Cytotoxic Saponins from Schefflera fagueti

• Abstract
• Introduction
• Materials and Methods
• General
• Plant material
• Extraction and isolation
• Basic hydrolysis of compounds 1 - 6
• Isolates
• Antiproliferative assay
• Results and Discussion
• References

Abstract

Six new lupane (1 - 4) and oleanane saponins (5 and 6) were isolated from the aerial parts of Schefflera fagueti Baill. (Araliaceae). Their structures were determined by 2D-NMR spectroscopy (DQF-COSY, 1D-TOCSY, 2D-HOHAHA, 1D-ROESY, HSQC, HMBC). The antiproliferative activity of compounds 1 - 6 and of their prosapogenins (1a - 6a) was evaluated using three continuous murine and human culture cell lines J774, HEK-293, WEHI-164. Oleanane saponins 5 and 6 were the most active, showing significant inhibitory effects on all cell lines, while their prosapogenins 5a and 6a demonstrated minor activity.

Introduction

Chemical and pharmacological investigations on plants belonging to Araliaceae have indicated that triterpenoid saponins are their important bioactive components [1], [2]. Plants of the Schefflera genus are used as folk remedies for the treatment of pain, rheumatic arthritis, fracture, sprains, and lumbago in Asian countries [3]. As a part of our continuing investigations on the Araliaceae plants from the Botanical Garden of Palermo [4], [5], we studied Schefflera fagueti Baill., a plant native of the Seychelles never before investigated from a phytochemical point of view. The present work deals with the isolation and structural elucidation from the aerial parts of S. fagueti, of six new saponins (1 - 6) (Figs. 1 and 2) together with the evaluation of their antiproliferative activity (cell lines J774, HEK-293, WEHI-164). Furthermore, to study some structure-activity relationships the saponins 1 - 6 were submitted to alkaline hydrolysis and the prosapogenins obtained (1a - 6a) were also tested in the antiproliferative activity biological assay.

Materials and Methods

General

Optical rotations were measured on a Perkin-Elmer 241 polarimeter using a sodium lamp operating at 589 nm. Elemental analysis was obtained from a Carlo Erba 1106 elemental analyzer. A Bruker DRX-600 NMR spectrometer, operating at 599.19 MHz for ¹H and 150.86 MHz for ¹³C, using the UXNMR software package was used for NMR experiments; chemical shifts are expressed in δ (ppm) referring to the solvent peaks, δ_H 3.34 and δ_C 49.0 for CD₃OD. 1D- and 2D-NMR experiments were carried out using the conventional pulse sequences as described in the literature [6]. FABMS were recorded in a glycerol matrix in the negative ion mode on a VG ZAB instrument (Xe atoms of energy of 2 - 6 kV). DCCC separations were performed on a Büchi apparatus, equipped with 300 tubes (Ø 2.7 mm). HPLC separations were performed on a Waters 515 series pumping system equipped with a Waters R401 refractive index detector and with a Waters μ-Bondapak C₁₈ column and U6K injector. Column chromatography was performed over Sephadex LH-20 (Pharmacia Fine Chemicals). TLC was conducted on silica 60 F₂₅₄ gel-coated glass sheets (Merck, Darmstadt, Germany), spray reagent Ce(SO₄)₂/H₂SO₄ (Sigma-Aldrich, Milano, Italy).

Plant material

The aerial parts of S. fagueti Baill. were obtained in September 1997 from plants cultivated at the Botanical Garden of Palermo, Italy, where a voucher specimen was deposited (No. 160).

