Brasiliensic acid: in vitro cytotoxic and genotoxic, in vivo acute toxicity and in silico pharmacological prediction of a new promising molecule.

Brasiliensic acid (Bras) is a chromanone isolated from Calophyllum brasiliense Cambèss. bark extracts with confirmed potential activity on gastric ulcer and Helicobacter pylori infection. This study aimed to investigate the in vitro and in vivo toxicity of Bras and molecular docking studies on its interactions with the H. pylori virulence factors and selected gastric cancer-related proteins. Cytotoxicity was evaluated by alamarBlue© assay, genotoxicity by micronucleus and comet assays, and on cell cycle by flow cytometry, using Chinese hamster epithelial ovary cells. Bras was not cytotoxic to CHO-K1 cells, and caused no chromosomal aberrations, nor altered DNA integrity. Furthermore, Bras inhibited damages to DNA by H2O2 at 1.16 µM. No cell cycle arrest was observed, but apoptosis accounted for 31.2% of the cell death observed in the CHO-K1 at 24 h incubation of the IC50. Oral acute toxicity by Hippocratic screening test in mice showed no relevant behavioral change/mortality seen up to 1,000 mg/kg. The molecular docking approach indicated potential interactions between Bras and the various targets for peptic ulcer and gastric cancer, notably CagA virulence factor of H. pylori and VEGFR-2. In conclusion, Bras is apparently safe and an optimization for Bras can be considered for gastric ulcer and cancer.Communicated by Ramaswamy H. Sarma.

Cytotoxicity and antibacterial activity of scutellarein and carajurone-enriched fraction obtained from the hydroethanolic extract of the leaves of Fridericia chica (Bonpl.) L.G. Lohmann.

The hydroethanolic extract obtained from the dry leaves of Fridericia chica (HEFc) underwent several fractionations by different chromatographic techniques. The ethyl acetate and dichloromethane fraction were subjected to phytochemical analysis, resulting in the identification and isolation of scutellarein (1) and in a fraction rich in carajurone (2). They were tested for cytotoxicity in CHO-K1 and the antibacterial activity and mode of action by in vitro assays. The HEFc and scutellarein (1) presented no cytotoxicity. The results showed good antibacterial effect of HEFc against Streptococcus pyogenes and Staphylococcus aureus and moderate activity for Staphylococcus epidermidis, Pseudomonas aeruginosa and Salmonella typhimurium. The fraction containing the compound carajurone (2) showed good activity against Staphylococcus aureus and Staphylococcus epidermidis and moderate activity against Streptococcus pyogenes. Scutellarein (1) showed no activity against the bacteria tested. HEFc antibacterial mode of action appeared to be associated with changes in the permeability of bacterial membranes and nucleotide leakage.

Comparative study of the use of miltefosine, miltefosine plus allopurinol, and allopurinol in dogs with visceral leishmaniasis.

Visceral leishmaniasis (VL) is an infectious disease caused by the protozoan parasite Leishmania (Leishmania) infantum, an intracytoplasmic parasite that affects humans and other species of domestic and wild mammals. In Brazil, the treatment of canine visceral leishmaniasis (CVL) with miltefosine has been implemented since 2016, and the reports on the clinical and immunological conditions of treated dogs are scarce. Thus, this study aimed to assess and monitor the clinical, laboratory, and immunological condition of dogs with CVL before (D0) and after (D29) using three pharmacotherapeutic protocols: miltefosine monotherapy (Milteforan™, Virbac) (G1), miltefosine plus allopurinol (G2), and allopurinol monotherapy (G3). Forty-five dogs with CVL were assigned to one of three treatment groups. The dogs were evaluated for clinical signs, was well as haematological, biochemical, serological, and cytokine levels. Significant reduction in clinical scores was observed in all protocols, with no differences between groups. We did not observe a clinical cure in any of the dogs in the groups. Haematological and biochemical parameters showed slow recovery, with better results observed in G2. Anti-Leishmania antibody titre remained increased in all groups. The quantification of serum cytokines demonstrated a mixed Th1/Th2 profile in CVL. The IL-2 levels decreased in all groups after treatment. Evaluation of IFN-y and IL-10 did not show changes in the groups analysed, and it did not contribute to short term therapeutic monitoring. All therapeutic protocols promoted, to varying degrees, an improvement in the general condition (clinical signs, haematological, and biochemical levels) of the animals. Through clinical-pathological exams, we found that the combination of miltefosine plus allopurinol promoted better effects in the short-term, representing the best choice for the treatment of CVL, even when compared to the only therapeutic protocol allowed in Brazil, miltefosine monotherapy. Through the quantification of cytokines, IL-2 proved to be a potential therapeutic marker for the monitoring and follow-up of dogs with CVL.

