**2. Evaluation of antibacterial activity of selected plant species**

Considering a large number of still insufficiently or incompletely examined species in the plant world; in inexhaustible possibilities of modifying natural substances, there is a need for systematic research and even greater affirmation of plant antimicrobial compounds. In this study the *in vitro* antibacterial activity of selected plant species was tested and evaluated the potential use of their extracts as a source of antibacterial compounds. The experiment involved water, ethanol, ethyl acetate and acetone extracts from 10 plant species belonging to 4 the most important plant families. The following plants were used: *Cychorium intybus* L. (Asteraceae); *Salvia officinalis* L., *Melissa officinalis* L., *Clinopodium vulgare* L. (Lamiaceae); *Torilis anthriscus* L. (Gmel), *Aegopodium podagraria* L. (Apiaceae); *Cytisus nigricans* L., *Cytisus capitatus* Scop.,

A great number of reports concerning the antibacterial screening of plant extracts have appeared in the literature. Examples of such articles include studies of medicinal plants from different geographical regions: Brazil (Alves et al., 2000), Argentina (Salvat et al., 2000), Columbia (López et al., 2001); India (Perumal et al., 1998; Ahmad & Beg, 2001), China (Zuo et al., 2008), Turkey (Sokman et al., 1999; Uzun et al., 2004); Greece (Skaltsa et al., 2003), Spain (Ríos et al., 1987; Recio et al., 1989), Serbia (Stefanovic et al., 2009a; Stefanovic et al., 2009b, Stanojevic et al., 2010a; Stanojevic et al., 2010b; Stojanovic-Radic et al., 2010; Stefanovic et al., 2011; Stefanovic et al., 2012); Africa (Atindehou et al., 2002; Konning et al., 2004; Chah et al., 2006); Australia (Palombo & Semple, 2001). According to published data, the plant extracts exhibited the activity against a great number of bacterial species (Gram positive and Gram negative strains; sensitive and resistant, pathogens and opportunistic pathogens). The experiments involved the standard strains as well as the clinical isolates of pathogenic bacteria, as more realistic test organisms for estimation of antibacterial activity. Plant extracts were prepared from fresh or dried plant material using conventional extraction methods (Soxhlet extraction, maceration, percolation). Extraction is process of separation of active compounds from plant material using different solvents. During extraction, solvents diffuse into the plant material and solubilise compounds with similar polarity. At the end of the extraction, solvents have been evaporated, so that an extract is a concentrated mixture of plant active compounds. Successful extraction is largely dependent on the type of solvent used in the extraction procedure. The most often tested extracts are: water extract as a sample of extract that primarily used in traditional medicine and extracts from organic solvents such as methanol, ethanol as well as ethyl acetate, acetone, chloroform, dichlormethane (Ncube et al., 2008).

Diffusion and dilution method are two types of susceptibility test used to determine the antibacterial efficacy of plant extracts. Diffusion method is a qualitative test which allows classification of bacteria as susceptible or resistant to the tested plant extract according to size of diameter of the zone of inhibition. In dilution method, the activity of plant extracts is determined as Minimum Inhibitory Concentration (MIC). MIC is defined as the lowest concentration able to inhibit bacterial growth. In broth-dilution methods, turbidity and redox-indicators are most frequently used for results reading. Turbidity can be estimated visually or spectofotometrically while change of indicator colour indicate inhibition of

Considering a large number of still insufficiently or incompletely examined species in the plant world; in inexhaustible possibilities of modifying natural substances, there is a need for systematic research and even greater affirmation of plant antimicrobial compounds. In this study the *in vitro* antibacterial activity of selected plant species was tested and evaluated the potential use of their extracts as a source of antibacterial compounds. The experiment involved water, ethanol, ethyl acetate and acetone extracts from 10 plant species belonging to 4 the most important plant families. The following plants were used: *Cychorium intybus* L. (Asteraceae); *Salvia officinalis* L., *Melissa officinalis* L., *Clinopodium vulgare* L. (Lamiaceae); *Torilis anthriscus* L. (Gmel), *Aegopodium podagraria* L. (Apiaceae); *Cytisus nigricans* L., *Cytisus capitatus* Scop.,

**2. Evaluation of antibacterial activity of selected plant species** 

• direct antibacterial activity showing effects on growth and metabolism of bacteria • indirect activity as antibiotic resistance modifying compounds which, combined with

antibiotics, increase their effectiveness.

bacterial growth (Cos et al., 2006).