Extraction and isolation

Dried aerial parts of S. fagueti (200 g) were defatted with petroleum ether then extracted with 80 % EtOH to give 6.4 g of residue. The ethanolic extract was partitioned between n-BuOH and H₂O to afford a n-BuOH-soluble portion (2.5 g). Part of the 1-butanol residue (2.0 g) was chromatographed over Sephadex LH-20 column (100 × 5 cm) with MeOH as eluent (600 mL). Fractions of 8 mL were collected and grouped by TLC results on silica 60 F₂₅₄ gel-coated glass sheets with n-BuOH-AcOH-H₂O (60 : 15 : 25) and CHCl₃-MeOH-H₂O (40 : 9 : 1). Fractions 22 - 30 (300 mg) were purified by DCCC (300 tubes, Ø 2.7 mm) with CHCl₃-MeOH-H₂O-i-PrOH (5 : 6 : 4 : 1), descending mode, flow 10 mL/h, to yield pure compound 5 (15.0 mg, elution volume 65 - 75 mL) together with three main fractions A (39 mg, elution volume 35 - 55 mL), B (57 mg, elution volume 100 - 200 mL), and C (40 mg, elution volume 220 - 330 mL). Fraction A was submitted to final separation by RP-HPLC on a C-18 μ-Bondapak column (30 cm × 7.8 mm, flow rate 4.0 mL/min) with MeOH-H₂O (7 : 3) as eluent to yield pure compound 6 (14.5 mg, t _R = 21 min). Fraction B was purified by RP-HPLC on a C-18 μ-Bondapak column (30 cm × 7.8 mm, flow rate 3.0 mL/min) with MeOH-H₂O (3 : 2) as eluent to give pure compounds 3 (12.0 mg, t _R = 8 min) and 4 (12.5 mg, t _R = 10 min). Fraction C was chromatographed over RP-HPLC on a C-18 μ-Bondapak column (30 cm × 7.8 mm, flow rate 3.0 mL/min) with MeOH-H₂O (1 : 1) as eluent to yield pure compounds 2 (13.2 mg, t _R = 10 min) and 1 (13.7 mg, t _R = 12 min).

Basic hydrolysis of compounds 1 - 6

Saponins 1 - 6 (9.0 mg each) in 0.5 M KOH (1 mL), were heated at 110 °C in a stoppered reaction vial for 2 h. Each reaction mixture was adjusted to pH 7 and then extracted with n-BuOH. The organic phase was evaporated to dryness, dissolved in CD₃OD, and analyzed by ¹H- and ¹³C-NMR spectroscopy. Hydrolysis of saponins 1 - 6 gave sapogenins 1a (4.5 mg), 2a (3.5 mg), 3a (3.5 mg), 4a (4.0 mg), 5a (3.7 mg), and 6a (3.0 mg).

Isolates

Compound 1 3β-O-(β-glucopyranosyl-(1→3)-α-rhamnopyranosyl-(1→2)-α-arabinopyranosyl)-lup-12-en-28-O-(α-rhamnopyranosyl-(1→4)-β-glucopyranosyl-(1→6)-β-galactopyranosyl) ester: white amorphous powder, [α]²⁵ _D: + 139°, (c 1, MeOH); FABMS: m/z = 1365 [M - H]^-, 1203 [(M - H) - 162]^-, 1057 [(M - H) - (162 + 146)]^-, 911 [(M - H) - (162 + 146 + 146)]^-, 749 [(M - H) - (162 + 146 + 146 + 162)]^-; elemental analysis found: C 57.00 %, H 7.83 %, O 35.17 %; calcd. for C₆₅H₁₀₆O₃₀: C 57.09 %, H 7.81 %, O 35.10 %; ¹H- and ¹³C-NMR for the aglycone moiety: see Table [1]; ¹H- and ¹³C-NMR data of the sugar moiety: see Table [2].

Compound 2 3β-O-(β-glucopyranosyl-(1→3)-α-rhamnopyranosyl-(1→2)-α-arabinopyranosyl)-23-hydroxylup-12-en-28-O-(α-rhamnopyranosyl-(1→4)-β-glucopyranosyl-(1→6)-β-galactopyranosyl) ester: white amorphous powder, [α]²⁵ _D: + 120°, (c 1, MeOH); FABMS: m/z = 1381 [M - H]^-, 1235 [(M - H) - 146]^-, 1073 [(M - H) - (146 + 162)]^-, 927 [(M - H) - (146 + 162 + 146)]^-; elemental analysis found: C 56.40 %, H 7.73 %, O 35.87 %; calcd. for C₆₅H₁₀₆O₃₁: C 56.43 %, H 7.72 %, O 35.85 %; ¹H- and ¹³C-NMR for aglycone moiety: see Table [1]; ¹H- and ¹³C-NMR data of the sugar moiety are superimposable with those reported for compound 1.

