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Citation: Jugreet, B.S.; Lall, N.; Anina
Lambrechts, I.; Reid, A.-M.;
Maphutha, J.; Nel, M.; Hassan, A.H.;
Khalid, A.; Abdalla, A.N.; Van, B.L.;
et al. In Vitro and In Silico
Pharmacological and Cosmeceutical
Potential of Ten Essential Oils from
Aromatic Medicinal Plants from the
Mascarene Islands. Molecules 2022,27,
8705. https://doi.org/10.3390/
molecules27248705
Academic Editors: Manuela
Labbozzetta and Kemal Husnu
Can Baser
Received: 5 November 2022
Accepted: 6 December 2022
Published: 8 December 2022
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molecules
Article
In Vitro and In Silico Pharmacological and Cosmeceutical
Potential of Ten Essential Oils from Aromatic Medicinal Plants
from the Mascarene Islands
Bibi Sharmeen Jugreet 1, Namrita Lall 2,3,4, Isa Anina Lambrechts 2, Anna-Mari Reid 2, Jacqueline Maphutha 2,
MarizéNel 2, Abdallah H. Hassan 5, Asaad Khalid 6,7 , Ashraf N. Abdalla 8, Bao Le Van 9,10 ,*
and Mohamad Fawzi Mahomoodally 1, 11,12
1Department of Health Sciences, Faculty of Medicine and Health Sciences, University of Mauritius,
Réduit 80837, Mauritius
2Department of Plant and Soil Sciences, University of Pretoria, Pretoria 0002, South Africa
3School of Natural Resources, University of Missouri, Columbia, MO 65211, USA
4College of Pharmacy, JSS Academy of Higher Education and Research, Mysuru 570015, India
5Chemistry Department, College of Education, Salahaddin University, Erbil 44002, Iraq
6Substance Abuse and Toxicology Research Center, Jazan University, Jazan 45142, Saudi Arabia
7Medicinal and Aromatic Plants and Traditional Medicine Research Institute, National Center for Research,
Khartoum P.O. Box 2404, Sudan
8Department of Pharmacology and Toxicology, College of Pharmacy, Umm Al-Qura University,
Makkah 21955, Saudi Arabia
9Institute of Research and Development, Duy Tan University, Da Nang 550000, Vietnam
10 Faculty of Natural Sciences, Duy Tan University, Da Nang 550000, Vietnam
11 Center for Transdisciplinary Research, Department of Pharmacology, Saveetha Institute of Medical and
Technical Science, Saveetha Dental College, Chennai 600077, India
12 Centre of Excellence for Pharmaceutical Sciences, North-West University, Private Bag X6001,
Potchefstroom 2520, South Africa
*Correspondence: vnble@duytan.edu.vn
Abstract: In this study, 10 essential oils (EOs), from nine plants (Cinnamomum camphora,Curcuma longa,
Citrus aurantium,Morinda citrifolia,Petroselinum crispum,Plectranthus amboinicus,Pittosporum senacia,
Syzygium coriaceum, and Syzygium samarangense) were assessed for their antimicrobial, antiaging and
antiproliferative properties. While only S. coriaceum,P. amboinicus (MIC: 0.50 mg/mL) and M. citrifolia
(MIC: 2 mg/mL) EOs showed activity against Cutibacterium acnes, all EOs except S. samarangense EO
demonstrated activity against Mycobacterium smegmatis (MIC: 0.125–0.50 mg/mL). The EOs were either
fungistatic or fungicidal against one or both tested Candida species with minimum inhibitory/fungicidal
concentrations of 0.016–32 mg/mL. The EOs also inhibited one or both key enzymes involved in skin
aging, elastase and collagenase (IC
50
: 89.22–459.2
µ
g/mL; 0.17–0.18 mg/mL, respectively). Turmerone,
previously identified in the C. longa EO, showed the highest binding affinity with the enzymes (binding
energy:
−
5.11 and
−
6.64 kcal/mol). Only C. aurantium leaf, C. longa,P. amboinicus,P. senacia,S. coriaceum,
and S. samarangense EOs were cytotoxic to the human malignant melanoma cells, UCT-MEL1 (IC
50
:
88.91–277.25
µ
g/mL). All the EOs, except M. citrifolia EO, were also cytotoxic to the human keratinocytes
non-tumorigenic cells, HaCat (IC
50
: 33.73–250.90
µ
g/mL). Altogether, some interesting therapeutic
properties of the EOs of pharmacological/cosmeceutical interests were observed, which warrants further
investigations.
Keywords: essential oils; antiacne; antimycobacterial; antiaging; antiproliferative
1. Introduction
Natural products derived from plants are part of traditional medicine and represent
therapeutic possibilities for treating a panoply of diseases. In recent years, their uses
in the development of new drugs have shown much visibility due to their efficiency
Molecules 2022,27, 8705. https://doi.org/10.3390/molecules27248705 https://www.mdpi.com/journal/molecules
Molecules 2022,27, 8705 2 of 16
and limited adverse effects. Essential oils (EOs), which are aromatic and volatile liquids
extracted from plants, are particularly indicated to possess a broad spectrum of curative
properties, such as antimicrobial, antiviral, antimutagenic, anticancer, antioxidant, anti-
inflammatory, immunomodulatory, and antiprotozoal properties [
1
]. In fact, they have
been used for centuries in medicine, perfumery, and cosmetics, and have been added to
foods as components of spices and herbs. Almost 3000 different EOs are known, and 300
are used commercially in the flavours and fragrances market [2].
Moreover, their molecular diversity, wide range of activity, structure–activity rela-
tionships, and capacity for targeting paradoxical responses triggered by different genes
and pathways, have been significantly appraised [
3
]. A greater understanding of EOs’
chemistry and penetrative capabilities via biological membranes make them important
treatment tools for the management of various neurological disorders. Essential oils in
combination with vegetable oils are used in massages, with some being reported to cure
one or more diseases and are used in para-medicinal practices [4,5].
Additionally, EOs have been shown to possess anticancer properties through various
mechanisms, including cancer preventative mechanisms, as well as acting on the established
tumor cell itself as well as interaction with the microenvironment [
6
]. Importantly, these
activity mechanisms of EOs lead to cellular and metabolic responses, thus making them
attractive in anticancer therapeutic strategies.
Furthermore, EOs, as complex mixtures of volatile compounds, can be regarded as a
powerful tool to reduce bacterial resistance. An important characteristic of EOs and their
components is their hydrophobicity, which enables them to partition with the lipids present
in the cell membrane of bacteria and mitochondria, rendering them more permeable
by disturbing the cell structures. This eventually results in the death of bacterial cells
due to substantial leakage of critical molecules and ions from the bacterial cells. Some
EO compounds have also been found to modulate drug resistance by targeting efflux
mechanisms in several species of Gram-negative bacteria [
7
]. Additionally, EOs have been
acknowledged to act as antifungal agents, and play an important role in blocking cell
communication mechanisms, fungal biofilm formation, and mycotoxin production [8].
Indeed, botanical products that can prevent or reduce the signs of aging skin include
products that offer photoprotection, decreased transepidermal water loss, increased skin
elasticity, collagen formation, and decreased facial pigmentation, or offer antioxidant effects
in the skin. Essential oils are no exception, as they have been found to counteract some
of the signs of skin aging [
9
]. Besides, most of these oils also confer powerful antioxidant
benefits, which means they have the power to scavenge free radicals to protect the skin
from damage [10].
In this study, 10 EOs extracted from nine endemic and exotic medicinal plants from
Mauritius, which have previously been found to possess multiple benefits
in vitro
and
showed promising results in silico [
11
–
13
], were subjected to further investigations. Notably,
they were explored for their antimicrobial, antiaging, and antiproliferative properties.
2. Results and Discussion
2.1. Antimicrobial
2.1.1. Antimycobacterial
Mycobacterium species are responsible for several diseases, particularly in immunocom-
promised individuals. The spread of resistance to antimycobacterial drugs is a significant
problem to public health and requires the search for a new and innovative alternative for
the treatment of drug-resistant mycobacterial strains [
14
]. The hydrophobic structure of
the cell wall is responsible for the innate antibiotic resistance of Mycobacterium species. It
has been suggested that they became more hydrophobic by increasing the proportion of
less polar lipids in their outer membrane. Importantly, such a change implies an enhanced
capability for aerosol transmission, affecting their virulence and pathogenicity [
15
]. Since it
is impermeable for commonly used antibiotics and can be attacked by substances with a
high affinity for lipid-rich cell surfaces, one successful route to overcoming the hydropho-
Molecules 2022,27, 8705 3 of 16
bic barrier of the mycobacterial outer membrane is to use hydrophobic biologically active
compounds, such as EOs [
16
]. Thus, in this study, the inhibitory effect of the EOs on
Mycobacterium smegmatis was evaluated. Indeed, M. smegmatis is a useful research surrogate
for pathogenic Mycobacterium species in a laboratory experimental setup [
17
], given that
working with some strains of Mycobacterium, such as M. tuberculosis, poses some biosafety
risks [18].
In the present study, with the only exception of S. samarangense EO, all EOs demon-
strated antimycobacterial potential against M. smegmatis (MIC range: 0.125–0.50 mg/mL).
