fmicb-08-00804 April 29, 2017 Time: 14:57 # 1 ORIGINAL RESEARCH published: 03 May 2017 doi: 10.3389/fmicb.2017.00804 Efficacy of (C)-Lariciresinol to Control Bacterial Growth of Staphylococcus aureus and Escherichia coli O157:H7 Vivek K. Bajpai1†, Shruti Shukla2†, Woon K. Paek3, Jeongheui Lim3*, Pradeep Kumar4*, Pankaj Kumar5 and MinKyun Na6* 1 Department of Applied Microbiology and Biotechnology, Microbiome Laboratory, Yeungnam University, Gyeongsan, South Korea, 2 Department of Energy and Materials Engineering, Dongguk University, Seoul, Seoul, South Korea, 3 National Edited by: Science Museum, Ministry of Science, ICT and Future Planning, Daejeon, South Korea, 4 Department of Forestry, North Luis Cláudio Nascimento da Silva, Eastern Regional Institute of Science and Technology, Nirjuli, India, 5 Department of Microbiology, Dolphin (PG) College of CEUMA University, Brazil Science & Agriculture, Fatehgarh Sahib, India, 6 College of Pharmacy, Chungnam National University, Daejeon, South Korea Reviewed by: Diego Garcia-Gonzalo, This study was undertaken to assess the antibacterial potential of a polyphenolic University of Zaragoza, Spain Dinesh Yadav, compound (C)-lariciresinol isolated from Rubia philippinensis against selected Deen Dayal Upadhyay Gorakhpur foodborne pathogens Staphylococcus aureus KCTC1621 and Escherichia coli University, India C Osmar Nascimento Silva, O157:H7. ( )-Lariciresinol at the tested concentrations (250 mg/disk) evoked a Universidade Católica Dom Bosco, significant antibacterial effect as a diameter of inhibition zones (12.1–14.9 mm) with Brazil minimum inhibitory concentration (MIC), and minimum bactericidal concentration values *Correspondence: of 125–250 and 125–250 mg/mL, respectively. Furthermore, (C)-lariciresinol at MIC Pradeep Kumar C [email protected] showed reduction in bacterial cell viabilities, efflux of potassium (K ) ions and release Jeongheui Lim of 260 nm materials against E. coli O157:H7 and S. aureus KCTC1621. Moreover, [email protected] MinKyun Na deteriorated cell wall morphology of E. coli O157:H7 and S. aureus KCTC1621 cells [email protected] treated with (C)-lariciresinol at MIC further confirmed its inhibitory effect against the †These authors have contributed tested pathogens, suggesting it to be an alternative means of antimicrobials. equally to this work. Keywords: antimicrobial effect, Rubia philippinensis,(C)-lariciresinol, foodborne pathogens, scanning electron Specialty section: microscopy This article was submitted to Antimicrobials, Resistance and Chemotherapy, INTRODUCTION a section of the journal Frontiers in Microbiology Food industry and regulatory authorities are more concerned about the safety of food, and its Received: 14 January 2017 contamination by foodborne pathogenic bacteria (Al-zorekya and Al–Taher, 2015). Consumer Accepted: 19 April 2017 awareness on the knowledge of quality food information has raised greater concern to use Published: 03 May 2017 high quality food products due to possible contamination by foodborne pathogenic bacteria. Citation: Among foodborne pathogens, Staphylococcus aureus and Escherichia coli O157:H7 have posed Bajpai VK, Shukla S, Paek WK, serious concerns to consumers due to their reported foodborne outbreaks and foodborne illnesses Lim J, Kumar P, Kumar P and Na M (Awaisheh et al., 2013). In the USA, about 75 and 68% cases account for foodborne outbreaks (2017) Efficacy of (C)-Lariciresinol and reported cases of foodborne illnesses, respectively (Scot, 2003). Meat associated foodborne to Control Bacterial Growth illnesses in Canada costs about five hundred million dollars for their cure (Oussalah et al., of Staphylococcus aureus and Escherichia coli O157:H7. 2007). Furthermore, failure to control foodborne pathogens using current preservation techniques Front. Microbiol. 8:804. has raised a serious concern to explore new and natural classes of antimicrobials to combat doi: 10.3389/fmicb.2017.00804 against serious foodborne pathogens (Awaisheh and Ibrahim, 2009). Recently, it has been Frontiers in Microbiology| www.frontiersin.org 1 May 2017| Volume 8| Article 804 fmicb-08-00804 April 29, 2017 Time: 14:57 # 2 Bajpai et al. Antibacterial Potential of (C)-Lariciresinol found that currently available preservation techniques have not and identified by Dr. Phuong Thien Thuong at the Department provided optimal results to control or retard the growth of of Pharmaceutical Analysis and Herbal Standardization, National foodborne and food spoilage pathogens in vivo (Aneja et al., Institute of Medicinal Materials, Vietnam. A voucher specimen 2014). was deposited at the herbarium of the National Institute of On the other hand, current scenario in drug discovery Medicinal Materials, Hanoi, Vietnam, as well as the Laboratory of and development has consistently showed development of Pharmacognosy at the College of Pharmacy, Chungnam