Biological Activities and Safety of Citrus spp. Essential Oils

Abstract

Citrus fruits have been a commercially important crop for thousands of years. In addition, Citrus essential oils are valuable in the perfume, food, and beverage industries, and have also enjoyed use as aromatherapy and medicinal agents. This review summarizes the important biological activities and safety considerations of the essential oils of sweet orange (Citrus sinensis), bitter orange (Citrus aurantium), neroli (Citrus aurantium), orange petitgrain (Citrus aurantium), mandarin (Citrus reticulata), lemon (Citrus limon), lime (Citrus aurantifolia), grapefruit (Citrus × paradisi), bergamot (Citrus bergamia), Yuzu (Citrus junos), and kumquat (Citrus japonica).

Full text

International Journal of
Molecular Sciences
Review
Biological Activities and Safety of Citrus spp.
Essential Oils
Noura S. Dosoky 1and William N. Setzer 1,2 ,*ID
1
Aromatic Plant Research Center, 230 N 1200 E, Suite 102, Lehi, UT 84043, USA; ndosoky@aromaticplant.org
2Department of Chemistry, University of Alabama in Huntsville, Huntsville, AL 35899, USA
*Correspondence: wsetzer@chemistry.uah.edu; Tel.: +1-256-824-6519
Received: 6 June 2018; Accepted: 3 July 2018; Published: 5 July 2018


Abstract:
Citrus fruits have been a commercially important crop for thousands of years. In addition,
Citrus essential oils are valuable in the perfume, food, and beverage industries, and have also
enjoyed use as aromatherapy and medicinal agents. This review summarizes the important biological
activities and safety considerations of the essential oils of sweet orange (Citrus sinensis), bitter
orange (Citrus aurantium), neroli (Citrus aurantium), orange petitgrain (Citrus aurantium), mandarin
(Citrus reticulata), lemon (Citrus limon), lime (Citrus aurantifolia), grapefruit (Citrus
×
paradisi),
bergamot (Citrus bergamia), Yuzu (Citrus junos), and kumquat (Citrus japonica).
Keywords:
sweet orange; bitter orange; neroli; orange petitgrain; mandarin; lemon; lime; grapefruit;
bergamot; yuzu; kumquat
1. Introduction
The genus Citrus (Rutaceae) is one of the ancient, most traded, and most popular crops.
The earliest records of its cultivation date back to 2100 BC [
1
]. The origin of Citrus is still controversial;
however, it is believed to have originated from Southeast Asia [
2
]. Citrus is grown widely all over the
world for its numerous health benefits. Citrus fruits are consumed as a fresh fruit desert or used for
making juice and jam. They are an excellent source of vitamins, especially vitamin C. Processing Citrus
fruits results in a significant amount of waste (peels, seeds, and pulps), which accounts for 50% of
the fruit [
3
]. Citrus waste is a valuable source of d-limonene, flavonoids, carotenoids, dietary fibers,
soluble sugars, cellulose, hemicellulose, pectin, polyphenols, ascorbic acid, methane, and essential
oils [
4
–
6
]. Interestingly, the essential oil (EO) is the most vital by-product of Citrus processing. Citrus
EOs are broadly used as natural food additives in several food and beverage products [
7
] because they
have been classified as generally recognized as safe (GRAS) [
8
]. Furthermore, Citrus EOs are used as
natural preservatives due to their broad spectrum of biological activities including antimicrobial and
antioxidant effects [
9
]. The presence of terpenes, flavonoids, carotenes, and coumarins is thought to
be responsible for the strong anti-oxidative and antimicrobial activities [
10
–
14
]. Due to their pleasant
refreshing smell and rich aroma, Citrus EOs are also used in air-fresheners, household cleaning
products, perfumes, cosmetics, and medicines.
Because of their high economic importance, numerous studies have investigated the chemical
composition of the peel, leaf, and flower essential oils of different Citrus species. It is worth noting that
there is a great variation in the chemical composition of Citrus oils due to differences in origin, genetic
background, season, climate, age, ripening stage, method of extraction, etc. [
15
–
19
]. The key volatile
components are presented in Figure 1. Sweet orange, bitter orange, mandarin, and grapefruit EOs are
rich in monoterpenes with the major component being d-limonene (65.3–95.9%) (Table 1) [
8
]. The main
components in the essential oil of bitter orange leaf are linalyl acetate and linalool [
16
], while the flower
EO contained linalool as the major component, followed by d-limonene and linalyl acetate [
20
]. Some
Int. J. Mol. Sci. 2018,19, 1966; doi:10.3390/ijms19071966 www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2018,19, 1966 2 of 25
of the Citrus EOs are prepared by expression, which results in the presence of non-volatile components
(Figure 2) that can cause photosensitivity and skin irritation [
8
]. The percentages of these non-volatile
constituents in expressed oils are given in Table 2.
Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 2 of 25
while the flower EO contained linalool as the major component, followed by d-limonene and linalyl
acetate [20]. Some of the Citrus EOs are prepared by expression, which results in the presence of non-
volatile components (Figure 2) that can cause photosensitivity and skin irritation [8]. The percentages
of these non-volatile constituents in expressed oils are given in Table 2.
Figure 1. Chemical structures of key volatile components in Citrus essential oils.
Figure 2. Chemical structures of key non-volatile components in expressed Citrus essential oils.
The objective of this review is to summarize the reported biological activities and safety of the
essential oils of sweet orange (Citrus sinensis L.), bitter orange (Citrus aurantium L.), neroli (Citrus
aurantium L.), orange petitgrain (Citrus aurantium L.), mandarin (Citrus reticulata Blanco), lemon
(Citrus limon Osbeck), lime (Citrus aurantifolia), grapefruit (Citrus × paradisi Macfady), bergamot
(Citrus bergamia Risso & Poit), Yuzu (Citrus junos Sieb. ex Tanaka), and kumquat (Citrus japonica
Thunb).
d-Limonene γ-Terpinene
OH
Linalool
OAc
Linalyl acetate
OH
α-Terpineol (E)-β-Ocimene Terpinolene β-Pinene
OO
O
O
8-Geranyloxypsoralen
OOO
O
Bergamottin
OOO
OO
Epoxybergamottin
OO
O
MeO
5-Geranyloxy-7-methoxycoumarin
OO
OMe
O
OMe
Isopimpinellin
OOO
O
OMe
5-Geranyloxy-8-methoxypsoralen
OO
O
O
OMe
O
Byakangelicol
OOO
O
O
Oxypeucedanin
OO
OMe
MeO
Citropten
OOO
OH
OOO
OMe
OOO
Bergaptol Bergapten Psoralen
Figure 1. Chemical structures of key volatile components in Citrus essential oils.
Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 2 of 25
while the flower EO contained linalool as the major component, followed by d-limonene and linalyl
acetate [20]. Some of the Citrus EOs are prepared by expression, which results in the presence of non-
volatile components (Figure 2) that can cause photosensitivity and skin irritation [8]. The percentages
of these non-volatile constituents in expressed oils are given in Table 2.
Figure 1. Chemical structures of key volatile components in Citrus essential oils.
Figure 2. Chemical structures of key non-volatile components in expressed Citrus essential oils.
The objective of this review is to summarize the reported biological activities and safety of the
essential oils of sweet orange (Citrus sinensis L.), bitter orange (Citrus aurantium L.), neroli (Citrus
aurantium L.), orange petitgrain (Citrus aurantium L.), mandarin (Citrus reticulata Blanco), lemon
(Citrus limon Osbeck), lime (Citrus aurantifolia), grapefruit (Citrus × paradisi Macfady), bergamot
(Citrus bergamia Risso & Poit), Yuzu (Citrus junos Sieb. ex Tanaka), and kumquat (Citrus japonica
Thunb).
d-Limonene γ-Terpinene
OH
Linalool
OAc
Linalyl acetate
OH
α-Terpineol (E)-β-Ocimene Terpinolene β-Pinene
OO
O
O
8-Geranyloxypsoralen
OOO
O
Bergamottin
OOO
OO
Epoxybergamottin
OO
O
MeO
5-Geranyloxy-7-methoxycoumarin
OO
OMe
O
OMe
Isopimpinellin
OOO
O
OMe
5-Geranyloxy-8-methoxypsoralen
OO
O
O
OMe
O
Byakan gelicol
OOO
O
O
Oxypeucedanin
OO
OMe
MeO
Citropten
OOO
OH
OOO
OMe
OOO
Bergaptol Be rgapten Psoralen
Figure 2. Chemical structures of key non-volatile components in expressed Citrus essential oils.
The objective of this review is to summarize the reported biological activities and safety of
the essential oils of sweet orange (Citrus sinensis L.), bitter orange (Citrus aurantium L.), neroli
(Citrus aurantium L.), orange petitgrain (Citrus aurantium L.), mandarin (Citrus reticulata Blanco),
lemon (Citrus limon Osbeck), lime (Citrus aurantifolia), grapefruit (Citrus
×
paradisi Macfady), bergamot
(Citrus bergamia Risso & Poit), Yuzu (Citrus junos Sieb. ex Tanaka), and kumquat (Citrus japonica Thunb).

