Effects of Bergamot（Citrus bergamia（Risso）Wright & Arn.）Essential Oil Aromatherapy on Mood States, Parasympathetic Nervous System Activity, and Salivary Cortisol Levels in 41 Healthy Females

Forschende Komplementarmedizin Original Article.Originalarbeit Wissenschaft.Praxis.Perspektiven Forsch Komplementmed 2015;22:43-49 Published online:February 19,2015 D0:10.1159/000380989 Effects of Bergamot (Citrus bergamia(Risso) Wright Arn.)Essential Oil Aromatherapy on Mood States,Parasympathetic Nervous System Activity, and Salivary Cortisol Levels in 41 Healthy Females Eri Watanabea Kenny Kuchtab Mari Kimuraa Hans Wilhelm Rauwaldc Tsutomu Kameid Jiro Imanishie a Department of Immunology,Kyoto Prefectural University of Medicine,Kyoto,Japan; bNatural Products Chemistry Research,Department of Food and Nutrition,Sanyo Gakuen University-College Okayama,Okayama,Japan; c Department of Pharmaceutical Biology,Leipzig University,Leipzig,Germany; dCenter for Industry,University and Government Cooperation,Nagasaki University,Nagasaki,Japan; eMUIM Center for Integrative Medicine,Meiji University of Integrative Medicine Kyoto,Kyoto,Japan Keywords Schlusselworter Bergamot essential oil (BEO).Citrus bergamia(Risso) Atherisches Bergamotte-Ol.Citrus bergamia(Risso)Wright Arn.. Wright Arn..Psycho-endocrinological study.Salivary cortisol. Psychoendokrinologische Studie.Speichel-Cortisol.Sympathikus Sympathetic nervous system.Randomized crossover clinical trial Randomisierte Crossover-Studie Summary Zusammenfassung Background:Bergamot essential oil (BEO)is commonly used Hintergrund:Atherisches Bergamotte Ol wird im Rahmen der Aro- against psychological stress and anxiety in aromatherapy.The pri- matherapie gegen psychischen Stress und bei Angstzustanden ein- mary aim of the present study was to obtain first clinical evidence gesetzt.Das primare Ziel der vorliegenden Studie war es,erste klini- for these psychological and physiological effects.A secondary aim sche Belege dieser Effekte zu generieren.Ein weiteres Ziel bestand was to achieve some fundamental understanding of the relevant darin,die pharmakologischen Wirkprinzipien der daran beteiligten pharmacological processes.Methods:Endocrinological,physiolog- Prozesse zu erfassen.Material und Methoden:Die endokrinologi- ical,and psychological effects of BEO vapor inhalation on 41 schen,physiologischen und psychologischen Effekte der Dampfinha- healthy females were tested using a random crossover study de- lation von atherischem Bergamotte-Ol wurden in einer randomisier- sign.Volunteers were exposed to 3 experimental setups (rest(R), ten Crossover-Studie an 41 gesunden Frauen getestet.Die Proban- rest water vapor(RW),rest water vapor bergamot essential oil dinnen wurden in 3 Versuchsgruppen aufgeteilt:(Ruhe(R),Ruhe (RWB))for 15 min each.Immediately after each setup,saliva sam- Wasserdampf(RW),Ruhe Wasserdampf atherisches Bergamotte- ples were collected and the volunteers rested for 10 min.Subse- Ol(RWB)).Jede Sequenz dauerte 15 min.Unmittelbar nach jedem quently,they completed the Profile of Mood States,State-Trait Set wurden den Studienteilnehmerinnen Speichelproben entnom- Anxiety Inventory,and Fatigue Self-Check List.High-frequency(HF) men und eine 10-minutige Ruhephase verordnet.Im Anschluss heart rate values,an indicator for parasympathetic nervous system daran fullten die Testpersonen das Profile of Mood States,das State- activity,were calculated from heart rate variability values measured Trait-Angstinventar und die Fatigue Self-Check-Liste aus.Die hohen both during the 15 min of the experiment and during the subse Herzfrequenzwerte,die als Indikator fur die Parasympathikus-Aktivi- quent 10 min of rest.Salivary cortisol(CS)levels in the saliva sam tat gelten,wurden anhand der Herzfrequenzvariabilitatswerte kalku- ples were analyzed using ELISA.Results:CS of all 3 conditions R, liert und wahrend des 15-minutigen Experiments sowie der anschlie- RW,and RWB were found to be significantly distinct (p =0.003).In Benden 10-minutigen Ruhephase erhoben.Die Level des Speichel- the subsequent multiple comparison test,the CS value of RWB was Cortisols (CS)aus den Speichelproben wurden mittels ELISA analy- significantly lower when compared to the R setup.When compar- siert.Ergebnisse:Die CS in allen 3 Bedingungen(R,RW und RWB) ing the HF values of the RWB setup during the 10 min of rest after unterschieden sich signifikant voneinander (p =0,003).Der CS-Wert the experiment to those of RW,this parameter was significantly in- bei RWB erwies sich im multiplen Vergleichstest mit R als signifikant creased (p=0.026)in the RWB setup for which scores for negative niedriger.Der Vergleich der HF-Werte im RWB-Versuchsaufbau mit emotions and fatigue were also improved.Conclusion:These re- den RW-Werten wahrend der 10-minutigen Ruhephase zeigte einen sults demonstrate that BEO inhaled together with water vapor ex- signifikanten Anstieg dieses Parameters unter der RWB-Kondition erts psychological and physiological effects in a relatively short (p =0,026).Dabei verbesserten sich auch die Scores fur negative time. Stimmungen und Mudigkeit.Schlussfolgerung:Die Ergebnisse zei- gen,dass die kombinierte Inhalation von atherischem Bergamotte-Ol und Wasserdampf sowohl psychisch als auch physisch positive Ef- fekte innerhalb einer relativ kurzen Zeitspanne hervorruft. KARGER 2015 S.Karger GmbH,Freiburg Eri Watanabe,M.D. 1661-4119/15/0221-0043539.50/0 Department of Immunology Fax+497614520714 Kyoto Prefectural University of Medicine Information@Karger.com Accessible online at: 465 Kajii-cho.Kawaramachi-Hirokoji Kamigyo-ku,Kyoto 602-8566.Japan www.karger.com www.karger.com/fok -wkoto.kpu-m.ac.jp

Fax +49 761 4 52 07 14 Information@Karger.com www.karger.com Accessible online at: www.karger.com/fok Original Article · Originalarbeit Forsch Komplementmed 2015;22:43–49 DOI: 10.1159/000380989 Effects of Bergamot (Citrus bergamia (Risso) Wright & Arn.) Essential Oil Aromatherapy on Mood States, Parasympathetic Nervous System Activity, and Salivary Cortisol Levels in 41 Healthy Females Eri Watanabea Kenny Kuchtab Mari Kimuraa Hans Wilhelm Rauwaldc Tsutomu Kameid Jiro Imanishie a Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto, Japan; b Natural Products Chemistry Research, Department of Food and Nutrition, Sanyo Gakuen University-College Okayama, Okayama, Japan; c Department of Pharmaceutical Biology, Leipzig University, Leipzig, Germany; d Center for Industry, University and Government Cooperation, Nagasaki University, Nagasaki, Japan; e MUIM Center for Integrative Medicine, Meiji University of Integrative Medicine Kyoto, Kyoto, Japan Schlüsselwörter Ätherisches Bergamotte-Öl · Citrus bergamia (Risso) Wright & Arn. · Psychoendokrinologische Studie · Speichel-Cortisol · Sympathikus · Randomisierte Crossover-Studie Zusammenfassung Hintergrund: Ätherisches Bergamotte Öl wird im Rahmen der Aro￾matherapie gegen psychischen Stress und bei Angstzuständen ein￾gesetzt. Das primäre Ziel der vorliegenden Studie war es, erste klini￾sche Belege dieser Effekte zu generieren. Ein weiteres Ziel bestand darin, die pharmakologischen Wirkprinzipien der daran beteiligten Prozesse zu erfassen. Material und Methoden: Die endokrinologi￾schen, physiologischen und psychologischen Effekte der Dampfinha￾lation von ätherischem Bergamotte-Öl wurden in einer randomisier￾ten Crossover-Studie an 41 gesunden Frauen getestet. Die Proban￾dinnen wurden in 3 Versuchsgruppen aufgeteilt: (Ruhe (R), Ruhe + Wasserdampf (RW), Ruhe + Wasserdampf + ätherisches Bergamotte- Öl (RWB)). Jede Sequenz dauerte 15 min. Unmittelbar nach jedem Set wurden den Studienteilnehmerinnen Speichelproben entnom￾men und eine 10-minütige Ruhephase verordnet. Im