Compound 3 3β-O-(α-rhamnopyranosyl-(1→2)-α-arabinopyranosyl)-23-hydroxylup-12-en-28-O-(α-rhamnopyranosyl-(1→4)-β-glucopyranosyl-(1→6)-β-galactopyranosyl) ester: white amorphous powder, [α]²⁵ _D: + 93°, (c 1, MeOH); FABMS: m/z= 1219 [M - H]^-, 1073 [(M - H) - 146]^-, 765 [(M - H) - (146 + 162)]^-; elemental analysis found: C 57.98 %, H 7.94 %, O 34.08 %; calcd. for C₅₉H₉₆O₂₆: C 58.02 %, H 7.92 %, O 34.06 %; ¹H- and ¹³C-NMR data of the aglycone moiety are almost superimposable to those of compound 2; ¹H- and ¹³C-NMR data of the sugar moiety: see Table [2].

Compound 4 3β-O-(α-rhamnopyranosyl-(1→2)-α-arabinopyranosyl)-lup-12-en-28-O-(α-rhamnopyranosyl-(1→4)-β-glucopyra-nosyl-(1→6)-β-galactopyranosyl) ester: white amorphous powder, [α]²⁵ _D: + 86°, (c 1, MeOH); FABMS: m/z = 1203 [M - H]^-, 911 [(M - H) - (146 + 146)]^-, 749 [(M - H) - (146 + 146 + 162)]^-; elemental analysis found: C 58.73 %, H 8.05 %, O 33.22 %; calcd. for C₅₉H₉₆O₂₅: C 58.79 %, H 8.03 %, O 33.18 %; ¹H- and ¹³C-NMR data of the aglycone moiety are identical to those of compound 1, ¹H- and ¹³C-NMR data of the sugar moiety to those of compound 3.

Compound 5 3β-O-(β-glucopyranosyl-(1→2)-β-glucopyranosyl-(1→3)-β-xylopyranosyl)-16α-hydroxyolean-12-en-28-O-(β-galactopyranosyl) ester-30-oic acid: white amorphous powder, [α]²⁵ _D: + 18°, (c 1, MeOH); FABMS: m/z = 1119 [M - H]^-, 1075 [(M - H) - 44]^-, 957 [(M - H) - 162)]^-, 633 [(M - H) - (162 + 162)]^-; elemental analysis found: C 56.74 %, H 7.56 %, O 35.70 %; calcd. for C₅₃H₈₄O₂₅: C 56.77 %, H 7.55 %, O 35.67 %; for ¹H- and ¹³C-NMR data of the aglycone moiety, see De Tommasi et al. [4]; ¹H- and ¹³C-NMR data of the sugar moiety: see Table [3].

Compound 6 3β-O-(β-glucopyranosyl-(1→3)-β-xylopyranosyl)-16α-hydroxyolean-12-ene-28,30-dioic acid 28-O-(β-galactopyra- nosyl) ester: white amorphous powder, [α]²⁵ _D: + 11°, (c 1, MeOH); FABMS: m/z = 957 [M - H]^-, 913 [(M - H) - 44]^-, 795 [(M - H) - 162]^-; elemental analysis found: C 58.84 %, H 7.79 %, O 33.37 %; calcd. for C₄₇H₇₄O₂₀: C 58.86 %, H 7.78 %, O 33.36 %; for ¹H- and ¹³C-NMR data of the aglycone moiety, see De Tommasi et al. [4]; ¹H- and ¹³C-NMR data of the sugar moiety: see Table [3].

Compound 1a 3β-O-(β-glucopyranosyl-(1→3)-α-rhamnopyranosyl-(1→2)-α-arabinopyranosyl)-lup-12-en-28-oic acid: white amorphous powder, [α]²⁵ _D: + 22°, (c 0.1, MeOH); the ¹H- and ¹³C-NMR data of the aglycone moiety are identical to those of compound 1 except for C-28 signal that resonated at δ = 180.5; ¹H- and ¹³C-NMR data of the sugar moiety: see Table [4].

