Antidepressant-like effect of Citrus sinensis (L.) Osbeck essential oil

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Chemistry and Biology of Aroma and Taste

Antidepressant-like effect of Citrus sinensis (L.) Osbeck essential oil and its main component limonene on mice Lulu Zhang, Ziyu Yang, Gang Fan, Jingnan Ren, Kai Jing Yin, and Siyi Pan J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 24 Mar 2019 Downloaded from http://pubs.acs.org on March 27, 2019

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Journal of Agricultural and Food Chemistry

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Antidepressant-like effect of Citrus sinensis (L.) Osbeck

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essential oil and its main component limonene on mice

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Lu-Lu Zhang, Zi-Yu Yang, Gang Fan*, Jing-Nan Ren, Kai-Jing Yin, Si-Yi Pan

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Key Laboratory of Environment Correlative Dietology, Ministry of Education, College of Food

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Science and Technology, Huazhong Agricultural University.

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Corresponding Author: Gang Fan, No.1, Shizishan Street, Hongshan District, Wuhan, Hubei

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Province, 430070, P.R.China, [email protected]; Tel.: +86-27-87282111, Fax:

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+86-27-87288373.

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ABSTRACT: The present study investigated the antidepressant-like effects of navel orange

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(Citrus sinensis (L.) Osbeck) essential oil (OEO) and its main components using the chronic

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unpredictable mild stress (CUMS) model mice, and explored its possible mechanisms. The results

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indicated that OEO inhalation significantly ameliorated the depression-like behaviors of CUMS

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mice with decreased body weight, sucrose preference, curiosity and mobility, as well as shortened

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immobile time, and attenuated dyslipidemia. Limonene was the most abundant compound in the

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sniffing OEO environment and mice brain after sniffing, and it was not metabolized immediately

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in the brain. In addition, limonene inhalation significantly restored CUMS-induced depressive

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behavior, hyperactivity of HPA axis and the decrease of monoamine neurotransmitters levels,

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down-regulation of BDNF and its receptor expression in the hippocampus. Thus, the study

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indicates that the improvements in neuroendocrine, neurotrophic, and monoaminergic systems are

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related to the antidepressant effects of limonene.

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KEYWORDS: essential oil from navel orange, chronic unpredictable mild stress model,

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limonene, antidepressive mechanism, olfactory pathway

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INTRODUCTION

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Physical and psychological stressors are deemed to cause depression. 1 Depression, a mental

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disorder, is distinguished by sleeplessness, anorexia, anhedonia, sorrow, helplessness, guilt, and

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the reduction of a person's ability to work and study. It could weaken health-related quality of life

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and increase mortality.

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imbalance of monoamines. Recent studies found that these antidepressants alleviate the symptoms

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for some patients but they aren't universally effective, and sometimes even lead to the serious

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adverse effects which limit their application.

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anti-depressive drugs.

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2

Current antidepressants are developed mainly based on the chemical

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Therefore, it is meaningful to develop the new

Aromatherapy is an important complementary and alternative medicine, which has been used 4

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by many cultures around the world.

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extracted from plant and flowers to treat different diseases.” 5 And it has not accompanied by side

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effects.

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natural agents.

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oils as a safe group A medicine.

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(OEO) is one of the most valuable by-products after Citrus fruits processing. It is widely used in

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the food, beverage, cosmetics and other industries, and widely used as aromatherapy and

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medicinal agents. 10 The pharmacological effects of OEO have been extensively studied, including

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antimicrobial, insecticidal, antioxidant and anticancer effects, as well as pain relief,

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hepatocarcinogenesis suppressant, food preservation, acne treatment, relaxation, anti-anxiety

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function.

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research shows that limonene is generally considered safe, low in toxicity, without causing

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Aromatherapy is “the controlled use of essential oils

Essential oils have recently become popular and raised scientific interest as potential

11-16

7, 8

And the U.S. Food and Drug Administration (FDA) has recognized essential 9

The navel orange (Citrus sinensis (L.) Osbeck) essential oil

OEO are rich in monoterpenes with the major constituent of d-limonene. Recent

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teratogenic, carcinogenic or mutagenic risk to humans.

