Avaliação do potencial efeito ansiolítico do aroma de laranja doce (Citrus sinensis)

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(1)UNIVERSIDADE FEDERAL DE SERGIPE PRÓ-REITORIA DE PÓS-GRADUAÇÃO E PESQUISA MESTRADO EM CIÊNCIAS DA SAÚDE. TIAGO COSTA GOES. AVALIAÇÃO DO POTENCIAL EFEITO ANSIOLÍTICO DO AROMA DE LARANJA DOCE (Citrus sinensis). ARACAJU 2011.

(2) TIAGO COSTA GOES. AVALIAÇÃO DO POTENCIAL EFEITO ANSIOLÍTICO DO AROMA DE LARANJA DOCE (Citrus sinensis) Dissertação apresentada ao Núcleo de PósGraduação em Medicina da Universidade Federal de Sergipe como requisito parcial à obtenção do grau de Mestre em Ciências da Saúde. Orientadora: Profa. Dra. Flavia Teixeira Silva. ARACAJU 2011.

(3) FICHA CATALOGRÁFICA ELABORADA PELA BIBLIOTECA DA SAÚDE UNIVERSIDADE FEDERAL DE SERGIPE. G598a. Goes, Tiago Costa Avaliação do potencial efeito ansiolítico do aroma de laranja doce (Citrus sinensis) / Tiago Costa Goes. – Aracaju, 2011. 49 f.: il. Orientador (a): Profa. Dra. Flávia Teixeira Silva. Dissertação (Mestrado em Ciências da Saúde) – Universidade Federal de Sergipe, Pró-Reitoria de Pós-Graduação e Pesquisa, Núcleo de Pós-Graduação em Medicina.. 1. Ansiedade 2. Rutaceae 3. Citrus sinensis (Laranja doce) 3. Aromaterapia 4. Farmacologia I. Título CDU 582.746.21:615.

(4) TIAGO COSTA GOES. AVALIAÇÃO DO POTENCIAL EFEITO ANSIOLÍTICO DO AROMA DE LARANJA DOCE (Citrus sinensis) Dissertação apresentada ao Núcleo de PósGraduação em Medicina da Universidade Federal de Sergipe como requisito parcial à obtenção do grau de Mestre em Ciências da Saúde. Aprovada em: _____/_____/_____. BANCA EXAMINADORA _________________________________________ Orientadora: Profa. Dra. Flavia Teixeira Silva – UFS ________________________________________________ 1º Examinador: Profa. Dra. Zenith Nara Costa Delabrida – UFS ________________________________________ 2º Examinador: Prof. Dr. Murilo Marchioro – UFS. PARECER ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________.

(5) Dedico esta conquista aos meus pais, José e Angela, a minha prima Magaly, a minha avó Ana Viana e a minha orientadora Profa. Dra. Flavia, que sempre se fizeram presentes nos momentos em que eu mais precisei. O carinho, a força e a dedicação que de vós recebi, me fortaleceram e me impulsionaram a continuar a minha caminhada..

(6) AGRADECIMENTOS Agradeço, primeiramente, a DEUS, por estar sempre ao meu lado, iluminando o meu caminho, me dando força e coragem para superar os obstáculos desta vida, que afinal de contas não foram poucos. Obrigado também Senhor, por ter permitido que pessoas maravilhosas cruzassem a minha vida. Pessoas estas, que de uma forma ou de outra, deram a sua contribuição para a realização deste momento. Aos meus pais, José e Angela, que sempre me ensinaram o verdadeiro sentido da vida e que, mesmo em meio a tantas dificuldades que a vida nos impôs, conduziram majestosamente os seus papéis de pai e mãe. A minha prima, Magaly, e a minha avó Ana Viana, pela presença constante e edificante em todos os momentos da minha vida. Aos meus irmãos, Lucas, Abraão, Thalita e Thaísa, por todo apoio e incentivo que sempre me deram. Aos que compõem o LAFICO (Laboratório de Fisiologia do Comportamento) que com o passar do tempo tornaram-se tão próximos que tempo algum vos apagará da minha memória: O médico Fabrício Dias, os alunos de iniciação científica Fábio, Thiago Henrique, Débora, Flávia Garcez, André e Ângelo e, as mestrandas Camila, Vânia e Pollyana com os quais pude compartilhar um pouco do meu conhecimento e também com os quais muito aprendi. A Profa. Dra. Virgínia Mara, com a qual não só dividi o mesmo espaço físico, como também os bons momentos que vivi.. A CAPES, pela concessão da bolsa de estudos. A FAPITEC, pelo apoio financeiro para realização deste projeto. Por fim e não menos importante, aos voluntários que participaram deste estudo sem os quais nada disto seria possível..

(7) AGRADECIMENTO ESPECIAL A minha orientadora, Profa. Dra. Flavia Teixeira, que com sua simplicidade, serenidade e sabedoria me mostrou que para ser um bom mestre não basta apenas adquirir conhecimento científico, é preciso também aprimorar os valores morais. Seus ensinamentos me fizeram progredir consideravelmente e hoje percebo o quanto os seus alertas (“Tiago, você precisa ensaiar esta apresentação para não ultrapassar o tempo”), a sua forma brilhante de corrigir os relatórios da minha iniciação científica e esta dissertação (corrigia me sugerindo e nunca impondo as correções e/ou sugestões), as oportunidades que me ofereceu no laboratório (onde pude participar de vários estudos pré-clínicos e outros, como este, que constituem uma das fases dos estudos clínicos), as suas explicações sobre os testes estatísticos, a sua forma sutil de me corrigir, o seu jeito de tomar uma decisão (ponderava sempre os prós e os contras), a forma pela qual se referia as ideias brilhantes que ela tinha (sempre se referia na primeira pessoa do plural e nunca na primeira do singular), enfim, o quanto esta sua maneira de conduzir a formação científica de um pequeno jovem, o torna um grande homem..

