THE JOURNAL OF ALTERNATIVE AND COMPLEMENTARY MEDICINE Volume 18,Number 8,2012,pp.798-804 Mary Ann Liebert.Inc. D0:10.1089/acm.2011.0551 Effect of Sweet Orange Aroma on Experimental Anxiety in Humans Tiago Costa Goes,MSc,Fabricio Dias Antunes,MD, Pericles Barreto Alves,PhD,and Flavia Teixeira-Silva,PhD' Abstract Objectives:The objective of this study was to evaluate the potential anxiolytic effect of sweet orange(Citrus sinensis)aroma in healthy volunteers submitted to an anxiogenic situation. Design:Forty (40)male volunteers were allocated to five different groups for the inhalation of sweet orange essential oil (test aroma:2.5,5,or 10 drops),tea tree essential oil (control aroma:2.5 drops),or water (non- aromatic control:2.5 drops).Immediately after inhalation,each volunteer was submitted to a model of anxiety, the video-monitored version of the Stroop Color-Word Test(SCWT). Outcome measures:Psychologic parameters(state-anxiety,subjective tension,tranquilization,and sedation)and physiologic parameters (heart rate and gastrocnemius electromyogram)were evaluated before the inhalation period and before,during,and after the SCWT. Results:Unlike the control groups,the individuals exposed to the test aroma(2.5 and 10 drops)presented a lack of significant alterations(p>0.05)in state-anxiety,subjective tension and tranquillity levels throughout the anxiogenic situation,revealing an anxiolytic activity of sweet orange essential oil.Physiologic alterations along the test were not prevented in any treatment group,as has previously been observed for diazepam. Conclusions:Although more studies are needed to find out the clinical relevance of aromatherapy for anxiety disorders,the present results indicate an acute anxiolytic activity of sweet orange aroma,giving some scientific support to its use as a tranquilizer by aromatherapists. Introduction the other hand,this therapy still does not have much scien- tific support.6.7 chatne diornders in the neral ppulon Double-blind,randomized,placebo-controlled clinical trials performed to evaluate the effect of essential oils on their treatment is still challenging,as the drugs used for the anxiety symptoms are gradually starting to appear in the relief of anxiety symptoms can have important side-effects, literature (for a systematic review,see Cooke and Ernst). promote therapeutic dependence,or present a delay in their However,in most of these studies,exposure to the essential onset of action.Furthermore,not all patients benefit from the oil odor was accompanied by massage.This makes it difficult available treatments,and only a few of them have a response to draw firm conclusions about the essential oil effect,as the near complete recovery.3 massage per se is able to reduce anxiety scores.s However,in These facts justify the growing search for alternative or a recent study performed with rats,animals submitted to complementary procedures for the relief of anxiety symp- two different experimental models of anxiety,after being toms.Among these procedures,one can find aromatherapy, exposed to Citrus sinensis aroma,showed less anxiety than which is the use of essential oils as an alternative treatment animals exposed to air only.This result could not be at- for medical purposes.4 tributed to massage,previous experience with the aroma, According to Charlesworth,5 about 60%of health com- therapeutic relationship,or even to an unspecific effect of plaints in the medical office are stress-related,and aro-any aroma,as animals exposed to Melaleuca alternifolia es- matherapy could be a great alterative to conventional sential oil did not behave differently from control animals. medication since it has shown positive emotional effects.On Thus,the probability of C.sinensis essential oil having a "Departamento de Fisiologia,Centro de Ciencias Biologicas e da Saude,Universidade Federal de Sergipe,Sergipe,Brazil. 2Departamento de Quimica,Centro de Ciencias Exatas e Tecnologicas,Universidade Federal de Sergipe,Sergipe,Brazil. 798
Effect of Sweet Orange Aroma on Experimental Anxiety in Humans Tiago Costa Goes, MSc,1 Fabrı´cio Dias Antunes, MD,1 Pe´ ricles Barreto Alves, PhD,2 and Flavia Teixeira-Silva, PhD1 Abstract Objectives: The objective of this study was to evaluate the potential anxiolytic effect of sweet orange (Citrus sinensis) aroma in healthy volunteers submitted to an anxiogenic situation. Design: Forty (40) male volunteers were allocated to five different groups for the inhalation of sweet orange essential oil (test aroma: 2.5, 5, or 10 drops), tea tree essential oil (control aroma: 2.5 drops), or water (nonaromatic control: 2.5 drops). Immediately after inhalation, each volunteer was submitted to a model of anxiety, the video-monitored version of the Stroop Color-Word Test (SCWT). Outcome measures: Psychologic parameters (state-anxiety, subjective tension, tranquilization, and sedation) and physiologic parameters (heart rate and gastrocnemius electromyogram) were evaluated before the inhalation period and before, during, and after the SCWT. Results: Unlike the control groups, the individuals exposed to the test aroma (2.5 and 10 drops) presented a lack of significant alterations ( p > 0.05) in state-anxiety, subjective tension and tranquillity levels throughout the anxiogenic situation, revealing an anxiolytic activity of sweet orange essential oil. Physiologic alterations along the test were not prevented in any treatment group, as has previously been observed for diazepam. Conclusions: Although more studies are needed to find out the clinical relevance of aromatherapy for anxiety disorders, the present results indicate an acute anxiolytic activity of sweet orange aroma, giving some scientific support to its use as a tranquilizer by aromatherapists. Introduction Anxiety disorders are the most prevalent class of psychiatric disorders in the general population.1 However, their treatment is still challenging, as the drugs used for the relief of anxiety symptoms can have important side-effects, promote therapeutic dependence, or present a delay in their onset of action.2 Furthermore, not all patients benefit from the available treatments, and only a few of them have a response near complete recovery.3 These facts justify the growing search for alternative or complementary procedures for the relief of anxiety symptoms. Among these procedures, one can find aromatherapy, which is the use of essential oils as an alternative treatment for medical purposes.4 According to Charlesworth,5 about 60% of health complaints in the medical office are stress-related, and aromatherapy could be a great alternative to conventional medication since it has shown positive emotional effects. On the other hand, this therapy still does not have much scientific support.6,7 Double-blind, randomized, placebo-controlled clinical trials performed to evaluate the effect of essential oils on anxiety symptoms are gradually starting to appear in the literature (for a systematic review, see Cooke and Ernst6 ). However, in most of these studies, exposure to the essential oil odor was accompanied by massage. This makes it difficult to draw firm conclusions about the essential oil effect, as the massage per se is able to reduce anxiety scores.8 However, in a recent study performed with rats, animals submitted to two different experimental models of anxiety, after being exposed to Citrus sinensis aroma, showed less anxiety than animals exposed to air only.9 This result could not be attributed to massage, previous experience with the aroma, therapeutic relationship, or even to an unspecific effect of any aroma, as animals exposed to Melaleuca alternifolia essential oil did not behave differently from control animals. Thus, the probability of C. sinensis essential oil having a 1 Departamento de Fisiologia, Centro de Cieˆncias Biolo´gicas e da Sau´de, Universidade Federal de Sergipe, Sergipe, Brazil. 2 Departamento de Quı´mica, Centro de Cieˆncias Exatas e Tecnolo´gicas, Universidade Federal de Sergipe, Sergipe, Brazil. THE JOURNAL OF ALTERNATIVE AND COMPLEMENTARY MEDICINE Volume 18, Number 8, 2012, pp. 798–804 ª Mary Ann Liebert, Inc. DOI: 10.1089/acm.2011.0551 798
