**4. Molecules involved in antimicrobial activity**

Plants, herbs, spices and other products of vegetable origin must be seen as multi ingredient mixtures. Thus, there is a high probability that a product may have different compounds

*bidentata* extract was ineffective against all bacterial strains. The largest inhibition area was

As refered before, the unavailability of conventional antibiotics drives people to search in their own local products solutions for bacterial contamination of food and for infectious diseases. In a study conducted in Algeria, the antibacterial properties of four types of local berries (*Crataegus azarolus*, *Crataegus monogyna*, *Ziziphus lotus* and *Eleagnus angustifolia*) and three date (*Phoenix dactylifera*) varieties were analyzed. The targets were seven strains of *Salmonella* isolated from poultry industry. The bacterial strains were shown to be resistant to ticarcillin, amoxicillin, chloramphenicol and co-trimoxazole. Despite local berries extracts were ineffective against the bacteria, the date fruits results have shown moderate inhibition of *Salmonella*. An *Escherichia coli* strain was used as control and its inhibition diameters were

*Cassia tora*, *Momordica charantia* and *Calendula officinalis* are herbs used by ayurvedic medicine to treat psoriasis and other dermatological episodes. Aqueous and organic extracts of these plants were tested against *Staphylococcus aureus*, *Bacillus subtilis*, *Pseudomonas aeruginosa* and *Escherichia coli*. Results have shown that aqueous extracts were more effective than extracts obtained using organic solvents. *Staphylococcus aureus* was the most susceptible bacteria to any of the four herbs tested. However, none of inhibition diameters produced was larger than the diameter produced by the antibiotic used for comparison, streptomycin

In Brazilian folk medicine, a very large number of plants is used for treatmente of various diseases. This is due not only to ancestral tribal tradition, but mainly to the extreme abundance of vegetable raw materials in tropical environment. One of the plants employed with therapeutic objectives is *Plectranthus ornatus*, one of the many species of *Plectranthus* genus. In a study conducted in Portugal aimed at isolating the antimicrobial components of this plant, MIC were determined by microdilution. Extracts obtained by using different solvents were tested against bacterial strains, Gram positive and Gram negative and *Candida albicans*. The main experimental results show an MIC of 31.25 μg/mL against *Streptococcus faecalis*, using an acetone extract and an MIC of 125 μg/mL against *Staphylococcus aureus*, using a methanol/water extract. Extracts obtained with other organic solvents were devoid

Beyond the traditional plants and herbs, other "vegetable" edible products are becoming object of attention. Seaweeds are part of oriental food habits for long, like the use of seaweeds in preparation of sushi-like dishes in Japanese cuisine, but the interest in its antimicrobial and other biological activity is recent. Methanolic extracts of six species of edible Irish seaweeds proved to be effective against food pathogenic bacteria like *Listeria monocytogenes*, *Salmonella abony*, *Enterococcus faecalis* and *Pseudomonas aeruginosa*, with brown

Plants, herbs, spices and other products of vegetable origin must be seen as multi ingredient mixtures. Thus, there is a high probability that a product may have different compounds

obtained against *Salmonella typhi* by *Eugenia caryophillata* extract (Joshi *et al*., 2011).

smaller than those of *Salmonella* (Ayachi *et al*., 2009).

(Roopashree *et al*., 2008).

**3.3 Others** 

of antimicrobial activity (Rijo *et al*., 2010).

algae more effective than green and red algae (Cox *et al*., 2010).

**4. Molecules involved in antimicrobial activity** 

that may act as antimicrobial agents, although present in low concentrations. However, we often relate a plant with a specific molecule. For instance, piperine in pepper, eugenol in clove, allicin in garlic, cinnamic aldehyde in cinnamon stick or thymol in thyme leafs. In other cases, even if the specific molecule is unknown, there seems to be a significant correlation between antibacterial activity and high content of phenolic components (Shan *et al*., 2007), flavonoids and terpenoids (Cowan, 1999; Rios *et al*., 1987) which, among other effects, may be associated with membrane disruption.

The membrane disruption, specifically a rupture of the phospholipid bilayer, is the main cause of cell death when natural antimicrobials are concerned. However, cell death can also be precipitated by other factors, including the disruption of enzyme systems. The inhibition of flagellin synthesis in *Escherichia coli* O157:H7, promoted by carvacrol, seems to be of relevance (Tajkarimi *et al*., 2010).

