Author’s Accepted Manuscript

Rosmarinus officinalis Essential oil: A review of its

phytochemistry, anti-inflammatory activity, and
mechanisms of action involved

Raphaelle Sousa Borges, Brenda Lorena Sánchez

Ortiz, Arlindo César Matias Pereira, Hady Keita,
José Carlos Tavares Carvalho
www.elsevier.com/locate/jep

PII: S0378-8741(18)31410-7
DOI: https://doi.org/10.1016/j.jep.2018.09.038
Reference: JEP11536
To appear in: Journal of Ethnopharmacology
Received date: 18 April 2018
Revised date: 27 September 2018
Accepted date: 28 September 2018
Cite this article as: Raphaelle Sousa Borges, Brenda Lorena Sánchez Ortiz,
Arlindo César Matias Pereira, Hady Keita and José Carlos Tavares Carvalho,
Rosmarinus officinalis Essential oil: A review of its phytochemistry, anti-
inflammatory activity, and mechanisms of action involved, Journal of
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 1

Rosmarinus officinalis Essential oil: A review of its phytochemistry,

anti-inflammatory activity, and mechanisms of action involved

Raphaelle Sousa Borges1,2, Brenda Lorena Sánchez Ortiz1, Arlindo César Matias Pereira1,
Hady Keita1,3, José Carlos Tavares Carvalho1,2*

1
Laboratório de Pesquisa em Fármacos, Departamento de Ciências Biológicas e da Saúde, Universidade Federal do Amapá, Rodo
via Juscelino Kubitschek, S/N, Campus Marco Zero, CEP 68903-419, Macapá, AP, Brazil.
2
Programa de Pós-Graduação em Inovação Farmacêutica, Departamento de Ciências Biológicas de da Saúde, Universidade Federal
do Amapá, Juscelino Kubitscheck, KM 02, S/N- Jardim Marco Zero, Macapá- AP, 68903-419, Brasil.
3
Division de Pós-Grado, Instituto de Investigación sobre la Salud Publica. Ciudad Universitaria, Universidad de la Sierra Sur, Calle
Guillermo Rojas Mijangos S/N, Miahuatlán de porfirio Díaz, Oaxaca, Mexico.
*Corresponding author e-mail: farmacos@unifap.br
 2

Abstract

Ethnopharmacological relevance

Plant species Rosmarinus officinalis L. (Lamiaceae; Synonyms: Salvia rosmarinus Schleid. and
Rosmarinus angustifolius Mill.) is a herb widely used worldwide. In local and traditional medicine, its
used for inflammation-related diseases. Currently, studies report anti-inflammatory activity in its
essential oil (EORO). However, to better understand EORO’s anti-inflammatory activity its necessary
to understand its phytochemistry and the signaling pathways affected by it. Hence, this review aimed
to describe EORO phytochemical profile, ethnopharmacological uses, some biological activities of
EORO will be described but emphasizing its anti-inflammatory potential and possible mechanisms of
action involved.

Materials and Methods

The research was performed using the databases Medline, Embase, BVS Regional Portal, Science
Direct, CAPES Journals, and Scopus; using the keywords “Rosmarinus officinalis”,
“anti-inflammatory” and “essential oil”. Additional information was gathered from related textbooks,
reviews, and documents.

Results and Discussion

Until now about 150 chemical compounds were identified in EORO samples, more frequently reported
molecules were 1,8-cineole, α-pinene, and camphor. Studies suggest that the anti-inflammatory
activity of EORO occur mainly through inhibition of NF-κB transcription and suppression of
arachidonic acid cascade. Its antioxidant activity also aids by preventing injury caused by the reactive
species of inflammation; its smooth muscle relaxant activity contributes to ameliorating airway
inflammatory diseases. Lastly, toxicity assessments indicate low toxicity to EORO.

Conclusions

Current evidence indicates anti-inflammatory activity in EORO, supporting its ethnopharmacological

uses in inflammatory-related diseases, and potential future applications. However, although
considerable acute inflammatory models were tested, more chronic inflammatory models are needed;
clinical studies are still absent, this may be due to the high doses needed for essential oils to exert
 3

pharmacological effects, but recent studies show this issue can be bypassed using the oil formulated as
nanoemulsions to improve its bioavailability.

Keywords: antioxidant, inflammation, monoterpenes, 1,8-cineole, camphor, α-pinene

Abbreviations:

AA: Arachidonic Acid;

ABST: 2,2′-Azino-Bis(3-ethylbenzothiazoline-6-Sulphonic Acid)

COX: Cyclooxygenase;

DAMP: Damage-Associated Molecular Pattern;

DMSO: Dimethyl Sulfoxide;

DPPH: 2,2-Diphenyl-1-Picryl-Hydrazyl-Hydrate;

EORO: Essential oil of Rosmarinus officinalis;

fMLP: N-formyl Methionyl Leucyl Phenylalanine;

ICAM: Intercellular Adhesion Molecule;

IFN: Interferons;

Ig: Immunoglobulin;

IL: Interleukin;

iNOS: Inducible Nitric Oxide Synthase;

ISO: International Organization for Standardization;

HMGB: High mobility group box;

JAK: Janus Kinase;

LOX: Lipoxygenase;

LPS: Lipopolysaccharides;
 4

LT: Leukotriene;

MAPKs: Mitogen-Activated Protein Kinases;

MCP: Monocyte Chemoattractant Protein;

MIC: Minimum Inhibitory Concentration;

MIP: Macrophage Inflammatory Protein;

MKP-1: Mitogen-Activated Protein Kinase Phosphatase-1;

MMP: Matrix Metalloproteinase;

MPO: Myeloperoxidase;

NF-κB: Nuclear Factor kappa B;

NLRP-3: Nod-Like Receptor P-3;

NO: Nitric Oxide;

NOS: Nitric Oxide Synthase;

p.o.: Per Oral;

PAMP: Pathogen-Associated Molecular Pattern;

PG: Prostaglandin;

ROS: Reactive Oxygen Species;

STAT: Signal Transducer and Activator Of Transcription;

TLR: Toll-Like Receptor;

TNBS: Trinitrobenzene Sulfonic Acid;

TNF-α: Tumor Necrosis Factor;

TPA: 12-O-Tetradecanoyl-Phorbol-13-Acetate;

TREM: Triggering Receptor Expressed on Myeloid Cells;

 5

TST: Tail Suspension Test;

VCAM: Vascular Cell Adhesion Molecule;

1. Introduction

Plants have long been used for therapeutic purposes. Rosmarinus officinalis Linnaeus (1753,
Lamiaceae), is also known by the synonyms: Salvia rosmarinus Schleid. and Rosmarinus angustifolius
Mill. (The Plant List, 2018). This species is a worldwide cultivated plant known for its nutritional
value and pharmacological properties that made it famous in local and traditional medicine. In the food
industry, it is used as a food flavoring and preservative due to its antioxidant and antimicrobial
potential. R. officinalis is also used in cosmetic products (Borrás-Linares et al., 2014).

Andrade et al. (2018) showed the growing interest in this plant, with an average number of 120
studies per year since 2010. In local and traditional medicine, R. officinalis is used as tincture or tea
from aerial parts of the plant for the treatment of gastric disorders, pain and inflammation-related
diseases (Marchiori, 2004). Another popular way to use R. officinalis is as its essential oil.

In the essential oil of R. officinalis (EORO), biological activities reported are attributed to
several molecules, mainly monoterpenes, like 1,8-cineole, borneol, pinene, limonene, camphene,
camphor, and myrcene (Borges et al., 2017; Selmi et al., 2017; Bajalan et al., 2017; Vilela et al., 2016;
Takayama et al., 2016; Chávez-González et al., 2016; Akbari et al., 2015; Machado et al., 2013; Faria
et al., 2011; Juhás et al., 2009). EORO is used in the treatment of dyspepsia and milder forms of
spasmodic gastrointestinal disorders, circulatory anomalies, as a complementary in the treatment of
muscular or articular pain, and inflammations (Rašković et al., 2014).

Inflammation is an adaptative physiological condition triggered by tissue injury, stress or

infection to recover tissue homeostasis. However, self-damage caused by inflammation is unavoidable,
and its non-resolution leads to pathological conditions (Ashley, Weil, & Nelson, 2012;
Schmid-Schönbein, 2006), making it necessary the development of anti-inflammatory agents to control
it. Besides the use of EORO in local and traditional medicine for treatment of inflammation-related
diseases, EORO’s anti-inflammatory activity is reported both in vitro and in vivo assays (Takaki et al.,
 6

2008, Inoue et al., 2005; de Melo et al., 2011).

However, to better understand the biological activities attributed to EORO, including its
anti-inflammatory activity, its necessary to know the molecules of its composition and how they act.
Hence, this review aimed to update our current knowledge of EORO chemical composition. An
overview of general biological activities reported to it will be given but emphasizing its
anti-inflammatory potential and the mechanism of actions involved.

2. Materials and Methods

The literature review was performed using the following databases: Medline/Pubmed
(www.ncbi.nlm.nih.gov/pubmed/), Embase (www.embase.com), BVS Regional Portal
(www.bvsalud.org), Science Direct (www.sciencedirect.com), Capes Periodicals
(www.periodicos.capes.gov.br) and Scopus (www.scopus.com); using the keywords (in English)
“Rosmarinus officinalis”, “anti-inflammatory”, “essential oil”, and their combinations. Among found
articles, repeated ones were excluded. Complementary information was gathered from related reviews,
documents cited in the surveyed articles (ex: reports, thesis, and others), and textbooks (ex: Rang et al.,
2011). Molecules in the test cited in the first time are followed by parenthesis indicating their ID
according to PubChem (2018).

3. Rosmarinus officinalis
Research numbers show the outstanding importance of Rosmarinus officinalis. A total of 944
items were found: 51 in Medline, 47 in Embase, 62 in BVS Regional Portal, 567 in Science Direct,
174 in CAPES Periodicals, and 43 in Scopus.

3.1. Botanical Description

Rosmarinus officinalis L. (1753) − Taxonomic Serial N°.: 32677 (ITIS, 2018) – belongs to
family Lamiaceae, formerly called Labiatae (Begum et al., 2013). Popularly known by the name of
Rosemary (English), Alecrim (Portuguese) and Romero (Spanish) (Muñoz-Centeno, 2002). It is a
shrub-type plant native of the Mediterranean; grows up to 2 meters tall and has perennial green leaves
(Ribeiro-Santos et al., 2015; Kokkini et al., 2003).