Anschluss daran füllten die Testpersonen das Profile of Mood States, das State￾Trait-Angstinventar und die Fatigue Self-Check-Liste aus. Die hohen Herzfrequenzwerte, die als Indikator für die Parasympathikus-Aktivi￾tät gelten, wurden anhand der Herzfrequenzvariabilitätswerte kalku￾liert und während des 15-minütigen Experiments sowie der anschlie- ßenden 10-minütigen Ruhephase erhoben. Die Level des Speichel￾Cortisols (CS) aus den Speichelproben wurden mittels ELISA analy￾siert. Ergebnisse: Die CS in allen 3 Bedingungen (R, RW und RWB) unterschieden sich signifikant voneinander (p = 0,003). Der CS-Wert bei RWB erwies sich im multiplen Vergleichstest mit R als signifikant niedriger. Der Vergleich der HF-Werte im RWB-Versuchsaufbau mit den RW-Werten während der 10-minütigen Ruhephase zeigte einen signifikanten Anstieg dieses Parameters unter der RWB-Kondition (p = 0,026). Dabei verbesserten sich auch die Scores für negative Stimmungen und Müdigkeit. Schlussfolgerung: Die Ergebnisse zei￾gen, dass die kombinierte Inhalation von ätherischem Bergamotte-Öl und Wasserdampf sowohl psychisch als auch physisch positive Ef￾fekte innerhalb einer relativ kurzen Zeitspanne hervorruft. Keywords Bergamot essential oil (BEO) · Citrus bergamia (Risso) Wright & Arn. · Psycho-endocrinological study · Salivary cortisol · Sympathetic nervous system · Randomized crossover clinical trial Summary Background: Bergamot essential oil (BEO) is commonly used against psychological stress and anxiety in aromatherapy. The pri￾mary aim of the present study was to obtain first clinical evidence for these psychological and physiological effects. A secondary aim was to achieve some fundamental understanding of the relevant pharmacological processes. Methods: Endocrinological, physiolog￾ical, and psychological effects of BEO vapor inhalation on 41 healthy females were tested using a random crossover study de￾sign. Volunteers were exposed to 3 experimental setups (rest (R), rest + water vapor (RW), rest + water vapor + bergamot essential oil (RWB)) for 15 min each. Immediately after each setup, saliva sam￾ples were collected and the volunteers rested for 10 min. Subse￾quently, they completed the Profile of Mood States, State-Trait Anxiety Inventory, and Fatigue Self-Check List. High-frequency (HF) heart rate values, an indicator for parasympathetic nervous system activity, were calculated from heart rate variability values measured both during the 15 min of the experiment and during the subse￾quent 10 min of rest. Salivary cortisol (CS) levels in the saliva sam￾ples were analyzed using ELISA. Results: CS of all 3 conditions R, RW, and RWB were found to be significantly distinct (p = 0.003). In the subsequent multiple comparison test, the CS value of RWB was significantly lower when compared to the R setup. When compar￾ing the HF values of the RWB setup during the 10 min of rest after the experiment to those of RW, this parameter was significantly in￾creased (p = 0.026) in the RWB setup for which scores for negative emotions and fatigue were also improved. Conclusion: These re￾sults demonstrate that BEO inhaled together with water vapor ex￾erts psychological and physiological effects in a relatively short time. Published online: February 19, 2015 Eri Watanabe, M.D. Department of Immunology Kyoto Prefectural University of Medicine 465 Kajii-cho, Kawaramachi-Hirokoji Kamigyo-ku, Kyoto 602-8566, Japan e-w@koto.kpu-m.ac.jp © 2015 S. Karger GmbH, Freiburg 1661–4119/15/0221–0043$39.50/0 Downloaded by: Jiaotong University 218.193.183.57 - 4/28/2015 7:01:25 AM

Introduction In the present randomized crossover study,41 healthy young adult women were given bergamot volatile oil-saturated water Bergamot (Citrus bergamia (Risso)Wright Arn.;syn.Citrus vapor to inhale,while their CS level was measured repeatedly.Both aurantium L.var.bergamia Loisel.),which belongs to the Citreae heart rate variability as an indicator for the activity of the autono- tribe in the Aurantioideae subfamily of the Rutaceae plant family, mous nervous system and emotional state were monitored. originates from the Mediterranean ecoregion,particularly from southern Italy and Greece.Its volatile oil,which is produced from the exocarp by means of cold pressing [1],is in high demand for a Material and Methods wide range of perfumes,cosmetics,and especially for aromather- apy[2,3. Study Participants Originally,42 healthy female graduate and undergraduate university stu- According to recent research in a combination of fields,such as dents,all between 20 and 23 years old and with an average age of 21.3+1.02 phytochemical analysis,pharmacology,and psychology,a variety years,were recruited for the present human trail.This number was based more of volatile oils have specific neuropharmacological effects [4].The on financial than on statistical considerations as we tried to recruit as many individual volatile oil compounds are detected by the nervus olfac- participants for the study as our budget did allow.As one of the recruited vol. torius on the back of the nasal cavity,which carries more than unteers did not show up on the day of the experiment,the study was carried out with the remaining 41 subjects.In order to prevent influences of age or gender 1,000 kinds of receptors and is directly connected via the intracra- on the data,age and gender of the test subjects had been equalized,and also nial olfactory bulb to the limbic system in the hypothalamus and their general living conditions as they are relatively similar among university thus to the autonomic nervous system.Accordingly,effects on the students.The experimental protocols were approved by the only currently es. endocrine and immune systems have been demonstrated [5].Fur- tablished Ethics Committee of the Kyoto Prefectural University of Medicine in thermore,volatile oil components inhaled during aromatherapy full accordance with the Declaration of Helsinki in the most recent revised and amended version of 2013. can pass from the alveoli into the capillary blood vessels [5].Even if All test subjects were informed in detail about the purpose of the experi- rubbed onto the skin,they can enter through the subcutaneous tis- ment and about the overall experimental setup.After receiving detailed infor- sue into the capillaries and pass the blood-brain barrier with the mation,such as a plan of the intended study similar to table 1,all recruited indi- bloodstream,thus affecting the entire central nervous system [5]. viduals manifested their intent to participate in the study by signing the respec- Volatile oils were shown to be especially effective in the therapy of tive test consent forms Each participant gave written informed consent and completed a medical chronic pain [6,7],depression [8,9],cognitive disorders [10,11], health questionnaire prior to participation.Before the study,the volunteers had insomnia [12],and stress-related ailments [13].They were further to fill out a questionnaire concerning sleep and wake-up time for the day of the demonstrated to exert antibacterial,antifungal,antiviral,and cyto- investigation itself,general health,mental stress,energy,time of their most re. toxic effects [14]. cent menstrual period,if they had eaten breakfast or not,and when they had Bergamot volatile oil is commonly regarded as an effective tra- eaten for the most recent time to make sure that all of them were in normal mental and physical state. ditional pharmaceutical and medicinal product that was used for many centuries against a variety of conditions.Today,it is widely Study Design used in aromatherapy for reducing stress and anxiety [3].Accord- The study was designed as a randomized crossover trial.In order to elimi- ing