In particular, S. coriaceum EO exhibited the most potent inhibition against M. smegmatis
(MIC: 0.125 mg/mL), although it was still lower compared with the antibiotic ciprofloxacin
(Table 1). Many EOs have also been reported to be effective against both tuberculous and
non-tuberculous mycobacteria [
14
,
16
,
19
,
20
], and thus they can be considered as important
antimycobacterial agents from natural products.
Table 1. Antimycobacterial and anti-acne activities of EOs.
MIC (mg/mL)
Bacteria
Tested
Essential Oils Antibiotics
CAF CAL CC CL MC PA PC PS SC SS CIP TRC
M.
smegmatis
(ATCC
MC2155)
0.25 0.25 0.50 0.25 0.25 0.25 0.25 0.50 0.125 NI1b3.13 ×10−4-
C. acnes
(ATCC
6919)
NI2cNI2cNI2cNI2c2 0.50 NI2cNI2c0.50 NI2c-7.8 ×10−4
CC: Cinnamomum camphora; CAL: Citrus aurantium (leaf); CAF: Citrus aurantium (fruit peel), CL: Curcuma longa,
MC: Morinda citrifolia, PC: Petroselinum crispum, PS: Pittosporum senacia, PA: Plectranthus amboinicus, SC: Syzygium
coriaceum, SS: Syzygium samarangense; MIC: Minimum inhibitory concentration;
b
No inhibition at the highest
concentration tested (1 mg/mL),
c
No inhibition at the highest concentration tested (2 mg/mL), CIP: ciprofloxacin,
TRC: tetracycline.
2.1.2. Anti-Acne
Acne is a common chronic inflammatory skin disease in both adolescents and adults
that mainly involves the epidermis and pilosebaceous units. The pathogenesis of acne
is complicated and the colonization of Cutibacterium acnes is considered a crucial factor
throughout the whole development of acne. Cutibacterium acnes promotes the abnormal
proliferation and differentiation of keratinocytes and increases sebum production [
21
].
Furthermore, while a direct involvement of C. acnes seems certain, it may not be involved
in the initiation of acne lesions, but instead would mediate later inflammatory events,
causing deterioration of the lesions while stimulating the production of host antimicrobial
peptides: small molecules with antimicrobial activity and immunomodulatory properties.
Moreover, a lipase secreted by the bacterium is responsible for the hydrolysis of sebum and
the subsequent release of free fatty acids, which results in an irritating and proinflammatory
effect [
22
]. The main therapy for acne is topical and oral antibiotics, which have been used
for decades against C. acnes. However, like many other bacteria, the increasing number of
strains of drug-resistant C. acnes due to antibiotic abuse in acne treatment has aroused wide
concern [23,24]; consequently, novel therapies are in high demand worldwide.
Medicinal plants, in particular, may present a unique source of new therapeutic
options. Many studies have been dedicated to the documentation of traditional uses of
medicinal plants for managing dermatological conditions, and these may represent a strong
armor in drug discovery in the search for effective acne treatment [
25
,
26
]. Many EOs from
medicinal plants have also been explored for their wide application in acne management,
especially as topical preparations [
27
–
29
], and hence continue to be the focus of many
anti-acne studies.
In the current investigation, only P. amboinicus,M. citrifolia and S. coriaceum EOs
showed anti-acne activity. P. amboinicus and S. coriaceum EOs showed higher potential
(MIC: 0.50 mg/mL) than M. citrifolia EO (2 mg/mL) (Table 1). This could be related to
Molecules 2022,27, 8705 4 of 16
the presence of specific compounds effective against C. acnes present in them. It is well
known that in acne, the reactive oxygen species (ROS) produced by neutrophils play a
critical role in the irritation and destruction of the follicular wall and are responsible for
the progression of acne [
22
,
30
]. In addition, C. acnes has been shown to survive for long
periods of time in human tissues with low oxidative potential [
31
]. Therefore, EOs with
antioxidant activity could have beneficial effects in acne management. In fact, in our recent
studies, P. amboinicus and S. coriaceum EOs have demonstrated good antioxidant activity in
various
in vitro
antioxidant assays performed [
12
,
13
]. As expected, the known antibacterial
drug used as a positive control, notably, tetracycline demonstrated prominent inhibitory
capacity (MIC: 7.8 ×10−4mg/mL).
2.1.3. Antifungal
Although Candida species are common commensals in the human microbiome, they can
nevertheless trigger opportunistic fungal infections despite their non-pathogenic character
when the immune system of the affected individuals is impaired or weakened. Moreover,
the emergence of antifungal drug resistance in Candida species has led to increased morbid-
ity and mortality in immunocompromised patients [
32
,
33
]. Thus, given the antifungal drug
resistance patterns making treatment of candidiasis difficult, it is essential to search for new
antifungals against these opportunistic human pathogens responsible for frequent nosoco-
mial infections. In this context, plant-derived products including EOs have demonstrated
promising results
in vitro
and
in vivo
against several Candida species and thus, could be
considered useful in the development of novel anticandidal drugs [34,35].
In the present study, while P. senacia EO was inactive, the other EOs showed var-
ied inhibitory potential against C. albicans (MIC range: 0.25–32 mg/mL). In particular,
M. citrifolia
EO was the most potent against C. albicans (0.25 mg/mL), followed by P. am-
boinicus (
2 mg/mL)
,P. crispum and C. aurantium (leaf) (4 mg/mL) EOs. Among them, M.
citrifolia,P. amboinicus and C. aurantium (leaf) EOs were found to be fungicidal at their MIC.
Both Syzygium EOs showed an MIC of 8 mg/mL, although S. coriaceum EO was fungicidal
while S. samarangense EO was only fungistatic at that concentration (Table 2).
Table 2. Antifungal activities of EOs against Candida spp.
MIC/MFC (mg/mL)
Fungi Tested
Essential Oils Antifungals
CAF CAL CC CL MC PA PC PS SC SS Nystatin Amphotericin B
C. albicans
(ATCC
10,231)
(32) (4) (16) (8) (0.25) (2) (4) - (8) (8) (1.56 ×10−3) (3.13 ×10−3)
FS FC FS FS FC FC FS FC FS FS FS
[ND] [32] [16] [16] (16) [3.13 ×10−3][ND]
C. tropicalis
(ATCC 750) (8) (0.016) (8) (1) (0.25) (2) (2) (32) (1) (1) (1.56 ×10−3) (1.25 ×10−2)
FS FS FC FC FS FS FC FS FS FC FS FS
[16] [0.0625] [1] [4] [ND] [2] [ND] [ND]
MIC: minimum inhibitory concentration, MFC: minimum fungicidal concentration, CC: Cinnamomum camphora;
CAL: Citrus aurantium (leaf); CAF: Citrus aurantium (fruit peel), CL: Curcuma longa, MC: Morinda citrifolia, PC:
Petroselinum crispum, PS: Pittosporum senacia, PA: Plectranthus amboinicus, SC: Syzygium coriaceum, SS: Syzygium
samarangense, - not active; ( ): MIC, FS: fungistatic at MIC, FC: fungicidal at MIC, [ ] new MFC in case not fungicidal
at MIC, [ND]: not determined if MFC > MIC ×4.
Moreover, C. tropicalis was found to be more sensitive to the EOs. For instance, seven EOs,
namely C. aurantium (leaf), M. citrifolia,C. longa,P. amboinicus,P. crispum and the two Syzygium
Eos, demonstrated MICs ranging from 0.016 to 2 mg/mL and MFCs ranging from 0.0625 to
2 mg/mL
. On the other hand, C. camphora and C. aurantium fruit peel EOs displayed an MIC
of 8 mg/mLand a MFC of 8 and 16 mg/mL, respectively. Clearly, C. aurantium leaf EO was the
most potent against C. tropicalis (MIC = 0.016 mg/mL and MFC = 0.0625 mg/mL), followed
by M. citrifolia EO (MIC = 0.25 mg/mL, MFC = 1 mg/mL). P. senacia EO showed the weakest
antifungal effect on C. tropicalis (MIC = 32 mg/mL) (Table 2). Interestingly, although the
Molecules 2022,27, 8705 5 of 16
standard antifungals, nystatin and amphotericin B, were very effective against both Candida
species, they were nevertheless fungistatic at their respective MIC values (Table 2).
In the present study, M. citrifolia EO was found to be effective against both Candida
species, with an MIC of 0.25 mg/mL. This was in agreement with the study by Holanda
et al. [
36
], which also reported the high efficacy of M. citrifolia fruit EO against Candida
species, namely C. albicans and C. utilise, with MIC values of 39 and 78
µ
g/mL, respectively.
Remarkably, the same major components, octanoic (38.7%) and hexanoic (20.0%) acids,
were revealed in the EO, as was previously reported by Jugreet and Mahomoodally (2020)
for
M. citrifolia
EO investigated in the present study [
11
], although they varied in their
percentages (octanoic acid (78.9%); hexanoic acid (11.3%)). Furthermore, the antimicrobial
potential of the oil was observed to decrease drastically after it was subjected to the
esterification reaction, suggesting that the carboxyl group is responsible for the potent oil
activity [
36
]. Hence, the good antifungal property obtained herein could be attributed to
the richness of the M. citrifolia EO in short-chain fatty acids.
