Chem. Senses 27: 225-230,
2002
© Oxford University Press 2002
Concentration Effects of Green Odor on Event-related Potential (P300) and Pleasantness
Soda Aromatic Co. Ltd Fragrance Laboratory, Tokyo 103-0023, Japan 1 MOA Health Science Foundation, Fukuoka 811-2302, Japan 2 Department of Integrative Physiology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582 Japan 3 Department of Biochemistry, Yamaguchi University, Yamaguchi, 753-8511, Japan
Correspondence to be sent to: Kota Sano, Soda Aromatic Co. Ltd Fragrance Laboratory, 4-15-9 Nihonbashi-honcho, Chuo-ku, 103-0023 Tokyo, Japan. e-mail: kouta_sano{at}soda.toray.co.jp
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Abstract |
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 Top Abstract IntroductionExperiment 1Experiment 2General discussionReferences |
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The effects of eight compounds, constituting the so-called `natural green odor', including leaf alcohol, on the event-related potential (P300) were investigated. In experiments with a series of single compounds, each of these eight compounds could be characterized by an overall change consisting predominantly of an increase, a decrease or no change in the amplitude of P300, whereas in experiments with a series of two-component mixtures, noticeable synergism could not be demonstrated, contrary to our expectation. Experiments with leaf alcohol (3Z-hexenol) performed at two concentrations showed a significantly different degree of pleasantness and an increase or decrease in the amplitude of P300 depending on their concentration, suggesting that concentration is important in odorant-presentation studies.
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Introduction |
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TopAbstractIntroductionExperiment 1Experiment 2General discussionReferences |
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The so-called `green odor' emanating from green leaves has been elucidated to be composed of eight odorants consisting of 6-carbon aliphatic alcohols and aldehydes including 3Z-hexenol (leaf alcohol) and 2E-hexenal (leaf aldehyde) (Hatanaka, 1999
). These compounds are of plant origin that are formed by a
biosynthetic reaction from the chloroplast membrane through linolenic acid
(Hatanaka, 1996) and are known
to have various physiological actions as plant-to-plant messengers in
allelopathy, insect-attracting pheromone-like substances, bactericides and
phytontids (Hatanaka, 1999).
Recently, research activity has expanded to cover the field of aromachology
including stress reduction (Aou et al.,
2002a,
b). We have revealed that the
odor characteristics of each compound can be attributed to various factors
such as differences in geometrical and positional isomerism of unsaturated
bond, terminal functional groups and carbon chain lengths through our studies
on the chemical structure—odor relationship of green odor
(Hatanaka et al.,
1992; Sano et al.,
1999,
2000,
2001). The effects of the odor
on the EEG were studied using the event-related potential, ERP (CNV, P300),
since some compounds, mainly natural essential oils, have been shown to affect
mental processes (Fukuda et al.,
1985; Koga, 1996).
The present study was therefore performed to clarify more precisely the
possible effects of green odor in humans and the possible relationship between
the mental effects caused by each of the green odorants and their odor
characteristics, using the ERP (P300) as an indicator in our aromachological
investigations.
Through their contact with plants, people come to associate green odor with
nature and thereby experience pleasantness on perceiving this odor. Another
purpose of the present study was to add further detailed data to our
previously published data (Sugano et
al., 1996) describing the changes in the ERP (P300) induced
by green odor. As a result, we have succeeded in identifying a pattern of
changes in the ERP (P300) caused by each green odorant. Furthermore, taking
account the fact that natural green odor is a multi-component mixture, a
series of two-component mixtures was studied with the expectation of their
potential synergistic effects. In addition, the ERP (P300) was recorded at
different concentrations of leaf alcohol to examine the relationship between
the mental action of green odor and its concentration level and
pleasantness.
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Experiment 1 |
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TopAbstractIntroductionExperiment 1Experiment 2General discussionReferences |
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Materials and methods
The subjects who participated in experiment 1 were 128 healthy women aged 18-22 years, without any abnormal olfactory or auditory perception. Among these 128 women, 39 and 89 were allocated to either the experiment with a series of single compounds or that with a series of two-component mixtures, respectively. The subjects were requested to avoid using fragrant cosmetics and taking strongly smelling meals on the day of the experiment.
