Nerolidol: A Sesquiterpene Alcohol with Multi-Faceted Pharmacological and Biological Activities

Abstract

Nerolidol (3,7,11-trimethyl-1,6,10-dodecatrien-3-ol) is a naturally occurring sesquiterpene alcohol that is present in various plants with a floral odor. It is synthesized as an intermediate in the production of (3E)-4,8-dimethy-1,3,7-nonatriene (DMNT), a herbivore-induced volatile that protects plants from herbivore damage. Chemically, nerolidol exists in two geometric isomers, a trans and a cis form. The usage of nerolidol is widespread across different industries. It has been widely used in cosmetics (e.g., shampoos and perfumes) and in non-cosmetic products (e.g., detergents and cleansers). In fact, U.S. Food and Drug Administration (FDA) has also permitted the use of nerolidol as a food flavoring agent. The fact that nerolidol is a common ingredient in many products has attracted researchers to explore more medicinal properties of nerolidol that may exert beneficial effect on human health. Therefore, the aim of this review is to compile and consolidate the data on the various pharmacological and biological activities displayed by nerolidol. Furthermore, this review also includes pharmacokinetic and toxicological studies of nerolidol. In summary, the various pharmacological and biological activities demonstrated in this review highlight the prospects of nerolidol as a promising chemical or drug candidate in the field of agriculture and medicine.

Full text

molecules
Review
Nerolidol: A Sesquiterpene Alcohol with
Multi-Faceted Pharmacological and
Biological Activities
Weng-Keong Chan 1,2, Loh Teng-Hern Tan 1,2, Kok-Gan Chan 3, Learn-Han Lee 1,2,4,*
and Bey-Hing Goh 1,2,4,*
1School of Pharmacy, Monash University Malaysia, 47500 Bandar Sunway, Selangor Darul Ehsan, Malaysia;
wkcha29@student.monash.edu (W.-K.C.); lttan13@student.monash.edu (L.T.-H.T.)
2Biomedical Research Laboratory, Jeffrey Cheah School of Medicine and Health Sciences,
Monash University Malaysia, 47500 Bandar Sunway, Selangor Darul Ehsan, Malaysia
3Division of Genetics and Molecular Biology, Institute of Biological Sciences, Faculty of Science,
University of Malaya, 50603 Kuala Lumpur, Malaysia; kokgan@um.edu.my
4Center of Health Outcomes Research and Therapeutic Safety (Cohorts), School of Pharmaceutical Sciences,
University of Phayao, 56000 Phayao, Thailand
*Correspondence: lee.learn.han@monash.edu or leelearnhan@yahoo.com (L.-H.L.);
goh.bey.hing@monash.edu (B.-H.G.); Tel.: +60-3-5514-5887 or +60-3-5514-4887 (L.-H.L. & B.-H.G.);
Fax: +60-3-5514-6364 (L.-H.L. & B.-H.G.)
Academic Editor: Luca Forti
Received: 24 February 2016; Accepted: 14 April 2016; Published: 28 April 2016
Abstract: Nerolidol (3,7,11-trimethyl-1,6,10-dodecatrien-3-ol) is a naturally occurring sesquiterpene
alcohol that is present in various plants with a floral odor. It is synthesized as an intermediate in the
production of (3E)-4,8-dimethy-1,3,7-nonatriene (DMNT), a herbivore-induced volatile that protects
plants from herbivore damage. Chemically, nerolidol exists in two geometric isomers, a trans and
acis form. The usage of nerolidol is widespread across different industries. It has been widely
used in cosmetics (e.g., shampoos and perfumes) and in non-cosmetic products (e.g., detergents
and cleansers). In fact, U.S. Food and Drug Administration (FDA) has also permitted the use of
nerolidol as a food flavoring agent. The fact that nerolidol is a common ingredient in many products
has attracted researchers to explore more medicinal properties of nerolidol that may exert beneficial
effect on human health. Therefore, the aim of this review is to compile and consolidate the data
on the various pharmacological and biological activities displayed by nerolidol. Furthermore, this
review also includes pharmacokinetic and toxicological studies of nerolidol. In summary, the various
pharmacological and biological activities demonstrated in this review highlight the prospects of
nerolidol as a promising chemical or drug candidate in the field of agriculture and medicine.
Keywords: cis-nerolidol; trans-nerolidol; sesquiterpene; essential oil; pharmacological activities
1. Introduction
Ever since ancient times, medicinal plants have been explored and used as herbal medicines
to treat many diseases [
1
]. With the advancement of technology, research on herbal medicine has
intensified on the efforts to identify the bioactive compounds in medicinal plants that are responsible
for their pharmacological and biological activities. Essential oils (EOs) are volatile, natural and complex
bioactive compounds which are characterized by a strong odour, and their biological effects are known
to be associated to a series of complex interactions with cells, tissues and whole organisms [
2
]. Besides
its well-known application in aromatherapy [
3
], the uses of EO have been extended into the food,
agriculture and pharmaceutical industries [
4
–
6
]. Among the plants that are rich in EOs are Baccharis
Molecules 2016,21, 529; doi:10.3390/molecules21050529 www.mdpi.com/journal/molecules

Molecules 2016,21, 529 2 of 40
dracunculifolia DC, Elettaria cardamomum (L.) Maton, Momordica charantia L., Piper aleyreanum C. DC and
Piper claussenianum (Miq.) C. DC [7–11].
Nerolidol (3,7,11-trimethyl-1,6,10-dodecatrien-3-ol), also known as peruviol, is a naturally
occurring sesquiterpene alcohol present in the EO of various plants with a floral odour [
12
,
13
].
Nerolidol was found to exist as one of the bioactive compounds responsible for the biological activities
demonstrated by the EOs of the aforementioned plants.
Statistics showed that the global usage of nerolidol per annum ranges from 10 to 100 metric
tonnes [
14
]. For instance, nerolidol is frequently incorporated in cosmetics (e.g., shampoos and
perfumes) and non-cosmetic products (e.g., detergents and cleansers) [
13
]. Besides, nerolidol is also
widely used in the food industry as a flavor enhancer in many food products since its approval by U.S.
Food and Drug Administration as a safe food flavoring agent.
Principally, this article aims to review the diverse range of pharmacological and biological
activities of nerolidol which include antioxidant, anti-microbial, anti-biofilm, anti-parasitic, insecticidal,
anti-ulcer, skin penetration enhancer, anti-tumor, anti-nociceptive and anti-inflammatory properties.
The review also covers the chemical structure, physical properties, and the biosynthesis pathway of
nerolidol as the intermediate involved in the mechanisms responsible for protection of plants against
herbivores and plant pathogens. This article also highlights the pharmacokinetic and toxicological
properties of nerolidol in both in vitro and in vivo experimental models.
2. Chemical Structure and Physical Properties
Nerolidol has four different isomeric forms which consist of two enantiomers and two geometric
isomers [
15
]. The existence of these isomeric forms is due to the presence of a double bond at the C-6
position and the asymmetric center at the C-3 position. These isomeric forms of cis- and trans-nerolidol
are illustrated in Figure 1. Besides, the synonyms for cis- and trans-nerolidol are listed in Table 1.
Molecules 2016, 21, 529 2 of 38
Baccharis dracunculifolia DC, Elettaria cardamomum (L.) Maton, Momordica charantia L., Piper aleyreanum
C. DC and Piper claussenianum (Miq.) C. DC [7–11].
Nerolidol (3,7,11-trimethyl-1,6,10-dodecatrien-3-ol), also known as peruviol, is a naturally occurring
sesquiterpene alcohol present in the EO of various plants with a floral odour [12,13]. Nerolidol was
found to exist as one of the bioactive compounds responsible for the biological activities demonstrated
by the EOs of the aforementioned plants.
Statistics showed that the global usage of nerolidol per annum ranges from 10 to 100 metric tonnes
[14]. For instance, nerolidol is frequently incorporated in cosmetics (e.g., shampoos and perfumes)
and non-cosmetic products (e.g., detergents and cleansers) [13]. Besides, nerolidol is also widely used
in the food industry as a flavor enhancer in many food products since its approval by U.S. Food and
Drug Administration as a safe food flavoring agent.
Principally, this article aims to review the diverse range of pharmacological and biological
activities of nerolidol which include antioxidant, anti-microbial, anti-biofilm, anti-parasitic, insecticidal,
anti-ulcer, skin penetration enhancer, anti-tumor, anti-nociceptive and anti-inflammatory properties.
The review also covers the chemical structure, physical properties, and the biosynthesis pathway of
nerolidol as the intermediate involved in the mechanisms responsible for protection of plants against
herbivores and plant pathogens. This article also highlights the pharmacokinetic and toxicological
properties of nerolidol in both in vitro and in vivo experimental models.
2. Chemical Structure and Physical Properties
Nerolidol has four different isomeric forms which consist of two enantiomers and two geometric
isomers [15]. The existence of these isomeric forms is due to the presence of a double bond at the C-6
position and the asymmetric center at the C-3 position. These isomeric forms of cis- and trans-nerolidol
are illustrated in Figure 1. Besides, the synonyms for cis- and trans-nerolidol are listed in Table 1.
Figure 1. Chemical structures of the two enantiomers both for cis- and trans-isomers of nerolidol.
Table 1. Synonyms of cis- and trans-nerolidol.
Cis-Nerolidol Trans-Nerolidol
(i) (±)-cis-nerolidol
(ii) (6Z)-3,7,11-trimethyl-1,6,10-dodecatrien-3-ol
(iii) (6Z)-3,7,11-trimethyldodeca-1,6,10-trien-3-ol
(iv) (6Z)-nerolidol
(v) 1,6,10-dodecatrien-3-ol, 3,7,11-trimethyl-, (6Z)-
(vi) (Z)-nerolidol
(i) (±)-trans-nerolidol
(ii) (6E)-3,7,11-trimethyl-1,6,10-dodecatrien-3-ol
(iii) (6E)-3,7,11-trimethyldodeca-1,6,10-trien-3-ol
(iv) (6E)-nerolidol
(v) 1,6,10-dodecatrien-3-ol, 3,7,11-trimethyl-, (6E)-
(vi) (E)-nerolidol
Figure 1. Chemical structures of the two enantiomers both for cis- and trans-isomers of nerolidol.
Table 1. Synonyms of cis- and trans-nerolidol.
Cis-Nerolidol Trans-Nerolidol
(i) (˘)-cis-nerolidol (i) (˘)-trans-nerolidol
(ii) (6Z)-3,7,11-trimethyl-1,6,10-dodecatrien-3-ol (ii) (6E)-3,7,11-trimethyl-1,6,10-dodecatrien-3-ol
(iii) (6Z)-3,7,11-trimethyldodeca-1,6,10-trien-3-ol (iii) (6E)-3,7,11-trimethyldodeca-1,6,10-trien-3-ol
(iv) (6Z)-nerolidol (iv) (6E)-nerolidol
(v) 1,6,10-dodecatrien-3-ol, 3,7,11-trimethyl-, (6Z)- (v) 1,6,10-dodecatrien-3-ol, 3,7,11-trimethyl-, (6E)-
(vi) (Z)-nerolidol (vi) (E)-nerolidol

Molecules 2016,21, 529 3 of 40
Like other sesquiterpene compounds, nerolidol has high hydrophobicity, thereby allowing
easier penetration across the plasma membrane and interaction with intracellular proteins and/or
intra-organelle sites [
16
]. The physical properties of nerolidol (isomer not specified) have been
described by Lapczynski et al. [13] as follows:
(i)
Physical description: A clear pale yellow to yellow liquid having a faint floral odor reminiscent
of rose and apple.
(ii) Chemical formula: C15H26O
(iii) Flash point: >212˝F; CC.
(iv) Boiling point: 276 ˝C.
(v) LogKow (calculated): 5.68.
(vi) Vapor pressure (calculated): 0.1 mm Hg 20 ˝C.
(vii) Specific gravity: 0.8744.
(viii) Water solubility (calculated): 1.532 mg/L at 25 ˝C.
3. Sources, Extraction and Analytical Methods of Nerolidol
Numerous extraction methods have been employed for extracting EOs from various plant
samples [
2
]. The hydrodistillation method using the Clevenger-type apparatus appears as the most
common method used for extracting nerolidol. Table 2summarizes the different extraction methods
and the yield of nerolidol from various parts of plants such as leaves, flowers, seeds, fruits, resins,
twigs and woods. Based on the literature references, leaves are the most common source for extraction
of nerolidol. In terms of the percentage of nerolidol in the leaf EO among different plant species,
Piper claussenianum (Miq.) C. DC. has the highest percentage of trans-nerolidol (81.4%), followed by
Zanthoxylum hyemale A.St.-Hil. (51.0%), Zornia brasiliensis Vogel (48.0%) and Swinglea glutinosa (Blanco)
Merr. (28.4%) (Table 2).
Microclimatic and environmental factors such as species, season, location, climate, soil type,
age of the leaves and the extraction method may influence the concentration of each constituents
in EOs [
17
]. Seasonal variation is one of the main factors that influences the composition of EOs in
plants [
18
–
20
] including the concentration of nerolidol. It was reported by Marques and Kaplan [
19
]
that the harvested leaves from Piper claussenianum (Miq.) C. DC. yielded variable amounts of nerolidol
during the year of 2009. The content of trans-nerolidol was higher during the Brazilian spring collection
period (September, October and November, 87.0%, 94.0%, 92.0%, respectively) as compared to that
during autumn collection period (March, April and May, 78.0%; 77.0%; 80.0%, respectively). Another
study conducted by de Sousa et al. [
20
] has shown that the mean concentration of trans-nerolidol in the
leaves of Baccharis dracunculifolia DC. was five fold higher in March 2005 (136.53 mg/100 g of plant)
than that in July 2004 (25.03 mg/100 g of plant). All these findings provide important information to
identify and determine the most appropriate harvest period to obtain the highest yield of nerolidol
from different plants.
Gas chromatography-mass spectrometry (GC-MS) is the analytical method that is most commonly
used to detect nerolidol [
21
]. This is because the boiling points of sesquiterpenes range from ~250 to
280
˝
C in which suitable for the gas-phase separation technique employed by GC-MS analysis [
22
,
23
].
Apart from GC-MS, liquid chromatography-mass spectrometry (LC-MS) method is also widely used
due to its high sensitivity and high accuracy [
24
]. Recently, He et al. [
25
] suggested that LC-MS could
be used for
in vivo
pharmacokinetic analysis of nerolidol due to its convenience and stability features.
The study demonstrated that the lower limit lower quantification (LLOQ) of nerolidol using LC-MS
was reported as 10 ng/mL [
25
]. On the other hand, another study reported the LLOQ of nerolidol as
3.5 ng/mL by using GC-MS [
22
]. These results suggest that GC-MS may be a more preferable detection
method as it was shown to have higher sensitivity than LC-MS in detecting nerolidol.