Furthermore, C. aurantium (leaf) EO was found to be the most potent against
C. tropicalis.
Interestingly, its major compound, sabinene, identified in our previous study [
11
], has also
been found to be effective against several Candida species (MIC: 0.25 mg/mL), including
C. tropicalis of the same ATCC strain used in this study and incubated for the same dura-
tion (24 h) [
37
]. However, the antifungal property of the EO was noted to be much more
prominent in the present study compared to that of its major compound, sabinene, reported
previously [
37
], which indicates that there could have been a synergistic interaction among
the different components in the EO. Besides, C. tropicalis was reported to be more sensitive
than C. albicans (same ATCC as the one used in this study), which was in agreement with
the results obtained here, whereby C. aurantium (leaf) EO showed weaker inhibitory activity
on C. albicans.
Similarly, some of the major components of the other EOs investigated here (ocimene,
myrcene, carvacrol, pinene, limonene, and 1,8-cineole), were also evaluated in the study
by ˙
I¸scan [
37
] and were found to display anticandidal effects with MICs ranging from
0.12 to
4 mg/mL
against C. albicans and C. tropicalis. The antifungal mechanisms of EOs
and their constituents are normally explained by membrane damage or disruption of its
integrity, increasing permeability, inhibition of ergosterol synthesis or binding to ergosterol
on the membrane, and ROS production by acting on mitochondria [
37
]. Moreover, it has
been suggested that EOs’ components accumulate in the lipophilic hydrocarbon molecules
of the cell lipid bi-layer, thereby allowing the easier transfer of other components to the
inner part of the cell [8].
2.2. Anti-Aging
The extracellular matrix (ECM) is the largest component of the dermis and provides
the structural framework essential for the growth and elasticity of the skin. The ECM
is composed of proteoglycans interwoven with macromolecules like collagen, elastin,
and fibronectin, which are formed by the fibroblasts of the dermis. Collagen, the most
abundant protein in the ECM, is responsible for the elasticity and strength of the skin and
for maintaining its flexibility, while elastin confers the unique property of elastic recoil,
which is vital for maintaining skin elasticity and resilience [
38
]. An increase in elastase
activity has been found in several disorders, including psoriasis, dermatitis, inflammatory
processes, and premature skin aging, which are closely associated with the formation of
wrinkles [
39
]. Thus, the degradation of ECM is mainly due to the enhanced activity of
proteolytic enzymes, such as collagenase and elastase.
The inhibition of these enzymatic activities by natural plant compounds is a promising
approach to prevent skin aging and represents a pool of increasingly important ingredients
in cosmetics and medications for the prevention of skin aging [
40
]. Of particular interest for
anti-aging applications are the EOs, possessing multiple beneficial functions, such as the
inhibition of aging-related enzymes and the capacity for scavenging free radicals [41–43].
Molecules 2022,27, 8705 6 of 16
In the present study, with the exception of four EOs (C. aurantium fruit peel, C. cam-
phora,M. citrifolia and P. amboinicus), the remaining EOs were found to inhibit elastase
(
IC50: 141.81–588.80 µg/mL
). On the other hand, all EOs showed interesting anti-collagenase
activity, with IC
50
values ranging from 0.17 to 1.54 mg/mL (Table 3). Remarkably, C. aurantium
(leaf), C. longa,P. crispum,P. senacia,S. coriaceum, and S. samarangense EOs were found to inhibit
both enzymes.
Table 3. Anti-elastase and anti-collagenase activity of studied EOs.
EO Elastase Inhibition Collagenase Inhibition
IC50 (µg/mL) IC50 (mg/mL)
CAF NI1000 1.46 ±0.19
CAL 275.95 ±13.86 1.54 ±0.40
CC NI1000 1.34 ±0.09
CL 89.22 ±23.72 0.17 ±0.01
MC NI1000 0.62 ±0.04
PA NI1000 0.33 ±0.02
PC 354.65 ±21.43 0.37 ±0.02
PS 233.47 ±21.45 0.77 ±0.17
SC 767.2 ±27.99 0.84 ±0.13
SS 459.2 ±21.24 0.18 ±0.04
Positive control
Ursolic acid 10.10 ±15.27 -
1,1 Phenanthroline - 4.20 ×10−3±0.00
CAL: Citrus aurantium leaf, CAF: Citrus aurantium fruit (peel), CC: Cinnamomum camphora; CL: Curcuma longa,
MC: Morinda citrifolia, PA: Plectranthus amboinicus, PC: Petroselinum crispum; PS: Pittosporum senacia; SC: Syzygium
coriaceum; SS: Syzygium samarangense; IC50: half-maximal inhibitory concentration.
Of all the active EOs, C. longa EO displayed the most potent activity. In another study,
the anti-aging effect of C. longa EO was also tested using a different strategy [
44
]. This was
determined using ultraviolet B (UVB)-induced skin aging assays, whereby C. longa EO was
seen to reduce cutaneous photoaging in a UVB-irradiated nude mouse model.
Indeed, EOs with anti-aging activities could be an interesting approach to combat
skin aging and could be incorporated in topical creams [
45
]; the EOs under the present
investigation provide good scope in this regard.
2.3. Molecular Docking
Molecular docking is one of the most applied virtual screening methods widely used
in drug discovery [
46
]. This method helps to predict the intermolecular framework formed
between a protein and a small molecule or a protein and protein and suggests the binding
modes accountable for the inhibition of the protein [
47
]. Thus, molecular docking was
selected as a suitable method to obtain useful information about the binding affinity of
the tested components to the active site of the enzymes and therefore understand the
interactions of the enzymes with the major oil components.
In the present study, the most abundant components of the EOs previously identified
by GC-MS/GC-FID (in Supplementary Materials) were docked with the enzymes’ active
sites, with the results of the docking scores listed in Table 4. The lower the binding energy,
the greater is the binding efficiency. As shown in Table 4, turmerone was revealed to be the
most potent inhibitor and displayed the highest binding affinity with both collagenase and
elastase (binding energy:
−
5.11 and
−
6.64 kcal/mol, respectively). The two-dimensional
interactions of turmerone with the enzymes are depicted in Figure 1.
Molecules 2022,27, 8705 7 of 16
Table 4. Docking scores of EOs compounds with target enzymes.
EOs Major Compounds Collagenase PP Elastase
CAF Limonene −3.87 a
(1.5 mM) b
−5.61
(77.7 µM)
CAL Sabinene −3.87
(1.5 mM)
−5.36
(118.8 µM)
CC 1,8-Cineole −4.07
(1.0 mM)
−5.30
(130.8 µM)
CL Turmerone −5.11
(179.0 µM)
−6.64
(13.6 µM)
MC Octanoic acid −3.67
(2.1 mM)
−4.91
(249.7 µM)
PA Carvacrol −4.55
(459.4 µM)
−5.45
(100.9 µM)
PC Myristicin −3.56
(2.4 mM)
−5.73
(63.0 µM)
PS Myrcene −3.32
(3.7 mM)
−4.68
(374.3 µM)
SC (E)-β-Ocimene −3.29
(3.9 mM)
−4.63
(401.0 µM)
SS β-Pinene −4.13
(938.4 µM)
−5.47
(97.0 µM)
CAL: Citrus aurantium leaf, CAF: Citrus aurantium fruit (peel), CC: Cinnamomum camphora; CL: Curcuma longa,
MC: Morinda citrifolia, PA: Plectranthus amboinicus, PC: Petroselinum crispum; PS: Pittosporum senacia; SC: Syzygium
coriaceum; SS: Syzygium samarangense.abinding free energy in kcal/mol; bcalculated inhibition constant.
Molecules 2022, 27, x FOR PEER REVIEW 8 of 17
Figure 1. Shows intramolecular interactions of turmerone displaying the highest binding affinity
with the studied enzymes.
Carvacrol follows turmerone, with a binding energy of −4.55 kcal/mol with colla-
genase enzyme (Table 4). Myristicin and limonene were the compounds with the second-
and third-highest binding affinity with elastase (−5.73 and −5.61 kcal/mol, respectively).
Interestingly, turmerone was also reported to be a potent enzyme inhibitor in previous in
silico studies [13], that could be related to the efficient binding interactions between the
compound and these enzymes.
2.4. Cytotoxic/Antiproliferative Evaluation of EOs
Currently there is a need for novel non-toxic and selective agents to prevent and/or
treat cancer and tumor malignancies. One method involves using synergistic molecules
that can block multiple pathways. The use of active components derived from natural
products is valuable in this regard [48]. Ancient practices using plants in the treatment of
many diseases are increasingly being used in recent years. Medicinal plants are a source
of compounds with biological activities as anticancer agents, and over 50% of the drugs
used in the clinical treatment of cancer, such as taxol, camptothecin, vincristine, and vin-
blastine, are obtained from natural sources [49].
Key hallmarks of cancer include resisting cell death, sustained proliferative signal-
ling, and escaping growth suppressors. Therefore, therapeutic strategies focused on in-
ducing apoptosis and cellular arrest are of great significance. Remarkably, EOs have been
shown to induce both the intrinsic (or mitochondria-dependent) and extrinsic (or death
receptor-dependent) apoptosis pathways [50]. Interestingly, specific EO constituents have
even been found to enhance the cytotoxic activity of chemotherapy drugs on various cell
lines, thus increasing the therapeutic window; that is, showing the same effectiveness with
reduced drug concentrations [51,52].
