Chemical constituents from the roots and stems of Litsea cubeba

Abstract A new monoterpene and a new lignan, named litsecols A and B (1 and 2), respectively, together with nine known compounds (3–11), were isolated in a continuous investigation on the roots and stems of Litsea cubeba. Their structures were elucidated on the basis of extensive spectroscopic data analysis, and the absolute configuration of 1 was resolved by X-ray diffraction analysis. Compounds 2–5 and 7–9 showed significant inhibitory activity against nitric oxide (NO) production in lipopolysaccharide (LPS)-induced murine microglial (Bv-2) cell line. Compounds 10 and 11 exhibited significant neuroprotective effect against hydrogen peroxide-induced oxidative damage in rat adrenal pheochromocytoma (PC12) cell line.


Introduction
Litsea cubeba is a small deciduous aromatic tree belonging to the family Lauraceae. It is widely cultivated in tropical or subtropical areas, including most southern regions of China [1]. The roots and stems of L. cubeba, 'Dou-chi-jiang' in Chinese, have been used for the treatment of various ailments. Modern pharmacological research has demonstrated its activities in cardiovascular diseases and its anticancer, asthma-relieving, and antioxidants effects [2,3]. We previously reported the isolation of aporphine alkaloids and phenolic constituents from the roots and stems of L. cubeba [4,5].
All compounds were tested for NO product inhibitory activity in Bv-2 cell line (Table 3) and neuroprotective effect in PC12 cell line ( Table 4). The results showed that compounds 2-5 and 7-9 exhibited significant inhibition against NO production in Bv-2 cells, with their IC 50 values of 15.8-50.9 μM. Compounds 10 and 11 showed significant neuroprotection against hydrogen peroxide-induced oxidative damage in PC12 cells.

Plant material
The roots and stems of Litsea cubeba were collected from Huaiji Town in Zhaoqin Country, Guangdong province, China, in Oct. 2012, and authenticated by Dr. Yuan Zhang (School of Chinese Materia Medica, Beijing University of Chinese Medicine, BUCM). A voucher specimen (LC201210) is deposited in Modern Research Center for Traditional Chinese Medicine, BUCM.

NO production inhibitory assay
Bv-2 cells were seeded in 96-well plates (1 × 10 5 cells/well). The cells were pretreated with drugs for 4 h and then incubated with LPS (1 μg/ml) for 24 h. The amount of NO was assessed by determining the nitrite concentration in the cultured Bv-2 cells with Griess reagent. The absorbance was recorded on a microplate reader at a wavelength of 540 nm. Cell viability was measured by an MTT assay, and the absorbance read at 570 nm with an ELISA analyzer. Quercetin was used as a positive control [24].

Neuroprotective activity assay
PC12 neuroblastoma cells were seeded in 96-well plates at a density of 2 × 10 5 cells/ml, 100 μl/well. Experiments were carried out 24 h after cells were seeded. The PC12 cells were pre-incubated with the different compounds 2 h before hydrogen peroxide (200 μM) was added. Cell survival was evaluated by reduction of MTT. Briefly, 24 h after hydrogen peroxide exposure, MTT solution in PBS (100 μl/well, 0.5 mg/ml) was added, and incubation was continued for 4 h. Finally, 150 μl of DMSO was added, and the amount of MTT formazan was quantified by determining the absorbance at 570 and 630 nm using a microplate reader. The values of cell survival were normalized against the values for the control group, which was set to 100%. Data were evaluated for statistical significance with a one-way ANOVA followed by an LSD test using a computerized statistical package. Vitamin E was used as a positive control [25].