International Journal of Phytomedicine 6 (2014) 177-181 http://www.arjournals.org/index.php/ijpm/index Original Research Article ISSN: 0975-0185 The effect of Cyclamen coum extract on pyocyanin production by Pseudomonas aeruginosa Zahra Ahmadbeigi1*, Azra Saboora1, Ahya Abdi-Ali1 *Corresponding author: Abs tract Researches have shown that some plants possess antimicrobial activity and the ability to overcome Zahra Ahmadbeigi drug-resistant pathogens. Their frequent used in treatment of microbial infections has been led to isolation of the active compounds and evaluation of their antimicrobial properties. Cyclamen coum Miller is one of these plants with a secondary metabolite called saponin which has antimicrobial 1Department of Biology, Faculty of activity. Pyocyanin is one of the virulence factors in Pseudomonas aeruginosa, an opportunistic Science, University of Alzahra, Tehran pathogen, causing lung diseases. The present study indicates the effect of cyclamen saponin 1993893973, Iran extracts on pyocyanin production by P. aeruginosa. We prepared three different types of plant extracts (ethanolic, aqueous and butanolic) from tuber of C. coum. The effect of 0, 10 and 20 mg of cyclamen saponin were tested by agar disk diffusion technique. Pyocyanin purification was done from microbial broth culture and the extracted pyocyanin was measured by spectrophotometric method. Results showed that the production of pyocyanin was remarkably reduced by ethanolic extract of saponin. In addition increased saponin concentration led to further decrease in pyocyanin content. Keywords: Pseudomonas aeruginosa; Cyclamen coum; Pyocyanin; Antimicrobial activity es Bacterial cells communicate with each other through producing Introduction signaling factors named inducers. When bacterial cell density increases, the inducers bind to the receptors and alter the Extensive In vitro studies on plants used in traditional medicine expression of certain genes. This type of bacterial communication have been carried out in the field of microbiology, especially on has been termed quorum sensing (QS). Quorum sensing enables pathogenic bacterial growth. Some plants are known as medicinal bacteria to respond quickly to environmental changes, such as the sources as they contain active substances that cause certain availability of nutrients, other microbes or toxins in their reactions, from diseases abatement to complete cure in human environment through auto inducers. Various bacterial physiological [1,2]. Infectious diseases still represent an important cause of activities including symbiosis, virulence factor expression, morbidity and mortality in human, especially in developing competence, conjugation, luminescence, antibiotic production, countries. Even though pharmaceutical industries have produced motility, sporulation, biofilm formation and pigment production are numbers of new antimicrobial drugs in the last years but controlled by QS [8,9,10]. The production of several extracellular microorganisms resistance to these drugs also has increased. virulence factors of P. aeruginosa such as pyoverdin, pyocyanin In general, bacteria have the genetic ability to transmit and acquire and biofilm are known to be controlled by quorum-sensing system resistance to drugs used as therapeutic agents [3]. Researchers [11,12]. For example, presence of some plant extracts in bacterial are trying to find phytochemicals that could be used for healing of growth media can control pyocyanin production. Level of this infectious diseases [4] and to produce the more effective virulence factor in extract containing media was detected to be antimicrobial agents for drug-resistant microorganisms. The human significantly lower than extract deficient media [13]. opportunistic pathogen, Pseudomonas aeruginossa is a gram- Saponin-containing plants such as ginseng, yucca, horse chestnut, negative bacterium that infects immune deficient or immune sarsaparilla, licorice and cyclamen have been used in traditional compromised patients and causes a wide range of infections [5]. system of medicine by various cultures for the prevention and Pathophysiological effects of P. aeruginosa are often associated treatment of various ailments [14,15,16].The antifungal, antiviral with a number of virulence factors secreted by this bacterium. and antibacterial activities of saponin are well documented Among these virulence factors, pyocyanin is a low molecular [17,18,19,20]. Characterization of the medicinal plants and their weight phenazine redox pigment [6]. In vivo studies have extracts points to the role of free and conjugated saponin with other suggested that some radicals created by pyocyanin, inhibit bioactive components in the observed health effects [15, 21]. epidermal cell growth; disrupt