstrte tht FO2 contine feruloyl rinosyl xylohexose (FAX6, MW1118), feruloyl rinosyl xylopentose (FAX5, MW986), n feruloyl rinosyl xylotetrose (FAX4, MW854). OH OH O O O O H O H O O O OH O H n H 3 C O O OH H O FAXn Fig. 1. Moleculr structures of feruloyl rinoxylns (FAXs). FO1 consists of feruloyl rinosyl xylopentose (FAX5), feruloyl rinosyl xylotetrose (FAX4), feruloyl rinosyl xylotriose (FAX3) n feruloyl rinosyl xyloiose (FAX2). FO2 consists of feruloyl rinosyl xylohexose (FAX6), FAX5 n FAX4. Yu et l. (2014). Feruloyl oligoscchrie, BioResources 9(4),

4 Anti-tumor ctivity test for FOs in vitro BGC-823 n HepG2 cells were culture in RPMI-1640 meium with 10% FBS. A549 cells were culture in F-12 nutrient mixture meium with 10% FBS, n L-02 n GES cells culture in IMDM meium with 10% FBS. BGC-823, HepG2 n A549 cells in logrithmic phse were igeste with 0.02% EDTA to count the living cells. They were culture in 96-well flt-ottom pltes with 100 μl 1x10 6 cells/ml suspension, incute (in triplicte) in mei contining 1000 μg/ml to 7.8 μg/ml of FO1 n FO2, t 37 C in n tmosphere contining 95% mient ir n 5% CO2. An equl volume of serum meium without e FOs ws e to the cells s the control group. A finl ose of 100 μg/ml 5-FU solution ws e s the positive group. The meium, MTT, n DMSO together were use s the zero control. The sornce vlue ws mesure y enzyme-lelling mesuring instrument (TECAN Infinite M200, Austri) t 570 nm. The inhiition rte of the cells ws clculte y the formul: (%) = (1-T/C) 100, where T n C re the verge sornce of the e FOs groups n control groups, respectively. BGC-823, HepG2 n A549 cells in logrithmic phse were igeste with 0.02% EDTA to count the living cells. They were culture in 6-well flt-ottom pltes with 1x10 6 cells/ml suspension. Mei contining 500, 1000, n 1500 μg/ml of FO1 n FO2 were introuce into the wells contining herent cells tht h een culture for 12 to 24 h. The group without FOs ws set up s the lnk. After incution t 37 C uner 5% CO2 for 24 h, the morphologicl chnge of cells ws oserve with n inverte microscope n then photogrphe t 20 (Wng et l. 2009; Wng et l. 2011). Soli tumor evelopment n rug ministrtion The ICR mice were inoculte with soli tumors ccoring to the portle tumor pproch (Xu et l. 2009; Co et l. 2011). Ech mouse ws inoculte in the right thigh of the lower lim with 0.2 ml of S180 cells n weighe fter 24 h. They were rnomize into five groups, with eight mice in ech group. Dose n ministrtion of physiologicl sline for norml control (mice without tumor cells inoculte) n moel control (mice inoculte with tumor cells) ws 0.4 ml/20 g of oy weight/ ministere orlly. The positive control ws 5-FU intrperitonelly ministrte once every other y t 0.4 ml per 20 g of oy weight (five times in totl). The tretment ose, issolve in sterile, 0.5% (w/v) CMC-N, ws ministere orlly t 50, 100, n 250 mg/kg of oy weight/ for the FO1 n FO2 groups. The first orl ose ws ministere fter 24 h (1) of the inocultion of tumors n repete once ily for totl of 10 times. Effect of FOs on tumor growth, weight gining rte, thymus n spleen inexes, onemrrow cell count, n peripherl white loo cell count (WBC) Mice were weighe n scrifice y cervicl isloction on Dy 11. Soli tumors, thymus, n spleen were ech collecte n weighe uner septic mnipultion, n were store in n ice th. The inhiition rte of tumor growth (%) ws clculte ccoring to the formul: (%) = (1-T/C) 100, where T n C re the verge tumor weight (g) of the e FOs tretment groups n the moel control groups, respectively. The weight gining rte ws clculte s (%) = [(Z-I)/I] 100, where I n Z re the initil n finl weight of the mice, respectively. The thymus n spleen inexes were expresse s the thymus or spleen weight (mg) reltive to its oy weight (g). Bone mrrow ws collecte from the left femur ones y flushing thoroughly with Hnk s Blnce Slt Solution (HBSS) using 1-mL syringe equippe with 28-guge neele. Yu et l. (2014). Feruloyl oligoscchrie, BioResources 9(4),

