Vaccine22(2004)
3882–3889
Adjuvant effect of Panax notoginseng saponins on the
immune responses to ovalbumin in mice
Hong-Xiang Sun a,b,?,Yi-Ping Ye c,Hang-Jun Pan a,Yuan-Jiang Pan b,?
a College of Animal Sciences,Zhejiang University,Hangzhou310029,China
b College of Science,Zhejiang University,Hangzhou310027,China
c Institute of Materia Medica,Zhejiang Academy of Medical Sciences,Hangzhou310013,China
Received6March2003;accepted19April2004
Available online20May2004
Abstract
In this study,the haemolytic activities of Panax notoginseng saponins(PNS)and its adjuvant potentials on the cellular and humoral immune responses of ICR mice against ovalbumin(OV A)were evaluated.We determined the haemolytic activity of PNS using0.5%rabbit red blood cell.PNS showed a slight haemolytic effect,with its haemolytic percents being11.59and3.60%at the concentrations of500and 250?g/ml,respectively.Furthermore,the adjuvant potential of PNS at three dose levels on the cellular and humoral immune responses of ICR mice against ovalbumin were investigated.ICR mice were immunized subcutaneously with OV A100?g alone or with OV A100?g dissolved in saline containing aluminum hydroxide gel(Alum)(200?g),Quil A(10and50?g)or PNS(50,100or200?g)on days1and 15.Two weeks later(day28),concanavalin A(Con A)-,pokeweed(PWM)-and OV A-stimulated splenocyte proliferation and OV A-speci?c antibodies in serum were measured.PNS signi?cantly enhanced the Con A-,PWM-,and OV A-induced splenocyte proliferation in the OV A-immunized mice at a dose of100?g(P<0.05or P<0.025).OV A-speci?c IgG,IgG1and IgG2b antibody levels in serum were signi?cantly enhanced by PNS compared with OV A control group(P<0.025).Moreover,enhancing effect of PNS on the OV A-speci?c IgG2b antibody responses to OV A in mice were more signi?cant than that of Quil A(P<0.025).In conclusion,the results suggest that PNS could be safely used as adjuvant with low or non-haemolytic effect.
?2004Elsevier Ltd.All rights reserved.
Keywords:Panax notoginseng;Saponins;Haemolysis;OV A;Adjuvants;Proliferation;Antibody
1.Introduction
New generations of vaccines,particularly those based on recombinant proteins and DNA,are likely to be less reactogenic and immunogenic than traditional vaccines. Therefore,there is an urgent need for the development of new and improved vaccine adjuvant[1].Although a vari-ety of adjuvants have been used in experimental vaccines, most of these materials only elicit an antibody response and/or have undesirable side effects that have limited their potential application in vaccines[1–3].
Adjuvants have signi?cant effects on the nature of the immune responses,and can tilt the immune system in favor to Th1or Th2type response[3].The Th1immune response, which is mediated by Th1helper cells,is characterized by ?Corresponding author.Tel.:+8657186971091;
fax:+8657186971316.
E-mail address:sunhx@https://www.doczj.com/doc/9e7712065.html,(H.-X.Sun).production of the cytokines IL-2,tumor necrosis factor-?(TNF-?)and interferon-?,and an enhanced production of IgG2a,IgG2b and IgG3in mice.A Th1immune response is a requisite for cytotoxic T lymphocyte(CTL)produc-tion.The Th2response is characterized by production of the cytokines IL-4,IL-5and IL-10,and an enhanced pro-duction of IgG1and secretary IgA.The Th1response is required for protective immunity against intracellular infec-tious agents,such as viruses,certain bacteria and protozoa, and presumably against cancer cells.Th2immunity is ef-fective for protection against most bacterial as well as cer-tain viral infections[3].The currently available adjuvants mainly stimulate the Th2type immune response,which is frequently ineffective against intracellular pathogens and malignant cells.For instance,the most commonly used ad-juvants including water/oil emulsions and Alum elicit only Th2immunity[3–5].While adjuvant such as lipid A and its derivatives are capable of modulating cytokine and IgG isotype pro?les characteristic of Th1immunity,they are
0264-410X/$–see front matter?2004Elsevier Ltd.All rights reserved. doi:10.1016/j.vaccine.2004.04.012
H.-X.Sun et al./Vaccine22(2004)3882–38893883
unable to stimulate the production of CTL against soluble or exogenous antigens,which is essential for the develop-ment of effective subunit vaccines against malignant cells or some particular infectious agents[3,6].
