A new ascorbic acid derivative and two new terpenoids from the leaves and twigs of Rhododendron decorum

Abstract The phytochemical study of the ethanol extract of the leaves and twigs of Rhododendron decorum afforded a new ascorbic acid derivative (1), a new ionone analogue (2), a new ursane-type triterpenoid glucoside (3), and four known compounds (4−7). The structures were elucidated by spectroscopic analyses, including HRESIMS, 1D, and 2D NMR. The anti-neuroinflammatory activities of the compounds were evaluated by measuring inhibitory effects of LPS-induced NO production in BV2 cells. GRAPHICAL ABSTRACT


Introduction
Rhododendron decorum is an evergreen shrub that distributes mainly in southwestern China and northeastern Burma. It is used as folk medicine in China to treat rheumatism and pain [1]. The family of Ericaceae is rich in terpenoids, lignans, and flavonoids, some of which show valuable anti-inflammatory, antinociceptive, antiviral, sodium channel antagonistic, and cytotoxic activities [2][3]. To explore the chemical diversity and bioactivity, leaves and twigs of R. decorum were collected from Yunnan Province. In the present investigation, three new compounds along with five known compounds were isolated ( Figure 1). Herein, the isolation and structural elucidation as well as the anti-neuroinflammatory activities of these compounds are described.
The 1 H and 13 C NMR data of 1 were similar to those of amarusine A (4) [4], which possesses a skeleton composed of a spirocyclic di-tetrahydrofuran fused to a butyrolactone ring and an aromatic ring. NMR data of 1 indicated the existence of a 3,4-dioxygenated aromatic ring, which is different from amarusine A (4). This was confirmed by the HMBC correlations of H-5 (d H 7.24) to C-1 (d C 131.0) and C-4 (d C 149.0). In the NOESY spectrum (  (d H 6.88 and 7.73). The 13 C NMR and DEPT spectra exhibited 20 carbon resonances, including five methyls, three methylenes, eight methines, and four quaternary carbons. The NMR data of 2 were consistent with an ionone-type glycoside and comparable to those of pisumionoside (5) [5]. The HMBC correlation from H 3 -14 (-OCH 3 , d H 3.30) to C-5 (d C 81.2) indicated that 5-OH is methylated. In the NOESY spectrum, the   In addition, the anomeric proton at H-1 0 (d H 6.29) was observed with a large coupling constant (8.1 Hz), which indicated the b-configuration of the glucose. Acid hydrolysis and GC analysis of the sugar moiety of 3 confirmed the sugar is D -glucose. Thus, the structure of 3 was elucidated as depicted and named rhododeoside B.
Compounds were assayed for their anti-neuroinflammatory activities by measuring inhibitory effects of LPS-induced NO production in BV2 cells. The inhibition rates of compounds 5À7 (10 lM) are 18.08%, 16.67%, and 17.37%, respectively.

Plant material
Twigs and leaves of Rhododendron decorum were collected from Chuxiong, Yunnan Province in April 2014. The plant was authenticated by associate Prof. Lin Ma of Peking Union Medical College. A voucher specimen (ID-s-2603) was deposited in the herbarium of our institute.

Acid hydrolysis and GC analysis of the sugar moieties of compounds 2 and 3
Compounds 2 and 3 (1.0 mg) were added to 2 N HCl (2 ml). The solution was heated at 90 C for 12 h. The reaction mixture was evaporated and partitioned with EtOAc and H 2 O. The aqueous layer was concentrated to furnish the sugar mixture, which was dissolved in dry pyridine and reacted with L -cysteine methyl ester hydrochloride (2 mg) at 80 C for 1 h. After removal of the solvent, N-trimethylsilylimidazole (1 ml) was added, and the mixture was heated at 80 C for 0.5 h. The mixture was evaporated to dryness, and the residue partitioned into n-hexane and H 2 O. The sample was analyzed on a GC system equipped with an FID (detector temperature, 300 C). Chromatography conditions: injection temperature, 280 C; column, HP-5 (60 m Â 0.32 mm Â 0.25 lm); initial column temperature, 200 C; column temperature increased to 280 C (10 C/min) and kept at 280 C for 40 min under N 2 carrier gas (1.8 ml/min).
In the GC chromatogram, the derivatives of the acid hydrolysate of 2, 3 and the D -glucose (standard) showed approximate retention times of 29.61, 29.61, and 29.62 min, respectively.

Anti-neuroinflammatory activity assays
Compounds (5À7) were tested for their anti-neuroinflammatory activity by measuring inhibitory effects of LPS-induced NO production in BV2 cells. Curcumin was used as the positive control. The BV2 macrophage cell line was obtained from the Cell Culture Center at the Institute of Basic Medical Sciences, Peking Union Medical College. LPS was bought from Sigma-Aldrich company. After preincubation for 24 h in a 96-well plate, the cells were treated with the test compounds (10 À5 mol/L), then stimulated with LPS for 24 h. The production of NO was determined via measuring the concentration of nitrite in the culture supernatant. NaNO 2 was utilized to generate a standard curve. The absorbances at 550 nm were measured.