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.

Structural Characterization and Anticancer Activity of a New Anthraquinone from Senna velutina (Fabaceae).

In this study, a novel compound was isolated, identified, and its chemical structure was determined from the extract of the roots of Senna velutina. In addition, we sought to evaluate the anticancer potential of this molecule against melanoma and leukemic cell lines and identify the pathways of cell death involved. To this end, a novel anthraquinone was isolated from the barks of the roots of S. velutina, analyzed by HPLC-DAD, and its molecular structure was determined by nuclear magnetic resonance (NMR). Subsequently, their cytotoxic activity was evaluated by the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) method against non-cancerous, melanoma, and leukemic cells. The migration of melanoma cells was evaluated by the scratch assay. The apoptosis process, caspase-3 activation, analysis of mitochondrial membrane potential, and measurement of ROS were evaluated by flow cytometry technique. In addition, the pharmacological cell death inhibitors NEC-1, RIP-1, BAPTA, Z-VAD, and Z-DEVD were used to confirm the related cell death mechanisms. With the results, it was possible to elucidate the novel compound characterized as 2'-OH-Torosaol I. In normal cells, the compound showed no cytotoxicity in PBMC but reduced the cell viability of all melanoma and leukemic cell lines evaluated. 2'-OH-Torosaol I inhibited chemotaxis of B16F10-Nex2, SK-Mel-19, SK-Mel-28 and SK-Mel-103. The cytotoxicity of the compound was induced by apoptosis via the intrinsic pathway with reduced mitochondrial membrane potential, increased levels of reactive oxygen species, and activation of caspase-3. In addition, the inhibitors demonstrated the involvement of necroptosis and Ca2+ in the death process and confirmed caspase-dependent apoptosis death as one of the main programmed cell death pathways induced by 2'-OH-Torosaol I. Taken together, the data characterize the novel anthraquinone 2'-OH-Torosaol I, demonstrating its anticancer activity and potential application in cancer therapy.

Effects of Probiotic Use on Quality of Life and Physical Activity in Constipated Female University Students: A Randomized, Double-Blind Placebo-Controlled Study.

Objectives: To evaluate the effect of a probiotic supplement containing two genera and five species of bacteria versus placebo on the quality of life (QoL) in female university students with intestinal constipation (IC). Design: A randomized, double-blind placebo-controlled study was conducted on female university students in a single study center. Settings/Location: Two phases of interventions were carried out, the pilot and main study. All participants were female students of Federal University of Mato Grosso, Brazil. Subjects: Female students whose ages ranged from 20 to 40 years and self-reported to be suffering from IC based on a questionnaire containing Rome III criteria were included. Interventions: Interventions occurred during a period of 30 days in the pilot phase (n = 32) and 15 days in the main study phase (n = 63). The subjects were numbered and randomly divided into experimental probiotic and placebo control groups. Therefore, neither the participants nor the researchers were aware of the allocations of the treatment groups. Outcome measures: The sociodemographic, Rome III, Patient Assessment of Constipation Quality of Life (PAC-QoL) and International Physical Activity questionnaires, and anthropometric measures were utilized. The relative risk (RR) treatment effect, absolute risk reduction (ARR), RR reduction, number needed to treat (NNT), and odds ratio were calculated. Results: Improvement in the QoL (ARR = 14% and p < 0.01) and satisfaction (ARR = 44% and p < 0.01) according to the PAC-QoL questionnaire was observed in the experimental group compared with the control group. For probiotic supplementation, an NNT = 7 was obtained. This implies that for every seven constipated women treated, a worsening in the QoL is prevented in one. An NNT = 1 was obtained concerning satisfaction in the same group of women with respect to the treatment. No clinically significant observations related to the safety of the product were reported. The authors did not detect the effect of exercise intensity on the QoL of participants. Conclusion: The probiotic supplementation had a positive impact on the QoL of constipated female university students.

Medicinal plants of the Caatinga, northeastern Brazil: Ethnopharmacopeia (1980-1990) of the late professor Francisco José de Abreu Matos.