Compound 2a 3β-O-(β-glucopyranosyl-(1→3)-α-rhamnopyranosyl-(1→2)-α-arabinopyranosyl)-23-hydroxylup-12-en-28-oic acid: white amorphous powder, [α]²⁵ _D: + 21°, (c 0.1, MeOH); the ¹H- and ¹³C-NMR data of the aglycone moiety are identical to those of compound 2 except for C-28 signal that resonated at δ = 180.8; ¹H- and ¹³C-NMR data of the sugar moiety are superimposable on those reported for compound 1a.

Compound 3a 3β-O-(α-rhamnopyranosyl-(1→2)-a-arabinopyranosyl)-23-hydroxylup-12-en-28-oic acid: yellowish amorphous powder, [α]²⁵ _D: + 9°, (c 0.1, MeOH); the ¹H- and ¹³C-NMR data of the aglycone moiety are identical to those of compound 2 except for C-28 signal that resonated at δ = 181.0; ¹H- and ¹³C-NMR data of the sugar moiety: see Table [4].

Compound 4a 3β-O-(α-rhamnopyranosyl-(1→2)-a-arabinopyranosyl)-lup-12-en-28-oic acid: whitish amorphous powder, [α]²⁵ _D: + 9°, (c 0.1, MeOH); the ¹H- and ¹³C-NMR data of the aglycone moiety are identical to those of compound 1 except for C-28 signal that resonated at δ = 180.8; ¹H- and ¹³C-NMR data of the sugar moiety are superimposable on those reported for compound 3a.

Compound 5a 3β-O-(β-glucopyranosyl-(1→2)-β-glucopyranosyl-(1→3)-β-xylopyranosyl)-16α-hydroxyolean-12-ene-28,30-dioic acid: white amorphous powder, [α]²⁵ _D: -5°, (c 0.1, MeOH); for the ¹H- and ¹³C-NMR data of the aglycone moiety, see De Tommasi et al. [4]; ¹H- and ¹³C-NMR data of the sugar moiety: see Table [5].

Compound 6a 3β-O-(β-glucopyranosyl-(1→3)-b-xylopyranosyl)-16α-hydroxyolean-12-ene-28,30-dioic acid: yellowish amorphous powder, [α]²⁵ _D: -3°, (c 0.1, MeOH); for ¹H- and ¹³C-NMR data of the aglycone moiety, see De Tommasi et al. [4]; ¹H- and ¹³C-NMR data of the sugar moiety: see Table [5].

Antiproliferative assay

J774.A1, murine monocyte/macrophage, WEHI-164, murine fibrosarcoma, and HEK-293, human epithelial kidney cells were grown as reported previously [7]. All reagents for cell culture were from Hy-Clone (Euroclone, Paignton Devon, U.K.); MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-phenyl-2H-tetrazolium bromide] and 6-mercaptopurine (6-MP) were from Sigma Chemicals (Milan, Italy). J774.Al, WEHI-164, and HEK-293 (3.4 × 10⁴ cells) were plated on 96-well microtiter plates and allowed to adhere at 37 °C in 5 % CO₂ and 95 % air for 2 h. Thereafter, the medium was replaced with 50 μL of fresh medium and a 75 μL aliquot of 1 : 4 serial dilution of each test compound was added and then the cells incubated for 72 h. In some experiments, serial dilutions of 6-MP were added. The cell viability was assessed through an MTT conversion assay [8], [9], [10]. The optical density (OD) of each well was measured with a microplate spectrophotometer (Titertek Multiskan MCC/340) equipped with a 620 nm filter. The viability of each cell line in response to treatment with tested compounds and 6-MP [11] was calculated as: % dead cells = 100 - (OD treated/OD control) × 100. Table [6] shows the results obtained expressed as an IC₅₀ value (μM), the concentration that inhibited cell growth by 50 % as compared to the control.

Results and Discussion

Compounds 1 - 6 were isolated from the ethanolic extract of S. fagueti aerial parts by Sephadex LH-20 column chromatography, DCCC, and RP-HPLC.

