Research Article The Immunological Enhancement Activity of Propolis Flavonoids Liposome In Vitro and In Vivo

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1 Hinwi Pulishing Corportion Eviene-Bse Complementry n Alterntive Meiine Volume 214, Artile ID , 8 pges Reserh Artile The Immunologil Enhnement Ativity of Propolis Flvonois Liposome In Vitro n In Vivo Yng To, 1 Deqing Wng, 2 Yunling Hu, 1 Yee Hung, 1 Yun Yu, 1 n Deyun Wng 1 1 Institute of Tritionl Chinese Veterinry Meiine, College of Veterinry Meiine, Nnjing Agriulturl University, Nnjing 2195, Chin 2 College of Life n Environment Siene, Hungshn University, Hungshn 24541, Chin Corresponene shoul e resse to Deyun Wng; ywng@nju.eu.n Reeive 3 April 214; Revise 1 July 214; Aepte 23 Septemer 214; Pulishe 14 Otoer 214 Aemi Eitor: Hruki Ym Copyright 214 Yng To et l. This is n open ess rtile istriute uner the Cretive Commons Attriution Liense, whih permits unrestrite use, istriution, n reproution in ny meium, provie the originl work is properly ite. The im of this stuy ws to investigte n ssess the effets of propolis flvonois liposome impose on the immune system y ompring it to propolis flvonois n lnk liposome. In vitro, the effets of the ove rugs on mrophges were ssesse y mesuring the phgoyti funtion n ytokine proution. In vivo, the immunologil juvnt tivity of propolis flvonois liposome ws ompre with those of propolis flvonois n lnk liposome. The results showe tht in vitro propolis flvonois liposome n signifintly enhne the phgoyti funtion of mrophges n the relese of IL-1β, IL-6, n IFN-γ. In ition, suutneous ministrtion of propolis flvonois liposome with ovlumin to mie oul effetively tivte the ellulr n humorl immune response, inluing inuing higher level onentrtions of IgG, IL-4, n IFN-γ in serum n the prolifertion rtes of spleni lymphoytes. These finings provie vlule informtion regring the immune moultory funtion of propolis flvonois liposome n inite the possiility of use of propolis flvonois liposome s potentil juvnt. 1. Introution Propolis is omplex mixture proesse y honeyees from the resins ollete from us, leves, n exutes of ifferent plnts, suh s poplr, irh, horse hestnut, ler, eeh, n onifer trees. It hs ttrte reserhers interest n lso extensively ppere in the omposition olumn s n itive in helth foos, everges, n nutritionl supplements for improvement of helth n prevention of severl iseses euse of its iologil n phrmologil properties, inluing immunomoultory, ntitumor, ntiinflmmtory, ntioxint, n ntiteril [1 6]. Over 15 onstitutions were ontine in propolis, suh s polyphenols (flvonois, phenoli is, n their esters), terpenois, steroi, n is, ut vry in the geogrphil n otnil origins [7]. Propolis from Chin ontins mny flvonois [1] n flvonois re thought to ount for muh of the iologil n phrmologil tivities. However, it mkes propolis flvonois iffiult to e utilize euse of the poor soluility in wter. Liposomes re mirosopi vesiles ompose of memrne-like lipi ilyers surrouning queous omprtment. The lipi lyers re omprise minly of phospholipis [8]. Propolis flvonois, mixes of vrious hyrophoi ompouns, n e interlte into the lipi ilyer n ultimtelyissolveinthequeoussolution.mostimportntly, liposomes re prove to e sfe n well tolerte, s emonstrte through the extensive reserhes n pplitions of liposome-se ntiner [9] n nti-infetive rugs [1]. In ition, effetive tive trgeting n eviently prolonge resienttimeinloonepurposefullyhievefter speifi moifition of liposomes s rug elivery systems [11]. Aientlly, the oxition proess of phospholipis n e elye ue to the ntioxint effet exerte y propolis. In ition, the reserh n evelopment of liposomes s juvnt hs gretly intensifie in the lst 1 yers, whih exhiits vntges of eliittion of oth humorl meite immunity n ell meite immunity n others esrie ove [12 14]. Similrly, the vine ontining propolis emonstrte fster, more effiient, n more urle

