Chemical Composition and Stimulating Effect of Citrus Hystrix Oil on Humans

FLAVOUR AND FRAGRANCE JOURNAL COMPOSITION AND EFFECTS OF CITRUS HYSTRIX OIL ON HUMANS 443 Flavour Fragr. J. 2007; 22: 443–449 Published online 18 July 2007 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/ffj.1820 Chemical composition and stimulating effect of Citrus hystrix oil on humans

Tapanee Hongratanaworakit1* and Gerhard Buchbauer2

1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Srinakharinwirot University, Nakhon-nayok 26120, Thailand 2 Department of Clinical Pharmacy and Diagnostics, Centre of Pharmacy, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria Received 12 January 2007; Revised 15 May 2007; Accepted 28 May 2007

ABSTRACT: Kaffir lime essential oil was obtained from fresh peels of Citrus hystrix (Rutaceae) by hydrodistillation and analysed by GC and GC–MS. The effects of kaffir lime oil on human autonomic and behavioural parameters after massage were investigated in this study. Forty healthy volunteers participated in the experiments. Autonomic parameters recorded were skin temperature, pulse rate, breathing rate and blood pressure. Behavioural parameters were assessed by means of visual analogue scales (VAS). The kaffir lime oil caused a significant increase in blood pressure and a significant decrease in skin temperature. Regarding the behavioural parameters, subjects in the kaffir lime oil group rated themselves more alert, attentive, cheerful and vigorous than subjects in the control group. These findings are likely to represent stimulating/activating effects of the kaffir lime oil and provide some evidence for the use of kaffir lime oil in aromatherapy, such as causing relief from depression and stress in humans. Copyright © 2007 John Wiley & Sons, Ltd.

KEY WORDS: physiological parameters; activating effect; behavioural evaluation; kafﬁr lime oil

Introduction patients by increasing blood flow to the prefrontal cortex.10 Stress-relieving properties could also be attrib- The essential oil of kaffir lime (Citrus hystrix DC.; uted to C. sinensis oil, measuring the saliva cortisol con- family Rutaceae) is increasingly used as a fragrance in centration.11 An investigation at the Middlesex Hospital the food, perfumery and cosmetic industries. Kaffir lime in London showed that foot massage with the essential oil has been associated with anti-proliferative activity on oil from the flowers of C. aurantium (Neroli oil) KB and P388 cell lines and has the potential for cancer positively affects the psychological state of patients.12 treatment.1 The repellency effect of kaffir lime oil has Clinical aromatherapy for agitated behaviour in dementia been demonstrated by many researchers.2,3 Kaffir lime oil patients has been reported.13–16 Furthermore, aromatherapy shows antioxidant as well as free radical scavenging massage is increasing in oncology and palliative care activity.4–6 Antiviral activity of kaffir lime extract has unit.17–18 been reported by Khan et al.7 In medicine, interest in Recently there have been a variety of measurements the usage of citrus oils as therapeutically active agents to evaluate the physiological and psychological effects has grown considerably. Especially in aromatherapy, of fragrances, measuring such parameters as: changes Citrus sinensis (sweet orange oil) has been used as an in autonomic parameters, e.g. heart rate, breathing rate, antidepressant in the case of depression. Lehrner et al.8 blood pressure, eye-blinks, skin temperature and skin reported that ambient odours of C. sinensis oil reduced conductance19–23; changes in brain wave activities, e.g. anxiety and improved mood in patients waiting for electroencephalogram; contingent negative variation24–25; dental treatment. A mixture of citrus oils helps to reduce changes in mood, cognitive performances and emotion.26–28 the therapeutic doses of antidepressants in depressive A study conducted at the Human Cognitive Neuroscience patients.9 Aromatherapy massage using C. sinensis oil Unit, University of Northumbria, Newcastle upon Tyne, every week for 4 weeks improves depressive states in UK,29 showed that aromas of rosemary and lavender oils differentially affected cognition and mood in healthy adults. Analysis of performance revealed that lavender oil produced a significant decrement in the performance * Correspondence to: T. Hongratanaworakit, Faculty of Pharmacy, of working memory and impaired reaction times for Srinakharinwirot University, Rangsit-Ongkharak Road, Nakhon-Nayok both memory- and attention-based tasks compared to 26120, Thailand. controls. In contrast, rosemary oil produced a significant E-mail: [email protected] Contract/grant sponsor: Srinakharinwirot University, Thailand; Contract/grant enhancement of performance for overall quality of number: 012/2005. memory and secondary memory factors, but also

