adjuvant action of chenopodium quinoa saponins on the induction of antibody responses to...

Adjuvant action ofChenopodium quinoasaponins on the induction of antibodyresponses to intragastric and intranasal

administered antigens in mice

Alberto Estrada *, Bing Li, Bernard Laarveld

Animal Biotechnology Centre, Department of Animal and Poultry Science, 72 Campus

Drive, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5B5

Received for publication 26 November 1997

Abstract

Saponins extracted from the seed of Chenopodium quinoa (quinoa) were studied for theirability to act as mucosal adjuvants upon their intragastric or intranasal administration

together with model antigens in mice. Quinoa saponins, co-administered intragastrically orintranasally with cholera toxin or ovalbumin, potentiated speci®c IgG and IgA antibody re-sponses to the antigens in serum, intestinal and lung secretions. The potentiating e�ect of

the saponins appeared, to some extent, mediated by increased permeability of the mucosa,allowing increased uptake of the antigen. The intragastric administration of 99mTc-radio-labeled human serum albumin together with quinoa saponins revealed an increased presenceof the radiolabeled protein in blood, liver, spleen and lungs of mice. This study indicates

the potential of quinoa saponins as adjuvants for mucosally administered vaccines. # 1998Elsevier Science Ltd. All rights reserved.

Keywords: Quinoa saponins; Adjuvant; Antibody responses; Mucosal immunity; Intestinal permeability

ReÂ sumeÂ

Le saponines extraites de la graine de Chenopodium quinoa (quinoa) ont eÂ teÂ eÂ tudieÂ es dans

leur possibiliteÂ d'agir comme adjuvants de muqueuses, quand elle sont administreÂ es par lesvoies intragastrique ou intranasales avec antigeÁ nes modeÁ les chez la souris. Les saponines dela quinoa, co-administreÂ es par les voies intragastriques ou intranasales avec les antigeÁ nes de

Com. Immun. Microbiol. & Infect. Dis. 21

(1998) 225±236

0911-6044/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved.

PII: S0147 -9571 (97)00030 -1

C OMPARATIVE

I MMUNOLOGY

M ICROBIOLOGY &

I NFECTIOUS

D ISEASESPERGAMON

* Author for correspondence: Tel.: (306) 966-8755; Fax: (306) 966-8542; E-mail: [email protected]

sask.ca.

la toxine choleÂ rique ou de l'albumine d'oeuf, ont intensi®eÂ les reÂ ponses d'anticorps speÂ ci®-que IgG et IgA dans le seÂ rum, l'intestin et les poumons. L'e�ect d'intensi®cation par les

saponines semble relieÂ , dans une certaine mesure a l'augmentation de la permeÂ abiliteÂ desmuqueuses, en favorisant la captation d'antigeÁ nes. L'administration intragastrique d'albu-mine humaine du seÂ rum 99mTc-radioactive, avec les saponines de la quinoa, a reÂ veÂ leÂ plus de

proteÂ ines radioactives dans le sang, le foie, la rate et les poumons des souris. Cette eÂ tude aindiqueÂ le potentiel des saponines de quinoa aÁ eÃ tre utiliseÂ es comme adjuvants pour vaccinsadministreÂ s par voie muqueuse. # 1998 Elsevier Science Ltd. All rights reserved.

Mots-cleÂfs: Saponines de la quinoa; Adjuvant; ReÂ ponses d'anticorps; ImmuniteÂ de muqueuses; PermeÂ a-

biliteÂ intestinale

1. Introduction

The majority of microorganisms enter the body through mucosal surfaces, andtherefore an e�ective mucosal immune response is highly desirable for protectionagainst many diseases. The development of e�ective mucosal vaccines has beenhampered by the lack of suitable adjuvants. Various adjuvants have been shownto potentiate immune responses at the mucosal level, including muramyldipeptide [1], lipopolysaccharides [2] and lipoidal amines [3]. However, the immuneresponses induced with these adjuvants have been quite variable, ranging frome�ective to ine�ective [4]. The toxicity of these compounds also restricts their useas mucosal adjuvants. Hence, there is a need for identi®cation of mucosal adju-vants that are both safe and e�cacious. One such potential adjuvant is a class ofcompounds extracted from plant sources, termed collectively as saponins.