Green odor is known to be composed of eight compounds including n-hexanol, n-hexanal, 3Z-hexenol (leaf alcohol), 3Z-hexenal, 2E-hexenol, 2E-hexenal (leaf aldehyde), 3E-hexenol and 3E-hexenal. Thus, these eight compounds and an odorless solvent, triethyl citrate, were used as test samples. An appropriate concentration corresponding to the greatest degree of pleasantness in each of the subjects was examined preliminarily using a 10-step dilution series for 3Z-hexenol (leaf alcohol) and 2E-hexenal (leaf aldehyde), and the results obtained demonstrated that such concentrations showed a normal distribution with a peak at 0.01 and 0.1% for the former and latter, respectively. Based on the results, a concentration of 0.03% was selected as the mean of these values calculated by logarithmic transformation.
Each of the eight compounds was dissolved in triethyl citrate at a concentration of 0.03% to prepare test solutions and triethyl citrate served as the control solution (Table 2). In the experiment with a series of two-component mixtures, equal amounts of each of the test solutions were mixed (w/w%) to prepare mixed solutions of 28 combinations as shown in Table 2.
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The experiment was carried out in a quiet and air conditioned room kept at
23°C, that was, particularly, kept dim during the experiment.
Subjects were asked to sit on a chair in a relaxed position keeping their eyes
closed and an EEG was recorded from Fz, Cz and Pz. A sheet of filter paper (50
150 mm) was impregnated with an aliquot of test solution containing 100 mg of
each odorant or control solution and held 2 cm from the tip of the nose for
presentation. The EEG was recorded with a Synafit 2500 EF 2514 (NEC Corp.),
with 0.5 Hz lowpass and 60 Hz highpass filters. The conventional Oddball
method was applied to record ERP (P300) and acoustic stimuli were given in the
form of the sound with a pressure level (SPL) of 70 dB at either 2000 or 1000
Hz, generated randomly in a ratio of 20 and 80% for the acoustic stimuli given
less frequently and those given more frequently, respectively. Each of the
acoustic stimuli was given at intervals of 1-1.5 s and the subjects were
instructed to push a button when they heard the target sound of 2000 Hz, which
occurred less frequently. P300 was obtained by averaging 20 stimuli after
excluding artefacts. The experiment was designed to avoid involvement of
sequence effects throughout the entire series of studies by scheduling
implementation of each session so as to make intervals long enough to avoid
the occurrence of olfactory fatigue.
Results
Comparison of the changes in amplitude of P300 in Fz, Cz and Pz was made
between each of the eight compounds and control. Tables
1 and
2 show the overall changes in
the amplitude of P300 observed with the series of single compounds and with
the series of two-component mixtures, respectively. The data compiled in each
table were subjected to 
2-testing of independence after having
been rearranged in a contingency table. The 2-value thus
obtained is represented in the legend for each table (just as reference data
because of small value of the expected frequency).
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Regarding the relation between each of the test compounds and the overall predominating changes in the amplitude of P300, statistically significant association could not be identified when the data compiled in each table were analysed as a whole, whereas some of the individual samples of data compiled in Tables 1 and 2 were found to indicate a particular predominating change. More precisely, neither of the two saturated compounds elicited change in amplitude and among the unsaturated compounds, 3Z-hexenol (leaf alcohol) and 3Z-hexenal predominantly elicited a decrease, whereas 2E-hexenal (leaf aldehyde) predominantly elicited an increase. On the other hand, changes in the amplitude elicited by other compounds were equivocal in terms of predominance of an increase or decrease. In other words, the eight compounds did not elicit a common change, but elicited a particular change in each compound, even though the eight compounds used in the study had similar chemical structures. Figure 1a,Figure 1b shows a representative example of decreased amplitude of P300 elicited with 3Z-hexenol (leaf alcohol).