Compound 1 had the molecular formula C₆₅H₁₀₆O₃₀, as determined by ¹³C-, ¹³C-DEPT NMR, negative-ion FABMS, and elemental analysis. The FAB-MS of 1 showed the [M - H]^- ion at m/z = 1365 and prominent fragments at m/z = 1203 [(M - H) - 162]^-, 1057 [(M - H) - (162 + 146)]^-, 911 [(M - H) - (162 + 146 + 146)]^-, and 749 [(M - H) - (162 + 146 + 146 + 162)]^-. The ¹³C-NMR spectrum showed 65 signals, of which 30 were assigned to a triterpenoid moiety and 35 to the saccharide portion. In the ¹H-NMR spectrum, singlets at δ = 0.85, 0.99 (integrating for six protons), 1.03, and 1.08 demonstrated the presence of five tertiary methyl groups, while two doublets appearing at δ = 0.90 (3H, d, J = 6.0 Hz) and 0.94 (3H, d, J = 6.0 Hz) were attributed to two secondary methyl groups. Further, the spectrum showed an olefinic proton at δ = 5.18 (1H, br s, J = 3.0 Hz) and one-proton double doublet at δ = 3.30 (1H, dd, J = 11.0, 4.5 Hz). All these features, together with the complete NMR analysis, that revealed the presence of a carboxylic group located at C-28, were completely in agreement with 3β-hydroxylup-12-en 28-oic acid as the aglycone of compound 1 [12], [13]. To our knowledge, the ¹³C-NMR data of compound 1 has not been reported in the literature. The sugar portion of 1 contained, in the ¹H NMR spectrum (Table [2]) six anomeric proton signals (δ = 4.38, d, J = 6.8 Hz; 4.44, d, J = 7.5 Hz; 4.56, d, J = 7.5 Hz; 5.17, d, J = 1.3 Hz; 5.26, d, J = 1.5 Hz; 5.28, d, J = 7.0 Hz). The remaining 1D sugar spectral region of 1 was complex since most of the chemical shifts were overlapped. 2D-HOHAHA experiment, together with the DQF-COSY spectrum, led us to establish the proton sequence within this sugar fragments that were identified as one α-arabinopyranoside, two α-rhamnopyranoside, one β-galactopyranoside, and two β-glucopyranoside. In the HSQC experiment glycosidation shifts were observed for C-2_ara (76.1 ppm), C-3_rhaI (82.5 ppm), C-6_gal (69.0 ppm), and C-4_glcII (79.6 ppm). The absence of any ¹³C glycosidation shifts for one glucopyranosyl and one rhamnopyranosyl moieties suggested that these sugars were terminal units. Chemical shifts of H-1_gal (5.25) and C-1_gal (95.5) indicated that this sugar was involved in an ester linkage with the C-28 carboxylic group [14]. The positions of the sugar units were unambiguously defined by the HMBC experiments: α-arabinose unit was linked at C-3 as shown by the cross peak between δ = 4.38 (H-1_ara) and 90.0 ppm (C-3); key correlations were observed between H-1_rhaI-C-2_ara, H-1_glcI-C-3_rhaI, H-1_gal-C-28, H-1_glcII-C-6_gal, H-1_rhaII-C-4_glcII. Thus, the structure of compound 1 was established as 3β-O-(β-glucopyranosyl-(1→3)-α-rhamnopyranosyl-(1→2)-α-arabinopyranosyl)-lup-12-en-28-O-(α-rhamnopyranosyl-(1→4)-β-glucopyranosyl-(1→6)-β-galactopyranosyl) ester.

