Abstract
For a better scientific understanding of the basics of aromatherapy, our research group attempted to clarify the effectiveness of the essential oil from Pelargonium graveolens (EOPG, geranium essential oil), which is used to try and balance the mind-body connection. In order to eliminate any possible placebo effect, we used animal experiments that are considered to be insensitive to the placebo effect. Measurements of blood pressure and heart rate in the mouse tail artery were used as a reflection of the mind-body balance. Thirty minutes after inhalation of EOPG (5 µL/L air) for 90 minutes, blood pressure and heart rate of the mice were measured. EOPG significantly reduced blood pressure and heart rate. To further clarify the factors responsible for these effects, gas chromatography analysis was performed in order to determine the components transferred into the brain after the EOPG inhalation. Linalool, citronellol, and geraniol were detected at concentrations around 0.1 nL/L tissue from the brain after 10 µL/L air inhalation of EOPG. However, these were not detected after a 5 µL/L air inhalation of EOPG, as the levels were below the detection limit. These results suggest EOPG inhalation might lower blood pressure and heart rate, with the expressed effects associated with the transfer of components such as linalool into the brain.
Keywords
Although aromatherapy has been the subject of research for a long time, scientific elucidation of the mechanisms involved has yet to be definitively established. Our research group has recently been conducting basic studies for the purpose of determining a scientific method for elucidating mechanisms of aromatherapy. 1,2 One of the factors that makes aromatherapy research difficult is the placebo effect caused by the aroma. To eliminate this placebo effect, we performed experiments using an animal model that made it possible to evaluate the scientific mechanism of aromatherapy.
Aromatherapy consists of both inhalation and treatment. There are 2 mechanisms of action involved with aromatherapy inhalation. These include olfactory stimulation (neurotransmission) pathways and pathways that specifically enter the body, such as the brain (pharmacological transmission). Our research group has been focusing on the pharmacological transmission, as this is thought to be both persistent and potent. Our current study additionally focused on the route of pharmacological transmission.
Studies of essential oil from Pelargonium graveolens (EOPG, geranium essential oil) have suggested that EOPG can be used to balance the mind-body connection. In the current study, assessment of the mind-body balance was performed using measurements of the blood pressure and heart rate in mice.
EOPG has been reported to have antiviral, antibacterial, and antifungal activities. Inhibitory effects of EOPG and essential oil from Cymbopogon citratus have been reported for the Ross River virus 3 ; antioxidant and antibacterial activities have been shown for the essential oil from Pelargonium asperum and Ormenis mixta 4 ; antifungal activity against pathogenic Candida strains has been shown for the essential oil from Cinnamomum verum, Thymus capitatus, Syzygium aromaticum, and Pelargonium graveolens 5 ; and protective antifungal activity has been reported for the essential oils from Buddleja perfoliata and Pelargonium graveolens, 6 among others. However, there have yet to be any basic research studies that have examined the effect of EOPG on the mind-body balance.
Presently, there has been no definitive overall mechanism established for the basis of aromatherapy. However, mechanisms of action have been reported for essential oils administered by inhalation, for the neurotransmission pathways associated with olfactory stimulation, 7 for the pharmacological transduction pathways associated with the periphery, 8,9 and for the pharmacological transduction pathways associated with the central nervous system. 1,2 Our current report presents information on a potential mechanism of action for essential oils in aromatherapy.
Experiment 1
The EOPG components tested included citronellol (21.9%), geraniol (13.3%), citronellyl formate (8.6%), and linalool (6.3%). Mice were lightly stressed by being individually housed for 1 to 3 days. Individual housing is carried out so that individual differences do not occur during the housing. After mice inhaled EOPG (5 µL/L air) for 90 minutes, the blood pressure and heart rate were measured using the caudal artery at 30 minutes after the administration of the anesthesia. Purified water was used as a negative control. Thus, EOPG inhalation resulted in significant reductions in the blood pressure and heart rate in mice as compared to the inhalation of purified water (Figure 1).
Blood pressure (a) and heart rate (b) after EOPG inhalation (5 µL/L air). Values are mean ± SE, n = 8. *P < 0.05. EOPG, essentialoil from Pelargonium graveolens.
Experiment 2
The results of Experiment 1 were considered to indicate the intracerebral transfer of the component, thereby making it possible to examine the intracerebral transferability of the component. Similar to Experiment 1, EOPG was also inhaled for 90 minutes, immediately thereafter the brain was removed and analyzed by gas chromatography (GC) for its hexane extract. Although none of the components could be detected in the brain after inhalation of 5 µL/L air EOPG, linalool (0.095 ± 0.002 nL/g tissue), citronellol (0.071 ± 0.001 nL/g tissue), and geraniol (0.097 ± 0.029 nL/g tissue) were detected in the brain after inhalation of 10 µL/L air EOPG (Figure 2).
Brain transferability of major components in inhaled EOPG (10 µL/L air). Values are mean ± SE, n = 3. EOPG, essentialoil from Pelargonium graveolens.
Experiment 3
The results of Experiment 2 clearly indicated that linalool, citronellol, and geraniol were transferred into the brain after inhalation of EOPG. Since the results for linalool were the most stably detected from the brain, we examined the effects of linalool inhalation on the blood pressure and heart rate. Under the same conditions as used in Experiment 1, we examined the influence of inhalation of linalool (5 µL/L air) on the blood pressure and heart rate of mice. Although the inhalation of linalool (5 µL/L air) exhibited no significant differences as compared to the negative control, purified water, it tended to decrease both blood pressure and heart rate (Figure 3).
Blood pressure (a) and heart rate (b) after inhalation of linalool (5 µL/L air). Values are mean ± SE, n = 8.