Saponins are glycosides occurring mainly in plants, and which are composed ofa ring structureÐthe aglycone, to which is attached one or more sugar chains [5].The saponins are grouped together based on several common properties. In par-ticular, saponins are surfactants which display hemolytic activity and form com-plexes with cholesterol. Although saponins share these properties, they arestructurally diverse. In particular, the aglycone can be a steroid or a triterpenoid,and the number of sugars attached to the glycosidic bonds varies greatly [5].

Saponins have been used as immunological adjuvants in parenterally adminis-tered veterinary vaccines [6]. The saponins typically used as immunological adju-vants are triterpene glycosides extracted from the South American tree, Quillajasaponaria Molina [7]. Saponins from Panax ginseng have been reported to exertnon-speci®c immunostimulatory activities, but they have not been used as vaccineadjuvants [8].

Although saponins are often highly hemolytic, their oral toxicity to mammals isvery low as a result of low absorption. A long-term toxicity study in mice fed quil-laja saponins daily for 84 weeks, showed that a saponin dose of 700 mg per kg ofbody weight did not produce any detrimental e�ect [9]. The long-term toxicity ofintranasally administered saponins to animals has not been evaluated. Orallyadministered quillaja saponins to mice have been shown to potentiate speci®c

A. Estrada et al. / Com. Immun. Microbiol. & Infect. Dis. 21 (1998) 225±236226

immune responses to fed inactivated rabies antigen [10, 11], and to induce a sys-temic non-speci®c immunostimulation [12, 13].

Chenopodium quinoa Willd. (quinoa) is a grain crop which formed a major partof the diet of the Incas [14]. In contrast to other Andean crops such as beans,corn and potato, quinoa had not been cultivated subsequently on a wide scale inother parts of the world. However, in recent years interest in quinoa as a valuablefood source has been renewed due to its high (14±18%) protein content, toleranceof the plant to unfavourable climatic conditions and pest resistance [15]. As a con-sequence, quinoa is now grown and marketed in North America and Europe forhuman consumption [16].

The quinoa grain contains signi®cant levels of saponins, these compounds pro-tect the crop against birds, insects and fungi [14]. Quinoa contains at least sixdi�erent saponins [16, 17] which can be divided into three di�erent saponin groupscontaining the aglycones: oleanolic acid, hederagenin or phytolaccagenic acid [18].No adjuvant or immunological properties have previously been reported for thesaponins extracted from quinoa.

The aim of the present study was to examine the potential adjuvant activity ofquinoa saponins in the induction of speci®c antibody responses to cholera toxinand ovalbumin antigens following intragastric or intranasal administration. Theresults of this study indicate that quinoa saponins enhance the antibody responsesto the co-administered proteins, possibly by increasing mucosal permeability. The®ndings indicate the potential for the use of quinoa saponins as adjuvants for oralor intranasal administered vaccines.

2. Materials and methods

2.1. Animals

Female BALB/c were obtained from Charles River Laboratories. The animalswere individually caged and housed in an isolation room. The mice were six-to-eight weeks old at the time of the experiments. The procedures applied to the micewere approved by the Animal Care Committee, University of Saskatchewan, in ac-cordance with the requirements of the Canadian Council on Animal Care.

2.2. Quinoa saponins

Quinoa saponins were extracted from the hulls of Chenopodium quinoa Willd.seeds obtained from plants grown in Kamsack, Saskatchewan, Canada. For thesaponin extraction, 100 ml of double-distilled water were added to 10 g of quinoahulls and kept under agitation, using a stirring bar, at 508C for 1 h; the mixturewas then centrifuged at 700 g for 15 min. The supernatant was ®ltered, and theprocess repeated on the pellet. The combined ®ltrates were extensively dialyzedagainst phosphate bu�ered saline (PBS) pH 7.2. The solution was freeze-dried and

A. Estrada et al. / Com. Immun. Microbiol. & Infect. Dis. 21 (1998) 225±236 227

stored at 48C. The water extraction and dialysis procedures yielded approximately10% of the initial dry weight of quinoa hulls. The saponins were tested for activityby a hemolytic test described below.