Int. J. Mol. Sci. 2018,19, 1966 3 of 25
Table 1. Major volatile components in essential oils of different Citrus spp.
Citrus EO Sweet
Orange [8,21]
Bitter
Orange [8]
Neroli
(Egyptian) [8]
Petitgrain
[8]
Mandarin
[8]
Lemon
(D) [8]
Lemon
(Ex) [8]
Lime (D)
[8,22,23]
Lime (Ex)
[8,22]
Bergamot
(FCF) [8,24]
Bergamot
(Ex) [8,24,25]
Grapefruit
[8,26]
Yuzu
[8,27]
Plant Part Fruit Peel Fruit Peel Flower Leaf Fruit Peel Fruit Peel Fruit Peel Fruit Peel Fruit Peel Fruit Peel Fruit Peel Fruit Peel Fruit Peel
Essential oil Composition
d-Limonene 83.9–95.9% 89.7–94.7% 6.0–10.2% 0.4–8.0% 65.3–74.2%
64.0–70.5% 56.6–76.0% 40.4–49.4%
48.2% 28.0–45.0% 27.4–52.0%
84.8–95.4%
63.1%
Linalool 0–5.6% 0.1–2.0% 43.7–54.3% 12.3–24.2% 4.0–20.0% 1.7–20.6% 2–8%
Linalyl acetate 3.5–8.6% 51.0–71.0% 18.0–28.0% 17.1–40.4%
β-Pinene 3.5–5.3% 0.3–2.7% 1.4–2.1% 8.2–14.0% 6.0–17.0% 2.0–2.9% 21.1% 4.0–11.0% 4.4–11.0% 1.1%
γ-Terpinene 16.4–22.7% 8.4–10.7% 3.0–13.3% 9.5–10.7% 8.1% 3.0–12.0% 5.0–11.4% 12.5%
α-Pinene 0.6–1.0% 2.0–2.7% 1.1–2.1% 1.3–4.4% 1.2–2.1% 2.5% 1.0–1.8% 0.7–2.2% 0.2–1.6% 2.7%
β-Myrcene 1.3–3.3% 1.6–2.4% 1.4–2.1% 0–2.0% 1.5–1.8% 1.4–1.6% tr–2.2% 1.3–2.1% 1.3% 0.6–1.8% 1.4–3.6% 3.2%
α-Terpineol 3.9–5.8% 2.1–5.2% 0.1–8.0% 5.4–12.7%
(E)-β-Ocimene 4.6–5.8% 0.2–2.2%
Sabinene 0.2–1.0% 0.4–1.6% 0.8–1.7% 0.5–2.4% 3.1% 0.4–1.0%
Neral 0–1.3% 0.5–1.5% 0.4–2.0% 1.4%
Geranial 0–1.8% 0.7–2.2% 0.5–4.3% 2.4%
Bicyclogermacrene
2.0%
(E)-
β
-Farnesene
1.3%
Geranyl acetate 3.4–4.1% 1.9–3.4%
Terpinolene 0.7–1.0% 8.1–8.7%
(E)-Nerolidol 1.3–4.0%
Geraniol 2.8–3.6% 1.4–2.3%
Nerol 1.1–1.3% 0.4–1.1%
p-Cymene 0.1–1.4% tr–2.3% 1.6–2.5%
(E,E)-Farnesol 1.6–3.2%
(E,Z)-Farnesol
Neryl acetate 1.7–2.1% 0–2.6% 0.1–1.5% 0.1–1.2%
Terpinen-4-ol tr–1.9% 0.7–1.9%
(Z)-β-Ocimene 0.7–1.0%
α-Thujene 0.7–1.0%
1,4-Cineole 2.0–3.0%
Terpinen-1-ol 1.0–2.3%
(Z)-β-Terpineol 0.5–2.2%
α-Terpinene tr–2.1%
β-Bisabolene 1.6–1.8% 1.8%
α-Fenchol 0.6–1.4%
Borneol 0.5–1.4%
Camphene 0.5–1.3%
γ-Terpineol 0.8–1.6%
(E)-
α
-Bergamotene
1.1%
β
-Caryophyllene
1.0%
(2E,6E)-
α
-Farnesene
1.0%
β-Phellandrene 5.4%
Nootkatone 0.1–0.8%
D = Distilled; Ex = Expressed; FCF = Furanocoumarin-Free.

Int. J. Mol. Sci. 2018,19, 1966 4 of 25
Table 2. Non-volatile components of some expressed Citrus oils.
Non-Volatile Components Bitter Orange [8,26] Lemon [8] Lime [8,26] Grapefruit [8,26] Bergamot [8,24,25] Bergamot (FCF) [8,24] Mandarin [8]
Bergamottin - 0.16–0.54% 1.7–3.0% <0.11% 0.68–2.75% 0–1.625% 0–0.001%
Bergapten 0.035–0.073% 0.0001–0.035% 0.17–0.33% 0.012–0.19% 0.11–0.33% 0–0.0091% 0–0.0003%
Oxypeucedanin - 0.09–0.82% 0.02–0.3% - - - -
5-Geranloxy-7-methoxycoumarin
- 0.18–0.28% 1.7–3.2% - 0.08–0.68% 0–0.19% -
Citropten - 0.05–0.17% 0.4–2.2% - 0.01–0.35% 0–0.0052% -
Byakangelicol - 0.006–0.16% - - - - -
8-Geranyloxypsoralen - 0.01–0.045% 0.10–0.14% - - - -
Isopimpinellin - 0–0.011% 0.1–1.3% - - - -
5-Geranoxy-8-methoxypsoralen - - 0.2–0.9% - - - -
Epoxybergamottin 0.082% - - 0.1126% - - -
Psoralen 0.007% - - - 0–0.0026% - -
Bergaptol - - - - 0–0.19% - -
FCF = Furanocoumarin-Free.