to most recent research,it counteracts anxiety behavior in rats nate effects caused by the mere sequence in which the experiments were per- via the stress response pathway,reducing the activity of the hypo- formed,the 42 study participants had originally been divided into 6 groups, consisting of 7 volunteers each,so that every 7-person group could perform 1 of thalamus-pituitary-adrenocortical axis (HPA)[3].Furthermore, the 6 possible permutations of the 3 setups-rest (R),rest water vapor(RW), most recent studies using the forced swim and open field tests have and rest water vapor bergamot oil (RWB).All study participants were as- demonstrated the taxonomically identical TCM drug fructus au- signed to I of these groups using a random number table generated in Micro- rantii (Chin.Ph.)to exert significant antidepressant effects via the soft Excel.The same applied for the sequence in which the individual groups monoaminergic mechanism in rats [15].However,despite the took part in the experiment.The person who did not show up at the day of the experiment had originally been assigned to the RWB.R,RW permutation, common use of bergamot volatile oil water vapor,its psychological which was therefore performed by only 6 volunteers,whereas each of the 5 and endocrinological effects in humans have not been subject to other permutations was performed by groups consisting of 7 test subjects.The experimental investigation up to now. first group took part in the setups with R,RW,RWB;the next group in the se. When a stressor reaches the cerebral cortex,adrenocortico- quence RWB,R,RW;the next in the sequence RW,RWB,R;and so forth,until tropic hormone (ACTH)is released from the hypothalamus stimu- all 6 groups had finished their respective permutation.Each participant took part in the study only once,completing all 3 setups in a row.The study was lating the secretion of cortisol from the adrenal cortex [16.Based conducted with I group per day under exactly identical measuring conditions on this knowledge,we have already used salivary cortisol (CS)lev- for all parameters. els as an indicator for stress reduction in previous research pro- The test persons were exposed to the 3 study setups R,RW,and RWB for 15 jects,demonstrating that during emotional improvement after re- min each (table 1)followed by 2 min of rest for the collection of saliva samples, laxation cortisol levels are also reduced [17,18].In a preliminary 10 min for continued recording of the heart rate variability,and 10 min for fill. ing out the questionnaires.In order to limit interferences in the data that can be study related to the present project,these changes in the CS levels caused by physical exercise or nervousness,the test subjects took a rest of 15 of individual test subjects under bergamot aroma therapy were min before the start.The experiments took 3 h from the first explanations to the shown to be closely linked to their individual personalities and life- volunteer until the disconnection of the heart rate recording device.On each styles [19]. day of the study,work began at exactly 1:00 pm and proceeded in the fashion 44 Forsch Komplementmed 2015;22:43-49 Watanabe/Kuchta/Kimura/Rauwald/Kamei/ Imanishi

Watanabe/Kuchta/Kimura/Rauwald/Kamei/ Imanishi 44 Forsch Komplementmed 2015;22:43–49 Introduction Bergamot (Citrus bergamia (Risso) Wright & Arn.; syn. Citrus aurantium L. var. bergamia Loisel.), which belongs to the Citreae tribe in the Aurantioideae subfamily of the Rutaceae plant family, originates from the Mediterranean ecoregion, particularly from southern Italy and Greece. Its volatile oil, which is produced from the exocarp by means of cold pressing [1], is in high demand for a wide range of perfumes, cosmetics, and especially for aromather￾apy [2, 3]. According to recent research in a combination of fields, such as phytochemical analysis, pharmacology, and psychology, a variety of volatile oils have specific neuropharmacological effects [4]. The individual volatile oil compounds are detected by the nervus olfac￾torius on the back of the nasal cavity, which carries more than 1,000 kinds of receptors and is directly connected via the intracra￾nial olfactory bulb to the limbic system in the hypothalamus and thus to the autonomic nervous system. Accordingly, effects on the endocrine and immune systems have been demonstrated [5]. Fur￾thermore, volatile oil components inhaled during aromatherapy can pass from the alveoli into the capillary blood vessels [5]. Even if rubbed onto the skin, they can enter through the subcutaneous tis￾sue into the capillaries and pass the blood-brain barrier with the bloodstream, thus affecting the entire central nervous system [5]. Volatile oils were shown to be especially effective in the therapy of chronic pain [6, 7], depression [8, 9], cognitive disorders [10, 11], insomnia [12], and stress-related ailments [13]. They were further demonstrated to exert antibacterial, antifungal, antiviral, and cyto￾toxic effects [14]. Bergamot volatile oil is commonly regarded as an effective tra￾ditional pharmaceutical and medicinal product that was used for many centuries against a variety of conditions. Today, it is widely used in aromatherapy for reducing stress and anxiety [3]. Accord￾ing to most recent research, it counteracts anxiety behavior in rats via the stress response pathway, reducing the activity of the hypo￾thalamus-pituitary-adrenocortical axis (HPA) [3]. Furthermore, most recent studies using the forced swim and open field tests have demonstrated the taxonomically identical TCM drug fructus au￾rantii (Chin.Ph.) to exert significant antidepressant effects via the monoaminergic mechanism in rats [15]. However, despite the common use of bergamot volatile oil water vapor, its psychological and endocrinological effects in humans have not been subject to experimental investigation up to now. When a stressor reaches the cerebral cortex, adrenocortico￾tropic hormone (ACTH) is released from the hypothalamus stimu￾lating the secretion of cortisol from the adrenal cortex [16]. Based on this knowledge, we have already used salivary cortisol (CS) lev￾els as an indicator for stress reduction in previous research pro￾jects, demonstrating that during emotional improvement after re￾laxation cortisol levels are also reduced [17, 18]. In a preliminary study related to the present project, these changes in the CS levels of individual test subjects under bergamot aroma therapy were shown to be closely linked to their individual personalities and life￾styles [19]. In the present randomized crossover study, 41 healthy young adult women were given bergamot volatile oil-saturated water vapor to inhale, while their CS level was measured repeatedly. Both heart rate variability as an indicator for the activity of the autono￾mous nervous system and emotional state were monitored. Material and Methods Study Participants Originally, 42 healthy female graduate and undergraduate university stu￾dents, all between 20 and 23 years old and with an average age of 21.3 ± 1.02 years, were recruited for the present human trail. This number was based more on financial than on statistical considerations as we tried to recruit as many participants for the study as our budget did allow. As one of the recruited vol￾unteers did not show up on the day of the experiment, the study was carried out with the remaining 41 subjects. In order to prevent influences of age or gender on the data, age and gender of the test subjects had been equalized, and also their general living conditions as they are relatively similar among university students. The experimental protocols were approved by the only currently es￾tablished Ethics Committee of