ISSN: 2690-0777
Open Journal of Environmental Biology
Research Article       Open Access      Peer-Reviewed

The effect of olfactory exposure on the heart rate of rabbits during transportc

Anouck Haverbeke1-3*, Stefania Uccheddu2, Dieter Everaert4, Maya Lagae2, Marie Lacoste2-6, Heidi Arnouts1-5 and Adinda Sannen1

1Odisee University of Applied Sciences, Salto Research Group, Hospitaalstraat 23, 9100 Sint-Niklaas, Belgium
2Vet Ethology, Leemveldstraat 44, 3090 Overijse, Belgium
3Department of Veterinary Medical Imaging and Small Animal Orthopaedics, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium
4Hagelandstraat 40, 3012 Wilsele, Belgium
5Department of Engineering Management, University of Antwerp, Prinsstraat 13, 2000 Antwerp, Belgium
6Vetagro Sup, Veterinary Campus of Lyon, 1 avenue Bourgelat, 69280 Marcy-L’Étoile, France
*Corresponding author: Anouck Haverbeke, Odisee University of Applied Sciences, Salto Research Group, Hospitaalstraat 23, 9100 Sint-Niklaas, Belgium, E-mail: anouck.haverbeke@vetethology.com
Received: 30 September, 2022 | Accepted: 14 November, 2022 | Published: 15 November, 2022
Keywords: Rabbits; Stress; Olfactory exposure, Essential oils; Heart rate; Transport

Cite this as

Haverbeke A, Uccheddu S, Everaert D, Lagae M, Lacoste M, et al. (2022) The effect of olfactory exposure on the heart rate of rabbits during transport. Open J Environ Biol 7(1): 033-039. DOI: 10.17352/ojeb.000032

Copyright

© 2022 Haverbeke A, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Rabbits can experience stress during transport. This study explores the effects of Essential Oils (EOs) on the heart rate of rabbits during transport. Rabbits were submitted to 3 different treatments: no olfactory exposure to EOs (treatment 1, control); olfactory exposure to 30 drops of Lavandula angustifolia EO (treatment 2); and olfactory exposure to 30 drops of a blend of 5 EOs (treatment 3, Cananga odorata, Citrus aurantium, Cupressus sempervirens, Lavandula angustifolia, Litsea citrata EOs) in a randomized controlled crossover study design. Treatment 1 and Treatment 2 did not induce a significant change in rabbits’ heart rates, however, treatment 3 did induce a significant decrease in rabbits’ heart rates after transport. These results suggest that olfactory exposure to this blend of 5 EOs decreased the rabbit’s heart rate after transport. More research is needed to further evaluate the effects of olfactory enrichment with EOs in rabbits during transport.

Abstract

The study explores the effects of Essential Oils (EOs) on the heart rate of rabbits during transport. Rabbits (n = 35) were individually transported three times by car in a transport box for 5 minutes. Each rabbit was subjected to three treatments: no olfactory exposure to EOs (treatment 1, control); olfactory exposure to 30 drops of Lavandula angustifolia EO (treatment 2); and olfactory exposure to 30 drops of a blend of 5 EOs (treatment 3, Cananga odorata, Citrus aurantium, Cupressus sempervirens, Lavandula angustifolia, Litsea citrata EOs) in a randomized controlled crossover study design. Heart rate was measured before and after transport. Compared to the control, treatment 3 showed a significant decrease in HR after transport (mean = -24.33, SD = 41.77; post hoc Tukey test p = 0.007). Results suggest that the blend of EOs might help rabbits to recover homeostasis quicker after a stressful event. This suggests interesting potential field applications not only for rabbit owners but also for veterinarians. There is more research needed regarding the specific effects of EOs on rabbits.

Introduction

Transport is considered stressful for rabbits [1,2]. During stress, the autonomic nervous system acts on various systems including the cardiovascular system by a modification of blood pressure and heart rate with tachycardia [3]. These indicators are especially interesting because the data can be collected non-invasively, avoiding the effect of the procedure. Rabbits, as a prey species, are very susceptible to stress and often disguise these signs of stress [1]. So far, the interest in travel-induced stress was specifically related to a rabbit as farm animals [2]. However, rabbits are widely accepted as pets [3] and they might accompany their owners during travel or, in case of illness, they might need to be transported to veterinary centers. Since rabbits might respond to road transport with a pronounced stress response, new treatments for travel-induced stress are still under research with specific characteristics of being cheap and not associated with adverse effects.

In literature, Essential Oils (EOs) have been already applied as a treatment for travel-induced stress in other domestic species and horses [4,5]. Particularly Lavender EO (Lavandula angustifolia) is recognized for its anxiolytic effects, already been studied in humans and animals [6,7].