Int. J. Mol. Sci. 2018,19, 1966 5 of 25
2. Biological Properties
A summary of the biological activities of different Citrus essential oils is presented in Table 3.
Table 3. Biological activities of different Citrus essential oils.
Essential Oil Biological Activity Ref.
Sweet orange
Anticarcinogenic [28,29]
Relaxant [30]
Anxiolytic [31–33]
Pain relief [34]
Hepatocarcinogenesis suppressant [35]
Anti-tumor [36]
Antioxidant [37]
Food preservative [38]
Acne treatment (with sweet basil oil) [39]
Antibacterial [40–43]
Antifungal [10,44,45]
Anti-aflatoxigenic (at 500 ppm) [44]
Larvicidal [46,47]
Insecticidal [48–50]
Anthelmintic [51]
Growth promoter (in Tilapia) [52]
Bitter orange
Mild sedative, hypnotic, soothing, calming, and motor relaxant [53]
Sleep inducer [54]
Anxiolytic and antidepressant [53,55–58]
Pain relief [34,59]
Antiseizure and anticonvulsant agent [54]
Anti-spasmodic and sexual desire enhancer [59]
Gastroprotective and ulcer healing [60]
Digestive disorders treatment [53]
Hepatocarcinogenesis suppressant [35]
Antioxidant [53,61]
Nephroprotective [62]
Antibacterial [53,63–65]
Pimple and acne treatment [53]
Antifungal [15,53,66]
Fumigant and anti-cholinesterase [67]
Larvicidal [46]
Neroli
Sedative, soothing, calming, and motor relaxant [55,68]
Anxiolytic and antidepressant [53,57,69,70]
Antiseizure and anticonvulsant [71,72]
Central and peripheral antinociceptive effects [73]
Anti-inflammatory [73]
Menopausal symptoms relief [74]
Premenstrual syndrome (PMS) relief [75]
Sexual desire enhancer [59]
Endothelium- and smooth muscle-dependent vasodilator [76]
Hypotensive [77]
Antioxidant [20,78]
Anti-amnesic [72]
Antibacterial [53,79]
Antifungal [20,53,75,79]
Orange petitgrain
Antioxidant [78,80]
Antibacterial [81]
Antifungal [81]

Int. J. Mol. Sci. 2018,19, 1966 6 of 25
Table 3. Cont.
Essential Oil Biological Activity Ref.
Mandarin
Anti-proliferative [82]
Chemoprotective [82]
Antioxidant [83]
Antibacterial [83,84]
Antifungal [84–87]
Lemon
Stress relief [88,89]
Cytotoxic [28,90]
Chemoprotective [91]
Anti-obesity [92]
Antioxidant [93]
Neuroprotective [94,95]
Anti-anxiety [96]
Creativity and mood enhancer [97]
Analgesic [98]
Relief of nausea and vomiting of pregnancy [99]
Anti-spasmodic [89]
Attention level, concentration, cognitive performance, mood,
and memory enhancer [89,100]
Skin penetration enhancer [101]
Antibacterial [102,103]
Antifungal [10]
Insect repellent [104]
Miticidal [105]
Lime
Anti-obesity [106]
Spasmolytic agent [107,108]
Selective acetylcholinesterase and buytrylcholinesterase inhibitor [109]
Antioxidant [109]
Anti-inflammatory [110]
Flavoring agent [111,112]
Antibacterial [111,113]
Antifungal [111,113]
Insecticidal [114]
Phytotoxic [113]
Grapefruit
Anti-obesity [92,115–117]
Cravings and hunger reducer (mixed with patchouli oil) [116]
Body cleansing promoter [116]
Cytotoxic [28,90]
Antibacterial [118,119]
Antifungal [118–120]
Larvicidal [121–124]
Bergamot
Melanogenic component in suntan preparations [125,126]
Pain relief [127–129]
Peripheral antinociceptive [129,130]
Antiallodynic [127,131]
Wound healing [132]
Cytotoxic [125,133–135]
Anti-tumor [136]
Neuroprotective [137,138]
Sedative, calming, and soothing [139]
Anxiolytic [139,140]
Mood enhancer [141]
Antioxidant [109]
Antibacterial [142–144]
Antifungal [142,143,145]
Anti-dermatophyte [146,147]
Antimycoplasmal [148]

Int. J. Mol. Sci. 2018,19, 1966 7 of 25
Table 3. Cont.
Essential Oil Biological Activity Ref.
Yuzu
Anti-carcinogenic [149]
Anti-inflammatory [150]
Anti-anxiety [151]
Mood disturbance, tension-anxiety, anger-hostility, and fatigue reducer [152,153]
Mind and body health promoter [152]
Odor suppressant [154]
Anti-cancer [155]
Hypocholesterolemic [156]
Anti-diabetic [157]
Anti-obesity [158]
Platelet aggregation inhibitor [159]
Heart failure treatment [160]
Kumquat
Antiproliferative [161]
Antioxidant [161,162]
Antibacterial [163]
Antifungal [163]
2.1. Sweet Orange (Citrus sinensis L.) Essential Oil
Sweet orange EO showed anticarcinogenic potential via inducing apoptosis in human leukemia
(HL-60) cells [
28
] and human colon cancer cells [
29
], and inhibiting angiogenesis and metastasis [
29
].
Olfactory stimulation using orange EO induced physiological and psychological relaxation. Inhalation
of orange EO for 90 s caused a significant decrease in oxyhemoglobin concentration in the right
prefrontal cortex of the brain which increases comfortable, relaxed, and natural feelings [
30
]. The odor
of sweet orange decreases the symptoms of anxiety and improves the mood [
31
]. The oil showed
strong anxiolytic activity in Wistar rats [
32
]. When female dental patients were exposed to sweet
orange odor diffused in the waiting room prior to a dental procedure, they showed lower levels of
state-anxiety compared to control patients who were exposed to air only [
33
]. Sweet orange EO in
combination with ginger and accompanied by a massage was effective in alleviating moderate to
severe knee pain among the elderly in Hong Kong [
34
]. Moreover, sweet orange EO suppressed
pre-neoplastic hepatic lesions during N-nitrosodiethylamine (DEN)-induced hepatocarcinogenesis
in rats by restoring the normal phenotype and upregulating junctional complexes [
35
]. Injections
of orange EO in mice 24 h after subcutaneous injections with dibenzo-[
α
]-pyrene (DBP) reduced
the tumor incidence to less than 50% after 30 weeks [
36
]. In addition, the oil was reported to have
a good radical-scavenging activity [
37
], mainly due to the high d-limonene content [
12
,
13
]. It is
used in combination with thyme oil to improve the quality traits of marinated chicken meat [
38
].
Moreover, formulations based on orange and sweet basil oils were effective in treating acne [
39
].
Improvement of the acne condition was observed with 43–75% clearance of lesions. It should be noted
that there were some side effects, such as burning and redness that disappeared within a few minutes of
completing the application [
39
]. Sweet orange EO was reported to inhibit the growth of several bacteria
including Staphylococcus aureus,Listeria monocytogenes,Vibrio parahaemolyticus,Salmonella typhimurium,
Escherichia coli, and Pseudomonas aeruginosa [
40
–
43
], as well as several fungal species, such as Aspergillus
flavus,A.fumigatus,A.niger,A.terreus,Alternaria alternata,Cladosporium herbarum,Curvularia lunata,
Fusarium oxysporum,Helminthosporium oryzae,Penicillium chrysogenum,P.verrucosum, and Trichoderma
viride [
10
,
44
,
45
]. It also showed a good anti-aflatoxigenic effects (inhibited aflatoxin B
1
) at 500 ppm [
44
].
In addition, it has an intense larvicidal activity against the malaria vector, Anopheles labranchiae [
46
],
and the vector of yellow and dengue fever, Aedes aegypti [
47
]. Sweet orange EO is a potent fumigant
against house flies, cockroaches, and mosquitoes [
48
,
49
]. It can be used for controlling subterranean
termites [
50
]. It is also an effective anthelmintic agent against gastrointestinal nematodes; five times
more effective on Haemonchus contortus eggs than tea tree EO [
51
]. Moreover, sweet orange EO acted