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Taking account of the particular importance of 3Z-hexenol (leaf alcohol) and 2E-hexenal (leaf aldehyde) among green odorants showing a unilateral change with predominantly either an increase or decrease in the study with the series of single compounds, a study was performed with a series of two-component mixtures of each of 3Z-hexenol and 2E-hexenal with other odorants. This showed that two of the two-component mixtures—2E-hexenal with 3Z-hexenol and 2E-hexenal with 2E-hexenol—predominantly elicited an increase. Overall evaluation of the data obtained from the 89 subjects, however, did not demonstrate any synergistic effects of mixtures, contrary to our expectation prior to the study.
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Experiment 2 |
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TopAbstractIntroductionExperiment 1Experiment 2General discussionReferences |
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Materials and methods
The procedure and method were the same as in experiment 1. The subjects who participated in experiment 2 were 34 healthy women aged 18-22 years.
Among the eight compounds composing green odor, 3Z-hexenol (leaf alcohol) was selected for further detailed investigation, as the dominant odorant of green odor. Concentrations of 10 and 0.1% (w/w%) were selected based on the outcomes of preliminary examination of pleasantness. The test compound was dissolved in triethyl citrate at concentrations of 10 and 0.1% to prepare test solutions; triethyl citrate served as the control solution.
Recording of P300 was carried out in the same manner as in experiment 1 in terms of the environment, equipment and procedure; measurements of the amplitude of P300 derived from Pz were averaged over the entire 20 stimuli and, after adjustment of the baseline, data contaminated with artefacts were excluded. Thus, cleaned-up data were subjected to analysis by paired t-test. Statistical analysis was performed using SPSS software. After completion of EEG recording, odor intensity (0-5), degree of pleasantness (-4 to +4) and impression of odor were surveyed through an interview. Odor intensity and the degree of pleasantness were each assessed based on the `6-rating classification of odor intensity' and `9-rating classification of pleasantness/unpleasantness', respectively, which are commonly used by the Ministry of the Environment, as shown in Table 3.
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Results
Among the data obtained from the 34 subjects, analysis was performed on the
data obtained from 31 subjects remaining after excluding three subjects in
whom P300 could not be identified. For analysis, the amplitude of P300 derived
from Pz was compared for each of 10% 3Z-hexenol, 0.1%
3Z-hexenol and triethyl citrate (control). As shown in
Figure 2, for 10%
3Z-hexenol and 0.1% 3Z-hexenol, the amplitude of P300 was
significantly smaller (P < 0.01) as compared with control. The
outcomes agreed with those observed in experiment 1 in that
3Z-hexenol predominantly elicited a decrease in amplitude.
Furthermore, it was found that the decrease in P300 amplitude was
significantly larger (P < 0.05) with 0.1% 3Z-hexenol as
compared with 10% 3Z-hexenol.
Figure 3 shows the results of
survey questions asked after completion of EEG recording, showing a
significantly greater degree of pleasantness (P < 0.01)
experienced with 0.1% 3Z-hexenol than with 10% 3Z-hexenol.
The results demonstrated that the degree of pleasantness experienced with the
same compound varied markedly depending on the concentration. The 31 subjects
were stratified into three subgroups: a group of 26 subjects who rated 0.1%
3Z-hexenol more pleasan smelling than 10% 3Z-hexenol; a
group of two subjects who rated 0.1% 3Z-hexenol and 10%
3Z-hexenol equally pleasant smelling; and a group of three subjects
who rated 10% 3Z-hexenol more pleasant smelling than 0.1%
3Z-hexenol. The values of P300 potential with 10% 3Z-hexenol
and 0.1% 3Z-hexenol were compared among these three subgroups. In the
subgroup of 26 subjects who rated 0.1% 3Z-hexenol more pleasant
smelling than 10% 3Z-hexenol, a finding of a lower potential with 0.1
than 10% was obtained in 20 subjects (77%), a comparable potential with
both 0.1 and 10% in one (4%) and a higher potential with 0.1 than 10% in
five (19%), whereas in the subgroup of three subjects who rated 10%
3Z-hexenol as more pleasant smelling than 0.1% 3Z-hexenol, a
finding of a lower potential with 10 than with 0.1% was obtained in all three
subjects.