R. officinalis has brown, erect, rarely crawling branches with bright dark-green linear leaves. Its
 7

flowers are small and arranged in axillary pauciflorus verticillasters, with slightly white to pink
corolla. Two distinctly exposed lateral stamens form the androecium, and its filaments have a small
lateral tooth (Ribeiro-Santos et al., 2015; Kokkini et al., 2003; Muñoz-Centeno, 2002; Tawfik et al.,
1998).

This plant grows in dry or moderately humid soil, does not tolerate anaerobic or soaked soil and
averagely tolerate salinity. Its flowering period often occurs between May and June in the
Mediterranean climate, and the fruiting period occurs between the spring and summer (Ribeiro-Santos
et al., 2015).

3.2 Ethnobotanical Relevance

There are two possible origins for the generic name Rosmarinus. According to one possibility,
the name derives from Latin “ros” (dew) and “marinus” (sea), sea dew; the other possibility is that the
name derives from the two Greek words “rhos” (shrub) and “mirrinos” (aromatic), due to its
characteristics. The species names “officinalis” is used to denote its application as a medicinal plant
(Morgan, 2005; Muñoz-Centeno, 2002).

3.2.1 History and Cultural Use

The use of Rosemary is as old as the human History and dates from 5000 BC. In the first
pharaonic dynasty of ancient Egypt, branches of Rosemary were put in the tombs of pharaohs to
perfume their trip to the afterlife (Ambrose et al., 2016; Muñoz-Centeno, 2002).

Greeks and Romans believed that Rosemary had mystical and healing power, protecting them
from evil spirits when cultivated in their gardens. According to them, this plant would grow only in the
garden of the righteous. Rosemary was a sacred plant that symbolized love and death. They used
Rosemary in weddings and funerals as a symbol of enduring love and eternal bond (Ambrose et al.,
2016; Santayana and Heinrich, 2006; Lax, 2014).

Greeks also used to burn Rosemary branches in their temples during rituals for Aphrodite, the
goddess of love. Students wore sprigs of Rosemary in their hair and around their neck while studying
to improve their memory. Arabians also believed that Rosemary helped their brain and memory. Until
today this herb is a symbol of remembrance (Lax, 2014; Santayana and Heinrich, 2006; Schiller and
 8

Schiller, 2008; Nozedar, 2008).

From the Romans, the use of Rosemary arrived in Britain, from there it spread out to the whole
of Europe, and posteriorly, to the New World. Ancient Europeans used Rosemary to improve blood
circulation and to strengthen vessels. In 1330, Ramon Llull obtained the essential oil of Rosemary for
the first time, and since then, it is used in perfumeries. In the 16th century, the Hungary Queen Isabell
used Rosemary through an infusion to treat its rheumatism, this preparation became known as “the
Queen of Hungary water,” which turned into a famous medicine in the court of Louis XIV (Ambrose
et al., 2016; Muñoz-Centeno, 2002).

Rosemary has a remarkable presence in historical events. During the Great Plague of 1665, this
herb was carried in pouches to inhale its vapors when traveling through infected areas. During The
World War II, mixtures of Rosemary leaves and Juniper berries were burned in French hospitals to kill
germs (Schiller and Schiller, 2008). Not only in History but until today Rosemary has exceptional
importance, either for therapeutic purposes or in cultural aspects. In the year 2000, Rosemary was
elected the Herb of the Year by The International Herbs Association (IHA, 2018).

3.2.2 Local and traditional medicine

Renaissance herbals recommend Rosemary for diverse purposes: as a digestif and carminative,
for wound healing, respiratory disorders, to enhance memory and others (Santayana and Heinrich,
2006). Current European phytotherapy still uses it as a circulatory stimulant, especially for those with
low blood pressure; to improve memory and concentration due to increased blood flow to the head.
Some known traditional and current uses of Rosemary also include: as a restorative for long-term
stress and chronic illness; to stimulate adrenal glands; to improve debilities caused by poor circulation
and digestion; and to relieve rheumatic muscles when applied as a lotion or diluted essential oil
(Chevallier, 2016).

Ethnopharmacological surveys show the therapeutic uses of Rosemary among traditional

communities in different world regions, they are summarized in Table 1.

Although some ethnopharmacological reports do not give proper details ‒ like the form of
treatment or even the part of plant used ‒ it is interesting to notice that some frequent medicinal uses of
R. officinalis are directly or indirectly linked to the inflammatory process; for example, chronic
 9

inflammation has a critical role in rheumatoid arthritis and others inflammatory rheumatic diseases
(Benatti and Pedersen, 2015). Chronic low-grade inflammation is also a primer for metabolic diseases
(such as diabetes) due to the induction of insulin resistance, and hence hyperglycemia (Suganami et al.,
2012).

Likewise, respiratory diseases such as bronchitis and asthma involve eosinophilic airway
inflammation (Brightling, 2011). Mild chronic vascular inflammation is a part of cardiovascular
diseases’ pathophysiology (Siti et al., 2015). According to Kiecolt-Glaser (2015), depression and
inflammation are also closely related due to the higher cytokine responses to stressors and pathogens
caused by depression, resulting in sickness behavior, depressive symptoms, and adverse health
behavior, leading to further inflammation and depression in a cycle. Other uses are more directly
related, like as a general anti-inflammatory agent.

Non-volatile terpenes from R. officinalis extracts are also known to have anti-inflammatory
activity, such as the carnosic acid (65126), carnosol (442009), rosmanol (13966122), and rosmarinic
acid (5281792) (Ulbricht et al., 2010). However, there is a high and growing interest in essential oils,
which are composed of volatile terpenes (mainly monoterpenes); hence, the anti-inflammatory potential
of EORO and its more abundant molecules will be discussed.

4. Essential oils
ISO defines essential oils as the product obtained from raw plant material by steam distillation,
including hydrodistillation, or alternative process with the same principle (de Groot and Schmidt,
2016). They are complex mixtures of volatile compounds, usually large amounts of terpenes, mainly
monoterpenes and sesquiterpenes (de Melo et al., 2011). Essential oils are purchased in significant part
by food and fragrance industries; it is also used in the development of cleaning products, cosmetics,
candles, and incenses. (de Groot and Schmidt, 2016).

Industrially the most commonly used extraction technique is the steam-distillation, which is
faster and more suitable for large quantities. On the other hand, the preferred technique in laboratories
is the hydrodistillation using Clevenger-type devices, which despite being slower, volatilizing the oil at
lower temperatures, it maintains the integrity of thermolabile compounds that could be degraded by
steam distillation (de Groot and Schmidt, 2016). Other methods include cold press distillation, dry
distillation, and microwave-assisted hydrodistillation, a modified hydrodistillation method which takes
 10

up to 65% less extraction time due to higher temperature gradient caused by microwaves (Patel, 2015;
Filly et al., 2014; Karakaya et al., 2014).

The chemical composition of essential oils depends not only of the plant species but also of its
age, variety, the part collected, origin, climate, soil, agrochemicals used, stocking time, preparation
and other factors. This variation is usually more quantitative than qualitative, and due to it, essays
performed with essential oils should always provide a biological characterization of the plant material
and the oil’s phytochemical profile, enabling the reproducibility and accuracy of data (Freires et al.,
2015). Currently, the standard method for the analysis of oil composition is through Gas
Chromatography coupled to Mass Spectroscopy (Waseen and Low, 2014).

5. Essential oil of Rosmarinus officinalis and its phytochemistry

Due to its application in pharmaceutical and food industries, the essential oil of R. officinalis
(EORO) – ISO N° 1342:2012 (ISO, 2018) – has high commercial importance (Kfoury et al., 2015). It
is a colorless or pale yellow liquid with an intense and spicy aroma. EORO represents about 1-2.5% of
the plant’s total weight and its chemical composition, like other essential oils, diverge according to the
geographical area where plant is collected, climate, part of the plant used, and the extraction method
(Mouahid et al., 2017; Tawfeeq et al., 2016; Yosr et al., 2013; Graber et al., 2010; Napoli et al.,
2010; Tawfik et al., 1998).

Some characteristic chemical components of this oil include 1,8-cineole (2758), α-pinene (6654),
camphor (2537), bornyl acetate (6448), borneol (64685), camphene (6616), α-terpineol (17100),
limonene (22311), β-pinene (14896), β-caryophyllene (5281515) and myrcene (31253)
(Chávez-González et al., 2016).

EORO from Morocco and Tunisia often shows a high content of 1,8-cineole, while EORO from
Spain shows low content of this molecule, and yields a high concentration of camphor and borneol
instead; EORO from France, in its turn, has a high concentration of verbenone (29025)
(Muñoz-Centeno, 2002; Republic Of South Africa, 2009). This data evidence the chemical
composition variation due to the geographical area where the plant is collected.

Tawfik (1998) reported that EORO obtained from leaves showed higher extraction yield during
the flowering phase (1.43%) compared to vegetative phase (1.23%), and plants collected in summer
 11

showed almost doubled yield compared to those collected in winter, evidencing chemical composition
variation due to plant phenological stage.

Likewise, there is a significant phytochemical variation of EORO according to the used part of
the plant. Yosr et al. (2013) reported EORO obtained from leaves had 1,8-cineole (35.8%) as the
principal component, while caryophyllene (5322111, 16.7%) was predominant in stem-extracted oil. In
the oil extracted from flowers, the major compound was caryophyllene oxide (1742210, 11.9%).
However, the leaves are most commonly used for EORO extraction (Rašković et al., 2014).

Lastly, as an example of influence of the extraction method over the oil composition, we cite the
study performed by Mouahid et al. (2017), who reported that EORO extraction by CO2 supercritical
fluid had increased concentration of some compounds, such as verbenone (34.16%) and bornyl acetate
(17.31%) when compared to EORO extraction by the traditional hydrodistillation method (Napoli et
al., 2010).

Terpenes – molecules formed by isoprene units – are usually the most abundant and diverse in
essential oils. Being accountable for the characteristic smell and flavor of aromatic and spicy plants
(Silveira e Sá et al., 2013). Napoli et al. (2015) surveyed phytochemical components found in EORO
from 40 different wild plant samples. The authors identified 82 molecules and classified them into
three categories: monoterpene hydrocarbons, oxygenated monoterpenes, and sesquiterpenes.
Components that did not fit in either group were classified as “others”.