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have

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antinociceptive, antiallergic, antiviral, anti-tumor, and anti-stress effects, as well as the treatments

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of some diseases such as diabetes, stomach ulcers, asthma, and airway inflammation.

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However, there are few reports on the antidepressant effect of OEO and its major compound.

multiple

beneficial

biological

effects

Besides, limonene has been shown to

including

anti-inflammatory,

antioxidant,

18, 19

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In addition, α-pinene, β-myrcene and linalool are also the dominant components of OEO. It

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has been reported that monoterpenes could be recognized as a fragrance by olfactory receptors.

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The monoterpenes exert activity of affecting emotions by directly acting on the central nervous

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system and olfactory nerve. 20, 21 There may be a link between the accumulation of monoterpenes

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in the brain and the influence of monoterpenes on emotional behavior. However, very little

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detailed research was found in this area.

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Therefore, the aim of this study is to use CUMS model and biochemical test to study the

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antidepressant effects of OEO by inhalation. And the accumulation of the main components of

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OEO (d-limonene, α-pinene, β-myrcene and linalool) in the mice brain was studied. Moreover, the

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antidepressant-like effects of limonene (main component of OEO) on the biochemical test, and the

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levels of monoamine neurotransmitters and hormones associated with the monoamine system and

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hypothalamic-pituitary-adrenal (HPA) axis in the prefrontal cortex and hippocampus were studied.

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We also investigated brain-derived neurotrophic factor (BDNF) and tropomyosin receptor kinase

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B (TrkB) expression in the hippocampus. The study could be used as a reference for the high

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efficiency use of essential oils and fragrance compounds to treat depression in aromatherapy.

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MATERIALS AND METHODS

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Animals

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Totally 155 male Kunming mice weighing 29-31 g each at 5 weeks after the birth (Hubei

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Provincial Center for Experimental Animal Research, Wuhan, Hubei, China) were group-housed

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at 24-26 °C and 60% relative humidity on a 12-h light/dark cycle. And the mice had ad libitum

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access to standard food and water. The study was performed in line with the regulation of the

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administration of affairs concerning experimental animals of P.R. China. The study was confirmed

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by the Laboratory Animal Research Center of Hubei province and the ethics committee of

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Huazhong Agricultural University (HZAUMO-2015-024).

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Materials

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Orange essential oils (Citrus sinensis (L.) Osbeck) with cold-pressed technique were

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purchased from Zigui County Qugu Food Co.,Ltd. in Hubei, China. Fluoxetine was purchased

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from Patheon France (Bourgoin-Jallieu, France) and was dissolved in saline. Syringe was

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purchased from Crown Medical Devices (Wuhan, Hubei, China). The solvents (i.e., ethanol,

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sodium chloride, sucrose, glacial acetic acid, Sinopharm Chemical Reagent Co., Ltd., Shanghai,

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China) were of analytical reagent grade. Standards of n-paraffins (C6–C25) were obtained from

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Sigma Chemical Company (Saint Louis, MO, USA). Aroma standards d-limonene, linalool and

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β-myrcene were obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan); α-pinene,

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α-phellandrene, cymene, β-terpinene, γ-terpinene and ocimene were purchased from Sigma

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Chemical Company (Saint Louis, MO, USA).

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CUMS procedure

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The CUMS procedure was conducted as described previously. 5


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Briefly, the animals were

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subjected to a variety of mild stressors. Stress included food and water deprivation (F), overhang

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(60 s-T1, 90 s-T2, 120 s-T3), swimming (5 min-S), shaking cage (1 time/s, 100 times-CS1, 150

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times-CS2, 200 times-CS3), whole-day light (W), 45-degrees cage (8 h-CT), housing in wet

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sawdust (8 h-M), no treatments (N). These stressors were applied in a random order to produce an

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unexpected mild stress effect, but the same stressor was not applied in two consecutive days.

