|Year : 2020 | Volume
| Issue : 3 | Page : 322-327
Trypanocidal potentialities of skimmianine an alkaloid isolated from Zanthoxylum pistaciifolium griseb leaves
Yamilé Heredia Diaz1, Julio César Escalona Arranz1, Rosalia González Fernández2, Ania Ochoa Pacheco1, Jesus García Díaz1, Lianet Monzote Fidalgo3, Denise da Gama Jaén Batista4, Cristiane Franca da Silva4, Paul Cos5
1 Department of Pharmacy, Faculty of Natural and Exact Sciences, University of Oriente, Cuba
2 Medical Toxicology Centre, Medical Sciences University, Santiago de Cuba, Cuba
3 Department of Parasitology, Institute of Tropical Medicine “Pedro Kourí”, La Habana, Cuba
4 Laboratório de Biologia Celular, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil
5 Laboratory for Microbiology, Parasitology and Hygiene, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Antwerp, Belgium
|Date of Submission||08-May-2019|
|Date of Acceptance||31-Dec-2019|
|Date of Web Publication||14-Aug-2020|
Prof. Yamilé Heredia Diaz
Department of Pharmacy, Faculty of Natural and Exact Sciences, University of Oriente, Avenida Patricio Lumumba s/n, 90500 Santiago de Cuba
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Zanthoxylum pistaciifolium Griseb (Rutaceae) is an endemic Cuban species traditionally used for respiratory ailment treatments and religious practices. Objective: The aim of this study was to evaluate the in vitro antimicrobial activity from Z. pistaciifolium leaves. Materials and Methods: Four phases obtained from the ethanolic extract were tested against bacteria (Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa), fungi (six Candida spp.) and parasites (Leishmania amazonensis and Trypanosoma cruzi) by microdilution methods. In addition, the cytotoxicity was determined against peritoneal macrophages from BALB/c mice and fibroblast cells L929 using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and resazurin assay. Results: The hexane-butanone phase was the most active against Candida spp. and exhibited moderate effect on T. cruzi trypomastigote (39.3 ± 5.5 μg/mL). The skimmianine isolated from this phase and characterized by nuclear magnetic resonance showed a median inhibitory concentration of 58.0 ± 4.0 μg/mL against T. cruzi trypomastigotes and 31.2% ± 6.5% of inhibition on amastigote form at 100 μg/mL. Antiprotozoal activity of this alkaloid was selective with a selectivity index of 9. Conclusion: The current study demonstrated for the first time the antimicrobial potential of the leaves of Z. pistaciifolium, supporting its ethnobotanical uses. The trypanocidal activity observed for the alkaloid skimmianine could contribute to development future of an herbal medicine for the treatment of Chagas diseases.
Keywords: Antifungal activity, hexane-butanone phase, skimmianine, trypanocidal, Zanthoxylum pistaciifolium
|How to cite this article:|
Diaz YH, Escalona Arranz JC, Fernández RG, Pacheco AO, Díaz JG, Fidalgo LM, Batista Dd, da Silva CF, Cos P. Trypanocidal potentialities of skimmianine an alkaloid isolated from Zanthoxylum pistaciifolium griseb leaves. Phcog Res 2020;12:322-7
|How to cite this URL:|
Diaz YH, Escalona Arranz JC, Fernández RG, Pacheco AO, Díaz JG, Fidalgo LM, Batista Dd, da Silva CF, Cos P. Trypanocidal potentialities of skimmianine an alkaloid isolated from Zanthoxylum pistaciifolium griseb leaves. Phcog Res [serial online] 2020 [cited 2020 Sep 24];12:322-7. Available from: http://www.phcogres.com/text.asp?2020/12/3/322/292053
- Zanthoxylum pistaciifolium Griseb, a Cuban endemic medicinal plant, has a trypanocidal effect. Hexane-butanone phase was the most active by their antifungal activity and moderate effect against Trypanosoma cruzi.
Abbreviations Used: NMR: Nuclear magnetic resonance; HBP: Hexane-butanone phase; DP: Dichloromethane phase; BP: Butanol phase; MP: Methanol phase; TLC: Thin-layer chromatography; ATCC: American Type Culture Collection; CFU: Colony-forming units; IC50: Median inhibitory concentration; SI: Selectivity index.
| Introduction|| |
Natural products have played a significant role in the drug discovery process mainly in the last century. Dissatisfied therapeutic needs in the treatment of infectious diseases have led to the search of new substances with therapeutic applications.
