Synergy of Plant Essential Oils in Antibiotic Therapy to Combat Klebsiella pneumoniae Infections.

Increased antibiotic resistance presents a health problem worldwide. The World Health Organization published a list of pathogens considered a priority for designing new treatments. Klebsiella pneumoniae (Kp) is a top-priority microorganism, highlighting the strains that produce carbapenemases. Developing new efficient therapies or complementing existing treatments is a priority, and essential oils (EOs) provide an alternative. EOs could act as antibiotic adjuvants and enhance antibiotic activity. Employing standard methodologies, the antibacterial activity of the EOs and their synergic effect with antibiotics were detected. A string test was used to identify the impact of the EOs over the hypermucoviscosity phenotype presented by Kp strains, and Gas Chromatography-Mass Spectrometry analysis identified EOs and the composition of EOs. The potential of EOs for designing synergistic therapies with antibiotics to combat the infection of KPC diseases was demonstrated. In addition, the alteration of the hypermucoviscosity phenotype was shown as the principal mechanism of a synergic action between EOs and antibiotics. The differential composition of the EOs lets us identify some molecules that will be analyzed. Synergic activity of EOs and antibiotics can provide a solid platform for combating multiresistant pathogens that represent a severe health sector problem, such as Kp infections.

Potent Anti-amoebic Effects of Ibogaine, Voacangine and the Root Bark Alkaloid Fraction of Tabernaemontana arborea.

Plants of Tabernaemontana species have several pharmacological activities including antimicrobial effects. Amoebiasis continues to be a public health problem, with increasing evidence of resistance to metronidazole. In this study, we assessed the effect of the alkaloid fraction of T. arborea root bark and the alkaloids ibogaine and voacangine on the viability and infectivity of Entamoeba histolytica trophozoites. Cultures were exposed to 0.1 - 10 µg/mL for 24, 48 and 72 h, and viability was then determined using a tetrazolium dye reduction assay and type of cellular death analyzed by flow cytometry. Results showed that the alkaloid fraction, but mainly ibogaine and voacangine alkaloids, exhibited potent dose-dependent anti-amoebic activity at 24 h post-exposure (IC50 4.5 and 8.1 µM, respectively), comparable to metronidazole (IC50 6.8 µM). However, the effect decreased after 48 and 72 h of exposure to concentrations below 10 µg/mL, suggesting that the alkaloids probably were catabolized to less active derivatives by the trophozoites. The treatment of trophozoites with the IC50 s for 24 h induced significant morphological changes in the trophozoites, slight increase in granularity, and death by apoptonecrosis. The capacity of T. arborea alkaloids to inhibit the development of amoebic liver abscesses in hamsters was evaluated. Results showed that even when the treatments reduced the number of amoebic trophozoites in tissue sections of livers, they were unable to limit the formation of abscesses, suggesting their rapid processing to inactive metabolites. This work leaves open the possibility of using Tabernaemontana alkaloids as a new alternative for amoebiasis control.

Five centuries of Cirsium ehrenbergii Sch. Bip. (Asteraceae) in Mexico, from Huitzquilitl to Cardo Santo: History, ethnomedicine, pharmacology and chemistry.

ETHNOPHARMACOLOGICAL RELEVANCE: Several medicinal plants, including the endemic herb Cirsum ehrenbergii (Asteraceae), have been documented in manuscripts, medical and botanical books written in Mexico since the XVI century until the present. This unique circumstance is a real window in the time that allows to investigate historical and contemporary ethnopharmacological knowledge. AIM OF THE STUDY: To examine the persistence, disappearance, and transformation of ethnomedicinal knowledge of C. ehrenbergii along time. Also, to investigate the chemistry and pharmacology of this species in relation to its historical and present day main ethnomedical applications related to Central Nervous System and inflammation. MATERIALS AND METHODS: A thorough review was performed of written sources of medicinal plants from XVI and onwards. For the pharmacological studies, the organic extracts were tested in mice models to assess its antidepressant and anti-inflammatory properties. The active extracts were studied chemically. The isolated compounds were identified by 1H, 13C NMR, or characterized by GC-MS. RESULTS: Cirsum ehrenbergii was illustrated for the first time (1552) in the Libellus de Medicinalibus Indorum Herbis (Booklet of Medicinal Plants of the Indians) and named in the Nahuatl native language as huitzquilitl (edible thistle). It was there recommended as nigris sanguinis remedium (remedy for black blood), and for the treatment of illnesses with an inflammatory component. Nigris sanguinis was well known in the European medicine of that time and currently it has been interpreted as "depression". At the present time, peasants and native population in Mexico mainly name C. ehrenbergii in Spanish as cardo Santo (holy thistle). Its original Nahuatl name has been almost forgotten. However, these communities use this species, among other maladies, to heal "nervios" (anxiety and/or depression) and for anti-inflammatory purposes. These ailments and treatments resemble those recorded in the Libellus and in several medicinal plant books along centuries. The ethanol extract of C. ehrenbergii roots showed antidepressant-like activity in mice administered at 300 mg/kg, as indicated by the forced swim test (FST). The glycosylated flavonoid linarin was identified as antidepressant principle and was active at the doses of 30 and 60 mg/kg in the FST. Regarding to anti-inflammatory activity, the most active was the methylene chloride extract of the aerial parts, which contains taraxasterol, pseudotaraxasterol, ß-sitosterol and stigmasterol. CONCLUSIONS: Cirsium ehrenbergii extracts possess antidepressant-like (roots, EtOH) and anti-inflammatory (aerial parts, CH2Cl2) properties, containing active compounds. Our results sustain historical and present day ethnomedical applications of this species documented along five centuries.

