Antibacterial and antibiotic-potentiation activities of the hydro-ethanolic extract and protoberberine alkaloids from the stem bark of Enantia chlorantha against multidrug-resistant bacteria expressing active efflux pumps

Enantia chlorantha is traditionally used to treat various ailments including rickettsia fever, cough and wounds, typhoid fever, infective hepatitis, jaundice, and urinary tract infections.To isolate the antibacterial constituents of the hydro-ethanolic extract of the stem bark of E. chlorantha (ECB) and to evaluate the antibacterial and antibiotic-modifying activities of ECB and its constituents against the multidrug-resistant (MDR) phenotypes.Chromatographic methods were used to isolate the constituents of ECB and Spectroscopic methods were used to elucidate the chemical structures of the isolated compounds. The antibacterial activity of samples was determined by the broth microdilution method while spectrophotometric methods were used to evaluate the effects of ECB and its most active constituent on bacterial growth. Their effects on bacterial proton-ATPase pumps was assessed through the acidification of the bacterial culture medium.Six protoberberine alkaloids were isolated and identified as columbamine (1), pseudocolumbamine (2), jathrorrhizine (3), palmitine (4), 4,13-dihydroxy-3,9,10-trimethoxyprotoberberine (5), and 13-hydroxy-2,3,9,10-tetramethoxyprotoberberine (6). The crude extract (ECB) inhibited the growth of all the tested MDR bacteria, with the minimal inhibitory concentration (MIC) values below 100 μg/mL obtained against Escherichia coli ATCC 10536, AG 102, Enterobacter aerogenes EA 27, Klebsiella pneumoniae ATCC 11296 and KP 55, Providencia stuartii NEA 16, and Staphylococcus aureus MRSA3 and MRSA6. Compound 1 had the best antibacterial effects with MIC values ranging from 16 to 64 μg/mL. The efflux pump inhibitor (EPI), phenylalanine-arginine-β naphthylamide (PAβN) significantly improved the activity of compounds 1-6. Compounds 1-3 significantly potentiated the antibacterial activity of antibiotics such norfloxacin (NOR), ciprofloxacin (CIP), and doxycycline (DOX) against the tested MDR bacteria.The crude extract (ECB) and its isolated compounds 1-6 are potential antibacterial products from Enantia chlorantha. They could be explored more to develop the antibacterial agents that could be used alone or in combination with antibiotics to overcome MDR phenotypes.