National resistance among the foodborne pathogens to the commercially University, Daejeon, Korea. available antibiotics (Militello et al., 2011). Though some synthetic antimicrobials have shown profound efficacy to control Extraction, Isolation, and foodborne pathogens (Shakiba et al., 2011; Shen et al., 2014), they Characterization of (C)-Lariciresinol have shown to exhibit several side effects as a serious human- (C)-Lariciresinol was obtained from the roots of R. philippinensis health threatening, including toxic, carcinogenic, and teratogenic by chromatographic methods. Briefly, the ethanol extract of effects (Hsouna et al., 2011). This has caused an alarming threat R. philippinensis (150 g) was suspended in H O (1.5 L) and to the consumers to acquire contamination free food or food 2 partitioned with CH Cl (2 L × 3) to yield CH Cl extract. products. 2 2 2 2 The CH Cl -soluble fraction (50 g) was subjected to silica gel For decades, plants or plant-based phyto-constituents have 2 2 VLC and eluted with n-hexane-EtOAc (20:1, 10:1, 5:1, 3:1, 2:1) been used as alternative means for the prevention and treatment and CHCl -MeOH (8:1) to afford six fractions (D-1 ! D-6). of various diseases, both in modern and traditional medicine 3 Fraction D-6 (10 g) was divided into 11 sub-fractions (D-6-1 ! system (Pandey and Rizvi, 2009; Vauzour et al., 2010; Bajpai et al., D-6-11) using MPLC with a gradient of MeOH-H O (10:90 ! 2015). Plant extracts from Trachyspermum ammi (L.) were found 2 100:0, 7 L). (C)-Lariciresinol (t 33.0 min, 31 mg) was obtained to be effectivive against different foodborne pathogens (Paul et al., R from D-6-3 (360 mg) by HPLC model 1200 using a Zorbax SB- 2011). Also, olive and oregano derived extracts found to effective C18 analytical column (4.6 mm × 150 mm, 5 mm particle size), against different foodborne pathogens includings, S. aureus, Agilent, Germany eluting with MeOH-H O (45:55, 4 mL/min, S. enterica, L. monocytogenes, and E. coli O157:H7 (Friedman 2 UV 254 nm). (C)-Lariciresinol: brownish amorphous powder, et al., 2013). Polyphenols and bioactive compounds extracted 1H NMR (300 MHz, methanol-d ): 2.38 (1H, m, H-8), 2.47 (1H, from Camellia sinensis showed significant inhibitory effects agaist 4 dd, J D 13.1, 11.7, H -7’), 2.72 (1H, m, H-8’), 2.91 (1H, dd, E. coli (Noormandi and Dabaghzadeh, 2015). In the present a J D 13.1, 4.5, H -7’), 3.63 (1H, dd, J D 10.8, 6.6, H -9), 3.72 study, a polyphenolic lignan, (C)-lariciresinol isolated from the b a (1H, dd, J D 8.1, 6.1, H -9’), 3.82 (1H, overlapped, H -9), 3.97 roots of Rubia philippinensis was assessed for its antimicrobial a b (1H, dd, J D 8.1, 6.1, H -9’), 4.75 (1H, d, J D 6.9, H-7), 6.63 efficacy against selected foodborne pathogens, Staphylococcus b (1H, dd, J D 8.0, 1.2, H-6’), 6.73 (1H, d, J D 8.0, H-5’), 6.78 (3H, aureus and Escherichia coli O157:H7. overlapped, H-6, H-5, H-2’), 6.91 (1H, d, J D 1.2, H-2); 13C NMR (75 MHz, methanol-d4): 135.7 (C-1), 110.6 (C-2), 148.9 (C-3), MATERIALS AND METHODS 146.9 (C-4), 116.0 (C-5), 119.8 (C-6), 83.9 (C-7), 53.9 (C-8), 60.4 (C-9), 133.5 (C-1’), 113.4 (C-2’), 148.9 (C-3’), 145.7 (C-4’), 116.2 Chemicals and Reagents (C-5’), 122.1 (C-6’), 33.6 (C-7’), 43.8 (C-8’), 73.4 (C-9’), 56.3, 56.3 × The nutrient broth (NB) medium was purchased from (2 OCH3-3,3’). Difco (USA). Other chemicals and reagents used were of very pure and high analytical grade. Test samples of (C)- Determination of Antibacterial Activity of lariciresinol isolated from R. philippinensis were prepared in (C)-Lariciresinol 1% dimethyl sulfoxide (DMSO) (Sigma-Aldrich, Germany). Standard agar diffusion method was used for antibacterial Spectrophotometric measurements were made using an enzyme- assay (Bajpai and Kang, 2010). Petri plates were prepared linked immunosorbent assay (ELISA) instrument (Tecan, by pouring 20 ml of LB medium and allowed to solidify. Infinite M200, Männedorf, Switzerland) was used. Plates were dried, and 100 mL of standardized inoculum containing approximately 107 CFU/mL of bacterial suspension Test Foodborne Pathogens was poured and uniformly spread. The inoculum was allowed Two selected foodborne pathogenic bacteria one Gram-positive to dry for 5 min. The compound was dissolved in 5% (Staphylococcus aureus KCTC1621), and one Gram-negative DMSO, and a Whatman No. 1 sterile filter paper disk (6 mm (Escherichia coli O157:H7, shiga-toxin producing bacterium) diameter) was impregnated with 50 mL of the compound (C)- were procured from the Korean Collection for Type Cultures lariciresinol corresponding to 250 mg/disk. Negative controls (KCTC, Korea) and used in this study. All the bacterial strains were prepared using the same solvent that was employed to were grown in the NB and incubated at 37◦C. The bacterial dissolve the sample. The plates were incubated at 37◦C for strains were maintained on nutrient agar slants at 4◦C.