Phytochemistry Letters 6 (2013) 633–639

A new complex triterpenoid saponin from Calliandra pulcherrima withhaemolytic activity and adjuvant effect

Bernadete Pereira da Silva, Jose Paz Parente *

Laboratorio de Quımica de Plantas Medicinais, Nucleo de Pesquisas de Produtos Naturais, Centro de Ciencias da Saude, Universidade Federal do Rio de Janeiro,

P.O. Box 68045, CEP 21941-971 Rio de Janeiro, Brazil

A R T I C L E I N F O

Article history:

Received 30 March 2013

Received in revised form 2 August 2013

Accepted 13 August 2013

Available online 3 September 2013

Keywords:

Calliandra pulcherrima

Leguminosae

Complex triterpenoid saponin

Haemolytic activity

Adjuvant effect

A B S T R A C T

A new complex triterpenoid saponin was isolated from the leaves of Calliandra pulcherrima by using

chromatographic methods. On the basis of chemical evidence, spectroscopic analyses and comparison of

known compounds its structure was established as 3-[(O-a-L-arabinopyranosyl-(1 ! 2)-O-a-L-

arabinopyranosyl-(1 ! 6)-2-(acetylamino)-2-deoxy-b-D-glucopyranosyl)oxy]-(3b)-olean-12-en-28-

oic acid O-b-D-xylopyranosyl-(1 ! 3)-O-b-D-xylopyranosyl-(1 ! 4)-O-[(b-D-glucopyranosyl-(1 ! 3)]-

O-6-deoxy-a-L-mannopyranosyl-(1 ! 2)-6-O-[(2E,6S)-6-[[2-O-[(2E,6S)-6-[[6-deoxy-2-O-[(2E,6S)-2,6-

dimethyl-1-oxo-6-(b-D-xylopyranosyloxy)-2,7-octadienyl]-b-D-glucopyranosyl]oxy]-2,6-dimethyl-1-

oxo-2,7-octadienyl]-b-D-xylopyranosyl]oxy]-2,6-dimethyl-1-oxo-2,7-octadienyl]-b-D-glucopyranosyl

ester (1). The haemolytic activity of the saponin was evaluated using in vitro assays, and its adjuvant

potential on the cellular immune response against ovalbumin antigen was investigated using in vivo

models

� 2013 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

Contents lists available at ScienceDirect

Phytochemistry Letters

jo u rn al h om ep ag e: ww w.els evier .c o m/lo c ate /p hyt ol

1. Introduction

Calliandra pulcherrima Benth. (Leguminosae) is native to Braziland it has been thoroughly dispersed throughout the TropicalAmerica. This evergreen plant is non-invasive, but a widespreadornamental plant often cultivated in gardens and parks (Lorenziand Souza, 1995). In Brazil, the aqueous extract of the branches ofthis plant is used as a remedy for malaria and leishmaniasis(Miliken, 1997). In a previous research, a complex triterpenoidsaponin named as pulcherrimasaponin with adjuvant activity wasisolated and identified (da Silva et al., 2005).

According to the literature, complex triterpenoid saponins areshown to possess several physiological properties depending ontheir chemical structures, such as haemolytic activity and capacityfor alteration of membrane permeability (Oda et al., 2000).Additionally, these compounds have been reported to possesstherapeutic potential for immune system modulation throughdifferent mechanisms (Lacaille-Dubois and Wagner, 1996). As partof our ongoing efforts in discovering potential bioactive com-pounds from natural sources, we describe the structural elucida-tion and evaluation of the haemolytic activity and immunological

* Corresponding author. Tel.: +55 21 2562 6791; fax: +55 21 2562 6513.

E-mail addresses: parente@pq.cnpq.br, parente@nppn.ufrj.br (J.P. Parente).

1874-3900/$ – see front matter � 2013 Phytochemical Society of Europe. Published by

http://dx.doi.org/10.1016/j.phytol.2013.08.009

adjuvant effect of a new complex triterpenoid saponin isolatedfrom the leaves of C. pulcherrima.

2. Results and discussion

The MeOH extract of C. pulcherrima was suspended in H2O andpartitioned with n-BuOH. The n-BuOH extract was subjected tochromatographic purification steps to afford compound 1, acolourless amorphous powder, which was positive to Lieber-mann–Burchard test. It revealed a quasi-molecular weight ionpeak at m/z 2590.9180 [M+Na]+ in the positive-ion mode MALDI-TOFMS. In the 13C NMR spectrum, one hundred twenty-two carbonsignals observed belong to sixteen methyl groups, twenty-eightmethylene groups (nine of which were oxygenated), sixty methinegroups (fifty of which were oxygenated) and eighteen quaternarycarbon atoms (five of which were oxygenated). The number ofhydrogen atoms attached to each individual carbon atom wascalculated by DEPT-45, DEPT-90 and DEPT-135 spectra. On thebasis of the above mentioned MS and 13C NMR spectral data(Table 1), compound 1 was assumed to be a triterpenoid saponinwith the molecular formula C122H191O56, bearing three mono-terpenes and eleven monosaccharide moieties. In addition to this,the IR spectrum showed an absorption at 1647 cm�1 which istypical of an a,b-unsaturated carbonyl group, supported by UVabsorption at 220 nm.

