Biological Activity of Essential Oils and Their Constituents

Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol.
21 571
© 2000 Elsevier Science B.V. All rights reserved
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
572 NAKATSUtftfA
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].
576 NAKATSUe/r//.
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 C 0 2
J
step 1 J heat pressure
Supercritical C 0 2
i
sample matrix
step 2 J Analytes in C 0 2 solution
pressure reduction
i
in analyte trap
Gaseous CQ2 vented
r )
Rinse solvent
]
step 3 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].
578 NA KATSU £**!/.
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].
580 NAKATSU etal.
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 Structure Odor Perception Threshold Value

(ppb) 1
{4R)-cts Floral-Green 0.5

Natural type Clean, sharp, metallic, light, rose-
green., diffusive and strong
~ ^
{4R)-trans
Natural type
(4S)-cis
£ Floral-Green
Green, minty herbal, and fruity
Herbal-Green-Floral
160
50
Synthetic Hay green, earthy, heavy
&
(4S)-trans
6 Herbal-Green-Floral 80
X
Synthetic Fruity, herbal rose, and citrusy-
bitter-peel
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.
582 NAKATSlItfn/.
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).
584 NAKATSUtffl/.
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 39 3
OH OH OH OH
40 41 42 43 44
HO" *> HOx%%

> %
45 46 47 48 49
50 51
Fig. (5).
\i S o-
H
52 53 54
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
63 64 65
CHO C02H
s
OH ^^ ^^ "OH
66 67 68
CHO
OAc
71
Fig. (7).
Melanin Formation Assay

The appropriate numbers of cells (4 xl0 4 per 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 Inhibition (%) Compound Inhibition (%)
Citronellol (56) 74.9 Dihydromyrcenol (62) 32.9
Geraniol (58) 66.1 Tetrahydrolinalool (63) 43.1
Tetrahydrogeraniol (59) 50.9 Farnesol (64) 56.9
Linalool (60) 50.5 Phytol (57) 41.6
Nerolidol (61) 50.0 Geranylgeraniol (65) 44.3

590 NAKATSUtffl/.
Table 3. Tyrosinase Inhibitory Activity of Acyclic Alcohol at 100 Jig/ml
Compound Inhibition (%)
Citronellol (56) 74^9
Octanol (66) 46.2
Decanol (67) 56.6
Table 4. Tyrosinase Inhibitory Activity of Acyclic Terpene Aldehyde and Acid at 100
|ig/ml
Compound Inhibition (%)
Citronellol (56) 74^9
Citronellal (68) 41.6
Citronellic acid (69) 88.6
Citronellie acetate (71) 51.5
Geraniol (58) 66.1
Citral (70) 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 Concentration (mM) Inhibition (%)
Citronellol (56) 0.25 25
Geraniol (58) 0.25 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 IC 5 0 (|iM) Compound I C 5 0 (|iM)
72 1.1 74 3^5
76 6.9 78 18.9 1
1
| 80 6.7 82 25.7 |
84
1 73
6.1 86 39.6 1
1 144.5 75 7.8 1
1 77 105.8 79 3.1 1
81
1 64.9 83 4.1 1
1 85 23.5 87 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
OH
72 73 74 75
76 77 78 79
80 81 82 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 Concentration (fiM) Inhibition (%)
1 ^ 584.0 73
1 85 670.1 79 1
1 86 608.8 55
87 625.0 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 IC 5 0 (uM) Compound IC 5 0 (u.M)
72 100.4 74 NAT*
76
1 40.4 78 300.7 1
1 80 49.3 82 118.0 1
84
1 73
20.4 86 115.7 1
1 77
>328.4 75 312.0 1
1 81
>330.6 79 28.3
1 332.8 83 28.4
1 85 180.9 87 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. Anti-
inflammatory 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 anti-
inflammatory 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].
CO 2 H L ^ ^ ^ .CO2H
8 9
88 90
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
91 92 93 94
)Me QMe
OAc
98
99 100 101
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
MIC Oig/ml)
S. aureus Methicillin Penicillin Polymyxin B Gentamicin Vancomycin
[~~ ATCC6538 250.00 7.81 31.25 7.81 o.98 n

1 ATCC 12598 250.00 7.81 31.25 15.62 1.95
1 ATCC 11632 7.81 31.25 62.50 62.50 3.90
1 ATCC 29247 7.81 > 1000.00 31.25 3.90 1.95
| JB 1108 2000.00 >2000.00 125.00 >500.00 1.95 ]
1 JB 87023 >2000.00 62.50 62.50 >500.00 1.95
Table 10. Effects Against Clinical Isolates of MRSA from Combining Totarol (102)
and Methicillin with Individual FIC and Combined FIC Values
Totarol (102) Methicillin
Lowest Lowest Combined*
MIC FIC MIC FIC FIC
| S. aureus )B 1108 0.975 0.50 0.015 0.0000075 0.5000075 1

1 S. aureus 3B S1023 3.90 0.50 0.06 <0.00003 O.50003
1.95 0.25 0.12 <0.00006 <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
Totarol (102) Penicillin
MIC FIC MIC FIC FIC
( S. aureus )B 1108 0.976 0.50 0.0075 O.00000375 <0.50000375 1

[ S. aureus }B 87023 0.976 0.25 15.625 0.25 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
Totarol (102) Gentamicin
MIC FIC MIC FIC FIC
1 5. aureus SB 1108 0.97 0.50 31.25 <0.0625 <0.5625
S. aureus HB&1023 3.91 0.50 1.95 <0.0039 <0.5039