There are other EOs appear to be interesting as well for their proven anxiolytic effects in humans and shelter dogs [6-10]. In line with previous literature, a recent study on olfactory enrichment in shelter dogs confirmed a significant anxiolytic effect of the following 5 EOs: Ylang-ylang (Cananga odorata), Orange (Citrus aurantium), Cypress (Cupressus sempervirens), Lavender (Lavandula angustifolia) and Litsea citrata.

Ylang-ylang EO has shown anxiolytic effects on mice in several behavioral tests based on instinctive responses to novel environments [11]. This EO has shown anti-depressant and anxiolytic effects on humans [12]. Ylang-ylang EO furthermore induced neuropathic pain relief and ameliorated pain-associated anxiety [13].

Orange EO has gained attention for its sedative and relaxing actions in mice [14,15] and humans [16]. During the test of the elevated plus maze, anxiolytic effects have been observed in mice exposed to the scent of Orange EO [13]. Carvalho-Freitas and colleagues have demonstrated the sedative and hypnotic activity of Citrus aurantium on mice [15].

Cypress is known for its antibacterial [17] as well as antioxidant effects. Antioxidant therapy with Cypress is regarded as a cornerstone in the treatment of several types of neurological disorders and depression [17,18]. Lim and colleagues have also pointed out the sedative effects of Cypress EO in mice [19].

The neuropharmacological properties of Litsea citrata EO (also called Litsea cubeba) have been studied in humans [20] and its anxiolytic effects have been demonstrated [6,21]. Male mice treated orally for seven days with Litsea cubeba EO showed increased exploration (time spent and the number of entries) in the open arms of the elevated plus-maze test and a decrease in total distance traveled in the open arms suggesting a sedative effect of Litsea cubeba EO [22].

Based upon this literature, we wanted to investigate further the effect of Lavender EO, as well as the effect of a blend of the 5 EOS described above on the heart rate of rabbits during transport. Each rabbit was subjected to 3 treatments: no olfactory exposure to EO (treatment 1, control); olfactory exposure to 30 drops of Lavender EOs (treatment 2); and olfactory exposure to 30 drops of a blend of 5 EOS (treatment 3, Cananga odorata, Citrus aurantium, Cupressus sempervirens, Lavandula angustifolia, Litsea citrata EOs) in a randomized controlled crossover study design. The difference between treatments was measured using the heart rate.

As suggested by Galindo and colleagues [23], the interactions between compounds often result in biological activity that is greater than the activity of the isolated compounds. Therefore, we did expect a higher decrease in the heart rate of the rabbits with the blend of EOs (treatment 3) compared to the control (treatment 1) or lavender only (treatment 2).

Materials and methods

Study design

Each rabbit was subjected to each of the three treatments in a randomized controlled crossover study design. The rabbits were divided into three groups respecting the homogeneity of the rabbit population (Table 1).

The study was carried out for 4 days at the shelter ‘Het Vrolijke Konijnenhol’ in Oostkamp, Flanders, Belgium on February 27th (day 1, pre-test), March 5th (day 2), March 12th (day 3) and March 19th (day 4) 2020.

Animals and housing conditions

Thirty-five rabbits participated in the study, all were mixed breeds. There were seventeen females and eighteen males. The rabbits aged between ten months and seven years. The exact breed and age of most of the rabbits were difficult to establish as several rabbits have been relinquished by the owners or found as stray. Thirteen rabbits had been in the shelter for more than 6 months.

The shelter housed about 40 rabbits of varying ages (between 1 month and 7 years). The average room temperature was 10 °C during the entire study. There were no changes in the housing conditions of the rabbits due to the research. The rabbits were housed individually or with two or three conspecifics. 18 rabbits were housed in a room where three blocks of three cages were placed next to each other. The size of these cages ranged from 0,291 to 0,434m3 (indoor cages) and from 0,367 to 1,634m3 (outdoor cages). Each cage included one or two water bottles, one food hopper and one or two litter boxes. In the evening (so after the experiment) food and water were provided. Litter boxes were cleaned every week.

Essential oils

The EOs were obtained by steam distillation and purchased from the company Alanine Laboratory (EODIS, Belgium). The Biochemical composition and relative proportion of the constituents have been assessed by Gas Chromatograph with Mass Selective Detector for each EO (see Annex for the complete analyses reports).

The principal components of Lavender EO were linalyl acetate (34%), linalool (30%) and Lavandula acetate (3%).

Next to the anti-inflammatory and stress-releasing effects of linalyl acetate, Shen and colleagues [24] have discussed its utility in a stress management treatment among repeatedly stressed Ulcerative Colitis patients.

Several studies have shown the anxiolytic [25] and sedative effects of linalool [26,27]. Harada and colleagues examined the anxiolytic effects of linalool odor with light/dark box test and with elevated plus maze (EPM) and found that linalool odor has an anxiolytic effect without motor impairment in mice [25].

Lavandulyl acetate has been described for its anti-repellent effects [28], but so far, no anxiolytic effects have been reported.

