**3.3 Antibacterial activity of** *Melissa officinalis*

The results of antibacterial activity of water, ethanol and ethyl acetate extract from *Melissa officinalis* are presented on the Figure 4. The tested extracts showed equable antibacterial activity, based on the results of statistic analysis, no difference was noted (*p*>0.05). Water extract acted in the interval from 0.31 mg/ml tо 20 mg/ml; ethanol extract from 0.62 mg/ml to >20 mg/ml, and ethyl acetate extract from 1.25 mg/ml to 20 mg/ml. Ethanol extract did not act on three species of Gram-negative bacteria. Antimicrobial activity of different *Melissa officinalis* extracts was also tested by other scientists who showed different level of antimicrobial activity with their research (Uzun et al., 2004; Ertürk, 2006; Iauk et al., 2003).

The tested bacteria showed sensitivity at different concentrations. The concentrations were ranged from 0.31 tо 20 mg/ml. The greatest sensitivity was noted for standard strains: *Staphylococcus aureus*; *Escherichia coli* and *Pseudomonas aeruginosa* only to water extract.

Antibacterial Activity of Naturally Occurring Compounds from Selected Plants 13

Fig. 5. Antibacterial activity of *Clinopodium vulgare* extracts expressed as MIC values (mg/ml)

Among the tested extracts, the most active was ethanol extract, then ethyl acetate, and the weakest water extract. Statistically significant difference was noted between the activity of ethanol extract, on one hand, and ethyl acetate and water extract on the other hand (*p*H20 = 0.008; *p*EtAc = 0.032). MIC values were ranged in the interval from 1.25 mg/ml to 20 mg/ml. The bacteria showed different level of sensitivity to the tested extracts (Figure 6.). They were the most sensitive to ethanol extract (MIC for most of the bacterium was 5 mg/ml), and then to ethyl acetate extract (MIC for most of the bacterium was 10 mg/ml). Most of the bacteria showed weak sensitivity to water extract with MIC values of 20 mg/ml. For *Staphylococcus aureus* ATCC 25923, *Pseudomonas aeruginosa* ATCC 27853 and *Staphylococcus aureus*, water extract acted at lower concentrations (MIC = 2.5 and 10 mg/ml). The most sensitive bacterium was *Staphylococcus aureus* ATCC 25923, growth inhibition of this bacterium was

Antibacterial activity of *T. anthriscus* extracts is not explored enough. Inhibitory effect was tested on phytopathogenic bacteria and especially the action of water, ethanol and ethyl acetate extract was shown on *Pseudomonas glycinea* (Brkovic et al., 2006). Methanol extract of fruits slowed germination of spores down and inhibited the growth of vegetative cells of

The results of antibacterial activity of water, ethanol and ethyl acetate extract from *Aegopodium podagraria* are presented on the Figure 7. The extracts showed low antibacterial

**3.5 Antibacterial activity of Torilis anthriscus** 

noted at 1.25 mg/ml and 2.5 mg/ml.

*Bacillus subtilis* (Cho et al., 2008).

**3.6 Antibacterial activity of** *Aegopodium podagraria* 

Fig. 4. Antibacterial activity of *Melissa officinalis* extracts expressed as MIC values (mg/ml)

The most tested bacteria showed sensitivity at 10 mg/ml for ethyl acetate extract. *Escherichia coli, Pseudomonas aeruginosa* and *Proteus mirabilis* showed resistance to ethanol extract. Weaker activity of lemon balm to mentioned Gram-negative bacterium was also noted in the work of Canadanović-Brunet et al., 2008.

#### **3.4 Antibacterial activity of** *Clinopodium vulgare*

The extracts manifested different level of antibacterial activity, the results are shown on the Figure 5. The ethanol extract acted in concentrations from 1.25 mg/ml tо >20 mg/ml, ethyl acetate and acetone extract from 0.62 mg/ml tо 20 mg/ml. Ethyl acetate and acetone extract acted better than ethanol extract. Statistically significant difference was noted (*p*ЕtAc=0.015 и *p*AcOH=0.018). Between ethyl acetate and acetone extract no statistically significant difference was noted in the activity (*p*=0.756).

The tested bacteria in most of the cases showed sensitivity at 10 mg/ml and 20 mg/ml. Gram-positive bacteria *Bacillus subtilis* and *Staphylococcus aureus* ATCC 25923 were the most sensitive to the action of *Clinopodium vulgare* extracts*.* MIC values were in the interval from 0.62 mg/ml tо 2.5 mg/ml. *Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecalis, Escherichia coli* and *Pseudomonas aeruginosa* manifested the resistance to the tested concentrations of ethanol extract*.* Opalchenova & Obreshkova, 1999 showed the action of ethanol extract to G+ and G- bacteria but only at 5% of extract's concentration, while Sarac & Ugur, 2007 did not notice the action of ethanol extract to the tested bacteria. These results are in accordance with the shown activity of ethanol extract in this work.

