Alkaloids with Nitric Oxide Inhibitory Activities from the Roots of Isatis tinctoria

As our ongoing research project on Ban Lan Gen (Isatis tinctoria roots), a total of 23 alkaloids were obtained. Compounds 1 and 2 contain an unusual C–C bond between the 2(1H)-quinolinone moiety and the phenol moiety and between the 2(1H)-quinolinone moiety and the 1H-indole moiety, respectively. Compound 3 possesses an unusual carbon skeleton and its putative biosynthetic pathway was discussed, and Compound 23 was deduced as a new indole alkaloid glycoside. Compounds 4–7 were identified as four new natural products by extensive spectroscopic experiments. Additionally, the anti-inflammatory activity was assessed based on nitric oxide (NO) production using Lipopolysaccharide-stimulated RAW264.7 macrophages. Compounds 9, 15, and 17 showed inhibitory effects with IC50 values of 1.2, 5.0, and 74.4 μM.


Inhibitory Assay of NO Production
Cytotoxicity was examined using the Cell Counting Kit-8 (CCK-8). RAW264.7 cells were taken from logarithmic growth stage and the cell density reached above 80%. And then, the cells were inoculated on 96-well cell culture plates at 100 microns/well, with 3×10 4 cells per well, cultured overnight at 37 °C with 5% CO2. After 24h, the culture medium in the 96-well plate was discarded, the solution was changed and different concentrations (25 μM, 50 μM and 100 μM) of drugs were added. The absorbance at 450 nm was measured using a microplate reader (Thermo Fisher Scientific). Viability was defined as the ratio (expressed as a percentage) of absorbance values of treated cells to untreated cells.
The results were listed in Table 3.
Compounds 1-23 were dissolved in dimethyl sulfoxide (DMSO) and diluted with complete medium to 6 degrees of concentration (Compound 9: 0.1, 0.5, 1, 5, 10 and 25 μM, while the others: 0.1, 1, 5, 25, 50 and 100 μM) for inhibition rate determination. RAW 264.7 cells were maintained in Dulbecco's modified Eagle's medium (high-glucose condition) supplemented with 10% fetal calf serum, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in 5% CO2. RAW 264.7 cells were pretreated with each tested compound for 30 min, and then stimulated with lipopolysaccharide (LPS) (100 ng/mL) for 24 h. Aminoguanidine hydrochloride (100 μM) was used as a positive control. The NO production was measured using the Griess reagent. Briefly, the cell culture supernatant was reacted with equal volumes of Griess reagent in a 96-well plate for 10 min, and then the absorbance at 540 nm was measured by a plate reader. All experiments were performed in triplicate. All tested compounds were prepared as stock solutions with a concentration of 10 mM in DMSO. The IC50 values of compounds 1-23 were calculated.

S5
The conformers of compound 23 were obtained using the MM2 force field with ChemBio3D software. Gaussian 09 software was utilized for the semiempirical PM3 quantum mechanical geometry optimizations and the time-dependent density functional theory (TDDFT) ECD was calculated at the b3lyp/6-31g(d) level. The ECD spectra conformers of compound 23 were obtained using SpecDis 1.62 and compared with the experimental data.

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