Molecules 2016,21, 529 4 of 40
Table 2. Plant sources of nerolidol along with its percentage of nerolidol and extraction method.
Plant
Part
Type of Nerolidol Found
in the Essential Oil
Nerolidol Purified from the Essential Oil of the
Respective Plants (%) Extraction Method Ref.
Aerial
parts trans-nerolidol
(i) Warionia saharae ex Benth. & Coss. (23.0%)
Hydrodistillation technique using the
Clevenger-type apparatus [10,26–28]
(ii) Scutellaria abida L. ssp. albida (9.03%)
(iii) Piper aleyreanum C. DC (1.2%)
(iv) Leonotis ocymifolia (Burm.f.) Iwarsson (0.41%)
Leaf
Nerolidol (n.s.)
(i) Capparis tomentosa Lam. (5.14%) Hydrodistillation technique using the
Clevenger-type apparatus [29,30]
(ii) Virola surinamensis (Rol. ex Rottb.) Warb. (3.0%)
Ginkgo biloba L. (0.12%)
Molecular distillation at a feed temperature of
60
˝
C, distillation temperature of 280
˝
C, feed flow
rate of 180 mL per hour, scraper rate of 300 rpm,
and operating pressure of 0.1–0.5 Pa
[31]
trans-Nerolidol
(i) Baccharis dracunculifolia DC. (33.51%)
Hydrodistillation technique using the
Clevenger-type apparatus [8,9,32–43]
(ii) Cassia fistula L. (2.2%)
(iii) Comptonia peregrina (L.) Coult. (2.11% and 3.43% after
0–30 min fraction and 30–60 min fraction respectively)
(iv) Melaleuca quinquenervia (Cav.) S.T.Blake (24.19%)
(v) Myrica rubra (Lour.) Siebold & Zucc. (2%)
(vi) Lantana radula Sw. (19.0%)
(vii) Peperomia serpens (Sw.) Loudon (38.0%)
(viii) Piper aduncum L. (0.2%)
(ix) Piper chaba Hunter (5.1%)
(x) Piper claussenianum (Miq.) C. DC. (81.4%)
(xi) Strychnos spinosa Lam. (0.7%)
(xii) Swinglea glutinosa (Blanco) Merr. (28.4%)
(xiii) Zanthoxylum hyemale A.St.-Hil. (51.0%)
(xiv) Zornia brasiliensis Vogel (48.0%)
Stem trans-Nerolidol Oplopanax horridus (Sm.) Miq. (54.5%) Steam distillation using a low pressure system
with an external steam source [44]
Flower trans-Nerolidol
(i) Achillea millefolium L. (11.6%–31.9%) Hydrodistillation technique using the
Clevenger-type apparatus [42,45,46]
(ii) Cananga odorata (Lam.) Hook.f. & Thomson (0.32%)
(iii) Cassia fistula L. (38.0%)

Molecules 2016,21, 529 5 of 40
Table 2. Cont.
Plant
Part
Type of Nerolidol Found
in the Essential Oil
Nerolidol Purified from the Essential Oil of the
Respective Plants (%) Extraction Method Ref.
Root trans-Nerolidol Oplopanax horridus (Sm.) Miq. (54.6%) Steam distillation using a low pressure system
with an external steam source [44]
Seed/grain
Nerolidol (n.s.) Magnolia denudata Desr. (2.18%) Hydrodistillation technique using the
Clevenger-type apparatus [47]
trans-Nerolidol (i) Elettaria cardamomum (L.) Maton (3.6%) Hydrodistillation technique using the
Clevenger-type apparatus [7,48]
(ii) Momordica charantia L. (61.6%)
Fruit trans-Nerolidol Swinglea glutinosa (Blanco) Merr. (19.1%) Hydrodistillation technique using the
Clevenger-type apparatus [43]
Resin trans-Nerolidol Canarium schweinfurthii Engl. (14%) Hydrodistillation technique using the
Clevenger-type apparatus [49]
Twig/wood
Trans-Nerolidol Cinnamomum osmophloeum Kaneh. (1.05%) Hydrodistillation technique using the
Clevenger-type apparatus [50]
Fokienia hodginsii (Dunn) A.Henry & H H.Thomas (34.8%)
Solid-phase microextraction [51]
cis-Nerolidol Myrocarpus fastigiatus Allemao (80.0%) Hydrodistillation technique using the
Clevenger-type apparatus [52]
Key: n.s. = not specified.

Molecules 2016,21, 529 6 of 40
In order to differentiatie the cis- and trans-isomers of nerolidol, the retention time of different
LC-MS and GC-MS chromatography columns as well as the major peaks of the mass spectra (m/z) are
the parameters used (Table 3). According to Table 3,cis-nerolidol displayed shorter retention times
than trans-nerolidol regardless of the type of GC or LC column used. Besides retention time, one
can also discriminate cis- from trans-nerolidol by referring to the retention indices (RIs) and RIs can
be used for comparison across different chromatographic systems [
53
]. RIs, also known as Kováts
retention indices, are frequently used along with mass spectrometry because the combination provides
a more accurate identification of isomers, which is often difficult to be achieved by mass spectrometry
alone [54]. The retention indices of different chromatographic columns of GC are shown in Table 3.
Table 3.
Retention indices of different chromatographic columns of GC and major peaks of mass
spectrometry to differentiate cis- and trans-nerolidol.
Types of Column/Equipment Used Cis-Nerolidol Trans-Nerolidol Ref.
(A) Retention time of different chromatographic columns of GC (minutes)
(i) A-100 or 154-C column 14 16 [22]
(ii) DB-5 capillary column n.a. 10.5 [21]
(iii) TR-5MS capillary column 5.87 5.98 [22]
(B) Retention time of different chromatographic columns of LC (minutes)
(i) Hypersil BDS C18 column 11.9 13.1 [25]
(C) Major peaks of mass spectrometry (MS) (m/z)
(i) M-80B gas chromatograph double
focusing mass spectrometer 41, 69, 134, 91, 93, 79 69, 41, 93, 43, 71, 55 [55]
(ii) Y2K ion trap (MS) PolarisQ System mass
spectrometer
93, 91, 67, 107, 79, 161, 121,
133, 55, 147, 189, 175
93, 121, 67, 107, 79, 161, 136,
55, 189, 148, 175 [22]
(D) Retention indices of different chromatographic columns of GC
(i) HP-101 n.a. 1564 [56]
(ii) HP-20M n.a. 2009 [56]
(iii) HP-FFAP n.a. 2055 [56]
(iv) Fused silica capillary column coated
with DB-5 n.a. 1564 [29]
(v) OV-101 1533 1549 [55]
(vi) PEG 20M 2028 2035 [55]
(vii) DB-5 1565 1539 [57]
(viii) DB-Wax 2010 2054 [57]
(ix) SPB-1 1543 n.a. [58]
(x) Dimethylsilicone (DIMS) 1524.4 (a) 1550.1 (a) [54]
(xi) Dimethylsilicone with 5% phenyl groups
(DIMS5P) 1543.6 (a) 1560.9 (a) [54]
(xii) Polyethylene glycol (PEG) 2007.3 (a) 2036.3 (a) [54]
Key: (a) = average value; n.a. = not available.
4. Industrial Synthesis of Nerolidol
Chemical synthesis of nerolidol is required to increase its production in order to meet the
growing industrial demand for nerolidol. Initially, nerolidol was synthesized as an intermediate
in the chemical synthesis of geranyl esters from linalool [
59
]. The process began with the treatment
of linalool with diketene or ethyl acetoacetate by the Carroll reaction to yield a mixture of (E)- and
(Z)-geranylacetone [
59
]. Addition of acetylene to both (E)- and (Z)-geranylacetone led to the production
of (E)- and (Z)-dehydronerolidol, respectively, which were selectively hydrogenated to trans- and
cis-nerolidol, respectively, using a Lindlar catalyst [
60
]. The overall chemical synthesis of (E)- and
(Z)-nerolidol is illustrated as shown in Scheme 1as described by Nigmatov et al. [59].

Molecules 2016,21, 529 7 of 40
Molecules 2016, 21, x 7 of 38
Scheme 1. The overall chemical synthesis pathway of nerolidol to fulfill the demand of nerolidol in
the industrial sector.
Although nerolidol can be obtained via the aforementioned chemical reaction or isolation from
natural sources, both methods suffer from the disadvantages that they are expensive and produce
low yields of end products. In order to overcome these limitations, researchers have utilized
eukaryotes such as yeast to produce higher yields of nerolidol. Therefore, a new method of nerolidol
production has been developed and patented [61]. The method involved the cultivation of a yeast
strain (particularly Saccharomyces cerevisiae, as it is a natural producer of farnesyl diphosphate (FDP))
lacking functional squalene synthase by modifying one ERG9 squalene synthase gene. This was
because the absence of functional squalene synthase prevented the conversion of FDP to squalene,
therefore causing FDP to accumulate. The next step involved modifying the yeast to overexpress
3-hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase using an inducible promoter such
as GAL1 HMG CoA reductase, lea ding to a higher t hroughput of FDP . The last step in volved growin g
the yeast in a synthetic medium which is lacking of uracil so that FDP can be fully hydrolyzed into
nerolidol. Further shifting towards nerolidol production can be also enhanced by adjusting the pH
of the medium to be more acidic either at the start, during or at the end of the growth cycle.
5. The Ecological Role and Biosynthesis of Nerolidol
Plant secondary metabolites (PSMs) are organic compounds that do not interfere with the
primary metabolism of plants. Given that they mediate many ecological functions, PSMs are mainly
secreted as plant defenses against herbivore and pathogen damages [62]. PSMs are stored either
constitutively in inactive forms or induced in response to insect or microbe attack. To thwart off
pathogens and herbivores, PSMs employ different chemical defensive strategies involving secondary
metabolite pathways [62]. The first strategy consists of an indirect defense mechanism in which the
Scheme 1.
The overall chemical synthesis pathway of nerolidol to fulfill the demand of nerolidol in the
industrial sector.
Although nerolidol can be obtained via the aforementioned chemical reaction or isolation from
natural sources, both methods suffer from the disadvantages that they are expensive and produce
low yields of end products. In order to overcome these limitations, researchers have utilized
eukaryotes such as yeast to produce higher yields of nerolidol. Therefore, a new method of nerolidol
production has been developed and patented [
61
]. The method involved the cultivation of a yeast
strain (particularly Saccharomyces cerevisiae, as it is a natural producer of farnesyl diphosphate (FDP))
lacking functional squalene synthase by modifying one ERG9 squalene synthase gene. This was
because the absence of functional squalene synthase prevented the conversion of FDP to squalene,
therefore causing FDP to accumulate. The next step involved modifying the yeast to overexpress
3-hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase using an inducible promoter such as
GAL1 HMG CoA reductase, leading to a higher throughput of FDP. The last step involved growing
the yeast in a synthetic medium which is lacking of uracil so that FDP can be fully hydrolyzed into
nerolidol. Further shifting towards nerolidol production can be also enhanced by adjusting the pH of
the medium to be more acidic either at the start, during or at the end of the growth cycle.
5. The Ecological Role and Biosynthesis of Nerolidol
Plant secondary metabolites (PSMs) are organic compounds that do not interfere with the primary
metabolism of plants. Given that they mediate many ecological functions, PSMs are mainly secreted as
plant defenses against herbivore and pathogen damages [
62
]. PSMs are stored either constitutively
in inactive forms or induced in response to insect or microbe attack. To thwart off pathogens and
herbivores, PSMs employ different chemical defensive strategies involving secondary metabolite
pathways [
62
]. The first strategy consists of an indirect defense mechanism in which the plants confront

Molecules 2016,21, 529 8 of 40
herbivores indirectly by secreting herbivore-induced plant volatiles (HIPVs) to attract parasitoids and
natural enemies of herbivores [
63
]. On the other hand, the direct defense mechanism employs another
strategy, that is, toxic, volatile and non-volatile metabolites which are stored in specialized cells to be
released or activated when plants are attacked by pathogens [64].
Among PSMs, terpenoids are the most structurally diverse group. For instance, monoterpenes and
sesquiterpenes are the major volatile terpenoids released from plants [
65
]. Their function are diverse
ranging from basic plant functions such as photosynthesis, respiration, growth and development, to
playing role in plant defense mechanism to protect plants against herbivore and pathogen attacks [
66
].
In general, terpenoids are formed from the universal C5 precursor isopentenyl diphosphate (IPP)
and its allylic isomer dimethylallyl diphosphate (DMAPP) [
67
]. Subsequently, condensation of IPP and
DMAPP by prenyltransferases leads to the production of linear isoprenyl diphosphate precursors of
many chain lengths such as geranyl diphosphate (GDP), FDP and geranylgeranyl diphosphate (GGDP).
The allylic prenyldiphosphates of GDP, FDP and GGDP are then converted by terpene synthases (TPSs)
to form monoterpenes (C10), sesquiterpenes (C15 ) and diterpenes (C20), respectively [68].
Molecules 2016, 21, x 8 of 38
plants confront herbivores indirectly by secreting herbivore-induced plant volatiles (HIPVs) to attract
parasitoids and natural enemies of herbivores [63]. On the other hand, the direct defense mechanism
employs another strategy, that is, toxic, volatile and non-volatile metabolites which are stored in
specialized cells to be released or activated when plants are attacked by pathogens [64].
Among PSMs, terpenoids are the most structurally diverse group. For instance, monoterpenes and
sesquiterpenes are the major volatile terpenoids released from plants [65]. Their function are diverse
ranging from basic plant functions such as photosynthesis, respiration, growth and development, to
playing role in plant defense mechanism to protect plants against herbivore and pathogen attacks [66].
In general, terpenoids are formed from the universal C5 precursor isopentenyl diphosphate (IPP)
and its allylic isomer dimethylallyl diphosphate (DMAPP) [67]. Subsequently, condensation of IPP and
DMAPP by prenyltransferases leads to the production of linear isoprenyl diphosphate precursors of
many chain lengths such as geranyl diphosphate (GDP), FDP and geranylgeranyl diphosphate (GGDP).
The allylic prenyldiphosphates of GDP, FDP and GGDP are then converted by terpene synthases
(TPSs) to form monoterpenes (C
10
), sesquiterpenes (C
15
) and diterpenes (C
20
), respectively [68].
Scheme 2. The biosynthesis pathway of (3S)-(E)-nerolidol as an intermediate product for the
production of DMNT as an herbivore-induced volatile to protect the plant against herbivore damage.
With regard to the production of nerolidol, (E)-nerolidol synthase was recently found to be
responsible for the conversion of FDP, the universal precursor of sesquiterpenes to (3S)-(E)-nerolidol.
In snapdragon (Antirrhinum majus L.), Nagegowda et al. have recently purified two nerolidol/linalool
synthases (AmNES/LIS-1/-2) that are responsible for the production of nerolidol and linalool.
AmNES/LIS-1 is found in the cytosol and is responsible for nerolidol biosynthesis, whereas
AmNES/LIS-2 is located in the plastids and is responsible for the formation of linalool [69]. Similar to
Scheme 2.
The biosynthesis pathway of (3S)-(E)-nerolidol as an intermediate product for the production
of DMNT as an herbivore-induced volatile to protect the plant against herbivore damage.
With regard to the production of nerolidol, (E)-nerolidol synthase was recently found to be
responsible for the conversion of FDP, the universal precursor of sesquiterpenes to (3S)-(E)-nerolidol.
In snapdragon (Antirrhinum majus L.), Nagegowda et al. have recently purified two nerolidol/linalool