The FABMS of compound 2 (C₆₅H₁₀₆O₃₁) displayed [M - H]^- at m/z = 381 and prominent fragments at m/z = 1235 [(M - H) - 146]^-, 1073 [(M - H) - (146 + 162)]^-, and 927 [(M - H) - (146 + 162 + 146)]^- due to the subsequent losses of a deoxyhexose, hexose, and deoxyhexose units. Analysis of NMR data of compound 2 and comparison with those of 1 showed that they both possessed the same saccaridic chains at C-3 and C-28 while the aglycone was the point of difference. Comparison of ¹H- and ¹³C-NMR data of the aglycone of 2 with those of 1 indicated structural similarity. The main difference was the presence of an AB doublet at δ = 3.40 (J = 12.0 Hz) and 3.58 (J = 12.0 Hz), indicating the presence of a -CH₂OH function that was placed at C-23 position of the lupane skeleton on the basis of the upfield shifts of C-3 (83.4 ppm), C-5 (48.4 ppm), and C-24 (13.3 ppm) and downfield shifts of C-4 (43.6 ppm) in the ¹³C-NMR spectrum. This hypothesis was confirmed unambiguously by DQF-COSY, HSQC, and HMBC experiments that permitted the full assignments of the proton and carbon signals of the aglycone part of 2. Thus, the aglycone of compound 2 was characterized as 3β,23-dihydroxylup-12-en-28-oic acid and is now reported for the first time in nature. On the basis of the above data, the structure of 2 was 3β-O-(β-glucopyranosyl-(1→3)-α-rhamnopyranosyl-(1→2)-α-arabinopyranosyl)-23-hydroxylup-12-en-28-O-(α-rhamnopyranosyl-(1→4)-β-glucopyranosyl-(1→6)-β-galactopyranosyl) ester.

Compound 3 was assigned C₅₉H₉₆O₂₆ as molecular formula, as shown by its FABMS data (m/z = 1219 [M - H]^-) in combination with the ¹³C-NMR spectrum. The FABMS of 3 displayed also other prominent fragments at m/z = 1073 [(M - H) - 146]^- and 765 [(M - H) - (146 + 162)]^-. Analysis of NMR data of compound 3 and comparison with those of 2 showed 3 to differ from 2 only in the absence of the terminal glucopyranosyl unit of the saccharidic chain linked at C-3 of the aglycone (Table [2]). Therefore, the structure 3β-O-(α-rhamnopyranosyl-(1→2)-α-arabinopyranosyl)-23-hydroxylup-12-en-28-O-(α-rhamnopyranosyl-(1→4)-α-glucopyranosyl-(1→6)-β-galactopyranosyl) ester was assigned to compound 3.

The FABMS of compound 4 (C₅₉H₉₆O₂₅) showed the [M - H]^- ion at m/z = 1203 and a fragmentation pattern due to the subsequent loss of two deoxyhexose units (m/z = 911 [(M - H) - (146 + 146)]^-) and one hexose unit (m/z = 749 [(M - H) - (146 + 146 + 162)]^-). Analysis of NMR data of compound 4 and comparison with those of 1 showed 4 to differ from 1 only in the absence of the terminal glucopyranosyl unit linked at C-3 of the rhamnopyranosyl unit (Table [2]). Thus, compound 4 was identified as 3β-O-(α-rhamnopyranosyl-(1→2)-α-arabinopyranosyl)-lup-12-en-28-O-(α-rhamnopyranosyl-(1→4)-β-glucopyranosyl-(1→6)-β-galactopyranosyl) ester.

