Identification of Functional Groups, Total Phenol and Flavonoids Contents, Antioxidant Potential and Antimicrobial Activity of Black Pepper (Piper nigrum L.) Aqueous Extract and Its Interactions with Chloramphenicol and Amphotericin B

 The addition of chemical preservatives increases the shelf life of food products, but prolonged and indiscriminate use of chemical preservatives increases the resistance of microorganisms and the health risks associated with theiruptake. Medicinal plants have a wide variety in the world as well as in Iran. In recent years, the use of natural preservatives such as plant extracts and essential oils, due to their importance and role in controlling the growth of pathogenic microorganisms, has been proposed as an alternative to chemical preservatives. Black pepper is an aromatic medicinal plant. The specific properties of black pepper essential oil, such as its antimicrobial and antioxidant activity, have also been confirmed. Amphotericin B is one of the effective antibiotics for treating infections caused by pathogenic fungi. The mechanism of action of amphotericin B is to destroy fungal cells in such a way that by binding to ergosterol in the cell membrane of fungi, it creates pores and eventually destroys them. One of the most important and common antibiotics used in the treatment of infections caused by pathogenic bacteria is chloramphenicol. This antibiotic is effective against gram-positive and gram-negative bacteria due to its broad spectrum. The aim of this study was to identify bioactive functional groups, antioxidant potential, phenol and total flavonoid compounds and to evaluate the antimicrobial activity of black pepper extract against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus coagulans and Aspergillus niger.

 In this study, the antimicrobial effect of black pepper aqueous extract was investigated against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus coagulans and Aspergillus niger by disc diffusion agar, well diffusion agar, minimum inhibitory concentration, and minimum bactericidal concentration methods. Total phenol and flavonoid contents of the species were determined by Folin-Ciocalteu and AlCl3 assays, respectively. Three biochemical assays, namely 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2-azinobis 3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) free radical scavenging and β- caroten/linoleic acid activity systems, were used to evaluate antioxidant activity. Identification of functional groups as well as the structure of organic compounds in black pepper extract was also performed by Fourier transform infrared spectroscopy (FTIR). To evaluate the synergistic effect of black pepper extract in combination with amphotericin B and chloramphenicol antibiotics, Sub-MIC was used.

 The peaks observed in aqueous black pepper extract confirmed the presence of O-H (3000-3500 cm-1), C-H (2800-3000 cm-1), C=O (1613.62-1633.52 cm-1) and C-O (100.57-1038.82 cm-1) functional groups of bioactive compounds. The total phenol and flavonoids content of the extract were 45.12 mg GAE/g extract and its flavonoid content was 29.66 mg QUE/g extract which had an important role in its antioxidant activity. The aqueous black pepper extract had remarkable DPPH free radical scavenging activity (IC50=32.37 μg/ml), ABTS free radical scavenging activity (IC50=28.45 μg/ml) and beta-carotene bleaching inhibitory effect (46.45%), revealing the electron/hydrogen donating ability of the essential oil. The results of measuring the antimicrobial activity of extract by disk diffusion and agar well showed that black pepper extract showed more antimicrobial effect on gram-positive bacteria Staphylococcus aureus and Bacillus coagulans than gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa. This could be due to the difference in their cell wall structure. Aspergillus niger is the most sensitive species to aqueous black pepper extract. The minimum inhibitory concentrations of extract for Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus coagulans and Aspergillus niger were equal to 32, 16, 4, 8 and 4 mg/ml, respectively. The minimum bactericidal concentration of black pepper extract for two bacterial species, Escherichia coli and Pseudomonas aeruginosa was more than 512 mg/ml. Also, the minimum bactericidal concentration for Staphylococcus aureus and Bacillus coagulans was 128 and 256 mg/ml, respectively, and 128 mg/ml for Aspergillus niger. The results of interaction of black pepper extract with chloramphenicol antibiotic showed that the Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus were synergistic, but antagonism was observed for the gram-positive Bacillus coagulans.