(8) RESUMO Avaliação do potencial efeito ansiolítico do aroma de laranja doce (Citrus sinensis), Tiago Costa Goes, Aracaju – SE, 2011. O presente estudo teve por objetivo avaliar o potencial efeito ansiolítico do aroma de laranja doce (C. sinensis) em voluntários saudáveis submetidos a um modelo experimental de indução de ansiedade. Para tanto, quarenta voluntários do sexo masculino, com idade entre 18 e 30 anos, foram alocados em cinco diferentes grupos (n=8) para inalação do óleo essencial de laranja doce (aroma teste: 2,5, 5 ou 10 gotas), do óleo essencial de Melaleuca alternifolia (controle aromático: 2,5 gotas) ou de água destilada (controle não-aromático: 2,5 gotas). Imediatamente após a inalação, cada voluntário foi submetido à situação ansiogênica do Teste de Stroop Monitorado por Vídeo (TSMV), sendo que antes, durante e depois do TSMV foram realizadas medidas psicológicas (níveis de ansiedade-estado, de tensão subjetiva, de tranquilização e sedação) e fisiológicas (frequência cardíaca e eletromiograma do músculo gastrocnêmio). Todos estes parâmetros também foram avaliados previamente ao tratamento. Os resultados dos parâmetros psicológicos foram analisados por ANOVA de Friedman, para cada grupo de tratamento, seguido de teste a posteriori tipo Tukey. Já os resultados dos parâmetros fisiológicos foram analisados por ANOVA de duas vias para medidas repetidas, seguido de teste a posteriori de Tukey. Ambas as análises estatísticas foram realizadas com nível de significância de 5%. A existência de efeito ansiolítico deveria refletir-se nos resultados na forma de ausência de diferenças significativas entre as situações de teste. Assim sendo, diferentemente dos grupos controles, os grupos tratados com óleo de laranja doce nas doses de 2,5 e 10 gotas não apresentaram alterações significativas (p > 0,05) nos níveis de ansiedadeestado, tensão subjetiva e tranquilização ao longo do TSMV. Já em relação aos parâmetros fisiológicos, este efeito não foi observado, assim como ocorreu com o clássico ansiolítico, diazepam, em estudo anterior. Embora mais estudos sejam necessários para constatar a relevância clínica da aromaterapia no tratamento dos transtornos de ansiedade, os resultados do presente estudo demonstraram um efeito ansiolítico agudo do aroma de laranja doce, trazendo certo apoio científico para sua utilização como tranquilizante pelos aromaterapeutas. Descritores: ansiedade; aromaterapia; Citrus sinensis..

(9) ABSTRACT Evaluation of the potential anxiolytic effect of sweet orange (Citrus sinensis) aroma, Tiago Costa Goes, Aracaju – SE, 2011. The aim of this study was to evaluate the potential anxiolytic effect of sweet orange aroma in healthy volunteers submitted to an anxiogenic situation. For this purpose, forty male volunteers, aged between 18 and 30 years, were allocated to five different groups (n=8) for the inhalation of sweet orange essential oil (test aroma: 2.5, 5 or 10 drops), Melaleuca alternifolia essential oil (control aroma: 2.5 drops) or distilled 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 Colour Word Test. Psychological parameters (state-anxiety, subjective tension, tranquilization and sedation) and physiological parameters (heart rate and gastrocnemius electromyogram) were evaluated before the inhalation period and before, during and after the test. The results of the psychological parameters were analyzed by Friedman’s ANOVA, for each treatment group, followed by Tukey-type test for post hoc comparisons. The results of the physiological parameters were analyzed using a two-factor analysis of variance (ANOVA) for repeated measures followed by Tukey’s test for post hoc comparisons. All significance tests were two-tailed and were performed at the 5% significance level. It’s 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. Unlike the control groups, the individuals exposed to the test aroma in the doses of 2.5 drops and 10 drops did not present 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. Physiological alterations along the test were not prevented in any treatment group, as has previously been observed for diazepam. 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. Keywords: anxiety; aromatherapy; sweet orange oil essential..

(10) SUMÁRIO 1 INTRODUÇÃO .............................................................................................. 08 2 ARTIGO ........................................................................................................ 12 3 CONCLUSÃO ............................................................................................... 43 4 REFERÊNCIAS ............................................................................................. 44 APÊNDICE A – TERMO DE CONSENTIMENTO LIVRE E ESCLARECIDO .. 47 ANEXO A – DECLARAÇÃO DO COMITÊ DE ÉTICA EM PESQUISA ENVOLVENDO SERES HUMANOS DA UNIVERSIDADE FEDERAL DE SERGIPE ......................................................................................................... 49.

(11) 8 . 1 INTRODUÇÃO. A ansiedade, apesar de ser um termo largamente empregado na literatura psicológica e psiquiátrica, ainda não possui uma definição exata, já que há pouca concordância entre as suas diferentes definições (PESSOTI, 1978), chegando mesmo a ser considerada como indefinível (LADER, 1972). Mesmo sem uma definição exata, ela pode ser facilmente notada. No homem, ela é percebida como uma emoção desagradável, caracterizada por um alerta tenso e fisicamente exaustivo, focalizado em um perigo ou emergência iminente e inevitável, ainda que não objetivamente aparente, com uma incerteza dolorosa sobre a possibilidade de se resolver a situação. Esta emoção é também acompanhada por uma ativação autonômica (MARTIN, 1998). Muitos acreditam que os efeitos nocivos do atual estilo de vida, resultado das enormes pressões de uma sociedade moderna, tecnológica e competitiva sejam fatores de grande importância no desenvolvimento da ansiedade (FISHER, 1990), a qual, seja como sintoma ou como transtorno, é, dentre os problemas psiquiátricos, o que se apresenta em maior prevalência na população geral (LEPINE, 2002). Dentre os diferentes grupos de drogas psicotrópicas, os benzodiazepínicos têm se destacado como os mais amplamente utilizados para o alívio de sintomas de ansiedade. No entanto, apesar do uso generalizado, mesmo em se tratando de drogas muito seguras, os benzodiazepínicos podem produzir, dentre outros efeitos colaterais, síndromes de dependência e de abstinência, com o risco de induzir o paciente a abuso (ANDREATINI et al., 2001; HOFFMAN; MATHEW, 2008). Outras drogas que têm sido utilizadas no tratamento dos transtornos de ansiedade, e que não produzem dependência, são os agentes que afetam o sistema serotonérgico, mas estes apresentam um grande atraso no início de ação (CUTLER et al., 1993; DEN BOER; SLAAP, 1998; HEDGES et al., 2007; POLLACK, 1999; RICKELS et al., 1993).. Ainda assim, nem todos os pacientes se beneficiam dos tratamentos. disponíveis, e somente poucos chegam próximo da recuperação completa (BALLENGER, 1999; RAVINDRAN & STEIN, 2010). Estes fatos têm justificado um.

