Monophyllidin, a new alkaloid l-proline derivative from zanthoxylum monophyllum

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / p h y t o l Monophyllidin, a new alkaloid L-proline derivative from Zanthoxylummonophyllum Laboratorio de Investigacio´n en Productos Naturales Vegetales, Departamento de Quı´mica, Facultad de Ciencias, Universidad Nacional de Colombia, AA 14490, KR 30 45-03,Bogota´, Colombia A new L-proline derivative, monophyllidin (1), together with four known compounds, thalifoline (2), berberine (3), jathrorrhizine (4) and 3b-glucositosterol (5) has been isolated from the bark of Zanthoxylum monophyllum. The structure of compound 1 was elucidated on the basis of extensive spectroscopic data analysis. Characterization of compounds 2–5 was based on spectral analysis andcomparison with reported data. Compound 3 showed antibacterial activity against the five bacterial strains used, while 1, 4 and 5 presented only antibacterial activity against Enterococcus faecalis.
Compounds 2 and 4 showed antifungal activity, with 100 mg against Fusarium oxysporum f. sp.
ß 2010 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
Antifungal activityAntibacterial activity including alkaloids, coumarins, lignans, benzenoids, triterpenes,sterols and fatty acids ( The current interest in natural products research can be attributed to several factors, including unmet therapeutic needs, has been used popularly as a colorant and in development of agricultural pests resistant to pesticides and the Venezuelan traditional medicine has been used to treat of runny lack of comprehensive biological and chemical studies of a large nose, jaundice, ophthalmia and as an anesthetic number of species. Additionally, the rapid extinction of species has increased considerably in the last 20 years, cause irretrievable loss its fractions from the bark of this plant showed significant of species and potentially useful compounds. This phytochemical antifungal activity against seven human pathogenic fungi ( research represents a valuable potential for finding new com- ). Continuing with our phytochemical studies on plants pounds to fight diseases where treatments are still insufficient, as of the genus Zanthoxylum growing in Colombia, the polar alkaloid biocontrol agents with low environmental impact to agriculture extract obtained from the bark of Z. monophyllum was investigated and/or as templates for the design of drugs and agrochemicals. The and a new alkaloid, (À)-monophyllidin (1), was isolated along with species of the genus Zanthoxylum have great importance in four known compounds (2–5). This paper describes the isolation ethnobotanical, phytochemistry and biological activity, addition- and characterization of the new alkaloid (1) and the antimicrobial ally are a promising source of alkaloids among which highlights benzophenanthridines and have shown interesting antimicrobialproperties. Z. monophyllum (Lam.) P. Wilson (syn. Fagara mono- phylla Lam.) is a shrub or tree of 4–12 m that grows in somecountries of America at elevations of 300–1000 m The ethanolic extract obtained from the air-dried and powdered bark of Z. monophyllum was subjected to acid–base extraction of Previous chemical studies of this plant (bark, stems, wood alkaloids with some modifications in the procedure of the literature and leaves) have indicated the isolation of several components, (with the use of ultrasound to facilitate theextraction process and theuse of the CHCl3 and CHCl3–EtOH 8:2 as solvents for the liquid–liquid extraction process, according to preliminary phytochemical * Corresponding authors. Tel.: +57 1 3165000x14476; fax: +57 1 3165220.
analysis previously performed that allowed to determine the types of alkaloids present in the extract (The polar 1874-3900/$ – see front matter ß 2010 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
doi: Please cite this article in press as: Patin ˜ o, O.J., Cuca, L.E., Monophyllidin, a new alkaloid L-proline derivative from Zanthoxylum monophyllum. Phytochem. Lett. (2010), doi: O.J. Patin˜o, L.E. Cuca / Phytochemistry Letters xxx (2010) xxx–xxx carboxyl group [d 14.52 (s, 1H, OH) for 1H NMR and d 170.4 for 13CNMR] were placed at N-1 and C-2, respectively, on basis of HMBCcorrelations. The deshielded secondary carbons at d 46.9 and 68.7,along with a methine carbon at d 56.2, supported the presence ofnitrogen in 1. The above-mentioned NMR signals and the residualmolecular formula of C6H10NO suggested that the residual moietywas the proline aminoacid with a methylene group on the nitrogenatom. Furthermore, the proline moiety was connected via amethylene group [d 4.24 (s, 2H)] to C-10 of the xanthoxylin moiety.
