Two New Oleanane-Type Saponins with Anti-Proliferative Activity from Camellia oleifera Abel. Seed Cake

Two new oleanane-type saponins, named oleiferasaponins C4 (1) and C5 (2), were isolated from Camellia oleifera Abel. seed cake residue. Their respective structures were identified as 16α-hydroxy-22α-O-angeloyl-23α-aldehyde-28-dihydroxymethylene-olean-12-ene-3β-O-[β-d-galacto-pyranosyl-(1→2)]-[β-d-glucopyranosyl-(1→2)-β-d-galactopyranosy-(1→3)]-β-d-glucopyranosid-uronic acid methyl ester (1) and 16α-hydroxy-22α-O-angeloyl-23α-aldehyde-28-dihydroxy-methylene-olean-12-ene-3β-O-[β-d-galactopyranosyl-(1→2)]-[β-d-galactopyranosyl-(1→3)]-β-d-glucopyranosiduronic acid methyl ester (2) through 1D- and 2D-NMR, HR-ESI-MS, and GC-MS spectroscopic methods. The two compounds exhibited potent cytotoxic activities against five human tumor cell lines (BEL-7402, BGC-823, MCF-7, HL-60 and KB).


Introduction
Camellia oleifera Abel. seed saponins are triterpenoidic natural compounds that have potent pharmacological and biological activities, including antimicrobial [1][2][3], antioxidant [4], and gastroprotective [5] effects. Camellia saponins are commercially used as biosurfactants [6], biopesticides [7] and detergents [8]. New saponin monomers continue to be isolated and identified from the seed cake of Camellia oleifera, with many showing potent anti-proliferative activity against human cancer cell lines [9][10][11]. C. oleifera seed cake, is an agricultural byproduct resulting from the extraction of the edible tea oil from the seeds. The seed cake contains approximately 10% saponins by weight, a fraction comprised of more than 30 types of saponins [3,12]. There are 10 saponins [1,9,10,[12][13][14] that have been recently identified in tea seed cake, including nine previously unreported from any source. Seven of the saponins have been reported to have anti-proliferative activity against human cancer cell lines. A previous study indicated that different tea saponin structures display different anti-tumor activity [15]. Some of the varied saponins in C. oleifera seed cake are more difficult to isolate and have complex chemical structures that are difficult to identify, hindering the study of their structure and function. In order to study the saponin structure-activity relationships, it is necessary to isolate and identify additional monomer compounds from the crude saponin fraction, especially triterpene saponins. Identification of the functions of oleanane-type saponins in C. oleifera and clear illustration of structure-activity relationship would increase the effective utilization of tea cake, particularly in pharmaceutical applications. As a part of our ongoing study of the constituents of the C. oleifera seed cake of, we recently isolated two new oleanane-type saponins. We report herein the isolation and structural elucidation of the new saponins, namely oleiferasaponins C 4 and C 5 (Figure 1), along with namely oleiferasaponins C4 and C5 (Figure 1), along with their anti-proliferative activity against five human tumor cell lines, namely BEL-7402, BGC-823, MCF-7, HL-60 and KB.

Isolation and Characterization of the Triterpenoid Saponins
The n-BuOH fraction obtained from the 70% EtOH extract of defatted seed cake of C. oleifera was further separated through silica gel column and gel permeation chromatography on Sephadex LH-20, and by repeated reversed-phase C18 column chromatography. Two new oleanane-type saponins were thus obtained. Their structures were determined mainly by 600 MHz NMR experiments and high resolution electro-spray ionization mass spectrometry (HR-ESI-MS).

Isolation and Characterization of the Triterpenoid Saponins
The n-BuOH fraction obtained from the 70% EtOH extract of defatted seed cake of C. oleifera was further separated through silica gel column and gel permeation chromatography on Sephadex LH-20, and by repeated reversed-phase C 18 column chromatography. Two new oleanane-type saponins were thus obtained. Their structures were determined mainly by 600 MHz NMR experiments and high resolution electro-spray ionization mass spectrometry (HR-ESI-MS).