(12) 9 . número considerável de estudos realizados mais recentemente, com o intuito de desenvolver drogas ou procedimentos psicoterápicos mais promissores para o controle dos quadros de ansiedade, e que não apresentem efeitos colaterais significativos. Procedimentos. complementares. como. fitoterápicos,. técnicas. de. relaxamento, massagem, meditação, entre outros, vêm cada vez mais sendo empregados por profissionais da saúde e pela população em geral, apesar das poucas evidências experimentais que comprovem sua eficácia (COOKE; ERNEST, 2000; ROBERTS et al., 2000).. No Reino Unido, pelo menos um em cada dez. médicos têm interesse pela medicina complementar. Muitos clínicos gerais têm se interessado por aromaterapia, sendo que, enquanto uns estão sendo treinados para praticá-la,. outros. estão. regularmente. encaminhando. pacientes. para. aromaterapeutas (HARRIS, 2003). Na França, a disciplina “Aromaterapia” é opcional no curso de Medicina, podendo os médicos optarem por esse tratamento para seus pacientes (COOKE; ERNEST, 2000). A aromaterapia é o uso de óleos essenciais extraídos de folhas, flores ou outras partes da planta para o tratamento de diversas doenças. Os proponentes da aromaterapia referem-se a ela como uma antiga tradição da medicina natural praticada em países como o Egito e a Índia há milhares de anos (COOKE; ERNEST, 2000), embora, o termo em si, só tenha sido criado por um químico francês, Maurice René de Gattefossê, em 1928. Não obstante, a definição exata de aromaterapia permanece problemática. Enquanto alguns autores sugerem a mudança do termo, para que ele se adeque melhor à definição (PERRY; PERRY, 2006), outros sugerem a mudança da definição, para que ela se adeque melhor ao termo (BUCHBAUER; JIROVETZ, 1994 apud VAND DER WATT et al., 2008).. Perry e Perry (2006). consideram que os termos “terapia com óleos essenciais” ou “farmacologia fitoessencial” seriam mais precisos, já que os efeitos não são necessariamente relacionados aos aromas. Já Buchbauer e Jirovetz (1994 apud VAND DER WATT et al., 2008 ) propõem que a definição universal de aromaterapia seja o uso terapêutico de fragrâncias ou de substâncias voláteis para curar ou prevenir doenças, infecções e indisposições, somente por meio de inalação..

(13) 10 . É empiricamente conhecido de tempos remotos que várias fragrâncias têm efeitos psicológicos e fisiológicos (FUJIWARA et al., 2002). Hoje, um certo número de referências começa a sugerir que odores liberados por cosméticos ou mesmo odores ambientais podem influenciar o humor, a cognição e a ansiedade, por meio de ação no sistema nervoso central (ALMEIDA et al., 2004; FATURI et al., 2010; FUJIWARA et al., 2002; LEHRNER et al., 2000; SCHIFFMAN, 1998). Por exemplo, já foi demonstrado, por meio de técnicas imunohistoquímicas, que áreas cerebrais relacionadas à ansiedade, como o núcleo central da amígdala e os núcleos paraventricular e dorsomedial do hipotálamo, são influenciadas pelo aroma de lavanda, o qual apresenta atividade ansiolítica (SHAW et al., 2011). As possíveis maneiras pelas quais as moléculas voláteis, presentes nos óleos essenciais, chegam ao cérebro são: 1) circulação sistêmica, já que a inalação pode conduzir as substâncias voláteis para os pulmões, onde elas podem rapidamente se difundir para o sangue (MADDOCKS-JENNINGS; WILKINSON, 2004); 2) estimulação eletrofisiológica dos receptores olfatórios, que além de ativarem áreas neocorticais, envolvidas na percepção olfatória, ativam também regiões do sistema límbico (BUCK, 2000), responsáveis pelas respostas emocionais; ou 3) via nariz-ao-cérebro, onde substâncias administradas intranasalmente podem alcançar o sistema nervoso central, atravessando a placa crivosa por meio da difusão entre as fibras do nervo olfatório, ou ainda, podem penetrar nas células olfatórias e serem transportadas até o soma do neurônio no bulbo olfatório (STOCKHORST; PIETROWSKY, 2004). Independentemente da via de ativação, é possível perceber que a aromaterapia, anteriormente fundamentada apenas no conhecimento tradicional de várias culturas, está atingindo uma nova etapa de desenvolvimento com os resultados das investigações na área aromacológica. O termo aromacologia foi criado em 1989, pela Fundação para Pesquisa do Olfato (Sense of Smell Institute, New York – EUA), para designar o estudo das interrelações entre compostos aromáticos e estados emocionais (CORAZZA, 2002). Apesar da aromaterapia ter ampla aceitação dentre as práticas alternativas, a aromacologia é uma ciência que está em fase de desenvolvimento.. Existem.

(14) 11 . pouquíssimos estudos que comprovem cientificamente os efeitos terapêuticos dos aromas. Até o momento, os poucos estudos originais, que visam avaliar a aromaterapia como tratamento para a ansiedade, são pequenos e apresentam problemas metodológicos (COOKE; ERNEST, 2000).. Contudo, a simples. desconsideração desse tipo de terapia, por falta de validação científica, poderia estar privando pacientes ansiosos de um tratamento eficaz. Dessa forma, torna-se de fundamental importância que, a partir das evidências empíricas apresentadas em textos de aromaterapia, sejam desenhados estudos científicos, com metodologia apropriada, a fim de se examinarem as propriedades ansiolíticas de determinados óleos essenciais. Entre esses óleos, ganha especial destaque o óleo de laranja, que além de ser considerado um tranquilizante, pelos aromaterapeutas (CORAZZA, 2002), também começa a aparecer na escassa literatura científica sobre aromaterapia. Recentemente, foi demonstrado o efeito ansiolítico agudo do óleo de laranja amarga (Citrus aurantium) em camundongos avaliados num modelo animal de ansiedade, o labirinto em cruz elevado (CARVALHO-FREITAS; COSTA, 2002). Utilizando o mesmo modelo, o Laboratório de Fisiologia do Comportamento da Universidade Federal de Sergipe demonstrou o efeito ansiolítico agudo do óleo essencial de laranja doce (Citrus sinensis) administrado a ratos por inalação (FATURI et al., 2010). Uma vez que o óleo essencial de laranja doce (C. sinensis) encontra-se na lista de substâncias reconhecidas como seguras do Food and Drug Administration Center (FDA’s GRAS List), o próximo passo na investigação das propriedades ansiolíticas desse óleo seria a administração do aroma a voluntários saudáveis..

(15) 12 . 2 ARTIGO. .