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OEO treatment alleviated depression-related behavior in CUMS model mice

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Mice were randomly assigned to five groups (n=7): control group (Con), negative control

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group (CUMS treated group, CUMS), positive control group (fluoxetine treated group, Flu),

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long-term sniffing group (Long), short-term sniffing group (Short). Flu group received daily

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gavage of 0.2 mL fluoxetine (30 mg/kg) for next 5 consecutive days. The control and CUMS

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group received daily 0.9% saline (0.2 mL). The mice of sniffing group were housed in a cage

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(cage size: 28 cm×21 cm×17.5 cm) that had a piece of filter paper with 1 mL OEO. Long and

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Short group received daily by inhalation OEO for 24 h and 1.5 h respectively, and lasted for 5

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days, as well as also received daily 0.9% saline (0.2 mL). The behavioral tests and serum

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biochemical indices were carried out at the end of the 5-day sniffing OEO. Figure 1A shows the

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details of the experimental procedure.

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Analysis of the aroma compounds of OEO in the sniffing environment

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Aroma compounds in the sniffing environment were identified and quantified by headspace

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GC-MS and SPME/GC-MS. The samples were placed on an enclosed space with size of

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28×21×17.5 cm3 which was covered with 1 mL OEO. After 1 h of equilibration, the gas (5 mL)

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was extracted from right over enclosed space using a manual GC syringe (Agilent Technologies,

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Palo Alto, CA, USA), and then it was measured by GC-MS. The experiments were performed in

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triplicate.

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SPME was conducted by a manual device equipped with a 50/30 μm DVB/CAR/PDMS fibre

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(Supelco, Bellfonte, PA, USA). The samples were placed on an enclosed space. The fibre was

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exposed for 40 min to extract the volatile constituents after 1 h of equilibration. And then the fiber

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was inserted through a septum at the injection port of GC for 5 min. The experiments were

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performed in triplicate.

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Volatile components were analyzed with an Agilent 6890N GC coupled with an Agilent

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5975B mass spectrometer (Agilent Technologies, Palo Alto, CA, USA). Analytes were separated

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using an HP-5 fused silica capillary column (30 m×320 μm×0.25 μm). The conditions of GC-MS

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were conducted as described previously.

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retention indices with authentic standards or comparing mass spectra with published data, and they

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were further identified by using the NIST 0.5 and Wiley 7.0 libraries. A mixture of n-paraffins

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(C6-C25) was used to calculate retention indices. The volatile constituents absorbed in the mice

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brain were quantitatively determined by using the external standard method.

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The accumulation of aromas in the mice brain

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In addition, the compounds were identified by using

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The mice were randomly divided into 2 groups: group I and group II. A piece of filter paper

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was soaked in 50 μL d-limonene, 50 μL α-pinene, 50 μL β-myrcene and 50 μL linalool

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respectively, and then it was placed on the upper side of the cage (cage size: 28 cm×21 cm×17.5

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cm). The animals were placed in a cage for 30 min prior to experimentation. The animals of group

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I were exposed to the sample at room temperature for 90 min. Group I was classified as follows:

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S1, the control group did not take any treatments. S2, the brains were collected and analyzed after

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90 min inhalation. S3, the brains were collected and analyzed after 90 min inhalation and 30 min

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in the feeding cage. In addition, the mice of group II were treated with the sample for 90 min per

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day for successively 7 days. Group II was classified as follows: S1, mice were given food and

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water ad libitum, and did not take any treatment for 7 consecutive days. S2, the brains were

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collected and analyzed after 90 min inhalation at the 7th day. S3, the brains were collected and

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analyzed after 90 min inhalation and followed a 30-min rest in the feeding cage at the 7th day. The

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metabolism time of the aroma compounds in the brain was evaluated in the S2 and S3 groups.