SWEET ORANGE AROMA AND ANXIETY 799 therapeutic action per se is quite high.However,whether this high and medium trait anxiety was set 1 SD above the mean, aroma has the same effect in humans is yet to be verified. with the result that scores greater than 48 were classified as Bringing all this into consideration,the aim of the present high anxiety and,therefore,met the exclusion criteria. work was to evaluate the potential anxiolytic effect of sweet orange (C.sinensis)essential oil inhalation in healthy vol- Visual Analogue Mood Scale (VAMS)15.This scale is unteers submitted to an anxiogenic situation.Moreover,in composed of 16 pairs of opposite adjectives.Each pair is order to determine the main components of the essential oils separated by a 10-cm line on which the subject is requested administered,a gas chromatography/mass spectrometry to mark the point that best represents his feelings.The 16 analysis was also carried out. items are distributed into four factors:(1)tranquilization;(2) mental sedation;(3)physical sedation,and(4)other feelings Materials and Methods and attitudes.The range of values,for each factor,is 0-40 cm Participants A Portuguese version,translated and validated by Zuardi and Karnioll6 and adapted by Del Porto et al.,17 was used. Forty (40)graduate student male volunteers,subjectively healthy,aged between 18 and 30,were selected for inclusion Physiologic measurements in the study using a structured clinical questionnaire and a translated and adapted version of the State-Trait Anxiety Heart rate.Heart rate is derived from two active Inventory (STAI)-Part II.10 Individuals possibly presenting Ag/AgCl electrodes placed across the chest,and one ground pathologies that could interfere with the results,such as Ag/AgCl electrode placed on the abdomen asthma,olfactory problems,psychiatric disorders,or chronic drug use,and individuals with trait-anxiety above 48 points Electromyogram of the gastrocnemius muscle.This is derived from two active Ag/AgCl electrodes placed on the were excluded. This study was conducted with approval from the Ethics gastrocnemius muscle(part of the fight/flight response)of Committee in Research with Humans of the Universidade the nondominant leg. Federal de Sergipe,Brazil.Written informed consent was The skin was cleaned with a mixture of alcohol/ether obtained from all participants. (90:10,vol/vol)before placement of the electrodes The recordings were made using a computerized system Anxiogenic condition for monitoring physiologic responses (I-330-C2+Physiolo- gical Monitoring System,J&J Engineering,USA). The video-monitored Stroop Color-Word Test,as stan- All tests were performed in a quiet room maintained at a dardized by Teixeira-Silva et al.was used to elicit anxiety temperature between 23C and 25C(73.4F and 77.0F). in the volunteers.In short,this test consists of presenting a board to the participant with 100 of the color-naming words Treatments blue,yellow,red,green,and violet organized randomly in a Test aroma.The test aroma consisted of essential oil of 10x10 matrix.Each word is printed in a color different from its meaning (for example,the word "red"printed in yellow sweet orange (C.sinensis,BioEssencia,Brazil),2.5,5,or 10 ink).This board corresponds to the "Color-Word"card of the drops (SO25,SOs,SO10). Stroop test.12 To perform the task,the subject has to say,as quickly as possible and in the sequence presented,the names Control aroma.The control aroma was essential oil of tea of the colors being seen (i.e.,the color of the ink),but not the tree(M.alternifolia,BioEssencia,Brazil),2.5 drops (TT). colors designated by the words.The task has to be per- Nonaromatic control.The nonaromatic control was dis- formed in 2 minutes(maximum),and any errors are signaled with a bell.Skipping a color's sequence,hesitation in saying tilled water,2.5 drops (H2O). All treatments were administered by inhalation.Each the color,and saying the color's "word"instead of its "ink" are all considered to be errors.The whole test is videoed and drop corresponded to 25 uL added to a surgical mask,which presented to the subject on a monitor during the test. was worn by the volunteers for 5 minutes. Instructions were given to the subject using a CD re- A randomized double-blind design was followed.For this, cording,which led them to believe that a group of profes- a random distribution of the subjects to the experimental sionals,located in another room,were observing them and groups(n=8)was made,using a draw,by a researcher who would evaluate their performance. had no direct participation in the test sessions.In addition, the volunteers were told that they would inhale nontoxic Psychologic measurements substances extracted from plants,which could or could not have a smell.The term "aromatherapy"was never used.A State-Trait Anxiety Inventory (STAI)13.This instrument control aroma,proven not to change anxiety levels in rats, is divided into two sections,each having 20 questions.The was used to confirm that the observed results were not due first subscale measures state anxiety,the second measures to unspecific effects of any aroma. trait anxiety.The range of scores,for each subscale,is 20-80, Throughout the entire study,the volunteers were kept the higher the score,the higher the anxiety.A validated ignorant of the therapeutic use of the essential oils being Portuguese version of the STAI was used.10 A cutoff score for evaluated and about the real purpose of the Stroop test, high trait anxiety was derived from Gama et al.;it provides which was to induce anxiety.Therefore,even if some vol- normative data for male university students from our city. unteers could recognize the orange smell,they had no idea The mean anxiety score for this normative group was 39.6, how the aroma was supposed to affect their performance in and the standard deviation (SD)was 9.2.The cutoff between the test
therapeutic action per se is quite high. However, whether this aroma has the same effect in humans is yet to be verified. Bringing all this into consideration, the aim of the present work was to evaluate the potential anxiolytic effect of sweet orange (C. sinensis) essential oil inhalation in healthy volunteers submitted to an anxiogenic situation. Moreover, in order to determine the main components of the essential oils administered, a gas chromatography/mass spectrometry analysis was also carried out. Materials and Methods Participants Forty (40) graduate student male volunteers, subjectively healthy, aged between 18 and 30, were selected for inclusion in the study using a structured clinical questionnaire and a translated and adapted version of the State-Trait Anxiety Inventory (STAI)–Part II.10 Individuals possibly presenting pathologies that could interfere with the results, such as asthma, olfactory problems, psychiatric disorders, or chronic drug use, and individuals with trait-anxiety above 48 points were excluded. This study was conducted with approval from the Ethics Committee in Research with Humans of the Universidade Federal de Sergipe, Brazil. Written informed consent was obtained from all participants. Anxiogenic condition The video-monitored Stroop Color-Word Test, as standardized by Teixeira-Silva et al.,11 was used to elicit anxiety in the volunteers. In short, this test consists of presenting a board to the participant with 100 of the color-naming words blue, yellow, red, green, and violet organized randomly in a 10 · 10 matrix. Each word is printed in a color different from its meaning (for example, the word "red" printed in yellow ink). This board corresponds to the ‘‘Color-Word’’ card of the Stroop test.12 To perform the task, the subject has to say, as quickly as possible and in the sequence presented, the names of the colors being seen (i.e., the color of the ink), but not the colors designated by the words. The task has to be performed in 2 minutes (maximum), and any errors are signaled with a bell. Skipping a color’s sequence, hesitation in saying the color, and saying the color’s ‘‘word’’ instead of its ‘‘ink’’ are all considered to be errors. The whole test is videoed and presented to the subject on a monitor during the test. Instructions were given to the subject using a CD recording, which led them to believe that a group of professionals, located in another room, were observing them and would