2.3. Saponins activity assay

To test the activity of quinoa saponins a hemolytic test was used. One hundredmicroliters of 2% sheep red blood cells in PBS were added to individual wells of a96-well microtiter plate. 1 mg of quinoa saponins in PBS were added to the topwells and serial double dilutions were performed. The saponin content was esti-mated as the last dilution presenting complete hemolysis of the red cells andexpressed as hemolytic units (HU) per mg of material. The hemolytic activity ofquinoa saponins was 64 HU per mg.

2.4. Immunization of mice

To examine the adjuvant activities of quinoa saponins, cholera toxin (CTX)from List Biological Laboratories (Campbell, CA) and ovalbumin (OVA) fromSigma Chemical Co. (St Louis, MO) were used to immunize eight-week-oldBALB/c mice by intragastric (IG) or intranasal (IN) routes, using a rigid steelfeeding tube and a 20 ml pipette, respectively. Mice were immunized IG with0.2 mg of CTX or 50 mg of OVA. IN immunizations were given with 0.05 mg ofCTX or 5 mg of OVA. Quinoa saponins were co-administered with CTX andOVA antigens at the doses of 1280 HU for IG immunizations, and 32 HU for INimmunizations. The antigen/saponins mixtures were administered in a PBS suspen-sion with volumes of 100 ml for IG immunizations, and 10 ml for IN immuniz-ations. Immunizations were performed twice at a 14-day interval and mice wereeuthanized 7 days after the last immunization. The immunization protocol isshown in Table 1.

2.5. Collection of samples

Mice were bled to collect serum samples and euthanized 21 days after primaryinoculation. Intestinal washes were collected by everting the entire small intestineover a capillary tube and exposing the intestinal mucosa to 5 ml of PBS bu�ercontaining 0.05 TIU/ml aprotinin, 2 mM phenylmethylsulfonyl ¯uoride, 5 mMethylene diamine tetraacetic acid and 0.02% NaN3 at 48C for 4 h. Lung washeswere collected by ¯ushing the lung through the trachea with 1 ml of above-men-tioned PBS bu�er containing protease inhibitors. All samples were centrifuged at800 g at 48C for 10 min and stored at ÿ208C until analysis by ELISA.


2.6. Measurement of anti-CTX and anti-OVA antibody responses

An enzyme-linked immunosorbent assay (ELISA) was used to measure IgG,IgG1, IgG2a and IgA anti-CTX and anti-OVA antibody levels in sera, intestinalwashes and lung washes. The wells of 96-well microtiter plates (Immulon 2,Dynatech Laboratories Inc., Chantilly, VA) were coated with 10 mg per ml ofeither CTX or OVA antigens in PBS pH 7.2 at 48C for 18 h. The wells werewashed with PBS bu�er containing 0.05% Tween-20 (PBS-T) and incubatedwith 1% bovine serum albumin in PBS at 378C for 1 h. The wells were washedwith PBS-T, 100 ml of serum samples diluted 1/100 or 1/10 of intestinal or lungwashes were added to the wells, and incubated at 378C for 2 h. The wells werethen washed with PBS-T and biotinylated goat anti-mouse IgG, IgG1, IgG2a orIgA antibodies (Southern Biotechnology Associates, Birmingham, AL) diluted1/1000 in PBS-T were added and incubated at 378C for 1 h. After washingwith PBS-T, 100 ml of streptavidin-alkaline phosphatase conjugate (Gibco BRL,Life Technologies Inc., Gaithersburg, MD) was added and incubated at 378Cfor 1 h. The plates were washed with PBS-T and 100 ml of the alkaline phos-phatase substrate solution added to each well. The substrate consisted of 1 mgper ml of p-nitrophenyl phosphate (104 phosphatase substrate tablets; Sigma)in 1 M diethanolamine bu�er, pH 9.8. The absorbance of each well at 405 nmwas measured using an automated spectrophotometer (Molecular Devices Vmax

Kinetic microplate reader; Molecular Devices, Mexico Park, CA). Antibodylevels were reported as the optical density (OD) readings, after the subtractionof the OD read-out of the mean plus three standard errors of the mean (SEM)of series of control wells with normal mouse serum, intestinal washes or lungwashes, and expressed as means2SEM for each group.

