90 1. Introduction In the modern age, people are forced to lead busy lives and are exposed to a state of stress (Lederbogen et al., 2011). Thus, measures to prevent and relieve this stress state are urgently needed. Recently, forest therapy has emerged as a method to address stress states, and much data on the physiological and psychological relaxing effects of forest environments have been accumulated. Previous studies have reported that viewing forest scenery or walking in forests can: increase parasympathetic nervous activity, which is en- hanced in relaxing situations and suppresses sympathetic nervous activity which is increased in stress states (Tsu- netsugu et al., 2007; Park et al., 2008; Lee et al., 2009; Park et al., 2009; Park et al., 2010; Lee et al., 2011; Park et al., 2012; Tsunetsugu et al., 2013; Lee et al., 2014); de- crease cerebral blood flow in the prefrontal cortex (Park et al., 2007); and decrease salivary cortisol concentration of stress hormone (Tsunetsugu et al., 2007; Park et al., 2007; Park et al., 2008; Lee et al., 2009; Park et al., 2010). In addition, visiting a forest enhanced natural killer-cell ac- tivity and improved immune function (Li et al., 2007; Li et al., 2008 a, b, c) and the effect lasted 30 days (Li et al., 2008 b). In subjective evaluations, it was reported that people feel more “comfortable,” “soothed,” and “natu- ral” when experiencing a forest environment (Park et al., 2007; Tsunetsugu et al., 2007; Park et al., 2008; Lee et al., 2009; Park et al., 2009; Lee et al., 2011; Park et al., 2011; Tsunetsugu et al., 2013; Lee et al., 2014), and that the “tension-anxiety,” “depression,” “anger-hostility,” “fa- tigue,” “confusion,” and “vigor” of the mood state profile (McNair and Lorr, 1964; McNair et al., 1992; Yokoyama, 2005) improved (Li et al., 2008 a, b, c; Park et al., 2010; Lee et al., 2011; Park et al., 2011; Tsunetsugu et al., 2013; Lee et al., 2014). Unfortunately, many people liv- ing in cities find it difficult to access forest environments. Thus, much attention has been focused on nature-based stimuli, such as walking in an urban park (Song et al., 2013), viewing rooftop forests (Matsunaga et al., 2011), the presence of plants, including dracaena (Igarashi et al., 2014) or roses (Ikei et al., 2014), and physical contact with wood (Sakuragawa et al., 2008), and the relaxing ef- fects of these stimuli have been reported. Nature-based stimuli are intuitively perceived through the five senses. Of these five senses, the physiological effects of olfactory stimulation have been characterized Physiological and psychological effects of olfactory stimulation with D-Limonene D. Joung (1) * , C. Song(1) **, H. Ikei**, T. Okuda**, M. Igarashi**, H. Koizumi**, B.J. Park*, T. Yamaguchi**, M. Takagaki**, Y. Miyazaki (2) ** * Department of Environment and Forest Resources, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon 305-764, Republic of Korea. ** Center for Environment, Health and Field Sciences, Chiba University, 6-2-1 Kashiwanoha, Kashiwa, Chiba 277-0882, Japan. Key words: heart rate, heart rate variability, limonene, physiological relaxation, semantic differential method. Abstract: Although D-Limonene can be considered an important component of nature-based stimuli, the physiological ef- fects of olfactory stimulation with D-Limonene have not been completely clarified by scientific studies. The physiological and psychological effects of olfactory stimulation with D-Limonene were studied measuring heart rate variability (HRV), heart rate, and subjective evaluation using a modified semantic differential method; thirteen Japanese female university students (mean age±SD, 21.5±1.0 years) participated in the study. A concentration of 60 µL of D-Limonene was used as olfactory stimulant and room air as control. Subjects were exposed for 90 s while sitting with eyes closed. During D-Limonene inhalation: (1) the high-frequency (HF) value of HRV, a marker of parasympathetic nervous activity that is enhanced in relaxing situations, was significantly higher; (2) the heart rate was significantly lower; and (3) subjects reported feeling significantly more comfortable during D-Limonene administration than control. The results obtained clearly indicate that olfactory stimulation with D-Limonene induced