the Kyoto Prefectural University of Medicine in full accordance with the Declaration of Helsinki in the most recent revised and amended version of 2013. All test subjects were informed in detail about the purpose of the experi￾ment and about the overall experimental setup. After receiving detailed infor￾mation, such as a plan of the intended study similar to table 1, all recruited indi￾viduals manifested their intent to participate in the study by signing the respec￾tive test consent forms. Each participant gave written informed consent and completed a medical health questionnaire prior to participation. Before the study, the volunteers had to fill out a questionnaire concerning sleep and wake-up time for the day of the investigation itself, general health, mental stress, energy, time of their most re￾cent menstrual period, if they had eaten breakfast or not, and when they had eaten for the most recent time to make sure that all of them were in normal mental and physical state. Study Design The study was designed as a randomized crossover trial. In order to elimi￾nate effects caused by the mere sequence in which the experiments were per￾formed, the 42 study participants had originally been divided into 6 groups, consisting of 7 volunteers each, so that every 7-person group could perform 1 of the 6 possible permutations of the 3 setups – rest (R), rest + water vapor (RW), and rest + water vapor + bergamot oil (RWB). All study participants were as￾signed to 1 of these groups using a random number table generated in Micro￾soft Excel. The same applied for the sequence in which the individual groups took part in the experiment. The person who did not show up at the day of the experiment had originally been assigned to the RWB, R, RW permutation, which was therefore performed by only 6 volunteers, whereas each of the 5 other permutations was performed by groups consisting of 7 test subjects. The first group took part in the setups with R, RW, RWB; the next group in the se￾quence RWB, R, RW; the next in the sequence RW, RWB, R; and so forth, until all 6 groups had finished their respective permutation. Each participant took part in the study only once, completing all 3 setups in a row. The study was conducted with 1 group per day under exactly identical measuring conditions for all parameters. The test persons were exposed to the 3 study setups R, RW, and RWB for 15 min each (table 1) followed by 2 min of rest for the collection of saliva samples, 10 min for continued recording of the heart rate variability, and 10 min for fill￾ing out the questionnaires. In order to limit interferences in the data that can be caused by physical exercise or nervousness, the test subjects took a rest of 15 min before the start. The experiments took 3 h from the first explanations to the volunteer until the disconnection of the heart rate recording device. On each day of the study, work began at exactly 1:00 pm and proceeded in the fashion Downloaded by: Jiaotong University 218.193.183.57 - 4/28/2015 7:01:25 AM

Table 1.Schematic de- Psychoendocrinological Parameters piction of the experiment Sequence min procedure.The sequence ◇rest 15 Salivary Cortisol Level:Cortisol,which is one of the hormones of the adrenal in which the rest,rest+ ◇rest(R) 15 cortex,controls carbohydrate metabolism,fat metabolism,and protein metabo- water vapor,and rest ★saliva collection 2 lism.Although it is an essential hormone for the body,there are reports indicat- water vapor bergamot ◇rest 10 ing that this hormone is related to psychological stress:When we feel stress,its oil settings were per 10 concentration increases within a few minutes but only returns to the initial level formed was randomized rest water vapour(RW) 15 after a few hours [20].The isolated cortisol,which was measured in the present (6 permutations)in order research project,spreads from the blood into urine and saliva within seconds, to exclude an influence ★saliva collection 2 of the experimental se- ◇rest 10 with its concentration exceeding more than 90%of the original concentration in the blood [20]. quence on the study out- O questionnaire 10 During each permutation of R,RW,and RWB,all in all 3 saliva samples come.The distribution ◇rest+water vapour 15 were collected from each of the volunteers-1 after R,1 after RW,and 1 after of the 41 volunteers be- bergamot oil (RWB) tween the 6 groups and ★saliva collection 3 RWB.Sample collection was performed as follows:All volunteers collected a saliva sample in a plastic tube exactly 90 s after completion of the respective the order in which the ◇rest 10 setup.After collection,all samples were immediately stored at-20C.After groups participated in ◎questionnaire 10 defrosting at 37C,the samples were centrifuged at 3000 rpm for 15 min using the test were randomized a KUBOTA 7000 high-speed refrigerated centrifuge in order to remove impuri. using a random number measurement of heart rate variability, sequence generated in saliva collection,O completed questionnaire. ties and mucus.Thereafter,CS levels were determined using the Salivary Cor- tisol EIA Kit(Salimetrics,USA)for the enzyme-linked immunosorbent assay Microsoft Excel. (ELISA). described above.The test subjects were advised not to eat anything from 1 h Heart Rate Variability:The heart rate (R-R interval)was measured with a before the start of the experiment.The experimental schedule was conducted as GMS AC-301A instrument produced by GMS (Tokyo,Japan).Data were col- depicted in table 1. lected without interruption from the very beginning to the very end of the experi- During the experiment,the test subjects were required to sit on a chair,keep ment,using the analysis software MemCalc for Windows version 1.2 (GMS, their hands on a table in front of them,and their body in a stable position.They Tokyo,Japan)in maximum entropy mode for the analysis of heart rate variability were forbidden to talk,move,drink,eat,or do anything else that was not related to by spectral analysis of R-R interval variability,calculating the high-frequency the experiment.Also when resting,the volunteers had to keep sitting on the chair. (HF)components (0.15-0.4 Hz)as an indicator of the activity of the parasympa- The applied diffusor (Tree of Life,Tokyo-Shibuya,Japan)diffuses water vapor thetic system [21].These indicator values were calculated individually as average into the air using ultrasound waves.When volatile oils are inserted into the sys- values over the 15 min of each of the experimental setups as well as over the sub- tem,they are dispersed in the air together with the water.During the experiment, sequent 10 min of rest. the apparatus was placed on the table in front of the volunteers 30 cm away from The differences between HF values during setups and subsequent rests were the face.During the R setup,it was switched on with the tank being empty,in the also calculated.Furthermore,low-frequency (LF)components(0.04-0.15 Hz), RW setup the tank was filled with water(purified tap water using a common car- normalized LF power (LFn.u.),normalized HF power (HFn.u.)as well as the bon adsorption filter)so that only water vapor was produced.In the RWB setup, LF/HF ratio as an indicator of the activity of sympathetic nervous system were bergamot essential oil (BEO)was applied by giving 400 ul of BEO into the 400 ml recorded. tank of the diffusor,dispersing it in the air together with the water.This dosage of BEO was determined on the basis of previous preliminary test results [19]. Psychological Measurements Measuring conditions for room temperature and overall humidity were kept In order to evaluate the psychological condition of the volunteers at this constant at 22C and 50%using a