The principal components of Ylang-Ylang EO were Germacrène D 21%, β-caryophyllène 17%, E,E-α- Farnesene 13% , Benzyl benzoate (6,85%) and linalool (4,42%).

Germacrenes are typically produced in a few plant species for their antimicrobial and insecticidal properties and can serve as a precursor for many other sesquiterpenes [29].

Beta-caryophyllene (BCP) has been identified as one of the best terpenes to reduce anxiety. Hwang and colleagues [30] observed that BCP improved chronic stress-related behavioral and biochemical changes in mice, concluding that BCP may be effective in treating depression and stress-related mental illnesses.

Farnesene act as a natural insect repellent [31], but its already field-reported sedative effects need still to be validated. Benzyl benzoate and linalool have shown individual anxiolytic effects in male mice [32].

The most relevant components of Orange EO were linalyl acetate (53%), linalool (21%), and α-terpinéol (6%). It is interesting to note that its 2 major components are similar to those of Lavender EO.

Litsea citrata was mainly composed of geranial (41%, geranial is considered as citral), neral (32 %), and d-limonene (12 %). While geranial and neral are mainly known for their anti-inflammatory activities [33], stress-reducing activities were reported for citral [34] and d-limonene [34,35]. 

Cypress EO was mainly composed of α-pinene (58%), δ-3-care (18%), and α-terpinolene (2%).

Weston-Green and colleagues reviewed the anti-anxiety and anti-depressant effects of α-pinene on humans [36].

D3-care is known to have hypnotic, anti-inflammatory, antioxidant, and anti-stress effects [37]. Terpinolene is known for its sedative effects through oral administration, as well as through nasal absorption [38].

The concentration of the 5 EOs in the blend (treatment 3) was proportional to the same, each EO was present for 20% in the blend.

Experimental set-up

The transport study was conducted by three experimenters (AH, DE and ML). The rabbit was placed in the transport box (45 L x 27 W x 30 H cm) and its heart rate was measured. The rabbit data was inside the transport box for 5 minutes in the experimental analysis, while being gently transported by car by one of the investigators. The investigator drove with an average speed of 30 km/h. Immediately after the transport, the heart rate was measured in the transport box and afterward in the cage.

All controllable conditions were similar for all the rabbits during the study. Nevertheless, due to field conditions, some variables did change like time of day, housing conditions, weather as well as some minor noises.

Treatment

Each rabbit was subjected to three treatments: no olfactory exposure to EOs (treatment 1, control); olfactory exposure to 30 drops of Lavandula angustifolia EO (treatment 2); and olfactory exposure to 30 drops of a blend of 5 EOs (treatment 3, Cananga odorata, Citrus aurantium, Cupressus sempervirens, Lavandula angustifolia, Litsea citrata EOs).

For every treatment another but similar transport, the box was used so the EOs did not interfere. For each rabbit, a new piece of cloth (10 x 10cm) was used. The pieces of cloth were taped inside the transport boxes at the top.

Data collection

The parameter heart rate was used to determine the effect of olfactory exposure with EOs on this group of rabbits. The measurements of the heart rate were taken by the same experimenter to guarantee homogeneity in counting. The heart rate was measured with a standard stethoscope. All the rabbits were always carried and held by the same experimenter. Heart rate was measured at the onset of the stressful event, when the rabbit was just placed in the transport box, before getting into the car for transportation (see HR1 below) and after the stressful event when the rabbit was back in his familiar environment (his cage; see HR2 below).

Experimenter 1 prepared the transport box for the rabbit. The transport box was disassembled, and the upper side was put upside down on the ground. Experimenter 1 taped a piece of cloth in the middle of the upper piece of the transport box and put nothing (treatment 1), 30 drops of lavender EO (treatment 2), or 30 drops of the blend of EOs (treatment 3) on the piece of cloth. The transport box was put back together.

Experimenter 2 carried the rabbit from its cage to the transport box inside the garage by holding him close to his body and supporting the hindquarters. The heart rate from the rabbit was measured in the transport box by experimenter 2 with the help of experimenter 3. Experimenter 2 held the rabbit and placed the stethoscope under the front legs. Experimenter 3 timed 30 seconds on a stopwatch.

Then experimenter 1 closed the transport box and gently carried it to the car for a 5-minute drive. During this time, experimenter 3 multiplied the counted heartbeats by two and documented this in the excel file (HR1).

Experimenter 1 came back from the transport. The heart rate was measured in the transport box by experimenter 2 while experimenter 1 timed 30 seconds on a stopwatch. The counted heartbeats were multiplied by two and documented in the excel file by experimenter 3.

Experimenter 3 took the piece of cloth from the transport box and placed it in the cage of the rabbit. The rabbit was carried to his cage in the transport box by experimenter 1 and the rabbit went by himself in his cage.

Experimenter 3 timed 30 seconds on a stopwatch while experimenter 2 counted the heart rate. The counted heartbeats were multiplied by two and experimenter 3 documented this in the excel file (HR2).