Antimycobacterial Activity of Alkaloids and Extracts from Tabernaemontana alba and T. arborea.

Tuberculosis is the main cause of death from a single infectious agent. Globally, according to the World Health Organization, in 2018, there were an estimated 1.2 million tuberculosis deaths. Moreover, there is a continuous appearance of drug-resistant strains. Thus, development of new antituberculosis medicines should receive high priority. Plant-derived natural products are promising candidates for this purpose. We therefore screened alkaloid extracts obtained from the root and stem barks of the Mexican Apocynaceae species Tabernaemontana alba and Tabernaemontana arborea, as well as the pure alkaloids ibogaine, voacangine, and voacamine, tested for activity against Mycobacterium tuberculosis H37Rv and cytotoxicity to mammalian Vero cells using the resazurin microtiter and the MTT assays, respectively. The extracts were analyzed by GC-MS and HPLC-UV. T. arborea root bark alkaloid extract showed the highest activity against M. tuberculosis (MIC100 = 7.8 µg/mL) of the four extracts tested. HPLC suggested that voacangine and voacamine were the major components. The latter was isolated by column chromatography, and its chemical structure was elucidated by 1H and 13C NMR, and MS. Unambiguous assignation was performed by HSQC, HMBC, and NOESY experiments. Voacamine is a dimeric bis-indole-type alkaloid and is 15 times more potent than the monomeric ibogan-type alkaloids ibogaine and voacangine (MIC100 = 15.6, 250.0, and 250.0 µg/mL, respectively). However, all of these compounds showed cytotoxicity to Vero cells, with a poor selectivity index of 1.00, 0.16, and 1.42, respectively. This is the first report of voacamine activity against M. tuberculosis.

New Insights into the Methylation of Mycobacterium tuberculosis Heparin Binding Hemagglutinin Adhesin Expressed in Rhodococcus erythropolis.

In recent years, knowledge of the role that protein methylation is playing on the physiopathogenesis of bacteria has grown. In Mycobacterium tuberculosis, methylation of the heparin binding hemagglutinin adhesin modulates the immune response, making this protein a subunit vaccine candidate. Through its C-terminal lysine-rich domain, this surface antigen interacts with heparan sulfate proteoglycans present in non-phagocytic cells, leading to extrapulmonary dissemination of the pathogen. In this study, the adhesin was expressed as a recombinant methylated protein in Rhodococcus erythropolis L88 and it was found associated to lipid droplets when bacteria were grown under nitrogen limitation. In order to delve into the role methylation could have in host-pathogen interactions, a comparative analysis was carried out between methylated and unmethylated protein produced in Escherichia coli. We found that methylation had an impact on lowering protein isoelectric point, but no differences between the proteins were found in their capacity to interact with heparin and A549 epithelial cells. An important finding was that HbhA is a Fatty Acid Binding Protein and differences in the conformational stability of the protein in complex with the fatty acid were observed between methylated and unmethylated protein. Together, these results suggest that the described role for this mycobacteria protein in lipid bodies formation could be related to its capacity to transport fatty acids. Obtained results also provide new clues about the role HbhA methylation could have in tuberculosis and point out the importance of having heterologous expression systems to obtain modified proteins.

