isolation and evaluation of immunological adjuvant activities of saponins from polygala senega l

Isolation and evaluation of immunologicaladjuvant activities of saponins from Polygala

senega L.

Alberto Estradaa,*, Georgios S. Katselisb, Bernard Laarvelda,Branka Barlb

aAnimal Biotechnology Centre, Department of Animal and Poultry Science, University of Saskatchewan,

72 Campus Drive, Saskatoon, Saskatchewan, Canada, S7N 5B5bDepartment of Plant Sciences, University of Saskatchewan, 51 Campus Drive, Saskatoon, Saskatchewan,

Canada, S7N 5A8

Abstract

We have identi®ed saponins in the root of Polygala senega L., a plant indigenous to the

Canadian prairies, which display immunopotentiation activity to protein and viral antigens.By two-step extraction and hemolytic activity-guided fractionation by silica ¯ushchromatography six saponin fractions were generated and their HPLC pro®les determined.

Two dominant fractions, designated as PS-1 and PS-2, were tested for adjuvant activity inmice immunized with ovalbumin, and hens immunized with rotavirus. The resultingadjuvant activity was compared with that of Quil A saponin. The P. senega saponinsincreased speci®c antibody levels to the antigens, in both mice and hens. In mice, there was

a preferential increase of the IgG2a subclass, and upon in vitro secondary antigenstimulation, high IL-2 and IFN-g levels were observed in spleen cell cultures from P. senegasaponins-immunized animals. The saponins were tested for their toxicity by lethality in mice

and were found to be less toxic at the same dose than their counterpart Quil A. The resultsof this study indicated the potential of P. senega saponins as vaccine adjuvants to increasespeci®c immune responses. # 1999 Elsevier Science Ltd. All rights reserved.

Keywords: Polygala senega L.; Saponins; Adjuvant; Immune responses

0147-9571/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.

PII: S0147 -9571 (99)00020 -X

Comparative Immunology, Microbiology

& Infectious Diseases 23 (2000) 27±43

www.elsevier.com/locate/cimid

* Corresponding author. Tel.: 1-306-966-8755; fax: 1-306-966-8542.

E-mail address: [email protected] (A. Estrada)

ReÂ sumeÂ

Nous avons identi®eÂ des saponines extraites de la racine de Polygala senega L., plante

originaire des prairies canadiennes, lesquelles preÂ sentent une activiteÂ d'immunostimulationvis aÁ vis d'antigeÁ nes proteÂ iques et viraux. La mise en place d'un proceÂ deÂ d'extractionapproprieÂ a permis de seÂ parer les saponines de l'extrait brut, au moyen d'une colonne aÁ

fractionnement rapide. Nous avons obtenu cinq fractions de saponines heÂ molytiques dont

nous avons deÂ termineÂ les pro®ls chromatographiques. Les deux fractions semblant contenirles saponines preÂ dominantes ont eÂ teÂ testeÂ es a®n d'eÂ valuer leur activiteÂ en tant qu'adjuvantschez des souris injecteÂ es avec de l'albumine d'oeuf et des poules immuniseÂ es avec le

rotavirus; nous avons ensuite compareÂ cette activiteÂ aÁ celle de Quil A. Les saponines de P.senega appeleÂ es PS-1 et PS-2 ont entraineÂ une augmentation du niveau des anticorpsspeÂ ci®ques chez les souris ainsi que chez les poules. Chez les souris, nous avons observeÂ une

augmentation de la sous-classe IgG2a en particulier; ulteÂ rieurement une autre stimulation invitro avec l'antigeÁ ne a entraineÂ de hauts niveaux de IL-2 et de IFN-g dans les cultures decellules de rates des animaux injecteÂ s avec les saponines de P. senega. La toxiciteÂ et la doseleÂ tale des saponines ont eÂ teÂ testeÂ es chez la souris; celles ci semblent eÃ tre moins toxiques que

les saponines correspondantes de Quil A. Les reÂ sultats obtenus indiquent le potentiel dessaponines de P. senega comme adjuvants des vaccins pour intensi®er les reÂ ponsesimmunitaires speÂ ci®ques. # 1999 Elsevier Science Ltd. All rights reserved.