Molecules 2016,21, 529 9 of 40
synthases (AmNES/LIS-1/-2) that are responsible for the production of nerolidol and linalool.
AmNES/LIS-1 is found in the cytosol and is responsible for nerolidol biosynthesis, whereas
AmNES/LIS-2 is located in the plastids and is responsible for the formation of linalool [
69
]. Similar
to snapdragon, (3S)-(E)-nerolidol synthase activities were demonstrated in maize [
70
]. Schnee et al.
isolated the terpene synthase 1 (TPS1) enzyme, which is encoded by the maize TPS1 gene to produce
both 3R- and 3S-enantiomer of (E)-nerolidol [
71
]. Due to the stimulation of herbivore damage, the
expression of tps1 was increased by almost 8-fold, followed by the conversion of (E)-nerolidol to
(3E)-4,8-dimethyl-1,3,7-nonatriene (DMNT). Taken all together, the conversion of (E)-nerolidol to
DMNT is crucial due to the fact that DMNT acts as an herbivore-induced volatile to protect the plant
against herbivore damage. The overall biosynthesis mechanism of nerolidol is illustrated in Scheme 2
as described by Bouwmeester et al. [72].
6. Pharmacological and Biological Activities of Nerolidol
With the knowledge that nerolidol plays a very active role in the defense system of some
plants, researchers have been interested to further explore various aspects of its pharmacological
and biological activities. To date, various pharmacological and biological activities of nerolidol
have been reported such as anti-microbial, anti-biofilm, anti-oxidant, anti-parasitic, skin-penetration
enhancer, skin-repellent, anti-nociceptive, anti-inflammatory and anti-cancer. Table 4summarizes the
important information on the pharmacological and biological activities of nerolidol in different
in vitro
and
in vivo
models. Besides the pharmacological and biological activities of nerolidol, the sources of
nerolidol extraction from various parts of plants are illustrated as well (Figure 2).
Molecules 2016, 21, x 9 of 38
snapdragon, (3S)-(E)-nerolidol synthase activities were demonstrated in maize [70]. Schnee et al.
isolated the terpene synthase 1 (TPS1) enzyme, which is encoded by the maize TPS1 gene to produce
both 3R- and 3S-enantiomer of (E)-nerolidol [71]. Due to the stimulation of herbivore damage, the
expression of tps1 was increased by almost 8-fold, followed by the conversion of (E)-nerolidol to
(3E)-4,8-dimethyl-1,3,7-nonatriene (DMNT). Taken all together, the conversion of (E)-nerolidol to
DMNT is crucial due to the fact that DMNT acts as an herbivore-induced volatile to protect the plant
against herbivore damage. The overall biosynthesis mechanism of nerolidol is illustrated in Scheme
2 as described by Bouwmeester et al. [72].
6. Pharmacological and Biological Activities of Nerolidol
With the knowledge that nerolidol plays a very active role in the defense system of some plants,
researchers have been interested to further explore various aspects of its pharmacological and
biological activities.
To date, various pharmacological and biological activities of nerolidol have been
reported such as anti-microbial, anti-biofilm, anti-oxidant, anti-parasitic, skin-penetration enhancer,
skin-repellent, anti-nociceptive, anti-inflammatory and anti-cancer. Table 4 summarizes the important
information on the pharmacological and biological activities of nerolidol in different in vitro and
in vivo models. Besides the pharmacological and biological activities of nerolidol, the sources of
nerolidol extraction from various parts of plants are illustrated as well (Figure 2).
Figure 2. The source of extraction of nerolidol and an overview of the biological activities of nerolidol.
Figure 2.
The source of extraction of nerolidol and an overview of the biological activities of nerolidol.

Molecules 2016,21, 529 10 of 40
Table 4. A summary of pharmacological and biological activities of nerolidol.
Bioactivity Type of Nerolidol Plant and Part of Plant
Used (If Any) Target Organism(s) Screening Assay and
Methods Used Results Possible Mechanisms
of Action Ref.
Antioxidant activity
cis-Nerolidol (Aldrich
Chemical Co.,
Milwaukee, WI, USA)
- - DPPH and hydroxyl
radical scavenging
activity
(i) Exhibited DPPH radical
scavenging activity Mediates antioxidant
activities via free
radical scavenging
activity
[73]
(ii) Exhibited scavenging activity
against hydroxyl radical with
IC50 = 1.48 mM
cis-Nerolidol
(Sigma-Aldrich,
St. Louis, MO, USA)
- - Thiobarbituric acid
reactive substances
(TBARS) assay
(i) Demonstrated 25.60% ˘0.98%
malonaldehyde (MDA) reduction in
hepatocytes at 1 mM under
physiological conditions
Mediates antioxidant
activity via lipid
peroxidation inhibitory
effect
[74]
(ii) Demonstrated higher MDA
reduction with value of
36.50% ˘4.47% at 1 mM in
hepatocytes under oxidative stress
induced by tert-BuOOH
Mixture of cis- and
trans-nerolidol (Sigma
Chemical Company,
St. Louis, MO, USA)
- - TBARS assay, nitrite
assay, superoxide
dismutase (SOD)
activity and catalase
activity
(i) At doses of 25, 50 and 75 mg/kg of
nerolidol caused a significant
decrease in lipid peroxidation by
59.97%, 74.79% and 91.31%
respectively when compared to
negative control
(i) Suggested to prevent
oxidation of
polyunsaturated fatty
acids
(ii) At doses of 25, 50 and 75 mg/kg
of nerolidol caused a significant
decrease in nitrite level by 71.1%,
66.6% and 63.35 % respectively when
compared to negative control
(iii) At doses of 25, 50 and 75 mg/kg
of nerolidol increased superoxide
dismutase activity by 31.1%, 34.8%
and 66.1%, respectively when
compared to negative control
(ii) Suggested to
inactivate the enzyme
nitric oxide synthase
[75]
(iv) At doses of 25, 50 and 75 mg/kg
of nerolidol increased catalase
enzymatic activity by 109%, 148% and
177.7%, respectively when compared
to negative control
Antibacterial activity
Mixture of cis- and
trans-nerolidol (Sigma
Chemical Company,
St. Louis, MO, USA)
-
Staphylococcus aureus
FDA 209P, 14 strains of
methicillin-susceptible
S. aureus (MSSA) and 20
strains of
methicillin-resistant
S. aureus (MRSA)
Broth-dilution with
shaking method (BDS)
Exhibited dose-related inhibition
against 34 clinical isolates of S. aureus.
Inhibitory dose 50% (ID50) ranged
from 5.0 to 22.0 µg/mL and from 2.6
to 10.6 µg/mL against MSSA and
MRSA respectively.
Suggested the aliphatic
chain of nerolidol
mediates the
antibacterial activity by
damaging the bacterial
cell membrane
[76]

Molecules 2016,21, 529 11 of 40
Table 4. Cont.
Bioactivity Type of Nerolidol Plant and Part of Plant
Used (If Any) Target Organism(s) Screening Assay and Methods
Used Results Possible Mechanisms
of Action Ref.
Mixture of cis- and
trans-nerolidol (Sigma
Chemical Company,
(St. Louis, MO, USA)
-Staphylococcus aureus
FDA209P
Broth dilution with shaking
(BDS) method and quantitation
of the leakage of K+ions using
K+-selective electrode
Treatment of nerolidol caused a
dose-dependent increase in amount of K+
ions leakage from bacterial cells.
Mediates the
antibacterial activity via
cell
membrane-distrupting
mechanism and hence
resulting in the leakage
of K+ions from
bacterial cells
[77]
Mixture of cis- and
trans-nerolidol (Sigma
Chemical Company,
St. Louis, MO, USA)
-Staphylococcus aureus
FDA209P
Broth dilution with shaking
(BDS) method and quantitation
of the leakage of K+ions using
K+-selective electrode
(i) Caused a dose-dependent increase in
K+ ions leakage from bacterial cells [78]
(ii) Exhibited minimum inhibitory
concentration at 40 µg/mL
trans-Nerolidol Momordica charantia
L., seed
Staphylococcus aureus
ATCC 6538
Broth microdilution method
(MIC)
(i) Exhibited anti-microbial activity with
MIC ranged from 125–500 µg/mL - [7]
Nerolidol (n.s.) Camellia sinensis (L.)
Kuntze, leaves
Staphylococcus aureus
and Streptococcus
mutans
Broth dilution method
Exhibited antibacterial activity against
S. aureus and S. mutans with MIC
measured at 200 and 25 µg/mL
respectively
- [79]
Nerolidol (n.s.) Ginkgo biloba L., leaves
Salmonella enterica,
Staphylococcus aureus
and Aspergillus niger
Disc-diffusion and broth dilution
methods
(i) Exhibited antibacterial activity against
S. enterica,S. aureus and A. niger with MIC,
MBC and MFC values measured ranging
from 3.9–15.6 µg/mL, 31.3–62.5 µg/mL
and 62.5 µg/mL respectively
- [31]
cis-Nerolidol and the
racemic mixture of cis-
and trans-nerolidol
(Aldrich Chemical Co.,
Milwaukee, WI, USA)
-Escherichia coli and
Staphylococcus aureus Agar-disc diffusion assay
Nerolidol (cis-nerolidol and the racemic
mixture of cis- and trans-isomers)
potentiated the action of antibiotics: -[80]
(i) amoxicillin/clavulanic acid against
S. aureus and
(ii) amoxicilline/clavulanic acid,
ceftadizine and imipenem against E. coli
Nerolidol (n.s.) (Sigma,
St. Louis, MO, USA)
Escherichia coli ATCC
25922 and
Staphylococcus aureus
Disc-diffusion assay
(i) Nerolidol concentrations ranged from
0.5 to 2 mM enhanced the susceptibility of
S. aureus to ciprofloxacin, clindamycin,
erythromycin, gentamicin, tetracycline,
and vancomycin
-[81]
(ii) Nerolidol (1 mM) enhanced the
susceptibility of E. coli to polymyxin B

Molecules 2016,21, 529 12 of 40
Table 4. Cont.
Bioactivity Type of Nerolidol Plant and Part of Plant
Used (If Any) Target Organism(s) Screening Assay and Methods
Used Results Possible Mechanisms
of Action Ref.
Racemic mixture of cis-
and trans-nerolidol (1:1)
(Aldrich, Madrid,
Spain)
-
Escherichia coli
ATCC 25922 and
Staphylococcus aureus
ATCC 25923
Antibiotic disc assay
Nerolidol (20 mM) potentiated the
susceptibility of E. coli and S. aureus
towards ciprofloxacin, erythromycin,
gentamicin and vancomycin
- [82]
Anti-biofilm activity Mixture of cis- and
trans-nerolidol
Black pepper, cananga,
and myrrh EOs (Berjé
(Bloomfield, NJ, USA),
Jin Aromatics (Anyang,
Gyeonggi Province,
Korea) and
Sigma-Aldrich
(St. Louis, USA))
Staphylococcus aureus Crystal violet biofilm assay
Cis-nerolidol at 0.01% (v/v) inhibited
S. aureus biofilm formation by > 80 %;
trans-nerolidol at similar
concentration exerted 45% inhibition
- [45]
trans-Nerolidol Piper claussenianum
(Miq.) C. DC., leaves Candida albicans MTT assay
Concentrations of 0.06%–1.0%
inhibited biofilm formation by 30%
and 50% after 24 and 48 h incubation
respectively
- [32]
cis,trans-Nerolidol and
cis-nerolidol (Sigma
Aldrich)
-Candida albicans MTT assay
1.0% of cis,trans-nerolidol exerted
76.1% reduction in the viability of
pre-formed biofilm while only 67.0%
reduction observed from 1.0%
cis-nerolidol
- [32]
Anti-fungal activity Nerolidol (n.s.)
Chamaecyparis obtusa
(Siebold & Zucc.) Endl.
(Japanese cypress)
Microsporum gypseum Broth microdilution method Skin
lesion scoring in guinea pig
model
(i) Exhibited MIC concentrations of
0.5%–2% against M. gypseum -[83]
(ii) Nerolidol-treated group exhibited
a significant improvement (p < 0.05)
in lesion as compared to eugenol and
econazole (positive control) treated
groups
trans-Nerolidol Piper claussenianum
(Miq.) C. DC.,
Piperaceae, leaves
Candida albicans Broth microdilution and trypan
blue exclusion method
(i) Exhibited anti-fungal activity with
MIC values ranging from 0.24% to
1.26%. -[32]
(ii) Exhibited inhibitory effect on
yeast-to-hyphae transition by 81%
Nerolidol (n.s.)
(Sigma-Aldrich, Yongin,
Korea)
-
Trichophyton
mentagrophytes
Agar dilution method
Inhibited the hyphal growth of
T. mentagrophytes at the concentration
of 0.4 mg/mL.
- [16]
Nerolidol (n.s.) Camellia sinensis (L.)
Kuntze, leaves Broth dilution method Inhibited the growth of
T. mentagrophytes at 12.5 µg/mL - [79]

Molecules 2016,21, 529 13 of 40
Table 4. Cont.
Bioactivity Type of Nerolidol Plant and Part of Plant
Used (If Any) Target Organism(s) Screening Assay and Methods
Used Results Possible Mechanisms
of Action Ref.
trans-Nerolidol Lantana radula Sw.,
leaves Corynespora cassiicola Poison food (PF) technique
(i) L. radula EO at the concentration of
1000 mg/L and 3000 mg/L inhibited
the growth of C. cassiicola by 17.2%
and 40.6% respectively
-
[33]
(ii) L. radula EO at the concentration
of 5000 mg/L and 10,000 mg/L
completely inhibited the growth of
C. cassiicola
trans-Nerolidol Piper chaba Hunter,
leaves
Fusarium oxysporum,
Phytophthora capsici,
Colletotrichum capsici,
Fusarium solani and
Rhizoctonia solani
Spore germination assay and
agar dilution method
Caused 55.1%–70.3% growth
inhibition at concentration ranging
from 125 to 500 µg/mL.
- [34]
trans-Nerolidol
Warionia saharae ex
Benth. & Coss., aerial
part
Alternaria sp.,
Penicillium expansum
and Rhizopus stolonifer
Poisoned food (PF) technique
and volatile activity (VA) assay
Inhibited the fungal spore production
of Alternaria sp., P. expansum and
R. stolonifera at 1, 2 and 2 µL/mL air
respectively
- [26]
Nerolidol Allium sativum L., bulb Sclerotium cepivorum Disc diffusion method; scanning
electron microscopy
(i) Nerolidol ranged from 2.0 to
5.0 µg/disc displayed fungistatic
property by inhibiting mycelial
growth by ~85% -
[84]
(ii) Nerolidol ranged from 2.0 to
5.0 µg/disc inhibited the production
of sclerotial by ~84%
(ii) Nerolidol at 4.0 µg/disc caused
morphological alterations such as
shorter branching, hyphal shrinkage
and partial distortion
Anti-trypanosomal
activity
trans-Nerolidol Strychnos spinosa Lam.,
leaves
Trypanosoma brucei
Alamar Blue™ assay.
Exhibited anti-trypanosomal activity
with IC50 measured at 1.7 µg/mL
(7.6 µM)
- [35]
cis-Nerolidol
Leonotis ocymifolia
(Burm.f.) Iwarsson,
aerial part
Trypanocidal and cytotoxic
assays
Exhibited anti-trypanosomal activity
with IC50 measured at 15.78 µg/mL - [27]
Mixture of ˘40%
cis-nerolidol and ˘55%
of trans-nerolidol
(Merck, Darmstadt,
Germany)
-
Trypanosoma evansi
Collection of blood samples from
T. evansi-infected mice for
observation using light and
electron microscopes
(i) Adverse morphological changes
observed in nerolidol-treated group.
The parasites lost their undulating
membrane after 23 day
post-treatment. -[85]
(ii) Total disfigurement observed after
27 day post-treatment

