Chemical Constituents of the Seeds of Raphanus sativus and their Biological Activity

As part of our ongoing search for bioactive constituents of natural Korean medicinal resources, a bioassay-guided fractionation and chemical investigation of the MeOH extract of Raphanus sativus (Brassicaceae) seeds resulted in the isolation and identification of fifteen compounds, including a new phenolic compound. The structure of the new compound was determined by extensive spectroscopic analysis and the Mosher’s method. One of the compounds has been recently reported as a synthetic product. Some compounds showed moderate antiproliferative activities against the tumor cell lines A549, SK-OV-3, SK-MEL-2, and HCT-15 with IC50 values in the range of 5.62 to 28.88 μM. Moreover, the anti-neuroinflammatory activities of the isolates were determined by measuring the nitric oxide (NO) levels in the medium using murine microglia BV-2 cells. With exception of one specific compound, all the others inhibited the lipopolysaccharide (LPS)-stimulated NO production (IC50 values < 200 μM).


Introduction
The edible root vegetable Raphanus sativus L. (Brassicaceae), commonly known as radish, is one of the most widely grown and consumed vegetables throughout the world.][8] In a previous work, 9,10 we found that the MeOH extract of Raphani Semen exhibits significant cytotoxic activity against the human tumor cell lines, A549, SK-OV-3, SK-MEL-2, and HCT-15, in addition to inhibitory effects on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells.This observation led to the isolation and identification of 4-methylthiobutanyl derivatives and phenylpropanoid sucrosides that correlated with the cytotoxic and anti-inflammatory activities. 9,10Using a bioassay-guided methodology, we are now reporting the isolation of fifteen compounds (1-15), including a new phenolic (1), from the most active CHCl 3 -soluble fraction of the MeOH extract (Figure 1).In addition, their antiproliferative activities against A549, SK-OV-3, SK-MEL-2, and HCT-15 cell lines, and their inhibitory effects on NO production in a LPS-activated BV2 cell line, have been evaluated.

Plant material
The seeds of R. sativus were purchased at the Kyungdong herbal market, Seoul, Korea, in January 2010, and identified by one of the authors (K.R. Lee).A voucher specimen (SKKU-2010-01) has been deposited in the herbarium of the School of Pharmacy, Sungkyunkwan University, Suwon, Korea.

Cytotoxicity assays
All tumor cell cultures were maintained using RPMI1640 cell growth medium (Gibco, Carlsbad, CA), supplemented with 5% fetal bovine serum (FBS) (Gibco), 100 units mL −1 penicillin and 100 μg mL −1 streptomycin.The human tumor cell lines, A549 (non-small cell lung carcinoma), SK-OV-3 (ovarian malignant ascites), SK-MEL-2 (skin melanoma), and HCT-15 (colon adenocarcinoma) were provided by the National Cancer Institute (NCI).The cytotoxicity of the isolates against cultured human tumor cell lines was evaluated by the sulforhodamine B (SRB) method.Each tumor cell line was inoculated into standard 96-well flat-bottom microplates and incubated for 24 h at 37 °C in a humidified atmosphere of 5% CO 2 .The attached cells were then incubated with the serially diluted compounds with the control cultures receiving the carrier solvent (0.1% dimethyl sulfoxide).After continuous exposure to the compounds for 48 h, the culture medium was removed from each well and the cells were fixed with 10% cold trichloroacetic acid at 4 °C for 1 h.After washing with tap water, the cells were stained with 0.4% SRB dye and incubated for 30 min at room temperature.The cells were washed again and solubilized with 10 mM unbuffered Tris base solution (pH 10.5).The absorbance was measured spectrophotometrically at 520 nm using a microtiter plate reader.Doxorubicin (purity ≥ 98%; Sigma) was used as a positive control.Tested compounds were demonstrated to be pure as evidenced by NMR and HPLC analysis (purity ≥ 95%).