5 The cells were collecte in sterile tue n ilute to 10 ml in HBSS. Bone mrrow cell concentrtion ws etermine y n Innovtis CASY Moel TT cell counter (Roche Innovtis AG; Bielefel, Germny) (Co et l. 2011). The loo smples were hrveste y enucleting eyes of mice on Dy 11, n mixe with 2.5 mg/ml EDTA. The peripherl white loo cell count (WBC) were etermine using Sysmex XE-2100 hemtology nlyzer (Sysmex Corportion; Koe, Jpn). Histologicl tumor oservtion Tumors of the mice were fixe in 10% neutrl uffere formlin solution. After the tumors were processe n emee in prffin, 4-mm sections were prepre n stine with hemtoxylin n eosin (H&E) for microscopic oservtion. Effect of FOs on helper T (Th) cytokines nlysis The concentrtion of cytokines incluing IFN-γ n IL-3 ws etecte y specific enzyme-linke immunosorent ssy (Diclone, Rt IFN-γ ELISpot Kit; Frnce). The mount of IFN-γ n IL-3 ws mesure y spectrophotometry t 450 nm using n enzyme-lelling mesuring instrument (TECAN Infinite M200, Austri). Sttisticl nlysis Experimentl t were expresse s the men ± SD. All the t were t lest triplicte. The SPSS 18.0 softwre (SPSS Inc., Chicgo, IL, USA) ws use for ANOVA sttisticl nlysis. When P < 0.05, the ifference ws consiere to e sttisticlly significnt. RESULTS AND DISCUSSION Anti-tumor Property of FOs in vitro Both FO1 n FO2 showe significnt (p < 0.05) inhiitory property ginst humn liver cells HepG2 (Fig. 2), humn gstric cncer cells BGC-823 (Fig. 2), n humn lung enocrcinom cells A549 (Fig. 2c) in vitro. The tumor inhiition rte increse with increse FO ose (Figs. 2, 2, n 2c). When the concentrtion of FOs reche 1000 μg/ml, tumor cell growth ws inhiite y pproximtely 50%. The inhiition effect of 5-FU, positive control, ginst cncer cell prolifertion ws goo, ut its toxic impct on norml cells ws lso notle. When norml cells were ministere FO1 n FO2, only the group ose with 1000 μg/ml FO2 showe inhiitory effect on cell prolifertion. Specificlly, t 1000 μg/ml FO2 the inhiition rte ginst the humn norml liver cell L-02 ws 10.9 ± 0.6% (Fig. 2), n the humn norml stomch cell GES ws 14.3 ± 0.4% (Fig. 2e). FO ose elow 500 μg/ml i not show inhiitory effect on cell growth. By contrst, they even promote the norml cell prolifertion. This result inictes tht FO1 n FO2 not only hve n inhiitory effect on tumor-cell prolifertion, ut lso re not toxic to norml cells. The toxic ifference etween tumor cells n norml cells y FOs my e cuse y the inuction of certin signling molecules insie tumor cells. Morphologiclly, it ws shown tht cncer cells without FOs, the control, h vigorous growth, cler cell outlines, compct connections, n ttche growth (t not shown). When trete with FO1 n FO2, cncer cells file to mintin the intercellulr contcts n ecme roune. Smller cell sizes, inictive of nonvile cells, resulte Yu et l. (2014). Feruloyl oligoscchrie, BioResources 9(4),

6 from n increse FOs ose. When the ose of FO1 n FO2 reche 500 μg/ml, ttchment of the cncer cells ecrese n the cells flote n ecme roune, inicting tht the cells h ie. At 1500 μg/ml, the inhiitory effect of FOs ws even more ovious. Hence, these t suggest FO1 n FO2 cn inhiit the ctivities of humn liver cell HepG2, humn gstric cncer cell BGC-823 n humn lung enocrcinom A549 cncer cells in vitro FO 1 FO Inhiition rte (%) c c e f e g f g h g FU Concentrtion ( g/ml) c Inhiition rte (%) c c e f ef f e g g g h h FU Concentrtion ( g/ml) Inhiition rte (%) c c c c e f ef e f g g FU Concentrtion ( g/ml) Inhiition rte (%) c e e e f f f g c h i FU Concentrtion ( g/ml) e 40 Inhiition rte (%) e e f f g g h h i i c c FU Concentrtion ( g/ml) Fig. 2. Inhiitory effects of FOs on () humn heptom cells HepG2, () humn gstric cncer cells BGC-823, (c) humn lung enocrcinom cells A549, () humn norml liver cells L-02, n (e) humn norml stomch cells GES. Both FO1 n FO2 ose rnge from 7.8 µg/ml to 1000 µg/ml. The positive control, 5-FU, ws use t 100 μg/ml. Different letters within the sme tretment inicte sttisticl ifference (P < 0.05). Yu et l. (2014). Feruloyl oligoscchrie, BioResources 9(4),