Copyright © 2007 John Wiley & Sons, Ltd. Flavour Fragr. J. 2007; 22: 443–449 DOI: 10.1002/ffj 444 T. HONGRATANAWORAKIT AND G. BUCHBAUER produced an impairment of speed of memory compared β-pinene, γ-terpinene). The correction factors of the monoterpene to controls. This study indicated that these essential oils alcohols for the other components, i.e. linalool, exo-fenchol, can produce objective effects on cognitive performance neo-isopulegol, isopulegol and borneol, were obtained from the as well as subjective effects on mood. However, up to average of the correction factors of two standards monoterpene α now, no experiments on the effects of kaffir lime oil on alcohols (terpinene-4-ol, -terpineol). The percentage compositions of the remaining components were calculated without the human autonomic nervous system and behavioural correction factors. measures after massage have been carried out. Therefore, the main objectives of the present study were: (a) to investigate the chemical composition of kaffir lime oil; Gas Chromatography–Mass Spectrometry (GC–MS) (b) to evaluate the effects of this fragrance compound on autonomic nervous system parameters and behavioural GC–MS was carried out on a Trace GC Ultra/Trace DSG parameters in healthy humans following transdermal Finnigan gas chromatograph fitted with a fused-silica BPX5 absorption. capillary column (30 m × 0.25 mm i.d., 0.25 µm film thickness); carrier gas, helium at 1.0 ml/min; injector temperature, 220 °C; split ratio, 1:20; transfer line temperature, 240 °C; column Experimental temperature, 60–246 °C at 3 °C/min; mass spectra obtained using an ionization energy of 70 eV and ion source temperature Fragrance Compound of 200 °C. The individual components were identified: (a) by comparison of their mass spectra with those stored in the MS database (NIST, WILEY libraries) and with mass spectra liter- Kaffir lime oil was obtained from the fresh fruit peel of 30 Citrus hystrix DC. by hydrodistillation for 2 h using a ature data; (b) by comparison of their linear retention indices Clevenger apparatus. The obtained oil was dried over anhydrous (LRI) on DB-5 column, calculated from the retention temperature/retention times of a homologous series of n-alkanes,31 with sodium sulphate and kept at 4 °C until analysed. The 32–35 average yield was 1.5% w/w. A voucher specimen has been those of authentic standards or with those of the literature. deposited at Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Srinakharinworit University, under Accession No. 4807295-/1307. Subjects

Forty healthy volunteers aged 18–48 (mean 23.55 ± 6.06) years Gas Chromatography (GC) took part in the experiments. Demographic data for the control group and the experimental group are presented in Table 1. GC–FID was performed using a Perkin-Elmer AutoSystem Subjects were tested in individual sessions and randomly gas chromatograph fitted with a fused-silca PB-WAX capillary assigned to either the control group or the kaffir lime oil group. column (60 m × 0.25 mm i.d., 0.32 µm film thickness); column Each group consisted of 20 subjects. They were fully briefed, temperature, 90 °C for 4 min, then 90–220 °C at 10 °C/min and gave written informed consent to all aspects of the study held isothermal at this temperature for 3 min; injector and (Srinakharinwirot University Ethics Committee) and were free detector temperatures, 190 °C and 220 °C, respectively; carrier to withdraw at any time. Forty-eight hours prior to testing gas, helium at 1.0 ml/min; samples (1.5 ul) injected using a split subjects had to abstain from food, beverages and toiletries con- ratio of 1:10. Quantitative analysis of the oil component taining the essential oil of Citrus species or its constituents (e.g. was calculated according to the correction factor, using stand- limonene), i.e. citrus fruits, apricots, peaches, fruit juices, basil, ards at least for the main components, i.e. limonene, β-pinene, coriander, thyme, cinnamon, geranium and bergamot, as well as γ-terpinene, terpinene-4-ol, α-terpineol, since the components of from tea, coffee and alcohol. the same or similar chemical class could give a similar correction factor. Therefore, the correction factors of the monoterpene hydrocarbons for the other components, i.e. α-thujene, α-pinene, Fragrance Administration camphene, sabinene, myrcene, α-phellandrene, α-terpinene and terpinolene, were obtained from the average of the correction In the experimental group, 1 ml 20% (w/w) solution of kaffir factors of three standard monoterpene hydrocarbons (limonene, lime oil in sweet almond oil was applied to the skin of the

