Ilhem Zebiri

Mohamed Haddad

Laurent Duca

Dominique Harakat

Billy Cabanillas

Lucie Paloque

Amandine Scandolera

Michel Sauvain

Elsa Rengifo

Laurence Voutquenne-Nazabadioko

Laurence Voutquenne- Nazabadioko

Zebiriosides A-L, oleanane saponins from the roots of Dendrobangia boliviana

Keywords: Dendrobangia boliviana, Cardiopteridaceae, Triterpenoid saponins, Cytotoxic activity

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Firstly classified in the family of Icacinaceae by Rusby in 1896, the genus Dendrobangia was moved in 2001 from Icacinaceae to Cardiopteridaceae by Kârehed who conducted a cladistic study based on morphological and biological characters (DNA sequences analysis), and divided the Icacinaceae "sensu lato" in four families and three order: Icacinaceae « sensu stricto » (Garryales), Cardiopteridaceae and Stemonuraceae (Aquifoliales) and Pennantiaceae (Apiales) [START_REF] Karehed | Multiple origin of the tropical forest tree family Icacinaceae[END_REF]. The Cardiopteridaceae is composed of 43 species in 6 genera and are located in tropical zones. The genus Dendrobangia is composed of three species: D. multinerva Ducke, D. tenuis Ducke and D. boliviana Rusby, large trees growing in the tropical area. No phytochemical studies on this genus have been reported so far.

Dendrobangia boliviana Rusby is a tree of 5 to 30 m high and 50 cm in diameter widely distributed in the Neotropical region of Costa Rica and Panama, Brazil and Bolivia. It grows in mountain forests and rarely in floodplain forests between 100 and 1200 m altitude, and tolerates a wide range of soils. In Colombia, the community of the region Hibito consumes the fine fruits like dried fruits. The wood of this tree is used for its strength in construction, furniture, sporting goods, tool handles, interior coverings [START_REF] De Stefano | Tratamiento taxonómico del género Dendrobangia Rusby Cardiopteridaceae o Icacinaceae[END_REF]. In order to discover previously undescribed secondary metabolites of biological importance from Peru's Amazon rainforest, we investigated the roots of D. boliviana. This paper deals with the isolation and structure elucidation of twelve previously undescribed oleanane saponins (1)(2)(3)(4)(5)(6)(7)(8)(9)[START_REF] Zebirioside | )-[β-Dglucopyranosyl-(1→2)-]β-D-glucuronopyranosyl serjanic acid (6): White powder[END_REF][START_REF] Zebirioside | )-[β-Dxylopyranosyl-(1→2)-]β-D-glucuronopyranosyl serjanic acid (7): White powder[END_REF][START_REF] Zebirioside | )-]β-D-glucuronopyranosyl-28-O-β-D-glucopyranosyl serjanic acid (8): White powder[END_REF] and two known compounds. The characterization of these 14 compounds from the roots of D. boliviana, studied for the first time in a chemical and biological point of view, leads us to explore the cytotoxic, antileishmanial and hemolytic activities of our different molecules.

Results and discussion

The hydromethanolic extract of the roots of D. boliviana was purified by multiple chromatographic steps over silicagel and reversed phase C18 and finally by semi-preparative HPLC, yielding twelve previously undescribed saponins, zebiriosides A to L (1-12), together with two known compounds, 3-O-β-D-glucuronopyranosyl serjanic acid (13) [START_REF] Jayasinghe | Glucuronides of Diploclisia glaucescens[END_REF] and talunùmoside I (14) [START_REF] Kohda | Saponins from Talinum triangulare[END_REF]) (Figure 1). Their structures were elucidated by a detailed analysis of their spectral data including 2D NMR experiments (COSY, TOCSY, HSQCJ-modulate, HMBC, and ROESY) and mass spectrometry.

Acid hydrolysis of the crude saponin precipitate afforded four sugar units in the aqueous layer, identified by analysis on a chiral column as D-glucuronic acid (GlcA), D-glucose (Glc), D-xylose (Xyl) and L-rhamnose (Rha).

Compound 1 was obtained as a white powder. The HRESIMS (positive ion mode) exhibited a pseudomolecular ion peaks at m/z 845.4290 ([M+Na] + , calcd for C43H66O15Na, 845.4299) indicating a molecular formula of C43H66O15. The 1 H NMR spectra of the aglycone part indicated the presence of six tertiary methyl groups at δH 0.82 (s, Me-26), 0.87 (s, Me-24), 0.97 (s, Me-25), 1.07 (s, Me-23), 1.16 (s, Me-29), and 1.19 (s, Me-27), one olefinic proton at δH 5.32 (J=3. 4 Hz,, one oxygenated methine protons at δH 3.19 (dd, J=11.8-4. 5 Hz, H-3) and the deshielded proton H-18 at δH 2.71 (dd, J=13.8-4. 5 Hz), in the carbonyl anisotropy cone of C-28. A methoxy group was also observed at δH 3.72. Its 13 The 1 H and 13 C-NMR spectra revealed the presence of two sugar units with anomeric protons at δH 5.19 ppm (d, J =1.7 Hz) and 4.41 ppm (d, J=7.9 Hz) and the corresponding carbons at δC 102.8, and 106.8 suggesting two osidic units [START_REF] Agrawal | NMR spectroscopy in the structural elucidation of oligosaccharides and glycosides[END_REF]. A methyl signal at δH 1.25 (d, J=6.2 Hz), and δC 17.8 indicated a 6-desoxy-hexose and a carbonyl carbon at δC 172.5 indicated a hexosuronic moiety. Analysis of COSY and ROESY correlations allowed us to assign complete spin systems of a β-D-glucuropyranose and a α-L-rhamnopyranose [START_REF] Bock | Carbon-13 Nuclear Magnetic Resonance Spectroscopy of Monosaccharides[END_REF] ) (Table 1). The carbons of each monosaccharide were attributed by analysis of HSQC spectra and indicated the presence of a terminal α-L-rhamnopyranose, and a 3-substitued β-D-glucuronic acid (δC 83.4) (Table 1). The linkage sites of the monosaccharide units were determined by analysis of HMBC spectrum. The HMBC correlations between the β-D-glucuropyranose anomeric proton H-1' (GlcA-H-1') at δH 4.41 and the carbon C-3 of the genin (δC 91.2) indicates the position of the sugar on the serjanic acid, and between the α-L-rhamnopyranose anomeric proton H-1'' (Rha-H-1'') at δH 5.19 and the β-D-glucuropyranose carbon C-3' (δC 83.4) (GlcA-C-3'). Consequently, the structure of saponin 1, zebirioside A, was concluded to be 3-O-α-L-rhamnopyranosyl-(1→3)-β-Dglucuronopyranosyl serjanic acid (Figure 1).

Compound 2, was obtained as a white powder. The positive HRESIMS spectra gave a pseudomolecular ion peaks at m/z 1007.4835 ([M+Na] + calcd for C49H76O20Na, 1007.4828) corresponding to the molecular formula C49H76O20. The 1 H-and 13 C-NMR spectra were very similar to those of compound 1, except for the presence of a supplementary hexose unit and an ester glycosidic linkage at C-28 (δC 177.6) of serjanic acid. From the anomeric proton at δH 5.36 (d, J=8.2 Hz) and the corresponding anomeric carbon at δC 95.8, a β-Dglucopyranose moiety was identify by analysis of COSY and HSQC spectra (Table 1) [START_REF] Bock | Carbon-13 Nuclear Magnetic Resonance Spectroscopy of Monosaccharides[END_REF]. The chemical shifts of C-3 (δC 91.2) and C-28 (δC 177.6) confirmed the attachment of the sugar units to the aglycone and showed that compound 2 was a bidesmosidic saponin. This was confirmed by the HMBC correlations between GlcA-H-1' (δH 4.59)/C-3 (δC 91.2) and Glc H-1''' (δH 5.36)/C-28 (δC 177.6). Another HMBC correlation between Rha-H-1'' (δH 5.19)/GlcA-C-3' (δC 78.3) revealed the sequencing in the disaccharide moiety. Thus, saponin 2, zebirioside B, was elucidated as the previously undescribed 3-O-α-L-rhamnopyranosyl-(1→3)-β-D-glucuronopyranosyl-28-O-β-D-glucopyranosyl serjanic acid (Figure 1).

Compound 3 was obtained as a white powder. The positive HRESIMS spectra gave a pseudomolecular ion peaks at m/z 1007.4832 ([M+Na] + , calcd for C49H76O20Na, 1007.4828) corresponding to the same molecular formula C49H76O20 as compound 2 and suggesting that these compounds were isomers. The 1 H-and 13 C-NMR spectra were very similar to those of compound 2, except of the chemical shift of C-28 (δC 181.3) of serjanic acid suggesting a free carboxylic acid. Thus the glucopyranose was not linked with an ester linkage in compound 3. This was confirmed by the deshielding carbons C-2' (δC 78.1) and C-3' (δC 86.0) of β-D-glucuropyranosyl moiety indicating that it is disubstituted on these positions (Table 1). The HMBC correlations between GlcA-C-3' (δC 86.0) and H-3 of the aglycone, Rha-H-1''/GlcA-C-3', and Glc-H-1'''/ GlcA-C-2', confirmed a trisaccharide chain attached to position C-3 of serjanic acid. Thus, compound 3, zebirioside C, was identified as 3-O-α-Lrhamnopyranosyl-(1→3)-[β-D-glucopyranosyl-(1→2)]-β-D-glucuronopyranosyl serjanic acid (Figure 1).

Compound 4 was obtained as a white powder and their positive HRESIMS spectra gave the molecular formula C49H76O20 by the pseudomolecular ion peaks at m/z 977.4727 ([M+Na] + , calcd for C48H74O19Na, 977.4722). Comparison of the 1 H-and 13 C-NMR spectra of compounds 3 and 4 showed three sugars linked to the hydroxyl in position 3 of serjanic acid and the absence of the glucopyranose unit replaced by a pentose unit in compound 4. The anomeric proton H-1''' of the third sugar is at δH 4.40 and its anomeric carbon C-1''' at δC to 104.8 (Table 1). The chemical shifts and coupling constants indicate a β-D-xylopyranose [START_REF] Bock | Carbon-13 Nuclear Magnetic Resonance Spectroscopy of Monosaccharides[END_REF]. The linkage was deduced by HMBC correlations between GlcA-H-1'/C-3, Rha-H-1''/GlcA-C-3', and Xyl-H-1'''/ GlcA-C-2'. Thus, compound 4, zebirioside D, was concluded to be 3

-O-α-L-rhamnopyranosyl-(1→3)-[β-D-xylopyranosyl-(1→2)]-β-D-
glucuronopyranosyl serjanic acid (Figure 1).

Compound 5 was obtained as a white powder. Its molecular formula C54H84O24 was deduced from the pseudomolecular ion peaks at m/z 1139.5260 ([M+Na] + , calcd for C54H84O24Na, 1139.5250) in the positive HRESIMS spectra indicating an additional hexose unit when compare to compound 4. The 1 H-and 13 C-NMR spectra were very similar to those of compound 4 with the same trisaccharide chain attached to C-3 of serjanic acid but this compound is glucosylated at position C-28 (δC 177.5) as in compound 2 (Table 1). This 1) or a β-D-xylopyranose in 7 (Table 2), and C-3' by a α-L-rhamnopyranose. The supplementary sugar unit was identifying as a β-D-xylopyranose in both compounds 6 and 7. This sugar was attached to the α-L-rhamnopyranose at C-3'' as suggested by the deshielding signal of C-3'' (δC 81.5) (Tables 1 and2). The HMBC correlations in compound 6 [GlcA-H-1'/C-3, Rha-H1''/GlcA-C-3', Glc-H-1'''/ GlcA-C-2' and Xyl-H-1 IV /Rha-C-3''] allowed us to confirm the point of linkages of the sugars each other and on the aglycone. Thus, saponin 6, zebirioside F, was identified as 3 (δC 64.6). Thus, the genin of these compounds is phytolaccinic acid [START_REF] Johnson | Phytolaccinic acid, a new triterpene from Phytolacca americana[END_REF].

-O-β-D-xylopyranosyl-(1→3)- α-L-rhamnopyranosyl-(1→3)-[β-D-glucopyranosyl-(1→2)]-β-D-glucuronopyranosyl serjanic acid. The structure of compound 7, zebirioside G, is also deduced from the HMBC correlations [GlcA-H-1'/C-3, Rha-H1''/GlcA-C-3', Xyl-H-1'''/GlcA-C-2' and Xyl-H-1 IV /Rha-C-3''] to 3-O-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→3)-[β-D-xylopyranosyl-(1→2)]-β-D- glucuronopyranosyl serjanic acid.
For compound 10, in addition to phytolaccinic acid, signals for two sugars units, a β-Dglucuronopyranose substituted at C-2' and an α-L-rhamnopyranose were observed (Table 2). The hemolytic activity is considered to be a typical characteristic of saponins because of their detergent-like properties, caused by a rapid cell lysis interacting with membrane cholesterol, although, some saponins show only weak or no hemolytic effect. The hemolytic activity of the isolated saponins was assessed using the method previously described [START_REF] Chwalek | Structureactivity relationships of some hederagenin diglycosides: Haemolysis, cytotoxicity and apoptosis induction[END_REF]. No hemolytic activity was observed for compounds 1-14.