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The whole brains were collected immediately and homogenized with ice. The extracts were

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prepared by using an ultrasonic disruptor (KQ-300DE, Kun Shan Ultrasonic Instruments Co., Ltd.,

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Kunshan, Jiangsu, China) with 4 mL of hexane, and were dehydrated by adding anhydrous sodium

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sulfate. The extracts were filtered by 0.22 μm filter membrane and then analyzed using GC/MS.

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Limonene treatment alleviated depression-related behavior in CUMS model mice

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Mice were randomly assigned to five groups (n=12): control group (Con), negative control

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group (CUMS treated group, CUMS), positive control group (fluoxetine treated group, Flu),

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long-term sniffing group (Long), short-term sniffing group (Short). Flu group received daily

146

gavage of 0.2 mL fluoxetine (30 mg/kg) for next 5 consecutive days. The control and CUMS

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group received daily 0.9% saline (0.2 mL). The mice of sniffing group were housed in a cage

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(cage size: 28 cm×21 cm×17.5 cm) including a piece of filter paper with 1 mL limonene. Long

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and Short group received daily limonene inhalation for 24 h and 1.5 h respectively, which lasted

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for 5 days, and the mice were also received daily 0.9% saline (0.2 mL). The behavioral tests were

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performed at the end of the 5-day sniffing of limonene. Furthermore, the brain was rapidly

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obtained from the sacrificed mice and was prepared for measurements of monoamine

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neurotransmitters and HPA axis hormone levels. Immunofluorescence technique and western blot

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were conducted to determine the expression of BDNF and TrkB in the hippocampus．Figure 1B

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shows the details of the experimental procedure.

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Behavioral Test

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Depression-related behaviors were assessed using sucrose preference test (SPT), open field test (OFT), and forced swimming test (FST).

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The SPT was conducted as described previously. 24 The animals were given free access to two

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bottles of sucrose solution (1%, m/v) for 24 h. The animals were subsequently presented with a

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bottle of sucrose solution and a bottle of pure water for 24 h. Finally the mice were deprived of

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water and feed for 24 h. SPT was then performed for 2 h that the mice were housed individually

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and given free access to a bottle of sucrose solution (150 g) and a bottle of pure water (150 g). The

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preference for the sucrose solution was calculated according to the following formula: 25

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sucrose preference (%) = (sucrose consumption/total fluid consumption) × 100%.

The OFT was performed as described previously.

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Experiment was conducted in a quiet

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environment. The mice were consistently placed in the center of the box (70×50×40 cm3) with a

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number of the same squares at the bottom, and they were allowed to explore the arena for the

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following 5 min. The number of crossings (with the four paws) and rearings (posture sustained

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with hind-paws on the floor) were counted manually for 5 min. The box was thoroughly cleaned

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with 90% ethanol after each experiment. 9


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The FST was carried out as described previously. 27 The mouse was individually placed into a

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glass beaker (diameter 11.2 cm, height 15.5 cm) containing 10 cm of water at room temperature.

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Each mouse was exposed to the swimming conditions for a period of 6 min. The duration of

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immobility of the last 4 minutes in the total 6 minutes of swimming time was recorded. The

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immobility was defined as the lack of motion of the whole body, except for small movements

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necessary to keep the animal’s head above the water. 28 Then the animals were removed from the

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water and dried in a warmed enclosure before being returned to their home cages.

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Serum biochemical indices

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After the behavior test, the blood was obtained by extracting the eyeball blood and sampled

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into plain tubes. The blood sera were obtained following a 2 min centrifugation at 3 000 r/min

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(Eppendorf, Saxony, Germany) at 4 °C, and then the serum biochemical indices were determined.

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The total cholesterol (TC) (A111-1), triglyceride (TG) (A110-1), low-density lipoprotein

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cholesterol (LDL-C) (A113-1), high-density lipoprotein cholesterol (HDL-C) (A112-1) levels

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were determined using the respective assay kits (Nanjing Jiancheng Bioengineering Institute,

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Nanjing, Jiangsu, China).