One of the issues that raises interest, not only scientific but also economic, is the possibility to obtain a source of new anti-retrovirals in the nature. Terpenes, phenols and polysaccharides obtained from several medicinal plants could act as inhibitors of HIV replication, the majority of them targeting HIV reverse transcriptase (Jung *et al*., 2000).

Some spices and herbs may have the same type of active molecule, and consequently the same bactericidal mechanism. Zaika *et al*. shown that oregano, rosemary, sage and thyme had the same effects. The resistance development observed in their test strains (lactic bacteria) when exposed to one of the herbs, allowed resistance to the other three. The same herbs, nonetheless, were considered to be among the most antimicrobial.

As said previously, plants have a great number of molecules that are responsible for the colour, odour and flavour of vegetable products. Most of these phytochemicals are secondary metabolites, and among those that have antimicrobial activity we find (Cowan, 1999): phenols and polyphenols, terpenoids, essential oils, alkaloids and, finally, lectins and polypeptides.

This list is an atempt to systematise the phytochemicals envolved in antimicrobial activity. From a strictly chemical point of vue, there are phytochemicals that could be put in different groups. As an example of the "confusing" chemical denomination, we have terpenoids, phenols and phenolic terpenoids. However, we chose to follow this list to better organize the available information.

The chemistry envolved in these antimicrobial mechanisms is complex and the diversity of molecules is great. There are other types of natural antimicrobials whose inclusion in any of above mentioned groups is not easy. A group of molecules, obtained from garlic, the thiosulfinates are active against Gram negative strains. Vegetables like broccoli, Brussels sprouts, cabbages but also mustard and horseradish are rich in glucosinolates that have a wide range of antibacterial and antifungal activity.

#### **4.1 Phenols and polyphenols**

This group comprises a large number of different molecules like simple phenols, phenolic acids, quinones, flavonoids, flavones, flavonols, tannins and coumarins. They have in common the fact of participating in the aromatic characteristics of plants and are very common.

Antimicrobial Activity of Condiments 119

Additionally, abyssinone has the ability to inhibit HIV reverse transcriptase, pointing out the therapeutic value that some phytochemicals could have for some infectious diseases (Cowan, 1999). Tannins are one of the most commom chemical groups present in plants and vegetable material. It is easily found in seeds, bark, and other parts of the plant. In terms of organoleptic characteristics, tannins are responsible for astringency. An example of tannins with antimicrobial activity is ellagitannin, and the mechanism of inhibition is related with their ability to bind to proteins, blocking some metabolic pathways. Finally, we have coumarins, like warfarin, whose medicinal/therapeutic use is related to the ability to interact with

Like phenols, terpenoids are one of the most common constituents in plants, herbs and spices. As plant metabolites, terpenoids play a role in growth and development but also in the process of resistance against environmental stresses (Figure 4). Their inhibition ability is based on membrane disruption, a mechanism widely refered in scientific literature, but yet not fully understood. Diterpene metabolites like totarol and abietic acid are active against Gram positive bacteria. In fact, diterpenes is one of the largest groups of plantderived natural products with anti-staphylococcal activity. This is justified by their ability to cross the bacterial cytoplasmatic membrane (due to their amphipathic character).

eucaryotic DNA, which implies some antiviral activity (Figure 3) (Cowan, 1999).

Fig. 3. Chemical structure of a coumarin, warfarin.

Fig. 4. Chemical structures of totarol and abietic acid.

**4.2 Terpenoids** 

Phenols like catechol and epicatechin or cinnamic acid (phenolic acid) participate in membrane disruption (Figure 1). Because phenols are very common, the antibacterial activity of plants and herbs is very often related with this phenomenon.

Fig. 1. Structural formulae of some phenols with antimicrobial properties.

The antibacterial activity of quinones, flavonoids, flavones and flavonols seems to be similar. Some examples are hyperacin, chrysin, and abyssinone (Figure 2). Their mechanism of inhibition includes ability to bind to adhesins and specially, enzymatic inhibition.

Fig. 2. Chemical structures of abyssinone and chrysin.

Phenols like catechol and epicatechin or cinnamic acid (phenolic acid) participate in membrane disruption (Figure 1). Because phenols are very common, the antibacterial

activity of plants and herbs is very often related with this phenomenon.

Fig. 1. Structural formulae of some phenols with antimicrobial properties.

Fig. 2. Chemical structures of abyssinone and chrysin.

