**1. Introduction**

Man is in constant contact with a large number of different bacteria which temporarily or permanently inhibit his body creating temporary or permanent community. Relations which are thus established are various and very complex, from those positive to those whose consequences for man are extremely negative. Very often, both on and in man's body, bacteria which have the ability to cause an infection are present. This ability of pathogenic bacteria is reflected in possession of certain pathogenicity factors. A set of factors which enable successful invasion and damage of the host are: toxins, surface structures and enzymes. Between the host and the pathogen very complex relations are established whose income depends on host's characteristics as well as on pathogen's characteristics.

Infections caused by bacteria can be prevented, managed and treated through anti-bacterial group of compounds known as antibiotics. Antibiotics are natural, semi-synthetic or synthetic compounds that kill or inhibit the growth of bacteria. When bacteria are exposed to an antibiotic, they doubly respond: i) they are sensitive what cause the inhibition of their growth, division and death or ii) they can remain unaffected or resistant.

The resistance of bacteria to antibiotics can be natural (intrinsic) or acquired. Natural resistance is achieved by spontaneous gene mutation. The acquired resistence occurs after the contact of bacteria with an antibiotic as a result of adaptation of a species to adverse environmental conditions. In such population, an antibiotic as a selective agent, acts on sensitive individuals, while resistant survive and become dominant. Bacteria gain antibiotic resistance due to three reasons namely: (i) modification of active site of the target resulting in reduction in the efficiency of binding of the drug, (ii) direct destruction or modification of the antibiotic by enzymes produced by the organism or, (iii) efflux of antibiotic from the cell (Sheldon, 2005). The evolution of antibacterial resistance in human pathogenic and commensal microorganisms is the result of the interaction between antibiotic exposure and the transmission of resistance within and between individuals. It is especially interesting the phenomenon of horizontally gene transfer. Extrachromosomal DNA material, so-called plasmids, often carry genes of resistance and can transfer information within and between the individuals of the same or related bacterial species, thus also spreading the resistance. Transformation, transduction and conjugation represent the horizontal gene transfer mechanisms of resistance between the bacteria.

Antibacterial Activity of Naturally Occurring Compounds from Selected Plants 3

group (-OH) attached to an aromatic phenolic group. The site(s) and number of hydroxyl groups on the phenol group are thought to be related to their relative toxicity to microorganisms, with evidence that increased hydroxylation results in increased toxicity (Geissman, 1963, as cited in Cowan, 1999). Quinones have aromatic rings with two ketone substitutions. Quinones have a potential to form irreversible complex with nucleophilic amino acids in proteins (Stern et al., 1996, as cited in Cowan, 1999). This could explain their antibacterial properties. Probable targets in the microbial cell are surface-exposed adhesins, cell wall polypeptides and membrane-bound enzymes. Flavones are phenolic structures containing one carbonyl group. The addition of a 3-hydroxyl group yields a flavonol. Flavonoids are also hydroxylated phenolic substances but occur as a C6-C3 unit linked to an aromatic ring. Flavones, flavonoids and flavonols have been known to be synthesized by plants in response to microbial infection so it is not surprising that they have been found, *in vitro*, to be effective antimicrobial substances against a wide array of microorganisms (Dixon et al., 1983, as cited in Cowan, 1999). Their activity is probably due to their ability to complex with extracellular and soluble proteins and to complex with bacterial cell walls. Lipophilic flavonoids may also disrupt microbial membranes (Tsuchiya et al., 1996, as cited in Cowan, 1999). Tanins, a group of polymeric phenolic substances, are found in almost every plant part: bark, wood, leaves, fruits and roots. They are divided into two groups, hydrolyzable and condensed tannins. In plant tissue, tannins have been synthesized and accumulated after microbial attack. Their mode of antimicrobial action may be related to their ability to inactivate microbial adhesins, enzymes, cell envelope transport proteins, because of a property known as astringency. Coumarins are benzo-α-pyrones and could be categorised as: simple coumarins and cyclic coumarins (furanocoumarins and pyranocoumarins) (Ojala, 2001). Coumarins have been found to stimulate macrophages, which could have an indirect negative effect on

infections (J. R. Casley-Smith & J. R.Casley-Smith, 1997, as cited in Cowan, 1999).

