Effect of Essential Oil of Cedarwood (Cedrus Atlantica) against Serum

Cortisol Levels in Rats Which Were Given Stressor

Jose Giovanny

Faculty Of Medicine, Widya Mandala Catholic University, Kalisari Raya 01 Street, Surabaya,East Java, Indonesia

Keywords: Essential Oil, Cedarwood Balm, Forced Swim Test, Serum Cortisol.

Abstract: Stress is a response of the body to any demand that can affect the body’s endocrine system such as the release

of cortisol into the bloodstream. Essential oils have been widely used for stress treatment because they have

a calming effect. One of them is cedarwood essential oil. This study was conducted to determine the effect of

cedarwood balm against serum cortisol levels in rats which were given stressor. In this study wistar male rats

were randomly selected. This animal was exposed to forced swim test as stressor and then they were given

cedarwood balm. We used 3 groups, the first group were given a daily forced swim test and applied cedarwood

balm on the shaved back, the second group were given a daily forced swim test only, the third group were

given nothing. Their serum cortisol levels were measured by ELISA test after 30 days. Result were analysed

by Kruskal Wallis test for all group and man whitney test as a post test (p < 0,05). Serum cortisol level was

significantly lower in cedarwood balm group then the other groups. When the all group were compared, serum

cortisol level was significantly different (p = 0,018). In conclusion this indicates that cedarwood balm affect

the endocrine regulatory mechanism to modulate stress responses.

1 INTRODUCTION

Stress is the body's response to mental, emotional and

/ or physical needs that exceed the body's regulatory

capacity (Cohen et al., 2013; Fink, 2016). Stress is

well known to change serum cortisol in animal

models (Hall, 2016). There are a variety of stress

delivery techniques, one of which is the forced swim

test. Forced swim test is a stressor technique that is

often used for cases of depression in experimental

animals, the forced swim test (FST), which is one of

the most commonly used assays for the study of

depressive-like behavior in rodents). The use of FST

as a stressor to increase cortisol hormone levels has

been demonstrated by various researchers (Khaleel

Jameel et al., 2014).

The central regulation of stress and cortisol

release occurs in the hypothalamus, which contains

multiple responses from the brain. This biological

response is the activation of three systems namely the

sensory system in the brain, the Sympathetic Adrenal

Medullary (SAM), and the hypothalamic-pituitary-

adrenal (HPA) axis. Stressors that are stimulated by

the sensory system in the brain will activate two

hormonal systems to help individuals cope with this

condition, the first of which is mediated by a

sympathetic nerve called a "fight-or-flight" response

which rapidly results in the release of epinephrine and

norepinephrine6. Second is a slower and longer

hormonal response, mediated by the hypothalamic-

pituitary-adrenal (HPA) axis. This response is

mediated by a group of neurons in the hypothalamic

paraventricular nucleus (PVN), which secretes

corticotrophin-releasing hormone (CRH) to give

pituitary signals so that the pituitary releases

adrenocorticotropic hormone (ACTH). ACTH

stimulates the adrenal glands to synthesize and

secrete Cortisol (Hall, 2016).

Cortisol is an indication of a stress condition.

Almost all types of stress that are physical or mental

cause an increase in ACTH secretion which increases

cortisol secretion within a period of several minutes.

Stimulation resulting from physical stress or tissue

damage will be delivered to the eminence of the

hypothalamic median through the brain stem which

causes the activation of the HPA axis so that there is

an increase in cortisol secretion into the blood (Cohen

et al., 2013; Fink, 2016). In rats given stressor by

Giovanny, J.

Effect of Essential Oil of Cedarwood (Cedrus Atlantica) against Serum Cortisol Levels in Rats Which Were Given Stressor.

DOI: 10.5220/0009954600180021

In Proceedings of the 2nd International Conference of Essential Oils (ICEO 2019), pages 18-21

ISBN: 978-989-758-456-5

forced swim test method has been shown to increase

levels of the hormone cortisol, but the signaling of

cortisol hormone levels is higher in acute exposure to

FST in mice, it increases stress hormone more than

chronic FST exposure (Khaleel Jameel et al., 2014).