Fig. 1. Chemical structure of compound 1.
The HMBC spectrum revealed 3J correlations NCH2 with C-2, C-5, C-20 and C-60 ), which further supported the argument that 1was a benzenoid-type derivative of proline named monophyllidin.
The negative rotation of monophyllidin (1) and comparison withliterature values of derivatives of proline indicated that it was aderivative of (À)-S-proline The known compounds were identified by comparison of physical and spectroscopic data with corresponding authenticsamples or literature values, and this included one isoquinolonealkaloid, thalifoline (2) twoberberine alkaloids, berberine (3) and Fig. 2. COSY and HMBC correlations of 1.
jathrorrhizine (4) and one sterol, 3b-glucositosterol (5) ().
fraction of alkaloids was subjected to purification by flash All compounds isolated (1–5) were tested for antibacterial chromatography over silica gel to yield 1–5, as shown in .
activity by the radial diffusion method reported by Compound 1 was obtained as a white crystalline solid with a mp against two Gram (+) standard strains: Staphylococcus aureus at 150–151 8C (CHCl3) and gave a positive test with Dragendorff´s 6538 and Enterococcus faecalis 29212 and three Gram (À): reagent. It showed a protonated molecular ion [M+H]+ peak at m/z Escherichia coli 25922 and Salmonella tiphymurium, strains 324.1343 (calcd. 324.1447) in the HRESIMS, and the resulting MS7953 and 14028s. Compound 3 showed antibacterial activity molecular formula was determined to be C16H21NO6, representing with diameters of inhibition zones greater than 3.5 mm against the seven degrees of unsaturation. The IR bands at 3451, 1725, 1680, five bacterial strains used, while the compounds 1, 4 and 5 showed 1600, 1498 and 1462 cmÀ1, respectively, indicated the presence of selective antibacterial activity against Enterococcus faecalis ).
carbonyl, aromatic and carboxylic acid functionalities in 1. The 1H The antifungal activity against Fusarium oxysporum f. sp.
and 13C NMR spectra of 1 gave signals including three methyl lycopersici of the isolated compounds from the bark of Z.
carbons, four methylenes, two methines and seven quaternary monophyllum was evaluated by means of direct bioautography carbons. The proton signal at d 5.99 (s, 1H) along with the carbon signals at d 86.1, 98.8, 105.7, 164.4, 164.8 and 164.9 indicated the The minimum amount required for the inhibition of fungal growth presence of pentasubstituted benzene to a ring in 1. The signals at d was appreciable at 100 mg for thalifoline (2) and jathrorrhizine (4), 2.60 (s, 3H), 3.93 (s, 3H) and 3.98 (s, 3H) for 1H NMR and at d 33.0, while the other compounds were inactive.
55.8 and 56.2 for 13C NMR indicated the presence of one acetyl andtwo methoxy groups attached to aromatic ring, which were placed at C-30, C-40 and C-60, respectively, on basis of HMBC correlations). The data suggested the partial structure of 1 to be a 6- substituted xanthoxylin moiety, an acetophenone isolated of Z.
monophyllum (and its structure was supported by Melting points were determined using a Fisher-Johns melting EIMS and CIMS with a peak at m/z 209 for a xanthoxylin moiety.
point apparatus. Optical rotations were measured on a Schmidt– The remaining signals in the 1H NMR spectrum at d 2.01–1.95 Haensch polarimeter. IR spectra were obtained on a PerkinElmer (m, 2H, H2-4), 2.34–2.21 (m, 1H, H-3b), 2.46–2.35 (m, 1H, H-3a), FT-IR Panagon 500 series 1000 spectometer as a thin film. 1H and 3.06–2.92 (m, 1H, H-5), 3.65–3.51 (m, 1H, H-5) and 3.85 (dd, J = 9.2, 13C NMR spectra as well as 2D spectra (COSY, HMQC, and HMBC) 3.7 Hz, 1H, H-2) supported by COSY correlations and EIMS mass were recorded on a Bruker Avance 400 spectrometer operating at fragmentations (characteristic peak at m/z 70 for pyrrolidines) 400 MHz for 1H and 100 MHz for 13C using the solvent peaks as indicated the presence of a 1,2-substituted pyrrolidine. A methy- internal references. HRMS were determined on a Shimadzu LCMS- lene [d 4.24 (s, 2H) for 1H NMR and d 46.9 for 13C NMR] and a IT-TOF mass spectrometer system with ESI in positive ion mode.