Saponins are chemically a heterogeneous group of sterol glycosides and triterpene glycosides.Several important bi-ological properties have been attributed to saponins.Their use as humoral and cellular immunomodulators has been intensi?ed in dogs,cattle,sheep,mice,pig and non-human primate experimental models[7–13].The saponins of Quil-laja saponaria,for instance,have been included as adjuvant in vaccine formulations against HIV-1,cytomegalovirus and Toxoplasma gondii,with potent activity and lack of toxicity [14–18].
Although the outstanding and speci?c adjuvant potential of the Quil A saponin and its reverse phase HPLC-released saponin QS-21has been extensively proved[4,19,20],their high toxicity and undesirable haemolytic effect has been pointed out as the main restriction to their use in human vaccination.[21–27].In what concern to toxicity,the Quil A was lethal to mice in the dose range of100–150?g, while QS-21was lethal only at500?g.Slight variations of the HD50(caused50%haemolysis)values were already reported for QS-21:7±2[28],13.3[29]and18?g/ml [30]using16,5and0.5%red blood cell suspensions, respectively.
Panax notoginseng(Burk.)F.H.Chen(Araliaceae),a well-known traditional Chinese medicinal plant indigenous to the mountains of Yunnan province and Guangxi province, is used for treatment of cardiovascular diseases,in?amma-tion,different body pains,trauma,and internal and exter-nal bleeding due to injury[31,32].It has also been used as a tonic and haemostatic agent.Extensive phytochemi-cal and pharmacological studies on this plant proved the dammarane-type saponins to be the main bioactive prin-ciples[33–38].The dammarane-type saponins of P.noto-ginseng,which include ginsenosides and notoginsenosides, account for12%of the total root content,and are com-posed of a protopanaxadiol and protopanaxatriol glucosides [31,32,39–41].
In view of the results reported by Takechi and Tanaka [42]and Santos et al.[30,43]that the haemolytic activity of the steroids was greater than that of the triterpenoids,with the aim of selecting non-haemolytic saponins which could be possibly used as adjuvant in future large-scaled stud-ies of vaccination,in previous work,the saponins from the root of Achyranthes bidentata,Astragalus membranaceus, Bupleurum chinense,Glycyrrhiza uralensis,Panax ginseng, P.notoginseng and the herb of Gynostemma pentaphyllum, which share a triterpenoid aglycone moiety,were screened for their haemolytic activity on rabbit erythrocytes,and their adjuvant potentials on the cellular and humoral immune re-sponse of IRC mice against ovalbumin(OV A)was com-pared.We found that the lest haemolytic and best adjuvant saponins were obtained from P.notoginseng.We wish to re-port here the haemolytic activity of P.notoginseng saponins (PNS)and its adjuvant potential on the cellular and humoral immune responses of IRC mice against OV A.
2.Materials and methods
2.1.Experimental animals
Male ICR mice(Grade II,5weeks old)weighing18–22g were purchased from Zhejiang Experimental Animal Center (Certi?cate No.22-2001001,Hangzhou,China)and accli-matized for1week prior to use.Rodent laboratory chow and tap water were provided ad libitum and maintained un-der controlled conditions with a temperature of24±1?C, humidity of50±10%,and a12/12h light/dark cycle.
2.2.Materials
OV A,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-lium bromide(MTT),concanavalin A(Con A),pokeweed (PWM),RPMI-1640medium,and rabbit anti-mouse IgG peroxidase conjugate were purchased from Sigma Chemical Co.,USA;goat anti-mouse IgG1and IgG2b peroxidase con-jugate were from Southern.Biotech.Assoc.,Birmingham, AL,USA;Quil A was from NOR-VET ApS,Denmark. Fetal calf serum(FCS)was provided by Hangzhou Sijiqing Corp;aluminum hydroxide gel(Alum)was by Zhejiang Wanma Pharm Co Ltd.All other chemicals were of grade AR.
