Chemical Analysis by LC-MS of Cannabis sativa Root Samples from Northeast Brazil and Evaluation of Antitussive and Expectorant Activities.

Cannabis sativa is a millenary medicinal plant. However, contrary to worldwide paradigm-shifting, countries like Brazil still prohibit C. sativa cultivation and its medicinal use, even though many populations use aerial parts and roots of this plant for healthcare. As such, the objective of this work was to identify substances in the samples of the C. sativa roots, tracing a correlation with antitussive and expectorant effects. Therefore, samples of C. sativa roots were donated by the Polícia Federal Brasileira, and its aqueous extract (AECsR) was prepared with subsequent lyophilization, to maintain the material stability. After that, the material was analyzed by LC-MS to observe its chemical profile. Four samples (AECsR-A, B, C, and D) were tested in animal models of citric acid-induced cough (0.4 M) and phenol red expectoration (500 mg/kg). Using LC-MS it was possible to identify 5 molecules in C. sativa roots: p-coumaroyltyramine, tetrahydrocannabinol-C4, feruoiltyramine, anhydrocanabisativine, and cannabisativine. In experimental protocols, male mice (Mus musculus) were treated with samples of AECsR at doses of 12.5, 25, or 50 mg/kg regardless of the pharmacological test. In these tests, all samples showed the potential to treat cough and promote fluid expectoration, differing only in the dose at which these effects were observed. Therefore, the data showed that the C. sativa roots of the Brazilian Northeast showed antitussive and expectorant effects, even with intense secondary metabolites' variation, which alters its potency, but not its effect. This highlights the importance of this medicinal plant for future therapy and corroborates to traditional use.

Antinociceptive activity of ethanol extract from Duguetia chrysocarpa Maas (Annonaceae).

The ethanol extract from the fruits of Duguetia chrysocarpa was evaluated for its antinociceptive activity in chemical and thermal models of nociception in mice. The intraperitoneal administration of the ethanol extract (100, 200, and 400 mg/kg body weight) showed a dose-dependent inhibition of acetic-acid-induced abdominal writhes. The extract also produced a significant inhibition of both phases of the formalin test in all doses tested and increased the reaction time in hot-plate test at dose of 200 mg/kg. The data obtained suggest that the antinociceptive effect of the extract may be mediated via both peripheral and central mechanisms. The phytochemical investigation yielded the isolation of the benzenoid derivative 3-methoxy-4-ethoxy benzoic acid which is being reported for the first time in this genus.

ß-carboline-independent antidepressant-like effect of the standardized extract of the barks of Mimosa tenuiflora (Willd) Poir. occurs via 5-HT2A/2C receptors in mice.

BACKGROUND: Depression is a psychiatric disorder with limited therapy options. Psychedelics are new antidepressant candidates, being the ayahuasca one of the most promising ones. A synergistic combination of N,N-dimethyltryptamine (DMT) and ß-carbolines allows ayahuasca antidepressant properties. Another psychedelic and DMT-containing beverage is the jurema wine used religiously by indigenous people from Northeastern Brazil. AIMS: To evaluate the antidepressant-like effect of standardized extract of Mimosa tenuiflora (SEMT), associated or not with harmine (ß-carboline), in behavioral models of depression. METHODS: The SEMT was submitted to (+) ESI-IT-LC/MS analysis for DMT quantification. To assess the antidepressant-like effect of SEMT, the open field (OFT), tail suspension (TST), and forced swim (FST) tests were performed. To verify the participation of serotonergic systems, the 5-hydroxytryptophan (5-HTP)-induced head twitch test was performed. RESULTS: The content of DMT found in SEMT was 24.74 ± 0.8 mg/g. Yuremamine was also identified. SEMT presented an antidepressant-like effect in mice submitted to the TST and FST, independent from harmine, with no significant alterations on the OFT. The sub-dose interaction between SEMT and ketamine also produced an anti-immobility effect in the TST, with no changes in the OFT. SEMT potentiated the head twitch behavior induced by 5-HTP and ketanserin prevented its antidepressant-like effect in the TST (p < 0.05). CONCLUSIONS: SEMT presented a harmine-independent antidepressant-like effect in mice submitted to the TST and FST. This effect occurs possibly via activation of serotonergic systems, particularly the 5-HT2A/2C receptors.

