Clinical Pharmacology of Citrus aurantium and Citrus sinensis for the Treatment of Anxiety

Objective The aim of this review is to analyze preclinical and clinical studies investigating the anxiety effects of Citrus aurantium or Citrus sinensis essential oils (EOs). Design The bibliographic research was made on the major scientific databases. Analysis included only articles written in English and published on peer-reviewed scientific journals describing preclinical experiments and clinical trials carried out to investigate the antianxiety effects of Citrus aurantium or Citrus sinensis EOs on anxiety disorders. Clinical studies reporting the antianxiety effects of products containing Citrus aurantium or Citrus sinensis EOs in combination with other active substances, including medicinal plants, were excluded. Nine clinical studies fulfilled the criteria adopted for analysis. Results Data show that Citrus aurantium or Citrus sinensis EOs produce anxiolytic effects both in preclinical experiments and in different clinical conditions. Citrus aurantium EO aromatherapy reduced anxiety level in the great part of stress conditions studied (subjects affected by chronic myeloid leukemia and preoperative patients) except for a sample of patients subjected to colonoscopy. Exposition to Citrus sinensis EO in clinical studies shows to be positive in reducing anxiety level in patients waiting for dental treatment as well as in healthy volunteers submitted to an anxiogenic situation. Conclusions Overview of clinical trials conducted with Citrus aurantium or Citrus sinensis on people with anxiety showed that inhalation or oral administration of Citrus aurantium and inhalation of Citrus sinensis can exert beneficial effects on anxiety; however, because of incomplete accuracy in the reporting of methodology, further more complete clinical studies are warranted.


Introduction
Citrus plants derived from the single genus Citrus are largely interbreedable. Among the common names given to the various members of the citrus family, orange often refers to the most popular Citrus sinensis and Citrus aurantium [1]. Chemical composition of Citrus plants is characterized by the presence of several polyphenolic classes, including flavones, flavanones, flavonols, flavans, and anthocyanins [2,3].
In folk medicine, products derived from the peel and/or whole dried immature fruit of orange plants have been eaten fresh or drunk as juice. Juice can be consumed directly or further processed into concentrate, and both derivatives are used in soda and cocktail drinks, punches, and liqueurs. Orange fruits and peels are also used in desserts, jams and marmalades, and candied peels, as well as cookies, cakes, and candies. EO derived from orange peels, flowers, leaves, and twigs is used in perfumes; orange seed oil may also be used in cooking or as a component in plastic industry [30].
The sweet orange tree is found more or less in the same places as the bitter orange tree. Sweet orange oil is extracted from the fruit of the tree via cold pressure; it is also possible to distill sweet orange oil [31].
Citrus sinensis contains several active secondary metabolites contributing to the pharmacological activities of the plant. In Citrus sinensis fruits, peel, leaves, juice, and roots, several types of chemical compounds including flavonoids [2,32], hydroxyamides, steroids, alkanes and fatty acids, coumarins, carbohydrates, peptides, carbamates and alkylamines, carotenoids, volatile compounds, and minerals such as potassium, magnesium, calcium, and sodium have been identified [12,33].
C. sinensis is a rich source of vitamin C, a natural antioxidant that support the immune system activity [33,34]. C. sinensis has been used traditionally, to treat intestinal disorders (such as cramps, constipation, colic, and diarrhea), respiratory disorders (such as cough, cold, bronchitis, and tuberculosis), obesity, menstrual disorder, cardiovascular disease (angina, hypertension), anxiety, depression, and stress [35].
Anxiety disorders are among the leading prevalent causes of global mental disorders [36,37]. They contribute to favour poor compliance with therapy [38] and insufficient patient adoption of healthy behaviors [39]. On the basis of their negative effects on the results of therapy, it is necessary to find effective interventions.
It is known that inhalation of volatile components of Citrus EO is able to influence the activity of brain areas such as the hypothalamus, hippocampus, and pyriform; preclinical and clinical research showed that citrus fragrance can restore stress-induced cortex [40,41] and immunosuppression [42] and may have potential antidepressant effects in rats [43,44].
