Asclepain cI, a proteolytic enzyme from Asclepias curassavica L., a south American plant, against Helicobacter pylori.

Helicobacter pylori is a Gram negative bacterium most frequently associated with human gastrointestinal infections worldwide. The increasing occurrence of antibiotic-resistant isolates of H. pylori constitutes a challenge. The eradication of the microorganism is currently being considered a "high priority" by the World Health Organization (WHO). In this context, bioactive compounds found in natural products seem to be an effective therapeutic option to develop new antibiotics against the pathogen. In this study, we investigated the effect of asclepain cI, the main purified proteolytic enzyme of the latex of petioles and stems from Asclepia curassavica L. (Asclepiadaceae), a South American native plant, against H. pylori; in order to obtain a natural therapeutic adjuvant and a safe nutraceutical product. Asclepain cI showed antibacterial activity against reference strains and drug-resistant clinical isolates of H. pylori in vitro. A range of minimal inhibitory concentration (MIC) from 1 to 2 µg/ml and minimal bactericidal concentration (MBC) from 2 to 4 µg/ml was obtained, respectively. The action of asclepain cI on the transcription of omp18, ureA, flaA genes showed a significantly decreased expression of the selected pathogenic factors. Furthermore, asclepain cI did not induce toxic effects at the concentrations assayed. Asclepain cI could be considered a highly feasible option to be used as a natural therapeutic adjuvant and a safe nutraceutical product against H. pylori.

Antimicrobial Effect of a Proteolytic Enzyme From the Fruits of Solanum granuloso-leprosum (Dunal) Against Helicobacter pylori.

Helicobacter pylori is a gram-negative, helix-shaped, and microaerophilic bacteria that colonizes the human gastric mucosa, causing chronic infections, gastritis, peptic ulcer, lymphomas associated with lymphoid mucosa tissue, and gastric cancer. H. pylori is considered a Type 1 human carcinogen by WHO. The prevalence of the infection is estimated in more than half of the world population. Treatment of H. pylori infection includes antibiotics and proton pump inhibitors, but the increasing antibiotic resistance promotes the research of novel, more effective, and natural antibacterial compounds. The aim of this work was to study the effect of the partially purified proteolytic extract (RAP) of the fruits from Solanum granuloso-leprosum (Dunal), a South American native plant, and a purified fraction named granulosain I, against H. pylori, to obtain natural food additives for the production of anti-H. pylori functional foods. Furthermore, granulosain I and RAP could be used as natural adjuncts to conventional therapies. Granulosain I and RAP antibacterial activity was evaluated as minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against H. pylori NCTC 11638 (reference strain) and twelve H. pylori wild strains, using a microdilution plating technique (Clinical and Laboratory Standards Institute). All the strains tested were susceptible to granulosain I with MIC from 156.25 to 312.5 µg/mL and MBC from 312.5 to 625 µg/mL, respectively. Besides, all the strains tested were susceptible to the RAP with MIC from 312.5 to 625 µg/mL and MBC from 625 to 1,250 µg/mL, respectively. The effect of granulosain I and RAP on the transcription of H. pylori genes encoding pathogenic factors, omp18, ureA, and flaA, with respect to a housekeeping gene (16S rRNA), was evaluated by RT-PCR technique. The band intensity between pathogenic factors and control gene was correlated under treated or untreated conditions, using the ImageJ program. Granulosain I and RAP significantly decreased the expression of pathogenic factors: omp18, ureA, and flaA. The combined inhibitory effect of granulosain I or RAP and an antibiotic such as, amoxicillin (AML, 10 µg), clarithromycin (CLA, 15 µg), levofloxacin (LEV, 5 µg), and metronidazole (MTZ, 5 µg) was evaluated, using the agar diffusion technique. Granulosain I and RAP showed significant synergistic effect on AML, CLA, and LEV, but no significant effect on MTZ was observed. Besides, granulosain I and RAP did not show toxicological effects at the concentrations studied. Finally, granulosain I and RAP could be used as safe natural food additives and as adjuvants for conventional therapies against H. pylori.

Gastroprotective effects and antimicrobial activity of Lithraea molleoides and isolated compounds against Helicobacter pylori.

ETHNOPHARMACOLOGICAL RELEVANCE: Lithraea molleoides (Vell.) Engl. (Anacardiaceae) is a medicinal plant traditionally used in South America to treat various ailments, including diseases of the digestive system. AIM OF THE STUDY: To evaluate the in vivo antiulcer and antimicrobial activities against Helicobacter pylori of L. molleoides and its isolated compounds. MATERIALS AND METHODS: Methanolic extract 250 and 500 mg/kg, (LmE 250 and LmE 500, respectively) and infusions, 10 g and 20 g en 100mL (LmI 10 and LmI 20, respectively) of L. molleoides was evaluated for antiulcer activity against 0.6N HCl, 0.2N NaOH, 200mg/kg acetilsalicilic acid and absolute ethanol-induced gastric ulcers in rats. The degree of erosion in the glandular part of the stomach was assessed from a scoring system. Acute toxicity in mice was also evaluated. The antiulcer effect of the isolated compounds (catechol, mannitol, rutin, gallic acid, ferulic acid and caffeic acid, 100mg/kg) was evaluated against absolute ethanol-induced gastric ulcers in rats. The anti-Helicobacter pylori activity of L. molleoides and isolated compounds was performed using broth dilution methods. RESULTS: The LmE 250, LmE 500, LmI 10 and LmI 20 produced significant inhibition on the ulcer index in 0.6N HCl, 0.2N NaOH, 200mg/kg acetilsalicilic acid and absolute ethanol- induced gastric ulcers in rats. The isolated compounds, catechol, mannitol, rutin, ferulic acid and caffeic acid were active in absolute ethanol- induced gastric ulcers in rats. L. molleoides and different compounds showed antimicrobial activity in all strains tested. The lowest MIC value (0. 5 µg/mL) was obtained with catechol in six of eleven strains assayed. No signs of toxicity were observed with doses up to 2g/kg in an acute toxicity assay. CONCLUSION: These findings indicate that L. molleoides displays potential antiulcerogenic and antimicrobial activities and the identification of active principles could support the use of this plant for the treatment of digestive affections.

Design of an internal amplification control for a duplex PCR used in the detection of Shiga toxin producing Escherichia coli in pediatric feces.

A conventional PCR targeted directly to the detection of Shiga toxin-producing Escherichia coli (STEC) in diarrheal stools of symptomatic patients may require the introduction of internal controls to detect false negative results. In the present study, we designed a competitive internal amplification control (IAC) to be included in a well-known PCR protocol used to amplify the stx1and stx2 genes from STEC isolates. The IAC was introduced in the PCR reaction and amplified when E. coli O157:H7 cultures and contaminated pediatric feces were assayed. When STEC concentration was 10(3) CFU ml(-1) in pure culture and 10(4) CFU g(-1) in contaminated stools, the IAC at concentration of 0.143 pg µl(-1) in the PCR reaction mixture was co-amplified with the stx2 sequence, producing bands of 279 and 349 bp, respectively. These STEC values were considered the detection limits of the duplex PCR. The specific detection of STEC by duplex PCR including IAC might be achieved directly on pediatric feces when the pathogen load reaches concentrations of at least 10(4) CFU g(-1).