2.3.Samples and preparation of saponins
Notoginseng Radix(roots of P.notoginseng)were pur-chased from drugstore in Hangzhou,and identi?ed by Professor Xiang-Ji Xue at College of Pharmacy,Zhejiang University according to the China Pharmacopoeia(CP). Saponins were prepared as follows.Brie?y,the powered samples(1kg)were extracted with70%EtOH three times under re?ux for2h,and then concentrated in vacuum (40?C)to evaporate the solvent to give a small volume. After extracting with ether(3×0.5l),the water layer portion was extracted with n-BuOH until n-BuOH layer became colorless.The n-BuOH solution was concentrated and dried in vacuum(60?C).The dried extract was sub-jected to D101resin column chromatography,washed with H2O,and eluted with EtOH to give PNS.PNS contained 61.76±1.24%of saponins determined by the spectropho-tometric method according with drug standards of Yunnan provincec and State Drug Administration[44].A stock PNS solution with a concentration of4mg/ml was prepared by dissolving in0.89%saline.The solution was sterilized by passing it through a0.22?m millipore?lter,and then analyzed for endotoxin level by a gel-clot Limulus amebo-cyte lysate assay(Zhejiang A and C Biological,Zhejiang, China).The endotoxin level in the PNS solution was less than0.5endotoxin units(EU)/ml.
3884H.-X.Sun et al./Vaccine22(2004)3882–3889
2.4.Haemolytic assay
Red blood cells were obtained from healthy New Zealand
rabbit(Zhejiang Experimental Animal Center,China).
Blood was collected with BD Vacutainer TM(NH143IU,
Belliver Industrial Estate,Plymouth,UK).Aliquots of7ml
of blood were washed three times with sterile saline solution
(0.89%,w/v NaCl,pyrogen free)by centrifugation at180×g for5min.The cell suspension was prepared by?nally diluting the pellet to0.5%in saline solution.A volume of
0.5ml of the cell suspension was mixed with0.5ml dilu-
ents containing5,10,25,50,100,250,500and1000?g/ml
concentrations of PNS in saline solution.The mixtures were
incubated for30min at37?C and centrifuged at70×g
for10min.The free haemogloblin in the supernatants was
measured spectrophotometrically at412nm[21].Saline
and distilled water were included as minimal and maximal
haemolytic controls.The haemolytic percent developed by
the saline control was subtracted from all groups.Each
experiment included triplicates at each concentration.
2.5.Immunization
Six-week-old male ICR mice were divided into eight
groups,each consisting of?ve mice.Animals were immu-
nized subcutaneously with OV A100?g alone or with OV A
100?g dissolved in saline containing Alum(200?g),Quil
A(10and50?g)or PNS(50,100or200?g)on day1.
Saline-treated animals were included as controls.A boosting
injection was given2weeks later.Sera and splenocytes were
collected2weeks after the second immunization for prolif-
eration assay and measurement of OV A-speci?c antibody.
2.6.Splenocyte proliferation assay in vivo
Spleen collected from the OV A-immunized ICR mice
under aseptic conditions,in Hank’s balanced salt solution
(HBSS,Sigma),was minced using a pair of scissors and
passed through a?ne steel mesh to obtain a homogeneous
cell suspension,and the erythrocytes were lysed with am-
monium chloride(0.8%,w/v).After centrifugation(380×
g at4?C for10min),the pelleted cells were washed three
times in PBS and resuspended in complete medium[RPMI
1640supplemented with12mM HEPES(pH7.1),0.05mM
2-mercaptoethanol,100IU/ml penicillin,100?g/ml strep-
tomycin,and10%FCS].Cell numbers were counted with
a haemocytometer by trypan blue dye exclusion technique.
