Introduction

In recent years, aromatic oil has attracted attention from various fields such as environmental and cosmetics. Aromatic oils are produced from various sources, including animal, plant, and synthetic fragrances. Research on the aromatic oil’s compounds and their effects on the human body are often conducted separately. Okada et al.1 mentioned the effect of aromatic oil toward autonomic nervous system during exercise. Their study showed that compared with aromatic oil, the heart rate of the participants showed a later recovery. At the same time, it would be better if the olfactory stimuli also contained information about the chemical compounds of the aroma to be used. Since the analysis of chemical and psychophysiological effects requires multidisciplinary skills, the combination of the two studies is rarely done. Therefore, our proposed study became a pilot study to explore the effect of aromatic oil on olfactory stimulation. We conducted a randomized crossover pilot study to determine how best to conduct a large-scale research project in an exploratory study of olfactory stimulation with aromatic oils and olfactory stimulation at rest with closed eyes. To understand what compounds are contained in the aromatic oil to be used in this olfactory stimulation, this study used TDU-GC-MS (thermal desorption device combined with gas chromatography and mass spectrometry). Thermal desorption is a technique that uses heat to increase the volatility of contaminants and separated them from a solid matrix without combusting the medium or contaminants2. This is the background why we used the technique to find the compound of aromatic oil and do the chemical profiling. Chemical profiling is needed to gain the understanding of what compound are contained in the aroma and to investigate the effect of aroma on human psychophysiological activity.

The aroma’s journey into the human body is first to the brain regions that process emotion and memory. Therefore, measuring brain activity becomes crucial to see the effect of white musk fragrance on human psychophysiology. Electroencephalography (EEG) becomes a suitable measurement device since it is a non-invasive method and has no side effects in the use of this electrical brain activity monitoring to the participants. The data of EEG its selves can be extracted into various features, from linear to nonlinear features3. The classification of EEG features based on its frequency could be referred to International Federation of Clinical Neurophysiology (IFCN) 20174. Where the theta (ø)band is in the range of 4 to less than 8 Hz, alpha band is in the range 8 to 13 Hz, beta band is in the range 14 to 30 Hz and gamma band is distinguish in the frequency more than 30 Hz to 80 Hz. Gamma band in this study is divided into two categories, low gamma (31–59 Hz) and high gamma (65–80 Hz). The frequency band features of EEG data and its dominant power activities electrode’s location often correlated to the mental states of the human. We also explored the nonlinear features of neural activity which is called the slope of 1/f fluctuations. Several studies found there are information hidden under the slope of 1/f instead just seeing it as a noise without neuronal information. Gao et al.5 and Waschke et al.6 mentioned that excitation (E) and inhibition (I) of neural circuit can be estimated from the slope of 1/f. Where Donoghue7 et al. providing the algorithm for 1/f analysis that could be used for aging related analysis. Voytek et al. in 20158, explored the correlation between the slope of 1/f with the age, founding of their study mentioned that aging is associated with a flatter 1/f. However, until now, there is no other research that investigates the effect of fragrance on the slope of 1/f. This represents our novelty point, as it will be the first pilot study to investigate the slope of 1/f change for aromatic oil inhalation.

To measure the effect of aromatic oil on human physiology, this study also monitored the autonomic activity of the heart. Electrocardiography (ECG) is used to monitor the electrical activity of the heart through the cycles of the heart using an electrode placed on the skin. The features from the ECG data consisted of the sympathetic nervous system (SNS) and parasympathetic nervous system (PNS), which are reflected in the low frequency (LF) and high frequency (HF) bands in heart rate variability (HRV), while the sympathovagal balance is shown on the ratio of the powers in these frequency bands, the so-called LF-HF ratio (LF/HF)9. Another sympathetic activity measurement that has correlation with the stress is salivary amylase. The significant differences were found between the stress and the rest condition in salivary alpha-amylase in Nater et al.10 research that created the change of alpha amylase as stress index biomarker11,12,13.