1.95 0.25 125 <0.25 <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 Ratio Compounds Ratio
2-Hexanol 0.23* Pulegone 0.90
Ocimene (21) and/or (22) 0.45* Perillaldehyde (103) 74.00
Benzaldehyde (105) 1.60 Perillyl alcohol (106) 0.26
Sabinene 0.40 a-Terpinyl acetate (98) 0.13*
P-Pinene (14) 0.60 Isoeugenol (55) 0.25
Myrcene trace P-Caryophyllene (52) 3.80
Pseudolimonene 0.20 a-Humulene (53) 0.13
Limonene (3) 12.80 a-Bergamotene (104) 3.50*
Terpinolene 0.10 Farnesene 0.10*
Linalool (60) 2.60 Aromadendrene 0.30*
Limonene oxide 0.10*
* 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 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
Table 14. Antimicrobial Activity of the Steam Distillate of P. frutescens and its
Constitutes
MIC ((ig/mL) Against Microbes
Org SD 103 3 52 60 105 14 106 53 10
A. na. 250 500 31.2 125 500 >1000 250 >1000 250 125
B. s. 500 500 >1000 >1000 1000 >1000 >I000 1000 >1000 >1000
1 P.&. 500 1000 125 >1000 >I000 > 1000 1000 >1000 250 125
P. ac. 3 .2 250 250 7.8 500 >1000 125 250 125 15.6
S. a. 125 1000 125 >1000 >1000 >1000 125 1000 500 125
S. m. 1000 500 125 >1000 1000 >1000 125 500 500 >1000
E. a. >I000 500 >1000 >I000 >1000 >I000 >1000 500 500 >1000
E. c. 1000 500 >1000 >1000 >1000 >1000 >1000 500 500 >1000
P. ae. >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000
S. ch. 500 1000 >1000 >1000 1000 1000 1000 500 500 >1000
A. ni. 500 250 >1000 >1000 1000 250 >1000 500 500 >1000
C. a. 500 500 >1000 >1000 >1000 >I000 2000 1000 500 >1000
C. u. 250 500 62.5 >1000 500 1000 1000 500 >1000 >1000
M. m. 62.5 250 >1000 >1000 500 250 1000 250 250 >1000
P. ch. 62 5 250 >1000 >I000 500 500 125 500 250 >1000
S. ce. 250 500 62.5 >1000 1000 1000 250 500 500 >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
CHO
A
103 107
Fig. (15).
610 NAKATSU <*«/.
Table 15. Antimicrobial Activity of Polygodial (107) and Perillaldehyde (103)
MIC Oig/mL) Against Microbes

Organism 107 103 1
B. subtilis 125 500

S. choleraesuis 2000 1000
P. aeruginosa >1000 >1000 1

C. albicans 3.9 500 1
C. utilis 7.8 500 I
M. mucedo 1.95 250
P. chrysogenum 7.8 250 1
S. cerevisiae 1.95 500 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
107 103
MIC Lowest MIC Lowest Combined*
FIC FIC F I C J
B. subtilis 31.25 0.25 125 0.25 O500
S. choleraesuis 250 0.125 250 0.25 0.375 1

P. aeruginosa 500 <0.25 500 <0.25 <0.5 1
C. albicans 1.95 0.25 15.6 0.031 0.281 ]
0.12 0.015 250 0.5 0.515
C. utilis 1.95 0.50 125 0.25 0.750 1

0.98 0.25 250 0.50 0.750
M. mucedo 1.95 0.25 7.81 0.031 0.281 1
0.98 0.126 31.25 0.125 0.251
P. chrysogenum 0.48 0.246 62.5 0.25 0.496 1