Compound 5 (C₅₃H₈₄O₂₅) showed in the FABMS a quasi-molecular ion peak at m/z = 1119 [M - H]^- and prominent fragments at m/z = 1075 [(M - H) - 44]^-, 957 [(M - H) - 162)]^-, and 633 [(M - H) - (162 + 162)]^-. The following NMR data suggested the structural features of 3β,16α-dihydroxyolean-12-ene-28,30-dioic acid for the aglycone of compound 5: olefinic hydrogen at δ = 5.24 (1H, br t, J = 3.5 Hz, H-12), signals for carbinolic protons attributed to H-3 at δ = 3.20 (1H, dd, J = 11.0, 5.0 Hz) and to H-16 at δ = 3.95 (1H, br s), two carbonyl carbon resonances at δ = 178.5 (C-28) and 180.0 (C-30). Full assignments of the proton and carbon signals of the aglycone part of 5 were secured by DQF-COSY, HSQC, and HMBC spectra. The proton and carbon signals due to the A, B, C, and D rings were completely in agreement with those reported for 3β,16α-dihydroxyolean-12-ene-28,30-dioic acid [4]. The structures of the oligosaccharide moieties of 5 were deduced using 1D-TOCSY and DQF-COSY experiments (Table [3]). Thus, the chemical shifts of the sugar resonances were attributable to one β-xylopyranosyl (δ_H-1xyl = 4.67), two β-glucopyranosyl (δ_{H-1glc I} = 4.49 and δ_{H-1glc II} = 4.45), and one β-galactopyranosyl (δ_{H-1glc I} = 5.21) units. The absence of any glycosidation shift for one β-glucopyranose and β-galactopyranose suggested that these sugars were the terminal units. Glycosidation shifts were observed for C-3_xyl (83.0) and C-2_glc I (81.7) (Table [3]). Chemical shifts of H-1_gal (δ = 5.21) and C-1_gal (96.0 ppm) indicated that this sugar unit was involved in an ester linkage with the C-28 carboxylic group [14]. A cross peak due to long-range correlation (HMBC) between C-3 (90.6 ppm) of the aglycone and H-1_xyl indicated that xylose was the residue linked to C-3 of the aglycone; a cross-peak between C-3_xyl (83.0 ppm) and H-1_glc I indicated that glucose I was the second unit of the trisaccharide chain at C-3 of the aglycone. Similarly, the cross peak between C-28 (178.5 ppm) and H-1_gal confirmed that galactose was the hexose unit linked to C-28 of the aglycone. Therefore, the structure of 3β-O-(β-glucopyranosyl-(1→2)-β-glucopyranosyl-(1→3)-β-xylopyranosyl)-16α-hydroxyolean-12-ene-28,30-dioic acid 28-O-(β-galactopyranosyl) ester was assigned to compound 5.

Compound 6 possessed an FAB-MS showing the [M - H]^- peak at m/z = 957 and a fragmentation pattern similar to that of 5. The NMR aglycone signals were similar to those of 5 allowing identification of the aglycone of 6 as 3β,16α-dihydroxyolean-12-en-28,30-dioic acid. Analysis of sugar chain NMR data of compound 6 and comparison with those of 5 showed that compound 6 differed from 5 only in the absence of the terminal glucopyranosyl unit linked at C-3 of the aglycone (Table [3]). Thus, compound 6 was determined to be 3β-O-(β-glucopyranosyl-(1→3)-β-xylopyranosyl)-16α-hydroxyolean-12-en-28-O-(β-galactopyranosyl) ester-30-oic acid.

The saponins 1 - 6 were submitted to basic hydrolysis to obtain prosapogenins 1a - 6a. Analysis of the NMR data of compounds 1a - 6a and comparison with those of 1 - 6 showed that 1a - 6a differed from 1 - 6 only in the absence of the saccharide chain linked at C-28 (see Materials and Methods). To obtain some structure-activity relationships, both saponins 1 - 6 and their prosapogenins 1a - 6a were tested in the antiproliferative activity assay against the J774.A1, WEHI-164, and HEK-293 cell lines. The IC₅₀ values obtained were reported in Table [6] and showed that the most active compounds were 5 and 6. These results showed that oleanane saponins were more active than lupane ones. Comparison of the data obtained for compounds 1 - 6 and their prosapogenins 1a - 6a showed that a saccharide chain esterified at C-28 seemed to be crucial for the antiproliferative activity, because most of the prosapogenins were inactive. These results were also in agreement with those reported before for similar compounds [7], showing that the hydroxyl group at C-16 of the aglycone seemed to be necessary for cytotoxicity. The presence of a carboxylic group at C-30 may play an important role in maintaining the antiproliferative activity of prosapogenis 5a and 6a in comparison with similar oleanane compounds previously tested [7].