Fig. 4. Antibacterial activity of *Melissa officinalis* extracts expressed as MIC values (mg/ml)

work of Canadanović-Brunet et al., 2008.

was noted in the activity (*p*=0.756).

in this work.

**3.4 Antibacterial activity of** *Clinopodium vulgare* 

The most tested bacteria showed sensitivity at 10 mg/ml for ethyl acetate extract. *Escherichia coli, Pseudomonas aeruginosa* and *Proteus mirabilis* showed resistance to ethanol extract. Weaker activity of lemon balm to mentioned Gram-negative bacterium was also noted in the

The extracts manifested different level of antibacterial activity, the results are shown on the Figure 5. The ethanol extract acted in concentrations from 1.25 mg/ml tо >20 mg/ml, ethyl acetate and acetone extract from 0.62 mg/ml tо 20 mg/ml. Ethyl acetate and acetone extract acted better than ethanol extract. Statistically significant difference was noted (*p*ЕtAc=0.015 и *p*AcOH=0.018). Between ethyl acetate and acetone extract no statistically significant difference

The tested bacteria in most of the cases showed sensitivity at 10 mg/ml and 20 mg/ml. Gram-positive bacteria *Bacillus subtilis* and *Staphylococcus aureus* ATCC 25923 were the most sensitive to the action of *Clinopodium vulgare* extracts*.* MIC values were in the interval from 0.62 mg/ml tо 2.5 mg/ml. *Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecalis, Escherichia coli* and *Pseudomonas aeruginosa* manifested the resistance to the tested concentrations of ethanol extract*.* Opalchenova & Obreshkova, 1999 showed the action of ethanol extract to G+ and G- bacteria but only at 5% of extract's concentration, while Sarac & Ugur, 2007 did not notice the action of ethanol extract to the tested bacteria. These results are in accordance with the shown activity of ethanol extract

Fig. 5. Antibacterial activity of *Clinopodium vulgare* extracts expressed as MIC values (mg/ml)

#### **3.5 Antibacterial activity of Torilis anthriscus**

Among the tested extracts, the most active was ethanol extract, then ethyl acetate, and the weakest water extract. Statistically significant difference was noted between the activity of ethanol extract, on one hand, and ethyl acetate and water extract on the other hand (*p*H20 = 0.008; *p*EtAc = 0.032). MIC values were ranged in the interval from 1.25 mg/ml to 20 mg/ml. The bacteria showed different level of sensitivity to the tested extracts (Figure 6.). They were the most sensitive to ethanol extract (MIC for most of the bacterium was 5 mg/ml), and then to ethyl acetate extract (MIC for most of the bacterium was 10 mg/ml). Most of the bacteria showed weak sensitivity to water extract with MIC values of 20 mg/ml. For *Staphylococcus aureus* ATCC 25923, *Pseudomonas aeruginosa* ATCC 27853 and *Staphylococcus aureus*, water extract acted at lower concentrations (MIC = 2.5 and 10 mg/ml). The most sensitive bacterium was *Staphylococcus aureus* ATCC 25923, growth inhibition of this bacterium was noted at 1.25 mg/ml and 2.5 mg/ml.

Antibacterial activity of *T. anthriscus* extracts is not explored enough. Inhibitory effect was tested on phytopathogenic bacteria and especially the action of water, ethanol and ethyl acetate extract was shown on *Pseudomonas glycinea* (Brkovic et al., 2006). Methanol extract of fruits slowed germination of spores down and inhibited the growth of vegetative cells of *Bacillus subtilis* (Cho et al., 2008).

#### **3.6 Antibacterial activity of** *Aegopodium podagraria*

The results of antibacterial activity of water, ethanol and ethyl acetate extract from *Aegopodium podagraria* are presented on the Figure 7. The extracts showed low antibacterial

Antibacterial Activity of Naturally Occurring Compounds from Selected Plants 15

activity. Only the ethanol extract activity stands out in relation to water extract, which was confirmed by statistis analysis (*p*=0.035). Ethanol extract inhibited the growth of most of the bacteria. Water extract only acted on two bacteria, and ethyl acetate extract on four bacteria.

The tested bacteria showed significant sensitivity to ethanol extract, exceptions were *Escherichia coli* ATCC 25922 and *Pseudomonas aeruginosa* ATCC 27853. Growth inhibition occurred at concentrations from 0.62 mg/ml tо 5 mg/ml. All bacteria, except *Pseudomonas aeruginosa* ATCC 27853 and *Staphylococcus aureus* ATCC 25923, were resistant to water extract, while *Escherichia coli* ATCC 25922 was resistant to all three extracts. Bacteria did not show any significant sensitivity to ethyl acetate extract. Ethyl acetate extract acted on *Enterobacter cloacae, Klebsiella pneumoniae, Pseudomanas aeruginosa* ATCC 27853 and *Staphylococcus aureus* ATCC 25923. *Staphylococcus aureus* ATCC 25923 was the most sensitive

and only partial effect on *Bacillus subtilis*, *Staphylococcus aureus*, *Staphylococcus epidermidis* and *Pseudomonas aeruginosa* was noted as well as on *Fusarium culmorum* and *Heterobasidion annosum*, phytopathogenic fungi. Brkovic et al., 2006 also noted the effect on phytopathogenic bacteria. Similar results were obtained for methanol extract (Ojala et al., 2000) and for ethanol extract in this study were expected since the methanol and ethanol are solvents of similar polarity and

