Aqueous extract from Ipomoea asarifolia (Convolvulaceae) leaves and its phenolic compounds have anti-inflammatory activity in murine models of edema, peritonitis and air-pouch inflammation.

ETHNOPHARMACOLOGICAL RELEVANCE: Ipomoea asarifolia (Desr.) Roem. and Schult.(Convolvulaceae), popularly known as salsa or salsa-brava, is a plant of which the decoction of leaves is used in folk medicine to treat various inflammatory disorders such of dermatitis, scabies, symptoms of syphilis, skin ulcers and external wounds. However, little is known about possible compounds and mechanisms of action of the plant to support the activities reported by popular use. AIM OF THE STUDY: The study aimed to identify bioactive molecules present in the crude extract of I. asarifolia leaves and investigate the anti-inflammatory potential of this extract in different experimental in vivo models to improve the understanding on that activity. MATERIAL AND METHODS: Aqueous extract of I. asarifolia leaves was prepared by decoction (1:10 m/v) and its chromatographic profile was obtained by high performance liquid chromatography coupled with diode array detector (HPLC-DAD) and liquid chromatography diode array detector coupled with mass spectrometry (LC-DAD-MS). The potential anti-inflammatory activity of the extract was assessed using the following in vivo models: xylene-induced ear edema (20, 30 and 40mg/kg), evaluating the degree of edema formation; carrageenan-induced peritonitis (10, 20 and 30mg/kg), evaluating leukocyte migration and cytokine levels (IL-1ß, IL-6, IL-12 and TNF-α) at 4h; zymosan-induced air pouch inflammation (20, 30 and 40mg/kg), evaluating the kinetics of leukocyte migration by total and differential counts at 6, 24 and 48h. The same tests were conducted using pure compounds identified in the aqueous extract from I. asarifolia leaves in different doses for each experimental model. RESULTS: The compounds identified in the aqueous extract of I. asarifolia leaves by HPLC-DAD and LC-DAD-MS were rutin, chlorogenic acid and caffeic acid. The extract significantly reduced ear edema induced by xylene (81%, 85% and 86% for doses of 20, 30 and 40mg/kg, respectively, p<0.001), as well as cell migration in experimental models of peritonitis (70%, 78% and 83% for doses of 10, 20 and 30mg/kg, respectively, p<0.001) and air pouch inflammation (58%, 67% and 53% for doses of 20, 30 and 40mg/kg, respectively, p<0.001). In addition, the extract demonstrated the ability to significantly inhibit the production of cytokines IL-1ß, IL-6, IL-12 and TNF-α (p<0.001). The secondary metabolites tested (rutin, chlorogenic acid and caffeic acid) also showed the ability to significantly (p<0.001) decrease the parameters analyzed above. CONCLUSION: This is the first study to identify and confirm these phenolic compounds in I. asarifolia leaves extract and to suggest that these compounds contribute to the anti-inflammatory activity in vivo, as reported by ethnomedicinal use of this plant. Through the different experimental models performed, we can conclude that the results obtained with the aqueous extract from I. asarifolia leaves support its popular use for the treatment of inflammatory disorders.

Release of Periplasmic Nucleotidase Induced by Human Antimicrobial Peptide in E. coli Causes Accumulation of the Immunomodulator Adenosine.

Previous work by our group described that human ß-defensin-2 induces accumulation of extracellular adenosine (Ado) in E. coli cultures through a non-lytic mechanism causing severe plasmolysis. Here, we investigate the presence of AMP as a direct precursor and the involvement of a bacterial enzyme in the generation of extracellular Ado by treated bacteria. Following hBD-2 treatment, metabolites were quantified in the supernatants using targeted HPLC-MS/MS analysis. Microbial growth was monitored by optical density and cell viability was determined by colony forming units counts. Phosphatase activity was measured using chromogenic substrate pNPP. The results demonstrate that defensin-treated E. coli strain W releases AMP in the extracellular space, where it is converted to Ado by a bacterial soluble factor. An increase in phosphatase activity in the supernatant was observed after peptide treatment, similar to the effect of sucrose-induced osmotic stress, suggesting that the periplasmic 5'nucleotidase (5'-NT) is released following the plasmolysis event triggered by the peptide. Ado accumulation was enhanced in the presence of Co2+ ion and inhibited by EDTA, further supporting the involvement of a metallo-phosphatase such as 5'-NT in extracellular AMP conversion into Ado. The comparative analysis of hBD-induced Ado accumulation in different E. coli strains and in Pseudomonas aeruginosa revealed that the response is not correlated to the peptide's effect on cell viability, but indicates it might be dependent on the subcellular distribution of the nucleotidase. Taken together, these data shed light on a yet undescribed mechanism of host-microbial interaction: a human antimicrobial peptide inducing selective release of a bacterial enzyme (E. coli 5'-NT), leading to the formation of a potent immunomodulator metabolite (Ado).