*Melilotus albus* Medic., *Dorycnium pentaphyllum* Vill. (Fabaceae). In general, the plants are annual or perennial, herbaceous or shrubby, widespread in Europe. They are rich in secondary metabolites from a group of phenols, flavonoids, coumarins, tannins, terpenes. It is known that different solvents extracted different groups of secondary metabolites, hence different types of extracts were prepared. The plants, as potential candidates for antibacterial agents, were selected on the base of three criteria: i) use in traditional medicine as antiseptic agents, ii) random selection followed by chemical screening (ii) insufficient antibacterial scientific data. Detailed description of known traditional uses, *in vitro* found biological activities and the chemical constituent is shown in Table 1.


Antibacterial Activity of Naturally Occurring Compounds from Selected Plants 7

Dried, ground plant material was extracted by direct maceration with water, ethanol, ethyl acetate and acetone. Briefly, 30g of plant material was soaked with 150ml of solvent for 24h at room temperature. During 24 hours, targeted compounds from plant material were extracted by the solvent. After that the resulting extract was filtered through filter paper (Whatman no.1). The residue from the filtration was extracted again, twice, using the same procedure. The filtrates obtained were combined and then evaporated to dryness using a rotary evaporator at 40°C, for water extracts heating on a water bath. The crude plant extracts are stored at -20°C. Before the testing, stock solutions of the crude extracts were obtained by dissolving in dimethyl sulfoxide (DMSO) and then diluted into nutrient liquid medium to achieve a concentration of 10% DMSO. The groups of secondary metabolites

Water extract Ethanol extract Ethyl acetate extract Acetone extract Simle phenols Simle phenols Simle phenols Simle phenols Phenolic acids Phenolic acids Phenolic acids Phenolic acids Flavonoids Flavonoids Flavonoids Flavonoids Quinones Quinones Quinones Quinones Tannins Tannins Tannins Tannins

Table 2. The expected groups of plant secondary metabolites (according to Kovacevic, 2004)

The following bacteria were used: *Staphylococcus aureus* ATCC 25923, *Escherichia coli* ATCC 25922, *Pseudomonas aeruginosa* ATCC 27853 and clinical isolate of *Staphylococcus aureus*  (PMFKg-B30), *Bacillus subtilis* (PMFKg-B2), *Enterococcus faecalis* (PMFKg-B22), *Enterobacter cloaceae* (PMFKg-B23), *Klebsiella pneumoniae* (PMFKg-B26), *Escherichia coli* (PMFKg-B32), *Pseudomonas aeruginosa* (PMFKg-B28) and *Proteus mirabilis* (PMFKg-B29). All clinical isolates were a generous gift from the Institute of Public Health, Kragujevac. Bacteria are stored in microbiological collection at the Laboratory of Microbiology (Faculty of Science, University

Bacterial suspension were prepared from overnight cultures by the direct colony method. Colonies were taken directly from the plate and suspent into 5ml of sterile 0,85% saline. The turbidity of initial suspension was adjusted comparing with 0,5 Mc Farland standard (0,5 ml 1,17% w/v BaCl2 × 2H2O + 99,5 ml 1% w/v H2SO4) (Andrews, 2001). When adjusted to the turbidity of a 0,5 Mc Farland standard, a suspension of bacteria contains about 108 colony forming units (CFU)/ml. Ten-fold dilutions of initial suspension were additionally prepared

Antibacterial activity was tested by determining the minimum inhibitory concentration (MIC) using microdilution plate method with resazurin (Sarker et al., 2007). Briefly, the 96-well

which are expected in prepared plant extracts are given in Table 2.

Coumarins Coumarins Saponins Saponins

into sterile 0,85% saline to achieve 106 CFU/ml.

**2.1.4 Microdilution method** 

**2.1.3 Microorganisms** 

of Kragujevac).

**2.1.2 Extraction** 


Table 1. Traditional uses, biological activities and chemical constituents of selected plant species