When comparing the cytotoxic activity and the hemolytic activity of different triterpene bidesmoside saponins [START_REF] Bader | Cytotoxicity of triterpenoid saponins. Part 1. Activities against tumor cells in vitro and hemolytic index[END_REF][START_REF] Chwalek | Structureactivity relationships of some hederagenin diglycosides: Haemolysis, cytotoxicity and apoptosis induction[END_REF], the conclusion was that these two activities were generally directly correlated. In this work, the cytotoxicity on fibroblasts of compounds 1-14 was evaluated at a concentration of 10 µg/mL (Table 3).

Zebirioside C (3) (monodesmosidic saponin) and I (9) (bidesmosidic saponin) were not hemolytic in the range of concentration tested whereas these compounds were found to be cytotoxic on fibrobasts (IC50 of 5.6 and 6.4 µM, respectively). These data suggest that another mechanism than permeabilization formation is responsible of cell cytotoxicity. It is also conceivable that considering the different plasma membrane composition between these two cell types, membrane penetration of saponins could be different leading to different biological effect. Indeed, saponins possess detergent-like properties, they can also increase the permeability of cell membranes without destroying them. They could affect the interaction with trans-membrane proteins or with cytoskeleton proteins of cell membrane that could lead to different biological effects considering the specific plasma membrane composition of different cell types. In the case of cytotoxicity, saponins activity could results from membrane alteration and/or apoptosis which is induced by the condensation of chromatin and DNA fragmentation at the nuclear level [START_REF] Chwalek | Structureactivity relationships of some hederagenin diglycosides: Haemolysis, cytotoxicity and apoptosis induction[END_REF]. It is consequently possible that such a process is induced in fibroblasts.To confirm this hypothesis and to check the action of saponins 3 and 9, biological tests such as annexin V/propidium iodide stainings, analysis of chromatin condensation and analysis of apoptosis specific cell signalling pathways could be of interest.

In addition, the evaluation of the antileishmanial activity of these saponins on promastigotes of Leishmania infantum gave no activity.

Conclusions

Phytochemical study of the roots of Dendrobangia boliviana, a Peruvian plant used mainly for the robustness of its wood, led to the isolation of twelve previously undescribed saponins, zebirioside A to L (1-12), and two known saponins (13)[START_REF] Zebirioside | O-α-L-rhamnopyranosyl-(1→3)-β-D-glucuronopyranosyl phytolaccinic acid (10): White powder[END_REF], which are glycosides of serjanic or phytolaccinic acid. Their chemical structures were determined by analyzing 1D and 2D NMR and MS data. This is the first report of the constituents of Dendrobangia species. These saponins were tested for their cytotoxic, leishmanicidal and hemolytic activities. No antileishmanial or hemolytic activity was revealed and only zebirioside C (3) and I (9) have shown cytotoxicity against fibroblasts (IC50 5.6 and 6.4 µM, respectively). This activity is interesting because these saponins have demonstrated no hemolytic activity, suggesting a specific action mode, depending on the cell type. This phytochemical investigation helps us to extend the knowledge about the constituents of the unstudied Dendrobangia genus.

Experimental

General experimental procedures

Optical rotations were measured in MeOH with a Perkin-Elmer 241 polarimeter. 1 H and 13 C NMR spectra were recorded in CD3OD on a Bruker Avance III-600 spectrometer equipped with a cryo plateform ( 1 H at 600 MHz and 13 C at 151 MHz). 2

Extraction and isolation of compounds 1-14

The powdered dried roots of D. boliviana (150 g) were extracted by maceration in MeOH/H2O (8/2 v/v, 1.5 L) at room temperature for 24 h. After filtration and evaporation to dryness under reduced pressure, the crude MeOH extract (18.6 g) was dissolved in MeOH (100 mL) and precipitated into acetone (1500 mL). After filtration the saponin precipitate (DBP) is dried up (11.4 g) under vacuum over KOH. A part (3.5 g) of the saponin rich precipitate were solubilized in H2O and passed through an IRN77 (H + ) Amberlite resin column (activation with HCl/H2O 30/60 and elution with H2O 100%) to give a protonated saponins fraction. This saponin mixture (2.7 g) was fractionated by vacuum liquid chromatography on RP-18 eluted successively with MeOH/H2O 2/8, 4/6, 6/4 and 100% MeOH (each 600 mL) to give nine fractions (DB-A to DB-I). The fraction DB-E (438 mg) was fractionated by High Performance Flash Chromatography (HPFC) on silicagel (4 g cartridge) using a binary gradient of CHCl3/MeOH (10/0 to 6/4 for 10 min, 6/4 for 20 min and 6/4 to 0/10 for 17 min) to give 6 fractions (DB-E1 to DB-E6). The fraction DB-E4 (245 mg) was fractionated in the same manner as DB-E to give 8 fractions DB-E4a to DB-E4H. Fractions DB-E4d (18 mg), DB-E4f (69 mg) and DB-E4G (30 mg) were purified by semi-preparative HPLC with an isocratic mixture of ACN/H2O-TFA 0.025% (35/65) for 20 min to give compounds 13 (Rt = 5.05 min, 54 mg,) and 10 (Rt = 5.6 min, 25 mg). Fraction DB-F (193 mg) was fractionated by HPFC on silica gel (4 g cartridge) with a binary gradient of CHCl3/MeOH (10/0 to 0/10) for 30 min to give 9 fractions (DB-F1 to DB-F9). Fractions DB-F5 (9 mg) and DB-F6 (35 mg) were purified by semi-preparative HPLC in isocratic conditions (ACN/H2O-TFA 0.025% (45/55) for 20 min) to afford compounds 8 (Rt = 8.7 min, 14 mg) and 4 (Rt = 7.4 min, 8 mg). The fraction BD-F7 (108 mg) was purified by semi-preparative HPLC using a gradient of ACN/H2O-0.025% TFA (3/7 to 38/62 for 30 min) to give the compounds 10 (Rt = 5.4 min, 13 mg), 11 (Rt = 5.8 min, 4 mg), 9 (Rt = 5.9 min, 3 mg), 4 (Rt = 6.5 min, 15 mg), 2 (Rt = 7.7 min, 2 mg), 14 (Rt = 11.1 min, 2 mg) and 12 (Rt = 12.1 min, 14 mg). Fraction DB-G (63 mg) was purified by semi-preparative HPLC using ACN/H2O-TFA 0.025% (1/9 to 4/6 for 5 min and 4/6 for 15 min) to give compounds 4 (Rt = 8.4 min, 11 mg), 2 (Rt = 8.8 min, 3 mg), 12 (Rt = 11.4 min, 7 mg) and 7 (Rt = 12.1 min, 5 mg). Fraction DB-H (197 mg) was fractionated by HPFC on silica gel (4 g cartridge) using CHCl3/MeOH (1/9 to 6/4 for 15 min, 6/4 for 10 min and 6/4 to 0/10 for 10 min) to give 9 fractions (DB-H1 to DB-H9). Fractions DB-H5 (15 mg) and DB-H6 (25 mg) were purified by semi-preparative HPLC with a gradient of ACN/H2O-0.025% TFA (45/55 to 5/5) for 15 min to give compounds 3 (Rt = 9.9 min, 13 mg) and 1 (Rt = 12.3 min, 4 mg).

Fraction DB-H7 (50 mg) was purified by semi-preparative HPLC using a gradient of ACN/H2O-0.025% TFA (4/6 to 45/55) for 30 min to give compounds 7 (Rt = 8.4 min, 13 mg), 5 (Rt = 8.8 min, 6 mg), 8 (Rt = 9.3 min, 7 mg), 6 (Rt = 9 8 min, 7 mg) and 3 (Rt = 14.4 min, 11 mg).

Acid hydrolysis and identification of sugars units

Saponins precipitate (1.08 g) was refluxed with 25 mL of 2 M TFA for 4h. After extraction with EtOAc (3 x 30 mL), the aqueous layer was neutralized to pH 6 with 50mM KOH and freezedried to provide the monosaccharide residue. The sugars profile was determined by comparison with authentic samples on TLC in MEtK/iso-Pro/Ac2O/H2O (20/10/7/6, v/v).

Detection was performed with α-naphthol. The identification of sugars was determined by HPLC on an analytical chiral column Chiralpack® ICA (5 µm, 4.6 x 250 mm) eluted with an isocratic mixture of Hexane/EtOH/TFA (50/50/0.1, v/v) at a flow rate of 0.5 mL/min and 35 °C. Chromatograms were monitored by a refractive index detector RI-410 (Waters) and retention times of the sugars were compared to standard sugars (L-xylose, D-xylose, Lglucose, D-glucose, L-rhamnose and D-glucuronic acid).

Zebirioside A, 3-O-α-L-rhamnopyranosyl-(1→3)-β-D-glucuronopyranosyl serjanic acid

(1): White powder; [α]D 20 +14.3° (c 0.87, MeOH); 1

  C NMR and HSQC spectra exhibited signals for six methyls groups at δC 15.9 (C-25), 17.0 (C-24), 17.7 (C-26), 26.4 (C-27), 28.5 (C-23), and 28.7 (C-29), one hydroxy group at C-3 (δC 91.2) and two olefinic carbons at δC 124.3 (C-12) and 144.7 (C-13), characteristic of an oleane-12-ene skeleton (Jayasinghe et al., 1998; Kohda et al., 1992; Mahato and Kundu, 1994); together with two carbonyl groups at δC 178.8 and 181.3 and a methoxy group at δC 52.3. The HMBC correlations observed between H-18 and the C-28 carbonyl carbon (181.3 ppm) indicate a carboxylic group in C-28. The HMBC correlations between the carbonyl carbon at δC 178.8 with protons Hβ-19 (δH 1.69), Hα-21 (δH 2.01), H-29 (δH 1.16) and methoxy group (δH 3.72) suggest an ester carbonyl at C-30. The 3β configuration of the hydroxyl group was confirmed from the ROESY experiment in which correlations were observed between H-3α, H-5α, and H-23α. These data are consistent with serjanic acid or 3β-hydroxy-30-methylcarboxylate oleane-12-ene-28-oic acid (Montoya Pelaez et al., 2013). The deshielded nature of C-3 (δC 91.2) suggested a monodesmosidic saponin with an osidic chain at this position.

compound 5 ,

 5 Zebirioside E, was identified as 3-O-α-L-rhamnopyranosyl-(1→3)-[β-Dxylopyranosyl-(1→2)]-β-D-glucuronopyranosyl-28-O-β-D-glucopyranosyl serjanic acid (Figure 1). Compounds 6 and 7 (Figure 1) were obtained as a white amorphous powder. The molecular formula C54H84O24 for 6 and C53H82O23 for 7 were determined separately for the both compounds from the pseudomolecular ion peak observed in positive HRESIMS at m/z 1139.5242 [M+Na] + for 6 and at m/z 1109.5149 [M+Na] + for 7, indicating a supplementary pentose unit comparing to those of compounds 3 and 4, respectively. The 1 H NMR and 13 C NMR spectra of 6 and 7 showed signals assignable to four sugars moieties. Analysis of COSY, TOCSY, HSQCJ-modulate, and HMBC experiments allowed us to identify three sugars moieties as in compounds 3 and 4, a β-D-glucuronopyranose substituted at positions C-2' by a β-D-glucopyranose in 6 (Table

Compounds 8 and 9 (

 9 Figure 1) were obtained as a white powder. Their molecular formula C60H94O29 for 8 and C59H92O28 for 9 obtained in positive HRESIMS, indicates a supplementary hexose unit by comparing to those of compounds 6 and 7. As in compounds 2 and 5, these compounds are bidesmoside saponins with a β-D-glucopyranose attached by an ester linkage to the C-28 of compounds 6 and 7, respectively (Table 2). Thus, their structures were established as 3-O-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→3)-[β-D-glucopyranosyl-(1→2)-]β-D-glucuronopyranosyl-28-O-β-D-glucopyranosyl serjanic acid (Zebirioside H) for saponin 8 and 3-O-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→3)-[β-D-xylopyranosyl-(1→2)-]β-D-glucuronopyranosyl-28-O-β-D-glucopyranosyl serjanic acid (Zebirioside I) for compound 9. Compounds 10, 11 and 12 (Figure 1) were obtained as white amorphous powders. Analysis of 1D ( 1 H, 13 C) and 2D NMR spectra (COSY, HSQC, and HMBC) indicated that the aglycone was different. Five methyl groups instead of six were observed at δH 0.60 (H-24), 0.70 (H-26), 0.87 (H-25), 1.04 (H-29) and 1.09 (H-27) and δC 13.4 (C-24), 17.9 (C-26), 16.4 (C-25), 28.7 (C-29) and 26.4 (C-27), respectively. The singlet corresponding to the methyl at C-23 has disappeared and the C-24 is shielded due to its environmental change. An additional methylene at δH 3.51 (d, J = 11.5 Hz) and 3.18 (d, J = 11.5 Hz), and δC 64.6 ppm indicate the presence of an hydroxyl group on the C-23. This is confirmed by the HMBC correlations between H-24 (δH 0.60) and the carbons C-4 (δC 44.0), C-5 (δC 48.0), C-3 (δC 83.4) and C-23

  The HMBC correlations observed between GlcA-H-1'/C-3 (δC 83.4) and Rha-H-1''/GlcA-C-3' (δC 83.4) confirm the glycosidic linkage and allow us to establish the structure of compound 10, zebirioside J, as 3-O-α-L-rhamnopyranosyl-(1→3)-β-D-glucuronopyranosyl-phytolaccinic acid. Compound 11 is the bidesmoside of compound 10 glucosylated in C-28. It is identified as 3-O-α-L-rhamnopyranosyl-(1→3)-β-D-glucuronopyranosyl-28-O-β-D-glucopyranosyl phytolaccinic acid (zebirioside K). For compound 12 signals for three sugars units, a β-D-glucuronopyranose substituted at C-3' (δC 83.1), a α-L-rhamnopyranose substituted at C-3'' (δC 82.3) and a β-D-xylopyranose were observed (Table 2). The HMBC correlations between GlcA-H-1'/C-3, Rha-H1''/GlcA-C-3', and Xyl-H-1'''/Rha-C-3'' indicated the site of linkages. Thus, compound 12, zebirioside L, was identified as 3-O-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→3)-β-Dglucuronopyranosyl phytolaccinic acid.The characterization of the twelve previously undescribed compounds, together with two known saponins from the roots of Dendrobangia boliviana, leads us to explore the cytotoxic, antileishmanial and hemolytic activities of our different molecules.

