Marylosides A-G, Norcycloartane Glycosides from Leaves of Cymbidium Great Flower ‘Marylaurencin’

Seven novel norcycloartane glycosides, maryloside A–G (1–7), were isolated from the leaves of Cymbidium Great Flower ‘Marylaurencin’, along with a known norcycloartane glycoside, cymbidoside (8). These structures were determined on the basis of mainly NMR experiments as well as chemical degradation and X-ray crystallographic analysis. The isolated compounds (1–6 and 8) were evaluated for the inhibitory activity on lipopolysaccharide (LPS) and interferon-γ (IFN-γ)-stimulated nitric oxide (NO) production in RAW 264.7 cells. Consequently, 1 and 3 exhibited moderate activity.


Introduction
Cymbidium Great Flower 'Marylaurencin' is a new breed produced by Kawano-Mericlone Corporation by mating. The seed is registered at the Ministry of Agriculture, Forestry and Fisheries of Japan. During our search for bioactive substances from C. Great Flower 'Marylaurencin', which is a cultivar, we have already reported some phenanthrene derivatives with antibacterial effect and cytotoxic activity, and some aromatic glycosides with antioxidant activity from its root, stem and flower [1][2][3][4]. The leaves of C. Great Flower 'Marylaurencin' are sold as an herb tea in this area. Therefore, we started exploring the active ingredients from the leaves of this plant to support peoples' health. Here, we report the isolation and structural determination of seven novel 30-norcycloartane-type triterpene glycosides (1-7) along with one known compound, cymbidoside (8) [5], from the leaves of C. Great Flower 'Marylaurencin'. Their structures were elucidated using high resolution (HR)-FAB-MS analyses, extensive two dimensional (2D)-heteronuclear NMR data interpretation and X-ray crystallographic analysis.
The negative FAB-MS of maryloside F (6) gave a molecular ion peak at m/z 753.4033 [M − H] − , corresponding to the molecular formula C 39 H 62 O 14 . The IR spectrum showed the presence of carbonyl group (1711 cm −1 ) and hydroxyl group (3150 cm −1 ). The 1 H-NMR and the 13 C-NMR spectra of 6 were similar to those of 2, but a few differences were also recognized. The 13 C-NMR spectrum of 6 showed the presence of two carboxylate esters at δ C 169.6 and 168.1. The HMBC correlation from anomeric proton H-1' at δ H 4.90 (d, J = 7.8 Hz) to C-28 indicated that the glucose connected to C-28. The HMBC correlations from H-6' to C-α and from H-β to C-α and C-γ indicated that malonyl moiety connected to C-6'. From the above data, the structure of maryloside F (6) was elucidated as 24-methyl-30-norcycloart-22R*, 24S*, 25, 28, 30-pentaol-3-one-28-O-(6 -O-malonyl) β-D-glucopyranoside ( Figure 4).
The molecular formula of maryloside G (7) was determined to be C 48 H 82 O 19 from the molecular ion peak [M − H]at m/z 961.5522 (calcd. for m/z 961.5524). The IR spectrum showed the presence of hydroxyl group (3367 cm −1 ). The 1 H-NMR and 13 C-NMR spectra also suggested that 7 was the cycloartane-type saponin with three sugar moieties (Tables 1 and 2 . The 13 C-NMR spectrum of 7 showed no carbonyl groups. HMBC correlations from a methyl group at δ H 1.41 to C-3, C-4 and C-5 showed that this methyl connected to C-4 position ( Figure 5). Moreover, an oxygen-bearing methine proton at δ H 3.45 revealed to be at C-3 position from 1D and 2D NMR data. From the coupling pattern of H-3 at δ H 3.45 (ddd, J = 10.6, 10.6, 4.6 Hz), the configurations of H-3 and H-4 found to be α-axial and β-axial protons, respectively. HMBC correlations from the anomeric proton at H-1' to C-3, from the anomeric proton at H-1"to at C-2' and from the anomeric proton at H-1 to at C-24 indicated that two glucose (S 1 in Figure 4 Marylosides A-G (1-7) and cymbidoside (8) were evaluated for the inhibitory activity of NO production in the LPS and IFN-γ-stimulated RAW 264.7 cells. Furthermore, 1 and 3 showed the inhibitory activity in dose dependently, the IC50 value was calculated as 17.8 ± 2.3 and 83.9 ± 4.8 µM, respectively, while IC50 of AG was calculated as 81.4 ± 2.6 µM ( Figure 6). Other compounds showed weak inhibitory activities (IC50 values were ≧100 µM). The result of MTT cell viability assay revealed that all compounds hardly affect to cell viability. Considering these results, the hydroxy group at position 25 reduced the activity. Additionally, the comparison of 1 and 4 suggests the presence of glucose moiety at position 30 which also reduced the NO producing inhibitory activity. Marylosides A-G (1-7) and cymbidoside (8) were evaluated for the inhibitory activity of NO production in the LPS and IFN-γ-stimulated RAW 264.7 cells. Furthermore, 1 and 3 showed the inhibitory activity in dose dependently, the IC 50 value was calculated as 17.8 ± 2.3 and 83.9 ± 4.8 µM, respectively, while IC 50 of AG was calculated as 81.4 ± 2.6 µM ( Figure 6). Other compounds showed weak inhibitory activities (IC 50 values were 100 µM). The result of MTT cell viability assay revealed that all compounds hardly affect to cell viability. Considering these results, the hydroxy group at position 25 reduced the activity. Additionally, the comparison of 1 and 4 suggests the presence of glucose moiety at position 30 which also reduced the NO producing inhibitory activity.

