Effects of bioconversion by Battus polydamas on the chemical composition of Aristolochia spp. and evaluation of antimicrobial activity and biocompatibility.

Aristolochia plants are emblematic from an ethnopharmacological viewpoint and are know to possess numerous biological properties, including antiseptic. However, the medicinal potential of these species is debatable because of their representative chemical constituents, aristolochic acids (AAs) and aristolactams (ALs), which are associated, for instance, with nephropathy and cancer. These contrasting issues have stimulated the development of approaches intended to detoxification of aristoloquiaceous biomasses, among which is included the bioconversion method using larvae of the specialist phytophagous insect Battus polydamas, previously shown to be viable for chemical diversification and to reduce toxicity. Thus, eleven Aristolochia spp. were bioconverted, and the antimicrobial activities of the plant methanolic extracts and its respective bioconversion products were evaluated. The best results were found for Aristolochia esperanzae, Aristolochia gibertii, and Aristolochia ringens against Bacillus cereus, with MIC ranging from 7.8 to 31.25 µg/mL. These three species were selected for chemical, antioxidant, cytotoxic, hemolytic, and mutagenic analyses. Chemical analysis revealed 65 compounds, 21 of them possible bioconversion products. The extracts showed potential to inhibit the formation and degradation of B. cereus biofilms. Extracts of A. gibertii and its bioconverted biomass showed antioxidant activity comparable to dibutylhydroxytoluene (BHT) standard. Bioconversion decreased the hemolytic activity of A. esperanzae and the cytotoxicities of A. esperanzae and A. gibertii. None of the extracts was found to be mutagenic. The bioactivities of the fecal extracts were maintained, and biocompatibility was improved. Therefore, the results obtained in this study reveal positive expectations about the natural detoxification process of the Aristolochia species.

Antinociceptive Effect of Hinokinin and Kaurenoic Acid Isolated from Aristolochia odoratissima L.

Aristolochia odoratissima L. is employed for the treatment of pain and as an antidote against the poison of venomous animals in traditional medicine. However, reports have not been found, to our knowledge, about the evaluation of the antinociceptive activity of extracts nor about the presence of compounds associated with this activity. Thus, the purpose of this work was to evaluate the antinociceptive activity of extracts and compounds isolated from the stems of Artistolochia odoratissima L. The extracts were obtained with solvents of increasing polarity and the compounds were isolated and characterized by column chromatography, HPLC, and NMR. The antinociceptive activity was carried out by the formalin test in mice. Ethyl acetate (AoEA) and methanolic (AoM) extracts decreased the paw licking in both phases of the formalin test. The isolated compounds (kaurenoic acid and hinokinin) from AoEA showed the highest antinociceptive activity in both phases of the formalin test. These results confirmed the analgesic effect of this specie described in traditional medicine and provided a base for a novel analgesic agent. They also allowed an approach for the development of standardized plant extracts with isolated metabolites.

Health risk associated with the oral consumption of "Chiniy-tref", a traditional medicinal preparation used in Martinique (French West Indies): Qualitative and quantitative analyses of aristolochic acids contained therein.

"Chiniy-tref" (CT) is a traditional preparation used in folk medicine in Martinique Island (French West Indies) that is nowadays mainly taken orally to prevent or act against any "manifestation of evil". CT is easily prepared at home by macerating larvae of the endemic swallowtail Battus polydamas (ssp.) cebriones (Dalman, 1823), sometimes accompanied by a leaf of its host-plant Aristolochia trilobata L., in commercial rum. We have previously reported the detection of nephrotoxic and carcinogenic aristolochic acids (AAs) I and II in CT, leading the Regional Health Agency (ARS) of Martinique to issue an alert regarding the potential risks associated with its consumption in 2015. In order to complete the toxicity risk assessment for oral consumption of CT, a full qualitative analysis of AAs and their analogues (AAAs) was performed, as well as a quantitative determination of the major AAs, namely AAs I and II. The phytochemical profiling of AAAs present in CT, that also corresponds to that of B. polydamas cebriones larvae feeding on A. trilobata, has been established for the first time by ultra-high performance liquid chromatography/electrospray ionization quadrupole time of-flight tandem mass spectrometry. AAs I and II were quantified in a small panel of tinctures by using a validated UHPLC/UV method, allowing us to estimate the probable daily intakes of these toxins by CT consumers. The results proved the existence of a real risk of renal toxicity and carcinogenicity associated with the chronic oral consumption of CT in Martinique, and more generally of similar "snake bottles" throughout the Caribbean.