  D NMR experiments were performed using standard Bruker microprograms (TopSpin version 3.2 software). HR-ESI-MS and ESI-MS experiments were performed using a Micromass Q-TOF micro-instrument (Manchester, UK). The samples were introduced by direct infusion in a solution of MeOH at a rate of 5 µM/min. TLC were carried out on precoated silica gel 60 F254 (Merck), with CHCl3/MeOH/H2O (70/30/5), and spots were visualizes by heating after spraying with 50 % H2SO4. Vacuum liquid chromatography was carried out on Lichroprep RP-C18 (40-63 µm, Merck). High Performance Flash Chromatography (HPFC) was realized on Grace® Reveleris System using Grace® cartridges (Silica gel or RP-C18) and a flow rate of 18 mL/min. The chromatograms were monitored at 205 and 210 nm. Analytical HPLC was performed on a Dionex apparatus equipped with a WPS-3000 automated sample injector, a TTC-3000 column oven, a LPG-3400SD pump, a diode array detector UVD-340U and the Chromeleon® software. Prepacked C18-2 reversed phase columns 4.6 x 250 mm, 5 µm, 100 A° (Interchim or Phenomenex) were used with a binary gradient elution (H2O-TFA 0.025%/ MeCN) and a flow rate of 1 mL/min. The chromatograms were monitored at 205 and 210 nm. The semi-preparative HPLC was performed on a Dionex apparatus equipped with an ASI-100 automated sampler injector, a STH 585 column oven, a LPG-3400A pump, a diode array detector UVD-340U and the Chromeleon® software. Prepacked C18 reversed phase columns Luna 5u C18, 250 x 10 mm, 5 µm, 100 A° (Phenomenex) or Luna 5u, C18, 250 x 15 mm, 5 µm, 100 A° (Interchim) were used with a binary gradient elution (H2O-TFA 0.025%/MeCN) and a flow rate of 5 mL/min. The chromatograms were monitored at 205 and 210 nm. The chiral chromatography was performed on a Waters apparatus equipped with an automated sampler injector 717 plus, a CO-965 column oven, a system controller 600E, a 60F extended pump diode array detector Waters-996 and a refractometer RI Waters-410 and the Empower® software. Prepacked column Chiralpack IC (Phenomenex) 4.6 x 250 mm, 5 µm was used with an isocratic solvent (Hexane/EtOH/TFA 50/50/0.1) and a flow rate of 0.5 mL/min. 4.2. Plant material Dendrobangia boliviana (roots) were collected and identified in Iquitos, Loreto district, Peru by C. Amasifusen, E. Rengifo and M. Haddad, in September 2011. A voucher specimen (No. CA3240) was carried out and deposed at the national Herbarium of the National University San Marcos in Lima, Peru (UNMSM).

  H NMR (600 MHz, Methanol-d4) of the aglycon: δH 5.32 (1H, t, J = 3.4 Hz, H-12), 4.41 (1H, d, J = 7.9 Hz, H-1'), 3.72 (3H, s, H-31), 3.19 (1H, dd, J = 11.8, 4.5 Hz , H-3), 2.71 (1H, dd, J = 13.8, 4.5 Hz, H-18), 2.03 (1H, td, J = 13.6, 3.5 Hz , Hα-16 ), 2.01 (1H, m, Hα-21), 1.96 (1H , m , Hα-19), 1.93 (2H, dd, J = 9.3, 3.6 Hz, H-11), 1.87 (1H, dd, J =13.5, 4.5 Hz, Hα-2), 1.78 (1H, td, J = 13.6, 4.3 Hz, Hα-15), 1.70 (1H, m, Hβ-2), 1.69 (1H, m , Hβ-19), 1.67 (1H , m , Hβ-16), 1.64 (1H, m, Hα-1), 1.63 (1H, m, H-22) , 1.62 (1H , m , H-9) , 1.58 (1H, m, Hα-6), 1.54 (1H , td, J = 12.4, 4.1 Hz , Hα-7), 1.44 (1H, m, Hβ-6), 1.39 (1H, td, J = 13.5, 4.4 Hz, Hβ-21), 1.33 (1H , m , Hβ-7), 1.19 (3H, s, H-27), 1.16 (3H, s, H-29), 1.12 (1H, dt, J = 13.6, 3.6 Hz, Hβ-15), 1.07 (3H, s, H-23), 1.01 (1H, td, J = 13.3, 3.6 Hz, Hβ-1), 0.97 (3H, s, H-25), 0.82 (4H, s, H-26 , H-5), 0.87 (3H, s, H-24); 13 C NMR (151 MHz, CD3OD) of the aglycon: δc 181.3 (COOH, C-28), 178.8 (COOCH3, C-30), 144.7 (HC=C, C-13), 124.3 (HC=C, C-12), 91.2 (CH-O, C-3), 57.0 (CH, C-5), 52.3 (COOCH3, C-31), 49.0 (CH, C-9), 47.0 (C, C-17), 45.0 (C, C-20), 44.0 (CH, C-18), 43.4 (CH2, C-19), 42.8 (C, C-14), 40.5 (C, C-8), 40.2 (C, C-4), 39.7 (CH2, C-1), 37.9 (C, C-10), 35.0 (CH2, C-22), 34.0 (CH2, C-7), 31.3 (CH2, C-21), 28.9 (CH2, C-15), 28.7 (CH3, C-29), 28.5 (CH3, C-23), 27.0 (CH2, C-2), 26.4 (CH3, C-27), 24.5 (CH2, C-11), 24.2 (CH3, C-16), 19.3 (CH2, C-6), 17.7 (CH3, C-26), 17.0 (CH3, C-24), 15.9 (CH3, C-25); 1 H and 13 C NMR of the glycosidic part, see Table 1; HRESIMS (positive-ion mode) m/z 845.4290 [M+Na] +(calculated for C43H66O15Na, 845.4299).4.6. Zebirioside B, 3-O-α-L-rhamnopyranosyl-(1→3)-β-D-glucuronopyranosyl-28-O-β-Dglucopyranosyl serjanic acid (2): White powder; [α]D 20 +7.0° (c 0.44, MeOH); 1 H NMR (600 MHz, Methanol-d4) of the aglycon: δH 5.33 (1H, t, J = 3.6 Hz, H-12), 3.72 (3H, s, H-31), 3.19 (1H, dd, J = 11.5, 4.3 Hz , H-3), 2.72 (1H, dd, J = 13.7, 4.0 Hz, H-18), 2.08 (1H, td, J = 15.0, 3.8 Hz , Hα-16 ), 2.02 (1H, m, Hα-21), 1.97 (1H, ddd, J = 13.7, 4.4, 2.6 Hz, Hα-19), 1.93 (2H, dd, J = 8.9, 3.4 Hz, H-11), 1.86 (1H, dq, J =13.5, 4.3 Hz, Hα-2), 1.80 (2H, m, Hα-15, Hβ-16), 1.74 (1H, dt, J =13.8, 3.4 Hz, Hα-22), 1.71 (1H, t, J = 13.7 Hz, Hβ-19), 1.70 (1H, m, Hβ-2), 1.64 (1H, dt, J =13.4, 3.5 Hz, Hα-1), 1.61 (1H, m, H-9), 1.56 (2H, m, Hβ-22/Hα-6), 1.51 (1H, m, Hα-7), 1.41 (1H, m, Hβ-6), 1.33 (1H, m, Hβ-7), 1.40 (1H, td, J = 9.9, 3.3 Hz, Hβ-21), 1.19 (3H, s, H-27), 1.16 (3H, s, H-29), 1.12 (1H, dt, J = 14.5, 3.4 Hz, Hβ-15), 1.07 (3H, s, H-23), 1.00 (1H, m, Hβ-1), 0.97 (3H, s, H-25), 0.86 (3H, s, H-5/H-24), 0.80 (3H, s, H-26); 13 C NMR (151 MHz, CD3OD) of the aglycon: δC 178.8 (COOCH3, C-30), 177.6 (COOR, C-28), 144.4 (HC=C, C-13), 124.4 (HC=C, C-12), 91.2 (CH-O, C-3), 52.4 (COOCH3, C-31), 49.0 (CH, C-9), 57.0 (CH, C-5), 47.4 (C, C-17), 45.0 (C, C-20), 43.9 (CH, C-18), 43.3 (CH2, C-19), 42.8 (C, C-14), 40.7 (C, C-8), 40.2 (C, C-4), 39.8 (CH2, C-1), 37.9 (C, C-10), 34.4 (CH2, C-22), 34.0 (CH2, C-7), 31.3 (CH2, C-21), 29.0 (CH2, C-15), 28.6 (CH3, C-29), 28.5 (CH3, C-23), 27.0 (CH2, C-2), 26.2 (CH3, C-27), 24.5 (CH2, C-11), 24.1 (CH2, C-16), 19.3 (CH2, C-6), 17.7 (CH3, C-26), 17.0 (CH3, C-24), 16.0 (CH3, C-25); 1 H and 13

4. 7 .

 7 Zebirioside C, 3-O-α-L-rhamnopyranosyl-(1→3)-[β-D-glucopyranosyl-(1→2)]-β-Dglucuronopyranosyl serjanic acid (3): White powder; [α]D 20 +7.5° (c 0.48, MeOH); 1 H NMR (600 MHz, Methanol-d4) of the aglycon: δH 5.32 (1H, t, J = 3.5 Hz, H-12), 3.72 (3H, s, H-31), 3.23 (1H, m, H-3), 2.72 (1H, dd, J = 13.5, 3.6 Hz, H-18), 2.03 (1H, td, J = 13.4, 3.3 Hz , Hα-16 ), 2.02 (1H, m, Hα-21), 1.95 (1H , m, Hα-19), 1.93 (2H, m, H-11), 1.91 (1H, m, Hα-2), 1.77 (1H, td, J = 13.5, 4.1 Hz, Hα-15), 1.75 (1H, m, Hβ-2), 1.69 (1H, t, J = 13.7 Hz, Hβ-19), 1.67 the metabolic activity of L. infantum promastigotes compared to the control. IC50 were calculated by non-linear regression analysis processed on dose-response curves, using TableCurve 2D V5 software. IC50 values represent the mean value calculated from three independent experiments.

Figure 1 :

 1 Figure 1: structures of compounds 1-14

Table 1 ;

 1 C NMR of the glycosidic part, see HRESIMS (positive-ion mode) m/z 1007.4835 [M+Na] + (calculated for C49H76O20Na, 1007.4828).

H (600 MHz) and13 C (151 MHz) NMR spectral data (δ in ppm) of osidic parts of saponins 7-12(MeOD)

Acknowledgment.

Financial support by CNRS, Conseil Regional Champagne Ardenne, Conseil General de la Marne, Ministry of Higher Education and Research (MESR) and EU-programme FEDER to the PlAneT CPER project is gratefully acknowledged.

then removed and DMSO (200 µL/well) was added. Absorbance was measured at 540 nm on a microplate spectrophotometer (Tecan infinite 200). All tests were performed in quadruplicate and results are expressed as inhibition % at 10 µg/mL or as IC50 (µM) when a dose effect was realized.

Hemolytic assay

The hemolytic activity of compounds 1-14 on sheep erythrocytes was measured on 96-wellmicroplate as previously reports with slight modifications [START_REF] Chwalek | Structureactivity relationships of some hederagenin diglycosides: Haemolysis, cytotoxicity and apoptosis induction[END_REF]. 25 µL of 10% sheep erythrocytes (Eurobio) suspension in phosphate-buffered saline (PBS, pH 7.4) was incubated with 500 µL of saponins dilutions (100, 75, 50, 40, 30, 25, 20, 10, 7.5, 5, 2.5 and 1 µg/ml) in PBS at 37 °C for 1h. The mixture was centrifuged (3000 rpm, 5 min) and the supernatant (200 µL) was transferred to 96-well microtiter plates. The hemoglobin content was measured at 540 nm by a multiwall spectrophotometer (FLUO STAR Omega).

Commercial saponins (Sigma-Aldrich) was used as positive control on the same conditions and all tests was performed in triplicate.

Antileishmanial assay

All molecules were first screened in vitro on the promastigote stage of L. infantum by determining their inhibitory concentrations 50% (IC50) and comparing them to the one of amphotericin B, chosen as antileishmanial reference-drug.