The antibacterial activity of quinones, flavonoids, flavones and flavonols seems to be similar. Some examples are hyperacin, chrysin, and abyssinone (Figure 2). Their mechanism of inhibition includes ability to bind to adhesins and specially, enzymatic inhibition.

Additionally, abyssinone has the ability to inhibit HIV reverse transcriptase, pointing out the therapeutic value that some phytochemicals could have for some infectious diseases (Cowan, 1999). Tannins are one of the most commom chemical groups present in plants and vegetable material. It is easily found in seeds, bark, and other parts of the plant. In terms of organoleptic characteristics, tannins are responsible for astringency. An example of tannins with antimicrobial activity is ellagitannin, and the mechanism of inhibition is related with their ability to bind to proteins, blocking some metabolic pathways. Finally, we have coumarins, like warfarin, whose medicinal/therapeutic use is related to the ability to interact with eucaryotic DNA, which implies some antiviral activity (Figure 3) (Cowan, 1999).

Fig. 3. Chemical structure of a coumarin, warfarin.

#### **4.2 Terpenoids**

Like phenols, terpenoids are one of the most common constituents in plants, herbs and spices. As plant metabolites, terpenoids play a role in growth and development but also in the process of resistance against environmental stresses (Figure 4). Their inhibition ability is based on membrane disruption, a mechanism widely refered in scientific literature, but yet not fully understood. Diterpene metabolites like totarol and abietic acid are active against Gram positive bacteria. In fact, diterpenes is one of the largest groups of plantderived natural products with anti-staphylococcal activity. This is justified by their ability to cross the bacterial cytoplasmatic membrane (due to their amphipathic character).

Fig. 4. Chemical structures of totarol and abietic acid.

Antimicrobial Activity of Condiments 121

Alkaloids constitute a chemical group that includes many molecules of vegetable origin that are very well known, such as caffein or cocaine. In terms of antimicrobial properties, molecules like berberine and piperine seem have the ability to intercalate into cell wall or DNA (deoxyrribonucleic acid) (Figure 6). Some activity against protozoa is also refered, mainly anti-*Plasmodium* and anti-trypanossome, although *Giardia* and *Entamoeba* infections, common in HIV patients, can also be eliminated through the consumption of some alkaloids. Solamargine, a glycoalkaloid extracted from *Solanum khaniasum* is helpeful in HIV

Antimicrobial activity of phytochemicals is not only against bacteria, fungi or protozoa. Lectins and polypeptides, whose main characteristic is the ability to form disulfide bridges, are targeted to virus. These molecules are able to block viral fusion or adsorption, probably through competition for cellular binding spots. An example of such phytochemicals is fabatin, found in fava beans (Cowan, 1999). There are vegetables (including potatos) and seeds producing protease inhibitors that are involved in the innate defense response of the host against phytopathogens. This antimicrobial activity could, in the future, be applied in clinical research. Antimicrobial proteins and peptides are produced by various plants and its presence is correlated with plant resistance to some microbial infections. These peptides

Medicinal use of plants is conducted through different ways, according to geographical availability, historic tradition and known efficacy and may include preparation of aqueous infusions and beverages using dried plants, tinctures (plants in alcoholic solutions), inhalation of steam from boiling preparations, ingestion of fresh or cooked parts as ingredients, preparation of topic ointments, essential oils, among others. However, the real scientific and therapeutic value of the plant properties, like antimicrobial potential, needs to be evaluated through proper techniques. The screening of antimicrobial activity may be

include lectins, protease inhibitors and antifungal peptides (Kim *et al*., 2009).


**4.4 Alkaloids** 

infections (Cowan, 1999).

Fig. 6. The piperine and berberine molecules.

**4.5 Lectins and polypeptides** 

**5. Methodologies** 

pursued on two different pathways:

Totarol also behaves as an efflux pump inhibitor, stopping one of the pathways bacteria can follow to resist antimicrobial molecules (Rijo *et al*., 2010). Other example of an antimicrobial terpenoid is capsaicin, a phytochemical found in chilli pepper seeds (Cowan, 1999).