Table 1

Immunization protocols

Experimental days

Immunization route AntigenÐdose Saponins dose (HU) 0 14 21

IG CTXÐ0.2 mg 1280 1 2 3

IG CTXÐ0.2 mg Ð 1 2 3

IN CTXÐ0.05 mg 32 1 2 3

IN CTXÐ0.05 mg Ð 1 2 3

IG OVAÐ50 mg 1280 1 2 3

IG OVAÐ50 mg Ð 1 2 3

IN OVAÐ5 mg 32 1 2 3

IN OVAÐ5 mg Ð 1 2 3

Groups of ®ve mice were immunized.

IGÐintragastric; INÐintranasal.1 Primary immunization.2 Secondary immunization.3 Serum, intestinal and lung washes samples.


2.7. Intestinal permeability studies

Human Serum Albumin (HSA) (Sigma) was radiolabeled with Technetium(99mTc) (Amersham Canada Ltd, Oakville, Ontario, Canada). One mg of HSA in0.1 M NaCl was incubated with 5 mCi of 99mTc at 258C for 30 min. Free radiola-bel was removed by chromatography through a Sephadex G-25 column(Pharmacia Biotech Inc., Baie d'Urfe, Quebec, Canada), the resultant HSA±99mTcconjugate was used immediately after this step. Studies on the e�ect on intestinalpermeability exerted by quinoa saponins were performed by the IG administrationto BALB/c mice of 100 ml of a 0.2 M NaHCO3 solution containing 1280 HU ofquinoa saponins and 100 mCi of 99mTc±HSA, or 100 mCi of 99mTc±HSA alone.Groups of four mice were bled and euthanized at 0.5, 1, 2, 4, 6, 8, 10 and 12 hafter IG administration, and liver, spleen and lungs organs were collected andweighed. The radioactivity in the samples was measured in a gamma counter(Apex Automatic; ICN Micromedic Systems, Huntsville, AL). Radioactivity levelswere expressed as the mean counts per minute (CPM) per gram of tissue2SEMfor each group.

2.8. Statistical analysis

Results were expressed as means2SEM and compared by the Student's t-test.

3. Results

3.1. Anti-CTX and anti-OVA antibody responses following IG administration

The e�ect of the administration of quinoa saponins by IG route on antigen-speci®c antibodies to CTX and OVA were evaluated. Tables 2 and 4 indicate theserum antibody responses to CTX and OVA, respectively. The administration ofsaponins by the IG route signi®cantly enhanced the levels of IgG, IgG1 and IgAanti-CTX antibodies (P < 0.05), as well as IgG, IgG1 (P < 0.001) and IgA(P < 0.05) anti-OVA antibody levels, compared to the groups that received anti-gen alone. Speci®c IgG2a anti-CTX and anti-OVA antibody levels were nota�ected by the saponin treatment. Tables 3 and 5 show the intestinal and lungIgG and IgA antibody responses to CTX and OVA, respectively. The intestinalwashes from saponin-administered mice contained signi®cantly higher levels ofIgA anti-CTX antibody (P < 0.05), and IgG (P < 0.01) and IgA (P < 0.05) anti-OVA antibodies, when compared to the control groups. A correlation betweenserum and intestinal antibody levels was noted. Mice with relatively high levels ofantigen-speci®c IgG and IgA antibodies in serum also had measurable levels of in-testinal antigen-speci®c IgG and IgA antibodies. Antibody responses in lung werenot a�ected by the IG administration of saponins. In general, serum and mucosalantibody responses to both CTX and OVA antigens were enhanced by their co-administration with saponins by the IG route.