This sequence was repeated for each rabbit in each treatment.

Ethical note

An ethical note has been approved by Odisee University of Applied Sciences confirming that the rabbits were not considered laboratory animals. The animals were constantly monitored. The experiment would immediately have been stopped, should a rabbit have shown any sign of distress, impaired welfare, or clinical problem. By the IACUC, all the following measurements had been taken to ensure that the proposed research respected Animal Welfare and didn’t involve any animal tests violating animal welfare.

The researchers didn’t conduct any invasive procedures on the rabbits. All the rabbits were monitored accurately during the entire experiment. The researchers did not observe any negative effects of these concentrations of EOs on these rabbits (no signs of distress that could be linked to the EOs). No procedures caused any pain or distress to the animals.

Statistical analysis

A mixed linear model was valued with the response (difference in heart rate: HR2 minus HR1) and random effects. Individuals, groups and days were included in the random effects. Post hoc test evaluated for pairwise comparisons between treatments. A Tukey correction for multiple tests was applied.

Results

Two heart rate measurements were used to determine the effect of olfactory stimulation with EOs on rabbits. The first heart rate (HR1) was measured at the onset of the stressful event, when the rabbit was just placed in the transport box, before getting into the car for transportation. The second heart rate (HR2) was measured after the stressful event when the rabbit was back in his familiar environment (their cage; Table 2).

The graph shows HR2 (after) minus HR1 (before). Positive values thus signify an increase in HR, and negative values a decrease in HR. The mean heart rate increased for treatment 1 (control, no olfactory exposure to EOs), but decreased for treatment 2 (olfactory exposure to 30 drops of Lavandula angustifolia EO) and treatment 3 (olfactory exposure to 30 drops of the blend of EOs). Compared to treatment 1, treatment 3 showed a significant decrease in HR (Figure 1).

Discussion

Compared to treatment 1 (control, no olfactory exposure to EOs), treatment 3 (olfactory exposure to 30 drops of the blend of EOs) showed a significant decrease in HR after transport. This is in line with previous research, suggesting that a blend of anxiolytic EOs might have more effect than the single use of Lavender EO [8]. These results suggest that this blend of EOs might help rabbits to recover homeostasis quicker after a stressful event. This presents interesting potential field applications not only for rabbit owners but also for veterinarians. Olfactory stimulation with EOs may be useful not only during transport but also during stressful events at the shelter or at the owner’s home as well as at the veterinary clinic.

Based on the literature [1,2], our premise was that transport induced stress in rabbits. Also in our study, results showed an increase in heart rate in the rabbits during transport in treatment 1 (control, not exposed to any EOs), supporting the assumption that transport was stressful to them.

The study was conducted in a real-life unstandardized environment. This means that some variables varied during the experiment. The result of the study may have been affected by for example the margin of error from the heart rate measurements, the time of the day, the housing conditions, the size of the rabbits, the weather on the days of the experiment, and some minor noise during the experiment. However, due to the repeated measures design of our experiment, we expect that the triggers mentioned above had little effect on the results.

Our results did not show a significant effect of the treatment with lavender EO on heart rate. Most studies on Lavender however pointed out its anxiolytic activity. Research conducted on humans showed that Lavender EO could decrease some agitation and emotional disorders in people suffering from dementia [39-41]. Research has shown that rodents became calmer and less agitated with the smell of lavender oil [42-44]. Gerbils exhibited less anxious behavior when exposed to the smell of lavender oil, which appeared to have a similar effect as diazepam [44]. Lavender EO was found to reduce anxiety and depression in rats. At a high concentration, a sedative effect has been induced [45]. An anticonvulsant effect of lavender EO was reported against seizures in male mice. Particularly, lavender EO inhibited the onset, shortened the duration, and reduced the intensity of seizure attacks [46]. Lavender EO appeared to decrease the incidence and severity of travel sickness in pigs but not overall levels of stress (as measured by concentrations of salivary cortisol) [47]. In dogs, lavender EO decreased barking and active behavior [9] and dogs barked less in the car when being exposed to lavender oil during transport [4]. Although its mechanism of action has not been completely determined yet, the antidepressant activities of lavender may be attributed at least in part to the NMDA receptor modulation as well as an inhibition of the SERT [48].

In contrast, some studies have shown that Lavender EO modulated motor movement and locomotion [49] and increased arousal levels [50]. It is not yet known whether the effect is concentration-dependent or individual-dependent. Following previous observations on individual coping styles [51,52], it might be interesting to investigate further if any genetic or temperamental differences might determine whether lavender EO alleviates or exacerbates anxiety in rabbits.

