Effect of inhalation of isomers, (+)-α-pinene and (+)-β-pinene on human electroencephalographic activity according to gender difference

European Journal of Integrative Medicine 17 (2018) 33–39

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European Journal of Integrative Medicine journal homepage: www.elsevier.com/locate/eujim

Research paper

Effect of inhalation of isomers, (+)-α-pinene and (+)-β-pinene on human electroencephalographic activity according to gender difference Minju Kima,1, Kandhasamy Sowndhararajana,1, Se Jin Parka, Songmun Kima,b, a b

MARK

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School of Natural Resources and Environmental Sciences, Kangwon National University, Chuncheon 24341, Gangwon-do, Republic of Korea Gangwon Perfume Alchemy Ltd. Co., Chuncheon 24341, Gangwon-do, Republic of Korea

A R T I C L E I N F O

A B S T R A C T

Keywords: Electroencephalography Inhalation Isomer (+)-α-pinene (+)-β-pinene

Introduction: Pinenes are the most abundant components in the essential oils of various plant species. (+)-αPinene and (+)-β-pinene are bicyclic monoterpene hydrocarbons with different fragrance qualities. The present study was carried out to investigate the effect of inhalation of isomeric aroma components, (+)-α-pinene and (+)-β-pinene on human electroencephalographic (EEG) activity according to gender difference. Methods: In this study, the EEG activity was evaluated by the measurement of EEG power spectrum in healthy participants - 10 women and 10 men. The EEG readings were recorded using QEEG-8 system from 8 scalp positions of the International 10–20 System. Results: The results indicated that the inhalation of (+)-α-pinene and (+)-β-pinene produced different EEG power spectrum changes as well as affect different brain regions. The gender difference played a major role in the EEG activity of both the components. Women highly responded to both the compounds when compared with men. In women, absolute alpha, absolute beta and absolute high beta activities significantly (P < 0.05) increased during the inhalation of (+)-α-pinene. In the case of (+)-β-pinene, absolute fast alpha and absolute high beta activities also significantly increased. Whereas in men, significant decrease of absolute waves such as theta, beta, low beta and high beta were observed during the inhalation of (+)-α-pinene but there were no significant changes in the absolute waves by (+)-β-pinene. Conclusion: The different EEG activities of these isomers might be due to the different fragrance quality and structural arrangement of the components.

1. Introduction In human beings, smell is one of the most important senses. With the help of olfactory receptors that are sensitive to odor molecule, we can identify even traces in relation to fragrance [1]. Among the various odor molecules from nature, essential oils and their components play a major role in the flavor and fragrance industries for a variety of purposes. In particular, the essential oils and their individual components have been used in the aromatherapy treatment for reducing stress and enhancing relaxation, mood and working capacity [2]. Further, recent studies have suggested that olfactory stimulation via fragrance inhalation significantly improves psychophysiological conditions of human [3,4]. The effect of inhalation of fragrances on brain function has been easily determined by using electroencephalograph (EEG). Recently, a number of authors have reported that the EEG recordings are extensively used to understand spontaneous brain wave changes due to the exposure of fragrances. In addition, the fragrances produce different

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EEG activity according to gender difference [5–8]. Monoterpenes are the major group of components in the essential oils with a wide range of different functional groups as well as isomers of particular components. The isomers of carvone, linalool and limonene have different fragrance qualities and intensities. In addition, the isomeric components also exhibit significantly different biological properties [8–10]. Romagni et al. [11] reported that two isomers of cineole appear to have different modes of action in their allelopathic effects. Weber and Mosandl [12] reported that the enantiomers of 3methylthiobutanal, one has a specific aroma and another one is odorless. Further, carvone is an important perfume ingredient, and its enantiomers, S(+)-carvone has spearmint like smell and R(−)-carvone has caraway like smell [13]. Studies on the synthesis of novel fragrant molecules have revealed that altering the spatial structure of molecules can also affect the fragrance qualities of compounds [14]. It is well known that the olfactory receptors react differently for each fragrant molecule [15]. In a recent study, isomeric components, (+)-limonene

Corresponding author: School of Natural Resources and Environmental Sciences, Kangwon National University, Chuncheon 24341, Republic of Korea. E-mail address: [email protected] (S. Kim). These authors contributed equally.

https://doi.org/10.1016/j.eujim.2017.11.005 Received 31 October 2017; Received in revised form 6 November 2017; Accepted 7 November 2017 1876-3820/ © 2017 Elsevier GmbH. All rights reserved.