evaluate their performance. Psychologic measurements State-Trait Anxiety Inventory (STAI)13. This instrument is divided into two sections, each having 20 questions. The first subscale measures state anxiety, the second measures trait anxiety. The range of scores, for each subscale, is 20–80, the higher the score, the higher the anxiety. A validated Portuguese version of the STAI was used.10 A cutoff score for high trait anxiety was derived from Gama et al.14; it provides normative data for male university students from our city. The mean anxiety score for this normative group was 39.6, and the standard deviation (SD) was 9.2. The cutoff between high and medium trait anxiety was set 1 SD above the mean, with the result that scores greater than 48 were classified as high anxiety and, therefore, met the exclusion criteria. Visual Analogue Mood Scale (VAMS)15. This scale is composed of 16 pairs of opposite adjectives. Each pair is separated by a 10-cm line on which the subject is requested to mark the point that best represents his feelings. The 16 items are distributed into four factors: (1) tranquilization; (2) mental sedation; (3) physical sedation, and (4) other feelings and attitudes. The range of values, for each factor, is 0–40 cm. A Portuguese version, translated and validated by Zuardi and Karniol16 and adapted by Del Porto et al.,17 was used. Physiologic measurements Heart rate. Heart rate is derived from two active Ag/AgCl electrodes placed across the chest, and one ground Ag/AgCl electrode placed on the abdomen. Electromyogram of the gastrocnemius muscle. This is derived from two active Ag/AgCl electrodes placed on the gastrocnemius muscle (part of the fight/flight response) of the nondominant leg. The skin was cleaned with a mixture of alcohol/ether (90:10, vol/vol) before placement of the electrodes. The recordings were made using a computerized system for monitoring physiologic responses (I-330-C2 + Physiological Monitoring System, J&J Engineering, USA). All tests were performed in a quiet room maintained at a temperature between 23C and 25C (73.4o F and 77.0o F). Treatments Test aroma. The test aroma consisted of essential oil of sweet orange (C. sinensis, BioEsseˆncia, Brazil), 2.5, 5, or 10 drops (SO2.5, SO5, SO10). Control aroma. The control aroma was essential oil of tea tree (M. alternifolia, BioEsseˆncia, Brazil), 2.5 drops (TT). Nonaromatic control. The nonaromatic control was distilled water, 2.5 drops (H2O). All treatments were administered by inhalation. Each drop corresponded to 25 lL added to a surgical mask, which was worn by the volunteers for 5 minutes. A randomized double-blind design was followed. For this, a random distribution of the subjects to the experimental groups (n = 8) was made, using a draw, by a researcher who had no direct participation in the test sessions. In addition, the volunteers were told that they would inhale nontoxic substances extracted from plants, which could or could not have a smell. The term ‘‘aromatherapy’’ was never used. A control aroma, proven not to change anxiety levels in rats,9 was used to confirm that the observed results were not due to unspecific effects of any aroma. Throughout the entire study, the volunteers were kept ignorant of the therapeutic use of the essential oils being evaluated and about the real purpose of the Stroop test, which was to induce anxiety. Therefore, even if some volunteers could recognize the orange smell, they had no idea how the aroma was supposed to affect their performance in the test. SWEET ORANGE AROMA AND ANXIETY 799
800 GOES ET AL. It is worth mentioning that both of the essential oils used Statistical analyses are considered to be safe.Sweet orange oil is on the U.S The data collected during the adaptation phase were not Food and Drug Administration's GRAS(Generally Regarded as Safe)List,while tea tree oil is generally considered to be analyzed as this phase was only intended to habituate the nontoxic and nonirritant,although it may cause sensitization participants to the environment and apparatus that would be in some individuals.19 used on the following day. All the psychologic data obtained during the test were analyzed by Friedman's analysis of variance(ANOVA),for Procedure each treatment group,followed by Tukey-type test for The selected volunteers attended the laboratory on 2 post hoc comparisons. consecutive days.The first day was used for adaptation,and All the physiologic data obtained were analyzed using a the second for the actual test. two-factor ANOVA for repeated measures followed by Tukey's test for post-hoc comparisons. Adaptation day.The subjects were taken to the experi- The STAI-Trait scores used in the selection process were ment room,which was already organized and equipped analyzed by Kruskal-Wallis ANOVA. with the necessary apparatuses for the execution of the test. The familiarity and hedonicity of the aromas were also Subsequently,the subjects were submitted to the psycho- compared by Kruskal-Wallis ANOVA.For this,the subjects logic evaluations and then to 5 minutes of physiologic who recognized the smell scored"1"and those who did not recordings. recognize the smell scored"0".In the same way,the subjects who found the smell pleasant scored "2,"the ones who Test day.The subjects were again taken to the experi- found the smell unpleasant scored "0,"and the ones who ment room,where they rested for 5 minutes,after which found the smell neither pleasant nor unpleasant scored "1." the adaptation procedure from the first day was repeated. All significance tests were two-tailed and were performed The psychologic and physiologic data collected were la- at the 5%significance level. beled as the "Pretreatment"experimental phase.Subse- quently,the volunteers were conducted to another room,in Results order to receive one of the treatments.Here,they watched a Aroma perception researcher pick up one of three identical amber flasks, identified as A,B,or C,and pipette 62.5,125,or 250 uL of its The aroma perception scores obtained for each treatment contents onto a surgical mask,which was gently attached to group are shown in Table 1. their face using just the top straps,so as to allow some air The ANOVA revealed no significant differences among circulation under the mask.Immediately after the inhala- the treatment groups regarding either familiarity(p=0.47)or tion period(5 minutes),the mask was removed,put into a hedonicity(p=0.32)of the aromas. sealed container and taken out of the room.The volunteers In the groups SO and TT,all the volunteers sad "yes"to were then asked the following questions:(1)Could you the question "Could you smell anything on the mask?" smell anything on the mask?(2)Can you identify what you while in the group H2O,only 2 subjects said "yes"to the smelled?and (3)Did you find the smell pleasant,unpleas-same question,although they could not identify the smell ant,or neither?They were then taken back to the experi- nor did they find it either pleasant or unpleasant. ment room and submitted to the anxiogenic condition as follows:Immediately before being given the test instruc- Psychologic measurements tions,the participant was submitted to psychologic and Data are presented as median()and interquartile range 30 seconds of physiologic evaluations.These data were la- beled as the "Before"experimental phase.After listening to (Q-Q3) the recorded instructions,the participant then performed the task,during which his physiologic measurements were TABLE 1.AROMA PERCEPTION recorded.After 50 words,a pause was made for a second Aroma perception scores set of psychologic evaluations.These new data were labeled as the"During"experimental phase.Immediately following Familiarity Hedonicity (Did you the evaluations,the test was restarted and continued up to (Can you identify find the smell pleasant, the last color or until the end of the scheduled time.The Treatment what you smelled?) unpleasant or neither?) participant then rested for 5 minutes,after which all the 2.0(1.8-2.0) physiologic and psychologic parameters were again evalu- S025 0.50.0-1.0) SOs 1.0(1.0-1.0) 2.02.0-2.0) ated.This final set of data was labeled as the "After"ex- S010 1.0(0.0-1.0) 2.0(1.0-2.0) perimental phase. 1.0(0.0-1.0) 2.01.8-2.0) The room where the aromas were administered to the volunteers was left ventilated for about 2 hours between Data are presented as median and interquartile range (Q-Q3). tests. SO25,SOs,SOo represent essential oil of sweet orange 2.5,5,or 10 drops. Gas chromatography/mass spectrometry(GC/MS) Familiarity scores:Subjects who identified the smell scored "1" and those who did not scored "0". analysis of the essential oils Hedonicity scores:Subjects who found the smell pleasant scored "2",the ones who found the smell unpleasant scored "0",and the GC/MS analysis was performed as described in a previ- ones who found the smell neither pleasant or unpleasant scored "1". ous article. SO,essential oil of sweet orange;TT,essential oil of tea tree