Our results clearly show that more research is needed to further evaluate the effects of olfactory stimulation with EO in rabbits. This research leads to other questions and pushes forward the investigation into the effects of EOs. For example (1) understanding the minimal concentration necessary to decrease anxiety in rabbits; (2) the impact of olfactory stimulation on multi-rabbit transportation, (3) different breeds and sizes of rabbits. To improve the welfare of rabbits, it is important to find tools to help them recover fast in stressful situations like for example transport, waiting at the veterinary or consultation room. In this study, we have focused on the individual transport of rabbits. It might be interesting to explore further the effects of increased transport on rabbits during commercial group transport and how EOs might support these rabbits.

Further research is needed to confirm our preliminary results, in which olfactory exposure to a blend of EOs has been more effective than olfactory exposure to one single EO. It would also be interesting to compare this blend of EOs to other blends of anxiolytic EOs. To evaluate the total effect of olfactory enrichment, future studies would benefit from a study that includes a standardized environment, behavioral observations as well as measurements of other physiological non-invasive and non-stressful parameters.

Conclusion

The primary purpose of this study was to evaluate the effect of olfactory exposure of EOs on the heart rate of shelter rabbits during transport. Results have shown that olfactory exposure to a blend of 5 EOs (Cananga odorata, Citrus aurantium, Cupressus sempervirens, Lavandula angustifolia, and Litsea citrata EOs) significantly decreased the rabbit’s heart rate during transport. No significant decrease has been observed in the control treatment or treatment with lavender EO. EOs can be useful to help rabbits to recover homeostasis after a stressful event. There is more research needed about the specific effects of EOs on rabbits.

Author contributions

Conceptualization, AH and AS; methodology, AH and AS; software, HA; validation, AH and AS; formal analysis, HA; investigation, AH, ML, DE; resources, AH, ML, DE; data curation, ML; writing—original draft preparation, AH, ML, ML; writing—review and editing, AH and AS; visualization, AH; supervision, AH and AS; project administration, AH; funding acquisition:/ All authors have read and agreed to the published version of the manuscript.

Institutional review board statement

Ethical review and approval were waived for this study, due to IACUC.

We would like to acknowledge the generosity and availability of the Belgian rabbit's shelter “Het Vrolijke Konijnenhol”.