Leishmania species used in this study were L. infantum MHOM/MA/67/ITMAP-263 (CNR Leishmania, Montpellier, France) expressing luciferase activity.

The effects of the tested compounds on the growth of L. infantum promastigotes were assessed by Luciferase Assay. Briefly, promastigotes in log-phase in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine and antibiotics (100U/mL penicillin, 100 µg/mL streptomycin and 50µg/mL geneticin), were incubated at an average density of 106 parasites/mL in sterile 96-well plates with various concentrations of compounds dissolved in DMSO or MeOH (final concentration less than 0.5% v/v), in duplicate. Appropriate controls treated by DMSO, MeOH and amphotericin B (reference drug purchased from Sigma Aldrich) were added to each set of experiments. After a 72h incubation period at 24°C, each plate-well was then microscope-examined for detecting possible precipitate formation. To estimate the luciferase activities of promastigotes, 80 µL of each well are transferred in white 96-well plates, Steady Glow reagent (Promega) was added according to manufacter's instructions, and plates were incubated for 2 min. The luminescence was measured in Microbeta Luminescence Counter (PerkinElmer). Inhibitory concentration 50% (IC50) was defined as the concentration of drug required to inhibit by 50%


Dr Laurence Voutquenne
email: laurence.voutquenne@univ-reims.fr

Cécile Kokougan

Catherine Lavaud

Isabelle Pouny

Marc Litaudon

Triterpenoid saponins and acylated prosapogenins from Harpullia austro-caledonica

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Harpullia austro-caledonica Baillon (Sapindaceae) is a tree or a shrub originating from New Caledonia and growing in the rain forest. In the phylogenetic and taxonomic systems of this genus, this isolated species is next to the most primitive species H. pendula and H. arborea [START_REF] Leenhouts | An attempt towards a natural system of Harpullia (Sapindaceae)[END_REF]. The leaves consist of four to seven pairs of leaflets and the inflorescence is composed of yellow unisexual flowers that show an unusually wide range of variability [START_REF] Leenhouts | A taxonomic revision of Harpullia (Sapindaceae)[END_REF]. This species was selected as a part of a screening program for potential cytotoxic compounds from plants collected in New Caledonia. Primary chemical studies showed the presence of alkaloids and saponins. The ethanolic extract from the stem bark of H. austro-caledonica exhibited in vitro a cytotoxic activity against KB cells (90% at 10g/ml). This paper reports on the isolation and structural elucidation of three new and structurally unusual saponins from the stem bark of this plant and of three prosapogenins obtained after acid hydrolysis of saponin crude extract.

Results and discussion

H. austro-caledonica was collected in the reserve of Amieu Pass and Table Unio in New Caledonia.

Dried and powered stem bark was extracted with boiling 80% methanol and the methanolic extract was concentrated and precipitated into acetone. The crude saponin precipitate was dialysed, chromatographed on a silicagel column and purified by reversed phase C-18 column chromatography. From the most polar fraction, three new compounds 1, 2 and 3 were obtained in a pure state after a final purification by preparative TLC. Acid hydrolysis of the saponin extract gave a mixture of three major prosapogenins 4, 5 and 6 purified by silica gel column chromatography. 1). The MS 3 fragmentation of ion at m/z 975 led to ions at m/z 829 [M-162-146]and 667 [M-162-308]attributed to the losses of the terminal 6-desoxy-hexose and of a disaccharide made of 6-desoxy-hexose and hexose, respectively. The MS 4 of ion fragment at m/z 829 gave rise to a product ion at m/z 505 [genin-H] -corresponding to the loss of a third hexose and suggesting a molecular formula of C 30 H 50 O 6 for the aglycone moiety (M r of 506 amu).

Protoaescigenin was identified as the aglycone of saponin 1 by analysis of 1 H and 13 C-NMR spectra (Table 2) and from observation of connectivities in COSY, HSQC and HMBC spectra. The set of data was in full agreement with those reported in the literature [START_REF] Chen | Studies on the constituents of Xanthoceras sorbifolia BUNGE. III. Minor Prosapogenins from the Fruits of Xanthoceras sorbifolia BUNGE[END_REF][START_REF] Mahato | 13 C NMR spectra of pentacyclic triterpenoids. A compilation and some salient features[END_REF]. The 1 H-NMR spectrum showed four methylene protons linked to oxygen bearing carbons which constituted two AX spin systems at  3. 67 and 4.13 (d,J=10 Hz,J=10 Hz,, two hydroxymethine protons at  3.27 (dd, coupling to two protons at  1.64 and 1.76, and at  4.57 (brs, W1/2 = 8 Hz, H-16) coupling to two protons at  1.89 (dd, J=15-4 Hz, H-15ax) and 1.43 (d, J=15 Hz, H-15eq), and two vicinal hydroxymethine protons corresponding to a trans-diequatorial diol which constituted an AB spin system at  3.79 (d,J=10 Hz,J=10 Hz,. In the ROESY experiment, the interaction observed between H-16 and H-28 confirmed the -equatorial orientation for H-16 and the observation of ROE between -axial H-2 at  1.76 and H-24 confirmed the configuration of C-4 with a -axial orientation for C-24. The -axial orientation of H-21 and the -axial position for H-As shown from analysis of ESI-MS, the sugar part of 1 consisted of four residues, with anomeric carbons at  100, 101.7, 102.4 and 103.4 in the 13 C NMR spectrum, attached to proton doublets at  5. 38,4.23,4.32 and 4.66 respectively (HSQC exp.). The proton system of each sugar was completely assigned on the basis of COSY and TOCSY experiments (Table 3). The methyl carbon at  16.9 which correlated in HSQC spectrum with a methyl proton doublet at  1.29 ppm (J=6 Hz) was assigned to an -L-rhamnose whose anomeric proton appeared as a narrow doublet at  5.38 (J=1.3 Hz). The sugars with their anomeric protons at  4.23 (J=7.7 Hz), 4.32 (J=7.7 Hz) and 4.66 (J=7.8 Hz) corresponded to three hexoses with hydroxymethyl carbons at  61.1, 61.4 and 60.6, respectively, and were identified as three -D-glucoses (Table 3). Sequencing of the two sugar chains was achieved by analysis of HMBC and ROESY experiments. HMBC spectrum showed correlations between C-28 (C 77) of protoaescigenin and H-1 of a glucose (H 4.23) and between C-2 ( 80.3) of this glucose and H-1 (H 4.66) of a second glucose unit, thus confirming the presence of a sophorose residue attached to C-28 of protoaescigenin. The HMBC cross-peaks observed between C-24 (C 71.1) of the protoaescigenin and H-1 of the third glucose unit (H 4.32) and between C-1 ( 100) of the rhamnose and H-2 of this glucose (H 3.41), allowed to locate a neohesperidosyl chain at C-24 of the aglycone. The C-3 position of the protoaescigenin part was not substituted as proven by its carbon chemical shift at  79 [START_REF] Chen | Studies on the constituents of Xanthoceras sorbifolia BUNGE. III. Minor Prosapogenins from the Fruits of Xanthoceras sorbifolia BUNGE[END_REF]. These particular sequences were confirmed by the observation of ROE interactions between protons involved in the interglycosidic linkages. Thus, the structure of saponin

1 is 24-O-[-L-rhamnopyranosyl-(1->2)-- D-glucopyranosyl]-28-O-[-D-glucopyranosyl-(1->2)--D-glucopyranosyl]-protoaescigenin.
Formulae 1, 2 and 3 near here Tables 1, 2 and 3 near here Saponin 2 exhibited an intense [M+Na] + ion peak at m/z 1145 in its positive ESI-MS and a molecular ion was detected at m/z 1121 in the negative mode corresponding to a molecular formula of C 54 H 90 O 24 (Mr of 1122). All the fragmentations observed in the MS/MS n experiments followed the pattern observed in saponin 1, thus suggesting the similarity of the sugar chains and a missing oxygen atom in the genin (Table 1).

The lack of a substitution of C-16 in 2 was suggested by the absence of NMR signal for any deshielded signal for a H-16 proton near  4.57 ppm and by the replacement of the C-16 hydroxymethine of 1 by a supplementary methylene at  42.2 (Table 2). Thus the genin was identified as 16-desoxy-protoaescigenin. Proton and carbon spectra, and 2D NMR experiments (COSY, HSQC and HMBC) permitted assignments of all proton and carbon signals for four sugars (Table 3). The TOCSY and ROESY experiments indicated that the osidic chains of saponins 1 and 2 were identical. The structure of the new saponin 2 was deduced to be 24

-O-[-L- rhamnopyranosyl-(1->2)--D-glucopyranosyl]-28-O-[-D-glucopyranosyl-(1->2)--D- glucopyranosyl]-16-desoxy-protoaescigenin.
The positive ESI-MS of the new compound 3 gave a quasi-molecular ion at m/z 1176 amu

[M+Na+H] + and the negative ESI-MS a molecular ion at m/z 1151 in agreement with a molecular formula of C 54 H 88 O 26 (C 54 H 90 O 25 for saponin 1). The fragmentations were identical to those observed for saponins 1 and 2 and suggested the presence of the same osidic part (Table 1).

Comparison of the 1 H NMR spectra of compounds 1 and 3 showed signals for six angular methyl groups and for four hydroxymethine protons H-3, H-16, H-21 and H-22 in both compounds (Table 2) and absence of the AX spin system of H-24 protons in saponin 3. HMBC spectrum of saponin 3 displayed a correlation between H-23 at  1.49 ppm and one carbonyl at  175 ppm suggesting that C-24 position of the genin was a carboxylic ester. The chemical shift of C-23 at  22.9 also suggested that this methyl group was in an -equatorial position. The observation of ROE between -axial H-3 and H-23 confirmed the configuration of C-4 with a -axial carboxylic ester C-24. The aglycone of saponin 3 was concluded to be the new 3, 16, 21, 22, 28-pentahydroxy-olean-12-en-24-oic acid named 24-oxo-camelliagenin D.

The 1 H and 13 C NMR spectra of 3 showed that this saponin contained four sugars with signals of anomeric carbons at C 93.9, 96, 102.1 and 104 with corresponding anomeric protons at H 5.65, 5.34, 4.24 and 4.66, respectively (Table 3). Analysis of 2D experiments (COSY, TOCSY and HSQC) permitted assignments of osidic 1 H and 13 C signals and identification of one -D-glucose with shielded anomeric carbon (93.9) and deshielded anomeric proton ( 5.65) engaged in an ester linkage with the C-24 of genin, of one -L-rhamnose and of two -D-glucoses. The ROESY experiment provided information about glycosidic linkages and showed that the sugar chains were identical as in saponins 1 and 2. Thus, saponin

3 was 24-O-[-L-rhamnopyranosyl-(1->2)--D- glucopyranosyl]-28-O-[-D-glucopyranosyl-(1->2)--D-glucopyranosyl]-24-oxo-camelliagenin D.
These three compounds 1, 2 and 3 represent a new type of bidesmosidic triterpene saponins and to the best of our knowledge, the presence of a single sugar chain at C-24 instead of C-3 is unique.

The positive ESI-MS of compound 4 gave a quasi-molecular ion peak at m/z 693 [M+Na] + corresponding to a Mr of 670 and a molecular formula of C 40 H 62 O 8 . The MS 2 displayed two fragmentation peaks at m/z 593 and 493 corresponding to the losses of two C5H8O2 units, identified as angelic acids by analysis of 1 H and 13 C NMR spectra (Table 4) [START_REF] Dizes | Harpuloside a triterpenoid saponin from Harpullia ramiflora[END_REF].

The presence of protoaescigenin as aglycone in the structure of prosapogenin 4 was deduced from the analysis of its 1 H and 13 C NMR spectra (Table 4). The deshielded vicinal H-21 and H-22 protons at  5.80 and 5.40 suggested that these positions were esterified by the angeloyl groups.

HMBC experiment confirmed this assumption by the correlations observed between H-21 (H 5.80) and the carbonyl (C 167.5) of the first angeloyl group and between H-22 (H 5.40) and the carbonyl (C 169.2) of the second angeloyl residue. Prosapogenin 4 was identified as 21, 22-O-diangeloylprotoaescigenin previously described in Harpullia ramiflora [START_REF] Dizes | Harpuloside a triterpenoid saponin from Harpullia ramiflora[END_REF].

Formulae 4, 5 and 6 near here [START_REF] Sati | Triterpenoids of Aesculus indica[END_REF] and Maesa ramentacea [START_REF] Tuntiwachwuttikul | A triterpenoid saponin from Maesa ramentacea[END_REF]. Its positive ESI-MS showed a quasi-molecular ion [M+Na] + at m/z 677 in agreement with a molecular formula of C 40 H 62 O 7 . The MS 2 fragmentation gave two ion fragments at m/z 577 and 477 attributed to the losses of two angelic acids as in prosapogenin 4. Comparison of 1 H NMR spectrum of compounds 4 and 5 indicated that 5 possessed one supplementary methyl singlet at  0.81. In the 13 C NMR spectrum of 5, only one hydroxymethylene was detected at  63.6 (C-28) and a supplementary methyl was observed at  15.8 (Table 4). Consequently, the genin was identified as barringtogenol C [START_REF] Ito | Two new triterpenes from Camellia sinensis and Camellia sasanqua[END_REF][START_REF] Mahato | 13 C NMR spectra of pentacyclic triterpenoids. A compilation and some salient features[END_REF] 4). The ROESY experiment confirmed this assignment with a ROE effect between axial H-3 and equatorial H-23 both in an -orientation. The configurations of C-16, C-21, C-22 and C-28 were consistent with those reported for camelliagenin D [START_REF] Ito | Two new triterpenes from Camellia sinensis and Camellia sasanqua[END_REF]. Thus, the new prosapogenin 6 was identified as 21, 22-O-diangeloyl camelliagenin D.