Int. J. Mol. Sci. 2018,19, 1966 8 of 25
as a growth promoter, increased immunity, and improved disease resistance to Streptococcus iniae
in Tilapia [52].
2.2. Bitter Orange (Citrus aurantium L.) Essential Oil
Bitter orange EO is used as a mild sedative and hypnotic for its soothing, calming, and motor
relaxant effects [
53
]. It also enhances sleeping time and is used to treat insomnia [
54
]
.
Bitter orange odor
decreases the symptoms of anxiety, improves mood, and creates a sense of well-being [
53
]. It showed
strong anxiolytic activity in rodents without any motor impairment, even after 15 consecutive days
of treatment [
55
]. It increased social interactions for rats (time spent in active social interaction),
and increased exploration time in the open arms of the elevated plus-maze (EPM) [
55
]. It was also
effective in treating the symptoms of anxiety in patients with chronic myeloid leukemia prior to the
collection of medullary material [
56
]. It exerted its antianxiety effects by regulating serotonin (5-HT)
receptors in rats [
57
] and its antidepressant effects through the monoaminergic system in mice [
58
].
Furthermore, bitter orange EO was effective in reducing the severity of first-stage labor pain and
anxiety in primiparous women [
59
], as well as in alleviating moderate and severe knee pain [
34
]. Bitter
orange EO is used as a natural antiseizure and anticonvulsant agent. It has been used in treating
epilepsy and seizures [
54
]. It has been reported to have anti-spasmodic effect and to enhance sexual
desire [
59
]. Due to the presence of limonene, bitter orange EO possesses its gastroprotective and ulcer
healing actions through increasing the gastric production of mucus, which is useful as a secondary
intervention in the treatment of chronic inflammatory diseases [
60
]. It is used as a treatment for
digestive disorders such as slow digestion, constipation, flatulence, gastric problems, etc. [
53
]. Bitter
orange EO suppressed preneoplastic hepatic lesions during DEN-induced hepatocarcinogenesis in
rats by restoring the normal phenotype and upregulating junctional complexes [
35
]. Bitter orange EO
showed good radical-scavenging activity [
53
], largely due to the high d-limonene content [
12
,
13
] and its
microencapsulated form, which was effective in reducing oxidative stress in acute otitis media rats [
61
].
Due to its free radical-scavenging properties, bitter orange extract showed nephroprotective effects
against gentamicin-induced renal damage [
62
]. The antibacterial activity of bitter orange EO was
manifested by inhibiting the growth of Listeria innocua,Salmonella enterica,Escherichia coli,Pseudomonas
fluorescens, and Aeromonas hydrophila [
53
,
63
,
64
]. It was also effective in controlling multi-species
biofilms [
65
]. Due to its antimicrobial effects, bitter orange EO is used for treating colds, dull skin, flu,
gums and mouth, and chronic bronchitis, as well as a food preservative [
53
]. The diluted oil is used to
treat pimples and acne [53]. In addition, bitter orange EO inhibits the growth of Penicillium digitatum,
and P.italicum [
15
,
53
]. The oil was mentioned as a topical treatment for skin fungal infections like
ringworm, jock itch, and athlete’s foot [
66
]. Furthermore, bitter orange EO showed potent fumigant
and anti-cholinesterase activities against the silverleaf whitefly, Bemisia tabaci [
67
]. It was also effective
against the larvae of the malaria vector, Anopheles labranchiae [46].
2.3. Neroli (Citrus aurantium L.) Essential Oil
Neroli EO is used as a sedative for its soothing, calming, and motor relaxant effects by healthcare
centers in Puerto Rico, Guatemala, Mexico, Italy, Martinique, and Spain [
55
]. Neroli EO is effective
for cardiac palpitations resulting from shock or fear [
68
]. Similar to the fruit peel oil, the odor of
neroli decreases the symptoms of anxiety, improves mood, and creates a sense of well-being [
53
].
It was proven to be effective in reducing preoperative anxiety before minor operations [
69
]. Neroli EO
reduced the mean anxiety scores in postmenopausal women [
70
]. It exerted its antianxiety effects by
regulating 5-HT receptors in rats [
57
]. Neroli EO is used as a natural antiseizure and anticonvulsant
agent [
71
]. It has been used in treating insomnia, epilepsy, and seizures [
72
]. Neroli EO has central
and peripheral antinociceptive effects which support the ethnomedicinal claims of its use in the
management of pain and inflammation [
73
]. Neroli EO possesses significant anti-inflammatory activity
against acute and chronic inflammation [
73
]. Neroli EO is effective in reducing stress and improving
the endocrine system. Inhalation of neroli EO helps in relieving menopausal symptoms, reducing