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presented in Table 1.

and terpinolene produced different EEG power spectrum changes and also affect different regions of the brain [8]. Pinenes are the most abundant components in a wide range of species including tropical, Mediterranean and coniferous plants, especially in the essential oils from the genera such as Pinus, Eucalyptus, Rosmarinus and Lavandula [16]. α-Pinene and β-pinene are also major components of commercial turpentine. Pinenes possess various pharmacological properties such as antimicrobial, hypertensive, antinociceptive and anti-inflammatory. In addition to various bioactive properties, pinenes have been used for centuries in the cosmetic industries especially for flavor and fragrance purpose [13,17]. α-Pinene is an important food flavoring ingredient and was recognized as a safe food additive by U.S. Food and Drug Administration [18]. Pinene has two structural isomers (α-pinene and β-pinene) and both isomers have enantiomers such as (−)-α-pinene, (+)-α-pinene, (−)-β-pinene and (+)-β-pinene [13]. It was reported that the optical isomers or enantiomers of pinenes do not overlap within their mirror images and they vary only in their interaction with polarized light. Further, toxicity and biological activities of these components are also varied [19,20]. (+)-α-Pinene [(1R,5R)-2,6,6-trimethylbicyclo[3.1.1]hept-2-ene] and (+)-β-pinene [(1R,5R)-6,6-dimethyl-2-methylenebicyclo[3.1.1] heptane] are bicyclic monoterpene hydrocarbons with different fragrance. Further, there has been no published report on the effect of these isomeric compounds on human EEG activity. Therefore, the aim of this study was to investigate the effect of inhalation of isomeric aroma components, (+)-α-pinene and (+)-β-pinene on human EEG activity according to gender difference.

2.3. Experimental design In the present study, a single group pre-test and post-test experimental design was used (10 men and 10 women subjects). All the subjects passed the preliminary olfactory evaluation test using commercial perfume strips. The subjects were instructed to sit quietly, close their eyes and to breathe normally during the EEG recordings. After the EEG recordings, preference and impression of the fragrances [(+)-αpinene and (+)-β-pinene] were obtained from each subject. Further, none of the participants indicated that they felt that the fragrance molecules had affected them in any way. 2.4. EEG recordings

2. Materials and methods

The EEG recordings were performed using QEEG-8 system (LXE3208, LAXTHA Inc., Daejeon, Republic of Korea). The electrodes (silver/silver chloride) were placed on the scalp at left prefrontal (Fp1), right prefrontal (Fp2), left frontal (F3), right frontal (F4), left temporal (T3), right temporal (T4), left parietal (P3) and right parietal (P4) according to the International 10–20 System. The electrodes were referenced to the ipsilateral earlobe electrodes. The EEG sampling rate of the measured subjects was 256 Hz, filtered in the range of 0.5–50 Hz, and the readings were stored in a computer by the 12-bit AD conversion. The ECI electrode gel (Electro-gel™, Electro-Cap International Inc., Eaton, Ohio, USA) was applied into each electrode to connect with the surface of the scalp in order to drop the electric resistance of the scalp below 5 kΩ.

2.1. Materials

2.5. Fragrance administration

(+)-α-Pinene (CAS No. 7785-70-8) and (+)-β-pinene (CAS No. 19902-08-0) were purchased from Sigma (St. Louis, MO, USA). The purchased chemicals were stored at 4 °C until used for EEG experiment.