S. cerevisiae 0.48 0.246 125 0.25 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 co-
administration 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 co-
administration 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 anti-
inflammatory 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
620 NAKATSUtftf/.
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.
REFERENCES
[I] Teranishi, R.; Kint, S. Bioactive Volatile Compounds from Plants', Teranishi,
R.; Kint, S„ Ed.; American Chemical Society: Washington, DC, 1993.
[2] Foderaro, S. J. New American Encyclopedia', Foderaro, S. J., Ed.; Grolier
International, 1991; Vol. 7, pp 245.
[3] Dash, D. B.; Kashap, V. L. Materia Medica of Ayurveda; Naurang Rai Concept
Publishing Company: New Delhi, 1980.
[4] Chinese Medicinal Herbs of Hong Kong Hong Kong, 1978.
[5] Nakatsu, T.; Stout, M., Unpublsihed results.
[6] Sticher, 0 . Plant Mono-, Di-, and Sesquiterpenoids with Pharmacological or
Therapeutical Activity; Sticher, O., Ed.; Springer-Verlag: New York, 1977, pp
137-178.
[7] Teranishi, R.; Buttery, R. G.; Sugisawa, H. Bioactive Volatile Compounds from
Plants', Teranishi, R.; Buttery, R. G.; Sugisawa, H., Ed.; American Chemical
Society: Washington, DC, 1993.
[8] Kubo, I. Antimicrobial Activity of Green Tea Flavor Components', Kubo, I., Ed.;
American Chemical Society, 1993, pp 57-70.
[9] Buchbauer, G.; Jager, W.; Jirovetz, L.; Ilmberger, J.; Dietrich, H. Therapeutic
Properties of Essential Oils and Fragrance', Buchbauer, G.; Jager, W.; Jirovetz,
L.; Ilmberger, J.; Dietrich, H., Ed.; Amrican Chemical Society: Washington,
DC, 1993, pp 162-165.
[10] Baker, J. R. JAMA 1997, 278, 1803.
[II] CDC JAMA 1997,275,891.
[12] Huang, Z.; Hankinson, S. E.; Colditz, G. A.; Stamfer, M. J.; Hunter, D. J.;
Manson, J. E.; Hennekens, C. H.; Rosner, B.; Speizer, F. E.; Willett, W. C.
JAMA 1991,278, 1407.
[13] Dugo, G.; d'Alcontres, I. S.; Cotroneo, A.; Dugo, P. J Essent. Oil Res. 1992, 4,
589.
[14] Lanuzza, F.; Micali, G.; Curro, P.; Calabro, G. Flavour Fragrance J. 1991, 6,
29.
[15] Ziegler, H.; Spiteller, G. J Essent. Oil Res. 1992, 4, 355.
[16] Njoroge, S. M.; Ukeda, H.; Kusunose, H.; Sawamura, M. Flavour Fragrance J.
1995, 70,341.
[17] Manthey, J. A.; Grohmann, K. J. Agric. Food Chem. 1996, 44, 811.
[18] Chen, J.; Montanari, A. M.; Widmer, W. W. J. Agric. Food Chem 1997, 45,
364.
624 NAKATSU <?/«/.
[19] Dugo, P.; Mondello, L.; Cogliandro, E.; Verzera, A.; Dugo, G. J. Agric. Food
Chem. 1996,44,544.
[20] Wannissorn, B.; Jarikasem, S.; Soontorntanasart, T. Phytother. Res. 1996, 10,
551.
[21] Souto Bachiller, F. A.; De Jesus Echevarria, ML; Cardenas Gonzalez, O. E.;
Acuna Rodriguez, M. F.; Melendez, P. A.; Romero Ramsey, L. Phytochemistry
1997,44, 1077.
[22] Tzakou, O.; Roussis, V.; Loukis, A.; Harvala, C ; Galati, E. M.; Germano, M.
P. Flavour Fragrance J. 1997, 12, 113.
[23] Sefidkon, F.; Khajavi, M. S.; Mirza, M. J. Essent. Oil Res. 1997, 9, 471.
[24] Asefa, A.; Dagne, E. Bull. Chem. Soc. Ethiop. 1997, / / , 47.
[25] Belanger, A.; Dextraze, L.; Goudmand, H.; Garneau, F.-X.; Collin, G. Essential
Oil composition fo Acorus calamus from Quebec, in 15th Journees Internationales
Huiles Essentielles. 1996, pp 529-534.
[26] Whish, J. P. M. J. Essent. Oil Res. 1996, 8, 405.
[27] Scheffer, J. J. C. Phytother. Res. 1996, 10, S6.
[28] Dionex-Corporation, Sunnyvale, CA.
[29] Likens, S. T.; Nickerson, G. B. J. Chromatogr. 1966, 2/, 1.
[30] Maignial, L.; Pibarot, P.; Bonetti, G.; Chaintreau, A.; Marion, J. P. J.
Chromatogr. 1992, 606, 87.
[31] Queckenberg, O. R.; Frahm, A. W. Pharmazie 1994, 49, 159.
[32] Kerrola, K. Food Rev. Int. 1995, / / , 547.
[33] Gawdzik, J.; Kawka, S.; Mardarowicz, M.; Suprynowicz, Z.; Wolski, T. J. High
Resolut. Chromatogr. 1995, 18, 781.
[34] Gawdzik, J.; Mardarowicz, M.; Suprynowicz, Z.; Kawaka, S.; Wolski, T. J.
High Resolut. Chromatogr 1996, 19, 237.
[35] Reverchon, E. J. Supercrit. Fluids 1997, 10, 1.
[36] Jarvis, A. P.; Morgan, E. D. Phytochem. Anal. 1997, 8, 217.
[37] Bandyopadhyay, C. . in Newer Trends Essential Oils Flavours. 1993: Tata
McGraw-Hill, pp 153.
[38] Oszagyan, M.; Simandi, B.; Sawinsky, J.; Kery, A. J. Essent. Oil Res. 1996, 8,
333.
[39] Vasudevan, P.; Tandon, M.; Pathak, N.; Nuennerich, P.; Mueller, F.; Mele, A.;
Lentz, H. J. Sci. Ind. Res. 1997, 56, 662.
[40] Sargenti, S. R.; Lancas, F. M. Chromatographia 1997, 46, 285.
[41] Naik, S. N.; Kumar, A.; Maheshwari, R. C. Indian Perfum. 1993, 37, 364.
[42] Catchpole, O. J.; Grey, J. B.; Smallfield, B. M. ; Catchpole, O. J.; Grey, J. B.;
Smallfield, B. M., Ed.; American Chemical Society: Washington, DC, 1997;
Vol. 670, pp 76.
[43] Bureau, S.; Razungles, A.; Baumes, R.; Bayonove, C. J. Food Sci 1996, 61,
557.
[44] Kim, Y. D.; Morr, C. V. J. Agric. Food Chem. 1996, 44, 1314.
[45] Boelens, M. H. Perfumer & Flavorist 1996, 21, 27.
[46] Majors, R. E.; Raynie, D. E. LC-GC 1997, 15, 1106.
[47] Surburg, H.; Guentert, M.; Harder, H. . in Recent Dev. Flavor Fragrance Chem.,
Proc. Int. Haarmann Reimer Symp., 3rd. 1993: VCH: Weinheim;, pp 103.
[48] Potter, T. L. J. Essent. Oil Res. 1995, 7, 347.
[49] Jakobsen, H. B. Mod. Methods Plant Anal. 1997, 19, 1.
[50] Awano, K.; Ichikawa, Y.; Tokuda, K.; Kuraoka, M. Flavour Fragrance J. 1997,
12, 327.
[51] Bestmann, H. J.; Winkler, L.; von Helversen, O. Phytochemistry 1997, 46, 1169.
[52] Tarantilis, P. A.; Polissiou, M. G. J. Agric. Food Chem. 1997, 45, 459.
[53] Malundo, T. M. M.; Baldwin, E. A.; Moshonas, M. G.; Baker, R. A.; Shewfelt,
R. L. J. Agric. Food Chem. 1997, 45, 2187.
[54] Song, J.; Gardner, B. D.; Holland, J. F.; Beaudry, R. M. J. Agric. Food Chem
1997, 45, 1801.
[55] Cu, J.-Q.; Zhang, Z.; Pu, F.; Perineau, F.; Delmas, M.; Gaset, A. J. Essent. Oil
Res. 1990,2, 1.
[56] Wilde, P. F.; McClory, P. G. Perfumer & Flavorist 1994, 19, 25.
[57] Chinn, J. W.; Nakatsu, T.; Perry, P. Unpublished results. 1997.
[58] Lee, M. L.; Yang, F. J.; Bartle, K. D. Open Tubular Column Gas
Chromatography: Theory and Practice', John Wiley & Sons: New York, 1984.
[59] Diehl, J. W.; Kleinjan, S. B.; Olson, E. S. Spectrosc. Int. J. 1990, 8, 43.
[60] Kubo, I.; Nakatsu, T. LC-GC 1990, 8, 933.
[61] Geribaldi, S.; et al. Flavour Fragrance J. 1990, 5, 147.
[62] Leibrand, R. J. Am. Lab. 1991, Feb., 73.
[63] Hedges, L. M.; Wilkins, C. L. J. Chromatogr. Sci. 1991, 29, 345.
[64] David, F.; Sandra, P. Phytochemical Analysis 1991, 3, 145.
[65] Listemenn, M. L. Spectroscopy 1993, 8, 40.
[66] Canigueral, S.; Iglesias, J.; Vila, R.; Virgili, A.; Ibanez, C. Flavour Fragrance
J. 1994, 9, 201.
[67] Chyau, C.-C.; Mau, J.-L.; Wu, C.-M. J. Agric. Food Chem. 1996, 44, 1096.
[68] Koenig, W. A.; Rieck, A.; Hardt, I.; Gehrcke, B.; Kubeczka, K. H.; Muhle, H. J.
High Resolut. Chromatogr. 1994, 77, 315.
[69] Hardt, I. H.; Rieck, A.; Fricke, C.; Koenig, W. A. Flavour Fragrance J. 1995,
10, 165.
[70] Koenig, W. A.; Rieck, A.; Saritas, Y.; Hardt, I. H.; Kubeczka, K.-H.
Phytochemistry 1996, 42, 461.
[71] Ravid, U.; Putievsky, E.; Katzir, I. Flavour Fragrance J. 1996, 11, 191.