2 2 Eviene-Bse Complementry n Alterntive Meiine immune response thn nonformulte one n furthermore less toxi thn white oil [15]. Therefore, if propolis flvonois re enpsulte with liposome, not only the wter soluility of propolis flvonois will e enhne, ut lso they mye hve synergisti effet. In this stuy, in vitro, the effet of the propolis flvonois liposome on phgoyti tivity n ytokine seretion of mrophges ws mesure. The effet of the propolis flvonois liposome on the humorl meite immunity nellmeiteimmunitywsomprewithpropolis flvonois, liposome, n Freun s Complete Ajuvnt (). The im of this strtegy is to investigte whether immunologil enhnement tivity of propolis flvonois n liposome formultions n further enhne or moulte the immune response ginst OVA vine ompre with the juvnt lone. 2. Mterils n Methos 2.1. Preprtion of Propolis Flvonois Liposome. Propolis ws purhse from Dhu Chinese tritionl meiine ompny in Nnjing, Jingsu provine. Propolis flvonois were prepre in our lortory Briefly, propolis ws extrte with 95% ethnol for three times n the ethnol solution ws retrieve. Then, the preipittion ws extrte with ethyl ette for three times n then the ethyl ette ws retrieve. Finlly, the preipittion ws rie in vuum n propolis flvonois were otine. Propolis flvnois re omplex mixture whih mostly ontins rutin, myrietin, queretin, kempferol, pigenin, pinoemrin, hrysin, n glnigin. Propolis flvnois ws purhse from Dhu Tritionl Chinese Meiine Compny in Nnjing, Jingsu provine, Chin. The ontent of rutin in the propolis flvnois ws mesure y UV-spetrophotometri metho n the ontents of hrysin n glnigin in the propolis flvnois were mesure y HPLC (Wters 2695, wter 2489 UV/Visile Detetor, wters Symmetry C18 Column, 1 Å, 5 μm, 4.6 mm 25 mm, 1/pkg. Milfor, Msshusetts, USA). The rutin, hrysin, n glnigin ontents of propolis flvonois use in our experiments were, respetively, 5.67%, 6.5%, n 22.68%, whih ws in or with the stnr of Chinese Phrmopoei. The propolis flvonois liposome (the verge size of propolis flvonois liposome ws out 1 nm n the enpsultion effiieny of propolis flvonois liposome ws 91%) ws prepre with the ethnol injetion metho oring to our previous reserh [16] Assy for Mrophges Ativtion y Propolis Flvonois Liposome Peritonel Mrophge Preprtion. Peritonel mrophges were isolte with minor moifitions, s esrie in previous report [17]. In rief, peritonel mrophges were hrveste from ICR mie (4 weeks ol) 2 ys fter n intrperitonel injetion of 1 ml 6% strh-roth meium. ICR mie were srifie n out 5mL of PBS ws injete into the ominl, then the omen ws mssge gently for 3 min, n the PBS ws rwn k with peritonel flui. After entrifugtion t 15 rpm for 1 min, the mrophges were ollete n wshe twie with PBS. Peritonel mrophges were resuspene n ilute to ml 1 with RPMI-164 with fetl ovine serum. The ells were trnsferre to 24-well pltes (Costr, Corning, NY, USA) n inute to here for 4 h in humi tmosphere (Thermo Sientifi, Wlthm, MA, USA) with 5% CO 2 t 37 C. Then remove nonherent ells n wsh gently twie with RPMI 164 meium Phgoytosis Assy. The effets of propolis flvonois liposome, propolis flvonois, n lnk liposome on phgoyti funtion of mrophges