Jorjani Biomedicine Journal: Spring 2018: Vol 6: No 1: P 22-32 Original article Evaluation of the Bactericidal Effects of Zingiber officinale, Aloysia citrodora and Artemisia dracunculus on the Survival of Standard Gram-Positive and Gram-Negative Bacterial Strains Mahdi Tajbakhsh1, Neda Soleimani2* 1. Bachelor of Plant Biology, Faculty of Life Sciences, Shahid Beheshti University, Tehran, Iran 2. Assistant Professor, Ph.D. in Medical Microbiology, Department of Microbiology and Microbial Biotechnology, Faculty of Life Science and Biotechnology, Shahid Beheshti University, Tehran, Iran Abstract Background and objectives: Historically, herbs and plants have been used for their therapeutic properties in the form of flavors and preservatives. Recently, the application of medicinal herbs has increased considering their numerous benefits and minimum side-effects. Treatment of bacterial infections is currently a major challenge in the healthcare systems across the world. The present study aimed to assess the bacterial effects of Zingiber officinale, Aloysia citrodora and Artemisia dracunculus essential oils on the survival of standard Gram-positive and Gram-negative bacterial strains. Methods: In the present experimental study, we evaluated the effects of Tarragon (A. dracunculus), Ginger (Z. officinale) and Lemon Beebrush (A. citrodora) essential oils on 6 strains of Staphylococcus aureus, Klebsiella spp, Salmonella typhimurium, Staphylococcus epidermidis, Proteus spp and Corynebacterium diphtheriae. The well-diffusion method was applied to assess the antibacterial properties of the essential oils. Moreover, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) tests were used to determine the bacterial and inhibitory concentrations of the extracts. Results: MIC and MBC results demonstrated that the ginger extract (0.125 mg/mL) had the most significant impact on Staphylococcus aureus, Salmonella typhimurium, and Staphylococcus epidermidis. Furthermore, tarragon extract (0.03125 mg/mL) had the most significant effect on Staphylococcus aureus, Proteus spp, and Corynebacterium diphtheriae. The antibacterial effects of these essential oils were not observed on other bacteria. Conclusion: Medicinal plants have long been used for their therapeutic properties. According to the results, ginger and tarragon extracts are effective combinations for the treatment of the infections caused by Gram-negative and Gram-positive bacteria. Keywords: Antibiotic resistance; Minimum bactericidal concentration; Minimum inhibitory concentration; Zingiber officinal; Aloysia citrodora; Artemisia dracunculus Corresponding Author: Neda Soleymani Address: Department of Microbiology and Microbial Biotechnology, Faculty of Science and Biotechnology, Shahid Beheshti University, Tehran, Iran E-mail: [email protected] 22 Jorjani Biomedicine Journal: spring 2017: Vol 6: No 1: P 22-32 Introduction Ginger (Zingiber officinale) belongs to the Historically, herbs and plants have been Zingiberales order and Zingiberaceae family. used as foods and medications. With the With a height of 20-100 centimeters, this advancement of pharmaceutical sciences, yellow plant has purple leaf-tints, and its main which has led to the increased application of properties lie in the underground stem, known chemical compounds and the subsequent side- as the rhizome. Some of the main compounds effects, special attention has been paid to the found in ginger are camphene, phellandrene, use of medicinal plants for healthcare zingiberene, cineole, citral and borneol (11). purposes (1). It is notable that gingerols and the Extensive research has been conducted on corresponding shogaols are the major pungent new antibacterial medications with high compounds in ginger. This perennial herb has effectiveness due to the increased drug 70 species across the world and is originated resistance associated with the growing use of in East Asia. In addition to its antifungal antibiotics against bacterial infections (2, 3). properties, ginger has been reported to exert Antibiotic resistance in bacteria is a global sedative effects. phenomenon, encompassing a wide range of According to the literature, constant human pathogens and antibiotic groups (3, 4). consumption of antibiotics has led to bacterial In general, bacteria become resistant to resistance to these agents. Therefore, further antibiotics through several patterns. While investigation has been focused on the use of some organisms are naturally resistant to natural antibacterial agents (11). For instance, some or all antibacterial agents, others lemon beebrush (Aloysia citrodora) is a gradually become resistant to antibacterial medicinal herb with various properties, which agents through molecular mechanisms (5, 6). could