Elsevier B.V. All rights reserved.

Table 113C NMR data of compound 1 (75 MHz, pyridine-d5).

Position dC (DEPTa) Position dC (DEPTa) Position dC (DEPTa)

1 38.5 (CH2) a-L-AraII-1 105.7 (CH) b-D-XylIII-1 97.3 (CH)

2 26.2 (CH2) 2 74.4 (CH) 2 74.7 (CH)

88.7 (CH) 3 76.8 (CH) 3 75.4 (CH)

4 38.9 (C) 4 70.0 (CH) 4 70.5 (CH)

5 55.6 (CH) 5 66.3 (CH2) 5 66.1 (CH2)

6 18.4 (CH2) b-D-GlcI-1 94.2 (CH) MTII-1 166.8 (C)

7 32.8 (CH2) 2 77.7 (CH) 2 127.7 (C)

8 39.7 (C) 3 76.9 (CH) 3 143.0 (CH)

9 47.8 (CH) 4 70.7 (CH) 4 23.2 (CH2)

10 36.7 (C) 5 75.1 (CH) 5 40.6 (CH2)

11 23.6 (CH2) 6 63.8 (CH2) 6 79.1 (C)

12 122.4 (CH) a-L-Rha-1 101.4 (CH) 7 143.2 (CH)

13 143.7 (C) 2 69.8 (CH) 8 114.5 (CH2)

14 41.7 (C) 82.8 (CH) 9 12.0 (CH3)

15 27.9 (CH2) 4 77.7 (CH) 10 23.3 (CH3)

16 23.3 (CH2) 5 68.4 (CH) b-D-Qui-1 96.3 (CH)

17 46.8 (C) 6 18.2 (CH3) 2 74.9 (CH)

18 41.5 (CH) b-D-GlcII-1 104.3 (CH) 3 75.1 (CH)

19 46.3 (CH2) 2 74.6 (CH) 4 76.2 (CH)

20 30.3 (C) 3 77.3 (CH) 5 72.1 (CH)

21 33.7 (CH2) 4 70.8 (CH) 6 18.0 (CH3)

22 31.8 (CH2) 5 77.3 (CH) MTIII-1 167.3 (C)

23 27.7 (CH3) 6 61.9 (CH2) 2 127.9 (C)

24 16.7 (CH3) b-D-XylI-1 103.7 (CH) 143.1 (CH)

25 15.3 (CH3) 2 73.8 (CH) 4 23.3 (CH2)

26 17.0 (CH3) 3 87.3 (CH) 5 40.3 (CH2)

27 25.5 (CH3) 4 68.8 (CH) 6 79.5 (C)

28 175.9 (C) 5 65.6 (CH2) 7 143.4 (CH)

29 32.9 (CH3) b-D-XylII-1 105.1 (CH) 8 115.0 (CH2)

30 23.6 (CH3) 2 74.4 (CH) 9 12.4 (CH3)

b-D-GlcNAc-1 104.0 (CH) 3 76.7 (CH) 10 23.5 (CH3)

2 57.0 (CH) 4 69.9 (CH) b-D-XylIV-1 99.7 (CH)

3 74.6 (CH) 5 66.2 (CH2) 2 74.6 (CH)

4 71.8 (CH) MTI-1 167.7 (C) 3 77.8 (CH)

5 75.2 (CH) 2 127.4 (C) 4 70.6 (CH)

6 68.8 (CH2) 3 142.5 (CH) 5 66.3 (CH2)

NHCOCH3 170.8 (C) 4 23.5 (CH2)

NHCOCH3 22.9 (CH3) 5 40.4 (CH2)

a-L-AraI-1 101.7 (CH) 6 79.2 (C)

2 79.5 (CH) 7 142.0 (CH)

3 71.7 (CH) 8 115.0 (CH2)

4 68.8 (CH) 9 11.9 (CH3)

5 63.7 (CH2) 10 23.5 (CH3)

a Multiplicities were assigned from DEPT-45, DEPT-90 and DEPT-135 spectra.