Zanthoxylum pistaciifolium Griseb, endemic of Cuba, known as “palo vencedor,” is a tree prevalent in high and dry coasts. The popular literature describes that it is used for aromatic baths, pulmonary infections, and cold. The activity of Z. pistaciifolium against different micro-organisms has not been determined; therefore, this study was designed to evaluate the antimicrobial potentialities of its leaves.
| Materials and Methods|| |
Leaves of Z. pistaciifolium were collected from “El Palenque” Siboney, Santiago de Cuba, in October 2016. A specimen of the plant was identified and authenticated by the taxonomist Ing. Felix Acosta Cantillo, and a voucher sample was deposited at the Herbarium of the Eastern Center of Ecosystems and Biodiversity (BIOECO, Spanish acronym), Santiago de Cuba, identified by number The taxonomic identification corresponded with the specie reported by Greuter and Rankin to be Z. pistaciifolium Griseb.
Phytochemical screening was carried out according to the methodology proposed by Harborne with minor modifications. The air-dried leaves were powdered and successively macerated with n-hexane, ethanol 95%, and water. The qualitative determination of secondary metabolites was carried out according to previously proposed methods to identify alkaloids, triterpene and steroids, quinones, coumarins, saponins, reducing sugar, carbohydrates, phenol and tannin, amino acids, amines, and flavonoids.
Extraction, isolation, and spectroscopy
An ethanol extract was prepared by percolation method during 9 days at room temperature, using air-dried and powdered leaves (1000 g). The extract obtained was filtered and evaporated under reduced pressure at 40°C until dryness was achieved. Later, this dry extract was extracted with n-hexane, to remove fat and waxes, alkalinized (NaOH 1 mol/L until pH 8.5), and finally partitioned. Partitioning was developed using successive extractions with n-hexane-butanone (50:50 v/v), dichloromethane, butanol, and methanol, obtaining the phases: hexane-butanone (HBP), dichloromethane (DP), butanol (BP) and methanol (MP). The remaining residue was discarded. A yellow solid crystalline precipitated obtained from hexane-butanone phase was purified in methanol, yielding 100 mg.
All phases were analyzed to determine their phytochemical composition by thin-layer chromatography (TLC), using n-hexane/chloroform/methanol (15:12:5, v/v/v) as a mobile phase for alkaloids. The plates were observed under ultraviolet (UV) light at 365 nm and under visible light after spraying with Dragendorff reagent. The crystals obtained from liquid-liquid partition were characterized by spectroscopic techniques.1 H-nuclear magnetic resonance (NMR) and13 C-NMR uni- and bidimensional spectra were obtained on a spectrometer (Varian, USA), operating at 500 MHz and 125 MHz for1 H and13 C analysis, respectively. As for the two-dimensional NMR experiments, correlation spectroscopy (COSY), heteronuclear single-quantum coherence (HSQC), and heteronuclear multiple-bond correlation (HMBC) NMR experiments were performed, revealing1 H-1 H, direct1 H-13 C, and indirect1 H-13 C connections, respectively. Chloroform-d (99.8% D) was used as a solvent, which was purchased from Sigma-Aldrich (St. Louis, MO, USA). Tetramethylsilane (TMS, Sigma-Aldrich, Germany) was used as an internal standard, while chemical shifts (δ) were expressed in ppm and coupling constants (J) in Hertz. NMR data processing was performed with software MestReNova version 6.1.0-6224 for Windows. UV–visible spectra on spectrophotometer (Shimadzu, model UVmini-1240, Japan) with an emission range from 190 to 1100 nm, infrared (IR) spectra in KBr pellets on spectrometer (WQF-510A, FTIR, Rayleigh, China), and melting points on a Fisatom apparatus (model 431D, Brazil) were also determined.
Micro-organism and reference drug
Thein vitro antimicrobial activity against three bacteria, six fungi, and two parasites was tested. Bacteria and fungi cultures were obtained from the culture collection of Laboratory of Microbiology, Parasitology and Hygiene of the University of Antwerp, Belgium. The antifungal activity was evaluated against Candida albicans ATCC B59630 (azole resistant), Candida glabrata ATCC B63155, Candida kefyr ATCC B46120, Candida krusei ATCC B68404, Candida parapsilosis ATCC J941058, and Candida tropicalis CDC49. The employed bacteria were Staphylococcus aureus ATCC 6538, Escherichia More Details coli ATCC 8739, and Pseudomonas aeruginosa ATCC 9027. All cultures and assays were conducted at 37°C. Miconazole and ampicillin (Sigma-Aldrich, USA) were used as reference drugs for fungi and bacteria, respectively. Parasites were the MHOM/77BR/LTB0016 Leishmania amazonensis strain provided by the Institute of Tropical Medicine “Pedro Kourí,” Havana, and Trypanosoma cruzi (Y and Tulahuen strains) supplied by the Laboratory of Cellular Biology, Oswaldo Cruz Institute/FIOCRUZ, Rio de Janeiro, Brazil. Pentamidine (Sigma-Aldrich, St. Louis, MO, USA) and benznidazole (LAFEPE, Brazil) were used as a reference drug for Leishmania and Trypanosoma, respectively.