Table 1. Demographic data for the control and experimental groups

Parameter Control group Kafﬁr lime oil group

Number of volunteers 20 20 Sex (M:F) 8:12 9:11 Height (cm; mean ± SD) Male 172.75 ± 6.01 171.66 ± 4.27 Female 159.91 ± 4.48 159.45 ± 4.63 Weight (kg; mean ± SD) Male 61.87 ± 7.29 62.55 ± 9.11 Female 53.16 ± 4.70 52.63 ± 6.05

Copyright © 2007 John Wiley & Sons, Ltd. Flavour Fragr. J. 2007; 22: 443–449 DOI: 10.1002/ffj COMPOSITION AND EFFECTS OF CITRUS HYSTRIX OIL ON HUMANS 445 lower abdomen of each subject, and subjects themselves ness, mood and alertness) were used to assess behavioural massaged the oil into the skin for 5 min. The massage area was parameters. All statistical calculations and data analyses were then covered with plastic ﬁlm in order to prevent evaporation of performed using the Statistical Package for the Social Sciences the oil. In the control group, 1 ml of placebo substance, i.e. pure (SPSS version 11.5). The effects of fragrances on autonomic sweet almond oil, was used. In both groups the subjects were parameters and ratings of behavioural parameters were deter- supplied with pure air via breathing masks (Inhalation Set mined by Mann–Whitney U-test analysis of variance. for Adult, No. 1500004020, B + P Beatmungsprodukte GmbH, Neunkirchen, Germany) in order to eliminate any olfactory stimulation by nose or mouth. Results

Experimental Design Chemical Composition of the Oil

The experimental design has been previously used by our The chemical compositions, their linear retention indices, group.36–41 One session consisted of two trials of 20 min each. the percentage composition, the correction factor, and At the beginning and end of each trial, behavioural parameters identification methods of the kaffir lime essential oil were assessed by visual analogue scales (VAS). Autonomic are summarized in Table 2. The oil mainly consisted of parameters were recorded continuously during each trial. In the monoterpene hydrocarbons, with limonene (30.73%) and first trial, which served as a control for influences of the experi- β-pinene (18.76%) as the main components. Other minor mental set-up, the placebo substance was administered to all components were terpinene-4-ol (10.63%), α-terpineol subjects. In the second trial the placebo was again administered (8.35%), γ-terpinene (6.18%), α-terpinene (5.09%) and to the control group, whereas in the experimental group the terpinolene (4.33%). appropriate fragrance was administered.

