Chemical characterisation and toxicity assessment in vitro and in vivo of the hydroethanolic extract of Terminalia argentea Mart. leaves.

ETHNOPHARMACOLOGICAL RELEVANCE: Terminalia argentea Mart. (Combretaceae), known mainly as "capitão", is a native tree, not endemic, that occurs in the Amazon, Caatinga, Cerrado and Atlantic Forest in Brazil. Leaf infusion is popularly mentioned by riverine communities that inhabit the microregion of Northern Araguaia (Mato Grosso, Brazil) for the treatment of gastric ulcer, bronchitis and haemorrhage. Considering the wide medicinal use, lack of studies that evaluate the safety of use and the scarcity of phytochemical studies of T. argentea leaves, this work was carried out with the objective of evaluating the toxicity of the hydroethanolic extract of the leaves of T. argentea Mart. (HETa) in experimental models in vivo and in vitro, as well as to advance the phytochemical analysis of HETa. MATERIALS AND METHODS: HETa was prepared by macerating the leaf powder in hydroethanolic solution. Phytochemical characterisation was carried out by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC) and mass spectrometry through direct flow infusion coupled with electrospray ionization and ion-trap analyzer (DFI-ESI-IT-MS analyses) The contents of phenols, flavonoids and phytosterols were analysed by colorimetric methods. Cytotoxicity was assessed by the Alamar blue assay on Chinese hamster ovary epithelial cells (CHO-K1) and human gastric adenocarcinoma cells (AGS). In vitro genotoxicity of HETa (10, 30 or 100 µg/mL) was assessed by micronucleus (MN) and comet tests using CHO-K1 cells. The acute toxicity assessment was performed by oral administration of HETa in single dose Swiss mice (males and females) up to 2000 mg/kg and sub-chronic toxicity by daily oral administration of HETa (50, 200 and 800 mg/kg) in Wistar rats for 30 days. The parameters related to the clinical and toxicological observations were determined every 6 days and at the end of the treatment the blood was collected for biochemical and haematological analysis, and some organs were removed for macroscopic and histopathological analysis. RESULTS: Preliminary phytochemistry and TLC analysis of HETa revealed the presence of phenolic compounds (18.8%), flavonoids (10.8%), saponins, tannins and phytosterols (19%). The HPLC data revealed the presence of gallic acid, rutin, ellagic acid, catechin, quercetin and kaempferol. In the analysis by DFI-ESI-IT-MS, the presence of gallic acid, rutin, ellagic acid and quercetin was confirmed and identified caffeic acid, quinic acid, galloylmucic acid, quercetin xyloside, quercetin rhamnoside, quercetin glucoside, caffeoyl ellagic acid, quercetin galloyl xyloside, terminalin, quercetin galloyl glucose, corilagin, quercetin digalloyl xyloside, quercetin digalloyl glucoside, punicalin and punicalagin. HETa showed no cytotoxic effect on CHO-K1 and AGS cells. In the MN assay, HETa increased the number of MNs and nuclear buds (NBUDs) in binucleate cells at the three concentrations tested and the nucleoplasmic bridges (NPBs) number at 30 µg/mL. In the comet test, HETa (10 and 100 µg/mL) alone showed a genotoxic effect on CHO-K1 cells. In pre-treatment, HETa at all concentrations tested prevented DNA damage induced by H2O2. In co-treatment with H2O2, HETa showed genotoxic effects at the three concentrations, and post-treatment DNA damage in exposed CHO-K1 cells to H2O2 was repaired in 22.5% with 10 µg/mL HETa. In the acute toxicity test, the HETa did not cause death in the mice, being verified only by piloerection and reversible in 2 h in males and in 4 days in females. No macroscopic changes were observed in the analysed organs. In the sub-chronic toxicity test, the HETa did not cause death in the rats after 30 days and the few changes were: absolute (103/mm3) and relative (%) values of basophils increased by 477.8% and 423% (p < 0.001), respectively, with 50 mg/kg; reduction in feed intake (23.6%, p < 0.01) only on day 18; total cholesterol concentration (13.1%, p < 0.05) and relative heart weight (13.2% %, p < 0.05) at a dose of 800 mg/kg. These effects were not dose-dependent nor followed by clinical signs and symptoms of intoxication, nor of macroscopic and histopathological changes in the organs of animals treated with HETa. CONCLUSIONS: The results demonstrated that HETa had no cytotoxic in vitro effects for CHO-K1 and AGS cells. In in vitro genotoxicity assays, the HETa induced different responses, according to concentration and experimental condition. In the MN test the HETa presented genotoxic potential by increasing the number of MNs, NBUDs and NPBs. In the comet assay, HETa was genotoxic by itself and in the co-treatment protocol with H2O2. In pre-treatment or post-treatment protocols with H2O2, HETa presented an antigenotoxic effect by preventing or repairing, respectively, the genotoxicity induced by H2O2. In the in vivo models, HETa was shown to be relatively safe after acute administration in mice [no-observed-adverse effect level (NOAEL) of 2000 mg/kg] and sub-chronic in rats (NOAEL of 800 mg/kg), confirming the riverine information that it is non-toxic in the dosage used. Phytochemical analysis of HETa revealed the presence of phenolic compounds, flavonoids, saponins, tannins and phytosterols. Among the flavonoids and tannins, we highlight gallic acid, rutin, ellagic acid, quercetin, caffeic acid, quinic acid, corilagin, punicalin and punicalagin. Thus, it can be stated that HETa has a good safety margin for therapeutic use.