Mots-cleÂf: Polygala senega L.; Saponines; Adjuvant; ReÂ ponses immunitaires

1. Introduction

Formulation of e�ective vaccines generally requires an appropriate adjuvant to

optimize protective humoral and cell-mediated immune responses. An important

consideration, in addition to the e�cacy of the adjuvant for eliciting a protective

immune response, is the toxicity of the adjuvant. Many adjuvants manifest

signi®cant side e�ects such as formation of granulomas or eliciting pyrogenic

responses upon injection. Complete Freund's adjuvant (CFA), which is used

extensively in research vaccines, induces excellent humoral and cell-mediated

immunities to antigens, but is unsuitable for use in human and veterinary vaccines

because of the adverse side e�ects [1]. Accordingly, there is a need for

identi®cation of adjuvants that are both safe and e�cacious for use in a variety of

vaccines.

Saponins are natural products that are a promising source of adjuvants.

Saponins are structurally constructed of aglycones, either triterpenoid or steroid,

and one or more sugar side chains [2]. They are main constituents of many plant

drugs and folk medicines, and are considered responsible for numerous

pharmacological properties, such as anti-in¯ammatory [3], anti-tumor [4], anti-

viral [5] and anti-fungal [6].

A. Estrada et al. / Comp. Immun. Microbiol. Infect. Dis. 23 (2000) 27±4328

Saponins have been used as immunological adjuvants in parenterallyadministered veterinary vaccines [7]. Triterpenoid saponins extracted from thebark of the South American tree, Quillaja saponaria Molina have long been usedas adjuvants for veterinary vaccines [8±10]. Crude preparations of Q. saponariasaponins have been used as adjuvants to increase the immune responses to avariety of antigens [7,11]. In addition, Quil A, a mixture of partially puri®edsaponins from Q. saponaria, has been used as an adjuvant for several proteinantigens, either alone [12,13] or associated with hydrophobic antigens and lipids inform of complexes termed iscoms [14,15]. Few saponins from sources other thanQ. saponaria have been identi®ed to act as adjuvants; saponins extracted from theseed of Chenopodium quinoa (quinoa) act as mucosal adjuvants potentiatingspeci®c antibody responses to oral or intranasal administered antigens [16];saponins puri®ed from the leaves of the plant Hedera taurica (crimean ivy)enhanced humoral responses to HIV envelope glycoproteins [17]; saponinsextracted from the plant Periandra mediterranea increased antibody responses toan antigen of Leishmania donovani [18]. Saponins from Panax ginseng have beenreported to exert non-speci®c immunostimulatory activities, but they have notbeen used as vaccine adjuvants [19].

The roots of the plants Polygala senega L., grown in the wild in Canadian

Fig. 1. Extraction scheme for saponins from P. senega root.

A. Estrada et al. / Comp. Immun. Microbiol. Infect. Dis. 23 (2000) 27±43 29

prairies, and P. senega L. var. latifolia Torry et Gray, cultivated in Japan, arecommonly known as senega root. Senega root has been used by North Americannatives as a cough remedy and for treatment of rattlesnake bites [20,21]. Presently,the dry or ¯uid extract of the root is used as an antitussive and expectorantremedy [22] and to treat chronic bronchitis and pharyngitis [23]. P. senega L. var.latifolia Torry et Gray has been reported to contain up to 12% saponins [24]. Itconsists of 6±10 di�erent saponins [25], the main ones being senegin II, III, IV,and senega-saponins a, b and c [26±29]. There are no reports on the chemicalcharacterization of saponins in P. senega grown in Canada. Noimmunostimulating uses for P. senega saponins have been previously reported.

In this study, we report the separation of saponins extracted from the root of P.senega and the evaluation of the adjuvant activity of these saponins on theinduction of speci®c immune responses to ovalbumin and rotavirus antigensfollowing immunization of mice and hens, respectively.

2. Materials and methods

2.1. Sources of saponins, extraction and isolation

Saponins, contained in the roots of P. senega plants grown in Saskatchewan,Canada, were extracted with methanol in Soxhlet apparatus. The extract wasconcentrated, freeze-dried and re-extracted with water and butanol saturated withwater (Fig. 1). The dried butanolic extract, dissolved in a minimal amount ofchloroform:methanol:water (64:26:10, lower phase solvent), was furtherfractionated by ¯ash chromatography on a silica (10±40 mm particle size, type H,Sigma, Chemical Co., St Louis, MO, USA) column (25 mm i.d. � 60 cm length).Column was eluted with a stepwise gradient of ascending polarity of

Fig. 2. Fractionation of P. senega saponins by ¯ash column chromatography using gradient elution.


chloroform:methanol:water solvent (64:26:10, 60:30:10, 55:35:10, 50:40:10, lowerphases) to ensure separation of saponins from compounds of similar polarity, andalso to achieve separation of individual saponin compounds from saponin mixture(Fig. 2). In total, 22 15 ml-fractions were collected according to the schematicelaborated in Fig. 2. All 22 fractions eluted were analysed by thin layerchromatography (TLC) and subjected to hemolysis test.