Procedure Autonomic Parameters

All experiments were conducted in a bright and quiet room with The mean and SEM of autonomic parameters of the con- ambient temperature of 24–26 °C. Upon arrival, the volunteers trol and experimental groups are presented in Table 3. were interviewed about their personal data. In addition, they The SBP of subjects in the control group changed only were asked about the rating of behavioural parameters. After marginally at the end of the second trial compared with completion of the interview and administration of the rating the end of the first trial. In contrast, the SBP of subjects scales, systolic and diastolic blood pressure (SBP, DBP) were in the kaffir lime oil group increased at the end of the measured. Subsequently, the subjects were informed about second trial compared with the end of the first trial. The the proceedings. Afterwards subjects were seated in a semi- difference scores of SBP between the second and first reclined position, providing easy access for attachment of trials for the control and the kaffir lime oil groups are the electrodes in suitable positions. The inhalation set was fitted shown in Figure 1. Comparison of these difference scores to the volunteer’s face to cover nose and mouth. The pure air was then supplied directly. The oil or the placebo substance revealed a significantly larger increase of SBP in the = were administered as described above. Then, the recording kaffir lime oil group than in the control group (p of the autonomic parameters was started. After completion of 0.019). The DBP of subjects in the control and kaffir the first trial, the subjects were asked to complete the rating lime oil groups increased at the end of the second trial scales. The SBP and DBP were measured at the end of the first compared with the end of the first trial. The difference trial. This procedure was repeated in the second trial. At the end scores of DBP between the second and first trials for the of each trial, the subjects were asked if they had smelled any control and kaffir lime oil groups are shown in Figure 1. odour during the experiment; all subjects stated that they had Comparison of these difference scores revealed a signifi- not. cantly larger increase of DBP in the kaffir lime oil group than in the control group (p = 0.044). SKT of subjects in the control group only marginally changed in the second Acquisition of Autonomic Parameters and trial compared with the first trial. In contrast, SKT of Statistical Analysis subjects in the kaffir lime oil group decreased in the second trial compared with the first trial. The difference Breathing rate (BR), pulse rate (PR) and skin temperature (SKT) scores of SKT between the second and first trials for the were measured using Power Lab/4SP hardware (ADInstruments control and kaffir lime oil groups are shown in Figure 1. Inc., NSW, Australia). The sampling rate was 100 Hz. Systolic and diastolic blood pressure (SBP and DBP) were determined Comparison of these difference scores revealed a signifi- by sphygmomanometry, using an automated system (Model cantly larger decrease of SKT in the kaffir lime oil group DS-155E, Digital Electronic, Japan). Details of the recording than in the control group (p = 0.019). No significant system and procedure have been described elsewhere.36–41 effects of the kaffir lime oil on BR and on PR were Visual analogue scales (relaxation, vigour, calmness, attentive- found (p > 0.05 for all).

Table 2. Chemical composition of the essential oil from Citrus hystrix DC

Component LRI on DB–5 (%) Correction factor Identiﬁcation References

α-Thujene 926 tr 1.20 MS, LRI 30, 35 α-Pinene 933 3.23 1.20 MS, LRI 30, 33, 35 Camphene 947 0.49 1.20 MS, LRI 30, 32, 35 Sabinene 973 1.74 1.20 MS, LRI 30, 32, 35 β-Pinene 977 18.76 1.18 MS, LRI 30, 33, 35 Myrcene 990 1.45 1.20 MS, LRI 30, 32, 33, 35 α-Phellandrene 1004 0.83 1.20 MS, LRI 30, 33,35 α-Terpinene 1016 5.09 1.20 MS, LRI 30, 35 p-Cymene 1024 0.39 — MS, LRI 30 Limonene 1030 30.73 1.29 MS, LRI 30, 32, 35 γ-Terpinene 1058 6.18 1.13 MS, LRI 30, 33, 35 cis-Linalool oxide 1077 0.81 — MS, LRI 30, 35 Terpinolene 1088 4.33 1.20 MS, LRI 30, 32 Linalool 1100 0.51 1.18 MS, LRI 30, 33, 35 exo-Fenchol 1112 0.41 1.18 MS, LRI 30 neo-Isopulegol 1144 1.60 1.18 MS, LRI 30, 32 β-Citronellal 1152 tr — MS, LRI 30, 32, 33, 35 iso-Isopulegol 1155 1.36 1.18 MS, LRI 30 Borneol 1164 0.32 1.18 MS, LRI 30, 32 Terpinene-4-ol 1178 10.63 1.17 MS, LRI 30, 32, 33, 35 α-Terpineol 1192 8.35 1.19 MS, LRI 30, 35 β-Citronellol 1227 0.17 — MS, LRI 30, 32, 33 Citronellyl acetate 1351 0.16 — MS, LRI 30 α-Cubebene 1372 0.64 — MS, LRI 30 Neryl acetate 1381 0.15 — MS, LRI 30 β-Cubebene 1388 tr — MS, LRI 30 (Z)-Caryophyllene 1416 0.33 — MS, LRI 30, 33, 35 Germacrene D 1478 tr — MS, LRI 30, 35 γ-Cadinene 1521 0.93 — MS, LRI 30, 35