conventional air conditioning system and specific time,psychological tests were conducted as described below in a 10- heating-type humidifier.Between the individual setups,all windows were min time frame directly after the end of each of the 3 measuring conditions. opened and the room vented with an industrial fan (Yamazen,45cm YKY-454) The used questionnaires were identical in all 3 conditions.The Profile of Mood until all participating experimenters unanimously judged the bergamot smell to State (POMS)assesses 6 mood subscales:tension-anxiety,depression,anger- have completely disappeared.Thereafter,the setups without bergamot oil vapor hostility,vigor,fatigue,and confusion.High vigor scores reflect good mood or were conducted. emotions,whilst in the other subscales low scores reflect good mood or emo- In the present experiment,the physiological and psychological parameters tions.The Total Mood Disturbance (TMD)score is computed by adding the 5 described below were analyzed in order to detect potential differences between negative subscale scores(tension-anxiety (T-A),depression(D),anger-hostility the 3 experimental setups. (A-H),fatigue(F),and confusion (C))and subtracting the vigour (V)score.In 1990,Yokoyama et al.[22]translated the 65-item scale POMS into Japanese Phytochemical Analysis of BEO (Kaneko Shobo Co.,Tokyo,Japan),and demonstrated the reliability and valid- BEO (Lot:LF1481211)was produced from original Italian bergamot fruits ity of the Japanese version of POMS in Japanese participants.This Japanese ver- and obtained via Laboratoire Sanoflore (Renens,Switzerland).BEO was charac- sion was used for the present study.The validated State-Trait Anxiety Inventory terized at the laboratory of Sanoflore using a Hewlett Packard GC-MS instru- (STAI)[23,24]was used to assess the response of the psychological stress val- ment with combined mass spectrometric and flame ionisation detectors to- ues on aroma treatment(STAI state).The STAI state subscales contain 20 items gether with an Agilent J&W HP-INNOWax polyethylene glycol(PEG)station- with subscale scores ranging from 20 to 80.In this score,higher scores indicate ary phase(ID 0.25 mm;length 60 m;film 0.50 um)and a temperature gradient increased anxiety.Using the Fatigue Self-Check List [25],which was put for- stating with 6 min at 50C,followed by an increase up to 250C at a rate of 2C ward by the Ministry of Education,Culture,Sports,Science and Technology of per min,and ending with a second stable phase for 10 min at 250C.Helium Japan for gathering information on consumer needs,10 items for mental fa- was chosen as vector gas and applied with a pressure of 23 psi.This measure- tigue and 10 items for physical fatigue(all in all 20 items)were evaluated by the ment revealed the tested brand of BEO to be composed of 45.45%limonene, volunteers themselves on a 5-step scale (0-4 points)to receive a numerical 23.10%linalyl acetate,8.05%y-terpinen,7.25%B-pinene,6.50%linalool,1.35% measurement of their subjective fatigue.The applied questionnaires were de- a-pinene,and 0.35%geranial (a huge variety of unidentified minor compounds signed for repeated use in a short time and have already been used in the same constitute the remaining 7.95%). way in several other clinical studies [26,27]. Human Trial on Bergamot Oil Aromatherapy Forsch Komplementmed 2015;22:43-49 45

Human Trial on Bergamot Oil Aromatherapy Forsch Komplementmed 2015;22:43–49 45 described above. The test subjects were advised not to eat anything from 1 h before the start of the experiment. The experimental schedule was conducted as depicted in table 1. During the experiment, the test subjects were required to sit on a chair, keep their hands on a table in front of them, and their body in a stable position. They were forbidden to talk, move, drink, eat, or do anything else that was not related to the experiment. Also when resting, the volunteers had to keep sitting on the chair. The applied diffusor (Tree of Life, Tokyo-Shibuya, Japan) diffuses water vapor into the air using ultrasound waves. When volatile oils are inserted into the sys￾tem, they are dispersed in the air together with the water. During the experiment, the apparatus was placed on the table in front of the volunteers 30 cm away from the face. During the R setup, it was switched on with the tank being empty, in the RW setup the tank was filled with water (purified tap water using a common car￾bon adsorption filter) so that only water vapor was produced. In the RWB setup, bergamot essential oil (BEO) was applied by giving 400 μl of BEO into the 400 ml tank of the diffusor, dispersing it in the air together with the water. This dosage of BEO was determined on the basis of previous preliminary test results [19]. Measuring conditions for room temperature and overall humidity were kept constant at 22 °C and 50% using a conventional air conditioning system and heating-type humidifier. Between the individual setups, all windows were opened and the room vented with an industrial fan (Yamazen, 45cm YKY-454) until all participating experimenters unanimously judged the bergamot smell to have completely disappeared. Thereafter, the setups without bergamot oil vapor were conducted. In the present experiment, the physiological and psychological parameters described below were analyzed in order to detect potential differences between the 3 experimental setups. Phytochemical Analysis of BEO BEO (Lot: LF1481211) was produced from original Italian bergamot fruits and obtained via Laboratoire Sanoflore (Renens, Switzerland). BEO was charac￾terized at the laboratory of Sanoflore using a Hewlett Packard GC-MS instru￾ment with combined mass spectrometric and flame ionisation detectors to￾gether with an Agilent J&W HP-INNOWax polyethylene glycol (PEG) station￾ary phase (ID 0.25 mm; length 60 m; film 0.50 μm) and a temperature gradient stating with 6 min at 50°C, followed by an increase up to 250°C at a rate of 2°C per min, and ending with a second stable phase for 10 min at 250°C. Helium was chosen as vector gas and applied with a pressure of 23 psi. This measure￾ment revealed the tested brand of BEO to be composed of 45.45% limonene, 23.10% linalyl acetate, 8.05% γ-terpinen, 7.25% β-pinene, 6.50% linalool, 1.35% α-pinene, and 0.35% geranial (a huge variety of unidentified minor compounds constitute the remaining 7.95%). Psychoendocrinological Parameters Salivary Cortisol Level: Cortisol, which is one of the hormones of the adrenal cortex, controls carbohydrate metabolism, fat metabolism, and protein metabo￾lism. Although it is an essential hormone for the body, there are reports indicat￾ing that this hormone is related to psychological stress: When we feel stress, its concentration increases within a few minutes but only returns to the initial level after a few hours [20]. The isolated cortisol, which was measured in the present research project, spreads from the blood into urine and saliva within seconds, with its concentration exceeding more than 90% of the original concentration in the blood [20]. During each permutation of R, RW, and RWB, all in all 3 saliva samples were collected from each of the volunteers – 1 after R, 1 after RW, and 1 after RWB. Sample collection was performed as follows: All volunteers collected a saliva sample in a plastic tube exactly 90 s after completion of the respective setup. After collection, all samples were immediately stored at –20 °C. After defrosting at 37°C, the samples were centrifuged at 3000 rpm for 15 min using a KUBOTA 7000 high-speed refrigerated centrifuge in order to remove impuri￾ties and mucus. Thereafter, CS levels were determined using the Salivary Cor￾tisol EIA Kit (Salimetrics, USA) for