Heteropterys tomentosa A. Juss: Toxicological and adaptogenic effects in experimental models.

BACKGROUND: The constant pursuit of improved athletic performance characterizes high-performance sport and the use of medicinal plants as dietary supplements is becoming widespread among athletes to enhance long-term endurance performance. AIM: The present study evaluated the toxicity of Heteropterys tomentosa (HEHt) and its acute adaptogenic effects. METHODS: The in vitro safety profile was evaluated on CHO-k1 cells using the alamar Blue assay, at concentrations ranging from 3.125 to 200 µg/mL. In vivo acute oral toxicity was conducted in male and female mice with oral administration of graded doses of HEHt from 400 to 2000 mg/kg. A subchronic oral toxicity study was completed by oral administration of HEHt (50, 200 or 1000 mg/kg) and vehicle for 30 days in male Wistar rats. Clinical observations and toxicological related parameters were determined. Blood was collected for biochemical and hematological analyses, while histological examinations were performed on selected organs. Thereafter, an adaptogenic test consisting of progressive loads until exhaustion was conducted in rats ( n = 5/group) orally pre-treated with the vehicle and HEHt (25, 100 or 400 mg/kg). RESULTS: HEHt exhibited no cytotoxic effects on the CHO-k1 cells and, apparently, no acute toxicity in mice and no subchronic toxicity in rats. An ergogenic effect was observed only at the dose of 25 mg/kg compared with the vehicle in relation to time to exhaustion and exercise load ( p = .011 and .019, respectively). HEHt is safe at up to 400 mg/kg, contains astilbin and taxifolin as the major phytochemical compounds, and exhibited a potential adaptogenic effect. CONCLUSIONS: These results justify its anecdotal usage as a tonic, show that the hydroethanolic maceration of the root does not cause toxicity, and provide scientific evidence of its potential as a source of new adaptogenic substance(s).

Chemical characterization, toxicology and mechanism of gastric antiulcer action of essential oil from Gallesia integrifolia (Spreng.) Harms in the in vitro and in vivo experimental models.

Gallesia integrifolia is a Brazilian Amazon tree whose bark decoction is popularly used to treat peptic ulcer. The essential oil from the inner stem bark of G. integrifolia (EOGi) was chemically characterized by GC/MS. The in vitro cytotoxicity and genotoxicity were evaluated in CHO-K1 cells, while the in vivo oral acute toxicity was performed in mice. The gastroprotective effect of EOGi was assessed in acidified ethanol and piroxicam and ulcer healing on acetic acid -induced ulcer models in rodents. Anti-secretory, mucus, K+-ATP channels, prostaglandins (PGs), nitric oxide (NO), TNF-α, IL-1ß, IL-10, catalase (CAT) and myeloperoxidase (MPO) activities and in vitro Helicobacter pylori action by EOGi were evaluated. EOGi exhibited cytotoxic effects only at 72h and no acute toxicity. EOGi showed gastroprotective and ulcer healing effects. EOGi gastroprotection was attenuated by indomethacin pre-treatment. Gastric volume and total acidity were reduced, while gastric pH was elevated. EOGi increased mucus and NO productions and CAT activity, and inhibited MPO activity, TNF-α and IL-1ß concentrations and augmented IL-10. EOGi was not active against H. pylori. These results indicated that EOGi is safe and exerts preventive and curative gastric ulcer effects by multitarget actions. Twenty compounds were identified and (-)-alpha-santalene was the main compound.