physiological and psychological relaxation, provid- ing important scientific evidence of the health benefits of D-Limonene. Adv. Hort. Sci., 2014 28(2): 90-94 (1) These authors equally contributed to the manuscript. (2) Corresponding author: ymiyazaki@faculty.chiba-u.jp Received for publication 31 March 2014 Accepted for publication 30 June 2014 91 in detail. Miyazaki et al. (1992) conducted a pioneer- ing study which revealed that olfactory stimulation with Chamaecyparis taiwanensis essential oil significantly decreased blood pressure. Furthermore, inhalation of rose oil odor was shown to suppress sympathetic nervous activity and decrease adrenaline concentration (Haze et al., 2002). Lavender oil has been shown to induce deep sleep (Goel et al., 2005) and improve concentration (Sakamoto et al., 2005). However, evidence-based research using the indices of autonomic nervous activity to clarify the effect of compo- nents of these essential oils is lacking. The essential oil components of Cryptomeria japonica and Pinus densiflora, representative forest trees, have been reported (Cimanga et al., 2002; Hong et al., 2004; Cheng et al., 2009). These oils are composed of various volatile organic compounds, including D-Limonene, α-Pinene, β-Pinene. D-Limonene is the main component of citrus peel oil (Bernhard, 1960; Attaway et al., 1968; Shaw, 1979; Chiralts et al., 2002; Yoo et al., 2004). The purpose of the present study was to investigate the physiological effect of olfactory stimulation with D- Limonene on autonomic nervous activity by measuring its effect on heart rate variability (HRV) (Camm et al., 1996; Kobayashi et al., 1999) and the heart rate. 2. Materials and Methods Subjects Thirteen Japanese female university students (age range, 21.5±1.0 years; mean±SD) participated in the study. Before beginning the experiment, a full explanation about the research aim, the experimental procedure, and all measured indices was provided. Informed consent was obtained from all subjects. This study was conducted in accordance with the regulations of the Ethics Committee of the Center for Environment, Health, and Field Sciences, Chiba University, Japan. Study protocol Physiological and psychological measurements were carried out in a chamber with an artificial climate main- tained at 25°C with 50% relative humidity and 230- lux illumination. D-Limonene (>95.0% purity, Tokyo Chemical Industry Co., Ltd., Japan) was used as an ol- factory stimulant, and room air was used as a control. A total of 60 µL D-Limonene was injected into a 24-L odor bag (polyethylene terephthalate film heat seal bag; NS-KOEN Co., Ltd., Kyoto, Japan) and the odors were presented to each subject by means of a device fitted on the chest and situated approximately 10 cm under the nose (Fig. 1). The flow rate of the odor was set at 3 L/ min. Subjective sensitivity to the odor was determined in a preliminary investigation. The subjects were exposed to the odor for 90 s while sitting with their eyes closed. The order of presentation of D-Limonene and control was counterbalanced. Heart rate variability and heart rate HRV was measured as the periods between consecutive R waves (R-R intervals) in an electrocardiogram recorded with a portable electrocardiograph (Activtracer AC-301A, GMS, Japan). In this study, two major spectral compo- nents of HRV, the low-frequency (LF; 0.04–0.15 Hz) band and the high-frequency (HF; 0.15-0.40 Hz) band were ob- tained by the maximum-entropy method (MemCalc/Win, GMS, Japan). The HF power was considered to reflect parasympathetic nervous activity, and the LF/HF power ratio was considered to reflect the sympathetic nervous ac- tivity (Camm et al., 1996; Kobayashi et al., 1999). Heart rate was also investigated using R-R interval data. Semantic differential method The subjects provided a subjective evaluation of the emotional impact of the odors according to a modified semantic differential (SD) method (Osgood et al., 1957). This method allowed the subject to assess a pair of ad- jectives, such as “comfortable-uncomfortable,” using a 13-point scale. The SD method was performed after ad- ministration of each odor. Statistical analysis All statistical analyses were performed using Statistical Package for Social Sciences software version 20.0 (IBM Corp., Armonk, NY, USA). A paired t-test was used to compare differences in the physiological responses over the 90 s of exposure to D-Limonene and air. Wilcoxon signed-rank test was applied to analyze differences in psy- chological response between D-Limonene and air. A one- sided test was used in this study. In all cases, the signifi- cance level was set at P < 0.05. 