Structural characterization of a novel peptide with antimicrobial activity from the venom gland of the scorpion Tityus stigmurus: Stigmurin.

A new antimicrobial peptide, herein named Stigmurin, was selected based on a transcriptomic analysis of the Brazilian yellow scorpion Tityus stigmurus venom gland, an underexplored source for toxic peptides with possible biotechnological applications. Stigmurin was investigated in silico, by circular dichroism (CD) spectroscopy, and in vitro. The CD spectra suggested that this peptide interacts with membranes, changing its conformation in the presence of an amphipathic environment, with predominance of random coil and beta-sheet structures. Stigmurin exhibited antibacterial and antifungal activity, with minimal inhibitory concentrations ranging from 8.7 to 69.5µM. It was also showed that Stigmurin is toxic against SiHa and Vero E6 cell lines. The results suggest that Stigmurin can be considered a potential anti-infective drug.

Human ß-defensin 2 induces extracellular accumulation of adenosine in Escherichia coli.

Human ß-defensins are host defense peptides performing antimicrobial as well as immunomodulatory functions. The present study investigated whether treatment of Escherichia coli with human ß-defensin 2 could generate extracellular molecules of relevance for immune regulation. Mass spectrometry analysis of bacterial supernatants detected the accumulation of purine nucleosides triggered by ß-defensin 2 treatment. Other cationic antimicrobial peptides tested presented variable outcomes with regard to extracellular adenosine accumulation; human ß-defensin 2 was the most efficient at inducing this response. Structural and biochemical evidence indicated that a mechanism other than plain lysis was involved in the observed phenomenon. By use of isotope ((13)C) labeling, extracellular adenosine was found to be derived from preexistent RNA, and a direct interaction between the peptide and bacterial nucleic acid was documented for the first time for ß-defensin 2. Taken together, the data suggest that defensin activity on a bacterial target may alter local levels of adenosine, a well-known immunomodulator influencing inflammatory processes.

Prosthecate sphingomonads: proposal of Sphingomonas canadensis sp. nov.

Two stalked, aerobic, catalase- and oxidase-positive rod-shaped isolates, VKM B-1508 ( = CB 258) and FWC47(T), were analysed using a polyphasic approach. While the morphology and the 16S rRNA gene sequence of strain VKM B-1508 were 100% identical to the ones of Sphingomonas leidyi DSM 4733(T), the morphology of FWC47(T) was different, and the closest recognized species were Sphingomonas oligophenolica S213(T) ( = DSM 17107(T)) and Sphingomonas leidyi DSM 4733(T) with 97.2% and 97.0% 16S rRNA gene sequence similarity, respectively. DNA-DNA hybridization studies supported the differentiation of strain FWC47(T) from S. oligophenolica and S. leidyi. Strain FWC47(T) grew optimally at 28-30 °C, and pH 6.0-8.0. The major respiratory quinone was Q10 and the major polyamine was sym-homospermidine. The major fatty acids were C(17:1)ω6c and C(18:1)ω7c and C(15:0) 2-OH was the major 2-hydroxy fatty acid. The major polar lipids were phosphatidylglycerol, diphosphatidylglycerol, phosphatidyldimethylethylamine and unidentified sphingoglycolipids. The G+C content of the genomic DNA of strain FWC47(T) was 67.1 mol%. Strain FWC47(T) differed from S. leidyi by its ability to assimilate l-alanine, maltose and sucrose, by the presence of ß-galactosidase and α-chymotrypsin, and the lack of valine arylamidase and ß-glucosidase activities. Contrary to S. leidyi, FWC47(T) did not reduce nitrate and could not use fructose, acetate and N-acetyl-glusosamine. In the genus Sphingomonas, the dimorphic life cycle involving a prosthecate sessile and a flagellated swarmer cell was hitherto only known from Sphingomonas leidyi. Therefore, strain FWC47(T) represents an additional distinct prosthecate species in this genus for which the name Sphingomonas canadensis is proposed. The type strain is FWC47(T) ( =LMG 27141(T) =CCUG 62982(T)).