B.P. Silva, J.P. Parente / Phytochemistry Letters 6 (2013) 633–639634

On acid hydrolysis, compound 1 gave a sapogenin 1a, arabinose,glucose, rhamnose, xylose, quinovose and 2-amino-2-deoxy-glucose as the component sugars. The structure of 1a (Fig. 1)was established as 3-hydroxy-(3b)-olean-12-en-28-oic acid (olea-nolic acid) (Halsall and Aplin, 1964) by comparing its physicalproperties ([a]D and m.p.), and 1H and 13C NMR spectra with thoseof an authentic sample. Analysis of the sugars by GC/MS indicatedthe presence of arabinose, glucose, rhamnose, xylose, quinovoseand 2-amino-2-deoxy-glucose in a ratio of 2:2:1:4:1:1, respec-tively (Kamerling et al., 1975). Their absolute configurations weredetermined by GC of their trimethylsilylated (�)-2-butylglyco-sides (Gerwig et al., 1978). L-Arabinose, D-glucose, L-rhamnose, D-xylose, D-quinovose and 2-amino-2-deoxy-D-glucose weredetected. The 1H NMR spectrum of compound 1 displayed elevenanomeric hydrogen atoms at d 5.84 (d, J = 7.3 Hz), 5.71 (brs), 5.28(d, J = 7.3 Hz), 5.18 (d, J = 7.3 Hz), 4.99 (d, J = 7.3 Hz), 4.93 (d,J = 5.5 Hz), 4.91 (d, J = 7.4 Hz), 4.81 (d, J = 6.7 Hz), 4.78 (d,J = 8.0 Hz), 4.76 (d, J = 8.6 Hz), 4.66 (d, J = 7.9 Hz), which gavecorrelations in the HSQC spectrum with eleven anomeric carbonatoms at d 94.2, 101.4, 103.7, 104.3, 105.1, 101.7, 104.0, 105.7, 96.3,97.3, 99.7, respectively. Evaluation of chemical shifts andspin–spin couplings allowed the identification of one a-rhamno-pyranosyl unit (a-Rha), one b-quinovopyranosyl unit (b-Qui), one2-(acetylamino)-2-deoxy-b-glucopyranosyl unit (b-GlcNAc), two

a-arabinopyranosyl units (a-AraI and a-AraII), two b-glucopyr-anosyl units (b-GlcI and b-GlcII) and four b-xylopyranosyl units(b-XylI, b-XylII, b-XylIII and b-XylIV). The attachments of thesugar moieties to the aglycone moiety were established by 1H–1HCOSY and HMBC experiments. The COSY spectrum was useful toestablish couplings and determine the connectivity information incarbohydrate sequences. The HMBC spectrum displayed longrange couplings between GlcNAc-H-1 at d 4.91 and triterpenoid-C-3 at d 88.7, between GlcI-H-1 at d 5.84 and triterpenoid C-28 at d175.9, which accounted for two saccharide part linkages to the C-3b-OH and C-28 COOH groups of oleanolic acid. In addition to this,long range couplings were observed between AraII-H-1 at d 4.81and AraI-C-2 at d 79.5, between AraI-H-1 at d 4.93 and GlcNAc-C-6at d 68.8, between XylII-H-1 at d 4.99 and XylI-C-3 at d 87.3,between XylI-H-1 at d 5.28 and Rha-C-4 at d 77.7, between GlcII-H-1 at d 5.18 and Rha-C-3 at d 82.8, between Rha-H-1 at d 5.71 andGlcI-C-2 at d 77.7, between XylIV-H-1 at d 4.66 and MTIII-C-6 at d79.5, between Qui-H-2 at d 5.45 and MTIII-C-1 at d 167.3,between Qui-H-1 at d 4.78 and MTII-C-6 at d 79.1, between XylIII-H-2 at d 5.46 and MTII-C-1 at d 166.8, betweenXylIII-H-1 at d 4.76and MTI-C-6 at d79.2 and between GlcI-H-6 at d 4.59, 4.80 andMTI-C-1 at d 167.7, which accounted for the elucidation ofcompound 1 (Fig. 1). The NMR signals of compound 1 wereassigned by 2D NMR experiments including COSY, HSQC and

Fig. 1. Chemical structures of compounds 1, 1a, 1b and 1c.

B.P. Silva, J.P. Parente / Phytochemistry Letters 6 (2013) 633–639 635

HMBC and by comparing the NMR data of 1 (Table 1 and Exper.Part) with those reported in the literature (Takeda et al., 1993;Nakamura et al., 1994; Tani et al., 1996, 1998; da Silva et al.,2005; Barbosa et al., 2008).

On alkaline hydrolysis compound 1 afforded compounds 1band 1c. By comparing 1H and 13C NMR and MS spectral data ofcompounds 1b and 1c with those reported in the literature(Takeda et al., 1993; Kiuchi et al., 1977), 1b was identified as 6-(6-deoxy-b-D-glucopyranosyloxy)-2,6-dimethyl-[S-(E)]-2,7-octadienoicacid (Fig. 1) and 1c was identified as 6-(b-D-xylopyranosyloxy)-2,6-dimethyl-[S-(E)]-2,7-octadienoic acid. The stereochemistryat C-6 of 1b and 1c was assigned to be S by comparing theiroptical activities, [a]D

25 _188 (c 0.3, MeOH) and [a]D25�268 (c 0.7,

MeOH), respectively, with those reported in the literature (Kiuchiet al., 1997).