[72] Pietsch, M.; Konig, W. A. J. High Resolut. Chromatogr. 1997, 20, 257.
[73] Matsuda, H.; Yamamoto, T.; Kanisawa, T. Synthesis and Odor Properties of
Optical Isomers of Rose oxide and Dihydrorose oxide, in The 13th International
Congress of Flavours, Fragrances and Essential Oils. 1995. Istanbul, Turkey, pp
85-91.
[74] Meckes, M.; Calzada, F.; Tortoriello, J.; Gonzalez, J. L.; Martinez, M.
Phytother. Res. 1996, 10, 600.
[75] Ansari, S. H.; Ali, M.; Qadry, J. S. Int. J. Pharmacogn. 1993, 31, 89.
[76] Van Beek, T. A.; Kleis, R.; Posthumus, M. A.; Van Veldhuizen, A.
[77] Hassan, A. ML; Muhtadi, F. J.; Al-Badr, A. A. J. Pharm. Sci. 1979, 68, 800.
[78] Kamel, A.; Sandra, P. Biochem. System. Ecol. 1994, 22, 529.
[79] Kamel, A. J. Nat. Prod. 1995, 58, 428.
[80] Miyazawa, M.; Shimamura, H.; Nakamura, S.; Kameoka, H. J. Aric. Food
Chem. 1996, 44, 1647.
[81] El-sayed, A. M.; Al-Yahya, M. A. Int. J. Crude Drug Res. 1989, 27, 185.
[82] Tressl, R.; Engei, K.; Kossa, K. J. Agric. Food Chem. 1983, 31, 892.
[83] Miyazawa, M.; Maruyama, H.; Kameoka, H. Agric. Biol. Chem. 1983, 47, 2925.
[84] Miyazawa, M.; Maruyama, H.; Kameoka, H. Agric. Biol. Chem. 1984, 48, 2847.
[85] Miyazawa, M.; Watanabe, H.; Kameoka, H. J. Agric. Food. Chem. 1997,45,
677.
626 NAKATSU tftf/.
86] Lam, L. K. T.; Zheng, B. J. Agric. Food Chem. 1991, 39, 660.
;87] Zheng, G.-O.; Kenney, P. K.; Lam, L. K. T. J. Nat. Prod 1992, 55, 999.
88] Valero, E.; Varon, R.; Garcia-Carmona, F. J. Agirc. Food. Chem. 1990,55,
1097.
89] McAlister, K. L.; Kang, R.; Honda, C ; Huang, J.; Nakatsu, T. Tyrosinase
Inhibitory Effects of Acyclic Terpneoids and Structure Activity Relationship, in
The 33rd Meeting of Society Cosmetic Chemist Japan. 1993. Tokyo, pp 17 - 22.
90] Valero, E.; Varon, R.; Garcia-Carmona, F. J. Agric. Food. Chem. 1990,35,
1097.
91] Attanasi, O. Chim. Oggi. 1983, 8, 11.
92] Adawadkar, P. D.; EI. Sohly, M. A. Fitoterapia 1981, 52, 129.
93] Kubo, I.; Kim, M.; Naya, K.; Komatsu, S.; Yamagiwa, Y.; Ohashi, K.;
Sakamoto, Y.; Hirakawa, S.; Kamikawa, T. Chem. Lett. 1987, 1101.
94] Gerhold, D. L.; Craig, R.; Mumma, R. O. J. Chem. Ecol. 1984, 10, 713.
95] Spencer, G., F.; Tjarks, L. W.; Kleiman, R. J. Nat. Prod 1980, 43, 724.
96] Shobha, S. V.; Ravindranath, B. J. Agric. Food Chem. 1991, 39, 2214.
[97] Barrero, A. F.; Sanchez, J. F.; Barron, A.; Corrales, F.; Rodriguez, I.
98] Sullivan, J. T.; Richards, C. S.; Lloyd, H. A.; Krishna, G. Planta Med. 1982,
44, 175.
'99] Kubo, I.; Komatsu, S.; Ochi, M. J. Agric. Food Chem. 1986, 34, 970.
100] Himejima, M.; Kubo, I. J. Agric. Food Chem. 1991, 39, 418.
•101] Irie, J.; Murata, M.; Homma, S. Biosci. Biotech. Biochem. 1996, 60, 240.
[102] Laurens, A.; Fourneau, C ; Hocquemiller, R.; Cave, A.; Bories, C ; Loiseau, P.
M. Phytother. Res. 1997, / / , 145.
! 103] Bhattacharya, S. K.; M. Mukhopadhyay, M.; Mohan Rao, R. J. R.; Bagchi, A.;
Ray, A. B. Phytother. Res. 1987, 7, 127.
[104] Toyomizu, M.; Sugiyama, S.; Jin, R. L.; Nakatsu, T. Phytother. Res. 1993, 7,
252.
[105] Tanaka, S.; Akimoto, A.; Tambe, Y.; Tabata, M. Phytother. Res. 1996, 10, 238.
[106] Benett, A.; Stamford, I. F.; Tavares, I. A.; Jacobs, S.; Capasso, F.; Mascolo, N.;
Autore, G.; Romano, V.; Di Carlo, G. Phytother. Res. 1990, 2, 124.
[107] Laekeman, G. M.; Van Hoof, L.; Haemers, A.; Vanden Berghe, D. A.; Herman,
A. G.; Vlietinck, A. J. Phytother. Res. 1990, 4, 90.
[108] Ozaki, Y.; Rui, J.; Tang, T.; Satake, M. Biol. Pharm. Bull. 1997, 20, 861.
[109] Obeng-Ofori, D.; Reichmuth, C ; Bekele, J.; Hassanali, A. J. Appl. Entomol.
1997,727,237.
[110] Rai, R. K.; Gupta, K. C ; Broophy, J. J.; Tripathi, D. N. Chem. Environ. Res.
1993,2,267.
[Ill] Mwangi, J. W.; Addae-Mensah, I.; Muriuki, G.; Munavu, R.; Lwande, W.;
Hassawnali, A. Int. J. Pharmacogn. 1992, 30, 9.
[112] Sharma, R. N.; Tare, V.; Pawar, P.; Vartak, P. H. Appl. Entomol Zool. 1992,
27, 285.
[113] Maini, P. N.; Morallo-Rejesus, B. Philipp. J. Sci. 1992, 727, 391.
[114] Juven, B. J.; Kanner, J.; Schved, F.; Weisslowicz, H. J. Appl. Bacteriol. 1994,
76, 626.
[115] Remmal, A.; Bouchikhi, T.; Tantaoui-Elaraki, A.; Ettayebi, M. J. Pharm. Belg.
1993, 48, 352.
[116] Omer, E. A.; El-Bazza, Z. E.; Eggbo, Z. G. Egypt. J. Hortic. 1997, 24, 175.
[117] Aboutabl, E. A.; El-Dahmy, S. I. Bull. Fac. Pharm. (Cairo Univ.) 1995, 33, 87.
Mueller-Riebau, F.; Berger, B.; Yegen, O. J. Agric. Food Chem. 1995, 43, 2262.
Lagouri, V.; Boskou, D. Dev. FoodSci. 1995, 37A, 869.
Taira, S.; Tawata, S.; Kobamoto, N.; Toyama, S.; Yasuda, M. Nippon Noyaku
Gakkaishi 1994, 19, 299.
Lattaoui, N.; Tantaoui-Elaraki, A. Riv. Ital. EPPOS 1994, 5, 13.
Panizzi, L.; Flamini, G.; Cioni, P. L.; Morelli, I. J. Ethnopharmacol. 1993, 39,
167.
Arras, G.; Grella, G. E. J. Hortic. Sci. 1992, 67, 197.
Zygadlo, J. A.; Grosso, N. R. Flavour Fragrance J. 1995, 10, 113.
Yegen, O.; Berger, B.; Heitefuss, R. Z Pflanzenkrankh. Pflanzenschutz 1992,
99, 349.
Kedzia, B.; Segiet-Kujawa, E.; Holderna, E.; Krzyzaniak, M. Herba.Pol. 1990,
36, 155.
Soliman, F. M.; El-Kashoury, E. A.; Fathy, M. M.; Gonaid, M. H. Flavour
Fragrance J. 1994,9,29.
Piccaglia, R.; Marotti, M.; Giovanelli, E.; Deans, S. G.; Eaglesham, E. Ind.
Crops Prod. 1993,2,47.
Charai, M.; Mosaddak, M ; Faid, M. J. Essent. Oil Res. 1996, 8, 657.
Montes, M. A.; Valenzuela, L.; Wilko-Mirsky, T.; Bello, H.; Valladares, G. An.
R. Acad. Farm. 1992, 58, 509.
Biondi, D.; Cianci, P.; Geraci, C ; Ruberto, G. Flavour Fragrance J. 1993, 8,
331.
Remmal, A.; Bouchikhi, T.; Rhayour, K.; Ettayebi, M.; Tantaoui-Elaraki, A. J.
Essent. Oil Res. 1993,5, 179.
Badei, A. Z. M. Chem. Mikrobiol. Technol. Lebensm. 1992, 14, 177.
Marotti, M.; Piccaglia, R.; Giovanelli, E.; Deans, S. G.; Eaglesham, E. J.
Essent. Oil Res. 1994,(5, 57.
Fadel, H. H. M.; Aeisa, M. Bull. Natl. Res. Cent. (Egypt) 1994, 19. 135.
El-Naghy, M. A.; Maghazy, S. N.; Fadl-Allah, E. M.; El-Gendy, Z. K.
Zentralbl. Mikrobiol. 1992, 147, 214.
Marotti, M.; Piccaglia, R.; Giovanelli, E.; Deans, S. G.; Eaglesham, E. Flavour
Fragrance J. 1994, 9, 125.
Tawata, S.; Taira, S.; Kobamoto, N.; Ishihara, M.; Toyama, S. Nippon Noyaku
Gakkaishi 1996, 21, 141.
Kuhnt, M.; Proebstle, A.; Rimpler, H.; Bauer, R.; Heinrich, M. Planta Med.
1995, 61, 227.
Perrucci, S.; Cecchini, S.; Pretti, C ; Varriale Cognetti, A. M.; Macchioni, G.;
Flamini, G.; Cioni, P. L. Phytother. Res. 1995, 9, 147.
Karawya, M. S.; Hashim, F. M.; El-Wahab, S. M. A.; El-Deeb, K. S.; Soliman,
S. N.; Salam, I. A.; Mokhtar, N.; El-Hossiny, S. Zagazig J. Pharm. Sci. 1994,
5,49.
Caccioni, D. R. L.; Guizzardi, M. J. Essent. Oil Res. 1994, 6, 173.
Soliman, F. M.; El-Kashoury, E. A.; El-Fishawy, A. M.; El-Kawy, M. A. A.
Bull. Fac. Pharm. (Cairo Univ.) 1994, 32, 387.
Pooter, H. L. D.; Aboutabl, E. A.; El-Shabrawy, A. O. Flavour Fragrance J.
1996, /0,63.
Singh, G.; Srivastava, P.; Mallavrapu, G. R.; Ramesh, S.; Rao, G. P. Parfuem.
Kosmet. 1993, 74,714.
Gundidza, M.; Deans, S. G.; Kennedy, A. I.; Mavi, S.; Waterman, P. G.; Gray,
A. I. J. Sci. Food Agric. 1993, 62, 361.
628 NAKATSU tffl/.
[147] Hammerschmidt, F. J.; Clark, A. M.; Soliman, F. M.; El-Kashoury, E. S. A.;