ETHNOPHARMACOLOGICAL IMPORTANCE: The Caatinga (semi-arid region), is an exclusively Brazilian biome. Considering the scarcity of ethnobotanical and ethnopharmacological studies in this region before the year 2000, this study presents data from ethnobotanical expeditions carried out between 1980 and 1990, by the late professor Francisco José de Abreu Matos (1924-2008). The information revealed in this present work are valuable and remained unpublished until now. MATERIALS AND METHODS: The objective was to organize, systematize and analyze ethnobotanical and ethnopharmacological data using ethnobotanical analytical techniques. The most cited native species in each use category were selected for literature review of the pharmacological studies related to their ethnomedicinal uses. RESULTS: Revision of the botanical nomenclature led to the botanical confirmation of 272 plants, of which 84 (30.9%) were reclassified. These represented 71 families and 220 genera that were cited 1957 times. 153 (56.3%) of these plant species are native to Brazil, of which 36 (23.4%) are endemic to the Caatinga. The use reports (RU) associated with these plants, according to the body systems (ICPC-2) in decreasing order of UR and the ICF values were respiratory system (93 species, 407 UR, ICF 0.77), digestive system (119 species, 373 UR, ICF 0.68), general and nonspecific symptoms (95 species, 219 UR, ICF 0.58), female genital system (60 species, 184 UR, ICF 0.68), skin (71 species, 156 UR, ICF 0.55), cardiovascular (50 species, 99 UR, ICF 0.50), blood and immune system diseases (46 species, 96 UR, ICF 0.53), urological (44 species, 88 UR, ICF 0.51), musculoskeletal (33 species, 80 UR, ICF 0.60), psychological (21 species, 71 UR, ICF 0.60), while others represent less than 10.0% of the UR. The most cited plants in the disease categories were Dysphania ambrosioides (28), Pombalia calceolaria (28) Hymenaea courbaril (26), Myracrodruon urundeuva (50), Brassica juncea subsp. integrifolia (16), Scoparia dulcis (22), Phyllanthus niruri (14), Egletes viscosa (25), Lippia alba (16), Erythroxylum vacciniifolium (9) and Salvia rosmarinus (21). The most prominent clades of the medicinal plants based on cluster analysis were the Lamiids (Euasterids)-497 UR and the Fabids (Eurosids I) - 468 UR. Association between certain phylogenetic clades and use-category were also observed and discussed. CONCLUSION: This study demonstrated a new approach in ethnopharmacology by mapping plant usages to diseases prevalent in a community from old ethnobotanical travel reports. In addition to revealing the therapeutic potential of Caatinga species using cluster analysis.

Cochlospermum regium (Mart. ex Schrank) Pilg.: Evaluation of chemical profile, gastroprotective activity and mechanism of action of hydroethanolic extract of its xylopodium in acute and chronic experimental models.

ETHNOPHARMACOLOGICAL RELEVANCE: Cochlospermum regium (Bixaceae) is a native shrub of Brazil and its xylopodium (infusion/decoction) is being used for the treatment of gastritis, ulcers, arthritis, intestinal infections, gynaecological infections, skin diseases, among others. The aim of the present study was to evaluate the gastroprotective/antiulcer activity and the mechanism of action of hydroethanolic extract of C. regium xylopodium (HECr), using in vitro and in vivo models. Additionally, phytochemical constituents were identified by high-performance liquid chromatography (HPLC). MATERIALS AND METHODS: C. regium xylopodium was macerated with ethanol/water to obtain the HECr. The phytochemical characterisation was carried out by HPLC. The antiulcer efficacy of HECr (25, 100 and 400 mg/kg, p.o.) was evaluated using acute acidified ethanol (HCl/EtOH), piroxicam and water immersion-induced experimental ulcer models. Chronic gastric ulcer healing activity of HECr was evaluated through acetic acid (99.8%) - induced model. Histological analysis and myeloperoxidase (MPO), glutathione (GSH), catalase (CAT) activities were also evaluated in chronic ulcer induced gastric tissues. The plausible mode of action of the HECr was assessed by estimation of gastric wall mucus production and the role of gastric secretion in pylorus ligature. The animals were also pre-treated with various inhibitors which includes indomethacin (10 mg/kg, p.o.) a selective inhibitor of cyclooxygenase, L-NAME (10 mg/kg, i.p.), an inhibitor of nitric oxide synthase, glibenclamide, a ATP-sensitive potassium channels (K+ATP) blocker (5 mg/kg, p.o.) or yohimbine (2 mg/kg, i.p.), an α2-adrenergic receptor antagonist. In vitro, Helicobacter pylori action was done by broth microdilution method. RESULTS: The HPLC analysis data revealed the presence of gallic acid, rutin, myricetin, morin and kaempferol. HECr promoted protective effect against acute ulcers induced by HCl/EtOH with inhibitions of 47.52% (p < 0.01) and 62.69% (p < 0.001) at 100 and 400 mg/kg, and in piroxicam by 34.11% (p < 0.05), 49.14% (p < 0.01) and 61.34% (p < 0.001), at 25, 100 or 400 mg/kg, respectively, and in water restraint stress by 78.26% inhibition, p < 0.001, at the dose of 400 mg/kg when compared to the vehicle control group respectively. In the chronic gastric ulcer model, HECr (25, 100 and 400 mg/kg p.o.) significantly (p < 0.001) decreased the injured area by 58.80%, 77.87% and 71.10% respectively. Histological examination indicated that oral treatment of HECr promoted healing of gastric lesions by regenerating gastric mucosa layer with less inflammatory cells. HECr augmented the GSH, CAT activities and reduced MPO level. The pre-treatment with HECr increased the gastric wall mucus production. It also significantly altered the gastric secretion parameters by causing the reduction in the gastric juice volume, elevated the pH level and reduced the total acidity at all doses tested when compared with the vehicle group. HECr at the most active dose (100 mg/kg) reversed completely the reduction of PGs, NO production, closure of K+ATP- channels and α2-adrenoreceptor blockage - induced damages. In microdilution assay, the HECr showed good anti-Helicobacter pylori effect with MIC = 100 µg/mL. CONCLUSION: The HECr presented preventive and curative effects in the experimental gastric ulcer models, besides good anti-Helicobacter pylori activity, which supports the traditional medicinal use of the xylopodium of this plant for gastrointestinal diseases. The underlying mechanisms of this antiulcerogenic/antiulcer action involve, at least, augmentation of mucus production, inhibition of gastric secretion, stimulation of PGs and NO synthesis. And that it involves activation of K+ATP channels and α-2-adrenergic receptors, in addition to an antioxidant activity, probably due to the presence of gallic acid and flavonoids in HECr.