Ethnobotanical study of medicinal plants used by Ribeirinhos in the North Araguaia microregion, Mato Grosso, Brazil.

ETHNOPHARMACOLOGICAL IMPORTANCE: Currently, in many traditional communities, such as the riverine community in the North Araguaia microregion (Mato Grosso, Brazil), plant knowledge and use represent the main, if not the only, therapeutic resource for the maintenance of health and/or treatment of diseases. This study aimed to identify and document species of medicinal plants used by local experts from riverine communities in the North Araguaia microregion in Mato Grosso State, and to further chemical and pharmacological studies on species selected based on searches in the relevant literature. MATERIALS AND METHODS: This is a cross-sectional ethnobotanical study, with non-probabilistic sampling (n =60), that applied the snowball method to select local riverine experts who understand medicinal plant use. Socio-demographic, ethnobotanical and ethnopharmacological data (vernacular name, uses, geographical origin, habit, method of preparation and part used) on medicinal plants were collected during semi-structured interviews. The results were analyzed by descriptive and quantitative means: indices of use-report (UR) were used to select plant species with therapeutic potential. RESULTS: In total, 309 plant species belonging to 86 botanical families were cited; 73% were native to Brazil, and Fabaceae was the most representative family (11.3%). Arboreal was the predominant life form (37.2%). The leaf was the most used part (28.9%). Infusion was the most commonly reported method of preparation (31.3%). The plants reported in the survey were indicated for 18 of the 22 ICD-10 disease categories. The disease categories most commonly cited were the infectious and parasitic diseases (IPD, 718 UR), digestive system diseases (DSD, 565 UR) and respiratory system diseases (RSD, 504 UR), representing 16.6%, 13.1% and 11.7%, respectively of the total UR. Dysphania ambrosioides L. was the most sighted in the IPD category 50 UR. Copaifera langsdorffii Desf. (133), Lafoensia pacari A. St.-Hil. (131), and Cecropia pachystachya Trécul (126) were the species with the highest UR. Bidens pilosa L., Vernonia ferruginea Less, and L. pacari, respectively, were the most cited native plants used to treat such diseases. Of the 8 investigated native plants, C. langsdorffii, and Brosimum gaudichaudii are the most prominent: in addition to having been widely studied, in terms of phytochemical and pharmacological, these species have been marketed as pharmaceutical products, with associated patent deposits. CONCLUSIONS: Local riverine experts from the North Araguaia microregion use a wide variety of medicinal plants in self-care health, especially those species used to treat IPD. The therapeutic potential of some of these plants has been scientifically validated; however, there are other species whose pharmacological effects and safety remain to be properly investigated. Thus, the present study, aside from being a basis for future chemical, pharmacological and agronomic bioprospecting studies, may contribute to the development of the management, conservation and sustainable use of medicinal flora in the microregion studied.