3. Results The results of the HRV data after exposure to D-Limo- nene and control were compared, and a significant differ- Fig. 1 - Olfactory stimulation setup. 92 ence was found in the HF value, which is a marker of para- sympathetic nervous activity, as shown in Figure 2. The HF value increased 26.4% during D-Limonene administration (827.2±191.3 ms2; mean±SE) compared with control (654.4 ±163.6 ms2), indicating that parasympathetic nervous activ- ity was significantly higher during D-Limonene administra- tion (P<0.05). However, no significant difference was found in the LF/HF power ratio for the two stimuli. Figure 3 shows a comparison of the heart rate during the administration of D-Limonene and control. Heart rate decreased during D-Limonene administration (72.8±2.3 bpm) compared with control (74.1±2.5 bpm), and this dif- ference was significant (P<0.05). Figure 4 shows the results for a “comfortable” feeling according to the subjective evaluation. Subjects reported significantly more comfortable ratings during D-Limo- nene administration than control (P<0.01). 4. Discussion and Conclusions D-Limonene is one of the most common volatile or- ganic compounds in nature (Sun, 2007). It is a major com- ponent of various citrus oils, such as lemon, orange, grape- fruit, and lime (Attaway et al., 1968; Bernhard, 1960; Chiralts et al., 2002; Shaw, 1979; Yoo et al., 2004), as well as essential oils from coniferous trees, such as Pinus densiflora, Pinus koraiensis, Chamaecyparis obtusa, and Cryptomeria japonica (Cimanga et al., 2002; Hong et al., 2004; Cheng et al., 2009). In addition, because of its citrus fragrance, D-Limonene is commonly added to perfumes, soaps, and cosmetics (Bakkali et al., 2008). Although D-Limonene is an important component of nature-based stimuli, the physiological effect of olfactory stimulation with D-Limonene has not been completely clarified. Previously, Tsunetsugu et al. (2012) investigated the physiological effect of olfactory simulation with D- Limonene on blood pressure and showed that olfactory simulation with a concentration of 10 μL D-Limonene decreases subjects’ systolic blood pressure. However, to our knowledge, no previous study has examined the physi- ological effect of olfactory stimulation with D-Limonene on HRV and heart rate. The present study shows that olfactory stimulation with D-Limonene induced (1) a significant increase in parasym- pathetic nervous activities, (2) a significant decrease in the Fig. 2 - Comparison of high-frequency power levels of heart rate vari- ability during olfactory stimulation with D-Limonene or con- trol (air). Data are expressed as mean ± SE; n = 13. *P < 0.05 by paired t-test. Fig. 3 - Comparison of the heart rate during olfactory stimulation with D-Limonene or control (air). Data are expressed as mean ± SE; n = 13. *P < 0.05 by paired t-test. Fig. 4 - Subjective evaluation of “comfortable” measured by a modified semantic differential questionnaire after olfactory stimulation with D-Limonene or control (air). Data are expressed as mean ± SE; n = 13. **P < 0.01 by Wilcoxon signed-rank test. 93 heart rate, and (3) a significant increase in a “comfortable” feeling. These results agree with previous studies of other nature-based stimuli (Tsunetsugu et al., 2007; Park et al., 2008; Park et al., 2009; Park et al., 2010; Lee et al., 2011; Park et al., 2012; Song et al., 2013; Tsunetsugu et al., 2013; Ikei et al., 2014, Lee et al., 2014). Park et al. (2012) showed that the HF value of HRV was significantly increased while viewing scenery of forests using the results of field experi- ments at 35 forests in Japan. Ikei et al. (2014) reported that the HF component was significantly increased by viewing roses. Song et al. (2013) revealed that parasympathetic ner- vous activity was enhanced and the heart rate was signifi- cantly lower after walking in an urban park than walking in a city area. 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