Visual analog scale (VAS) was used to measure the participants’ impression of the presented aroma. The mood changes of the participants are observed by using Profile of Mood States (POMS) questionnaire. VAS is a visual-based scale that typically measures participant impressions of stimuli and participant feelings based on past references14. At the same time, The Profile of Mood States Second Edition (POMS2) Japanese version is used to evaluate the mood states of the participant that also been used by several studies such as evaluation of mood15,16 .

Due to the many types and characteristics of aromatic oils, in this study we focused on exploring the effects of white musk aromatic oil. White musk aromatic oil was chosen as the experimental material because this aromatic oil is popular among the public. From the various evaluation literature, ours is the first pilot study to investigate the effects of white musk aromatic oils, including investigating the effects of chemical compounds contained in aromatic oils on human psychophysiology. We are also the first team to investigate the changes in 1/f slope in response to fragrance.

Results

Chemical profiling in fragrance olfactory stimulus

This study used white musk aromatic oil as an olfactory stimulus. In the qualitative analysis, several peaks were detected in the white musk aromatic oil. This study used diethylene glycol monoethyl ether (15.7%) and isopropyl myristate (4.7%) as solvents. The component with the largest peak area was linalool (11.9%; detected peak area in the total peak area), and the others were hedione (8.3%), galaxolide(5.2%), α-hexylcinnamaldehyde (4.6%), benzyl acetate (3.7%), and tonalide (1.6%). Galaxolide and tonalide, were selected for quantitative analysis because these components are the main characteristic of white musk aromatic oil. The concentrations of galaxolide and tonalide were 34.8 ± 1.9 mg/mL and 15.0 ± 0.8 mg/mL, respectively.

Psychological evaluation (survey)

Participant impression toward presented aromatic oil

The VAS was used to quantify the fragrance impression of a room using a total of 21 items, including adjectives and verbs expressing fragrance characteristics (Table 1). In the before-and-after comparison of the white musk aromatic oil group, the impression of “sour” was significantly reduced after compared to before (z = -2.251, p = 0.028). On the other hand, no significant difference was found in the before-after comparison of “sour” in the unscented group and in the comparison of the amount of change between the unscented group and the white musk aromatic oil group.

Table 1 Statistic value of 21 VAS variables.
Full size table

In a before and after comparison of the white musk group, the impressions of the three items “like”, “sweet”, and “gorgeous” increased significantly after compared to before (t(9) = -4.071, p = 0.003, t(9) = -3.184, p = 0.011, t(9) = -3.283, p = 0.009). On the other hand, no significant differences were found in the before and after comparisons of the unscented group on these three items. In addition, when comparing the unscented group and the aromatic oil group in terms of the amount of change in each item, the amount of change in the aromatic oil group was significantly greater than the amount of change in the unscented group for the three items “like”, “sweet”, and “gorgeous”. (t (9) = -3.232, p = 0.010, t(9) = -2.657, p = 0.026, t(9) = -2.397, p = 0.040). These results showed that the white musk that be used as aromatic oil has strong impressions of “like”’, “sweet” and “gorgeous”.

Mood changes in response to aromatic oil

Seven mood scales and the associated Total Mood Disturbance (TMD) score were quantified using the short version of the POMS2 (Table 2). A pre-test comparison within each condition revealed that the Confusion-Embarrassment (CB) and Fatigue-Inertia (FI) scales were measured in both the unscented and aromatic oil groups, and post-test scores were compared with pre-test scores. showed a significant decrease (CB: t(9) = 2.290, p = 0.048; t(9) = 2.363, p = 0.042, FI: z = 2.201, p = 0.036; t(9) = 4.348, p = 0.002). The ``Tension-Axiety (TA)’’ and ``Total Mood Disturbance (TMD)’’ scales showed a significant decrease in post-test scores compared to pre-test scores in the white musk (aromatic oil) group (TA: t (9) = 2.941, p = 0.016, TMD: t(9) = 2.580, p = 0.030). There was no significant difference between the unscented group and the white musk aromatic oil group in terms of the amount of change in each item.