Sobre el primer libro de plantas medicinales escrito en el Continente Americano: El Libellus Medicinalibus Indorum Herbis de México, 1552. Una revisión / On the first book of medicinal plants written in the American Continent: The Libellus Medicinalibus Indorum Herbis from Mexico, 1552. A review

Resumen: El "Libellus de Medicinalibus Indorum Herbis" (Librito de las Hierbas Medicinales de los Indios) fue elaborado por los sabios indígenas Martín De la Cruz y Juan Badiano, 31 años después de la caída del imperio azteca. El primero es su autor, el segundo tradujo el manuscrito del Náhuatl al latín. Contiene numerosas recetas para tratar enfermedades humanas y 185 dibujos a color de las plantas utilizadas. En 1939 se publicó por primera vez como "Un Herbario Azteca". Empero, también contiene enfermedades y prácticas médicas europeas del siglo XVI. Presentamos una revisión actualizada de este hermoso códice, su historia, concepción, creadores y botánica; además, la química y farmacología de cinco plantas ahí citadas. El Libellus es una ventana en el tiempo que permite la investigación científica del antiguo conocimiento etnofarmacológico en Mesoamérica y documentar su persistencia, desaparición o transformación. Sin embargo, esto requiere superar desafíos lingüísticos, pero también derivados de su contexto histórico, antropológico, cultural, botánico y médico.

Abstract: The "Libellus de Medicinalibus Indorum Herbis" (Little Book of Indian Medicinal Plants) was composed by the indigenous sages Martín De la Cruz and Juan Badiano, 31 years after the Aztec Empire fall. The former was the author, and the latter translated the manuscript from the Nahuatl language to Latin. It contains numerous recipes for treating human diseases and 185 colored drawings of the prescribed plants. In 1939 it was first published as "An Aztec Herbarium". However, it also contains XVI century European diseases and medical practices. We present an updated review of this beautiful codex, its history, conception, creators, and botany; as well as, the chemistry and pharmacology of five plants therein cited. The Libellus is a window in the time that allows the scientific research of ancient ethnopharmacological knowledge in Mesoamerica and document its persistence, disappearance, or transformation. However, this requires overcoming linguistic defies, but also derived from its historical, anthropological, cultural, botanical, and medical context.

Diacetylcurcumin: Its Potential Antiarthritic Effect on a Freund's Complete Adjuvant-Induced Murine Model.

The present study aims to evaluate the antiarthritic activity of diacetylcurcumin (DAC), a synthetic derivative where the free phenolic groups of curcumin are derivatized by acetylation, thereby conferring greater lipophilicity to the parent molecule and partially overcoming the limited systemic bioavailability of curcumin. Antiarthritic activity was evaluated on a Freund's complete adjuvant (FCA)-induced murine model of arthritis. Oral administration of DAC (60 and 120 mg/kg) resulted in a significant inhibition of inflammation in the acute and chronic phases, respectively, demonstrating an improved and sustained anti-inflammatory effect, comparable to that of curcumin (150 mg/kg) in the chronic stage at a lower dose. Phenylbutazone (80 mg/kg) was used as a reference drug. The pharmacological consequence of DAC or curcumin treatment is the prevention of secondary lesions commonly associated with this biological model.

Trypanocidal and toxicological assessment in vitro and in silico of three sesquiterpene lactones from Asteraceae plant species.

We report the effect of the Sesquiterpene Lactones Ambrosin, Incomptine B and Glaucolide E against seven strains of Trypanosoma cruzi, the etiological agent of Chagas Disease. These compounds were isolated from Parthenium hysterophorus, Decachaeta incompta, and Vernonia liatroides, respectively. We evaluated by flow cytometry the viability of epimastigotes. Ambrosin was the most effective, then Incomptine B, and Glaucolide E (IC50 = 67.1, 123.7, and 215.1 µM, respectively). These compounds were more potent than the drugs Benznidazole (IC50 > 400 µM) and Nifurtimox (IC50 = 199.7 to >400 µM). Toxicity to mammalian Vero and Jurkat cells was also determined in vitro. All the compounds had a poor selective index (0.003-1.859). Toxicoinformatics is useful to forecast in silico toxicological and pharmacokinetic properties. Ambrosin and Incomptine B may not possess mutagenic, tumorigenic, or reproductive effects. Glaucolide E could possess a low mutagenic and high tumorigenic effects, and probably target the Amine Oxidase A, Prostaglandin and G/H Synthase I. Interestingly, Ambrosin, Incomptine B and Glaucolide E, comply with Lipinsky Rule of Five, indicating a suitable pharmacokinetic profile. Ambrosin and Incomptine B possess high trypanocidal activity, and pharmaceutical properties suitable for development; however, their safety profile should be optimized by structural modifications.