were ssesse y Vyrnt PhgoytosisAssyKit(MoleulrProes,In,Eugene,OR, USA) n ultimtely the totl men fluoresene intensity (MFI) ws etermine y flow ytometry (BD FACSVerse, Sn Jose, CA). Briefly, peritonel mrophges ( /ml in RPMI omplete meium, 2 ml per well) were expose to ifferent onentrtions of propolis flvonois liposome (6 μg/ml, 3μg/mL, n 15μg/mL; oring to previous results, the 5% ytotoxi onentrtion of the propolis flvonois ws 181.3μg/mL n the mximl sfety onentrtion of the propolis flvonois 6 μg/ml), propolis flvonois (6 μg/ml, 3 μg/ml, n 15 μg/ml), lnk liposomes, LPS (eing from Esherihi oli 55:B5, 1 μg/ml, purity > 97%, Sigm, St. Louis, MO, USA), n RPMI- 164 meium for 48 h. Then the superntnts were remove from the 24-well pltes refully n 6 μl oftheprepre fluoresent BioPrtile suspensions ws e to ll the wells.afterinutionfor2hourst37 Cin5%CO 2,the BioPrtile loing suspensions were remove n 6 μl of theprepretrypnluesuspensionswsefor1minute t room temperture. The exess trypn lue suspensions were remove n.5 ml.25% trypsin ws e to eh smple for 2 minutes. Finlly, the mrophges were wshe twie with PBS, fixe with 6μL 4% prformlehye, n exmine y flow ytometry Mesurement of Cytokine Proution. After peritonel mrophge ws prepre s Setion 2.2.1, propolis flvonois liposome t series of onentrtions (6 μg/ml, 3μg/mL, 15μg/mL), propolis flvonois (6 μg/ml, 3 μg/ml, n 15 μg/ml), lnk liposomes, n RPMI-164 meium were, respetively, e in finl volume of 15 μl per well, four wells eh onentrtion. The pltes were inuteinhumitmospheret37 Cwith5%CO 2 for 48 h. The superntnt ws hrveste n the tumor nerosis ftor lph (TNF-α), interleukin 1 et (IL-1β), interleukin 6 (IL-6), interleukin 12 (IL-12), n interferon-gmm (IFN-γ) proution were nlyze y using the Quntikine Mouse ytokines ELISA kits (R&D Systems, In, Minnepolis, MN, USA), respetively, oring to the mnufturer s instrutions Ajuvnt Ativity In Vivo Animl Tretment Protool. The sme quntity of femlenmleicrmie(4weeksol)werepurhse from Comprtive Meiine Centre of Yngzhou University

3 Eviene-Bse Complementry n Alterntive Meiine 3 n limtize for 7 ys prior to use. The mie were mintine uner ontrolle onitions t temperture of 24 ± 1 C, humiity of 5 ± 1%, n 12/12-h light rk yle with free ess to foo n wter. Eh mouse ws use one n trete in orne with the Ntionl Institutes of Helth guie lines for the re n use of lortory nimls Immuniztion. Mie were injete suutneously in theorslskinfolonysny14with.5mlpropolis flvonois liposome (.5 mg/ml), propolis flvonois (.5 mg/ml), juvnt, n lnk liposome, eh formultion ontining OVA (2 μg).on weeks 1,2,3,4,5,n 6 fter the first vintion, mie were euthnize n the peripherl loo smples n the spleni lymphoyte were ollete to etermine the onentrtions of OVA-speifi IgG, IL-4, IFN-γ, n spleen lymphoyte prolifertion Mesurements of Serum IL-4 n IFN-γ. Collete peripherl loo smples were ple in inutor (37 C) for 2-3 h n then were entrifuge t 1, rpm t 4 Cfor 1mintoollettheserum.TheonentrtionsofIL-4n IFN-γ in the serum were etermine with ELISA kit (BD Biosienes, Frnklin Lkes, New Jersey, USA) in orne with mnufturer s instrutions Mesurements of OVA-Speifi IgG. OVA-speifi IgG in serum ws etete y n iniret ELISA [18 2]. In rief, mirotiter plte wells were ote with 1 μl OVA solution (oting uffer-pbs ontining.4 μg OVA)n inute for 12 h t 4 C. The wells were wshe three times with wshing uffer ontining.12% (w/w) Tris n.85% (w/w) NCl in eionize wter n loke with 1% (v/v) geltin/pbs t 37 Cfor1h.Afterthreetimesofwshing, 1 μl of series of ilute