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- MATERIALS and METHODS
The volatile oils of black pepper [Piper nigrum L. (Piperaceae)], clove [Syzygium aromaticum (L.) Merr. & Perry (Myrtaceae)], geranium [Pelargonium graveolens L'Herit (Geraniaceae)], nutmeg [Myristica fragrans Houtt. (Myristicaceae), oregano [Origanum vulgare ssp. hirtum (Link) Letsw. (Lamiaceae)] and thyme [Thymus vulgaris L. (Lamiaceae)] were assessed for antibacterial activity against 25 different genera of bacteria. These included animal and plant pathogens, food poisoning and spoilage bacteria. The volatile oils exhibited considerable inhibitory effects against all the organisms under test while their major components demonstrated various degrees of growth inhibition.
The antiseptic qualities of aromatic and medicinal plants and their extracts have been recognized since antiquity, while attempts to characterize these properties in the laboratory date back to the early 1900s ( Martindale 1910; Hoffman & Evans 1911). Plant volatile oils are generally isolated from nonwoody plant material by distillation methods, usually steam or hydrodistillation, and are variable mixtures of principally terpenoids, specifically monoterpenes [C10] and sesquiterpenes [C15] although diterpenes [C20] may also be present, and a variety of low molecular weight aliphatic hydrocarbons (linear, ramified, saturated and unsaturated), acids, alcohols, aldehydes, acyclic esters or lactones and exceptionally nitrogen- and sulphur-containing compounds, coumarins and homologues of phenylpropanoids. Terpenes are amongst the chemicals responsible for the medicinal, culinary and fragrant uses of aromatic and medicinal plants. Most terpenes are derived from the condensation of branched five-carbon isoprene units and are categorized according to the number of these units present in the carbon skeleton ( Dorman 1999).
The aims of the present investigation were to assess the antimicrobial activities of the test volatile oils and compare these to the effect of the antibiotics upon bacterial growth; to assess the components determined to be present in the volatile oils where available; to use these data to deduce which components are likely to contribute to the activities of the whole oils and to determine any structural relationships between the components and their antibacterial activity.
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- MATERIALS and METHODS
The activity of the oils would be expected to relate to the respective composition of the plant volatile oils, the structural configuration of the constituent components of the volatile oils and their functional groups and possible synergistic interactions between components. A correlation of the antimicrobial activity of the compounds tested and their relative percentage composition in the plant volatile oils used in this study, with their chemical structure, functional groups and configuration, suggests a number of observations.
The importance of the hydroxyl group in the phenolic structure was confirmed in terms of activity when carvacrol was compared to its methyl ether. Furthermore, the relative position of the hydroxyl group exerted an influence upon the components effectiveness as seen in the difference in activity between carvacrol and thymol against Gram-negative and Gram-positive bacteria. Furthermore, the significance of the phenolic ring was demonstrated by the lack of activity of the monoterpene cyclic hydrocarbon p-cymene. The high activity of the phenolic components may be further explained in terms of the alkyl substitution into the phenol nucleus, which is known to enhance the antimicrobial activity of phenols ( Pelczar et al. 1988 ). The introduction of alkylation has been proposed to alter the distribution ratio between the aqueous and the nonaqueous phases (including bacterial phases) by reducing the surface tension or altering the species selectivity. Alkyl substituted phenolic compounds form phenoxyl radicals which interact with isomeric alkyl substituents ( Pauli & Knobloch 1987). This does not occur with etherified/ esterified isomeric molecules, possibly explaining their relative lack of activity.
The presence of an acetate moiety in the structure appeared to increase the activity of the parent compound. In the case of geraniol, the geranyl acetate demonstrated an increase in activity against the test microorganisms ( Table 2). Only Cl. sporogenes was found to be more resistant to the acetate. A similar tendency was identified in the case of borneol and bornyl acetate ( Table 2). Borneol was less active then the acetate except against Aeromonas hydrophila, Bacillus subtilis, Beneckea natriegens, Escherichia coli, Flavobacterium suaveolens and Serratia marcescens but only the acetate was capable of affecting the growth of the bacterium Micrococcus luteus.
Alcohols are known to possess bactericidal rather than bacteriostatic activity against vegetative cells. The alcohol terpenoids in this study did exhibit activity against the test microorganisms, potentially acting as either protein denaturing agents ( Pelczar et al. 1988 ), solvents or dehydrating agents.
Aldehydes, notably formaldehyde and glutaraldehyde, are known to possess powerful antimicrobial activity. It has been proposed that an aldehyde group conjugated to a carbon to carbon double bond is a highly electronegative arrangement, which may explain their activity ( Moleyar & Narasimham 1986), suggesting an increase in electronegativity increases the antibacterial activity ( Kurita et al. 1979 , 1981). Such electronegative compounds may interfere in biological processes involving electron transfer and react with vital nitrogen components, e.g. proteins and nucleic acids and therefore inhibit the growth of the microorganisms. The aldehydes cis + trans citral displayed moderate activity against the test microorganisms while citronellal was only active against B. subtilis, Cl. sporogenes, Fl. suaveolens, M. luteus and Pseudomonas aeruginosa ( Table 2).