the host immune system and Cyclamen is a genus of tuberous plants, traditionally classified in possess antibiotic properties against other microorganisms [7]. the family Primulaceae, but in recent years reclassified in the family This work is licensed under a Creative Commons Attribution 3.0 License. Ahmadbeigi et al. International Journal of Phytomedicine 6 (2) 177-181 [2014] of Myrsinaceae [22]. One of the Cyclamen species is C. coum controls). The samples were then incubated at 37C for 17 to 24 Miller which is widely distributed in north of Iran. Çali et al. (1997) hrs with aeration.The cells were harvested by centrifugation (5000 reported antimicrobial activities of isolated saponins from C. g, 30 min), and pyocyanin in supernatant was extracted by mirabile and C. coum. repeated extractions with 15ml of chloroform until the medium At this time, little is known about the possibilities of regulation role became colorless. The chloroform layer was transferred to a glass of saponins on the virulence factors production. In this study we cuvette and the extracted pyocyanin was measured by checking extracted saponins from tuber of C. coum and added the extract in the absorbance at 690nm and using an extinction coefficient of l% P. aeruginosa growth medium in order to investigate its E 1 = 164 [26]. antibacterial property and inhibition effect on pyocyanin production. Statistical Tests Experimental Section All the results represented are the average of three independent experiments. One-way analysis of variance (ANOVA) was used to Samples collection and Extraction determine the effect of different saponin treatments on pyocyanin Cyclamen coum Miller were collected from north of Iran (Golestan production. All statistical analyses were done by using the SPSS province, Gorgan, Naharkhoran). Slices of underground stems software version 12, SPSS Inc., Chicago. Statistical significance (tubers) were dried at 70oC for 48hrs and blended into a fine dry was accepted at P < 0.05. Averages were presented as mean μ powder. The powdered samples (200g) were extracted with 70% standard deviation (SD). ethanol (3120ml) by soxhlet method for 6 hrs and then concentrated at reduced pressure in a rotary evaporator to remove Results and Discussion the solvent to get a small volume (crude extract) and subsequently We designed this experiment to investigate the effect of saponin as the crude extract was defatted with ether (315 ml). The aqueous quorum sensing inhibitor and its effect on pyocyanin virulence layer was extracted with n-butanol until n-butanol layer became factor. We measured the level of extracellular pyocyanin in colorless. At the end of this process, two phases were obtained; P.aeruginosa culture medium. Results showed significant changes upper phase (n-butanolic extract) and lower phase (aquatic in pyocyanin production without reduction in bacterial growth. extract). Both extracts were concentrated up to 5 ml. Based on our observations, the largest measured zone of growth inhibition was recorded around the disk containing 20mg n- Agar disk diffusion assay butanolic extract. The bacterial growth was observed around the disks containing different concentrations of aqueous and ethanolic Agar disk diffusion technique (Kirby-Bauer Method), as qualitative saponin extracts with decrease in the green color of pyocyanin test, has been used to assay plant extracts for their antimicrobial (Figure. 1). High activity of crude extract (ethanolic extract) has activities. P. aeruginosa (ATCC 27853) was grown in nutrient Agar different reasons. It may contain conjugated saponin such as (NA) for 24hrs at 37oC. A microbial broth culture adjusted to 0.5 glycosidic forms or it occurred because of existence of different McFarland turbidity standard has been prepared for inoculating into kind of extracted saponin in different fractions. a Mueller Hinton agar (MHA) medium. In this method, 6 mm paper disks were saturated with sterilized aquatic, n-butanolic and 70% ethanolic saponin extracts or solvents (controls) and dried at room temperature. After drying the disks contained 0, 10 and 20mg cyclamen saponins. Disks were placed on the surface of a MHA medium which was pre-inoculated with the P. aeruginosa. Each diffusion plate had inoculum size of 1-2 x 108 bacteria/ml. The plates were then incubated at 37oC for 24hrs. Zones of growth inhibition were then measured from the circumference of the disks to the circumference of the inhibition zone or recorded as the difference in diameter between the disks and the growth free zones around the disks [23]. Figure 1. Inhibition of P. aeruginosa pyocyanin production by Pyocyanin Assay various extracts of cyclamen in agar disk diffusion assay. For pyocyanin purification, 1ml of microbial broth culture