*Cytisus nigricans* extracts showed weaker antibacterial activity than other tested plants. They mostly acted at the highest tested concentration (20 mg/ml). Acetone extract did not act on *Escherichia coli*. There is no statistically significant action difference between extracts (*p*<0.05). Bacteria sensitivity to tested extracts is shown on the Figure 8. The most significant results showed the following bacteria: *Bacillus subtilis* (2.5 mg/ml, 5 mg/ml), *Staphylococcus aureus* ATCC 25923 (2.5 mg/ml, 5 mg/ml) and *Pseudomonas aeruginosa* ATCC 27853 (1.25 mg/ml, 5 mg/ml, 10 mg/ml). Growth of other bacteria was inhibited at approximately same concentration (20 mg/ml), and *Escherichia coli* also showed the resistance to *Cytisus nigricans*

extracts. Antibacterial activity of this plant was tested for the first time in this study.

*Cytisus capitatus* extracts showed equable antibacterial activity. MICs were in the interval from 5 mg/ml tо >20 mg/ml for ethanol extract, from 1.25 mg/ml tо >20 mg/ml for ethyl acetate extract and from 1.25 mg/ml to >20 mg/ml for acetone extract. Based on statistic analysis no difference was noted in acting between extracts (*p*<0.05). The obtained results

The most sensitive bacteria to tested extracts were *Bacillus subtilis* and *Staphylococcus aureus* ATCC 25923. MIC for *Bacillus subtilis* showed at 5 mg/ml for ethanol extract, 2.5 mg/ml for ethyl acetate extract and 1.25 mg/ml for acetone extract. For *Staphylococcus aureus* ATCC 25923, MIC of ethanol extract was 10 mg/ml, ethyl acetate 1.25 mg/ml and acetone extract 2.5 mg/ml. *Escherichia coli* showed resistance to all three extracts. The results for

bacteria, yeasts and molds

Ethanol turned the best extractant of active compounds from this plant.

Ojala et al., 2000 tested methanol extract of *A. podagraria* on G+, G-

that there are similar groups of secondary metabolites isolated in extracts.

bacterium to *A. podagraria* extracts.

**3.7 Antibacterial activity of** *Cytisus nigricans* 

**3.8 Antibacterial activity of** *Cytisus capitatus* 

are presented on the Figure 9.

Fig. 6. Antibacterial activity of *Torilis anthriscus* extracts expressed as MIC values (mg/ml)

Fig. 7. Antibacterial activity of *Aegopodium podagraria* extracts expressed as MIC values (mg/ml)

Fig. 6. Antibacterial activity of *Torilis anthriscus* extracts expressed as MIC values (mg/ml)

Fig. 7. Antibacterial activity of *Aegopodium podagraria* extracts expressed as MIC values

(mg/ml)

activity. Only the ethanol extract activity stands out in relation to water extract, which was confirmed by statistis analysis (*p*=0.035). Ethanol extract inhibited the growth of most of the bacteria. Water extract only acted on two bacteria, and ethyl acetate extract on four bacteria. Ethanol turned the best extractant of active compounds from this plant.

The tested bacteria showed significant sensitivity to ethanol extract, exceptions were *Escherichia coli* ATCC 25922 and *Pseudomonas aeruginosa* ATCC 27853. Growth inhibition occurred at concentrations from 0.62 mg/ml tо 5 mg/ml. All bacteria, except *Pseudomonas aeruginosa* ATCC 27853 and *Staphylococcus aureus* ATCC 25923, were resistant to water extract, while *Escherichia coli* ATCC 25922 was resistant to all three extracts. Bacteria did not show any significant sensitivity to ethyl acetate extract. Ethyl acetate extract acted on *Enterobacter cloacae, Klebsiella pneumoniae, Pseudomanas aeruginosa* ATCC 27853 and *Staphylococcus aureus* ATCC 25923. *Staphylococcus aureus* ATCC 25923 was the most sensitive bacterium to *A. podagraria* extracts.

Ojala et al., 2000 tested methanol extract of *A. podagraria* on G+, G bacteria, yeasts and molds and only partial effect on *Bacillus subtilis*, *Staphylococcus aureus*, *Staphylococcus epidermidis* and *Pseudomonas aeruginosa* was noted as well as on *Fusarium culmorum* and *Heterobasidion annosum*, phytopathogenic fungi. Brkovic et al., 2006 also noted the effect on phytopathogenic bacteria. Similar results were obtained for methanol extract (Ojala et al., 2000) and for ethanol extract in this study were expected since the methanol and ethanol are solvents of similar polarity and that there are similar groups of secondary metabolites isolated in extracts.