(16) Page 1 of 1. Journal of Alternative and Complementary Medicine - Decision on Manuscript ID JACM-2011-0551.R2 DE:. Quarta-feira, 21 de Setembro de 2011 13:24. PARA: CC:. 21-Sep-2011 Dear Dr. Teixeira-Silva: We are pleased to accept your manuscript entitled "Effect of Sweet Orange Aroma on Experimental Anxiety in Humans" for publication in Journal of Alternative and Complementary Medicine. Your paper is tentatively scheduled to be published in our July 2012 issue. Please be sure to cite this article to ensure maximum exposure of your work. You will receive page proofs electronically approximately one month prior to publication from Jason Schappert (jschappert@liebertpub.com) and may receive additional correspondence related to production from Ms. Billie Spaight (BSpaight@liebertpub.com). Please add these as well as MCanning@liebertpub.com and RGordon@liebertpub.com to your address book so correspondence from them is not caught in your spam filter. All authors will get a follow-up email with instructions on how to complete our online Copyright Agreement form. FAILURE BY ALL AUTHORS TO SUBMIT THIS FORM MAY RESULT IN A DELAY OF PUBLICATION. The corresponding author is responsible for communicating with coauthors to make sure they have completed the online copyright form. Authors not permitted to release copyright must still return the form acknowledging the statement of the reason for not releasing the copyright. The corresponding author will receive notification when all copyright forms have been submitted. Consider Liebert Open Option to have your paper made Free Online immediately upon publication for a one-time fee. If the paper has NIH funding, it will also be uploaded onto PubMedCentral on behalf of the author. Benefits of Liebert Open Option include: fast track publication; email message highlighting the article; increased readers, citations, and downloads; and an identifying icon in the table of contents if published in print. Subsequent accepted papers are eligible for a reduced fee for Open Option. Please contact Karen Ballen at kballen@liebertpub.com or at (914) 740-2194 for more information. If your institution is not currently subscribing to this journal, please ensure that your colleagues have access to your work by recommending this title (http://www.liebertpub.com/mcontent/files/lib_rec_form.pdf ) to your Librarian. Thank you for your contribution. On behalf of the Editors of Journal of Alternative and Complementary Medicine, we look forward to your continued contributions to the Journal. Sincerely, Barbara Perrin Journal of Alternative and Complementary Medicine Editorial Office JACM-editorial@sbcglobal.net. Responder para JACM-editorial@sbcglobal.net. http://36ohk6dgmcd1n.yom.mail.yahoo.net/om/api/1.0/openmail.app.invoke/36ohk6dgmc.... 28/9/2011.

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(27) Page 1 of 27. ee. rP. Fo ev rR w. ie. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60. Journal of Alternative and Complementary Medicine. Mary Ann Liebert Inc., 140 Huguenot Street, New Rochelle, NY 10801.

(28) Journal of Alternative and Complementary Medicine 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60. Page 2 of 27. Effect of Sweet Orange Aroma on Experimental Anxiety in Humans. Tiago Costa Goes (BSc)a, Fabrício Dias Antunes (MD)a , Péricles Barreto Alves (PhD)b, Flavia Teixeira-Silva (PhD)a. Fo. a. Departamento de Fisiologia - Centro de Ciências Biológicas e da Saúde –. b. rP. Universidade Federal de Sergipe. 49100-000 - SE - Brazil. Departamento de Química - Centro de Ciências Exatas e Tecnológicas –. ee. Universidade Federal de Sergipe. 49100-000 - SE - Brazil.. ev rR ie. w. Running Head: Sweet Orange Aroma and Anxiety. ϭ . Mary Ann Liebert Inc., 140 Huguenot Street, New Rochelle, NY 10801.

(29) Page 3 of 27. ABSTRACT Objective: To evaluate the potential anxiolytic effect of sweet orange (Citrus sinensis) aroma in healthy volunteers submitted to an anxiogenic situation. Design: Forty 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,. Fo. the video-monitored version of the Stroop Colour-Word Test (SCWT).. rP. Outcome measures: Psychological parameters (state-anxiety, subjective tension, tranquilization and sedation) and physiological parameters (heart rate and. ee. gastrocnemius electromyogram) were evaluated before the inhalation period and before, during and after the SCWT.. ev rR. 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,. ie. revealing an anxiolytic activity of sweet orange essential oil. Physiological. w. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60. Journal of Alternative and Complementary Medicine. 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. Key Words: sweet orange, essential oil, aromatherapy, anxiety, anxiolytic Ϯ . Mary Ann Liebert Inc., 140 Huguenot Street, New Rochelle, NY 10801.

(30) Journal of Alternative and Complementary Medicine. INTRODUCTION Anxiety disorders are the most prevalent class of psychiatric disorders in general population1. 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 action2. Furthermore, not all patients benefit from the available treatments, and only a few of them have a. Fo. response near to complete recovery3. These facts justify the growing search for alternative or complementary procedures. rP. 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 purposes4.. ee. ev rR. According to Charlesworth5, 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. ie. therapy still doesn’t have much scientific support6,7.. w. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60. Page 4 of 27. Double blind, randomised, 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 & Ernst6). However, in most of these studies, exposure to the essential oil odour 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 scores8. However, in a recent study performed with rats, animals submitted to two different experimental models of. ϯ . Mary Ann Liebert Inc., 140 Huguenot Street, New Rochelle, NY 10801.

(31) Page 5 of 27. anxiety, after being exposed to Citrus sinensis aroma, showed less anxiety than animals exposed to air only9.. 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 alternifólia essential oil did not behave differently from control animals. So, the probability of C. sinensis essential oil having a therapeutic action per se is quite high. However, whether this aroma has the same effect in humans is yet to be verified.. Fo. Bringing all this into consideration, the aim of the present work was to evaluate the. rP. potential anxiolytic effect of sweet orange (C. sinensis) essential oil inhalation in healthy volunteers submitted to an anxiogenic situation. Moreover, in order to. ee. determine the main components of the essential oils administered, a Gas. ev rR. chromatography/mass spectrometryanalysis was also carried out.. MATERIALS AND METHODS Participants. ie. Forty graduate student male volunteers, subjectively healthy, aged between 18 and. w. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60. Journal of Alternative and Complementary Medicine. 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 II10. 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.. ϰ . Mary Ann Liebert Inc., 140 Huguenot Street, New Rochelle, NY 10801.

(32) Journal of Alternative and Complementary Medicine. 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 Colour-Word Test, as standardized by Teixeira-Silva et al.11, was used to elicit anxiety in the volunteers. In short, this test consists of. Fo. presenting a board to the participant with one hundred of the color naming words blue, yellow, red, green and violet organized randomly in a 10 X 10 matrix. Each. rP. word is printed in a colour different to its meaning, for example the word "red". ee. printed in yellow ink. This board corresponds to the “Colour-Word” card of the Stroop test12. To perform the task, the subject has to say, as quickly as possible. ev rR. and in the sequence presented, the names of the colours being seen (i.e. the colour of the ink), but not the colours designated by the words. The task has to be performed in 2 minutes (maximum) and any errors are signalled with a bell.. ie. Skipping a colour’s sequence, hesitation in saying the colour, and saying the. w. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60. Page 6 of 27. colour’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.. ϱ . Mary Ann Liebert Inc., 140 Huguenot Street, New Rochelle, NY 10801.

