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Química Nova

Print version ISSN 0100-4042

Quím. Nova vol.34 no.2 São Paulo  2011 



Triterpenes and new saponins from Ilex chamaedryfolia: chemotaxonomic tool to ilex species differentiation



Claiton L. LencinaI; Mariana C. de CardosoI; Ivomar ZancanaroI; Grace GosmannI, *; Viviane S. PiresII; Pascal SonnetII; Dominique GuillaumeII, #; Eloir P. SchenkelIII

IFaculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Av. Ipiranga, 2752, 90610-000 Porto Alegre - RS, Brasil
IIUMR-CNRS 6219, Laboratoire des glucides, Faculté de Pharmacie, Université de Picardie Jules Verne, 1 rue des Louvels, 80037 Amiens, France
IIICentro de Ciências da Saúde, Universidade Federal de Santa Catarina, 88040-900 Florianópolis - SC, Brasil




Three saponins were isolated from leaves of Ilex chamaedryfolia. Their structures were established by spectroscopic and mass spectrometry data as the new saponin 3β-O-β-D-glucopyranosyl-(1-3)-α-L-arabinopyranosyl-20( S)-19α-hydroxyurs-12-en-28-oic acid 28-O-β-D-glucopyranosyl-(1-3)-β-D-glucopyranosyl ester, the new saponin 3β-O-β-D-glucopyranosyl-(1-3)-α-L-arabinopyranosyl-20( S)-19α-hydroxyurs-12-en-28-oic acid 28-O-β-D-glucopyranosyl ester and the known saponin 3β-O-β-D-glucuronopyranosyl-20(R )-19α-hydroxyurs-12-en-28-oic acid 28-O-β-D-glucopyranosyl ester. Ursolic acid and α-amyrin were also isolated.

Keywords: Ilex chamaedryfolia; Aquifoliaceae; triterpenes.




We initiated some years ago a program aimed to identified saponins from South American Ilex species1 considering the importance of this genera which includes Ilex paraguariensis A. St. Hil., named yerba mate. This species is widely used in South Brazil, Argentina, Paraguay and Uruguay to obtain the raw material used to prepare the traditional beverage called maté which is a very important economical crop. Hence, we already reported the structure of several saponins isolated from maté and other South American Ilex species: I. affinis. I. argentina. I. brevicuspis. I. buxifolia. I. dumosa. I. integerrima. I. microdonta. I. psammophila. I. pseudobuxus. I. taubertiana and I. theezans.1-8

Ilex chamaedryfolia Reissek is a native shrub in Southern Brazil, found in the states of Rio Grande do Sul, Paraná and Santa Catarina, where it is known as "congonha miúda", "congonhinha" or "congonha brava".2,9 I. chamaedryfolia is one of the species eventually reported as an adulterant of the I. paraguariensis. Herein it is described the isolation and structural elucidation of three saponins and two triterpenes (ursolic acid and α-amyrin) from the leaves of I. chamaedryfolia. As far as we know, the saponins reported herein have not been yet described in the literature.



Plant material

Leaves from I. chamaedryfolia Reissek were collected in Guaratuba, State of Paraná, Brazil. A specimen is on deposit in the Herbarium of the Department of Botany (ICN) at the Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil (M. Sobral and E. P. Santos 9308).


Optical Rotation was measured on a Perkin-Elmer® 341 polarimeter. FAB-MS analysis was performed in positive mode on a Kratos MS 80 instrument and HRMS MS spectra were recorded on a Q-Tof micro Waters® high resolution mass spectrometer, operating on eletronspray ionization mode. NMR spectra (1H, 500 MHz; 13C, 125 MHz) were recorded in CD3OD and CDCl3 on a Bruker® Avance 500 spectrometer. Thin-layer chromatography (TLC) was on Si gel GF254 Merck® or Aldrich® using CH2Cl2:MeOH (98:2, v/v) and CHCl3:MeOH:H2O (80:40:5, v/v) as eluents for sapogenins and saponins, respectively. Compounds were visualised using anysaldehyde-sulphuric acid reagent and heating (100 °C).

Extraction and isolation

Air-dried powdered leaves (475 g) were submitted to maceration in aqueous 80% EtOH (1:10 plant:solvent, m/v) at room temperature (2 x 10 days). The ethanol extract was evaporated to dryness under reduced pressure and the residue (85 g, 18%) was suspended in water (1500 mL) and extracted successively with dichloromethane (3 x 500 mL), ethyl acetate (3 x 500 mL) and n-butanol (3 x 500 mL). Each organic phase was separately evaporated to obtain 5 g (0.7%), 5 g (0.7%) and 45 g (9.5%) of each fraction, respectively. The aqueous residue was lyophilised to obtain 27 g (5.6%).