Chemical Constituents and Pharmacology properties of Aristolochia triangularis: a south brazilian highly-consumed botanical with multiple bioactivities.

Aristolochia triangularis Cham., is one of the most frequently used medicinal plant in Southern Brazil. Preparations containing the leaves and/or stems are traditionally used as anti-inflammatory, diuretic, as well as antidote against snakebites. This study screened A. triangularis extracts, fractions and isolated compounds for different bioactivities. A weak antiproliferative activity against human lung cancer cell line (A549) was observed only for chloroform fraction obtained from stems (CFstems - CC50: 2.93 µg/mL). Also, a moderate antimicrobial activity against Staphylococcus aureus was detected just for chloroform fraction obtained from leaves (CFleaves -13-16 mm inhibition zone). Additionally, two semi-purified fractions (CFstems-4 and CFleaves-4) selectively inhibited HSV-1 replication (IC50 values of 0.40 and 2.61 µg/mL, respectively), while only CFleaves showed promising results against Leishmania amazonensis. Fractionation of extracts resulted in the isolation of one neolignan (-) cubebin and one lignan (+) galbacin. However, these compounds are not responsible for the in vitro bioactivities herein detected. The presence of aristolochic acid I and aristolochic acid II in the crude ethanol extract of stems (CEEstems) and leaves (CEEleaves) was also investigated. The HPLC analysis of these extracts did not display any peak with retention time or UV spectra comparable to aristolochic acids I and II.

Acute Toxicity and Sub-lethal Effects of the Essential Oil of Aristolochia trilobata and Its Major Constituents on Nasutitermes corniger (Termitidae: Nasutitermitinae).

Nasutitermes corniger (Motschulsky, 1855) (Termitidae: Nasutitermitinae) is an important pest in urban environments and bioinsecticides can be an alternative to its control. Here, we determined the toxicity and repellence of the essential oil (EO) prepared from stems of Aristolochia trilobata L. (Aristolochiaceae) and its major constituents on N. corniger. We also investigated behavioral changes of individuals exposed to limonene. The lethal dose required to kill 50% of N. corniger population (LD50) of EO of A. trilobata was 2.44 µg mg-1. Limonene was the most toxic compound to N. corniger followed by linalool (LD50 = 1.02 and 1.29 µg mg-1, respectively). In addition, all treatments presented median lethal time (LT50) less than 11 h. A. trilobata EO and its constituents showed irritability activity, but only limonene repelled soldiers more than workers. The negative behaviors of N. corniger groups were higher in individuals treated with limonene. A. trilobata EO and its constituents, especially the limonene, are promising for the control of N. corniger due the high toxicity, repellence, and possible disturbance in the colonies.

Chemical Composition, Antiprotozoal and Cytotoxic Activities of Indole Alkaloids and Benzofuran Neolignan of Aristolochia cordigera.

This is a comparative study on the intraspecific chemical variability of Aristolochia cordigera species, collected in two different regions of Brazil, Biome Cerrado (semiarid) and Biome Amazônia (coastal). The use of GC-MS and statistical methods led to the identification of 56 compounds. A higher percentage of palmitone and germacrene-D in the hexanes extracts of the leaves of plants from these respective biomes was observed. Phytochemical studies on the extracts led to the isolation and identification of 19 known compounds, including lignans, neolignans, aristolochic acids, indole-ß-carboline, and indole alkaloids. In addition, two new indole alkaloids, 3,4-dihydro-hyrtiosulawesine and 6-O-(ß-glucopyranosyl)hyrtiosulawesine, were isolated and a new neolignan, cis-eupomatenoid-7, was obtained in a mixture with its known isomer eupomatenoid-7. Their structures were determined by spectroscopic methods, mainly by 1D- and 2D-NMR. The occurrence of indole alkaloids is being described for the first time in the Aristolochiaceae family. Moreover, the in vitro susceptibility of intracellular amastigote and promastigote forms of Leishmania amazonensis to the alkaloids and eupomatenoid-7 were evaluated. This neolignan exhibited low activity against promastigotes (IC50 = 46 µM), while the alkaloids did not show inhibitory activity. The new alkaloid 6-O-(ß-glucopyranosyl)hyrtiosulawesine exhibited activity in the low micromolar range against Plasmodium falciparum, with an IC50 value of 5 µM and a selectivity index higher than 50.