In this survey, monoterpene hydrocarbons ranged from 20.9 to 65.6% of samples content; among
then, most abundant molecules were α-pinene (11.8 - 39.8%) and camphene (3.2 - 12.1%); other
monoterpene hydrocarbons identified were limonene, β-pinene, terpinolene (11463), and β-myrcene
(31253). Compounds classified as oxygenated monoterpenes ranged from 27.7 to 74.3% of EORO
composition, these compounds were represented majorly by 1,8-cineole (0.1 – 62.7%) followed by
camphor (2.6 – 30.5%); borneol, linalool, and verbenone were also identified in a significative
quantity. Lastly, sesquiterpenes ranged from 0.6 to 7.2% of EORO composition and β-caryophyllene
was the molecule with the highest quantity; no sesquiterpene alone exceeded 1%. The group named
“others” represented from 0.2 to 2.2% of total sample content; no compound of this group exceeded
1%, and octan-3-one was the most representative molecule.

EORO samples are usually classified into chemotypes according to its most abundant chemical
 12

component, such as cineoliferous (1,8-cineole as the major component), camphoriferous (camphor as

the major component), and α-pinene chemotype (α-pinene as the major component) (Napoli et al,
2015; Yosr et al., 2013; Afolous et. al., 2013; Tomi et al., 2016; Tucker and Maciarello, 1986).

In this review was noticed that until now, about 150 different compounds were identified in
EORO (Table 3). Linear, oxygenated and bicyclic monoterpenes are the most frequently reported
terpenes, such as 1,8-cineol, -pinene, camphene, -pinene, camphor, borneol, bornyl acetate,
p-cymene (7463), -myrcene, limonene, -terpinene (7462), verbenone, -terpineol (8748), linalool
(6549) and terpinen-4-ol (11230) (Figure 1). Many of these monoterpenes are the most representative
of EORO, as they are often present, and may represent biochemical markers of this oil.

These monoterpenes (10 carbon atoms) are formed by two isoprene units (Tawfik et al., 1998).
In the plant metabolism, 1,8-cineole is an epoxidized monoterpene biosynthesized from α-terpineol
through the formation of an ether bond between carbons C1 and C8, followed by reduction. The
bicyclic monoterpene α-pinene is formed through an electrophilic cyclization of α-terpineol, resulting
in the cation pinyl, which undergo a deprotonation forming α-pinene. Camphor, an oxygenated
monoterpene, is biosynthesized from the bicyclic monoterpene borneol through oxidation of its
hydroxyl group; these pathways are shown in Figure 2 (Wiart, 2014; Dewick, 2009).

5.1. Biological activities attributed to EORO and its major compounds

The chemical composition of EORO affects its biological activities. Among the main
components of EORO which were attributed its pharmacological activities are 1,8-cineole, camphor,
and α-pinene. Biological activities attributed to 1,8-cineole include: anti-depressive, antimicrobial,
anti-oxidant, anti-allergic, smooth muscle relaxant effect, and anti-inflammatory activity; to α-pinene
was attributed anti-oxidant, anti-fungal, anti-bacterial, and anti-inflammatory activities. Lastly,
was attributed to camphor anti-mutagenic, anti-oxidant, anti-allergic and anti-inflammatory activity
(Table 2).

In a research conducted by Machado et al. (2013), orally administered EORO had antidepressant
activity on the TST assay by reducing the immobilization time of treated rats compared to a negative
control group (vehicle); fluoxetine was used to treat the positive control group. This activity was
 13

associated with 1,8-cineole, which consisted of 45.1% of the used oil. Among the tested doses (ranging
from 0.1 to 100 mg/kg), EORO at 100 mg/kg had the highest immobilization decreasing activity.

Mekonnen et al. (2016), using α-pinene-type R. officinalis oil (50.8% of α-pinene), tested the
antimicrobial activity of this oil against several bacteria strains (from genera Salmonella, Shigella,
Pseudomonas, Staphylococcus and Escherichia) and fungi (from genera Trichophyton and
Aspergillus). For this, they used agar diffusion and agar dilution tests with Gentamicin as a positive
control and DMSO as a negative control. The zone of inhibition of EORO ranged from 6 mm
(Escherichia coli) to 32 mm (Staphylococcus epidermis), and MIC values ranged from <15.75 mg/ml
(Staphylococcus epidermis) to 36.33 mg/ml (Pseudomonas aeruginosa). Overall, their results show a
moderate antimicrobial activity by EORO, but the values significantly varied according to the strain.

In addition, Bajalan et al. (2017), using EORO from seven different populations of Rosemary
from Western Iran, assessed their antimicrobial activity against two gram-positive and two
gram-negative bacteria strains through agar disc diffusion. The inhibition zone ranged from 9.18 mm
(Stafilococus agalactiae) to 18.51 mm (Escherichia coli) – this is in contrast with the results reported
by Mekonnen et al. (2016), where E. coli was the less affected by the oil – the MIC was not
determined. They also assessed samples’ antioxidant activity through the DPPH assay. In brief, they
conclude that the oil had noteworthy antimicrobial activity and moderate antioxidant activity. The
antioxidant potential of EORO is reported not only in vitro but also in vivo; as will be further detailed
in the next section.

These antimicrobial and antioxidant activity of EORO (and other essential oils) are some of the
favorable characteristics favoring its use as a food additive; they are also shelf-life enhancers and
toxicity-reducers, as stated by Patel (2015). In a review, Sears (2015) discusses the benefits of an
anti-inflammatory diet, including the prevention of metabolic disorders such as obesity, metabolic
syndrome, and diabetes. Considering the anti-inflammatory potential of EORO, it is plausible to
consider that this characteristic as an additional benefit of EORO as a food additive.

A considerable number of other biological activities was assessed in R. officinalis and its oil;
describing all of them is out of the scope of this article. For additional information about them, we
recommend the review article by Andrade et al. (2018). Here, instead of describing general biological
 14

activities studied in R. officinalis, will be emphasized the anti-inflammatory activity of EORO, and the
mechanisms of action involved in it.

5.2. Anti-inflammatory activity of EORO and mechanisms of action involved

Among EORO biological activities, its anti-inflammatory capacity stands out. It is important to
notice that other monoterpenes besides 1,8-cineole, α-pinene, and camphor may also contribute to this
property of EORO – like limonene and myrcene (Rufino et al., 2015) – but given the greater abundance
of those former three molecules, they will be further detailed.

Faria et al. (2011) tested EORO (doses ranging from 100 to 1000 mg/kg, p.o) in rat models of
carrageenan-induced paw edema, cotton-induced granulomatous tissue formation, and croton
oil-induced ear edema. In the paw edema test, the ED50 ‒ determined as 300 mg/kg ‒ significantly
inhibited edema formation similarly to indomethacin treated group (positive control, 10 mg/kg, p.o) in
a dose-dependent manner. Granulomatous tissue formation was inhibited by 59% compared to the
negative control group (saline solution, 0.5 ml, p.o), and ear edema was inhibited by 77%.

Interestingly, in this study, the treatment with EORO had 64% less gastric damage compared to
the group treated with indomethacin; this is highly relevant because the continuous use of nonsteroidal
anti-inflammatory drugs leads to gastric damage and peptic ulcer (Drini, 2017). Prolonged therapy with
steroidal anti-inflammatory drugs also has adverse effects such as growth retardation in children,
immunosuppression, hypertension, inhibition of wound repair, osteoporosis, and metabolic
disturbances (Rhen and Cidlowski, 2005). Hence, there is a demand for novel anti-inflammatory
agents.

Moreover, Takaki et al. (2008) evaluated EORO (doses ranging from 250 to 500 mg/kg, p.o)
anti-inflammatory activity by appraising exudate’s volume and leukocyte migration in
carrageenan-induced pleurisy and carrageenan-induced paw edema, both in rats. EORO treatment
significantly inhibited carrageenan-induced edema formation 1-4 hours after carrageenan treatment
compared to the control group; also, significantly reduced the volume of pleural inflammatory exudate
and the number of migrated cells.

EORO was also evaluated in the form of a nanoemulsion. Borges et al. (2017) compared the
anti-inflammatory activity of EORO, both pure and formulated as a nanoemulsion, in
 15

carrageenan-induced rat paw edema assay. EORO and its nanoemulsion were orally administered 30
minutes before carrageenan treatment. The former was effective at 300 mg/kg (ED50 reported by Faria
et al., 2011) and inhibited 50% of edema formation; the latter was administered at 498 µg/kg and
inhibited 46% of edema formation.

That is highly relevant, considering that similar results were obtained with a 600 times smaller
dose of EORO when administered as a nanoemulsion. Borges et al. (2018), further studied this same
dose of EORO nanoemulsion in carrageenan-induced abdominal edema in zebrafish; compared to the
non-treated group, the treatment with EORO’s nanoemulsion inhibited 78% of edema formation, which
was more significant than the inhibition of Diclofenac at 0.5 mg/kg and Dexamethasone at 0.5 mg/kg.
They also studied the toxicity of this nanoemulsion in zebrafish through histopathology analysis and
behavior alterations.

Essential oils are a mixture of secondary metabolites, and they are also hydrophobic/lipophilic.
Nanoemulsion, differently of emulsions, are more thermodynamically stable, preventing the physical
separation of phases. Also, as droplets size decreases, the biological activity of lipophilic compounds in
the form of nanoemulsions increases due to enhanced transport of molecules through biological
membranes and the increased surface/volume ratio, leading to improved reactivity and bioavailability
(Donsì and Ferrari, 2016; Gupta et al., 2016). This can explain the improved EORO’s pharmacological
activity reported by Borges et al. (2017).

Inhibition arachidonic acid cascade enzymes: In the study of Takaki et al. (2008), authors proposed
that the anti-inflammatory effect of EORO was due to the presence of 1,8-cineole with possibly
synergistic action with myrcene, and at least partially due to inhibition of prostaglandin synthesis or
release of other endogenous mediators. This hypothesis is illustrated in Figure 3. Moreover, Juergens,
(2014) proposed the inhibition of 5-LOX and COXs by 1,8-cineol, preventing the formation of
inflammatory arachidonic acid metabolites, such as LTB4 and PGE2 (Figure 3).

An additional way of direct interference in the arachidonic acid cascade was proposed by Borges
et al. (2017), who showed through a molecular modeling study (docking) of EORO molecules that
camphor had the highest number of interactions with therapeutic targets of inflammation, such as
COX-2 (Figure 3). Nonetheless, only in silico study support this claim.
 16

Cutillas et al. (2018) reported the inhibition of LOX by samples of EORO (150 µg/ml) in vitro. In
this study, the camphoriferous oil had the higher inhibition capacity. Lastly, Kohoude et al. (2017),
proposed the inhibition of 5-LOX by α-pinene (Figure 3).