1997, as cited in Cowan, 1999).

of cell respiration (Kovacevic, 2004).

**1.1.2 Plant extracts as potential antibacterial agents** 

Terpenes are a large and varied class of organic compounds built up from isoprene subunits, while the terpenoids are oxygen-containing analogues of the terpenes. According to number of isoprene subunits, there are monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), triterpenes (C30), tetraterpenes (C40) and polyterpenes (Kovacevic, 2004). Monoterpenes, diterpenes and sesquiterpenes are the primary constituents of the essential oils. The mechanism of antibacterial action of terpenes is not fully understood but is speculated to involve membrane disruption by the lipophilic compounds. Accordingly, Mendoza et al., 1997 found that increasing the hydrophilicity of kaurene diterpenoids by addition of a methyl group drastically reduced their antimicrobial activity. (Mendoza et al.,

Alkaloids, one of the earliest isolated bioactive compounds from plants, are heterocyclic nitrogen compounds. They are derived from amino acids, and the nitrogen gives them alkaline properties. The mechanism of antibacterial action is attributed to their ability to intercalate with DNA, inhibition of enzymes (esterase, DNA-, RNA-polymerase), inhibition

The screening of plant extracts has been of great interest to scientists for the discovery of

new compound effective in the treatment of bacterial infection. Plant extracts exhibit:

Having in mind the current progress of resistance spreading and resilience of larger and larger number of bacteria to traditional antibiotics as well as a way of transmitting the gene of resistance, above all via plasmids, one can conclude that the ability of obtaining bacterium resistance to antibiotics represents a very dynamic and unpredictable phenomenon. For that reason, bacterial resistance to antibiotics represents a major health problem. Solving this problem and search for new sources of antimicrobial agents is a worldwide challenge and the aim of many researches of scientific and research teams in science, academy institutions, pharmaceutical companies. One of the approaches in solving this issue is testing the biologically active compounds of plant origin.

## **1.1 Plants as potential antibacterial agents**

Healing potential of plants has been known for thousands of years. Medicinal use of plants and their products was passed down from generation to generation in various parts of the world throughout its history and has significantly contributed to the development of different traditional systems of medicine. Even today, the World Health Organization (WHO) has estimated that approximately 80% of the global population relies on traditional herbal medicines as part of standard healthcare (Foster et al., 2005). Many drugs presently prescribed by physicians are either directly isolated from plants or are artificially modified versions of natural products. In Western countries, approximately 25% of the drugs used are of natural plant origin (Payne et al., 1991).

Plants produce a whole series of different compounds which are not of particular significance for primary metabolism, but represent an adaptive ability of a plant to adverse abiotic and biotic environmental conditions. They can have a remarkable effect to other plants, microorganisms and animals from their immediate or wider environment. All these organic compounds are defined as biologically active substances, and generally represent secondary metabolites, given the fact that they occur as an intermediate or end products of secondary plant metabolism. These secondary metabolites, apart from determining unique plant traits, such as: colour and scent of flowers and fruit, characteristic flavour of spices, vegetables, they also complete the functioning of plant organism, showing both biological and pharmacological activity of a plant. Therefore, medicinal properties of plants can be attributed to secondary metabolites (Hartmann, 2008).

#### **1.1.1 Antibacterial secondary metabolites**

It is known and proved by *in vitro* experiments that plants produce a great number of secondary metabolites that have antimicrobial activity (Iwu et al., 1999; Cowan, 1999; Rios & Recio, 2005; Cos et al., 2006). These plant-formed antibiotics have been classified as phytoanticipins, which are preformed inhibitory compounds, or phytoalexins, which are derived from precursors via *de novo* synthesis in response to microbial attack (VanEtten et al., 1994). Antibacterial secondary metabolites are usually classified in three large molecule families: phenolics, terpenes and alkaloids.

