Biological Activity of Essential Oils And

Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 21 © 2000 Elsevier Science B.V. All rights reserved

571

BIOLOGICAL ACTIVITY OF ESSENTIAL OILS AND THEIR CONSTITUENTS

TETSUO NAKATSU*, ANDREW T. LUPO, JR., JOHN W. CHINN, JR. and RAPHAEL K.L. KANG

Takasago Institute for Interdisciplinary Science, 4 Volvo Dr., Rockleigh, NJ07647U.S. A.

ABSTRACT: Recent work in the field of biologically active, essential oils is reviewed. Essential oil extraction methods that are covered include cold pressing, extraction with other essential oils, steam distillation, solvent extraction, supercritical fluid extraction, and solid phase extraction. Separation methods for the isolation of individual constituents that are covered include GC, LC, and distillation. Biological activities of essential oils and their components, including antiallergic, enzyme inhibitory, psychological, anti-inflammatory, antimutagenic, anticarcinogenic, antiviral, insect repellent, molluscicidal, and antimicrobial are also reviewed. In particular, several examples of our own and others' work in this area that are discussed include, 1) the structure and antimutagenic activity of new sesquiterpenoid eudesmol derivatives, 2) the biological activity and odor perception of optically active rose oxides, 3) the polyphenol oxidase inhibitory activity of acyclic terpene alcohols, commonly found in essential oils, that are used in cosmetic applications, 4) the effects of the diterpene phenol, totarol, in combination with known antibiotics, on a methicillin resistant Staphylococcus aureus (MRSA) strain, and 5) the synergistic antimicrobial activity of the combination of perillaldehyde and polygodial, constituents of two herbs found in traditional Japanese foods. Also reviewed are the various uses and applications of essential oils that have been reported recently; examples included in the discussion are insect and animal repellents, oral care products, pharmaceuticals, drug delivery systems, topical applications, cosmetics, food preservatives, antioxidants, industrial applications, and the uses in packaging and consumer household products.

INTRODUCTION

Essential oils are well accepted and recognized in academia, in chemical industry and, more recently, even in everyday life [1]. In general, essential oils are known as aromatic substances produced by specific plant species. Most of these oils have been used as fragrance raw materials and flavoring agents since ancient times. They are called essential because it was once thought that each oil represented the essence of the original plant [2]. Essential oils also have been used as medicines since ancient times. From this standpoint, essential oils are considered the most widely used natural products in many areas because many traditional folk medicines are based mainly on plant materials. Ayurveda in India [3], Jamoo in Indonesia, and

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Zhong Yo in China [4] are well-known traditional collections of pharmacological preparations. In India, not only traditionally prescribed medicines, but also many kinds of spices have been used for medicinal applications. Chinese medicine has a more than several thousand year history. Even though a very limited number of the active components of these Chinese medicines have been identified, the fact that many Chinese medicines are consumed with hot water, or are extracted with hot water, leads to the conclusion that some of the active components may be essential oils, materials that can be partitioned into water by heating. Besides these well-organized, traditional medicines, many people in different geographical regions around the world are still using plant-based folk medicine, which is sometimes more effective than modern medicine. Aromatic plants or herbs are now becoming very popular and common in the developed countries, but, compared with the amount of consumption of medicinal herbs and spices in these countries, our understanding and investigation of the active components and action mechanisms are quite limited.

Surprisingly, the definition of an essential oil is ambiguous in academia and natural products study even though essential oils were among the first targets investigated in chemistry. In the late 19th and early 20th centuries, most studies in essential oils were focused on the isolation and structure determination of aromatic molecules from these oils. Essential oils are composed of many kinds or classes of molecules including terpenoids, phenolics, aromatics, cyclic and acyclic compounds, acetonides, and sulfur- and nitrogen-containing compounds, depending on the plant and the extraction method. Terpenoids comprise the largest organic chemical group, not only in essential oils, but also in natural products. Terpenoids identified in essential oils include from hemiterpenes (5 carbons) to triterpenes (30 carbons). Phenolic compounds are exemplified by eugenol (1) and vanillin (2). In general, essential oil components are less than 500 daltons in molecular weight and contain only 1-3 oxygen atoms. Although, sometimes in the literature [2] an essential oil is defined as an aromatic and volatile plant extract, many essential oils contain non-odoriferous and/or non-volatile compounds such as the triterpene lupeol identified in tea extract [5]. For the purpose of this discussion, we define essential oils as mixtures of compounds extractable by steam distillation, non-polar solvents (such as pentane, hexane and essential oils), supercritical carbon dioxide (SFE), and fluorocarbons. Essential oils can be extracted from many plant parts including flowers, leaves, fruits, fruit peels, seeds, twigs, stems, and roots.

The roles of essential oils have not been well studied, particularly for their physiological effects on the plants themselves. On the other hand, it has been known for many years that some of the components have important roles for the plant as insect attractants or repellents. Mono-, sesqui-, and diterpenoids, the major classes of materials of essential oils,

ESSENTIAL OILS AND THEIR CONSTITUENTS 573

have also been reported to have pharmacological or therapeutical activity [6]. Biologically active volatile compounds from plants have been disclosed [7] that possess antimicrobial activity [8] and therapeutic effects [9]. In the following discussion, we will be focusing on the recent progress in studies on the extraction of essential oils, the isolation and identification of essential oil constituents, and the biological activities of essential oils and their individual constituents. It is very important to understand the relationship between essential oils and biological activity, because the use of essential oils is becoming more popular and important for many practical applications in everyday life. Furthermore, we are consuming considerable quantities of essential oils from a variety of food sources and are being exposed to volatiles from plants when we are in gardens and forested areas.

From many reported investigations, the use of essential oils can be potentially applied to improve many disorders. Based on our current focus, we are interested in the following issues. 1) Asthma and allergies are the most common human disorders, affecting approximately 15 - 25% of the total population. Although not necessarily life threatening, treatments for these conditions require billions of dollars and result in the loss of a significant amount of time from work and school [10]. The application of essential oils could have great potential for moderately controlling these disorders. 2) Food poisoning is not an issue of the past, even in highly developed countries. For example, the recent case of infection caused by Escherichia coli 0157:H7 was very serious and resulted in the loss of many lives. This serious case caused by ground beef contamination was recently examined [11]. Hygienic concern in daily environments is becoming more and more important. 3) Obesity is not only an aesthetic issue for some, it is also highly related to many diseases such as cardiovascular disorders, cancers and diabetes. Recently, the relationship between weight and the risk of breast cancer was reported [12]. Consumption of fruits and vegetables may contribute to suppress weight gain, not only because of calorie control, but also because of the control of key metabolic enzymes.

With these considerations, we primarily review the biological activities of essential oils for enzyme activity control, antiallergy and antimicrobial efficacy.

EXTRACTION OF ESSENTIAL OILS

The methods of extraction of essential oils from plants significantly affect the chemical constituents and composition of the essential oil. The most appropriate and convenient method to concentrate the targeted biologically active compound into the essential oil should be selected. If the activity is based on a mixture, not a single compound, then all the active components should be concentrated from the extract. Since, in general, most

574 NA KATSU etal.

constituents of essential oils are small, volatile and lipophilic, a key consideration is the need to separate these compounds from aqueous plant materials. Several methods have been developed, and we review only the most recent reports describing methods used to extract essential oils.

Cold Press Extraction

This technique is the simplest, least harmful, and best method to maintain the integrity of the essential oil. Limonene (3), and other citrus oil components are typically extracted from citrus peelings using the cold press method [13]. It has most recently been used to great advantage to isolate oxygen-containing species, which tend to rearrange or degrade when heat is applied [14-18]. Nevertheless, even when this method is used, certain chemical species are difficult to isolate. For example, meranzin (4), isolated from orange peel, contains a very reactive epoxide group and can be easily converted to other materials in slightly acidic media [19]. Many essential oils from seeds, grains, kernels and fruits have been obtained by the cold press method. The cashew nut, one of the most popular nuts, is contained within a very hard shell that contains large amounts of essential oils referred to collectively as cashew shell oil. Cashew shell oil is conveniently obtained by this method and has been well studied because of its very unique biological activity, the details of which will be discussed later.

Fig. (l).

Extraction of One Oil with Another

This is another simple, economical and harmless process for increasing the yield of essential oils from plant materials. Cashew shell oils are extracted via this method on an industrial scale. In this process the cashew shells are heated with cashew shell oil and, after a certain period of time, some of the oil is removed and the process is repeated with fresh cashew shells.


Steam Distillation

This method is most commonly used for industrial scale extractions but also has widespread use in laboratory studies. The simple apparatus design makes this technique readily available to the global community [20-25]. Despite its simplicity, however, several groups are still working to improve the design, especially for the rapid, small-scale screening of plant materials [26]. Although a very efficient process, the applied heat, water acidity/basicity, or trace metals in the sample or apparatus can cause saponifications, isomerizations, or other undesired reactions that can affect the odor and/or flavor balance of the original essential oil [27].

Solvent Extraction

This is by far the simplest method and is most often used in the lab. Indeed, it requires little or no apparatus, making it an ideal technique for both field research and sample preparation for analysis. The main drawback is the contamination of the sample with the solvent (or impurities in the solvent) that must be completely removed either to characterize the olfactory qualities of the oil or to study its biological activity. Unfortunately, often many low-molecular-weight species are lost during solvent evaporation, thereby changing, in some cases very dramatically, the aroma balance of the essential oil.

Recently, a modified version of this method, accelerated solvent extraction (ASE), has been commercialized for laboratory use [28]. Using high pressure and slightly elevated temperatures, the technique achieves similar results to traditional solvent extraction but at a considerably faster rate.

Simultaneous Distillation-Solvent Extraction (SDE)

Frequently, steam distillation is combined with solvent extraction to obtain a more complete oil balance. The technique uses a Likens-Nickerson-type apparatus to isolate the oil as it is removed from the substrate, minimizing contact of the oil with the hot water [29]. Thermal artifact formation, though significantly reduced, is not completely eliminated by this technique [27]. A modification of the technique uses vacuum conditions to isolate the volatiles, thereby reducing the operating temperature to between 20°C and 40°C. It appears that this procedure more effectively eliminates some of the more commonly observed artifacts [30].

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Supercritical Fluid Extraction (SFE)

Developed in the 1980's, this technology is becoming more popular today for solventless extractions [31-36]. The most well-known commercial application uses supercritical carbon dioxide to decaffeinate coffee and tea. The process leaves no residue and, thus, does not affect the aroma or taste of the essential oil. The generalized procedure is illustrated in Scheme 1. Usually, the extraction is done at ambient temperature (although heat can be used carefully to assist the extraction), reducing effects to thermally labile components. Very volatile components are also efficiently isolated. Varying the temperature and pressure of the extracting fluid changes the fluid's density, thereby changing the composition of the essential oil isolated. Such flexibility, however, makes compositional comparison to other extraction techniques difficult [27,37-40].

r Liquid C0 2 J

step 1 J heat pressure

Supercritical C0 2 i sample matrix

step 2 J Analytes in C0 2 solution i pressure in analyte

r

reduction trap

Gaseous CQ2 vented

step 3

Rinse solvent ] )

< analyte trap

Analytes in rinse solution J Collection vial J Scheme 1. Flow diagram for supercritical fluid extraction process.


The main drawback for laboratory use is equipment cost (primarily to manipulate the high-pressure fluid), but that is slowly decreasing. Laboratory systems are quite compact and often have carousels to process multiple samples in an automated mode. Although the extracting fluid is normally used at supercritical conditions, operating at subcritical temperatures and pressures can give useful extracts as well [41,42]. Carbon dioxide is the most commonly used gas (inexpensive, readily available, nonpolluting), however, some groups are investigating other gases such as nitrogen dioxide, zsobutane and fluorocarbons for use in supercritical fluid extractions.

Microwave Oven Extraction

This technique is relatively new, and not enough examples appear in the literature to determine its usefulness [43]. The method appears to give compositions similar to steam distillation, but it is subject to the generation of thermal artifacts, an inherent disadvantage of any method employing heat [27].