A number of the components tested are ketones. The presence of an oxygen function in the framework increases the antimicrobial properties of terpenoids ( Naigre et al. 1996 ). From this study, and by using the contact method, the bacteriostatic and fungistatic action of terpenoids was increased when carbonylated. Menthone was shown to have modest activity, Cl. sporogenes and Staphphyloccus aureus being the most significantly affected ( Table 2).
An increase in activity dependent upon the type of alkyl substituent incorporated into a nonphenolic ring structure appeared to occur in this study. An alkenyl substituent (1-methylethenyl) resulted in increased antibacterial activity, as seen in limonene [1-methyl-4-(1-methylethenyl)-cyclohexene], compared to an alkyl (1-methylethyl) substituent as in p-cymene [1-methyl-4-(1-methylethyl)-benzene]. As shown in Table 2, the inclusion of a double bond increased the activity of limonene relative to p-cymene, which demonstrated no activity against the test bacteria. In addition, the susceptible organisms were principally Gram-negative, which suggests alkylation influences Gram reaction sensitivity of the bacteria. The importance of the antimicrobial activity of alkylated phenols in relation to phenol has been previously reported ( Pelczar et al. 1988 ). Their data suggest that an allylic side chain seems to enhance the inhibitory effects of a component and chiefly against Gram-negative organisms.
Furthermore, the stereochemistry had an influence on bioactivity. It was observed that α-isomers are inactive relative to β-isomers, e.g. α-pinene; cis-isomers are inactive contrary to trans-isomers, e.g. geraniol and nerol; compounds with methyl-isopropyl cyclohexane rings are the most active; or unsaturation of the cyclohexane ring further increases the antibacterial activity, e.g. terpinolene, terpineol and terpineolene ( Hinou et al. 1989 ).
Investigations into the effects of terpenoids upon isolated bacterial membranes suggest that their activity is a function of the lipophilic properties of the constituent terpenes ( Knobloch et al. 1986 ), the potency of their functional groups and their aqueous solubility ( Knobloch et al. 1988 ). Their site of action appeared to be at the phospholipid bilayer, caused by biochemical mechanisms catalysed by the phospholipid bilayers of the cell. These processes include the inhibition of electron transport, protein translocation, phosphorylation steps and other enzyme-dependent reactions ( Knobloch et al. 1986 ). Their activity in whole cells appears more complex ( Knobloch et al. 1988 ). Although a similar water solubility tendency is observed, specific statements on the action of single terpenoids in vivo have to be assessed singularly, taking into account not only the structure of the terpenoid, but also the chemical composition of the cell wall ( Knobloch et al. 1988 ). The plant extracts clearly demonstrate antibacterial properties, although the mechanistic processes are poorly understood. These activities suggest potential use as chemotherapeutic agents, food preserving agents and disinfectants.
Chemotherapeutic agents, used orally or systemically for the treatment of microbial infections of humans and animals, possess varying degrees of selective toxicity. Although the principle of selective toxicity is used in agriculture, pharmacology and diagnostic microbiology, its most dramatic application is the systemic chemotherapy of infectious disease. The tested plant products appear to be effective against a wide spectrum of microorganisms, both pathogenic and nonpathogenic. Administered orally, these compounds may be able to control a wide range of microbes but there is also the possibility that they may cause an imbalance in the gut microflora, allowing opportunistic pathogenic coliforms to become established in the gastrointestinal tract with resultant deleterious effects. Further studies on therapeutic applications of volatile oils should be undertaken to investigate these issues, especially when considering the substantial number of analytical studies carried out on these natural products.
The volatile oils and their component volatility and lack of solubility make these plant extracts less appealing for general disinfectant applications. However, a role as disinfectants of rooms has been reportedly investigated in a classical study ( Kellner & Kober 1954). Their volatility would be a distinct advantage in lowering microbial contamination in air and on difficult to reach surfaces. Although the minimum inhibitory concentrations for a selection of oils tested in a closed chamber were lower in the vapour phase ( Inouye et al. 1983 ), evidence suggests that such applications may have merit ( Taldykin 1979; Makarchuk et al. 1981 ).
As food preservatives, volatile oils may have their greatest potential use. Spices, which are used as integral ingredients in cuisine or added as flavouring agents to foods, are present in insufficient quantities for their antimicrobial properties to be significant. However, spices are often contaminated with bacterial and fungal spores due to their volatile oil content, often with antimicrobial activity, being enclosed within oil glands and not being released onto the surface of the spice matter. Volatile oils, which often contain the principal aromatic and flavouring components of herbs and spices, if added to foodstuffs, would cause no loss of organoleptic properties, would retard microbial contamination and therefore reduce the onset of spoilage. In addition, small quantities would be required for this effect. Furthermore, evidence suggests that these oils possess strong antioxidant activities ( Dorman 1999; Youdim et al. 1999 ), which are favourable properties to combat free radical-mediated organoleptic deterioration.