Int J Clin Exp Med 2019;12(1):573-580 www.ijcem.com /ISSN:1940-5901/IJCEM0076819 Original Article Inhibitory effect of plumbagin on Helicobacter pylori growth and urease activity Min Ho Lee 1,2* , Hyun Jun Woo 1* , Yoonjung Cho 2 , Cheol Moon 3 , Sa-Hyun Kim 3 , Jong-Bae Kim 1 1 Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, Wonju 26493, Republic of Korea; 2 Forensic DNA Division, National Forensic Service, Wonju 26460, Republic of Korea; 3 Department of Clinical Laboratory Science, Semyung University, Jaecheon 27136, Republic of Korea. * Equal con- tributors and co-frst authors. Received August 28, 2017; Accepted September 10, 2018; Epub January 15, 2019; Published January 30, 2019 Abstract: Helicobacter pylori primarily colonizes in the human stomach and induces various gastric diseases, in- cluding gastric cancer. H. pylori utilizes various bacterial proteins, such as toxins, ureases, and adhesion and fagella proteins, for successful colonization and pathogenesis. In particular, urease is an indispensable virulence factor for the pathogenesis and survival of H. pylori in the acidic conditions of the stomach. In this study, therefore, we investigated the anti-urease activity of a natural compound, plumbagin, and found that it inhibited urease activity of various urease-producing bacteria. We determined the minimal inhibitory concentration of plumbagin against H. pylori, and demonstrated the inhibitory effect of plumbagin on H. pylori urease by using a urease activity assay. The inhibitory effect of plumbagin on urease was confrmed by using purifed jack bean urease as the reference mol- ecule, with acetohydroxamic acid as the reference inhibitor. Furthermore, we also confrmed the binding affnity of plumbagin to urease proteins. Collectively, our results suggest that plumbagin is a potent inhibitor of urease, which may possess potential therapeutic effcacy as a supportive agent against H. pylori infection. Keywords: Helicobacter pylori, plumbagin, urease, anti-microbial effect, natural compound Introduction Helicobacter pylori is a gram-negative curved bacterium that primarily colonizes on the human stomach; approximately half of the world’s population is reported to be infected with H. pylori [1]. Infection with H. pylori is asso- ciated with various gastric diseases, including infammation of the gastric mucosa, peptic ulcers, and gastric adenocarcinoma [2]. In par- ticular, the WHO has classifed H. pylori as a group I carcinogen based on the epidemiologi- cal evidence in gastric cancer development [3]. Urease is a major virulence factor of H. pylori, because it is indispensable for colonization on the gastric epithelium. Typically, H. pylori colo- nizes at the acidic mucosa of the stomach, and urease produces NH 3 to increase the pH where it is colonized to survive in the hostile environ- ment [4]. The structure of H. pylori urease is known to be a dodecameric complex of the form ((αβ) 3 ) 4 , which is composed of two sub- units, α (61-66 kDa) and β (26-31 kDa) [4]. Plumbagin is a natural compound found in Plumbago indica. Many reports have suggested that plumbagin inhibits the growth of various tumor cell lines through the induction of apop- tosis or cell cycle arrest [5, 6]. Moreover, plum- bagin exhibits an anti-bacterial effect [7]. In particular, some studies have reported that plumbagin has an inhibitory effect on H. pylori, although the mechanism was not clarifed [8, 9]. In this study, we determined the minimal inhibi- tory concentration (MIC) of plumbagin against H. pylori (ATCC 49503) and investigated the inhibitory effect of plumbagin on the urease expression and activity, which is indispensable for the colonization of the bacteria on the gas- tric mucosa. Materials and methods Materials H. pylori reference strain was purchased from ATCC (ATCC49503, Manassas, VA, USA). Mue-