The undiluted (+)-α-pinene and (+)-β-pinene were used as the fragrance stimuli. During the EEG measurement, the subjects were seated in a comfortable chair with a constant room temperature at 23 °C and humidity of 50%. The fragrance stimulus (10 μL) was separately spotted on the filter paper then placed about 5 cm in front of the subject's nose. EEG readings were recorded 45 s before and 45 s during the inhalation of each stimulus. First, EEG was recorded before and during the inhalation of (+)-α-pinene. After resting for 3 min, EEG was again recorded before and during inhalation of (+)-β-pinene.

2.2. Subjects The study followed the Declaration of Helsinki on Biomedical Research Involving Human Subjects. The study was approved by the ethics committee from the Kangwon National University, Chuncheon, Republic of Korea (IRB No. KWNUIRB-2017-05-001-003). A total of 20 healthy volunteers (10 men and 10 women) aged 20–25 years participated in this study. We advertised the EEG study details on our university notice board in order to recruit the volunteer subjects. The inclusion criteria for the subjects were non-smokers and right-handed without any abnormalities in the sense of smell. The purpose of this study was clearly informed to the subjects. None of the subjects had olfactory diseases or abused drugs. Alcohol consumption or medications was prohibited from 2 days before the experiment. Further, the subjects were informed not to consume any caffeinated drinks on the day of the experiment. There were no statistically significant differences between two groups. Prior to experiment, the subjects were screened for an olfactory evaluation test by using the commercial perfumes. The subjects who unable to recognize the familiar fragrance types were excluded from this study. Informed consent was obtained from all the subjects before participation. The basic characteristics of subjects used in the present study are

2.6. Data analysis Two EEG segments were selected for each fragrance condition, 45 s before fragrance presentation and 45 s during the fragrance presentation. Fast Fourier transform was used to calculate the mean power values [microvolt square (μV2)] of each segment. The mean power spectrum values were calculated for 25 EEG indices including absolute and relative power spectra of theta (4–8 Hz), alpha (8–13 Hz), beta (13–30 Hz), and gamma (30–50 Hz) waves [21]. Data were analyzed using SPSS statistical package 18 (SPSS, Inc., Chicago, IL, USA). The EEG power spectrum values before and during the inhalation of (+)-αpinene and (+)-β-pinene were analyzed by a paired Student’s t-test and the P value < 0.05 was considered significant. Based on the significant changes, the t-mapping of EEG power spectra was constructed by Telescan software package (LXSMD61, LAXTHA Inc., Daejeon, Republic of Korea).

Table 1 Basic characteristics of subjects used in the study. Gender

Number of subjects

Age

Handedness

Nationality

Job

Education

Marital status

Men Women

10 10

22 ± 3.2 21 ± 4.8

Right Right

Korean Korean

Students Students

Undergraduate Undergraduate

Single Single

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Table 2 Effect of inhalation of (+)-α-pinene on EEG activity in women. EEG indices

Site

Before inhalation (μV2)

During inhalation (μV2)

t-test

P value*

Absolute alpha Absolute beta Absolute high beta Relative gamma Relative high beta Ratio of SMR to theta Ratio of mid beta to theta Ratio of SMR-mid beta to theta Ratio of alpha to high beta

P4 T3 T3 T4 T3 T3 T3 T3 T3

48.3314 11.6858 5.9464 0.1499 0.1383 0.4972 0.5750 1.0647 3.7167

52.9984 16.6303 8.9281 0.1752 0.1598 0.7342 0.8248 1.5486 2.8166

−2.3725 −2.5567 −2.7400 −2.4606 −2.2795 −2.2775 −2.8151 −2.7983 2.3536

0.0417 0.0308 0.0228 0.0361 0.0485 0.0487 0.0202 0.0207 0.0430

P4, right parietal; T3, left temporal; T4, right temporal * Significant difference (P < 0.05); number of subjects, 10.

