Genetic diversity and verbascoside content in natural populations of Pyrostegia venusta (Ker Gawl.) Miers.

BACKGROUND: Pyrostegia venusta (Ker Gawl.) Miers occurs in threatened biodiversity hotspots of Cerrado and Atlantic forest biomes in Brazil and is used in traditional medicine to treat various respiratory and skin diseases. METHODS AND RESULTS: This study (i) examined the genetic diversity and structure of six natural populations of P. venusta from different Brazilian regions using sequence-related amplified polymorphism (SRAP) markers; and (ii) compared the intra- and inter-populational levels of the bioactive component verbascoside using high-performance liquid chromatography. The population from Nova Mutum, Mato Grosso, presented the highest genetic variability (Nei index H = 0.2759; Shannon index I = 0.4170; 85.14% polymorphic loci), whereas the population from Araxá, Minas Gerais, presented the lowest genetic variability (H = 0.1811; I = 0.2820; 70.27% polymorphic loci). The intra-populational variability (79%) was significantly higher (p = 0.001) than the inter-populational variability (21%). The populations were clustered into two groups but their genetic differentiation was not associated with geographical origin (Mantel test, r = 0.328; p > 0.05). The verbascoside content significantly differed (p > 0.05) among the six populations and between the individuals from each population. The highest verbascoside levels (> 200 µg/mg extract) were detected in populations from Araxá and Serrana, while the lowest verbacoside levels were detected in populations from Paranaíta and Sinop. CONCLUSIONS: This is the first report on the use of SRAP markers to analyze genetic variability in the family Bignoniaceae. Our findings shall help to better understand the genetic and chemical diversity of P. venusta populations, as well as provide useful information to select the most appropriate individuals to prepare phytomedicines.

Anticancer Activities of the Quinone-Methide Triterpenes Maytenin and 22-ß-hydroxymaytenin Obtained from Cultivated Maytenus ilicifolia Roots Associated with Down-Regulation of miRNA-27a and miR-20a/miR-17-5p.

Natural triterpenes exhibit a wide range of biological activities. Since this group of secondary metabolites is structurally diverse, effects may vary due to distinct biochemical interactions within biological systems. In this work, we investigated the anticancer-related activities of the quinone-methide triterpene maytenin and its derivative compound 22-ß-hydroxymaytenin, obtained from Maytenus ilicifolia roots cultivated in vitro. Their antiproliferative and pro-apoptotic activities were evaluated in monolayer and three-dimensional cultures of immortalized cell lines. Additionally, we investigated the toxicity of maytenin in SCID mice harboring tumors derived from a squamous cell carcinoma cell line. Both isolated molecules presented pronounced pro-apoptotic activities in four cell lines derived from head and neck squamous cell carcinomas, including a metastasis-derived cell line. The molecules also induced reactive oxygen species (ROS) and down-regulated microRNA-27a and microRNA-20a/miR-17-5p, corroborating with the literature data for triterpenoids. Intraperitoneal administration of maytenin to tumor-bearing mice did not lead to pronounced histopathological changes in kidney tissue, suggesting low nephrotoxicity. The wide-ranging activity of maytenin and 22-ß-hydroxymaytenin in head and neck cancer cells indicates that these molecules should be further explored in plant biochemistry and biotechnology for therapeutic applications.

Curcuma longa L. ameliorates asthma control in children and adolescents: A randomized, double-blind, controlled trial.

ETHNOPHARMACOLOGICAL RELEVANCE: Roots of Curcuma longa L. are used as medicine for millennia. They possess several pharmacological properties, including anti-inflammatory action, and can be suitable for asthma treatment. AIM OF THE STUDY: We aimed to test the hypothesis that, in children and adolescents with persistent asthma, the administration of powdered roots of C. longa for 6 months, in addition to standard treatment, compared to placebo, will result in better disease control. PATIENTS AND METHODS: We conducted a randomized, double-blind, placebo-controlled, phase II clinical trial. Patients were randomly assigned to receive 30 mg/kg/day of C. longa for 6 months, or placebo. Data were collected prospectively. All patients were categorized for asthma severity and control according to GINA-2016 and underwent pulmonary function tests. RESULTS: Overall, both groups experienced amelioration of their frequency of symptoms and interference with normal activity, but no differences were found between the two treatment groups. However, patients receiving C. longa experienced less frequent nighttime awakenings, less frequent use of short-acting ß-adrenergic agonists, and better disease control after 3 and 6 months. CONCLUSION: The powdered roots of C. longa led to less frequent nighttime awakenings, less frequent use of short-acting ß-adrenergic agonists, and better disease control after 3 and 6 months, when compared to placebo.

