Chirality Influences the Effects of Linalool on Physiological Parameters of Stress

• Abstract
• Introduction
• Materials and Methods
• Fragrance inhalation
• Subjects
• Experimental procedure
• Determined parameters
• Data and statistical analyses
• Supporting information
• Results
• Discussion
• Acknowledgements
• References

Abstract

The specific physiological responses induced by inhaling R-(-)- as well as S-(+)-linalool in 24 human subjects undergoing experimental stress were investigated in this study. Various physiological parameters of the autonomous nervous system (heart rate, blood pressure, electrodermal activity) as well as the endocrine system (salivary cortisol) were monitored. The study clearly indicated that odorants can modulate salivary cortisol levels, with both linalool enantiomers exerting relaxing effects. Concerning blood pressure and heart rate, S-(+)-linalool acted as an activating agent in contrast to electrodermal activity. R-(-)-linalool proved to be stress-relieving as determined by heart rate. In conclusion, the results revealed that (1) chirality crucially influences the physiological effects of odorants and that (2) odorants may act differently on certain physiological parameters.

Introduction

Sedative and stress-relieving properties have been attributed to various essential oils (EOs) traditionally used in phytotherapy. Consequently, several studies have shown that odorant inhalation exhibits physiological effects in mammals as well as humans. For instance, inhaling fragrant substances significantly attenuated hypothalamus-pituitary-adrenal (HPA) activity as indicated by salivary cortisol (SC) levels [1] as well as blood pressure (BP) [2] following different types of stressors which is in accord with the existence of a close functional connection between the olfactory system and the hypothalamus [3].

The odorants chosen for the present investigation were the enantiomers of linalool, a major EO component of plants traditionally used as sedative and anxiolytic agents as well as for prophylaxis and intermission of epileptic seizures. It has dose-dependent sedative effects on the central nervous system (CNS), among which are hypnotic, anticonvulsive and hypothermic activities, respectively [4]. Linalool exerts anticonvulsive effects by modulation of glutamatergic and GABAergic transmission [4], influencing GABA-mediated neuronal inhibition and potential effects on GABA liberation and reuptake. It significantly reduces glutamate liberation and uptake and is a competitive antagonist of L-glutamate binding [5]. Additionally, inhaling linalool affected norepinephrine and dopamine levels that decrease after stress due to accelerated turnover of catecholamines in the CNS; the catecholamine levels were restored to nearly normal. These observations may support that linalool inhibits the accelerated turnover of catecholamine metabolism in the CNS and indirectly suggest that the increase of dopaminergic or noradrenergic neuron activity via the inhalation of linalool may induce a concomitant decrease in ACTH levels in animals [6]. Moreover, R-(-)- in contrast to S-(+)-linalool shows sedative effects on beta waves of the electroencephalogram in human subjects exposed to pleasant environmental sounds [7] as well as on both autonomous nervous system (ANS) and mood states: It significantly decreased heart rate (HR) and increased a high-frequency component and produced calm and vigorous mood states in humans at rest, reducing tension, depression and hostility [8].

Therefore, the aim of the present study was to evaluate a possible stress-relieving potential of inhaling the naturally occurring monoterpene linalool on humans during a standardised stressor by determination of a variety ANS parameters and of SC reflecting endocrine activity. The influence of chirality on these parameters was of further interest.

Materials and Methods

Fragrance inhalation

R-(-)-Linalool (95 % [Art. no. 62 139]) was purchased from Sigma Aldrich Chemie GmbH (Steinheim, Germany). S-(+)-Linalool was provided by Kurt Kitzing GmbH (Wallerstein, Germany). The purity was 94.3 % for R-(-)- and 86.9 % for S-(+)-linalool, as determined by chiral chromatography. Ethanolic solutions of the substances were atomized in both experimental rooms. The applied concentrations were 2.7 mg/m³ for R-(-)- and 9.8 mg/m³ for S-(+)-linalool, equivalent to hundred-fold concentrations of the substances’ odour thresholds [9]. Odorants can affect physiological parameters such as patterns of EEG activity and subjective mood states without being consciously perceived [10]. Then again, the same can be stated for the suggestion of EO effects [11]. Therefore, the subjects were not told about being exposed to the odorants during the entire testing session to obviate influences of their subjective expectations.