Solid Phase Extraction (SPE)

New developments in adsorbent technology this decade have led to an explosion in products designed to simplify extraction of many chemical types (drugs, biochemicals, pollutants) from many complex material matrices such as air, water, soil, and biological tissue [44-46]. Adsorption from air is typically termed "headspace extraction," of which purge-and-trap is one example among many [47-53]. Only very small amounts of solvent are needed to elute the trapped material from the adsorbent. In general, however, sample loading is a severe limitation, so that SPE is most often used primarily for analytical applications. For the purge-and-trap method, solvent elution is not necessary, and samples can be desorbed directly into a chromatograph inlet [54].

Fluorocarbon Extraction

1,1,1,2-Tetrafluoroethane (hydrofluorocarbon-134a or HFC-134a, b.p. -25°C), which was developed as a replacement for the chlorofluorocarbons that were banned because of ozone-depleting effects, is approved in the UK for the production of natural food flavor extracts. It can be applied to optimize the extraction of plant materials and provides an environmental advantage, as well as health and safety benefits [55,56].

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SEPARATION AND IDENTIFICATION OF ESSENTIAL OIL COMPONENTS

In the study of essential oils, the separation and isolation of the individual chemical constituents is critical in understanding the origin of the biological activity of these oils. This process also can eliminate undesirable compounds such as colorants and other materials, which may impart toxicity or have a detrimental effect on the biological activity or quality of the essential oil products. In follow-up studies, complete structural determination, in particular the relative and absolute stereochemical assignments, is critical for a complete understanding of the active compounds and their structure-activity-relationships. In this section, a brief general review of separation, isolation, and structure determination methods will be discussed.

Separation

Fractional Distillation

Fractional distillation is frequently used to purify the essential oils or to concentrate the desirable parts of the essential oil for use. In general, fractional distillation is used primarily for the pre-purification or pre-treatment of the sample for the essential oil study, because this method of purification usually cannot achieve the separation resolution necessary to obtain pure, single, minor components of the essential oil. Since the purification of these minor components is critical to the study of the biological activity of the essential oils, the following chromatographic techniques have been applied extensively.

Gas Chromatography (GC)

GC can achieve the highest resolution of the essential oils, but there are some significant limitations with regards to preparative scale separations. Typically, as the sample capacity is increased, the resolution of the chromatographic separation is reduced. On a lab scale, equipment is available that permits 24-hour automated and unattended separations, however, the recovery yield and sample resolution are still problematic [57]. Capillary column GC has become so routine for essential oil analysis that one rarely finds a lab without that capability. A multitude of detectors exist for GC: thermal conductivity (TCD), flame ionization (FID), flame photometric (FPD), thermionic specific (TSD), photoionization (PID), electron capture (ECD), atomic emission (AED), mass spectrometry (MS), and infrared spectroscopy (FTIR) [58,59]. The TCD is used primarily with preparative-GC (packed column) because it is


a nondestructive detector, but sensitivity and dynamic range are low. Trapping of individual components from preparative-GC for full structural characterization can be very valuable and success depends mostly on the patience of the investigator.

Liquid Chromatography (LC)

Silica gel liquid column chromatography is suitable to separate classes of compounds that have significant differences in polarity such as hydrocarbons and alcohols, and alcohols and ketones. Nevertheless, it is not as useful for the separation of functionally similar compounds and/or isomeric mixtures. Reversed-phase-bonded columns have given, in many cases, very good separations and recovered yields. Many re versed-phase adsorbents have been introduced for a variety of different applications. The introduction of recycling HPLC has been very helpful in separating structurally similar molecules, which are poorly resolved in a single-pass chromatography system [60]. The disadvantage of reversed-phase chromatography in the study of essential oils is the use of a water-based solvent system, which may cause other problems in the final purification of the volatile compound.

Identification of Compounds

GC-mass spectrometry (GC-MS) is most frequently and effectively used to identify the essential oil constituents by using database libraries of both retention indices and mass spectral fragmentation patterns. LC-mass spectrometry is less frequently used for the identification of the essential oil constituents due to increased experimental complexity. One of the recent technological developments is the combined use of GC-MS and FTIR spectrometries which can provide additional real time information for molecular identification without the need for macroscopic separation of mixtures [55,61-67].

Despite all that we know of the essential oil components, many new compounds are constantly being isolated and their structures determined. The determination of the two-dimensional structures of small molecules is becoming much easier based on the intensive development of NMR spectroscopy techniques. On the other hand, the most important breakthrough in the past decade for essential oil study has probably been the development of various chiral stationary phases useful for the separation of enantiomeric mixtures [68-72].

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ODOR PERCEPTION, PSYCHOLOGICAL EFFECTS AND BIOLOGICAL ACTIVITY

In the following section, the odor perception, psychological effects, and biological activity of a variety of essential oils are discussed. It is well known that the odors of naturally derived and chemically synthesized samples of the same compound may be quite different and that it is very difficult to reproduce or imitate the aroma profiles of an essential oil or a constituent natural material. These differences between a natural and a synthetic sample are due primarily to the relative ratios of geometric, chemical and stereochemical isomers constituting what might commonly be referred to as a "single" compound or material.

Chiral Molecules and Odor Perception

The monoterpene ether rose oxide was isolated from Bulgarian rose oil. Since rose oxide has two asymmetric centers, there are four possible

Table 1. Threshold Value of Rose Oxides

Rose Oxide

{4R)-cts Natural type

{4R)-trans Natural type

(4S)-cis Synthetic

(4S)-trans Synthetic

Structure

~ ^

£ &

X6

Odor Perception

Floral-Green Clean, sharp, metallic, light, rose-

green., diffusive and strong

Floral-Green Green, minty herbal, and fruity

Herbal-Green-Floral Hay green, earthy, heavy

Herbal-Green-Floral Fruity, herbal rose, and citrusy-

bitter-peel

Threshold Value (ppb) 1

0.5

160

50

80


stereoisomers. Although two of the four were found in nature, the odor perception and biodegradability were studied by the synthesis of all four isomers ( 5 - 8 ) [73].

Besides the differences in odor perception, preliminary biodegradation tests show that the two natural (4i?)-enantiomers (5, 6) were biodegraded 90% within 28 days, compared with no biodegradation of the (4S)~ enantiomers (7, 8). In further studies, the biodegradation of the (47?)-cis isomer 5 is much more facile than that of the (47?)-trans isomer 6.

Depressant Activity on the Central Nervous System (CNS)

The ingestion of Psidium guajava Linn. (Myrtaceae), native to tropical Africa, India, southeast Asia, Central and South America, is associated with the treatment of diarrhea, abdominal pain, convulsions, epilepsy, chorea and insomnia. The ground, dried leaves of A guajava Linn, growing in Moleros, Mexico, were extracted by percolation with w-hexane. The active components in the hexane fraction, structurally distinct from quercetin, a material found in the methanol extract, were isolated by bioassay-guided fractionation. The fractions were assayed by the suppressive effect on the contractions induced by transmural electrical stimulation in isolated guinea-pig ileum. The protective effect of the hexane extract and the active fractions against convulsions induced by strychnine and leptazol was analyzed in mice. The biological activity was assessed by the potentiation of the hypnotic effect induced with barbiturates on mice [74].

The fractionation of the hexane extract, 5.2% of dried leaves, using silica-gel chromatography gave three main fractions having a strong contraction-inhibiting effect in vitro in isolated guinea-pig ileum eccentrically stimulated. The least polar fraction, Fl, was the most active; it increased the time appearance of convulsion in both clonic (72.86%) and tonic (56.75%) responses compared to the control. The second and third less polar fractions, F2 and F3, enhanced the latency of the tonic convulsions evoked by leptazol by 47.68% and 19.40% respectively. Fl was again shown to be the most active in the animal test with sodium pentobarbital, where the latency period was increased by 61.75% with respect to the control.

Fl and F2 contain (3-caryophyllene-oxide (9) at a 0.025% yield, (3-selinene (10) (0.09%), selin-ll-en-4oc-ol (11) (0.004%), and squalene (12) (0.017%). Compounds 9 and 10 potentially increased the time of hypnosis in the animals treated with pentobarbital. The administration of the compounds enhanced sleeping time up to 100% compared to the control group.

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t ? <Pr *Y 9 10 11

Fig. (2).

The steam-distilled oil derived from the galls of Pistacia integerrima was analyzed by chromatography and spectral techniques. The oil was found to be rich in oc-pinene (13) (21.8%), (3-pinene (14) (16.2%), oc-phellandrene (15) (15.5%), and A3-carene (16) (11.1%). The other main constituents characterized were (3-phellandrene (17), y-terpinene (18), oc-terpineol (19), and (3-terpineol (20) as well as oc-ocimene (21) and p-ocimene (22). This steam-distilled oil was shown to possess CNS-depressant activity [75].

13 14 15 16 17

18 19 20 21 22

Fig. (3).


New Sesquiterpenoids and Antimutagenic Activity

New Eudesmols

This class of compounds may be found in essential oils extracted with both polar solvents and non-polar solvents. The structure of a sesquiterpene alcohol, newly isolated from the oil extracted from the wood of Amyris balsamifera (Rutaceae), has been reported [76]. These investigators either identified by GC-MS or isolated a total of 23 sesquiterpenoids, including valerianol (23) (21.5% of oil), 7-epi-a-eudesmol (24) (10.7%), lO-epz-y-eudesmol (25) (9.7%), elemol (26) (9.1%), (3-eudesmol (27) (7.9%), y-eudesmol (28) (6.6%), a-eudesmol (29) (4.8%) and (3-sesquiphellandrene (30) (4.7%) as the major constituents. 7-

Fig. (4).

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epz-a-Eudesmol (24), had not been isolated before and its structure was determined by NMR spectroscopy and mass spectrometry.

Two new sesquiterpene diols from Teucrium polium L. (Lamiaceae), which grows in the region of the St. Caterine Mountains, Sinai, Egypt, were reported. In Egypt, it is used as an appetizer, expectorant, hypoglycemic, and for stomachache and promotion of wound healing as a folk medicine. The essential oil of T. polium is reported to have antispasmodic activity [77]. Several sesquiterpenoids were isolated as the major constituents (78.61%) of the volatile oil extracted with hexane and were identified by GC-MS [78]. These sesquiterpenoids include (3-eudesmol (27), 10-cadinol (31), and the tentatively identified patchouli alcohol (32) and represent 41.21% of the oil. Further investigation of this oil, by passing it through an ODS-C18 cartridge, silica gel column chromatography, and reversed-phase, preparative, thin layer chromatography (TLC) gave two alcohols. These compounds were identified as 7-ep/-eudesm-4(15)-ene-lp,6a-diol (33) and 7-epz-eudesm-4(15)-ene-l(3,6(3-diol (34) by high resolution mass spectrometry (HRMS), NMR, 2D NMR and CMR [79].

Antimutagenic Activity

While we are exposed to many carcinogenic and mutagenic chemicals in the environment, the foods we consume contain many anticarcinogenic and antimutagenic compounds. It is vital to learn from nature and investigate which compounds in food can contribute to these neutralizing effects. As mentioned in the previous section, while the biological activity of these newly isolated sesquiterpene eudesmols have not been reported, the antimutagenic activity of (3-eudesmol (27) and a phenolic compound, paeonol (35) was recently reported [80]. Found in the Chinese yam, Dioscorea japonica, these compounds can be used to treat diarrhea, asthma, polyuria and diabetes. The antimutagenic activity of these compounds was monitored by the umu test system and Ames test. The umu test evaluates the genotoxic activities of carcinogens and mutagens, using the expression of one of the SOS genes to detect DNA-damaging agents. In this study, furylfuramide [2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide] was used as the SOS-inducing active. A hot methanol extract from the dried D. japonica was partitioned and fractionated to give an active methylene chloride fraction. Two active compounds, (+)-(3-eudesmol (27) and paeonol (35), were identified after the purification using repeated silica gel column chromatography. (+)-p-Eudesmol (27) and paeonol (35) had previously been found in the essential oils of Chenopodium botrys [81] and Humulus lupulus (Hop) [82]. In addition, paeonol (35) was identified as a constituent of the essential oils of Paeon mouton and Paeon laciflora [83,84]. At less than 0.18 |Limol/mL of (+)-p-eudesmol (27), the SOS-inducing activity of furylfuramide was suppressed


by 80%. Paeonol (35) suppressed the SOS-induced activity by 60% at less than 0.12 (imol/mL. The two compounds also indicated antimutagenic activity in the Ames test using Salmonella typhymurium TA100. (+)-(3-Eudesmol (27) has been reported to have a preventative activity against experimental ulceration, antianoxic activity, and possible antitumor promoter activity indicated by the Epstein-Barr virus early-antigen-activation test.