Table 2 Statistic value of POMS 2.
Full size table

Physiological evaluation

Salivary amylases result in response to aromatic oil inhalation

A before and after comparison of the saliva test results showed a significant difference in the post-measurement of the unscented group t (9) = 2.262, p = 0.012 as shown in Fig. 1. No significant difference was observed in the White Musk group t(9) = 2.262, p = 0.56.

Fig. 1
figure 1

Alpha amylase activity in unscented and White Musk. In this experiment, we confirmed that there was no significant change under aromatic oil inhalation, but on the other hand, there were significant changes in salivary alpha-amylase when the participant under the unscented condition.

Full size image

Autonomic activities change after the aromatic oil inhalation

Since ECG has a slower response compared to brain activity, we found that after 10 min of white musk aromatic oil presentation, LF/HF in the white musk (white musk aromatic oil) group was significantly decreased compared to the unscented group (t (8) = 3.518, p = 0.024), as shown in Table 3.

Table 3 Electrocardiogram result during experiment is conducted.
Full size table

Brain analysis result

Power spectrum frequency band (PSD)

We performed EEG power spectrum frequency analysis and mapped the potential distribution of specific EEG activity across the scalp from 8 representative channels. As a result of visual examination of areas with specific changes between groups in each brain wave frequency band after the Bonferroni correction, changes were observed in the beta band of the left central region (C3 region) (t (8) = 3.972, p = 0.032) and the gamma band of the right occipital region (O2 region) ((t (8) = -3.757, p = 0.048) as shown in Fig. 2. This study also presented the brain topography mapping of 8 representative channels of the brain area in Fig. 3. The brain topography mapping based on the subtraction of each individual average power spectrum for easier interpretation since the electrode used are 8 channels. The color indicates the brain location that has dominant activity with red-ish color showed the higher power activities in white musk aromatic oil where the blue-ish color showed a lower power activity under white musk aromatic oil inhalation. Where the statistical part shows the paired t-test comparison between power spectrum of white musk inhalation and unscented with Bonferroni comparison to each electrode location. All electrode placement measurement values and their changes to experimental conditions can be seen in Table 4.

Fig. 2
figure 2

The changes of beta and gamma band in specific brain location. The left picture shows the changes on the beta band on the left central area (C3) between unscented and white musk for 10 min inhalation and the right picture shows the increase on the gamma band on the right hemisphere of the occipital between unscented and white musk for 10 min inhalation.

Full size image
Fig. 3
figure 3

Brain topography from 8 channels between white musk- unscented. Subtraction between white musk-unscented PSD from 8 channel EEG, the red dot on the statistic (left image) means where the value is significant, red-ish color in the brain topography showed higher power, where blue-ish showed lower power under aromatic oil inhalation (right).

Full size image
Table 4 The changes of brain activity during experiment.
Full size table
1/f fluctuations result

1/f is a fitting line (dotted line) of the power spectral density (PSD) versus frequency, and Fig. 4 shows the average PSD results and 1/f from all electrodes of the nine participants analysed. During olfactory stimulus, the slope of 1/f in the White Musk (white musk aromatic oil) group was significantly gentler than that in the unscented group starting from 37 Hz oscillation (t(8) = -2.354, p = 0.046).

Fig. 4
figure 4

1/f slope and PSD during inhalation of aromatic oil within 10 min inhalation. 1/f activity was shown to be gentler when participants exposed with the aromatic oil comparing with the unscented condition. This phenomenon may occur under the influence of dopamine. When humans like something, they release the hormone dopamine, which causes an increase in vibrational activity in the brain. The larger the oscillation, the greater the force, and the slope tends to be gentler.