serum smples ws e into triplite wells. The pltes were inute for 2 h t 37 C, followe y three times wshing. At this point, 1 μl of peroxise (HRP)-onjugte AffiniPure got nti-mouse IgG (H + L) ilute to 1 : 5 with PBS ontining.5% (v/v) Tween 2 (PBST) ws ispense into eh well n inute t 37 C for 1 h. After three times of wshing, 1 μl of 3,3,5,5 -tetrmethyl enziine (TMB, Tingen Bioteh Co., Lt) ws e to eh well. The pltes were in the rk t 37 C for 15 min. The retion ws terminte y the ition of 1 μl ofstoppinguffer(2mol/lh 2 SO 4 solution). The sorne ws mesure t 45 nm. The stnr urve of IgG, on whih the onentrtions of the stnr IgG were s siss n the reltive A 45 s orinte, ws estlishe. The onentrtion of IgG of eh smple serum ws lulte oring to the stnr urve Spleni Lymphoyte Prolifertion Assy. Spleni lymphoyte prolifertion ws mesure s esrie [21, 22]. The spleen ells were seee into 96-well ulture pltes n phytohemgglutinin (PHA, purity > 97%, Sigm, 1 μg/ml) or lipopolyshrie (LPS, eing from Esherihi oli 55:B5, 1 μg/ml, purity > 97%, Sigm, St. Louis, MO, USA) ws e to stimulte T or B lymphoyte prolifertion. Then 2 μl of RPMI-164 meium ws e into the left wells s the ontrol. After 44 h inution t 37 C, 3 μl ofmtt solution (5 mg/ml) ws e to eh well n inute for n itionl 4 h. The superntnt ws remove, n then the forme formzn slts were issolve y 1 μl of DMSO in shking pltes for 1 min. Asorne ws mesure t 57 nm using n ELISA reer (Multiskn FC Miroplte photometer, Thermo sientifi, USA). The prolifertion rte (%) ws lulte oring to the following eqution: Prolifertion rte (%) = [A (rug group) A (ontrol group) ]. (1) A (ontrol group) 2.4. Sttistil Anlysis. Dt re expresse s men ± stnr errors (S.E.). Dunn n LSD s multiple rnge test were use to etermine the ifferene mong groups. P vlues of less thn.5 were onsiere to e sttistilly signifint. 3. Results 3.1. Propolis Flvonois Liposome Enhne the Phgoytosis Funtion of Mrophges. The proess of phgoyti is essentil for mrophge s importnt ntigen present ells n the first line of efense. The effets of the propolis flvonois liposome, propolis flvonois, lnk liposome, nlpsonmrophgesweressesse.asshowninfigure1, phgoyti funtion of mrophges fter ing propolis flvonois liposome ws signifintly enhne n tene to e ose-epenent. The results showe tht not only propolis flvonois liposome ut lso propolis flvonois, lnk liposome, n LPS enhne the phgoyti funtion of mrophges Propolis Flvonois Liposome Stimulte Cytokines Proution of Murine Peritonel Mrophges. The murine peritonel mrophges were stimulte with propolis flvonois liposome for 24 h n the ytokines ontents in the ulture superntnt were mesure y ELISA. As result, propolis flvonois liposome (6 μg/ml, 3 μg/ml, n 15 μg/ml) oul signifintly inrese the proution of the IFN-γ, whih ws signifint ifferene etween with propolis flvonois n lnk liposome (Figure 2()). The proution of the IL-1β of propolis flvonois liposome (6 μg/ml, 3μg/mL, n 15μg/mL) groups ws signifintly higher thn those of propolis flvonois n lnk liposome (Figure 2()). The IL-6 onentrtions of propolis flvonois liposome groups in 3 μg/ml n 6 μg/ml were signifintly higher thn those in other groups (P <.5) (Figure2()). However, they file to reproue the similr trens ove in terms of IL-12 n TNF-α (Figures 2() n 2(e)) Propolis Flvonois Liposome Inrese the Spleni Lymphoyte Prolifertion. To ompre the ility of propolis flvonois liposome, propolis flvonois, liposomes, n to inue ellulr immune response, the spleni lymphoyte prolifertion of vinte mie ws quntifie fter weeks1,2,3,4,5,n6ofimmuniztion.theresultsseen in Figures 3() n 3() is s follows: signifintly higher mgnitue of spleni prolifertion with PHA ws otine