Table 2

Serum antibody responses at day 21 post-immunization in mice immunized IG or IN with CTX

co-administered with quinoa saponins

Serum anti-CTX (OD405) levels

Immunization

route

Quinoa

saponins IgG IgG1 IgG2a IgA

IG + 1.3820.13* 1.6120.15* 0.3320.07 1.9220.25

IG ÿ 0.8620.11 1.0220.16 0.2920.09 0.9720.23

IN + 1.1920.20** 0.1020.02* 0.0420.02 0.1420.03*

IN ÿ 0.3720.09 0.0420.01 0 0.0620.01

Mean values of groups of ®ve mice2SEM.

* P < 0.05, **P< 0.01 versus groups receiving no quinoa saponins.

Table 3

Intestine and lung antibody responses at day 21 post-immunization in mice immunized IG or IN with

CTX co-administered with quinoa saponins

Intestine and lung washes anti-CTX (OD405) levels

Intestine Lung

Immunization

route

Quinoa

saponins IgG IgA IgG IgA

IG + 0.5920.13 1.8720.22* 0.0520.02 0

IG ÿ 0.3320.16 1.1520.20 0 0

IN + 0.1620.04* 0.1520.02* 0.1720.03** 0.0920.01**

IN ÿ 0.0620.01 0.0620.02 0.0320.01 0.0320.01


* P < 0.05, **P< 0.01 versus groups receiving no quinoa saponins.

Table 4

Serum antibody responses at day 21 post-immunization in mice immunized IG or IN with OVA

co-administered with quinoa saponins

Serum anti-OVA (OD405) levels

Immunization

route

Quinoa

saponins IgG IgG1 IgG2a IgA

IG + 1.3120.22*** 1.9220.18*** 0.2420.06 0.1220.04*

IG ÿ 0.0620.01 0.0820.02 0.1320.01 0.0120.01

IN + 1.8620.14** 1.9120.10** 0.1820.02 0.1120.02*

IN ÿ 1.1720.05 1.3720.07 0.1420.02 0.0420.01


* P < 0.05, **P< 0.01, ***P< 0.001 versus group receiving no quinoa saponins.


3.2. Anti-CTX and anti-OVA antibody responses following IN administration

To further investigate whether quinoa saponins could enhance mucosal immuneresponses in the respiratory tract, mice were immunized with CTX and OVA anti-gens co-administered with saponins by IN route and the antigen-speci®c antibodieswere evaluated. Tables 2 and 4 denote the serum antibody responses to CTX andOVA, respectively. The administration of saponins by the IN route signi®cantlyenhanced the levels of IgG (P < 0.01), IgG1 and IgA (P < 0.05) anti-CTX anti-bodies. IgG, IgG1 (P < 0.01) and IgA (P < 0.05) anti-OVA antibody levels werealso signi®cantly increased in serum, compared to the groups that received antigenalone. Mucosal antibody responses were assessed in intestinal and lung washes ofIN-immunized mice. Tables 3 and 5 summarize these responses. IgG and IgAanti-CTX antibody levels in intestinal washes proved to be signi®cantly augmented(P < 0.05) in mice administered IN saponins, compared to the control group. TheIN administration of saponins together with OVA did not a�ect the intestinalantibody responses. The lung washes of IN-saponin-administered mice containedsigni®cantly higher levels of IgG and IgA anti-CTX and anti-OVA antibodies(P < 0.01), when compared to the control groups. Similar to the e�ects of IG im-munization with antigen±saponin formulations, the enhancement of antigen-speci®c IgG responses following IN administration of saponins with CTX or OVAantigens, was characterized by the preferential stimulation of IgG1 over IgG2a sub-class (Tables 2 and 4).