General Procedures
Melting points were determined with a Yanagimoto micro melting point apparatus (Yanaco, Kyoto, Japan). Optical rotation was performed on JASCO DIP-1000 polarimeter (JASCO, Tokyo, Japan). IR spectra were measured on a JASCO FT/IR-5300. NMR spectra were obtained on a Varian UNITY 600 NMR spectrometer (Varian, CA, USA) with deuterated solvents (MeOH-d4 and pyridine-d5), and the solvent chemical shifts were taken as the internal standard. The chemical shifts are given in δ (ppm), and coupling constants are reported in Hz. HR-FAB-MS spectra were obtained on a JEOL JMS-700 instrument (JEOL, Tokyo, Japan). Kieselgel 60 (230-400 mesh, Merck) and Sephadex LH-20 (GE Healthcare Life Sciences, IL, USA) were used for column chromatography. HPLC separation was performed on a JASCO PU 1580 pump with a JASCO UV-970 detector with a CAPCELL PAK C18 AQ (SHISEIDO, Tokyo, Japan, 20 mm i.d. × 250 mm).

Plant Material
Plant growth conditions of inside a plastic greenhouse of Kawano-Mericlone Corporation in Tokushima Prefecture, Japan. Insolation; Cymbidium was grown in a plastic greenhouse. A 50% shaded net was used in summer. The average temperature was 12-30 °C. The average humidity was 40-90%.
The   Table 2. 13 C-NMR (150 MHz) spectral data of compounds 1-7, 1a, and 8a (δ in ppm).  The powder of leaves of C. Great Flower 'Marylaurencin' were supplied by Kawano-Mericlone Corporation in Tokushima Prefecture, Japan. C. Great Flower 'Marylaurencin' (Ministry of Agriculture, Forestry and Fisheries of Japan, seed registration No. 2841) was cultivated and harvested in November 2008 at Kawano-Mericlone Co., Ltd. and were identified by one of the authors (Dr. S. Kawano). A voucher specimen (TB 5430) has been deposited in the Herbarium of Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima, Japan.

Extraction and Isolation
The leaves were dried after harvest and made into powder and stored at −20 degrees until we started the research. The powder of leaves of C. Great Flower 'Marylaurencin' (2.0 kg) were extracted with 70% EtOH aq. at room temperature for 3 months. The 70% EtOH aq.

Determination of Nitric Oxide (NO) Production and MTT Cell Viability Assay
The concentration of nitrite in the medium was measured as indicator of NO production according to the Griess method [7]. Cells were cultured in F-12 HAM medium with 10% fetal bovine serum, 100 U/mL penicillin G and 0.1 mg/mL streptomycin. Cells were seeded on 96-well plate at 2.4 × 10 5 cell/well and incubated at 37 • C for 2 h. Then, medium was changed to one with LPS (final concentration: 100 ng/mL), IFN-γ (final concentration: 0.33 µg/mL) in each sample. After 24 hr of incubation at 37 • C, supernatants were collected, and NO concentrations were measured by using the Griess method. Aminoguanidine was used as positive control (final concentration: 100 µM). Cell viability was assessed by MTT assay [8]. After removal of supernatant for measurement of NO concentration, 10 µL of MTT solution (5 mg/mL in phosphate buffered saline) was added. MTT-formazan was measured by the following procedure. Data represent the mean value ± standard error of mean of experiments performed in triplicated.