Abd El-Kawy, M. M.; El-Fishawy, A. M. Planta Med. 1993, 59, 68.
148] Belaiche, T.; Tantaoui-Elaraki, A.; Ibrahimy, A. Sci. Aliments 1995, 15, 571.
149] El-Fishawy, A. M.; El-Kashoury, E. S. A.; Abd El-Kawy, M. A.; Soliman, F.
M. ZagazigJ. Pharm. Sci. 1993, 2, 150.
150] Rodriguez, M.; Garcia, D.; Garcia, M.; Pino, J.; Hernandez, L. Alimentaria
(Madrid) 1996,274, 107.
151] Bourrel, C ; Vilarem, G.; Gaset, A.; Mitjavila, S.; Fernandez, Y. Riv. Ital.
EPPOS 1996, 7,429.
152] Fournier, G.; Hadjiakhoondi. A.; Charles, B.; Fourniat, J.; Leboeuf, M.; Cave,
A. Planta Med 1994, 60, 283.
153] Fournier, G.; Hadjiakhoondi, A.; Leboeuf, M.; Cave, A.; Fourniat, J.; Charles,
B. J. Essent. Oil Res. 1993, 5, 403.
154] Deans, S. G.; Kennedy, A. 1.; Gundidza, M. G. M., S.Waterman, P. G.; Gray,
A. 1. Flavour Fragrance J. 1994, 9,245.
155] Bamba, D.; Bessiere, J. M.; Marion, C ; Pelissier, Y.; Fouraste, I. Planta Med.
1993, 59, 184.
156] El-Tantawy, M. E.; Hamauda, M. S. M.; Azzam, A. S. Bull. Fac. Pharm.
(Cairo Univ.) 1994,52, 113.
157] Garg, A. P. J. Indian Bot. Soc. 1992, 71, 251.
158] Esterfieled, T. H.; McDowell, J. C. Trans New Zealand Inst. 1911, 43, 55.
159] Esterfieled, T. H.; McDowell, J. C. Trans New Zealand Inst. 1915, 48, 518.
160] Hasegawa, S.; Hirose, Y. Phytochemistry 1981, 20, 508.
161] Nakatsu, T.; McAlister, K.; Reitz, G., Unpublished results.
162] Kubo, I.; Muroi, H.; Himejima, M. J. Nat. Prod. 1992, 55, 1436.
163] Kang, R.; Komazaki, S.; Helms, R.; Chen, Z.; L. McAlister, K. L.; Nakatsu, T.
A Possible New Agent for the Control of Methicillin Resistant Staphylococcus
aureus, in The 4th Tohwa University International Symposium. 1994. Fukuoka,
Japan, pp 71 - 76.
164] Duke, D. A. Handbook of Medicinal Herbs', Duke, D. A., Ed.; CRC Press: Boca
Raton, 1988, pp 354.
165] Honda, G.; Koga, K.; Koezuka, Y.; Tabata, M. Shoyakugaku Zasshi 1984, 38,
12.
166] Terao, J.; Suzuki, H.; Yamazak, H. i. J. Agric. Food Chem. 1991, 39, 1477.
167] Hirose, M.; Masuda, A.; Ito, N.; Kana, K.; Harumi, O. Carciongenesis 1990, / / ,
731.
168] Kang, R.; Helms, R.; Stout, M. J.; Hasan, J.; Chen, Z.; Nakatsu, T. J. Agric.
Food Chem. 1992, 40, 2328.
169] Ito, H. Yakugaku Zasshi 1970, 7, 883.
170] Koezuka, Y.; Honda, G.; Tabata, M. Phytochemistry 1986, 25, 859.
171] Nabeta, K.; Oda, T.; Fujimura, T.; Sugisawa, H. Agric. Biol. Chem. 1985, 49,
276.
172] Kubo, I. J. Nat. Prod. 1988, 51, 859.
173] Kubo, I.; Himejima, M. J. Agirc. Food Chem. 1991, 39, 2290.
174] Berenbaum, M. C. J. Infect. Dis. 1978, 137, 122.
175] Guhr, G.; Lachance, P. A. Role of phytochemicals in chronic disease
prevention.', Guhr, G.; Lachance, P. A., Ed.; Food & Nutrition Press: Trumbull,
Conn., 1997, pp 311-364.
[176] Craig, W. J. J. Am. Diet. Assoc. 1997, 97, S199.
[177] Barel, A. 0.; Clarys, P.; Manou, I. Objective evaluation of the cosmetic use of
some selected essential oils as "active ingredients" in skin care products, in In-
Cosmet. 1997. 1997: Verlag fuer Chemische Industrie H. Ziolkowsky,
Augsburg, Germany., pp 109-121.
[178] Rao, A. V.; Koratkar, R. Anticarcinogenic effects of saponins and phytosterols.;
Rao, A. V.; Koratkar, R., Ed.; American Chemical Society: Washington, DC,
1997, pp 313-324.
[179] Friedman, M. Potato polyphenols: role in the plant and in the diet.; Friedman,
M., Ed.; American Chemical Society: Washington, DC, 1997, pp 61-93.
[180] Wargovich, M. J. Cancer Lett (Shannon, Irel.) 1997, 114, 11.
[181] Kabara, J. J. Cos met. Sci. Technol. Ser. 1997, 16, 181.
[182] Hedin, P. A.; Hollingworth, R. M. New applications for phytochemical pest-
control agents.; Hedin, P. A.; Hollingworth, R. M., Ed.; American Chemical
Society: Washington, DC, 1997, pp 6156.
[183] Gould, G. W. J. Food Prod 1996, Issue Suppl, 82.
[184] Johns, T.; Chapman, L. Recent Adv. Phytochem. 1995, 29, 161.
[185] Suttisri, R.; Lee, I.-S.; Kinghorn, A. D. J. Ethnopharmacol. 1995, 47, 9.
[186] Hudson, J. B. The phytochemical approach to antiviral chemotherapy.', Hudson,
J. B., Ed.; CRC Press: Boca Raton, Fla, 1995, pp 161-74.
[187] Wink, M. Proc. Phytochem. Soc. Eur. 1993, 34, 171.
[188] Pickett, J. A.; Wadhams, L. J.; Woodcock, C. M. Proc. Phytochem. Soc. Eur.
1993,54,62.
[189] Escoubas, P.; Lajide, L.; Mizutani, J. Insecticidal and antifeedant activities of
plant compounds: potential leads for novel pesticides.', American Chemical
Scociety: Washington, DC, 1994; Vol. 551, pp 162-71.
[190] Tsao, R.; Yu, Q. Toxicity of monoterpenoids against economically important
nematodes in agriculture, in 214th ACS National Meeting,. 1997. Las Vegas:
ACS.
[191] Trigg, J. K. J. Am. Mosq. Control Assoc. 1996, 12, 243.
[192] Sridevi, D.; Dhingra, S. J. Entomol. Res. 1996, 20, 335.
[193] Okuyama, J. Patent JP 07097304, 1995.
[194] Ofer, Y. Patent IL 105109, 1996.
[195] Perrucci, S.; Macchioni, G.; Cioni, P. C ; Flamini, G.; Morelli, I.; Taccini, F.
Phytother. Res. 1996, 10, 5.
[196] Don-Pedro, K. N. Pestic. Sci. 1996, 46, 71.
[197] Kominato, J.; Nishimura, S.; Takeyama, Y.; Nishii, K.; Nishimi, T.; Oohira, H.
Patent AT 400395, 1995.
[198] Kominato, J.; Nishimura, S.; Takeyama, Y.; Nishii, K.; Nishimi, T.; Oohira, H.
Patent JP 07285821, 1995.
[199] Verdicchio, R. Patent CA 2111909, 1994.
[200] Pan, P.; Buch, R. M.; Volpe, F.; Rubin, M. Patent WO 9730685, 1997.
[201] Pan, P. C ; Rubin, M.; Leung, S.-H. S. Patent WO 9726855, 1997.
[202] Nabi, N.; Gaffar, A.; Lucchesi, S. Patent DE 4418796, 1994.
[203] Moran, J.; Addy, M.; Newcombe, R. J. Clin. Periodontol. 1997, 24, 636.
[204] Pelissier, Y.; Marion, C ; Casadebaig, J.; Milhau, M.; Kone, D.; Loukou, G.;
Nanga, Y.; Bessiere, J.-M. J. Essent. Oil Res. 1994, 6, 623.
[205] Sodis, M.; Sodis, G. Patent US 5637290, 1997.
[206] Okada, T. Patent JP 07165547, 1995.
[207] Medenica, R. D. Patent US 5660833, 1997.
[208] Ishizuka, Y.; Sato, S.; Amo, Y.; Kobayashi, M. Patent JP 08333244, 1996.
630 NAKATSUtftf/.
Santos, F. A.; Rao, V. S. N.; Silveira, E. R. Fitoterapia 1997, 68, 65.