3. Results

pinene. Based on the results, the isomers significantly affect different EEG waves as well as different brain regions. Further, the EEG changes highly varied among the different genders (Figs. 1 and 2). Fig. 3 illustrates a schematic representation of EEG activity due to the exposure of (+)-α-pinene and (+)-β-pinene according to the gender difference. In women, absolute alpha (48.3314–52.9984 μV2 at P4), absolute beta (11.6858–16.6303 μV2 at T3) and absolute high beta (5.9464–8.9281 μV2 at T3) waves significantly increased during the inhalation of (+)-α-pinene than before inhalation. On the other hand, absolute fast alpha (4.8116–7.8914 μV2 at Fp1, 4.6448–7.5589 μV2 at Fp2, 6.0231–9.8769 μV2 at F3, 5.7987–9.5249 μV2 at F4 and 4.7922–7.0038 μV2 at T3) and absolute high beta (6.8619–8.1628 μV2 at P3) waves significantly increased during the inhalation of (+)-βpinene when compared with before inhalation. Apart from absolute waves, significant changes of relative gamma, relative high beta, ratio of SMR to theta, ratio of mid beta to theta, ratio of SMR ∼ mid beta to theta and ratio of alpha to high beta were observed during the inhalation of (+)-α-pinene in women. In regards to the inhalation of (+)-β-pinene in women, relative fast alpha, relative high beta, ratio of SMR to theta, ratio of mid beta to theta, ratio of SMR ∼ mid beta to theta and spectral edge frequency 50% of alpha significantly increased with the exception of relative theta. In general, both the components highly affected the left temporal region (T3) than other regions in women. In the case of men, absolute theta (12.5633–9.4554 μV2 at Fp2, 14.1270–11.9835 μV2 at μV2 at F4, 12.2993–10.3241 μV2 at P3 and 11.5944–9.4047 μV2 at P4), absolute beta (9.0659–7.8745 μV2 at Fp2), absolute low beta (2.6135–2.1621 μV2 at T4) and absolute mid beta (3.2367–2.8422 μV2 at F4) significantly decreased during the inhalation of (+)-α-pinene. However, there was no significant change of absolute wave in men during the inhalation of (+)-β-pinene (Table 4). Further, relative theta and relative low beta significantly decreased during the inhalation of (+)-α-pinene. In addition, relative low beta significantly increased at Fp2 and F4 regions and ratio of mid beta to theta significantly decreased at Fp1 and Fp2 regions due to the exposure of (+)-β-pinene.

In this study, the olfactory stimulation analysis was evaluated with the inhalation of (+)-α-pinene and (+)-β-pinene at a constant concentration (undiluted). The EEG power spectrum values were recorded from 8 electrode sites placed on the scalp at Fp1, Fp2, F3, F4, T3, T4, P3 and P4 according to the International 10–20 System. Out of 25 EEG indices analyzed from 8 electrode sites, significant changes (P < 0.05) were observed in 9 indices during the inhalation of each component in women. In men, (+)-α-pinene exhibits significant changes in 6 indices and (+)-β-pinene exhibits significant changes in 2 indices. Tables 2–4 show the significant changes in EEG power spectrum values before and during the inhalation of (+)-α-pinene and (+)-β-pinene in women and men. The t-mapping of brain wave changes clearly expressed the alteration of EEG waves due to the exposure of (+)-α-pinene and (+)-βTable 3 Effect of inhalation of (+)-β-pinene on EEG activity in women. EEG indices

Site

Before inhalation (μV2)

During inhalation (μV2)

t-test

P value*

Absolute fast alpha

Fp1 Fp2 F3 F4 T3 P3 Fp1 F3 F4 T3 P3 P4 Fp1 Fp2 F3 F4 T3 T4 P3 P4 T3 F3 T3 T3

4.8116 4.6448 6.0231 5.7987 4.7922 6.8619 0.2003 0.2246 0.2152 0.2001 0.2106 0.2048 0.0898 0.0855 0.0949 0.0956 0.0971 0.0848 0.1614 0.1734 0.1312 0.3149 0.5356 0.6440

7.8914 7.5589 9.8769 9.5249 7.0038 8.1628 0.1572 0.1760 0.1810 0.1627 0.1541 0.1559 0.1282 0.1255 0.1392 0.1364 0.1351 0.1186 0.2307 0.2281 0.1570 0.4582 0.7652 0.8197