Subjects

A collective of 24 subjects (age 26.59 ± 1.87 years, body mass index [BMI] 22.01 ± 1.59 kg/m²) participated and completed the study. Oral informed consent was obtained after briefing of the experimental protocol. Participation was voluntary, and the subjects could drop out at any time. Subjects were asked to abstain from eating, consuming low pH drinks and sportive exercise at least one hour prior to the beginning of the experimental session. They confirmed avoidance of caffeine and nicotine consumption and were not suffering from acute diseases at the time of investigation. Exclusion criteria were: age < 18 or > 35 years, abnormal body weight (BMI < 18.0 or > 25.0 kg/m²), chronic, particularly endocrine, diseases, allergies, consumption of nicotine and intake of medication. Female participants confirmed not to use hormonal contraceptives and to be in the luteal phase of their menstrual cycle at the experimental day, providing a reaction to the experimental stressor similar to that of males [12].

Experimental procedure

Two experimental groups and one control group, each consisting of four female and four male participants, were investigated. Each participant performed once a standardised stress task in an individual session starting at 2.30 p. m. and lasting about 90 minutes. The assignment to the experimental groups was randomised. Subjects sojourned in an isolated and quiet experimental room during the resting phase at the beginning, in the following preparation phase and in the completing recreation phase. As required by the experimental protocol, the testing phase took place in an adjacent room equipped with a tape recorder and a video camera. The stress task was conducted according to [13], except for minor modifications. Following a short explanatory introduction about the test procedure, the participant had to fill out a questionnaire assessing habitual parameters. The continuous recording of electrocardiogram (ECG) and electrodermal activity (EDA) was started. After a resting phase of ten minutes, a first saliva sample was taken and BP was measured. The subject was given a preparation period of ten minutes to prepare an oral ad-lib presentation lasting five minutes out of a scientific text. After ten minutes, a second saliva sample was gained and BP was determined. Upon entrance of the subject into the adjacent room, tape and video recording were started. Once the participant interrupted speech for more than twenty seconds, the psychologist asked detailed questions about the text or demanded that the participant lay out his/her personal opinion about this topic. The subject performed mental arithmetic for five minutes as a second part of the stress test. Back in the experimental room, BP was determined a third time. In intervals of ten minutes, another four saliva samples were collected and BP was measured three times. 15 minutes after beginning of the recreation phase, ECG and EDA recording was stopped. Finally, the participant was fully debriefed.

Determined parameters

Saliva sample collection and determination of SC by DELFIA® (Wallac Oy; Turku, Finland) were conducted as described [14]. BP was periodically measured on the non-dominant upper arm by means of an automatic BP monitor (Hartmann Digital HG-160; Paul Hartmann GmbH; Wiener Neudorf, Austria). ECG and EDA were continuously recorded using a PhysioLogger® (Rimkus Medizintechnik; Riemerling, Germany). Skin conductance reactivity (SCR) of the non-dominant palm was determined using non-polarising Ag/AgCl electrodes filled with electrode paste (Signa® Creme; Parker Laboratories; Orange, NJ, USA). Electrodes were placed on the thenar and hypothenar of the non-dominant hand. Sampling rate was 10/s. ECG was recorded using Ag/AgCl single-use electrodes (Schiller 30 EKG; Schiller Handels GmbH; Linz, Austria). Electrodes were placed between the third and fourth intercostal left and between fifth and sixth intercostal right, respectively. The reference electrode was placed on the middle of sternum. Sampling rate was 100/s.

Data and statistical analyses

SC levels were computed using SigmaPlot® 2000 (SPSS Inc.; Chicago, IL, USA, 1986 - 2000). All statistical analyses were performed with SPSS® (SPSS Inc.; Chicago, IL, USA, 1989 - 1999). Group differences were examined for statistical significance using the non-parametric one-tailed Mann-Whitney-U test. A significance level of p < 0.05 was used for all analyses.

Supporting information

Technical data of chiral chromatography and a chiral gas chromatogram of a sample of linalool distilled from the EO of Coriandrum sativum L. are presented in the Supporting Information.