The sequence of sugar chain of compound 1 was confirmedby methylation analysis (Parente et al., 1985) which furnished1,5-di-O-acetyl-2,3,4-tri-O-methyl xylitol, 1,5-di-O-acetyl-2,3,4-tri-O-methyl arabinitol, 1,5-di-O-acetyl-2,3,4,6-tetra-O-methylglucitol, 1,2,5-tri-O-acetyl-3,4-di-O-methyl xylitol, 1,3,5-tri-O-acetyl-2,4-di-O-methyl xylitol, 1,2,5-tri-O-acetyl-3,4-di-O-methyl arabinitol, 1,2,5-tri-O-acetyl-3,4-di-O-methyl quinovitol,1,5,6-tri-O-acetyl-2-deoxy-N,N-dimethyl-3,4-di-O-methyl glu-cosaminitol, 1,3,4,5-tetra-O-acetyl-2-O-methyl rhamnitol, and1,2,5,6-tetra-O-acetyl-3,4-di-O-methyl glucitol. Consequently,on the basis of the results described above, the structure ofcompound 1 was established as 3-[(O-a-L-arabinopyranosyl-(1 ! 2)-O-a-L-arabinopyranosyl-(1 ! 6)-2-(acetylamino)-2-de-oxy-b-D-glucopyranosyl)oxy]-(3b)-olean-12-en-28-oic acid

O-b-D-xylopyranosyl-(1 ! 3)-O-b-D-xylopyranosyl-(1 ! 4)-O-[(b-D-glucopyranosyl-(1 ! 3)]-O-6-deoxy-a-L-mannopyranosyl-(1 ! 2)-6-O-[(2E,6S)-6-[[2-O-[(2E,6S)-6-[[6-deoxy-2-O-[(2E,6S)-2,6-dimethyl-1-oxo-6-(b-D-xylopyranosyloxy)-2,7-octadienyl]-b-D-glucopyranosyl]oxy]-2,6-dimethyl-1-oxo-2,7-octadienyl]-b-D-xylopyranosyl]oxy]-2,6-dimethyl-1-oxo-2,7-octadienyl]-b-D-glucopyranosyl ester.

According to the literature, complex triterpenoid saponins areshown to possess several physiological properties, mainly thecapacity for alteration of membrane permeability. Additionally,these compounds have been reported to possess therapeuticpotential for immune system modulation through differentmechanisms (Lacaille-Dubois and Wagner, 1996). Because theoriginal observation that certain saponins cause substantialenhancement of immune responses when given together with anantigen in a vaccine, their use as adjuvants received specialattention (Sun et al., 2009). However, this activity is sometimesaccomplished by an undesirable haemolytic effect (Oda et al.,2000). Generally, these compounds possess powerful haemolyticactivity due to its higher affinities for cholesterol on erythrocytemembranes, as a consequence of its amphipathic structurecontaining a hydrophobic nucleus and a hydrophilic carbohy-drate moiety (Oda et al., 2000).

In order to determine the safety and tolerability of compound 1,the haemolytic activity was evaluated (Fig. 2) and a toxicity assaywas performed in comparison with an adjuvant commonly used inanimal and human experimental models, the purified saponin QS-21obtained from commercial extracts of Quillaja saponaria (Santoset al., 1997). In spite of the fact that both substances possess a

Fig. 2. Haemolytic activity (mg/mL) of compound 1 and commercial adjuvants

commonly used in animal and human experimental models. The adjuvant

concentration inducing 50% of the maximal haemolysis was considered the

median haemolytic dose (HD50; graphical interpolation). Each experiment included

triplicates at each concentration. Results are mean � S.E.M. (n = 10); *p < 0.05,

**p < 0.01 significantly different from the control.

Fig. 3. Immunological adjuvant activity of compound 1 and commercial adjuvants on the

responses after two subcutaneous immunizations with 100 mg of ovalbumin and 100 mg

Results are mean � S.E.M. (n = 5); *p < 0.05, **p < 0.01 significantly different from the salin

Adjuvant; FIA: Freund’s Incomplete Adjuvant; CQS: Quillaja saponaria saponin control; CC

B.P. Silva, J.P. Parente / Phytochemistry Letters 6 (2013) 633–639636

powerful haemolytic potential, no lethality was detected in thetoxicity assay after the complete treatment. Only local pain, swellingand loss of hair at the injection site were observed after eachinjection, with complete spontaneous recovery in both cases.

In addition, the immunological property of compound 1 wasinvestigated and its adjuvant potential on the cellular immuneresponse against ovalbumin (OVA) antigen was evaluated (Fig. 3).Delayed type hypersensitivity (DTH) reaction was measured as anin vivo assay of cellular immune response. This type of immunity istypically elicited by soluble protein antigens that are introducedwith adjuvants. In the classical experimental model, the animal isfirst immunized by the administration of the antigen conjugatedwith adjuvants, which is called sensitization. After, in theelicitation stage, the animal is challenged subcutaneously withthe same antigen and the subsequent reaction is analyzed. In thisstudy, mice immunized with OVA conjugated with compound 1showed response greater than those when the antigen wascombined with commercial adjuvants. This response developedrapidly after immunization and persisted at high levels for at leastthree days (Mowat et al., 1991).

When the animals were immunized only with the antigen orthe respective adjuvants without conjugation, the cellular immuneresponse was practically abolished, discarding the endotoxincontamination in the experiment. Additionally, a possible contri-bution of proinflammatory properties of the adjuvants is unlikely,since these compounds were not capable to induce an effectiveimmune response when administered alone, which implies the

cellular immune response against ovalbumin antigen. Delayed type hypersensitivity

of each adjuvant. Control animals received only ovalbumin or saponins, as indicated.

e control. Student’s t-test. Abbreviations: SAL: saline solution; FCA: Freund’s Complete

P: Calliandra pulcherrima saponin control.

