**4. Herbal extracts as antimicrobial agent**

Even before the role of microorganisms in disease pathogenesis was understood, plant-based medicines were used for treating such illnesses. It is recognized that plant molecules have antimicrobial properties; especially, EOs exhibit broad spectrum inhibitory activities against various Gram-positive and Gram-negative bacteria pathogens [20, 38, 57].

However, herbal extract medicine diminished as soon as modern antibiotics were discovered. Renewed recent interest in their use has been attributed to several factors, including the desire for antimicrobial compounds with even better safety and toxicity profiles [80]. Also, severity of bacterial infections has gone up even after the discovery of many antibiotics, mainly due to the reduced susceptibility to conventional antimicrobial agents shown by many important pathogens. Therefore, infectious diseases caused by bacteria are still one of the leading causes of deaths [57, 81]. In addition, toxicity due to side effects limits the prolonged use of high concentrations of available antibacterial drugs [57].

Most EOs possess at least some degree of antibacterial activity. Generally, EOs with phenolics and aldehydes exhibit better antibacterial efficacies [38]. In few cases, a main component of the oil has been observed to possess activity better than the EO. For example, carvacrol and eugenol from clove oil, terpinen-4-ol in *Melaleuca alternifolia* (tea tree oil), or thymol from oregano oil displays greater efficacy than specific oil. Many of the plant molecules are effective against drug-sensitive as well as drug-resistant strains [58, 82].

The methods used for establishing antibacterial activities are, usually, disc diffusion methods or agar or broth dilution methods. Although disc diffusion methods are popular, the data they offer are less useful than others. Agar and broth dilution methods, in which serial dilutions of the test oil in agar or broth media are inoculated with a known concentration of test organism, allow minimum inhibitory concentrations (MICs) to be determined [83]. The MIC is generally defined as the lowest concentration of EO that inhibits growth of the test organism. Although solubilization or dispersion in these systems may be problematic, MICs can help establish safe and effective final concentrations in formulated products.

Two principal reasons for performing the in vitro tests are as follows:

**•** Identification of antimicrobially active compounds

**•** Control of microbial susceptibilities toward approved antibiotics and antimicotics

The procedure from identification of antimicrobially active compounds for their use in humans to treat infectious disease is a multistep pathway, which includes pharmacological (concentration of the active compound at the site of action, half-life time, serum levels, dose– response relationship, etc.) and toxicological (e.g. toxicity, allergic responses, and interac‐ tions) aspects [84].

#### **4.1. Mode of action of bioactive compounds**

The health benefits of medicinal plants are ascribed to their bioactive compounds, known as phytochemicals. It has been estimated that 74% of pharmacologically active plant-derived compounds were discovered after following up on ethnomedicinal use of the plants [85]. Various phytochemicals are recognized to possess antimicrobial, anti-inflammatory, analgesic, anesthetic, antioxidant, neuroprotective, and antitumor activity, thus providing medicinal plants with great therapeutic as well disease-preventive potential [86].

However, detailed knowledge about the mode of action of EOs and other bioactive compounds is still lacking. In general, the mechanism of action of EOs is to alter the structure of the cytoplasmic membrane, disrupting the proton motive force, electron flow, active transport, and coagulation of cell contents [87, 88]. The mode of action of phytochemicals depends on the type of microorganism and is generally related to their outer membrane arrangement as well as cell wall structure. For example, antimicrobial action of EOs depends on their hydro‐ philic or lipophilic character. Certain components of EOs can act as uncouplers, which interfere with proton translocation over a membrane vesicle and subsequently interrupt ADP phos‐ phorylation [89]. Phytochemicals may also modulate transcription factors, redox-sensitive transcription factors, redox signaling, and inflammation [90].