4 4 Eviene-Bse Complementry n Alterntive Meiine Averge MFI e Conentrtions of rugs (μg/ml) Propolis flvonois liposome Blnk liposome Cell ontrol Propolis flvonois LPS Figure 1: The MFI of the phgoytosis response on eh group. LPS, three ifferent onentrtions of propolis flvonois liposome, propolis flvonois, n lnk liposome were e to peritonel mrophges. The mrophges were hnle with Vyrnt Phgoytosis Assy Kit fter 48 h inution. The phgoytosis funtion ws finlly ssesse y etermining the verge MFI. Vlues re men ± SEM, n=6. e The supersripts without the sme letters ove the olumns iffer signifintly (P <.5) from eh other in the sme onentrtion group. in propolis flvonois liposome group from week 2 to week 4 fter first vintion ompre with propolis flvonois, lnk liposome,, n lnk ontrol groups (P <.5). In ition, from week 2 to week 6 fter first vintion, signifintly higher mgnitue of spleni prolifertion with LPS ws otine in propolis flvonois liposome group ompre with propolis flvonois, lnk liposome,, n lnk ontrol groups (P <.5) Propolis Flvonois Liposome Enhne the Proution of IgG. The humorl immune response ginst most exogenous ntigens n e evlute y etermining the immunogloulin in loo, espeilly IgG, the mjor onstitution. As shown in Figure 4, signifintly greter onentrtion of IgG ws oserve in mie vinte with propolis flvonois liposome (P <.5) s ompre to propolis flvonois n liposome. Propolis flvonois liposome ws lso more effiient (P <.5) t inuing the onentrtion of IgG ompre to mie vinte with on weeks 4 n 5 fter first vintion Propolis Flvonois Liposome Promote the IFN-γ n IL-4 Proutions. The mssive seretion of ytokines y T- helper ells ws essentil in the proess of speifi immunity. IFN-γ n IL-4, s Th1 ytokine n Th2 ytokine, were ssesse. The onentrtions of IFN-γ in mouse vinte with propolis flvonois liposome were signifintly higher thn mouse vinte with propolis flvonois, liposome, n on weeks 5 n 6 fter primry vintion (P <.5) (Figure5()). Similrly, propolis flvonois liposome resulte in signifintly higher mount of IL-4 thn others on weeks 3 to 6 fter first vintion (P <.5)(Figure5()). 4. Disussion An iel vine juvnt is omponent tht not only n improve the effetiveness of vines y inuing roust immune responses [23] ut lso shoul e sfe enough uring vintion. Therefore, the suess of vines my lie in their ssoition with selete juvnts, espeilly to the innovtive vines with poor immunogeniity. As vrious vines re evelope, mny types of juvnts re lso sought out n unergo evlution in terms of seurity n vilility. For exmple, lthough lum is wiely use s vine juvnt, its role in nephrotoxiity, Alzheimer s isese, suutneous retion, epitope moifition, its inilitytoinueellulrresponse,nitslimiteeffet on polyshrie ntigens highlighte the nee for new juvnts. In ition, espite the ft tht Freun s Complete Ajuvnt n le to immune responses hrterize y mixe Th1/Th2 response n is useful for inuing ell-meite response, its high toxiity, potentil verse retion, n high ost mke it unfit to e use s vine juvnt for humns [24 27]. In view of shortges of syntheti ompouns, some nturl prouts eome fertile soures for new meiines [28]. The in vivo prelinil investigtions highly reommen further pplitions [15]. The immunomoultory effets of nturl sustnes, suh s propolis, ginsenosie, n QS21, hve een onsiere s lterntive juvnt therpies in the tretment of vrious iseses [29, 