Int. J. Mol. Sci. 2018,19, 1966 9 of 25
blood pressure, and increasing sexual desire in postmenopausal women [
74
]. It also decreased
the overall symptoms of premenstrual syndrome (PMS) in university students. It showed positive
effects on the mood, blood pressure, pain, inflammation, bloating, and indigestion in addition to
its anti-depressant effects [
75
]. Inhaling neroli odor enhances sexual desire [
59
]. Neroli EO is an
endothelium- and smooth-muscle-dependent vasodilator that can alleviate cardiovascular symptoms.
The endothelial component is mediated by the nitric oxide to soluble guanylyl cyclase pathway,
while the smooth muscle component involves inhibiting extracellular Ca2+ influx and store-operated
Ca
2+
release mediated by the ryanodine receptor (RyR) signaling pathway [
76
]. Inhaling a mixture of
lavender, ylang-ylang, marjoram, and neroli (20:15:10:2) decreased systolic and diastolic blood pressure,
as well as
the concentration of salivary cortisol in prehypertensive and hypertensive subjects [
77
].
These positive
effects were immediate and continuous [
77
]. Furthermore, neroli EO is a strong
antioxidant. It showed a 100% singlet oxygen scavenging activity at all concentrations between
0.1 and 2% [
20
,
78
]. Interestingly, the C.aurantium flower extract showed anti-amnesic and repairing
effects on memory, learning impairments, and behavioral disorders induced by scopolamine, and
has the potential to treat Alzheimer’s disease [
72
]. Neroli EO inhibits the growth of several bacteria
including Bacillus subtilis,B.cereus,Staphylococcus aureus,S.epidermis,Enterococcus faecalis,Micrococcus
luteus,Listeria monocytogenes,Salmonella enteritidis,Escherichia coli,Pseudomonas aeruginosa, and Klebsiella
pneumonia [
53
,
79
], as well as several fungi including Aspergillus niger,A.flavus,A.nidulans,A.fumigatus,
Fusarium graminearum,F.oxysporum,F. culmorum, and Alternaria alternata [20,53,75,79].
2.4. Orange Petitgrain (Citrus aurantium L.) Essential Oil
Orange petitgrain EO showed a remarkable radical-scavenging activity, higher than the flower
oil (neroli) and fruit peel oil (bitter orange) from the same plant [78,80]. The potent antioxidant effect
could be attributed to the high d-limonene content [
12
,
13
]. It also inhibited the growth of Bacillus
subtilis,Staphylococcus aureus,Escherichia coli,Saccharomyces cerevisiae,Mucor ramannianus, and Fusarium
culmorum [81].
2.5. Mandarin (Citrus reticulata Blanco) Essential Oil
Citrus reticulata EO showed an anti-proliferative effect against human embryonic lung fibroblasts
(HELFs) and showed protective effects against bleomycin (BLM)-induced pulmonary fibrosis in rats.
The mechanism is thought to be through adjusting the unbalance of oxidation and antioxidation,
down-regulating the expressions of connective tissue growth factor (CTGF) and mRNA in lung
tissues, and reducing collagen deposition and fibrosis [
82
]. C. reticulata EO showed a moderate
radical scavenging activity [
83
] mainly due to the high d-limonene content [
12
]. Mandarin oil is
well known for its broad spectrum antibacterial and antifungal actions. It inhibits the growth of
several bacteria including Escherichia coli,Bacillus subtilis,Pseudomonas aeruginosa, and Staphylococcus
aureus [
83
,
84
], as well as several fungi including Penicillium italicum,P.digitatum,P.chrysogenum,
Aspergillus niger,A. flavus,Alternaria alternata,Rhizoctonia solani,Curvularia lunata,Fusarium oxysporum,
and Helminthosporium oryzae [84–87].
2.6. Lemon (Citrus limon Osbeck) Essential Oil
Lemon EO is a natural stress reliever. Inhaling lemon EO causes anti-stress effects through
modulating the 5-HT and dopamine (DA) activities in mice [
88
,
89
]. Lemon EO showed cytotoxic
effects against human prostate, lung, and breast cancer cells [
90
]. It also induced apoptosis in HL-60
cells due to the presence of citral, decanal, and octanal [
28
]. Oral administration of lemon EO
inhibited 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced neoplasia of the lungs
and forestomach of female mice [
91
]. Lemon EO causes activation of the sympathetic nerve activity
innervating the white adipose tissue (WAT), which increases lipolysis and results in the suppression
of body weight gain [
92
]. Lemon EO significantly reduces lipid peroxidation levels and nitrile
content, but increases reduced glutathione (GSH) levels, as well as superoxide dismutase, catalase,

Int. J. Mol. Sci. 2018,19, 1966 10 of 25
and glutathione peroxidase activities in mouse hippocampus [
93
]. The neuroprotective effect of lemon
EO is attributed to its remarkable radical-scavenging activity [
94
,
95
]. Prolonged exposure (for 2 weeks)
to lemon EO induces significant changes in neuronal circuits involved in anxiety and pain in rats [
96
].
Lemon EO improves creativity and mood, and is thought to affect heart rhythm [
97
]. The analgesic
effect of lemon EO is induced by dopamine-related activation of anterior cingulate cortex (ACC) and
the descending pain inhibitory system [
98
]. Inhalation of lemon EO reduces the intensity of nausea
and vomiting of pregnancy (NVP) by 33% [
99
]. It also showed anti-spasmodic activity [
89
]. Lemon
EO significantly enhanced attention level, concentration, cognitive performance, mood, and memory
of students during the learning process [
100
]. Rats exposed to lemon EO were able to find a target
point faster than a control group [
89
]. Lemon EO is a safe and effective penetration enhancer for
topical administration of lipid- and water-soluble vitamins which are critical issues for the protection of
anti-ageing formulations. It significantly enhances the trans-epidermal release of
α
-tocopherol (vitamin
E), retinyl acetate (vitamin A), pyridoxine (vitamin B
6
), and ascorbic acid (vitamin C) from topical
emulsions in reconstructed human epidermis [
101
]. In addition, lemon EO is a potent antibacterial
against Bacillus cereus,Mycobacterium smegmatis,Listeria monocytogenes,Lactobacillus curvatus,L.sakei,
Micrococcus luteus,Escherichia coli,Klebsiella pneumoniae,Pseudococcus aeruginosa,Proteus vulgaris,
Enterobacter gergoviae,E.ammnigenus,Staphylococcus aureus,S.carnosus,
and S.xylosus
[
102
,
103
],
and a strong antifungal against Aspergillus niger,A.flavus,Penicillium verrucosum,P.chrysogenum,
Kluyveromyces fragilis,Rhodotorula rubra,Candida albicans,Hanseniaspora guilliermondii, and Debaryomyces
hansenii [
10
]. Lemon EO has insect repellent effects against the malaria vector, Anopheles stephensi [
104
].
It also showed remarkable miticidal activity against Sarcoptes scabiei var. cuniculi, both
in vitro
and
in vivo
. When lemon EO was tested at 20% and applied topically on the infected parts of rabbits once
a week for four successive weeks, the infected rabbits completely recovered after the second week of
treatment [105].
2.7. Key Lime (Citrus aurantifolia) Essential Oil
Lime EO has been used to relieve common cold, flu, asthma, arthritis, and bronchitis [
111
,
164
].
It could be useful in weight loss and the treatment of drug-induced obesity and related diseases.
It displayed a reduction in body weight and food consumption in ketotifen-induced obese mice [
106
].
It has been reported as a potent spasmolytic agent [
107
,
108
]. Lime EO could also be useful in
treating Alzheimer’s disease since it is a strong selective acetylcholinesterase and buytrylcholinesterase
inhibitor [
109
]. It has a remarkable radical-scavenging activity (IC
50
= 19.6
µ
g/mL) [
109
] due to
the high d-limonene content [
12
,
13
]. Lime EO exhibited anti-inflammatory effects by reducing cell
migration, cytokine production, and protein extravasation induced by carrageenan [
110
]. Lime EO is
used as a flavoring agent in syrups and suspensions [
111
,
112
]. In addition, it is a potent antibacterial
against Escherichia coli,Listeria monocytogenes,Bacillus subtilis,Enterococcus durans,E.hirae,Staphylococcus
epidermidis,S.aureus,Enterobacter cloacae,Pseudomonas aeruginosa,Serratia marcescens,Shigella flexnerii,
Streptococcus faecalis,Citrobacter spp., Klebsiella pneumoniae,and Salmonella typhi [
111
,
113
]. It also inhibits
the growth of many fungi including Colletotrichum gloeosporioides,Rhizopus stolonifer,Aspergillus niger,
A.parasiticus,Rhizoctonia solani,Candida albicans, and C.parapsilosis [
111
,
113
]. Lime EO has insecticidal
activity (contact, fumigation, and feeding deterrent activities) against the maize weevil, Sitophilus
zeamais [
114
]. It showed phytotoxic activities against Avena fatua L., Echinochloa crus-galli (L.) Beauv,
Allium cepa L., and Phalaris minor Retz [113].
2.8. Grapefruit (Citrus ×paradisi Macfady) Essential Oil
Because of its anti-obesity effects, grapefruit EO is called the “dieter’s friend” [
116
]. The fragrance
of grapefruit EO causes activation of the sympathetic nerve activity innervating the WAT,
which facilitates lipolysis, then results in a suppression of body weight gain [
92
,
115
]. It efficiently
inhibits adipogenesis via inhibiting the accumulation of triglycerides [
117
]. When mixed with patchouli
oil, grapefruit EO is known to lower cravings and hunger, which makes it a great tool to lose weight