7 Anti-tumor Function of FOs in vivo All three oses (50, 100, n 250 mg/kg oy weight) of FO2 h significnt (P < 0.05) inhiitory effect ginst soli tumor formtion in S180 tumor-ering mice, with inhiitory rtes of 29.39%, 34.85%, n 44.85%, respectively (Tle 1). Tretment with the positive control (5-FU) showe the highest tumor inhiitory effect (62.12%), with tumor weight significntly (P < 0.05) lower thn tht of FO1 t the sme ose. But the oy weight of mice ecrese. Collectively, FO2 showe more efficient nti-tumor ctivity thn FO1. In ition, no toxic symptoms were oserve in tretments with FOs (Tle 1). Tle 1. Inhiitory Effects of FOs on Srcom S180 in Mice Group Dose (mg/kg) Weight gining Tumor inhiiting Tumor weight (g) rte (%) rtes (%) Blnk control 19.63±1.46 Moel control 16.47± ±0.18 FO1 FO ± ± ± ± ±0.09 c 16.36±0.08 c ± ±0.07 c 22.42±0.06 c ± ±0.11 c 29.39±0.10 c ± ±0.13 c 34.85±0.11 c ± ±0.08 e 44.85± FU ±1.72 c 0.42±0.08 e ±0.07 Different letters in the sme column inicte sttisticl ifference (P < 0.05) Figure 3 isplys the histologicl morphology of stine tumor tissue. The moel control (Fig. 3) ws lrge hyperchromtic tumor group, with vigorous tumor growth. The positive control of 5-FU (Fig. 3) h numer of white cvities with holes suggesting tumor cells poptosis. However, there ws no re ye structure in the 5-FU tretment (Fig. 3). FO1 group t ose of 50 mg/kg (Fig. 3c1) ppere to contin some net-like eosinophilic res n few white cvities, ut h no re-stine structure. Apoptotic cells hve istinctive morphologicl ppernce, such s shrinking n chromtin conenstion. The cells with white cvities were ientifie s poptotic cell oies with holes. The presence of poptotic cells uner FO1 tretment were less frequently foun thn the positive control. At FO1 ose of 100 mg/kg (Fig. 31), tumor cells showe the mss of necrosis n white cvities ut no re ye structure. The rte of tumor cell poptosis in FO1 t 100 mg/kg group ws higher thn FO1 t 50 mg/kg, ut less thn the positive control. When FO1 ose increse to 250 mg/kg (Fig. 3e1), numer of eosinophilic res n white cvities showe, inicting the mss necrosis of tumor cells mixe with isintegrtion, n egenertion of nucleoli. In contrst, for FO2, t ose of 50 mg/kg (Fig. 3c2), lrge tumor group evelope in the mile of cells, with eosinophilic res n few white cvities on the ege, perhps cuse y inflmmtion. Thus, few poptotic cells were foun n some of them were phgocytize y other tumor cells. Incresing FO2 ose to 100 mg/kg (Fig. 32) cuse only little tumor group, ut numer of white cvities, inicting tumor cell isintegrtion, n egenertion of nucleoli. When FO2 ws further increse to 250 mg/kg (Fig. 3e2), lrge re of re ye n few white cvities were oserve, Yu et l. (2014). Feruloyl oligoscchrie, BioResources 9(4),

8 inicting tumor cell poptosis. In generl, t 250 mg/kg, FO2 ws more effective in inhiiting tumor growth thn FO1. c1 c2 1 2 e1 e2 Fig. 3. Histologicl morphology of tumor tissue. () Moel control, () Positive control, (c1) FO1, 50 mg/kg, (1) FO1, 100 mg/kg (e1) FO1, 250 mg/kg, (c2) FO2, 50 mg/kg, (2) FO2, 100 mg/kg, n (e2) FO2, 250 mg/kg Yu et l. (2014). Feruloyl oligoscchrie, BioResources 9(4),