Anti-Proliferative Assay on Human Tumor Cells
The isolated Compounds 1, 2 and total seed cake saponins (C. oleifera) were tested against five human tumor cell lines (human hepatocellular carcinoma cells BEL-7402, human gastric carcinoma cells BGC-823, human breast cancer cells MCF-7, human promyelocytic leukemia cells HL-60 and human oral epidermoid carcinoma cells KB) using the MTT in vitro assay. Taxol, an anticancer agent, was used as the positive control. Both oleanane-type saponins exhibited effective anti-proliferative activity against the human tumor cell lines tested, especially compound 2. Compound 2 showed potent anti-proliferative activities, with IC 50 values ranging from 1.8 to 5.5 µM, while compound 1 displayed anti-proliferative activity with IC 50 values between 6.5 and 15.1 µM. Total saponins showed anti-proliferative activity with IC 50 values between 6.0 and 19.8 µM (Table 2). Taxol was used as a positive control.
The structure-activity relationships were inferred by comparison of the structure and the anti-proliferative activities on five human tumor cell lines. Compounds 1 and 2 have the same aglycone, but compound 2 lacks the glucose sugar moiety at position Gal-C-2 111 . The higher anti-proliferative activities of 2 compared with those of 1 on all of the tested tumor cell lines suggested that three saccharide units (rather than four saccharide units) leads to an increase in the biological potency. The results are consistent with Mu et al. [15]. In comparison with those of previously reported compounds [10], it seems that the Ang group at position C-22 and the free hydroxy group at C-28 in compound 2 may also play important roles in cytotoxicity. These results further support the idea that the anti-proliferative activity of saponins depends not only on isolated structural factors but also on combinatorial properties of both the aglycone and the sugar moieties.

General Information
The following spectroscopic instruments were used to obtain physical data on the two new isolated saponins: 1 H-and 13 C-NMR, 1

Acid hydrolysis and GC-MS Analysis of the Sugar Moieties in 1 and 2
Each saponin (0.8 mg) was dissolved in 1 M HCl (1 mL) for 3 h at 90˝C. The reaction mixture was extracted with chloroform, and the supernatant was evaporated to dryness under N 2 flow. The residue was dissolved in 0.2 mL of pyridine containing L-cysteine methyl ester hydrochloride (10 mg/mL) and reacted at 70˝C for 1 h. This reaction was evaporated under N 2 flow, after which 0.2 mL trimethylsilylimidazole (Adamas Reagent Co., Ltd, Shanghai, China) was added. The mixture was heated at 70˝C for another 1 h, and then partitioned between n-hexane and water. The organic phase was analyzed by GC-MS. Temperature conditions were as follows: injector temperature at 280˝C; the initial oven temperature was 160˝C for 1 min, then linearly increased to 200˝C at 6˝C/min. A further linear increase at 3˝C/min was performed to 280˝C, which was held for 5 min. The standard sugar samples were subjected to the same reaction and GC-MS conditions. The sugar units of compounds 1 and 2 were identified by comparison with authentic samples: D-xylose (t R 16.93 min), D-glucose (t R 21.67 min), D-galactose (t R 22.31 min), D-glucuronic acid methyl ester (t R 23.34 min). D-glucose, D-galactose and D-glucuronic acid methyl ester were identified in a ratio of 1:2:1 for compound 1, D-galactose and D-glucuronic acid methyl ester were identified in a ratio of 2:1 for 2.

Anti-Proliferative Activity Assay in Vitro
The procedure for the anti-proliferative activity assay was performed according to the MTT reduction method using the human tumor cell lines BEL-7402, BGC-823, MCF-7, HL-60 and KB (Nanjing KeyGEN BioTECH Co., LTD, Jiangsu, China). In brief, the human tumor cell lines in culture medium (100 µL) were placed in a cell of a 96-well plate at a concentration of 4ˆ10 3 cells/mL and incubated at 37˝C in 5% CO 2 for 24 h. After 24 h, an additional 100 µL of complete medium with either: no additions (negative control), 0.1% DMSO (solvent control), 10 µg/mL Taxol (positive control), or different concentrations (0.391, 0.781, 1.562, 3.125, 6.25, 12.5, 25 and 50 µM) of 1 or 2 or total saponins (0.391, 0.781, 1.562, 3.125, 6.25, 12.5, 25 and 50 µg/mL). The treated cells were incubated as above for 72 h. Then, 20 µL of MTT solution (5 mg/mL) were added to the culture medium, and the reaction mixture was incubated as above for 4 h. After 4 h, the medium was discarded and 150 µL DMSO were added. The optical density (OD) of each well was measured at 490 nm using a Tunable Microplate Reader (EL-x800, BioTek Instruments, Winooski, VT, USA). The results were expressed as concentrations of compound producing 50% toxicity (IC 50 value).

Conclusions
Two new oleanane-type saponins, namely oleiferasaponins C 4 and C 5 (1, 2), were isolated from the seed cake of Camellia oleifera Abel. and identified. The anti-proliferative activity of the two compounds were investigated on five human tumor cell lines (BEL-7402, BGC-823, MCF-7, HL-60 and KB) and exhibited potent cytotoxic activities, especially compound 2.