Int. J. Mol. Sci. 2018,19, 1966 11 of 25
in a healthy way [
116
]. The bright, refreshing scent of grapefruit EO energizes and uplifts the senses.
Grapefruit EO promotes body cleansing and removal of toxins and excess fluids [
116
]. Grapefruit EO
was cytotoxic against human prostate and lung cancer cells [
90
]. It also induced apoptosis in HL-60
cells due to the presence of citral, decanal, and octanal [
28
]. Moreover, it showed a strong antibacterial
activity against Bacillus cereus,Enterococus faecalis,Escherichia coli,Klebsiella pneumoniae,Pseudococcus sp.,
Salmonella thyphimurium,Shigella flexneri, and Staphylococcus aureus [
118
,
119
], and a strong antifungal
activity against Aspergillus niger,Candida albicans,Cladosporium cucumerinum,Penicillium digitatum,
P.italicum, and P.chrysogenum [
118
–
120
]. Grapefruit EO was 95% lethal to eggs and larvae of Anastrepha
fraterculus and Ceratitis capitata [
121
]. It completely inhibited the viability of Aedes aegypti eggs exposed
at 400 ppm, and inhibits its larval development at 100 ppm [
122
]. Also, grapefruit EO is a potent
larvicide against Anopheles stephensi at 80 ppm [
123
]. It caused an 89.6% decrease of Eimeria-induced
coccidiosis contamination with 5 mg/kg for 30 days [124].
2.9. Bergamot (Citrus bergamia Risso & Poit) Essential Oil
Bergamot EO is widely used in the perfumery, pharmaceutical, cosmetic, and food industries [
125
].
It is used in suntan preparations due to the presence of bergapten, which is the active melanogenic
component [
126
]. Bergamot EO is used in complementary medicine to treat chronic nociceptive and
neuropathic pain via modulating sensitive perception of pain [
127
–
129
]. Intraplantar injection of
bergamot EO, linalool, and linalyl acetate showed a peripheral antinociception effect in the capsaicin
test mediated by a peripheral opioid mechanism [
129
,
130
]. A combination of a low dose of morphine
with inactive doses of bergamot oil or linalool was sufficient to induce antiallodynic effects in mice
via inhibiting spinal extracellular signal-regulated protein kinase (ERK) phosphorylation [
127
,
131
].
The oil is used to facilitate wound healing [
132
]. Bergamot EO was reported to be cytotoxic against
SH-SY5Y human neuroblastoma cells, suppressing their growth rate through a mechanism related to
both apoptotic and necrotic cell death [
133
,
134
]. Bergamottin and 5-geranyloxy-7-methoxycoumarin
were identified as the bioactive molecules responsible for the cytotoxic effect of bergamot EO [
133
].
Bergamot EO inhibited tumor formation by the carcinogen NDMA
in vitro
by more than 70% [
136
].
Bergamot oil and its d-Limonene were reported to modulate autophagic pathways in SH-SY5Y
cells [
125
]. Liposomal bergamot oil showed improved anticancer activity against SH-SY5Y cells
because of its higher stability and higher bioavailability [
135
]. In addition, it has been shown to
reduce neuronal damage caused
in vitro
by excitotoxic stimuli by preventing an injury-induced
decrease of phosphorylated protein kinase B (phospho-Akt) and phosphorylated glycogen synthase
kinase 3
β
(phospho-GSK-3
β
) levels [
137
,
138
]. Bergamot EO is used as a mild sedative that acts by
calming and soothing the nervous system [
139
]. In rodent experiments, the pleasant, refreshing
odor of bergamot decreased the symptoms of stress-induced anxiety and minimized behavior-related
depressive disorders in chronic stressed rats [
139
]. Inhalation of bergamot EO was reported to increase
the release of amino acid neurotransmitters (glutamate, gamma-aminobutyric acid (GABA), aspartate,
glycine, and taurine) in rat hippocampuses, both
in vivo
and
in vitro
, which suggested that the oil may
interfere with exocytosis [
165
]. Similar to diazepam, bergamot oil exerted anxiolytic-like behaviors
and attenuated hypothalamic-pituitary-adrenal (HPA) axis activity via reducing the corticosterone
response to acute stress caused by EPM [
140
]. A pilot study performed in the waiting room of a
mental health treatment center (Utah, USA) revealed that inhalation of bergamot EO for 15 minutes
improves positive feelings [
141
]. Furthermore, bergamot EO showed a good radical scavenging
activity evaluated by
β
-carotene bleaching test (IC
50
= 42.6
µ
g/mL) [
109
] due to the high d-limonene
content [
12
,
13
]. Bergamot EO inhibits the growth of several bacteria including Escherichia coli,
Staphylococcus aureus,Bacillus cereus,Salmonella enterica,S.typhimurium,Pseudomonas putida,Arcobacter
butzleri,Enterococcus faecium,E.faecalis, and Listeria monocytogenes [
142
–
144
]. Several studies showed a
broad spectrum antifungal activity of bergamot EO against Hanseniaspora guilliermondii,Debaryomyces
hansenii,Kluyveromyces fragilis,Rhodotorula rubra,Candida albicans,Aspergillus niger,A.flavus,Penicillium
italicum,Fusarium solani,F.sporotrichioides,F.oxysporum,Curvularia lunata,Verticillium dahliae,Phomopsis