B.P. Silva, J.P. Parente / Phytochemistry Letters 6 (2013) 633–639 637

necessity of the antigen conjugation to elicit an immunologicalreaction in the sensitization stage. In conclusion, the results of thetoxicity analysis and the effects on immune response demonstrat-ed that compound 1 formulation showed a safety and significantadjuvant activity against ovalbumin antigen.

The structural similarities between compound 1 and otherbioactive complex triterpenoid saponins isolated from medicinalplants may help to explain its immunological activity (Lacaille-Dubois and Wagner, 1996). For example, the sugar side chain at C-28 may be responsible for the activation of the cellular immuneresponse, since this residue is shared by several adjuvant saponinsand its removal by hydrolysis reactions abolished this activity,indicating that the integrity of the carbohydrate moiety attached atthat position is mandatory for these functions (Sun et al., 2009).Moreover, it was proved that the remarkable property of Quillaja

saponaria to stimulate lymphocyte proliferation appears to dependon their lipophilic acylated moiety in molecular structure, sincethis property was significantly diminished after the removal of themonoterpene units, which implies that these residues play apivotal role in the adjuvant activity (Marciani, 2003). Specially, theoverall conformation harmoniously constructed by both hydro-philic and hydrophobic functional groups, rather than eachindividual functional group itself, is the most essential elementfor the consideration of adjuvant activity (Sun et al., 2009).Echinocystic acid (OH at C-16) and oleanolic acid (1a) are thesapogenins of pulcherrimasaponin (da Silva et al., 2005) andcompound 1, respectively. Relatively little is the differencebetween both compounds, but sufficiently crucial to show acritical sapogenin structure required for adjuvant function.Compound 1 is the first complex triterpenoid saponin containingin its structure monoterpenoid moieties and oleanolic acid assapogenin with immunoadjuvant activity against ovalbuminantigen, although some oleanane-type saponins with slighthaemolytic and significant adjuvant effects are related (Sunet al., 2009). In conclusion, the investigation of the biologicalproperties of compound 1 indicated that this substance may be thepotential therapeutic agent involved in the immunomodulatoryactivity, justifying the use of C. pulcherrima in the traditionalmedicine.

3. Experimental

3.1. General experimental procedures

Carbohydrate content was analyzed by gas-chromatography-electron impact mass spectrometry (GC-EIMS) of the alditolacetates (Sawardeker et al., 1965). The experimental data weretested for statistical differences using the Student’s t-test. Meltingpoints were determined by an Electrothermal 9200 micro-melting point apparatus and are uncorrected. The opticalrotations were measured on a Perkin Elmer 243B polarimeter.IR spectra were measured on a Perkin Elmer FT-IR 1600spectrometer. 1H and 13C NMR, DEPT, COSY, HSQC and HMBCexperiments were performed in deuterated pyridine on aMercury-300 NMR spectrometer (300 MHz for dH and 75 MHzfor dC). All chemical shifts (d) are given in ppm units with referenceto tetramethylsilane (TMS) as the internal standard and thecoupling constants (J) are in Hz. Gas chromatography (GC) wascarried out with flame ionization detector (FID), using a glasscapillary column WCOT SE-30 (0.31 mm � 25 m; 0.25 mm filmthickness) using the following temperature programme forsplitless injection mode: 60–250 8C (5 8C/min), and the detectortemperature at 270 8C. GC-EIMS were taken on a VG Auto SpecQspectrometer operating at 70 eV. The MALDI-TOFMS was obtainedusing a Perseptive Voyager RP mass spectrometer. Silica gelcolumns (230–400 mesh ASTM, Merck) and Sephadex LH-20

(Pharmacia) were used for column chromatography (CC). Thin-layer chromatography (TLC) was performed on silica gel coatedplates (Merck) using the following solvent systems: (A) CHCl3-MeOH-H2O (55: 45: 5, v/v/v) for triterpenoid saponin 1, (B) CHCl3-MeOH (96: 4, v/v) for sapogenin 1a, (C) CHCl3–MeOH (5: 1, v/v) formonoterpene glycosides 1b and 1c and (D) n-BuOH-acetone-H2O(4: 5: 1, v/v/v) for monosaccharides. Spray reagents were orcinol/H2SO4 for compounds 1, 1b, 1c and monosaccharides, and CeSO4

for compound 1a.

3.2. Plant material

Fresh leaves of C. pulcherrima Benth. were collected from theBotanical Garden of the Federal University of Rio de Janeiro (Rio deJaneiro, Brazil) in May 2012.