Essential Oil of Aristolochia trilobata: Synthesis, Routes of Exposure, Acute Toxicity, Binary Mixtures and Behavioral Effects on Leaf-Cutting Ants.

Plants of the genus Aristolochia have been frequently reported as important medicinal plants. Despite their high bioactive potential, to date, there are no reports of their effects on leaf-cutting ants. Therefore, the present study aimed to evaluate the insecticidal activity of the essential oil of Aristolochia trilobata and its major components on Atta sexdens and Acromyrmex balzani, two species of leaf-cutting ants. The bioassays were performed regarding routes of exposure, acute toxicity, binary mixtures of the major components and behavioral effects. Twenty-five components were identified in the essential oil of A. trilobata using a gas chromatographic system equipped with a mass spectrometer and a flame ionization detector. The components found in higher proportions were sulcatyl acetate, limonene, p-cymene and linalool. The essential oil of A. trilobata and its individual major components were efficient against A. balzani and A. sexdens workers when applied by fumigation. These components showed fast and efficient insecticidal activity on ants. The components acted synergistically and additively on A. balzani and A. sexdens, respectively, and caused a strong repellency/irritability in the ants. Thus, our results demonstrate the great potential of the essential oil of A. trilobata and its major components for the development of new insecticides.

Antinociceptive effect of Aristolochia trilobata stem essential oil and 6-methyl-5-hepten-2yl acetate, its main compound, in rodents.

Aristolochia trilobata L. is an aromatic plant, popularly known as "mil-homens", and its essential oil (EO) is generally used to treat colic, diarrhea and dysentery disorders. We evaluated the antinociceptive effect of A. trilobata stem EO and of its major compound, the (R)-(-)-6-methyl-5-hepten-2-yl acetate (sulcatyl acetate: SA), using acetic acid (0.85%)-induced writhing response and formalin-induced (20 µL of 1%) nociceptive behavior in mice. We also evaluated the EO and SA effect on motor coordination, using the rota-rod apparatus. EO (25, 50 and 100 mg/kg) or SA (25 and 50 mg/kg) reduced nociceptive behavior in the writhing test (p<0.001). EO (100 mg/kg) and SA (25 and 50 mg/kg) decreased the nociception on the first phase of the formalin test (p<0.05). On the second phase, EO (25: p<0.01; 50: p<0.05 and 100 mg/kg: p<0.001) and SA (25 and 50 mg/kg; p<0.001) reduced the nociceptive response induced by formalin. EO and SA were not able to cause changes in the motor coordination of animals. Together, our results suggest that EO has an analgesic profile and SA seems to be one of the active compounds in this effect.

Detection of aristolochic acids I and II in "Chiniy-trèf", a traditional medicinal preparation containing caterpillars feeding on Aristolochia trilobata L. in Martinique, French West Indies.

"Chiniy-trèf" is a traditional medicinal preparation used in Martinique, French West Indies, for the prevention of all kinds of attempted poisoning and hex. It is produced by the maceration in alcohol (mostly rum) of larvae (caterpillars) of the butterfly Battus polydamas ssp. cebriones, feeding on the leaves of Aristolochia trilobata. Aristolochic acids I and II that are well-known nephrotoxic and carcinogenic substances were identified on two samples of "chiniy-trèfl" by chromatographic methods.

Temporal Variation of Aristolochia chilensis Aristolochic Acids during Spring.