be considered as a proper alternative to Currently, multidrug resistance of bacteria synthetic antibiotics. Lemon verbena has to antibiotics has been reported in many narrow lanceolate leaves of approximately 7- studies. Synthetic antibiotics play a pivotal 10 centimeters and a knot-shaped base with role in the treatment of infectious diseases (7- triplets per node. The plant has 21 seeds and 9). However, the resistance of consists of an unbranched inflorescence of microorganisms to these agents and the stalked flowers in the form of narrow clusters, associated complications is a major concern with a small flower pot and two lips of pale among researchers, encouraging the use of purple. alternative herbal and organic compounds (6, Considering the wide application and 7). numerous benefits of medicinal plants in Tarragon (Artemisia dracunculus) belongs clinical settings and in order to comprehend to the flowering plant order Asterales and their effects, the present study aimed to Asteraceae family. This perennial plant grows evaluate the sensitivity of standard Gram- to an average height of 30 centimeters to one negative and Gram-positive bacterial strains meter and has simple green leaves. The basal to the herbal extracts of ginger, tarragon and leaves of tarragon are cleft and three-lobed, lemon beebrush as new antibacterial and the middle lobe is larger than the side compounds. lobes, and the cluster-shaped capitols are small and green. Tarragon contains various chemical compounds, including estragole, ocimene, methyl chavicol, and a significantly small amount of tannin (10). Jorjani Biomedicine Journal: spring 2017: Vol 6: No 1: P 22-32 Materials and Methods (MHA) using a swap, and wells were formed on the agar surface using sterile pipettes Preparation of Herbal Extracts (Pasteur Institute, Iran). Afterwards, 25 μL of In this laboratory experiment, tarragon, the extracts was injected into the wells, lemon beebrush and ginger were purchased followed by the incubation of the plates in an from FZ Biotech Company. The plants were inverted position at the temperature of 37°C completely washed, naturally dried, and for 18 hours. By the end of this stage, the powdered. The powder was placed in a one- diameter of the bacterial growth inhibition liter flask with 300 milliliters of sterile, zone around the wells was measured in distilled water in order to extract the herbal millimeters. In addition, physiological serum essences using the Clevenger (Borosil, India) and gentamicin were used as negative and apparatus. positive control, respectively. It is notable that Standard Bacterial Strains three replications of the test were carried out (13). The standard strains of Corynebacterium diphtheriae (ATCC 13812), Staphylococcus Minimum Inhibitory Concentration (MIC) epidermidis (PTCC 1114), Salmonella The broth dilution method was applied to typhimurium (ATCC 14028), Staphylococcus determine the minimum inhibitory aureus (ATCC 25923), Proteus mirabilis, and concentration (MIC) in the bacterial strains Klebsiella pneumoniae were purchased from (14). To this end, 300 μL of MHA (Merck, Pasteur Institute of Iran and used to evaluate Germany) was added to the 96-well the antibacterial effects of the tarragon, lemon microplate (dilution ratio: 1:2). After adding beebrush and ginger extracts. The identity of 300 μL of the 1.2 concentration to the first the evaluated strains was confirmed using well, 150 μL of the mixture was collected differential, selective, dedicated media and from the first well and added to the second biochemical tests, including catalase, oxidase, well, resulting in the dilution series in the and glucose fermentation. Afterwards, the wells. strains were cultured on tryptic soy broth In the next stage, 10 μL of the bacterial (TSB) at the temperature of 37°C for 24 suspension (108 CFU/mL) was separately hours. Fresh bacterial culture was applied in added to the wells. Finally, the microplate the following stages of the study. was incubated at the temperature of 37°C for Antibacterial Effects of Herbal Extracts on 18 hours. MIC was defined as the lowest Bacterial Strains concentration required to inhibit bacterial growth. In this process, the well containing no Bacterial Sensitivity Test Using the Disc- herbal essence and bacterial suspension was Diffusion Method considered as the negative control (14). MIC was determined in triplications. The well-diffusion method was applied to assess the antibacterial activities of tarragon, Minimum Bactericidal Concentration lemon beebrush and ginger (5). In addition, (MBC) the bacterial inhibition zone was measured to At this stage, 10 μL of the contents of the determine the antibacterial effects. To do so, a wells was cultured on MHA (Merck, bacterial