Inhibition of NF-κB: Some terpenes, including monoterpenes, are known to inhibit NF-κB
transcription, as shown in Figure 4 (Santana Souza et al., 2014; Silveira e Sá et al., 2013; Heras et al.,
2009). It is an established fact that NF-κB plays a crucial role in inflammation (Hoesel and Schmid,
2013; Baker et al., 2011; Zhang et al., 2017), since it acts as transcription factor of several molecules
involved in this process, such as cytokines (ex: TNF-α, IL-1, IL-6), metalloproteinases (ex: MMP-9),
inflammatory enzymes (ex: COX-2, iNOS, 5-LOX), adhesion molecules of diapedesis (ex: ICAM-1,
VCAM-1), chemokines (ex: MIP-1, MIP-2), among others (Gilmore, 2018).

Melo et al. (2011) reported the anti-inflammatory activity of EORO both in vitro (chemotaxis)
and in vivo (leukocyte migration). These authors discuss that the presence of terpenes, and their
potential to inhibit NF-κB transcription may contribute to EORO anti-inflammatory potential. The
results of these authors are in accordance to NF-κB inhibition since this transcription factor is involved
in the synthesis of chemokines (hence in chemotaxis) and adhesion molecules (hence in leukocyte
migration).

In TNBS-induced colitis model in mice, dietary supplement with cineoliferous EORO (1250,
2500 and 5000 ppm) significantly decreased MPO activity, IL-6 levels, and had protective effects in
colon mucosa, significantly decreasing macroscopic scores of colon inflammation (Juhás et al., 2009).
These authors also used the carrageenan-induced paw edema model, where EORO anti-inflammatory
activity was dependent on time and dose used in this study. Note that the reduction of IL-6 levels is in
accordance with the inhibition of NF-κB, considering its transcription products; if this is the case, the
decreased activity of MPO would be indirectly caused by it (downstream).

Antioxidant activity: Another mechanism is involved in the anti-inflammatory potential of EORO

besides the inhibition of NF-κB transcription and blockade of the AA-metabolites formation.
Considering that reactive oxygen (or nitrogen) species are significantly accountable for the harmful
 17

activity of inflammation (Arulselvan et al., 2016), EORO can alternatively act by attenuating the
damage caused by those reactive species due to its high antioxidant activity.

In vitro, Cutillas et al. (2018) tested several EORO molecules for antioxidant and chelating
capacity. They reported oxygen radical absorption capacity by β-pinene, limonene, γ-terpinene,
linalool, terpin-4-ol, α-terpineol, and β-caryophyllene; authors also reported radical sequestering
activity of α-terpinene and γ-terpinene; and chelating activity of Fe2+ ions by α-pinene, camphene, and
1,8-cineol. Bozin et al. (2007) assessed EORO antioxidant activity through lipid peroxidation induced
by Fenton reaction with Fe2+/ascorbate and Fe2+/H2O2, where EORO significantly inhibited lipid
peroxidation (Figure 5).

Antioxidant activity of plants’ extract or oil in vitro alone has low considerable relevance since
such potential is present virtually in most of the plants (see Gafner (2018) for discussion), but we cite
these reports to illustrate its antioxidant mechanisms in Figure 5. However, the antioxidant activity of
EORO is reported not only in vitro but also in vivo.

Takayama et al. (2016) tested a pre-treatment with EORO (50 mg/kg, p.o.) in animals with
ethanol-induced gastric injury. The treatment significantly decreased lipid peroxidation and ROS
formation (Figure 5), reduced MPO activity, inhibited the formation of superoxide anions and
decreased mucosal injury. According to authors, this happened probably through the modulation of
enzymes superoxide dismutase and glutathione peroxidase, with a concomitant increase or upkeeping
of glutathione levels. This is because ethanol-induced oxidative stress induces the formation of
superoxide ion (O2-), which is converted to hydrogen peroxide (H2O2) by superoxide dismutase (SOD),
and this H2O2 is subsequently inactivated by glutathione peroxidase (GTX). Glutathione, in its turn, is
the main ROS scavenger inside cells. The EORO sample used in this study had a high concentration of
1,8-cineol, camphor, and α-pinene, and the authors attributed the antioxidant effect of treatment to the
combined action of these monoterpenes.

Other authors report the antioxidant activity of EORO in vivo models. Selmi et al. (2017) tested
the treatment with EORO in rats with alloxan-induced diabetes and oxidative stress. The treatment
with EORO corrected all alloxan-induced biochemical alterations, exerted a protective effect against
alloxan-induced hyperglycemia, and protected liver and kidney from oxidative stress. The positive
 18

effect of this treatment was attributed to be at least partially due to EORO antioxidant potential. This
study also supports some of the traditional uses of R. officinalis (anti-hyperglycemic, anti-diabetic).

Smooth muscle relaxant activity: In respiratory airways inflammation, where the limited flow of air
causes excessive contraction of smooth muscle cells, it is plausible to consider that EORO’s smooth
muscle relaxant activity can indirectly aid the improvement of this condition along with its
anti-inflammatory potential. This activity of EORO was showed by Aqel (1992), who reported that
EORO inhibited rabbits tracheal smooth muscle contractions induced by acetylcholine, histamine and
high concentration of K+; this study suggests that EORO perform an antagonistic activity to calcium
(Figure 6).

These results are supported by Coelho-de-Souza et al. (2005) who showed that 1,8-cineole
(EORO major compound) reduced K+-induced guinea-pig tracheal smooth muscle contraction in a
concentration-dependent manner.

Overall, available data about EORO anti-inflammatory mechanism of action is based mainly on
the inhibition of arachidonic acid metabolites formation (Figure 3), inhibition of NF-κB transcription
(Figure 4) and antioxidant activity (Figure 5). Additionally, smooth muscle relaxant effect contributes
to aid airway inflammatory conditions (Figure 6).

5.2.1 Anti-inflammatory action of 1,8-cineole

The molecule 1,8-cineole (also known as eucalyptol) was identified in various plant genera oil,
such as Eucalyptus, Rosmarinus, Psidium, Croton and Salvia (Juergens, 2014). In this review was
noticed that this molecule is often the most abundant in EORO, and the most studied isolated.

In vitro, Juergens et al. (1998) reported a significant inhibition of leukotrienes and prostaglandins
production in bronchial asthma patients’ monocytes. Juergens et al. (2004) further reported that
1,8-cineole decreased the production of TNF-α, IL-1β, IL-4, IL-5 in lymphocytes and TNF-α IL-1β,
IL-6, IL-8 in monocytes. Khan et al. (2014) using amyloid beta-induced inflammation of PC-12 cells,
 19

showed that treatment with this molecule decreased the concentrations of the inflammatory cytokines
TNF-α, IL-1β, and IL-6; and decreased the expression of NOS-2, COX-2, and NF-κB.

In vivo, orally administered 1,8-cineole significantly reduced carrageenan-induced inflammatory

edema in the rat paw edema test, reduced the cotton pellet-induced granuloma formation and decreased
inflammation-induced increase of vascular permeability (Santos and Rao, 2000). Zhao et al. (2014)
tested 1,8-cineole in LPS-induced acute pulmonary inflammation and reported that the treatment
reduced the amount of proteins and inflammatory cells in the bronchoalveolar fluid. Moreover, the
authors reported significantly decreased levels of the pro-inflammatory cytokines TNF-α and IL-1β,
increased levels of the anti-inflammatory cytokine IL-10 in lung tissues, reduced expression of
NF-κB’s subunit p65, reduced expression of TLR4 (PAMP receptor involved in inflammation
signaling), and lastly, reduced activity of myeloperoxidase.

Bastos et al. (2011) further tested inhaled 1,8-cineole treatment in airway inflammatory
parameters of ovalbumin-challenged guinea pigs. The acute treatment with 1,8-cineole decreased
levels of the pro-inflammatory cytokines TNF-α and IL-1β, and decreased myeloperoxidase activity.

In a double-blind placebo-controlled clinical study with 1,8-cineole (Juergens et al., 2003).

Groups of patients with bronchial asthma were treated with this molecule. From a total of 16 patients
treated, 12 had significant improvement of this inflammatory-induced condition. Currently, 1,8-cineole
is the active principle of Soledum®, which is used in the treatment of airway obstructive inflammatory
diseases (Greiner et al., 2013).

Greiner et al. (2013) demonstrated that 1,8-cineole could inhibit NF-κB transcription by
preventing its translocation into the nucleus (Figure 4). Alternatively, Juergens (2014) proposed a
mechanism of action of 1,8-cineole through reduction of two arachidonic acid pathways, involving
5-LOX and COXs, impairing the production of AA-derived pro-inflammatory mediators, such as LTB4
and PGE2 (Figure 3). However, this should be demonstrated, and considering that NF-κB is involved in
the transcription of these inflammatory enzymes, their impaired activity could be a consequence of
NF-κB inhibition, instead of direct inhibition of these enzymes by 1,8-cineole.
 20

5.2.2 Anti-inflammatory action of α-pinene

Alpha-pinene is monoterpene found in EORO, and the anti-inflammatory activity of several oils
was attributed to it. This molecule is abundant in the essential oils of Helichrysum dasyanthum, H.
excisum, and H. petiolare. In a study by Lourens et al. (2004), these oils managed to inhibit in vitro
5-LOX activity, which catalyzes the formation of the inflammatory mediator LTA4 (Figure 3). The oil
of Boswellia dalzielii also has α-pinene as a major component and could inhibit 5-LOX, as reported by
Kohoude et al. (2017).

In the rat paw edema test (induced by carrageenan and PGE1), the essential oil of Bupleurum
fruticescens inhibited the edema formation. This activity was attributed to its major components,
including α-pinene. Curiously, it was noticed this anti-inflammatory effect depended on the integrity of
the adrenal glandules since it was ineffective in adrenalectomized animals (Martin et al., 1993). The
oleoresin of Pistacia vera α-pinene as the major compound also showed
anti-inflammatory activity, reducing carrageenan-induced rat paw edema (Orhan et al. 2006).