It is worth mentioning that both of the essential oils used are considered to be safe. Sweet orange oil is on the U.S. Food and Drug Administration’s GRAS (Generally Regarded as Safe) List,18 while tea tree oil is generally considered to be nontoxic and nonirritant, although it may cause sensitization in some individuals.19 Procedure The selected volunteers attended the laboratory on 2 consecutive days. The first day was used for adaptation, and the second for the actual test. Adaptation day. The subjects were taken to the experiment room, which was already organized and equipped with the necessary apparatuses for the execution of the test. Subsequently, the subjects were submitted to the psychologic evaluations and then to 5 minutes of physiologic recordings. Test day. The subjects were again taken to the experiment room, where they rested for 5 minutes, after which the adaptation procedure from the first day was repeated. The psychologic and physiologic data collected were labeled as the ‘‘Pretreatment’’ experimental phase. Subsequently, the volunteers were conducted to another room, in order to receive one of the treatments. Here, they watched a researcher pick up one of three identical amber flasks, identified as A, B, or C, and pipette 62.5, 125, or 250 lL of its contents onto a surgical mask, which was gently attached to their face using just the top straps, so as to allow some air circulation under the mask. Immediately after the inhalation period (5 minutes), the mask was removed, put into a sealed container and taken out of the room. The volunteers were then asked the following questions: (1) Could you smell anything on the mask? (2) Can you identify what you smelled? and (3) Did you find the smell pleasant, unpleasant, or neither? They were then taken back to the experiment room and submitted to the anxiogenic condition as follows: Immediately before being given the test instructions, the participant was submitted to psychologic and 30 seconds of physiologic evaluations. These data were labeled as the ‘‘Before’’ experimental phase. After listening to the recorded instructions, the participant then performed the task, during which his physiologic measurements were recorded. After 50 words, a pause was made for a second set of psychologic evaluations. These new data were labeled as the ‘‘During’’ experimental phase. Immediately following the evaluations, the test was restarted and continued up to the last color or until the end of the scheduled time. The participant then rested for 5 minutes, after which all the physiologic and psychologic parameters were again evaluated. This final set of data was labeled as the ‘‘After’’ experimental phase. The room where the aromas were administered to the volunteers was left ventilated for about 2 hours between tests. Gas chromatography/mass spectrometry (GC/MS) analysis of the essential oils GC/MS analysis was performed as described in a previous article.9 Statistical analyses The data collected during the adaptation phase were not analyzed as this phase was only intended to habituate the participants to the environment and apparatus that would be used on the following day. All the psychologic data obtained during the test were analyzed by Friedman’s analysis of variance (ANOVA), for each treatment group, followed by Tukey-type test for post hoc comparisons. All the physiologic data obtained were analyzed using a two-factor ANOVA for repeated measures followed by Tukey’s test for post-hoc comparisons. The STAI-Trait scores used in the selection process were analyzed by Kruskal-Wallis ANOVA. The familiarity and hedonicity of the aromas were also compared by Kruskal-Wallis ANOVA. For this, the subjects who recognized the smell scored ‘‘1’’ and those who did not recognize the smell scored ‘‘0’’. In the same way, the subjects who found the smell pleasant scored ‘‘2,’’ the ones who found the smell unpleasant scored ‘‘0,’’ and the ones who found the smell neither pleasant nor unpleasant scored ‘‘1.’’ All significance tests were two-tailed and were performed at the 5% significance level. Results Aroma perception The aroma perception scores obtained for each treatment group are shown in Table 1. The ANOVA revealed no significant differences among the treatment groups regarding either familiarity ( p = 0.47) or hedonicity ( p = 0.32) of the aromas. In the groups SO and TT, all the volunteers sad ‘‘yes’’ to the question ‘‘Could you smell anything on the mask?’’, while in the group H2O, only 2 subjects said ‘‘yes’’ to the same question, although they could not identify the smell nor did they find it either pleasant or unpleasant. Psychologic measurements Data are presented as median (x˜) and interquartile range (Q1–Q3). Table 1. Aroma Perception Aroma perception scores Treatment Familiarity (Can you identify what you smelled?) Hedonicity (Did you find the smell pleasant, unpleasant or neither?) SO2.5 0.5 (0.0–1.0) 2.0 (1.8–2.0) SO5 1.0 (1.0–1.0) 2.0 (2.0–2.0) SO10 1.0 (0.0–1.0) 2.0 (1.0–2.0) TT 1.0 (0.0–1.0) 2.0 (1.8–2.0) Data are presented as median and interquartile range (Q1–Q3). SO2.5, SO5, SO10 represent essential oil of sweet orange 2.5, 5, or 10 drops. Familiarity scores: Subjects who identified the smell scored ‘‘1’’ and those who did not scored ‘‘0’’. Hedonicity scores: Subjects who found the smell pleasant scored ‘‘2’’, the ones who found the smell unpleasant scored ‘‘0’’, and the ones who found the smell neither pleasant or unpleasant scored ‘‘1’’. SO, essential oil of sweet orange; TT, essential oil of tea tree. 800 GOES ET AL
SWEET ORANGE AROMA AND ANXIETY 801 ANOVA results are described here,while post hoc results STAl-state.Friedman's ANOVA revealed no significant are shown in Table 2. differences among experimental phases for the groups SO25 It is worth calling attention to the fact that the treatment's (p=0.063)and SO1o (p=0.098),although differences were effect is revealed by the lack of significant alterations in the found for groups SOs (p<0.001),TT (p=0.001)and H2O observed parameters throughout the test. (p=0.005) STAl-trait.Kruskal-Wallis ANOVA found no significant VAMS-tranguilization.Friedman's ANOVA revealed no differences in trait-anxiety(p=0.79)among treatment groups significant differences among experimental phases for the [(Q1-Q3:S025=34.0(31.0-38.0:S05=32.0(30.8-38.0: group SO10(p=0.062),although differences were found for S010=33.5(29.8-35.8):TT=35.5(32.8-37.3:H20=36.0 groups SO2.5(p=0.015),SOs(p=0.003),TT(p=0.007,and 33.0-37.5)1 H2O(p=0.018). TABLE 2.SUMMARY OF THE PSYCHOLOGIC RESULTS Experimental phase Parameter Treatment Pretreatment Before During After STAI-state(points) S02.5 30.5(25.0-32.5) 26.5(23.5-31.5) 37.0(30.0-40.2) 33.0(30.0-34.0) SOs 31.5(30.3-33.0) 28.5(26.5-31.0) 36.5(30.3-41.3) 31.5(28.5-35.3)b,c S010 32.5(30.7-36.2) 32.0(27.5-33.2) 39.0(35.241.2) 33.0(32.5-34.5) TT 30.0(27.0-33.2) 28.0(24.0-29.2) 36.5(31.7-39.0 28.5(26.2-34.2),c H2O 32.5(30.5-36.0) 32.5(30.5-35.8) 41.0(32.3-43.3 36.0(30.3-42.3b.c Tranquilization (cm) S025 34.4(31.9-37.3) 35.9(33.0-37.9 25.8(23.8-30.7 33.1(30.9-34.9,e SOs 33.8(32.3-35.6) 35.1(33.438.5) 31.0(24.0-33.0 32.127.8-36.5,c S010 34.3(25.1-38.1) 32.1(29.0-38.8) 23.3(21.5-30.4) 33.6(24.0-37.8) TT 35.0(29.7-36.8) 35.6(31.4-36.1) 27.0(23.3-30.6) 34.3(29.4-36.2) H,O 31.8(27.8-35.2) 29.2(27.3-33.5 26.8(19.3-28.7 29.023.2-30.1,e Tranquilization:tense/ S02.5 8.7(7.6-9.3) 9.1(8.8-9.2) 6.2(4.2-7.9) 8.1(7.3-8.4) relaxed item(cm) SOs 8.6(8.2-9.6) 8.6(7.9-9.5) 7.1(5.8-8.1) 7.8(7.4-8.9e S010 8.1(6.3-10.0) 8.6(7.3-9.9) 