Jatropha mutabilis, a new source of vitexin: HPLC quantification and pharmacological evaluation.

Jatropha mutabilis (Pohl) Baill is an endemic species of the Caatinga biome, little studied in terms of chemical composition. The objective of this work was to develop an analytical methodology to quantify vitexin in the ethanolic extract of J. mutabilis and to evaluate the expectorant and antitussive activities in mice. The expectorant activity was performed by measuring the phenol red obtained from the bronchoalveolar fluid in animals and the antitussive activity was evaluated by the cough method induced by citric acid (0.4 M). The method developed by HPLC-DAD proved to be simple, linear, precise, accurate, robust and specific. Besides, both vitexin (0.2, 1 and 5 mg/kg) and the extract of J. mutabilis (20, 102, 510 mg/kg) showed efficacy in decrease cough and increase aqueous mucus in mice, but vitexin was more potent. Lastly, the identification of vitexin opens the possibility of new studies for J. mutabilis.

Cannabis roots: Pharmacological and toxicological studies in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: There are many studies and therapeutic properties attributed to the flowers and leaves of the Cannabis species, but even with few pharmacological studies, Cannabis sativa L. (Cannabaceae) roots presents several therapeutic indications in folk medicine. AIM OF THE STUDY: This study aimed to evaluate the anti-inflammatory and spasmolytic effects as well as the toxicological profile of the aqueous extract of Cannabis sativa roots (CsAqEx) in mice. MATERIALS AND METHODS: We assessed the anti-inflammatory effect with carrageenan-induced leukocyte migration assay, and carrageenan and histamine-induced paw edema methods; The spasmolytic effect was assessed through in vitro assays with isolated mice trachea. To assess motor coordination and mobility, mice went through the rotarod and open field tests, respectively. For the single-dose toxicity study, we administered CsAqEx at the dose of 1000 mg/kg by gavage. In a repeated dose toxicity study, animals received CsAqEx at doses of 25 mg or 100 mg/kg for 28 days. RESULTS: The CsAqEx inhibited the migration of leukocytes at the doses of 25, 50, and 100 mg/kg. The CsAqEx showed anti-inflammatory activity after the intraplantar injection of carrageenan, presenting a reduction in edema formation at all tested doses (12.5, 25, 50 and 100 mg/kg). The dose of 12.5 mg/kg of CsAqEx prevented edema formation after intraplantar injection of histamine. In an organ bath, 729 µg/mL of CsAqEx did not promote spasmolytic effect on isolated mice tracheal rings contracted by carbachol (CCh) or potassium chloride (KCl). We did not observe clinical signs of toxicity in the animals after acute treatment with CsAqEx, which suggested that the median lethal dose (LD50) is greater than 1000 mg/kg. Repeated dose exposure to the CsAqEx did not produce significant changes in hematological, biochemical, or organ histology parameters. CONCLUSIONS: The results suggest that the anti-inflammatory effect of CsAqEx is related to the reduction of vascular extravasation and migration of inflammatory cells, without effects on the central nervous system. Moreover, there was no spasmolytic effect on airway smooth muscle and no toxicity was observed on mice.

Investigation of antinociceptive, antipyretic, antiasthmatic, and spasmolytic activities of Brazilian Cannabis sativa L. roots in rodents.