3]. When use s vine juvnt, propolis hs een shown to inrese the sfety of the ssoite vine, in ition to inresing its protetive inex, eliiting higher ntioy titer, eliiting high n persistent muosl immunity, enhning the ellulr response, offering higher phgoyti tivity, inresing leukoyti retion, promoting peripherl lymphoytes prolifertion, extening vine protetion, inuing erly protetion, reuing the optimum ose onentrtion, n enhning nonspeifi immunity regrless of the types of vine preprtion [31, 32]. Moreover, these effets were usully orrelte to the flvonois ontent [33, 34]. However, propolis flvonois iffiultly issolve into wter. Liposomes, ioegrle n essentilly nontoxi vehiles, n enpsulte oth hyrophili n hyrophoi mterils [35]. In ition, liposomes re themselves the immunologil juvnt n hve een onfirme n extene to inlue wie rnge of ntigens from teri, protozo, viruses, tumors, n spermtozo [36, 37]. Therefore, if propolis flvonois re enpsulte with liposome, not only soluility of propolis flvonois will e inresingly promote, ut lso the immunologil juvnt will e synergisti. In this stuy, the propolis flvonois were enpsulte with liposome n we ompre the immunologil enhnement tivity with propolis flvonois in vitro n in vivo. As mononuler phgoyti ells erive from peripherl loo monoytes n re resient in most tissues, mrophges funtion s professionl ntigen presenting ells (APCs) n s effetor ells in humorl n ellulr immunity. They t s the rige etween the innte immune system n the pt immune system y ifferentiting into ells to exert iverse funtions fter eing tivte y

5 Eviene-Bse Complementry n Alterntive Meiine 5 IFN-γ (pg/ml) IL-6 (pg/ml) Conentrtions of rugs (μg/ml) Propolis flvonois liposome Propolis flvonois () Blnk liposome Cell ontrol Conentrtions of rugs (μg/ml) Propolis flvonois liposome Propolis flvonois () TNF-α (pg/ml) Blnk liposome Cell ontrol IL-1β (pg/ml) IL-12 (pg/ml) Conentrtions of rugs (μg/ml) Propolis flvonois liposome Propolis flvonois Conentrtions of rugs (μg/ml) () Blnk liposome Cell ontrol Conentrtions of rugs (μg/ml) Propolis flvonois liposome Propolis flvonois () Blnk liposome Cell ontrol Propolis flvonois liposome Propolis flvonois (e) Blnk liposome Cell ontrol Figure 2: The ytokines proution of the peritonel mrophges on eh group (pg/ml). The effets of propolis flvonois liposome, propolis flvonois, n lnk liposome on peritonel mrophges were ssye. () TNF-α, () IL-1β, () IL-6, () IL-12, n (e) IFN-γ. Vlues re men ± SEM, n=6. The supersripts without the sme letters ove the olumns iffer signifintly (P <.5) from eh other in the sme onentrtion group. ifferent stimuli or omintion of stimuli [38, 39]. Consequently, propolis flvonois liposome, s stimuli, ws inute with peritonel mrophge. The phgoyti pility of mrophges ws signifintly enhne when expose to propolis flvonois liposome s shown in Figure 1. The proessofphgoytosisisessentilformrophgetoler inves n present ntigens to effetive T ells. In ition, signifintly greter onentrtions of IL-1β,IL-6,nIFN-γ with ing PFL were foun s ompre to ing PF or liposome. However, the effets of propolis flvonois liposome re not lwys ose-epenent. The effet of propolis flvonois liposome on IL-6 (Figure 2()) ppere to e ose-epenent, wheres the releses of IL-1β n IFN- γ (Figures 2() n 2()) were more ovious on the mile onentrtion of propolis flvonois liposome. In ition, propolis flvonois liposome oul not oviously promote the seretion of IL-12 n TNF-α.Tht lso inite tht the propolis flvonois liposome only ffete seretion of prtil ytokine. IL-1β, IL-6,nIFN-γ n promote the immune responses, inluing strikingly enhning ntigen-riven responses of CD4 n CD8 T ells [4], etermining the pth of T ell ifferentition, inuing B ells to ifferentite