While the efficacy of natural products towards certain cancer types is encouraging,
their toxicity towards normal healthy cells must remain low to obtain the highest level of
efficacy and specificity towards cancer cells [53]. Thus, in the present study, the cytotoxic
effects of EOs were investigated in vitro on both cancerous and non-cancerous cells using
the MTT assay. In the current study, a human keratinocyte non-tumorigenic cell line (Ha-
Cat) and a human malignant melanoma cell line (UCT-MEL1) were used. The results are
presented in Table 5.
Figure 1.
Shows intramolecular interactions of turmerone displaying the highest binding affinity
with the studied enzymes.
Carvacrol follows turmerone, with a binding energy of
−
4.55 kcal/mol with collage-
nase enzyme (Table 4). Myristicin and limonene were the compounds with the second-
and third-highest binding affinity with elastase (
−
5.73 and
−
5.61 kcal/mol, respectively).
Interestingly, turmerone was also reported to be a potent enzyme inhibitor in previous in
silico studies [
13
], that could be related to the efficient binding interactions between the
compound and these enzymes.
2.4. Cytotoxic/Antiproliferative Evaluation of EOs
Currently there is a need for novel non-toxic and selective agents to prevent and/or
treat cancer and tumor malignancies. One method involves using synergistic molecules
that can block multiple pathways. The use of active components derived from natural
Molecules 2022,27, 8705 8 of 16
products is valuable in this regard [
48
]. Ancient practices using plants in the treatment of
many diseases are increasingly being used in recent years. Medicinal plants are a source of
compounds with biological activities as anticancer agents, and over 50% of the drugs used
in the clinical treatment of cancer, such as taxol, camptothecin, vincristine, and vinblastine,
are obtained from natural sources [49].
Key hallmarks of cancer include resisting cell death, sustained proliferative signalling,
and escaping growth suppressors. Therefore, therapeutic strategies focused on inducing
apoptosis and cellular arrest are of great significance. Remarkably, EOs have been shown
to induce both the intrinsic (or mitochondria-dependent) and extrinsic (or death receptor-
dependent) apoptosis pathways [
50
]. Interestingly, specific EO constituents have even been
found to enhance the cytotoxic activity of chemotherapy drugs on various cell lines, thus
increasing the therapeutic window; that is, showing the same effectiveness with reduced
drug concentrations [51,52].
While the efficacy of natural products towards certain cancer types is encouraging,
their toxicity towards normal healthy cells must remain low to obtain the highest level of
efficacy and specificity towards cancer cells [
53
]. Thus, in the present study, the cytotoxic
effects of EOs were investigated
in vitro
on both cancerous and non-cancerous cells using
the MTT assay. In the current study, a human keratinocyte non-tumorigenic cell line
(HaCat) and a human malignant melanoma cell line (UCT-MEL1) were used. The results
are presented in Table 5.
Table 5. Cytotoxic effects of EOs on HaCat and UCT-MEL1 cell lines.
EOs IC50 ±SD (µg/mL)
HaCat UCT-MEL1
CAF 182.70 ±3.54 NI400
CAL 33.73 ±7.06 277.25 ±1.48
CC 250.90 ±0.57 NI400
CL 56.1 ±1.90 88.91 ±5.83
MC NI400 NI400
PA 49.12 ±2.58 189.50 ±1.41
PC 104.50 ±4.24 NI400
PS 50.33 ±1.43 95.52 ±0.77
SC 34.17 ±5.32 95.37 ±4.34
SS 54.70 ±3.59 94.09 ±1.85
ActinomycinD 2.85 ×10−2±8.49 ×10−48.65 ×10−3±1.13 ×10−4
Cytotoxicity is expressed as the concentration of the EOs inhibiting cell growth by 50% (IC
50
); NI
400
: no inhibition
at the highest concentration tested of 400 µg/mL.
All EOs except M. citrifolia EO, showed cytotoxicity towards HaCat cells (IC
50
: 34.17–
250.90
µ
g/mL). The highest cytotoxic effect was exhibited by C. aurantium (leaf) and
S. coriaceum
EOs (IC
50
: 33.73
±
7.06
µ
g/mL and 34.17
±
5.32
µ
g/mL, respectively). This was fol-
lowed by EOs such as P. amboinicus,P. senacia,S. samarangense and C. longa EOs respec-
tively (IC
50
: 49.12–56.10
µ
g/mL). On the other hand, a lower cytotoxic potential was ob-
served by
C. camphora
,C. aurantium (fruit peel) and P. crispum EOs on the HaCat cells (IC
50
:
104.50–
250.90 µg/mL
). The positive control Actinomycin D, a known anti-tumor drug, in-
stead showed a significantly higher cytotoxic effect on HaCat compared to the EOs (IC
50
:
2.85 ×10−2±8.49 ×10−4µg/mL
). Out of the tested EOs, only C. longa,C. aurantium (leaf),
P. senacia
,P. amboinicus,S. coriaceum, and S. samarangense EOs showed inhibitory activity
against the UCT-MEL1 cells, with IC
50
values ranging from 88.91 to 277.25
µ
g/mL, although
the values were significantly higher than the positive control actinomycin D (IC
50
:
8.65 ×10−3
±
1.13
×
10
−4µ
g/mL). On the other hand, M. citrifolia EO did not exert inhibitory effect on
any of the cell lines at the highest tested concentration (400
µ
g/mL) (Table 5). Furthermore,
while some of the tested EOs demonstrated cytotoxicity towards the tumorigenic cells, they
were however not selective towards them as they also showed cytotoxicity towards the normal
non-tumorigenic cells.
Molecules 2022,27, 8705 9 of 16
Indeed, as reported, the cytotoxic properties of EOs result from the complex interaction
between the different classes of compounds, such as phenols, alcohols, esters, aldehydes,
ketones, ethers, or hydrocarbons [
54
]. Additionally, in some cases, the cytotoxic activity
is closely related to a few of the main oil components and it has also been found that
some of these isolated compounds exert considerable cytotoxic properties when tested
individually [
55
]. Nevertheless, the scarcer compounds could also be of importance, as
the various molecules could synergistically act with the major compounds [
56
,
57
], while
antagonistic interactions of the compounds have been recognized as well [
58
]. The wide
variation in the chemical profile of EOs also means a great diversity in the mechanisms of
action and molecular targets, whereby each compound can modulate or alter the effects
of another compound. The main mechanisms that mediate the cytotoxic effects of EOs
include the induction of cell death by activating apoptosis and/or necrosis processes, cell
cycle arrest, and loss of function of vital organelles. Several of these effects are due to
the lipophilic nature and low molecular weight of the main components, allowing them
to cross cell membranes, alter membrane composition, and increase membrane fluidity,
causing leakage of ions and cytoplasmic molecules [
54
]. Interestingly, all the three major
types of EO constituents namely, phenols, aldehydes, and alcohols, have been reported
to exert cytotoxic effects in this way [
59
]. Hence, the presence of numerous constituents
that simultaneously interfere with multiple signaling pathways might be the key for
overcoming the current limit of chemotherapeutic agents and particularly, the development
of multidrug resistance [60].
Some of the EOs investigated in this study have also been previously subjected to
cytotoxic studies using different cancer cell lines. In the study by Jacob and Toloue [
48
], the
purified turmeric oil fractions containing
α
,
β
and ar-turmerones showed growth inhibitory
activity against breast (SKBR-3), pancreatic (PANC-1), and prostate (PC-3) cancers, and
reduced activity against a non-cancerous cell line (WI-38). Percent inhibition was suggested
to be associated with the structural parameters of the turmerones. Furthermore, turmeric
EO was found to have significant
in vitro
cytotoxic activity against Dalton’s lymphoma
ascites cells (DLA) and Ehrlich ascites carcinoma (EAC) cancer cell lines (IC
50
8
µ
g and
18 µg
, respectively). Oral administration of turmeric EO was found to significantly increase
the life span (56.25%) of DLA-induced ascites tumour bearing mice, as well as signifi-
cantly reducing the solid tumours [
61
]. Furthermore, M. citrifolia EO was reported to be
cytotoxic to human colorectal carcinoma (HCT-116) and human breast carcinoma (MCF-7)
cell lines, exhibiting IC
50
values of 91.46
µ
g mL
−1
and 78.15
µ
g mL
−1
, respectively [
62
].
The chemotherapeutic activity of the P. amboinicus EO on C57BL/6 mice injected with
B16F-10 melanoma cell line was also revealed in a former study, whereby P. amboinicus EO
(
50 µg/dose
) via i.p. was used as a treatment for 21 days [
63
]. Additionally, the daily topical
treatment with C. camphora EO in the study of Moayedi et al. [
64
], was found to induce
dramatic regression of pre-malignant skin tumors and a two-fold reduction in cutaneous
squamous cell carcinoma
in vivo
. Interestingly, C. camphora EO was found to stimulate
calcium signaling, resulting in calcineurin-dependent activation of nuclear factor for acti-
vated T cells; in cultured keratinocytes and
in vivo
, it induced transcriptional variations in
immune-related genes, resulting in cytotoxic T cell-dependent tumor regression.