Molecules 2016,21, 529 14 of 40
Table 4. Cont.
Bioactivity Type of Nerolidol Plant and Part of Plant
Used (If Any) Target Organism(s) Screening Assay and Methods
Used Results Possible Mechanisms
of Action Ref.
Anti-leishmanial
activity
A mixture of cis- and
trans-nerolidol -
Leishmania (L.)
amazonensis,
L. braziliensis, and
L. chagasi
MTT assay and metabolic
labeling with [2-14C] mevalonic
acid, [1-14C] acetic acid, [1(n)-3H]
farnesyl pyrophosphate and
L-[35S]methionine
(i) Inhibited the growth of
L. amazonensis, L. braziliensis and
L. chagasi promastigotes, and
L. amazonensis amastigotes with IC50
of 85, 74, 75, and 67 µM respectively
Inhibition of the
isoprenoid biosynthesis
pathway [86]
(ii) Nerolidol at 100 µM reduced the
percentage of intracellular parasitism
of L. amazonensis by 95% from the
pre-infected macrophages culture
trans-Nerolidol Baccharis dracunculifolia
DC., leaves Leishmania donovani
Parasite lactate dehydrogenase
(pLDH) assay, antileishmanial
assay, schistosomicidal assay and
cytotoxicity assay using the
mammalian cells Vero.
Exhibited anti-leishmanial activity
against promastigotes of L. donovani
with IC50 and IC90 values of 42 and
85 µg/mL respectively.
- [8]
Nerolidol Piper claussenianum
(Miq.) C. DC.,
Piperaceae, leaves
Leishmania amazonensis Protozoal arginase activity,
nitrite determination and
cytotoxicity assay using L929
fibroblast cells (mouse) and Raw
cells (mouse macrophages)
(i) Nerolidol inhibited the arginase
activity by 62.17% in the
promastigotes of
Leishmania amazonensis
Interferes with
parasite-host cell
interaction
[9]
(ii) Nerolidol caused an increase in
NO production (20.5%)
Nerolidol (n.s.) (Acros
Organics, Geel,
Belgium)
-Promastigotes of
Leishmania amazonensis
Anti-proliferative activity assay
and electron paramagnetic
resonance (EPR) spectroscopy of
the spin-labeled 5-doxyl stearic
acid
Nerolidol modulated the molecular
dynamics of the lipid component in
the Leishmania plasma membrane
Insertion of nerolidol
into the lipid bilayer
increased the fluidity of
membranes, thus
causing leakage of
cytoplasmic content
and eventually the
death of Leishmania cells
[87]
Anti-schistosomal
activity
Nerolidol (n.s.)
Baccharis dracunculifolia
DC. (Asteraceae),
leaves Schistosoma mansoni
Schistosomicidal assay
100% mortality of S. mansoni adult
worms after 24 h incubation with 10
to 100 mg/mL of EO containing
nerolidol as the main constituent
- [8]
Racemic mixture of cis-
and trans-nerolidol (1:1)
(Sigma-Aldrich,
St. Louis, MO, USA)
-In vitro anti-schistosomal assay
and microscopy studies
Exhibited anti-schistosomal activity
by reducing worm motor activity and
caused 100% mortality of male and
female schistosomes at concentration
of 31.2 and 62.5 µM respectively
(i) Induced severe
tegumental damage in
adult schistosomes. [88]
(ii) Caused alterations
on the tubercles of male
parasites

Molecules 2016,21, 529 15 of 40
Table 4. Cont.
Bioactivity Type of Nerolidol Plant and Part of Plant
Used (If Any) Target Organism(s) Screening Assay and Methods
Used Results Possible Mechanisms
of Action Ref.
Anti-malarial activity
Nerolidol (n.s.) Virola surinamensis (Rol.
ex Rottb.) Warb., leaves
Plasmodium falciparum
In vitro anti-plasmodial assay
Treatment with 100 µg/mL of nerolidol
caused 100% inhibition in the
development of young trophozoite to the
schizont stage after 48 h
- [29]
trans-Nerolidol Piper claussenianum
(Miq.) C. DC., leaves
Exerted anti-malarial activity with IC50 of
11.1 µg/mL - [89]
Nerolidol (n.s.) (Sigma,
St. Louis, MO, USA) -Immunoprecipitation assays and
metabolic labeling
Exhibited inhibitory activity on the
biosynthesis of the isoprenic side chain of
the benzoquinone ring in ubiquinones
during the schizont stage
Interferes with the
elongation of isoprenic
chains via inhibition of
isoprenyl diphosphate
synthases
[90]
Nerolidol (n.s.) (Sigma,
St. Louis, MO, USA) -
Nerolidol at 50 nM inhibited the synthesis
of the isoprenic chain attached to
coenzyme Q at all intraerythrocytic stages
- [91]
Nerolidol (n.s.) - Isobolographic analysis
Nerolidol mediated supra-additive (the
sum of the fractions of IC50 of < 1)
interaction with fosmidomycin and
squalestatin with average IC50 values of
0.57 and 0.62 µM, respectively in the
inhibition of plasmodial isoprenoid
pathway
- [92]
Other anti-parasite
activities
Mixture of cis- and
trans-nerolidol
(Sigma-Aldrich,
St. Louis, MO, USA)
-
Four Babesia species (B.
bovis, B. bigemina, B.
ovata, and B. caballi)
In vitro growth inhibition assay
Inhibited in vitro growth of B. bovis,B.
bigemina,B. ovata, and B. caballi with IC50
values of 21 ˘1, 29.6 ˘3, 26.9 ˘2, and
23.1 ˘1µM respectively
Inhibits the isoprenoid
biosynthesis pathway
in a similar mechanism
with that of P. falciparum
[93]
Mixture of cis- and
trans-nerolidol (Sigma
Chemical Company,
St. Louis, MO, USA)
-Caenorhabditis elegans Mortality assay against
Caenorhabditis elegans
Caused 74.0% mortality of C. elegans at
50 µg/mL - [94]
Nerolidol (n.s.)
-
L3larvae of Anisakis In vitro and in vivo larvicidal
activity
(i) Nerolidol at both 31.5 and 62.5 µg/mL
resulted in 100% mortality of L3larvae of
Anisakis type I after 4 h. -[95]
(ii) Only 20% of nerolidol-treated rats were
affected by gastric wall lesions caused by
Anisakis larvae in comparison to 86% of
the control rats
Insecticidal activity
trans-Nerolidol
Siam-wood (Fokienia
hodginsii (Dunn)
A.Henry & H
H.Thomas), wood
Mosquito and house
flies House fly toxicity test Exhibited insecticidal activity with LD50
measured at 0.17 µmol/fly - [51]
Combination of
nerolidol (n.s.) and
linalool
Capparis tomentosa,
leaves
Maize weevil
(Sitophilus zeamais)
Repellency assay using a glass
Y-tube Olfactometer
Exhibited mean repellency value of 58.23%
˘2.95% against S. zeamais at 2 µL- [30]

Molecules 2016,21, 529 16 of 40
Table 4. Cont.
Bioactivity Type of Nerolidol Plant and Part of Plant
Used (If Any) Target Organism(s) Screening Assay and Methods
Used Results Possible Mechanisms
of Action Ref.
Nerolidol (n.s.)
(Moellhausen
SpA,Vimercate, Milano,
Italy)
Melaleuca alternifolia
(Maiden & Betche)
Cheel (tea tree oil)
Pediculus capitis (head
lice) and its eggs
Pediculicidal and ovicidal
activities
Nerolidol in combination with tea tree oil
with ratio of 1:2 (tea tree oil 0.5% plus
nerolidol 1%), exerted a total killing effect
of lice within 30 min and abortive effect of
louse eggs after 5 days.
- [96]
Nerolidol (n.s.) Magnolia denudata Desr.,
seeds
Culex pipiens pallens,
Aedes aegypti,Aedes
albopictus and Anopheles
sinensis
Direct-contact mortality bioassay
Exerted larvacidal activity against
Culex pipiens pallens,Aedes aegypti,
Aedes albopictus and Anopheles sinensis with
LD50 value of 9.84, 13.85, 16.34 and
20.84 mg/L respectively
- [47]
trans-Nerolidol
Melaleuca quinquenervia
(Cav.) S.T.Blake, leaves Aedes aegypti Larvicidal activity test
Exerted larvicidal activity with ě95% and
> 80% mortality of A. aegypti at 0.1 mg/mL
and 0.05mg/mL respectively
- [36]
Piper aduncum L., leaves Tetranychus urticae Koch Fumigant, contact, repellency
and two-choice assay
Exerted acaricidal activity with repellency
value of 83.2% ˘0.59 % at 9.8 µg/mL - [37]
Nerolidol (n.s.) Baccharis dracunculifolia
DC., leaves Rhipicephalus microplus Larval packet test (LPT) and
engorged female immersion test
(i) Exerted acaricidal activity when
concentration more than 5mg/mL and
100% mortality of larvae at 15 mg/mL -[97]
(ii) Reduced the quality of the egg and
larval hatching rate with increasing
concentration from 20 to 50 mg/mL
Antiulcer activity Nerolidol (n.s.) Baccharis dracunculifolia
DC., leaves -
In vivo antiulcer activity in male
Wistar rat ulcer models induced
with ethanol, indomethacin and
stress
Nerolidol displayed gastroprotective
activity by inhibiting the formation of
ulcers induced by all physical and
chemical agents in dose-dependent
manner (50, 250, 500 mg/kg)
- [98]
Skin penetration
enhancer activity
Nerolidol (n.s.)
(Aldrich, Gillingham,
UK)
- -
In vitro diffusion studies and
stratum corneum-water
partitioning studies
Increased diffusion rate by over 20-fold for
transdermal delivery of drugs such as
5-fluorouracil
Nerolidol exhibits a
chemical structure that
allows it to align within
the lipid lamellae of the
stratum corneum in
order to disrupt the
organization of stratum
corneum
[99]
Nerolidol (n.s.) (Alfa
Aesar Ltd., Haverhill,
MA, USA)
- -
Solubility studies, ex vivo
permeation studies and
histopathological studies
The enhancement effect is increased with
the increasing lipophilicity; the rank of
order (nerolidol > farnesol > limonene >
linalool > geraniol > carvone > fenchone >
menthol) in facilitating transdermal
delivery of alfuzosin hydrochloride
[100]
Nerolidol (n.s.)
(Merck-Schuchardt,
Hohenbrunn,
Germany)
- - In vitro permeation studies
Exhibited the highest permeation
enhancing ability with a 3.2-fold increase
in permeation of selegiline hydrochloride
across the rat skin, followed by the effect
of carvone (2.8-fold increase) and anethole
(2.6-fold increase)
- [101]
Nerolidol (n.s.)
(Aldrich Chemical Co.
Milwaukee, WI, USA)
- - In vitro skin permeability studies
Most effective terpene enhancer for
percutaneous permeation of four different
drug models (nicardipine hydrochloride,
hydrocortisone, carbamazepine, and
tamoxifen) when compared to fenchone,
thymol and limonene
- [102]

Molecules 2016,21, 529 17 of 40
Table 4. Cont.
Bioactivity Type of Nerolidol Plant and Part of Plant
Used (If Any) Target Organism(s) Screening Assay and Methods
Used Results Possible Mechanisms
of Action Ref.
Anti-nociceptive and
anti-inflammatory
activities
trans-Nerolidol
Peperomia serpens (Sw.)
Loudon, leaves -
(i) Chemical (acetic acid and
formalin) and thermal (hot plate)
models of nociception trans-Nerolidol could be responsible for
the anti-inflammatory and
anti-nociceptive effects displayed by
essential oils of both Peperomia serpens
(Sw.) Loudon and Piper aleyreanum C. DC
-
[38]
(ii) Carrageenan- and
dextran-induced paw edema
tests in rats croton oil-induced
ear edema
(iii) Cell migration, rolling and
adhesion activities
trans-Nerolidol Piper aleyreanum C. DC,
aerial parts -
(i) Nociception induced by
formalin -
[10]
(ii) Evaluation of locomotor
activity
(iii) Induction of acute gastric
lesions
Nerolidol (n.s.) (Sigma,
St. Louis, MO, USA) - -
(i) Rotarod, acetic acid-induced
writhing, formalin and hot-plate
tests
(ii) Involvement of ATP-sensitive
opioid and GABAergic K+
channels
(iii)Carrageenan-induced paw
edema
(iv) Analysis of leukocytes,
tumor necrosis factor (TNF-α),
interleukin 1 beta (IL-1β) and
interleukin 6 in peritoneal lavage
(i) For acetic acid-induced writhing test, at
the doses of 200, 300 and 400 mg/kg,
nerolidol reduced the frequency of acetic
acid-induced writhing at all three doses
tested compared to the mice in the control
group (55% ˘1.1%, 53% ˘4.5%, and 41%
˘2.4%, respectively)
(ii) For formalin test, at the doses of 200,
300 and 400 mg/kg, nerolidol significantly
inhibited licking time by 20% ˘3.3%, 33%
˘5.9% and 37% ˘4.8%, respectively
when compared to the control mice.
(iii) For hot-plate test, no increase in the
reaction time to painful stimulation in the
mice treated with nerolidol when
compared to the control mice.
(iv) Reduced leukocytes level by 51% ˘
0.7%, 37% ˘0.5% and 57% ˘0.4% at
doses of 200, 300 and 400 mg/kg
respectively
(v) Reduced the level of tumor necrosis
factor (TNF-α) at doses of 300 (59.3% ˘
30.2%) and 400 (62.2% ˘13.7%) in
peritoneal lavage.
(vi) IL-1βproduction was inhibited after
treatment with nerolidol (1, 10, 50 and
100 µM) whereas IL-6 level was
unchanged
(i) Anti-nociceptive
activtity of nerolidol
was indicated to be
mediated by GABAA
receptors, as the use of
bicuculline, a GABAA
antagonist inhibited the
effect of nerolidol in
reducing the paw
licking times
(ii) Anti-inflammatory
activity of nerolidol
was suggested to be
mediated by inhibiting
the production or the
activity of
pro-inflammatory
cytokines such as
TNF-αanalgesic and
IL-1β
[103]

Molecules 2016,21, 529 18 of 40
Table 4. Cont.
Bioactivity Type of Nerolidol Plant and Part of Plant
Used (If Any)
Target
Organism(s) Screening Assay and Methods Used Results Possible Mechanisms
of Action Ref.
Anti-cancer or
anti-tumor activity
Nerolidol (a
combination of
cis-nerolidol 40.7%,
trans-nerolidol 58.3%,
cis-dihydronerolidol
0.4% and
trans-dihydro-nerolidol)
(Kurt Kitzing Co.
Wallerstein, Germany)
- - Cytotoxicity assay on HeLa cell lines using
CytoTox-96®-assay
Exhibited anticancer effect against HeLa
cells with CC50 value at 1.5 ˘0.7 µM- [104]
cis-Nerolidol (Charabot
S.A. Grasse, France) - -
Cytotoxicity and cytoproliferative activity
on HeLa cell lines using Cytotoxicity
Detection Kit (LDH) and the Cell
Proliferation Reagent WST-1, respectively
Exhibited cytotoxic effect (16.5 ˘6.7 µM)
against HeLa cells - [105]
Nerolidol (n.s.) Camellia sinensis (L.)
Kuntze, leaves - MTT assay
Exhibited cytotoxic effect with IC50 value
of 2.96 and 3.02 µg/mL against BT-20
breast carcinoma and HeLa cells
respectively
- [106]
trans-Nerolidol
Zornia brasiliensis Vogel,
leaves -In vitro cytotoxic activity assay using
Alamar blue assay, and in vivo antitumor
activity assay
(i) trans-Nerolidol induced cytotoxic effect
on B16-F10, HepG2, HL-60 and K562 cells
with IC50 value of >25, >25, 21.99 and
17.58 µg/mL respectively
-[39]
(ii) The EO at dose of 100 mg/kg
containing trans-nerolidol as major
constituent reduced the weight of tumor
in mice injected with B16-F10 melanoma
by 38.61%
Myrica rubra (Lour.)
Siebold & Zucc.,leaves -
Neutral red uptake (NRU) test, MTT assay
and
21,71-dichlorodihydrofluorescein-diacetate
(H2DCF-DA) oxidation
Potentiated the action of doxorubicin, an
anticancer drug in the modulation of
CaCo-2 cancer cells
- [40]
Nerolidol (n.s.) (Sigma
Aldrich Chemical
Company)
- - In vivo anti-cancer study
(i) Reduction of incidence of intestinal
neoplasia from 82% to 33% in rats fed with
nerolidol
Modulation of nerolidol
on protein prenylation
which responsible for
the formation of cancer
[107]
(ii) Reduction of number of tumors/rat
from 1.5 to 0.7 in rats fed with nerolidol
Combination of
farnesol and nerolidol
(n.s.)
- - In vitro anti-cancer study
The combination suppressed the
proliferation of human HL-60 acute
promyelocytic leukemia (HL-60) cells by
20%. Meanwhile, farnesol isomers
(2.5 µmol/L) and nerolidol (5 µmol/L)
individually suppressed the proliferation
of HL-60 cells by 4 and 9%, respectively
Nerolidol induced cell
cycle arrest at the
G0-G1/S interphase in
HL-60 cells and
eventually lead to
apoptotic cell death
[108]
trans-Nerolidol Myrica rubra (Lour.)
Siebold & Zucc.,leaves -Cell adhesion and apoptosis luminescent
assays
(i) Reduced adhesion of HT29 to collagen. Nerolidol induced
apoptosis in cancer cells [109]
(ii) Suppressed cell adhesion of HT29 cells
in the presence TNFαcytokines
(iii) Decreasing the phosphorylation of
NF-κB and increased the activity of
caspases
Key: n.s. = not specified.