Int. J. Mol. Sci. 2018,19, 1966 12 of 25
sp., Phoma sp., and Myrothechium verrucaria [
142
,
143
,
145
]. It was also reported to have antifungal effects
against dermatophytes of the genera Trichophyton,Microsporum, and Epidermophyton [
146
]. It could be
used in the treatment of dermatophytosis in animals [
147
]. The mechanism underlying its antimicrobial
and antifungal effect is thought to be via increasing reactive oxygen species (ROS) production,
relevant to its action in human polymorphonuclear leukocytes [
132
]. Bergamot EO also showed
strong antimycoplasmal activity against Mycoplasma hominis,M.fermentans, and M.pneumoniae [148].
2.10. Yuzu or Yuja (Citrus junos Sieb. ex Tanaka) Essential Oil
Yuzu EO inhibited the formation of the carcinogen N-nitrosodimethylamine (NDMA) in
vegetables (by 22–59%) and saliva (by 24–62%) [
149
]. Yuzu EO is useful in treating bronchial asthma
due to its anti-inflammatory activities. It inhibits the production of cytokines and ROS, and reduces
eosinophil migration [
150
]. Yuzu odor was reported to decrease maternal anxiety for a sick child
receiving an infusion at a pediatric clinic [
151
]. A 10 min inhalation of the yuzu odor significantly
decreased the heart rate and increased the high frequency power of heart rate variability reflecting
parasympathetic nervous system activity, regardless of menstrual phase. Inhalation of the yuzu oil
decreased total mood disturbance, tension-anxiety, anger-hostility, and fatigue, which are common
premenstrual symptoms [
152
,
153
]. Yuzu odor promotes mind and body health in Japan [
152
]. It is also
used to suppress the odor of Niboshi soup stock [
154
]. Yuzu peel ethanol extract is useful in preventing
colitis and colorectal cancer through reducing cyclooxygenase-2 (COX-2) expression [
155
]. This extract
also showed hypocholesterolemic effect both
in vitro
and
in vivo
by reducing the weight gain, lipid
accumulation, liver fat content, liver weight, total cholesterol, and low-density lipoprotein (LDL)
cholesterol [
156
]. Yuzu extract was reported to exert anti-diabetic activity through increasing glucose
uptake in C
2
C1
2
myotubes by modulating the AMP-activated protein kinase (AMPK) and peroxisome
proliferator-activated receptor gamma (PPAR-
γ
) signaling pathways. It improved insulin resistance
(IR) in mice that were fed a high-fat diet [
157
]. Moreover, yuzu peel extract showed anti-obesity
effects in a zebrafish model via activating hepatic PPAR-
α
and adipocyte PPAR-
γ
pathways [
158
].
The methanol extract of yuzu could be beneficial for individuals at high risk of cardiovascular disease
because it inhibits platelet aggregation [
159
]. Yuzu extract could be useful in treating heart failure as it
prevents myocardial infarction (MI)-induced ventricular dysfunction and structural remodeling of
myocardium [160].
2.11. Kumquat (Citrus japonica Thunb) Essential Oil
Kumquat EO showed antiproliferative action against human prostate cancer (LNCaP) cells
via inducing apoptosis and inhibition of inflammation [
161
]. The oil also showed a considerable
radical-scavenging activity evaluated by a 2,2-diphenyl-1-picrylhydrazyl (DPPH) test [
161
,
162
] due
to the high d-limonene content [
12
,
13
]. Kumquat EO exhibits potent antibacterial effects against
Escherichia coli,Staphylococcus aureus,Bacillus cereus,Bacillus subtilis,Bacillus laterosporus,Salmonella
typhimurium, and Lactobacillus bulgaricus, as well as antifungal effects against Candida albicans [163].
3. Safety of Citrus Oils
Generally speaking, Citrus EOs are non-toxic, non-mutagenic, and non-carcinogenic [
8
]. They are
not hazardous in pregnancy and do not alter the maternal reproductive outcome [
8
,
166
]. Sweet orange,
bitter orange, neroli, petitgrain, lemon, lime (both distilled and expressed), bergamot, and grapefruit
oils have GRAS status [
8
]. However, there is a possible skin sensitization issue if old or oxidized oil
is used. The distilled oils are not phototoxic, while the expressed oils carry a low to moderate risk
of phototoxicity (Table 4) [
167
] due to the presence of furanocoumarins [
168
]. In case of applying
expressed EOs to the skin in a dose higher than the maximum dermal use level, it is recommended
to avoid exposure to sunlight for at least 12 h [
8
]. Neroli and yuzu oils are neither irritating nor
sensitizing [
167
]. Expressed sweet orange oil was neither irritating nor sensitizing to 25 volunteers
when tested at 8 and 100% [
167
], whereas it caused sensitivity to 0.13% of total dermatitis patients

Int. J. Mol. Sci. 2018,19, 1966 13 of 25
when tested at 2% [
169
]. Bitter orange EO was neither irritating nor sensitizing to 25 volunteers when
tested at 10% [
167
], while it caused sensitivity to 1.5% of total dermatitis patients when tested at
2% [
169
]. Lemon oil was neither irritating nor sensitizing to volunteers when tested at 10% [
167
],
and similar results were observed for distilled lime oil when tested at 15 and 100% [
167
]. No irritation
or sensitization data were found for the expressed lime oil. The high citral content of lime EO causes
potential toxic and myelotoxic effects [
110
]. Grapefruit oil was neither irritating nor sensitizing to
volunteers when tested at 10 and 100% [
167
]. Mandarin EO was neither irritating nor sensitizing
to 25 volunteers when tested at 5 and 8% [
167
]. The expressed bergamot oil was neither irritating
nor sensitizing to 25 volunteers when tested at 10% [
167
]. It caused no irritation when tested at
2% on 1200 dermatitis patients, with only two (0.17%) patients showing sensitivity reaction [
170
],
whereas when tested at 10% in 590 eczema patients, 0.5% of the patients had reactions [
171
]. Expressed
bergamot oil caused severe phototoxic effects in hairless mice and pigs using simulated sunlight,
and in humans using natural sunlight and may be photocarcinogenic [
167
]. When applied to mice,
then irradiated with UV light, bergamot oil showed a carcinogenic action due to the presence of
bergapten [
172
]. Chronic skin pigmentation (also known as berloque dermatitis, bergapten dermatitis,
or photophytodermatitis) can also develop. Increased exposure to UV light can lead to serious burns [
8
].
In the absence of UV light, bergamot oil is not carcinogenic and even low concentration sunscreens
can completely inhibit bergapten-enhanced phototumorigenesis [
172
]. No hazards found for the
furanocoumarin-free (FCF) or rectified bergamot oil. The rectified oil was not sensitizing when tested
at 30% on 25 volunteers [173].
Table 4.
Phototoxicity risk, irritation of the undiluted oil, acute dermal LD
50
in rabbits, acute oral LD
50
in rats, and maximum dermal use level for different essential oils from Citrus species.
Acute Toxicity Phototoxicity
Risk [167]Irritation of Undiluted Oil [8]
Acute Dermal
LD50 in Rabbits
(g/kg) [167]
Acute Oral
LD50 in Rats
(g/kg) [167]
Maximum
Dermal Use
Level [8]
Sweet orange
EO Low risk Moderately irritating to rabbits
but not irritating to mice >5 >5 -
Bitter orange
EO low risk Moderately irritating to rabbits >10 >5 1.25%
Neroli EO Not phototoxic Not irritating >5 4.55 -
Petitgrain EO Not phototoxic Slightly irritating to rabbits, but
not irritating to mice or pigs <2 >5 -
Lemon EO
(distilled) Not phototoxic Moderately irritating to rabbits
and slightly irritating to mice >5 >5 20%
Lemon EO
(expressed) Low risk Not irritating >5 >5 2%
Lime EO
(distilled) Not phototoxic Slightly irritating to rabbits >5 >5 -
Lime EO
(expressed) moderate risk No data available >5 >5 0.7%
Grapefruit EO Low risk Slightly irritating to rabbits, but
not irritating to mice or pigs >5 >5 4%
Bergamot EO
(FCF) Not phototoxic Mildly irritating to rabbits >20 >10 0.4%
Bergamot EO
(expressed) Moderate risk Moderately irritating to rabbits - - -
Yuzu EO Not phototoxic Not irritating - - -
Mandarin Not phototoxic
Moderately irritating (produces
slight edema and erythema) to
rabbits, mice, and pigs
>5 >5 30%
To avoid oxidation of d-limonene, Citrus oils should be stored in a dark air-tight container and
placed at 4
◦
C [
8
]. The use of old or oxidized oils should be avoided. To avoid any possible adverse