3.3. Intestinal permeability

The uptake of 99mTc±HSA antigen was studied to assess the ability of quinoasaponins to enhance intestinal permeability following IG administration. Thesaponins dose of 1280 HU used in the experiment corresponded to the dose usedfor the IG administration of mice. Figure 1 illustrates the distribution of 99mTc±HSA in the blood, liver, spleen and lungs of mice. The mean concentrations of

Table 5

Intestine and lung antibody responses at day 21 post-immunization in mice immunized IG or IN with

OVA co-administered with quinoa saponins

Intestine and lung washes anti-OVA (OD405) levels

Intestine Lung

Immunization

route

Quinoa

saponins IgG IgA IgG IgA

IG + 0.1520.03** 0.0720.01* 0 0.0320.01

IG ÿ 0.0320.01 0.0320.01 0 0

IN + 0.0520.01 0 0.2220.03** 0.0920.01**

IN ÿ 0.0520.01 0 0.0920.01 0.0320.01


* P < 0.05, **P< 0.01 versus group receiving no quinoa saponins.


99mTc±HSA, at the di�erent time-points, were overall higher in mice given quinoasaponins together with the radiolabeled protein than in the control group, whichreceived radiolabeled protein alone. The time course experiment showed a signi®-cant increase of 99mTc±HSA in blood (P < 0.01), liver (P < 0.001), spleen(P < 0.05) and lungs (P < 0.05), 30 min after administration. The peak levels of99mTc±HSA in blood and organs occurred at 1 h post-administration of the pro-tein. In blood liver and spleen of groups of mice administered saponins, the levelsof 99mTc±HSA returned to the levels of control groups at 12 h after adminis-tration, while in the lungs this occurred at 8 h post-administration of the radio-labeled protein.

4. Discussion

This study reports the ®rst use of saponins extracted from quinoa as immuno-logical adjuvants. Our results demonstrate that quinoa saponins are capable of

Fig. 1. E�ect of quinoa saponins on the passage of radiolabeled antigen into tissues of mice sacri®ced

at di�erent time points following oral administration of 99mTc-labelled HSA alone or together with a

1280 HU dose of quinoa saponins. ., 99mTc±HSA+ saponins; w, 99mTc±HSA alone. Each point in the

®gure represents the mean CPM per gram of tissue of four mice2SEM. *P< 0.05, **P < 0.01,

***P< 0.001 versus group receiving no quinoa saponins.


enhancing both systemic and mucosal antigen-speci®c antibody responses follow-

ing IG or IN administration. Previous studies have shown that saponins extracted

from quillaja bark potentiate speci®c immune responses after oral [10] or

intraperitoneal [13] immunization of mice with inactivated rabies virus. Another

study [12] indicated that the oral administration of quillaja saponins to mice

enhanced the non-speci®c resistance against rabies infection.

CTX and OVA proteins, co-administered with quinoa saponins, were used as

model antigens to study the systemic and mucosal immune responses induced by

the IG and IN immunization of mice. The saponins co-administered with the anti-

gens evoked enhanced antibody responses to both CTX and OVA, when com-

pared to the responses elicited with the administration of the antigens alone.

Furthermore, the antigen-speci®c IgG antibodies were characterized by the

increase of the IgG1 subclass, but not IgG2a. The IgG1 subclass is associated with

the stimulation of CD4+ T cells of the Th2 subset, which prime for the production

of secretory IgA responses at mucosal sites [19]. It remains to be determined,

whether or not the preferential stimulation of IgG1 over IgG2a by quinoa saponins

correlates with the induction of Th2 type immune responses.

Generally, the production of systemic and mucosal immune responses by oral

or intranasal immunization requires the repeated administration of large amounts

of antigen [20]. In contrast, we demonstrated that quinoa saponins could enhance

antibody responses to low doses of CTX and to the poorly immunogenic OVA

antigen, when mucosally administered.

There are no previous reports on the administration of saponins by the intrana-

sal route as vaccine adjuvants. However, immunostimulating complexes (iscoms),

particles composed of quillaja saponins, cholesterol, phosphatidyl choline and

antigen, have been shown to have adjuvant activity following intranasal immuniz-

ation of mice [21].

One possible mechanism by which quinoa saponins exert their adjuvant activity

when administered by the mucosal route is by increasing permeability of the

mucosal epithelium, although it is likely that other mechanisms also are involved.