Dichloromethane fraction (1 g) was submitted to CC on silica gel eluting with gradients of ciclohexane:ethyl acetate mixtures. Fractions were pooled according to TLC. Fractions 35-41 (88 mg) and 82-85 (30 mg) were subjected to crystallisation in yielding compounds 1 (19 mg) and 2 (25 mg) in a pure form, respectively. Part of the n-butanol fraction (10 g) was submitted to successive CC on silica gel eluting with gradients of CH2Cl2:EtOH:H2O mixtures, or CC on LiChroprep™ RP-18 using H2O:MeOH. Fractions were pooled according to their TLC profiles. Compounds 3 (13 mg), 4 (10 mg) and 5 (13 mg) were isolated (Figure 1). One fraction containing mainly compound 3 was acetylated6 to obtain pure 3a (14 mg).



α-amyrin (1). This compound was identified after comparison of its physical-chemical data with the literature10 and by co-TLC with an authentic sample.

Ursolic acid (2). (3β)-3-hydroxyurs-12-en-28-oic acid. It was identified by co-TLC with an authentic sample.11

Compound 3 was identified as the deacylated form of 3a after comparison of both NMR data.

Peracetylated 3β-O-β-D-glucopyranosyl-(1-3)- α-L-arabinopyranosyl-20(S)-19α-hydroxyurs-12-en-28-oic acid 28-O-β-D-glucopyranosyl-(1-3)-β-D-glucopyranosyl ester (3a). Amorphous powder. FABMS (positive mode) m/z: 1659 [M+Na]+ and HRMS m/z 1659.7831 [M+Na]+ (C79H112O36). [α]25D +14.3º (CHCl3, c 0.35). 1H NMR and 13C NMR data (CDCl3): see Table 1.

3β-O-β-D-glucopyranosyl-(1-3)- α-L-arabinopyranosyl-20(S)-19α-hydroxyurs-12-en-28-oic acid 28-O-β-D-glucopyranosyl ester (4).

Amorphous powder. FABMS (positive mode) m/z: 951 and HRMS m/z 951.4899 [M+Na]+ (C47H76O18). [α]25D -1.67º (MeOH, c 0.6). 1H NMR and 13C RMN data (CD3OD): see Table 1.

3β-O-β-D-glucuronopyranosyl-20(R)-19 α-hydroxyurs-12-en-28-oic acid 28-O-β-D-glucopyranosyl ester (5). Amorphous powder. FABMS (positive mode) m/z: 833.6 [M+Na]+. 1H NMR and 13C NMR data (CD3OD): see Table 1.



Solvent partition and chromatographic procedures allowed the isolation of five compounds from leaves of I. chamaedryfolia whose identification was accomplished through spectroscopic methods.

Compound 1 was identified as α-amyrin after comparison of its physical-chemical data with literature and by co-TLC with an authentic sample. Compound 2 was rapidly identified as ursolic acid by co-TLC analysis using authentic sample.

Along with the signals corresponding to acetyl groups, {1H}-13C-NMR spectrum of 3a showed 53 signals, which after comparison with its DEPT-13C-NMR spectrum, allow to recognize signals attributed to 7 methyl, 13 methylene and 25 methine groups, and 8 quaternary carbon atoms. The 13C-NMR of this compound showed characteristic signals due to one carboxyl group (δ 175.7), a double bond (δ 128.0; δ 137.0) and two oxygenated carbons (δ 72.9; δ 89.9) beyond the acetyl characteristic signals.12 A signal at δH 2.77 (s, 1H, H-18) suggesting the presence of a 19-O-substituted urs-12-en skeleton, specifically a 19α-hydroxyursolic acid derivative.13 Chemical shifts of the C-18, C-22 and C-23 were found at upfield shift and indicate an unusual 20 S configuration to E ring of aglycone (Table 1).8,12 The observation of chemical shifts to C-28 (δ 175.7) and C-3 (δ 89.9) dislocated to upfield and downfield respectively, cleared bisdesmosidic features. Thus, the compound 3a (and 3) has as aglycone ilexgenin B (Figure 1).12

Its 1H-NMR and 13C-NMR data indicated the presence of four sugar moieties. The sugar residue δH 5.55 (H-1'''); δC 91.7 (C-1''') evidenced an ester linkage between the anomeric carbon and the C-28 of the aglycone.14 For the sugar chain, HH and HC correlation (COSY, HMQC, HMBC) (Figure 2) allowed to assign all carbon and proton signals. HMBC correlation spectrum allowed indicated the interglycosidic linkages, between the terminal glucose (δH 4.73, H-1; δC 99.7, C-1), attached at C-3''' of glucose II at C-28, as well as, glucose I and arabinose unit substituted at C-3. This spectrum showed a correlation between C-3' of arabinose and H-1'' of glucose I, C-3''' of glucose II and H-1'''' of glucose III (Figure 2). Besides H-1' of arabinose and C-3 of aglycone, H-1''' of glucose II and C-28 of aglycone. Thus 3a was identified to be peracetylated form of 3β-O-β-D-glucopyranosyl-(1-3)-α-L-arabinopyranosyl-20( S)-19α-hydroxyurs-12-en-28-oic acid 28-O-β-D-glucopyranosyl-(1-3)-β-D-glucopyranosyl ester (Figure 1).