−2.8663 −2.5639 −2.4810 −2.2814 −2.9730 −2.4909 2.3219 2.9117 2.6355 4.0490 2.8279 2.8872 −3.5313 −3.6474 −3.4620 −3.2662 −2.6060 −2.6160 2.8704 −2.4664 −2.9836 −2.4968 −3.0357 −2.2691

0.0185 0.0306 0.0369 0.0484 0.0156 0.0343 0.0453 0.0173 0.0271 0.0029 0.0198 0.0180 0.0064 0.0053 0.0071 0.0097 0.0285 0.0280 0.0185 0.0358 0.0153 0.0340 0.0141 0.0494

F3 T3

0.7138 1.1706

0.9660 1.5711

−2.7559 −3.1819

0.0222 0.0111

Fp2

10.3188

10.5438

−2.4246

0.0383

Absolute high beta Relative theta

Relative fast alpha

Relative high beta Ratio of SMR to theta Ratio of mid beta to theta Ratio of (SMR ∼ mid beta) to theta Spectral edge frequency 50% of alpha

4. Discussion The isomeric aroma components produced significantly different EEG power spectrum activities in different brain regions. In the present study, absolute wave activities such as alpha, fast alpha, beta and high beta significantly increased during the inhalation of (+)-α-pinene in women. The fast alpha wave activity significantly increased during the exposure of agarwood incense [5]. In another study, theta and alpha wave activities significantly increased during the inhalation of lavender oil in all the regions. The increase of alpha wave activity is highly associated with relaxation state of the brain [22]. In addition, alpha 1 wave activity significantly changed after the exposure of lavender oil, eugenol and chamomile [23]. Kamei et al. [24] found that yoga practice

Fp1, left prefrontal; Fp2, right prefrontal; F3, left frontal; F4, right frontal; T3, left temporal; T4, right temporal; P3, left parietal; P4, right parietal. * Significant difference (P < 0.05); number of subjects, 10.

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Fig. 2. The t-Mapping of EEG power spectrum changes before and during the inhalation of (+)-β-pinene in women and men. Fp1, left prefrontal; Fp2, right prefrontal; F3, left frontal; F4, right frontal; T3, left temporal; T4, right temporal; P3, left parietal; P4, right parietal. Arrows show the significant changes in the regions during the inhalation of (+)-β-pinene.

enhances relaxation state of the brain by increasing EEG alpha wave activity in the frontal region. Further, previous studies suggested that the alpha wave activity is decreased under emotional tension and stress states [22,25,26]. The results suggest that the increase of absolute alpha (at P4) and fast alpha (at Fp1, Fp2, F3, F4 and T3) during the inhalation of (+)-α-pinene and (+)-β-pinene, respectively may enhance relaxation state of the brain function in women. The data indicated that both the components specifically produce significant changes in the left temporal region than other regions in women (Tables 2 and 3). In the present study, the inhalation of (+)-αpinene exhibited the significant increase of absolute beta and absolute high beta at the left temporal region. The high beta wave activity is mainly linked with high awareness/alertness state of the brain, whereas less beta wave activity is mainly associated with drowsiness state of the brain. The beta waves generate during active concentration and the activation of beta waves is highly related to the improvement of cognitive performances [4,27]. The higher beta wave activity was also associated with the increase of positive emotions such as feeling active, fresh and romantic [28]. In addition, when chewing the flavored standard gum with inhaling the flavored aromatic oil may enhance the concentration with a harmonious high arousal state by increasing alpha and beta activities [29]. Kimura et al. [30] studied the effect of

Fig. 1. The t-Mapping of EEG power spectrum changes before and during the inhalation of (+)-α-pinene in women and men. Fp1, left prefrontal; Fp2, right prefrontal; F3, left frontal; F4, right frontal; T3, left temporal; T4, right temporal; P3, left parietal; P4, right parietal. Arrows show the significant changes in the regions during the inhalation of (+)-α-pinene.