Xiao et al. (2014), using rodents, tested the anti-inflammatory activity of Senecio flammeus oil –
with α-pinene among major compounds – in carrageenan-induced paw edema, TPA-induced ear
edema, and cotton pellet-induced granuloma. In this study, the essential oil (10, 30 and 90 mg/kg)
significantly reduced paw edema in a dose-dependent manner (17.4%, 52.9% and 66.4%), reduced
MPO activity, reduced TPA-induced ear edema in a dose-dependent manner (20.27%, 33.0% and
53.9%), and reduced cotton pellet-induced granuloma.

Not just in essential oils, the anti-inflammatory activity of isolated α-pinene was also assessed. In
vitro, Rufino et al. (2014) stimulated human chondrocytes with the pro-inflammatory cytokine IL-1β,
then treated then with α-pinene. In this study the authors reported that α-pinene exerted both
anti-inflammatory and anti-catabolic activity, inhibiting NF-κB signaling pathway and the expression
of iNOS. In this same study, the authors tested the treatment with α-pinene in LPS-stimulated mice
macrophages and reported that the treatment reduced the synthesis of IL-6 and TNF-α in a
dose-dependent manner, decreased the expression of iNOS and predictably the synthesis of nitric
oxide.

Kin et al. (2015) tested α-pinene in LPS-induced mouse peritoneal macrophages. The authors
reported that the treatment suppressed mitogen-activated protein kinases (MAPKs) and NF-κB
 21

pathway, which indicates two possible mechanisms by this molecule. Moreover, the authors reported
significantly decreased production of IL-6, TNF-α, nitric oxide, and inhibition of enzymes iNOS and
COX-2.

In vivo, Nam et al. (2014) tested intranasal treatment of α-pinene in rats with
ovalbumin-sensitized allergic rhinitis ‒ an inflammatory allergy. These authors reported that the
treatment decreased the levels of IL-4, IG-E, TNF-α, ICAM-1, and MIP-2; also, inhibited eosinophils
infiltration, and decreased the presence of mast cells in rats’ nasal mucosa. In vitro, the authors
observed that the treatment inhibited NF-κB activity in human mast cells.

Bae et al. (2012) tested α-pinene in rats with cerulein-induced acute pancreatitis. The treatment
reduced pancreatic average weigh – evidencing decreased organ edema – and reduced levels of
biochemical markers of pancreatic damage. Histologically, they observed that treatment decreased
organ injury. Through mRNA analysis, the authors observed a decreased synthesis of the
pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 both in vivo and in vitro cultured cells. Lastly, the
treatment also decreased MPO activity. Even though the activity of NF-κB was not evaluated, this
anti-inflammatory activity could occur due to NF-κB inhibition, which would explain the decreased
synthesis of TNF-α, IL-1β, and IL-6.

5.2.3 Anti-inflammatory action of camphor

Camphor is present in some essential oils with anti-inflammatory activity; it is a primary
compound of Artemisia Judaica L. essential oil. Abu-Darwish et al. (2016) showed in vitro the
potential of this oil to reduce the production of NO in LPS-induced macrophages. Corroborating these
results, Farghadan et al. (2016) also tested the oil of Artemisia Judaica L. in LPS-induced RAW264.7
macrophages, and the treatment inhibited iNOS expression without showing toxicity at the higher
doses.

Chen et al. (2017) tested a camphor-rich oil of Artemisia argy in LPS-induced RAF264.7
macrophages. The treatment dose-dependently suppressed the production of inflammatory mediators
such as NO, PGE2, ROS, IL-6, TNF-α, IFN-β, and MCP-1; and downregulated mRNA expression of
iNOS and COX-2 without directly affecting their activity. The authors also reported inhibition of JAK2
and STAT1/3 phosphorylation. Curiously, the treatment did not inhibit the activation of MAPKs or
 22

NF-κB, suggesting a different mechanism of action of other monoterpenes. In this study, oral
administration of this oil reduced TPA-induced ear edema and COX-2 levels. The authors concluded
that this oil suppressed the inflammatory response via down-regulation of the JAK/STAT signaling and
ROS scavenging. This is plausible considering that JAK/STAT signaling pathway is related to the
release of various cytokines and inflammatory mediators such as IL-6, IL-10, iNOS, and HMGB1 (Cai
et al., 2015).

In vivo, El Jemli et al. (2017) tested the essential oil of Tetraclinis articulate – whose
composition had camphor as one of the major components – in carrageenan-induced rat paw edema and
reported a significant antioxidant activity by this oil through scavenging activity measured by DPPH,
ferric-reducing power assay, among others. They proposed a possible inhibition of COX to these results
given its importance in inflammation. However, direct inhibition of COX is not the only possible
anti-inflammatory mechanism of action, and this should be considered.

In a study performed by Zhang et al. (2017), the essential oil from the roots of Curcuma
kwangsiensis significantly decreased the expression of the pro-inflammatory cytokine TNF-α and the
inflammatory enzyme COX-2 in TPA-induced mouse ear edema assay. Camphor was one of the major
components of the oil used in this study.

The anti-inflammatory effect of isolated camphor was tested by Silva-Filho et al. (2014). At
doses of 3, 10 and 30 μg/ml, camphor reduced leukocytes migration toward fMLP in vitro. Topical
treatment did not significantly reduce croton oil-induced ear edema in mice, neither MPO activity.
However, oral treatment at doses 100 and 200 mg/kg significantly reduced both. Despite not showing
cytotoxicity at 3, 10, 30 and 90 μg/ml doses and showing anti-inflammatory activity with low doses
(p.o.), the authors reported irritant effect at higher doses of pure camphor. That is the only study found
that assessed the anti-inflammatory activity of isolate camphor, and although its anti-inflammatory
activity is evidenced, a precise mechanism of action cannot be entirely inferred.

Through all these studies surveyed, it can be noticed that the most common in vivo methods used
to evaluate the anti-inflammatory activity of EORO is through the paw edema and ear edema models
(in rat or mouse). They are models of acute inflammatory response.
 23

The paw edema test is usually performed by injecting carrageenan solution in the rodents’ paw.
After this injection, the paw shows the cardinal signs of inflammation ‒ heat, swelling, redness, pain,
and loss of tissue function (Ashley et al., 2012). The inflammation response is quantified by measuring
the edema volume with a plethysmometer. This model has a vital role in novel drug development, and
the carrageenan-induced inflammation is well-researched, and highly reproducible (Morris, 2003).

The ear edema test, in its turn, is induced often by a solution of croton oil that triggers an acute
phlogistic response, which is quantified by removing and weighing the ear six hours later. Other
pro-inflammatory substances used to induce inflammation are TPA, cantharidin, mustard oil,
arachidonic acid, dithranol (anthralin), capsaicin, ethyl phenyl propiolate (EPP), interleukin-1 (IL-1),
zymosan, and carrageenan. This method is one of the most suitable to evaluate the topical
anti-inflammatory activity of a substance (Colorado et al., 1991; Gábor, 2002).

5.3. EORO safety assessments

To evaluate EORO toxicity, Faria et al. (2011) tested doses up to 2000 mg/kg in mice and
observed them for seven days to calculate the LD50. No death occurred after administration of EORO,
and no stereotypical symptoms of acute toxicity were observed. The authors concluded that the median
lethal dose was higher than 2000 mg/kg. Most abundant compounds of the oil used were 1,8-cineole
(28.1%), α-pinene (17.4%), and an unidentified terpene (23.9%).

Takaki et al. (2008) also evaluated the toxicity of EORO to obtain the LD50 value. For this, they
tested EORO intraperitoneally (up to 1,000 mg/kg) and orally (3,000 mg/kg), but no lethality or sign of
toxicity was observed. The used oil had myrcene (24.6%), 1,8-cineole (19.8%), and an unidentified
terpene (19.6%) as main compounds.

Fahim et al. (1999) reported an LD50 value of 5.5 g/kg BW. The authors tested EORO (doses
from 2 to 9 g/kg BW) through intragastric administration in Swiss albino rats. The authors also
reported the value of LD100 (9 g/kg BW) and LD10 (1.1 g/kg BW). Their oil had bornyl acetate (20.8%),
L-camphor (14.0%) and borneol (8.2%) as major compounds.

According to a document of Baker and Grant (2008), the acute oral toxicity of EORO is reported
as being 5 ml/kg in rats, and the acute dermal toxicity is reported as being > 10 ml/kg in rabbits.
Moderate acute dermal irritation occurs only with 24 hours of direct contact, and skin sensitization was
 24

reported as being negative. Acute inhalation, acute eye irritation, or sub-chronic toxicity were not
appraised according to this document.

Considering the ED50 of 300 mg/kg reported by Faria et al. (2011) and the high doses to induce
toxicity, we can deduce that EORO treatment is quite safe according to the currently available data.
However, it must be considered that the chemical variation of essential oils could influence not only in
the ED50 value but also in toxicity values. It would be beneficial to compare these values among
different oil samples to correlate their activity and toxicity to their chemical composition.

6. Concluding Remarks and Future Perspectives

In this article, we reviewed studies of Rosmarinus officinalis and its essential oil, its
ethnopharmacological importance, phytochemistry, and some biological activities reported,
emphasizing its anti-inflammatory activity and the underlying mechanisms of action of its major
molecules.

It is interesting to notice that the traditional use of R. officinalis (and EORO) in several countries
worldwide; this influenced the execution of phytochemical studies to elucidate its pharmacological
properties. Researches provided so far, the identification of about 150 molecules found in EORO
samples. However, some of them are frequently more reported and at higher concentration, such as
1,8-cineole, α-pinene, and camphor mainly, but this can vary according to the oil’s chemotype. To
these molecules (mainly monoterpenes) were attributed some pharmacological activities including
smooth muscle relaxant effect, anti-microbial, antioxidant, and mainly anti-inflammatory activity.

This anti-inflammatory activity of EORO can be attributed mainly to 1,8-cineole and α-pinene,
which are often more abundant, and whose mechanisms are better understood. Camphor was already
tested isolated, exerting anti-inflammatory activity in vivo and impairing leukocyte migration in vitro,
but the underlying mechanisms responsible for this is not entirely elucidated. Depending on the oil
chemotype, limonene and myrcene can contribute as well if their concentration is high enough.

Overall, these major monoterpenes found in EORO are reported to exert anti-inflammatory
activity through the decreased activity of the transcription factor NK-κB. There is evidence of this
 25

activity by 1,8-cineol, α-pinene, limonene, and myrcene, which impedes the synthesis of
pro-inflammatory mediators, such as TNF-α, IL-1β, subsequently blocking the formation of the
AA-metabolites downstream, due to the inhibited synthesis of AA-enzymes (ex: COX-2, which
transcription is dependent of NK-κB).

