Growth-Inhibiting, Bactericidal, and Urease Inhibitory Effects of Paeonia lactiflora Root Constituents and Related Compounds on Antibiotic-Susceptible and -Resistant Strains of Helicobacter pylori Luong Thi My Ngan, † Joon-Kwan Moon, § Takayuki Shibamoto, ∥ and Young-Joon Ahn* ,‡ † Interdisciplinary Program in Agricultural Biotechnology, College of Agriculture and Life Sciences, and ‡ WCU Biomodulation Major, Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Republic of Korea § Department of Plant Life and Environmental Sciences, Hankyong National University, Ansung 456-749, Republic of Korea ∥ Department of Environmental Toxicology, University of California, Davis, California 95616, United States ABSTRACT: An assessment was made of the growth-inhibiting, bactericidal, and urease inhibitory activities of paeonol (PA), benzoic acid (BA), methyl gallate (MG), and 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose (PGG) identified in Paeonia lactiflora root, structurally related compounds, and four antibiotics toward three reference strains and four clinical isolates of Helicobacter pylori using broth dilution bioassay and Western blot. BA and PA showed strong bactericidal effect at pH 4, while MG and PGG were effective at pH 7. These constituents exhibited strong growth-inhibiting and bactericidal activity toward the five strains resistant to amoxicillin (minimal inhibitory concentration (MIC) 12.5 mg/L), clarithromycin (64 mg/L), metronidazole (64 mg/ L), or tetracycline (15 mg/L), indicating that these constituents and the antibiotics do not share a common mode of action. Structural characteristics, such as types of functional groups and carbon skeleton, and hydrophobicity appear to play a role in determining the anti-H. pylori activity. H. pylori urease inhibitory activity of PGG was comparable to that of acetohydroxamic acid, while MG was less potent at inhibiting urease than thiourea. The UreB band disappeared at 250 mg/L PGG on Western blot, while the UreA bands were faintly visible at 1000 mg/L PGG. These constituents showed no significant cytotoxicity. Global efforts to reduce the level of antibiotics justify further studies on P. lactiflora root-derived materials containing MG, PA, and PGG as potential antibacterial products or lead molecules for the prevention or eradication from humans from diseases caused by H. pylori. KEYWORDS: Helicobacter pylori, Paeonia lactif lora, natural bactericide, structure−activity relationship, antibiotic resistance, urease ■ INTRODUCTION In humans, Helicobacter pylori is highly associated with a number of the most important diseases of the upper gastrointestinal tract, including chronic superficial gastritis, duodenal and gastric ulcers, gastric adenocarcinoma, and non- Hodgkin’s lymphomas of the stomach. 1,2 Infections are prevalent worldwide and common in both developed and developing countries. In developing countries, 70−90% of the population carries H. pylori, while the prevalence of infection in developed countries is lower, ranging from 25% to 50%. 1,3 Most infections by H. pylori are acquired in childhood and persist lifelong if not eradicated effectively. H. pylori eradication has been achieved principally by the use of conventional antibacterial drugs, including potent triple therapies consisting of a mixture of two antibiotics such as amoxicillin, clarithromycin, and/or metronidazole with bismuth or a proton pump inhibitor. Triple therapy has a success rate of 80−90%, 4 but sometimes serious side effects occur, such as taste disturbances, nausea, diarrhea, dyspepsia, headache, and angioedema, 2 as well as disturbance of human gastrointestinal microflora. 5 The cost of combination therapy is also significant. In addition, widespread use of antimicrobial agents has often resulted in the development of resistance. 2,3,6 There is therefore a critical need for the development of new improved antibacterial agents with novel target sites to establish a rational resistance management strategy based on all available information on the extent and nature of resistance in H. pylori because a commercial vaccine is still not available. Natural compounds extracted from plants, particularly higher plants, have been suggested as alternative sources for anti-H. pylori products. This approach is appealing, in part because they constitute a potential source of bioactive chemicals that have been perceived by the general public as relatively safe and often act at multiple and novel target sites, thereby reducing the potential for resistance. 7 Much effort has been focused on plant preparations and their constituents as potential sources of commercial antibacterial products for prevention or eradication of H. pylori. In particular, it was reported that Paeonia lactiflora Pallas (Paeoniaceae) root had growth inhibitory activity toward human intestinal bacteria 8 and antitumor activity. 9 P. lactiflora root is composed of the monoterpene glycosides (albiflorin, benzoylpaeoniflorin, oxypaeoniflorin, and paeoniflorin), the monoterpenes (lactoflorin, paeoniflorigenone, and paeonilac- tones), benzoic acid and its esters, and gallotannins. 9 No information has been obtained concerning the potential of P. lactiflora root-derived materials to control antibiotic-resistant H. pylori, although pharmacological actions of the genus Paeonia have been well described by He et al. 9 Received: March 8, 2012 Accepted: August 14, 2012 Published: August 14, 2012 Article pubs.acs.org/JAFC © 2012 American Chemical Society 9062 dx.doi.org/10.1021/jf3035034 | J. Agric. Food Chem. 2012, 60, 9062−9073