Antinociceptive, anti-inflammatory, and central nervous system (CNS) effects of the natural coumarin soulattrolide.

Soulattrolide is a natural coumarin synthesized by the leaves of species of Calophyllum (Calophyllaceae) rain forest trees, including the American C. brasiliense, and the Asian C. teysmanii. Soulattrolide is a potent inhibitor of the reverse transcriptase from HIV-1 (RT-HIV-1), and active against Mycobacterium tuberculosis. However, the effects of this coumarins on other systems, remains to be evaluated. C. brasiliense is used in traditional medicine for the treatment of pain and inflammation. Therefore, we decided to explore the antinociceptive, anti-inflammatory activity of soulattrolide in mice, as well as, some of its possible effects on the CNS. Soulattrolide showed antinociceptive effects in the writhing test (ED50 = 33.8 mg/kg), as well as, in the formalin test with an ED50 = 7.9, and 22.1 mg/kg for Phases 1 and 2, respectively. The highest dose of soulattrolide (50 mg/kg) induced 40% of antinociception in the hot plate test. Regarding to anti-inflammatory activity, in the 12-O-Tetradecanoylphorbol-13-acetate (TPA) test, soulattrolide showed an IC50 = 1.81 µmol/ear, whereas in the myeloperoxidase assay, it showed an inhibition of 87% (1 µmol/ear). Soulattrolide showed sedative effects on the pentobarbital-induced sleeping time test, and the rotarod test, but lacked antidepressant activity on the tail suspension test. In conclusion, we report for the first time, the antinociceptive effects of soulattrolide in mice, like those of naproxen; soulattrolide also showed mild anti-inflammatory activity, as well as mild sedative and anxiolytic properties, therefore, it has also activity on the CNS.

Mexican Propolis: A Source of Antioxidants and Anti-Inflammatory Compounds, and Isolation of a Novel Chalcone and ε-Caprolactone Derivative.

The propolis produced by bees are used in alternative medicine for treating inflammation, and infections, presumably due to its antioxidant properties. In this context, five propolis from México were investigated to determine their inhibitory lipid peroxidation properties. The ethyl acetate extract from a red propolis from Chiapas State (4-EAEP) was the most potent (IC50 = 1.42 ± 0.07 µg/mL) in the TBARS assay, and selected for further studies. This extract afforded two new compounds, epoxypinocembrin chalcone (6), and an ε-caprolactone derivative (10), as well as pinostrobin (1), izalpinin (2), cinnamic acid (3), pinocembrin (4), kaempherol (5), 3,3-dimethylallyl caffeate in mixture with isopent-3-enyl caffeate (7a + 7b), 3,4-dimethoxycinnamic acid (8), rhamnetin (9) and caffeic acid (11). The HPLC profile, anti-mycobacterial, and antioxidant properties of this extract was also determined. Most of the isolated compounds were also tested by inhibition of reactive oxygen species (ROS) in challenged mouse bone marrow-derived mast cells (BMMCs), and DPPH. Their anti-inflammatory activity was evaluated by TPA, and MPO (myeloperoxidase) activity by ear edema test in mice. The most potent compounds were 7a + 7b in the TBARS assay (IC50 = 0.49 ± 0.06 µM), and 2 which restored the ROS baseline (3.5 µM). Our results indicate that 4-EAEP has anti-oxidant, and anti-inflammatory properties due to its active compounds, suggesting it has anti-allergy and anti-asthma potential.

Heliopsis longipes: anti-arthritic activity evaluated in a Freund's adjuvant-induced model in rodents

ABSTRACT This study assesses the anti-arthritic effect of the affinin-enriched (spilanthol, main alkamide) hexane extract from the roots of Heliopsis longipes (A. Gray) S.F. Blake, Asteraceae, on a Freund adjuvant-induced arthritis model in rodents. The extract was orally administered at a dose of 2, 6.6, or 20 mg/kg; a significant edema-inhibitory activity in the acute and chronic phases was observed with a dose of 2 and 20 mg/kg, respectively. The extract showed higher anti-inflammatory and anti-arthritic effects than the reference drug phenylbutazone (80 mg/kg). Moreover, the extract prevented the occurrence of secondary lesions associated to this pharmacological model.