Cedrus atlantica plant is one species that comes

from the family Pinaceae. Oil from the cedarwood

plant is extracted from the Cedrus atlantica tree by

distilling it from shavings and splinters from the

Cedrus atlantica tree. especially in α-atlantone which

can help calm the mind (Rhind, 2012). The high

sesquiterpene alcohol content in cedarwood oil has a

sedative effect that can overcome tension, fear,

anxiety, and depression (Fradelos and Komini, 2015;

K G Stiles, 2017). In previous studies the use of

cedarwood oil to reduce stress is still unclear, because

the method is not clearly explained and there are still

many cases of stress management with the use of

synthetic drugs such as psychotropic drugs. The use

of drugs with this class is still often misused,

therefore researchers are interested in further research

on the use of natural ingredients such as cedarwood

oil to see the effectiveness of cedarwood oil in dealing

with stress conditions, with the hormone cortisol as a

marker of stress conditions.

2 METHODS AND MATERIALS

2.1 Animal

Rats in this study were male Wistar strain rats

weighing 120-150gr, aged 2-3 months, and healthy.

The experiments were performed after the animal had

been habituated to the experimental environment for

1 week.. foods were given 60 gram and water were

given 45 ml every day. These animals were

individually housed in cage. The male rat were

divided into 3 groups, the first group is negative

control group who were not given balms and stressors

), the second group is positive control group who

were given forced swim test but were not given balms

), and the treatment groups who were given

cedarwood oil balms and given stressors (T

). Rats

will be shaved feathers on the back to apply

cedarwood oil balm to the group to be given balm.

Mice will be given a forced swim test for 30 days.

Experiments were conducted between 1 to 4pm every

day.

2.2 Materials

10 % Cedarwood balm consist of 5 ml virgin coconut

oil, 1-gram beeswax, and 0,625 gram cedarwood

essential oil (Cedrus atlantica). Cedarwood balm will

be applied at the back of the rats after getting forced

swim test.

2.3 Forced Swim Test and Cedarwood

Balm

Forced swim tests were given to control group two

and treatment group for 30 days, every day on

cylinder tubes with a diameter of 20 cm and a height

of 30 cm. This cylinder tube will be filled with water

as high as 20 cm then the mouse will be placed in a

cylindrical tube filled with water and will be

quenched for 10 seconds then the rat will be removed

and dried by wiping with a towel and allowed to stand

for 30 minutes at room temperature. After that the

treatment group will be applied cedarwood oil balm

with a concentration of 10%.

2.4 Measurement of Serum Cortisol

Level

Blood was collected from all the study group animals

after 30 days at 4 pm, all blood samples were taken at

the same time to get the same result. 3ml of blood was

collected by intra cardiac and then the serum was

separated by centrifugation at 3000rpm for 5 minutes

and stored at -20c. The serum sample was analyzed

with ELISA KIT to determine the cortisol level. This

assay has high sensitivity and specificity for

estimation of cortisol levels in wistar rats.

2.5 Statistic

Data were analyzed by statistical test and the Kruskal

Wallis followed Whitney test man as a post hoc test

with SPSS 25.0 software to indicate a statistically

significant difference between the control group and

the treatment group with a significant value of p

<0.05.

3 RESULTS

The effect of cedarwood balm against serum cortisol

levels in the treatment group shown in Figure 1.

Comparison of serum cortisol level between

Effect of Essential Oil of Cedarwood (Cedrus Atlantica) against Serum Cortisol Levels in Rats Which Were Given Stressor

treatment group (T

), positive control group (C

), and

negative control group (C

) was done in study group

animals. Statistical tests using Kruskal Wallis show

that the data is significant and has significant

differences. It was observed that serum cortisol level

was decrease more after given cedarwood balm

(Table 3).

Post hoc test (Mann-Whitney U test) showed that

treatment control group significantly decreasing

serum cortisol level in wistar rats compared to

positive control group and negative control group.

While the serum cortisol level was 706.85 ng/ml in

treatment group, 728.34 ng/ml in positive control

group, and 713.04 in negative control group (Table 4

and Table 5).

Result showed no significant different in serum

cortisol level between negative control group and

positive control group. Mean ± SD of serum cortisol

level in negative control group were 713.04 ±

129.533 and positive group were 728.34 ± 48.12

(Table 2).

Figure 1: Comparison of serum cortisol level

Table 1: Serum cortisol levels.

Groups N Serum cortisol

9 728.34

9 713.04

9 706.95

N: number of samples

: forced swim test only

: no stressor and no treatment were given

: forced swim test and 10% cedarwood essential oil

balm were given

Table 2: Comparison of serum cortisol levels between

groups.