the enzyme-linked immunosorbent assay (ELISA). Heart Rate Variability: The heart rate (R-R interval) was measured with a GMS AC-301A instrument produced by GMS (Tokyo, Japan). Data were col￾lected without interruption from the very beginning to the very end of the experi￾ment, using the analysis software MemCalc for Windows version 1.2 (GMS, Tokyo, Japan) in maximum entropy mode for the analysis of heart rate variability by spectral analysis of R-R interval variability, calculating the high-frequency (HF) components (0.15–0.4 Hz) as an indicator of the activity of the parasympa￾thetic system [21]. These indicator values were calculated individually as average values over the 15 min of each of the experimental setups as well as over the sub￾sequent 10 min of rest. The differences between HF values during setups and subsequent rests were also calculated. Furthermore, low-frequency (LF) components (0.04–0.15 Hz), normalized LF power (LFn.u.), normalized HF power (HFn.u.) as well as the LF/HF ratio as an indicator of the activity of sympathetic nervous system were recorded. Psychological Measurements In order to evaluate the psychological condition of the volunteers at this specific time, psychological tests were conducted as described below in a 10- min time frame directly after the end of each of the 3 measuring conditions. The used questionnaires were identical in all 3 conditions. The Profile of Mood State (POMS) assesses 6 mood subscales: tension-anxiety, depression, anger￾hostility, vigor, fatigue, and confusion. High vigor scores reflect good mood or emotions, whilst in the other subscales low scores reflect good mood or emo￾tions. The Total Mood Disturbance (TMD) score is computed by adding the 5 negative subscale scores (tension-anxiety (T-A), depression (D), anger-hostility (A-H), fatigue (F), and confusion (C)) and subtracting the vigour (V) score. In 1990, Yokoyama et al. [22] translated the 65-item scale POMS into Japanese (Kaneko Shobo Co., Tokyo, Japan), and demonstrated the reliability and valid￾ity of the Japanese version of POMS in Japanese participants. This Japanese ver￾sion was used for the present study. The validated State-Trait Anxiety Inventory (STAI) [23, 24] was used to assess the response of the psychological stress val￾ues on aroma treatment (STAI state). The STAI state subscales contain 20 items with subscale scores ranging from 20 to 80. In this score, higher scores indicate increased anxiety. Using the Fatigue Self-Check List [25], which was put for￾ward by the Ministry of Education, Culture, Sports, Science and Technology of Japan for gathering information on consumer needs, 10 items for mental fa￾tigue and 10 items for physical fatigue (all in all 20 items) were evaluated by the volunteers themselves on a 5-step scale (0–4 points) to receive a numerical measurement of their subjective fatigue. The applied questionnaires were de￾signed for repeated use in a short time and have already been used in the same way in several other clinical studies [26, 27]. Sequence min ۍ rest 15 ۍ rest(R) 15 ۻ saliva collection 2 ۍ rest 10 ۔ questionnaire 10 ۍ rest + water vapour (RW) 15 ۻ saliva collection 2 ۍ rest 10 ۔ questionnaire 10 ۍ rest + water vapour + bergamot oil (RWB) 15 ۻ saliva collection 2 ۍ rest 10 ۔ questionnaire 10 ۍ measurement of heart rate variability, ۻ saliva collection, ۔ completed questionnaire. Table 1. Schematic de￾piction of the experiment procedure. The sequence in which the rest, rest + water vapor, and rest + water vapor + bergamot oil settings were per￾formed was randomized (6 permutations) in order to exclude an influence of the experimental se￾quence on the study out￾come. The distribution of the 41 volunteers be￾tween the 6 groups and the order in which the groups participated in the test were randomized using a random number sequence generated in Microsoft Excel. Downloaded by: Jiaotong University 218.193.183.57 - 4/28/2015 7:01:25 AM

Statistical Analysis POMS In order to understand if there is a statistically significant difference be- When comparing the 3 measured setups,significant difference tween the 3 conditions R,RW,and RWB,the one-way Repeated Measures could be seen in the item scores T-A (F 3.863,p =0.025),A-H Analysis of Variance(rANOVA)was used.When comparing R and RW,R and RWB,and RW and RWB,the multiple comparison analysis according to Bon- (F=3.238,p=0.044),V(F=17.838,p<0.001,F(F=21.389, ferroni was applied.The differences were regarded as significant at 25%and P<0.001,C(F=6.479,p=0.002),and TMD(F=16.274, calculated using the SPSS version 15.0 statistics software.A comparison be- p<0.001).In the subsequent multiscale analysis,differences were tween the 3 groups(R,RW,RWB)was performed,and the null hypothesis con- found especially between the scores of the R and RWB setups for T-A stituted a plurality.The more commonly applied t-test,which is typically used for the comparison of2 groups,could not be applied. (p=0.040),V(p<0.001),F(p<0.001),C(p=0.008),and TMD (p<0.001)with the latter values indicating that negative mood of the study participants has decreased.Furthermore,in the case of V,a sig- Results nificant difference could also be detected between the RW and RWB condition(p=0.049),demonstrating that the test subjects'vigor Changes in the Salivary Cortisol Level between the R,RW,and could indeed be significantly increased by the pharmacological action RWB Setup of the volatile oil as opposed to the effect of water vapor alone.There For the average CS levels of the 41 volunteers after the setups R, were also significant differences between the R and RW setups for the RW,and RWB,see table 2.In the rANOVA test,significant dif- items V(p=0.001),F(p<0.001),and TMD(p=0.001). ferences between the setups R,RW,and RWB could be found (F=6.090,p=0.003).In the subsequent multiple comparison,sig- STAI nificant differences in CS levels could be detected comparing R+ The STAI scores were different for all 3 measured setups RW setup(p=0.049)and R+RWB setup(p=0.004).The average (F=9.969,p<0.001).In the subsequent multiscale analysis,this CS level decreased from the highest values at R,followed by RW,to difference was significant for both the comparisons of R and RWB the lowest values at RWB.It could be demonstrated that these dif- (p =0.001)as well as RW and RWB(p <0.001).The average values ferences were independent from the sequence in which the experi- decreased in the sequence from the highest values for R,to the ments were performed. lower RW,and finally the lowest RWB values. Heart Rate Variability Fatigue Self-Check List Performing a spectral analysis with the heart rate analysis soft- Concerning both physical fatigue (F=3.124,p=0.049)and psy- ware MemCalc,the HF component,which is an indicator for the chological fatigue (F 3.259,p 0.044),all 3 conditions gave function of the parasympathetic nervous system,and the LF/HF ra- diverting results.In the subsequent multiscale analysis,a signifi- tion,which is an indicator of the sympathetic nervous system,could cant difference between the R and RWB condition (p=0.041)was be calculated (table 2).During the 15 min of measurement for each detected.For both physical and psychological fatigue the values condition as well as during the 10 min of rest afterwards,average decreased from the highest values for R,to RW,and finally to the heart rate variability was calculated and compared between R,RW, lowest values for RWB and RWB.As far as HF is concerned,no significant differences could be detected in any condition during the 15 min of exposure.How- Side Effects ever,during the 10 min of rest,the comparison of the HF between None of the study participants did report any kid of side effect, the 3 setups