On the other hand, some authors propose a direct inhibition of AA-enzymes by the monoterpenes
found in EORO, but more evidence is needed to assume this. Even if some studies report the inhibited
activity of inflammatory enzymes like iNOS, COX-2, and LOX-5 by oil components, this could be an
indirect effect caused by the impairment of NK-κB transcription, since it acts in the transcription of
these enzymes.

The anti-oxidant activity of EORO molecules may also attenuate inflammation-induced injury by
neutralizing the reactive species produced in inflammation. In the case of air-way inflammatory
conditions, besides the anti-inflammatory activity of EORO, its smooth muscle relaxant effects may
also contribute for the treatment.

Several factors can affect essential oils’ composition (including EORO): the collecting season,
geographical area, extraction method, plant variety, among others. The oil composition, in its turn,
affects its biological activities. Hence, it would be of great applicability to standardize the parameters
involved, whenever possible, for the obtention of oils’ samples according to the biological activity
intended. That could not only avoid conflicting results and discrepancies of effective doses but could
also optimize the oil activity to its purpose. Still due to the variation of biological activity according to
the oil composition, the phytochemical characterization of the oil assessed is indispensable.

It is important to remember that plants’ extracts and oils contain a considerable mixture of
secondary metabolites that can mask the activity of the major compounds; this could explain the higher
doses of essential oil needed to exert a given biological activity when compared to its isolated
molecules. Moreover, the lipophilic nature of essential oils contributes to this decreased activity, as
discussed previously, but recent studies suggest this can be bypassed using the oil in the form of
nanoemulsions as an alternative to improve its bioavailability and efficacy; this is especially relevant
for systemic administrations. This technology could also improve essential oils’ commercial potential
due to lower doses of oil needed to exert its pharmacological activity.
 26

Finally, the available data provide substantial evidence to consider R. officinalis’ essential oil in
the treatment of acute inflammatory conditions, considering its efficacy and high safety of use.
However, more chronic inflammation models are needed, and clinical studies are still absent. Overall
the studies reviewed support the ethnopharmacological uses of EORO against inflammation-related
diseases, and the mechanisms described here provide a theoretical framework for the anti-inflammatory
effect of EORO.

Acknowledgments

The authors acknowledge CAPES (Pró-Amazonia, No. 3292/2013 AUXPE), and CNPq Proc.
402332/2013-0 – RANAF) for the financial support.

Conflict of Interest

The authors declare that they have no conflict of interest.

Authors Contribution

All authors contributed similarly, and JCTC acted as project supervisor.

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42
43
 44

Figure 1. Compounds found in the essential oil of Rosmarinus officinalis L. (EORO).

 45

Figure 2. Biosynthesis of the monoterpenes 1,8-cineole, α-pinene and camphor (mevalonate

route), major compounds of EORO.

Figure 3. Arachidonic acid cascade pathway and mechanism of action proposed by some authors
to camphor, α-pinene, and 1,8-cineol isolated or combined with myrcene; 1: G protein-coupled
receptors (GPCR) are stimulated by agonists; 2: Guanosine diphosphate (GDP) is activated into
guanosine triphosphate (GTP); 3: Calcium ions concentration (Ca2+) increases in the cytoplasm;
4: These processes activate phospholipase A2 (PLA2); 5: PLA2 acts on membrane phospholipids
releasing linoleic acid (AL), which is transformed into arachidonic acid (AA); 6: AA is oxidized
 46

by Lipoxygenase (LOX); 7: 5-lipoxygenase (5-LOX) converts hydroxyhexanoatetetraenoic acid

(5-HPETE) to leukotriene A4 (LTA4); 8: LTA4 is converted to leukotriene B4 (LTB4), acting on
macrophages stimulating the production of proinflammatory cytokines; 9: AA is oxidized by
cyclooxygenase (COX-1 and COX-2); 10: COXs form prostaglandin H2 (PGH2); 11: From PGH2
the catalysis of enzymes results in prostaglandins (PGE2, PGF2, PGI2, PGD2) and thromboxane
(TXA2); 12: 1,8-cineol and α-pinene block the action of 5-LOX by preventing the formation of
proinflammatory cytokines; 13: 1,8-cineol alone or combined with myrcene acts on the blockade
of COX-1 and COX-2; 14: Camphor has a high number of possible interactions with PGI2 and
COX-2.
 47

Figure 4: Evidenced mechanism of action of 1,8-cineol, α-pinene and other terpenes in the
NF-κB signaling pathway; 1: The PAMP, DAMP or cytokine-receptor is activated by its
correspondent agonist molecule; 2: Receptor activation stimulus is transduced by coupled
proximal adaptor protein (PAP), which in turn activates IκB-kinase (IKK) by phosphorylation;
3: Activated IKK phosphorylates the inhibitor of κB (IκB), bounded to nuclear factor-κB
(NF-κB); 4: Phosphorylation of IκB signalize its consequent polyubiquitination (u); 5: IκB
polyubiquitination causes its degradation by proteasome; 6: Without its inhibitor, NF-κB is able
to translocate into nucleus; 7: In the nucleus, NF-κB binds to DNA, acting as transcription
factor of several pro-inflammatory mediators; 8: NF-κB transcription products include, among
others, IL-1, IL-6, TNF-α, COX-2, MIP-1, MIP-2, ICAM-1, and VCAM-1; 9: 1,8-cineole,
α-pinene, and other terpenes can act by preventing NF-κB translocation into nucleus, and the
consequent formation of pro-inflammatory mediators.

Figure 5. Antioxidant mechanism of action performed by EORO components in lipid

peroxidation and ROS formation; 1: Hydroxyl (OH•) sequesters a hydrogen atom from
polyunsaturated fatty acids (AGP-LH) from the cell membrane, forming a lipidic radical (L•) and
water (H2O); 2: L• reacts with oxygen (O2) resulting in a peroxyl radical (LOO•); 3: LOO•
sequesters another hydrogen from AGP-LH forming a new L• radical and hydroperoxide
 48

(LOOH); 4: L and LOO• form a stable radical (LOOL), which is also formed by two LOO•; 5:
EORO compounds (oxygenated monoterpenes, mono- and sesquiterpene hydrocarbons) manage
to neutralize OH•, avoiding membrane deterioration; 6: In the inner membrane (IM) of
mitochondria, the I complex (CI) releases electrons, generating superoxide radicals (O2•) to
mitochondrial matrix; 7: Complex III (CIII) releases electrons, generating O2• in matrix and
mitochondrial intermembrane space, O2• undergo the action of superoxide dismutase 1 (SOD1)
from intermembrane space and superoxide dismutase 2 (SOD2) from matrix, forming hydrogen
peroxide (H2O2); 8. Enzyme glutathione peroxidase (GPX) reduces H2O2 into H2O and O2; 9:
Fe2+ ions react with H2O2 to form Fe3+ and OH (Fenton reaction); 10: EORO compounds
(oxygenated monoterpenes, mono- and sesquiterpene hydrocarbons) react with OH•, avoiding
lipid peroxidation; 11: EORO compounds (1,8-cineole, camphor, α-pinene, and other molecules)
increase the amount of glutathione, resulting in decreased levels of H2O2 by the action of GPX.

Figure 6. Smooth muscle mechanism of action and the effects of 1,8-cineol in calcium channel
blockade; 1: Differences of ions concentration between intra- and extracellular media causes
membrane depolarization, which promotes the opening of voltage-operated calcium channels
 49

(VOCC) and passage of calcium ions (Ca2+) into the sarcoplasm; 2: G protein-coupled receptors
(GPCR) are stimulated by an agonists, activating guanosine diphosphate (GDP); 3: GDP is
activated into guanosine triphosphate (GTP), which results in the release of α-GTP domain to
activate Phospholipase C (PLC); 4: PLC cleaves Phosphatidylinositol biphosphate (PIP2) into
Diaglycerol (DAG) and Inositol triphosphate (IP3); 5: IP3 binds to the IP3 receptor (IP3R) from the
sarcoplasmic reticulum membrane, inducing the opening of the receptor-operated calcium channel
(ROCC), which promotes the release of Ca2+ to the sarcoplasm; 6: Increased Ca2+ concentration in
sarcoplasm induces the activation of the VOOC bound ryanodine receptor (RYR); 7: VOOC-RYR
release more Ca2+ into the sarcoplasm; 8. Four free calcium ions bind to Calmodulin forming the
Ca2+-calmodulin complex (CaCM); 9: CaCM activates myosin light chain kinase (MLCK); 10:
MLCK activates the ATPase enzyme to hydrolyze adenosine triphosphate (ATP) molecule into
adenosine diphosphate (ADP) and inorganic phosphate (P), which will be assigned to the myosin
light chain (MLC); 11: MLCK phosphorylates MLC, which promotes phosphorylation of the
globose region of Myosin (M); 12: The CaCM complex binds to Caldesmon (Cd), liberating the
binding site of Actin; 13: Muscle contraction; 14: 1,8-cineol acts as an antagonist to calcium by
blocking the calcium channels, reducing its concentration in sarcoplasm and hence preventing the
process of muscle contraction.

Table 1. Ethnopharmacological uses of Rosmarinus officinalis in traditional communities.

Ailment/uses Geographical Area References

Kidney stones, high blood-sugar
levels Middle Eastern Lev (2006)

Abdominal colic Jordan Aqel (1991)

Allergies, dermatological ailments, Morrocco Jamila and Mostafa

gastrointestinal disorders (2014)
 50

Asowata-Ayodele et al.
Appetite loss South-Africa
(2016)
Menstrual problems, fever, stomach
ache, hypertension, improving
Algeria Benarba (2016)
memory

Throat antiseptic, hypotension,

rheumatism, boldness Center of Portugal Gaspar et al. (2002)

To improve memory
United Kingdom Perry et al. (1999)

Sore throat, stomach aches South Italy Montesano et al. (2012)

Dandruff, fortifier,
Ecuadorian Andes Cerón Martínez (2006)
anti-inflammatory agent

Stomach ache, menstrual pain, Guatemalan Caribbean Girón et al. (1991)

cough

Sinusitis, depression, dyspnea, flu,

Pasa et al. (2011); Silva
inflammations, cardiovascular Center of Brazil
and Faria (2014)
diseases

Asthma, bronchitis, sinusitis,

cough, stomachache, flatulence,
blood circulation, rheumatism, Northeast of Brazil Vieira (2012)
diabetes, depression, to improve
memory
 51

Southeast of Brazil Garcia et al. (2010)

Muscle pain

Correa (1984)
Inflammatory diseases Brazilian Amazon

Table 2. Major compounds of Rosmarinus officinalis L. essential oil and some of the biological
activities attributed to them.

