**2.1.2 Extraction**

6 Antimicrobial Agents

antibacterial, antifungal activity ( Ojala et al., 2000; Garrod et al.,

antibacterial activity ( Cho et al., 2008), anti-protozoal activity ( Youn et

al., 2004)

2002)

2003)

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

The aerial parts of *Clinopodium vulgare, Aegopodium podagraria, Torilis anthriscus, Dorycnium pentaphyllum, Melilotus albus, Cytisus nigricans* and *Cytisus capitatus* were collected from the different regions of Serbia, during summer 2006 and 2009 while *Cichorium intybus* (root), *Salvia officinalis* (leaves) and *Melissa officinalis* (leaves) were supplied from the commercial source. Identification and classification of the plant material was performed at the Faculty of Science, University of Kragujevac. The voucher specimens of the plants are deposited in the Herbarium of the Faculty of Science, University of Kragujevac. The collected plant materials were air-dried under shade at room temperature and then ground into small pieces which

antibacterial activity (Acamovic-Djokovic et al.,

activity (Stamatis,

No data No data alkaloids ( Wink et al., 1983)

No data No data alkaloids (l-sparteine,

1979)

**Biological activity** 

**Chemical constituents** 

furano-coumarins ( Ojala et al.,

sesquiterpenoids (guaiane, humulene, germacrane, eudesmane) (Kitajima et al.,

coumarins (Stoker, 1964), saponins (Khodakov et al.,

phenylbutanone glucosides (dorycnioside), flavonoids, (Kazantzoglou et al., 2004)

sarothamnine, genisteine, lupanine, oxysparteine) (Wink

et al., 1983)

2000), polyacetilenes, falcarindiol ( Christensen & Brandt, 2006), flavonoids

(Cisowski, 1985)

coumarins (3),

2002 )

1996)

**Plant species** 

Fam. Apiaceae *Aegopodium podagraria*

*Torilis anthriscus*  syn. *Torilis japonica*

Fam. Fabaceae *Melilotus albus*

*Dorycnium pentaphyllum* 

*Cytisus capitatus* 

*Cytisus nigricans* 

species

**2.1 Materials and methods** 

were stored into paper bags at room temperature.

**2.1.1 Plant material** 

**Traditional use** 

for gout and sciatics

an expectorant and tonic (Duke et al., 2002), for flatulence (Manandhar,

as ointments for external

as an anticoagulant agent

No data antibacterial

(Saric, 1989)

2002)

ulcers,

(Saric, 1989)

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


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

## **2.1.3 Microorganisms**

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 of Kragujevac).

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

### **2.1.4 Microdilution method**

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

Antibacterial Activity of Naturally Occurring Compounds from Selected Plants 9

bacteria, plant species and the type of extract. The MIC values were in range from 0.019 mg/ml to >20mg/ml. In relation to positive control (cephalexin MIC 0. 00156 - >1mg/ml), the extracts showed lower activity. In general, according to obtained results, the following

• Detectable MICs were noticed in 100% of tested bacteria for *Cichorium intybus*, 70% for *Salvia officinalis*, 90% for *Melissa officinalis*, 83, 33% for *Clinopodium vulgare*, 100% for *Torilis anthriscus*, 33, 33% for *Aegopodium podagraria*, 96, 67% for *Cytisus nigricans*, 76, 67% for *Cytisus capitatus*, 60% for *Melilotus albus* and 96, 67% for *Dorycnium*

• Among tested plants, the best inhibitory effects showed acetone extract from *Salvia officinalis*, ethyl acetate and acetone extract from *Cichorium intybus* and ethanol extract from *Aegopodium podagraria*. Moderate antibacterial activity exhibited *Melissa officinalis*, *Clinopodium vulgare* (ethyl acetate and acetone extract), *Torilis anthriscus*, *Cytisus nigricans*, *Cytisus capitatus* and *Dorycnium pentaphyllum* while low activity showed *Clinopodium vulgare* (ethanol extract), *Melilotus albus* and *Aegopodium podagraria* (water

• The antibacterial activity of the tested extracts was closely associated with present

• The Gram-positive bacteria were more sensitive than the Gram-negative bacteria. The reason for higher sensitivity of the Gram-positive bacteria than Gram-negative bacteria could be ascribed to their differences in cell wall constituents and their arrangement. The Gram-positive bacteria contain a peptidoglycan layer, which is an ineffective permeability barrier while Gram-negative bacteria are surrounded by an additional outer membrane carrying the structural lipopolysaccharide components, which makes it impermeable to lipophilic solutes and porins constitute a selective barrier to the

• Among tested bacteria, the most sensitive bacteria were Gram positive bacteria: *B. subtilis* and *S. aureus* ATCC 25923. Susceptibility of *E. cloaceae*, *Ent. faecalis*, *K. pneumoniae*, *S. aureus*, *Ps. aeruginosa* ATCC 27853 was moderate. Clinical isolate of Gram negative bacteria, *Ps. aeruginosa*, *P. mirabilis*, *E. coli*, exhibited low susceptibility or

The results of antibacterial activity of ethanol, ethyl acetate and acetone extract from *Cichorium intybus* are presented on the Figure 2. Extracts showed different activity. Ethanol extract acted at concentrations of 2.5 mg/ml to 20mg/ml; ethyl acetate from 1.09 mg/ml to 8.75 mg/ml; acetone extract from 2.5 mg/ml to 5 mg/ml. Antibacterial activity of ethyl acetate and acetone extract is more pronounced than ethanol extract. Similar results were obtained by Nandagopal & Ranjitha Kumari, 2007, among tested extracts, ethyl acetate was one of the more active. Statistic analysis confirms the presented results. Activity of ethyl acetate (*p*EtAc=0.001) and acetone (*p*AcOH=0.002) extract was statistically significantly higher

The tested bacteria manifested different sensitivity level to the tested extracts. MIC values were ranged from 1.09 mg/ml to 20 mg/ml. The most sensitive bacterium to the tested

that the activity of ethanol extract. Acetone extract was the most active (*p*=0.018).

• Water extracts were less active than ethanol, ethyl acetate and acetone extracts.

remarks could be made:

*pentaphyllum*.

and ethyl acetate extract).

secondary metabolites.

resistance.

hydrophilic solutes (Nikaido, 2003).

**3.1 Antibacterial activity of** *Cichorium intybus* 

microplate was prepared by dispensing 100 μL of Mueller-Hinton broth (Torlak, Belgrade) into each well. A 100 μL from the stock solution of tested extract (concentration of 40mg/ml) was added into the first row of the plate. Then, two-fold, serial dilutions were performed by transferring 100 μl of solution from one row to another, using a multichannel pipette. The obtained concentration range was from 20 mg/ml to 0.156 mg/ml. Ten microlitres of each 106 CFU/ml bacterial suspension was added to wells. Finally, 10 μL of resazurin solution was added. Resazurin is an oxidation-reduction indicator used for the evaluation of microbial growth. It is a blue non-fluorescent dye that becomes pink and fluorescent when reduced to resorufin by oxidoreductases within viable cells (Figure 1.). The inoculated plates were incubated at 37°C for 24h. MIC was defined as the lowest concentration of the tested plant extracts that prevented resazurin color change from blue to pink.

Antibiotic cephalexin, dissolved in Mueller-Hinton broth, was used as positive control. Solvent control test was performed to study an effect of 10% DMSO on the growth of bacteria. It was observed that 10% DMSO did not inhibit the growth of bacteria. Each test included growth control and sterility control. All tests were performed in duplicate and MICs were constant.