Enzyme Inhibitory Activity

Acetylcholinesterase Inhibitors

17 common monoterpenes, including hydrocarbons: p-cymene (37), oc-terpinene (38), y-terpinene (18), (+)-/?-menth-l-ene (39), (+)-limonene (3), and (-)-limonene (40); alcohols: (+)-menthol (41), (-)-menthol (42), (+)-isomenthol (43), (-)-isopulegole (44), (+)-terpinen-4-ol (45), and (-)-terpinen-4-ol (46); and ketones: (+)-pulegone (47), (+)-carvone (48), (-)-carvone (49), (-)-menthone (50), and (+)-isomenthone (51), were tested for acetylcholinesterase inhibitory activity. Acetylcholine has been shown to have an important role in the CNS and in Alzheimer dementia progression. In general, the ketones (IC50 in the range of 0.89-1.85 mM) are more active than the alcohols and hydrocarbons, with the exception of oc-terpinene (38) and (+)-/?-mentha-l-ene (39) [85].

Glutathione S-Transferase (GST) Activity and Anticarcinogenic Activity

The enhancement of GST activity has been suggested to increase the host's ability to detoxify xenobiotics, including carcinogens. Thirty common essential oils and sixteen related materials were tested for GST activity. Caraway oil, celery seed oil, clove terpenes, dillweed oil, dillweed terpenes, eucalyptus terpenes, fennel terpenes, lemon grass oil, and spearmint residues are significantly more active than the control. Lesser in activity, although more so than the control, are ambrette musk residues, angelica root oil, chamomile oil, fennel oil, galanga root oil, hops oil, lime oil tails, origanum oil, and parsley leaf oil. Bergamot oil, ginger oil, lemon oil terpenes, lime oil terpenes, orange oil residue, peppermint tail fractions, sweet basil oil, and thyme oil are the least active in comparison with control. Since <i-limonene (3) and d-carvone (48) are known to have GST activity, the activity of caraway oil, which includes more than 95% of d-limonene (3), is presumed to be due to d-limonene (3). The active principals of the other oils were previously unknown [86]. Four sesquiterpenes and eugenol (1) were isolated from clove, Eugenia caryophyllata, by bioassay-guided fractionation based on GST activity. The most active compound was p-caryophyllene oxide (9). Other actives

586 NAKATSUtftf/.

were P-caryophyllene (52), a-humulene (53), a-humulene epoxide (54), and eugenol (1) [87].

37 38 18 3 9 3

OH OH OH OH

40 4 1 42 43 44

HO" *> H O x % %

O

> %

4 5 46 47 48 4 9

O

Fig. (5). 5 0 5 1


\i H

S o-

5 2 5 3 5 4

OMe OMe

Fig. (6).

Polyphenol Oxidase Inhibitors

Tyrosinase, a polyphenol oxidase, is widely distributed in nature and is considered to play an important role in the browning of fruit and molting of insects. Therefore, the inhibition of tyrosinase may work to prevent browning and control molting in insects. As tyrosinase catalyses both the oxidation of /-tyrosine to /-dopa and the oxidation of /-dopa to dopaquinone, inhibitors may inhibit both oxidation steps. With these considerations, tyrosinase inhibitors would be expected to be useful in cosmetics and pharmaceuticals to whiten the skin and remove melasma and ephelides. The many potential tyrosinase inhibitors screened include fusaric acid, flavonoids, arbutin, kojic acid, Chinese drugs, hydroquinone, and honey. Some of these, such as arbutin and kojic acid, already are in the marketplace as skin-whitening cosmetics in Japan. Lower aliphatic alcohols such as butanol were shown to inhibit tyrosinase at very high concentrations [88]. Higher aliphatic alcohols such as citronellol (56), a major constituent of many essential oils, also showed significant tyrosinase inhibitory activity [89].

Tyrosinase Activity Assay

For our own work [89] in this area, mushroom tyrosinase was used for biological assay in a protocol modified from a standard assay technique. Since the assay is a water-based system, while the compounds are lipophilic, sample solutions were prepared and diluted a minimum of 48

588 NAKATSU etai

hours in advance to maximize the solubility of these non-polar compounds in aqueous buffer.

OH

OH

6 3 6 4 6 5

CHO

sOH ^^ ^^ "OH

6 6 6 7 6 8

C0 2 H

CHO OAc

71 Fig. (7).


Melanin Formation Assay

The appropriate numbers of cells (4 xl04per well) of a mouse melanoma cell line were cultured in 6-well tissue culture plates. After incubation, the melanin pellets were mixed with 1 N NaOH for dissolution, and the absorbance at 475 nm was measured.

Enzvme Activity Inhibition

Acyclic terpenoids showed strong inhibitory effects on tyrosine activity and the results are shown in Table 2. Monoterpene alcohols such as citronellol (56) were stronger inhibitors than higher acyclic terpene alcohols such as phytol (57). This indicates that the length of the carbon chain of the molecule is an important factor in suppressing tyrosinase activity. The isoprenyl group in citronellol (56) appears to enhance the inhibitory activity when compared to tetrahydrogeraniol (59). In addition, primary alcohols such as citronellol (56), geraniol (58) and farnesol (64) more strongly inhibited tyrosinase than tertiary alcohols such as linalool (60) and nerolidol (61).

Lower acyclic alcohols such as butanol inhibited tyrosinase at very high concentrations [90], but the activity of higher, straight-chain alcohols had not been previously reported. In our in-house tests, both octanol (66) and decanol (67) showed weaker inhibitory activity than citronellol (56) (Table 3) but were much more active than lower alcohols [90]. From these results, the length of the alkyl chain appears to be the most critical factor for the attainment of significant activity. The addition of pendant methyl groups appears to enhance the activity.

Compounds with similar carbon skeletons but different oxygen-containing functional groups were tested (Table 4). Citronellic acid (69) was the most active, however, no other trends could be determined from the remaining data.

Table 2. Tyrosinase Inhibitory Activity of Acyclic Terpene Alcohol at 100 Jig/ml

Compound

Citronellol (56)

Geraniol (58)

Tetrahydrogeraniol (59)

Linalool (60)

Nerolidol (61)

Inhibition (%)

74.9

66.1

50.9

50.5

50.0

Compound

Dihydromyrcenol (62)

Tetrahydrolinalool (63)

Farnesol (64)

Phytol (57)

Geranylgeraniol (65)

Inhibition (%)

32.9

43.1

56.9

41.6

44.3

590 NAKATSUtffl/.

Table 3. Tyrosinase Inhibitory Activity of Acyclic Alcohol at 100 Jig/ml

Compound

Citronellol (56)

Octanol (66)

Decanol (67)

Inhibition (%)

74^9

46.2

56.6

Table 4. Tyrosinase Inhibitory Activity of Acyclic Terpene Aldehyde and Acid at 100 |ig/ml

Compound

Citronellol (56)

Citronellal (68)

Citronellic acid (69)

Citronellie acetate (71)

Geraniol (58)

Citral (70)

Inhibition (%)

74^9

41.6

88.6

51.5

66.1

71.8

The Inhibition of Melanin Formation on Melanoma Cells

The tyrosinase inhibitory effect of citronellol (56) and geraniol (58) was also shown by the inhibition of melanin production in melanoma cells (Table 5).

Table 5. Melanin Formation Inhibitory Activity of Tyrosinase Inhibitors on Melanoma Cells

Compound

Citronellol (56)

Geraniol (58)

Concentration (mM)

0.25

0.25

Inhibition (%)

25

30

Glucosidase, Invertase and Aldose Reductase Inhibitory Activity

Alkylphenols with long alkyl groups have been identified from many plants [91]. Anacardic acids (Ginkgolic acids) are the most intensively


studied because of their unique, broad-spectrum biological activity. Anacardic acids and related long-chain alkylphenols were extracted with ethanol from Ginkgo biloba fruit [92], with methanol from Ozoroa mucronata, (an African medicinal plant) [93], by direct extraction from the leaves of mite-resistant geraniums {Pelargonium hortorum) [94], with petrol (bp 35-60°C) from finely ground seeds of Knema elegans (Myristiaceae) [95], with supercritical carbon dioxide from cashew nut (Anacardium occidentale) shell [96], with chloroform from the flowers of Ononis speciosa [97], and with various other methods from cashew nut shell.

The long-chain alkylphenols showed a variety of interesting biological activities such as molluscicidal activity [98,99], antimicrobial activity [92,100], prostaglandin synthetase inhibitory activity [93], g l y c e r o phosphate dehydrogenase inhibitory activity [101], and antivectorial activity [102]. In addition, pharmacological effects of anacardic acid sodium salts and acetates have been reported. The effects observed on the behavior of mice include spontaneous locomotor activity, hexobarbitone hypnosis, pentylenetetrazol-induced convulsions, the anticonvulsant action of phenobarbitone sodium against maximal electroshock-induced seizures, morphine-, haloperidol-, and bradykinin-induced catalepsy, antinociceptive activity, anti-inflammation, antipyretic activity, and acute toxicity [103].

In general, the crude extracts of long-chain alkylphenols are complicated mixtures of different alkyl chain lengths. In an investigation of the anti-obesity activity of the compounds, twelve alkylphenols (76 - 87) were isolated and identified through spectroscopic analysis. Pentadecyl derivatives (72 - 75) were detected in the extract, but the amounts of compounds obtained were not enough for bioassay [104]. Recycling HPLC on an ODS column was used to separate the complicated mixture.

Table 6. a-Glucosidase Inhibitory Activity of Compounds 72 - 87

Compound

72

1 76

| 80

1 84

1 73

1 77

1 81

1 85

IC 5 0 (|iM)

1.1

6.9

6.7

6.1

144.5

105.8

64.9

23.5

Compound

74

78

82

86

75

79

83

87

IC 5 0 (|iM)

3^5

18.9 1

25.7 |

39.6 1

7.8 1

3.1 1

4.1 1

6.0

592 NAKATSUtfrt/.

g-Glucosidase Inhibition

The oils were examined for activity against obesity and related complications. The results of ot-glucosidase inhibitory activity of compounds 72 - 87 are shown in Table 6. Among them, 6-alkylsalicylic acids (anacardic acids) (72, 76,80,84) and 5-alkylresorcinols (cardols) (75, 79, 83, 87) show stronger inhibition than 3-alkylphenols (cardanols) (73, 77, 81, 85) and 2-methy 1-5-alkylresorcinols (methylcardols) (74, 78, 82, 86). Among these inhibitors 6-pentadecylsalicylic acid (72) is the strongest inhibitor followed by 5-pentadecenylresorcinol (79). The other compounds from the groups described above also inhibited the enzyme activity, but only at higher concentrations.

HO. ^ R HO.

R Group O H

72 73 74 75

76 77

80 81

78

82

79

83

84 85 86 87

72: 6-pentadecylsalicylic acid; 73: 3-pentadecylphenol; 74: 2-methyl-5-pentadecylresorcinol; 75: 5-pentadecylresorcinol; 76: 6-[8(Z)-pentadecenyl]salicylic acid; 77: 3-[8(Z)-pentadecenyl]-phenol; 78: 2-methyl-5-[8(Z)-pentadecenyl]resorcinol; 79: 5-[8(Z)-pentadecenyl]resorcinol; 80: 6-[8(Z),l 1(Z)-pentadecadienyl]salicylic acid; 81: 3-[8(Z),l l(Z)-pentadecadienyl]phenol; 82: 2-methyl-5-[8(Z),l 1(Z)-pentadecadienyljresorcinol; 83: 5-[8(Z),l 1(Z)-pentadecadienyl]-resorcinol; 84: 6-[8(Z),l 1 (Z), 14-pentadecatrienyljsalicylic acid; 85: 3-[8(Z),l l(Z),14-pentadecatrienyl]resorcinol; 86: 2-methyl-5-[8(Z),1 l(Z),14-pentadecatrienyl]resorcinol; 87: 5-[8(Z),l l(Z),14-pentadeca-trienyl]resorcinol.