Table 1Results antibacterial activity of compounds isolated.
Please cite this article in press as: Patin ˜ o, O.J., Cuca, L.E., Monophyllidin, a new alkaloid L-proline derivative from Zanthoxylum O.J. Patin˜o, L.E. Cuca / Phytochemistry Letters xxx (2010) xxx–xxx EIMS and CIMS were determined on a Shimadzu GCMS-QP2010 deposited in 3 mL of soy trypticase (TSB) for Gram (+) strains and mass spectrometer system. Flash chromatography (FC) was carried Luria Broth (LB) for Gram (À) strains, and were incubated at 37 8C out with silica gel (230–400 mesh, Merck), and analytical with stirring, until the microorganisms were in the logarithmic chromatography was performed using silica gel 60 PF254 phase. The supernatant was removed and the sediment obtained (0.25 mm). The visualization of the compounds was carried out was re-suspended in phosphate buffer (PBS), followed by washes with iodine vapor, UV light or Dragendorff’s reagent.
with PBS and centrifuging. Finally the sediment was re-suspended inPBS and the optical density determined at 620 nm to calculate the number of CFU (colony forming units) per milliliter. It disperses100 mL that contain 4 Â 107 CFU in each dish. The measured volume The bark of Z. monophyllum (Lam.) P. Wilson was collected in was mixed and homogenized in 15 mL agarose fused to more or less the town of San Bernardo, Cundinamarca department, Colombia, 45 8C. This bacterial suspension was dispensed in Petri dishes and during February 2007 by Juan D. Guzma´n. The Plant material was left to solidify at room temperature, after which 2 mm diameter identified by Adolfo Jara. A voucher specimen (COL 517520) has holes were made with a sterile punch.
been deposited at Herbario Nacional Colombiano, Instituto de The test samples were prepared dissolving 1 mg of the pure Ciencias Naturales, Universidad Nacional de Colombia.
compound in 500 mL of DMSO, which are placed 8 of the sample induplicate and incubated at 37 8C for 30 min. After this time the nutrient medium was added, which contains molten agar–agar andTSB, incubated for 18 h at 37 8C and then diameter of inhibition Air-dried and powdered bark of Z. monophyllum (750.5 g) was zones was measured by the activity of the compound. Positive exhaustively extracted with 96% ethanol by maceration at room controls used were different antibiotics, ampicillin (50 mg/mL), temperature. The solvent was evaporated under vacuum to afford kanamycin (10 mg/mL) and tetracycline (4.12 mg/mL) at a dilution 80.5 g of the crude extract. A part of this residue (78 g) was 1:100 in PBS and was used as negative controls DMSO and PBS, extracted with H2O–Et2O 1:1 mixture (800 mL) assisted by each control 8 mL be served by each well. The diameters of ultrasound and acidified with 2N HCl to pH 2.0. The organic inhibition zones were measured in millimeters.
phase was separated and the aqueous layer was extracted withEt2O (3 Â 80 mL). The Et2O phases were not analyzed. The aqueous acidic solution was basified with NH4OH to pH 9.0 and successivelypartitioned with CHCl3 (4 Â 150 mL) to sive 4.0 g of extract and The antifungal activity of the isolated compounds against F.
CHCl3–EtOH (8:2) mixture (4 Â 150 mL) to sive 2.6 g of extract.
oxysporum f. sp. lycopersici was determined using the bioauto- The CHCl3–EtOH fraction (2.3 g) was submitted to silica gel flash chromatography (FC) and eluted with CHCl3–MeOH (9:1 to The microorganism used in the antifungal assay, F. oxysporum f. sp.