The TLC analysis was performed with the method of Wagner et al. [30]. Thedeveloping solvent was n-butanol:acetic acid:water (40:10:50; upper phase).Fractions were dissolved in the developing solvent and applied to aluminum-backed plates of silica gel 60 F254 (layer thickness 0.2 mm, 20 � 20 cm; E. Merck,Darmstadt, Germany). After development, plates were air-dried, observed underUV light, sprayed with methanol:acetic acid:sulphuric acid:anisaldehyde

Fig. 3. HPLC chromatograms of P. senega fractions (a) PS-1 and (b) PS-2. Column, Waters Nova-

Pak1 60 AÊ 4 mm (3.9 mm i.d. � 15 cm) C18; eluent, 25% solution A [10% ammonium acetate bu�er

(pH 6.9, 50 mM)Ð90% acetonitrile] and 75% solution B [90% ammonium acetate bu�er (pH 6.9,

50 mM)Ð10% acetonitrile]; ¯ow rate, 1 ml/min; detection at 315 nm.


(85:10:5:0.1) and heated at 1008C for 5 min. On heating, saponin bands turned redand were readily visualized.

Fractions 17±22 containing a mixture of saponins were analyzed by high-performance liquid chromatography (HPLC) on a Beckman liquid chromatograph(Beckman Instruments, Inc., Fullerton CA, USA) equipped with a variablewavelength UV detector and a system Gold

2

, version 5.0, software package.Separations were performed on a Nova-Pak

1

, (Waters, Milford, MA, USA) 4 mmparticle size, 60 AÊ pore size, (3.9 mm i.d. � 15 cm) C18 column. The mobile phaseconsisted of 25% solution A [10% ammonium acetate bu�er (pH 6.9, 50 mM)Ð90% acetonitrile] and 75% solution B [90% ammonium acetate bu�er (pH 6.9,50 mM)Ð10% acetonitrile]. Saponin peaks, detected by absorbance at 315 nm,were eluted isocratically at a ¯ow rate of 1 ml/min. Fractions 17 and 19, whichwere used for the adjuvanticity studies, were designated as P. senega fractions 1(PS-1) and 2 (PS-2), respectively. The HPLC pro®les of these fractions areillustrated in Fig. 3.

Quil A saponins from Q. saponaria (Superfos Biosector, Vedbaek, Denmark)were used to compare the e�ectiveness of P. senega saponins as adjuvants in theimmunization studies.

2.2. Saponin hemolytic activity assay

The hemolytic activity of P. senega and Quil A saponins was tested as follows.One hundred ml of 2% sheep red blood cells in phosphate bu�ered saline (PBS)pH 7.2, were added to individual wells of a 96 well microtiter plate. One mg ofeach saponin tested, dissolved in 100 ml of PBS, were added to the wells and serialdilutions in duplicate were performed. The saponin content was estimated fromthe highest dilution causing complete hemolysis of the red cells and expressed ashemolytic units (HU) per mg of material.

2.3. Adjuvant activity of P. senega saponins in mice

2.3.1. AnimalsCD-1 mice were obtained from the Animal Resources Centre, University of

Saskatchewan (Saskatoon, SK, Canada). The mice were 6±8 weeks of age when®rst used. The procedures applied to the mice were approved by the Animal CareCommittee, University of Saskatchewan, in accordance with the requirements ofthe Canadian Council on Animal Care.

2.4. Saponins toxicity test

The toxicity of PS-1, PS-2 and Quil A saponins was tested in mice. Saponindoses from 6.4 to 51.2 HU in PBS were injected subcutaneously to groups of ®vemice. The mice were observed for 96 h after injections and the mortality in eachgroup was recorded. Determination of 50% lethal dose (LD50) of the saponinswas performed by probit analysis [31].


2.5. Immunization of mice

To examine the adjuvant activities of PS-1, PS-2 and Quil A saponins,ovalbumin (OVA) (Sigma) was used as model antigen. Groups of ®ve mice wereinjected subcutaneously with 50 mg of OVA alone in PBS or mixed with P. senegaor Quil A saponins in 100 ml of PBS. Two di�erent doses of P. senega saponinswere used: PS-1, 1.6 and 6.4 HU; PS-2, 3.2 and 12.8 HU; Quil A, 6.4 HU. ThePS-1 and PS-2 saponin doses used corresponded to HU amounts four and sixteen-fold below the respective HU amount causing mortality of mice; Quil A was usedat a HU dose two-fold below the dose which showed toxic e�ects to mice (Table3). Immunizations were performed twice at a 10-day interval and mice wereeuthanized 10 days after the last immunization. Serum samples obtained fromanimals on days 10 and 20 were used for measurement of anti-OVA IgG, IgG1

and IgG2a by ELISA. The immunization protocol is shown in Table 1.