Dilodendron bipinnatum Radlk. inhibits pro-inflammatory mediators through the induction of MKP-1 and the down-regulation of MAPKp38/JNK/NF-κB pathways and COX-2 in LPS-activated RAW 264.7 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: The stem bark of Dilodendron bipinnatum Radlk. (Sapindaceae), a tree native to Pantanal of Mato Grosso, Brazil, popularly known as "mulher-pobre" (poor woman), has been historically used by the locals, after decoction and maceration, for the treatment of inflammatory conditions. We have recently shown that these preparations indeed possess anti-inflammatory properties, which are mediated by the inhibition of cell migration and the modulation of Th1 and Th2 cytokines. The NO pathway was not affected. AIM OF THE PRESENT STUDY: The aim of the present study was to further investigate the mechanisms responsible for the anti-inflammatory properties of the hydroethanolic extract of the stem bark of Dilodendron bipinnatum (HEDb). MATERIALS AND METHODS: HEDb was obtained by maceration of the stem bark of D. bipinnatum as previously described. The corresponding effects on a macrophage-like cell line, RAW 264.7, were investigated. The apoptosis of RAW 264.7 upon treatment with LPS, HEDb and N-acetyl-L-cysteine (NAC) was assessed by flow cytometry, using an Annexin V-PE kit. The production of inflammatory cytokines (TNF-α, IL-1ß and IL-10) and PGE2 were evaluated by ELISA, after cell challenge with LPS. The intracellular redox state and changes in mitochondrial membrane potential were also assessed by flow cytometry, using DCFH-DA and JC-1 as probes. The protein expression levels of MAPK p-p38, p-ERK, p-JNK, MKP-1 and COX-2 were analysed by western blotting. Nuclear translocation of NF-қB was assessed by immunofluorescence microscopy. The quantified results are presented as a nuclear:cytoplasmic ratio. RESULTS: LPS, HEDb and NAC did not appear to decrease the number of viable cells in comparison to control treatment. HEDb attenuated the production of pro-inflammatory cytokines (IL-1ß and TNF-α) and PGE2 induced by LPS but did not affect IL-10. The production of ROS was also inhibited by HEDb (1, 5 or 20µg/mL), even at the lowest concentration; at 20µg/mL, HEDb was more effective than NAC, which was used as a positive control (74.1% and 66.2% inhibition of LPS's effect, respectively). LPS induced an increase in ΔΨm (19.2%, p<0.001), while HEDb inhibited the change in ΔΨm (7.7% at 20µg/mL, p<0.001). The results of western blotting showed that HEDb inhibited the expression of MAPK p-p38, p-JNK and COX-2, while the expression of MKP-1 was increased. p-ERK was not affected. LPS promoted the nuclear translocation of NF-қB p65 (67%, p<0.01) in RAW 264.7 cells, in comparison to baseline (33%). Pre-treatment with HEDb inhibited this translocation of NF-κB p65 (58% at 20µg/mL, p<0.001). CONCLUSION: HEDb has a potent anti-inflammatory activity and acts on multiple targets and biological pathways of potential therapeutic relevance.

Gallesia integrifolia (Spreng.) Harms: In vitro and in vivo antibacterial activities and mode of action.

ETHNOPHARMACOLOGICAL RELEVANCE: Gallesia integrifolia (Phytolaccaceae) is commonly known as "pau-d'alho" in Brazil or "garlic plant" due to the strong scent of garlic peculiar to all parts of the plant. The bark decoction is used for the treatment of microbial infections among other diseases by different ethnic groups in Brazil, Peruvian Amazonians, Bolivia and Mosetene Indians. This study aimed to advance in the antibacterial activity and characterize the mode of action of the hydroethanolic extract of the inner stem bark of G. integrifolia (HEGi) using in vivo and in vitro experimental models. MATERIALS AND METHODS: The qualitative and quantitative phytochemical analyzes of HEGi were carried out using colorimetric and HPLC technique. The cytotoxic potential of HEGi was evaluated against CHO-K1 cells by Alamar blue assay and its acute toxicity was assessed by the Hippocratic screening test using Swiss-Webster mice. The antibacterial activity was evaluated by micro- dilution method against ten strains of Gram-positive and Gram-negative bacteria. The mode of action of HEGi was investigated by outer membrane permeability, nucleotide leakage and potassium efflux assays. In vivo infection model was established by using Staphylococcus aureus infection model Wistar rats. RESULTS: Qualitative phytochemical analysis of HEGi revealed the presence of saponins, alkaloids, phenolic compounds and flavonoids. Phytochemical quantification of HEGi showed that higher total phenolic (80.10±0.62mg GAE/g) and flavonoid (16.10±0.03mg RE/g) contents. HPLC fingerprint analysis revealed the presence of gallic acid, rutin, and morin. In the Alamar blue assay no cytotoxic effect of HEGi in CHO-K1 cells was observed up to 200µg/mL, and no signs or symptoms of acute toxicity were observed in mice of both sexes at higher doses of up to 2000mg/kg, p.o. HEGi demonstrated bacteriostatic effect against selected Gram positive and Gram negative bacterial pathogens. Its mode of action is associated, at least partly, with changes in the permeability of bacterial membranes, evidenced by the increased entry of hydrophobic antibiotic in Pseudomonas aeruginosa, intense K(+) efflux and nucleotides leakage in Shigella flexneri, Streptococcus pyogenes and S. aureus. HEGi attenuated the experimental blood borne S. aureus infection in rats at all the tested doses levels (10, 50 and 250mg/kg). CONCLUSION: HEGi is safe at the dose tested when used acutely, and it presented broad antibacterial effect, which support its traditional use in the treatment of bacterial infections. It contains well known important phytochemicals, recognized to be active against bacterial pathogens in vitro and might be collectively responsible for the antibacterial activity of HEGi. It is bacteriostatic in nature, with membrane perturbation being one of it mode of action. HEGi represent a potential phytotherapic antibacterial agent.