6 6 Eviene-Bse Complementry n Alterntive Meiine Spleen lymphoyte prolifertion rtes with PHA Weeks fter first vintion Spleen lymphoyte prolifertion rtes with LPS Weeks fter first vintion Propolis flvonois liposome Propolis flvonois Blnk ontrol Blnk liposome Propolis flvonois liposome Propolis flvonois Blnk ontrol Blnk liposome () () Figure 3: The ynmi hnge of spleen lymphoyte prolifertion in synergisti stimultion of rugs with PHA (A 57 )()nlps(a 57 )() fter vintion. ICR mouse ws inoulte twie on ys n 14 with OVA enpsulte in propolis flvonois liposome, lnk liposome, n Freun s Complete Ajuvnt or mixe in propolis flvonois n the prolifertion rtes of spleen lymphoyte mix with PHA or LPS were mesure on weeks, 1, 2, 3, 4, 5, n 6 fter first vintion. Vlues re men ± SEM, n=4mie/group. The supersripts without the sme letters ner the urves iffer signifintly (P <.5) from eh other in the sme week group. Conentrtions of IgG (ng/ml) Weeks fter first vintion Propolis flvonois liposome Propolis flvonois Blnk ontrol Blnk liposome Figure 4: The ynmi hnge of IgG fter vintion. ICR mouse ws inoulte twie on ys n 14 with OVA enpsulte in propolis flvonois liposome, lnk liposome, n Freun s Complete Ajuvnt or mixe in propolis flvonois. The onentrtions ofigginserumweremesureonweeks,1,2,3,4,5,n6 fter first vintion. Vlues re men ± SEM, n = 4 mie/group. The supersripts without the sme letters ner the urves iffer signifintly (P <.5) from eh other in the sme week group. into plsm ells, n riving the proution of Th1 ells. It emonstrte tht the propolis flvonois liposome, similr to PAMP, oul e reognize y innte immune system through interting with the mrophges n the results might e expline to e relte to the tivtion of TLRs signl pths [41, 42]. In vivo, results showe tht sustntil effet of PFL on the IgG onentrtion ws seen. At lter perio of immuniztion (from 4 to 5 weeks), the effet of propolis flvonois liposome ws signifintly superior to PF n liposomes, whih inite tht the PF with enpsulte liposomes ws ontrolle relese. Moreover, signifint ifferene ws seen etween propolis flvonois liposome n. From week 2 to week 4, the spleen lymphoyte prolifertion rtes of propolis flvonois liposome group were signifintly higher thn those of other groups. It inite tht ellulr immunity response oul e ooste when propolis flvonois liposomeserve s juvntn high levels ofifn-γ in propolis flvonois liposome group were lso improve. High levels of IL-4 n IgG in propolis flvonois liposome group showe tht propolis flvonois liposome oul effiiently inue the humorlimmunityresponses. In wor, propolis flvonois liposome not only oul promote the phgoyti pility n the ytokine proution of murine peritonel mrophges in vitro, ut lso oulenhnethehumorlimmunityresponsenellulr immunity response. Moreover, in ontrst to n lum, propolis flvonois liposome ws nontoxi n h few verseretionsnsuperioreffet,whihisfittertoeuse s vine juvnt. Arevitions OVA: Ovlumin DMSO: Dimethyl sulfoxie PHA: Phytohemgglutinin : Freun s Complete Ajuvnt LPS: Lipopolyshrie MTT: 3-(4,5-Dimethylthizol-2-yl)-2,5- iphenyltetrzolium romie PBS: Phosphte uffere sline ELISA: Enzyme linke immunosorent ssy IgG: Immunogloulin G IFN-γ: Interferon-γ IL-12: Interleukin-12 IL-6: Interleukin-6 IL-1β: Interleukin-1β IL-4: Interleukin-4 TNF-α: Tumor nerosis ftor-α.