Natural Compounds from Mexican Medicinal Plants as Potential Drug Leads for Anti-Tuberculosis Drugs

ABSTRACT In Mexican Traditional Medicine 187 plant species are used in the treatment of respiratory conditions that may be associated with tuberculosis. In this contribution, we review the ethnobotany, chemistry and pharmacology of 63 species whose extracts have been assayed for antimycobacterial activity in vitro. Among these, the most potent is Aristolochia brevipes (MIC= 12.5 µg/mL), followed by Aristolochia taliscana, Citrus sinensis, Chrysactinia mexicana, Persea americana, and Olea europaea (MIC<64 µg/mL). Other potent extracts (inhibition > 95%, 50 µg/mL) include: Amphipterygium adstringens, Larrea divaricata, and Phoradendron robinsoni. Several active compounds have been identified, the most potent are: Licarin A (isolated from A. taliscana), and 9-amino-9-methoxy-3,4-dihydro-2H-benzo[h]-chromen-2-one (transformation product of 9-methoxytariacuripyrone isolated from Aristolochia brevipes), both with MIC= 3.125 µg/mL, that is 8-fold less potent than the reference drug Rifampicin (MIC= 0.5 µg/mL). Any of the compounds or extracts here reviewed has been studied in clinical trials or with animal models; however, these should be accomplished since several are active against strains resistant to common drugs.

Natural Compounds from Mexican Medicinal Plants as Potential Drug Leads for Anti-Tuberculosis Drugs.

In Mexican Traditional Medicine 187 plant species are used in the treatment of respiratory conditions that may be associated with tuberculosis. In this contribution, we review the ethnobotany, chemistry and pharmacology of 63 species whose extracts have been assayed for antimycobacterial activity in vitro. Among these, the most potent is Aristolochia brevipes (MIC= 12.5 µg/mL), followed by Aristolochia taliscana, Citrus sinensis, Chrysactinia mexicana, Persea americana, and Olea europaea (MIC<64 µg/mL). Other potent extracts (inhibition > 95%, 50 µg/mL) include: Amphipterygium adstringens, Larrea divaricata, and Phoradendron robinsoni. Several active compounds have been identified, the most potent are: Licarin A (isolated from A. taliscana), and 9-amino-9-methoxy-3,4-dihydro-2H-benzo[h]-chromen-2-one (transformation product of 9-methoxytariacuripyrone isolated from Aristolochia brevipes), both with MIC= 3.125 µg/mL, that is 8-fold less potent than the reference drug Rifampicin (MIC= 0.5 µg/mL). Any of the compounds or extracts here reviewed has been studied in clinical trials or with animal models; however, these should be accomplished since several are active against strains resistant to common drugs.

Antimycobacterial and HIV-1 Reverse Transcriptase Activity of Julianaceae and Clusiaceae Plant Species from Mexico.

The extracts of 14 Julianaceae and 5 Clusiaceae species growing in Mexico were tested in vitro (50 µg/mL) against Mycobacterium tuberculosis H37Rv and HIV reverse transcriptase (HIV-RT). The Julianaceae bark and leaf extracts inhibited M. tuberculosis (>84.67%) and HIV-RT (<49.89%). The Clusiaceae leaves extracts also inhibited both targets (>58.3% and >67.6%), respectively. The IC50 values for six selected extracts and their cytotoxicity (50 µg/mL) to human macrophages were then determined. Amphipterygium glaucum, A. molle, and A. simplicifolium fairly inhibited M. tuberculosis with IC50 of 1.87-2.35 µg/mL; but their IC50 against HIV-RT was 59.25-97.83 µg/mL. Calophyllum brasiliense, Vismia baccifera, and Vismia mexicana effect on M. tuberculosis was noteworthy (IC50 3.02-3.64 µg/mL) and also inhibited RT-HIV (IC50 26.24-35.17 µg/mL). These 6 extracts (50 µg/mL) presented low toxicity to macrophages (<23.8%). The HPLC profiles of A. glaucum, A. molle, and A. simplicifolium indicated that their antimycobacterial activity cannot be related to masticadienonic, 3α, or 3ß-hydromasticadienonic acids, suggesting that other compounds may be responsible for the observed activity or this might be a synergy result. The anti-HIV-RT and antimycobacterial activities induced by C. brasiliense can be attributed to the content of calanolides A, B, as well as soulatrolide.

Linalool and ß-pinene exert their antidepressant-like activity through the monoaminergic pathway.