LRI, linear retention indices relative to C9–C20 n-alkanes on DB-5 column; MS, identiﬁcation based on comparison of mass spectra; %, percentage composition; tr, trace (peak area <0.1%).

Table 3. Mean and SEM of autonomic parameters of the control and experimental groups

Autonomic C (mean ± SEM) KFL (mean ± SEM) parameters Trial 1 Trial 2 Trial 1 Trial 2

SBP 101.06 ± 3.02 101.69 ± 3.16 96.94 ± 2.52 101.56 ± 2.48 DBP 59.54 ± 2.19 61.46 ± 2.44 58.46 ± 1.20 64.15 ± 1.28 ST 36.92 ± 0.19 36.64 ± 0.21 36.13 ± 0.17 35.17 ± 0.31 BR 17.00 ± 0.84 16.96 ± 0.90 14.61 ± 0.77 14.27 ± 0.71 PR 68.06 ± 1.75 66.20 ± 1.83 62.99 ± 2.10 59.04 ± 2.40

C, control group; KFL, kafﬁr lime oil group; SBP, systolic blood pressure; DBP, diastolic blood pressure; ST, skin temperature; BR, breathing rate; PR, pulse rate.

Behavioural Parameters group felt slightly attentive at the end of the second trial compared with the end of the first trial. On the other The mean and SEM of behavioural parameters of the hand, subjects in the kaffir lime oil group judged them- control and experimental groups are presented in Table 4. selves more attentive at the end of the second trial com- Subjects in the control group felt slightly alert at the end pared with the end of the first trial. The difference scores of the second trial compared with the end of the first of subjective attentiveness between the second and first trial. In contrast, subjects in the kaffir lime oil group trials for the control and kaffir lime oil groups are shown judged themselves more alert at the end of the second in Figure 2. Comparison of these difference scores re- trial compared with the end of the first trial. The differ- vealed a significant increase of subjective attentiveness in ence scores of subjective alertness between the second the kaffir lime oil group compared with the control group and first trials for the control and kaffir lime oil groups (p = 0.039). Furthermore, subjects in the control group are shown in Figure 2. Comparison of these difference felt less cheerful at the end of the second trial compared scores revealed a significant increase of subjective alert- with the end of the first trial. On the other hand, subjects ness in the kaffir lime oil group compared with the con- in the kaffir lime oil group judged themselves more trol group (p = 0.029). In addition, subjects in the control cheerful at the end of the second trial compared with the

Figure 1. The difference scores of systolic blood Figure 2. The difference scores of subjective alertness, pressure (SBP), diastolic blood pressure (DBP) and skin subjective attentiveness, subjective mood and subjec- temperature (SKT) for the control and kafﬁr lime oil tive vigour for the control and kafﬁr lime oil groups groups

Table 4. Mean and SEM of behavioural parameters of the control and experimental groups

C (Mean ± SEM) KFL (Mean ± SEM)

Trial 1 Trial 2 Trial 1 Trial 2

AT 24.35 ± 3.23 24.17 ± 2.74 27.02 ± 3.26 21.95 ± 2.76 AL 40.00 ± 3.00 39.65 ± 3.83 34.85 ± 3.09 27.87 ± 3.45 C 23.20 ± 2.74 25.15 ± 3.40 24.52 ± 3.04 21.15 ± 2.31 R 30.25 ± 3.65 29.40 ± 3.48 28.65 ± 3.05 23.65 ± 3.23 M 33.90 ± 3.22 35.25 ± 3.45 33.55 ± 2.87 27.15 ± 2.88 V 47.45 ± 3.37 45.65 ± 3.86 41.07 ± 2.99 30.82 ± 3.11