In vitro antibacterial and antifungal activity
In vitro antibacterial and antifungal activity was determined by the microdilution method with resazurin (redox indicator, at 50 μg/mL) (Sigma-Aldrich, Germany) in sterile 96-well microplates (Costar, USA). Stock solution of phases was prepared in 100% dimethyl sulfoxide (DMSO, BDH, Inglaterra) at 1 g/mL. In each well, 10 μL of the two-fold water compound dilutions (0.25–4 mg/mL) was added together with 190 μL of bacterial inoculum (5 × 105 CFU/mL) and fungal inoculum (5 × 103 CFU/mL). In the microplates were included untreatedcontrol wells (medium plus cells,100% cell growth) and control wells with medium without cells (0% cell growth). Then, the microplates were incubated at 37°C for 17 h (for bacteria) and 24 h (for fungi). Afterward, 20 μL of resazurin at 50 μg/mL per well (for bacteria) and 10 μL (for fungi) was added. The microplates were incubated under the same temperature conditions (bacteria: 30 min and fungi: 4 h). Microbial growth was determined by fluorimetry method (λex= 550 nm, λem= 590 nm) using a microplate reader (TECAN GENios, Germany). The phases were classified as active, if the bacterial growth inhibition (%) was >50%. The results are expressed as a percentage reduction in bacterial growth/viability compared to control wells. Each experiment was performed by triplicate, and the results were expressed as the mean ± standard deviation.
In vitro antileishmanial activity
Stock solution of phases was diluted in 100% DMSO at 20 mg/mL, and serial dilutions were made to obtain final concentrations between 12.5 and 200 μg/mL. In each well of the 96-well microplate, 2 μL of compound dilutions was added to 98 μL of Schneider's medium (Sigma, St. Louis, MO, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Sigma, St. Louis, MO, USA) and antibiotics. Afterward, 50 μL of promastigotes was added at 4 × 105 parasite/mL in logarithmic phase. Microplates were sealed with Parafilm and incubated at 26°C for 72 h. Then, 20 μL of a solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma, St. Louis, MO, USA) at 5 mg/mL was added in each well. An additional incubation of 4 h was performed, the supernatant was eliminated, and the formazan crystals were dissolved with 100 μL of DMSO. Finally, the microplate was read in an ELISA microplate reader (Sirio S Reader, 2.4-0, Italy) at 540 nm and 620 nm as reference wavelengths. The median inhibitory concentration (IC50) was determined using SPSS v. 19 software (IBM, Armonk, NY, USA) by lineal dose–response regression analysis. Each experiment was performed by duplicated, and the results were expressed as the mean ± standard deviation.
In vitro trypanocidal activity
Trypomastigotes forms (Y strain, T. cruzi) were incubated in 96-well microtiter plates. One hundred microliter of suspension in RPMI (Gibco, France) and 5% FBS (Invitrogen Corporation, UK) containing 107 parasites/mL was added to an equal volume of samples (phases and skimmianine) (diluted into RPMI + 5% FBS) at twice the desired final concentration (from 0 to 300 μg). After 2 and 24 h of treatment at 37°C, the number of living parasites was determined by microscopy light quantification in a Neubauer chamber. Controls were performed with parasites kept under the same conditions in the absence of treatment. All the experiments were run in triplicate, and the activity was expressed by calculating the IC50 value for 2- and 24-h treatment. IC50 values were calculated based on the drug concentration that reduced 50% of parasites using SPSS v. 19 software by linear regression.
The trypomastigotes of Tulahuen used in this step were obtained from L929 cell lineages (ATCC, CCL-1, originated from a C3H mouse, which were provided by Dr. Maria de Nazaré Correia Soeiro of Laboratory of Cellular Biology, IOC, Fiocruz) with tissue culture-derived trypomastigotes (Tulahuen strain expressing the E. coli β-galactosidase gene), following previously established protocols. In a 96-well plate, 4 × 103 cells/well (L929) was seeded in 80 μL RPMI medium (Gibco, BRL) supplemented with 10% FBS, 2% L-glutamine, and phenol red. The plate was incubated “overnight” in an oven at 37°C and 5% CO2. Cells were infected with 4 × 103 trypomastigotes/well, and 20 μL was diluted in RPMI supplemented with phenol red. The plate was incubated at the same conditions, and after 2 h, the free parasites were removed without phenol-red supplemented RPMI medium. The plate was incubated under the same conditions for 48 h until infection was stablished. Afterward, the culture medium was exchanged for 180 μL medium supplemented RPMI without phenol red plus 20 μL of phase (at 10 μg/mL) or skimmianine (up to 100 μg/mL). The following controls were used per plate: uninfected cells without addition of compounds, infected cells without addition of compounds, cells infected and treated with benznidazole, and infected cells treated with DMSO 1%. The plate was incubated at 37°C and 5% CO2 for 96 h. Following this period, 50 μL/well of chlorophenol red-β-D-galactopyranoside (Sigma-Aldrich, St. Louis, MO, USA) substrate was added, and the plate reading was performed after 16–20 h in ELISA reader (VersaMax tunable microplate reader; Molecular Devices, USA) with 570 nm filter. Benznidazole at concentration of 2.6 μg/mL was employed as a reference drug.