Full size image

Discussion

This pilot study experiment used white musk as an olfactory stimulus. The choice of using this fragrance among other fragrances such as lavender and fir that we used to confirm the olfactory function of the participants because white musk is one of the best-selling products in the fragrance category. To get the right amount of white musk in a 4.2 m3 room, researchers in this team was conducted several aroma tests when the amount of white musk was in 5 µL, 10 µL, 20 µL, 100 µL before the experiment is conducted to participants. This study found that the fragrance of 10 µL below was appropriate to avoid strong aroma within that specific room size. In the component analysis of white musk, we found that the white musk used in the test contained galaxolide (HHCB) and tonalide (AHTN). In general, Galaxolide gives a pleasant aroma impression based on other research17, which is also showed in our current result based on participant answer on VAS survey. While Tonalide is a compound also often used in other fragrances18. Knowing the chemical profile of white musk aromatic oils is expected to expand the hypothesis about the possibility of other similar white musk aromatic oils on changes in psychophysiological activity. Some common product white musk aromatic oils can have different chemical profiles, therefore mentioning the compounds contained in the white musk aromatic oil is necessary for clear validation conditions. Other studies sometimes only mention the name of the white musk aromatic oil, but how much white musk aromatic oil is presented is often not mentioned. Through this preliminary study, we hope to be a guide for larger experiments in the future.

Salivary amylase activity is known to have a high correlation with plasma norepinephrine concentration, reflecting sympathetic nerve activity and usually reflecting both mental and physical stress10,13. Result in our study showed that under unscented condition, there is significant difference in alpha amylase. But there is no significant changes when the participant inhaling the white musk aromatic oil. This might be correlated with the change of mood of participant under white musk aromatic oil condition. As mentioned in the results, under the condition of white musk aromatic oil, FI (Fatigue-Inertia), TA (Tension-Anxiety), TMD (Total mood disturbance) have decreased significantly. The decreasing of alpha amylase in unscented could be correlated to post awakening effect. The salivary alpha amylase was taken prior the electrode placement and after the olfactory experiment finished. During the experiment, participants closed their eyes for 16 min. Because there was no stimulus from the fragrance that induced aroma awareness, participants could feel sleepier compared to when there was a white musk aromatic oil present in the room. Since the salivary alpha amylase is taking within 10–15 min windows after olfactory experiment finish, this could be the reason why in unscented condition the alpha amylase is significant decrease. Some studies mentioned the decrease of saliva alpha amylase within 10–60 min post awakening and increase or flattened after that window19,20,21. The decrement was not corresponded to the relaxing effect itself is confirm by the participant self-assessment via VAS (Visual analogue scale), where the relaxation variables was not significant difference (p = 0.545) in unscented condition. At the same time, Electroencephalogram (EEG) measurements reflect neural oscillations in the central nervous system (CNS) and are directly related to various higher-order cognitive processes, including emotion. Emotion recognition based on brain waves shows great potential because it is difficult to intentionally hide, or unscented internal neural oscillations compared to approaches based on facial expressions or speech. Studies that more comprehensively and systematically explored a wide range of EEG features have shown that data in high frequency bands are more influenced by emotion recognition compared to lower frequency bands. Soleymani et al.22. performed a cross-subject emotion recognition task using EEG and gaze data and extracted the power spectral density (PSD) of the EEG and found that the most discriminative feature regarding fluctuations in arousal level was found at the occipital electrodes. Emotional features were found in the beta and gamma bands of temporal electrodes. In this study, fluctuations were observed in the beta waves from central electrodes and the low gamma waves from occipital electrodes. This study also found there is increasing value in positive impressions such as “gorgeous”, “sweet” and “like” white musk aromatic oil toward white musk in the VAS survey. Which this finding is consistent with the report that positive emotional recognition affects low gamma band in right hemisphere23. Enhancing the previous sentence, beta band activity during white musk aromatic oil olfactory stimulus showed significantly lower activity in the left central region (area C3) compared to unscented. The lower power in the power spectrum of the β band in left hemisphere area during white musk inhalation comparing with unscented observed in this study probably were a response to the participants’ recognition of the white musk scent during fragrance presentation which has pleasant aroma. Regarding the results of the LF/HF index obtained by frequency analysis of heart rate fluctuations, LF/HF was significantly lower than unscented. This is consistent with the fact that within 10 min white musk presentation, beta-band activity in left hemisphere (C3) area was lower compared to unscented and gamma band is higher in right hemisphere (O2). It was suggested that the scent of white musk that is consisted of galaxolide (HHCB) and tonalide (AHTN) may influence the balance of the autonomic nervous system and give a pleasant feeling24 and as indication of wellbeing25.