C, control group; KFL, kaffir lime oil group; AT, attentiveness; AL, alertness; C, calmness; R, relaxation; M, mood; V, vigour. end of the first trial. The difference scores of subjective the other hand, subjects in the kaffir lime oil group mood between the second and first trials for the control judged themselves more vigorous at the end of the and kaffir lime oil groups are shown in Figure 2. Com- second trial compared with the end of the first trial. The parison of these difference scores revealed a significant difference scores of subjective vigour between the second increase of subjective mood in the kaffir lime oil group and first trials for the control and kaffir lime oil groups compared with the control group (p = 0.023). Subjects in are shown in Figure 2. Comparison of these difference the control group felt slightly vigorous at the end of the scores revealed a significant increase of subjective vigour second trial compared with the end of the first trial. On in the kaffir lime oil group compared with the control

Copyright © 2007 John Wiley & Sons, Ltd. Flavour Fragr. J. 2007; 22: 443–449 DOI: 10.1002/ffj 448 T. HONGRATANAWORAKIT AND G. BUCHBAUER group (p = 0.018). No significant effects of the kaffir methyl chavicol, which possibly mediate the stimulating lime oil on subjective calmness and relaxation were effect on sympathetic activity. Although our findings found (p > 0.05 for all). agree with other reports, it is important to investigate those active components that contribute to the stimulating effect on the sympathetic activity in future studies. Discussion Correlation analysis between the ANS and behaviour parameters showed that the increase of blood pressure In the present investigation kaffir lime oil was adminis- was not correlated with changes in behavioural responses. tered transdermally to healthy subjects. Autonomic These findings suggest the effectiveness of pharmacologi- parameters, i.e. blood pressure, pulse rate, breathing rate cal mechanisms, e.g. direct interactions between fragrance and skin temperature, were recorded as indicators of molecules and receptor sites which are involved in the the arousal level of the autonomic nervous system. In regulation of ANS arousal. Due to their high lipophilicity, addition, subjects had to rate their mental and emotional fragrance molecules easily penetrate the blood–brain condition in terms of relaxation, vigour, calmness, atten- barrier43 and enter the brain following inhalation or tiveness, mood and alertness in order to assess subjective massage.44,45 Therefore, one possibility that explains the behavioural arousal. The kaffir lime oil caused a signifi- stimulating effect of kaffir lime oil could be that the oil cant increase of blood pressure. Since blood pressure possibly stimulates the locus coeruleus in the brain into is determined by the activity of the sympathetic branch releasing noradrenalin, a neurotransmitter that creates a of the autonomic nervous system (ANS), an increase stimulating/activating effect. The locus coeruleus is also of blood pressure shows an increase of sympathetic tone, involved in arousal and activation.46,47 Another possibil- i.e., an increase of autonomic arousal. A significantly ity that explains its effect could be that essential kaffir larger decrease of skin temperature in the kaffir lime oil lime oil exerts its effects by an interaction with central group compared with the control group was found. Skin (e.g. hypothalamic, limbic, thalamic) structures which temperature