Compound Biological activity References

1,8-cineole Anti-inflammatory (Juhás et al., 2009)
(Faria et al., 2011) (Machado
Anti-depressive
et al., 2013)
Antialgic
(Vilela et al., 2016)
Antioxidant (Takayama et al., 2016)
Smooth muscle relaxant activity (Bajalan et al., 2017)
(Selmi et al., 2017)
(Souza et al., 2005)

- pinene Anti-inflammatory (Nam et al., 2014)

Antifungal (Bae et al., 2012)

Antioxidant (Lin et al. 2016) (Mekonnen

et al. 2016)
Antibacterial
(Takayama et. al, 2016)

Camphor Anti-inflammatory (Silva-Filho et al., 2014)

(Fahin et. al, 1999) (Faria et.
Antialgic al, 2011)
Anti-mutagenic (Melo et al. 2011)
(Takayama et. al, 2016)
Antioxidant (Bajalan et. al, 2017)
 52

(Borges et. al, 2017)

Table 3. Compounds identified in the essential oil of Rosmarinus officinalis L. (OERO).

Number Compounds References

(Tucker and Maciarello, 1986)

(Fahim et al. 1999) (Takaki et al.
2008) (Juhás et al. 2009) (Graber
et al. 2010) (Napoli et al. 2010)
(Melo et al. 2011) (Faria et al.
1,8 cineole 2011) (Machado et al. 2013)
1 (Yosr et al. 2013) (Akbari et al.
2015) (Kfoury et al. 2015) (Lin
et al. 2016) (Mekonnen et al.
2016) (Takayama et al. 2016)
(Tawfeeq et al. 2016) (Vilela et
al. 2016) (Bajalan et al. 2017)
(Borges et al. 2017) (Mouahid et
al. 2017) (Selmi et al. 2017 )

(Tucker and Maciarello, 1986)

(Fahim et al. 1999) (Juhás et al.
2009) (Graber et al. 2010)
(Napoli et al. 2010) (Faria et al.
2011) (Melo et al. 2011)
(Machado et al. 2013) (Yosr et
-pinene al. 2013) (Afoulous et al. 2013)
2
(Akbari et al. 2015) (Kfoury et
al. 2015) (Lin et al. 2016)
(Mekonnen et al. 2016)
(Tawfeeq et al. 2016)
(Takayama et al. 2016) (Vilela et
al. 2016) (Bajalan et al. 2017)
 53

(Borges et al. 2017) (Mouahid et

al. 2017) (Selmi et al. 2017)

(Tucker and Maciarello, 1986)

(Fahim et al. 1999) (Juhás et al.
2009) (Graber et al. 2010)
(Napoli et al. 2010) (Faria et al.
2011) (Melo et al. 2011)
(Machado et al. 2013) (Yosr et
Camphene al. 2013) (Akbari et al. 2015)
3 (Kfoury et al. 2015) (Lin et al.
2016) (Mekonnen et al. 2016)
(Takayama et al. 2016)
(Tawfeeq et al. 2016) (Vilela
et al. 2016) (Bajalan et al. 2017)
(Borges et al. 2017) (Mouahid et
al. 2017) (Selmi et al. 2017)

(Tucker and Maciarello, 1986)

(Fahim et al. 1999) (Juhás et al.
2009) (Graber et al. 2010)
(Napoli et al. 2010) (Faria et al.
2011) (Afoulous et al. 2013)
(Machado et al. 2013) (Yosr et
-pinene al. 2013) (Kfoury et al. 2015)
4
(Lin et al. 2016) (Mekonnen et
al. 2016) (Takayama et al. 2016)
(Tawfeeq et al. 2016) (Vilela et
al. 2016) (Bajalan et al. 2017)
(Borges et al. 2017) (Mouahid et
al. 2017) (Selmi et al. 2017)

(Tucker and Maciarello, 1986)

(Fahim et al. 1999) (Juhás et al.
2009) (Napoli et al. 2010) (Faria
et al. 2011) (Melo et al. 2011)
(Machado et al. 2013) (Yosr et
Camphor al. 2013) (Akbari et al. 2015)
5
(Lin et al. 2016) (Mekonnen et
al. 2016) (Takayama et al. 2016)
(Tawfeeq et al. 2016) (Vilela et
al. 2016) (Borges et al. 2017)
(Selmi et al. 2017)

(Tucker and Maciarello, 1986)

(Fahim et al. 1999) (Juhás et al.
2009) (Napoli et al. 2010)
(Machado et al. 2013) (Yosr et
al. 2013) (Akbari et al. 2015)
6 Borneol (Mekonnen et al. 2016)
(Takayama et al. 2016)
(Tawfeeq et al. 2016) (Bajalan et
al. 2017) (Borges et al. 2017)
(Mouahid et al. 2017) (Selmi et
al. 2017) (Vilela et al. 2016)

(Tucker and Maciarello, 1986)

(Fahim et al. 1999) (Napoli et al.
7 Bornyl acetate 2010) (Yosr et al. 2013) (Akbari
et al. 2015) (Lin et al. 2016)
(Mekonnen et al. 2016)
 54

(Takayama et al. 2016)

(Tawfeeq et al. 2016) (Vilela
et al. 2016) (Bajalan et al. 2017)
(Mouahid et al. 2017) (Selmi et
al. 2017)

(Tucker and Maciarello, 1986)

(Fahim et al. 1999) (Graber et
al. 2010) (Napoli et al. 2010)
(Melo et al. 2011) (Afoulous et
al. 2013) (Yosr et al. 2013)
-caryophyllene (Kfoury et al. 2015) (Lin et al.
8 2016) (Mekonnen et al. 2016)
(Tawfeeq et al. 2016) (Bajalan et
al. 2017) (Borges et al. 2017)
(Mouahid et al. 2017) (Selmi et
al. 2017)

(Tucker and Maciarello, 1986)

(Fahim et al. 1999) (Juhás et al.
2009) (Graber et al. 2010)
p-cymene (Napoli et al. 2010) (Melo et al.
9 2011) (Machado et al. 2013)
(Yosr et al. 2013) (Kfoury et al.
2015) (Mouahid et al. 2017) (Lin
et al. 2016) (Takayama et al.
2016) (Vilela et al. 2016)

(Graber et al. 2010) (Napoli et

al. 2010) (Melo et al. 2011)
(Machado et al. 2013) (Yosr et
al. 2013) (Lin et al. 2016)
-myrcene (Mekonnen et al. 2016)
10
(Takayama et al. 2016)
(Tawfeeq et al. 2016) (Vilela et
al. 2016) (Bajalan et al. 2017)
(Borges et al. 2017)

(Tucker and Maciarello, 1986)

(Fahim et al. 1999) (Graber et al.
2010) (Napoli et al. 2010) (Melo
et al. 2011) (Yosr et al. 2013)
Limonene (Kfoury et al. 2015) (Lin et al.
11 2016) (Mekonnen et al. 2016)
(Tawfeeq et al. 2016) (Vilela et
al. 2016) (Borges et al. 2017)
(Mouahid et al. 2017)

(Tucker and Maciarello, 1986)

(Graber et al. 2010) (Napoli et
al. 2010) (Yosr et al. 2013) (Lin
-terpinene et al. 2016) (Mekonnen et al.
12 2016) (Tawfeeq et al. 2016)
(Vilela et al. 2016) (Bajalan et
al. 2017) (Borges et al. 2017)
(Mouahid et al. 2017)

(Tucker and Maciarello, 1986)

13 -terpinene (Graber et al. 2010) (Napoli et
 55

al. 2010) (Yosr et al. 2013)

(Kfoury et al. 2015)
(Mekonnen et al. 2016)
(Takayama et al. 2016)
(Tawfeeq et al. 2016) (Vilela et
al. 2016) (Bajalan et al. 2017)
(Borges et al. 2017) (Mouahid et
al. 2017)

(Fahim et al. 1999) (Graber et al.

2010) (Napoli et al. 2010) (Melo
et al. 2011) (Akbari et al. 2015)
(Lin et al. 2016) (Mekonnen et
14 Verbenone al. 2016) (Tawfeeq et al. 2016)
(Vilela et al. 2016) (Bajalan et
al. 2017) (Borges et al. 2017)
(Mouahid et al. 2017)

(Fahim et al. 1999) (Graber et al.

2010) (Napoli et al. 2010)
(Machado et al. 2013) (Lin et al.
-terpineol 2016) (Mekonnen et al. 2016)
15 (Vilela et al. 2016) (Tawfeeq et
al. 2016) (Bajalan et al. 2017)
(Borges et al. 2017) (Mouahid et
al. 2017)

(Tucker and Maciarello, 1986)

(Fahim et al. 1999) (Graber et al.
2010) (Napoli et al. 2010) (Yosr
et al. 2013) (Kfoury et al. 2015)
16 Linalool (Mekonnen et al. 2016)
(Tawfeeq et al. 2016) (Borges et
al. 2017) (Mouahid et al.
2017)

(Tucker and Maciarello, 1986)

(Fahim et al. 1999) (Graber et al.
2010) (Napoli et al. 2010)
Terpinen-4-ol (Yosr et al. 2013) (Lin et al.
17
2016) (Tawfeeq et al. 2016)
(Vilela et al. 2016) (Bajalan et
al. 2017)

(Tucker and Maciarello, 1986)

(Graber et al. 2010) (Napoli et
al. 2010) (Yosr et al. 2013) (Lin
Terpinolene et al. 2016) (Mekonnen et al.
18 2016) (Mouahid et al. 2017)
(Borges et al. 2017) (Takayama
et al. 2016)

(Tucker and Maciarello, 1986)

(Fahim et al. 1999) (Graber et al.
2010) (Napoli et al. 2010) (Lin
19 -thujene et al. 2016) (Tawfeeq et al.
2016) (Borges et al. 2017)
(Mouahid et al. 2017)

(Tucker and Maciarello, 1986)

20 -phellandrene (Napoli et al. 2010) (Yosr et al.
 56

2013) (Lin et al. 2016) (Tawfeeq

et al. 2016) (Bajalan et al. 2017)
(Borges et al. 2017) (Mouahid et
al. 2017)

(Tucker and Maciarello, 1986)

(Fahim et al. 1999) (Takaki et al.
Myrcene 2008) (Napoli et al. 2010)
21
(Kfoury et al. 2015) (Mouahid et
al. 2017)

(Graber et al. 2010) (Napoli et

al. 2010) (Vilela et al. 2016)
22 Myrtenol (Bajalan et al. 2017) (Mouahid
et al. 2017)

(Tucker and Maciarello, 1986)

(Fahim et al. 1999) (Graber et al.
23 Methyl eugenol 2010) (Bajalan et al. 2017)
(Mouahid et al. 2017)

(Graber et al. 2010) (Lin et al.