Cytotoxicity assays were performed in parallel to the above antiparasitic assays in order to assess their selectivity. The cytotoxicity effect of phases was assessed on peritoneal macrophages from BALB/c mice (for anti-Leishmania assay) and L929 cells from fibroblast mice (for anti-Trypanosoma assay) using the tetrazolium dye (MTT, Sigma, St. Louis, MO, USA) colorimetric method and resazurin assay, respectively. Isolated compound was evaluated with resazurin assay on L929 cells.
Peritoneal macrophages: Resident macrophages from the peritoneal cavity of healthy BALB/c mice were collected and washed with RPMI 1640 medium (Sigma, St. Louis, MO, USA), supplemented with antibiotics (penicillin 200 IU and streptomycin 200 μg/mL). Later, 1–3 × 106 macrophages/mL was seeded in 24-well plates (Costar, USA) and incubated for 2 h at 37°C in 5% CO2 atmosphere. The nonadherent cells were removed by washing with phosphate buffer solution. Subsequently, RPMI medium and phases at the tested concentrations (12.5–200 μg/mL) were added per well. The plates were incubated for 72 h under the same conditions, and macrophages treated with DMSO were included as a control. The cellular viability was determined by colorimetric assay with MTT (15 μL MTT solution added per well) as described above. Median cytotoxic concentration (CC50) was determined by dose–response linear regression analysis. Each experiment was performed in duplicated, and the results were expressed as the mean ± standard deviation.
L929 cells: Monolayers of mice L929 fibroblasts were cultivated (4 × 103 cells/well in 96-well microplates) at 37°C in RPMI 1 640 medium (pH 7.2–7.4) without phenol red supplemented with 10% FBS and 2 mM glutamine (RPMIS). Uninfected cultures (L929 cells) were exposed to the compounds, and the cytotoxicity level was evaluated using alamarBlue (Thermo fisher, Brazil) reagent. The microplates were incubated during 96 h at 37°C with different concentrations (diluted in RPMI without phenol red) of phases and the isolated compound (up to 1200 μg). Morphology was evaluated by light microscopy. In each well, 10 μL de resazurin was added and the microplates were incubated for 5 h. Finally, the cellular viability was determined by colorimetric bioassay at reference wavelengths of 570 and 600 nm. As negative controls, L929 cells in RPMI medium and RPMI containing each tested compound were included. The results were expressed as the percent differences in the decreases between sample-treated and vehicle-treated cells. The level of CC50 was determined by dose–response linear regression analysis.
A calculated selectivity index (SI) allowed examining the relationship between cytotoxicity and a chosen activity. The SI was then calculated by dividing the CC50(for macrophages or L929 cells) by the IC50(for protozoan). Phases or compounds with a SI ≥10 were considered as selective.
| Results|| |
The three extracts generated, i.e., n-hexane, ethanol 95%, and water, demonstrated the common presence of alkaloids, tannins, and flavonoids but with higher abundance in the ethanol extract. Coumarins, quinones, reducing sugars, and triterpenes/steroids were other chemical principles detected. Considering these results, the extract selected to be fractioned was the ethanol extract.
The extract on ethanol 95% yielded 60.1 g of soluble substances after dryness under reduced pressure. Alkaloid fractioning process yields a total of four phases: HBP (18.98 g), DP (7.16 g), MP (2.41 g), and BP (0.65 g). The HBP showed intense positive evidence for alkaloid and phenols qualitatively, followed by DP and BP, whereas MP was negative. Other detected metabolites were flavonoids, coumarins, quinones, triterpenes, and steroids.
Consequently, HBP and DP were analyzed on TLC plates. Six spots were detected, one of them clearly in majority with an intense fluorescence blue and positive responses to the Dragendorff reagent, for the HBP. As a result of the purification, the skimmianine alkaloid was obtained and recrystallized in methanol from HBP (113.2 mg). Its structure was determined by UV, IR,1 H, and13 C NMR spectra and finally compared to data from existing literature, [Figure 1].