2.6. Collection of samples

Sera were prepared from blood obtained at 10 and 20 days after the primaryimmunization. All sera were clari®ed by centrifugation and stored at ÿ208C untilanalysis by ELISA. At day 20, the mice were euthanized and the spleens wereremoved. Spleen cells were prepared by grinding the tissues through a 70 mm cellstrainer (Falcon, Becton Dickinson Labware, Franklin Lakes, NJ, USA) andrinsing with RPMI-1640 containing 10% fetal bovine serum (Gibco BRL, LifeTechnologies Inc., Gaithersburg, MD, USA) (RPMI-FCS). Spleen cells werecentrifuged at 300 g at 48C for 5 min, the supernatant discarded, and the cellpellet treated with 1 ml of a 1% ammonium chloride solution. The cells werewashed by centrifugation twice, suspended in RPMI-FCS, counted and adjustedto a cell concentration of 107 cells/ml

Table 1

Immunization protocol of mice: Groups of ®ve were immunizeda

Experimental days

Group Saponins dose (HU) 0 10 20

PS-1 1.6 1 2, 3 3, 4

PS-1 6.4 1 2, 3 3, 4

PS-2 3.2 1 2, 3 3, 4

PS-2 12.8 1 2, 3 3, 4

Quil A 6.4 1 2, 3 3, 4

Control ± 1 2, 3 3, 4

a 1, primary immunization; 2, secondary immunization; 3, serum samples; 4, spleen cell samples.


2.7. Immunological assays

ELISA was used to measure serum IgG, IgG1 and IgG2a anti-OVA. The wellsof 96-well microtiter plates (Immulon 2; Dynatec, Laboratories Inc., Chantilly,VA, USA) were coated with 10 mg/ml of OVA in PBS at 48C for 18 h. The wellsof all plates were washed three times with PBS containing 0.05% Tween-20 (PBS-T) and incubated with PBS containing 1% bovine serum albumin (BSA) at 378Cfor 30 min. The plates were then washed with PBS-T. To the plates, 100 ml of1:100 mouse serum dilutions for IgG anti-OVA or 1:10 for IgG2a and IgG1 inPBS-T were added and incubated at 378C for 1 h. After washing with PBS-T,biotinylated goat anti-mouse IgG, IgG2a or IgG1 (Southern BiotechnologyAssociates, Birmingham, AL, USA) diluted 1:1000 in PBS-T were added andincubated at 378C for 1 h. After washing with PBS-T, 100 ml of streptavidin-alkaline phosphatase conjugate (Gibco) was added and incubated at 378C for 1 h.The plates were washed with PBS-T and 100 ml of the alkaline phosphatasesubstrate solution added to each well. The substrate consisted of 1 mg/ml of p-nitrophenyl phosphate (104 phosphatase substrate tablets; Sigma) in 1 Mdiethanolamine bu�er, pH 9.8. The absorbance of each well at 405 nm wasmeasured using an automated spectrophotometer (Molecular Devices Vmax Kineticmicroplate reader; Molecular Devices, Menlo Park, CA, USA). Anti-OVA levelswere reported as the optical density (OD) readings, after the subtraction of theOD read-out of the mean plus 3 standard errors of the mean (SEM) of a series ofcontrol wells with normal mouse serum added, and expressed as means2SEM foreach group.

