A New Triterpene Hexaglycoside from the Bark of Kalopanax septemlobus (Thunb.) Koidz

The new triterpene glycoside 3-O-β-D-xylopyranosyl-(1→4)-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosylhederagenin 28-O-β-D-gluco-pyranosyl-(1→6)-β-D-glucopyranoside, named septemoside A (1), and the known 3-O-α-L-rhamnopyranosyl-(1→2)-O-α-L-arabinopyranoside-28-O-β-D-glucopyran-osyl-(1→6)-O-β-D-glucopyranosyl ester of hederagenin (2), were isolated from the bark of Kalopanax septemlobus. The structure elucidation of the compounds was based on spectroscopic evidence, including HRESIMS, 1D and 2D-NMR analysis.

Our investigation on the constituents of the ethanol extract of the plant, led to the isolation of the new triterpenoidal saponin septemoside A (1), along with a known 3- [15]. Herein, we describe the isolation and structure elucidation of 1. The acid hydrolysis of 1 yielded hederagenin [5], and a sugar mixture consisting of D-xylose, D-glucose, L-rhamnose, and L-arabinose, which were identified by TLC and HPLC using authentic samples as references, as well as from the analysis of the 1 H-and 13 C-NMR chemical shifts. The alkaline hydrolysis of 1 only yielded D-glucose, which was identified by comparison of its TLC and NMR spectra, with those of an authentic sample. The β configuration of the glucose unit was inferred from the coupling constant (J = 8.0 Hz) of the anomeric proton, and the chemical shift of the anomeric carbon (δ C 95.8).

Results and Discussion
Evaluation of J HH couplings allowed the identification of one α-L-arabinopyranosyl unit, one α-Lrhamno-pyranosyl, two β-D-glucopyranosyl, and two β-D-xylopyranosyl units. The protons of the monosaccharide residues were assigned starting from the anomeric protons by means of TOCSY experiment. On the basis of extensive 2D NMR experiments, along with the ESI-MS fragmentation pathway which displayed main fragment ions at m/z 326 attributable to a bissaccharide [glc + glc] + , 679 attributable to [aglycone + glc + Na] + , 905 attributable to [aglycone + ara + rha + xyl] + , 927 attributable to [aglycone + ara + rha + xyl + Na] + , and 943 attributable to [aglycone + ara + rha + xyl + K] + , the structure of 1 suggested a bisdesmosidic saponin with a tetrasacharide linked at C-3 (δ 81.2), and a disaccharide linked at C-28 (δ 176.6) through an ester bond (Figure 1). This was further confirmed by the HMBC experiment ( Figure 2). The identities of the sugar units linked to C-3 and C-28 were confirmed as L-arabinose and D-glucose, respectively, from the HMBC correlations of the anomeric protons at δ 4.86 and δ 6.12, with carbons at δ 81.2 and δ 176.6, respectively. Thus, the new structure of 1 was determined as 3 Compound 2 was isolated first time from the barks of this plant. Compounds 1 and 2 were evaluated for cytotoxicity by the MTT method, and showed a marginal activity against the human tumor cell lines HepG2 (hepatoma carcinoma), A549 (lung carcinoma), and Hela (cervical cancer).

Experimental
General NMR spectra were recorded on a Bruker 400 NMR spectrometer with TMS as an internal standard. ESI-MS data were measured on a Agilent 1200 LC-MS spectrometer. HRESIMS and HRFAB-MS were obtained on Bruker Daltonics APEX II 47e and MAT 95XP (Thermo) mass spectrometers, respectively. The silica gel GF254 used for TLC were supplied by the Qingdao Marine Chemical Factory, Qingdao, China. Analytical HPLC was performed on a Hitachi L-2400 HPLC system, using a YMC ODS-H80 column (250 × 4.6 mm i.d., 4 μm) coupled to an Alltech ELSD 800 detector; semipreparative HPLC was performed on a Hitachi L-2400 HPLC system, using a YMC ODS-H80 column (250 × 10 mm i.d., 4 μm) coupled to an Alltech ELSD 800 detector with flow-splitter valve (Parker: NS) set at a split ratio of 20:1 (collector: detector). Spots were detected on TLC under UV light or by heating after spraying with 5% H 2 SO 4 in EtOH (v/v).

Plant material
The

Acid hydrolysis of 1
A solution of 1 (3.0 mg) in 2.0 M trifluoroacetic acid (2 mL) was heated at 110 °C for 6 h (kept sealed). After cooling, the reaction mixture was extracted with EtOAc saturated with H 2 O (3 mL × 2). The EtOAc extract was evaporated to yield an aglycon, which was identified as hederagenin by comparison of spectroscopic and physical data with the reported values in the literature [5]. The H 2 O residue was concentrated under reduced pressure to yield a mixture of D-glucose, D-xylose, Lrhamnose, and L-arabinose which were identified by TLC and HPLC using authentic samples as references, as well as from the analysis of NMR spectrum [19].

Alkaline hydrolysis of 1
A solution of 1 (2.0 mg) in 5% NaOH (2 mL) was heated at 100 °C for 4 h (kept sealed). After cooling, the reaction mixture was evaporated under vacuum, then distilled water was added and the mixture extracted with BuOH saturated with H 2 O (3 mL × 3). The BuOH extract was evaporated to yield an aglycon, which was identified as hederagenin by comparison of spectroscopic and physical data with the reported values in the literature [5]. The H 2 O residue was concentrated under reduced pressure, yielding D-glucose, which was identified by comparison of its TLC and NMR spectra, with those of an authentic sample. 1339.6539, calcd. for 1339.6534)); 1 H-NMR and 13 C-NMR spectral data: see Table 1.

Conclusions
A phytochemical investigation on the bark of Kalopanax septemlobus led to the isolation of the new triterpene glycoside 1 and the known one 2. Compound 2 was isolated from the bark of this plant for the first time. Compounds 1 and 2 showed marginal activity against the human tumor cell lines HepG2, A549, and Hela cell lines.