9 Immunity Functions of FOs in S180 Tumor-ering Mice The immunopotentition property of FO1 n FO2 on S180 tumor ering mice ws supporte y the fct tht spleen inexes of mice ering srcom were higher thn those of lnk control group, except for the 5-FU group (Tle 2). Spleen inexes of FO2 t 250 mg/kg were significntly lower (p < 0.05) thn those of FO1, inicting tht FO2 ws more efficient thn FO1 in improving immunity function of the mice ering srcom S180. All the thymus inexes of mice ering srcom S180 either with or without FO tretment were lower thn tht of the lnk control group. Thymus inexes increse with FO oses (Tle 2). Thymus inexes of FO2 t 100 n 250 mg/kg i not show significnt ifferences from lnk control group, ut were significntly higher (P< 0.05) thn those of FO1 t the sme ose. Thus, FO2 h more effects on immunity justment thn FO1. Thymus inex of positive control, 5-FU group, ws significntly lower (P < 0.05) thn tht of other groups, suggesting the thymus ws trophie. Tle 2 shows tht IFN-γ n IL-3 contents of mice ering srcom S180 fe with FO1 n FO2 were higher thn those of moel control group, especilly when the ose reche 100 mg/kg, inicting tht oth FO1 n FO2 improve the immunity function of mice ering srcom S180. When the ose of FO2 reche 250 mg/kg, IFNγ n IL-3 contents were significntly higher thn those of positive control 5-FU n FO1 feeing group. It ws therefore conclue tht FO2 ws more efficient in moulting immunity function for the mice thn FO1. Tle 2. Effects of FOs on the Immunity Functions of the Tumor-ering Mice Group Blnk control Moel control FO1 FO2 Dose (mg /kg) Spleen inex (mg/g) Thymus inex (mg/g) IFN-γ concentrtion (pg/ml) IL-3 concentrtion (pg/ml) 7.70±1.09 c 6.50± ± ±8.51 ef 9.50± ±0.56 c 1.06± ±8.51 f ± ±0.68 c 9.57±6.38 c 32.98±12.77 ef ± ±0.50 c 20.92±4.43 c 47.87±6.38 e ± ±0.92 c 24.47±4.26 c 64.89±8.51 c ± ±0.53 c 15.25±7.47 c 35.11±12.77 ef ± ± ± ±8.51 c ±0.79 c 6.35± ± ± FU ±1.72 c 1.70± ± ±8.51 Different letters in the sme column inicte sttisticl ifference (P < 0.05) Effect of FOs on WBC Count n Bone-mrrow Cellulrity in S180 Tumorering Mice As shown in Tle 3, the one-mrrow cell concentrtion in S180 tumor-ering mice increse significntly t 250 mg/kg of oth FO1 n FO2 tretment compre with the moel control group (P < 0.05). The peripherl WBC cell concentrtion in S180 tumor-ering mice lso increse y 250 mg/kg of FO1, 100 n 250 mg/kg of FO2, ut ecrese significntly y 5-FU ministrtion compre with moel control group (p < Yu et l. (2014). Feruloyl oligoscchrie, BioResources 9(4),

10 0.05). The effects of FO1 n FO2 on WBC count n one-mrrow cellulrity in S180 tumor-ering mice were not significntly ifferent (p >0.05). Tle 3. Effects of FOs on Peripherl White Bloo Cell (WBC) Count n Bonemrrow Cell Count in S180 Tumor-ering Mice Femorl one-mrrow cell WBC count Group Dose (mg/kg) count ( 10 7 /ml) ( 10 9 cells/l) Moel control 4.37±0.99 c 5.94±1.28 c FO1 FO ±1.01 c 6.23±1.22 c ±1.53 c 7.69±1.05 c ± ± ±1.02 c 6.74±1.31 c ±0.98 c 8.27± ± ± FU ± ±0.71 Different letters in the sme column inicte sttisticl ifference (P < 0.05) In the present stuy, FO1 n FO2 not only inhiite the growth of humn liver cells HepG2, humn gstric cncer cells BGC-823, n humn lung enocrcinom A549 cncer cells in vitro, ut lso inhiite tumor evelopment n improve immunity function in the mice ering srcom S180 in vivo. Moreover, pthologiclly they chnge the histopthologicl profile of the tumor mss compre with moel control group. Since FOs h no effect on norml humn liver cells L-02 n stomch cells GES, it cn e presume tht the nti-tumor ctivity of FOs ws not ue to cytotoxicity, ut its inhiitory effect on cncer cell prolifertion n immunostimultory properties. The norml immunity function cn e compromise y the evelopment n formtion of tumors (Willims et l. 1996; Mio et l. 2013; Pn et l. 2013). The importnt immunity orgns, spleen, n thymus, prticipte in moulting immunity function through their prouction of lymphocytes n mcrophges (Willims et l. 1996). Tumors usully chnge the weight of immunity orgns, while immunity promoters cn restore the immunity orgn weight to norml levels (Zheng 1999). Here, in the current stuy, the effect of FO1 n FO2 prepre y ifferent processes on thymus n spleen inex of mice ering srcom ws stuie comprtively. For the chemotherpy rug 5-FU, the S180 tumor inhiition rte ws higher thn tht of FO1 or FO2, ut the mice lost weight, n the thymus n spleen inex of the mice were significntly reuce. Thus while 5-FU coul inhiit the growth of tumor cells, it simultneously inhiite the growth of norml cells. The effects of 5-FU in vitro lso showe the sme results. For FO1 n FO2, they not only inhiite cncer cell growth in the mice ering srcom S180, ut simultneously increse thymus n spleen inexes for the mice. Therefore, FO1 n FO2 might function s ntitumor gents inirectly y improving the host s immunity function. As importnt ntineoplstic constituent of orgnisms, cytokine IFN-γ n IL-3 cuse chnge of cell ehvior to enhnce cell ility for killing tumor cells (Liu et l. 2003). In this stuy, FO1 n FO2 cte s immunity ccelertors, which stimulte n recovere low immunity function of the mice ering srcom S180 to norml level, n effectively reverse the low level of IFN-γ n IL-3 ue to tumor growth. These Yu et l. (2014). Feruloyl oligoscchrie, BioResources 9(4),