showed significant differences(F=6.176,p=0.003).In such as cough,during the present study. the subsequent multiple comparisons,the HF values measured dur- ing the 10 min of rest after the RW and RWB setups were signifi- cantly different(p=0.026).The average HF value decreased in the Discussion sequence RWB,RW,to R showing the lowest values.As far as the LF/ HF ratio is concerned,no differences between any of the 3 setups In a randomized crossover clinical trial,41 healthy adult women could be found.The LF components measured during the 15 min of were exposed to the 3 testing conditions R,RW,and RWB for the setups did not display any significant effects in the subsequent 15 min each,elucidating significant differences between the 3 multiple comparisons.However,during the last 10 min of the setups compared setups concerning CS,HF components of heart rate var- R and RWB significant effects(p =0.048)could be observed. iability as well as psychological mood indicators,such as anxiety For the other measured parameters,such as LFn.u.,HFn.u.,and and fatigue.When inhaling BEO dissolved in water vapor,CS was LF/HF ratio,no significant differences between any of the 3 setups significantly reduced and the HF component of the heart rate vari- could be found. ability,which is an indicator for the function of the parasympa- thetic nervous system,significantly increased. Psychological Measurements However,whilst an effect on the CS level could be shown,this dif- Immediately after each of the 3 setups was measured,the volun- ference was observed only between the R and RW setups as well as teers completed a questionnaire concerning their mood,feelings, between the R and RWB setups,but unexpectedly not between the anxiety,and fatigue.The results are shown in table 2. RW and RWB setups.Another surprising observation was that the HF Forsch Komplementmed 2015;22:43-49 Watanabe/Kuchta/Kimura/Rauwald/Kamei/ Imanishi

Watanabe/Kuchta/Kimura/Rauwald/Kamei/ Imanishi 46 Forsch Komplementmed 2015;22:43–49 Statistical Analysis In order to understand if there is a statistically significant difference be￾tween the 3 conditions R, RW, and RWB, the one-way Repeated Measures Analysis of Variance (rANOVA) was used. When comparing R and RW, R and RWB, and RW and RWB, the multiple comparison analysis according to Bon￾ferroni was applied. The differences were regarded as significant at ˰5% and calculated using the SPSS version 15.0 statistics software. A comparison be￾tween the 3 groups (R, RW, RWB) was performed, and the null hypothesis con￾stituted a plurality. The more commonly applied t-test, which is typically used for the comparison of 2 groups, could not be applied. Results Changes in the Salivary Cortisol Level between the R, RW, and RWB Setup For the average CS levels of the 41 volunteers after the setups R, RW, and RWB, see table  2. In the rANOVA test, significant dif￾ferences between the setups R, RW, and RWB could be found (F = 6.090, p = 0.003). In the subsequent multiple comparison, sig￾nificant differences in CS levels could be detected comparing R + RW setup (p = 0.049) and R + RWB setup (p = 0.004). The average CS level decreased from the highest values at R, followed by RW, to the lowest values at RWB. It could be demonstrated that these dif￾ferences were independent from the sequence in which the experi￾ments were performed. Heart Rate Variability Performing a spectral analysis with the heart rate analysis soft￾ware MemCalc, the HF component, which is an indicator for the function of the parasympathetic nervous system, and the LF/HF ra￾tion, which is an indicator of the sympathetic nervous system, could be calculated (table 2). During the 15 min of measurement for each condition as well as during the 10 min of rest afterwards, average heart rate variability was calculated and compared between R, RW, and RWB. As far as HF is concerned, no significant differences could be detected in any condition during the 15 min of exposure. How￾ever, during the 10 min of rest, the comparison of the HF between the 3 setups showed significant differences (F = 6.176, p = 0.003). In the subsequent multiple comparisons, the HF values measured dur￾ing the 10 min of rest after the RW and RWB setups were signifi￾cantly different (p = 0.026). The average HF value decreased in the sequence RWB, RW, to R showing the lowest values. As far as the LF/ HF ratio is concerned, no differences between any of the 3 setups could be found. The LF components measured during the 15 min of the setups did not display any significant effects in the subsequent multiple comparisons. However, during the last 10 min of the setups R and RWB significant effects (p = 0.048) could be observed. For the other measured parameters, such as LFn.u., HFn.u., and LF/HF ratio, no significant differences between any of the 3 setups could be found. Psychological Measurements Immediately after each of the 3 setups was measured, the volun￾teers completed a questionnaire concerning their mood, feelings, anxiety, and fatigue. The results are shown in table 2. POMS When comparing the 3 measured setups, significant difference could be seen in the item scores T-A (F = 3.863, p = 0.025), A-H (F = 3.238, p = 0.044), V (F = 17.838, p < 0.001), F (F = 21.389, p < 0.001), C (F = 6.479, p = 0.002), and TMD (F = 16.274, p < 0.001). In the subsequent multiscale analysis, differences were found especially between the scores of the R and RWB setups for T-A (p = 0.040), V (p < 0.001), F (p < 0.001), C (p = 0.008), and TMD (p < 0.001) with the latter values indicating that negative mood of the study participants has decreased. Furthermore, in the case of V, a sig￾nificant difference could also be detected between the RW and RWB condition (p = 0.049), demonstrating that the test subjects’ vigor could indeed be significantly increased by the pharmacological action of the volatile oil as opposed to the effect of water vapor alone. There were also significant differences between the R and RW setups for the items V (p = 0.001), F (p < 0.001), and TMD (p = 0.001). STAI The STAI scores were different for all 3 measured setups (F = 9.969, p < 0.001). In the subsequent multiscale analysis, this dif ference was significant for both the comparisons of R and RWB (p = 0.001) as well as RW and RWB (p < 0.001). The average values decreased in the sequence from the highest values for R, to the lower RW, and finally the lowest RWB values. Fatigue Self-Check List Concerning both physical fatigue (F = 3.124, p = 0.049) and psy￾chological fatigue (F = 3.259, p = 0.044), all 3 conditions gave diverting results. In the subsequent multiscale analysis, a signifi￾cant difference between the R and RWB condition (p = 0.041) was detected. For both physical and psychological fatigue the values decreased from the highest values for R, to RW, and finally to the lowest values for RWB. Side Effects None of the study participants did report any kid of side effect, such as cough, during the present study. Discussion In a randomized crossover clinical trial, 41 healthy adult women were exposed to the 3 testing conditions R, RW, and RWB for 15 min each, elucidating significant differences between the 3 compared setups concerning CS, HF components of heart rate var￾iability as well as psychological mood indicators, such as anxiety and fatigue. When inhaling BEO dissolved in water vapor, CS was significantly reduced and the HF component of the heart rate vari￾ability, which is an indicator for the function of the parasympa￾thetic nervous system, significantly increased. However, whilst an effect on the CS level could be shown, this dif￾ference was observed only between the R and RW setups as well as between the R and RWB setups, but unexpectedly not between the RW and RWB setups. Another surprising observation was that the HF Downloaded by: Jiaotong University 218.193.183.57 - 4/28/2015 7:01:25 AM