Fig. (8).


Invertase Inhibition

Only the pentadecatrienyl derivatives (84 - 87), which are the most abundant in each group of the essential oil constituents, inhibited invertase moderately (more than 30%) at a concentration of approximately 600 |LiM (Table 7).

Table 7. Invertase Inhibitory Activity of Compounds 72 - 87

Compound

1 ^ 1 85

1 86

87

Concentration (fiM)

584.0

670.1

608.8

625.0

Inhibition (%)

73

79 1

55

32 1

The other compounds not listed have shown no significant inhibition at the concentration of approximately 600 uM.

Aldose Reductase Inhibition

Compounds 72 - 87, with the exception of compound 74, were tested against aldose reductase (Table 8). 6-Pentadecatrienylsalicylic acid (84) is the strongest inhibitor followed by 5-pentadecenylresorcinol (79) and 5-pentadecadienylresorcinol (83).

Table 8. Aldose Reductase Inhibitory Activity of Compounds 72 - 87

Compound

72

1 76

1 80

1 84

1 73

1 77

1 81

1 85

IC5 0(uM)

100.4

40.4

49.3

20.4

>328.4

>330.6

332.8

180.9

Compound

74

78

82

86

75

79

83

87

IC 5 0 (u.M)

NAT*

300.7 1

118.0 1

115.7 1

312.0 1

28.3

28.4

57.2

N/T = Not tested.

Among the salicylic acids, 6-pentadecylsalicylic acid (72) was the strongest inhibitor of oc-glucosidase, whereas 6-pentadecatrienylsalicylic acid (84) was the most active inhibitor of aldose reductase. Compound 84

594 NAKATSUtffl/.

is also a moderate inhibitor of invertase. The structure-activity relationship with respect to the exact nature of the alkyl group within each class (anacardic acids, cardols, cardanols, and methylcardols) is unclear at present, but an enzyme kinetic study indicated that compound 72 was a competitive inhibitor against oc-glucosidase (K/ = 6.2x106M).

Antiallergic Activity

The unique antiallergic activity of Zyumi-Haidoku-San-Ka-Rengyo, a traditional herbal prescription medication from the Sino-Japanese traditional medicine, Kampo, was recently investigated [105]. This medication is traditionally used for the treatment of eczyma, urticaria, purulent dermatitis and recently, atopic diathesis. Actually, this prescription includes 11 Sino-Japanese crude drugs that have a variety of biological and pharmacological activities such as anti-inflammatory and antiallergic activity. The extract of the licorice root, one of these crude drugs, contains glycyrrhetinic acid as the main component and has been shown to have antiallergic activity against type I and IV allergies. Nevertheless, it is not known whether the mixture of these crude drugs has antiallergic activity against type I allergy.

An ovalbumin-induced, homologous passive cutaneous anaphylaxis (PCA, type I allergy) test in rats was used to evaluate antiallergic activity. Oral administration of the hot water extract from the crude drug decreased the dye leakage due to the rat dorsal PCA by approximately 40%. In comparison, a known antiallergic agent, glycyrrhetinic acid, showed approximately 50% reduction. A yellowish, fluorescent, odorant oil was obtained from the hot water extract by distillation. The essential oil obtained in this manner showed inhibition. The distillate was fractionated and the highest boiling fraction was found to be the most active. This high-boiling fraction was further separated into 7 fractions (A - G) by TLC. Fractions E and G (50mg/Kg) significantly inhibited the anaphylactic reaction. Two sesquiterpene hydrocarbons in these fractions were identified as (3-caryophyllene (52) and ot-humulene (53). (3-Caryophyllene (52) was more active than ot-humulene (53), and the inhibitory activity of 52 was dose dependent on the mouse ear PCA.

Anti-inflammatory Activity

Eugenol (1), the major constituent of the essential oil of nutmeg, Myristica fragrance, showed prostaglandin synthesis inhibitory activity, reduced the tone of isolated gut muscle and myometrium, reduced the rate of intestinal transit, reduced the intestinal accumulation of fluid induced by prostaglandin E2, and reduced diarrhea induced by castor oil. Antiinflammatory activity in the rat paw carrageenin edema assay has been


reported [106]. Eugenol (1) (IC50 = 3.0 x 10"7 M) and isoeugenol (55) (IC50 = 7.2 x lO-7 M) both inhibited platelet aggregation at a similar activity level to indomethacin (IC5o = 2.2 x 10"7 M), a commonly used antiinflammatory agent [107].

The 70% methanol extract from the dried fruit of Forsythia suspensa VAHL, a Chinese medicine, and uRengyo" in Japanese medicine, showed anti-inflammatory activity. Even though the active was not identified, it was partitioned into the hexane fraction and, therefore, the active is believed to be one of the essential oil components. The anti-inflammatory activities of the hexane fraction on acetic acid-induced vascular permeability, writhing symptoms in mice, carrageenin-induced edema, and the cotton pellet-induced granuloma formation in rats, as well as analgesic activity, were also at approximately the same level activity as indomethacin [108].

Insecticidal and Molluscicidal Activity

Insecticidal Activity

1,8-cineole (88), found in the essential oil of Ocimum kenyense (Ayobangira), is active against stored-product beetles [109]. The essential oil of Cymbopogan winterianus root oil consisting of oc-eudesmol (29), (5-eudesmol (27), and elemol (26), has insecticidal activity against the rice weevil Sitophyllus oryzae [110], while maize weevils are repelled by the essential oils of Lippia ukambensis. Other promising repellents are the essential oils of L. javanica, L. dauensis, L. somalensis and L. grandifolia. The essential oil of L. wilmsii has been shown to be very active as a larvicide. Individual hydrocarbon monoterpene constituents were generally more active than the oxygenated constituents of these essential oils [111]. Terpenes and other oxygenated constituents of Acorus calamus (calamus) oil, including terpineol (18), farnesol (64), cineole (88), citral (70), capric acid (89), lauric acid (90), and carvone (48), demonstrate an insecticidal activity toward the psyllid H. cubana [112].

CO2H L ^ ^ ^ . C O 2 H

88 8 9 9 0 Fig. (9).

596 NAKATSUtftf/.

Molluscicidal Activity

Sassafras (Sassafras), star anise (Illicium verum) and oregano (Origanum vulgare) essential oils demonstrate biotoxicity to golden snails at 1-100 ppm and are active at 10 and 20 ppm against young snails [113].

ANTIMICROBIAL ACTIVITY OF ESSENTIAL OILS AND THEIR COMPONENTS

The focus of the work of many of the articles published in the last 10 years by researchers in the field of essential oil biological activity is on the activity to inhibit microorganisms. Over a third (out of 220) of the articles obtained from a recent literature search dealt with antimicrobial activity, either against bacteria or fungi or both. Essential oil activity for the inhibition of bacteria and/or fungi was determined, usually followed by an investigation of the activity of the individual components. Sometimes the activities associated with the essential oils from various plant varieties, with different amounts of constituents, were compared.

Methods

For antimicrobial assays, there are several common methods employed. Due to its ease of operation, the most common method used is the disk diffusion method, which involves the application of a material onto a filter paper disk, and then the disk is placed onto solid medium previously seeded with the test microorganism of interest. Sometimes, the sample is dissolved in an appropriate solvent before application onto the paper disk. This method is very common in the evaluation of antibiotics and is the method adopted by the National Committee for Clinical Laboratory Standards (NCCLS). The method depends on the aqueous solubility of the antibiotics in order to facilitate diffusion through the solid medium. Essentials oils, however, are generally hydrophobic, do not readily diffuse through an aqueous medium and, therefore, the prevalence of false negatives or reduced activity might then be anticipated.

Another common method is the incorporation of materials into cooled agar or broth either in the presence or absence of a solvent. Without a solvent, the poor solubility of the essential oil becomes a significant issue. To solve this problem, solvents or detergents are typically used. Tween and ethanol are common solvents for lipophilic materials, such as essential oils, to aid dissolution into an aqueous environment. Unfortunately, many of these materials affect activity. For example, when thyme oil or thymol (91) is tested in the presence of Tween 80, its activity is markedly decreased when compared with a system without Tween [114]. Furthermore, minimal inhibition concentrations (MICs) and minimal lethal


concentrations (MLCs) for different bacterial species, in the presence of agar, were significantly lower than those observed in the presence of Tween 80 or ethanol [115]. This demonstrates the need to select an appropriate solvent with care and to ensure that it is being used at appropriate concentrations.

The issue is then to devise a system that will allow lipophilic materials to disperse in an aqueous environment using a solvent that does not interfere with antimicrobial activity. Dimethylformamide (DMF) and dimethylsulfoxide (DMSO) are two solvents that appear to be able to achieve this. Some authors have incorporated essential oils into DMSO, adding an aliquot into growth medium followed by serial dilutions in growth medium. This method causes both the active material and the solvent to be serially reduced. A better approach might be to dissolve the essential oils in solvent first, make serial dilutions in the same solvent, and then add aliquots to the growth medium. In this way, all the tubes contain the same amount of solvent. Our proposed protocol is the following:

The sample is dissolved in an appropriate amount of dimethylformamide (DMF) to give a concentration equivalent to 100 mg/mL. Five or more twofold serial dilutions are made from this concentrate by adding an aliquot to an equal volume of DMF. 30 \\L of each concentration is added to 3 mL of the appropriate media, followed by 60 |iL (or a small piece in the case of filamentous fungi) of an overnight culture. Therefore, each tube contains 1% DMF, which we have shown does not affect the growth of the microorganism; DMF concentrations need to be more than 4% before the microorganisms are affected. The tubes are incubated for 48 hours at the appropriate conditions. Growth after 48 hours is observed as an increase in turbidity read at 660 nm. The minimum inhibition concentration (MIC) is determined as the lowest concentration that gives rise to no observable growth. Combination mixtures are prepared by using aliquots of appropriate concentrations and using DMF as the diluent to correct the final concentrations before inoculating into the media.

Activity

Essential oils are active in the inhibition of Gram positive and Gram negative bacteria, yeast, and fungi. These oils usually show weak to moderate activity when compared with chemical biocides such as antibiotics, quaternary ammonium salts, or chlorinated phenols such as triclosan. When the major components are isolated, they usually show improved activity compared to the essential oils. The test methods employed commonly determine inhibition activity via an MIC, but do not usually address the issue of MLC (minimum lethal concentration) or how quickly viable organisms are reduced over a short period of time. In order to determine this, other test methods need to be employed. Differences in

598 NAKATSUtftf/.

mechanism of action of these essential oils will determine the relationship between biostasis and biocidal activity.

Furthermore, what is considered active is subjective. While one researcher might consider microbial inhibition at 1% as active, another would only consider activities of less than 0.025% to be active. Since test methods vary greatly, it is difficult to compare the results directly. In addition, the different methods tend to lead to different results due to many issues including, but not limited to, solubility and solvent choice. When standards are used, it is important to consider structurally related standards with similar solubilities in water.

Plant Materials

The selection of the plant species for study is in large part because of the traditional interest in the use of these materials. The materials studied range from common herbs used in cooking to plants used for medicinal purposes in ancient times as well as today. The presence of plants and their varieties that are found in the local region is also another selection criteria for study. Here, there is interest in looking at differences in the varieties and related species to compare their biological activity as well as their composition.

Spices

Spices are often simply considered condiments to incorporate flavor in cooking. The power of spices to impart biological activity is now slowly reemerging as an area of interest. The discussion of spices with biological activity does not necessarily imply exotic varieties and uncommon species. This, in fact, is usually not the case as many common spices have activities, that include the inhibition of microorganisms. There has also been keen interest in examining local varieties of spices and seasonal variations.