Evaluation of the safety, gastroprotective activity and mechanism of action of standardised leaves infusion extract of Copaifera malmei Harms.

ETHNOPHARMACOLOGICAL RELEVANCE: Copaifera malmei Harms (Fabaceae) is a plant that occurs in the central region of Brazil, where the plant's leaves infusion is popularly used to treat gastric ulcer and inflammatory diseases. This study was aimed to investigate the gastroprotective activity and mode of action of the plants' leaves infusion in order to establish the scientific basis for such usage, and to assess its potential as a source of an anti-ulcer agent. MATERIALS AND METHODS: Leaves infusion extract of the plant (SIECm) was prepared, freeze dried and lyophilised. Its qualitative and quantitative phytochemical constituents were investigated using TLC and HPLC techniques. The safety profile was evaluated on CHO-k1 epithelial cells viability using the Alamar blue assay, and by acute toxicity test in mice. The gastroprotection and anti-ulcer efficacy of the SIECm (25, 100 and 400mg/kg, p.o.) were tested using acute (acidified ethanol, piroxicam and water restrain stress), and chronic (acetic acid) experimental ulcer models. The plausible mode of action of the SIECm was assessed using gastric secretion, gastric barrier mucus, nitric oxide, and its antioxidant (myeloperoxidase and catalase) effects in mice and rats. The histopathological analyses of the ulcerated tissues as well as the extract's activity on Helicobacter pylori were also investigated. RESULTS: Phytochemical tests indicated the presence of mainly phytosterols, phenolics and flavonoids. The SIECm exhibited no cytotoxic effects on the CHO-k1 cells, and no oral acute toxicity in mice. It prevented against the acute induced ulcerations by enhancing gastroprotection through gastric mucus production, NO modulation, antioxidant, reduced gastric secretion and enhanced chronic ulcers healing process, as shown by reduction/prevention of epithelial and vascular damage, in addition to reduction in leucocyte infiltration. The SIECm however did not exhibit activity against H. pylori. CONCLUSION: The SIECm is safe, contain useful phytochemicals and exhibited significant gastroprotective/anti-ulcer effects. The results justify its folkloric usage, and provided scientific evidence of its potential as a source of new phytodrug to treat gastric ulcers.

Antiinflammatory, antinociceptive and antipyretic effects of hydroethanolic extract from Macrosiphonia velame (A. St. -Hil. ) M. Arg. in animal models / Efeitos antiinflamatório, antinociceptivo e antipirético do extrato hidroetanólico de Macrosiphonia velame (A. St. - Hil. ) M. Arg. em modelos animais

Macrosiphonia velame (Apocynaceae), popularly known as "velame-branco", is mainly used for treating inflammatory conditions. The antiinflammatory, antinociceptive and antipyretic effects of the hydroethanolic extract of the xylopodium from M. velame (HEMv) were evaluated using several animal models. HEMv showed low acute oral toxicity with LD50 of 4.176 ± 218.5 mg/kg in mice. In tests of carrageenan and dextran-induced paw edema and carrageenan-induced pleurisy in rats, and croton oil-induced cutaneous dermatitis in mice, HEMv presented systemic and topical antiinflammatory activities. In experiments of nociception induced by acetic acid, formalin and capsaicin in mice, the HEMv evidenced an antinociceptive effect, being active against both inflammatory and neurogenic pain. Additionally, the HEMv prevented brewer's yeast-induced pyrexia in rats. It is likely that the pharmacologic mechanism of HEMv may involve the inhibition of different mediators of the inflammatory response, such as histamine, serotonin, prostaglandins and leukotrienes. A preliminary phytochemical study was also undertaken on HEMv, which revealed the presence of flavonoids, phenolic compounds, pentacyclic triterpenoids, saponins, coumarins, catechins, tannins, and alkaloids. Taken together, these results suggest that M. velame extract has antiinflammatory, antinociceptive and antipyretic properties and further validate the traditional use of this plant in inflammatory conditions.