3.3. Extraction and isolation

Fresh leaves of C. pulcherrima (1 kg) were extracted with MeOH(4 L) for 72 h at r. t. and the extract was concentrated underreduced pressure. The residue (33.4 g) was suspended in water(500 mL), the suspension was extracted with n-BuOH (500 mL).The resulting organic phase was evaporated in vacuo to give acrude material (12.7 g). This residue was dissolved in MeOH(400 mL) and EtOAc (2 L) was added to the MeOH solution to give aprecipitate. After setting for 72 h at r. t., the supernatant wasdecanted off. The precipitate was suspended in MeOH (200 mL)and concentrated in vacuo to give a dry residue (3.8 g). It wasdissolved in MeOH (30 mL) and chromatographed by columnchromatography over Sephadex LH-20 (3.8 cm � 67 cm) usingMeOH as eluent to yield 50 fractions (23 mL each one). Thefractions containing saponin (18–20) were evaporated in vacuo togive a residue (693 mg). It was further purified by columnchromatography over silica gel (2.8 cm � 95 cm), using CHCl3–MeOH–H2O (55:45:5, v/v/v) as eluent afforded 25 fractions (23 mLeach one). The fractions 14–16 (243 mg) less polar thanpulcherrimasaponin (da Silva et al., 2005) were chromatographed(three times) to give compound 1 (83 mg; Rf 0.52). The QS-21saponin was isolated from Riedel De Haen, saponin pure1 (8047-15-2) EINECE (West Germany) according to da Silva et al. (2005).

3.4. Compound 1

3-[(O-a-L-arabinopyranosyl-(1 ! 2)-O-a-L-arabinopyranosyl-(1 ! 6)-2-(acetylamino)-2-deoxy-b-D-glucopyranosyl)oxy]-(3b)-olean-12-en-28-oic acid O-b-D-xylopyranosyl-(1 ! 3)-O-b-D-xylopyranosyl-(1 ! 4)-O-[(b-D-glucopyranosyl-(1 ! 3)]-O-6-de-oxy-a-L-mannopyranosyl-(1 ! 2)-6-O-[(2E,6S)-6-[[2-O-[(2E,6S)-6-[[6-deoxy-2-O-[(2E,6S)-2,6-dimethyl-1-oxo-6-(b-D-xylopyra-nosyloxy)-2,7-octadienyl]-b-D-glucopyranosyl]oxy]-2,6-dimeth-yl-1-oxo-2,7-octadienyl]-b-D-xylopyranosyl]oxy]-2,6-dimethyl-1-oxo-2,7-octadienyl]-b-D-glucopyranosyl ester (1). Colourlessamorphous powder (83 mg); m.p. 234–240 8C (dec.); [a]D

25 �32(c 0.3, MeOH); UV lmax (nm): 220; IR (KBr) nmax cm�1: 3420 (OH),2931 (CH), 1708 (C55O), 1647 (C55O), 1377, 1281, 1077, 640; 1HNMR (C5D5N, 300 MHz) d 5.84 (1H, d, J = 7.3 Hz, GlcI-H-1), 5.71(1H, brs, Rha-H-1), 5.46 (1H, t, J = 7.3 Hz, XylIII-H-2), 5.45 (1H, t,J = 8.0 Hz, Qui-H-2), 5.28 (1H, d, J = 7.3 Hz, XylI-H-1), 5.18 (1H, d,J = 7.3 Hz, GlcII-H-1), 4.99 (1H, d, J = 7.3 Hz, XylII-H-1), 4.93 (1H, d,J = 5.5 Hz, AraI-H-1), 4.91 (1H, d, J = 7.4 Hz, GlcNAc-H-1), 4.81 (1H,d, J = 6.7 Hz, AraII-H-1), 4.80 (1H, m, GlcI-H-6b), 4.78 (1H, d,J = 8.0 Hz, Qui-H-1), 4.76 (1H, d, J = 8.6 Hz, XylIII-H-1), 4.66 (1H, d,J = 7.9 Hz, XylIV-H-1), 4.59 (1H, m, GlcI-H-6a), 2.10 (3H, s, NHCO-Me), 1.87 (6H, s, MTII-Me-9 and MTIII-Me-9), 1.82 (3H, s, MTI-Me-9), 1.67 (3H, s, Me-27), 1.56 (3H, d, J = 6.1 Hz, Rha-Me-6), 1.47(3H, d, J = 6.1 Hz, Qui-Me-6), 1.42 (3H, s, MTII-Me-10), 1.40 (6H, s,

B.P. Silva, J.P. Parente / Phytochemistry Letters 6 (2013) 633–639638

MTI-Me-10 and MTIII-Me-10), 1.11 (3H, s, Me-23), 1.01 (3H, s, Me-30), 0.98 (3H, s, Me-26), 0.90 (6H, s, Me-24 and Me-29), 0.84 (3H, s,Me-25). 13C NMR (C5D5N, 75 MHz): see Table 1. MALDI-TOFMSm/z: 2590.9180 [M+Na]+ (calcd for C122H191NNaO56

+, 2590.8454).

3.5. Acid hydrolysis of compound 1

Compound 1 (25 mg) was dissolved in 2 N H2SO4 (5 mL) in asealed tube at 100 8C during 6 h. After dilution with water (5 mL),the reaction mixture was extracted with diethyl ether. The etherlayer was evaporated to dryness. The residue was recrystallizedfrom EtOH to give the sapogenin (1a, 3 mg) as colourless prisms,m.p. 306–308 8C, [a]D

25 +808 (c 0.1, MeOH). The water layer waspassed through an Amberlite IRA-410 column. The eluate wasconcentrated to give a residue containing the monosaccharidemixture (12 mg). A sample of the monosaccharide mixture (1 mg)was dissolved in pyridine (100 mL) and analyzed by TLC andcompared with standards of sugars.