Antibacterial mode of action of the hydroethanolic extract of Leonotis nepetifolia (L.) R. Br. involves bacterial membrane perturbations.

ETHNOPHARMACOLOGICAL RELEVANCE: Leonotis nepetifolia (L) R. Br., Lamiaceae, a pantropical shrub, popularly known in Brazil as "cordão-de-frade", "rubim", is reportedly used in Brazilian ethnomedicine as well as in different countries in the treatments of ailments such as infections, inflammations, wounds, stomach disorders, among others. AIM OF THE STUDY: To evaluate its potential cytotoxicity and antibacterial mode of action of the hydroethanolic extract of L. nepetifolia (HELn) leaves, including phytochemical analysis. MATERIALS AND METHODS: The cytotoxicity of HELn was investigated by Alamar blue assay, using CHO-K1 cells. Antibacterial activity of HELn was tested by broth microdilution methods against a panel of bacteria of clinical interest. The mode of action of L. nepetifolia was studied by targeting bacterial membranes. Phytochemical analysis was performed by determining total secondary metabolites with spectrophotometric assays and HPLC. RESULTS: HELn is not cytotoxic in the in vitro evaluation (IC50>200 µg/mL). It demonstrated a good spectrum of antibacterial activity with major activity against Shigella flexneri, Enterococcus faecalis, Staphylococcus aureus and Bacillus subtilis with MIC=6.25 µg/mL, Helicobacter pylori with MIC of 25 µg/mL and Streptococcus pyogenes with MIC of 50 µg/mL. Its mode of action is associated, at least partly, with changes in the permeability of bacterial membranes, as evidenced by the increased entry of hydrophobic antibiotics in Shigella flexneri and intense efflux of K(+) and nucleotide leakage in E. faecalis and Shigella flexneri. In addition, the presence of phenols, flavonoids and carotenoids, described in the literature to possess antibacterial effects, were detected in the composition of HELn, with high phenol content (11.55%), especially the flavonoids (6.47%). CONCLUSION: The results indicate that HELn has low cytotoxicity and potent antibacterial activity. It is bacteriostatic in nature, possibly acting at the level of bacterial membranes, especially on the cytoplasmic membrane and outer membrane, thus supporting its popular use in infectious processes. In addition, the presence of phenols, flavonoids, carotenoids, fatty acids and steroids, described in the literature as possessing antimicrobial activity, were detected in the composition of HELn.

Helicteres sacarolha A. St.- Hil. et al.: gastroprotective and possible mechanism of actions in experimental animals.

ETHNOPHARMACOLOGICAL RELEVANCE: Helicteres sacarolha A. St.- Hil. et al. popularly known in Brazil as 'semente-de-macaco', is widely employed in the popular medicine in many of parts of Brazil in the alleviation of symptoms of ailments such as peptic ulcer and inflammation. Up to the present, there is no study addressing the gastroprotective activity of the hydroethanolic extract of H. sacarolha and its possible mechanism of actions. MATERIALS AND METHODS: The hydroethanolic (70%) extract of H. sacarolha (HEHs) was obtained by maceration. The gastroprotective activity was assessed using gastric ulcer models induced by acidified ethanol, piroxicam, and water restraint stress in mice and rats at doses of 20, 50 and 250mg/kg p.o. Mechanistic studies involved the antisecretory assay evaluated with pylorus ligation in rats and pre-treatments with appropriate antagonists/inhibitors such as yohimbine, glibenclamide, indomethacin and l-NAME, effect on catalase and myeloperoxidase activities and gastric mucus determination using acidified ethanol- induced ulcer in mice. RESULTS: HEHs at all doses tested demonstrated potent gastroprotective activities in the acute ulcer models. The gastroprotective activity of HEHs was attenuated by pre-treatments with yohimbine, glibenclamide, indomethacin and l-NAME. HEHs effectively reduced basal gastric juice production without any effect on the free and total acidity. The gastroprotective action of HEHs involved increasing the antioxidant enzyme catalase and mucus secretion and inhibition of neutrophyl infiltration as reflected by the reduction in the myeloperoxidase activity. CONCLUSION: The results of this study gave a scientific support for the popular use of the leaves of H. sacarolha in the treatment of gastric ulcers and that it has a multi-targeted action.

Evaluation of toxicity of Calophyllum brasiliense stem bark extract by in vivo and in vitro assays.