Molecules 2016,21, 529 19 of 40
6.1. Antioxidant Activity
Reactive oxygen species (ROS) are formed by the incomplete reduction of oxygen during aerobic
metabolism [
110
]. Superoxide anion (O
2–
), hydrogen peroxide (H
2
O
2
), and hydroxyl radicals (OH
‚
)
are some examples of ROS. Under normal circumstance or low level of oxidative stress, an in-built
antioxidant defense system in the body helps the cells to counteract with any potential damages by
detoxifying ROS with appropriate enzymes such as glutathione (GSH) reductase, GSH peroxidase,
superoxide dismutase (SOD) and catalase. However, an imbalance in the antioxidant defense system
or overproduction of free radicals which exceeds the detoxification capacity of cell may contribute to
the onset of oxidative stress [
111
]. During oxidative stress, an elevation of intracellular levels of ROS
was found to cause damage on biomolecules (lipids, proteins and DNA) [
112
]. Thus, high level of ROS
is detrimental to cells. It is mainly due to the formation and accumulation of cellular damage resulted
from oxidative stress and subsequently leading to the loss of cellular functions. If left untreated, it
will result in many complications such as cancer, cardiovascular diseases and neurodegenerative
disorders [113].
Given the fact that chemical compounds belonging to the sesquiterpene group are well known for
their antioxidant properties [
113
,
114
], perhaps antioxidant activity may be expected from nerolidol.
Furthermore, EOs containing nerolidol derived from medicinal plants were found to exhibit antioxidant
activity, suggesting its plausible utilization as an antioxidant agent. Indeed, it has been demonstrated
that nerolidol exhibits potent antioxidant properties in counterbalancing the effect of ROS by
protecting the cells against oxidative damage to lipids, proteins and DNA [
73
–
75
]. According to
a study conducted by Vinholes et al., the antioxidant activity of cis-nerolidol was evaluated using
1,1-diphenyl-2-picrylhydrazine (DPPH) radical scavenging assay. The study revealed that cis-nerolidol
exhibited DPPH scavenging activity [
73
]. In another study, cis-nerolidol was found to possess higher
scavenging activity towards hydroxyl radicals with IC
50
measured at 1.48 mM [
73
]. Due to the ability
in scavenging several types of free radicals, cis-nerolidol was evidenced to protect Caco-2 cells against
oxidative stress induced by tert-butyl hydroperoxide (tert-BuOOH), suggesting that nerolidol is a
good antioxidant that exerts protection against oxidative damage [
73
]. Another study conducted by
Vinholes et al. reported that cis-nerolidol mediated its strong antioxidant activity in protecting the
hepatocytes through the inhibition of lipid peroxidation induced by tert-BuOOH, thereby 1mM of
cis-nerolidol resulted in 36.50% ˘4.47% of malonaldehyde (MDA) reduction [74].
Besides the in vitro evidences displaying the antioxidant properties of nerolidol, an in vivo study
by Nogueira Neto et al. demonstrated the neuroprotective effects of nerolidol (a mixture of cis- and
trans-nerolidol) in adult male Swiss albino mice hippocampus against neuronal damages induced
by oxidative stress [
75
]. The study demonstrated that significant decrease in MDA and nitrite levels
were observed for the nerolidol group at doses of 25, 50 and 75 mg/kg when compared to saline,
the negative control. Beside that, nerolidol also increased the antioxidant enzymatic activities of
superoxide dismutase and catalase at doses of 25, 50 and 75 mg/kg. These observations suggest
that nerolidol mediates a potent antioxidant activity by scavenging free radicals, preventing lipid
peroxidation and enhancing the production of antioxidant enzymes in cells for protection against
oxidative stress [115,116].
6.2. Antibacterial Activity
The decrease in the effectiveness of many antibiotics due to the rise of antimicrobial resistance is a
global concern faced by the pharmaceutical, medical and food industries. Consequently, the number
of infections caused by multidrug-resistant bacteria is increasing globally, leading to increased risk
of mortality and morbidity [
117
]. Due to this, a large proportion of investments by pharmaceutical
industries are being put into drug discovery research of new inhibitory compounds of microbiological,
plant, or animal origin to be developed into potentially new anti-microbial drugs.
The studies showed that nerolidol exhibited potent antimicrobial activity against
Staphylococcus aureus FDA 209P, 14 strains of methicillin-susceptible S. aureus (MSSA) and 20 strains of

Molecules 2016,21, 529 20 of 40
methicillin-resistant S. aureus (MRSA) with MIC values ranging from 512 to over 1024
µ
g/mL [
76
].
Besides, nerolidol possessed antibacterial activity against various strains of Staphylococcus aureus
including MRSA by disrupting the cell membranes as indicated by the increased leakage of K
+
ions from the bacterial cells [
76
–
78
]. The observed effects could be due to the presence of the long
aliphatic chain in chemical structure of nerolidol. The hypothesis may comply with the findings of
Togashi et al. [118]
that the terpene alcohols with carbon chains of C10 to C12 (the numbering is started
from the carbon atom connected to a hydroxyl group) was found to exhibit a strong antibacterial
activity against S. aureus FDA209P. Since the carbon chain length of nerolidol is C12, it was shown to
cause damage to the cell membrane, leading to the leakage of macromolecules and eventually cell
lysis [
78
]. Besides causing membrane disruption, nerolidol was also found to interfere with genes
which regulate the pathogenicity of the pathogens. For example, a study conducted by Lee et al.
reported that cis-nerolidol present in the black pepper oil was responsible for the down-regulation of
the α-hemolysin gene hla expression in S. aureus via quantitative real-time PCR analyses [45].
In a similar way, EOs of Momordica charantia L. seed was found to exhibit strong antibacterial
activity against S. aureus ATCC 6538 with MIC value of 125
µ
g/mL. The high content of trans-nerolidol
was suggested to be responsible for the antibacterial activity demonstrated by the EOs of Momordica
charantia L. seed [
7
]. Likewise, the study conducted on the antimicrobial activity of green tea flavor
components by Kubo [
79
] has also shown that nerolidol as one of the ten major green tea flavor
compounds, exhibited anti-microbial activity against S. aureus with MIC values of 200
µ
g/mL. Besides,
nerolidol was also found to exert the strongest antibacterial activity against Streptococcus mutans when
compared among the ten green tea flavor compounds with a MIC value of 25
µ
g/mL [
79
]. Meanwhile,
nerolidol derived from the leaf EO of Ginkgo biloba (L.) demonstrated the highest antibacterial activity
against Salmonella enterica, and S. aureus when compared to other EOs (isophytol, linalool,
β
-sitosterol
acetate,
β
-sitosterol, stigmasterol, ergosterol,
β
-sitosterol-3-O-
β
-D-gluco-pyranoside and Ginkgo biloba
polyprenols (GBP)) [
31
]. The evidences presented above may suggest nerolidol can be a good
antibacterial agent particularly against S. aureus.
Besides the direct antibacterial action of nerolidol, nerolidol (cis-nerolidol and the racemic
mixture of cis- and trans-isomers (1:1)) was found to potentiate the action of antibiotics, namely
amoxicilline/clavulanic acid against S. aureus and amoxicilline/clavulanic acid, ceftadizine and
imipenem against Escherichia coli [
80
]. The sensitization effect was also observed when nerolidol
enhanced the susceptibility of S. aureus to ciprofloxacin, clindamycin, erythromycin, gentamicin,
tetracycline, and vancomycin. Other than S. aureus, nerolidol was also found to enhance the
susceptibility of E. coli ATCC 25922 to polymyxin B [
81
]. These findings were further supported
by the experiment conducted by Simões et al. as it revealed that the treatment of nerolidol (racemic
mixture of the cis- and trans-nerolidol) (1:1) potentiated the susceptibility of E. coli and S. aureus
towards the antibiotics (ciprofloxacin, erythromycin, gentamicin and vancomycin), thereby resulted
in significantly lower MIC concentrations [
82
]. Moreover, the study also demonstrated a moderate
correlation between cell killing and permeabilization effects of nerolidol against both S. aureus and
E. coli, suggesting that nerolidol exerted its action by modifying the bacterial outer layer as evidenced
by the increased propidium iodide uptake [
82
]. All these observations suggest that nerolidol provides
an alternative therapeutic option for the development of drug combinations that may be more effective
in controlling multi-drug resistant bacteria.
6.3. Anti-Biofilm Activity
Many bacteria are known to possess the ability to produce biofilm, which is defined as a
community of microorganisms held together by a self-produced extracellular matrix and attached to
living or inert surfaces such as polystyrene, glass, stainless steel and blood components in different
environments [
119
]. Due to the complexity of the biofilm structure formation, microbial biofilms
represent a significant challenge to the medical and pharmaceutical industries. The formation of
biofilm induces microbial resistance to anti-microbial agents as well as to the body’s immune system. It

Molecules 2016,21, 529 21 of 40
has also been associated with the increased antibiotic resistance, thereby leading to biofilm-associated
infections which complicate the treatment procedure. Therefore, there is a new trend in recent studies
focusing on the evaluation of essential oils as potential inhibitors of biofilm formation. In the meantime,
nerolidol was found to exhibit anti-biofilm activity against a number of pathogens. For example, a
study conducted by Lee et al. has revealed that the EO of Cananga odorata (Lam.) Hook.f. & Thomson
exhibited strong biofilm activity in a dose-dependent manner against the biofilm formation of S. aureus
ATCC 6538 [
45
]. The anti-biofilm activity was attributed to the presence of cis- and trans-nerolidol
in the essential oil of C. odorata. It was demonstrated that the cis-nerolidol at 0.01% (v/v) inhibited
S. aureus biofilm formation by more than 80%, whereas trans-nerolidol at similar concentration exerted
45% inhibition. Another study conducted by Curvelo et al. revealed that the EO of Piper claussenianum
(Miq.) C. DC., Piperaceae leaf (trans-nerolidol identified as the main component (81.4%) in this EO)
decreases the formation of biofilm by Candida albicans for 30% and 50% after 24 and 48 incubation hours,
respectively [
32
]. The same study also compared the anti-biofilm activities of the cis,trans-nerolidol
and cis-nerolidol on the pre-formed biofilm by C. albicans. The study indicated that cis,trans-nerolidol
resulted a stronger reduction in the viability of the mature biofilm than that of cis-nerolidol. Thus,
it was suggested that trans-nerolidol, which was the main constituent in EO of Piper claussenianum
(Miq.) C. DC., Piperaceae leaf, may responsible for the observed anti-biofilm activity in reducing the
viability of the pre-formed biofilms.
6.4. Anti-Fungal Activity
Various anti-fungal agents have been developed to control the spread of fungal diseases such as
candidiasis [
120
]. However, there are serious questions concerning the safety of these drugs due to
their well-known side-effects as well as the possible development of antifungal drug resistance [
121
].
Therefore, the attention has been shifted to bio-prospecting the natural products to overcome or control
fungal infections. In fact, EOs have been extensively studied and have been proven to be effective
against fungal infections [122].
There are many evidences that support the effectiveness of nerolidol in exhibiting anti-fungal
activity. Trans-nerolidol, which is a major component of leaf EO of Piper claussenianum (Miq.) C.
DC., Piperaceae (81.4%), has been shown to exhibit fungicidal activity against Candida albicans with
MIC values measured ranging from 0.24%–1.26% [
32
]. Similarly, the leaf EO also exerted a strong
activity in the inhibition of germ-tube transformation of Candida albicans by 81% [
32
]. In another
study, nerolidol has also been found to possess strong antifungal activity by distorting the hyphal
growth of Trichophyton mentagrophytes at the concentration of 0.4 mg/mL [
16
]. Also, the growth of
T. mentagrophytes was inhibited by nerolidol derived from green tea flavor with MIC value measured
at 12.5 µg/mL [79].
Lee et al. reported a strong anti-fungal effect of nerolidol against Microsporum gypseum that causes
dermatophytosis, a superficial infection in keratinized tissues including hair, nail and stratum corneum
of skin [
83
]. Although nerolidol (0.5%–2%) was found to exhibit lower anti-fungal activity as compared
to eugenol (0.01%–0.03%), the study showed that nerolidol was more effective in reducing the skin
lesion than eugenol in guinea pig model. Moreover, histopathologic analysis revealed that animals
treated with nerolidol had a lower degree of hyperkeratosis and inflammatory cell infiltration than
non-treated animals.
Besides its anti-fungal effect against human pathogens, nerolidol also shows promising outcomes
in controlling fungal infections in plants caused by phytopathogenic fungi. Trans-nerolidol, extracted
from EO of Lantana radula Sw., has demonstrated to exhibit stronger fungistatic activity against the
the phytopathogenic fungi Corynespora cassiicola than the EO extracted from Lantana camara [
33
].
Similarly, the leaf EO of Piper chaba Hunter which contained the trans-nerolidol as one of the major
constituents, exhibited antifungal activity against phytopathogenic fungi such as Fusarium oxysporum,
Phytophthora capsici,Colletotrichum capsici,Fusarium solani and Rhizoctonia solani with 55.1 to 70.3%
growth inhibition and a MIC ranging from 125 and 500 µg/mL [34].