Table 2.Statistics for salivary cortisol,high frequency and low frequency/high frequency components,mood,anxiety,and fatigue after rest,rest +vapor,and rest vapor bergamot oil (n=41) Repeated ANOVA Bonferroni multiple test Parameter Mean±SE F(R) p value P value RW RWB (RW) (RWB) (R)- (R)- (RW)- (RW) (RWB) (RWB) Cortisol CS level 0.16±0.01 0.13±0.01 0.12±0.01 6.09 0.003 0.049 0.004 n.s. HF(ms2) setting a 630.92±110.15 499.34±61.51 589.13±105.32 1.395 n.s. n.s. n.s. n.s. setting b 600.27±112.18 557.19±91.58 534.76±101.75 0.914 n.s. n.s. n.s. n.s. resta 475.71±61.62 456.44±56.50 540.94±74.11 6.176 0.003 n.s. 0.067 0.026 settingc -37.66±52.04 -35.64±47.57 3.51±72.16 0.822 n.s. n.s. n.s. n.s. Setting d -20.52±52.04 -19.22±47.57 -1328±72.16 0.028 n.s. n.s. n.s. n.s. HFn.u. Settinga 28.87±1.74 30.21±2.12 30.23±2.02 0.484 n.s. n.s. n.s. n.s. Setting b 28.54±1.77 29.99±2.29 29.89±1.94 0.248 n.s. n.s. n.s. n.s. rest a 29.21±2.10 27.95±1.92 27.65±1.88 0.619 n.s. n.s. n.s. n.s. setting c 0.93±1.51 -1.87±1.68 -2.05±1.40 1.154 n.s. n.s. n.s. n.s. setting d 1.06±1.58 -1.88±1.76 -2.23±1.43 1.229 n.s. n.s. n.s. n.s. LF(ms2) setting a 1242.33±116.40 1213.50±122.24 1109.86±104.13 1.552 n.s. n.s. n.s. n.s. setting b 1259.02±116.46 1226.79±133.75 1085.81±104.54 1.027 n.s. n.s. 0.048 n.s. rest a 1122.48±100.80 1158.82±83.48 1141.16±91.18 0.012 n.s. n.s. n.s. n.s. setting c -56.03±101.47 -54.68±90.15 31.30±92.95 0.754 n.s. n.s. n.s. n.s. setting d -73.15±103.19 -67.96±101.02 55.35±95.91 1.059 n.s. n.s. n.s. n.s. LFn.u setting a 71.13±1.74 69.79±2.12 69.03±2.11 0.484 n.s. n.s. n.s. n.s. setting b 71.46±1.77 70.01±2.29 70.11±1.94 0.436 n.s. n.s. n.s. n.s. rest a 70.79±2.10 72.00±1.92 71.99±1.88 0.619 n.s. n.s. n.s. n.s. setting c -0.93±1.51 2.21±1.68 2.22±1.40 1.154 n.s. n.s. n.s. n.s. settingd -1.06±1.58 2.22±1.76 2.40±1.43 1.229 n.s. n.s. n.s. n.s. LF/HF setting a 3.53±0.41 3.43±0.46 3.72±0.63 0177 n.s n.s. n.s. s. setting b 3.59±0.37 3.79±0.49 3.53±0.43 0200 n.s. ns. n.s. ns rest 3.60±037 3.72±0.36 3.66±0.51 0.109 n.s. n.s. n.s. n.s. setting a -0.06±0.24 0.12±0.38 0.14±0.31 0.097 n.s. n.s. n.s. n.s. setting b -0.16±024 0.00±0.44 0.12±0.33 0.097 n.s. n.s. n.s. n.s. POMS(mood) T-A 5.97±0.65 4.90±0.63 4.51±0.56 3.863 0.025 n.s. 0.040 n.s. D 4.95±1.19 4.09±1.13 3.68±1.01 2.339 0.097 n.s. n.s. n.s. A-H 1.68±0.44 1.12±0.32 0.78±0.24 3.238 0.044 n.s. 0.097 n.s. 4.95±0.61 8.14±1.03 10.97±0.90 17.838 <0.001 0.001 <0.001 0.049 E 7.82±0.73 4.78±0.71 3.39±0.55 21.389 <0.001 <0.001 <0.001 n.s. C 6.60±0.51 6.04±0.54 5.14±0.49 6.479 0.002 n.s. 0.008 n.S. TMD 22.10±3.02 12.80±3.39 6.53±2.82 16.274 <0.001 0.001 <0.001 n.s. Anxiety STAI 41.36±1.19 37.12±1.32 35.60±1.32 9.969 <0.001 0.001 0.001 n.s. Fatigue body 4.04±0.64 3.48±0.50 3.07±0.53 3124 0.049 n.s. 0.041 n.s. mind 7.82±0.97 7.75±0.95 6.21±0.79 3.259 0.044 n.s. n.s. n.s. body+ 1187±1.54 11.24±136 9.29±1.20 3571 0.033 n.s. 0.048 n.s. mind p<0.050. Setting a:15 min;setting b:10 min,rest a:10 min rest after setting:setting c:rest a-setting a;setting d:rest a-setting b. SE=standard error;R=rest;RW=rest vapor;RWB=rest vapor +bergamot oil;salivary cortisol;n.s.not significant;HF=high frequency components; LF/HF=low frequency/high frequency components;HFn.u.normalized HF power;LFn.u.=normalized LF power;POMS=Profile of Mood State; TMD Total Mood Disturbance;STAI=State-Trait Anxiety Inventory. Negative subscale scores:T-A=tension-anxiety;D=depression;A-H=anger-hostility;V=vigor;F=fatigue;C=confusion. Human Trial on Bergamot Oil Aromatherapy Forsch Komplementmed 2015;22:43-49 47

Human Trial on Bergamot Oil Aromatherapy Forsch Komplementmed 2015;22:43–49 47 Table 2. Statistics for salivary cortisol, high frequency and low frequency/high frequency components, mood, anxiety, and fatigue after rest, rest + vapor, and rest + vapor + bergamot oil (n = 41) Repeated ANOVA Bonferroni multiple test Parameter Mean ± SE F (R) p value p value R RW RWB (RW) (RWB) (R) - (RW) (R) - (RWB) (RW) - (RWB) Cortisol CS level 0.16 ± 0.01 0.13 ± 0.01 0.12 ± 0.01 6.09 0.003* 0.049* 0.004* n.s. HF (ms2 ) setting a 630.92 ± 110.15 499.34 ± 61.51 589.13 ± 105.32 1.395 n.s. n.s. n.s. n.s. setting b 600.27 ± 112.18 557.19 ± 91.58 534.76 ± 101.75 0.914 n.s. n.s. n.s. n.s. rest a 475.71 ± 61.62 456.44 ± 56.50 540.94 ± 74.11 6.176 0.003* n.s. 0.067 0.026* setting c –37.66 ± 52.04 –35.64 ± 47.57 3.51 ± 72.16 0.822 n.s. n.s. n.s. n.s. Setting d –20.52 ± 52.04 –19.22 ± 47.57 –13.28 ± 72.16 0.028 n.s. n.s. n.s. n.s. HFn.u. Setting a 28.87 ± 1.74 30.21 ± 2.12 30.23 ± 2.02 0.484 n.s. n.s. n.s. n.s. Setting b 28.54 ± 1.77 29.99 ± 2.29 29.89 ± 1.94 0.248 n.s. n.s. n.s. n.s. rest a 29.21 ± 2.10 27.95 ± 1.92 27.65 ± 1.88 0.619 n.s. n.s. n.s. n.s. setting c 0.93 ± 1.51 –1.87 ± 1.68 –2.05 ± 1.40 1.154 n.s. n.s. n.s. n.s. setting d 1.06 ± 1.58 –1.88 ± 1.76 –2.23 ± 1.43 1.229 n.s. n.s. n.s. n.s. LF (ms2 ) setting a 1242.33 ± 116.40 1213.50 ± 122.24 1109.86 ± 104.13 1.552 n.s. n.s. n.s. n.s. setting b 1259.02 ± 116.46 1226.79 ± 133.75 1085.81 ± 104.54 1.027 n.s. n.s. 0.048* n.s. rest a 1122.48 ± 100.80 1158.82 ± 83.48 1141.16 ± 91.18 0.012 n.s. n.s. n.s. n.s. setting c –56.03 ± 101.47 –54.68 ± 90.15 31.30 ± 92.95 0.754 n.s. n.s. n.s. n.s. setting d –73.15 ± 103.19 –67.96 ± 101.02 55.35 ± 95.91 1.059 n.s. n.s. n.s. n.s. LFn.u. setting a 71.13 ± 1.74 69.79 ± 2.12 69.03 ± 2.11 0.484 n.s. n.s. n.s. n.s. setting b 71.46 ± 1.77 70.01 ± 2.29 70.11 ± 1.94 0.436 n.s. n.s. n.s. n.s. rest a 70.79 ± 2.10 72.00 ± 1.92 71.99 ± 1.88 0.619 n.s. n.s. n.s. n.s. setting c –0.93 ± 1.51 2.21 ± 1.68 2.22 ± 1.40 1.154 n.s. n.s. n.s. n.s. setting d –1.06 ± 1.58 2.22 ± 1.76 2.40 ± 1.43 1.229 n.s. n.s. n.s. n.s. LF/HF setting a 3.53 ± 0.41 3.43 ± 0.46 3.72 ± 0.63 0.177 n.s. n.s. n.s. n.s. setting b 3.59 ± 0.37 3.79 ± 0.49 3.53 ± 0.43 0.200 n.s. n.s. n.s. n.s. rest 3.60 ± 0.37 3.72 ± 0.36 3.66 ± 0.51 0.109 n.s. n.s. n.s. n.s. setting a –0.06 ± 0.24 0.12 ± 0.38 0.14 ± 0.31 0.097 n.s. n.s. n.s. n.s. setting b –0.16 ± 0.24 0.00 ± 0.44 0.12 ± 0.33 0.097 n.s. n.s. n.s. n.s. POMS (mood) T-A 5.97 ± 0.65 4.90 ± 0.63 4.51 ± 0.56 3.863 0.025* n.s. 0.040* n.s. D 4.95 ± 1.19 4.09 ± 1.13 3.68 ± 1.01 2.339 0.097 n.s. n.s. n.s. A-H 1.68 ± 0.44 1.12 ± 0.32 0.78 ± 0.24 3.238 0.044* n.s. 0.097 n.s. V 4.95 ± 0.61 8.14 ± 1.03 10.97 ± 0.90 17.838 <0.001* 0.001* <0.001* 0.049* F 7.82 ± 0.73 4.78 ± 0.71 3.39 ± 0.55 21.389 <0.001* <0.001* <0.001* n.s. C 6.60 ± 0.51 6.04 ± 0.54 5.14 ± 0.49 6.479 0.002* n.s. 0.008* n.s. TMD 22.10 ± 3.02 12.80 ± 3.39 6.53 ± 2.82 16.274 <0.001* 0.001* <0.001* n.s. Anxiety STAI 41.36 ± 1.19 37.12 ± 1.32 35.60 ± 1.32 9.969 <0.001 0.001 0.001 n.s. Fatigue body 4.04 ± 0.64 3.48 ± 0.50 3.07 ± 0.53 3.124 0.049 n.s. 0.041 n.s. mind 7.82 ± 0.97 7.75 ± 0.95 6.21 ± 0.79 3.259 0.044 n.s. n.s. n.s. body + mind 11.87 ± 1.54 11.24 ± 1.36 9.29 ± 1.20 3.571 0.033 n.s. 0.048 n.s. * p < 0.050. Setting a: 15 min; setting b: 10 min, rest a: 10 min rest after setting; setting c: rest a – setting a; setting d: rest a – setting b. SE = standard error; R = rest; RW = rest + vapor; RWB = rest + vapor + bergamot oil; salivary cortisol; n.s. = not significant; HF = high frequency components; LF/HF = low frequency/high frequency components; HFn.u. = normalized HF power; LFn.u. = normalized LF power; POMS = Profile of Mood State; TMD = Total Mood Disturbance; STAI = State-Trait Anxiety Inventory. Negative subscale scores: T-A = tension-anxiety; D = depression; A-H = anger-hostility; V = vigor; F = fatigue; C = confusion. Downloaded by: Jiaotong University 218.193.183.57 - 4/28/2015 7:01:25 AM