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• 7 De Tommasi N, Autore G, Bellino A, Pinto A, Pizza C, Sorrentino R, Venturella P. Antiproliferative triterpene saponins from Trevesia palmata .  J Nat Prod. 2000;  63 308-14
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• 8 Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxic assay.  J Immunol Methods. 1983;  65 55-63
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• 9 Green L M, Reade J L, Ware C F. Rapid colorimetric assay for cell viability: application to the quantitation of cytotoxic and growth inhibitory lymphokines.  J Immunol Methods. 1984;  70 257-68
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• 10 Opipari A WJ, Hu H M, Yabkowitz R, Dixit Y M. The A20 zinc finger protein protects cell from tumor necrosis factor cytoxicity.  J Biol Chem. 1992;  267 12 424-7
  PubMedSearch in Google Scholar
• 11 Kusumoto H, Maehara Y, Sakaguchi Y, Kohnoe S, Kumashiro R, Sugimachi K. Modulation of cytotoxic effect of anticancer drugs by dipyridamole in HeLa cells in vitro .  Anticancer Res. 1990;  10 1643-5
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• 12 Misra T N, Singh R S, Upadhyay J. Triterpenoids from Hyptis suaveolens roots.  Phytochemistry. 1983;  22 603-5
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• 13 Bhan S, Kumar R, Kalla A K, Dhar K L. Triterpenoids from Swertia petiolata .  Phytochemistry. 1988;  27 539-42
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• 14 Piacente S, Pizza C, De Tommasi N, De Simone F. New dammarane-type glycosides from Gymnostemma penthaphyllum .  J Nat Prod. 1995;  58 512-9
  CrossrefPubMedSearch in Google Scholar

Dr. Alessandra Braca

Dipartimento di Chimica Bioorganica e Biofarmacia

Università di Pisa

Via Bonanno, 33

56126 Pisa

Italy

Fax: +39-050-43321

Email: braca@farm.unipi.it

References

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• 4 De Tommasi N, Pizza C, Bellino A, Venturella P. Triterpenoid saponins from Schefflera divaricata .  J Nat Prod. 1997;  60 663-8
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• 5 Cioffi G, Bellino A, Pizza C, Venturella F, De Tommasi N. Triterpene saponins from Tupidanthus calyptratus.  J Nat Prod. 2001;  64 750-3
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• 6 Braca A, De Tommasi N, Morelli I, Pizza C. New metabolites from Onopordon illyricum .  J Nat Prod. 1999;  62 1371-5
  CrossrefPubMedSearch in Google Scholar
• 7 De Tommasi N, Autore G, Bellino A, Pinto A, Pizza C, Sorrentino R, Venturella P. Antiproliferative triterpene saponins from Trevesia palmata .  J Nat Prod. 2000;  63 308-14
  CrossrefPubMedSearch in Google Scholar
• 8 Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxic assay.  J Immunol Methods. 1983;  65 55-63
  CrossrefPubMedSearch in Google Scholar
• 9 Green L M, Reade J L, Ware C F. Rapid colorimetric assay for cell viability: application to the quantitation of cytotoxic and growth inhibitory lymphokines.  J Immunol Methods. 1984;  70 257-68
  CrossrefPubMedSearch in Google Scholar
• 10 Opipari A WJ, Hu H M, Yabkowitz R, Dixit Y M. The A20 zinc finger protein protects cell from tumor necrosis factor cytoxicity.  J Biol Chem. 1992;  267 12 424-7
  PubMedSearch in Google Scholar
• 11 Kusumoto H, Maehara Y, Sakaguchi Y, Kohnoe S, Kumashiro R, Sugimachi K. Modulation of cytotoxic effect of anticancer drugs by dipyridamole in HeLa cells in vitro .  Anticancer Res. 1990;  10 1643-5
  PubMedSearch in Google Scholar
• 12 Misra T N, Singh R S, Upadhyay J. Triterpenoids from Hyptis suaveolens roots.  Phytochemistry. 1983;  22 603-5
  CrossrefPubMedSearch in Google Scholar
• 13 Bhan S, Kumar R, Kalla A K, Dhar K L. Triterpenoids from Swertia petiolata .  Phytochemistry. 1988;  27 539-42
  CrossrefPubMedSearch in Google Scholar
• 14 Piacente S, Pizza C, De Tommasi N, De Simone F. New dammarane-type glycosides from Gymnostemma penthaphyllum .  J Nat Prod. 1995;  58 512-9
  CrossrefPubMedSearch in Google Scholar

Dr. Alessandra Braca

Dipartimento di Chimica Bioorganica e Biofarmacia

Università di Pisa

Via Bonanno, 33

56126 Pisa

Italy

Fax: +39-050-43321

Email: braca@farm.unipi.it