Chemical characterization and evaluation of gastric antiulcer properties of the hydroethanolic extract of the stem bark of Virola elongata (Benth.) Warb.

ETHNOPHARMACOLOGICAL RELEVANCE: Virola elongata is a tree species belonging to the Myristicaceae family, distributed in the North and Midwest regions of Brazil, in the phytogeographic domain of the Amazon. The aqueous infusion or the hydroethanolic macerate of the stem bark of V. elongata are used in Brazilian and Ecuadorian indigenous folk medicine for several ethnopharmacological purposes, principally, in the treatment of stomach pain, indigestions, and gastric ulcers. This study was aimed to investigate the gastroprotective activity of this plant in order to support its popular use with scientific evidence. MATERIALS AND METHODS: The stem bark hydroethanolic extract of the plant (HEVe) was prepared by maceration. Its qualitative and quantitative phytochemical constituents were investigated by classical colorimetric techniques, HPLC, and electrospray ionization-multiple stage fragmentation (ESI-MSn). The gastroprotective and antiulcer activity of HEVe at doses of 100, 300 and 900 mg/kg p.o. were tested using three acute (acidified ethanol, piroxicam, and in-water-restrain stress), and one chronic (acetic acid) animal ulcer models. The probable mode of action of the HEVe was evaluated by analyzing gastric acid secretion, mucus content, nitric oxide effect, and its antioxidant properties (on catalase, myeloperoxidase, and GSH content) in experimental rodents. The direct extract's activity on the growth of Helicobacter pylori was also investigated. RESULTS: Total phenolic content in the HEVe was of 146.20 ±â€¯1.07 mg, being flavonoids about 50% (71.79 ±â€¯0.70 mg) of it. Comparative HPLC fingerprint analysis revealed the presence of known phenolic antiulcer compounds, such as gallic acid, catechin, and rutin. Also, methanol/water fractionation and ESI-MSn analysis of the HEVe reveals the presence of quinic acid, 3,3',4-trihydroxystilbene, juruenolid D, one catechin dimer, one C-glycosyl flavonoid, one polyketide and two neolignans as the major components of the extract. The HEVe attenuated gastric ulceration in all the different models of acute gastric ulcer, by enhancing gastroprotection through its antioxidant properties in vivo, and reducing also considerably the gastric secretion and total acidity. The HEVe also presented healing properties against the induced chronic ulceration process. On the other hand, the HEVe did not exhibit direct activity against H. pylori. CONCLUSION: The HEVe exhibited significant gastroprotective/antiulcer effects and contain a relative high proportion of phenolic compounds, especially flavonoids, that could likely account, at least in part, for its pharmacological properties. The results justify its traditional usage and provided scientific evidence for its potential as a new herbal medicine to treat gastric ulcers.

Sphenodesme involucrata var. paniculata (C.B. Clarke) Munir.: Chemical characterization, anti-nociceptive and anti-inflammatory activities of methanol extract of leaves.