2.8. Antigen-speci®c IL-2, IL-4 and IFN-gg cytokine responses

Splenic lymphocytes in RPMI-FCS were added in 100 ml volumes containing107 cells/ml to each of the wells of 96-well round-bottom cell culture microtiterplates (Corning Glass Works, Corning, NY, USA). To stimulate the cells, 100 mgof OVA in 100 ml of RPMI-FCS was added to each well in triplicate cultures.Cells cultured in growth medium only were used as negative controls. The plateswere placed at 378C in an atmosphere containing 5% CO2 for 72 h. The spleenculture supernatants were collected and tested for IL-2, IL-4 and IFN-g cytokinesby ELISA. The wells of 96-well microtiter plates were coated with 5 mg of anti-IL-2, IL-4 and IFN-g cytokines monoclonal antibodies (PharMingen, San Diego, CA,USA) in 50 ml of PBS at 48C for 18 h. The plates were washed with PBS-T anddilutions of culture supernatants from 1:2 to 1:128 in PBS-T were added to thewells and incubated at 378C for 2 h. After washing with PBS-T, 50 ml ofbiotinylated anti-IL-2, IL-4 and IFN-g monoclonal antibodies (PharMingen),diluted 1:1000 in PBS-T, were added to the wells and incubated at 378C for 2 h.The plates were washed with PBS-T and 50 ml of streptavidin-alkaline phosphateconjugate, diluted 1:1000 in PBS-T, were added to the wells and incubated at378C for 1 h. The plates were washed with PBS-T and 100 ml of the alkalinephosphatase substrate solution added to each well. The absorbance of each well at


405 nm was measured in an automated spectrophotometer. The culturesupernatant samples were analysed individually. The IL-2, IL-4 and IFN-gamounts were calculated by standard curves using recombinant murine IL-2, IL-4and IFN-g. Data were expressed as the amount of IL-2, IL-4 and IFN-g cytokinesin culture supernatants in pg/ml and expressed as means2SEM for each group(®ve mice).

2.9. Adjuvant activity of P. senega saponins in poultry

Isobrown laying hens from the Poultry Research Unit of the University ofSaskatchewan (Saskatoon, SK, Canada) were used in the experiment. The henswere housed in individual cages. The procedures applied to the hens wereapproved by the Animal Care Committee, University of Saskatchewan, inaccordance with the requirements of the Canadian Council on Animal Care.

2.10. Immunization of hens

To examine the adjuvant activities of P. senega and Quil A saponins in poultry,groups of ten hens were injected intramuscularly in the breast muscle with killedporcine rotavirus particles containing 1 � 108 focus forming units (�u) per ml; thevirus was obtained as a gift from Animal Biotechnology and NutritionCorporation (Saskatoon, SK, Canada). One ml of rotavirus particles was mixedwith PS-1, PS-2 or Quil A saponins in 1 ml of PBS bu�er. The immunizationprotocol is indicated in Table 2.

2.11. Collection of samples

Eggs were collected on days 28, 42, 56 and 70 after the primary immunizationfor analysis of egg IgG anti-rotavirus antibodies. The eggs were cracked and thewhites and yolks were homogenized using a polytron apparatus. Homogenized egg(100 ml) were then mixed with 500 ml of citrate-phosphate bu�er pH 5.0 in a 96

Table 2

Immunization protocol of hens: Groups of ten were immunizeda

Experimental days

Group Saponins dose (HU) 0 28 42 56 70

PS-1 32 1 2, 3 3 3 3

PS-2 64 1 2, 3 3 3 3

Quil A 38.4 1 2, 3 3 3 3

Control ± 1 2, 3 3 3 3

a 1, primary immunization; 2, secondary immunization; 3, egg collection (egg IgG anti-rotavirus).


deep-well sample block and stored at ÿ208C until analysis by ELISA at the endof the experiment.

2.12. Immunological assays

Anti-rotavirus IgG levels in the egg samples were analyzed by ELISA using thefollowing method. All samples were thawed and diluted to 1:600 using citrate-phosphate bu�er pH 5.0. The samples were then centrifuged at 3000 g for 15 minto remove fat and precipitate proteins. The wells of 96-well microtiter plates were®lled with 50 ml of a 1:100 dilution of the rotavirus antigen used for vaccinepreparation and incubated at 48C for 18 h. The wells of all plates were washedthree times with PBS-T and incubated with PBS containing 1% BSA at 378C for30 min. The plates were washed with PBS-T and 50 ml of the egg samples werethen added into duplicate wells of the microtiter plates and incubated at 378C for2 h. The plates were washed with PBS-T and 50 ml of a 1:10,000 dilution of goatanti-chicken IgG horseradish peroxidase conjugate (Southern BiotechnologyAssociates) were added to the wells. After incubation at 378C for 1 h, the plateswere washed with PBS-T and 50 ml of tetramethylbenzidine (TMB) substrate wereadded to the wells and incubated at room temperature for 20 min. Theabsorbance of the wells at 650 nm was measured using an automatedspectrophotometer. Anti-rotavirus levels were reported as the OD readings, afterthe subtraction of the OD read-out of the mean plus 3 SEM of a series of controlwells with control normal egg from a non-immunized hen, and expressed asmeans2SEM for each group.