Regarding the fluctuations in γ waves observed when presented with fragrances, studies related to various types of meditation have reported that meditation activates of gamma band (> 30 Hz) mainly in the parietal and occipital regions. Lutz et al.26 found increased activity in the low gamma band (25–42 Hz) in the lateral frontal lobe and occipital region during non-directed meditation. Cahn et al.27. reported that during Vipassana meditation, one of India’s oldest meditation methods, the power value of the low gamma band (35–45 Hz) in the parietal and occipital regions increased significantly. Berkovic et al.28 reported an increase in the power value of the low gamma wave band (25–45 Hz) in the occipital region during mindfulness meditation, which has recently attracted attention in the medical and business communities. In this experiment, the white musk fragrance showed higher low gamma wave band activity in the right occipital region (O2 region) compared to unscented. This suggests that the patient may have been induced into a deep state of mental activity. At the same time, by high gamma band (65–80 Hz) did not show significant difference when using statistical comparison with Bonferroni correction between electrode placement strengthen the statement that the activity gamma band is not only induced by the aroma, but because the aroma is like (likeable) by the participants. Since the experiment in this study has been done within 16 min with closed eyes, the changes of low gamma band also indicated correlation with the meditation states as mentioned Lutz et al.26 that long term meditator high amplitude of gamma band (25–42 Hz), which we specified it as low gamma band in this article. The higher amplitude of gamma band in occipital area is indicate with meditation is also explored by Cahn et al.27 and Ferrareli et al.29.

Until now, the 1/f power spectrum of brain waves has been regarded as background noise, but the possibility that it contains useful information is being investigated. Dave et al.30. state that this 1/f neural noise may reflect the distribution of power spectral density over a wide frequency range, and that it is useful in discussing neuronal activation and aging. Erik J Peterson et al.31. state that in diagnosing schizophrenia and predicting the severity of symptoms, the steepness of 1/f can better predict the condition than conventionally focusing on vibration power in each frequency band. In this study, we conducted an evaluation of the effects of white musk scent on generally healthy and young participants, so we cannot address the aging and health issues mentioned above, but we do discuss physiological effects from a wide frequency range. It is possible. In a previous study on the 1/f slope, Gao et al.5. also noted that the 1/f slope became negative under anaesthesia compared to the awake state. Freeman et al.32. showed that the slope of PSD during sleep is steeper than during wakefulness. The present pilot study differs from the previously mentioned article in that instead of comparing unconscious and conscious states (sleep and wakefulness), this research protocol is the first to explore non-periodic brain activity in the wakeful state between different scent conditions. The result showed that the slope of 1/f was significantly gentler when white musk aromatic oil was presented compared with unscented condition. It has been reported that when humans feel that they “like” an object, brain wave activity is activated, the brain wave period becomes shorter, the power spectrum in the high frequency region becomes larger, and the slope of 1/f becomes gentler. In our current assumption, this phenomenon may occur under the influence of dopamine. Based on literature, hormone dopamine related with liking or rewards33,34. At the same time, the activation of dopamine causes an increase brain oscillation activity in the brain35. The larger the oscillation, the greater the force, and the slope tends to be flatter. Indeed, further study need to be done to explore more the effect of olfactory stimulus toward aromatic oil since this study was applied to small number of participants and limited to white musk aromatic oil. This pilot study is expected can be the future direction for the large-scale experiment.

Methods

Ethical matters

This study proposed a pilot study on the effect of olfactory stimulus with white musk, as white musk aromatic oil and evaluated the effects of an original blend fragrance using artificial musk developed by Next Day Co., Ltd. and Kyushu University (hereinafter referred to as “white musk”) toward the psychological and physiological responses of humans. This study was conducted with the approval of the Ethics Committee of the Faculty of Agriculture, Kyushu University (application number: 23 − 004, approval number: 110, project title: Exploratory Research on the Functionality of Fragrances, Approval Date: July 28, 2023. We also registered this study UMIN Clinical Trials Registry with register ID : UMIN000051972, August 24th, 2023 (24/08/2023). All measurements involving human participants were conducted in accordance with the Declaration of Helsinki. Prior to participation, participants were fully informed of the purpose and content of this study. After confirming that the participants fully understood and agreed with the content, we obtained their free and written informed consent to participate in this study.