Thymus sp. (thyme) is a common spice that has been extensively studied [116-123]. Thyme is one of the earliest medicinal plants in western herbal medicine. The essential oil isolated from this spice is active in the inhibition of Gram positive and Gram negative bacteria as well as yeast and filamentous fungi. A major constituent of thyme oil is thymol (91), which has been implicated as the molecule responsible for the activity of this essential oil. Other materials isolated from thyme oil that possess biological activity include carvacrol (92), borneol (93), /?-cymene (37), oc-pinene (13) and camphene (94). Thymol (91) was shown to be the most active, followed by carvacrol (92), borneol (93), /?-cymene (37), oc-pinene (13), and camphene (94) [121].


Salvia sp. (sage) essential oils have been recently studied for their activity to inhibit fungi [118,124-126]. Cineole (88) can be found in the essential oil of sage and is a major contributor of activity. Rosmarinus sp. (rosemary) [122,127] essential oil inhibits bacteria and oxidation [128]. The essential oil of Origanum sp. (oregano), with the phenolic carvacrol (92) as the major constituent, has antimicrobial activity [129-132] and antioxidant activity [119]. Elettaria sp. (cardamon) essential oil, containing terpenes and cineole (88), inhibits fungi [133]. Anethole (96), limonene (3), and fenchone (95), found in fennel (Foeniculum vulgare), are active to inhibit fungi, especially Aspergillus niger [134]. Mentha sp. (mint) essential oil has antimicrobial [135], antifungal [116,136], and antioxidant activity [137]. The major constituent of mint is menthol (41), which plays a significant role in the activity. The essential oils of sweet marjoram {Origanum marjorana), spearmint (Mentha spicata), and thyme (Thymus vulgaris) are active in the inhibition of Aspergillus sp., as well as the Gram positive, spore-forming Bacillus sp. [116].

Thymol (91)

Thymol (91), its isomer carvacrol (92) (isothymol), and their derivatives have been implicated as the major constituents responsible for the biological activity of many essential oils. Thymol (91) and carvacrol (92) are not only found in thyme essential oil but also in many other spices such as oregano, and other plants such as Alpinia speciosa [138] and Hyptis verticillata [139]. Thymol (91) is active for the inhibition of many microorganisms [114,117,121,122,125,128,129,131,132,138-142] including both Gram positive and Gram negative bacteria as well as yeast and fungi. It is perhaps one of the most active, broad-spectrum chemicals isolated from common plant essential oils. In study after study, it is shown to be the most active constituent of the essential oil. In fact, it has been registered with the United States Environmental Protection Agency as an active ingredient biocide and is used in commercial products to sanitize and disinfect. Additionally, it also has antioxidant activity [119] preventing the breakdown of fats and lipids.

Cineole (88)

Found in eucalyptus essential oil, cineole (88) is also found in rosemary (Rosmarinus officinalis) [127], cardamom (Elettaria) [133], sage (Salvia) [126], Laurus nobilis [143], Alphinia speciosa [144], Aegle marmelos [145], Heteropyxis natalensis [146], Jasonia candicans, and J. montana [147]. It is active in the inhibition of fungi [118,133,143,145] but is reported to be inactive against the Gram positive bacteria S. aureus [148].

600 NAKATSUtffl/.

Eugenol (1) and Linalool (60)

Eugenol (1), commonly found in clove (Eugenia) essential oil, and the structurally related estragole (97), found in Feronia elephatum [149], both have been shown to inhibit yeast. Clove and peppermint oils have been suggested to be incorporated in antidermatophytic drugs [136] because of the antifungal activity of eugenol (1) and estragole (97) [150]. Additionally, it is highly active against bacteria [148], especially against S aureus.

Linalool (60) has high antioxidant activity, especially in the presence of phenolic materials [151], and is active in the inhibition of S. aureus [148]. The interaction of these two materials, discussed below, is interesting and merits further study.

Hydrocarbons

Essential oils rich in hydrocarbons, such as those obtained from Salvia gilliessi, inhibit fungi via an interference with spore germination and mycelia growth [124]. The essential oils ofXylopia aromatica [152] andX. longifolia [153], both being hydrocarbon-rich, inhibit both bacteria and fungi.

Terpenes

Terpenes such as a-terpinyl acetate (98) can be found in large amounts in spices such as cardamom. As much as 51.15% of the essential oil of cardamom is made up of these terpenes. oc-Terpinyl acetate (98), the most active material, is followed by linalool (60), which makes up 6.95% of this essential oil [133]. Heteromorpha trifoliata [154] essential oil also contains high amounts of terpenes that inhibit bacteria and fungi. Terpenes found in Eupatorium odoratum, amounting to 88% of the essential oil and demonstrating no activity against Staphylococcus aureus, exhibited notable activity against gram negative bacteria [155]. Gram negative bacteria such as Salmonella typhi and Escherichia coli, are easily controlled by these terpenes both in vitro and in vivo [156].

Antivirus Activity

The main constituents of the essential oil of Tagetes erecta leaves are monoterpenes and long-chain hydrocarbons. The oil includes terpinene (38) (13.83%), limonene (3) (13.74%), /7-decane (99) (10.28%), /?-tridecane (100) (9.78%), undecane (101) (9.19%), c/s-ocimene (21, 22) (6.92 %), and /r<my-ocimene (21, 22) (0.99%). The propagation of both


"OH

9 1 9 2 9 3 9 4

)Me QMe

OAc 9 8

9 9 100 101

Fig. (10).

Adeno 127 virus (DNA group) and Newcastle virus (RNA group) are inhibited by this essential oil [156].

Synergies, Antagonisms and other Interactions

As mentioned before, of the constituents of thyme oil, thymol (91) was the most active, followed by carvacrol (92), borneol (93), p-cymene (37), oc-pinene (13) and camphene (94). When mixtures of these 6 materials were made and the antibacterial activity evaluated, the results were quite unexpected. The antibacterial activity of the mixtures was less than that of the essential oil of thyme. This suggests that the minor compounds play a significant role in the biological activity [121].

The addition of Desferal to nutrient agar counteracted the antibacterial effects of both thyme oil and thymol (91). In spite of this effect, no further conclusive evidence was obtained for a possible role of iron in the oxygen-related antibacterial action of the thyme oil and thymol (91). In the presence of thymol (91), the viable counts of Salmonella typhimurium

602 NAKATSl! etai

obtained on a minimal medium were lower than those obtained on nutrient agar. Addition of bovine serum albumin also neutralized the antibacterial action of thymol (91). It was suggested that the effects of bovine serum albumin or Desferal are due to their ability to bind phenolic compounds through their amino and hydroxylamine groups, thus preventing complexation reactions between the phenolic constituents of the oil and bacterial membrane proteins. This hypothesis is supported by the marked decrease in the viable counts of Salmonella typhimurium caused by either thyme oil or thymol (91), when the pH of the medium was changed from 6.5 to 5.5 or the concentration of Tween 80 in the medium was reduced [114].

Amla, cantharidine, coconut, and mustard oils are fungicidic toward Trichophyton rubrum, Microsporum canis, M. gypseum, and T. mentagrophytes. There is a relationship between the toxicity of oils and the toxicity of their constituent fatty acids. Saturated fatty acids with less than 12-carbon chains were more active than saturated fatty acids with longer carbon chains. In general, the activity of saturated fatty acids decreased with increasing carbon number (with the exception of undecanoic acid, which showed the highest fungal toxicity). Fatty acids containing an odd number of carbons were slightly more toxic than the corresponding one-carbon-less, even-numbered fatty acids. Unsaturated fatty acids were more active than the corresponding saturated fatty acids. The activity of polyunsaturated fatty acids increased with increasing degrees of unsaturation [157].

It seems pardoxical, that while two major constituents of plants would have activity by themselves, combining them would eliminate their activity. This, however, has been observed with the combination of eugenol (1) and linalool (60), both active by themselves, which show an antagonistic effect toward one another [148].

Actives Against Methicillin Resistant Staphylococcus aureus (MRSA)

Totarol (102), first isolated from the heartwood of a tree indigenous to New Zealand, Podocarpus totara G. Benn (Podocarpaceae) [158,159], is widely distributed throughout the plant kingdom in various species such as P. nagi (Nagi in Japanese), P. macrophyllus (Southern Yew, Inumaki in

Fig. (11).


Japanese), and Thujopsis dolabrata (Aomori Hiba in Japanese) [160,161]. The antibacterial activity of totarol (102) has been studied [162]. A further detailed study of totarol (102), in combination with antibiotics against MRS A, was also reported [163].

Table 9. Profile of Antibiotic Resistance of MRSA Strains Used

S. aureus

[~~ ATCC6538

1 ATCC 12598

1 ATCC 11632

1 ATCC 29247

| JB 1108

1 JB 87023

MIC Oig/ml)

Methicillin

250.00

250.00

7.81

7.81

2000.00

>2000.00

Penicillin

7.81

7.81

31.25

> 1000.00

>2000.00

62.50

Polymyxin B

31.25

31.25

62.50

31.25

125.00

62.50

Gentamicin

7.81

15.62

62.50

3.90

>500.00

>500.00

Vancomycin

o.98 n 1.95

3.90

1.95

1.95 ]

1.95

Table 10. Effects Against Clinical Isolates of MRSA from Combining Totarol (102) and Methicillin with Individual FIC and Combined FIC Values

| S. aureus )B 1108

1 S. aureus 3B S1023

Totarol (102)

Lowest

MIC

0.975

3.90 1.95

FIC

0.50

0.50 0.25

Methicillin

Lowest

MIC

0.015

0.06 0.12

FIC

0.0000075

<0.00003 <0.00006

Combined*

FIC

0.5000075 1

O.50003 <0.25006

* Combined FIC = Lowest FIC of 1 + Lowest FIC of 2

FIC= Combined MIC of 1 + Combined MIC of 2 Individual MIC of 1 Individual MIC of 2

Table 11. Effects Against Clinical Isolates of MRSA from Combining Totarol (102) and Penicillin with Individual FIC and Combined FIC Values

( S. aureus )B 1108

[ S. aureus }B 87023

Totarol (102)

Lowest

MIC

0.976

0.976

FIC

0.50

0.25

Penicillin

Lowest

MIC

0.0075

15.625

FIC

O.00000375

0.25

Combined*

FIC

<0.50000375 1

0.50 J

604 NAKATSU^fl/.

Table 12. Effects Against Clinical Isolates of MRSA from Combining Totarol (102) and Gentamicin with Individual FIC and Combined FIC Values

1 5. aureus SB 1108

S. aureus HB&1023

Totarol (102)

Lowest

MIC

0.97

3.91 1.95

FIC

0.50

0.50 0.25

Gentamicin

Lowest

MIC

31.25

1.95 125

FIC

<0.0625

<0.0039 <0.25

Combined*

FIC

<0.5625

<0.5039 <0.500

Four strains of Methicillin resistant Staphylococcus aureus (MRSA) are inhibited in the concentration range of 0.78 |ig/mL to 7.8 |ig/mL of totarol (102). This level is lower than most antibiotics. More interestingly, totarol (102) was effective as a bactericide as shown in Figures 12 and 13. Totarol (102) reduced the bacterial cell count of the clinical isolate strains in only 5-10 minutes from 109 cfu/mL to 103cfu/mL. In contrast, mupirocin could not reduce the bacterial cell count of MRSA to the same extent in this short time period.

Critical Time Kill of Totarol 1 mg/ml on MRSA

Fig. (12). Time(min)

The combination of totarol (102) with methicillin and penicillin demonstrated synergistic antibacterial activity as shown in Tables 10, 11 and 12. Totarol (102) was involved in the interruption of cell wall integrity. This, in turn, facilities cell lysis. In addition, totarol (102) appeared to interfere with septum formation and the final stages of cellular division; the cells treated with totarol (102) were unable to complete cell division (Photos 1 and 2).

ESSENTIA!, OILS AND THEIR CONSTITUENTS 605

Critical Time Kill of Compounds on MRSA JB1108

Time (hours) Fig. (13).

Photo 1. Photo 2.