Work is in progress to identify the saponins containing these prosapogenins.

Experimental

General

Plant material

Stem bark of H. austro-caledonica was collected in rain forest at an elevation of 600 m in the reserve of Amieu Pass and Table Unio, New Caledonia, in March 1997. The specimen of the plant (LIT 0250) is deposited in the herbarium of the botanical laboratory at the CNRS centre of Noumea (New Caledonia).

Extraction and isolation

Dried and powered stem bark (1110 g) was macerated in 20% aq. MeOH (10 l) for 17 h and boiled for 3 h. The hydromethanolic extract was filtered, evaporated and freeze-dried to give a residue (106 g) which was suspended in MeOH (400 ml). The methanolic fraction was added to 2 l of Me2CO and the ppt. was filtered and dried over KOH in vacuo. This dried ppt. (57 g) was dissolved in pure H2O and dialysed against H2O in seamless cellulose tubing under agitation during 48 h. The contents of the tubes were freeze-dried to afford 32 g of a saponin mixt. (yield 3%).

Two aliquotes of the saponin mixt. (1 g and 3 g) were fractionated on a silica gel CC, using a gradient of CHCl3-MeOH-H 2 O (8:2:0 to 15:10:1) for the first and (7:3:0 to 60:40:5) for the second.

Saponin 1

White powder.  21°D : -13.6° (MeOH, c 0.45); 1 H and 13 C NMR (CD 3 OD) see Tables 2 and3 Table 1.

Saponin 2

White powder.  21°D : -7.46° (MeOH, c 0.25); 1 H and 13 C NMR (CD 3 OD) see Tables 2 and3 1.

Saponin 3

White powder.  21°D : 0° (MeOH, c 0.108,); 1 H and 13 C NMR (CD 3 OD) see Tables 2 and3 1.

Acid hydrolysis of saponins

The crude saponin (500 mg) was dissolved in 16 ml of a mixture (1:1) of 6.5 % HClO 4 and H 2 SO 4 0.02N, and heated at 140°C in a sealed tube for 2 h. After cooling, the sapogenin ppt. was filtered, rinsed with H 2 O and dried in vacuo over P 2 O 5 . The acid aq. layer was neutralised with KOH 0.5 M and freeze-dried. Four sugars were identified with authentic samples by TLC in MeCOEt-iso-PrOH-Me 2 CO-H 2 O (20:10:7:6) as glucose, galactose, rhamnose and xylose. The sapogenin mixt.

(260mg) was purified by silica gel CC using a gradient of CHCl3-MeOH (100:0 to 93:7).

Prosapogenins 5 (3.7 mg) and 6 (3 mg) were found in frs. [7][8][9][10][11][12][13] eluted with CHCl3 and purified by silica gel CC using a gradient of hexane-AcOEt (9:1 to 7:3). Frs. [27][28][29][30] eluted with (99:1) contained prosapogenin 4 (33.2 mg).

21,22-O-diangeloyl protoaescigenin 4

 21°D : +14.9° (CHCl 3, c 1); 1 H and 13 C NMR (CDCl 3 ) see table 4;

O CH 3 H H 3 C O CH 3 H H 3 C 21 22 4 R = CH 2 OH 5 R = CH 3 6 R = CHO
2 ([M-H] -) [M-H-rha] - 991 975 - [M-H-glc] - 975 959 989 [M-H-rha-glc] - 829 813 843 MS 3 ([M-H-rha] -) [M-H-rha-glc] - 829 813 [M-H-rha-2glc] - 667 651 MS 3 ([M-H-glc] -) [M-H-rha-glc] - 829 [M-H-rha-2glc] - 667 MS 3 ([M-H-rha-glc] -) [M-H-rha-2glc] - 681 [genin-H] - 519 MS 4 ([M-H-rha-glc] -) [M-H-rha-2glc] - 667 651 [genin-H] - 505 489 MS 4 ([M-H-rha-2glc] -) [genin-H] - 519 a rha, rhamnopyranosyl (C6H11O4); glc, glucopyranosyl (C6H11O5)

  The sugars were identified by TLC as D-glucose, D-galactose, L-rhamnose, L-arabinose and D-xylose. The positive ESI-MS of saponin 1 gave a quasi-molecular ion peak at m/z 1161 [M+Na] + and in the negative ESI-MS a molecular ion was detected at m/z 1137 [M-H]in agreement with a M r of 1138 amu (C 54 H 90 O 25 ). The MS 2 experiment of [M-H]ion gave negative fragments at m/z 991 [M-146]and 975 [M-162]attributed to the losses of a terminal 6-desoxy-hexose and a terminal hexose, respectively (Table

  also named theasapogenol B, which was substituted by two angeloyl groups on C-21 and C-22 in prosapogenin 5. Prosapogenin 6 presented in positive ESI-MS an ion peak [M+Na] + at m/z 691 (C 40 H 60 O 8 ) and two ion fragments due to the losses of two angeloyl groups (m/z 591 and 491). The 1 H and 13 C-NMR spectra of prosapogenin 6 showed six quaternary methyls, two hydroxymethines at C-3 and C-16 positions, two hydroxymethines at C-21 and C-22 positions which were deshielded by esterification with angeloyl groups, one AX spin system (H-28) at H 2.92 and 3.27 (d, J=10.9 Hz) and C 63.7. Deshielded signals at H 9.80 and C 207.9 were detected and analysed for an aldehydic fonction at position C-24 (Table

1

  H and 13 C NMR spectra were recorded on Bruker Avance DRX 500 ( 1 H at 500 MHz and 13 C at 125 MHz); 2D experiments were performed using standard Bruker microprograms. ESI-MS and MS-MS experiments were recorded on Bruker Esquire LC-MS instrument. Optical rotations were measured on a Perkin-Elmer 241 Polarimeter.

  Frs.[15][16][17][18][19][20][21] of the first column and frs.[10][11][12][13][14][15][16][17][18][19][20][21] of the second column eluted with CHCl3-MeOH (7:3) were similar in comparing by TLC (CHCl3-MeOH-H 2 O 60:40:5) and were purified on a reversed-phase RP-18 CC using a gradient of MeOH-H 2 O (55:45 to 8:2). Frs. [14-16] eluted with MeOH-H2O (55:45) was purified by preparative TLC in CHCl3-MeOH-H 2 O (60:40:5) to give saponin 1 (7.8 mg). Frs.[19][20][21][22][23][24][25][26][27][28][29] eluted with (55:45), contained saponins 2 (4.7 mg) and 3 (2.4 mg), which were purified by silica gel CC eluted with CHCl3-MeOH-H 2 O and followed by prep. TLC (CHCl3-MeOH-H 2 O, 60:40:5).

  ; ESI-MS (positive ion mode) m/z 1161 [M+Na] + ; ESI-MS-MS : MS 2 (1161) m/z 1015 [(M+Na)rha] + , 997 [(M+Na)-glc] + , 853 [(M+Na)-rha-glc] + ; MS 3 (1015) m/z 691 [(M+Na)-rha-2glc] + ; ESI-MS (negative ion mode) m/z 1137 [M-H] -, 991 [M-H-rha] -, 975 [M-H-glc] -; ESI-MS-MS : see

  ; ESI-MS (positive ion mode) m/z 1145 [M+Na] + , ESI-MS-MS : MS 2 (1145) m/z 999 [(M+Na)rha] + , 983 [(M+Na)-glc] + , 837 [(M+Na)-rha-glc] + , 675 [(M+Na)-rha-2glc] +; ESI-MS (negative ion mode) m/z 1122 [M] -. ; ESI-MS-MS : see Table

  ; ESI- MS (positive ion mode) m/z 1176 [M+Na+H] + ; ESI-MS-MS : MS 2 (1176) m/z 1129 [(M+Na)rha] + , 867 [(M+Na)-rha-glc] + , MS 3 (867) m/z 705 [(M+Na)-rha-2glc] +, MS 4 (705) m/z 543 [genine+Na] + ; ESI-MS (negative ion mode) m/z 1152 [M] -. ; ESI-MS-MS : see Table

  ESI-MS (positive ion mode) m/z 677 [M+Na] + ; ESI-MS-MS (677) m/z 577 [(M+Na)-Ang] + , 477 [(M+Na)-2Ang] + . 3.9. 21,22-O-diangeloyl barringtogenol C 5  21°D : +11° (CHCl 3 , c 0.308); 1 H and 13 C NMR (CDCl 3 ) see table 4; ESI-MS (positive ion mode) m/z 691 [M+Na] + ; ESI-MS-MS (691) m/z 591 [(M+Na)-Ang] + , 491 [(M+Na)-2Ang] + ; ESI-MS (negative ion mode) m/z 667 [M-H] -. 3.10. 21,22-O-diangeloyl camelliagenin D 6  21°D +27° (CHCl 3 , c 0.1); 1 H and 13 C NMR (CDCl 3 ) see table 4; ESI-MS (positive ion mode) m/z 693 [M+Na] + ; ESI-MS-MS (693) m/z 593 [(M+Na)-Ang] + , 493 [(M+Na)-2Ang] + ; ESI-MS (negative ion mode) m/z 669 [M-H] -.

Table 4

 4

	near here

Table 1

 1 ESI-MS and MS n data of saponins 1-3 (m/z) a

	MS n (parent ion)	Fragments	1	2	3
	ESI-MS +	[M+Na] +	1161	1145	1176
	ESI-MS -	[M-H] -	1137	1121 (obs. at 1122)	1151 (obs. at 1152)
	MS

Table 2 1

 2 H and 13 C NMR data of genin part of saponins 1-3 in CD3OD

				1				2				3
	Genin	 H (ppm)	m	J (Hz)	C (ppm)	 H (ppm)	m	J (Hz)	C (ppm)	 H (ppm)	m	J (Hz)	C (ppm)
	1 eq 1 ax 2 eq	1,70 1.03 1.64	m m m	---	38.7 26.6	1.69 1.03 1.65	m m m	---	38.5 26.6	1.77 1.10 1.69	m m dm	--12	39 na a
	2 ax	1.76	m	-		1.76	m	-		2.30	dd	12/3.7
	3	3.27	dd	12/4.8	79	3.27	m	-	79	3.17	dd	12/4.5	78.5
	4	-	-	-	42.6	-	-	-	42.6	-	-	-	49
	5 6 ax	0.86 1.61	d m	8.3 -	56.1 19.2	0.93 1.62	d m	12 -	56 19	1.03 1.84	m m	--	56.7 na a
	6 eq	1.75	m	-		1.76	m	-		1.58	m	-
	7eq	1.42	m	-	33	1.40	m	-	33.8	1.42	m	-	na a
	7ax	1.59	m	-		1.54	m	-		1.60	m	-
	8	-	-	-	39.4	-	-	-	39.6	-	-	-	40
	9	1.68	m	-	46.7	1.62	m	-	47.7	1.66	dd	15/8.5	47
	10	-	-	-	36.5	-	-	-	36.5	-	-	-	37.1
	11	1.93	m	-	23.5	1.94	m	-	23.3	1.94	m	-	23.5
	12	5.31	brt	3	122.9	5.27	brt	3.5	123.2	5.31	brt	3	123
	13	-	-	-	142.1	-	-	-	143	-	-	-	142.3
	14	-	-	-	41.1	-	-	-	41.4	-	-	-	41.2
	15 ax	1.89	dd	15/4	33	-	-	-	25	1.90	dd	14.5/5	32.7
	15 eq	1.43	d	15	-	-	-	-		1.44	dm	14.5
	16	4.57	brs	W1/2 = 8	66.3	-	-	-	42.2	4.58	brs	W1/2 = 8	66.3
	17	-	-	-	45.7	-	-	-	na a	-	-	-	na a
	18	2.18	dd	13.5/4.5	41.3	2.16	dd	15/4.1	41.5	2.17	dd	14/7.7	41.1
	19 ax	2.52	t	13.5	47.4	1.98	m	-	46	2.51	t	13.5	47.3
	19 eq	1.06	m	-	-	1.15	dd	14/4.1		1.05	dd	13.5/4.5
	20	-	-	-	35.1	-	-	-	35.4	-	-	-	35.1
	21	3.95	d	10	77.5	3.29	d	10	76.2	3.95	d	9.6	77.6
	22	3.79	d	10	77.7	3.74	d	10	76.2	3.79	d	9.6	77.6
	23	1.22	s	-	22.4	1.22	s	-	22.4	1.49	s	-	22.9
	24	4.13	d	10	71.1	4.14	d	10	70.7	-	-	-	175
	24'	3.67	d	10	-	3.67	d	10
	25	1.04	s	-	14.7	1.03	s	-	14.6	0.96	s	-	13
	26	0.97	s	-	16	1.01	s	-	15.9	0.99	s	-	15.9
	27	1.44	s	-	26	1.21	s	-	25	1.45	s	-	25.9
	28	3.66	d	10	77	3.75	d	10	75	3.65	d	10.5	77.2
	28'	3.45	d	10	-	3.66	d	10		3.46	d	10.5
	29	0.97	s	-	28.7	0.98	s	-	28.6	0.93	s	-	28.7
	30	0.94	s	-	18	0.92	s	-	17.8	0.97	s	-

a na : not assigned

Acknowledgments

The authors are grateful to Dr T. Sevenet, ICSN, Gif-sur-Yvette, France, for providing plant material and to Dr G. Massiot, Laboratoires Pierre Fabre, France, for revising the manuscript and for arranging mass spectra.