Participants

The total participants were 10 people, 5 man and 5 women (23.2 ± 2.3 years). The study design was a randomized, single-blind, crossover study. Participants were healthy adults without physical or mental problems. Participants were randomly assigned to two groups of 5 people and participated in the study twice. In Group A, the unscented was presented on the first test, and white musk was presented on the other day. In Group B, white musk was presented on the first test and the unscented on the other day. The washout period for the previous and subsequent tests was about 2 weeks. During the washout period, one participant was found to have the effects of COVID-19, and this participant is excluded from the physiological analysis in this study. The study was conducted from August 28 to October 4, 2023.

Olfactory function confirmation

Prior to the experiment, an interview session was held with the participants to explain the experiment and to test olfactory function of the participant with three different aromas: lavender, white musk, and fir. During this meeting, participants reported that they were able to distinguish between the aromas. In addition, the aromas presented were preferred by all participants. During this session, we did not inform the participant what kind of aroma will be presented during the experiment to avoid the bias.

Experimental room and olfactory simulation presentation

In the human test, a soundproof room (width 1,700 mm, depth 1,260 mm, height 1,965 mm, manufactured by Yamaha Corporation) (hereinafter referred to as the laboratory) installed in Room 457 of West Building 5 on the Ito Campus of Kyushu University was used (Fig. 5). The test was conducted in a 4.2 m3 experimental space under conditions that suppressed external sound and light stimulation. The temperature and humidity before the start of the test were 22.4 ± 1.4℃ and 63.1 ± 5.5% (TR-72wf T&D Temperature and Humidity Data Logger, T&D Co., Ltd.), and the carbon dioxide concentration was 567.6 ± 118.8 ppm (testo 160 IAQ - Cloud monitoring logger). The white musk (undiluted solution) used in this study’s intervention is widely used in household detergents, perfumes, and cosmetics, and in product manufacturing, the undiluted solution is added to sprays at 1% and diffusers at 2–3%. To detect the scent, a cotton swab was soaked with 10 µL of white musk and inserted into the room through a hole made in the laboratory (Fig. 5). No fans were used during the test, and doors were not opened or closed to eliminate the effects of external sound and light. We divided the test condition into unscented and white musk aromatic oil inhalation. In addition, our participants were blinded to the type of scent presented, they did not know what scents were in the air in the room.

Fig. 5
figure 5

Experimental room and olfactory stimulus method.

Full size image

Experimental procedure

Participants changed into a designated uniform before the start of the experiment to ensure uniform scent conditions. After being fitted with bioelectrodes, participants entered the experimental room and were seated in a chair. Two types of questionnaires were administered to all participants at pre- and post-measurement: a visual analog scale (VAS) of mood and a short version of the Profile of Mood States Second Edition (POMS2) Japanese version. An examination was performed. During the electroencephalogram and electrocardiogram measurements in the laboratory, participants maintained a resting state with their eyes closed for a total of 16 min. In the white musk aroma condition, the conditions were divided into 3 conditions, they are: 3 min before the olfactory stimulus, 10 min during the olfactory stimulus, and 3 min after the olfactory stimulus was removed from the room wall. Electrical activity was recorded. Salivary amylase measurements were also taken before the electrodes were applied to the participants and after the electrodes were removed as shown in Fig. 6. Room temperature before the start of the experiment was monitored and the result showed the average condition as follows: 22.4 ± 1.4℃, humidity: 63.1 ± 5.5%.

Fig. 6
figure 6

Experiment timeline.