3. Materials and Methods
3.1. Plant Materials
Nine plants, of which two are endemic species; namely, Syzygium coriaceum J. Bosser
& J. Gueho and Pittosporum senacia Putterl. subsp. Senacia, and seven exotic species;
namely, Cinnamomum camphora (L.) Nees & Eberm, Citrus aurantium L., Curcuma longa
L., Morinda citrifolia L., Petroselinum crispum (Mill.) Fuss, Plectranthus amboinicus (Lour.)
Sprengel, and Syzygium samarangense (Blume) Merr. & L. M. Perry, were used based on
their importance in traditional medicine, as documented in a previous publication [
11
].
The plant specimens were authenticated by a local botanist at the Mauritius Sugarcane
Molecules 2022,27, 8705 10 of 16
Industry Research Institute (MSIRI) Herbarium, at Réduit, whereby an identification code
was assigned for each of them.
The leaves of Syzygium coriaceum (MAU 0027510) and C. camphora (MAU 0027508),
were collected from Monvert nature park (20
◦
20
0
35.2
00
S, 57
◦
31
0
21.2
00
E) in the month of
October 2018, while C. aurantium (leaves or fruit peel; MAU 0027511), M. citrifolia (fruits;
MAU 0027506), P. amboinicus (leaves; MAU 0027507) and P. senacia (fruits; MAU 0027512)
was obtained from the university farm, Réduit (20
◦
13
0
39
00
S, 57
◦
29
0
33
00
E) in the month of
May to September 2018. The leaves of S. samarangense (MAU 0027509) was collected in
March 2018 from Mont-fertile, a southern region in Mauritius (20
◦
24
0
31.0
00
S,
57◦36049.100 E
).
Rhizomes of C. longa (MAU 0027514) were harvested from Plaine-Magnien, a southern
region in Mauritius (latitude 20
◦
25
0
46.81
00
S, longitude 57
◦
40
0
10.85
00
E) in May 2018. Lastly,
P. crispum (aerial parts; MAU 0027505) was purchased in July 2018, from the local market.
3.2. Extraction of EOs
Fresh plant material, cut into small pieces, was subjected to the process of hydrodistil-
lation using a Clevenger-type apparatus for 3 h. The EO distillates, once yielded (% yield
in Table S1), were dried over anhydrous magnesium sulfate, filtered, and then stored in
dark vials at −4◦C until further analysis [65].
3.3. Antimicrobial Assay
3.3.1. Anti-Mycobacterium
The minimum inhibitory concentration (MIC) values of all the EOs were determined
according to the method used by Lall et al. [
66
]. The EOs were dissolved in 20% DMSO and
sterile Middlebrook 7H9 media, and two-fold dilution was performed to produce final test
concentrations ranging from 31.25 to 1000
µ
g/mL. The bacterial suspension was adjusted to
0.5 McFarland standard (1.5
×
10
8
colony-forming units/mL [CFUs/mL]). The inoculum of
M. smegmatis (ATCC
®
(American Type Culture Collection, Rockville, MD, USA) MC
2
155)
was further diluted 50-fold to obtain the final test concentration of (1.5
×
10
6
CFUs/mL).
After the addition of the bacterial inoculum (100
µ
L), the final assay volume in each well was
200 µL
. Ciprofloxacin (Sigma-Aldrich, Saint Louis, MO, USA) (0.078 to 10
µ
g/mL) was used
as standard drug. The plates were left to incubate at 37
◦
C for 24 h, followed by the addition
of the viability indicator PrestoBlue
®
(Invitrogen Corporation, San Diego, CA, USA) (20
µ
L)
to each well, and after a further 2 h incubation, the colour change was observed. The MIC
value was defined as the lowest concentration at which no colour change from blue to pink
could be observed.
3.3.2. Anti-Acne
The
in vitro
microdilution method as described by Kamatou [
67
] was followed to
determine the MIC of the EOs against Cutibacterium acnes (ATCC
®
6919). The EOs were
prepared in 100% acetone, with a final starting concentration of 2000
µ
g/mL. Tetracycline
(Sigma-Aldrich, Saint Louis, MO, USA) served as the positive control, with a final starting
concentration of 50
µ
g/mL. The sample dilutions were prepared in a 96-well flat-bottom
microtiter plate. Brain–heart infusion broth (BHI; 100
µ
L) was added to all the wells,
followed by 100
µ
L of the EOs in the first wells in triplicate. The EOs were diluted through
a series of two-fold dilutions. The bacterial suspension was prepared from 72 h-old bacterial
cultures grown on BHI agar at 37
◦
C under anaerobic conditions. The inoculated BHI broth
was diluted to 6
×
10
6
CFU/mL and added to all the wells (100
µ
L) except the broth control
wells. After 72 h incubation under anaerobic conditions, 20
µ
L of PrestoBlue
®
reagent was
added to all the wells and incubated at 37
◦
C for 60 min. The MIC value was determined
by observing the colour change.
Molecules 2022,27, 8705 11 of 16
3.3.3. Antifungal
Microdilution Broth Susceptibility Assay
The MIC of the EOs was determined as previously described by Seebaluck-Sandoram
et al. [
68
], with minor modifications. Each EO (100
µ
L) was serially diluted two-fold, in
triplicate, with Mueller–Hinton broth (MHB) in 96-well microtitre plates. Fresh inoculums
of C. albicans (ATCC 10231) and C. tropicalis (ATCC 750) were then prepared and adjusted
to 0.5 Mc Farland standard, which were further diluted at a ratio of 1:100 with fresh broth
in order to yield starting inoculums of approximately 10
6
CFU/mL. Next, 100
µ
L of fungal
culture was added to each well of the plates. Nystatin and amphotericin B (from Sigma-
Aldrich Co., Steinheim, Germany) (100
µ
g/mL) were used as standard antifungal drugs.
After 24 h incubation at 37
◦
C, 40
µ
L of iodonitrotetrazolium chloride (0.2 mg/mL) was
added to each well and the plates were incubated for another 20 min. The well containing
the lowest concentration in which no pinkish-red coloration was observed was regarded to
be the MIC.
Minimum Fungicidal Concentration
The minimum fungicidal concentration (MFC) of the EOs was determined according to
Aumeeruddy-Elalfi et al. [
65
]. Briefly, 10
µ
L of broth from the uncoloured wells (where no
growth was observed in the earlier MIC assay), corresponding to the MIC value
MIC ×2
(one dilution higher than MIC), and MIC
×
4 (one dilution higher than MIC
×
2), were
inoculated on Sabouraud dextrose agar (SDA) and incubated at 37
◦
C for 24 h. The MFC
was defined as the lowest recorded EO concentration of the MIC wells in which fungi
failed to grow on the SDA. Alternatively, if growth was observed following inoculation on
SDA, the concentration of the corresponding well used for inoculation (MIC, MIC
×
2, and
MIC
×
4) was referred to as the fungistatic (FS) concentration. Both negative and positive
controls were included for comparison.
3.4. Antiaging Assay
3.4.1. Anti-Elastase
The method used to determine the anti-elastase potential of the EOs was that de-
scribed by Lall et al. [
69
], with modifications. All reagents used were purchased from
Sigma-Aldrich (Johannesburg, South Africa). Each EO and ursolic acid (positive control)
were serially diluted in DMSO to obtain a final concentration range of 31.25–1000
µ
g/mL
and
0.94–60 µg/mL
, respectively. In a 96-well plate, 155
µ
L of potassium phosphate buffer
(
pH 8
) was added, whereafter, 5
µ
L of each dilution was added (in triplicate) to the re-
spective wells. Afterwards, 20
µ
L of 4.942 mU porcine pancreatic elastase enzyme was
added and incubated at 37
◦
C for 5 min. Following incubation, the reaction was initiated by
adding 20
µ
L of 4.4 mM N-succinyl-Ala-Ala-Ala-
ρ
-nitroanilide substrate. The absorbance
values were measured using a BIO-TEK Power-Wave XS plate reader (Analytical and
Diagnostic Products CC, Roodepoort, South Africa) at a wavelength of OD
405 nm
for 15 min.
The percentage inhibition was calculated using the following Equation (1) and GraphPad
Prism 4 was used to determine 50% inhibitory concentration (IC50) for each sample:
% Inhibition =100 −(Absorbance sample at 15 min −absorbance at 0 min
Absorbance control at 15 min −absorbance at 0 min)×100 (1)
3.4.2. Anti-Collagenase
The anti-collagenase assay was performed as described previously by Aumeeruddy-
elalfi et al. [
43
], with slight modifications using the EnzCheck
®
Gelatinase/Collagenase
Assay Kit (Molecular Probes Inc., Eugene, OR, USA). A 1
×
reaction buffer was prepared
from 10
×
buffer provided in the kit. The enzyme collagenase from Clostridium histolyticum
(Type IV) obtained in the kit was dissolved in the 1
×
reaction buffer for use at a concen-
tration of 0.2 U/mL. Dye-quenched (DQ) gelatin from pigskin and fluorescein conjugate,
used as the substrate, was diluted to a final concentration of 150
µ
g/mL. To constitute
Molecules 2022,27, 8705 12 of 16
the reaction mixture, 80
µ
L of each EO dissolved in a 1
×
reaction buffer at different con-
centrations was distributed in Nunc 96-well microtitre plates. An amount of 100
µ
L of
collagenase enzyme were added to the wells and allowed to incubate for 15 min at 37
◦
C.