Effect of Costus spiralis (Jacq.) Roscoe Leaves, Methanolic Extract and Guaijaverin on Blood Glucose and Lipid Levels in a Type II Diabetic Rat Model.

This study aimed to isolate and identify flavonoids with hypoglycemic activity in Costus spiralis leaves. The methanolic extract (ME) was rich in flavonoids, while the powdered leaves (PL) contained considerable amounts of macro- and microelements. Oral acute treatment of streptozotocin (STZ)-induced diabetic rats for 18 h with the C. spiralis PL, ME and isolated guaijaverin (GUA) lowered glycemia, improved oral glucose tolerance and inhibited liver lipid peroxidation. GUA and ME lowered plasma levels of low-density and non-high density lipoproteins; GUA also lowered total cholesterol levels. PL, ME and GUA did not significantly alter the plasma levels of triglycerides, high-density lipoproteins, very low-density lipoproteins, creatinine and aspartate transaminase, and the total protein levels in the kidney and liver tissues. Therefore, C. spiralis leaves are promising raw materials and rich sources of bioactive flavonoids for the development of novel antidiabetic drugs due to their hypoglycemic, antidyslipidemic and antioxidant actions.

Genetic structure and chemical diversity in natural populations of Uncaria guianensis (Aubl.) J.F.Gmel. (Rubiaceae).

Uncaria guianensis is native to the Amazon and is used traditionally as an anti-inflammatory. Natural populations of the species have declined markedly in recent times because of strong anthropic pressure brought about by deforestation and indiscriminate collection. The aim of the present study was to assess the genetic and chemical diversity among eight natural populations of U. guianensis located in the Brazilian states of Acre, Amapá and Amazonas. A set of four primer combinations was employed in sequence-related amplified polymorphism (SRAP) amplifications of leaf DNA, and the fragments were analyzed in an LI-COR model 4300 DNA Analyzer. Genetic variability within the populations (81%) was substantially greater than that detected between them (19%). The highest percentage of polymorphic loci (90.21%) and the largest genetic variability were observed in the population located in Mazagão, Amapá. Genetic differentiation between populations was high (Fst = 0.188) and the studied populations formed three distinct genetic groups (K = 3). The population located in Assis Brasil, Acre, presented the highest average content of the mitraphylline (0.60 mg/g dry weight,). However, mitraphylline and isomitraphylline not detected in most individuals in the studied populations, and it is questionable whether they should be considered as chemical markers of the species. The genetic data confirm the urgent need for conservation programs for U. guianensis, and for further studies aimed at ascertaining the genetic basis and heritability of alkaloid accumulation.

Atividade antibiofilme de substâncias de Croton urucurana em Staphylococcus aureus isolado de mastite bovina / Antibiofilm activity of Croton urucurana compounds against Staphylococcus aureus isolated from mastitis in dairy cattle

A mastite bovina é a enfermidade que causa maior impacto na produção leiteira, sendo o microrganismo Staphylococcus aureus o mais prevalente. Este gênero possui a capacidade de produzir biofilmes que é um importante mecanismo de resistência aos antibióticos. Considerando a capacidade terapêutica das plantas, a espécie Croton urucurana, nativa do Cerrado, foi alvo do presente estudo, que teve como objetivo avaliar a atividade antibiofilme in vitro do extrato vegetal e substâncias isoladas desta espécie, frente Staphylococcus aureus, isolados de leite de vacas com mastite, bem como dos antibióticos gentamicina e vancomicina. A atividade antibiofilme foi avaliada por meio do cristal violeta e da contagem de unidades formadoras de colônia. As imagens foram obtidas por microscopia eletrônica de varredura. O extrato bruto e frações de C. urucurana apresentaram atividade antibiofilme superior à gentamicina e semelhante à vancomicina, enquanto a substância isolada α-Costol foi significativamente mais ativa quando comparada aos demais tratamentos avaliados, reduzindo cerca de 6 ciclos logarítmicos da população bacteriana em biofilme. Conclui-se que os fitocomplexos e a substância α-Costol isolados de Croton urucurana são promissores no combate a um dos principais agentes etiológicos da mastite bovina.(AU)

Mastitis in dairy cattle is the disease that impacts dairy production the most; Staphylococcus aureus is the main causative agent of this condition. The genus Staphylococcus has the ability to produce biofilms, an important mechanism of antibiotic resistance. Bearing in mind that plants have therapeutic action, this study investigated the in vitro antibiofilm activity of the plant extract and compounds isolated from the species Croton urucurana, native to the Brazilian Cerrado, against Staphylococcus aureus isolated from the milk of cows with mastitis, as well as the antibiotic gentamycin and vancomicyn. The antibiofilm activity was evaluated by means of violet crystal and the counting of Colony Forming Units. The images were obtained by scanning electron microscopy. The C. urucurana crude extract and fraction displayed better antibiofilm effect than gentamycin; their antibiofilm action was similar to the action of vancomycin. Compared with all the assessed treatments, the isolated compound α-Costol was significantly more active it reduced six logarithmic cycles of the bacterial population composing the biofilm. The phytocomplexs and the α-Costol substance isolated from Croton urucurana are promising in the fight against one of the main etiological agents of bovine mastitis.(AU)

Costus spiralis (Jacq.) Roscoe: A Novel Source of Flavones with α-Glycosidase Inhibitory Activity.

