The preservative properties of the volatile oils and extracts of aromatic and medicinal plants have been recognised since Biblical times, while attempts to characterise these properties in the laboratory date back to the early 1900s (e.g. Hoffman and Evans 1911). Martindale (1910) included ‘Eucalyptus amygdalina’ (probably the phellandrene variant of Eucalyptus dives) and Eucalyptus globulus oils, as well as eucalyptol (1,8-cineole), in his study of the antiseptic powers of essential oils and although the ‘carbolic coefficients’ of eucalyptus oils were not as great as those for oils containing large amounts of phenolics – such as origanum (carvacrol), cinnamon leaf (eugenol) and thyme (thymol) – they did, nevertheless, give some quantitative measure of the antiseptic properties of eucalyptus leaf oils.
Many volatile oils – particularly those of herbs and spices, but including those from Eucalyptus – have been used to extend the shelf-life of foods, beverages and pharmaceutical and cosmetic products; their antimicrobial and antioxidant properties have also pointed to a role in plant protection. Such a wide variety of applications, actual or potential, has meant that the antimicrobial properties of volatile oils and their constituents from a large number of plants have been assessed and reviewed (). In recent years attempts have been made to identify the component(s) of the oils responsible for such bioactivities (). Bhalla () has listed the organisms against which oils from Indian eucalypts have been tested.
The level of interest in the antimicrobial properties of volatile oils is just one aspect of the practical potential that such oils have in various protective roles. There also appears to be a revival in the use of traditional approaches to livestock welfare and food preservation in which essential oils play a part (). The use of Eucalyptus and its oils in protecting stored food products against insect pests is discussed elsewhere (), as are the nonvolatile constituents of Eucalyptus, some of which have antibacterial properties (). It is intended here to examine the antimicrobial activity of eucalyptus oils and, where it exists, to assess its potential application to human health care, food preservation and plant protection.
Antimicrobial activity of eucalyptus leaf oils
Unfortunately, much of the research involving the antimicrobial activity of volatile oils, including that of Eucalyptus, has been empirical with many of the oils tested simply because they have been readily at hand. Often, little or nothing has been done to determine the composition of the oils. Occasionally, the botanical source of the oil is not stated or, where it is, the existence of chemical variants (chemotypes) is not acknowledged so that even the compositional type cannot be known with certainty. When commercially available essential oils are tested it is not particularly helpful simply to refer to them by name. As Lis-Balchin et al. () have found, oils of the same name can have widely different activities. Testing of ‘eucalyptus oil’, without any indication of geographic or botanical source, or composition, is therefore of limited value. As far as possible, the examples cited below have been chosen only when the eucalyptus oil tested is from a named species of Eucalyptus. Compositional data are also quoted where possible.
A discussion of the antimicrobial activity of individual eucalyptus oil constituents such as cineole and citronellal, and the question of possible structure–activity relationships which might enable one to focus further research on particular types of eucalyptus oil, are deferred till later, after the results of testing whole oils are examined.
Composition and structure–activity relationships
The antimicrobial activity of eucalyptus oils and other volatile oils would be expected to reflect their composition, the structural configuration of the constituents and their functional groups, along with potential synergistic interactions between the constituents. Aqueous solubility, and the ability of toxic compounds to penetrate the fungal or bacterial cell wall, is also likely to be an important factor and this, too, will be influenced by the chemical nature of individual compounds within the oil. However, while some general observations can be made about the antimicrobial activity of different classes of terpenes, detailed structure–activity relationships are still not well understood. Carbonylation of terpenoids, for example, is known to increase their bacteriostatic activity but not necessarily their bactericidal activity (), while alcohols possess bactericidal rather than bacteriostatic activity against vegetative bacterial cells.
In a study by Dorman and Deans (), a correlation of the antimicrobial activity of the compounds tested, and their relative percentage composition in the oils, with their chemical structure, functional groups and configuration has confirmed a number of observations concerning structure–activity relationships made by others. Constituents with phenolic structures, for example, such as carvacrol and thymol, were highly active against test bacteria, despite their low capacity to dissolve in water. This may be due to the relative acidity of the hydroxyl group – carvacrol was more active than its methyl ester and, in tests carried out by Knobloch et al. (), methyl and acetyl eugenol were less inhibitory than eugenol; alcohols such as geraniol and nerol were also less active than phenolic ones. However, Kurita et al. () found that geraniol was very similar to eugenol (and citronellol) in terms of antifungal activity and only slightly less active than thymol and isoeugenol. The fact that compounds often have markedly different responses towards different organisms sometimes makes generalisations unwise. Citronellol has been found to be relatively inactive towards Escherichia coli and Pseudomonas aeruginosa but it is strongly active against Staphylococcus aureus (). Some organisms are also more sensitive to relatively small changes in structure than others: terpinen-4-ol and α-terpineol, identical p-menthane tertiary alcohols except for the position of the hydroxyl group, have identical activity profiles against E. colt, S. aureus and Candida albicans, but the former retains activity against P. aeruginosa while α-terpineol loses it ().
The antimicrobial activity of phenolic compounds is further enhanced by alkyl substitution in the aromatic ring (). An allylic side chain appears to enhance the inhibitory effects, chiefly against Gram-negative bacteria. It has been suggested that alkylation alters the distribution ratio between the aqueous and non-aqueous 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 (). This does not occur with etherified or esterified isomers, possibly explaining their relative lack of activity.