  1. Trocino, A., Zomeño, C., Birolo, M., Di Martino, G., Stefani, A., Bonfanti, L., Bertotto, D., Gratta, F., Xiccato, G. (2018). Impact of preslaughter transport conditions on stress response, carcass traits and meat quality in growing rabbits. Meat Science, 146, 68-74.
  2. Fazio F, Casella S, Giudice E, Giannetto C, Piccione G. Evaluation of secondary stress biomarkers during road transport in rabbit. Livestock Science. 2015; 173:106-110. https://doi.org/10.1016/j.livsci.2015.01.006
  3. Guiblin-Legent H. Essential oils: interest in pet rabbits to reduce stress in veterinary consultation (veterinary doctorate). National Veterinary School of Alfort. 2018.
  4. Wells DL. Aromatherapy for travel-induced excitement in dogs. J Am Vet Med Assoc. 2006 Sep 15;229(6):964-7. doi: 10.2460/javma.229.6.964. PMID: 16978115.
  5. Ferguson CE, Kleinman HF, Browning J. Effect of Lavender Aromatherapy on Acute-Stressed Horses. Journal of Equine Veterinary Science. 2013; 33 (1): 67-69. https://doi.org/10.1016/j.jevs.2012.04.014
  6. Bagetta G, Cosentino M, Sakurada T. Aromatherarpy – Basic mechanisms and Evidence-Based Clinical Use. CRC Press, Taylor & Francis Group, ISBN. 2016; 13-978-1-4822-4663-6:225-227.
  7. de Sousa DP, de Almeida Soares Hocayen P, Andrade LN, Andreatini R. A Systematic Review of the Anxiolytic-Like Effects of Essential Oils in Animal Models. Molecules. 2015 Oct 14;20(10):18620-60. doi: 10.3390/molecules201018620. PMID: 26473822; PMCID: PMC6332383.
  8. Uccheddu S, Mariti C, Sannen A, Vervaecke H, Arnout H, Gutierrez Rufo J, Haverbeke A. Behavioural and cortisol responses of shelter dogs to a cognitive bias test after olfactory enrichment with essential oils. Dog Behavior. 2018; 2:1-14. https://doi.org/10.4454/db.v4i2.87
  9. Graham L, Wells DL, Hepper PG. The influence of olfactory stimulation on the behaviour of dogs housed in a rescue shelter. Applied Animal Behaviour Science. 2005; 91:143-153. https://doi.org/10.1016/j.applanim.2004.08.024
  10. Haverbeke A, Uccheddu S, Arnouts H, Sannen A. A Pilot Study on Behavioural Responses of Shelter Dogs to Olfactory Enrichment. Veterinary Science Research. 2019; 1(1). https://doi.org/10.30564/vsr.v1i1.1147
  11. Zhang N, Zhang L, Feng L, Yao L. Cananga odorata essential oil reverses the anxiety induced by 1-(3-chlorophenyl) piperazine through regulating the MAPK pathway and serotonin system in mice. J Ethnopharmacol. 2018 Jun 12;219:23-30. doi: 10.1016/j.jep.2018.03.013. Epub 2018 Mar 12. PMID: 29545208.
  12. Hongratanaworakit T, Buchbauer G. Evaluation of the harmonizing effect of ylang-ylang oil on humans after inhalation. Planta Med. 2004 Jul;70(7):632-6. doi: 10.1055/s-2004-827186. PMID: 15303255.
  13. Borgonetti V, López V, Galeotti N. Ylang-ylang (Cananga odorata (Lam.) Hook. f. & Thomson) essential oil reduced neuropathic-pain and associated anxiety symptoms in mice. J Ethnopharmacol. 2022 Aug 10;294:115362. doi: 10.1016/j.jep.2022.115362. Epub 2022 May 10. PMID: 35551977.
  14. Pultrini Ade M, Galindo LA, Costa M. Effects of the essential oil from Citrus aurantium L. in experimental anxiety models in mice. Life Sci. 2006 Mar 6;78(15):1720-5. doi: 10.1016/j.lfs.2005.08.004. Epub 2005 Oct 25. PMID: 16253279.
  15. Carvalho-Freitas MI, Costa M. Anxiolytic and sedative effects of extracts and essential oil from Citrus aurantium L. Biol Pharm Bull. 2002 Dec;25(12):1629-33. doi: 10.1248/bpb.25.1629. PMID: 12499653.
  16. Pimenta FC, Alves MF, Pimenta MB, Melo SA, de Almeida AA, Leite JR, Pordeus LC, Diniz Mde F, de Almeida RN. Anxiolytic Effect of Citrus aurantium L. on Patients with Chronic Myeloid Leukemia. Phytother Res. 2016 Apr;30(4):613-7. doi: 10.1002/ptr.5566. Epub 2016 Jan 20. PMID: 26787366.
  17. Selim SA, Adam ME, Hassan SM, Albalawi AR. Chemical composition, antimicrobial and antibiofilm activity of the essential oil and methanol extract of the Mediterranean cypress (Cupressus sempervirens L.). BMC Complement Altern Med. 2014 Jun 2;14:179. doi: 10.1186/1472-6882-14-179. PMID: 24890383; PMCID: PMC4052795.
  18. van Dooren FE, Pouwer F, Schalkwijk CG, Sep SJ, Stehouwer CD, Henry RM, Dagnelie PC, Schaper NC, van der Kallen CJ, Koster A, Denollet J, Verhey FR, Schram MT. Advanced Glycation End Product (AGE) Accumulation in the Skin is Associated with Depression: The Maastricht Study. Depress Anxiety. 2017 Jan;34(1):59-67. doi: 10.1002/da.22527. Epub 2016 Jun 6. PMID: 27271340.            