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General discussion |
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TopAbstractIntroductionExperiment 1Experiment 2General discussionReferences |
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The results of experiment 1 revealed that the eight compounds composing green odor can be classified into three categories: those that increase, those that do not change and those that decrease the amplitude of P300, regardless of their high similarity in chemical structure and odor characteristics. Experience of various feelings of, for example, refreshment and relaxation in association with perception of a natural green odor reflects the fact that odors of natural origin are due to multi-component mixture of compounds, each with a different mental action. Our findings that saturated compounds did not elicit any change in amplitude, while many unsaturated compounds elicited changes that were predominantly either an increase or decrease help us to understand that with respect to differences in chemical structure, unsaturated-bond-containing compounds favor perception of an odor in humans.
The results of experiment 2 revealed that the amplitude of P300 was
significantly decreased with 3Z-hexenol (leaf alcohol) Although
various interpretations have been proposed for the changes in the amplitude of
P300, a widely accepted interpretation is that its change reflects possible
influence on the cognitive function
(Intriligator and Polich,
1994). A decrease in the amplitude of P300 is considered to
reflect the onset of a kind of sedative action
(Yagyu et al., 1992;
Yamadera et al.,
1996) and, thus, the series of compounds including
3Z-hexenol is considered to be involved in the feeling of relaxation
induced by green odor. An interesting finding obtained through the results of
experiment 2 was that the amplitude of P300 was certainly decreased with
3Z-hexenol; nevertheless, the decrease was not necessarily larger in
response to a higher concentration. The finding that the amplitude-decreasing
effect was larger with a concentration of 0.1 than with 10% agreed with the
findings obtained in survey questions on the degree of pleasantness, as shown
by the findings that the decrease in amplitude of P300 was greater with
3Z-hexenol at the concentration corresponding to a greater degree of
pleasantness. A report describing that, when different substances were
compared, not pleasant but unpleasant odors elicited decrease in the amplitude
of P300 is available (Koga,
1996), whereas, in our present study in which two different
concentration levels of an identical substance were compared, the
concentration level corresponding to higher degree of pleasantness was found
to elicit larger decrease in the amplitude of P300. On the other hand, the
findings showed that the amplitude of P300 was generally lower with 0.1%
3Z-hexenol as compared with 10% 3Z-hexenol (as shown in
Figure 2), whereas, in the
subgroup of three subjects who rated the 3Z-hexenol solution more
pleasant smelling at a concentration of 10% than at 0.1%, the potentials were
lower with 10% 3Z-hexenol as compared with 0.1% 3Z-hexenol
in all three of these subjects. These findings suggest that the
amplitude-decreasing effect does not depend on the concentration but rather on
the degree of pleasantness, in that a pleasant smell may enhance and an
unpleasant smell may attenuate the sedative effect. Consequently, green odor
has been demonstrated to constitute an element of aromachology by bringing
about stress reduction in humans. Further study to investigate any
relationship between EEG, particularly alpha wave and ERP (P300) each recorded
under comparative conditions, is to be planned.
In recent years, selection of appropriate concentration levels of test samples in designing study protocols has been suggested to be a matter of importance, through investigations on the relation between odor and EEG conducted at various research institutions. The present paper suggests that data on the degree of pleasantness is important as a rationale for concentration selection.
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Acknowledgments |
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We gratefully acknowledge Dr Nobushige Sato, Emeritus Professor and Dr Shigeki Kitajima, Associate Professor at the University of Occupational and Environmental Health, Japan, for their cooperation and recruitment of volunteers for the study.
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References |
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TopAbstractIntroductionExperiment 1Experiment 2General discussionReferences |
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Accepted November 28, 2001
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