Antimicrobial Furoquinoline Alkaloids from Vepris lecomteana (Pierre) Cheek & T. Heller (Rutaceae)

Three new prenylated furoquinoline alkaloids named lecomtequinoline A (1), B (2), and C (3), together with the known compounds anhydroevoxine (4), evoxine (5), dictamnine (6), N-methylflindersine (7), evoxanthine (8), hesperidin, lupeol, β-sitosterol, stigmasterol, β-sitosterol-3-O-β-d-glucopyranoside, stearic acid, and myristyl alcohol, were isolated by bioassay-guided fractionation of the methanolic extracts of leaves and stem of Vepris lecomteana. The structures of compounds were determined by spectroscopic methods (NMR, MS, UV, and IR) and by comparison with previously reported data. Crude extracts of leaves and stem displayed high antimicrobial activity, with Minimum Inhibitory Concentration (MIC) (values of 10.1–16.5 and 10.2–20.5 µg/mL, respectively, against Escherichia coli, Bacillus subtilis, Pseudomonas agarici, Micrococcus luteus, and Staphylococcus warneri, while compounds 1–6 showed values ranging from 11.1 to 18.7 µg/mL or were inactive, suggesting synergistic effect. The extracts may find application in crude drug preparations in Western Africa where Vepris lecomteana is endemic, subject to negative toxicity results in vivo.

Since species of Vepris are used in traditional medicine for the treatment of bacterial infections related to forms of pneumonia, ocular diseases, cardiac pains, coughs, colds, angina, and fever [2], antibacterial properties of leaves and stem crude extracts, fractions, and some of the isolated compounds, were investigated. The microdilution assay gave high activities for the methanolic extract of leaves and stem, with MIC values of 10.1-16.5 and 10.2-20.5 µg/mL, respectively. While fraction C of the leaf extract showed enhanced activity against Micrococcus luteus with MIC = 4.5 µg/mL, fraction C of the stem extract showed good activities, with MIC values against Escherichia coli, Micrococcus luteus, and Staphylococcus warneri of 10.5, 10.7, and 13.5 µg/mL, respectively. Lecomtequinoline A-C (1-3) and anhydroevoxine (4) isolated from the leaf extract, and evoxine (5) and dictamnine (6) from the stem extract, however, gave slightly lower activities, with MIC values ranging from 11.1 to 18.7 µg/mL, or were inactive (Table 2). Previously, the water-soluble alkaloid fraction of Vepris louisii occurring in Western Africa had shown significant antibacterial activity, and delivered the dihydrofuroquinoline alkaloid veprisinium chlorid exhibiting broad and high activity against a number of clinical bacterial isolates [4]. In the following, Vepris lanceolata, endemic to Mauritius, delivered from its hexane and methanol/chloroform fractions of the stem MICs of 32 and 16 mg/mL, respectively, against Pseudomonas aeruginosa as well as against S. aureus, which is around 1000-fold less active than MICs received against human pathogen strains reported here from Vepris lecomteana. In addition, the plant's methanol/chloroform fraction from leaves was not active against Pseudomonas aeruginosa at all, and displayed a very low MIC of 16 mg/mL against Staphylococcus aureus [21]. Furthermore, flindersine isolated from the chloroform/methanol extract of the wood of Vepris punctata, occurring in the Madagascar rain forest, was later reported, as well, from the ethyl acetate extract of the leaves of another medicinal plant belonging to the Rutaceae family and tested against Bacillus subtilis, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Pseudomonas aeruginosa, and Acinetobacter baumannii, delivering low MICs of 31.25, 62.5, 62.5, 31.25, 250, and 125 µg/mL, respectively [22,23]. Interestingly, antibacterial testing of an epoxidized prenylated cinnamaldehdye derivative from Vepris glomerata, East Africa-named glomeral, provided significant MICs of 2 µg/mL and 0.4 µg/mL against standard strains of Staphylococcus aureus and Salmonella dysentrieae, respectively, giving a rationale for the use of this plant in the treatment of bacterial infections [12]. It should be noted that Vepris species are widely used in traditional African medicine against multiple diseases, including various bacterial infections, probably indicating a medically valuable metabolite spectrum still to be detected from this genus.

General
Optical rotation indices were determined in methanol on a JASCO DIP-3600 digital polarimeter (JASCO, Tokyo, Japan) using a 10 cm cell. CD spectra were measured on a JASCO J-810 spectropolarimeter (JASCO). IR spectra were determined on a JASCO Fourier transform IR-420 spectrometer (JASCO). Ultraviolet spectra were recorded on a Hitachi UV 3200 spectrophotometer in MeOH and infrared spectra on a JASCO 302-A spectrophotometer (Thermo Scientific, Waltham, MA, USA). ESI-HR mass spectra were measured on Agilent Techn. 6220 TOF LCMS mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) and EI-MS on a Finnigan MAT 95 spectrometer (70 ev) (Thermo Fischer Scientific, Darmstadt, Germany) with perfluorokerosene as reference substance for ESI-HR-MS. The 1 H-and 13 C-NMR spectra were recorded at 500 MHz and 125 MHz, respectively, on Bruker DRX 500 NMR spectrometers (Bruker Corporation, Brussels, Belgium) in CDCl 3 . Methyl, methylene, and methine carbons were distinguished by DEPT experiments. Homonuclear 1 H connectivities were determined by using the COSY experiment. One-bond 1 H-13 C connectivities were determined with HMQC gradient pulse factor selection, and two-and three-bond 1 H-13 C connectivities by HMBC experiments. Chemical shifts are reported in δ (ppm) using Tetramethylsilane (TMS) (Sigma-Aldrich, Munich, Germany) as internal standard, while coupling constants (J) were measured in Hz. Column chromatography was carried out on silica gel 230-400 mesh, Merck, (Merck, Bielefeld, Germany) and silica gel 70-230 mesh (Merck). Thin layer chromatography (TLC) was performed on Merck precoated silica gel 60 F 254 aluminum foil (Merck), and spots were detected using ceric sulfate spray reagent after heating. The degree of purity of the positive control compounds was ≥98%, while that of the isolated compound was >95%. The molecular composition of the isolated compounds was identified by exact mass determinations. Gentamycin was purchased from Jinling Pharmaceutic (Group) Corp. All reagents used were of analytical grade.