3.6. Alkaline hydrolysis of compound 1

Compound 1 (30 mg) was hydrolyzed with 1 N KOH (3 mL) atr.t. for 24 h. The reaction mixture was acidified with 10% HCl andextracted with EtOAc. The EtOAc solution was washed with H2Oand evaporated to dryness. The residue (8.3 mg) was chromato-graphed on a silica gel column (1 cm � 30 cm) eluted with CHCl3–MeOH (5:1, v/v) to afford 1b (1.8 mg) as a colourless syrup, [a]D

25

�188 (c 0.3, MeOH) and 1c (4.1 mg) as a colourless syrup, [a]D25

�268 (c 0.7, MeOH).

3.7. Molar carbohydrate composition and D,L configurations

The molar carbohydrate composition of compound 1 (1 mg)was determined by GC–MS analyses of their monosaccharides astheir trimethylsilylated methylglycosides obtained after metha-nolysis (0.5 M HCl in MeOH, 24 h, 80 8C) and trimethylsilylation(Kamerling et al., 1975). The configurations of the glycosides wereestablished by capillary GC and GC-EIMS of their trimethylsilylated(�)-2-butylglycosides (Gerwig et al., 1978).

3.8. Methylation analysis

Compound 1 (1 mg) was dissolved in dimethylsulfoxide(200 mL) in a Teflon-lined screw-cap tube. Lithium methylsulfinylcarbanion (200 mL) was added to the solution under an inertatmosphere and the mixture was sonicated for 60 min. Aftercooling to �4 8C, cold methyl iodide (400 mL) was added.Sonication was conducted in a sonication bath (20 8C) for45 min. The methylation was terminated by addition of water(4 mL) containing sodium thiosulfate, and the permethylatedproduct extracted with chloroform (3 � 2 mL) and evaporated(Parente et al., 1985). The methyl ethers were obtained afterhydrolysis (4N TFA, 2 h, 100 8C) and analyzed as alditol acetates byGC-EIMS (Sawardeker et al., 1965).

3.9. Haemolytic activity

Normal human red blood cell suspension (0.5 mL of 0.5%) wasmixed with 0.5 mL of diluent containing 5, 10, 20, 30, 40, 50, 100,250 and 500 mg/mL of compound 1, Al(OH)3, purified Quillaja

saponaria saponin (QS-21), and 5–500 mg/mL of Freund’s CompleteAdjuvant (FCA) and Freund’s Incomplete Adjuvant (FIA) in salinesolution. Mixtures were incubated for 30 min at 37 8C andcentrifuged at 70 � g for 10 min. The free haemoglobin in thesupernatant was measured by absorbance at 412 nm. Saline anddistilled water were included as minimal and maximal haemolytic

controls, respectively. The haemolytic percents developed by thesaline control were subtracted from all groups. The adjuvantconcentration inducing 50% of the maximal haemolysis wasconsidered the median haemolytic dose (HD50; graphical interpo-lation). Each experiment included triplicates at each concentration(Santos et al., 1997).

3.10. Immunological adjuvant activity

Male Swiss mice (three months old) were subcutaneouslyimmunized twice at weekly intervals with 100 mg ovalbuminantigen (OVA) dissolved in 100 mL sterile saline (SAL) as the controlgroup or with 100 mg of OVA conjugated with 100 mg of compound1 or Freund’s Complete Adjuvant (FCA) or Freund’s IncompleteAdjuvant (FIA) as the positive control groups, each one dissolved in100 mL of saline as vehicle. A reference compound, the commercialpurified Quillaja saponaria saponin (QS-21) 100 mg was conjugatedwith 100 mg of the antigen (OVA) and dissolved in 100 mL of saline,for comparison. Compound 1 (100 mg) or QS-21 (100 mg) dissolvedin saline without the antigen were included as negative controls.The delayed-type hypersensitivity (DTH) responses were assessedby measuring the increment in the right footpad thickness aftersubcutaneous challenge with 100 mg OVA in 100 mL saline a weekafter the second immunization. The footpad thickness wasmeasured with a spring-loaded dial gauge (Mitutoyo Corp., Tokyo,Japan) before and 24, 48 and 72 h after injection. Injecting eachanimal with 100 mL saline in the left hind footpad served ascontrol. The ovalbumin specific responses were obtained bysubtracting the response to OVA challenge in unimmunizedcontrol mice (Mowat et al., 1991).

3.11. Toxicity assays

Toxicity (assessed by lethality, local pain in response toinjection, local swelling, loss of hair and skin lesion) was testedin swiss mice. Aliquots of 200 mg of compound 1 or QS-21dissolved in 100 mL sterile saline were injected subcutaneously onthe back of the mice (n = 5), as three doses at weekly intervals. Themice were monitored during seven days after the last dose. Sterilesaline solution treated animals were included as control group.

Acknowledgements

This work was financially supported by FINEP, CAPES and CNPq.