Cell viability exceeded95%.Splenocyte proliferation was
assayed as previously described[45].Brie?y,Splenocytes
were seeded into4-5wells of a96-well?at-bottom mi-
crotiter plate(Nunc)at5×106cell/ml in100?l complete
medium,thereafter Con A(?nal concentration5?g/ml),
PWM(?nal concentration10?g/ml),OV A(?nal concentra-
tion10?g/ml),or medium were added giving a?nal volume
of200?l.The plates were incubated at37?C in a humid
atmosphere with5%CO2.After68h,50?l of MTT solu-tion(2mg/ml)were added to each well and incubated for 4h.The plates were centrifuged(1400×g,5min)and the untransformed MTT was removed carefully by pipetting. To each well200?l of a DMSO working solution(192?l DMSO with8?l1N HCl)was added,and the absorbance was evaluated in an ELISA reader at570nm with a630nm reference after15min.The stimulation index(SI)was calcu-lated based on the following formula:SI=the absorbance value for mitogen-cultures divided by the absorbance value for non-stimulated cultures.
2.7.Measurement of OVA-speci?c antibody
OV A-speci?c IgG IgG1and IgG2b antibodies in serum were detected by an indirect ELISA.In brief,microtiter plate wells(Nunc)were coated with100?l OV A so-lution(50,25and50?g/ml,respectively,in50mM carbonate–bicarbonate buffer,pH9.6)for24h at4?C.The wells were washed three times with PBS containing0.05% (v/v)Tween20(PBS/Tween),and then blocked with5% FCS/PBS at37?C for1h.After three washings,100?l of diluted serum sample(IgG,1:400;IgG1,1:100;IgG2b, 1:200)or0.5%FCS/PBS as control were added to tripli-cate wells.The plates were then incubated for1h at37?C, followed by three times of washing.Aliquots of100?l of rabbit anti-mouse IgG horseradish peroxidase conjugate di-luted1:50,000,goat anti-mouse IgG1peroxidase conjugate 1:16,000or IgG2b peroxidase conjugate1:8000with0.5% FCS/PBS were added to each plate.The plates were further incubated for1h at37?C.After washing,the peroxidase ac-tivity was assayed as following:100?l of substrate solution (10mg of o-phenylenediamine and37.5?l of30%H2O2in 25ml of0.1M citrate–phosphate buffer,pH5.0)was added to each well.The plate was incubated for10min at37?C, and enzyme reaction was terminated by adding50?l/well of2N H2SO4.The optical density(OD)was measured in an ELISA reader at490nm with a595nm reference.Data were expressed as the mean OD value of the samples minus the mean OD value of the control.Where sets of serum samples have been subjected to within and between group comparisons,ELISA assays were performed on the same day for all of the samples.
2.8.Statistical analysis
Statistical signi?cance was evaluated by Wilcoxon–Mann–Whitney test.P-values of less than0.05were considered to be statistically signi?cant.
3.Results
3.1.Haemolytic activities
The haemolytic activity of PNS on rabbit red blood cells was shown in Table1.Haemolytic percents of red blood cell
H.-X.Sun et al./Vaccine 22(2004)3882–3889
3885
Table 1
Haemolytic activities of Panax notoginseng saponins (PNS)Group Absorbance value Haemolytic percent (%)Distilled water 1.637±0.040100.00±2.45??Saline 0.094±0.0090.000±5.19PNS
500?g/ml 0.284±0.01911.59±1.44?250?g/ml 0.153±0.007 3.60±1.30125?g/ml 0.090±0.015?0.22±0.5550?g/ml 0.072±0.009?1.32±0.6625?g/ml 0.073±0.011?1.28±0.8612.5?g/ml 0.078±0.007?1.00±0.532.5mg l ?1
0.061
±0.005
?2.02
±0.38
Haemolytic percents of saline and distilled water were included as mini-mal and maximal haemolytic controls.All values represent the mean ±standard deviation (n =3test).
?Signi?cant differences with saline group was designated as (P <0.05).
??Signi?cant differences with saline group was designated as (P <0.001).
treated with PNS were 11.59and 3.60%at concentrations of 500and 250?g/ml,respectively.PNS showed no haemolytic activity at concentrations of 2.5–125?g/ml.However,the HD 50value of Quil A was 19.91±0.57?g/ml determined on the same condition.