Molecules 2016,21, 529 22 of 40
Znini et al. have also reported strong anti-fungal activity of trans-nerolidol extracted from
EOs of aerial parts of Warionia saharae ex Benth. & Coss. against the three apple phytopathogenic
fungi, Alternaria sp., Penicillium expansum and Rhizopus stolonifer causing the deterioration of apple by
significantly inhibiting the mycelial growth of all strains tested. It was also found to inhibit the fungal
spore production of Alternaria sp., P. expansum and R. stolonifera at the dosage of 1, 2 and 2
µ
L/mL air,
respectively [
26
]. Besides this result, another study conducted by Pontin et al. has revealed strong
antifungal activity of nerolidol in inhibiting mycelial growth and sclerotial production by ~85% and
~84%, respectively [
84
]. Nerolidol was also found to cause alterations in hyphal morphology and
membrane permeability as demonstrated by hyphal shrinkage and partial distortion [
84
]. In addition,
the study revealed an increase in the level of nerolidol in garlic (Allium sativum L.) tissues in response to
fungal attack by Sclerotium cepivorum [
84
]. Based on the number of studies reported on the anti-fungal
activity of trans-nerolidol, it could be suggested that trans-nerolidol is a good candidate for the
development of anti-fungal drugs.
6.5. Anti-Parasitic Activity
Parasitic diseases such as malaria, leishmaniasis, sleeping sickness and Chagas’ disease continue
to affect hundreds of millions of people around the world with a majority of them living in tropical
regions [
123
]. However, most of them live in countries where the prospects of any financial return
on investment are too low to support market-driven drug discovery and development of new drugs
on parasitic diseases. Moreover, the emergence of parasites resistant to current anti-parasitic drugs
thwarts the effort in treating the parasitic diseases. All these challenges underline the importance of
plant EO as potential novel anti-parasitic agents [89,124].
6.5.1. Anti-Leishmaniasis
Leishmaniasis is a vector-borne infection caused by protozoan parasites from the genus of
Leishmania. Leishmaniasis affects approximately 350 million people in 88 tropical and subtropical
countries. The clinical syndromes and manifestations of leishmaniasis vary widely but are often
divided into the three clinically distinct syndromes, the visceral leishmaniasis, cutaneous leishmaniasis
(CL), and mucosal leishmaniasis (ML), depending on the parasite species and the host’s immune
response [
125
]. CL has affected mankind for centuries, mainly affecting the skin or mucous membranes
and is distinguished by the presence of ulcerative skin lesions. On the other hand, VL is fatal if left
untreated and the cutaneous forms are disfiguring and mutilating. Although pentavalent antimonials
are still widely used to treat leishmaniasis, they are toxic, poorly tolerated and become increasingly
ineffective to cure drug-resistant parasites [126]. Therefore, the search of alternative drugs continues.
Recently, trans-nerolidol purified from the leaf EO of Baccharis dracunculifolia DC has been found
to mediate strong anti-leishmanial activity against promastigotes of Leishmania (L.)donovani with
an IC
50
and IC
90
values of 42 and 85
µ
g/mL, respectively [
8
]. Besides this study, nerolidol also
exhibited anti-leishmaniasis activity by inhibiting the growth of L. amazonensis,L. braziliensis, and
L. chagasi promastigotes and L. amazonensis amastigotes with
in vitro
IC
50
of 85, 74, 75, and 67
µ
M,
respectively. Moreover, L. amazonensis-infected macrophages treated with 100
µ
M nerolidol resulted
in 95% reduction in the rate of infection. Arruda et al. suggested that nerolidol at 30
µ
M mediated
anti-leishmaniasis activity through the inhibition of isoprenoid biosynthesis in L. amazonensis, as
demonstrated by the reduced incorporation of [2-
14
C] mevalonic acid or [1-
14
C] acetic acid precursors
into dolichol, ergosterol and ubiquinone in the mevalonate pathway [
86
]. However, nerolidol did not
reduce the incorporation of [1(n)-
3
H] farnesyl pyrophosphate into dolichol and ergosterol, suggesting
that nerolidol could be an inhibitor at the early step in the mevalonate pathway [
86
]. Previously, the
inhibition of isoprenoid biosynthesis pathway was shown to result in the arrest of development of
Plasmodium falciparum during the intraerythrocytic stages [
127
]. Marques et al. have also observed
similar growth inhibition of promastigotes of L. amazonensis after being treated with trans-nerolidol
purified from the leaves of Piper claussenianum (Miq.) C. DC., Piperaceae [
9
]. Trans-nerolidol was also

Molecules 2016,21, 529 23 of 40
found to induce (1) a significant inhibition (62.17%) on the arginase activity of L. amazonensis and (2) an
increase in the production of nitric oxide (NO) in L. amazonensis-infected macrophages. These results
indicated that trans-nerolidol was able to interfere with parasite-host cell interaction, thus reducing the
percentage of infected cells. Another study conducted by Camargos et al. have shown that through
electron paramagnetic resonance (EPR) spectroscopy, nerolidol was able to increase the molecular
dynamics of the lipid component in the Leishmania plasma membrane at IC
50
of 0.008
µ
M [
87
]. This
could be possibly due to the insertion of nerolidol into the lipid bilayer that act as spacers to increase
the fluidity of membranes since nerolidol has high hydrophobicity, thus causing major reorganization
in cell membranes [
128
]. Subsequently, this will lead to an increase in the overall molecular dynamics
of the membrane, causing leakage of cytoplasmic content and eventually the death of Leishmania cells.
6.5.2. Anti-Trypanosomal Activity
Trypanosomiasis, also known as sleeping sickness, is caused by protozoan parasites of African
trypanosomes (e.g., Trypanosoma brucei subspecies) and is fatal if left untreated. Its symptoms include
swollen lymph nodes, fever, extreme fatigue and rash. Trans-nerolidol purified from the aerial part of
Leonotis ocymifolia (Burm.f.) Iwarsson and leaves of Strychnos spinosa Lam. showed anti-trypanosomal
activity with IC
50
of 15.78
µ
g/mL and 1.7
µ
g/mL, respectively on bloodstream forms of T. brucei
brucei [
27
,
35
]. Mohd-Shukri et al. conducted an in-depth study about the effects of nerolidol (containing
the mixture of
˘
40% cis-nerolidol and
˘
55% of trans-nerolidol) compared to a positive control, berenil
(a standard anti-trypanosomal drug) on the morphological changes of a protozoan parasite Trypanosoma
evansi in mice by using light and electron microscopy [
85
]. Berenil elicited immediate adverse
morphological changes after 2–3 h post-treatment as demonstrated by stiffening and tapering at
both ends of the parasite as well as distorted flagella and loss of undulating membranes. On the
other hand, nerolidol only induced adverse morphological changes beginning from 23rd to 25th
day post-treatment when the parasites became stiff, lost their undulating membrane. At the 27th
day post-treatment, total disfigurement was observed, indicating that nerolidol exhibited promising
trypanosomatidal activity against the morphology of T. evansi in mice.
6.5.3. Anti-Schistosomal Activity
Schistosomiasis is caused by a trematode blood fluke of the genus Schistosoma and is one of the
most significantly neglected tropical diseases in the world [
129
]. Schistosome transmission involves
the contamination of water by faeces or urine containing eggs with a specific freshwater snail as
intermediate host, followed by human contact with water inhabited by the freshwater snail [
130
].
Its acute symptoms include fever, urticaria, diarrhea and eosinophilia. However, schistosomiasis,
if left untreated, can progress to its chronic stage, leading to inflammatory and obstructive disease
in the urinary system (S. haematobium) or intestinal disease, hepatosplenic inflammation, and liver
fibrosis [
130
]. According to a study by Parreira et al., the EO of Baccharis dracunculifolia DC. (Asteraceae)
possessed high schistosomicidal activity since all pairs of Schistosoma mansoni adult worms were
dead after 24 h incubation with the EO at concentrations of 10, 50, and 100
µ
g/mL [
8
]. However,
trans-nerolidol did not display any significant schistosomicidal activity in the tested assays with the
concentration ranging from 10 to 100
µ
M. In contrast, another experiment conducted by
Silva et al.
have revealed that nerolidol in the form of cis- and trans-nerolidol racemic mixture (1:1) exerted
anti-schistosomal activity by reducing worm motor activity and causing the death of all male and
female schistosomes of Schistosoma mansoni at concentrations of 31.2 and 62.5
µ
M, respectively [
88
].
The differences between these two results could be due to the fact that trans-nerolidol isomer is less
active than the racemic mixture of cis- and trans-nerolidol [
88
]. The study also found that nerolidol
induced (1) severe tegumental damage in adult schistosomes and (2) alterations on the tubercles of
male parasites in a concentration-dependent manner. With the available findings, a mixture of cis- and
trans-nerolidol was shown to be a promising candidate to treat schistosomiasis.

Molecules 2016,21, 529 24 of 40
6.5.4. Anti-Malarial Activity
Malaria is an infection caused by the protozoan parasites belonging to the genus of Plasmodium
and is transmitted via the bite of Anopheles mosquito [
131
]. Its symptoms are fever, headache, vomiting,
sweating and fatigue. If left untreated, it can cause organ failure, abnormal blood coagulation and
ultimately death. According to a study conducted by Lopes et al., nerolidol was found to exhibit strong
anti-malarial activity since treatment of Plasmodium falciparum with 100 mg/mL of nerolidol extracted
from the leaf EO of Virola surinamensis (Rol. ex Rottb.) Warb. for 48 h resulted in 100% of the inhibition
on the development of young trophozoite to the schizont stage without pigment formation [
29
].
Similarly, nerolidol (23.7%), which is one of the major volatile components extracted from inflorescences
oil of Piper claussenianum (Miq.) C. DC., has been demonstrated to exert anti-malarial activity with
IC
50
of 11.1
µ
g/mL whereas the crude oil of P. claussenianum showed IC
50
of 7.9
µ
g/mL [
89
]. The
study suggested that nerolidol may exert the inhibition of glycoprotein biosynthesis by repressing the
biosynthesis of N-glycoproteins that are otherwise observable in P. falciparum mainly at the ring and
young trophozoite stages of the intra-erythrocytic cycles [
29
]. Besides this mechanism of action, another
study conducted by Rodrigues Goulart et al. has shown that the nerolidol inhibited the biosynthesis of
the isoprenoid chain attached to the benzoquinone ring in the intraethryocytic stages of Plasmodium
falciparum [
90
]. It was evidenced that nerolidol interfered with isoprenoid biosynthesis of apicoplast
by disrupting the elongation of isoprenic chains via inhibition of isoprenyl diphosphate synthases,
an enzyme that is responsible for the formation of isoprenoid compounds such as dolichols. Beside
isoprenyl diphosphate synthase, nerolidol also inhibited the enzyme octaprenyl phosphate/phytoene
synthase which is localized in the cytoplasm and also in mitochondria at the intra-erythrocytic stages
of P. falciparum [
132
]. Moreover, treatment with nerolidol at doses 2.2 times below the IC
50
of 0.12
µ
M
was shown to inhibit the production of isoprenic chain attached to coenzyme Q at all intraerythrocytic
stages of P. falciparum [
91
]. These findings indicated that nerolidol possesses strong anti-malarial
activity by inhibiting the development of the intraerythrocytic stages of the parasites.
Besides displaying anti-malarial activity alone, nerolidol has also been found to exhibit a
synergistic effect with either fosmidomycin or squalestatin against malarial parasites. The combination
of nerolidol with either fosmidomycin or squalestatin resulted in strong supra-additive (the sum of the
fractions of IC
50
of <1) interaction in mediating inhibition of plasmodial isoprenoid pathway against
P. falciparum with strong combinatorial IC50 of 0.57 and 0.54 µM respectively [92].
6.5.5. Other Anti-Parasitic Activities
Nerolidol demonstrated strong nematicidal activity against a nematode, Caenorhabditis elegans
with its LC
50
value of 12
µ
g/mL as well as 74.0% mortality at 50
µ
g/mL [
94
]. Besides nematicidal
activity, nerolidol (a mixture of cis- and trans-nerolidol) inhibited the
in vitro
growth of four Babesia
species with IC
50
values of 21
˘
1, 29.6
˘
3, 26.9
˘
2, and 23.1
˘
1
µ
M for B. bovis,B. bigemina, B. ovata,
and B. caballi, respectively. This anti-parasitic activity could be due to inhibition of the isoprenoid
pathway by nerolidol by a similar mechanism similar to that found with Plasmodium falciparum [
93
].
Nerolidol was also found to be the most active compound among the tested sesquiterpenes (nerolidol,
farnesol and elemol) that caused the death of nematodes, L
3
larvae of Anisakis simplex type I with the
mortality at 4 hours of 100% at the concentrations of 31.5 and 62.5
µ
g/mL [
95
]. Moreover, only 20% of
nerolidol-treated rats were affected by gastric wall lesions caused by Anisakis larvae in comparison to
86% of the control rats [95].
6.6. Insect Repellent Activity
There is a growing concern about the usage of current commercial synthetic insecticides due to the
increasing difficulty in the management of pesticide resistance [
133
]. For this reason, the researchers
have focused on research of EOs that have been traditionally used as repellants. Studies in several
countries have shown that certain plant EOs are effective not only in repelling insects, but have contact

Molecules 2016,21, 529 25 of 40
and fumigant insecticidal activity against specific pests without harmful side-effects to humans and
animals [134,135].
The combination of nerolidol and linalool (that are purified from EO of Capparis tomentosa fresh
leaves) showed significant repellence activity against maize weevil Sitophilus zeamais at all tested doses
(0.002, 0.02, 0.2 and 2
µ
L) [
30
]. In another study, trans-nerolidol derived from the EO of Siam-wood
(Fokienia hodginsii (Dunn) A.Henry & H H.Thomas) was shown to possess insecticidal activity with
LD50 value at 0.17 µmol/fly [51].
A mixture of nerolidol and tea tree oil with a ratio of 2:1 (tea tree oil 0.5% plus nerolidol 1%) was
shown to exert insecticidal and ovicidal activity against Pediculus capitis (head lice) and its eggs [
96
].
Besides, nerolidol purified from the seeds of Magnolia denudata Desr. also showed larvacidal activity
against third-instar larvae of insecticide-susceptible Culex pipiens pallens and Aedes aegypti as well as
the wild Aedes albopictus and Anopheles sinensis with lethal dose (LD)
50
values of 9.84, 13.85, 16.34 and
20.84 mg/L respectively [
47
]. Similarly, trans-nerolidol, which is one of the components of EO from the
leaves of Melaleuca quinquenervia (Cav.) S.T.Blake, at its concentration of 0.1 mg/mL exhibited strong
larvicidal activity with ě95% mortality against Aedes aegypti [36].
Meanwhile, the trans-nerolidol that was purified from the leaves of Piper aduncum L. possessed
strong acaricidal activity due to its highest repellency of 83.2%
˘
0.59% compared to
α
-humulene
(73.3%
˘
0.83%) and
β
-caryophyllene (70.7%
˘
0.88%) against the two-spotted spider mite, Tetranychus
urticae Koch that causes damage to many agricultural crops [
37
]. The EO of aerial parts of Baccharis
dracunculifolia DC. containing nerolidol as one of the major components was discovered to demonstrate
strong acaricidal activity by causing 100% mortality of Rhipicephalus microplus larvae (cattle tick that
infests cattle) at 20.0 mg/mL [
97
]. Meanwhile, a 100% mortality of Rhipicephalus microplus larvae
was achieved at a lower concentration of nerolidol (15.0mg/mL). The study also demonstrated that
nerolidol reduced the quality of the egg and larval hatching rate with increasing concentration from
20 to 50mg/mL [97].
6.7. Anti-Ulcer Activity
Gastric ulcer affects thousands of people around the world and is known to be caused by an
imbalance between aggressive (acid, pepsin) and protective factor (secretion and action of mucus
and bicarbonate) in the stomach [
98
]. It is induced by several factors, such as stress, smoking,
nutritional deficiencies and ingestion of non-steroidal anti-inflammatory drugs (NSAIDs). The current
therapy for ulcers usually involves the use of histamine H
2
-antagonists, proton pump inhibitors
and anti-muscarinics for the inhibition of gastric acid secretion. However, these drugs pose severe
side-effects, particularly hypersensitivity, arrhythmia and impotence [
136
]. With this in mind, plant
EOs have recently been exploited as they have been shown to produce promising results for alternative
therapies to treat gastric ulcers with lesser side-effects.
A study has been conducted by Klopell et al. on the anti-ulcer property of nerolidol using different
experimental models such as ethanol-, indomethacin- and stress-induced ulceration in rat [
98
]. In the
stress-induced ulceration model of experiment, nerolidol treatment at 50, 250 and 500 mg/k caused
a significant reduction in the ulcerative lesion index (ULI) by 41.22, 51.31 and 56.57, respectively
when compared to the control group animals. With regard to the ethanol-induced ulceration model
of experiment, treatment with nerolidol at 250 and 500 mg/kg significantly inhibited the formation
of ulcer at 52.63% and 87.63%, respectively as compared to the control group. On the other hand,
indomethacin-induced ulceration model of experiment, the treatment at 250 and 500 mg/kg of nerolidol
had significantly inhibited the gastric ulcer for 51.02% and 46.93%. These findings indicate that
nerolidol could be used as an active component in gastroprotective and anti-ulcer treatments.
6.8. Skin Penetration Enhancer Activity
Transdermal delivery has gained a lot of attention as an attractive alternative route to intravenous
and oral drug delivery systems [
137
]. However, the application of transdermal delivery is limited