#### *4.1.1. Terpenoids*

Specific terpenoids with functional groups, such as phenolic alcohols or aldehydes, interfere with membrane-integrated or associated enzyme proteins stopping their production or activity. It was found that some phenolic alcohols (e.g. carvacrol and thymol) cause a disrup‐ tion of the lipopolysaccharide outer layer followed by partial disintegration of the outer membrane [89]. The interaction of thymol with the membrane affects membrane permeability and results in the release of K+ ions and ATP [91]. In some cases, thymol can induce the release of lipopolysaccharides, but it does not affect chelating cations [92]. Thymol integrates within the polar head groups of the lipid bilayer, inducing alterations of the cell membrane. Zengin and Baysal [93] found that α-terpineol 1,8-cineole and linalool alter the function of cell membranes and the permeability of outer membranes of *Staphylococcus aureus* and *E. coli*. Bard *et al.* [94] reported that geraniol enhances the permeability of whole cells of *Candida albicans* and also increases the fluidity of both *C. albicans* membranes and dipalmitoyl phosphatidyl‐ choline DPPC liposomal membranes. Mendanha *et al.* [95] revealed that all the tested terpe‐ noids (nerolidol, menthol, pulegone, carvone, (+)-limonene, α-terpineol, and 1,8-cineole) increase the fluidity of cell membrane and exert cytotoxic effects on fibroblast cells. Yin *et al.* [96] found that borneol increases the fluidity of DPPC bilayer membranes. Terpenoid, isolated from purple prairie clover, petalostemumol, has significant activity against *Bacillus subtilis* and *S. aureus* [97]. Carvone is capable to disrupt pH gradient and membrane potential of cells. With increasing amount of carvone, Oosterhaven *et al.* [98] reported a decrease in the growth rate of *E. coli*, *Streptococcus thermophilus*, and *Lactococcus lactis* was caused by disturbing the metabolic energy status of cells.

#### *4.1.2. Phenolics*

Phenolic toxicity to microorganism is often explained by enzyme inhibition with the oxidized compounds, probably through reaction with sulfhydryl groups or in more nonspecific interactions with the proteins. The induced defense response includes formation of a lesion that limits the growth of the pathogen, where polyphenols and other antibiotic compounds accumulate [85].

Hydroxyl groups number as well sites in phenolics are thought to be related to their relative toxicity to microorganisms (increased hydroxylation results in increased toxicity) [85]. Thus, eugenol is considered bacteriostatic against both fungi and bacteria [88]. It was found that eugenol alters the membrane, affects the transport of ions and ATP, and changes the fatty acid profile of different bacteria [99]. It also acts against different bacterial enzymes, including ATPase, histidine carboxylase, amylase, and protease [100, 101]. Cinnamaldehyde is usually less powerful than eugenol [102], but extremely effective against *E. coli* and Salmonella *typhimurium* [92]. Catechin acts on different bacterial strains belonging to different species (*E. coli, Salmonella choleraesis, Klebsiella pneumonie, Serratia marcescens, B. subtilis, Pseudomonas aeruginosa*, and *S. aureus*) by generating hydrogen peroxide and by altering the permeability of the microbial membrane [85, 103]. It was found that polyphenols obstruct bacterial quorum sensing, that is, the production of small signal molecules by *E. coli, Pseudomonas putida,* and *Burkholderia cepacia* cells that trigger the exponential growth of a bacterial population [104, 105].

#### *4.1.3. Other compounds*

Flavonoids have been shown in vitro to be effective antimicrobial substances against a wide array of microorganisms [106]. Their activity is probably due to their ability to disrupt microbial membranes or to complex with extracellular and soluble proteins in bacterial cell walls [107, 108]. Moreover, Arora *et al.* [109] demonstrated that some flavonoids (naringenin and rutin) and isoflavonoids (genistein) decrease the membrane fluidity. Selvaray *et al*. [110] correlated bioactivities of flavonoids to their membrane localization and their induced changes in membrane fluidity. Thus, catechins inhibited in vitro *Vibrio cholerae*, *Streptococcus mutans*, *Shigella*, and other bacteria and microorganisms [97]. Several studies have documented the effectiveness of flavonoids such as swertifrancheside, glycyrrhizin (from licorice), and chrysin against multiple viruses [97, 106, 111]. It was found that membrane-interacting properties of flavonoids to modify permeability of cellular membranes could inhibit *E. coli* growth [112].

The mode of antimicrobial action of tannins could be related to their capability to inactivate enzymes, cell envelope, transport proteins, microbial adhesins, etc. Previous studies have shown that tannins can be toxic to filamentous fungi, yeasts, and bacteria. It was found that condensed tannins prevent growth and protease activity by binding the cell walls of ruminal bacteria [97].

The potential range of quinones' antimicrobial effects is great, due to its ability to complex irreversibly with nucleophilic amino acids in proteins, often leading to inactivation of the protein and loss of function. Possible targets in the microbial cell are cell wall polypeptides, surface-exposed adhesins, and membrane-bound enzymes. Quinones may also render substrates unavailable to the microorganism [85]. Hypericin, an anthraquinone from *Hyperi‐ cum perforatum*, has received much attention lately as an antimicrobial agent [97].