The phenolics and polyphenols are one of the largest groups of secondary metabolites that have exhibited antimicrobial activity. Important subclasses in this group of compounds include phenols, phenolic acids, quinones, flavones, flavonoids, flavonols, tannins and coumarins. Phenols are a class of chemical compounds consisting of a hydroxyl functional

Having in mind the current progress of resistance spreading and resilience of larger and larger number of bacteria to traditional antibiotics as well as a way of transmitting the gene of resistance, above all via plasmids, one can conclude that the ability of obtaining bacterium resistance to antibiotics represents a very dynamic and unpredictable phenomenon. For that reason, bacterial resistance to antibiotics represents a major health problem. Solving this problem and search for new sources of antimicrobial agents is a worldwide challenge and the aim of many researches of scientific and research teams in science, academy institutions, pharmaceutical companies. One of the approaches in solving

Healing potential of plants has been known for thousands of years. Medicinal use of plants and their products was passed down from generation to generation in various parts of the world throughout its history and has significantly contributed to the development of different traditional systems of medicine. Even today, the World Health Organization (WHO) has estimated that approximately 80% of the global population relies on traditional herbal medicines as part of standard healthcare (Foster et al., 2005). Many drugs presently prescribed by physicians are either directly isolated from plants or are artificially modified versions of natural products. In Western countries, approximately 25% of the drugs used are

Plants produce a whole series of different compounds which are not of particular significance for primary metabolism, but represent an adaptive ability of a plant to adverse abiotic and biotic environmental conditions. They can have a remarkable effect to other plants, microorganisms and animals from their immediate or wider environment. All these organic compounds are defined as biologically active substances, and generally represent secondary metabolites, given the fact that they occur as an intermediate or end products of secondary plant metabolism. These secondary metabolites, apart from determining unique plant traits, such as: colour and scent of flowers and fruit, characteristic flavour of spices, vegetables, they also complete the functioning of plant organism, showing both biological and pharmacological activity of a plant. Therefore, medicinal properties of plants can be

It is known and proved by *in vitro* experiments that plants produce a great number of secondary metabolites that have antimicrobial activity (Iwu et al., 1999; Cowan, 1999; Rios & Recio, 2005; Cos et al., 2006). These plant-formed antibiotics have been classified as phytoanticipins, which are preformed inhibitory compounds, or phytoalexins, which are derived from precursors via *de novo* synthesis in response to microbial attack (VanEtten et al., 1994). Antibacterial secondary metabolites are usually classified in three large molecule

The phenolics and polyphenols are one of the largest groups of secondary metabolites that have exhibited antimicrobial activity. Important subclasses in this group of compounds include phenols, phenolic acids, quinones, flavones, flavonoids, flavonols, tannins and coumarins. Phenols are a class of chemical compounds consisting of a hydroxyl functional

this issue is testing the biologically active compounds of plant origin.

**1.1 Plants as potential antibacterial agents** 

of natural plant origin (Payne et al., 1991).

attributed to secondary metabolites (Hartmann, 2008).

**1.1.1 Antibacterial secondary metabolites** 

families: phenolics, terpenes and alkaloids.

group (-OH) attached to an aromatic phenolic group. The site(s) and number of hydroxyl groups on the phenol group are thought to be related to their relative toxicity to microorganisms, with evidence that increased hydroxylation results in increased toxicity (Geissman, 1963, as cited in Cowan, 1999). Quinones have aromatic rings with two ketone substitutions. Quinones have a potential to form irreversible complex with nucleophilic amino acids in proteins (Stern et al., 1996, as cited in Cowan, 1999). This could explain their antibacterial properties. Probable targets in the microbial cell are surface-exposed adhesins, cell wall polypeptides and membrane-bound enzymes. Flavones are phenolic structures containing one carbonyl group. The addition of a 3-hydroxyl group yields a flavonol. Flavonoids are also hydroxylated phenolic substances but occur as a C6-C3 unit linked to an aromatic ring. Flavones, flavonoids and flavonols have been known to be synthesized by plants in response to microbial infection so it is not surprising that they have been found, *in vitro*, to be effective antimicrobial substances against a wide array of microorganisms (Dixon et al., 1983, as cited in Cowan, 1999). Their activity is probably due to their ability to complex with extracellular and soluble proteins and to complex with bacterial cell walls. Lipophilic flavonoids may also disrupt microbial membranes (Tsuchiya et al., 1996, as cited in Cowan, 1999). Tanins, a group of polymeric phenolic substances, are found in almost every plant part: bark, wood, leaves, fruits and roots. They are divided into two groups, hydrolyzable and condensed tannins. In plant tissue, tannins have been synthesized and accumulated after microbial attack. Their mode of antimicrobial action may be related to their ability to inactivate microbial adhesins, enzymes, cell envelope transport proteins, because of a property known as astringency. Coumarins are benzo-α-pyrones and could be categorised as: simple coumarins and cyclic coumarins (furanocoumarins and pyranocoumarins) (Ojala, 2001). Coumarins have been found to stimulate macrophages, which could have an indirect negative effect on infections (J. R. Casley-Smith & J. R.Casley-Smith, 1997, as cited in Cowan, 1999).