  19. Asgary S, Naderi GA, Shams Ardekani MR, Sahebkar A, Airin A, Aslani S, Kasher T, Emami SA. Chemical analysis and biological activities of Cupressus sempervirens var. horizontalis essential oils. Pharm Biol. 2013 Feb;51(2):137-44. doi: 10.3109/13880209.2012.715168. Epub 2012 Nov 20. PMID: 23167275.
  20. Lim WC, Seo JM, Lee CI, Pyo HB, Lee BC. Stimulative and sedative effects of essential oils upon inhalation in mice. Arch Pharm Res. 2005 Jul;28(7):770-4. doi: 10.1007/BF02977341. PMID: 16114490.
  21. Wang YS, Wen ZQ, Li BT, Zhang HB, Yang JH. Ethnobotany, phytochemistry, and pharmacology of the genus Litsea: An update. J Ethnopharmacol. 2016 Apr 2;181:66-107. doi: 10.1016/j.jep.2016.01.032. Epub 2016 Jan 23. PMID: 26812679.
  22. Chen CJ, Tseng YH, Chu FH, Wen TY, Cheng WW, Chen YT, Tsao NW, Wang SY. Neuropharmacological activities of fruit essential oil from Litsea cubeba Persoon. J. Wood Sci. 2012; 58:538–543. https://doi.org/10.1007/s10086-012-1277-3
  23. Galindo LA, Pultrini Ade M, Costa M. Biological effects of Ocimum gratissimum L. are due to synergic action among multiple compounds present in essential oil. J Nat Med. 2010 Oct;64(4):436-41. doi: 10.1007/s11418-010-0429-2. Epub 2010 Jun 18. PMID: 20559750.
  24. Shin YK, Kwon S, Hsieh YS, Han AY, Seol GH. Linalyl acetate restores colon contractility and blood pressure in repeatedly stressed-ulcerative colitis rats. Environ Health Prev Med. 2022;27(0):27. doi: 10.1265/ehpm.22-00041. PMID: 35753805; PMCID: PMC9283910.
  25. Harada H, Kashiwadani H, Kanmura Y, Kuwaki T. Linalool Odor-Induced Anxiolytic Effects in Mice. Front Behav Neurosci. 2018 Oct 23;12:241. doi: 10.3389/fnbeh.2018.00241. PMID: 30405369; PMCID: PMC6206409.
  26. Linck VM, da Silva AL, Figueiró M, Piato AL, Herrmann AP, Dupont Birck F, Caramão EB, Nunes DS, Moreno PR, Elisabetsky E. Inhaled linalool-induced sedation in mice. Phytomedicine. 2009 Apr;16(4):303-7. doi: 10.1016/j.phymed.2008.08.001. Epub 2008 Sep 27. PMID: 18824339.
  27. Liu J, Xu JT. The effect of lavender essential oil on sedative hypnotic effects in mice. Journal of Clinical and Experimental Medicine. 2012; 11(18):1440-1441. 10.3969/j.issn.1671-4695.2012.18.004
  28. Govindarajan M, Benelli G. Eco-friendly larvicides from Indian plants: Effectiveness of lavandulyl acetate and bicyclogermacrene on malaria, dengue and Japanese encephalitis mosquito vectors. Ecotoxicol Environ Saf. 2016 Nov;133:395-402. doi: 10.1016/j.ecoenv.2016.07.035. Epub 2016 Aug 6. PMID: 27504617.
  29. Liu J, Chen C, Wan X, Yao G, Bao S, Wang F, Wang K, Song T, Han P, Jiang H. Identification of the sesquiterpene synthase AcTPS1 and high production of (-)-germacrene D in metabolically engineered Saccharomyces cerevisiae. Microb Cell Fact. 2022 May 18;21(1):89. doi: 10.1186/s12934-022-01814-4. PMID: 35585553; PMCID: PMC9115970.
  30. Hwang ES, Kim HB, Lee S, Kim MJ, Kim KJ, Han G, Han SY, Lee EA, Yoon JH, Kim DO, Maeng S, Park JH. Antidepressant-like effects of β-caryophyllene on restraint plus stress-induced depression. Behav Brain Res. 2020 Feb 17;380:112439. doi: 10.1016/j.bbr.2019.112439. Epub 2019 Dec 17. PMID: 31862467.
  31. Cui LL, Dong J, Francis F, Liu YJ, Heuskin S, Lognay G, Chen JL, Bragard C, Tooker JF, Liu Y. E- β-farnesene synergizes the influence of an insecticide to improve control of cabbage aphids in China. 2012. https://doi.org/10.1016/j.cropro.2012.01.003
  32. Zhang N, Zhang L, Feng L, Yao L. The anxiolytic effect of essential oil of Cananga odorata exposure on mice and determination of its major active constituents. Phytomedicine. 2016 Dec 15;23(14):1727-1734. doi: 10.1016/j.phymed.2016.10.017. Epub 2016 Oct 27. PMID: 27912874.
  33. Liao PC, Yang TS, Chou JC, Chen J, Lee SC, Kuo YH, Ho CL, Chao LKP. Anti-inflammatory activity of neral and geranial isolated from fruits of Litsea cubeba Lour. Journal of Functional Foods. 2015; 19(A):248-258. https://doi.org/10.1016/j.jff.2015.09.034
  34. Dobetsberger C, Buchbauer G, Actions of essential oils on the central nervous system: an updated review. Flavour Fragrance Journal. 2011; 26:300-316. https://doi.org/10.1002/ffj.2045
  35. d'Alessio PA, Bisson JF, Béné MC. Anti-stress effects of d-limonene and its metabolite perillyl alcohol. Rejuvenation Res. 2014 Apr;17(2):145-9. doi: 10.1089/rej.2013.1515. Epub 2014 Apr 8. PMID: 24125633.