2.13. Statistical analysis

Results were expressed as means2SEM and compared by the analysis ofvariance (ANOVA) Tukey test.

Table 3

Lethality of saponins to micea

Saponin dose (HU)

Group 6.4 12.8 25.6 51.2 LD50

PS-1 0/5 0/5 2/5 5/5 30.3

PS-2 0/5 0/5 0/5 3/5 49.7

Quil A 0/5 1/5 4/5 5/5 23.3

a Results are expressed as number of deaths per group of ®ve mice within 96 h after subcutaneous

injection of saponins.


3. Results

3.1. Extraction and isolation of P. senega saponins

The root of P. senega contains a relatively high lipid content, approximately5.0% [23] and defatting was desirable prior to saponin extraction. The yield ofmethanolic extract was 34.7% of the root dry weight.

The crude methanolic extract was suspended in water and further extracted withbutanol saturated with water to separate water-soluble carbohydrates and otherpolar compounds from saponins. The yield of butanolic extract enriched insaponins was 16.8% with respect to the initial dry weight of root powder.

As illustrated in Figs. 2, 22 fractions were eluted by silica gel open columnchromatography. The analysis of all fractions by TLC revealed the presence ofsaponins (bright red band upon spraying with methanol/acetic acid/sulphuric acid/anisaldehyde reagent) [30] in fractions 17±22 only. Fractions 17±22 were thusdesignated as a mixture of isolated saponins. They accounted for 4.5% of thestarting root dry weight.

Fractions 17±22 were the only fractions that displayed hemolytic activity, whichis a typical characteristic of saponins. The HPLC pro®les of fractions 17±22 wereobtained at 315 nm, an absorption maximum of 3, 4-dimethoxycinnamic acid, achromophore that was reported to be present in the sugar side chains of P. senegavar. latifolia saponins [26]. The HPLC analysis of fractions 17 (Fig. 3a) and 18revealed the presence of four saponin peaks. Comparison of retention times of theeluted peaks suggested that both fractions were identical. Fraction 19 consisted ofnine saponin components (Fig. 3b). A total of ten saponin peaks were detected infractions 20, 21 and 22 by HPLC. Fractions 17 (PS-1) and 19 (PS-2) were selectedfor the adjuvanticity studies, since they displayed the highest hemolytic activity ofall saponin fractions tested. The hemolytic activities of PS-1 and PS-2 were 32 and64 HU per mg, respectively, whereas the hemolytic activity of Quil A saponins,used for comparison purposes, was 128 HU per mg.

3.2. Toxicity of saponins in mice

The toxic levels of PS-1 and PS-2 saponin fractions were assessed by thelethality of di�erent HU saponin doses subcutaneously injected to mice. Theresults indicated that P. senega saponins were less toxic than Quil A saponin(Table 3). LD50 doses of PS-1, and PS-2 and Quil A saponins were 30.3, 49.7 and23.3 HU, respectively (Table 3).

3.3. Adjuvant activity of P. senega saponins in mice

The immunization with OVA-saponins mixture resulted in signi®cant increases(P < 0.05±P < 0.001) of anti-OVA IgG levels at 20 days after primaryimmunization, when compared to the control group that received OVA alone. Nosigni®cant e�ect of P. senega saponins was detectable at day 10 post-


immunization. IgG anti-OVA levels induced by the higher doses of PS-1 and PS-2fractions, at day 20, were similar to those induced by Quil A (Table 4).

The serum IgG1 and IgG2a anti-OVA responses at day 20 following primaryimmunization are presented in Table 5. IgG2a levels were signi®cantly enhanced(P < 0.01) with higher doses of PS-1 (6.4 HU) and PS-2 (12.8 HU) saponins andwith Quil A (6.4 HU) as compared to the control mice immunized with OVAalone. IgG1 isotype levels showed no di�erences among treatment groups.

The e�ects of P. senega and Quil A saponins on the in vitro secretion of IL-2,IL-4 and IFN-g cytokines by spleen cells of mice in response to OVA antigen arepresented in Table 6. The IL-2 secretion was signi®cantly increased by the higherdoses of P. senega saponins (PS-1, P < 0.001; PS-2, P < 0.01) and by Quil A(P < 0.05) in comparison to the OVA in PBS control group. The IFN-g secretionwas signi®cantly augmented (P < 0.05±0.001) in all saponin-administered groupsin relation to the control group receiving no saponin. No saponin e�ect wasobserved on the induction of IL-4 cytokine.