11 results inicte tht FOs nti-tumor effects my e meite through improving the prouction of cytokine in the oies. The inhiition effect of FO2 on tumor ws higher thn tht of FO1 t the sme ose. Previous reserch showe tht there is close reltionship mong the iologicl ctivity of oligoscchries n their compositions, egrees of polymeriztion, n structures (Ktpois et l. 2003; Chen n Yn 2005). For exmple, strong ntioxint ctivity ws foun in gr oligoscchrie with polymeriztion egree of 6 (Chen n Yn 2005). The effects of xylo-oligoscchries on prolifertion of lctic ci cteri n Bifiocterium re ifferent ecuse of ifferent polymeriztion egrees (Mour et l. 2007). For FOs, t polymeriztion egree of 3 to 4, they were cple of inhiiting the growth of Stphylococcus ureus (Yun 2006). Xie (2010) inicte tht FOs with polymeriztion egrees of 3 to 6 coul significntly inhiit the growth of the colon cncer cell line HCT-116. To te, t hve revele tht polymeriztion egree of 2 to 8 is eneficil to the improvement of iologicl ctivity of oligoscchries. In the present reserch, polymeriztion egree of FOs rnge from 2 to 6. For FOs, the inhiitory effect on tumor growth in vivo n in vitro ws relte with their purity (numers of ifferent compositions) s well, except polymeriztion egree. The purity n verge polymeriztion egree of FO2 were higher thn tht of FO1. Although oth of them h significnt inhiitory effect on tumor growth in vivo n in vitro, FO2 ws more efficient thn FO1. The current t imply tht the ifferent inhiitory function of FOs ginst cncer cells is relte to the specific structure tht FOs contin. Peripherl WBC count n one-mrrow cellulrity re two frequently stuie clinicl prmeters tht ccurtely reflect chemotherpeutic injury (Co et l. 2011). In the present stuy, tretments with FOs increse peripherl WBC counts n one mrrow cellulrity in S180 tumor-ering mice. The current t further confirme tht FOs coul stimulte the hemopoietic system n prolifertion of stem cells. Orl ministrtion of FOs lso h n immunostimultory effect, suggesting tht the immunostimultory ctivity of FOs might e cuse y their metolites. In the future, more etile stuies re require to further explore how FOs contriute to helth n the iomoleculr mechnisms unerlying their ntitumorous n immunostimultory functions. CONCLUSIONS 1. In this stuy, WB ws fermente y A. pullulns through one-stge n two-stge controlling temperture n ph process, which prouce FO1 n FO2, respectively. Results showe tht oth FO1 n FO2 significntly inhiite the in vitro growth of cncer cells n trnsplnte tumors in mice without toxicity to norml cells. 2. FO tretments increse the levels of IFN-γ, serum IL-3, WBC, n one-mrrow cellulrity in tumor-ering mice. At the sme ose, FO2 exhiite more efficient ntitumor functions thn FO1. 3. Structure of FOs ffects their immunity responses n functions. The ntitumor ctivity of FOs might e meite through n improvement in the immune response. Yu et l. (2014). Feruloyl oligoscchrie, BioResources 9(4),

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