Vitellin- and hemoglobin-digesting enzymes in Rhipicephalus (Boophilus) microplus larvae and females.

The aim of the present study was to address the involvement of Rhipicephalus microplus larval cysteine endopeptidase (RmLCE) in protein digestion in R. microplus larvae and adult females. In this work, an improved purification protocol for native RmLCE was developed. Partial amino acid sequence of the purified enzyme indicates that it is the same enzyme as Boophilus microplus cathepsin-L1 (BmCL1). When vitellin (Vt) degradation by egg and larval enzymes was analyzed, stage-specific differences for RmLCE activity in comparison to vitellin-degrading cysteine endopeptidase (VTDCE) were observed. RmLCE is also able to degrade host hemoglobin (Hb). In agreement, an acidic cysteine endopeptidase activity was detected in larval gut. It was shown that cysteine and aspartic endopeptidases are involved in Vt and Hb digestion in R. microplus larvae and females. Interestingly, we observed that the aspartic endopeptidase Boophilus yolk cathepsin (BYC) is associated with a cysteine endopeptidase activity, in larvae. Synergic hemoglobin digestion by BYC and RmLCE was observed and indicates the presence of an Hb-degrading enzymatic cascade involving these enzymes. Our results suggest that RmLCE/BmCL1 has a continued role in vitellin and hemoglobin digestion during tick development.

Combining Biofilm-Controlling Compounds and Antibiotics as a Promising New Way to Control Biofilm Infections.

Many bacteria grow on surfaces forming biofilms. In this structure, they are well protected and often high dosages of antibiotics cannot clear infectious biofilms. The formation and stabilization of biofilms are mediated by diffusible autoinducers (e.g. N-acyl homoserine lactones, small peptides, furanosyl borate diester). Metabolites interfering with this process have been identified in plants, animals and microbes, and synthetic analogues are known. Additionally, this seems to be not the only way to control biofilms. Enzymes capable of cleaving essential components of the biofilm matrix, e.g. polysaccharides or extracellular DNA, and thus weakening the biofilm architecture have been identified. Bacteria also have mechanisms to dissolve their biofilms and return to planktonic lifestyle. Only a few compounds responsible for the signalling of these processes are known, but they may open a completely novel line of biofilm control. All these approaches lead to the destruction of the biofilm but not the killing of the pathogens. Therefore, a combination of biofilm-destroying compounds and antibiotics to handle biofilm infections is proposed. In this article, different approaches to combine biofilm-controlling compounds and antibiotics to fight biofilm infections are discussed, as well as the balance between biofilm formation and virulence.

Novel approaches to control biofilm infections.

Biofilms are matrix-enclosed microbial aggregations that adhere to biological or non-biological surfaces. They represent a significant and incompletely understood mode of growth for bacteria and fungi. Biofilm infections cause many deaths and high health costs worldwide. Biofilm infections on indwelling devices or implants are difficult to eradicate because of their much better protection against macrophages and antibiotics, compared to free living cells, leading to severe clinical complications often with lethal outcome. One promising approach to combat biofilm infections independent from the conventional control by antibiotics is the generation of functional surfaces preventing the attachment of bacteria. Another aim is the communication machinery used by bacteria to establish a biofilm, the so called quorum-sensing. Here, small diffusible compounds are produced and sensed by the producing cells to measure their concentration and hence cell density. Natural compounds and synthetic analogues have been used successfully to prevent biofilm formation by quorum-quenching. These compounds are still in the preclinical phase, often struggling with toxicity. A principal problem of quorum-quenchers is their high species specificity, resulting in the control of only some pathogenic strains leaving other pathogens untouched. A field still in its infancy is the control of virulence factors expression not preventing the biofilm but suppressing its virulence. This review will give an overview over the pros and cons of the individual targets and an outlook of future developments.