Results

Fig. [1] illustrates means ± standard deviations of the response of SC, systolic (SBP) and diastolic blood pressure (DBP), HR and SCR to the experimental stressor in the R-(-)- and S-(+)-linalool groups compared to the control group.

The SC concentration peaked 10 minutes after testing in all experimental groups. Between resting and preparation period, SC increased conspicuously in the control group, whereas it hardly changed in the R-(-)- and S-(+)-linalool groups. The difference between the R-(-)-linalool and the control group was significant (p = 0.042). Consequently, between preparation period and 30 and 40 minutes of recreation, respectively, SC decreased in the control group unlike the other groups, resulting in significant differences to R-(-)- (p = 0.026/n. s.) and S-(+)-linalool groups (p = 0.007/0.043). The alleviated reaction of HPA to the stressor points to a slightly stress-relieving effect for both R-(-)- and S-(+)-linalool.

Maximum SBP was measured immediately after testing phase in all experimental groups. The increase of SBP between resting period and preparation as well as testing period was clearer in the S-(+)-linalool group compared to the other experimental groups, but not significant. Between testing period and after 10, 20 and 30 minutes of recreation, the decline of SBP in the S-(+)-linalool group was significantly greater than in the R-(-)-linalool (p = 0.042/0.007/0.02) and control groups (p = 0.013/0.027/0.031). The same can be stated for the decrease of SBP between preparation after 20 minutes of recreation (p = 0.034/0.044). The results indicate an activating effect for S-(+)-linalool whereas no differences between the R-(-)-linalool and the control group were observed. Maximum DBP was determined immediately after testing in all experimental groups. Comparing the values measured after 10 as well as 20 minutes of recreation to those after 30 minutes, the decrease of DBP was significantly greater in the S-(+)-linalool than in the control group (p = 0.035/0.046). Since the increase of DBP between resting and testing period is evident, although insignificant, an activating effect of S-(+)-linalool inhalation can be assumed. Results for R-(-)-linalool did not point to a certain effect on DBP.

The maximum HR values were determined during mental arithmetic in all experimental groups. Comparing the first 5 minutes of resting period to the first as well as the second 5 minutes of preparation periods, HR increased evidently greater in the S-(+)-linalool group than in the other experimental groups. Thereby, the differences between the linalool enantiomers were significant (p = 0.027/0.044). Then again, the decline of HR between the first 5 minutes of preparation as well as recreation period and the last five minutes of recreation period was more distinct in the S-(+)-linalool group than in the other experimental group, revealing significant differences to control group (p = 0.031/0.027). Comparing the first 5 minutes of resting to the first 5 minutes of the testing period, HR increase was significantly smaller in the R-(-)-linalool group than in the S-(+)-linalool as well as in the control group (p = 0.048 and p = 0.03). Whereas a distinct decrease of HR between the first 5 minutes of resting and recreation periods was observed in the R-(-)-linalool group, HR declined only slightly in the control group and increased in the S-(+)-linalool group, showing significant differences to the R-(-)-linalool group (p = 0.037/0.03). Thus, an activating effect of S-(+)-linalool on HR can be stated, whereas the opposite results were observed for R-(-)-linalool.

The maximum SCR was measured during oral presentation in the R-(-)-linalool as well in the control group, whereas in the S-(+)-linalool group it peaked during the second 5 minutes of the preparation period. The results for the R-(-)-linalool group did not significantly differ from the ones of the control group, except for a stronger decrease between the first and second 5 minutes of the resting phase (p = 0.026). Thus, results for R-(-)-linalool cannot be clearly attributed to a relaxing or activating effect. Concerning inhalation of S-(+)-linalool, the autonomous reaction as determined by SCR explicitly differed from the other experimental group. Between the first and second 5 minutes of the preparation period, SCR increased in the S-(+)-linalool group in contrast to the R-(-)-linalool and control groups (p = 0.028/0.01), thus providing evidence of an activating effect of S-(+)-linalool during the early phase of the stressor. Then again, comparing the second 5 minutes of the preparation to the testing period, SCR declined in the S-(+)-linalool group, whereas the converse results were observed in both the R-(-)-linalool (p = 0.008/0.003) and control groups (p = 0.008/0.005). The findings for the difference between the second 5 minutes of resting and testing periods were similar, however only the R-(-)- and S-(+)-linalool groups proved to be significantly different (p = 0.012). Concerning the comparison of the second 5 minutes of the preparation period to the recreation period, SCR decreased more markedly in the S-(+)-linalool group than in the R-(-)-linalool (p = 0.009/0.012/0.021) as well as in the control group (p = 0.049/0.041/0.035). In conclusion, S-(+)-linalool apparently acted as a stress-relieving agent during testing and recreation periods on SCR.

