(±)-Zanthonitidine A, a Pair of Enantiomeric Furoquinoline Alkaloids from Zanthoxylum nitidum with Antibacterial Activity

Abstract A pair of new enantiomeric furoquinoline alkaloids, (±)-zanthonitidine A (1), together with nine known ones (2–10) were isolated from the radix of Zanthoxylum nitidum. Their chemical structures were elucidated based on the extensive spectroscopic analysis. The racemic mixture of 1 was separated by chiral column chromatography, and the absolute configurations of (+)-1 and (−)-1 were determined by the comparison of experimental and calculated electronic circular dichroism spectra. Antibacterial activities of compounds 1–9 were evaluated, and compounds (+)-1, (−)-1, 3, 7 and 8 showed antibacterial activities against Bacillus subtilis, Enterococcus faecalis or Staphylococcus aureus. Graphical Abstract Electronic supplementary material The online version of this article (10.1007/s13659-018-0169-7) contains supplementary material, which is available to authorized users.


Results and Discussion
; three methoxyl groups at d C 59.5 (q), 56.7 (q), 56.7 (q). Based on these data, 1 was presumed to be a furoquinoline containing glycerol and benzene moieties.
The structure was elucidated by detailed interpretation of 2D NMR correlations (Fig. 2). The HMBC correlations from d H 7.56 (H-a) to d C 163.8 (C-2) and d C 102.    The antimicrobial activities of compounds 1-9 was tested on the gram-positive strains Bacillus subtilis, Enterococcus faecalis, and Staphylococcus aureus ( In summary, the phytochemical investigation of the radix of Zanthoxylum nitidum in this study led to the identification of ten alkaloids (1-10) including a pair of

General Experimental Procedures
Optical rotations were measured with a Horiba SEPA-300 polarimeter. UV spectra were obtained using a Shimadzu UV-2401A spectrophotometer. CD spectra were tested using Chirascan Circular Dichroism spectrometer. A Tenor 27 spectrophotometer was used for scanning IR spectroscopy with KBr pellets. MS data were measured on Agilent G6230 TOF Mass spectrometer. 1D-NMR and 2D-NMR spectra were measured on a Bruker AM-400, DRX-500 or AVANCE III-600 at 298 K. Chemical shifts (d) were expressed in parts per million (ppm) with reference to the solvent signals. Semi-preparative HPLC was performed on Waters HPLC system (1525 pump with 2998 photodiode array detector and 2707 autosampler) coupled with Zorbax Eclipse-C18 (9.4 mm 9 250 mm; 5 lm) for purification or DAICEL Chiralpak ID column (4.6 mm 9 250 mm; 5 lm) for chiral analysis.

Extraction and Isolation
The air-dried and milled radix of Z. nitidum (10 kg) was extracted three times with methanol (3 9 20 L) under reflux, and the resulting solution was evaporated under reduced pressure to yield the methanol extract (688.4 g). All amount of the extract was separated by a silica gel column chromatography (CC) (100-200 mesh), eluted with a gradient of

ECD Calculation
Conformational analysis was initially performed using Confab at MMFF94 force field for two configurations for 1. Room-temperature equilibrium populations were calculated according to Boltzmann distribution law. The conformers with Boltzmann-population of over 1% were subjected to ECD calculations. The theoretical calculation was carried out using Gaussian 09 [20]. The comformers was initially optimized at PM6 using semiempirical theory method, and then optimized at the B3LYP/6-311G (d, p) in MeOH using the IEFPCM model. The theoretical calculation of ECD was conducted in MeOH using Time-dependent Density Functional Theory (TD-DFT) at the same theory level.

Antibacterial Assay
Bacillus subtilis, Enterococcus faecalis, and Staphylococcus aureus were cultured in Luria-Bertani (LB) broth. The broth microdilution assay was applied for the antibacterial activity screening according to CLSI guidelines (CLSI 2015). Bacillus subtilis, E. faecalis, and S. aureus were propagating in the Mueller-Hinton broth (0.20%, w/v, beef extract; 1.75%, w/v, acid digest of casein; 0.15%, w/v, starch). After incubation with various concentrations of 1-9 at 37°C for 24 h, the 96-well plates were checked by visual inspection; penicillin was used as the positive control. The MICs were determined as the lowest concentration for no visible growth of bacteria.