6.0(4.4-6.7) 8.4(6.2-9.5) TT 8.5(7.0-9.4) 8.9(7.9-9.3)a 5.9(4.0-7.9 8.6(7.9-9.2)c H,O 8.4(8.0-9.6) 7.9(7.3-8.8)a 7.0(3.7-75) 6.6(4.7-7.8) Mental sedation(cm) S02.5 12.2(7.2-17.7) 11.3(4.7-13.6) 9.1(6.5-10.5) 10.1(6.1-12.4) S05 9.5(5.0-14.0) 9.6(6.2-14.9) 9.2(5.4-11.7) 10.7(7.6-12.7) S010 11.1(9.9-12.2) 14.0(9.2-20.2) 9.4(3.2-13.1) 8.6(3.4-15.5) TT 10.3(7.5-14.0) 8.3(5.3-11.9) 10.5(6.8-13.5) 8.7(5.0-12.0) H2O 12.8(6.4-16.9) 7.0(4.8-13.0) 9.9(7.6-13.3) 10.6(6.4-12.6) Physical sedation(cm) S02.5 12.1(7.8-14.5 5.7(5.0-9.6) 62(4.2-82) 9.4(7.0-10.9) S05 6.2(5.5-10.8) 7.2(5.8-11.0) 7.8(5.8-11.6) 7.1(5.7-13.0) SO10 8.6(2.4-12.5) 9.3(2.3-15.6) 7.3(0.7-11.2) 7.7(1.2-12.9P,e TT 8.0(6.7-10.6) 7.2(5.8-12.1) 8.6(6.5-12.1) 7.1(4.5-11.0) H2O 10.9(6.5-14.0) 7.6(3.6-13.8) 8.0(5.5-10.3) 7.9(3.1-10.1) Physical sedation: S02.5 3.6(1.6-4.6) 2.1(1.5-2.6) 1.8(1.3-2.1) 2.3(1.7-3.0) lethargic/energetic S05 2.8(1.2-3.7 2.9(1.8-4.2) 2.9(1.94.5) 2.71.3-3.8) item (cm) S010 3.2(2.1-3.5) 3.1(1.7-3.8) 1.7(0.2-2.9) 1.5(0.4-3.4)b TT 2.6(1.5-4.3) 2.5(2.1-3.6) 2.5(1.2-4.1) 1.7(0.9-3.1) H,O 3.4(1.9-4.9) 2.4(1.5-3.1) 1.9(1.2-3.1) 2.2(0.9-3.1) Others feelings S02.5 4.2(3.3-6.5) 4.1(3.1-7.3) 4.5(3.7-7.5) 3.6(3.5-5.9) and attitudes (cm) SOs 4.1(2.9-6.3) 4.4(2.0-7.6) 5.2(2.0-8.2) 5.7(3.2-7.4) SO10 5.8(2.7-7.3) 7.5(4.0-10.1)° 7.3(3.2-10.3) 6.7(2.6-9.9) TT 4.5(3.0-10.3) 4.4(3.2-9.7 7.1(5.1-9.2) 5.5(3.2-8.8) H2O 5.8(3.5-11.7 9.2(4.1-11.3) 7.7(4.2-10.9) 7.1(3.6-11.5) Others feelings and S02.5 0.7(0.3-1.6) 0.7(0.4-1.5) 1.1(0.6-1.7) 0.8(0.3-1.3) attitudes: SOs 0.5(0.4-1.6) 0.5(0.2-0.9) 1.0(0.6-1.2) 0.9(0.4-1.3) withdrawn/ S010 1.2(0.2-1.9) 1.5(0.3-2.2) 1.7(0.4-3.0) 1.3(0.2-2.6) gregarious item(cm) TT 1.4(0.7-1.8) 1.2(0.4-1.9) 1.8(1.2-2.3) 1.5(0.7-2.2) H2O 1.8(0.5-2.9) 1.8(0.9-2.8) 1.7(0.6-2.3) 1.5(0.6-2.3) Data are presented as median and interquartile range (O -Q3).The treatments in bold are those that showed no significant differences in state-anxiety among the experimental phases.SO25,SOs,SOio represent essential oil of sweet orange 2.5,5,or 10 drops. Significantly different from "Pretreatment." PSignificantly different from "Before." y different from“During.” p<0.05
ANOVA results are described here, while post hoc results are shown in Table 2. It is worth calling attention to the fact that the treatment’s effect is revealed by the lack of significant alterations in the observed parameters throughout the test. STAI-trait. Kruskal-Wallis ANOVA found no significant differences in trait-anxiety ( p = 0.79) among treatment groups [x˜ (Q1–Q3): SO2.5 = 34.0 (31.0–38.0); SO5 = 32.0 (30.8–38.0); SO10 = 33.5 (29.8–35.8); TT = 35.5 (32.8–37.3); H2O = 36.0 (33.0–37.5)]. STAI-state. Friedman’s ANOVA revealed no significant differences among experimental phases for the groups SO2.5 ( p = 0.063) and SO10 ( p = 0.098), although differences were found for groups SO5 ( p < 0.001), TT (p = 0.001) and H2O ( p = 0.005). VAMS–tranquilization. Friedman’s ANOVA revealed no significant differences among experimental phases for the group SO10 ( p = 0.062), although differences were found for groups SO2.5 ( p = 0.015), SO5 ( p = 0.003), TT ( p = 0.007), and H2O ( p = 0.018). Table 2. Summary of the Psychologic Results Experimental phase Parameter Treatment Pretreatment Before During After STAI-state (points) SO2.5 30.5 (25.0–32.5) 26.5 (23.5–31.5) 37.0 (30.0–40.2) 33.0 (30.0–34.0) SO5 31.5 (30.3–33.0) 28.5 (26.5–31.0)a 36.5 (30.3–41.3)b 31.5 (28.5–35.3)b,c SO10 32.5 (30.7–36.2) 32.0 (27.5–33.2) 39.0 (35.2–41.2) 33.0 (32.5–34.5) TT 30.0 (27.0–33.2) 28.0 (24.0–29.2)a 36.5 (31.7–39.0)b 28.5 (26.2–34.2)b,c H2O 32.5 (30.5–36.0) 32.5 (30.5–35.8) 41.0 (32.3–43.3)b 36.0 (30.3–42.3)b,c Tranquilization (cm) SO2.5 34.4 (31.9–37.3) 35.9 (33.0–37.9)a 25.8 (23.8–30.7)b 33.1 (30.9–34.9)b,c SO5 33.8 (32.3–35.6) 35.1 (33.4–38.5)a 31.0 (24.0–33.0)b 32.1 (27.8–36.5)b,c SO10 34.3 (25.1–38.1) 32.1 (29.0–38.8) 23.3 (21.5–30.4) 33.6 (24.0–37.8) TT 35.0 (29.7–36.8) 35.6 (31.4–36.1) 27.0 (23.3–30.6)b 34.3 (29.4–36.2)c H2O 31.8 (27.8–35.2) 29.2 (27.3–33.5)a 26.8 (19.3–28.7)b 29.0 (23.2–30.1)b,c Tranquilization: tense/ relaxed item (cm) SO2.5 8.7 (7.6–9.3) 9.1 (8.8–9.2) 6.2 (4.2–7.9) 8.1 (7.3–8.4) SO5 8.6 (8.2–9.6) 8.6 (7.9–9.5) 7.1 (5.8–8.1)b 7.8 (7.4–8.9)b,c SO10 8.1 (6.3–10.0) 8.6 (7.3–9.9) 6.0 (4.4–6.7) 8.4 (6.2–9.5) TT 8.5 (7.0–9.4) 8.9 (7.9–9.3)a 5.9 (4.0–7.9)b 8.6 (7.9–9.2)b,c H2O 8.4 (8.0–9.6) 7.9 (7.3–8.8)a 7.0 (3.7–7.5)b 6.6 (4.7–7.8) Mental sedation (cm) SO2.5 12.2 (7.2–17.7) 11.3 (4.7–13.6) 9.1 (6.5–10.5) 10.1 (6.1–12.4) SO5 9.5 (5.0–14.0) 9.6 (6.2–14.9) 9.2 (5.4–11.7) 10.7 (7.6–12.7) SO10 11.1 (9.9–12.2) 14.0 (9.2–20.2) 9.4 (3.2–13.1) 8.6 (3.4–15.5) TT 10.3 (7.5–14.0) 8.3 (5.3–11.9) 10.5 (6.8–13.5) 8.7 (5.0–12.0) H2O 12.8 (6.4–16.9) 7.0 (4.8–13.0) 9.9 (7.6–13.3) 10.6 (6.4–12.6) Physical sedation (cm) SO2.5 12.1 (7.8–14.5) 5.7 (5.0–9.6) 6.2 (4.2–8.2) 9.4 (7.0–10.9) SO5 6.2 (5.5–10.8) 7.2 (5.8–11.0) 7.8 (5.8–11.6) 7.1 (5.7–13.0) SO10 8.6 (2.4–12.5) 9.3 (2.3–15.6)a 7.3 (0.7–11.2)b 7.7 (1.2–12.9)b,c TT 8.0 (6.7–10.6) 7.2 (5.8–12.1) 8.6 (6.5–12.1) 7.1 (4.5–11.0) H2O 10.9 (6.5–14.0) 7.6 (3.6–13.8) 8.0 (5.5–10.3) 7.9 (3.1–10.1) Physical sedation: lethargic/energetic item (cm) SO2.5 3.6 (1.6–4.6) 2.1 (1.5–2.6) 1.8 (1.3–2.1) 2.3 (1.7–3.0) SO5 2.8 (1.2–3.7) 2.9 (1.8–4.2) 2.9 (1.9–4.5) 2.7 (1.3–3.8) SO10 3.2 (2.1–3.5) 3.1 (1.7–3.8) 1.7 (0.2–2.9)b 1.5 (0.4–3.4)b,c TT 2.6 (1.5–4.3) 2.5 (2.1–3.6) 2.5 (1.2–4.1) 1.7 (0.9–3.1) H2O 3.4 (1.9–4.9) 2.4 (1.5–3.1) 1.9 (1.2–3.1) 2.2 (0.9–3.1) Others feelings and attitudes (cm) SO2.5 4.2 (3.3–6.5) 4.1 (3.1–7.3) 4.5 (3.7–7.5) 3.6 (3.5–5.9) SO5 4.1 (2.9–6.3) 4.4 (2.0–7.6) 5.2 (2.0–8.2) 5.7 (3.2–7.4) SO10 5.8 (2.7–7.3) 7.5 (4.0–10.1)a 7.3 (3.2–10.3) 6.7 (2.6–9.9)c TT 4.5 (3.0–10.3) 4.4 (3.2–9.7) 7.1 (5.1–9.2) 5.5 (3.2–8.8) H2O 5.8 (3.5–11.7) 9.2 (4.1–11.3) 7.7 (4.2–10.9) 7.1 (3.6–11.5) Others feelings and attitudes: withdrawn/ gregarious item (cm) SO2.5 0.7 (0.3–1.6) 0.7 (0.4–1.5) 1.1 (0.6–1.7) 0.8 (0.3–1.3) SO5 0.5 (0.4–1.6) 0.5 (0.2–0.9) 1.0 (0.6–1.2) 0.9 (0.4–1.3) SO10 1.2 (0.2–1.9) 1.5 (0.3–2.2)a 1.7 (0.4–3.0)b 1.3 (0.2–2.6)c TT 1.4 (0.7–1.8) 1.2 (0.4–1.9) 1.8 (1.2–2.3) 1.5 (0.7–2.2) H2O 1.8 (0.5–2.9) 1.8 (0.9–2.8) 1.7 (0.6–2.3) 1.5 (0.6–2.3) Data are presented as median and interquartile range (Q1–Q3). The treatments in bold are those that showed no significant differences in state-anxiety among the experimental phases. SO2.5, SO5, SO10 represent essential oil of sweet orange 2.5, 5, or 10 drops. a Significantly different from ‘‘Pretreatment.’’ b Significantly different from ‘‘Before.’’ c Significantly different from ‘‘During.’’ a,b,cp < 0.05. SWEET ORANGE AROMA AND ANXIETY 801
802 GOES ET AL. VAMS-tranquilization:Tense/relaxed item.Friedman's ing"in relation to "Before"(p=0.001).The treatment effect ANOVA revealed no significant differences among experi- was not significant (p=0.126). mental phases for groups SO25 (p=0.078)and SO10 (p=0.126),although differences were found for groups SOs CG/MS analysis.The volatile composition of sweet or- (p=0.044),TT(p=0.003),andH20(p=0.003). ange and tea tree essential oils is shown in Tables 4 and 5, respectively. VAMS-mental sedation.Friedman's ANOVA revealed no significant differences among test phases for all of the Discussion groups[SO2.5(p=0.327):S05(p=0.985:SO1o(p=0.060):TT (p=0.832):andH20(p=0.369)l. The aim of this work was to evaluate the potential anxi- olytic effect of sweet orange (C.sinensis)essential oil in VAMS-physical sedation.Friedman's ANOVA revealed healthy volunteers submitted to an anxiogenic situation.The no significant differences among test phases for the groups experimental model of anxiety,as employed here,was S02.5(p=0.615),S05(p=0.403),TT(p=0.138),andH20 shown to be valid,since the subjects from the control groups (p=0.271).However,differences were found for the group behaved as expected,significantly increasing their anxiety SOo(p=0.009).Separate analyses of the individual items of signs/symptoms during the Stroop task. this category,for this group,showed differences among test The obtained results showed the anxiolytic properties of phases for the lethargic/energetic item(p=0.008). sweet orange essential oil through the lack of a significant increase in state-anxiety and tension,and the lack of a sig- VAMS-other feelings and