Groups Groups Sig (P<0.05)

C- 0.200

T1 0.047

C+ 0.200

T1 0.007

C+ 0,047

C- 0,007

*: statistically significance

**: statistically not significance

: forced swim test only

: no stressor and no treatment were given

: forced swim test and 10% cedarwood essential oil

balm were given

Table 3: Comparison of negative control group and

treatment group.

Parameter C

Sig

(p<0.05)

Serum

Cotisol

(mean ±

SD)

713.04

129.533

728.34

48.12

706.95

37.78

0.018

: no stressor and no treatment were given

: forced swim test and 10% cedarwood essential oil

balm were given

Table 4: Comparison of negative control group and

treatment group.

Parameter

Sig

(p<0.05)

Serum Cortisol

(mean ± SD)

713.04 ±

129.533

706.95 ±

37.78

0.007

: no stressor and no treatment were given

: forced swim test and 10% cedarwood essential oil

balm were given

Table 5: Comparison of negative control group and

treatment group.

Parameter C

Sig

(p<0.05)

Serum Cortisol

(mean ± SD)

728.34 ±

48.12

706.95 ±

37.78

0.047

: forced swim test only

: forced swim test and 10% cedarwood essential oil

balm were given

4 DISCUSSION

In the present study we investigated the effects of

cedarwood balm on serum cortisol level in male

wistar rats. Several studies have said that cedarwood

can reduce stress with the hormone cortisol as an

695

700

705

710

715

720

725

730

Cortisol(ng/ml)

Group

Serum cortisol levels

C+

C‐

ICEO 2019 - 2nd International Conference of Essential Oil Indonesia

indication. However, the mechanisms is not clearly

explained and the species used is different

(Worwood, 2016).

Our result showed that cedarwood balm has an

effect to reduce stress by reducing the level of the

hormone cortisol in male wistar rats. This happen

when we compare treatment group and positive

control group. There was significant different

between serum cortisol level, which is a decrease in

the level of the serum cortisol, which is thought to be

caused by the content of cedarwood oil.

There are four major constituent in cedarwood

essential oil such as cedrol, α-atlantone, α-pinene, and

himachalol (Aberchane and Fechtal, 2004; Tisserand

and Young, 2013). The composition of essential oils

differ according to the part of the plant used,

according to the region or origin of the plant, the stage

of germination and its extraction methods (Ainane et

al., 2018; Fidah et al., 2016; Satrani et al., 2015). All

of this major constituent has an effect on stress. The

high content of sesquiterpene alcohol in cedarwood

oil has a sedative effect that can overcome tension,

fear, anxiety, and depression10,13.

Cedrol works by activating GABA so that it

causes a sedative effect that can cope with stress

conditions8,30,31. In research conducted by Ryuji et

al. (2016) cedrol is given inhaled in male wistar rats

and provides a sedation and relaxation effect that is

thought to be caused by the mechanism of GABA

inhibition (Kagawa et al., 2003). In another study

cedrol improve sleep in young women by heightening

parasympathetic activity (Takeda et al., 2017).

Another constituent that can cause sedation and

relaxation effect is α-pinene that produces cinnamon

scent which can help calm the mind and works on

GABAergic transmission like cedrol (Aoshima and

Hamamoto, 1999; Rhind, 2012).

The mechanism of action of cedarwood essential

oils to produce sedative and relaxing effects remains

to be determined. These effect may occur through

inhibition of the activity of am-aminobutyric acid

(GABA) transaminases which are enzymes for

GABA metabolism in synapses. This inhibitory

activity causes an increase in GABA levels and a

decrease in glutamate levels which will cause a

sedative effect (Franz and Novak, 2015). Previous

studies have suggested that there is a barrier to the

activity of the HPA axis after injecting GABA-A

agonists by inhibiting the production of CRH in the

parvocellular paraventricular nucleus (Herman et al.,

2004). As a result of HPA axis suppression and the

cessation of CRH production, the serum cortisol level

will be reduced.

5 CONCLUSIONS

Our study showed that 10% cedarwood balm has the effect

of reducing stress by reducing the level of the hormone

cortisol in male wistar rats given the stressor. Cedarwood

balm showed potential to be used as alternative treatment to

relief stress condition but further studies will be needed to

have more conclusive evidence on this aromatherapy.

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Effect of Essential Oil of Cedarwood (Cedrus Atlantica) against Serum Cortisol Levels in Rats Which Were Given Stressor