3.2.Effect of PNS on mitogen-and OVA-stimulated splenocyte proliferation in OVA-immunized mice
The effects of PNS on mitogen-and OV A-stimulated splenocyte proliferation in mice immunized with OV A are shown in Fig.1.Con A-stimulated splenocyte proliferation in the mice immunized with OV A/PNS (50,100?g)and OV A/Quil A (10?g)was signi?cantly higher than that
in
Fig.1.Effect of Panax notoginseng saponins (PNS)on mitogen-and OV A-stimulated splenocyte proliferation in vivo.Groups of ?ve male ICR mice were immunized sc with OV A 100?g alone or with OV A 100?g dissolved in saline containing Alum (200?g),Quil A (10and 50?g)or PNS (50,100or 200?g)on days 1and 15.Splenocytes were prepared 2weeks after the last immunization and cultured with Con A,PWM,or OV A or RPMI 1640for 72h.Splenocyte proliferation was measured by the MTT method as described in the text,and shown as a stimulation index.The values are presented as mean ±S.E.(n =5).Signi?cant differences with OV A groups were designated as ?(P <0.05)and ??(P <0.025).
the OV A control group (P <0.05or P <0.025).Spleno-cytes isolated from OV A/PNS (100?g)-and OV A/Quil A (10?g)-immunized mice and stimulated by PWM or OV A show a greater proliferative response than that observed for the mice immunized with OV A alone (P <0.05or P <0.01).However,no signi?cant differences (P >0.05)were observed between the OV A group and OV A/Alum group.3.3.Effect of PNS on the OVA-speci?c serum antibody response
To investigate the effect of PNS on the induction of humoral immune responses against OV A in mice,groups of mice were immunized two times by S.C.routes.The OV A-speci?c IgG,IgG1and IgG2b antibody levels in the sera were measured 2weeks after the last immunization using ELISA and shown in Fig.2.The serum IgG level in mice immunized with OV A was signi?cantly enhanced by Alum,Quil A (50?g),and PNS (50,100and 200?g),as compared with control group (P <0.025).Alum,Quil A (10and 50?g)and PNS (50?g)also signi?cantly en-hanced the total serum IgG1levels in OV A-immunized mice (P <0.025).There were,however,no signi?cant differences among the total serum IgG and IgG1levels in mice groups immunized with OV A/Alum,OV A/Quil A,and OV A/PNS.
Signi?cant enhancements in total serum IgG2b levels were observed in all group of PNS-immunized mice com-pared with control group.Moreover,the serum IgG2b levels were signi?cantly higher than those of mice groups immu-nized with OV A/Alum and OV A/Quil A (P <0.025).Thus,?ndings indicate that PNS at suitable dose signi?cantly en-hanced serum antibody production in mice immunized with OV A.
3886H.-X.Sun et al./Vaccine 22(2004)
3882–3889
Fig.2.Effect of Panax notoginseng saponins (PNS)on OV A-speci?c IgG,IgG1and IgG2b antibody.Groups of ?ve male ICR mice were immunized sc with OV A 100?g alone or with OV A 100?g dissolved in saline containing Alum (200?g),Quil A (10and 50?g)or PNS (50,100or 200?g)on days 1and 15.Sera were collected 2weeks after the last immunization.OV A-speci?c IgG,IgG1and IgG2b antibodies in the sera were measured by an indirect ELISA as described in the text.The values are presented as mean ±S.E.(n =5).Signi?cant differences with OV A groups were designated as ??(P <0.025);those with OV A/Alum and OV A/Quil A groups as aa (P <0.025).