Discussion

In the present study, the modulating effects of inhalation of chiral monoterpenes on human stress reaction was investigated by assessing both endocrine and ANS parameters. The experimental stressor used, TSST, has been shown to significantly elicit HPA activation followed by significantly elevated levels of related hormones such as ACTH and cortisol and to increase ANS activity in a variety of studies. This study clearly confirmed former experiments by demonstrating significant reactions to the selected stressor. The results indicate that S-(+)-linalool has activating effects on BP and HR as well as on SCR during the preparation period; however, it acts conversely on SCR during the testing and recreation periods. On the contrary, R-(-) indeed exerted a sedating effect on HR but the effects on BP as well as on SCR were not markedly different from the control. HPA activity as indicated by SC levels was influenced by both R-(-)- and S-(+)-linalool in a stress-relieving way thus indicating that odorants exert actions on endocrine parameters. The results largely coincide with other studies [6], [7], evidencing principally relaxing effects for R-(-)-linalool as well as activating effects of S-(+)-linalool. However, responses provoked by linalool inhalation apparently also depend on the tasks presented to the subjects: for example when applied before and after mental work, R-(-)-linalool increased agitation and alertness as opposed to S-(+)-linalool [7], which is contradictory to the results of the present investigation. Generally, all studies investigating the enantiomers of linalool proved opposite responses to R-(-)- and S-(+)-linalool, probably on account of the significantly different odour impressions of the enantiomers [7].

The mechanisms of the effects of these fragrances on sympathetic and endocrine activity are not clearly known. Similar to drug molecules, chirality has a crucial influence not only on perceived odour impression but also on odour effects [15]. Effects of fragrance inhalation on ANS or HPA are possibly affected by mental and emotional conditions. The psychological responses to an odorant are also influenced by acquired experience and memory. Pleasant odour impressions conceivably induce relaxation and decrease sympathetic activity, contrary to unpleasant ones as indicated by the unfavourable appreciation of S-(+)-linalool accompanied by increased beta waves [7]. Another possibility is a pharmacological action of EO components, i. e., interaction with receptors mediating physiological responses. As formerly suggested [8], unperceivable odorants may have merely pharmacological effects. However, this theory is partially disproved by the concept of blind smell [10]. Probably, physiological effects of odorant substances can be assigned to both pharmacological actions of odour molecules and psychological influences. Thereby, the extent of these concepts contributing to odorant action may be vary due to applied concentrations and thus perceived odour intensity.

In conclusion, linalool revealed actions on physiological parameters when applied during an experimental stressor that were essentially dependent on chirality. Whether the effects of odorants on parameters selected for this study are influenced by odour quality or intensity and whether using topical application and thus excluding hedonic effects would give different results, should be proven by further experiments.

Acknowledgements

Thanks to Erich Schmidt from Kurt Kitzing GmbH for providing S-(+)-linalool and chiral GC data.

References

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• 15 Heuberger E, Hongratanaworakit T, Böhm C, Weber R, Buchbauer G. Effects of chiral fragrances on human autonomic nervous system parameters and self-evaluation.  Chem Senses. 2001;  26 281-92
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Dr. Martina Höferl