Extraction and Isolation
The air dried and powdered leaves (335.0 g) of Vepris lecomteana were extracted with methanol at room temperature for 72 h. After filtration and evaporation under reduced pressure at 40 • C, 24.8 g of dried crude extract were obtained and fractionated using vacuum liquid chromatography (VLC) with a mixture of petrol ether, ethyl acetate, and methanol vacuum liquid chromatography on the basis of TLC analysis, to afford fractions A (1.1 g) (20% of petrol ether in EtOAc), B (5.7 g) (50% of petrol ether in EtOAc), and C (8.5 g) (70% of petrol ether in EtOAc and 100% EtOAc). Fractions were subjected to column chromatography over silica gel 60 C (0.04-0.063 mm), and eluted with petrol ether followed by a mixture of petrol ether/EtOAc, using gradients of increasing polarity, and finally by EtOAc.
The air-dried and powdered stem (2.3 kg) of the plant was likewise extracted with methanol at room temperature for 72 h. After filtration and evaporation under reduced pressure at 40 • C, 45.2 g of dried crude extract were obtained, and fractionated with a mixture of petrol ether, ethyl acetate, and methanol, using VLC on the basis of TLC analysis, to afford fractions D (10.5 g) (20% of petrol ether in EA) and E (27.6 g) (70% of petrol ether in EA and 100% EA). The fractions were subjected to column chromatography over silica gel 60 C (0.04-0.063 mm), and eluted with petrol ether followed by a mixture of petrol ether/EtOAc using gradients of increasing polarity, and finally, by EtOAc. During the extraction of the stem powder, a white solid precipitated. After filtration and recrystallization using a mixture of petrol ether/EtOAc (1/3), hesperidin (401.5 mg) was obtained. Fraction D (10.5 g) was also treated-using the same approach as applied for fraction A-to receive 62 subfractions of around 100 mL each, which were collected and combined on the basis of TLC analysis.

Antimicrobial Activities
The minimum inhibition concentrations (MICs) of test samples and the positive control drug gentamycin were measured by the microdilution broth susceptibility assay [24] against the bacteria Escherichia coli (DSMZ 1058), Bacillus subtilis (DSMZ 704), Pseudomonas agarici (DSMZ 11810), Micrococcus luteus (DSMZ 1605), and Staphylococcus warneri (DSMZ 20036), obtained from DSMZ, Germany. The inocula of bacterial strains were prepared from 12 h broth cultures, and suspensions were adjusted to 0.5 McFarland standard turbidity. The samples were dissolved in 10% DMSO and diluted twofold in sterile 96-well microtiter plates, in duplicate, using BHI broth. Standardized inocula of test strains were added, and after incubation at 37 • C for 24 h on a rotary shaker at 200 rpm, MICs were read as the lowest concentration with inhibition of the growth of the test organisms, compared to the positive control gentamycin and medium containing 10% DMSO as negative control.

Conclusions
To the best of our knowledge, this is the first study on phytochemical and pharmacological properties of Vepris lecomteana. We report here, the isolation and structural elucidation of new furoquinoline alkaloids named lecomtequinoline A (1), B (2), and C (3), and their antibacterial activities, together with those of anhydroevoxine (4), evoxine (5), and dictamnine (6). The microdilution assay concerning antibacterial activity against Escherichia coli, Bacillus subtilis, Pseudomonas agarici, Micrococcus luteus, and Staphylococcus warneri resulted in MIC values displaying decreasing activities from crude extracts over fractions towards isolated compounds, suggesting synergistic effects of compounds, potentially involving 1-6, as well as N-methylflindersine (7), evoxathine (8), lupeol, stigmasterol, β-sitosterol, β-sitosterol-3-O-β-D-glucopyranoside, and myristic alcohol. In accordance with other Vepris species, Vepris lecomteana might find applications in crude drug medicines, especially in Western African countries where the plant occurs endemically. Subject to determination of their in vivo toxicity profile, extracts of the leaves and stem might turn out to be valuable for treatment of bacterial infections caused by Gram-negative Escherichia coli responsible for certain forms of diarrhea; the Gram-positive Bacillus subtilis causing diarrhea, enteritis, and dermatosis; the Gram-positive Micrococcus luteus causing skin infections in immunosuppressed patients; as well as the Gram-positive Staphylococcus warneri suggested to be linked to spontaneous abortion, urinary tract infection, meningitis, and endocarditis.
Supplementary Materials: The spectra of compounds (1-3) are available online.