4.Discussion
Since the original observation of Espinet [46]that cer-tain saponins act as adjuvant when given together with an antigen in a vaccine,considerable efforts have been made for the identi?cation of the active components responsible for this effect.One of the major steps forward in this re-spect was the identi?cation of a puri?ed fraction of saponin from Q.Saponaria Molina,including the Quil A saponin,a crudely enriched saponin prepared from an aqueous ex-tract and the reverse phase HPLC-released saponins called QS-7,17,18and 21[28].The unique capacity of Quil A to stimulate both the Th1immune response and the produc-tion of cytotoxic T lymphocyte against exogenous antigens makes them ideal for use in subunit vaccines and vaccines directed against intracellular pathogens as well as for thera-peutic cancer vaccines [47,48].In effect,there are a series of commercial veterinary vaccines as well as human vaccines formulated with this kind of adjuvants undergoing clinical evaluation.QS-21has been evaluated in a large number of vaccines in Phases I and II human clinical trials [49].These vaccines include cancer immunotherapeutics [50–54],HIV recombinant envelope [55,56],and malarial antigens [57].However,Quillaja saponins have serious drawbacks in-cluding high toxicity and undesirable haemolytic effect,which limit their use as adjuvant.Kensil et al.[28]reported that the Quil A saponin was lethal to mice in the dose range of 100–125?g,while QS-21is lethal only at 500?g,and that saponins QS-17,18and 21caused 50%haemolysis at concentrations of 25,15and 7?g/ml using 16%red blood cell suspensions,respectively.Oda et al.[29]and Santos et al.[30]also reported that the HD 50values for QS-21were 13.3and 18?g/ml using 5and 0.5%red blood cell suspensions,respectively.It was claimed in the past that saponins should not be used as adjuvant due to their intrinsic haemolytic proper-ties.Nevertheless,there are saponins that show low or no haemolytic effect.The hemolytic activities of saponins are related to their chemical composition.Takechi and Tanaka analyzed the haemolytic activity of 27saponins out of 75synthetic steroid and triterpene glycosides and found that the haemolytic activity of the steroids were greater than that of the triterpenoids [42].Santos et al.also reported that saponins of Agave sisalana and Smilax of?cinalis shared a steroid aglycone moiety were the most haemolytic,while saponins obtained from Bredemeyera ?oribunda and Perian-dra mediterranea with a triterpenoid aglycone moiety the less haemolytic [30,43].
QS-21however,although a triterpenoidal saponin is still hemolytic.The degree of hemolytic activity was shown to relate to the presence of side chains bearing aglycone (sugar chains)[43],acyl residues or the epoxy framework system [29].All QS-fractions isolated from Q.saponaria showed a monomer size consistent with the molecular weight pre-dicted for a triterpene with 8–10monosaccharide residues [58–65].The hemolytic potential of Quil A mixture or QS-21could be related then to their increased number of monos-sacharides and the complexity of their glycidic moieties.Indeed,QS-21consists of quillaic acid with one branched trisaccharide and one unbranched tetrasaccharide attached,and a dimeric fatty acyl group attached to the ?rst sugar of the tetrasaccharide by an ester linkage.An eighth sugar is attached to the fatty acyl group [23].The presence of fatty acids could also favor interactions between the saponin and membrane cholesterol promoting the haemolysis.
In this investigation,we found that PNS showed a slight haemolytic effect,with 11.59and 3.60%haemoly-sis at concentrations of 500and 250?g/ml,respectively
H.-X.Sun et al./Vaccine22(2004)3882–38893887
(Table1).Guided by an adjuvanticity assay in vivo,?ve
protopanaxadiol-type saponins:ginsenoside Rb1,Rb2,Rd,
Rg3and notoginsenoside K,and eight protopanaxatriol-type
saponins:ginsenoside Rh1,Rg1,Rg2and notoginsenoside
R1,R2,R3,R4,U(new compound)were isolated and
identi?ed from the PNS used in this study using silica
gel column chromatography with CHCl3–MeOH–H2O as
eluent and Rp-18column chromatography with aqueous
methanol as eluent(submitted elsewhere).Among these
saponins,ginsenoside Rb1,Rg2,and notoginsenoside R1
were the most main components,accounting for more than
60%of total PNS.The haemolytic assay showed the HD50
values for above thirteen saponins were all bigger than
300?g/ml using0.5%rabbit red blood cell suspensions
by the method in the text,while that of Quil A was19.91±0.57?g/ml.Meng et al.[66]analyzed the haemolytic activity of ginsenoside Rb1,Rb2,Rb3,Rd,Rg1and Rg2
using2%rabbit red blood cell suspensions for3h at37?C
and found that the minimum haemolytic concentrations of
ginsenoside Rg1and Rg2were200and160?g/ml,and
that the other saponins showed no haemolytic activity at
200?g/ml concentration.Different from all QS-fractions
from Q.saponaria,which had branched sugar chains,8–10
monosaccharide residues,distinctive acyl domain,and alde-
hyde group in its molecule[13–17],the above saponins
obtained from PNS have a single or two unbranched sugar
chains attached to the carbon C-3,C-6or C-20in pro-
topanaxadiol and protopanaxatriol via oxygen,with each
chain being composed of1–2monosaccharide residues,
and without distinctive acyl domain and aldehyde group in
its molecule[31,32,39–41].This fact could account for the
less haemolytic activity of PNS.