In this communication, we report the springtime variation of the composition of aristolochic acids (AAs) in Aristolochia chilensis leaves and stems. The dominant AA in the leaves of all samples, which were collected between October and December, was AA-I (1), and its concentration varied between 212.6±3.8 and 145.6±1.2 mg/kg and decreased linearly. This decrease occurred in parallel with the increase in AA-Ia (5) concentration from 15.9±0.8 mg/kg at the beginning of October to 96.8±7.8 mg/kg in mid-December. Both acids are enzymatically related by methylation-demethylation reactions. Other AAs also showed important variations: AA-II (2) significantly increased in concentration, reaching a maximum in the first two weeks of November and subsequently decreasing in mid-December to approximately the October levels. The principal component in the AA mixture of the stems was also AA-I (1); similar to AA-II (2), its concentration increased beginning in October, peaked in the second week of November and subsequently decreased. The concentrations of AA-IIIa (6) and AA-IVa (7) in the leaves and stems varied throughout the study period, but no clear pattern was identified. Based on the variation of AAs in A. chilensis leaves and stems during the study period, the reduced contents of non-phenolic AAs and increased concentrations of phenolic AAs are likely associated with a decrease in this plant's toxicity during the spring.

Neolignans from Aristolochia elegans as antagonists of the neurotropic effect of scorpion venom.

ETHNOPHARMACOLOGICAL RELEVANCE: The high frequency of poisoning by sting or bite from venomous animals has begun to be a serious public health problem in Mexico where scorpion sting is the most common. Because of this, there is the need to seek active substances in plant species with an antagonistic effect against neurotropic activity of scorpion venom. The aim of this work was to demonstrate which of the compounds contained in the n-hexane extract from Aristolochia elegans roots display activity against scorpion venom. MATERIAL AND METHODS: Antagonist activity displayed by extract, fractions and isolated compounds obtained from Aristolochia elegans was guided by the inhibition of smooth muscle contraction induced by scorpion venom (Centruroides limpidus limpidus) in a model of isolated guinea pig ileum. The neolignans obtained from this extract were isolated and analyzed by chromatographic methods including HPLC. The chemical characterization of these compounds was performed by the analysis of (1)H and (13)C NMR spectra. RESULTS: The bio-guided chromatographic fractionation allowed us to isolate 4 known neolignans: Eupomatenoid-7 (1), licarin A (2), licarin B (3), eupomatenoid-1 (4) and other new neolignan which was characterized as 2-(3'-hydroxy-4'-methoxyphenyl)-3-methyl-5-[(E)-α-propen-γ-al]-7-methoxy-benzo [b] furan (5). This compound was named as eleganal. Compounds 1 and 2 were purified from the most active fraction AeF3 (EC50 of 149.9µg/mL, Emax of 65.66%). A doses-response analysis of eupomatenoid-7(1) and licarin A(2) allowed us to establish EC50 values (65.96µg/mL and 51.96µg/mL) respectively. CONCLUSIONS: The antagonistic effect against Centuroides limpidus limpidus scorpion venom displayed by the n-hexane extract from Aristolochia elegans roots is due to the presence of neolignans 1-2 contained in the fraction AeF3. Chemical analysis of fraction AeF2 allowed the isolation of a new compound which was identified as 2-(3'-hydroxy-4'-methoxyphenyl)-3-methyl-5-[(E)-α-propen-γ-al]-7-methoxy-benzo[b]furan (5), denominated as eleganal.

Phenolic aristolactams from leaves and stems of Aristolochia chilensis / Aristolactamas fenólicas de hojas y tallos de Aristolochia chilensis

Three phenolic aristolactams, aristolactam AII (3), velutinam (4) and piperolactam A (5), were identified from the leaves and stems of Aristolochia chilensis Bridges ex Lindl. The structures of these compounds were elucidated using a combination of HPLC-DAD, GC-MS and NMR experiments.

Tres aristolactamas fenólicas aristolactama AII(3), velutinam(4) y piperolactama A(5), se identificaron en hojas y tallos de Aristolochia chilensis Bridges ex Lindl. Las estructuras de estos compuestos se determinaron por combinación de CLAE-DAD, CG-EM y experimentos de RMN.

Antitubercular activity and the subacute toxicity of (-)-Licarin A in BALB/c mice: a neolignan isolated from Aristolochia taliscana.