Macrosiphonia velame (Apocynaceae), conhecida popularmente como velame-branco, é utilizada no tratamento de inflamações. Avaliou-se nesse estudo, os efeitos antiinflamatório, antinociceptivo e antipirético do extrato hidroetanólico do xilopódio de M. velame (HEMv) em modelos animais. O HEMv apresentou baixa toxicidade aguda oral, com DL50= 4.176 ± 218,5 mg/kg nos camundongos. Nos testes de edema de pata por carragenina e dextrana e pleurisia por carragenina em ratos e dermatite cutânea por óleo de croton em camundongos, o HEMv apresentou atividade antiinflamatória sistêmica e tópica. Nos experimentos de nocicepção induzida por ácido acético, formalina e capsaicina em camundongos, o HEMv inibiu a resposta nociceptiva nos três modelos, mostrando-se ativo tanto na dor de origem inflamatória como neurogênica. Adicionalmente, o HEMv bloqueou a hipertermia induzida por levedura de cerveja nos ratos. É provável que os efeitos farmacológicos observados para o HEMv decorram da inibição de mediadores da resposta inflamatória produzidos pelas vias da COX e/ou LOX bem como da inibição da liberação de histamina e serotonina. A análise fitoquímica do HEMv revelou a presença de flavonóides, compostos fenólicos, triterpenóides pentacíclicos, saponinas, cumarinas, catequinas, taninos catéquicos e alcalóides. Estes resultados validam sob o ponto de vista pré-clínico, o uso popular de M. velame em processos inflamatórios.

Influência do aloe vera e própolis na contração de feridas em dorso de ratos / The Influenceaf Aloe vera and propolis in the contraction of wounds on dorsal area of rats

Os produtos naturais Aloe vera e própolis vêm se destacando na indústria farmacêutica. Para uma melhor compreensão desses agentes, o estudo objetivou comparar clinicamentea influência do Aloe vera in naturae um extrato hidroalcoólicode própolis na contração de feridas cutâneas em dorso de ratas. Depois de anestesiados os animais, produziu-se uma ferida com área de 1cm2 no dorso de cada um deles até atingiro tecido subcutâneo, preservando-se o tecido muscular. Imediatamenteapós a cirurgia, iniciaram-se as aplicações dos agentes cinco vezes por semana, uma vez ao dia, no mesmo horáriodurante toda a fase experimental. As mensurações das áreasdas feridas foram realizadas nos períodos de 1,3,7, 12 e 15dias, através de um paquímetro digital. Após obter-se o valor das áreas, foi avaliada a contração das feridas através da seguinte fórmula: (área inicial - área do dia da medida) / área inicial x 100 = percentual de contração no dia da medida. Efetuaram-se as médias dos grupos em cada tempo experimentale comparam-nas (Anova e corretivo Bonferroni). Os resultados evidenciaram que nos dias 3 e J2 as contrações daslesões no grupo Aloe vera e própolis foram maiores, se comparado-as ao grupo teste (p<0,05). No tempo experimental de 1, 7 e 15 dias, não houve diferença estatística (p>0,05) entre os grupos. Diante de tais resultados, observou-se que clinicamente, em dorso de ratos, Aloe vera in naturae própolis foram auxiliaresem um reparo mais rápido da lesão.