**3.2 Ethnic products**

114 Antimicrobial Agents

There are some generally accepted conclusions about the antimicrobial abilities of these natural products: spices are less effective in foods than in culture media, probably because of the higher concentration *in vitro* and non-existing co-factors, and Gram-positive bacteria are more sensible than Gram negatives, although some Gram negative strains may be extremely affected as well (Jay, 1982; Zaika *et al*., 1983), as *Escherichia coli* to varying concentrations of cumin, for example (Allahghadri *et al*., 2010). Within the Gram positive

Confirming the greater susceptibility to herbs and condiments of Gram positive bacteria when compared with Gram negative, an experiment using forty-six methanolic extracts of dietary spices and medicinal herbs reached results in which *Staphylococcus aureus* was the most sensitive bacteria and *Escherichia coli* the less sensitive bacteria to the extracts tested. The other test species included *Bacillus cereus*, *Listeria monocytogenes* and *Salmonella anatum*. The experiment also has shown a strong positive correlation between antibacterial activity

Garlic and clove are widely used for culinary purposes due to their strong flavour and aroma. They are also some of the most widely refered condiments in scientific literature when comes to antibacterial activity. Some bacteria that show resistance to conventional antibiotics are susceptible to decoctions or extracts of garlic and clove. Garlic, in particular, was able to inhibit *Staphylococcus epidermidis* and *Salmonella typhi* after one and three hours, respectively. In terms of yeast inhibition, it took one hour to achieve it, and in terms of inhibition diameter, reached larger areas of inhibition for *Candida* than those produced by nystatin. Clove took five hours to completely inhibit yeasts, including *Candida* species

There are indications that some spices, or their components may be active not only against resistant strains, but also against clinical isolates (usually studies are carried on academic collection strains). Rahman *et al*., proved the effectiveness of spice extracts on multi-resistant *Escherichia coli*. In spite the fact each extract, alone, caused no inhibition, the combination of three different aqueous extracts (1:1:1) of ginger, lime and garlic had a synergistic effect, inhibiting the bacteria. The incorporation of 0.3% in culture media was sufficient to inhibit 21 of the 24 Gram positive bacteria tested (Shelef *et al*., 1980). In a study conducted by Masood et al*.,* using oral isolates as test targets, black pepper decoction shown bacterial toxicity against 75% of the samples, bay leaf was effective against 53% and aniseed against

Many herbs and spices also have antifungal activity, which is important to relate, as fungi and its toxins also have a role as origin of food spoilage and food contaminants. Out of 29 spices and herbs studied, clove (genus *Caryophyllus*), star anise (*Illicium anisatum*) and allspice (genus *Pimenta*) completely inhibited the growth of 3 different *Aspergillus* species (*Aspergillus ochraceus*, *Aspergillus versicolor* and *Aspergillus flavus*) and also inhibited the toxin production. Eugenol extracted from clove and anethol extracted from star anise were incorporated in PDA (Potato Dextrose Agar) medium to test its growth inhibition ability. The concentrations required for total inhibition were 0.4 and 2.0 mg/mL, respectively (Hitokoto *et al*., 1980).

Dietary and medicinal plants and spices have been constantly studied for their antimicrobial properties, in particular for their antibacterial activity. In a study aimed at finding solutions against diarrhoegenic bacteria, onion, garlic, ginger, black pepper, clove, mint, cumin and

group, lactic bacteria are considered the less sensitive (Zaika *et al*., 1983).

and the total phenolic content of the herbs (Shan *et al*., 2007).

18%. Coriander did not show any inhibiting activity.

(Arora & Kaur, 1999).

Beyond the more conventional vegetable products known worldwide, there is a great number of plants and herbs that have a great importance in cooking and medicinal practices, especially in Eastern Asian countries. For some plants, the biological properties described include not only antimicrobial activity but also the ability to influence the immune system.

Among these properties, there is the ability to stimulate the production of cytokines, increase the activation of macrophages, lymphocytes and NK cells (Natural killer cells). Chinese traditional medicine often uses plants like *Aloe vera*, *Angelica* spp., *Astragalus membranaceus*, *Ganoderma lucidum*, *Panax ginseng*, *Scutellari*a spp. and *Zingiber officinale*, some of which, beyond the immunomodulatory activity refered, also show some antimicrobial activity, like toxicity to *Aspergillus candidus* (*Angelica* spp.) and toxicity to MRSA (methicillinresistant *Staphylococcus aureus*), shown by *Scutellaria* spp. (Tan & Vanitha, 2004).

The purpose of using plants and herbs may not be only a medicinal or therapeutic purpose. The presence of some herbs with antimicrobial activity in some dishes may act as food preservatives. Curry leaf (*Murraya koenigii*) and Vietnamese coriander (*Persicaria odorata*) have shown to be effective against some bacteria isolated from fish. Methanolic extracts of curry leaf and Vietnamese coriander were effective against *Streptococcus agalactiae* (MIC= 0.39 mg/mL) and *Staphylococcus aureus*, respectively (MIC= 3.13 mg/mL) (Najiah et al., 2011).

Asafoetida (*Ferula assafoetida*), a spice used in Indian cuisine, was tested against endemic pathogens in India and shown antibacterial activity against *Salmonella typhi* and *Escherichia coli* O157, leading the authors of the study to conclude that the presence of some spices and condiments acts not only as flavouring agents but also as some sort of protection against gastroentestinal diseases (Vaishnavi *et al*., 2007).

In Thai cuisine and traditional medicine, galangal (genus *Alpinia*) has a special relevance. Studies have shown that the ryzome of this plant has antimicrobial properties, in particular against *Vibrio paraheamolyticus*. Bacterial inhibition was performed using disks with 10 μL freshly squeezed galangal, although the chloroform extract also shown activity. One of the molecules envolved in the inhibition mechanism was identified as being 1'-acetoxychavicol acetate (Vuddhakul *et al*., 2006).

Ayurvedic medicinal tradition uses many plants and herbs to treat several diseases, including those of infectious nature. *Ocimum sanctum*, *Eugenia caryophillata*, *Achyranthes bidentata* and *Azadirachta indica* are plants used in Nepal and India and were tested for their antibacterial properties against *Escherichia coli*, *Salmonella typhi*, *Salmonella paratyphi*, *Staphylococcus aureus*, *Klebsiella pneumoniae* and *Pseudomonas aeruginosa* through cup diffusion method. The experimental results have shown that both *Klebsiella pneumoniae* and *Pseudomonas aeruginosa* were resistant to any of the four plant extracts, and *Achyranthes* 

Antimicrobial Activity of Condiments 117

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

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

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

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

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 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

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

herbs, nonetheless, were considered to be among the most antimicrobial.

effects, may be associated with membrane disruption.

relevance (Tajkarimi *et al*., 2010).

polypeptides.

common.

the available information.

**4.1 Phenols and polyphenols** 

wide range of antibacterial and antifungal activity.

*bidentata* extract was ineffective against all bacterial strains. The largest inhibition area was obtained against *Salmonella typhi* by *Eugenia caryophillata* extract (Joshi *et al*., 2011).

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

*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 (Roopashree *et al*., 2008).

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