(33) Page 7 of 27. Psychological measurements State-Trait Anxiety Inventory (STAI)13. This instrument is divided into two sections, each having twenty 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 used10. A cut-off score for high trait anxiety was derived from Gama et. Fo. al.14, which 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. rP. (SD) was 9.2. The cut-off between high and medium trait anxiety was set 1 SD. ee. above the mean, with the result that scores greater than 48 were classified as high anxiety and, therefore, met the exclusion criteria.. ev rR. Visual Analogue Mood Scale (VAMS)15. This scale is composed by 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. ie. distributed into four factors: 1) tranquilization; 2) mental sedation; 3) physical. w. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60. Journal of Alternative and Complementary Medicine. 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. Physiological measurements Heart rate (HR): derived from two active Ag/AgCl electrodes placed across the chest, and one ground Ag/AgCl electrode placed on the abdomen.. ϲ . Mary Ann Liebert Inc., 140 Huguenot Street, New Rochelle, NY 10801.

(34) Journal of Alternative and Complementary Medicine. Electromyogram of the gastrocnemius muscle (EMG): 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, v/v) before placement of the electrodes. The recordings were made using a computerized system for monitoring. Fo. physiological responses (I-330-C2+ Physiological Monitoring System, J&J Engineering, USA).. rP. All tests were performed in a quiet room maintained at a temperature between 23 and 25 ºC (73.4 and 77.0 oF).. ee. Treatments. ev rR. Test aroma: Essential oil of sweet orange (C. sinensis, BioEssência, Brazil) – 2.5, 5 or 10 drops (SO2.5, SO5, SO10). ie. Control aroma: Essential oil of tea tree (M. alternifólia, BioEssência, Brazil) – 2.5 drops (TT). w. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60. Page 8 of 27. Non-aromatic control: distilled water – 2.5 drops (H2O) All treatments were administered by inhalation. Each drop corresponded to 25 Pl added to a surgical mask, which was worn by the volunteers for 5 minutes. A randomized double-bind design was followed. For this, a random distribution of the subjects to the experimental groups (n= 8) was made, using a draw, by a. ϳ . Mary Ann Liebert Inc., 140 Huguenot Street, New Rochelle, NY 10801.

(35) Page 9 of 27. researcher who had no direct participation in the test sessions. In addition, the volunteers were told that they would inhale non-toxic 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 rats9, 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. Fo. 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. rP. recognize the orange smell, they had no idea how the aroma was supposed to affect their performance in the test.. ee. It’s worth mentioning that both the essential oils used are considered to be safe.. ev rR. Sweet orange oil is on the FDA’s GRAS (Generally Regarded as Safe) List18, while tea tree oil is generally considered to be nontoxic and non irritant, although it may cause sensitization in some individuals19. Procedure. w. ie. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60. Journal of Alternative and Complementary Medicine. The selected volunteers attended the laboratory on two 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 psychological. evaluations and then to 5 minutes of physiological recordings.. ϴ . Mary Ann Liebert Inc., 140 Huguenot Street, New Rochelle, NY 10801.

(36) Journal of Alternative and Complementary Medicine. Test day: The subjects were again taken to the experiment room, where they rested for five minutes, after which the adaptation procedure from the first day was repeated. The psychological and physiological data collected were labelled as the “Pre-treatment” 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 Pl of its contents onto a surgical mask, which. Fo. was gently attached to their face using just the top straps, so to allow some air. rP. circulation under the mask. Immediately after the inhalation period (5 min), the mask was removed, put into a sealed container and taken out of the room. The. ee. volunteers were then asked the following questions: 1) Could you smell anything. ev rR. on the mask?; 2) Can you identify what you smelled?; 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 psychological. ie. and 30 seconds of physiological evaluations. These data were labelled as the “Before” experimental phase.. w. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60. Page 10 of 27. After listening to the recorded instructions, the. participant then performed the task, during which his physiological measurements were recorded.. After 50 words, a pause was made for a second set of. psychological evaluations.. These new data were labelled as the “During”. experimental phase. Immediately following the evaluations, the test was restarted and continued up to the last colour or until the end of the scheduled time. The. ϵ . Mary Ann Liebert Inc., 140 Huguenot Street, New Rochelle, NY 10801.

(37) Page 11 of 27. participant then rested for 5 min, after which all the physiological and psychological parameters were again evaluated. This final set of data was labelled as the “After” experimental phase. The room where the aromas were administered to the volunteers was left ventilated for about two hours between tests. Gas chromatography/mass spectrometry (GC/MS) analysis of the essential oils. rP. Fo. GC/MS analysis was performed as described in a previous article9. Statistical analyses. ee. The data collected during the adaptation phase were not analyzed as this phase. ev rR. was only intended to habituate the participants to the environment and apparatus that would be used on the following day.. All the psychological data obtained during the test were analyzed by Friedman’s. ie. ANOVA, for each treatment group, followed by Tukey-type test for post hoc comparisons.. w. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60. Journal of Alternative and Complementary Medicine. All the physiological data obtained were analyzed using a two-factor analysis of variance (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.. ϭϬ . Mary Ann Liebert Inc., 140 Huguenot Street, New Rochelle, NY 10801.

(38) Journal of Alternative and Complementary Medicine. 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. ee. rP. Fo ev rR. 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.. ie. In the groups SO and TT, all the volunteers sad “yes” to the question “Could you. w. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60. Page 12 of 27. smell anything on the mask?”, while in the group H2O, only two subjects sad “yes” to the same question, although they could not identify the smell nor did they find it either pleasant or unpleasant. Psychological measurements Data are presented as median ([Ѻ) and interquartile range (Q1-Q3).. ϭϭ . Mary Ann Liebert Inc., 140 Huguenot Street, New Rochelle, NY 10801.

(39) Page 13 of 27. ANOVA results are described here, while post hoc results are shown in Table 2. It’s 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 [[Ѻ (Q1 - Q3): SO2.5 = 34.0 (31.0 - 38.0); SO5 = 32.0 (30.8 –. Fo. 38.0); SO10 = 33.5 (29.8 – 35.8); TT = 35.5 (32.8 – 37.3); H2O = 36.0 (33.0 – 37.5)].. rP. STAI-state. ee. Friedman’s ANOVA revealed no significant differences among experimental phases for the groups SO2.5 (p = 0.063) and SO10 (p = 0.098).. ev rR. Although,. differences were found for groups SO5 (p < 0.001), TT (p = 0.001) and H2O (p = 0.005). VAMS – Tranquilization. ie. Friedman’s ANOVA revealed no significant differences among experimental phases for the group SO10 (p = 0.062).. w. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60. Journal of Alternative and Complementary Medicine. Although, differences were found for. groups SO2.5 (p = 0.015), SO5 (p = 0.003), TT (p = 0.007) and H2O (p = 0.018). 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).. ϭϮ . Mary Ann Liebert Inc., 140 Huguenot Street, New Rochelle, NY 10801.