Al-Zuhair, H.; El-Sayeh, B.; Ameen, H. A.; Al-Shoora, H. Pharmacol. Res.
1996, 34, 79.
Goebel, H.; Schmidt, G.; Dworschak, M.; Stolze, H.; Heuss, D. Phytomedicine
1995,2,93.
Gattu, M.; Boss, K. L.; Terry, A. V. J.; Buccafusco, J. J. Pharmacol. Biochem.
Behav. 1997,57,793.
Elisabetsky, E.; De Souza, G. P. C ; Dos Santos, M. A. C ; Siquieira, I. R.;
Amador, T. A.; Nunes, D. S. Fitoterapia 1995, 66, 407.
Benet, L. Z.; Wacher, V. J.; Benet, R. M. Patent WO 9640192, 1996.
Thacharodi, D.; Rao, K. P. Int. J. Pharm. 1994, / / / , 235.
Carson, C. F.; Hammer, K. A.; Riley, T. V. J. Antimicrob. Chemother. 1996,
37, 1177.
Koyama, S.; Yamaguchi, Y. Patent JP 08169839, 1996.
Tkachenko, K. G.; Platonov, V. G.; Satzyperova, I. F. Rastit. Resur. 1995, 31,
9.
Dupont, P. Patent FR 2709964, 1995.
Singh, S.; Majumdar, D. K. Int. J. Pharmacogn. 1995, 33, 288.
Onwukaeme, N. D. J. Ethnopharmacol. 1995, 46, 121.
Chan, M. M.-Y. Biochem. Pharmacol. 1995, 49, 1551.
Recsan, Z.; Pagliuca, G.; Piretti, M. V.; Penzes, L. G.; Youdim, K. A.; Noble,
R. C ; Deans, S. G. J. Essent. Oil Res. 1997, 9, 53.
Lee, J. M.; Lee, E. J.; Bahn, K. N.; Kim, J. O.; Ha, Y. L. Han'guk Nonghwa
Hakhoechi 1995, 38, 468.
Bautz, J. Patent FR 2719221, 1995.
Elder, H. V.; Molina, L.; Perez, A.; Cardell, D. Cosmetologic uses of essential
oil of Lippia alba (Miller) N.E. Brown (Lipia). in 15th Journees Internationales
Huiles Essentielles. 1996, pp 712-714.
Staton, J. A.; Rowe, J. S. Patent WO 9735597, 1997.
Arraudeau, J.-P.; Aubert, L. L. R. Patent EP 756866, 1997.
Kinley, J. S.; Moan, J.; Dall'Aqua, F.; Young, A. R. J. Invest. Dermatol. 1994,
103, 97.
David, N.; Delorme, B. Patent WO 9625409, 1996.
Dupont, P. Patent FR 2710266, 1995.
Bekele, A. J.; Obeng-Ofori, D.; Hassanali, A. J. Appl. Entomol. 1997, 121, 169.
Shaaya, E.; Kostjukovski, M.; Eilberg, J.; Sukprakarn, C. J. Stored Prod. Res.
1997,55,7.
Hasan, H. A. H. Pak. J. Sci. Ind. Res. 1994, 37, 471.
Singh, M.; Srivastava, S.; Srivastava, R. P. Int. J. Food Sci. Nutr. 1995, 46,
225.
Maestri, D. M.; Zygadlo, J. A.; Lamarque, A. L.; Labuckas, D. O.; Guzman, C.
A. Grasas Aceites (Seville) 1996, 47, 397.
King, G. A. Patent WO 9616140, 1996.
Walters, M. T.; Hughes, P. S.; Bamforth, C. W. The evaluation of natural
antioxidants in beer and its raw materials, in Conv. - Inst. Brew. (Asia Pac.
Sect.). 1996, pp 103-109.
Lis-Balchin, M.; Deans, S. G. J. Appl. Microbiol. 1997, 82, 759.
Inada, N.; Katayama, T. Patent JP 08113723, 1996.
Ismail, M. H. Egypt. J. Bot. 1996, 34, 167.
Ishikawa, H.; Suzuki, Y.; Sakai, A.; Ishizuka, S. Patent JP 07145398, 1995.
Driskill, C. R. Patent US 5658498, 1997.