-O-[-L-rhamnopyranosyl-(1->2)--D-glucopyranosyl]-28-O-[-D-glucopyranosyl-(1->2)--D-glucopyranosyl]-16desoxyprotoaescigenin, 24-O-[-L-rhamnopyranosyl-(1->2)--D-glucopyranosyl]-28-O-[-Dglucopyranosyl-(1->2)--D-glucopyranosyl]-24-oxo-camelliagenin D. of the saponin extract together with a new prosapogenin identified as 21,22-di-O-angeloyl camelliagenin D.

The structures were established using one-and two-dimensional NMR and mass spectrometry.


Dr Laurence Voutquenne
email: laurence.voutquenne@univ-reims.fr

Pauline Guinot

Clément Froissard

Odile Thoison

Marc Litaudon

Catherine Lavaud

Haemolytic acylated triterpenoid saponins from Harpullia austro-caledonica

Keywords: Harpullia austro-caledonica, Sapindaceae, acylated saponins, protoaescigenin

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

1 Nine acylated triterpenoid saponins were isolated from the stem bark of Harpullia austro-caledonica. Their structures were established by spectroscopic methods. The saponin mixture and major compounds showed high haemolytic activity.

Haemolytic acylated triterpenoid saponins from Harpullia austro-caledonica

Laurence Voutquenne* a , Pauline Guinot a, b , Clément Froissard a , Odile Thoison c , Marc Litaudon c , Catherine Lavaud a a Laboratoire de Pharmacognosie, FRE CNRS 2715, Bât. 18, BP 1039, 51097

Introduction

In a continuation of our study on saponin constituents of plants of the Sapindaceae family and particularly on the chemotaxonomy of the genus Harpullia, we have examined the stem bark of Harpullia austro-caledonica Baillon (Sapindaceae). The genus Harpullia consists at least of 37 species distributed in Indo-Malaysia, Australia and the Pacific islands [START_REF] Mabberley | The Plant-Book[END_REF]. Three species have been studied previously, H. pendula [START_REF] Khong | Chemical constituents of Harpullia pendula[END_REF], H. ramiflora [START_REF] Dizes | Harpuloside, a triterpenoid saponin from Harpullia ramiflora[END_REF] and H. cupanioides [START_REF] Voutquenne | Saponins from Harpullia cupanioides[END_REF]. H. austro-caledonica is a tree or a shrub originating from New Caledonia and growing in the tropical rain forest [START_REF] Morat | The flora of new Caledonia's calcareous substrates[END_REF]. In the phylogenetic and taxonomic systems of this genus, this isolated species is next to the most primitive species H. pendula and H. arborea [START_REF] Leenhouts | An attempt towards a natural system of Harpullia (Sapindaceae)[END_REF]. The leaves consist of four to seven pairs of leaflets and the inflorescence is composed of yellow unisexual flowers that show an unusually wide range of variability [START_REF] Leenhouts | A taxonomic revision of Harpullia (Sapindaceae)[END_REF]. This species was selected as a part of a screening program for potential cytotoxic compounds from plants collected in New Caledonia. In a previous chemical study, we isolated three unusual bidesmosidic saponins, along with three prosapogenins, obtained after acid hydrolysis (Voutquenne et al. 2002a). This paper reports on the isolation and structural elucidation of eight new monodesmosidic saponins (1-8) from the stem bark of this plant along with the known saponin, harpuloside 9, previously isolated from H. ramiflora [START_REF] Dizes | Harpuloside, a triterpenoid saponin from Harpullia ramiflora[END_REF]. The ethanolic extract from the stem bark of H. austro-caledonica exhibited in vitro cytotoxic activity against KB cells (90% at 10g/ml). The haemolytic activity of the saponin mixture was tested and showed an activity 10 fold higher than the dialysed reference saponin Sigma ® . Pure saponins 2, 3 and the mixture of saponins 67 were tested and are highly haemolytic.

Results and discussion

H. austro-caledonica was collected in the special fauna reserve of Amieu Pass and Table Unio in New Caledonia. Dried and powered stem bark was extracted with boiling 80% methanol and the methanolic extract was concentrated and precipitated in acetone. The crude saponin precipitate was dialysed, chromatographed on a silica gel column and purified by reversed phase C-18 column chromatography. Eight new compounds 18 were obtained, accompanied by impure harpuloside 9 [START_REF] Dizes | Harpuloside, a triterpenoid saponin from Harpullia ramiflora[END_REF]. Acid hydrolysis of the saponin extract gave the previously isolated mixture of prosapogenins and sugars identified by TLC and by measurement of optical rotation as D-glucose, D-galactose, L-rhamnose, L-arabinose and D-xylose (Voutquenne et al., 2002a). compound was previously isolated from Harpullia ramiflora [START_REF] Dizes | Harpuloside, a triterpenoid saponin from Harpullia ramiflora[END_REF].

The positive ESI-LC-MS spectrum of compounds 1, 2 and 3 gave the same ion fragment at m/z 677

[prosapogenin+Na] + , attributed to the loss of the glycosidic chain at position 3 and in agreement with a molecular formula of C40H62O7Na. The prosapogenin was identified as 21,22-di-Oangeloylbarringtogenol C from analysis of its 1 H and 13 C-NMR spectra (Table 1) and from observation of connectivities in COSY, HSQC and HMBC spectra. The set of data was in full agreement with those reported in the literature [START_REF] Tuntiwachwuttikul | A triterpenoid saponin from Maesa ramentacea[END_REF][START_REF] Sati | Triterpenoids of Aesculus indica[END_REF]. The sugar part of 1 consisted of two residues with anomeric carbons at  105.5 and 106.2 in the 13 C NMR spectrum, attached to proton doublets at  4.5 and 4.56, respectively (HSQC). The proton system of each sugar was completely assigned on the basis of COSY and TOCSY experiments (Table 1). The sugar with its anomeric proton at  4.56 (J = 7.6 Hz) corresponded to a -Dgalactose with a hydroxymethyl carbon at  62.7 and characterised by an equatorial proton H-4 at  3.89 (J3,4 = 5 Hz). The second sugar with its anomeric proton at  4.5 (J = 7.1 Hz) was identified as a -D-glucuronic acid on the basis of its carbon resonances (Table 1). The deshielding of C-2' ( 83) of glucuronic acid suggested the point of linkage of the galactose. Sequencing of the disaccharidic chain was achieved by analysis of a ROESY experiment which showed ROE interactions between H-3 ( 3.22) of barringtogenol C and H-1' of the glucuronic acid ( 4.5), and between H-2' ( 3.57) of the glucuronic acid and H-1'' ( 4.56) of the galactose unit. Thus, saponin experiment. The sugar with its anomeric proton as a broad doublet at 5.08 (J 1.8 Hz) was identified as an -L-rhamnose, with a methyl proton doublet at  1.27 (J  6.2 Hz) which correlated in HSQC spectrum with a methyl carbon at  16.9. The sugars with their anomeric protons at  4.57 (J  7.4 Hz) and 4.61 (J  7.1 Hz) corresponded to a -D-galactose and a -D-glucuronic acid as in saponin 1 (Table 1). The downfield shifts of C-2' ( 78.6) and C-3' ( 85.9) of the glucuronyl moiety suggested the points of linkage of the trisaccharide chain (Table 1). The HMBC spectrum showed cross peaks between C-3 (C 92.2) of barringtogenol C and H-1' of the glucuronic acid (H 4.61) and between C-2' and C-3' of this glucuronic acid and H-1'' (H 4.57) of the galactose unit and H-1''' (H 5.08) of the rhamnose unit, respectively. This sequence was confirmed by the observation of ROE interactions between the protons involved in the interglycosidic linkages. Thus, with anomeric protons at  4.69 (J  7.6 Hz) and 4.51 (J 7.5 Hz), respectively (Table 1). The deshielded chemical shifts of the anomeric proton (H 5.29) and carbon (C 110.6) of the third glycosidic unit indicated a furanosyl ring. This third sugar was identified as an -L-arabinose as shown by the 13 C NMR data, which were in good agreement with those reported for -Larabinofuranoside [START_REF] Tezuka | Kinmoonosides AC, Three New Cytotoxic Saponins from the Fruits of Acacia concinna, a Medicinal Plant Collected in Myanmar[END_REF]. The cross peaks observed in the HMBC experiment between C-3 ( 91.9) of aglycone and H-1' of glucuronic acid and between C-2' ( 79.1) and C-3' ( 86.5) of this glucuronic acid and H-1" of galactose and H-1"' of arabinofuranose, respectively,

1 is 3-O--D-galactopyranosyl-(12)--D-glucuronopyranosyl-21,22-di-O-angeloyl barringtogenol C.
saponin 2 is 3-O--L-rhamnopyranosyl-(13)-[-D-galactopyranosyl-(12)]--D- glucuronopyranosyl-21,22-di-O-angeloylbarringtogenol C.
showed that saponin 3 is 3-O--L-arabinofuranosyl-(13)-[-D-galactopyranosyl-(12)]--D- glucuronopyranosyl-21,22-di-O-angeloylbarringtogenol C.
As previously isolated after acidic hydrolysis [START_REF] Dizes | Harpuloside, a triterpenoid saponin from Harpullia ramiflora[END_REF], Voutquenne et al. 2002a), from analysis of 1 H and 13 C-NMR, COSY, HSQC and HMBC spectra (Table 2). 3).

Thus, saponin with anomeric carbons at C 110.9 and 110.1 (HSQC), and of two CH2OH at C 63 indicated the presence of two terminal -L-arabinofuranosyl moieties (Table 3). The sugar attached to the genin was identified as a -D-glucuronic acid (H 4.56, C 105) disubstituted in positions 2' (C 80.9) and 3' (C 86.5) as in saponins 2and 3. The observation of ROE connectivities between H-3 of protoaescigenin and H-1' of glucuronic acid and between H-2' and H-3' of this glucuronic acid and H-1" (H 5.3) of the first arabinofuranose and H-1"' (H 5.19) of the second arabinofuranose

4 is 3-O--L-arabinofuranosyl-(12)--D-glucuronopyranosyl-21,22-di-O-
identified saponin 5 as 3-O--L-arabinofuranosyl-(13)-[-L-arabinofuranosyl-(12)]--D- glucuronopyranosyl-21,22-di-O-angeloylprotoaescigenin.
Compounds 6 and 7 were difficult to obtain in a pure state. The best separation was obtained by semi-prep. HPLC with an isocratic elution with 47 to 48% MeCN in H2O at pH 2.4 (TFA). The retention times were 24.9 min for saponin 6 and 25.8 min for saponin 7. 3).

The sequencing of the triglycosidic chain was achieved by analysis of HMBC correlations observed between H-1' of the glucuronic acid (H 4.51) and C-3 (C 92.2) of the genin, between H-1" of the arabinofuranose (H 5.28) and C-3' (C 86.8) of the glucuronic acid, and between H-1"' of the xylose (H 4.65) and C-2' (C 78.5) of the glucuronic acid (Table 3). Thus, saponin

6 is 3-O--L- arabinofuranosyl-(13)-[-D-xylopyranosyl-(12)]--D-glucuronopyranosyl-21,22-di-O- angeloylprotoaescigenin.
The 3). The analysis of HMBC correlations showed that saponin 7 is 3-

O--L-arabinofuranosyl-(13)-[-D-glucopyranosyl-(12)]--D-glucuronopyranosyl-21,22-di- O-angeloylprotoaescigenin.
As observed with ESI-MS experiments, saponins 4 and 8 were isomers. The fragmentation showed the presence of a terminal pentose identified by NMR as a -D-xylose (H 4.66), and of a hexosuronic acid identified as a -D-glucuronic acid (H 4.52) monosubstituted in position 2' (C 80.8). The ROE correlations showed that saponin

8 is 3-O--D-xylopyranosyl-(12)--D- glucuronopyranosyl-21,22-di-O-angeloylprotoaescigenin.
The haemolytic activity of the decoloured saponin mixture and of pure saponins 2, 3 and the mixture of 67 was assessed on sheep erythrocytes (10% suspension in phosphate buffer saline) using the method previously described (Voutquenne et al., 2002b). The saponin mixture was more active than the dialysed reference saponin mixture from Sigma ® (Sigma ® D) and than the pure tested saponins. 25% Haemolysis was obtained at 1 g/ml. The HD100 was determined at 5 g/ml and the HD50 was estimated at 2 g/ml. Saponins 2, 3 and the mixture 67 showed HD100 at 10, 5

and 10 g/ml and HD50 at 5, 2.5 and 3 g/ml, respectively (Figure 1). The haemolytic activity of saponin 3 was twice that of saponin 2, suggesting that an arabinofuranosyl moiety attached to position 3 of glucuronic acid is more effective than a rhamnosyl moiety. More saponins need to be tested to confirm this result. The quantities of the isolated pure saponins 1, 4, 5, and 8 were insufficient to allow the measurement of their individual haemolytic activities in order to complete structure-activity relationships.

