Adjuvant activities of saponins from the root of Polygala senega L. 1 Georgios S. Katselis, Alberto Estrada, Dennis K.J. Gorecki, and Branka Barl Abstract: Eight pure triterpenoid saponin compounds isolated from the root of Polygala senega L., a plant indigenous to the Canadian prairies, were evaluated for their immunological activity in mouse models. The specific antibody responses of the IgG 2a subclass increased significantly when isolated P. senega saponins were used as adjuvants in the immunization of mice with OVA antigen. In addition, increased IL-2 levels were observed in spleen cell cultures from P. senega sapo- nin-immunized mice after in vitro secondary antigen stimulation. The saponins were tested for their toxicity in mice by us- ing a haemolytic activity assay and found to be less toxic than Quillaja saponaria saponins that have long been used as adjuvants in vaccine formulations. This study has shown the potential of P. senega saponins to be considered as a natural source of vaccine adjuvants with biological activity equivalent to the current commercially available saponin adjuvants. Key words: senega root, Polygala senega, saponins, immunological activity, adjuvant, Quil A. Re ´sume ´: Huit saponines extraites de la racine de Polygala senega L., plante originaire des prairies ont e ´te ´e ´value ´s pour leur activite ´ immunologique comme adjuvants des vaccins chez des mode `les de souris. Les saponines de P. senega, isole ´es et utilise ´es en tant qu’adjuvants vacciniques, ont produit une augmentation pre ´fe ´rentielle du niveau des anticorps spe ´cifi- ques de la sous-classe d’IgG2a apre `s l’immunisation avec l’antige `ne de l’albumine d’œuf, et sur la stimulation in vitro avec le me ˆme antige `ne a entraı ˆne ´ de hauts niveaux de IL-2 dans les cultures de cellules de rates des souris injecte ´s avec les saponines de P. senega. Les saponines ont e ´te ´ examine ´es pour leur toxicite ´ en e ´valuant la le ´talite ´ chez les souris et se sont ave ´re ´es moins toxiques a ` la me ˆme dose que les saponines de Quillaja saponaria lesquelles ont e ´te ´ longtemps em- ploye ´es comme adjuvants dans des formulations de vaccins. Cette e ´tude a montre ´ le potentiel des saponines de P. senega comme adjuvants des vaccins pour intensifier les re ´ponses immunitaires spe ´cifiques. Mots-cle ´s : racine senega, Polygala senega, saponines, re ´ponses immunitaires, adjuvant, Quil A. Introduction Polygala senega L., known as senega root, is an indige- nous plant to the Canadian prairies traditionally used as an expectorant. The root has been used over the years by North America First Nations people as a cough remedy and to treat rattlesnake bites (Kindscher 1992; Barl et al. 1996). It gained popularity in European medicine in the early 18th century as a treatment for pleurisy and certain types of pneumonia (Briggs 1988). Presently, it is ethically harvested in the wild (wildcrafted) from Manitoba and Saskatchewan and the dry or fluid extract of the root is used, especially in Japan and parts of Europe, as an antitussive and expectorant remedy and to treat bronchitis and pharyngitis (Bradley 1992; Kenkel and Turcotte 1996). These pharmacological properties have been attributed to saponins, a mixture of nat- urally occurring triterpene glycosides that constitute up to 12% of the root (Bradley 1992). Saponins also play a major role in other pharmacological properties of senega (Yoshi- kawa et al. 1995a, 1995b; Lacaille-Dubois 2000) including immunostimulation and as adjuvants in vaccine formulations (Estrada et al. 2000). Adjuvants control the efficacy of vac- cines by enhancing immune responses to vaccine antigens and are typically used in newer vaccine formulations (So et al. 1998; Vogel 2000; Edelman 2001). Saponins are a promising source of adjuvants (Campbell 1995; Hostettmann and Marston 1995; Sun et al. 2004). Tri- terpenoid saponins extracted from the bark of the South American tree Quillaja saponaria Molina have been widely studied as adjuvants to increase immune response to a vari- ety of antigens (Dalsgaard 1974; Campbell and Peerbaye 1992). Quil A, a mixture of partially purified saponins from Q. saponaria has been used extensively in a number of ex- perimental animal vaccines as an adjuvant, but its high tox- icity still poses problems (Kensil et al. 1991; Campbell 1995). Research on the role of plant-derived adjuvant com- pounds in vaccine formulations has intensified in recent years with particular emphasis on safety. There is still a Received 16 February 2007. Published on the NRC Research Press Web site at cjpp.nrc.ca on 21 November 2007. G.S. Katselis 2 and D.K.J. Gorecki. College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, SK S7N 5C9, Canada. A. Estrada. Health Canada, Medical Devices Bureau, Ottawa, ON K1A 0K9, Canada. B. Barl. 3,4 Department of Plant Sciences, University of Saskatchewan, Saskatoon, SK S7N 5A8, Canada. 1 This article is one of a selection of papers published in this special issue (part 2 of 2) on the Safety and Efficacy of Natural Health Products. 2 Present address: Beckman Research Institute, Division of Immunology, City of Hope National Medical Center, Duarte, CA 91010, USA. 3 Corresponding author (e-mail: branka.barl@shaw.ca). 4 Present address: Barl and Associates, 18-10235, 111 Street, Edmonton, AB T5K 2V5, Canada. 1184 Can. J. Physiol. Pharmacol. 85: 1184–1194 (2007) doi:10.1139/Y07-109 # 2007 NRC Canada