#### **4.3 Essential oils**

Essential oils do not constitute a separate molecular group. In fact, many of the molecules present in essential oils are terpenoids. However, they are treated separately because scientific literature studies them intensively and they are commercially traded as medicinal products, perfume ingredients or food additives. Essential oils are aromatic oily liquids extracted from plants. As said, essential oils are mixtures, but among the most common molecules present in some essential oils we find carvacrol, thymol, eugenol, perillaldehyde, cinnamaldehyde, cinnamic acid, camphor or linalool (Figure 5). Different plants can be used for the extraction of essential oil, like coriander, cinnamon, oregano, rosemary, sage, clove or thyme, among others. The methods of oil extraction include the traditional steam distillation but also hydrodistillation and more recent supercritical fluid extraction. Some essential oils constituents like carvacrol, thymol, eugenol, perillaldehyde, cinnamaldehyde and cinnamic acid have been shown to have antibacterial activity against such food pathogens like *Listeria monocytogenes*, *Salmonella typhimurium*, *Escherichia coli* O157:H7, *Shigella dysenteriae*, *Bacillus cereus* and *Staphylococcus aureus* with MIC ranging 0.05 μL/mL to 5 μL/mL *in vitro*. In terms of application on foods (as preservative agents) or as constituents in washing solutions for fruits and vegetables, however, MIC of essential oils increases, with values ranging 0.5 to 20 μL/g in first case and 0.1 to 10 μL/mL, in the second (Burt, 2004).

Fig. 5. Chemical structures of some essential oils components.

#### **4.4 Alkaloids**

120 Antimicrobial Agents

Totarol also behaves as an efflux pump inhibitor, stopping one of the pathways bacteria can follow to resist antimicrobial molecules (Rijo *et al*., 2010). Other example of an antimicrobial terpenoid is capsaicin, a phytochemical found in chilli pepper seeds

Essential oils do not constitute a separate molecular group. In fact, many of the molecules present in essential oils are terpenoids. However, they are treated separately because scientific literature studies them intensively and they are commercially traded as medicinal products, perfume ingredients or food additives. Essential oils are aromatic oily liquids extracted from plants. As said, essential oils are mixtures, but among the most common molecules present in some essential oils we find carvacrol, thymol, eugenol, perillaldehyde, cinnamaldehyde, cinnamic acid, camphor or linalool (Figure 5). Different plants can be used for the extraction of essential oil, like coriander, cinnamon, oregano, rosemary, sage, clove or thyme, among others. The methods of oil extraction include the traditional steam distillation but also hydrodistillation and more recent supercritical fluid extraction. Some essential oils constituents like carvacrol, thymol, eugenol, perillaldehyde, cinnamaldehyde and cinnamic acid have been shown to have antibacterial activity against such food pathogens like *Listeria monocytogenes*, *Salmonella typhimurium*, *Escherichia coli* O157:H7, *Shigella dysenteriae*, *Bacillus cereus* and *Staphylococcus aureus* with MIC ranging 0.05 μL/mL to 5 μL/mL *in vitro*. In terms of application on foods (as preservative agents) or as constituents in washing solutions for fruits and vegetables, however, MIC of essential oils increases, with values ranging 0.5 to

20 μL/g in first case and 0.1 to 10 μL/mL, in the second (Burt, 2004).

Fig. 5. Chemical structures of some essential oils components.

(Cowan, 1999).

**4.3 Essential oils** 

Alkaloids constitute a chemical group that includes many molecules of vegetable origin that are very well known, such as caffein or cocaine. In terms of antimicrobial properties, molecules like berberine and piperine seem have the ability to intercalate into cell wall or DNA (deoxyrribonucleic acid) (Figure 6). Some activity against protozoa is also refered, mainly anti-*Plasmodium* and anti-trypanossome, although *Giardia* and *Entamoeba* infections, common in HIV patients, can also be eliminated through the consumption of some alkaloids. Solamargine, a glycoalkaloid extracted from *Solanum khaniasum* is helpeful in HIV infections (Cowan, 1999).

Fig. 6. The piperine and berberine molecules.

#### **4.5 Lectins and polypeptides**

Antimicrobial activity of phytochemicals is not only against bacteria, fungi or protozoa. Lectins and polypeptides, whose main characteristic is the ability to form disulfide bridges, are targeted to virus. These molecules are able to block viral fusion or adsorption, probably through competition for cellular binding spots. An example of such phytochemicals is fabatin, found in fava beans (Cowan, 1999). There are vegetables (including potatos) and seeds producing protease inhibitors that are involved in the innate defense response of the host against phytopathogens. This antimicrobial activity could, in the future, be applied in clinical research. Antimicrobial proteins and peptides are produced by various plants and its presence is correlated with plant resistance to some microbial infections. These peptides include lectins, protease inhibitors and antifungal peptides (Kim *et al*., 2009).