BACKGROUND AND AIMS: Tuberculosis remains a worldwide health problem and requires long-term treatment with several antibiotics; therefore, compliance problems and the emergence of multidrug resistance (MDR) are involved. (-)-Licarin A (LA) was isolated from diverse plants such as Aristolochia taliscana and possesses antimycobacterial, antiinflammatory, trypanocidal, and neuroprotective activities. The aim of the study was to determine the antitubercular and subacute toxicity of LA isolated from A. taliscana in BALB/c mice. METHODS: The antitubercular activity of LA was tested in a TB murine model inducing disease with M. tuberculosis H37Rv or MDR. Mice were treated with LA (5 mg/kg) for 30 and 60 days; post/treatment, lung bacilli loads and pneumonia percentage were determined. The subacute toxicity of LA (21 days) was evaluated in healthy mice. After treatment, biochemical and hematological parameters were determined and main organs were analyzed histologically. RESULTS: In animals infected with drug-sensitive or MDR strains, LA produced a significant decrease of pulmonary bacillary burdens at day 30 of treatment, and a significant pneumonia reduction at days 30 and 60 of treatment. Regarding subacute toxicity, LA administration during 21 days showed no abnormalities in main-organ macro- and microarchitecture. Biochemical and hematological parameters analyzed showed no statistical differences between control and treated groups. CONCLUSIONS: (-)-Licarin A reduces pneumonia of mice infected with both mycobacterium strains. Also, subacute toxicity of LA exhibits no major signs of damage. Biochemical and hematological parameters and histological analyses indicate that LA caused no significant changes at the doses assayed.

Are Aristolochic Acids Responsible for the Chemical Defence of Aposematic Larvae of Battus polydamas (L.) (Lepidoptera: Papilionidae)?

Aristolochic acids (AAs) are thought to be responsible for the chemical protection of the aposematic larvae Battus polydamas (L.) (Papilionidae: Troidini) against predators. These compounds are sequestered by larvae from their Aristolochia (Aristolochiaceae) host plants. Studying the role of the chemical protection of the second and fifth instars of B. polydamas against potential predators, we found that the consumption of larvae by the carpenter ant Camponotus crassus Mayr and young chicks Gallus gallus domesticus was dependent on larval developmental stage. Second instars were more preyed upon than fifth instars; however, the assassin bug Montina confusa Stål was not deterred by chemical defences of the fifth instar B. polydamas. Laboratory bioassays with carpenter ants and young chicks using palatable baits topically treated with a pure commercial mixture of AAs I and AAs II in concentrations up to 100 times those previously found in B. polydamas larvae showed no activity. Similar results were found in field bioassays, where palatable baits treated as above were exposed to the guild of predators that attack B. polydamas larvae and were also consumed irrespective of the commercial AA concentration used. These results suggest that the mixture of AAs I and AAs II have no defensive role against predators, at least against those investigated in the present work. Other compounds present in Aristolochia host plants such as O-glycosylated AAs; benzylisoquinoline alkaloids; and mono-, sesqui-, di-, and triterpenes, which can be sequestered by Troidini, could act as deterrents against predators.

Sequestration of aristolochic acid I from Aristolochia pilosa by Mapeta xanthomelas Walker, 1863.

Sequestration of secondary plant chemicals and brightly colored bodies occur in a number of unpalatable insects. The utilization of toxic plant chemicals has been proposed as a strategy of chemical defense, while aposematic coloration may advertise unpalatability. Here, we tested for the presence of aristolochic acid I in leaves of Aristolochia pilosa and female bodies of Mapeta xanthomelas, obtained from larvae feeding on the plant, using high performance liquid chromatography with photodiode array detection and electrospray ionization mass spectrometry. The presence of aristolochic acid I in females of this conspicuous diurnal moth, an oligophagous herbivore of Aristolochia, is the first report of sequestration of aristolochic acids by an herbivore other than a species of Papilionidae.

Antibacterial activity of Aristolochia brevipes against multidrug-resistant Mycobacterium tuberculosis.