ETHNOPHARMACOLOGICAL RELEVANCE: Sphenodesme involucrata var. paniculata (C. B. Clarke) Munir is native as well as endemic to South India. Its leaves are used in folklore medicine to treat pain and rheumatism. OBJECTIVE: This study was aimed to investigate the chemical characterization, anti-nociceptive and mode of action underlying the anti-inflammatory effects of methanol extract of S. involucrata leaves (MESi). METHODS: Phytoconstituents of MESi was analyzed using colorimetric and liquid chromatography-mass spectrometry (LC-MS) methods, and the oral acute toxicity was evaluated in mice up to 2000 mg/kg. The anti-nociceptive effect was evaluated in hot plate and writhing tests; whereas the anti-inflammatory effect was investigated using carrageenan, cotton pellet and lipopolysaccharide (LPS)-induced peritonitis models at doses of 100, 200 and 400 mg/kg. Additionally nitric oxide (NO) and inflammatory cytokines levels were also evaluated. RESULTS: MESi exhibited the high content of phenolics and flavonoids as well as compounds like austricine, benzylglucosinolate, gossypin, justicidin B and cirsimarin were detected in LC-MS. In the acute toxicity study, oral administration of MESi did not cause any toxic effect and mortality up to 2000 mg/kg body weight in mice. In the anti-nociceptive tests, MESi augmented the latency period at higher dose (400 mg/kg), on the other hand attenuated writhings at the dose of 400 mg/kg by 87.87% (p < 0.001). In the carrageenan induced paw oedema MESi significantly inhibited the oedema formation at dose 400 mg/kg by 32.1%; besides, anti-inflammatory effect was registered in the cotton pellets-induced inflammation model at doses 200 and 400 mg/kg by 27.09% (p < 0.001) and 35.47% (p < 0.001) respectively. On the other hand, MESi appreciably reduced leukocyte, neutrophils infiltration, nitric oxide, TNF-α and IL-1ß levels and increased the IL-10 level in the (LPS)-induced peritonitis model. CONCLUSION: The results conclude that MESi has no acute toxic effect and it demonstrated potent anti-nociceptive and anti-inflammatory activities. Its anti-nociceptive activities are probably mediated through peripheral and central mechanisms. The anti-inflammatory effect of MESi involved the inhibition of neutrophils migration and the modulation of Th1 and Th2 cytokines, besides the attenuation of production of PGE2 and NO. LC-MS analysis revealed the predominant presence of the austricine, benzylglucosinolate, gossypin, justicidin B and cirsimarin compounds, which are possibly involved in the anti-nociceptive and anti-inflammatory effects of MESi. The current study provided supportive evidence for the folklore use of S. involucrata in the treatment of pain and inflammatory conditions.

Assessment of toxicity and differential antimicrobial activity of methanol extract of rhizome of Simaba ferruginea A. St.-Hil. and its isolate canthin-6-one.

ETHNOPHARMACOLOGICAL RELEVANCE: Simaba ferruginea A. St.-Hil., Simaroubaceae, popularly known as "calunga" is a typical subtropical shrub used in Central Brazil mainly for infection, anti-inflammatory, analgesic and gastric duodenal-ulcers. It presents in its composition the alkaloid canthin-6-one, an alkaloid indole ß-carboxylic. AIM: This study aims to investigate the toxicity, antimicrobial activities of methanol extract of Simaba ferruginea (MESf) and canthin-6-one by using different experimental models. METHODS: The present study evaluated the phytochemical analysis by high performance liquid chromatography (HPLC), toxicological potential of MESf and canthin-6-one, using the cytotoxicity, genotoxicity assays with CHO-K1 cells and in vivo acute test in mice. Antimicrobial activity was evaluated by the broth microdilution assays, while the antimicrobial mechanism of action was also assessed using different in vitro bacterial and fungal models. RESULTS: The HPLC analysis of MESf revealed the presence of canthin-6-one, kaempferol and morin. Differential in vitro toxicities were observed between MESf and canthin-6-one. In the cytotoxicity assay, MESf presented toxicity against CHO-K1, while canthin-6-one did not. In the case of in vitro genotoxicity, both showed to be potentially genotoxic. In the in vivo toxicity study, both MESf (up to 1000 mg/kg) and cantin-6-one (up to 100 mg/kg) caused no toxicologically relevant alterations and are thus considered not to be toxic. MESf was shown to be relatively safe with NOAEL (100 mg/kg) when administrate in mice. Both MESf and canthin-6-one also showed differential antimicrobial activities. On one hand, MESf demonstrated good spectrum of antibacterial action against Staphylococcus aureus (MIC 12.5 µg/mL) and Escherichia coli (MIC 25 µg/mL) and moderate activity against Enterococcus faecalis and Shigella flexneri (MIC 200 µg/mL) but no antifungal effect. On the hand, canthin-6-one showed no antibacterial activity, except against Staphylococcus aureus (100 µg/mL), but potent in vitro fungicidal activity against clinically important Aspergillus niger and Candida species at MFC intervals ranging from 3.12 to 25 µg/mL. Both MESf and canthin-6-one were bacteriostatic in action. MESf antimicrobial mechanism of actions are associated with changes in the permeability of bacterial membranes, evidenced by the increased entry of hydrophobic antibiotic in Shigella flexneri, intense K+ efflux (Shigella flexneri, Staphylococcus aureus) and nucleotides leakage (Staphylococcus aureus). In the antifungal mode of action, canthin-6-one inhibited Saccharomyces cerevisiae growth and including alteration in the cell membrane of Neurospora crassa. CONCLUSION: The results of this work demonstrated the differential antimicrobial activities of MESf and its alkaloid isolate, canthin-6-one with antibacterial and antifungal activities, respectively. The present study support the popular use of Simaba ferruginea in combatting afflictions related to bacterial infections, and demonstrate that canthin-6-one as a promising antifungal agent. Both MESf and canthin-6-one are considered non-toxic based on the in vitro toxicological study.