ETHNOPHARMACOLOGICAL RELEVANCE: Calophyllum brasiliense Camb., Clusiaceae, is commonly known as "guanandi" and its stem bark is used in Brazilian traditional medicine to treat rheumatism, vein problems, hemorrhoids and gastric ulcers. The aim of this study was to evaluate the toxicity of hexane extract of Calophyllum brasiliense stem bark (HECb) using in vitro and in vivo experimental models. MATERIALS AND METHODS: In vitro toxicity was evaluated by Alamar Blue cytotoxicity assay and micronucleus test, using Chinese hamster ovary (CHO-k1) epithelial cells. in vivo toxicity was evaluated by oral acute and subchronic toxicity assays. In the oral acute toxicity screening, a single dose of HECb was administered to mice at doses ranging from 250 to 1000 mg/kg. In the subchronic study, HECb was administered orally for 30 days to Wistar rats at doses of 100 mg/kg and 500 mg/kg. Phytochemical analyses were performed by HPLC/UV-vis, secondary metabolites were quantified by spectrophotometric methods. RESULTS: HECb presented IC50=119.94±4.31 µg/mL after a 24 h cytotoxicity test using CHO-k1 cells, showing low cytotoxicity. However, when the cells were exposed to HECb for 72 h, the IC50 value was 8.39±2.00 µg/mL, showing in this case, a pronounced cytotoxic effect. In the oral acute toxicity studies, doses up to 500 mg/kg of HECb did not cause any changes in both male and female mice. At 1000 mg/kg, male mice showed signs typical of depression and stimulation that were reversed at 72 h. Besides, female mice were more sensitive to the toxic effect of HECb at 1000 mg/kg, which initially presented typical agitation signals, followed by depression signals, leading to death of all the animals at 24h. In subchronic assay with rats, HECb administered orally at doses of 100 and 500 mg/kg did not cause significant changes in all clinical parameters evaluated. Histopathological analyses showed no deleterious effect in the vital organs of rats. Preliminary phytochemical analysis revealed the presence of phenolic compounds, steroids, and volatile coumarins. Analysis by HPLC showed two major peaks characteristic of chromanones. CONCLUSIONS: In vitro toxicological tests showed that HECb exhibited cytotoxicity especially after 72 h of exposition, and mutagenicity on the highest tested dose. The in vivo studies demonstrated that HECb produced some toxicity signs at the highest dose tested, particularly, in the acute toxicity test but showed no significant signs of toxicity in the subchronic assay. Based on these and previous pharmacological studies, it is possible to say that HECb did not exhibit significant toxicity at its effective dose. This suggests that HECb is relatively safe in humans at its effective dose.

Toxicological evaluation of the hydroethanolic extract of Dilodendron bipinnatum Radlk.

ETHNOPHARMACOLOGICAL RELEVANCE: Dilodendron bipinnatum Radlk., Sapindaceae, is popularly known as 'mulher-pobre'. Its stem bark macerate or decoction is popularly used mainly to treat uterine inflammation and bone fractures. Acute oral and subchronic toxicities of the hydroethanolic extract of Dilodendron bipinnatum (HEDb) were investigated as well as the classes of phytochemical present in the extract. MATERIALS AND METHODS: Acute toxicity of HEDb was investigated by hippocratic screening in Swiss mice, cytotoxicity and potential genotoxic effect were evaluated with micronucleus test in the CHO-k1cells. Subchronic oral toxicity of HEDb was assessed in Wistar rats with graded doses of HEDb repeatedly administered for 30 days, in order to evaluate the behavioral changes, weight gain, water and feed consumption, urine and feces excretion, hematological and biochemical parameters and histopathological examinations of vital organs. RESULTS: In hippocratic screening doses up to 5000 mg/kg p.o. did not cause any changes in female mice, while signs of reduction in motility, increased respiratory rate and tail erection were observed in male mice but were all reversed within an hour. In cytotoxicity assay, IC50 was 118±1.55 µg/mL in CHO-k1 cells. Micronucleus test demonstrated that the plant extract is non-genotoxic. In subchronic toxicity studies, minor change was observed as an increase in the urine output in the last 18 days of treatment with 500 mg/kg of HEDb, while other parameters remained unchanged. All alterations observed were discrete and minor in nature and were not accompanied by any relevant clinical signs or any toxicologically significant biochemical, hematological and histopathological changes. Preliminary phytochemical analysis revealed the presence of aurones, chalcones, coumarins, flavonones, flavononoids, phenols, and saponins. CONCLUSION: The results demonstrate high safety profile of HEDb in both experimental animals and cell models assays. However, there may be risk of hepatotoxicity in the use of the extract at high doses for a prolonged period.

Evaluation of anti-inflammatory activity of hydroethanolic extract of Dilodendron bipinnatum Radlk.