Following incubation, 20
µ
L of DQ gelatin was added to the reaction mixture. Fluorescence
was read with parameters set for an excitation wavelength at 485 nm and emission at
515 nm
. 1,10-phenanthroline was used as the positive control. Blank wells were prepared
in the microplate, consisting of 100 µL 1×reaction buffer and 100 µL collagenase enzyme.
The inhibition percentage of the collagenase enzyme was calculated using Equation (2)
below [70]:
Collagenase inhibition (%) = {[(A−B)-(C−D)]/(A−B)} ×100 (2)
where A is the fluorescent intensity without the test sample (control), B is the fluorescent
intensity without the test sample and enzyme (blank of A), C is the fluorescent intensity
with the test sample, and D is the fluorescent intensity with the test sample without enzyme.
The anti-collagenase activities of all EOs were finally expressed as an IC
50
value using
GraphPad Prism 7 software.
3.5. Molecular Docking
The chemical structures of the most abundant compound in each EO (limonene,
sabinene, 1,8-cineole, octanoic acid, carvacrol, myristicin, myrcene, (E)-
β
-ocimene
β
-pinene
and turmerone) previously identified by GC-MS/GC-FID [
11
] (Table S1), were downloaded
as a mol file from Chemspider and ZINC database [
71
]. The AM1 method was applied
using Gaussian09 software to optimize the structures [72]. The optimized structures were
then each docked with the active sites of the enzymes elastase and collagenase.
The enzymes’ crystal structures were downloaded as a pdb format from the database,
Protein Databank RCSB PDB. The pdb codes of the enzymes were 2Y6I for the collagenase
and 1BRU for the structure of the porcine pancreatic elastase enzyme. Preparation of the
protein structures for docking calculations was done as previously described [
12
]. All possi-
ble conformations were docked at the active site of the enzyme via the Lamarckian genetic
algorithm of the AutoDock software, with 250 runs for each inhibitor. Visualisation and the
docking results’ analysis were accomplished using the Discovery studio 5.0 visualizer.
3.6. Cell Culture
The human keratinocyte (HaCat) and human malignant melanoma (UCT-MEL-1)
cells were maintained in T75 tissue-culture flasks containing Dulbecco’s Modified Eagles
Medium (DMEM) supplemented with 1% antibiotics (100 U/mL penicillin and 100
µ
g/mL
streptomycin), 250
µ
g/L fungizone, and 10% heat-inactivated and gamma-irradiated fetal
bovine serum (FBS). The cells were grown in an incubator set to 37
◦
C and 5% CO
2
. The cells
were passaged using 0.25% trypsin-EDTA after an 80% confluent monolayer had formed.
In Vitro Antiproliferative Activity
The
in vitro
antiproliferative activity of the EO samples was determined using the
PrestoBlue viability assay, as described by Lall et al. [
66
], on human keratinocyte (HaCat)
and human malignant melanoma (UCT-MEL-1) cell lines, donated by Dr Lester Davids
from the University of Cape Town, Department of Human Biology (South Africa). The cells
were seeded in 96-well microtitre plates at concentrations of 1
×
10
6
cells/mL (UCT-MEL-1)
and 4
×
10
4
cells/mL (HaCat), and incubated for 24 h at 37
◦
C and 5% CO
2
to allow
for cell attachment. A stock solution of the EOs (40 mg/mL in DMSO) and the positive
control, actinomycin D (Sigma-Aldrich, Saint Louis, MO, USA) (1 mg/mL in distilled
water), was prepared, followed by serial dilutions to obtain final concentrations ranging
from
12.5–400 µg/mL
for the EOs, and 0.05–3.9
×
10
−4µ
g/mL for actinomycin D in the
96-well plates. The plates were incubated for 72 h, thereafter 20 µL PrestoBlue was added
to each of the wells. After 2 h incubation, the fluorescence was measured at an excitation
wavelength of 560 nm and an emission wavelength of 590 nm, using the VICTORNivo
Molecules 2022,27, 8705 13 of 16
Multimode plate reader (Perkin Elmer, Midrand, South Africa). The samples were tested
in triplicate to calculate percentage of cell viability using Equation (3) below and the IC
50
values were determined using GraphPad Prism 9 software.
% Cell viability =Fluor.sample −Fluor.0%
Fluor.vehicle control −Fluor.0% (3)
where Fluor.
sample
is the fluorescence of (PrestoBlue + sample or the positive control)
and Fluor
.0%
is the fluorescence of (PrestoBlue + media), and Fluor.
vehicle control
is the
fluorescence of (PrestoBlue + DMSO).
4. Conclusions
This study emphasized the antimicrobial, antiaging, and antiproliferative effects of
plant-derived EOs. Interestingly, the EOs showed fungistatic and fungicidal effects on
both tested Candida species. Additionally, some of the EOs were observed to also possess
antimycobacterial and anti-acne properties against M.smegmatis and C. acnes, respectively.
Indeed, natural anti-aging skincare products have been in great demand in recent
years, owing to their claimed effectiveness in delaying skin aging. Remarkably, the findings
presented in the current study demonstrated that EOs could inhibit one or both key enzymes
involved in skin aging, and thus could be regarded as attractive anti-aging ingredients.
Additionally, molecular docking revealed the compound turmerone from C. longa EO to be
the most potent inhibitor of the enzymes, which was also supported by the results from the
in vitro anti-aging assays.
Furthermore, the
in vitro
antiproliferative properties of the EOs were tested using a hu-
man keratinocyte non-tumorigenic (HaCat) cell line and human malignant melanoma (UCT-
MEL1) cell line. While most EOs showed a cytotoxic effect on the UCT-MEL1 melanoma
cell line, they were not selective against the cancer cell and were quite cytotoxic to the
HaCat cells. As EOs are complex mixtures of compounds, it would be interesting to test
the cytotoxicity of only selected major components from the active EOs, as different compo-
nents may exert antagonistic effects altogether, and thus decrease their overall effect on the
cancer cells.
Taken together, this study revealed some interesting pharmacological and cosmeceuti-
cal attributes of the tested EOs that could be further exploited in
in vivo
assays to assess
their mechanisms of action and unveil compounds of interest.
Supplementary Materials:
The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/molecules27248705/s1, Table S1: Percentage yields and chemical
composition of major components of the studied essential oils (EOs).
Author Contributions:
Conceptualization, B.S.J., M.F.M., A.H.H., A.N.A., N.L.; methodology, N.L.,
I.A.L., A.-M.R., J.M., M.N.; software, A.H.H.; validation, B.S.J., N.L., A.K., A.H.H., B.L.V., M.F.M.; formal
analysis, B.S.J.; investigation, M.F.M.; resources, N.L.; data curation, all authors;
writing—original
draft
preparation, all authors.;
writing—review
and editing, all authors. All authors have read and agreed to
the published version of the manuscript.
Funding:
The authors would like to thank the Deanship of Scientific Research at Umm Al-Qura
University for supporting this work by Grant Code: (22UQU4331128DSR05).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
Molecules 2022,27, 8705 14 of 16
References
1.
Cardia, G.F.E.; de Souza Silva-Comar, F.M.; da Rocha, E.M.T.; Silva-Filho, S.E.; Zagotto, M.; Uchida, N.S.; Do Amaral, V.;
Bersani-Amado, C.A.; Cuman, R.K.N. Pharmacological, medicinal and toxicological properties of lavender essential oil: A review.
Res. Soc. Dev. 2021,10, e23310514933. [CrossRef]
2.
Burt, S. Essential oils: Their antibacterial properties and potential applications in foods—A review. Int. J. Food Microbiol.
2004
,94,
223–253. [CrossRef] [PubMed]
3.
Saad, N.Y.; Muller, C.D.; Lobstein, A. Major bioactivities and mechanism of action of essential oils and their components. Flavour
Fragr. J. 2013,28, 269–279. [CrossRef]
4.
Perry, N.S.; Bollen, C.; Perry, E.K.; Ballard, C. Salvia for dementia therapy: Review of pharmacological activity and pilot tolerability
clinical trial. Pharmacol. Biochem. Behav. 2003,75, 651–659. [CrossRef] [PubMed]
5.
Ayaz, M.; Sadiq, A.; Junaid, M.; Ullah, F.; Subhan, F.; Ahmed, J. Neuroprotective and anti-aging potentials of essential oils from
aromatic and medicinal plants. Front. Aging Neurosci. 2017,9, 168. [CrossRef]
6.
Blowman, K.; Magalhães, M.; Lemos, M.F.L.; Cabral, C.; Pires, I.M. Anticancer properties of essential oils and other natural
products. Evid. Based Complement. Alternat. Med. 2018,2018, 3149362. [CrossRef]
7.
Chouhan, S.; Sharma, K.; Guleria, S. Antimicrobial activity of some essential oils—Present status and future perspectives.
Medicines 2017,4, 58. [CrossRef]
8. Nazzaro, F.; Fratianni, F.; Coppola, R.; Feo, V.D. Essential oils and antifungal activity. Pharmaceuticals 2017,10, 86. [CrossRef]
9. Campa, M.; Baron, E. Anti-aging effects of select botanicals: Scientific evidence and current trends. Cosmetics 2018,5, 54. [CrossRef]
10.
Ziosi, P.; Manfredini, S.; Vertuani, S.; Ruscetta, V.; Radice, M.; Sacchetti, G. Evaluating essential oils in cosmetics: Antioxidant
capacity and functionality. Cosmet. Toilet. 2010,125, 32–40.