The Quil A and QS-21were lethal to mice in the
dose range of100–125?g and at500?g,respectively
[28],while the LD50(ip)of PNS to mice was825.6
(730.7–934.8)mg/kg[67].In addition,we have checked the
effect of PNS on viability of mice splenocytes in vitro and
observed that up to100?g/ml concentration of this product
has no adverse effect on cell viability when exposed for
72h(data not shown).The results suggested that PNS was
much safer than Quil A and its components in clinical use.
We also evaluated whether PNS could enhance the cellu-
lar immune responses to OV A in mice when given together
with OV A.As shown in Fig.1,PNS and Quil A signi?cantly
enhanced the mitogen-and OV A-stimulated splenocyte pro-
liferations in OV A-immunized mice at a dose of100and
10?g,respectively,as comparison with OV A control group
(P<0.05or P<0.025).However,no signi?cant differ-
ences(P>0.05)were observed between the OV A group and
OV A/Alum group.The results indicated that PNS could sig-
ni?cantly increase the activation potential of T and B cells
in mice immunized with OV A.
The OV A-speci?c IgG,IgG1and IgG2b antibody levels
in the sera of OV A-immunized mice were shown in Fig.2.
PNS signi?cantly enhanced OV A-speci?c IgG and IgG2B
levels in mice immunized with OV A at three doses of10,50and100?g,as compared with controls(P<0.025).Total serum IgG1level was also signi?cantly enhanced by PNS at a dose of10?g(P<0.025).Moreover,the adjuvant po-tential of PNS on OV A-speci?c IgG2b in OV A-immunized mice was superior to that of Quil A for the studied dose (P<0.025).Thus,it is likely that PNS at suitable dose is effective on Th1and Th2cell,as associated sensitively with an enhancement of IgG1and IgG2b levels.The saponiins obtained from PNS differs from all QS-fractions from Quil A in the chemical structure,which had branched sugar chains,distinctive acyl domain,and aldehyde group in its molecule[59–66].Therefore,the immune response of Quil A must not be compared easily with PNS.To clarify the chemically structural factors regulating the adjuvanticity of PNS,we are comparing the pro?le of the cellular and hu-moral immune responses against OV A in mice of the above thirteen puri?ed saponins.The main saponins obtained from P.notoginseng were similar to saponins present in other well-known species in the same plant genus–P.ginseng, both mainly including ginsenosides[31,32,39–41].It has been proven that total ginseng saponins potentiate antibody response[11–13,68],and exerts a number of effects on the cells of the immune system,e.g.promotes natural killer cell activity[68,69]and the phagocytic activity of macrophages and polymorphonuclear leucocytes[70,71];induces inter-feron production[69];stimulates the activity of cytotoxic T-lymphocytes[72]and the Th1immune response[73].In general,Al(OH)3adjuvanted vaccines included higher lev-els of IgG1antibodies compared to IgG2a,but the Al(OH)3 adjuvanted vaccines supplemented with ginseng saponins favoured IgG2[12].Rivera et al.also reported that gin-senosides Rb1stimulated similar or in most cases higher antibody titres than Al(OH)3-adjuvanted vaccines,and con-cluded that Rb1is the most important active adjuvant frac-tion in the ginseng saponins[12].These previous reports could account for adjuvanticity of PNS to some extent.
In our conditions PNS showed a slight haemolytic effect and enhanced signi?cantly a speci?c antibody and cellular response against OV A in mice.The results suggested PNS could be safely used as adjuvant with low side effect. Acknowledgement
This work was supported by Grant-in-Aid from the Zhe-jiang Provincial Natural Science Foundation of China(No. M303749).
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