Skimmianine appeared as colorless prism crystals, m.p. 178–179°C. UV λmax nm (log ε), MeOH: 246 (4.9), 320 (4.08) 330 (4.09). IR (KBr): 3147, 3118, 3068, 3006, 2981, 2944, 2835, 1616, 1577, 1490, 1092 cm−1.1 H-NMR (500 MHz, CDCl3) δH: 7.89 (1H, d, J = 9.4 Hz, H-5), 7.47 (1H, d, J = 2.8 Hz, H-2), 7.13 (1H, d, J = 9.4 Hz, H-6), 6.92 (1H, d, J = 2.8 Hz, H-3), 3.95 (3H, s, 7-OCH3), 4.04 (3H, s, 8-OCH3), 4.31 (3H, s, 4-OCH3);13 C-NMR (125 MHz, CDCl3) δC: 164.3 (C/C-9a), 152.1 (C/C-7), 157 (C/C-4), 142.9 (CH/C-2), 141.8 (C/C-8), 141.3 (C/C-8a), 118.2 (CH/C-5), 114.7 (C/C-4a), 111.9 (CH/C-6), 104.6 (CH/C-3), 101.9 (C/C-3a), 56.7 (7-OCH3), 61.6 (8-OCH3), 58.9 (4-OCH3). HSQC1 H-13 C1 J (125 MHz, CDCl3) δH/δC: 6.92/104.6, 7.13/111.9, 7.89/118.2, 7.47/142.9, 3.95/56.7, 4.04/61.6, 4.31/58.9. HMBC1 H-13 Cn J (n = 2,3) (125 MHz, CDCl3) δH/δC: 4.04/141.8 (3 J), 3.94/152.1 (3 J), 4.31/157 (3 J), 7.88–141.23 (3 J)/152.07 (3 J)/157,10 (3 J), 7.47–101.83 (3 J)/104.60 (2 J)/164.28 (3 J), 7.13–114.76 (3 J)/141.85 (3 J)/152.11 (2 J), 6.92–101.88 (2 J)/142.84 (2 J)/164.29 (3 J). COSY1 H-1 H1 J (500 MHz, CDCl3) δH/δH: 7.89/7.13,7.47/6.92.
The inhibitory effect on the micro-organisms (bacteria and fungi) was in the range from 250 μg/mL until 4000 μg/mL. The activity was observed mainly on non-polar phases, while the highest levels of concentration were observed on the polar phases [Table 1].
|Table 1: Antibacterial and antifungal activities of Zanthoxylum pistaciifolium leaves phases|
Click here to view
The phases showed inhibitory effect on six of the evaluated microbial strains, especially against Candida spp. HBP and DP demonstrated antifungal activity against C. albicans, C. kefyr, C. krusei, and C. parapsilosis at concentration levels below 250 μg/mL, being less active against bacteria. The most susceptible micro-organisms were C. kefyr, C. glabrata, C. krusei, and S. aureus, whereas C. tropicalis and E. coli were resistant to all phases.
The antileishmanial and trypanocidal activity, cytotoxicity, and SI of the phases are shown in [Table 2]. The DP shows moderate inhibitory activity, with an IC50 of 54.4 μg/ml against L. amazonensis, whereas HBP, BP, and MP were inactive.
|Table 2: Antiprotozoal activity, cytotoxicity, and selectivity index of Zanthoxylum pistaciifolium leaf extracts|
Click here to view
When macrophages were treated with MP and BP, the CC50 was higher than 200 μg/mL; therefore, it did not exhibit cytotoxicity. In contrast, for DP and HBP, the CC50 was 45.2 and 122.36 μg/mL, respectively [Table 2]. The SI was calculated using the IC50 and CC50(for peritoneal macrophages) values obtained from antileishmanialin vitro assay. The SI was calculated for the most active phase (DP) and classifies as non-selective due to the small difference between the active and toxic concentrations.
[Table 2] shows the activity against trypomastigotes and amastigotes of T. cruzi. The highest activity was found in the HBP against trypomastigote form with IC50 value of 39.3 μg/mL at 24 h. A SI higher than two was observed after 24 h of treatment but still insufficient regarding the standard protocols. On the other hand, the activity against intracellular form of parasite (Tulahuen strain) was weak for all the phases, since the growth inhibition was <50%. Nevertheless, once again the HBP was the most active.
Considering the isolated compound (skimmianine), an IC50 of 58 ± 4 μg/mL for trypomastigote form was noticed after 24 h, when the highest dead parasites percentage at 150 μg/mL was exhibited, while benznidazole did so at 83 μg/mL [Figure 2]. This alkaloid was less active than the reference drug but more active than any other phase. At the same time, it shows less cytotoxicity (CC50 =513 ± 56.7 μg/mL) and therefore a good SI with a value of 8.9. Nevertheless, it was inactive when faced to the intracellular forms of the parasite.
|Figure 2: Effect of skimmianine (a) and benznidazole (b) upon bloodstream trypomastigotes (Y strain) Trypanosoma cruzi after 24 h of incubation|
Click here to view
| Discussion|| |
Preliminary phytochemical analysis is in agreement with the previous reports of other Zanthoxylum species in which alkaloids and phenol compounds are frequently reported.