ETHNOPHARMACOLOGICAL RELEVANCE: Dilodendron bipinnatum Radlk. (Sapindaceae), popularly known as "mulher-pobre", is a native tree of the Pantanal of Mato Grosso, Brazil. The stem bark of Dilodendron bipinnatum is used by the population, in the forms of decoction and maceration in the treatment of inflammatory conditions. There is no information in the literature demonstrating the anti-inflammatory activity of Dilodendron bipinnatum and its respective mechanism of action. This study aimed to evaluate the anti-inflammatory activity and mechanism of action of the hydroethanolic extract of the stem bark of Dilodendron bipinnatum (HEDb) using in vivo and in vitro experimental models. MATERIALS AND METHODS: The stem bark of Dilodendron bipinnatum was macerated in 70% hydroethanolic solution (1:3, w/v) for 7 days, filtered, concentrated on a rotary evaporator and the residual solvent removed in oven at 40°C, thus obtaining HEDb. Cytotoxicity of HEDb in RAW 264.7 was assessed by the Alamar blue assay. in vivo anti-inflammatory activity of HEDb was evaluated with carrageenan and dextran-induced paw edemas and lipopolysaccharide (LPS)-induced peritonitis in mice. Effects of HEDb on the inflammatory cytokines (TNF-α, IL-1ß and IL-10) concentrations in the peritoneal fluid were evaluated using commercial ELISA kits. The in vitro anti-inflammatory activity was evaluated using RAW 264.7 cells stimulated with LPS and/or INF-γ, while a Griess method was employed to determine nitric oxide (NO) concentrations in the peritoneal lavage and in the supernatants of RAW 264.7 cells. Preliminary phytochemical analysis was carried out using classical methods and secondary metabolites detected on HEDb were analyzed and confirmed by high performance liquid chromatography (HPLC). RESULTS: HEDb showed very low cytotoxicity with IC50>200±0.38 µg/mL. HEDb effectively inhibited paw edema by carrageenan in the 2nd hour at 20 mg/kg (36%, p<0.001), and by dextran in the 1st hour at 100 mg/kg (46%, p<0.01), after induction with the phlogistic agents. Furthermore, HEDb reduced total leukocytes and neutrophils migration at all doses tested producing maximum effect at 20 mg/kg (45% and 64%, p<0.001 respectively). HEDb also attenuated increases in the concentrations of the pro-inflammatory cytokines (IL-1ß and TNF-α) and increased the level of the anti-inflammatory cytokine IL-10 in the peritonitis model. However, it had no effect on NO production in activated RAW 264.7 cells. Preliminary phytochemical analysis revealed the presence of phenolic compounds, chalcones, flavones, flavonones, flavonoids, saponins and coumarins. HPLC analyses identified some tannins, with epigallocatechin gallate being the major compound. CONCLUSIONS: Our findings provide evidence for the popular use of the stem bark of Dilodendrum bipinnatum in inflammation. Its anti-inflammatory action was due, at least in part, to the inhibition of cell migration, of the inflammatory mediators and Th1 cytokines and an increase in Th2 cytokines, without affecting NO pathway. It can be suggested that tannins account at least in part for the anti-inflammatory activity of HEDb.

Evaluation of anti-inflammatory and mechanism of action of extract of Macrosiphonia longiflora (Desf.) Müll. Arg.