Molecules 2016,21, 529 26 of 40
by poor drug permeability as the stratum corneum plays as a rate-limiting lipophilic barrier against
the uptake of chemical and biological agents [
138
]. Therefore, terpenes are often used as topical skin
penetration enhancers due to their wide range of physicochemical properties such as low cutaneous
irritancy and good toxicological profile as well as adsorption enhancement ability [139].
Nerolidol has been found to be a potent skin penetration enhancer. It was found to increase the
diffusion rate by over 20-fold for transdermal delivery of several drugs especially on 5-fluorouracil [
99
].
This high permeation-enhancing ability was attributed to the structure of nerolidol which is suitable
for the alignment within lipid lamellae of the stratum corneum in order to disrupt the organization of
stratum corneum. This view has been further supported by Prasanthi and Lakshmi [
100
] who reported
that nerolidol with highest lipophilicity (log P = 5.36
˘
0.38) have the highest enhancement effect with
its rank of order of nerolidol > farnesol > limonene > linalool > geraniol > carvone > fenchone > menthol
in facilitating transdermal delivery of alfuzosin hydrochloride. Similarly, nerolidol has the highest
permeation enhancing ability with a 3.2-fold increase in permeation of selegiline hydrochloride across
the rat skin, followed by carvone (2.8-fold increase) and anethole (2.6-fold increase) [
101
]. Another
study conducted by El-Kattan et al. has shown that nerolidol was the most effective percutaneous
permeation enhancer for four model drugs (nicardipine hydrochloride, hydrocortisone, carbamazepine,
and tamoxifen) when compared to other terpenes (fenchone, thymol, nerolidol and D-limonene) [
102
].
6.9. Anti-Nociceptive and Anti-Inflammatory Activity
Pain is an unpleasant sensation and emotional experience that is associated with actual or
potential tissue damage [
140
]. On the other hand, the stimulation of nociception is associated
with the detection of real tissue injury or a potentially damaging event by nociceptors as a stimuli
(transduction) followed by its transmission of encoded information to the brain [
141
]. Under normal
circumstances, the presence of an injury activates the inflammatory response as follows: firstly the
inflammatory mediators are released from damaged cells such as ions (K
+
, H
+
), bradykinin, histamine,
5-hydroxytryptamine (5-HT), ATP and nitric oxide. Subsequent activation of arachidonic acid pathway
leads to the production of prostanoids and leukotrienes that would then lead to the release of more
inflammatory mediators such as cytokines and growth factors. These mediators will ultimately
activate peripheral nociceptors directly, resulting in spontaneous pain. Beside this action, they also act
to convert responses of primary afferent neurons to subsequent stimuli (peripheral sensitization) to be
transmitted to the brain. Due to exacerbated physiological response, chronic exposure to pain is very
harmful to an individual as it can cause organ damage and ultimately death, if left untreated [142].
Non-steroid anti-inflammatory drugs (NSAIDs) are well-known analgesic drugs that act to reduce
inflammation and pain by acting as an inhibitor of cyclooxygenases (COXs) in the arachidonic acid
pathway. However, one major disadvantage of administration of NSAIDs is their serious side-effects
such as significant gastrointestinal upset, gastritis, ulceration, hemorrhage, and even death [
143
].
In order to address this issue, EOs extracted from various medicinal plants have been increasingly
explored as alternative traditional medicines to treat inflammation and pain without posing harmful
side-effects.
Pinheiro et al. investigated the anti-nociceptive and anti-inflammatory effects of EO of
Peperomia serpens (Sw.) Loudon in rodents [
38
]. The EO has been found to possess anti-inflammatory
and anti-nociceptive activities which could possibly be mediated by one of its major compounds,
trans-nerolidol (38.0%). In a similar experiment, Lima et al. reported anti-nociceptive and
anti-inflammatory activities of the EO of the aerial parts of Piper aleyreanum C. DC which is attributable
to the presence of trans-nerolidol (1.2%) [
10
]. In order to strengthen the findings, Fonsêca et al.
investigated the anti-nociceptive and anti-inflammatory activities of nerolidol using mouse models
of pain [
103
]. The study found that nerolidol has no effect on the locomotor activity. Meanwhile,
anti-nociceptive activity was evaluated via acetic acid-induced writhing and the formalin tests. The
results demonstrated that oral administration of nerolidol was able to cause lesser acetic acid-induced
abdominal contractions and also inhibition in paw licking behavior in the respective tests when

Molecules 2016,21, 529 27 of 40
compared to the control group (Table 4). These results implied that nerolidol modulates its effect on
neuropathic pain and inflammatory processes as demonstrated by the formalin test [
103
]. However,
the anti-nociceptive effect of nerolidol did not involve the thermal stimulation of centrally mediated
nociception as shown by the negative hot-plate test result. In addition, the researchers also further
elucidated the possible anti-nociceptive mechanisms of nerolidol by examining its effects on the
parameters GABAergic system, opioidergic and ATP-sensitive K+ channels. The results have shown a
positive association of nerolidol with the GABAergic system but not with opioidergic or ATP-sensitive
K+ channels, implying that the anti-nociceptive activtity of nerolidol is mediated through GABA
A
receptors [
103
]. In order to evaluate the anti-inflammatory activity of nerolidol, carrageenan-induced
paw edema was used as a model of inflammation. It was found that nerolidol exhibited inhibitory
effect on inflammation. Further investigation of carrageenan-induced peritonitis model revealed
that nerolidol decreased the levels of polymorphonuclear cells and tumor necrosis factor (TNF-
α
)
in peritoneal lavage as well as interleukin 1 beta (IL-1b) in LPS-stimulated, peritoneal macrophages
(Table 4) [
103
]. Taken these results together, nerolidol has been shown to demonstrate promising
analgesic and anti-inflammatory activities.
6.10. Anti-Cancer and Anti-Tumor Activity
Cancer is one of the most alarming causes of death, with an estimated over six million deaths
have been reported around the world annually. It is a multifactorial disease that leads to uncontrolled
growth and invasion of abnormal cells, ultimately leading to the formation of tumor. Chemotherapy,
radiosurgery and surgery are some of the effective treatments against various type of tumors. Despite
that, these treatments still pose many side-effects that lead to acute and chronic organ damage such as
bone marrow suppression, hepatic, pulmonary, cardiac, renal and gastrointestinal toxicities [
144
,
145
].
Moreover, the development of drug resistance in tumors have also been reported during the courses
of chemotherapy. This may be due to the occurrence of mutations in the tumor cells that negates
the apoptotic pathway during cancer treatment [
146
]. These drawbacks of chemotherapy treatment
have urged researchers to find alternative treatments without harming the growth of normal cells and
triggering anti-tumor drug resistance. Among the alternative approaches, plant phytochemicals have
been recently explored for their possible beneficial (anti-proliferative and cytotoxic) effects on cancer
cells in vitro or in vivo models [147,148].
6.10.1. In Vitro Studies
Several studies have shown anti-tumor properties of nerolidol on cancer cell lines. A study
conducted by Ryabchenko et al. has demonstrated strong anti-tumor effects of nerolidol (a combination
of cis-nerolidol 40.7%, trans-nerolidol 58.3%, cis-dihydronerolidol 0.4% and trans-dihydronerolidol).
It was found to reduce the viability of HeLa cells at its concentration (CC
50
) of less than 5
µ
M
(1.5 ˘0.7 µM)
[
104
]. Beside this study, nerolidol (isomer not specified), which is one of the ten
major compounds found in the green tea flavor, exhibited strong cytotoxicity effect with an IC
50
value of 2.96 and 3.02
µ
g/mL against BT-20 breast carcinoma cells and HeLa cells, indicating
that nerolidol potentially exhibit strong anti-cancer activity against the two tumor cell lines [
106
].
This has been further supported by the fact that trans-nerolidol, which was purified from the leaf
EO of Zornia brasiliensis Vogel, had a strong cytotoxicity activity against cancer cell lines such as
B16-F10 (mouse melanoma), HepG2 (human hepatocellular carcinoma), HL-60 (human promyelocytic
leukemia) and K562 (human chronic myelocytic leukemia) with IC
50
values of >25, >25, 21.99 and
17.58
µ
g/mL, respectively using Alamar blue assay [
39
]. The study also reported no cytotoxicity effect
of trans-nerolidol on non-tumor cells, particularly the peripheral blood mononuclear cells (PBMCs) [
39
].
Another study conducted by Boris et al. have shown that cis-nerolidol exhibited the strongest cytotoxic
activity (16.5
˘
6.7
µ
M) against HeLa cells among other sesquiterpene alcohols such as
α
-bisabolol,
cedrol, patchoulol, and santalol [
105
]. Beside green tea, trans-nerolidol extracted from leaf EO of
Comptonia peregrina (L.) Coult. has been found to induce strong cytotoxic effect against human

Molecules 2016,21, 529 28 of 40
lung carcinoma A-549 and colon adenocarcinoma DLD-1 cell lines with IC
50
values of 6.4
˘
0.4 and
5.8
˘
0.4
µ
g/mL, respectively [
149
]. Another anti-proliferative study conducted by Ambrož et al. has
shown that trans-nerolidol potentiated the action of doxorubicin, an anticancer drug, by increasing
killing of CaCo-2 cancer cells [
40
]. The study also reported no cytotoxicity effect of trans-nerolidol
on rat hepatocytes that serve as non-tumor cells [
145
]. Based on the literature, it can be suggested
nerolidol is a good candidate for the development of anticancer agent that selectively targets specific
cancerous cells with no cytotoxicity towards PBMCs and rat hepatocytes.
When examining the cytotoxic effect of nerolidol with regard to the apoptotic pathway, the
combination of two acylic isoprenoids, farnesol and nerolidol was found to suppress the proliferation
of human HL-60 acute promyelocytic leukemia (HL-60) cells by 20%, which was slightly synergistic
(slightly exceeding the 13% suppression obtained from the sum of both compounds); farnesol isomers
(2.5
µ
mol/L) and nerolidol (5
µ
mol/L) individually suppressed the proliferation of HL-60 cells by
4% and 9%, respectively [
108
]. The mechanism of action involved prolonging the cell cycle arrest of
HL-60 cells at the G
0
-G
1
/S interphase and lead to apoptotic cell death. Another study conducted by
Hanušová et al. focused on the anti-proliferative effect of EO from leaves of Myrica rubra (Lour.) Siebold
& Zucc. (MEO) and its major compound, trans-nerolidol on the adhesion, expression of adhesion
molecules (ICAM-1; E-cadherin;
β
-catenin and apoptotic molecules (NF-
κ
B, caspases) in colorectal
cancer cell line HT29 [
147
]. The study showed that only the MEO reduced the cell adhesion to collagen.
Meanwhile, both MEO and trans-nerolidol (30
µ
g/mL) significantly suppressed cell adhesion of HT29
cells in the presence of TNF
α
and it was suggested due to the down-regulation of ICAM-1 [
109
].
Besides, trans-nerolidol (30
µ
g/mL) also significantly increased the expression of E-cadherin [
109
], a
cell adhesion molecule that mediates the suppression of epithelial cell tumor invasiveness [
150
]. MEO
and trans-nerolidol were also found to decrease the phosphorylation of NF-
κ
B and activate caspases
activity in TNFα-induced HT29, thereby leading to apoptosis of cancer cells [109].
6.10.2. In Vivo Studies
In animal studies, nerolidol has been found to possess strong anti-tumor activity by inhibiting the
intestinal carcinogenesis induced by azoxymethane (15 mg/kg body weight) administered twice per
week for a duration of three weeks in male F344 rats [
107
]. The result showed the reduction of incidence
of intestinal neoplasia from 82% to 33% in rats fed with nerolidol. Moreover, the number of tumors/rat
was reduced from 1.5 to 0.7 in rats fed with nerolidol. The improvement of intestinal carcinogenesis
was possibly due to the modulatory effect nerolidol on protein prenylation, a post-translational process
that is required to cause cancer [
151
]. Another study conducted by Costa et al. has demonstrated
that the EO of Zornia brasiliensis Vogel leaf at dose of 100 mg/kg containing trans-nerolidol as major
constituent reduced the weight of tumor in mice injected with B16-F10 melanoma by 38.61% when
compared to the untreated group [39].
7. Pharmacokinetic Studies
Although there is an increasing popularity of herbal medicines and essential oils, they are not
properly screened for purity and potency which may raise serious questions regarding their possible
herb-drug interaction with conventional medicine that may cause serious adverse effects on human
health [
152
]. In order to address this issue, more pharmacokinetic and toxicological research have been
conducted to examine the efficacy and safety of essential oils.
7.1. In Vitro Studies
In order to determine the recovery of nerolidol, Saccharomyces cerevisiae prototrophic haploid strain
IWD72 was incubated in YEPD medium comprised of yeast extract, bacteriological peptone, glucose,
adenine, and uracil. Nerolidol (100
µ
g/mL) was subsequently added to the 50 mL bacteria culture in
YEPD medium. The aerobic culture cells were harvested by centrifugation after 24 h, and the cells were
collected for the recovery of nerolidol. Residual nerolidol recovered at 24 h was 79.0 µg/mL [14,153].