Phytochemical screening of HBP and DP indicated the presence of alkaloids, phenols, flavonoids, and coumarins. Those metabolites have been associated with antimicrobial activity in other species of the genus. In addition, some isolated alkaloids from species of Zanthoxylum genus have been found to possess antifungal activity. Therefore, the antifungal effect observed for HBP and DP could be related to alkaloid presence. Moreover, there is a direct correlation between the antimicrobial effect and the presence of alkaloids because the most active phase (HBP) was the one with higher intensity to alkaloids in the qualitative assays and with the largest amount of spots (reactive with Dragendorff reagent) on the TLC analysis. Three alkaloids isolated of Zanthoxylum nitidum had good antifungal activity, while the ones isolated from Zanthoxylum rhoifolium (eight) were active against ten micro-organisms. These studies indicated a higher activity for alkaloids of the benzophenanthridine kind over those of the fluoroquinolonic one but confirm that both types are indeed active. Regarding their solubility profile, it looks like they are clearly involved in the antifungal activity.
The results against fungi were also similar to Zanthoxylum americanum extracts, with a broad spectrum of antifungal activity associated with the furanocoumarins. Precisely, coumarins are one of the metabolites identified in the HBP and DP and therefore could be also responsible for the antimicrobial activity detected for Z. pistaciifolium leaf extracts in a direct or synergistic way.
The essential oils are other of nonpolar metabolites that could be related to the antimicrobial activity in the species, whereas essential oils extracted from the leaves of Zanthoxylum alatum and Zanthoxylum monogynum show potent antimicrobial activity. At last, phenol compounds had a wide range of biological activities including the antimicrobial, antiviral, antioxidant, anticancer, and other remarkable properties. These pharmacological activities are usually associated to their free radical scavenging efficacies and their ability of binding proteins with a high specificity degree. All these facts suggest us that the antifungal activity observed in HBP and DP could be related to their chemical composition, mainly alkaloids and phenols. Regarding the antibacterial activity, all the studied phases were active only against S. aureus. This is a fact already previously reported but in leaf extracts of Zanthoxylum acanthopodium. Alkaloids can be also associated to this activity, considering the results reported from isolates of two species of Zanthoxylum, being chelerythrine the most active compound.
The activity of Zanthoxylum spp. extracts against L. amazonensis has been less explored. In Costa Rica, extracts from 67 plant species were evaluated and Zanthoxylum juniperinum was one of the 16 that showed activity. In Brazil, extracts of Z. rhoifolium were also identified as active against promastigote forms. Recently, extracts from Zanthoxylum armatum exhibited good antileishmanial activity. The results obtained in this work showed that DP of Z. pistaciifolium is the only active and attending their alkaloid composition, and they emerged as possible responsible. Benzophenanthridine alkaloids isolated from Z. rhoifolium root bark were active against both parasite forms: promastigotes and amastigotes as well as inin vivo studies of cutaneous leishmaniasis in mice. The same occurs to the alkaloids isolated from Z. chiloperone against L. amazonensis. On the other hand,in vitro experiments show that skimmianine alkaloid isolated from Spiranthera odoratíssima (Rutaceae) had a significant effect against Leishmania braziliensis. Moreover, this alkaloid extracted from Adiscanthus fusciflorus showed inhibitory activity against the enzyme adenine phosphoribosyltransferase (APRT) from Leishmania of 68%. The authors showed that its alkaloid crystallizes in the centrosymmetric space group P21/c, with one molecule in the asymmetric unit, and has at least two C-H…O intermolecular interactions, leading to the formation of centrosymmetric dimers. The structural characterization of skimmianine provided important information regarding the interaction ways of this compound with APRT. Further investigations will allow to study the interactions between this compound and the APRT active site to explain its action mechanism against Leishmania spp. as well as the inhibition capacity of other PRTases. Furanoquinolines alkaloids (common in species from Rutaceae) have been reported as typical DNA alkylation compounds excerpting cytotoxic and antiparasitic properties. Typical DNA damage occurs when DNA-alkylating compounds form covalent bonds with DNA bases. If the DNA enzymes do not repair these alkylations, errors will occur after the next round of replication. Consequences are point mutations and sometimes deletions and frameshift mutations. If such mutations occur in important protein-coding genes, they can lead to the death of a parasite. Considering the presence of alkaloids in the most active phase (DP), it is likely that there is an association between antileishmanial activity and some of the present alkaloids. Nevertheless, skimmianine, the furanoquinoline alkaloid isolated for the first time in this work for Z. pistaciifolium, was not active at the doses tested for the HBP where it was isolated. This discrepancy between its inactivity and the previous reports for the skimmianine, opens a gap for new structural activity relationship studies for the furanoquinoline alkaloids against Leishmania spp.