Experimental

General experimental procedures

Plant material

Stem bark of H. austro-caledonica was collected in the rain forest at an elevation of 600 m in the special fauna reserve of Amieu Pass and

Extraction and isolation

Dried and powered stem bark (1110 g) was macerated in 20% aq. MeOH (10 l) for 17 h and boiled for 3 h. The hydromethanolic extract was filtered, evaporated and freeze-dried to give a residue (106 g) which was suspended in MeOH (400 ml). The methanolic solution was added to 2 l of Me2CO and the ppt. was filtered and dried over KOH in vacuo. This dried ppt. (57 g) was dissolved in pure H2O and dialysed against H2O in seamless cellulose tubing under agitation during 48 h. The contents of the tubes were freeze-dried to afford 32 g of a saponin mixt. (yield 3%).

Two aliquots of the saponin mixt. (1 g and 3 g) were fractionated on a silica gel CC, using a gradient of CHCl3-MeOH-H 2 O (8:2:0 to 15:10:1) for the first sample and (7:3:0 to 12:8:1) for the second sample. HPLC, using the same conditions, to give saponin 2 (rt  13.9 min, 2.2 mg).

Frs Frs. [22][23][24][25][26][27][28][29][30] (302 mg) of the second column, eluted with CHCl3-MeOH-H2O (30:20:1), were purified by silica gel CC, eluting with a gradient of CHCl3-MeOH-HCOOH (90:10:1 to 60:40:1).

Frs. [49][50][51][52][53][54][55][56][57], [62-68] and [76-88], eluted with (85:15:1), were purified by semi-prep. HPLC with 49 % B in 30 min to give saponins 6 (rt 19.7 min, 3.2 mg), 7 (rt 20.1 min, 3.2 mg), 8 (rt 21 min, 2 mg), 3 (rt 21.8 min, 1.2 mg), 1 (rt 25.9 min, 2 mg) and 2 (rt 27.9 min, 2 mg).

The different fractions containing the mixture of saponins 6-7 were gathered together and purified in twice by semi-prep. HPLC with 47% to 48% B in 30 min to give saponins 6 (rt = 24.9 min, 3.3 mg) and 7 (rt = 25.8 min, 1.3 mg).

Saponin

Saponin 7

1 H and 13 C NMR (CD 3 OD), see Tables 2 and3

Saponin 8

-3.76 (MeOH; c 0.13); 1 H and 13 C NMR (CD 3 OD), see Tables 2 and3; ESI-MS (negative

ion mode) m/z 977 [M-H] -; ESI-MS-MS : MS 2 (997) m/z 877 [M-H-100] -; 845 [M-H-132] -; 669 [M-H-308] -.

Haemolytic activity

This assay was performed as described previously (Voutquenne et al., 2002b). The 10% sheep erythrocyte suspension was obtained by dilution of a commercial 50% suspension from Biomerieux ® Lyon with phosphate buffer saline (PBS). 30 mg of the saponin mixt. was decoloured by VLC with CHCl3-MeOH-H 2 O (70:30:5) to eliminated the tannins. The purified saponin mixt.

and saponins 2, 3 and 67 were dissolved in PBS; the samples were prepared in triplicate with concentrations ranging from 1 to 50 g/ml. 25 l of erythrocytes diluted suspension were added to 1 ml of the sample and rapidly stirred. Absorbance of the supernatant was measured at 540 nm after 60 min of incubation and 5 min of centrifugation at 3000 rpm. HD50 and HD100 were the concentrations of sample which cause 50% and 100% of haemolysis, respectively. 1.84 q t (1.5) 20.9 1.84 q t (1.4) 20.9 1.84 q t (1.5) 20.9 1.84 q t (1.5) 20.9 1.84 q t (1.5) 20.9 6.10 qq (7.3-1.5) 139.5 6.10 qq (7.4-1.5) 139.5 6.10 qq (7.3-1.5) 139.5 6.10 qq (7.3-1.5) 139.5 6.10 qq (7.3-1.5) 139.5 4 1.95 dq (7.3-1.5) 16.0 1.95 dq (7.4-1.5) 16.0 1.95 dq (7.3-1.5) 16.0 1.95 dq (7.3-1.5) 16.0 1.95 dq (7.3-1.5) 16.0 5 1.86 q t (1.5) 20.9 1.86 q t (1.5) 20.9 1.86 q t (1.5) 20.9 1.86 q t (1.5) 20.9 1.86 q t (1.5) 20.9

Saponin 9 ,

 9 molecular formula C 57 H 88 O 22 (ESI-MS -: m/z 1123 [M-H] -) was identified as harpuloside, 3-O--L-rhamnopyranosyl-(13)-[-D-xylopyranosyl-(12)]--Dglucuronopyranosyl-21,22-di-O-angeloylprotoaescigenin, on the basis of its spectral data. This

Saponin 3

 3 exhibited a [M-H]molecular ion peak at m/z 1123 in the negative ESI-MS, in agreement with an M r of 1124 amu (C 57 H 88 O 22 ). The MS 2 experiment gave negative fragments at m/z 1023 [M-H-C5H8O2] -, 991 [M-H-132]and 923 [M-H-200]due to the losses of two angeloyl groups, and of a terminal pentose. These results indicated that saponin 3 contained a supplementary pentose unit relative to saponin 1. The 13 C NMR spectrum revealed the presence of three anomeric carbons at  104.5, 105.5 and 110.6, and their corresponding proton doublets were detected at  4.69, 4.51 and 5.29, respectively in the HSQC experiment. Analysis of 2D experiments (COSY, TOCSY and HSQC) revealed the presence of a terminal -D-galactose and a disubstituted -D-glucuronic acid

  observed with saponins 1-3, the positive ESI-LC-MS experiments of compounds 5-7 gave a common ion fragment at m/z 693 [prosapogenin+Na] + attributed to the loss of the glycosidic part. In addition, in the negative ESI-MS-MS of saponins 4-8, the MS 2 experiment of the [M-H]ion gave fragment at m/z 669 [M-H-glycosidic part] -, indicating a molecular formula of C 40 H 62 O 8 for the prosapogenin moiety, which was identified as 21,22-di-O-angeloylprotoaescigenin,

Saponin 4

 4 was found to have the molecular formula C 51 H 78 O 18 as deduced from the [M-H]molecular ion at m/z 977 (M r 978 amu) in the negative ESI-MS spectrum. The MS 2 experiment of this ion gave fragments at m/z 877 [M-H-C5H8O2] -, 845 [M-H-132]and 669 [M-H-308]corresponding to the losses of an angeloyl group, a terminal pentose and a disaccharide chain (C11H16O10) consisting of a pentose and a hexosuronic acid. The 1 H NMR spectrum of saponin 4 showed two anomeric protons at  4.53 (d, J  7.2 Hz) and 5.42 (brs) which correlated in the HSQC experiment with the anomeric carbons at  105 and 109.6, respectively. Analysis of 2D experiments (COSY, TOCSY and HSQC) permitted the identification of a terminal -L-arabinofuranose (H 5.42) linked to a -D-glucuronic acid (H 4.53) monosubstituted in position 2' (C 80.6) (Table

  angeloylprotoaescigenin.Comparison of the 1 H and 13 C NMR spectra of saponins 5 and 4 indicated that compound 5 possessed one supplementary glycosidic unit. The molecular ion peak observed at m/z 1109 [M-H]in the negative ESI-MS experiment, in agreement with an M r of 1110 amu (C 56 H 86 O 22 ), and the negative fragment at m/z 977 [M-H-132]showed that this sugar was a pentose. In the 1 H NMR and 13 C NMR spectra, the detection of two anomeric protons at H 5.19 (brs) and5.3 (d, J = 3.6 Hz)

  The negative ESI-MS experiment of compound 6 gave a molecular ion at m/z 1109 [M-H] -(C 56 H 86 O 22 ). The MS 2 of this ion gave the same negative fragments at m/z 977 [M-H-132]and 669 [M-H-440]observed for saponin 5 suggesting that 5 and 6 were isomers. Analysis of COSY and TOCSY experiments identified an -L-arabinofuranose (H 5.28, d, J = 2.2 Hz), a -D-xylose (H 4.65, d, J = 7.8 Hz), and a disubstituted -D-glucuronic acid (H 4.51, d, J = 7.6 Hz) (Table

  ESI-MS-MS experiments and the 1 H and 13 C NMR spectra showed for saponin 7 a set of signals corresponding to the major saponin (75 %) and signals of smaller intensity (25%) due to the occurrence of residual saponin 6. The negative ESI-MS experiment gave an intense molecular ion at m/z 1139 [M-H]for compound 7 (C 57 H 88 O 23 ) accompanied by a minor ion at m/z 1109 [M'-H]for the residue of 6. This result indicated that the difference corresponded to the presence of one hexose instead of a pentose in the glycosidic chain of saponin 7. The MS 2 of [M-H]at m/z 1139 gave negative fragments at m/z 1007 [M-H-132]due to the loss of a terminal pentose, and the MS 3 of this ion gave fragment at m/z 845 [M-H-132-162]suggesting a loss of a terminal hexose. Analysis of COSY, TOCSY and HSQC experiments showed the presence of a terminal -Larabinofuranose (H 5.24), a terminal -D-glucose (H 4.81, d, J = 7.9 Hz), and a disubstituted -Dglucuronic acid (H 4.49) (Table

1

  H and 13 C NMR spectra were recorded on Bruker Avance DRX 500 in CD3OD ( 1 H at 500 MHz and 13 C at 125 MHz); 2D experiments were performed using standard Bruker microprograms. ESI-LC-MS, ESI-MS and MS-MS experiments were recorded on a Finningan LCQ deca ion trap mass spectrometer (Finnigan MAT, San Jose, USA); For MS n experiments, the samples were introduced by direct infusion of a methanolic solution at a flow rate of 5µl min -1 . MALDI-TOF Voyager DE STR, Applied Biosystemes, Les Ulis-France Optical rotations were measured in MeOH with a Perkin-Elmer 241 Polarimeter. CC was carried out on Kieselgel 60 (63-200 mesh) Merck or LiChroprep RP-18 (40-63 m) Merck. HPLC was performed on a DIONEX apparatus equipped with an ASI-100 autosampler, a P580 pump, a diode array detector UVD 340S (212nm) and Chromeleon ® software. A C-18 DIONEX VYDAC (201SP510, 250  10 mm, 5 m) was used for semi-preparative HPLC with a binary eluent (solvent A, H2O (pH 2.4 with 0.025 % TFA); solvent B, MeCN) and a flow rate of 3 ml min -1 .

Frs. [ 11

 11 -14] (90 mg) of the first column and frs.[8][9] (200 mg) of the second column eluted with CHCl3-MeOH (7:3) were similar on TLC (CHCl3-MeOH-H 2 O, 12:8:1) and then were purified on a reversed-phase RP-18 CC using a gradient of MeOH-H 2 O (55:45 to 8:2). Frs. [10-25] eluted with MeOH-H2O (65:35) were purified by silica gel CC, eluting with a gradient of CHCl3-MeOH-HCOOH (90:10:1 to 70:30:1) and then finally purified by semi-prep. HPLC with a linear gradient of 50 to 51 % B in 20 min to give saponins 9 (rt  15.1 min, 4 mg), 6-7 (rt  15.6 min, 3.2 mg) and 5 (rt 17.9 min, 1.5 mg); Frs.[33][34][35][36][37][38] eluted with MeOH-H2O (65:35) were purified by semi-prep.