Aldehydes generally possess powerful antimicrobial activity. The highly electronegative arrangement of an aldehyde group conjugated to a carbon–carbon double bond appears to enhance activity () and cinnamaldehyde is much more strongly antifungal than benzaldehyde (). Such electronegative compounds may interfere in biological processes involving electron transfer and react with vital nitrogen components such as proteins and nucleic acids, thereby inhibiting growth of the microorganisms. Essential oils rich in cinnamaldehyde or citral have been linked with consistently high antimicrobial activity in vitro (). Kurita et al. () and Dorman () also found citral to be moderately active; citronellal, on the other hand, the major constituent of Eucalyptus citriodora oil, was only weakly active. Griffin et al. () found citronellal to be active against S. aureus and C. albicans but relatively ineffective against E. coli and P. aeruginosa.
Using the contact method, Naigre et al. () showed that the bacteriostatic and fungistatic action of terpenoids was increased when there was a keto group present. Griffin et al. (), however, have found that ketones are variable in their activity – carvone was strongly active against the organisms tested, verbenone was moderately active and menthone was relatively inactive (the latter in some contrast to the work of Dorman and Deans () who found that menthone exhibited modest antibacterial activity, particularly against Clostridium sporogenes and Staphylococcus aureus). Interestingly, piperitone, a major constituent of Eucalyptus dives oil (piperitone variant), was also quite strongly active (and more so than carvone against E. coli and S. aureus).
Although Lis-Balchin et al. () demonstrated significant antimicrobial activity for several Myrtaceae oils, including eucalyptus, they found no correlation between activity and 1,8-cineole content. Eucalyptus globulus oil (91 per cent cineole) was less active towards bacteria than E. radiata (84 per cent cineole) and neither was particularly effective against the fungi tested (Aspergillus niger, A. ochraceus and Eusarium culmorum). Eucalyptus citriodora oil (<1 per cent cineole but high in citronellal) showed much greater antifungal activity.
Terpene hydrocarbons such as α- and β-pinene, limonene and/>-cymene have been found to be inactive towards a variety of organisms (), as have terpene acetates such as geranyl, linalyl, neryl and α-terpinyl acetates (). Inactivity has been attributed to low water solubility and low hydrogen bonding capacity.
An increase in activity dependent upon the type of alkyl substituent incorporated into a non-phenolic ring structure was observed by Dorman and Deans (). 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 that alkylation influences Gram reaction sensitivity of the bacteria.
Stereochemistry also has an influence on bioactivity. It has been observed that in some cases α-isomers are inactive relative to β-isomers (e.g. α-pinene cf. β-pinene) and m-isomers are inactive compared to the trans-isomers (e.g. geraniol cf. nerol). Compounds with methyl-isopropyl cyclohexane rings are most active and unsaturation in the cyclohexane ring further increases the antibacterial activity, as in terpinolene, α-terpineol and terpinen-4-ol ().
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 (), the potency of their functional groups and their aqueous solubility (). As noted earlier, the importance of water solubility and hydrogen bonding has been pointed out by others (). These interacting factors are not easy to unravel and different researchers – using different procedures and different organisms – sometimes obtain results that are difficult to reconcile. In the test conditions used by Knobloch et al. (), β-pinene showed moderate antibacterial activity towards Rhodopseudomonas sphaeroides although it is insoluble in water; cineole was only slightly active, despite having about the same water solubility as citronellal, which was very active; and carvacrol and thymol were also highly active although they were less water soluble than citronellal. The site of action of the terpenoid appears to be at the phospholipid bilayer of the cell and to be caused by biochemical mechanisms catalysed by the bilayers. These processes include the inhibition of electron transport, protein translocation, phosphorylation steps and other enzyme-dependent reactions (). Terpenoid activity in whole cells appears to be more complex ().
Chemotherapeutic agents, used topically 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 diseases. Plant products which have been tested appear to be effective against a wide spectrum of microorganisms, both pathogenic and non-pathogenic. 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 opportunist pathogenic bacteria, such as coliforms, to become established in the gastrointestinal tract with resultant deleterious effects. Further studies on therapeutic applications of volatile oils, including those from Eucalyptus, are needed to investigate these issues, and to complement the substantial number of analytical and in vitro bioactivity studies that are being carried out on these natural products.
The potential of eucalyptus oils for use as practical antimicrobial agents remains to be proven. Some results have been encouraging but others have been less so. The variability is as much a reflection of the widely differing conditions, procedures and test organisms used by different workers as it is of the compositional variation in the oils themselves. Mixtures of oils, as used by Chao et al. (), may be more effective than single oils, although the choice of which oils to combine is no easy matter. Prediction of activity in whole oils (or mixtures) based on that of individual constituents is complicated by the existence of synergistic effects, as noted earlier ().
In vitro studies have shown that oils from some Eucalyptus species are effective against a range of pathogens, non-pathogens and spoilage organisms. More comprehensive (and standardised) tests of oils from a greater number of Eucalyptus species are needed to determine whether such oils, or formulations containing them, have a major role to play as antimicrobial agents. If they have, then in vivo studies are needed to assess their efficacy under clinical conditions. With an increasing public awareness of ‘green issues’, plant volatile oils, including those from Eucalyptus, offer a more eco-friendly alternative to conventional formulations in a number of sectors where antimicrobial action is desirable.
Selections from the book: “Eucalyptus. The Genus Eucalyptus”. Edited by John J.W. Coppen. Series: “Medicinal and Aromatic Plants — Industrial Profiles”. 2002.