3.4. Adjuvant activity of P. senega saponins in poultry

The trend of IgG anti-rotavirus levels in eggs of hens immunized with porcinerotavirus along with P. senega or Quil A saponins as adjuvants is shown in Fig. 4.The results indicate that PS-1, PS-2 and Quil A saponins increased the IgGresponse to rotavirus compared to the hens that were immunized with rotavirusantigen alone. The groups receiving P. senega and Quil A saponins showedsigni®cantly enhanced (PS-1, P < 0.01; PS-2, P < 0.001; Quil A, P < 0.05)antibody responses than the control (with no saponin) at day 28 after primaryimmunization. At day 42, only the groups receiving P. senega demonstrated higher(PS-1, P < 0.01; PS-2, P < 0.001) IgG anti-rotavirus levels than the controlgroup. At day 56, signi®cantly enhanced (P < 0.001) antibody levels, in relationto the control group, were observed only in the PS-2 group. By day 70 after

Table 4

Serum IgG anti-OVA antibody responses at days 10 and 20 post-immunization in mice immunized with

OVA-saponin mixturesa

Serum IgG anti-OVA (OD405) levels

Saponin groupÐdose (HU) Day 10 Day 20

PS-1±1.6 0.03520.015 0.32020.073 �

PS-1±6.4 0.02820.014 0.48720.058 ��

PS-2±3.2 0.03520.017 0.31420.103�

PS-2±12.8 0.04020.023 0.48420.131��

Quil A±6.4 0.05720.035�� 0.51720.096��

Control±0 0.00620.009 0.12920.051

a Serum samples diluted 1:100. Mean values of groups of ®ve mice2SEM.� P < 0.05, ��P < 0.01, ��P < 0.001 vs control group receiving no saponins.


primary immunization, the IgG antibody responses in the saponin-administeredgroups were no di�erent from the control group immunized with rotavirus antigenalone.

4. Discussion

Phytochemical studies on Polygala species in the 1970s revealed the presence oftriterpenoid saponins as major constituents. While saponins from Polygala senegaL. var. latifolia, cultivated in Japan, were partially chemically characterized [26±29], to the best knowledge of the authors, there have been no reports on chemicaland immunological studies on saponins from P. senega grown in the wild inSaskatchewan.

Table 5

Serum IgG1 and IgG2a anti-OVA antibody responses at day 20 post-immunization in mice immunized

with OVA-saponin mixturesa

Serum IgG1 and IgG2a anti-OVA (OD405) levels

Saponin groupÐdose (HU) IgG1 IgG2a

PS-1±1.6 0.89520.040 1.01520.726

PS-1±6.4 0.89720.146 1.90620.844��

PS-2±3.2 0.94320.048 0.78020.657

PS-2±12.8 0.96420.053 1.98420.746 ��

Quil A±6.4 0.78820.251 1.96320.828 ��

Control±0 0.74620.136 0.01220.007

a Serum samples diluted 1:10. Mean values of groups of ®ve mice2SEM.��P < 0.01 vs control group receiving no saponins.

Table 6

IL-2, IL-4 and IFN-g cytokine concentrations in spleen cells culture supernatants in response to OVA

at day 20 post-immunization in mice immunized with OVA-saponin mixturesa

Cytokine concentration (pg/ml)

Saponin groupÐdose (HU) IL-2 IL-4 IFN-g

PS-1±1.6 213821625 63238 1620021450��

PS-1±6.4 509722187�� 57221 1627021270��

PS-2±3.2 115521058 68222 1397021800�

PS-2±12.8 360821325�� 92256 1524021210��

Quil A±6.4 31362851� 58270 161402790��

Control±0 325284 16247 1042023180

a Mean values of groups of ®ve mice2SEM. �P < 0.05, ��P< 0.01, ��P < 0.001 vs control group

receiving no saponins.


An HPLC analysis of six saponin fractions (fractions 17±22) obtained from theroot of wild Saskatchewan P. senega revealed the presence of diverse saponincomponents. P. senega fractions 17 (PS-1) and 19 (PS-2) were selected for testingfor adjuvant activity because they displayed the highest hemolytic activity andcontained dominant saponin peaks. The adjuvant e�ects of P. senega saponinswere compared to those of Quil A, a mixture of Q. saponaria saponins, which iswidely used as an adjuvant [7].

Since mice are generally used in immunological research we evaluated theadjuvanticity e�ect of P. senega saponins in these animals. The poultry model as anon-mammal animal model was used to verify the adjuvant e�ect using a vaccineprepared with a viral antigen.

