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 vol￾unteers 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 stan￾dardized 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 per￾formed 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 re￾cording, which led them to believe that a group of profes￾sionals, 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 + Physiolo￾gical 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 dis￾tilled 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 vol￾unteers 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