  /z 1161 [M+Na] + , 677 [M+Na-484] + ; ESI-MS (negative ion mode) m/z 1137 [M-H] -; ESI-MS-MS : MS 2 (1137) m/z 991 [M-H-146] -; ESI-MS (positive ion mode) m/z 1183 [(M-H+Na)+Na] + , 677 [M+Na-484] + ; ESI-MS-MS : MS 2 (1183) m/z 1083 [(M-H+Na)+Na-100] + , 1037 [(M-H+Na)+Na-146] + , 1021 [(M-H+Na)+Na-162] + , 999 [M+Na-162] + , 899 [M+Na-162-100] + , 853 [M+Na-162-146] + . 3.6. Saponin 3 -10.9 (MeOH; c 0.53); 1 H and 13 C NMR (CD 3 OD), see Table 1; ESI-LC-MS (positive ion mode) m/z 1147 [M+Na] + , 677 [M+Na-470] + ; ESI-MS (negative ion mode) m/z 1123 [M-H] -; ESI-MS-MS : MS 2 (1123) m/z 1023 [M-H-100] -, 991 [M-H-132] -, 923 [M-H-200] -; ESI-MS (positive ion mode) m/z 1169 [(M-H+Na)+Na] + , 677 [M+Na-470] + ; ESI-MS-MS : MS 2 (1169) m/z 1069 [(M-H+Na)+Na-100] + , 1037 [(M-H+Na)+Na-132] + , 1007 [(M-H+Na)+Na-162] + , 985 [M+Na-162] + , MS 3 (1069) m/z 969 [(M-H+Na)+Na-200] + , 937 [(M-H+Na)+Na-100-132] + , 907 [(M-H+Na)+Na-100-162] + , 675 [(M-H+Na)+Na-200-294] + , 577 [M+Na-100-470] + , MS 3 (1007) m/z 907 [(M-H+Na)+Na-162-100] + , 875 [(M-H+Na)+Na-162-132] + , 807 [(M-H+Na)+Na-162-200] + . 3.7. Saponin 4 -25.5 (MeOH; c 0.11); 1 H and 13 C NMR (CD 3 OD), see Tables 2 and 3; ESI-LC-MS (positive ion mode) m/z 1001 [M+Na] + , 693 [M+Na-308] + ; ESI-MS (negative ion mode) m/z 977 [M-H] -; ESI-MS-MS : MS 2 (977) m/z 877 [M-H-100] -, 845 [M-H-132] -, 669 [M-H-308] -; ESI-MS (positive ion mode) m/z 1023 [(M-H+Na)+Na] + , 923 [(M-H+Na)+Na -100] + , 693 [M+Na-308] + .3.8. Saponin 5-32.3 (MeOH; c 0.13); 1 H and 13 C NMR (CD 3 OD), see Tables2 and 3; ESI-LC-MS (positive ion mode) m/z 1133 [M+Na] + , 1001 [M+Na-132] + , 869 [M+Na-264] + , 693 [M+Na-440] + ; ESI-MS (negative ion mode) m/z 1109 [M-H] -, 977 [M-H-132] -; ESI-MS-MS : MS 2 (1109) m/z 977 [M-H-132] -, 669 [M-H-440] -; ESI-MS (positive ion mode) m/z 1155 [(M-H+Na)+Na] + , 693 [M+Na-440] + ; ESI-MS-MS : MS 2 (1155) m/z 1055 [(M-H+Na)+Na-100] + , 1023 [(M-H+Na)+Na-132] + , 715 [(M-H+Na)+Na-440] + , MS 3 (1023) m/z 923 [(M-H+Na)+Na-132-100] + , 891 [(M-H+Na)+Na-132-132] + , 693 [M+Na-440] + . MeOH; c 0.25); 1 H and 13 C NMR (CD 3 OD), see Tables 2 and 3; ESI-LC-MS (positive ion mode) m/z 1133 [M+Na] + , 1001 [M+Na-132] + , 693 [M+Na-440] + ; ESI-MS (negative ion mode) m/z 1109 [M-H] -; ESI-MS-MS : MS 2 (1109) m/z 977 [M-H-132] -, 669 [M-H-440] -; ESI-MS (positive ion mode) m/z 1155 [(M-H+Na)+Na-H)] + , 693 [M+Na-440] + ; ESI-MS-MS : MS 2 (1155) m/z 1055 [(M-H+Na)+Na-100] + , 1023 [(M-H+Na)+Na-132] + , 955 [(M-H+Na)+Na-200] + , 715 [(M-H+Na)+Na-440] + , MS 3 (1055) m/z 955 [(M-H+Na)+Na-200] + , 923 [(M-H+Na)+Na-100-132] + , MS 3 (1023) m/z 1005 [(M-H+Na)+Na-132-H2O] + , 923 [(M-H+Na)+Na-132-100] + , 715 [(M-H+Na)+Na-440] + , 693 [M+Na-440] + .

  ; ESI-LC-MS (positive ion mode) m/z 1163 [M+Na] + , 693 [M+Na-470] + ; ESI-MS (negative ion mode) m/z 1139 [M-H] -; ESI-MS-MS : MS 2 (1139) m/z 1007 [M-H-132] -, MS 3 (1007) m/z 907 [M-H-132-100] -, 845 [M-H-132-162] -, 669 [M-H-470] -; ESI-MS (positive ion mode) m/z 1185 [(M-H+Na)+Na] + , 693 [M+Na-470] + ; ESI-MS-MS : MS 2 (1185) m/z 1085 [(M-H+Na)+Na-100] + , 1053 [(M-H+Na)+Na-132] + , 715 [(M-H+Na)+Na-470] + , MS 3 (1085) m/z 985 [(M-H+Na)+Na-200] + , 953 [(M-H+Na)+Na-100-132] + , MS 3 (1053) m/z 1035 [(M-H+Na)+Na-132-H2O] + , 953 [(M-H+Na)+Na-132-100] + , 891 [(M-H+Na)+Na-132-162] + , 693 [M+Na-470] + .

Fig. 1 -

 1 Fig.1-Haemolytic activity of the saponin mixture of H. austro-caledonica relative to the pure saponins 2, 3 and the mixture of 6-7, and the commercial saponin from Sigma ® .

  The positive ESI-LC-MS of saponin 1 gave a quasi-molecular ion peak at m/z 1015 [M+Na] + and in the negative ESI-LC-MS a molecular ion was detected at m/z 991 [M-H]in agreement with a M r of 992 amu (C 52 H 80 O 18 ). The positive ESI-MS experiment showed a quasi-molecular ion peak at

	m/z 1037 [(M-H+Na)+Na] + and the MS 2 experiment of this ion gave positive fragments at m/z 937
	[(M-H+Na)+Na-100] + , 853 [M+Na-162] + and 677 [M+Na-338] + attributed to the losses of an
	angeloyl group (C5H8O2), a terminal hexose and a disaccharide moiety C12H18O11, consisting of a
	hexosuronic acid and a hexose, respectively.

Table Unio ,

 Unio New Caledonia, in March 1997. The specimen of the plant (LIT 0250) is deposited in the herbarium of the Botany and Plant Ecology Department at the Research Institute for the Development (IRD) of Noumea (New Caledonia).

Table 2 1

 2 H and 13 C NMR data of the prosapogenin part of saponins 48

		4			5			6			7			8
		H		C	H		C	H		C	H		C	H		C
	Protoaescigenin
	3	3.45 m	91.7	3.42 m	92.8	3.34 m	92.2	3.42 m	92.8	3.37 m	91.7
	12 5.42 m (w1/2 =11.5)	125.2	5.41 m (w1/2 =11.5) 125.2 5.41 brt (3.4)	125.1 5.41 m (w1/2 =11.5)	125.2	5.41 brt (3.5)	125.2
	13	-		143.0	-		143.1	-		143.0	-		143.0	-		143.0
	15 1.38	dd (11-3.8)	34.8	1.39	brd (14.5)	34.8	1.39	brd (15.2)	34.8	1.39	brd (15)	34.8	1.39	brd (14.5)	34.8
		1.71	dm (11)		1.71	brd (14.5)		1.72	m		1.72	m		1.71	dd (14.2-4.3)
	16 4.01 m (w1/2 =7.6)	69.7	4.01 m	69.7	4.01 m (w1/2 =6.7)	69.7	4.02 m (w1/2 =6.6)	69.7	4.02 m (w1/2 =7.4) 69.7
	18 2.66 dm (11.8)	40.8	2.66 dm (13.7)	40.8	2.66 dd (13.7-2.8)	40.8	2.66 dd (12-4.2)	40.8	2.66 dm (12.1)	40.8
	19 1.22	dm (11.8)	47.8	1.22	dm (13.7)	47.9	1.22	m	47.8	1.22	m	47.8	1.22	dd (12.1-11.4)	47.8
		2.72	t (11.8)		2.72	t (13.7)		2.72	t (13.3)		2.72	t (12)		2.72	t (11.4)
	21 6.02 d (10.1)	79.8	6.02 d (10)	79.8	6.03 d (10.2)	79.8	6.02 d (10.1)	79.8	6.02 d (10.2)	79.8
	22 5.61 d (10.1)	74.3	5.61 d (10)	74.3	5.60 d (10.2)	74.3	5.60 d (10.1)	74.3	5.61 d (10.2)	74.3
	23 1.26 s	23.1	1.26 s	23.1	1.21 s	22.6	1.21 s	22.8	1.21 s	22.7
	24 3.37	d (11.7)	64.5	3.36	d (11.7)	64.4	3.23	d (11.4)	63.8	3.24	d (11.1)	64.1	3.25	d (11.8)	63.8
		4.09	d (11.7)		4.10	d (11.7)		4.09	d (11.4)		4.09	d (11.1)		4.09	d (11.8)
	25 0.96 s	16.3	0.95 s	16.3	0.90 s	16.1	0.91 s	16.2	0.91 s	16.1
	26 0.96 s	17.2	0.95 s	17.2	0.95 s	17.2	0.96 s	17.2	0.96 s	17.2
	27 1.52 s	27.7	1.52 s	27.7	1.52 s	27.7	1.52 s	27.7	1.52 s	27.7
	28 2.98	d (11.2)	64.5	2.98	d (11.7)	64.5	2.98	d (11.1)	64.5	2.98	d (11.2)	64.5	2.98	d (11.2)	64.5
		3.29	d (11.2)		3.29	d (11.7)		3.29	d (11.1)		3.29	d (11.2)		3.29	d (11.2)
	29 0.90 s	29.7	0.89 s	29.7	0.90 s	29.7	0.90 s	29.7	0.90 s	29.7
	30 1.12 s	20.3	1.11 s	20.3	1.11 s	20.3	1.11 s	20.3	1.11 s	20.3
	21-Angeloyl
	1	-		169.2	-		169.2	-		169.3	-		169.3	-		169.2
	2	-		129.4	-		129.4	-		129.4	-		129.4	-		129.4
	3	6.07 qq (7.3-1.5)	138.8	6.08 qq (7.4-1.4)	138.8 6.08 qq (7.3-1.5)	138.8 6.08 qq (7.3-1.5)	138.8	6.08 qq (7.2-1.5)	138.8
	4	1.93 dq (7.3-1.5)	16.0	1.93 dq (7.4-1.4)	16.0	1.93 dq (7.3-1.5)	16.0	1.93 dq (7.3-1.5)	16.0	1.92 dq (7.2-1.5)	16.0
	5

Table 3 1

 3 H and 13 C NMR data of the glycosidic part of saponins 48

		4			5			6			7			8
		H		C	H		C	H		C	H		C	H		C
	3--D-GlcA
	1' 4.53 d (7.2)	105.0 4.56 d (7.2)	105.0 4.51 d (7.6)	105.0 4.49 d (7.8)	105.0 4.50 d (7.2)	105.0
	2' 3.44 m	80.6	3.55 dd (8.5-7.2)	80.9	3.64 dd (8.9-7.7)	78.5	3.70 m	78.6	3.52 t (7.9)	80.8
	3' 3.58 m	78.6	3.67 m	86.5	3.72 t (8.7)	86.8	3.70 m	87.0	3.65 t (8.1)	78.3
	4' 3.50 m	73.6	3.63 m	72.6	3.69 m	72.7	3.58 t (9)	72.5	3.53 m	73.3
	5' 3.89 m	77.8	3.57 m	na a	3.59 m	78.0	3.61 d (9)	77.9	3.87 m	77.8
	6'	-		na a	-		na a	-		176.3	-		176.3	-		na a
	2'--L-Ara(f)		2'--L-Ara(f)		2'--D-Xyl		2'--D-Glc		2'--D-Xyl
	1" 5.42 brs	109.6 5.30 d (3.6)	110.1 4.65 d (7.8)	104.6 4.81 d (7.9)	104.0 4.66 d (7.7)	105.0
	2" 4.05 m	83.0	4.02 dd (6-3.6)	83.3	3.19 dd (9.2-7.8)	75.4	3.19 t (8.1)	75.4	3.22 dd (9.3-7.9)	75.5
	3" 3.85 t(6.1)	77.3	3.88 t (6)	76.9	3.30 t (9.2)	78.0	3.38 t (8.7)	78.0	3.30 t (8.8)	78.1
	4" 4.08 m	85.0	4.07 ddd (6-5-2.3) 84.6	3.53 ddd (10.7-9.2-5.5) 70.9	3.53 t (8.9)	70.0	3.52 ddd (9.9-8.8-5.1) 70.8
	5" 3.59	dd (11.8-4.8)	63.1	3.58	dd (12.4-5)	63.0	3.17	t (11.1)	66.8	3.23	m	78.2	3.15	t (11)	66.9
		3.75	dd (11.8-2.4)		3.76	dd (12.4-2.3)		3.84	dd (11.5-5.5)					3.84	dd (11-5.3)
	6"										3.78 3.80	m m	61.5
	3'--L-Ara(f)
	1'"				5.19 brs (w1/2 =5) 110.9 5.28 d (2.2)	110.9 5.24 d (2.2)	110.8
	2'"				4.13 m	83.2	4.13 dd (4.6-2.2)	83.3	4.13 dd (4.5-2.2) 83.4
	3'"				3.88 t (6)	77.8	3.86 dd (6.7-4.6)	77.8	3.85 dd (7-4)	77.9
	4'"				4.13 m	85.5	4.12 m	85.4	4.09 m	85.3
	5'"				3.65	dm (11.5)	63.0	3.80	dd (11.7-2.3)	63.0	3.78	dd (12-2.5)	62.9
					3.79	dm (11.5)		3.62	dd (11.7-6.3)		3.63	dd (12-4)

a na : not assigned

Acknowledgements

The authors are very grateful to Dr Tanguy Jaffré and Dr Jean-Marie Veillon of the Botany and Plant Ecology Department, Research Institute for the Development (IRD), Noumea (New Caledonia), for their help in the identification of the plant, and to Dr Thierry Sevenet (ICSN, CNRS), Laurent Ghnassia and Pelenato Maituku (IRD, Noumea) for the collection of the plant.

. activity and caused 100% haemolysis of a 10% suspension of sheep erythrocytes.