is controlled indirectly by the sympathetic control the level of autonomic and/or behavioural arousal. division of the ANS via the contraction or relaxation of All our findings indicate that differential effects of the the smooth muscles which surround the blood vessels and essential oils depend on mode/route of administration. regulate blood supply to distinct skin areas. When these Both pharmacological and psychological effects are muscles are contracted, skin temperature is reduced active simultaneously when the oils are administered by because less blood reaches there. On the other hand, means of inhalation and olfactory processing occurs. when these muscles are relaxed, skin temperature rises In contrast, percutaneous administration gives evidence because more blood is supplied there. Therefore, the of a pure pharmacological effect and exclusion of decrease of skin temperature in the kaffir lime oil group olfactory processing. Therefore, in order to differentiate indicates an increase of ANS arousal. Among the behavi- between pharmacological and psychological effects of oural parameters, subjects in the kaffir lime oil group fragrances, subjective evaluation of the odours must be rated themselves more alert, more attentive, more cheer- prevented.36–41,48,49 ful, and more vigorous than subjects in the control group. In conclusion, our investigations are likely to represent This finding points towards an increase of arousal in the stimulating/activating effects of kaffir lime oil and terms of self-evaluation. Transdermal absorption of kaffir provide some evidence for the use of kaffir lime oil in lime oil enhanced the level of arousal of the ANS and medicines, such as causing reduction in blood pressure or led to activation at the behavioural parameters, i.e. the in relieving depression and stress in humans. subjects felt more alert, attentive, cheerful and vigorous Acknowledgements—This work was supported by Grant No. 012/2005 than before the administration of the oil. Thus, the effects (to T.H.) from Srinakharinwirot University, Thailand. The authors are of kaffir lime oil by means of percutaneous administra- grateful to Dr E. Heuberger for experimental design suggestions and tion may be characterized by the concept of stimulating/ Dr L. Jirovetz, University of Vienna, Austria, for suggestions regarding essential oil analysis. activating effects, which have also been described for sandalwood essential oil37 and for the essential oil 39 36 of Citrus sinensis. In addition, our previous study re- References ported that limonene, the main component of kaffir lime oil, enlarged the level of arousal of the ANS, i.e. increase 1. Manosroi J, Dhumtanom P, Manosroi A. Cancer Lett. 2006; 235: of blood pressure, as well as leading to activation at the 114–120. behavioural parameters, i.e. the subjects felt more alert 2. Amer A, Mehlhorn H. Parasitol. Res. 2006; 99: 478– 490. 3. Tawatsin A, Wratten SD, Scott RR, Thavara U, Techadamrongsin and less calm than before the administration of the oil. Y. J. Vector Ecol. 2001; 26: 76–82. Furthermore, essential oils with a stimulating effect on 4. Huttadilok-Towatana N, Chaiyamutti P, Panthong K, sympathetic activity have been reported, e.g. pepper oil, Mahabusarakam W, Rukachaisirikul V. Pharmaceut. Biol. 2006; 42 44: 221–228. oestragon oil and grapefruit oil. These essential oils 5. Lertsatitthanakorn P, Taweechaisupapong S, Aromdee C, consist of some components, such as limonene, pinene or Khunkitti W. Int. J. Aromather. 2006; 16: 43–49.