2016) (Bajalan et al. 2017)
24 Chrysanthenone (Mouahid et al. 2017) (Selmi et
al. 2017)

(Tucker and Maciarello, 1986)

(Graber et al. 2010) (Napoli et
25 -humulene al. 2010) (Mekonnen et al. 2016)
(Bajalan et al. 2017)

(Tucker and Maciarello, 1986)

(Napoli et al. 2010) (Afoulous et
26 -terpineol al. 2013) (Takayama et al. 2016)
(Mouahid et al. 2017)

(Fahim et al. 1999) (Napoli et al.

2010) (Yosr et al. 2013) (Vilela
27 Caryophyllene oxide et al. 2016) (Bajalan et al. 2017)

(Tucker and Maciarello, 1986)

28 Geraniol (Napoli et al. 2010) (Lin et al.
2016) (Mouahid et al. 2017)

(Graber et al. 2010) (Yosr et al.

29 -bisabolol 2013) (Tawfeeq et al. 2016)
(Bajalan et al. 2017)

(Fahim et al. 1999) (Graber et al.

30 -copaene 2010) (Napoli et al. 2010)
(Selmi et al. 2017)

(Graber et al. 2010) (Napoli et

31 Germacrene-D al. 2010) (Yosr et al. 2013)
(Selmi et al. 2017)

(Graber et al. 2010) (Napoli et

32 -cadinene al. 2010) (Yosr et al. 2013)
(Selmi et al. 2017)

(Graber et al. 2010) (Napoli et

 57

33 Pinocarvone al. 2010) (Bajalan et al. 2017)

(Mouahid et al. 2017)

(Tucker and Maciarello, 1986)

34 Sabinene (Graber et al. 2010) (Napoli et
al. 2010) (Tawfeeq et al. 2016)

(Tucker and Maciarello, 1986)

35 cis--ocimene (Graber et al. 2010) (Napoli et
al. 2010)

(Napoli et al. 2010) (Borges et

36 -campholenal al. 2017) (Mouahid et al. 2017)

37 -muurolene (Graber et al. 2010) (Napoli et

al. 2010) (Yosr et al. 2013)

(Graber et al. 2010) (Napoli et

38 Verbenene al. 2010) (Mouahid et al. 2017)

(Fahim et al. 1999) (Graber et al.

39 Carvacrol 2010) (Napoli et al. 2010)

(Machado et al. 2013) (Vilela et

40 -linalool al. 2016) (Borges et al. 2017)

(Napoli et al. 2010) (Afoulous et

41 -muurolene al. 2013) (Yosr et al. 2013)

(Tucker and Maciarello, 1986)

(Vilela et al. 2016) (Mouahid et
42 Nerol al. 2017)

(Graber et al. 2010) (Napoli et

43 -cadinol al. 2010) (Yosr et al. 2013)

(Tucker and Maciarello, 1986)

44 Isoborneol (Fahim et al. 1999) (Bajalan et
al. 2017)

(Graber et al. 2010) (Napoli et

45 3-octanone al. 2010) (Bajalan et al. 2017)

(Fahim et al. 1999) (Machado et

46 4-terpineol al. 2013) (Mouahid et al. 2017)

(Graber et al. 2010) (Napoli et

47 Isopulegol al. 2010)

(Tucker and Maciarello, 1986)

48 trans--ocimene (Graber et al. 2010)

(Graber et al. 2010) (Napoli et

49 Thymol al. 2010)
 58

50 - bisabolene (Graber et al. 2010) (Napoli et

al. 2010)

(Graber et al. 2010) (Yosr et al.

51 -amorphene 2013)

(Takaki et al. 2008) (Faria et al.

52 Genanyl acetate 2011)

(Graber et al. 2010) (Yosr et al.

53 Calamenene 2013)

(Graber et al. 2010) (Yosr et al.

54 -calacorene 2013)

(Graber et al. 2010) (Yosr et al.

Cadalene 2013)
55

(Borges et al. 2017) (Tawfeeq et

56 o-cymene al. 2016)

(Tucker and Maciarello, 1986)

57 Eugenol (Napoli et al. 2010)

(Tucker and Maciarello, 1986)

58 -2-carene (Tawfeeq et al. 2016)

Ferruginol (Yosr et al. 2013) (Mouahid et

59 al. 2017)
(Napoli et al. 2010) (Yosr et al.
60 -cubebene 2013)

(Napoli et al. 2010) (Mouahid et

61 Thuja-2,4(10)-diene al. 2017)

(Napoli et al. 2010) (Mouahid

62 Pinocarveol et al. 2017)

(Akbari et al. 2015) (Tawfeeq et

63 E-caryophyllene al. 2016)

(Yosr et al. 2013) (Selmi et al.

64 -humulene 2017)

(Fahim et al. 1999) (Yosr et al.

65 Spathulenol 2013)

(Tucker and Maciarello, 1986)

 59

66 Linalyl acetate (Fahim et al. 1999)

(Graber et al. 2010) (Bajalan et

67 Piperitone al. 2017)

(Vilela et al. 2016) (Bajalan et

-terpinolene al. 2017)
68
-phellandrene (Borges et al. 2017)
69
cis-verbenol (Mouahid et al. 2017)
70
Camphre (Mouahid et al. 2017)
71
Nopol (Mouahid et al. 2017)
72
1-octan-3-ol (Mouahid et al. 2017)
73
octan-3-ol (Mouahid et al. 2017)
74
terpin-1-en-4-ol (Mouahid et al. 2017)
75
α-campholenol (Mouahid et al. 2017)
76
Widdrol (Bajalan et al. 2017)
77
trans-sabinene hydrate (Bajalan et al. 2017)
78
-thujone (Selmi et al. 2017)
79
-thujone (Selmi et al. 2017)
80
3-carene (Vilela et al. 2016)
81
-thujene (Vilela et al. 2016)
82
2,4(10)-thujadiene (Vilela et al. 2016)
83
Italicene (Tawfeeq et al. 2016)
84
E-isocitral (Tawfeeq et al. 2016)
85
Citronellal (Kfoury et al. 2015)
86
(E)- -farnesene (Afoulous et al. 2013)
87
 60

88 Epicedrol (Afoulous et al. 2013)

Cararene (Yosr et al. 2013)
89
90 Caryophylla-4.8-diene-5-ol (Yosr et al. 2013)
T-cadinol (Yosr et al. 2013)
91
-selinenol (Yosr et al. 2013)
92
2-ethyl-4,5-dimethylphenol (Faria et al. 2011)
93
-fenchene (Graber et al. 2010)
94
p-cymenene (Graber et al. 2010)
95
Citronellol (Graber et al. 2010)
96
-terpineneol (Graber et al. 2010)
97
(+)-pulegone (Graber et al. 2010)
98
Isonocamphone (Graber et al. 2010)
99
Trans-sabinen hydrate (Graber et al. 2010)
100
-sesquiphellandrene (Graber et al. 2010)
101
Naphthalene, (Graber et al. 2010)
102 1,2,3,4,4a,7-hexahydro-1,6-dimethyl-4-(1-methylethyl)

-cadinene (Graber et al. 2010)

103
-cubenene (Graber et al. 2010)
104
Epizonarene (Graber et al. 2010)
105
1-octene 3-ol (Graber et al. 2010)
106
4-(4´-methyl-3´-pentenyl) -3-ciclohexenil propyl (Graber et al. 2010)
107 ketone

trans-pinene hydrate (Napoli et al. 2010)

108
p-cymenen-8-ol (Napoli et al. 2010)
109
110 -citronellol (Napoli et al. 2010)
 61

111 Tricyclene (Napoli et al. 2010)

Trans-3-pinanone (Napoli et al. 2010)
112
Cis-3-pinanone (Napoli et al. 2010)
113
Geranyl acetone (Napoli et al. 2010)
114
Eugenol methyl ether (Napoli et al. 2010)
115
Estragole (Napoli et al. 2010)
116
Benzene acethaldheyde
117 (Napoli et al. 2010)

14-hydroxy-9-epi-E-Caryophyllene (Napoli et al. 2010)

118
allo-aromandrene (Napoli et al. 2010)
119
epi--muurolol (Napoli et al. 2010)
120
-muurolol (Napoli et al. 2010)
121
cis-cadinen-4-en-7-ol (Napoli et al. 2010)
122
Bisabolol-4-ol (Napoli et al. 2010)
123
Valencene (Napoli et al. 2010)
124
-amorphene (Napoli et al. 2010)
125
-copaene (Napoli et al. 2010)
126
Ylangene (Napoli et al. 2010)
127
Piperitenone (Napoli et al. 2010)
128
cis-pulegol (Napoli et al. 2010)
129
Thymol methyl ether (Napoli et al. 2010)
130
cis-carveol (Napoli et al. 2010)
131
trans-carveol (Napoli et al. 2010)
132
Myrtenal (Napoli et al. 2010)
133
 62

Cymen-8-ol (Napoli et al. 2010)

134
Camphene hydrate (Napoli et al. 2010)
135
Chrysantenone (Napoli et al. 2010)
136
exo-fenchol (Napoli et al. 2010)
137
endo-fenchol (Napoli et al. 2010)
138
cis-sabinene hydrate (Napoli et al. 2010)
139
p-mentha-7,8-diene (Napoli et al. 2010)
140
1-O-menthen-8-ol (Takaki et al. 2008)
141
phenylacetic acid (Takaki et al. 2008)
142
2-ethyl-4,5-dimethylphenol (Takaki et al. 2008)
143
Methyl chavicolf (Fahim et al. 1999)
144
Terpinyl acetatec (Fahim et al. 1999)
145
Carveol (Fahim et al. 1999)
146
Thymole (Fahim et al. 1999)
147
Carvone (Fahim et al. 1999)
148
-thujone + 1-octen-3-ol (Tucker and Maciarello, 1986)
149
150 Isobornyl acetate (Tucker and Maciarello, 1986)