Comparative analysis of the 13C-NMR-{1H} and 13C-NMR-DEPT spectra of 4 was also used to identify the presence of signals attributed to 7 methyl, 12 methylene and 20 methine groups and 8 quaternary carbon atoms. The NMR data (13C; 1H) of compound 4 were very similar to compound 3a excepting the absence of the acetyl characteristic signals and those corresponding to one glucose. After detailed analysis of the spectroscopic data of 4 it was possible to conclude that this compound contained a single glucose residue in the ester chain at C-28, contrasting to 3a and 3 that contained two glucose units. Thus, 4 was identified as 3β-O-β-D-glucopyranosyl-(1-3)-α-L-arabinopyranosyl-20( S)-19α-hydroxyurs-12-en-28-oic acid 28-O-β-D-glucopyranosyl ester (Figure 1). The HH and HC correlation (COSY, HMQC and HMBC) allowed to assign all carbon and proton signals and to identify all interglycosidic linkages (Figure 2). The 20R enantiomer of this compound has already been reported in Ilex kudincha15 and Randia formosa.16 Thus, 4 is the epimer of ilekudinoside E and randiasaponin III.

In relation to 5, through comparison of its 13C-NMR-{1H} and 13C-NMR-DEPT spectra, it was possible to identify the presence of 7 methyl, 10 methylene and 16 methine groups and 8 quaternary carbon atoms. 1H-NMR joined to 13C-NMR data indicated the presence of two sugar residues. The later spectrum also evidenced the presence of one glucose residue linked to C-28 via an ester linkage (anomeric carbon at δC 95.8) and the presence of glucuronic acid moiety (δC 106.7) attached to C-3. Comparison of the NMR data of compound 5 to those of the literature allowed the identification of its aglycone as pomolic acid, one 19α-hydroxyursolic acid derivative possessing a 20R-configuration.17 The β configuration for the glucopyranosyl and glucuronopyranosyl units and the α configuration for the arabinopyranosyl residues were inferred from their 13C-NMR data and J values.18 These data suggested that 5 is the 3β-O-β-D-glucuronopyranosyl-20(R )-19α-hydroxyurs-12-en-28-oic acid 28-O-β-D-glucopyranosyl ester (Figure 1) which was already isolated from Ilex kudincha by Nishimura et al.15 as ilekudinoside B.

To the best of our knowledge, the saponins 3 and 4 reported herein have not been yet described in the literature. Moreover, excepting few reports,8,19 the co-occurrence of triterpenes with configuration 20R and 20S is not a common feature to the Ilex species already studied. Indeed, it is possible to differentiate I. chamaedryfolia from other South-American Ilex considering their saponins.8



Several works have demonstrated that the saponins isolated from leaves of I. paraguariensis (maté) are different from those found in other South-American Ilex species.1,3-6,8 The aglycone pattern 19-hydroxyursolic and 29-hydroxyursolic acid found in saponins isolated from South-American Ilex species investigated up to now, allowed to differentiate all this species from I. paraguariensis leaves whose saponins are glycosides of ursolic or oleanolic acid. As suggested by our previous results,8 saponin content of South-American Ilex species can be used as a chemotaxonomic tool to the genera. This was confirmed herein considering that saponins so far isolated from I. chamaedryfolia are based on 19-hydroxyursane triterpenes which are different from those presented in the leaves of I. paraguariensis.



The authors are grateful to M. Sobral (Universidade Federal do Rio Grande do Sul) and S. A. L. Bordignon (Centro Universitário La Salle, Canoas-RS) for collecting and identifying the plant material and to Unidade de Química de Proteínas e Espectrometria de Massas (Uniprote-MS) do Centro de Biotecnologia/UFRGS for HRMS. This work was supported by the Brazilian agencies: CNPq, CAPES and FAPERGS. The authors are also grateful to the Programa de Pós-Graduação em Ciências Farmacêuticas/UFRGS (Brazil).



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Recebido em 16/2/10; aceito em 13/9/10; publicado na web em 8/12/10



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# Present address: Faculté de Pharmacie, Université de Reims, Chimie Thérapeutique, 51 rue Cognacq Jay, 51100 Reims, France

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