Lafoensia pacari A. St.-Hil.: Wound healing activity and mechanism of action of standardized hydroethanolic leaves extract.

ETHNOPHARMACOLOGICAL IMPORTANCE: Lafoensia pacari A. St.-Hil., (Lythraceae) is a native tree of Brazilian Cerrado and commonly known in Brazil as "mangava-brava". Its leaves are used in Brazilian folk medicine in wound healing, cutaneous mycoses, and in the treatment of gastritis and ulcers. AIM OF THE STUDY: The present study was designed to evaluate the wound healing activity and mechanism of action of the hydroethanolic extract of Lafoensia pacari A. St.-Hil. leaves (HELp), and to advance in its chemical profiling. MATERIALS AND METHODS: HELp was prepared by maceration in 70% hydroethanolic solution (1:10, w/v). The phytochemical analyses were investigated using colorimetry and electrospray ionization/mass spectrometric detection (ESI-MSn). Its in vitro cytotoxicity was evaluated in CHO-K1 and L929 cells, while the in vivo acute toxicity was performed in mice. The potential in vivo wound healing activity was assessed using excision and incision rat models and histopathology of the wounded skin (excision model) was carried out. The in vitro wound healing activity of HELp was demonstrated by scratch assay in L-929 cells, by measuring proliferation/migration rate and p-ERK 1/2 protein expression using western blot analysis. HELp's in vivo anti-inflammatory activity was evaluated by lipopolysaccharide (LPS) induced peritonitis in mice, along with the determination of nitric oxide (NO) and cytokines (TNF-α and IL-10) in the peritoneal lavages. Its potential in vitro antibacterial activity was performed using microbroth dilution assay, while in vitro antioxidant activities was by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, and ferric reducing antioxidant power (FRAP) assays. RESULTS: The phytochemical analysis of HELp revealed the presence of polyphenols with ellagic acid, punicalagin, punicalin, kaempferol, quercetin-3-O-xylopyranoside and quercetin-3-O-rhamnopyranoside being the most prominent. HELp showed no toxicity on CHO-k1 and L929 cell lines. Topical treatment with HELp (10 and 30 mg/g of gel) presented increased rates of wound contraction at all the days evaluated with complete wound re-epithelialization at 22.0 ±â€¯1.5 (p < 0.05) and 21.7 ±â€¯1.6 (p < 0.01) days, respectively. Topical application of HELp (10, 30 or 100 mg/g of gel) in incised wounds caused an increase in tensile break strength at all concentrations resulting in moderate re-epithelialization and neovascularization, increased cell proliferation an accelerated remodeling phase of the wound, in a manner comparable to standard drug (Madecassol®, 10 mg/g). In the scratch assay with L929 cells, HELp (0.1 and 0.03 mg/mL) and PDGF (5 ng/mL) resulted in the increased proliferation/migration rate of fibroblasts and higher expression of p-ERK 1/2 protein. In LPS-induced peritonitis, HELp (100 and 200 mg/kg p.o.) decreased total leukocyte migration, comparable to the dexamethasone (0.5 mg/kg p.o.). In RAW 264.7 macrophages activated by LPS, HELp produced anti-inflammatory activity dependent on increased concentrations of IL-10, reduction in NO production, without altering the TNF-α levels. HELp also presented potent antioxidant activity in the DPPH and FRAP, but lacks in vitro antibacterial activity. CONCLUSION: The present study results support the popular use of the leaves of L. pacari in the treatment of wounds. Its wound healing activity is multi-targeted and involves inhibition of the proliferative and anti-inflammatory phases, antioxidant and positive modulation of the remodeling phase that might be involved different secondary metabolites, with emphasis on the ellagic acid, punicalagin, punicalin, kaempferol, quercetin-3-O-xylopyranoside and quercetin-3-O-rhamnopyranoside.