Full size image

Chemical profiling analysis in the white musk aromatic oil

A gas chromatography mass spectrometry system (GC-MS; 7890 A/5975 C, Agilent Technologies, Santa Clara, CA) with a thermal desorption unit (TDU; Gerstel, Mülheiman der Ruhr, Germany) was used for analysis of the white musk aromatic oil. TDU-GC-MS analysis condition is shown in Table 4. Diluted sample solution was injected in the splitless mode. GC-MS was equipped with a DB-5MS column (60 m×0.25 mm; 0.25 μm film thickness; Agilent Technologies). The oven temperature program was held at 70 °C for 1 min, gradually increased (40 °C/min) to 150 °C and held for 3 min, and then to 200 °C (5 °C/min) and increased (20 °C/min) to 300 °C and held for 3 min. Helium was used as the carrier gas at a flow rate of 1.2 mL/min. The synthetic musk compounds were identified by comparing the mass spectrum with the National Institute of Standards and Technology mass spectral library (NIST 11.0).

For quantitative analysis, the calibration curve was created by using standard substances and internal standard substance (IS). The standard substance galaxolide was purchased from BLD Pharmatech Ltd. (Shanghai, China), tonalide from Fluorochem Ltd. (Hadfield, UK), and IS phenanthrene-d10 from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). One µL of the mixture of the standard substances and IS prepared to the predetermined concentration was added to the glass wool at the tip of the glass liner for TDU (length 60 mm, outer diameter 6 mm, inner diameter 5 mm, Gerstel, Mülheiman der Ruhr, Germany) with a micro-syringe and analyzed under the conditions shown in Table 5. One µL of the white musk solution diluted to 1000 times in hexane (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) with IS was analyzed under the same conditions as above. The concentrations of galaxolide and tonalide were calculated using the internal standard method. The process of chemical profiling analysis was shown in Fig. 7. As shown in the GC-MS chromatogram in Fig. 7, several peaks were detected in the white musk aromatic oil that were combination from various synthetic fragrances. In this study, galaxolide and tonalide, which are the characteristic components of white musk, were selected for quantitative analysis.

Table 5 TDU-GC-MS analysis condition.
Full size table
Fig. 7
figure 7

Chemical profiling analysis process of the white musk aromatic oil.

Full size image

Psychological evaluation

An overview of the two questionnaire tests conducted is shown below.

Impression survey toward presented aroma

VAS (Visual analogue scale) was used to investigate the impression of the scent in the room. VAS is a widely used assessment method for estimating the degree of pain and determining the degree of pain relief. Healthcare professionals ask patients to measure the degree of pain on a line drawn between two ends. It is a continuous measure consisting of horizontal or vertical lines, usually 100 mm long. In recent years, it has been widely used in the medical field to measure health-related quality of life. It has also been used to measure post-sleep alertness, quality of life, anxiety, breathlessness, nausea, dyspnea, pruritus intensity, and attitude toward the environment. In addition, its usefulness has been reported in numerous applications in research on education, sound environments, and scents.

In this test, the left end of a 10 cm straight line was ``no impression’’ (0), and the right end was ``impression’’ (100). Participants placed a mark on the line for each item as a breaking point. Table 6 shows the adjectives used as evaluation words in this test. The evaluation words were adjectives and verbs expressing fragrance impressions, and responses to a total of 21 items were estimated as a VAS score (%).

Table 6 VAS spatial scent impression questionnaire.
Full size table

Mood states change survey towards presented aroma

The Profile of Mood States Second Edition (POMS2) Japanese version (hereinafter referred to as POMS2 abbreviated version) (Kaneko Shobo) was used to evaluate mood states and compare the states before and after fragrance presentation. This is a revised version of POMS, and POMS2 is a questionnaire that can quickly assess not only relatively long-lasting emotions, but also fluctuating and transient emotions. “Anger – Hostility (AH)”, “Confusion – Bewilderment (CB)”, “Depression – Depressed (DD)”, “Fatigue – Intertia (FI)”, “Tension – Anxiety (TA)”, “Vigor – Activity (VA)”, “ The mood state in a predetermined time frame is evaluated from the 7 scales of “friendship (F)” and the “Total Mood Disturbance (TMD) score,” which comprehensively represents negative mood states. In this study, we used the shortened version of POMS2 to reduce the burden on participants. Participants responded to a total of 35 questions on 7 scales, 5 items each, asking how they felt right now, ranging from ``Not at all’’ (0 points) to ``Very much’’ (4 points).) Answers were given on a five-point scale. The total score for each subscale was calculated, and the score for “Vibrance-Vitality” was subtracted from the total score for “Anger-Hostility,” “Confusion-Embarrassment,” “Depression-Depressed,” “Fatigue-Apathy,” and “Tension-Anxiety.” A “TMD score” was calculated. POMS2 short version scores were converted into standardized scores (T-scores) to assess participants’ mood states.