AIMS: Linalool and ß-pinene are two volatile monoterpenes that possess antidepressant-like activity. These are components of many aromatic plants used in folk medicine around the world to relieve anxiety and depression. In this contribution, we focused on examining the mechanism of action of these compounds. MAIN METHODS: We used mice in the forced swimming test (FST) and antagonist drugs (i.p.) to receptors related to the depression process such as 5-HT1A. To assess the possible contribution of the serotoninergic system, animals were pre-treated with WAY 100635 (a 5-HT1A receptor antagonist) and PCPA (a serotonin synthesis inhibitor).To assess the participation of the noradrenergic system, the animals were pre-treated with yohimbine (an α2 receptor antagonist), propranolol (a ß receptor antagonist) and neurotoxin DSP-4 (a noradrenergic neurotoxin). In the dopaminergic system, we used SCH23390 (a D1 receptor antagonist). KEY FINDINGS: WAY 100635 blocked the antidepressant-like effect of linalool and ß-pinene. In contrast, pretreatment of mice with PCPA did not modify reductions in the immobility time elicited by the two monoterpenes. The yohimbine modified the effect of linalool on immobility time. Propranolol and neurotoxin DSP-4 reversed the anti-immobility effect of ß-pinene; also, SCH23390 blocked the antidepressant-like effect of ß-pinene. SIGNIFICANCE: Our results indicate that linalool and ß-pinene produce an antidepressant-like effect through interaction with the monoaminergic system.

Anthraquinones from Vismia mexicana.

Vismia mexicana (Clusiaceae) is a small tropical tree found from Mexico to Honduras. The CH2Cl2/MeOH extract from the leaves has been reported to have inhibitory properties against reverse transcriptase of human immunodeficiency virus type 1 (HIV-1 RT). In order to characterize some of its chemical constituents, the EtOAc-soluble fraction of this extract was subjected to column chromatography. A new natural product was isolated and designated vismiaquinone D [1-hydroxy-6-methoxy-7,8-(3',3'-dimethyl-pyrano) anthraquinone]. In addition, vismiaquinone was obtained. The structures of vismiaquinone and vismiaquinone D were determined by 1H and 13C NMR spectroscopy, unambiguous assignments were achieved with DEPT, HSQC, and HMBC experiments, and corroborated by X-ray diffraction studies. The isolated anthraquinones were tested against HIV-1 RT. However, none showed relevant activity, suggesting that other compounds in this extract may be responsible for its HIV-1 RT inhibitory properties.

Pharmacological exploration of the sedative mechanism of hesperidin identified as the active principle of Citrus sinensis flowers.

The infusion of flowers of several species of Citrus genera is used as a sedative to treat insomnia in Mexican traditional medicine. The aims of this study were to investigate the sedative effect of different extracts of flowers of Citrus sinensis (L.) Osbeck (Rutaceae) and describe the pharmacological action mechanism of the sedative active compounds of this plant. The methanol and dichloromethane extracts, obtained from the flowers of Citrus sinensis (L.) Osbeck (Rutaceae), showed a dose-dependent sedative effect in the exploratory cylinder model in mice, with ED50 (ip) values of 47.04+/-12.03 mg/kg and 129.15+/-21.25 mg/kg, respectively. Hesperidin (ED50=11.34+/-2.48 mg/kg) was identified in the methanol extract as the sedative active principle of this plant. The pre-treatment with atropine (1 mg/kg I. P.), flumazenil (2 mg/kg I. P.), clonidine (0.01 mg/kg I. P.), isoproterenol (0.3 mg/kg I. P.), haloperidol (0.3 mg/kg I. P.), WAY 100 635 (3 mg/kg I. P.), P-chlorophenylalanine (250 mg/kg I. P., twice per day for 2 days), forskolin (3 mg/kg I. P.) and rolipram (0.173 mg/kg I. P.) did not modify the sedative effect of 30 mg/kg hesperidin. However, the sedative effect of this compound was potentiated by yohimbine (1.25 mg/kg I. P.) and buspirone (1 mg/kg I. P.), and reverted by pretreatment with aminophylline (30 mg/kg I. P.), caffeine (30 mg/kg I. P.) and several doses of 1,3-dimethyl-8-phenylxanthine (10, 30 and 54.7 mg/kg I. P.). These results suggest that adenosine receptors might be involved in the sedative action of hesperidin, identified as the active principle of the flowers of Citrus sinensis.