ETHNOPHARMACOLOGICAL RELEVANCE: Many studies are performed with the aerial parts of Cannabis sativa L. (Cannabaceae). However, roots remain poorly studied, despite citations in the scientific literature. The C. sativa roots are indicated for the treatment of pain, inflammation, fever, among other health problems. AIM OF THE STUDY: This study aimed to evaluate the antinociceptive, antipyretic, antiasthmatic, and spasmolytic activities of C. sativa roots in experimental models using mice and rats. MATERIAL AND METHODS: The chemical composition of the aqueous extract of C. sativa roots (AECsR) was evaluated by LC-MS. The antinociceptive activity was assessed in mice by the induction of writhing with acetic acid, paw licking with formalin, and reactivity in the hot plate test. Fever was induced by the administration of a suspension of Saccharomyces cerevisiae in young rats. The asthmatic activity was performed with ovalbumin (OVA)-immunized mice with cellular and histological analysis. Finally, the spasmolytic activity was performed using mice isolated trachea. For in vivo studies, the doses were 12.5, 25, or 50 mg/kg whereas for in vitro, the concentration of AECsR was 729 µg/mL. RESULTS: From the LC-MS data, we identified p-coumaroyltyramine, feruloyltyramine canabissativine in AECsR. The extract promoted a reduction of writhing in all tested doses (12.5, 25, or 50 mg/kg). Similarly, it reduced the pain in the formalin test at doses of 12.5 and 50 mg/kg (first phase) and 12.5 and 25 mg/kg (second phase). In the hot plate test, the doses of 12.5, 25, and 50 mg/kg promoted antinociceptive effect at different times, and the lowest dose maintained its action in the analyzes performed at 60, 90, and 120 min after administration. The anti-inflammatory activity of AECsR was observed in the mouse model of asthma, reducing the total leukocyte count in the bronchoalveolar fluid (BALF) at a dose of 25 mg/kg, as well as reducing eosinophilia in all tested doses (12.5, 25, and 50 mg/kg). Histological analysis of lungs stained with H&E and PAS showed a reduction in the number of inflammatory cells in the perivascular and peribronchial region, as well as reduced mucus production. CONCLUSION: The results suggest that AECsR promotes pain control, either by a central or inflammatory mechanism, and has antiasthmatic activity. However, there was no antipyretic or spasmolytic effect.

Lippia alnifolia essential oil induces relaxation on Guinea-pig trachea by multiple pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Lippia alnifolia Mart. & Schauer, known as "alecrim-do-mato", "alecrim-de-vaqueiro" and "pedrécio", is used in folk medicine as antiseptic and to treat diseases that affect respiratory system, like bronchitis and asthma. AIM OF THE STUDY: The aim of this work was to investigate the spasmolytic activity and relaxant mechanism of the Lippia alnifolia essential oil (EOLA) on isolated guinea-pig trachea and to correlate with its use in folk medicine. MATERIALS AND METHODS: Leaves from L. alnifolia were collected in Pico das Almas, Chapada Diamantina, situated in the city of Rio de Contas, Bahia, Brazil. EOLA was extracted by hydrodistillation, analyzed by GC/FID and GC/MS and the volatile constituents were identified. Spasmolytic activity was assayed in isolated guinea-pig trachea pre-contracted with carbachol 1 µM or histamine 10 µM. Relaxant mechanism of EOLA was determined comparing concentration-response curves in the presence or absence of different blockers. RESULTS: Chemical analysis revealed the presence of carvone (60 ±â€¯0.8%) as major constituent. EOLA (1-243 µg/mL) relaxed isolated guinea-pig trachea pre-contracted with carbachol 1 µM [EC50 = 53.36 (44.75-63.51) µg/mL] or histamine 10 µM [EC50 = 5.42 (4.42-6.65) µg/mL]. The pre-incubation of 4-aminopyridine in histamine-induced contractions did not alter significantly the relaxant effect of EOLA. However, the presence of cesium chloride, glibenclamide, tetraethylammonium, propranolol, indomethacin, dexamethasone, hexamethonium, atropine, L-NAME, methylene blue or ODQ reduced EOLA relaxant effect. EOLA 18 µg/mL pre-incubation in calcium-free medium reduced histamine-evoked contractions, but did not alter histamine contractions in the presence of nifedipine. CONCLUSIONS: Lippia alnifolia essential oil has spasmolytic activity on isolated guinea-pig trachea and its mechanism of action possibly involves the activation of multiple signal transduction pathways, which culminate in potassium channels activation and cytosolic calcium reduction.

Analytical and pharmacological validation of the quantification of phenol red in a mouse model: An optimized method to evaluate expectorant drugs.