6:4) to give 15 fractions (1–15). The fractions 2–4 were combined lycopersici, has been maintained at the Universidad Nacional de (153.2 mg) and purified by FC eluting with CHCl3–MeOH 9:1 to Colombia - Bogota´ (Laboratorio de Investigacio´n en Productos afford 2 (14 mg). The fractions 7–8 (1250 mg) were subjected to Naturales Vegetales, Departamento de Quı´mica, Facultad de successive FC using CHCl3–MeOH 9:1 and CH2Cl2–MeOH 9:1 to Ciencias). Ten microliters of the solutions were prepared, in obtain 3 (574 mg). The fractions 9–10 (324.2 mg) were purified by different concentrations, corresponding to 100, 50, 25, 10, 5, 2 and successive FC eluting with CHCl3–MeOH 8:2 and CH2Cl2–MeOH 1 mg of pure compounds. The samples were applied to TLC plates, 9:1 to yield 4 (7.4 mg). The fractions 12–14 (296.8 mg) were and then were sprayed with a spore suspension of fungi in glucose subjected to FC eluting with CHCl3–MeOH 85:15 to afford 9 and salt solution and incubated for 72 h in the darkness in a subfractions (9a–9i). The subfractions 9c (25.3 mg) and 9i moistened chamber at 25 8C. Exposure of TLC plates to UV light (36.4 mg) were individually purified using FC with CH2Cl2–MeOH (254 nm) and iodine vapor significantly enhanced contrast in order 9:1 to give 5 (4.3 mg) and 1 (12.6 mg), respectively.
to detect inhibition zones, indicating the minimal amount of (À)-Monophyllidin 1: white crystalline solid; mp 150–151 8C compound required for the inhibition of fungal growth. Benomyl was used as positive control (1 mg), and the solvents used to D : À40:3 (c 0.28, CHCl3); IR (film) nmax = 3415, 2919, 2851, 1725, 1690, 1600, 1498, 1462, 1275, 1250 and 1231 cmÀ1. 1H dissolved the samples were the negative controls.
NMR (400 MHz, CDCl3): d 14.52 (s, 1H, OH), 5.99 (s, 1H, H-50), 4.24 (s,2H, NCH2), 3.98 (s, 3H, OCH3), 3.93 (s, 3H, OCH3), 3.85 (dd, J = 9.2, 3.7 Hz, 1H, H-2), 3.65–3.51 (m, 1H, H-5), 3.06–2.92 (m, 1H, H-5), 2.60(s, 3H, CH3CO), 2.46–2.35 (m, 1H, H-3a), 2.34–2.21 (m, 1H, H-3b), The authors thank to Colciencias and to the Universidad Nacional 2.01–1.95 (m, 2H, H-4); 13C NMR (100 MHz, CDCl3): d 203.7 (CO), de Colombia for financial support. Also thank to NMR Laboratory and 170.4 (COOH), 164.9 (C-40), 164.8 (C-60), 164.4 (C-20), 105.8 (C-30), LCMS Laboratory at Universidad Nacional de Colombia-Bogota´ for 98.9 (C-10), 86.0 (C-50), 68.7 (C-5), 56.2 (C-2), 55.8 (OCH3), 54.0 (OCH3), the support in recording of NMR spectra and HRESIMS, respectively.
46.9 (NCH2), 33.1 (CH3), 29.0 (C-4), 24.2 (C-3); HRESIMS [M+H]+ m/z Our gratitude is extended to Dr. J. Isaza for the EIMS and ICMS 324.1343 (calcd. for C16H22NO6 324.1447); EIMS m/z (%) 278 (4), 210 records on a spectrometer at Universidad Tecnolo´gica de Pereira (16), 209 (54), 193 (39), 163 (14), 70 (100), 69 (16), 68 (16), 51 (15); (Colombia) and to Dr. J. Lozano at FIDIC (Foundation Institute of CIMS m/z (%) 324 (2), 278 (3), 210 (16), 209 (100), 70 (50).
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maleate at 4 mg/mL [Fenistil injectable solution, Novartis]), Anaphylaxis to Dimenhydrinate Caused by the 2 piperazines (cetirizine hydrochloride at 10 mg/mL [Zirtek Theophylline Component tablet in normal saline, UCB] and levocetirizine dihydrochloride at 5mg/mL [Xozal tablet in normal saline, UCB]) and a piperidine (desloratadine at 5mg/mL [Aerius tablet in normal Allergy Departmen


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