ETHNOPHARMACOLOGICAL RELEVANCE: Macrosiphonia longiflora (Desf.) Müll. Arg (Apocynaceae), popularly known as 'velame' and 'velame branco', is a native subshrub that grows in the Brazilian Cerrado. This plant is widely used in traditional medicine in the form of decoction and infusion, particularly as anti-inflammatory, depurative, anti-rheumatic, antisyphilitic and antiulcer remedy. There is no available information in the literature that has addressed its pharmacological activity and phytochemical analysis. AIM OF THE STUDY: This study aimed to evaluate the anti-inflammatory pharmacological profile of the hydroethanolic extract of Macrosiphonia longiflora, using in vivo and in vitro acute inflammation experimental models, as well as investigate the roles of cytokines and nitric oxide in its mechanism of action, and including phytochemical analysis constitution of its hydroethanolic extract. MATERIALS AND METHODS: Hydroethanolic (70%) extract of Macrosiphonia longiflora (HEMl) was prepared by maceration. The preliminary phytochemical analysis was performed according to procedures described in the literature. Selected secondary metabolites detected were quantified by spectrophotometry and high performance liquid chromatography (HPLC). Its cytotoxic potential in Chinese hamster ovary (CHO-k1) epithelial cell lines was evaluated using Alamar Blue. in vivo anti-inflammatory activity was evaluated with carrageenan- and dextran-induced paw edemas, carrageenan-induced pleurisy in rats and lipopolysaccharide (LPS)-induced peritonitis in mice. The in vitro anti-inflammatory activity was evaluated using RAW 264.7 cells stimulated with LPS and interferon (INF)-γ. Effects of HEMl on the inflammatory cytokines (IL-1ß, IL-10, IL-17, INF-γ and TNF-α) concentrations in the peritoneal lavage were evaluated using commercial ELISA kits, while the Griess method was employed to determine nitric oxide (NO) concentrations in the peritoneal lavage, as well as in the supernatants of RAW 264.7 cells. RESULTS: Preliminary phytochemical analysis, revealed the presence of phenolics compounds, terpenoids, alkaloids and flavonoids. Spectrophotometric analysis revealed the presence of relatively high content of phenolics and flavonoids in HEMl. HPLC analysis confirmed the presence of the quantified compounds and demonstrated the presence of ellagic acid in the detected matrix of compounds. HEMl appeared to be non-cytotoxic. It effectively inhibited (p<0.05) paw edema induced by carrageenan and dextran. Furthermore, HEMl also significantly reduced exudates volume and leukocyte migration in the carrageenan-induced pleurisy and LPS-induced peritonitis, neutrophils counts in LPS-induced peritonitis. HEMl also acts by effectively inhibiting the following inflammatory cytokines: IL-1ß and IL-10 levels in the peritoneal lavage, but had no effect on IL-17 level in the peritonitis model. In addition, HEMl had no effect on the levels of tumor necrosis factor alpha (TNF-α) present in the peritoneal lavage and cells supernatants. The concentration of NO, as assessed by measurement of nitrite (NO2(-)), showed that pretreatment with HEMl reduced NO significantly in the peritoneal lavage and in RAW 264.7 cells co-stimulated with LPS and INF-γ. CONCLUSION: The results obtained in this study indicate that HEMl possesses very low cytotoxic potential. In addition, it demonstrated a potent anti-inflammatory activity in both the in vivo and in vitro models of acute inflammation. The anti-inflammatory effect is partly related to the inhibition of IL-1ß, IL-10, and nitric oxide releases, but independent of TNF-α and IL-17 modulation. Phytochemical analysis revealed the predominant presence of the flavonoids (naringin, rutin, myricetin, morin, quercetin, (±)-naringenin, and luteolin) and phenols (ellagic acid), which are possibly involved in the anti-inflammatory effect of HEMl. The current study provided supportive evidence for the popular use of HEMl in the treatment of inflammatory conditions, and shed more light on the possible roles of the inflammatory cytokines in its mechanisms of action as anti-inflammatory agent.

Evaluation of acute toxicity, antibacterial activity, and mode of action of the hydroethanolic extract of Piper umbellatum L.

ETHNOPHARMACOLOGICAL RELEVANCE: Piper umbellatum L., Piperaceae, is a shrub that grows up to 3m high. It is commonly known as "capeba" or "pariparoba" in Brazil. Tea prepared using the leaves of this plant is employed in the treatment of infections and inflammatory processes in different countries. Approximately 50 compounds, notably from the flavonoid, alkaloid, terpene, and sterol classes, have been isolated from the leaves of Piper umbellatum. To evaluate the acute toxicity, antibacterial activity, and mode of action of the hydroethanolic extract of Piper umbellatum leaves (HEPu). MATERIALS AND METHODS: Acute toxicity of HEPu against CHO-K1 cells was evaluated using a cytotoxicity assay with Alamar Blue and that against mice was assessed by the Hippocratic test. Antibacterial activity of HEPu was tested using the broth microdilution method using a panel of clinically relevant bacteria, and the effects of HEPu on the bacterial membrane were analyzed in detail. A preliminary phytochemical analysis based on coloration/precipitation was performed according to procedure described in the literature. Secondary metabolites detected were analyzed and confirmed by thin layer chromatography (TLC), spectrophotometry, and high performance liquid chromatography (HPLC). RESULTS: Piper umbellatum did not appear to be toxic in the in vitro (IC50>200 µg/mL) cytotoxicity test. When administered in vivo at doses up to 2000 mg/kg p.o., HEPu did not cause any signs or symptoms of toxicity in mice. It demonstrated a good spectrum of antibacterial activity and its mode of action appeared to be associated with changes in the permeability of bacterial membranes; it led to increased entry of hydrophobic antibiotics, efflux of K(+), and nucleotide leakage. Preliminary phytochemical analysis revealed the presence of flavonoids, alkaloids, terpenes, and sterols in the extract. Spectrophotometric and HPLC analysis revealed the presence of the flavonoids rutin and quercetin. CONCLUSION: In summary, HEPu has antibacterial activity and low acute toxicity in vitro and in vivo. Its mode of action appears to be associated with changes in the permeability of the bacterial cell wall and cytoplasmic membrane, which can at least be partly attributed to the flavonoids present in the extract.

Ethnopharmacology of medicinal plants of the pantanal region (mato grosso, Brazil).