Evaluation of genotoxicity and subchronic toxicity of the standardized leaves infusion extract of Copaifera malmei Harms in experimental models.

ETHNOPHARMACOLOGICAL RELEVANCE: Copaifera malmei Harms (Fabaceae), known mainly as óleo-mirim, is a native and endemic plant found in the states of Mato Grosso and Goiás of Brazil. The plant's leaves infusion is popularly used by riverine communities of the northern Araguaia microregion, Mato Grosso, Brazil, for the treatment of gastric ulcers and inflammatory diseases of the respiratory tract. The gastric antiulcer activity of the standardized leaves infusion extract of Copaifera malmei (SIECm) in rodents has been reported. The objective of this study was to advance the investigation of the safety profile of SIECm by evaluating the genotoxicity and subchronic toxicity using in vitro and in vivo experimental models. MATERIALS AND METHODS: SIECm was prepared by infusion, by incubating the powdered dried leaves material in boiled water for 15min. In vitro genotoxicity of SIECm (10, 30 or 100µg/mL) was assessed by micronucleus and comet tests using Chinese hamster ovary (CHO-k1) epithelial cells. The evaluation of subchronic toxicity profile was performed by daily oral administration of SIECm (100, 400 or 1000mg/kg) to Wistar rats for 30 days. Clinical observations of toxicological related parameters were done every 6 days. After the treatment period, blood was collected for hematological and biochemical analysis, and some organs were removed for macroscopic and histopathological analysis. RESULTS: In the micronucleus assay, SIECm demonstrated anti-mutagenic activity. In the comet assay, SIECm presented anti-genotoxic effect preventing DNA damage at all the three concentrations tested with pre-treatment, while the same effect was only observed in the co-treatment at the lowest concentration. Post-treatment with SIECm increased the genetic damage induced by hydrogen peroxide (H2O2) at the highest concentration. In the subchronic toxicity test, few changes were observed, such as increase in feed consumption in the group of animals treated with 100mg/kg of the SIECm, which reversed after 6 days. There were no macroscopic, histological and relative weights changes in the organs of animals treated with SIECm. No toxicologically relevant changes were observed in the hematological analysis. Subchronic administration of SIECm reduced levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in animals treated with 100mg/kg and serum triglyceride levels at 400 and 1000mg/kg. However, the hematological and biochemical changes observed are within the physiological ranges for this animal species. CONCLUSION: The results demonstrate that SIECm is not genotoxic, and does not present toxicity when used orally for up to 30 days. In addition, it showed protection to the genetic damage induced by H2O2. The SIECm therefore has a high safety margin for therapeutic use.

Vitexin inhibits inflammation in murine ovalbumin-induced allergic asthma.

Vitexin is an important component of various medicinal plants frequently used to treat asthma, such as Crataegus spp., Vitex spp., Passiflora spp., and Echinodorus spp. However, there is no information about the vitexin potential as anti-asthmatic. The aim of the present work was to evaluate the anti-hypersensitive activity of vitexin in a murine ovalbumin (OVA)-induced allergic asthma model. Mice were sensitized to OVA by i.p. injection on days 1st and 10th, followed by a daily challenge with OVA using a nebulizer, from days 19th to 24th. Vitexin or dexamethasone were orally administered 1h before each OVA challenge. Vitexin attenuates migration induced by OVA-hypersensitivity of eosinophil, neutrophil, and mononuclear cells in bronchoalveolar lavage fluid (BALF). Histological analysis of the lungs shows that vitexin suppressed leukocyte infiltration, mucus production and pulmonary edema. Increases in Th2 cytokines in BALF in OVA-induced asthma is also attenuated by vitexin, as well as plasma levels of IgE. Overall, these results suggest that vitexin can suppress OVA-induced allergic inflammation in mice and provide a strong rationale for further developing vitexin as a candidate treatment for allergic hypersensitivity. These data corroborate the popular use of vitexin-rich plants for asthma treatment.

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.

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.