In light of the abovementioned findings, we tried to assess if the potential health effects of both Citrus aurantium and Citrus sinensis are really effective in the treatment of anxiety conditions. With this aim, we summarized the published reports of preclinical and clinical studies regarding the use of Citrus aurantium-or Citrus sinensis-based products in conditions related to anxiety disorders.

Research Method and Inclusion Criteria of Clinical Trials.
A bibliographic research was carried out independently by two researchers (blinded to the authors and initially on results) in the major scientific databases and search engines of peer-reviewed literature from 2000 to July 2018, on life sciences and biomedical topics (PubMed, Scopus, Embase, Web of Science, and Google Scholar). The following Evidence-Based Complementary and Alternative Medicine 3 Numbers of records identified through database searching n = 94 Number of additional records identified through other sources n = 0 Records a er duplicates removed n = 84 Records excluded for title and abstract n = 63 Records excluded a er full review n = 4 (association products) Full text articles assessed for eligibility n = 21 Articles included in the study n = 17 Pre-clinical studies n = 8 Clinical studies n = 9 keywords or combination of keywords "Citrus anxiety", "Citrus aurantium axiety", "Citrus sinensis anxiety", and "clinical trials" were used.  [46] and the Jadad Scale [47]. From the eligible articles, two investigators independently extracted data by using a standard data extraction form. Data were considered for therapeutic indication, design of the study, number, sex, and age of subjects, endpoints, adverse effects, and outcome. In all the studies, reporting of adverse reactions was monitored. The absence of adverse reactions was defined as "not reported." All the authors reviewed all the eligible articles and resolved by discussion any uncertainty regarding the statistical method used to handle the missing data.

Results
A collection of 94 scientific articles was selected from our bibliography research. Only 17 articles describing effects of Citrus aurantium or Citrus sinensis treatment for anxiety were corresponding to the inclusion criteria. 63 articles were excluded for title and abstract and 4 were excluded because the products object of the studies were combinations including Citrus aurantium.
Nine clinical studies were included in the review (Figure 1). In eight clinical studies, Citrus aurantium or Citrus sinensis were administered for inhalation as aromatherapy, and in one study Citrus aurantium was orally administered. Table 1 summarizes for each preclinical study the authors, the route of administration, animal species, experimental model to study anxiety, dose, and observed effects of Citrus aurantium or Citrus sinensis administration. Tables 2 and 3 summarize authorship of the paper, therapeutic indication, study design, subjects involved, endpoints, adverse effects, and outcome of all the clinical trials regarding use of Citrus aurantium or Citrus sinensis, respectively. Tables 4 and 5 report CONSORT items for trials with herbal medicine interventions applied to clinical studies, for Citrus aurantium and Citrus sinensis, respectively. Table 6 reports quality assessment of randomized controlled trials by the Jadad scoring system.  Rotarod test Citrus aurantium essential oil 0.5 or 1.0 g/kg in a volume of 10 ml/kg.
In light-dark box test, single treatment with essential oil (0.5 or 1.0 g/kg) increased the time spent by mice in the light chamber and the number of transitions between the two compartments.
Single and repeated treatments with essential oil (0.5 or 1.0 g/kg) were able to suppress marble-burying behavior.
No impairment on rotarod procedure after both single and repeated treatments with essential oil was observed, denoting absence of motor deficit.
Evidence-Based Complementary and Alternative Medicine 5  Participants were randomly divided into three groups. Group 1 received 10 mg diazepam as oral dose; Group 2 received C. aurantium essential oil (EO) 10 mL diffused in the room through an electric dispenser. Group 3 (placebo) was exposed to the vaporization of saline solution.
In the last two groups, the exposure lasted 30min.  Patients of the fragrance condition were exposed to 5 drops (0.25 mL) of C. aurantium essential oil poured in 5 L of water and diffused using an electrical dispenser. Patients in the control condition were exposed in the same environment to diffusion of water without fragrance. All the patients (control and experimental) waited about 10 min in the waiting room. The dental anxiety scale (DAS) questionnaire was used to determine the anxiety level of the patients prior to surgery Mean blood pressure, respiratory rate, and pulse rate were also evaluated.