Terpenes are a large and varied class of organic compounds built up from isoprene subunits, while the terpenoids are oxygen-containing analogues of the terpenes. According to number of isoprene subunits, there are monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), triterpenes (C30), tetraterpenes (C40) and polyterpenes (Kovacevic, 2004). Monoterpenes, diterpenes and sesquiterpenes are the primary constituents of the essential oils. The mechanism of antibacterial action of terpenes is not fully understood but is speculated to involve membrane disruption by the lipophilic compounds. Accordingly, Mendoza et al., 1997 found that increasing the hydrophilicity of kaurene diterpenoids by addition of a methyl group drastically reduced their antimicrobial activity. (Mendoza et al., 1997, as cited in Cowan, 1999).

Alkaloids, one of the earliest isolated bioactive compounds from plants, are heterocyclic nitrogen compounds. They are derived from amino acids, and the nitrogen gives them alkaline properties. The mechanism of antibacterial action is attributed to their ability to intercalate with DNA, inhibition of enzymes (esterase, DNA-, RNA-polymerase), inhibition of cell respiration (Kovacevic, 2004).

#### **1.1.2 Plant extracts as potential antibacterial agents**

The screening of plant extracts has been of great interest to scientists for the discovery of new compound effective in the treatment of bacterial infection. Plant extracts exhibit:

Antibacterial Activity of Naturally Occurring Compounds from Selected Plants 5

*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

> **Biological activity**

antibacterial, antifungal activity (Petrovic et al., 2004; Rani & Khullar, 2004; Mares et al., 2005)

antibacterial, antifungal, antiviral activity ( Velickovic et al., 2003; Nolkemper et al., 2006; Horiuchi et al., 2007; Weckesser et

al., 2007)

2006)

antibacterial activity

(Opalchenova & Obreshkova, 1999)

antibacterial, antifungal, antiviral activity ( Iauk et al., 2003; Ertürk, 2006; Nolkemper et al.,

**Chemical constituents** 

simple phenols, phenolic acids,

tannins, terpenoids (Lu & Foo, 1999; 2002; Durling et al., 2007)

flavonoids ( Herodež et al., 2003; Patora & Klimek , 2002), phenolic acids (Herodež et al., 2003; Canadanović –Brunet et al., 2008), simple phenols, tannins (Hohmann et al., 1999)

polyphenols ( Kratchanova et al., 2010), *cis*-cinnamic, *trans*cinnamic, *p*-coumaric and ferulic acid (Obreshkova et al., 2001), saponins ( Miyase & Matsushima, 1997 )

flavonoids, coumarins,

sesquiterpene lactones (Zidorn, 2008), inulin , flavonoids, coumarins (Dem'yanenko & Dranik, 1971), tannins, phenolic acids ( Sareedenchai & Zidorn, 2010 )

chemical constituent is shown in Table 1.

(Saric, 1989)

1989)

*officinalis* to reduce indigestion and flatulence,

> as a mild sedative, to treat headache,

and insomnia,

as a heart tonic, an expectorant,

migraine, nervous tension

to treat cold, fever and cough (Saric, 1989)

as a diuretic (Saric, 1989) as an antiseptic for wounds and injuries (Opalchenova and Obreshkova, 1999).

for disorders of the digestive system, as antiseptic for sore throats, ulcers,

**Traditional use** 

for cleansing the liver and benefiting the gallbladder

to treat insect bites, mouth and gum infections and vaginal discharge for night sweats (Saric,

improving digestion, for a diarrhea, as diuretic,

**Plant species** 

Fam. Asteraceae *Cichorium intybus*

Fam. Lamiaceae *Salvia officinalis*

*Melissa* 

*Clinopodium vulgare* 


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 bacterial growth (Cos et al., 2006).