INTRODUCTION: The evaluation of expectorant activity has been extensively studied in murine models, involving the secretion of phenol red in the trachea or bronchus to estimate the secretory capacity of lower airway mucosa. However, differences in the experimental protocols of several studies evidenced the need of to standardize the quantification of phenol red in the bronchoalveolar fluid (BALF). METHODS: The analytical methodology for the quantification of phenol red in the BALF was optimized by investigation of pH influence, quantity of the alkali agent added and appropriate wavelength for quantification of phenol red by UV-VIS spectroscopy. Different phenol red suspensions (0.05, 0.5, 1.25, 2.5 and 5%) were prepared and administered intraperitoneally in mice at doses 5, 25, 50, 250 or 500 mg/kg. RESULTS: It was shown that phenol red should be used at dose 500 mg/kg and intraperitoneal administration should be performed from a suspension at 1.25% (w/v). Furthermore, the alkalinizing agent of choice would be NaOH (0.1 M). The pharmacological validation of the analytical method showed that ambroxol (30, 60 or 120 mg/kg), guaifenesin (100 mg/kg), NH4Cl (2000 mg/kg) or salbutamol (4 mg/kg) can be used as positive controls. DISCUSSION: The phenol red quantification in the BALF is a rapid and low cost assay for the discovery of new expectorant drugs. Thus, it was proposed a standardization of the analytical and pharmacological methods to ensure the reliability of BALF processing and reproducibility of phenol red quantification for data analysis.

Influence of light intensity, fertilizing and season on the cirsiliol content, a chemical marker of Leonotis nepetifolia (Lamiaceae).

BACKGROUND: Leonotis nepetifolia (Family Lamiaceae) is a medicinal plant from which the flavonoid cirsiliol with sedative, hypnotic, anti-inflammatory and cytotoxic activity has been extracted. METHODS: Seedlings were cultivated under different levels of shade in native or fertilized modes. The content of cirsiliol was measured monthly by high-performance liquid chromatography and the total phenolic content by the Folin-Ciocalteu method. Monitoring of growth was carried out with the weekly measurement of height until the stabilization of growth. RESULTS: The application of fertilizing and/or shading does not alter significantly the cirsiliol content. However, this content varies throughout the year, reaching the peak production in the summer, independently of the treatment applied. This same profile, with production in the summer, was also verified for phenolic compounds, reaching 58.15 ± 9.35 mg of equivalents of gallic acid per g of extract in the summer, content 1.84 times greater than the content verified in winter (31.56 ± 4.09 mg of gallic acid/g of extract). Although shading and fertilizing had no effect on cirsiliol content, the results also showed a positive influence on the height and biomass of the plant, which can causes a higher yield of extractable material. DISCUSSION: Biotic and abiotic stresses are able to increase or decrease the production of secondary metabolites, including phenolic compounds in medicinal plants and, as the stress response is peculiar to each species, cultivation studies become necessary. The present study reports by the first time the influence of shading, fertilizing and seasons in cirsiliol content in L. nepetifolia. Among analyzed variables, the seasons showed a larger influence in expression of cirsiliol and among seasons, our results showed that the summer is the ideal season for collections. In summer, the photoperiod is larger than in other seasons of the year and due to that, the plants need greater protection against the long photoperiod. For this, the plants increase the production of phenolic compounds as observed in this study. Although they do not influence the production of cirsiliol, the shading and nutrients in soil favor growth and leaf area of several plants, explaining, thus, the higher height and biomass obtained.

Anti-inflammatory and healing action of oral gel containing borneol monoterpene in chemotherapy-induced mucositis in rats ( Rattus norvegicus )

ABSTRACT The aim of this study was to investigate the effect of gels containing the monoterpene borneol in induced oral mucositis using an animal model. Gels were prepared with borneol at 1.2% and 2.4% (w/w). Oral mucositis was induced by administration of three doses of 5-fluorouracil (30 mg/kg, i.p.) and injury with acetic acid (50%, v/v) soaked in filter paper applied to right cheek mucosa for 60s. Four subgroups comprising 12 animals each were formed. Six animals from each group were sacrificed at days seven and fourteen after oral mucositis induction. Mucous samples were processed and stained with hematoxylin-eosin and Masson's Trichrome. The semiquantitative evaluation involved observation of inflammatory parameters. ImageJ® software was used in the quantitative evaluation. For statistical analyses, Two-way ANOVA, followed by Tukey's post-test (p <0.05), were employed. Borneol 2.4% gel proved effective in the treatment of oral mucositis with statistically significant differences between groups for angiogenesis control, inflammatory cell count reduction and percentage neoformed collagen increase. The confirmation of anti-inflammatory and healing action of borneol in oral mucositis in rats renders it a good marker for predicting this activity for plant extracts rich in this substance