Not reported Orange fragrance is effective in reducing anxiety linked to surgical removal of impacted mandibular third molar.
Mean blood pressure, respiratory rate, and pulse rate, during surgery, were significantly reduced.  2 ml of orange essence was placed in a dispenser activated for 2 min every 10 min.
Anxiety of children was assessed with salivary cortisol level and pulse rate before and on completion of each of two dental treatment appointments.

Not reported
Statistically significant reduction of salivary cortisol level and pulse rate in aromatherapy group compared to control group. Not reported 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 a dose-dependent anxiolytic activity of sweet orange essential oil.   [48]. The acute administration induced an anxiolytic-like effect in the light/dark transition tests (increased time spent in the light side, and in the number of transitions) and in marble burying (decreased number of marbles buried) without any motor impairment, while repeated administration showed effects in the marble-burying test only. Repeated diazepam administrations did not increase light/dark transitions. Thus, the results suggest an anxiolytic-like effect following acute and repeated Citrus aurantium EO administration [49].
Similar results were obtained in another experiment showing that anxiolytic-like effect of oral administration of Citrus aurantium EO was reversed by 5-HT1A antagonist WAY100635 but not by flumazenil, a benzodiazepine antagonist, suggesting serotonergic mediation [44].
In another study, inhalation of Citrus aurantium EO increased social interactions (time spent in active social interaction) in rats and increased exploration time in the open arms of the elevated plus-maze, suggesting an anxiolytic-like effect at a dose that did not impair motor activity in the openfield test [50].
Acute intraperitoneal administration of Citrus aurantium EO, similar to fluoxetine, increased open arm explorations (percentage of time spent and percentage of entries) in the elevated plus-maze in male mice. The authors suggested that the effect of Citrus aurantium L. is linked to serotonergic transmission based on a fluoxetine + Citrus aurantium EO interaction. However, Citrus aurantium EO did not change the anxiolytic effect of fluoxetine in the elevated plus-maze, suggesting no drug interaction [51].
Khosrovi et al. investigated the effect of intraperitoneal injection of Citrus aurantium EO on anxiety and its interaction with GABAergic pathways, evaluating the coadministration effects of Citrus aurantium and diazepam.
Evidence-Based Complementary and Alternative Medicine 13 The JADAD scoring system was used for the assessment of randomized controlled trials with the following 5 items: Was the study described as randomized? ( Flumazenil did not alter the effect of Citrus aurantium; however, authors suggest that Citrus aurantium L. may exert an anxiolytic-like effect acting as partial agonist at the GABA-A receptor/benzodiazepine site [52]. Preclinical antianxiety effects of Citrus aurantium are summarized in Table 1.
Complex six studies investigating Citrus aurantium EO effects on anxiety levels during different medical conditions were found. All of the six studies in which Citrus aurantium was investigated were randomized/controlled clinical trials.
One randomized clinical trial described the effects of Citrus aurantium EO in reducing anxiety during labor in a group of Iranian pregnant women [53].
Before the aromatherapy, both groups had the same levels of anxiety; the levels of anxiety evaluated at dilations of 3-4 and 6-8 cm were significantly lower in the aromatherapy group with Citrus aurantium compared with the control group, thus suggesting that aromatherapy with Citrus aurantium EO could reduce anxiety during labor [53].
A trial was carried out on patients proposed for colonoscopy and divided into two groups. Aromatherapy was performed by inhalation of Sunflower oil (control group) and Neroli oil (experimental group). Results showed that there was no significance difference of procedural anxiety measured by State Trait Anxiety Inventory state (STAI-S) score and procedural pain evaluated by visual analogue scale (VAS), before and after aromatherapy in patients subjected to colonoscopy. Significant lower pre-and postprocedural systolic blood pressure in Neroli group than control group were observed. The authors concluded that the inhalation aromatic agent had an effect on lowering procedural anxiety-related (excessive fear of medical, dental, or surgical procedures that results in acute distress or interference) blood pressure [54].