11.
Jugreet, B.S.; Mahomoodally, M.F. Essential oils from 9 exotic and endemic medicinal plants from Mauritius shows
in vitro
antibacterial and antibiotic potentiating activities. S. Afr. J. Bot. 2020,132, 355–362. [CrossRef]
12.
Jugreet, B.S.; Kouadio Ibrahime, S.; Zengin, G.; Abdallah, H.H.; Mahomoodally, M.F. GC/MS profiling,
in vitro
and in silico
pharmacological screening and principal component analysis of essential oils from three exotic and two endemic plants from
Mauritius. Chem. Biodivers. 2021,18, e2000921.
13.
Jugreet, B.S.; Mahomoodally, M.F.; Sinan, K.I.; Zengin, G.; Abdallah, H.H. Chemical variability, pharmacologicalpotential, multivariate
and molecular docking analyses of essential oils obtained from four medicinal plants. Ind. Crops Prod. 2020,150, 112394.
14.
El Omari, K.; Hamze, M.; Alwan, S.; Osman, M.; Jama, C.; Chihib, N.E. In-vitro evaluation of the antibacterial activity of the
essential oils of Micromeria barbata,Eucalyptus globulus and Juniperus excelsa against strains of Mycobacterium tuberculosis (including
MDR), Mycobacterium kansasii and Mycobacterium gordonae.J. Infect. Public Health 2019,12, 615–618. [CrossRef] [PubMed]
15.
Jankute, M.; Nataraj, V.; Lee, O.Y.C.; Wu, H.H.; Ridell, M.; Garton, N.J.; Barer, M.R.; Minnikin, D.E.; Bhatt, A.; Besra, G.S. The role
of hydrophobicity in tuberculosis evolution and pathogenicity. Sci. Rep. 2017,7, 1315.
16.
Polyudova, T.V.; Eroshenko, D.V.; Pimenova, E.V. The biofilm formation of nontuberculous mycobacteria and its inhibition by
essential oils. Int. J. Mycobacteriol. 2021,10, 43. [PubMed]
17.
Lelovic, N.; Mitachi, K.; Yang, J.; Lemieux, M.R.; Ji, Y.; Kurosu, M. Application of Mycobacterium smegmatis as a surrogate to
evaluate drug leads against Mycobacterium tuberculosis.J. Antibiot. 2020,73, 780–789. [CrossRef]
18.
Xu, Y.; Wang, G.; Xu, M. Biohazard levels and biosafety protection for Mycobacterium tuberculosis strains with different virulence.
Biosaf. Health 2020,2, 135–141. [CrossRef]
19.
Bueno, J.; Escobar, P.; Martínez, J.R.; Leal, S.M.; Stashenko, E.E. Composition of three essential oils, and their mammalian cell
toxicity and antimycobacterial activity against drug resistant-tuberculosis and nontuberculous mycobacteria strains. Nat. Prod.
Commun. 2011,6, 1934578X1100601143. [CrossRef]
20.
Chraibi, M.; Farah, A.; Lebrazi, S.; El Amine, O.; Houssaini, M.I.; Fikri-Benbrahim, K. Antimycobacterial natural products from
Moroccan medicinal plants: Chemical composition, bacteriostatic and bactericidal profile of Thymus satureioides and Mentha
pulegium essential oils. Asian Pac. J. Trop. Biomed. 2016,6, 836–840.
21.
Zhang, N.; Yuan, R.; Xin, K.Z.; Lu, Z.; Ma, Y. Antimicrobial susceptibility, biotypes and phylotypes of clinical Cutibacterium (formerly
Propionibacterium)acnes strains isolated from acne patients: An observational study. Dermatol. Ther. 2019,9, 735–746. [CrossRef]
22.
Juliano, C.; Marchetti, M.; Pisu, M.L.; Usai, M.
In vitro
antimicrobial activity of essential oils from Sardinian flora against
Cutibacterium (Formerly Propionibacterium)acnes and its enhancement by chitosan. Sci. Pharm.
2018
,86, 40. [CrossRef] [PubMed]
23. Humphrey, S. Antibiotic resistance in acne treatment. Skin Ther. Lett. 2012,17, 1–3.
24.
Sardana, K.; Gupta, T.; Garg, V.K.; Ghunawat, S. Antibiotic resistance to Propionobacterium acnes: Worldwide scenario, diagnosis
and management. Expert Rev. Anti Infect. Ther. 2015,13, 883–896. [CrossRef] [PubMed]
25.
Cavero, R.Y.; Akerreta, S.; Calvo, M.I. Medicinal plants used for dermatological affections in Navarra and their pharmacological
validation. J. Ethnopharmacol. 2013,149, 533–542. [PubMed]
26.
Nelson, K.; Lyles, J.T.; Li, T.; Saitta, A.; Addie-Noye, E.; Tyler, P.; Quave, C.L. Anti-acne activity of Italian medicinal plants used
for skin infection. Front. Pharmacol. 2016,7, 425. [CrossRef]
27.
Orafidiya, L.O.; Agabani, E.O.; Oyedele, A.O. Preliminary clinical tests on topical preparations of Ocimum gratissimum Linn leaf
essential oil for the treatment of acne vulgaris. Clin. Drug Investig. 2002,22, 313–319. [CrossRef]
28.
Viyoch, J.; Pisutthanan, N.; Faikreua, A.; Nupangta, K.; Wangtorpol, K.; Ngokkuen, J. Evaluation of
in vitro
antimicrobial activity of
Thai basil oils and their micro-emulsion formulas against Propionibacterium acnes.Int. J. Cosmet. Sci. 2006,28, 125–133. [CrossRef]
Molecules 2022,27, 8705 15 of 16
29.
Bhatt, D.; Sachan, A.K.; Jain, S.; Barik, R. Studies on inhibitory effect of Eucalyptus oil on sebaceous glands for the management
of acne. Indian J. Nat. Prod. Resour. 2011,2, 345–349.
30.
Briganti, S.; Picardo, M. Antioxidant activity, lipid peroxidation and skin diseases. What’s new. J. Eur. Acad. Dermatol. Venereol.
2003,17, 663–669. [CrossRef]
31.
Mayslich, C.; Grange, P.A.; Dupin, N. Cutibacterium acnes as an opportunistic pathogen: An update of its virulence-associated
factors. Microorganisms 2021,9, 303. [CrossRef]
32.
Szabó, K.; Miskei, M.; Farkas, I.; Dombrádi, V. The phosphatome of opportunistic pathogen Candida species. Fungal Biol. Rev.
2021,35, 40–51.
33.
Verma, R.; Pradhan, D.; Hasan, Z.; Singh, H.; Jain, A.K.; Khan, L.A. A systematic review on distribution and antifungal resistance
pattern of Candida species in the Indian population. Med. Mycol. 2021,59, 1145–1165. [CrossRef] [PubMed]
34.
Asong, J.A.; Amoo, S.O.; McGaw, L.J.; Nkadimeng, S.M.; Aremu, A.O.; Otang-Mbeng, W. Antimicrobial activity, antioxidant potential,
cytotoxicity and phytochemical profiling of four plants locally used against skin diseases. Plants 2019,8, 350. [CrossRef] [PubMed]
35.
Pedroso, R.D.S.; Balbino, B.L.; Andrade, G.; Dias, M.C.P.S.; Alvarenga, T.A.; Pedroso, R.C.N.; Pimenta, L.P.; Lucarini, R.; Pauletti, P.M.;
Januário, A.H.; et al.
In vitro
and
in vivo
anti-Candida spp. activity of plant-derived products. Plants
2019
,8, 494. [CrossRef] [PubMed]
36.
Holanda, L.; Bezerra, G.B.; Ramos, C.S. Potent antifungal activity of essential oil from Morinda citrifolia fruits rich in short-chain
fatty acids. Int. J. Fruit Sci. 2020,20, S448–S454. [CrossRef]
37. ˙
I¸scan, G. Antibacterial and anticandidal activities of common essential oil constituents. Rec. Nat. Prod. 2017,11, 374–388.
38.
Osorio, E.; Bravo, K.; Cardona, W.; Yepes, A.; Osorio, E.H.; Coa, J.C. Antiaging activity, molecular docking, and prediction of
percutaneous absorption parameters of quinoline–hydrazone hybrids. Med. Chem. Res. 2019,28, 1959–1973. [CrossRef]
39.
Era, B.; Floris, S.; Sogos, V.; Porcedda, C.; Piras, A.; Medda, R.; Fais, A.; Pintus, F. Anti-aging potential of extracts from Washingtonia
filifera seeds. Plants 2021,10, 151. [CrossRef]
40.
Binic, I.; Lazarevic, V.; Ljubenovic, M.; Mojsa, J.; Sokolovic, D. Skin ageing: Natural weapons and strategies. Evid. Based
Complement. Alternat. Med. 2013,2013, 827248. [CrossRef]
41.
Mori, M.; Ikeda, N.; Kato, Y.; Minamino, M.; Watabe, K. Inhibition of elastase activity by essential oils
in vitro
.J. Cosmet. Dermatol.
2002,1, 183–187. [CrossRef]
42.