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Fig. 3. A schematic representation of inhalation of (+)-α-pinene and (+)-β-pinene on human EEG activity.

of the brain. Hence, this study clearly suggests that the increase of beta and high beta activity due to the inhalation of (+)-α-pinene and (+)-βpinene may increase the alertness state of brain function in women (Figs. 1 and 2). In the case of men, theta waves significantly decreased in 4 out of 8 regions due to the exposure of (+)-α-pinene. The theta wave activity has been considered to maintain attention during the performance of difficult tasks. Further, less theta activity is mainly related to the memory formation [31,32]. The decreased theta wave activity may enhance the attention and concentration state of brain. However, significant decrease of absolute beta, low beta and mid beta activities were observed during the inhalation of (+)-α-pinene in men (Fig. 1). The decrease of beta wave activities is mainly associated with the drowsiness state of the brain function [27]. In the current investigation, (+)-α-pinene and (+)-β-pinene produced different EEG changes according to gender variation. When compared with the inhalation of (+)-β-pinene, significant changes of absolute waves were observed in both men and women only during the inhalation of (+)-α-pinene. It was reported that gender variation highly influenced on the EEG activity of resting, stimulus and non-stimulus conditions [33]. Corsi-Cabrera et al. [34] found that higher relative beta activity in men and higher relative alpha activity in women during the cognitive function. Wada et al. [35] found that gender differences exist in the EEG activity during stimulus as well as non-stimulus conditions. In addition, the gender variations occurred in event-related oscillations during simple visual stimulation [36]. Doty and Cameron [37] also extensively reviewed the influences of sex differences and reproductive hormone on human odor perception. Radulescu and Mujica-Parodi [38] stated that women possess a better sense of smell than men in many species and the increased sensitivity may be based on biological meanings. In women, the cerebral lateralization develops sooner when compared to men [39,40]. Furthermore, Bishop and Wahlsten [41] mentioned that a large neural fiber tract (corpus callosum) connecting the two hemispheres of the brain is larger in women when compared with men. The larger corpus callosum allows more free flow of communication between the hemispheres. Hence, more synapses are generated between the hemispheres of the brain in women [42].

Table 4 Effect of inhalation of (+)-α-pinene and (+)-β-pinene on EEG activity in men. EEG indices

(+)-α-Pinene Absolute theta

Absolute beta Absolute low beta Absolute mid beta Relative theta Relative low beta (+)-β-Pinene Absolute theta

Absolute beta Absolute low beta Absolute mid beta Relative low beta Ratio of mid beta to theta

Site

Before inhalation (μV2)

During inhalation (μV2)

t-test

P value*

Fp2 F4 P3 P4 Fp2 T4

12.5633 14.1270 12.2993 11.5944 9.0659 2.6135

9.4554 11.9835 10.3241 9.4047 7.8745 2.1621

2.5718 2.6315 2.4776 2.8755 2.3354 3.3613

0.0300 0.0272 0.0351 0.0183 0.0443 0.0083

F4

3.2367

2.8422

2.9251

0.0168

T3 P3 T3

0.1938 0.1842 0.0960

0.1609 0.1564 0.0852

2.6689 2.4631 2.4844

0.0256 0.0359 0.0347

Fp2 F4 P3 P4 Fp2 T4

10.9239 13.0082 11.4357 9.7354 9.2463 2.5384

11.3581 14.1311 11.7404 11.1581 9.2964 2.8180

−0.685 −0.835 −0.378 −1.789 −0.057 −0.908

0.511 0.425 0.714 0.107 0.956 0.388

F4

3.4446

3.3370

0.677

0.515

Fp2

0.0604

0.0777

−2.4949

0.0341

F4 Fp1 Fp2

0.0582 0.3277 0.3056

0.0755 0.2683 0.2600

−2.4399 2.9955 3.3761

0.0373 0.0150 0.0081

Fp1, left prefrontal; Fp2, right prefrontal; F4, right frontal; T3, left temporal; T4, right temporal; P3, left parietal; P4, right parietal. * Significant difference (P < 0.05); number of subjects, 10.

peppermint fragrance on EEG activity and reported that the higher beta wave activity may stimulate the brain to remain active and vigilant. Based on the previous studies, the higher beta wave activity is mainly associated with the enhancement of alertness and concentration states 37

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recorded for a short duration (45 s before and during the exposure). Therefore, future studies should focus on EEG recording time, concentration of fragrance molecules, EEG measurement after stimulation with placebo control, and optimization of EEG feature extraction methods.