Elges, W. Patent DE 19537120, 1997.
Helms, A.-K. Patent DE 4421141, 1995.
Morikawa, T.; Tabuchi, Y.; Inoe, T. Patent JP 08105827, 1996.
Habar, G.; Le Pape, A.; Descusse, C. Patent EP 714786, 1996.
Zimin, A., Sr.; Caputo, P. A. Patent US 5603735, 1997.
Suzuki, Y.; Yagishita, Y. Patent JP 09235490, 1997.
Minagawa, F. Patent JP 09169401, 1997.
Nakahara, M.; Nakahara, T.; Sasaki, Y.; Kai, S. Patent JP 07207165, 1995.
Nakanishi, M.; Suzuki, H. Patent JP 08027624, 1996.
Sakagami, T.; Sakagami, K. Patent JP 07243172, 1995.
Aoyanagi, S.; Maeda, T. Patent JP 06298619, 1994.
Kawaguchi, T.; Kida, N.; Kunugino, T. Patent JP 08026910, 1996.
Comer, D. K.; Berry, M. F.; Monfredi, A. J.; Lew, C. W. Patent WO 9517816,
1995.
Arai, H.; Myagawa, K.; Sekiguchi, K.; Ootaki, T.; Takahashi, J. Patent JP
07145598, 1996.
Romano, R ; Trani, M.; Minervini, G. Patent WO 9731092, 1997.
Romano, R ; Trani, M.; Minervini, G. Patent WO 9731093, 1997.
Barranx, A.; Barsaco, M.; Dufau, G.; Lauilhe, J.-P. Patent WO 9616549, 1996.
Smid, E. J.; De Witte, Y.; Vrees, O.; Gorris, L. G. M. Use of secondary plant
metabolites for the control of postharvest fungal diseases on flower bulbs, in Acta
Hortic. (1994), 368 (International Symposium on Postharvest Treatment of
Horticultural Crops. 1993, pp 523-530.
[266] Fedida, J. Patent EP 758634, 1997.
[267] Shuey, M. W.; Custer, R. S. Patent US 5650000, 1997.
[268] Shuey, M. R.; Custer, R. S. Patent WO 9533798, 1995.
[269] Hill, R. A.; Rohitha, B. H. Patent WO 9507807, 1995.
[270] Zhao, H.; Ivic, L.; Otaki, J. M.; Hashimoto, M.; Mikoshiba, K.; Firestein, S.
Science 1998, 279, 237.

