In this investigation we pursued the analysis of the adjuvant potentials of CA3 and CA4 saponins of C. alba aiming to identify if the addition of one sugar unit has any impact on the immunoprotective potential of the saponin. All mouse studies followed the guidelines set by the National Institutes of Health, USA and the

Institutional Animal Care and Use Committee approved the animal protocols (Biophysics Compound C nmr Institute-UFRJ, Brazil, protocol IMPPG-007). Samples of C. alba were collected in Nova Friburgo, Rio de Janeiro, Brazil. The botanical identification was made by Dr. Sebastião Neto, and a voucher specimen (RB395399) has been deposited in the Herbarium of the Rio de Janeiro Botanical Garden. Air-dried and powdered roots of C. alba (400 g) were extracted with ethanol. The extract was evaporated and the residue obtained (12 g) was suspended in water and successively partitioned with methylene chloride and butanol. The butanol fractions were combined, evaporated and the residue (4 g) was suspended in methanol and subjected to controlled precipitation with diethyl ether. The precipitate (2 g) was fractionated by column

chromatography (octadecylsilane, SB431542 mw 60 cm × 20 cm) using H2O with increasing proportions of methanol (0–100%) to obtain 10 fractions. TLC tests carried out with Liebermann–Bouchard and sulfuric orcinol reagents together with the observation of an abundant foam formation, allowed the identification of the saponin enriched fractions. Further purification was carried out with reversed-phase (octadecylsilane) preparative HPLC using methanol: 0.02% aqueous trifluoroacetic acid

(60:40; v/v) to obtain 48 mg of CA3 (Chiococca saponin II) and 78 mg of CA4 (Chiococca saponin I) [28]. We also collected and identified two other saponins of C. alba to be used as controls: the CA2 (18 mg) and the CA3X (10 mg) ( Fig. 1). Dichloromethane dehalogenase All saponins (CA4, CA3, CA3X and CA2) share a triterpene nucleus to which a glucuronic acid is attached at C-3 and a rhamnose and arabinose containing chain is attached at C-28 ( Fig. 1). The CA3X and CA3 have a third sugar attached 1 → 4 to the rhamnose unit. This third sugar is xylose in CA3X and apiose in CA3. The CA4 saponin has, in addition to the 1 → 4 linked apiose present in CA3, a fourth apiose unit, 1 → 3 linked to the rhamnose unit of the C-28 carbohydrate chain ( Fig. 1). The hydrophile–lipophile balance (HLB) value of the saponins was calculated theoretically by the Davies and Riedel method [30] considering their chemical structure as previously described by Borges et al. [28] and represented in Fig. 1. The value was calculated by integrating the number of each functional group composing the saponin molecule with the group unit defined by the Davies method (HLB = 7 + ∑ hydrophilic groups − ∑ lipophilic groups) [30]. Normal human red blood cell suspension (0.1 ml of 0.5%) was mixed with 0.