The increased incidence of Multidrug-Resistant Mycobacterium tuberculosis (MDR-MT) requires the search for alternative antimycobacterial drugs. The main aim of this study was to evaluate the dichloromethane extract from Aristolochia brevipes (Rhizoma) and the compounds isolated from this extract against several mycobacterial strains, sensitive, resistant (monoresistant), and clinical isolates (multidrug-resistant), using the alamarBlue™ microassay. The extract was fractionated by column chromatography, yielding the following eight major compounds: (1) 6α-7-dehydro-N-formylnornantenine; (2) E/Z-N-formylnornantenine; (3) 7,9-dimethoxytariacuripyrone; (4) 9-methoxy-tariacuripyrone; (5) aristololactam I; (6) ß-sitosterol; (7) stigmasterol; and (8) 3-hydroxy-α-terpineol. The structures of these compounds were elucidated by 1H- and 13C- (1D and 2D) Nuclear Magnetic Resonance (NMR) spectroscopy. This study demonstrates that the dichloromethane extract (rhizome) of A. brevipes possesses strong in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv (Minimum Inhibitory Concentration value [MIC], 12.5 µg/mL). The most active compound against all mycobacterial strains tested was the compound aristolactam I (5), with MIC values ranging between 12.5 and 25 µg/mL. To our knowledge, this the first report of antimycobacterial activity in this plant.

Aristolactams and Alkamides of Aristolochia gigantea.

A new aristolactam, aristolactam 9-O-ß-D-glucopyranosyl-(1→2)-ß-D-glucoside, and two alkamides, N-cis- and N-trans-p-coumaroyl-3-O-methyldopamine, were isolated from stems of Aristolochia gigantea, together with the known compounds allantoin, E-nerolidol, ß-sitosterol, (+)-kobusin, (+)-eudesmin, trans-N-feruloyltyramine, trans-N-coumaroyltyramine, trans-N-feruloyl-3-O-methyldopamine, aristolactam Ia-N-ß-D-glucoside, aristolactam Ia 8-ß-D-glucoside, aristolactam IIIa, and magnoflorine. Their structures were determined by spectroscopic analyses.

Antitrypanosomal activity of a diterpene and lignans isolated from Aristolochia cymbifera.

Bioguided fractionation of extract from the leaves of Aristolochia cymbifera led to the isolation of the furofuran lignans fargesin, epieudesmin, and sesamin; the dibenzylbutyrolactone lignans hinokinin and kusunokinin; and an ENT-labdane diterpene named copalic acid. Our data demonstrated that copalic acid and kusunokinin were the most active compounds against trypomastigotes of Trypanosoma cruzi. Additionally, copalic acid demonstrated the highest parasite selectivity as a result of low toxicity to mammalian cells, despite a considerable hemolytic activity at higher concentrations. Among the isolated compounds, kusunokinin could be considered the most promising candidate, as it displayed significant activity against intracellular amastigotes (IC(50) = 17 µM) and trypomastigotes (IC(50) = 51 µM) without hemolytic activity. Fargesin, hinokinin, epieudesmin, and sesamin were also effective against trypomastigotes, but these compounds were highly toxic to mammalian cells and no parasite selectivity could be identified. The need for novel drugs for American trypanosomiasis is evident, and these secondary metabolites from A. cymbifera represent a useful tool for drug design.

Antimycobacterial neolignans isolated from Aristolochia taliscana.

Tuberculosis (TB - Mycobacterium tuberculosis) is an ancient infectious disease that has appeared once again as a serious worldwide health problem and now comprises the second leading cause of death resulting from a single infection. The prevalence of multidrug resistance (MDR) TB is increasing and therapeutic options for treatment are not always accessible; in fact, some patients do not respond to the available drugs. Therefore, there is an urgent need to develop novel anti-TB agents. The aim of the present study was to screen extracts of Aristolochia taliscana, a plant used in traditional Mexican medicine to treat cough and snake bites, for antimycobacterial activity. The hexanic extract of A. taliscana was tested by microdilution alamar blue assay against Mycobacterium strains and bioguided fractionation led to the isolation of the neolignans licarin A, licarin B and eupomatenoid-7, all of which had antimycobacterial activity. Licarin A was the most active compound, with minimum inhibitory concentrations of 3.12-12.5 microg/mL against the following M. tuberculosis strains: H37Rv, four mono-resistant H37Rv variants and 12 clinical MDR isolates, as well as against five non-tuberculous mycobacteria (NTM) strains. In conclusion, licarin A represents a potentially active anti-TB agent to treat MDR M. tuberculosis and NTM strains.