Patients with a mandibular third molar with B II classification of impacted teeth and American Society of Anaesthesiologists (ASA) class I patients (healthy subjects with no organic pathology), with moderate and high anxiety levels measured through the dental anxiety scale (DAS) questionnaire, were included in another randomized and controlled study.
The ASA clinical status classification system assessed the fitness of patients before surgery. The outcome variables were physiologic measures related to anxiety, including mean blood pressure, respiratory rate, and pulse rate.
After aromatherapy, mean blood pressure, pulse rate, and respiratory rate were significantly lower in the fragrance group during surgery (from the time of sitting in the dental chair to the end of surgery). Vital signs measurements showed significant differences between the two groups, which may be due to the sedative effect of the orange fragrance during surgery. Results suggest that ambient orange fragrance could be helpful in reducing dental anxiety during dental surgical removal [55].
Another study was carried out on a Brazilian volunteers' cohort with chronic myeloid leukemia (CML), treated with standard therapy. The study compared Citrus aurantium EO with diazepam anxiolytic effects, in the moment that precedes the collection of medullary material in patients with CML. Systolic and diastolic blood pressure and cardiac and respiratory frequencies were measured. C. aurantium EO decreased both systolic and diastolic blood pressure while with diazepam, only systolic blood pressure decreased. The Citrus aurantium EO did not decrease the respiratory frequencies.
The use of STAI-S revealed an anxiolytic effect of Citrus aurantium EO group of patients with CML but not in the CML diazepam and placebo groups. In conclusion, study results showed an anxiolytic effect of Citrus aurantium EO in patients with CML with an improvement of psychological and physiologic parameters. For the authors, this effect has a great clinical relevance, because patients with cancer go through various stressful phases during the disease, and the standard therapy significantly contributes to improve the anxiety level and physiological parameters during a procedure that is a cause of distress [13].
Chaves Neto et al. (2017) studied the anxiolytic effects of Citrus aurantium EO in patients experiencing crack withdrawal. Based on the fact that individuals who experience crack withdrawal present a high anxiety trait, anxiety status was induced with the Simulated Public Speaking (SPS) method (subject is requested to deliver a speech in front of a video camera with its image being displayed on a TV screen). Anxiety levels were assessed by the Inventário de Ansiedade Traço-Estado (IDATE), the Brazilian version of STAI-S.
The results demonstrated that subjects in the groups treated with Citrus aurantium EO maintained controlled anxiety levels during SPS when compared to the control group (no treatment). Subjects who were administered Citrus aurantium EO also maintained states of discomfort and cognitive impairment often associated with anxiety during SPS. However, nebulization of the EO of Citrus aurantium provided an acute anxiolytic effect in crack cocaine users exposed to SPS.
Authors' conclusions were that Citrus aurantium EO, administered by nebulization, produced anxiolytic effects in crack users in abstinence thus indicating the possibility of Citrus aurantium EO use as an alternative complementary therapy in the control of anxiety in users who are abandoning drugs abuse [56].
In a randomized double-blind design, the effect of oral administration of Citrus aurantium blossom distillate (CABd) on preoperative anxiety was evaluated. Preoperative anxiety was assessed using STAIS and Amsterdam Preoperative Anxiety and Information Scale (APAIS). The main finding of this study was the confirmation of the anxiolytic effect obtained with oral administration of blossom distillate Citrus aurantium. Both STAI-state and APAIS scores were decreased by CABd. On the other hand, neither STAI-state nor APAIS was changed in the placebo group [57].
Results of the studies using Citrus aurantium EO showed that inhalation of the oil produces significant anxiolytic effects. Methods for diffusion of EO used in the studies are by direct inhalation with hand-hold nebulizers generally with doses of EO ranging 10 -50 mL or by dilution of EO in distilled water and successive diffusion through an electric dispenser or by gauzes impregnated with 4 mL of Citrus aurantium EO.