Several alkaloids have the properties to interact with artificial and biological membranes to change the fluidity in association with their pharmacological effects [113]. Berberine is one of isoquinoline alkaloids, which have been considered to possess anti-inflammatory and antimicrobial effects [114]. Also, it has been found that alkaloid sanguinarine possesses antibacterial activity against bacteria and pathogenic fungi [115].

The bacterial resistance is conferred by multidrug resistance pumps (MDRs), membrane translocases that extrude structurally unrelated toxins from the cell. These protect microbial cells from both synthetic and natural antimicrobials [116]. The mechanism of action of EOs depends on their chemical composition, amount of the single components, and their antimi‐ crobial activity is not attributable to a unique mechanism but is instead a cascade of reactions involving the entire bacterial cell [20]. Also, the synergistic effects of antibiotics and herbal extracts can provide successful therapy against drug-resistant bacteria. The use of herbal extracts and phytochemicals can be of great significance in therapeutic applications and could help control the problem of multidrug-resistant organisms [85].

#### **4.2. Effect of some herbal extracts on selected food-borne microorganisms**

Here, some original results of antibacterial activity of EO and supercritical extracts from aromatic herbs (from Montenegro) – sage (*Salvia officinalis*), rosemary (*Rosmarinus officinalis*), oregano (*Origanum vulgare*), and savory (*Satureja montana*) – against some pathogenic bacteria important in food industry are presented.

Leaves from selected herbs were collected in the central southern part of Montenegro, air-dried and stored in double-layer paper bags at the room temperature, until further analysis.

The selected test organisms, used to evaluate the antimicrobial activity of the herbal extracts, were as follows: Gram-positive (*Bacillus cereus* ATCC 11778, *S. aureus* ATCC 25923, and *Listeria innocua* ATCC 33090) and Gram-negative (*Salmonella enteritidis* ATCC 13076 and *E. coli* ATCC 25922).

To obtain EOs, herb material was subjected to hydrodistillation in a Clevenger-type apparatus for 2 hours according to Yugoslav Pharmacopoeia IV. Supercritical CO2 extraction (SCE) procedure is previously described in detail [117], while the extraction conditions were: temperature 40 °C, pressure 100 bar, extraction time 360 min; and CO2 flow rate 0.3 kg CO2/h. MIC values were determined for extracts displaying antimicrobial properties in screening studies, using a modified microdilution broth method [118]. Briefly, the extracts were first dissolved in DMSO, then diluted in sterile water and tested over a range of concentrations from 0.09 to 25 mg/ml against overnight broth cultures of selected bacteria grown to a population of 106 CFU/ml in tryptic soy broth (TSB). Microplates were incubated at optimum growth temperature for each bacterial strain, and growth was monitored by measuring absorbance at 600 nm every 45 min over 18 hours, using a microplate reader.

The antibacterial activity, for investigated herbal extracts against some pathogenic bacteria important in food industry, are summarized in Table 2.


**Table 2.** Antibacterial activity of isolates obtained by hydrodistillation and SCE from selected herbs against some pathogenic bacteria important in food industry

According to the results presented in Table 2, sage extracts had the highest antibacterial efficiency against tested bacteria strains (MIC=0.09–3.13 mg/ml) followed by oregano (MIC=0.09–3.13 mg/ml) extracts. In this study, the carriers of antimicrobial activity of the sage oil were probably α-thujone and camphor. High efficiency of oregano extracts could be attributed to the high content of compounds with known antimicrobial activity in examinated samples, such as phenolic components, thymol and its isomer carvacrol, as well as its precur‐ sors, γ-terpinene and p-cymene. Rosemary extracts showed somewhat smaller activity than expected, probably because rosemary is a cultivated herb, whereas all other examined herbs were wild growing.

Among tested bacteria, *B. cereus* and *S. aureus* were the most sensitive to presence of all tested extracts, especially to presence of sage extracts. Thus, Gram-positive bacteria seemed to be more susceptible to all tested herb extracts.

The results of the bioassays show that tested extracts obtained by SC-CO2 extraction from different pretreated herb matrices exhibited the same or weaker antimicrobial activity when compared to the EO obtained by hydrodistillation.

The presented results in this study confirm the facts that plant molecules have significant antibacterial activity and therefore can be used as a strong antimicrobial agent. The use of EOs in foods as preservatives is limited due to toxicological aspects, but also possible reasons for this limitation may be the strong smell and taste of these substances when used at effective doses. The SC-CO2 extracts bear the closest natural smell and taste of original material, thus further investigation should point to various combinations of investigated extracts, which should improve the level of inhibition due to synergistic effects.