Physiological evaluation

Physiological evaluation was performed by salivary amylase and by measuring electrocardiograms (ECG) and electroencephalograms (EEG). During the analysis of ECG and EEG, one participant has been excluded due to the recovery from illness. An overview of each is shown below.

Measurement of salivary amylase activity

Salivary α-amylase was measured by salivary α-amylase monitor (Nipro Co.). Salivary α-amylase was used as an indicator of stress marker because it increases with the acceleration of sympathetic nerve activity.

Measurement of autonomic system

ECG was measured by PolymateV (Miyuki Giken Co.) with sampling frequency 1000 Hz. High frequency components (HF) and low frequency components (LF) of electrocardiogram (ECG) were calculated by power spectral analysis of R-R intervals and used as an indicator of autonomic nerve activity. In this experiment, frequency analysis was performed from the RR interval data obtained by measuring electrocardiograms, HF and LF/HF were calculated, and the influence of fragrance on sympathetic and parasympathetic nerve activity was evaluated. The changes in RRI of the electrocardiogram every 10 s were calculated, and HF and LF/HF values were calculated using a CD method R-R interval analysis program (NoruPro Light Systems, R-R Interval Analysis).

Brain activity measurement

In this study, a biological amplifier (Polymate V, Miyuki Giken) was used to measure brain waves as in electrocardiogram measurements. The sampling rate was 1000 Hz. Based on the international 10-20 method, recording electrodes were placed at F3 (left side of the frontal region), F4 (right side of the frontal region), C3 (left side of the central region), and C4 to confirm the distribution of EEG on the scalp with a small number of electrodes. (right side of the center), P3 (left side of the parietal region), P4 (right side of the parietal region), O1 (left side of the occipital region), and O2 (right side of the occipital region) (Fig. 8). Reference electrodes were placed on A1 (left earlobe) and A2 (right earlobe), and A1 and A2 were connected. Biological signal active electrodes with an amplifier were used to record low-noise brain waves even at high impedance. The electrodes were fixed to the scalp with bioelectric signal measurement paste so that the resistance was 20 kΩ or less. In this experiment, participants maintained a resting state with their eyes closed during the EEG measurements. To observe whether blink noise was mixed into the brain waves, electrodes were placed on the upper, lower, and left sides of the left eye and on the right side of the right eye, and electrooculograms (EOGs) were measured. An electrooculogram measures the corneal-retinal potential of the eyeball (the corneal side is positively charged, and the retinal side is negatively charged), which changes as the eyeball moves. A vertical electrooculogram (VEOG) and a horizontal electrooculogram (HEOG) were recorded. During EEG recording, clothlike electromagnetic shields (Noi Cut Sheet 44552, GE Healthcare Japan, Tokyo) were placed around the participant’s feet, chair, and EEG monitor to suppress commercial power noise from the participant’s feet. Brain analysis was performed in the MATLAB 2013b36 environment using the EEGlab37 toolbox to plot brain topography (such as Fig. 3) and a homemade script to detect 1/f fluctuations, the process of detection as shown in Supplementary Fig. 1.

Fig. 8
figure 8

EEG electrode placement.

Full size image

Statistical analysis

Regarding between-group comparisons of before-after differences and changes in white musk presentation and unscented (no fragrance presentation) groups, normality was confirmed using the Shapiro-Wilk test, and paired t was used for comparison pairs where normality could be assumed. A two-tailed test was conducted. For comparison pairs for which normality could not be assumed by the Shapiro-Wilk test, the Wilcoxon signed-rank test was performed. The significance level was set at 5% using a two-tailed test.