Costus spiralis, a plant used in traditional Brazilian medicine for the treatment of complications in diabetes, was investigated. Assay of hexane, ethyl acetate, methanol, and aqueous fractions obtained by partition of a crude methanol extract of dried leaves of C. spiralis revealed that AGI activity was confined to the ethyl acetate fraction. Purification of this fraction yielded schaftoside and isoschaftoside. The AGI activities of the two flavones were lower than, but comparable with, that of the anti-diabetic drug acarbose. In contrast, the IC50 value of the ethyl acetate fraction was 1.95-, 2.34-, and 2.22-fold higher than those of acarbose, schaftoside, and isoschaftoside, respectively. The results demonstrate for the first time that schaftoside and isoschaftoside are responsible, in part, for the AGI activity of C. spiralis. Our study suggests that further investigations into C. spiralis may lead to the discovery of additional compounds with antihyperglycemic activity.

Genetic and chemical diversity of Uncaria tomentosa (Willd. ex. Schult.) DC. in the Brazilian Amazon.

Uncaria tomentosa (Willd. ex Schult.) DC., a plant native to the Amazon region, is used widely in popular medicine and by the pharmaceutical industry because of its anti-inflammatory activity. However, the survival of this species is endangered by deforestation and indiscriminate collection, and a preservation plan is urgently required. The objectives of this study were to determine the genetic and chemical variability between and within eight populations of U. tomentosa from the Brazilian states of Acre, Pará and Amapá, and to investigate possible correlations between genetic and geographical distances, and between geographical distances or altitude and the accumulation of bioactive oxindole alkaloids. Three sequence-related amplified polymorphism (SRAP) markers were employed to fingerprint genomic DNA, and the amounts of mitraphylline and isomitraphylline in leaf samples were established by high-performance liquid chromatography. Although significant divergence existed between the tested populations (FST = 0.246), the largest genetic diversity and the highest percentage of polymorphism (95.68%) was found within the population from Mâncio Lima, Acre. Gene flow was considered rather limited (Nm = 1.57), and no correlations between genetic and geographical distances were detected, suggesting that population structure followed an island model. Accumulations of mitraphylline and isomitraphylline varied in the range 32.94 to 0.57 and 3.75 to 0.36 mg g-1 dry weight, respectively. The concentration of isomitraphylline was positively influenced by altitude, such that the population collected at the site with the highest elevation (Tarauacá, Acre) exhibited the greatest alkaloid content. SRAP markers were very efficient in fingerprinting genomic DNA from U. tomentosa populations and clearly showed that genetic variability within populations was greater than between populations. A conservation and management plan should prioritize the creation of germplasm banks to prevent the loss of existing genetic variability, particularly within alkaloid-rich populations such as those of Tarauacá.

Mevalonate-derived quinonemethide triterpenoid from in vitro roots of Peritassa laevigata and their localization in root tissue by MALDI imaging.

Biosynthetic investigation of quinonemethide triterpenoid 22ß-hydroxy-maytenin (2) from in vitro root cultures of Peritassa laevigata (Celastraceae) was conducted using (13)C-precursor. The mevalonate pathway in P. laevigata is responsible for the synthesis of the quinonemethide triterpenoid scaffold. Moreover, anatomical analysis of P. laevigata roots cultured in vitro and in situ showed the presence of 22ß-hydroxy-maytenin (2) and maytenin (1) in the tissues from transverse or longitudinal sections with an intense orange color. MALDI-MS imaging confirmed the distribution of (2) and (1) in the more distal portions of the root cap, the outer cell layers, and near the vascular cylinder of P. laevigata in vitro roots suggesting a role in plant defense against infection by microorganisms as well as in the root exudation processes.

An ent-kaurane-type diterpene in Croton antisyphiliticus mart.