In mice, P. senega saponins augmented the production of speci®c anti-OVA IgG(Table 4) and of the IgG2a subclass (Table 5); the in vitro production of IL-2 andIFN-g by lymphocytes in response to OVA antigen was also enhanced (Table 6).Thus, it appears that the e�ect of P. senega saponins on the levels of IgG2a andIgG1 subclasses and IFN-g and IL-4 cytokines is consistent with the induction ofa Th1 type cellular immune response. The enhancement of IgG2a subclass andIFN-g production is associated with the stimulation of CD4+ T cells of the Th1subset [32]. The IgG2a subclass pro®le observed with P. senega saponins di�ersfrom a study that utilized a crude Q. saponaria saponin mixture [33], whichelicited a predominantly IgG1 anti-OVA response to immunization of mice.However, others using puri®ed Q. saponaria saponin as adjuvant to modelantigens also observed a preferential IgG2a antibody response [8,34] suggesting theinduction of Th1 rather than Th2 helper T-cell subsets [32].

Fig. 4. Time response of egg-yolk IgG anti-rotavirus responses following immunization at days 0 and

28 with rotavirus co-administered with PS-1, PS-2 P. senega saponins or Quil A. The control group

was immunized with rotavirus alone. The bars represent the mean values of groups of ten hens2SEM.�P < 0.05, ��P < 0.01, ��P < 0.001 vs control group.


The immunization of hens with porcine rotavirus, using P. senega saponins as

adjuvant, also demonstrated that these saponins increase speci®c IgG immune

responses in an avian model. Whether or not similar cytokine pro®les to those

observed in mice are elicited by P. senega saponins in poultry remains to be

elucidated.

Administration of an appropriate adjuvant enhances the potency of a vaccine,

while decreasing the amount of antigen and the number of injections required to

obtain protective immunity. There is a considerable interest in adjuvants and

other agents with immunostimulatory properties to elicit greater immune

responses to weak antigens such as soluble and subunit vaccines [35,36]. E�orts

are being made to develop novel adjuvants capable of potentiating humoral and

cellular immune responses. Our results show that parenterally administered

saponins from P. senega root potentiated immune responses in mice and hens in

response to OVA and rotavirus antigens, respectively, in a manner comparable to

Quil A saponins, which are recognized potent adjuvants inducing both humoral

and cell-mediated immune responses including class I-restricted cytotoxic T

lymphocytes [37], and have been extensively used in a wide variety of experimental

animal vaccines [38].

Saponins appear to be among the few adjuvants that stimulate both humoral

and cellular responses. Among a wide range of adjuvants that included muramyl

dipeptide, alum, Freund's incomplete adjuvant, Corynebacterium parvum and

squalene, only Q. saponaria saponin promoted cell-mediated immunity against a

glycoprotein of the protozoa Trypanosoma cruzi [39]. CFA adjuvant stimulates

strong humoral and cellular immune responses. However, the intense

in¯ammatory reaction at the site of inoculation and the additional immunization

to mycobacteria antigens is a severe limitation to its use as a vaccine adjuvant [1].

The use of saponins as adjuvants has been associated with toxicity, assessed by

lethality [13]. Quil A has been described to be lethal to mice at the dose of 100 mg[8], a dose that corresponds to 12.8 HU and which was still toxic to mice in this

study (Table 3). PS-1 and PS-2 saponins were devoid of toxicity at the same HU

dose as Quil A. PS-1 fraction showed toxicity at the dose of 25.6 HU, a dose that

produced no ill e�ects in mice with the administration of PS-2 fraction. As the

adjuvant activities of both fractions proved to be comparable, it appears that

there is no relationship between relative adjuvant activity and relative lethality.

The P. senega saponin doses used in the immunization studies did not appear to

produce any signs of distress or injury in the mice or hens. In mice, post-mortem

morphological examination of the subcutaneous tissue at the injection site failed

to reveal any lesions, as a consequence of saponin administration.

The results presented here show that adjuvant activity is not con®ned to Q.

saponaria saponins but is also present in saponins from P. senega. Since mixtures

of P. senega saponins were used for this study, the precise structural requirements

for adjuvanticity cannot be de®ned at present. Work on puri®cation and chemical

characterization of dominant saponin compounds from P. senega root responsible

for adjuvant activity is in progress.


Acknowledgements

This work was supported by the Saskatchewan Agriculture Development Fund,Saskatchewan Department of Agriculture and Food, Canada. The authors aregrateful to Bing Li and Shirley Hauta for their excellent technical assistance.

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isolation and evaluation of immunological adjuvant activities of saponins from polygala senega l

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