Measurement of NO production
The murine microglial BV2 cell line was generously provided by PhD E. Choi from Korea University (Seoul, Korea), and maintained in Dulbecco's modified Eagle (DMEM) medium supplemented with 5% FBS, 100 units mL −1 penicillin, and 100 μg mL −1 streptomycin.All cells were incubated at 37 °C in a humidified incubator with 5% CO 2 .BV-2 cells were plated into a 96-well plate (3 × 10 4 cells well −1 ).Following culture for 24 h, cells were pretreated with the compounds for 30 min, and subsequently stimulated with 100 ng mL −1 of LPS for a further 24 h.Control cultures received the carrier solvent (0.1% dimethyl sulfoxide).Nitrite, a soluble oxidation product of NO, was measured in the culture media using the Griess reaction.The supernatant (50 μL) was harvested and mixed with an equal volume of Griess reagent (1% sulfanilamide, 0.1% N-1-napthylethylenediamine dihydrochloride in 5% phosphoric acid).After 10 min, the absorbance at 570 nm was measured using a microplate reader.Sodium nitrite was used as a standard to calculate the NO 2 − concentration.N G -monomethyl-L-arginine (NMMA, Sigma, St. Louis, MO, USA), a well-known NO synthase inhibitor, was tested as a positive control.
Compounds 1-15 were also examined for their antineuroinflammatory activities by measuring the NO levels produced in LPS-activated BV-2 cells, a microglial cell line.The results (Table 3) showed that all the compounds, with the exception of compound 6, inhibited NO production (IC 50 values < 200 μM).These compounds had no effect on cell viability in LPS-treated BV-2 cells at their respective IC 50 values (data not shown).Among these, compounds 2, 4, and 12 significantly inhibited NO levels with IC 50 values of 18.99, 18.69, and 15.60 μM, respectively.In particular, compound 12 showed potent inhibition of NO in LPSstimulated NO production with an IC 50 of 15.60 µM, which is a lower concentration than that displayed by the positive control N G -nonomethyl-L-arginine (IC 50 19.15µM).Excess production of NO by activated microglia induces neuronal cell death, 26,27 which consequently leads to various neurodegeneration disorders of the CNS.Therefore, the active compounds 2, 4, and 12 could be considered potential candidates in the prevention of the progressive damage resulting from neurodegenerative diseases.

Conclusions
The bioassay-guided fractionation and chemical investigation of the MeOH extract of R. sativus seeds resulted in the isolation and identification of fifteen  compounds (1-15), including a new phenolic compound, methyl (3R)-hydroxy-3-(4-hydroxy-3,5-dimethoxyphenyl) propanoate (1).Among the isolates, three indole alkaloids (9-11) showed good antiproliferative effects against all the tumor cells tested, with IC 50 values ranging from 5.62 to 28.88 μM.In addition, compounds 2, 4, and 12 significantly inhibited LPS-stimulated NO production in murine microglia BV-2 cells with IC 50 values of 18.99, 18.69, and 15.60 μM, respectively.These results suggest that the above mentioned active compounds with cytotoxic or anti-neuroinflammatory activities, could be considered as lead molecules for drug development related to various cancers or neurodegenerative diseases.
1 (Table 1) exhibited a total of 12 carbons including six aromatic carbons in the range d C 147.1-102.3,one oxygenated carbon at d C 70.5, one methylene carbon at d C 43.4, one carbonyl carbon at d C 172.8, and three methoxy carbons at d C 56.3 (× 2) and 51.9.Interpretation of the 2D NMR spectra correlations, including

Table 3 .
Inhibitory effects of compounds 1

Table 2 .
Antiproliferative activities of compounds 2, 3, and 9-11 isolated from R. sativus IC 50 value of compounds against each cancer cell line.The IC 50 value was defined as the concentration (μM) causing 50% inhibition of cell growth in vitro; b Doxorubicin as positive control. a