ISSN: 2601 – 6141, ISSN-L: 2601 – 6141 Acta Biologica Marisiensis 2018, 1(1): 5-9 ORIGINAL PAPER ANTIBACTERIAL ACTIVITY OF SPRUCE BARK (PICEA ABIES L.) EXTRACT AGAINST ESCHERICHIA COLI Corneliu TANASE1, Irina BOZ2,3*, Silvia OROIAN1, Sanda COȘARCĂ1, Felicia TOMA1, Anca MARE1, Adrian MAN1 1University of Medicine and Pharmacy of Tîrgu Mureș, Gheorghe Marinescu, 38, 540139, Tîrgu Mureș, Mureș, Romania 2NIRDBS - Institute of Biological Research, Department of Experimental and applied biology, Lascăr Catargi 47,700107, Iași, Romania 3Integrated Centre for Environmental Science Studies in the North-East Development Region – CERNESIM, Alexandru Ioan Cuza University, Carol I 20 A, 700505 Iași, Romania *Correspondence: Irina BOZ [email protected] Received: 17 May 2018; Accepted: 17 June 2018; Published: 15 July 2018 Abstract: The increase of antibiotic resistant bacteria in lasts years resulted in limited options for treatment of bacterial diseases. Escherichia coli is one of the most common Gram-negative bacterial pathogen and a cause of both community and hospital acquired infections. Medicinal plants are alternative rich sources of useful antibacterial agents. The antimicrobial activities of the spruce (Picea abies L.) bark polyphenolic extracts were examined for their capacity to inhibit the growth of Escherichia coli. Spruce bark extract was obtained by conventional aqueous extraction and with ultrasounds. The minimum inhibitory concentration was determined by microdilution method. The antibacterial effect of both extracts was strong against Escherichia coli. The antimicrobial effect of polyphenolic extracts on Escherichia coli was expressed at a concentration of 15 mg/ml. Antimicrobial activity of spruce bark compounds suggest a possible use of spruce bark in pharmaceutical preparations. Keywords: antimicrobial agents, Escherichia coli, polyphenols, spruce bark, Picea abies L. 1. Introduction The drug resistance in human pathogenic Balchin and Deans 1997; Dorman and Deans, bacteria and many adverse effects of antibiotics 2000; Sathasivampillai et al., 2017). has led to a search for new antimicrobial agents According to the classification system with plant origin. It is known that plants from “Vascular plants from Romania - Field produce antimicrobial compounds. Thus, crude Illustrative Determinator” (Sârbu et al., 2013), plant extracts have been used for a various Picea abies (L.) H. Karst has the following purposes for a very long time (Moradi et al., systematic classification: Vegetable Kingdom, 2016; Sharif et al., 2016). The antimicrobial Pinophyta Division, Pinatae Class, Pinales activity of crude plant extracts was the basis of Order, Pinaceae Family, Picea Genus, abies various applications, such as food preservation, species. Norway spruce is very widespread in pharmacy, natural therapies and medicine (Lis- the Romanian forests. Spruce present great 5 Corneliu Tanase et al. economic importance, being used as Ultrasound assisted extraction was performed construction wood, paper manufacturing, and under ultrasounds action in accord with method in phytotherapy. previously described (Tanase et al., 2018a). The literature data show many studies on volatile oil from spruce, with different 2.3 Minimum inhibitory concentration antimicrobial activity and intensity depending of E. coli (MIC) on tested strains. The inhibitory effect was To determine the MIC of the obtained noticed against the Gram-positive and fungal extract against E. coli, the microdilution strains (Radulescu et al., 2011; Chauhan and method was used, as previously described Dahiya, 2016). In the spruce bark alcoholic (Tanase et al., 2018a). Shortly, from fresh extract, was identified gallic acid, catechin and bacterial culture, a standard inoculum was vanillic acid in high concentration (Ignat et al., prepared in liquid culture medium. One- 2013). The results of Ignat et al. (2013) showed hundred microliters of the tested extracts were that the spruce bark ethanol extracts exerted mixed in the first well of the microplate with antibacterial activity against Staphylococcus 100 µl of bacterial inoculum. We evaluated the aureus and for Escherichia coli and MIC of the tested extracts in the first well with Pseudomonas aeruginosa being less no bacterial growth. The MICs were calculated susceptible. in mg/ml, by adjusting the obtained The aim of this study was to evaluate the concentrations with the dilution factor and antimicrobial activity of Picea abies L. bark processed volumes. aqueous crude extracts against E. coli. E. coli growth rate 2. Materials and Methods For E. coli growth rate determination, the method described in a previously paper was 2.1 Plant/bacteria materials used (Tanase et al., 2018a). To determine if the Spruce (Picea abies L.) bark it is a waste tested solutions affect the E. coli growth rate, product from a wood processing company stock solutions of the both extracts was (Vatra Dornei, Romania). The spruce bark was prepared. The concentration of the stock air-dried