Our recent studies also clearly demonstrated that the gender variation highly influenced on the EEG activity. Further, women highly responded to essential oils and fragrant molecules when compared with men [8,20]. Recently, Haehner et al. [43] reported that men and women responded differently during the exposure of rose or grapefruit or a combination of orange, lime and lemon fragrances. In their study, women were more likely to observe the presence of a fragrance and altered their behavior accordingly. In a similar line, the results of the present study also clearly demonstrated that the exposure of (+)-αpinene and (+)-β-pinene exhibited different EEG activities in men and women. It is well known that fragrances exhibit instant effect on brain function. In the previous reports, the authors followed 2 to 3 min wash out time between each stimulus in order to reduce the influence of previously presented odor. Previously, Iijima et al. [5] used 3 min resting time between rose and incense odor exposures. In another study, Skoric et al. [7] investigated the EEG response to lemon, vanilla and peppermint with 2 min interval time. Further, Masago et al. [23] studied the effect of lavender, chamomile, sandalwood and eugenol on EEG activity with 3 min interval time between each stimulus. Therefore, we used 3 min wash out time between (+)-α-pinene and (+)-βpinene exposures. In the present study, the data clearly suggest that the isomers produce different EEG activities. It is well known that isomeric aroma components have different fragrance qualities and intensities for humans. The chirality of fragrance molecules plays an important role in the bioactivity of fragrances [44]. Sugawara et al. [9,45] investigated the sedative properties of optically active linalools and their effect on humans in order to determine their odor distinctiveness by chiral isomers ((R)-(−)-linalools and (S)-(+)-linalools). The authors demonstrated that enantiomeric stereospecificity of linalools produced different odor perception and also with chiral and task dependence. The structure–activity relationships of several isomeric components were investigated in order to determine their characteristic fragrance properties [46]. Sowndhararajan et al. [8] studied the effect of inhalation of isomeric components, (+)-limonene and terpinolene on EEG activity and found that the isomers exhibit different EEG activity. Tsunetsugu et al. [47] reported that olfactory stimulation of α-pinene decreased systolic blood pressure in male students. In another study, olfactory stimulation of α-pinene induces physiological relaxation by significantly decreasing heart rate [48]. Furthermore, inhalation of αpinene exhibits anxiolytic activity in mice and increased rapid eye movement sleep in rats [49,50]. Based on the results of this study, the isomers possess fragrance distinctiveness, (+)-α-pinene has coniferous and turpentine like smell, whereas (+)-β-pinene has fresh and woody like smell. These isomers produce different activities on the brain because of dissimilarity in their sensitivity of olfactory receptors. In general, olfactory receptors are specifically tuned to individual fragrant properties. The olfactory system discriminates fragrances from several small molecules as the brain analyses signals from olfactory receptors [51]. The findings of the present study clearly suggest that these isomeric compounds have fragrance distinctiveness and produce EEG changes in different indices and different brain regions. Our findings has some limitations. In the present study, fast Fourier transform method was applied to extract EEG features before and during the exposure of (+)-α-pinene and (+)-β-pinene. However, there are many techniques available for extracting the features from EEG signals (time frequency distributions, eigenvector methods, wavelet transform, Hilbert Huang transform, the autoregressive method, nonlinear dynamics analysis, etc.). Another limitation is subject’s eye movement during the EEG recordings. In addition, we observed more deviations in the EEG results between the individuals (10 men and 10 women). In this study, undiluted (+)-α-pinene and (+)-β-pinene were used as fragrance stimuli. The results may be varied when changing the concentration of fragrance molecules. Furthermore, EEG readings were

