Antibacterial, Antifungal and Cytotoxic Isoquinoline Alkaloids from Litsea cubeba

Five novel isoquinoline alkaloids (+)-N-(methoxylcarbonyl)-N-nordicentrin (1), (+)-N-(methoxylcarbonyl)-N-norpredicentrine (2), (+)-N-(methoxylcarbonyl)-N-norbulbodione (3), and (+)-N-(methoxylcarbonyl)-N-norisocorydione (4), and (+)-8-methoxyisolaurenine-N-oxide (5) were isolated, together with one known compound, (+)-N-(methoxylcarbonyl)-N-norglaucine (6), from a 70% EtOH extract of the barks of Litsea cubeba. Structural elucidation of all the compounds were performed by spectral methods such as 1D- and 2D-NMR, IR, UV, and HRESIMS. Alkaloids 1, 2 and 6 showed antimicrobial activity against the bacterium S. aureus and two fungi (A. alternata and C. nicotianae). Compounds 3,4 exhibited significant cytotoxicity against all of six tested tumor cell lines.

Compound 4 was obtained as a violet amorphous powder. Its positive HRESIMS spectrum showed a quasimolecular ion peak at m/z = 420.1055 [M+Na] + , consistent with the molecular formula C 21 H 19 NO 7 , accounting for 13 degrees of unsaturation. The general features of its IR and NMR spectra closely resembled those of 3, except for the chemical shifts of two methoxyls in 4 taking the place of the methylenedioxyl between C-1 and C-2 in 3, which was confirmed by HMBC correlations from the two methoxyl signal ( H 3.65 and 3.90) with C-1 ( C 143.8) and C-2 ( C 151.9), respectively. The stereochemistry of C-6a was established as S as inferred from its positive specific rotation ([α] 23.3 D = +87.3) [19]. Thus, the structure of 4 was assigned the name (+)-N-(methoxycarbonyl)-N-norisocorydione.

Plant Material
The barks of L. cubeba was collected in the Tongren, Guizhou Province, China, in July 2011. A specimen (201107001A) was identified by one of the authors (Q.C. Zhao) and deposited in the Herbarium of Shengyang Medicine College, Shengyang, China.

Extraction and Isolation
The barks of L. cubeba (9.6 kg) were cut into small pieces and were extracted with 70% EtOH (

Antimicrobial Activity Bioassay
All compounds (purity > 90%) were screened for their antimicrobial activity in vitro using the disk-diffusion method as described in the literature with minor modifications [19]. To each agar plate, an inoculum containing 10 7 bacteria/mL or a 0.5 optical density of the McFarland Scale was incorporated. The plates were solidified and sterile filter paper disks (6-mm diameter) were done on each one. Solution of each compound (5 mM) in DMSO, antibacterial agents (rifampicin 5 μM/mL), antifungal agents (nystatin 10 μM/mL), control vehicles (DMSO) were added into too. The plates were aerobically incubated at 37 °C for S. aureus during 18 h, for the five species of fungi during 24 h and for M. tuberculosis during 15-45 days, and four assays under identical conditions were carried out for each one. The diameter of the inhibition zone was measured for testing of antibacterial and antifungal activities. Experiments were performed in triplicate, and the results are presented as the mean values of the diameters of the inhibitory zones from three runs. The MIC values of the most active compounds, in the previous experiment, were determined using the dilution method in 96-hole plates. The diameters of the inhibitory zones and the MIC value were used as criteria to judge the antimicrobial activity (active: the diameters of the inhibitory zones ≥16 mm, MIC ≤ 5 mM; moderately active: the diameters of the inhibitory zones are visible, MIC > 5 mM; not active: the diameters of the inhibitory zones are invisible). All strains of bacteria and fungi were purchased from Shanghai Institute of Biochemistry & Cell Biology, Chinese Academy of Sciences (Shanghai, China).

Cytotoxicity Assay in Vitro
The cytotoxic activities of the isolated compounds were determined using the revised MTT method [21,22] against BGC-823 cells (human gastric carcinoma), HepG2 cells (Human hepatocellular carcinoma), MCF-7 cells (human breast cancer), SGC-7901 cells (human gastric adenocarcinoma), SK-MEL-2 (human skin cancer), and with SK-OV-3 (ovarian), with doxorubicin (DOX, adriamycin, Sigma Chemical Co., St. Louis, MO, USA) as positive control. Cancer cells (4 × 10 3 cells) suspended in 100 μL/well of DMEM medium containing 10% fetal calf serum were seeded onto a 96-well culture plate. After 24 h pre-incubation at 37 °C in a humidified atmosphere of 5% CO 2 /95% air to allow cellular attachment, various concentrations of test solution were added and cells were incubated for 48 h under the above conditions. At the end of the incubation, 10 μL of tetrazolium reagent was added into each well followed by further incubation at 37 °C for 4 h. The supernatant was decanted, and DMSO (100 μL/well) was added to allow formosan solubilization. The optical density (OD) of each well was detected using a microplate reader at 550 nm and for correction at 595 nm. Each determination represented the average mean of six replicates. The 50% inhibition concentration (IC 50 value) was determined by curve fitting and was used as criteria to judge the cytotoxicity (active: IC 50 ≤ 20 μM; moderately active: 20 μM < IC 50 ≤ 70 μM; not active: IC 50 > 70 μM). All cell lines were purchased from Cell Bank of Shanghai Institute of Biochemistry & Cell Biology, Chinese Academy of Sciences. Other reagents were purchased from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China)