Traditional knowledge is an important source of obtaining new phytotherapeutic agents. Ethnobotanical survey of medicinal plants was conducted in Nossa Senhora Aparecida do Chumbo District (NSACD), located in Poconé, Mato Grosso, Brazil using semi-structured questionnaires and interviews. 376 species of medicinal plants belonging to 285 genera and 102 families were cited. Fabaceae (10.2%), Asteraceae (7.82%) and Lamaceae (4.89%) families are of greater importance. Species with the greater relative importance were Himatanthus obovatus (1.87), Hibiscus sabdariffa (1.87), Solidago microglossa (1.80), Strychnos pseudoquina (1.73) and Dorstenia brasiliensis, Scoparia dulcis L., and Luehea divaricata (1.50). The informant consensus factor (ICF) ranged from 0.13 to 0.78 encompassing 18 disease categories,of which 15 had ICF greater than 0.50, with a predominance of disease categories related to injuries, poisoning and certain other consequences of external causes (ICF = 0.78) having 65 species cited while 20 species were cited for mental and behavioral disorders (ICF = 0.77). The results show that knowledge about medicinal plants is evenly distributed among the population of NSACD. This population possesses medicinal plants for most disease categories, with the highest concordance for prenatal, mental/behavioral and respiratory problems.

Pharmacological mechanisms underlying the anti-ulcer activity of methanol extract and canthin-6-one of Simaba ferruginea A. St-Hil. in animal models.

RELEVANCE: Simaba ferruginea A. St-Hil. (Simaroubaceae) is a subshrub typical of the Brazilian Cerrado, whose rhizomes are popularly used as infusion or decoction for the treatment of gastric ulcers, diarrhea and fever. AIM OF THE STUDY: To evaluate the pharmacological mechanism(s) of action of the antiulcer effects of the methanol extract of Simaba ferruginea and its alkaloid canthin-6-one. MATERIALS AND METHODS: Rhizome of Simaba ferruginea was macerated with methanol to obtain the methanol extract (MESf) from which was obtained, the chloroform fraction. Canthin-6-one alkaloid (Cant) was purified and then isolated from the chloroform fraction (CFSf). The isolated Cant was identified by HPLC. Anti-ulcer assays were determined using ethanol and indomethacin-induced ulcer models in mice and rats respectively. In order to determine the probable mechanisms of actions of MESf and Cant animals were pretreated with l-NAME prior to anti-ulcer agent treatments and ulcer induction and nitric oxide (NO) level determined in order to assess NO involvement in the gastroprotective effects. Assays of malondialdehyde (MDA), myeloperoxidase (MPO), pro-inflammatory cytokines: interleukin 8 (IL-8) and tumor necrosis factor-alpha (TNF-α) and prostaglandin E(2) (PGE(2)) were also carried out according to previously described methods. RESULTS: The results indicate that the antiulcerogenic effects of MESf and Cant in ethanol-induced ulcer is mediated in part through increase in the production of protective endogenous NO as the antiulcerogenic activity of MESf and Cant was reduced in animals pre-treated with l-NAME. In indomethacin-induced ulcer pre-treatment with MESf and Cant showed reduction in the levels of MPO and MDA in the gastric tissue, thus indicating the participation of the antioxidant mechanisms on the gastroprotective effects. The plasma levels of IL-8 in ulcerated rats with indomethacin were also reduced by Cant, but not by MESf, indicating that inhibition of this cytokine contributes to the gastroprotective effect of Cant. However MESf and Cant had no effect on the mucosal membrane levels of PGE(2), indicating that the gastroprotective effects of these agents is independent of PGE(2) modulation. CONCLUSION: The results obtained in this study with MESf and Cant added insights into the pharmacological mechanisms involved in their mode of antiulcer action. The results indicate that Cant is one of the compounds responsible for these effects. Such findings are of extreme importance in the strive for future development of potent, safer and effective antiulcer agent. The efficacy of MESf and Cant in gastroprotection shows that Simaba ferruginea might be a promising antiulcer herbal medicine, in addition to confirming the popular use of this plant against gastric ulcer models utilised in this study.

Phytochemical and antiulcerogenic properties of rhizomes from Simaba ferruginea St. Hill. (Simaroubaceae).

Simaba ferruginea (Simaroubaceae) is a Brazilian medicinal plant used in traditional medicine to treat several ailments, including gastric ulcers, fever, diarrhea, and dolorous and inflammatory processes. This study examines the chemical composition and antiulcerogenic effects of rhizomes from this plant. Bioassay-guided fractionation led to the isolation of two bioactive indole alkaloids called canthin-6-one (1) and 4-methoxycanthin-6-one (2). The alkaloid fraction and both alkaloids demonstrated potent antiulcerogenic effects when evaluated in gastric lesion-induced animals, as well as significant antinociceptive activity in mice. These results confirm and justify the popular use of S. ferruginea against gastric ulcers and dolorous processes.