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

Abstract

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.

Keywords: Helicobacter pylori; Solanum granuloso-leprosum; antimicrobial proteolytic enzyme; granulosain; natural adjuvant against H. pylori; safe food additive.

Figure 1

Native PAGE and zymogram of partially purified proteolytic extract (RAP) of Solanum granuloso-leprosum and granulosain I. Lane 1, RAP, stained gel with Coomassie brilliant blue-R-250; Lane 2, zymogram of RAP; Lane 3, zymogram of granulosain I; Lane 4, granulosain I, stained gel with Coomassie brilliant blue-R-250.

Figure 2

(A) Electrophoretic gels: Helicobacter pylori amplicons resulting from RT-PCR in 1.8% agarose gels stained with Gel Red®. (T) Treated cultures and (UT) untreated cultures of H. pylori with the partially purified proteolytic extract (RAP) of the fruits of Solanum granuloso-leprosum and granulosain I. (B) Relative quantification of the expression of the H. pylori pathogenic factors, before and after treating H. pylori cultures with RAP and granulosain I. *Represents significant differences (p ≤ 0.05) with respect to 16S rRNA, according to the Student's t-test.

Figure 3

Synergistic effect against Helicobacter pylori after combining the partially purified proteolytic extract (RAP) of the fruits of Solanum granuloso-leprosum or granulosain I and antibiotics such as (A) AML, (B) CLA, (C) LEV and (D) MTZ.

Figure 4

Citotoxicity assays. Effect of the partially purified proteolytic extract (RAP) of the fruits of Solanum granuloso leprosum and the main purified fraction (granulosain I) on: (A) The activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and (B) Creatinine in serum. Results represent the Mean ± SD of three independent experiments. PBS was administered to the control group.