Ocimum gratissimum L. leaf flavonoid-rich fraction suppress LPS-induced inflammatory response in RAW 264.7 macrophages and peritonitis in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Ocimum gratissimum L. is a herbaceous plant that has been reported in several ethnopharmacological surveys as a plant readily accessible to the communities and widely used for the treatment of inflammatory diseases. The main goal of this study was to investigate the in vitro and in vivo anti-inflammatory activity and mechanism of action of the ethylacetate fraction of O. gratissimum leaf (EAFOg) and to chemically characterize this fraction. MATERIALS AND METHODS: EAFOg was obtained from a sequential methanol extract. The safety profile was evaluated on RAW 264.7 cells, using the alamarBlue® assay. Phenolic contents were determined by spectrophotometry, and metabolites quantified by high performance liquid chromatography. The anti-inflammatory activity of EAFOg and its ability to acts on leucocytes infiltration, inflammatory mediators as NO, IL-1ß, TNF-α, and IL-10 in lipopolysaccharide-induced peritonitis in mice and LPS-stimulated RAW 264.7 macrophage were evaluated. In addition, the anti-inflammatory activity of EAFOg was also investigated in arachidonic acid-related enzymes. RESULTS: Total phenolic and flavonoid contents of EAFOg were 139.76±1.07mg GAE/g and 109.95±0.05mg RE/g respectively. HPLC analysis revealed the presence of rutin, ellagic acid, myricetin and morin. The fraction exhibited no cytotoxic effects on the RAW 264.7 cells. The EAFOg (10, 50 and 200mg/kg) significantly reduced (p<0.05) neutrophils (38.8%, 58.9%, and 66.5%) and monocytes (38.9%, 58.0% and 72.8%) in LPS-induced peritonitis. Also, EAFOg (5, 20 and 100µg/mL) produced significant reduction in NO, IL-1ß, and TNF-α in RAW 264.7 cells. However, IL-10 level was not affected by the EAFOg, and it preferentially inhibits COX-2 (IC50 =48.86±0.02µg/mL) than COX-1 and 15-LO (IC50 >100µg/mL). CONCLUSION: The flavonoid-rich fraction of O. gratissimum leaves demonstrated anti-inflammatory activity via mechanisms that involves inhibition of leucocytes influx, NO, IL-1ß, and TNF-α in vivo and in vitro, thus supporting its therapeutic potential in slowing down inflammatory processes in chronic diseases.

Hydroethanolic extract from Echinodorus scaber Rataj leaves inhibits inflammation in ovalbumin-induced allergic asthma.

ETHNOPHARMACOLOGICAL RELEVANCE: Echinodorus scaber, Alismataceae, is popularly known in Brazil as "chapéu-de-couro". The plant leaves are used by the population as decoction, infusion, or maceration in bottled spirits, to treat inflammatory respiratory diseases. AIM OF THE STUDY: To investigate the anti-inflammatory mechanism of the hydroethanolic extract of leaves of Echinodorus scaber (HEEs) in allergic asthma. A phytochemical analysis of the extract was performed as well. MATERIALS AND METHODS: The leaves of Echinodorus scaber were prepared by maceration in 75% ethanol. Preliminary phytochemical analysis was carried out using basic classical methods, and the secondary metabolites detected in HEEs were analyzed and confirmed by high-performance liquid chromatography (HPLC). The in vivo anti-inflammatory activity of HEEs was evaluated in Swiss male albino mice sensitized and challenged by OVA. The HEEs (1, 5 and 30mg/kg, p.o.) was administered to mice twice a day, 1h before the challenge, from days 19 through 24. The mechanism of action of HEEs was studied by evaluating the levels of TH2 cytokines (IL-4, IL-5 and IL-13) in bronchoalveolar lavage fluid (BALF) and IgE production in blood plasma. Histopathological changes triggered by OVA-sensitization/challenge in the lung tissue were also investigated. RESULTS: HEEs reduced total leukocyte, eosinophil, neutrophil, and mononuclear cell counts at all doses tested, with maximum effect at 30mg/kg (73.9%, 75.9%, 75.5%, and 65.2% reduction, p<0.001, respectively). Increases in TH2 cytokine secretion (IL-4, IL-5 and IL-13) and in IgE levels were also attenuated by HEEs. Preliminary phytochemical screening seems to indicated the presence of phenolic compounds, flavonoids and alkaloids. HPLC analyses evidenced the presence of phenolic compounds, such as gallic acid, rutin and vitexin. CONCLUSION: Our findings provided pharmacological preclinical evidence for the popular use of the leaves of Echinodorus scaber in allergic inflammation. Its anti-inflammatory effect was dependent on the decrease in migratory inflammatory cells, and both TH2 cytokines and IgE levels. It is suggested that vitexin, gallic acid and rutin, known anti-inflammatory compounds, may participate in the anti-asthamtic effect of the HEEs, by acting jointly along with other components present in the extract.

Piper umbellatum L.: A medicinal plant with gastric-ulcer protective and ulcer healing effects in experimental rodent models.