5. Conclusion The results revealed that the isomeric aroma components, (+)-αpinene and (+)-β-pinene showed different EEG power spectrum activities. Further, gender difference highly influenced on the EEG activity of these isomers. When compared with men, both the components produce positive effect in women by enhancing relaxation and alertness states of the brain. The present study clearly demonstrates that the activity of isomers on EEG activity is mainly associated with the fragrance type of the component as well as gender difference. Funding source Not applicable. Conflict of interest The authors declare that they have no potential conflict of interest. Acknowledgements This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through High Value-added Food Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (317044-03) and the research grant from Kangwon National University, Chuncheon, Republic of Korea. References [1] K. Touhara, L.B. Vosshall, Sensing odorants and pheromones with chemosensory receptors, Annu. Rev. Physiol. 71 (2009) 307–332. [2] R.S. Herz, Aromatherapy facts and fictions: a scientific analysis of olfactory effects on mood, physiology and behavior, Int. J. Neurosci. 119 (2009) 263–290. [3] F.L. Angelucci, V.V. Silva, C. Dal Pizzol, L.G. Spir, C.E. Praes, H. Maibach, Physiological effect of olfactory stimuli inhalation in humans: an overview, Int. J. Cosmet. Sci. 36 (2014) 117–123. [4] K. Sowndhararajan, S. Kim, Influence of fragrances on human psychophysiological activity: with special reference to human electroencephalographic response, Sci. Pharm. 84 (2016) 724–751. [5] M. Iijima, N. Osawa, Effects of incense on brain function: evaluation using electroencephalograms and event–related potentials, Neuropsychobiology 59 (2009) 80–86. [6] E. Matsubara, M. Fukagawa, T. Okamoto, K. Ohnuki, K. Shimizu, R. Kondo, The essential oil of Abies sibirica (Pinaceae) reduces arousal levels after visual display terminal work, Flavour Frag. J. 26 (2011) 204–210. [7] M.K. Skoric, I. Ivan Adamec, A.B. Jerbic, T. Gabelic, S. Hajnšek, M. Habek, Electroencephalographic response to different odors in healthy individuals: a promising tool for objective assessment of olfactory disorders, Clin. EEG Neurosci. 46 (2015) 370–376. [8] K. Sowndhararajan, H. Cho, B. Yu, S. Kim, Effect of olfactory stimulation of isomeric aroma compounds, (+)-limonene and terpinolene on human electroencephalographic activity, Eur. J. Integr. Med. 7 (2015) 561–566. [9] Y. Sugawara, C. Hara, T. Aoki, N. Sugimoto, T. Masujima, Odor distinctiveness between enantiomers of linalool: difference in perception and responses elicited by sensory test and forehead surface potential wave measurement, Chem. Senses 25 (2000) 77–84. [10] E. Brenna, C. Fuganti, S. Serra, Enantio selective perception of chiral odorants, Tetrahedron: Asymmetry 14 (2003) 1–42. [11] J.G. Romagni, S.N. Allen, F.E. Dayan, Allelopathic effects of volatile cineoles on two weedy plant species, J. Chem. Ecol. 26 (2000) 303–313. [12] B. Weber, A. Mosandl, Sterioisomeric flavor compounds LXXV: Synthesis and structure–function relationship of 3–methylthiobutanol enantiomers, Z. Lebensm. Unters. F. A. 204 (1997) 194–197. [13] P.M. Rivas da Silva, D.C. Barros de Azevedo, C.S. Costa, D.S. Alviano, Biological activities of α-pinene and β-pinene enantiomers, Molecules 17 (2012) 6305–6316. [14] K. Winska, B. Potaniec, W. Maczka, M. Grabarczyk, M. Aniol, C.Z. Wawrzenczyk, Isomers and odor or nose as stereochemist, Chemik 68 (2014) 83–90. [15] M. Laska, P. Teubner, Olfactory discrimination ability of human subjects for ten

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