Croton antisyphiliticus is a medicinal plant widely used in the treatment of microbial infections, especially those affecting the genital tract. Crude extract, fractions and pure compound isolated from roots of this species were investigated to validate their antimicrobial activity against Escherichia coli and Staphylococcus aureus. The compound ent-kaur-16-en-18-oic acid was isolated as a major component (0.7% of crude extract), and its MIC value determined against S. aureus (ATCC 6538) was 250 µg/mL. This is the first phytochemical work on the species monitored with antimicrobial assay.

Antimicrobial activities of indole alkaloids from Tabernaemontana catharinensis.

Tabernaemontana catharinensis root bark ethanol extract, EB2 fraction and the MMV alkaloid (12-methoxy-4-methylvoachalotine) were evaluated for their antimicrobial activities. T. catharinensis ethanol extract was effective against both strains of the dermatophyte Trichophyton rubrum at concentrations of 2.5 mg/mL (wild strain) and 1.25 mg/mL (mutant strain), while the EB2 fraction and MMV alkaloid showed a strong antifungal activity against wild and mutant strains with MIC values of <0.02 and 0.16 mg/mL, respectively. The EB2 fraction showed a strong antibacterial activity against ATCC strains of S. aureus, S. epidermidis, E. coli and P. aeruginosa with MICs from <0.02 to 0.04 mg/mL, as well as against resistant clinical isolates species of Enterococcus sp, Klebsiella oxytoca, Citrobacter, K. pneumoniae, P. mirabilis, S. aureus, S. epidermidis, E. coli and P. aeruginosa with MIC values ranging from 0.04 to 0.08 mg/mL. The MMV alkaloid presented a MIC of 0.16 mg/mL against the strains of S. aureus and E. coli ATCC. For the resistant clinical isolates Enterococcus sp, Citrobacter, S. aureus, S. epidermidis, E. coli and P. aeruginosa the MIC of MMV ranged from 0.08 to 0.31 mg/mL. The chromatography analysis of the EB2 fraction revealed the presence of indole alkaloids, including MMV, possibly responsible for the observed antimicrobial activity.

Molecular evaluation of the incidence of Ehrlichia canis, Anaplasma platys and Babesia spp. in dogs from Ribeirão Preto, Brazil.

Canine monocytic ehrlichiosis caused by Ehrlichiacanis is endemic in many regions of Brazil. Since thrombocytopenia is a common finding in infected dogs, many clinicians tend to use it as an indication for antibiotic treatment. Polymerase chain reaction (PCR) and nested PCR were used to study the presence of E. canis, Anaplasma platys and Babesia spp. in thrombocytopenic and non-thrombocytopenic dogs from Ribeirão Preto, Brazil. Despite the high prevalence of E. canis infection among thrombocytopenic dogs, 46.7% of the thrombocytopenic dogs studied were either infected with Babesia spp. or A.platys or not infected with any of the three pathogens. There was a high incidence (25.4%) of E. canis infection in non-thrombocytopenic dogs. Although infection with E. canis should be considered in thrombocytopenic dogs, the final diagnosis needs to be confirmed by complementary tests such as blood smears and PCR to avoid the unnecessary use of antibiotics.

Antihemorrhagic, antinucleolytic and other antiophidian properties of the aqueous extract from Pentaclethra macroloba.

Several Brazilian plants have been utilized in folk medicine as active agents against various effects induced by snake venoms. The inhabitants of the Amazon region use, among others, the macerated bark of a plant popularly named "Pracaxi" (Pentaclethra macroloba Willd) to combat these effects. We report now the antihemorrhagic properties against snake venoms of the aqueous extract of Pentaclethra macroloba (EPema). EPema exhibited full inhibition of hemorrhagic and nucleolytic activities induced by several snake venoms. Additionally, partial inhibition of myotoxic, lethal, phospholipase and edema activities of snake venoms and its isolated PLA(2)s by EPema is reported. In vivo tests showed that EPema is able to totally inhibit a Bothrops jararacussu metalloprotease (BjussuMP-I) induced hemorrhage, suggesting interaction of the extract compounds with this high molecular weight protein. The extract did induce neither hemorrhage nor death in mice when administered alone by i.m. route. When administered separately by i.m. route, the extract did not induce death in mice at 12.5--300 mg/kg doses. Other assays demonstrated that EPema was unable to inhibit fibrinogenolytic and coagulant activities of Bothrops atrox venom. Although the mechanism of action of EPema is still unknown, the finding that no visible change was detected in the electrophoretic pattern of snake venom after incubation with the extract excludes proteolytic degradation as a potential mechanism. The search for new inhibitors of venom metalloproteases and DNAases are a relevant task. Investigation of snake venom inhibitors can provide useful tools for the elucidation of the action mechanisms of purified toxins. Furthermore, these inhibitors can be used as molecular models for development of new therapeutical agents in the treatment of ophidian accidents.