(40) Journal of Alternative and Complementary Medicine. VAMS – Mental sedation Friedman’s ANOVA revealed no significant differences among test phases for all 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. Fo. 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. rP. analyses of the individual items of this category, for this group, showed differences among test phases for the lethargic/energetic item (p = 0.008).. ee. VAMS – Other feelings and attitudes. ev rR. 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. ie. individual items of this category, for this group, showed differences among test phases for the withdrawn/gregarious item (p = 0.002).. w. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60. Page 14 of 27. Physiological measurements Data are presented as mean and standard error of the mean (mean ± SEM) in Table 3. Heart rate. ϭϯ . Mary Ann Liebert Inc., 140 Huguenot Street, New Rochelle, NY 10801.

(41) Page 15 of 27. 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 < 0.001), in that the heart rate was greater During in relation to Before (p < 0.001). The treatment effect was not significant (p = 0.525). Gastrocnemius electromyogram The ANOVA revealed that the interaction between test phase and treatment was. Fo. not significant (p = 0.653); therefore the two main effects were analyzed. The test phase effect was significant (p < 0.001), in that the muscular tension was greater. rP. During in relation to Before (p = 0.001). The treatment effect was not significant (p = 0.126). CG/MS analysis. ee ev rR. The volatile composition of sweet orange and tea tree essential oils are shown in Tables 4 and 5, respectively.. DISCUSSION. w. ie. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60. Journal of Alternative and Complementary Medicine. The aim of this work was to evaluate the potential anxiolytic effect of sweet orange (C. sinensis) essential oil in healthy volunteers submitted to an anxiogenic situation. The experimental model of anxiety, as employed here, was shown to be valid, since the subjects from the control groups behaved as expected11, significantly increasing their anxiety signs/symptoms during the Stroop task.. ϭϰ . Mary Ann Liebert Inc., 140 Huguenot Street, New Rochelle, NY 10801.

(42) Journal of Alternative and Complementary Medicine. The obtained results showed the anxiolytic properties of sweet orange essential oil through the lack of a significant increase in state-anxiety and tension, and the lack of a significant decrease in tranquility, presented by the individuals exposed to the aroma. Of the two doses (2.5 and 10 drops) that demonstrated anxiolytic activity, the higher one seemed to be better at preventing anxious symptoms, as its results were further from reaching statistical significance and were observed in a greater number of parameters. It’s reasonable to question the fact that the intermediate. Fo. dose failed to produce an effect. Yet, this activity profile was observed before,. rP. when the same aroma was tested in animals9. This lack of a dose/effect relation is common when dealing with a mixture of compounds instead of a pure substance.. ee. Interestingly, the observed anxiolytic effects were not followed by sedative/hypnotic effects.. On the contrary, at the highest dose, sweet orange oil made the. ev rR. volunteers feel more energetic, while performing the test. They also felt more introverted. It’s tempting to speculate that these two symptoms together mean that the subjects were more concentrated on the task, which would be a welcome side. ie. effect, especially as tranquilizers tend to cause concentration loss. Nevertheless, this should be further investigated.. w. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60. Page 16 of 27. The GC/MS analysis determined that limonene corresponded to 54.48% of the volatile components of our sweet orange oil sample. Previous studies, performed with rodents, using essential oils which were practically pure limonene (> 97%)9,20 have shown very clear anxiolytic effects, indicating this monoterpene as the responsible constituent for the anxiolytic activity of citrus oils.. Therefore, it’s. possible that, in the present study, a purer sweet orange oil sample (around 90% ϭϱ . Mary Ann Liebert Inc., 140 Huguenot Street, New Rochelle, NY 10801.

(43) Page 17 of 27. limonene) could have led to more robust results. It’s 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,. Fo. in which female patients exposed to this oil, diffused in a waiting room before a. rP. dental procedure, showed lower levels of state anxiety compared to control patients, exposed to air only.. ee. 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. ev rR. 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 wasn’t a very good aromatic control, as it may not have. ie. been as familiar to the volunteers as the orange smell, and familiarity might have provided comfort to the subjects.. w. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60. Journal of Alternative and Complementary Medicine. 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 smell was, it was not unfamiliar to them. Another characteristic of the oils that could have interfered with the results is their hedonicity, as pleasant smells may induce a state of well ϭϲ . Mary Ann Liebert Inc., 140 Huguenot Street, New Rochelle, NY 10801.

(44) Journal of Alternative and Complementary Medicine. being in people. But again, there were no significant differences between orange and tea tree, with both smells generally being considered pleasant. Therefore, it’s 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 favour of this idea being the fact that not all doses of this oil were effective. Regarding the physiological parameters, there was no prevention of the stress response to the Stroop test in any of the treatment groups. However, this result. Fo. does not weaken the case for sweet orange aroma as an anxiety reducer, since. rP. diazepam, a classic anxiolytic drug, is also unable to prevent physiological alterations provoked by anxiogenic situations11, 22, 23.. ee. Nevertheless, it’s worth pointing out that this research does present some sample-related limitations. First, all subjects were graduate students, under 30 years of age.. ev rR. This was not only because it was convenient to recruit these. volunteers, but also because the model of anxiety used was validated for this population11. Still, it would be valuable to test the effects of sweet orange aroma in. ie. subjects with different levels of education and in different age groups. Second,. w. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60. Page 18 of 27. only men were selected for this study and, despite the fact that the anxiolytic effect of sweet orange aroma in women has been shown before21, 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.. ϭϳ . Mary Ann Liebert Inc., 140 Huguenot Street, New Rochelle, NY 10801.

(45) Page 19 of 27. 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 can not be accredited to expectancy biases, massage or therapeutic relationship which, according to some authors8, 24 could be responsible for most of the aromatherapy outcome.. Fo. ACKNOWLEDGMENTS. rP. This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e. ee. Tecnológico (CNPq) and Fundação de Apoio a Pesquisa e à Inovação Tecnológica. ev rR. do Estado de Sergipe (FAPITEC/SE). AUTHOR DISCLOSURE STATEMENT No competing financial interests exist.. w. ie. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60. Journal of Alternative and Complementary Medicine. ϭϴ . Mary Ann Liebert Inc., 140 Huguenot Street, New Rochelle, NY 10801.