Int. J. Mol. Sci. 2018,19, 1966 14 of 25
skin reactions, it is recommended to dilute Citrus oils with a carrier oil before topical use [
174
]. Also,
adding an antioxidant to preparations containing Citrus oils is recommended [8].
4. Bioactivity and Safety of Individual Key Components
4.1. d-Limonene
d-Limonene has been shown to possess antioxidant, anti-inflammatory [
12
],
and anticarcinogenic
[
8
]
effects. It is not acutely toxic, nephrotoxic, or carcinogenic, but the oxidized d-limonene may carry
some toxicity. Unoxidized d-limonene is listed as an allergen by the EU, and moderately allergenic
in Germany [
8
]. Unoxidized d-limonene was allergenic in 0.2% of dermatitis patients when tested at
2–3% [
8
]. No positive skin reactions were observed when testing the 98% pure d-limonene at 20% in
dermatitis patients [
175
]. Undiluted d-limonene was moderately irritating to rabbits [
167
]. d-Limonene
was irritating at concentrations of 70–80%, a weak irritant at 50%, and a non-irritant at concentrations
of 20–30%. The acute dermal LD
50
of d-limonene was >5 g/kg in rabbits, while the acute oral LD
50
was >5 g/kg in rats [167].
4.2. γ-Terpinene
γ
-Terpinene is an antioxidant [
176
]. It is neither irritating nor sensitizing [
167
]. It possesses
minimal toxicity. Depending on concentration, it may be mutagenic or non-mutagenic [
8
]. The acute
dermal LD
50
of
γ
-terpinene was >5 g/kg in rabbits, while the acute oral LD
50
was 3.65 g/kg in
rats [167].
4.3. Linalool
Linalool is a sedative, an antidepressant, and an anticancer, antifungal, and pesticidal
EO [177–180]
. It is neither toxic nor irritable to skin. It presents an extremely low risk of skin
sensitization [
8
]. No positive skin reactions were observed when testing the 97% pure linalool at 20%,
or to oxidized linalool tested at 1% in dermatitis and eczema patients [
175
,
181
]. Linalool does not cause
photo-irritation or photo-allergy because it does not absorb UV light in the range of 290–400 nm [
182
].
No fetal toxicity was observed [
8
]. No carcinogenic, mutagenic, or genotoxic activities were found [
8
].
The acute dermal LD
50
was 5.61 g/kg in rabbits, while the acute oral LD
50
was 2.79 g/kg in rats [
183
]
and 2.2–3.92 g/kg in mice [184]. High doses of linalool cause ataxia and narcosis [185].
4.4. Linalyl Acetate
Linalyl acetate has narcotic effects [
177
]. It is non-toxic, and is very minimally skin reactive [
8
].
When tested at 5–20%, no skin reaction was observed [
186
]. Similar to linalool, linalyl acetate
does not cause photo-irritation or photo-allergy because it does not absorb UV light in the range
of 290–400 nm [
182
]. It has no carcinogenic activity [
8
]. The acute dermal LD
50
was higher than 5 g/kg
in rabbits, while the acute oral LD50 was 14.5 g/kg in rats and 13.5 g/kg in mice [184].
4.5. α-Terpineol
α
-Terpineol has anticarcinogenic activity [
187
]. It is a non-irritant at 1–15%,
and non-phototoxic
[
188
].
It is not mutagenic or genotoxic. The acute dermal LD
50
of the mixed isomer terpineol was >3 g/kg in
rabbits, while the acute oral LD50 was 4.3 g/kg in rats [167].
4.6. Geranyl Acetate
Geranyl acetate has anti-inflammatory [
189
], antifungal [
189
], and antimicrobial properties [
190
].
It is a very weak skin sensitizer [
167
]. It is neither toxic nor carcinogenic [
8
]. It was not mutagenic
in the Ames test [
191
], and had no genotoxic effect [
192
]. The acute oral LD
50
of geranyl acetate is
6.33 g/kg in rats [183].

Int. J. Mol. Sci. 2018,19, 1966 15 of 25
4.7. Terpinolene
Terpinolene is an antioxidant [
193
]. It is neither irritating nor sensitizing at 20% [
167
]. Limited
data suggests minimal toxicity. The acute oral LD
50
was 4.4 mL/kg in rats and mice [
167
]. Thresholds
of terpinolene skin sensitization are not known.
4.8. β-Pinene
β
-Pinene showed antiproliferative and cytotoxic effects [
19
,
194
]. It is not mutagenic or
genotoxic [
8
]. It is generally a non-irritant and non-sensitizing. Undiluted
β
-pinene was moderately
irritating to rabbits [
8
].
β
-pinene was irritating at concentrations of 70–80%, a weak irritant at 50%,
and a non-irritant at concentrations of 25–30% to dermatitis patients [
195
].
β
-Pinene was classified as
a category B substance in Germany, meaning it is considered moderately allergenic [
196
]. The acute
dermal LD
50
of
β
-pinene was >5 g/kg in rabbits, subcutaneous LD
50
was 1.42 g/kg in mice, and the
acute oral LD50 was >5 g/kg in rats [167].
5. Conclusions
Citrus essential oils are well known for their flavor and fragrance properties, as well as numerous
aromatherapeutic and medicinal applications. With the exception of some phototoxicity of expressed
oils, they are generally safe to use with negligible toxicity to humans. These readily available essential
oils will undoubtedly continue to play important roles in the food and beverage industries, as well as
for medicinal, cosmetic, and “green” pest-control uses.
Author Contributions: Writing-Original Draft Preparation, N.S.D.; Writing-Review & Editing, N.S.D. & W.N.S.
Funding:
This work was carried out as part of the activities of the Aromatic Plant Research Center. (APRC, https:
//aromaticplant.org/). The authors are grateful to d
¯
oTERRA International (https://www.doterra.com/US/en)
for financial support of the APRC.
Conflicts of Interest:
The authors declare no conflict of interest. d
¯
oTERRA International had no role in the writing
or the decision to publish this manuscript.
Abbreviations
5-HT serotonin
ACC anterior cingulate cortex
AMPK AMP-activated protein kinase
BLM bleomycin
COX-2 cyclooxygenase-2
CTGF connective tissue growth factor
DA dopamine
DBP Dibenzo-[α]-pyrene
DENA N-nitrosodiethylamine
DPPH 2,2-diphenyl-1-picrylhydrazyl
EO essential oil
EPM elevated plus-maze
ERK extracellular signal-regulated protein kinase
FCF furanocoumarin-free
GABA gamma-aminobutyric acid
GRAS generally recognized as safe
GSH glutathione
HELFs human embryonic lung fibroblasts
HL-60 human leukemia cells
HPA hypothalamic-pituitary-adrenal
IC50 median inhibitory concentration

Int. J. Mol. Sci. 2018,19, 1966 16 of 25
IR insulin resistance
LD50 median lethal dose
LDL low-density lipoprotein
LNCaP human prostate acedocarcinoma cells
MI myocardial infarction
NDMA N-nitrosodimethylamine
NNK 4-(methylnitrosoamine)-1-(3-pyridyl)-1-butanone
NVP nausea and vomiting of pregnancy
phospho-Akt phosphorylated protein kinase B
phospho-GSK-3βphosphorylated glycogen synthase kinase 3 beta
PMS premenstrual syndrome
PPAR-γperoxisome proliferator-activated receptor gamma
ppm parts per million
ROS reactive oxygen species
RyR ryanodine receptor
SH-SY5Y human neuroblasoma cells
WAT white adipose tissue
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