For T. cruzi, not too many reports for activity of Zanthoxylum spp. extracts exist. Nevertheless, extracts of Zanthoxylum chalybeum stem bark exhibited antitrypanosomal activity at very low doses. The canthin-6-one alkaloid isolated from the ethanol extract of Zanthoxylum chiloperone reached the same level ofin vitro activity against trypomastigote and amastigote forms of T. cruzi than the reference drug benznidazole. In this study, HBP and the skimmianine alkaloid both exhibit good levels of activity against T. cruzi parasite. Moreover, the SI of skimmianine (close to ten) is considered acceptable, being close to tenfold less toxic than active and opening opportunities for futurein vivo studies. Michael previously suggested the activity of skimmianine in his review paper about quinoline, quinazoline, and acridone alkaloids. This activity makes the skimmianine producing plants a valuable source of ethnomedicinal formulations.
| Conclusion|| |
The phytochemical screening revealed the presence of alkaloids, triterpenes, and steroids, reducing sugars, flavonoids, coumarins, phenols, and tannins in Z. pistaciifolium leaves. The HBP did not prove to be active against promastigotes of L. amazonensis but shows an antifungal potential and moderate effect on T. cruzi. On the other hand, DP shows promising activities against fungi and L. amazonensis.In vitro trypanocidal activity is attributable to skimmianine, a furoquinoline alkaloid isolated in this specie. These findings suggest for the first time that Z. pistaciifolium specie could be a potential candidate for the treatment of several infectious diseases in ethnobotanical practices.
Authors would like to thanks the Belgian Development Cooperation through VLIR-UOS project (Flemish Interuniversity Council-University Cooperation for Development), especially to the P-3 project “Biopharmaceutical Products from Natural Sources in the Development of Biotechnology”, and the project CAPES-MES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-Ministerio de Educación Superior) 144/2011, as well as to the Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) n° E-26/102.830/2011.
Financial support and sponsorship
This work has been supported by the Belgian Development Cooperation through VLIR-UOS project (Flemish Interuniversity Council-University Cooperation for Development) in the context of the Institutional University Cooperation Program with Universidad de Oriente, especially by means of the P-3 project “Biopharmaceutical Products from Natural Sources in the Development of Biotechnology” and the project CAPES-MES 144/2011, as well as Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) n° E-26/102.830/2011.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Fournet A, Muñoz V. Natural products as trypanocidal, antileishmanial and antimalarial drugs. Curr Top Med Chem 2002;2:1215-37.
Roig JT. Medicinal, Aromatic and Poisonous Plants from Cuba. Volume II. Havana: Editorial Científico Técnica; 2012. p. 736-7.
Prieto S, Molina J, González JA. Contribution to the natural therapy for antiviral treatment. Rev Latinoam Quím 2000;18:108-9.
Garden JM, Hause SK, Hoskin FC, Roush AH. Comparison of DFP-hydrolyzing enzyme purified from head ganglion and hepatopancreas of squid (Loligo pealei
) by means of isoelectric focusing. Comp Biochem Physiol C 1975;52:95-8.
Harborne JB. Phytochemical Methods. A Guide to Modern Techniques of Plant Analysis. New York: Chapman and Hall; 1973. p. 6.
Falkenberg MD, Santos RD, Simões CM. Introduction to phytochemical analysis. In: Oliveira CM Pharmacognosia: from Plant to Medicine. 6a
ed. Porto Alegre: UFRGS Publisher; Florianópolis: UFSC Publisher; 2007. p. 229-44.
Wagner H, Bladt S. Plant Drug Analysis: A Thin Layer Chromatography Atlas. 2nd
ed. Munich: Springer; 1996. p. 334
Cos P, Vlietinck AJ, Berghe DV, Maes L. Anti-infective potential of natural products: How to develop a strongerin vitro
'proof-of-concept'. J Ethnopharmacol 2006;106:290-302.
Sladowski D, Steer SJ, Clothier RH, Balls M. An improved MTT assay. J Immunol Methods 1993;157:203-7.
Romanha AJ, Castro SL, Soeiro Mde N, Lannes-Vieira J, Ribeiro I, Talvani A, et al
experimental models for drug screening and development for Chagas disease. Mem Inst Oswaldo Cruz 2010;105:233-8.
Tiuman TS, Ueda-Nakamura T, Garcia Cortez DA, Dias Filho BP, Morgado-Díaz JA, de Souza W, et al
. Antileishmanial activity of parthenolide, a sesquiterpene lactone isolated from Tanacetum parthenium
. Antimicrob Agents Chemother 2005;49:176-82.
Chatelain E. Chagas disease drug discovery: Toward a new era. J Biomol Screen 2015;20:22-35.
Brown NM, Grundon MP, Harrison DM, Surgenor SA. Quinoline alkaloids and related prenylquinolines. Tetrahedron 1980;36:3579-84.
Parhoodeh P, Rahmani M, Mohd N, Mohd H, Sukari A, Cheng GL. Alkaloid constituents of Haplophyllum laeviusculum
). Sains Malaysiana 2012;41:47-52.
Krohn K, Cludius-Brandt S, Schulz B, Sreelekha M, Shafi PM. Isolation, structure elucidation, and biological activity of a new alkaloid from Zanthoxylum rhetsa
. Nat Prod Commun 2011;6:1595-6.