Molecules 2016,21, 529 29 of 40
7.2. In Vivo Studies
Rats were fed 20 and 40 mg of nerolidol that were mixed with 1 mL of cottonseed oil and 30–35 mL
of evaporated milk per day, for eight days. The average daily excretion on the 1st to 4th and 4th to 8th
day was monitored. Rats fed with 20 mg/day of nerolidol excreted 0.3 and 0.7 mg of nerolidol per
day with a maximum average of 0.9 mg. On the other hand, the rats fed with 40 mg/day of nerolidol
excreted 1.0 and 1.6 mg per day with a maximum average of 2.1 mg [14,154].
Based on a recent
in vivo
pharmacokinetic study conducted by Saito et al., nerolidol
(cis-/trans-nerolidol, 1:3; w/w) was quantitatively determined in mouse plasma using GC-MS
method [
22
]. Three BALB/c mice weighing 20
˘
2 g were firstly fed orally with a single oral dose
of 1000 mg/kg of nerolidol. Blood samples were then taken at 30 min, 1, 2, 3, 4, 5, 6, 8, and 12 h
after oral administration, followed by separation of plasma from blood via centrifugation and GC-MS
analysis. The level of nerolidol was observed in the plasma with its maximum concentration of
„
0.27
˘
0.07
µ
g/mL within 30 min after oral administration and remained constant for up to 3 h after
administration, reaching a maximum concentration of ~0.35
˘
0.05
µ
g/mL after 6 h of administration.
The concentration of nerolidol in plasma after 6 hours was twice the IC
50
concentration of
in vitro
nerolidol administration on P. falciparum (0.169
µ
g/mL) [
155
]. Moreover, this maximum concentration
was detected to be ~1460 times lower than the concentration required to induce 50% hemolysis
(
„
511
µ
g/mL). The concentration of nerolidol in plasma decreased to near zero 12 h after oral
administration. These results indicated that the maximum concentration in mouse plasma after oral
administration was below the hemolytic concentration, thus the safe oral dose is up to 1000 mg/kg.
In another recent study, He et al. utilized LC-MS instead of GC-MS method to quantitatively
determine the
in vivo
pharmacokinetics of nerolidol (cis-/trans-nerolidol, 2:3) in rat plasma [
25
].
Sprague-Dawley rats weighing 250–300 g were firstly administered only once with 25 mg/kg of
nerolidol via intraperitoneal injection. Blood samples were then collected at 10, 20, 30, 60, 90, 120, 240
and 360 min after injection, followed by separation of plasma from blood via centrifugation. Through
the LC-MS analysis, they revealed that the maximum concentration of nerolidol observed in rat plasma
was 8.30
˘
1.07
µ
g/mL at 20 min after single intraperitoneal injection. The concentration of nerolidol
in plasma decreased to near zero two hours after intraperitoneal injection.
Table 5.
Comparison of pharmacokinetic studies of nerolidol conducted by Saito et al. and He et al. [22,25]
Parameters Saito et al. [22] He et al. [25]
Type of nerolidol Mixture of cis- and
trans-nerolidol (1:3)
Mixture of cis- and
trans-nerolidol (2:3)
Analytical method used GC-MS LC-MS
Animal used BALB/c mice Sprague-Dawley rats
Route of administration Oral Intraperitoneal injection
Dosage (mg/kg) 1000 25
Type of sample used Plasma
Time collection taken (min) 30, 60, 120, 180, 240, 300,
360, 480 and 720
10, 20, 30, 60, 90, 120, 240
and 360
Peak plasma concentration (Cmax) (µg/mL) ~0.27 ˘0.07 8.30 ˘1.07
Peak time (Tmax) (min) 30 20
Elimination half life (T1/2) (min) n.a. 20.98 ˘7.71
Mean residence time (MRT) (min) n.a. 27.72 ˘2.14
Clearance (L/min/kg) n.a. 0.082 ˘0.012
Time for drug to be eliminated to almost near zero 12 ~2
Human equivalent dose a(HED) (mg/kg) 81.08 4.05
Key: (i) n.a. = data not available; (ii) a = HED values were calculated based on the formula for dose translation
based on body surface area (BSA) [
156
] as follows: Human equivalent dose (HED) (mg/kg) = Animal dose
(mg/kg) ˆAnima l Km
Huma n Km ; with adult human, rat and mouse Kmfactors of 37, 6 and 3 respectively [156].
A comparative pharmacokinetic study between work by Saito et al. [
22
] and He et al. [
25
] revealed
that the oral administration of nerolidol exhibited lower peak plasma concentration than that of
intraperitoneal administration (Table 5). This is because orally administered nerolidol must first

Molecules 2016,21, 529 30 of 40
undergo first-pass effect by which it must pass through the intestinal wall and then to the portal
circulation and liver [
157
]. As a result, a portion of the oral dose of nerolidol was lost during the
first pass metabolism in the liver, thus contributing to low bioavailability as compared to that of
intraperitoneal injection [
158
]. Another key point of the pharmacokinetic comparison between the two
studies is that the human equivalent doses (HEDs) of both administration calculated in Table 5can be
used as appropriate starting doses of nerolidol in human clinical trials.
7.3. Toxicological Studies
Despite the long history of therapeutic uses of EOs and its general acknowledgment by the public,
the assumption that the HEDs can be used for the first clinical trial has however seldom been verified,
thus raising the concern of safety issues pertaining the usage of EOs [
159
]. For that reason, toxicological
screenings are required to assess the potential toxicities induced by the EOs.
7.3.1. Acute Toxicity
The dermal LD
50
values of nerolidol (isomer not specified) in rabbit was found to be higher than
2000 mg/kg body weight, thus indicating low acute toxicity via transdermal route [
13
]. In terms of
oral administration, the oral LD
50
values of nerolidol in rats and mice were higher than 2000 mg/kg
body weight (>5000 and 9976 mg/kg body weight, respectively), indicating low acute toxicity via oral
route [13].
7.3.2. Skin Irritation and Sensitization Studies
In human studies, 4% nerolidol (isomer not specified) administration in a pre-test for a
maximization study with single occlusive application for 48 h did not cause skin irritation [
13
].
On the other hand, the application of undiluted nerolidol (isomer not specified) on intact and abraded
skin caused well-defined erythema or slight edema in rabbits after 48 h, whereas 5% nerolidol in
diethylphthalate caused very slight edema in one animal and was cleared after 48 h [
13
]. With regard
to skin sensitization, human studies have shown that nerolidol (isomer not specified) (4%) did not
cause any positive reactions in maximization tests or repeated insult patch tests. In animal studies,
the administration of nerolidol in guinea pigs resulted in weak reactions in two adjuvant tests with
concentrations of 3% and 10% [
160
,
161
]. However, no cross-sensitization was observed when guinea
pigs induced with farnesyl acetate were cross-sensitized with nerolidol.
7.3.3. Mucous Membrane Irritation
No human data is available for mucous membrane irritation studies [
13
]. However, in animal
studies, undiluted nerolidol initially caused very slight redness, but was cleared by 2 h. On the other
hand, 5% nerolidol in DEP poses no mucous membrane irritation [13].
7.3.4. Phototoxicity and Photoallergenicity
UV spectra for nerolidol indicated that it did not absorb UVB light (290–320 nm) [
162
]. However,
nerolidol peaked at the UVC range (220–240 nm) with a very slight absorption at 250–300 nm range,
thus do not possibly induce phototoxicity or photoallergy under the current conditions of use as
fragrance ingredients [13].
7.3.5. Reproductive and Developmental Toxicity
In order to investigate the development of fetal epidermal permeability barrier
in vitro
, the
activators of the receptors for vitamin D
3
and retinoids, and of the peroxisome proliferator activated
receptors (PPARs) and the farnesoid X-activated receptor (FXR) were examined. Sprague-Dawley rats
were firstly impregnated (plug date = day 0) in order for the skin of 17-day old fetus to be cultured
for the measurement of barrier function. The effect of activators of FXR, isoprenoid precursors and

Molecules 2016,21, 529 31 of 40
metabolites on the development of epidermal barrier was monitored. Explants were incubated in the
presence of 100
µ
M nerolidol for two days. Full-thickness flank skin was excised from fetal rats for skin
analysis with light and electron microscopy. The results have shown that nerolidol did not activate the
FXR, thus did not alter the epidermal barrier during skin development [14,163].
7.3.6. Cytotoxicity and Genotoxicity
In Vitro Studies
Due to nerolidol being used as a potent skin permeation enhancer with low toxicity, a number
of studies were conducted on the toxicological properties of nerolidol. Mendanha et al. compared
the hemolytic and toxic effects of nerolidol and various monoterpenes on fibroblast cells as well
as their effect on erythrocyte membrane fluidity [
155
]. By using the 3T3 NRU assay to evaluate
the cytotoxicity of nerolidol and various monoterpenes (
α
-terpineol, L-(
´
)-carvone, (+)-limonene,
L-menthone, D,L-menthol, pulegone and 1,8-cineole) on fibroblast cells, nerolidol was found to be
the most cytotoxic with its IC
50
value of 0.06
˘
0.01 mM. Besides, nerolidol caused 50% hemolysis at
2.3
˘
0.8 mM and induced a significant increase in the fluidity of erythrocyte membrane at 2.5
ˆ
10
9
molecules/cell, indicating that the nerolidol possessed the highest hemolytic effect on erythrocyte
membrane fluidity when compared to terpenes. In addition, electron paramagnetic resonance (EPR)
spectroscopy of the spin label 5-doxyl stearic acid (5-DSA) was used to investigate the effect of terpenes
on membrane fluidity in erythrocyte and fibroblast cells. Nerolidol was found to be more potent than
terpenes that caused an increase in the membrane fluidity. The results implied that nerolidol was able
to increase membrane fluidity but also had increased ability to disrupt the membrane and had higher
cytotoxic potential.
Ferreira et al. further examined the toxicity of nerolidol (a racemic mixture of cis- and trans-isomers)
(1:1) on mitochondrial and cellular energetics in
in vivo
model using Wister rat liver mitochondria and
in vitro
model using HepG2 (human hepatocellular liver carcinoma) cells [
12
]. In the
in vitro
study,
nerolidol exertes hepatic cell cytotoxicity due to a decrease in ATP/ADP levels by negatively interfering
with hepatic mitochondrial bioenergetics in concentrations lower than 2.4
µ
M. Consequently, nerolidol
induced cell arrest and cell death which is possibly due to the inhibition of F
0
F
1
-ATP synthase in a
concentration-dependent manner. In the
in vivo
study, nerolidol (low doses up to 2.4
µ
M) induced a
decrease in transmembrane electric potential in the mitochondrial membrane isolated from rat liver in a
concentration-dependent manner [
12
]. By decreasing transmembrane electric potential, nerolidol could
negatively affect the hepatic mitochondrial bioenergetics as it would cause mitochondrial dysfunction,
thus leading to hepatic cell cytotoxicity and eventually cell death [164].
Marques et al. investigated the cytotoxicity effect of EO extracted from leaves of P. claussenianum
and reported no toxicity effect in the fibroblasts nor macrophages cell lines in any concentration tested
(ranging from 40 to 0.56 mg/mL) [
9
]. Similarly, the EO extracted from leaves of P. claussenianum did not
induce toxicity on the L929 mouse fibroblast cells [
78
]. Peres et al. investigated the cytotoxicity effect of
EO purified from Piper gaudichaudianum Kunth in which its major compounds were trans-nerolidol,
α
-humulene, (E)-caryophyllene and bicyclogermacrene [
165
]. Although dose-dependent cytotoxicity
effect of the EO as well as single-strand DNA breakage were observed in the Chinese hamster lung
fibroblast cells (V79 cells), however, no double-strand breaks occurred. Furthermore, the EO induced a
significant increase in lipid peroxidation at higher dose. The results indicated that the EO possessed
strong cytotoxicity, genotoxic and mutagenic effects which were attributed to the role of nerolidol. Due
to this reason, Sperotto et al. further investigated the cytotoxic and mutagenic properties of the EO of
P. gaudichaudianum as well as its major compound trans-nerolidol using Saccharomyces cerevisiae
as a model organism [
166
]. P. gaudichaudianum EO was found to induce cytotoxic effects in the
XV185-14c and N123 strains of S. cerevisiae but induced mutagenesis only at the lys1 locus at the
highest concentration of 100
µ
g/mL. On the other hand, nerolidol (a racemic mixture of cis- and
trans-isomers) (1:1) was found to be cytotoxic in XV185-14c at concentration of 25, 50 and 100
µ
g/mL

Molecules 2016,21, 529 32 of 40
and did not significantly cause any induction of mutagenicity at the three loci evaluated. Moreover, EO
and nerolidol were discovered to generate ROS via DCF-DA probing assay in superoxide dismutase
(Sod)-deficient strains. Based on these findings, Sperotto et al. [
166
] claimed that nerolidol shows a
weak mutagenic effect but exerts strong cytotoxicity effect which could be attributed to the formation
of reactive oxygen species (ROS) and the formation of single-strand breaks. Despite that, more in
depth analysis involving molecular techniques could be conducted to elucidate the mechanisms
that are responsible for the strong cytotoxic but weak mutagenic effects of nerolidol. Furthermore,
toxicogenomics could be an option to be considered in further evaluating the safety of nerolidol. Briefly,
toxicogenomic is based on the integration of genomics and toxicology with the aim of studying the
toxicity of xenobiotics on the biological systems via global analysis of genome-wide mRNA expression
(transcriptomics), protein (proteomics) and metabolite patterns (metabonomics) [
167
]. Due to its
wide usage in the research of plant-based medicinal natural products, particularly in traditional
Chinese medicine (TCM) [
168
], toxicogenomics could perhaps be an effective tool in evaluating the
safety of nerolidol at the genomic level to ensure that it is safe for humans. Besides that, more
in vitro
experiments investigating the induction of micronuclei, chromosome aberration or telomere shortening
effect of nerolidol may also be performed in order to confirm the genotoxicity status of nerolidol in
human or mammalian cells.
In Vivo Studies
Pículo et al. had investigated the genotoxicity and clastogenicity (ability to cause DNA damage
by inducing chromosomal aberrations) effects of trans-nerolidol in blood and liver cells of 12-week-old
male Swiss albino mice (Mus musculus) using comet and micronucleus assays respectively [
169
]. The
experiment was based on the cytotoxicity effect analysis by scoring 200 consecutive total polychromatic
(PCE) and normochromatic (NCE) erythrocytes (PCE:NCE ratio) in bone marrow cells and no
significant decrease in PCEs:NCEs ratios at the three doses tested (250, 500 and 2000 mg kg
´1
) was
observed, indicating the absence of cytotoxic effects of trans-nerolidol at these doses. Nevertheless,
weak genotoxic effects of trans-nerolidol in the blood and liver cells were observed and a slight increase
in the DNA damage at higher doses. Based on the available studies, it can be deduced that the
non-toxic dose of trans-nerolidol for animal is up to 2000 mg/kg. Moreover, no DNA damage was
observed in the peripheral blood cells and liver of the animals. In order to determine the safety of
nerolidol consumption in humans, more clinical trials should be conducted to assess and validate the
toxicity and side effects of nerolidol in humans.
8. Conclusions
Nerolidol is one of the common components found in the essential oil of various medicinal plants.
A majority of the studies reveal that nerolidol is the major constituent in many plants that accounts
for their pharmacological and biological activities such as anti-microbial, anti-parasitic, anti-biofilm,
anti-oxidant, anti-nociceptive, anti-inflammatory, anti-ulcer, skin penetration enhancer, insect repellent
and anti-cancer properties. Based on pharmacokinetic and toxicological data available, the dosage
of nerolidol is considered safe to be translated from animal to clinical studies in order to evaluate its
efficacy. Taken all together, nerolidol has a great potential to be used as a new chemical or therapeutic
drug in the field of agriculture and medicine, respectively and sufficient baseline information is
available for guiding future works and commercial exploitation.
Acknowledgments:
This work was supported by the Monash University Malaysia ECR Grant (5140077-000-00),
Ministry of Science, Technology and Innovation Malaysia (MOSTI), eScience Funds (02-02-10-SF0215 &
06-02-10-SF0300), University of Malaya for High Impact Research Grant (UM-MOHE HIR Nature Microbiome
Grant No. H-50001-A000027 and No.A000001-50001), External Industry Grants from Biotek Abadi Sdn Bhd (vote
no. GBA-808138 and GBA-808813).
Author Contributions: All authors contributed equally.
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2016,21, 529 33 of 40
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