Biological Activity of Essential Oils and Their Constituents

Uploaded by

Copyright:

Available Formats

Biological Activity of Essential Oils and Their Constituents

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Biological Activity of Essential Oils and Their Constituents

Uploaded by

Copyright:

Available Formats

Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol.

BIOLOGICAL ACTIVITY OF ESSENTIAL OILS AND

TETSUO NAKATSU*, ANDREW T. LUPO, JR.,

Takasago Institute for Interdisciplinary Science, 4 Volvo Dr., Rockleigh,

Essential oils are well accepted and recognized in academia, in chemical

Zhong Yo in China [4] are well-known traditional collections of

have also been reported to have pharmacological or therapeutical activity

EXTRACTION OF ESSENTIAL OILS

The methods of extraction of essential oils from plants significantly affect

constituents of essential oils are small, volatile and lipophilic, a key

Cold Press Extraction

Extraction of One Oil with Another

Simultaneous Distillation-Solvent Extraction (SDE)

Frequently, steam distillation is combined with solvent extraction to

Supercritical Fluid Extraction (SFE)

Developed in the 1980's, this technology is becoming more popular today

step 2 J Analytes in C 0 2 solution

Analytes in rinse solution

The main drawback for laboratory use is equipment cost (primarily to

Microwave Oven Extraction

Solid Phase Extraction (SPE)

New developments in adsorbent technology this decade have led to an

1,1,1,2-Tetrafluoroethane (hydrofluorocarbon-134a or HFC-134a, b.p.

SEPARATION AND IDENTIFICATION OF ESSENTIAL OIL

Fractional distillation is frequently used to purify the essential oils or to

Gas Chromatography (GC)

a nondestructive detector, but sensitivity and dynamic range are low.

Liquid Chromatography (LC)

Silica gel liquid column chromatography is suitable to separate classes of

GC-mass spectrometry (GC-MS) is most frequently and effectively used

ODOR PERCEPTION, PSYCHOLOGICAL EFFECTS AND

In the following section, the odor perception, psychological effects, and

Chiral Molecules and Odor Perception

Rose Oxide Structure Odor Perception Threshold Value

{4R)-cts Floral-Green 0.5

Depressant Activity on the Central Nervous System (CNS)

The ingestion of Psidium guajava Linn. (Myrtaceae), native to tropical

The steam-distilled oil derived from the galls of Pistacia integerrima

New Sesquiterpenoids and Antimutagenic Activity

This class of compounds may be found in essential oils extracted with

While we are exposed to many carcinogenic and mutagenic chemicals in the

by 80%. Paeonol (35) suppressed the SOS-induced activity by 60% at

Enzyme Inhibitory Activity

17 common monoterpenes, including hydrocarbons: p-cymene (37), oc-

Glutathione S-Transferase (GST) Activity and Anticarcinogenic Activity

The enhancement of GST activity has been suggested to increase the

were P-caryophyllene (52), a-humulene (53), a-humulene epoxide (54),

HO" *> HOx%%

Polyphenol Oxidase Inhibitors

Tyrosinase, a polyphenol oxidase, is widely distributed in nature and is

Tyrosinase Activity Assay

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

Melanin Formation Assay

Enzvme Activity Inhibition

Acyclic terpenoids showed strong inhibitory effects on tyrosine activity

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

Compound Inhibition (%) Compound Inhibition (%)

Citronellol (56) 74.9 Dihydromyrcenol (62) 32.9

Geraniol (58) 66.1 Tetrahydrolinalool (63) 43.1