attitudes.Friedman's ANOVA nificant decrease in tranquility,presented by the individuals exposed to the aroma.Of the two doses(2.5 and 10 drops) revealed no significant differences for the groups SO2.5 (p=0.145),SOs(p=0.814),TT(p=0.110),andH20 that demonstrated anxiolytic activity,the higher one seemed (p=0.618).However,differences were found for the group to be better at preventing anxious symptoms,as its results SOo(p=0.027).Separate analyses of the individual items of were further from reaching statistical significance and were this category,for this group,showed differences among test observed in a greater number of parameters.It is reasonable phases for the withdrawn/gregarious item(p=0.002). to question the fact that the intermediate dose failed to produce an effect.However,this activity profile was ob- Physiologic measurements served before,when the same aroma was tested in animals.9 This lack of a dose/effect relation is common when dealing Data are presented as mean and standard error of the with a mixture of compounds instead of a pure substance. mean in Table 3. Interestingly,the observed anxiolytic effects were not followed by sedative/hypnotic effects.On the contrary,at Heart rate.The ANOVA revealed that the interaction the highest dose,sweet orange oil made the volunteers feel between test phase and treatment was not significant more energetic while performing the test.They also felt more (p=0.641);therefore the two main effects were analyzed.The introverted.It is tempting to speculate that these two test phase effect was significant (p97%),9,20 have shown very clear anxiolytic effects, TABLE 3.SUMMARY OF PHYSIOLOGIC RESULTS Experimental phase Parameters Treatment Pretreatment Before During After Heart rate (beats/min) S025 76.7±3.5 70.5±3.2 108.8±9.4 77.7±3.7 SOs 71.5±45 69.9±3.6 95.7±7.6 72.1±5.1 S010 76.5±5.2 74.9±5.3 107.1±10.8 74.5±5.0 TT 80.3±7.0 79.5±7.4 127.1±20.8 75.1±4.9 H2O 69.5±2.9 69.9±3.9 103.1±10.3 70.6±2.5 Electromyogram of the S025 1.1±0.3 0.9±0.2 2.3±0.5 0.9±0.3 gastrocnemius muscle (uV) SO5 3.1±1.0 2.1±0.6 3.7±0.8 1.7±0.7 S010 1.5±0.4 0.8±0.2 3.6±1.5 1.0±0.3 TT 1.0±0.2 1.7±0.5 2.2±0.6 1.6±0.5 H2O 2.7±0.7 1.8±0.4 4.0±0.6 2.5±0.5 Data are presented as means+standard error of the mean. SO2,SOs,SOo represent essential oil of sweet orange 2.5,5,or 10 drops.TT,essential oil of tea tree
VAMS–tranquilization: Tense/relaxed item. Friedman’s ANOVA revealed no significant differences among experimental phases for groups SO2.5 ( p = 0.078) and SO10 ( p = 0.126), although differences were found for groups SO5 ( p = 0.044), TT ( p = 0.003), and H2O ( p = 0.003). VAMS–mental sedation. Friedman’s ANOVA revealed no significant differences among test phases for all of the groups [SO2.5 ( p = 0.327); SO5 ( p = 0.985); SO10 ( p = 0.060); TT (p = 0.832); and H2O ( p = 0.369)]. VAMS–physical sedation. Friedman’s ANOVA revealed no significant differences among test phases for the groups SO2.5 ( p = 0.615), SO5 ( p = 0.403), TT ( p = 0.138), and H2O ( p = 0.271). However, differences were found for the group SO10 ( p = 0.009). Separate analyses of the individual items of this category, for this group, showed differences among test phases for the lethargic/energetic item ( p = 0.008). VAMS–other feelings and attitudes. Friedman’s ANOVA revealed no significant differences for the groups SO2.5 ( p = 0.145), SO5 ( p = 0.814), TT ( p = 0.110), and H2O ( p = 0.618). However, differences were found for the group SO10 ( p = 0.027). Separate analyses of the individual items of this category, for this group, showed differences among test phases for the withdrawn/gregarious item ( p = 0.002). Physiologic measurements Data are presented as mean and standard error of the mean in Table 3. Heart rate. The ANOVA revealed that the interaction between test phase and treatment was not significant ( p = 0.641); therefore the two main effects were analyzed. The test phase effect was significant ( p 97%),9,20 have shown very clear anxiolytic effects, Table 3. Summary of Physiologic Results Experimental phase Parameters Treatment Pretreatment Before During After Heart rate (beats/min) SO2.5 76.7 – 3.5 70.5 – 3.2 108.8 – 9.4 77.7 – 3.7 SO5 71.5 – 4.5 69.9 – 3.6 95.7 – 7.6 72.1 – 5.1 SO10 76.5 – 5.2 74.9 – 5.3 107.1 – 10.8 74.5 – 5.0 TT 80.3 – 7.0 79.5 – 7.4 127.1 – 20.8 75.1 – 4.9 H2O 69.5 – 2.9 69.9 – 3.9 103.1 – 10.3 70.6 – 2.5 Electromyogram of the gastrocnemius muscle (lV) SO2.5 1.1 – 0.3 0.9 – 0.2 2.3 – 0.5 0.9 – 0.3 SO5 3.1 – 1.0 2.1 – 0.6 3.7 – 0.8 1.7 – 0.7 SO10 1.5 – 0.4 0.8 – 0.2 3.6 – 1.5 1.0 – 0.3 TT 1.0 – 0.2 1.7 – 0.5 2.2 – 0.6 1.6 – 0.5 H2O 2.7 – 0.7 1.8 – 0.4 4.0 – 0.6 2.5 – 0.5 Data are presented as means – standard error of the mean. SO2.5, SO5, SO10 represent essential oil of sweet orange 2.5, 5, or 10 drops. TT, essential oil of tea tree. 802 GOES ET AL
SWEET ORANGE AROMA AND ANXIETY 803 TABLE 4.VOLATILE COMPOSITION female patients exposed to this oil,diffused in a waiting OF CITRUS SINENSIS ESSENTIAL OIL room before a dental procedure,showed lower levels of state Peak RT (min) Compounds (%) RI anxiety compared to control patients who were exposed to air only. 1 9.958 Limonene 54.481027 It could be argued that Lehrner's results were only due to 2 13.342 trans-p-Mentha-2,8-dien-1-ol 2.001120 unspecific effects of any aroma,as his study did not use any 3 13.892 cis-p-Menth-2,8-dienol 2.971135 aromatic control and tested only a single oil dose,which was 4 15.458 0.831177 not very precise.The results presented here serve to rule out 5 16.100 NI 1.181195 this possibility,as tea tree essential oil did not show any 6 16.233 2.041198 anxiolytic activity.Still,it could be argued that tea tree was 7 16.942 trans-Carveol 5.59 1218 not a very good aromatic control,as it may not have been as 8 17.425 cis-Carveol 1.98 1231 9 17.842 Carvona 7.65 1243 familiar to the volunteers as the orange smell,and familiarity 10 19.017 NI might have provided comfort to the subjects.However,sta- 0.68 1275 11 19.517 NI 1.12 1289 tistical analysis showed no differences between the orange 2 21.475 Limonene-1,2-diol 13.47 1345 and tea tree essential oils in this respect.This result was 13 23.558 4.67 1405 probably due to the fact that tea tree essential oil has an 4 24.842 NI 1.34 1444 astringent,camphorous scent,similar to eucalyptus and Total 100.00 other medicinal plants.Thus,despite the fact that the vol- unteers may not have known exactly what the scent was,it RT,retention time;RI,retention index;NI, nonidentified com- was not unfamiliar to them.Another characteristic of the oils pounds. that could have interfered with the results is their hedonicity, as pleasant aromas may induce a state of well-being in indicating this monoterpene as the responsible constituent people.But again,there were no significant differences be- for the anxiolytic activity of citrus oils.Therefore,it is pos- tween orange and tea tree,with both aromas generally being sible that,in the present study,a purer sweet orange oil considered pleasant.Therefore,it is reasonable to believe sample (around 90%limonene)could have led to more ro- that the anxiolytic effects observed in this study were due to bust results.It is also possible that the duration of exposure sweet orange essential oil per se,with the strongest argument to the essential oil needs adjustments,as aromatherapists' in favor of this idea being the fact that not all doses of this oil recommendations vary from a few breaths to a few minutes. were effective. For these reasons,future studies must be designed in order Regarding the physiologic parameters,there was no to optimize the posology of sweet orange aroma for the relief prevention of the stress response to the Stroop test in any of of anxiety symptoms. the treatment groups.However,this result does not weaken Nevertheless,the present study makes it clear that sweet the case for sweet orange aroma as an anxiety reducer, orange essential oil has anxiolytic properties in humans, since diazepam,a classic anxiolytic drug,is also unable to corroborating a previous study by Lehrner et al.2 in which prevent physiologic alterations provoked by anxiogenic 112223 situations. Nevertheless,it is worth pointing out that this research TABLE 5.VOLATILE COMPOSITION does present some sample-related