Current experimental results show that EOPG lowered blood pressure and heart rate in mice. In addition, results suggested that the effect occurred due to the transfer of components such as linalool into the brain. Thus, EOPG is thought to regulate the autonomic nervous system, as EOPG inhalation lowers both the blood pressure and heart rate. These results can be considered to be scientific evidence documenting the effect of EOPG on the mind-body balance.
Prior to this study, there has been little research on the effects of EOPG on the blood pressure and heart rate. However, there have been several reports on linalool. For example, clinical studies on the inhalation of linalool have documented antioxidant activity and reductions in the blood pressure and heart rate. 10 Furthermore, it has also been reported that olfactory stimulation affects the anxiolytic-like activity of linalool after inhalation in mice. 7 Linalool and essential oil from Citrus bergamia have been reported to induce vasorelaxation in mice. 8,9 Other previous studies have suggested that the inhalation effect of essential oils is mainly dependent on the transfer of individual components into the brain. 1,2 Anxiolytic-like effects of intraperitoneal administration of lavender essential oil and linalool have also been reported. 11
Although we can conclude that transfer of the components to the brain was responsible for observed changes in our experimental study, we cannot completely rule out the potential effects of olfactory stimulation and changes in the peripheral blood vessels. A further detailed study will need to be performed in the future. Our current and expected future findings suggest that EOPG may be able to be clinically applied for therapeutic treatments in patients.
Experimental
Analysis Using GC
Component analysis was performed using GC-MS-QP2010 (Shimadzu Corporation, Kyoto, Japan) and GC-2010 (Shimadzu Corporation, Kyoto, Japan). Samples were prepared by diluting EOPG with n-hexane. The capillary column for analysis is a DB-5ms capillary column (30 m x 0.25 mm ID, 0.25 µm, non-polar column; Agilent Technologies Inc., Tokyo, Japan). Helium (99.99995%, 1.82 mL/min) was used as the carrier gas. Inlet line temperature and source temperature are 250°C. The temperature of column oven is 40°C for 2 minutes, 40°C to 200°C at 5°C/min, and then 200°C for 2 minutes. The voltage of electron impact (electron ionization) was 70 eV. Components were detected by comparing standard compounds or the GC-MS NIST 02 spectral library (National Institute of Standards and Technology, Gaithersburg, MD, USA) and retention indices. 12 GC-FID was analyzed under the same conditions as GC-MS.
Essential Oil and Components
EOPG extracted from the leaves of Pelargonium graveolens (Geraniaceae) by steam distillation was purchased from Green Flask Co., Ltd. (Tokyo, Japan). The components of EOPG primarily include citronellol (21.9%), geraniol (13.3%), citronellyl formate (8.6%), and linalool (6.3%) (Table 1).
Concentration of the Components of EOPG.
EOPG, essential oil from Pelargonium graveolens; LRI, Linear retention index.
(-)-Linalool was purchased from Tokyo Chemical Industry (Tokyo, Japan).
Animals
Male ICR mice (Kumagai-Shigeyasu Co., Ltd., Miyagi, Japan) that were 5 weeks of age at the start of each experiment were used in the study. All mice were individually housed in cages for 1 to 3 days. The cages were placed in a room that was artificially illuminated by fluorescent lamps on a 12 hours light and dark schedule (light period from 8:00 to 20:00) and maintained at 24°C ± 5°C. The mice had free access to food (Labo MR Stock, Nosan Corporation, Kanagawa, Japan) and water. One mouse is used only for 1 experiment. The number of mice used in this experiment is 41. For this experiment, it was assumed that mice that were independently housed experienced more stress as compared to mice kept in group cages. All experiments were conducted in accordance with the guidelines regarding the care of experimental animals as approved by the Animal Research Committee at the International University of Health and Welfare.
Procedure
A mouse was placed in a stainless-steel container (10 L, φ240 × 240 mm, As One Corporation, Osaka, Japan). A filter paper (GE Healthcare Japan, Tokyo, Japan) soaked in either EOPG (5, 10 µL/L air), linalool (5 µL/L air), or water (5 µL/L air) was placed on the upper side of the stainless-steel container. Mice inhaled EOPG, linalool, or water for 90 minutes. The temperature in the room was 24°C ± 5°C.
Three types of mixed anesthesia (IP) were used. The temperature of the mouse was maintained at 37°C, and the blood pressure and heart rate were measured after 30 minutes using a noninvasive blood pressure monitor (MK-2000, Muromachi Kikai, Tokyo, Japan) (n = 8). The 3 types of anesthesia used in the mixture were medetomidine hydrochloride (0.3 mg/kg mouse, Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan), butorphanol tartrate (5 mg/kg mouse, Meiji Seika Pharma Co., Ltd., Tokyo, Japan), and midazolam (4 mg/kg mouse, Astellas Pharma Inc., Tokyo, Japan). 13
Quantitative Analysis of the EOPG Components in the Brain
As linalool, citronellol, and geraniol were detected in the brain after inhalation of EOPG, a measurement index was constructed based on linalool. Immediately after inhalation, the mice were decapitated, and whole brains were removed (n = 3). Each brain was homogenized in 1 mL n-hexane with an ultrasonic crusher (THU-80, As One Corporation). This GC analysis was similar to the analysis performed for the EOPG components. The obtained results are quantitatively calculated for wet brain using the standard compound linalool.
Statistical Analysis
Results are expressed as the means ± SE (n = 8). Statistical differences were determined by a 2-sided Student’s t-test using a bell curve in Excel (Social Survey Research Information Co., Ltd., Tokyo, Japan). Differences with P < 0.05 were considered significant.
Footnotes
Acknowledgments
We are grateful to Mr Shinichiro Hayashi of Green Flask Laboratory for providing the EOPG.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.