  36. Weston-Green K, Clunas H, Jimenez Naranjo C. A Review of the Potential Use of Pinene and Linalool as Terpene-Based Medicines for Brain Health: Discovering Novel Therapeutics in the Flavours and Fragrances of Cannabis. Front Psychiatry. 2021 Aug 26;12:583211. doi: 10.3389/fpsyt.2021.583211. PMID: 34512404; PMCID: PMC8426550.
  37. Woo J, Yang H, Yoon M, Gadhe CG, Pae AN, Cho S, Lee CJ. 3-Carene, a Phytoncide from Pine Tree Has a Sleep-enhancing Effect by Targeting the GABAA-benzodiazepine Receptors. Exp Neurobiol. 2019 Oct 31;28(5):593-601. doi: 10.5607/en.2019.28.5.593. PMID: 31698551; PMCID: PMC6844839.
  38. Ito K, Ito M. The sedative effect of inhaled terpinolene in mice and its structure-activity relationships. J Nat Med. 2013 Oct;67(4):833-7. doi: 10.1007/s11418-012-0732-1. Epub 2013 Jan 22. PMID: 23339024.
  39. Sakamoto Y, Ebihara S, Ebihara T, Tomita N, Toba K, Freeman S, Arai H, Kohzuki M. Fall prevention using olfactory stimulation with lavender odor in elderly nursing home residents: a randomized controlled trial. J Am Geriatr Soc. 2012 Jun;60(6):1005-11. doi: 10.1111/j.1532-5415.2012.03977.x. Epub 2012 May 30. Erratum in: J Am Geriatr Soc. 2012 Nov;60(11):2193. PMID: 22646853.
  40. Fujii M, Hatakeyama R, Fukuoka Y, Yamamoto T, Sasaki R, Moriya M, Kanno M, Sasaki H. Lavender aroma therapy for behavioral and psychological symptoms in dementia patients. Geriatr Gerontol Int. 2008 Jun;8(2):136-8. doi: 10.1111/j.1447-0594.2008.00461.x. PMID: 18713168.
  41. Lin PW, Chan WC, Ng BF, Lam LC. Efficacy of aromatherapy (Lavandula angustifolia) as an intervention for agitated behaviours in Chinese older persons with dementia: a cross-over randomized trial. Int J Geriatr Psychiatry. 2007 May;22(5):405-10. doi: 10.1002/gps.1688. PMID: 17342790.
  42. Buchbauer G, Jirovetz L, Jäger W, Dietrich H, Plank C. Aromatherapy: evidence for sedative effects of the essential oil of lavender after inhalation. Z Naturforsch C J Biosci. 1991 Nov-Dec;46(11-12):1067-72. doi: 10.1515/znc-1991-11-1223. PMID: 1817516.
  43. Lim WC, Seo JM, Lee CI, Pyo HB, Lee BC. Stimulative and sedative effects of essential oils upon inhalation in mice. Arch Pharm Res. 2005 Jul;28(7):770-4. doi: 10.1007/BF02977341. PMID: 16114490.
  44. Bradley BF, Starkey NJ, Brown SL, Lea RW. Anxiolytic effects of Lavandula angustifolia odour on the Mongolian gerbil elevated plus maze. J Ethnopharmacol. 2007 May 22;111(3):517-25. doi: 10.1016/j.jep.2006.12.021. Epub 2006 Dec 27. PMID: 17289317.
  45. Shaw D, Annett JM, Doherty B, Leslie JC. Anxiolytic effects of lavender oil inhalation on open-field behaviour in rats. Phytomedicine. 2007 Sep;14(9):613-20. doi: 10.1016/j.phymed.2007.03.007. Epub 2007 May 4. PMID: 17482442.
  46. Arzi A, Ahamehe M, Sarahroodi S. Effect of hydroalcoholic extract of Lavandula officinalis on nicotine-induced convulsion in mice. Pak J Biol Sci. 2011 Jun 1;14(11):634-40. doi: 10.3923/pjbs.2011.634.640. PMID: 22235504.
  47. Bradshaw RH, Marchant JN, Meredith MJ, Broom DM. Effects of lavender straw on stress and travel sickness in pigs. J Altern Complement Med. 1998 Fall;4(3):271-5. doi: 10.1089/acm.1998.4.3-271. PMID: 9764765.
  48. López V, Nielsen B, Solas M, Ramírez MJ, Jäger AK. Exploring Pharmacological Mechanisms of Lavender (Lavandula angustifolia) Essential Oil on Central Nervous System Targets. Front Pharmacol. 2017 May 19;8:280. doi: 10.3389/fphar.2017.00280. PMID: 28579958; PMCID: PMC5437114.
  49. Cline M, Taylor JE, Flores J, Bracken S, McCall S, Ceremuga TE. Investigation of the anxiolytic effects of linalool, a lavender extract, in the male Sprague-Dawley rat. AANA J. 2008 Feb;76(1):47-52. PMID: 18323320.
  50. Duan X, Tashiro M, Wu D, Yambe T, Wang Q, Sasaki T, Kumagai K, Luo Y, Nitta S, Itoh M. Autonomic nervous function and localization of cerebral activity during lavender aromatic immersion. Technol Health Care. 2007;15(2):69-78. PMID: 17361051.
  51. Koolhaas JM, Korte SM, De Boer SF, Van Der Vegt BJ, Van Reenen CG, Hopster H, De Jong IC, Ruis MA, Blokhuis HJ. Coping styles in animals: current status in behavior and stress-physiology. Neurosci Biobehav Rev. 1999 Nov;23(7):925-35. doi: 10.1016/s0149-7634(99)00026-3. PMID: 10580307.
  52. Hawken PA, Fiol C, Blache D. Genetic differences in temperament determine whether lavender oil alleviates or exacerbates anxiety in sheep. Physiol Behav. 2012 Mar 20;105(5):1117-23. doi: 10.1016/j.physbeh.2011.12.005. Epub 2011 Dec 13. PMID: 22192707.