(46) Journal of Alternative and Complementary Medicine. 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 .. Fo. 3. Ravindran LN, Stein MB. The pharmacologic treatment of anxiety disorders: a review of progress.J Clin Psychiatry 2010;71:839–854 .. rP. 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 .. ee. ev rR. 5. Charlesworth H. Aromatherapy and stress related health disorders. The Aromatherapist 1995;2:9–23 .. 6. Cooke B, Ernest E. Aromatherapy: a systematic review. Br J Gen Pract 2000;50:493–496 .. ie. 7. Roberts L, Wilson S, Greenfield S. Why aromatherapy works. Br J Gen Pract 2000;50:825–826 .. w. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60. Page 20 of 27. 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.. ϭϵ . Mary Ann Liebert Inc., 140 Huguenot Street, New Rochelle, NY 10801.

(47) Page 21 of 27. 10. Biaggio AMB, Natalício L. Manual para o Inventário de Ansiedade Traço-Estado (IDATE). 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.. Fo. 13. Spielberger CD, Gorsuch RL, Lushene RE. Manual for the state-trait anxiety. rP. inventory (“self-evaluation questionnaire”). California: Consulting-Psychologists Press, 1970.. ee. 14. Gama MMA, Moura GS, Araújo RF, Teixeira-Silva F. Ansiedade-traço em estudantes universitários de Aracaju (SE). Rev Psiquiatr RS 2008;30:19-24.. ev rR. 15. Norris H. The action of sedatives on brain stem oculomotor system in man. Neuropharmacology 1971;10:181–191 .. 16. Zuardi AW, Karniol IG. Estudo transcultural de uma escala de auto-avaliação. ie. para estudos subjetivos. J Bras Psiquiatr 1981;30:403–406 .. w. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60. Journal of Alternative and Complementary Medicine. 17. Del Porto JA, Laranjeiras RR, Masur J. Escalas de auto-avaliação de estados subjetivos – influência das instruções. J Bras Psiquiatr 1983;32:87–90. 18. FDA’s GRAS (Generally Regarded as Safe) List. Online document at: http://www.fda.gov/Food/FoodIngredientsPackaging/FoodAdditives/ucm191033. htm. Acessed August 29th, 2011. 19. Clarke S. Composition of essential oils. In: Clarke S., ed. Essential Chemistry for Safe Aromatherapy. London: Churchill Livingstone, 2003:111-164. ϮϬ . Mary Ann Liebert Inc., 140 Huguenot Street, New Rochelle, NY 10801.

(48) Journal of Alternative and Complementary Medicine. 20. Leite MP, Fassim Jr J, Baziloni EMF, et al. Behavioral effects of essential oil of Citrus aurantium L. inhalation in rats. Rev Bras Farmacogn 2008;18 (Supl.):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. Guimarães FS, Kohem G, Fillmann HS, et al. A simple simulated public. Fo. speaking test for evaluating anxiolytic drugs. Braz J Med Biol Res. rP. 1989;22:1083–1089.. 23. Albus M, Zellner A, Bondy B, et al. Influence of CGP361A, propranolol and diazepam. on. ee. autonomous. reactions. to. different. stressors.. Prog. Neuropsychopharmacol Biol Psychiatry 1989;13:87–97.. ev rR. 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:. w. ie. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60. Page 22 of 27. Flavia Teixeira-Silva, PhD Departamento de Fisiologia - Centro de Ciências Biológicas e da Saúde – Universidade Federal de Sergipe. 49100-000 - SE - Brazil. Tel: (+55) 79-2105-6645 FAX: (+55) 79-2105-6414 e-mail: teixeira_silva@terra.com.br Ϯϭ . Mary Ann Liebert Inc., 140 Huguenot Street, New Rochelle, NY 10801.

(49) Page 23 of 27. 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. Fo. 1.0 (0.0 – 1.0). 2.0 (1.8 – 2.0). Data are presented as median and interquartile range (Q1–Q3). 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”.. ee. rP. ev rR w. ie. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60. Journal of Alternative and Complementary Medicine. ϭ . Mary Ann Liebert Inc., 140 Huguenot Street, New Rochelle, NY 10801.

(50) Journal of Alternative and Complementary Medicine. ee. rP. Fo. 1 2 3 4 5 6 7 8 9 Table 2 - Summary of the psychological results. 10 11 EXPERIMENTAL PHASE 12 PARAMETER TREATMENT 13 14 Pre-treatment Before During 15 16 SO2.5 30.5 (25.0 – 32.5) 26.5 (23.5 – 31.5) 37.0 (30.0 – 40.2) 17 18 § SO5 31.5 (30.3 – 33.0) 28.5 (26.5 – 31.0) 36.5 (30.3 – 41.3)* 19 20 SO10 32.5 (30.7 – 36.2) 32.0 (27.5 – 33.2) 39.0 (35.2 – 41.2) 21 STAI-state (points) 22 § TT 30.0 (27.0 – 33.2) 28.0 (24.0 – 29.2) 36.5 (31.7 – 39.0)* 23 24 H2O 32.5 (30.5 – 36.0) 32.5 (30.5 – 35.8) 41.0 (32.3 – 43.3)* 25 26 § 27 SO2.5 34.4 (31.9 – 37.3) 35.9 (33.0 – 37.9) 25.8 (23.8 – 30.7)* 28 § 29 SO5 33.8 (32.3 – 35.6) 35.1 (33.4 – 38.5) 31.0 (24.0 – 33.0)* Tranquilization 30 31 SO10 34.3 (25.1 – 38.1) 32.1 (29.0 – 38.8) 23.3 (21.5 – 30.4) (cm) 32 33 TT 35.0 (29.7 – 36.8) 35.6 (31.4 – 36.1) 27.0 (23.3 – 30.6)* 34 § 35 H2O 31.8 (27.8 – 35.2) 29.2 (27.3 – 33.5) 26.8 (19.3 – 28.7)* 36 37 SO2.5 8.7 (7.6 – 9.3) 9.1 (8.8 – 9.2) 6.2 (4.2 – 7.9) 38 39 Tranquilization: SO5 8.6 (8.2 – 9.6) 8.6 (7.9 – 9.5) 7.1 (5.8 – 8.1)* 40 41 tense/relaxed item SO10 8.1 (6.3 – 10.0) 8.6 (7.3 – 9.9) 6.0 (4.4 – 6.7) 42 § 43 (cm) TT 8.5 (7.0 – 9.4) 8.9 (7.9 – 9.3) 5.9 (4.0 – 7.9)* 44 § 45 H2O 8.4 (8.0 – 9.6) 7.9 (7.3 – 8.8) 7.0 (3.7 – 7.5)* 46 47 SO2.5 12.2 (7.2 – 17.7) 11.3 (4.7 – 13.6) 9.1 (6.5 – 10.5) 48 49 SO5 9.5 (5.0 – 14.0) 9.6 (6.2 – 14.9) 9.2 (5.4 – 11.7) 50 Mental sedation 51 SO10 11.1 (9.9 – 12.2) 14.0 (9.2 – 20.2) 9.4 (3.2 – 13.1) 52 (cm) 53 TT 10.3 (7.5 – 14.0) 8.3 (5.3 – 11.9) 10.5 (6.8 – 13.5) 54 55 H2O 12.8 (6.4 – 16.9) 7.0 (4.8 – 13.0) 9.9 (7.6 – 13.3) 56 57 58 59 60 Mary Ann Liebert Inc., 140 Huguenot Street, New Rochelle, NY 10801. Page 24 of 27. After 33.0 (30.0 – 34.0) 31.5 (28.5 – 35.3)*. #. 33.0 (32.5 – 34.5) 28.5 (26.2 – 34.2)*. #. 36.0 (30.3 – 42.3)*. #. 33.1 (30.9 – 34.9)*. #. 32.1 (27.8 – 36.5)*. #. ev rR. 33.6 (24.0 – 37.8) 34.3 (29.4 – 36.2). #. 29.0 (23.2 – 30.1)* 8.1 (7.3 – 8.4). w. ie. 7.8 (7.4 – 8.9)*. #. 8.4 (6.2 – 9.5) 8.6 (7.9 – 9.2)* 6.6 (4.7 – 7.8) 10.1 (6.1 – 12.4) 10.7 (7.6 – 12.7) 8.6 (3.4 – 15.5) 8.7 (5.0 – 12.0) 10.6 (6.4 – 12.6). ϭ. #. #.