7 Eviene-Bse Complementry n Alterntive Meiine 7 Conentrtions of IFN-γ (ng/ml) Weeks fter first vintion Conentrtions of IL-4 (ng/ml) Weeks fter first vintion Propolis flvonois liposome Propolis flvonois Blnk ontrol Blnk liposome Propolis flvonois liposome Propolis flvonois Blnk ontrol Blnk liposome () () Figure 5: The ynmi hnge of onentrtions of IFN-γ n IL-4 fter vintion. On weeks, 1, 2, 3, 4, 5, n 6 fter first vintion, the onentrtions of IFN-γ n IL-4 in serum were etermine y ELISA kits. Vlues re men ± SEM, n=4mie/group. The supersripts without the sme letters ove the olumns iffer signifintly (P <.5) from eh other in the sme onentrtion group. Conflit of Interests The uthors elre tht there is no onflit of interests regring the pulition of this pper. Authors Contriution Yng To n Deqing Wng ontriute eqully to this work. Aknowlegments The projet ws supporte y Ntionl Nturl Siene Fountion of Chin (Grnt no ), Speil Fun for Agro-Sientifi Reserh in the Puli Interest (Grnt nos n ), n Projet Fune y the Priority Aemi Progrm Development of Jingsu Higher Eution Institutions (PAPD). The uthors re grteful to ll the other stff in the Institute of Tritionl Chinese Veterinry Meiine of Nnjing Agriulturl University for their ssistne in the experiments. Referenes [1] V. S. Bnkov, S. L. e Cstro, n M. C. Mrui, Propolis: reent vnes in hemistry n plnt origin, Apiologie, vol. 31,no.1,pp.3 15,2. [2] N. Oršolić, A. H. Knežević, L. Šver, S. Terzić, n I. Bšić, Immunomoultory n ntimetstti tion of propolis n relte polyphenoli ompouns, Journl of Ethnophrmology,vol.94,no.2-3,pp ,24. [3] T. Kimoto, M. Ag, K. Hino et l., Apoptosis of humn leukemi ells inue y Artepillin C, n tive ingreient of Brzilin propolis, Antiner Reserh, vol. 21, no. 1, pp , 21. [4] L. Wng, S. Mineshit, I. G, T. Shigemtsu, n T. Mtsuno, Antiinflmmtory effet of propolis, Jpnese Journl of Phrmologil Therpeutis,vol.24,pp ,1993. [5] T. Hmsk, S. Kumzw, T. Fujimoto, n T. Nkym, Antioxint tivity n onstituents of propolis ollete in vrious res of Jpn, Foo Siene n Tehnology Reserh, vol.1,no.1,pp.86 92,24. [6] F. Szzohio, F. D. D Auri, D. Alessnrini, n F. Pntnell, Multiftoril spets of ntimiroil tivity of propolis, Miroiologil Reserh,vol.161,no.4,pp ,26. [7] M. I. N. Moreno, M. I. Isl, A. R. Smpietro, n M. A. Vttuone, Comprison of the free ril-svenging tivity of propolis from severl regions of Argentin, Journl of Ethnophrmology, vol. 71, no. 1-2, pp , 2. [8] D. Meisner n M. Mezei, Liposome oulr elivery systems, Avne Drug Delivery Reviews,vol.16,no.1,pp.75 93,1995. [9] T. Nkmur, D. Ymzki, J. Ymuhi, n H. Hrshim, The nnoprtiultion y otrginine-moifie liposome improves α-gltosylermie-meite ntitumor therpy vi systemi ministrtion, Journl of Controlle Relese,vol. 171, no. 2, pp , 213. [1] T. M. Allen n P. R. Cullis, Liposoml rug elivery systems: from onept to linil pplitions, Avne Drug Delivery Reviews,vol.65,no.1,pp.36 48,213. [11] H.-W. Wng, P.-L. Jing, S.-F. Lin et l., Applition of gltose-moifie liposomes s potent ntigen presenting ell trgete rrier for intrnsl immuniztion, At Biomterili,vol.9,no.3,pp ,213. [12] W. Yn, W. Chen, n L. Hung, Mehnism of juvnt tivity of tioni liposome: phosphoryltion of MAP kinse, ERK n inution of hemokines, Moleulr Immunology, vol. 44,no.15,pp ,27. [13] M. Henriksen-Ley, K. S. Korsholm, P. Anersen, Y. Perrie, n D. Christensen, Liposoml vine elivery systems, Expert Opinion on Drug Delivery,vol.8,no.4,pp ,211. [14] A. Henerson, K. Propst, R. Kel, n S. Dow, Muosl immuniztion with liposome-nulei i juvnts genertes effetive humorl n ellulr immunity, Vine,vol.29,no. 32, pp , 211. [15]E.S.H.ElAshrynT.A.Ahm, Theuseofpropoliss vine s juvnt, Vine,vol.31,no.1,pp.31 39,212.

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