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Enzyme-linked immunosorbent assay (ELISA)

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The brain was obtained from the sacrificed mice rapidly after the behavioral tests. The

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prefrontal cortex and hippocampus tissues were dissected on the ice and frozen in liquid nitrogen.

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The samples were stored at -80 °C until assay. The content of serotonin (5-HT) (KJ-3179A),

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dopamine (DA) (KJ-3092A), norepinephrine (NE) (KJ-3269A), corticosterone (CORT)

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(KJ-2797A), glucocorticoid receptor (GR) (KJ-3067A), corticotropin-releasing factor (CRF)

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(KJ-4033A) were determined in the hippocampus and prefrontal cortex using commercially

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available ELISA kits (Jiangsu KeJing Biological Technology Co., Ltd, Yancheng, Jiangsu,

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China).

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Western blot analysis

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The protein was obtained from the hippocampus of mice using modified RIPA buffer

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(CWBio, Beijing, China) and quantified by the BCA protein kit (Yeasen Biotech Co., Ltd.,

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Shanghai, China). The proteins were separated by sodium dodecyl sulfate polyacrylamide gel

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electrophoresis and then transferred to a polyvinylidene fluoride membrane. The membranes were

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blocked for 2 h with blocking solution (1× TBS supplemented with 0.1% Tween-20 and 5% skim

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milk), and were washed three times with washing buffer (1×TBS containing 0.1% Tween-20).

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Subsequently, the membranes were incubated at 4 ℃ overnight with rabbit anti-TrkB polyclonal

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antibody (1:1000, OM127017, Omnimabs, Alhambra, CA, USA), and then incubated with

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horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin G secondary antibodies

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(1:1000) for 2 h at room temperature. Blots were washed three times with washing buffer, and

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immunoreactive bands were visualized using an ECL detection system (Bio-Rad, Hercules, CA,

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USA).

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Immunofluorescence staining

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The brain tissue of mouse was fixed in 4% paraformaldehyde solution for 24 h and was

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dehydrated through the routine serial treatment of samples with graded alcohol and xylene. Then

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all the samples were embedded in paraffin and 5 μm sections were obtained using a rotary

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microtome (RM2016, Lecia Microsystems, Wetzlar and Mannheim, Germany), then dried at 60℃

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for 1 h. After deparaffinization of the slices, antigen unmasking was performed by heating slices

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in 0.01mol/L sodium citrate buffer (pH 6.0) using a microwave oven for 10 min. Subsequently,

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tissue sections were dipped in 3% H2O2 for 10 min to block endogenous peroxidase activity and

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then blocked with 10% normal donkey serum at room temperature for 30 min. Slices were

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incubated overnight at 4℃ with antibody (anti-BDNF antibody, DF6387, Affinity Biosciences,

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Cincinnati, OH, USA) diluted 1:500. Goat anti-rabbit antibody (1:2000, K5007, Dako, Glostrup,

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Denmark) was used as secondary antibody and then dropped on the sections for 1 h at 4℃. Tissue

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sections were subsequently washed with PBS for three times. A mounting medium containing

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DAPI was added to the slides and then covered with coverslips for observation. Slides were

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observed under a fluorescence microscope (Nikon Eclipse Ti-SR, Nikon, Tokyo, Japan). Images

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were taken at 200× magnification.

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Statistical analysis

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Mean values and standard deviations (SD) of experimental data of serum biochemical indices

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were calculated using MS Excel 2013 (Microsoft Inc., Seattle, WA, USA). Data in the figures

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were presented as the means ± standard deviation of mean (SEM). Statistical analysis was

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performed using Mann-whitney test, followed by the student’s t-test using Prism 5.0 (GraphPad

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Software, Inc. USA). Significant differences were indicated at levels of p < 0.05, p < 0.01, p

Antidepressant-like effect of Citrus sinensis (L.) Osbeck essential oil

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