"Shiso" and "Akame", Japanese Herbs Consumed with Raw Fish

The leaves as well as the seeds of Perilla frutescens (Labiatae) are part of popular and traditional Chinese herb medicines that are prescribed for colds, coughs and promoting digestion [164]. This fast-growing herb is used in a wide variety of applications including foods, food coloring, flavoring, and as a sweetening agent. The edible, green leaves of P. frutescens, Japanese general name "shiso" or "aoziso" , in particular, are

606 NAKATStltffl/.

commonly used when preparing raw fish and shellfish in Japanese dishes such as "sushi" and "sashimi", which have become popular worldwide.

Table 13. Constituents of Steam Distillate from the Leaves of P. frutescens

Compounds

2-Hexanol

Ocimene (21) and/or (22)

Benzaldehyde (105)

Sabinene

P-Pinene (14)

Myrcene

Pseudolimonene

Limonene (3)

Terpinolene

Linalool (60)

Limonene oxide

Ratio

0.23*

0.45*

1.60

0.40

0.60

trace

0.20

12.80

0.10

2.60

0.10*

Compounds

Pulegone

Perillaldehyde (103)

Perillyl alcohol (106)

a-Terpinyl acetate (98)

Isoeugenol (55)

P-Caryophyllene (52)

a-Humulene (53)

a-Bergamotene (104)

Farnesene

Aromadendrene

Ratio

0.90

74.00

0.26

0.13*

0.25

3.80

0.13

3.50*

0.10*

0.30*

* Identification is tentative.

Many of the medicinal properties, including antidermatophytic, of P. frutescens have been reported [164-167]. A steam distillate was obtained from partially dried P. frutescens leaves commercially cultivated in California using simultaneous steam distillation-extraction. Finely macerated leaves (39.2 grams) were combined with approximately 600 mL of distilled water in a round bottom flask. The flask was then connected to a Likens-Nickerson distillation-extraction head. Pentane was used as the extraction solvent and the distillation was allowed to proceed for approximately 90 minutes. This process yielded 0.3 grams (0.77%) of an aroma concentrate possessing a characteristic "shiso" aroma. The antimicrobial activity of shiso, in relation to its traditional use in sashimi dishes, has been studied [168].

Antimicrobial Activity of the Extracts

The steam distillate and methanol extracts of P. frutescens have been tested against microbes. The methanol extract did not remarkably inhibit


any microbes at 500 |Lig/mL, instead the crude steam distillate exhibits broad spectrum activity in the range of 125 to 1000 |Lig/mL for both Gram positive and Gram negative bacteria as well as fungi (Table 14). It is particularly active against filamentous fungi, for which the MICs for M mucedo and P. chrysogenum are as low as 62.5 |Lig/mL. More interestingly, the steam distillate inhibits Gram negative Salmonella choleraesuis, which is one of the major bacterial species responsible for food poisoning from eating raw foods such as fish and eggs. Recognizing these results, we are encouraged to investigate the volatile constituents of the P. frutescens essential oil.

Antimicrobial Constituents

The constituents of the essential oils of P. frutescens, as well as their genetic control, have also been extensively studied [169,170]. They indicate that the constituents of P. frutescens vary widely depending on the variety of the strains and the location of cultivation. In addition, the constituents of the steam distillate of P. frutescens harvested and distributed in the US had not been previously reported. The GC-MS data (Table 13) shows that the steam distillate of the leaves of P. frutescens harvested in California consisted mainly of perillaldehyde (103) (74%), (3-caryophyllene (52), limonene (3), and oc-bergamotene (104). This composition is slightly different from other samples harvested in Japan or from callus tissues [171]. In particular, the extremely high concentration of perillaldehyde (103), compared to other steam distillates of P. frutescens reported, the existence of a-bergamotene (104) and the ratio of (3-caryophyllene (52) to perillaldehyde (103) are significantly different with a seasonal variation of the constituents.

The most abundant component, perillaldehyde (103), has moderate and broad-spectrum activity against all microbes tested. It has an MIC against many microorganisms of between 125 and 1000 |ig/mL, close to that of the steam distillate. In the case of Gram positive bacteria and fungi, the activity is generally weaker when compared to the steam distillate, while the activity is generally equivalent to that of the oil for Gram negative bacteria. The high concentration of perillaldehyde (103) indicates that it is the major compound responsible for the activity of the crude distillate. The second major component, limonene (3) inhibits gram positive anaerobes, particularly A. naeslundii, and yeast. The other minor components, perillyl alcohol (106), isoeugenol (55), and linalool (60), show weak but broad spectrum activity with isoeugenol (55) as the most active and linalool (60) as the least active of the three. (3-Caryophyllene (52) also shows activity against Gram positive anaerobic bacteria, especially against P. acnes with an MIC of 7.8 |ig/mL, while (3-pinene (14) shows activity against both Gram positive bacteria and fungi.

608 NAKATSDtffl/.

CHO

6 ^ 6 103 104 105 106

Fig. (14).

Synergistic Effects

The leaves of P. frutescens are commonly served with the red bud leaves of Polygonum hydropiper, an herb containing the hot-tasting constituent, polygodial (107). This tradition suggests an interaction between the constituents of both herbs, and it can be surmised that they would not interact in an antagonistic way. In fact, when exploring combinations, none of these materials were shown to be antagonistic towards the others. Polygodial (107) has been shown to exhibit synergy with both actinomycin D [172] and anethole (96) [173] against fungi, but, in general, these are not consumed together. The possible synergy between polygodial (107) and perillaldehyde (103), which are consumed together, was explored. The individual MICs of the two compounds are shown in Table 15, while the synergistic effects of combining these two compounds are clearly shown in Table 16.

The amount of polygodial (107) needed to inhibit the yeast C. utilis and S. cerevisiae is reduced to one quarter of the uncombined MIC at the same time that the concentration of perillaldehyde (103) is reduced fourfold from its uncombined MIC. A similar but more dramatic combination effect occurs against C. albicans and M. mucedo where a fourfold decrease from the uncombined MIC of polygodial (107) at 1.95 |ig/mL reduces the amount of perillaldehyde (103) required for inhibition by as much as 32-fold and 16-fold, respectively. Further limited reduction in polygodial (107) concentration will maintain the synergistic effect, provided there is a corresponding increase in perillaldehyde (103) concentration, as seen with C utilis, C. albicans, M. mucedo, and P. chrysogenum.

Polygodial (107) has been reported to be a potentiator in combinations against fungi but has not been considered one against bacteria. Nevertheless, our antibacterial test data for the gram negative bacteria S. choleraesuis shows it to be inhibited synergistically with this combination of perillaldehyde (103) and polygodial (107). The MIC of polygodial (107) is decreased eightfold at the same time that the MIC of perillaldehyde (103) is decreased fourfold from their individual MICs of

6


Table 14. Antimicrobial Activity of the Steam Distillate of P. frutescens and its Constitutes

Org

A. na.

B. s.

1 P.&. P. ac.

S. a.

S. m.

E. a.

E. c.

P. ae.

S. ch.

A. ni.

C. a.

C. u.

M. m.

P. ch.

S. ce.

MIC ((ig/mL) Against Microbes

SD

250

500

500

3 .2

125

1000

>I000

1000

>1000

500

500

500

250

62.5

62 5

250

103

500

500

1000

250

1000

500

500

500

>1000

1000

250

500

500

250

250

500

3

31.2

>1000

125

250

125

125

>1000

>1000

>1000

>1000

>1000

>1000

62.5

>1000

>1000

62.5

52

125

>1000

>1000

7.8

>1000

>1000

>I000

>1000

>1000

>1000

>1000

>1000

>1000

>1000

>I000

>1000

60

500

1000

>I000

500

>1000

1000

>1000

>1000

>1000

1000

1000

>1000

500

500

500

1000

105

>1000

>1000

> 1000

>1000

>1000

>1000

>I000

>1000

>1000

1000

250

>I000

1000

250

500

1000

14

250

>I000

1000

125

125

125

>1000

>1000

>1000

1000

>1000

2000

1000

1000

125

250

106

>1000

1000

>1000

250

1000

500

500

500

>1000

500

500

1000

500

250

500

500

53

250

>1000

250

125

500

500

500

500

>1000

500

500

500

>1000

250

250

500

10

125

>1000

125

15.6

125

>1000

>1000

>1000

>1000

>1000

>1000

>1000

>1000

>1000

>1000

>1000

SD: Steam Distillate; perillaldehyde (103); limonene (3); p-caryophyllene (52); linalool (60); benzaldehyde (105); P-pinene (14); perillyl alcohol (106); isoeugenol (55); oc-luimulene (53) . Org: Microorganism. A. na.:A. naeshmdii; B. s:B. subtilus; P. gi.:P. gingivalis; P. ac.:P. acnes; S. a.: S. aureus; S. m.:S. mutans; E. a.: E. aerogenes; E. c: E. tali; P. ac: P. aeruginosa; S. ch.: S. choleraesuis; A. ni.: A. niger; C. a.: C. albicans; ('.. u.: (.". ulilis; M. in.: M. muceth; P. ch.: P. chrysogcnuin; S. ce.: S. cerevisiae.

CHO CHO

A

CHO

103 107

Fig. (15).

610 NAKATSU <*«/.

Table 15. Antimicrobial Activity of Polygodial (107) and Perillaldehyde (103)

Organism

B. subtilis

S. choleraesuis

P. aeruginosa

C. albicans

C. utilis

M. mucedo

P. chrysogenum

S. cerevisiae

MIC Oig/mL) Against Microbes

107

125

2000

>1000

3.9

7.8

1.95

7.8

1.95

103 1

500

1000

>1000 1

500 1

500 I

250

250 1

500 1

250 |ig/mL. Equally unexpected is the synergy of both polygodial (107) and perillaldehyde (103) (individual MICs of 500 |ig/mL each) against another Gram negative bacteria, P. aeruginosa, which is one of the two most resistant bacterial strains selected in this study. The Gram positive, spore-forming B. subtilis is inhibited at a concentration fourfold less than the uncombined MIC of either compound alone.

Table 16. Synergistic Effects Against Microbes of Polygodial (107) and Perillaldehyde (103) with Individual FIC and Combined FIC Values

B. subtilis

S. choleraesuis

P. aeruginosa

C. albicans

C. utilis

M. mucedo

P. chrysogenum

S. cerevisiae

107

MIC

31.25

250

500

1.95 0.12

1.95 0.98

1.95 0.98

0.48

0.48

Lowest FIC

0.25

0.125

<0.25

0.25 0.015

0.50 0.25

0.25 0.126

0.246

0.246

103

MIC

125

250

500

15.6 250

125 250

7.81 31.25

62.5

125

Lowest FIC

0.25

0.25

<0.25

0.031 0.5

0.25 0.50

0.031 0.125

0.25

0.25

Combined* F I C J

O500

0.375 1

<0.5 1

0.281 ] 0.515

0.750 1 0.750

0.281 1 0.251

0.496 1

0.496 1

•Combined FIC = Lowest FIC of 107 + Lowest FIC of 103

Combined MIC of 107 or 103 Lowest FIC 107 or 103 =

Individual MIC of 107 or 103


Evaluation of the Synergistic Effect

The fractional inhibitory concentration (FIC) method for calculating synergy or antagonism is used to further analyze the data [174]. The FIC values of 0.5 or less indicate unequivocal synergy. Using this method, there is true synergy (FIC value <0.5) for all the combinations tested, except against C. utilis. The synergy is most effective (lowest FIC value) on C. albicans and M mucedo for the microorganisms tested. None of the combinations tested showed an antagonistic effect which would be demonstrated by an FIC above 1.0 (Table 16).

THE USE AND APPLICATION OF ESSENTIAL OILS

The use and application of essential oils, as well as the individual constituent chemical entities, is commonplace and widespread in our society [175-188]. In addition to the extensive use of essential oils for their organoleptic qualities, which will not be reviewed here, they are employed as preservatives for flavor, fragrance, and cosmetic products, for prevention of the progression of various disease states, as biodegradable solvents, etc. The reviews referenced above provide excellent coverage of the uses of essential oils and phytochemicals in industry prior to the mid 1990s. Selected recent developments occurring during the last several years are summarized below.