Terezan AP, Rossi RA, Almeida RN, Freitas TG, Fernandes JB, Silva MF, et al
. Activities of extracts and compounds from Spiranthera odoratissima St
) in leaf-cutting ants and their symbiotic fungus. J Braz Chem Soc 2010;21:882-86.
Yang G, Chen D. Alkaloids from the roots of Zanthoxylum nitidum
and their antiviral and antifungal effects. Chem Biodivers 2008;5:1718-22.
Tavares Lde C, Zanon G, Weber AD, Neto AT, Mostardeiro CP, Da Cruz IB, et al
. Structure-activity relationship of benzophenanthridine alkaloids from Zanthoxylum rhoifolium
having antimicrobial activity. PLoS One 2014;9:e97000.
Bafi-Yeboa NF, Arnason JT, Baker J, Smith ML. Antifungal constituents of northern prickly ash, Zanthoxylum americanum
mill. Phytomedicine 2005;12:370-7.
Guleria S, Tiku AK, Koul A, Gupta S, Singh G, Razdan VK. Antioxidant and antimicrobial properties of the essential oil and extracts of Zanthoxylum alatum
grown in North-Western Himalaya. Scientific World J 2013;2013:790580.
Silva FB, Santos NO, Pascon RC, Vallim MA, Figueiredo CR, Martins RC, et al
. Chemical composition andin vitro
cytotoxic and antimicrobial activities of the essential oil from Leaves of Zanthoxylum monogynum
St. Hill (Rutaceae
). Medicines (Basel) 2017;4. pii: E31.
Barkatullah IM, Muhammad N, Tahir L. Antimicrobial evaluation, determination of total phenolic and flavonoid contents in Zanthoxylum armatum
DC. J Med Plant Res 2012;6:2105-10.
Devi OZ, Rao KS, Bidalia A, Wangkheirakpa R, Mukherjee OS. GC-MS Analysis of phytocomponents and antifungal activities of Zanthoxylum acanthopodium
DC. Collected from Manipur, India. European J Med Plants 2015;10:1-9.
Patiño OJ, Prieto JA, Lozano JM, Lesmes LS, Cuca LE. Antibacterial in vitro
properties of isolated secondary metabolites from two Zanthoxylum
genus specie (Rutaceae
). Rev Cub Farm 2011;45:431-8.
Chinchilla CM, Valerio CI, Sánchez PR, Bagnarello MV, Martínez EL, González PA. Anti-Leishmania
sp. (Kinetoplastida: Trypanosomatidae) activity of plants from a Biological Reserve of Costa Rica. Rev Biol Trop 2014;62:1229-40.
Moura-Costa GF, Nocchi SR, Ceole LF, de Mello JC, Nakamura CV, Dias Filho BP, et al
. Antimicrobial activity of plants used as medicinals on an indigenous reserve in Rio das Cobras, Paraná, Brazil. J Ethnopharmacol 2012;143:631-8.
Alam F, Us Saqib QN. Evaluation of Zanthoxylum armatum
Roxb forin vitro
biological activities. J Tradit Complement Med 2017;7:515-8.
Castillo D, Sauvain M, Rivaud M, Jullian V.In vitro
activity of benzo[c] phenanthridines against Leishmania amazonensis
. Planta Med 2014;80:902-6.
Ferreira ME, Rojas de Arias A, Torres de Ortiz S, Inchausti A, Nakayama H, Thouvenel C, et al
. Leishmanicidal activity of two canthin-6-one alkaloids, two major constituents of Zanthoxylum chiloperone
. J Ethnopharmacol 2002;80:199-202.
Dos Santos RA, Batista J Jr., Rosa SI, Torquato HF, Bassi CL, Ribeiro TA, et al
. Leishmanicidal effect of Spiranthera odoratíssima (Rutaceae
) and its isolated alkaloid skimmianine occurs by a nitric oxide dependent mechanism. Parasitology 2011;138:1224-33.
Napolitano HB, Silva M, Ellena J, Rocha WC, Vieira PC, Thiemann OH, et al
. Redetermination of skimmianine: A new inhibitor against the leishmania APRT enzyme. Acta Crystallographica Sect E: Struct Rep Online 2003;59:o1503-05.
Wink M. Medicinal plants: A source of anti-parasitic secondary metabolites. Molecules 2012;17:12771-91.
Freiburghaus F, Jonker SA, Nkunya MH, Mwasumbi LB, Brun R.In vitro
trypanocidal activity of some rare Tanzanian medicinal plants. Acta Trop 1997;66:79-83.
Ferreira ME, Cebrián-Torrejón G, Corrales AS, Vera de Bilbao N, Rolón M, Gomez CV, et al
. Zanthoxylum chiloperone
leaves extract:First sustainable Chagas disease treatment. J Ethnopharmacol 2011;133:986-93.
Michael JP. Quinoline, quinazoline and acridone alkaloids. Nat Prod Rep 2007;24:223-46.
[Figure 1], [Figure 2]
[Table 1], [Table 2]