Department of Clinical Pharmacy and Diagnostics

Centre of Pharmacy

University of Vienna

Althanstrasse 14

1090 Wien

Austria

Phone: +43-1-4277-555-52

Fax: +43-1-4277-9551

Email: martina.hoeferl@univie.ac.at

References

• 1 Tsuchiya T, Inoue K, Tonida M, Hosoi J, Horii I, Shoji K. et al .Effects of odorant inhalation on plasma cortisol level in humans. Chem Senses 2002; 27 JASTS XXXV Abstracts: 168-9
  Search in Google Scholar
• 2 Nagai M, Wada M, Usui N, Tanaka A, Hasebe Y. Pleasant odors attenuate the blood pressure increase during rhythmic handgrip in humans.  Neurosci Lett. 2002;  289 227-9
  PubMedSearch in Google Scholar
• 3 Lathe R. Hormones and the hippocampus.  J Endocrinol. 2001;  169 205-31
  CrossrefPubMedSearch in Google Scholar
• 4 Brum L F, Elisabetsky E, Souza D. Effects of linalool on [³H] MK801 and [³H] muscimol binding in mouse cortical membranes.  Phytother Res. 2001;  15 422-5
  CrossrefPubMedSearch in Google Scholar
• 5 Silva Brum L F, Emanuelli T, Souza T O, Elisabetsky E. Effects of linalool in glutamate release and uptake in mouse cortical synaptosomes.  Neurochem Res. 2001;  26 191-4
  CrossrefPubMedSearch in Google Scholar
• 6 Yamada K, Mimaki Y, Sashida Y. Effects of inhaling the vapor of Lavandula burnatii super-derived essential oil and linalool on plasma adrenocorticotropic hormone (ACTH), catecholamine and gonadotropin levels in experimental menopausal female rats.  Biol Pharm Bull. 2005;  28 378-9
  CrossrefPubMedSearch in Google Scholar
• 7 Sugawara Y, Hara C, Aoki T, Sugimoto N, Masujima T. Odor distinctiveness between enantiomers of linalool: difference in perception and responses elicited by sensory test and forehead potential wave measurement.  Chem Senses. 2000;  25 77-84
  PubMedSearch in Google Scholar
• 8 Kuroda K, Inoue N, Ito Y, Kubota K, Sugimoto A, Kakuda T. et al . Sedative effects of the jasmine tea odor and R-(-)-linalool, one of its major odor components, on autonomic nerve activity and mood states.  Eur J Appl Physiol. 2005;  95 107-14
  PubMedSearch in Google Scholar
• 9 Boelens M H, Boelens H, van Gemert L J. Sensory properties of optical isomers.  Perfum Flavor. 1993;  18 1-15
  PubMedSearch in Google Scholar
• 10 Sobel N, Prabhakaran V, Hartley C A, Desmond J E, Glover G H, Sullivan E V. et al . Blind smell: brain activation induced by an undetected air-borne chemical.  Brain. 1999;  122 209-17
  CrossrefPubMedSearch in Google Scholar
• 11 Campenni C E, Crawley E J, Meier M E. Role of suggestion in odor-induced mood change.  Psychol Rep. 2004;  94 1127-36
  CrossrefPubMedSearch in Google Scholar
• 12 Kirschbaum C, Kudielka B M, Gaab J, Schommer N C, Hellhammer D H. Impact of gender, menstrual cycle phase, and oral contraceptives on the activity of the hypothalamus-pituitary-adrenal axis.  Psychosom Med. 1999;  61 154-62
  CrossrefPubMedSearch in Google Scholar
• 13 Kirschbaum C, Pirke K M, Hellhammer D H. The ‘Trier Social Stress Test’: a tool for investigating psychobiological stress responses in a laboratory setting.  Neuropsychobiology. 1993;  26 76-81
  PubMedSearch in Google Scholar
• 14 Höferl M, Krist S, Buchbauer G. Adaptation of DELFIA™ cortisol kit for determination of salivary cortisol concentration.  Arch Pharm. 2005;  238 493-7
  PubMedSearch in Google Scholar
• 15 Heuberger E, Hongratanaworakit T, Böhm C, Weber R, Buchbauer G. Effects of chiral fragrances on human autonomic nervous system parameters and self-evaluation.  Chem Senses. 2001;  26 281-92
  CrossrefPubMedSearch in Google Scholar

Dr. Martina Höferl

Department of Clinical Pharmacy and Diagnostics

Centre of Pharmacy

University of Vienna

Althanstrasse 14

1090 Wien

Austria

Phone: +43-1-4277-555-52

Fax: +43-1-4277-9551

Email: martina.hoeferl@univie.ac.at

• Supplementary Material