at room temperature and milled in a solution was adjusted to correspond to the MIC GRINDOMIX GM 2000 mill (0.5 mm that was assessed by the microplate method. diameter). The biomass was used without any The total number of colony forming units/ml pre-treatments. (CFU/ml) at time 0 (H0), 3 hours (H1), 6 hours To determine the antibacterial activity on (H2) was determined inoculating 50 µl from Escherichia coli, ATCC 25922 strain was 1/100 diluted working solution and from 1/100 selected from the collection of Laboratory of diluted control on Mueller Hinton agar plates. Microbiology, Virology and Parasitology After 24 hours of incubation, the number of the (Faculty of Medicine - University of Medicine colonies was counted using the colony counter and Pharmacy, Tîrgu Mureș). "IUL Flash & Grow" and mathematically adjusted in order to be expressed as CFU/ml. 2.2 Aqueous extraction For extraction was used 20 g of ground 2.4 Statistical analysis spruce bark over which added 125 mL distilled Using the calculated CFU/ml numbers from water. The mixture was incubated for 45 min in each time point, absolute growth curves were a water bath at 85-90°C (Tanase et al., 2018a). plotted. The following formula was used in 6 ISSN: 2601 – 6141, ISSN-L: 2601 – 6141 Acta Biologica Marisiensis 2018, 1(1): 5-9 order to assess the growth rate (r) for the tested extract induced the bactericidal effect on E. sample and control, after 6 hours of incubation. coli. The statistical significance was assessed by The biological activity of P. abies and GraphPad InStat 3 software, at a significance other species of the Pinaceae family was threshold value of p<0.05. previously tested on other strains. For the aqueous extract of P. abies bark, LN(CFU / ml for H 2 CFU / ml for H 0 Staphylococcus aureus, methicillin-resistant S. r no. hours for H 2 aureus, Klebsiella pneumoniae and Pseudomonas aeruginosa were used (Tanase et 3. Results and discussions al., 2018b). For the spruce bark ethanol extracts, Staphylococcus aureus, Escherichia 3.1 Extract characterization coli and Pseudomonas aeruginosa were tested The previous results summarized that spruce (Ignat et al., 2013). The ethereal extracts from bark extracts (EAM and USM) contain Pinus nigra, Pinus halepensis, Abies equi- considerable quantities of bioactive aromatic trojani, Abies bornmulleriana, Abies cilicica, compounds (EAM - 0.135 mg GAE/mL and Abies nordmanniana, Cedrus libani and Picea USM 0.114 mg GAE/mL). The compounds orientalis were examined against identified in samples by HPLC were vanillic Staphylococcus aureus, methicillin-resistant acid and taxifolin in small amounts (Tanase et Staphylococcus aureus, Bacillus subtilis, al., 2018b). Escherichia coli, Pseudomonas aeruginosa, 3.2 Minimum inhibitory concentration Klebsiella pneumoniae and Candida albicans (Eryilmaz et al., 2015). The authors concluded of E. coli (MIC) The results indicate that the two aqueous that all the tested extracts, except Abies extracts (EAM and USM) of different bornmulleriana, Cedrus libani and Pinus concentrations have antibacterial activity halepensis showed weak antibacterial activity against E. coli. The minimum inhibitory against the various tested bacteria comparing concentrations (MIC) of polyphenolic extracts with the standards. required for growth inhibition of E. coli was 15 From previous studies, it is known that mg/mL both for EAM and for USM. spruce bark contains vanillic acid and taxifolin (Tanase et al., 2018). Moon et al., (2006) E. coli growth rate concluded that vanillic acid accelerated the As shown in Table 1, when the growth death of E. coli. It was also found that taxifolin medium was enriched with both tested have antibacterial action against human solutions (EAM and USM), the bacterial pathogens inclusive E. coli (Asmi et al., 2017). growth was significantly inhibited, compared Thus, the vanillic acid and taxifolin identified with control. After three hours of incubation, in the spruce bark crude extract can have a role the growth of E. coli was significantly reduced in antibacterial activity against E. coli. The (Table 1, Fig. 1 and Fig. 2). At H2 time, EAM results of the present investigation suggest that inhibited the growth of E. coli (Fig. 1), while the spruce bark crude extracts can be used as USM presented bactericidal effect (Fig. 2). As potential leads to discover new antibacterial a research direction, it would be interesting to agents to control E. coli bacterial infections. investigate which component of the USM 7 Corneliu Tanase et al. Table 1. Data presenting the growth rate of E. coli in presence of EAM and USM (p<0.0001) Experimental CFU/ml Growth rate (h-1) Generation time variants (min) H0 H1 H2 r g EAM 1.4 x 104 2.8 x 104 3.1 x 107 1.28 32.42 Control 5.6 x 104 5.3 x 106 3.6 x 108 1.46 28.47 USM 2.8 x 104 2.1 x 105 0 N/A N/A Control 1.4 x 104 2.1 x 106 3.3 x 108 1.68 24.76 Fig. 1. The graphics representation of the growth rate for Escherichia coli in the presence of EAM comparing to the Control Fig.