New 30-Noroleanane Triterpenoid Saponins from Holboellia coriacea Diels

Three new 30-noroleanane triterpenoid saponins, akebonoic acid 28-O-β-d-glucopyranosyl-(1′′→6′)-β-d-glucopyranosyl ester (1), akebonoic acid 28-O-(6′′-O-caffeoyl)-β-d-glucopyranosyl-(1′′→6′)-β-d-glucopyranosyl ester (Holboelliside A, 2) and 3β,20α,24-trihydroxy-29-norolean-12-en-28-oic acid 3-O-(6′-O-caffeoyl)-β-d-glucopyranoside (Holboelliside B, 3) were isolated from the stems of Holboellia coriacea Diels, together with five known compounds, eupteleasaponin VIII (4), 3α-akebonoic acid (5), quinatic acid (6), 3β-hydroxy-30-norhederagenin (7) and quinatoside A (8). The structures of these compounds were determined on the basis of spectral and chemical evidence. Compounds 1–5 were evaluated for their inhibitory activity against three human tumors HepG2, HCT116 and SGC-7901 cell lines in vitro.


Introduction
Holboellia coriacea Diels belonging to family Lardizabalaceae, is an evergreen woody vines mainly distributed in Qinling Mountain region of China at altitudes of 500-2000 m [1]. The fruit of H. coriacea, edible berries, can be used to wine, while the stems and roots of the plant have been used as Chinese folk medicine for treating arthritis and rheumatism paralysis [1,2]. To date there are very few reports about the chemical constituents of this species. Recently, a phytochemical study revealed six triterpenoid saponins, including two noroleanane-type ones from a methanol extract of root of H. coriacea [2]. Noroleanane triterpenoids are recognized as characteristic constituents of the family Lardizabalaceae, some of which were revealed to show significant bioactivities [3][4][5]. Our interest in the chemistry of noroleanane triterpenoids prompted us to continue the phytochemical study of H. coriacea, whereby three new 30-noroleanane triterpenoid saponins (1-3) and five known ones (4)(5)(6)(7)(8) were obtained from the stem of the plant. Herein, we report the isolation, structure elucidation, and cytotoxic activity of these compounds.

Results and Discussion
The dried stems of H. coriacea were extracted using 95% aq. EtOH. The EtOH extract residue was suspended in water and then partitioned successively with petroleum ether and EtOAc. Column chromatography of the EtOAc-soluble fraction yielded three new triterpenoid saponins (1-3), which were identified by NMR techniques and HRESIMS, and five known compounds 4-8 that were identified as by comparison of their NMR and MS data with those reported in the literature as eupteleasaponin
Compound 3 was obtained as a light yellow powder. The molecular formula was shown to be C 44 H 62 O 13 by its HR-ESIMS and 13 C-NMR data. Its 1 H-and 13 C-NMR spectra (  [12]. The deshielded signal of the protons H-6 1 (4.95 (dd, J = 11.4, 4.7 Hz)/5.03 (br d, J = 10.1 Hz)) of the glucose moiety and the correlation between H-6 1 and C=O (δ C 167.4) in the HMBC spectrum indicated that the trans-caffeoyl moiety is attached to C-6 1 via an ester linkage. Moreover, In the NOESY spectrum ( Figure S21), Hα-5 was correlated with H-3 and H 3 -23, and Hβ-18 was correlated with H 3 -29, confirming the stereostructures of the aglycone should be 3β,20α,24-trihydroxy-29-norolean-12-en-28-oic acid [13]. Consequently, the structure of 3 was determined to be 3β,20α,24-trihydroxy-29-norolean-12-en-28-oic acid 3-O-(6 1 -O-caffeoyl)-β-D-glucopyranoside, a cafeoyl ester of 4 that we have named Holboelliside B.  It is noteworthy that the caffeoylation of nortriterpenoid saponins (2 and 3) were isolated from family Lardizabalaceae for the first time. This kind of esterification is particularly important for saponins for increasing their solubility in water (and hence to favor its lymphatic transport), thus the esterification of many secondary metabolites generally represents one of the last steps of their biosynthesis [12]. Because noroleanane triterpenoids are recognized as characteristic constituents of the family Lardizabalaceae [3], the occurrence of those caffeoylation derivatives in Holboellia coriacea may have a role as chemotaxonomic markers for Holboellia, which is worthy of further study.

Cytotoxicity Assay
The cytotoxicity of compounds 1-5 against three human tumors HepG2, HCT116 and SGC-7901 cell lines were assessed, using a sulforhodamine B (SRB) method. The resulting IC 50 values are displayed in Table 4. Only compound 5 showed interesting cytotoxicity against HCT-116, HepG2 and SGC-7901 cell lines, with IC 50 values of 9.1, 15.2 and 41.0 µM, respectively. Previous cytotoxicity studies on this type of nortriterpenoids suggested that the exocyclic double bond at C-20(29) might be an important active center to "maintain" their potential cytotoxicity [5], while the present result suggested that the saccharide unit attached at C-28 could greatly reduce the in vitro cytotoxicity.

Plant Material
The stems of H. coriacea were collected from Xiangxi, Hunan Province, China, in December 2012, and identified by Prof. Zhang Dai-gui (Key Laboratory of Plant Resources Conservation and Utilization, Jishou University, Jishou, China). A voucher specimen (zdg-20121203) has been deposited at the Hunan Agricultural University.

Chemical Hydrolysis of 1-3
Alkaline and Acid Hydrolysis of 1 and 2: A solution of saponins (1 or 2, 10 mg each) in 5% KOH (3 mL) was stirred at 85˝C for 4 h. The reaction mixture was acidified to pH 4.0 with 10% HCl and extracted with CHCl 3 (3ˆ10 mL). The CHCl 3 layer was dried (anhydr. MgSO 4 ) and concentrated under reduced pressure. The residue was subjected to Sephadex LH-20 CC using MeOH to afford akebonoic acid (1a) for the aglycone (5 mg from 1, 3.5 mg from 2). 1a was identified on the basis of 1 H and 13 C-NMR spectrum comparisons with that of published data for akebonoic acid [7]. The aqueous layer was further concentrated and the residue in 5%-10% H 2 SO 4 (2 drops) was heated in a boiling H 2 O bath for 1 h. The solution was extracted with n-BuOH (3ˆ20 mL). The n-BuOH solution was concentrated in vacuo and desalted (Sephadex LH-20, MeOH) to afford the sugar residue (1.0 mg). The sugar residue was derivatized with Sigma Sil-A for 35 min at 70˝C and analyzed by GC-MS with only one peak detected (Rt = 12.19 min, EI-MS m/z: M + 540) [14], which was identified as trimethylsilyl derivative of D-glucose.
Alkaline and Acid Hydrolysis of 3: Compounds 3 (8.0 mg) in 1 N HCl (5 mL, 1,4-dioxane-H 2 O, 1:1) was heated under reflux for 8 h. After removal of the solvent, the residue was partitioned between CHCl 3 and H 2 O. The CHCl 3 -soluble portion was evaporated and subjected to ODS column chromatography using 90% MeOH to yield 4 mg of the aglycone (3a) which was identified as