Citrus sinensis (Sweet Orange) Pre-Clinical and Clinical
Anti-Anxiety Effects. In comparison to findings published about investigation on Citrus aurantium, a reduced number of preclinical and clinical experiments were conducted on Citrus sinensis. Inhalation of Citrus sinensis EO (sweet orange, containing 97% limonene) in rats submitted to elevated plus-maze followed by the light/dark paradigm produced an increase in open arm exploration (% time spent and % number of entries) in the elevated plus-maze, and of the time spent in the lit chamber of the light/dark test. No effect on motor activity, as measured by the total distance travelled in the elevated plus-maze, was detected [58].
Anxiolytic-like, sedative, and antidepressant-like potential effects of inhalation of both Citrus aurantium and Citrus sinensis EOs were evaluated through behavioral tests and measurement of corticosterone and melatonin plasma levels in mice.
Results of behavioural tests showed an anxiolytic-like and sedative effect of Citrus sinensis EO 10% inhalation, without affecting melatonin and corticosterone physiological levels. Inhalation of 10% Citrus aurantium EO did not show neither anxiolytic-like effects nor change in melatonin and corticosterone plasma levels [59].
Three randomized and controlled studies investigating on the effects of Citrus sinensis were found. Two studies describe the effects of Citrus sinensis EO on anxiety in adults and children, respectively, subjected to dental treatments.
A randomized controlled trial was carried out on adult patients waiting for dental treatment to evaluate the anxiolytic effects of Citrus sinensis. Orange odor was diffused in the waiting room of odor group through an electrical dispenser whereas in the control group no odor was diffused. For assessing cognitive functions, patients completed the Wortschatz test (WST) [60]. For assessing trait and state anxiety, patients were given the State-Trait Anxiety Inventory (STAI) [61]. The Mehrdimensionale Befindlichkeitsfragebogen (MDBF) [62] were used for assessment of current mood, alertness, and calmness.
Results of the study showed that exposure to ambient orange odor has a relaxant effect compared to control group. Results reported also a sex difference of Citrus sinensis effects, showing that women exposed to sweet orange EO had a lower level of state anxiety, a more positive mood, and a higher level of calmness [63].
The effects of orange odor (Citrus sinensis) on child anxiety during dental treatment were also evaluated in another study by using salivary cortisol and pulse rate as index of patients' anxiety.
The results of this study showed that the salivary cortisol level and pulse rate significantly decreased in intervention groups by using aromatherapy with Citrus sinensis EO [64]. The potential anxiolytic effect of Citrus sinensis EO was evaluated in a double-blind, randomized, placebo-controlled clinical trials carried out on healthy volunteers submitted to an anxiogenic situation. Immediately after inhalation, each subject was submitted to the video-monitored version of the Stroop Color-Word Test (SCWT), a method to induce anxiety. During the text, a board with 100 of the color-naming words blue, yellow, red, green, and violet organized randomly in a 10 ⋅ 10 matrix is presented to each participant. The color of each word is different from its own meaning (for example, the word "green" printed in red). The participant has to say quickly (in two minutes) the sequence presented, the color of the ink, but not the colors described by the single words. The whole test is recorded and presented to the subject on a monitor during the test [65].
State-Trait Anxiety Inventory (STAI) and Visual Analogue Mood Scale (VAMS) were used to evaluate psychologic parameters. Physiologic parameters were evaluated before the inhalation period and before, during, and after the SCWT. Results showed dose-dependent anxiolytic properties of sweet orange EO [65].
These three studies indicate that Citrus sinensis EO exerts anxiolytic effects in anxiogenic situations. One of the studies show that Citrus sinensis EO exerts its anxiolytic effects also in children.

Discussion
EOs physiological and psychological effects are known in folk medicine and aromatherapy for a long time [14,66]. Aromatherapists have proposed that Citrus fragrances have mood-enhancing properties. These effects were confirmed by successful clinical study carried out with citrus fruits oils on patients affected by stress symptoms or depression [67,68].