ETHNOPHARMACOLOGICAL RELEVANCE: Piper umbellatum L. (Piperaceae) is a shrub found in the Amazon, Savannah and Atlantic Forest region of Brazil. It is widely used in folk medicine in many countries primarily for the treatment of gastric disorders. The aim of this study was to evaluate the gastroprotective and anti-ulcer effects of hydroethanolic extract of P. umbellatum (HEPu) leaves in experimental rodents. In addition, the anti-Helicobacter pylori activity of the extract was assessed. MATERIALS AND METHODS: The leaves of P. umbellatum were macerated in 75% (1:3w/v) hydroethanolic solution to obtain HEPu. The gastroprotective and ulcer healing activities of HEPu were evaluated using acidified ethanol (acute) and acetic acid (chronic) gastric ulcer models in rodents. The anti-H. pylori activity was evaluated by in vitro broth microdilution assay using H. pylori cagA+ and vacA+ strain. The probable mechanism of action of HEPu was evaluated by determining gastric secretory parameters, antioxidant enzyme (catalase), non-protein sulfhydryl (glutathione) and malondialdehyde levels in gastric tissue, including pro-inflammatory (IL-1ß, TNF-a, IL -17, RANTES, IFN-γ and MIP-2) and anti-inflammatory (IL-10) cytokines. RESULTS: HEPu demonstrated potent gastroprotection against acute ulcer induced by acidified ethanol and excellent healing effect of the chronic ulcer induced by acetic acid. The gastroprotective activity in acidified ethanol is partly attributed to the antioxidant mechanisms, while anti-secretory, anti-inflammatory and regeneration of the gastric mucosa are evoked as part of its antiulcer mechanism of action. The gastric ulcer healing of HEPu also involves restoration of the altered cytokines levels to near normal. However, it has no in vitro anti-H. pylori activity. CONCLUSION: The results of this study showed that HEPu possesses preventive and curative effects in experimental models of gastric ulcers in animals. These effects are partially dependent on antioxidant, antisecretory, anti-inflammatory and mucosa regeneration. It is independent of anti-H. pylori activity, with substances probably responsible for the pharmacological activity being flavonoids, quercetin and rutin. These results support the popular use of P. umbellatum leaves in the treatment of peptic ulcers.

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.

Vitexin reduces neutrophil migration to inflammatory focus by down-regulating pro-inflammatory mediators via inhibition of p38, ERK1/2 and JNK pathway.

BACKGROUND: Vitexin is a flavonoid found in plants of different genus such as Vitex spp. and Crataegus spp. Despite being an important molecule present in phytomedicines and nutraceuticals, the mechanisms supporting its use as anti-inflammatory remains unclear. PURPOSE: To investigate the cellular and molecular mechanisms involved in acute anti-inflammatory effect of vitexin with regard to neutrophil recruitment and macrophages activation. METHODS: Anti-inflammatory properties of vitexin were evaluated in four models of neutrophil recruitment. The regulation of inflammatory mediators release was assessed in vivo and in vitro. Vitexin (5, 15 and 30 mg/kg p.o) effects on leukocytes migration to peritoneal cavity induced by zymosan (ZY), carrageenan (CG), n-formyl-methionyl-leucyl-phenylalanine (fMLP) and lipopolysaccharide (LPS) were evaluated in Swiss-Webster mice and the effects on the levels of TNF-α, IL-1ß and IL-10 cytokines, and NO concentration were in the LPS-peritonitis. RAW 264.7 macrophages viability were determined by Alamar Blue assay as well as the capacity of vitexin in directly reducing the concentrations of TNF-α, IL-1ß, IL-10, NO and PGE2. Additionally, vitexin effects upon the transcriptional factors p-p38, p-ERK1/2 and p-JNK were evaluated by western blotting in cells activated with LPS. RESULTS: Vitexin was not cytotoxic (IC50 > 200 µg/ml) in RAW 264.7 and at all doses tested it effectively reduced leukocyte migration in vivo, particularly neutrophils in the peritoneal lavage, independently of the inflammatory stimulus used. It also reduced TNF-α, IL-1ß and NO releases in the peritoneal cavity of LPS-challenged mice. Vitexin had low cytotoxicity and was able to reduce the releases of TNF-α, IL-1ß, NO, PGE2 and increase in IL-10 release by LPS activated RAW 264.7 cells. Vitexin was also able to regulate transcriptional factors for pro-inflammatory mediators, reducing the expression of p-p38, p-ERK1/2 and p-JNK in LPS-elicited cells. CONCLUSIONS: Vitexin presented no in vitro cytotoxicity. Inhibition of neutrophil migration and pro-inflammatory mediators release contributes to the anti-inflammatory activity of vitexin. These effects are associated with the inactivation of important signaling pathways such as p38, ERK1/2 and JNK, which act on transcription factors for eliciting induction of inflammatory response.

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.