Cytotoxic and Antimalarial Amaryllidaceae Alkaloids from the Bulbs of Lycoris radiata

Phytochemical investigation of the 80% ethanol extract of the bulbs of Lycoris radiata resulted in the isolation of five new Amaryllidaceae alkaloids: (+)-5,6-dehydrolycorine (1), (+)-3α,6β-diacetyl-bulbispermine (2), (+)-3α-hydroxy-6β-acetyl-bulbispermine (3), (+)-8,9-methylenedioxylhomolycorine-N-oxide (5), and 5,6-dihydro-5-methyl-2-hydroxyphenanthridine (7), together with two known compounds, (+)-3α-methoxy-6β-acetylbulbispermine (4) and (+)-homolycorine- N-oxide (6). Structural elucidation of all the compounds were performed by spectral methods such as 1D and 2D (1H-1H COSY, HMQC, and HMBC) NMR spectroscopy, in addition to high resolution mass spectrometry. Alkaloid 1 showed potent cytotoxicity against astrocytoma and glioma cell lines (CCF-STTG1, CHG-5, SHG-44, and U251), as well as HL-60, SMMC-7721, and W480 cell lines with IC50 values of 9.4–11.6 μM. Additonally, compound 1 exhibited antimalarial activity with IC50 values of 2.3 μM for D-6 strain and 1.9 μM for W-2 strain of Plasmodium falciparum.

Compound 3 corresponded to the molecular formula C 18 H 19 NO 6 , which was established by a quasimolecular ion peak in the HRESIMS. The general features of its IR and NMR spectra closely resembled those of 2, except that the acetyl at C-3 in 2 were replaced by a hydroxyl in 3, and an upfield shift of the signals for C-3 relative to that of 2 was observed. On the basis of the observation of NOESY data similar to those of 2, the stereochemistry of 3 was expected to be the same. Accordingly, compound 3 was elucidated to be (+)-3α-hydroxy-6β-acetylbulbispermine.
Malaria is one of the most common vector-borne infectious diseases. This disease is caused by parasites of the genus Plasmodium and causes such symptoms as anemia, fever, chills, nausea, and in severe cases, coma and death. The effects of isolated alkaloids in vitro antimalarial activity were evaluated by using the drug-resistant D-6 strain and the drug-sensitive W-2 strain of P. falciparum. Lycorine-type alkaloid 1 possessed high antimalarial activities with low IC 50 values (D-6: 2.3 μM; W-2 strain: 1.9 μM) ( Table 3)

General
Optical rotations were determined with a JASCO P2000 digital polarimeter (Tokyo, Japan). Ultraviolet (UV) and infrared (IR) spectra were obtained on JASCO V-650 and JASCO FT/IR-4100 spectrophotometers (Tokyo, Japan), respectively. The NMR spectra were measured in CDCl 3 on a Bruker AM-600 spectrometer (Fällanden, Switzerland). Chemical shifts were reported using residual CDCl 3 (δ H 7.26 and δ C 77.0 ppm) and CD 3

Plant Material
The bulbs of L. radiata were collected in April of 2011 in Lishui, a city of Zhejiang Province in China, and identified by one of the authors (Q.-J. Zhao). A specimen (201104001L) was deposited in the Herbarium of School of Pharmacy, Second Military Medical University, Shanghai, China.

Extraction and Isolation
The bulbs of L. radiata (10.5 kg) were cut into small pieces and were extracted with 80% ethanol (10 L) three times under reflux for 15 h and then concentrated under reduced pressure to give a crude extract (618.5 g). The crude extract was partitioned between equal volumes of chloroform and water to provide a chloroform-soluble fraction (110.6 g) and an aqueous layer. The chloroform-soluble fraction was further fractionated through a silica gel column (200-300 mesh) using increasing volumes of acetone in petroleum ether (100:1, 50:1, 30:1, 15:1, 10:1, 7:1, 5:1, 3:1, 1:1, V/V) as eluents to give 12 fractions according to TLC analysis.

Cytotoxicity Assay in Vitro
The cytotoxic activities of the isolated compounds were determined using the revised MTT method [26,27] against BEN-MEN-1 (meningioma), CCF-STTG1 (astrocytoma), CHG-5 (glioma), SHG-44 (glioma), U251 (glioma), HL-60 (human myeloid leukemia), SMMC-7721 (hepatocellular carcinoma), and W480 (colon cancer). Doxorubicin was used as the 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 concentrations of the assayed compounds were 0.04, 0.2, 1.0, 5, 25, and 125 μM, respectively. 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 non-linear regression with Graphpad Prism software version 4.0 (GraphPad Software, Inc., San Diego, CA, USA) and was used as criteria to judge the cytotoxicity. All the IC 50 results represent an average of a minimum of three experiments and were expressed as means ± standard deviation (SD). All cell lines were purchased from the Cell Bank of the Shanghai Institute of Biochemistry & Cell Biology, Chinese Academy of Sciences (Shanghai, China). Other reagents were purchased from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd (Shanghai, China).

Assay for in Vitro Antimalarial Activity against Plasmodium Falciparum
P. falciparum strains D-6 (drug-resistant) and W-2 (drug-sensitive) were cultured in human erythrocytes in RPMI medium (RPMI-1640 with 25 mM HEPES buffer, 24 mM NaHCO 3 , 0.2% glucose, 0.05% L-glutamine, 50 μg/mL hypoxanthine, and 25μg /mL gentamicin) supplemented with 10% human plasma at 37 °C, under 93% N 2 , 4% CO 2 , and 3% O 2 [4,10]. Antimalarial activity of the test compound was determined from the dose-response curve using the parasite lactate dehydrogenase (pLDH) assay according to the method of Makler [4,10]. The concentrations of the assayed compounds were 0.02, 0.1, 0.5, 2.5, 12.5, and 62.5 μM, respectively. One hundred and ninety microliters of asynchronous parasites (2.0% hematocrit and 0.5 or 1% parasitemia) was seeded into a 96-well microplate and 10 μL of test compound solution (dissolved in 25% ethanol or 5% DMSO) was added. After incubating at 37 °C for 72 h under 93% N 2 , 4% CO 2 , and 3% O 2 , the microplate was immediately frozen at −20 °C for 18 h. Then, the microplate was thawed at 37 °C and 20 μL of the hemolyzed parasite suspension was transferred to another microplate containing 100 μL of Malstat reagent. The plate was further incubated for 15 min at room temperature, and 20 μL of a 1:1 mixture of nitroblue tetrazolium and phenazine ethosulfate (2 mg and 0.1 mg/mL, respectively) was added to each well. After incubation for 2 h at room temperature in the dark, the blue formazan product was measured at 655 nm with iEMS microplate reader MF (Labsystems, Helsinki, Finland). The 50% inhibitory concentration (IC 50 ) value was determined by non-linear regression with Graphpad Prism software version 4.0 (GraphPad Software, Inc.). It was used as criteria to judge the activity (active: IC 50 ≤ 10 μM; moderately active: 10 μM < IC 50 ≤ 30 μM; not active: IC 50 > 30 μM). P. falciparum strains were purchased from the Cell Bank of the Shanghai Institute of Biochemistry & Cell Biology, Chinese Academy of Sciences.