limitations.First,all OF MELALEUCA ALTERNIFOLIA ESSENTIAL OIL subjects were graduate students,under 30 years of age.This was not only because it was convenient to recruit these Peak RT(min) Compounds (%) RI volunteers,but also because the model of anxiety used was 6.575 x-Tujene 0.58 924 validated for this population.Still,it would be valuable to 2 6.800 g-Pinene 2.17 931 test the effects of sweet orange aroma in subjects with dif- 3 8.208 B-Pinene 0.55 976 ferent levels of education and in different age groups.Sec- 8.600 Myrcene 0.65 988 ond,only men were selected for this study and,despite the 5 9.550 x-Terpinene 6.97 1016 fact that the anxiolytic effect of sweet orange aroma in 6 9.825 p-Cymene 5.30 1024 women has been shown before,21 it would be interesting to 9.983 Limonene 1.02 1027 test women in the conditions presented here,especially if the 8 10.100 1,8-Cineole 4.31 1031 menstrual cycle was controlled.Third,the sample size was 11.050 -Terpinene 17.69 1058 0 12.017 Terpinolene 2.76 1084 rather small and,although it was large enough to give the 11 15.633 Terpinen-4-ol 49.83 1082 experiment sufficient power to detect statistical differences 12 16.108 a-Terpineol 3.79 1194 future studies with larger sample sizes could reinforce the 23.992 (E)-Caryophyllene 0.37 1417 results. 14 24.617 Aromadendrene 1.19 1437 15 25.325 Allo-aromadendrene 0.49 1458 16 26.350 Viridiflorene 0.59 1489 Conclusions 17 26.500 NI 0.37 1494 In conclusion,although more investigations are necessary 18 27.217 -Cadinene 0.95 1517 in order to clarify the clinical relevance of sweet orange es 19 29.350 Globulol 0.42 1585 sential oil as an anxiety treatment,the present work has Total 99.26 strengthened the idea that this oil has anxiolytic properties RT,retention time;RI,retention index;NI,nonidentified com- that cannot be accredited to expectancy biases,massage, pounds. or therapeutic relationship which,according to some
indicating this monoterpene as the responsible constituent for the anxiolytic activity of citrus oils. Therefore, it is possible that, in the present study, a purer sweet orange oil sample (around 90% limonene) could have led to more robust results. It is also possible that the duration of exposure to the essential oil needs adjustments, as aromatherapists’ recommendations vary from a few breaths to a few minutes. For these reasons, future studies must be designed in order to optimize the posology of sweet orange aroma for the relief of anxiety symptoms. Nevertheless, the present study makes it clear that sweet orange essential oil has anxiolytic properties in humans, corroborating a previous study by Lehrner et al.,21 in which female patients exposed to this oil, diffused in a waiting room before a dental procedure, showed lower levels of state anxiety compared to control patients who were exposed to air only. It could be argued that Lehrner’s results were only due to unspecific effects of any aroma, as his study did not use any aromatic control and tested only a single oil dose, which was not very precise. The results presented here serve to rule out this possibility, as tea tree essential oil did not show any anxiolytic activity. Still, it could be argued that tea tree was not a very good aromatic control, as it may not have been as familiar to the volunteers as the orange smell, and familiarity might have provided comfort to the subjects. However, statistical analysis showed no differences between the orange and tea tree essential oils in this respect. This result was probably due to the fact that tea tree essential oil has an astringent, camphorous scent, similar to eucalyptus and other medicinal plants. Thus, despite the fact that the volunteers may not have known exactly what the scent was, it was not unfamiliar to them. Another characteristic of the oils that could have interfered with the results is their hedonicity, as pleasant aromas may induce a state of well-being in people. But again, there were no significant differences between orange and tea tree, with both aromas generally being considered pleasant. Therefore, it is reasonable to believe that the anxiolytic effects observed in this study were due to sweet orange essential oil per se, with the strongest argument in favor of this idea being the fact that not all doses of this oil were effective. Regarding the physiologic parameters, there was no prevention of the stress response to the Stroop test in any of the treatment groups. However, this result does not weaken the case for sweet orange aroma as an anxiety reducer, since diazepam, a classic anxiolytic drug, is also unable to prevent physiologic alterations provoked by anxiogenic situations.11,22,23 Nevertheless, it is worth pointing out that this research does present some sample-related limitations. First, all subjects were graduate students, under 30 years of age. This was not only because it was convenient to recruit these volunteers, but also because the model of anxiety used was validated for this population.11 Still, it would be valuable to test the effects of sweet orange aroma in subjects with different levels of education and in different age groups. Second, only men were selected for this study and, despite the fact that the anxiolytic effect of sweet orange aroma in women has been shown before,21 it would be interesting to test women in the conditions presented here, especially if the menstrual cycle was controlled. Third, the sample size was rather small and, although it was large enough to give the experiment sufficient power to detect statistical differences, future studies with larger sample sizes could reinforce the results. Conclusions In conclusion, although more investigations are necessary in order to clarify the clinical relevance of sweet orange essential oil as an anxiety treatment, the present work has strengthened the idea that this oil has anxiolytic properties that cannot be accredited to expectancy biases, massage, or therapeutic relationship which, according to some Table 4. Volatile Composition of Citrus sinensis Essential Oil Peak RT (min) Compounds (%) RI 1 9.958 Limonene 54.48 1027 2 13.342 trans-p-Mentha-2,8-dien-1-ol 2.00 1120 3 13.892 cis-p-Menth-2,8-dienol 2.97 1135 4 15.458 NI 0.83 1177 5 16.100 NI 1.18 1195 6 16.233 NI 2.04 1198 7 16.942 trans-Carveol 5.59 1218 8 17.425 cis-Carveol 1.98 1231 9 17.842 Carvona 7.65 1243 10 19.017 NI 0.68 1275 11 19.517 NI 1.12 1289 12 21.475 Limonene-1,2-diol 13.47 1345 13 23.558 NI 4.67 1405 14 24.842 NI 1.34 1444 Total 100.00 RT, retention time; RI, retention ı´ndex; NI, nonidentified compounds. Table 5. Volatile Composition of Melaleuca alternifolia Essential Oil Peak RT (min) Compounds (%) RI 1 6.575 a-Tujene 0.58 924 2 6.800 a-Pinene 2.17 931 3 8.208 b-Pinene 0.55 976 4 8.600 Myrcene 0.65 988 5 9.550 a-Terpinene 6.97 1016 6 9.825 p-Cymene 5.30 1024 7 9.983 Limonene 1.02 1027 8 10.100 1,8-Cineole 4.31 1031 9 11.050 c-Terpinene 17.69 1058 10 12.017 Terpinolene 2.76 1084 11 15.633 Terpinen-4-ol 49.83 1082 12 16.108 a-Terpineol 3.79 1194 13 23.992 (E)-Caryophyllene 0.37 1417 14 24.617 Aromadendrene 1.19 1437 15 25.325 Allo-aromadendrene 0.49 1458 16 26.350 Viridiflorene 0.59 1489 17 26.500 NI 0.37 1494 18 27.217 d-Cadinene 0.95 1517 19 29.350 Globulol 0.42 1585 Total 99.26 RT, retention time; RI, retention ı´ndex; NI, nonidentified compounds. SWEET ORANGE AROMA AND ANXIETY 803