Saponins are a class of compounds, not a single compound, and diverse biological

activities have been demonstrated in preparations from di�erent plant

sources [5, 17, 22]. A study in mice with orally provided quillaja saponins [12] indi-

cated that the saponins exerted a non-speci®c immunostimulation against rabies

infection; the saponin mechanism on this e�ect, although unknown, may not be

related with an increased intestinal permeability.

Studies on the e�ect of quinoa saponins on intestinal permeability indicated

that the saponins enhanced the uptake of 99mTc±HSA from the gastrointestinal

tract into the bloodstream. Since the IG co-administration of the saponins with

CTX and OVA also enhanced systemic antibody responses to the antigens, we

may assume that the saponins facilitate the entry of antigen into the bloodstream.

It may be, therefore, that one of the mechanisms of action of the saponins in the

gut is to increase the intestinal permeability thus facilitating the antigen uptake. In

the present study we assessed the e�ect of quinoa saponins on the permeability of


the intestine, whether or not similar e�ects occur in the respiratory tract epi-thelium remains to be elucidated.

Diverse saponins from plants have been reported to increase intestinal per-meability in vitro or in vivo [10, 23, 24]. Saponins have been described as toxiccompounds [17] when given intravenously. However, orally fed saponins are welltolerated at much higher concentrations [9]. Saponins are probably not absorbedby the intestinal epithelium, but interact with the epithelial cells by forming com-plexes with cholesterol in the cell membrane, and therefore do not enter thecirculation [24].

However, the passage of antigen into the bloodstream is not an absoluterequirement for the initiation of immune responses. Antigens in the intestine mayinteract with local macrophages, dendritic cells and lymphocytes, and this mayoccur in the follicular associated lymphoid tissue of the Peyer's patches, or in thelamina propria, an e�ector site of IgA responses [19]. Saponins, therefore, mayincrease the permeability of the overlying epithelial layer facilitating the inter-action between the mucosal associated lymphoid tissue and antigen.

Mice tolerated the e�ective saponins doses without any visible signs of distressor injury. Morphological examination of small intestines of the mice used in theintestinal permeability studies failed to reveal any mucosal lesions at the saponinconcentration administered.

Since unpuri®ed mixtures of quinoa saponins were used in these studies, thesaponin(s) responsible for the adjuvanticity and/or intestinal permeability remainto be elucidated. Further studies are required to establish the exact mechanism ofaction and to evaluate the adjuvant/immunostimulating activities of the individualsaponins.

In conclusion, the present study indicated that the administration of quinoasaponins enhanced both systemic and mucosal speci®c antibody when IG or INadministered. These saponins may prove to be a useful adjuvant system for gener-ating systemic and mucosal responses.

Acknowledgements

We are grateful to Dr Gilbert Matte of the Nuclear Medicine Department,University of Saskatchewan, for experimental assistance with the intestinal per-meability studies. This work was supported by a grant from the AgricultureDevelopment Fund, Province of Saskatchewan, Canada.

References

[1] Michalek SM, Morisaki I, Gregory RL, Kiyono H, Hamada S, McGhee JR. Oral adjuvants

enhance IgA responses to Streptococcus mutans. Mol. Immunol. 1983;20:1009±18.

[2] Keren DF, McDonald RA, Carey JL. Combined parenteral and oral immunization results in an

enhanced mucosal immunoglobulin. A response to Shigella ¯exneri. Infect. Immun. 1988;56:910±5.


[3] Bergmann KC, Waldman RH. Enhanced murine respiratory tract IgA antibody response to oral

in¯uenza vaccine when combined with a lipoidal amine (avridine). Int. Arch. Allergy Appl.

Immunol. 1988;87:334±5.

[4] Elson CO and Dertzbaugh MT, Mucosal adjuvants. In Handbook of Mucosal Immunology, Edited

by Ogra P, Mestecky J, Lamm M, Strober W, McGhee J and Bienenstock J, pp. 391±402.

Academic Press Inc., San Diego, CA (1994).

[5] Price KR, Johnson IT, Fenwick GR. The chemistry and biological signi®cance of saponins in

foods and feedingstu�s. CRC Crit. Rev. Food Sci. Nutr. 1987;26:27±135.