X-ray Crystallographic Analysis
Single crystals of aglycone of maryloside A (1a), obtained from MeOH solution were selected and fitted onto a glass fiber and measured at −173 • C with a Bruker Apex II ultra-diffractometer using MoKα radiation. Data correction and reduction were performed with the crystallographic package Apex II. The structure was solved by direct methods using SHELXS-97 and refined by means of full matrix least-squares based on F 2 using SHELXL-97 (Sheldrick, 1997). All non-hydrogen atoms were refined anisotropically, and hydrogen atoms were positioned geometrically. A total of 325 parameters were finally considered. Final disagreement indices were R 1 = 0.0495, wR 2 = 0.1269 (I > 2 sigma(I)). The

Conclusions
Seven novel 30-norcycloartane-type triterpene glycosides (1-7) along with one known compound (8) were isolated from the leaves of C. Great Flower 'Marylaurencin'. Although their relative structures were estimated by using NOE correlations and J values, it was difficult to determined relative configuration from them regarding side chains of these compounds. The configuration at the C-20 position of these compounds was suggested as S* configuration according to biosynthetic pathway [9][10][11]. The configuration at C-20 position of 1a was suggested as S* configuration according to NOE correlations between H-12β-equatorial and H-21, and between H-18 and H-20 and the relative configuration of side chain of 1 was definitely determined as 20S*, 22R* and 24R* by X-ray crystallographic analysis of 1a, eventually. The relative structure of the side chain from C-20 to C-30 of 3 was fully the same as 1. The chemical shift values of 13 C-NMR of 3 and 4 almost superimposable with those reported for 1. Therefore, the relative configuration of side chain of 3 and 4 was decided the same as 1. The same thing as 3 and 4 could be said for 2, 5 and 6 compared with 8. The relative configuration of 8 was determined as 20S*, 22R* and 24S* by the X-ray crystallographic analysis of aglycon of 8 (Figures 7 and 8). The chemical shift values of 13 C-NMR of 2, 5 and 6 almost superimposable with those of 8. Therefore, configuration of side chain of 2, 5 and 6 was decided as 20S*, 22R* and 24S*. configuration according to NOE correlations between H-12β-equatorial and H-21, and between H-18 and H-20 and the relative configuration of side chain of 1 was definitely determined as 20S*, 22R* and 24R* by X-ray crystallographic analysis of 1a, eventually. The relative structure of the side chain from C-20 to C-30 of 3 was fully the same as 1. The chemical shift values of 13 C-NMR of 3 and 4 almost superimposable with those reported for 1. Therefore, the relative configuration of side chain of 3 and 4 was decided the same as 1. The same thing as 3 and 4 could be said for 2, 5 and 6 compared with 8. The relative configuration of 8 was determined as 20S*, 22R* and 24S* by the X-ray crystallographic analysis of aglycon of 8 (Figures 7 and 8). The chemical shift values of 13 C-NMR of 2, 5 and 6 almost superimposable with those of 8. Therefore, configuration of side chain of 2, 5 and 6 was decided as 20S*, 22R* and 24S*.  In NO production inhibitory activity assay, 1 and 3 exhibited moderate activity whereas the result of the MTT cell viability assay revealed that all compounds hardly affect cell viability. To the best of our knowledge, this is the first report of the anti-inflammatory effect of norcycloartane glycosides while norcycloartanes are reported to show anti-bacterial activity against Micrococcus luteus and Bacillus subtilis or anti-human immunodeficiency virus activity [12,13]. Inflammation by over-production of NO was related to tissue toxicity, aging and occurrence of adenocarcinoma. Therefore, continuous drinking of tea of C. Great Flower 'Marylaurencin' may contribute to a human's health life. Taking into consideration of all other bioactivities those we already reported about C. Great Flower 'Marylaurencin', it is worth to study C. Great Flower 'Marylaurencin' further. In NO production inhibitory activity assay, 1 and 3 exhibited moderate activity whereas the result of the MTT cell viability assay revealed that all compounds hardly affect cell viability. To the best of our knowledge, this is the first report of the anti-inflammatory effect of norcycloartane glycosides while norcycloartanes are reported to show anti-bacterial activity against Micrococcus luteus and Bacillus subtilis or anti-human immunodeficiency virus activity [12,13]. Inflammation by over-production of NO was related to tissue toxicity, aging and occurrence of adenocarcinoma. Therefore, continuous drinking of tea of C. Great Flower 'Marylaurencin' may contribute to a human's health life. Taking into consideration of all other bioactivities those we already reported about C. Great Flower 'Marylaurencin', it is worth to study C. Great Flower 'Marylaurencin' further.