Anxiety disorders are the most common type of psychiatric disorders in the general population [69]. Their treatment is difficult because of the important side effects of the drugs used to improve anxiety symptoms, which generally promote dependence [70]. Moreover, common treatments do not benefit all patients and only few of them have a slight resumption [71]. These conditions justify the increasing interest and search for alternative or complementary procedures aimed at improving anxiety symptoms. One of these is aromatherapy, a procedure that uses EOs by inhalation as a treatment for medical purposes [72].
Citrus aurantium and Citrus sinensis are rich in flavonoids and polyphenolic compounds with numerous pharmacological properties, such as the inhibition of the oxidation of lowmolecular weight proteins and platelet accumulation thus contributing to immune cell stability.
They are also used in treatment of mental disorders, inflammation, viral infections, and allergies [73,74]. Flavonoids effects in reducing anxiety are due to their action as benzodiazepine receptor agonists [57].
A series of review articles regarding the safety and efficacy of bitter orange has been published [75][76][77][78]. However, this is the first analysis of clinical studies using Citrus aurantium or Citrus sinensis for the treatment of anxiety. We analyzed all clinical studies according to the recommendations described in the checklist developed by the Consolidated Standards of Reporting Trials [46] for the reporting of clinical trials using herbal medicinal products (Tables 4 and 5). The clinical trials included in this review have different levels of methodological accuracy. All of studies were randomized and controlled. Only one study regarding Citrus aurantium use was carried out with a double-blind methodology ( Table 2). Two of the studies regarding Citrus sinensis use were carried out with a double-blind methodology, and only one was performed as a crossover study (Table 3).
Since it is a common source of selection bias, a point of weakness in all the studies is the lack of description of the methods adopted to generate random allocation sequences [78] (Table 6). Moreover, all the studies were poor in number of subjects recruited. In four of them, the sample of people recruited did not exceed 50 patients, in four studies, the samples exceeded 50 participants, and only in one study the sample was more than 100 patients (Tables 2 and 3).
All the studies reported statistical data analysis but no one reported sample size calculation. Latin binomial name of the plant was always correctly reported, although not all articles provided an exhaustive description of the characteristics of the product. Procedure adopted to obtain the EO and description of the raw material used to produce the herbal preparations were reported only in one study (Tables 4 and  5). The highest standards in methodology for clinical trials in herbal medicine strongly recommend the reporting of the characteristics of the product to facilitate the reproducibility of the studies. The presence of these data are fundamental to establish a link between the putative efficacy and safety to the single product [46].
Only few studies report qualitative testing producing the chemical fingerprint of the herbal products (Table 4). Finally, one study [13] compares the effects of Citrus aurantium against diazepam, a well-established drug prescribed for the treatment of anxiety (Table 2).

Conclusions
Antianxiety effects of Citrus aurantium and Citrus sinensis EOs were previously demonstrated through behavioral experiments carried out on laboratory animals. Complex clinical studies considered for this overview suggest that Citrus aurantium or Citrus sinensis EOs, used for anxiolytic therapy of people prevalently in conditions in which stress is dominating, produce positive effects against anxiety. In particular, Citrus aurantium EO aromatherapy reduced anxiety level in the great part of stress conditions studied (subjects affected by chronic myeloid leukemia and preoperative patients) except for a sample of patients subjected to colonoscopy. Exposition to Citrus sinensis EO in clinical studies is shown to be positive in reducing anxiety level in patients waiting for dental treatment as well as in healthy volunteers submitted to an anxiogenic situation. However, a definitive conclusion needs further studies because of the reduced number of studies and the small number of patients for each study. Regarding data reporting of clinical studies with Citrus aurantium, they appear without description of the characteristics of the herbal product and lack qualitative testing. From the methodological point of view, most of the clinical studies show the lack of important items such as treatment allocation conceal and description of double-blind procedure. On the basis of the present overview, we can conclude that the use of Citrus aurantium and Citrus sinensis EOs could be useful to reduce anxiety caused by clinical stress conditions; however, results are impaired by the poor accuracy and methodology applied in clinical studies.

EO:
Essential oil STAI-S: State Trait Anxiety Inventory score VAS: Visual