References

Barbosa, A.P., da Silva, B.P., Parente, J.P., 2008. Brevifoliasaponin with adjuvantactivity from Calliandra brevifolia. Z. Naturforsch. 63b, 894–902.

da Silva, B.P., Soares, J.B.R.C., de Souza, E.P., Palatnik, M., de Sousa, C.B.P., Parente, J.P.,2005. Pulcherrimasaponin, from the leaves of Calliandra pulcherrima, as adju-vant for immunization in the murine model of visceral leishmaniasis. Vaccine23, 1061–1071.

Gerwig, G.J., Kamerling, J.P., Vliegenthart, J.F.G., 1978. Determination of the D and L

configuration of neutral monosaccharides by high-resolution capillary G.L.C.Carbohyd. Res. 62, 349–357.

Halsall, T.G., Aplin, R.T., 1964. A pattern of development in the chemistry ofpentacyclic triterpenes. Fortschritte d. Chem. org. Naturst. 32, 153–202.

Kamerling, J.P., Gerwig, G.J., Vliegenthart, J.F.G., Clamp, J.R., 1975. Characterizationby gas–liquid chromatography–mass spectrometry and proton-magnetic-res-onance spectroscopy of pertrimethylsilyl methyl glycosides obtained in themethanolysis of glycoproteins and glycopeptides. Biochem. J. 151, 491–495.

Kiuchi, F., Gafur, M.A., Obata, T., Tachibana, A., Tsuda, Y., 1977. Acacia concinasaponins. II. Structures of monoterpenoid glycosides in the alkaline hydrolysateof the saponin fraction. Chem. Pharmaceut. Bull. 45, 807–812.

Lacaille-Dubois, M.A., Wagner, H., 1996. A review of the biological and pharmaco-logical activities of saponins. Phytomedicine 2, 363–383.

Lorenzi, H., Souza, H.M., 1995. Plantas Ornamentais no Brasil: Arbustivas, Herbacease Trepadeiras. Editora Plantarum, Sao Paulo.

Marciani, D.J., 2003. Vaccine adjuvants: role and mechanisms of action in vaccineimmunogenicity. Drug Discov. Today 8, 934–943.

B.P. Silva, J.P. Parente / Phytochemistry Letters 6 (2013) 633–639 639

Miliken, M., 1997. Plants for Malaria, Plants for Fever. Medicinal Species in LatinAmerica—A Bibliographic Study. Balogh Scientific Book, New York.

Mowat, A.M., Donachie, A.M., Reid, G., Jarrett, O., 1991. Immune-stimulating com-plexes containing Quil A and protein antigen prime class I MHC-restricted Tlymphocytes in vivo and are immunogenic by the oral route. Immunology 72,317–322.

Nakamura, T., Takeda, T., Ogihara, Y., 1994. Studies on the constituents of Calliandraanomala (Kunth) Macbr. II. Structure elucidation of four acylated triterpenoidalsaponins. Chem. Pharmaceut. Bull. 42, 1111–1115.

Oda, K., Matsuda, H., Murakami, T., Katayama, S., Ohgitani, T., Yoshikawa, M., 2000.Adjuvant and haemolytic activities of saponins derived from medicinal and foodplants. Biol. Chem. 381, 67–74.

Parente, J.P., Cardon, P., Leroy, Y., Montreuil, J., Fournet, B., Ricart, G., 1985. Aconvenient method for methylation of glycoprotein glycans in small amountsby using lithium methylsulfinyl carbanion. Carbohyd. Res. 141, 41–47.

Santos, W.R., Bernardo, R.R., Pecanha, L.M.T., Palatnik, M., Parente, J.P., de Sousa,C.B.P., 1997. Haemolytic activities of plant saponins and adjuvants. Effect of

Periandra mediterranea saponin on the humoral response to the FML antigen ofLeishmania donovani. Vaccine 15, 1024–1029.

Sun, H.-X., Xie, Y., Ye, Y.-P., 2009. Advances in saponin-based adjuvants. Vaccine 27,1787–1796.

Takeda, T., Nakamura, T., Takashima, S., Yano, O., Ogihara, Y., 1993. Studies onthe constituents of Calliandra anomala (Kunth) Macbr. I. Structure elucidationof two acylated triterpenoidal saponins. Chem. Pharmaceut. Bull. 41,2132–2137.

Tani, C., Ogihara, Y., Mutuga, M., Nakamura, T., Takeda, T., 1996. Studies on theconstituents of Calliandra anomala (Kunth) Macbr. III. Structure elucidation ofsix acylated triterpenoidal saponins. Chem. Pharmaceut. Bull. 44, 816–822.

Tani, C., Ogihara, Y., Takeda, T., 1998. Studies on the constituents of Calliandraanomala (Kunth) Macbr. IV. Structure analysis by HPLC retention time and FAB-MS spectrum. Chem. Pharm. Bull. 46, 723–725.

Sawardeker, J.S., Sloneker, J.H., Jeanes, A., 1965. Quantitative determination ofmonosaccharides as their alditol acetates by gas liquid chromatography. Anal.Chem. 37, 1602–1604.