[6] Campbell JB, Peerbaye YA. Saponin. Res. Immunol. 1992;143:526±30.

[7] Kensil CR, Patel U, Lennick M, Marciani D. Separation and characterization of saponins with

adjuvant activity from Quillaja saponaria Molina cortex. J. Immunol. 1991;146:431±7.

[8] Kenarova B, Neychev H, Hadjiivanova C, Petkov V. Immunomodulating activity of ginsenoside

Rg1 from Panax ginseng. Japan J. Pharmacol. 1990;54:447±54.

[9] Phillips JC, Butterworth KR, Gaunt IF, Evans JG and Grasso P, Long-term toxicity study of

Quillaja extract in mice, Vol. 17, Edited by Cosmet. Toxicol., pp. 23±27 (1979).

[10] Maharaj I, Froh KJ, Campbell JB. Immune responses of mice to inactivated rabies vaccine admi-

nistered orally: potentiation by Quillaja saponins. Can. J. Microbiol. 1986;32:414±20.

[11] Chavali SR, Campbell JB. Adjuvant e�ects of orally administered saponins on humoral and cellu-

lar immune responses in mice. Immunobiol. 1987;174:347±59.

[12] Chavali SR, Campbell JB. Immunomodulatory e�ects of orally-administered saponins and nonspe-

ci®c resistance against rabies infection. Int. Archs. Allergy appl. Immun. 1987;84:129±34.

[13] Chavali SR, Barton LD, Campbell JB. Immunopotentiation by orally-administered Quillaja sapo-

nins: e�ects in mice vaccinated intraperitoneally against rabies. Clin. Exp. Immunol. 1988;74:339±

43.

[14] Risi JC, Galwey NW. The Chenopodium grains of the Andes: Inca crops for modern agriculture.

Adv. Appl. Biol. 1984;10:145±216.

[15] Galwey NW, Leakey CL, Price KR, Fenwick GR. Chemical composition and nutritional charac-

teristics of quinoa (Chenopodium quinoa). Human Nutr. Food Sci. Nutr. 1990;42:245±61.

[16] Ridout CL, Price KR, DuPont SM, Parker ML, Fenwick RG. Quinoa saponinsÐanalysis and

preliminary investigations into the e�ects of reduction by processing. J. Sci. Food Agric.

1991;54:165±76.

[17] Heinstein PF, McLaughlin JL. Additional toxic, bitter saponins from the seeds of Chenopodium

quinoa. J. Nat. Prod. 1989;52:1132±5.

[18] Mizui F, Kasai R, Ohtani K, Tanaka O. Saponins from brans of quinoa, Chenopodium quinoa.

Willd. Chem. Pharm. Bull. 1988;36:1415±8.

[19] Kramer DR, Sutherland RM, Bao S, Husband AJ. Cytokine mediated e�ects in mucosal immu-

nity. Immunol. Cell Biol. 1995;73:389±96.

[20] Aizpurua HJ, Russell-Jones GJ. Oral vaccination. Identi®cation of classes of proteins that provoke

an immune response upon oral feeding. J. Exp. Med. 1988;167:440±51.

[21] Lovgren K, Kaberg H, Morein B. An experimental in¯uenza subunit vaccine (iscom): Induction of

protective immunity to challenge infection in mice after intranasal or subcutaneous administration.

Clin. Exp. Immunol. 1990;82:435±9.

[22] Domon B, Hostettmann K. New saponins from Phytolacca dodecandra. Helv. Chim. Acta

1984;67:1310±5.

[23] Onning G, Wang Q, Westrom BR, Asp NG, Karlsson BW. In¯uence of oat saponins on intestinal

permeability in vitro and in vivo in the rat. Brit. J. Nutr. 1996;76:141±51.

[24] Johnson IT, Gee JM, Price K, Curl C, Fenwick GR. In¯uence of saponins on gut permeability

and active nutrient transport in vitro. J. Nutr. 1986;116:2270±7.


adjuvant action of chenopodium quinoa saponins on the induction of antibody responses to...

Documents