6. Jamilah B, Che-Man YB, Ching TL. J. Food Lipids 1998; 5: 149– 30. Adams RP. Identiﬁcation of Essential Oil Components by Gas 157. Chromatography/Quadrupole Mass Spectroscopy. Allured: Carol 7. Khan MTH, Ather A, Thompson KD, Gambari R. Antiviral Res. Stream, IL, 2004. 2005; 67: 107–119. 31. Dool VDH, Kratz PD. J. Chromatogr. 1963; 11: 463. 8. Lehrner J, Marwinski G, Lehr S, Johren P, Deecke L. Physiol. 32. Madruga M. LRI Values of Authentic compounds on DB5 column Behav. 2005; 86: 92–95. (GC–MS). Department of Food Science and Technology, Univer- 9. Komori T, Fujiwara R, Tanida M, Nomura J, Yokoyama MM. sity of Reading, UK, 1993. Neuroimmunomodulation 1995; 2: 174–180. 33. Hognadottir A, Rouseff RL. J. Chromatogr. A 2003; 998: 201– 10. Okamoto A, Kuriyama H, Watanabe S et al. Psychiat. Clin. 211. Neurosci. 2005; 59: 363. 34. Smadja J, Rondeau P, Sing ASC. Flavour Fragr. J. 2005; 20: 11. Hoeferl M, Krist S, Buchbauer G. Planta Med. 2006; 72: 1188– 399–402. 1192. 35. Ngassoum MB, Ousmaila H, Ngamo LT et al. J. Food Compos. 12. Stevensen C. Complement. Therap. Med. 1994; 2: 27–35. Anal. 2004; 17: 197–204. 13. Ballard CG, O’Brien JT, Reichelt K, Perry EK. J. Clin. Psychiat. 36. Heuberger E, Hongratanaworakit T, Boehm C, Weber R, 2002; 63: 553–558. Buchbauer G. Chem. Senses 2001; 26: 281–292. 14. Holmes C, Hopkins V, Hensford C et al. Int. J. Geriatr. Psychiat. 37. Heuberger E, Hongratanaworakit T, Buchbauer G. Planta Med. 2002; 17: 305–308. 2006; 72: 792–800. 15. Smallwood J, Brown R, Coulter F, Irvine E, Copland C. Int. J. 38. Hongratanaworakit T, Buchbauer G. Phytother. Res. 2006; 20: Geriatr. Psychiat. 2001; 16: 1010–1013. 758–763. 16. Snow LA, Hovanec L, Brandt J. J. Altern. Complement. Med. 39. Hongratanaworakit T, Buchbauer G. Acta Hort. 2005; 679: 75– 2004; 10: 431– 437. 81. 17. Dunwoody L, Smyth A, Davidson R. Int. J. Palliat. Nurs. 2002; 40. Hongratanaworakit T, Buchbauer G. Planta Med. 2004; 70: 632– 8: 497–504. 636. 18. Wilcock A, Manderson CA, Weller R et al. Palliat. Med. 2004; 41. Hongratanaworakit T, Heuberger E, Buchbauer G. Planta Med. 18: 287– 290. 2004; 70: 3–7. 19. Bensaﬁ M, Rouby C, Farget V et al. Chem. Senses 2002; 27: 42. Haze S, Sakai K, Gozu Y. Jpn J. Pharmacol. 2002; 90: 247–253. 703–709. 43. Buchbauer G, Jirovetz L, Czejka M, Naßel C, Dietrich H. Paper 20. Brauchli P, Ruegg PB, Etzweiler F, Zeier H. Chem. Senses 1995; presented at the 24th International Symposium on Essential Oils 20: 505–515. (ISEO), 21–24 July 1993, TU Berlin, Germany. 21. Inoue N, Kuroda K, Sugimoto A, Kakuda T, Fushiki T. Biosci. 44. Kovar KA, Gropper B, Friess A, Ammon HPT. Planta Med. Biotechnol. Biochem. 2003; 67: 1206–1214. 1987; 53: 315–318. 22. Kuroda K, Inoue N, Ito Y et al. Eur. J. Appl. Physiol. 2005; 95: 45. Buchbauer G, Jirovetz L, Jaeger W, Plank C, Dietrich H. J. 107–114. Pharm. Sci. 1993; 82: 660–664. 23. Saeki Y. Complement. Ther. Med. 2000; 8: 2–7. 46. Kalat JW. Biological Psychology. Brooks/Cole: Washington, DC, 24. Diego MA, Jones NA, Field T et al. Int. J. Neurosci. 1998; 96: 1995. 217–224. 47. Hugdahl K. Psychophysiology: The Mind–Body Perspective. 25. Goel N, Lao RP. Biol. Psychol. 2006; 71: 341–349. Harvard University Press: Cambridge, MA, USA, 1995. 26. Field T, Diego M, Hernandez-Reif M et al. Int. J. Neurosci. 2005; 48. Hongratanaworakit T, Buchbauer G. Proceedings of the 23rd 115: 207–222. International Federal Society Cosmetics Chemists, 24–27 October 27. Warrenburg S. Chem. Senses 2005; 30(suppl 1): 248–249. 2004, Orlando, FL, USA. 28. Degel J, Koester EP. Chem. Senses 1999; 24: 317–325. 49. Hongratanaworakit T, Heuberger E, Buchbauer G. Abstract from 29. Moss M, Cook J, Wesnes K, Duckett P. Int. J. Neurosci. 2003; the 31st International Symposium on Essential Oils (ISEO), Ham- 113: 15–38. burg, Germany, 10–13 September 2000; A39.