(51) Page 25 of 27. EXPERIMENTAL PHASE PARAMETER. TREATMENT Pre-treatment. Physical sedation (cm). Before. During. After. 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). 7.3 (0.7 – 11.2)*. 7.7 (1.2 – 12.9)*. 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). 10.9 (6.5 – 14.0). 7.6 (3.6 – 13.8). 8.0 (5.5 – 10.3). 7.9 (3.1 – 10.1). 3.6 (1.6 – 4.6). 2.1 (1.5 – 2.6). 1.8 (1.3 – 2.1). 2.3 (1.7 – 3.0). 2.8 (1.2 – 3.7). 2.9 (1.8 – 4.2). 2.9 (1.9 – 4.5). 2.7 (1.3 – 3.8). H2O SO2.5. §. SO5. lethargic/energetic. SO10. 3.2 (2.1 – 3.5). 3.1 (1.7 – 3.8). 1.7 (0.2 – 2.9)*. 1.5 (0.4 – 3.4)*. 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). 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). Others feelings. 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). and. SO10. 5.8 (2.7 – 7.3). 7.3 (3.2 – 10.3). 6.7 (2.6 – 9.9). TT. 4.5 (3.0 – 10.3). 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). 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). 1.7 (0.4 – 3.0)*. 1.3 (0.2 – 2.6). 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). item (cm). withdrawn/gregari ous item (cm). 4.4 (3.2 – 9.7). §. w. and attitudes:. 7.5 (4.0 – 10.1). ie. Others feelings. ev rR. attitudes (cm). ee. Physical sedation:. rP. Fo. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60. Journal of Alternative and Complementary Medicine. §. #. #. #. #. 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. Significantly different from Pre-treatment. *Significantly # § # different from Before. Significantly different from During. * p < 0.05. Ϯ . Mary Ann Liebert Inc., 140 Huguenot Street, New Rochelle, NY 10801.

(52) Journal of Alternative and Complementary Medicine. Table 3 - Summary physiological results.. PARAMETERS. EXPERIMENTAL PHASE. TREATMENT Pre-treatment. Before. During. After. 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. SO2.5. 1.1 ± 0.3. 0.9 ± 0.2. 2.3 ± 0.5. 0.9 ± 0.3. Heart rate (beats/min). Electromyogram of the gastrocnemius. 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. 1.0 ± 0.2. 1.7 ± 0.5. 2.2 ± 0.6. 1.6 ± 0.5. 2.7 ± 0.7. 1.8 ± 0.4. 4.0 ± 0.6. 2.5 ± 0.5. TT H2O. ee. muscle (µV). rP. Fo Data are presented as means ± SEM.. ev rR w. ie. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60. Page 26 of 27. ϭ . Mary Ann Liebert Inc., 140 Huguenot Street, New Rochelle, NY 10801.

(53) Page 27 of 27. 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. ee. rP. Fo. Total. NI: non-identified compounds. w. RI: retention index. 100.00. ie. RT: retention time. ev rR. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60. Journal of Alternative and Complementary Medicine. ϭ . Mary Ann Liebert Inc., 140 Huguenot Street, New Rochelle, NY 10801.

(54) Journal of Alternative and Complementary Medicine. Table 5 - Volatile composition of Melaleuca alternifolia essential oil. Peak. RT (min). Compounds. (%). RI. 1. 6.575. D-tujene. 0.58. 924. 2. 6.800. D-pinene. 2.17. 931. 3. 8.208. E-pinene. 0.55. 976. 4. 8.600. myrcene. 0.65. 988. 5. 9.550. D-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. 1031. 9. 11.050. J-terpinene. 17.69. 1058. 10. 12.017. rP. 4.31. terpinolene. 2.76. 1084. 11. 15.633. terpinen-4-ol. 49.83. 1082. 12. 16.108. D-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. 17. 26.500. NI. 0.37. 18. 27.217. G-cadinene. 0.95. 19. 29.350. globulol. 0.42. Total. 99.26. Fo. ee. ev rR. ie 0.59. 1489. w. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60. Page 28 of 27. 1494 1517 1585. RT: retention time RI: retention index NI: non-identified compounds. ϭ . Mary Ann Liebert Inc., 140 Huguenot Street, New Rochelle, NY 10801.

(55) 43 . 3 CONCLUSÃO. Embora mais estudos sejam necessários para constatar a relevância clínica do óleo essencial de laranja doce no tratamento dos transtornos de ansiedade, os resultados do presente estudo indicam um efeito ansiolítico agudo do aroma de laranja doce, trazendo certo apoio científico para sua utilização como tranquilizante pelos aromaterapeutas..