Insect and Animal Control

An area where essential oils have been traditionally used very extensively is in products for the control of insects and rodents. The need for alternatives to toxic and non-biodegradable synthetic pesticides is a strong incentive for developing new products that employ the natural biological activities of essential oils. The insecticidal and antifeedant activities of a number of plant compounds against a variety of insect species including crop pests, medically important insects, and wood-destroying insects have been reported [189]. Plants such as Xylopia aethiopica (Annonaceae), Zanthoxylum xanthoxyloides (Rutaceae), and Detarium microcarpum (Leguminosae), among others, were shown to be interesting sources of bioactive compounds. Recently, an evaluation of a number of monoterpenoids of natural origin as nematicides was conducted [190]. Also, the results of a study were published in which a eucalyptus-based insect repellent, with the principal active ingredient /?-menthane-3,8-diol (108), was evaluated in the field in comparison with DEET [191]. This repellent was just as effective as DEET in terms of efficacy and duration of protection from biting by the insects tested.

612 NAKATSUtffl/.

Learning from nature, a considerable amount of work is being invested in finding combinations of natural materials, as well as combinations of natural and synthetic materials, which synergize each other's activities in order to find effective compositions for insect repellency. The results of an investigation were reported recently in which several nontoxic vegetable oils were evaluated as synergists for different synthetic pyrethroids in mixed formulations against Tribolium castaneum [192]. In many combinations, the desired toxicity of a particular insecticide tested was maintained, even though the amount of toxicant (pyrethroid) was reduced as much as ninefold. Recently, a patent was granted for the use of the combination of essential oils and acetic acid as insect repellents [193]. It was reported that the repellent activities of eucalyptus oil and citronella oil are enhanced by the presence of acetic acid. In addition, another patent was granted for a synergistic composition for the control of insects, their larvae and eggs, in the order Hymenoptera, which contains citrus peel essential oils and a C9-C11 alkanoic acid [194].

Studies into the method of action of essential oils as insect repellents are currently underway that may further help in the development of new products. Recently, the results of an investigation were reported on the method of action of the acaricidal properties of Lavandula angustifolia Miller essential oil and of linalool (60), one of its main components, against Psoroptes cuniculi [195]. The study confirms the anti-mite properties of lavender essential oil and of linalool (60) by inhalation, indicating an additional route for possible use of these substances both for prophylactic and therapeutic purposes. Also, the method of action of the toxicity of citrus peel oils to several insect species has recently been investigated [196]. The results indicated that an efficient way to use citrus peel essential oils to control insects would be as a fumigant in relatively enclosed or air-tight systems.

Search of the recent literature reveals many more miscellaneous examples of the use of essential oils as insect and rodent repellents. Recently, a patent has been granted for the use of essential oils as insect repellents and conditioners for the coat, skin, and hooves of horses [197]. These essential oils are applied to the coat, skin, and hooves of horses to replace lipids lost due to washing and are also useful as conditioners for the hooves and as repellents for horseflies and other stinging insects. A rodent repellent based on components found in garlic oil was also recently disclosed [198]. It was shown that the odor of refined garlic oil, or the high-boiling components, such as diallyl sulfide, allyl methyl sulfide, and allyl propyl sulfide, repels mice. In addition, a patent for insect repellent compositions for human skin, which have long-lasting activity and contain natural insect-repelling fragrance materials such as cedarwood oil, citronella oil, and geraniol (58), was published [199].


Oral Care Products

Mouth care is an another application for which essential oils have traditionally been used extensively, and work in this area continues to be quite active. Recently, several patents have been granted for antibacterial mouthwash compositions that contain essential oils as their active ingredients. Reduced alcohol antiplaque and anticalculus mouthwash compositions, containing essential oils and polyols, were shown to be useful in the prevention and reduction of bad breath, plaque and related gum diseases [200]. Another antimicrobial mouthwash composition, containing thymol (91) and one or more other active essential oils, was also shown to be useful for these applications [201]. In addition, a patent was granted for oral compositions containing terpenoids for the treatment of plaque and gingivitis [202]. It was reported that thymol (91) and eugenol (1) each inhibited the growth of Actinomyces viscosus in vitro at 25 ppm and that four days treatment with a mouthwash containing these two actives inhibited plaque growth by almost 60%. Also, in a direct comparison of commercial antiplaque mouth rinse products, a product containing an essential oil as the active ingredient was found to be more effective and longer lasting in its antiplaque activity than another mouthrinse that contained triclosan, a commonly used, synthetic antibacterial as its active ingredient [203].

Recently, the results of an investigation were published on the antibacteriological and toxicological effects of the essential oil of Lippia multiflora mold [204]. The results indicated the leaf oil of L. multiflora, containing primarily (Z)- and (£)~tagetone (110), was highly active against isolated buccal flora microorganisms, was active as a mouthwash solution (500-fold dilution of the essential oil), and showed negative results in toxicological evaluation. A patent for the use of natural mastic from chios (or extracted mastic oil), for the production of toothpaste, mouth wash, mouth deodorizers, as well as other products has recently been granted [205]. It was claimed that these extracts improve the efficiency of the natural tissue defense system, situated between the teeth and the gums, acting against plaque and the formation of gum disease. Dentifrices

HO'

109 HO

614 NAKATSU £*<?/.

containing rutin or its glycosides and essential oils for treatment of periodontal diseases have also been recently disclosed [206]. The coadministration of rutin (50 mM) and limonene (3) (1 mM) was almost twice as effective as rutin (50 mM) alone in the prevention of collagen degradation and inflammation associated with periodontal diseases.

Pharmaceuticals

The pharmaceutical applications of essential oils and their individual chemical constituents are many and varied. Much of the recent work in this area is inspired by the knowledge gained through the investigation of traditionally used folk medicines. Recently, a patent has been granted for an antitussive composition containing, among other ingredients, essence of anise, senega dry extract, and pure licorice root extract as a method of treating cough in humans [207]. Also for application in the buccopharyngeal region, a patent has been granted for snoring-inhibiting compositions containing essential oils [208]. The active ingredients in these compositions are selected from either essential oils such as eucalyptus oil, mint oil, and peppermint oil, or individual chemical constituents such as menthol and cineole.

Other recent developments have indicated that essential oils can have significant analgesic effects, as well as effects on CNS-related disorders. The results of an investigation reported recently suggest the presence of analgesic, as well as anti-inflammatory, constituents in the essential oil of Psidium guianense and support the popular use of Psidium leaf oils in the control of local inflammatory pain [209]. In addition, the oil extracted from Elettaria cardamomum seeds showed significant anti-inflammatory, analgesic, and antispasmodic activity in a variety of assays [210]. Also, the use of several essential oils for the alleviation of headache has been implicated by the results of a recently published investigation. The effects of peppermint oil and eucalyptus oil preparations, applied topically to the forehead of human subjects, on neurophysiological, psychological, and experimental algesimetric parameters were investigated [211]. Among other findings, there was observed a significant analgesic effect with a reduction in sensitivity to headache by the combination of peppermint oil and ethanol. Other effects on the brain, which have been linked to the presence of essential oils, or their individual chemical constituents, have included cognitive-enhancement and sedation. Recently, the effects of the seed oil of Celastrus paniculatus, a medicinal plant from India that has been reputed to be useful as a pharmaceutical aid for learning and memory, were evaluated [212]. The results showed that the seed oil of Celastrus paniculatus, when administered chronically, selectively reversed the impairment in spatial memory produced by acute central muscarinic receptor blockade, supporting the possibility that one or more constituents of the oil may offer cognitive-enhancing properties. Also, the


results of an investigation were published on the sedative properties of linalool (60) [213]. The results showed that this compound has dose-dependent effects on the CNS, including hypnotic, anticonvulsant and hypothermic. The effects of linalool (60) revealed by this evaluation are useful in understanding the traditional medical use of several plant species on different continents and point to the validity of exploring terpenes as sources of new anticonvulsant agents.

Drug Delivery Systems

Essential oils have also been shown to be useful for the delivery and to improve the bioavailability of pharmaceuticals. Recently, a patent was granted that describes a method for increasing the bioavailability of an orally administered hydrophobic pharmaceutical compound by coadministration with an essential oil (anise, basil, bergamot, etc.) [214]. In addition, the results of an investigation were reported recently on the use of oil-water microemulsions for the transdermal absorption of nifedipine, which employed essential oils (ylang ylang oil, lavender oil, cinnamon oil) and natural materials [cineole (88), menthone (50), menthol (41)] as lipophilic skin penetration enhancers [215].

Wound Healing

Wound healing and disinfectancy is another area where essential oils have been shown to be of use. The results of a study have been published on the in vitro activity of the essential oil of Melaleuca alternifolia against a variety of Streptococcus species [216]. The activity of this essential oil was such that it was suggested that the oil might be effective against streptococci when used topically as a wound disinfectant. In addition, a patent was granted for the use of aromatic, neutral resin oils extracted from Cupressaceae in medicinal and pharmaceutical compositions [217]. The neutral oils isolated from Cupressaceae {Thujopsis sp.} Chamaecyparis sp.), by extraction and subsequent processing to remove the acid and tar components, were shown to stimulate epithelium formation and promote wound healing.

Antiviral

Essential oils have even shown potential for the treatment of viral diseases. Recently, the results of an investigation were reported on the antiviral and antibacterial activity of essential oils from the fruit of some species of the genus Heracleum L. (Apiaceae) [218]. As well as having antibacterial activity against S. aureus and other bacteria, the essential oils from Heracleum L. species also showed considerable antiviral activity

616 NAKATSUtffl/.

against influenza virus type A and type B. In addition, a patent was granted for the use of extracts of the Luaraceae family, such as Agatophyllum aromaticum and Melaleuca quinquenervia, for the treatment of AIDS and herpes [219].

Anti-inflammatory

Essential oils have also been shown to be useful as anti-inflammatory agents. The anti-inflammatory and antipyretic activities of Ocimum sanctum fixed oil have been evaluated recently [220]. The results reported are consistent with the folk medicine use of different parts of this plant for the treatment of acute and chronic inflammation. The results of an investigation on the anti-inflammatory activities of flavonoids of Baphia nitida, another plant used in folk medicine, were recently reported [221], The flavonoid-rich fraction of the leaf, obtained by a chromatographic process, was formulated into an ointment and exhibited significant antiinflammatory activity in several rodent inflammation models. The inhibition of tumor necrosis factor (TNF-a) and interleukin-ip (IL-1), mediators in many acute and chronic inflammatory diseases, by curcumin (111), a phytochemical isolated from the plant Curcuma longa Linn, was recently reported [222]. This report shows that, in vitro, curcumin (111), at 5 |iM, inhibited lipopolysaccharide (LPS)-induced production of TNF-a and IL-1 by a human monocytic macrophage cell line.

Others

There were also several other miscellaneous pharmaceutical uses of essential oils or their individual chemical constituents, which were disclosed during the past several years. The results of a study were reported recently on the effects of the dietary administration of a selection of volatile oils from medicinal plants on the polyunsaturated fatty acid composition in the retina of aged (28 months) rats [223]. The possible efficacy for the application of the oils from such medicinal plants, through their antioxidant capacities, in the prevention of age-related macular degeneration was discussed. Recently, the results of an investigation were published on the antimutagenic activity of caryophyllene oxide (9), a phytochemical present in the essential oils of traditionally used, folk medicinal plants and herbal spices [224]. It was suggested that the level of activity detected in the Ames pre-incubation test indicates that caryophyllene oxide (9) could be developed as a potent antimutagenic flavoring agent. In the cardiovascular area, a patent has been granted for the use of the essential oil obtained from Juniper communis as an antiatherosclerotic and hypocholesterolemic agent [225]. Oral administration of an aqueous/alcoholic extract of dried Juniper communis


resulted in the reduction of the blood cholesterol level of a patient by almost 20%.