Tu, P.T.B.; Tawata, S. Anti-oxidant, anti-aging, and anti-melanogenic properties of the essential oils from two varieties of Alpinia
zerumbet.Molecules 2015,20, 16723–16740. [CrossRef]
43.
Aumeeruddy-Elalfi, Z.; Lall, N.; Fibrich, B.; Van Staden, A.B.; Hosenally, M.; Mahomoodally, M.F. Selected essential oils inhibit
key physiological enzymes and possess intracellular and extracellular antimelanogenic properties
in vitro
.J. Food Drug Anal.
2018,26, 232–243. [CrossRef] [PubMed]
44.
Zheng, Y.; Pan, C.; Zhang, Z.; Luo, W.; Liang, X.; Shi, Y.; Liang, L.; Zheng, X.; Zhang, L.; Du, Z. Antiaging effect of Curcuma longa
L. essential oil on ultraviolet-irradiated skin. Microchem. J. 2020,154, 104608. [CrossRef]
45.
Lohani, A.; Verma, A.; Hema, G.; Pathak, K. Topical delivery of geranium/calendula essential oil-entrapped ethanolic lipid
vesicular cream to combat skin aging. BioMed Res. Int. 2021,2021, 4593759. [CrossRef]
46.
Wang, G.; Zhu, W. Molecular docking for drug discovery and development: A widely used approach but far from perfect. Future
Med. Chem. 2016,8, 1707–1710. [CrossRef] [PubMed]
47.
Sethi, A.; Joshi, K.; Sasikala, K.; Alvala, M. Molecular docking in modern drug discovery: Principles and recent applications. In
Drug Discovery and Development—New Advances; IntechOpen: London, UK, 2019; Volume 2, pp. 1–21.
48.
Jacob, J.N.; Toloue, M. Biological studies of turmeric oil, Part 1: Selective
in vitro
anticancer activity of turmeric oil (TO) and
TO-paclitaxel combination. Nat. Prod. Commun. 2013,8, 1934578X1300800632. [CrossRef]
49.
Pérez-González, C.; Pérez-Ramos, J.; Méndez-Cuesta, C.A.; Serrano-Vega, R.; Martell-Mendoza, M.; Pérez-Gutiérrez, S. Cytotoxic
activity of essential oils of some species from Lamiaceae family. In Cytotoxicity-Definition, Identification, and Cytotoxic Compounds;
IntechOpen: London, UK, 2019; p. 29. [CrossRef]
50.
Cabral, C.; Efferth, T.; Pires, I.M.; Severino, P.; Lemos, M.F. Natural products as a source for new leads in cancer research and
treatment. Evid. Based Complement. Alternat. Med. 2018,2018, 8243680. [CrossRef]
51.
Legault, J.; Pichette, A. Potentiating effect of
β
-caryophyllene on anticancer activity of
α
-humulene, isocaryophyllene and
paclitaxel. J. Pharm. Pharmacol. 2007,59, 1643–1647. [CrossRef]
52.
Rabi, T.; Bishayee, A. D-Limonene sensitizes docetaxel-induced cytotoxicity in human prostate cancer cells: Generation of reactive
oxygen species and induction of apoptosis. J. Carcinog. 2009,8, 9.
53.
Lall, N.; Chrysargyris, A.; Lambrechts, I.; Fibrich, B.; Blom Van Staden, A.; Twilley, D.; De Canha, M.N.; Ooshuizen, C.B.; Bodiba,
D.; Tzortzakis, N. Sideritis perfoliata (subsp. perfoliata) nutritive value and its potential medicinal properties. Antioxidants
2019
,8,
521.
54.
Sharifi-Rad, J.; Sureda, A.; Tenore, G.C.; Daglia, M.; Sharifi-Rad, M.; Valussi, M.; Tundis, R.; Sharifi-Rad, M.; Loizzo, M.R.;
Ademiluyi, A.O.; et al. Biological activities of essential oils: From plant chemoecology to traditional healing systems. Molecules
2017,22, 70. [CrossRef] [PubMed]
55.
Prashar, A.; Locke, I.C.; Evans, C.S. Cytotoxicity of lavender oil and its major components to human skin cells. Cell Prolif.
2004
,37,
221–229. [CrossRef] [PubMed]
56.
Bayala, B.; Bassole, I.H.; Scifo, R.; Gnoula, C.; Morel, L.; Lobaccaro, J.M.A.; Simpore, J. Anticancer activity of essential oils and
their chemical components—A review. Am. J. Cancer Res. 2014,4, 591. [PubMed]
Molecules 2022,27, 8705 16 of 16
57.
Bhalla, Y.; Gupta, V.K.; Jaitak, V. Anticancer activity of essential oils: A review. J. Sci. Food Agric.
2013
,93, 3643–3653. [CrossRef]
58.
Katiki, L.M.; Barbieri, A.M.E.; Araujo, R.C.; Veríssimo, C.J.; Louvandini, H.; Ferreira, J.F.S. Synergistic interaction of ten essential
oils against Haemonchus contortus in vitro. Vet. Parasitol. 2017,243, 47–51. [CrossRef]
59.
Sharma, M.; Grewal, K.; Jandrotia, R.; Batish, D.R.; Singh, H.P.; Kohli, R.K. Essential oils as anticancer agents: Potential role in
malignancies, drug delivery mechanisms, and immune system enhancement. Biomed. Pharmacother.
2022
,146, 112514. [CrossRef]
60.
Russo, R.; Corasaniti, M.T.; Bagetta, G.; Morrone, L.A. Exploitation of cytotoxicity of some essential oils for translation in cancer
therapy. Evid. Based Complement. Alternat. Med. 2015,2015, 397821. [CrossRef]
61.
Liju, V.B.; Jeena, K.; Kuttan, R. Article details cytotoxicity, antitumour and anticarcinogenic activity of Curcuma longa essential oil.
Indian Drugs 2014,51, 28–34. [CrossRef]
62.
Piaru, S.P.; Mahmud, R.; Abdul Majid, A.M.S.; Ismail, S.; Man, C.N. Chemical composition, antioxidant and cytotoxicity activities
of the essential oils of Myristica fragrans and Morinda citrifolia.J. Sci. Food Agric. 2012,92, 593–597. [CrossRef]
63.
Manjamalai, A.; Grace, V.B. The chemotherapeutic effect of essential oil of Plectranthus amboinicus (Lour) on lung metastasis
developed by B16F-10 cell line in C57BL/6 mice. Cancer Investig. 2013,31, 74–82. [CrossRef] [PubMed]
64.
Moayedi, Y.; Greenberg, S.A.; Jenkins, B.A.; Marshall, K.L.; Dimitrov, L.V.; Nelson, A.M.; Owens, D.M.; Lumpkin, E.A. Camphor white
oil induces tumor regression through cytotoxic T cell-dependent mechanisms. Mol. Carcinog. 2019,58, 722–734. [CrossRef] [PubMed]
65.
Aumeeruddy-Elalfi, Z.; Gurib-Fakim, A.; Mahomoodally, F. Antimicrobial, antibiotic potentiating activity and phytochemical
profile of essential oils from exotic and endemic medicinal plants of Mauritius. Ind. Crops Prod. 2015,71, 197–204. [CrossRef]
66.
Lall, N.; Henley-Smith, C.J.; De Canha, M.N.; Oosthuizen, C.B.; Berrington, D. Viability reagent, PrestoBlue, in comparison with
other available reagents, utilized in cytotoxicity and antimicrobial assays. Int. J. Microbiol. 2013,2013, 420601. [CrossRef]
67.
Kamatou, G.P.P. Indigenous Salvia Species: An Investigation of Their Pharmacological Activities and Phytochemistry. Ph.D.
Thesis, University of the Witwatersrand, Johannesburg, South Africa, 2006.
68.
Seebaluck-Sandoram, R.; Lall, N.; Fibrich, B.; Van Staden, A.B.; Mahomoodally, F. Antibiotic-potentiating activity, phytochemical
profile, and cytotoxicity of Acalypha integrifolia Willd.(Euphorbiaceae). J. Herb. Med. 2018,11, 53–59. [CrossRef]
69.
Lall, N.; Kishore, N.; Fibrich, B.; Lambrechts, I.A.
In vitro
and
in vivo
activity of Myrsine africana on elastase inhibition and
anti-wrinkle activity. Pharmacogn. Mag. 2017,13, 583–589. [CrossRef]
70.
Pientaweeratch, S.; Panapisal, V.; Tansirikongkol, A. Antioxidant, anti-collagenase and anti-elastase activities of Phyllanthus emblica,
Manilkara zapota and silymarin: An
in vitro
comparative study for anti-aging applications. Pharm. Biol.
2016
,54, 1865–1872. [CrossRef]
71.
Irwin, J.J.; Sterling, T.; Mysinger, M.M.; Bolstad, E.S.; Coleman, R.G. ZINC: A free tool to discover chemistry for biology. J. Chem.
Inf. Model. 2012,52, 1757–1768. [CrossRef]
72.
Frisch, M.J.; Trucks, G.W.; Schlegel, H.B.; Scuseria, G.E.; Robb, M.A.; Cheeseman, J.R.; Scalmani, G.; Barone, V.; Mennucci, B.;
Petersson, G.A.; et al. Gaussian 09 Revision D. 01; Gaussian Inc.: Wallingford, CT, USA, 2009; p. 106.