804 GOES ET AL. authors,8,24 could be responsible for most of the aromather-13.Spielberger CD,Gorsuch RL,Lushene RE.Manual for the apy outcome state-trait anxiety inventory ("self-evaluation question- naire").Palo Alto,CA:Consulting-Psychologists Press,1970 Acknowledgments 14.Gama MMA,Moura GS,Aratijo RF,Teixeira-Silva F.Trait This work was supported by Coordenacao de Aperfei- anxiety in Brazilian University Students from Aracaju.Rev coamento de Pessoal de Nivel Superior (CAPES),Conselho Psiquiatr RS 2008:30:19-24. Nacional de Desenvolvimento Cientifico e Tecnologico(CNPq) 15.Norris H.The action of sedatives on brain stem oculomotor and Fundacao de apoio a Pesquisa e a Inovacao Tecnologica system in man.Neuropharmacology 1971;10:181-191 do Estado de Sergipe(FAPITEC/SE). 16.Zuardi AW,Karniol IG.Transcultural study of a self-rating scale for subjective states.J Bras Psiquiatr 1981;30:403-406. Disclosure Statement 17.Del Porto JA,Laranjeiras RR,Masur J.Self-rating scales for subjective states-influence of instructions.J Bras Psiquiatr No competing financial interests exist. 1983;32:87-90. 18.FDA's GRAS (Generally Regarded as Safe)List.Online References document at:www.fda.gov/Food/FoodIngredientsPackaging/ 1.Kessler RC,Chiu WT,Demler O,et al.Prevalence,severity, FoodAdditives/ucm191033.htm Posted December 28,2011. and comorbidity of 12-month DSM-IV disorders in the Na- Accessed July,2012. tional Comorbidity Survey Replication.Arch Gen Psychiatry 19.Clarke S.Composition of essential oils.In:Clarke S,ed. 2005:62:617-627 Essential Chemistry for Safe Aromatherapy.London: 2.Starcevic V.Issues in the pharmacological treatment of Churchill Livingstone,2003:111-164. anxiety disorders.Australas Psychiatry 2005;13:371-374. 20.Leite MP,Fassim I Ir,Baziloni EMF,et al.Behavioral effects 3.Ravindran LN,Stein MB.The pharmacologic treatment of of essential oil of Citrus aurantium L.inhalation in rats.Rev anxiety disorders:A review of progress.IClin Psychiatry 2010; Bras Farmacogn 2008;18(suppl):661-666. 71:839-854. 21.Lehrner J,Eckersberg C,Walla P,et al.Ambient odor of 4.Kuriyama H,Watanabe S,Nakaya T,et al.Immunological orange in a dental office reduces anxiety and improves and psychological benefits of aromatherapy massage.Evid mood in female patients.Physiol Behav 2000:71:83-86 Based Complement Alternat Med 2005;2:179-184. 22.Guimaraes FS,Kohem G,Fillmann HS,et al.A simple 5.Charlesworth H.Aromatherapy and stress related health simulated public speaking test for evaluating anxiolytic disorders.Aromatherapist 1995;2:9-23. drugs.Braz J Med Biol Res 1989;22:1083-1089. 6.Cooke B,Ernest E.Aromatherapy:A systematic review.BrI 23.Albus M,Zellner A,Bondy B,et al.Influence of CGP361A, Gen Pract200050:493-496. propranolol and diazepam on autonomous reactions to 7.Roberts L,Wilson S,Greenfield S.Why aromatherapy different stressors.Prog Neuropsychopharmacol Biol Psy- works.Br J Gen Pract 2000:50:825-826. hiatry1989,13:87-97. 8.Vickers A.Why aromatherapy works(even if it doesn't)and 24.Dunning T.Applying a quality use of medicines framework why we need less research.Br J Gen Pract 2000:50:444-445. to using essential oils in nursing practice.Complement Ther 9.Faturi CB,Leite IR,Alves PB,et al.Anxiolytic-like effect of Clin Pract2005,11:172-181. sweet orange aroma in Wistar rats.Prog Neuropsycho- pharmacol Biol Psychiatry 2010:34:605-609. 10.Biaggio AMB,Natalicio L.Manual for the State-Trait Anxi- Address correspondence to: ety Inventory (STAD).Rio de Janeiro:Centro Editor de Psi- Flavia Teixeira-Silva,PhD cologia Aplicada-CEPA,1979:58. Departamento de Fisiologia 11.Teixeira-Silva F,Prado GB,Ribeiro LCG,Leite JR.The Centro de Ciencias Biologicas e da saude anxiogenic video-recorded Stroop Color-Word Test:Psy- Universidade Federal de Sergipe chological and physiological alterations and effects of diaz- 49100-000-SE Sergipe epam.Physiol Behav 2004;82:215-230. Brazil 12.Stroop JR.Studies of interference in serial verbal reactions. JExp Psychol 1935;18:643-662. E-mail:teixeira silva@terra.com.br
authors,8,24 could be responsible for most of the aromatherapy outcome. Acknowledgments This work was supported by Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES), Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) and Fundac¸a˜o de Apoio a Pesquisa e a` Inovac¸a˜o Tecnolo´gica do Estado de Sergipe (FAPITEC/SE). Disclosure Statement No competing financial interests exist. References 1. Kessler RC, Chiu WT, Demler O, et al. Prevalence, severity, and comorbidity of 12-month DSM-IV disorders in the National Comorbidity Survey Replication. Arch Gen Psychiatry 2005;62:617–627. 2. Starcevic V. Issues in the pharmacological treatment of anxiety disorders. Australas Psychiatry 2005;13:371–374. 3. Ravindran LN, Stein MB. The pharmacologic treatment of anxiety disorders: A review of progress. J Clin Psychiatry 2010; 71:839–854. 4. Kuriyama H, Watanabe S, Nakaya T, et al. Immunological and psychological benefits of aromatherapy massage. Evid Based Complement Alternat Med 2005;2:179–184. 5. Charlesworth H. Aromatherapy and stress related health disorders. Aromatherapist 1995;2:9–23. 6. Cooke B, Ernest E. Aromatherapy: A systematic review. Br J Gen Pract 2000;50:493–496. 7. Roberts L, Wilson S, Greenfield S. Why aromatherapy works. Br J Gen Pract 2000;50:825–826. 8. Vickers A. Why aromatherapy works (even if it doesn’t) and why we need less research. Br J Gen Pract 2000;50:444–445. 9. Faturi CB, Leite JR, Alves PB, et al. Anxiolytic-like effect of sweet orange aroma in Wistar rats. Prog Neuropsychopharmacol Biol Psychiatry 2010;34:605–609. 10. Biaggio AMB, Natalı´cio L. Manual for the State-Trait Anxiety Inventory (STAI). Rio de Janeiro: Centro Editor de Psicologia Aplicada-CEPA, 1979:58. 11. Teixeira-Silva F, Prado GB, Ribeiro LCG, Leite JR. The anxiogenic video-recorded Stroop Color-Word Test: Psychological and physiological alterations and effects of diazepam. Physiol Behav 2004;82:215–230. 12. Stroop JR. Studies of interference in serial verbal reactions. J Exp Psychol 1935;18:643–662. 13. Spielberger CD, Gorsuch RL, Lushene RE. Manual for the state-trait anxiety inventory (‘‘self-evaluation questionnaire’’). Palo Alto, CA: Consulting-Psychologists Press, 1970. 14. Gama MMA, Moura GS, Arau´jo RF, Teixeira-Silva F. Trait anxiety in Brazilian University Students from Aracaju. Rev Psiquiatr RS 2008;30:19–24. 15. Norris H. The action of sedatives on brain stem oculomotor system in man. Neuropharmacology 1971;10:181–191. 16. Zuardi AW, Karniol IG. Transcultural study of a self-rating scale for subjective states. J Bras Psiquiatr 1981;30:403–406. 17. Del Porto JA, Laranjeiras RR, Masur J. Self-rating scales for subjective states-influence of instructions. J Bras Psiquiatr 1983;32:87–90. 18. FDA’s GRAS (Generally Regarded as Safe) List. Online document at: www.fda.gov/Food/FoodIngredientsPackaging/ FoodAdditives/ucm191033.htm Posted December 28, 2011. Accessed July, 2012. 19. Clarke S. Composition of essential oils. In: Clarke S, ed. Essential Chemistry for Safe Aromatherapy. London: Churchill Livingstone, 2003:111–164. 20. Leite MP, Fassim J Jr, Baziloni EMF, et al. Behavioral effects of essential oil of Citrus aurantium L. inhalation in rats. Rev Bras Farmacogn 2008;18 (suppl):661–666. 21. Lehrner JJ, Eckersberg C, Walla P, et al. Ambient odor of orange in a dental office reduces anxiety and improves mood in female patients. Physiol Behav 2000;71:83–86. 22. Guimara˜es FS, Kohem G, Fillmann HS, et al. A simple simulated public speaking test for evaluating anxiolytic drugs. Braz J Med Biol Res 1989;22:1083–1089. 23. Albus M, Zellner A, Bondy B, et al. Influence of CGP361A, propranolol and diazepam on autonomous reactions to different stressors. Prog Neuropsychopharmacol Biol Psychiatry 1989;13:87–97. 24. Dunning T. Applying a quality use of medicines framework to using essential oils in nursing practice. Complement Ther Clin Pract 2005;11:172–181. Address correspondence to: Flavia Teixeira-Silva, PhD Departamento de Fisiologia Centro de Cieˆncias Biolo´gicas e da Sau´de Universidade Federal de Sergipe 49100-000 - SE Sergipe Brazil E-mail: teixeira_silva@terra.com.br 804 GOES ET AL
This article has been cited by: 1.Lillehei Angela S.,Halcon Linda L..2014.A Systematic Review of the Effect of Inhaled Essential Oils on Sleep.The Journal of Alternative and Complementary Medicine 20:6,441-451.[Abstract][Full Text HTML][Full Text PDF][Full Text PDF with Links 2.Susanna Stea,Alina Beraudi,Dalila De Pasquale.2014.Essential Oils for Complementary Treatment of Surgical Patients:State of the Art.Evidence-Based Complementary and Alternative Medicine 2014,1-6.[CrossRef]
This article has been cited by: 1. Lillehei Angela S., Halcon Linda L.. 2014. A Systematic Review of the Effect of Inhaled Essential Oils on Sleep. The Journal of Alternative and Complementary Medicine 20:6, 441-451. [Abstract] [Full Text HTML] [Full Text PDF] [Full Text PDF with Links] 2. Susanna Stea, Alina Beraudi, Dalila De Pasquale. 2014. Essential Oils for Complementary Treatment of Surgical Patients: State of the Art. Evidence-Based Complementary and Alternative Medicine 2014, 1-6. [CrossRef]