Cosmetics

Essential oils in cosmetic products exert their effects through a variety of mechanisms that include, but are not limited to, moisturization, cell growth stimulation, and antimicrobial effects. The essential oil obtained from the leaves and flowers of Lippia alba, mixed with biological creams, has been shown to be excellent for treating aged and dry skins [226]. It contributes to the skin cell cohesion by forming a barrier that regulates trans-epidermic moisture loss. In addition, new compositions for the treatment of acne were patented containing, among other components, tea tree oil [227]. Individual materials isolated from essential oils also are quite useful in a variety of skin-related applications. A patent has been granted for the use of gentisic acid (2,5-dihydroxybenzoic acid) (109) in cosmetic compositions useful for stimulating the epidermis and for treating the skin [228]. In addition, the results of an investigation were reported on the assessment of the effectiveness of treatment with topical furocoumarins from bergamot oil and UVA radiation for epidermal melanogenesis [229]. The greatest activity found was that of 6,4,4'-trimethylangelicin (112), which induced a 6-fold increase in the production of melanocytes compared to all other UVA-only treated groups. Naturally derived triglycerides and oils also are used to a great extent in cosmetic preparations. Cosmetic or pharmaceutical compositions that contain ozonide-enriched natural vegetable oils, which can be used for the treatment of skin diseases such as acne, have recently been disclosed [230]. Recently, a patent was granted for cosmetic compositions useful for the treatment of corns and warts that comprise plant tinctures and essential oils such as Thuya occidentalis tincture, Thymus vulgaris oil, and Lavandula spica oil [231].

Food Preservatives

The use of essential oils for the protection of food and related products has also received considerable attention recently. An investigation of the bioactivity of materials derived from the leaves and succulent stems of Ocimum kenyense as a source of repellents, toxicants and protectants in storage against three major stored product insect pests was recently conducted [232]. Furthermore, it was shown that a large number of other essential oils (i.e. anise, basil, oregano, etc.) also could be employed as fumigants and contact insecticides for the control of these stored-product insects [233]. It was suggested that these materials could be very useful on the farm level in developing countries and could play an important role in

618 NAKATSUtftf/.

stored-grain production and reduce the need for, and risks associated with, the use of insecticides. In addition, the results of an investigation were published on the effect of anise, cinnamon, clove, marjoram and peppermint spice oils on the inhibition of mycoflora and zearalenone production on rice grains [234]. The population of fungi was reduced by spice oils, used as low as 0.1%, and were completely inhibited by cinnamon oil at 1%. Japanese mint {Mentha arvensis) oil has also been evaluated as a fumigant on stored sorghum [235]. The Japanese mint oil was effective as a fumigant against Sitophilus oryzae in sorghum, however, treated samples of boiled sorghum scored significantly lower values for sensory quality when compared to untreated samples and, as such, this technique was recommended only for seed sorghum preservation.

Antioxidants

Recently, the results of an investigation were reported showing that a variety of essential oils, particularly oils from Origanum majorana, Acantholippia seriphioides and Tagetes filifolia, exhibited a pronounced antioxidative activity in peanut oil [236]. In addition, a patent was granted for a natural antioxidant that is used to prolong the shelf life of animal fats and vegetable oils [237]. This antioxidant solution contains up to 20 weight percent of carnosic acid (113) and is miscible with fats and oils up to a concentration of 500 ppm of carnosic acid (113). An evaluation of several natural antioxidants, useful for the prevention of oxidation of materials in beer, was recently completed [238]. Two potential natural antioxidants, catechin (114) and ferulic acid (115), were tested for their ability to suppress the formation of aldehydes during the force-aging of beer. Several carbonyl compounds decreased in level in the beer dosed with catechin (114), whereas there was no decrease in the level of carbonyl compounds in beer dosed with ferulic acid (115).

The results of an investigation of the screening of many different commercial essential oils for activity against 20 Listeria monocytogenes strains, implicated in food poisoning, were reported [239]. The possible use of a number of essential oils in a dual role as flavors and antimicrobials was also discussed. Recently, a patent was granted for a process for the manufacture of a stable formulation of lycopene, a natural food colorant, which contains terpenoids such as d-limonene (3) used to stabilize the natural pigment [240]. Geranium, lemongrass, peppermint, and spearmint essential oils were also recently evaluated for the inhibition of the potato virus Y [241]. Almost complete inhibition of this virus was observed with as little as 500 ppm essential oil for time periods as small as one hour. Recently, a patent was granted for a method of improving flavoring in consumer products by the addition of as little as 1 ppm polygodial (107) [242]. It was reported that gum with flavoring containing polygodial (107)


had a longer lasting, fresher taste than gum with mint flavoring not containing polygodial (107).

Industrial Applications

In light of the increased awareness of the environmental consequences of industrial activities, it should not be surprising that essential oils, materials that are, by definition, natural and biodegradable, are finding increased use for industrial applications. Essential oils are finding increased use as solvents and cleaning fluids for a variety of applications. Recently, a patent has been granted for cleaning fluids for retarding electrolysis between dissimilar metals in electrical circuits that contain, among other ingredients, varying amounts of rosemary oil, cypress oil, mint oil, eucalyptus oil, and/or clove oil [243]. Also, the use of lavender oil, pelargonium oil, and/or rose oil for the removal of paints from polypropylene automobile parts, enabling the recycling of the cleaned parts, has been claimed [244]. Another patent has been granted for a solvent mixture useful for separating latex from carpets for recycling which comprises Gaultheria procumbens oil, Thymus vulgaris oil, anise oil, Foeniculum oil, and methyl salicylate [245]. A universal cleaner for the removal of oils, fats, lubricants and other similar contaminants has also been disclosed [246]. Among other naturally derived materials, the cleaning composition contains natural solvents such as citrus peel oil or terpene alcohols. Yet another patent was granted for an environmentally safe graffiti remover [247]. The patent describes a formulation, containing primarily Gaultheria procumbens essential oil (80%), which can be used for the removal of synthetic coating materials with no damage to the substrate. The recycling of plastics is yet another application where essential oils are being used to significant advantage. Recently, a patent was granted for a process for the separation and recycling of plastics, which is based on the solubility of these materials in various natural essential oils [248]. Plastic articles without granulation are passed through a series of dissolution stages until they dissolve, and inorganic fillers and reinforcements can then be separated by filtration. Solvents containing monoterpenes, useful for preparing tissue samples for histopathological examinations, were recently disclosed [249]. These solvents, containing a variety of terpenes, including oc-pinene (13) and p-pinene (14), show good performance based on several criteria; tissue hardness was suitable for slicing after solvent replacement, transparency of the tissue was good, odor was low, and there was no skin irritation. Yet another example of the use of essential oils as industrial solvents is provided by the disclosure of a process for the preparation of microcapsules, containing terpene or abietic acid derivatives as biodegradable solvents, for use on chemical copying papers and pressure-sensitive papers [250]. Water-reducible dye compositions comprising dyes and citrus solvents (citrus peel oils and

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terpenes) has also been claimed [251]. The dye solution is dispersible in water and infinitely reducible, and is especially useful in inks.

116 117 118

Fig. (17).

Packaging

The incorporation of essential oils, as well as the individual chemical constituents, into packaging and construction materials, in order to take advantage of a variety of biological activities, is yet another way that essential oils can be put to good use. Recently, a number of products were patented that were described as being useful for food packages, medical goods, or sanitary goods, and which have antibacterial coating layers containing cyclodextrin-hiba oil mixtures as antibacterial agents [252]. Trash bag compositions that contain, in addition to the ethylene polymers used to comprise the bag, animal repellents that are chosen from acidic and/or spicy (mustard) components, bitters (horseradish) and/or


stimulants (capsaicin), and essential oils from citrus [d-limonene (3)] have also been disclosed [253]. Also, antibacterial and worm-repellent thermoplastic compositions containing essential oils have been described [254]. These compositions, useful for packaging materials, health instruments, transportation and storage containers, etc., are prepared from thermoplastic resins and cyclodextrin microcapsules of antibacterial and worm-repellent essential oils such as cedar leaf oil.

Consumer Household Products

Incorporation of essential oils into fabrics, useful for a variety of applications, has also been employed to take advantage of the antimicrobial and insect repellency of these natural materials. Recently, a patent was granted for tickicidal odor-absorbing antibacterial fibers and their uses for stockings and medical-care products [255]. A solution containing 1,8-cineole (88), as the active ingredient, was used to give encapsulated materials suitable for the manufacture of these odor-absorbing, antibacterial, tickicidal fibers. Another patent discloses a method of producing fabrics with antibacterial and anti-tick activity by the incorporation of microencapsulated hinokitiol (116) or hiba oil [256]. Fabrics impregnated with a mixture of encapsulated hiba oil and a polyurethane showed bactericidal activity even after 10 launderings. Yet another patent was granted for the use of fabrics containing cedar (Thujopsis dorablata) oil, hinokitiol, or camphor tree (Cinnamonum camphora) oil [257]. The claims include the use of these essential oil-impregnated fabrics for the manufacture of domestic items, such as carpets and filter bags for vacuum cleaners, to control fungi and mites.

Many other miscellaneous examples of the incorporation of essential oils into packaging and construction materials can be found in the recent literature. The manufacture of a solution for fireproofing and pesticidal and microbicidal protection of waste paper-based insulation materials, which contains almost 20% Thymus vulgaris oil, has been patented [258]. Antifouling agents that contain cinnamaldehyde (117), useful for coating tools for culturing aquatic animals have also been disclosed [259]. These antifouling agents are designed for the control of algae, mollusks, etc., and showed good effects on fishing nets that were placed in seawater for two months following application of the agent. Recently, a patent was granted for an encapsulated mite repellent useful for food-packaging materials [260]. The terpene mite repellents were encapsulated into a shell made of cellulose derivatives, gums, waxes, oils and/or glycerides and applied to food-packaging materials. Yet another patent was granted for sheet products made from tissue paper impregnated with herbal and essential oils [261]. The products described, useful for disinfecting, insect repellent, and air-freshening applications, contain any one of a number of essential oils such as eucalyptus oil, peppermint oil, or rosemary oil.

622 NAKATStJtffl/.

Miscellaneous Uses

A search of the literature for the use and application of essential oils reveals many other examples that are not covered by inclusion in the categories discussed above. Essential oils also have found use in a variety of miscellaneous antimicrobial applications. Recently, several patents have been granted for compositions, useful for disinfecting surfaces, which contain essential oils as the active ingredient [262,263]. Another patent has been granted for the use of a combination of at least one terpene alcohol or pine oil and at least one non-bactericidal surfactant as a synergistic bactericidal mixture [264]. The bactericidal mixture may be used in disinfectants (particularly cleaning agents) and antiseptics as it contains no phenol derivatives or quaternary ammonium salts. Secondary plant metabolites have also been claimed as being useful for the control of postharvest fungal diseases on flower bulbs [265]. Carvone (48), cuminaldehyde (118), perillaldehyde (103), among others, were the most potent inhibitors of in vitro growth of the fungi Penicillium hirsutum and P. allii.

Recently, a patent has been granted for incapacitating agents that contain a mixture of piperidides (Piperacea family) and capsaicinoids (Salanacea family) [266]. This combination of the piperidides and capsaicinoids is synergistic in its effect and is biodegradable. In addition, several patents were granted for emulsifier compositions (dry, powder form or in aqueous solution) that comprise a mixture of a Natural Vegetable Polyphenols Extract (NVPE) [267,268]. These NVPE emulsifiers are particularly useful in producing stable, anionic asphalt-in-water emulsions that can be used as is or with a wide variety of fillers, additives and pigments. Methods and compositions of materials, containing essential oils, which address problems associated with wood such as sapstain, wood degradation and pests have also been disclosed recently [269]. The methods comprise the incorporation or application of compositions containing terpene derivatives such as those obtained from the processing of a-pinene (13) and P-pinene (14), unsaturated aldehydes and ketones, and mono- and diphenols. Different combinations of techniques and compositions were described which can be used to provide short- to long-term protection to wood and wood-based materials.

CONCLUSION

Even though essential oils are most commonly used in our everyday life as fragrance and flavoring materials, and are major components in fruits, vegetables, beans and other plant food products, the actual role in the maintenance of our health is still under investigation. A significant difficulty in the study of the biologically active essential oils is that the


remarkable biological activity often does not depend on only one active compound. In addition, the essential oils are usually mixtures of structurally very similar compounds and, therefore, the biological activity of essential oils cannot necessarily be explained with a simple, single molecule - single activity approach. A recent study of a mammalian odorant receptor [270] indicates that the investigation of the relationship between essential oil constituents and receptors is the key to understanding the organoleptic and biological activities of essential oils in the future.

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